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。     

九龙江口肥水-缢蛏养殖塘厌氧氨氧化微生物活性研究

缢蛏串联养殖经由饵料藻类养殖塘(俗称肥水塘)投放适量含氮化合物以提高生产效率,了解此过程中的沉积物氮转化对调控和管理河口养殖塘氮的收支具有重要意义。以福建九龙江口肥水-缢蛏养殖塘为研究对象,运用同位素示踪技术和高通量测序技术探究缢蛏塘和肥水塘沉积物厌氧氨氧化速率及微生物群落结构,并分析其环境调控机制。结果表明,肥水塘沉积物厌氧氨氧化速率为(1.76±0.11) nmol/(g·h),相对贡献为9.16%±0.40%,显著高于缢蛏塘(P<0.05)。缢蛏塘和肥水塘厌氧氨氧化微生物主要由Candidatus Scalindua、Ca.Kuenenia、Ca.Brocadia和Ca.Anammoximicrobium属组成。其中,Ca.Kuenenia属为养殖塘的优势菌属。沉积物厌氧氨氧化微生物多样性和丰富度呈显著的空间分布特征,肥水塘中生物多样性更高(P<0.05)。厌氧氨氧化微生物活性和群落结构差异主要受养殖土地利用方式、溶解氧(DO)、NH₄⁺和NO₃^-含量等环境因子的调控。该研究在一定...     

用于海洋沉积物中厌氧氨氧化细菌分子生态学研究的引物比较

为比较厌氧氨氧化细菌16S rRNA基因的2对特异性引物(Amx368F/Amx820R、Brod541F/Amx820R)在其分子生态学研究方面的优点与不足,本研究采用高通量测序和qPCR技术对长江口及其邻近海域沉积物中厌氧氨氧化细菌细菌的群落组成、多样性和丰度进行比较分析,结果表明,引物Amx368F/Amx820R特异性较高,能够较为完整地反映沉积物中厌氧氨氧化细菌的多样性信息,且其覆盖的厌氧氨氧化细菌丰度与功能基因hzo丰度正相关(P0.01);而引物Brod541F/Amx820R特异性较低,覆盖部分厌氧氨氧化细菌及其他多个门的细菌,其量化的厌氧氨氧化细菌与功能基因hzo丰度无明显相关关系(P0.05)。因此,引物Amx368F/Amx820R能够更加真实地反映厌氧氨氧化细菌在海洋沉积物中的群落特征,在其分子生态学研究中更具优势。     

海洋沉积物氨氧化菌群落结构的测序分析

本文以氨氧化细菌(ammonia-oxidizing bacteria,AOB)特征基因氨单加氧酶(ammonia monooxygenase,AMO)α亚基基因(amoA)为目标基因(～491bp),分别采用克隆文库、454高通量测序和illumina测序技术对其群落结构进行了比较研究。结果表明, 454高通量测序和illumina测序技术得到的菌群多样性及丰富度均高于克隆文库技术。在菌群组成方面,虽然3种测序技术检测到的优势类群均为亚硝化螺菌,但在菌群丰度方面存在差异。3种测序技术中,克隆文库技术在很大程度上可能高估物种丰度,且对部分低丰度类群的检测能力有限;illumina单端测序由于其测序片段较短,可能存在物种注释错误、不能准确区分各物种间序列差异等问题;比较而言, 454高通量测序技术能较为全面和准确地反映海洋沉积物中AOB的菌群结构特征。     

海洋环境中的厌氧氨氧化细菌与厌氧氨氧化作用

厌氧氨氧化是细菌在厌氧条件下将氨氮氧化成氮气的过程,主要应用在污水处理反应器中,最近几年发现在海洋环境中也广泛存在,并在海洋氮循环过程中发挥了重要作用,代表了海洋中一个巨大的氮汇,对碳循环和全球气候变化也有重要影响。梯烷膜脂结构独特,是厌氧氨氧化细菌的化学生物标志物,具有化学分类与古海洋学应用潜力。厌氧氨氧化作用及厌氧氨氧化细菌已成为海洋生物地球化学、微生物学、有机地球化学等研究领域的热点。     

