Relationship between nitrifier and denitrifier community composition and abundance in predicting nitrous oxide emissions from ephemeral wetland soils

The link between differences in the community composition of nitrifiers and denitrifiers to differences in the emission of nitrous oxide (N₂O) from soils remains unclear. Nitrifier and denitrifier community composition, abundance and N₂O emission activity were determined for two common landscapes characteristic of the North American “prairie pothole region”: cultivated wetlands (CW) vs. uncultivated wetlands (UW). The hypotheses of this study were: (1) landscape selects for different nitrifier and denitrifier communities, (2) denitrification was the dominant N₂O emitting process, and (3) a relationship exists between nitrifier and denitrifier community composition, their abundance, and N₂O emission. Comparisons were made among soils from three CW and three UW at the St. Denis National Wildlife Area. Denaturing gradient gel electrophoresis was used to compare community composition, and quantitative polymerase chain reaction was used to estimate community size. Incubation experiments on re-packed soil cores with ¹⁵N-labeled nitrate were performed to assess the relative contributions of nitrification and denitrification to total N₂O emission. Results indicate: (1) nitrification was the primary source of N₂O emission, (2) cultivation increased nitrifier abundance but decreased nitrifier richness, (3) denitrifier abundance was not affected by cultivation but richness was increased by cultivation, and (4) differences in nitrifier and denitrifier communities composition and abundance between land-use and landform did not correspond to differences in N₂O emission.     

凉水国家自然保护区不同林型红松林土壤nosZ型反硝化微生物群落组成和多样性分析

【目的】分析凉水国家级自然保护区内的3种原始红松林(云冷杉红松林、椴树红松林和枫桦红松林)、红松人工林和红松天然次生林5种林型的土壤nosZ型反硝化微生物的群落组成和多样性特征,为全面了解不同林型红松林土壤的反硝化潜势和氮循环过程提供数据支持。【方法】以选取的5种林型红松林林下土壤为研究对象,以反硝化过程中的关键酶——氧化亚氮还原酶的编码基因nosZ为标记基因,采用高通量测序和生物信息学分析技术进行研究。【结果】从5种林型红松林15个土壤样品中一共得到nosZ基因631 878条有效序列,579 871条优质序列,长度分布在178~383 bp之间,主要分布在260 bp。5种林型红松林土壤nosZ型反硝化微生物主要门类为变形菌门和拟杆菌门,核心属为伯霍尔德杆菌属、黄杆菌属、慢生根瘤菌属、假单胞菌属、Dechloromonas属、芽单胞菌属、无色杆菌属和中华根瘤菌属。nosZ型反硝化微生物α多样性分析显示:除枫桦红松林的Shannon和Simpson指数显著高于红松天然次生林外,5种林型红松林之间土壤nosZ型反硝化菌群的4种α多样性指数(Shannon、Chao1、ACE和Simpson指数)差异不显著。β多样性分析显示:5种林型土壤nosZ型反硝化微生物群落组成差异显著(R=0.387,P=0.006),但3种原始红松林之间差异不显著。土壤铵氮和全氮含量是显著影响nosZ型反硝化微生物群落的主要因子(P﹤0.05)。【结论】5种林型红松林土壤nosZ型反硝化微生物多数α多样性指数无显著差异,但β多样性差异显著,引起不同林型之间nosZ型反硝化微生物组成和丰度的主要环境因子是土壤铵氮和全氮含量。     

Advantages of a diluted nutrient broth medium for isolating N2-producing denitrifying bacteria of α-Proteobacteria in surface and subsurface upland soils

This research aimed to determine whether a diluted nutrient broth (DNB) medium was different from a conventional nutrient broth (NB) medium when counting and isolating denitrifying bacteria in surface and subsurface upland soils. To this end, we investigated populations of denitrifying bacteria isolated from the surface to a depth of 4 m of subsurface upland soil that had received slurry. The DNB medium gave higher viable counts of denitrifying bacteria than the NB medium and a higher isolation ratio of denitrifying bacterial isolates. In total, 74 isolates from the DNB medium (D-isolates) and 26 isolates from the NB medium (N-isolates) were collected. We characterized their denitrifying activity and analyzed the diversity of 16S rDNA and denitrifying-related genes. Seventy-three percent of the D-isolates were oligotrophic denitrifying bacteria. The N₂-producing, oligotrophic denitrifying bacteria, largely of α-Proteobacteria, increased in the D-isolates. The D-isolates and the N-isolates had some taxonomic overlapping on a phylogenetic tree based on 16S rDNA. It was not possible to identify the denitrification phenotype (N₂-producing or N₂O-producing) on the phylogenetic tree. Phylogenetic groups of isolates corresponded to nirK groups, except in some isolates in which horizontal gene transfer might have occurred. The terminal gas emission of the isolates was consistent with the existence of the nosZ gene. The DNB medium may be very useful in isolating N₂-producing denitrifying α-Proteobacteria. Its use highlights the ecological significance of oligotrophic isolates and the different viable counts resulting from the selectivity of conventional and diluted media.     

