Carola Matthies

Hydrogenotrophic Methanogenesis by Moderately Acid-Tolerant Methanogens of a Methane-Emitting Acidic Peat

ABSTRACT The emission of methane (1.3 mmol of CH4 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 CH4 (0.4 to 1.7 μmol g [dry wt] of soil−1 day−1) under anoxic conditions. At in situ pH, supplemental H2-CO2, ethanol, and 1-propanol all increased CH4 production rates while formate, acetate, propionate, and butyrate inhibited the production of CH4; methanol had no effect. H2-dependent acetogenesis occurred in H2-CO2-supplemented bog peat only after extended incubation periods. Nonsupplemented bog peat initially produced small amounts of H2 that were subsequently consumed. The accumulation of H2 was stimulated by ethanol and 1-propanol or by inhibiting methanogenesis with bromoethanesulfonate, and the consumption of ethanol was inhibited by large amounts of H2; these results collectively indicated that ethanol- or 1-propanol-utilizing bacteria were trophically associated with H2-utilizing methanogens. A total of 109 anaerobes and 107 hydrogenotrophic methanogens per g (dry weight) of bog peat were enumerated by cultivation techniques. A stable methanogenic enrichment was obtained with an acidic, H2-CO2-supplemented, fatty acid-enriched defined medium. CH4 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 CH4-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 H2 that could be used by hydrogenotrophic methanogens. These results indicated that in this acidic bog peat, (i) H2 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.

N2O-Producing Microorganisms in the Gut of the Earthworm Aporrectodea caliginosa Are Indicative of Ingested Soil Bacteria

ABSTRACT 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 (N2O) and (ii) to characterize the N2O-producing bacteria of the earthworm gut. The production of N2O 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 N2O were transient products when supplemental nitrate was reduced to N2 by gut content homogenates. In contrast, nitrite and N2O 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 N2O-producing isolates that reduced either nitrate or nitrite were obtained from high serial dilutions of gut homogenates. Of the 25 representative N2O-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 N2O or N2. The initial N2O 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 N2O via nitrite-utilizing denitrifiers in the gut. The results of this study suggest that most of the N2O emitted by earthworms is due to the activation of ingested denitrifiers and other nitrate-dissimilating bacteria in the gut lumen.

Clostridium akagii sp. nov. and Clostridium acidisoli sp. nov. : acid-tolerant, N2-fixing clostridia isolated from acidic forest soil and litter

Two anaerobic acid-tolerant bacteria, CK58T and CK74T, were isolated from acidic beech litter and acidic peat-bog soil, respectively. Both bacteria were spore-forming, motile rods with peritrichous flagella. The capacity to sporulate decreased with prolonged cultivation. Cells of CK58T formed chains or aggregates and were linked by a connecting filament that consisted of a core and a surrounding sheath. Cellobiose, glucose, xylose, arabinose, maltose, mannose and salicin supported growth of CK58T. These substrates, as well as mannitol, lactose, sucrose, glycerol, melezitose, raffinose and rhamnose, supported growth of CK74T. Sorbitol, trehalose, H2/CO2, CO/CO2, vanillate, Casamino acids, peptone, and various purines and pyrimidines did not support the growth of either organism. Growth of CK58T and CK74T on glucose yielded butyrate, lactate, acetate, formate, H2 and CO2 as end products. Growth of CK58T and CK74T was observed at pH 3.7-7.1 and 3.6-6.9, respectively. CK58T and CK74T grew in nitrogen-free medium at pH 3.7 under an N2 atmosphere and reduced acetylene at rates approximating 1 nmol min-1 (mg protein)-1. CK58T and CK74T did not contain carbon monoxide dehydrogenase or cytochromes, produce methane, or dissimilate nitrate or sulfate. Thus, CK58T and CK74T were characterized as nonacetogenic, N2-fixing, fermentative chemo-organotrophs. The G + C contents of CK58T and CK74T were 31.4 and 30.7 mol%, respectively. CK58T and CK74T were phylogenetically most closely related to Clostridium pasteurianum. The 16S rRNA gene sequence similarity values of CK58T and CK74T to C. pasteurianum and each other did not exceed 96.5%, and it is proposed that strains CK58T and CK74T be named Clostridium akagii CK58T (DSM 12554T) and Clostridium acidisoli CK74T (DSM 12555T), respectively. These results suggest that previously uncharacterized clostridial species reside and might fix N2 in the annoxic microzones of acidic forest soil and litter.

Fumarate dissimilation and differential reductant flow by Clostridium formicoaceticum and Clostridium aceticum

Methanol and the O-methyl group of vanillate did not support the growth of Clostridium formicoaceticum in defined medium under CO2-limited conditions; however, they were growth supportive when fumarate was provided concomitantly. Fumarate alone was not growth supportive under these conditions. Fumarate reduction (dissimilation) to succinate was the predominant electron-accepting, energy-conserving process for methanol-derived reductant under CO2-limited conditions. However, when both reductant sinks, i.e., fumarate and CO2, were available, reductant was redirected towards CO2 in defined medium. In contrast, in undefined medium with both reductant sinks available, C. formicoaceticum simultaneously engaged fumarate dismutation and the concomitant usage of CO2 and fumarate as reductant sinks. With Clostridium aceticum, fumarate also substituted for CO2, and H2 became growth supportive under CO2-limited conditions. Fumarate dissimilation was the predominant electron-accepting process under CO2-limited conditions; however, when both reductant sinks were available, H2-derived reductant was routed towards CO2, indicating that acetogenesis was the preferred electron-accepting process when reductant flow originated from H2. Collectively, these findings indicate that fumarate dissimilation, not dismutation, is selectively used under certain conditions and that such usage of fumarate is subject to complex regulation.

