若尔盖高原泥炭沼泽湿地土壤细菌群落空间分布及其驱动机制

了解高原泥炭沼泽湿地生态系统土壤微生物群落结构组成、多样性及空间分布特征对认识高原湿地生态特征及演化过程至关重要。利用高通量测序技术，在局域尺度上研究了四川若尔盖高原泥炭沼泽湿地土壤细菌群落结构与多样性特征。通过进一步测定土壤及植物基本理化指标，量化采样点之间的地理距离，比较了细菌群落不同成员（稀有种和丰富种）的空间周转差异，分析了土壤环境变量和空间因子对细菌群落结构的相对贡献。结果表明：若尔盖泥炭土壤细菌群落主要由绿弯菌门（Chloroflexi）（26.25%）、变形菌门（Proteobacteria）（23.21%）、厚壁菌门（Firmicutes）（10.56%）等优势物种门类组成；土壤细菌群落结构表现出较强的空间依赖关系，群落结构相似性随采样点地理距离增加而逐渐降低，细菌群落的周转速率表现为总细菌群落 > 丰富种 > 稀有种；Mantel检验结果显示，地上生物量与细菌群落呈极显著相关性（P<0.01），其中，影响稀有种空间分布特征的环境因子还包括土壤硫含量、活性磷、Mn和土壤pH值；方差分解分析表明，局域尺度上的土壤因子对若尔盖高原泥炭沼泽土壤细菌群落构建的相对贡献大于空间因子，土壤异质性是影响微生物空间分布特征的关键因素。研究为开展高原湿地泥炭土壤微生物多样性调查及揭示微生物群落构建机制提供了重要参考。     

人工遮光和营养添加对河流反硝化活性和反硝化细菌群落结构的影响

河流生态系统受到人类活动例如河岸带森林植被毁损和农业活动施肥等的干扰日益加剧,而这些活动使河流接收的光照增多、河流的氮磷营养盐浓度增加。微生物的反硝化作用是河流去除氮的有效途径。在汉江的一级支流金水河上游核心保护区内选取6条溪流开展野外控制实验,利用营养添加模拟河流中营养的增加,遮盖河面来模拟源头溪流的隐蔽状态,来研究河流沉积物中微生物的反硝化作用对光照和营养改变的响应,并利用高通量测序(Mi Seq)技术研究在两种处理下河流沉积物中nir S型反硝化细菌的群落结构变化。结果显示:营养元素添加促进了沉积物的反硝化活性,河面遮盖抑制了沉积物的反硝化活性。营养添加和遮盖两种处理均降低了控制实验区域内脱氯单胞菌属(优势菌属)的相对丰度,同时也降低了该区域nir S型反硝化菌群落的Chao多样性。本研究初步证实了光照增加和河流的营养增加提高了河流沉积物反硝化活性,并为提高河流的脱氮能力提供科学依据。     

