echanais
Fermne n
T h i s paper discusses the chemical mechanisms b3 which
methane may be produced by bacteria in nature. The
early history and bacteriology are briefly reviewed. Jleth-
ane formation has been found to occur both with and
without the reduction of carbon dioxide. Hydrocarbons,
ethers, and lignin have not been found to be fermentable.
With these exceptions the fermentation appears to pro-
ceed smoothly and continuously regardless of the chemical
composition of the substrate. Complete conversion of the
ETHANE formation in nature has interested bacteriolo-
gists and chemists for more than sixty years. The studies
in this field have progressed to a point where sufficient methane for
fuel for power plants of several thousand horsepower is produced
by anaerobic fermentation.
The literature in t,his field was summarized some years ago b>-
McBeth and Scales ( 1 3 ) and by Stephenson (59). Briefly, it xms
known that when moist organic matter \\-as allowed to dccom-
pose under restricted oxygen conditions it yielded hydrogen,
carbon dioxide, methane, and a variety of organic acids in greater
or less amounts. Little or no quantitative data o n the yield of
the various products were available, and little had been done with
pure compounds although Sohngen (23) had shown that lovcer
fatty acids with an even number of carbon atoms could he de-
composed by mixed cultures, giving methane and carbon dioxide.
Whether hydrogen and fatty acids were necessary intermediates
in the process of methane formation was not known. The in-
formation on the susceptibility of various natural products to
methane fermentation was conflicting. Cellulose vas known to
be fermented to some extent (22, 65) but so-called lignocellulose
was said to be resistant (16). One author held that grease nould
decompose anaerobically (24j, while another held that it \?-odd
not ferment to any substantial extent (33). The addition of
lime to favor methane production was preferred hy one group of
workers and opposed by another.
Of the earlier systematic studies of this fermentation, those
of Sohngen were the most extensive (29). More recent studies are
those of Fowler and Joshi (15),Sen, Pal, and Ghosh ( U ) ,Fischer,
Lieske, and Winzer (141, and Buswell et nl. (8).
Engineering design and loading factors have been discussed
previously ( 7 ) .
Characteristics of MothaKe Fernmenitation
Mixed Culture. The anaerobic fermcntations as carried out for
the production of methane differ in many respects from other
types of fermentations. The most important difference is perhaps
the fact that it is not necessary t o use a pure culture of organisms
in order to obtain uniform results, nor is it necessary to maintain
purified cultures for inoculation or reinoculation. The bacteria
which are capable of producing methane are found almost uni-
versally in nature, although in preponderating numbers in black
mud and decaying vegetable matter. Under proper conditions
these bacteria can be cultivated to a high degree of activity within
a few days. The culture can then be maintained a t this high
degree of activity indefinitely, providing a few simple rules
concerning chemical and physical environment are followed.
In fact, microscopic and subculture studies of this ferment'at>ion
reveal such a mixed and variable flora that it appears a t present,
550 INDUSTRIAL AND ENGINEERING CHEMISTRY
A. M. BUSWELL and H. F. MUELLEPI
INZZRQZSS t a t e W a f e rSurvog, Urbana, IRL
substrate to the two simple products, methane and carbon
dioxide, is a unique characteristic of the process. The re-
action is an oxidation-reduction one involving water, rep-
resented by the empirical equation
Cn&Ob + ( n - a/4 - b/2)H?O +
(n/2 - a/8 f b / 4 ) C O z + ( n / 2 + u / 8 - b/4)CHI
With a little care, it is possible to get 95 to 100% yields cal-
culated from this equation.
to be environment rather than flora which determines the result
obtained.
Continuous Process. This situation makes possible the coli-
tinuous operation of the fermentation, a procedure which i p at,
least very unusual in fermentology. It is possible t o carry on
this process in apparatus arranged to allow the subst,rate to enter
continuously at one point and the exhausted or inert residue t,o
be discharged continuously at, another, while the products,
methane and carbon dioxide, are given off at a steady rate.
There is apparently n o limit to the size of apparatus which can
be used. Large tanks yielding several hundred thousand cubic
feet of gas a day operate as snioothly as laboratory-size flasks.
Independent of Substrate. A third characterigtic of thw(1
fermentations is t'hat practically any sort or kind of organic mat-
ter may be used as a substrate. Nearly 100 different pur('
substances ( 1 , 3 4 , S b ) and some 30 or 40 natural plant and animal
products ( 4 , 5 , Q), such as cornstalks, milk whey, etc., have been
used successfully as fermentation material. There is apparently
no decomposition of hydrocarbons, ethers are not fermented, and
lignin, when isolated, is attacked not a t all or with difficulty
( 6 , 18, 21).
