171-175 Copyright © 1967 American Society for Microbiology

Vol. 94, No. 1 Printed in U.S.A.

Inhibition of Rumen Methanogenesis by Methane Analogues

Departmenzt of Bacteriology,

University of California, Davis, California 95616

Received for publication 20 March 1967

Extremely low concentrations of chloroform and carbon tetrachloride and somewhat larger concentrations of methylene chloride inhibited the formation of methane by the rumen microbiota in the presence or absence of added substrate. The accumulation of hydrogen at these low concentrations indicates a selective inhibition of methanogenesis. Presumably, these inhibitors affect one or more of the reactions by which methane is formed from hydrogen and carbon dioxide.
The chief pathway of methane production in the bovine rumen is the reduction of carbon dioxide with molecular hydrogen (1, 3). In both rumen liquid and pure culture preparations (4), methane production is extremely sensitive to inhibition by oxygen. Wolin et al. (6) found that methane production by cell-free preparations of the culture known as Methanobacillus omelianskii was similarly affected by oxygen. Later, Wolin et al. (5) found that these same preparations were inhibited also by low concentrations of the viologen dyes, benzyl and methyl viologen. The present work shows an inhibition by the chlorinated methanes.
MATERIALS AND METHODS Rumen. Rumen contents were incubated in an 800-ml cylindrical gas washing bottle (Corning Glass

Works, Corning, N.Y.) with a sintered-glass base through which gas was continuously circulated by means of a small pump. Controlled minute quantities of gas or liquid reagent could be injected via syringe through injection ports closed with a butyl rubber recessed stopper. Rumen contents were freshly obtained from a cow fed on alfalfa hay. Rumen liquid was obtained by collecting rumen contents and incubating at 39 C until the particulate material either settled or was carried to the top by fermentation gases. The middle liquid containing bacteria with relatively few protozoa or food particles was drawn off and used, with or without addition of solids. When both liquid and solids were used, the material was designated rumen

(Bellco, Glass, Inc., Vineland, N. J.) sealed anaerobically with recessed rubber stoppers. Substrates or inhibitors (0.1 ml) were pipetted into the tubes first, following the anaerobic culture-tube techniques of Hungate (3). A 10-ml amount of rumen liquid or rumen contents was then added to each tube which was stoppered anaerobically. The mixed microbiota protected the methanogenic bacteria during these operations, and the samples produced methane without appreciable lag. Except where indicated in the text, the gas phase in the tubes and in the recirculation apparatus was carbon dioxide. In some experiments, 1 g of total contents (solids) was substituted for 1 ml of the rumen liquid. Tubes were incubated at 39 C. In the tube experiments, 0.25 ml of gas was removed by syringe for analysis. Larger samples could be withdrawn from the recirculation experiments. Gases were analyzed at room temperature on a Perkin-Elmer Vapor Fractometer with a silica gel column and N2 as carrier gas. When formate was used, 0.1 ml of 1 M sodium formate was added to each tube. Solutions of inhibitors. Carbon tetrachloride and methylene chloride were dissolved in absolute alcohol. Because 0.1 ml of ethyl alcohol occasionally stimulated methane production, a control tube with only ethyl alcohol added was run. Usually, the rate of methane production was identical with and without ethyl alcohol.

Small-scale experiments. To increase the number of experiments beyond those possible with the single gas recirculation apparatus, samples of rumen contents were also incubated in 150 by 16 mm roll culture tubes I Present address: National Center for Primate Biology, University of California, Davis 95616.

RESULIS AND DISCUSSION The studies evolved from an accidental finding during an investigation of methane production by bovine rumen contents in vitro. Excessive foaming of the rumen liquid occasionally occurred in the gas circulation apparatus. On one such occasion, a small quantity of Antifoam A spray (Dow Corning, Corp. Midland, Mich.) was added. This reduced the foaming, but subsequent measurements disclosed that methane was no longer formed. The gas phase in this experiment con-

