Citation
Kinetics of swine waste assimilation by phototrophic sulfur bacteria

Material Information

Title:
Kinetics of swine waste assimilation by phototrophic sulfur bacteria
Creator:
Earle, Jonathan F. K., 1940- ( Dissertant )
Koopman, Ben ( Thesis advisor )
Lincoln, Edward P. ( Thesis advisor )
Zoltek, John ( Reviewer )
Nordstedt, Roger A. ( Reviewer )
Smerage, Glen H. ( Reviewer )
Place of Publication:
Gainesville, Fla.
Publisher:
University of Florida
Publication Date:
Copyright Date:
1985
Language:
English
Physical Description:
xiv, 229 leaves : ill. ; 28 cm.

Subjects

Subjects / Keywords:
Acetates ( jstor )
Bacteria ( jstor )
Carbon dioxide ( jstor )
Kinetics ( jstor )
Methane ( jstor )
Photosynthetic bacteria ( jstor )
Species ( jstor )
Sulfides ( jstor )
Sulfur ( jstor )
Swine ( jstor )
Animal waste ( lcsh )
Dissertations, Academic -- Environmental Engineering Sciences -- UF
Environmental Engineering Sciences thesis Ph. D
Photosynthetic bacteria ( lcsh )
Sewage -- Purification ( lcsh )
Sulfur bacteria ( lcsh )
Swine ( lcsh )
Genre:
bibliography ( marcgt )
non-fiction ( marcgt )

Notes

Abstract:
The degree of waste treatment achieved in anaerobic processes is usually much lower than that of aerobic biological processes. However, anaerobic waste stabilization 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, completelymixed 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 steadystate conditions or washout of the phototrophic bacteria had occurred. All experiments were conducted at a controlled temperature of 27 + 1°C, 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 SRT.
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

Source Institution:
University of Florida
Holding Location:
University of Florida
Rights Management:
Copyright [name of dissertation author]. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
Resource Identifier:
029451558 ( AlephBibNum )
AEG6832 ( NOTIS )
014393184 ( OCLC )

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Full Text













KINETICS OF SWINE WASTE ASSIMILATION
BY PHOTOTROPHIC SULFUR BACTERIA







By

JONATHAN F. K. EARLE


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

UNIVERSITY OF FLORIDA


1985























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.















ACKNOWLEDGEMENTS


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

iii









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.

















TABLE OF CONTENTS

Page

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
2.3.2.1 Hydrolytic Bacteria......... 26
2.3.2.2 Acetogenic Bacteria......... 28
2.3.2.3 Methanogenic Bacteria...... 30
2.3.3 The Phototrophic Anaerobes.......... 35
2.3.3.1 General Description........ 35
2.3.3.2 Classification ............. 36
2.3.3.3 Photometabolism............ 39
2.3.3.4 Energetics.................. 45
2.3.3.5 Ecology of Phototrophic
Bacteria.................... 53
2.3.3.6 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
2.5.3.1 Microbial Growth........... 66
2.5.3.2 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
3.4.2.1 Phototrophs................ 84
3.4.2.2 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
4.2.2.1 5-d SRT ..................... 96
4.2.2.2 7-d SRT ..................... 98
4.2.2.3 10-d SRT .................... 98
4.2.2.4 15-d SRT... ................ 103
4.2.2.5 20-d SRT. .................. 103
4.2.2.6 30-d SRT.................... 106









4.2.3 Continuous/Continuous Mode Trials... 109
4.2.3.1 8.5-d SRT................... 109
4.2.3.2 15-d SRT. .................. 110
4.2.3.3 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

CHAPTER 6. CONCLUSIONS AND RECOMMENDATIONS.......... 148

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

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

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

APPENDIX C. MISCELLANEOUS TABLES OF RESULTS.......... 207

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

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


vii
















LIST OF TABLES


Page


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
production..............................

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
investigations..........................

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

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


viii









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
loading.................................

COD available for biomass synthesis....


115


128



129

132















LIST OF FIGURES


Figure

Figure


2-1.

2-2.


Figure 2-3.


Figure 2-4.



Figure 2-5.


Figure

Figure


2-6.

2-7.


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
photosystems..........................

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

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

Schematic diagram of experimental
apparatus..............................

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...............


Page

11


13


15



19



20

25


40



50


52


67


79





97









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..........................

xi


99



100



102



104



105



107



108



111



112



113


116


117


118



121










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 .............................


xii


122



123


125


126


131



134


135




142















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

KINETICS OF SWINE WASTE ASSIMILATION
BY PHOTOTROPHIC SULFUR BACTERIA

By

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


xiii









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

SRT.


xiv















CHAPTER 1
INTRODUCTION


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

operations.









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








5

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







7

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.







8

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

(84,179).

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

treatment.



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

process.



4. Develop a mathematical model for waste degradation

and biomass production in a waste treatment system

incorporating phototrophic sulfur bacteria.














CHAPTER 2
LITERATURE REVIEW


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
0I
Uz


r-
,-4

,(

N -,






u
u






0




(U
El-4


.(










-4
Cr)


N L-
O c.
u0<


co
0


00

, E

0.
UE


O
UI


E




C
"0



> 0
-_ E






12

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).






14
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
2)
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












I COMPLEX ORGANICSI



GROUP 1
Hydrolytic and
Fermentative
Bacteria



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



GROUP 2
H2- Producing
Acetogenic
Bacteria

Acetate! H2 H CO2

GROUP 3
H2-Utilizi ng
Acetogenic
Bacteria


GROUP 4 GROUP 4
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
(200).



















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,
propanol.

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

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

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


Source: Zeikus 1980 (209).




















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


End products


Proteins and other
nitrogenous compounds










Carbohydrates





Fats and related
substances





Nucleic acids, purines,
pyrimidines


Amino acids
Ammonia
Hydrogen sulfide
Methane
CO
H
Alcohols
Organic acids
Phenols
Indole

CO
H
Alcohols
Fatty acids
Neutral compounds

Fatty acids
Glycerol
CO2
H
Alcohols
Lower fatty acids

Amino acids
Lower fatty acids
PO
NH3
CO2


Substrate









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












C\J
0 0
, U
-- E --- --- ----u
L I


L C
C00

u u
m


c J
0 I


o _


o 0r


0 I
>, r



Ul
i 00
.0


S I



o




uu
rof
-. a


UC~
o e'
0




r,4 ,


nq a


j u N

























































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







21

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
m
and higher V ) (149). In such environments, the final
max
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






24

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






/1







EI







CQ
()



*r-

fc|


U
I-



I-,-
w




OU
I <
am


zU

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Z



ma-


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OU
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am









2.3.2 The Nonphototrophic Anaerobes


2.3.2.1. 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.


Group


Generic identity and description


Hydrolytic bacteria




Hydrogen-producing
acetogenic bacteria

Homoacetogenic
bacteria

Methanogens


Sulfate reducers


Majority unidentified Gram-
negative rods;
Clostridium
Eubacterium

Unidentified Gram-negative rods.


Acetobacterium
Clostridium

Methanobacterium
Methanospirillum
Methanococcus
Methanosarcina
Methanothrix


Desulfovibrio
Desulfatomaculum


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.


2.3.2.2. 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

manner:



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.


2.3.2.3. 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
















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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.


Compound


Methanogenic species


H /CO or
Formate/CO2


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


Acetate


Methanobacterium soehngenii
Methanosarcina methanica
Methanosarcina barkeri
Methanococcus mazei

Methanosarcina methanica

Methanosarcina barkeri

Methanosarcina barkeri


Butyrate

Methanol


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

follows:









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


2.3.3.1 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).


2.3.3.2 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.


2.3.3.3 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







40







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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

photosynthesis



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







42











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



Substrate Bacteria Metabolic role


H2
H2S
Na2S203
S
S22


S, NS

S


H donor

H donor


CH4

N2

N2

NH3

Simple organic acids

Amino acids

Peptones

Fats, oils

Sugars

Alcohols

Ketones

Aromatics


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.


Compound


Acetate


Pyruvate




Glucose


Intermediates of
TCA Cycle

Fructose


Fumarate

Glycerol

Malate


Succinate

Propionate


Amino acids

Butyrate

Lactate

Glutamate

Peptone

Ethanol

Formate


Bacterial Genera and Species


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


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

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

Chromatium vinosum


Thiocapsa roseopersicina, Amoebobacter,
Ectothiorhodospira, Chlorobium, Thiocystis

Thiocapsa roseopersicina

Thiocapsa roseopersicina, Chloropseudomonas

Thiocapsa roseopersicina, Amoebobacter,
Ectothiorhodospira

Thiocapsa roseopersicina

Thiocapsa pfenigii, Ectothiorhodospira,
Chlorobvium, Pelodictyon.

Amoebobacter

Ectothiorhodospira

Ectothiorhodospira, Chloropseudomonas

Chlorobium

Chlorobium

Chloropseudomonas

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.


2.3.3.4 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.


Process


Bacterial energy budget for cells grown on
glucose.


Percent Energy (ATP) expended
on each process.


Synthesis

Polysaccharide

Protein

Lipid

Nucleic Acid

Transport into cells


6.5

61.1

0.4

13.5

18.3


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

reaction



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

instances.

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





in

O0
a -^ I r
E m


17O O


0\ z 0
- n nO \ -

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


3- 0 >
IC L.

0

CCM
0I
0 4

:r 41








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




)-V
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

2-9.

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

O O
0
04
+
U)
I 0


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






U c4

., -\

0

0r

00
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.


2.3.3.5 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

(176).

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.


2.3.3.6 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


Municipal



Swine manure





Cattle feedlot

Poultry processing

Fellmongery


Meat-packing




Domestic and
industrial


Lagoon


Lagoon

Lagoon

Lagoon


Activated
sludge


Lagoon





Lagoon

Lagoon

Lagoon


Lagoon




Lagoon


Thiopedia rosea
Chromatium

Chromatium

Thiopedia rosea

Thiocystis
Thiopedia rosea

Chromatium vinosum
Thiocapsa
roseopersicina

Thiopedia rosea
Rhodothece
Chromatium
Thiocapsa
roseopersicina

Thiopedia rosea

Chromatium

Thiocapsa
roseopersicina

Chromatium
Thiopedia rosea
Thiocapsa
roseopersicina

Thiopedia rosea
Chromatium
Thiocapsa
roseopersicina


34, 35


34, 35

35

35


133



98, 141





174

102

33, 98


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

(208).

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

inhibition.



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

(136).










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


(2-9)


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


where


b

dX/dt =


Substrate

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


(2-10)









dS/dt = qX (2-11)



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



where dS/dt = substrate utilization rate,
mass/volume-time

q = specific substrate utilization rate,
time-


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

utilization

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
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-

organisms



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


where p = specific growth rate, time-
1
pmax = maximum specific growth rate, time- con-
stant

K = half velocity constant
s


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


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


(2-19)










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

be neglected and the equation becomes


dS/dt = KSX


where


(2-20)


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


2.5.3. Completely-Mixed Continuous
Culture Model Without Recycle


2.5.3.1. Microbial Growth

Consider the schematic of a continuous culture reactor

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

yields

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


or stated directly


accumulation


= 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,
mass/volume

X = microorganism concentration in reactor
mass/volume

Q = flowrate, volume/time


(2-21)









67







4j'j








U,

X

















-l





0
U







0
0
C:





ro
Q)





Q)
4,
W






'-q
04




0
rr







X, N


0
arl
TT






68


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
c

= 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.






69


From equation 2-27


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


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


Yobs = p (2-29)
q


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)
cC
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
biomass

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/


2.5.3.2. 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
XV


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







71


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

expression



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

1
where P = specific growth rate, time-
1
max= maximum specific growth rate, time-

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

UVA = concentration of un-ionized volatile acids,
mass/volume.


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.







72


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/
volume.

K. = inhibition coefficient for ammonia, mass/-
volume.

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
fermenter.day.










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
V
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
c










X = Y (S Sl
1 b 1-


where S1

K
s
b

8
c
Y

k


X

S
o


= 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.


(2-40)















CHAPTER 3
MATERIALS AND METHODS


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

forces.

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

reactor.

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.


Ingredients


Percent


Basic

Corn Meal

Soybean Meal

Dynafos

Limestone

Iodized Salt

Trace Mineral

Vitamin Mix

Additives

Antibiotics

Selenium

Potassium

Magnesium

Lysine


77 88

9.4 20

0.1 2.8

0.8 1.0

0.2 0.5

0.1

0.1


0.15

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


3.4.2.1 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.


3.4.2.2 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.




Full Text

PAGE 1

KINETICS OF SWINE WASTE ASSIMILATION BY PROTOTROPHIC SULFUR BACTERIA By JONATHAN F. K. EARLE A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 1985

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UNIVERSITY OF FLORIDA , niiiiiu nil " 3 1262 08552 4428

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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.

