Project description:Propionate is an important intermediate of the degradation of organic matter in many anoxic environments. In methanogenic environments, due to thermodynamic constraints, the oxidation of propionate requires syntrophic cooperation of propionate-fermenting proton-reducing bacteria and H(2)-consuming methanogens. We have identified here microorganisms that were active in syntrophic propionate oxidation in anoxic paddy soil by rRNA-based stable-isotope probing (SIP). After 7 weeks of incubation with [(13)C]propionate (<10 mM) and the oxidation of approximately 30 micromol of (13)C-labeled substrate per g dry weight of soil, we found that archaeal nucleic acids were (13)C labeled to a larger extent than those of the bacterial partners. Nevertheless, both terminal restriction fragment length polymorphism and cloning analyses revealed Syntrophobacter spp., Smithella spp., and the novel Pelotomaculum spp. to predominate in "heavy" (13)C-labeled bacterial rRNA, clearly showing that these were active in situ in syntrophic propionate oxidation. Among the Archaea, mostly Methanobacterium and Methanosarcina spp. and also members of the yet-uncultured "rice cluster I" lineage had incorporated substantial amounts of (13)C label, suggesting that these methanogens were directly involved in syntrophic associations and/or thriving on the [(13)C]acetate released by the syntrophs. With this first application of SIP in an anoxic soil environment, we were able to clearly demonstrate that even guilds of microorganisms growing under thermodynamic constraints, as well as phylogenetically diverse syntrophic associations, can be identified by using SIP. This approach holds great promise for determining the structure and function relationships of further syntrophic or other nutritional associations in natural environments and for defining metabolic functions of yet-uncultivated microorganisms.
Project description:Propionate is one of the major intermediary products in the anaerobic decomposition of organic matter in wetlands and paddy fields. Under methanogenic conditions, propionate is decomposed through syntrophic interaction between proton-reducing and propionate-oxidizing bacteria and H(2)-consuming methanogens. Temperature is an important environmental regulator; yet its effect on syntrophic propionate oxidation has been poorly understood. In the present study, we investigated the syntrophic oxidation of propionate in a rice field soil at 15°C and 30°C. [U-(13)C]propionate (99 atom%) was applied to anoxic soil slurries, and the bacteria and archaea assimilating (13)C were traced by DNA-based stable isotope probing. Syntrophobacter spp., Pelotomaculum spp., and Smithella spp. were found significantly incorporating (13)C into their nucleic acids after [(13)C]propionate incubation at 30°C. The activity of Smithella spp. increased in the later stage, and concurrently that of Syntrophomonas spp. increased. Aceticlastic Methanosaetaceae and hydrogenotrophic Methanomicrobiales and Methanocellales acted as methanogenic partners at 30°C. Syntrophic oxidation of propionate also occurred actively at 15°C. Syntrophobacter spp. were significantly labeled with (13)C, whereas Pelotomaculum spp. were less active at this temperature. In addition, Methanomicrobiales, Methanocellales, and Methanosarcinaceae dominated the methanogenic community, while Methanosaetaceae decreased. Collectively, temperature markedly influenced the activity and community structure of syntrophic guilds degrading propionate in the rice field soil. Interestingly, Geobacter spp. and some other anaerobic organisms like Rhodocyclaceae, Acidobacteria, Actinobacteria, and Thermomicrobia probably also assimilated propionate-derived (13)C. The mechanisms for the involvement of these organisms remain unclear.
