引用本文 ↓

陈柔雯,王可欣,何媛秋,等.一份南海深海沉积物样品中可培养细菌的多样性[J].生物资源, 2018, 40(4): 321-333.

Chen R W, Wang K X, He Y Q, et al. Diversity of cultured bacteria isolated from a deep see sediment in South China Sea [J]. Biotic Resources, 2018, 40(4): 321-333.

    摘要

    海洋沉积环境蕴含丰富的微生物资源。对深海难培养微生物的分离培养,不仅有利于深海微生物资源的挖掘与利用,也有利于对深海微生物学的研究。本研究采用多种培养基分离获得细菌菌株纯培养,并通过16S rRNA基因序列鉴定,对我国南海海域1个4 000 m水深的深海表层沉积物样品的可培养细菌多样性进行初探。共设计23种分离培养基,经过选择性分离培养最终获得612株细菌菌株,分别隶属于厚壁菌门(Firmicutes)、放线菌门(Actinobacteria)和拟杆菌门(Bacteroidetes)的9目10科27个属级类群,可培养优势类群为厚壁菌门,占所有分离物种数量的85.8%,包含13个16S rRNA基因序列相似性低于98%的潜在新物种。海洋琼脂类培养基适合培养不同种类的海洋细菌类群,放线菌选择性分离类合成培养基对放线菌类群的分离效果较好。最终获得一些与具有产抗生素、细胞毒素、高效酶活、耐受不良环境、降解污染物等特殊功能微生物相近的菌株。研究结果表明,该深海沉积物样品的可培养微生物资源、潜在新物种和微生物生理特性丰富多样,研究深海环境难培养微生物的分离策略及其微生物适应生理特性对研究极端环境微生物打下了基础。

    Abstract

    Microbial resources are abundant in marine sedimentary environment. Researches on the isolation strategies for uncultured groups not only promote the microbial resource exploitation from the unusual environments, but also help to understand their role in deep sea. In this study, a deep sea sediment, collected from South China Sea at a water depth of 4 000 m, was used for bacterial diversity analysis based on cultured-dependent method and 16S rRNA gene sequencing. By using 23 designed media, 612 strains in total were isolated and identified, which affiliated to 9 orders, 10 families and 27 genera in three phyla: Firmicutes, Actinobacteria and Bacteroidetes. Strains in phylum Firmicutes were the easiest group to be recovered from the deep-sea sediment and covered 85.8% of the total isolates, with 13 potential novel species. The diluted marine agar (MA) medium was suitable to isolate normal single-cell bacteria, and actinobacterial isolation agar (AIA) dilution medium was more suitable for actinobacteria growth. Many strains in this study had the highest similarities with the known species with the characteristics of antibiotic production, cytotoxin production, highly effective enzyme activity, tolerance to adverse environments, and degradation of pollutants. The preliminary results indicate that the cultivatable bacteria, potential novel species and microbial physiologic features are abundant and diverse in the deep-sea sediment. Research on the isolation strategies for uncultured groups and the adaptability of microorganisms to marine habitats will promote the microorganism recovery from the extreme environments.

  • 0 引 言

    0

    深海沉积环境是海洋生态系统的一个重要组成部分,也是地球生物圈最大的碳源储库。与陆地沉积环境相比,深海沉积环境有以下6个主要特征:(1)低温,除了海底热液口及其附近环境温度可高达100~400 ℃外,深海大部分环境温度稳定在1~4 ℃之间;(2)高压,水深每增加10 m,海水静压力约增加1个大气压[1],因此深海中生存的生物需要承受很大的静水压力;(3)低光照或黑暗,水深超过200 m属于无光区,占整个海洋面积的80%;(4)高盐,大部分深海区域的盐度约为3%~3.5%,因此深海中的生物理论上都能耐受至少3%的盐度[2];(5)低溶解氧,透光层下方缺乏光合作用的氧气补充,加上细菌分解作用和动物呼吸作用消耗部分氧气,因此深海沉积环境中的溶解氧含量比陆地表层沉积环境的低[3];(6)寡营养,大部分有机物在深海沉降过程中被海水中的生物消耗分解,因此仅有不到1%的营养物质沉积于深海底层。深海沉积环境主要由矿物质及有机碎屑组成,表层沉积物与海水不断进行物质与能量的交换,细菌数量和新陈代谢活动比底层海水要大得多[4]

    深海环境中蕴藏着数量庞大、丰富多样、功能特殊的微生物资源,是目前研究的热点之一。大约75 %的细菌生物量存在于表层10 cm的深海沉积物中,占全球微生物总量的13%[5]。由于长期适应于高压、高盐、贫营养等极端环境,深海微生物演化出嗜压、嗜盐、嗜酸碱、嗜冷热等独特生理特性,具有显著区别于陆源微生物的代谢途径,以及适应环境的信号转导功能和化学防御机制。在深海环境中,可培养微生物的比例被普遍认为不到1%。Takami等[6]通过平板培养方法在10 898 m深的海底沉积物中发现微生物,包括放线菌、真菌、非极端菌,以及各种极端菌如嗜碱菌、嗜热菌、嗜压菌、嗜冷菌等,它们是各种极端酶的产生菌。深海微生物是产生各种极端酶的重要来源,获得纯培养菌株对深入研究其生态功能、应用潜力等方面具有重要的意义。

    南海海底地形地貌独特而复杂,似一个半封闭的深海盆地,四周分布着环形阶梯状的周边陆坡,中央平坦的深海盆地水深约4 000 m[7]。南海北部盆地平原为粘土类沉积物,沉积速率约为11.8 cm/ka,受海流和深水团影响,南海深海沉积物具有半远洋沉积物性质,由于物质补给源比较丰富,而具有沉积物堆积速度较快的沉积环境[8]。本研究以一个南海深海沉积物为对象,采用传统的纯培养分离方法,对其中的可培养细菌多样性进行初步分析研究。

  • 1 材料与方法

    1
  • 1.1 材料

    1.1
  • 1.1.1 沉积物样品:

    1.1.1

    本次实验的南海深海沉积物样品,来自中国科学院南海海洋研究所2016年9~10月组织的国家自然科学基金委南海中部开放共享航次。样品采集于站位S16(118.800o E,18.295o N,4 000 m)使用抓斗采集器获得,无菌采样勺取表层0~3 cm的新鲜沉积泥样,-20 ℃保藏备用。

  • 1.1.2 分离培养基:

    1.1.2

    设计以下23种培养基,并根据培养基的主要成分将其分成海洋琼脂类培养基(MA)、放线菌选择性分离类合成培养基(AIA)、淀粉类培养基、天然成分培养基、高盐类培养基和其他培养基(表1),每种培养基中琼脂终浓度为15.0 g/mL,含超纯水1 000 mL。

    表1 不同培养基的类型及其成分

    Table 1 Types and ingredients of different media

    培养基类型培养基种类与成分
    海洋琼脂类培养基

    ①海洋细菌培养基2216E(MA;BD DifcoTM

    ②50%MA培养基(MAB)

    ③20%MA培养基(MAE)

    ④10%MA培养基(MAJ)

    放线菌选择性分离类合成培养基

    ①放线菌选择培养基(AIA;BD DifcoTM

    ②50%AIA培养基(AIAB)

    ③20%AIA培养基(AIAE)

    天然成分培养基

    ①酸微菌培养基(AM):MgSO4·7 H2O 0.50 g,(NH4)2SO4 0.40 g,K2HPO4 0.20 g,KCl 0.10 g,FeSO4·7H2O 0.01 g,酵母提取物0.25 g

    ②麦芽糖-酵母-蛋白胨培养基(MYP):麦芽糖提取物5.0 g,酵母提取物5.0 g,蛋白胨5.0 g,NaCl 3.0 g

    ③营养培养基(R):蛋白胨10.0 g,酵母提取物5.0 g,麦芽糖提取物5.0 g,酪蛋白氨基酸5.0 g,牛肉浸膏2.0 g,甘油2.0 g,吐温80 50.0 mg,MgSO4·7H2O 1.0 g

    ④50%R2A培养基(R2AB;BD DifcoTM)

    ⑤5%R2A培养基(R2AJ)

    淀粉类培养基

    ①MA淀粉培养基(MAS):MA,1 %(m/V)可溶性淀粉

    ②50%MA淀粉培养基(MABS):MAB,1 %(m/V)可溶性淀粉

    ③20%MA淀粉培养基(MAES):MAE,1 %(m/V)可溶性淀粉

    ④10%MA淀粉培养基(MAJS):MAJ,1 %(m/V)可溶性淀粉

    ⑤5%MA淀粉培养基(MATS):5 % MA,1 %(m/V)可溶性淀粉

    高盐类培养基

    ①高盐AIA培养基(AIAS):AIA 10.0 g,NaCl 100.0 g,海盐粗提物5.0 g,SrCl2 2.0 g

    ②高盐牛肉膏培养基(BFSM):牛肉浸膏2.0 g,碳酸钙1.0 g,海盐粗提物5.0 g,钼酸钠5.0 g,可溶性淀粉2.0 g,NaCl 100.0 g

    ③高盐酪蛋白培养基(CAAM):酪蛋白水解物1.0 g,KCl 2.0 g,MgSO4·7H2O 2.0 g,NaCl 100.0 g,海盐粗提物10.0 g,葡萄糖酸盐1.0 g;柠檬酸三钠1.0 g,酵母提取物1.0 g,高锰酸钾2.0 g(单独灭菌)

    ④高盐含铁培养基(YJSF):MA 15.0 g,碳酸钙5.0 g,NaCl 100.0 g,FeCl2 0.5 g(过滤除菌),硫酸亚铁0.5 g(过滤除菌)

    其他培养基

    ①SN:NaNO3 0.75 g,K2HPO4 0.015 9 g,EDTA二钠0.005 6 g,Na2CO3 0.010 4 g,50% 海水,Vitamin B12 0.001 g(过滤除菌),Cyano trace metal solution 1×10-6单独灭菌(乙酸6.25 g,柠檬酸铁铵6.0 g,MnCl2·4H2O 1.4 g,Na2MoO4·2H2O 0.39 g,Co(NO32·6H2O 0.025 g,ZnSO3·7H2O 0.222 g)

    ②ZANT:NaHCO32.0 g,NaH2PO4·2H2O 0.05 g,NaNO30.5 g,CaCl20.02 g,MgSO4·7H2O 0.05 g,KCl 0.1 g,A5溶液1×10-6(H3BO3 2.86 g,MnCl·4H2O 1.80 g,ZnSO4·7H2O 0.22 g,Na2MoO4·2H2O 0.3 g,CuSO4·5H2O 0.08 g)

    表1
                    不同培养基的类型及其成分
  • 1.1.3 实验试剂与仪器:

    1.1.3

    Taq DNA聚合酶(北京全式金);PCR仪(Eppendorf, German);凝胶成像系统(Bio-rad, USA);小型台式高速离心机和恒温箱(ESCO, Singapore)。

  • 1.2 菌株的分离、纯化与保藏

    1.2

    称取2.5 g沉积物样品至7.5 mL无菌超纯水中,常温混匀,取200 μL悬浊液至分离培养基,涂布,置于28 oC恒温培养箱培养20 d。挑取不同形态的单菌落至海洋细菌培养基2216E平板,四线法纯化,于25%(m/V)甘油管中-80 ℃保藏。

