Title Page
 Table of Contents
 List of Tables
 List of Figures
 Literature review
 Materials and methods
 Conclusions and recommendation...
 Appendix A: Raw data
 Appendix B: Steady state resul...
 Appendix C: Miscellaneous tables...
 Biographical sketch

Title: Kinetics of swine waste assimilation by phototrophic sulfur bacteria /
Full Citation
Permanent Link: http://ufdc.ufl.edu/UF00097406/00001
 Material Information
Title: Kinetics of swine waste assimilation by phototrophic sulfur bacteria /
Physical Description: xiv, 229 leaves : ill. ; 28 cm.
Language: English
Creator: Earle, Jonathan F. K., 1940-
Publication Date: 1985
Copyright Date: 1985
Subject: Sewage -- Purification   ( lcsh )
Swine   ( lcsh )
Animal waste   ( lcsh )
Sulfur bacteria   ( lcsh )
Photosynthetic bacteria   ( lcsh )
Environmental Engineering Sciences thesis Ph. D
Dissertations, Academic -- Environmental Engineering Sciences -- UF
Genre: bibliography   ( marcgt )
non-fiction   ( marcgt )
Thesis: Thesis (Ph. D.)--University of Florida, 1985.
Bibliography: Bibliography: leaves 209-227.
Additional Physical Form: Also available on World Wide Web
General Note: Typescript.
General Note: Vita.
Statement of Responsibility: by Jonathan F. K. Earle.
 Record Information
Bibliographic ID: UF00097406
Volume ID: VID00001
Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
Resource Identifier: alephbibnum - 000869778
notis - AEG6832
oclc - 014393184


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Table of Contents
    Title Page
        Page i
        Page i-a
        Page ii
        Page iii
        Page iv
    Table of Contents
        Page v
        Page vi
        Page vii
    List of Tables
        Page viii
        Page ix
    List of Figures
        Page x
        Page xi
        Page xii
        Page xiii
        Page xiv
        Page 1
        Page 2
        Page 3
        Page 4
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    Literature review
        Page 10
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    Materials and methods
        Page 75
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    Conclusions and recommendations
        Page 148
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    Appendix A: Raw data
        Page 151
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    Appendix B: Steady state results
        Page 197
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    Appendix C: Miscellaneous tables of results
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    Biographical sketch
        Page 228
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Full Text







This dissertation is dedicated to the loving memory of my
parents, Stanford and Eunice Earle, whose vision, sacrifice,
and guidance during my formative years were instrumental in
the accomplishment of this work.


Sincere appreciation is expressed to my committee

chairman Dr. Ben Koopman for his guidance and encouragement

from the very outset of my studies at this University and

throughout this series of investigations. His keen sense of

observation and attention to detail contributed greatly to

the successful completion of this undertaking. Thanks are

also extended to committee cochairman Dr. Edward Lincoln

whose extensive knowledge of photosynthetic systems was

always available to be tapped; to committee member Dr. John

Zoltek Jr. for his friendship, encouragement, and support

during my tenure at Black Hall, and for his guidance during

my studies; to committee member Dr. Roger Nordstedt, an

authority on anaerobic systems, whose knowledge, personal

library, and laboratory facilities were made freely avail-

able to me during these studies; to committee member

Dr. Glen Smerage whose comments and detailed critique have

greatly enhanced the quality of this document.

My wife Yvonne and my children Kevin, Celia, and Jeremy

sacrificed greatly in enabling me to achieve a lifetime

ambition. Their love and understanding provided the motiva-

tional force for the undertaking and completion of this

endeavour, and for this I am extremely grateful. My siblings


have been towers of strength to me throughout my studies,

providing unselfish support at critical times. My col-

leagues in the firm of Earle & Associates Limited have been

extremely generous to me throughout this period, and this

has been greatly appreciated.

Finally, appreciation is expressed to my fellow

travellers through Black Hall who have helped to make this

journey so pleasant. Laboratory partners Chang-Won Kim, Ho

Kang, Sang-Ill Lee, and Chan-Won Kim; also Robert Ryczak,

Lisa Drinkwater, Rick Meston, and Joe Angley have each,

in some way, contributed to the achievement of this goal.

Appreciation is also expressed to Dane Bernis of the Swine

Research Unit, Veronica Campbell for her assistance with

laboratory analyses, Susan Scherer for preparing the

drawings, and Barbara Smerage for editing, final typing, and

compilation of this document. I would also like to thank

the office staff at Black Hall, especially Eleanor Humphreys

and Jo David, for the very efficient manner in which they

have dealt with my affairs.



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

LIST OF TABLES....................................... viii

LIST OF FIGURES...................................... x

ABSTRACT............................................. xiii

CHAPTER 1. INTRODUCTION............................. 1

1.1 Problems Created by Animal Wastes.......... 1
1.2 Waste Management Considerations............ 3
1.3 Anaerobic Processing of Animal Wastes...... 4
1.4 Application of Phototrophic Sulfur Bacteria
to Waste Treatment Problems................ 6
1.5 Research Objectives......................... 9

CHAPTER 2. LITERATURE REVIEW........................ 10

2.1 Mechanism of Anaerobic Decomposition....... 10
2.2 Biochemistry of the Anaerobic Process...... 12
2.3 Microbiology of Anaerobic Environments..... 22
2.3.1 Anaerobic Microbial Community....... 22
2.3.2 The Nonphototrophic Anaerobes ...... 26 Hydrolytic Bacteria......... 26 Acetogenic Bacteria......... 28 Methanogenic Bacteria...... 30
2.3.3 The Phototrophic Anaerobes.......... 35 General Description........ 35 Classification ............. 36 Photometabolism............ 39 Energetics.................. 45 Ecology of Phototrophic
Bacteria.................... 53 Natural Occurrence and Role
in Waste Treatment Systems. 5C
2.4 Process Inhibition.......................... 59

2.5 Kinetics of the Anaerobic Process.......... 61
2.5.1 Basic Considerations................ 61
2.5.2 Relationship Between Microbial
Growth and Substrate Utilization
in Batch Culture .................... 63
2.5.3 Completely-Mixed Continuous Culture
Model Without Recycle............... 66 Microbial Growth........... 66 Substrate Utilization...... 70
2.5.4 Anaerobic Kinetic Models............ 70

CHAPTER 3. MATERIALS AND METHODS.................... 75

3.1 Rationale for Experimental Design........... 75
3.2 Summary of Investigations.................. 78
3.3 Experimental Apparatus..................... 78
3.4 Materials ................................... 81
3.4.1 Substrate ............................ 81
3.4.2 Bacterial Inocula................... 84 Phototrophs................ 84 Methanogens and Other
Anaerobes................... 84
3.5 Experimental Methods....................... 84
3.5.1 Start-up Batch Cultures............. 84
3.5.2 Continuous Mode...................... 86
3.6 Analytical Techniques...................... 88
3.6.1 Bacteriochlorophyll a............... 88
3.6 2 Sulfide .............................. 89
3.6.3 Protein............................. 90
3.6.4 BOD COD, TS, VS and TSS........... 90
3.6.5 Kjeldahl-N, NH3-N and Total P........ 91
3.6.6 pH.................................. 91
3.6.7 Absorbance .......................... 91
3.5.8 Gas Quantity and Quality............ 91

CHAPTER 4. RESULTS.................................. 94

4.1 Identification of Phototrophic Bacteria.... 94
4.2 Temporal Variation of Phototrophic
Bacterial Population, Gas Production
and pH During Experimental Trials.......... 95
4.2.1 Experimental Series................. 95
4.2.2 Batch/Continuous Mode Trials........ 96 5-d SRT ..................... 96 7-d SRT ..................... 98 10-d SRT .................... 98 15-d SRT... ................ 103 20-d SRT. .................. 103 30-d SRT.................... 106

4.2.3 Continuous/Continuous Mode Trials... 109 8.5-d SRT................... 109 15-d SRT. .................. 110 30-d SRT. .................. 110
4.3 Growth Characteristics of
Phototrophic Bacteria...................... 110
4.3.1 Batch Growth Characteristics........ 110
4.3.2 Steady State Growth Kinetics......... 114
4.3.3 Biomass Productivity................ 120
4.4 Waste Conversion ........................... 124
4.4.1 Gas Production and Quality........... 124
4.4.2 Oxygen Demand........................ 130
4.4.3 Nitrogen and Phosphorus............. 133

CHAPTER 5. DISCUSSION............................... 136

5.1 Substrate Characteristics.................. 136
5.2 Bacterial Species in Laboratory Cultures... 137
5.3 Impact of Phototrophs on the
Anaerobic Digestion Process................. 140
5.3.1 Gas Quantity and Quality............. 140
5.3.2 Waste Treatment and Nutrient Uptake. 144
5.4 Kinetic Parameters and Mathematical Model.. 145
5.5 Application of Results to
Field Operations............................ 146


6.1 Conclusions ............................... 148
6.2 Recommendations for Further Research....... 150

APPENDIX A. RAW DATA ................................. 152

APPENDIX B. STEADY STATE RESULTS ..................... 198


REFERENCES........................................... 209

BIOGRAPHICAL SKETCH .................................. 228




Table 2-1.

Table 2-2.

Table 2-3.

Table 2-4.

Table 2-5.

Table 2-6.

Table 2-7.

Table 2-8.

Table 2-9.

Table 3-1.

Table 3-2.

Table 3-3.

Metabolic pattern of nonphotosynthetic
anaerobic bacteria.....................

Representative end products of
anaerobic microbial degradation of
organic wastes..........................

Identification of anaerobic bacterial
populations in sewage digesters........

Methanogenic bacteria isolated in pure
cultures from digesting sludge...........

Compounds utilized by methanogenic
bacteria as energy sources for methane

Role of compounds metabolized
by the phototrophic bacteria...........

Some organic compounds photoassimilated
by phototrophic bacteria...............

Bacterial energy budget for cells grown
on glucose.............................

Species of phototrophic bacteria
identified in waste treatment systems..

Composition of grower/finisher ration
used at the University of Florida's
Swine Research Unit during

Principal characteristics of swine
waste used as substrate in
investiga cions.........................

Operating conditions for chromato-
graphic analysis of gas samples........


Table 4-1.

Table 4-2.

Table 4-3.

Table 4-4.

Batch growth characteristics of
phototrophic sulfur bacteria cultured
in swine waste medium...................

Steady state gas production at STP
related to COD destroyed................

Steady state gas production at STP
related to volatile solids and COD

COD available for biomass synthesis....










Figure 2-3.

Figure 2-4.

Figure 2-5.





Figure 2-8.

Figure 2-9.

Figure 2-10

Figure 3-1.

Figure 4-1.

The anaerobic cycle in nature..........

Three-stage biochemical scheme for
anaerobic biodegradation..............

Nonphotosynthetic bacterial groups
involved in anaerobic biodegradation..

Interrelationships between methane
bacteria and metabolites of the
anaerobic carbon cycle.................

Approximate percentage distribution of
carbon in metabolic end products of
anaerobic biodegradation..............

The sulfur cycle in nature.............

The reductive tricarboxylic acid
cycle of green sulfur bacteria........

Simplified comparative illustrations
of oxygenic and anoxygenic

Scheme for photosynthetic NAD(P)
reduction in purple sulfur bacteria...

Schematic of completely-mixed
reactor without solids recycle.........

Schematic diagram of experimental

Temporal variation of bchl a, biogas
production, and pH during the 5-d SRT
trial, Series 1. ER = experimental
(illuminated) reactor, CR = control
(nonilluminated) reactor...............














Figure 4-2.

Figure 4-3.

Figure 4-4.

Figure 4-5.

Figure 4-6.

Figure 4-7.

Figure 4-8.

Figure 4-9.

Figure 4-10.

Figure 4-11.

Figure 4-12.

Figure 4-13.

Figure 4-14.

Figure 4-15.

Temporal variation or bchl a, biogas
production, and pH during the 7-d SRT
trial, Series 1........................