Project description:Background:Paddies are an important anthropogenic source of methane emissions to the atmosphere, and they are impacted by heavy metal pollution. Nickel (Ni) and cobalt (Co) pollution might either enhance or mitigate CH4 emission from paddy soils due to the total amounts of metals, bioavailability and functional microbial activity and composition. Methods:An incubation experiment was conducted, and different Ni and Co concentrations were added to test the effects of trace metals on methane production in paddy soil. The archaea community structure and the abundance of methanogen functional groups in the paddy soil with added Ni and Co were detected using high-throughput sequencing and quantitative PCR based on the 16S rRNA and mcrA (methyl coenzyme M reductase) genes, respectively. Results:The highest methane production rate was 561 mg CH4 kg-1 dry soil d-1 with the addition of 50 mg kg-1 Ni and 684 mg CH4 kg-1 dry soil d-1 with the addition of 25 mg kg-1 Co. Accordingly, the mcrA gene was most abundant in the 50 mg kg-1 Ni addition (3.1 × 106 ± 0.5 × 106 copies g-1 dry soil). The lowest mcrA gene abundance was detected in the 500 mg kg-1 Co addition (9.2× 105 ± 0.4 × 105 copies g-1 dry soil). The dominant methanogens were Methanobacterium, Methanosarcina, Methanocella, Methanomassiliicoccus, Bathyarchaeota, and Rice Cluster I (RC-I), and the relative abundances of these groups were higher than 1% in the Ni and Co treatments. Additionally, the archaeal compositions differed significantly in the soils with various Ni and Co additions. The most abundant Methanococcus spp. represented 51.3% of the composition in the 50 mg kg-1 Ni addition, which was significantly higher than that of the control (12.9% to 17.5%). Discussion:Our results indicated that the contamination of soil by Ni and Co significantly affected total methanogens abundance and specific methanogen functional groups. Ni and Co additions to paddy soil promoted methanogenic activity at low concentrations, while they had inhibitory effects at high concentrations. Because paddy soils largely contribute to methane emissions and are increasingly exposed to heavy metal pollution, our results show that future assessments of greenhouse gas flux from paddy soils should take into account the effects of pollution by Ni and Co.
Project description:Waterlogged paddy soils possess anoxic zones in which microbes actively induce reductive nitrogen transformation (RNT). In the present study, a shotgun RNA sequencing analysis (metatranscriptomics) of paddy soil samples revealed that most RNT gene transcripts in paddy soils were derived from Deltaproteobacteria, particularly the genera Anaeromyxobacter and Geobacter. Despite the frequent detection of the rRNA of these microbes in paddy soils, their RNT-associated genes have rarely been identified in previous PCR-based studies. This metatranscriptomic analysis provides novel insights into the diversity of RNT microbes present in paddy soils and the ecological function of Deltaproteobacteria predominating in these soils.
Project description:Iron reduction is an important biogeochemical process in paddy soils, yet little is known about the microbial coupling between nitrogen and iron reduction. Here, we investigated the shift of acetate-metabolizing iron-reducers under long-term nitrogen fertilization using (13)C-acetate-based ribosomal RNA (rRNA)-stable isotope probing (SIP) and pyrosequencing in an incubation experiment, and the shift of putative iron-reducers in original field samples were investigated by 16S rRNA gene-based pyrosequencing. During SIP incubations, in the presence of iron(III) oxyhydroxides, more iron(II) formation and less methane production were detected in nitrogen-fertilized (N) compared with non-fertilized (NF) soil. In (13)C-rRNA from microcosms amended with ferrihydrite (FER), Geobacter spp. were the important active iron-reducers in both soils, and labeled to a greater extent in N (31% of the bacterial classified sequences) than NF soils (11%). Pyrosequencing of the total 16S rRNA transcripts from microcosms at the whole community level further revealed hitherto unknown metabolisms of potential FER reduction by microorganisms including Pseudomonas and Solibacillus spp. in N soil, Dechloromonas, Clostridium, Bacillus and Solibacillus spp. in NF soil. Goethite (GOE) amendment stimulated Geobacter spp. to a lesser extent in both soils compared with FER treatment. Pseudomonas spp. in the N soil and Clostridium spp. in the NF soil may also be involved in GOE reduction. Pyrosequencing results from field samples showed that Geobacter spp. were the most abundant putative iron-reducers in both soils, and significantly stimulated by long-term nitrogen fertilization. Overall, for the first time, we demonstrate that long-term nitrogen fertilization promotes iron(III) reduction and modulates iron-reducing bacterial community in paddy soils.
