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Microbial utilization of cathodic hydrogen and related corrosion

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Microbial utilization of cathodic hydrogen and related corrosion
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Sifontes, Jose Rafael, 1949-
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English
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xiii, 185 leaves : ill. ; 29 cm.

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Subjects / Keywords:
Bacteria ( jstor )
Bioreactors ( jstor )
Corrosion ( jstor )
Coupons ( jstor )
Flasks ( jstor )
Hydrogen ( jstor )
Microbial corrosion ( jstor )
Microorganisms ( jstor )
Minerals ( jstor )
Steels ( jstor )
Agricultural Engineering thesis Ph.D
Dissertations, Academic -- Agricultural Engineering -- UF
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bibliography ( marcgt )
non-fiction ( marcgt )

Notes

Thesis:
Thesis (Ph. D.)--University of Florida, 1994.
Bibliography:
Includes bibliographical references (leaves 170-181).
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by Jose Rafael Sifontes.

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University of Florida
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University of Florida
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Copyright [name of dissertation author]. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
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33032691 ( OCLC )
AKL5727 ( NOTIS )

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MICROBIAL UTILIZATION OF CATHODIC HYDROGEN
AND RELATED CORROSION



















By

JOSE RAFAEL SIFONTES


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA


1994


































To the memory of my father, Pedro Rafael Sifontes Lopez.















ACKNOWLEDGEMENTS


I wish to thank the following persons for their

contributions throughout my graduate studies at the

Agricultural Engineering Department. My sincere appreciation

goes to Dr. David P. Chynoweth, chairman of my supervisory

committee, for his friendship, concern, and economic support

throughout this work. I also would like to thank Dr. Henry

Aldrich, Dr. Edward Lincoln, Dr. Mark Orazem, Dr. Roger

Nordstedt, and Dr. Ellis Verink for always being there when I

needed them and for access to their laboratories.

My special thanks go to all my friends and departmental

staff who encouraged me, particularly to Dr. K.T. Shanmugam,

Jose Moratalla, Paul Lane, Brian Ferber, Emo Crews, Larry

Miller, and Hyung-Jib Lee for their help and encouragement

during my stay at the Agricultural Engineering Department.

This work was initially supported by the Gas Research

Institute.

And most important, I want to thank God in the name of my

Lord Jesus Christ for allowing this to happen. To HIM be all

the Glory!


iii















TABLE OF CONTENTS


ACKNOWLEDGEMENTS ....................................iii

LIST OF TABLES ...................................... vi

LIST OF FIGURES .....................................vii

ABSTRACT ................................ ........... x

CHAPTERS

1 INTRODUCTION .............. .................... 1

Objectives ...................................... 1
Justification ..................................... 2
Background ............................................... 7
The Defective and Nonuniform Nature of
Metallic Surfaces ........................ 10
The Molecular Fluctuations of the
Electrical Double Layer ................. 12
The Mass Transport Perturbation in the
Diffusion Layer .......................... 13
Statement of the Problem ....................... 14

2 REVIEW OF LITERATURE .......................... 17

Historical Overview ........................... 17
Other Aspects of Microbial Corrosion ........... 24
Microorganisms Involved in Microbial Corrosion.. 27
Mechanisms of Microbial Corrosion .............. 31
Theory of Cathodic Depolarization .............. 34
Previous Studies in Microbial Corrosion ........ 37
Microbial Corrosion Control ................... 42
Research Approach .............................. 47

3 MATERIALS AND METHODS .......................... 49

Introduction ................................... 49
Flowthrough Bioreactor ......................... 50
Description ....................... ....... 50
Operation ................................. 54
Batch Bioreactor ............................... 58
Description ............................... 58
Operation ............................... 59









Electrochemical Cells Description .............. 60
Electrochemical Cells Operations ............. 62
Single Flask Electrochemical Cell ......... 62
Dual Flask Electrochemical Cell ........... 64
Triad Flask Electrochemical Cell .......... 65
Preparation of Bacterial Suspensions ........... 67
Preparation of Mineral Solution Electrolyte..... 69
Metal Coupon .................................. 70
Parameter Setting ............................. 71
Measurement Procedures .................... 73
Analytical Procedures ..................... 75
Gases ..................................... 76
Scanning Electron Microscopy .............. 77
Light Microscopy .......................... 77

4 RESULTS AND DISCUSSION ....................... 78

Flowthrough Bioreactor ......................... 78
Batch Bioreactor .............................. 92
Effect of Head Space Gas Composition ...... 93
Effect of Bacteria Combination .......... 95
Selection of Media ....................... 95
Selection of a Reducing Agent ............. 96
Selection of a Sterilization Method ....... 97
Experimental Results ..................... 98
The Electrochemical Cell .......................106
Single Flask Electrochemical Cell ..............113
Dual Flask Electrochemical Cell ..............117
Triad Flask Electrochemical Cell ...............125
Final Discussion .......................... 137
New Proposed Microbial Corrosion Mechanism .....154
Capabilities of the Triad Cell ................156

5 SUMMARY AND CONCLUSIONS .......................161

Summary ........................................161
Conclusions ....................................165
Suggested Future Research......................168

LITERATURE CITED ....................................170

BIOGRAPHICAL SKETCH ............................... 182














LIST OF TABLES


Table page

2-1 Chemistry of the cathodic depolarization theory... 35

3-1 Protocol for metallic surface fixation for
scanning electron microscopy examinations ........ 55

3-2 Glucose Lactate Yeast Extract (GLYE)
Medium Composition ............................... 56

3-3 Trace Vitamins Solution Composition .............. 57

3-4 Mineral Solution Composition .................... 57

4-1 Batch bioreactor volatile fatty acids
measurements from experiments using steel
coupons and bacterial triculture in mineral
solution ......................................... 99

4-2 Batch bioreactor head-space pressure drop in
GLYE and mineral solution in the absence of
bacteria ........................ ....... .........102

4-3 Hydrogen production by filings of Mg, Zn,
and SA106 steel in mineral solution in the
absence of bacteria after 3 days ................. 107

4-4 Free corrosion potential of SA106 steel
coupons at different surface polishing grades ....108

4-5 Galvanic series of selected metals and alloys
in mineral solution at 30C vs Ag/AgCl
reference electrode in the absence of bacteria ...110

4-6 Effect of media composition on the hydrogen
uptake of E. coli at 300C during 6 hours .........112

4-7 Bacteria hydrogen uptake capabilities in
mineral solution at 300C during 6 hours ..........113

4-8 Effect of different bacteria on the free
corrosion potential of Mg, Cu, and SA106 steel
coupons in mineral solution at 30 C ..............115








4-9 Dual cell schedule of experiments and
corresponding corrosion rates, using E. coli
(JW111) and SA106 steel coupons ..................118

4-10 Triad cell schedule of runs and related
corrosion USING E. coli (JW111) and SA106
steel coupons ....................................127


vii














LIST OF FIGURES


Figure page

3-1 Flowthrough bioreactor general description of
components ........................... .......... 51
3-2 Metal coupon and fluorescence probe setup ........ 53
3-3 Metal coupon, fluorescence probe and reference
electrode setup ............................. ..... 53
3-4 Flowthrough bioreactor components ................. 54

3-5 Batch Bioreactor ................................ 59

3-6 Schematic of electrochemical cell and data
acquisition system .............................. 61

3-7 Single flask electrochemical cell ................ 62

3-8 Dual flask electrochemical cell ................ 64

3-9 Triad flask electrochemical cell ................. 65

4-1 Flowthrough bioreactor results on bacteria
attachment to carbon steel coupons.
a) SEM micrograph showing bacteria within
the outer and inner structure of the
biofilm ....................................... 81
b) SEM micrograph indicating the nature of the
outer biofilm structure ....................... 81
c) SEM micrograph showing bacteria on inner
biofilm ....................................... 82

4-2 SEM characterization of bacterial species from
triculture.
a) Enterobacter aerogenes ......................... 82
b) Clostridium acetobutylicum ................ 83
c) Desulfovibrio desulfuricans ................... 83

4-3 Flowthrough bioreactor SEM results of microbial
corrosion of carbon steel coupons.
a) Control surface of coupon unexposed to
bacteria and polished to 240 grid ............. 85
b) Localized pitting near biofilm after exposure
to bacteria triculture ........................ 85
c) SEM micrograph showing microbial corrosion
near biofilm .................................. 86


viii










d) SEM micrograph showing generalized metal
deterioration after exposure to bacteria ...... 86
e) SEM micrograph showing elongated pits ......... 87

4-4 Flowthrough bioreactor results of EDXA analysis.
a) Control EDXA spectrum of a steel coupon
unexposed to bacteria indicating the metallic
components of the SA106 alloy ................. 87
b) EDXA spectrum of a metal surface exposed to
bacteria showing the sulfur peak resulting
from metal sulfides deposition by
Desulfovibrio desulfuricans ................... 88

4-5 Flowthrough bioreactor biofilm appearance
under the stereo microscope.
a) Flat-side view .............................. 88
b) Edge-side view .............................. 89

4-6 Flowthrough bioreactor volatile fatty acids
and pH profile ................................... 89

4-7 Flowthrough bioreactor-second experiment.
a) SEM micrograph showing appearance of biofilm .. 90
b) SEM micrograph indicating massive pitting ..... 91

4-8 Flowthrough bioreactor EDXA spectrum of metal
coupon exposed to bacteria ...................... 91

4-9 Batch bioreactor biofilm formed within
24 hours .................................... ... 100

4-10 Batch bioreactor SEM and EDXA results
on bacterial attachment to carbon steel.
a) Bacteria colonization within 24 hours .........100
b) EDXA spectrum of metal coupon exposed
to bacteria for 24 hours ......................101

4-11 Batch bioreactor-formation of a crystalline
film on the metal surface after one week of
exposure to the mineral solution in the absence
of bacteria.
a) SEM micrograph showing characteristics of the
crystalline film ..............................104
b) EDXA spectrum showing elemental distribution
of crystalline film on metal surface ..........104

4-12 Batch bioreactor-formation of crystalline film
on metal surface after exposure to bacteria.
a) SEM micrograph showing a transition zone
from biofilm to crystalline film ..............105








b) SEM micrograph showing a deteriorated
polymeric biofilm structure and pitting
after bacteria have died ...................... 105

4-13 Potential-time curve for SA106 steel in the
absence of bacteria ..............................109

4-14 Growth curve for E. coli at 600 nm, 370C and
trypticase soy broth medium ......................111

4-15 Potential-time curves for SA106 steel in the
presence of E. coli ...........................114

4-16 Potential-time curves for Mg in the presence
of E. coli ......... ............................... 114

4-17 Dual cell run potential-time curves.
a) Effect of E. coli, 6 hr old ...................121
b) Effect of E. coli, 15 hr old .................122
c) Effect of E. coli, 23 hr old ..................123
d) Effect of E. coli, 27 hr old and fumarate .....124

4-18 Dual cell run dissolved iron profiles.
a) Effect of E. coli, 6 hr old ..................121
b) Effect of E. coli, 15 hr old ................. 122
c) Effect of E. coli, 23 hr old ..................123
d) Effect of E. coli, 27 hr old and fumarate .....124

4-19 Triad cell run potential-time curves.
a) Effect of E. coli, 8 hr old ...................129
b) Effect of E. coli, 20 hr old and fumarate .....130
c) Effect of E. coli, >6 days old ...............132

4-20 Triad cell run dissolved iron profiles.
a) Effect of E. coli, 8 hr old ...................129
b) Effect of E. coli, 20 hr old and fumarate .....130
c) Effect of E. coli, >6 days old ................132

4-21 Triad cell artificial Hydrogen uptake test .......134

4-22 Artificial hydrogen uptake in a triad cell
using Cu and Mg electrodes .....................136

4-23 Hydrogen embrittlement of SA106 steel in
mineral solution electrolyte.
a) SEM micrograph of crack at a
low magnification ...........................148
b) SEM micrograph of crack at a
high magnification ............................148
c) EDXA micrograph of corrosion products
inside the crack .............................149








d) EDXA micrograph of uncracked control
surface .................................... 149

4-24 Proposed anaerobic mechanism for the on-set of
microbial corrosion of steel by hydrogen
oxidizing bacteria .............................155














Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

MICROBIAL UTILIZATION OF CATHODIC HYDROGEN
AND RELATED CORROSION

By

Jose Rafael Sifontes

August 1994

Chairman: David P. Chynoweth
Major Department: Agricultural Engineering

Microbial corrosion is the deterioration of a material by

corrosion processes that occur directly or indirectly as a

result of the activity of microorganisms. Although it is a

widely recognized phenomenon in many industrial processes

worldwide, the fundamental corrosion mechanisms are not

understood, it is not well defined, and the microbial effect

has not been quantified. The objective of this research was

to show a relationship between cathodic hydrogen utilization

by bacteria and corrosion at its onset for the purpose of

improving its understanding and attempting to quantify the

biological component of corrosion.

Two new experimental flowthrough and batch bioreactor

systems were evaluated to reproduce anaerobic microbial

corrosion. Both reactors were able to reproduce microbial

corrosion in 24 hr. However, the batch system offered the


xii








better alternative to study microbial corrosion at an

attractive economic operation. Since microbial corrosion is

difficult to separate from pure electrochemical corrosion and

electrochemical measurements offers a nondestructive technique

to measure corrosion, the batch bioreactor was instrumented

for electrochemical measurements and for physicochemical

analysis.

Results from potential-time curves, dissolved iron

profiles, and differential corrosion currents indicated that

metals were reactive to the presence of bacteria. However,

the results were not adequate to quantify the biological

component of corrosion. Significant increases in corrosion

rates, observed during the addition of a terminal electron

acceptor, suggested a catalytic effect on the bacterial

hydrogenase system. The observation of cracks on steel

samples suggested the occurrence of hydrogen embrittlement.

The results of this research are important because they

offer a new approach for the investigation of microbial

corrosion, the triad cell offers an opportunity to quantify

the biological component of corrosion, and a new mechanism was

proposed for the understanding of the onset of anaerobic

microbial corrosion.


xiii














CHAPTER 1
INTRODUCTION


Objectives

The overall objective of this research was to show a

direct relationship between corrosion of metals and hydrogen

utilization by bacteria during the onset of microbial

corrosion, for the purpose of improving our understanding of

the phenomenon. A new approach to the investigation of

microbial corrosion was selected to break ground in an

alternative direction to the one established over the past

century and for a better comprehension of the fundamental

microbial corrosion mechanisms.

The specific objectives of the study were to

1) evaluate new experimental flowthrough and batch bioreactors

to adequately reproduce anaerobic microbial corrosion,

2) quantify the biological component of corrosion at its onset

by measuring indirectly hydrogen uptake by bacteria and

correlating it to total metal corrosion, using nondisrupting

electrochemical techniques,

3) incorporate into a comprehensive microbial corrosion model

all concepts developed during the investigation.










Justification


The ability of microorganisms to induce or influence the

deterioration of metals has been known for over a century, and

now it is a widely recognized problem in most industrial

processes worldwide. The involvement of microorganisms in

metal deterioration has led to the question of how biological

agents affect the classical corrosion mechanisms.

The true economic impact of metals deterioration is very

difficult to determine. However, some estimates have been

made in the USA and the UK. In the USA, the cost of corrosion

estimated by Poff (1985) and a projection by the National

Bureau of Standards for the year 1985 was approximately $140

billion and $170 billion, respectively. As for microbial

metal deterioration, studies performed by Butlin et al.

(1952), Allred et al. (1959), Booth (1964), and the National

Corrosion Service (Wakerly, 1969) estimated that metallic

corrosion caused by microbial intervention in a wide range of

industrial cases in the UK was on the order of 70%, 77%, 50%,

and 10%, respectively, of the total corrosion problem. In the

USA, Paternaude (1985) indicated that 50% of the steel culvert

pipe corrosion in Wisconsin was due to sulfate reducing

bacteria. If the lowest 10% is selected, the cost of

microbial corrosion represents a $17 billion per year problem

in the USA alone. Although such calculations may be open to

objections on matters of detail, the magnitude is likely to








3

stay high whatever route is followed to arrive at a final cost

because the above costs include replacement, prevention, and

maintenance but exclude losses of time, money, natural

resources, human suffering, and death due to equipment

failure.

Metallic corrosion is a natural process in which metals

return to their natural, oxidized states. It is an

interdisciplinary field of engineering and science where a

knowledge of the metallurgy of the metal, the environmental

conditions, the chemical composition, and the electrochemistry

of the system, are essential to understanding the process.

For example, hydrogen embrittlement is too complex for one of

the above disciplines to explain, and despite several decades

of investigation it is still unclear why hydrogen embrittles

some metals and alloys and not others (Oriani, 1987; Wilde and

Kim, 1986). Most corrosion processes are essentially surface

electrochemical mechanisms common to most metals in aqueous,

or at least humid, environments. This conclusion was first

reached by Whitney (1903). During the manufacturing process

of metals from their ores, metals are converted to a reduced

state, which makes them thermodynamically unstable in the

presence of oxygen, except gold.

Microbial corrosion is defined as the deterioration of

materials by natural processes that are directly or indirectly

related to the activity of microorganisms. The microbial

corrosion phenomenon has been known for nearly a century. It









4
has been reported to exist almost anyplace that microorganisms

colonize. In the process industry, it can happen inside and

outside of equipment and in aerobic or anaerobic environments.

Microbial corrosion is a diverse and complex phenomenon, and

the literature associated with it tends to be just as diverse

and complex. Microbial corrosion was well recognized and

established as a serious problem in the 1970s (Tatnall, 1981).

Microbial corrosion is difficult to separate from pure

electrochemical corrosion. Microorganisms involved in

microbial corrosion generally do not lead to a new form of

corrosion, but to a stimulation of the normal electrochemical

corrosion process. Consequently, if the microbes interfere

with an electrode process, with their own metabolism or

presence, it is a direct effect, otherwise it is an indirect

effect.

To demonstrate microbial corrosion, the presence of

microorganisms must be shown to induce or influence metal loss

during corrosion. Unfortunately, microbial corrosion in real

cases is very difficult to reproduce, extremely complex, and

difficult to model (Tatnall, 1988). Laboratory-based

controlled experiments using defined media and characterized

pure cultures do not often yield the expected results because

a broad range of variables makes it difficult to simulate the

dynamic natural environment (Allsopp and Seal, 1986).

The study of microbial corrosion has had a tendency to

consider processes in isolation rather than actual microbial









5
corrosion cases. For instance, there are many studies dealing

with the effect of a single species of microorganisms on metal

corrosion (Tatnall, 1988). While such investigations are

valuable in elucidating mechanisms, they give little insight

into the wider biological/metal/fluid interactions. Although

recent reappraisal of the role of bacterial consortia together

with studies of the effect of such consortia have improved our

knowledge of the microbial corrosion process, few studies of

microbial corrosion look beyond bacterial consortia and

surface corrosion effects to wider biological/metal/fluid

interactions. Furthermore, any living or indeed dead organism

that becomes associated with a metal surface immersed in an

electrolyte has the potential to influence the corrosion of

that metal. Edyvean (1988) in his recent work discusses the

interaction of both bacteria and macro- and micro-algae in the

fouling community on steel substrata in sea water.

Most engineering materials in general use are susceptible

to some form of microbial corrosion. Several of the metals

and alloys reported as being susceptible to microbial

corrosion include iron, copper, aluminum, nickel, cobalt, and

zinc and their alloys (Gabrielli, 1988; Zamanzadeh et al.,

1989; Griffin et al., 1989). The industries affected by

microbial corrosion have been identified as wastewater

facilities, water flood control systems, petrochemical

equipment, cooling water systems, underground structures and

pipelines, aircraft fuel systems, ships and marine structures,








6

chemical process industries, power generation industries, and

paper mills.

From case histories of microbial corrosion, the forms of

corrosion that are stimulated by the interaction of

microorganisms with metals range from general pitting

corrosion, crevice corrosion, and stress corrosion cracking to

enhancement of corrosion fatigue, intergranular stress

cracking, and hydrogen embrittlement with cracking (Sifontes

and Block, 1991).

The fundamental mechanisms that drive microbial corrosion

are not properly understood nor have they been well defined.

Consequently, additional research to understand them is

required. Most work to date in microbial corrosion has dealt

with reports of case histories or observations incidental to

the main study, with poor documentation regarding the

physical, chemical, and microbiological conditions under which

it occurred (Tatnall, 1981, 1988).

Data from the literature surveyed suggest that

qualitative relationships exist between the surface properties

of the various metals and their alloys and the extent of

biological response in relation to corrosion. The fact is

that it is not well known why some metals are more susceptible

to microbial corrosion than are others and why the microbial

corrosion effect of a particular microorganism is different

from the microbial corrosion effect of similar or different

species.










Background



The literature indicates that the first time a mechanism

for microbial corrosion was proposed was in 1934 by von

Wolzogen Kuhr and van der Vlught. This theory initiated

systematic studies on microbial corrosion and identified

formally the components of the microbial corrosion phenomenon,

namely, the metal surface, the suspended fluid, and the

microorganisms. They proposed that sulfur reducing bacteria

accelerated the corrosion of ferrous metals by cathodic

depolarization, that is, by removing adsorbed hydrogen from

the cathodic surfaces of the metal.

The theory appeared to be relatively simple to confirm by

conventional electrochemical techniques. However, qualitative

confirmation has been obtained only for specific hydrogenase-

positive species of microorganisms such as sulfate reducing

bacteria (Booth and Tiller, 1960) and methanogens (Daniels et

al., 1987). Belay and Daniels (1990) reported that besides

iron, other metals (Al, Zn, Ni, and As) can also be oxidized

via cathodic depolarization.

In the work performed by Daniels and coworkers (1987), it

was demonstrated that methanogenic bacteria use either pure

elemental iron or iron from mild steel as a source of

electrons in the reduction of carbon dioxide to methane.

These bacteria use the oxidation of iron for energy generation

and growth. The mechanism of iron oxidation is cathodic








8

depolarization, in which electrons from iron and hydrogen ions

from water produce molecular hydrogen, which is then released

for use by the methanogens.

The extent of bacterial adhesion is determined by the

surface properties of the phases involved and the need of

bacteria to locate energy sources. Bacterial cells will stick

to most surfaces, whether the surfaces are those of other

cells or merely inert material such as metals. In nutrient-

poor environments (e.g., water transmission pipelines), most

bacteria grow attached to surfaces mainly due to

hydrophobicity.

It is the belief of some researchers that initially,

sessile bacteria adhere randomly to metal surfaces by means of

their production of extracellular polysaccharides. The

continued production of the polysaccharide and the

reproduction of the bacteria lead to the development of

biofilms in which a consortium of cells interact in a hydrated

matrix of anionic polysaccharide polymers that provide

protection from natural or synthetically produced

antimicrobial agents (Costerton and Geesey, 1985).

As a bacterium begins to proliferate within biofilms, its

metabolic products stimulate the growth of other organisms.

As the different microorganisms develop, molecular or proton

exchanges occur; consortia such as these have been detected

and associated with microbial corrosion of metallic surfaces.








9

In many neutral solutions the corrosion of the common

structural metals appears to be associated with the flow of

electric currents between various parts of the metal surface

at finite distances from one another (Mears and Brown, 1941).

This statement is supported by much evidence in the case of

steels, where the quantities of current flowing during

corrosion account for the amount of corrosion that occurs. In

other words, the corrosion of metals and their alloys in

neutral solutions is electrochemical in nature. Furthermore,

for corrosion to occur, it requires all four components of an

electrochemical cell which include: 1) electrodes, 2)

electrolyte, 3) potential difference, and 4) electrical

continuity. When one of these conditions is regulated, then

the corrosion process is controlled.

Among some of the nonbiological factors of microbial

corrosion that corrosion engineers and scientists have

associated with corrosion and have known for several decades

are 1) impurities in the corroding metal, 2) grain boundaries,

3) orientation of grains, 4) differential grain size, 5)

differential thermal treatment, 6) surface roughness, 7) local

scratches and abrasions, 8) difference in shape, 9)

differential strain, 10) differential pre-exposure to air or

oxygen, 11) differential concentration or composition of the

corroding solution, 12) differential aeration, 13)

differential heating, 14) differential illumination, 15)

differential agitation, 16) contact with dissimilar metals,








10

17) externally applied potential, and 18) complex cells (Mears

and Brown, 1941). Those factors are closely related with the

so-colled "eight forms" of corrosion, which include uniform,

galvanic, crevice, pitting, intergranular, dealloying,

erosion, and stress corrosion (Fontana and Green, 1987).

In addition to the above, there is always a lack of

homogeneity at the metal-electrolyte interface during the

microbial corrosion of metals. Consequently, several of the

following specific interactions need to be recognized (Sato,

1987).



The Defective and Nonuniform Nature of Metallic Surfaces

During the formation of a metal or an alloy, different

elements compose the molten metal. When solidification

starts, metal is formed of crystalline grains that grow in

size and they meet each other to form grain boundaries until

the metal is all solid. Once the grains are formed, the

overall energy of the individual metal atoms

lowers and the grain boundaries remain as sites of higher

energy or higher thermodynamic instability (Smith, 1986).

The process of electron transfer is suspected to take

place at metallic surface locations of higher thermodynamic

instability such as the grain boundaries. This fact may be

explained by the nature of the metallic bond where the metal

ions occupy positions in the crystal lattice of the grains and

the outer valence electrons are shared by the surrounding









11
metal atoms. These electrons are like a cloud that moves

freely throughout the lattice and binds the crystal together.

Consequently, microorganisms can take advantage of these

electrons as their energy source for growth and affect the

stability of the metal atoms.

In addition, the steel-making process and treatment

influences the microstructure of the alloy by producing

microcompositional differences that have different

electrochemical behavior, which leads to the formation of

localized corrosion. Other changes in the alloy's internal

structure, such as aging and welding, may occur after it has

been established during fabrication.

Among other metallurgical factors to be considered at the

onset of microbial corrosion are the following

a) Composition of alloying elements and impurities

(inclusions), cooling conditions and post heat treatments have

a marked influence on their size, shape, number, and

distribution. These constituents are never uniformly

distributed throughout the alloy and certain elements tend to

congregate in localized concentrations as second phase

particles in grain boundaries, as pure compounds or as

intermetallic compounds (Godard,1980). These particles play

a major role in the corrosion behavior of alloys, especially

in the case of pitting corrosion.

b) Surface contamination by mill scale during the rolling

of the steel causes severe localized corrosion at breaks and








12

imperfections in the surface and may need to be removed before

service. Steel can also be contaminated during forging with

particles of metal from the forming equipment and consequently

produce serious localized corrosion, too.



The Molecular Fluctuations of the Electrical Double Layer

For iron, there exists a spatial fluctuation of the

electrical double layer potential on the metal surface in the

order of 0.8 V for every 2-3 atomic distances (Sato, 1987).

One possible reason is that, in addition to the heterogeneous

metal surface, water dipoles tend to point nearly

perpendicular to the metal surface, while the two nearest

dipoles tend to align antiparallel to each other in order to

minimize their dipole interaction energy. Consequently,

reorientation of water dipoles in the electrical double layer

occur in a time scale much greater than the time scale

relevant to the electron fluctuation. In addition, water

dipoles require greater time to reorient at surface defects

sites where the adsorption energy of water dipoles is greater

(Sato, 1987). This fact causes the electron transfer to take

place unevenly on the metal surface. At certain surface

defect sites, local enhancement of the electrical field in the

electrical double layer may be maintained for a sufficiently

long time so that the electrode reaction takes place

preferentially at these sites, making them active reaction

sites. Other sources of the spatial non-uniform electrical








13

double layer potential are specific ion adsorption. In this

case, the ions divest of their hydration water molecules and

come into direct contact with the metal surface. This causes

a local enhancement (approximately 3 times) of the electrical

field in the inner part of the electrical double layer. This

phenomenon takes place preferentially at certain lattice or

defect sites and will generate active spots for some electrode

reactions.



The Mass Transport Perturbation in the Diffusion Layer

This dynamic perturbation causes the local ion

concentration and the local electric field to fluctuate in the

diffusion layer. In addition, when the corroding metal

surface is usually covered with a porous corrosion precipitate

film of hydrated metal oxides or insoluble salt, selective

mass transport occurs, and this film either accelerates or

decelerates further corrosion of the underlying metal. When

those interactions are favorable to corrosion, chemical and/or

electrochemical active surface sites occur that result in a

corrosion activity. The nonuniform nature of the metal-

electrolyte interface, whether passive or not, implies that

the corrosion process takes place preferentially at specific

sites that somehow differ energetically from other parts of

the metal surface. These specific sites may be permanently

localized or spatially fluctuate during the progress of

corrosion.








14

As a result of the above, the microbial corrosion

phenomenon is presumably initiated by the interaction of the

metal, the bacteria, and the electrolyte at the onset of the

process, followed by the formation of either differential

concentration cells or local electrochemical corrosion cells

within the biofilm, which further complicates the corrosion

mechanism.

Microorganisms selected for this study had to contain a

hydrogenase enzyme. Such microbes belong to the facultative

lithotrophs, sulfur oxidizing bacteria, phototrophics,

methanogens, and denitrifying bacteria. These bacteria are

all hydrogen oxidizing bacteria that use molecular hydrogen as

their electron donor. In anaerobic environments hydrogen is

generated from the processes of fermentation or metal

oxidation. Hydrogen oxidizing bacteria may grow

autotrophically on hydrogen carbon dioxide as an electron

acceptor.



Statement of the Problem



The cathodic depolarization theory of von Wolzegen Kuhr

and van der Vlugt (1934) has become the center of attention of

researchers in the field because it proposes a logical

separation of the components of corrosion: biological,

metallic, and fluid. As a result, much of the literature on








15

microbial corrosion has been influenced by this theory and has

referred to it, either to criticize or prove it.

In work performed by Daniels and coworkers (1987), it was

demonstrated that methanogenic bacteria used hydrogen produced

from iron oxidation of carbon dioxide to methane. The

mechanism of iron oxidation is cathodic depolarization, in

which electrons from iron, and hydrogen ions from water

produce molecular hydrogen that is then released and used by

methanogens.

Most important, according to the literature reviewed,

nobody has been able to determine how microorganisms induce or

influence the corrosion process. Inspired by the cathodic

depolarization theory and by Daniels and coworkers'

demonstration, this research indicated that it might be

feasible to measure and correlate total metal corrosion and

hydrogen uptake by bacteria.

The project was initiated while evaluating some new

bioreactor systems. In an attempt to understand and quantify

the biological component of corrosion, an experimental system

based on the batch bioreactor, which includes electrochemical

measurement devices to study the utilization of cathodic

hydrogen by bacteria and its relation to the corrosion at its

onset, was implemented. Further along, data acquisition

hardware and computer software were adapted to the system.

The hypothesis is as follows: The corrosion rate of a

metal, under anaerobic conditions, in the presence of








16

bacteria, is a function of its free corrosion potential and

the ability of microorganisms to utilize cathodic hydrogen.














CHAPTER 2
REVIEW OF LITERATURE

Historical Overview


Microorganisms can exist almost anywhere and so can

microbial corrosion. In process industries, it can occur

inside and outside of equipment. In soil and water, it can

happen in aerobic or anaerobic environments.

Microbial corrosion of steel by sulfate reducing bacteria

was probably the first area investigated in this field of

study and remains one of the most important. In an earlier

work (Sifontes and Block, 1991), the authors covered

extensively a historical review on the subject. Here some of

the main historical aspects are presented here along with a

fresh look of microbial corrosion.

Microbial corrosion was first reported before the turn of

the century by Garret (1891). He ascribed the corrosion of

the lead sheathed cable to the action of bacterial metabolites

(ammonia, nitrates, and nitrites). In 1910, Gaines defined

the problem more clearly, providing evidence that iron and

sulfur bacteria were involved in the corrosion of the inside

and the outside of water pipes by making evident the presence

of abnormally large quantities of sulfur. He showed that

Gallionella, Sphaerotilus, and sulfate reducing bacteria were








18

responsible for the corrosion of ferrous alloys buried in

soil.

In 1924, Bengough and May demonstrated the effect of

ammonia produced by bacteria on the corrosion of copper

alloys. Later, von Wolzogen Kuhr and van der Vlugt (1934)

reported on the anaerobic corrosion of ferrous metals by

sulfate reducers. They proposed for the first time a

mechanism for microbial corrosion, which actually initiated

the systematic studies on microbial corrosion and

differentiated the various components of the microbial

corrosion process (metal, liquid, and microorganisms).

Details on the theory are presented later in this chapter.

Evidence for this bacterially influenced corrosion

continued to accumulate from around the world and was reviewed

by Starkey and Wright (1945). In 1953, Uhlig reported that

the primary role of slime-forming microorganisms was the

production of differential aeration and concentration cells

type corrosion. The first studies that demonstrated cathodic

depolarization with sulfate reducing bacteria was conducted by

Booth and Tiller (1960). They added a new dimension to the

problem, indicating that depolarization occurred with a

hydrogenase positive strain of Desulfovibrio bulgaris and did

not occur with a pure strain of hydrogenase negative

Desulfovibrio orientis. Furthermore, they demonstrated an

additional phenomenon that complicated matters: 1)

depolarization was observed when the culture was in active








19

growth, 2) the stimulation of corrosion was approximately

similar for both microbes, 3) the FeS film that formed on the

iron samples had an apparent inhibitory effect on corrosion

rates, and 4) the corrosion rates reported were much lower

than reported rates for similar alloys in natural anaerobic

environments in the presence of sulfate reducing bacteria.

Booth and Tiller (1962) and Booth, Robb, and Wakerly

(1967) demonstrated also that cathodic depolarization was

affected by FeS precipitate, presented experimental evidence

that the structure of the FeS film was instrumental in the

corrosion process, and indicated that once formed its

depolarizing activity continued even in the absence of

bacteria.

Since the energy crisis of the 1970s, several reviews on

the subject have been published. The results reported by Mara

and Williams (1972), King, Miller, and Smith (1973), Smith and

Miller (1975), and King, Dittmer, and Miller (1976) suggest

that precipitated FeS may initially form a protective film on

a ferrous metal surface in the presence of sulfate reducing

bacteria. As the microbial corrosion process continues, the

film thickens and changes stoichiometrically. As the ratio

Fe/S in the film changes from a sulfur deficient to a sulfur

rich structure, the film becomes less protective and

eventually spalls. Once spalled, the film does not reform and

vigorous anodic activity proceeds at the exposed metal

surfaces. According to Smith and Miller (1975), the FeS film,








20

regardless of structure, is cathodic to iron and the corrosion

process continues galvanically. Smith (1980) indicated that

the FeS film would not remain permanently cathodic in the

absence of bacteria. The role of the sulphate reducing

bacteria, he suggested, could be either to depolarize the FeS,

enabling it to remain cathodic, or to produce more FeS by

their metabolism.

Iverson (1981) discounted the FeS argument in his paper.

He indicated corrosion rates were above 210 mpy for mild steel

specimens exposed to filtered media from actively growing

culture of Desulfovibrio (API strain). He also suggested that

SRB produced a highly corrosive compound in addition to

hydrogen sulfide. The process appeared to depend on whether

FeS formed a protective film before the highly corrosive

product contacted the metal surface. Thus, it was apparent

that a number of factors from the metal and the solution were

involved in the process of microbial corrosion by sulphate

reducing bacteria.

During the 1980s, researchers found evidence of more

factors involved during anaerobic microbial corrosion.

Volatile metabolites such as phosphine have been reported to

be responsible for microbial corrosion of steels in

environments free of sulfate and sulfide (Iverson, 1985).

Since iron phosphide was detected among the corrosion products

found, it seemed that the amount of the chemical reaction was

the result of the competence between the sulfide that would








21

passivate the metal and the volatile phosphorous (phosphine)

that would replace the sulfide. There was no experimental

evidence of the chemical nature of the corrosion product,

excluding the direct contact between the bacteria and the

metal. Postgate (1979) rather concisely accounted for the

variety of factors involved in the microbial corrosion

process: nature of metal surface, dissolved ions and/or

organic matter, biofilm formation, FeS precipitate forms, and

other ions (sodium, chlorine) present.

