Effects of a transient oxic period on mineralization of organic matter to CH4 and CO2 in anoxic peat incubations

Rates of organic matter mineralization in peatlands, and hence production of the greenhouse gases CH₄ and CO₂, are highly dependent on the distribution of oxygen in the peat. Using laboratory incubations of peat, we investigated the sensitivity of the anoxic production of CH₄ and CO₂ to a transient oxic period of a few weeks’ duration. Production rates during 3 successive anoxic periods were compared with rates in samples incubated in the presence of oxygen during the second period. In surface peat (5–10‐cm depth), with an initially high level of CH₄ production, oxic conditions during period 2 did not result in a lower potential CH₄ production rate during period 3, although production was delayed ～1 week. In permanently anoxic, deep peat (50–55‐cm depth) with a comparatively low initial production of CH₄, oxic conditions during period 2 resulted in zero production of CH₄ during period 3. Thus, the methanogens in surface peal—but not in deep peat—remained viable after several weeks of oxic conditions. In contrast to CH₄ production, the oxic period had a negligible effect on anoxic CO₂ production during period 3, in surface as well as deep peat. In both surface and deep peat, CO₂ production was several times higher under oxic than under anoxic conditions. However, for the first 2 weeks of oxic conditions, CO₂ production in the deep peat was very low. Still, deep peat obviously contained facultative microorganisms that, after a relatively short period, were able to maintain a considerably higher rate of organic matter mineralization under oxic than under anoxic conditions.     

Methanogenic archaea and CO2-dependent methanogenesis on washed rice roots

Washed excised roots of rice (Oryza sativa) immediately started to produce CH4 when they were incubated in phosphate buffer under anoxic conditions (N2 atmosphere), with initial rates varying between 2 and 70nmolh(-1)g(-1) dry weight of root material (mean +/- SE: 20.3 +/- 5.9 nmol h(-1) g(-1) dry weight; n = 18). Production of CH4 continued for at least 500 h, with rates usually decreasing slowly. CH4 production was not significantly affected by methyl fluoride, an inhibitor of acetoclastic methanogenesis. Less than 0.5% of added [2-14C]-acetate was converted to 14CH4, and conversion of 14CO2 to 14CH4 indicated that CH4 was almost exclusively produced from CO2. Occasionally, however, especially when the roots were incubated without additional buffer, CH4 production started to accelerate after about 200h reaching rates of > 100 nmol h(-1) g(-1) dry weight. Methyl fluoride inhibited methanogenesis by more than 20% only in these cases, and the conversion of 14CO2 to 14CH4 decreased. These results indicate that CO2-dependent rather than acetoclastic methanogenesis was primarily responsible for CH4 production in anoxically incubated rice roots. Determination of most probable numbers of methanogens on washed roots showed highest numbers (10(6)g(-1) dry roots) on H2 and ethanol, i.e. substrates that support CH4 production from CO2. Numbers on acetate (10(5) g(-1) dry roots) and methanol (10(4)g(-1) dry roots) were lower. Methanogenic consortia enriched on H2 and ethanol were characterized phylogenetically by comparative sequence analysis of archaeal small-subunit (SSU) ribosomal RNA-encoding genes (rDNA). These sequences showed a high similarity to SSU rDNA clones that had been obtained previously by direct extraction of total DNA from washed rice roots. The SSU rDNA sequences recovered from the H2/CO2-using consortium either belonged to a novel lineage of methanogens that grouped within the phylogenetic radiation of the Methanosarcinales and Methanomicrobiales or were affiliated with Methanobacterium bryantii. SSU rDNA sequences retrieved from the ethanol-using consortium either grouped within the genus Methanosarcina or belonged to another novel lineage within the phylogenetic radiation of the Methanosarcinales and Methanomicrobiales. Cultured organisms belonging to either of the two novel lineages have not been reported yet.     

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.     

Effect of inhibition of acetoclastic methanogenesis on growth of archaeal populations in an anoxic model environment

Methyl fluoride is frequently used to specifically inhibit acetoclastic methanogenesis, thus allowing determination of the relative contribution of acetate versus H2/CO2 to total CH4 production in natural environments. However, the effect of the inhibitor on growth of the target archaeal population has not yet been studied. Therefore, we incubated rice roots as an environmental model system under anoxic conditions in the presence and absence of CH3F, measured the activity and Gibbs free energy (DeltaG) of CH4 production, and determined the abundance of individual archaeal populations by using a combination of quantitative (real-time) PCR and analysis of terminal restriction fragment length polymorphism targeting the 16S rRNA gene. It was shown that CH3F specifically inhibited not only acetoclastic methanogenic activity but also the proliferation of Methanosarcina spp, which were the prevalent acetoclastic methanogens in our environmental model system. Therefore, inhibition experiments with CH3F seem to be a suitable method for quantifying acetoclastic CH4 production. It is furthermore shown that the growth and final population size of methanogens were consistent with energetic conditions that at least covered the maintenance requirements of the population.     