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ACKNOWLEDGEMENTS 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 available 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 motivational force for the undertaking and completion of this endeavour, and for this I am extemely grateful. My siblings iii

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have been towers of strength to me throughout my studies, providing unselfish support at critical times. My colleagues 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. IV

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TABLE OF CONTENTS Page 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 2.3.2.1 Hydrolytic Bacteria 26 2.3.2.2 Acetogenic Bacteria 28 2.3.2.3 Methanogenic Bacteria 30 2.3.3 The Phototrophic Anaerobes 3 5 2.3.3.1 General Description 35 2.3.3.2 Classification 36 2.3.3.3 Photometabolism 39 2.3.3.4 Energetics 45 2.3.3.5 Ecology of Phototrophic Bacteria 53 2.3.3.6 Natural Occurrence and Role in Waste Treatment Systems. 5' 2 . 4 Process Inhibition 59

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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 2.5.3.1 Microbial Growth 66 2.5.3.2 Substrate Utilization 70 2.5.4 Anaerobic Kinetic Models 70 CHAPTER 3 . MATERIALS AND METHODS 7 5 3.1 Rationale for Experimental Design 75 3 . 2 Summary of Investigations 78 3 . 3 Experimental Apparatus 7 8 3.4 Materials 81 3.4.1 Substrate 81 3.4.2 Bacterial Inocula 84 3.4.2.1 Phototrophs 84 3.4.2.2 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 9 3.6.5 Kjeldahl-N, NH -N and Total P 91 3.6.6 pH 7 91 3.6.7 Absorbance 91 3.5.8 Gas Quantity and Quality 91 CHAPTER 4 . RESULTS 94 4.1 Identification of Phototrophic Bacteria. 4.2 Temporal Variation of Phototrophic Bacterial Population, Gas Production and pH During Experimental Trials , 4.2.1 Experimental Series , 4.2.2 Batch/Continuous Mode Trials...., 4.2.2.1 5-d SRT , 4.2.2.2 7-d SRT 4.2.2.3 10-d SRT , 4.2.2.4 15-d SRT 4.2.2.5 20-d SRT , 4.2.2.6 30-d SRT , 94 95 95 96 96 98 98 103 103 106 VI

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4.2.3 Continuous/Continuous Mode Trials... 109 4.2.3.1 8.5-d SRT 109 4.2.3.2 15-d SRT 110 4.2.3.3 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 CHAPTER 6. CONCLUSIONS AND RECOMMENDATIONS 14 8 6 . 1 Conclusions 148 6.2 Recommendations for Further Research 150 APPENDIX A. RAW DATA 152 APPENDIX B. STEADY STATE RESULTS 198 APPENDIX C. MISCELLANEOUS TABLES OF RESULTS 207 REFERENCES 209 BIOGRAPHICAL SKETCH 228 vn

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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 LIST OF TABLES Metabolic pattern of nonphotosynthetic anaerobic bacteria 16 Representative end products of anaerobic microbial degradation of organic wastes 17 Identification of anaerobic bacterial populations in sewage digesters 27 Methanogenic bacteria isolated in pure cultures from digesting sludge 31 Compounds utilized by methanogenic bacteria as energy sources for methane production 33 Role of compounds metabolized by the phototrophic bacteria 42 Some organic compounds photoassimilated by phototrophic bacteria 43 Bacterial energy budget for cells grown on glucose 46 Species of phototrophic bacteria identified in waste treatment systems.. 57 Composition of grower/finisher ration used at the University of Florida's Swine Research Unit during investigations 82 Principal characteristics of swine waste used as substrate in investigations 83 Operating conditions for chromatographic analysis of gas samples 93 vm

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Table 4-1. Table 4-2 Table 4-3 Table 4-4 Batch growth characteristics of phototrophic sulfur bacteria cultured in swine waste medium 115 Steady state gas production at STP related to COD destroyed 128 Steady state gas production at STP related to volatile solids and COD loading 129 COD available for biomass synthesis.... 132 IX

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LIST OF FIGURES Pa^e Figure 2-1. The anaerobic cycle in nature 11 Figure 2-2. Three-stage biochemical scheme for anaerobic biodegradation 13 Figure 2-3. Nonphotosynthetic bacterial groups involved in anaerobic biodegradation. . 15 Figure 2-4. Interrelationships between methane bacteria and metabolites of the anaerobic carbon cycle 19 Figure 2-5. Approximate percentage distribution of carbon in metabolic end products of anaerobic biodegradation 20 Figure 2-6. The sulfur cycle in nature 25 Figure 2-7. The reductive tricarboxylic acid cycle of green sulfur bacteria 40 Figure 2-8. Simplified comparative illustrations of oxygenic and anoxygenic photosystems 50 Figure 2-9. Scheme for photosynthetic NAD(P) reduction in purple sulfur bacteria. . . 52 Figure 2-10. Schematic of completely-mixed reactor without solids recycle 67 Figure 3-1. Schematic diagram of experimental apparatus 79 Figure 4-1. 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 97

PAGE 12

Figure 4-2. Temporal variation or bchl a, biogas production, and pH during the 7-d SRT trial, Series 1 99 Figure 4-3. Temporal variation of bchl a, biogas production, and pH during the 10-d SRT trial, Series 1 100 Figure 4-4. Temporal variation of bchl a, biogas production, and pH during the 10-d SRT trial, Series 2 102 Figure 4-5. Temporal variation of bchl a, biogas production, and pH during the 15-d SRT trial, Series 1 104 Figure 4-6. Temporal variation of bchl a, biogas production, and pH during the 20-d SRT trial, Series 1 105 Figure 4-7. Temporal variation of bchl a, biogas production, and pH during the 20-d SRT trial, Series 2 107 Figure 4-8. Temporal variation of bchl a, biogas production, and pH during the 30-d SRT trial, Series 1 108 Figure 4-9. Temporal variation of bchl a, biogas production, and pH during the 8.5-d SRT trial, Series 2 Ill Figure 4-10. Temporal variation of bchl a, biogas production, and pH during the 15-d SRT trial, Series 2 112 Figure 4-11. Temporal variation of bchl a, biogas production, and pH during the 30-d SRT trial, Series 2 113 Figure 4-12. Relationship of bchl a to solids retention time 116 Figure 4-13. Relationship of protein to solids retention time 117 Figure 4-14. Relationship of solids concentration to solids retention time 118 Figure 4-15. Relationship of productivity in term of bchl a and protein to dilution rate 121 xi

PAGE 13

Figure 4-16. Relationship of productivity in terms of total solids and volatile solids to dilution rate 122 Figure 4-17. Relationship of productivity in terms of total suspended solids to dilution rate 123 Figure 4-18. Effect of solids retention time on gas production and quality 125 Figure 4-19. Effect of solids retention time on methane production 126 Figure 4-20. Soluble COD and soluble BOD removals related to solids retention time 131 Figure 4-21. Relationship of soluble Kjeldahl nitrogen and soluble phosphorus to solids retention time 134 Figure 4-22. Relationship of ammonia uptake to solids retention time 135 Figure 5-1. Suggested schematic of bacterial interactions during phototrophic anaerobic degradation of organic compounds 14 2 xn

PAGE 14

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 KINETICS OF SWINE WASTE ASSIMILATION BY PHOTOTROPHIC SULFUR BACTERIA By 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 stabilization 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 xm

PAGE 15

conditions was investigated. Two daily fed, completelymixed 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 steadystate conditions or washout of the phototrophic bacteria had occurred. All experiments were conducted at a controlled temperature of 27 + 1°C, 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 SRT. xiv

PAGE 16

CHAPTER 1 INTRODUCTION 1 . 1 Problems Created By Animal Wastes Improper management of the high-strength wastes generated 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, agricultural 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 operations . 1

PAGE 17

2 In a 1978 survey (181), it was indicated that approximately 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 sulfide (H„S), ammonia (NH.,), carbon dioxide (CO„), and methane (CH.) (126,165). Of these gases, H 2 S and NH 3 may be toxic to both man and livestock and are also associated with odor of f ensiveness . 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, trimethylamine, and ethylamine (194).

PAGE 18

1. 2 Waste Management Considerations Conventional techniques of municipal waste management are not appropriate for the very highly concentrated livestock wastes which are encountered. Swine wastes may have chemical oxygen demand (COD) values in excess of 80 000 mg/L, and biochemical oxygen demand (BOD-) 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 available for the management of livestock wastes are (1) utilization 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 characteristics are, in turn, influenced by animal-type, feedtype, 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.

PAGE 19

1 . 3 Anaerobic Processing of Animal Wastes Application of anaerobic biotechnology to the stabilization of organic solids and the treatment of highly concentrated 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 conventional anaerobic digestion as a stabilization process for sewage sludge and for the generation of methane gas. Currently, the technology is being investigated for general application to waste treatment problems. Because of the high total solids (TS) and BODconcentrations 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

PAGE 20

5 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 operated within the mesophilic range, usually at a temperature of 35-37° 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 consequently 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 stabilization 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

PAGE 21

6 from such lagoons can be eliminated by encouraging the development of phototrophic anaerobes (7). 1. 4 Application of Phototrophic Sulfur Bacteria to Waste Treatment Problems Waste stabilization ponds receiving municipal, industrial, 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 situations 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 organics, hydrosulf ides, thiosulf ates , 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 BODj. levels (2000 to greater than 10,000 mg/L) have been successfully treated in photobiological treatment plants utilizing phototrophic bacteria and algae. These include wastes from the

PAGE 22

7 starch, woolwashing, canned food, and pharmaceutical industries (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 phototrophic 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 H ? S is used as electron 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.

PAGE 23

8 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 detoxifiers (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 (84,179). 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 culture as commonly observed in waste stabilization lagoons, must be determined.

PAGE 24

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 treatment. 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 biogasif ication in the anaerobic digestion process. 4. Develop a mathematical model for waste degradation and biomass production in a waste treatment system incorporating phototrophic sulfur bacteria.

PAGE 25

CHAPTER 2 LITERATURE REVIEW 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 microorganisms 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 (CH-), carbon dioxide (CO-), 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 stabilization 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 (C0~). 10

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11 M O a E o V a o o a

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12 In an anaerobic environment, the various microbiological 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 CO~ , 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 1kg ultimate BOD ( BOD ) or COD being 0.348 m (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 system, degradation of complex organics may be conveniently represented by the three-stage biochemical scheme (113,136) illustrated in Figure 2.2 (113,135). The complex organics comprising cellulose, hemicellulose, xylanes, and lignins, together with proteins, lipids and nucleic acids, are hydrolysed, fermented, and mineralized by at least four

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13 ACETATE Acetate Decarboxylation CH 4 . CQ 2 COMPLEX ORGANICS Carbohydrate Protein Lipid Hydrolysis and Permentat ion Patty Ac ids Acetogemc Dehydrogenation Acetogen ic Hydrogenat ion STAGE 1 STAGE 2 H 2 CQ 2 STAGE 3 Reduct i ve Methane Pormati on CH • CQ 2 Figure 2-2 Three-stage biochemical scheme for anaerobic biodegradation (113).

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14 different groups of anaerobes, as shown schematically in Figure 2.3 (209), and represented in Table 2-1. First stage enzymatic hydrolysis of complex organics by inducible peripheral enzymes leads to the production of a range of intermediates, which are subsequently used for energy production 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, CO ? , hydrogen (H-), acetate, longer chain fatty acids, ammonia (NH. ), and 2 ) sulfide (S (4). The fermented end products are selectively metabolized by the second and third groups of bacteria, which together are responsible for the activities of the second stage. These are the obligate H„-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 H„, CO™, H^S, and ammonia (NH. ) (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

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15 iCOMPLEX ORGANICSi I 1 GROUP 1 Hydrolytic and Fermentative Bacteria FERMENTED END PRODUCTS J Longer-chain Fatty Acids, Organic Acids Alcohol NH, ",C0 2 GROUP 2 H 2 Produc i ng Acetogenic Bacteria GROUP 3 Hp-Uti lizi ng Acetogenic Bacteria GROUP 4 Acetate-Utilizing Methanogenic Bacteria GROUP 4 H 2 /C0 2 Utilizing Metha nogen ic Bacteria |ch. I CO 2J i CH 4 * C0 2 | Figure 2-3 Wonphotosynthetic bacterial groups involved in anaerobic biodegradation (200).

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16 Table 2-1. Metabolic patterns of nonphotosynthetic anaerobic bacteria. Bacterial Metabolic Group Substrates utilized Metabolic end products 1. Hydrolytic and fermentative bacteria Polysaccharides Lipids Proteins Acetic acid, H-/C0 2 , buyrate, propionate, methanol, ethanol, propanol. 2 . Hydrogenproducing acetogenic bacteria. butyrate, propionate ethanol, propanol hydrogen, acetate 3 . Homoacetogenic bacteria multior one carbon compounds acetic acid 4 . Methanogenic bacteria acetate, H 2 /C0 2 , methanol carbon monoxide methylamine methane, C0 2 , H 2 Source: Zeikus 1980 (209).

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17 Table 2-2. Representative end products of anaerobic microbial degradation of organic wastes. Substrate End products Proteins and other nitrogenous compounds Amino acids Ammonia Hydrogen sulfide Methane COV Alcohols Organic acids Phenols Indole Carbohydrates Fats and related substances Nucleic acids, purines, pyrimidines COV Alcohols Fatty acids Neutral compounds Fatty acids Glycerol C0 ? V Alcohols Lower fatty acids Amino acids Lower fatty acids P0 4 nh* CO.

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18 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 stabilization 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 methanogenic and sulf atereducing bacteria which utilize H„ , 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, H» and CO ? 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 distribution 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

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19

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20 COMPLEX ORGANICS 76V. 20V. ACETIC ACID HIGHER ORGANIC ACIDS 4 V. 52 V. HYDROGEN CO2 72V. 28 V.. METHANE CH„ Figure 2-5 Approximate percentage distribution of carbon in metabolic end products of anaerobic biodegradation (162).

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21 consume these substrates as energy sources. These are the methanogens, the sulfate reducers (29) and the homoor H„ -consuming acetogens. Phototrophic sulfur bacteria utilize H~ as an electron donor and certain of the other substrates, notably acetate, as carbon source (140). The sulf ate-reducing bacteria will outcompete the methanogens 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 max stages of conversion of organic molecules into COwill be primarily dependent on these organisms. It has been demonstrated (143) that certain species of the sulf ate-reducing bacteria will oxidize longand short-chain fatty acids and some aromatic compounds to CO.,. For these organisms, elemental 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 CO_ by H 2 (76). In sludge fermentation, most of the methane is formed from acetate (162), although formate 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 responsible for production of acetate from which most of the

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22 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 produced by the first three trophic groups above (139). Their metabolic activities also effectively remove H 2 S ' a potent toxicant, from the environment. Like methanogens , phototrophic bacteria are terminal organisms and, under suitable environmental conditions, will be in direct competition with the former for certain substrates, primarily acetate, Hand CO. 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 + H„ in the presence of an H 2 ~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 characteristics of the organic substrate and environmental factors such as pH, light, temperature and oxygen tension. The

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23 bacterial population of such ecosystems may be conveniently divided into two groups based upon their energy metabolism: (1) nonphotosynthetic anaerobes and (2) photosynthetic anaerobes. The nonphotosynthetic anaerobes include the hydrolytic and fermentative, acetogenic, methanogenic, and sulfate-reducing bacteria. The photosynthetic anaerobes include species of the families Rhodospirillaceae, Chromatiaceae, Chlorobiaceae and Chlorof lexaceae. Identification of the various bacterial species encountered in these environments has been based on the isolation, characterization, and enumeration of predominant microbial populations of bottom muds, anaerobic sludge digesters (103105), 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 phototrophic sulfur bacteria which convert COto cell material using H~S as reductant (139). Acetate and other simple organic compounds are readily assimilated by phototrophic

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24 nonsulfur bacteria (140), and certain species of phototrophic sulfur bacteria. In some environments, these phototrophic 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 CO, H~ , NH, , organic acids and alcohols (208). The H~ and some of the other fermentative products are anaerobically oxidized by sulf ate-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 sulf ate-reducing bacterial species Desulf ovibrio . Anaerobic oxidation performed by sulfate reducers results in the formation of H~S and acetate, both utilizable in turn by phototrophic bacteria. The combined activities of sulf ate-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 rot undergo valence changes but alternates between NH-. and the amino groups (R-NH 9 ) in nitrogenous cell material.

