United States
Environmental Protection
Agency
Municipal Environmental Research
Laboratory
Cincinnati OH 45268
EPA-600 2-80-093
August 1980
Research and Development
Studies of
Methanogenic
Bacteria in Sludge
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RESEARCH REPORTING SERIES
Research reports of the Office of Research and Development, US. Environmental
Protection Agency, have been grouped into nine series. These nine broad cate-
gories were established to lacilitate further dev&opment and application of en-
vironmental technology. Elimination of traditional grouping was consciously
planned to foster technology transfer and amaxi mum interface in related fields.
The nine series are:
1. Environmental Health Effects Research
2. Environmental Protection Te hnoIogy
3. Ecological Research
4. Environmental Monitoring
5. Socioeconomic Environmental Studies
6. Scientific and Technical Assessment Reports (STAR)
7. Interagency Energy-Environment Research and Development
8. Special” Reports
9. Miscellaneous Reports
This report has been assigned to the ENVIRONMENTAL PROTECTION TECH-
NOLOGY series. This series describes research performed to develop and dem-
onstrate instrumentation, equipment, and methodology to repair or prevent en-
vironmental degradation from point and non-point sources of pollution. This work
provides the new or improved technology required for the control and treatment
of pollution-sources to meet environmental quality standards.
This document is available to the public through the National Technical Informa-
tion Service, Springfield, Virginia 22161.
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EPA-600/2-80-093
August 1980
STUDIES OF METHANOGENIC
BACTERIA IN SLUDGE
by
P. H. Smith
University of Florida
Gainesville, Florida 32601
Grant No. 17070-DJV
Project Officer
A. D. Venosa
Wastewater Research Division
Municipal Environmental Research Laboratory
Cincinnati, Ohio 45268
MUNICIPAL ENVIRONMENTAL RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
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DISCLAIMER
This report has been reviewed by the Municipal Environmental Research
Laboratory, U. S. Environmental Protection Agency, and approved for publica-
tion. Approval does not signify that the contents necessarily reflect the
views and policies of the Li. S. Environmental Protection Agency, nor does
mention of trade names or coninercial products constitute endorsement of recom-
mendation for use.
11
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FOREWORD
The Environmental Protection Agency was created because of increasing
public and government concern about the dangers of pollution to the health
and welfare of the American people. The complexity of that environment and
the interplay between its components require a concentrated and integrated
attack on the problem.
Research and development is that necessary first step in problem solution
and it involves defining the problem, measuring its impact, and searching for
solutions. The Municipal Environmental Research Laboratory develops new and
improved technology and systems for the prevention, treatment, and management
of wastewater and solid and hazardous waste pollutant discharges from munici-
pal and con-iiiunity sources, for the preservation and treatment of public drink-
ing water supplies, and to minimize the adverse economic, social, health, and
aesthetic effects of pollution. This publication is one of the products of
that research; a most vital coniiiunications link between the researcher and the
user comunity.
This report describes fundamental studies on methanogenic and hydrogeno-
genic microorganisms. Results demonstrate the existence of a new physiolo-
gical group of bacteria which play a central role in anaerobic digestion of
domestic waste.
Francis T. Mayo
Director
Municipal Environmental Research
Laboratory
111
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ABSTRACT
Methanogenic bacteria were isolated from mesophilic anaerobic digesters.
The isolates were able to utilize H 2 and CO 2 acetate, formate and methanol,
but were not able to metabolize propionate ai d butyrate. It was shown the
propionate and butyrate are not substrates for methanogenic bacteria but are
converted to hydrogen, carbon dioxide and acetate by a hydrogenogenic micro-
flora. The reactions leading to methane were quantitatively analyzed. It was
shown that acetate, propionate and butyrate metabolism were inhibited by
hydrogen. The formation of acetate and propionate were shown to be rate
limiting in the digestion process, and that sludge digestion was not inhib-
ited by hydrogen under conditions of excess substrate.
This report was submitted in fulfillment of Grant No. 17070-DJV by the
University of Florida under the sponsorship of the U. S. Environmental Pro-
tection Agency. This report covers the period September 1, 1966 to
October 15, 1979 and work was completed as of October 15, 1979.
,iv
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FOREWORD.
ABSTRACT
FIGURES
TABLES
ACKNOWLEDGEMENT
• iii
iv
vii
xi
xii
1
3
5
6
ISOLATION OF PURE CULTURES
DETERMINATION OF METHANOGENIC INTERMEDIATES.
Formate
Acetate
Propionate
n-Butyrate
i so-Butyrate
Ethanol
iso-Valeric Acid
CONVERSION OF PROP IONATE AND BUTYRATE TO ACETATE
HYDROGEN EFFECTS ON SLUDGE DIGESTION
Inhibition of Volatile Acid Turnover
Determination of Inhibitory Concentrations of
Volatile Acids
Effect of Molecular Hydrogen on Volatile
Organic Acid Dissimilation
Rates of Methanogenesis in the Absence and Presence
of Added Molecular Hydrogen
MPN Analysis for Methanogenic Bacteria
Hydrogen Stripping Experiments
Pure Culture Isolations
• . . 58
59
59
64
65
69
CONTENTS
1. INTRODUCTION
2. CONCLUSION
3. RECOMMENDATIONS
4. RESULTS AND DISCUSSION
... 6
8
10
10
10
18
18
18
23
• 23
• 23
23
Hydrogen Uptake Capacity as a Parameter for Predicting
Digester Failure
Digestion Inhibition Studies and Hydrogen Effects .
Long-Term Hydrogen Inhibition
HYDROGEN PRODUCTION FROM VOLATILE ACiDS
• 30
• 39
• 45
• 45
V
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ACETATE AND PROPIONATE FEED RATE EXPERIMENTS 72
BIBLIOGRAPHY 76
APPENDICES
A. MATERIALS AND METHODS 79
B. FERMENTATION OF RUM SLOPS 87
vi
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FIGURES
Eigure Page
1. Conventional scheme for the formation of methane from
insoluble organic matter based on previously known facts 2
2. Scheme for the formation of methane from insoluble organic
matter based on the data presented in this report 4
3. Change in counts per minute in the formic acid fraction
from a constant volume of sludge, after addition of
radioactive formic acid 11
4. Manometric changes in control flasks during the period
of the experiment 12
5. Changes in the acetic acid concentration of sludge during
an eleven hour period following the feeding of the digester 13
6. Change in counts per minute in the acetic acid fraction
from a constant volume of sludge, after addition of
radioactive acetic acid 14
7. Change in counts per minute in the acetic acid fraction
from a constant volume of sludge, after addition of
radioactive acetic acid 15
8. Change in the counts per minute in the propionic acid
fraction from a constant volume of sludge, after addition
of radioactive propionic acid 16
9. Change in the counts per minute in the propionic acid
fraction from a constant volume of sludge, after addition
of radioactive propionic acid 17
10. Change in the counts per minute in the n-butyric acid
fraction from a constant volume of sludge, after addition
of radioactive n-butyric acid 19
11. Change in the counts per minute in the n-butyric acid
fraction from a constant volume of sludge, after addition
of radioactive n-butyric acid 20
vii
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Figure Page
12. Change in the counts per minute in the iso-butyric acid
fraction from a constant volume of sludge, after addition 21
13. Change in the concentration of ethanol added to sludge, after
addition of ethanol 22
14. Counts per minute lost from propionic acid, and found in
acetic acid, in a constant volume of sludge, following
addition of radioactive propionic acid 24
15. Counts per minute lost from butyric acid, and found in
acetic acid, in a constant volume of sludge, following
addition of radioactive butyric acid 25
16. Change in counts per minute in the acetic acid fraction
from a constant volume of sludge after addition of
radioactive acetic acid to sludge exposed’to hydrogen,
and sludge not exposed to hydrogen 27
17. Change in counts per minute in the propionic fraction
from a constant volume of sludge after addition of
radioactive propionic acid to sludge exposed to hydrogen,
and sludge not exposed to hydrogen 28
18. Changes in the acetic acid fraction from a constant volume of
sludge following addition of radioactive propionic acid
and exposure to various concentrations of propionic acid 29
19. Feeding schedule of digesters being induced to fail 31
20. Methane production rates in digesters being induced to fail 32
21. Methane production rates in digesters being induced to fail 33
22. Acetate concentration in digesters being induced to fail 34
23. Propionate concentration in digesters being induced to fail 35
24. n-Butyrate concentration in digesters being induced to fail 36
25. iso-Butyrate concentration in digesters being induced to fail.... 37
26. iso-Valerate concentration in digesters being induced to fail.... 38
27. Change in the counts per minute in the acetate fraction
from constant volumes of sludge following addition of
radioactive acetate to digesters being induced to fail 40
vii 1
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Figure Page
28. Change in the counts per minute in the propionate fraction
from constant volumes of sludge following addition of
radioactive propionate to digesters being induced to fail 41
29. Methane production from sludge not exposed to hydrogen,
sludge exposed to hydrogen and sludge exposed to hydrogen
with add hydrogen as substrate 42
30. Acetate concentration in sludge exposed to hydrogen, and
sludge not exposed to hydrogen 43
31. Propionate concentration in sludge exposed to hydrogen, and
sludge not exposed to hydrogen 44
32. Methane production from sludge exposed to hydrogen, and
sludge not exposed to hydrogen 46
33. Methane production from added hydrogen by sludge previously
exposed to hydrogen for periods of time up to 55 days 47
34. Methane production from added hydrogen by sludge previously
exposed to hydrogen for periods of time up to 55 days 48
35. Acetate pools in sludge exposed to hydrogen for periods of
time up to 55 days 49
36. Acetate pools in sludge exposed to hydrogen for periods of
time up to 55 days 50
37. Propionate pools in sludge exposed to hydrogen for periods
of time up to 55 days 51
38. Propionate pools in sludge exposed to hydrogen for periods
of time up to 55 days 52
39. n-Butyrate and n-valerate pools in sludge exposed to
hydrogen for periods of time up to 55 days 53
40. n-Butyrate and n-valerate pools in sludge exposed to
hydrogen for periods of time up to 55 days 54
41. iso-Butyrate and iso-valerate pools in sludge exposed to
hydrogen for periods of time up to 55 days 55
42. iso-Butyrate and iso-valerate pools in sludge exposed to
hydrogen for periods of time up to 55 days 56
ix
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Figure Page
43. Inhibition of gas production by methanogenic enrichments
maintained at pH 6.9 to 7.0. Acetate enrichments with added
acetate, propionate enrichments with added propionate and
butyrate enrichments with added butyrate 60
44. Degradation of acetic acid in the absence and presence
of added molecular hydrogen 61
45. Degradation of propionic acid in the absence and presence
of added molecular hydrogen 62
46. Degradation of butyric acid in the absence and presence
of added molecular hydrogen 63
47. Gas production obtained from a propionate enrichment with
acetate as the substrate, when flushed with 100 percent CO 2 66
48. Gas production obtained from a propionate enrichment with
propionate as the substrate when flushed with 100 percent CO 2 .... 67
49. Gas production obtained from a butyrate enrichment with
butyrate as the substrate when flushed with 100% CO 2 68
50. Gas produced from digesting sludge when flushed with 100% CO 2 .... 70
51. Gas produced from digesting sludge diluted with sludge supernate
when flushed with 100% CO 2 71
52. Changes in concentration, with time, of acetic acid in
digesting sludge fed acetic acid at feed rates from
0.002 pmoles/ml/min to 0.014 itmoles/mi/min 74
53. Changes in concentration, with time, of propionic acid in
digesting sludge fed propionic acid at feed rates from
0.0012 jimoles/mi/min to 0.0033 pmoles/ml/min 75
B-i. Acetic acid and propionic acid concentrations each day
24 hours after feeding 93
B-2. n-Butyric acid, iso-butyric acid and n-valeric acid
concentrations each day 24 hours after feeding 94
B-3. Ionic strength each day 24 hours after feeding 95
B-4. Liters of gas produced each day 24 hours after feeding 96
B—5. Daily methane produced 5 hours after feeding 98
x
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TABL ES
Table Page
1 CHARACTERISTICS OF ISOLATES 7
2 TURNOVER OF ETHANOL IN SLUDGE 18
3 TURNOVER OF ISO-VALERIC ACID IN SLUDGE 23
4 HYDROGEN UTILIZATION BY DOMESTIC SLUDGE 26
5 EFFECT OF HYDROGEN GAS ON SLUDGE METHANOGENESIS 30
6 EQUATIONS AND FREE ENERGY CHANGES FOR THE ANAEROBIC
OXIDATION OF PROPIONATE AND BUTYRATE, AND THE REDUCTION
OF CO 2 TO METHANE 57
7 METHANE PRODUCTION AND HYDROGEN UTILIZATION
BY ENRICHMENT 64
B-i IONS AND THEIR CONCENTRATIONS IN RUM SLOP 88
B-2 pH CHANGES OF RUM SLOPS DUE TO ADDITION OF SLUDGE 88
B-3 GAS PRODUCTION FROM DIGESTING SLUDGE DILUTED WITH RUM SLOPS 90
B-4 PRODUCTS FORMED BY SLUDGE DILUTED WITH RUM SLOPS 91
xi
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ACKNOWLEDGEMENTS
I thank Dr. R. A. Mah for his useful help in carrying out these
experiments and acknowledge the contributions of Dr. EL Boone,
Mr. F. Bordeaux, Dr. P. Shuba and Mrs. J. Ward to the project.
x l ’
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SECTION 1
INTRODUCT ION
In 1776 the Italian physicist, Alessandro Volta, observed a combustible
gas formed from plant material in lake sediments. Since that time many inves-
tigators have endeavored to elucidate the biological features of the formation
of such gas. The gas has been shown to be methane. Practical interest in the
rnethanogenic process later centered primarily on the formation of methane in
the rumen of herbivorous animals and the utilization of the process in anae-
robic waste treatment. Recently, further interest in the fermentation has
been stimulated by its possible application to production of methane as a fuel
source.
Understanding of methanogenesis as a dissimilation process remained in a
primative state compared to our understanding of other major microbiological
processes, because of the fastidious growth characteristics of the bacteria
involved and our misunderstanding of the substrates used by these organisms
and the products they produce. In addition, the taxonomy of these organisms
is complicated by the paucity of their identifying characteristics.