海洋环境中的厌氧氨氧化细菌与厌氧氨氧化作用

厌氧氨氧化是细菌在厌氧条件下将氨氮氧化成氮气的过程,主要应用在污水处理反应器中,最近几年发现在海洋环境中也广泛存在,并在海洋氮循环过程中发挥了重要作用,代表了海洋中一个巨大的氮汇,对碳循环和全球气候变化也有重要影响.梯烷膜脂结构独特,是厌氧氨氧化细菌的化学生物标志物,具有化学分类与古海洋学应用潜力.厌氧氨氧化作用及厌氧...     

渤海和南黄海沉积物中氨氧化微生物对硝化潜势的相对贡献

硝化作用是海洋氮循环的核心过程。作为硝化过程关键步骤的氨氧化过程的主要参与者,氨氧化古菌和氨氧化细菌对硝化作用的相对贡献是海洋氮循环关注的热点问题之一。本文选取渤海和南黄海20个站位的表层沉积物,通过微宇宙培养实验研究了沉积物中氨氧化古菌和氨氧化细菌对硝化潜势的相对贡献。结果表明,渤海和南黄海海域表层沉积物中潜在硝化速率(以氮计,下同)为0.004 6～0.283 1μmol/(g·d),其中氨氧化古菌潜在硝化速率为0.004 3～0.274 3μmol/(g·d),氨氧化细菌潜在硝化速率为0.000 4～0.056 0μmol/(g·d)。氨氧化古菌是硝化潜势的主要贡献者,在渤海海域的贡献率为59.79%～97.95%,在南黄海海域的贡献率为18.47%～94.26%。渤海海域潜在硝化速率显著高于南黄海海域。此外,本研究海域中盐度是影响潜在硝化速率的关键环境因子,对渤海海域的分析则表明越高的NO₃^-浓度可能指示着越高的硝化潜势。在河口及近海沉积物中,氨氧化古菌在硝化过程中起着更加重要的作用;河口和近岸沉积物硝化潜势总体高于远海。本研究为进一...     

珠江口沉积柱中厌氧氨氧化活动的梯烷脂记录

为了反演珠江口过去环境的厌氧氨氧化活动状况,本文分析了来自珠江口3个站位的柱状沉积物中梯烷脂的含量,在所有的样品中均检测到了梯烷脂,表明珠江口地区存在厌氧氨氧化活动。P03沉积柱的梯烷脂总含量为0.16~4.13μg/g,L1为0.15~1.3μg/g,C2T为0.07~2.19μg/g。梯烷脂的垂直分布特征表明3个沉积柱中厌氧氨氧化细菌的来源可能不同,P03沉积柱中梯烷脂可能主要来源于沉积物中的厌氧氨氧化细菌,而L1梯烷脂可能来自上层水体和沉积物,C2T的厌氧氨氧化活动可能主要发生在底层缺氧水体中。将梯烷脂与总有机碳(TOC)作相关性分析,结果显示在TOC含量高的柱状样中,两者相关性好,TOC校准后的梯烷脂与原梯烷脂的分布趋势的一致性,表明梯烷脂含量能够指示厌氧氨氧化活动。对珠江口过去的厌氧氨氧化活动的重建表明P03站位沉积物中厌氧氨氧化活动自1970—1990增强,L1站位的厌氧氨氧化活动近十年来亦逐年增强,C2T站位上覆水体的厌氧氨氧化活动则呈现近几年快速增强的趋势。虽然本研究仅初步探讨了近些年来珠江口厌氧氨氧化活动的变化规律,但为进一步研究珠江口氮循环过程提供了良好的基础。     

