不同培养基凝固剂对水体沉积物中可培养细菌多样性的影响
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摘要
【目的】研究琼脂和结冷胶两种培养基凝固剂对水体沉积物中可培养细菌多样性的影响,为挖掘环境微生物资源提供科学依据。【方法】以LB为基础培养基,分别以琼脂和结冷胶为凝固剂,利用纯培养法对江苏省太湖水体沉积物中的细菌进行分离培养,结合16S rRNA序列分析法对分离细菌进行多样性分析。【结果】以琼脂和结冷胶为凝固剂,分离获得的细菌总量分别为2.4×10~5和2.1×10~5CFU/g沉积物;16S r RNA序列分析剔除重复菌株后,对应得到20和21株细菌,分别隶属于11和17个细菌属,均归属于厚壁菌门(Firmicutes)、放线菌门(Actinobacteria)和变形菌门(Proteobacteria)三大类群。两种不同凝固剂培养基分离获得的细菌属组成差异明显,在LB琼脂培养基分离获得的20株细菌属为芽孢杆菌属(Bacillus,9株)、动性杆菌属(Planomicrobium,2株)、房间芽孢杆菌属(Domibacillus,1株)、类芽孢八叠球菌属(Paenisporosarcina,1株)、土壤芽孢杆菌属(Solibacillus,1株)、红球菌属(Rhodococcus,1株)、棒状杆菌属(Corynebacterium,1株)、微杆菌属(Microbacterium,1株)、无色杆菌属(Leucobacter,1株)、柠檬球菌属(Citricoccus,1株)和不动杆菌属(Acinetobacter,1株);在LB结冷胶培养基上分离获得的21株细菌属为芽孢杆菌属(4株)、赖氨酸芽孢杆菌属(Lysinibacillus,2株)、微小杆菌属(Exiguobacterium,1株)、鸟氨酸芽孢杆菌属(Ornithinibacillus,1株)、葡萄球菌属(Staphylococcus,1株)、虚构芽孢杆菌属(Fictibacillus,1株)、红球菌属(1株)、迪茨氏菌属(Dietzia,1株)、微杆菌属(1株)、考克氏菌属(Kocuria,1株)、四球菌属(Tessaracoccus,1株)、短杆菌属(Brevibacterium,1株)、两面神菌属(Janibacter,1株)、肠杆菌属(Enterobacter,1株)、依格纳季氏菌属(Ignatzschineria,1株)、副球菌属(Paracoccus,1株)和普罗维登斯菌属(Providencia,1株)。【结论】结冷胶作为凝固剂在一定程度上提高了可培养细菌的种类数,结冷胶和琼脂两种凝固剂联合使用可获得更多的细菌种类。
【Objective】The effects of two medium coagulators(agar and gellan gum)on diversity of cultivable bacteria in lake sediment were investigated to provide scientific basis for exploring the environmental microbial resources.【Method】By pure culture method on LB medium,agar and gellan gum were used as coagulants to isolate and culture the bacteria from sediments of Taihu Lake in Jiangsu. Diversity of the isolated bacteria was analyzed by 16 S rRNA sequencing.【Result】The bacterial quantities were detected as 2.4×10~5 and 2.1×10~5 CFU/(g sediment)on agar and gellan gum media respectively. Based on the 16S rRNA sequencing analysis,after the same strains were removed,20 and 21 strains were obtained respectively. Phylogenetic analysis results showed that they belonged to 11 and 17 genera,including Firmicutes,Actinobacteria and Proteobacteria. However,their bacteria compositions isolated by the two media were muchdifferent. The 20 strains isolated from LB agar medium were Bacillus(9 strains),Planomicrobium(2 strains),Domibacillus(1 strain),Paenisporosarcina(1 strain),Solibacillus(1 strain),Rhodococcus(1 strain),Corynebacterium(1 strain),Microbacterium(1 strain),Leucobacter(1 strain),Citricoccus(1 strain)and Acinetobacter(1 strain). And the 21 strains isolated on LB gellan gum medium were Bacillus(4 strains),Lysinibacillus(2 strains),Exiguobacterium(1 strain),Ornithinibacillus(1 strain),Staphylococcus(1 strain),Fictibacillus(1 strain),Rhodococcus(1 strain),Dietzia(1 strain),Microbacterium(1 strain),Kocuria(1 strain),Tessaracoccus(1 strain),Brevibacterium(1 strain),Janibacter(1 strain),Enterobacter(1 strain),Ignatzschineria(1 strain),Paracoccus(1 strain)and Providencia(1 strain).【Conclusion】Using gellan gum medium as coagulator can increase types of culturable bacteria at certain extents. Combining use of gellan gum and agar can obtain more becteria types.