Nitrous oxide from aerated dairy manure slurries: Effects of aeration rates and oxic/anoxic phasing

Small-scale laboratory research was conducted to compare the effects of different aeration rates and oxic/anoxic phasing on nitrous oxide (N(2)O) formation from dairy manure slurries. Manure slurry samples were incubated in triplicate for three-weeks under a range of continuous sweep gas flows (0.01-0.23Lmin(-1)kg(-1) slurry) with and without oxygen (air and dinitrogen gas). The net release of N(2)O-N was affected by both aeration rates and oxic/anoxic conditions, whereas ammonia volatilization depended mainly on gas flow rates. Maximum N(2)O-N losses after three-weeks incubation were 4.2% of total slurry N. Major N losses (up to 50% of total slurry N) were caused by ammonia volatilization that increased with increasing gas flow rates. The lowest nitrous oxide and ammonia production was observed from low flow phased oxic/anoxic treatment.     

Microbial carbon monoxide consumption in salt marsh sediments

We have examined sediments from a fringing salt marsh in Maine to further understand marine CO metabolism, about which relatively little is known. Intact cores from the marsh emitted CO during dark oxic incubations, but emission rates were significantly higher during anoxic incubations, which provided evidence for simultaneous production and aerobic consumption in surface sediments. CO emission rates were also elevated when cores were exposed to light, which indicated that photochemical reactions play a role in CO production. A kinetic analysis of marsh surface sediments yielded an apparent K(m) of about 82 ppm, which exceeded values reported for well-aerated soils that consume atmospheric CO (65nM). Surface (0-0.2 cm depth interval) sediment slurries incubated under oxic conditions rapidly consumed CO, and methyl fluoride did not inhibit uptake, which indicated that neither ammonia nor methane oxidizers contributed to the observed activity. In contrast, aerobic CO uptake was inhibited by additions of readily available organic substrates (pyruvate, glucose and glycine), but not by cellulose. CO was also consumed by surface and sub-surface sediment slurries incubated under anaerobic conditions, but rates were less than during aerobic incubations. Molybdate and nitrate or nitrite, but not 2-bromoethanesulfonic acid, partially inhibited anaerobic uptake. These results suggest that sulfidogens and acetogens, but not dissimilatory nitrate reducers or methanogens, actively consume CO. Sediment-free plant roots also oxidized CO aerobically; rates for Spartina patens and Limonium carolinianum roots were significantly higher than rates for Spartina alterniflora roots. Thus plants may also impact CO cycling in estuarine environments.     

Activation of methanogenesis in arid biological soil crusts despite the presence of oxygen

Methanogenesis is traditionally thought to occur only in highly reduced, anoxic environments. Wetland and rice field soils are well known sources for atmospheric methane, while aerated soils are considered sinks. Although methanogens have been detected in low numbers in some aerated, and even in desert soils, it remains unclear whether they are active under natural oxic conditions, such as in biological soil crusts (BSCs) of arid regions. To answer this question we carried out a factorial experiment using microcosms under simulated natural conditions. The BSC on top of an arid soil was incubated under moist conditions in all possible combinations of flooding and drainage, light and dark, air and nitrogen headspace. In the light, oxygen was produced by photosynthesis. Methane production was detected in all microcosms, but rates were much lower when oxygen was present. In addition, the δ(13)C of the methane differed between the oxic/oxygenic and anoxic microcosms. While under anoxic conditions methane was mainly produced from acetate, it was almost entirely produced from H(2)/CO(2) under oxic/oxygenic conditions. Only two genera of methanogens were identified in the BSC-Methanosarcina and Methanocella; their abundance and activity in transcribing the mcrA gene (coding for methyl-CoM reductase) was higher under anoxic than oxic/oxygenic conditions, respectively. Both methanogens also actively transcribed the oxygen detoxifying gene catalase. Since methanotrophs were not detectable in the BSC, all the methane produced was released into the atmosphere. Our findings point to a formerly unknown participation of desert soils in the global methane cycle.     

