Characterization of microbial NO production, N2O production and CH4 oxidation initiated by aeration of anoxic rice field soil

Intermittent drainage of rice fields isdiscussed as an option to mitigate emission ofCH₄, an important greenhouse gas. HoweverN₂O, a potentially more effective greenhouse gas,may be emitted during the aeration phase. Therefore,the metabolism of NO, N₂O, NH ,NO and NO and the kinetics ofCH₄ oxidation were measured after aeration ofmethanogenic rice field soil. Before aeration, thesoil contained NH in relatively highconcentrations (about 4 mM), while NO andNO were almost undetectable. Immediatelyafter aeration both NO and N₂O were produced withrates of about 15 pmol h^-1 gdw^-1 and 5 pmolh^-1 gdw^-1, respectively. Simultaneously,NH decreased while NO accumulated. Later on, NO was depletedwhile NO concentrations increased.Characteristic phases of nitrogen turnover wereassociated with the activities of ammonium oxidizers,nitrite oxidizers and denitrifiers. Oxidation ofNH and production of NO and N₂O wereinhibited by 10 Pa acetylene demonstrating thatnitrification was obligatory for the initiation ofnitrogen turnover and production of NO and N₂O.Ammonium oxidation was not limited by the availableNH and thus, concomittant production of NOand N₂O was not stimulated by addition ofNH . However, addition of NO stimulated production of NO and N₂O in bothanoxic and aerated rice soil slurries. In this case,10 Pa acetylene did not inhibit the production of NOand N₂O demonstrating that it was due todenitrification which was obviously limited by theavailability of NO . In the aerated soilslurries CH₄ was only oxidized if present atelevated concentrations >50 ppmv CH₄). Atatmospheric CH₄ concentrations (1.7 ppmv)CH₄ was not consumed, but was even slightly produced.CH₄ oxidation activity increased afterpreincubation at 20% CH₄, and then CH₄was also oxidized at atmospheric concentrations. CH₄oxidation kinetics exhibited sigmoid characteristicsat low CH₄ concentrations presumably because ofinhibition of CH₄ oxidation by NH .     

Effects of short-term drainage and aeration on the production of methane in submerged rice soil

Emission rates of CH₄ were measured in microcosms of submerged soil which were planted with rice. Drainage of the rice microcosms for 48 h resulted in drastically decreased CH₄ emission rates which only slowly recovered to the rates of the undrained controls. Drainage also resulted in drastically increased sulphate concentrations which only slowly decreased to nearly zero background values after the microcosms were submerged again. The mechanisms responsible for the decrease of CH₄ production by aeration were investigated in slurries of a loamy and a sandy Italian rice soil. Incubation of the soil slurries under anoxic conditions resulted first in the reduction of nitrate, sulphate and ferric iron before CH₄ production started. Incubation of the soil slurries for 48 h under air resulted in immediate and complete inhibition of CH₄ production. Although the soil slurries were then again incubated under anoxic conditions (N₂ atmosphere), the inhibition of CH₄ production persisted for more than 30 days. The redox potential of the soil increased after the aeration but returned within 15 days to the low values typical for CH₄ production. However, the concentrations of sulphate and of ferric iron increased dramatically after the aeration and stayed at elevated levels for the period during which CH₄ production was inhibited. These observations show that even brief exposure of the soil to O₂ allowed the production of sulphate and ferric iron from their reduced precursors. Elevated sulphate and ferric iron concentrations allowed sulphate-reducing and ferric iron-reducing bacteria to outcompete methanogenic bacteria on H₂ as common substrate. Indeed, concentrations of H₂ were decreased as long as sulphate and ferric iron were high so that the Gibbs free energy of CH₄ production from H₂/CO₂ was also increased (less exergonic). On the other hand, concentrations of acetate, the more important precursor for CH₄, were not much affected by the short aeration of the soil slurries, and the Gibbs free energy of CH₄ production from acetate was highly exergonic suggesting that acetotrophic methanogens were not outcompeted but were otherwise inhibited. Aeration also resulted in increased rates of CO₂ production and in a short-term increase of N₂O production. However, these increases were < 10% of the decreased production of CH₄ and did not represent a trade-off in terms of CO₂ equivalents. Hence, short-term drainage and aeration of submerged paddy fields may be a useful mitigation option for decreasing the emission of greenhouse gases.     

