不同水分条件下格陵兰岛冻土活性甲烷氧化菌群落分异规律

揭全球气候变化导致丹麦格陵兰岛形成了旱地和间歇淹水的土壤景观,采用稳定性同位素核酸探针技术和高通量测序16S r RNA及pmoA基因的分析方法,开展了格陵兰岛旱地和间歇淹水土壤微宇宙培养试验,探究不同水分条件下冻土的甲烷氧化潜力及活性好氧甲烷氧化菌群落演替规律。结果表明:与旱地土壤相比,淹水土壤氧化高浓度甲烷的速率呈现降低趋势,分别为12.38和12.17μg/(g·d),但后者对甲烷碳同化利用效率显著高于前者,土壤~(13)C-有机碳原子百分比从自然丰度1.08%,分别增加至1.64%和1.99%。超高速密度梯度离心分析~(13)C-DNA发现甲烷氧化菌群落发生演替,旱地土壤中Crenothrix甲烷氧化菌16S rRNA基因丰度仅为0.04%,而在间歇淹水土壤中为23.78%,增幅高达557倍;类型Ⅱ甲烷氧化菌Methylosinus则从33.76%增至44.38%。然而,类型Ⅰ甲烷氧化菌Methylocaldum的丰度明显降低,从旱地土壤10.15%显著降低为间歇淹水0.14%;进一步通过pmo A基因高通量测序分析,也得到了类似的结果,特别是类型Ⅰ甲烷氧化菌RPCs从旱地土壤15.61%显著降低至间歇淹水土壤的0.13%。这些结果表明:尽管格陵兰冻土中经典的类型Ⅱ甲烷氧化菌主导了旱地土壤和间歇性淹水土壤好氧甲烷氧化过程,但水分可能是甲烷氧化菌群落演替的重要环境驱动力,水分增加导致活性的类型Ⅰ种群丰度降低,同时显著刺激了新型甲烷氧化菌Crenothrix的大量生长并可能在间歇淹水土壤中发挥了重要作用。     

水稻土好氧甲烷氧化菌对大气CO2浓度升高的适应规律

CH4是仅次于CO2的第二大温室气体,而稻田是CH4的主要排放源,但未来大气CO2浓度升高情景下（elevated CO2, eCO2）,水稻土好氧甲烷氧化过程及其功能微生物群落适应规律尚不清楚。本研究依托中国FACE（Free Air CO2 Enrichment）水稻田试验平台,通过13C-CH4示踪的室内微宇宙培养实验,采用稳定性同位素核酸探针（DNA-SIP）和高通量测序技术,研究了未来大气CO2浓度升高对水稻土甲烷氧化活性及其功能微生物的影响规律。研究结果表明：与常规大气CO2浓度（ambient CO2, aCO2）相比,eCO2条件下的甲烷氧化活性显著增加,从243 nmol g-1 d.w.s h-1增加至302 mol g-1 d.w.s h-1,增幅高达24.3%,甲烷氧化菌数量则增加了1.1~1.2倍。通过超高速离心获得活性甲烷氧化菌同化13CH4后合成的13C-DNA,高通量测序发现,未来大气CO2升高情景下水稻土活性好氧甲烷氧化微生物群落极可能发生明显演替,与对照相比,类型I甲烷氧化菌甲基杆菌属Methylobacter的相对丰度增加16.2%~17.0%,而甲基八叠球菌属Methylosarcina的相对丰度下降4.7%-11.1%;同时刺激了食酸菌属Acidovorax和假单胞菌属Pseudomonas等非甲烷氧化菌的活性。这些研究结果表明：未来大气CO2升高情景下,水稻土好氧甲烷氧化微生物群落结构发生分异,促进了甲烷氧化通量,而甲烷氧化的代谢产物可能引发土壤中微生物食物网的级联反应,是土壤碳储存和周转的重要功能微生物群。     

