纳米氧化锌和菲对长江口附近海域沉积物反硝化作用及功能菌群落结构的影响

本研究在长江口附近海域采集表层沉积物,采用实验室模拟培养与分子生物学手段相结合的方法,通过测定纳米氧化锌(ZnO NPs)和菲(Phe)胁迫下沉积物中NO^-₃-N和NO^-₂-N浓度和反硝化还原酶活性及反硝化细菌基因丰度和群落多样性变化,目的是比较研究ZnO NPs和Phe对河口区沉积物反硝化作用及功能菌群落结构的影响,并探讨其作用过程和可能的作用机制。结果表明:ZnO NPs和Phe对沉积物硝酸盐还原能力和亚硝酸还原能力均产生抑制作用,浓度越高,抑制作用越强,其中亚硝酸盐还原过程受到2种污染物抑制更强烈,加重了沉积物亚硝酸盐的累积。ZnO NPs对沉积物硝酸盐还原能力、硝酸还原酶活性、narG基因丰度的抑制程度大于Phe,Phe对沉积物亚硝酸盐还原能力、亚硝酸还原酶和nirS基因丰度的抑制程度大于ZnO NPs,表明对反硝化还原酶和反硝化功能基因的抑制是外源污染物胁迫影响反硝化过程的主要机制。ZnO NPs和Phe降低了沉积物反硝化菌群落多样性水平,增加沉积物中Halomonas的优势度,降低了Bacillus的优势度,但Phe对沉积物群落多样性和组成的影响更加明显,说明Phe对长江口海区的生态影响大于ZnO-NPs。     

湛江湾沉积物中反硝化和厌氧氨氧化细菌的丰度、多样性及分布特征

采用实时荧光定量PCR、高通量测序等方法对湛江湾沉积物中四个月份的反硝化细菌与厌氧氨氧化细菌的多样性和丰度进行了分析。结果表明:湛江湾沉积物中反硝化细菌和厌氧氨氧化细菌丰度在四个月份的变化和空间分布趋势为:nirS型反硝化细菌在二月份最高,四月份最低,且其平均丰度有从湛江湾湾内向湾口附近呈现先升高再降低的趋势;nirK型反硝化细菌丰度在九月份最高,十一月份最低;nosZ型反硝化细菌在四月份最高,其余月份变化不大;厌氧氨氧化细菌丰度在九月份最高,二月份最低。通过相关性分析结果表明,亚硝酸盐、铵盐等共同调控着湛江湾沉积物中反硝化和厌氧氨氧化细菌丰度变化。系统发育分析表明:湛江湾中存在着一些广泛分布的反硝化细菌,但也生活着一些新奇的nirK型和nosZ型反硝化细菌。对于厌氧氨氧化细菌而言,其主要属于浮霉菌门及Candidatus Scalindua属,具有较高的耐盐性,另外湛江湾海区的厌氧氨氧化细菌也生活着一类在其他地方没有的新分支。典范对应分析分析结果表明:硝酸盐显著影响湛江湾反硝化细菌和厌氧氨氧化细菌的群落结构。湛江湾沉积物中反硝化细菌和厌氧氨氧化细菌存在特殊的竞争与共存的关系,且由亚硝酸盐、硝酸盐、pH等多种环境因子共同驱动。     

辽河口沉积物反硝化细菌数量及多样性的研究

对辽河口沉积物中反硝化细菌的数量、多样性以及群落结构进行了研究.采用MPN-Griess分析了春夏秋三季的反硝化细菌数量,结果表明其数量春季最多,秋季次之,夏季最低,且从辽河下游及河口向远海区域逐渐降低.采用PCR-RFLP对反硝化细菌的亚硝酸盐还原酶基因(nirS)进行分析,发现沉积物中的反硝化细菌类型较新,大多数细...     