Anaerobic consumers of monosaccharides in a moderately acidic fen

16S rRNA-based stable isotope probing identified active xylose- and glucose-fermenting Bacteria and active Archaea, including methanogens, in anoxic slurries of material obtained from a moderately acidic, CH(4)-emitting fen. Xylose and glucose were converted to fatty acids, CO(2), H(2), and CH(4) under moderately acidic, anoxic conditions, indicating that the fen harbors moderately acid-tolerant xylose- and glucose-using fermenters, as well as moderately acid-tolerant methanogens. Organisms of the families Acidaminococcaceae, Aeromonadaceae, Clostridiaceae, Enterobacteriaceae, and Pseudomonadaceae and the order Actinomycetales, including hitherto unknown organisms, utilized xylose- or glucose-derived carbon, suggesting that highly diverse facultative aerobes and obligate anaerobes contribute to the flow of carbon in the fen under anoxic conditions. Uncultured Euryarchaeota (i.e., Methanosarcinaceae and Methanobacteriaceae) and Crenarchaeota species were identified by 16S rRNA analysis of anoxic slurries, demonstrating that the acidic fen harbors novel methanogens and Crenarchaeota organisms capable of anaerobiosis. Fermentation-derived molecules are conceived to be the primary drivers of methanogenesis when electron acceptors other than CO(2) are absent, and the collective findings of this study indicate that fen soils harbor diverse, acid-tolerant, and novel xylose-utilizing as well as glucose-utilizing facultative aerobes and obligate anaerobes that form trophic links to novel moderately acid-tolerant methanogens.     

Effect of temperature on anaerobic ethanol oxidation and methanogenesis in acidic peat from a northern wetland

The effects of temperature on rates and pathways of CH4 production and on the abundance and structure of the archaeal community were investigated in acidic peat from a mire in northern Scandinavia (68 degrees N). We monitored the production of CH4 and CO2 over time and measured the turnover of Fe(II), ethanol, and organic acids. All experiments were performed with and without specific inhibitors (2-bromoethanesulfonate [BES] for methanogenesis and CH3F for acetoclastic methanogenesis). The optimum temperature for methanogenesis was 25 degrees C (2.3 micromol CH4.g [dry weight](-1) . day(-1)), but the activity was relatively high even at 4 degrees C (0.25 micromol CH4. g [dry weight](-1) . day(-1)). The theoretical lower limit for methanogenesis was calculated to be at -5 degrees C. The optimum temperature for growth as revealed by real-time PCR was 25 degrees C for both archaea and bacteria. The population structure of archaea was studied by terminal restriction fragment length polymorphism analysis and remained constant over a wide temperature range. Hydrogenotrophic methanogenesis accounted for about 80% of the total methanogenesis. Most 16S rRNA gene sequences that were affiliated with methanogens and all McrA sequences clustered with the exclusively hydrogenotrophic order Methanobacteriales, correlating with the prevalence of hydrogenotrophic methanogenesis. Fe reduction occurred parallel to methanogenesis and was inhibited by BES, suggesting that methanogens were involved in Fe reduction. Based upon the observed balance of substrates and thermodynamic calculations, we concluded that the ethanol pool was oxidized to acetate by the following two processes: syntrophic oxidation with methanogenesis (i) as an H2 sink and (ii) as a reductant for Fe(III). Acetate accumulated, but a considerable fraction was converted to butyrate, making volatile fatty acids important end products of anaerobic metabolism.     

Methane, oxygen, photosynthesis, rubisco and the regulation of the air through time

Rubisco I's specificity, which today may be almost perfectly tuned to the task of cultivating the global garden, controlled the balance of carbon gases and O(2) in the Precambrian ocean and hence, by equilibration, in the air. Control of CO(2) and O(2) by rubisco I, coupled with CH(4) from methanogens, has for the past 2.9 Ga directed the global greenhouse warming, which maintains liquid oceans and sustains microbial ecology.Both rubisco compensation controls and the danger of greenhouse runaway (e.g. glaciation) put limits on biological productivity. Rubisco may sustain the air in either of two permissible stable states: either an anoxic system with greenhouse warming supported by both high methane mixing ratios as well as carbon dioxide, or an oxygen-rich system in which CO(2) largely fulfils the role of managing greenhouse gas, and in which methane is necessarily only a trace greenhouse gas, as is N(2)O. Transition from the anoxic to the oxic state risks glaciation. CO(2) build-up during a global snowball may be an essential precursor to a CO(2)-dominated greenhouse with high levels of atmospheric O(2). Photosynthetic and greenhouse-controlling competitions between marine algae, cyanobacteria, and terrestrial C3 and C4 plants may collectively set the CO(2) : O(2) ratio of the modern atmosphere (last few million years ago in a mainly glacial epoch), maximizing the productivity close to rubisco compensation and glacial limits.     