A generalized scheme for the formation of methane from insoluble organic
matter is shown in Figure 1. Prior to this investigation there have been no
definitive studies showing whether this scheme is or is not correct. The
studies reported here show the scheme to be incorrect. The investigations to
be reported deal primarily with the question of the microbiology of methane
production shown in the last two boxes. If the scheme shown in Figure 1 were
correct the following would be possible:
1. Volatile acids and hydrogen should exist as intermediates in the pro-
cess and it should be possible to chemically demonstrate their exis-
tence.
2. It should be possible to isolate, in pure culture, bacteria capable
of converting the postulated intermediates to methane.
3. It should be possible to quantitate the contribution of each volatile
acid to total methane formation and it should not be possible to
quantitatively identify intermediates in an external pool between
volatile acids and methane gas.
This investigation deals with the above considerations in methane produc-
tion by mesophilic digestion of domestic organic wastes. In addition the in-
vestigation deals with the identification of the steps in the reaction, and
considers the matter of digestion failure under conditions of excess substrate.
1
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1L_J LUBL 1______$ . VOLATILE ACIDS CH +C0 2
“ 1 ORGANICS J H 2 +C02
Figure 1. Conventional scheme for the formation of methane from insoluble organic matter
based on previously known facts.
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SECTION 2
CONCLUSIONS
1. The scheme shown in Figure 1 is incorrect. A correct scheme is shown in
Figure 2.
2 . A large and diverse methanogenic bacterial population exists in domestic
sludge digesters. These organisms are restricted in their substrate util-
ization to the utilization of hydrogen, formate, methanol and acetate.
3. The results of this work demonstrate the existence of a new physiological
group of bacteria which play a central role in anaerobic digestion of
domestic waste. There exists hydrogenogenic bacteria which produce hydro-
gen from propionic acid and butyric acid. The dissimilation of these
acids is not a methanogenic process. These hydrogenogenic organisms dis-
simulate as much as 70% of the organic matter in the eventual formation
of methane.
4. Acetate and hydrogen plus carbon dioxide are the direct precursors of
methane during anaerobic digestion.
5. Propionate and butyrate are the primary precursors of acetate and hydro-
gen during anaerobic digestion.
6. Hydrogen can inhibit the digestion process but is not the cause of diges-
tion failure induced by excesses of organic substrates.
7. During normal digestion processes the rate of methane formation is limit-
ed by the rate of volatile acid production from other organic substrates.
8. Ethanol and iso-valerate are not intermediates in the digestion.
9. Iso-butyrate is quantitatively a minor intermediate.
10. The following contributions of volatile acids to methane as a final pro-
duct in mesophilic digestion of domestic sludge were observed, calculated
on maximum and minimum contributions of propionate and butyrate.
a. Acetate* - 53% : 24%
b. Propionate - 23% : 30%
c. n-Butyrate - 10% : 40%
d. iso-Butyrate - 1.5%
* Acetate not formed from propionate or n-butyrate.
3
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Figure 2. Scheme for the formation of methane from insoluble organic matter based on the
data presented in this report.
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SECTION 3
RECOMMEN DAT IONS
1. It is recoim ended that research be initiated to determine the biological
characteristics of the hydrogenogenic microflora which function in
anaerobic digestion with emphasis on those factors which stimulate
growth and survival of these unique organisms.
5
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SECTION 4
RESULTS AND DISCUSSION
ISOLATION OF PURE CULTURES
Materials and Methods used are presented in Appendix A. During the course
of this investigation a major effort was made to isolate, in pure culture,
those bacteria which were responsible for methane production in anaerobic di-
gestors. At the beginning of these studies it was assumed that the imediate
precursors for methanogenesis were short chain volatile acids, hydrogen, and
carbon dioxide. It was assumed that bacteria such as Methanobacterium pro-
pionicum and Methanobacterium suboxydans did in fact exist, and their isolation
in pure culture awaited only the application of proper techniques. Hydrogen
utilizing methanogens and an acetate utilizing methanogen were readily iso-
lated. All effort to isolate propionate and butyrate dissimilating methano-
gens were unsuccessful. Each effort in this regard ended with the isolation
of a hydrogen oxidizing methanogen, in most cases an organism now know as
Methanospirillum hungatii . The failure of this massive attempt led to an ex-
perimental study of the possibility that short chain volatile organic acids
were indirectly metabolized by a hycirogenogenic and a methanogenic microflora.
If this were the case, then organisms such as Methanobacterium propionicum
and Methanobacterium suboxydans would not exist. Data presented later in the
report demonstrates that this is the case. Propionate and butyrate are metab-
olized by a hydrogenogenic microflora.
The isolation and characterization of methanogen has been unsatisfactory
until very recently because of the fact that substantial distinguishing fea-
tures could not be obtained for these organisms. The biology of this question
has been recently reviewed by Wolfe and Higgins ( 27 ). The basis for the
confusion associated with the taxonomy of these organisms has recently been
identified by Balch eta]. ( 1 ). Application of the 16 5 r RNA analysis
methods developed by Woese have shown that these organisms are only distantly
related to typical bacteria. They are different from other forms of micro-
organisms studied in the past.
An effort was made to obtain conventional characters for classification
of these organisms. Initial experiments were positive, but later results
failed to confirm the initial observations. Isolation results are shown in
Table 1. Isolate 2 is now known in the literature as Methanobacterium
ruminantium strain P.S. This is the most prevalent methanogen in digesting
sludge.
6
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TABLE 1. CHARACTERISTICS OF ISOLATES
Isolate
Characteristics 1 2 3 4 5 6
Morphology Rod Rod Sarcina Rod Coccus Rod
Length Variable 1.8 p 4 p 5 p 1.5 p 2.0
Width 0.4 p 0.7 p 4 p 1.0 p 1.5 p 0.5 p
Motility - - - - - -
Capsule - + - — - -
Spore formation - - - - - -
Anaerobic growth + + +. + + +
Aerobic growth - - - - - -
Catalase production - - - - - -
Nitrate reduction - - - - - -
Sulfate reduction - - - - - -
CO 2 reduction + + + + + +
Gram reaction + + + + + +
Growth on hydrogen + + + + + +
Growth on formate + + - + + +
Growth on acetate - - + - - -
Growth on methanol - - + - - -
Growth on ethanol - - - - - -
Growth on sugars - - - - - -
Growth on mineral medium + - - - +
Growth at 55°C - - - - - -
Growth at 45°C + + + + + +
Growth at 30°C + + + + ÷ +
Growth at 25°C - - - - - -
CH4 from CO 2 and H 2 + + + + + +
CH 4 from formate ÷ + - + + ÷
CH 4 from acetate - - + - - -
CH 4 from methanol - - + - - -
CH4 from sugar - - - - - -
CH 4 from glycine - - - - - -
CH 4 from alanine - - - - - -
Cl -I 4 from serine - - - - - -
CH 4 from glycerol - - - - - -
Cl-I 4 from tartrate - - - - - -
CH4 from isovalerate - - - - - -
CH 4 from raw sludge - - - - - -
+ *, Varies with different strains.
7
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Isolate 4 is now known as Methanspirillum hungatil ( 7 ).
Isolates 1 and 6 were cultures of Methanobacterium formicicum .
Isolate 3is a strain of Methanosarcina barkeri .
Isolate 5 has been lost. Efforts will be made to re-isolate.
In addition to the above isolates, a salt tolerant coccus was isolated
which was believed to be Methanococcus vannielii . It has, however, turned out
to be a species distinct from M. vannielii .
These organisms, along with organisms isolated by other investigators,
are to be characterized shortly in a new taxonomic scheme based on the
16S r RNA concept (Personal comunication R.S. Wolfe). This taxonomy will,
for the first time, place the methanogenic bacteria in a rational scheme.
Three of the fourteen organisms to be characterized will be organisms isolated
during the course of this study. They are isolate 2 and 4 and the salt toler-
ant coccus. Isolates 2 and 4 were obt4ined from digesting sludge and are pre-
sent there in numbers exceeding 1 x lO’/ml. The coccus was isolated from an
estuary and is not believed to be an inhabitant of digesting sludge.
The results show that hydrogen utilizing methanogens are present in large
numbers in digesting sludge. As shown later in this report, they function to
maintain a low hydrogen concentration in digesting sludge.
DETERMINATION OF METHANOGENIC INTERMEDIATES
Kluyver ( 11 ) elucidated the biological principle that the metabolism of
living organisms involves a continuous and directed flow of electrons from
electron doners to electron acceptors. Metabolic processes terminate when
electron transfers cease. Considered from this point of view sludge digestion
consists of a continuous and orderly flow of electrons which produces methane,
carbon dioxide, and cells, since these are the terminal products of electron
transfer under the operating conditions of a sludge digestor. These same pro-
ducts are produced during the dissimilation of organic matter in other anaero-
bic environments. The reactions in a sludge digestor occur in a habitat fairly
constant in temperature, pH, anaerobiosis, and substrate availability. The
dissimilation rate is rapid, the pathways varied, and the microflora diverse
but fairly constant. These characteristics make sludge digestors attractive
as a source material for ecological studies, with the prospect that information
obtained from this source will be applicable to the understanding of other im-
portant environments such as swamps and lake sediments.
Sludge digestion has been generally believed to consist of three steps:
hydrolysis of complex materials, acid production, and methane formation. The
microbiology and biochemistry of these steps have not been clearly elucidated.
Jeris and McCarty ( 10 ) have shown that methane evolved from enrichments
could be accounted for primarily on the basis of the dissimilation of acetic
and propionic acid.
The rapid reaction rates of sludge methanogenesis make sludge an attrac-
tive material to analyze directly using isotope dilution techniques. The
8
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possible role of acetate in sludge methanogenesis was investigated, using
these techniques, by Smith and Mah ( 22 ). Some of the data from these
studies are included for clarity, because they were conducted under a previous
portion of this grant.
A brief discussion of theoretical considerations is included here because
erroneous interpretations have been recently published. Strayer and Tiedje
( 21 ) concluded that, using this technique, the H 2 contribution to methane
projection could be lost in the gas headspace. They failed to recognize that
this event would cause the digestion to deviate from a steady state and since
steady state conditions were maintained, H 2 loss could not have occurred. An-
alysis of the gas phase from these systems had in fact been analyzed, but not
reported because the question had been eliminated with the control showing a
steady state. The H 2 concentration in the digester vessels varied from 0.003%
to 0.009%, for gases containing 60% to 66% CH 4 .
The rate of dissimilation of an intermediate in methane formation is the
product of pool size times the turnover rate. The turnover rate can be deter-
mined, under ce ’tain circumstances, from the rate of dilution of added radio-
active tracer. This is governed by the following consideration.
d t=time
= —kx x = total radioactivity in
the intermediate
k = the rate constant
then: Xt
-k [ dt
tx
x , to
and: lnxt =-kt+lnx 0
This equation is simply the equation for a straight line with a slope
of m.
y = mx + b.
The rate constant k equals minus m.
This then means that for experiments to be valid, the reactions must
occur with gas evolution being constant, the pool size of the intermediates
constant, and the plot of in radioactivity (in some form proportional to
specific activity) against time linear. Controls must be included in the
experimentation to show that such is the case. If dilution of the inter-
mediate occurs by some mechanism other than the formation of non-radioactive
molecules from non-radioactive precursors, the experiments are not valid.
Deviation may occur from an exchange reaction, from a change in pooi size
due to the addition of the intermediate, or from alterations in the steady
state of the reaction sequences. In the work reported here steady states
were maintained, except as noted.
9
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Fo rmate
It was postuated that formate could play an important role in sludge
methanogenesis. An effort was made to evaluate this possibility using isotope
dilution techniques. Results are shown in Figure 3. Linear dilution of for-
mate was not observed. The formate exchanged rapidly with CO 2 . The method
is therefore not applicable. Efforts to redesign the experimental procedure
failed because. the rate of the exchange reaction was the same as the rate of
formate dilution, within the limits of our experimental procedures. The pos-
sible role of formate in sludge methanogenesis is not known.
Acetate
Acetate turnover was calculated using the procedures described in the
methods section. Figure 4 shows that the rate of gas evolution during the
experiments was constant. Figure 5 shows that our batch fed digesters had
constant pool sizes of acetic acid during a period of from 3 to 7 hours after
feeding. Figure 6 shows that the rate of isotope dilution was linear. Mano-
metric measurements, and direct analysis of the gas phase showed that gas
evolution was constant during the course of the experiment. Similar results
were observed for the other experiments.
The rate of methane production during the experiment was 0.033 moles/
ml/min. The rate constant from Figure 6 is 0.0052/mm and the acetic acid
pool was 4.7 pmoles/ml. Assuming 1 mole of methane from 1 mole of acetate,
the acetic acid would account for approximately 73% of the methane produced.
The results of a similar experiment, with similar controls, are shown in
Figure 7. In this experiment the rate of methane production was 0.042 umoles/
mi/mm. The rate constant from Figure 7 is 0.0078/mm and the acetic acid
tool was 3.8. pmoies/ml. Again calculating from methane rate and pool size
times the rate constant, approximately 71% of the methane produced would pass
through acetate.
Propionate
Propionate turnover was calculated using the same procedures as used for
acetate. Figure 8 shows the results of such an experiment. The pool size
during the experiment was 0.87 i.imoles/mi, the rate constant from the graph
is 0.0041/mm and the rate of methane formation during the experiment was
0.021 ano1e/ml/min. The rate of turnover of propionate calculated from this
is 0.0036 j.imoles/ml/min. Assuming 1.75 moles of methane per mole of pro-
pionate, metabolized propionic acid would contribute 30% of the total methane
formed, 13% via C02 reduction and 17% via acetate.
Figure 9 shows the results of a second experiment. In this experiment
the pool size was 1.4 pmoles/mi, the rate constant 0.0039/mm and the rate
of methane formation during the experiment 0.042 pmoles/ml/min. The rate
of propionate turnover calculated from this is 0.0055 pmoles/ml/min. Assum-
ing 1.75 pmoles of methane from one mole of propionate, propionate would
contribute 23% of the total methane formed, 10% via CO 2 reduction and 13%
via acetate. Another experiment gave results showing 27% of the total
10
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I ii
I —
uJ<
a-c-)
zc
D0
QLL
oz
0
z
I 3.8-
11.5-
9.2-
6.9-
4.6-
60
Figure 3. Change in counts per minute in the formic acid fraction from a constant
volume of sludge, after addition of radioactive formic acid.
