南海深海氮循环微生物的原位培养与多样性分析
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摘要
借助自主研发的深海水体原位定植培养系统,在南海3 300 m的深海水体中进行了氮循环微生物的原位培养.通过向富集仓中投加缓释肥(铵盐、硝酸盐和尿素)进行了为期17个月的原位富集.结合高通量测序技术和分离培养的方法,对深海原位富集物及实验室二次富集物进行了微生物多样性分析. 16S rRNA基因高通量测序结果表明,在南海原位富集样品中,细菌以变形菌门丰度最高,富集仓内脱脂棉附着介质和仓内水样中的最优势属分别为希瓦氏菌属(Shewanella)和科韦尔氏菌属(Colwellia);古菌以奇古菌门为主,其中氨氧化古菌(AOA)所占比例很高.通过平板培养分离到17株细菌,主要包括盐单胞菌属(Halomonas)、海杆菌属(Marinobacter)和亚硫酸盐杆菌属(Sulfitobacter)等.通过不同起始氮源(氨氮、硝酸盐、亚硝酸盐)和培养温度(28℃和10℃),对原位富集样品进行实验室二次富集,获得了5个富集菌群.分析发现,它们均具有较好的脱氮效果,可以产生N_2O或N_2;这些菌群均以盐单胞菌属占绝对优势,其次为海源菌属(Idiomarina)或海杆菌属,其中盐单胞菌和海源菌成功获得分离.单菌功能验证表明,分离获得的盐单胞菌具有好氧反硝化能力,其在原位环境下反硝化活性尚有待确认.
In this study,an in-situ enrichment in depth of 3 300 m in the South China Sea was newly developed for17 months by adding slowly releasing ammonium,nitrate and urea into a circulation chamber of a deep-sea water incubation system. The response of microbial community was analyzed combining with Miseq high-throughput sequencing technology and the lab seperation. Our results showed that γ-Proteobacteria is the most abundant in thebacterial population. The bacterial composition on genus level differed obviously in the cotton samples and water samples in the circulation chamber,where Shewanella spp. and Colwellia spp. are the main members,respectively. Thaumarchaeota dominated in the archaea domain with a high proportion of the ammonia oxidizing archaea( AOA). Culture results showed that a total of 17 bacterial strains were isolated from the in-situ samples,including Halomonas,Marinobacter,Sulfitobacter and so on. The in-situ enrichment in the South China Sea was further selectively enriched in laboratory under different initial nitrogen sources( ammonia,nitrate,nitrite) and temperatures( 28℃ and 10℃). A total of five secondary enrichments were obtained. The enriched consortia exhibited a strong nitrogen removal capacity,by releasing N_2O or N_2 as the end products,indicating their denitrification ability. Bacterial community analysis of the secondary enrichments showed that Halomonas became the dominant bacteria,followed by Marinobacter and Idiomarina. Among them Halomonas and Idiomarina were successfully isolated and Halomonas was further confirmed of aerobic denitrification ability.
In this study,an in-situ enrichment in depth of 3 300 m in the South China Sea was newly developed for17 months by adding slowly releasing ammonium,nitrate and urea into a circulation chamber of a deep-sea water incubation system. The response of microbial community was analyzed combining with Miseq high-throughput sequencing technology and the lab seperation. Our results showed that γ-Proteobacteria is the most abundant in thebacterial population. The bacterial composition on genus level differed obviously in the cotton samples and water samples in the circulation chamber,where Shewanella spp. and Colwellia spp. are the main members,respectively. Thaumarchaeota dominated in the archaea domain with a high proportion of the ammonia oxidizing archaea( AOA). Culture results showed that a total of 17 bacterial strains were isolated from the in-situ samples,including Halomonas,Marinobacter,Sulfitobacter and so on. The in-situ enrichment in the South China Sea was further selectively enriched in laboratory under different initial nitrogen sources( ammonia,nitrate,nitrite) and temperatures( 28℃ and 10℃). A total of five secondary enrichments were obtained. The enriched consortia exhibited a strong nitrogen removal capacity,by releasing N_2O or N_2 as the end products,indicating their denitrification ability. Bacterial community analysis of the secondary enrichments showed that Halomonas became the dominant bacteria,followed by Marinobacter and Idiomarina. Among them Halomonas and Idiomarina were successfully isolated and Halomonas was further confirmed of aerobic denitrification ability.
引文
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[17] WANG Q,GARRITY G M,TIEDJE J M,et al. Naive Bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy[J]. Applied and Environmental Microbiology,2007,73(16):5 261-5 267.
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[21] SAUDER L A,ALBERTSEN M,ENGEL K,et al. Cultivation and characterization of Candidatus Nitrosocosmicus exaquare,an ammonia-oxidizing archaeon from a municipal wastewater treatment system[J]. The ISME Journal,2017,11(5):1 142-1 157.