Diversity of denitrifying microflora and ability to reduce N2O in two soils

 The ozone-depleting gas N₂O is an intermediate in denitrification, the biological reduction of NO₃ ^– to the gaseous products N₂O and N₂ gas. The molar ratio of N₂O produced (N₂O/N₂O+N₂) varies temporally and spatially, and in some soils N₂O may be the dominant end product of denitrification. The fraction of NO₃ ^–-N emitted as N₂O may be due at least in part to the abundance and activity of denitrifying bacteria which possess N₂O reductase. In this study, we enumerated NO₃ ^–-reducing and denitrifying bacteria, and compared and contrasted collections of denitrifying bacteria isolated from two agricultural soils, one (Auxonne, soil A) with N₂O as the dominant product of denitrification, the other (Chalons, soil C) with N₂ gas as the dominant product. Isolates were tested for the ability to reduce N₂O, and the presence of the N₂O reductase (nosZ)-like gene was evaluated by polymerase chain reaction (PCR) using specific primers coupled with DNA hybridization using a specific probe. The diversity and phylogenetic relationships of members of the collections were established by PCR/restriction fragment length polymorphism of 16s rDNA. The two soils had similar numbers of bacteria which used NO₃ ^– as a terminal electron acceptor anaerobically. However, the soil A had many more denitrifiers which reduced NO₃ ^– to gaseous products (N₂O or N₂) than did soil C. Collections of 258 and 281 bacteria able to grow anaerobically in the presence of NO₃ ^– were isolated from soil A and soil C, respectively. These two collections contained 66 and 12 denitrifying isolates, respectively, the others reducing NO₃ ^– only as far as NO₂ ^–. The presence of nosZ sequences was generally a poor predictor of N₂O reducing ability: there was agreement between the occurrence of nosZ sequences and the N₂O reducing ability for only 42% of the isolates; 35% of the isolates (found exclusively in soil A) without detectable nosZ sequences reduced N₂O whereas 21% of the isolates carrying nosZ sequences did not reduce this gas under our assay conditions. Twenty-eight different 16S rDNA restriction patterns (using two restriction endonucleases) were distinguished among the 78 denitrifying isolates. Two types of patterns appeared to be common to both soils. Twenty-three and three types of patterns were found exclusively among bacteria isolated from soils A and C, respectively. The specific composition of denitrifying communities appeared to be different between the two soils studied. This may partly explain the differences in the behaviour of the soils concerning N₂O reduction during denitrification. Received: 31 October 1997     

沿海滩涂土壤中反硝化细菌群落随盐分梯度的更迭

To better understand the effect of salinity on denitrification communities,soils along a salinity gradient (ranging from 7.32 to 1.70 mS cm 1) in a wetland along the Yellow Sea coastline in Jiangsu Province,China,were studied using both culture-dependent and-independent methods.Culture efforts yielded 82 isolates in total,81.7% of which were close relatives of Bacillus sp.based on partial sequences of their 16S rRNA genes.Denaturing gradient gel electrophoresis (DGGE) analysis based on 16S rRNA sequences suggested possible existence of bacterial community succession along the salinity gradient.Clone library analysis based on nosZ gene sequences (coding nitrous oxide reductase) showed that operational taxonomic units (OTUs) associated with α-proteobacteria dominated in all three soils,whereas those associated with β-and γ-subdivisions showed a clear succession.In the high salinity soil,only the OTUs associated with α-subdivision were found.In the medium salinity soil,small proportions of β-(6.5%) and γ-associated (19.6%) OTUs were found.In the low salinity soil,the proportions were further increased to 33% and 25% for β-and γ-Proteobacteria,respectively.Statistic analysis using Unifrac P test showed that nosZ-communities in different saline soils were significantly different from each other.It could be concluded that α-subdivision of nosZ-community tended to be sustained in high salinity environments whereas β-and γ-subdivisions,especially the former,tended to be sustained in low salinity environments.Salinity was the key determinant of nosZ-community composition in the environment.     