【Objective】The effects of two medium coagulators(agar and gellan gum)on diversity of cultivable bacteria in lake sediment were investigated to provide scientific basis for exploring the environmental microbial resources.【Method】By pure culture method on LB medium,agar and gellan gum were used as coagulants to isolate and culture the bacteria from sediments of Taihu Lake in Jiangsu. Diversity of the isolated bacteria was analyzed by 16 S rRNA sequencing.【Result】The bacterial quantities were detected as 2.4×10~5 and 2.1×10~5 CFU/(g sediment)on agar and gellan gum media respectively. Based on the 16S rRNA sequencing analysis,after the same strains were removed,20 and 21 strains were obtained respectively. Phylogenetic analysis results showed that they belonged to 11 and 17 genera,including Firmicutes,Actinobacteria and Proteobacteria. However,their bacteria compositions isolated by the two media were muchdifferent. The 20 strains isolated from LB agar medium were Bacillus(9 strains),Planomicrobium(2 strains),Domibacillus(1 strain),Paenisporosarcina(1 strain),Solibacillus(1 strain),Rhodococcus(1 strain),Corynebacterium(1 strain),Microbacterium(1 strain),Leucobacter(1 strain),Citricoccus(1 strain)and Acinetobacter(1 strain). And the 21 strains isolated on LB gellan gum medium were Bacillus(4 strains),Lysinibacillus(2 strains),Exiguobacterium(1 strain),Ornithinibacillus(1 strain),Staphylococcus(1 strain),Fictibacillus(1 strain),Rhodococcus(1 strain),Dietzia(1 strain),Microbacterium(1 strain),Kocuria(1 strain),Tessaracoccus(1 strain),Brevibacterium(1 strain),Janibacter(1 strain),Enterobacter(1 strain),Ignatzschineria(1 strain),Paracoccus(1 strain)and Providencia(1 strain).【Conclusion】Using gellan gum medium as coagulator can increase types of culturable bacteria at certain extents. Combining use of gellan gum and agar can obtain more becteria types.
引文
蔡莹,李志江,田蕴,郑天凌.2010.虾池沉积物细菌多样性分析和若干可培养技术的优化探讨[J].厦门大学学报(自然科学版),49(5):731-737.[Cai Y,Li Z J,Tian Y,Zheng T L.2010.Bacterial diveisity in aquaculture environment and optimization of culturing technique[J].Journal of Xiamen University(Natural Science),49(5):731-737.]
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金一荻,冯福应,赵宇龙,张晓军.2012.基于结冷胶培养基的乌梁素海水体中可培养细菌的多样性研究[J].内蒙古农业大学学报,32(2):160-165.[Jin Y D,Feng FY,Zhao Y L,Zhang X J.2012.Cultivable bacteria diversity of Wuliangsuhai Lake by using gellan gum medium[J].Journal of Inner Mongolia Agricultural University,32(2):160-165.]
李晓丹,屈建航,周佳,张璐洁,李海峰,田海龙.2017.泥浸汁对太湖沉积物中的好氧可培养细菌多样性的影响[J].微生物学通报,44(3):554-560.[Li X D,Qu J H,Zhou J,Zhang L J,Li H F,Tian H L.2017.Effect of sediment extract on the culturable aerobic bacterial diversity in the sediments of Taihu Lake[J].Microbiology China,44(3):554-560.]