Temporal variation of bchl a, biogas
production, and pH during the 10-d SRT
trial, Series 1........................

Temporal variation of bchl a, biogas
production, and pH during the 10-d SRT
trial, Series 2 .......................

Temporal variation of bchl a, biogas
production, and pH during the 15-d SRT
trial, Series 1........................

Temporal variation of bchl a, biogas
production, and pH during the 20-d SRT
trial, Series 1........................

Temporal variation of bchl a, biogas
production, and pH during the 20-d SRT
trial, Series 2 .......................

Temporal variation of bchl a, biogas
production, and pH during the 30-d SRT
trial, Series 1........................

Temporal variation of bchl a, biogas
production, and pH during the 8.5-d
SRT trial, Series 2 ...................

Temporal variation of bchl a, biogas
production, and pH during the 15-d SRT
trial, Series 2.......................

Temporal variation of bchl a, biogas
production, and pH during the 30-d SRT
trial, Series 2.......................

Relationship of bchl a to solids
retention time ........................

Relationship of protein to solids
retention time.........................

Relationship of solids concentration
to solids retention time...............

Relationship of productivity in
term of bchl a and protein to
dilution rate..........................
















Figure 4-16.

Figure 4-17.

Figure 4-18.

Figure 4-19.

Figure 4-20.

Figure 4-21.

Figure 4-22.

Figure 5-1.

Relationship of productivity in
terms of total solids and volatile
solids to dilution rate...............

Relationship of productivity in
terms of total suspended solids
to dilution rate......................

Effect of solids retention time on gas
production and quality................

Effect of solids retention time on
methane production.....................

Soluble COD and soluble BOD removals
related to solids retention time......

Relationship of soluble Kjeldahl
nitrogen and soluble phosphorus
to solids retention time...............

Relationship of ammonia uptake to
solids retention time.................

Suggested schematic of bacterial
interactions during phototrophic
anaerobic degradation of organic
compounds .............................










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



Jonathan F. K. Earle

December 1985

Chairman: Ben Koopman
Cochairman: Edward P. Lincoln
Major Department: Environmental Engineering Sciences

The degree of waste treatment achieved in anaerobic

processes is usually much lower than that of aerobic

biological processes. However, anaerobic waste stabiliza-

tion is accompanied by the evolution of valuable gases as

end products. Use of anaerobic photobiological processes

for waste stabilization has not been exploited, although

their potential for such application has been recognized for

some time.

Among the metabolic end products of anaerobic

processes, are the gases hydrogen, hydrogen sulfide, and

ammonia. Several of these potentially toxic end products

are readily assimilated by phototrophic bacteria under

suitable conditions.

In a series of bench-scale continuous culture studies,

the anaerobic processing of swine waste under illuminated


conditions was investigated. Two daily fed, completely-

mixed 4.0 L reactors, one illuminated (ER) and the other

nonilluminated (CR), were inoculated with phototrophic

bacterial culture and operated at retention times of 5, 7,

8.5, 10, 15, 20, and 30 days until achievement of steady-

state conditions or washout of the phototrophic bacteria had

occurred. All experiments were conducted at a controlled

temperature of 27 + 1C, using waste with a volatile solids

concentration of 1.0 + 0.1 percent. Kinetic parameters were

determined for bacterial growth, biomass productivity, and

substrate uptake. The reaction rate coefficient was found

to be 0.200 L/g-day.

Presence of phototrophic bacteria enhanced the waste

treatment capability of the anaerobic digestion process,

achieving reductions of 84 to 90 percent in soluble COD

levels, and 66 to 74 percent in soluble Kjeldahl-N levels.

Uptake of ammonia-N and soluble P was 68 percent and 46

percent, respectively. Minimum biological solids retention

time was determined to lie between 8.5 and 10 days.

Specific methane yield was 0.173 L/L vol-d in the ER

and 0.181 L/L vol-d in the CR. The methane content of the

biogas from the ER ranged from 64 to 71 percent. Biomass

productivity and waste treatment were optimized at 15-days




1.1 Problems Created By Animal Wastes

Improper management of the high-strength wastes gener-

ated by agricultural operations, including feedlots, has

frequently resulted in the overloading and pollution of

rivers, streams, and lakes, with other adverse consequential

impacts on the environment. In reports submitted to the

Environmental Protection Agency (EPA) (182) in 1977, agri-

cultural sources accounted for 93 of 503 fish-kills. Of

this number, manure-silage drainage was directly implicated

in 32 incidents.

With the continued trend towards industrialization of

livestock farming, effective and efficient disposal of the

large volumes of highly concentrated waste generated under

confined conditions each day, therefore, becomes a problem

of major proportions (99,129,191). Traditionally, such

wastes have been disposed of by land-spreading to serve

as soil conditioner and fertilizer. The increased volume of

collectible waste generated under confined conditions,

coupled with the reduction in area available for

land-disposal, now makes this method unsuitable for such


In a 1978 survey (181), it was indicated that approxi-

mately 158 million metric tonnes of dry animal manure was

produced annually in the United States. Confined livestock

operations accounted for 61 million tonnes (39 percent). Of

this latter volume, swine operations contributed a total of

5.5 million tonnes (9 percent). The annually-produced

volume of waste contains approximately 7.0 million tonnes of

nitrogen, 1.7 million tonnes of phosphorus, and 3.8 million

tonnes of potassium, indicating the pollutional potential of

the waste.

The environmental impact of these wastes is evident,

not only in their observed effects on waterways, but also in

the atmosphere. Various gases are produced by microbial

degradation of stored wastes. These include hydrogen sulfi-

de (H2S), ammonia (NH3), carbon dioxide (C02), and methane

(CH4) (126,165). Of these gases, H2S and NH3 may be toxic

to both man and livestock and are also associated with odor

offensiveness. Odors have been observed to increase with

increasing concentrations of volatile fatty acids (VFA),

phenol, p-cresol and skatole (196). Other recorded

contributors to air pollution and offensive odor of animal

waste slurries include methanethiol, dimethyl sulfide,

diethyl sulfide, propyl acetate, n-butyl acetate, trime-

thylamine, and ethylamine (194).

1.2 Waste Management Considerations

Conventional techniques of municipal waste management

are not appropriate for the very highly concentrated live-

stock wastes which are encountered. Swine wastes may have

chemical oxygen demand (COD) values in excess of 80 000

mg/L, and biochemical oxygen demand (BOD5) values in excess

of 30 000 mg/L, compared with 350-450 mg/L, and 250-300

mg/L, respectively, for municipal waste. The options avail-

able for the management of livestock wastes are (1) utiliza-

tion and (2) treatment and disposal. Wastes may be used as

plant nutrients (157,195), as feed ingredients for farm

animals and fish (69,79,200,205), as a substrate for

microbial and insect protein synthesis (16,28,118,124), and

as a substrate for microbial methane production (51,85,168).

Waste treatment and disposal techniques are largely

dependent on the characteristics of the waste. These char-

acteristics are, in turn, influenced by animal-type, feed-

type, and method of confinement. Available waste treatment

and/or disposal techniques include composting (153,169),

dehydration and incineration (47), use of oxidation ditches

or other aeration processes (56,57), photosynthetic

reclamation (89,91,146,150), facultative and anaerobic

lagoons (62,125,193), and anaerobic digestion (168). Of

these, anaerobic processes are most commonly used in the

management of swine waste.

1.3 Anaerobic Processing of Animal Wastes

Application of anaerobic biotechnology to the stabili-

zation of organic solids and the treatment of highly concen-

trated liquid wastes has been investigated and implemented

for several years (70,148,201). Wide interest in the

development of this naturally-occurring stabilization

process for waste treatment was stimulated at the beginning

of the twentieth century. Early interest was in conven-

tional anaerobic digestion as a stabilization process for

sewage sludge and for the generation of methane gas. Cur-

rently, the technology is being investigated for general

application to waste treatment problems.

Because of the high total solids (TS) and BOD5 concen-

trations of swine waste, anaerobic digestion has been the

preferred method of processing where energy recovery in the

form of methane has been a prime consideration (63,72,199,

207). Similar production of energy does not occur in

aerobic waste treatment processes. Several studies on swine

waste digestion have been conducted (66,74,204), and the

literature contains numerous references to the advantages

and disadvantages of the process (19,33,73). The main

advantages of the process are production of a useful product

in the form of methane gas, and low levels of microbial

cells. Referenced disadvantages include high initial

capital outlay, high operation and maintenance costs, and


process instability (13,25,104). In addition, a high degree

of treatment usually is not achieved by this method.

The two types of digestion systems commonly in use

today are the conventional or standard rate digester

(71,105), which is used primarily for the stabilization of

thicker sludges, and the anaerobic filter or fixed bed

reactor (184,206)), used for the treatment of more dilute or

settled wastes. In standard rate digestion, the digester

may either be mixed or unmixed, heated or unheated, and it

usually is operated at retention times in excess of 10

days. Operating temperature may be within the mesophilic or

thermophilic range, but the majority of digesters are oper-

ated within the mesophilic range, usually at a temperature

of 35-370 C. Fixed bed reactors are packed with a solid

medium, such as wood chips, to which the bacterial cells

attach. Washout of cells is thereby minimized and conse-

quently liquid retention times may be reduced.

Because of the very long retention times which are

possible, anaerobic waste stabilization ponds, which have

been applied to the disposal of swine waste for several

years (62,125,193), provide operators with an inexpensive

but very effective alternative to anaerobic digestion. The

initial capital outlay required is low, and such stabiliza-

tion ponds are virtually free of operation and maintenance

costs. However, they suffer the disadvantage of being

odorous at times. Indications are that the odors emanating

from such lagoons can be eliminated by encouraging the deve-

lopment of phototrophic anaerobes (7).

1.4 Application of Phototrophic Sulfur Bacteria to
Waste Treatment Problems

Waste stabilization ponds receiving municipal, indus-

trial, or agricultural wastes, and exhibiting anaerobic

characteristics, are often distinctly colored by a large

population of phototrophic sulfur bacteria (39,40,110,116).

Successful efforts have been made to study and apply these

bacteria to the treatment of certain wastes under controlled

conditions. These bacteria are particularly useful in situ-

ations where wastes containing high levels of sulfide must

be treated. This is the case with fellmongery wastes

resulting from the unhairing of hides prior to tanning.

These wastes are highly saline, have pH levels of 12 to 13,

and contain sulfide concentrations ranging from 80 to over

400 mg/L. Other waste constituents include insoluble

organic, hydrosulfides, thiosulfates, and chlorides of

sodium, calcium, and ammonium, as well as free ammonia

(38). Effective treatment of these wastes has been achieved

in lagoon systems designed for utilization of purple sulfur

bacteria (111,112).

A variety of industrial wastes with high BOD5 levels

(2000 to greater than 10,000 mg/L) have been successfully

treated in photobiological treatment plants utilizing photo-

trophic bacteria and algae. These include wastes from the


starch, woolwashing, canned food, and pharmaceutical indus-

tries (88,90). It has been indicated that purple sulfur

bacteria produce substances which inactivate some animal and

human pathogenic viruses (88). Successful removal of amines

(putrescene and cadaverine) was also noted. Use of photo-

trophic bacteria in the treatment of hazardous wastes (86),

sewage sludge and cattle feedlot effluent (173), and the

effluent from anaerobic waste treatment systems has also

been reported (87).

In lagoons in which these microorganisms proliferate, a

marked reduction in odors has been noted (7,39,189). This

has been attributed to photosynthetic metabolism of the

phototrophic sulfur bacteria, in which H2S is used as elec-

tron donor for photosynthesis and consequently is oxidized

to elemental sulfur. The phototrophic sulfur bacteria are

divided into two groups, purple sulfur bacteria and green

sulfur bacteria (139). The majority of purple sulfur

bacteria store elemental sulfur internally, whereas the

green sulfur bacteria deposit sulfur externally. Studies on

lagoons treating organic industrial wastes (40,90,110) have

confirmed that, in addition to oxidizing inorganic sulfur

compounds, phototrophic sulfur bacteria utilize a number of

metabolic end products which would otherwise accumulate

under anaerobic conditions, with negative effects. Notable

among these are certain organic acids.