On the other hand, the role of microorganisms in aerobic

corrosion was postulated by Olsen and Szaybalski (1949) to be

due in part to the formation of tubercles in conjunction with

microbial growth, which initiates oxygen concentration cells.

This mechanism, along with others, was proposed as the cause

of the worldwide problem of microbiologically associated

aluminum aircraft wing-tank corrosion that surfaced in the

late 1950s and early 1960s. Both commercial and military

aircraft were affected. Many microorganisms were reported to

be present in significant number in the fuel tank sludge

(Churchill, 1963). The same year, Leathen and Kinsel obtained

184 isolates of microorganisms from jet fuel-storage tanks at

nine Air Force bases. Results indicated the presence of

bacteria and fungi. The predominant bacteria were species of

Pseudomonas, and the most prevalent fungi were species

Hormodendrum. This case indicated the presence of a variety

of organisms including fungi, bacteria, and yeast at a site








22

of microbial corrosion. The medium was also complex due to

the presence of two liquids water and fuel phases.

Later work suggested Cladosporium resinae was the most

important fungus encountered in wing tanks and considered it

responsible for filter blockage and metal corrosion (Berner

and Ahearn, 1977). In 1976, Hill indicated that Cladosporium

resinae is only found in subsonic aircraft. In supersonic

aircraft, a higher temperature prevails and the predominant

flora found were Aspergillus fumigatus, gram negative

bacteria, yeast, and other fungi.

Recent evidence appears to indicate that organic acids,

produced by fungi, were primarily involved in this corrosion

(Miller, 1981).

Other problems due to the activities of SRB have arisen

in offshore oil operations (Hamilton and Sanders, 1986). They

were identified in the legs and storage cells of offshore

structures and include the production of hydrogen sulfide,

which is a serious personnel hazard, and the production of

bacterial metabolites that give rise to accelerated concrete

deterioration (Wilkinson, 1983). Furthermore, internal and

external microbial corrosion of long large-capacity subsea

pipelines, which transport oil and gas from offshore

production fields to shore, are of a major concern due to the

high cost associated with their failures and the harm to their

surrounding environment (King et al., 1986).








23

A considerable amount of research on microbial corrosion

in relation to sulphate reducing bacteria has been done in the

UK, especially in the 1960s. Most of this research was

carried out at the National Chemical Laboratory at Teddington

(presently the National Physical Laboratory) by a group of

well-known researchers in the areas of microbiology and

corrosion science. Despite the disbandment of the group in

1968 as a result of a decision of the British Government that

microbiology should not be conducted at that institution, the

researchers have continued to dominate the literature on

microbial corrosion (Tiller, 1985). Among some of the

original members of the group are G.H. Booth, D.S. Wakerly,

A.K. Tiller, J.R. Postgate, R.A. Kim, W.A. Hamilton, J.A.

Hardy, E.C. Hill, and B.N. Herbert.

In the USA, interest in the field has increased in the

past 15 years, but the scope has been limited. The most

noteworthy efforts began 12 years ago when a microbial

corrosion program was initiated by the Materials Technology

Group and a separate symposium and technical committee on

microbial corrosion was created by the National Association

of Corrosion Engineers (NACE) for their annual meetings.

Among some of the principal investigators in the USA are D.C.

White, R. Mitchell, G. Geesey, W. Characklis, W. Lee, N.

Dowling, R. Tatnall, G. Licina, B. Little, and J. LeGall.

During the last 10 years interest in the area has spread

in other countries.










Other Aspects of Microbial Corrosion



Despite advancement in the area of microbial corrosion,

some researchers have detected some common mistakes in the

practice of this type of investigation. Bryant and Laishley

(1989, 1993) indicated that the result of uncertain

conclusions and overestimates of corrosion due to influence of

microorganisms is the failure to account for effects of media

constituents such as phosphate. They found that phosphate was

one factor among many that together contribute to the overall

corrosion rate and that corrosion studies should include

controls that account for the constituents of the growth

media. There was little or no information on the levels of

phosphates on corroding metals. However, sea water contains

normally a concentration of 3 pM of phosphate, and dead cells

may release some phosphate that may raise the concentration in

spacial locations such as crevices and pitts and influence

corrosion. Booth and Tiller (1968) did not recommend the use

of batch cultures of sulfate reducing bacteria directly to

study their effect on the cathodic reaction. They suggested

that batch bioreactors were undesirable because the

composition of the media would be changing continuously due to

the release of metabolites including sulfide, which was known

to have a cathodic effect on the corrosion of steel and in the

presence of dissolved iron results in the precipitation of

FeS.








25

Hardy (1983) indicated that many attempts of researchers

to demonstrate the utilization of cathodic hydrogen by

sulphate reducing bacteria and its stimulatory effect on the

cathodic reaction have failed because the evidence was based

exclusively on measured electrochemical effects. The observed

depolarization might have been due to the effect of sulfides

present and could not be attributed exclusively to the effect

of hydrogenase. He also indicated that there was some

evidence that the periplasmic hydrogenase enzyme that appears

to be the hydrogen uptake enzyme constituted a greater

proportion of the total hydrogenase present in hydrogen-grown

cells than of the hydrogenase in lactate-grown cells. This

feature, as explained earlier, is a characteristic of bacteria

adaptation that is seldom considered during microbial

corrosion research. Researchers most of the time use bacteria

that have been cultured in standard media rather than wild

type cultures. Wild type species of Desulfovibrio have been

shown to lose their adhesion properties when they are

transferred from the wild to laboratory and grow in standard

medium.

Rajagopal and LeGall (1989) suggested that the ability of

some sulphate reducing bacteria to reduce nitrate or nitrite

with hydrogen as sole energy source provides a better

experimental system to study the cathodic depolarization

phenomenon, since problems with measurements of dissolved iron









26
and growth in the presence of precipitated sulfides resulting

from the customary sulphate reduction can be avoided.

The presence of organic electron donors has been

considered during cathodic depolarization of steel. Widdel

(1988) indicated that most Desulfovibrio spp are able to grow

on acetate and carbon dioxide as carbon sources and hydrogen

as sole energy source. Cord-Ruwisch and Widdel (1986) found

that the availability of organic electron donors appears to be

an important factor that influences the removal of cathodic

hydrogen. If a favorable organic energy source was present,

sulphide was produced, indicating that iron alone did not

allow sulphate reduction. They suggested either that cathodic

hydrogen was preferentially oxidized with the organic

substrate or that sulphide from sulphate reduction with the

lactate reacts with the remaining ferrous ions or ferrous

hydroxide from the corrosion process to produce more FeS.

In addition to the above observations, some other

important scientific areas related to the microbial corrosion

phenomenon need to be addressed in order to envision the

complexity of the problem. Those areas include bacterial

adhesion, surface thermodynamics, interfacial chemistry,

hydrogen embrittlement, interspecies hydrogen, bioenergetics,

microbial ecology, and applied fundamentals of metallurgy,

electrochemistry and chemistry. Information on those subjects

is referred to in the cited literature. Specific topics

include the prevailing mass transfer conditions and the









27
surface properties of the phases involved, which include

molecular fluctuations at the electrical double layer (Liu,

1983), the adhering bacteria, the substrate, and the suspended

media (Absolom et al., 1983; David and Misra, 1985), chemical

interaction at metal surfaces and at the liquid interface

(Sato, 1987), and metallurgical aspects such as

microstructure, grain boundaries, inclusions, surface

contamination, and sources of corrosion currents (Godard,

1980; Mears and Brown, 1941).



Microorganisms Involved in Microbial Corrosion



The microorganisms that have been associated with

microbial corrosion include many genera and species.

Microorganisms are anatomically simple yet biochemically

complex. They may be divided into three groups: (1) algae and

fungi, (2) protozoa, and (3) monera (e.g., bacteria). Many of

these organisms have been firmly established in laboratories

and field sites as having roles in the corrosion process,

whereas others have merely been isolated from suspected

corrosion sites. Microorganisms are sustained by chemical

reactions by ingesting reactants and eliminating waste

products. Thus these processes can influence corrosion

general by 1) directly influencing anodic and cathodic

reactions, 2) influencing protective surface films,









28
3) producing deposits, 4) producing corrosive metabolites, and

5) feeding on corrosion inhibitors.

Several characteristics of microorganisms enhance their

involvement in corrosion. They are generally very small,

starting from less than 0.2 pm, which allows them to penetrate

crevices very easily, to several hundred pm in length. Some

of them are motile, which aids in their migration to more

favorable environmental conditions. Microorganisms are able

to establish in sites that encourage their growth. For

instance, microbes establish and colonize surfaces in cooling

water systems where food sources concentrate at metal surfaces

because of their hydrophobic properties. They can withstand

a wide range of environmental conditions: pH values from 0 to

11 (Brock and Madigan, 1991), temperatures from -30 to 1100C,

and oxygen concentrations from 0 to 100% (Morgan and Dow,

1986).

Microorganisms can adhere to a surface and form colonies

of different species. These consortia, once formed, can

sustain survival under adverse conditions. They can reproduce

themselves to a great number in a short time. This fact

allows them to bloom and take over an environment quickly.

They are easily dispersed in air, water, animals, etc., and

adapt to other environments in which it may be easier for them

to grow. Many can adapt easily to a wide variety of

substrates, such as the Pseudomonas, some species of which

can use well over 100 different kinds of food as sole carbon









29
and energy sources. Many can produce extracellular

polysaccharides or slime layers where a consortia of bacteria

can develop and consequently influence corrosion (White et

al., 1986). These layers attract food and other

microorganisms and cause several other well-known problems in

the process industry, such as poor heat transfer.

Some microbes produce spores that resist the most severe

environmental conditions and are capable of surviving for long

periods of time by remaining dormant. They can quickly

colonize surfaces when the environment changes to their

liking. Some resist antimicrobial agents by virtue of their

ability to degrade them or by resisting permeability through

the cell wall or by the extracellular polysaccharide

protection. Such resistance may be acquired by mutation or

acquisition of a plasmid. Many species produce a wide variety

of organic acids that may promote corrosion of many alloys

even at low concentrations (e.g., Clostridium acetobutylicum

produces acetic acid).

Some species produce mineral acids that are extremely

corrosive. Thiobacillus thiooxidans produces sulfuric acid,

which is of economic importance in biohydrometallurgy because

it makes possible the leaching of metal sulfide ores. In the

case of pyrite, it oxidizes both the sulfur and ferrous

moiety. Boes and Kuenen (1983) have recently reviewed the

sulfur oxidizing bacteria and their relationship to corrosion

and leaching. Several bacteria metabolize nitrate, sometimes









30
used as a corrosion inhibitor (e.g., Pseudomonas spp reduce

nitrate and nitrite to nitrogen gas). Other organisms convert

nitrate to nitrite or ammonia to nitrite (e.g., Nitrosomonas)

and others turn nitrite into nitrate (e.g., Nitrobacter).

Many organisms form ammonia from the metabolism of amino

acids. This forms ammonia ions in basic solution, which may

be corrosive to copper alloys.

Microorganisms such as sulfate reducing bacteria produce

enzymes, which may be excreted outside the cell and which can

act on substances outside the cells. For instance,

hydrogenase has been reported as being responsible for

depolarizing cathodic sites during the microbial corrosion of

iron and steel. Many organisms can produce carbon dioxide and

hydrogen as a result of their fermentative metabolism. Carbon

dioxide in acidic solutions becomes carbonic acid, which is

highly corrosive, and hydrogen can polarize metal surfaces of

stainless steel and may cause hydrogen embrittlement.

Many genera of bacteria that normally use organic

compounds as carbon and energy sources can use hydrogen gas as

their energy source and carbon dioxide as their carbon source

and live chemoautotrophically. This can cause depolarization

of cathodic sites on steel and promote corrosion (e.g.,

Methanogens).

Some bacteria can oxidize or reduce metals or metallic

ions directly. For example, the iron-oxidizing bacteria

(Gallionella, Sphaerotilus) oxidize ferrous ion to ferric ion.









31
The ferric compounds precipitate in a sheath around the cells

and form tubercles in pipes and cause plugging. Thus,

concentration of cells is easily formed under those deposits.

On the other hand, ferric ion can be reduced to ferrous ion by

Pseudomonas spp from oil wells and marine sediments. It has

been suggested that the ferric film, which normally stabilizes

the surface of mild steel from corrosion, are destroyed

leaving the surface susceptible to corrosion attack. Other

bacteria can oxidize or reduce metals such as manganese.

Microorganisms can form synergistic communities (e.g.,

algae and bacteria). These consortia can accomplish things

that individually would be difficult if not impossible, such

as the case of fungi and Desulfovibrio spp. The fungi break

down wood to organic acids and consume oxygen, thus providing

the food and anaerobic conditions for Desulfovibrio spp..

Communities providing protection for individuals can also

change structure, dominant species, etc. by genetic mutation,

and can adapt to environmental changes, even to deliberate

chemical changes intended to kill them. More details on this

subject may be found in Sifontes and Block (1991).



Mechanisms of Microbial Corrosion


For proper selection of methods to prevent or control

microbial corrosion, it is necessary to know the mechanisms by

which microbial activity affect the deterioration of metals.








32

The mechanisms of microbial corrosion in some cases are well

defined, but where environments encourage the activity of

bacteria, the corrosion processes are more complex and still

not fully understood. The mechanisms of microbial corrosion

can be subdivided in direct and indirect mechanisms. If a

microbe interlinks an electrode process with its own

metabolism or presence, it is a direct effect (i.e.,

differential aeration cell produced by sessile bacteria),

otherwise it is an indirect effect (i.e., corrosive

metabolites produced by planktonic bacteria).

Even though the mechanisms of microbial corrosion are not

well understood, microbes that cause or influence corrosion

have been classified previously (Kobrim, 1976) into the

following groups:

1) Acid production. Some microbes can oxidize sulfur

compounds to sulfuric acid. Very low pH has been reported in

places where sulfur oxidizing bacteria are active. Many

species produce a wide variety of organic acids (e.g. acetic,

butyric, succinic, and formic) which may promote corrosion of

many metals and their alloys.

2) Protective coating destruction. Protective coatings

ranging from polymeric materials to passive films can be

broken by the activity of microorganisms and corrosion of bare

metal starts rapidly.

3) Production of corrosion cells. Differential aeration and

ion concentration cells are notable examples such as the case









33
of a metallic surface that is accessible to oxygen. If a

deposit such as a biofilm or a corrosion product covers it,

the surface under the deposit is shielded from oxygen and the

surface outside of the deposit is not. This results in a

corrosion cell.

4) Sulfur reduction and oxidation. Sulfate reducing bacteria

are the most publicized class of corrosive microbes. They

reduce sulfates to sulfides and can depolarize cathodic sites

by consuming hydrogen. Most sulfur oxidizing bacteria known

fall in the category of acid producers.

5) Concentration of anions and/or cations. Iron and manganese

bacteria are examples of this category. They generally form

thick, bulky deposits which create concentration cells or

harbor other corrosive microbes. This group is also known as

metal ion oxidizers or reducers.

6) Hydrocarbon utilization. Certain microorganisms have been

observed that destroy organic coatings or linings in the

presence of hydrocarbon fuels. Some others destroy metals

such as aluminum and feed on hydrocarbon fuels.

7) Slime formation. Certain algae, yeast, bacteria, and fungi

may form deposits which foul heat transfer equipment and

produce concentration cells on metal surfaces.

The above mechanisms can be grouped in three general

modes of microbial attack based on their metabolism: corroded

material serves as substrate for microbial growth, microbes








34

colonize material surface but feed on something else, and

microbes produce metabolites that corrode material.



Theory of Cathodic Depolarization



The theory of cathodic depolarization was postulated in

1934 by von Walzogen Kuhr and van der Vlught. The idea of the

authors came from their interpretation from the

electrochemical point of view of the anaerobic corrosion of a

cast iron pipe in wet soils near Amsterdam. Since the pipe

was under clay at a near neutral pH, the severely corroded pipe

did not allow them to explain the phenomena using the

reduction of oxygen as the cathodic reaction. The alternative

cathodic reaction was the reduction of hydrogen that was

feasible under those conditions.

The theory proposes that when iron is immersed in water,

a natural equilibrium is set up between the ferrous cations

released at the anode, and the metal surface negatively

charged at the cathode by the remaining electrons. The

dissolving process continues only if the electrons are

removed. Under aerobic conditions oxygen serves as an

electron acceptor resulting in rust formation. Under

anaerobic conditions free protons from the dissociation of

water are reduced on the cathodic metallic surface by the

remaining electrons, to form a protective hydrogen polarized

envelope that protects the iron metal from further








35

dissociation. A dynamic equilibrium is established which

keeps the iron polarized. The theory suggests that the

principle mechanism of anaerobic corrosion is cathodic

depolarization of the iron surface by hydrogen oxidizing

microorganisms such as sulfate reducing bacteria and

methanogen. These organisms disturb the equilibrium by

oxidation of the cathodically formed hydrogen with sulphate

and carbon dioxide as electron acceptors respectively, via the

hydrogenase enzyme. In an attempt to re-establish the

anodic/cathodic equilibrium more iron is oxidized, the end

result of which is pitting formation.


Table 2-1. Chemistry of the cathodic depolarization theory.



component reaction


1)Metal dissolution 4Fe --> 4Fe+2 + 8e

2)Hydrogen reduction 8H+ + 8e --> 8H --> 4H2

3)Water dissociation 8H20 --> 8H+ + 80H

4)Microbial activity SO4-2 + 8H --> S-2 + 4H20

5)Corrosion product Fe+2 + S-2 -> FeS

6)Corrosion product 3Fe+2 + 60H --> 3Fe(OH)2

7)Total rxn 4Fe + SO4-2 + 4H20 --> 3Fe(OH)2 + FeS + 2(OH)


The importance of this theory lies in the fact that it

separates for the first time the three components of the








36

microbial corrosion system which include the microorganisms,

the metal, and the suspended medium.

A large volume of literature up to the last decade has

been influenced by the theory of cathodic depolarization and

has referred to it, either to prove or to disprove it.

However, the use of electrochemical techniques allowed Horvath

and Solti (1959) to discover an anodic effect in addition to

the cathodic effect of the theory. They studied this effect

as a function of pH, environmental regulatory conditions and

the concentration of FeS present. According to those results,

SRB had an indirect role, which would be the stabilization of

the sulfide compounds over the metal surface by modification

of the redox potential.

One of the most conclusive findings was reported by

Costello (1974) who indicated a cathodic effect due to the

hydrogen sulfide produced by the sulfate reducing bacteria,

suggesting that hydrogen utilization by the sulfate reducing

bacteria became secondary and so the participation of those

bacteria in the corrosion process. Furthermore, It has been

suggested as an amendment to the corrosion theory that solid

ferrous sulfide, in contact with iron, acts as a cathode

(Booth et al., 1968). Other authors proposed that reduced

phosphorous compounds, too, are involved in the anaerobic

corrosion process (Iverson and Olson, 1984). Recently it was

reported that phosphate and hydrogenase can affect the

corrosion of mild steel. Phosphate reacts on mild steel with








37

concomitant production of hydrogen gas and the formation of an

iron/phosphate complex (vivianite) Bryant and Laishley, 1990;

Weimer, et al., 1988). The enzyme hydrogenase was reported to

accelerate cathodic depolarization by oxidizing the hydrogen

produced (Bryant and Laishley, 1993). Although many studies

have clearly demonstrated the involvement of sulphate reducing

bacteria and methanogens in corrosion, only a few have shown

the influence of those microorganisms using the electrons from

the metal as energy source.

On the other hand, some authors have provided evidence in

support of the cathodic depolarization theory (Cord-Ruwisch

and Widdel, 1986; Hardy, 1983; Tiller and Booth, 1962; Booth

and Tiller, 1960, 1962). Few, however have actually

demonstrated that this phenomenon is coupled to microbial

growth (Belay and Daniels, 1990; Rajagopal and LeGall, 1989;

Daniels et al., 1987; Pankhania et al., 1986; and Tomei and

Mitchell, 1986). In conclusion, research indicates that the

described theoretical mechanism is not entirely correct.



Previous Studies in Microbial Corrosion



Despite the large amount of literature in microbial

corrosion, it is still not an easy subject to understand,

because of the multiplicity of factors at play. Some authors

have indicated that microbial corrosion is a newly discovered

problem or an emerging science, possibly still in its infancy









38
(Tatnall, 1988). Others have confirmed by surveys that the

continued incidence of microbial corrosion could be due to a

general lack of awareness of the problem (Wakerly, 1979). The

fact is that its scientific study began more than 80 years ago

(Tiller, 1982) and after the work of von Wolzogen Kuhr and van

der Vlugt in 1934, who established the classical mechanism of

anaerobic corrosion, the subject of microbial corrosion in

general and of anaerobic corrosion in particular has become

recognized of prime importance (Bessems, 1983; Hamilton,

1985).

Most of the literature on microbial corrosion, before

1960, was concerned basically with observations of the effect

of bacteria on the environment. During the 1960s, there was

considerable activity on the field and most of the research

was focused on gaining understanding at the different

mechanisms. Some notable papers of this period belong to

Postgate (1960), Horvath (1960), Sorokin (1966), Iverson

(1966), Booth, Elford and Wakerly (1968), Booth and Tiller

(1968), and Costello (1969).

During the 1970s the interest in the subject broadened

the scope of research involving scientists from other fields

including plant engineers, corrosion scientists,

microbiologists, and biochemists (Sequeira and Carrasquinho,

1988). This has brought improved understanding of the

ecology, nutrition and physiological requirements of the

microbes involved in microbial corrosion, which also improved








39

the biochemical techniques used in both the field and the

laboratory. In addition, the publication of case histories of

a diverse range of failed industrial equipment has enhance

understanding of the problem. This period is well described

in the articles by Iverson (1972), King and Miller (1973),

Mara and Williams (1972), Miller and King (1975), Kobrin

(1976), Widdel and Pfennig (1977), Jorgensen (1977, 1978,

1980), Wakerly (1979), and Postgate (1979).

It was only within the last decade that microbial

corrosion was recognized as a serious problem in the chemical

industry by the leading corrosion society in the world (NACE)

and substantial advancement has been achieved specially in the

areas of microbiology and corrosion. In microbiology, among

other findings, the biochemistry of dissimilatory sulfate

reduction in Desulfovibrio has revealed enzymes and electron

carriers of special character and structure whose function and

distribution within the cell are just beginning to be

revealed, indicating new discoveries in the peculiar energy

generation systems in these bacteria.

In corrosion, sophisticated methods to study it, have

evolved; such as new methods in electrochemistry,

metallography, macroanalysis, and microanalysis. A

significant part of the research in that period is described

in the articles by Hamilton (1985), Iverson (1987), White et.

al. (1986), Tatnal (1981), Odom and Peck (1984), Cord-Ruwisch

and Widdel (1986), Tiller (1982), Miller (1981), Hardy (1981,








40

1983), Rajagopal and LeGall (1989), Bryant and Laishley

(1989), Daniels et al. (1987), and Widdel and Pfennig (1981).

Today, microbial corrosion is well recognized as a

serious problem in most industries, particularly in oil and

gas, the power generating and the process industries. In the

chemical industry alone, multimillion dollar failures due to

microbial corrosion have been reported involving cooling water

systems (Felzin et al., 1988).

While the above studies have yielded a wealth of

information, the literature reveals that the corrosion

community still knows very little about how microorganisms

influence or induce corrosion, and what the role of

microorganisms is in the corrosion process. In general, most

of the research deals with observations related to the basic

problem, case histories, biological research, and reviews of

literature. It is important to notice that this was not

usually the fault of the investigator; in the majority of

instances the specialist lacked interdisciplinary help

required to tackle such complex studies.

Microbial corrosion is by definition an interdisciplinary

field that requires among others, the understanding of

microbiology, corrosion science, metallurgy, electrochemistry,

transport phenomena, and surface chemistry. Several factors

have cause a further hindrance in the development of this

microbial corrosion. Among some of the factors are: the lack

of awareness; the difficulty of growing, isolating, and








41

identifying anaerobic microorganisms; the adaptation of

techniques from clinical microbiology to handle the microbes

involved; the fact that to the average researcher microbial

corrosion spans the boundary of traditional specialties; and

the idea that many microbiologists have of microbial corrosion

as a rather specialized even esoteric field, and the lack of

acceptance of the role of bacteria in corrosion by corrosion

engineers.

Actually, many theories on how microorganisms influence

or induce corrosion have been proposed. However, none of them

are fully proven but rather equal number of existing papers on

the subject claim to support or refute these theories.

Consequently, it seems fair to say that there is little

uniform agreement among those working on this field about what

is really going on (Tatnall, 1988).

Customary practices, used during the investigation of

microbial corrosion, include separated studies of either the

microorganisms, the metal or the fluid but ignore their

interaction. In addition, most laboratory microbial corrosion

experiments use flowthrough type bioreactors which are run for

weeks or even months. The most common corrosion measurement

methods used are adopted from classical corrosion science such

as Tafel plots and weight loss techniques. In many cases,

these practices have failed to give reproducible results

because of the length of the experiments, nature of the metal

surface, and fluid chemistry in addition to microbial









42
ecological considerations of such a complex systems. The

application of external potentials to the system, a common

practice in microbial corrosion experiments, may produce

significant thermodynamic changes known to affect the

stability of the system, and constitute a source of

uncontrolled variables.

The practical separation of the three components of the

microbial corrosion systems as suggested by the cathodic

depolarization theory, and the work performed by Daniels et.

al. (1987), set the stage for studying the microbial

utilization of cathodic hydrogen during corrosion. If hydrogen

oxidizing bacteria can take up the hydrogen produced

cathodically from steel in well defined experimental systems

then there exists the possibility to further explore in this

field and increase our understanding of these microbes and

their mechanisms of attack on metals.



Microbial Corrosion Control


Prior to 1970 the control of microbial corrosion focused

on the use of cathodic protection and antimicrobial agents.

This practice was complemented by improvements in surface

coatings, in particular antifouling systems such as tapes and

wrappings. Because of the limited ability of those methods to

control bacterial activity and the inefficacy of the

methodology to asses antimicrobial agents; the main driving








43
force after 1975 has been the consolidation of several

methodologies and assessment procedures to improve the

standard practice (Tiller, 1985).

In 1964, Saleh and co-workers evaluated nearly 200

antimicrobial agents and concluded that laboratory evaluations

of those chemicals can only be considered as an introductory

sign and should be supplemented with trials on the field. In

1983 Bessems recommended the importance of assessment

procedures and Gaylarde and Johnston emphasized the

deficiencies in current methodology and the need for

improvement. During last decade a joint venture of the

Institute of Corrosion Science and Technology and the National

Association of Corrosion Engineers has reviewed and updated

the current recommended practice for monitoring bacterial

growth. Topics such as killing time, the importance between

planktonic and sessile bacterial consortia and their

attachment properties are now important issues in the control

of microbial corrosion (Tiller, 1985).

Due to the complexity of the environmental factors that

results in microbial corrosion, the success of a control

program depends more in our outlook on the available

information about the problem, and becomes a challenge to

identify the most practical and economic solution.

In general, the control of microbial corrosion requires

a sound strategy. This involves a diagnosis of the microbial

corrosion problem that begin with determining the cause and








44

the mechanisms associated with the corrosion problem. A sound

diagnosis include: comprehensive current system diagrams,

materials of construction, fabrication methods, operating

history, chemical analysis, site specific environmental

conditions, biological history, and historical and current

chemical treatment. Unfortunately, if this is not followed,

adverse consequences may result. For example, a strong

oxidant may have a good killing power but may produce severe

local corrosion if applied to a particular system.

Fellers (1989) suggested that the best strategy is the

product of a multi-disciplinary team which looks at all

aspects of the problem, examines root cases, and objectively

evaluates alternatives. He also indicated in a later paper

that if the main strategy is to maintain the system clean of

microbial growth, instead of looking for independent solutions

to several problems, several issues may be avoided including

microbial corrosion (Fellers, 1990).

The essential strategies to control microbial corrosion

include: detection, prevention, and mitigation. Detection

techniques are basic to prevention and mitigation. They

include electrochemical methods, microbiological methods,

physical and metallurgical methods, in system monitoring

techniques, and other laboratory techniques. Details on these

techniques are found in earlier work of the author (Sifontes

and Block, 1991). Additional detection techniques include

visual inspections to identify suspected indications of









45
microbial attack and side stream monitors for determination of

corrosion and fouling rates.

Prevention and mitigation techniques hold the highest

payback in plant protection and preservation. There is no

universal approach to the prevention and mitigation of

microbial corrosion since it is almost impossible to use a

single preventive method. The prevention and mitigation

techniques offered by the current technology include planning

considerations such as material selection, design, and non-

metallic materials; physical-chemical control such as

corrosion inhibitors, selection and control of the

environment, protective coatings, electrochemical protection,

maintenance cleaning, and chemical control; and biological

control such as antimicrobial agents, and environmental system

control. Details on above prevention techniques are found in

earlier work of the author (Sifontes and Block, 1991). Other

prevention and mitigation techniques include general corrosion

control such as prevention of scales which enhances

biofouling, suspended solids dispersion such as the use of

penetrants of other surface active agents which re-disperse

fouling materials (Fellers, 1990).

Finally, an effective control of microbial corrosion

involves a combination of selected techniques to solve an

specific problem. Numerous history cases that include the

results of the applied techniques are available throughout the

literature cited. A reported case history of successful








46

corrosion control in a cooling water system used a combination

of biodispersant/biocide. It employed an organic corrosion

inhibitor, a polyacrylate/phosphonate dispersant, and a

combination of two microbiocides used simultaneously

(Honneysett et al., 1985).

the use of surfactants penetrantss /biodispersants) has

improved the effectiveness of microbial corrosion control.

Chemically, these substances are composed of nontoxic organic

compounds with penetrating and dispersing properties. The

biodispersants allow the sessile colonies to be penetrated by

the antimicrobial agents, thus used at lower dosages with

improved effectiveness. They inhibit the biomass produced

from becoming so massive that antimicrobial agents can not

penetrate the consortia of microbes. Some penetrants are

hydrophobic to the extent that a film forms on the metal

surfaces allowing less deposition of sessile colonies

associate or not with microbial corrosion.

Other references that contain a variety of history cases

include: Microbial Corrosion proceedings by the Metals

Society of London, 1983; International Conference on

Biologically Induced Corrosion proceedings by the NACE, 1985;

Microbial Corrosion 1 proceedings by the First European

Federation of Corrosion workshop on Microbial Corrosion, 1988;

and Microbially Influenced Corrosion and Biodeterioration

proceedings by the International Congress on Microbially

Influenced Corrosion, 1990.










Research Approach



The following tasks were considered in order to comply

with the objective of this research. The first task included

the evaluation of some new experimental flowthrough and batch

bioreactors to reproduce and study the anaerobic microbial

corrosion phenomena. The initial task consisted of runs of

bioreactors with the object of reproducing microbial corrosion

in the laboratory. The second task involved the use of the

batch bioreactor to study the onset of microbial corrosion,

using conditions that resemble the inside of gas transmission

pipelines. The operation of the batch bioreactor was examined

concerning practical functioning for head space composition,

sterilization method, reducing agent use, metal coupon

preparation, and bacteria handling. Different metals and

their alloys were tested for electroactivity and their ability

to produce hydrogen; and different hydrogen oxidizing bacteria

were also tested to determine their ability to uptake

hydrogen. A third task involved the development of an

experimental system that included the redesign of the batch

bioreactor, development of a working electrode preparation of

bacterial suspensions, preparation of mineral solution

electrolyte, and the implementation of a data acquisition

system to accommodate electrochemical measurements, in an

attempt to quantify the biological component of corrosion.

The redesign of the batch bioreactor consisted of improvements









48
to the experimental system that resulted in the development of

the single, dual, and triad flask electrochemical cells. A

forth task involved the general setting of parameters to

assure simpler and reliable results such as the selection and

setting of fixed conditions and analytical procedures.














CHAPTER 3
MATERIALS AND METHODS


Introduction


The overall objective of this research was to study

anaerobic microbial corrosion at its on-set and to quantify

cathodic hydrogen utilization by bacteria. For this purpose,

a total of seven experimental set-ups were utilized. These

include: the 1) flowthrough bioreactor, 2) batch bioreactor,

3) single flask electrochemical cell or single cell, 4) dual

flask electrochemical cell or dual cell, 5) triad flask

electrochemical cell or triad cell, 6) artificial hydrogen

uptake triad cell, and 7) galvanic couple triad cell.

Initially, the flowthrough bioreactor was used to

replicate microbial corrosion and to get familiar with the

laboratory techniques and analyses required for the

investigation. This traditional bioreactor design used a

known bacteria triculture commonly found at microbial

corrosion sites and a glucose lactate yeast extract (GLYE)

medium that allowed the three bacteria to grow rapidly. Runs

of this bioreactor were performed using carbon steel coupons

in the presence of the bacteria triculture in GLYE medium.

Then, since the corrosion reaction and the biofilm formation

developed relatively fast in the flowthrough bioreactor, a









50
simpler batch bioreactor to study the on-set of the reaction

was used. This new bioreactor offered the best alternative to

study the initiation of the microbial corrosion process and

was able to reproduce microbial corrosion of carbon steel

within 24 hours in GLYE and in mineral solution media. The

batch bioreactor was redesigned and instrumented to include

electrochemical measurements and became the single flask

electrochemical cell. The single cell was used to measure

free corrosion potential in the presence and absence of

bacteria. It was improved later to the dual cell and the

triad cell. The dual cell allowed bacteria to avoid direct

contact with toxic metals and the triad cell provided for

measurement of a differential corrosion current. The last two

experimental set-ups were identical to the triad cell, except

they were used to study the effect of an artificial hydrogen

uptake and to determine the reliability of the electrochemical

measuring system respectively.

All electrochemical set-ups used the mineral electrolyte

solution developed for the purpose of studying microbial

corrosion at its on-set and hinder the formation of a biofilm



Flowthrough Bioreactor


Description

The 3-L flowthrough bioreactor system, used for the

preliminary work of this research was designed in house and









51

built out of 5 cm 4 PVC pipe components. It consisted of two

sections for the study of sessile and planktonic bacteria

activity and their effect on metal deterioration. A basic

diagram showing its components is illustrated in Figure 3-1.









FEED


INOCULUM PORT


A'


w


SESILE ACTIVITY
TESTING


NAD(P)H PROBE PORT







. '-- METAL SPECIMEN
PORT



x\ "
\ ,



'Ni'


PLANKTONIC
ACTIVITY
TESTING




CLEAR PLASTIC
FOR LEVEL
CONTROL


,,f





/

1L
u


Figure 3-1. Flowthrough bioreactor general description of
components


FLOW
CONTROL
VALVE









52
The first section, for sessile activity testing,

consisted of eight side screw cap ports that fit a

fluorescence probe, developed by Biochem Technology, Inc.,

King of Prussia, Pennsylvania, and four top screw caps, to

which metal coupons and reference electrodes are attached.

The second section, for planktonic activity testing, contained

two side screw caps ports for attachment of the fluorescence

probe, and a clear section at the end of the reactor for the

purpose of controlling the bioreactor head space volume.

The bioreactor was fed from a 4-L stoppered flask through

a tubing, Tygon R-3603, and an effluent flow of 125 mL/hr was

controlled using a hose clamp on a 30 cm tubing, Tygon R-3603,

at its outlet.

A known bacteria triculture composed of Entrobacter

aergenes, Desulfovibrio desulfuricans, and Clostridium

acetobutylicum were used as the inoculum. Bacteria were

obtained from the American Type Culture Collection (ATCC).