Depth distribution of microbial production and oxidation of methane in northern boreal peatlands

The depth distributions of anaerobic microbial methane production and potential aerobic microbial methane oxidation were assessed at several sites in both Sphagnum- and sedge-dominated boreal peatlands in Sweden, and compared with net methane emissions from the same sites. Production and oxidation of methane were measured in peat slurries, and emissions were measured with the closed-chamber technique. Over all eleven sites sampled, production was, on average, highest 12 cm below the depth of the average water table. On the other hand, highest potential oxidation of methane coincided with the depth of the average water table. The integrated production rate in the 0–60 cm interval ranged between 0.05 and 1.7 g CH4 m ^–2 day^– and was negatively correlated with the depth of the average water table (linear regression: r ² = 0.50, P = 0.015). The depth-integrated potential CH₄-oxidation rate ranged between 3.0 and 22.1 g CH₄ m^–2 day^–1 and was unrelated to the depth of the average water table. A larger fraction of the methane was oxidized at sites with low average water tables; hence, our results show that low net emission rates in these environments are caused not only by lower methane production rates, but also by conditions more favorable for the development of CH₄-oxidizing bacteria in these environments. Correspondence to: I. Sundh.     

Thermophilic anaerobic oxidation of methane by marine microbial consortia

The anaerobic oxidation of methane (AOM) with sulfate controls the emission of the greenhouse gas methane from the ocean floor. AOM is performed by microbial consortia of archaea (ANME) associated with partners related to sulfate-reducing bacteria. In vitro enrichments of AOM were so far only successful at temperatures ⩽25 °C; however, energy gain for growth by AOM with sulfate is in principle also possible at higher temperatures. Sequences of 16S rRNA genes and core lipids characteristic for ANME as well as hints of in situ AOM activity were indeed reported for geothermally heated marine environments, yet no direct evidence for thermophilic growth of marine ANME consortia was obtained to date. To study possible thermophilic AOM, we investigated hydrothermally influenced sediment from the Guaymas Basin. In vitro incubations showed activity of sulfate-dependent methane oxidation between 5 and 70 °C with an apparent optimum between 45 and 60 °C. AOM was absent at temperatures ⩾75 °C. Long-term enrichment of AOM was fastest at 50 °C, yielding a 13-fold increase of methane-dependent sulfate reduction within 250 days, equivalent to an apparent doubling time of 68 days. The enrichments were dominated by novel ANME-1 consortia, mostly associated with bacterial partners of the deltaproteobacterial HotSeep-1 cluster, a deeply branching phylogenetic group previously found in a butane-amended 60 °C-enrichment culture of Guaymas sediments. The closest relatives (Desulfurella spp.; Hippea maritima) are moderately thermophilic sulfur reducers. Results indicate that AOM and ANME archaea could be of biogeochemical relevance not only in cold to moderate but also in hot marine habitats.     

Microbial mechanism for rice variety control on methane emission from rice field soil

Rice variety is one of the key factors regulating methane (CH₄) production and emission from the paddy fields. However, the relationships between rice varieties and populations of microorganisms involved in CH₄ dynamics are poorly understood. Here we investigated CH₄ dynamics and the composition and abundance of CH₄‐producing archaea and CH₄‐oxidizing bacteria in a Chinese rice field soil planted with three types of rice. Hybrid rice produced 50–60% more of shoot biomass than Indica and Japonica cultivars. However, the emission rate of CH₄ was similar to Japonica and lower than Indica. Furthermore, the dissolved CH₄ concentration in the rhizosphere of hybrid rice was markedly lower than Indica and Japonica cultivars. The rhizosphere soil of hybrid rice showed a similar CH₄ production potential but a higher CH₄ oxidation potential compared with the conventional varieties. Terminal restriction fragment length polymorphism analysis of the archaeal 16S rRNA genes showed that the hydrogenotrophic methanogens dominated in the rhizosphere whereas acetoclastic methanogens mainly inhabited the bulk soil. The abundance of total archaea as determined by quantitative (real‐time) PCR increased in the later stage of rice growth. However, rice variety did not significantly influence the structure and abundance of methanogenic archaea. The analysis of pmoA gene fragments (encoding the α‐subunit of particulate methane monooxygenase) revealed that rice variety also did not influence the structure of methanotrophic proteobacteria, though variable effects of soil layer and sampling time were observed. However, the total copy number of pmoA genes in the rhizosphere of hybrid rice was approximately one order of magnitude greater than the two conventional cultivars. The results suggest that hybrid rice stimulates the growth of methanotrophs in the rice rhizosphere, and hence enhances CH₄ oxidation which attenuates CH₄ emissions from the paddy soil. Hybrid rice is becoming more and more popular in Asian countries. The present study demonstrated that planting of hybrid rice will not enhance CH₄ emissions albeit a higher grain production than the conventional varieties.     