  • 1.3 DNA的提取与PCR扩增

    1.3

    菌株基因组DNA的提取采用Chelex提取法[9],16S rRNA基因序列的扩增参照Rainey等[10]的方法,使用通用引物27 F (5’-AGAGTTTGATCCTGGCTCAG-3’)和1492 R(5’-CGGTTACCTTGTTACGACTT -3’)。PCR产物经琼脂糖凝胶电泳检测后送往广州天一辉远测序公司进行16S rRNA基因测序。

  • 1.4 系统进化分析

    1.4

    菌株16S rRNA序列片段使用SeqMan软件(Version 7.1.0) 进行拼接,获得近1 500 bp的16S rRNA基因序列在EzBioCloud[11](https://www.ezbiocloud.net/identify) 进行相似性比对分析,选取同源性较高的模式菌株的16S rRNA基因序列作为参比序列,使用CLUSTAL_X软件进行多序列比对,使用MEGA7.0软件[12],根据Kimura模型[13]估算系统进化矩阵,采用邻近法进行聚类分析,设置1 000次重采样构建系统发育树。

  • 2 结果与分析

    2
  • 2.1 细菌物种多样性

    2.1

    通过23种培养基平板,共分离得到612株细菌,经16S rRNA基因序列比对分析,以98%的16S rRNA基因序列相似性作为原核微生物物种的界限[14],合并相似性高于98%的菌株为同一个种,新分离菌株的16S rRNA基因序列比对分析显示它们分别与113个已知物种相近,这些物种分属于厚壁菌门(Firmicutes)、放线菌门(Actinobacteria)和拟杆菌门(Bacteroidetes)的9目10科27属。图1为分离获得的27个属级类群所具有的菌株数量和物种数量的分布图,描述了分离获得菌株在属级水平上的丰富度和多样性。图2为每个属选取一个最相似种菌株的16S rRNA基因序列构建的系统发育树。对获得的3个门级类群纯培养菌株的多样性分别进行描述。

    图1
                            27个属级类群中菌株数量(A)和物种数量(B)的分布图

    图1 27个属级类群中菌株数量(A)和物种数量(B)的分布图

    Fig.1 Numbers of strains (A) and species (B) in 27 genera

    图2
                            基于16S rRNA基因序列构建的海洋沉积物中27个属代表菌株的系统发育树,>50%的自举值(1 000次重复的百分比)在节点处显示

    图2 基于16S rRNA基因序列构建的海洋沉积物中27个属代表菌株的系统发育树,>50%的自举值(1 000次重复的百分比)在节点处显示

    Fig. 2 Phylogenetic tree of the selected reference strains in 27 genera and their related species based on 16S rRNA gene sequences and neighbor-joining method, bootstrap value (expressed as percentage of 1 000 replications) > 50% are shown at branch points

  • 2.1.1 厚壁菌门

    2.1.1

    厚壁菌门包含芽胞杆菌纲的2个目,18个属的97个物种,占所有分离物种数量的85.8%,是该沉积物样品中最容易获得纯培养一大类群。乳杆菌目仅包含1个属级类群Chungangia,其最相近物种Chungangia koreensis CAU 9163T同样分离自海洋沉积物[15]。芽胞杆菌目包括芽胞杆菌科、类芽胞杆菌科和动球菌科的3个科17个属级类群,其中芽胞杆菌科中假芽胞杆菌属(Fictibacillus)是实现纯培养频率最高的类群之一,共获得纯培养菌株276株,分布于6个种,其中出现频率最高的物种是假芽胞杆菌属的F. enclensis,共发现107株菌株,其次是嗜磷假芽胞杆菌(F. phosphorivorans)物种,共发现86株。258株纯培养菌株隶属于芽胞杆菌属(Bacillus),该类群获得物种数最多,分布于59个种,该属级类群的可培养物种最丰富,其中发现频率最高的物种是印度芽胞杆菌(B. indicus),共发现40个菌株。芽胞杆菌为该环境最优势可培养类群之一,这一结果与其他作者对南海深海沉积物可培养细菌的研究结果一致[16]。我国学者采用分子生物学手段,检测到厚壁菌门在个别北极海洋沉积物[17]和南海北部陆坡深水沉积物[18]中为优势类群,其相对丰度高达38%~40 %。可能由于厚壁菌门的细菌多为好氧异养型,容易在营养较丰富的表层沉积物中繁殖而占据优势。另外,芽胞杆菌纲内的物种大多可形成抗逆性极强的芽胞,抵抗脱水和不良环境,具有极强的环境适应性,因此在各类生态系统中发挥着重要的生物学作用[19]

  • 2.1.2 放线菌门

    2.1.2

    放线菌是一类高G + C含量的革兰氏阳性细菌,因产生丰富的活性次生代谢产物而著名,它是微生物中一个重要的类群,广泛分布在海洋各种环境中,有研究发现放线菌占南海西沙海槽沉积环境微生物的7%[20]。本研究中,该类群包含放线菌亚纲(Actinobacteria)和红色杆菌亚纲(Rubrobacteria)的6个目,7个科,8个属的14个物种,占所有分离物种数量的12.3%。这些菌种中有与红色杆菌属的Rubrobacter radiotolerans物种相近的菌株SCSIO 54388,其16S rRNA基因序列相似度为99.86%,该物种最初分离自日本放射性温泉[21];有与海绵来源短状杆菌属放线菌Brachybacterium paraconglomeratum[22]相近菌株SCSIO 52782,也有4株隶属于微球菌目细杆菌属菌株分别与分离自日本墓地土壤的嗜中温放线菌Microbacterium aoyamense[23]、分离自玉米浸出液的M. aurum KACC 15219T[24]、分离自石油储存洞穴的M. oleivorans NBRC 103075T[25]、分离自极性淋巴细胞白血病的孩童血液中的病原菌之一的M. paraoxydans NBRC 103076T[26]相近。2株小单孢菌目的菌株,分别与分离自空气的青铜小单孢菌(Micromonospora chalcea) DSM 43026T[27]以及分离自海南红树林根际表面的内生继生菌(Jishengella endophytica) 202201T[28]相近。4株隶属于链孢囊菌目拟诺卡氏菌属的菌株与分离自新疆盐渍土样的盐诺卡氏菌(Nocardiopsis salina) YIM 90010T[29]相近。1株假诺卡氏菌目普氏菌属的菌种,与分离自新疆盐湖的艾丁湖普氏菌(Prauserella aidingensis) DSM 45266T[30]相近。1株棒状杆菌目分支杆菌属菌株SCSIO 50209,与分离自分支杆菌菌血症病人血液中的菌血症分支杆菌(Mycobacterium bacteremicum) ATCC 25791T[31]相近。表明深海是一个相对稳定的区域,从中发现的海洋放线菌显示出功能多样性特征,特别是嗜中温放线菌和病原菌的生境与深海环境极其不同,产生这种物种多样性的现象或机制值得研究。

  • 2.1.3 拟杆菌门

    2.1.3

    拟杆菌门广泛存在于深海环境中,南极洲罗斯陆缘冰下的海洋沉积物[32]、印度洋深海热液硫化物区沉积物[33]等环境中均检测到拟杆菌门类群。研究人员对南海北部表层沉积物的16S rRNA克隆文库分析,发现拟杆菌门占5.2%[34]。本研究仅发现该类群的3株菌,与金黄杆菌属(Chryseobacterium)的分离自大西洋深海沉积物(2 577 m)的C. profundimaris DY46T[35]和分离自西藏高加隆冰川藻苔的C. takakiae DSM 26898T[36]相近,16S rRNA基因序列相似度达98.5%~100%。金黄杆菌属菌株是深海环境的常见菌株,为革兰氏阴性菌,专性好氧,产黄色素,不产生内生孢子,具有强烈的芳香气味以及过氧化氢酶和氧化酶活性。该属的物种生境来源多种多样,如分离自水体环境、深海环境、污染土壤、鱼类、乳制品和人类临床标本等,且多数菌株对许多抗生素都具有抗性[37]。海洋环境中的拟杆菌门微生物偏好附着于颗粒表面或藻类细胞而生长,具有降解聚合物的能力以及环境适应性基因,如大量的肽酶、糖苷水解酶、糖基转移酶、粘附蛋白以及滑动运动等相关基因[38],但该类群的纯培养菌株获得较少,其在深海环境中实际发挥的功能作用尚未可知。

  • 2.2 不同培养基获得细菌多样性

    2.2

    利用MA、AIA、淀粉类培养基、天然成分培养基、高盐类培养基和其他培养基分离培养后,挑取平板上的不同形态菌落进行纯化和鉴定,不同培养基上获得的属级类群数目和菌株数目如表2。MA获得菌株数目和属级类群最多,分别是260株和厚壁菌门的14个细菌属级类群,该类培养基对厚壁菌门的细菌具有较好的选择性。其次是AIA类合成培养基,分别获得173株菌株和17个属级类群,从该类培养基中获得放线菌属级类群和菌株数目最多,分布于4个属的10株菌株,包括短状杆菌属、继生菌属、细杆菌属和拟诺卡氏菌属。淀粉类培养基上共分离获得12个细菌属级类群和55株菌,获得的放线菌类群有细杆菌属和小单孢菌属,拟杆菌门的金黄杆菌属和厚壁菌门的显核菌属的菌株,淀粉类培养基获得的菌种类群跨3个门,推测该类培养基可能对淀粉利用菌株有较大的选择性。分支杆菌属和少盐芽胞杆菌属的菌株仅在天然成分培养基上获得。高盐类培养基获得30株耐盐菌株,能够耐受10%(m/V)以上的NaCl浓度,如喜盐芽胞杆菌属、拟诺卡氏菌属、普氏菌属和红色杆菌属的多数物种被发现。芽胞杆菌属和假芽胞杆菌属在所有培养基中均能分离获得。放线菌类群在AIA类合成培养基、高盐类培养基、淀粉类培养基和天然成分培养基中均能获得纯培养,其中AIA类合成培养基对放线菌类群的分离培养效果最好。

    表2 不同培养基获得的细菌属级类群数目和菌株数目

    Table 2 Numbers of genera and strains recovered on the six isolation media

    培养基类型属级类群数/菌株数目

    菌株

    总数目

    厚壁

    菌门

    拟杆

    菌门

    放线

    菌门

    海洋琼脂类培养基14/2600/00/0260
    AIA类合成培养基13/1630/04/10173
    淀粉类培养基9/491/32/355
    天然成分培养基7/810/02/283
    高盐类培养基5/240/03/630
    其他培养基3/110/00/011
    表2
                    不同培养基获得的细菌属级类群数目和菌株数目
  • 2.3 可培养细菌的生理功能多样性

    2.3
  • 2.3.1 海洋来源特性的菌株

    2.3.1

    所分离部分菌种,其最相近物种(>98%)具有明显的深海来源或海洋特性。已报道的近似菌种中,有的产抗生素、细胞毒素,有的具有高效酶活、耐受不良环境、降解难降解污染物等生理特性。