Quantitative Yields. The nearly quantitative yields of the two
simple products carbon dioxide and methane are somewhat
unique. It is true that in the various commercial fermentat,ions
of grains the starch is practically quantitatively recovered in the
products, but, the fats, proteins, and fiber are not attacked a t all.
The methane fermentation converts the entire grain, with the
possible exception of a small amount of fiber, to carbon dioxide
and methane within 24 to 7 2 hours. The reaction is of t,he oxida-
tion-reduction type involving water, represented by the empirical
equation
CnH,Ob + ( n - a / 4 - b/2)H20 ---f
( n / 2 - a/8 + b/4)C02 + ( n / 2 + a/8 - h/4)CH4
With a little cme, it is possible to get 95 to 100% yields calculated
from this e q u t i o n .
The uniformity of the end products, carbon dioxide and meth-
ane, was a t first puzzling. A study of the energy (34) of the re-
action of various compounds with water showed the maximum
free energy when the final products were carbon dioxide and
methane. In experience in this laboratory, neither carbon mon-
oxide nor higher hydrocarbons havc bcen observed among the
products. A few earlier reports in the literature stating that
ethane was formed have not been verified and the amounts re-
ported are within the limits of analytical error in the experiments
referred to.
In more recent investigations Grosse and Libby (16) found the
gas from a sludge digestion tank to contain methane free from
Vol. 44, No. 3

higher hydrocarbons within the limits detectable by a mass
spectrograph. Table I, furnished by Grosse but not included
in the abstract referred to, shows a comparison of the composi-
tion of petroleum methane and sewage methane.
Table I. Composition of Baltimore Sewage Gas after
Purification and Comparison with Petroleum Methane
Petroleum Methane, Sewage Methane,
Constituent VOl. % Vol. %
Methane 98 0 99 2
Ethane 0 6 <o 002
Propylene 0 1 <o 002
Nitrogen 0 7 0 2
Carbon dioxide 0 4 0 1
Air 0 2 0 5
Wide Temperature Range. A fifth characteristic is the
absence of any narrow optimum temperature range. The rate
of fermentation increases with an increase, in temperature from
0" C., or perhaps below, t o 55" C. or a little higher. Maxima
have been reported a t 26", 37", and about 50" to 55" C. (IS).
These maxima are not very pronounced and have not been noticed
by some observers.
Hydrogen Formation. Hydrogen is not formed in detectable
amounts during the methane fermentation of the lower fatty
acids, although it may well be an intermediate. When carbohy-
drates are used as substrates, the hydrogen may be as high as
30% by volume (M), although with proper control the hydrogen
can be held to about 1%.
There appear t o be only two types of fermentations yielding
hydrogen which have been subjected to quantitative investiga-
tion. One is the fermentation of sugars by the coli group (17, a6)
of organisms and the other the butanol-acetone fermentation.
pH Range. The methane fermentation proceeds smoothIy and
continuously at a p H value of 6.5 to about 8.0. An accumulation
of volatile acids and their salts precedes an unfavorable drop in
p H by several days.
Mechanism of Methane Fermentation
References on the mechanism of the bacteriological degrada-
tion of cellulose and cellulosic materials and especially those
dealing with the anaerobic fermentation of these materials are
scarce and far from enlightening. Some information may be
found in the periodic literature, in texts, and in Stephenson's
book, "Bacterial Metabolism" (3.9). For a review of the litera-
ture, the reader is referred to (37)and (38)
Since acetic acid is always found during methane fermentation,
one is inclined to regard it as an important if not universal pre-
cursor of methane. Three possible mechanisms for the methane
fermentation of acetic acid are suggested by the findings and
reasoning of previous workers in this field. From the work of
Qmelianskil (22,23) and Sohngen (68,29) a preliminary de-
composition of the acetic acid to hydrogen and carbon dioxide,
with subsequent reduction of carbon dioxide to methane by the
hydrogen, would be expected. Barker's earlier work (2)was en-
tirely in support of the theory proposed by van Niel that meth-
ane is formed biologically by the reduction of carbon dioxide.
The reasoning of Buswell and Neave (11) was that methane
originates primarily by a simple decarboxylation of acetic acid.