Effect of rice plants on methane production and rhizospheric metabolism in paddy soil

In order to elucidate the effects of rice plants on CH₄ production, we conducted experiments with soil slurries and planted rice microcosms. Methane production in anoxic paddy soil slurries was stimulated by the addition of rice straw, of unsterile or autoclaved rice roots, and of the culture fluid in which rice plants had axenically been cultivated. The addition of these compounds also increased the concentrations of acetate and H₂, precursors of CH₄ production, in the soil. Planted compared to unplanted paddy soil microcosms exhibited lower porewater CH₄ concentrations but higher CH₄ emission rates. They also exhibited higher sulfate concentrations but similar nitrate concentrations. Concentrations of acetate, lactate and H₂ were not much different between planted and unplanted microcosms. Pulse labeling of rice plants with¹⁴CO₂ resulted during the next 5 days in transient accumulation of radioactive lactate, propionate and acetate, and after the second day of incubation in the emission of¹⁴CH₄. Most of the radioactivity (40–70%) was incorporated into the above-ground biomass of rice plants. However, during a total incubation of 16 days about 3–6% of the applied radioactivity was emitted as¹⁴CH₄, demonstrating that plant-derived carbon was metabolized and significantly contributed to CH₄ production. The sequence of the appearance of radioactive products and their specific radioactivities indicate that CH₄ was produced from root exudates by a microbial community consisting of fermenting and methanogenic bacteria.     

Butachlor inhibits production and oxidation of methane in tropical rice soils under flooded condition

In laboratory incubation experiments, application of a commercial formulation of the herbicide butachlor (N-butoxymethyl-2-chloro-2',6'-diethyl acetanilide) to three tropical rice soils, widely differing in their physicochemical characteristics, under flooded condition inhibited methane (CH4) production. The inhibitory effect was concentration dependent and most remarkable in the alluvial soil. Thus, following application of butachlor at 5, 10, 50 and 100 microg g(-1) soil, respectively, cumulative CH4 production in the alluvial soil was inhibited by 15%, 31%, 91% and 98% over unamended control. Since CH4 production was less pronounced in the sandy loam and acid sulfate soil, the impact of amendment with butchalor, albeit inhibitory, was less extensive than the alluvial soil. Inhibition of CH4 production in butachlor-amended alluvial soil was related to the prevention in the drop in redox potential as well as low methanogenic bacterial population especially at high concentrations of butachlor. CH4 oxidation was also inhibited in butachlor-amended alluvial soil with the inhibitory effect being more prevalent under flooded condition. Inhibition in CH4 oxidation was related to a reduction in the population of soluble methane monooxygenase producing methanotrophs. Results demonstrate that butachlor, a commonly used herbicide in rice cultivation, even at very low concentrations can affect CH4 production and its oxidation, thereby influencing the biogeochemical cycle of CH4 in flooded rice soils.     