Steel coupons, 5 mm x 20 mm x 10 mm, polished to 240

grid, and tied plastic strips to a 1/4 0 teflon rods were

attached to the upper screw caps of section one. Figures 3-lb

and 3-1c illustrate the assembly combinations of metal

coupon/fluorescence probe and a metal coupon/fluorescence

probe/reference electrode, respectively. Figure 3-1d shows an

actual set-up of the flowthrough bioreactor.













SCREW CAP


METAL SPECIMEN


Figure 3-2. Flowthrough bioreactor metal coupon and
fluorescence probe setup.


SCREW CAP


IONIC BRIDGE


REFERENCE
ELECTRODE


METAL SPECIMEN


Figure 3-3. Flowthrough bioreactor metal coupon, fluorescence
probe and reference electrode setup.



































Figure 3-4. Flowthrough bioreactor components.


Operation

The flowthrough bioreactor system was operated at a 1-day

retention time (125 mL/hr). Medium was fed to the bioreactor

by gravity from the 4-L reservoir under a nitrogen atmosphere

to assure anoxic conditions. The feed, a glucose-lactate

yeast-extract (GLYE) media, was prepared fresh daily,

sterilized and neutralized prior to use.

Effluent samples for pH and volatile fatty acids were

taken directly from the effluent. Metal coupons samples for

scanning electron microscopy analysis were sampled under

aseptic conditions while sparging with N2 gas and placed









55
immediately into 10 mL vials containing a 0.5% glutaraldehyde,

2% formaldehyde, cacodylate buffer, pH 7.2. Metal samples

were further processed, according to protocol described in

Table 3-1, for scanning electron microscopy examinations.



Table 3-1. Protocol for metallic surface fixation for
scanning electron microscopy examinations.


1) Place metal coupon in a 0.5% glutaraldehyde, 2.0%

formaldehyde, cacodylate buffer, pH 7.2 solution for 5 min at

room temperature, then on ice for 10 min.

2) Wash coupon in ice-cold buffer two times for 5 min each.

3) Wash coupon in ice-cold ethanol solutions for 5 min each,

in the following ethanol concentrations: 25%, 50%, 75%, and

95%.

4) Wash coupon at room temperature in 100% ethanol.

5) Wash coupon in acetone for 15 min at room temperature.

6) Wash coupon in fresh acetone at room temperature for 30

min.

7) Treat coupon with solutions of epon-araldite 30%, 70%, and

100% for 1 hr each at room temperature, except the 100% which

is done at 60C in oven.



Bacteria inoculations were done as follows for initial

run. Ten mL of E. aerogenes were inoculated first in order to

reduce the media. After 24 hours, 15 mL each of D.

desulfuricans and C. acetobutylicum were inoculated. For








56

other runs, bacteria were inoculated within six hours in

amounts 10 times larger than the volumes used in the initial

run. All runs were temperature controlled at 35"C inside a

walk in incubator.

GLYE media was prepared using ingredients outlined in

Table 3-2, Table 3-3, and Table 3-4. Media was prepared in 4-

L feed reservoir flasks and autoclaved at 15 psig for 15 min

(Balch et al., 1979).



Table 3-2. Glucose Lactate Yeast Extract Medium Composition.



Yeast Extract 0.5 g

Glucose 0.5 g

Sodium Lactate 0.5 g

Mineral Solution 1 (table 3) 25.0 mL

Mineral Solution 2 (table 3) 25.0 mL

Trace Mineral Solution 3 (table 3) 5.0 mL

Trace Vitamins Solution 4 (table 2) 5.0 mL

L-Cysteine HC1.H20 0.5 g

Na2S.9H20 0.5 g

Resazurin (0.5 mg/mL) 1.0 mL

Complete to 1-L with distilled deionized water, adjust pH to

7.5 with 5N NaOH and autoclave for 15 min at 15 psig.














Table 3-3. Trace Vitamins Solution 4 Composition.

Biotin 2 mg

Folic Acid 2 mg

Pyridoxine HC1 10 mg

Thiamine HC1 5 mg

Riboflavin 5 mg

Nicotinic Acid 5 mg

DL-Calcium Pantothenate 5 mg

Vitamin B12 0.1 mg

p-Amino Benzoic Acid 5 mg

Lipoic Acid 5 mg

Complete with distilled and deionized water to 1-L


Table 3-4. Mineral Solutions Composition.

1) Mineral solution 1

K2HPO4 6.0 g/L

2) Mineral solution 2

KH2PO4 6.0 g/L

(NH) 2SO, 6.0 g/L

NaCl 2.0 g/L

MgSO4.7H20 2.6 g/L

CaC12.2H20 0.16 g/L


J










3) Trace Minerals solution 3

N (CH2CO2H)3

MgSO4. 7H20

MnSO4.2H20

NaCl

FeSO4 7H20

CoSO,

CaC12 2H20

ZnSO4

CuSO4 5H20

AlK(SO4)2

H3Bo

Na2MoO4.2H20


1.5 g/L

3.0 g/L

0.5 g/L

1.0 g/L

0.1 g/L

0.1 g/L

0.1 g/L

0.1 g/L

0.01 g/L

0.01 g/L

0.01 g/L

0.01 g/L


Batch Bioreactor



Description

The batch bioreactor system consists of a 250 mL bioassay

bottle with the metal coupon suspended in 150 mL of liquid

media by a nylon string attached to a crimp top butyl rubber

septum to preserve bottle anaerobically. A typical batch

bioreactor experiment is shown in Figure 3-2. This set-up was

used to study microbial corrosion under a known growth

environment, medium, metal coupon, and head space, using

different bacteria combinations.


















nylon string

-- metal coupon

suspended medium


Figure 3-5. Batch bioreactor.



Prior to the tests, the batch bioreactor was assembled

using a 250 mL bioassay bottle to which 150 mL of GLYE media

was added and a metal coupon is suspended in the media. The

metal coupon was fabricated from ASTM-ASME SA106 grade B1

steel pipe 2.5 cm 4, schedule 80, obtained from Texas Eastern,

Louisiana. Coupons were machine ground to a size of 1.5 cm x

2.0 cm x 3 mm with a side hole of 3mm 4 for the attachment of

the nylon string that holds them to the butyl rubber stoppers.

The inoculum used for the batch bioreactor was the same as

used for the flowthrough bioreactor and it is described

therein.



Operation

Batch bioreactors were intended for one day runs;

however, some experiments were run for several days. Their

100 mL head spaces were maintained with N2 at 7 psig to assure









60
anoxic conditions. Bioassay bottles, including medium and

metal coupon, were autoclaved at 15 psig for 15 min prior to

each run or inoculation. Bacteria were inoculated at room

temperature and placed in a Fisher low temperature incubator,

model 307, at 370C under aseptic conditions. One mL of E.

aerogenes was inoculated first, then after 12 hrs one mL of

each D. desulfuricans and C. acetobutylicum was added.

Samples for pH and volatile fatty acids analysis were taken

via the rubber stopper with a 5 mL syringe. Metal coupons

were sampled aseptically and placed in 10 mL vials containing

a cacodylate buffer, pH 7.2, to be further processed according

to the protocol described in Table 3-1 for scanning electron

microscopy examinations.

The GLYE media were prepared using ingredients outlined

in Table 3-2, Table 3-3, and Table 3-4.



Electrochemical Cells Description



The new design of the batch bioreactor, here called the

electrochemical cell, consisted of a modified 1-L erlenmeyer

flask including an extra side opening that fit #8 butyl rubber

stoppers. At the top opening port, an assembly of electrodes

was fitted that included the working electrode (metal

coupon), the pH combination Ag/AgCl reference electrode,

polymer body unit/gel filled by Fisher Scientific, Co., and









61
pressure transducer by Setra Systems, Inc., model 205-2 and

digital pressure indicator, model 300C.

The new system was instrumented for automated data

acquisition of free corrosion potential, pH, oxidation-

reduction potential, and head space pressure. Prior to

filling the electrochemical cell to the 1-L mark with the

medium electrolyte, the cell was outgased in an atmosphere of

80% N2/20% CO2. The flask was then sealed under a positive

head space pressure and placed in a precision water bath by

GCA corporation at 300C. A head space pressure in the range

of 20 to 40 mm of Hg above atmospheric pressure was used

throughout the investigation. This was a pressure that

allowed stoppers not to blow away, it was economical and

allowed an adequate gas supply to the head space. A schematic

of the electrochemical cell is detailed in Figure 3-3.



REDOX POTENTIAL

pH : METRABYTE'S
METRABUS
g A/D DATA
POTENTIAL C ACQUISITION
SYSTEM
PRESSURE






UQUID 1 L
SAMPLUNG 1
PORT

4 EFC E METAL
:ECpODE IBM PC
WATER BATH

Figure 3-6. Schematic of electrochemical cell and data
acquisition system.









62
The system set-up employed an IBM compatible computer

with a four channel MetraByte's MetraBus A/D data acquisition

system that interfaced the four parameters. Custom software,

written in Quickbasic, was used to acquire pH and the three

other parameters, free corrosion potential, oxidation-

reduction potential, and pressure in the form of voltages.

The liquid sampling port consisted of a Pasteur pipet

connected to a 10 mL syringe via a clear plastic tube with an

adjustable tube clamp.



Electrochemical Cells Operations



Single Flask Electrochemical Cell

The first modification or redesign of the batch

bioreactor was the single flask electrochemical cell. A

detailed schematic of the single cell is shown in Figure 3-4.

POTENTIAL


pH PRESSURE





S1L


REFERENCE
ELECTRODE
/ METAL
COUPON

Figure 3-7. Single flask electrochemical cell.








63

Experiments using the electrochemical cells were run at

constant temperature in a water bath at 30C, described

earlier. Anaerobic conditions were maintained throughout the

experiments using gases treated in a 5.0 cm 4 and 75 cm long

copper column that was electrically heated to approximately

3500C and reduced with pure hydrogen. A gas mixture

consisting of 80% N2/20% CO2 was used during outgasing,

preparation of the mineral electrolyte, and preparation of the

bacteria suspensions as described earlier in this chapter.

After outgasing the single cell, the electrochemical cell

was filled with the mineral solution electrolyte to the 1

liter mark. Then, it was sealed with the stopper containing

the metal coupon and electrode assembly, leaving a head space

of approximately 200 mL at a pressure in the range of 20 to 40

mm of Hg above atmospheric pressure. At this stage, the metal

coupon was held clear of the solution electrolyte and the

complete cell was placed in the water bath. Resting cells

harvested by centrifugation were supplied to the single cell

by injecting them through the rubber stopper. Once the data

acquisition system was connected to the SC and data started to

be collected, the metal coupon was lowered into the

electrolyte by pushing the insulated copper wire that holds

the epoxy-mounted coupon through the rubber stopper.

Experiments using this electrochemical cell allowed

fitting 4 single cells in the water bath for each run, one

control and triplicate samples. Gas and liquid samples were








64

removed under sterile conditions, using appropriate syringes

through the septums provided at the top and side flask

openings.



Dual Flask Electrochemical Cell

It consisted of the single cell (flask I) interconnected

to an additional flask II through their head spaces as

described in Figure 3-5. The objective of flask II was to

overcome some anticipated inconveniences experienced in the

single cell such as metal toxicity and/or any stress imposed

on bacteria during the harvesting procedure. Now flask II

could allow bacteria to grow freely and out of contact with

the metal to avoid metal toxicity. The head space connection

allowed cathodically produced hydrogen to be transferred from

flask I to flask II.
PRESSURE
REDOX
POTENTIAL -I

POTENTIAL
1 L VALVE


pH



1L
UQUID
SAMPLING
PORT
METAL
REFERENCE COUPON
ELECTRODE


Figure 3-8. Dual flasks electrochemical cell.









65

Dual cell experimental runs followed a procedure similar

to the setting and operation of the single cell and described

above, except that flask II could be used to grow bacteria in

their optimum media. The setting of the dual cell did not

allow more than one dual cell in the water bath, for which

only single experiments were run.



Triad Flask Electrochemical Cell

This final design consisted of a set-up similar to the

dual cell including an additional flask III, which is

interconnected to flask I via an ionic bridge made out of 5%

agar, KC1 saturated. Flask III was similar to flask II except

the latter was connected to flask I by the head space. A

detailed schematic is shown in Figure 3-6.


POTENTIAL
DIFFERENTIAL II
CORROSION
PRESSURE CURRENT
REDOX
POTENTIAL I
^ = POTENTIAL
1 L VALVE I 1 L

I|
pH


I L
II 1L III
LIUQUID 4 G
SAMPLNG GEL
PORT 1 IONIC
BRIDGE
METAL
REFERENCE COUPOt --
ELECTRODE


Figure 3-9. Triad flasks electrochemical cell.









66
This design was considered capable of satisfying the

objectives of this research because it allowed the measurement

of a differential corrosion current (DCC) induced by bacteria

in addition to other parameters mentioned earlier in the

single cell. A home-made zero resistance ammeter was

incorporated in the data acquisition system in order to

measure and acquire the new DCC parameter. The triad cell as

well as the dual cell allowed bacteria to be grown in flask II

or injected in as suspended cells.

The four available data acquisition system channels were

used to acquire data on the following parameters: pH in flask

I, free corrosion potential in flasks I and III, and DCC

between metal coupons exposed and unexposed to bacteria in

flasks I and III respectively. Triad cell runs used the

selected metal AS106 and the selected bacteria Escherichia

coli (JW11).

Liquid samples for dissolved iron determinations were

taken simultaneously from flask I and flask III, every ten

minutes. Only one triad cell fit the 300C water bath.

Other experiments using the triad cell include a run in

which a vacuum was applied to flask I and then to flask III in

order to exaggerate a case of hydrogen uptake. Near the end

of the test a positive hydrogen pressure was applied to flask

III in order to observe its effect on the potential and

differential corrosion current measurements. The last

experiment consisted of a galvanic metal couple of Mg working








67

electrode in flask I and a Cu working electrode in flask III,

to which vacuums were applied correspondingly to test the

triad cell response to hydrogen uptakes in both flasks. The

purpose of this test was to check the measurement capacity of

the electrochemical system since a significant differential

corrosion current was supposed to be developed.



Preparation of Bacterial Suspensions



The organisms used in this investigation were

Desulfovibrio desulfuricans (ATCC 7757), Entrobacter aerogenes

(ATCC 13048), Clostridium acetobutvlicum (ATCC 824),

Escherichia coli (ATCC 8739), Alcaliaenes eutrophus (ATCC

29597), and Escherichia coli (JW 111), provided by Dr. K. T.

Shanmugam. The first three bacteria have been found often in

real MC cases and all of them represented species of hydrogen

oxidizing bacteria.

The first three bacteria D. desulfuricans, E. aerogenes,

and C. acetobutylicum were grown in glucose lactate yeast

extract (GLYE) medium, as shown in Tables 3-2, Table 3-3, and

Table 3-4. The rest of the bacteria were grown in Trypticase

Soy Broth media. All chemicals used to prepare the media were

reagent grade and were obtained from Fisher Scientific or

Sigma Chemical Co. All media were prepared under an

atmosphere of 80% N/20% CO2. However, when microorganisms

used for MC experiments were inoculated in their respective








68

medium, an atmosphere of 80% Hz/20% CO2 was used to activate

their hydrogen uptake enzymatic systems.

Stock cultures from ATCC were prepared at 30C from

freeze-dried ampules of the organisms in their recommended

growth media indicated above. Cultures were maintained

inoculating agar slants (monthly) and liquid media (weekly).

20 mL serum vials were used for facultative bacteria, and 25

mL crimp-top tubes, sealed with butyl rubber septa, for

anaerobic bacteria.

Resting-cell suspensions were used regularly only in

experimental set-ups that included electrochemical

measurements; other experiments used bacteria inoculated

directly from their grow media. The batch bioreactor also used

washout from slants in some of its runs. Resting-cell

suspensions avoid electrolyte contamination with media used to

grow bacteria.

Cell suspensions were prepared as follows: 1 mL of

subculture of facultative microorganisms less than 1 week old

or 2 mL of subculture of anaerobic microorganisms were

inoculated in 400 mL of appropriate growth media and incubated

in a Fisher low temperature incubator, model 307, at 35C

under a 80% Hz/20% CO2 headspace for 1 day for facultative

bacteria and 3 days for anaerobic bacteria. Once cells were

grown, they were harvested by centrifugation in 50 mL screw

cap tubes for 10 min at 10,000 g.








69

Bacteria pellets from tubes were then resuspended in 5 mL

of the mineral solution electrolyte to give an approximate

concentration of the cell suspension of 65 mg (dry weight

basis).


Preparation of Mineral Solution Electrolyte



In order to provide the electrochemical cell with minimum

nutrient conditions as encountered inside gas transmission

pipelines and still allow bacteria to survive for few days, a

mineral solution electrolyte was developed to be used in the

experimental system to study the onset of microbial corrosion.

The ingredients of the mineral solution electrolyte consisted

of 10% of the concentration of the components of solutions

specified in Table 3-4 including 1 mg/L of resazurine and

deionized distilled water to complete the required volume

concentration. The ingredients were heated to the maximum mark

on the dial on a Fisher Scientific hot plate, model 210T and

stirred under an 80% N/20% CO2 atmosphere. After heating for

0.5 hr, the flask was cooled down to 300C surrounded by

crushed ice in a plastic container for approximately 1 hr. At

this time, the electrolyte was transferred to previously

cleaned electrochemical cells and filled to the l-L marks.

Remaining electrolyte was used to resuspend bacteria and other

routine tests.










Metal Coupon


From an original collection of 33 different metals and

alloys, only 18 different metal samples were cut and coupons

fabricated. Samples were prepared based on availability and

ease of cutting to the maximum size allowed in the molds used

for epoxy mounting. Metal coupons used in the flowthrough and

batch bioreactors were not epoxy mounted.

The availability of SA106 steel coupons, its non-toxic

effects on bacteria, its successful surface colonization and

observed surface deterioration, and the popular use of the

alloy in gas production pipelines suggested its selection for

the electrochemical experiments. In addition, it has a

moderate hydrogen production and it is relatively easy to

polish.

A technique was developed to fabricate the working

electrodes used in the electrochemical cells. It consisted in

cutting metal coupons from ASTM-ASME SA106 grade Bl steel, 2.5

cm diameter schedule 80 17-24 H.T. 2500 psi pipe, obtained

from Texas Eastern, Louisiana. The metal coupons were machine

ground to a size of 1 cm x 1.9 cm x 0.02 cm, and attached to

30 cm long 12 THWN insulated hard copper wire by Cerrowire

using nickel print paint (cat. No. 22-207) by GC Electronics.

Coupons were centrally mounted on epoxide resin 20-8130-128

and hardener 20-8132-032 from Buehler, leaving an area of 1.0

cm x 1.9 cm exposed for the microbial corrosion studies. The








71

hard wire served to support and to electrically connect the

coupon and was thereby insulated from contact with the

electrolyte in which the working electrode was immersed.

Prior to the start, the working electrode was held clear of

the solution contained in the flask and lowered into the

solution at will by pushing the hard copper wire through the

butyl rubber stopper that holds the electrode assembly.

After preparation, the working electrodes were polished

with 600 grit, rinsed with ethanol and blotted dried with

Kimwipes EX-L delicate task wipers, then stored in either a

desiccator glass container or a desiccator cabinet by Labconco

Co., model 55300, under vacuum until ready to be used. During

the polishing of the working electrodes, they were initially

sanded with a 240 emery grid paper to remove any

inconsistencies on the metal surface, then with a 400 grid,

and finally with a 600 grid. The working electrodes were

placed in an ethanol bath in the ultrasonic cleaner to ensure

the removal of metal particles left from previous polishing.

The polishing procedure was done using a Buehler rotary

polishing wheel and care was taken so that no cross-hatching

was left on the metal surface.



Parameters Setting



In an effort to assure consistency during the

electrochemical measurements, several parameters were fixed








72

based on equipment capabilities, typical inside conditions of

gas transmission pipelines, and other anaerobic stagnant

conditions to make measurements simpler and more reliable.

This task involved the use of the batch bioreactor and

consisted of the study of several determining factors that

formed the basis for the development of the electrochemical

system described herein. Experiments done included

determination of head space gas composition, selection of

reducing agent, selection of sterilization method, and other

related studies.

The head space gases used were methane, hydrogen,

nitrogen, carbon dioxide, and helium. The reducing agents

tested were sodium sulfide, cysteine, thyoglycolate/ascorbate,

and Enterobacter aerogenes as a natural reducing agent. Among

the sterilization methods used were: 1) all components of

batch bioreactor autoclaved at 15 psig during 15 min, 2) all

components of batch bioreactor autoclaved except metal-coupon-

rubber-stopper assembly that was oven treated at 105C for 12

hrs, 3) media autoclaved as before and metal-coupon-rubber-

stopper assembly sterilized in acetone and ethyl alcohol for

15 min then blotted dry prior the set-up in bioassay bottle,

and 4) media components autoclaved and metal coupon flamed.

The basic anaerobic and stagnant conditions included fixed

temperature of 35C and pH in the range of 6.0 and 6.5.










Measurement Procedures

All biocorrosion experiments, using electrochemical

cells, were performed at 30C. Flasks using electrode

assemblies include a rectangular metal coupon (working

electrode) with surface areas of 1.9 cm2 mounted in epoxy

resin and abraded to a 600 grit finish. Metal coupons were

connected to 30 cm of insulated hard copper wire that served

as electrical conductors and support in the stopper.

Electrode assemblies included Ag/AgC1 reference electrodes,

polymer body unit/gel filled, from Fisher Scientific.

Reference electrodes were continuously calibrated against each

other using the data acquisition system prior to the

experimental runs. The side opening of flasks contained

platinum electrodes for oxidation-reduction potential

measurement and Pasteur pipets attached to a 10 mL syringe for

liquid sampling.

The four data acquisition channels were connected to the

triad cell as follows: channel four was wired to a pH

reference electrode for pH readings. Channel one and channel

two were wired to the working electrode and reference

electrode of flask I for free corrosion potential

measurements. Channel two was wired to the working electrode

and reference electrode of flask III for free corrosion

potential measurements, and channel three was wired to the

working electrodes of flasks I and III for differential

corrosion current measurements. Current was measured with a









74
homemade zero resistance ammeter with a 10 nA sensitivity,

made of a #308 operational amplifier and a high precision

10,000 0 resistor. This device has the property of converting

current into voltage out with a gain determined by the high

precision resistor, allowing the data acquisition system to

measure and acquire data in volts. A digital pressure

indicator, model 300C by Setra Systems was also adapted to

monitor the headspace pressure in flask I. Once the data

acquisition system was started the working electrodes were

immersed in their respective electrolytes and data was stored

in computer floppy diskettes.

Samples of bacteria, in their growth medium, in their

resuspended medium, and in the liquid discarded after

centrifugation, were homogenized using a vortex Fisher brand,

model Genie 2, and taken to a spectrophotometer Spectronic 21D

by Milton Roy for determination of transmittance, absorbance,

and concentration at the start of each experiment. After

spectrophotometer determinations, samples were vacuum filtered

using 47 Mm pore size membrane filters, preweighted in a

Metler AE100 balance. Wet mounts were also prepared regularly

to check bacteria viability under a light microscope by Nikon,

model LABOPHOT-2. At intervals of 10 minutes, 10 mLs aliquots

were withdrawn from the electrolytes from the flasks I and III

under stagnant conditions and analyzed for dissolved Fe at the

IFAS Soil Science Analytical Research Laboratory. Last runs

were sampled only at the beginning and at the end, shaking








75

flasks to avoid stagnant conditions for dissolved iron. The

duration of each electrochemical experiment was approximately

1 hour. Experiments were continued until the corrosion

potentials leveled off.



Analytical Procedures

Dissolved iron analyses were performed at the IFAS Soil

Science Analytical Research Laboratory. Samples were

collected in 10 mL vials and preserved using 1 drop of

concentrated H2SO and analyzed on an atomic absorption unit

by Perkin Elmer.

Molecular hydrogen was measured on a gas chromatograph by

Gow-Mac Instruments Co., series 580, equipped with a thermal

conductivity detector. 50 pL samples were collected in a gas

tight syringe and injected onto a molecular sieve type 5A

column. Samples were injected at an inlet temperature of 400C

with column temperature of 35C and a detector temperature of

93C. The carrier gas used was nitrogen and the detector

current was 50 mA.

Transmittance, absorbance and concentration were measured

on a spectrophotometer Spectronic 21D by Milton Ray. The

samples were collected in 10 mL Hatch COD tubes and

homogenized in a vortex mixer by Fisher, model Gene 2, prior

to measurements.

Volatile fatty acids were measured on a FID gas

chromatograph by Shimadzu, model GC-9AM. The samples were









76
prepared by centrifugation after acidification with 20%

phosphoric acid. The samples were injected onto a 2m long by

2mm 0 glass column packed with 80/100 chromosorb 1200 WAW

coated with 3% H3PO, and carried with nitrogen gas. IpL

volumes were injected at an inlet temperature of 180C with

column temperature ramped from 130C to 170C over 5 min and

a detector temperature of 2000C.

Dry weights of resting cells were determined by vacuum

filtering known volumes of bacterial suspensions. Filtration

was performed in a 47 mm magnetic Gelman Filter funnel (cat.

09-735). The samples were filtered using 0.45 pm pore size

membrane filters, 47 mm plain Supor 450, by Gelman Sciences,

Co.. Vacuum was achieved with a precision belt-driven vacuum

pump by Fisher Scientific, model D-75. Membrane filters were

then oven dried at 105C for 12 hrs. Weights were measured in

a Metler balance, model AE100.



Gases

The gases used during the investigation were: Nitrogen

(UN1066), Helium (UN1046), Hydrogen (UN1049), Nitrogen/Carbon

Dioxide (80/20, UN1956), and Hydrogen/Carbon Dioxide (80/20,

UN1954) supplied by Liquid Air Corporation. For anaerobic

experiments all gases were passed through a heated copper

column (5 cm 4 and 75 cm long) to free gases from traces of

oxygen. The column was heated electrically to about 350C by

a coil of electrical wire wrapped around the column.








77

Reduction of the column was achieved by passing hydrogen gas

through the column.



Scanning Electron Microscopy

Metal coupons, after exposure to microorganisms, were

treated with a buffered glutaraldehyde solution to fix the

morphology of the bacteria, and then dehydrated through a

graded ethanol series of increasing concentration ranged from

25% to 100% using procedure outline in Table 3-1, page 93.

Samples were then dried and sputter coated with gold

palladium, then examined in a Hitachi S-450 SEM, that included

an Energy Dispersive X-ray Analyzer (EDXA). Coupons were

analyzed throughout and micrographs and spectra were taken at

sites of major interest.



Light Microscopy

Metal coupons were observed and photographed under a

Nikon stereomicroscope, model SMZ-2T and a Nikon microscope,

model LABOPHOT-2, for biofilm and corrosion examination. Pure

cultures and liquid samples from bioreactors were examined

periodically for contamination via wet mounts.














CHAPTER 4
RESULTS AND DISCUSSION


Flowthrough Bioreactor


Two experimental runs were performed in this bioreactor

in order to reproduce microbial corrosion and to study the

effect of a bacterial triculture on the corrosion of carbon

steel coupons and the formation of biofilm. Its set-up is

described in Figure 3-1. Both runs reproduced microbial

corrosion and their results are shown in Figures 4-1, 4-2, 4-

3, 4-5, and 4-6. Metal coupons exposed to a bacterial

triculture, grown in glucose lactate yeast extract (GLYE)

media under anaerobic conditions, were examined under the

light and the scanning electron microscope (SEM). The results

indicated extensive bacteria colonization as well as metal

deterioration. Careful observations of biofilm micrographs

have suggested a mechanism for understanding its formation.

The first experiment was run for a period of four weeks.

Metal coupons and liquid media were sampled weekly for routine

analyses. First week scanning electron microscopy (SEM)

micrograph results indicated that the bacterial triculture

developed a biofilm consisting of a separated polymeric double








79

layer structure; an inner structure attached to the metal

surface, and an outer structure that covers it. It appears

that Enterobacter aeroaenes, the bacterium inoculated first,

was responsible for the production of the inner structure and

perhaps the outer structure of the biofilm, see Figures 4-la

and 4-1b. This bacterium, besides reducing the media to

accommodate the strict anaerobes, synthesized a considerable

amount of extracellular polymeric material for attachment

purposes. This material is believed to overcome the natural

surface repulsion caused by the negatively charged bacteria

and metal surfaces (Beveridge and Doyle, 1989). Figure 4-1c

shows the characteristics of the abundant polymeric network-

like inner structure produced by the bacterial triculture. E.

aerogenes is probably responsible for building this structure

because it is the most prolific of the three species and

reaches the log phase of the growth curve in less than 6 hrs

as compared to the other two bacteria which require often more

than two days. Wachenheim and Patterson (1992) reported that

anaerobic production of extracellular polysacharides is

enhanced generally with any improvement of the conditions of

bacterial growth and that this material is only produced

during the log phase of growth where its production is also

exponential. In our work, the flowthrough bioreactor was run

using GLYE medium which must have enhanced the production of

the polymeric material. This fact, if compared to experiments

run in a medium with limited nutrient composition (mineral








80

solution only), indicates that the amount of biofilm formed on

the metal surface is proportional to the amount of organic

nutrient in the medium.

Desulfovibrio desulfuricans and Clostridium

acetobutylicum were inoculated one day after the initial

inoculation of Enterobacter aerogenes. The components of the

bacteria triculture were cultured separately. Figure 4-2

shows scanning electron microscopy micrographs of each species

for identification purposes. After inoculation, the strict

anaerobes probably displaced E. aerogenes from the inner

structure of the biofilm so it moved upward and started

producing the outer biofilm layer. This mechanism, suggested

in Figure 4-la, indicates the presence of D. desulfuricans and

C. acetobutylicum in between the inner and outer structure and

Figure 4-1c shows that most bacteria in the outside are

represented by E. aerogenes and C. acetobutylicum. These

figures also show that the outer biofilm structure is rougher

on the inside than on the outside. The inner surface seems

like a woven net of polymeric material attached to the metal

surface.

Metal coupon surfaces for this experiment were polished

to 240 grid. Figure 4-3a represents a micrograph of the

control surface, unexposed to bacteria and magnified 1200

times. Figure 4-4a shows the corresponding energy dispersive

x-ray analysis (EDXA) spectrum of the metal surface.








81
After anaerobic exposure of the metal coupons to the

bacterial triculture for a week, surface colonization

including pitting corrosion was observed. Figures 4-3b, 4-3c,

4-3d, and 4-3e show localized pitting and polymeric biofilm,










a)










Figure 4-1. Flowthrough bioreactor results on bacteria
attachment to carbon steel coupons. a) SEM micrograph showing
bacteria within the outer and the inner structures of the
biofilm. b) SEM micrograph indicating the nature of the outer
biofilm structure.


























c)










Figure 4-1.
biofilm.


c) SEM micrograph showing bacteria on inner


Figure 4-2. SEM characterization of bacterial species from
triculture. a) Enterobacter aeroqenes.

































Figure 4-2. b) Clostridium acetobutylicum.


Figure 4-2. c) Desulfovibrio desulfuricans.








84

metal deterioration near the edge of the biofilm, generalized

metal deterioration, and elongated pits respectively. Figure

4-4b shows the EDXA spectrum of a metal coupon after exposure

to the bacterial triculture. This analysis presents

additional evidence of microbial corrosion. It shows the

presence of an additional peak (if compared to the control),

corresponding to a sulfur compound, which is probably due to

the deposition of ferrous sulfide resulting from the activity

of D. desulfuricans.

Stereomicroscopic observation of coupon surfaces after

exposure to the bacterial triculture suggest the nature of the

biofilm to be slimy. Figure 4-5a and 4-5b are photographs of

the face and edge of a metal coupon respectively.

During the initial run of the flowthrough bioreactor,

volatile fatty acids and pH were measured weekly for one

month. Results, shown in Figure 4-6, indicate that the only

VFAs produced were acetic and butyric acids. During the first

week, only acetic acid was produced and its concentration

ranged between 300 and 400 ppm until the 3rd week and almost

double by the end of the fourth week. Butyric acid appeared

after the second week and increased at a rate of 200 ppm per

week to a concentration very close to 600 ppm at the end of

the 4th week. Figure 4-6 also shows that pH dropped to 6.5,

probably due to the accumulation of volatile fatty acids.

During the first three weeks the pH ranged between 7.0 and 7.5

then dropped to 6.5 during the 4th week.


































Figure 4-3. Flowthrough bioreactor SEM results of microbial
corrosion of carbon steel coupons. a) Control surface of
coupon unexposed to bacteria and polished to 240 grid.


Figure 4-3. b) Localized pitting near biofilm after exposure
to bacterial triculture.


































Figure 4-3.
near biofilm.


c) SEM micrograph showing microbial corrosion


Figure 4-3. d) SEM micrograph showing generalized metal
deterioration after exposure to bacteria.
































Figure 4-3. e) SEM micrograph showing elongated pits.


Figure 4-4. Flowthrough bioreactor results of EDXA analysis.
a) Control EDXA spectrum of a steel coupon unexposed to
bacteria indicating metallic components of the SA106 alloy.




Full Text
MICROBIAL UTILIZATION OF CATHODIC HYDROGEN
AND RELATED CORROSION
By
JOSE RAFAEL SIFONTES
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1994

To the memory of my father, Pedro Rafael Sifontes Lopez.

