浅表层水稻土N2O消耗能力及其与N2O还原微生物的耦合关系

土壤不仅能够产生、排放温室气体N_2O,还具有截留、吸收、转化N_2O的能力。土壤消耗N_2O已经成为很重要的一种降低大气N_2O浓度的途径,但目前关于土壤N_2O消耗过程及其微生物调控机制的系统研究较为缺乏。试验以浅表层水稻土柱(0—5 cm)为研究对象,通过外源添加N_2O气体研究N_2O迁移通过淹水土柱的动态过程,以及N_2O消耗能力与氧化亚氮还原酶基因丰度变化和其他土壤养分含量变化的联系,揭示浅表层水稻土N_2O消纳量与N_2O还原微生物之间的耦合关系。结果显示,淹水厌氧条件下5 cm土壤深度外源添加的N_2O迁移通过浅表层土柱后,仅有7.17—9.80%部分逸散出土表,表明0—5 cm淹水水稻土层具有极强的N_2O截留能力(90%以上)而减少N_2O净排放量。排放出土表的N_2O也可被淹水土柱继续吸收消耗,且吸收转化速率随N_2O浓度增加而大幅提高,最高可达到3896.75μg N m~(-2) h~(-1)。与此同时,土壤DOC含量大量消耗,含nosZⅠ基因的反硝化微生物数量显著增长(P0.01),而nosZⅡ基因丰度的无显著变化。说明高浓度N_2O添加能够促进淹水土壤N_2O吸收消耗能力,此刺激作用可能主要由含nosZⅠ基因的N_2O还原微生物进行调控。浅表层土壤强大的N_2O吸收消耗功能可进一步深入系统研究,为实践温室气体减排提供理论基础。     

一株兼性氧化亚氮还原菌的还原N2O能力

【目的】从水稻土中分离筛选出一株兼性氧化亚氮还原菌,并探索其在不同条件下还原N_2O的能力,为减少温室气体N_2O的排放提供重要依据。【方法】通过微生物富集培养分离技术从水稻土中分离得到纯菌;利用nosZ基因和16S rRNA的测序分析鉴定菌株;通过测定菌株在不同条件下N_2O的还原量,分析该菌株还原N_2O的能力及调控因子。【结果】经鉴定,该菌株含有nos Z基因,属于假单胞菌属,在温度30°C、厌氧条件下还原N_2O速率高达0.0219μmol/min以上,改变不同温度和氧气浓度后其能力相对减弱,但仍具备较强的还原N_2O作用。【结论】从水稻土中分离筛选得到的兼性氧化亚氮还原菌为假单胞菌,它在不同环境条件下都具备较强的还原N_2O能力,该菌株可能为减少土壤N_2O排放提供新途径,对保障生态环境安全具有重要的应用价值。     

长期缺素施肥及石灰石膏施用对江西鹰潭红壤反硝化微生物功能基因丰度的影响

反硝化功能基因丰度是决定温室气体氧化亚氮(N2O)排放潜力的重要生物因素.反硝化功能基因主要包括产生N2O的关键基因nirK和nirS,以及将N2O还原成氮气的基因nosZⅠ和nosZⅡ.本研究利用实时荧光定量PCR,研究了32年缺施氮(N)、磷(P)或钾(K)肥,以及施用石灰、石膏处理下江西鹰潭红壤反硝化功能基因的丰...     

采伐剩余物管理对杉木人工林土壤nosZ型反硝化细菌群落多样性的影响

反硝化细菌是土壤氧化亚氮(N₂O)排放的关键因子。以杉木人工林为研究对象,设置4种采伐剩余物处理方式(RF:对照;RB:火烧;MT:粉碎;NR:移除),采用高通量测序技术,以nosZ为标记基因,测定了自2018年9月—2020年9月,2年期间土壤nosZ型反硝化细菌群落的组成和丰度。研究结果显示,4种采伐剩余物处理中的土壤nosZ型反硝化细菌90%以上来自变形菌门,优势菌属包括固氮螺菌属、中慢生根瘤菌属、动胶菌属、伯克霍尔德菌属、嗜酸菌属、慢生根瘤菌属、假单胞菌属、固氮弧菌属以及无色杆菌属;样本间差异物种的显著性分析表明,在处理完成半年时,火烧相较于对照于β-变形菌纲水平显著增加了nosZ基因丰度;在处理完成一年时,火烧分别于红螺菌目、红螺菌科、固氮螺菌属水平显著高于粉碎;粉碎相较于移除在处理完成一年时,于γ-变形菌纲和产碱菌科水平显著增加了nosZ基因丰度;在处理完成两年时,粉碎处理的nosZ基因丰度在变形菌门水平显著高于对照和火烧。α多样性数据显示,处理完成一年时,粉碎处理相较于对照和移除显著增加了Shannon和Simpson指数;处理完成两年时,粉碎和火烧...     

