过量施肥对设施菜田土壤菌群结构及N2O产生的影响

【背景】N_2O是一种很强的温室气体,其温室效应强度大约是CO_2的265倍。土壤氮肥施加量是影响N_2O排放的重要因素,而厌氧条件下微生物反硝化则是N_2O产生的重要途径。【目的】研究过量施肥条件下蔬菜大棚土壤菌群结构变化及其对N_2O气体排放的影响。【方法】利用自动化培养与实时气体检测系统(Robot)监测土壤厌氧培养过程中N_2O和N_2排放通量,比较过量施肥和减氮施肥模式下土壤N_2O排放模式的差异。通过Illumina二代测序平台对这2种不同施肥处理的土壤微生物群落进行高通量测序,研究不同施肥量对土壤菌群组成的影响。【结果】过量施肥土壤中硝酸盐的含量大约是减氮施肥土壤的2倍,通过添加硝酸盐使2种土壤的硝酸盐含量均为60 mg/kg或为200 mg/kg时,过量施肥土壤在厌氧培养前期N_2O气体的产生量及产生速度都明显高于减氮施肥土壤。另外,过量施肥导致土壤菌群结构发生显著改变,并且降低了土壤微生物的多样性。相对于减氮施肥,过量施肥方式富集了Rhodanobacter属的微生物。PICRUSt预测结果显示,传统施肥没有显著改变反硝化功能基因相对丰度。【结论】长期过量氮肥施用显著增加了土壤N_2O的排放,可能原因是施肥改变了包括氮转化相关微生物在内的土壤菌群组成,从而影响了土壤N_2O气体的形成与还原过程。     

设施菜田不同碳氮管理对反硝化菌结构和功能的影响

【目的】通过6年长期定位试验,比较设施菜田不同碳氮管理下反硝化菌结构和功能的差异。【方法】采用末端限制性片段多态性(T-RFLP)和变性梯度凝胶电泳(DGGE)方法分别分析nir K/nir S和nos Z型反硝化菌群结构特征,利用自动连续在线培养监测体系(Robot系统)测定分析NO/(NO3-+NO2-)和N2O/(N2O+N2)产物比,并通过乙炔抑制法测定反硝化酶活性。【结果】传统施肥处理(CN)显著改变了nir K和nos Z型反硝化菌的结构,增加了NO/(NO3-+NO2-)和N2O/(N2O+N2)产物比。nir S型菌受碳氮管理影响较小。减氮(RN)和添加秸秆处理(RN+S)的nir K和nos Z型反硝化菌结构与CN处理的差异性显著,且会显著降低NO/(NO3-+NO2-)和N2O/(N2O+N2)产物比;与CN和RN相比,RN+S显著增加反硝化酶活性。【结论】设施菜田长期传统施肥措施改变了反硝化菌的结构和功能,增加土壤自身的NO产生能力并减弱了N2O还原N2的能力。减氮和添加秸秆管理能形成自身的反硝化菌群结构,并降低NO和N2O排放风险;秸秆的添加会促进反硝化潜在速率,降低菜田NO3-淋洗风险。     

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。     

分离于河口区芦苇湿地1株好氧反硝化菌的鉴定及其反硝化特性

【目的】筛选高效脱氮且N_2O释放量少的好氧反硝化细菌,并对菌株的反硝化特性进行研究,可为河口湿地富营养化水体的生物修复提供技术支撑。【方法】经BTB培养基初筛和反硝化能力测定,从辽河河口区芦苇湿地土壤中分离得到1株具有较高反硝化能力的好氧反硝化菌C3。经形态观察、生理生化鉴定和16S rRNA序列分析,对菌株进行鉴定。研究温度、碳源、pH及C/N对其生长量、反硝化能力及N2O释放的影响。【结果】筛选得到的高效好氧反硝化细菌C3,经鉴定属于假单胞菌属(Pseudomonas sp.)。反硝化特性研究结果表明,该菌最适碳源为柠檬酸三钠,在温度为30°C、pH为7.0、C/N为10时生长速率和脱氮效率最高且N_2O释放量较少。在此条件下,该菌在36 h内使NO_3~–由179.55 mg/L降至5.08 mg/L,脱氮率高达97.17%。该菌株在整个反硝化过程中中间产物N_2O的最大累积量较低,为0.22 mg/L。【结论】从湿地土壤中分离所得好氧反硝化菌C3为假单胞菌属的1个种(Pseudomonas sp.),该菌株在高效除氮和低N_2O累积方面均具有明显优势,对后续河口湿地富营养化水体治理具有重要意义。     