ACKNOWLEDGEMENTS
I wish to thank the following persons for their
contributions throughout my graduate studies at the
Agricultural Engineering Department. My sincere appreciation
goes to Dr. David P. Chynoweth, chairman of my supervisory
committee, for his friendship, concern, and economic support
throughout this work. I also would like to thank Dr. Henry
Aldrich, Dr. Edward Lincoln, Dr. Mark Orazem, Dr. Roger
Nordstedt, and Dr. Ellis Verink for always being there when I
needed them and for access to their laboratories.
My special thanks go to all my friends and departmental
staff who encouraged me, particularly to Dr. K.T. Shanmugam,
Jose Moratalla, Paul Lane, Brian Ferber, Emo Crews, Larry
Miller, and Hyung-Jib Lee for their help and encouragement
during my stay at the Agricultural Engineering Department.
This work was initially supported by the Gas Research
Institute.
And most important, I want to thank God in the name of my
Lord Jesus Christ for allowing this to happen. To HIM be all
the Glory!
iii

TABLE OF CONTENTS
ACKNOWLEDGEMENTS iii
LIST OF TABLES vi
LIST OF FIGURES vii
ABSTRACT X
CHAPTERS
1 INTRODUCTION 1
Objectives 1
Justification 2
Background 7
The Defective and Nonuniform Nature of
Metallic Surfaces 10
The Molecular Fluctuations of the
Electrical Double Layer 12
The Mass Transport Perturbation in the
Diffusion Layer 13
Statement of the Problem 14
2 REVIEW OF LITERATURE 17
Historical Overview 17
Other Aspects of Microbial Corrosion 24
Microorganisms Involved in Microbial Corrosion.. 27
Mechanisms of Microbial Corrosion 31
Theory of Cathodic Depolarization 34
Previous Studies in Microbial Corrosion 37
Microbial Corrosion Control 42
Research Approach 47
3 MATERIALS AND METHODS 49
Introduction 49
Flowthrough Bioreactor 50
Description 50
Operation 54
Batch Bioreactor 58
Description 58
Operation 59
iv

Electrochemical Cells Description 60
Electrochemical Cells Operations 62
Single Flask Electrochemical Cell 62
Dual Flask Electrochemical Cell 64
Triad Flask Electrochemical Cell 65
Preparation of Bacterial Suspensions 67
Preparation of Mineral Solution Electrolyte 69
Metal Coupon 70
Parameter Setting 71
Measurement Procedures 73
Analytical Procedures 75
Gases 76
Scanning Electron Microscopy 77
Light Microscopy 77
4 RESULTS AND DISCUSSION 78
Flowthrough Bioreactor 78
Batch Bioreactor 92
Effect of Head Space Gas Composition 93
Effect of Bacteria Combination 95
Selection of Media 95
Selection of a Reducing Agent 96
Selection of a Sterilization Method 97
Experimental Results 98
The Electrochemical Cell 106
Single Flask Electrochemical Cell 113
Dual Flask Electrochemical Cell 117
Triad Flask Electrochemical Cell 125
Final Discussion 137
New Proposed Microbial Corrosion Mechanism 154
Capabilities of the Triad Cell 156
5 SUMMARY AND CONCLUSIONS 161
Summary 161
Conclusions 165
Suggested Future Research 168
LITERATURE CITED 170
BIOGRAPHICAL SKETCH 182
v

LIST OF TABLES
Table page
2-1 Chemistry of the cathodic depolarization theory... 35
3-1 Protocol for metallic surface fixation for
scanning electron microscopy examinations 55
3-2 Glucose Lactate Yeast Extract (GLYE)
Medium Composition 56
3-3 Trace Vitamins Solution Composition 57
3-4 Mineral Solution Composition 57
4-1 Batch bioreactor volatile fatty acids
measurements from experiments using steel
coupons and bacterial triculture in mineral
solution 99
4-2 Batch bioreactor head-space pressure drop in
GLYE and mineral solution in the absence of
bacteria 102
4-3 Hydrogen production by filings of Mg, Zn,
and SA106 steel in mineral solution in the
absence of bacteria after 3 days 107
4-4 Free corrosion potential of SA106 steel
coupons at different surface polishing grades ....108
4-5 Galvanic series of selected metals and alloys
in mineral solution at 30°C vs Ag/AgCl
reference electrode in the absence of bacteria ...110
4-6 Effect of media composition on the hydrogen
uptake of E. coli at 30°C during 6 hours 112
4-7 Bacteria hydrogen uptake capabilities in
mineral solution at 30°C during 6 hours 113
4-8 Effect of different bacteria on the free
corrosion potential of Mg, Cu, and SA106 steel
coupons in mineral solution at 30°C 115
vi

4-9 Dual cell schedule of experiments and
corresponding corrosion rates, using E. coli
(JW111) and SA106 steel coupons 118
4-10 Triad cell schedule of runs and related
corrosion USING E. coli (JW111) and SA106
steel coupons 127
vii

LIST OF FIGURES
Figure page
3-1 Flowthrough bioreactor general description of
components 51
3-2 Metal coupon and fluorescence probe setup 53
3-3 Metal coupon, fluorescence probe and reference
electrode setup 53
3-4 Flowthrough bioreactor components 54
3-5 Batch Bioreactor 59
3-6 Schematic of electrochemical cell and data
acquisition system 61
3-7 Single flask electrochemical cell 62
3-8 Dual flask electrochemical cell 64
3-9 Triad flask electrochemical cell 65
4-1 Flowthrough bioreactor results on bacteria
attachment to carbon steel coupons.
a) SEM micrograph showing bacteria within
the outer and inner structure of the
biofilm 81
b) SEM micrograph indicating the nature of the
outer biofilm structure 81
c) SEM micrograph showing bacteria on inner
biofilm 82
4-2 SEM characterization of bacterial species from
triculture.
a) Enterobacter aerogenes 82
b) Clostridium acetobutvlicum 83
c) Desulfovibrio desulfuricans 83
4-3 Flowthrough bioreactor SEM results of microbial
corrosion of carbon steel coupons.
a) Control surface of coupon unexposed to
bacteria and polished to 240 grid 85
b) Localized pitting near biofilm after exposure
to bacteria triculture 85
c) SEM micrograph showing microbial corrosion
near biofilm 86
viii

d) SEM micrograph showing generalized metal
deterioration after exposure to bacteria 86
e) SEM micrograph showing elongated pits 87
4-4 Flowthrough bioreactor results of EDXA analysis.
a) Control EDXA spectrum of a steel coupon
unexposed to bacteria indicating the metallic
components of the SA106 alloy 87
b) EDXA spectrum of a metal surface exposed to
bacteria showing the sulfur peak resulting
from metal sulfides deposition by
Desulfovibrio desulfuricans 88
4-5 Flowthrough bioreactor biofilm appearence
under the stereo microscope.
a) Flat-side view 88
b) Edge-side view 89
4-6 Flowthrough bioreactor volatile fatty acids
and pH profile 89
4-7 Flowthrough bioreactor-second experiment.
a) SEM micrograph showing appearence of biofilm .. 90
b) SEM micrograph indicating massive pitting 91
4-8 Flowthrough bioreactor EDXA spectrum of metal
coupon exposed to bacteria 91
4-9 Batch bioreactor biofilm formed within
24 hours 100
4-10 Batch bioreactor SEM and EDXA results
on bacterial attachment to carbon steel.
a) Bacteria colonization within 24 hours 100
b) EDXA spectrum of metal coupon exposed
to bacteria for 24 hours 101
4-11 Batch bioreactor-formation of a crystalline
film on the metal surface after one week of
exposure to the mineral solution in the absence
of bacteria.
a) SEM micrograph showing characteristics of the
crystalline film 104
b) EDXA spectrum showing elemental distribution
of crystalline film on metal surface 104
4-12 Batch bioreactor-formation of crystalline film
on metal surface after exposure to bacteria,
a) SEM micrograph showing a transition zone
from biofilm to crystalline film 105
ix

b)SEM micrograph showing a detriorated
polymeric biofilm structure and pitting
after bacteria have died 105
4-13 Potential-time curve for SA106 steel in the
absence of bacteria 109
4-14 Growth curve for E. coli at 600 nm, 37°C and
trypticase soy broth medium Ill
4-15 Potential-time curves for SA106 steel in the
pressence of E. coli 114
4-16 Potential-time curves for Mg in the presence
of E. coli 114
4-17 Dual cell run potential-time curves.
a) Effect of E. coli. 6 hr old 121
b) Effect of E. coli. 15 hr old 122
c) Effect of E. coli. 23 hr old 123
d) Effect of E. coli. 27 hr old and fumarate 124
4-18 Dual cell run dissolved iron profiles.
a) Effect of E. coli. 6 hr old 121
b) Effect of E. coli. 15 hr old 122
c) Effect of E. coli. 23 hr old 123
d) Effect of E. coli. 27 hr old and fumarate 124
4-19 Triad cell run potential-time curves.
a) Effect of E. coli. 8 hr old 129
b) Effect of E. coli. 20 hr old and fumarate 130
c) Effect of E. coli. >6 days old 132
4-20 Triad cell run dissolved iron profiles.
a) Effect of E. coli. 8 hr old 129
b) Effect of E. coli. 20 hr old and fumarate 130
c) Effect of E. coli. >6 days old 132
4-21 Triad cell artificial Hydrogen uptake test 134
4-22 Artificial hydrogen uptake in a triad cell
using Cu and Mg electrodes 136
4-23 Hydrogen embrittlement of SA106 steel in
mineral solution electrolyte.
a) SEM micrograph of crack at a
low magnification 148
b) SEM micrograph of crack at a
high magnification 148
c) EDXA micrograph of corrosion products
inside the crack 149
x

d) EDXA micrograph of uncracked control
surface 149
4-24 Proposed anaerobic mechanism for the on-set of
microbial corrosion of steel by hydrogen
oxidizing bacteria 155
xi

Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
MICROBIAL UTILIZATION OF CATHODIC HYDROGEN
AND RELATED CORROSION
By
Jose Rafael Sifontes
August 1994
Chairman: David P. Chynoweth
Major Department: Agricultural Engineering
Microbial corrosion is the deterioration of a material by
corrosion processes that occur directly or indirectly as a
result of the activity of microorganisms. Although it is a
widely recognized phenomenon in many industrial processes
worldwide, the fundamental corrosion mechanisms are not
understood, it is not well defined, and the microbial effect
has not been quantified. The objective of this research was
to show a relationship between cathodic hydrogen utilization
by bacteria and corrosion at its onset for the purpose of
improving its understanding and attempting to quantify the
biological component of corrosion.
Two new experimental flowthrough and batch bioreactor
systems were evaluated to reproduce anaerobic microbial
corrosion. Both reactors were able to reproduce microbial
corrosion in 24 hr. However, the batch system offered the
xii

better alternative to study microbial corrosion at an
attractive economic operation. Since microbial corrosion is
difficult to separate from pure electrochemical corrosion and
electrochemical measurements offers a nondestructive technique
to measure corrosion, the batch bioreactor was instrumented
for electrochemical measurements and for physicochemical
analysis.
Results from potential-time curves, dissolved iron
profiles, and differential corrosion currents indicated that
metals were reactive to the presence of bacteria. However,
the results were not adequate to quantify the biological
component of corrosion. Significant increases in corrosion
rates, observed during the addition of a terminal electron
acceptor, suggested a catalytic effect on the bacterial
hydrogenase system. The observation of cracks on steel
samples suggested the occurrence of hydrogen embrittlement.
The results of this research are important because they
offer a new approach for the investigation of microbial
corrosion, the triad cell offers an opportunity to quantify
the biological component of corrosion, and a new mechanism was
proposed for the understanding of the onset of anaerobic
microbial corrosion.
xiii

CHAPTER 1
INTRODUCTION
Objectives
The overall objective of this research was to show a
direct relationship between corrosion of metals and hydrogen
utilization by bacteria during the onset of microbial
corrosion, for the purpose of improving our understanding of
the phenomenon. A new approach to the investigation of
microbial corrosion was selected to break ground in an
alternative direction to the one established over the past
century and for a better comprehension of the fundamental
microbial corrosion mechanisms.
The specific objectives of the study were to
1) evaluate new experimental flowthrough and batch bioreactors
to adequately reproduce anaerobic microbial corrosion,
2) quantify the biological component of corrosion at its onset
by measuring indirectly hydrogen uptake by bacteria and
correlating it to total metal corrosion, using nondisrupting
electrochemical techniques,
3) incorporate into a comprehensive microbial corrosion model
all concepts developed during the investigation.
1

2
Justification
The ability of microorganisms to induce or influence the
deterioration of metals has been known for over a century, and
now it is a widely recognized problem in most industrial
processes worldwide. The involvement of microorganisms in
metal deterioration has led to the question of how biological
agents affect the classical corrosion mechanisms.
The true economic impact of metals deterioration is very
difficult to determine. However, some estimates have been
made in the USA and the UK. In the USA, the cost of corrosion
estimated by Poff (1985) and a projection by the National
Bureau of Standards for the year 1985 was approximately $140
billion and $170 billion, respectively. As for microbial
metal deterioration, studies performed by But1in et al.
(1952), Allred et al. (1959), Booth (1964), and the National
Corrosion Service (Wakerly, 1969) estimated that metallic
corrosion caused by microbial intervention in a wide range of
industrial cases in the UK was on the order of 70%, 77%, 50%,
and 10%, respectively, of the total corrosion problem. In the
USA, Paternaude (1985) indicated that 50% of the steel culvert
pipe corrosion in Wisconsin was due to sulfate reducing
bacteria. If the lowest 10% is selected, the cost of
microbial corrosion represents a $17 billion per year problem
in the USA alone. Although such calculations may be open to
objections on matters of detail, the magnitude is likely to

3
stay high whatever route is followed to arrive at a final cost
because the above costs include replacement, prevention, and
maintenance but exclude losses of time, money, natural
resources, human suffering, and death due to eguipment
failure.
Metallic corrosion is a natural process in which metals
return to their natural, oxidized states. It is an
interdisciplinary field of engineering and science where a
knowledge of the metallurgy of the metal, the environmental
conditions, the chemical composition, and the electrochemistry
of the system, are essential to understanding the process.
For example, hydrogen embrittlement is too complex for one of
the above disciplines to explain, and despite several decades
of investigation it is still unclear why hydrogen embrittles
some metals and alloys and not others (Oriani, 1987; Wilde and
Kim, 1986). Most corrosion processes are essentially surface
electrochemical mechanisms common to most metals in aqueous,
or at least humid, environments. This conclusion was first
reached by Whitney (1903). During the manufacturing process
of metals from their ores, metals are converted to a reduced
state, which makes them thermodynamically unstable in the
presence of oxygen, except gold.
Microbial corrosion is defined as the deterioration of
materials by natural processes that are directly or indirectly
related to the activity of microorganisms. The microbial
corrosion phenomenon has been known for nearly a century. It

4
has been reported to exist almost anyplace that microorganisms
colonize. In the process industry, it can happen inside and
outside of equipment and in aerobic or anaerobic environments.
Microbial corrosion is a diverse and complex phenomenon, and
the literature associated with it tends to be just as diverse
and complex. Microbial corrosion was well recognized and
established as a serious problem in the 1970s (Tatnall, 1981).
Microbial corrosion is difficult to separate from pure
electrochemical corrosion. Microorganisms involved in
microbial corrosion generally do not lead to a new form of
corrosion, but to a stimulation of the normal electrochemical
corrosion process. Consequently, if the microbes interfere
with an electrode process, with their own metabolism or
presence, it is a direct effect, otherwise it is an indirect
effect.
To demonstrate microbial corrosion, the presence of
microorganisms must be shown to induce or influence metal loss
during corrosion. Unfortunately, microbial corrosion in real
cases is very difficult to reproduce, extremely complex, and
difficult to model (Tatnall, 1988). Laboratory-based
controlled experiments using defined media and characterized
pure cultures do not often yield the expected results because
a broad range of variables makes it difficult to simulate the
dynamic natural environment (Allsopp and Seal, 1986).
The study of microbial corrosion has had a tendency to
consider processes in isolation rather than actual microbial

5
corrosion cases. For instance, there are many studies dealing
with the effect of a single species of microorganisms on metal
corrosion (Tatnall, 1988). While such investigations are
valuable in elucidating mechanisms, they give little insight
into the wider biological/metal/fluid interactions. Although
recent reappraisal of the role of bacterial consortia together
with studies of the effect of such consortia have improved our
knowledge of the microbial corrosion process, few studies of
microbial corrosion look beyond bacterial consortia and
surface corrosion effects to wider biological/metal/fluid
interactions. Furthermore, any living or indeed dead organism
that becomes associated with a metal surface immersed in an
electrolyte has the potential to influence the corrosion of
that metal. Edyvean (1988) in his recent work discusses the
interaction of both bacteria and macro- and micro-algae in the
fouling community on steel substrata in sea water.
Most engineering materials in general use are susceptible
to some form of microbial corrosion. Several of the metals
and alloys reported as being susceptible to microbial
corrosion include iron, copper, aluminum, nickel, cobalt, and
zinc and their alloys (Gabrielli, 1988; Zamanzadeh et al.,
1989; Griffin et al., 1989). The industries affected by
microbial corrosion have been identified as wastewater
facilities, water flood control systems, petrochemical
equipment, cooling water systems, underground structures and
pipelines, aircraft fuel systems, ships and marine structures,

6
chemical process industries, power generation industries, and
paper mills.
From case histories of microbial corrosion, the forms of
corrosion that are stimulated by the interaction of
microorganisms with metals range from general pitting
corrosion, crevice corrosion, and stress corrosion cracking to
enhancement of corrosion fatigue, intergranular stress
cracking, and hydrogen embrittlement with cracking (Sifontes
and Block, 1991).
The fundamental mechanisms that drive microbial corrosion
are not properly understood nor have they been well defined.
Consequently, additional research to understand them is
required. Most work to date in microbial corrosion has dealt
with reports of case histories or observations incidental to
the main study, with poor documentation regarding the
physical, chemical, and microbiological conditions under which
it occurred (Tatnall, 1981, 1988) .
Data from the literature surveyed suggest that
qualitative relationships exist between the surface properties
of the various metals and their alloys and the extent of
biological response in relation to corrosion. The fact is
that it is not well known why some metals are more susceptible
to microbial corrosion than are others and why the microbial
corrosion effect of a particular microorganism is different
from the microbial corrosion effect of similar or different
species.

7
Background
The literature indicates that the first time a mechanism
for microbial corrosion was proposed was in 1934 by von
Wolzogen Ruhr and van der Vlught. This theory initiated
systematic studies on microbial corrosion and identified
formally the components of the microbial corrosion phenomenon,
namely, the metal surface, the suspended fluid, and the
microorganisms. They proposed that sulfur reducing bacteria
accelerated the corrosion of ferrous metals by cathodic
depolarization, that is, by removing adsorbed hydrogen from
the cathodic surfaces of the metal.
The theory appeared to be relatively simple to confirm by
conventional electrochemical techniques. However, qualitative
confirmation has been obtained only for specific hydrogenase-
positive species of microorganisms such as sulfate reducing
bacteria (Booth and Tiller, 1960) and methanogens (Daniels et
al., 1987). Belay and Daniels (1990) reported that besides
iron, other metals (Al, Zn, Ni, and As) can also be oxidized
via cathodic depolarization.
In the work performed by Daniels and coworkers (1987) , it
was demonstrated that methanogenic bacteria use either pure
elemental iron or iron from mild steel as a source of
electrons in the reduction of carbon dioxide to methane.
These bacteria use the oxidation of iron for energy generation
and growth. The mechanism of iron oxidation is cathodic

8
depolarization, in which electrons from iron and hydrogen ions
from water produce molecular hydrogen, which is then released
for use by the methanogens.
The extent of bacterial adhesion is determined by the
surface properties of the phases involved and the need of
bacteria to locate energy sources. Bacterial cells will stick
to most surfaces, whether the surfaces are those of other
cells or merely inert material such as metals. In nutrient-
poor environments (e.g., water transmission pipelines), most
bacteria grow attached to surfaces mainly due to
hydrophobicity.
It is the belief of some researchers that initially,
sessile bacteria adhere randomly to metal surfaces by means of
their production of extracellular polysaccharides. The
continued production of the polysaccharide and the
reproduction of the bacteria lead to the development of
biofilms in which a consortium of cells interact in a hydrated
matrix of anionic polysaccharide polymers that provide
protection from natural or synthetically produced
antimicrobial agents (Costerton and Geesey, 1985).
As a bacterium begins to proliferate within biofilms, its
metabolic products stimulate the growth of other organisms.
As the different microorganisms develop, molecular or proton
exchanges occur; consortia such as these have been detected
and associated with microbial corrosion of metallic surfaces.

9
In many neutral solutions the corrosion of the common
structural metals appears to be associated with the flow of
electric currents between various parts of the metal surface
at finite distances from one another (Mears and Brown, 1941).
This statement is supported by much evidence in the case of
steels, where the quantities of current flowing during
corrosion account for the amount of corrosion that occurs. In
other words, the corrosion of metals and their alloys in
neutral solutions is electrochemical in nature. Furthermore,
for corrosion to occur, it requires all four components of an
electrochemical cell which include: 1) electrodes, 2)
electrolyte, 3) potential difference, and 4) electrical
continuity. When one of these conditions is regulated, then
the corrosion process is controlled.
Among some of the nonbiological factors of microbial
corrosion that corrosion engineers and scientists have
associated with corrosion and have known for several decades
are 1) impurities in the corroding metal, 2) grain boundaries,
3) orientation of grains, 4) differential grain size, 5)
differential thermal treatment, 6) surface roughness, 7) local
scratches and abrasions, 8) difference in shape, 9)
differential strain, 10) differential pre-exposure to air or
oxygen, 11) differential concentration or composition of the
corroding solution, 12) differential aeration, 13)
differential heating, 14) differential illumination, 15)
differential agitation, 16) contact with dissimilar metals,

10
17) externally applied potential, and 18) complex cells (Mears
and Brown, 1941). Those factors are closely related with the
so-colled "eight forms" of corrosion, which include uniform,
galvanic, crevice, pitting, intergranular, dealloying,
erosion, and stress corrosion (Fontana and Green, 1987) .
In addition to the above, there is always a lack of
homogeneity at the metal-electrolyte interface during the
microbial corrosion of metals. Consequently, several of the
following specific interactions need to be recognized (Sato,
1987).
The Defective and Nonuniform Nature of Metallic Surfaces
During the formation of a metal or an alloy, different
elements compose the molten metal. When solidification
starts, metal is formed of crystalline grains that grow in
size and they meet each other to form grain boundaries until
the metal is all solid. Once the grains are formed, the
overall energy of the individual metal atoms
lowers and the grain boundaries remain as sites of higher
energy or higher thermodynamic instability (Smith, 1986).
The process of electron transfer is suspected to take
place at metallic surface locations of higher thermodynamic
instability such as the grain boundaries. This fact may be
explained by the nature of the metallic bond where the metal
ions occupy positions in the crystal lattice of the grains and
the outer valence electrons are shared by the surrounding

11
metal atoms. These electrons are like a cloud that moves
freely throughout the lattice and binds the crystal together.
Consequently, microorganisms can take advantage of these
electrons as their energy source for growth and affect the
stability of the metal atoms.
In addition, the steel-making process and treatment
influences the microstructure of the alloy by producing
microcompositional differences that have different
electrochemical behavior, which leads to the formation of
localized corrosion. Other changes in the alloy's internal
structure, such as aging and welding, may occur after it has
been established during fabrication.
Among other metallurgical factors to be considered at the
onset of microbial corrosion are the following
a) Composition of alloying elements and impurities
(inclusions), cooling conditions and post heat treatments have
a marked influence on their size, shape, number, and
distribution. These constituents are never uniformly
distributed throughout the alloy and certain elements tend to
congregate in localized concentrations as second phase
particles in grain boundaries, as pure compounds or as
intermetallic compounds (Godard,1980). These particles play
a major role in the corrosion behavior of alloys, especially
in the case of pitting corrosion.
b) Surface contamination by mill scale during the rolling
of the steel causes severe localized corrosion at breaks and

12
imperfections in the surface and may need to be removed before
service. Steel can also be contaminated during forging with
particles of metal from the forming equipment and consequently
produce serious localized corrosion, too.
The Molecular Fluctuations of the Electrical Double Layer
For iron, there exists a spatial fluctuation of the
electrical double layer potential on the metal surface in the
order of 0.8 V for every 2-3 atomic distances (Sato, 1987).
One possible reason is that, in addition to the heterogeneous
metal surface, water dipoles tend to point nearly
perpendicular to the metal surface, while the two nearest
dipoles tend to align antiparallel to each other in order to
minimize their dipole interaction energy. Consequently,
reorientation of water dipoles in the electrical double layer
occur in a time scale much greater than the time scale
relevant to the electron fluctuation. In addition, water
dipoles require greater time to reorient at surface defects
sites where the adsorption energy of water dipoles is greater
(Sato, 1987) . This fact causes the electron transfer to take
place unevenly on the metal surface. At certain surface
defect sites, local enhancement of the electrical field in the
electrical double layer may be maintained for a sufficiently
long time so that the electrode reaction takes place
preferentially at these sites, making them active reaction
sites. Other sources of the spatial non-uniform electrical

13
double layer potential are specific ion adsorption. In this
case, the ions divest of their hydration water molecules and
come into direct contact with the metal surface. This causes
a local enhancement (approximately 3 times) of the electrical
field in the inner part of the electrical double layer. This
phenomenon takes place preferentially at certain lattice or
defect sites and will generate active spots for some electrode
reactions.
The Mass Transport Perturbation in the Diffusion Laver
This dynamic perturbation causes the local ion
concentration and the local electric field to fluctuate in the
diffusion layer. In addition, when the corroding metal
surface is usually covered with a porous corrosion precipitate
film of hydrated metal oxides or insoluble salt, selective
mass transport occurs, and this film either accelerates or
decelerates further corrosion of the underlying metal. When
those interactions are favorable to corrosion, chemical and/or
electrochemical active surface sites occur that result in a
corrosion activity. The nonuniform nature of the metal-
electrolyte interface, whether passive or not, implies that
the corrosion process takes place preferentially at specific
sites that somehow differ energetically from other parts of
the metal surface. These specific sites may be permanently
localized or spatially fluctuate during the progress of
corrosion.

14
As a result of the above, the microbial corrosion
phenomenon is presumably initiated by the interaction of the
metal, the bacteria, and the electrolyte at the onset of the
process, followed by the formation of either differential
concentration cells or local electrochemical corrosion cells
within the biofilm, which further complicates the corrosion
mechanism.
Microorganisms selected for this study had to contain a
hydrogenase enzyme. Such microbes belong to the facultative
lithotrophs, sulfur oxidizing bacteria, phototrophics,
methanogens, and denitrifying bacteria. These bacteria are
all hydrogen oxidizing bacteria that use molecular hydrogen as
their electron donor. In anaerobic environments hydrogen is
generated from the processes of fermentation or metal
oxidation. Hydrogen oxidizing bacteria may grow
autotrophically on hydrogen carbon dioxide as an electron
acceptor.
Statement of the Problem
The cathodic depolarization theory of von Wolzegen Kuhr
and van der Vlugt (1934) has become the center of attention of
researchers in the field because it proposes a logical
separation of the components of corrosion: biological,
metallic, and fluid. As a result, much of the literature on

15
microbial corrosion has been influenced by this theory and has
referred to it, either to criticize or prove it.
In work performed by Daniels and coworkers (1987), it was
demonstrated that methanogenic bacteria used hydrogen produced
from iron oxidation of carbon dioxide to methane. The
mechanism of iron oxidation is cathodic depolarization, in
which electrons from iron, and hydrogen ions from water
produce molecular hydrogen that is then released and used by
methanogens.
Most important, according to the literature reviewed,
nobody has been able to determine how microorganisms induce or
influence the corrosion process. Inspired by the cathodic
depolarization theory and by Daniels and coworkers'
demonstration, this research indicated that it might be
feasible to measure and correlate total metal corrosion and
hydrogen uptake by bacteria.
The project was initiated while evaluating some new
bioreactor systems. In an attempt to understand and quantify
the biological component of corrosion, an experimental system
based on the batch bioreactor, which includes electrochemical
measurement devices to study the utilization of cathodic
hydrogen by bacteria and its relation to the corrosion at its
onset, was implemented. Further along, data acquisition
hardware and computer software were adapted to the system.
The hypothesis is as follows: The corrosion rate of a
metal, under anaerobic conditions, in the presence of

16
bacteria, is a function of its free corrosion potential and
the ability of microorganisms to utilize cathodic hydrogen.

CHAPTER 2
REVIEW OF LITERATURE
Historical Overview
Microorganisms can exist almost anywhere and so can
microbial corrosion. In process industries, it can occur
inside and outside of equipment. In soil and water, it can
happen in aerobic or anaerobic environments.
Microbial corrosion of steel by sulfate reducing bacteria
was probably the first area investigated in this field of
study and remains one of the most important. In an earlier
work (Sifontes and Block, 1991) , the authors covered
extensively a historical review on the subject. Here some of
the main historical aspects are presented here along with a
fresh look of microbial corrosion.
Microbial corrosion was first reported before the turn of
the century by Garret (1891). He ascribed the corrosion of
the lead sheathed cable to the action of bacterial metabolites
(ammonia, nitrates, and nitrites). In 1910, Gaines defined
the problem more clearly, providing evidence that iron and
sulfur bacteria were involved in the corrosion of the inside
and the outside of water pipes by making evident the presence
of abnormally large quantities of sulfur. He showed that
Gallionella. Sphaerotilus, and sulfate reducing bacteria were
17

18
responsible for the corrosion of ferrous alloys buried in
soil.
In 1924, Bengough and May demonstrated the effect of
ammonia produced by bacteria on the corrosion of copper
alloys. Later, von Wolzogen Kuhr and van der Vlugt (1934)
reported on the anaerobic corrosion of ferrous metals by
sulfate reducers. They proposed for the first time a
mechanism for microbial corrosion, which actually initiated
the systematic studies on microbial corrosion and
differentiated the various components of the microbial
corrosion process (metal, liquid, and microorganisms).
Details on the theory are presented later in this chapter.
Evidence for this bacterially influenced corrosion
continued to accumulate from around the world and was reviewed
by Starkey and Wright (1945). In 1953, Uhlig reported that
the primary role of slime-forming microorganisms was the
production of differential aeration and concentration cells
type corrosion. The first studies that demonstrated cathodic
depolarization with sulfate reducing bacteria was conducted by
Booth and Tiller (1960). They added a new dimension to the
problem, indicating that depolarization occurred with a
hydrogenase positive strain of Desulfovibrio bulqaris and did
not occur with a pure strain of hydrogenase negative
Desulfovibrio orientis. Furthermore, they demonstrated an
additional phenomenon that complicated matters: 1)
depolarization was observed when the culture was in active

19
growth, 2) the stimulation of corrosion was approximately
similar for both microbes, 3) the FeS film that formed on the
iron samples had an apparent inhibitory effect on corrosion
rates, and 4) the corrosion rates reported were much lower
than reported rates for similar alloys in natural anaerobic
environments in the presence of sulfate reducing bacteria.
Booth and Tiller (1962) and Booth, Robb, and Wakerly
(1967) demonstrated also that cathodic depolarization was
affected by FeS precipitate, presented experimental evidence
that the structure of the FeS film was instrumental in the
corrosion process, and indicated that once formed its
depolarizing activity continued even in the absence of
bacteria.
Since the energy crisis of the 1970s, several reviews on
the subject have been published. The results reported by Mara
and Williams (1972), King, Miller, and Smith (1973), Smith and
Miller (1975), and King, Dittmer, and Miller (1976) suggest
that precipitated FeS may initially form a protective film on
a ferrous metal surface in the presence of sulfate reducing
bacteria. As the microbial corrosion process continues, the
film thickens and changes stoichiometrically. As the ratio
Fe/S in the film changes from a sulfur deficient to a sulfur
rich structure, the film becomes less protective and
eventually spalls. Once spalled, the film does not reform and
vigorous anodic activity proceeds at the exposed metal
surfaces. According to Smith and Miller (1975), the FeS film,

20
regardless of structure, is cathodic to iron and the corrosion
process continues galvanically. Smith (1980) indicated that
the FeS film would not remain permanently cathodic in the
absence of bacteria. The role of the sulphate reducing
bacteria, he suggested, could be either to depolarize the FeS,
enabling it to remain cathodic, or to produce more FeS by
their metabolism.
Iverson (1981) discounted the FeS argument in his paper.
He indicated corrosion rates were above 210 mpy for mild steel
specimens exposed to filtered media from actively growing
culture of Desulfovibrio (API strain). He also suggested that
SRB produced a highly corrosive compound in addition to
hydrogen sulfide. The process appeared to depend on whether
FeS formed a protective film before the highly corrosive
product contacted the metal surface. Thus, it was apparent
that a number of factors from the metal and the solution were
involved in the process of microbial corrosion by sulphate
reducing bacteria.
During the 1980s, researchers found evidence of more
factors involved during anaerobic microbial corrosion.
Volatile metabolites such as phosphine have been reported to
be responsible for microbial corrosion of steels in
environments free of sulfate and sulfide (Iverson, 1985).
Since iron phosphide was detected among the corrosion products
found, it seemed that the amount of the chemical reaction was
the result of the competence between the sulfide that would

21
passivate the metal and the volatile phosphorous (phosphine)
that would replace the sulfide. There was no experimental
evidence of the chemical nature of the corrosion product,
excluding the direct contact between the bacteria and the
metal. Postgate (1979) rather concisely accounted for the
variety of factors involved in the microbial corrosion
process: nature of metal surface, dissolved ions and/or
organic matter, biofilm formation, FeS precipitate forms, and
other ions (sodium, chlorine) present.
On the other hand, the role of microorganisms in aerobic
corrosion was postulated by Olsen and Szaybalski (1949) to be
due in part to the formation of tubercles in conjunction with
microbial growth, which initiates oxygen concentration cells.
This mechanism, along with others, was proposed as the cause
of the worldwide problem of microbiologically associated
aluminum aircraft wing-tank corrosion that surfaced in the
late 1950s and early 1960s. Both commercial and military
aircraft were affected. Many microorganisms were reported to
be present in significant number in the fuel tank sludge
(Churchill, 1963). The same year, Leathen and Kinsel obtained
184 isolates of microorganisms from jet fuel-storage tanks at
nine Air Force bases. Results indicated the presence of
bacteria and fungi. The predominant bacteria were species of
Pseudomonas. and the most prevalent fungi were species
Hormodendrum. This case indicated the presence of a variety
of organisms including fungi, bacteria, and yeast at a site

22
of microbial corrosion. The medium was also complex due to
the presence of two liquids water and fuel phases.
Later work suggested Cladosporium resinae was the most
important fungus encountered in wing tanks and considered it
responsible for filter blockage and metal corrosion (Berner
and Ahearn, 1977). In 1976, Hill indicated that Cladosporium
resinae is only found in subsonic aircraft. In supersonic
aircraft, a higher temperature prevails and the predominant
flora found were Aspergillus fumigatus, gram negative
bacteria, yeast, and other fungi.
Recent evidence appears to indicate that organic acids,
produced by fungi, were primarily involved in this corrosion
(Miller, 1981).
Other problems due to the activities of SRB have arisen
in offshore oil operations (Hamilton and Sanders, 1986). They
were identified in the legs and storage cells of offshore
structures and include the production of hydrogen sulfide,
which is a serious personnel hazard, and the production of
bacterial metabolites that give rise to accelerated concrete
deterioration (Wilkinson, 1983). Furthermore, internal and
external microbial corrosion of long large-capacity subsea
pipelines, which transport oil and gas from offshore
production fields to shore, are of a major concern due to the
high cost associated with their failures and the harm to their
surrounding environment (King et al., 1986).

23
A considerable amount of research on microbial corrosion
in relation to sulphate reducing bacteria has been done in the
UK, especially in the 1960s. Most of this research was
carried out at the National Chemical Laboratory at Teddington
(presently the National Physical Laboratory) by a group of
well-known researchers in the areas of microbiology and
corrosion science. Despite the disbandment of the group in
1968 as a result of a decision of the British Government that
microbiology should not be conducted at that institution, the
researchers have continued to dominate the literature on
microbial corrosion (Tiller, 1985). Among some of the
original members of the group are G.H. Booth, D.S. Wakerly,
A.K. Tiller, J.R. Postgate, R. A. Kim, W.A. Hamilton, J.A.
Hardy, E.C. Hill, and B.N. Herbert.
In the USA, interest in the field has increased in the
past 15 years, but the scope has been limited. The most
noteworthy efforts began 12 years ago when a microbial
corrosion program was initiated by the Materials Technology
Group and a separate symposium and technical committee on
microbial corrosion was created by the National Association
of Corrosion Engineers (NACE) for their annual meetings.
Among some of the principal investigators in the USA are D.C.
White, R. Mitchell, G. Geesey, W. Characklis, W. Lee, N.
Dowling, R. Tatnall, G. Licina, B. Little, and J. LeGall.
During the last 10 years interest in the area has spread
in other countries.

24
Other Aspects of Microbial Corrosion
Despite advancement in the area of microbial corrosion,
some researchers have detected some common mistakes in the
practice of this type of investigation. Bryant and Laishley
(1989, 1993) indicated that the result of uncertain
conclusions and overestimates of corrosion due to influence of
microorganisms is the failure to account for effects of media
constituents such as phosphate. They found that phosphate was
one factor among many that together contribute to the overall
corrosion rate and that corrosion studies should include
controls that account for the constituents of the growth
media. There was little or no information on the levels of
phosphates on corroding metals. However, sea water contains
normally a concentration of 3 /¿M of phosphate, and dead cells
may release some phosphate that may raise the concentration in
spacial locations such as crevices and pitts and influence
corrosion. Booth and Tiller (1968) did not recommend the use
of batch cultures of sulfate reducing bacteria directly to
study their effect on the cathodic reaction. They suggested
that batch bioreactors were undesirable because the
composition of the media would be changing continuously due to
the release of metabolites including sulfide, which was known
to have a cathodic effect on the corrosion of steel and in the
presence of dissolved iron results in the precipitation of
FeS.

