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。     

生物标志物梯烷脂指示胶州湾厌氧氨氧化活动

厌氧氨氧化是重要的氮脱除途径,对海洋中的氮循环,尤其是人类活动影响下海湾的氮循环起重要作用。梯烷脂作为厌氧氨氧化的生物标志物,能指示不同生态系统中的厌氧氨氧化活动,但其在海湾环境中的来源以及对人类活动影响的响应尚未明确。本文对人类活动影响下的典型海湾——山东半岛的胶州湾海域进行研究,通过分析胶州湾表层沉积物中梯烷脂及其他有机质指标,来研究海湾中有机质及厌氧氨氧化菌的来源及梯烷脂对环境因子的响应。研究显示厌氧氨氧化活动在胶州湾海域普遍存在。对总有机质参数(TOC,δ~(13)C和TOC/TN)与有机质来源贡献指标(TMBR和BIT)的分析显示胶州湾海域表层沉积物有机质以海源为主(BIT指示为83%、TMBR指示为54%),但各有机质来源指标与海湾中梯烷脂含量无明显相关。对梯烷脂总量及不同种类梯烷脂占比的分析显示,梯烷脂主要来自夏季水体中的颗粒物,且在梯烷脂总量较高的区域,梯烷脂可能主要受河流输入及污水排放的影响。主成分分析显示海湾沉积物中梯烷脂与营养盐及溶解氧含量受相同因子所调控,表明梯烷脂可以在胶州湾指示人类活动对海水环境质量的影响。这也为沉积物中梯烷脂在海湾环境,乃至其他边缘海环境中对人类活动的指示作用提供了新思路。     

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16SrRNA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(P<0.01);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(P>0.05)。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。     

厌氧氨氧化细菌物种组成对环境因子响应的研究进展

分析海洋水体及沉积物中厌氧氨氧化细菌所处的环境条件,比较海洋与淡水环境中其物种组成的差异,总结了其物种组成、丰度及厌氧氨氧化活性对环境因子的响应规律。认为厌氧氨氧化细菌物种组成受多种环境因子的影响。其中,盐度为影响其物种组成的最显著因素,如Candidatus Brocadia,Candidatus Kuenenia及Candidatus Scalindua分别适应淡水、寡盐和海水环境;同一地区,其物种组成在不同季节表现出明显差异,夏季的多样性高于冬季的;根据陆源厌氧氨氧化细菌多样性及海洋沉积物中浮霉菌门多样性随深度增加而减小的趋势,推断海洋沉积物中厌氧氨氧化细菌多样性与深度成反比。厌氧氨氧化细菌丰度、厌氧氨氧化活性与温度、NO-2及NO-3浓度之间成正相关;丰度与季节的关系,在不同地区的研究结果存在差异。未来厌氧氨氧化细菌研究工作重点应在其对氧浓度的耐受范围、海洋中厌氧氨氧化细菌多样性随深度的变化规律、导致其物种组成、丰度及厌氧氨氧化活性季节性差异的具体环境因子。     

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。     

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...     

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...     

兼性厌氧海洋细菌Shewanella sp.N3B_R的分离鉴定及产电性能分析研究

本研究的目的是利用聚苯胺修饰双室微生物燃料电池(MFC),从海水养殖废水中分离筛选阳极海洋产电菌,并利用响应面Box-Behnken设计优化各产电因子,提高产电性能.从海水养殖池塘的废水和污泥中分离出一株兼性厌氧海洋菌株,利用16SrRNA基因序列和API20NE生化分析进行鉴定,采用扫描电镜进行菌株形态的观察.采用循...     

柄海鞘共附生细菌的分离培养与系统发育多样性研究

通过纯培养和16SrRNA基因序列的相似性比对,并构建系统发育树对威海海域柄海鞘共附生细菌的多样性进行了研究。用2216E培养基从柄海鞘样品中分离到78株细菌,通过形态学特征和TCBS培养基选择性筛选后,选取30株具有代表性的菌株进行了16SrRNA基因测序和基于16SrRNA基因序列的系统发育多样性分析。结果表明,30株菌株分别属于细菌域的4个系统发育类群(Actinobacteria,Bacteroidetes,Firmicutes,Gro-teobacteria)的11个科,11个属。根据16SrRNA基因序列,大多数菌株与其系统发育关系最密切的模式菌株之间存在一定的遗传差异(16SrRNA基因序列相似性为91.82%～98.93%)。其中菌株HQW7,HQYD1,HQWD4,HQE1和HQE2与相关模式菌株的16SrRNA基因最高相似度仅分别为91.82%,92.51%,92.99%,93.52%,93.87%,这5株菌可能代表新的分类单元。威海海域柄海鞘共附生细菌存在着较为丰富的物种多样性和系统发育多样性,并可能存在新的物种。     