1). Normally. 3. Gas phase = CO2. Other experimental details as for Fig. CH4 and H2 production from formate by rumen liquid. 5 -J a -480 w 1 z w -J 0 0 a 0 I. The Antifoam 2 5 A spray.ever. 1. Although the active principle was O IDJ 5/ not chloroform.caused accumulation of the precursor hydrogen. BACTERIOL. howfurther. In rumen liquid.2 .172 BAUCHOP J. This suggested inhibition at a site in the metabolic sequence between hydrogen production and its 2. D LOX o 0. MINUTES FIG. 1.2 ymoles per ml per hr. consisted of a silicone ingredient and a volatile Freon (CC12F2) propellant. Chloroform added where indicated by the arrow.) The structural resemblance to 0 methane supported the idea that chloroform 0~ C_ might be the inhibitory agent in the antifoam. 2. the verted to methane. Effect ofAntifoam A on CH4 and H2 production from formate. an odor resembling chloroform was detected. Effect of chloroform on CH4 and H2 production from rumen liquid plus solids. The Antifoam A preparation addition of formate at this rate caused a constant selectively inhibited methane formation and rate of methane production while the concentra.umoles per ml per hr. When a sample of Antifoam A was transferred to a beaker to quantitate its inhibitory ability. (This tj~ was in fact an incorrect conclusion. hydrogen is normally conthen fed into the incubation mixture at a constant rate of 10.0 conversion into methane. w MINUTES z FIG. 2). 1. Chloroform = 20 mi. . Formate feed rate = 10. Sodium formate was accumulated (Fig.5 J - uJ -J 0 H4 D~~~~~~~ CI T H2 800 z CH4 25 35 0 5 15 ° 640. Experimental details as for Fig. Gas circulation apparatus.C H2 320 H2 cX 0n w 0 0 0 cr 1600 E 0 4-J CH4 40 0 10 20 30 40 50 MINUTES FIG. No formate added. no methane formed and hydrogen gas placed by carbon dioxide. the gas mixture was flushed out and re. it is probable that Freon acts in a similar manner. it was later learned. tion of hydrogen in the gas phase remained relaIn an attempt to investigate the antifoam effect tively constant (Fig. With Antifoam A. sisted of 80%c hydrogen and 20% carbon dioxide.

212 0 0.5 2.umoles per ml per hr) Inhibition (%O) of CH4 production H2 CH4 None 114 CHC13 CC14 Rumen solids CH2CI2 15.1 ml of CHCI3 solution 0.5 2.200 CH2CI2 CHC13 CC14 15.07 1.15 3 49 1.5 ml of chloroform to 300 ml of rumen contents.043 0. 0 Methanogenesis was completely inhibited and w LJ hydrogen accumulated (Fig.012 0.0 15.92 4 0. Effect of chloroform on the!quantities of CH4 and H2 produced from solidIs in roll tubes Incubation time (hr) The accumulation of hydrogen was not stoi- Addition Inhibitor concn (pM) Gas produced _ (pmoles/ml) H.5 2.006 0.88 9. In experiment 1.064 6.160 0.5 2.25 15. 3). liquid plus solids.144 0. 'r C) LLI :D V) TABLE 2.0 2.080 0 0. except in the hydrogen experiment for which 80% H2 and 20% CO2 was used. This suggests that chloroform.04 97 100 98 100 100 100 Hydrogen CH2C12 CHCI3 CC14 145 145 145 145 1.3 .72 0 0 0 91 100 86 100 100 100 CHC13 CC14 Sodium formate CH2CI2 0 0.66 0 11 0.umoles/ml. .0 15. 13. Sodium formate concentration was 10 mm.8 0. 1967 INHIBITION OF METHANOGENESIS 173 TABLE 1.8 0.007 0.848 0.120 0. -i This was tested by adding 0. at these concentrations. Table 1 shows the MM CC14 H2 and CH4 produced during the incubation of rumen contents in tubes with and without chloroFIG.0 114 114 114 180 180 180 180 155 155 155 155 0.1 ml of H20 0.16 0. also partially inhibits hydrogen production.umoles/ml was obtained.31 2. the total hydrogen needed to account for the methane formed in the control was 35. Effect of low concentrations of carbon tetraform. Also. Effect of chlorinated methanes oni CH4 and H2 production from different substratesSubstrate added to rumen fluid Inhibitor Concn (mm) Duration of expt (min) Gas produced (.018 0.46 -i LL z z LLJ 2 490 7.36 a: x 2 0. cumulation.6 . 0). With the inhibitor.1 ml of CHCI3 solution 0 0 490 0 chiometric with the equation CO2 + 4H2 -+ CH4 + 2H20. Gas production was linear during at least the 1 st hr of the incubation periods used. in these experiments the chloroform chloride onz CH4 and H2 production from formate.127 0.VOL.1 ml of CHCI3 solution 0.8 0. 94. inhibited methane production with hydrogen ac. 4.8 0. An altemative explanation that the accumulated hydrogen ca cr D 0 1: 0 Z) 4.06 0 0. Similar results were obtained in experiments ZL E with rumen contents in tubes.026 CH4 8.30 0 0 0 a Incubation was in roll tubes under an atmosphere of CO2.Incubation was in tubes for a period of I hr during which time the rates ofgas production were linear.120 1.