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25 I

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26 2.3.2 The Nonphototrophic Anaerobes 2.3.2.1. Hydrolytic Bacteria Hydrolytic bacteria may be Gram-negative or Gram-positive, non-spore-forming or endospore-f orming, 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 H^-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 magnitude greater than in the water column (81). Bacterial species identified include the proteolytic Clostridium spp. , Streptococcus spp. and Eubacterium spp. (19).

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27 Table 2-3. Identification of anaerobic bacterial populations in sewage sludge digesters. Group Generic identity and description Hydrolytic bacteria Majority unidentified Gramnegative rods; Clostridium Eubacterium Hydrogen-producing acetogenic bacteria Unidentified Gram-negative rods Homoacetogenic bacteria Acetobacterium Clostridium Methanogens Methanobacterium Methanospirillum Methanococcus Methanosarcina Methanothrix Sulfate reducers Desulf ovibrio Desulf atomaculum Source: Zeikus 1980 (209).

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28 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 biopolymer destruction by these bacteria. 2.3.2.2. The Acetogenic Bacteria The microbial groups central to anaerobic activity comprise the H~-producing acetogenic bacteria and the H--Utilizing (homo-acetogenic ) bacteria (209) which convert fatty acids and other compounds to acetate, H„, and CO. In order for them to do this, the hydrogen concentration must be kept very low by the methanogens and other H„-utilizing organisms (128). Only a few species of H„producing acetogenic bacteria have been isolated. This group degrades propionate and longer-chain fatty acids, alcohols, aromatic and other organic acids which are produced 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 + H~. The H„ is used by M_;_ omelianskii to reduce COto CH. in this syntrophic relationship. Other examples of obligate hydrogen-producing acetogenic bacteria which can metabolize only

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29 in the presence of H -scavenging bacteria are Syntrophobacter wolinii (14) which will oxidize propionate to acetate + H 2 only if coupled with a ^-utilizing organism such as a methanogen or a sulf ate-reducing bacterium, and Syntrophomonas wolf ei (115) which metabolizes fatty acids of chain lengths up to C„ by 8-oxidation when cocultured with a ^-utilizing organism. Fatty acids with even numbers of carbon atoms such as butyrate, caproate, and caprylate are oxidized to acetate + H_ by this bacterium, and those with an odd number of carbon atoms such as valerate and heptanoate are oxidized to acetate + propionate + H_ (115). Strains of Desulf ovibrio desulf uricans and Desulf ovibrio vulgaris produce H 2 from lactate or ethanol when grown without sulfate in the presence of H~-utilizing methanogens (20). Lactate is degraded to acetate in the following manner : CH 2 CHOHCOO+ 2H 2 > CH 3 C00+ HCO,+ H + + 2H 2 (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 H„+C0-, methanol, and/or multicarbon compounds to acetate (6), have been identified. Not all species of Clostridium , however, are capable of metabolizing H_ (209).

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30 By consuming H, , these bacteria lower the partial pressure of hydrogen in the anaerobic environment sufficiently for other metabolic activities to be continued. 2.3.2.3. Methanogenic Bacteria Methanogens are a diverse group of bacteria with considerable 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 formate (21). In general, they metabolize oneand two-carbon compounds, utilizing H„ , C0 2 , and acetate in the production of CH. and CO-. 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 CH. and C0 2 , while the other produces CH 4 by reducing C0 2 , utilizing H„ or formate (208). The principal users of acetate are the genera Methanosarcina and Methanothrix (161), which produce methane by cleaving the acetate molecule with the formation of CH. from the CH, -group and C0 2 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 (lb2). Species of methanogenic bacteria which have been isolated from digesting sludge are listed in Table 2.4 (201). These organisms include the Methanobacterium species

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31 0) Cn Ti 3 cn 0) P rO P p U) X 3 CO (N
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32 which have minimal nutrient requirements and grow autotrophically on H-+COwith 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 H 2 as an energy source in reducing CO~ to CH. and influence the carbon and electron flow in anaerobic habitats by an interaction termed interspecies hydrogen transfer. Compounds utilized by methanogenic 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 Hin reducing C0 2 to CH 4 , will also utilize formate 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 nitrogen source, phosphate as the source of phosphorus, and sulfide or cysteine as the source of sulfur (102).

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33 Table 2-5. Compounds utilized by Methanogenic bacteria as energy sources for methane production. Compound Methanogenic species H /C0_ or Formate/CO, Acetate Methanobacterium formicicum Methanobacterium thermoautotrophicum Methanobacterium ruminantium Methanobacterium mobile Methanosarcina barkeri Methanococcus vanniellii Methanobacterium soehngenii Methanosarcina methanica Methanosarcina barkeri Methanococcus mazei Butyrate Methanol CO Methanosarcina methanica Methanosarcina barkeri Methanosarcina barkeri Source: Mah and Smith 1981 (102)

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34 Methanogens are extremely sensitive to environmental factors such as oxygen (0~) and require a highly reduced environment for growth, the required oxidation/reduction potential (E ) being in the range -520 to -530 mV (43). For swine waste studied under minimal aeration, it was found that methane production ceased at E of -360 mV. The r c 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 NH-. is considered inhibitory at concentrations of 1500-3000 mg/L, and above 3000 mg/L the ammonium ion becomes toxic regardless 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, 35-40° 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 COproduced. Biodegradable protein is deaminated to produce ammonia which reacts with water as follows :

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35 NH 3 + HOH > NH 4+ + OH(2-2) The OHreacts with COin water to form bicarbonate ions C0 2 + HOH > H 2 C0 3 > H + + HCO (2-3) H 2 C0 3 + OH> HC0 3 + HOH (2-4) 2.3.3 The Phototrophic Anaerobes 2.3.3.1 General Description Phototrophic anaerobes are a physiologically diverse group of Gram-negative aquatic bacteria which perform an anoxygenic type of photosynthesis under anaerobic conditions, 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 bacteriochlorophyll 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

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36 species contain cytochromes, ubiquinones, and nonheme iron proteins as components of their electron transport systems (140). 2.3.3.2 Classification In the classification of phototrophic bacteria, Order Rhodospirillales is divided into two sub-orders, Rhodospirillineae, 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 sulf ide-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 nongas-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

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37 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 COfixation 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 photoassimilate acetate and pyruvate (139). These bacteria are Gram-negative, frequently gas-vacuolated , spherical, ovoid, rod-, vibrio-, or spiral-shaped cells which display heterogeneous guanosine + cytosine (G + C) ratios ranging from 45-70.4 percent. T he family Chromatiaceae comprises two main physiological-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 vertically 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 blooms of Chromatiaceae observed in lagoons, shallow pools and estuarine environments (139).

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38 Suborder Chlorobiineae also contains two families, the Chlorobiaceae (green sulfur bacteria) and the Chloroflexaceae (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 addition, 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 Chloropseudomonas , are nonmotile, frequently gas-vacuolated, spherical, ovoidor rod-shaped cells (140). They are obligately 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).

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39 Chlorof lexaceae (175) are Gram-negative, filamentous, gliding, anoxygenic phototrophs with flexible cell walls. Of this family, only one species, Chlorof lexus 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. 2.3.3.3 Photometabolism During autotrophic growth, photosynthetic fixation of COby the Chromatiaceae is primarily by the reductive pentose phosphate cycle (139). This cycle is of limited importance to Chlorobiaceae which utilize a cycle of reactions involving f erredoxin-dependent carboxylations, catalyzed by pyruvateand 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 COfixation 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 f erredoxin-dependent carboxylations. One complete turn of the cycle produces a molecule of oxaloacetate from four molecules of COp (155). Intermediates of the cycle include precursors for lipids and amino

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40 w i< a. UJ U < o

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41 acids; carbohydrate is formed from pyruvate by reversed glycolysis. 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 COfixation has been made by Van Niel (190) who postulated the unitary concept of photosynthesis C0 2 + 2H 2 A > CH 2 + H 2 + 2A (2-5) where H~A may represent either water, as in the case of green plant photosynthesis, or hydrogen sulfide, as in bacterial photosynthesis. Cell mass is represented by CH_0. 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 compounds 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

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42 Table 2-6. Role of compounds metabolized by the phototrophic bacteria Substrate Bacteria Metabolic role H 2 Na 2 S 2 3 CH N, N 2 NH 3 Simple organic acids S, NS Amino acids Peptones Fats, oils Sugars Alcohols Ketones Aromatics s,

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43 Table 2-7. Some organic compounds photoassimilated by phototrophic bacteria. Compound Bacterial Genera and Species Acetate Chromatium , Chloropseudomonas , Thiocystis Lamprocystis , Thiospirillum , Thiocapsa , Amoebobacter , Ectothiorhodospira , Pyruvate Glucose Intermediates of TCA Cycle Fructose Fumarate Glycerol Malate Succinate Propionate Amino acids Butyrate Lactate Glutamate Peptone Ethanol Formate Chromatium , Thiocystis , Thiocapsa , Lamprocystis , Thiodictyon , Chlorobium , Amoebobacter , Ectothiorhodospira , Chloro pseudomonas . Chromatium , Amoebobacter , Ectothiorhodo spira , Chloropseudomonas , Thiocystis . Chromatium vinosum Thiocapsa roseopersicina , Amoebobacter , Ectothiorhodospira , Chlorobium , Thiocystis Thiocapsa roseopersicina Thiocapsa roseopersicina , Chloropseudomonas Thiocapsa roseopersicina , Amoebobacter , Ectothiorhodospira Thiocapsa roseopersicina Thiocapsa pf eniqii , Ectothiorhodospira , Chlorobvium , Pelodictyon . Amoebobacter Ectothiorhodospira Ectothiorhodospira , Chloropseudomonas Chlorobium Chlorobium Chloropseudomonas Chloropseudomonas Source: Man and Smith 1981 (102)

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44 utilized (185). Sulfide uptake during the metabolism of C0 2 is indicated in the following reaction: 40CO 2 + 21H 2 S + 8NH 3 + 20H 2 O > 8(C 5 Hg0 2 N) + 21H 2 S0 4 (2-6) and for acetate 40C„H.0 o + H„S + 8NH-, > 8(_ c H Q o N) z 4 2. 2 J to o 2 + H 2 S0 4 + 20H 2 O (2-7 From these equations it may be observed that sulfide uptake by phototrophic sulfur bacteria is 21 times greater during COmetabolism than during acetate metabolism (185). The key enzymes involved in photoassimilation of C0„ via the reductive pentose cycle are ribulose 1, 5-diphosphate 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 H 2 S with 2 (82). Ribulose bisphosphate carboxylase of the latter organisms possesses oxygenase activity, and it has been

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45 demonstrated (170) that the cells excrete glycolate in the presence of 0_ . 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 proportional to either the sulfide or the bicarbonate concentration when either is growth limiting. 2.3.3.4 Energetics Microbial growth results from coordinated synthesis of a range of complex macromolecules utilizing an energy source appropriate to the particular organism. During biological oxidations, the energy present in an organic substrate is released by successive dehydrogenations of the carbon chain. Reducing equivalents are removed in pairs and transferred to a final acceptor which may be 0_, in the case of aerobic respiration, inorganic compounds other than 0in 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 phototrophs an energy source external to their environment,

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46 Table 2-8. Bacterial energy budget for cells grown on glucose. Process Percent Energy (ATP) expended on each process. Synthesis Polysaccharide Protein Lipid Nucleic Acid Transport into cells 6.5 61.1 0.4 13.5 18.3 Source: Stouthamer 1973(167)

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47 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 reaction ADP + P. > ATP + H-0 (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.molcannot 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 (oxidative) phosphorylation. In substrate level phosphorylation, in which a phosphate molocule is first added to a substrate, and is then subsequently transferred to ADP to form ATP, one molecule of ATP is synthesized by phosphorylation of one molecule of substrate in the cell (97). Oxidative phosphorylation, which had been hypothesized for some time (119,120) but only recently has been demonstrated

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48 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 potential and a pH gradient across the membrane. These energy-transducing systems in the membrane convert 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 CO-, a major waste product of their cohabitants, using H„S, 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 cyanobacteria, these organisms carry out an anoxygenic photosynthesis 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.

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49 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 bacteria obtain their energy for maintenance by fermentation of storage polysaccharides (11). A comparative illustration of the two types of photosystems is given in Figure 2-8 (50). In bacterial photosynthesis, 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 Photosystem 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 instances . 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

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50 ID d o i C\J d o d d d d + 00 d

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51 2-8) or by a process of reverse electron transfer effected by an external electron donor (5). This is shown in Figure 2-9. 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, H 2 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 biosynthesis must be provided via ATP-dependent reversed electron transport (139). The role of ATP in photometabolism is two-fold. It is required either in forming an "activated" substrate, to bring COfixation 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

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52 a 3 c o •H +J o 3 QJ U Q < a u •H -P im in — O +J (0 O -H a cd -p H u o fC 4-1 X! e 3 x: .h O 3 U3 W I (N
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53 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 bacteriochlorophyll a, and where electron transfer reactions occur (133). In these bacteria, ATP generation by cyclic photophosphorylation is not directly linked to substrate utilization, as is the case with heterotrophic bacteria. 2.3.3.5 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 temperatures ranging from below zero to about 80° 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 (176). Shallow ponds, mud flats, and similar locations which are rich in organic matter, C0 2' H ? ' anc ^ H 2 S ' are i dea -'habitats for phototrophic bacteria that photometabolize all

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54 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 wavelength 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 properties of such bodies of water are important regulatory parameters in the physiology and behaviour of organisms which reside therein. Very dense populations of phototrophic bacteria have been found at various depths in a number of meromictic lakes (132,171,176), which are characterized 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 bluegreen 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). Phototrophs identified in such environments include Chlorobium

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55 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). COis fixed by purple and green sulfur bacteria using H~S or H~ 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 primary production on an annual basis (9). In addition to being grazed by protozoans in the water column (42), organic constituents of dead cells of phototrophic bacteria are decomposed by fermentative bacteria with the formation of CO-, H~, NH^, organic acids and alcohols. When sufficient sulfate is present, sulf ate-reducing bacteria oxidize H„ with formation of H_S and acetate which are subsequently used by phototrophic bacteria. Other anaerobic oxidations are performed by methanogenic bacteria which oxidize H_ and convert CO~ 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.