TIME IN SECONDS
-4
.
I I I I I
0 10 20 50
30
40
-------
200
E
E
L J
C,)
4
w
C-)
z
- LU
U)
U)
LU
0
TIME IN MINUTES
Figure 4. Manometric changes in duplicate control flasks during the period of the
experiment. These flasks contained no add isotopes.
-------
4
0 2 4 6 8 10 12
TIME IN HOURS
Figure 5.
Changes in the acetic acid concentration of sludge during an eleven hour
period following the feeding of the digester.
z
0
WE
002
0
F-
W
0
0
-a
-------
9.10
w
I—
z
a 3 8.98
zo
z8.87
LL
0
z
-J
8.75
Figure 6.
Change in counts per minute in the acetic acid fraction from a constant
volume of sludge, after addition of radioactive acetic acid to duplicate
flasks containing sludge.
-J
20 30
TIME IN MINUTES
-------
U i
I—
z
LLi
a- 0
I-Ui
Z()
0
U-
0
2
-J
Figure 7. Change in counts per minute in the acetic acid fraction from a constant
volume of sludge, after addition of radioactive acetic acid to duplicate
flasks containing sludge.
01
20
TIME IN MINUTES
-------
8.52
w
I-
W 8.41
Q-z
8.17
Figure 8.
Change in the counts per minute in the propionic acid fraction from a
constant volume of sludge, after addition of radioactive propionic acid
to duplicate flasks containing sludge.
—
0 t
TIME IN MINUTES
-------
TIME IN MINUTES
Figure 9.
Change in the counts per minute in the propioriic acid fraction from a
constant volume of sludge, after addition of radioactive propionic acid
to duplicate flasks containing sludge.
U i
I-
Ui 0
0
0
z
-J
-a
0 10 20 30 40
-------
methane formed through propionate, presumably 16% via acetate and 11% via CO 2
reduction.
n-Butyrate
Experiments were conducted with n-butyric acid, utilizing the methods
previously described, with results shown in Figure 10. In this experiment
the pool size of n-butyrate was 0.14 moles/ml, the rate constant 0.033/mm,
and the rate of methanogenesis 0.029 umoles/mi/min. The butyrate turnover
would then be 0.0046 tmoles/m1/min. Assuming 2.5 moles of methane per mole
of n-butyrate, n-butyrate would contribute 40% of the total methane formed,
8% by CO 2 reduction and 32% by way of acetate.
A similar experiment was conducted with sludge from a digester producing
methane at a rate of 0.O04 1 imoles/ml/min, with results shown in Figure 11.
The pool size was 0.067 pmoles/ml, and the rate constant 0.026/nun. These
results give a turnover of butyrate of 0.0017 pmoles/ml/min, which calculate
to a 10% contribution of butyrate to methane, approximately 3.3% by CO 2 re-
duction and 6.6% by way of acetate.
i so-Butyrate
Low, but consistant, pools of iso -butyrate were observed. An experiment
was conducted, with considerable difficulty as shown by the spread of points
on Figure 12. The sludge used produced methane at a rate of 0.047
umoles/ml/min. The pool size was 0.021 mo1es/m1. The rate constant calcu—
lated from Figure 12 was 0.013/mm giving a turnover rate for iso-butyrate
of 0.00027. Assuming 2.5 iimoles of methane per mole of iso-butyrate, iso-
butyrate would contribute 1.5% of the total methane formed.
Ethanol
Ethanol added to sludge disappeared very rapidly as shown in Figure 13.
However, when radioactive ethanol was added, no appreciable change in spe-
cific activity was observed over a 35 minute period as shown in Table 2.
Therefore, ethanol is not produced in this fermentation and does not contrib-
ute to the total methane formed.
TABLE 2. TURNOVER OF ETHANOL IN SLUDGE
Time
(nun.)
Specific Activity
picocuries/pmo le
1
6.2
10
6.7
20
7.0
35
6.6
The variation observed was assumed to be due to analytical error.
18
-------
8.52
8.29
F-
8.06
L)
C/)I— 7.83
za
3 - 7.5W
0
z
7.14
Figure 10.
Change in the counts per minute in the n-butyric acid fraction from a
constant volume of sludge, after addition of radioactive n-butyric
acid to duplicate flasks containing sludge.
-J
TIME IN MINUTES
-------
8.51
U i
I—
8.41
a:
Ui
cm—
8.33
0c
C)
0
z
-J
8.3
8.1
Figure 11.
TIME IN MINUTES
Change in the counts per minute in the n-butyric acid fraction from a
constant volume of sludge, after addition of radioactive n-butyric acid.
r.’)
0
0 4 8 12 16 20 24 28
-------
6.22
w
I-.
>-
(1)1 -
z
-J
Figure 12.
5.98
5.75.
5.52
5.30
TIME IN MINUTES
Change in the counts per minute in the iso-butyric acid fraction from a
constant volume of sludge, after addition of radioactive iso-butyric acid.
-J
0 10 20 30 40 50
-------
8.0
Change in tne concentration of ethanol added to sludge, after
addition of ethanol.
7.0
E
0
E
z
0
I—
z
U i
0
z
36.0
-J
0
z
I—
Ui
5.0
TIME IN MINUTES
Figure 13.
-------
iso-Valeric Acid
Iso-valeric acid was observed in low concentrations in the digesting
sludge. However, as with ethanol, there was no change in specific activity
upon addition of label as shown in Table 3. This demonstrates that iso-
valeric acid does not contribute appreciably to the production of methane.
TABLE 3. TURNOVER OF ISO-VALERIC ACID IN SLUDGE
Time (mm.)
Counts/mm/mi
in
iso-valerate
0
5
75
4.9
5.0
4.8
X
X
X
io
io
1O
CONVERSION OF PROPIONATE AND BUTYRATE TO ACETATE
Experiments were conducted using techniques similar to those used for
the turnover experiments to evaluate the valiçtjty of the concept of conver-
sion of propionate and butyrate to acetate. ‘ C-labeled propionate was added.
Samples were removed and the amount of label in acetate and propionate per
unit of volume was determined with the results shown in Figure 14. A similar
experiment was conducted with 14 C-butyrate with the results shown in
Figure 15. The expected values were calculated from the rates of the prev-
ious turnover experiments. The results are consistent with the conversion of
propionate and butyrate to acetate.
HYDROGEN EFFECTS ON SLUDGE DIGESTION
Inhibition of Volatile Acid Turnover
The large number of methanogenic bacteria in digesting sludge suggested
that the capacity of digesting sludge for hydrogen metabolism should be great.
The fact that all isolates utilized hydrogen suggested that addition of hydro-
gen to digesting sludge should stimulate the methanogenic process since add-
ition of hydrogen should result in an increase in the population of the meth-
anogenic bacteria. Capacity of digesting sludge for hydrogen metabolism was
determined by incubating digesting sludge under a gas phase of 70% hydrogen
and 30% carbon dioxide. Hydrogen uptake and methane formation were deter-
mined quantitatively. Methane formation was also quantitatively determined
for digesting sludge incubated under a gas phase of 70% nitrogen and 30%
carbon dioxide. The results are shown in Table 4. Taking average values,
447 imoles of hydrogen were utilized in the hydrogen flasks. Assuming
1 umole of methane from 4 iimoles of hydrogen, 112 umoles of methane would
have been produced from added hydrogen and carbon dioxide, leaving 98
pmoles of methane formed from the sludge. However 134 pmoles of methane
were produced in the nitrogen flasks. The difference between the 134
iimoles of methane produced from sludge in the absence of added hydrogen and
23
-------
1,400-
1,200-
U i
I,000-
z
Ui
a-
z
0
C)
800-
600-
400-
200-
Figure 14.
0 tO
TIME IN MINUTES
Counts per minute lost from propionic acid, and found in acetic acid, in a
constant volume of sludge, following addition of radioactive propionic acid.
N)
• LOST
• FOUND
• ANTICIPATED
20
I I I
30 40 50
60
-------
2500-
2000-
z
LU 1500-
0
C ’,
F-
U, z
0 -
0
500-
100— I U I I
0 5 10 15 20 25
TIME IN MINUTES
Counts per minute lost from butyric acid, and found in acetic acid, in a
constant volume of sludge, following addition of radioactive butyric acid.
O LOST
• FOUND
• ANTICIPATED
I I
30 35
Figure 15.
-------
the 98 moles calculated from sludge in the presence of hydrogen indicates
that either hydrogen inhibited methane production from other sludge precur-
sors, or hydrogen and carbon dioxide were converted to molecules other than
methane. The rate of methane formation was determined for sludge samples
which had been equilibrated for two hours with a gas phase of 70% hydrogen
and 30% carbon dioxide. This rate was compared to the rate of methanogenesis
from sludge which had been equilibrated for two hours with a gas phase of
70% nitrogen ar d 30% carbon dioxide. The rates were calculated from the
methane evolved during seventy-five minutes iimiiediately following the equi-
libration period. The results are shown in Table 5. The lowered rate of
methane evolution following exposure to hydrogen gas shows that an inhibition
of methanogenesis had occurred. Calculations with average values from
Table 5 indicate a 15% inhibition following exposure to a 70% hydrogen atmos-
phere. Calculations from average values of gas utilization and production
given in Table 4 show an inhibition of 30% during exposure to a 70% hydrogen
atmosphere. The difference between these two figures can be explained on the
basis of recovery from inhibition following the brief exposure to molecular
hydrogen during the experiment reported in Table 5.
The initial data obtained suggested that hydrogen gas should ‘stimulate
anaerobic digestion of sludge. However, initial experiments did not produce
the anticipated result. In fact, hydrogen had an inhibitory effect. The
inhibition was approximately 30%, consistent with the hypothesis that the
inhibition was an inhibition of propionate, without an inhibition of acetate.
To test this hypothesis, experiments were conducted to determine the effect
of hydrogen on the rate constants for propionate and butyrate turnover with
the results shown in Figure 16 and Figure 17. Hydrogen inhibited propionate
turnover strongly but had little if any effect on acetate turnover. These
experiments were conducted under a gas phase of 70% hydrogen, 30% carbon
dioxide.
An experiment was then conducted to determine the concentration of hydro-
gen which inhibited the reaction. In this experiment the inhibition was
followed by measuring the appearance of labeled acetate from labeled pro-
pionate in sludge incubated in equilibration with various concentrations of
hydrogen. The gas mixtures used were 30% carbon dioxide, with a balance of
hydrogen plus nitrogen. The results are shown in Figure 18. The fermentation
was inhibited by 18% hydrogen but not 9% hydrogen.
TABLE 4. HYDROGEN UTILIZATION BY DOMESTIC SLUDGE
Gas
70% H2,
U
Phase
30% C02
CH 4
in
Formed
mole
207
214
Initial H 2
in pmole
1650
1695
Fi
in
nal H2
mole
1220
1230
70% N2,
“
30% CO 2
“
132
136
0
0
3
3
26
-------
8.37
w
I-
z
w4
8.33
co
0
Li..
8.28
z
-J
8.23
Figure 16.
TIME IN MINUTES
Change in counts per minute in the acetic acid fraction from a constant
volume of sludge after addition of radioactive acetic acid to duplicate
flasks containing sludge exposed to hydrogen, and duplicate flasks
containing sludge not exposed to hydrogen.
N.,
—1
0 10 20 30 40
-------
TIME IN MINUTES
Figure 17.
Change in counts per minute in the propionic fraction from a constant
volume of sludge after addition of radioactive propionic acid to
duplicate flasks containing sludge exposed to hydrogen, and duplicate
flasks containing sludge not exposed to hydrogen.
LU
I —
c r : 4
CflQ
0
C-)
0
z
-J
0 tO 20 30 40
-------
w
1— 6.45-
z
a 5
w
0
:D<
5.53-
LL
0
z
-J
5.07
I
I
I
I
I
I
I
I
I
5
10
15 20
TIME
25 30 35
IN MINUTES
40
45
,\)
0
618% H 2
S 9% H 2
IN
IN
IN
GAS PHASE
GAS PHASE
GAS PHASE
-------
TABLE 5. EFFECT OF HYDROGEN GAS ON SLUDGE METHANOGENESIS
Treatment Prior
to Inoculation
CH4
in
Formed
pmole
Rate of
in
CH4 Formation
uTflOle/rnin
Exposed to H2 gas*
62
0.017
U
66
0.018
Exposed to N2 gas+
75
0.020
II II II U
78
0.021
*Equilibrated with 70% H2 and
30%
C02
for two
hours.
+Equilibrated with 70% N2 and
(Incubation time 75 minutes)
30%
C02
for two
hours.
Hydrogen Uptake Capacity as a Parameter for
Predicting Digester Failure
During the course of our studies it was observed that poorly digesting
sludge had a limited capacity for hydrogen utilization. These observations
suggested that hydrogen uptake by sludge might be a useful parameter for ascer-
taining digester functioning and in the assay of effects of toxic substances
on the digestion process. Long-term experiments were designed to provide in-
formation on the feasibility of these assays.
Two four-liter digesters were established using domestic sludge as feed.
Both digesters were fed ten grams of solids per day with a hydraulic retention
time of twenty days. Both digesters were then induced to fail. “Digester A”
was induced to failure by maintaining the hydraulic retention time while the
amount of solids fed was increased. “Digester B” was induced to failure by
maintaining the amount of solids fed while the hydraulic retention time was
decreased. The feeding schedule is shown in Figure 19.
Sludge samples were transferred to Warburg flasks and methane production
was determined under an atmosphere of 70% N 2 and 30% C0 2 , and under an atmos-
phere of 70% H2 and 30% CO2. Methane production from hydrogen-carbon dioxide
atmosphere and total methane produced under a nitrogen-carbon dioxide atmos-
phere. The results are shown in Figures 20 and 21.
Volatile organic acid concentrations were determined for sludge from both
digesters, with the results shown in Figures 22 to 26.
The changes in retention time and feeding rate were slow, producing slow
changes in methane production during the initial stages of the experiment, as
shown in Figures 20 and 21. Volatile organic acid concentrations increased as
methane production decreased in both digesters. The organic acid concentra-
tion in digester A was quite different from the organic acid concentration in
digester B at the time of failure. For these experiments failure was consid-
ered to have occurred when the sludge could not produce methane from hydrogen
and carbon dioxide.