Project description:Evidence for anaerobic ammonium oxidation in a paddy field was obtained in Southern China using an isotope-pairing technique, quantitative PCR assays and 16S rRNA gene clone libraries, along with nutrient profiles of soil cores. A paddy field with a high load of slurry manure as fertilizer was selected for this study and was shown to contain a high amount of ammonium (6.2-178.8? mg ?kg(-1)). The anaerobic oxidation of ammonium (anammox) rates in this paddy soil ranged between 0.5 and 2.9 ?nmol N per gram of soil per hour in different depths of the soil core, and the specific cellular anammox activity observed in batch tests ranged from 2.9 to 21 ?fmol per cell per day. Anammox contributed 4-37% to soil N2 production, the remainder being due to denitrification. The 16S rRNA gene sequences of surface soil were closely related to the anammox bacteria 'Kuenenia', 'Anammoxoglobus' and 'Jettenia'. Most of the anammox 16S rRNA genes retrieved from the deeper soil were affiliated to 'Brocadia'. The retrieval of mainly bacterial amoA sequences in the upper part of the paddy soil indicated that nitrifying bacteria may be the major source of nitrite for anammox bacteria in the cultivated horizon. In the deeper oxygen-limited parts, only archaeal amoA sequences were found, indicating that archaea may produce nitrite in this part of the soil. It is estimated that a total loss of 76 ?g ?N ?m(-2) per year is linked to anammox in the paddy field.
Project description:Paddy fields represent a unique ecosystem in which regular flooding occurs, allowing for rice cultivation. However, the taxonomic identity of the microbial functional guilds that catalyze soil nitrification remains poorly understood. In this study, we provide molecular evidence for distinctly different phylotypes of nitrifying communities in a neutral paddy soil using high-throughput pyrosequencing and DNA-based stable isotope probing (SIP). Following urea addition, the levels of soil nitrate increased significantly, accompanied by an increase in the abundance of the bacterial and archaeal amoA gene in microcosms subjected to SIP (SIP microcosms) during a 56-day incubation period. High-throughput fingerprints of the total 16S rRNA genes in SIP microcosms indicated that nitrification activity positively correlated with the abundance of Nitrosospira-like ammonia-oxidizing bacteria (AOB), soil group 1.1b-like ammonia-oxidizing archaea (AOA), and Nitrospira-like nitrite-oxidizing bacteria (NOB). Pyrosequencing of 13C-labeled DNA further revealed that 13CO2 was assimilated by these functional groups to a much greater extent than by marine group 1.1a-associated AOA and Nitrobacter-like NOB. Phylogenetic analysis demonstrated that active AOB communities were closely affiliated with Nitrosospira sp. strain L115 and the Nitrosospira multiformis lineage and that the 13C-labeled AOA were related to phylogenetically distinct groups, including the moderately thermophilic "Candidatus Nitrososphaera gargensis," uncultured fosmid 29i4, and acidophilic "Candidatus Nitrosotalea devanaterra" lineages. These results suggest that a wide variety of microorganisms were involved in soil nitrification, implying physiological diversification of soil nitrifying communities that are constantly exposed to environmental fluctuations in paddy fields.