    本研究中获得的部分菌株在其他海洋环境中也被报道,且具有独特的海洋生境适应性特征,如与菌株SCSIO 52782的16S rRNA基因序列相似度为99.78%的海洋放线菌(Brachybacterium paraconglomeratum) IFO 15224T,该菌最初来源于海绵内生环境,浙江大学的王钰从太平洋海山富钴结壳区样品中也分离了该物种,且具有耐受钴锰特性[39],其相似度为100%;与菌株SCSIO 51636相近的嗜冷芽胞杆菌(Psychrobacillus psychrodurans)最初来源于深海环境,该物种细胞壁的肽链中含有鸟氨酸,具有耐寒特性,能在-2 oC下良好生长[40]。本研究在4 000 m水深发现的菌株与文献描述的其他海域不同深度的沉积环境中发现的物种相近,包括与菌株SCSIO 50459最相似的芽胞杆菌科的Domibacillus indicus[41],最初分离自印度拉克沙群岛深度5 m水深的海洋沉积物,与菌株SCSIO 53896最相近的海洋诺卡氏菌(Nocardiopsis oceani[42],同样来源于南海海域深度2 460 m水深的深海沉积物,以及与菌株SCSIO 09998最相近的深海类芽胞杆菌(Paenibacillus abyssi[43],最初来源于印度洋海域深度3 636 m水深的深海沉积物;以上物种显示出其在海洋沉积环境广泛分布,且具有适应深海环境的独特生理特征。

  • 2.3.2 产抗生素或细胞毒素

    2.3.2

    小单孢菌科(Micromonosporaceae)中海洋“土著”放线菌-盐孢菌属及其高效抗肿瘤活性次级代谢产物salinisporamide A受到海洋微生物研究者的强烈关注[44],小单孢菌是仅次于链霉菌的发现新生物活性物质的优良微生物资源。本研究中获得小单孢菌科的菌株SCSIO 52804和SCSIO 53876分别与青铜小单孢菌(Micromonospora chalcea)和内生继生菌(Jishengella endophytica)2个物种显示出99%~100%的相似性。青铜小单孢菌KY11091T菌株能产生抗肿瘤化合物——四制癌素[45],以及对大肠杆菌有抑菌活性的二肽化合物[46],从青铜小单孢菌中还发现产新霉素的氨基糖苷乙酰转移酶基因[47]和能裂解多种酵母细胞的胞外酶[48]等生物活性物质。内生继生菌最初分离自海南红树林根际表面,它的一株分离自红树林的内生继生菌161111能产生物碱和1-hydroxy-β-carboline,后者具有抗H1N1病毒活性[49]。本研究发现的深海小单孢菌株的活性代谢产物值得深入研究。

    本次研究中获得的厚壁菌门的产芽孢类群部分菌株,其相近物种也被报道能产生抗生素或细胞毒素等生物活性物质,如菌株SCSIO 50439最相近菌种解硫胺素芽胞杆菌属的Aneurinibacillus migulanus DSM 2895T产广谱的短杆菌肽抗生素,对革兰氏阴性和阳性菌以及植物病原真菌均具有良好的抑制效果[50],该物种基因组中有多达11个次生代谢物生物合成基因簇,包括细菌素、微囊素、非核糖体肽、聚酮、萜烯、膦酸盐、拉索肽和linaridins等基因簇[51]。另外,还发现与产生新的热稳定肽类抗生素paenibacillin P和paenibacillin N的蜂房芽胞杆菌(Paenibacillus alvei) NP75[52],产新型广谱羊毛硫氨酸抗生素-甲醛乙酰胺的类地衣芽胞杆菌(B. paralicheniformis) APC 1576[53]和产河豚毒素的芽胞杆菌属的B. horikoshii S184相近的菌株,显示出深海微生物产生独特稀有活性代谢产物的巨大潜力。

  • 2.3.3 产生物酶特性

    2.3.3

    本研究中通过16S rRNA基因序列比对发现许多菌种的相近物种具有产生生物酶的特性,如与放线菌菌株SCSIO 52933相近的细杆菌属物种M. aurum中发现α-淀粉酶能降解不同类型的淀粉颗粒[54]。产芽孢类群物种的一些菌株如嗜几丁质类芽胞杆菌(Paenibacillus chitinolyticus) CKS1具有β-淀粉酶活性[55]与菌株SCSIO 50395相近,与菌株SCSIO 51512相近的海水芽胞杆菌(B. aquimaris) MKSC 6.2除了分泌α-淀粉酶[56],还能分泌碱性纤维素酶[57]。与菌株SCSIO 53540相近的芽胞杆菌属的B. altitudinis GVC11能生产丝氨酸碱性蛋白酶[58],这种碱性蛋白酶具有较强的分解蛋白质的能力[59],在该物种的其他菌株中还发现热碱稳定的木聚糖酶[60]和耐热的β-1,3-1,4-葡聚糖酶[61]。SDS-稳定碱性蛋白酶[62]、产几丁质酶[63]、细胞色素P-450 单氧酶[64]、用于去除食品或饲料加工中非营养因子的中性耐热植酸酶[65]、参与催化岩藻糖基化的α-L-岩藻糖苷酶[66]β-D-半乳糖苷酶[67]、特异性吡哆醛磷酸酶和核苷二磷酸酶[68]等,均在本研究中芽胞杆菌属或类芽胞杆菌属菌株的最相近物种中发现,这些生物酶被广泛应用于生物、医药、食品、酿造、丝绸、制革等行业。

  • 2.3.4 耐盐、耐受不良环境特性

    2.3.4

    本研究发现的部分菌株与一些耐受高盐的菌种具有最相近的亲缘关系,如与菌株SCSIO 51512相近的芽胞杆菌属的B. aquimaris、与菌株SCSIO 09878相近的芽胞杆菌属的B. haikouensis等,以及与菌株SCSIO 53860的16S rRNA基因序列相似性为99%,最初分离自新疆高盐区高耐盐特性(耐受20%)的盐诺卡氏菌(Nocardiopsis salina) YIM 90010T[29]。另外,还发现与菌株SCSIO 53855相近、最适生长NaCl浓度为10%~15%(m/V)的普氏菌属的Prauserella alba YIM 90005T,该菌株能够利用5-羟基四氢嘧啶作为相容性溶质,维持其在高盐浓度条件下的渗透调节功能[69]。我们还发现一些芽胞杆菌菌株与能耐受重金属环境的芽胞杆菌属的B. dabaoshanensis GSS04T[70],能耐受UV和γ射线和过氧化氢和干燥等不良条件的芽胞杆菌属的B. nealsonii FO-92T[71]菌种显示出最高的16S rRNA基因序列相似性,表明深海微生物可能存在迥异于陆生微生物的特殊的生理特性。

  • 2.3.5 环境修复相关特征

    2.3.5

    本研究分离到的一些与石油降解、耐正丁醇等功能菌种相近的菌株,可能对深海沉积环境中环境有害物质消除和海洋自身净化方面发挥着重要作用。如发现3株菌株SCSIO 52784、SCSIO 50382和SCSIO 52782分别与能利用原油作为碳源基质降解石油的食石油微杆菌(Microbacterium oleivorans) BAS69T[25]和土壤嗜冷芽胞杆菌(Psychrobacillus soli) NHI-2T[72],以及降解多环芳烃化合物的副凝聚短状杆菌(Brachybacterium paraconglomeratum) IFO 15224T[73]的相似度为99%~100%。发现菌株SCSIO 09895和SCSIO 50370分别与耐受有机溶剂且能降解柴油的芽胞杆菌属的B. oleivorans JC228T[74]和能利用正丁醇作为唯一碳源生长、耐正丁醇的芽胞杆菌属的B. butanolivorans K9T[75]相近;以及发现参与降解低密度聚乙烯的物种波茨坦短芽胞杆菌(Brevibacillus parabrevis) PL-1[76]和水解木糖赖氨酸芽胞杆菌(Lysinibacillus xylanilyticu) XDB9T[77]等物种相近的一些菌株。另外,本研究中还发现菌株SCSIO 52784与最初分离自油矿的食石油微杆菌(Microbacterium oleivorans) BAS69T有最高相似性,该菌株能还原有致癌作用的六价铬,是潜在的铬酸盐解毒剂[78]。这些微生物对于评价其在环境修复中的应用潜力有提供了微生物资源。

  • 2.4 潜在新物种信息

    2.4

    以98%作为区分两个原核物种的“金标准”[14],即菌株16S rRNA基因序列最大相似度低于98%的视为潜在新物种,同时合并相互之间的16S rRNA 基因全序列相似度大于98%的菌株为同一物种,合并后本研究发现与已知最相似菌种的最高相似性范围在95.45%~97.99%之间的有13个(16S rRNA基因序列EzBioCould比对结果见表3),分别隶属于厚壁菌门的芽胞杆菌属、显核菌属、假芽胞杆菌属、类芽胞杆菌属和类芽胞八叠球菌属。本研究未发现放线菌门和拟杆菌门类群的潜在新物种,在后续研究中应调整分离培养基的选择性,拓展对海洋放线菌稀有资源的发现。

    表3 13个潜在新物种的16S rRNA基因序列EzBioCould比对结果

    Table 3 EzBioCould BLAST results of 16S rRNA gene sequences of 13 potential new species

    潜在新物种菌株号最相似菌种名称最相似菌种拉丁名称最相似菌种序列登录号最高相似度/%
    SCSIO 09913洞穴芽胞杆菌L5TBacillus cavernae L5TKT18624497.47 (35/1 384)
    SCSIO 52384沙漠芽胞杆菌ZLD-8TBacillus deserti ZLD-8TGQ46504197.94 (29/1 406)
    SCSIO 51743钻特省芽胞杆菌LMG 21831TBacillus drentensis LMG 21831TAJ54250696.92 (42/1 365)
    SCSIO 09896稻壳芽胞杆菌R1TBacillus oryzaecorticis R1TKF54848097.74 (24/1 060)
    SCSIO 50865土壤芽胞杆菌NBRC 102451TBacillus soli NBRC 102451TBCVI0100012197.99 (28/1 395)
    SCSIO 53557装备显核菌DSM 14152TCaryophanon tenue DSM 14152TMASJ0100002597.57 (34/1 402)
    SCSIO 52372嗜磷假芽胞杆菌Ca7TTFictibacillus phosphorivorans Ca7TTJX25892497.98 (29/1 404)
    SCSIO 52353井水假芽胞杆菌WPCB074TFictibacillus rigui WPCB074TEU93968997.86 (30/1 401)
    SCSIO 50833井水假芽胞杆菌WPCB074TFictibacillus rigui WPCB074TEU93968995.47 (62/1 370)
    SCSIO 50852盐渍土假芽胞杆菌YC1TFictibacillus solisalsi YC1TEU04626897.98 (28/1 388)
    SCSIO 50395分解几丁质类芽胞杆菌NBRC 15660TPaenibacillus chitinolyticus NBRC 15660TBBJT0100002995.45 (63/1 386)
    SCSIO 50246沼泽类芽胞杆菌N3/975TPaenibacillus uliginis N3/975TFN55646797.90 (29/1 380)
    SCSIO 50238垃圾类芽胞八叠球菌属SK55TPaenisporosarcina quisquiliarum SK55TDQ33389797.43 (36/1 403)
    表3
                    13个潜在新物种的16S rRNA基因序列EzBioCould比对结果

    括号中数字表示潜在新物种与其16S rRNA基因序列最相近菌株序列比对的差异碱基数目与碱基总数

    numbers in brackets represent the different number and total number of bases of potential new species compared with the most related strains based on the 16S rRNA gene sequences