Responses of oxidation rate and microbial communities to methane in simulated landfill cover soil microcosms

CH4 oxidation capacities and microbial community structures developed in response to the presence of CH4 were investigated in two types of landfill cover soil microcosms, waste soil (fine material in stabilized waste) and clay soil. CH4 emission fluxes were lower in the waste soil cover over the course of the experiment. After exposure to CH4 flow for 120 days, the waste soil developed CH4 oxidation capacity from 0.53 to 11.25-13.48micromol CH4gd.w.(-1)h(-1), which was ten times higher than the clay soil. The topsoils of the two soil covers were observed dried and inhibited CH4 oxidation. The maximum CH4 oxidation rate occurred at the depth of 10-20cm in the waste soil cover (the middle layer), whereas it took place mainly at the depth of 20-30cm in the clay soil cover (the bottom layer). The amounts of the phospholipid fatty acid (PLFA) biomarks 16:1omega8c and 18:1omega8c for type I and II methanotrophs, respectively, showed that type I methanotrophic bacteria predominated in the clay soil, while the type II methanotrophic bacteria were abundant in the waste soil, and the highest population in the middle layer. The results also indicated that a greater active methanotrophic community was developed in the waste soil relative to the clay soil.     

Assimilation of methane and inorganic carbon by microbial communities mediating the anaerobic oxidation of methane

The anaerobic oxidation of methane (AOM) is a major sink for methane on Earth and is performed by consortia of methanotrophic archaea (ANME) and sulfate-reducing bacteria (SRB). Here we present a comparative study using in vitro stable isotope probing to examine methane and carbon dioxide assimilation into microbial biomass. Three sediment types comprising different methane-oxidizing communities (ANME-1 and -2 mixture from the Black Sea, ANME-2a from Hydrate Ridge and ANME-2c from the Gullfaks oil field) were incubated in replicate flow-through systems with methane-enriched anaerobic seawater medium for 5–6 months amended with either ¹³CH₄ or H¹³CO₃^-. In all three sediment types methane was anaerobically oxidized in a 1:1 stoichiometric ratio compared with sulfate reduction. Similar amounts of ¹³CH₄ or ¹³CO₂ were assimilated into characteristic archaeal lipids, indicating a direct assimilation of both carbon sources into ANME biomass. Specific bacterial fatty acids assigned to the partner SRB were almost exclusively labelled by ¹³CO₂, but only in the presence of methane as energy source and not during control incubations without methane. This indicates an autotrophic growth of the ANME-associated SRB and supports previous hypotheses of an electron shuttle between the consortium partners. Carbon assimilation efficiencies of the methanotrophic consortia were low, with only 0.25–1.3 mol% of the methane oxidized.     

Vertical distribution of the methanotrophic community after drainage of rice field soil

Anoxic soils, such as flooded rice fields, are major sources of the greenhouse gas CH(4) while oxic upland soils are major sinks of atmospheric CH(4). Nevertheless, CH(4) is also consumed in rice fields where up to 90% of the produced CH(4) is oxidized in a narrow oxic zone around the rice roots and in the soil surface layer before it escapes into the atmosphere. After 1 day drainage of rice field soil, CH(4) oxidation was detected in the top 2-mm soil layers, but after 8 days drainage the zone of CH(4) oxidation extended to 8 mm depth. Simultaneously, the potential for CH(4) production decreased, but some production was still detectable after 8 days drainage throughout the soil profile. The vertical distribution of the methanotrophic community was also monitored after 1 and 8 days drainage using denaturing gradient gel electrophoresis after PCR amplification with primer sets targeting two regions on the 16S rRNA gene that are relatively specific for methylotrophic alpha- and gamma-Proteobacteria, and targeting two functional genes encoding subunits of key enzymes in all methanotrophs, i.e. the genes for the particulate methane monooxygenase (pmoA) and the methanol dehydrogenase (mxaF). Drainage stimulated the methanotrophic community. Eight days after drainage, new methanotrophic populations appeared and a distinct methanotrophic community developed. The population structure of type I and II methanotrophs was differently affected by drainage. Type II methanotrophs (alpha-Proteobacteria) were present throughout the soil core directly after drainage (1 day), and the community composition remained largely unchanged with depth. Only two new type II populations appeared after 8 days of drainage. Drainage had a more pronounced impact on the type I methanotrophic community (gamma-Proteobacteria). Type I populations were not or only weakly detected 1 day after drainage. However, after 8 days of drainage, a large diversity of type I methanotrophs were detected, altough they were not evenly distributed throughout the soil core but dominated at different depths. A distinct type I community structure had developed within each soil section between 0 and 20 mm soil depth, indicating the widening of suitable habitats for methanotrophs in the rice field soil within 1 week of drainage.     