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56 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 H 2 S 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, H^S, and minimal 0concentration are most favorable for their multiplication (50). Due to the higher tolerance of green sulfur bacteria to H 2 S, they are usually located in habitats below the purple sulfur bacteria. 2.3.3.6 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 intermediates, and benzoates (139). Odors are generally minimized in systems in which they occur due to their metabolism of H„S and other odorous compounds. Phototrophic bacteria, by their ability to utilize potentially toxic products of

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57 Table 2-9. Species of phototrophic bacteria identified in waste treatment systems. Type of Waste Treatment System Phototrophic Bacteria Reference Rendering plant Municipal Swine manure Meat-packing Domestic and industrial Lagoon Petroleum refinery Lagoon Hide-washing plant Lagoon Poultry manure Lagoon Activated sludge Lagoon Cattle feedlot Lagoon Poultry processing Lagoon Fellmongery Lagoon Lagoon Lagoon Thiopedia rosea 34, 35 Chromatium Chromatium 34, 35 Thiopedia rosea 3 5 Thiocystis 3 5 Thiopedia rosea Chromatium vinosum 133 Thiocapsa roseopersicina Thiopedia rosea 98, 141 Rhodothece Chromatium Thiocapsa roseopersicina Thiopedia rosea 174 Chromatium 102 Thiocapsa 33, 98 roseopersicina Chromatium 9 8 Thiopedia rosea Thiocapsa roseopersicina Thiopedia rosea 64, 98 Chromatium Thiocapsa roseopersicina

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58 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. Occurrence of phototrophic sulfur bacteria in a lagoon treating fellmongery wastes (38) correlated with the recorded reduction 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 26.5° C and optimum pH of 7.5 (116), but growth of these phototrophs has been recorded at temperatures ranging from 10-30° 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 8 6 and 85 percent, respectively, were achieved.

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59 2.4. Process Inhibition Varying levels of certain inorganic and organic substances 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 combinations 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 concentrations 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

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60 been observed to have a negative effect on methanogenesis (208). 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 associated 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 sulf ate-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 (10 9) found that volatile acid levels below 2000 mg/L were desirable. This observation was confirmed 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, however, 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

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61 (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 detrimental 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 inhibition. 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.

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62 The theory of growth kinetics in biological systems, is based on growth rate, growth yield, and the relationship between growth rate and an essential nutrientMathematical 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 disadvantage of Monod type models is that the kinetic parameters cannot be obtained for certain complex substrates (136).

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63 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 (2-9) where X = organism concentration, mass/unit volume t = time U = specific growth rate, timeAccounting for the effect of endogenous respiration on the overall growth rate, equation 2-9 becomes dX/dt = (u b)X (2-10) where b = endogenous decay coefficient, timedX/dt = net growth rate of microorganisms per unit volume of reactor, mass/volume-time Substrate utilization rate is considered proportional to the biomass present, as expressed by the equation

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64 dS/dt = qX (2-11) from which q = dS/dt (2-12) X where dS/dt = substrate utilization rate, mass /volume -time q = specific substrate utilization rate, timeTrue growth yield, Y_, is defined as Y = weight of organisms formed T weight of limiting substrate utilized Mathematically, Y^ = dX/dS (2-13) where Y = growth yield constant, mass/mass Combination of equations 2-10, 2-11 and 2-12 provides the relationship between biological growth and substrate utilization dX/dt = Y (dS/dt) bX (2-14) Hence q = u/Y T (2-15) Rate of substrate utilization may also be related to concentration of microorganisms in the reactor and concentration of the growth-limiting substrate surrounding the organisms as

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65 dS = kSX (2-16) dt K + S 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 (l/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 microorganisms \i = u S (2-17) max K + S s where u = specific growth rate, timeumax = maximum specific growth rate, time, constant K = half velocity constant s 1 Combining equations 2-14 and 2-16 yields the expression u = Y_kS b (2-18) s where \i = (dX/dt)/X, net specific growth rate When S is very much greater than K in equation 2-16, K g can be neglected and the equation becomes dS/dt = kX (2-19)

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66 When S is very much less than K , S in the denominator can 1 s be neglected and the equation becomes dS/dt = KSX (2-20) where K = k/K , specific substrate utilization rate constant, volume/mass . time 2.5.3. Completely-Mixed Continuous Culture Model Without Recycle 2.5.3.1. Microbial Growth Consider the schematic of a continuous culture reactor shown in Figure 2-10. A materials balance for bacteria yields microorganism rate of net growth of net rate of accumulation = microorganism + microorganism microorganism within reactor inflow within reactor outflow or stated directly accumulation = inflow + net growth outflow i.e. (dX/dt)V = QX q + u VX QX (2-21) where V = volume of microbial culture in reactor X = microorganism concentration in influent, mass /volume X = microorganism concentration in reactor mass/volume Q = flowrate, volume/time

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to X o" 67 o o o (/I i rH 0) +»
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68 Neglecting microorganisms in influent, assuming steady-state conditions (dX/dt = 0), and substituting for u from equation 2-18, equation 2-21 reduces to 2 " _K T k§ b = 1 (2-22) V K + § 6 s e where = hydraulic retention time, V/Q At steady state, the specific growth rate is equal to the dilution rate, and u 1 1 = _YJcS b (2-23) n 6 6 K + S c s e where 8 = biological solids retention time, time 1/8 = dilution rate, timefrom which we obtain the expression 1 = Y (dS/dt) b (2-24) 8 1 X c = Y T q b (2-25) From equation 2-24 dS 1 . X + bX (2-26) at Y m e T c Net microbial growth may be described by the expression (96) dX = Y . dS (2-27) dt ODS dt where y k s = var i a frl e observed yield coefficient.

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69 From equation 2-27 Y nhQ = (dX/dt) (2-28) S (dS/dt) multiplying the right hand side of expression by X/X gives Yobs = u (2-29) q Substituting dS/dt from equation 2-27 in equation 2-26 _1 . dX = 1 . X + bX (2-30) obs T c This expression reduces to b6 + 1 (2-31) — c Y Y Y obs T T The substrate utilized by an organism may be considered to comprise a variable portion for biosynthesis and a relatively constant portion for maintenance. The total specific substrate utilization rate can therefore be expressed as q = a, + a_ (2-32) ^ lu 2 where a, = substrate utilization to form a unit of biomass U = specific growth rate of biomass a„ = substrate utilized for maintenance, function per unit biomass per unit time, timeq = specific substrate utilization rate, time-

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70 Substituting for q in equation 2-32 from equation 2-29 gives Y , 1 (2-33) obs a, + a 2 /u 2.5.3.2. Substrate Utilization A similar materials balance may be written for substrate within the reactor change within = influent consumption effluent the reactor or V(dS/dt) = QS V(KXS ) QS (2-34) o e e where KXS = 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) — °iar e e 2.5.4 Anaerobic Kinetic Models The anaerobic digestion process has been described by several kinetic models, which have been developed to optimize 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

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71 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 considering un-ionized volatile acids as the rate-limiting substrate and inhibitory agent. This approach yielded the expression M = 1 (2-36) u 1 + K + UVA l where u = specific growth rate, time. ^ m = v = maximum specific growth rate, time. IllaX K = saturation constant, mass/volume. K. = inhibition coefficient of un-ionized volatile acids, mass/volume. UVA = concentration of un-ionized volatile acids, mass/volume. This model was further developed and expanded by Andrews and Graef (3), who considered the effect of interactions between volatile acids, pH, alkalinity, gas production 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.

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72 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 methane-producing bacteria. The resulting expression was U 1 (2-37) u 1 + K + UVA + NH, maX UV*A K~ iCl l i2 where NH-. = concentration of un-ionized ammonia, mass/ volume. K.„ = inhibition coefficient for ammonia, mass/volume. 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 = B S [1 K (2-38) — e *v 1 + K where B = volumetric methane production rate L CH./L f ermenter.day.

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73 S = influent total volatile solids (VS) concentration g/L. B = ultimate CH. yield, L CH./gm VS added 8 = retention time, day. u = 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 60° C. The equation relates the daily volume of methane produced with loading rate (S /6 ) , material biodegradability (B ) and kinetic parameters y and K. For livestock wastes, B r m o 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( 9 c) ] (2-39) 1 6 S (Yk-b)-l c

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74 Y (S S, ) (2-40) where S, = effluent waste concentration, mass/volume. K = half velocity coefficient, mass/volume, s b = microorganism decay coefficient, time9 = biological solids retention time, time, c Y = growth yield coefficient, mass/mass. k = maximum rate of substrate utilization per unit weight of microorganisms, timeX = microbial mass concentration, mass/volume. S = influent waste concentration, mass/volume. In a comparative evaluation of the kinetic constants reported for methanogenic bacteria by several researchers, Scharer and Moo Young (14 8) 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.

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CHAPTER 3 MATERIALS AND METHODS 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 75

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76 the hydraulic retention time. A low mixing speed was selected to achieve gentle continuous turnover of the contents of the reactor without introduction of high shear forces. 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 concentration 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 concentration, 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 suitable 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

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77 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 interpreting 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 relatively stable level of bchl a had been attained. This was considered to be an indication of steady state conditions 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 biogasif ication.

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78 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 temperature of 27 + 1°C 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), biochemical oxygen demand (BOD-), total solids ( TS ) , volatile solids (VS), ammonia-nitrogen (NH,-N), total k jeldahl-nitrogen (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 wherezas 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

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79 a c 0> a 0)

PAGE 95

80 30R20, Westinghouse Electric Corp., Bloomfield, New Jersey). The lights in each bank were 12 cm apart vertically, with the lower light being 12 cm above the base of the reactor. The banks were spaced 135 degrees apart horizontally from each other, and 30 cm from the side of the reactor. The illuminated reactor was cooled by Dayton Model 4C004A air blowers (Dayton Electric Manufacturing Co., Chicago, 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 + 1°C. The required temperature of the nonilluminated 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.

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81 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 experiments 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 urn screen (U. S. Standard Sieve No. 140, Soiltest Inc., Evanston, Illinois), analyzed, and diluted with tap water to a concentration of 1.0 + 0.1 percent VS. Typical characteristics of the waste used in these experiments are shown in Table 3.2.

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82 Table 3-1. Composition of grower/finisher ration used at the University of Florida's Swine Research Unit during investigations. Ingredients Percent Basic Corn Meal 77 88 Soybean Meal 9.4 2 Dynafos 0.1 2.8 Limestone 0.8 1.0 Iodized Salt 0.2 0.5 Trace Mineral 0.1 Vitamin Mix 0.1 Additives Antibiotics 0.15 Selenium 0.05 3.0 Potassium 0.5 1.0 Magnesium 1.4 2.8 Lysine 0.1 0.3 Note: Additives were not all used at the same time.

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83 Table 3-2. Principal characteristics of swine waste collected from the confinement units. Parameter Unit Concentration COD

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84 3.4.2 Bacterial Inocula 3.4.2.1 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 phototrophic sulfur bacteria. Their presence was recognized by the vivid red to purple-pink color imparted to the lagoon. 3.4.2.2 Methanogens and Other Anaerobes Methanogens and other anaerobes used as inoculum were 3 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 Septemoer 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,

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85 which was indicated by a levelling-of f 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 reactors 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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86 Gas production and temperature were monitored daily, and bacteriochlorophyll a (bchl a) concentration and pH were measured at intervals of two days, or as considered appropriate. Upon attainment of the stationary growth phase of phototrophic bacteria, the experiment was continued by loading the reactors once daily with swine waste. 3.5.2 Continuous Mode Solids residence times (SRTs) of 5, 7, 8.5, 10, 15, 20, and 30 days with respective volumetric loading rates of 700, 500, 411.8, 350, 233.3, 175, and 116.7 ml/d were used in these experiments. Each reactor was loaded and unloaded on a daily basis with feedstock which was kept refrigerated and brought to room temperature prior to loading. Each reactor was first unloaded by pumping the required volume of effluent from the reactor. When enough gas was available in the gas collectors, it was drawn into the reactor simultaneously as the effluent was withdrawn, to reduce the possibility of air being drawn into the system. The measured volume of feedstock was then pumped into the reactor from the reservoir, simultaneously replacing the gas which had been drawn in during the unloading process. This arrangement ensured that the space above the liquid in the reactors was saturated with reactor gas. This was not always possible, and it sometimes became necessary to draw a certain volume of air into the reactor during unloading due

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87 to shortage of reactor gas. This volume was subsequently displaced from the reactor during loading. The volume of gas produced by each reactor in the 24-hour interval between loading was monitored daily by recording the volume of water displaced from a graduated cylinder. Reactor temperature was monitored at least twice daily; pH and bchl a concentration were generally measured at intervals of two days, except in situations where it became necessary to measure these parameters daily, or at other intervals. Gas was sampled and analyzed at intervals during both batch and continuous phases. The samples were withdrawn via a septum in the stopper of the gas collector using 1 cc Tuberculin 26G 3/8 disposable hypodermic syringes (Becton, Dickinson and Company, Rutherford, New Jersey 07070). The gas-filled syringes were sealed immediately upon withdrawal from the septum by inserting the tip of the needle into a rubber plug. They were removed from this plug immediately prior to injection of the gas sample into a gas chromatograph . Phototrophic bacterial activity was monitored by bchl a concentration. Upon attainment of steady state conditions in the illumiated reactor, which was identified by a leveling-off of bchl a concentration over a period of several days, analyses were carried out on samples from each reactor. Four complete sets of analyses were carried out for

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88 each steady state condition over a period of 8 days. With the exception of phosphorus, analyses of all samples were initiated within one hour of withdrawal from the reactors. Samples for phosphorus determination were filtered and acidified to pH 2 . and stored at 4°C until analysed. Gas samples were also analysed for methane content at these times. 3 . 6 Analytical Techniques 3.6.1 Bacteriochlorophyll a Bchl a was measured using a modified version (44) of the procedure described by Siefert et al. (151). For analysis, 10 mL of a ten-fold diluted sample was centrifuged for 15 minutes at 2400xg and the centrate decanted. The pellet was washed by adding 10 mL of distilled water to the centrifuge tube, resuspending the pellet by manual shaking and centrifuging again for 15 minutes. The centrate was decanted and 10 mL of a 7+2 (v/v) solution of acetone/methanol was added to the pellet in the centrifuge tube and the tube shaken vigorously to resuspend the particulate matter. Extraction was carried out in the dark at room temperature for 30 minutes. The sample was then centrifuged again for 15 minutes, and absorbance of the centrate was measured at a wavelength of 770 nm, with an additional reading at 850 nm to correct for turbidity. The bchl a concentration, in mg/L, was computed from the following equation:

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89 bchl a = 12.1 x (D ?70 D 85Q ) x F (3-1) where D 77Q = absorbance at 770 nm, D 85Q = absorbance at 850 nm, and F = (v/V) x 1 in which v = volume of acetone/methanol extract (mL), V = sample volume (mL) , and 1 = path length (cm) . 3.6.2 Sulfide Sulfide concentrations were measured by the methylene blue method (Section 427C) of Standard Methods (166). Samples were analyzed immediately following collection. A . 5 mL volume of an amine-sulfuric acid reagent was added to 7.5 mL of sample followed by addition of 0.15 mL FeCl., solution. The presence of sulfide was indicated by the appearance of a blue color. Five to ten minutes were allowed for color development then 1.6 mL of diammonium hydrogen phosphate solution was added. This produced a white precipitate leaving a clear blue supernatant for color determination, the absorbance of which was read at 664 nm. A blank was prepared at each analysis and used for standardization. This was done by substituting . 5 mL 1 + 1 H-SO. for the amine-sulfuric acid reagent in a measured 7.5 mL sample and following all other procedures as above. Sulfide concentrations were determined from a standard curve. At least one standard was analyzed with each batch of samples.