珠江口表层沉积物nirS型反硝化微生物多样性

本研究以nirS基因为分子标记,将PCR、克隆文库构建与测序和典范对应分析相结合,对珠江口表层沉积物nirS型反硝化微生物的群落多样性进行了研究。3个站位共获得180个nirS基因克隆子,隶属于62个OTUs,氨基酸序列的相似性在50%—100%之间。各站位OTU分布格局差异明显,范围在19—33之间,表现出高度的多样性。系统进化分析表明,62个OTUs形成了5个类群,分别与河口、海洋沉积物、海岸养殖排放废水、富营养化海湾及海水养殖沉积物等的反硝化微生物聚类在一起,表明珠江口作为海淡水交汇区具有独特的反硝化微生物群落分布格局,同时也指示了珠江口氮污染及富营养化程度。典范对应分析结果表明,盐度、氮相关营养盐水平(PON/TN、NH4-N、NO-N和NO-N)可能是影响其分布格局的重要因素。     

基于次溴酸钠氧化-氨基磺酸还原测定沉积物15N加富培养样品中的15NH+4的方法探索

沉积物中的异化硝酸盐还原过程是海洋中活性氮转化的关键过程之一.不同于反硝化和厌氧铵氧化,异化硝酸盐还原为铵(DNRA)是将硝酸盐直接还原为铵,而不是以氮气的形态移除,这有可能会加重水体富营养化和缺氧.目前测定沉积物中异化硝酸盐还原过程的主要手段是15N标记培养技术.为了准确评估DNRA的潜在速率,首先要准确测定加富样品...     

辽河口沉积物中参与主要生物地化循环的微生物功能基因分析

辽河口区域丰富的生物多样性在生物地球化学循环和维持生态系统功能等方面发挥着极其重要的作用。研究辽河口表层沉积物中参与常见地球化学循环的功能基因及其关键影响因子,对揭示和预测辽河口沉积物微生物的功能代谢潜力和受环境变化的影响具有重要意义。应用功能基因芯片(GeoChip 4.0)技术开展微生物功能基因多样性研究。结果表明:在6个沉积物样品中,检测到各类型功能基因48742个,反映了9个微生物介导的关键生物过程。其中,碳循环相关功能基因最多,其次是氮循环和硫循环,而磷循环相关功能基因较少。各站位生物过程相关的功能基因数量和荧光信号值整体呈由远海到近海上升的趋势,即河相区的微生物功能基因较海相区与混合区普遍丰富。碳循环中参与碳固定的基因荧光信号值最高。氮循环中反硝化功能基因荧光信号值最高。总磷与硝酸盐是影响微生物功能基因多样性分布格局的重要因素。该研究有助于了解环境变化对辽河口沉积物生态系统结构和功能的影响,为研究辽河口表层沉积物微生物介导的生物地球化学循环提供科学依据。     

海洋沉积物中反硝化细菌的分离及去除硝酸盐氮的模拟试验

从长江口沉积物中筛选分离出了海洋反硝化细菌,模拟了该细菌对不同浓度水平硝酸盐氮的去除效率。研究结果表明,分离出的海洋反硝化细菌能有效去除海水中硝酸盐氮,在硝酸盐氮的初始浓度为 1 mg/L,1 d 内硝酸盐氮去除率就达到了 70 %;在 100 mg/L 硝酸盐氮模拟试验中,约在一周内能将 90 % 硝酸盐氮去除。试验证明反硝化细菌的生长与水体中硝酸盐氮浓度有一定的相关关系,一旦生物修复过程完成,反硝化细菌就会大量死亡,水体重新恢复到清澈透明状况。     