30
-------
C’)
w
I-
z
0
p
z
w
I-
w
I ii
( )
-J
0
C,)
C /)
4
CD
—
0 0 20 30 40 50 60 70
DAYS
Figure 19. Feeding schedule of digesters being induced to fail.
-------
‘ Ii
Oc
wcl)
U i
DAYS
0 10 20 30 40 50 60 70
Figure 20.
Methane production rates in digesters being induced to fail.
-------
0
w
0
0
0
LU
z
I
I-
LU
0 10 20 30 40 50 60 70
DAYS
Figure 21. Methane production rates in digesters being induced to fail.
-------
0 10 20 30 40 50 60 70
DAYS
I-
z
w
0
z
0
0
w
F-
w
0
U)
.3
E
Figure 22. Acetate concentration in digesters being induced to fail.
-------
E
20
E
DAYS
40
30
z
0
I-
z
w
0
2
0
0
w
z
0
0
0
0
(A)
0 ,
0 10 20 30 40 50 60 70
Figure 23.
Propionate concentration in digesters being induced to fail.
-------
z
Q
C-,
0 10 20 30 40 50 60 70
DAYS
Figure 24.
n-Butyrate concentration in digesters being induced to fail.
-------
z
0
F-
z
w
C-)
0
0
w
>-
F-
C D
c i)
E
U)
a)
0
E
c )
0 10 20 30 40 50 60 70
DAYS
Figure 25. iso-Butyrate concentration in digesters being induced to fail.
-------
z
Q
I —
z
LU
ZE
0u
.5 ?
0
C t)
DAYS
( )
0 10 20 30 40 50 60 70
Figure 26. iso-Valerate concentration in digesters being induced to fail.
-------
An effort was made to quantitate the relative contributions of acetate
and propionate to methane production by a failing digester. Samples were ana-
lyzed when methane production was reduced to approximately 6% of the initial
rate. Our attempts to obtain rate constants were unsuccessful as shown in
Figures 27 and 28. Linearity could not be obtained.
The results obtained show that under the conditions imposed the capacity
of sludge to produce methane from hydrogen and carbon dioxide reflects the
capacity of sludge to produce methane from other sources. Due to the diffi-
culty of the assay, and the fact that during failure decrease in methane pro-
duction from hydrogen did not precede a decrease in methane formation from
other sources, it is concluded thatthe assay of hydrogen conversion to
methane is not a promising assay for predicting digester failure.
Digestion Inhibition Studies and Hydrogen Effects
We previously concluded that hydrogen gas specifically inhibited the
metabolism of propionic acid in sludge. We continued these studies in the
hopes of elucidating the roles of various physiological types of bacteria in
the total fermentation. The initial studies dealt with short-term effects of
hydrogen on the fermentation. These studies have been extended to include
long-term effects with emphasis on the organic acids.
Small digesters were established and fed domestic sludge with a retention
time of twenty days. Three experimental digesters were equilibrated with a
gas phase of 70% hydrogen and 30% carbon dioxide. A fourth digester was equi-
librated was a gas phase of 70% nitrogen and 30% carbon dioxide. Methane pro-
duction and organic acid concentrations were followed with the results shown
in Figures 29 to 31. Methane production from added hydrogen was determined by
direct analysis of gases produced in the experimental digesters. Methane pro-
duction from digesting sludge was determined in the experimental digesters by
flushing out all hydrogen with a nitrogen-carbon dioxide mixture, and then
directly assaying the gases produced.
The results showed that in the presence of hydrogen gas production of
methane from digesting sludge was inhibited. The inhibition was very marked
during the first twenty-four hours and was accompanied by a rapid increase in
the concentration of propionic acid. On continued exposure to hydrogen, the
propionic acid concentration continued to rise, followed after four days by an
increase in concentration of acetic acid. After ten days exposure, methane
production from the sludge was essentially zero. The small residual value
shown is interpreted to have been formed from reserve materials in the hydro-
gen oxidizing microflora which developed on the added hydrogen.
From these, and our other reported results, we have concluded that the
role of the hydrogen oxidizing methanogenic bacteria in anaerobic digestion
is the maintenance of a hydrogen concentration low enough to prevent the inhi-
bition of short chain organic acid metabolism and the cessation of the fermen-
tation.
39
-------
U i
I-
D
z
0
z
-J
Figure 27. Change in the counts per minute in the acetate fraction from constant
volumes of sludge following addition of radioactive acetate to
digesters being induced to fail.
0
TIME IN MINUTES
-------
w
I-
cI,
C)
0
2
-J
Figure 28. Change in the counts per minute in the propionate fraction from
constant volumes of sludge following addition of radioactive
propionate to digesters being induced to fail.
-I
80 100
TIME IN MiNUTES
-------
0.20-
I SLUDGE NOT EXPOSED TO HYDROGEN
£ SLUDGE EXPOSED TO HYDROGEN
0.16- • SLUDGE EXPOSED TO HYDROGEN: PLUS
ADDED HYDROGEN
0.12-
c E
Z.i 0.08-
l\)
Li
0.04-
O-0
4 6 8 10 12
DAYS
Figure 29. Methane production from sludge not exposed to hydrogen, sludge exposed
to hydrogen and sludge exposed to hydrogen with added hydrogen as
substrate.
-------
z
0
I —
z—
wE
0’ -
2”
WI.
w
0
Figure 30.
Acetate concentration in sludge exposed to hydrogen, and sludge
not exposed to hydrogen.
(. )
0 2 4 6 8 10 12
DAYS
-------
z
0
I-
z
LU
0
C)
LU
z
0
0
0
a-
C
E
E
II
Figure 31.
Propionate concentration in sludge exposed to hydrogen, and sludge
not exposed to hydrogen.
• SLUDGE
• SLUDGE
A SLUDGE
O SLUDGE
EXPOSED TO HYDROGEN. EXPERIMENT I
EXPOSED TO HYDROGEN. EXPERIMENT 2
EXPOSED TO HYDROGEN. EXPERIMENT 3
NOT EXPOSED TO HYDROGEN
DAYS
12
-------
Long-Term Hydrogen Inhibition
The previous experiments were conducted for twelve days. The experiments
were then repeated over a sixty-five day period to determine which physiolo-
gical groups of bacteria survived this form of inhibition. The experiments
were conducted with duplicate samples. Figure 32 shows the results of hydro-
gen exposure over a 65-day period. Figures 33 and 34 show the rates of
methane production from added hydrogen by sludge which had been exposed to
hydrogen. Figures 35 and 36 show the acetate pools in sludge exposed to hy-
drogen. Figures 37 and 38 show the propionate pool sizes of sludge exposed to
hydrogen. Figures 39 and 40 show the n-butyrate and the n-valerate pools of
sludge exposed to hydrogen. Figures 41 and 42 show the iso-butyrate and iso-
valerate pooi sizes in sludge exposed to hydrogen.
These results show that hydrogen inhibits the dissimilation of propio-
nate, n-butyrate, iso-butyrate, n-valerate and iso-valerate. Acetate is in-
hibited, assuming no formation of acetate from hydrogen and carbon dioxide,
after about five days but after approximately twelve days the inhibition is
reversed and the acetate pool size decreases rapidly. The results show that
the inhibition by hydrogen affects a different microflora than the inhibition
caused by reduced retention time, or increased feed rate, since in the former
case the sludge retains the ability to produce methane from hydrogen and
carbon dioxide, and acetate inhibition is temporary.
HYDROGEN PRODUCTION FROM VOLATILE ACIDS
Radioactive tracer studies (10, 19) of methanogenic enrichments and the
data reported here have shown that acetate is an intermediate in the methano-
genic dissimilation of propionate and butyrate. Using highly purified cul-
tures which were unable to dissimilate acetate, Stadtman and Barker (20) de-
monstrated that in the presence of H 14 C03, the methane produced from pro-
pionate and butyrate had the same specific activity as bicarbonate, indicating
that all methane was produced by carbon dioxide reduction. These reactions
can therefore be rewritten as the sum of an oxidation reaction and a reduction
(methanogenic) reaction (Table 6, equations A through D).
The highly purified cultures used by Stadtman and Barker are generally
believed to have been impure cultures (3) but direct evidence is lacking. No
demonstrably pure methanogenic cultures have been shown capable of dissimilat-
ing either propionate or butyrate. The inability to use these substrates
directly suggests that non—methanogenic bacteria may oxidize propionate and
butyrate and provide hydrogen to methanogenic bacteria (4). Because molecular
hydrogen is the preferred substrate for carbon dioxide reduction by methano-
gens (26), hydrogen is a likely candidate for a role in interspecies electron
carrier. If this were the case, the dissimilation of propionate and butyrate
could be written as the sum of a hydro9en producing and hydrogen utilizing re-
action (Table 6, equations E through H). It can be seen from the free energy
changes of equations E and G (Table 6) that the partial pressure of hydrogen
must be maintained at a very low level to allow exergonic hydrogen production
from propionate and butyrate. This could presumably be accomplished by the
45
-------
0.04
.E 0.0
LU u)002
0.01
Figure 32.
Methane production from sludge exposed to hydrogen, and sludge
not exposed to hydrogen.
0•i
30
DAYS
-------
DAYS
Methane production from added hydrogen by sludge previouslY exposed
to hydrogen for periods of time up to 55 days.
C-)
0
w
z
I-
U i
C
E
E
U,
I
0 15 30 45 60
Figure 33.
-------
0 15 30 45
DAYS
Figure 34.
Methane production from added hydrogen by sludge previously exposed
to hydrogen for periods of time up to 55 days.
D c
LU
I
cc
60
-------
-J
0
0
0
w
I—
U i
0
E
U)
0
E
0 15 30 45 60
DAYS
Figure 35.
Acetate pools in sludge exposed to hydrogen for periods of time up to 55 days.
-------
0
0
Wv
20
U,
0
0 15 30 45 60
DAYS
Figure 36.
Acetate pools in sludge exposed to hydrogen for periods of time up to 55 days.
-------
-J
0
0
wE
U)
0
a-
DAYS
(n
-J
0 $5 30 45 60
Figure 37.
Propionate pools in sludge exposed to hydrogen for periods of time up to 55 days.
-------
-J
0
0
WE
zi’
0
0.
0.
DAYS
0,
0 15 30 45 60
Figure 38. Propionate pools in sludge exposed to hydrogen for periods of time up to 55 days.
-------
0
ou)
—w
z-
0
Figure 39. n-Butyrate and n-valerate pools in sludge exposed to hydrogen for
periods of time up to 55 days.’
DAYS
-------
U)
3E
0 (l)
—4)
(9
Figure 40.
0 (5 30 45 60
DAYS
n-Butyrate and n-valerate pools in sludge exposed to hydrogen for
periods of time up to 55 days.
01
-------
C l )
OE
QU)
z-
Figure 41.
iso-Butyrate and iso-valerate pools in sludge exposed to hydrogen for
periods of time up to 55 days.
01
0 ,
0 15 30 45 60
DAYS
-------
C,)
=3.
QE
Qu)
2
Figure 42.
0 15 30 45
60
DAYS
iso-Butyrate and iso-valerate pools in sludge exposed to hydrogen for
periods of time up to 55 days.
U,
0 i
-------
TABLE 6. EQUATIONS AND FREE ENERGY CHANGES FOR THE
ANAEROBIC OXIDATION OF PROPIONATE AND BUTYRATE,
AND THE REDUCTION OF CO 2 TO METHANE
t G° 1
Equation (kcal/react.)a
b
(kcal/react.)
A
4CH 3 CH 2 COO + 8H 2 0 4CH 3 C00 + 4C0 2 + 24H +
24e
--
--
B
24e + 24 H + 3C0 2 - 3CH 4 + 6 H 2 0
-—
--
A
+ B 4CH 3 CH 2 COO + 2H 2 0 4CH 3 C00 + CO 2 + 3CH 4
-25.23
-23.72
C
2CH 3 CH 2 CH 2 COO + 4H 2 0 - 4CH 3 C00 + 8H + 8e
--
--
0
8e + 8H + CO 2 CH + 2I i2O
--
--
C
+ D 2CH 3 CH 2 CH 2 COO + 2H 2 0 + CO 2 - 4CH 3 C00 + CH 4
-8.16
-19.65
E
4CH 3 CH 2 COO + 8H 2 O - 4CH 3 C00 + 4C0 2 + 12H 2
+68.52
-1.86
F
12H 2 + 3C0 2 -* 3CH 4 + 6H 2 0
-93.75
-21.86
E
+ F 4CH 3 CH 2 C00 + 2H 2 0 4CH 3 COO + CO 2 + 3CH 4
-25.23
-23.72
G
2CH 3 CH 2 CH 2 COO + 4H 2 O 4CH 3 COO + 4H 2
+23.09
-12.37
H
4H 2 + CO 2 - CH 4 + 2H 2 0
-31.25
-7.28
G
+ H 2CH 3 CH 2 CH 2 COO + 2H 2 0 + CO 2 - 4CH 3 C00 + CH 4
-25.23
-23.72
a. H2, CH 4 , and C02 in gaseous state; H at i0 mole activity per kg;
all other substances at 1 mole activity per kg. The free-energy data
have been calculated using free energies of formation from the elements
compiled by Thauer et al. (16).
b. AG calculated at the following activities: Propionate, butyrate, and
acetate at io3 moles/kg, C02 at 0.3 atm, CH 4 at 0.7 atm, and H 2 at
5 x iO atm.
57
-------
large numbers of hydrogen oxidizing methanogens present in methanogenic eco-
systems (26).
Growth yield studies of hydrogen-grown methanogens indicate 2.3 to 3.3 g
of cells per mole of methane produced (16, 18, 32), or (assuming YATp of
10.5 g/mole ATP) 0.21 to 0.31 moles ATP/mole CH 4 . Thauer et al., (24) indi-
cate that, including allowances for inefficiencies, 10 to 12 Kcal/mole are
required for ATP production from ADP and inorganic phosphate. Taking a con-
servative view, allowing 12 Kcal/mole ATP and 0.31 moles ATP/mole CH 4 , one
can calculate that 3.72 Kcal/mole CH 4 must be coupled to ATP production. If
one assumes that methane production from hydrogen and carbon dioxide approach-
es equilibrium in methanogenic ecosystems, one can substitute into the free
energy equation for methane production according to equation H in Table 6:
[ CH 4 ]
= G° + RT ln
[ CO 2 ] [ H 2 ]
If one sets the partial pressure of CH 4 and CO 2 at values comon to methano-
genic ecosystem (.7 atm and .3 atm respectively), solving the equation for the
partial pressure of hydrogen gives a value of 1.1 X 10-5 atm, well below the
level necessary to allow exergonic hydrogen production from propionate or
butyrate.