施肥制度对水稻土壤nosZ型反硝化细菌群落的影响

由含氧化亚氮还原酶(NOS)的反硝化细菌驱动的氧化亚氮(N2O)还原成氮气(N2)的过程是N2O排放的重要调控途径。为探明施肥对稻田土壤nosZ型反硝化细菌群落的影响,采用荧光定量PCR和高通量测序等方法,研究了湖南省宁乡县长达30年的定位试验条件下4种施肥制度[不施肥(CK)、化肥(CF)、70%化肥+30%有机肥(CFM1)和40%化肥+60%有机肥(CFM2)]对水稻土壤nosZ型反硝化细菌数量和群落结构的影响。结果表明:不同施肥处理nosZ基因丰度为2.14×10~8～6.09×10~8copies·g~(-1)干土,施肥处理nosZ基因拷贝数比对照低47.3%～64.8%(P0.05),但不同有机肥配施比例处理间nosZ基因拷贝数差异不显著;变形菌门是优势门水平类群,占总序列的60.2%～77.5%; Bacteria_unclassified、Proteobacteria_unclassified、Betaproteobacteria_unclassified和根瘤菌目为优势目水平类群,占总序列的93.6%～95.9%。施肥显著降低了Proteobacteria_unclassified的相对丰度(P0.01),但显著提高了根瘤菌目、environmental_samples和红环菌目的相对丰度(P0.05);施肥显著改变nosZ型反硝化细菌的群落结构,但有机肥配施比例对其影响较弱;除碳氮比外,其他土壤理化性质均显著影响nosZ型反硝化细菌的数量和群落结构,其中,硝态氮和土壤p H是驱动nosZ型反硝化细菌群落变化的主要因子;施肥显著影响nosZ型反硝化细菌数量和群落结构,有机肥配施比例对nosZ型反硝化细菌群落的影响较弱,研究结果为进一步阐述施肥制度对土壤反硝化微生物的影响提供依据。     

海洋N2O的排放及其关键微生物过程作用机制研究进展

氧化亚氮(N_2O)是一种重要的温室效应气体,同时也是造成平流层臭氧损耗的主要化合物。海洋是大气中N_2O的重要排放源,海洋中的N_2O产生和释放主要由微生物的代谢过程介导。本文对海洋N_2O的释放通量、海水N_2O的分布特征、环境影响因素以及海洋N_2O产生的微生物调控机制等几个方面的最新研究进展进行综述,并结合低氧与N_2O产生的关系以及近岸海域低氧区的扩大等科学问题,对河口近岸生态系统N_2O的释放通量以及其关键微生物过程进行展望。     

真菌反硝化过程及其驱动的N2O产生机制研究进展

真菌反硝化过程的发现打破了反硝化过程只发生在原核生物中的传统认识,是对全球微生物氮循环过程的重要补充。真菌参与的反硝化过程由于缺乏N_2O还原酶,其终产物为具有强辐射效应的温室气体N_2O。真菌在环境中分布广泛,生物量巨大,故真菌反硝化作用对全球N_2O释放通量的贡献是不容忽视的。近年来许多研究表明,真菌反硝化过程是自然环境中N_2O产生的重要途径。本文对反硝化真菌的发现、多样性及分布、产生N_2O的机制和活性测定方法等几个方面进行综述,并对未来的研究提出展望。     

UV-B增强下施硅对稻田CH4和N2O排放及其增温潜势的影响

大气平流层臭氧损耗导致的地表紫外辐射增强作为全球变化重要问题之一,受到广泛关注。硅是水稻生长有益元素,但施硅是否影响稻田CH_4和 N_2O排放,迄今相关报道尚不多见。通过大田试验,研究UV-B增强下施硅对水稻生长、稻田甲烷(CH_4)和氧化亚氮( N_2O)排放及其增温潜势的影响。UV-B辐照设2水平,即对照(A,自然光)和增强20%(E);施硅量设2水平,即对照(Si0,0 kg SiO_2/hm2)和施硅(Si1,200 kg SiO_2/hm2)。结果表明,UV-B增强降低了成熟期水稻地上部和地下部生物量,而施硅能缓解UV-B增强对水稻生长的抑制作用,使水稻地上部和地下部生物量增加。UV-B增强可显著提高稻田CH_4和 N_2O排放通量和累积排放量,增加稻田CH_4和 N_2O排放的综合增温潜势。施硅能明显降低稻田CH_4排放,促进 N_2O排放,降低稻田CH_4和 N_2O排放的综合增温潜势。研究表明,施硅显著降低稻田CH_4和 N_2O的全球增温潜势,缓解UV-B增强对稻田CH_4和 N_2O的全球增温潜势的促进作用。     