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唐婧,徐小蓉,商传禹,牛晓娟,张习敏,乙引.2015.南明河城区河段细菌多样性与环境因子的关系[J].微生物学报,55(8):1050-1059.[Tang J,Xu X R,Shang C Y,Niu X J,Zhang X M,Yi Y.2015.Association of bacterial diversity in city area of Nanming River with environmental factors[J].Acta Microbiologica Sinica,55(8):1050-1059.]
王福芳.2013.水体表层沉积物中细菌群落结构研究[D].郑州:河南工业大学.[Wang F F.2013.Study on characteristics of bacterial community structure of the interfacial water-sediment[D].Zhengzhou:Henan University of Technology.]
薛银刚,刘菲,江晓栋,耿金菊,滕加泉,谢文理,张皓,陈心一.2018.太湖不同湖区冬季沉积物细菌群落多样性[J].中国环境科学,38(2):719-728.[Xue Y G,Liu F,Jiang X D,Geng J J,Teng J Q,Xie W L,Zhang H,Chen X Y.2018.The diversity of bacterial communities in the sediment of different lake zones of Lake Taihu in winter[J].China Environmental Science,38(2):719-728.]
杨江科,程占冰.2016.富营养化城市湖泊武汉东湖沉积物微生物群体结构及空间变化研究[J].微生物学报,56(6):943-955.[Yang J K,Cheng Z B.2016.Community composition and spatial variation of bacteria in the sediments of a eutrophic fresh water urban lake,East Lake,Wuhan,China[J].Acta Microbiologica Sinica,56(6):943-955.]
张瑞杰,张琼琼,黄兴如,郭逍宇.2016.基于分离培养方法的香蒲根内生细菌多样性[J].湿地科学,14(2):201-210.[Zhang R J,Zhang Q Q,Huang X R,Guo X Y2016.Diversity of endophytic bacteria in roots of Typha orientalis estimated by culture method[J].Wetland Science,14(2):201-210.]
Amann R I,Ludwig W,Schleifer K H.1995.Phylogenetic identification and in situ detection of individual microbia cells without cultivation[J].Microbiological Reviews,59(1):143-169.
Bollmann A,Palumbo A V,Lewis K,Epstein S S.2010Isolation and physiology of bacteria from contaminated subsurface sediments[J].Applied and Environmental Microbiology,76(22):7413-7419.
Cho J C,Giovannoni S J.2004.Cultivation and growth characteristics of a diverse group of oligotrophic marine Gammaproteobacteria[J].Applied and Environmental Microbiology,70(1):432-440.
Davis K E R,Shayne J,Janssen P H.2005.Effects of growth medium,inoculum size,and incubation time on culturability and isolation of soil bacteria[J].Applied and Environmental Microbiology,71(2):826-834.
Hashimoto T,Koga M,Masaoka Y.2009.Advantages of a diluted nutrient broth medium for isolating N2-producing denitrifying bacteria ofα-proteobacteria in surface and subsurface upland soils[J].Soil Science and Plant Nutrition,55(5):647-659.
Janssen P H,Yates P S,Grinton B E,Taylor P M,Sait M2002.Improved culturability of soil bacteria and isolation in pure culture of novel members of the divisions Acidobacteria,Actinobacteria,Proteobacteria,and Verrucomicrobia[J].Applied and Environmental Microbiology,68(5):2391-2396.
Moreno C,Romero J,Espejo R T.2002.Polymorphism in repeated 16S rRNA genes is a common property of type strains and environmental isolates of the genus Vibrio[J].Microbiology,148(4):1233-1239.
Ringeisen B R,Rincon K,Fitzgerald L A,Fulmer P A.Wu P K.2015.Printing soil:A single-step,high-throughput method to isolate micro-organisms and near-neighbour microbial consortia from a complex environmental sample[J].Methods in Ecology and Evolution,6(2):209-217.
Rygaard A M,Th?gersen M S,Nielsen K F,Gram L,Bentzontilia M.2017.Effects of gelling agent and extracellular signaling molecules on the culturability of marine bacteria[J].Applied and Environmental Microbiology,doi:10.1128/AEM.00243-17.