Functional-Structural Analysis of Nitrogen-Cycle Bacteria in a Hypersaline Mat from the Omani Desert

Anaerobic microbial activity in northern peat soils most often results in more carbon dioxide (CO ₂ ) production than methane (CH₄) production. This study examined why methanogenic conditions (i.e., equal molar amounts of CH₄ production and CO₂ production) prevail so infrequently. We used peat soils from two ombrotrophic bogs and from two rheotrophic fens. The former two represented a relatively dry bog hummock and a wet bog hollow, and the latter two represented a forested fen and a sedge-dominated fen. We quantified gas production rates in soil samples incubated in vitro with and without added metabolic substrates (glucose, ethanol, H₂/CO₂). None of the peat soils exhibited methanogenic conditions when incubated in vitro for a short time (< 5 days) and without added substrates. Incubating some samples > 50 days without added substrates led to methanogenic conditions in only one of four experiments. The anaerobic CO₂:CH₄ production ratio ranged from 5:1 to 40:1 in peat soil without additions and was larger in samples from the dry bog hummock and forested fen than the wet bog hollow and sedge fen. Adding ethanol or glucose separately to peat soils led to methanogenic conditions within 5 days after the addition by stimulating rates of CH₄ production, suggesting CH₄ production from both hydrogenotrophic and acetoclastic methanogenesis. Our results suggest that methanogenic conditions in peat soils rely on a constant supply of easily decomposable metabolic substrates. Sample handling and incubation procedures might obscure methanogenic conditions in peat soil incubated in vitro.     

Abundant Trimethylornithine Lipids and Specific Gene Sequences Are Indicative of Planctomycete Importance at the Oxic/Anoxic Interface in Sphagnum-Dominated Northern Wetlands

Northern wetlands make up a substantial terrestrial carbon sink and are often dominated by decay-resistant Sphagnum mosses. Recent studies have shown that planctomycetes appear to be involved in degradation of Sphagnum-derived debris. Novel trimethylornithine (TMO) lipids have recently been characterized as abundant lipids in various Sphagnum wetland planctomycete isolates, but their occurrence in the environment has not yet been confirmed. We applied a combined intact polar lipid (IPL) and molecular analysis of peat cores collected from two northern wetlands (Saxnäs Mosse [Sweden] and Obukhovskoye [Russia]) in order to investigate the preferred niche and abundance of TMO-producing planctomycetes. TMOs were present throughout the profiles of Sphagnum bogs, but their concentration peaked at the oxic/anoxic interface, which coincided with a maximum abundance of planctomycete-specific 16S rRNA gene sequences. The sequences detected at the oxic/anoxic interface were affiliated with the Isosphaera group, while sequences present in the anoxic peat layers were related to an uncultured planctomycete group. Pyrosequencing-based analysis identified Planctomycetes as the major bacterial group at the oxic/anoxic interface at the Obukhovskoye peat (54% of total 16S rRNA gene sequence reads), followed by Acidobacteria (19% reads), while in the Saxnäs Mosse peat, Acidobacteria were dominant (46%), and Planctomycetes contributed to 6% of the total reads. The detection of abundant TMO lipids in planctomycetes isolated from peat bogs and the lack of TMO production by cultures of acidobacteria suggest that planctomycetes are the producers of TMOs in peat bogs. The higher accumulation of TMOs at the oxic/anoxic interface and the change in the planctomycete community with depth suggest that these IPLs could be synthesized as a response to changing redox conditions at the oxic/anoxic interface.     

