Dechloromonas denitrificans sp. nov., Flavobacterium denitrificans sp. nov., Paenibacillus anaericanus sp. nov. and Paenibacillus terrae strain MH72, N2O-producing bacteria isolated from the gut of the earthworm Aporrectodea caliginosa.

Earthworms emit nitrous oxide (N(2)O) via the activity of bacteria in their gut. Four N(2)O-producing facultative aerobes, ED1(T), ED5(T), MH21(T) and MH72, were isolated from the gut of the earthworm Aporrectodea caliginosa. The isolates produced N(2)O under conditions that simulated the microenvironment of the earthworm gut. ED1(T) and ED5(T) were Gram-negative, motile rods that carried out complete denitrification (i.e. the reduction of nitrate to N(2)) and contained membranous c-type cytochromes. ED1(T) grew optimally at 30 degrees C and pH 7. ED1(T) oxidized organic acids and reduced (per)chlorate, sulfate, nitrate and nitrite. The closest phylogenetic relative of ED1(T) was Dechloromonas agitata. ED5(T) grew optimally at 25 degrees C and pH 7. ED5(T) grew mainly on sugars, and nitrate and nitrite were used as alternative electron acceptors. The closest phylogenetic relatives of ED5(T) were Flavobacterium johnsoniae and Flavobacterium flevense. MH21(T) and MH72 were motile, spore-forming, rod-shaped bacteria with a three-layered cell wall. Sugars supported the growth of MH21(T) and MH72. Cells of MH21(T) grew in chains, were linked by connecting filaments and contained membranous b-type cytochromes. MH21(T) grew optimally at 30-35 degrees C and pH 7.7, grew by fermentation and reduced low amounts of nitrite to N(2)O. The closest phylogenetic relatives of MH21(T) were Paenibacillus borealis and Paenibacillus chibensis. Based on morphological, physiological and phylogenetic characteristics, ED1(T) (= DSM 15892(T) = ATCC BAA-841(T)), ED5(T) (= DSM 15936(T) = ATCC BAA-842(T)) and MH21(T) (=DSM 15890(T) = ATCC BAA-844(T)) 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.

Lactovum miscens gen. nov., sp. nov., an aerotolerant, psychrotolerant, mixed-fermentative anaerobe from acidic forest soil.

An aerotolerant, psychrotolerant anaerobe, anNAG3, was isolated from an acidic forest floor solution (in situ pH of 4.5). Cells of anNAG3 stained Gram-positive did not form spores, and were not motile. Cells were ovoid, approximately 1 microm long and 0.7 microm wide, mostly in pairs, and contained a multi-layered cell wall and intracytoplasmic membranes. Growth was observed at pH 3.5-7.5 and 0-35 degrees C. Glucose, galactose, fructose, mannitol, glucosamine, N-acetylglucosamine, cellobiose, and maltose supported growth. Lactate, ethanol, formate, and acetate were end products. H(2) and CH(4) were not detected, and only very minor amounts of CO(2) were produced. The relative amount of a particular product was dependent on the substrate utilized, and product profiles indicated that (i) sugars were initially metabolized to pyruvate via glycolysis, and (ii) lactate dehydrogenase and pyruvate-formate lyase were responsible for the subsequent metabolism of pyruvate. O(2) was not significantly utilized and was not toxic to growth. anNAG3 did not contain detectable membranous or cytoplasmic cytochromes. Nitrate, sulfate, and Fe(III) were not dissimilated. Thus, anNAG3 was characterized as an aerotolerant, non-acetogenic chemoorganotroph with a mixed-fermentative metabolism. The G + C content of the DNA was 37.6 mol%. The similarity of the 16S rRNA gene sequence of anNAG3 to that of its closest phylogenetic relatives (which were in the genera Lactococcus and Streptococcus) approximated 88-89%, indicating that anNAG3 constitutes the type species of a new genus. Based on the collective properties of anNAG3, it is proposed that anNAG3 be termed Lactovum miscens.

N2O-producing microorganisms in the gut of the earthworm Aporrectodea caliginosa are indicative of ingested soil bacteria.

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.

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

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.

Ecological consequences of the phylogenetic and physiological diversities of acetogens.

Acetogens reduce CO2 to acetate via the acetyl-CoA pathway and have been classically thought of as obligately anaerobic bacteria. Nearly 100 acetogenic species from 20 different genera have been isolated to date. These isolates are able to use very diverse electron donors and acceptors, and it is thus very likely that the in situ activities of acetogens are very diverse and not restricted to acetogenesis. Since acetogens constitute a very phylogenetically diverse bacteriological group, it should be anticipated that they can inhabit, and have impact on, diverse habitats. Indeed, they have been isolated from a broad range of habitats, including oxic soils and other habitats not generally regarded as suitable for acetogens. Although the ecological impact of acetogens is determined by the in situ manifestation of their physiological potentials, assessing their in situ activities is difficult due to their physiological and phylogenetic diversities. This mini-review will highlight a few of the physiological and ecological realities of acetogens, and will focus on: (i) metabolic diversities and regulation, (ii) phylogenetic diversity and molecular ecology, and (iii) the capacity of acetogens to cope with oxic conditions under both laboratory and in situ conditions.