Methane production potential and methanogenic archaeal community structure in tropical irrigated Indian paddy soils

Soil characteristics regulate various belowground microbial processes including methanogenesis and, consequently, affect the structure and function of methanogenic archaeal communities due to change in soil type which in turn influences the CH₄ production potential of soils. Thus, five different soil orders (Alfisol, Entisol, Inceptisol, Podzol and Vertisol) were studied to assess their CH₄ production potential and also the methanogenic archaeal community structure in dryland irrigated Indian paddy soils. Soil incubation experiments revealed CH₄ production to range from 178.4 to 431.2 μg CH₄ g^-1 dws in all soil orders as: Vertisol<Inceptisol<Entisol<Podzol<Alfisol. The numbers of methanogens as quantified using real-time quantitative polymerase chain reaction (qPCR) targeting mcrA genes varied between 0.06 and 72.97 (×10⁶ copies g^-1 dws) and were the highest in Vertisol soil and the least in Alfisol soil. PCR-denaturing gradient gel electrophoresis (DGGE)-based approach targeting 16S rRNA genes revealed diverse methanogenic archaeal communities across all soils. A total of 43 DGGE bands sequenced showed the closely related groups to Methanomicrobiaceae, Methanobacteriaceae, Methanocellales, Methanosarcinaceae, Methanosaetaceae and Crenarchaeota. The composition of methanogenic groups differed among all soils and only the Methanocellales group was common and dominant in all types of soils. The highest diversity of methanogens was found in Inceptisol and Vertisol soils. Methane production potential varied significantly in different soil orders with a positive relationship (p?<?0.05) with methanogens population size, permanganate oxidizable C (POXC) and CO₂ production. The present study suggested that CH₄ production potential of different soils depends on physicochemical properties, methanogenic archaeal community composition and the population size.     

Dynamics of the methanogenic archaeal community in anoxic rice soil upon addition of straw

Addition of rice straw, which is a common practice in rice agriculture, generally results in enhanced production and emission of the greenhouse gas methane (CH₄). However, it is unclear whether straw addition affects only the activity or also the composition of the methanogenic microbial community. It is also unclear to what extent methanogenic archaea would be able to proliferate in the soil. Anoxic slurries of Italian rice‐field soil produced CH₄ after a lag, during which ferric iron and sulfate were reduced. Addition of rice straw slightly decreased this lag and greatly enhanced the subsequent production of CH₄. At the same time, addition of rice straw enhanced the intermediate production of H₂ and acetate that served as the methanogenic substrates. Compared with the unamended control, the addition of rice straw resulted in an increased concentration of phospholipid fatty acids in the soil. Quantitative ‘real‐time’ PCR targeting the 16S rRNA gene also showed increased copy numbers of both Bacteria and Archaea in the straw‐amended soil at the end of the experiment. The composition of the archaeal community was followed over time by terminal restriction length polymorphism (T‐RFLP) analysis of the archaeal 16S rRNA genes extracted from straw‐amended soil and the control. Rice Cluster‐I (RC‐I) methanogens and Methanosarcinaceae were the most abundant methanogenic populations, followed by Methanobacteriales, Methanomicrobiales and Methanosaetaceae. Addition of rice straw resulted in a relative increase of Methanosarcinaceae and Methanobacteriales and a relative decrease of RC‐I methanogens and Methanomicrobiales. Our results revealed a dynamic methanogenic community in anoxic rice‐field soil and showed that addition of organic matter selectively enhanced the growth of particular methanogenic populations, which were apparently better adapted to the presence of straw than the others. The extent of archaeal growth was consistent with that expected theoretically from the ambient Gibbs free energies of hydrogenotrophic and acetoclastic methanogenesis.     