In the management of swine wastes, the coupling of

anaerobic digestion with accelerated photosynthetic systems

offers operators an attractive option for waste treatment

and fuel and feed production. The photosynthetic stage may

involve the use of phototrophic sulfur bacteria as detoxi-

fiers (88,90) followed by algal cultures. By their ability

to oxidize sulfide, the bacteria remove a primary toxicant

and thus condition the medium for growth of the algae.

Both the bacterial and algal cells may then be harvested

and used as a protein source. The protein content of the

bacterial cells is reported to be in excess of 70 percent


In a series of laboratory-scale batch studies, purple

sulfur bacteria were cultured in a swine waste medium (45).

From these studies it was concluded that the presence of

these microorganisms was advantageous to the anaerobic

treatment process. It is therefore suggested that the

design of waste treatment systems incorporating phototrophic

sulfur bacteria could result in enhanced treatment of highly

concentrated organic wastes. Before such design can be

undertaken, the kinetic parameters influencing growth and

substrate uptake by these bacteria, in mixed undefined cul-

ture as commonly observed in waste stabilization lagoons,

must be determined.

1.5 Research Objectives

This laboratory-scale research project was designed to

define the kinetic parameters which influence the growth

and substrate uptake of phototrophic sulfur bacteria. The

specific objectives were to

1. Conduct laboratory-scale, continuous culture

anaerobic studies with phototrophic sulfur

bacteria and determine their impact on waste


2. Determine the kinetic parameters which influence

the anaerobic degradation of swine waste by these

microorganisms, and the loading rate and retention

time required for optimum treatment.

3. Assess the effect of phototrophic sulfur bacteria

on biogasification in the anaerobic digestion


4. Develop a mathematical model for waste degradation

and biomass production in a waste treatment system

incorporating phototrophic sulfur bacteria.


2.1 Mechanism of Anaerobic Decomposition

In natural aquatic systems, the decay of organic matter

occurs either aerobically, in the presence of oxygen, or

anaerobically, in the absence of oxygen. These processes

are mediated by aerobic, facultative, or anaerobic microor-

ganisms which degrade the organic matter, producing new cell

mass, maintenance energy and stabilized end products. The

anaerobic cycle of decomposition in nature, with emphasis on

the elements carbon, nitrogen and sulfur, is illustrated in

Figure 2-1 (117). The stabilized end products of this cycle

are methane (CH4), carbon dioxide (C02), and humus.

In biological waste treatment systems, the environment

of microorganisms is controlled to achieve optimum metabolic

activity, resulting in maximum stabilization of organic

matter. In such systems, as well as in nature, this stabil-

ization is accomplished by a combination of two metabolic

processes, oxidation and synthesis. In aerobic processes,

dissolved oxygen is the ultimate hydrogen acceptor, whereas

in anaerobic processes the ultimate hydrogen acceptor may be

oxidized organic matter, nitrates, nitrites, sulfates or

carbon dioxide (C02).

N0 m



N -,






N L-
O c.



, E





> 0
-_ E


In an anaerobic environment, the various microbiologi-

cal and biochemical reactions which occur result in an

orderly and controlled degradation of the complex organic

materials present. Current understanding of the reactions

leads to the conceptual development of a staged sequential

process (17,19,27), in which organic biodegradation is

mediated by the coordinated metabolic activities of groups

of facultative and obligate anaerobic bacteria (18,114,

209). The end products of complete anaerobic metabolism are

the gaseous compounds methane (CH4) and CO2, together with a

relatively small amount of cell mass (18,114,209). Waste

stabilization is directly linked to methane production, the

theoretical methane production from Ikg ultimate BOD (BODL)

or COD being 0.348 m3 (105). In anaerobic ecosystems

exposed to light, bacterial photosynthesis may occur, with

resulting primary production of organic matter which becomes

an essential part of the microbial food chain (35,42).

2.2 Biochemistry of the Anaerobic Process

In a well-buffered, actively-operating anaerobic sys-

tem, degradation of complex organic may be conveniently

represented by the three-stage biochemical scheme (113,136)

illustrated in Figure 2.2 (113,135). The complex organic

comprising cellulose, hemicellulose, xylanes, and lignins,

together with proteins, lipids and nucleic acids, are

hydrolysed, fermented, and mineralized by at least four

CH4 + C02 CH4 CO2
4 C02

Figure 2-2. Three-stage biochemical scheme for
anaerobic biodegradation (113).

different groups of anaerobes, as shown schematically in

Figure 2.3 (209), and represented in Table 2-1. First stage

enzymatic hydrolysis of complex organic by inducible

peripheral enzymes leads to the production of a range of

intermediates, which are subsequently used for energy pro-

duction and growth by the various bacterial species present

in the environment. Representative end products of

anaerobic microbial degradation of organic wastes are listed

in Table 2-2.

Organic polymers such as cellulose, proteins and lipids

are first reduced to individual monomers which are then

fermented to organic acids, alcohols, CO2, hydrogen (H2),

acetate, longer chain fatty acids, ammonia (NH4 ), and
sulfide (S ) (4). The fermented end products are selec-

tively metabolized by the second and third groups of bac-

teria, which together are responsible for the activities of

the second stage. These are the obligate H2-producing and

the homoacetogenic bacteria (209). In this acid-forming

stage, the metabolic end products include organic acids,

aldehydes, alcohols, mercaptans, and amines. Also produced

are H2, CO2, H2S, and ammonia (NH4 ) (4,19). High molecular

weight compounds, such as lignin, are not readily

metabolized by anaerobic bacteria. Stabilization of wastes

containing cellulose surrounded by polymeric lignin is

therefore restricted. Increased anaerobic degradation can,

however, be achieved by physico-chemical pretreatment which


Hydrolytic and

SLonger-chain Fatty Acids, Organic Acids5
Alcohol, NH4, S2-,CO2 I

H2- Producing

Acetate! H2 H CO2

H2-Utilizi ng

Acetate-Utilizing H2 CO2 Utilizing
Methanogenic Methanogenic
Bacteria Bacteria

-- I
LCH4* CO2 I iCH4 CO2"
L-J ---- ----

Figure 2-3. Nonphotosynthetic bacterial groups
involved in anaerobic biodegradation

Table 2-1. Metabolic patterns of nonphotosynthetic
anaerobic bacteria.

Bacterial Metabolic Substrates utilized Metabolic end
Group products

1. Hydrolytic and Polysaccharides Acetic acid, H2/CO2,
fermentative Lipids buyrate, propionate,
bacteria Proteins methanol, ethanol,

2. Hydrogen- butyrate, hydrogen,
producing propionate acetate
acetogenic ethanol, propanol

3. Homoacetogenic multi- or one acetic acid
bacteria carbon compounds

4. Methanogenic acetate, H2/CO2, methane,
bacteria methanol CO2, H20
carbon monoxide

Source: Zeikus 1980 (209).

Table 2-2. Representative end products of anaerobic
microbial degradation of organic wastes.

End products

Proteins and other
nitrogenous compounds


Fats and related

Nucleic acids, purines,

Amino acids
Hydrogen sulfide
Organic acids

Fatty acids
Neutral compounds

Fatty acids
Lower fatty acids

Amino acids
Lower fatty acids


separates lignin from cellulose or solubilizes the lignin

into digestible substrates. The large quantities of

ethanol and acetic, formic, and lactic acids produced by

acid-forming bacteria become toxic if allowed to accumulate

(209), resulting in inhibition of the anaerobic stabiliza-

tion process. This is prevented by the metabolic activities

of terminal trophic groups which transform the metabolites

of the first two stages. Principal species are the methano-

genic and sulfatereducing bacteria which utilize H2,

one-carbon substrates formate and methanol), and two-carbon

substrates (acetate) as energy sources or electron donors.

Phototrophic bacteria may also be included with the terminal

organisms in anaerobic environments.

In ecosystems containing low sulfate concentration, the

terminal stage of anaerobic degradation is controlled by the

activities of methanogenic bacteria (208) which utilize

the acetate, H2 and CO2 produced in the earlier stages to

form methane and carbon dioxide (101,122,123). Estimates

vary as to the relative importance of these substrates

(162). The interrelationship between methanogenic bacteria

and substances of the anaerobic carbon cycle is shown in

Figure 2-4 (145), and the approximate percentage distribu-

tion of carbon in the metabolic end products of all stages

is shown in Figure 2-5.

Single-carbon compounds are readily metabolized in the

anaerobic environment. Three principal bacterial groups

0 0
, U
-- E --- --- ----u


u u

c J
0 I

o _

o 0r

0 I
>, r

i 00



-. a

o e'

r,4 ,

nq a

j u N

Figure 2-5. Approximate percentage distribution of
carbon in metabolic end products of
anaerobic biodegradation (162).


consume these substrates as energy sources. These are the

methanogens, the sulfate reducers (29) and the homo- or

H2-consuming acetogens. Phototrophic sulfur bacteria

utilize H2 as an electron donor and certain of the other

substrates, notably acetate, as carbon source (140).

The sulfate-reducing bacteria will outcompete the meth-

anogens for common energy sources when sulfate is in excess

(29), as they have been shown to possess hydrogen metabolism

activity with more favorable kinetic properties (lower K
and higher V ) (149). In such environments, the final
stages of conversion of organic molecules into CO2 will be

primarily dependent on these organisms. It has been demons-

trated (143) that certain species of the sulfate-reducing

bacteria will oxidize long- and short-chain fatty acids and

some aromatic compounds to CO2. For these organisms, ele-

mental sulfur can also act as electron acceptor in place

of sulfate (143).

The principal pathway of methane production is

dependent on the anaerobic environment. In the rumen,

methane is primarily produced through reduction of CO2 by H2

(76). In sludge fermentation, most of the methane is formed

from acetate (162), although format is also used by some of

these microorganisms. In the latter environment, lipids

comprise approximately 28 percent of the organic compounds,

and the lipid fraction was found to be primarily respon-

sible for production of acetate from which most of the

methane is formed. This correlates with the high degree of

lipid degradation, 65.2 to 90.3 percent, reported by several

investigators (34).

In illuminated anaerobic environments, phototrophic

anaerobes also play a significant role in the degradative

process (139). This they do through the assimilatory

metabolism of several of the intermediary metabolites pro-

duced by the first three trophic groups above (139).

Their metabolic activities also effectively remove H2S, a

potent toxicant, from the environment. Like methanogens,

phototrophic bacteria are terminal organisms and, under

suitable environmental conditions, will be in direct compe-

tition with the former for certain substrates, primarily

acetate, H2 and CO2.

The trophic divisions and metabolic stages outlined

above are not rigidly defined. Certain bacteria bypass the

intermediary fermentative stage and metabolize carbohydrates

directly to acetate + H2 in the presence of an H2-scavenging

bacterium (113).

2.3 Microbiology of Anaerobic Environments

2.3.1 Anaerobic Microbial Community

Species composition and methane production within an

anaerobic environment are greatly influenced by the charac-

teristics of the organic substrate and environmental fac-

tors such as pH, light, temperature and oxygen tension. The

bacterial population of such ecosystems may be conveniently

divided into two groups based upon their energy metabolism:

(1) nonphotosynthetic anaerobes and (2) photosynthetic anae-

robes. The nonphotosynthetic anaerobes include the hydro-

lytic and fermentative, acetogenic, methanogenic, and

sulfate-reducing bacteria. The photosynthetic anaerobes

include species of the families Rhodospirillaceae,

Chromatiaceae, Chlorobiaceae and Chloroflexaceae.