Sauvain L,Bueche M,Junier T,Matthieu,Wunderlin T,Milleret R K,Diez E G,Loizeau G L,Waeber M L T,Junier P.2014.Bacterial communities in trace metal contaminated lake sediments are dominated by endosporeforming bacteria[J].Aquatic Sciences,76(1):33-46.
Sipkema D,Schippers K,Maalcke W J,Yu Y,Salim S,Blanch H W.2011.Multiple approaches to enhance the cultivability of bacteria associated with the marine sponge Haliclona(gellius)sp.[J].Applied and Environmental Microbiology,77(6):2130-2140.
Suzuki S I,Okuda T,Komatsubara S.1999.Selective isolation and distribution of sporichthya strains in soil[J].A-pplied and Environmental Microbiology,65(5):1930-1935.
Tamaki H,Hanada S,Sekiguchi Y,Tanaka Y,Kamagata Y.2009.Effect of gelling agent on colony formation in solid cultivation of microbial community in lake sediment[J].Environmental Microbiology,11(7):1827-1834.
Zhang L G,Du J H,Yang J.2018.Low acyl gellan gum as a gelling agent in medium of saccharomyces yeasts[J].International Journal of Food Engineering,doi:10.1515/ijfe-2017-0292.
陈泽斌,夏振远,雷丽萍,黄鹤平,张永福,莫丽玲,靳松.2014.云南烟草内生细菌菌群密度及分布特征[J].西南农业学报,27(2):682-687.[Chen Z B,Xia Z Y,Lei LP,Huang H P,Zhang Y F,Mo L L,Jin S.2014.Study on population density and distribution characteristics of endophytic bacteria in tobacco in Yunnan[J].Southwest China Journal of Agricultural Sciences,27(2):682-687.]
金一荻,冯福应,赵宇龙,张晓军.2012.基于结冷胶培养基的乌梁素海水体中可培养细菌的多样性研究[J].内蒙古农业大学学报,32(2):160-165.[Jin Y D,Feng FY,Zhao Y L,Zhang X J.2012.Cultivable bacteria diversity of Wuliangsuhai Lake by using gellan gum medium[J].Journal of Inner Mongolia Agricultural University,32(2):160-165.]
李晓丹,屈建航,周佳,张璐洁,李海峰,田海龙.2017.泥浸汁对太湖沉积物中的好氧可培养细菌多样性的影响[J].微生物学通报,44(3):554-560.[Li X D,Qu J H,Zhou J,Zhang L J,Li H F,Tian H L.2017.Effect of sediment extract on the culturable aerobic bacterial diversity in the sediments of Taihu Lake[J].Microbiology China,44(3):554-560.]
萨姆布鲁克J,拉塞尔D W.2002.分子克隆实验指南[M].第3版.黄培堂,译.北京:科学出版社:27.[Sambrook J,Russell D W.2002.Molecular Cloning:A Laboratory Manual[M].The 3rdEdition.Translated by Huang P T.Beijing:Science Press:27.]
唐婧,徐小蓉,商传禹,牛晓娟,张习敏,乙引.2015.南明河城区河段细菌多样性与环境因子的关系[J].微生物学报,55(8):1050-1059.[Tang J,Xu X R,Shang C Y,Niu X J,Zhang X M,Yi Y.2015.Association of bacterial diversity in city area of Nanming River with environmental factors[J].Acta Microbiologica Sinica,55(8):1050-1059.]
王福芳.2013.水体表层沉积物中细菌群落结构研究[D].郑州:河南工业大学.[Wang F F.2013.Study on characteristics of bacterial community structure of the interfacial water-sediment[D].Zhengzhou:Henan University of Technology.]
薛银刚,刘菲,江晓栋,耿金菊,滕加泉,谢文理,张皓,陈心一.2018.太湖不同湖区冬季沉积物细菌群落多样性[J].中国环境科学,38(2):719-728.[Xue Y G,Liu F,Jiang X D,Geng J J,Teng J Q,Xie W L,Zhang H,Chen X Y.2018.The diversity of bacterial communities in the sediment of different lake zones of Lake Taihu in winter[J].China Environmental Science,38(2):719-728.]