三亚天涯海角潮间带趋磁细菌特性

在三亚天涯海角潮间带沉积物中发现了一定量的海洋趋磁细菌,最大丰度可达100个/cm3。透射电子显微镜(TEM)观察发现该区域趋磁细菌绝大多数为趋磁球菌。磁小体形状单一,皆为棱柱形;磁小体排列方式多样,以链状排列为主,包括单链、双链,也有少数成簇排列。高分辨率透射电子显微镜(HRTEM)与X-射线能谱仪(EDXS)结果表明,磁小体成分为Fe3O4。趋磁细菌的铁含量平均为2.05%(0.70%~7.53%)。通过16S r RNA基因文库的构建与测序,在78个阳性克隆中得到了16个趋磁细菌序列,分属9个OTU。系统发育分析结果表明,其中8个OTU属于α-变形菌纲,1个OTU属于γ-变形菌纲。在全部9个OTU中,有8个OTU与已知趋磁细菌序列的相似性低于97%,有5个OTU与已知趋磁细菌序列的相似性低于93%,说明该区域存在可观的趋磁细菌资源。     

Nitrogen uptake,ammonium oxidation and nitrous oxide (N2O) levels in the coastal waters of western Cook Strait,New Zealand

Vertical measurements of $\text{NH}{}_4{}^{\mathrm{+}}$, $\text{NO}{}_3{}^{\mathrm{?}}$ and N₂O concentrations, $\text{NO}{}_3{}^{\mathrm{?}}$ and $\text{NH}{}_4{}^{\mathrm{+}}$ uptake, and $\text{NH}{}_4{}^{\mathrm{+}}$ oxidation rates were measured at 5 sites in western Cook Strait, New Zealand, between 31 March and 3 April 1983. Nitrate increased with depth at all stations reaching a maximum of 10.5 μg-atom $\text{NO}{}_3{}^{\mathrm{?}}\text{N l}{}^{\mathrm{?1}}$ at the most strongly stratified station whereas $\text{NH}{}_4{}^{\mathrm{+}}$ was relatively constant with depth at all stations (～0.1 μg-atom $\text{NH}{}_4{}^{\mathrm{+}}\text{N l}{}^{\mathrm{?1}}$). The highest rates of $\text{NH}{}_4{}^{\mathrm{+}}$ oxidation generally occurred in the near surface waters and decreased with depth. N₂O levels were near saturation with respect to the air above the sea surface and showed no obvious changes during 24 h incubation. $\text{NH}{}_4{}^{\mathrm{+}}$ oxidation by nitrifying bacteria may account for about 30% of the total $\text{NH}{}_4{}^{\mathrm{+}}$ utilization (i.e. bacterial+agal) and, assuming oxidation through to $\text{NO}{}_3{}^{\mathrm{?}}$, may supply about 40% of the algal requirements of $\text{NO}{}_3{}^{\mathrm{?}}$ in the study area. These results suggest that bacterial nitrification is of potential importance to the nitrogen dynamics of the western Cook Strait, particularly with respect to the nitrogen demands of the phytoplankton.     

青岛潮间带沉积物可培养厌氧细菌多样性的研究

为了验证设计的简易厌氧培养方法,作者以青岛潮间带沉积物为研究对象,采用5种培养基,从青岛潮间带沉积物共分离获得138株厌氧细菌。16S r DNA序列分析表明,这些细菌分属3个门15个属32个种,其中γ-变形菌纲64株18个种,在种类上处于优势地位;此外还包括δ-变形菌纲(δ-Proteobacteria)16株2个种,ε-变形菌纲4株1个种,拟杆菌门(Bacteroidetes)29株8个种,梭杆菌门(Fusobacteria)25株3个种。在属水平上,弧菌属(Vibrio)、Marinifilum属、泥杆菌属(Ilyobacter)、脱硫弧菌属(Desulfovibrio)、希瓦氏菌属(Shewanella)在数量上占优势。此外,有26株8种细菌(占总菌株数的18.84%)与已知菌的同源性介于89.38%~94.22%,为潜在的海洋细菌新科或新属,表明此简易厌氧菌培养方法在获得新菌方面具有较大优势。另外,研究结果还表明,不同培养基对特定的类群具有选择性:2216E培养基对γ-变形菌纲分离培养效率较高;SPG培养基在获得新菌方面具有优势(占新菌数62.5%),且这些新菌大多属于拟杆菌门和梭杆菌门,其中SPG-1培养基对于分离硫酸盐还原菌和难培养的ε-变形菌纲细菌具有优势,SPG-4培养基对分离硝酸盐还原菌具有优势。     

Growth of marine bacteria and ammonium regeneration from substrates in different C: N ratios