Activity and composition of the methanogenic archaeal community in soil vegetated with wild versus cultivated rice

Wild rice (Oryza rufipogon) is a problematic weed in fields of cultivated rice (Oryza sativa). We hypothesized that the composition and/or the activity of the methanogenic microbial communities might be different in soil grown with cultivated versus wild rice. We used samples from Hainan, China, where wild rice grew on a field adjacent to cultivated rice. The composition of the methanogenic archaeal community was analyzed in samples of rice soil by targeting the 16S rRNA gene. Analysis of the terminal restriction fragment length polymorphism (T-RFLP) showed similar patterns in soil from wild versus cultivated rice. Sequences of archaeal 16S rRNA genes also showed similar composition in soil from wild versus cultivated rice, revealing the presence of Methanosarcinaceae, Methanosaetaceae, Methanobacteriales, Methanocellales (Rice Cluster I), Rice Cluster II, Crenarchaeota Group I.3 and Crenarchaeota Group I.1b. Incubation of soil samples under anoxic conditions generally resulted in vigorous CH₄ production after a lag phase of 7-8 days. Production of CH₄ was partially inhibited by methyl fluoride, a specific inhibitor of acetoclastic methanogenesis, resulting in nearly stoichiometric accumulation of acetate. CO₂ was produced without lag phase. The δ¹³C of the produced CO₂ was slightly lower in soil grown with cultivated rice versus wild rice, reflecting the δ¹³C of organic matter, which was about −29‰ for cultivated rice soil and about −24‰ for wild rice soil. The δ¹³C of the produced CH₄ and the acetate that accumulated in the presence of CH₃F was much more negative in cultivated versus wild rice soil, mainly since the isotopic fractionation factors for hydrogenotrophic methanogenesis were higher for soil from cultivated rice (α = 1.054) versus wild rice (α = 1.039). However, the percentage contribution of hydrogenotrophic methanogenesis to total CH₄ production was similar in both soils (27-35%). In conclusion, although the two soils exhibited different δ¹³C values of soil organic matter and derived products, they were similar with respect to rates and composition of the methanogenic communities.     

Mechanisms of biochar decreasing methane emission from Chinese paddy soils

Paddy fields are one of the largest anthropogenic sources of global CH₄ emission. A decrease in paddy CH₄ emission can contribute significantly towards the control of global warming. Recent studies have demonstrated that the application of biochar in paddy soils has such a capability, but its underlying mechanism has yet to be elucidated. In this investigation, we studied CH₄ emission, methanogenic archaeal, as well as methanotrophic proteobacterial communities, from microcosms derived from two paddy soils, Inceptisol and Ultisol. Both soils were amended with biochar at different pyrolysis temperatures (300 °C, 400 °C and 500 °C) at field condition. The soil CH₄ flux was monitored across whole rice season in 2010; the functional guilds communities were analyzed by PCR–DGGE and real-time quantitative PCR (qPCR). It is found that paddy CH₄ emissions significantly decreased under biochar amendments, which, interestingly, didn't result from the inhibition of methanogenic archaeal growth. qPCR further revealed that biochar amendments (1) increased methanotrophic proteobacterial abundances significantly, and (2) decreased the ratios of methanogenic to methanotrophic abundances greatly. These results shed insight on the underlying mechanism of how biochar decreases paddy CH₄ emission. This knowledge can be applied to develop a more effective greenhouse gas mitigation process for paddy fields.     

Temporal variation in methanogenic community structure and methane production potential of tropical rice ecosystem

Temporal variations in diversity of methanogenic community and CH₄ production potential were analyzed in an Indian tropical rice ecosystem. Laboratory incubations showed that methane production varied from 20.86 to 134.11 μg CH₄ g⁻¹ d.w.s. during the two consecutive years, 2009 and 2010. CH₄ production potential was high at the flowering stage of the rice crop followed by ripening, tillering, post-harvest and pre-plantation stage. Phylogenetic analysis of 16S rRNA genes of methanogenic community indicated that flowering and ripening stages comprised of Methanomicrobiaceae, Methanosarcinaceae, Methanosaetaceae and RC I methanogenic groups, while only the members of Methanomicrobiaceae and RC I were present in the remaining stages. Further, the dominance of RC I was observed in all stages. This study demonstrates that flowering and ripening stages of rice crop offer relatively favorable ecological niche for methanogenic community. The overall analyses suggest that the temporal change in diversity of methanogens regulates CH₄ production potential in rice field soils.     