稻田水分管理和秸秆还田对甲烷排放的微生物影响

为了探讨不同水分管理和秸秆还田对稻田甲烷排放的影响机理,依托中国科学院亚热带农业生态研究所长沙农业环境监测研究站长期定位试验,选择长期淹水(CF)、秸秆还田+长期淹水(HS+CF)、常规灌溉(IF)、秸秆还田+常规灌溉(HS+IF)4个处理,应用限制性片段长度多态技术(T-RFLP)和实时荧光定量PCR技术在DNA和c DNA水平对比研究不同水分管理和秸秆还田影响稻田土壤甲烷排放的微生物分子机理。结果表明:长期淹水处理的甲烷排放量显著高于常规灌溉处理,秸秆还田处理对甲烷排放的影响不显著。产甲烷菌和甲烷氧化菌群落结构只对土壤水分管理产生响应,对秸秆还田没有响应。水分管理和秸秆还田显著影响产甲烷菌的数量,却对甲烷氧化菌数量没有影响。产甲烷菌和甲烷氧化菌群落组成和表达群落组成存在明显的分异,c DNA水平上产甲烷菌和甲烷氧化菌表达群落组成对水分管理的响应比DNA水平群落组成更敏感。稻田甲烷排放通量与mcr A和pmo A基因丰度和表达丰度均没有显著相关性,只与DNA水平基因丰度比值(mcr A/pmo A)呈显著正相关关系。可以看出,长期淹水处理可以通过改变土壤中产甲烷菌和甲烷氧化菌的群落结构和数量及两者数量的比例来调控甲烷的排放量。     

大于80年自然风干保存土壤中甲烷氧化菌的复苏与富集

本研究通过对长期保存的土壤标本中甲烷氧化菌的复苏和富集培养，明确了活性甲烷氧化菌类群及其氧化甲烷潜力，同时分析了对长期干旱胁迫有较强抗逆性的甲烷氧化菌种类。研究主要针对自然风干保存83～87年的采集自福建龙岩荒地、江西南昌水田、四川华阳旱地、甘肃榆中林地、青海湟源和青海都兰牧场的6个土壤标本进行了高浓度甲烷条件下的微宇宙培养，利用气相色谱法测定了甲烷气体浓度动态变化情况以研究其甲烷氧化能力；在获得富集培养物后提取基因组DNA，利用16S rRNA和pmo A基因高通量测序分析了整体微生物和甲烷氧化菌的群落结构；同时，利用两种基因的长片段克隆测序对优势甲烷氧化菌序列进行了系统发育分析。结果显示：经过短暂的复苏，6例土壤档案样品均表现出强烈的甲烷氧化活性，前两代富集液甲烷氧化速率较慢，甲烷氧化速率仅为1.4～3.8μg/(mL·h)，而第三代富集液的甲烷氧化速率达到了4.9～7.7μg/(mL·h)。其中，福建龙岩、江西南昌、青海都兰3个样品的富集物还可以将高浓度甲烷持续氧化到大气甲烷浓度(1.8μL/L)以下。高通量测序结果表明，6种富集物中甲烷氧化菌在细菌总量中的占比为39%～85%;...     

模拟干湿交替对水稻土古菌群落结构的影响

干湿交替是自然界普遍存在的现象,但长期以来由于技术的限制,复杂土壤中微生物对水分变化的响应规律仍不清楚。针对我国江苏常熟湖泊底泥发育的典型水稻土,在室内开展湿润-风干以及风干-湿润各三次循环,每次循环中湿润、风干状态各维持7d,利用微生物核糖体rRNA的通用引物进行PCR扩增,通过高通量测序分析土壤古菌多样性变化,同时结合实时荧光定量PCR技术,在DNA和RNA水平研究古菌数量对干湿交替过程的响应规律。结果表明:水稻土湿润-风干过程中,在DNA水平土壤古菌数量降幅约为149倍~468倍,而在RNA水平降幅最高仅为2.06倍;水稻土风干-湿润过程中,在DNA水平古菌数量增幅在147倍~360倍之间,而在RNA水平增幅最高仅为2.95倍。表明在干湿交替过程中,DNA水平的古菌16S rRNA基因数量变化远高于RNA水平。基于高通量测序多样性的结果表明,在DNA和RNA水平,湿润土壤3次风干、以及风干土壤3次加水湿润7d恢复后,土壤古菌群落结构均发生统计显著性改变。在微生物门、纲、目、科和属的不同分类水平下,水稻土古菌主要包括3、10、13、14、10种不同的类群,在RNA和DNA水平的结果基本一致。干湿交替导致部分古菌类群发生显著变化,其中在微生物分类学目水平发生显著变化的古菌最高达到6种,主要包括产甲烷古菌和氨氧化古菌,如Methanobacteriales、Methanosarcinales、Methanomicrobiales和Nitrososphaerales等。这些研究结果表明,反复的干湿交替并未显著改变水稻土中古菌的主要类群组成,古菌类群的绝对数量和相对丰度发生了一定程度的变化,但这些变化与微生物生理作用的联系仍需进一步研究;风干土壤中古菌RNA序列极可能来自于完整的古菌细胞,暗示了这些古菌细胞能够较好地适应水稻土中水分的剧烈变化,风干状态的土壤在一定程度也可用于土壤古菌群落组成研究。     