Interspecies transfer of electrons via molecular hydrogen has been demon-
strated in a number of fermentative bacteria (5, 6, 9, 12, 14, 15, 17, 26,
29). When co-inoculated with methanogens, the soluble products of these fer-
mentative bacteria are generally more oxidized than when grown in pure cul-
ture. Large amounts of hydrogen (measured as methane production) are the
major reduced product when cocultured with methanogens, whereas little or no
hydrogen is produced by the pure cultures. This phenomenon of interspecies
hydrogen transfer has recently been reviewed by Mah et al. (13).
If interspecies hydrogen transfer is to occur, then there must be a
hydrogenogenic microflora which produces hydrogen from volatile organic acids.
The production of hydrogen from volatile acids is demonstrated by the experi-
ments which follow. This unique form of hydrogen production was first report-
ed by Smith and Shuba (23); however, in those experiments there was an inh bi—
tion of the reaction rates. The results could, therefore, be explained on the
basis of a side reaction caused by the inhibition. In the experiments now re-
ported the inhibition has been eliminated.
Determination of Inhibitory Concentrations of Volatile Acids
To insure that the levels of volatile organic acids used in subsequent
experiments were not inhibitory, enrichments were incubated in the presence
of various amounts of volatile organic acids, and the rate of gas production
was monitored.
Warburg vessels were gased out with a gas mixture of 30% CO 2 - 70% N2,
and 50 ml of enrichment culture (acetate, propionate, or butyrate) were added
58
-------
to vessels with varying amounts of volatile organic acids (oxygen-free solu-
tion neutralized with NaOH). Manometric readings were taken hourly, and at
the end of each experiment liquid samples were taken for volatile organic acid
analysis. Figure 43 illustrates that for these enrichment cultures, volatile
organic acid concentrations up to 50 mM were not inhibitory. All subsequent
experiments were performed with the volatile organic acid concentrations be-
tween 5 and 50 mM.
Effect of Molecular Hydrogen on Volatile Organic Acid Dissimilation
The free energy change which accompanies the production of molecular
hydrogen from propionate and butyrate is positive at partial pressures of
hydrogen above atm, so if this is the mechanism for the dissimilation of
these acids, then hydrogen should inhibit the dissimilation of these sub-
strates by the enrichment cultures used.
Acetate, propionate, and butyrate enrichments were incubated in Warburg
flasks under 70% N 2 and 30% CO 2 . At the beginning of the experiment, the
volatile organic acid corresponding to each enrichment was added anaerobically
t 9 give a final substrate concentration of 5 to 10 mM, and carboxy-labeled
1 ’C volatile organic acid were added to the pool. Liquid samples were removed
at the beginning of the experiment, and at hourly intervals. After 2 hours
incubation the gas phase was changed to 70% H 2 and 30% C02, and incubation was
continued for 2 additional hours. The specific activity of the substrate was
then determined. In each case, the specific activity remained constant
throughout the experiment (+4%) indicating that no substrate was produced
during the experiments. Therefore, changes in substrate levels could be in-
terpreted as substrate utilization by the enrichments. Figures 44 to 46 show
that acetate dissimilation was slightly inhibited by molecular hydrogen, while
propionate dissimilation and butyrate dissimilation were completely shut off.
The propionate data represents a corroboration of evidence reported by Smith
and Shuba (23).
Rates of Methanogenesis in the Absence and Presence of Added Molecular
Hydrogen
Studies in the lab of van den Berg (25) indicated that when enriching
with acetate, the ability to utilize hydrogen as a methanogenic substrate
diminished as the original inoculum was washed out. This indicates that hy-
drogen is not an intermediate in acetate dissimilation, for otherwise there
would have been an enrichment for hydrogen oxidizing methanogens during en-
richment for acetate utilizers. To determine the ability of the enrichments
in the present study to utilize hydrogen, the methanogenic rates in the ab-
sence and presence of hydrogen were determined and compared. Three fifty ml
samples from each of the enrichment cultures were added anaerobically to War-
burg vessels with 70% N2 and 30% C02 atmospheres, and gas production from each
was measured hourly. After 6 hours the atmospheres were replaced with 70% H 2
and 30% C02, and gas uptake was measured every 10 mm. for 1 hour (Table 7).
Gas samples taken at the end of this hour always contained within 8% of the
methane expected from hydrogen uptake by the stoichiometry of the reduction
of carbon dioxide with hydrogen. To establish that the hydrogen uptake
59
-------
VOA CONCENTRATION (mM)
Figure 43.
Inhibition of gas production by methanogenic enrichments maintained at
pH 6.9 to 7.0. Acetate enrichments with added acetate, propionate
enrichments with added propionate, and butyrate enrichments with added
butyrate.
E
z
Q
I-
0
0
0
C,)
o )
-------
U)
Figure 44.
Degradation of acetic acid ifl the absence and presence of added
molecular hydrogen.
HOURS
-------
-g
U)
:1
Figure 45.
HOURS
Degradation of propionic acid in the absence and presence of
added molecular hydrogen.
N.)
0 I 2 3 4
-------
HOURS
Degradation of butyric acid in the absence and presence of
added molecular hydrogen.
0 i
I 2
3 4
Figure 46.
-------
resulted in carbon dioxide reduction, 50 ml aliquots of acetate enrichment
were incubated in two Warburg vessels with atmospheres of 70% H2 and 30% Co 2 .
Labeled sodium bicarbonate was added, and flasks were incubated for 1 hour at
300 C while monitoring gas uptake manometrically. At the end of the incuba-
tion period, 10 ml of 1 M NaOH solution were added slowly with a syringe.
The NaOH solution caused the carbon dioxide to be absorbed by the liquid in
the flasks. The 10 ml volume approximately replaced the volume of gas absorb-
ed, so the total volume of liquid plus gas remained approximately the same.
Ten ml of gas was then removed for quantitation of the radioactivity. Based
on the specific activity of the bicarbonate and the amount of hydrogen utiliz-
ed, the amount of labeled methane was calculated (assuming that all hydrogen
utilized resulted in carbon dioxide reduction). In the duplicate flasks, 93%
and 96% of the expected radioactivity was found in the gas samples. This dem-
onstrates that nearly all of the hydrogen absorbed in these experiments was
used for carbon dioxide reduction.
The high rates of hydrogen oxidation exhibited by propionate and butyrate
enrichments demonstrate that enriching for these substrates also enriches for
hydrogen oxidizing methanogens, suggesting that hydrogen may be an intermed-
iate in the dissimilation of these substrates.
TABLE 7. METHANE PRODUCTION AND HYDROGEN UTILIZATION BY ENRICHMENT
Enrichment
Gas Production from VOA H2 Utilization
(rmi/hr); N 2 /C0 2 atm. (m/hr); H 2 /CO 2 atm
Ratio
to
of H 2
gas pro
from
Utilization
duction
VOA
Acetate
16.6
(+1.2)
39.2
(+2.5)
2.4
(+ 0.23)
Propionate
18.9
(+1.9)
88.8
(+11.8)
4.7
(+0.61)
Butyrate
15,3
(+1.0)
82.8
(+4.8)
5.4
(+0.31)
MPN Analysis for Methanogenic Bacteria
The results of the previous experiment were corroborated by MPN analysis.
Serial ten-fold dilutions of each enrichment were made using liquid media with
70% N 2 and 30% C02, and from these dilutions were inoculated in triplicate
into roll tubes having a gas phase of 70% H 2 and 30% CO 2 . The tubes were in-
cubated at 300 C for 3 weeks, and then checked for deveTopment of a vacuum.
Negative pressure development plus the presence of greater than 10% methane
constituted a positive test for the presence of methanogens. NuWbers of meth-
anogens in the enrichments were calculated as follows: 1.1 X 10’ per ml in
the acetate enrichment, 4.4 X 108 per ml in the propionate enrichment, and
4.2 X 108 per ml in the butyrate enrichment.
64
-------
Hydrogen Stripping Experiments
Hydrogen is generally not detectable in gases produced from healthy
anaerobic digestors. Hydrogen was not produced by the enrichments used in
this study, presumably because of the rapid oxidation of hydrogen by methan-
ogenic bacteria present. Attempts were therefore made to remove dissolved
hydrogen from enrichments before it could be utilized in methanogenesis.
A 500 ml gas scrubber was gased out with oxygen-free 100% C0 2 , and
500 ml of enrichment culture was added. To prevent a decrease in pH during
equilibration with 100% C02, 6 g NaHCO 3 was added. The pH, after equilibrium
with 100% carbon dioxide, was between 6.8 and 6.9. Enrichments were maintain-
ed at room temperature under 100% CO 2 . Carbon dioxide was then vigorously
bubbled through the enrichments and the effluent gas was collected. Carbon
dioxide was absorbed using the alkaline gas collector. Collections were made
at various sparging rates, and the alkali insoluble gases analyzed for methane
and hydrogen. The procedure was repeated on samples from each enrichment.
In the case of the propionate and butyrate enrichments, feed to the enrich-
rnents was withheld 24 hours prior to its use in a sparging experiment, result-
ing in the substrate being dissimilated. Detectable volatile organic acids
were not present. At the beginning of an experiment the desired substrate
was added. This was done to limit acetate dissimilation during the experi-
ments in which propionate and butyrate dissimilation were examined.
Figure 47 shows gases produced by the propionate enrichment when only
acetate was present as substrate. Even at sparging rates approaching 1 1/mm,
little hydrogen was produced. Acetate enrichments dissimilating acetate gave
similar results. Figure 48 shows that a different aliquot of the same pro-
pionate enrichment used in Figure 47 produced large amounts of hydrogen when
propionate was present as substrate. The decrease in methane production at
high sparge rates can be accounted for by the loss of methane which would have
been produced from the hydrogen which had been stripped from solution.
Figure 49 shows gases produced by the butyrate enrichment dissimilating buty-
rate, and, as with propionate dissimilation, large amounts of hydrogen were
produced at high sparging rates. The decrease in the methane production at
high sparge rates can again be accounted for by the loss of methane which
would have been produced from the hydrogen which had been stripped from solu-
tion.
To determine the quantity of methane and hydrogen remaining in the alka-
line solution of the collector, 100% nitrogen was added to the collector
through the septum after collected gas had been removed at the end of a
sparging experiment. The nitrogen gas was then shaken in the collector with
the alkaline solution for 1 hour and removed for analysis. The gas that had
remained in solution was calculated according to Henry’s law and was found to
represent less than 0.5% of the methane and less than 0.5% of the hydrogen
produced during the spargirig experiment. This was deemed insignificant, and
routine analysis included only undissolved gases.
Thirty liters from each tank of 100% carbon dioxide used for sparging
experiments were absorbed using NaOH solution in the gas collector. No
hydrogen and no methane was detected in the alkali insoluble gas.
65
-------
0 200 400 600 800
(mi/rn in)
SPARGE RATE
Gas production obtained from a propionate enrichment with acetate as the
substrate, when flushed with 100 percent C02.
U i
z
0
I-
U
a-
‘I)
o l
I000
Figure 47.
-------
0 200 400 600 800
SPARGE RATE (mi/mm)
1000
Gas production obtained from a propionate enrichment with propionate as
substrate when flushed with 100 percent C02.
I
U i
z
0
I—
0
0
0
cr
Figure 48.
-------
II
Gas production obtained from a butyrate enrichment with butyrate as the
substrate when flushed with 100% CO 2 .
w
a:
z
Q
I.-
C-)
0
a:
C /)
(9
co
HOURS
Figure 49.
-------
Sludge from a conventional laboratory scale anaerobic digestor was also
sparged, as well as sludge diluted 1:1 with sludge supernatent. The super-
natent was prepared by adding sludge to a gazed out flask and cooling in an
ice-water bath to halt gas production which might stir up sediment. Sludge
was allowed to settle for 50 mm. and the supernatent was siphoned off into
the gas scrubber. It was then warmed to 350 C, and an equal volume of sludge
was added from the digestor. As with the enrichment sparging experiments, 6 g
of NaHCO 3 was added to maintain pH at 6.8 to 6.9. Sludge sparging experiments
were performed at 35° C and always began exactly 1 hour after the daily feed-
ing of the digesters. Figure 50 shows the results with sludge, and Figure 51
shows the results with sludge diluted with an equal volume of sludge superna-
tant.
The relative small amount of hydrogen removed from the sludge may be due
to high levels of solids present. Bacteria adhering to the solids may accom-
plish interspecies hydrogen transfer in the confines of microenvironments,
preventing the removal of hydrogen by sparging.
Pure Culture Isolations
Serial ten-fold dilutions were made from each enrichment using liquid
media with a nitrogen and carbon dioxide gas phase. Solid media with hydrogen
and carbon dioxide gas phase and solid media with nitrogen and carbon dioxide
gas phase with 25 rrtl VOA (corresponding to the enrichment) were inoculated
from these dilutions and incubated 3 weeks at 30° C.
Slowgrowing colonies picked from the solid media, with a nitrogen and
carbon dioxide gas phase, produced no colonies on attempted subculturing in
identical media. Colonies picked from solid media with hydrogen and carbon
dioxide gas yielded 16 methanogenic isolates after subculturing. These were
isolated from original dilutions of l07 or greater. None of the isolates
were able to utilize propionate or butyrate when inoculated into media with
either nitrogen and carbon dioxide, or hydrogen and carbon dioxide in the gas
phase.
Methanogenic propionate and butyrate enrichments were shown to have the
ability to rapidly oxidize hydrogen. This allows the production of hydrogen
in enrichments without its accumulation, an important consideration since
thermodynamic calculations indicate that hydrogen accumulation could inhibit
hydrogen production from propionate or butyrate. This possibility was con-
firmed by demonstrating inhibition of propionate and butyrate dissimilation in
the presence of added hydrogen.