青岛潮间带趋磁细菌的垂直分布特征及与环境因子的关系

趋磁细菌是一类能够沿着磁力线运动的革兰氏阴性细菌, 主要生活在淡水和海洋的有氧-无氧过渡区。本调查通过对青岛汇泉湾潮间带沉积物柱状样分层样品中趋磁细菌的计数和沉积物理化因子的分析, 研究了趋磁细菌的垂直分布特征及其与环境因子的关系。结果显示趋磁细菌以趋北型的趋磁球菌占优势, 主要分布环境的氧化还原电位为179.7～-107.0 mV, 为弱还原-还原性质, 最大丰度(94～169个/cm³)出现在氧化还原跃层(表层下5～9cm黄-黑沙界面处), 暗示有氧-无氧过渡区; 趋磁细菌的垂直变化趋势与粒径、含水率、硫酸盐的变化趋势基本一致。     

海水反硝化和厌氧氨氧化速率同步测定的15N示踪法及其应用

反硝化与厌氧氨氧化作用是海洋结合态氮迁出的主要途径,15N示踪法是定量反硝化和厌氧氨氧化作用的新方法,它具有灵敏度高、可同步测量反硝化与厌氧氨氧化速率的优点.本研究开展了一系列方法学实验,以确定高纯氦气驱赶水样所含气体的时间、超声赶气时间、同位素比值质谱仪测量N2含量的精度、N2含量及其同位素组成测定的工作曲线以及示踪剂的最佳添加量等,从而确立起15N示踪同步测定海水反硝化和厌氧氨氧化速率的方法.所建立方法测量N2含量的相对标准偏差小于8%,可满足海洋反硝化与厌氧氨氧化速率测定的需要.应用该方法实测了厦门筼筜湖富氧与缺氧水体的反硝化与厌氧氨氧化速率,结果表明,缺氧底层水的反硝化速率[4.10～5.47μmol/（dm3·d）]比富氧表层水[0.43～0.46μmol/（dm3·d）]明显来得高,但缺氧底层水的厌氧氨氧化速率[0.00～0.12μmol/（dm3·d）]则比富氧表层水的速率[0.52～0.67μmol/（dm3·d）]低得多.     

Rates and regulation of nitrogen cycling in seasonally hypoxic sediments during winter (Boknis Eck,SW Baltic Sea): Sensitivity to environmental variables

This study investigates the biogeochemical processes that control the benthic fluxes of dissolved nitrogen (N) species in Boknis Eck – a 28 m deep site in the Eckernförde Bay (southwestern Baltic Sea). Bottom water oxygen concentrations (O_2−BW) fluctuate greatly over the year at Boknis Eck, being well-oxygenated in winter and experiencing severe bottom water hypoxia and even anoxia in late summer. The present communication addresses the winter situation (February 2010). Fluxes of ammonium (NH₄⁺), nitrate (NO₃⁻) and nitrite (NO₂⁻) were simulated using a benthic model that accounted for transport and biogeochemical reactions and constrained with ex situ flux measurements and sediment geochemical analysis. The sediments were a net sink for NO₃⁻ (−0.35 mmol m⁻² d⁻¹ of NO₃⁻), of which 75% was ascribed to dissimilatory reduction of nitrate to ammonium (DNRA) by sulfide oxidizing bacteria, and 25% to NO₃⁻ reduction to NO₂⁻ by denitrifying microorganisms. NH₄⁺ fluxes were high (1.74 mmol m⁻² d⁻¹ of NH₄⁺), mainly due to the degradation of organic nitrogen, and directed out of the sediment. NO₂⁻ fluxes were negligible. The sediments in Boknis Eck are, therefore, a net source of dissolved inorganic nitrogen (DIN = NO₃⁻ + NO₂⁻ + NH₄⁺) during winter. This is in large part due to bioirrigation, which accounts for 76% of the benthic efflux of NH₄⁺, thus reducing the capacity for nitrification of NH₄⁺. The combined rate of fixed N loss by denitrification and anammox was estimated at 0.08 mmol m⁻² d⁻¹ of N₂, which is at the lower end of previously reported values. A systematic sensitivity analysis revealed that denitrification and anammox respond strongly and positively to the concentration of NO₃⁻ in the bottom water (NO₃⁻_BW). Higher O_2−BW decreases DNRA and denitrification but stimulates both anammox and the contribution of anammox to total N₂ production (%R_amx). A complete mechanistic explanation of these findings is provided. Our analysis indicates that nitrification is the geochemical driving force behind the observed correlation between %R_amx and water depth in the seminal study of Dalsgaard et al. (2005). Despite remaining uncertainties, the results provide a general mechanistic framework for interpreting the existing knowledge of N-turnover processes and fluxes in continental margin sediments, as well as predicting the types of environment where these reactions are expected to occur prominently.     

