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.     

Methanogenic archaeal communities developed in paddy fields in the Kojima Bay polder, estimated by denaturing gradient gel electrophoresis, real-time PCR and sequencing analyses

Methanogenic archaeal communities inhabiting the paddy field soils in the Kojima Bay polder were investigated using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE), real-time PCR and sequencing analyses. Soil samples of the plow and subsoil layers were collected in 2006 from four paddy fields that were reclaimed between 1692 and 1954. The DGGE band patterns of the targeted 16S rRNA genes amplified from the extracted DNA from the samples were different from the patterns from the paddy field soils in diluvial and alluvial areas. The numbers of targeted 16S rRNA genes, which were involved with methanogenic archaeal and other archaeal sequences, were approximately 10⁷–10⁸ and 10⁶ g⁻¹ dry soil in the plow and subsoil layers, respectively. Sequences of methanogenic archaeal 16S rRNA genes belonging to Methanocellales (Rice cluster I), Methanosarcinales and Methanobacteriales were obtained from the major DGGE bands. Whereas sequences in Methanomicrobiales, which were predominant methanogens in the diluvial and alluvial paddy fields, were not recovered. Known halophilic and methylotrophic methanogens, which are characteristic of saline and marine environments, were not detected. These results indicate that distinctive methanogenic archaeal communities have developed in the paddy field soils in the Kojima Bay polder.     

Community structure of methanogenic archaea in paddy field soil under double cropping (rice-wheat)

Community structure of methanogenic archaea in paddy field soil under double cropping (rice [Oryza sativa L.] and wheat [Triticum aestivum L.]) was studied by the denaturing gradient gel electrophoresis (DGGE) method. Soil samples under flooded and upland conditions were collected 7 and 6 times, respectively, from two paddy fields throughout a year, and two primer sets, 0357F-GC/0691R and newly designed 1106F-GC/1378R, were used for DGGE analysis. The 25 and 29 different bands were observed on the DGGE gels with the primers 0357F-GC/0691R and 1106F-GC/1378R, respectively. DGGE band patterns of the methanogenic archaeal community were stable throughout a year including the cultivation periods of rice under flooded conditions and of wheat under upland conditions. Cluster analysis and principal component analysis suggested that the difference in the soil type (sampling region) largely influenced the community structures of methanogenic archaea in paddy field soil, while the effects of sampling period and different fertilizer treatments on them were small. Most of the sequences obtained from the DGGE bands were closely related to Methanomicrobiales, Methanosarcinaceae, Methanosaetaceae and Rice cluster-I.     

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.     

Bt-transgenic rice straw affects the culturable microbiota and dehydrogenase and phosphatase activities in a flooded paddy soil

There have been few investigations of the possible effects of genetically engineered plants on the microbiota and enzyme activities in flooded soil. We studied the influence of the transgenic rice KeMingDao (KMD) straw on the culturable microbiota and enzymatic activities in a flooded paddy soil under laboratory conditions. KMD contained a synthetic cry1Ab gene from Bacillus thuringiensis under the control of a maize ubiquitin promoter and linked in tandem with the gusA and hpt genes. The results showed that there were only some occasional significant differences (P<0.05) in the number of Colony forming units of aerobic bacteria, actinomycetes and fungi and in the number of anaerobic fermentative bacteria, denitrifying bacteria, hydrogen-producing acetogenic bacteria, and methanogenic bacteria between the paddy soil amended with Bt-transgenic rice straw and with the non-Bt parental rice straw during the early stages of incubation. From d14 to d84 there were significant increases (P<0.05) in soil dehydrogenase and soil neutral phosphatase activity in soils amended with rice straw compared to soil without added straw. The dehydrogenase activity was significantly greatly (almost 1.95-fold) in soil amended with Bt-transgenic straw from d7 to d14 but from d21 to d49 there was significantly greater activity (about 1.47-fold) in the soil amended with non-Bt-straw. There were no apparent differences between the activity of soil neutral phosphatase in the soils to which non-Bt-straw and Bt-straw had been added. However, both soils to which rice straws were added demonstrated significant differences in the number of microorganisms except for aerobic bacteria and enzymatic activities with respect to the control soil throughout the incubation. The above results indicated that the Bt-straw from KMD transgenic rice is not toxic to a variety of culturable microorganisms in the studied flooded paddy soil.     