Under normal conditions the hydrogen produced in methanogenic enrich-
ments is rapidly oxidized by the large numbers of methanogenic bacteria
present. In these experiments hydrogen was removed before it could be utiliz-
ed, by rapidly flushing the enrichments with carbon dioxide. Effluent gas,
containing mostly C02, was greatly reduced in volume by absorbing the C02
with NaOH for ease of analysis. Using this technique, propionate and butyrate
degrading enrichments were shown to produce large amounts of hydrogen.
69
-------
- 0-
w
w •METHANE
U HYDROGEN
z
0
04-
0
0
0 .2-
U)
(9 U
I I
0 200 400 600 800
HOURS
Figure 50. Gas produced from digesting sludge when flushed with 100% Co 2 .
-------
•METHANE
3. S HYDROGEN
z
0
C) 2-
0
0 200 400 600 800
HOURS
Figure 51. Gas produced from digesting sludge diluted with sludge supernatent
when flushed with 100% C02.
-------
In sumary, the evidence that molecular hydrogen is an extracellular
intermediate in the methanogenic dissimilation of propionate and butyrate is:
1) Enrichments are able to produce large amounts of hydrogen from these sub-
strates. 2) Hydrogen inhibits propionate and butyrate dissimilation. 3) Hy-
drogen can be utilized by these enrichments in large amounts. 4) Enriching
cultures with propionate or butyrate at the same time enriches for large
numbers of hydrogen oxidizing methanogenic bacteria which cannot themselves
utilize these substrates.
Models for methanogenic propionate and butyrate degradation based on the
organisms known as Methanobacterium propionicum and Methanobacterium
suboxydans are inconsistant with the hydrogen production demonstrated by pro-
pionate and butyrate enrichments.
A model for propionate and butyrate dissimilation involving interspecies
hydrogen transfer is consistent with these results: Enrichments are able to
produce large amounts of hydrogen from these substrates. Hydrogen is rapidly
oxidized by methanogens present in the enrichments, so that the partial pres-
sure of hydrogen is maintained at a level low enough to allow exergonic hy-
drogen production from propionate and butyrate. Only when hydrogen is rapid-
ly removed from solution by sparging can significant quantities be detected.
Artifically increased hydrogen concentrations halt propionate and butyrate
dissimilation. Finally, in enriching cultures with propionate or butyrate,
one at the same time enriches for hydrogen oxidizing methanogens, which cannot
themselves utilize these substrates.
It is concluded that methane production from propionate and butyrate
involves a hydrogenogenic and a methanogenic microflora.
ACETATE AND PROPIONATE FEED RATE EXPERIMENTS
It is generally accepted that short chain volatile acids are major inter-
mediates in the formation of methane during sludge digestion. Work previously
presented in this report further confirms this fact, quantitating the contri-
bution of acetate, propionate, and butyrate to methane formation, and showing
the interplay of hydrogen in this process. The significance of these acids
and their balance in the process suggest that the pool size and turnover of
these acids may limit the reaction rate under steady state conditions. If
this were true an increase in the rate of formation of the acids would result
in a continued increase in the pool size. If this were not the case, an in-
crease in the rate of formation of the acids would result in an initial in-
crease in pool size followed by the establishment of a new stable pool size,
and it should also be possible to quantitatively determine the capacity of the
existing microflora to metabolize additional substrate. Experiments were con-
ducted to evaluate these questions, for acetate and propionate, using the
methods previously described.
The digesting sludge used in these experiments was obtained from a seven
liter laboratory digester being maintained on domestic waste with a retention
time of 20 days. The digesting sludge was obtained from the laboratory
72
-------
digester 2.5 hours after feeding. The rate of methane formation in control
flasks was 0.017 i.imoles/ml/min at 90 minutes and 0.017 mole/ml/min at 380
minutes.
Results for acetate are shown in Figure 52. New pool sizes were estab-
lished at pumping rates below 0.011 pmoles/ml/min. There results show that
the sludge microflora had a capacity to metabolize acetate at a rate approxi-
mately twice the rate at which acetate was in fact being metabolized under
steady state conditions 2.5 hours after feeding. The rate of methane forma-
tion from acetate, therefore, was limited by the rate of acetate formation.
The sludge had a substantial ability to metabolize additional acetate under
steady state conditons.
Results for propionate are shown in Figure 53. The capacity of the
micro-flora to metabolize additional propionate was less than its capacity to
metabolize additional acetate, as would be expected from their relative rates
of metabolism in the system. New pool sizes were established at pumping rates
below 0.0013 pmoles/ml/min. The results show that, as with acetate, the
sludge microflora had a capacity to metabolize propionate at a rate approxi-
mately 40% faster than propionate was being metabolized under steady state
conditons, assuming that 30% of the methane formed was formed through pro-
pionate. The rate of methane formation from propionate was limited by the
rate of propionate formation. The sludge had a substantial ability to metabo-
lize additional propionate under steady state conditions.
73
-------
6.8-
I I I I
40 80 120 160
FEEDING TIME
I T •
200 240 280 320
IN MINUTES
Changes in concentration, with time, of acetic acid in digesting sludge
fed acetic acid at feed rates from 0.002 iimoles/ml/min to
0.014 i.tmoles/ml/min.
FEED RATE — moles/m1/m n
• 0.014
£ 0.011
• 0.0075
o 0.005
A 0.002
00.0
—4
z
Q 6.0
I-
WE
0
0.!
0
I-
W
0
II 3.6-.
Figure 52.
D
2.
0
-------
0 100 200 300 400
FEEDING TIME IN MINUTES
Figure 53.
Changes in concentration, with time, of propionic acid in digesting sludge
fed propionic acid at feed rates from 0.0012 .tmoles/ml/min to
0.0033 pmoles/nil/min.
I.0c
z
0
0::
I-
z
LU
C.,
z
0
0
0
0
z
Q
0
0
0
E
s.
w
0
E
0,
500
-------
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78
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APPENDIX A
MATERIALS AND METHODS
ISOLATION TECHNIQUES
The equipment used included the following: A copper column consisting
of an 80 X 5 cm glass tube packed with fine copper turnings and containing a
400-watt iniiiersion heating element. The outlet from the column was connected
by rubber tubing to 10 cm (4 inch), 20 gauge syringe needles. The tubing
preceding the needles was packed with 5.1 cm (2 inches) of sterile cotton.
A syringe pipetting assembly with a 5 ml capacity and 2 ml capacity auto-
matic pipetting syringes was fitted with 3.8 cm (1.5 inch) number 19 needles
having Huber points.
Reinforced 150 X 16 n ii culture tubes were used. The culture tubes were
sealed with number 0 rubber stoppers.
A 19 X 25 cm press was used to hold the stoppers in place during auto-
ctaving. Media were prepared in a water bath adjusted to 450 C + 1°C.
The final percentage composition of the basic medium was NaC1, 0.1;
NH 4 C1, 0.05; CaC12, 0.005; NaHCO 3 , 0.5; MgC1 2 .6H 2 0, 0.005; KC1, 0.005;
(NH4)6Mo70 24 . 41- 120, 0.001; C0C1 2 .6H20, 0.001; resazurin, 0.001; L-cysteine
hydrochloride hydrate, 0.05; Na 2 S 9H 2 O, 0.05; agar, 1.5; ruiiien fluid, 30. The
30 percent rumen fluid was added as a source of nutrients. Substitution of
30 percent sludge supernatant, 0.3 percent yeast extract, 0.3 percent beef
extract, or 0.3 percent trypticase soy broth for the 30 percent rumen fluid
was not satisfactory.
Medium was prepared containing all ingredients except rumen fluid,
cysteine, Na2S9H20, and NaHCO 3 . The medium was boiled to drive off dissolved
oxygen. The atmosphere above the medium was maintained free of oxygen by
flushing with a gas mixture of 70 percent hydrogen and 30 percent carbon
dioxide. The gas mixture was freed of oxygen by passing it through a hot cop-
per column. Rumen fluid, cysteine, and NaHCO 3 were added after the medium had
been cooled to 45° C. The medium was distributed in 5 ml quantities into the
culture tubes using the automatic pipetting assembly. The gas in culture
tubes was freed of oxygen by flushing with the gas mixture prior to the intro-
duction of the medium. The culture tubes were maintained free of oxygen and
sealed with rubber stoppers. This was done by passing the gas mixture through
a syringe needle inserted into the culture tube. The gas mixture flowed
through the needle at a rate sufficient to keep the tube anaerobic. The
79
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rubber stopper was firmly seated as the syringe needle was removed. After
the stoppers were inserted the culture tubes were placed in test tube racks,
clamped in the press, and sterilized at 1200 C for 15 minutes. After cooling
at 45° C in a water bath, 0.05 ml of 5 percent Na2S 9H 0 was injected asep-
tically through the stoppers using an automatic pipetting syringe. Dilution
blanks were prepared in the same way and had the same composition as the med-
ium except that agar was omitted.
The substrates in the medium described above were either hydrogen or an
organic substrate added to give a final concentration of 0.3 percent. The
sodium sulfide was added immediately before the medium was inoculated. A
5 percent NaS•9H 2 0 solution was prepared under helium and injected into each
tube immediately prior to inoculation.
Samples to be inoculated were suspended in dilution blanks and then in-
jected into culture tubes containing growth medium and agar. Appropriate di-
lutions were rolled in an ice bath until the agar gelled. Inoculated tubes
were then incubated at the proper temperature.
DETERMINATION OF TURNOVER RATES
Procedures used were similar to those described by Smith and Mah ( 22 )
or as described below.
Fifty ml aliquots of sludge from a laboratory digester were placed in
Warburg respirometers. The respirometers were 100 ml nominal volume and
equipped with sampling ports closed with rubber serum caps. The sludge was
kept anoxic during transfer by flushing the digester and respirometers with
70% N2 : 30% C0 2 , and filling the transfer pipet with the same gas mixture.
The gas mixture was freed of traces of oxygen by passage through a column
packed with hot, freshly reduced copper turnings. The sludge in the respiro-
meters was equilibrated with the gas mixture for 15 minutes static, and 15
minutes at a shake rate of 105 strokes/mm. The respirometers were then
brought to atmospheric pressure and closed, and an elapsed time clock started.
Clock time was recorded for each addition to the respirometers of each sample
taken from them. One-half ml aliquots of radioisotope solutions were added
with a syringe through the serum caps. Radioisotope was added to two respiro-
meters, two respirometers were used for measurement of methane production, and
one respirometer was used for fatty acid pool measurements. The respirometer
was used for fatty acid pool measurements. The respirometers were maintained
at 35° C and a shake rate of 105/mm, in a Warburg water bath. Manometer ex-
cursions were recorded over the experimental period, and then gas samples were
taken for methane analysis. Samples were taken from the respirometers with
added labelled compounds at approximately 10 mm. intervals over a 60 mm.
period. Syringes in metal pipetting holders, fitted with 19 gauge needles,
were used to remove 1 ml samples from the respirometers, in the case of ace-
tate and propionate. The syringes were flushed with 70% N 2 : 30% C02 prior
to sampling. Samples were taken through the rubber serum caps and injected
into test tubes containing 0.5 ml of carrier acid in H 3 P0 4 , and pre-chilled
in an ice bath. Samples were kept in the ice bath until the end of the ex-
periment, and then they were placed in a freezer at -20° C until analyzed.
80
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In the case of butyrate, where the pool size was very low, the procedure
was changed. Butyrate samples were taken by removing the respirometer from
the Warburg bath, removing the serum cap, and pipetting out two 20 ml au-
quots. In this case a large number of respirometers was used. Anoxic con-
ditions were maintained with a flow of 70% N 2 : 30% 02 during the operation.
The 20 ml aliquots were run into duplicate 50 ml, teflon-lined, stainless
steel centrifuge tubes. The centrifuge tubes were in an ice bath, and con-
tained 2.5 ml 0.25N NaOH each. Immediately after the experiment, they were
centrifuged for 15 nun, at 37,000 x g and 00 C. The supernatent liquid was
then decanted and stored at -20° C overnight. The duplicate butyrate samples
were combined and 25 ml of each combined sample were pipetted into 500 ml
round-bottom flasks. Two glass boiling beads and 2.00 ml of carrier plus in-
ternal standard solution were added to each. The samples were then dried on
a rotary evaporator, under reduced pressure at 60-70° C. The residue was
dissolved in 2.00 ml 20% H 3 P0 4 prior to analysis. Resulting samples analyzed
for internal standard (n-valerate) concentration showed a maximum variation
of less than 4%; hence the measured radioactivity in the samples was not
corrected for concentration differences.
Organic acid pool samples were taken and chilled in the same manner as
samples for 14 C-acetate and 14 C-propionate samples (carrier and acid were
omitted from the test tubes), and the samples were frozen immediately after
chilling 2-5 mm. Response was used to calculate concentrations of methane
in samples, based on response when known amounts were injected.
Organic acid pools were measured by centrifuging sludge samples for 10
mm. at 27,000 x g and 0° C, acidifying 2.00 ml of the supernatent liquid with
0.20 ml of 20% H 3 P04, and injecting 2 il of the acidified solutions into the
chromatograph. A Hamilton 10 pl syringe, equippped with a Chaney constant-
volume adapter, was used to inject the solutions. Organic acids were separated
on a 2.4 m x 3.2 mm glass column, packed with 15% SP-l000 (Supelco Inc. Belle-
fonte, Pa.) and 2% H 3 P04 on 70/80 mesh anakrom A (AnalobsInc, Handen, Conn.)
Injection flash heater temperature was 145° C, column oven temperature was
130° C, and nitrogen flow rate was 25 mi/mm. Acetate and propionate were
measured at 3 x 10-10 amps/mv sensitivity and butyrate at 3 x 10-11 amps/mv.
All other conditions were the same as for methane analysis. Standards for
each acid were injected immediately after the samples.
When radioactive fractions were to be trapped, the SP-1000 column was re-
placed with a 1.5 m x 6 mm glass column packed with 15% FFAP (Varian-Aero-
graph, Walmit Creek, Ca.) and 2% I-1 3 PO 4 on 70/80 mesh anakrom A. Samples were
centrifuged at 27,000 x g and 0° C for 15 mm., and 8 i1 of the supernatant
were injected into the chromatograph. Column oven temperature was 1050 C and
nitrogen flow rate was 50 mi/mm. A 16 : 1 stream splitter was connected to
the exit end of the column, with the minor portion of flow going to the de-
tector, and the major portion to a heated outlet tube. The outlet tube was
2 mm diameter stainless steel, heated to 1600 C by means of a surrounding
jacket. The outlet end of the tube extended 10 mm beyond the end of the
jacket. Fractions were trapped by observing the chart trace and connecting a
trap to the end of the outlet tube when the desired organic acid was emerging.