  • 3 讨 论

    3

    本研究主要采用纯培养方式对深海沉积环境微生物多样性进行研究,共使用23个培养基,完成612株菌的16S rRNA基因测序及分析,显示共获得3个门9个目10科27属113个相近物种,其中厚壁菌门获得总菌株数的87.3%,占发现物种总数的85.8%,是深海沉积环境最容易获得纯培养菌株最多的一类细菌类群,也是南海深海沉积环境纯培养物种最丰富多样的原核微生物类群之一。其余的放线菌门和拟杆菌门获得菌株较少,放线菌门占本研究所获得菌种数的12.3%,与文献报道深海沉积环境高通量测序获得的放线菌的比例14.4%相近[20];而拟杆菌门仅获得3株菌2个物种,占比较少,其他细菌门更是没有发现。

    沉积物中的微生物群落组成随着氧浓度、温度、营养元素的种类和含量以及沉积物的深度等物理化学参数的变化而变化。有氧的表层沉积环境微生物的多样性相对较高,有文献报道在海洋沉积环境中变形菌门(Proteobacteria)是优势细菌类群之一[79],但在本次研究中并未获得该类群的纯培养菌株,可能由于分离培养方法的选择性和局限性,影响到深海沉积物样品中变形菌门微生物的纯培养;另外,本研究使用的样品中实际是否存在活的这类微生物也值得深入思考和验证。因此,免培养的分子技术与纯培养方法应相互结合、取长补短,才能更加准确的评价深海沉积环境微生物多样性情况;同时,在免培养结果指导下,可深度优化和改进纯培养的方法和技术,更好地做到“有的放矢”地挖掘深海微生物稀有资源。

    从生理功能多样性方面来看,本研究从深海沉积物环境中获得的大部分菌株,具有产抗生素、细胞毒素、高效酶活、耐受不良环境、降解难降解污染物等各种适应独特海洋极端环境的特性。深海沉积环境营养贫乏,它承纳了海洋表层不易利用的各式各样难降解营养物,该类营养物的利用需要微生物产生特殊的降解酶,或以共代谢的方式实现对其同化。同时也有部分微生物产生特殊的活性代谢物质通过抑制其他微生物的生长,获取对营养物、环境因子或在空间上的竞争优势。因此,在深海独特的生态系统中生存的微生物需进化出维持其与该环境生存相适应的各种特异生理功能。

    微生物的分离培养是海洋微生物学和微生物生态学研究的重要一环,由于微生物自身的营养缺陷或对极端环境的依赖等原因,导致许多难培养优势类群尚未获得纯培养。一般的分离培养方法仅能适用于筛选某些对环境耐受性强,能快速适应实验室条件的易培养类群,而对于某些难培养的特殊类群需要进行针对性的分析和研究。例如,适应能力差或处于休眠状态的海洋细菌[80],对分离条件的敏感和不耐受性,使其容易遭受营养、毒素、生长抑制因子等外源压力的损害而无法实现纯培养。另

    一方面,由于自身营养缺陷或受到外压限制而生长缓慢的微生物,在培养基上仅形成微小菌落,无法被肉眼发现或很快被繁殖速度快的微生物覆盖而无法获得纯培养。针对这类群难培养和未培养微生物,设计针对性的分离方案、富集培养策略和检测技术,以更好地研究深海微生物的多样性与发掘稀有的微生物资源。

    • 1

      Fang J R, Huang W Z. Recent progress in research on deep-sea microorganisms [J]. Mar Sci Bull, 1995(2): 65-69.

      方金瑞, 黄维真. 深海微生物的研究进展[J]. 海洋通报, 1995(2): 65-69.

    • 2

      Chen X L, Zhang Y Z, Gao P J. Progress in deep-sea microbiology [J]. Mar Sci, 2004, 28(1):61-66.

      陈秀兰, 张玉忠, 高培基. 深海微生物研究进展[J]. 海洋科学, 2004, 28(1): 61-66.

    • 3

      Bai J, Li H Y, Zhang J, et al. Diversity of bacterial community in the sediments of the Northern Yellow Sea [J]. China Environ Sci, 2009, 29(12): 1277-1284.

      白洁, 李海艳, 张健, 等. 黄海西北部沉积物中细菌群落16S rDNA多样性解析[J]. 中国环境科学, 2009, 29(12): 1277-1284.

    • 4

      Zhang S, Zhang C S, Tian X P, et al. The study of diversities of marine microbes in China [J]. Bull Chin Acad Sci, 2010, 25(6): 651-658.

      张偲, 张长生, 田新朋, 等. 中国海洋微生物多样性研究[J]. 中国科学院院刊, 2010, 25(6): 651-658.

    • 5

      Turley C. Bacteria in the cold deep-sea benthic boundary layer and sediment-water interface of the NE Atlantic [J]. Fems Microbiol Ecol, 2000, 33(2): 89-99.

    • 6

      Takami H.Isolation and characterization of microorganisms from deep-sea mud[M]//Extremophiles in Deep-Sea Environments. Tokyo: Springer Japan, 1999: 3-26.

    • 7

      Liu Z S, Fan S Q, Zhao H T, et al.Geology of the South China Sea[M]. Beijing: Science Press, 2002.

      刘昭蜀, 范时清, 赵焕庭.南海地质[M]. 北京:科学出版社, 2002.

    • 8

      Li C Z. 14C dating of abyssal sediment in the South China Sea and the study on sedimentation rates of modern sediments [J]. Acta Oceanol Sin,1990, 3(12): 340-346.

      李粹中. 南海深海沉积物14C测年和近代沉积速率的研究[J]. 海洋学报, 1990, 3(12): 340-346.

    • 9

      Walsh P S, Metzger D A, Higuchi R. Chelex 100 as a medium for simple extraction of DNA for PCR-based typing from forensic material [J]. Bio Tech, 1991, 10(4): 506-513.

    • 10

      Rainey F A, Wardrainey N, Kroppenstedt R M, et al. The genus Nocardiopsis represents a phylogenetically coherent taxon and a distinct actinomycete lineage: proposal of Nocardiopsaceae fam. nov. [J]. Int J Syst Bacteriol, 1996, 46(4): 1088.

    • 11

      Kim O S, Cho Y J, Lee K, et al. Introducing EzTaxon-e: a prokaryotic16S rRNA gene sequence database with phylotypes that represent uncultured species [J]. Int J Syst Evol Microbiol, 2012, 62(Pt 3): 716-721.

    • 12

      Kumar S, Stecher G, Tamura K.MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets [J]. Mol Biol Evol, 2016, 33(7): 1870.

    • 13

      Kimura M. A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences [J]. J Mol Evol, 1980, 16(2): 111-120.

    • 14

      Stackebrandt E. Taxonomic parameters revisited : tarnished gold standards [J]. Microbiol Today Nov, 2006, 6(4): 152-155.

    • 15

      Kim W, Traiwan J, Park M H, et al. Chungangia koreensis gen. nov., sp. nov., isolated from marine sediment [J]. Int J Syst Evol Microbiol, 2012, 62(8): 1914-1920.

    • 16

      Yu Q W, Hu L Q, Li F, et al. Diversity and bacteriostatic activity of cultivable marine bacteria from South China Sea sediment at low temperature [J]. Southwest China J Agric Sci, 2015, 28(6): 2803-2808.

      于清武, 胡丽琴, 李菲, 等. 低温环境下南海深海沉积物中可培养细菌的多样性及其抑菌活性分析[J]. 西南农业学报, 2015, 28(6): 2803-2808.

    • 17

      Lin X Z, Zhang L, Liu Y G, et al. Bacterial and archaeal community structure of pan-Arctic Ocean sediments revealed by pyrosequencing [J]. Acta Oceanol Sin, 2017, 36(8): 146-152.

      林学政, 张良, 刘焱光, 等. 北极海洋沉积物细菌和古菌群落结构分析[J]. 海洋学报, 2017, 36(8): 146-152.

    • 18

      Wang P, Li T. Phylogenetic analysis of bacterial community in deep-sea sediment from northern slope of the South China Sea [J]. Mar Sci, 2008, 32(4): 36-39.

      王鹏, 李涛. 南海北部陆坡深水区沉积物细菌多样性调查[J]. 海洋科学, 2008, 32(4): 36-39.

    • 19

      Song Z Q, Wang L, Liu X H, et al. Diversities of Firmicutes in four hot springs in Yunnan and Tibet [J]. Biotechnol, 2015, 25(5): 481-486.

      宋兆齐, 王莉, 刘秀花, 等. 云南和西藏四处热泉中的厚壁菌门多样性[J]. 生物技术, 2015, 25(5): 481-486.

    • 20

      Li T, Wang P, Wang P X. A preliminary study on the diversity of bacteria in the Xisha trough sediment, the South China Sea [J]. Adv Earth Sci, 2006, 21(10): 1058-1062.

      李涛, 王鹏, 汪品先. 南海西沙海槽沉积物细菌多样性初步研究[J]. 地球科学进展, 2006, 21(10): 1058-1062.

    • 21

      Yoshinaka T, Yano K, Yamaguchi H. Isolation of highly radioresistant bacterium, Arthrobacter radiotolerans nov. sp. [J]. Agric Biol Chem, 1973, 37(10): 2269-2275.

    • 22

      Takeuchi M, Fang C X, Yokota A. Taxonomic study of the genus Brachybacterium: proposal of Brachybacterium conglomeratum sp. nov., nom. rev., Brachybacterium paraconglomeratum sp. nov., and Brachybacterium rhamnosum sp. nov. [J]. Int J Syst Evol Microbiol, 1995, 45(1): 160-168.

    • 23

      Kageyama A, Takahashi Y, Ōmura S. Microbacterium deminutum sp. nov., Microbacterium pumilum sp. nov. and Microbacterium aoyamense sp. nov. [J]. Int J Syst Evol Microbiol, 2006, 56(9): 2113-2117.

    • 24

      Yokota A, Takeuchi M, Weiss N. Proposal of two new species in the genus Microbacterium: Microbacterium dextranolyticum sp. nov. and Microbacterium aurum sp. nov. [J]. Int J Syst Evol Microbiol, 1993, 43(3): 549-554.

    • 25

      Schippers A, Bosecker K, Spröer C, et al. Microbacterium oleivorans sp. nov. and Microbacterium hydrocarbonoxydans sp. nov., novel crude-oil-degrading Gram-positive bacteria [J]. Int J Syst Evol Microbiol, 2005, 55(2): 655-660.

    • 26

      Laffineur K, Avesani V, Cornu G, et al. Bacteremia due to a novel Microbacterium species in a patient with leukemia and description of Microbacterium paraoxydans sp. nov [J]. J Clin Microbiol, 2003, 41(5): 2242-2246.

    • 27

      Suarez J E, Hardisson C. Morphological characteristics of colony development in Micromonospora chalcea [J]. J Bacteriol, 1985, 162(3): 1342-1344.

    • 28

      Xie Q Y, Wang C, Wang R, et al. Jishengella endophytica gen. nov., sp. nov., a new member of the family Micromonosporaceae [J]. Int J Syst Evol Microbiol, 2011, 61(5): 1153-1159.

    • 29

      Li W J, Park D J, Tang S K, et al. Nocardiopsis salina sp. nov., a novel halophilic actinomycete isolated from saline soil in China [J]. Int J Syst Evol Microbiol, 2004, 54(5): 1805-1809.

    • 30

      Li Y, Tang S K, Chen Y G, et al. Prauserella salsuginis sp. nov., Prauserella flava sp. nov., Prauserella aidingensis sp. nov. and Prauserella sediminis sp. nov., isolated from a salt lake [J]. Int J Syst Evol Microbiol, 2009, 59(12): 2923-2928.

