A culture-dependent survey of thermophilic bacteria from hot springs in Xiamen area in China

Microbes are believed to play important roles in ecosystem function in many environments. The hot springs of Xiamen Island are close to the Xiamen Sea, and may have some characteristics different from those of inland hot springs. Microbes living in the hot springs of Xiamen may have new characteristics. However, little is known about microbial communities of hot springs close to the Xiamen Sea. A culturedependent survey of microbial population in the Xiamen hot springs was performed by using an approach combining total cellular protein profile identification and 16S rRNA gene sequencing. A total of 328 isolates of bacteria were obtained from liquid and sediment samples from the Xiamen hot springs, including neutrophilic thermophilic bacteria and moderately thermophilic acidophiles. Neutrophilic thermophilic bacteria, which grow at a temperature range of 55—90℃ including Rhodothermus marinus (Strain 1), Thermus thermophilus (Strain 2), Thermus thiopara (Strain 3), Geobacillus stearothermophilus (Strain 4), Geobacillus thermoleovorans (Strain 5), and Pseudomonas pseudoalcaligenes (Strain 6), were recovered by 2216E plates. Moderately thermophilic acidophiles, which can grow at temperatures above 50℃ and a pH range of 1.8—3.5 such as Alicyclobacillus acidoterrestris (Strain 8), Sulfobacillus acidophilus (Strain 9), and Sulfobacillus thermosulfidooxidans (Strain 10), were isolated on selective solid medium containing sulfur and Fe2+. Among these strains, Rhodothermus marinus, Thermus thermophilus and Geobacillus stearothermophilus are not only thermophiles, but also halophiles. One bacterium strain (Strain 6) shared 99% nucleotide sequence homology with Pseudomonas pseudoalcaligenes on the 16S rRNA gene sequence, but was quite different from Pseudomonas pseudoalcaligenes in biological characteristics, suggesting that it may represent a novel thermophilic species. Results indicated that various species of neutrophilic thermophiles and moderately thermophilic acidophiles were widely distributed in the Xiamen hot springs and that Rhodothermus marinus and Thermus thermophilus dominated the cultivable microbial community.     

印度尼西亚卡利安达岛近岸热泉微生物和氢酶基因的多样性

研究对2011年7月采自印度尼西亚卡利安达岛近岸热泉的样品,进行了细菌和古菌16S rRNA基因和细菌氢酶基因克隆文库的构建、序列测定和多样性分析。结果表明:热泉系统的细菌和古菌由16个门组成,Proteobacteria为热液流体和沉积物的优势类群,丰度分别为59.5%和73.3%,Cyanobacteria为菌苔的优势类群(丰度,56.5%);具有氨氧化作用的Crenarchaeota为古菌的优势类群。热泉系统中,70%以上的种类与海洋细菌的亲缘关系最近,说明该系统具有海洋特性;约20%的种类与陆地热泉中嗜热或中等嗜热细菌的亲缘关系最近,这些种类可能来源于高温地层内部,并参与了铁氧化还原、氢氧化、硫氧化和硝酸还原等过程。热泉系统中,NAD(P)-关联的双向NiFe-氢酶基因和FeFe-氢酶基因的组成分布也受到温度和盐度分布变化的影响。研究为揭示浅海热液系统提供理论参考。     