Cyanobacterial communities of rice straw left on the soil surface of a paddy field

To estimate diversity, seasonal variation, and phylogeny of the cyanobacterial communities in rice straw placed in nylon mesh bags and left on the soil surface of a paddy field, total DNA was extracted from straw, amplified by polymerase chain reaction targeting 16S rRNA genes of cyanobacteria, and the amplicons were separated by denaturing gradient gel electrophoresis (DGGE). These DGGE bands were sequenced. The paddy field was under flooded condition after transplanting of rice (Experiment 1) and under drained conditions after harvest (Experiment 2). The residual samples on the soil surface under upland conditions were collected just before spring plowing and were placed again on the soil surface after transplanting under flooded conditions. DGGE band patterns of cyanobacterial communities of rice straw were different under drained conditions, under flooded conditions when fresh rice straw samples were placed (Experiment 1), and under flooded conditions when residual rice straw samples were replaced (Experiment 2), indicating that the communities were influenced by both water regime of the paddy field and the degree of the rice straw decomposition. Sequence analysis of DGGE bands indicated that most of the cyanobacteria in rice straw on the soil surface in the paddy field were filamentous members belonging to Subsections III and IV. Filamentous cyanobacterial cells were observed in rice straw under flooded conditions by epifluorescence microscopy.     

Rhodanese activity of flooded and nonflooded soils

Rhodanese activity (RA) was studied in 4 soils, incubated under flooded and nonflooded (60% water-holding capacity) conditions. RA in 3 soils including an acid sulphate soil pokkali increased 2.5–6.0-fold (over respective nonflooded soils), while activity of the enzyme decreased markedly in flooded alluvial soil. Similarly, anaerobic incubation of nonflooded soils under N₂ decreased RA in an alluvial soil, but increased it in pokkali soil. RA was negligible in soils, that had been reduced by flooding for 30 days and then sterilized by autoclaving. Rice rhizosphere soil exhibited significantly higher RA than the nonrhizosphere soil samples under flooded or nonflooded conditions. RA in aerobic soils was related to the microbial oxidation of S° to SO^2?₄. But, no relationship could be established between RA and S-oxidation in flooded soils and in rhizosphere soil suspensions of flooded rice plants.     

Effects of rice‐straw and phosphorus application on production and emission of methane from tropical rice soil

Rice‐straw amendment increased methane production by 3‐fold over that of unamended control. Application of P as single superphosphate at 100 μg (g soil)^–1 inhibited methane (CH₄) production distinctly in flooded alluvial rice soil, in the absence more than in the presence of rice straw. CH₄ emission from rice plants (cv. IR72) from alluvial soil treated with single superphosphate as basal application, in the presence and absence of rice straw, and held under non‐flooded and flooded conditions showed distinct variations. CH₄ emission from non‐flooded soil amended with rice straw was high and almost similar to that of flooded soil without rice‐straw amendment. The cumulative CH₄ efflux was highest (1041 mg pot^–1) in rice‐straw‐amended flooded soil. Appreciable methanogenic reactions in rice‐straw‐amended soils were evident under both flooded and non‐flooded conditions. Rice‐straw application substantially altered the balance between total aerobic and anaerobic microorganisms even in non‐flooded soil. The mitigating effects of single‐superphosphate application or low‐moisture regime on CH₄ production and emission were almost nullified due to enhanced activities of methanogenic archaea in the presence of rice straw.     

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.     

Temporal shifts in diversity and quantity of nirS and nirK in a rice paddy field soil

Denitrification is an important part of the nitrogen cycle in the environment, and diverse bacteria, archaea, and fungi are known to have denitrifying ability. Rice paddy field soils have been known to have strong denitrifying activity, but the microbes responsible for denitrification in rice paddy field soils are not well known. Present study analyzed the diversity and quantity of the nitrite reductase genes (nirS and nirK) in a rice paddy field soil, sampled four times in one rice-growing season. Clone library analyses suggested that the denitrifier community composition varied over sampling time. Although many clones were distantly related to the known NirS or NirK, some clones were related to the NirS from Burkholderiales and Rhodocyclales bacteria, and some were related to the NirK from Rhizobiales bacteria. These denitrifiers may play an important role in denitrification in the rice paddy field soil. The quantitative PCR results showed that nirK was more abundant than nirS in all soil samples, but the nirK/nirS ratio decreased after water logging. These results suggest that both diversity and quantity changed over time in the rice paddy field soil, in response to the soil condition.     