不同季节艾比湖湿地盐角草根际nirS-型与nirK-型反硝化细菌群落组成分析

旨在了解艾比湖湿地盐生植物盐角草根际与非根际中不同类型反硝化细菌的分布及其随季节变化情况,为温带干旱地区荒漠盐化生态系统的代表-艾比湖湿地在生态植被恢复过程中,由微生物推动的土壤氮素循环过程提供数据支撑。采集了艾比湖湿地夏、秋、春三个季节的盐角草根际和非根际土壤样本,通过高通量测序技术,比较分析了nirS-型和nirK-型两种类型的反硝化细菌的多样性和群落结构特点;利用RDA (redundancy analysis)探究了土壤理化因素对反硝化细菌多样性及群落结构的影响。艾比湖湿地盐角草根际与非根际中,nirS-型和nirK-型反硝化细菌多样性最高的为秋季根际土壤样本;各土壤样本中的反硝化细菌多样性均呈现根际>非根际。盐角草各土壤样本中的nirS-型反硝化细菌在门分类水平上隶属于变形菌门(Proteobacteria),厚壁菌门(Firmicutes)和放线菌门(Actinobacteria),而nirK-型反硝化细菌在门水平上分类仅包括了Proteobacteria和Firmicutes。Proteobacteria在各土壤样本中的占比均较高;其中Gamma-Proteobacteria的盐单胞菌属(Halomonas)和假单胞菌属(Pseudomonas)是各土壤样本所共有的nirS-型反硝化菌的优势菌属,但它们在每个土壤样本中的相对丰度各有差异。Alpha-Proteobacteria的根瘤菌属(Rhizobium)是盐角草各土壤样本中较为广泛存在的nirK-型反硝化细菌。艾比湖湿地盐角草各土壤样本中的反硝化细菌群落结构存在着一定的差异。RDA结果显示含水量、有机质、全氮和铵态氮等对各土壤样本中的nirS-型反硝化细菌的多样性影响较大,含水量、有机质、全氮、碱解氮等是nirK-型反硝化细菌多样性的主要影响因素。土壤电导率、全磷、全钾、全氮和碱解氮协同影响nirS-型反硝化细菌的群落结构,有机质、速效钾、速效磷、pH和硝态氮是nirK-型反硝化细菌群落结构组成的主要影响因素。艾比湖湿地反硝化细菌呈现季节性变化,nirS-型和nirK-型反硝化细菌以不同的主要菌属,共同推进湿地反硝化作用。而对于湿地生态系统的保护,则需要进行长期而广泛的土壤状态评估和土壤反硝化微生物菌群的动态监测。     

基于宏基因组分析纳帕海高原湿地微生物及其碳氮代谢多样性

由于地理位置特殊和生态系统类型的复杂多样性,高原湿地在水源供给、温室气体调节、生物多样性保护等方面具有不可忽视的生态作用。纳帕海高原湿地是特殊的低纬度高海拔湿地类型,目前关于其微生物多样性的研究较少。文中基于宏基因组学方法对纳帕海高原湿地微生物的基因组进行测序,在细菌域中鉴定出184个门、3 262个属、24 260个种;在古菌域中检测到13个门、32个属;在真菌域中共有13个门、47个属。土壤和水体的物种多样性具有明显差异：在门水平上,土壤中酸杆菌门、变形菌门和放线菌门为优势菌门,水体则以变形菌门和拟杆菌门为主。通过宏基因组分析获得纳帕海微生物群落的多样性数据,并对高原湿地中固碳途径和氮代谢途径进行了初步分析。研究表明碳、氮代谢基因丰度较高,湿地微生物固碳途径主要以卡尔文循环、还原性三羧酸循环和3-羟基丙酸循环为主,变形菌门、绿弯菌门、泉古菌门为主要固碳菌群;对于氮循环,水中以固氮和异化硝酸盐还原过程为主,土壤则以硝化和反硝化过程为主,变形菌门、硝化螺旋菌门、疣微菌门、放线菌门、奇古菌门和广古菌门的微生物为氮循环的主要贡献者。本研究揭示的纳帕海高原湿地微生物多样性,为湿地环境的综合治理和保护提供了新的知识。     

长期施肥制度对稻田土壤反硝化细菌群落活性和结构的影响

以中国科学院桃源农业生态试验站长期定位施肥试验为平台,研究了3种长期施肥制度(对照不施肥-CK,化学施肥-NPK,化学施肥+有机肥-NPKOM)下土壤反硝化速率的差异.同时,以硝酸还原酶基因(narG)作为反硝化细菌的功能标志物,分析了施肥对反硝化细菌群落结构和多样性的影响.结果表明,长期施用有机肥的土壤反硝化速率,反硝化菌多样性都高于对照和施用化肥处理.从3个处理的土壤样品中共获得35个narG基因的可操作分类单元(OTU)主要分布在两个簇,与变形菌门(Proteobacteria)和放线菌门(Actinobacteria)的反硝化细菌有一定的亲缘关系,均为首次从土壤中克隆.Shannon多样性指数显示,NPKOM处理的narG基因多样性最高,CK处理次之,NPK处理最低.LUBSHUFF软件对narG基因群落组成的分析显示,施有机肥后含narG基因的细菌群落组成与CK之间有显著性差异(P<0.05),而化肥(NPK)没有产生显著影响.实验结果为进一步研究亚热带地区水稻土反硝化作用及反硝化功能菌提供了重要的依据.     

