Hydrogenotrophic methanogenesis by moderately acid-tolerant methanogens of a methane-emitting acidic peat

Abstract

The emission of methane (1.3 mmol of CH(4) m(-2) day(-1)), precursors of methanogenesis, and the methanogenic microorganisms of acidic bog peat (pH 4.4) from a moderately reduced forest site were investigated by in situ measurements, microcosm incubations, and cultivation methods, respectively. Bog peat produced CH(4) (0.4 to 1.7 micro mol g [dry wt] of soil(-1) day(-1)) under anoxic conditions. At in situ pH, supplemental H(2)-CO(2), ethanol, and 1-propanol all increased CH(4) production rates while formate, acetate, propionate, and butyrate inhibited the production of CH(4); methanol had no effect. H(2)-dependent acetogenesis occurred in H(2)-CO(2)-supplemented bog peat only after extended incubation periods. Nonsupplemented bog peat initially produced small amounts of H(2) that were subsequently consumed. The accumulation of H(2) was stimulated by ethanol and 1-propanol or by inhibiting methanogenesis with bromoethanesulfonate, and the consumption of ethanol was inhibited by large amounts of H(2); these results collectively indicated that ethanol- or 1-propanol-utilizing bacteria were trophically associated with H(2)-utilizing methanogens. A total of 10(9) anaerobes and 10(7) hydrogenotrophic methanogens per g (dry weight) of bog peat were enumerated by cultivation techniques. A stable methanogenic enrichment was obtained with an acidic, H(2)-CO(2)-supplemented, fatty acid-enriched defined medium. CH(4) production rates by the enrichment were similar at pH 4.5 and 6.5, and acetate inhibited methanogenesis at pH 4.5 but not at pH 6.5. A total of 27 different archaeal 16S rRNA gene sequences indicative of Methanobacteriaceae, Methanomicrobiales, and Methanosarcinaceae were retrieved from the highest CH(4)-positive serial dilutions of bog peat and methanogenic enrichments. A total of 10 bacterial 16S rRNA gene sequences were also retrieved from the same dilutions and enrichments and were indicative of bacteria that might be responsible for the production of H(2) that could be used by hydrogenotrophic methanogens. These results indicated that in this acidic bog peat, (i) H(2) is an important substrate for acid-tolerant methanogens, (ii) interspecies hydrogen transfer is involved in the degradation of organic carbon, (iii) the accumulation of protonated volatile fatty acids inhibits methanogenesis, and (iv) methanogenesis might be due to the activities of methanogens that are phylogenetic members of the Methanobacteriaceae, Methanomicrobiales, and Methanosarcinaceae.

FIG. 1.

Effects of supplemental H₂ on the production of CH₄ (A) and volatile fatty acids (B) in bog peat microcosms. Solid symbols indicate microcosms amended with H₂; open symbols indicate controls (unamended); •, ○, H₂; ▪, □, CH₄; ▴, ▵, acetate; ▾, ▿, propionate; ⧫, ◊, butyrate. A detailed view of the production of CH₄ during the first 2 days is shown in the inset.

FIG. 2.

Effects of supplemental formate (A and B) or ethanol (C and D) on the production of CH₄ (A and C), H₂ (B and D), and CO₂ (B and D) in bog peat microcosms. Solid symbols indicate amended microcosms; open symbols indicate controls (unamended); ⧫, ◊, formate; ▪, □, CH₄; ▾, ▿, ethanol; •, ○, H₂; ▴, ▵, CO₂. A detailed view of the production of CH₄ during the first 3 days is shown in the insets (A and C).

FIG. 3.

Effects of 20 mM bromoethanesulfonate (A) or easily fermentable carbon (TSB containing glucose) (B) on the production of H₂ and CH₄ in bog peat microcosms. Solid symbols indicate amended microcosms; open symbols indicate controls (unamended); •, ○, H₂; ▪, □, CH₄.

FIG. 4.

Effect of 5 mM acetate on the production of CH₄ from H₂-CO₂ by the methanogenic enrichment culture at pH 4.5 (<pK_a^{acetic acid}) (A) and pH 6.5 (≫pK_a^{acetic acid}) (B). Values are the means of triplicates. Symbols: •, amended with acetate; ○, controls (unamended).

FIG. 5.

Phylogenetic positions of bacterial and representative archaeal sequences obtained in this study (bold) as inferred from comparative analysis of 16S rRNA gene sequence data. Values in parentheses are the number of sequences obtained for a given group. The codes used for the sequences obtained in this study are as follows: A, archaeal; B, bacterial; M, from highest CH₄-positive MPN dilution of bog peat; E, from methanogenic enrichment culture; C, sequences (1,400 bp) from clone library; D, sequences (approximately 400 bp) from excised bands of DGGE analyses. Sequences that are almost complete (i.e., approximately 1,400 bp) are marked with an asterisk. The bar represents a 0.1 estimated change per nucleotide.