Laboratory Investigation of the Geochemical Behavior of Metals in Paddy Soils Contaminated with Mine Tailings

The geochemical behavior of metals, including Fe, Mn, Pb and Zn, in contaminated paddy soils was investigated during the cultivation of rice crops through laboratory microcosm experiments. From the two paddy fields contaminated by mine tailings, Siheung and Deokeum in Korea, paddy soils were collected and analyzed for their geochemical characteristics. The Siheung paddy soil showed higher levels of heavy metals, whereas the higher potential for the release of metals was anticipated due to the extremely acidic conditions at Deokeum. In microcosm experiments of flooded paddy soils over 18 weeks, Fe and Mn were released in subsurface pore waters by reductive dissolution, and Pb and Zn were dissolved in high amounts at the surface by oxidation of sulfides. Although amorphous Fe oxide-rich layers were formed at the surface of both paddy soils, the release of Pb and Zn were controlled at the surface by these layers only under slightly alkaline conditions at Siheung. Lead and Zn were associated with the reducible and carbonate fractions at the surface paddy soil of Siheung from the sequential extraction on core samples collected during the flooded period. In the acidic conditions at Deokeum, Pb and Zn were continuously released until the late stage of flooding. A great increase in the exchangeable fraction of metals was observed after the soils had drained. The bioavailability of metals for rice crops would be high under acidic conditions at Deokeum, despite the lower levels of heavy metal contamination.     

An approach to soil testing with special reference to the zinc requirement of rice paddy soils

Abstract

Experiments were conducted to seek a better basis for soil testing of rice paddy soils. Soils were incubated under variable conditions of simulated flooding, and then extracted with DTPA⁵ . The amounts of Cu, Zn, Mn and Fe extracted were sensitive to the imposed soil conditions. Good correlations between Zn extracted from simulated flooded soils and Zn uptakes by rice from flooded soils in pots, suggest that this approach to soil testing may be more useful for paddy soils than existing tests on air dried soils.     

Biodegradation of Cry1Ab protein from Bt transgenic rice in aerobic and flooded paddy soils

Degradation of Cry1Ab protein from Bt transgenic rice was examined under both aerobic and flooded conditions in five paddy soils and in aqueous solutions. The hydrolysis rate of Cry1Ab protein in aqueous solutions was correlated inversely with the solution pH in the range of 4.0 to 8.0, and positively with the initial concentration of Cry1Ab protein. Rapid degradation of Cry1Ab protein occurred in paddy soils under aerobic conditions, with half-lives ranging from 19.6 to 41.3 d. The degradation was mostly biotic and not related to any specific soil property. Degradation of the Cry1Ab protein was significantly prolonged under flooded conditions compared with aerobic conditions, with half-lives extended to 45.9 to 141 d. These results suggest that the toxin protein, when introduced into a paddy field upon harvest, will probably undergo rapid removal after the field is drained and exposed to aerobic conditions.     

Denitrification in drained and rewetted minerotrophic peat soils in Northern Germany (Pohnsdorfer Stauung)

This study was conducted to assess the nitrogen removal potential of a minerotrophic peatland in Northern Germany, where hydrological conditions were partly restored in the beginning of the 1990s. Actual denitrification and the effect of nitrate (NO₃^—) and glucose additions on denitrification rates were determined in two flooded and one drained histosols in spring and summer 1998. In the flooded soils, denitrification was insignificant, but the drained field emitted significant rates. Additions of NO₃^— stimulated denitrification at all sites in spring and summer, whereas glucose additions had no effect. Low NO₃^— concentration in floodwater was obviously limiting denitrification in the flooded soils. In the drained soil, a coupled nitrification/denitrification might explain the low but significant denitrification rates. No spontaneous production of nitrous oxide occurred in the flooded soils, whereas at the drained site an increase in spontaneous nitrous oxide concentration was measured during incubation in the summer samples. The suggested introduction of NO₃^— rich water from a stream flowing through the area would apparently induce denitrification in the flooded fields.     