Benthic metabolism and the fate of dissolved inorganic nitrogen in intertidal sediments

We determined patterns of benthic metabolism and examined the relative importance of denitrification (DNF) and dissimilatory nitrate reduction to ammonium (DNRA) as sinks for nitrate (NO₃⁻) in intertidal sediments in the presence and absence of benthic microalgal (BMA) activity. By influencing the activity of BMA, light regulated the metabolic status of the sediments, and, in turn, exerted strong control on sediment nitrogen dynamics and the fate of inorganic nitrogen. A pulsed addition of ¹⁵N-labeled NO₃⁻ tracked the effect and fate of dissolved inorganic nitrogen (DIN) in the system. Under illuminated conditions, BMA communities influenced benthic fluxes directly, via DIN uptake, and indirectly, by altering the oxygen penetration depth. Under dark hypoxic and anoxic conditions, the fate of water column NO₃⁻ was determined largely by three competing dissimilatory reductive processes; DNF, DNRA, and, on one occasion, anaerobic ammonium oxidation (anammox). Mass balance of the added ¹⁵N tracer illustrated that DNF accounted for a maximum of 48.2% of the ¹⁵NO₃⁻ reduced while DNRA (a minimum of 11.4%) and anammox (a minimum of 2.2%) accounted for much less. A slurry experiment was employed to further examine the partitioning between DNF and DNRA. High sulfide concentrations negatively impacted rates of both processes, while high DOC:NO₃⁻ ratios favored DNRA over DNF.     

自组装膜进样质谱系统及其在砂质沉积物异化硝酸盐还原研究中的应用

沉积物中的异化硝酸盐还原过程对于海洋氮循环起着至关重要的作用。基于15N标记的培养技术是目前测定沉积物异化硝酸盐还原的主要手段。准确快速测定15N标记的产物(29N2、 30N2)是量化异化硝酸盐还原各个过程速率的关键。本研究自行组装膜进样质谱系统用于29N2和30N2的测定,对其测量条件进行了优化。结果表明,进样蠕动泵进样流速0.80 mL/min,进样时间3~3.5 min,恒温槽温度20~25℃,同时铜还原炉温度在300~600℃的条件下,^29N2/^28N2和^30N2/^28N2的测试精密度分别可以控制在0.1%和1%以内,比较适合29N2和30N2的测定。利用自组装的膜进样质谱系统结合15N标记的培养技术研究了青岛石老人沙滩沉积物中的异化硝酸盐还原过程。石老人沙滩沉积物不存在将硝酸盐完全还原为氮气好氧的反硝化。厌氧铵氧化、厌氧反硝化和异化硝酸盐还原为铵(Dissimilatory Nitrate Reduction to Ammonium,DNRA)的潜在速率(以湿沉积物N计)分别为(0.05±0.01) nmol/(cm^3·h),(2.32±0.21) nmol/(cm^3·h)和(1.02±0.15) nmol/(cm^3·h)。厌氧反硝化是硝酸盐异化还原主要的贡献者,其比例接近70%,其次是DNRA,比例可达30%,而厌氧铵氧化的贡献最低,仅为1%。在N2产生过程中,主要贡献者是反硝化,厌氧铵氧化的贡献仅为2%。     

Profiling Planctomycetales diversity with reference to anammox‐related bacteria in a South China Sea,deep‐sea sediment

