UNITED STATES ENVIRONMENTAL PROTECTION AGENCY
"SUBJECT:" xne Meeiianicm'Whe.reby Methane Gas is Generated in an DATR: August'16. 1974
Anaerobic Digestion of Organic Material
FROM: Edniond P. Lomasney Ł?V,
Region IV, Research & Development Program
TO: Regional Personnel
SUMMARY
We take it for granted that most everything worth-knowing about
anaerobic digestion of organics has appeared in the literature and
that the process is well understood. This is not so—in fact, a good
deal of the published literature regarding tho behavior of this process
'may be misleading, especially as concerns the mechanism for generation
of methane.
In January of this year, the attached paper pertaining to this
subject was presented at an agricultural seminar in Atlanta. At that
time there unfortunately were no printed copies available, though
many requests were mads for copies. Recently, we have obtained
copies and as a consequence I am forwarding the attached for your
edification.
ACTION
None—the paper is only intended for educational purposes; relating
to a biological process for the generation of energy.
BACKGROUND . • ' .
Our program has had research projects which utilized anaerobic
digestion procedure for organics and have yielded strange or unusual
results. Some of the reasons for this behavior are indirectly related
to the subject matter of the attachment. Though in many respects the
generation of methane by this process was considered s known fact,
there \»ere times when a specific i-naerobic process performed in a
relatively inefficient manner for no apparent reason; methane production
ceased with, the system going sour. With the information generated by
Dr. Paul H. Smith at the University of Florida, Gainesville, \ve wers
able to have a more thorough understanding of the anaerobic system for
generation of methane, and consequently avoid the pitfalls experienced
in the past.
EPA Form 1320.-6 (Rev. 6-72)
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Distribution List;
Dr, Helen McCammon, Region I, Boston, MA
.Dr. Robert W. Mason, Region II, New York, NY
Albert Montague, Region III, Philadelphia, PA
Clifford Risley-, Jr., Region V, Chicago, IL
Mildred Smith, Region VI, Dallas, TX
Aleck Alexander, Region VII, Kansas City, MO
Russell W. Fitch, Region VIII, Denver, CO
>Vern Tenney, Region IX. San Francisco, CA
Robert Courson, Region X, Seattle, V/A
William T. Willis, Alabama Department of Health, Montgomery, AL
Peter P. Baljet, Florida Department of Pollution-Control.
Tallahassee, FL ' .
R. S. Howard, Jr., Department of Natural Resources, Atlanta, GA
Herman D. Regan, Department of Natural Resources & Environmental
Protection, Frankfort, KY
Glen Wood, Jr. , Mississippi Air & V/ater Pollution Control Commission,
Jackson, MS
Earl C. Hubbard, North Carolina Department of Natural & Economic
Resources, Raleigh, NC
Dr. E. Kenneth Aycock, South Carolina Department of Health &
Environmental Control, Columbia, SC
Dr. Eugene W. Fowinkle, Department of Public Health, Nashville, TN
Dr. John F. Andrews, Clemson University. .Clemson. SC
Dr. Robert Inpols, Geoi-gia Institute of Technology, Atlanta, GA
Dr. Paul H. Smith, University of Florida, Gainesville, FL
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11581
SOUTHEASTERN POULTRY AND EGG ASSOCIATION SEMINAR
Atlanta, Georgia
January 30, 1974
TECHNOLOGY OF METHANE CAS PRODUCTION RELATED TO
ANAEROBIC DICESTION OF ORGANIC MATERIAL
Edmond P. Lomasney
Research and Development Program Representative
Environmental Protection Agency
Southeast Region IV
Presented through the courtesy of:
Dr. Paul H. Smith, Project Director, University of Florida,
Gainesville, Florida
Cecil Chambers, Project Officer, National Environmental Research
Center, Environmental Protection Agency, Cincinnati, Ohio
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For Figures, Charts, Graphs, Tables and Pictures
see Appendix
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Anaerobic Digestion of Organic Material
•
This presentation is a summary of work conducted under an EPA
(
Grant on the breakdown of organic material under anaerobic conditions.