纳帕海高原湿地土壤细菌群落多样性初步研究

目的对云南省纳帕海高原湿地土壤中的细菌群落结构进行初步研究,为揭示纳帕海高原湿地生态环境变化提供理论依据。方法运用Illumina Miseq高通量测序平台,对纳帕海高原湿地不同生境类型土壤的细菌群落进行宏基因组测序,检测了纳帕海高原湿地淤泥、湿地和泥炭三种土壤样本细菌群落结构的变化。结果通过对Silva 119数据库进行对比,共获得475 288条序列,可由91 069条非冗余序列代表。对细菌群落结构的分析,结果表明,纳帕海湿地淤泥、湿地和泥炭三种土壤样本的优势菌群均为Proteobacteria、Chloroflexi和Actinobacteria,但Unclassified bacteria仍占有一定比例。结论本研究首次揭示了纳帕海高原湿地土壤细菌群落的多样性,阐明了三种土壤类型中细菌群落的结构变化。     

牦牛排泄物输入对滇西北高寒泥炭沼泽湿地土壤氮转化的影响

牦牛放牧对滇西北高寒湿地土壤环境产生严重影响，改变土壤氮的迁移转化过程，影响湿地生态系统初级生产。然而，关于牦牛排泄物输入对滇西北高寒泥炭沼泽湿地土壤氮转化过程的影响尚不清楚。本研究以滇西北高原典型泥炭沼泽湿地为对象，采用原位土芯室内控制实验方法，研究牦牛排泄物输入对泥炭沼泽湿地土壤氮转化的影响。结果表明，粪便和尿液输入初期促进土壤铵态氮(NH₄⁺-N)积累，但整个培养期则表现为消耗NH₄⁺-N,积累硝态氮(NO₃^--N),表明该过程以硝化作用为主。粪便和尿液输入提高土壤脲酶活性(P<0.05),降低反硝化酶活性(P<0.05)。粪便输入提高过氧化氢酶活性(P<0.05)和N-乙酰氨基葡萄糖苷酶活性(P<0.05),尿液输入降低N-乙酰氨基葡萄糖苷酶活性(P<0.05)。粪便输入对土壤的矿化和硝化作用无显著性影响，尿液输入对土壤硝化作用影响显著。粪便输入抑制土壤反硝化作用，而尿液输入促进反硝化作用。牦牛排泄物输入通过影响泥炭沼...     

干湿交替对生物滞留系统中氮素功能微生物群落的影响

【目的】为探究生物滞留系统干湿交替下环境因子对氮素功能微生物群落的影响。【方法】应用高通量测序技术(Illumina MiSeq PE300),并以amoA和nirS功能基因为分子标记,对无植物型和植物型生物滞留系统在干湿交替下不同土壤空间位置(种植层、淹没层)的硝化和反硝化细菌的多样性和群落结构进行研究,并对微生物群落与环境因子的相互关系进行相关性分析。【结果】微生物种群的功能基因存在显著的空间差异,相比淹没层,种植层的功能细菌更丰富。种植层的OTUs高于淹没层,而进水再湿润促使两种功能基因在种植层和淹没层的OTUs占比差异性增大。群落组成分析表明,amoA型硝化细菌和nirS型反硝化细菌的优势细菌门均为变形菌门(Proteobacteria)。虽然植物根系对氮素功能微生物的多样性指数影响不显著,但在属水平上,植物系统种植层的反硝化菌群种类高于淹没层,而无植物系统则刚好相反。CCA/RDA分析表明,土壤空间位置是影响硝化和反硝化菌群结构的最重要环境因子。【结论】本研究证实干湿交替运行下生物滞留系统中的氮素功能微生物群落受土壤空间位置、水分含量和植物根系的共同调控,其机制有待进一步研究。     