基于实时荧光定量PCR技术的油气微生物勘探样品保存研究

针对一个油气藏和两个非油气藏上方的土壤剖面,围绕典型油气指示微生物甲烷氧化细菌,采用分子生态学技术比较了新鲜、自然风干和冷冻干燥3种处理下土壤中甲烷氧化细菌标靶基因pmoA的变化规律,研究了自然风干土壤能否用于油气资源微生物勘探.与新鲜土壤相比,自然风干和冷冻干燥显著降低了不同土壤剖面微生物总DNA含量,甲烷氧化细菌pmoA基因数量最多分别下降了90.7％和77.5％;然而,与非油气藏上方土壤剖面相比,自然风干和冷冻干燥处理后,油气藏上方不同土壤剖面依然检测到了大量的甲烷氧化细菌pmoA基因.本研究中,自然风干或冷冻干燥处理后的土壤适合于油气微生物勘探.     

土壤CH4氧化与土壤甲烷营养菌及主要研究方法

甲烷营养菌(methanotrophs)是一类以CH4为唯一碳源和能源的细菌,广泛分布在水稻土、森林土、苔原土、泥炭地、海洋与湖泊底泥、堆肥、垃圾填埋场及地下水等环境中,并作为大气甲烷(CH4)唯一的生物汇(库),在全球温室效应研究中备受关注。目前,关于土壤甲烷营养菌的研究主要包括菌株的多样性、生态分布以及环境因素对微生物氧化CH4过程的影响。本文从甲烷营养菌的分类入手,概述稻田土壤CH4的氧化与释放、旱地土壤CH4的氧化以及影响土壤CH4氧化的因素等方面的研究进展,同时介绍了土壤甲烷营养菌研究领域的几种主要的分子研究技术,以期为甲烷营养菌相关的研究提供参考。     

生物炭与氮肥对稻田甲烷产生与氧化菌数量和潜在活性的影响

基于稻田中氮肥配施生物炭的田间定位试验,研究了施用生物炭与氮肥对旱季稻田土壤理化性质、甲烷氧化与产生潜势及甲烷氧化菌和产甲烷菌丰度的影响。田间试验共设置5个处理:单施生物炭、单施氮肥、氮肥配施生物炭(生物炭设置两个水平)以及对照。结果表明:施用生物炭三年后显著提高了有机碳和微生物生物量碳含量(p﹤0.05),与单施氮肥处理相比,氮肥配施生物炭后可显著提高土壤p H。与对照相比,单施生物炭显著提高土壤甲烷氧化潜势。在施氮条件下,甲烷氧化潜势与生物炭施用量之间存在正相关关系,与氮肥配施20 t hm-2处理相比,40 t hm-2生物炭处理甲烷氧化潜势增长53.8%。氮肥配施高倍生物炭与配施低倍生物炭处理相比产甲烷潜势由0.001提高至0.002 mg kg-1 h-1;氮肥施用一定程度上抑制了甲烷氧化菌数量的增长,单施氮肥处理中产甲烷菌数量较对照处理显著增加了3.0%;单施或配施低水平生物炭显著增加土壤甲烷氧化菌数量。氮肥显著降低了甲烷氧化菌与产甲烷菌基因丰度比(pmo A/mcr A)。而在同氮肥水平下施加生物炭显著增加了土壤pmo A/mcr A比值,即生物炭对甲烷氧化菌的促进作用显著高于产甲烷菌,提高了旱季稻田土壤的甲烷氧化能力,因此有助于减少稻田土壤甲烷的排放。     