The work deals with the conversion of complex organic matter such as
the material found in domestic and agricultural wastes to methane
and carbon dioxide. The conversion is attractive for waste disposal
because the products are innocuous, the conversion rates rapid, and
the process is economical. The process requires little energy input
and produces a product, methane, which may be utilizable as a secondary
energy source'. The anaerobic digestion processes, however, are made
unattractive by the current limited ability for control and regulation:
It is probable that our limited abilities to control and regulate the
process is based on our limited knowledge of the critical biological
steps involved. I shall deal largely with this subject.
The first figure, one (1) , illustrates the generally accepted
concept of the steps involved in the conversion of insoluble organic
material to methane and carbon dioxide. In this scheme there is a
conversion of insoluble organic material to soluble organic material,
catalyzed by a wide variety of extra-cellular enzymes. The existence
of this step has been convincingly demonstrated by a large amount of
experimental evidence. Organisms and enzymes capable of carrying out
this step have been isolated and studied over a long period of time.
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Similarly step two has been definitely established. In this step
soluble organics are converted to volatile acids, plus hydrogen and
carbon dioxide. Step three however has never been experimentally
verified. Volatile acids, primarily acetate, propionate, and butyrate,
have been isolated, but repeated efforts in many laboratories to
culture organisms capable of metabolizing these acids have all failed.
The reason these efforts have failed is that the supposition of the
scheme is not correct. Contrary to the general view, the digestion
of complex organic substances consists of four stages, as shown in
the second figure, two (2). Digestion consists of: hydrolysis of
complex organic molecules, acid production, hydrogenogenesis from
acids, and methane formation. The second scheme differs from the
first scheme in that some fatty acids are converted to hydrogen and
carbon dioxide prior to the formation of methane, by a microflora that
does not produce methane. What evidence, you may ask, exists in
support of the second scheme. I shall now discuss the shaded part of
the four stage scheme since this is the portion which differs from
the three stage scheme. If the new scheme is correct, it should be
possible to quantitatively demonstrate volatile acids including
acetate, as intermediates in the process using isotope dilution
techniques. With this technique a rate constant is calculated from
a linear change in specific activity of added radioactive label.
This constant multip"ica by the fatty acid pool size gives the rate
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-3-
of formation of the acid which is then used to calculate the percentage
contribution of the specific acid to total methanogenesis. Such rate
constants were experimentally determined as shown in the third
figure, three (3). This graph shows a linear change in a plot of
the log of counts pur minutes in acetate against time in a fermentation
producing methane at a rate of 0.033 micromoles per milliliter per
minute. From the da«.a on this graph a rate constant of 0.0052 per
minute Is obtained. The acetic acid pool size in the samples was
4.7 micromoies/mllli iter. The product of the rate constant times
the pool size gives a rate of formation of acetic ccid of 0.024
micromoles per milliliter per minute. Assuming that 1 mole of methane
is formed from 1 mole of acetic acid, the acetic acid would account
for approximately 73% of the methane produced during the fermentation.
In the fourth figux\,, four (4) , similar experiments with propionic
acid, n-butyric acid and isobutyric acid show that 93% * 5% of the
methane formed during digestion of domestic waste, had these volatile
acids as intermediates. The results show a mintdial contribution of
the fatty acids to methane of approximately 90% leaving 10% for
hydrOfodn and other oxidizable substrates. In the fifth figure, five (5),
the experiments also show that acetate was formed 40% from butyrate
and 23% from propionate.