[22] KNNEKE M,BERNHARD A E,JR D L T,et al. Isolation of an autotrophic ammonia-oxidizing marine archaeon[J]. Nature,2005,437(7 058):543-546.
[23] XIAO X,WANG P,ZENG X,et al. Shewanella psychrophila sp. nov. and Shewanella piezotolerans sp. nov.,isolated from west Pacific deep-sea sediment[J]. International Journal of Systematic and Evolutionary Microbiology,2007,57(1):60-65.
[24] KUSUBE M,KYAW T S,TANIKAWA K,et al. Colwellia marinimaniae sp. nov.,a hyperpiezophilic species isolated from an amphipod within the Challenger Deep,Mariana Trench[J]. International Journal of Systematic and Evolutionary Microbiology,2017,67(4):824-831.
[25] XU Y,NOGI Y,KATO C,et al. Moritella profunda sp. nov. and Moritella abyssi sp. nov.,two psychropiezophilic organisms isolated from deep Atlantic sediments[J]. International Journal of Systematic and Evolutionary Microbiology,2003,53(2):533-538.
[26] GUO Y,ZHOU X,LI Y,et al. Heterotrophic nitrification and aerobic denitrification by a novel Halomonas campisalis[J]. Biotechnology Letters,2013,35(12):2 045-2 049.
[27] DECLEYRE H,HEYLEN K,VAN COLEN C,et al. Dissimilatory nitrogen reduction in intertidal sediments of a temperate estuary:small scale heterogeneity and novel nitrate-to-ammonium reducers[J]. Frontiers in Microbiology,2015,6:1 124.
[28] LIU Y,AI G M,MIAO L L,et al. Marinobacter strain NNA5,a newly isolated and highly efficient aerobic denitrifier with zero N2O emission[J]. Bioresource Technology,2016,206:9-15.
[29] CHOI A,CHO H,KIM S H,et al. Rates of N2,production and diversity and abundance of functional genes associated with denitrification and anaerobic ammonium oxidation in the sediment of the Amundsen Sea Polynya,Antarctica[J]. Deep Sea Research Part II Topical Studies in Oceanography,2016,123(4):113-125.
[30] GONZLEZDOMENECH C M,MARTNEZCHECA F,BJAR V,et al. Denitrification as an important taxonomic marker within the genus Halomonas[J]. Systematic and Applied Microbiology,2010,33(2):85-93.
[2]郑天凌,田蕴,苏建强.海洋微生物研究的回顾与展望[J].厦门大学学报(自然科学版),2006,45(s2):150-157.
[3] COLLIER B. Analysis of particulate matter collected by sediment trapsand from sediment gores[J]. Geophysical Monograph,1991,63:235-242.
[4] SMITH C R,HOOVER D J,DOAN S E. Phytodetritus at the abyssal sea-floor across 10°of latitude in the central equatorial Pacific[J]. Deep-Sea Research Part II-Topical Studies in Oceanography,1996,43:1 309-1 338.
[5] VOSS M,BANGE H W,DIPPNER J W,et al. The marine nitrogen cycle:recent discoveries,uncertainties and the potential relevance of climate change[J]. Philosophical Transactions of the Royal Society of London,2013,368(1 621):20130121.
[6] XU M,ZHANG Q,XIA C,et al. Elevated nitrate enriches microbial functional genes for potential bioremediation of complexly contaminated sediments[J]. ISME Journal,2014,8(9):1 932-1 944.
[7] HARRISON W G,DOUGLAS D,FALKOWSKI P,et al. Summer nutrient dynamics of the Middle Atlantic Bight:nitrogen uptake and regeneration[J]. Journal of Plankton Research,1983,5(4):539-556.
[8] LAM P,LAVIK G,JENSEN M M,et al. Revising the nitrogen cycle in the Peruvian oxygen minimum zone[J]. Proceedings of the National Academy of Sciences,2009,106(12):4 752-4 757.
[9] PADILLA C C. unrecognized diversity of microbes linking methanotrophy to nitrogen loss in marine oxygen minimum zones[D].Atlanta:Georgia Institute of Technology,2017.
[10] YU T,LI M,NIU M,et al. Difference of nitrogen-cycling microbes between shallow bay and deep-sea sediments in the South China Sea[J]. Applied Microbiology and Biotechnology,2018. 102(1):447-459.
[11]白洁,李海艳,张健,等.黄海西北部沉积物中细菌群落16S r DNA多样性解析[J].中国环境科学,2009,29(12):1 277-1 284.