Thermophilic methane production and oxidation in compost

Methane cycling within compost heaps has not yet been investigated in detail. We show that thermophilic methane oxidation occurred after a lag phase of up to one day in 4-week old, 8-week old and mature (>10-week old) compost material. The potential rate of methane oxidation was between 2.6 and 4.1 micromol CH4(gdw)(-1)h(-1). Profiles of methane concentrations within heaps of different ages indicated that 46-98% of the methane produced was oxidised by methanotrophic bacteria. The population size of thermophilic methanotrophs was estimated at 10(9) cells (gdw)(-1), based on methane oxidation rates. A methanotroph (strain KTM-1) was isolated from the highest positive step of a serial dilution series. This strain belonged to the genus Methylocaldum, which contains thermotolerant and thermophilic methanotrophs. The closest relative organism on the basis of 16S rRNA gene sequence identity was M. szegediense (>99%), a species originally isolated from hot springs. The temperature optimum (45-55 degrees C) for methane oxidation within the compost material was identical to that of strain KTM-1, suggesting that this strain was well adapted to the conditions in the compost material. The temperatures measured in the upper layer (0-40 cm) of the compost heaps were also in this range, so we assume that these organisms are capable of effectively reducing the potential methane emissions from compost.     

Quantification of methane oxidation in the rice rhizosphere using 13C-labelled methane

In this paper isotope ratio mass spectrometry is used to determine the methane (CH₄) oxidation fraction in the rhizosphere of intact rice plant-soil systems. Earlier studies on quantification of the methane oxidation were based on inhibition or incubation procedures which strongly interfered with the plant-soil system and resulted in a large variability of the reported fractions, while other studies considered stable isotopes at natural abundance levels to investigate methanotrophy in the rhizosphere of rice. The current work is the first that used ¹³C-labelled CH₄ as additive and calculated the oxidation fraction from the ratio between the added ¹³C-labelled CH₄ and its oxidation product ¹³CO₂. Both labelled gases could be distinguished from the natural abundance percentages. The oxidation fraction for methane was found to be smaller than 7%, suggesting that former approaches overestimate the methane oxidation fraction.     

典型草地土壤好氧甲烷氧化的微生物生态过程

【目的】针对我国甘肃三个典型生态区草地土壤(玛曲MQ、临泽LZ和环县HX),研究其甲烷氧化潜力、甲烷氧化菌(methane-oxidizingbacteria,MOB)丰度及可能存在的群落分异规律。【方法】通过原位分析、室内高浓度甲烷模拟培养三种典型土壤及实时荧光定量、高通量测序的方法研究甲烷氧化菌标靶基因pmoA序列的组成及其丰度变化规律。【结果】三种典型草地土壤的原位甲烷氧化菌的丰度存在显著差异,表现为MQ>HX>LZ,其数量范围为为0.18–6.86×10^7g/d.w.s.;甲烷氧化潜力也表现出类似规律,其通量为109–169mg/(m^2·h);甲烷氧化潜力与原位土壤中甲烷氧化菌丰度有正相关。三种草地土壤甲烷氧化菌存在明显的空间异质性,采用高通量测序的方法,发现三种草地原位土壤中的优势类群为USCγ(Upland Soil Cluster gamma,USCγ);然而,室内高浓度甲烷氧化过程中,传统的甲烷氧化菌均发生明显增加,MQ土壤中TypeⅡ的Methylocystis为优势类群,而LZ和HX土壤的优势类群均为TypeⅠ型Methylosarcina。【结论】这些研究结果表明,我国甘肃典型草地土壤中也存在难培养的大气甲烷氧化菌和经典的可培养甲烷氧化菌,这些微生物极可能氧化极低浓度的大气甲烷,也可能利用闭蓄于土壤中的高浓度甲烷生长。未来应采用先进技术原位观测大气甲烷氧化过程并分离相应微生物类群,研究草地土壤甲烷氧化菌地理分异规律及其环境驱动机制。     

