Biodiversity Science ›› 2013, Vol. 21 ›› Issue (1): 28-37.doi: 10.3724/SP.J.1003.2013.10097

• Orginal Article • Previous Article     Next Article

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:2013-02-04
  • Wang Jianxin

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

Fig. 1

Direct observation of the sediment from East China Sea surface by the method of buried piece to find the distribution of microorganisms and organics in situ. Red arrows indicate microorganisms, and green arrows indicate organics."

Table 1

Statistics of colony-forming units in four kinds of medias by the dilution method of plate counting"

Dilution multiple
No. of plate culture count
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

Fig. 2

Distribution of the different genus. Sb, Sporolac- tobacillus; Hb,Halobacillus; Bb, Brevibacillus; Bi, Bacillus; Pb, Paenibacillus; Plb, Pimelobacter; Bbm, Brachybacterium; Ab, Arthrobactor; Sl, Staphylococcus; Eo, Enterococcus; Mo, Marinococcus; Pt, Pasteurella; Vb, Vibrio; Ao, Alteromonas; Pd, Pseudomonas; Ep, Erysipelothrix; Am, Agromyces; Aib, Aneurinibacillus; St, Streptococcus; Aom, Actinomyces."

Table 2

Identification of pure-cultured bacteria isolated from East China Sea surface sediment by bacterial 16S rDNA gene"

Strain no.
Accession no.
Closest species (Accession no.)
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

Fig. 3

Phylogenetic tree based on 16S rDNA sequences of pure-cultured clones from East China Sea surface sediment"

Table 3

Taxonomic affiliation of clones recovered in the bacterial clone libraries from East China Sea surface sediment and analysis of closest species origin"

Clones no.
Accession no.
Closest species (Accession no.)
Closest species origin
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

Fig. 4

Phylogenetic tree based on 16S rDNA sequences of uncultured clones from the sediment"

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