    • 31

      Brown-Elliott B A, Wallace R J, Petti C A, et al. Mycobacterium neoaurum and Mycobacterium bacteremicum sp. nov. as causes of mycobacteremia [J]. J Clin Microbiol, 2010, 48(12): 4377-4385.

    • 32

      Carr S A, Vogel S W, Dunbar R B, et al. Bacterial abundance and composition in marine sediments beneath the Ross Ice Shelf, Antarctica [J]. Geobiol, 2013, 11(4): 377-395.

    • 33

      Wu Y H, Cao Y, Wang C S, et al. Microbial community structure and nitrogenase gene diversity of sediment from a deep-sea hydrothermal vent field on the Southwest Indian Ridge [J]. Acta Oceanol Sin (in Chinese), 2014, 33(10): 94-104.

      吴月红, 曹佚, 王春生, 等. 西南印度洋深海热液硫化物区沉积物微生物群落结构和固氮基因多样性[J]. 海洋学报 (中文版), 2014, 33(10): 94-104.

    • 34

      Zhang H, Wu H B, Wang G H, et al. Prokaryote diversity in the surface sediment of northern South China Sea [J]. Acta microbiol Sin, 2013, 53(9): 915-926.

      张浩, 吴后波, 王广华, 等. 南海北部表层沉积物中原核微生物多样性[J]. 微生物学报, 2013, 53(9): 915-926.

    • 35

      Xu L, Huo Y Y, Li Z Y, et al. Chryseobacterium profundimaris sp. nov., a new member of the family Flavobacteriaceae isolated from deep-sea sediment [J]. Antonie van Leeuwenhoek, 2015, 107(4): 979-989.

    • 36

      Zhao R, Chen X Y, Li X D, et al. Chryseobacterium takakiae sp. nov., a member of the phylum Bacteroidetes isolated from Takakia lepidozioides [J]. Int J Syst Evol Microbiol, 2015, 65(1): 71-76.

    • 37

      Vandamme P, Bernardet J F, Segers P, et al. New perspectives in the classification of the Flavobacteria: description of Chryseobacterium gen. nov., Bergeyella gen. nov., and Empedobacter nom. rev. [J]. Int J Syst Bacteriol, 1994, 44(4): 827-831.

    • 38

      Fernandez-Gomez B, Richter M, Schuler M, et al. Ecology of marine bacteroidetes: a comparative genomics approach [J]. ISME J, 2013, 7(5): 1026-1037.

    • 39

      Wang Y.Analysis of microbial diversity in deep sea sediments and taxonomy of a bacterium from marine sediment[D]. Hangzhou: Zhejiang University, 2010.

      王钰. 深海沉积物微生物多样性研究及一株近海沉积物细菌多相分类鉴定[D].杭州:浙江大学, 2010.

    • 40

      Krishnamurthi S, Ruckmani A, Pukall R, et al. Psychrobacillus gen. nov. and proposal for reclassification of Bacillus insolitus Larkin & Stokes, 1967, B. psychrotolerans Abd-El Rahman et al., 2002 and B. psychrodurans Abd-El Rahman et al., 2002 as Psychrobacillus insolitus comb. nov., Psychrobacillus psychrotolerans comb. nov. and Psychrobacillus psychrodurans comb. nov. [J]. Syst Appl Microbiol, 2010, 33(7): 367-373.

    • 41

      Sharma A, Dhar S K, Prakash O, et al. Description of Domibacillus indicus sp. nov. isolated from ocean sediments and emended description of the genus Domibacillus [J]. Int J Syst Evol Microbiol, 2014, 64(9):3010-3015.

    • 42

      Pan H Q, Zhang D F, Li L, et al. Nocardiopsis oceani sp. nov. and Nocardiopsis nanhaiensis sp. nov., actinomycetes isolated from marine sediment of the South China Sea [J]. Int J Syst Evol Microbiol, 2015, 65(10): 3384-3391.

    • 43

      Huang X F, Wang F Z, Zhang W, et al. Paenibacillus abyssi sp. nov., isolated from an abyssal sediment sample from the Indian Ocean [J]. Antonie van Leeuwenhoek, 2014, 106(6): 1089-1095.

    • 44

      Lei X L, Hong K, Ruan J S.Micromonosporaceae and their important role in marine drug development [J]. Biotechnol Bull, 2006(S1):87-90.

      雷湘兰, 洪葵, 阮继生. 小单孢菌及其在海洋药物开发中的前景[J]. 生物技术通报, 2006(S1):87-90.

    • 45

      Tamaoki T, Kasai M, Shirahata K, et al. Tetrocarcins, novel antitumor antibiotics [J]. J Antibiot (Tokyo), 1980, 33(9): 946-950.

    • 46

      Shoji J, Hinoo H, Kato T, et al. Isolation of n-(2,6-diamino-6-hydroxymethylpimelyl)-l-alanine from Micromonospora chalcea [J]. J Antibiot (Tokyo), 1981, 34(4): 374-380.

    • 47

      Salauze D, Perez-Gonzalez J A, Piepersberg W, et al. Characterisation of aminoglycoside acetyltransferase-encoding genes of neomycin-producing Micromonospora chalcea and Streptomyces fradiae [J]. Gene, 1991, 101(1): 143-148.

    • 48

      Gacto M, Vicente‐Soler J, Cansado J, et al. Characterization of an extracellular enzyme system produced by Micromonospora chalcea with lytic activity on yeast cells[J]. J Appl Microbiol, 2000, 88(6): 961-967.

    • 49

      Wang P, Kong F, Wei J, et al. Alkaloids from the mangrove-derived actinomycete Jishengella endophytica 161111 [J]. Mar Drugs, 2014, 12(1): 477-490.

    • 50

      Berditsch M, Afonin S, Ulrich A S. The ability of Aneurinibacillus migulanus (Bacillus brevis) to produce the antibiotic gramicidin S is correlated with phenotype variation [J]. Appl Environ Microbiol, 2007, 73(20): 6620-6628.

    • 51

      Alenezi F N, Weitz H J, Belbahri L, et al. Draft genome sequence of Aneurinibacillus migulanus NCTC 7096 [J]. Genome A, 2015, 3(2): e00234-15.

    • 52

      Anandaraj B, Vellaichamy A, Kachman M, et al. Co-production of two new peptide antibiotics by a bacterial isolate Paenibacillus alvei NP75 [J]. Biochem Biophys Res Commun, 2009, 379(2): 179-185.

    • 53

      Collins F W J, O’Connor P M, O’Sullivan O, et al. Formicin-a novel broad-spectrum two-component lantibiotic produced by Bacillus paralicheniformis APC 1576 [J]. Microbiol, 2016, 162(9): 1662-1671.

    • 54

      Valk V, Eeuwema W, Sarian F D, et al. Degradation of granular starch by the bacterium Microbacterium aurum strain B8. A involves a modular α-amylase enzyme system with FNIII and CBM25 domains [J]. Appl Environ Microbiol, 2015, 81(19): 6610-6620.

    • 55

      Mihajlovski K R, Radovanović N R, Veljović Đ N, et al. Improved β-amylase production on molasses and sugar beet pulp by a novel strain Paenibacillus chitinolyticus CKS1 [J]. Ind Crops Prod, 2016, 80: 115-122.

    • 56

      Puspasari F, Radjasa O K, Noer A S, et al. Raw starch-degrading α-amylase from Bacillus aquimaris MKSC 6.2: isolation and expression of the gene, bioinformatics and biochemical characterization of the recombinant enzyme [J]. J Appl Microbiol, 2013, 114(1): 108-120.

    • 57

      Trivedi N, Gupta V, Kumar M, et al. Solvent tolerant marine bacterium Bacillus aquimaris secreting organic solvent stable alkaline cellulase [J]. Chemosphere, 2011, 83(5): 706-712.

    • 58

      Kumar E V, Srijana M, Kumar K K, et al. A novel serine alkaline protease from Bacillus altitudinis GVC11 and its application as a dehairing agent [J]. Bioprocess Biosyst Eng, 2011, 34(4): 403-409.

    • 59

      Madhuri A, Nagaraju B, Harikrishna N, et al. Production of alkaline protease by Bacillus altitudinis GVC11 using castor husk in solid-state fermentation [J]. Appl Biochem Biotechnol, 2012, 167(5): 1199-1207.

    • 60

      Adhyaru D N, Bhatt N S, Modi H A. Enhanced production of cellulase-free, thermo-alkali-solvent-stable xylanase from Bacillus altitudinis DHN8, its characterization and application in sorghum straw saccharification [J]. Biocatal Agric Biotechnol, 2014, 3(2): 182-190.

    • 61

      Mao S, Lu Z, Zhang C, et al. Purification, characterization, and heterologous expression of a thermostable β-1,3-1,4-glucanase from Bacillus altitudinis YC-9 [J]. Appl Biochem Biotechnol, 2013, 169(3): 960-975.

    • 62

      Joo H S, Choi J W. Purification and characterization of a novel alkaline protease from Bacillus horikoshii [J]. J Microbiol Biotechnol, 2012, 22(1): 58-68.

    • 63

      Song Y S, Seo D J, Kim K Y, et al. Expression patterns of chitinase produced from Paenibacillus chitinolyticus with different two culture media [J]. Carbohydr Polym, 2012, 90(2): 1187-1192.

    • 64

      Narhi L O, Fulco A J. Characterization of a catalytically self-sufficient 119,000-dalton cytochrome P-450 monooxygenase induced by barbiturates in Bacillus megaterium [J]. J Biol Chem, 1986, 261(16): 7160-7169.

    • 65

      Yu P, Chen Y.Purification and characterization of a novel neutral and heat-tolerant phytase from a newly isolated strain Bacillus nealsonii ZJ0702 [J]. BMC biotechnol, 2013, 13(1): 78.

    • 66

      Benešová E, Lipovová P, Dvořáková H, et al. α-L-fucosidase from Paenibacillus thiaminolyticus: its hydrolytic and transglycosylation abilities [J]. Glycobiol, 2013, 23(9): 1052-1065.

    • 67

      Benešová E, Lipovová P, Dvořáková H, et al. β-D-Galactosidase from Paenibacillus thiaminolyticus catalyzing transfucosylation reactions [J]. Glycobiol, 2009, 20(4): 442-451.

    • 68

      Tirrell I M, Wall J L, Daley C J, et al. YZGD from Paenibacillus thiaminolyticus, a pyridoxal phosphatase of the HAD (haloacid dehalogenase) superfamily and a versatile member of the Nudix (nucleoside diphosphate x) hydrolase superfamily [J]. Biochem J, 2006, 394(3): 665-674.

    • 69

      Li Y, Dong L, Wang L, et al. Cloning and characterization of gene cluster for biosynthesis of ectoine and 5-hydroxyectoine from extreme halotolerant actinomycete strain Prauserella alba YIM 90005T [J]. Acta microbiol Sin, 2011, 51(5): 603-608.

      李岩, 董雷, 王磊, 等. 极端耐盐放线菌白色普氏菌YIM 90005 T四氢嘧啶及5-羟基四氢嘧啶合成相关基因的克隆[J]. 微生物学报, 2011, 51(5): 603-608.

    • 70

      Cui X, Wang Y, Liu J, et al. Bacillus dabaoshanensis sp. nov., a Cr (VI)-tolerant bacterium isolated from heavy-metal-contaminated soil [J]. Arch Microbiol, 2015, 197(4): 513-520.

    • 71

      Venkateswaran K, Kempf M, Chen F, et al. Bacillus nealsonii sp. nov., isolated from a spacecraft-assembly facility, whose spores are γ-radiation resistant [J]. Int J Syst Evol Microbiol, 2003, 53(1): 165-172.