杨江科,程占冰.2016.富营养化城市湖泊武汉东湖沉积物微生物群体结构及空间变化研究[J].微生物学报,56(6):943-955.[Yang J K,Cheng Z B.2016.Community composition and spatial variation of bacteria in the sediments of a eutrophic fresh water urban lake,East Lake,Wuhan,China[J].Acta Microbiologica Sinica,56(6):943-955.]
张瑞杰,张琼琼,黄兴如,郭逍宇.2016.基于分离培养方法的香蒲根内生细菌多样性[J].湿地科学,14(2):201-210.[Zhang R J,Zhang Q Q,Huang X R,Guo X Y2016.Diversity of endophytic bacteria in roots of Typha orientalis estimated by culture method[J].Wetland Science,14(2):201-210.]
Amann R I,Ludwig W,Schleifer K H.1995.Phylogenetic identification and in situ detection of individual microbia cells without cultivation[J].Microbiological Reviews,59(1):143-169.
Bollmann A,Palumbo A V,Lewis K,Epstein S S.2010Isolation and physiology of bacteria from contaminated subsurface sediments[J].Applied and Environmental Microbiology,76(22):7413-7419.
Cho J C,Giovannoni S J.2004.Cultivation and growth characteristics of a diverse group of oligotrophic marine Gammaproteobacteria[J].Applied and Environmental Microbiology,70(1):432-440.
Davis K E R,Shayne J,Janssen P H.2005.Effects of growth medium,inoculum size,and incubation time on culturability and isolation of soil bacteria[J].Applied and Environmental Microbiology,71(2):826-834.
Hashimoto T,Koga M,Masaoka Y.2009.Advantages of a diluted nutrient broth medium for isolating N2-producing denitrifying bacteria ofα-proteobacteria in surface and subsurface upland soils[J].Soil Science and Plant Nutrition,55(5):647-659.
Janssen P H,Yates P S,Grinton B E,Taylor P M,Sait M2002.Improved culturability of soil bacteria and isolation in pure culture of novel members of the divisions Acidobacteria,Actinobacteria,Proteobacteria,and Verrucomicrobia[J].Applied and Environmental Microbiology,68(5):2391-2396.
Moreno C,Romero J,Espejo R T.2002.Polymorphism in repeated 16S rRNA genes is a common property of type strains and environmental isolates of the genus Vibrio[J].Microbiology,148(4):1233-1239.
Ringeisen B R,Rincon K,Fitzgerald L A,Fulmer P A.Wu P K.2015.Printing soil:A single-step,high-throughput method to isolate micro-organisms and near-neighbour microbial consortia from a complex environmental sample[J].Methods in Ecology and Evolution,6(2):209-217.
Rygaard A M,Th?gersen M S,Nielsen K F,Gram L,Bentzontilia M.2017.Effects of gelling agent and extracellular signaling molecules on the culturability of marine bacteria[J].Applied and Environmental Microbiology,doi:10.1128/AEM.00243-17.
Sauvain L,Bueche M,Junier T,Matthieu,Wunderlin T,Milleret R K,Diez E G,Loizeau G L,Waeber M L T,Junier P.2014.Bacterial communities in trace metal contaminated lake sediments are dominated by endosporeforming bacteria[J].Aquatic Sciences,76(1):33-46.
Sipkema D,Schippers K,Maalcke W J,Yu Y,Salim S,Blanch H W.2011.Multiple approaches to enhance the cultivability of bacteria associated with the marine sponge Haliclona(gellius)sp.[J].Applied and Environmental Microbiology,77(6):2130-2140.
Suzuki S I,Okuda T,Komatsubara S.1999.Selective isolation and distribution of sporichthya strains in soil[J].A-pplied and Environmental Microbiology,65(5):1930-1935.
Tamaki H,Hanada S,Sekiguchi Y,Tanaka Y,Kamagata Y.2009.Effect of gelling agent on colony formation in solid cultivation of microbial community in lake sediment[J].Environmental Microbiology,11(7):1827-1834.
Zhang L G,Du J H,Yang J.2018.Low acyl gellan gum as a gelling agent in medium of saccharomyces yeasts[J].International Journal of Food Engineering,doi:10.1515/ijfe-2017-0292.