Natural assemblages of marine bacteria were chosen in a batch culture experiments. The impact of varying nitrogen substrate concentrations and the substrate C:N ratios (C:N_S) on the bacterial C:N ratio (C:N_B), the bacterial growth efficiency (BGE) and ammonium regeneration was mainly examined. The C:N_S ratios varied from 5:1 (carbon limitation) to 40:1 (nitrogen limitation) with varying combinations of glucose and NO₃^-. The C:N_B ratio had positive relationship with the C:N_S ratio (r=0.93, n=8), whose value was 3.77 when the C:N_S ratio was 5:1 but increased to 6.47 when the C:NS ratio was 40:1. These results indicate that the C:N_B ratio is a potential diagnostic tool for determining the bacterial growth in natural waters controlled by either, carbon or nitrogen. BGE decreased with the declining nitrate concentration and negatively related to C:N_s (r=-0.51, n=8). The average value of BGE was 0.20. This value was a little lower than other reports, which could be induced by the nitrogen source used in our experiments. Finally, regeneration time of ammonium delayed with the increasing C:N_S ratio, which indicates that there were different metabolism mechanisms when bacterial growth was limited by carbon source and nitrogen source.     

厦门近岸水域海洋附着细菌的群落结构及其动态变化

为研究海洋附着细菌的群落结构及动态变化,在厦门近岸海区进行挂板实验.将无菌玻璃板浸没于海水中,连续放置14 d.分别于放置1 h和7、14 d后取玻璃板上附着生物样品.用细菌通用引物构建16S rRNA基因克隆文库,每个克隆文库随机挑选约40个克隆子测序,序列同源性分析和系统进化分析结果表明,所有的克隆子可分为六大类群:γ-变形菌纲(γ-Proteobacteria)、拟杆菌门(Bacteroidetes)、厚壁菌门(Firmicutes)、α-变形菌纲(α-Proteobacteria)、蓝细菌门(Cyanobacteria)和真核硅藻类叶绿体,各类群分别占42.0%、4.5%、2.2%、2.2%、1.1%和45.0%.γ-变形菌纲变形斑沙雷氏菌(Serratia proteamaculans)为优势附着细菌,占测序克隆子的31.5%.这类细菌在1 h样品中的比例超过一半,说明变形斑沙雷氏菌在生物膜形成初期发挥着重要作用.随着挂板时间延长,检测到的细菌类群有所增加:附着7 d后检测到拟杆菌门细菌,附着14 d后检测到厚壁菌门细菌.γ-变形菌纲细菌所占比例随挂板时间的延长而逐渐降低,从挂板1 h的81%降至7 d的21%,14 d的18%.另外,在各阶段的附着样品中,都检测到较多的真核克隆子序列,约占16%～64%.本研究为进一步阐明海洋附着细菌的附着动态及其在生物膜形成过程中的作用奠定了基础.     

Analysis of cultivable aerobic bacterial community composition and screening for facultative sulfate-reducing bacteria in marine corrosive steel

Anaerobic, aerobic, and facultative bacteria are all present in corrosive environments. However, as previous studies to address corrosion in the marine environment have largely focused on anaerobic bacteria, limited attention has been paid to the composition and function of aerobic and facultative bacteria in this process. For analysis in this study, ten samples were collected from rust layers on steel plates that had been immersed in seawater for diff erent periods (i.e., six months and eight years) at Sanya and Xiamen, China. The cultivable aerobic bacterial community structure as well as the number of sulfate-reducing bacteria (SRB) were analyzed in both cases, while the proportion of facultative SRB among the isolated aerobic bacteria in each sample was also evaluated using a novel approach. Bacterial abundance results show that the proportions are related to sea location and immersion time;abundances of culturable aerobic bacteria (CAB) and SRB from Sanya were greater in most corrosion samples than those from Xiamen, and abundances of both bacterial groups were greater in samples immersed for six months than for eight years. A total of 213 isolates were obtained from all samples in terms of CAB community composition, and a phylogenetic analysis revealed that the taxa comprised four phyla and 31 genera. Bacterial species composition is related to marine location;the results show that Firmicutes and Proteobacteria were the dominant phyla, accounting for 98.13% of the total, while Bacillus and Vibrio were the dominant genera, accounting for 53.06% of the total. An additional sixfacultative SRB strains were also screened from the isolates obtained and were found to encompass the genus Vibrio (four strains), Staphylococcus (one strain), and Photobacterium (one strain). It is noteworthy that mentions of Photobacterium species have so far been absent from the literature, both in terms of its membership of the SRB group and its relationship to corrosion.     