Identification of the various bacterial species encoun-

tered in these environments has been based on the isolation,

characterization, and enumeration of predominant microbial

populations of bottom muds, anaerobic sludge digesters (103-

105), animal manure digesters (74,77), gastrointestinal

tracts, and the rumen of cud-chewing animals (77,202).

Principal bacterial groups identified are the hydrolytic,

acetogenic, methanogenic, phototrophic, and sulfate reducing

bacteria (122).

Provided that light can penetrate the anaerobic

environment, microorganisms which exist therein are able to

achieve an almost completely closed anaerobic cycle of

matter, by their ability to metabolize the waste products

generated in their ecosystem. Primary synthesis of organic

matter under these conditions is mediated by the photo-

trophic sulfur bacteria which convert CO2 to cell material

using H2S as reductant (139). Acetate and other simple

organic compounds are readily assimilated by phototrophic


nonsulfur bacteria (140), and certain species of photo-

trophic sulfur bacteria. In some environments, these photo-

trophic cells are grazed by protozoans. Also, upon death of

the phototrophic bacteria, their organic cell components are

decomposed by Clostridia and other fermentative anaerobes,

with the formation of C02, H2, NH3, organic acids and

alcohols (208). The H2 and some of the other fermentative

products are anaerobically oxidized by sulfate-reducing and

methane-producing bacteria.

The sulfide on which the phototrophic sulfur bacteria

are dependent for metabolism is produced by the reduction of

sulfate and/or the breakdown of proteins into amino acids

and subsequent degradation of the amino acids cysteine,

cystine and methionine. Proteolytic bacteria responsible

for protein degradation include Proteus, Bacteroides spp.

and some Clostridium spp. Most of the sulfide is produced

by the sulfate-reducing bacterial species Desulfovibrio.

Anaerobic oxidation performed by sulfate reducers results

in the formation of H2S and acetate, both utilizable in turn

by phototrophic bacteria. The combined activities of sul-

fate-reducing bacteria and phototrophic bacteria are

reflected in the completely closed sulfur cycle illustrated

in Figure 2.6. In anaerobic environments, the nitrogen

cycle is also closed as the nitrogen atom does not undergo

valence changes but alternates between NH3 and the amino

groups (R-NH2) in nitrogenous cell material.

0 o








I <


I r







2.3.2 The Nonphototrophic Anaerobes Hydrolytic Bacteria

Hydrolytic bacteria may be Gram-negative or Gram-posi-

tive, non-spore-forming or endospore-forming, facultative

or obligately anaerobic rods or cocci. They are responsible

for initiating the anaerobic degradation of complex organic

molecules in the first stage of anaerobic digestion. They

produce extracellular or membrane-bound hydrolytic enzymes

which hydrolyse polymers of carbohydrates, proteins and,

lipids to their soluble monomers which are subsequently

fermented to various end products (24). These bacteria are

usually coupled to H2-utilizing bacteria. Representative

genera and species which have been identified are listed in

Table 2.3 (209).

Distribution of hydrolytic bacteria has been examined

by plate counts, and their population has been shown to

be highest near the sediment/water interface where the rate

of exoenzyme activity correlates with the counts of

exoenzyme-producing bacteria (93). The activities of

amylase, protease, lipase, and glucosidase in the surface

layer of sediment was found to be several orders of magni-

tude greater than in the water column (81). Bacterial

species identified include the proteolytic Clostridium spp.,

Streptococcus spp. and Eubacterium spp. (19).

Table 2-3. Identification of anaerobic bacterial
populations in sewage sludge digesters.


Generic identity and description

Hydrolytic bacteria

acetogenic bacteria



Sulfate reducers

Majority unidentified Gram-
negative rods;

Unidentified Gram-negative rods.




Source: Zeikus 1980 (209).

Because of the position of hydrolytic bacteria in the

sequentially staged biodegradative process, it has been

observed (209) that the rate of methane production in

anaerobic digesters is often limited by the rate of bio-

polymer destruction by these bacteria. The Acetogenic Bacteria

The microbial groups central to anaerobic activity

comprise the H2-producing acetogenic bacteria and the H2-u-

tilizing (homo-acetogenic) bacteria (209) which convert

fatty acids and other compounds to acetate, H2, and CO2.

In order for them to do this, the hydrogen concentration

must be kept very low by the methanogens and other

H2-utilizing organisms (128). Only a few species of H2-

producing acetogenic bacteria have been isolated. This

group degrades propionate and longer-chain fatty acids,

alcohols, aromatic and other organic acids which are pro-

duced in the first stage of fermentation (20).

Indications are that some of these organisms can only

be cultured in the presence of hydrogen-metabolizing species

(203). Included in this group is the "S organism" isolated

from Methanobacillus omelianskii by Bryant and his coworkers

(21), which catabolizes ethanol to acetate + H2. The H2 is

used by M. omelianskii to reduce CO2 to CH4 in this

syntrophic relationship. Other examples of obligate hydro-

gen-producing acetogenic bacteria which can metabolize only

in the presence of H2-scavenging bacteria are Syntropho-

bacter wolinii (14) which will oxidize propionate to acetate

+ H2 only if coupled with a H2-utilizing organism such as a

methanogen or a sulfate-reducing bacterium, and Syntropho-

monas wolfei (115) which metabolizes fatty acids of chain

lengths up to C8 by 6-oxidation when cocultured with a

H2-utilizing organism. Fatty acids with even numbers of

carbon atoms such as butyrate, caproate, and caprylate are

oxidized to acetate + H2 by this bacterium, and those with

an odd number of carbon atoms such as valerate and heptano-

ate are oxidized to acetate + propionate + H2 (115).

Strains of Desulfovibrio desulfuricans and Desulfovibrio

vulgaris produce H2 from lactate or ethanol when grown

without sulfate in the presence of H2-utilizing methanogens

(20). Lactate is degraded to acetate in the following


CH CHOHCOO- + 2H20 --------> CH COO- + HCO3-

+ H+ + 2H2 (2-1)

Homoacetogenic or hydrogen-consuming acetogenic

bacteria have been identified in sewage sludge (15,127).

Species of Acetobacterium and Clostridium which are able to

metabolize H2+CO2, methanol, and/or multicarbon compounds to

acetate (6), have been identified. Not all species of

Clostridium, however, are capable of metabolizing H2 (209).

By consuming H2, these bacteria lower the partial pressure

of hydrogen in the anaerobic environment sufficiently for

other metabolic activities to be continued. Methanogenic Bacteria

Methanogens are a diverse group of bacteria with con-

siderable variation in nutritional requirements (6). They

are strictly anaerobic bacteria which are not able to

catabolize alcohols other than methanol or organic acids

other than acetate and format (21). In general, they meta-

bolize one- and two-carbon compounds, utilizing H2, CO2, and

acetate in the production of CH4 and CO2.

Over 12 genera and several dozen species and strains of

these bacteria have been described (102). They are broadly

classified into two groups, one of which ferments acetic

acid to CH4 and CO2, while the other produces CH4 by reduc-

ing CO2, utilizing H2 or format (208). The principal users

of acetate are the genera Methanosarcina and Methanothrix

(161), which produce methane by cleaving the acetate mole-

cule with the formation of CH4 from the CH3-group and CO2

from the carboxyl group.

As illustrated in Figure 2-5, acetate accounts for

about 70 percent of the methane produced in digesters or in

nature (162). Species of methanogenic bacteria which have

been isolated from digesting sludge are listed in Table 2.4

(201). These organisms include the Methanobacterium species

N -l
O (
u e
= 44



0 )

+ E





0 0






+ E



- i





















which have minimal nutrient requirements and grow autotroph-

ically on H2+CO2 with sulfide and ammonia as sole sources of

sulfur and nitrogen, respectively.

Methanogens are terminal organisms in the sequential

anaerobic degradative chain. They are nutritionally simple

(104,208) and utilize potentially toxic compounds produced

from anaerobic fermentation or respiration of organic

material. Most of these bacteria use H2 as an energy source

in reducing CO2 to CH4 and influence the carbon and electron

flow in anaerobic habitats by an interaction termed inter-

species hydrogen transfer. Compounds utilized by methano-

genic bacteria as energy sources for methane production are

listed in Table 2.5. Methanogens are primarily autotrophic,

but the rumen species Methanobacterium ruminantium and

Methanobacterium mobile require various fatty acids, amino

acids, co-factors and B-vitamins for growth (104,208).

All species, except Methanobacterium arbophilicum,

Methanobacterium thermoautrophicum, and Methanobacterium

barkeri, in addition to utilizing H2 in reducing CO2 to CH4'

will also utilize format as an energy source for this

purpose (104,208). Methanosarcina barkeri is the only known

species which will form methane from both methanol and

acetate (104,208). Ammonia is generally used as the nitro-

gen source, phosphate as the source of phosphorus, and

sulfide or cysteine as the source of sulfur (102).

Table 2-5.

Compounds utilized by Methanogenic bacteria
as energy sources for methane production.


Methanogenic species

H /CO or

Methanobacterium formicicum
Methanobacterium thermoautotrophicum
Methanobacterium ruminantium
Methanobacterium mobile
Methanosarcina barkeri
Methanococcus vanniellii


Methanobacterium soehngenii
Methanosarcina methanica
Methanosarcina barkeri
Methanococcus mazei

Methanosarcina methanica

Methanosarcina barkeri

Methanosarcina barkeri



Source: Mah and Smith

1981 (102).

Methanogens are extremely sensitive to environmental

factors such as oxygen (02) and require a highly reduced

environment for growth, the required oxidation/reduction

potential (Ec) being in the range -520 to -530 mV (43). For

swine waste studied under minimal aeration, it was found

that methane production ceased at Ec of -360 mV. The

required pH range for methane production is 6.6-7.6 with

an optimum of 7.0-7.2, but acid production will continue

to pH 4.5 (106). At pH > 7.4, ammonia in the form of NH3 is

considered inhibitory at concentrations of 1500-3000 mg/L,

and above 3000 mg/L the ammonium ion becomes toxic regard-

less of pH (93,107). It has, however, been reported (37)

that under certain conditions, methane may be produced at

ammonia concentrations in excess of 3000 mg/L.

Two optimum levels of temperature have been established

for methane production, 35-400 C in the mesophilic range,

and 55-60 C in the thermophilic range. Sulfides can be

extremely toxic, but concentrations of up to 200 mg/L may be

tolerated by methanogens with some acclimation (95).

Operational stability of a methane-producing system

is also largely dependent on the buffering of the system.

The bicarbonate buffering capacity of a system will be

influenced by the protein content of the substrate as well

as the amount of CO2 produced. Biodegradable protein is

deaminated to produce ammonia which reacts with water as


NH3 + HOH ------> NH4+ + OH- (2-2)

The OH- reacts with CO2 in water to form bicarbonate ions

CO2 + HOH ----> H2CO3 ---> H' + HCO3 (2-3)

2CO3 + OH- ----> HCO 3- + HOH (2-4)

2.3.3 The Phototrophic Anaerobes General Description

Phototrophic anaerobes are a physiologically diverse

group of Gram-negative aquatic bacteria which perform an

anoxygenic type of photosynthesis under anaerobic condi-

tions, using only one photosystem (142). These anaerobes,

which may be broadly divided into two groups, the

phototrophic sulfur bacteria, and the phototrophic nonsulfur

bacteria, contain photosynthetic pigments of the bacterio-

chlorophyll type, and typical carotenoid pigments (139).

Phototrophic sulfur bacteria are obligate anaerobes which

are dependent upon the presence of oxidizable external

electron donors such as reduced sulfur compounds, molecular

hydrogen, or organic carbon compounds, primarily acetate,

for their metabolism (138). Assimilatory sulfate reduction

is lacking in these organisms and sulfide is required as a

source of reduced sulfur for biosynthesis (140). All

species contain cytochromes, ubiquinones, and nonheme iron

proteins as components of their electron transport systems

(140). Classification

In the classification of phototrophic bacteria, Order

Rhodospirillales is divided into two sub-orders, Rhodo-

spirillineae, and Chlorobiineae (141).