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。     

土壤释放的 N_2O 的原位测定

N_2O 是大气成分之一,它由微生物的硝化-反硝化作用、燃烧和大气闪电等过程产生,其中土壤微生物反硝化作用是最主要的来源。土壤微生物反硝化作用产生N_2O,不仅导致土壤中肥料氮素的损失,而且由于其“温室效应”和对臭氧层的破坏,受到国内外研究者     

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。     

丹麦森林土壤反硝化作用的动力学分析

本项研究将乙炔和氯霉素抑制技术结合起来 ,对丹麦一森林土壤的反硝化作用进行了研究 ,并考察温度对其还原酶活性的影响 .反硝化还原酶活性和合成过程受O2 的抑制 ,厌氧培养时 ,需要一定时间消耗系统中残余的O2 来解除这种抑制作用 .在无抗生素抑制蛋白质合成时 ,硝酸还原酶只有少量合成 ,而N2 O还原酶却显著地诱导产生 .这一结果对土壤吸收N2 O能力的研究具有重要意义 .在各处理下 ,系统中未发生亚硝酸盐的明显积累 ,表明亚硝酸还原酶活性大于硝酸还原酶 .外加葡萄糖加速了反硝化作用 ,并能促进酶的合成和消除还原过程中的电子竞争 .供试土壤表现出很强的厌氧呼吸作用 ,并受外加C源的促进 .反硝化作用的活化能低于土壤厌氧呼吸的活化能 ,因此反硝化作用的Q1 0值较低 ,CO2 和N2 O的产生比例随温度升高而加大 .     

蓝藻堆积对河蚬N2O释放通量及其肠道细菌群落结构的影响

【背景】底栖动物是湖泊生态环境的重要组成部分,在泥水界面氮迁移转化中扮演着重要的角色,是一个潜在的N_2O释放源,其N_2O释放量与栖息环境有关。蓝藻堆积区通常是湖泊N_2O释放的热点区域,其水质环境不同于无藻区。【目的】为了解蓝藻堆积与无藻堆积时底栖动物河蚬N_2O释放情况。【方法】采用室内微宇宙实验,以河蚬为研究对象,通过气相色谱和现代分子生物学相结合的技术测定蓝藻堆积区域底栖动物N_2O释放特征及其内在微生物学机制。【结果】蓝藻堆积区河蚬N_2O释放通量为447.2pmol/(ind·h),相比无藻区河蚬减少63%左右。河蚬肠道内总细菌及反硝化细菌对蓝藻堆积有不同的响应。454测序结果显示,无藻区河蚬肠道内细菌以变形菌门(β-和δ-变形菌纲)、绿弯菌门和拟杆菌门为主,三者相对丰度为67.3%,而蓝藻堆积区河蚬肠道细菌优势类群为变形菌门(α-和β-变形菌纲),相对丰度达到85.8%。Chao1和Shannon指数表明,蓝藻堆积区河蚬肠道细菌丰富度和多样性低于无藻区河蚬。对河蚬肠道内具有反硝化功能细菌进一步分析发现,蓝藻堆积区河蚬肠道内反硝化菌相对丰度可达到22.6%,是无藻区河蚬的2.3倍,反硝化作用得到增强。【结论】蓝藻堆积导致河蚬肠道内细菌丰富度和多样性降低,反硝化功能菌丰度增加,完全反硝化作用得以强化,导致河蚬N_2O释放通量降低。研究结果有助于揭示富营养化中湖泊底栖动物的环境效应,为湖泊温室气体控制提供理论支撑,具有重要的理论和现实意义。     

典型草原区不同生境反硝化菌群的空间特征

【背景】锡林河-河滨湿地-阶地草原是蒙古高原典型草原区代表性的水生-湿生-陆生生境,但不同生境中反硝化菌群的空间分布特征尚不明晰。【目的】阐明典型草原区不同生境反硝化菌群的组成、丰度、空间分布特征及异质性成因。【方法】利用16S rRNA基因测序研究锡林河流域水生、湿生、陆生生境6个样带沉积物/土壤细菌群落组成及相对丰度。基于2014年及以前文献报道的反硝化细菌及16S rRNA基因信息构建参比菌库,筛选生境关联的反硝化菌属。通过典范对应分析等探究反硝化菌群空间异质性成因。【结果】参比菌库包含80种反硝化细菌(65个属),6个样带测序获得的469个细菌属中36个为反硝化细菌属。3种生境共存的反硝化细菌有14个属,其中黄杆菌属(1.65%-14.17%)和噬氢菌属(1.56%-1.69%)是水生和湿生生境共有的优势菌,假单胞菌属(1.85%)是低河漫滩样带的优势菌。空间分布特征显示反硝化菌群沿水生-湿生-陆生生境呈现先升后降的分布趋势,在低河漫滩湿地达到最高值。典范对应分析表明:黄杆菌属、噬氢菌属、气单胞菌属、鞘氨醇单胞菌属等与pH值、水分及沙粒含量呈正相关关系,而芽孢杆菌属、链霉菌属、马杜拉放线菌属等与粘粒、粉粒、有机质、总氮含量等呈正相关关系。【结论】典型草原区反硝化菌群组成及丰度具有明显的生境异质性,低河漫滩湿地是反硝化细菌生长繁殖的最佳生境,由颗粒组成、水分含量和pH等环境因子共同驱动。     