Fermentation pattern of methanogenic degradation of rice straw in anoxic paddy soil

The anaerobic degradation of different fractions of rice straw in anoxic paddy soil was investigated. Rice straw was divided up into stem, leaf sheath and leaf blade. The different straw fractions were mixed with paddy soil and incubated under anoxic conditions. Fermentation of straw components started immediately and resulted in transient accumulation of acetate, propionate, butyrate, isobutyrate, valerate, isovalerate and caproate with much higher concentrations in the presence than in the absence of straw. Also some unidentified compounds with UV absorption could be detected. The maximum concentrations of these compounds were different when using different straw fractions, suggesting differences in the degradation pathway of these straw fractions during the early phase of incubation, i.e. with Fe(III) and sulfate serving as oxidants. When concentrations of the intermediates decreased to background values, CH(4) production started. Rates of CH(4)unamended soil. During the methanogenic phase, the percentage contribution of fermentation products to CH(4) production was determined by inhibition with 2-bromoethanesulfonate (BES). Acetate (48-83%) and propionate (18-28%) were found to be the main intermediates of the carbon flow to CH(4), irrespective of the fraction of the rice straw or its absence. Mass balance calculations showed that 84-89% of CH(4) was formed via acetate in the various incubations. Radiotracer experiments showed that 11-27% of CH(4) was formed from H(2)/CO(2), thus confirming that acetate contributed 73-89% to methanogenesis. Our results show that the addition of rice straw and the fraction of the straw affected the fermentation pattern only in the early phase of degradation, but had no effect on the degradation pathway during the later methanogenic phase.     

Soil microorganisms as controllers of atmospheric trace gases (H2, CO, CH4, OCS, N2O, and NO).

Production and consumption processes in soils contribute to the global cycles of many trace gases (CH4, CO, OCS, H2, N2O, and NO) that are relevant for atmospheric chemistry and climate. Soil microbial processes contribute substantially to the budgets of atmospheric trace gases. The flux of trace gases between soil and atmosphere is usually the result of simultaneously operating production and consumption processes in soil: The relevant processes are not yet proven with absolute certainty, but the following are likely for trace gas consumption: H2 oxidation by abiontic soil enzymes; CO cooxidation by the ammonium monooxygenase of nitrifying bacteria; CH4 oxidation by unknown methanotrophic bacteria that utilize CH4 for growth; OCS hydrolysis by bacteria containing carbonic anhydrase; N2O reduction to N2 by denitrifying bacteria; NO consumption by either reduction to N2O in denitrifiers or oxidation to nitrate in heterotrophic bacteria. Wetland soils, in contrast to upland soils are generally anoxic and thus support the production of trace gases (H2, CO, CH4, N2O, and NO) by anaerobic bacteria such as fermenters, methanogens, acetogens, sulfate reducers, and denitrifiers. Methane is the dominant gaseous product of anaerobic degradation of organic matter and is released into the atmosphere, whereas the other trace gases are only intermediates, which are mostly cycled within the anoxic habitat. A significant percentage of the produced methane is oxidized by methanotrophic bacteria at anoxic-oxic interfaces such as the soil surface and the root surface of aquatic plants that serve as conduits for O2 transport into and CH4 transport out of the wetland soils. The dominant production processes in upland soils are different from those in wetland soils and include H2 production by biological N2 fixation, CO production by chemical decomposition of soil organic matter, and NO and N2O production by nitrification and denitrification. The processes responsible for CH4 production in upland soils are completely unclear, as are the OCS production processes in general. A problem for future research is the attribution of trace gas metabolic processes not only to functional groups of microorganisms but also to particular taxa. Thus, it is completely unclear how important microbial diversity is for the control of trace gas flux at the ecosystem level. However, different microbial communities may be part of the reason for differences in trace gas metabolism, e.g., effects of nitrogen fertilizers on CH4 uptake by soil; decrease of CH4 production with decreasing temperature; or different rates and modes of NO and N2O production in different soils and under different conditions.     

Detecting active methanogenic populations on rice roots using stable isotope probing