Production, oxidation and emission of methane in rice paddies

Abstract Production and emission of methane from submerged paddy soil was studied in laboratory rice cultures and in Italian paddy fields. Up to 80% of the CH₄ produced in the paddy soil did not reach the atmosphere but was apparently oxidized in the rhizosphere. CH₄ emission through the rice plants was inhibited by an atmosphere of pure O₂ but was stimulated by an atmosphere of pure N₂ or an atmosphere containing 5% acetylene. Gas bubbles taken from the submerged soil contained up to 60% CH₄, but only < 1% CH₄ after the bubbles had passed the soil-water interface or had entered the intercellular gas space system of the rice plants. CH₄ oxidation activities were detected in the oxic surface layer of the submerged paddy soil. Flooding the paddy soil with water containing > 0.15% sea salt (0.01% sulfate) resulted in a strong inhibition of the rates of methanogenesis and a decrease in the rates of CH₄ emission. This result explains the observation of relatively low CH₄ emission rates in rice paddy areas flooded with brackish water.     

Intensities of microbial production and oxidation of methane in bottom sediments and water mass of the Black Sea

Intensities of biogeochemical (microbial) processes of methane production and methane oxidation were determined in bottom sediments and water column of the Black Sea. Aerobic bacterial oxidation of methane is confined to the upper 20-30 cm of Holocene bottom sediments of the shelf (0.7-259 ng C/(dm3 day)) and oxygenated waters (0.2-45 ng C/(dm3 day)). In reduced sediments of the deep-sea zone and in the hydrogen sulfide-containing water column, considerable intensities of anaerobic methane oxidation were recorded, comparable to or exceeding the intensities of methane oxidation in oxygenated layers. From one fourth to one half of the methane formed in bottom sediments was oxidized immediately therein. The major part of the remaining methane was oxidized in the water column, and a smaller portion arrived in the atmosphere.     

Molecular analyses of the methane-oxidizing microbial community in rice field soil by targeting the genes of the 16S rRNA, particulate methane monooxygenase, and methanol dehydrogenase

Rice field soil with a nonsaturated water content induced CH4 consumption activity when it was supplemented with 5% CH4. After a lag phase of 3 days, CH4 was consumed rapidly until the concentration was less than 1.8 parts per million by volume (ppmv). However, the soil was not able to maintain the oxidation activity at near-atmospheric CH4 mixing ratios (i.e., 5 ppmv). The soil microbial community was monitored by performing denaturing gradient gel electrophoresis (DGGE) during the oxidation process with different PCR primer sets based on the 16S rRNA gene and on functional genes. A universal small-subunit (SSU) ribosomal DNA (rDNA) primer set and 16S rDNA primer sets specifically targeting type I methylotrophs (members of the gamma subdivision of the class Proteobacteria [gamma-Proteobacteria]) and type II methylotrophs (members of the alpha-Proteobacteria) were used. Functional PCR primers targeted the genes for particulate methane monooxygenase (pmoA) and methanol dehydrogenase (mxaF), which code for key enzymes in the catabolism of all methanotrophs. The yield of PCR products amplified from DNA in soil that oxidized CH4 was the same as the yield of PCR products amplified from control soil when the universal SSU rDNA primer set was used but was significantly greater when primer sets specific for methanotrophs were used. The DGGE patterns and the sequences of major DGGE bands obtained with the universal SSU rDNA primer set showed that the community structure was dominated by nonmethanotrophic populations related to the genera Flavobacterium and Bacillus and was not influenced by CH4. The structure of the methylotroph community as determined with the specific primer sets was less complex; this community consisted of both type I and type II methanotrophs related to the genera Methylobacter, Methylococcus, and Methylocystis. DGGE profiles of PCR products amplified with functional gene primer sets that targeted the mxaF and pmoA genes revealed that there were pronounced community shifts when CH4 oxidation began. High CH4 concentrations stimulated both type I and II methanotrophs in rice field soil with a nonsaturated water content, as determined with both ribosomal and functional gene markers.     

Coupling of microbial processes of methane and ammonium oxidation in soils

The effect of ammonium ions on the activity of methane oxidation in soils was studied. The degree of inhibition of the methanotrophic activity in the presence of ammonium in the soil solution was quantitatively assessed as dependent on ammonium concentration and the properties of different types of soils of the European part of Russia.