Suborder Rhodospirillineae is characterized by those

bacteria which contain bacteriochlorophyll a or b as the

major bacteriochlorophyll, and carry their photopigments

in intracytoplasmic membrane systems continuous with the

cytoplasmic membrane. This suborder contains the two

families Rhodospirillaceae (purple nonsulfur bacteria), and

Chromatiaceae (purple sulfur bacteria).

Rhodospirillaceae are facultative heterotrophs, in

addition to being phototrophs. They do not grow well in a

sulfur or sulfide-containing environment and are generally

unable to use hydrogen sulfide (188). The single exception

is Rhodopseudomonas sulfidophila which grows well with

sulfide (61). They are unicellular, Gram-negative,

straight-, curved-, or helical-rods which are usually non-

gas-vacuolated. They have well defined guanine plus

cytosine (G + C) ratios ranging from 61-70 percent and are

flagellated when motile (10,178). This family primarily uses

simple organic compounds such as alcohols and acids which

act as electron donors and are photoassimilated (140).

All species of the family Chromatiaceae are capable of

photolithotrophic CO2 fixation in the presence of sulfide

and sulfur (9) during which, with the exception of the genus

Ectothiorhodospira, sulfur is deposited inside the cells

(178). Molecular hydrogen is also used as electron donor

by many species of this family. Some species will photo-

assimilate acetate and pyruvate (139). These bacteria are

Gram-negative, frequently gas-vacuolated, spherical, ovoid,

rod-, vibrio-, or spiral-shaped cells which display het-

erogeneous guanosine + cytosine (G + C) ratios ranging from

45-70.4 percent.

The family Chromatiaceae comprises two main physio-

logical-ecological groups representing ten genera and 26

species of bacteria (140). One group which includes

Amoebobacter, Lamprocystis, Thiodictyon, and Thiopedia

possesses gas vacuoles, thus enabling them to migrate verti-

cally within the water column (139). The second group,

which includes small Chromatium, Thiocystis, Thiocapsa,

Thiosarcina, and Ectothiorhodospira does not possess gas

vacuoles. All of these bacteria are able to develop either

in single cell or nonmotile aggregates aggregates of cells.

Both forms are features of the purple-red bloomns of

Chromatiaceae observed in lagoons, shallow pools and

estuarine environments (139).

Suborder Chlorobiineae also contains two families, the

Chlorobiaceae (green sulfur bacteria) and the Chloroflex-

aceae (gliding bacteria). In these families, the major

bacteriochlorophylls are c, d, or e along with small amounts

of bchl a in the photosynthetic reaction centers. In addi-

tion, the green-colored species of this family possess the

carotenoids chlorobactene and OH-chlorobactene, whereas the

brown-colored species possess the carotenoids isorenieratene

and -isorenieratene (98) which contribute to their color

and the broader absorption range between 480 and 550 nm.

The in vivo long wavelength absorption maxima of the major

bacteriochlorophylls (bchl) are: bchl a 830-890 nm, bchl b

835-850 and 1020-1040 nm, bchl c 745-755 nm, bchl d 705-740

nm, and bchl e 719-726 nm. Suborder Chlorobiineae is also

characterized by the chlorobium vesicles which contain the

photosynthetic apparatus and occur as special organelles

underlying and firmly fixed to the cytoplasmic membrane

(138). Chlorobiaceae, with the exception of the genus Chlo-

ropseudomonas, are nonmotile, frequently gas-vacuolated,

spherical, ovoid- or rod-shaped cells (140). They are obli-

gately anaerobic organisms which utilize sulfide as an

electron donor, deposit elemental sulfur extracellularly,

and are incapable of assimilatory sulfate reduction (178).

Their G + C ratios are well defined, ranging from 48.5-58.1

percent (142). They metabolize certain organic compounds,

notably acetate and propionate (138,139).

Chloroflexaceae (175) are Gram-negative, filamentous,

gliding, anoxygenic phototrophs with flexible cell walls.

Of this family, only one species, Chloroflexus aurantiacus

has been studied in pure culture (144). These bacteria

exhibit anoxygenic photosynthesis using reduced sulfur

compounds as electron donors, but their best growth occurs

in the light when using fixed carbon compounds. Photometabolism

During autotrophic growth, photosynthetic fixation of

CO2 by the Chromatiaceae is primarily by the reductive

pentose phosphate cycle (139). This cycle is of limited

importance to Chlorobiaceae which utilize a cycle of reac-

tions involving ferredoxin-dependent carboxylations,

catalyzed by pyruvate- and a-ketoglutarate synthase (22).

Other reactions involving carboxylic acid enzymes, have also

been shown to be of significance (23) for this family. In

the green sulfur bacteria a mechanism of CO2 fixation

involving a reverse tricarboxylic acid (TCA) cycle has been

proposed (49,154,156) and accepted as the major route of

carbon fixation (53).

This cycle, which is illustrated in Figure 2-7,

involves two ferredoxin-dependent carboxylations. One

complete turn of the cycle produces a molecule of

oxaloacetate from four molecules of CO2 (155). Intermedi-

ates of the cycle include precursors for lipids and amino


w a,
I a cJ

w LL

(NV 0 ::
0 Iu 0 -
cu u:0

0 a:
I- U

0 1 u
Ln 0

w a

4 4 -4l
0 -
I--j >1
0 a
C C.,

I- 4.J
00 w 0
U I a,

a. 1-
U cr
O aa.

0 mC

1 0
w rU
0 U
u c

acids; carbohydrate is formed from pyruvate by reversed gly-

colysis. Species of the family Rhodospirillaceae possess a

complete TCA cycle which functions oxidatively in the dark

as well as anaerobically in the light (54).

Primary contribution to current understanding of the

biochemistry of photosynthetic CO2 fixation has been made

by Van Niel (190) who postulated the unitary concept of


CO2 + 2H2A -----------> CH20 + H20 + 2A (2-5)

where H2A may represent either water, as in the case of

green plant photosynthesis, or hydrogen sulfide, as in

bacterial photosynthesis. Cell mass is represented by CH20.

Detailed studies on the photoassimilation of carbon

have been carried out in only a few of the more than 50

known species of phototrophic bacteria. The role of com-

pounds metabolized by phototrophic bacteria is indicated

in Table 2.6 (140), and some of the known organic compounds

metabolized by these bacteria are listed in Table 2.7 (41).

The photometabolic activity of sulfur bacteria is dependent

on the presence of oxidizable external electron donors such

as reduced sulfur compounds (139). The amount of sulfide

removed from the environment by these organisms during

metabolic activity is dependent on the carbon source being


Table 2-6. Role of compounds metabolized by the phototrophic

Substrate Bacteria Metabolic role




H donor

H donor





Simple organic acids

Amino acids


Fats, oils





C source, H donor

C source

N source

N source

growth substrates

S, sulfur bacteria (Chromatiaceae, Chlorobiaceae)
NS, nonsulfur bacteria (Rhodospirillaceae)
Source: Crofts A.R. 1971 (41).

Some organic compounds photoassimilated by
phototrophic bacteria.





Intermediates of
TCA Cycle







Amino acids







Bacterial Genera and Species

Chromatium, Chloropseudomonas, Thiocystis
Lamprocystis, Thiospirillum, Thiocapsa,
Amoebobacter, Ectothiorhodospira,

Chromatium, Thiocystis, Thiocapsa,
Lamprocystis, Thiodictyon, Chlorobium,
Amoebobacter, Ectothiorhodospira, Chloro-

Chromatium, Amoebobacter, Ectothiorhodo-
spira, Chloropseudomonas, Thiocystis.

Chromatium vinosum

Thiocapsa roseopersicina, Amoebobacter,
Ectothiorhodospira, Chlorobium, Thiocystis

Thiocapsa roseopersicina

Thiocapsa roseopersicina, Chloropseudomonas

Thiocapsa roseopersicina, Amoebobacter,

Thiocapsa roseopersicina

Thiocapsa pfenigii, Ectothiorhodospira,
Chlorobvium, Pelodictyon.



Ectothiorhodospira, Chloropseudomonas





Source: Mah and Smith 1981 (102).

Table 2-7.

utilized (185). Sulfide uptake during the metabolism of CO2

is indicated in the following reaction:

40CO2 + 21H2S + 8NH3 + 20H20 ---> 8(C5H802N)

+ 21H2S04 (2-6)

and for acetate

40C2H402 + H2S + 8NH3 ---> 8(C5H802N)

+ H2SO4 + 20H20 (2-7)

From these equations it may be observed that sulfide uptake

by phototrophic sulfur bacteria is 21 times greater during

CO2 metabolism than during acetate metabolism (185).

The key enzymes involved in photoassimilation of CO2

via the reductive pentose cycle are ribulose 1, 5-diphos-

phate carboxylase and ribulose 5-phosphate kinase (142).

Enzymes of the Calvin cycle are repressed in certain species

of the Chromatiaceae when they are grown anaerobically in

light with acetate as the sole carbon source. At such

times, metabolism is via the glyoxalate cycle. Green sulfur

bacteria are very sensitive to oxygen, but purple sulfur

bacteria are not killed by oxygen. In certain environments,

some species of purple sulfur bacteria are able to grow

chemoautotrophically in the dark, oxidizing H2S with 02

(82). Ribulose bisphosphate carboxylase of the latter

organisms possesses oxygenase activity, and it has been

demonstrated (170) that the cells excrete glycolate in the

presence of 02.

Certain simple organic substrates are photoassimilated

by Chlorobiaceae in the presence of sulfide. These include

acetate, propionate, butyrate, lactate and some amino acids

(83). The amount of acetate assimilated is directly propor-

tional to either the sulfide or the bicarbonate concentra-

tion when either is growth limiting. Energetics

Microbial growth results from coordinated synthesis

of a range of complex macromolecules utilizing an energy

source appropriate to the particular organism. During bio-

logical oxidations, the energy present in an organic sub-

strate is released by successive dehydrogenations of the

carbon chain. Reducing equivalents are removed in pairs and

transferred to a final acceptor which may be 02, in the case

of aerobic respiration, inorganic compounds other than 02 in

the case of anaerobic respiration, or organic compounds

in the case of fermentation. Transfer proceeds via electron

transport systems.

Approximately 80 percent of a cell's energy budget is

expended on biosynthetic processes, as indicated in Table

2.8 (167). In chemotrophic organisms, this energy is

obtained from nutrients in their environment. In photo-

trophs an energy source external to their environment,

Table 2-8.


Bacterial energy budget for cells grown on

Percent Energy (ATP) expended
on each process.





Nucleic Acid

Transport into cells






Source: Stouthamer 1973(167).

in the form of light of suitable wavelength, is required.

About 60 percent of the biosynthetic energy requirement is

utilized for protein synthesis, and nutrient transport

accounts for about 18 percent (167).

In all cells, the main energy-coupling agent is

adenosine triphosphate (ATP) (97), which is generated by the


ADP + P. -----> ATP + H20 (2-8)

Adenosine inorganic Adenosine
diphosphate phosphate triphosphate

When hydrolyzed, ATP yields a standard free energy change

AG of -7.0 kcal.mol-. This energy drives solute

transport across the cytoplasmic membrane. Reactions with a

AG less than -7.0 kcal.mol-1 cannot be coupled directly to

ATP generation (163).

ATP synthesis in bacteria occurs either by substrate

level phosphorylation or by chemiosmotic energy-generating

processes (172), commonly referred to as electron transport

oxidativee) phosphorylation. In substrate level phosphory-

lation, in which a phosphate molecule is first added to a

substrate, and is then subsequently transferred to ADP to

form ATP, one molecule of ATP is synthesized by phosphoryla-

tion of one molecule of substrate in the cell (97). Oxida-

tive phosphorylation, which had been hypothesized for some

time (119,120) but only recently has been demonstrated

experimentally (158), provides no direct generation of ATP.