印尼热泉中产嗜热碱性蛋白酶菌株筛选及酶学性质研究

采用MSM寡营养培养基从印度尼西亚Pantai cermin,Kalianda和Banyuwedang三个地区的热泉水样、泥样以及沉积物样品中分离获得细菌菌株,通过检测蛋白酶产生透明圈和福林酚蛋白酶活性测定相结合的方法,从中筛选出1株产嗜热蛋白酶的菌株PBI,该菌株经初步鉴定为短杆菌属(Brevibacillus),并对其酶学性质进行了研究。结果表明,菌株PBI产蛋白酶的最适酶活温度为60℃,最适pH值为8.0～9.0,具有较好的热稳定性和pH稳定性,60℃时酶的半衰期为30min,70℃条件下20min仍保持46.1%的酶活,该酶在pH值为5.0～9.0范围的缓冲液中酶活相对稳定。其产嗜热蛋白酶的酶活力最高可达到60.53U/mL,在100℃条件下仍能保留26.37%的相对酶活。Fe2+,Fe3+,Cu2+和Zn2+对嗜热蛋白酶活性具有明显的抑制作用。该嗜热蛋白酶对EDTA敏感,苯甲基磺酰氟(PMSF)、亮抑酶肽(Leupeptin)、苄眯(Benzamidine)和胃蛋白酶抑制剂(Pepstain A)对该嗜热蛋白酶都有一定的抑制作用,说明其酶活性受到丝氨酸、半胱氨酸残基的影响。结果表明,该菌株是1株具有进一步改造利用价值的产蛋白酶菌株。     

海洋微生物对甲胺磷农药的降解作用

作者从沿岸海域分离了38株有机磷农药的耐药菌,用分批培养法进行富集培养,得到有机磷农药的降解菌,并着重研究了其中两株菌对甲胺磷农药的降解情况,其结果表明,在10d内,降解菌株1对甲胺磷的降解率为59．1％,降解菌株2的降解率为53．3％,在各自的安磷培养液中,降解菌株1的数量大于降解菌株2;在甲胺降解过程中,其毒性逐渐减弱,降解菌株1所引起的毒性下降幅度大于降解菌株2。     

Complete genome sequence and proteomic analysis of a thermophilic bacteriophage BV1

Viruses of thermophiles are of great interest due to their roles in gene transfer, global geochemical cycle and evolution of life on earth. However, the thermophilic bacteriophages have not been studied extensively. In this investigation, a typical bacteriophage BV1 was obtained from a thermophilic bacterium Geobacillus sp. 6k512, which was isolated from an inshore hot spring in Xiamen of China. The BV1 contained a double-stranded linear DNA of 35 055 bp, which encodes 54 open reading frames (ORFs). Interestingly, eight of the 54 BV1 ORFs shared sequence similarities to genes from human disease-relevant bacteria. Seven proteins of the purified BV1 virions were identified by proteomic analysis. Determination of BV1 functional genomics would facilitate the better understanding of the mechanism for virus-thermophile interaction.     

季风环流影响下的南海海洋细菌多样性特征初探

季风驱动下的南海海洋动力过程有着季节性的多尺度变化特征, 显著影响南海海洋生态系统的演化进程。海洋细菌作为海洋生态系统中物质循环和能量流动的重要组成部分, 对环境变化和海洋多尺度动力过程有着积极的响应。将微生物生态过程与海洋动力过程相结合, 研究微生物的群落结构、动态变化及其与海洋环境过程的耦合, 是目前国际海洋科学多学科交叉研究的热点之一。文章以国家自然科学基金重点项目“季风环流影响下的南海海洋细菌多样性特征及其生物海洋学意义”的主要研究成果为基础, 从南海北部上升流、海洋锋面、中尺度涡旋、次中尺度过程及其对海洋细菌多样性和生态系统可能的影响等几个方面, 探讨当前的研究进展和初步研究成果。     

来自大黄鱼(Pseudosciaena crocea)肠道的弧菌拮抗菌的筛选与鉴定

采用点种法并以副溶血弧菌(Vp1.2164)和溶藻弧菌(Va-Y)为弧菌指示菌,对从健康的大黄鱼肠道内分离的115株细菌进行体外拮抗试验。并对筛选出的拮抗菌进行抗菌谱测定,最后利用VITEK-32细菌快速鉴定系统和16SrRNA基因序列对拮抗菌进行鉴定。结果表明,从中筛选到3株有较强拮抗作用的菌株,编号分别为X1402-1、X1108和X3914,且菌株X1402-1的抗菌作用最强,抗菌谱最广,21株病原指示菌中,对16株均能产生较强的抑制作用,其中对Vp-1.2164的抑菌直径最大,达到28.5mm,对Va-Y的抑菌直径为8.7mm;其次为菌株X1108和X3914,对两种指示弧菌的抑菌直径分别为:12.0mm、9.0mm和0mm、7.2mm。经VITEK-32细菌快速鉴定系统鉴定,3株拮抗菌分别与铜绿假单胞菌,恶臭假单胞菌,地衣芽孢杆菌的相似性均为99%。通过16SrRNA基因序列分析表明,3株拮抗菌分别与类产碱假单胞菌Pseudomonas pseudoalcaligenes(EU419918)、恶臭假单胞菌Pseudomon asputida(FJ440105)和地衣芽孢杆菌Bacillus licheniformis(DQ988136)的16SrRNA基因同源性最高,分别为100.0%、97.5%、99.0%。综合分析试验结果,X1402-1、X1108和X3914分别被确定为类产碱假单胞菌、恶臭假单胞菌和地衣芽孢杆菌。     