Project description:Identification of the carbon (C) sources of methane (CH?) and methanogenic community structures after organic fertilization may provide a better understanding of the mechanism that regulate CH? emissions from paddy soils. Based on our previous field study, a pot experiment with isotopic 13C labelling was designed in this study. The objective was to investigate the main C sources for CH? emissions and the key environmental factor with the application of organic fertilizer in paddies. Results indicated that 28.6%, 64.5%, 0.4%, and 6.5% of 13C was respectively distributed in CO?, the plants, soil, and CH? at the rice tillering stage. In total, organically fertilized paddy soil emitted 3.51 kg·CH? ha-1 vs. 2.00 kg·CH? ha-1 for the no fertilizer treatment. Maximum CH? fluxes from organically fertilized (0.46 mg·m-2·h-1) and non-fertilized (0.16 mg·m-2·h-1) soils occurred on day 30 (tillering stage). The total percentage of CH? emissions derived from rice photosynthesis C was 49%, organic fertilizer C < 0.34%, and native soil C > 51%. Therefore, the increased CH? emissions from paddy soil after organic fertilization were mainly derived from native soil and photosynthesis. The 16S rRNA sequencing showed Methanosarcina (64%) was the dominant methanogen in paddy soil. Organic fertilization increased the relative abundance of Methanosarcina, especially in rhizosphere. Additionally, Methanosarcina sp. 795 and Methanosarcina sp. 1H1 co-occurred with Methanobrevibacter sp. AbM23, Methanoculleus sp. 25XMc2, Methanosaeta sp. HA, and Methanobacterium sp. MB1. The increased CH? fluxes and labile methanogenic community structure in organically fertilized rice soil were primarily due to the increased soil C, nitrogen, potassium, phosphate, and acetate. These results highlight the contributions of native soil- and photosynthesis-derived C in paddy soil CH? emissions, and provide basis for more complex investigations of the pathways involved in ecosystem CH? processes.
Project description:Carboxylated multiwalled carbon nanotubes (MWCNTs-COOH) have become a growing concern in terms of their fate and toxicity in aqueous environments. Methane (CH4) is a major product of organic matter degradation in waterlogged environments. In this study, we determined the effect of MWCNTs-COOH on the production of CH4 from propionate oxidation in paddy soil enrichments. The results showed that the methanogenesis from propionate degradation was accelerated in the presence of MWCNTs-COOH. In addition, the rates of CH4 production and propionate degradation increased with increasing concentrations of MWCNTs-COOH. Scanning electron microscopy (SEM) observations showed that the cells were intact and maintained their structure in the presence of MWCNTs-COOH. In addition, SEM and fluorescence in situ hybridization (FISH) images revealed that the cells were in direct contact with the MWCNTs and formed cell-MWCNTs aggregates that contained both bacteria and archaea. On the other hand, nontoxic magnetite nanoparticles (Fe3O4) had similar effects on the CH4 production and cell integrity as the MWCNTs-COOH. Compared with no nanomaterial addition, the relative abundances of Geobacter and Methanosarcina species increased in the presence of MWCNTs-COOH. This study suggests that MWCNTs-COOH exerted positive rather than cytotoxic effects on the syntrophic oxidation of propionate in paddy soil enrichments and affected the bacterial and archaeal community structure at the test concentrations. These findings provide novel insight into the consequences of nanomaterial release into anoxic natural environments.
Project description:Background:Chinese milk vetch (Astragalus sinicus L.) can improve paddy soil fertility and ecology through nitrogen fixation, but it can also increase greenhouse gas emissions. Our primary objective was to investigate how Chinese milk vetch, rice straw, and nitrogen fertilization affect the methane and microbial components of the rice rhizosphere. Methods:We examined the rhizosphere's methane emissions and microbial abundance and diversity after incorporating Chinese milk vetch and rice straw into paddy soil. We used high-throughput sequencing of the 16s rRNA and ITS1 genes to study changes in the bacterial and fungal communities, respectively. Over the course of our experiment, we applied seven different treatments to the paddy soil: conventional fertilization (the control treatment) for winter fallow crops, three levels of nitrogen in Chinese milk vetch, and three levels of nitrogen in Chinese milk vetch combined with rice straw. Results:Rice yield and methane emissions increased during cultivation when the soil was treated with Chinese milk vetch with and without added straw. The nitrogen application also affected the methane fluxes. Alpha diversity measurements showed that Chinese milk vetch increased the diversity of the soil fungal community but did not significantly affect the bacterial community. Chinese milk vetch affected the rhizosphere microorganism communities by increasing the number of Methanomicrobia.