环境因子对土壤微生物产生和还原N_2O过程的影响

探究环境因子对土壤微生物反硝化气体产生的影响,选取培养温度、氧气含量、亚硝酸盐含量、葡萄糖含量、p H值、Cu~(2+)含量6个因素,分别设计3个水平,设计7因素3水平一共18组的正交实验,对土壤有机物含量和培养条件进行调控,在密闭环境下进行培养,同时监测顶空气体中反硝化气体含量,连续监测10 d,随后对土壤中氧化亚氮还原酶基因nos Z进行定量PCR检测。培养实验显示在18组条件中一共出现了3种的反硝化气体产生类型,分别为快速产N_2类型、N_2O累积类型和无气体产生类型,表明环境条件的差异导致了土壤反硝化微生物不同的产气过程。对定量PCR的方差分析显示单个环境因素并没有对nos Z基因拷贝数产生显著影响,表明反硝化气体产生的差异并不是通过影响nos Z基因拷贝数而产生的。     

设施菜田与棚外粮田土壤菌群和反硝化气体产生的比较分析

【目的】对比设施菜田与棚外粮田土壤菌群以及N2O产生模式的差异。【方法】采用变性梯度凝胶电泳(DGGE)和反硝化功能基因(nirS,nosZ)方法分别比较两种土壤细菌群落以及功能基因类群丰度的差异,利用自动连续在线培养监测体系(Robot系统)测定两种土壤在好氧、厌氧阶段N2O等反硝化相关气态产物产生模式,分析N2O/(N2+N2O+NO)产物比。【结果】设施菜田与棚外粮田具有不同的土壤细菌群落结构,并且土壤细菌总量得到了显著的提升,然而两种反硝化功能基因(nirS,nosZ)丰度并没有显著变化。与设施菜田相比,棚外粮田有相对低的N2O积累量以及产物比,并且在厌氧初期气体产生模式有所不同。培养后铵态氮和亚硝态氮含量上升。【结论】设施菜田长期有别于棚外粮田的管理方式造成了土壤细菌群落的显著改变,增大了活跃微生物总量,造成土壤酸化,并导致N2O在气态产物中的比例升高。设施菜田土壤微生物进行了与棚外粮田不同的硝酸盐呼吸过程,异化硝酸盐还原成铵(DNRA)过程有可能贡献了两种土壤的部分厌氧N2O产生量。     

Denitrification by fungi

Many fungi in the centre of the group of Fusarium and its teleomorphs were shown to be capable of reducing nitrite anaerobically to form nitric oxide (NO), nitrous oxide (N2O), and/or dinitrogen (N2). Several strains could reduce nitrate as well. Nitrous oxide was the major product of the reduction of nitrate or nitrite. Several fungi could also form N2. When [15]nitrite was used as substrate for the N2-forming denitrification, 15N2O, 15NO, and 14N15N were obtained as the products. These results demonstrated that, unexpectedly, many fungi have denitrifying abilities. It was also shown that the fungal system contains a unique reaction, formation of a hybrid dinitrogen.     

Phenotypic and genotypic heterogeneity among closely related soil-borne N2 - and N2 O-producing Bacillus isolates harboring the nosZ gene

Little is known about the genetic and phenotypic diversity of Gram-positive denitrifying bacteria. We compared the production of gaseous denitrification products for 14 closely related Bacillus soil isolates at pH 6 and 7 during 48-h batch incubations using a robotic gas-sampling apparatus. Primers targeting the nosZ gene encoding the nitrous oxide reductase were designed to confirm the presence of this gene in the isolates. The variation in the production of gaseous nitrogen products was compared with the genetic variation based on 16S rRNA gene sequences, genomic fingerprinting and nosZ sequences. The nosZ gene was detected in all isolates and all produced N(2) as the dominant end product at pH 7. Production of gaseous nitrogen products was more variable at pH 6, with different levels of NO and N(2) O production among the isolates, although minimal variation was observed among the 16S rRNA and nosZ gene sequences. One isolate was more divergent from the others based on genomic fingerprinting, and had two different nosZ gene copies, which coincided with the highest production of N(2) at pH 7 and the lack of intermediates at pH 6. Overall, our analysis suggests that genetic variation plays a role in the variation in N(2) O and N(2) production, but the variation in activity caused by acidification can be substantially greater than genotypic variation among closely related Bacillus.     