25
Hardy (1983) indicated that many attempts of researchers
to demonstrate the utilization of cathodic hydrogen by
sulphate reducing bacteria and its stimulatory effect on the
cathodic reaction have failed because the evidence was based
exclusively on measured electrochemical effects. The observed
depolarization might have been due to the effect of sulfides
present and could not be attributed exclusively to the effect
of hydrogenase. He also indicated that there was some
evidence that the periplasmic hydrogenase enzyme that appears
to be the hydrogen uptake enzyme constituted a greater
proportion of the total hydrogenase present in hydrogen-grown
cells than of the hydrogenase in lactate-grown cells. This
feature, as explained earlier, is a characteristic of bacteria
adaptation that is seldom considered during microbial
corrosion research. Researchers most of the time use bacteria
that have been cultured in standard media rather than wild
type cultures. Wild type species of Desulfovibrio have been
shown to lose their adhesion properties when they are
transferred from the wild to laboratory and grow in standard
medium.
Rajagopal and LeGall (1989) suggested that the ability of
some sulphate reducing bacteria to reduce nitrate or nitrite
with hydrogen as sole energy source provides a better
experimental system to study the cathodic depolarization
phenomenon, since problems with measurements of dissolved iron

26
and growth in the presence of precipitated sulfides resulting
from the customary sulphate reduction can be avoided.
The presence of organic electron donors has been
considered during cathodic depolarization of steel. Widdel
(1988) indicated that most Desulfovibrio spp are able to grow
on acetate and carbon dioxide as carbon sources and hydrogen
as sole energy source. Cord-Ruwisch and Widdel (1986) found
that the availability of organic electron donors appears to be
an important factor that influences the removal of cathodic
hydrogen. If a favorable organic energy source was present,
sulphide was produced, indicating that iron alone did not
allow sulphate reduction. They suggested either that cathodic
hydrogen was preferentially oxidized with the organic
substrate or that sulphide from sulphate reduction with the
lactate reacts with the remaining ferrous ions or ferrous
hydroxide from the corrosion process to produce more FeS.
In addition to the above observations, some other
important scientific areas related to the microbial corrosion
phenomenon need to be addressed in order to envision the
complexity of the problem. Those areas include bacterial
adhesion, surface thermodynamics, interfacial chemistry,
hydrogen embrittlement, interspecies hydrogen, bioenergetics,
microbial ecology, and applied fundamentals of metallurgy,
electrochemistry and chemistry. Information on those subjects
is referred to in the cited literature. Specific topics
include the prevailing mass transfer conditions and the

27
surface properties of the phases involved, which include
molecular fluctuations at the electrical double layer (Liu,
1983), the adhering bacteria, the substrate, and the suspended
media (Absolom et al., 1983; David and Misra, 1985), chemical
interaction at metal surfaces and at the liquid interface
(Sato, 1987), and metallurgical aspects such as
microstructure, grain boundaries, inclusions, surface
contamination, and sources of corrosion currents (Godard,
1980; Mears and Brown, 1941).
Microorganisms Involved in Microbial Corrosion
The microorganisms that have been associated with
microbial corrosion include many genera and species.
Microorganisms are anatomically simple yet biochemically
complex. They may be divided into three groups: (1) algae and
fungi, (2) protozoa, and (3) monera (e.g., bacteria). Many of
these organisms have been firmly established in laboratories
and field sites as having roles in the corrosion process,
whereas others have merely been isolated from suspected
corrosion sites. Microorganisms are sustained by chemical
reactions by ingesting reactants and eliminating waste
products. Thus these processes can influence corrosion
generaly by 1) directly influencing anodic and cathodic
reactions, 2) influencing protective surface films,

28
3) producing deposits, 4) producing corrosive metabolites, and
5) feeding on corrosion inhibitors.
Several characteristics of microorganisms enhance their
involvement in corrosion. They are generally very small,
starting from less than 0.2 /un, which allows them to penetrate
crevices very easily, to several hundred /un in length. Some
of them are motile, which aids in their migration to more
favorable environmental conditions. Microorganisms are able
to establish in sites that encourage their growth. For
instance, microbes establish and colonize surfaces in cooling
water systems where food sources concentrate at metal surfaces
because of their hydrophobic properties. They can withstand
a wide range of environmental conditions: pH values from 0 to
11 (Brock and Madigan, 1991), temperatures from -30 to 110°C,
and oxygen concentrations from 0 to 100% (Morgan and Dow,
1986).
Microorganisms can adhere to a surface and form colonies
of different species. These consortia, once formed, can
sustain survival under adverse conditions. They can reproduce
themselves to a great number in a short time. This fact
allows them to bloom and take over an environment quickly.
They are easily dispersed in air, water, animals, etc., and
adapt to other environments in which it may be easier for them
to grow. Many can adapt easily to a wide variety of
substrates, such as the Pseudomonas. some species of which
can use well over 100 different kinds of food as sole carbon

29
and energy sources. Many can produce extracellular
polysaccharides or slime layers where a consortia of bacteria
can develop and consequently influence corrosion (White et
al., 1986). These layers attract food and other
microorganisms and cause several other well-known problems in
the process industry, such as poor heat transfer.
Some microbes produce spores that resist the most severe
environmental conditions and are capable of surviving for long
periods of time by remaining dormant. They can quickly
colonize surfaces when the environment changes to their
liking. Some resist antimicrobial agents by virtue of their
ability to degrade them or by resisting permeability through
the cell wall or by the extracellular polysaccharide
protection. Such resistance may be acquired by mutation or
acquisition of a plasmid. Many species produce a wide variety
of organic acids that may promote corrosion of many alloys
even at low concentrations (e.g., Clostridium acetobutvlicum
produces acetic acid).
Some species produce mineral acids that are extremely
corrosive. Thiobacillus thiooxidans produces sulfuric acid,
which is of economic importance in biohydrometallurgy because
it makes possible the leaching of metal sulfide ores. In the
case of pyrite, it oxidizes both the sulfur and ferrous
moiety. Boes and Kuenen (1983) have recently reviewed the
sulfur oxidizing bacteria and their relationship to corrosion
and leaching. Several bacteria metabolize nitrate, sometimes

30
used as a corrosion inhibitor (e.g., Pseudomonas spp reduce
nitrate and nitrite to nitrogen gas). Other organisms convert
nitrate to nitrite or ammonia to nitrite (e.g., Nitrosomonas)
and others turn nitrite into nitrate (e.g., Nitrobacter).
Many organisms form ammonia from the metabolism of amino
acids. This forms ammonia ions in basic solution, which may
be corrosive to copper alloys.
Microorganisms such as sulfate reducing bacteria produce
enzymes, which may be excreted outside the cell and which can
act on substances outside the cells. For instance,
hydrogenase has been reported as being responsible for
depolarizing cathodic sites during the microbial corrosion of
iron and steel. Many organisms can produce carbon dioxide and
hydrogen as a result of their fermentative metabolism. Carbon
dioxide in acidic solutions becomes carbonic acid, which is
highly corrosive, and hydrogen can polarize metal surfaces of
stainless steel and may cause hydrogen embrittlement.
Many genera of bacteria that normally use organic
compounds as carbon and energy sources can use hydrogen gas as
their energy source and carbon dioxide as their carbon source
and live chemoautotrophically. This can cause depolarization
of cathodic sites on steel and promote corrosion (e.g.,
Methanooens).
Some bacteria can oxidize or reduce metals or metallic
ions directly. For example, the iron-oxidizing bacteria
(Gallionella. Sphaerotilus) oxidize ferrous ion to ferric ion.

31
The ferric compounds precipitate in a sheath around the cells
and form tubercles in pipes and cause plugging. Thus,
concentration of cells is easily formed under those deposits.
On the other hand, ferric ion can be reduced to ferrous ion by
Pseudomonas spp from oil wells and marine sediments. It has
been suggested that the ferric film, which normally stabilizes
the surface of mild steel from corrosion, are destroyed
leaving the surface susceptible to corrosion attack. Other
bacteria can oxidize or reduce metals such as manganese.
Microorganisms can form synergistic communities (e.g.,
algae and bacteria). These consortia can accomplish things
that individually would be difficult if not impossible, such
as the case of fungi and Desulfovibrio spp.. The fungi break
down wood to organic acids and consume oxygen, thus providing
the food and anaerobic conditions for Desulfovibrio spp. .
Communities providing protection for individuals can also
change structure, dominant species, etc. by genetic mutation,
and can adapt to environmental changes, even to deliberate
chemical changes intended to kill them. More details on this
subject may be found in Sifontes and Block (1991).
Mechanisms of Microbial Corrosion
For proper selection of methods to prevent or control
microbial corrosion, it is necessary to know the mechanisms by
which microbial activity affect the deterioration of metals.

32
The mechanisms of microbial corrosion in some cases are well
defined, but where environments encourage the activity of
bacteria, the corrosion processes are more complex and still
not fully understood. The mechanisms of microbial corrosion
can be subdivided in direct and indirect mechanisms. If a
microbe interlinks an electrode process with its own
metabolism or presence, it is a direct effect (i.e.,
differential aeration cell produced by sessile bacteria),
otherwise it is an indirect effect (i.e., corrosive
metabolites produced by planktonic bacteria).
Even though the mechanisms of microbial corrosion are not
well understood, microbes that cause or influence corrosion
have been classified previously (Kobrim, 1976) into the
following groups:
1) Acid production. Some microbes can oxidize sulfur
compounds to sulfuric acid. Very low pH has been reported in
places where sulfur oxidizing bacteria are active. Many
species produce a wide variety of organic acids (e.g. acetic,
butyric, succinic, and formic) which may promote corrosion of
many metals and their alloys.
2) Protective coating destruction. Protective coatings
ranging from polymeric materials to passive films can be
broken by the activity of microorganisms and corrosion of bare
metal starts rapidly.
3) Production of corrosion cells. Differential aeration and
ion concentration cells are notable examples such as the case

33
of a metallic surface that is accessible to oxygen. If a
deposit such as a biofilm or a corrosion product covers it,
the surface under the deposit is shielded from oxygen and the
surface outside of the deposit is not. This results in a
corrosion cell.
4) Sulfur reduction and oxidation. Sulfate reducing bacteria
are the most publicized class of corrosive microbes. They
reduce sulfates to sulfides and can depolarize cathodic sites
by consuming hydrogen. Most sulfur oxidizing bacteria known
fall in the category of acid producers.
5) Concentration of anions and/or cations. Iron and manganese
bacteria are examples of this category. They generally form
thick, bulky deposits which create concentration cells or
harbor other corrosive microbes. This group is also known as
metal ion oxidizers or reducers.
6) Hydrocarbon utilization. Certain microorganisms have been
observed that destroy organic coatings or linings in the
presence of hydrocarbon fuels. Some others destroy metals
such as aluminum and feed on hydrocarbon fuels.
7) Slime formation. Certain algae, yeast, bacteria, and fungi
may form deposits which foul heat transfer equipment and
produce concentration cells on metal surfaces.
The above mechanisms can be grouped in three general
modes of microbial attack based on their metabolism: corroded
material serves as substrate for microbial growth, microbes

34
colonize material surface but feed on something else, and
microbes produce metabolites that corrode material.
Theory of Cathodic Depolarization
The theory of cathodic depolarization was postulated in
1934 by von Walzogen Kuhr and van der Vlught. The idea of the
authors came from their interpretation from the
electrochemical point of view of the anaerobic corrosion of a
cast iron pipe in wet soils near Amsterdam. Since the pipe
was under clay at a near neutral pH, the severly corroded pipe
did not allow them to explain the phenomena using the
reduction of oxygen as the cathodic reaction. The alternative
cathodic reaction was the reduction of hydrogen that was
feasible under those conditions.
The theory proposes that when iron is immersed in water,
a natural equilibrium is set up between the ferrous cations
released at the anode, and the metal surface negatively
charged at the cathode by the remaining electrons. The
dissolving process continues only if the electrons are
removed. Under aerobic conditions oxygen serves as an
electron acceptor resulting in rust formation. Under
anaerobic conditions free protons from the dissociation of
water are reduced on the cathodic metallic surface by the
remaining electrons, to form a protective hydrogen polarized
envelope that protects the iron metal from further

35
dissociation. A dynamic equilibrium is established which
keeps the iron polarized. The theory suggests that the
principle mechanism of anaerobic corrosion is cathodic
depolarization of the iron surface by hydrogen oxidizing
microorganisms such as sulfate reducing bacteria and
methanogen. These organisms disturb the equilibrium by
oxidation of the cathodically formed hydrogen with sulphate
and carbon dioxide as electron acceptors respectively, via the
hydrogenase enzyme. In an attempt to re-establish the
anodic/cathodic equilibrium more iron is oxidized, the end
result of which is pitting formation.
Table 2-1. Chemistry of the cathodic depolarization theory.
component reaction
1)Metal dissolution
4Fe
—> 4Fe+2
+ 8e
2)Hydrogen reduction
8H+
+ 8e —>
8H —> 4H2
3)Water dissociation
8H20
—> 8H+ +
8 OH'
4)Microbial activity
so*'2
+ 8H —>
S'2 + 4H20
5)Corrosion product
Fe+2
+ s'2 —>
FeS
6)Corrosion product
3Fe+2
+ 60H -
-> 3Fe(OH)2
7)Total rxn 4Fe + SO* 2 +
4H20 —
> 3Fe(OH)2 +
FeS + 2(OH)
The importance of this theory lies in the fact that it
separates for the first time the three components of the

36
microbial corrosion system which include the microorganisms,
the metal, and the suspended medium.
A large volume of literature up to the last decade has
been influenced by the theory of cathodic depolarization and
has referred to it, either to prove or to disprove it.
However, the use of electrochemical techniques allowed Horvath
and Solti (1959) to discover an anodic effect in addition to
the cathodic effect of the theory. They studied this effect
as a function of pH, environmental regulatory conditions and
the concentration of FeS present. According to those results,
SRB had an indirect role, which would be the stabilization of
the sulfide compounds over the metal surface by modification
of the redox potential.
One of the most conclusive findings was reported by
Costello (1974) who indicated a cathodic effect due to the
hydrogen sulfide produced by the sulfate reducing bacteria,
suggesting that hydrogen utilization by the sulfate reducing
bacteria became secondary and so the participation of those
bacteria in the corrosion process. Furthermore, It has been
suggested as an amendment to the corrosion theory that solid
ferrous sulfide, in contact with iron, acts as a cathode
(Booth et al., 1968). Other authors proposed that reduced
phosphorous compounds, too, are involved in the anaerobic
corrosion process (Iverson and Olson, 1984) . Recently it was
reported that phosphate and hydrogenase can affect the
corrosion of mild steel. Phosphate reacts on mild steel with

37
concomitant production of hydrogen gas and the formation of an
iron/phosphate complex (vivianite) Bryant and Laishley, 1990;
Weimer, et al., 1988). The enzyme hydrogenase was reported to
accelerate cathodic depolarization by oxidizing the hydrogen
produced (Bryant and Laishley, 1993). Although many studies
have clearly demonstrated the involvement of sulphate reducing
bacteria and methanogens in corrosion, only a few have shown
the influence of those microorganisms using the electrons from
the metal as energy source.
On the other hand, some authors have provided evidence in
support of the cathodic depolarization theory (Cord-Ruwisch
and Widdel, 1986; Hardy, 1983; Tiller and Booth, 1962; Booth
and Tiller, 1960, 1962). Few, however have actually
demonstrated that this phenomenon is coupled to microbial
growth (Belay and Daniels, 1990; Rajagopal and LeGall, 1989;
Daniels et al., 1987; Pankhania et al., 1986; and Tomei and
Mitchell, 1986) . In conclusion, research indicates that the
described theoretical mechanism is not entirely correct.
Previous Studies in Microbial Corrosion
Despite the large amount of literature in microbial
corrosion, it is still not an easy subject to understand,
because of the multiplicity of factors at play. Some authors
have indicated that microbial corrosion is a newly discovered
problem or an emerging science, possibly still in its infancy

38
(Tatnall, 1988). Others have confirmed by surveys that the
continued incidence of microbial corrosion could be due to a
general lack of awareness of the problem (Wakerly, 1979). The
fact is that its scientific study began more than 80 years ago
(Tiller, 1982) and after the work of von Wolzogen Ruhr and van
der Vlugt in 1934, who established the classical mechanism of
anaerobic corrosion, the subject of microbial corrosion in
general and of anaerobic corrosion in particular has become
recognized of prime importance (Bessems, 1983; Hamilton,
1985) .
Most of the literature on microbial corrosion, before
1960, was concerned basically with observations of the effect
of bacteria on the environment. During the 1960s, there was
considerable activity on the field and most of the research
was focused on gaining understanding at the different
mechanisms. Some notable papers of this period belong to
Postgate (1960) , Horvath (1960), Sorokin (1966) , Iverson
(1966), Booth, Elford and Wakerly (1968), Booth and Tiller
(1968), and Costello (1969).
During the 1970s the interest in the subject broadened
the scope of research involving scientists from other fields
including plant engineers, corrosion scientists,
microbiologists, and biochemists (Sequeira and Carrasquinho,
1988). This has brought improved understanding of the
ecology, nutrition and physiological requirements of the
microbes involved in microbial corrosion, which also improved

39
the biochemical techniques used in both the field and the
laboratory. In addition, the publication of case histories of
a diverse range of failed industrial equipment has enhance
understanding of the problem. This period is well described
in the articles by Iverson (1972), King and Miller (1973),
Mara and Williams (1972), Miller and King (1975), Kobrin
(1976), Widdel and Pfennig (1977), Jorgensen (1977, 1978,
1980), Wakerly (1979), and Postgate (1979).
It was only within the last decade that microbial
corrosion was recognized as a serious problem in the chemical
industry by the leading corrosion society in the world (NACE)
and substantial advancement has been achieved specially in the
areas of microbiology and corrosion. In microbiology, among
other findings, the biochemistry of dissimilatory sulfate
reduction in Desulfovibrio has revealed enzymes and electron
carriers of special character and structure whose function and
distribution within the cell are just beginning to be
revealed, indicating new discoveries in the peculiar energy
generation systems in these bacteria.
In corrosion, sophisticated methods to study it, have
evolved; such as new methods in electrochemistry,
metallography, macroanalysis, and microanalysis. A
significant part of the research in that period is described
in the articles by Hamilton (1985), Iverson (1987), White et.
al. (1986), Tatnal (1981), Odom and Peck (1984), Cord-Ruwisch
and Widdel (1986), Tiller (1982), Miller (1981), Hardy (1981,

40
1983) , Rajagopal and LeGall (1989) , Bryant and Laishley
(1989), Daniels et al. (1987), and Widdel and Pfennig (1981).
Today, microbial corrosion is well recognized as a
serious problem in most industries, particularly in oil and
gas, the power generating and the process industries. In the
chemical industry alone, multimillion dollar failures due to
microbial corrosion have been reported involving cooling water
systems (Felzin et al., 1988).
While the above studies have yielded a wealth of
information, the literature reveals that the corrosion
community still knows very little about how microorganisms
influence or induce corrosion, and what the role of
microorganisms is in the corrosion process. In general, most
of the research deals with observations related to the basic
problem, case histories, biological research, and reviews of
literature. It is important to notice that this was not
usually the fault of the investigator; in the majority of
instances the specialist lacked interdisciplinary help
required to tackle such complex studies.
Microbial corrosion is by definition an interdisciplinary
field that requires among others, the understanding of
microbiology, corrosion science, metallurgy, electrochemistry,
transport phenomena, and surface chemistry. Several factors
have cause a further hindrance in the development of this
microbial corrosion. Among some of the factors are: the lack
of awareness; the difficulty of growing, isolating, and

41
identifying anaerobic microorganisms; the adaptation of
techniques from clinical microbiology to handle the microbes
involved; the fact that to the average researcher microbial
corrosion spans the boundary of traditional specialties; and
the idea that many microbiologists have of microbial corrosion
as a rather specialized even esoteric field, and the lack of
acceptance of the role of bacteria in corrosion by corrosion
engineers.
Actually, many theories on how microorganisms influence
or induce corrosion have been proposed. However, none of them
are fully proven but rather equal number of existing papers on
the subject claim to support or refute these theories.
Consequently, it seems fair to say that there is little
uniform agreement among those working on this field about what
is really going on (Tatnall, 1988).
Customary practices, used during the investigation of
microbial corrosion, include separated studies of either the
microorganisms, the metal or the fluid but ignore their
interaction. In addition, most laboratory microbial corrosion
experiments use flowthrough type bioreactors which are run for
weeks or even months. The most common corrosion measurement
methods used are adopted from classical corrosion science such
as Tafel plots and weight loss techniques. In many cases,
these practices have failed to give reproducible results
because of the length of the experiments, nature of the metal
surface, and fluid chemistry in addition to microbial

42
ecological considerations of such a complex systems. The
application of external potentials to the system, a common
practice in microbial corrosion experiments, may produce
significant thermodynamic changes known to affect the
stability of the system, and constitute a source of
uncontrolled variables.
The practical separation of the three components of the
microbial corrosion systems as suggested by the cathodic
depolarization theory, and the work performed by Daniels et.
al. (1987), set the stage for studying the microbial
utilization of cathodic hydrogen during corrosion. If hydrogen
oxidizing bacteria can take up the hydrogen produced
cathodically from steel in well defined experimental systems
then there exists the possibility to further explore in this
field and increase our understanding of these microbes and
their mechanisms of attack on metals.
Microbial Corrosion Control
Prior to 1970 the control of microbial corrosion focused
on the use of cathodic protection and antimicrobial agents.
This practice was complemented by improvements in surface
coatings, in particular antifouling systems such as tapes and
wrappings. Because of the limited ability of those methods to
control bacterial activity and the inefficacy of the
methodology to asses antimicrobial agents; the main driving

43
force after 1975 has been the consolidation of several
methodologies and assessment procedures to improve the
standard practice (Tiller, 1985).
In 1964, Saleh and co-workers evaluated nearly 200
antimicrobial agents and concluded that laboratory evaluations
of those chemicals can only be considered as an introductory
sign and should be supplemented with trials on the field. In
1983 Bessems recommended the importance of assessment
procedures and Gaylarde and Johnston emphasized the
deficiencies in current methodology and the need for
improvement. During last decade a joint venture of the
Institute of Corrosion Science and Technology and the National
Association of Corrosion Engineers has reviewed and updated
the current recommended practice for monitoring bacterial
growth. Topics such as killing time, the importance between
planktonic and sessile bacterial consortia and their
attachment properties are now important issues in the control
of microbial corrosion (Tiller, 1985).
Due to the complexity of the environmental factors that
results in microbial corrosion, the success of a control
program depends more in our outlook on the available
information about the problem, and becomes a challenge to
identify the most practical and economic solution.
In general, the control of microbial corrosion requires
a sound strategy. This involves a diagnosis of the microbial
corrosion problem that begin with determining the cause and

44
the mechanisms associated with the corrosion problem. A sound
diagnosis include: comprehensive current system diagrams,
materials of construction, fabrication methods, operating
history, chemical analysis, site specific environmental
conditions, biological history, and historical and current
chemical treatment. Unfortunately, if this is not followed,
adverse consequences may result. For example, a strong
oxidant may have a good killing power but may produce severe
local corrosion if applied to a particular system.
Fellers (1989) suggested that the best strategy is the
product of a multi-disciplinary team which looks at all
aspects of the problem, examines root cases, and objectively
evaluates alternatives. He also indicated in a later paper
that if the main strategy is to maintain the system clean of
microbial growth, instead of looking for independent solutions
to several problems, several issues may be avoided including
microbial corrosion (Fellers, 1990).
The essential strategies to control microbial corrosion
include: detection, prevention, and mitigation. Detection
techniques are basic to prevention and mitigation. They
include electrochemical methods, microbiological methods,
physical and metallurgical methods, in system monitoring
techniques, and other laboratory techniques. Details on these
techniques are found in earlier work of the author (Sifontes
and Block, 1991). Additional detection techniques include
visual inspections to identify suspected indications of

45
microbial attack and side stream monitors for determination of
corrosion and fouling rates.
Prevention and mitigation techniques hold the highest
payback in plant protection and preservation. There is no
universal approach to the prevention and mitigation of
microbial corrosion since it is almost impossible to use a
single preventive method. The prevention and mitigation
techniques offered by the current technology include planning
considerations such as material selection, design, and non-
metallic materials; physical-chemical control such as
corrosion inhibitors, selection and control of the
environment, protective coatings, electrochemical protection,
maintenance cleaning, and chemical control; and biological
control such as antimicrobial agents, and environmental system
control. Details on above prevention techniques are found in
earlier work of the author (Sifontes and Block, 1991). Other
prevention and mitigation techniques include general corrosion
control such as prevention of scales which enhances
biofouling, suspended solids dispersion such as the use of
penetrants of other surface active agents which re-disperse
fouling materials (Fellers, 1990).
Finally, an effective control of microbial corrosion
involves a combination of selected techniques to solve an
specific problem. Numerous history cases that include the
results of the applied techniques are available throughout the
literature cited. A reported case history of successful

46
corrosion control in a cooling water system used a combination
of biodispersant/biocide. It employed an organic corrosion
inhibitor, a polyacrylate/phosphonate dispersant, and a
combination of two microbiocides used simultaneously
(Honneysett et al., 1985).
the use of surfactants (penetrants /biodispersants) has
improved the effectiveness of microbial corrosion control.
Chemically, these substances are composed of nontoxic organic
compounds with penetrating and dispersing properties. The
biodispersants allow the sessile colonies to be penetrated by
the antimicrobial agents, thus used at lower dosages with
improved effectiveness. They inhibit the biomass produced
from becoming so massive that antimicrobial agents can not
penetrate the consortia of microbes. Some penetrants are
hydrophobic to the extent that a film forms on the metal
surfaces allowing less deposition of sessile colonies
associate or not with microbial corrosion.
Other references that contain a variety of history cases
include: Microbial Corrosion proceedings by the Metals
Society of London, 1983; International Conference on
Biologically Induced Corrosion proceedings by the NACE, 1985;
Microbial Corrosion 1 proceedings by the First European
Federation of Corrosion workshop on Microbial Corrosion, 1988;
and Microbially Influenced Corrosion and Biodeterioration
proceedings by the International Congress on Microbially
Influenced Corrosion, 1990.

47
Research Approach
The following tasks were considered in order to comply
with the objective of this research. The first task included
the evaluation of some new experimental flowthrough and batch
bioreactors to reproduce and study the anaerobic microbial
corrosion phenomena. The initial task consisted of runs of
bioreactors with the object of reproducing microbial corrosion
in the laboratory. The second task involved the use of the
batch bioreactor to study the onset of microbial corrosion,
using conditions that resemble the inside of gas transmission
pipelines. The operation of the batch bioreactor was examined
concerning practical functioning for head space composition,
sterilization method, reducing agent use, metal coupon
preparation, and bacteria handling. Different metals and
their alloys were tested for electroactivity and their ability
to produce hydrogen; and different hydrogen oxidizing bacteria
were also tested to determine their ability to uptake
hydrogen. A third task involved the development of an
experimental system that included the redesign of the batch
bioreactor, development of a working electrode preparation of
bacterial suspensions, preparation of mineral solution
electrolyte, and the implementation of a data acquisition
system to accommodate electrochemical measurements, in an
attempt to quantify the biological component of corrosion.
The redesign of the batch bioreactor consisted of improvements

48
to the experimental system that resulted in the development of
the single, dual, and triad flask electrochemical cells. A
forth task involved the general setting of parameters to
assure simpler and reliable results such as the selection and
setting of fixed conditions and analytical procedures.

CHAPTER 3
MATERIALS AND METHODS
Introduction
The overall objective of this research was to study
anaerobic microbial corrosion at its on-set and to quantify
cathodic hydrogen utilization by bacteria. For this purpose,
a total of seven experimental set-ups were utilized. These
include: the 1) flowthrough bioreactor, 2) batch bioreactor,
3) single flask electrochemical cell or single cell, 4) dual
flask electrochemical cell or dual cell, 5) triad flask
electrochemical cell or triad cell, 6) artificial hydrogen
uptake triad cell, and 7) galvanic couple triad cell.
Initially, the flowthrough bioreactor was used to
replicate microbial corrosion and to get familiar with the
laboratory techniques and analyses required for the
investigation. This traditional bioreactor design used a
known bacteria triculture commonly found at microbial
corrosion sites and a glucose lactate yeast extract (GLYE)
medium that allowed the three bacteria to grow rapidly. Runs
of this bioreactor were performed using carbon steel coupons
in the presence of the bacteria triculture in GLYE medium.
Then, since the corrosion reaction and the biofilm formation
developed relatively fast in the flowthrough bioreactor, a
49

50
simpler batch bioreactor to study the on-set of the reaction
was used. This new bioreactor offered the best alternative to
study the initiation of the microbial corrosion process and
was able to reproduce microbial corrosion of carbon steel
within 24 hours in GLYE and in mineral solution media. The
batch bioreactor was redesigned and instrumented to include
electrochemical measurements and became the single flask
electrochemical cell. The single cell was used to measure
free corrosion potential in the presence and absence of
bacteria. It was improved later to the dual cell and the
triad cell. The dual cell allowed bacteria to avoid direct
contact with toxic metals and the triad cell provided for
measurement of a differential corrosion current. The last two
experimental set-ups were identical to the triad cell, except
they were used to study the effect of an artificial hydrogen
uptake and to determine the reliability of the electrochemical
measuring system respectively.
All electrochemical set-ups used the mineral electrolyte
solution developed for the purpose of studying microbial
corrosion at its on-set and hinder the formation of a biofilm
Flowthrough Bioreactor
Description
The 3-L flowthrough bioreactor system, used for the
preliminary work of this research was designed in house and

51
built out of 5 cm PVC pipe components. It consisted of two
sections for the study of sessile and planktonic bacteria
activity and their effect on metal deterioration. A basic
diagram showing its components is illustrated in Figure 3-1.
FEED
INOCULUM PORT
FLOW
CONTROL
VALVE
\
Í
NAD(P)H PROBE PORT
r \«
SESILE ACTIVITY
TESTING
\ %y'\\
METAL SPECIMEN
PORT
W
\K\
\
PLANKTONIC
ACTIVITY
TESTING
v
x \ r
CLEAR PLASTIC
FOR LEVEL
CONTROL
ft
Figure 3-1. Flowthrough bioreactor general description of
components

52
The first section, for sessile activity testing,
consisted of eight side screw cap ports that fit a
fluorescence probe, developed by Biochem Technology, Inc.,
King of Prussia, Pennsylvania, and four top screw caps, to
which metal coupons and reference electrodes are attached.
The second section, for planktonic activity testing, contained
two side screw caps ports for attachment of the fluorescence
probe, and a clear section at the end of the reactor for the
purpose of controlling the bioreactor head space volume.
The bioreactor was fed from a 4-L stoppered flask through
a tubing, Tygon R-3603, and an effluent flow of 125 mL/hr was
controlled using a hose clamp on a 30 cm tubing, Tygon R-3603,
at its outlet.
A known bacteria triculture composed of Entrobacter
aergenes, Desulfovibrio desulfuricans. and Clostridium
acetobutvlicum were used as the inoculum. Bacteria were
obtained from the American Type Culture Collection (ATCC).
Steel coupons, 5 mm x 20 mm x 10 mm, polished to 24 0
grid, and tied plastic strips to a 1/4 " teflon rods were
attached to the upper screw caps of section one. Figures 3-lb
and 3-lc illustrate the assembly combinations of metal
coupon/fluorescence probe and a metal coupon/fluorescence
probe/reference electrode, respectively. Figure 3-Id shows an
actual set-up of the flowthrough bioreactor.

53
METAL SPECIMEN
Figure 3-2. Flowthrough bioreactor metal coupon and
fluorescence probe setup.
REFERENCE
ELECTRODE
Figure 3-3. Flowthrough bioreactor metal coupon, fluorescence
probe and reference electrode setup.

54
Figure 3-4. Flowthrough bioreactor components.
Operation
The flowthrough bioreactor system was operated at a 1-day
retention time (125 mL/hr). Medium was fed to the bioreactor
by gravity from the 4-L reservoir under a nitrogen atmosphere
to assure anoxic conditions. The feed, a glucose-lactate
yeast-extract (GLYE) media, was prepared fresh daily,
sterilized and neutralized prior to use.
Effluent samples for pH and volatile fatty acids were
taken directly from the effluent. Metal coupons samples for
scanning electron microscopy analysis were sampled under
aseptic conditions while sparging with N2 gas and placed

55
immediately into 10 mL vials containing a 0.5% glutaraldehyde,
2% formaldehyde, cacodylate buffer, pH 7.2. Metal samples
were further processed, according to protocol described in
Table 3-1, for scanning electron microscopy examinations.
Table 3-1. Protocol for metallic surface fixation for
scanning electron microscopy examinations.
1) Place metal coupon in a 0.5% glutaraldehyde, 2.0%
formaldehyde, cacodylate buffer, pH 7.2 solution for 5 min at
room temperature, then on ice for 10 min.
2) Wash coupon in ice-cold buffer two times for 5 min each.
3) Wash coupon in ice-cold ethanol solutions for 5 min each,
in the following ethanol concentrations: 25%, 50%, 75%, and
95%.
4) Wash coupon at room temperature in 100% ethanol.
5) Wash coupon in acetone for 15 min at room temperature.
6) Wash coupon in fresh acetone at room temperature for 30
min.
7) Treat coupon with solutions of epon-araldite 30%, 70%, and
100% for 1 hr each at room temperature, except the 100% which
is done at 60°C in oven.
Bacteria inoculations were done as follows for initial
run. Ten mL of E. aeroaenes were inoculated first in order to
reduce the media. After 24 hours, 15 mL each of D.
desulfuricans and C. acetobutvlicum were inoculated. For

56
other runs, bacteria were inoculated within six hours in
amounts 10 times larger than the volumes used in the initial
run. All runs were temperature controlled at 35°C inside a
walk in incubator.
GLYE media was prepared using ingredients outlined in
Table 3-2, Table 3-3, and Table 3-4. Media was prepared in 4-
L feed reservoir flasks and autoclaved at 15 psig for 15 min
(Balch et al., 1979).
Table 3-2. Glucose Lactate Yeast Extract Medium Composition.
Yeast Extract 0.5 g
Glucose 0.5 g
Sodium Lactate 0.5 g
Mineral Solution 1 (table 3) 25.0 mL
Mineral Solution 2 (table 3) 25.0 mL
Trace Mineral Solution 3 (table 3) 5.0 mL
Trace Vitamins Solution 4 (table 2) 5.0 mL
L-Cysteine HC1*H20 0.5 g
Na2S*9H20 0.5 g
Resazurin (0.5 mg/mL) 1.0 mL
Complete to 1-L with distilled deionized water, adjust pH to
7.5 with 5N NaOH and autoclave for 15 min at 15 psig.

57
Table 3-3. Trace Vitamins Solution 4 Composition.
Biotin
2
mg
Folic Acid
2
mg
Pyridoxine HC1
10
mg
Thiamine HC1
5
mg
Riboflavin
5
mg
Nicotinic Acid
5
mg
DL-Calcium Pantothenate
5
mg
Vitamin B12
0.1
mg
p-Amino Benzoic Acid
5
mg
Lipoic Acid
5
mg
Complete with distilled and deionized water to 1-L
Table 3-4. Mineral Solutions Composition.
1) Mineral solution 1
K2HP04 6.0 g/L
2) Mineral solution 2
KH2P04
(NH*)2SO*
NaCl
MgSCV7H20
CaCl2* 2HzO
6.0 g/L
6.0 g/L
2.0 g/L
2.6 g/L
0.16 g/L

58
3) Trace Minerals solution 3
N(CH2C02H)3
1.5 g/L
MgS04*7H20
3.0 g/L
MnS(V2H20
0.5 g/L
NaCl
1.0 g/L
FeS 0.1 g/L
CoS04
0.1 g/L
CaCl2* 2H20
0.1 g/L
ZnS04
0.1 g/L
CuS04* 5H20
0.01 g/L
A1K(S0J2
0.01 g/L
H3Bo
0.01 g/L
Na2MoCV2H20
0.01 g/L
Batch Bioreactor
Description
The batch bioreactor system consists of a 250 mL bioassay
bottle with the metal coupon suspended in 150 mL of liquid
media by a nylon string attached to a crimp top butyl rubber
septum to preserve bottle anaerobically. A typical batch
bioreactor experiment is shown in Figure 3-2. This set-up was
used to study microbial corrosion under a known growth
environment, medium, metal coupon, and head space, using
different bacteria combinations.