大气CO2浓度缓增对稻田土壤甲烷氧化过程的影响

微生物介导的甲烷好氧氧化,对控制稻田甲烷排放起着重要作用。本文从基因、群落、活性等多个层次上解析CO₂浓度缓增对稻田土壤甲烷好氧氧化过程的影响及其作用机理。依托于田间CO₂浓度自动调控平台,在背景CO₂浓度(AC)基础上,设置了CO₂浓度缓增处理(每年增加40μL·L^-1,持续4年)(EC)。采用室内泥浆培养以及高通量测序和定量PCR技术,对不同CO₂处理下水稻关键生育期(分蘖期、拔节期、扬花期和乳熟期)土壤中的甲烷氧化潜势及其功能微生物的丰度和群落结构进行了系统研究。结果表明：大气CO₂浓度升高促进了稻田甲烷氧化潜势和甲烷氧化菌丰度的增加;CO₂浓度升高还使得土壤中甲烷氧化菌的群落结构发生了显著变化,其优势菌从Ⅱ型菌转变为Ⅰ型菌。CO₂浓度升高所致的土壤中甲烷、氧气浓度以及氮素水平等的改变很可能对稻田甲烷氧化过程产生了重要影响。综合本研究发现,稻田甲烷氧化过程对大气CO₂     

枯草芽孢杆菌B11基因文库的构建及与拮抗物质的生物合成相关基因的克隆

枯草芽孢杆菌菌株B 11对广泛的植物病原真菌和细菌都具有拮抗作用。以柯斯质粒pW EB∷TNC为载体构建了枯草芽孢杆菌菌株B 11的基因文库,文库含9 000个克隆。文库克隆中插入的DNA片段平均为42.1 kb,该文库含有菌株B 11基因组中任一基因的概率为99.99%。采用平板活性检测法筛选文库,筛选到1个对茄青枯假单胞菌菌株P 13具有拮抗活性的文库克隆GXN 9527,该克隆的重组质粒pGXN 9527含有50 kb的菌株B 11的DNA。文库克隆对革兰氏阴性植物病原细菌如水稻黄单胞菌水稻变种也具有拮抗活性,而对革兰氏阳性细菌如地衣芽孢杆菌和植物病原真菌如尖孢镰刀菌西瓜专化型、立枯丝核菌、水稻稻灰梨孢菌则没有拮抗活性。分别含有pGXN 9527的18、12、9、8 kb B amHⅠ片段的亚克隆对P 13均没有拮抗活性,说明编码该拮抗物质的生物合成基因很可能成簇存在。     

Recovery of paddy soil methanotrophs from long term drought

Air-dried paddy soils stored for 1–18 years were used to examine the resistance of methanotrophs to drought. Older air-dried soils representing longer-lasting drought events reduced methanotrophic diversity, and adversely affected methane oxidation rate after re-wetting. In early incubations the type II methanotrophs are outperformed by the less abundant type I.     

The differential effects of ammonium and nitrate on methanotrophs in rice field soil

It has been known that nitrogenous fertilizers can either stimulate or inhibit methane oxidation in soils. The mechanism, however, remains unclear. Here we conducted laboratory incubation experiments to evaluate the effects of ammonium versus nitrate amendment on CH₄ oxidation in a rice field soil. The results showed that both N forms stimulated CH₄ oxidation. But nitrate stimulated CH₄ oxidation to a greater extent than ammonium per unit N base. The 16S rRNA genes and the pmoA genes were analyzed to determine the dynamics of total bacterial and methanotrophic populations, respectively. The methanotrophic community consisted of type I and type II methanotrophs and was dominated by type I group after two weeks of incubation. Nitrate promoted both types of methanotrophs, but ammonium promoted only type I. DNA-based stable isotope probing confirmed that ammonium stimulated the incorporation of ¹³CH₄ into type I methanotrophs but not type II, while nitrate caused almost homogenous distribution of ¹³CH₄ in type I and type II methanotrophs. Our study suggests that nitrate can promote CH₄ oxidation more significantly than ammonium and is probably a better N source for both types of methanotrophs in rice field soil. More investigations, e.g. using ¹⁵N labeling, are necessary to elucidate this possibility.     

Changes in methanotrophic community composition after rice crop harvest in tropical soils

Four selected tropical field sites from India were studied to assess the diversity and community structure of methanotrophs in rice fields following crop harvest. The rate of methane oxidation ranged from 0.04 to 0.11 μmol L⁻¹ h⁻¹ g⁻¹ dry weight in soils. Methanotrophic population size was high for the agriculture farm of Banaras Hindu University (BHU) site followed by agriculture farm of the Indian Institute of Vegetable Research (IIVR), Ghazipur, and Barkachcha. The cloning, restriction fragment length polymorphism, and sequence analyses of the pmoA gene fragment amplified from soil DNA extracts revealed the presence of type I and type II methanotrophs. The phylogenetic affiliation and community analysis based on the presence or absence of sequences showed that methanotrophs community composition in Barkachcha and Ghazipur soils was similar. IIVR soils, however, were quite different, while BHU soils were intermediate among the sites with regard to methanotrophic community composition. Diversity index of the methanotrophic community was high at the IIVR site. The study revealed that the rice harvest led to a change in type I methanotrophs from all the sites while type II community composition was almost uniform.     