The first mechanism was based on the similarity of the hydro-
gen and methane fermentations, but the absence of hydrogen
in the gas from the methane fermentation of acetic acid is evi-
dence against this mechanism. If the data of Symons and Bus-
well (34) is examined, i t is found that only 3.5 liters of methane
were formed over a period of 100 days with regular circulation of
hydrogen and carbon dioxide through a culture of 2.0 liters total
volume. In the same time, such a culture fermenting acetic
acid could be expected to form 7 5 liters of methane (8,36).
Stadtman and Ba&er (81)noted in more recent work that the
production of methane from the reduction of carbon dioxide is a
very slow process.
The second mechanism avoids this weakness by implying a
direct reduction of the carbon dioxide, without the intermediate
March 1952 INDUSTRIAL AND ENGINEERING CHEMISTRY
-LZquZd Indus&Zal Wastes-
stage of free hydrogen. This mechanism seems rather indirect
and involved, but parallels that found by Barker in the fermenta-
tive oxidation of alcohols.
The last mechanism appears to be the simplest and most direct.
Evidence against this mechanism is found in Thayer's work (36).
He reasoned that this reaction was a decarboxylation reaction and
that the fermentation of propionic and butyric acids should yield
ethane and propane. However, his results were entirely negative,
for no hydrocarbon other than methane was found. This has
been confirmed in all work where the gas was analyzed. There-
fore, if this mechanism of simple decarboxylation for acetic acid is
accepted, it must be as a special case, not applicable to the higher
fatty acids.
Barker, Ruben, and Kamen (3) found evidence for the reduc-
tion of carbon dioxide in the fermentation of acetic acid through
the use of C110~,but they stated that the radioactive methane
found might have been due to the presence of methanol carried
over with the inoculum. These workers were using a pure culture
of Methanosarcina methanica, and even if methane should be
formed by reduction of carbon dioxide in that case, that might
not be the predominant mechanism in the usual methane fer-
mentation where a mixed culture is used.
With the availability of radioactive carbon, it was possible
to determine the manner in which methane originates from
acetic acid. Buswell and Sollo (12) used C14-labeled carbon di-
oxide to investigate the anaerobic fermentation of acetic acid by
enrichment cultures. In this fermentation only a small portion
of the isotope was recovered in the resulting methane. By ob-
serving the relation of the specific activity of the methane to the
time of incubation, a correlation was found which would indicate
a slow reduction of the carbon dioxide unassociated with the
main course of fermentation, such as the formation of cellular
material and subsequent autolysis. From these observations it
was concluded that the methane produced was derived mainly
from the acetic acid and only to a slight extent from the carbon
dioxide.
Stadtman and Barker (30) in subsequent experiments using
C14-labeled carbon dioxide and acetic acid confirmed the results
of Buswell and Sollo. They further showed that methane is
derived mainly from the methyl group of acetate, whereas the
carboxyl group gives rise to carbon dioxide. These investiga-
tions were carried out using a partially purified culture of a
Methanococcus species. Results obtained in more recent ex-
periments by the same authors on the acetate fermentation by a
species of the Methanosarcina ( 3 2 ) are also in agreement with
those previously reported using enrichment cultures and a species
of the Methanococcus. On the other hand, the data obtained by
Stadtman and Barker (30) on the fermentation of ethyl alcohol
in the presence of C14-labeled carbon dioxide by Mb. omelianski
clearly show that 82 to 100% of the methane produced was
derived from carbon dioxide.
Recently Stadtman and Barker ( S I ) investigated the methaiie
fermentation of the lower fatty acids using CWabeled com-
pounds. The organisms used in t,hese experiments differed from
the methane-producing organisms previously described in that
they are able to carry out the incomplete anaerobic oxidation of
fatty acids. Methanobacterium suboxydans, by means of beta
oxidation, quantitatively converts caproic and butyric acids to
acetic acid. Valeric acid is oxidized to propionic and acetic acids.
Methanobacterium propionicum metabolizes propionic acid in
such a manner that the alpha and beta carbons give rise to acetic
acid, whereas the carboxyl carbon is converted to carbon dioxide.
The methane produced in these fermentations was derived en-
tirely from the biological reduction of carbon dioxide.