    • 72

      Jeong S W, Kim J. Psychrobacillus soli sp. nov., capable of degrading oil, isolated from oil-contaminated soil [J]. Int J Syst Evol Microbiol, 2015, 65(9): 3046-3052.

    • 73

      Lily M K, Bahuguna A, Bhatt K K, et al. Degradation of anthracene by a novel strain Brachybacterium paraconglomeratum BMIT637C (MTCC 9445) [J]. Int J Environ Sci, 2013, 3(4): 1242.

    • 74

      Azmatunnisa M, Rahul K, Subhash Y, et al. Bacillus oleivorans sp. nov., a diesel oil-degrading and solvent-tolerant bacterium [J]. Int J Syst Evol Microbiol, 2015, 65(4): 1310-1315.

    • 75

      Kuisiene N, Raugalas J, Spröer C, et al. Bacillus butanolivorans sp. nov., a species with industrial application for the remediation of n-butanol [J]. Int J Syst Evol Microbiol, 2008, 58(2): 505-509.

    • 76

      Pramila R, Padmavathy K, Ramesh K V, et al. Brevibacillus parabrevis, Acinetobacter baumannii and Pseudomonas citronellolis — potential candidates for biodegradation of low density polyethylene (LDPE) [J]. Afr J Bacteriol Res, 2012, 4(1): 9-14.

    • 77

      Esmaeili A, Pourbabaee A A, Alikhani H A, et al. Biodegradation of low-density polyethylene (LDPE) by mixed culture of Lysinibacillus xylanilyticus and Aspergillus niger in soil [J]. PLOS One, 2013, 8(9): e71720.

    • 78

      Sarkar A, Sar P, Islam E. Hexavalent chromium reduction by Microbacterium oleivorans A1: a possible mechanism of chromate-detoxification and-bioremediation [J]. Recent Pat Biotechnol, 2015, 9(2): 116-129.

    • 79

      Li T, Wang P, Wang P X. Microbial diversity in surface sediments of the Xisha Trough, the South China Sea [J]. Acta Ecol Sin, 2008, 28(3):1166-1173.

      李涛, 王鹏, 汪品先. 南海西沙海槽表层沉积物微生物多样性[J]. 生态学报, 2008, 28(3):1166-1173.

    • 80

      Xiao W, Yang Y L, Liu H W, et al. Culturable bacterial diversity of the ancient salt deposits in the Kunming Salt Mine, P. R. China [J]. Acta microbiol Sin, 2006, 46(6): 967-972.

      肖炜, 杨亚玲, 刘宏伟,等. 昆明盐矿古老岩盐沉积中可培养细菌多样性研究[J]. 微生物学报, 2006, 46(6):967-972.

陈柔雯

工作单位:1中国科学院南海海洋研究所 中国科学院热带海洋生物资源与生态重点实验室,广东省海洋药物重点实验室,中国科学院海洋微生物研究中心,广东 广州 510301

工作单位:2中国科学院大学,北京 100049

王可欣

工作单位:1中国科学院南海海洋研究所 中国科学院热带海洋生物资源与生态重点实验室,广东省海洋药物重点实验室,中国科学院海洋微生物研究中心,广东 广州 510301

工作单位:2中国科学院大学,北京 100049

何媛秋

工作单位:1中国科学院南海海洋研究所 中国科学院热带海洋生物资源与生态重点实验室,广东省海洋药物重点实验室,中国科学院海洋微生物研究中心,广东 广州 510301

工作单位:2中国科学院大学,北京 100049

田新朋

工作单位:1中国科学院南海海洋研究所 中国科学院热带海洋生物资源与生态重点实验室,广东省海洋药物重点实验室,中国科学院海洋微生物研究中心,广东 广州 510301

联系方式:E-mail: xinpengtian@scsio.ac.cn

龙丽娟

工作单位:1中国科学院南海海洋研究所 中国科学院热带海洋生物资源与生态重点实验室,广东省海洋药物重点实验室,中国科学院海洋微生物研究中心,广东 广州 510301

培养基类型培养基种类与成分
海洋琼脂类培养基

①海洋细菌培养基2216E(MA;BD DifcoTM

②50%MA培养基(MAB)

③20%MA培养基(MAE)

④10%MA培养基(MAJ)

放线菌选择性分离类合成培养基

①放线菌选择培养基(AIA;BD DifcoTM

②50%AIA培养基(AIAB)

③20%AIA培养基(AIAE)

天然成分培养基

①酸微菌培养基(AM):MgSO4·7 H2O 0.50 g,(NH4)2SO4 0.40 g,K2HPO4 0.20 g,KCl 0.10 g,FeSO4·7H2O 0.01 g,酵母提取物0.25 g

②麦芽糖-酵母-蛋白胨培养基(MYP):麦芽糖提取物5.0 g,酵母提取物5.0 g,蛋白胨5.0 g,NaCl 3.0 g

③营养培养基(R):蛋白胨10.0 g,酵母提取物5.0 g,麦芽糖提取物5.0 g,酪蛋白氨基酸5.0 g,牛肉浸膏2.0 g,甘油2.0 g,吐温80 50.0 mg,MgSO4·7H2O 1.0 g

④50%R2A培养基(R2AB;BD DifcoTM)

⑤5%R2A培养基(R2AJ)

淀粉类培养基

①MA淀粉培养基(MAS):MA,1 %(m/V)可溶性淀粉

②50%MA淀粉培养基(MABS):MAB,1 %(m/V)可溶性淀粉

③20%MA淀粉培养基(MAES):MAE,1 %(m/V)可溶性淀粉

④10%MA淀粉培养基(MAJS):MAJ,1 %(m/V)可溶性淀粉

⑤5%MA淀粉培养基(MATS):5 % MA,1 %(m/V)可溶性淀粉

高盐类培养基

①高盐AIA培养基(AIAS):AIA 10.0 g,NaCl 100.0 g,海盐粗提物5.0 g,SrCl2 2.0 g

②高盐牛肉膏培养基(BFSM):牛肉浸膏2.0 g,碳酸钙1.0 g,海盐粗提物5.0 g,钼酸钠5.0 g,可溶性淀粉2.0 g,NaCl 100.0 g

③高盐酪蛋白培养基(CAAM):酪蛋白水解物1.0 g,KCl 2.0 g,MgSO4·7H2O 2.0 g,NaCl 100.0 g,海盐粗提物10.0 g,葡萄糖酸盐1.0 g;柠檬酸三钠1.0 g,酵母提取物1.0 g,高锰酸钾2.0 g(单独灭菌)

④高盐含铁培养基(YJSF):MA 15.0 g,碳酸钙5.0 g,NaCl 100.0 g,FeCl2 0.5 g(过滤除菌),硫酸亚铁0.5 g(过滤除菌)

其他培养基

①SN:NaNO3 0.75 g,K2HPO4 0.015 9 g,EDTA二钠0.005 6 g,Na2CO3 0.010 4 g,50% 海水,Vitamin B12 0.001 g(过滤除菌),Cyano trace metal solution 1×10-6单独灭菌(乙酸6.25 g,柠檬酸铁铵6.0 g,MnCl2·4H2O 1.4 g,Na2MoO4·2H2O 0.39 g,Co(NO32·6H2O 0.025 g,ZnSO3·7H2O 0.222 g)

②ZANT:NaHCO32.0 g,NaH2PO4·2H2O 0.05 g,NaNO30.5 g,CaCl20.02 g,MgSO4·7H2O 0.05 g,KCl 0.1 g,A5溶液1×10-6(H3BO3 2.86 g,MnCl·4H2O 1.80 g,ZnSO4·7H2O 0.22 g,Na2MoO4·2H2O 0.3 g,CuSO4·5H2O 0.08 g)

/html/swzy/201804005/alternativeImage/71b61749-2085-4297-8111-1359a2599646-F001.jpg
/html/swzy/201804005/media/71b61749-2085-4297-8111-1359a2599646-image002.jpg
培养基类型属级类群数/菌株数目

菌株

总数目

厚壁

菌门

拟杆

菌门

放线

菌门

海洋琼脂类培养基14/2600/00/0260
AIA类合成培养基13/1630/04/10173
淀粉类培养基9/491/32/355
天然成分培养基7/810/02/283
高盐类培养基5/240/03/630
其他培养基3/110/00/011
潜在新物种菌株号最相似菌种名称最相似菌种拉丁名称最相似菌种序列登录号最高相似度/%
SCSIO 09913洞穴芽胞杆菌L5TBacillus cavernae L5TKT18624497.47 (35/1 384)
SCSIO 52384沙漠芽胞杆菌ZLD-8TBacillus deserti ZLD-8TGQ46504197.94 (29/1 406)
SCSIO 51743钻特省芽胞杆菌LMG 21831TBacillus drentensis LMG 21831TAJ54250696.92 (42/1 365)
SCSIO 09896稻壳芽胞杆菌R1TBacillus oryzaecorticis R1TKF54848097.74 (24/1 060)
SCSIO 50865土壤芽胞杆菌NBRC 102451TBacillus soli NBRC 102451TBCVI0100012197.99 (28/1 395)
SCSIO 53557装备显核菌DSM 14152TCaryophanon tenue DSM 14152TMASJ0100002597.57 (34/1 402)
SCSIO 52372嗜磷假芽胞杆菌Ca7TTFictibacillus phosphorivorans Ca7TTJX25892497.98 (29/1 404)
SCSIO 52353井水假芽胞杆菌WPCB074TFictibacillus rigui WPCB074TEU93968997.86 (30/1 401)
SCSIO 50833井水假芽胞杆菌WPCB074TFictibacillus rigui WPCB074TEU93968995.47 (62/1 370)
SCSIO 50852盐渍土假芽胞杆菌YC1TFictibacillus solisalsi YC1TEU04626897.98 (28/1 388)
SCSIO 50395分解几丁质类芽胞杆菌NBRC 15660TPaenibacillus chitinolyticus NBRC 15660TBBJT0100002995.45 (63/1 386)
SCSIO 50246沼泽类芽胞杆菌N3/975TPaenibacillus uliginis N3/975TFN55646797.90 (29/1 380)
SCSIO 50238垃圾类芽胞八叠球菌属SK55TPaenisporosarcina quisquiliarum SK55TDQ33389797.43 (36/1 403)

表1 不同培养基的类型及其成分

Table 1 Types and ingredients of different media

图1 27个属级类群中菌株数量(A)和物种数量(B)的分布图

Fig.1 Numbers of strains (A) and species (B) in 27 genera

图2 基于16S rRNA基因序列构建的海洋沉积物中27个属代表菌株的系统发育树,>50%的自举值(1 000次重复的百分比)在节点处显示

Fig. 2 Phylogenetic tree of the selected reference strains in 27 genera and their related species based on 16S rRNA gene sequences and neighbor-joining method, bootstrap value (expressed as percentage of 1 000 replications) > 50% are shown at branch points

表2 不同培养基获得的细菌属级类群数目和菌株数目

Table 2 Numbers of genera and strains recovered on the six isolation media

表3 13个潜在新物种的16S rRNA基因序列EzBioCould比对结果

Table 3 EzBioCould BLAST results of 16S rRNA gene sequences of 13 potential new species

image /

无注解

无注解

无注解

无注解

括号中数字表示潜在新物种与其16S rRNA基因序列最相近菌株序列比对的差异碱基数目与碱基总数

numbers in brackets represent the different number and total number of bases of potential new species compared with the most related strains based on the 16S rRNA gene sequences

    • 1

      Fang J R, Huang W Z. Recent progress in research on deep-sea microorganisms [J]. Mar Sci Bull, 1995(2): 65-69.