海岸带沉积物中脱氮作用的研究进展

脱氮作用是硝酸盐的反硝化过程之一 ,它是海岸带沉积物中氮循环的一个关键过程。在这个过程中 ,异氧菌在呼吸中利用硝酸盐作为电子接受者 ,将硝酸盐还原为气体。由于脱氮作用的气体产物 (Ｎ２ 或Ｎ２Ｏ)最终扩散进入大气层中 ,从而减小了海岸带水体中初级生产者可利用氮的数量。另一方面 ,脱氮作用也可转移人类活动向海岸带输送的过量氮 ,这有助于缓解这些环境中的富营养化趋势 ,对氮浓度升高起到重要的缓冲作用。自１９７６年Ｎｉｘｏｎ发现美国Ｎａｒｒａｇａｎｓｅｔｔ湾存在微生物脱氮作用以来 ,海岸带沉积物中活跃的脱氮作用已成为海洋…     

南极纳尔逊冰川土壤中微生物的群落结构及其代谢特征研究

冰川土壤中的微生物是冰冻圈生态系统中的重要组成部分。南极纳尔逊冰川四周环海,临近海洋的物质输送和其他因素扰动改变了近岸土壤中部分理化因子,从而对土壤中的微生物群落产生影响。本研究采集了南极纳尔逊冰川不同近海距离处的土壤样品,并对其进行了细菌和古菌V4区扩增子测序以及宏基因组测序,探讨了不同近海距离的冰川土壤中的微生物群落结构和代谢潜能。物种多样性结果显示,不同位点的土壤微生物群落组成有所差异,但变形菌门、放线菌门、拟杆菌门等在冰川土壤样品中普遍存在且相对丰度较高。宏基因组分析结果显示,不同近海距离的冰川土壤微生物群落的功能基因分布不同,且能量代谢和跨膜运输等代谢途径的基因的丰度随着采样位点远离海洋而降低。冰川土壤中碳、氮、硫代谢分别以还原性柠檬酸循环、反硝化、同化硫酸盐还原途径为主,其中反硝化途径基因在所有样品中丰度较高。通过分箱组装获得了含有反硝化功能基因的基因组bin_71,并重构了其核心的代谢通路。本研究初步揭示了南极纳尔逊冰川土壤中微生物的群落结构及代谢潜能,为后续南极冰川土壤新物种的发现、功能基因的挖掘、以及探究全球气候变暖下海洋对沿海生态系统的影响提供了基础数据。     

辽河口海域N2O分布特征和海气通量研究

于2007年5月份(枯水期)和8月份(丰水期)采集辽河口区域大气样品及水样,应用气相色谱及静态顶空法测定大气及海水中溶解N₂O的浓度,并计算不同站位的N₂O通量。结果表明:辽河口区域水体中溶解N₂O浓度存在着明显的时空变化,5月份河口区域水体中N₂O浓度高于8月份,且河流段溶解N₂O浓度大于近海岸海域。水中溶解N₂O浓度及溶解氧浓度呈显著的负相关关系。在枯水期和丰水期,所有站位的N₂O均处于过饱和状态,N₂O气体从水体向大气排出,辽河口区域是大气中N₂O的一个排放源。     

Seasonal and spatial patterns of sedimentary denitrification rates in the Chukchi sea

Recent constructions of the global nitrogen budget estimate that at least half of the ocean's fixed nitrogen is lost by sedimentary denitrification, the majority of which occurs in continental shelves. The Arctic contains approximately 20% of the world's continental shelf, suggesting it is a substantial contributor to the global sedimentary denitrification rate. During two cruises in the summer and spring of 2002 and 2004, respectively, denitrification rates were calculated from the downward diffusive flux of nitrate in the shelf and slope sediments of the Chukchi Sea in the western Arctic. Additionally, in the spring of 2004, denitrification rates were determined by whole-core incubations in which the flux of nitrogen gas out of the sediments was measured. Measurements were made along three transects crossing the shelf and slope (50–3000 m), each transect having different overlying water characteristics. Denitrification rates generally decreased with increasing water depth: rates varied from about 1.6 mmol N m⁻² d⁻¹ for the shallow-water sediments to undetectable in deep-water sediments. Rates showed little variation between the two seasons. However, rates were found to correspond with differences in annual overlying primary productivities and particulate organic carbon export fluxes. An extrapolation to the whole Arctic yielded an average Arctic sedimentary denitrification rate of 13 Tg N yr⁻¹. Taken in the context of the global nitrogen budget, it is about 4–13% of the total sink of fixed nitrogen in the ocean.     