Instead, an electrochemical proton gradient is developed

(119, 120), and electrogenic proton pumps translocate

protons across the cytoplasmic membrane from the cytoplasm

to the external medium; this generates an electrical poten-

tial and a pH gradient across the membrane.

These energy-transducing systems in the membrane con-

vert chemical energy or light energy into electrochemical

energy which is used to drive energy-requiring processes

(158). In anaerobic environments, fermentations are carried

out by a variety of heterotrophic bacteria. These result in

the generation of ATP by substrate level phosphorylation,

and formation of several intermediate stage metabolic end

products. Photoautotrophs derive their energy from an

external source not utilized by heterotrophs with which they

share the habitat (140). They are able to fix CO2, a major

waste product of their cohabitants, using H2S, another waste

product as electron donor.

Phototrophic sulfur bacteria obtain their energy from

light, transform it via cytochromes, and finally store it as

ATP (139). Unlike eucaryotic phototrophs and the cyano-

bacteria, these organisms carry out an anoxygenic photo-

synthesis using only one photosystem. They therefore

require electron donors of lower redox potential than water,

and generally utilize reduced sulfur compounds, molecular

hydrogen, or simple organic compounds for this purpose.

These compounds are either present in the environment or are

produced by metabolic activities of other organisms (139).

Under anaerobic conditions in the dark, phototrophic bac-

teria obtain their energy for maintenance by fermentation of

storage polysaccharides (11).

A comparative illustration of the two types of photo-

systems is given in Figure 2-8 (50). In bacterial photo-

synthesis, the electron absorbed by the reaction center

chlorophyll P870 is raised to an acceptor designated X

(186). It then passes down through an electron transport

system involving ubiquinones, generating ATP during this

cycle. In the oxygenic system, ATP is generated by cyclic

photophosphorylation in Photosystem I, and NAD(P)H is also

produced by this photosystem. ATP is generated in Photo-

system 2 by noncyclic photophosphorylation, this being the

normal process in green plants. When NAD(P)H is produced in

Photosystem I, the electrons diverted to NAD(P) are

replaced from Photosystem 2. In anoxygenic photosynthesis,

NAD(P)H cannot be produced directly by Photosystem I in most


In green sulfur bacteria, the redox potential of X

is apparently lower than that of the purple sulfur bacteria,

enabling the former to reduce ferredoxin (Fd) and NAD(P)

directly (128). Purple bacteria apparently are unable to

reduce NAD(P) directly by the photosystem. In such cases

NAD(P) reduction occurs either by expending ATP (Figure

e cu vv o
o5 6 6 o 6 6
O d d
iI I I i I I I


a -^ I r
E m

17O O

0\ z 0
- n nO \ -

UA < < o C,
0 ('A __--_ 0

3- 0 >


0 4

:r 41

0 0 0 ( C)
Q) I
o 4n >i

n 7.

I I 0
L E 0 M 04

00 04Ln
0 0

d 0 d 0 o.
SI + +
/ 4\, a a

U, \d

6 6 0 6 0

I I + +
~ !-1
iciu O cvi q ^ o
d~~~~~r d d o d '
'a i

2-8) or by a process of reverse electron transfer effected

by an external electron donor (5). This is shown in Figure


By utilizing an external energy source, phototrophs

are able to make opportunistic use of any organic matter

available to them in their environment. The presence of a

functional TCA cycle indicates a potential for acetate

metabolism (139).

The electron donors utilized by these organisms are

oxidized by different enzyme complexes and pathways. Thus,
H2 reduces NAD and the resulting NADH is oxidized via the

electron transport chain. In some phototrophic bacteria,

reduced sulfur compounds release electrons upon oxidation

at potentials too high to reduce NAD hence NADH for bio-

synthesis must be provided via ATP-dependent reversed elec-

tron transport (139).

The role of ATP in photometabolism is two-fold. It is

required either in forming an "activated" substrate, to

bring CO2 fixation into the Calvin cycle, or an "activated"

carbon source such as acetyl-CoA from acetal and co-enzyme A

(CoA) (48). The activated compounds are then involved in

synthetic reactions, which are catalyzed by specific enzyme

systems which function in the dark.

Phototrophic bacteria differ amongst themselves in the

nature of their light-harvesting or antenna pigments. The

electron transport systems and the light harvesting pigments


I 0

x+ 0
U) H.
S\ /
z ()

U c4

., -\



44 .4()

L 0 TI
0) a

L. 4) U-l
f ^

are associated with chromatophores, the small vesicles

formed by the invaginated intracytoplasmic membrane (133).

Light energy harvested by antenna pigments migrates to the

bacterial reaction center, which contains bacteriochloro-

phyll a, and where electron transfer reactions occur (133).

In these bacteria, ATP generation by cyclic photophosphory-

lation is not directly linked to substrate utilization, as

is the case with heterotrophic bacteria. Ecology of Phototrophic Bacteria

Phototrophic bacteria are restricted in ecological

distribution by their need for anaerobic conditions in the

light (139). Their presence has been catalogued in a wide

range of aquatic habitats including lakes, ponds, sewage

ponds and lagoons treating other high strength wastes, salt

and freshwater pools, mud flats, sulfur springs, and paddy

soils. They have been found in environments with salt

content ranging from near zero to saturation and at tempera-

tures ranging from below zero to about 800 C (26). The

existence of purple and green bacteria in these habitats

is generally indicated by the pink and green blooms, which

are observed especially below the thermocline in lakes


Shallow ponds, mud flats, and similar locations which

are rich in organic matter, CO2, H2, and H2S, are ideal

habitats for phototrophic bacteria that photometabolize all

end products of fermentative reactions (139). Except near

the air/water interface, such environments are free of

oxygen, and phototrophic bacteria can grow near the water

surface where light intensity is high. Here the ability of

phototrophic anaerobes to absorb light of very long wave-

length is critical to their survival. Such far red and

infrared light is transmitted by the overlying phototrophs

and is absorbed by the bacteria.

In the photic zones of freshwater lakes and seas, the

dominant organisms are primary producers. The optical prop-

erties of such bodies of water are important regulatory

parameters in the physiology and behaviour of organisms

which reside therein. Very dense populations of photo-

trophic bacteria have been found at various depths in a

number of meromictic lakes (132,171,176), which are charac-

terized by permanent stratification of the water. In such

lakes, the aerobic upper layer is underlain by a cold

oxygen-free zone in which anoxygenic phototrophs exist,

normally in a narrow band just within the anaerobic layer

(187). At this depth, the overlying water column becomes

an effective light filter, transmitting only green and blue-

green light of wavelengths between 450 and 550 nm. At such

times, the role of light harvesting is largely assumed by

carotenoids and not by bacteriochlorophylls (187). Photo-

trophs identified in such environments include Chlorobium

limicola, Chromatium, Prosthecochloris, Thiopedia,

Thiocystis, and Chlorobium phaeobacteroides.

Primary synthesis of organic material in bodies of

water which are not directly exposed to serious pollution,

is mediated by the phototrophic bacteria (35). CO2 is fixed

by purple and green sulfur bacteria using H2S or H2 as

reductant, while acetate and simple organic compounds are

assimilated by purple nonsulfur bacteria (139) and some

species of the purple sulfur bacteria. These bacteria have

been found to contribute significantly to primary production

in such habitats (35,42), their contribution ranging from a

fraction of a percent to over 80 percent of the total pri-

mary production on an annual basis (9).

In addition to being grazed by protozoans in the water

column (42), organic constituents of dead cells of photo-

trophic bacteria are decomposed by fermentative bacteria

with the formation of CO2, H2, NH3, organic acids and alco-

hols. When sufficient sulfate is present, sulfate-reducing

bacteria oxidize H2 with formation of H2S and acetate which

are subsequently used by phototrophic bacteria. Other

anaerobic oxidations are performed by methanogenic bacteria

which oxidize H2 and convert CO2 and acetate to methane.

Some of this methane is oxidized in the aerobic region of

the water body by aerobic methane oxidizers; the remainder

escapes to the atmosphere, constituting a net loss of carbon

from the system.

The depth at which the purple sulfur bacteria occur in

the water column of stratified lakes varies throughout the

day (163). On sunny days they consume H2S in the upper

hypolimnion during the morning and migrate downwards later

in the day (128). When this occurs, light becomes the

limiting factor (163). Similarly, it has been found that

the growth of green sulfur bacteria in stratified lakes is

strongly light-limited (8). In holomictic lakes where

seasonal stratification can occur, phototrophic sulfur

bacteria are located in a narrow horizontal plate where

light, H2S, and minimal 02 concentration are most favorable

for their multiplication (50). Due to the higher tolerance

of green sulfur bacteria to H2S, they are usually located

in habitats below the purple sulfur bacteria. Natural Occurrence and Role
in Waste Treatment Systems

The natural occurrence of phototrophic sulfur bacteria

in systems treating a variety of organic wastes is recorded

in the literature (Table 2-9). Their presence is directly

attributable to the wide range of substrates metabolized by

theml40). These include simple sugars, alcohols, volatile

fatty acids (VFA), tricarboxylic acid (TCA) cycle inter-

mediates, and benzoates (139). Odors are generally mini-

mized in systems in which they occur due to their metabolism

of H2S and other odorous compounds. Phototrophic bacteria,

by their ability to utilize potentially toxic products of

Table 2-9.

Species of phototrophic bacteria identified in
waste treatment systems.

Type of Waste Treatment Phototrophic Reference
System Bacteria

Rendering plant

Petroleum refinery

Hide-washing plant

Poultry manure


Swine manure

Cattle feedlot

Poultry processing



Domestic and












Thiopedia rosea


Thiopedia rosea

Thiopedia rosea

Chromatium vinosum

Thiopedia rosea

Thiopedia rosea



Thiopedia rosea

Thiopedia rosea

34, 35

34, 35




98, 141



33, 98


64, 98

anaerobic metabolism, play a beneficial role in the

anaerobic degradative process. Studies of lagoons treating

organic industrial wastes (38,40,75) have confirmed that

purple sulfur bacteria oxidize inorganic sulfur compounds

and certain short-chain fatty acids.

As shown in Table 2-9, several species of phototrophic

bacteria have been identified in waste treatment systems.

Thiocapsa roseopersicina and Chromatium vinosum were found

in a domestic waste treatment lagoon (75) which had been

overloaded by introduction of highly concentrated potato

processing waste. In a study of this lagoon, it was found

that levels of acetate, VFA, and sulfides were reduced as

the population of phototrophic anaerobes increased. Occur-

rence of phototrophic sulfur bacteria in a lagoon treating

fellmongery wastes (38) correlated with the recorded reduc-

tion of 89-98 percent in sulfide levels.

Growth studies on species of Chromatium removed from a

poultry processing waste lagoon have indicated an optimum

temperature of 26.50 C and optimum pH of 7.5 (116), but

growth of these phototrophs has been recorded at tempera-

tures ranging from 10-300 C (38) and pH levels of up to

9.2. In pilot scale waste treatment studies (112) with

photobiological systems, COD and sulfide removals of 86 and

85 percent, respectively, were achieved.

2.4. Process Inhibition

Varying levels of certain inorganic and organic sub-

stances may have an adverse effect on the anaerobic

process. The degree of inhibition by inorganic substances

varies depending on whether the substances act singly or in

combination with other potential toxicants. Certain combi-

nations of alkaline earth salts have been found to have

synergistic effects, while others display antagonistic

effects (93). Thus the toxic effects of sodium at 7,000

mg/L may be reduced by 80 percent by addition of 300 mg/L of

potassium. It may be completely eliminated by an addition

of 150 mg/L of calcium (93).

Digestion of ammonia-rich wastes, such as those from

swine and poultry, may be inhibited by either ammonia gas

or ammonium ion (37,152). McCarty (107) reported that

ammonia gas can become inhibitory at 1500 mg/L to 3000 mg/L

total ammonia concentrations and pH greater than 7.4.