若尔盖高原湿地土壤-植物系统有机碳的分布与流动

 湿地碳素变化对全球气候变化的影响一直是国内外湿地研究的热点。国内对沼泽湿地碳循环的研究主要集中在三江平原,其它地区则鲜见报道。若尔盖高原位于全球气候变化最敏感的区域之一——青藏高原的东北部,冷湿的气候条件下沼泽十分发育,泥炭贮量丰富,沼泽面积和泥炭资源贮量均居中国首位。为了评估该区湿地在全球气候变化中的作用,作者以该区分布最为广泛的3种沼泽植物群落——木里苔草（Carex muliensis）群落、乌拉苔草（Carex meyeriana）群落和藏嵩草（Kobresia tibetica）群落以及最为典型的3种湿地土壤——泥炭土、泥炭沼泽土和草甸沼泽土为对象,采用田间腐解试验方法,系统研究了高原湿地植物——土壤系统中有机碳的分布与流动,其目的在于：1)探明该区湿地土壤有机碳的数量与分布状况;2)了解植物碳在向土壤流动过程中的消失与残留情况。结果表明,若尔盖高原湿地土壤的有机碳含量一般较高且随土层加深而降低;在植物由活体—立枯—残落物的不同阶段,植物不同化学组分中碳的消失率各异,其中易分解组分碳的消失率最大（3种群落分别为61.37%、69.59%和66.34%）,木质素碳的消失率（44.53%～52.98%）略大于纤维素碳的消失率（38.23%～43.86%）,3种群落植物碳的总消失率分别为53.8%、60.03%和55.18%;3种群落的植物残落物在土壤中分解一年和两年后的残留碳量分别为30 g·m﹣2和25.5 g·m﹣2,而植物残根的相应数值则分别高达179～223 g·m﹣2和161～208 g·m﹣2,说明若尔盖高原湿地生态系统中植物残根是形成土壤有机碳的主要来源。由于该区湿地的生物量较高,有机碳的流动量也相应较大。     

闽江河口互花米草不同入侵阶段湿地土壤nirK型反硝化微生物群落结构及多样性

为了明确闽江河口互花米草海向不同入侵阶段湿地土壤nirK型反硝化微生物的群落结构及多样性,在鳝鱼滩东部的互花米草分布区,由陆向海方向选择互花米草海向入侵前的光滩(MF)、入侵1～2年(SAN)和入侵6～7年的互花米草(SA)湿地为研究对象,基于高通量测序技术,测定并分析了不同互花米草入侵阶段湿地土壤nirK型反硝化微生物群落结构及多样性的差异。结果表明:互花米草海向入侵降低了土壤中nirK型反硝化微生物群落的多样性及丰富度。不同入侵阶段湿地土壤的nirK型反硝化微生物主要包括变形菌门和放线菌门,其中变形菌门占绝对优势地位。互花米草海向入侵整体改变了湿地土壤中nirK型反硝化菌属的组成特征,MF、SAN、SA湿地土壤中相对丰度最高的nirK基因菌属分别为慢生根瘤菌属、中慢生根瘤菌属和产碱杆菌属。互花米草海向入侵增加了土壤中nirK型反硝化微生物群落组成的空间异质性,尤其是在SAN样地,这主要与样地本身的环境扰动性较大及互花米草海向入侵加大了样地环境因子的空间异质性有关。互花米草海向入侵主要是通过显著改变土壤理化因子(粒度组成、pH值和含水量)和氮养分条件(全氮、NH₄⁺-N、NO₃^--N)来影响nirK型反硝化微生物的群落结构及多样性。本研究结果有助于揭示互花米草海向入侵对湿地土壤反硝化过程影响的微生物机制。     