Abundance of narG, nirS, nirK, and nosZ genes of denitrifying bacteria during primary successions of a glacier foreland

Quantitative PCR of denitrification genes encoding the nitrate, nitrite, and nitrous oxide reductases was used to study denitrifiers across a glacier foreland. Environmental samples collected at different distances from a receding glacier contained amounts of 16S rRNA target molecules ranging from 4.9 x 10(5) to 8.9 x 10(5) copies per nanogram of DNA but smaller amounts of narG, nirK, and nosZ target molecules. Thus, numbers of narG, nirK, nirS, and nosZ copies per nanogram of DNA ranged from 2.1 x 10(3) to 2.6 x 10(4), 7.4 x 10(2) to 1.4 x 10(3), 2.5 x 10(2) to 6.4 x 10(3), and 1.2 x 10(3) to 5.5 x 10(3), respectively. The densities of 16S rRNA genes per gram of soil increased with progressing soil development. The densities as well as relative abundances of different denitrification genes provide evidence that different denitrifier communities develop under primary succession: higher percentages of narG and nirS versus 16S rRNA genes were observed in the early stage of primary succession, while the percentages of nirK and nosZ genes showed no significant increase or decrease with soil age. Statistical analyses revealed that the amount of organic substances was the most important factor in the abundance of eubacteria as well as of nirK and nosZ communities, and copy numbers of these two genes were the most important drivers changing the denitrifying community along the chronosequence. This study yields an initial insight into the ecology of bacteria carrying genes for the denitrification pathway in a newly developing alpine environment.     

Genetic characterization of denitrifier communities with contrasting intrinsic functional traits

Microorganisms capable of denitrification are polyphyletic and exhibit distinct denitrification regulatory phenotypes (DRP), and thus, denitrification in soils could be controlled by community composition. In a companion study (D?rsch et al., 2012) and preceding work, ex situ denitrification assays of three organic soils demonstrated profoundly different functional traits including N(2) O/N(2) ratios. Here, we explored the composition of the underlying denitrifier communities by analyzing the abundance and structure of denitrification genes (nirK, nirS, and nosZ). The relative abundance of nosZ (vs. nirK + nirS) was similar for all communities, and hence, the low N(2) O reductase activity in one of the soils was not because of the lack of organisms with this gene. Similarity in community composition between the soils was generally low for nirK and nirS, but not for nosZ. The community with the most robust denitrification (consistently low N(2) O/N(2) ) had the highest diversity/richness of nosZ and nirK, but not of nirS. Contrary results found for a second soil agreed with impaired denitrification (low overall denitrification activity, high N(2) O/N(2) ). In conclusion, differences in community composition and in the absolute abundance of denitrification genes clearly reflected the functional differences observed in laboratory studies and may shed light on differences in in situ N(2) O emission of the soils.     

Actinobacterial nitrate reducers and proteobacterial denitrifiers are abundant in N2O-metabolizing palsa peat