Methane is formed on rice roots mainly by CO2 reduction. The present study aimed to identify the active methanogenic populations responsible for this process. Soil-free rice roots were incubated anaerobically under an atmosphere of H2/(13CO2) or N2/(13CO2) with phosphate or carbonate (marble) as buffer medium. Nucleic acids were extracted and fractionated by caesium trifluoroacetate equilibrium density gradient centrifugation after 16-day incubation. Community analyses were performed for gradient fractions using terminal restriction fragment polymorphism analysis (T-RFLP) and sequencing of the 16S rRNA genes. In addition, rRNA was extracted and analysed at different time points to trace the community change during the 16-day incubation. The Methanosarcinaceae and the yet-uncultured archaeal lineage Rice Cluster-I (RC-I) were predominant in the root incubations when carbonate buffer and N2 headspace were used. The analysis of [13C]DNA showed that the relative 16S rRNA gene abundance of RC-I increased whereas that of the Methanosarcinaceae decreased with increasing DNA buoyant density, indicating that members of RC-I were more active than the Methanosarcinaceae. However, an unexpected finding was that RC-I was suppressed in the presence of high H2 concentrations (80%, v/v), which during the early incubation period caused a lower CH4 production compared with that with N2 in the headspace. Eventually, however, CH4 production increased, probably because of the activity of Methanosarcinaceae, which became prevalent. Phosphate buffer appeared to inhibit the activity of the Methanosarcinaceae, resulting in lower CH4 production as compared with carbonate buffer. Under these conditions, Methanobacteriaceae were the prevalent methanogens. Our study suggests that the active methanogenic populations on rice roots change in correspondence to the presence of H2 (80%, v/v) and the type of buffer used in the system.     

Turnover of glucose and acetate coupled to reduction of nitrate, ferric iron and sulfate and to methanogenesis in anoxic rice field soil

Turnover of glucose and acetate in the presence of active reduction of nitrate, ferric iron and sulfate was investigated in anoxic rice field soil by using [U-(14)C]glucose and [2-(14)C]acetate. The turnover of glucose was not much affected by addition of ferrihydrite or sulfate, but was partially inhibited (60%) by addition of nitrate. Nitrate addition also strongly reduced acetate production from glucose while ferrihydrite and sulfate addition did not. These results demonstrate that ferric iron and sulfate reducers did not outcompete fermenting bacteria for glucose at endogenous concentrations. Nitrate reducers may have done so, but glucose fermentation may also have been inhibited by accumulation of toxic denitrification intermediates (nitrite, NO, N(2)O). Addition of nitrate resulted in complete inhibition of CH(4) production from [U-(14)C]glucose and [2-(14)C]acetate. However, addition of ferrihydrite or sulfate decreased the production of (14)CH(4) from [U-(14)C]glucose by only 70 and 65%, respectively. None of the electron acceptors significantly increased the production of (14)CO(2) from [U-(14)C]glucose, but all increased the production of (14)CO(2) from [2-(14)C]acetate. Uptake of acetate was faster in the presence of either nitrate, ferrihydrite or sulfate than in the unamended control. Addition of ferrihydrite and sulfate reduced (14)CH(4) production from [2-(14)C]acetate by 83 and 92%, respectively. Chloroform completely inhibited the methanogenic consumption of acetate. It also inhibited the oxidation of acetate, completely in the presence of sulfate, but not in the presence of nitrate or ferrihydrite. Our results show that, besides the possible toxic effect of products of nitrate reduction (NO, NO(2)(-) and N(2)O) on methanogens, nitrate reducers, ferric iron reducers and sulfate reducers were active enough to outcompete methanogens for acetate and channeling the flow of electrons away from CH(4) towards CO(2) production.     

Activity, structure and dynamics of the methanogenic archaeal community in a flooded Italian rice field

Methane production was studied in an Italian rice field over two consecutive years (1998, 1999) by measuring the rates of total and acetate-dependent methanogenesis in soil and root samples. Population dynamics of methanogens were followed by terminal restriction fragment length polymorphism and real-time PCR targeting archaeal SSU rRNA genes. Rates of total and acetate-dependent methanogenesis in soil increased during the season, reached a maximum at about 70-80 days after flooding and then decreased again. In contrast, the size of the archaeal community remained relatively constant. Therefore, the seasonal changes in the methanogenic processes were probably not caused by changes in the size of the methanogenic community but in its activity. During the 1998/1999 winter period, a slight decrease in archaeal cell numbers was found. In both years, the dominant groups were methanogens affiliated with Rice cluster I, Methanosaetaceae, Methanosarcinaceae and Methanobacteriaceae. Correspondence analysis showed, however, that the archaeal community structure was different in 1998 and 1999. Methanogens with potential acetoclastic activity made up a larger fraction of the total archaeal community in 1999 (32-53%) than in 1998 (20-32%). Furthermore, the frequency of Methanosaetaceae relative to Methanosarcinaceae was significantly higher in 1999 than in 1998. This difference could be explained by the much lower soil acetate concentrations in 1999, to which Methanosaetaceae are physiologically better adapted than Methanosarcinaceae. Over the season, however, the composition of the archaeal community remained relatively constant and thus did not reflect the observed seasonal change in CH(4) production activity. The analysis of rice root samples in 1999 showed that the archaeal community structure on the roots was similar to that in soil but with acetoclastic methanogens being relatively less common. This observation is in agreement with domination of CH(4) production by H(2)/CO(2)-dependent methanogenesis on roots. Our study provided a link between size, structure and function of the methanogenic community in an Italian rice field.     