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90 3.6.3 Protein Crude protein was computed on the basis that protein comprised 16 percent nitrogen. It was determined from the relationship: Crude protein = 6.25 x particulate TKN. (3-2) 3.6.4 BOD ,. , COD, TS, VS and TSS These parameters were determined by the procedures given in Standard Methods (166). BOD,, was determined by the procedure in Section 507, using the azide modification of the titrimetric iodometric method (Section 421B), and the dichromate reflux method (Section 508A) was used for COD determinations. TS, VS and TSS were determined by the procedures of Sections 209A, 209E and 209D, respectively. A double filter technique entailing use of a 1.2 p pore size Whatman GF/C filter (Fisher Scientific Company, 711 Forbes Avenue, Pittsburgh, Philadelphia 15219) overlying a 0.45 urn pore size GN-6 Metricel membrane filter (Gelman Sciences, Inc., Ann Arbor, Michigan 48106), was used in preparation of the filtered samples. A similar technique was used for particulate determinations.

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91 3.6.5 Kjeldahl-N, NH -, -N and Total P Total K jeldahl-nitrogen and ammonia-nitrogen were determined by adaptation (44) of the micro-K jeldahl procedure (Sections 351.2 and 350.2 respectively) of the EPA's Methods for Chemical Analysis of Water and Wastes (183). Total phosphorus was determined by the procedures outlined in Standard Methods (166), using persulfate digestion (Section 424C-III) and ascorbic acid (Section 424F) methods. 3.6.6 p_H pH was monitored by an electrode-analyzer system (701A, Orion Research Inc., Cambridge, Massachusetts). 3.6.7 Absorbance Optical density at wavelengths specified by the colorimetric procedures was measured by absorption spectrometry (Bausch and Lomb Spectronic 70, Bausch and Lomb, Marietta, Georgia) . 3.6.8 Gas Quantity and Quality Daily gas production was measured by water displacement. Gas samples were analysed on a Gow Mac Series 550 gas chromatograph (GO equipped with a thermal conductivity detector (TCD) (Gow Mac Instrument Company, Madison, New Jersey). Bottled methane and carbon dioxide gas were used

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92 for preparation of standard curves. Peak heights were recorded on a nonintegrating chart recorder (Omniscribe Model 5710-5, Houston Instrument Company, Bellaire, Texas). Operating conditions of the GC are listed in Table 3-3.

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93 Table 3-3. Operating conditions for chromatographic analysis of gas samples. Parameter Characteristic Column 6.3 5 mm x 2.4 4mm Packing Poropak Q Support 50-80 Mesh Detector Thermal conductivity (TCD) Carrier Gas Helium Gas flowrate 70 ml/min 2 Gas pressure 28124 kg/m Injection port temperature 90 C Column temperature 75 C Detector temperature 135 C Bridge Current 195 mA

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CHAPTER 4 RESULTS 4.1. Identification of Phototrophic Bacteria The illuminated laboratory cultures varied in color from a vivid purple-red to reddish-brown at peak bchl a concentrations. Microscopic examination of these cultures revealed small non-motile coccoid cells approximately 1-2.5 Urn in diameter as the dominant species of phototrophic bacteria. These were tentatively identified as Thiocapsa roseopersicina . Under the microscope the dominant species of phototrophs in the lagoon effluent used as inoculum were observed to be in sheet-like cell clusters which is a typical characteristic of Thiopedia rosea . Enrichment of samples from the lagoon and the laboratory reactors were carried out at Southern Illinois University by Professor M.T. Madigan. An enrichment medium containing citric acid was used. Examination of the enriched cultures from the lagoon confirmed the presence of cells in tetrads which were free of gas vacuoles and were encased in a slimy outer layer. These were considered to be Thiopedia / Thiocapsa type organisms. Examination of an enriched laboratory culture indicated the presence of spherical cells with no evidence of internal sulfur 94

PAGE 110

95 granules. These were considered to be sulfur-free cells of Thiocapsa . In addition the laboratory sample contained large numbers of organisms similar in appearance and characteristics to the nonsulfur purple bacteria species Rhodopseudomonas palustris and Rhodopseudomonas sphaeroides (Professor M.T. Madigan, personal communication 1985). This enrichment was made from the reactor contents a few days after termination of the final experiment. Indications are that sulfide had been depleted at the time the sample was taken, leading to the proliferation of the nonsulfur species of bacteria. 4 . 2 Temporal Variation of Phototrophic Bacterial Population, Gas Production and pH During Experimental Trials 4.2.1 Experimental Series Variation of bchl a, gas production and pH during the experimental trials is described in this section. The trials were conducted in two series, the first of which lasted from September 1983 to March 1985, and the second from April 1985 to September 1985. Most of the trials consisted of an initial batch phase followed by a continuous loading phase (batch/continuous mode trials). In the first series of experiments all trials were of this type and these results are described first. The retention times for some trials were achieved by a change of loading rate between one continuous phase and another (continuous/continuous mode

PAGE 111

96 trials). Most of the experiments in the second series were of this type, and these results are presented separately. It should be noted that in the second series, which also included batch/continuous trials, no control reactors were operated. 4.2.2 Batch/Continuous Mode Trials 4.2.2.1 5-d SRT Plots of bchl a, gas production and pH in the experimental and control reactors during the 5-d SRT trial (Series 1) are shown in Figure 4-1. Following inoculation, the batch phase was continued for 22 days. From an inoculum level of 16.3 mg bchl a/L, the bchl a concentration attained a maximum level of 58.5 mg/L in the experimental reactor (ER) while declining to 10.9 mg/L in the control reactor (CR). Gas production during the batch phase peaked at 25.8 mL/h in the ER and 28.9 mL/h in the CR. Maximum pH values attained during this phase were 7.2 and 7.0 in the ER and CR, respectively. Upon commencement of continuous loading, bchl a concentrations declined exponentially in both reactors until the trial was terminated. Final bchl a levels were 2.42 mg/L and 1.06 mg/L in the ER and CR respectively. pH fell in both reactors also, ending at a value of 5.9. In the continuous mode, gas production in the ER reached a maximum of 16.6 mL/h 3 days after commencement of daily loading but

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97 5.0 Batch Phase i Continuous Phase >\< 30.020.0< 10.0o.o 1 60.0o ER 40.0 £ i u CD 20.0 0.0«Figure 4-1. TIME. d. 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).

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98 subsequently declined to zero. Gas production in the CR increased to a peak of 27.8 mL/h one day after commencement of daily loading but declined to zero 16 days later. 4.2.2.2 7-d SRT Bchl a, gas production and pH are plotted versus time in Figure 4.2. The initial bchl a concentration was 8.5 mg/L. The bchl a concentrations at the end of the batch phase were 5 9.3 mg/L in the ER and 6.7 mg/L in the CR. Upon commencement of daily loading, these values declined to 4.2 mg/L and 0.6 mg/L in the ER and CR, respectively, over a period of 19 days. In the batch phase, gas production peaked at 20.2 mL/h in the ER and 32.8 mL/h in the CR after 12 days. Gas production then declined sharply, but this decline was arrested on commencement of continuous loading. During the latter phase, gas production peaked at 3 9.5 mL/h in the ER and 6 4.1 mL/h in the CR. pH ranged between 6.6 and 7.2 in both reactors throughout the experiment. 4.2.2.3. 10-d SRT Two experimental trials involving both batch and continuous phases were conducted at the 10-d SRT. In the first trial (Figure 4-3), bchl a in the ER increased from an initial level of 9.5 mg/L to a peak of 45.6 mg/L at the end of the batch phase. With continuous loading, bchl a

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99 7.5 6.5 60.0 ' 40.0 < 20.00.0 L Continuous Phase < H 60.040.0o o ER a a CR ^QQon^^ i j_ Figure 4-2 20 30 40 TIME, d. Temporal variation of bchl a, biogas production and pH during the 7-d SRT trial, series 1.

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100 7.5 5 70 6.5 L 60.0 Batch Phase | < > \
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101 declined to a steady state value of 28.5 mg/L. In the CR, bchl a remained relatively stable at the inoculum level during the batch phase but subsequently declined to a value of 0.6 mg/L after continuous loading was begun. Gas production was relatively low in each reactor during the batch phase. Following respective peaks of 16.6 mL/h and 25 mL/h, production in the ER and CR declined to 0.0 and 3.5 mL/h, respectively, at the end of the batch phase. On commencement of continuous loading, rapid gas evolution was observed in both reactors, and respective production peaks of 35.5 mL/h and 46.0 mL/h were attained in the ER and CR. Steady state gas production was 21.0 mL/h in the ER and 36.3 mL/h in the CR. pH ranged 7.4 to 6.6 in the ER and from 7.2 to 6.7 in the CR. In the second trial at this SRT, no control reactor was operated. Bchl a increased from the inoculum level of 4.8 mg/L to a batch phase peak of 36.8 mg/L at day 12 (Figure 4-4). Following an initial period of decline at the start of continuous loading, bchl a increased further to a peak of 40.5 mg/L at day 31, eventually stabilizing at a steady state value of 35.5 mg/L. P H declined from 7.4 at the start of the trial to 6.7 at the end. There was no gas production during this trial.

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102 I a 6.5 Batch Phase 5.0 -* u) 0.0 < Continuous Phase 40.0 30.0 E °l 20.0 I U CD 10.0 0.0 L Figure 4-4 / ER o ER 10 20 30 40 50 TIME, d. Temporal variation of bchl a, biogas production and pH during the 10-d SRT trial, series 2.

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103 4.2.2.4 15-d SRT The temporal variation of bchl a, gas production and pH during the 15-d SRT trial (Series 1) is shown in Figure 4-5. Bchl a in the ER increased from an inoculum level of 13.5 mg/L to a peak of 4 3.6 mg/L after 13 days of batch operation. Bacterial growth continued with the commencement of continuous loading, attaining a peak of 52.8 mg/L which proved to be the steady state concentration. In the CR, bchl a remained near the inoculum level during the batch phase. On commencement of continuous loading, bchl a decreased gradually to 1.2 mg/L. Peak gas production rates of 26.0 mL/h and 30.0 mL/h were attained in the ER and CR, respectively, during the batch phase. During the continuous phase, the corresponding peak values were 44.2 mL/h and 51.0 mL/h. Steady state values were 40.0 mL/h and 47.0 mL/h, respectively. The pH in both reactors declined from an initial value of 7.5 to a steady state value of 7.0. 4.2.2.5 20-d SRT Two experimental trials involving both batch and continuous phases were conducted at the 20-d SRT. During the batch mode of the first trial, bchl a increased to a maximum of 4 3.2 mg/L in the ER and 5.5 mg/L in the CR (Figure 4-6). The phototrophic inoculum level was 4 . 2 mg bchl a/L. Bacterial growth in the ER continued with

PAGE 119

104 — r 7.5I a 7.0 6.6 L "0-°--o--o— -o.-a_.o-. \ CR 60.0Batch Phase Continuous Phase 40.010 < 20.0/"Vv ^fVW 0.0 60.0-o ER a O CR TD— a. a— a~ '^o.-a— r -o— o o 0.0 Ll_ a— q-o | Figure 4-5 10 20 30 40 50 TIME, d. Temporal variation of bchl a, biogas production and pH during the 15-d SRT, series 1.

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105 o ER ^^^*o-trOoq t Joo OGo _ o _ o ^.^ ( ^ 0.0 LL o-o-o-a-a — aoa j_ 10 20 30 40 50 Figure 4-6 TIME, a. Temporal variation of bchl a, biogas production and pH during the 20-d SRT trial, series 1.