Palsa peats are characterized by elevated, circular frost heaves (peat soil on top of a permanently frozen ice lens) and are strong to moderate sources or even temporary sinks for the greenhouse gas nitrous oxide (N(2)O). Palsa peats are predicted to react sensitively to global warming. The acidic palsa peat Skalluvaara (approximate pH 4.4) is located in the discontinuous permafrost zone in northwestern Finnish Lapland. In situ N(2)O fluxes were spatially variable, ranging from 0.01 to -0.02 μmol of N(2)O m(-2) h(-1). Fertilization with nitrate stimulated in situ N(2)O emissions and N(2)O production in anoxic microcosms without apparent delay. N(2)O was subsequently consumed in microcosms. Maximal reaction velocities (v(max)) of nitrate-dependent denitrification approximated 3 and 1 nmol of N(2)O per h per gram (dry weight [g(DW)]) in soil from 0 to 20 cm and below 20 cm of depth, respectively. v(max) values of nitrite-dependent denitrification were 2- to 5-fold higher than the v(max) nitrate-dependent denitrification, and v(max) of N(2)O consumption was 1- to 6-fold higher than that of nitrite-dependent denitrification, highlighting a high N(2)O consumption potential. Up to 12 species-level operational taxonomic units (OTUs) of narG, nirK and nirS, and nosZ were retrieved. Detected OTUs suggested the presence of diverse uncultured soil denitrifiers and dissimilatory nitrate reducers, hitherto undetected species, as well as Actino-, Alpha-, and Betaproteobacteria. Copy numbers of nirS always outnumbered those of nirK by 2 orders of magnitude. Copy numbers of nirS tended to be higher, while copy numbers of narG and nosZ tended to be lower in 0- to 20-cm soil than in soil below 20 cm. The collective data suggest that (i) the source and sink functions of palsa peat soils for N(2)O are associated with denitrification, (ii) actinobacterial nitrate reducers and nirS-type and nosZ-harboring proteobacterial denitrifiers are important players, and (iii) acidic soils like palsa peats represent reservoirs of diverse acid-tolerant denitrifiers associated with N(2)O fluxes.     

Reduction in denitrification activity in field soils exposed to long term contamination by 2,4,6-trinitrotoluene (TNT)

Terrestrial sites contaminated with 2,4,6-trinitrotoluene (TNT) are a widespread and persistent problem and often contain non-vegetated areas with TNT concentrations well in excess of 1000 mg kg(-1). In this study, we examined the effect of TNT on denitrification activity in field soils, and compared the sensitivity of denitrifying enzymes to TNT. DNA probes assessed the prevalence of nirS, nirK and nosZ (encoding cd(1) or copper nitrite reductase and nitrous oxide reductase, respectively), denitrifying genotypes in the culturable and total microbial community. The nitrate (NaR), nitrite (NiR) and nitrous oxide (N(2)OR) reductase activities in field soil and in isolates were assessed by gas chromatography. The relative occurrence of the nirK, nirS or nosZ genotypes increased in the cultured community and in total uncultured community DNA as nitroaromatic concentrations increased. However, denitrifying activity decreased in response to increasing TNT concentrations, with an IC(50) for NaR+NiR+nitric oxide reductase (NOR) of 400 mg TNT kg(-1) soil and for N(2)OR of 26 mg TNT kg(-1) soil. The denitrifying activity of four soil isolates also decreased in response to TNT, with N(2)OR activity being three times more sensitive to TNT than NaR+NiR+NOR activity. Interestingly, there were 118 times more nirK isolates than nirS isolates in uncontaminated soil but only 1.5 times more in soil containing 17400 mg kg(-1) TNT. The results from this study indicated that TNT reduced denitrification activity in field soils, and N(2)OR was much more sensitive to TNT than NaR+NiR+NOR.     

Phylogenetic analysis of nitrite, nitric oxide, and nitrous oxide respiratory enzymes reveal a complex evolutionary history for denitrification

Denitrification is a facultative respiratory pathway in which nitrite (NO2(-)), nitric oxide (NO), and nitrous oxide (N2O) are successively reduced to nitrogen gas (N(2)), effectively closing the nitrogen cycle. The ability to denitrify is widely dispersed among prokaryotes, and this polyphyletic distribution has raised the possibility of horizontal gene transfer (HGT) having a substantial role in the evolution of denitrification. Comparisons of 16S rRNA and denitrification gene phylogenies in recent studies support this possibility; however, these results remain speculative as they are based on visual comparisons of phylogenies from partial sequences. We reanalyzed publicly available nirS, nirK, norB, and nosZ partial sequences using Bayesian and maximum likelihood phylogenetic inference. Concomitant analysis of denitrification genes with 16S rRNA sequences from the same organisms showed substantial differences between the trees, which were supported by examining the posterior probability of monophyletic constraints at different taxonomic levels. Although these differences suggest HGT of denitrification genes, the presence of structural variants for nirK, norB, and nosZ makes it difficult to determine HGT from other evolutionary events. Additional analysis using phylogenetic networks and likelihood ratio tests of phylogenies based on full-length sequences retrieved from genomes also revealed significant differences in tree topologies among denitrification and 16S rRNA gene phylogenies, with the exception of the nosZ gene phylogeny within the data set of the nirK-harboring genomes. However, inspection of codon usage and G + C content plots from complete genomes gave no evidence for recent HGT. Instead, the close proximity of denitrification gene copies in the genomes of several denitrifying bacteria suggests duplication. Although HGT cannot be ruled out as a factor in the evolution of denitrification genes, our analysis suggests that other phenomena, such gene duplication/divergence and lineage sorting, may have differently influenced the evolution of each denitrification gene.