南极罗斯海区域可培养微生物分离鉴定及产低温酶能力初步筛选

为了利用南极微生物资源、探索南极罗斯海区域可培养微生物多样性,利用传统平板培养法对中国第33次南极科学考察采自南极罗斯海6个站位的海洋沉积物样品进行了细菌、真菌的分离培养。经细菌16S rRNA、真菌ITS基因序列检测及系统发育分析,共获得5个属的36株细菌和6个属的29株真菌。其中嗜冷杆菌属为优势细菌类群,枝孢属为优势真菌类群。该结果表明南极罗斯海区域具有丰富的微生物多样性。细菌API 20 NE生理生化及真菌产胞外酶活性实验显示,分离得到的细菌和真菌绝大多数都有低温酶活性。研究结果为南极罗斯海区域可培养微生物的多样性认识和低温酶产生菌资源获取与开发利用提供了支撑。     

南沙深海沉积物中石油降解菌的分离鉴定和多样性分析

为了解我国南沙海域中石油降解菌的多样性,认识其在环境自净中作用并获得用于石油污染生物修复的菌种资源,以不同链长烷烃及支链烷烃对南沙深海沉积物样品进行富集与烃降解菌的分离鉴定,并结合16S rRNA基因文库和DGGE技术对降解菌群的结构进行了分析.结果表明：通过16S rRNA基因文库构建及PCR-DGGE分析发现,不同链长烷烃富集后的菌群中,食烷菌（Alcanivorax）在各降解菌群中都是绝对优势菌;另外,除烃海杆菌（Marinobacter hydrocarbonoclasticus）、海绵假单胞菌（Pseudomonas pachastrellae）,以及Idiomarina与Kangiella属的细菌也是菌群中较重要的优势菌.此外,通过2种平板培养基从烷烃富集的降解菌群中分离获得了8个不同属的细菌.本研究还首次发现Kangiella属（食烷菌科）的细菌可能参与了烷烃降解.南沙沉积物烷烃降解菌多为γ-Proteobacteria;其中,食烷菌科（Alcanivoraceae）（Alcanivorax与Kangiella属）的细菌在降解菌群中是绝对优势菌,假单胞菌与海杆菌在烷烃降解过程中也起着重要作用.     

海洋石油降解微生物的分离鉴定

以柴油为惟一碳源,从胜利油田黄河码头和厦门储油码头两个海水样品中富集得到两组柴油降解菌,共9株细菌,1株真菌,它们对柴油都有降解能力.16SrDNA鉴定结果表明这9株细菌中有3株摩加夫芽孢杆菌、1株施氏假单胞菌、1株腊样芽孢杆菌、1株反硝化产碱杆菌.1株阿氏葡萄球菌、1株食烷菌和1株类似很小海旋菌的未知新菌(M-5),其中B-5对柴油的降解能力最强,已报道的食烷菌属中其他种的同源性最高,为95.2%,表明它是该属中的一个新种.实验中获得的惟一一株真菌M-3属于假丝酵母,对柴油有较强的降解和乳化能力.实验中还从B5和施氏假单胞菌M-2中扩增到了烷烃降解的限速酶烷烃单加氧酶的基因片断,其中B5编码了一种新的烷烃单加氧酶.这些菌在石油污染的海水自净中起着重要作用,可用于海洋石油污染的生物修复.     