[12]龚骏,张晓黎.微生物在近海氮循环过程的贡献与驱动机制[J].微生物学通报,2013,40(1):44-58.
[13] LIU J,FU B,YANG H,et al. Phylogenetic shifts of bacterioplankton community composition along the Pearl Estuary:the potential impact of hypoxia and nutrients[J]. Frontiers in Microbiology,2015,6:64.
[14] STEFFEN M M,LI Z,EFFLER T C,et al. Comparative metagenomics of toxic freshwater cyanobacteria bloom communities on two continents[J]. PLOS ONE,2012,7(8):e44002.
[15] MAUGHAN H,WANG P W,CABALLERO J D,et al. Analysis of the cystic fibrosis lung microbiota via serial Illumina sequencing of bacterial 16S rRNA hypervariable regions[J]. PLOS ONE,2012,7(10):e45791.
[16] OBERAUNER L,ZACHOW C,LACKNER S,et al. The ignored diversity:complex bacterial communities in intensive care units revealed by 16S pyrosequencing[J]. Scientific Reports,2013,3:1 413.
[17] WANG Q,GARRITY G M,TIEDJE J M,et al. Naive Bayesian classifier for rapid assignment of rRNA sequences into the new bacterial taxonomy[J]. Applied and Environmental Microbiology,2007,73(16):5 261-5 267.
[18]国家海洋局.海洋调查规范第四部分:海水化学要素调查:GB/T 12763. 4—2007[S].北京:中国标准出版社,2007.
[19] LEHTOVIRTA-MORLEY L E,STOECKER K,VILCINSKAS A,et al. Cultivation of an obligate acidophilic ammonia oxidizer from a nitrifying acid soil[J]. Proceedings of the National Academy of Sciences of the United States of America,2011,108(38):15 892-15 897.
[20] SANTORO A E,DUPONT C L,RICHTER R A,et al. Genomic and proteomic characterization of“Candidatus Nitrosopelagicus brevis”:An ammonia-oxidizing archaeon from the open ocean[J]. Proceedings of the National Academy of Science of the United States of America,2015,112(4):1 173.
[21] SAUDER L A,ALBERTSEN M,ENGEL K,et al. Cultivation and characterization of Candidatus Nitrosocosmicus exaquare,an ammonia-oxidizing archaeon from a municipal wastewater treatment system[J]. The ISME Journal,2017,11(5):1 142-1 157.
[22] KNNEKE M,BERNHARD A E,JR D L T,et al. Isolation of an autotrophic ammonia-oxidizing marine archaeon[J]. Nature,2005,437(7 058):543-546.
[23] XIAO X,WANG P,ZENG X,et al. Shewanella psychrophila sp. nov. and Shewanella piezotolerans sp. nov.,isolated from west Pacific deep-sea sediment[J]. International Journal of Systematic and Evolutionary Microbiology,2007,57(1):60-65.
[24] KUSUBE M,KYAW T S,TANIKAWA K,et al. Colwellia marinimaniae sp. nov.,a hyperpiezophilic species isolated from an amphipod within the Challenger Deep,Mariana Trench[J]. International Journal of Systematic and Evolutionary Microbiology,2017,67(4):824-831.
[25] XU Y,NOGI Y,KATO C,et al. Moritella profunda sp. nov. and Moritella abyssi sp. nov.,two psychropiezophilic organisms isolated from deep Atlantic sediments[J]. International Journal of Systematic and Evolutionary Microbiology,2003,53(2):533-538.
[26] GUO Y,ZHOU X,LI Y,et al. Heterotrophic nitrification and aerobic denitrification by a novel Halomonas campisalis[J]. Biotechnology Letters,2013,35(12):2 045-2 049.
[27] DECLEYRE H,HEYLEN K,VAN COLEN C,et al. Dissimilatory nitrogen reduction in intertidal sediments of a temperate estuary:small scale heterogeneity and novel nitrate-to-ammonium reducers[J]. Frontiers in Microbiology,2015,6:1 124.
[28] LIU Y,AI G M,MIAO L L,et al. Marinobacter strain NNA5,a newly isolated and highly efficient aerobic denitrifier with zero N2O emission[J]. Bioresource Technology,2016,206:9-15.
[29] CHOI A,CHO H,KIM S H,et al. Rates of N2,production and diversity and abundance of functional genes associated with denitrification and anaerobic ammonium oxidation in the sediment of the Amundsen Sea Polynya,Antarctica[J]. Deep Sea Research Part II Topical Studies in Oceanography,2016,123(4):113-125.
[30] GONZLEZDOMENECH C M,MARTNEZCHECA F,BJAR V,et al. Denitrification as an important taxonomic marker within the genus Halomonas[J]. Systematic and Applied Microbiology,2010,33(2):85-93.