一株嗜碱兼性好氧反硝化菌Marinobacter sp.B3的分离鉴定及脱氮性能研究

为获得反硝化脱氮效率较好的菌株,实验从海水螺旋藻培养体系中分离获得一株嗜碱兼性好氧反硝化菌, 通过观察细菌形态以及16S rRNA基因序列的同源性分析, 鉴定该菌株为海杆菌属, 命名为Marinobacter sp. B3。为明确该海杆菌的反硝化性能及氮转化途径, 研究开展了溶解氧(DO), 碳氮摩尔比(C/N), pH和温度等不同单因素对反硝化性能影响实验和氮平衡实验。单因素影响实验结果表明, 当硝酸钾(KNO₃)作为唯一氮源, NO₃^--N的初始浓度为100 mg/L, 盐度32, 振荡速度为150 r/min (初始DO质量浓度是5.6 mg/L), C/N=10, pH=8.0±0.2, 温度为35 °C时, 可获得最大脱氮效果。氮平衡实验结果得出, 在好氧环境下, 有20.11%的NO₃^--N转化为胞内氮, 5.58 mg/L的NO₃^--N转化为其他形态(NO₂^--N、NO₄⁺-N和有机氮), 74.72%转化为N₂释放; 厌氧环境下, 有26.65%的NO₃^--N转化为胞内氮, 72.86%的NO₃^--N转化为气态产物释放。最终实验结果表明, Marinobactersp. B3在好氧和厌氧条件下, 48 h对NO₃^--N的去除率分别为99.89%和93.80%, 具有较好的反硝化脱氮能力, 且在好氧条件下NO₃^--N去除效率更高, 在海水工厂化循环水养殖尾水处理方面具有良好的应用前景。     

Application of chemical tracers to an estimate of benthic denitrification in the Okhotsk Sea

To estimate benthic denitrification in a marginal sea, we assessed the usefulness of \({\text{N}}_{2}^{*}\) , a new tracer to measure the excess nitrogen gas (N₂) using dissolved N₂ and argon (Ar) with N* in the intermediate layer (26.6–27.4σ _θ ) of the Okhotsk Sea. The examined parameters capable of affecting \({\text{N}}_{2}^{*}\) are denitrification, air injection and rapid cooling. We investigated the relative proportions of these effects on \({\text{N}}_{2}^{*}\) using multiple linear regression analysis. The best model included two examined parameters of denitrification and air injection based on the Akaike information criterion as a measure of the model fit to data. More than 80 % of \({\text{N}}_{2}^{*}\) was derived from the denitrification, followed by air injection. Denitrification over the Okhotsk Sea shelf region was estimated to be 5.6 ± 2.4 μmol kg^?1. The distribution of \({\text{N}}_{2}^{*}\) was correlated with potential temperature (θ) between 26.6 and 27.4σ _θ (r = ?0.55). Therefore, we concluded that \({\text{N}}_{2}^{*}\) and N* can act complementarily as a quasi-conservative tracer of benthic denitrification in the Okhotsk Sea. Our findings suggest that \({\text{N}}_{2}^{*}\) in combination with N* is a useful chemical tracer to estimate benthic denitrification in a marginal sea.     