59
nylon string
metal coupon
suspended medium
Figure 3-5. Batch bioreactor.
Prior to the tests, the batch bioreactor was assembled
using a 250 mL bioassay bottle to which 150 mL of GLYE media
was added and a metal coupon is suspended in the media. The
metal coupon was fabricated from ASTM-ASME SA106 grade B1
steel pipe 2.5 cm , schedule 80, obtained from Texas Eastern,
Louisiana. Coupons were machine ground to a size of 1.5 cm x
2.0 cm x 3 mm with a side hole of 3mm for the attachment of
the nylon string that holds them to the butyl rubber stoppers.
The inoculum used for the batch bioreactor was the same as
used for the flowthrough bioreactor and it is described
therein.
Operation
Batch bioreactors were intended for one day runs;
however, some experiments were run for several days. Their
100 mL head spaces were maintained with N2 at 7 psig to assure

60
anoxic conditions. Bioassay bottles, including medium and
metal coupon, were autoclaved at 15 psig for 15 min prior to
each run or inoculation. Bacteria were inoculated at room
temperature and placed in a Fisher low temperature incubator,
model 307, at 37°C under aseptic conditions. One mL of E.
aerooenes was inoculated first, then after 12 hrs one mL of
each D. desulfuricans and C. acetobutvlicum was added.
Samples for pH and volatile fatty acids analysis were taken
via the rubber stopper with a 5 mL syringe. Metal coupons
were sampled aseptically and placed in 10 mL vials containing
a cacodylate buffer, pH 7.2, to be further processed according
to the protocol described in Table 3-1 for scanning electron
microscopy examinations.
The GLYE media were prepared using ingredients outlined
in Table 3-2, Table 3-3, and Table 3-4.
Electrochemical Cells Description
The new design of the batch bioreactor, here called the
electrochemical cell, consisted of a modified 1-L erlenmeyer
flask including an extra side opening that fit #8 butyl rubber
stoppers. At the top opening port, an assembly of electrodes
was fitted that included the working electrode (metal
coupon), the pH combination Ag/AgCl reference electrode,
polymer body unit/gel filled by Fisher Scientific, Co., and

61
pressure transducer by Setra Systems, Inc., model 205-2 and
digital pressure indicator, model 300C.
The new system was instrumented for automated data
acquisition of free corrosion potential, pH, oxidation-
reduction potential, and head space pressure. Prior to
filling the electrochemical cell to the 1-L mark with the
medium electrolyte, the cell was outgased in an atmosphere of
80% N2/20% C02. The flask was then sealed under a positive
head space pressure and placed in a precision water bath by
GCA corporation at 30°C. A head space pressure in the range
of 20 to 40 mm of Hg above atmospheric pressure was used
throughout the investigation. This was a pressure that
allowed stoppers not to blow away, it was economical and
allowed an adequate gas supply to the head space. A schematic
of the electrochemical cell is detailed in Figure 3-3.
METRABYTE'S
METRABUS
A/D DATA
ACQUISITION
SYSTEM
UQUID
SAMPUNG
PORT
WATER BATH
Figure 3-6. Schematic of electrochemical cell and data
acquisition system.
REDOX POTENTIAL
pH
POTENTIAL
PRESSURE

62
The system set-up employed an IBM compatible computer
with a four channel MetraByte's MetraBus A/D data acquisition
system that interfaced the four parameters. Custom software,
written in Quickbasic, was used to acquire pH and the three
other parameters, free corrosion potential, oxidation-
reduction potential, and pressure in the form of voltages.
The liquid sampling port consisted of a Pasteur pipet
connected to a 10 mL syringe via a clear plastic tube with an
adjustable tube clamp.
Electrochemical Cells Operations
Single Flask Electrochemical Cell
The first modification or redesign of the batch
bioreactor was the single flask electrochemical cell. A
detailed schematic of the single cell is shown in Figure 3-4.
POTENTIAL
Figure 3-7. Single flask electrochemical cell.

63
Experiments using the electrochemical cells were run at
constant temperature in a water bath at 30°C, described
earlier. Anaerobic conditions were maintained throughout the
experiments using gases treated in a 5.0 cm and 75 cm long
copper column that was electrically heated to approximately
350°C and reduced with pure hydrogen. A gas mixture
consisting of 80% Nz/20% C02 was used during outgasing,
preparation of the mineral electrolyte, and preparation of the
bacteria suspensions as described earlier in this chapter.
After outgasing the single cell, the electrochemical cell
was filled with the mineral solution electrolyte to the 1
liter mark. Then, it was sealed with the stopper containing
the metal coupon and electrode assembly, leaving a head space
of approximately 200 mL at a pressure in the range of 20 to 40
mm of Hg above atmospheric pressure. At this stage, the metal
coupon was held clear of the solution electrolyte and the
complete cell was placed in the water bath. Resting cells
harvested by centrifugation were supplied to the single cell
by injecting them through the rubber stopper. Once the data
acquisition system was connected to the SC and data started to
be collected, the metal coupon was lowered into the
electrolyte by pushing the insulated copper wire that holds
the epoxy-mounted coupon through the rubber stopper.
Experiments using this electrochemical cell allowed
fitting 4 single cells in the water bath for each run, one
control and triplicate samples. Gas and liquid samples were

64
removed under sterile conditions, using appropriate syringes
through the septums provided at the top and side flask
openings.
Dual Flask Electrochemical Cell
It consisted of the single cell (flask I) interconnected
to an additional flask II through their head spaces as
described in Figure 3-5. The objective of flask II was to
overcome some anticipated inconveniences experienced in the
single cell such as metal toxicity and/or any stress imposed
on bacteria during the harvesting procedure. Now flask II
could allow bacteria to grow freely and out of contact with
the metal to avoid metal toxicity. The head space connection
allowed cathodically produced hydrogen to be transferred from
flask I to flask II.
PRESSURE
REDOX
I
Figure 3-8. Dual flasks electrochemical cell.

65
Dual cell experimental runs followed a procedure similar
to the setting and operation of the single cell and described
above, except that flask II could be used to grow bacteria in
their optimum media. The setting of the dual cell did not
allow more than one dual cell in the water bath, for which
only single experiments were run.
Triad Flask Electrochemical Cell
This final design consisted of a set-up similar to the
dual cell including an additional flask III, which is
interconnected to flask I via an ionic bridge made out of 5%
agar, KC1 saturated. Flask III was similar to flask II except
the latter was connected to flask I by the head space. A
detailed schematic is shown in Figure 3-6.
POTENTIAL
Figure 3-9. Triad flasks electrochemical cell.

66
This design was considered capable of satisfying the
objectives of this research because it allowed the measurement
of a differential corrosion current (DCC) induced by bacteria
in addition to other parameters mentioned earlier in the
single cell. A home-made zero resistance ammeter was
incorporated in the data acquisition system in order to
measure and acquire the new DCC parameter. The triad cell as
well as the dual cell allowed bacteria to be grown in flask II
or injected in as suspended cells.
The four available data acquisition system channels were
used to acquire data on the following parameters: pH in flask
I, free corrosion potential in flasks I and III, and DCC
between metal coupons exposed and unexposed to bacteria in
flasks I and III respectively. Triad cell runs used the
selected metal AS106 and the selected bacteria Escherichia
coli (JW111).
Liquid samples for dissolved iron determinations were
taken simultaneously from flask I and flask III, every ten
minutes. Only one triad cell fit the 30°C water bath.
Other experiments using the triad cell include a run in
which a vacuum was applied to flask I and then to flask III in
order to exaggerate a case of hydrogen uptake. Near the end
of the test a positive hydrogen pressure was applied to flask
III in order to observe its effect on the potential and
differential corrosion current measurements. The last
experiment consisted of a galvanic metal couple of Mg working

67
electrode in flask I and a Cu working electrode in flask III,
to which vacuums were applied correspondingly to test the
triad cell response to hydrogen uptakes in both flasks. The
purpose of this test was to check the measurement capacity of
the electrochemical system since a significant differential
corrosion current was supposed to be developed.
Preparation of Bacterial Suspensions
The organisms used in this investigation were
Desulfovibrio desulfuricans (ATCC 7757) , Entrobacter aeroqenes
(ATCC 13048), Clostridium acetobutvlicum (ATCC 824) ,
Escherichia coli (ATCC 8739), Alcaliaenes eutrophus (ATCC
29597), and Escherichia coli (JW 111), provided by Dr. K. T.
Shanmugam. The first three bacteria have been found often in
real MC cases and all of them represented species of hydrogen
oxidizing bacteria.
The first three bacteria D^. desulfuricans, E. aeroqenes.
and C_^ acetobutvl icum were grown in glucose lactate yeast
extract (GLYE) medium, as shown in Tables 3-2, Table 3-3, and
Table 3-4. The rest of the bacteria were grown in Trypticase
Soy Broth media. All chemicals used to prepare the media were
reagent grade and were obtained from Fisher Scientific or
Sigma Chemical Co. All media were prepared under an
atmosphere of 80% N2/20% C02. However, when microorganisms
used for MC experiments were inoculated in their respective

68
medium, an atmosphere of 80% H2/20% C02 was used to activate
their hydrogen uptake enzymatic systems.
Stock cultures from ATCC were prepared at 30°C from
freeze-dried ampules of the organisms in their recommended
growth media indicated above. Cultures were maintained
inoculating agar slants (monthly) and liquid media (weekly).
20 mL serum vials were used for facultative bacteria, and 25
mL crimp-top tubes, sealed with butyl rubber septa, for
anaerobic bacteria.
Resting-cell suspensions were used regularly only in
experimental set-ups that included electrochemical
measurements; other experiments used bacteria inoculated
directly from their grow media. The batch bioreactor also used
washout from slants in some of its runs. Resting-cell
suspensions avoid electrolyte contamination with media used to
grow bacteria.
Cell suspensions were prepared as follows: 1 mL of
subculture of facultative microorganisms less than 1 week old
or 2 mL of subculture of anaerobic microorganisms were
inoculated in 400 mL of appropriate growth media and incubated
in a Fisher low temperature incubator, model 307, at 35°C
under a 80% H2/20% C02 headspace for 1 day for facultative
bacteria and 3 days for anaerobic bacteria. Once cells were
grown, they were harvested by centrifugation in 50 mL screw
cap tubes for 10 min at 10,000 g.

69
Bacteria pellets from tubes were then resuspended in 5 mL
of the mineral solution electrolyte to give an approximate
concentration of the cell suspension of 65 mg (dry weight
basis).
Preparation of Mineral Solution Electrolyte
In order to provide the electrochemical cell with minimum
nutrient conditions as encountered inside gas transmission
pipelines and still allow bacteria to survive for few days, a
mineral solution electrolyte was developed to be used in the
experimental system to study the onset of microbial corrosion.
The ingredients of the mineral solution electrolyte consisted
of 10% of the concentration of the components of solutions
specified in Table 3-4 including 1 mg/L of resazurine and
deionized distilled water to complete the reguired volume
concentration. The ingredients were heated to the maximum mark
on the dial on a Fisher Scientific hot plate, model 210T and
stirred under an 80% N2/20% C02 atmosphere. After heating for
0.5 hr, the flask was cooled down to 30°C surrounded by
crushed ice in a plastic container for approximately 1 hr. At
this time, the electrolyte was transferred to previously
cleaned electrochemical cells and filled to the 1-L marks.
Remaining electrolyte was used to resuspend bacteria and other
routine tests.

70
Metal Coupon
From an original collection of 33 different metals and
alloys, only 18 different metal samples were cut and coupons
fabricated. Samples were prepared based on availability and
ease of cutting to the maximum size allowed in the molds used
for epoxy mounting. Metal coupons used in the flowthrough and
batch bioreactors were not epoxy mounted.
The availability of SA106 steel coupons, its non-toxic
effects on bacteria, its successful surface colonization and
observed surface deterioration, and the popular use of the
alloy in gas production pipelines suggested its selection for
the electrochemical experiments. In addition, it has a
moderate hydrogen production and it is relatively easy to
polish.
A technique was developed to fabricate the working
electrodes used in the electrochemical cells. It consisted in
cutting metal coupons from ASTM-ASME SA106 grade B1 steel, 2.5
cm diameter schedule 80 17-24 H.T. 2500 psi pipe, obtained
from Texas Eastern, Louisiana. The metal coupons were machine
ground to a size of 1 cm x 1.9 cm x 0.02 cm, and attached to
30 cm long 12 THWN insulated hard copper wire by Cerrowire
using nickel print paint (cat. No. 22-207) by GC Electronics.
Coupons were centrally mounted on epoxide resin 20-8130-128
and hardener 20-8132-032 from Buehler, leaving an area of 1.0
cm x 1.9 cm exposed for the microbial corrosion studies. The

71
hard wire served to support and to electrically connect the
coupon and was thereby insulated from contact with the
electrolyte in which the working electrode was immersed.
Prior to the start, the working electrode was held clear of
the solution contained in the flask and lowered into the
solution at will by pushing the hard copper wire through the
butyl rubber stopper that holds the electrode assembly.
After preparation, the working electrodes were polished
with 600 grit, rinsed with ethanol and blotted dried with
Kimwipes EX-L delicate task wipers, then stored in either a
desiccator glass container or a desiccator cabinet by Labconco
Co., model 55300, under vacuum until ready to be used. During
the polishing of the working electrodes, they were initially
sanded with a 24 0 emery grid paper to remove any
inconsistencies on the metal surface, then with a 400 grid,
and finally with a 600 grid. The working electrodes were
placed in an ethanol bath in the ultrasonic cleaner to ensure
the removal of metal particles left from previous polishing.
The polishing procedure was done using a Buehler rotary
polishing wheel and care was taken so that no cross-hatching
was left on the metal surface.
Parameters Setting
In an effort to assure consistency during the
electrochemical measurements, several parameters were fixed

72
based on equipment capabilities, typical inside conditions of
gas transmission pipelines, and other anaerobic stagnant
conditions to make measurements simpler and more reliable.
This task involved the use of the batch bioreactor and
consisted of the study of several determining factors that
formed the basis for the development of the electrochemical
system described herein. Experiments done included
determination of head space gas composition, selection of
reducing agent, selection of sterilization method, and other
related studies.
The head space gases used were methane, hydrogen,
nitrogen, carbon dioxide, and helium. The reducing agents
tested were sodium sulfide, cysteine, thyoglycolate/ascorbate,
and Enterobacter aeroqenes as a natural reducing agent. Among
the sterilization methods used were: 1) all components of
batch bioreactor autoclaved at 15 psig during 15 min, 2) all
components of batch bioreactor autoclaved except metal-coupon-
rubber-stopper assembly that was oven treated at 105°C for 12
hrs, 3) media autoclaved as before and metal-coupon-rubber-
stopper assembly sterilized in acetone and ethyl alcohol for
15 min then blotted dry prior the set-up in bioassay bottle,
and 4) media components autoclaved and metal coupon flamed.
The basic anaerobic and stagnant conditions included fixed
temperature of 35°C and pH in the range of 6.0 and 6.5.

73
Measurement Procedures
All biocorrosion experiments, using electrochemical
cells, were performed at 30°C. Flasks using electrode
assemblies include a rectangular metal coupon (working
electrode) with surface areas of 1.9 cm2 mounted in epoxy
resin and abraded to a 600 grit finish. Metal coupons were
connected to 30 cm of insulated hard copper wire that served
as electrical conductors and support in the stopper.
Electrode assemblies included Ag/AgCl reference electrodes,
polymer body unit/gel filled, from Fisher Scientific.
Reference electrodes were continuously calibrated against each
other using the data acquisition system prior to the
experimental runs. The side opening of flasks contained
platinum electrodes for oxidation-reduction potential
measurement and Pasteur pipets attached to a 10 mL syringe for
liquid sampling.
The four data acquisition channels were connected to the
triad cell as follows: channel four was wired to a pH
reference electrode for pH readings. Channel one and channel
two were wired to the working electrode and reference
electrode of flask I for free corrosion potential
measurements. Channel two was wired to the working electrode
and reference electrode of flask III for free corrosion
potential measurements, and channel three was wired to the
working electrodes of flasks I and III for differential
corrosion current measurements. Current was measured with a

74
homemade zero resistance ammeter with a 10 nA sensitivity,
made of a #308 operational amplifier and a high precision
10,000 0 resistor. This device has the property of converting
current into voltage out with a gain determined by the high
precision resistor, allowing the data acquisition system to
measure and acquire data in volts. A digital pressure
indicator, model 300C by Setra Systems was also adapted to
monitor the headspace pressure in flask I. Once the data
acquisition system was started the working electrodes were
immersed in their respective electrolytes and data was stored
in computer floppy diskettes.
Samples of bacteria, in their growth medium, in their
resuspended medium, and in the liquid discarded after
centrifugation, were homogenized using a vortex Fisher brand,
model Genie 2, and taken to a spectrophotometer Spectronic 2ID
by Milton Roy for determination of transmittance, absorbance,
and concentration at the start of each experiment. After
spectrophotometer determinations, samples were vacuum filtered
using 47 nm pore size membrane filters, preweighted in a
Metier AE100 balance. Wet mounts were also prepared regularly
to check bacteria viability under a light microscope by Nikon,
model LABOPHOT-2. At intervals of 10 minutes, 10 mLs aliquots
were withdrawn from the electrolytes from the flasks I and III
under stagnant conditions and analyzed for dissolved Fe at the
IFAS Soil Science Analytical Research Laboratory. Last runs
were sampled only at the beginning and at the end, shaking

75
flasks to avoid stagnant conditions for dissolved iron. The
duration of each electrochemical experiment was approximately
1 hour. Experiments were continued until the corrosion
potentials leveled off.
Analytical Procedures
Dissolved iron analyses were performed at the IFAS Soil
Science Analytical Research Laboratory. Samples were
collected in 10 mL vials and preserved using 1 drop of
concentrated H2S04 and analyzed on an atomic absorption unit
by Perkin Elmer.
Molecular hydrogen was measured on a gas chromatograph by
Gow-Mac Instruments Co., series 580, equipped with a thermal
conductivity detector. 50 /iL samples were collected in a gas
tight syringe and injected onto a molecular sieve type 5A
column. Samples were injected at an inlet temperature of 40°C
with column temperature of 35°C and a detector temperature of
93°C. The carrier gas used was nitrogen and the detector
current was 50 mA.
Transmittance, absorbance and concentration were measured
on a spectrophotometer Spectronic 2ID by Milton Ray. The
samples were collected in 10 mL Hatch COD tubes and
homogenized in a vortex mixer by Fisher, model Gene 2, prior
to measurements.
Volatile fatty acids were measured on a FID gas
chromatograph by Shimadzu, model GC-9AM. The samples were

76
prepared by centrifugation after acidification with 20%
phosphoric acid. The samples were injected onto a 2m long by
2mm glass column packed with 80/100 chromosorb 1200 WAW
coated with 3% H3P04 and carried with nitrogen gas. 1¿iL
volumes were injected at an inlet temperature of 180°C with
column temperature ramped from 130°C to 170°C over 5 min and
a detector temperature of 200°C.
Dry weights of resting cells were determined by vacuum
filtering known volumes of bacterial suspensions. Filtration
was performed in a 47 mm magnetic Gelman Filter funnel (cat.
09-735). The samples were filtered using 0.45 ¿¿m pore size
membrane filters, 47 mm plain Supor 450, by Gelman Sciences,
Co.. Vacuum was achieved with a precision belt-driven vacuum
pump by Fisher Scientific, model D-75. Membrane filters were
then oven dried at 105°C for 12 hrs. Weights were measured in
a Metier balance, model AE100.
Gases
The gases used during the investigation were: Nitrogen
(UN1066), Helium (UN1046), Hydrogen (UN1049), Nitrogen/Carbon
Dioxide (80/20, UN1956), and Hydrogen/Carbon Dioxide (80/20,
UN1954) supplied by Liquid Air Corporation. For anaerobic
experiments all gases were passed through a heated copper
column (5 cm and 75 cm long) to free gases from traces of
oxygen. The column was heated electrically to about 350°C by
a coil of electrical wire wrapped around the column.

77
Reduction of the column was achieved by passing hydrogen gas
through the column.
Scanning Electron Microscopy
Metal coupons, after exposure to microorganisms, were
treated with a buffered glutaraldehyde solution to fix the
morphology of the bacteria, and then dehydrated through a
graded ethanol series of increasing concentration ranged from
25% to 100% using procedure outline in Table 3-1, page 93.
Samples were then dried and sputter coated with gold
palladium, then examined in a Hitachi S-450 SEM, that included
an Energy Dispersive X-ray Analyzer (EDXA). Coupons were
analyzed throughout and micrographs and spectra were taken at
sites of major interest.
Light Microscopy
Metal coupons were observed and photographed under a
Nikon stereomicroscope, model SMZ-2T and a Nikon microscope,
model LAB0PH0T-2, for biofilm and corrosion examination. Pure
cultures and liquid samples from bioreactors were examined
periodically for contamination via wet mounts.

CHAPTER 4
RESULTS AND DISCUSSION
Flowthrough Bioreactor
Two experimental runs were performed in this bioreactor
in order to reproduce microbial corrosion and to study the
effect of a bacterial triculture on the corrosion of carbon
steel coupons and the formation of biofilm. Its set-up is
described in Figure 3-1. Both runs reproduced microbial
corrosion and their results are shown in Figures 4-1, 4-2, 4-
3, 4-5, and 4-6. Metal coupons exposed to a bacterial
triculture, grown in glucose lactate yeast extract (GLYE)
media under anaerobic conditions, were examined under the
light and the scanning electron microscope (SEM). The results
indicated extensive bacteria colonization as well as metal
deterioration. Careful observations of biofilm micrographs
have suggested a mechanism for understanding its formation.
The first experiment was run for a period of four weeks.
Metal coupons and liquid media were sampled weekly for routine
analyses. First week scanning electron microscopy (SEM)
micrograph results indicated that the bacterial triculture
developed a biofilm consisting of a separated polymeric double
78

79
layer structure; an inner structure attached to the metal
surface, and an outer structure that covers it. It appears
that Enterobacter aerogenes, the bacterium inoculated first,
was responsible for the production of the inner structure and
perhaps the outer structure of the biofilm, see Figures 4-la
and 4-lb. This bacterium, besides reducing the media to
accommodate the strict anaerobes, synthesized a considerable
amount of extracellular polymeric material for attachment
purposes. This material is believed to overcome the natural
surface repulsion caused by the negatively charged bacteria
and metal surfaces (Beveridge and Doyle, 1989). Figure 4-lc
shows the characteristics of the abundant polymeric network¬
like inner structure produced by the bacterial triculture. E.
aerogenes is probably responsible for building this structure
because it is the most prolific of the three species and
reaches the log phase of the growth curve in less than 6 hrs
as compared to the other two bacteria which require often more
than two days. Wachenheim and Patterson (1992) reported that
anaerobic production of extracellular polysacharides is
enhanced generally with any improvement of the conditions of
bacterial growth and that this material is only produced
during the log phase of growth where its production is also
exponential. In our work, the flowthrough bioreactor was run
using GLYE medium which must have enhanced the production of
the polymeric material. This fact, if compared to experiments
run in a medium with limited nutrient composition (mineral

80
solution only), indicates that the amount of biofilm formed on
the metal surface is proportional to the amount of organic
nutrient in the medium.
Desulfovibrio desulfuricans and Clostridium
acetobutvlicum were inoculated one day after the initial
inoculation of Enterobacter aerogenes. The components of the
bacteria triculture were cultured separately. Figure 4-2
shows scanning electron microscopy micrographs of each species
for identification purposes. After inoculation, the strict
anaerobes probably displaced E. aerogenes from the inner
structure of the biofilm so it moved upward and started
producing the outer biofilm layer. This mechanism, suggested
in Figure 4-la, indicates the presence of D. desulfuricans and
C. acetobutvlicum in between the inner and outer structure and
Figure 4-lc shows that most bacteria in the outside are
represented by E. aerogenes and C. acetobutvlicum. These
figures also show that the outer biofilm structure is rougher
on the inside than on the outside. The inner surface seems
like a woven net of polymeric material attached to the metal
surface.
Metal coupon surfaces for this experiment were polished
to 240 grid. Figure 4-3a represents a micrograph of the
control surface, unexposed to bacteria and magnified 1200
times. Figure 4-4a shows the corresponding energy dispersive
x-ray analysis (EDXA) spectrum of the metal surface.

81
After anaerobic exposure of the metal coupons to the
bacterial triculture for a week, surface colonization
including pitting corrosion was observed. Figures 4-3b, 4-3c,
4-3d, and 4-3e show localized pitting and polymeric biofilm,
&
CO
. >» :*
' 1
r‘i>
00
% K
S'
*
c
cn
c
$
Figure 4-1. Flowthrough bioreactor results on bacteria
attachment to carbon steel coupons, a) SEM micrograph showing
bacteria within the outer and the inner structures of the
biofilm, b) SEM micrograph indicating the nature of the outer
biofilm structure.

82
Figure 4-1. c) SEM micrograph showing bacteria on inner
biofilm.
Figure 4-2. SEM characterization of bacterial species from
triculture. a) Enterobacter aerogenes.

83
Figure 4-2.
b) Clostridium acetobutvlicum.
Figure 4-2
c) Desulfovibrio desulfuricans

84
metal deterioration near the edge of the biofilm, generalized
metal deterioration, and elongated pits respectively. Figure
4-4b shows the EDXA spectrum of a metal coupon after exposure
to the bacterial triculture. This analysis presents
additional evidence of microbial corrosion. It shows the
presence of an additional peak (if compared to the control),
corresponding to a sulfur compound, which is probably due to
the deposition of ferrous sulfide resulting from the activity
of D. desulfuricans.
Stereomicroscopic observation of coupon surfaces after
exposure to the bacterial triculture suggest the nature of the
biofilm to be slimy. Figure 4-5a and 4-5b are photographs of
the face and edge of a metal coupon respectively.
During the initial run of the flowthrough bioreactor,
volatile fatty acids and pH were measured weekly for one
month. Results, shown in Figure 4-6, indicate that the only
VFAs produced were acetic and butyric acids. During the first
week, only acetic acid was produced and its concentration
ranged between 300 and 400 ppm until the 3rd week and almost
double by the end of the fourth week. Butyric acid appeared
after the second week and increased at a rate of 200 ppm per
week to a concentration very close to 600 ppm at the end of
the 4th week. Figure 4-6 also shows that pH dropped to 6.5,
probably due to the accumulation of volatile fatty acids.
During the first three weeks the pH ranged between 7.0 and 7.5
then dropped to 6.5 during the 4th week.

85
Figure 4-3. Flowthrough bioreactor SEM results of microbial
corrosion of carbon steel coupons. a) Control surface of
coupon unexposed to bacteria and polished to 240 grid.
Figure 4-3. b) Localized pitting near biofilm after exposure
to bacterial triculture.

86
Figure 4-3.
near biofilm.
Figure 4-3.
deterioration
c) SEM micrograph showing microbial corrosion
d) SEM micrograph showing generalized metal
after exposure to bacteria.

87
Figure 4-3. e) SEM micrograph showing elongated pits.
Figure 4-4. Flowthrough bioreactor results of EDXA analysis,
a) Control EDXA spectrum of a steel coupon unexposed to
bacteria indicating metallic components of the SA106 alloy.

88
Figure 4-4. b) EDXA spectrum of a metal surface exposed to
bacteria showing the sulfur peak resulting from metal sulfides
deposition by EK. desulfuricans. .
Figure 4-5. Flowthrough bioreactor biofilm appearance under
stereo microscope. a) Flat-side view.

VFA, ppm
u\i
Figure 4-5. b) Biofilm edge-side view.
- pH ACETIC BUTYRIC
Figure 4-6. Flowthrough bioreactor volatile fatty acids and
pH profile.

90
A second experiment with the flowthrough bioreactor
suggested that biocorrosion initiates within 24 hours.
Figures 4-7 and 4-8 show SEM micrograph and EDXA spectrum of
a metal coupon sampled one day after the start of the run.
Results of this experiment were similar to the previous one.
Figure 4-7a shows the presence of the extracellular polymeric
material attached to the metal surface and Figure 4-7b shows
metal deterioration, including pitting corrosion comparable to
previous results. Figure 4-8 indicates a similar peak
corresponding to sulfur compounds which may be attributed to
deposition of metal sulfides by D. desulfuricans. In general,
the results of the second experiment showed that the effect of
the bacterial triculture on the corrosion of carbon steel
produced similar types of biofilm and pitting but to a lesser
degree.
Figure 4-7. Flowthrough bioreactor-second experiment, a) SEM
micrograph showing appearance of biofilm.

•JI
Figure 4-7. b) SEM micrograph indicating massive pitting
corrosion.
OAY 1 PPT , :-i*
P*« IMS 1MSEC « ihT
H-10KEM 1 • IH M-IIKEU 1H
' i r
Figure 4-8. Flowthrough bioreactor EDXA spectrum of metal
coupon exposed to bacteria showing the sulfide peak.

92
Volatile fatty acids analysis indicated levels of 140 ppm for
acetate after one day and reached a value of 340 after one
week while pH remained constant in the range of 7.5 and
7.2.
Batch Bioreactor
Based on the results of the flowthrough bioreactor, the
theory of cathodic depolarization, and the literature reviewed
on organic acids'effect on corrosion (Soraco et al., 1988;
Little and Wagner, 1987; and Beveridge et al., 1983), a new
batch bioreactor system based on the principles of the
bioassay bottle experiment was used. The rationale here was
to study the initial steps of the microbial corrosion process
that could set the stage for the development of an
experimental system that would be able to quantify the
biological component of corrosion, in addition of using a
simpler bioreactor. It was necessary in this system to avoid
the formation of a biofilm allowing bacteria to live for at
least six hours, and then detect the effect of their activity
on the metal surface.
Using the batch bioreactor set-up described in chapter 3,
a series of experiments were performed to reproduce microbial
corrosion at conditions resembling the inside of gas
transmission steel pipelines. The research included a

93
preliminary study of carbon and energy sources required by the
bacterial triculture to survive one day.
Results show that the batch bioreactor reproduced
microbial corrosion within 24 hours. The system allows for
visual inspection of exposure of metal coupons to living
bacteria and to check for changes of parameters such as head
space gas and media composition. It is less prone to
bacterial contamination and medium does not need to be
replenished as in the flowthrough bioreactor. These
characteristics showed the batch bioreactor to be superior to
the flowthrough bioreactor, in addition to being more
attractive economically.
Effect of Head Space Gas Composition
Head space gases used were H2, CH4, 50% H2 / 50% CHA, N2,
80% N2 / 20% C02/ 8 0% CH4 / 20% C02, and He at a pressure of 7
psig. Head space gases were used in combination with carbon
steel coupons and variations of GLYE media compositions, the
bacterial triculture, and tar-condensate product from an
offshore gas production platform.
Results show that the head space affected the
bacteria/metal/fluid components. Results using GLYE media
indicate that for all head space except the controls, the
bacteria triculture caused the formation of a black
precipitate that adhered to the steel surface and also
remained suspended in the liquid. Bottles with methane in the

94
head space produced an additional shiny, black, uniform film
at the liquid interface. In the case of hydrogen, the
interfacial black precipitate was not formed. Results using
a GLYE media containing 10% of the original concentration of
glucose and lactate, show similar results to the flowthrough
bioreactor experiment except that a thicker black film was
formed on coupons under the nitrogen head space. Results
using GLYE media, excluding glucose and lactate, also
produced a black precipitate on the metal surface and in the
bulk of the fluid.
Coupons in bottles with nitrogen/carbon dioxide and
methane head space were uniformly covered with the black film.
Results using the mineral components of the GLYE media
produced similar results to the previous experiment.
When the medium was composed of only yeast extract and
vitamins, no black precipitate was formed either on the metal
surface or in the fluid. Results of the control experiments
using distilled water for the medium showed no black
precipitate either. Other control experiments using the GLYE
media composition combinations mentioned above, excluding only
yeast extract and vitamins in the absence of bacteria,
produced a slight darkening of the fluid after a week and the
production of a crystalline film on the surface of the metal
coupons.

95
Effect of Bacterial Combination
Based on previous results and the ability of sulphate
reducing bacteria to reduce sulphate to sulphide, an
experiment was set-up to determine the bacterial combination
responsible of the black precipitate. Of the seven bacteria
combinations tested, only the ones containing D. desulfuricans
produced the black precipitate in both the metal and the
fluid. In order to reduce the chances of production of the
black precipitate, washings of bacteria cultured on agar
slants were used to minimize bacterial metabolites and other
undesirable substances contained in the culture medium during
inoculation.
Selection of Media
Some complementary experiments to the head space
experiment were performed in order to select the medium that
will resemble inside conditions of gas transmissions pipelines
and allow the study of cathodic hydrogen utilization at the
onset of the microbial corrosion process. The medium
composition included combinations of the following components;
yeast extract and vitamins, minerals, glucose and lactate, and
minerals excluding phosphates and/or sulphates. A nitrogen
head space and a bacteria combination, D. desulfuricans and E.
aerogenes. were used for all combinations of the media
components. Results indicate that medium excluding phosphates
and sulphates did not allow the black precipitate to occur in

96
either the liquid or the on metal surface. The amount of
organic nutrient was directly related to the amount of black
film deposited on the metal coupons. Consequently, bottles
with GLYE medium produced the darkest fluid and the most
biofilm on the coupons. Results with 10% strength mineral
components of the GLYE medium, less organic components
produced the most acceptable results. It produced the least
turbidity and the least amount of biofilm. Further tests with
the 10% strength mineral solution showed that it could
maintain bacteria alive for over 6 hours, its normal pH is 6.5
but can be easily lowered to 4.5 with C02. Based on the above
facts and other reasons to be discussed, the 10% strength
mineral solution was selected as the electrolyte solution to
be used in all the electrochemical experiments.
Selection of a Reducing Agent
Early in the use of the batch bioreactor systems, it was
necessary to select a reducing agent for the media that would
assure the low redox conditions required by the strict
anaerobes but not react or interfere with the components of
the microbial corrosion system. Several reducing agents
normally used in anaerobic studies including a natural
bacterial reducing agent were tested to study their effects on
the metal and on the liquid media. Three chemical reducing
agents tested included sodium sulphide, cysteine,
thyoglycolate/ascorbate, and a selected facultative anaerobic

97
bacterium, E. aeroaenes. were tested in the mineral
electrolyte solution.
Results indicate that all chemical reducing agents were
able to reduce the medium at their recommended low
concentrations, but they produced a black film on the metal
coupon upon contact with the liguid media. The bacterial
reducing agent was also able to reduce the media, but it was
concentration dependent. Five mL of bacteria per 150 ml of
mineral solution were able to reduce the media in 6 hours.
However, 3 mL / 150 mL reduced the media in two days. The
bacterial reducing agent developed some turbidity after a
week.
Selection of a Sterilization Method
The selection of a sterilization method was another task
that required attention at the beginning of the tests with the
batch bioreactor. The most practical method was autoclaving
of the coupon and the medium together in a final set-up, ready
for inoculation. This method was inappropriate because it
allowed the steel coupon to rust, and rust accumulated at the
bottom of the bottle. For this reason two additional methods
were tested. They were: a) autoclaving medium and treating
the stopper holding the metal coupon in an oven at 105°C for
12 hours and b) autoclaving media and treating the stopper
holding the metal coupon in an acetone-alcohol bath for 15

98
minutes then blotting it dry. Autoclaving in all cases was
done at 15 psig and 15 minutes.
Results indicated that the most convenient method that
assures less bacterial contamination and prevents the metal
from corroding was autoclaving the medium and separately
sterilizing the metal coupon in an oven. The sterilized
coupon was then aseptically suspended in the medium. This
method was selected and after several trials, as the metal
coupons remained clean and shiny inside a clear mineral
electrolyte solution for over 3 days.
Experimental Results
The batch bioreactor experiments were analyzed for
volatile fatty acids, SEM, EDXA, head space gas composition
and pressure. Table 4-1 shows the results for volatile fatty
acids from experiments using the mineral solution, metal
coupon, bacterial triculture and head spaces containing N2 /
C02, H2, CH4, and CH4 / H2. Volatile fatty acids results were
expected to be zero. However, values in the range of 9 to 195
ppm were obtained from these experiments after two days.
Detectable values might be attributed to volatile fatty acids
already present in the inocula as they were taken from their
normal cultures that used media rich in nutrients and/or to
gas chromatograph calibration sensitivity.