Dung application increases CH<Subscript>4</Subscript> production potential and alters the composition and abundance of methanogen community in restored peatland soils from Europe

Peatland restoration via rewetting aims to recover biological communities and biogeochemical processes typical to pristine peatlands. While rewetting promotes recovery of C accumulation favorable for climate mitigation, it also promotes methane (CH₄) emissions. The potential for exceptionally high emissions after rewetting has been measured for Central European peatland sites previously grazed by cattle. We addressed the hypothesis that these exceptionally high CH₄ emissions result from the previous land use. We analyzed the effects of cattle dung application to peat soils in a short- (2 weeks), a medium- (1 year) and a long-term (grazing) approach. We measured the CH₄ production potentials, determined the numbers of methanogens by mcrA qPCR, and analyzed the methanogen community by mcrA T-RFLP-cloning-sequencing. Dung application significantly increased the CH₄ production potential in the short- and the medium-term approach and non-significantly at the cattle-grazed site. The number of methanogens correlated with the CH₄ production in the short- and the long-term approach. At all three time horizons, we found a shift in methanogen community due to dung application and a transfer of rumen methanogen sequences (Methanobrevibacter spp.) to the peatland soil that seemed related to increased CH₄ production potential. Our findings indicate that cattle grazing of drained peatlands changes their methanogenic microbial community, may introduce rumen-associated methanogens and leads to increased CH₄ production. Consequently, rewetting of previously cattle-grazed peatlands has the potential to lead to increased CH₄ emissions. Careful consideration of land use history is crucial for successful climate mitigation with peatland rewetting.     

Saccharolytic activity and its role as a limiting step in methane formation during the anaerobic degradation of rice straw in rice paddy soil

Rice straw polysaccharides are one of the major C sources for CH₄ formation in anoxic rice paddy soils. We investigated the initial step of straw degradation by measuring the substrate-saturated activities of the polysaccharolytic enzymes #-glucosidase, exo-#-1,4-glucanase and xylosidase using substrates labelled with methylumbelliferone (MUF). The actual activity of the enzymes was measured by the release of reducing sugars after the inhibition of microbial carbohydrate uptake by toluene. The substrate-saturated enzyme activities increased during the first 11 days of incubation, while the actual activities decreased, presumably due to the decreasing access of straw polysaccharides to hydrolytic enzymes. The temporal progress of polysaccharide hydrolysis, transient accumulation of fermentation products and CH₄ production indicated five distinct phases. In phase I (<8 h), the fermentation of sugar monomers released by hydrolysis of polysaccharides was limiting. In phase II (2 and acetate accumulated since the activity of methanogens was low, though increasing exponentially. In phase III (days 3-10), H₂ was also consumed by respiratory processes (e.g. SO₄^2- reduction) so that H₂-dependent methanogenesis sometimes became substrate limited. However, acetate still accumulated, probably due to the limiting activity of acetoclastic methanogens. In phase IV (days 10-18), methanogenic activity was no longer limited and acetate was depleted to low concentrations. In phase V (>day 18), the methanogenic degradation of straw reached a quasi-steady state, when polysaccharide hydrolysis became the rate-limiting step for CH₄ formation.     

间隙灌溉和控释肥施用对稻田土壤产甲烷微生物的影响

间隙灌溉和控释肥施用影响稻田CH_4的产生和排放,然而其微生物机理尚不清楚。本研究通过采集稻季田间原位试验新鲜土样,采用核酸定量技术(qPCR)和末端限制性片段长度多态性(T-RFLP)技术,研究间隙灌溉和控释肥施用对稻田土壤产甲烷微生物群落丰度和结构的影响。结果表明,稻季CH_4排放量与古菌、产甲烷菌(mcr A基因)和甲烷氧化菌(pmo A基因)数量均呈极显著正相关关系(P0.01),而与细菌数量无显著相关性。间隙灌溉显著影响产甲烷菌和甲烷氧化菌数量的季节变化,其中烤田抑制产甲烷菌生长,而对甲烷氧化菌数量没有显著影响。与尿素相比,施用控释肥增加了稻田土壤细菌、古菌和产甲烷菌数量,降低了甲烷氧化菌数量。土壤古菌群落的优势T-RFs片段为184bp和391bp,其中184bp片段的相对丰度随着间隙灌溉的进行由45%~55%降低到23%~30%;而391bp片段则相反,其相对丰度由12%~18%增加到23%~26%。典型相关性分析(CCA)表明间隙灌溉显著影响土壤古菌群落结构(P0.001),而控释肥施用对土壤古菌群落结构没有明显影响。     

Long-term submergence of non-methanogenic oxic upland field soils helps to develop the methanogenic archaeal community as revealed by pot and field experiments