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106 commencement of daily loading, attaining a peak value of 56.2 mg/L, before stabilizing at 53.5 mg/L. Bchl a concentration in the CR declined during the continuous loading phase to a value of 2.1 mg/L. pH values in the ER and CR followed almost similar trends during the first 20-d SRT trial. In the ER, pH increased gradually fron an initial value of 7.2 to a final value of 7.4. In the CR, pH declined initially from 7.2 to 6.9, then increased to a final value of 7.2. Gas production increased steadily during both the batch and continuous phases. Peak rates of 41.5 mL/h and 3 6.5 mL/h were attained in the ER and CR, respectively. In the second trial conducted at 20-d SRT, no control reactor was monitored (Figure 4-7). From an inoculum of 4.8 mg/L, bchl a concentration increased to a peak of 36.5 mg/L during the batch phase. After several days of continuous loading, a further increase of bchl a was noted. The steady state bchl a concentration was 45.3 mg/L. No gas production was detected during this trial. The pH was relatively steady during this experiment, averaging 7.2 over the period of steady state analyses. 4.2.2.6. 30-d SRT Results of the 30-d SRT, Series 1 trial are plotted in Figure 4-8. During the batch phase, which lasted for 18 days, bchl a peaked at 61.1 mg/L in the ER while declining

PAGE 122

107 7.5 •* 40.030.0 20.0 Continuous Phase en E 1 u
PAGE 123

108 7.5 I 7.0 ER \ 6.5 L 40.0 Pa-cr P j/^^P°° a o^ri aP^ a a 20.0 to < /W*k^ 0.0 80.0 60.0 E °l 40.0I U 20.0 o ER Figure 4-i 20 40 60 TIME, d. Temporal variation of bchl a, biogas production and pH during the 30-d SRT trial, series 1

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109 to 2.4 mg/L in the CR. Inoculum concentration was 5.8 mg bchl a/L. During the batch phase, pH increased to 7.5 in the ER, and declined 6.6 in the CR. Initial pH was 6.9. Gas production in the ER was poor, peaking at 5.2 mL/h and declining subsequently to zero after 17 days of batch operation. Gas production in the CR peaked at 22 mL/h then declined to 8.5 mL/h at the end of the phase. In the continuous phase bchl a in the ER peaked at 85.1 mg/L, then declined to a steady state bchl a concentration of 66 mg/L. In the CR bchl a fell gradually to 0.6 mg/L by the end of the trial. After an initial climbing trend, pH was relatively steady, averaging 7.3 in the ER and 7.1 in the CR over the period of steady state analyses. There was no gas production in the ER during the continuous phase of this trial. In the CR, gas production peaked at 46.5 mL/h and declined subsequently to 29.5 mL/h. 4.2.3. Continuous/Continuous Mode Trials 4.2.3.1. 8.5-d SRT Evaluation of bacterial response to continuous loading at this residence time was commenced from the steady state conditions which existed at the end of the 10-d SRT trial in the second series of experiments. After the loading rate was increased, bchl a declined from the 10-d steady state value of 34.5 mg/L to 2.5 mg/L over a period of 33 days

PAGE 125

110 (Figure 4-9). pH fell from 6.5 to 5.9 during this period. No gas production was observed. 4.2.3.2. 15-d SRT In the second trial at 15-d SRT, continuous loading commenced at the end of the 20-d SRT (Series 2) trial. After the loading rate was increased bchl a decreased from an initial level of 45.5 mg/L to a steady state value of 38.8 mg/L (Figure 4-10). pH stabilized at an average of 6.9 after starting at 7.2. No gas production was evident during this trial. 4.2.3.3. 30-d SRT The 30-d Series 2 trial followed at the end of the 15-d Series 2 trial. Bchl a was 3 8.8 mg/L and pH was 6.9 before the loading rate was changed. After the decrease in loading rate, bchl a increased to a value of 6 0.5 mg/L (Figure 4-11). pH fluctuated somewhat, eventually stabilizing at 6.7. No gas production was observed during this trial. 4 . 3 Growth Characteristics of Phototrophic Bacteria 4.3.1. Batch Growth Characteristics In trials having a batch mode, bchl a at first increased exponentially with time. The rate constant characterizing initial growth was determined by fitting an

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Ill I a. 7.0r 6.5 6.05.5 5.0 E 0.0 to < ER 40.0 Cont i nuous Phase 30.0 E a| 20.0 X u (0 10.0o ER 0.04: 10 30 40 Figure 4-9 20 TIME, d. Temporal variation of bchl a, biogas production and pH during the 8.5-d SRT trial, series 2.

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112 7.5r TIME, d. Figure 4-10 Temporal variation of bchl a, biogas production and pH during the 15-d SRT trial, series 2.

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113 TIME, d. Figure 4-11 Temporal variation of bchl a, biogas production and pH during the 30-d SRT trial, series 2.

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114 exponential relationship to the initial growth data. This relationship had the form; y = y Q e (4-i) where t = time (days), y = bchl a concentration (mg/L) at time t, y = bchl a concentration at time t , and u = rate o — o m constant characterizing initial (exponential) growth. The parameter u is an estimate of the maximum specific growth rate of the phototrophic bacteria under conditions (illumination, waste composition, temperature, pH). Estimates of maximum specific growth rate are given in Table 4-1. Values tended to fall either in a lower range (0.04-0.05 d1 ) or a higher range (0.10-0.12 d1 ). The theoretical washout SRT for the higher range of would be 3 m equal to 1/0.11 dor 9 . 1 days. The overall average for U m was 0.083 d, which corresponds to a theoretical washout SRT of 12.0 days. 4.3.2. Steady State Growth Kinetics Steady state biomass concentrations measured in terms of bchl a and protein, total and volatile solids, and suspended solids are plotted versus solids retention time in Figures 4-12 and 4-13 and 4-14 respectively. Bchl a in the ER, averaged over the two trials, increased from 32 mg/L to 63 mg/L for detention periods 10 to 30 days and showed a

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115 Table 4-1. Batch growth characteristics of phototrophic sulfur bacteria cultured in swine waste medium. SRT Series daypH range Bchl a range mg/L 5

PAGE 131

70.0 116 60.0 50.0 40.0 e i u CO 30-0 20-010.00.0o oER o Series 1 • Series 2 CR a Series 1 a a r _i_ 10 15 20 SOLIDS RETENTION TIME,d 25 J J 30 Figure 4-12. Relationship of bchl a to solids retention time .

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117 C71 z UJ Io ir Q. 6.0 5.0 4.0 3.0 2.0 1.0 0.0 _l_ — oER

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118 15. Or O.O 1 10.0TSS 5.0 O.oLl 10 ER o Series 1 • Series 2 CR a Series 1 jl 15 20 25 SOLIDS RETENTION TIME, d J_l. 30 Figure 4-14 Relationship of solids concentration to solids retention time.

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119 sharp decline below a detention time of 10 days (Figure 4-12). With washout occurring at the 8.5 days SRT trial, the minimum detention time lies between 8.5 and 10 days. Bchl a in the CR was nearly zero over the range of SRTs investigated. Protein in the ER increased significantly with retention time in both series of experiments. In Series 1, protein increased from 3.0 g/L at 10 days to 4.6 g/L at 30 days. Corresponding values for Series 2 were 4.4 g/L and 4.9 g/L (Figure 4-13). In the CR this parameter remained constant at approximately 1.9 mg/L. Total, volatile and suspended solids variations all indicated similar response patterns to varying SRTs (Figure 4-14). During the first trial, TS in the ER, declined initially from 10 g/L at 10 days to 8.4 g/L at 15 days, followed by an increase to a maximum value of 13.9 g/L at 30 days. Likewise, VS decreased initially from 7.1 g/L at 10 days, to 6 . 3 g/L at 15 days and then increased to 10.5 g/L at 30 days. The corresponding TSS values were 8.4, 6.2, and 13.1 g/L at 10, 15, and 30 days, respectively. In the CR, the pattern of behavior was similar, with the exception that the maximum values for all parameters occurred at the minimum SRT. Greatest divergence between these parameters in the ER and CR occurred at 30-d SRT where differences of 5.5, 5.7, and 6.1 g/L in TS, VS, and TSS concentrations, respectively, were observed. Variation of solids concentration in the ER with SRT was not significant

PAGE 135

120 in the Series 2 experiments. TS concentration varied from 11.8 g/L at 10 days to 13.3 g/L at 30 days. Correspondingly, VS ranged 9.7 to 10.2 g/L, and TSS ranged 11.8 to 12.6 g/L. 4.3.3. Biomass Productivity Productivity expressed in terms of bchl a and protein, total and volatile solids, and suspended solids is plotted versus dilution rate in Figures 4-15, 4-16 and 4-17, respectively. In Series 1 experiments, bchl a productivity in the ER increased from 7.7 mg/d (2.2 mg/L-d ) at a dilution rate of 0.033 d1 to a peak of 12.2 mg/d (3.5 mg/L-d) at dilution rate 0.067 d, subsequently declining to 2.9 mg/d at dilution rate 0.10 d(Figure 4-15). All other parameters showed increased productivity from the lowest to the highest dilution rate during Series 1. In the ER, protein increased from 0.5 g/d (0.14g/L-d) to 1 . g/d (0.29 g/L-d ) , TS productivity ranged 1.6 to 3.5 g/d (0.46 to 1.0 g/L-d), VS productivity ranged 1.2 to 2.4 g/d (0.34 to 0.69 g/L-d), and TSS productivity ranged from 1.5 to 2.9 g/d (0.43 to 0.83 g/L-d). In the CRs only bchl a showed no increase in productivity with increased dilution rate. Productivity was generally less in the CR than in the ER, the greatest differences being observed at the lower dilution rates. At the higher dilution rates, TS, VS and TSS productivities in the ER and the CR are close.

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121 ER

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122 3.0 2.0"O > 1.0 0.0 4.0 3.0 en 2. Olio 1.0o.o 1 ER Series 1 • Series 2 CR a Series 1 0.0 Figure 4-16 J_ 0.025 0.05 -1 0.075 0.10 DILUTION RATE, d" Relationship of productivity in terms of total solids and volatile solids to dilution rate.

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123 5.04.0 3.0 T3 C7I IS) 2.0 1.0ER o Series 1 • Series 2 CR a Series 1 0.0 _l_ 000 Jj 0.025 0.050 0.075 DILUTION RATE, d~ 1 0.10 Figure 4-17 Relationship of productivity in terms of total suspended solids to dilution rate.

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124 In Series 2 experiments, productivity in the ER increased for all parameters from the lowest dilution rate to the highest. Bchl a productivity increased from 7.10 mg/d (2.03 mg/L-d ) at dilution rate 0.033 dto 10.02 mg/d (2.86 mg/L-d) at dilution rate 0.10 d. Corresponding ranges for protein, TS, VS and TSS were 0.57 to 1.55 g/d (0.16 to 0.44 g/L-d), 1.56 to 4.47 g/d (0.45 to 1.28 g/l-d), 1.19 to 3.40 g/d (0.34 to 0.97 g/L-d) and 1.47 to 4.14 g/d (0.42 to 1.18 g/L-d), respectively. 4.4. Waste Conversion 4.4.1. Gas Production and Quality. Plots of steady state gas production and quality are shown in Figure 4-18 for both the ER and CR. All values were adjusted to a pressure of 1 atmosphere and temperature 0°C. Gas production in the CR was greater than that in the ER at every SRT used, although the volume of methane produced in each reactor (Figure 4-19) was very nearly the same. There is similarity between both curves, each showing a peak at about 15-d SRT. Peak gas production values were 1.07 L/d (0.30 L/L-d) in the CR and 0.90 L/d (0.26 L/L-d ) in the ER, equivalent to 0.63 L/d (0.18 L/L-d) and 0.61 L/d (0.17 L/L-d) methane, respectively. Gas production in the ER declined to zero at 30 days, while that in the CR levelled off to a rate of 0.65 L/d (0.19 L/L-d). Methane content of the reactor gas increased with retention time for

PAGE 140

125 60.0 < 40.0 I u 4 20.0 0.0 ER o Series 1 • Series 2 CR O Series 1 1-00 to < _i < o 0.50 0.00 -L _1_ 10 15 20 25 SOLIDS RETENTION TIME, d 30 Figure 4-18. Effect of solids retention time on gas production and quality.

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126 0.25 r 0.20z o Iu 3 Q o a. u z < I tUJ 0.150.100.050.010 15 20 25 SOLIDS RETENTION TIME, d. 30 Figure 4-19. Effect of solids retention time on methane production.

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127 both reactors, ranging 64-71 percent in the ER and 53-70 percent in the CR. The peak value of 71 percent occurred in the ER at 20 days. Percentages of C0~ showed only small variation over the range of detention times, lying between 22 and 30 percent in the CR and between 20 and 21 percent in the ER. Total gas production related to soluble COD destroyed is tabulated in Table 4-2. For the ER the values ranged from 0.08-0.29 L gas/g COD destroyed, and in the CR, the corresponding values were 0.18-0.40 L gas/g COD destroyed, corresponding to 0.05-0.21 and 0.10-0.28 L methane/g COD destroyed, respectively. Methane production related to COD and VS loading is indicated in Table 4-3. For COD loading which decreased from 6.7 g/d at 10-d SRT to 2 . 3 g/d at 30-d SRT, methane production in the ER increased from 0.04 L/g COD added (0.01 L/L-g) at 10 days to 0.18 L/g COD added (0.05 L/L-g) at 20 days reducing to zero at 30 days. In the CR, the values ranged from 0.06 L/g COD added (0.02 L/L-g) at 10 days, to 0.20 L/g COD added (0.06 L/L-g) at 30 days. VS loading ranged from 3.2 g/d at 10 days to 1.2 g/d at 30 days. Methane production in the ER was 0.0 9 L/g VS added (0.03 L/L-g) at 10 days increasing to o.36 L/g VS added (0.10 L/L-g) at 20 days, and reducing to zero at 30 days. In the CR, methane production values were 0.14 L/g VS added (0.04

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128 Table 4-2. Steady state gas production at STP related to COD destroyed. Parameter Reactor

PAGE 144

129 Table 4-3. Steady state gas production at STP* related to volatile solids and COD loading Parameter

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130 L/L-g) at 10 days, increasing to 0.39 L/g VS added (0.11 L/L-g) . 4.4.2. Oxygen Demand Comparison of soluble BOD,. and COD removals in the ER and CR at various dilution rates is given in Figure 4-20. In each reactor soluble removal rates increased with retention time. At 10 days, with influent COD level of 19200 mg/L, corresponding to a loading of 6.71 gCOD/d (1.92 gCOD/L-d), percentage removals were 84 and 66 for the ER and CR, respectively, representing the maximum total daily COD removals of 5.6 3 gCOD/d (1.61 gCOD/L-d) and 4.43 gCOD/d (1.26 gCOD/L-d ) , respectively, attained in these studies. Steady state methane production in the ER at 10-d SRT was 0.30 L/d (0.085 L/L-d ) and in the CR was 0.43 L/d (0.12 L/L-d ) . Since two moles of 0_ are required to oxidize one mole of methane gas, the COD equivalent of methane is 64 gCOD/mole methane (1 mole =22.4 L STP). From this relationship the theoretical value of the COD consumed in the production of methane was computed as shown in Table 4-4. These values indicate that at the time of optimum gas production at 15-d SRT 1.8 gCOD/day were available for cell synthesis and maintenance, and 1.1 gCOD/day were available for these activities in the CR.

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131 90Q U 80O K i— 10 UJ Q in § 70 60 90Q Ui 5 80 K Ii/l UJ Q Q S 70 60 OER o Series 1 • Series 2 CR Scrips 1 10 15 20 25 SOLIDS RETENTION T I M E, d 30 Figure 4-20. Soluble COD and soluble BOD removals related to solids retention time.