Methanotrophic communities in a landfill cover soil as revealed by [13C] PLFAs and respiratory quinones: Impact of high methane addition and landfill leachate irrigation

The soil microbial communities of a landfill cover substrate, which was treated with landfill gas (100 l CH₄ m^?2 d^?1) and landfill leachate for 1.5 years, were investigated by phospholipid fatty acid (PLFA), ergosterol and respiratory quinone analyses. The natural ¹³C depletion of methane was used to assess the activity of methanotrophs and carbon turnover in the soil system. Under methane addition, the soil microbial community was dominated by PLFAs (14:0 and 16:1 isomers) and quinones (ubiquinone-8 and 18-methylene-ubiquinone-8) related to type I methanotrophs, and 18:1 PLFAs contained in type II methanotrophs. While type I methanotrophic PLFAs were ¹³C depleted, i.e. type I methanotrophs were actively oxidising and assimilating methane, ¹³C depletion of 18:1 PLFAs was low and inconsistent with their abundance. This, possibly reflects isotopic discrimination, assimilation of carbon derived from type I methanotrophs and a high contribution of non-methanotrophic bacteria to the 18:1 isomers. Landfill leachate irrigation caused the methanotrophic community to shift closer to the soil surface. It also decreased 18:1 PLFAs, while type I methanotrophs were probably stimulated. Gram positive bacteria, but not fungi, were also ¹³C depleted and consequently involved in the secondary turnover of carbon originating from methanotrophic bacteria. Cy17:0 PLFA was ¹³C depleted in deep soil layers, indicating anaerobic methane oxidation.     

Diversity, abundance and vertical distribution of methane-oxidizing bacteria (methanotrophs) in the sediments of the Xianghai wetland, Songnen Plain, northeast China

Purpose

Methanotrophs in wetlands are of great importance because up to 90 % of the methane (CH₄) produced in such wetlands could be oxidized by methanotrophs before reaching the atmosphere. The Xianghai wetland of Songnen Plain represents an important ecosystem in northeast China. However, methanotrophic characteristics in this ecosystem have not been studied in detail. The aim of this study is to give an overview of methanotrophic diversity and vertical distribution in the sediments of this important wetland.

Materials and methods

Sediment cores were collected from three freshwater marshes, each dominated by a particular vegetation type: Carex alata, Phragmites australis and Typha orientalis. The diversity of methanotrophs was studied by phylogenetic analysis of both the 16S rRNA gene and the particulate methane monooxygenase (pmoA) gene. Methanotroph abundance was determined by quantitative PCR (qPCR) targeting the pmoA gene; group-specific pmoA gene quantification was also used to estimate the abundance of each methanotrophic group.

Results and discussion

16S rRNA and pmoA gene homological analysis revealed the presence of type Ia, Ib and II methanotrophs. Novel pmoA sequences distantly affiliated to cultured Methylococcus sp. were detected, implying the existence of novel methanotrophs in the wetland. Most obtained representatives of Methylobacter genus (both 16S rRNA and pmoA genes) were closely clustered in relation to sequences acquired from the Zoige wetland, Tibet and Siberia permafrost soils, therefore suggesting methanotrophs belonging to Methylobacter genus shared characteristics with methanotrophs in cold areas. The dominance of type I methanotrophs (especially the Methylobacter genus) was detected by both clone library analysis and group-specific qPCR assay. The relatively high methanotroph diversity and pmoA copy numbers measured in the T. orientalis marsh sediments indicated that vegetation type played an important role during CH₄ oxidation in the wetland.

Conclusions

We present the first data set on methanotroph diversity and vertical distribution in the sediments of the Xianghai wetland. DNA sequences information of Methylococcus-like methanotrophs in the wetland will facilitate the isolating of novel methanotrophs from the wetland. In a worldwide context, our study has enriched the database of genotypic diversity of methanotrophs, which will help in the understanding of the geographical distribution of methanotrophic communities.     