The community structure of methanogenic archaea is relatively stable,i.e.,it is sustained at a high abundance with minimal changes in composition,in paddy field soils irrespective of submergence and drainage.In contrast,the abundance in non-methanogenic oxic soils is much lower than that in paddy field soils.This study aimed to describe methanogenic archaeal community development following the long-term submergence of non-methanogenic oxic upland field soils in pot and field experiments.In the pot experiment,a soil sample obtained from an upland field was incubated under submerged conditions for 275 d.Soil samples periodically collected were subjected to culture-dependent most probable number(MPN)enumeration,polymerase chain reaction-denaturing gradient gel electrophoresis(PCR-DGGE)analysis of archaeal 16 S r RNA gene,and quantitative PCR analysis of the methyl-coenzyme M reductase alpha subunit gene(mcr A)of methanogenic archaea.The abundance of methanogenic archaea increased from 102 to 103 cells g-1 dry soil and 104 to 107 copies of mcr A gene g-1 dry soil after submergence.Although no methanogenic archaeon was detected prior to incubation by the DGGE analysis,members from Methanocellales,Methanosarcinaceae,and Methanosaetaceae proliferated in the soils,and the community structure was relatively stable once established.In the field experiment,the number of viable methanogenic archaea in a rice paddy field converted from meadow(reclaimed paddy field)was monitored by MPN enumeration over five annual cycles of field operations.Viability was also determined simultaneously in a paddy field where the plow layer soil from a farmer’s paddy field was dressed onto the meadow(dressed paddy field)and an upland crop field converted from the meadow(reclaimed upland field).The number of viable methanogenic archaea in the reclaimed paddy field was below the detection limit before the first cultivation of rice and in the reclaimed upland field.Then,the number gradually increased over five years and finally reached 103–104 cells g-1 dry soil,which was comparable to that in the dressed paddy field.These findings showed that the low abundance of autochthonous methanogenic archaea in the non-methanogenic oxic upland field soils steadily proliferated,and the community structure was developed following repeated and long-term submergence.These results suggest that habitats suitable for methanogenic archaea were established in soil following repeated and long-term submergence.     

Modeling aerobic decomposition of rice straw during the off-rice season in an Andisol paddy soil in a cold temperate region of Japan: Effects of soil temperature and moisture

Submerged rice paddies are a major source of methane (CH₄) which is the second most important greenhouse gas after carbon dioxide (CO₂). Accelerating rice straw decomposition during the off-rice season could help to reduce CH₄ emission from rice paddies during the single rice-growth season in cold temperate regions. For understanding how both temperature and moisture can affect the rate of rice straw decomposition during the off-rice season in the cold temperate region of Tohoku district, Japan, a modeling incubation experiment was carried out in the laboratory. Bulk soil and soil mixed with 2% of δ¹³C-labeled rice straw with a full factorial combination of four temperature levels (?5 to 5, 5, 15, 25°C) and two moisture levels (60% and 100% WFPS) were incubated for 24 weeks. The daily change from ?5 to 5°C was used to model the freezing–thawing cycles occurring during the winter season. The rates of rice straw decomposition were calculated by (i) CO₂ production; (ii) change in the soil organic carbon (SOC) content; and (iii) change in the δ¹³C value of SOC. The results indicated that both temperature and moisture affected the rate of rice straw decomposition during the 24-week aerobic incubation period. Rates of rice straw decomposition increased not only with high temperature, but also with high moisture conditions. The rates of rice straw decomposition were more accurately calculated by CO₂ production compared to those calculated by the change in the SOC content, or in its δ¹³C value. Under high moisture at 100% WFPS condition, the rates of rice straw decomposition were 14.0, 22.2, 33.5 and 46.2% at ?5 to 5, 5, 15 and 25°C temperature treatments, respectively. While under low moisture at 60% WFPS condition, these rates were 12.7, 18.3, 31.2 and 38.4%, respectively. The Q₁₀ of rice straw decomposition was higher between ?5 to 5 and 5°C than that between 5 and 15°C and that between 15 and 25°C. Daily freezing–thawing cycles (from ?5 to 5°C) did not stimulate rice straw decomposition compared with low temperature at 5°C. This study implies that to reduce CH₄ emission from rice paddies during the single rice-growth season in the cold temperate regions, enhancing rice straw decomposition during the high temperature period is very important.     

