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 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.

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The main objectives of this study were (i) to determine if gut wall-associated microorganisms are responsible for the capacity of earthworms to emit nitrous oxide (N 2 O) and (ii) to characterize the N 2 O-producing bacteria of the earthworm gut. The production of N 2 O in the gut of garden soil earthworms (Aporrectodea caliginosa) was mostly associated with the gut contents rather than the gut wall. Under anoxic conditions, nitrite and N 2 O were transient products when supplemental nitrate was reduced to N 2 by gut content homogenates. In contrast, nitrite and N 2 O were essentially not produced by nitrate-supplemented soil homogenates. The most probable numbers of fermentative anaerobes and microbes that used nitrate as a terminal electron acceptor were approximately 2 orders of magnitude higher in the earthworm gut than in the soil from which the earthworms originated. The fermentative anaerobes in the gut and soil displayed similar physiological functionalities. A total of 136 N 2 O-producing isolates that reduced either nitrate or nitrite were obtained from high serial dilutions of gut homogenates. Of the 25 representative N 2 O-producing isolates that were chosen for characterization, 22 isolates exhibited >99% 16S rRNA gene sequence similarity with their closest cultured relatives, which in most cases was a soil bacterium, most isolates were affiliated with the gamma subclass of the class Proteobacteria or with the gram-positive bacteria with low DNA G؉C contents, and 5 isolates were denitrifiers and reduced nitrate to N 2 O or N 2 . The initial N 2 O production rates of denitrifiers were 1 to 2 orders of magnitude greater than those of the nondenitrifying isolates. However, most nondenitrifying nitrate dissimilators produced nitrite and might therefore indirectly stimulate the production of N 2 O via nitrite-utilizing denitrifiers in the gut. The results of this study suggest that most of the N 2 O emitted by earthworms is due to the activation of ingested denitrifiers and other nitrate-dissimilating bacteria in the gut lumen.

Earthworms emit nitrous oxide (N2O) via the activity of bacteria in their gut. Four N2O-producing facultative aerobes, ED1T, ED5T, MH21T and MH72, were isolated from the gut of the earthworm Aporrectodea caliginosa. The isolates produced N2O under conditions that simulated the microenvironment of the earthworm gut. ED1T and ED5T were Gram-negative, motile rods that carried out complete denitrification (i.e. the reduction of nitrate to N2) and contained membranous c-type cytochromes. ED1T grew optimally at 30 °C and pH 7. ED1T oxidized organic acids and reduced (per)chlorate, sulfate, nitrate and nitrite. The closest phylogenetic relative of ED1T was Dechloromonas agitata. ED5T grew optimally at 25 °C and pH 7. ED5T grew mainly on sugars, and nitrate and nitrite were used as alternative electron acceptors. The closest phylogenetic relatives of ED5T were Flavobacterium johnsoniae and Flavobacterium flevense. MH21T and MH72 were motile, spore-forming, rod-shaped bacteria with a three-layered cell wall. Sugars supported the growth of MH21T and MH72. Cells of MH21T grew in chains, were linked by connecting filaments and contained membranous b-type cytochromes. MH21T grew optimally at 30–35 °C and pH 7·7, grew by fermentation and reduced low amounts of nitrite to N2O. The closest phylogenetic relatives of MH21T were Paenibacillus borealis and Paenibacillus chibensis. Based on morphological, physiological and phylogenetic characteristics, ED1T (=DSM 15892T=ATCC BAA-841T), ED5T (=DSM 15936T=ATCC BAA-842T) and MH21T (=DSM 15890T=ATCC BAA-844T) are proposed as type strains of the novel species Dechloromonas denitrificans sp. nov., Flavobacterium denitrificans sp. nov. and Paenibacillus anaericanus sp. nov., respectively. MH72 is considered a new strain of Paenibacillus terrae.

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