生物多样性 ›› 2013, Vol. 21 ›› Issue (1): 28-37.doi: 10.3724/SP.J.1003.2013.10097

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东海表层沉积物纯培养与非培养细菌多样性

何建瑜, 刘雪珠, 赵荣涛, 吴方伟, 王健鑫*()   

  1. 浙江海洋学院海洋生物资源与分子工程实验室, 浙江舟山 316000
  • 收稿日期:2012-04-11 接受日期:2012-08-10 出版日期:2013-01-20
  • 通讯作者: 王健鑫 E-mail:zswjx2575@163.com
  • 基金项目:
    国家自然科学基金(31270160);浙江省自然科学基金(LY12C03003);浙江省科技厅公益性技术应用研究项目(2011C31017);舟山市科技局海洋类项目(10248);浙江省大学生科技创新项目(2012R411002)

Diversity of cultured and uncultured bacteria in surface layer sediment from the East China Sea

Jianyu He, Xuezhu Liu, Rongtao Zhao, Fangwei Wu, Jianxin Wang*()   

  1. Laboratory for Marine Living Resources and Molecular Engineering, Zhejiang Ocean University, Zhoushan, Zhejiang 316000
  • Received:2012-04-11 Accepted:2012-08-10 Online:2013-01-20
  • Contact: Wang Jianxin E-mail:zswjx2575@163.com

于2011年7月采集了浙江舟山沿岸海底沉积物样品(122°10′41″ E, 29°49′7″ N), 通过埋片原位观察、荧光显微计数、纯培养菌分离以及非培养细菌构建克隆文库的方法, 分析和研究了东海表层沉积物细菌群落结构及其多样性。埋片原位观察和荧光显微计数法的结果表明: 沉积物样品中细菌的细胞数量为(9.30±3.44) ×107 cells/g; 通过分离纯化共获得313株细菌, 分属于20个属, 4种培养基对细菌的分离效果依次为RO>M1>Zobell 2216>MR2A; 常规形态学与生理生化鉴定结果显示芽孢杆菌属(Bacillus)和海球菌属(Marinococcus)从数量和种类上皆为优势菌属, 分别占分离菌株总数的21.08%和17.25%; 对73株典型海洋细菌进行16S rDNA分子鉴定发现, 厚壁菌门(57.5%)、γ-变形杆菌纲(32.9%)、黄杆菌纲(4.1%)和放线菌纲(5.5%)等为主要类群。非培养细菌克隆文库序列分析发现: 细菌克隆子主要属于厚壁菌门和变形杆菌门, 而芽孢杆菌纲和γ-变形杆菌纲是上述两个门的优势类群。综合纯培养与非培养数据得出东海海域表层沉积物细菌多样性丰富, 具有进一步开发研究的价值。

关键词: 东海海域, 海洋细菌, 16S rDNA, 群落结构, 多样性

Marine bacteria are an extremely important component of marine bioresources. We studied the community structure and diversity of cultured and uncultured bacteria in surface layer sediment sampled from the East China Sea (122°10′41″ E, 29°49′7″ N) by buried piece observation, fluorescence enumeration, pure culture techniques and 16S rDNA clone library analysis. The average abundance of bacteria was (9.30±3.44)×107 cells/g according to fluorescence microscopy enumeration and observation of buried slides. A total of 313 bacterial strains belonging to 20 genera was obtained from four kinds of culture medium and the separation efficiency of bacteria ranked as RO>M1>Zobell 2216>MR2A. In terms of morphology, physiology and biochemistry, Bacillus (21.08%) and Marinococcus (17.25%) were shown to be the most dominant and highly diverse bacteria. The analysis of 16S rDNA sequence from 73 representative bacteria showed most clones were affiliated with Firmicutes (57.5%): the other marine bacteria including gamma-Proteobacteria (32.9%), Flavobacteria (4.1%), and Actinobacteria (5.5%). Most clones were related to uncultivated bacterial clones retrieved from surface layer sediment and contained several highly divergent 16S rDNA gene sequences. After constructing a molecular library and phylogenetic analysis, the sequenced clones were shown to fall into two phyla of the domain Bacteria, Firmicutes and Proteobacteria, in which Bacillus and gamma-proteobacteria were the dominant groups, respectively. The results revealed that the species and community structure of bacteria in surface layer sediment from the East China Sea continental shelf were abundant and diverse, thus warranting further research and exploitation.

Key words: East China Sea, bacteria, 16S rDNA, community structure, diversity

图1

采用埋片观察法得到的东海表层沉积物样品中微生物和有机质的原位分布图。红色箭头为微生物菌体, 绿色箭头为有机杂质。"

表1

在4种培养基中利用稀释平板计数的方法统计纯培养的菌落单元总量"

培养基
Medium
稀释倍数
Dilution multiple
平板菌落数
No. of plate culture count
菌落单元数量
Numbers of colony- forming units(CFU/g)
M1 1,000 73 (7.30±.0.14)×104
Zobell 2216 1,000 71 (7.10±0.08)×104
MR2A 1,000 59 (5.90±0.09)×104
RO 10,000 49 (4.90±0.17)×105