Water-soluble organic materials in paddy soil ecosystem

Abstract

Column experiments were conducted to analyze the effect of the temperature on the amounts of organic materials in the leachate, especially organic acids and methane, from samples of the plow layer soil amended with rice straw. Total amount of inorganic carbon in the leachate during the 30-d period of incubation in relation to the temperature was 18°C < 25°C ≤ 30°C > 37°C > 45°C. Total amount of organic carbon in the leachate was signiicantly larger under 45°C incubation than that at other temperatures.

Acetic acid was the dominant organic acid in the leachate regardless of the temperature. Butylic and propionic acids were also present in large amounts in the early and the late period of incubation of temperatures ranging between 18 and 37°C, while only acetic acid was the dominant organic acid during the 30-d period of incubation at 45°C.

The total amount of methane in leachate during the 30-d period of incubation was very small at 18°C, while very large at 25, 30, and 37°C. It decreased nearly to one half at 45°C compared with that at 30°C. Based on the values of δ¹³CH₄ in the leachate, 3 different stages were recognized in the predominant processes of methane production in the submerged paddy soil amended with rice straw: the stage when methane production from CO₂-B₂ was predominant followed by the stages of methane production from acetic acid and from CO₂-H₂ in this order. The second stage coincided with the time of decrease of the organic acid contents in the leachate. Under 45°C incubation, methane production from CO₂-H₂ was predominant throughout the 30-d period of incubation.     

Effect of warming on the temperature dependence of soil respiration rate in arctic,temperate and tropical soils

We examined the response of the temperature coefficient (Q₁₀) for soil respiration rate to changes in environmental temperature through a laboratory incubation experiment. Soil samples were collected from three climatic areas: arctic (Svalbard, Norway), temperate (Tsukuba, Japan) and tropical (Pasoh, Malaysia). The arctic and temperate soils were incubated at 8 °C (control), 12 °C (4 °C warming) and 16 °C (8 °C warming) for 17 days. The tropical soil was incubated at 16 °C (8 °C cooling), 24 °C (control) and 32 °C (8 °C warming). Before and after the incubation experiment, the temperature dependence of soil microbial respiration was measured using an open-airflow method with IRGA by changing the temperature in a water bath. The initial Q₁₀ before the incubation experiment was larger in the soils from higher latitudes: 3.4 in the arctic soil, 2.9 in the temperate soil, and 2.1 in the tropical soil. The response of the microbial respiration rate to change in temperature differed among the three soil types. The temperature dependence of respiration rate in the arctic soil did not change in response to warming by 4 and 8 °C with a Q₁₀ of about 3. On the other hand, the Q₁₀ in the temperate soil decreased with increasing incubation temperature: from 2.8 in soils incubated at 8 °C to 2.5 at 12 °C and 2.0 at 16 °C. In the tropical soil, the Q₁₀ was not changed even by the 8 °C warming with a value of 2.1, whereas the Q₁₀ was increased from 2.1 to 2.7 by the 8 °C cooling. These results suggest that the response of microbial respiration to climatic warming may differ between soils from different latitudes.     

Growth of hydrogenotrophic and acetoclastic methanogens on substrate from rice plant callus cells in anaerobic soil: an estimation to the role of slough-off root cap cells to their growth

Flooded paddy fields are the major anthropogenic sources of methane (CH₄) emission, and organic materials of rice plant origin were estimated to be important as its source. This study used rice (Oryza sativa L. cv, Yukihikari) callus cells as a model material for slough-off root cap cells, and carbon-13 (¹³C)-labelled callus cells were subjected to decomposition in aerobic and anaerobic soil microcosms for 56 days. DNA was extracted from a soil incubated with carbon-12 (¹²C)- and ¹³C-callus cells and subjected to buoyant density gradient centrifugation to identify methanogenic archaeal species that assimilated carbon from the callus cells. ¹³C-labelled 16S rRNA gene (16S rDNA) fragments from methanogenic archaea were not polymerase chain reaction (PCR)-amplified in heavy fractions under aerobic soil conditions, while they were successfully done from day 3 onwards under anaerobic soil conditions. Eighty-four denaturing gradient gel electrophoresis (DGGE) bands in heavy fractions were sequenced, revealing that they were members of Methanosarcina spp. (20 clones), Methanosaeta spp. (18 clones), Methanocella spp. (25 clones), Methanomicrobiales (10 clones), Methanobacterium spp. (7 clones) and Cluster ZC-I (2 clones). They included hydrogenotrophic and acetoclastic methanogens and were phylogenetically different from those residing in rice roots and, presumably, from those assimilating root exudate and mucilage-derived carbon. This study indicates that carbon of slough-off root cap cells propagates specific methanogenic species in rice rhizosphere under anaerobic soil conditions and thus augments the diversity of the total rhizospheric methanogenic community.     