In view of the fact that considerable variation exists as to the
origin of methane, further experiments have been carried out to
determine how methane originates in the decomposition of other
compounds. Buswell et al. (10)investigated the action of enrich-
ment cultures on propionates having the carbons 1, 2, and 3
551
-Liquid Industrial Was$es-
labeled, respectively. The distirbution of the radioactivity in Inhibiting Agents. McNary et a;.( $ 0 )reported that the anaero-
the evolved gases indicated that propionic acid is oxidiaed in bic fermentation of citrus fruit wastes was inhibited by traces of
such a manner that both gases may arise from all three carbons, peel oil or D-limonene, the principal constituent of this essential
and that carbon dioxide is, in part a t least, a precursor of the oil. The authors later observed the inhibitory effect on gas
methane formed. This latter result could occur by either of two production by the addition of D-limonene to active methane-pro-
paths ducing cultures, using acetic acid as the substrate.
4CH3CHzCOOH +
4Ha0 +4CR3CIIzOH 4HCOOH + Literature Cited
2CH8CH20H +
COz --++ CH, +
2CHaCOOH (1)
Ardern, E., Ann. Rept. Revenue Dept., Manchester, England,
as suggested by Buswell et al. ( I O ) , or for year ending March 25, 1931.
CH3CHzCOOH +
2H20 +CHaCOOH COz + + 6[H] Barker, H. A., Arch. Mikrobzol., 7, 404 (1936).
Barker, H. A,, Ruben, S., and Kamen, M . D., Proc. N a t l . i l c a d .
(COz +
4H2 +CH4 2H20) + (2) Sci. U . S., 26,426 (1940).
Boruff, C . S.,and Buswell, A. hl., IND.ENG.CHEX, 21, 1181
as suggested by Stadtman and Barker (51). The reasoning that a
preliminary hydrolysis of propionic acid to ethyl alcohol and
.----,
il929\.
Ibid., 22, 931 (1930).
formic acid (Equation 1) may occur was in part confirmed by Boruff, C. S., and Buswell, A. M., J . Am. Chem. Soc., 56. 886
simultaneous adaptation experiments. A considerable yield of (1934).
Buswell, A. hI.,IND. ENG.CHEM., 42, 605 (1950).
acetic acid was regarded by Stadtman and Barker as evidence Buswell, A. hI., et al., Illmoze State Water SurLeg, Bull. No. 32,
favoring path of Equation 2. (1936).
In the fermentation of methanol by a species of the Methano. Buswell, A. M., Boruff, C. S., and Weisman, C. K., Ibid., 24,
sarczna, Stadtman and Barker (52) showed by tracer experiments 1423 (1932).
Buswell,‘ A. k., Fina, L., Mueller, H. F., and Yahiro, A,, J .
that over 98% of the methane originates from the alcohol and Am. Chem. Soc., 73, 1809-11 (1951).
not more than 2% is derived from carbon dioxide. In this Buswell, A. M., and Neave, S. L., Illinois State Water Survey,
fermentation, as in the case of the acetate fermentation, carbon Bull. KO.30 (1930).
dioxide utilization is of little importance. From the information Buswell, -4.PI.,and Sollo, F. IT., J . Am. Chein. Soc., 70, 1778
(1948).
now available, these investigators conclude that a common inter- Fair, G. M., and Moore, E. T.,Sewage W o r k s J . , 6, 3 (1934).
mediate is involved in the formation of methane from acetic acid, Fischer, F., Lieske, R., and TTinzer, K., Biochem. Z., 236, 247
methanol, and carbon dioxide. (1931).
Fowler, G. J., and Joshi, G. V.,J . Indian I n s t . Sei., C3, 39
Buswell et al. (10) in studies now in progress observe that in (1920).
the complete anaerobic fermentation of butyric acid by enrich- Grosse, A. V.,and Libby, W. F., Abstracts, 113th Meeting
ment cultures, methane is in part derived from the reduction of AM,CHEM.SOC., 13R-14R (1948).
carbon dioxide. These results are in agreement with the findings Kluyver, A. J., “Chemical Activities of Microorganisms,”p. 55,
of Stadtman and Barker (51) represented by the equation London, London University Press, 1931.
Levine, M., Nelson, G. H., Anderson, D. Q., and Jacobs, P. B.,
2CHsCHzCHzCOOH + 2Hz0 + COz +4CH3COOH + CH, I N D . ENG.CHEM., 27,195 (1935).
McBeth, I. G., and Scales, F. M., U . S.Dept. Agr., Plant I n d .