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132 Table 4-4. COD available for biomass production. Parameter

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133 4.4.3. Nitrogen and Phosphorus Nutrient uptake in the ER at steady state is shown in Figures 4-21 and 4-22. Uptake of soluble P, NH 3 ~N and soluble Kjeldahl-N increased with SRT. Soluble P uptake was not very great, increasing from an average of 44 percent at 10 days to 46 percent at 30 days (Figure 4-21). In the first series, NH^-N removals increased from 24 percent at 10-d SRT to 66 percent at 30-d SRT (Figure 4-22). In the second series, the NH..-N removals were a constant 72.5 percent. Soluble Kjeldahl-N removals also differed in the two series, but the removal patterns were similar. In the first series, removals ranged 70-77 percent, while in the second series, the corresponding values were 80-87 percent.

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r 50 40 134 -oQ. UJ _l m -I o to 30 < > O 2 u en 20 10L 80r 60 2 40 20Figure 4-21 u £R o Series 1 • Series 2 CR a Series 1 10 _L x 15 20 SOLIDS RETENTION TIME d. 25 Relationship of soluble TKN and soluble phosphorus uptake to solids retention time,

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135 100rr 80 60 40 _i < > O 2 w z n 20 I z 20 -40 Ll a N ER o Series 1 • Series 2 CR o Series 1 J_ 10 15 20 sol;ds retention time, d. 25 30 Figure 4-22 Relationship of soluble ammonia uptake to solids retention time.

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CHAPTER 5 DISCUSSION 5 . 1 Substrate Characteristics The relatively poor conversion efficiency of feed protein to meat protein (25-30 percent) in hogs results in the excretion of waste products containing high levels of protein, amino acids, and urea. In addition, inadequate digestion of the carbohydrate fraction of feed results in excretion of starch and cellulosic materials. Swine waste is considered a nutritionally well-balanced, easily biodegradable substrate suitable for treatment by anaerobic processes with the formation of methane as a useful by-product (168). Waste characteristics are dependent on a number of factors including feed composition, type and weight of animals, and conditions of housing. In an earlier series of batch studies (45), it was established that purple sulfur bacteria grow well in swine waste and that their presence results in enhanced removals of soluble BOD , soluble COD, Kjeldahl-N, ammonia-N, and total P. The characteristics of the waste collected for these studies from the experimental pig barns of the Swine Research Unit were influenced by the feeding experiment being conducted at the time of collection. Feeding 136

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137 experiments conducted during these studies had varying levels of protein, phosphorus, potassium, magnesium, trace minerals, and selenium. Trace minerals included calcium, manganese, zinc, iron, copper, and iodine. Selenium levels were, at times, increased to 3.0 percent by weight of feed, this amount being equivalent to 60 times the normally recommended level. Feed components which were not fully metabolized were excreted. The swine waste used in the laboratory experiments was collected directly from the floor of the pig barns and, being undiluted at the time of collection, contained high concentrations of excreted chemicals which were potentially harmful to microorganisms. In this respect, a flushed, diluted waste would be less hazardous. In Table 3.2 it is observed that the waste characteristics varied over widely. This attributable in part to the growth stage of the hogs at the time of waste collection. 5. 2 Bacterial Species in Laboratory Cultures The dominant species of phototrophic bacteria observed in the laboratory cultures comprised email coccoid cells, approximately 1-2.5 urn in diameter, which were identified as Thiocapsa roseopersicina . The dominant species of phototrophs in the lagoon effluent used as inoculum was Thiopedia rosea , a large spherical microorganism, usually 4-6 urn in diameter, which contains gas vacuoles, and stores sulfur

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138 granules inside the cell. Presence of these internal sulfur granules was confirmed in both the inoculum and the laboratory culture. This observation indicated that there was a major shift in bacterial species, from the larger cells growing under natural conditions in the lagoon, to much smaller cells of the same family which proliferated under laboratory conditions. A similar occurrence was observed during laboratory-scale batch studies using effluent from the same lagoon as phototrophic inoculum (45). Towards the end of the experiments, samples from the laboratory culture and from the lagoon were enriched for identification of the species. Unfortunately, the laboratory sample was taken from the reactor a few days after loading had ceased following termination of an experimental trial. At the time the sample was taken, sulfide would possibly have been depleted, and this therefore accounts for the sulfur-free cells observed by Professor Madigan. The experimental reactor was still being illuminated at the time the sample was taken, and this factor, coupled with a relatively sulfide-free environment, rich in organic compounds, would suggest ideal conditions for growth of the nonsulfur purple bacteria detected in the sample. Variation in species composition of phototrophic bacteria in natural cultures, and laboratory cultures inoculated from such sources has been investigated (131,187). Early work proposed that light quality might be

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139 an important factor in determining species composition among phototrophic bacteria (176). This was demonstrated to some extent in the laboratory (131) where it was observed that green and yellow light enriched for purple sulfur bacteria, whereas blue light favored green sulfur bacteria. In a series of continuous culture laboratory studies performed under various light regimes, van Gemerden (187) demonstrated that interspecies competition among the phototrophic sulfur bacteria, and hence species dominance, was influenced by the sulfide affinity of each species, the period of illumination, and the dilution rate. It was observed that in sulf ide-limited defined mixed cultures containing two Chromatium species, and subjected to continuous illumination, the smaller Chromatium vinosum species outcompeted the larger Chromatium weissei . Long dark periods coupled with short periods of illumination resulted in a dominant population of the larger-celled Chromatium weissei . van Gemerden observed that immediately following a dark period, the larger C. weissei had a sulf ide-uptake rate 2.5 times that of C. vinosum . This rate of uptake rapidly removed a required nutrient from the environment, and under these conditions, the smaller cells could not successfully compete with the larger cells. Occurrence of the smallercelled Thiocapsa roseopersicina instead of the larger-celled Thiopedia rosea in the laboratory cultures studied during

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140 these investigations would be explained by van Gemerden's observations . 5 . 3 Impact of Phototrophic Bacteria on the Anaerobic Digestion Process 5.3.1 Gas Quantity and Quality Successful efficient operation of anaerobic digestion is normally evaluated by the quantity and quality of the gas produced. The volume of methane produced per kilogram of volatile solids or COD added or removed is used as a reference for satisfactory methanogenic metabolism. The quantity and quality of gas produced is dependent on the type and characteristics of the digester feedstock, environmental parameters such as temperature and pH, and the retention time used. Gas production below certain levels is usually indicative of digestion problems. Phototrophic bacteria metabolize H„ , CO-, and acetate, the substrates normally used by the methanogens in their metabolic activities and from which most of the methane is produced. In addition, the phototrophs utilize H„S, a product of anaerobic metabolism which could be inhibitory to the methanogens. The methanogens and the phototrophs of natural aquatic environments, such as ponds and lakes, are normally spatially separated, the phototrophs locating in the water column where they have access to light and H„S gas released

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141 from the sediments, while the methanogens are normally found in bottom muds, where there is a more highly reduced environment. Competition for substrates between these two species of anaerobes would therefore not be a critical factor in their interrelationships. In the completely mixed reactor used in these experiments, the two species are brought into intimate contact by the mixing process, and interspecies competition becomes a factor of greater importance. Such competition could be both beneficial and detrimental to the organisms involved, and survival will be dependent largely on metabolic rate and, ultimately, growth rate of each organism. Under these conditions, the schematic diagram illustrated in Figure 5-1 could be used to describe the interspecies interrelationships which exist in an illuminated anaerobic ecosystem. In these investigations, the impact of the phototrophs was reflected in the lower gas production rates recorded for the illuminated reactor in which the phototrophs flourished, compared with that of the control reactor which was not illuminated, and which had no significant population of phototrophic bacteria. In experiments in which gasification occurred, the quantity of gas produced at steady state was lower in the ER than in the CR. However, the quality of the gas from the ER was better than that from the CR. The peak gas production rate of the CR, 1.066 L/d (STP), was 18.7 percent greater than that of the ER. The observed

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x42

PAGE 158

143 difference was most likely attributable to the assimilation of methane precursors by the phototrophic bacteria. ethane content of the reactor gas was 64-71 percent for the ER and 53-70 percent for the CR. The net result of the difference in gas quality in the two reactors is that the volume of methane produced in each was almost identical. Peak volumetric methane production values were 0.18 L/L-d in the CR, and 0.17 L/L-d in the ER, when the reactors were being loaded at a rate of 0.64 g VS/L. Using flushed screened swine waste in a digester loaded at the rate of 45 g VS/L, Hill and Bolte (66) recorded gas quality of 55.5 percent methane, and average volumetric production of 0.88 L CH./L-d. Loading a digester with swine waste at the rate of 60 g VS/L, Fischer et al. (52) achieved gas quality of 59 percent methane and average volumetric methane production rate of 1.36 L CH./L-d. When examined in terms of VS added, the results of Hill and Bolte yield methane production of 0.02 L CH./L vol-g VS added, and those of Fischer et al. yield a value of 0.02 L CH./L vol-g VS added. The corresponding average methane production values obtained from these studies were 0.02 L CH./L vol-g VS added, for both the ER and the CR. Phototrophic bacteria therefore do not have an adverse impact on methane production. Apparently, the reduction in overall gas quantity is compensated by the improved quality of the gas.

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144 The very high steady state concentration of phototrophic bacteria in the ER, 66 mg bchl a/L, attained at 30-d SRT, most likely contributed to the lack of gas production in the ER at this retention time. In addition to this, the feedstock contained only 1.2 g VS/d (0.34 g VS/L-d) and 2.3 8 g COD/d (0.68 g COD/L-d) at this dilution rate. These levels of substrate would be inadequate to support any significant gas production in addition to the high biomass levels observed. 5.3.2 Waste Treatment and Nutrient Uptake In anaerobic digesters, the degree of organic reduction achieved is not usually very high, and the effluent stream normally requires additional treatment prior to being discharged into receiving waters. From these experiments, it has been confirmed that a high degree of organic removal can be achieved in anaerobic systems incorporating phototrophic bacteria. However, because of the very high initial organic concentrations, it will still not be possible to discharge the effluent into receiving waters without further treatment. Steady state values of COD reduction ranged from 84 to 91 percent in the illuminated reactor, compared with 66 to 71 percent xn the nonilluminated reactor. COD loading was found to have very little effect on removal rates. Reductions of 66 to 75 percent for TKN, 54 to 68 percent for

PAGE 160

145 NH,-N and 44 to 46 percent for phosphorus were obtained for the illuminated reactor. These levels of nutrient removal exceed those normally attained in conventional aerobic biological waste treatment processes. Corresponding removals in the CR were much below these levels. By removing the sulfides which would otherwise cause odor problems in the anaerobic digester, or become toxic to the methanogens if allowed to accumulate above 200 mg/L, the phototrophic bacteria assume a positive synergistic role in their environment. 5 . 4 Kinetic Parameters and Mathematical Model The minimum biological solids retention time for the phototrophic anaerobes was found to exist between 8.5 and 10 days, indicating maximum specific growth rate between 0.10 and 0.12 d, and corresponding with COD loadings between 6.8 and 8.0 g COD/day. Using soluble COD as substrate parameter, Equation 2-35 was used to determine the reaction rate coefficient K, considering volatile solids as the measure of biomass. The value of K was determined to be 0.200 L/g-day, and the nondegradable portion of the COD was 2.010 g/L. The resulting design equation for COD removal is therefore given by (S S )/X t = 0.200(S 2.010) (5-1) o e ' v e

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146 The biomass production coefficients a, and a~ in Equation 2-32 were determined by plotting (S S )/X vs SRT. J * r o e v Values of 0.0678 and 1.92, respectively, for the acidogenic phase and 0.0542 and 3.25, respectively, for the methane fermentation stage were determined for these coefficients. The resulting equation governing biomass production then becomes X = (S S ) (5-2) 3.25 + 070542/u and the equation governing the observed yield is Y obs = 1 (5 " 3) 3.25 + 0.0542/u 5. 5 Application of Results to Field Operations With suitable modifications, the results of these investigations may be applied to the solution of a number of problems which are currently being experienced in the management of livestock and poultry wastes. Foremost among these are the noxious odors which tend to create a nuisance in the communities in which the operations are located. Phototrophic sulfur bacteria are extremely effective in removing most of the odor-causing compounds from wastes. With the knowledge now available from these studies,

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147 effective odor-free anaerobic lagoons can be designed to achieve high rates of organic removal. The studies have indicated that gas quality is enhanced by the presence of phototrophic bacteria. These organisms also assist in maintaining a well-buffered anaerobic system. Incorporation of these bacteria in an illuminated anaerobic digester would eliminate the need for very high solids concentration in the feedstock, as is now considered necessary for efficient digester operation. One area of application with great potential is use of phototrophic bacteria in an integrated system of biogas generation and biomass production. These organisms have a protein content of between 70 and 80 percent. Large-scale production in an illuminated anaerobic reactor and subsequent harvesting for use as a protein source certainly appears feasible. Protein productivity of 3-5 g/d was obtained in these studies. Optimization of the process under field conditions could result in even greater yields.

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CHAPTER 6 CONCLUSIONS AND RECOMMENDATIONS 6 . 1 Conclusions The kinetics of swine waste assimilation in an anaerobic photobiological reactor were investigated in laboratory-scale studies, utilizing waste generated at the University of Florida's Swine Research Unit as a substrate. The studies were designed to (1) assess the impact of phototrophic sulfur bacteria on the anaerobic digestion process, (2) to determine the kinetic parameters pertaining to uptake of substrate, and (3) to generate data for the design and operation of pilotand full-scale units which could be used for waste treatment and large-scale biomass production. From these studies it was concluded that use of photobiological treatment of swine waste is technically feasible and offers long-term potential for exploitation under suitable conditions. Specific conclusions are that 1. Swine waste is treatable by anaerobic photobiological processes, achieving comparable removal of organic compounds, as measured soluble COD and BOD^, and better removal of nitrogen and phosphorus than is commonly achieved in standard biological waste treatment processes, and producing good 148

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149 yields of high-protein photosynthetic biomass which may be harvested and used for a variety of purposes . 2. The minimum biological solids retention time (SRT) lies between 8.5 and 10 days, corresponding to specific growth rate between 0.12 and 0.10-day, under the experimental conditions in which these studies were conducted. 3. Washout of phototrophic anaerobes under conditions similar to those used in these studies will be experienced at volatile solids loading in excess of 4.1 g VS/day, and COD loading in excess of 7.8 g COD/day. 4. Substrate uptake increases with retention time, but gas production was optimized at 15-days SRT. 5. Gasification is not an essential prerequisite for the achievement of high levels of treatment. 6. Presence of phototrophic bacteria in anaerobic digestion systems enhances organic removals and gas quality, results in lower levels of total gas production, but a higher quality of gas.