Combined effect of fertilizer and herbicide applications on the abundance, community structure and performance of the soil methanotrophic community

The use of agrochemicals, such as mineral fertilizers and herbicides in agricultural systems, may affect the potential of soils to act as a sink for methane. Typically, the effect of each agrochemical on soil methane oxidation is investigated separately whereas in the field these agrochemicals are used together to form one comprehensive land management system. Here we report the results of field experiments that assessed the combined effect of multiple fertilizer and herbicide (nicosulfuron, dimethenamide and atrazine) applications on the soil methanotrophic community. Soils treated with organic fertilizer had three times higher methane oxidation rates compared to soils receiving mineral fertilizers. These higher oxidation rates were positively reflected in a significantly enhanced abundance of methanotrophs for the organic fertilized soils. In contrast, herbicide application did not alter significantly the soil methane oxidation rate or the methane-oxidizing population abundance. Subsequently, the methanotrophic community structure was analyzed with group-specific DGGE of 16S rRNA genes. Cluster analysis of the methanotrophic patterns clearly separated the mineral from organically fertilized soils. Less pronounced clustering differentiated between chemical and manual weed control. Furthermore, cluster analysis of the methanotrophic community revealed that soil type was the primary determinant of the community structure. Our results indicate that fertilizer type had the greatest influence on methane oxidizer activity and abundance. Soil type had the most pronounced effect on the microbial community structure.     

Methanotrophy-driven accumulation of organic carbon in four paddy soils of Bangladesh

Biological methane oxidation is a crucial process in the global carbon cycle that reduces methane emissions from paddy fields and natural wetlands into the atmosphere.However,soil organic carbon accumulation associated with microbial methane oxidation is poorly understood.Therefore,to investigate methane-derived carbon incorporation into soil organic matter,paddy soils originated from different parent materials(Inceptisol,Entisol,and Alfisol) were collected after rice harvesting from four major rice-producing regions in Bangladesh.Following microcosm incubation with 5%(volume/volume)¹³ CH₄,soil¹³ C-atom abundances significantly increased from background level of 1.08% to 1.88%–2.78%,leading to a net methane-derived accumulation of soil organic carbon ranging from 120 to 307 mg kg^-1.Approximately 23.6%–60.0% of the methane consumed was converted to soil organic carbon during microbial methane oxidation.The phylogeny of¹³ C-labeled pmoA(enconding the alpha subunit of the particulate methane monooxygenase) and 16 S rRNA genes further revealed that canonical α(type II) and γ(type I) Proteobacteria were active methane oxidizers.Members within the Methylobacter-and Methylosarcina-affiliated type Ia lineages dominated active methane-oxidizing communities that were responsible for the majority of methane-derived carbon accumulation in all three paddy soils,while Methylocystis-affiliated type IIa lineage was the key contributor in one paddy soil of Inceptisol origin.These results suggest that methanotroph-mediated synthesis of biomass plays an important role in soil organic matter accumulation.This study thus supports the concept that methanotrophs not only consume the greenhouse gas methane but also serve as a key biotic factor in maintaining soil fertility.     

Methane turnover and temperature response of methane-oxidizing bacteria in permafrost-affected soils of northeast Siberia

The abundance, activity, and temperature response of aerobic methane-oxidizing bacteria were studied in permafrost-affected tundra soils of northeast Siberia. The soils were characterized by both a high accumulation of organic matter at the surface and high methane concentrations in the water-saturated soils. The methane oxidation rates of up to 835 nmol CH₄ h⁻¹ g⁻¹ in the surface soils were similar to the highest values reported so far for natural wetland soils worldwide. The temperature response of methane oxidation was measured during short incubations and revealed maximum rates between 22 °C and 28 °C. The active methanotrophic community was characterized by its phospholipid fatty acid (PLFA) concentrations and with stable isotope probing (SIP). Concentrations of 16:1ω8 and 18:1ω8 PLFAs, specific to methanotrophic bacteria, correlated significantly with the potential methane oxidation rates. In all soils, distinct 16:1 PLFAs were dominant, indicating a predominance of type I methanotrophs. However, long-term incubation of soil samples at 0 °C and 22 °C demonstrated a shift in the composition of the active community with rising temperatures. At 0 °C, only the concentrations of 16:1 PLFAs increased and those of 18:1 PLFAs decreased, whereas the opposite was true at 22 °C. Similarly, SIP with ¹³CH₄ showed a temperature-dependent pattern. When the soils were incubated at 0 °C, most of the incorporated label (83%) was found in 16:1 PLFAs and only 2% in 18:1 PLFAs. In soils incubated at 22 °C, almost equal amounts of ¹³C label were incorporated into 16:1 PLFAs and 18:1 PLFAs (33% and 36%, respectively). We concluded that the highly active methane-oxidizing community in cold permafrost-affected soils was dominated by type I methanotrophs under in situ conditions. However, rising temperatures, as predicted for the future, seem to increase the importance of type II methanotrophs, which may affect methane cycling in northern wetlands.     