黄海北部海域沉积物反硝化细菌数量及反硝化速率的季节变化

用乙炔抑制法和最大或然数(most probable number, MPN)法对黄海北部海域沉积物反硝化速率及反硝化细菌数量的季节变化进行了研究, 结果表明, 该海域反硝化速率在夏季最大, 范围在3.2～7.5μmol/(m²·h)之间, 平均值为4.85μmol/(m²·h); 而在春、秋季其范围分别为0.26～2.65μmol/(m²·h)和1.21～4.12μmol/(m²·h)。该研究海域3 个季节反硝化细菌数量差别较大, 春、夏、秋季分别在1.78×10⁴～8.12×10⁴, 1.18×10⁶～6.18×10⁶ 和0.72×10⁵～4.50×10⁵ 个/g 之间。春、秋两季反硝化速率和反硝化细菌数量之间呈显著性正相关, 相关系数分别为0.759 和0.750(P<0.05)。本结果可为黄海北部海域氮循环机制研究提供重要参考。     

Isotopic fractionation of dissolved nitrate during denitrification in the eastern tropical north pacific ocean

The isotopic composition (δ¹⁵N) of dissolved nitrate was measured at five stations within the oxygen-deficient region of the eastern tropical North Pacific Ocean (ETNP) and at one station 900 km northeast of Hawaii, which was considered to be representative of all major water masses of the Pacific. At this last station, the δ¹⁵N composition of dissolved nitrate decreased systematically from about +6‰ at 400 m to approximately +5‰ at 5,000 m; these results are consistent with other estimates from the western Pacific.In contrast, vertical profiles of δ¹⁵N of dissolved nitrate from the ETNP showed marked departure from the above observed trend and correlated with losses of nitrate arising from denitrification. Instantaneous fractionation factors (α) were estimated, using the one dimensional vertical diffusion-advection model. These results suggest that ¹⁴NO₃⁻ is consumed 3–4% faster than ¹⁵NO₃⁻, significantly larger than fractionations (2%) observed under laboratory conditions.Maximum rates of denitrification at 100 m were also evaluated and ranged from 0.6 to 8 μg-at 1⁻¹ yr⁻¹ for the stations investigated. The above upper limit is probably excessive, but the average maximum for the four stations analyzed is estimated to be 3.5 μg-at NO₃⁻ 1⁻¹ yr⁻¹. These results compare favorably with suitably corrected oxygen utilization rates derived from electron transport activity measurements.     

黄海北部海域沉积物反硝化细菌数量及反硝化速率的季节变化

用乙炔抑制法和最大或然数（most probable number,MPN）法对黄海北部海域沉积物反硝化速率及反硝化细菌数量的季节变化进行了研究,结果表明,该海域反硝化速率在夏季最大,范围在3．2～7．5μmol／（m^2·h）之间,平均值为4．85μmol／（m^2·h）;而在春、秋季其范围分别为0．26～2．65μmol／（m^2·h）和1．21～4．12μmol／（m^2·h）。该研究海域3个季节反硝化细菌数量差别较大,春、夏、秋季分别在1．78×10^4～8．12×10^4,1．18×10^6～6．18×10^6和0．72×10^5～4．50×10^5个／g之间。春、秋两季反硝化速率和反硝化细菌数量之间呈显著性正相关,相关系数分别为0．759和0．750（P〈0．05）。本结果可为黄海北部海域氮循环机制研究提供重要参考。     

Evaluation of the N2 flux approach for measuring sediment denitrification

Direct gas chromatographic measurement of denitrification rates via N₂ fluxes from aquatic sediments can avoid some of the artifacts and complexities associated with indirect approaches and tracer techniques. However, measurement protocols have typically been determined based upon initial results or previous studies. We present a process-level study and simulation model for evaluating and optimizing N₂ gas flux approaches in closed chamber incubations. Experimental manipulations and simulations of both artificial and natural sediments were used to conduct sensitivity analyses of key design parameters in N₂ flux measurements. Experimental results indicated that depletion of labile organic matter during the long incubations required by common protocols (for diffusive off-gassing of porewater N₂) may result in underestimates of denitrification rates in some systems. Simulations showed that the required incubation time was primarily a function of sediment thickness. The best approach found to minimize incubation time and reduce errors was to select the minimum sediment thickness necessary to include the entire depth distribution of nitrification–denitrification for a particular sediment system. Attempts to increase measurement sensitivity and shorten incubation times by reducing the headspace thickness to 1–2 cm generally cause denitrification to be underestimated by 3–13% for gas headspaces, and up to 80% for water headspaces. However, errors were negligible with gas and water headspace thicknesses of 10 cm and 15 cm, respectively. Anaerobic cores to control for non-denitrification N₂ fluxes shortened incubation time, but introduced artifacts in sediments with extensive macrofaunal irrigation.     