牛场肥水灌溉对土壤nirK、nirS型反硝化微生物群落结构的影响

以徐水县梁家营长期定位施肥试验田为研究对象,利用末端限制性片段长度多态性(T-RFLP)分析和克隆文库构建,研究了5种施肥处理(清水灌溉CK、无机肥灌溉CF、牛场肥水不同浓度、不同次数灌溉T4、T5和T11)下土壤中nirK、nirS型反硝化细菌群落多样性及其群落结构的演变。结果表明,不同施肥处理下nirK、nirS型反硝化细菌群落多样性无显著差异,但群落结构却有明显变化:nirK型反硝化细菌群落结构既受施肥种类又受施肥量影响,优势种群尤其对施肥种类和施肥量响应显著;nirS型反硝化细菌则主要受施肥种类影响,施肥量影响微弱。牛场肥水处理和无机肥处理分别促进和抑制不同的nirS型反硝化细菌,群落主成分受无机肥促进、牛场肥水抑制。系统发育分析结果表明,土壤中nirK型反硝化细菌主要与假单胞菌属(Pseudomonas)、产碱杆菌属(Alcaligenes)和根瘤菌属(Rhizobium)的反硝化细菌具有较近的亲缘关系;nirS型反硝化细菌主要与劳尔氏菌(Ralstonia)和红长命菌属(Rubrivivax)有较近的亲缘关系。试验土壤中反硝化微生物多与目前已报道的好氧反硝化细菌亲缘关系较近,这可能与微生物分析取自表层土有关。     

Plant presence and species combination, but not diversity, influence denitrifier activity and the composition of nirK-type denitrifier communities in grassland soil

To explore potential links between plant communities, soil denitrifiers and denitrifier function, the impact of presence, diversity (i.e. species richness) and plant combination on nirK -type denitrifier community composition and on denitrifier activity was studied in artificial grassland plant assemblages over two consecutive years. Mesocosms containing zero, four and eight species and different combinations of two species were set up. Differences in denitrifier community composition were analysed by canonical correspondence analyses following terminal restriction fragment length polymorphism analysis of PCR-amplified nirK gene fragments coding for the copper-containing nitrite reductase. As a measure of denitrifier function, denitrifier enzyme activity (DEA) was determined in the soil samples. The presence as well as the combination of plants and sampling time, but not plant diversity, affected the composition of the nirK -type denitrifier community and DEA. Denitrifier activity significantly increased in the presence of plants, especially when they were growing during summer and autumn. Overall, we found a strong and direct linkage of denitrifier community composition and functioning, but also that plants had additional effects on denitrifier function that could not be solely explained by their effects on nirK -type denitrifier community composition.     

Diversity of Nitrite Reductase (nirK and nirS) Gene Fragments in Forested Upland and Wetland Soils

The genetic heterogeneity of nitrite reductase gene (nirK and nirS) fragments from denitrifying prokaryotes in forested upland and marsh soil was investigated using molecular methods. nirK gene fragments could be amplified from both soils, whereas nirS gene fragments could be amplified only from the marsh soil. PCR products were cloned and screened by restriction fragment length polymorphism (RFLP), and representative fragments were sequenced. The diversity of nirK clones was lower than the diversity of nirS clones. Among the 54 distinct nirK RFLP patterns identified in the two soils, only one pattern was found in both soils and in each soil two dominant groups comprised >35% of all clones. No dominance and few redundant patterns were seen among the nirS clones. Phylogenetic analysis of deduced amino acids grouped the nirK sequences into five major clusters, with one cluster encompassing most marsh clones and all upland clones. Only a few of the nirK clone sequences branched with those of known denitrifying bacteria. The nirS clones formed two major clusters with several subclusters, but all nirS clones showed less than 80% identity to nirS sequences from known denitrifying bacteria. Overall, the data indicated that the denitrifying communities in the two soils have many members and that the soils have a high richness of different nir genes, especially of the nirS gene, most of which have not yet been found in cultivated denitrifiers.     