中性厌氧环境中硫酸盐还原菌导致锌的腐蚀行为研究

以锌为研究对象,使用表面表征方法和电化学测试,研究了一个培养周期内硫酸盐还原菌(Sulfatereducing bacteria, SRB)Desulfovibrio vulgaris (D. vulgaris)引起锌(Zn)试样腐蚀行为的影响。实验结果表明,SRB导致Zn试样发生了严重的均匀腐蚀和点蚀。浸泡7天后,SRB体系中的Zn试样平均失重为32.2 mg/cm²,是无菌培养基中试样平均失重的42倍,腐蚀产物主要为ZnS。生物膜和腐蚀产物膜的累积在培养初期可以减缓金属基体与溶液界面的电子传输过程,导致腐蚀速率减缓。培养中后期,由于生物膜和腐蚀产物膜的阻隔作用,导致混合膜层底部有机碳源缺乏,SRB转向Zn获取自身所需电子,表现为腐蚀速率上升。     

Phylogenetic analysis and biological characteristic tests of marine bacteria isolated from Southern Ocean (Indian sector) water

Fifty-seven bacteria were isolated from Southern Ocean(Indian sector) water samples which were collected from different latitude and longitude of the ocean. All the isolates were able to grow at 4°C, 20°C, 37°C and tolerable Na Cl concentration up to 13.5%(w/v). 29 out of 57 isolates were identified using 16 S rDNA amplification and the sequences were submitted to National Center for Biotechnology Information(NCBI). All the isolates were classified by using Ribosomal Database Project(RDP) and found that isolates belongs to Proteobacteria and Bacteriodes. The average G+C content was 56.4%. The isolates were screened for the presence of extracellular enzymes, viz. amylase, catalase, urease, esterase, lipase and protease. The disc diffusion method is used to screen antibiotic production by the isolates against four pathogenic bacteria, viz. Salmonella typhimurium(NCIM 2501),Staphylococcus aureus(NCIM 2122), Bacillus subtilis(NCIM 2193), and Pseudomonas aeruginosa(NCIM 2036).Nine out of 29 were found to be antibiotic producer.     

海洋沉积物中金属依赖型甲烷厌氧氧化作用研究进展及展望

海洋沉积物中大部分甲烷会通过甲烷厌氧氧化作用（anaerobic oxidation of methane, AOM）而被消耗。早期研究表明,AOM可与硫酸盐、硝酸盐和亚硝酸盐的还原作用相耦合,从而有效减少甲烷向大气的排放。最近,金属依赖型AOM（metal-AOM,活性金属氧化物还原反应驱动的AOM）被证实存在于自然界沉积物和富集培养的样品中。但是,目前仍未从自然海洋环境中分离获得能够介导metal-AOM的微生物。对海洋沉积物中metal-AOM的研究大多聚焦于热液或冷泉等海洋特殊生境,一系列研究表明地质流体在这些海底化能自养生态系统的维持和演化方面起到了重要作用,并深刻影响全球地球化学循环,因此,该科学问题研究吸引了越来越多的注意力。本文讨论了可能参与海洋沉积物中metal-AOM的微生物类群及其地球化学证据,并在前人工作基础上,以冲绳海槽冷泉-热液共生区为例,提出一种新的metal-AOM作用机制。认为在全球冷泉-热液系统相互作用地区的调查有助于更好地探讨metal-AOM的发生机制及微生物在深海生境中分布的连通性问题。     

Phylogenetic diversity of planktonic bacteria in the Chukchi Borderland region in summer

Planktonic bacteria are abundant in the Chukchi Borderland region. However, little is known about their diversity and the roles of various bacteria in the ocean. Seawater samples were collected from two stations K2S and K4S where sea ice was melting obviously. The analysis of water samples with fluorescence in situ hybridization (FISH) showed that DMSP-degrading bacteria accounted for 13% of the total bacteria at the station K2S. No aerobic anoxygenic phototrophic (AAP) bacteria were detected in both samples. The bacterial communities were characterized by two 16S rRNA gene clone libraries. Sequences fell into four major lineages of the domain Bacteria, including Proteobacteria (Alpha, Beta and Gamma subclasses), Bacteroidetes, Actinobacteria and Firmicutes. No significant difference was found between the two clone libraries. SAR11 and Rhodobacteraceae clades of Alphaproteobacteria and Pseudoalteromonas of Gammapro-teobacteria constituted three dominant fractions in the clone libraries. A total of 191 heterotrophic bacterial strains were isolated and 76% showed extracellular proteolytic activity. Phylogenetic analysis reveals that the isolates fell into Gammaproteobacteria, Bacteroidetes, Actinobacteria and Firmicutes. The most common genus in both the bacterial isolates and protease-producing bacteria was Pseudoalteromonas. UniFrac data showed suggestive differences in bacterial communities between the Chukchi Borderland and the northern Bering Sea.