      方金瑞, 黄维真. 深海微生物的研究进展[J]. 海洋通报, 1995(2): 65-69.

    • 2

      Chen X L, Zhang Y Z, Gao P J. Progress in deep-sea microbiology [J]. Mar Sci, 2004, 28(1):61-66.

      陈秀兰, 张玉忠, 高培基. 深海微生物研究进展[J]. 海洋科学, 2004, 28(1): 61-66.

    • 3

      Bai J, Li H Y, Zhang J, et al. Diversity of bacterial community in the sediments of the Northern Yellow Sea [J]. China Environ Sci, 2009, 29(12): 1277-1284.

      白洁, 李海艳, 张健, 等. 黄海西北部沉积物中细菌群落16S rDNA多样性解析[J]. 中国环境科学, 2009, 29(12): 1277-1284.

    • 4

      Zhang S, Zhang C S, Tian X P, et al. The study of diversities of marine microbes in China [J]. Bull Chin Acad Sci, 2010, 25(6): 651-658.

      张偲, 张长生, 田新朋, 等. 中国海洋微生物多样性研究[J]. 中国科学院院刊, 2010, 25(6): 651-658.

    • 5

      Turley C. Bacteria in the cold deep-sea benthic boundary layer and sediment-water interface of the NE Atlantic [J]. Fems Microbiol Ecol, 2000, 33(2): 89-99.

    • 6

      Takami H.Isolation and characterization of microorganisms from deep-sea mud[M]//Extremophiles in Deep-Sea Environments. Tokyo: Springer Japan, 1999: 3-26.

    • 7

      Liu Z S, Fan S Q, Zhao H T, et al.Geology of the South China Sea[M]. Beijing: Science Press, 2002.

      刘昭蜀, 范时清, 赵焕庭.南海地质[M]. 北京:科学出版社, 2002.

    • 8

      Li C Z. 14C dating of abyssal sediment in the South China Sea and the study on sedimentation rates of modern sediments [J]. Acta Oceanol Sin,1990, 3(12): 340-346.

      李粹中. 南海深海沉积物14C测年和近代沉积速率的研究[J]. 海洋学报, 1990, 3(12): 340-346.

    • 9

      Walsh P S, Metzger D A, Higuchi R. Chelex 100 as a medium for simple extraction of DNA for PCR-based typing from forensic material [J]. Bio Tech, 1991, 10(4): 506-513.

    • 10

      Rainey F A, Wardrainey N, Kroppenstedt R M, et al. The genus Nocardiopsis represents a phylogenetically coherent taxon and a distinct actinomycete lineage: proposal of Nocardiopsaceae fam. nov. [J]. Int J Syst Bacteriol, 1996, 46(4): 1088.

    • 11

      Kim O S, Cho Y J, Lee K, et al. Introducing EzTaxon-e: a prokaryotic16S rRNA gene sequence database with phylotypes that represent uncultured species [J]. Int J Syst Evol Microbiol, 2012, 62(Pt 3): 716-721.

    • 12

      Kumar S, Stecher G, Tamura K.MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets [J]. Mol Biol Evol, 2016, 33(7): 1870.

    • 13

      Kimura M. A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences [J]. J Mol Evol, 1980, 16(2): 111-120.

    • 14

      Stackebrandt E. Taxonomic parameters revisited : tarnished gold standards [J]. Microbiol Today Nov, 2006, 6(4): 152-155.

    • 15

      Kim W, Traiwan J, Park M H, et al. Chungangia koreensis gen. nov., sp. nov., isolated from marine sediment [J]. Int J Syst Evol Microbiol, 2012, 62(8): 1914-1920.

    • 16

      Yu Q W, Hu L Q, Li F, et al. Diversity and bacteriostatic activity of cultivable marine bacteria from South China Sea sediment at low temperature [J]. Southwest China J Agric Sci, 2015, 28(6): 2803-2808.

      于清武, 胡丽琴, 李菲, 等. 低温环境下南海深海沉积物中可培养细菌的多样性及其抑菌活性分析[J]. 西南农业学报, 2015, 28(6): 2803-2808.

    • 17

      Lin X Z, Zhang L, Liu Y G, et al. Bacterial and archaeal community structure of pan-Arctic Ocean sediments revealed by pyrosequencing [J]. Acta Oceanol Sin, 2017, 36(8): 146-152.

      林学政, 张良, 刘焱光, 等. 北极海洋沉积物细菌和古菌群落结构分析[J]. 海洋学报, 2017, 36(8): 146-152.

    • 18

      Wang P, Li T. Phylogenetic analysis of bacterial community in deep-sea sediment from northern slope of the South China Sea [J]. Mar Sci, 2008, 32(4): 36-39.

      王鹏, 李涛. 南海北部陆坡深水区沉积物细菌多样性调查[J]. 海洋科学, 2008, 32(4): 36-39.

    • 19

      Song Z Q, Wang L, Liu X H, et al. Diversities of Firmicutes in four hot springs in Yunnan and Tibet [J]. Biotechnol, 2015, 25(5): 481-486.

      宋兆齐, 王莉, 刘秀花, 等. 云南和西藏四处热泉中的厚壁菌门多样性[J]. 生物技术, 2015, 25(5): 481-486.

    • 20

      Li T, Wang P, Wang P X. A preliminary study on the diversity of bacteria in the Xisha trough sediment, the South China Sea [J]. Adv Earth Sci, 2006, 21(10): 1058-1062.

      李涛, 王鹏, 汪品先. 南海西沙海槽沉积物细菌多样性初步研究[J]. 地球科学进展, 2006, 21(10): 1058-1062.

    • 21

      Yoshinaka T, Yano K, Yamaguchi H. Isolation of highly radioresistant bacterium, Arthrobacter radiotolerans nov. sp. [J]. Agric Biol Chem, 1973, 37(10): 2269-2275.

    • 22

      Takeuchi M, Fang C X, Yokota A. Taxonomic study of the genus Brachybacterium: proposal of Brachybacterium conglomeratum sp. nov., nom. rev., Brachybacterium paraconglomeratum sp. nov., and Brachybacterium rhamnosum sp. nov. [J]. Int J Syst Evol Microbiol, 1995, 45(1): 160-168.

    • 23

      Kageyama A, Takahashi Y, Ōmura S. Microbacterium deminutum sp. nov., Microbacterium pumilum sp. nov. and Microbacterium aoyamense sp. nov. [J]. Int J Syst Evol Microbiol, 2006, 56(9): 2113-2117.

    • 24

      Yokota A, Takeuchi M, Weiss N. Proposal of two new species in the genus Microbacterium: Microbacterium dextranolyticum sp. nov. and Microbacterium aurum sp. nov. [J]. Int J Syst Evol Microbiol, 1993, 43(3): 549-554.

    • 25

      Schippers A, Bosecker K, Spröer C, et al. Microbacterium oleivorans sp. nov. and Microbacterium hydrocarbonoxydans sp. nov., novel crude-oil-degrading Gram-positive bacteria [J]. Int J Syst Evol Microbiol, 2005, 55(2): 655-660.

    • 26

      Laffineur K, Avesani V, Cornu G, et al. Bacteremia due to a novel Microbacterium species in a patient with leukemia and description of Microbacterium paraoxydans sp. nov [J]. J Clin Microbiol, 2003, 41(5): 2242-2246.

    • 27

      Suarez J E, Hardisson C. Morphological characteristics of colony development in Micromonospora chalcea [J]. J Bacteriol, 1985, 162(3): 1342-1344.

    • 28

      Xie Q Y, Wang C, Wang R, et al. Jishengella endophytica gen. nov., sp. nov., a new member of the family Micromonosporaceae [J]. Int J Syst Evol Microbiol, 2011, 61(5): 1153-1159.

    • 29

      Li W J, Park D J, Tang S K, et al. Nocardiopsis salina sp. nov., a novel halophilic actinomycete isolated from saline soil in China [J]. Int J Syst Evol Microbiol, 2004, 54(5): 1805-1809.

    • 30

      Li Y, Tang S K, Chen Y G, et al. Prauserella salsuginis sp. nov., Prauserella flava sp. nov., Prauserella aidingensis sp. nov. and Prauserella sediminis sp. nov., isolated from a salt lake [J]. Int J Syst Evol Microbiol, 2009, 59(12): 2923-2928.

    • 31

      Brown-Elliott B A, Wallace R J, Petti C A, et al. Mycobacterium neoaurum and Mycobacterium bacteremicum sp. nov. as causes of mycobacteremia [J]. J Clin Microbiol, 2010, 48(12): 4377-4385.

    • 32

      Carr S A, Vogel S W, Dunbar R B, et al. Bacterial abundance and composition in marine sediments beneath the Ross Ice Shelf, Antarctica [J]. Geobiol, 2013, 11(4): 377-395.

    • 33

      Wu Y H, Cao Y, Wang C S, et al. Microbial community structure and nitrogenase gene diversity of sediment from a deep-sea hydrothermal vent field on the Southwest Indian Ridge [J]. Acta Oceanol Sin (in Chinese), 2014, 33(10): 94-104.

      吴月红, 曹佚, 王春生, 等. 西南印度洋深海热液硫化物区沉积物微生物群落结构和固氮基因多样性[J]. 海洋学报 (中文版), 2014, 33(10): 94-104.

    • 34

      Zhang H, Wu H B, Wang G H, et al. Prokaryote diversity in the surface sediment of northern South China Sea [J]. Acta microbiol Sin, 2013, 53(9): 915-926.

      张浩, 吴后波, 王广华, 等. 南海北部表层沉积物中原核微生物多样性[J]. 微生物学报, 2013, 53(9): 915-926.

    • 35

      Xu L, Huo Y Y, Li Z Y, et al. Chryseobacterium profundimaris sp. nov., a new member of the family Flavobacteriaceae isolated from deep-sea sediment [J]. Antonie van Leeuwenhoek, 2015, 107(4): 979-989.

    • 36

      Zhao R, Chen X Y, Li X D, et al. Chryseobacterium takakiae sp. nov., a member of the phylum Bacteroidetes isolated from Takakia lepidozioides [J]. Int J Syst Evol Microbiol, 2015, 65(1): 71-76.

    • 37

      Vandamme P, Bernardet J F, Segers P, et al. New perspectives in the classification of the Flavobacteria: description of Chryseobacterium gen. nov., Bergeyella gen. nov., and Empedobacter nom. rev. [J]. Int J Syst Bacteriol, 1994, 44(4): 827-831.

    • 38

      Fernandez-Gomez B, Richter M, Schuler M, et al. Ecology of marine bacteroidetes: a comparative genomics approach [J]. ISME J, 2013, 7(5): 1026-1037.

    • 39

      Wang Y.Analysis of microbial diversity in deep sea sediments and taxonomy of a bacterium from marine sediment[D]. Hangzhou: Zhejiang University, 2010.

      王钰. 深海沉积物微生物多样性研究及一株近海沉积物细菌多相分类鉴定[D].杭州:浙江大学, 2010.