Assuming the acetic acid in the above equation to be further de- Bull. 266 (1913).
graded to methane and carbon dioxide, the total yield of methane McNary, R. R., Walford, R. W., and Patton, V. D., Food Tech-
and carbon dioxide is theoretically the same as that previously noZ., 5, 320-3 (1951).
Norman, A. G., Science Progress, 30,442 (1936).
calculated by Buswell et al. (8). A comparison of the final specific OmelianskiI, W., Zentr. Bakt., Parasitenk., A b t . 11, 8 , 193, 225,
activities of the methane and carbon dioxide in this investigation 257, 289, 321, 353,385 (1902).
indicates that methane is derived from the substrate as well as Ibid., 11, 369 (1904).
from the reduction of carbon dioxide. Similar results have been O’Shaughnessy,F. R., J . Soc. Chem. I n d . ( L o n d o n ) , 33, 3 (1914).
Popoff, L., Arch. ges. Physiol. (PflUgers),10, 113 (1876).
observed in experiments on the fermentations of various acids, Rogers, L. A., Clark, NT.H., and Evans, A. C., J . Infectious
carbohydrates, and alcohols. Diseases. 17. 137 (1915).
Sen, H. K.; Pai, P. P., and Ghosh, S. B., J . I n d i a n Chem. Soc., 6,
Summary 673 (1929).
Sohngei, N,‘ L., Proc. Acad. Sci. Amsterdam, 8 , 327 (1905).
Reactions Shown to Occur in the Methane Fermentation. Sohngen, N. L., Rec. trav. chim., 29, 238 (1910).
Empirical equation: Stadtman, T. C., and Barker, H. A., Arch. Biochem., 21, 256
CnHaOb + (n- ~ / -4 b/2)Hz0 --+ (1949).
Stadtman, T. C., and Barker, H. A., J . Bact., 61, 67-80 (1951),
( n / 2 - a / 8 + b/4)C02 + (n/2 + a / 8 - b/4)CH4 Ibid., pp. 81-6.
Stephenson, Marjory, “Bacterial Metabolism,” 3rd ed.. New
NOT INVOLVING
1. REACTIONS REDUCTIONOF CARBON York, Longmans, Green and Co., 1949.
DIOXIDE.
CHsCOOH +COS CIla + Symons, G. E., and Buswell, A. M., J . Am. Chem. SOC.,55,2028
(1933).
4CHaOH +3CH4 COz 2Hz0 + + Tarvin, D., and Buswell, A. M., Ibid., 56, 1751 (1934).
Thayer, L. A,, BUZZ.Am. Assoc. Petroleum Geol., 15, 441 (1931).
2. REDUCTION DIOXIDEWITHOUT SET
OF CARBON DECREASE. Thaysen, A. C., and Bunker, H. J., “The Microbiology of Cellu-
4HCOOH ---+ CH4 + 3COz + 2H20 lose, Hemicelluloses, Pectin and Gums,” London, Oxford
4CH3CHzCOOH 2H20 + +7CH4 + 3201 University Press, 1927.
Thaysen, A. C., and Gallway, L. D., “The Microbiology of
AND NET DECREASE
3. REDUCTION OF CARBON
DIOXIDE. Starch and Sugars,” London, Oxford University Press, 1930.
Thumm, K., ViierteZjahrssch. gerichtl. Med. u. 6ffentl. Sanitatsw..
4H2 +
COz +CH, 2H20 + 48, Suppl. 2, 73 (1914).
2CHaCHzOH +
CO2 --+ CHa 2CH8COOH + RECEIVEDfor review September 6, 1961. ACCEPTED
January 14, 1952
+
2CHa(CHn)zCHZOH COz --+ CH, 2CHs(CHz)ZCOOH+
+
4CH3CHOHCH3 COP --f CHI 4CH3-CO-CHs + 2HzO + END OF SYMPOSIUM
2CH,(CH2)2COOH COz + +
2H20 + CH, 4CHsCOOH +
2CHa(CH2)3COOH COz + +
2H20 +
CH, +-2CH3CHzCOOH 2CH3COOH + Reprints of this symposium may be purchased for 75 cents
Nonfermentable Substances. Hydrocarbons and ethers have each from the Reprint Department, AMERICANCHEMICAL
not been fermented to methane. Lignin is attacked slowly if at all. SOCIETY, 1155 Sixteenth St., N.W., Washington 6, D. C.
552 INDUSTRIAL AND ENGINEERING CHEMISTRY Vol. 44, No. 3