Abundance and community composition of methanotrophs in a Chinese paddy soil under long-term fertilization practices

Background, aim, and scope  As the second most important greenhouse gas, methane (CH₄) is produced from many sources such as paddy fields. Methane-oxidizing bacteria (methanotrophs) consume CH₄ in paddy soil and, therefore, reduce CH₄ emission to the atmosphere. In order to estimate the contribution of paddy fields as a source of CH₄, it is important to monitor the effects of fertilizer applications on the shifts of soil methanotrophs, which are targets in strategies to combat global climate change. In this study, real-time polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE) based on 16S rRNA and pmoA genes, respectively, were used to analyze the soil methanotrophic abundance and community diversity under four fertilization treatments: urea (N), urea and potassium chloride (NK), urea, superphosphate, and potassium chloride (NPK), and urea, superphosphate, potassium chloride, and crop residues (NPK+C), compared to an untreated control (CON). The objective of this study was to examine whether soil methanotrophs responded to the long-term, different fertilizer regimes by using a combination of quantitative and qualitative molecular approaches. Materials and methods  Soil samples were collected from the Taoyuan Experimental Station of Agro-ecosystem Observation at Changde (28°55′ N, 111°26′ E), central Hunan Province of China, in July 2006. Soil DNAs were extracted from the samples, then the 16S rRNA genes were quantified by real-time PCR and the pmoA genes were amplified via general PCR followed by DGGE, cloning, sequencing, and phylogenetic analysis. The community diversity indices were assessed through the DGGE profile. Results  Except for NPK, other treatments of N, NK, and NPK+C showed significantly higher copy numbers of type I methanotrophs (7.0–9.6 × 10⁷) than CON (5.1 × 10⁷). The copy numbers of type II methanotrophs were significantly higher in NPK+C (2.8 × 10⁸) and NK (2.5 × 10⁸) treatments than in CON (1.4 × 10⁸). Moreover, the ratio of type II to type I methanotrophic copy numbers ranged from 1.88 to 3.32, indicating that the type II methanotrophs dominated in all treatments. Cluster analyses based on the DGGE profile showed that the methanotrophic community in NPK+C might respond more sensitively to the environmental variation. Phylogenetic analysis showed that 81% of the obtained pmoA sequences were classified as type I methanotrophs. Furthermore, the type I-affiliated sequences were related to Methylobacter, Methylomicrobium, Methylomonas, and some uncultured methanotrophic clones, and those type II-like sequences were affiliated with Methylocystis and Methylosinus genera. Discussion  There was an inhibitory effect on the methanotrophic abundance in the N and a stimulating effect in the NK and NPK+C treatments, respectively. During the rice-growing season, the type II methanotrophs might be more profited from such a coexistence of low O₂ and high CH₄ concentration environment than the type I methanotrophs. However, type I methanotrophs seemed to be more frequently detected. The relatively complex diversity pattern in the NPK+C treatment might result from the strong CH₄ production. Conclusions  Long-term fertilization regimes can both affect the abundance and the composition of the type I and type II methanotrophs. The inhibited effects on methanotrophic abundance were found in the N treatment, compared to the stimulated effects from the NK and NPK+C treatments. The fertilizers of nitrogen, potassium, and the crop residues could be important factors controlling the abundance and community composition of the methanotrophs in the paddy soil. Recommendations and perspectives  Methanotrophs are a fascinating group of microorganisms playing an important role in the biogeochemical carbon cycle and in the control of global climate change. However, it is still a challenge for the cultivation of the methanotrophs, although three isolates were obtained in the extreme environments very recently. Therefore, future studies will be undoubtedly conducted via molecular techniques just like the applications in this study.