Temperature and substrate effects on C & N mineralization and microbial community function of soils from a hybrid poplar chronosequence

Substrate input as well as climatic factors affect C and N cycling and microbial properties in forest soils. We used a microcosm approach to investigate the response of CO₂ efflux, net N mineralization, and microbial community-level physiological profile (CLPP) to temperature (5 vs. 15 °C) and substrate (with and without sucrose addition) addition in surface mineral soils collected from 4-, 6-, 13-, and 15-year old (ages in 2007) hybrid poplar (Populus deltoides × Populus × petrowskyana var. Walker) stands in northern Alberta. In the early stage of incubation (0–2 h), CO₂ efflux was higher at 5 °C than at 15 °C with little effect from substrate addition, while 24 h after the addition of substrate, CO₂ efflux became higher under the 15 °C incubation. After 72 h incubation, temperature and substrate addition effects on CO₂ efflux subsided and CO₂ efflux rates tended to converge among the treatments. Net N mineralization was significantly affected by substrate addition and stand age, while rates of net ammonification were higher at 5 °C than at 15 °C. Net N mineralization occurred without sucrose addition while net immobilization occurred with sucrose addition. The soil from the youngest stand had the lowest N mineralization rate among the stands for each corresponding substrate-incubation temperature treatment. We used Ecoplates from Biolog™ to study sole-carbon-source-utilization profiles of microbial communities at the end of the incubation. Principal component analysis of C utilization data separated microbial communities with respect to substrate addition, incubation temperature and stand age. Our data showed that organic matter mineralization and microbial substrate utilization were affected by incubation temperature, substrate availability and stand age, indicating that the responses of microbial communities in the studied hybrid poplar plantations to temperature changes were strongly mediated by labile C availability and stand development.     

Effect of winter-flooding on methanogenic archaeal community structure in paddy field under organic farming

Paddy field is a major emission source of methane. Methane is the terminal product of anaerobic decomposition of organic matter and generated by methanogenic archaea under flooded conditions in paddy fields. This study aimed to reveal the effect of winter flooding on methanogenic archaeal community structure in paddy fields of Andosols under organic farming. Soil samples were collected from experimental paddy fields in the Field Science Center, Tohoku University, for two years. They were under flooding conditions during winter with organic farming, under non-flooding conditions during winter with organic farming and under non-flooding conditions during winter with conventional farming (non-organic farming). Polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) analysis of methanogenic archaeal 16S rRNA gene revealed that the DGGE patterns were nearly the same irrespective of the treatment and sampling times. Twenty-three bands were observed from each treatment and 4, 13 and 6 sequences were closely related to Methanomicrobiales, Methanosarcinales and Methanocellales, respectively. Real-time quantitative PCR analysis indicated that the abundance of methanogenic archaeal 16S rRNA gene and mcrA gene, encoding α subunit of methyl-coenzyme M reductase, was not significantly different among the paddy fields. This study first revealed a methanogenic archaeal community in an Andosol paddy field and showed that the community was not affected by winter flooding under organic farming.     

The impact of sludge amendment on methanogen community structure in an upland soil

In the EU, municipal sewage sludge application to agricultural land has increased dramatically since the ban on dumping at sea came into effect in 1998. There are many concerns related to potential contamination and reduction in plant productivity. In this study, the aim was to assess the impact of repeated long-term soil amendment with anaerobically digested sewage sludge on methanogen diversity in an upland soil ecosystem. Sludge-treated and untreated upland soil samples as well as samples of the sludge used, were analysed for the diversity of methanogens using TGGE, PCR-RFLP and DNA sequence analysis of approximately 490 bp of the mcrA operon. PCR analysis using mcrA specific oligonucleotide primers confirmed the presence of methanogen DNA in treated and untreated soil samples and in sewage sludge. TGGE was used to describe the diversity of methanogen mcrA sequences and the differences in community structure between samples. Ninety-six mcrA gene PCR products were screened using RFLP analysis representing methanogen DNA amplified from anaerobically digested sewage sludge, control soils and sludge treated soils. Fourteen RFP's were detected in all treatments, five of which were common to all three treatments. Thirty-eight cloned amplimers were selected for sequencing and phylogenetic analysis. These included representatives of each RFP. From control soils, sludge and sludged soil samples 15, 16 and 7 clones were sequenced, respectively. Phylogenetic analysis suggested that they represented hitherto uncharacterised mcr genes; 35 of the clones fell into 7 clusters supported by moderate to high bootstrap values. The diversity of methanogens in an upland soil (treated and untreated) and sludge was evaluated and marked differences in the diversity of the methanogen communities was observed between the treatments. Our results indicate that sludge application may reduce soil methanogen community diversity.     