Dynamics of the nitrous oxide reducing community during adaptation to Zn stress in soil

Laboratory studies show that the nitrous oxide (N₂O) reduction rate in soil is strongly inhibited by trace metal contamination; however, this effect appears transient. Here we assess if this recovery is due to microbial adaptation associated with shifts in community composition. Soils were spiked with zinc chloride (0-5000 mg Zn kg⁻¹) in a factorial design with 3 application rates of organic matter (OM), i.e. 0, 2 and 4 g milled hay kg⁻¹, to accelerate growth and, potentially, adaptation rate. The soil treatments were incubated outdoors with free drainage during 1 year and periodically sampled. The potential N₂O reduction rate, measured in an anaerobic laboratory assay, was inhibited by Zn during the first 2 months after spiking with 50% inhibition at 500-1000 mg Zn kg⁻¹. After 6 months exposure, the N₂O reduction rate recovered to at least 80% of the rate in the control treatment in the series receiving OM up to the largest Zn dose, but strong inhibition remained in the series which did not receive OM. In this series recovery was only observed after 12 months exposure. Soil pore water Zn concentrations did not explain the recovery of the N₂O reduction rate in the control series suggesting that recovery is due to adaptation and not to reduced Zn bioavailability. The faster recovery in the series receiving OM was partially, but not fully related to the effects of OM on Zn bioavailability. The recovery at all Zn and OM treatments co-varied with a recovery of nosZ gene abundance from about 1 × 10⁷ copies g⁻¹ soil in the soil treatments with decreased activity to 5 × 10⁸ copies g⁻¹ soil in the other soil treatments. The nosZ gene DGGE profile of the soil microbial communities revealed minor changes in the nosZ containing community. This study strongly suggests that the transient effects of trace metal inhibition of N₂O reduction is due to the development of a Zn tolerant denitrifying community.     

添加水铁矿对水稻土N_2O释放及反硝化微生物的影响

水稻土氧化还原状态的变化与N_2O的释放有密切关系。为揭示水稻土中Fe对N_2O释放及反硝化功能微生物的影响,本研究选取第四纪红壤发育的水稻土,设置3个水铁矿添加水平(Fe 0,10,40μmol g~(-1)土)和两个土壤质量含水量(50%,80%)进行土壤培养试验,利用实时荧光定量PCR(real time flourescent quantification polymerase chain reaction,q PCR)和末端限制性片段长度多态性(terminal-restricted fragment length polymorphism,T-RFLP)分析技术开展研究。结果表明,N_2O排放速率升至高峰期的过程中,外源铁处理尤其是添加高量铁(40μmol g~(-1))处理导致硝态氮含量显著高于对照,而N_2O排放速率却明显低于对照;然而,高峰期后添加高量铁处理却维持了较对照显著高的N_2O排放速率;与此同时,添加水铁矿对硝酸还原酶基因(narG)和氧化亚氮还原酶基因(nosZ)丰度的影响表现出与N_2O排放相同的趋势,即N_2O排放速率升至高峰期的过程中,外源铁处理明显抑制了反硝化微生物的生长与繁殖,而高峰期后外源铁对反硝化微生物的抑制作用不明显。因此,水稻土中添加Fe(Ⅲ)对N_2O释放影响的主要原因可能是前期抑制了反硝化功能微生物的种群数量,从而减少了硝酸根的还原和N_2O的产生,而后期由于反硝化微生物数量的恢复和NO_3~-等含氮化合物的残留,使得外源铁处理的N_2O释放量明显高于对照。     

M.hydrocarbonoclasticus NY4中nosZ基因的克隆与异源表达

选取反硝化菌株Marinobacter hydrocarbonoclasticus NY4为试验对象,首先通过反硝化气态产物分析,证实了该菌株中N_2OR的存在;随后设计特异性引物并通过PCR扩增获取了nosZ的全长基因(1 896bp),该基因与来源于Geobacillus thermodenitrificans的nosZ基因仅保持了46.9%的相似性;实现了nosZ基因在大肠杆菌中的异源表达,并成功检测到了N_2OR酶活,其比活力达到(0.2±0.01)U/mg。