    • 40

      Krishnamurthi S, Ruckmani A, Pukall R, et al. Psychrobacillus gen. nov. and proposal for reclassification of Bacillus insolitus Larkin & Stokes, 1967, B. psychrotolerans Abd-El Rahman et al., 2002 and B. psychrodurans Abd-El Rahman et al., 2002 as Psychrobacillus insolitus comb. nov., Psychrobacillus psychrotolerans comb. nov. and Psychrobacillus psychrodurans comb. nov. [J]. Syst Appl Microbiol, 2010, 33(7): 367-373.

    • 41

      Sharma A, Dhar S K, Prakash O, et al. Description of Domibacillus indicus sp. nov. isolated from ocean sediments and emended description of the genus Domibacillus [J]. Int J Syst Evol Microbiol, 2014, 64(9):3010-3015.

    • 42

      Pan H Q, Zhang D F, Li L, et al. Nocardiopsis oceani sp. nov. and Nocardiopsis nanhaiensis sp. nov., actinomycetes isolated from marine sediment of the South China Sea [J]. Int J Syst Evol Microbiol, 2015, 65(10): 3384-3391.

    • 43

      Huang X F, Wang F Z, Zhang W, et al. Paenibacillus abyssi sp. nov., isolated from an abyssal sediment sample from the Indian Ocean [J]. Antonie van Leeuwenhoek, 2014, 106(6): 1089-1095.

    • 44

      Lei X L, Hong K, Ruan J S.Micromonosporaceae and their important role in marine drug development [J]. Biotechnol Bull, 2006(S1):87-90.

      雷湘兰, 洪葵, 阮继生. 小单孢菌及其在海洋药物开发中的前景[J]. 生物技术通报, 2006(S1):87-90.

    • 45

      Tamaoki T, Kasai M, Shirahata K, et al. Tetrocarcins, novel antitumor antibiotics [J]. J Antibiot (Tokyo), 1980, 33(9): 946-950.

    • 46

      Shoji J, Hinoo H, Kato T, et al. Isolation of n-(2,6-diamino-6-hydroxymethylpimelyl)-l-alanine from Micromonospora chalcea [J]. J Antibiot (Tokyo), 1981, 34(4): 374-380.

    • 47

      Salauze D, Perez-Gonzalez J A, Piepersberg W, et al. Characterisation of aminoglycoside acetyltransferase-encoding genes of neomycin-producing Micromonospora chalcea and Streptomyces fradiae [J]. Gene, 1991, 101(1): 143-148.

    • 48

      Gacto M, Vicente‐Soler J, Cansado J, et al. Characterization of an extracellular enzyme system produced by Micromonospora chalcea with lytic activity on yeast cells[J]. J Appl Microbiol, 2000, 88(6): 961-967.

    • 49

      Wang P, Kong F, Wei J, et al. Alkaloids from the mangrove-derived actinomycete Jishengella endophytica 161111 [J]. Mar Drugs, 2014, 12(1): 477-490.

    • 50

      Berditsch M, Afonin S, Ulrich A S. The ability of Aneurinibacillus migulanus (Bacillus brevis) to produce the antibiotic gramicidin S is correlated with phenotype variation [J]. Appl Environ Microbiol, 2007, 73(20): 6620-6628.

    • 51

      Alenezi F N, Weitz H J, Belbahri L, et al. Draft genome sequence of Aneurinibacillus migulanus NCTC 7096 [J]. Genome A, 2015, 3(2): e00234-15.

    • 52

      Anandaraj B, Vellaichamy A, Kachman M, et al. Co-production of two new peptide antibiotics by a bacterial isolate Paenibacillus alvei NP75 [J]. Biochem Biophys Res Commun, 2009, 379(2): 179-185.

    • 53

      Collins F W J, O’Connor P M, O’Sullivan O, et al. Formicin-a novel broad-spectrum two-component lantibiotic produced by Bacillus paralicheniformis APC 1576 [J]. Microbiol, 2016, 162(9): 1662-1671.

    • 54

      Valk V, Eeuwema W, Sarian F D, et al. Degradation of granular starch by the bacterium Microbacterium aurum strain B8. A involves a modular α-amylase enzyme system with FNIII and CBM25 domains [J]. Appl Environ Microbiol, 2015, 81(19): 6610-6620.

    • 55

      Mihajlovski K R, Radovanović N R, Veljović Đ N, et al. Improved β-amylase production on molasses and sugar beet pulp by a novel strain Paenibacillus chitinolyticus CKS1 [J]. Ind Crops Prod, 2016, 80: 115-122.

    • 56

      Puspasari F, Radjasa O K, Noer A S, et al. Raw starch-degrading α-amylase from Bacillus aquimaris MKSC 6.2: isolation and expression of the gene, bioinformatics and biochemical characterization of the recombinant enzyme [J]. J Appl Microbiol, 2013, 114(1): 108-120.

    • 57

      Trivedi N, Gupta V, Kumar M, et al. Solvent tolerant marine bacterium Bacillus aquimaris secreting organic solvent stable alkaline cellulase [J]. Chemosphere, 2011, 83(5): 706-712.

    • 58

      Kumar E V, Srijana M, Kumar K K, et al. A novel serine alkaline protease from Bacillus altitudinis GVC11 and its application as a dehairing agent [J]. Bioprocess Biosyst Eng, 2011, 34(4): 403-409.

    • 59

      Madhuri A, Nagaraju B, Harikrishna N, et al. Production of alkaline protease by Bacillus altitudinis GVC11 using castor husk in solid-state fermentation [J]. Appl Biochem Biotechnol, 2012, 167(5): 1199-1207.

    • 60

      Adhyaru D N, Bhatt N S, Modi H A. Enhanced production of cellulase-free, thermo-alkali-solvent-stable xylanase from Bacillus altitudinis DHN8, its characterization and application in sorghum straw saccharification [J]. Biocatal Agric Biotechnol, 2014, 3(2): 182-190.

    • 61

      Mao S, Lu Z, Zhang C, et al. Purification, characterization, and heterologous expression of a thermostable β-1,3-1,4-glucanase from Bacillus altitudinis YC-9 [J]. Appl Biochem Biotechnol, 2013, 169(3): 960-975.

    • 62

      Joo H S, Choi J W. Purification and characterization of a novel alkaline protease from Bacillus horikoshii [J]. J Microbiol Biotechnol, 2012, 22(1): 58-68.

    • 63

      Song Y S, Seo D J, Kim K Y, et al. Expression patterns of chitinase produced from Paenibacillus chitinolyticus with different two culture media [J]. Carbohydr Polym, 2012, 90(2): 1187-1192.

    • 64

      Narhi L O, Fulco A J. Characterization of a catalytically self-sufficient 119,000-dalton cytochrome P-450 monooxygenase induced by barbiturates in Bacillus megaterium [J]. J Biol Chem, 1986, 261(16): 7160-7169.

    • 65

      Yu P, Chen Y.Purification and characterization of a novel neutral and heat-tolerant phytase from a newly isolated strain Bacillus nealsonii ZJ0702 [J]. BMC biotechnol, 2013, 13(1): 78.

    • 66

      Benešová E, Lipovová P, Dvořáková H, et al. α-L-fucosidase from Paenibacillus thiaminolyticus: its hydrolytic and transglycosylation abilities [J]. Glycobiol, 2013, 23(9): 1052-1065.

    • 67

      Benešová E, Lipovová P, Dvořáková H, et al. β-D-Galactosidase from Paenibacillus thiaminolyticus catalyzing transfucosylation reactions [J]. Glycobiol, 2009, 20(4): 442-451.

    • 68

      Tirrell I M, Wall J L, Daley C J, et al. YZGD from Paenibacillus thiaminolyticus, a pyridoxal phosphatase of the HAD (haloacid dehalogenase) superfamily and a versatile member of the Nudix (nucleoside diphosphate x) hydrolase superfamily [J]. Biochem J, 2006, 394(3): 665-674.

    • 69

      Li Y, Dong L, Wang L, et al. Cloning and characterization of gene cluster for biosynthesis of ectoine and 5-hydroxyectoine from extreme halotolerant actinomycete strain Prauserella alba YIM 90005T [J]. Acta microbiol Sin, 2011, 51(5): 603-608.

      李岩, 董雷, 王磊, 等. 极端耐盐放线菌白色普氏菌YIM 90005 T四氢嘧啶及5-羟基四氢嘧啶合成相关基因的克隆[J]. 微生物学报, 2011, 51(5): 603-608.

    • 70

      Cui X, Wang Y, Liu J, et al. Bacillus dabaoshanensis sp. nov., a Cr (VI)-tolerant bacterium isolated from heavy-metal-contaminated soil [J]. Arch Microbiol, 2015, 197(4): 513-520.

    • 71

      Venkateswaran K, Kempf M, Chen F, et al. Bacillus nealsonii sp. nov., isolated from a spacecraft-assembly facility, whose spores are γ-radiation resistant [J]. Int J Syst Evol Microbiol, 2003, 53(1): 165-172.

    • 72

      Jeong S W, Kim J. Psychrobacillus soli sp. nov., capable of degrading oil, isolated from oil-contaminated soil [J]. Int J Syst Evol Microbiol, 2015, 65(9): 3046-3052.

    • 73

      Lily M K, Bahuguna A, Bhatt K K, et al. Degradation of anthracene by a novel strain Brachybacterium paraconglomeratum BMIT637C (MTCC 9445) [J]. Int J Environ Sci, 2013, 3(4): 1242.

    • 74

      Azmatunnisa M, Rahul K, Subhash Y, et al. Bacillus oleivorans sp. nov., a diesel oil-degrading and solvent-tolerant bacterium [J]. Int J Syst Evol Microbiol, 2015, 65(4): 1310-1315.

    • 75

      Kuisiene N, Raugalas J, Spröer C, et al. Bacillus butanolivorans sp. nov., a species with industrial application for the remediation of n-butanol [J]. Int J Syst Evol Microbiol, 2008, 58(2): 505-509.

    • 76

      Pramila R, Padmavathy K, Ramesh K V, et al. Brevibacillus parabrevis, Acinetobacter baumannii and Pseudomonas citronellolis — potential candidates for biodegradation of low density polyethylene (LDPE) [J]. Afr J Bacteriol Res, 2012, 4(1): 9-14.

    • 77

      Esmaeili A, Pourbabaee A A, Alikhani H A, et al. Biodegradation of low-density polyethylene (LDPE) by mixed culture of Lysinibacillus xylanilyticus and Aspergillus niger in soil [J]. PLOS One, 2013, 8(9): e71720.

    • 78

      Sarkar A, Sar P, Islam E. Hexavalent chromium reduction by Microbacterium oleivorans A1: a possible mechanism of chromate-detoxification and-bioremediation [J]. Recent Pat Biotechnol, 2015, 9(2): 116-129.

    • 79

      Li T, Wang P, Wang P X. Microbial diversity in surface sediments of the Xisha Trough, the South China Sea [J]. Acta Ecol Sin, 2008, 28(3):1166-1173.

      李涛, 王鹏, 汪品先. 南海西沙海槽表层沉积物微生物多样性[J]. 生态学报, 2008, 28(3):1166-1173.

    • 80

      Xiao W, Yang Y L, Liu H W, et al. Culturable bacterial diversity of the ancient salt deposits in the Kunming Salt Mine, P. R. China [J]. Acta microbiol Sin, 2006, 46(6): 967-972.

      肖炜, 杨亚玲, 刘宏伟,等. 昆明盐矿古老岩盐沉积中可培养细菌多样性研究[J]. 微生物学报, 2006, 46(6):967-972.