99
Table 4-1. Batch bioreactor volatile fatty acids measurements
from experiments using steel coupons and bacterial triculture
in mineral solution.
Volatile fatty acids, ppm
Gas head space Acetate propionate isobutvrate butyrate
N2/C02
190
43
93
—
n2
81
—
—
—
cha
112
114
—
—
h2
81
45
—
—
ch4/h2
126
46
—
23
Figure 4-9 shows the structure of the biofilm formed in
the GLYE medium within 24 hours. Bacterial counts per area
(mm2) of metal surface in a 4 hour sample, indicated
concentrations of 15,000 bacteria cells. Figure 4-10 shows a)
bacteria colonization within 24 hours and, b) its
corresponding EDXA spectrum indicating the sulfur peak
characteristic of the activity of D. desulfuricans.

100
Figure 4-9. Batch bioreactor biofilia formed within 24 hours.
Figure 4-10. Batch bioreactor SEM and EDXA results on
bacteria attachment to carbon steel, a) Bacteria colonization
within 24 hours.

101
Figure 4-10. b) EDXA spectrum of metal coupon exposed to
bacteria for 24 hours.
Head Space Gas and Pressure.
Head space gas composition and pressure were analyzed and
measured periodically to check anaerobic conditions and detect
gas leaks. Gas analysis aided in the explanation of observed
abnormal surface conditions resulting from air leaks. During
the experiments with the batch bioreactor, it was observed
that in 2% of the runs the head space became contaminated with
air. All cases had bad stoppers and as a result coupons
rusted from the start of the runs. Gas analysis in all cases
showed an oxygen concentration in the range of 6 to 9%.
Results in Table 4-2 indicated that head space gases show
pressure drops within one day and then stabilize thereafter.

102
Those results suggested the utilization of head space gases by
bacteria in their metabolic processes to different degrees
during their contact with the metal coupon. The head space
gas utilization was greater in experiments using mineral
solution than in experiments using GLYE medium. It was
interesting to notice that, in all bottles containing
nitrogen, the pressure drop was 100%. However, in the case of
the GLYE media, the pressure drop for hydrogen was 48.0% and
for methane 47.8%. In the case of the mineral solution, the
hydrogen pressure drop was 72.0% and the methane pressure drop
was 56.0%. Control experiments containing hydrogen in their
head space and no bacteria had a pressure drop of 36.0%.
Table 4-2. Batch bioreactor head-space pressure drop in GLYE
and mineral solution media after two days.
Head Space Gas
N2 / C02
N2
h2
ch4
Control
Pressure drop. %
GLYE
Mineral So
100.0
100.0
100.0
—
48.0
72.0
47.8
56.0
36.0
36.0
These results suggested that bacteria in the nutrient limited
medium metabolized more head-space gas than in the medium rich
in nutrients which might constitute a source of more readily
available energy source. These results were further confirmed

103
in other experiments performed to study hydrogen uptake by
hydrogen oxidizing bacteria. Results of those experiments
indicated larger hydrogen uptake when the medium lacked
organic nutrients.
Crystalline Films.
Figure 4-lla shows the SEM micrograph of the structure of
a crystalline film that formed after one week in the absence
of bacteria. Figure 4-llb represents the corresponding EDXA
spectrum showing the peak corresponding to phosphorus. The
spectrum suggested the presence of a phosphorous compound in
the crystalline film that formed on the metal surface.
Crystalline films were observed on metal surfaces
approximately one week after exposure to the mineral solution
in the absence of bacteria, or after one week bacteria had
died. Figure 4-12a shows a transition from the biofilm to the
crystalline film where the crystalline film formed several
days after all the bacteria had died and decayed. Figure 4-
12b shows a pitting corrosion and deterioration of the
polymeric network like biofilm material after a gentle removal
of the crystalline film with a nylon brush.
The results of the anaerobic batch bioreactor system were
used as the basis for the development of an experimental
system in which an attempt was made to guantify the biological
component of corrosion during bacterial utilization of
cathodic hydrogen.

104
Figure 4-11. Batch bioreactor - formation of a crystalline
film on the metal surface after one week of exposure to the
mineral solution in the absence of bacteria. a) SEM
micrograph showing characteristics of the crystalline film.
Figure 4-11. b) EDXA spectrum showing elemental distribution
of crystalline film on metal surface.

105
Figure 4-12. Batch bioreactor - formation of crystalline film
on metal surface after exposure to bacteria. a) SEM
micrograph showing a transition zone from biofilm to
crystalline film.
Figure 4-12. b) SEM micrograph showing a deteriorated
polymeric biofilm structure and pitting after bacteria had
died.

The Electrochemical Cell
The electrochemical cell was basically a modified batch
bioreactor equipped for making electrochemical measurements
integrated with a data acquisition system as described in
Figure 3-3, page 93. Using this set-up, several experiments
were performed excluding bacteria and their results were used
to select the parameters for the runs with the final modified
electrochemical cells. The theory for all electrochemical
cells is described in Capabilities of the Triad Cell, page
198.
The experiments included qualitative and quantitative
determinations of hydrogen production by some metals, free
corrosion potentials at different metal-surface polishing
grades, a galvanic series of some metals in the mineral
electrolyte solution, bacterial growth characteristics, and
hydrogen uptake by some hydrogen oxidizing bacteria.
Table 4-3 shows the results of hydrogen production by
three different metals in the mineral electrolyte solution.
Mg, the most active, produced hydrogen almost immediately and
in 1 hour reached an average hydrogen concentration in the
head space of 24.4%. For the other two metals, Zn and SA106
steel, hydrogen was not detected even six hours after the test
was begun. After one day, hydrogen production was appraised
by the head space pressure which was measured for several

107
Table 4-3. Hydrogen production by filings of Mg, Zn, and
SA106 steel in mineral solution in the absence of bacteria
after three days.
Dav
1
Dav 3
Metal
wt, qr
Press
H-, cone
Press
mm Hg
%
mm Hg
Mg (1)
0.5808
230
29
1870
Mg (2)
0.5811
410
21
1545
Mg (3)
0.5807
12
23
1680
Zn (1)
1.6847
90
0
170
Zn (2)
1.6843
130
0
190
Zn (3)
1.6842
146
0
210
SA106(1)
1.2199
149
0
160
SA106(2)
1.2195
147
0
150
SA106(3)
1.2193
124
0
135
CONTROL(1)
0
178
0
175
CONTROL(2)
0
183
0
181
CONTROL(3)
0
178
0
175
days. A noticeable increase in the head space pressure, for
the case of steel, started after three days.
Table 4-4 presents the free corrosion potentials of SA106
steel metal coupons polished at diffent grades in the mineral
electrolyte solution. Results indicated that the free
corrosion potential was not affected substantially in that
range of surface polishing grades tested. For the purpose of
this research, the 600 grid polish was selected. This grade

108
of polish provided a combination of a little roughness and a
fine surface.
Table 4-4. Free corrosion potential
different surface polishing grades.
Polishina arade
of SA106 steel coupons at
Free Corrosion Potential
grid #
Volts (Aa/AcfCl)
180
-0.714
320
-0.684
400
-0.701
600
-0.680
1200
-0.680
Results of the free corrosion potentials of selected
metals and alloys in the absence of bacteria are presented in
Table 4-5. The free corrosion potentials are arranged in a
galvanic series in order to compare their reactivities in the
mineral electrolyte solution. Potential-time curves obtained
during free corrosion potential measurements are shown in
Figure 4-13.

109
-0.250-
-0.300-
-0.350-
(0
% -0-400^
>
¿ -0-450
z -0.500-
t¡
O -0.550Í
Ql
-0.600-1
-0.650"
*0.700"1 r 1 1—
09:36 AM 12:00 AM 02:24 PM
TIME, hours
Figure 4-13. Potential-time curve in the absence of bacteria.
Measurement of the growth of pure cultures including
absorbance, transmittance, density, and dry weight were
performed to complement research in the development of the
experimental system. Figure 4-14 shows the growth curve at
37°C for E. coli (JW111) in tryptic soy broth media by Difco.
Results indicated that E. coli grew exponentially between 4
and 7 hours and reaches a constant growth after 8 hours. The
usefulness of Figure 4-14 finds its place in experiments that
require rapid interpretation such as results that require
normalization using the weight of bacteria. This way,

110
Table 4-5. Galvanic series of selected metals and alloys in
mineral solution at 30°C vs Ag/AgCl reference electrode in the
absence of bacteria.
Free corrosion potential
Metal coupon
Mg
Zn
A1
Steel
SX52(T)
Steel
SX7 0
Steel
SX60
Steel
SA106
Steel
SX42
Steel
SX42(B)
Steel
SX52(B)
Steel
SHP9430
Steel
SGRB
A1 20
Steel
SS430
Steel
S430
Cu-Ni
7030
Cu
Ag-Zn
Cu-Ni
9010
Volts (Aq/AqCl)
-1.520
-0.960
-0.750
-0.680
-0.680
-0.680
-0.680
-0.675
-0.670
-0.670
-0.560
-0.550
-0.540
-0.170
-0.040
-0.010
0.016
0.022
0.025

Ill
bacteria dry weight is correlated with absorbance which is
measured relatively rapidly. The drawback is that these
growth curves are valid only for cases involving the same
growth conditions.
Figure 4-14. Growth curve for E. coli at 600 nm, 37°C, and
trypticase soy broth medium.
The next two groups of experiments of this section
consisted of: 1) determining the effect of media on E. coli
during hydrogen uptake and 2) comparing the hydrogen uptake of
different hydrogen oxidizing bacteria in the medium that E.
coli had maximum hydrogen uptake. Table 4-7 shows that E.
coli takes up over twice as much hydrogen in the mineral
solution than in other media rich in organic nutrients. These
results suggested that E. coli were forced to use more

112
hydrogen as the energy source in an anaerobic environment poor
in nutrients.
Table 4-7. Effect of media composition on the hydrogen uptake
by E. coli at 30°C during 6 hours.
Media Hydrogen uptake, % Bacteria dry wt. mq/L
Mineral Solution 24 350
Nutrient Broth 9 600
GLYE 11 500
Control 4 0
Table 4-8 indicates that despite the density of the
different cultures their hydrogen uptake was very similar.
These results agree with earlier experiments and has been
suggested in the literature (Booth and Tiller, 1968).
Table 4-8. Bacteria hydrogen uptake capabilities in mineral
solution at 30°C during 6 hours.
Bacteria Hydrogen uptake, % Dry weight, mq/L
D. desulfuricans
27
20
E. aerogenes
20
344
A. eutroohus
26
60
C. acetobutvlicum
27
120
E. coli
24
350
Control
4
0

113
Single Flask Electrochemical Cell
The objective of this single cell was to measure the free
corrosion potential of metals in the presence and absence of
bacteria.
Using the setup indicated in Figures 3-6 and 3-7, a
combination of experiments were performed to determine the
effect of four different hydrogen oxidizing bacteria on the
free corrosion potential of three different metals. Results
of potential time curves showed that the presence of bacteria
positively influenced the corrosion of the metals, except
copper.
Figure 4-15 shows triplicates of potential-time curves of
SA106 steel in the presence of A. eutrophus and the control,
excluding bacteria. The potential differences during the
tests were on the order of 150 mV. Figure 4-16 shows the free
corrosion potential characterization of Mg in mineral solution
in the presence of E.coli.
Metal coupons experienced a decrease in their free
corrosion potentials when compared to the control experiments
unexposed to bacteria. These results suggest the utilization
of cathodic hydrogen by bacteria. Table 4-8 summarizes the
results of this combination experiment using the single cell.
An overall analysis of this experiment indicated that the free
corrosion potential differences were independent of the type
and amount of bacteria used. The free corrosion potentials

114
o
>
HI
I-
O
o.
-0.350-r
-0.400-
-0.450-
-0.500-
-0.550-
-0.600-
-0.650-
-0.700-
-0.750-
-0.800 1 1 1 1 1 1
03:36 PM 06:00 PM 08:24 PM 10:48 PM
04:48 PM 07:12 PM 09:36 PM 12:00 PM
TIME, hours
CONTROL —t— ALCALIGENS 1 —— ALCALIGENS 2 ALCALIGENS 3
Figure 4-15. Potential-time curves for SA106 steel in the
presence of A. eutrophus.
TIME, hours
Figure 4-16. Potential-time curves for Mg in the presence of
D. desulfuricans.

115
differences among the three metals tested were due to their
reactivities which are related to their different capacities
to produce cathodic hydrogen. The results with copper
indicate that this metal might have been toxic to bacteria as
suggested in the literature review section (Beveridge and
Doyle, 1989) .
Table 4-8. Effect of different bacteria on the free corrosion
potential of Mcf, Cu, and SA106 steel coupons in mineral
solution and 305C.
METAL
BACTERIA FCP. Volts
delta FCP
Mg
A. eutroohus
-1.495
0.195
E. coli
-1.475
0.175
E. aeroaenes
-1.545
0.245
D. desulfuricans
-1.515
0.215
SA106
A. eutroohus
-0.655
0.095
E. coli
-0.565
0.015
E. aeroaenes
-0.675
0.125
D. desulfuricans
-0.645
0.085
Cu
A. eutroohus
-0.046
0.086
E. coli
-0.005
0.035
E. aeroaenes
-0.047
0.087
D. desulfuricans
-0.046
0.086
An important observation during the anaerobic experiments
with the single flask electrochemical cell was the occurrence

116
of longitudinal cracks along the surface of the SA106 steel
coupons which otherwise did not occur when metal coupons were
in aerobic conditions. The cracks occurred after coupons were
used several times and occurred in the direction the pipe was
drawn. The cracks were first observed when unusual large
bubbles appeared at the metal surface on the cracks. The
cracks were easy to recognize when rust formed on them, after
the test and out of the mineral solution, but were difficult
to detect if the surface had been polished.
The discovery of this cracking phenomena and reports on
some unaccountable cathodic hydrogen loss during
electrochemical measurements in microbial corrosion
experiments (Booth and Tiller, 1968), suggested that part of
the cathodic hydrogen being produced was absorbed on the
surface of the steel with concomitant production of hydrogen
embrittlement. Figure 4-24, page 148, shows the effects of
hydrogen embrittlement on SA106 steel.
Despite some encouraging results, the single cell was
considered inappropriate to accomplish the objectives of the
overall research for the following reasons: 1) the similarity
of values of the free corrosion potentials for each metal,
despite the amount and the bacterial species used, 2) the
inability to handle metal toxicity as in the case of copper,
3) lack of confidence with the results of the potential-time
curves, 4) the inability to measure corrosion current, the
ideal parameter, 5) the possibility of suspended bacteria to

117
behave differently due to the imposed stress during
centrifugation, and 6) instrumental failures that made
difficult the maintenance of fixed experimental conditions.
In reference to the amount of bacteria inoculated, E.
aeraenes and E. coli outnumbered A. eutroohus by a factor of
12, and if compared to the amount of D. desulfuricans. the
factor was even larger because of the very low dry weight that
could be measured in a growing culture of the bacteria.
An overall review of the results and the components of
the single flask electrochemical cell suggested that the
source of the controversies might be due to the following
factors: a) reference electrodes were calibrated individually
instead of calibrated against each other b) The technique
used to mount metal coupons in epoxy was not appropriate in
some cases (some coupons experienced corrosion on the edges
after the first run and others experienced galvanic coupling
with the connecting copper wire) , and c) the cracks that
developed on the steel coupons could have influenced the
results of the potential-time curves.
Dual Flask Electrochemical Cell
The dual flask is an improvement of the single cell. Its
arrangement is described in Figure 3-5. This electrochemical
cell included an additional flask (II) interconnected to the
initial single cell, flask (I), through the head space in

118
order to allow bacteria to grow in their best growth media.
This flask allowed bacteria to avoid direct contact with the
metal and, consequently any metal effects such as toxicity.
The new set-up permitted the utilization of the other channels
of the data acquisition system for the measurement of other
parameters such as pH, oxidation-reduction potential, and head
space pressure.
Table 4-9 summarizes the results of five experiments that
evaluated effects of bacteria and the addition of a terminal
electron acceptor (fumarate) on the corrosion of SA106 steel.
These results include calculation of average corrosion rates
based on dissolved iron analysis. In addition, potential-
Table 4-9. Dual cell schedule of experiments and
corresponding corrosion rates, using E.coli (JW111) and SA106
steel coupons.
Run Bacteria
I
Age
Drv wt
Fe cone
Corrosion
hr
mg/L
(mg/L)xlOO
mg/dm2D
1
control
-
-
7.4
47.0
2
6
46.7
6.3
40.0
3
15
63.3
7.5
48.0
4
23
59.4
7.4
47.0
5
fumarate
27
69.8
15.0
95.0
Note: Control run contained no bacteria.

119
time curves for these experiments are presented to show the
effect of bacteria on free corrosion potential.
Figure 4-17 shows the effects of E. coli at different
ages on the free corrosion potential of SA106 steel coupons.
The potential-time control curve is included in every figure
for comparison. Figure 4-18 indicates the corresponding
dissolved iron profiles. Both curves show that the presence
of E. coli. one of the most prolific bacteria in most aqueous
environments, increased the corrosion of the steel. It is
interesting to notice that this is the first time E. coli has
been shown to influence the corrosion of metals; this hydrogen
uptaking strain was able to cause an effect on the free
corrosion potential at the onset of the corrosion process.
Figures 4-17a, b, c, and d and Figures 4-18a, b, c, and
d represent the effect of E. coli. harvested by
centrifugation and corresponding to 6, 15, 2 3 and 27 hours old
and 46.7, 63.3, 59.4 and 69.8 mg/100 mL dry weight
respectively. The bacterial effect on the potential-time
curves and dissolved iron profiles was consistent with the
rest of the research and influenced the corrosion of SA106
steel. In all experiments the potential-time curve was
affected by the bacteria in the horizontal portion by 10 mV in
the direction of the more active potential if compared to the
control. The dissolved iron profile appeared to be unaffected
for the first four experiments except for the case when
fumarate was added. Results of the dual cell were still

120
inadequate to determine the influence of bacteria in the
corrosion of steel because of the insignificant potential
differences and the inaccuracy of the iron analysis from
stagnant fluids.
Figures 4-17d and 4-18d represent probably the most
important finding from an experiment done with the dual cell.
They show the effect of fumarate, a terminal electron
acceptor. Results of the potential-time curve show a dual
effect on both segments of the curve if compared to the
control. The effects are comparable to the observed in the
cases of an induced mass transfer and a bacterial effect
combined. Results of the dissolved iron profile showed a two
fold increase after the addition of 40 mM of fumarate to the
single cell. These results suggested that the reducible
substrate activated the bacteria hydrogenase system during
their uptake of cathodic hydrogen. During the dual cell runs
other parameters including pH, oxidation-reduction potential,
and head space pressure were measured in order to determine
their trend. All experiments had a similar trend: pH
increased during the first 0.5 hour 0.3 pH units and then
stabilized at a rate of 0.05 pH units every 2 hours.
Oxidation-reduction potential of flask I decreased linearly
140 mV during the first 3 hours and oxidation-reduction
potential of flask II increased exponentially 140 mV during
the same time period. Head space pressure decreased very

121
Figure 4-17. Dual cell run potential-time curves, a) Effect
of E. coli. 6 hr old .
Figure 4-18. Dual cell run dissolved iron profiles. a)
Effect of E. coli. 6 hr old.

Dissolved Iron, mg/L Corrosion Potential, Volts
122
CONTROL
BACTERIA
7. b) Effect of E. coli, 15 hr old
Figure 4-18
b) Effect of E. coli. 6 hr old

123
Figure 4-17. c) Effect of E. coli. 23 hr old .
Figure 4-18
c) Effect of
E. coli
23 hr old

124
Figure 4-17. d) Effect of E. coli. 27 hr old, and fumarate .
Figure 4-18. d) Effect of E. coli. 27 hr old, and fumarate

125
slowly until samples were taken, then it decreased
proportionally to the sample volume drawn.
During the dual cell runs it was observed, as in the
single cell runs, that SA106 steel coupons developed
longitudinal cracks along their surfaces after coupons were
used more than four times. These observations confirmed the
development of cracks and clearly suggest the occurrence of
hydrogen embrittlement. Results are shown in Figure 4-24,
page 188.
An overall review of the dual cell experimental results
indicated that the dual cell was also inadequate to quantify
the effect of bacteria during the corrosion of steel. The
reasons considered here were the same ones presented for the
single cell. The insignificant potential difference resulting
from the activity of the bacteria led to abandoning the idea
of attempting to correlate the potential differences with
their biological corrosion effect. Fortunately, the effect of
fumarate on bacterial activity encouraged the search for a new
idea to incorporate the measurement of a corrosion related
current parameter into the dual cell which resulted in the
triad flask electrochemical cell.
Triad Flask Electrochemical Cell
The purpose of the triad flask electrochemical cell was
to incorporate the measurement of a corrosion related current,

126
into the dual cell to determine the influence of bacteria
during the corrosion process. A detailed schematic of the
triad cell is presented in Figure 3-6. The triad cell
included a third flask (III) similar to flask I, except
bacteria was not added to it. The set-up allowed the
measurement of a differential corrosion current (DCC) that
could be correlated to the corrosion influenced by bacteria.
The triad cell is comparable with a microbial corrosion
syatem and should be able to measure the utilization of
cathodic hydrogen by bacteria. The head space connection
between flasks I and II could be associated with the hydrogen
diffusion restriction between the bacteria and the metal
through a membrane or biofilm. Flask II could be associated
with a growing environment such as the one found within a
biofilm where bacteria could perform at optimum capacity.
Here bacteria could be protected from toxic effects and grow
under their optimum conditions, and could control the
corrosion reaction mechanisms (Little and Wagner, 1993; de
Beer et al., 1992). The triad cell also allowed the use of a
resting cell either in flasks I or III and the measurement of
a differential corrosion current (DCC) between flasks I and
III that may be correlated with the bacterial utilization of
cathodic hydrogen. Furthermore, the triad cell could allow
the measurement of cathodic hydrogen utilization by bacteria
indirectly via metabolism of a terminal electron acceptor such
as fumarate. Additionally, the effect of bacteria on the

127
corrosion of metals might also be calculated from dissolved
iron differences between flasks I and III. In general the
triad cell could offer several alternatives to determine the
biological component of corrosion.
A total of six experiments were performed using the triad
cell. The first four shown in Table 4-10 include DCC
measurements with an electrometer. On the last two
experiments a zero resistance ammeter was used in order to
improve inconsistent current measurements obtained with the
electrometer.
Table 4-10. Triad cell schedule of runs and related corrosion
using E. coli (JW111) and SA106 steel coupons.
1
1
2
3
4
Run
Age
hr
control
8
fumarate 20
>6D
Bacteria
Dry wt
mg/L
67.5
64.3
71.9
Fe cone
(mg/L) xlOO
0.75
0.42
2.58
0.08
Corrosion
mg/dm2Day
4.7
2.7
16.3
0.5
The initial four experiments evaluated the effects of E.
coli on the corrosion of SA106 steel through changes of the
shape of the potential-time curve, the dissolved iron profile,
and the DCC-time curve. The last two experiments portray an

128
exaggerated case of hydrogen uptake and hydrogen production
using SA106 steel, Cu, and Mg coupons.
Results of Figure 4-19 and Figure 4-20 show the effect of
E. coli on the anaerobic corrosion of SA106 steel in mineral
solution. Potential-time curves and dissolved iron profiles
indicate that the presence of bacteria influenced the
corrosion of the steel.
Figures 4-19a and 4-20a indicate the effect of bacteria,
harvested after 8 hr, on the corrosion of SA106 steel coupons.
Results showed a shift of 5 mV if compared to the control, in
the direction of the more active potential, on the horizontal
portion of the potential-time curve. Dissolved iron analyses
showed an average corrosion rate of 2.7 mg/dm2D and the DCC
indicated an average current of 215 nA. Potential-time curve
and dissolved iron profile of the control run showed
characteristics similar to their equivalent dual cell curves.
The magnitude of the differential corrosion current (DCC)
results was very small for the control run, on the order of 1
nA as expected. Figures 4-19b and 4-20b show the effect of
bacteria harvested after 20 hours including the addition of 40
mM of fumarate. Results showed a dual effect on the shape of
the potential-time curve similar to the corresponding run of
the dual cell (Figures 4-17d and 4-18d). The presence of the
terminal electron acceptor increased corrosion rate over three
times the value of the control run, to an average corrosion
rate of 16.3 mg/dm2D calculated from the dissolved iron

Corrosion Potential, Volts
129
Figure 4-19. Triad cell potential-time curves. a) Effect of
E. coli. 8 hr old.
Figure 4-20. Triad cell dissolved iron profiles,
of E. coli. 8 hr old.
a) Effect
Diff.corr.current, A

Corrosion Potential, Volts
130
Figure
t-19. b) Effect of E. coli. 20 hr old and fumarate.
Figure 4-20. b) Effect of E. coli. 20 hr old and fumarate
Diff.corr.current, A*10**(-07)

131
profile. A similar result was observed with the DCC that
jumped 20 nA as soon as the fumarate was added. This
important observation suggested once again that the addition
of fumarate
increased the activity of the bacteria and hence the microbial
corrosion process. Figures 4-19c and 4-20c show the effect of
bacteria harvested after 6 days. Results indicated that
potential-time curve had a similar effect as with bacteria
harvested within 20 hours. However, the corrosion rate
calculated from the dissolved iron profile was very low, 0.5
mg/dm2D.
The DCC was not accurately measured due to the continuous
inconvenients with the eletrometer calibration and chart
recorder signal noise. In order to improve DCC measurements,
a home made zero resistance ammeter was implemented and
instrumented to the data acquisition system. The last two
experiments were performed using the new device.
Overall results of the first four experiments indicated
that potential-time curves, dissolved iron profiles and DCCs
were not significant; due to the low potential differences
produced by the presence of bacteria, the unreliable dissolved
iron analysis as a result of the stagnant conditions of the
triad cell, and the inaccuracy of measurements provided by the
electrometer. The first of the last two experiments of the
triad cell depict the exaggerated conditions of hydrogen

Corrosion Potential, Volts
uptake using SA106 steel coupons as in previous experiments.
The purpose of this test was to determine the sensitivity of
Figure 4-19. c) Effect of E. coli. > 6 days old.
Figure 4-20. c) Effect of E. coli. > 6 days old.
Diff.corr.current, A*10**(-07)

133
the triad cell by applying artificial vacuums to flask I and
injecting volumes of hydrogen gas to flask III. The
artificial vacuums represented larger than normal hydrogen
uptakes by bacteria.
Figure 4-21 shows the behavior of the potential-time and
differential current-time curves under forced conditions of
hydrogen uptake and hydrogen injection. Potential I
represents the potential of the working electrode in flask I,
potential III represents the potential of working electrode in
flask III, and current represents the differential corrosion
current (DCC).
As indicated in the figure, two initial vacuums, of 3mm
Hg each, were applied to flask I. These forced vacuums were
equivalent to exaggerated hydrogen uptakes by bacteria.
Results indicate that none of those vacuums produced any
effect either in the DCC curve or in any of the two potential¬
time curves for flasks I and III. However, when two 50 mL of
hydrogen gas were injected in flask III, some changes were
observed on the curves. The initial volume injected caused
an instantaneous decrease in the DCC in the order of 1 ¿tA and
the second injection increased potential III by 12 mV and the
DCC stabilized. Then, after 20 minutes, potential III
returned to its original value and the DCC level off until the
end of the test.
The second of the last two experiments of the triad cell
represents a modified Daniel cell, a very early form of

134
battery which consisted of Cu and Zn electrodes in a solution
of their salts except in this experiment the Zn electrode was
replaced by a Mg electrode in order to maximize the cell
voltage and consequently the differential corrosion current
Figure 4-21. Triad cell artificial hydrogen uptake test.
<
3
4-1
C
0>
L.
1_
3
O
TO
C
0)
1—
5=
â–¡
(DCC). The electrolyte solution used in this last experiment
was the mineral solution utilized throughout this
investigation. The purpose of this experiment was to examine
the measurement capabilities of the triad cell by comparing it
to a known electrochemical model cell as the Daniel battery
that produces a significant amount of current. Also, attempt
to explain the controversial measuring difficulties of

135
previous triad cell runs in anaerobic conditions and further
demonstrate the capabilities of the triad cell.
Figure 4-22 shows the results of forced vacuums of 3 mm
Hg applied to flasks I and III and their effects on the DCC
and electrode potentials during the corrosion process of Mg.
The forced vacuums represented artificial hydrogen uptakes.
In this cell the SA106 steel electrodes were replaced by the
Cu and Mg electrodes. The first vacuum, applied to the flask
containing the Cu electrode, produced an increase in the Mg
potential on the order of 200 mV and a decrease of 30 ¿iA in
the DCC. The Cu potential remained almost unchanged for the
rest of the experiment. A second vacuum applied 20 minutes
later produced an additional increase of 300 mV in the Mg
potential and a decrease of 5 pA in the differential corrosion
current. A third vacuum was later applied to the flask
containing the Mg electrode produced an increase in the order
of 2 nA in the differential corrosion current. After 10
minutes a fourth vacuum was applied to the flask containing
the Mg coupon and significant changes were observed: the Mg
potential decreased to its original potential, by 500 mV, and
remained constant until the end of the test; and, the DCC
increased by 35 ¿¿A within 5 minutes, then decreased steadily
by 15 /¿A to a constant value of 3 5 ^A until the end of the
experiment.
Despite the evidence of experimental results that
demonstrate microbial utilization of cathodic hydrogen during

136
the corrosion of SA106 steel, the inaccuracy and inconsistency
of some of the results carried over from previous experiments
and the sensitivity of the electrometer and the zero
resistance ammeter did not provide accurate enough information
to quantitatively determine the biological component of
corrosion. However, results suggest that the triad flask
electrochemical cell may be used to study the anaerobic
utilization of cathodic hydrogen, provided some improvements
be made in the following areas: measurement of the DCC, data
acquisition system, rigidity of the cell, mechanical handling
of metal coupons, gas and liquid sampling, anaerobic
conditions, and water bath.
<
3
C
0
3
O
ra
c
0)
1—
3=
Q
Figure 4-22. Artificial hydrogen uptake in a triad cell using
Cu and Mg electrodes.

137
Final Discussion
In general this investigation presents a new approach to
studying anaerobic microbial corrosion. The research shows
evidence of metal deterioration by bacteria using two new
flowthrough and batch bioreactors. It shows proof of the
occurrence of hydrogen embrittlement and the effect of a
terminal electron acceptor in the activity of bacteria during
corrosion. A new microbial corrosion mechanism is proposed
to provide a better insight to the overall understanding of
this complex phenomena.
Figures 4-1 through Figure 4-8 show evidence of anaerobic
corrosion of carbon steel by a known bacteria triculture,
using the flowthrough bioreactor. Figures 4-9 through Figure
4-12 show evidence of microbial corrosion by single and
combinations of the bacteria triculture, using a new batch
bioreactor system. The batch bioreactor was able to reproduce
microbial corrosion within one day and resulted practical to
study the on-set of the corrosion process. This bioreactor
allowed visual inspection of coupons and was less prone to
bacterial contamination. In addition, further results of the
batch bioreactor indicate that head space gas composition
affected the bacteria/metal/fluid system. The presence of a
black precipitate at the metal surface and in the bulk of the
fluid was determined to be caused by both the activity of the

138
bacteria and the composition of the media. Other results
suggest bacteria metabolism of nitrogen, hydrogen and methane.
The batch bioreactor was further implemented to resemble
inside conditions of gas transmission pipelines, incorporate
electrochemical measurements, and improve control of fixed
parameters. The new experimental system became the
electrochemical cells. An effort was made to set the stage
for the study of microbial corrosion at its on-set. This
included the use of suspended bacteria cells harvested by
centrifugation instead of inoculum from their standard growth
media and the use of the mineral electrolyte solution that
provide the environmental conditions for hydrogen evolution
from metals. The bacteria triculture used in early
experiments was effective in the reproduction of microbial
corrosion but it was not appropriate to for this study because
of the concomitant effect of many variables resulting from
their use. Effects such as the production of extracellular
polymeric materials, bacteria age, ecological succession of
bacteria involved, environmental growth conditions for
different bacteria. The mineral solution developed did not
allow the formation of a biofilm within two days because of
the absence of organic nutrients that enhance the production
of polysaccharides (Wachenheim and Patterson, 1992) and
provided the chemical conditions for hydrogen evolution.
Suspended cells were used to avoid retention of metabolites
and other substances usually present in the growth media

139
contained in inoculum from standard medium. Iverson (1968)
demonstrated that resting cells of Desulfovibrio vulgaris and
Desulfotomaculum orientis grown in standard medium act as
depolarizing agents of mild steel. However, his experimental
procedure was criticized because it was found that these
microorganisms often retain FeS in their cell walls (Tiller,
1982) .
Microorganisms in general grow under many different
environmental surroundings. However they can also adapt, and
bacteria would make differential changes for a different
physiological state. Those changes could be negative such as
diminishing their biosynthetic activities, stopping their
reproduction capabilities, and increasing their catabolic
processes with production of secondary metabolites; or could
be positive such as improvement of their growth and regaining
optimal physiological conditions as under a biofilm.
Researchers have found different pH profiles inside
biofilms than in the bulk of the liquid (Wagner and Little,
1993). pH changes have been reported on bacteria to resist
heavy metal toxicity because of their effects on the
hydrolyzed forms of the heavy metals and their toxic effects
(Collins and Stozky, 1992). It has been reported also that
different bacteria species have different metal-oxidizing
capacities (Corstjens et al., 1992).
The utilization of cathodic hydrogen by bacteria is
directly related to their optimal growth conditions which are

140
not always the most favorable for hydrogen formation (Cord-
Ruwish and Widdel, 1986), low pH values stimulate the proton
hydrogen equilibrium in the direction of hydrogen formation.
Due to the electrochemical nature of this research and
the need for simplification, one bacterium was selected to
continue this research. The selection of the proper bacterium
used in the electrochemical experiments was based on
availability, ease of growth, amount of bacteria harvested at
the log phase, and hydrogen uptake capabilities. A literature
search on the hydrogenase enzyme, also suggested the bacteria
contain the proper hydrogenase. After contacting some
researchers in the area, it was found that Dr. K.T. Shanmungam
from the University of Florida had a mutated strain of E. coli
(JW111), containing only hydrogenase for hydrogen uptake.
Since this strain met all the above requirements, it was the
best selection. It is a facultative anaerobic microbe that
reaches maximum growth after 8 hours, as compared to D.
desulfuricans that requires over 70 hours. When harvested by
centrifugation at 10,000 g it yields a dry weight cell
concentration in the range of 70 mg/ 100 mis of culture,
compared to d. desulfuricans and C. acetobutvlicum that yield
a dry weight bacterium concentration around 5 mg/100 mis. In
addition this particular bacteria has been tested in hydrogen-
fumarate medium and results indicate that the bacteria uses
100% of fumarate during hydrogen uptake.