渤海沉积物中细菌群落结构和基因丰度的空间分布特征

This study investigated differences in the community structure and environmental responses of the bacterial community in sediments of the Bohai Sea.Illumina high-throughput sequencing technology and real-time PCR were used to assay the bacterial 16S rRNA genes in the surface sediments of 13 sampling stations in the Bohai Sea.The results showed that sediments at the majority of the 13 sampling stations were contaminated by heavy metal mercury.The main phyla of bacteria recorded included Proteobacteria(52.92%),Bacteroidetes(11.76%),Planctomycetes(7.39%),Acidobacteria(6.53%)and Chloroflexi(4.97%).The genus with the highest relative abundance was Desulfobulbus(4.99%),which was the dominant genus at most sampling stations,followed by Lutimonas and Halioglobus.The main factors influencing bacterial community structure were total organic carbon,followed by depth and total phosphorus.The content of lead,cadmium,chromium,copper and zinc had a consistent effect on community structure.Arsenic showed a negative correlation with bacterial community structure in most samples,while the impact of mercury on community structure was not significant.The bacterial community in sediment samples from the Bohai Sea was rich in diversity and displayed an increase in diversity from high to low latitudes.The data indicated that the Bohai Sea had abundant microbial resources and was rich in bacteria with the potential to metabolize many types of pollutants.     

南海沉积物细菌胞外蛋白酶在家族水平上的多样性

海洋产蛋白酶细菌及其分泌的胞外蛋白酶在降解有机氮以推动海洋氮循环进行方面发挥着重要作用,但目前对这二者多样性的认识非常有限。本研究自南海沉积物中筛选得到90株产蛋白酶细菌,并通过N-端氨基酸序列,分析了其胞外蛋白酶在家族水平上的多样性。16S rRNA基因序列分析的结果表明,筛选的产蛋白酶细菌均属于Gammaproteobacteria纲,且大多数属于Alteromonadales目和Vibrionales目的不同属。对其中14株菌株的14个胞外蛋白酶的N-端氨基酸序列分析表明,所有这些蛋白酶属于金属蛋白酶的M4家族或丝氨酸蛋白酶的S8家族。本研究提供了海洋沉积物产蛋白酶细菌在类群及其胞外蛋白酶在类型上的新细节,这将有助于全面了解微生物酶促降解海洋沉积物有机氮的过程和机理。     

基于低压变频电场技术对带鱼(Trichiurus lepturus)保鲜过程中微生物群落影响分析

带鱼(Trichiurus lepturus)在低温条件运输和贮藏过程中,易受微生物和内源性蛋白酶的影响而产生胺类和醛类物质导致腐败。为了研究低压变频电场对微冻带鱼在贮藏过程中的微生物群落特征,采用Miseq基因组测序技术,对贮藏至40d的舟山带鱼的微生物群落进行碱基信息测序。将产生的碱基序列通过OTU聚类、多样性分析、相关性分析等手段分析了影响带鱼保鲜效果的微生物群落组成和发育信息。在此基础上,研究了不同强度电场(0,2,2.5,3 kV/m)处理对带鱼微生物群落组成的影响。根据菌属相对丰度结果,推测希瓦氏菌属(Shewanella)、假单胞菌属(Pseudomonas)、紫色杆菌属(Janthinobacterium)是带鱼微生物腐败主要致腐菌。结果表明,40 d后,经3 kV/m处理的样品其OTU6嗜冷菌属和OTU13假单胞菌属c的相对丰度均小于1%,而且OTU7、OTU14、OTU8、OTU9等菌属未被OTU聚类。结果表明,经3kV/m电场强度的低压静电场处理的抑菌保鲜效果最好,表现为在贮藏过程中杂菌种类最少,样本微生物群落物种丰度和多样性均最低,腐败程度最低。     