细菌反硝化法测试硝酸盐氮、氧同位素的研究与应用

为了验证细菌反硝化法对水体中硝酸盐氮、氧同位素组成测定的适用性、重现性及准确性, 在不同时间(2019年7月28日、8月19日、8月26日)利用反硝化细菌分别将海水、湖水和自来水样品中的硝酸盐转化为氧化亚氮（N₂O）, 并进行氮、氧同位素测定。结果表明, 不同时间段3个批次实验的硝酸盐氮同位素校准曲线斜率都接近理论值1, 相关性系数均高于0.999, 说明反硝化细菌在将样品中的硝酸盐全部还原为N₂O的过程中氮同位素分馏效应很小; 同一样品3个批次测定的硝酸盐的氮同位素值基本相同, 表明细菌反硝化法对硝酸盐氮同位素的测定在长时间周期内具有很好的重现性和准确性。3个批次氧同位素校准曲线斜率稳定在0.61～0.63之间, 相关性系数均高于0.99, 单批次内海水、湖水和自来水3类样品中硝酸盐氧同位素比值的标准偏差范围在0.18‰～0.69‰之间, 表明经过氧同位素校准曲线的校正, 可以准确反映样品中硝酸盐氧同位素组成; 同一样品3个批次测定的氧同位素值差异较大, 其变化范围为1.33‰～16.38‰, 可能是由于样品储存过程中硝酸盐与水之间发生的氧同位素交换作用所致。     

Anoxia over the western continental shelf of India: bacterial indications of intrinsic nitrification feeding denitrification

Studies on the Arabian Sea coastal anoxia have been of immense interest, but despite its ecological significance there is sparse understanding of the microbes involved. Hence, observations were carried out off Goa (15 degrees 30'N, 72 degrees 40'E to 15 degrees 30'N, 72 degrees 59'E) to understand the processes that mediate the changes in various inorganic nitrogen species in the water column during anoxia. Water column chemistry showed a clear distinct oxic environment in the month of April and anoxic condition in October. Our study based on microbial signatures indicated that oxygen deficit appeared as a well-defined nucleus almost 40 km away from the coast during the oxic period (April) and spreads there after to the entire water column synchronizing with the water chemistry. Striking results of net changes in inorganic nitrogen species in nitrification blocked and unblocked experimental systems show that denitrification is the predominant process in the water column consuming available nitrate ( approximately 0.5 microM) to near zero levels within approximately 72 h of incubation. These observations have been supported by concomitant increase in nitrite concentration ( approximately 4 microM). Similar studies on denitrification-blocked incubations, demonstrate the potential of nitrification to feed denitrification. Nitrification could contribute almost 4.5 microM to the total nitrate pool. It was found that the relation between ammonium and total dissolved inorganic nitrogen (DIN) pool (r=0.98, p<0.001, n=122) was significant compared to the latter with nitrite and nitrate. The occurrence of high ammonium under low phosphate conditions corroborates our observations that ammonium does not appear to be locked under low oxygen regimes. It is suggested that ammonium actively produced by detrital breakdown (ammonification) is efficiently consumed through nitrification process. The three processes in concert viz. ammonification, nitrification and denitrification appear to operate in more temporal and spatial proximity than hitherto appreciated in these systems and this gives additional cues on the absence of measurable nitrate at surface waters, which was earlier attributed only to efficient algal uptake. Hence we hypothesize that the alarming nitrous oxide input into the atmosphere could be due to high productivity driven tighter nitrification-denitrification coupling, rather than denitrification driven by extraneous nitrate.