Responses of the methanogenic pathway and fraction of CH4 oxidization in a flooded paddy soil to rice planting

Rice planting (RP) is significant to methane (CH₄) emissions from paddy fields, but its effect on the relative contribution of the acetoclastic methanogenesis to total CH₄ production (F_ac) and the fraction of CH₄ oxidized (F_ox) is poorly understood. To quantify the responses of the F_ac and F_ox to RP, we investigated CH₄ fluxes, CH₄ production and oxidation potentials, dissolved CH₄ concentrations, and their stable carbon isotopes in a flooded paddy soil. The mcrA and pmoA gene copies were also determined by quantitative polymerase chain reaction (qPCR). Compared with the unplanted soil (control, CK), the seasonal CH₄ emissions from the planted soil were significantly enhanced, 13.6 times, resulting in large decreases in the CH₄ concentrations in the soil solution. This indicated that much more CH₄ was released into the atmosphere by the RP than was stored in the soils. Acetoclastic methanogenesis became more important from the tillering stage (TS) to the ripening stage (RS) for the CK, with F_ac values increased from 17%–20% to 46%–55%. With RP, the F_ac values were enhanced by 10%–20%, and it significantly increased the copy numbers of the mcrA gene at the four rice stages (TS, booting stage (BS), grain-filling stage (GS), and RS). Furthermore, the effect of the RP on the abundance of the mcrA gene was highly concurrent with the effect on the F_ac values. At the TS, the F_ox values at the soil-water interface were around 50%–75% for the CK, being 15%–20% lower than those of the RP in the rhizosphere. It increased to 65%–100% at the GS, but was reduced by 20%–30% after the RP. These differences might be because the copy numbers of the pmoA gene were significantly raised at the TS while lowered at the GS by the RP. This was further demonstrated by the strong correlations between the effect of the RP on the abundance of the pmoA gene and the effect on the F_ox values. These findings suggest that RP markedly impacts on the abundances of the mcrA and pmoA genes, affecting the pathway of CH₄ production and the fraction of CH₄ oxidization, respectively.     

Characters of humus formed under rice cultivation

To characterize the nature of humus in paddy soils, a comparison was made between the properties of humus in paddy soils and those in adjacent unflooded arable soils.

Rice cultivation generally brought about a considerable increase in organic matter and in the PQ-value, with the exception of Andosol-paddy soils in which organic matter tended to decrease somewhat and the PQ-value remained virtually unchanged. The humification degree of humic acid as judged from Δ log k and RF values was generally lowered by rice cultivation except in the case of Yellow soil-paddy soil in which humic acid was originally low in the degree of humification.

The accumulation of poorly humified humic acid may be a characteristic feature common to all paddy soils. These changes by rice cultivation are observed only in the upper part of the profiles, and seem to be associated with seasonally flooded conditions ot paddy soils. Iron oxides accumulated in subsurface soil have virtually no effect on the properties of humus.     

Comparison of community structures of microbiota at main habitats in rice field ecosystems based on phospholipid fatty acid analysis

The present study compares the community structures of microbiota at different habitats in Japanese rice fields by comparing their phospholipid fatty acid (PLFA) compositions to understand the contribution of different habitats to microbiological diversity. The data were collected from four neighboring rice fields. Comparison was made for the PLFA compositions extracted from the floodwater, percolating water, rice soils under flooded and drained conditions, rice straw (RS) placed in flooded and drained rice soils, RS in the composting process, and RS compost placed in a flooded rice field. Average amounts of PLFAs were 33 μg L⁻¹ in the floodwater, 17.1 μg L⁻¹ in the percolating water from plow layers, 34.6 μg L⁻¹ in the percolating water from subsoil layers, 108 μg g⁻¹ dry weight basis (dw) in flooded rice soils, 382 μg g⁻¹ dw in RS materials, 2,510 μg g⁻¹ dw in RS composts, 2,850 μg g⁻¹ dw in RS composts after application to a flooded rice soil, 222 μg g⁻¹ wet weight basis (ww) in RS in drained rice soils, and 284 μg g⁻¹ ww in RS in flooded rice soils. The total amount of PLFAs to the soil depth of 10 cm was estimated to be about 12 g m⁻². The PLFA compositions were different from each other depending on the habitats. Rice soils were characterized by the predominance of actinomycetes and Gram-positive bacteria in comparison with the other habitats. In contrast, the microbial communities in the floodwater and percolating water were characterized by the predominance of Gram-negative bacteria and eukaryotes (presumably algae), and Gram-negative bacteria, respectively. The microbial community of RS materials was dominated by fungi. Gram-positive bacteria became predominant in RS after application to flooded rice soils, while RS placed in a drained rice field after harvesting rice was characterized by the predominance of Gram-negative bacteria and fungi. The community structures at respective habitats were stable and specific, irrespective of the season of sampling and the duration of decomposition of RS.     

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.