Characterization of the membranous denitrification enzymes nitrite reductase (cytochrome cd1) and copper-containing nitrous oxide reductase from Thiobacillus denitrificans

Cytochrome cd ₁-nitrite reductase and nitrous oxide reductase of Thiobacillus denitrificans were purified and characterized by biochemical and immunochemical methods. In contrast to the generally soluble nature of the denitrification enzymes, these two enzymes were isolated from the membrane fraction of T. denitrificans and remained active after solubilization with Triton X-100. The properties of the membrane-derived enzymes were similar to those of their soluble counterparts from the same organism. Nitrous oxide reductase activity was inhibited by acetylene. Nitrite reductase and nitrous oxide reductase cross-reacted with antisera raised against the soluble enzymes from Pseudomonas stutzeri. The nirS, norBC, and nosZ genes encoding the cytochrome cd ₁-nitrite reductase, nitric oxide reductase, and nitrous oxide reductase, respectively, from P. stutzeri hybridized with genomic DNA from T. denitrificans. Cross-reactivity and similar N-terminal amino acid and gene sequences suggest that the primary structures of the Thiobacillus enzymes are homologous to the soluble proteins from P. stutzeri. Received: 18 August 1995 / Accepted: 30 October 1995     

Analysis of denitrification genes and comparison of nosZ, cnorB and 16S rDNA from culturable denitrifying bacteria in potato cropping systems

Bacterial denitrification in agricultural soils is a major source of nitrous oxide, a potent greenhouse gas. This study examined the culturable bacterial population of denitrifiers in arable field soils in potato (Solanum tuberosum L.) production and denitrification genes (nir, nor and nos) and 16S rDNA in those isolates. Enrichments for culturable denitrifiers yielded 31 diverse isolates that were then analysed for denitrification genes. The nitrous oxide reductase (nosZ) gene was found in all isolates. The majority of isolates ( approximately 90%) contained the cnorB nitric oxide reductase gene, with the remainder containing the qnorB gene. Nitrite reductase genes (nirS and nirK) were amplifiable from most of the isolates, and were segregated between species similar to previously isolated denitrifiers. Isolated strains were preliminarily identified using fatty acid methyl ester analysis and further identified using 16S rDNA sequencing. The majority of isolates (21) were classified as Pseudomonas sp., with smaller groups of isolates being most similar to Bosea spp. (4), Achromobacter spp. (4) and two isolates closely related to Sinorhizobium/Ensifer spp. Phylogenetic trees were compared among nosZ, cnorB and 16S rDNA genes for a subset of Pseudomonas strains. The trees were mostly congruent, but some Pseudomonas sp. isolates grouped differently depending on the gene analysed, indicating potential horizontal gene transfer of denitrification genes. Although Bosea spp. are known denitrifiers, to the best of our knowledge this is the first report of isolation and sequencing of denitrification genes from this bacterial genus.     

Phylogenetic analysis of nitric oxide reductase gene homologues from aerobic ammonia-oxidizing bacteria

Nitric oxide (NO) and nitrous oxide (N2O) are climatically important trace gases that are produced by both nitrifying and denitrifying bacteria. In the denitrification pathway, N2O is produced from nitric oxide (NO) by the enzyme nitric oxide reductase (NOR). The ammonia-oxidizing bacterium Nitrosomonas europaea also possesses a functional nitric oxide reductase, which was shown recently to serve a unique function. In this study, sequences homologous to the large subunit of nitric oxide reductase (norB) were obtained from eight additional strains of ammonia-oxidizing bacteria, including Nitrosomonas and Nitrosococcus species (i.e., both beta- and gamma-Proteobacterial ammonia oxidizers), showing widespread occurrence of a norB homologue in ammonia-oxidizing bacteria. However, despite efforts to detect norB homologues from Nitrosospira strains, sequences have not yet been obtained. Phylogenetic analysis placed nitrifier norB homologues in a subcluster, distinct from denitrifier sequences. The similarities and differences of these sequences highlight the need to understand the variety of metabolisms represented within a "functional group" defined by the presence of a single homologous gene. These results expand the database of norB homologue sequences in nitrifying bacteria.     