一株群体感应抑制活性海洋放线菌的筛选与鉴定 *

群体感应抑制剂(Quorum Sensing Inhibitors, QSIs)可以通过干扰群体感应(Quorum Sensing, QS)而有效降低病菌的感染性和毒力, 并且不会胁迫致病菌使其产生耐药性, 是具有前景的抗生素替代品。海洋微生物是新型QSIs的潜在来源。本研究利用紫色杆菌(Chromobacterium violaceum 026, CV026)模型评价QS抑制活性, 在来源于南海中西部和印度洋深海沉积物样品的放线菌中筛选QS抑制活性菌株, 并且根据形态学特征和16S rRNA基因序列鉴定活性菌株。通过筛选获得了菌株SCSIO 53717, 其提取物在CV026模型中能够显著降低指示菌株的紫色菌素产量, 并且在0~1000mg·mL^-1的有效浓度范围内不影响紫色杆菌的生长。菌株SCSIO 53717被鉴定为Kocuria属, 是首次报道的具有QS抑制活性的Kocuria属菌株, 因而具有进一步深入研究的价值。     

厦门海区潮间带海洋动植物共附生微生物的抗菌活性

从厦门海区潮间带石纯、浒苔、江蓠、海兔、海葵及鲨鱼肠道分离得２８９株共附生微生物,以８种敏感菌为指示菌,从中获得拮抗活性的幼苗２８株,放线菌１７株,真菌８株。拮抗细菌以假单胞菌（３９％）、弧菌（２５％）和芽直菌（１４％）为主,主要为抗革兰氏阳性细菌,抗真菌的菌株比例较低。拮抗放线菌分属于链霉菌和小单孢菌,其抗革兰氏阳性菌、阴性菌和真菌的菌株比例分别为６５％、４７％和２３％。拮抗真菌分青霉属和芽枝霉     

白令海和楚科奇海表层沉积物中多环芳烃降解微生物多样性

为了研究白令海(海盆及陆架)至楚科奇海陆架表层沉积物中多环芳烃(PAHs)降解菌的多样性,并获得新的PAHs降解菌资源。在GC-MS分析沉积物中PAHs种类和含量的基础上,以萘、菲和芘的混合物为唯一碳源和能源对表层沉积物样品进行富集,通过平板分离鉴定可培养菌株,并验证其降解能力;同时利用变性梯度凝胶电泳(DGGE)和Illumina高通量测序技术分析降解菌群结构。GC-MS测定结果表明,14个表层沉积物中PAHs总干质量介于32.99~276.97ng/g。富集菌群中共分离获得51株可培养细菌,平板纯培养、PCR-DGGE及Illumina测序结果均表明,菌群中优势的降解菌是γ-proteobacteria的Marinobacter,Pseudoalteromonas,Pseudomonas和Actinobacteria门的Dietzia菌。此外,Illumina测序结果还表明14个降解菌群在菌群结构组成上,可分为海盆区和陆架区两种类群;同时检测到一些低丰度的海洋专属PAHs降解菌,如Cycloclasticus,Alteromonas和Neptunomonas等。本文结果将加深对白令海及楚科奇海表层沉积物中PAHs降解菌资源与生物多样性的认识。     

Molecular analyses of bacterioplankton communities with highly abundant Vibrio clades: a case study in Bohai Sea coastal waters

We investigated the bacterioplankton abundance, community composition and the associated Vibrio clades of natural seawater in Bohai Sea coastal waters. Seawater samples (10 L in triplicate) were collected at 0.5, 3, and 5 m depths near the coastal aquaculture zone of the Bohai Sea on May 12, 2016. Real-time PCR and 16S rRNA gene amplicon high-throughput sequencing methods were employed by which 485 operational taxonomic units (OTUs) at a 97% sequence similarity level were generated. Gammaproteobacteria, Alphaproteobacteria, and Bacteroidetes were the most abundant groups, accounting for 49.5%, 23.5%, and 18.9% of the total assemblage, respectively. Obvious variations in Pseudoalteromonas, Vibrio, and Octadecabacter , which were the most abundant genera, could be observed among diff erent samples. Notably, the results of Vibrio -specifi c real-time PCR indicated that Vibrio had extremely high 16S rRNA gene copy numbers. The 16S rRNA gene sequencing results across all the samples also indicated that they occupied a large proportion of the total assemblage. Both the alpha diversity and major bacterioplankton group Pseudoalteromonas had significant correlations with the concentration of PO4^3-. Overall, studies on bacterioplankton communities with highly abundant Vibrio clades can provide interesting insight into the microbial function and health assessment of the Bohai Sea coastal ecosystem.     