Traps were 100 mm x 6 mm teflon tubes, packed to a depth of 45 nun with
81
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Porapak Q, and closed at the bottom with a plug of glass wool and a perforated
silicone rubber plug. Traps were connected to the outlet tube by inserting
the free end of the tube through the rubber plug. Trapped samples were mine-
diately washed off the Porapak with ethanol which had been dried over anhy-
drous Na 2 SO4. The trap was connected to a short piece of 2 nm diameter stain-
less steel tubing inserted through a rubber stopper. The stopper was also
penetrated with a 19 gauge hypodermic needle connected to a aspirator pump.
The stopper was inserted into a scintillation vial, the aspirator turned on,
and 5 ml ethanol passed through the trap into the vial. Ten ml of scintil-
lation fluid (0.4% PPO, 0.01% POPOP in toluene) were added to the ethanol
solution in the vial, and the mixture counted in an Packard Tri-Carb scintil-
lation counter. Counting efficiency of the system was 70%. Samples were
counted from 3 to 10 minutes, depending on activity observed.
RADIOISOTOPE, CARRIER AND STANDARD SOLUTIONS
2- 14 C-labelled acids were supplied at 99% radiopurity, as sodium salts.
Acetate was obtained from Calbiochem, Los Angeles, Ca., at a specific activity
of 22.5 pci/umole. Propionate was obtained from ICN, City of Industry, Ca.,
at a specific activity of 52 pci/umole. Butyrate was obtained from Volk
Radiochemical, Burbank, Ca., at a specific activity of 19 pci/itmole. Solu-
tions containing 170 i.ici/ml 2- 14 C-acetate, 57 iici/ml 2- C-propionate and 1.3
ci/ml 2-’ 4 C-butyrate were prepared, using boiled distilled water and sealed
under nitrogen gas with serum caps. Solutions were prepared on the same day
they were used.
Carrier for acetate and propionate samples was 15 pmole/ml propionic
acid in 1.8 N H3P04 solution. Carrier (and internal standard) solution for
butyrate was 4.3. pmole/ml n-butyric acid and 8.5 iimole/ml n-valeric acid,
in water.
Standards for organic acid analyses were prepared fresh, imediately
prior to analysis. Stock solutions of approximately 100 pmole/ml acetic, pro-
pionic, and butyric acids (titrated against standard NaOH) were diluted to
give standards containing 9.89 niole/ml acetic acid, 3.92 imole/ml propionic
acid and 0.432 and 0.216 umole/ml butyric acid. Error approximations, based
only on tolerances of glassware used in all dilutions and titrations, and on
probable error in weighing primary standard (potassium acid phthalate), indi-
cate final concentrations in standards should be accurate to better than + 2%.
ANALYSIS AND RADIOISOTOPE ASSAYS
Duplicate 1 ml gas samples from the respirometers were analyzed for
methane using a Packard model 7829 gas chromatograph. Samples were taken and
injected into the chromatograph with a Hamilton 1 ml gas-tight syringe.
Methane was separated on a 1.5 m x 6 nm glass column, packed with 50/80 mesh
Porapak Q (Waters Assoc., Framingham, Mass.) and detected with a flame ioniza-
tion detector. The column oven temperature was 105° C, carrier (N2) flow rate
was 20 ml/min., hydrogen flow rate was 20 ml/min. and air flow rate was
500 ml/min. A sensitivity of 1 x 10-7 amps for 1 my output was used. The
82
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output signal was displayed on a 28 cm (11 in.), lmv recorder, equipped with
a Disc integrator.
DIGESTOR EXPERIMENTS
Eight—liter glass bottles served as laboratory scale anaerobic digestors.
These digestors were totally sealed from the atmosphere. They had inlet and
outlet ports and were stirred with internal 5 inn teflon tubes which were
driven by low speed motors connected to low speed high torque motors. The
digesters were fed raw sludge which was stored in a frozen state. Gas pro-
duction was measured by water displacement.
HYDROGEN TRAPPING EXPERIMENTS
Anaerobic Digestor
An 8-1 glass bottle served as a laboratory scale anaerobic sludge di-
gestor. Anaerobically digested domestic wastewater sludge obtained from
the Gainesville, Florida, Wastewater Treatment Plant served as an inoculum.
The sludge was collected and handled in containers previously gassed out with
oxygen-free nitrogen gas. The laboratory scale digestor was maintained at
350 C in a water bath, and was mixed 15 mm. every hr. Each day 250 ml of
sludge were removed and 250 ml of raw sludge added; digestor liquid volume
was maintained at 7 1. Raw sludge was obtained from the Gainesville plant,
blended in a Waring blender for 5 mm., and frozen in polypropylene bottles
for storage. Gas production, measured by displacement of water, increased
slightly over the first week of incubation and then stabilized at 4.2 to
4.8 1 per day. Evolved gas was composed of 63 to 72% methane.
Gases
All gases were obtained from the Matheson Gas Company. Gas mixtures
for routine digestor, manometry, and culture work were Purified grade. Traces
of oxygen were removed by passing gases over freshly reduced hot copper fil-
ings. Carbon dioxide for sparging experiments (Anaerobe grade) was passed
through two gas scrubbers containing 500 ml of 0.018 M titanous chloride
solution which removed traces of oxygen ( 22 ), and saturated the gas with
water. Gases for chromatography were Purified grade. Standards used in the
quantitative analysis of gases were prepared from Ultra-high purity gases.
All gas mixtures were 30% carbon dioxide, with the balance either nitrogen
or hydrogen.
Determination of Volatile Organic Acids
Volatile organic acids were quantitatively determined using a Packard
800 Series gas chromatograph with a 1.8 ni by 2 m glass column packed with
10% SP1000 (Supelco, Bellefonte, Pa.) and 1% phosphoric acid coated on 70/80
mesh Anakrorn AW (Supelco). Temperatures were: inlet 150° C, column 122° C,
detector 132° C, and outlet 148° C. The carrier gas (100% nitrogen) flow
rate was 25 ml per mm. A flame ionization detector was used with hydrogen
83
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and air flow rates to the detector of 30 mi/mm and 300 mi/mm, respectively.
The signal from the detector was connected to an Autolab Minigrator which
was used for comparison to standards. Samples were prepared for analysis by
mixing 0.9 ml of sample with 0.1 ml of 30% phosphoric acid, followed by cen-
trifugation to remove cells. The injection volume was 0.4 p 1 .
Determination of Radioactivity in Volatile Organic Acids
VOA were separated chromatographically in a manner similar to that de-
scribed above. The injection volume was increased to 4 p1 and a stream
splitter (ratio Ca. 18:1) was inserted in the line of the column effluent, so
that one part of the effluent went to the detector and eighteen parts were
diverted to a port protruding through the column oven wall. Pasteur pipets
were lightly packed with glass wool, and through-hole septa (Supeico, Bele-
fonte, PA) were placed in the top. Just prior to use, the glass wool was
wetted with a solution of 1 N KOH in anhydrous ethanol. The septum was in-
serted into the side port of the chromatograph just prior to the time the de-
sired volatile organic acid was eluting from the column and removed when
elution was complete. The acid trapped on the alkaline glass wool was washed
into a scintillation vial with 10 ml of scintillation fluid (4 g 2,5-Dipheny-
loxazole, 0.1 g i,4_bis_ [ 2_(5_Phenyloxazolyi)]-benzefle, and 250 ml of dry
ethanol per liter of scintillation grade toluene). The amount of volatile
organic acid sent to the detector was quantitated to determine by difference
the amount diverted to the collector. Recovery of standard solutions of
labeled volatile organic acids was greater than 90%. Samples were counted in
a Beckman LS-l33 Liquid Scintillation System. Counting efficiency was greater
than 90%. For each sample, six replicate analyses were performed and aver-
aged.
Manometry
All manometric experiments were performed in 100 ml Warburg flasks having
a single side arm fitted with a septum. An 18 gauge needle was inserted into
the septum to provide an air exit while the flasks were being gased out with
oxygen-free gas. The septa were removed for introduction of 50 ml sample
enrichment cultures, which were transferred in volumetric pipets under anaer-
obic conditions. The septa were imediately replaced, and gasing continued.
After 5 mm. the enrichment cultures were shaken at a rate of 70 strokes per
mm. for 5 mm. to allow equilibration of the gas with the liquid. Needles
were then removed, and the flasks were equilibrated to atmospheric pressure
and sealed. Experiments were then started. To change atmospheres in the
vessels during an experiment, needles were re-inserted through the septa and
the flasks gased out with the second gas mixture.
For volatile organic acid analysis, liquid samples of 0.9 ml were re-
moved using syringes previously gased out with oxygen-free gas.
Collection of Sparge Gases
Effluent gas from sparging experiments consisted primarily of 100%
carbon dioxide. Large volumes of the gas were absorbed quickly by bubbling
84
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through excess NaOH solution in a one-liter Erlenmeyer flask sealed with a
three-hole stopper. The sparge gases entered the flask through one hole, via
a glass tube reaching to the bottom of the flask. As the gas bubbled up,
liquid was displaced through a second hole that had another glass tube reach-
ing to the bottom of the flask. This tube connected to a reservoir contain-
ing NaOH solution. The third hole was filled by a short piece of glass tubing
sealed at the top by a septum, through which alkali insoluble gases were re-
moved by a syringe at the end of an experiment. At very high sparge rates
the flask was placed on a reciprocal shaker to increase the rate of carbon
dioxide absorption. At the end of the collection period the flask was shaken
5 additional minutes to allow complete absorption of the carbon dioxide.
Quantitation of Radioactivity in Gases
A 250 ml Cary-Tolbert Ionization Chamber was used in conjunction with a
Cary 401 Vibrating Reed Electrometer for quantitation of radioactivity in
gases. The Rate-of-Charge method was used to determine the current through
the ionization chamber with an applied voltage of 90 V. A standard curve was
prepared using various amounts of Ba 14 CO with a specific activity of
1.16 x 103 dpm/mg (New England Nuclear, Boston). The Ba 14 C0 3 was converted
to gas using a carbon dioxide generator (Applied Physics Corporation,
No. 3120000).
Quantitation of Gases
Gases were quantitated using a Packard 800 Series Gas Chromatograph. A
1.8 m by 2 nm glass column was packed with Carboseive B (Supelco, Bellefonte,
Pa.). Temperatures were: inlet 100° C, column 70° C, detector 82° C, and
outlet 96° C. Carrier gas flow rate was 30 ml/min of nitrogen. Detection
was by thermal conductivity with a bridge current of 200 mA. The signal from
the detector was connected to an Autolab Minigrator which was used to quanti-
tate by comparison with prepared standards.
ACETATE AND PROPIONATE FEED RATE EXPERIMENTS
Stock 1.0 M solutions of sodium acetate and sodium propionate were pre-
pared. Feed solutions were made by dilution of the stock solutions. All so-
lutions were prepared with boiled distilled water which had been cooled in an
atmosphere of 70% Nitrogen-30% CO 2 . All solutions were flushed with the same
gas mixture if exposure to air had occurred, e.g., while filling syringes, etc.
The concentrations of feed solutions were measured on the gas chromatograph,
and syringe pumps were individually calibrated by pumping solutions into
aliquots of H 3 P0 4 for 3 hours and then measuring the resulting concentrations.
Disposable 2.5 ml syringes were filled with the feed solutions and
attached to a Harvard infusion pump (model 600-950). With the syringes and
pump rate setting used, the delivery rate was 0.0038 + 0.0001 mi/minute.
Incubation vessels were 125 ml respirometer vessels with sampling ports
closed with rubber serum caps. Feed solutions were delivered through 21 gauge
85
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needles inserted through the serum caps. Teflon capillary was attached to the
needles to deliver the solutions below the liquid level in the flasks. Fifty
ml aliquots of sludge from a 7-liter laboratory digestor (20 day detention
time), were transferred, into the incubation vessels under a stream of 02-free
N2-C0 2 .
The Harvard pump was started prior to t = 0 for the experimental vessels.
The teflon delivery tubes were carefully wiped immediately prior to fitting
the stoppers into the incubation vessels. Zero-time for each sample was noted
as the time when that flask was stoppered.
The flasks were incubated in a shaking water bath (new Brunswick model
RW-150) at 35° C and 100 strokes/minute. At various time intervals, 1 ml
samples were removed through the serum caps with 2 ml automatic pipetting
syringes equipped with 3.8 cm 19-gauge needles. The syringe was flushed
thoroughly with 70%-30% N 2 —C0 2 prior to sampling. Samples were killed by in-
jection into pre—chilled test tubes containing 0.5 ml 20% H3P04 and kept in
an ice bath. All samples were then frozen and kept in the freezer until ana-
lyzed, at which time they were thawed and filtered.
86
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APPENDIX B
FERMENTATION OF RUM SLOPS
In early 1970 EPA had an interest in determining the feasibility of fer-
menting Puerto Rico rum slop. A problem was encountered in establishing a
viable fermentation and advice was requested from this project, with the re-
suits which follow.
OBJECTIVE AND PROCEDURES
Rum slop samples obtained from Mr. Edmond Lomasney (U. S. EPA Region IV)
was examined to determine its fermentability characteristics. The slop was
assayed for chemical oxygen demand, ionic strength, ion composition, volatile
organic content, normality, pH, and buffering capacity. Short-term ferment-
ability was examined manometrically. Long-term fermentability was examined
by feeding an established digester.
RESULTS
S l p pos i t ion
Chemical Oxygen Demand (COD)--
The average COD for the samples in our lab was 88,800 mg/i (-i- 3,100).
This is a good agreement with the reported values determined in Puerto Rico.
We therefore assumed the sample was representative.
Ionic Strength
Conductivity measurements on the slop indicated an ionic strength
equivalent to 0.25 M NaCl. The slop is probably hypertonic for most non-
halophilic bacteria. The ionic strength of digesting sewage sludge in our
laboratory digesters is equivalent to 0.075 - 0.078 M NaCl. The high ionic
strength may be inhibitory to a rapid fermentation.