Clostridium uliginosum sp. nov., a novel acid- tolerant, anaerobic bacterium with connecting filaments

An anaerobic, acid-tolerant bacterium, CK55T, was isolated from an acidic forest bog. Cells of CK55T stained Gram-negative but did not have an outer membrane. Cells were spore-forming, motile rods with peritrichous flagella, formed chains or aggregates and were linked by connecting filaments that were composed of a core and outer sheath. Cellobiose, glucose, xylose, mannose, mannitol, sucrose and peptone supported growth. Arabinose, lactose, raffinose, H2/CO2, CO/CO2, vanillate, Casamino acids and various purines and pyrimidines did not support growth. Growth on carbohydrates yielded acetate, butyrate, lactate, formate and H2 as end-products. Growth was observed at pH 4.0-9.0, with an optimum at pH 6.5, and at 10-30 degrees C, with an optimum at 20-25 degrees C. At 20 degrees C, doubling times were 4 and 6 h at pH 6.5 and 4.0, respectively. Hydrogenase activity in cell-free extracts was 12 U (mg protein)-1. CK55T did not: (i) contain detectable levels of CO, formate, lactate dehydrogenases or cytochromes; (ii) carry out dissimilatory reduction of nitrate or sulfate; or (iii) produce methane. Thus, CK55T was characterized as a non-acetogenic, fermentative chemo-organotroph. The G+C content of CK55T was 28.0 mol%. CK55T was phylogenetically most closely related to Clostridium botulinum (types B, E and F), Clostridium acetobutylicum and other saccharolytic clostridia; the 16S rRNA gene sequence similarity values to the nearest relatives of CK55T were approximately 97%. Based on morphological, physiological and phylogenetic properties of CK55T, it is proposed that CK55T be termed Clostridium uliginosum sp. nov. (= DSM 12992T = ATCC BAA-53T).

Pelospora glutarica gen. nov., sp. nov., a glutarate-fermenting, strictly anaerobic, spore-forming bacterium

The strictly anaerobic, Gram-negative, spore-forming bacterium strain WoGl3T had been enriched and isolated in mineral medium with glutarate as the sole source of energy and organic carbon. Glutarate was fermented to a mixture of butyrate, isobutyrate, CO2 and small amounts of acetate. Strain WoGl3T grew only with the dicarboxylates glutarate, methylsuccinate and succinate. 16S rDNA sequence analysis revealed an affiliation of strain WoGl3T to the family Syntrophomonadaceae. This monophyletic group is comprised of strain WoGl3T and the genera Syntrophomonas, Syntrophospora and Thermosyntropha, within the phylum of Gram-positive bacteria with a low DNA G + C content. Overall intra-group 16S rRNA sequence similarities of 89.2-93.9% document a separate phylogenetic status for strain WoGl3T. Strain WoGl3T (= DSM 6652T) is described as the type strain of a new species within a new genus, Pelospora glutarica gen. nov., sp. nov.

Acetogenesis: Reality in the Laboratory, Uncertainty Elsewhere

This chapter focuses on recent work in our research group that further extends our awareness of the diverse metabolic potentials of acetogens and, consequently, broadens our uncertainty in making accurate predictions of the role acetogens actually play at the ecosystem level (i.e., “elsewhere” per the title of this chapter). Without debating what ecosystems are, acetogens are difficult to study in their natural habitat. This difficulty stems largely from the fact that the main product we think they make (i.e., acetate) is not easily assessed (a gaseous product minimizes this complication) and likely turns over rapidly in vivo. Likewise, many of the substrates they may consume are also problematic to assess. In addition, approaches such as the [3H]thymidine incorporation method to assess the productivity of acetogens may greatly underestimate their magnitude (Winding, 1992; Wellsbury et al., 1993). Thus, although enrichment and physiological studies have been somewhat elegant in recent years relative to defining acetogenic potentials in the laboratory, comparatively little is known about what they really do “elsewhere” (as emphasized in Chapter 7). Clearly, native ecosystems such as forests have little in common with test-tube cultures. In the present chapter and those that follow in Part IV these realities and uncertainties are addressed.

Metabolism of aromatic aldehydes as cosubstrates by the acetogen Clostridium formicoaceticum

When the acetogen Clostridium formicoaceticum was cultivated on mixtures of aromatic compounds (e.g., 4-hydroxybenzaldehyde plus vanillate), the oxidation of aromatic aldehyde groups occurred more rapidly than did O-demethylation. Likewise, when fructose and 4-hydroxybenzaldehyde were simultaneously provided as growth substrates, fructose was utilized only after the aromatic aldehyde group was oxidized to the carboxyl level. Aromatic aldehyde oxidoreductase activity was constitutive (activities approximated 0.8 U mg–1), and when pulses of 4-hydroxybenzaldehyde were added during fructose-dependent growth, the rate at which fructose was utilized decreased until 4-hydroxybenzaldehyde was consumed. Although 4-hydroxybenzaldehyde inhibited the capacity of cells to metabolize fructose, lactate or gluconate were consumed simultaneously with 4-hydroxybenzaldehyde, and lactate or aromatic compounds lacking an aldehyde group were utilized concomitantly with fructose. These results demonstrate that (1) aromatic aldehydes can be utilized as cosubstrates and have negative effects on the homoacetogenic utilization of fructose by C. formicoaceticum, and (2) the consumption of certain substrates by this acetogen is not subject to catabolite repression by fructose.