Ammonia toxicity may be experienced at total ammonia concen-

trations above 3000 mg/L irrespective of the pH level.

Stable methane production was achieved however at total

ammonia concentrations in excess of 3000 mg/L (37,52). An

investigation of ammonia inhibition in which a urea and

ecetic acid substrate were used, indicated progressive

inhibition to commence at total ammonia concentration of

2000 mg/L, but toxicity did not occur even at total ammonia

nitrogen concentration of 7000 mg/L. Nitrates have also

been observed to have a negative effect on methanogenesis


Sulfide has varying effect on methanogenesis. Low

concentrations of sulfide may be stimulatory to the process

(208), whereas high concentrations can be inhibitory (198).

Sulfide concentrations above 200 mg/L are considered toxic

to the anaerobic process (107). This is of significance

only when sulfides are in soluble form and not when associ-

ated with heavy metals. It has also been observed that high

concentrations of sulfates inhibited methanogenesis (197).

A number of investigators (20,100,198) have proposed that

this inhibition results from interspecies competition

between the methanogens and sulfate-reducing bacteria for

available hydrogen, in which the latter organisms are able

to outcompete the former for this substrate.

Levels of organic acids in an anaerobic environment are

critical for the efficient operation of the process.

McCarty and McKinney (109) found that volatile acid levels

below 2000 mg/L were desirable. This observation was con-

firmed by other investigators (34,60,210). Very small

concentrations of heavy metals may be toxic to anaerobic

microorganisms in the absence of sulfides with which they

form innocuous precipitates (107). Some metals may, how-

ever, have a stimulatory effect (164). The impact of a

variety of other substances on the anaerobic process has

been reported. These include organic priority pollutants

(80), bisulfate (46), trimethylamine (12), sulfur (92), and

certain industrial toxicants (130).

Antibiotics and growth promoters are widely used in

livestock production. Some of them may adversely affect the

anaerobic process; it will be minimized upon acclimation of

the microorganisms to the particular chemicals. The detri-

mental effect of antibiotic lincomycin on the anaerobic

process has been recorded (130).

The inhibitory effect of these toxicants will be

reflected in reaction rates, and, hence, the kinetic

parameters of the anaerobic process (92). While complete

allowance cannot be made for all inhibitory substances,

several kinetic models now include factors of process


2.5 Kinetics of the Anaerobic Process

2.5.1 Basic Considerations

In a completely mixed anaerobic reactor, various steps

of the staged sequential biodegradative process are assumed

to occur simultaneously. Consequently, overall kinetics

of this complex process are considered to be controlled by

the kinetics of a rate limiting step. Identification of

this rate-limiting step and determination of its kinetic

characteristics are considered essential to the development

of overall process kinetics.

The theory of growth kinetics in biological systems,

is based on growth rate, growth yield, and the relationship

between growth rate and an essential nutrient. Mathematical

descriptions of these relationships may be derived, thus

enabling development of kinetic equations or mathematical

models, for describing microbial activities under particular

growth conditions.

The Monod model (121) has successfully described the

kinetics of biological waste treatment systems and has

provided the basis for several kinetic models of the

anaerobic digestion process. This model describes the

growth of homogeneous cultures on simple substrates but not

heterogeneous cultures on complex substrates as found in

anaerobic environments. In spite of apparent limitations,

the Monod model has been adapted to the anaerobic digestion

kinetics of sewage sludge and animal manures (65,96).

Prediction of maximum biological activity and washout of

microbial cells from a reactor are possible with this

model. One other advantage of this type of model is that

the kinetic parameters (microorganism maximum specific

growth rate and half-velocity constant) fully describe the

microbial processes, although different parameters must be

used for short and long retention times (31,32). A dis-

advantage of Monod type models is that the kinetic parame-

ters cannot be obtained for certain complex substrates


Disadvantages of Monod models were overcome with the

development of various forms of first-order kinetic models

(1,2,3), which were simple to use and gave good fit of

experimental data. They are, however, limited in their

ability to predict the conditions for maximum biological

activity and system failure.

2.5.2. Relationship Between Microbial Growth and
Substrate Utilization in Batch Culture

In the logarithmic phase of bacterial growth, the

growth rate of organisms can be expressed by the equation

dX/dt = uX


where X = organism concentration, mass/unit volume

t = time

p = specific growth rate, time-

Accounting for the effect of endogenous respiration on the

overall growth rate, equation 2-9 becomes

dX/dt = (p b)X



dX/dt =


to the biomass

endogenous decay coefficient, time-

net growth rate of microorganisms per unit
volume of reactor, mass/volume-time

utilization rate is considered proportional

present, as expressed by the equation


dS/dt = qX (2-11)

from which q = dS/dt (2-12)

where dS/dt = substrate utilization rate,

q = specific substrate utilization rate,

True growth yield, YT, is defined as

YT= weight of organisms formed
weight of limiting substrate utilized

Mathematically, Y, = dX/dS (2-13)

where YT = growth yield constant, mass/mass

Combination of equations 2-10, 2-11 and 2-12 provides the

relationship between biological growth and substrate


dX/dt = Y (dS/dt) bX (2-14)

Hence q = p/Y, (2-15)

Rate of substrate utilization may also be related to

concentration of microorganisms in the reactor and concen-

tration of the growth-limiting substrate surrounding the

organisms as

dS = kSX (2-16)
dt K + S
where k = maximum rate of substrate utilization per
unit of microorganisms.

S = concentration of growth-limiting substrate
surrounding the microorganisms, mass/volume

K = half-velocity constant, equal to the
substrate concentration when (dS/dt)/X =
(1/2)k, mass/volume

This expression is in similar form to the Monod equation

(121) which describes the rate of bacterial growth as a

function of substrate concentration surrounding the micro-


1 = p S (2-17)
K + S

where p = specific growth rate, time-
pmax = maximum specific growth rate, time- con-

K = half velocity constant

Combining equations 2-14 and 2-16 yields the expression

n = Y kS b (2-18)
K + S

where n = (dX/dt)/X, net specific growth rate

When S is very much greater than Ks in equation 2-16, Ks can

be neglected and the equation becomes

dS/dt = kX


When S is very much less than Ks, S in the denominator can

be neglected and the equation becomes

dS/dt = KSX



K = k/K specific substrate utilization rate
constant, volume/mass.time

2.5.3. Completely-Mixed Continuous
Culture Model Without Recycle Microbial Growth

Consider the schematic of a continuous culture reactor

shown in Figure 2-10. A materials balance for bacteria


microorganism rate of net growth of net rate of
accumulation = microorganism + microorganism microorganism
within reactor inflow within reactor outflow

or stated directly


= inflow

+ net growth

- outflow

i.e. (dX/dt)V = QX + nVX QX

where V = volume of microbial culture in reactor

X = microorganism concentration in influent,

X = microorganism concentration in reactor

Q = flowrate, volume/time













X, N



Neglecting microorganisms in influent, assuming steady-state

conditions (dX/dt = 0), and substituting for pn from equa-

tion 2-18, equation 2-21 reduces to

Q = YkS b = 1 (2-22)
V K + e6
s e
where 8 = hydraulic retention time, V/Q

At steady state, the specific growth rate is equal to the

dilution rate, and

n = 1 1 = = YkS b (2-23)
n -T--
6 K +
c s e

where 8 = biological solids retention time, time

1/8 = dilution rate, time-

from which we obtain the expression

1 = Y (dS/dt) b (2-24)
8 X

= Y q b (2-25)

From equation 2-24

dS = 1 X + bX (2-26)
dt YT c

Net microbial growth may be described by the expression (96)

dX = bY dS (2-27)
d- obs"
dt dt

where Yobs = variable observed yield coefficient.


From equation 2-27

Yobs = (dX/dt) (2-28)

multiplying the right hand side of expression by X/X gives

Yobs = p (2-29)

Substituting dS/dt from equation 2-27 in equation 2-26

1 dX = 1 X + bX (2-30)
Y dt Y 6
obs T c

This expression reduces to

1 = be + 1 (2-31)
obs T T

The substrate utilized by an organism may be considered to

comprise a variable portion for biosynthesis and a rela-

tively constant portion for maintenance. The total specific

substrate utilization rate can therefore be expressed as

q = alp + a2 (2-32)

where a, = substrate utilization to form a unit of

u = specific growth rate of biomass

a = substrate utilized for maintenance function
per unit biomass per unit time, time-

q = specific substrate utilization rate, time-

Substituting for q in equation 2-32 from equation 2-29 gives

Y = 1 (2-33)
obs a + -
al + a2/ Substrate Utilization

A similar materials balance may be written for sub-

strate within the reactor

change within = influent consumption effluent
the reactor

or V(dS/dt) = QS V(KXSe) QSe (2-34)

where KXSe = substrate consumed by organisms

At steady state dS/dt = 0, and equation 2-34 becomes

Q(S S ) = V.KS
o e e

and Q(S S ) = KS = q (2-35)
o,---- e e

2.5.4 Anaerobic Kinetic Models

The anaerobic digestion process has been described by

several kinetic models, which have been developed to opti-

mize gas production (30) rather than waste treatment, as is

the case with activated sludge kinetics. None of these

models have been developed for the treatment of animal

manures by the anaerobic photosynthetic process. McFarlane

and Melcer (111) have, however, demonstrated the


applicability of Monod-type kinetics to the treatment of

industrial wastes by the anaerobic photosynthetic process.

Andrews (1,2) was among the first to introduce the

dynamic modeling of the anaerobic digestion process. He

incorporated an inhibition function into the model by con-

sidering un-ionized volatile acids as the rate-limiting

substrate and inhibitory agent. This approach yielded the


p = 1 (2-36)
max 1 + K + UVA
maxU K.

where P = specific growth rate, time-
max= maximum specific growth rate, time-

K = saturation constant, mass/volume.
K. = inhibition coefficient of un-ionized
volatile acids, mass/volume.

UVA = concentration of un-ionized volatile acids,

This model was further developed and expanded by

Andrews and Graef (3), who considered the effect of inter-

actions between volatile acids, pH, alkalinity, gas produc-

tion rate and gas composition on the process. This

resulted in the development of a model capable of predicting

process performance under transient conditions. Municipal

and industrial wastes were used as the influent material in

the development of this model.


Using computer simulation, Hill and Barth (65) modified

the model of Andrews and Graef (3) by substituting animal

manure, with its higher content of organic material and

nitrogen, as the influent substrate and considering the

inhibitory effect of free (un-ionized ) ammonia on the meth-

ane-producing bacteria. The resulting expression was

p = 1 (2-37)
m 1 + K + UVA + NH
Umax A K. K.
1 12

where NH3 = concentration of un-ionized ammonia, mass/

K. = inhibition coefficient for ammonia, mass/-

The Monod (121) kinetic model has been adapted to

describe anaerobic digestion kinetics of sewage sludge

(96). The disadvantages of this model, as noted above were

minimized with the development of first-order kinetic models

(58,59). Applying Monod (121) kinetics, Ghosh and Pohland

(55) investigated the kinetics of substrate assimilation and

product formation in anaerobic digestion. The Contois (36)

.kinetic model was adapted by Chen and Hashimoto (30) to

describe the kinetics of methane fermentation in the form

B = BSo[l K (2-38)
v 0mp -1 + K

where B = volumetric methane production rate L CH4/L

S = influent total volatile solids (VS)
concentration g/L.

B = ultimate CH4 yield, L CH4/gm VS added

8 = retention time, day.

A = maximum specific growth rate of organisms,
day 1

K = kinetic parameter, dimensionless.

This model was used to predict B of pilot and full-scale
systems fermenting livestock wastes at 35, 55 and 600 C.

The equation relates the daily volume of methane produced

with loading rate (S /e ), material biodegradability (B )

and kinetic parameters u and K. For livestock wastes, B

depends on type, animal ration and age of the manure, the

collection and storage method, and the amount of foreign

material incorporated in the manure.