T,.-se results are in agreement with both schemes. Sludge would
vapicuy metabolise toe fatvy acids, and hydrogen and carbon dioxide
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were also rapidly converted to methane. Efforts to isolate fatty
acid and hydrogen utilizing methanogenic bacteria resulted in the
isolation of n Itu^o Dumber of methanogenic bacteria from the sludge.
la the sixth figure, six (6), the picture shows some bacterial species
isolated from hign dilutions of sludge. All of these isolates were
present in digestiiij sludge in numbers exceeding one million per
•llliliter. The large sarcina converts acetate to methane and
carbon dioxide. Vi.c others all form methane from hydrogen and carbon
dioxide but will now produce methane from fatty acids. Figure seven (7)
shows bacteria representing other isolates from sludge. They all form
methane from hydrogen and carbon dioxide but do not metabolize fatty
acids. Figure eight (8) shows a spirillum, isolated from a high
dilution of a propionate enrichment, which is methanogenic and
metabolizes hydrogen and carbon dioxide but not fatty acids.
Figure nine (9) represents a colony of the spirillum and is Included
to show its beauty.
The large mui.~ar of hydrogen oxiax^ing raethanogenic bacteria in
digesting sludge suggested that the capacity of digesting sludge for
hydrogen luetaboli^r.t should bo great. Cupacity of digesting sludge
for hydrogen metabolism was detc,k-ttined by incubating digesting sludge
under a gas phase ox' 70% hycu-ogen ^»d 30% carbon dioxide. Hydrogen
uptake and methane z'or~.ation -ere ^jCo»-ciined quantitatively. Methane
formation was uli,. quantitatively determined for digesting sludge
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incubated under & gas phase of 70% nitrogen and 30% carbon dioxide.
The results are shown In figure ten (10). Taking average value*,
447 mlcromoles of hydrogen were utilized in the hydrogen flasks and
210 micromoles of methane was formed. Assuming 1 micromole of
methane from 4 raicroraoles of hydrogen, 112 raicromoles of methane
would have been produced from added hydrogen and carbon dioxide,
leaving 98 micromoles of methane formed from the sludge. However,
134 raicromoles of methane were produced from sludge in the nitrogen
flasks. The difference between the 134 micromoles of methane
produced from sludge in the absence of added hydrogen and the 98
micromoles calculated from sludge in the presence of hydrogen indicates
that either hydrogen inhibited methane production from other sludge
precursors, 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 ?s?om sludge which had been
equilibrated for two hours with a gas phase of 70% nitrogen and 30%
carbon dioxide as shown on the next figure, eleven (11). The rates
were calculated from the methane evolved during the seventy-five
minutes iomediately following the equilibration period. The lowered
rate of methane evolution following exposure to hydrogen gas shows
that an inhibition of raethaaoganesis had occurred. Calculations from
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-6-
average values oi c.;.^.-> utilization and production given in next to
last table show „.. inhibition of 30% during exposure to a 70%
hydrogen atmospnezv.
The data suggests that hydrogen inhibits the methane formation
from some substrates other than hydrogen. In figure twelve (12),
the 30% inhibition corresponds to the total methane expected from
propionate. This graph shows that turnover of propionic acid
metabolism was stvoagly inhibited by hydrogen since in the presence
of molecular hyarcgen labeled propionic acid was not diluted,
suggesting that a^. important ecc jgical role of the hydrogen oxidising
methanogenic bacteria in dissimulation processes is the maintenance
of a hydrogen concentration low enough to prevent the inhibition of
propionic acid metabolism, and the concurrent cessation of the
fermentation.
Efforts were made to isolate methanogenic bacteria from propionic
acid enrichments. In every case the enrichments were found to contain
large numbers of i. .irogen oxidizing raethanogenic bacteria. These
organism^, could dot metabolize propionate, sugge^ing that propionate
metabolism result-*- in the formation of i^ee molecular hydrogen which
served as tl,.; • suo.,..rate for the growth of the hydrogen oxidizing
methanogens which v._re isolated.
Propionate Jxci . ou ri c.v..ife.'. »s wevc u..e;\ ustabi^shed under an
atmosphere «f lix/t carbon dioxiae. The enrichments were ihen fed
propionate and vi,. ously ga&ed with 100% carbon dioxide.