图2

不同菌属菌株数量分布图。Sb: 芽孢乳杆菌属; Hb: 盐芽孢杆菌属; Bb: 短芽孢杆菌属; Bi: 芽孢杆菌属; Pb: 类芽孢杆菌属; Plb: 脂肪杆菌属; Bbm: 短状杆菌属; Ab: 节杆菌属; Sl: 葡萄球菌属; Eo: 肠球菌属; Mo: 海球菌属; Pt: 巴斯德氏菌属; Vb: 弧菌属; Ao: 交替单胞菌属; Pd: 假单胞菌属; Ep: 丹毒丝菌属; Am: 壤霉菌属; Aib: 硫胺素芽孢杆菌属; St: 链球菌属; Aom: 放线菌属"

表2

通过细菌16S rDNA基因序列对东海表层沉积物分离得到的纯培养细菌鉴定结果"

代表菌株
Strain no.
登录号
Accession no.
相似菌株(登录号)
Closest species (Accession no.)
相似性
Homology (%)
芽孢杆菌纲 Bacilli
DHC01 JQ904712 Bacillus pumilus strain PR13 (JQ435673) 99
DHC02 JQ904713 Bacterium YCG4 (JF775413) 97
DHC03 JQ904714 Bacillus tequilensis strain KM35 (JF411314) 99
DHC04 JQ904715 Bacillus sp. M71_S53 (FM992830) 100
DHC06 JQ904717 Bacillus cereus strain CB-10 (JF496530) 96
DHC07 JQ904718 Bacillus subtilis strain B7 (JQ086378) 100
DHC08 JQ904719 Bacillus horikoshii strain NBSL26 (FJ984529) 99
DHC10 JQ904720 Bacillus sp. RHS-str.320 (HE586887) 97
DHC11 JQ904721 Bacillus infantis strain AL10 (JF411305) 99
DHC12 JQ904722 Bacillus aryabhattai isolate PSB59 (HQ242772) 100
DHC13 JQ904723 Bacillus barbaricus strain P2 (GQ200827) 99
DHC14 JQ904724 Bacillus gibsonii strain S-2 (AY737309) 99
DHC15 JQ904726 Paenibacillus sp. GP26-03 (AM162316) 99
黄杆菌纲 Flavobacteria
DHC05 JQ904716 Cloacibacterium normanense strain tu33 (FJ544402) 100
放线菌纲 Actinobacteria
DHC09 JQ904725 Brevibacterium sp. 210_14 (GQ199716) 99
γ-变形菌纲 Gamma-proteobacteria
DHC16 JQ904727 Alteromonas sp. SCSWC10 (FJ461444) 99
DHC17 JQ904728 Pseudoalteromonas sp. B296 (FN295786) 98
DHC18 JQ904729 Pseudoalteromonas sp. VS-2 (FJ497709) 97
DHC19 JQ904730 Vibrio algoinfesta strain NBRC 14665 (AB680645) 99
DHC20 JQ904731 Vibrio harveyi EHP8 (FJ227114) 99
DHC21 JQ904732 Vibrio azureus strain CAIM 1457 (JN603238) 99
DHC22 JQ904733 Vibrio parahaemolyticus strain CM12 (EU660326) 99

图3

根据东海表层沉积物中纯培养得到的细菌菌株16S rDNA基因序列构建的系统发育树"

表3

东海表层沉积物非培养细菌的16S rDNA序列在数据库中的同源分类比较以及相似菌株的分离源分析"

代表克隆子
Clones no.
登录号
Accession no.
相似菌株(登录号)
Closest species (Accession no.)
相似菌株分离源
Closest species origin
相似性
Homology (%)
芽孢杆菌纲 Bacilli
DHUC01 JQ904734 Bacillus cereus strain E22(GU826149 ) 巴西土壤 Brazilian soils 99
DHUC02 JQ904735 Bacillus sp. C1(JN571045) 人造草坪土壤 Chinampa soils 98
DHUC03 JQ904736 Bacillus aquimaris strain T172 (HQ647277) 东乡野生水稻 Dongxiang wild rice 99
DHUC04 JQ904737 Bacillus subtilis strain MSI-9 (JN993708) 人参土壤 Ginseng soil 98
DHUC05 JQ904738 Bacillus megaterium strain XAS5-1 (JF496312) 生物土壤结皮 Biological soil crusts 100
DHUC06 JQ904739 Bacillus subterraneus strain DSM13966T (FR733689) 普通土壤 Normal soil 97
DHUC07 JQ904746 Bacillus barbaricus strain P2 (GQ200827) 盐田 Solar salterns 94
DHUC08 JQ904740 Uncultured Bacillus sp. (HQ397100) 普通土壤 Normal soil 100
DHUC09 JQ904741 Geobacillus stearothermophilus strain DoB50 (JQ359102) 铁皮石斛 Dendrobium officinale 99
DHUC10 JQ904747 Brevibacillus sp. 100-5 (FJ959373) 麦田土壤 Wheat field soil 94
α-变形菌纲 Alpha-proteobacteria
DHUC11 JQ904742 Uncultured alpha-proteobacterium (FJ205181) 不活动的热液区沉积物 Inactive hydrothermal field sediments 95
γ-变形菌纲 Gamma-proteobacteria
DHUC12 JQ904743 Uncultured Alteromonas sp. (HQ012278) 海洋 Marine 100
DHUC13 JQ904744 Vibrio harveyi strain EHP8(FJ227114) 枪乌贼发光器官 Loliginid squid light organs 100
DHUC14 JQ904745 Vibrio hepatarius (NR025491) 水生动物和海洋环境 Aquatic animals and the marine environment 98

图4

由东海表层沉积物中非培养细菌菌株的16S rDNA基因序列构建系统发育树"

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