A systematic assessment of Planctomycetales diversity in a South China Sea, deep‐sea sediment (1657 m) was conducted using the 16S rRNA gene analysis approach. PCR amplification of the samples from seven sediment layers (0.1, 1, 3, 5, 7, 9 and 11 m below the surface sediment) using the primer set Pla‐46‐F/1392‐R showed that the Planctomycetales existed within a limited range of sediment depths (≤ 5 m), and had a decreasing trend in diversity with increasing depth. The majority of the retrieved Pla‐46‐F/1392‐R sequences belonged to Pirellula‐related Planctomycetales, and two sequences retrieved from the 0.1‐m layer (GenBank accession numbers: DQ996944 and DQ996945 ) shared the same anammox‐related signature oligonucleotides and were closely related to commonly recognized anammox organisms. To identify new anammox‐related biomarkers, three primer sets were designed for amplifying the fragments of hydroxylamine oxidoreductase and S‐adenosylmethionine radical enzyme genes, but no related sequences were found. Our multiple 16S rRNA gene primer sets (Journal of Rapid Methods and Automation in Microbiology, 2008, in press) revealed even an higher diversity of Planctomycetales in the 0.1‐m layer of the sediment, especially at genus level. Our data profiled the distribution pattern of Planctomycetales diversity along sediment depths, and provided molecular evidence for the existence of anammox‐related bacteria in a new location, which broadens our understanding of Planctomycetales diversity in deep sea sediments.     

Microbial Biomass and Organic Nutrients in the Deep-Sea Sediments of the Central Indian Ocean Basin

In order to assess the impact of deep-sea mining on the in situ benthic life, we measured the microbial standing stock and concentration of organic nutrients in the deep-sea sediments of the Central Indian Ocean Basin in the Indian pioneer area. Sediments were collected using box core and grab samples during September 1996. The total bacterial numbers ranged from 10 10 -10 11 cells per g -1 dry weight sediment. There was a marginal decrease in the number of bacteria from surface to 30 cm depth, though the subsurface section registered a higher number than did the surface. The highest numbers were encountered at depths of 4-8 cm. The retrievable number of bacteria were two orders less in comparison with the direct total counts of bacteria. An almost homogeneous distribution of bacteria, total organic carbon, living biomass, and lipids throughout the depth of cores indicates active microbial and benthic processes in the deep sea sediments. On the other hand, a uniform distribution of total counts of bacteria, carbohydrates, and total organic carbon in all the cores indicates their stable nature and suggests that they can serve as useful parameters for long-term monitoring of the area after the benthic disturbance. Further studies on temporal variability in this region would not only verify the observed norms of distribution of these variables but would also help to understand restabilization processes after the simulated benthic disturbance.     

Assessing the Validity of Seismo-Acoustic Predictor Equations for Obtaining Seabed and Sub-Surface Sediment Physical Properties

The interdependence between the seismo-acoustic properties of a marine sediment and its geotechnical/physical parameters has been known for many years, and it has been postulated that this should allow the extraction of geotechnical information from seismic data. Though in the literature many correlations have been published for the surficial layer, there is a lack of information for greater sediment depths. In this article, a desktop study on a synthetic seafloor model illustrates how the application of published near-surface prediction equations to subsurface sediments (up to several tens of meters burial depth) can lead to spurious predictions. To test this further, acoustic and geotechnical properties were measured on a number of sediment core samples, some of which were subjected to loading in acoustically-equipped consolidation cells (oedometers) to simulate greater burial depth conditions. For low effective pressures (representing small burial depths extending to around 10 meters subsurface), the general applicability of established relationships was confirmed: the prediction of porosity, bulk density, and mean grain size from acoustic velocity and impedance appears generally possible for the investigated sedimentary environments. As effective pressure increases through, the observed relationships deviate more and more from the established ones for the near-surface area. For the samples tested in this study, in some instances increasing pressure even resulted in decreasing velocities. There are several possible explanations for this abnormal behavior, including the presence of gas, overconsolidation, or bimodal grain size distribution. The results indicate that an appropriate depth correction must be introduced into the published prediction equations in order to obtain reliable estimates of physical sediment properties for greater subsurface depths.