Nitrite reductase genes as functional markers to investigate diversity of denitrifying bacteria during agricultural waste composting

The purpose of this study was to investigate the diversity of denitrifier community during agricultural waste composting. The diversity and dynamics of the denitrifying genes (nirK and nirS) were determined using polymerase chain reaction–denaturing gradient gel electrophoresis (PCR-DGGE). Relationships between physico-chemical parameters and denitrifying genes structures were simultaneously evaluated by redundancy analysis (RDA). Phylogenetic analysis indicated that nirK clones grouped into six clusters and nirS clones into two major clusters, respectively. The results showed a very high diversity of nir gene sequences within composting samples. RDA showed that the nirK and nirS gene structures were significantly related to pH and pile temperature (P?<?0.05). Significant amounts of the variation (49.2 and 38.3 % for nirK and nirS genes, respectively) were explained by pH and pile temperature, suggesting that those two parameters were the most likely ones to influence, or be influenced by the denitrifiers harboring nirK and nirS genes.     

Isolation of functional single cells from environments using a micromanipulator: application to study denitrifying bacteria

We developed a novel method to isolate functionally active single cells from environmental samples and named it the functional single-cell (FSC) isolation method. This method is based on a combination of substrate-responsive direct viable counts, live-cell staining with 5-carboxyfluorescein diacetate acetoxymethyl ester, and micromanipulation followed by cultivation in a medium. To evaluate this method, we applied it to study a denitrifying community in rice paddy soil. Similar denitrifier counts were obtained by the conventional most probable number analysis and our FSC isolation method. Using the FSC isolation method, 37 denitrifying bacteria were isolated, some of which harbored copper-containing nitrite reductase gene (nirK). The 16S rRNA gene analysis showed that members belonging to the genera Azospirillum and Ochrobactrum may be the major denitrifiers in the rice paddy soil. These results indicate that the FSC isolation method is a useful tool to obtain functionally active single cells from environmental samples.     

Diversity of nitrite reductase (nirK and nirS) gene fragments in forested upland and wetland soils

The genetic heterogeneity of nitrite reductase gene (nirK and nirS) fragments from denitrifying prokaryotes in forested upland and marsh soil was investigated using molecular methods. nirK gene fragments could be amplified from both soils, whereas nirS gene fragments could be amplified only from the marsh soil. PCR products were cloned and screened by restriction fragment length polymorphism (RFLP), and representative fragments were sequenced. The diversity of nirK clones was lower than the diversity of nirS clones. Among the 54 distinct nirK RFLP patterns identified in the two soils, only one pattern was found in both soils and in each soil two dominant groups comprised >35% of all clones. No dominance and few redundant patterns were seen among the nirS clones. Phylogenetic analysis of deduced amino acids grouped the nirK sequences into five major clusters, with one cluster encompassing most marsh clones and all upland clones. Only a few of the nirK clone sequences branched with those of known denitrifying bacteria. The nirS clones formed two major clusters with several subclusters, but all nirS clones showed less than 80% identity to nirS sequences from known denitrifying bacteria. Overall, the data indicated that the denitrifying communities in the two soils have many members and that the soils have a high richness of different nir genes, especially of the nirS gene, most of which have not yet been found in cultivated denitrifiers.     