Abundance of transcripts of functional gene reflects the inverse relationship between CH<Subscript>4</Subscript> and N<Subscript>2</Subscript>O emissions during mid-season drainage in acidic paddy soil

Agricultural management significantly affects methane (CH₄) and nitrous oxide (N₂O) emissions from paddy fields. However, little is known about the underlying microbiological mechanism. Field experiment was conducted to investigate the effect of the water regime and straw incorporation on CH₄ and N₂O emissions and soil properties. Quantitative PCR was applied to measure the abundance of soil methanogens, methane-oxidising bacteria, nitrifiers, and denitrifiers according to DNA and mRNA expression levels of microbial genes, including mcrA, pmoA, amoA, and nirK/nirS/nosZ. Field trials showed that the CH₄ and N₂O flux rates were negatively correlated with each other, and N₂O emissions were far lower than CH₄ emissions. Drainage and straw incorporation affected functional gene abundance through altered soil environment. The present (DNA-level) gene abundances of amoA, nosZ, and mcrA were higher with straw incorporation than those without straw incorporation, and they were positively correlated with high concentrations of soil exchangeable NH₄⁺ and dissolved organic carbon. The active (mRNA-level) gene abundance of mcrA was lower in the drainage treatment than in continuous flooding, which was negatively correlated with soil redox potential (Eh). The CH₄ flux rate was significantly and positively correlated with active mcrA abundance but negatively correlated with Eh. The N₂O flux rate was significantly and positively correlated with present and active nirS abundance and positively correlated with soil Eh. Thus, we demonstrated that active gene abundance, such as of mcrA for CH₄ and nirS for N₂O, reflects the contradictory relationship between CH₄ and N₂O emissions regulated by soil Eh in acidic paddy soils.     

Dynamics of sulfate reduction and sulfate-reducing prokaryotes in anaerobic paddy soil amended with rice straw

Incorporation of rice straw to soil is a common agricultural practice in rice cultivation. In anaerobic paddy soil, the complete mineralization of organic matter to CH₄ and CO₂ is accomplished by the sequential reduction of nitrate, ferric iron, sulfate, and methanogenesis. In order to estimate the temporal changes of sulfate-reducing prokaryotes (SRP) as decomposers of organic matters, the effects of rice straw amendment on the dynamics of sulfate reduction and SRP were investigated by combining the monitoring of CH₄, sulfate, and organic acids with molecular tools such as soil DNA extraction, real-time PCR, cloning, sequencing, and phylogenetic analysis. The incorporation of rice straw into paddy soil significantly increased concentrations of sulfate, formate, acetate, propionate, and lactate and CH₄ production. The rate of sulfate reduction in the straw-amended slurries was significantly higher than that in the unamended slurries. The dsrAB gene copy numbers of SRP in the straw-amended soil slurries ranged from 4.26 × 10⁶ to 1.96 × 10⁸ per gram of dry soil, which were significantly higher than those in the unamended control ranging from 1.99 × 10⁶ to 7.90 × 10⁷ per gram of dry soil. Significant correlations were observed between SRP dsrAB gene copy numbers and the concentrations of sulfate and acetate. Cloning and sequencing analyses showed a clear shift of SRP community structure between treatments and time. In the straw-amended slurries, Clostridia-like SRP significantly increased, while Deltaproterobacteria-like SRP (Sytrophobacter, Desulfobacterium, Desulfovibrio, and Desulfomonile) decreased during the incubation period. Novel uncultured SRP were abundant in the straw-amended slurries and changed during the incubation period.