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150 7. A mathematical model comprising three equations was developed to describe the process. The equations are Substrate uptake (S S )/X t = 0.200CS 2.010) (5-!) o e v e Biomass production X = (S S ) (5-2) v 3.25 •~~6T0§42/u Observed yield Y obs (5-3) 3.25 + 0.0542/u 6 . 2 Recommendations for Further Research In continuation of these studies it is recommended that 1. The design parameters determined in this series of laboratory-scale experiments should be evaluated in pilot-scale investigations under natural and artificial lighting. 2. Field studies should be conducted to determine the start-up requirements and environmental controls required for successful operation of large-scale anaerobic photobiological systems for the treatment of swine waste.

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APPENDIX A RAW DATA

PAGE 167

Appendix A-l. 5-d SRT: temperature, pH and bchl a. Day Reactor Temp °C Effluent pH Bchl a mg/L ER CR ER CR ER CR 6.97 6.97 16.30 16.30

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153 A-l continued Day

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154 Appendix A-2. 5-d SRT: barometric pressure and reactor gas . Day

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155 A-2 continued Day Barom Press. in Hg Reactor Gas Production rate mL/h ER CR Quality % ER CH CO, CR CH CO, 21 22* 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 30.15 30.05 30.07 30.10 30.09 30.10 30.03 30.13 30.20 30.19 30.17 30.12 30.17 30.07 30.06 30.12 30.22 30.18 30.19 30.20 30.18 2.83 0.65 16.46 16.46 14.84 12.24 11.02 8.25 12.39 10.11 5.34 9.07 7.91 5.77 5.31 9.40 8.98 6.67 0.00 0.00 0.00 0.00 6.96 5.22 27.63 26.46 25.15 21.84 18.78 15.70 14.56 15.58 15.00 16.28 15.58 6.54 8.57 10.60 11.84 9.25 0.00 0.00 0.00 0.00 48 22 52 32 48 54 23 18 54 55 22 26 55 18 56 28

PAGE 171

156 Appendix A-3. 7-d SRT: temperature, pH, bchl a. Day

PAGE 172

A-3. continued 157 Day

PAGE 173

158 Appendix A-4. 7-d SRT: barometric pressure and gas Day

PAGE 174

A-4. continued 159 Day Barom Press. in Hg Reactor Gas Production rate mL/h ER CR Quality % ER CH CO, CR CH, CO, 20

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160 Appendix A-5. 8.5-d SRT: temperature, pH, bchl a. Day Reactor Temp °C Effluent pH Bchl a mg/L ER CR ER CR ER CR 6.72 35.20 6.63 30.25 6.63 26.62 6.60 22.99 6.46 22.59 6.18 20.57 6.08 19.97 6.06 14.92 6.05 14.12 6.05 11.50 5.97 9.08 0*

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161 A-5. continued Day Reactor Temp. °C Effluent pH Bchl a mg/L ER CR ER CR ER CR 5.98 7.04 21

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162 Appendix A-6. 10-d SRT — Series 1: temperature, pH, bchl a Day

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A-6. continued 163 Day

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A-6. continued 164 Day

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165 Appendix A-7. 10-d SRT — Series 1: barometric pressure and gas production. Day

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166 A-7. continued Day

PAGE 182

167 A-7.

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168 Appendix A-8. 10-d SRT — Series 2: temperature, pH and bchl a. Day Reactor Temp. °C Effluent pH Bchl a mg/L ER CR ER CR ER CR 7.40 4.84 7.26 7.26 7.23 13.52 7.13 20.05 7.24 27.72 7.19 33.45 7.16 36.60 7.18 36.42 7.16 35.20 7.16 32.02 7.14 31.46 7.05 31.46 7.02 31.46 7.02 31.46

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A-8. continued 169 Day Reactor Temp. °C ER CR Effluent pH ER CR Bchl a mg/L ER CR 21

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A-8. continued 170 Day Reactor Temp. °C ER CR Effluent pH ER CR Bchl a mg/L ER CR 42

PAGE 186

171 Appendix A-9. 15-d SRT--Series 1: temperature, pH and bchl a. Day

PAGE 187

172 A-9. continued Day Reactor temp. °C Effluent pH Bchl a mg/L ER CR ER CR ER CR 20 27.0 27.8 7.26 7.16 39.33 9.68 21 27.8 27.4 22 26.6 26.8 7.22 7.13 40.84 9.68 23 26.6 26.6 24 26.6 26.8 7.06 7.00 40.54 8.18 25 27.5 27.0 26 27.4 27.2 7.12 7.05 41.33 7.26 27 27.0 27.0 28 27.5 27.3 7.10 6.99 43.86 6.05 29 27.0 26.8 30 27.5 26.4 7.08 6.93 45.68 5.45 31 26.2 26.2 42.05 32 27.0 27.3 33 27.0 27.8 7.10 6.98 45.98 6.05 34 27.0 27.6 35 28.0 27.5 7.05 6.93 45.68 4.84 36 27.2 27.2 37 27.0 27.0 7.06 6.97 48.75 3.03 38 27.0 27.2 39 27.8 27.0 7.10 6.93 51.24 3.63 40 27.0 27.0 -

PAGE 188

A-9. continued 173 Day

PAGE 189

174 Appendix A-10. 15-d SRT--Series 1: barometric pressure and gas production. Day

PAGE 190

175 Appendix A-10. continued Day

PAGE 191

176 A-10. continued Day

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177 Appendix A-ll. 15-d SRT — Series 2: temperature, pH and bchl a . Day Reactor Temp. °C Effluent pH Bchl a mg/L ER CR ER CR ER CR 7.16 45.36 7.10 45.62 7.02 45.98 6.85 44.77 6.82 44.17 6.88 42.35 6.82 39.93 6.88 39.38 6.85 39.32 6.89 39.32 6.85 38.96 * denotes start of continuous loading phase. 0*

PAGE 193

178 A-ll. continued Day Reactor Temp. °C Effluent pH Bchl a mg/L ER CR ER CR ER CR 20

PAGE 194

179 Appendix A-12. 20-d SRT — Series 1: temperature, pH and bchl a. Day

PAGE 195

180 A-12. continued Day

PAGE 196

A-12. continued 181 Day

PAGE 197

182 Appendix A-13. 20-d SRT — Series 1: barometric pressure and gas production. Day Barom Reactor gas Press. Production Quality % rate mL/h ER CR in Hg ER CR CH 4 CC> 2 CH CO,

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183 A-13. continued Day

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184 A-13. continued Day

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185 Appendix A-14. 20-d SRT — Series 2: temperature, pH and bchl a Day Reactor Temp °C Effluent pH Bchl a mg/L ER CR ER CR ER CR 7.40 4.84 7.30 10.12 7.20 16.07 7.13 24.22 7.16 29.50 7.18 35.20

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186 A-14. continued Day Reactor Temp °C Effluent pH Bchl a mg/L ER CR ER CR ER CR 20

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187 A-14. continued Day

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188 Appendix A-15. 30-d SRT--Series 1: temperature, pH and bchl a. Day

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A-15. continued 189 Day

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190 A-15. continued Day

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191 Appendix A-16. 30-d SRT — Series 1: barometric pressure and gas production. Day

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192 A-16. continued Day

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193 A-16. continued Day

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194 Appendix A-17. 30-d SRT — Series 2: temperature, pH and bchl a. Day Reactor Temp °C Effluent pH Bchl a mg/L ER CR ER CR ER CR 6.90 38.70 6.78 41.44 6.73 41.44 6.78 43.72 6.85 45.22 7.07 48.70 7.07 48.66 7.01 49.54 7.00 49.54 7.04 50.22 0*

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195 A-17. continued Day Reactor Temp °C Effluent pH Bchl a mg/L ER CR ER CR ER CR 20

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196 A-17. continued Day Reactor Temp °C Effluent pH Bchl a mg/L ER CR ER CR ER CR 41

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APPENDIX B STEADY STATE RESULTS

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Appendix B-l. Steady state gas quality—Series 1 trials SRT Reactor ER CR d % CH 4 % C0 2 % CH 4 % C0 2 10 64 20 55 30 64 22 53 29 64 21 53 31 65 21 54 29 0.96 30 + 15 20 30 64 +

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199 Appendix B-2 Steady state effluent solids concentration — Series 1. SRT

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200 Appendix B-3. Summary of steady state effluent solids concentration. SRT Reactor Series TS VS TSS mg/L mg/L mg/1 10 ER 1 9966 + 138 7106 + 48 9436 + 49 2 12763 + 129 9708 + 90 11823 + 98 CR 1 9027 + 305 6864 + 162 8467 + 13 2 15 ER 1 8419 + 86 6341 + 44 6987 + 66 2 12439 + 69 9542 + 314 11786 + 53 CR 1 6834 + 24 4530 + 56 4543 + 95 2 20 ER 1 9747 + 87 7336 + 38 7321 + 31 2 11918 + 43 8968 + 176 8374 + 140 CR 1 7869 + 40 5619 + 200 5731 + 59 2 30 ER 1 13902 + 58 10501 + 290 13146 + 166 2 13332 + 110 10177 + 117 12579 + 328 CR 1 8397 + 34 4803 + 106 7058 + 279 2 -

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201 Appendix B-4. Summary of influent solids concentration. SRT Series TS VS mg/L mg/L 10 12.443+0.266 9.1564+0.088 9.703 +0.088 9.625+0.256 9.788+0.178 1

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202 Appendix B-5. 10-d SRT steady state parameters. Parameter Influent Effluent Series 1 Series 2 Series 1 Series 2 ER CR ER CR pH

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203 Appendix B-6. 15-d SRT steady state parameters. Parameter Influent Effluent Series 1 Series 2 Series 1 Series 2 ER CR ER CR pH 6.98 6.93 7.00 6.99 6.89 +0.02 +0.05 +0.03 +0.03 +0.02 bchl a 0.0 0.0 52.32 1.67 38.72 mg/L +0.3 4 +0.5 8 +0.26 COD* 18.022 18.484 2.760* 5.407* 2.102 g/L +0.48 +0.62 +0.085 +0.049 +0.035 BOD.* 6.430 7.261 1.125 2.345 1.416 g/L +0.397 +0.495 +0.102+0.234 +0.155 TKN* 0.82 0.88 0.26 0.49 0.163 g/L +0.011 +0.007 +0.006+0.059 +0.005 NH-.-N* 0.425 0.201 0.231 0.391 0.040 g/L +0.006 +0.004 +0.004 +0.056 +0.001 P* 0.441 0.464 0.245 0.353 0.251 g/ L +0.065 +0.044 +0.023 +0.060 +0.032 * soluble effluent

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204 Appendix B-7. 20-d SRT steady state parameters Parameters Influent Effluent Series 1 Series 2 Series 1 Series 2 ER CR ER CR PH

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205 Appendix B-8. 30-d SRT steady state parameters. Parameter Influent Effluent Series 1 Series 2 Series 1 Series 2 ER CR ER CR P H

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APPENDIX C MISCELLANEOUS TABLES OF RESULTS

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Appendix C-l. Calculation of organic removal rates by phototrophic bacteria in ER--Series 1. Parameter SRT 10 15 20 30 S mg/1 19 165 18 022 17 988 19 683 o S mg/L 3 083 2 760 2 455 2 333 e S -S mg/L 16 082 15 262 15 533 17 350 o e ^ X mg/L 7 588 6 141 7 336 10 498 S -S 2.119 2.485 2.117 1.653 -q— e v S -S 0.2119 0.1657 0.1058 0.0551 t C -$-re v S = influent COD, mg/L S° = effluent filtered COD, mg/L X e = average steady volatile solids concentration, mg/L t V = SRT, days Appendix C-2. Biomass productivity related to bchl a. SRT Bchl a mg/L Productivity mg/d d Series 1 Series 2 Series 1 Series 2 10 28.62 35.48 10.02 12.42 + + 15 52.32 38.72 12.19 9.02 + + 20 53.33 43.26 9.33 7.57 + 30 65.91 60.70 7.71 7.10 + + 207

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208 Appendix C-3. Soluble BODand COD removals. SRT

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BIOGRAPHICAL SKETCH Jonathan F. K. Earle was born April 12, 1940, in St. James, Jamaica. He pursued undergraduate engineering studies in England, where he received his bachelor's degree in civil engineering from the University of London in June 1965. Following graduation, he joined the Metropolitan Water Board, London, where he completed his training as an apprentice engineer in the design, construction, and management of waterworks systems. His civil engineering experience includes the design and construction of water distribution pipelines and soft earth tunnels, reservoirs, pumping stations, water treatment plants, and other civil engineering works. On his return to Jamaica in 1970, he entered the field of consulting engineering and was appointed managing director of the Jamaica branch of the United Kingdom based firm of Howard Humphreys & Sons, international consulting engineers, assuming complete technical and administrative responsibility for all local operations. In 1973 he established the firm of Earle & Associates Limited, Consulting Engineers. This company has since grown to be one of the leading firms in the island in the field of water supply engineering . 228

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229 Jonathan F. K. Earle is a member of the Jamaica Institution of Engineers, the Association of Consulting Engineers, Jamaica (Past President), the Institution of Civil Engineers (UK), the American Society of Civil Engineers, the American Water Works Association, the Water Pollution Control Federation, and the Royal Society of Health (UK); and a Fellow of the Institution of Water Engineers and Scientists (UK). He commenced graduate studies at the University of Florida in the Fall of 1981 and was awarded a Master of Engineering degree in environmental engineering in August 1983, majoring in water supply and water pollution control. His thesis was entitled "Potential for Utilization of Purple Sulfur Bacteria in the Management of Livestock Wastes." He pursued further research in this field, culminating in the presentation of this doctoral dissertation.

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I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. Ben Koopman, Chairrfgj^ Associate Professor of Environmental Engineering Sciences I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. Edward P. Lincoln, Cochairman Associate Professor of Agricultural Engineering I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. Jonri Zoltek, Jr. Professor of Environmental Engineering Sciences

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I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. Rog-e'r A. Nordstedt Associate Professor of Agricultural Engineering I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. A Glen H. Smerage Associate Professor of Agricultural Engineering This dissertation was submitted to the Graduate Faculty of the College of Engineering and to the Graduate School and was accepted as partial fulfillment of the requirements for the degree of Doctor of Philosophy. December 1985 Dean, Dean, Graduate School