Leachates from municipal solid waste disposal sites harbor similar, novel nitrogen-cycling bacterial communities

High emissions of nitrous oxide (N(2)O) have recently been documented at municipal solid waste (MSW) landfills. However, the biodiversity of the bacterial populations involved remains unexplored. In this study, we investigated communities of ammonia-oxidizing bacteria (AOB) and denitrifying bacteria associated with the leachates from three MSW disposal sites by examining the diversity of the ammonia monooxygenase structural gene amoA and the nitrous oxide reductase gene nosZ, respectively. Cloning and phylogenetic analysis of the functional genes revealed novel and similar groups of prokaryotes involved in nitrogen cycling in the leachates with different chemical compositions. All amoA sequences recovered grouped within the Nitrosomonas europaea cluster in the Betaproteobacteria, with the vast majority showed only relatively moderate sequence similarities to known AOB but were exclusively most similar to environmental clones previously retrieved from wastewater treatment plants. All nosZ sequences retrieved did not cluster with any hitherto reported nosZ genes and were only remotely related to recognized denitrifiers from the Gammaproteobacteria and thus could not be affiliated. Significant overlap was found for the three denitrifying nosZ leachate communities. Our study suggests a significant selection of the novel N-cycling groups by the unique environment at these MSW disposal sites.     

Expression of the nos operon proteins from Pseudomonas stutzeri in transgenic plants to assemble nitrous oxide reductase

Nitrous oxide (N(2)O) is a stable greenhouse gas that plays a significant role in the destruction of the ozone layer. Soils are a significant source of atmospheric N(2)O. It is important to explore some innovative and effective biology-based strategies for N(2)O mitigation. The enzyme nitrous oxide reductase (N(2)OR), naturally found in soil bacteria, is responsible for catalysing the final step of the denitrification pathway, conversion of N(2)O to dintrogen gas (N(2)). To transfer this catalytic pathway from soil into plants and amplify the abundance of this essential mechanism (to reduce global warming), a mega-cassette of five coding sequences was assembled to produce transgenic plants heterologously expressing the bacterial nos operon in plant leaves. Both the single-gene transformants (nosZ) and the multi-gene transformants (nosFLZDY) produced active recombinant N(2)OR. Enzymatic activity was detected using the methyl viologen-linked enzyme assay, showing that extracts from both types of transgenic plants exhibited N(2)O-reducing activity. Remarkably, the single-gene strategy produced higher reductase capability than the whole-operon approach. The data indicate that bacterial N(2)OR expressed in plants could convert N(2)O into inert N(2) without involvement of other Nos proteins. Silencing by homologous signal sequences, or cryptic intracellular targeting are possible explanations for the low activities obtained. Expressing N(2)OR from Pseudomonas stutzeri in single-gene transgenic plants indicated that such ag-biotech solutions to climate change have the potential to be easily incorporated into existing genetically modified organism seed germplasm.     

微生物驱动的滨海湿地N2O产生及其机制研究进展

滨海湿地位于海陆交界,具有初级生产力高、生物多样性丰富以及微生物驱动的营养元素循环活跃等特点,同时也是大气中一氧化二氮(N_2O)的重要排放源。N_2O是仅次于二氧化碳(CO2)和甲烷(CH4)的第三大温室气体,而全球90%以上的N_2O排放由微生物主导,并与滨海湿地氮循环的微生物群落多样性及功能密切相关。因此,滨海湿地系统中N_2O的产生与转化逐渐受到关注。本文综述了滨海湿地生态系统中微生物驱动下N_2O的产生过程,以及氮元素及其与碳、硫和金属元素耦合过程中产生N_2O的代谢途径,N_2O排放的时空变化与微生物调控,并对未来相关研究方向进行了展望,旨在揭示微生物驱动的N_2O产生及环境调控机制,为减缓全球变暖提供科学依据。     

Nitrous oxide reductase of Flexibacter canadensis: a unique membrane-bound enzyme

Abstract The subcellular distribution of nitrous oxide reductase was studied in the gliding soil bacterium Flexibacter canadensis . Nitrous oxide reductase activity, as measured by the methyl viologen-nitrous oxide oxidoreductase assay, was associated entirely with the membrane fraction of cell-free extracts. The enzyme was liberated from the membranes with use of detergents but not by high-salt concentrations, thus implying that nitrous oxide reductase is an integral membrane protein. The nitrous oxide reductase of F. canadensis is the first reported example of a membrane-bound form of this respiratory enzyme.