In a general presentation, Lawrence (94) outlined a

kinetic approach to the design of biological waste treatment

processes employing suspended cultures of microorganisms in

completely mixed process configurations. The relationships

developed for concentrations of effluent waste (Equation

2-38) and microbial biomass in the reactor (Equation 2-39)

would be applicable to anaerobic digestion. These

expressions are

S = K [ 1 + b(Sc) ] (2-39)
6 (Yk b) 1

X = Y (S Sl
1 b 1-

where S1






= effluent waste concentration, mass/volume.

= half velocity coefficient, mass/volume.

= microorganism decay coefficient, time-

= biological solids retention time, time.

= growth yield coefficient, mass/mass.

= maximum rate of substrate utilization per unit
weight of microorganisms, time-

= microbial mass concentration, mass/volume.

Sinfluent waste concentration, mass/volume.

In a comparative evaluation of the kinetic constants

reported for methanogenic bacteria by several researchers,

Scharer and Moo Young (148) found wide variations in the

parameters. These discrepancies were attributable to both

experimental conditions and methods of data analysis used.

They observed that kinetic information determined from

single substrates could not be used for predicting methane

generation from complex substrates.

The development of mathematical models and simulation

techniques for anaerobic digestion of animal waste have

been described by Hill and Barth (65), and Hill and

Nordstedt (67). These models were later refined (64,68) to

reflect new assumptions, thus enabling more accurate

prediction of process to be made.



3.1 Rationale for Experimental Design

The present research was designed to generate data for

the determination of kinetic parameters which influence

phototrophic biomass production and substrate uptake in a

swine waste medium. These parameters are represented in

Equations 2-33 and 2-35.

Two laboratory-scale anaerobic reactors, one

illuminated, and the other nonilluminated, were operated

in parallel in order to assess the impact of bacterial

photosynthesis on the anaerobic digestion process. In such

a configuration the nonilluminated reactor would yield

results consistent with a conventional anaerobic digester,

and would be considered as the control. Retention times,

which ranged from 5 to 30 days were selected on the basis of

published growth characteristics of methanogenic bacteria

and on the retention times normally used for standard rate

digesters. Uniform distribution of the medium throughout

the reactor was achieved by continuous mixing with magnetic

stirrers, ensuring that the bacteria within the reactor

would always be in intimate contact with the medium, and

that the biological solids retention time (SRT) was equal to

the hydraulic retention time. A low mixing speed was

selected to achieve gentle continuous turnover of the con-

tents of the reactor without introduction of high shear


The medium was blended from raw swine waste collected

directly from floors of the pig barns. This method of

preparation effected greater control over the solids concen-

tration used in the experiments, avoiding dilution which

would be experienced by flushing of the wastes. It also

eliminated any prior fermentation which would occur in

storage tanks. Before being blended to the required con-

centration, the waste was screened to remove coarse

cellulosic or inorganic materials, yielding a more easily

biodegradable medium. The experiments were started with

waste of total solids concentration in the range 0.4 to

0.6 percent, this range having been found (45) to be suit-

able for batch growth of phototrophic bacteria. Waste with

volatile solids (VS) concentration of 1.0 percent was used

for continuous loading of the reactors. Selection of this

concentration was based on the results of earlier batch

studies (45).

The contents of both reactors were maintained at a

temperature of 27 1 C in all experiments. Phototrophic

sulfur bacteria were observed to grow very well at this

temperature (45). The pH was not controlled in any of the

experiments conducted, but was allowed to vary as dictated

by the buffering capacity of the system. The reactors were

kept airtight to exclude oxygen, and sulfide levels were

maintained above 20 mg/L in the reactors by addition of

sodium sulfide as found necessary. The addition of sulfide

ensured that this nutrient did not become limiting during

the experiments. Illumination was provided by 120 watts of

incandescent lighting. Optimum light level was not known at

the time the experiments were designed.

Several parameters were measured and used in interpret-

ing the performance of the reactors. Measurements were made

throughout the operation of the reactors, but particular

emphasis was placed on sampling and analysis once a rela-

tively stable level of bchl a had been attained. This

was considered to be an indication of steady state condi-

tions in the reactors. Measurement of pH was made to assess

the buffering capability of illuminated and nonilluminated

systems. Bacterial growth and biomass production were

monitored by measurement of bacteriochlorophyll a (bchl a)

concentration. Biomass productivity was also determined by

measurement of total, volatile and suspended solids, and

protein. Chemical oxygen demand and biochemical oxygen

demand were used in monitoring substrate uptake in the

system. Nitrogen (N) and phosphorus (P) were determined in

order to compare nutrient uptake in both reactors. Gas

quantity and quality were monitored to determine impact of

phototrophic bacteria on biogasification.

3.2 Summary of Investigations

The performance of two bench-scale anaerobic reactors,

an illuminated experimental reactor and a nonilluminated

control reactor was monitored at SRTs of 5, 7, 8.5, 10, 15,

20, and 30 days. The reactors were operated at a tempera-

ture of 27 + 10C and were loaded with screened and blended

swine waste having a volatile solids concentration of 1.0 +

0.1 percent. Parameters measured included temperature, pH,

bacteriochlorophyll a, chemical oxygen demand (COD), bio-

chemical oxygen demand (BOD5), total solids (TS), volatile

solids (VS), ammonia-nitrogen (NH3-N), total kjeldahl-nitro-

gen (TKN), total suspended solids (TSS), total sulfide,

total phosphorus (P), and gas quantity and quality.

3.3 Experimental Apparatus

The apparatus is shown schematically in Figure 3-1.

Two identical 4.0 L glass bottles, each sealed with a rubber

stopper, were used as anaerobic reactors. The stoppers were

fitted with inlet/outlet, venting, and gas transfer tubing.

Each was also fitted with a thermometer. The reactors were

operated with liquid volume of 3.5 L. The control reactor

was completely covered with aluminum foil to exclude light

whereas the experimental reactor was continuously exposed

to illumination.

Illumination was supplied by two banks of incandescent

lights each containing 2 x 30-W floodlights (Westinghouse

,I U)
0 -14w
41 0 4 a-
U-- >00 X
(a C4 a 4 4-J > U) H4

ZJ-a: WOa )()() Q -
C) --I (n 3 x (1
E 4 --I C4 r4 () 0 -4
a H00 0 Z (n r U-44-J0
4 4J 4U (n -)CO (1 F
)) C)4-J) Q) '0 04 C r- m4

) W 0U (a(a zH*Ha

30R20, Westinghouse Electric Corp., Bloomfield, New

Jersey). The lights in each bank were 12 cm apart verti-

cally, with the lower light being 12 cm above the base of

the reactor. The banks were spaced 135 degrees apart hori-

zontally from each other, and 30 cm from the side of the


The illuminated reactor was cooled by Dayton Model

4C004A air blowers (Dayton Electric Manufacturing Co., Chi-

cago, Illinois) placed 12 cm from the reactor, one 8 cm and

the other 20 cm above its base. Each blower had a maximum

capacity of 36 L/s at a speed of 2880 rev/min. Airflow

control was effected by a sliding window arrangement which

allowed the temperature to be maintained within the required

range of 27 + 10C. The required temperature of the non-

illuminated reactor was achieved by heating with a single

150-W incandescent floodlight supported on an adjustable

base, which enabled its distance from the reactor to be

varied as necessary.

Continuous mixing was accomplished by magnetic stirrers

(Fisher Thermix Stirrer Model 620T, Fisher Scientific

Company, Fair Lawn, New Jersey). Gas was collected by

displacement of an acidified 5 percent solution of sodium

chloride from 1200 ml nominal capacity graduated gas

collector bottles equipped with septa in the stoppers for

gas sampling.

Reactors were loaded and unloaded by Masterflex

peristaltic pumps (Cole-Parmer, Chicago, Illinois), as

described in section 3.4.2.

3.4 Materials

3.4.1 Substrate

The substrate was swine waste obtained from the

University of Florida's Swine Research Unit (SRU). The

waste was collected from solid floors of barns housing

finishing hogs. These pigs were kept on a high grain

finishing ration of corn and soybean meal. The composition

of the feed at any particular time depended on the experi-

ments being conducted by the SRU. Details of the ration are

included in Table 3-1. Following collection, the waste was

dispensed into 250 mL containers and frozen until ready for

use. When required, the waste was thawed at 4/C, mixed with

tap water and screened by passing through a 105 pm screen

(U.S.Standard Sieve No. 140, Soiltest Inc., Evanston,

Illinois), analyzed, and diluted with tap water to a concen-

tration of 1.0 + 0.1 percent VS. Typical characteristics of

the waste used in these experiments are shown in Table 3.2.

Table 3-1.

Composition of grower/finisher ration used at
the University of Florida's Swine Research Unit
during investigations.




Corn Meal

Soybean Meal



Iodized Salt

Trace Mineral

Vitamin Mix







77 88

9.4 20

0.1 2.8

0.8 1.0

0.2 0.5




0.05 3.0

0.5 1.0

1.4 2.8

0.1 0.3

Note: Additives were not all used at the same time.

Table 3-2. Principal characteristics of swine waste
collected from the confinement units.

Parameter Unit Concentration

COD g/L 17.8 21.6

BOD5 g/L 6.1 8.3

Kjeldahl-N g/L 0.86 1.015

Ammonia-N g/L 0.20 0.45

Total Phosphorus g/L 0.413 0.566

Total Solids g/L 12.1 13.53

Total Volatile Solids g/L 9.1 10.8

Total Suspended Solids g/L 8.5 10.6

Total Sulfide g/L 0.04 0.07

pH 6.8 7.0

Temperature 0C 24 26

3.4.2 Bacterial Inocula Phototrophs

Phototrophic bacterial inoculum for each experiment

consisted of a blend of equal parts of laboratory-cultured

organisms and effluent obtained from the anaerobic lagoon at

the University of Florida's Swine Research Unit. This

lagoon, which receives waste from an average population

of 260 pigs housed on solid concrete slab and slatted

floors, normally contained a dense population of photo-

trophic sulfur bacteria. Their presence was recognized by

the vivid red to purple-pink color imparted to the lagoon. Methanogens and Other Anaerobes

Methanogens and other anaerobes used as inoculum were

obtained from a 20 m capacity standard rate anaerobic

digester treating swine waste. This digester was located

at the SRU.

3.5 Experimental Methods

3.5.1 Start-up Batch Cultures

The experiments were conducted in two series. The

first series extended from September 1983 to March 1985, and

included SRTs of 5, 7, 10, 15, 20, and 30 days. Each trial

consisted of a batch phase which continued until onset of

the stationary period of phototrophic bacterial growth,

which was indicated by a levelling-off of the growth curve.

The batch phase was followed by a continuous-loading phase

during which the reactors were loaded daily at a volumetric

rate consistent with the retention time of the trial. Both

experimental (illuminated) and control (nonilluminated)

reactors were operated throughout each trial in this series.

The second series of trials extended from April 1985 to

September 1985, and included SRTs of 8.5, 10, 15, 20, and

30 days. The 10-d SRT and 20-d SRT trials involved an

initial batch phase followed by a continuous loading phase,

as in the first series. However, only experimental reac-

tors were operated; there were no controls. Following

attainment of steady state conditions, the loading rate of

the 10-d SRT reactor was increased to give a SRT of 8.5-d,

and the loading rate of the 20-d SRT was increased to give a

SRT of 15-d. A final trial at a SRT of 30-d was conducted

by reducing the loading rate of the 15-d SRT reactor after

it had reached steady state.

The growth medium of experiments which started with a

batch growth phase comprised 62.5 percent v/v of swine waste

(prediluted to 0.4-0.6 percent total solids concentration),

25 percent v/v of phototrophic bacterial inoculum, and 12.5

percent v/v of methanogenic inoculum. Temperature and pH

were recorded, and 3.5 L of this growth medium was dispensed

to each reactor. Initial total solids and volatile solids

concentrations were also measured.

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