Impact of Plant Functional Group, Plant Species, and Sampling Time on the Composition of nirK-Type Denitrifier Communities in Soil

We studied the influence of eight nonleguminous grassland plant species belonging to two functional groups (grasses and forbs) on the composition of soil denitrifier communities in experimental microcosms over two consecutive years. Denitrifier community composition was analyzed by terminal restriction fragment length polymorphism (T-RFLP) of PCR-amplified nirK gene fragments coding for the copper-containing nitrite reductase. The impact of experimental factors (plant functional group, plant species, sampling time, and interactions between them) on the structure of soil denitrifier communities (i.e., T-RFLP patterns) was analyzed by canonical correspondence analysis. While the functional group of a plant did not affect nirK-type denitrifier communities, plant species identity did influence their composition. This effect changed with sampling time, indicating community changes due to seasonal conditions and a development of the plants in the microcosms. Differences in total soil nitrogen and carbon, soil pH, and root biomass were observed at the end of the experiment. However, statistical analysis revealed that the plants affected the nirK-type denitrifier community composition directly, e.g., through root exudates. Assignment of abundant T-RFs to cloned nirK sequences from the soil and subsequent phylogenetic analysis indicated a dominance of yet-unknown nirK genotypes and of genes related to nirK from denitrifiers of the order Rhizobiales. In conclusion, individual species of nonleguminous plants directly influenced the composition of denitrifier communities in soil, but environmental conditions had additional significant effects.     

Metabolic Profiles and Genetic Diversity of Denitrifying Communities in Activated Sludge after Addition of Methanol or Ethanol

External carbon sources can enhance denitrification rates and thus improve nitrogen removal in wastewater treatment plants. The effects of adding methanol and ethanol on the genetic and metabolic diversity of denitrifying communities in activated sludge were compared using a pilot-scale plant with two parallel lines. A full-scale plant receiving the same municipal wastewater, but without external carbon source addition, was the reference. Metabolic profiles obtained from potential denitrification rates with 10 electron donors showed that the denitrifying communities altered their preferences for certain compounds after supplementation with methanol or ethanol and that methanol had the greater impact. Clone libraries of nirK and nirS genes, encoding the two different nitrite reductases in denitrifiers, revealed that methanol also increased the diversity of denitrifiers of the nirS type, which indicates that denitrifiers favored by methanol were on the rise in the community. This suggests that there might be a niche differentiation between nirS and nirK genotypes during activated sludge processes. The composition of nirS genotypes also varied greatly among all samples, whereas the nirK communities were more stable. The latter was confirmed by denaturing gradient gel electrophoresis of nirK communities on all sampling occasions. Our results support earlier hypotheses that the compositions of denitrifier communities change during predenitrification processes when external carbon sources are added, although no severe effect could be observed from an operational point of view.     

Abundance and Structure Composition of nirK and nosZ Genes as Well as Denitrifying Activity in Heavy Metal-polluted Paddy Soils

Denitrification is an important microbial process in soils and leads to the emission of nitrous oxide (N₂O). However, studies about the microbial community involved in denitrification processes in polluted paddy fields are scarce. Here, we studied two rice paddies which had been polluted for more than three decades by metal mining and smelter activities. Abundance and community composition were determined using real-time polymerase chain reaction (PCR) assay and denaturing gradient gel electrophoresis of nitrite reductase and nitrous oxide reductase gene amplicons (nirK and nosZ), while denitrifying activities were assessed by measuring potential denitrifier enzyme activity. We found that the community structure of both nirK and nosZ containing denitrifiers shifted under pollution in the two rice paddies. All the retrieved nirK sequences did not group into either α- or β-proteobacteria, while most of the nosZ species were affiliated with α-proteobacteria. While the abundance of both nirK and nosZ was significantly reduced in the polluted soils at “Dexing” (with relatively higher Cu levels), these parameters did not change significantly at “Dabaoshan” (polluted with Cd, Pb, Cu, and Zn). Furthermore, total denitrifying activity and N₂O production and reduction rates also only decreased under pollution at “Dexing.” These findings suggest that nirK and nosZ containing denitrifier populations and their activities could be sensitive to considerable Cu pollution, which could potentially affect N₂O release from polluted paddy soils.