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A carbon dioxide absorbing train was placed at the outlet of the
fermentation vessel. The carbon dioxide was absorbed in potassium
hydroxide and the remaining gas analyzed. The concept here is that
under the experimental conditions created where hydrogen was formed
from propionate, most of it would be removed from the fermentation
liquid before it could be converted to methane and would then be
present in the collected gas. This would be consistent with the new
scheme. If propionate were converted directly to methane (as proposed
in the old scheme) then there would be no hydrogen in the collected gas.
The results are shown in figure thirteen (13). A large amount of
hydrogen was obtained. This definitely demonstrates a formation of
molecular hydrogen from propionate. Similar experiments are in progress
with butyrate. Butyrate enrichments yield hydrogen utilizing
methanogenic bacteria which lack the capacity to metabolize butyrate.
The data demonstrates a four step process in methane formation
from organic natter.
Every bit of this constitutes basic considerations in terms of
the methane production via biological processes. Much of the past
information relating to the methanogenic bacterial process has been
misleading. The correct interpretation of this basic phenomena must
be understood before we can tap this source of gaseous energy.
The information and data disclosed in this presentation is the result
of long-term work by Dr. Paul H. Smith at the University of Florida,
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-.5
e, under funding provisions by the Environmental Protuc ic
Agency, Research and Development Program. At present, Dr. Smith
»v-.ntinues to investigate this subjtct, and further disclosures on
;1'(.3 i^chaniso oi: fe-ciogici?.! ssathane production will u -.doubter^; '
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Appendix
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FIGURE 1
INSOLUBLE
ORGANICS
SOLUBLE
ORGANICS
VOUTILE
CO.
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FIGURE 2
INSOLUBLE
ORGANICS
I
SOLUBLE
ORGANICS
VOLATIte
OTtfSR
ACSTATC
^ C0a
AND
OH
^
(*co8
!
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ACETIC ACID
TURNOVER NUMBER DETERMINATION
395
UJ
H-
Z
5
CC
UJ O
O UJ
O O
,,_< 385
e>
o
380
FLA5K-2
10 20 30 40
TIME IN MINUTES
50
FIGURE 3
PRECURSORS OF METHANE IN DOMESTIC SLUDGE
of Total Methane
Acetic Acid
Propionic Acid
n-Butyric Acid
Isobutyric Acid
Formic Acid
73
13
TOTAL
FIGURE
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PRECURSORS OF ACETIC ACID IN DOMESTIC SLUDGE
Precursor
Butyric acid
Propionic acid
Others
% of total acetic acid
40
23
37
I i
FIGURE 5
FIGURE 6
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\
FIGURE 7
I a.
FIGURE 8
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FIGURE 9
HYDROGEN UTILIZATION BY DOMESTIC SLUDGE
Gas Phase
70%"H2, 30% C02
,.
7,.:/0 N2, 30% C02
ii
CH4 Formed
in p.mole
207
214
132
136
Initial H2
ia ;i mole
1650
1695
0
f
i
Final H2
in u^nole
1220
1230
3
3
FIGURE 10
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EFFECT OF HYDROGEN GAS ON SLUDGE METHANOGENESIS
Treatment Prior
to Inoculation
Exposed to H2 gas*
u it 11
Exposed to N2 gas+
u u M
i.'ll_j l-\>t nu-d
in :i mole
62
66
75
78
in |i mole/ml min"l
0.017
0.018
0.020
0.021
* Equilibrated with 70% H2 ahd 30% C02 for two hours.
+ Equilibrated with 70% N_ and 30% CO2 for two hours.
FIGUflE H
w u
d H
CO O
2
8
,4
5 10 15 i'; 20
TIMK IN MINUTES
x HT in 9'TS phase
o N2 in gas phase
FIGURE J2
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TIME IN
MINUTES
1 -10
10- 20
20-30
30-40
40 - 50
50 -60
PERCENT
HYDROGEN
0.9
40
43
43
43
43
PERCENT
METHANE
99
60
57
5 7
5 7
57
FIGUE 15
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