Individual Ions Present--
The slop was assayed for individual ions by atomic absorption spectro-
photometry, colorimetric assay, and the use of specific ion electrodes. The
results are listed in Table B-i. Chloride is quite high.
Volatile Compounds--
Gas chromatographic analyses of the slop demonstrated the following con-
centrations of volatile organic acids: acetic, 14 mole per ml; propionic,
iso—butyric, n-butyric, iso—valeric, and n-valeric, all less than 0.1 mole
87
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TABLE B-i. IONS AND THEIR CONCENTRATIONS IN RUM SLOP
Ion
mg/I
Percent
Fe
76.2
0.0076
Zn
3.5
0.00035
Cu
7.5
0.00075
K
8,300
0.83
AL
25
0.0025
Ca
1,900
0.19
Mg
1,400
0.14
P
130
0.013
Cl
27,000
2.7
TABLE
B-2.
pH
CHANGES
OF RUM SLOPS
DUE
TO ADDITION
OF SLUDGE
Manometer
Ml
Ml
Initial
Number
Sludge
Slop
pH
1
25
0
7.2
2
25
1
7.2
3
25
2
7.1
4
25
5
6.9
5
25
10
6.6
6
25
15
6.4
7
25
20
6.2
8
10
25
5.2
9
5
25 .
5.0
10
0
25
4.9
88
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per ml. The ethanol concentration was 0.54 i moles per ml. (0.02 percent).
These substances, at these concentrations, should have no adverse effect on
the fermentation.
Normality, pH and Buffering Capacity--
The pH of the slop sample was 4.9. Titration of a sample indicated a
normality of 0.05. This indicated that the slop could lower the pH of sludge
and inhibit the fermentation. Various combinations of sludge and slop were
mixed together to determine the pH change (see Table B-2).
Short-term Manometric Measurements
The short-term fermentability of the slop was determined by adding slop
to digesting sludge and measuring the resulting fermentation over a five day
period. Conditions and results are shown in Tables B-3 and B-4.
Warburg flasks were used as fermentation vessels. Anaerobic techniques
were employed in all phases of the experiment. Vessels and slop were gased
with 70 percent N2: 30 percent C02 prior to adding sludge.
After adding sludge to the manometer vessels, they were closed and shaken
in a 35 degree C water bath. The excursions resulting from gas production
were followed and at the intervals listed below gas samples were analyzed for
methane by gas chromatography.
Results Obtained the First Day of Incubation--
After 150 minutes of shaking, the gas produced was analyzed for methane.
In Table B-3, methane production for the first day represents the percent
methane produced in relation to that produced by the controls.
Second Day Results--
The manometers were opened and the gas produced for 18 hours was allowed
to escape through a 27 gauge needle. On the second day the manometers were
closed and shaken for 150 minutes. Methane in each vessel was measured; the
gas was released; and vessels were closed again and shaken for 230 minutes.
The gas produced was again assayed for methane. The methane production re-
ported in Table 8-3 is the difference between the levels found at 150 minutes
and the levels found after 230 minutes of additional shaking.
Fifth Day Results--
The mixtures of sludge and slop were then allowed to incubate without
shaking for an additional 65 hours. Gas produced during this interval was
allowed to escape through a 27 gauge needle. The manometers were then closed
and the gas was analyzed for methane. After 330 minutes of shaking, the gas
produced in each manometer vessel was again assayed for methane. The methane
levels reported in Table B-3 are differences between the levels found at the
time of closing the vessels and 330 minutes of shaking. After 5 days of incu-
bation, final pH, percent hydrogen, and final VOA pools were checked. These
properties are given in Table B-4.
89
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TABLE B-3. GAS PRODUCTION FROM DIGESTING SLUDGE DILUTED WITH RUM SLOPS
Manometer
Ml
Ml
Methane
percent
produced as
of the controls
Day
Day
Day
Number
Sludge
Slop
1
2
5
1 (Control
)
25
0
100
100
100
2
25
1
106
110
0
3
25
2
130
114
100
4
25
5
118
122
115
5
25
10
96
153
133
6
25
15
84
69
0
7
25
20
55
25
4
8
10
25
0
0
0
9
5
25
0
0
0
10
0
25
0
0
0
90
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TABLE B-4. PRODUCTS FORMED BY SLUDGE DILUTED WITH RUM SLOPS
‘ .O
* Less than 0.1 p mole per ml.
Manometer
Ml Ml
Final
Percent
VOA (p moles per
ml)
Number
Sludge Slop
pH
H 2
Acetic
Propionic
i-butyric
n-butyric
i-valeric
n-valeric
1
25 0
7.15
0.02
*
*
*
*
*
*
2
25 1
7.15
0.02
*
*
*
*
*
*
3
25 2
7.15
0.02
0.2
*
*
*
*
*
4
25 5
7.15
0.02
0.3
3.0
0.7
*
0.2
*
5
25 10
6.85
0.02
4.0
29.0
3.0
5.0
3.0
22.0
6
25 15
5.45
0.05
93.0
30.0
3.0
10.0
3.0
22.0
7
25 20
5.25
0.05
99.0
43.0
1.0
16.0
0.4
5.0
8
10 25
4.60
6.0
66.0
4.0
* *
2.0
*
0.5
9
5 25
4.70
4.0
45.0
0.8
* *
*
*
*
10
0 25
4.30
0
22.0
*
* *
*
*
*
11
25 0
7.15
0.02
*
*
*
*
*
*
* * Two large peaks coinciding with the iso-butyrate peak.
-------
Long-term Feed Experiments
The long-term experiments involved feeding slop to an anaerobic digester.
A 4 liter digester, containing 3.5 liters of digesting sewage sludge, was
used. The digester had been fed 175 ml of raw domestic sludge per day prior
to changing to slop feed. Gas production ranged between 5.8 to 6.2 ml/min for
the first two hours after feeding during a two week period prior to the change
over. During this same period the total gas produced during the first five
hours after feeding ranged from 1.4 - 1.6 liters with a composition of 69 -
74 percent methane. Total gas produced for twenty-four hours after feeding
was 5.8 - 6.2 liters. The following parameters were measured in this part of
the study: a) rate of gas evolution for the first five hours after feeding;
b) percent methane and the average p moles methane per minute per ml liquid
for the first five hours; c) total gas produced in twenty-four hours; d) pH
twenty-four hours after feeding; e) ionic strength twenty-four hours after
feeding; f) VOA concentrations five and/or twenty-four hours after feeding.
Because of the high ionic strength i’t was decided to dilute the slop
with water. The dilution rates were:
Days ml Slop ml Water
1-5 100 100
6—11 150 50
12—16 170 60
On the seventeenth day the feed was changed to 100 ml slop and 100 ml
raw sludge in an attempt to increase gas production and the percent methane.
Figures B-i and B-2 show the increase in VOA each day 24 hours after
feeding. Up to the eighth day the concentration of all VOA was less than
0.1 p mole per ml. On the tenth day acetate, propionate and n-butyrate be-
gan to increase and continued to increase throughout the period of feeding.
Iso-valerate is not included in Figure B-2 because its concentration never
exceeded 1.0 p mole per ml.
Figure B-3 shows the daily increase in ionic strength (measured by con-
ductivity and equivalent to the molarity of NaCl). The increase in VOA pools
beginning on the tenth day is correlated to an ionic strength of 0.140 M.
Figure 8—4 shows the total gas produced 5 and 24 hours after feeding.
Gas production for the first 5 hours was greater than, or equivalent to, the
gas produced from raw sludge feed. However, total gas produced in 24 hours
was always less than on raw sludge. Gas production for 24 hours did increase
between 6 - 10 days. This is correlated to an increase in the amount of slop
fed (see feed schedule). The general trend for total gas in 24 hours, how-
ever, is a constant decrease. Since gas production was good for the first 5
hours, the slop probably contains readily degradable substrates.
92
-------
TIME IN DAYS
Acetic acid and propionic acid concentrations each day,
24 hours after feeding.
Io
z
0
I—
L i i—
Ou,
2
a:
0
0
0 4 8 12 16 20
24
Figure B—i.
-------
10.0-
z
Q
F-
z
ou,
C-),
0
C-)
z
4
0
Figure B-2.
7.5-
5.0-
2.5-
0
£ n-BUTYR1C ACiD
• n-VALERIC ACID
• iso-BUTYRIC ACID
I I I I • I I
0 4 8
TIME IN DAYS
24
n-Butyric acid, iso-butyric acid and n-valeric acid concentrations each day
24 hours after feeding.
‘ 0
I I I I I
12 16 20
-------
=
I -
(9
z
U i
0
z
0
0
01
0 4 8 12 16 20 24
DAYS
Figure B-3. Ionic strength each day, 24 hours after feeding.
-------
w
0
D
S
0
Li
0
C I)
Figure B-4. Liters of gas produced each day 5 and 24 hours after feeding.
0 4 8 12 16 20 24
DAYS
-------
Figure B-5 illustrates the percent methane and the moles of methane
per mm per ml of liquid 5 hours after feeding. The percent methane in the
gas decreases to 50 percent and remains relatively stable for 8 days when it
begins to decrease. Except for an initial increase, the p moles of methane
per mm per ml constantly decreases. -
Assuming the slop consists mostly of readily degradable carbohydrates
(C6H1206)x, a gas phase of about 50 percent methane would be expected from
the available hydrogen. This could explain the decrease in the percent
methane for six days and a leveling off for the next eight days.
The pH of the digested slop did not decrease markedly as the VOA pools
increased. The starting pH was 7.1. It decreased to 6.9 by the fifteenth
day and went down to 6.4 by the twenty-third day.
The long term feed experiments indicated that the slop will not maintain
the activity of an anaerobic digester having a microflora developed on do-
mestic waste.
97
-------
(I,
a:
0
I
U,
LU
z
4
I
I—
LU
I.-
z
LU
C )
0
I
E
E
4
=
I-
LU
• MICROMOLES
•PERCENT
0.02
I -J
0 4 8 12 16 20 24
DAYS
‘1’
Figure B-5.
Daily methane produced 5 hours after feeding.
-------
Comments and Recommendations
The results of the study with a single sample of the rum slop suggest
that some difficulty will be encountered in establishing and maintaining a
fermentation of this material using a seed of sludge from a domestic digester.
The data suggest that the developed ionic strength in the fermentation is a
major problem, although other factors may be involved. I project that the
ionic strength of undiluted fermenting slop would develop to a value exceed-
ing 0.5 M NaC1. This is based on the fact that the slop fed was diluted, and
measurements were minimal values.
Feeding diluted slop the ionic strength developed to that equivalent to
0.20 M NaC1 in sixteen days, in contrast to the calculated value of 0.14 M
NaC1.
It is recommended that a digester be established with a domestic sludge
seed, or a marine sediment seed.
Domestic Seed--Start with 3.5 L of domestic sludge. Feed daily 125 ml
H 0, 75 ml slop and 25 ml of Metracal. Maintain for 3 weeks on this feed and
t en slowly increase the slop (5 mi/day), decrease the H 2 0 (5 ml/day), and
decrease the Metracal (2 ml/day) to the desired retention time. It may be
possible to maintain the fermentation on slop with no additions. This would
have to be arrived at empirically.
Marine Sediment Seed--Obtain 3.5 liters of black bay mud. Obtain the
mud from under decaying sea weed, if possible. Add 300 ml of slop and allow
to stand until gas evolution occurs. Then start adding slop at an increasing
rate. Start at 20 mi/day and increase to 200 ml/day over a period of approx-
imately 20 days. The maximum feed rates could be arrived at empirically.
If conventional digester systems do not work, I would recommend that
the feasibility of an anaerobic filter be tested.
99
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TECHNICAL REPORT DATA
(Please read Inwuctions on the reverse before completing)
1. REPORT NO. 2.
EPA-600/2-80-093
3. RECIPIENT’S ACCESSIOFNO.
4. TITLE AND SUBTITLE
STUDIES OF METHANOGENIC BACTERIA IN SLUDGE
5. REPORT DATE
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
P. H. Smith
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
University of Florida
Gainesville, Florida 32601
10. PROGRAM ELEMENT NO.
35 BIC
11.CONTRACT/GRANTNO.
17070 -_DJV
12. SPONSORING AGENCY NAME AND ADDRESS
Municipal Environmental Research Laboratory--Cm., OH
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, Ohio 45268
13. TYPE OF REPORT AND PERIOD COVERED
Final Report, 9/66 - 10/79
14.SPONSORING AGENCY CODE
EPA/600/14
15. SUPPLEMENTARY NOTES
Project Officer: Albert 0. Venosa, (513) 684-7668
16. ABSTRACT
Methanogenic bacteria were isolated from mesophilic anaerobic digesters.
The isolates were able to utilize H and CO 2 acetate, formate and methanol,
but were not able to metabolize pro ionate af d butyrate. It was shown the pro-
pionate and butyrate are not substrates for methanogenic bacteria but are con-
verted to hydrogen, carbon dioxide and acetate by a hydrogenogenic microflora.
The reactions leading to methane were quantitatively analyzed. It was shown
that acetate, propionate and butyrate metabolism were inhibited by hydrogen.
The formation of acetate and propionate were shown to be rate limiting in the
digestion process, and that sludge digestion was not inhibited by hydrogen
under conditions of excess substrate.
This report was submitted in fulfillment of Grant No. 17070-DJV by the
University of Florida under the sponsorship of the U. S. Environmental Pro-
tection Agency. This report covers the period September 1, 1966 to
October 15, 1979 and work was completed as of October 15, 1979.
17. KEY WOROS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.IDENTIFIERS/OPEN ENDED TERMS_-
C. COSATI Field/Group
Anaerobic processes, Anaerobic bacteria,
Dissolved gases, Methane, Hydrogen,
Acetic acid, Butyric acid, Propionic
Acid, Fatty Acids, Sludge digestion
Volatile organic acids,
Methanogenesis,
Anaerobic digestion,
Hydrogenogenic bacteria
138
6M
18. DISTRIBUTION STATEMENT
Release to Public
19. SECURITY CLASS (This Report)
Unclassified
21. NO. OF PAGES
112
20. SECURITY CLASS (This page)
Unclassified
22. PRICE
EPA Form 2220-1 19-73)
100
* U.S. GOVERNMENT PRINTING OFFICE: 1980--657/0130
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