Formate feed rate = 10. Hydrogen normally formed from ethyl alcohol accumulated in greater quantities when methane formation was inhibited by the viologen dyes.2 . Methylene chloride was less effective. 5) with the gas circulation apparatus.012 0 0. with sodium formate. or simply the rumen solids which are the normal substrate. 5. A 1-ml amount of 40 nLm benzyl viologeni was added at the time shown by the arrow to give a final concentrationi of 133 Am.0005 0. Gas circulation apparatus. chloroform.0 200 a LA :c -J a -j 160 E 3.0k 40 =cm en w CHC13 0 0. demonstrated that low concentrations of methyl or benzyl viologen inhibited the formation of methane from ethyl alcohol and from a gas mixture of hydrogen and carbon dioxide. With "solids" as substrate.40 0. Gas phase = C02.UM CC14. The related compounds methylene chloride and carbon tetrachloride produced effects similar to those obtained with chloroform (Table 2). A concentration of 10 AM benzyl viologen had no effect on the methane production rate. . 5 AM benzyl viologencompletely in- 4. The accumulation of hydrogen from "solids" thus further illustrated the selectivity of this group of inhibitors for the methane-forming reactions. In one experiment (Fig. The inhibitors were effective also when methanogenesis was increased by adding the above substrates. In each case.90 1. 4 except where indicated in the table. Inhibition by viologen dyes. Effect of low conicentrations of chlorinated methanes on CH4 and H2 production from formate Addition and carbon tetrachloride. BACTERIOL. production from formate. Rumen fluid was incubated with added solids. methane production was inhibited and hydrogen gas accumulated. Addition of benzyl viologen to rumen fluid produced similar effects. 4.86 2.54 28 50 88 27 46 79 9 32 76 98 0 w 2 0 0 I 2. (5).0H2 w 0 0 0 a 80 a 0L . again the amount of hydrogen evolved was less than that expected if it had been simply diverted fiom methane production. With increasing concentrations. was utilized in some other metabolic sequence was not explored. (5).002 0.001 79 79 79 79 60 60 60 60 60 C/) w -lJ 0 0 E 40 80 MINUTES CC14 0. CH4 w z z w 2 Concn (MM) Duain CH4 produced Douf reptn umolper per Inhibition (% (min) cr -J 120 2 mlprhr) CH2C12 0 1. in the experiments of Wolinetal. Effect of benzyl viologen on CH4 and H2 Details as for Fig. formate and hydrogen were probably still the immediate precursors of methane.174 BAUCHOP J.0049 0.0. Table 3 shows the inhibition of methanogenesis from formate by methylene chloride.13 2. Diversion of hydrogen to propionate could be of considerable economic importance.12 0.12 0.4 x 103 AtM CH2CI2.06 120 0 FIG. The effect of adding low concentrations of carbon tetrachloride to rumen fluid is shown in Fig. 7. Very low concentrations of chloroform and carbon tetrachloride were inhibitory.60 1.006 0. benzyl viologen was added to the reaction mixture 36 min after the start of the experiment. Wolin et al. TABLE 3. The rate of methane production was immediately depressed. omelianskii. hydrogen.81 2. and 2.02 1.003 a 3. and at the same time the rate of hydrogen production increased.74 0. working with M. The concentrations of these compounds required to produce 50% inhibition of methane production were: 1.6 2.33 2. or under an atmosphere of 80% hydrogen and 20% carbon dioxide (Table 2).0 90 90 90 90 2. However. whereas.Amoles per ml per hr.8 piM CHCl. The rate of methane production by rumen fluid can be increased by the addition of formate. the rate of methane production decreased and was accompanied by an increase in the rate of hydrogen production.4 .0 4.

1967 INHIBITION OF MIETHANOGENESIS 175 hibited methane production by washed cells of M. 1963. omelianskii. A. AND R. Viologen dye inhibition of methane formation by Methanobacillus omelianskii. WOLFE.VOL. J. 1966. The rumen and its microbes. . J. 75:713-718. AND R. E. Catabolic reactions of mixed suspensions of bovine rumen bacteria. Hungate for the stimulating experience of working in his laboratory. Inc. 6. 3. New York. WOLIN. Academic Press. E. Q. 1964. R.. AND R. E. E. R. E. R. WOLIN. P. SMITH. Bacteriol.... DOETSCH. S. Biol. 87:993-998. J. BROWN. sp. 5. R. This investigation was supported by Public Health Service grant AI-10266 to R. 1955. or of the lower sensitivity of the rumen methanogenic bacteria themselves. AND M.. E. HUNGATE. Hungate from the National Institute of Allergy and Infectious Diseases. Biochem. H. ACKNOWLEDGMENTS It is a pleasure to thank R. 1953. J. Isolation and characterization of Methanobacterium ruminantium n. 36:825-831. S. 56: 525-536. WOLIN. A. J. 4.. Formation of methane by bacterial extracts. HUNGATE. E. Bacteriol. HUNGATE. C. CARROLL. 94. WOLFE. J. J. Biophys. The difference may be a function of the mass of nonmethanogenic bacteria in the preparation. Dairy Sci. E. N. 238:2882-2886. AND J. M. WOLIN. rumen contents. LITERATURE CITED 1. Formate dissimilation and methane production in bovine 2. SHAW. ROBINSON. R. 1958. E. Chem. Arch.

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