Isolation and diversity analysis of heterotrophic bacteria associated with sea anemones

A study was undertaken to investigate the heterotrophic bacterial flora associated with the sea anemones. Samples of the sea anemones Anthopleura midori were collected from the coast of Weihai and bacteria were isolated from these samples. Additionally, high numbers of viable bacteria were obtained from the celom wall and surface of anemone, the community of cultivable bacteria was very diverse. As a result of this isolation, 60 strains were obtained, 56 of them were selected for identification and characterization by 16S rRNA gene sequence analysis and limited phenotypic testing. Among these isolates, 16 strains were phylogenetically related to members of the genus Pseudoalteromonas and neighboring taxa. Other isolates included members of the genera Colwellia, Vibrio, Acinetobacter, Pseudomonas, Endozoicomonas, Roseovarius, Paracoccus, Loktanella, Leisingera, Sulfitobacter, Bacillus, Staphylococcus, Plantibacter, Microbacterium, Micrococcus, Joostella, Psychroserpens, Cellulophaga, Krokinobacter, Polaribacter and Psychrobacter. Seven potential novel species were found. Among 60 strains, 17 of them can produce proteolytic exoenzyme, 20 can produce lipolytic exoenzyme. Strain NQ8 has strong antagonistic effects on some Vibrio strains. This study demonstrates that the culturable fraction of bacteria from the sea anemones Anthopleura midori is diverse and appears to possess much potential as a source for the discovery of novel bioactive materials.     

三份南海岛礁珊瑚砂样品中可培养细菌多样性

岛礁珊瑚砂环境中存在着大量未被培养和利用的微生物资源, 微生物多样性研究是了解微生物生态功能、开发和利用微生物资源的基础。文章采用多种寡营养培养基选择性分离南海岛礁珊瑚砂中的可培养细菌, 共获得纯培养细菌菌株349株, 通过16S rRNA基因序列分析, 发现它们隶属于4门(Actinobacteria、Proteobacteria、Firmicutes和Bacteroidetes)、6纲、26目、43科、73属、134种, 可培养细菌的优势类群为放线菌门, 占所有分离菌株数量的60%; 而且还发现18个16S rRNA 基因序列相似性低于97%的潜在新种。本研究使用改良优化的寡营养培养基进行分离, 较好地显示出样品中微生物的群落组成, 且获得了大量潜在的稀有新物种资源。研究结果表明, 岛礁珊瑚砂样品的可培养细菌资源十分丰富、细菌群落所涉及的生态功能完整、潜在新种比例较高, 为后期岛礁微生物资源挖掘打下了良好的基础, 也为后期开发应用积累了丰富且稀有的菌种资源。     

胶州湾沉积物中石油降解菌的降解能力及多样性研究

2013年青岛输油管道爆炸,大量石油污染了附近海岸。课题组采集了污染的沉积物样品,以原油为唯一碳源和能源,富集了四个石油降解菌群。生物多样性和群落分析表明,Luteibacter、Parvibaculum 和属于食烷菌科的一个属是降解菌群的主要优势菌,都属于变形菌门。从石油降解菌群中分离筛选,获得了9株具有不同16S rRNA基因序列的降解菌,分别属于8个属。重量法测定降解菌的石油降解率,其中5株的石油降解率大于30%。GC-MS分析结果表明,石油降解菌多倾向于降解烷烃,对多环芳烃的降解能力较差,其中5株细菌的烷烃降解率较大,仅1株菌D2对多环芳烃的降解率较大,其降解率在34.9%到77.5%。通过对高通量数据的分析,表明食烷菌属是菌群A和菌群E的主要降解菌群,其中筛选获得的菌株E4可能是菌群E的一株优势降解菌。本研究所筛选菌株证明了其石油降解潜力,为油污染海滩生物修复提供了菌株资源。