Phosphate inhibits acetotrophic methanogenesis on rice roots

The contribution of acetate- and H(2)/CO(2)-dependent methanogenesis to total CH(4) production was determined in excised washed rice roots by radiolabeling, methyl fluoride inhibition, and stable carbon isotope fractionation. Addition of > or = 20 mM phosphate inhibited methanogenesis, which then was exclusively from H(2)/CO(2). Otherwise, acetate contributed about 50 to 60% of the total methanogenesis, demonstrating that phosphate specifically inhibited acetotrophic methanogens on rice roots.     

Temperature Dependence of Methane Production in Tropical Rice Soils

Although CH 4 production is sensitive to temperature, it is not clear how temperature controls CH 4 production directly versus the production of organic substrates that methanogens convert into CH 4 . Therefore, this study was done to better understand how CH 4 production in rice paddy soil responded to temperature when the process was not limited by the availability of substrates. In a laboratory-incubation study using three Indian rice soils under flooded conditions, the effect of temperature on CH 4 production was examined. CH 4 production in acid sulphate, laterite, and alluvial soil samples under flooded conditions distinctly increased with increase in temperature from 15°C to 35°C. Laterite and acid sulphate soils produced distinctly less CH 4 than alluvial soils. CO 2 production increased with increase in temperature in all the soils. The readily mineralizable carbon C and Fe 2+ contents in soils were least at 15°C and highest at 35°C, irrespective of soil type. Likewise, a significant correlation existed between microbial population (methanogens and sulphate reducers) and CH 4 production. Comparing the temperature coefficients ( Q 10 ) for methane production within each soil type at low (15°C-25°C) and medium (25°C-35°C) temperature intervals revealed that these values were not uniform for both alluvial and laterite soils. But acid sulphate soil had Q 10 values that were near 2 at both temperature intervals. When these soil samples were amended with substrates (acetate, H 2 -CO 2 , and rice straw), there were stimulatory effects on methane production rates and consequently on the Q 10 values. The pattern of temperature coefficients was characteristic of the soil type and the nature of substrates used for amendment.     

Thermoanaerobacteriaceae oxidize acetate in methanogenic rice field soil at 50°C

Rice field soils contain a thermophilic microbial community. Incubation of Italian rice field soil at 50°C resulted in transient accumulation of acetate, but the microorganisms responsible for methane production from acetate are unknown. Without addition of exogenous acetate, the δ(13)C of CH(4) and CO(2) indicated that CH(4) was exclusively produced by hydrogenotrophic methanogenesis. When exogenous acetate was added, acetoclastic methanogenesis apparently also operated. Nevertheless, addition of [2-(13)C]acetate (99% (13)C) resulted in the production not only of (13)C-labelled CH(4) but also of CO(2), which contained up to 27% (13)C, demonstrating that the methyl group of acetate was also oxidized. Part of the (13)C-labelled acetate was also converted to propionate which contained up to 14% (13)C. The microorganisms capable of assimilating acetate at 50°C were targeted by stable isotope probing (SIP) of ribosomal RNA and rRNA genes using [U-(13)C] acetate. Using quantitative PCR, (13)C-labelled bacterial ribosomal RNA and DNA was detected after 21 and 32 days of incubation with [U-(13)C]acetate respectively. In the heavy fractions of the (13)C treatment, terminal restriction fragments (T-RFs) of 140, 120 and 171 bp length predominated. Cloning and sequencing of 16S rRNA showed that these T-RFs were affiliated with the bacterial genera Thermacetogenium and Symbiobacterium and with members of the Thermoanaerobacteriaceae. Similar experiments targeting archaeal RNA and DNA showed that Methanocellales were the dominant methanogens being consistent with the operation of syntrophic bacterial acetate oxidation coupled to hydrogenotrophic methanogenesis. After 17 days, however, Methanosarcinacea increasingly contributed to the synthesis of rRNA from [U-(13)C]acetate indicating that acetoclastic methanogens were also active in methanogenic Italian rice field soil under thermal conditions.