141
During all experiments standardization procedures were
carefully followed for all electrochemical and analytical
measurements in order to attain reliable measurements. A
series of experiments were performed using the experimental
setup described in Materials and Methods to assure the
satisfactory performance of the measuring instruments. The
experiments consisted of calibrations of the instruments and
experimental runs following recommended standard procedures.
The corrosion of a metal in an aqueous environment is
well recognized as a surface electrochemical phenomenon where
part of the metal, represented as the anode, is oxidized and
dissolved into the solution. The balancing cathodic reaction
is composed of simultaneous reductions of some components of
the environment (mineral solution) affecting the metal. The
dissolved metal ions may either precipitate as insoluble
compounds that may be loose or bulky or may attach firmly to
the surface.
The role of the bacteria, on the other hand, is not as
well defined as the role of the metal and the aqueous
solution, since it may participate directly or indirectly in
one or both of the electrochemical reactions on the metal,
thereby influencing the reaction.
Once the SA106 steel coupon is immersed in the acidic
electrolyte solution, the dominant reaction is iron
dissolution which leaves behind an excess of electrons:
Fe > Fe++ + 2e" (1)

142
the excess electrons are consumed by the balancing reaction at
cathodic sites. In the anaerobic set-up of the
electrochemical cells the dominant balancing reaction is the
reduction of hydrogen:
H30+ + e' > 1/2 H2 + H20 (2)
At pH < 7, which is the case of the mineral solution, hydrogen
evolution usually predominates in the absence or presence of
oxygen.
The theory of cathodic depolarization predicts that a
protective hydrogen envelop forms on the metal surface and
polarizes it. However, this was not the condition for the
metals tested. In the case of experiments involving SA106
steel, Zn, and Mg, results shown in Table 4-3 indicate that
the amount of cathodic hydrogen production is dependent on the
type of metal. Furthermore, it depends also on the surface
area of the metal, electrolyte composition, pH, and surface
orientation. In all cases a protective hydrogen film never
formed. In the Mg coupons the hydrogen bubbles evolved from
the surface for several days and in the Zn and steel coupons
fewer bubbles formed but left the surface also. In the case
of metal filings of the same metals results show that for Mg,
hydrogen bubbles emerge from the metal continuously for a
period of time undetermined, then, after a while the flow of
bubbles apparently stops for a moment and then larger bubbles
arise bringing the metal filings with them. A similar event
happened with the Zn filings but over longer time periods.

143
The steel filings produced bubbles after 3 days and required
some shaking to observe hydrogen evolution. It was observed
that the lower the pH used, as a result of carbon dioxide
diffusion into the mineral solution, the more hydrogen evolved
from the metal coupons. Initial experiments had the metal
coupons oriented horizontally right below the tip of the
reference electrodes. This practice was a source of faulty
potential measurements because it allowed hydrogen bubbles to
fill up the tip of the electrodes. Further experiments used
coupons reoriented vertically as shown in Figure 3-4.
After the dual flask experiments by Belay and Daniels
(1990), Rajagopal and LeGall (1989), and the implication of a
phosphate compound in the corrosion process (Weimer et al.,
1988) ; it has been suggested that there might be some
component in the media that interact with the metal to induce
the production of hydrogen. Bryant and Laishley (1993) were
puzzled with the results of the dual flask experiment of Belay
and Daniels, as how the interaction of the metal and the media
could generate enough hydrogen to actually promote corrosion
in view of the cathodic depolarization theory. The theory
states that after metal is polarized, it requires the bacteria
to depolarize it. They suggested that the interaction of
phosphate with the steel promotes the release of cathodic
hydrogen by an unknown mechanism. They based their
declaration on experiments that demonstrated that an increased
phosphate concentration in the media influenced the corrosion

144
of carbon steel and the production of black iron phosphates
such as vivianite.
The evolution of hydrogen may be explained from a
chemical point of view. Equation (2) above shows the overall
cathodic reaction via the formation of atomic hydrogen which
may be absorbed in the metal surface. Normally, molecular
hydrogen is produced by a combination reaction which is slow
and often becomes the rate limiting step in the corrosion
process (Miller, 1981).
2 H > H2
(3)
H + H+ + e > H2
(4)
During corrosion the anodic and cathodic reactions are in
balance, this is one of the reasons why corrosion current can
not be measured directly. The center of attention of the
corrosion process is often the anodic reaction because of the
metal loss. However, the cathodic reaction can be slow or
fast implying a control of the process and the metal loss.
Consequently, any component of the corrosion system that
influences reactions (3) and (4) will influence the cathodic
reaction and hence the metal loss. These would involve
compounds like phosphate that is included in most growth media
in concentrations between 5 and 100 mM, and in the
microorganisms themselves. The mineral solution used during
this research contained 0.2 mM of phosphate. Results of the
batch bioreactor shown in Figure 4-11 evidence the presence of
a phosphorous compound in the form of a crystalline black

145
scale. These results agree with the above discussion and the
findings of Iverson (1984) who suggested that a highly
corrosive metabolic product during the microbial corrosion of
steel by sulfate reducing bacteria was responsible for the
blackening of the solution under a hydrogen atmosphere. X-ray
powder pattern analysis of precipitated corrosion products
indicated the presence of iron phosphide, he also indicated
that the black film on the coupon was due to FeS. Results of
the batch bioreactor also indicated the presence of a black
precipitate suspended in the solution and attached to the
metal caused by the activity of D. desulfuricans in the
mineral solution. Figure 4-8 shows the corresponding EDXA
spectrum that exhibits the presence of FeS. These results
were very similar to the ones of pit coatings of FeS on mild
steel in media containing sulfide ions found by Otero and
Achucarro (1993). The results of this research and other
findings elsewhere suggest that iron phosphate complexes and
iron sulfide occur both in the bulk of the fluid and on the
surface of mild steel.
The observation of cracks on SA106 steel coupons during
experiments with electrochemical cells suggests the occurrence
of hydrogen embrittlement. Figure 4-23 shows SEM (a) and b) )
and EDXA (c) and d)) photomicrographs of SA106 steel coupons
affected by longitudinal cracks along the surface. Steel
coupons were used during the runs of the electrochemical cells

146
and developed cracks after more than four consecutive during
the experiments.
The above results and the fact that hydrogen evolution
from the steel proceeds via formation of atomic hydrogen, and
that hydrogen may be absorbed on the steel surface, suggest
that the cracks were the result of hydrogen embrittlement.
Booth and Tiller (1968) reported that they were not able to
account an apparent removal of hydrogen from mild steel and
attributed this effect to a cathodic hydrogen evolution into
the solution. This observation might have been associated
with hydrogen embrittlement but they concluded that it was
difficult for them to imagine another cathodic reaction
available. Figures 4-24a and 4-24b are SEM photomicrographs
of a characteristic crack at low and at high magnifications
respectively. Figures 4-24c and 4-24d indicate EDXA
photomicrographs showing the elemental distribution of
corrosion products inside the crack and on the surface of the
metal away from the crack.
The addition of fumarate to some of the electrochemical
experiments accidentally elucidated the effects of a terminal
electron acceptor on the hydrogen uptake activity of bacteria.
Because of the difficulty of measuring the relatively small
amounts of cathodic hydrogen produced by SA106 steel,
fumarate was initially used as an alternative way to
indirectly account for the amount of hydrogen uptake by the
bacteria. It indirectly measures hydrogen uptake by means of

147
succinate formation during hydrogen uptake. Succinate is a
more stable compound than fumarate (Anaerobe Laboratory
Manual, 1975) and is easily measured in an ion chromatograph.
It can also be measured in a gas chromatograph but requires a
methylation procedure. Chromatograms show a very well defined
peak for succinate after approximately 15 min of the injection
of the sample. Fumarate has been tested successfully with E.
coli with results showing 100% conversion to succinate during
hydrogen uptake (Lee et al., 1985).
Experiments using methyl viologen (paraquat) or its
analog benzyl viologen have demonstrated that those artificial
electron acceptors may preferentially accept electrons from
the atomic hydrogen first generated instead of electron from
the hydrogen gas. This was demonstrated by adding methyl
viologen to flasks that had a constant rate of hydrogen
evolution. The results showed that as soon as the methyl
viologen was added, the hydrogen evolution temporarily ceased
until all methyl viologen was reduced. Thereafter, the
hydrogen evolution approached the hydrogen evolution rate of
the control. This effect was directly proportional to the
concentration of methyl viologen used (Bryant and Laishley,
1989) .
Results of this investigation presented in Figures 4-6
and Figure 4-8 have suggested that potential-time curves and
hydrogen uptake characteristics are independent of the type
and amount of five different hydrogen oxidizing bacteria.

148
However, when fumarate was added to some of the experiments
with E. coli. it was observed that bacterial activity
.
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solution electrolyte. a) SEM micrograph of crack at a low
magnification
Figure 4-23.
magnification.
b) SEM micrograph of crack at a high

149
Figure 4-23. c) EDXA micrograph of corrosion products inside
the crack.
JI¬
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Figure 4-23. d) EDXA micrograph of uncracked control surface

150
increased with corresponding increase in the differential
corrosion current (DCC) and the concentration of dissolved
iron. This fact suggested that in the absence of fuxnarate the
hydrogen uptake rate is independent of the hydrogenase
activity of the bacteria. This finding is supported by
results of other researchers in the area (Booth and Tiller,
1968).
The hydrogen-fumarate medium has been recommended in the
literature to be a defined medium used to test the ability of
some bacteria to grow under anaerobic conditions utilizing
hydrogen as electron source and fumarate as terminal electron
acceptor (Lee et al., 1985). Its transformation is given in
the following equation:
COOHCHCHCOO + 2 H+ + 2 e~ > COOHCH2CH2COO' (5)
Fumarate Succinate
Data reported from several investigators has shown that
the hydrogenase enzyme influences the anodic reaction of the
corrosion process by oxidizing the hydrogen produced.
However, there is still much to be learned about hydrogenase
at the physiological and biochemical levels. Hydrogenase is
typically associated with the oxidation-reduction of various
electron carriers. Positive evidence of cathodic
depolarization by hydrogenase resulted from an experiment by
Iverson (1966) in which a AISI1010 steel coupon in contact
with masses of hydrogenase positive sulfate reducing bacteria
on a reduced buffer agar surface containing benzyl viologen in

151
place of sulphate and electrically connected to another coupon
isolated from bacterial cells through a micro ammeter. After
17 hours under a nitrogen atmosphere, the benzyl viologen
under the coupon with the cells was reduced from colorless to
purple color and iron dissolved under coupon isolated from
bacteria cells suggested a catalytic effect on the activity of
bacteria. In a similar manner, results of this research
suggest that the reduction of fumarate influenced the activity
of bacteria during their utilization of cathodic hydrogen
because the fumarate as a terminal electron acceptor must have
interacted with the bacterial hydrogenase system in order to
increase their hydrogen uptake. Sadana and Morey (1961) in
experiments with hydrogenase from sulfate reducing bacteria
and methyl viologen proposed that the reduced form of the
hydrogenase enzyme is more stable than its oxidized form.
Based on the fact that the chemical transformation of a
thermodynamically stable compound to a less stable form
involves a slower rate of reaction, and the fact that the rate
of reaction of hydrogenase is faster in the presence of
fumarase than in its absence; the presence of fumarate causes
the enzyme to oxidize faster and hence uptake more hydrogen.
Consequently, this may explain the observed increased
bacterial activity in the presence of the terminal electron
acceptor.
Hydrogenases are enzymes which are able to reversible
catalyze the reactions involving hydrogen:

152
H2 <===> 2 H <===> 2 H+ + 2 e (6)
The metabolism of hydrogen oxidizing bacteria is regulated by
a combination of reversible hydrogenases located in the
periplasm and cytoplasm of the organisms. It was after 1986
that three different hydrogenases were recognized for the
sulfate reducing bacteria. A highly active Fe-containing
periplasmic hydrogenase, a Fe-Ni-Se-containing periplasmic
hydrogenase and a Fe-Ni-containing cytoplasmic hydrogenase.
The activity of hydrogenase is affected by the concentration
of Fe in the media (Czechowsky et al., 1990). The Fe-
containing hydrogenases are believed to be restricted in
distribution to strict anaerobic bacteria where information is
limited or lacking (Adams, 1990). Recent research in the area
indicates that different hydrogenases have different
capabilities for hydrogen evolution and utilization and
similar hydrogenases have different capabilities depending on
their location in the cell. For example, the dimeric Ni-
containing hydrogenase is periplasmic for Desulfovibrio oigas
and has an specific activity of 1400 for hydrogen utilization
and 400 for hydrogen evolution. In the case of E. coli this
hydrogenase type is transmembranic and has an specific
activity of 38 for hydrogen utilization versus 76 for hydrogen
evolution (Przybyla et al., 1992). Those findings throw new
light on the understanding of the complex microbial corrosion
phenomena.

153
In addition to the above discussion most researcher fail
to consider the myriad of different factors that can affect
cathodic depolarization. For instance when surface scaling
adheres to metal surfaces depolarization of the cathode is not
as easily reversible as hydrogen depolarization. The study of
microbial utilization of cathodic hydrogen would be much
easier avoiding the formation of such products. This
research, besides providing conditions to avoid the formation
of any film and/or electrochemical external effect, also
focused on the onset of the corrosion process. The beginning
of the corrosion process may predict how the complex multiple
dynamic microbial corrosion process will influence the later
stages. In practice, experimental determinations of corrosion
currents always induce electrochemical changes in the
corrosion system and this is undesirable. On top of that, it
is important to consider that steel corrodes spontaneously in
aqueous environments such as the mineral solution; and the
presence of mild scale, irregularities in the crystalline
structure, and any other variation in the physical chemical
nature of the surface such as grain boundaries and inclusions,
will result in the metal structure becoming micro anodic and
cathodic areas that may result in some forms of localized
corrosion.

154
New Proposed Microbial Corrosion Mechanism
Overall results of this research afford evidence of
cathodic hydrogen utilization by hydrogen oxidizing bacteria
during anaerobic corrosion of carbon steel. They provide
evidence of several findings that allows the proposition of a
comprehensible new microbial corrosion model. This model
focus on the on-set of the microbial corrosion process and
builds on the theory of cathodic depolarization described in
Chapter 2. Figure 4-24 depicts the proposed new microbial
corrosion mechanism. The most important findings of this
investigation are enclosed in the model and include the
evidence of: 1) hydrogen production by metals in the mineral
electrolyte solution, Table 4-3, 2) positive influence of
bacterial activity in the corrosion of metals indicated in the
potential-time curves, dissolved iron profiles and
differential corrosion current measurements, see Figures 4-15
through 4-20, 3) SEM and EDXA spectra of corrosion products
such as FeS and acetate, resulted from the activity of
bacteria, Figures 4-1 through 4-12 and Table 4-1, 4) hydrogen
utilization by bacteria, Tables 4-6 and 4-7, 5) effect of a
terminal electron acceptor in the activity of bacteria
indicated in the results of experiments with the dual cell and
with the triad cell, Table 4-9 and Table 4-10, 6) hydrogen
embrittlement observed on SA106 steel coupons during
experiments with the electrochemical cells, Figure 2-23, and

155
7) approach to measure the biological component of corrosion
suggested by the triad cell results.
The new anaerobic corrosion mechanism suggests that
metallic iron oxidizes and dissolves in the acidic mineral
H+ + OH-
H20
r\ ,''0, H | 2H+ A
Lj /
FUMARATE
SUCCINATE
H+
p°l
1 G
â– Hy""
a
1 s
2H 1
1 0!
A I
1 ,o,
H+ 1 OH'
1 X 2e 1
IT
H + |<-
bT__
1
Figure 4-24. Proposed anaerobic mechanism for the on-set of
microbial corrosion of steel by hydrogen oxidizing bacteria.

156
electrolyte solution. During the process electrons from the
metal are released to cathodic sites. Hydrogen ions resulting
from water dissociation migrate to the negatively charged
metallic surfaces and become reduced with the excess electrons
accumulated at cathodic sites. Atomic hydrogen being produced
at the surface is either absorbed into the metallic
crystalline structure or recombines itself with additional
atomic hydrogen and produces molecular hydrogen. Absorbed
hydrogen may cause hydrogen embrittlement and molecular
hydrogen may either cause cathodic polarization or diffuse
into the bulk of the fluid and head space. Molecular hydrogen
is then taken up by the hydrogenase system of the hydrogen
oxidizing bacteria. The presence of a terminal electron
acceptor increases the hydrogenase activity and hence
increases the hydrogen uptake by bacteria. As a result, more
cathodic hydrogen is produced and consequently more metallic
iron is oxidized to maintain the overall electroneutrality.
Capabilities of the Triad Cell
The triad cell was designed with the objective of
simulating the on-set to study microbial utilization of
cathodic hydrogen from mild steel and for the determination of
most of the parameters suggested in the proposed microbial
corrosion model described in Figure 4-24. It is the
conviction of the author that with the proper determination of

157
those parameters and some auxiliary analysis such as SEM,
metallography, and others; it may be feasible to define the
microbial corrosion mechanism of a particular situation to be
studied in the triad cell, and the correlation of the measured
parameters elucidate quantitatively the biological component
of corrosion.
The triad cell could be compared to a short circuited
battery between flasks I and III where the differential
corrosion current (DCC) measured is the result of the
potential difference caused by the activity of the bacteria
influencing the corrosion of the metal coupon in flask I.
Potentials are practical indications of the corrosion
state of a metal in an electrolyte and depends on the
electrode reactions. It is a simple measurement that requires
a millivoltmeter, with a high impedance to prevent current
drain during measurement, and a reference electrode capable of
providing a stable reproducible potential. It is impossible
to measure absolute potential, for this reason an arbitrary
reference electrode standard is chosen such as the hydrogen
electrode that is assigned a potential of 0.00 V when hydrogen
activity is 1.0 M and its partial pressure is one atmosphere.
The free corrosion potential is measured by placing the metal
coupon and the reference electrode in the electrolyte and
reading the meter in volts. In practice, the free corrosion
potential, is the voltage developed between the corroding
metal and the reference electrode in contact with the

158
corrosive environment. While one reaction is dominant in the
boundary layer of the reference electrode, more electrode
reactions may be proceeding in the boundary layer of the
corroding metal coupon. As a result, the measured voltage is
the sum of two voltages developed in the double layers of
both electrodes. It is really a mixed potential in the
electrochemical sense. The free corrosion potential is also
a dependent variable in corrosion and electrochemical
measurements and it is connected to corrosion rate by simple
functions under fixed specific conditions. If a consistent
free corrosion potential difference between a metal coupon
exposed and unexposed to bacteria could be determined, this
might be correlated with the biological component of
corrosion. In the case of the single and dual electrochemical
cells, the rationale was to quantify a free corrosion
potential difference which can be attributed to the influence
of bacteria during the corrosion of a metal, and correlate
that to corrosion rates (corrosion current density) via
classical electrochemical corrosion theory such as the Butler-
Volmer and Nernst relationships. In this manner, a
theoretical corrosion rate might be calculated, avoiding the
classical electrochemical procedures that require
potentiostatic and/or potentiodynamic techniques that usually
introduce external effects to the microbial corrosion system.
Corrosion rate or corrosion current is probably the most
informative and most desirable parameter to know about the

159
deterioration of a metal. However, the literature indicate
that it is not possible as yet to measure such a parameter
directly, but experimentally as a function of potentials.
Some experimental techniques include mass loss measurements,
linear polarization, and impedance spectroscopy. Some new
technologies such as the quartz microbalance could give mass
loss information in real time. The triad cell offers to
measure a differential corrosion current (DCC) directly, and
this may be able to be correlated to the total corrosion rate
of the metal and to the biological component of corrosion.
Total corrosion is the integrated function of corrosion rate
over the time period considered for the experiment. The units
of corrosion rate are equivalent to current density by
Faraday's law. Total corrosion in a circumstance of microbial
corrosion includes the corrosion of the metal in the absence
of bacteria and the corrosion influenced by microorganisms.
The triad cell allows for the measurement and data
acquisition of the following parameters: pH, potential,
oxidation-reduction potential, temperature, DCC, and head
space pressure. It also allows the sampling of the head space
gas, liquid media and metal coupon for supplemental analysis
such as gas composition, volatile fatty acids, dissolved iron
and other ions, absorbance, metallography, microscopy, and
analysis of corrosion products. A list of recommended
implementations to improve the triad cell are included in the
suggested research section.

160
The experimental results shown in Figure 4-22 may be
analyzed by comparing it with a Daniel Cell in which the Zn
electrode is replaced with a Mg electrode and the solution of
the salts for the mineral solution electrolyte. The
electrical current produced by the corrosion reaction may be
compared to the differential corrosion current measured in the
triad cell. "The Daniel Cell was a very early form of battery
which consisted of Cu and Zn immersed in a solution of their
salts" (Trethewey and Chamberlain, 1988).

CHAPTER 5
SUMMARY AND CONCLUSIONS
Summary
Microbial corrosion is a pervasive and widely recognized
problem of massive proportions in many industrial processes
worldwide. In particular anaerobic corrosion of steel is of
great economic importance. Much remains to be learned about
basic mechanisms and measurement of the influence of those
organisms in the corrosion process if we are to mitigate and
control its catastrophic effects. The techniques available to
control the organisms and/or protect the metal are in many
cases inadequate. Microbial corrosion requires the
understanding of several scientific disciplines in order to
provide insight into its study. There is a need for
experimental systems which provide quantitative measurements
of the influence of bacteria in the corrosion process. Non-
disruptive electrochemical techniques combined with physical-
chemical analysis, in a new batch bioreactor, appears capable
of measuring the biological component of anaerobic corrosion
from the very early stages of the process. The technique
involves the use of a "triad flask" electrochemical cell,
Figure 3-6, that indirectly measures microbial utilization of
cathodic hydrogen using a terminal electron acceptor and
relates it to free corrosion potentials, differential
161

162
corrosion currents, and corrosion rates calculated from
dissolved iron analysis.
The overall objective of this investigation was to
develop a method to study microbial corrosion from its very
early stages by measuring the microbial utilization of
cathodic hydrogen. It is known that certain microorganisms
consume hydrogen in their metabolic processes. Since hydrogen
embrittlement of metallic structures is a corrosion
consequence of inmense economic proportions, the present study
was designed to explore the feasibility of integrating
hydrogen embrittlement (by consuming the hydrogen) through
microbiological processes. This secondary task focused on
attempting to quantify the biological component of corrosion.
Two new flowthrough and batch bioreactor systems were
employed. The flowthrough bioreactor is able to reproduce
microbial corrosion of carbon steel in the presence of a known
bacteria triculture and glucose-lactate yeast extract media.
During the first week, scanning electron microscopy revealed
that the bacteria triculture composed of Enterobacter
aerooenes. Clostridium acetobutvlicum and Desulfovibrio
desulfuricans produced a thick, and dark biofilm that
consisted of a double layer polymeric structure and a random
distribution of pitting on the metal surface. Electron
dispersion X-ray analysis (EDXA) analysis provided elemental
analysis of the corrosion products resulting from the
interaction of metabolites produced by bacteria on the surface

163
of the steel. Volatile fatty acids analysis indicated the
presence of acetate, a metabolite, reported to be responsible
for the corrosion of steel even in very low concentrations.
The batch bioreactor produced microbial corrosion within
24 hours. The various microbial species present are similar
to those encountered in natural environments. SEM analysis
confirmed that the bacteria triculture caused pitting
corrosion, formed a biofilm on the metal surface and produced
a black precipitate of FeS on the bulk of the fluid, and on
the metal surface. Among other advantages, it allowed for
visual inspection, it was less prone to contamination than the
flowthrough bioreactor, and it offered a better control of the
experiments. Experiments with the batch system to study the
carbon and energy sources for the bacteria triculture show
that head space gas composition affects the
bacteria/metal/fluid system. For example, bacteria metabolize
the head space gases nitrogen, hydrogen and methane in their
metabolic processes. Results show that desulfuricans and
phosphate were responsible for the black precipitate suspended
the liquid and deposited on the metal surface.
An experimental system based on the batch bioreactor is
intended to simulate conditions inside of gas transmission
pipelines. Electrochemical measurement capabilities was
provided to attempt to quantify the biological component of
corrosion. The experimental system was instrumented to a five
channel data acquisition system which recorded on-line data

164
including pH, free corrosion potential, oxidation-reduction
potential, head space gas pressure, and diferential corrosion
current in the presence and absence of bacteria under
controlled environmental conditions. Conventional physical-
chemical analyses were also performed periodically using
"grab" sample.
The experimental system results indicate that the
response of the potential-time curves to the presence of
bacteria were more reactive, but were not significant enough
to determine the influence of bacteria in the corrosion of
steel. Among the reasons were the small magnitudes of the
potential differences when compared to control runs without
bacteria, the similarities of results obtained using five
different bacteria, and different bacterial concentrations at
different stages of growth. Results of dissolved iron
concentration profiles were consistent with the potential-time
curves, indicating a higher corrosion for experiments with
bacteria.
Final experiments using the triad cell included
measurements of a differential corrosion current. These
experiments produced comparable results to the potential-time
curves and measurable DCCs were observed when the bacteria
were present. The results of this experiment were also
inadequate to calculate the biological component of corrosion.
During the electrochemical experiments, two important
findings were observed: the detection of longitudinal cracks

165
along the surface of the steel coupon due to hydrogen
embrittlement, and the increased activity of bacteria during
the addition of a terminal electron acceptor that suggest a
catalytic effect on the hydrogenase activity of bacteria
during the utilization of cathodic hydrogen. These findings
along with other ideas were incorporated in a new proposed MC
model. Significant increases in the calculated corrosion
values from dissolved iron analysis and the DCC, were observed
during the addition of the terminal electron acceptor.
Despite the difficulties in quantifying the biological
component of corrosion, this research brings about deeper
insight into the overall understanding of the phenomena
involved in microbial utilization of cathodic hydrogen; and
concepts developed herein provide another stepping stone on
the path towards a full understanding of microbial corrosion.
Conclusions
An attempt has been made to determine the influence of
bacteria during the anaerobic corrosion of carbon steel at its
onset, using a new triad electrochemical cell. Preliminary
experiments using two new bench scale flowthrough and batch
bioreactors reproduced microbial corrosion. The batch system
resulted in superior operations and was redesigned to include
electrochemical measurements. this then evolved into the
triad electrochemical cell. Despite the small and

166
inconsistent potential and deferential corrosion current
differences of steel coupons exposed and unexposed to
bacteria, which discourage the quantification of the
biological component of corrosion; the overall results afford
strong evidence of the influence of bacteria on the cathodic
depolarization of carbon steel. Furthermore, the observation
of hydrogen embrittlement in carbon steel coupons in the
electrochemical cells, and the closed examination of the
catalytic effect of a terminal electron acceptor on the
activity of bacteria during utilization of cathodic hydrogen
at the onset of corrosion suggested a new microbial corrosion
mechanism. In general, this research brings about a new
approach to the investigation of microbial corrosion and the
concepts developed herein provide better insight to the
overall understanding of this pervasive and costly problem.
Some detailed conclusions follow:
1) The flowthrough bioreactor reproduced anaerobic corrosion
of carbon steel. Of particular interest was the formation of
the biofilm that involved the formation of a double layered
structure.
2) The batch bioreactor was able to reproduce microbial
corrosion within 24 hours and resulted in a superior system to
study bacterial utilization of cathodic hydrogen at its onset,
of particular interest were: a) the head space pressure drop
measurements that suggested microbial metabolism of nitrogen,
hydrogen and methane, b) a black FeS precipitate formed on the

167
surface of the metal and in the bulk of the liquid as a result
of sulphate reduction by Desulfovibrio desulfuricans and the
corresponding increase of phosphate contained in the media.
3) Potential-time curves results afford strong evidence
regarding the influence of bacteria in the deterioration of
carbon steel.
4) The differential corrosion current measurements
corroborated previous reults of potential-time curves.
5) SA106 steel is susceptible to hydrogen embrittlement.
6) The use of fumarate as terminal electron acceptor in the
electrochemical cell suggested an increased activity of the
bacteria. Differential corrosion current and dissolved iron
concentrations were at least doubled immediately after its
addition.
7) The concepts developed during this investigation were
included in a new anaerobic mechanism that descrives the onset
of microbial corrosion.

168
Suggested Future Research
The author suggest continuation of the research effort in
the following specific areas.
1) Bacteria triculture metabolism of the head-space gases
nitrogen, hydrogen and methane in rich and poor nutrient media
at basic, neutral, and acidic pH values.
2) The effect of terminal electron acceptors on potential-time
curves for SA106 steel in mineral solutions in the presence of
different cell concentrations and age of E. coli and their
hydrogenase mutants, including continuous culture.
3) Effect of terminal electron acceptors on the activity of
hydrogenase from dead cells of hydrogenase positive bacteria,
during the corrosion of SA106 steel in mineral solution.
4) Test of the triad cell provided with the following
instrumentation: a) 15 channel data acquisition system able to
measure pH, pressure, temperature, oxidation-reduction
potential, potential, and current; b) a rigid triad flask
glass cell; c) an IBM compatible computer with a hard drive;
d) a mechanical device that allows the raising and lowering of
the metal coupons in the electrolyte; e) precise liquid and
gas sampling ports; f) leak proof system to assure anaerobic
conditions throughout the experiments; and g) a larger water
bath with temperature control.
5) Effect of phosphate in the microbial corrosion of SA106

169
steel in the presence of sulphate reducing bacteria and
emphasis on the formation of the black precipitate.
6) Effect of metallic microstructure on the production of
cathodic hydrogen.
7) Effect of grain boundaries on bacterial adhesion.
8) Hydrogen embrittlement of SA106 steel in mineral solution.
9) Effects of head space changes in the triad cell, using
similar and dissimilar metals.
10) Correlation of differential corrosion current, hydrogen
uptake, succinate formation, total corrosion and potential
changes in the triad cell using SA106 steel and a hydrogenase
positive bacteria.
11) Item 10 using Mg.

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BIOGRAPHICAL SKETCH
Jose Rafael Sifontes Garcia was born February 16, 1949,
in Santa Rosa, Anzoategui, Venezuela, to Mr. and Mrs. Pedro
Rafael Sifontes as the first born of two sons. Since early in
school he has shown to be an exceptionally gifted person.
From primary school to high school, he demonstrated his
scholastic excellence by achieving the #1 rank in all his
classes, receiving also the highest high school award during
his graduation in 1967 from Pedro Emilio Coll High School,
Caracas, Venezuela, for having attained the highest academic
performance. The same year he was awarded the Mobil Oil Co.
National Fellowship to study abroad, but he could not accept
it because of family sentimental values. He started his
university career at the Universidad Central de Venezuela,
where he advanced for two years until the 1969 political
turmoil that led the government to close down the institution.
Mobil Oil Co. offered the fellowship again, and he transferred
to the University of Kansas (KU), as an advanced student, to
continue his studies in chemical engineering. In four years
at KU he learned English and obtained bachelor's degrees in
chemical engineering and in chemistry, and a Master of Science
in Petroleum Engineering with a minor in business
administration. During his stay at KU, he also was on the
182

183
dean's list, participated in the leadership of the AICHE, and
was recognized by the Kansas Geological Survey. Upon
completion of his education he started working for Mobil Oil
Co. El Palito refinery in Venezuela in 1974. Months later he
also started teaching at the University of Carabobo during the
evenings. In 1978 he was offered a management position with
the Dowell Schlumberger Industrial Services Division. He
advanced positions rapidly in those organizations: at Mobil he
advanced from process to planning engineer; at Dowell from
sales engineer to national sales manager; and at the
University of Carabobo from part-time instructor to department
chairman and full professor. During all this time, his
involvement with industry, community, and concern for the
environment awoke his desire to pursue a multidisciplinary
education to help understand and solve the challenging
problems of our times.
During his high school years, Jose started competing in
athletics, baseball and soccer. He continue playing soccer
after high school and joined a professional soccer team in
Caracas. He also played the guitar for the high school group
Estudiantina del Colegio San Antonio. He was a member of the
Christian group Juventud Católica, which interacted with the
community in sports and musical events during Christmas.
While in the USA, he became involved in sports at KU, where he
learned and represented the university in judo, fencing, and
ping pong. He also practiced tennis, volley ball, and golf

184
for fun. He work for the KU police department under the
supervision of Mr. Ian Davis and joined student organizations
at KU and held leadership positions in AICHE.
While working at the Mobil Oil Co. refinery, he played
soccer and tennis, representing also the Universidad de
Carabobo. While teaching, he became aware of environmental
issues. Among his contributions were development of the
course Environmental Conservation for Chemical Engineers and
help with the initiation of a national environmental movement,
in accord with professors in other Venezuelan universities.
This movement produced the Environmental Federation, which
later led to the approval of the actual environmental laws for
air and water in Venezuela. It was after visiting the
University of Florida in 1981 that he decided to continue his
education. Since his arrival at the University of Florida, he
has polished and matured his knowledge and experience in life,
proving his outstanding scholastic record and his admirable
example of service and leadership in and out of the
university. He has graduated twice from the Environmental
Engineering Department, receiving M.E. and B.S. degrees with
honors, and from the Materials Science and Engineering
Department, receiving an M.E. degree. In June 1989, he
started pursuing the Doctor of Philosophy degree in the
Agricultural Engineering Department. He concentrated his
course work and research in the area of microbial corrosion.
His project work included the development and implementation

185
of a triad flask electrochemical cell to measure the
biological effect in the corrosion process and a new microbial
corrosion model. His overall graduate GPA is 3.6.
Among his contributions to the University of Florida, he
has served in the Mentor Program as peer mentor, on the Silver
and Gold Committee representing Golden Key National Honor
Society, in the SECME Olympiad as a UF representative, in the
Environmental Graduate Student Association as social director
and treasurer, with the Affirmative Action Round-table as
student representative, in the Hispanic Assembly as
ambassador, and in the Hispanic Engineering Society as
Engineering Fair chairman. Among his most recent awards are
the 1992 National Bill Nuanes scholarship from the Society of
Hispanic Professional Engineers; 1992 and 1993 UF Presidential
Recognition; 1992 Minority Student Recognition; 1991 and 1992
Florida College Student of the Year recognition; 1992 and 1993
Who's Who Among Students in American Universities and
Colleges; 1991, 1992, and 1993 Highest Honors awards from the
Southeastern Bible Institute. Jose is also a member of the
following campus honorary organizations: Omicron Delta Kappa,
the National Leadership Honor Society; Alpha Sigma Mu, the
International Metallurgical Honor Society; Alpha Epsilon, the
Honor Society of Agricultural Engineering; Golden Key National
Honor Society; Gamma Sigma Delta, the Honor Society of
Agriculture; and Tau Beta Pi, the National Engineering Honor
Society.

I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the deqree of Doctor of Philosophy.
David P. Chynoveth, Chairman
Professor of Agricultural
Engineering
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.
Y f‘
Henry
Aldrich
Professor of Microbiology and
Cell Science
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.
Edward P. Lincoln
Asociate Professor of
Agricultural Engineering
I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.
Roger7 A. Nordstedt
Professor of Agricultural
Engineering

I certify that I have read this study and that in my
opinion it conforms to acceptable standards of scholarly
presentation and is fully adequate, in scope and quality, as
a dissertation for the degree of Doctor of Philosophy.
Mark E. Orazeja
Professor orChemical
Engineering
This dissertation was submitted to the Graduate Faculty
of the College of Engineering and to the Graduate School and
was accepted as partial fulfillment of the requirements for
the degree of Doctor of Philosophy.
August 1994
Winfred M. Phillips
Dean, College of Engineering
Karen A. Holbrook
Dean, Graduate School

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INGEST IEID ELG8EIP8W_EORM7U INGEST_TIME 2011-09-29T20:31:19Z PACKAGE AA00004727_00001
AGREEMENT_INFO ACCOUNT UF PROJECT UFDC
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