Collaborative Research: Microbial Communities at the Cariaco Redox Interface: Coupling of Sulfur, Carbon and Metal Cycles

富氧和缺氧环境之间的界面在海洋中许多元素的循环中起着重要作用，特别是具有多种氧化还原状态的元素，包括碳、硫、氮、锰和铁。界面位于氧化还原过渡区，其中还原（硫化物，还原金属，有机）和氧化（氧，硝酸盐，金属氧化物）化学物质以相反的浓度梯度共存。过渡区的许多重要反应都是生物介导的（例如，硫化物氧化和MnO2还原），这些反应支持无法在其他任何地方存在的微生物种群。卡里亚科盆地是世界上第二大远洋缺氧系统，也是唯一一个真正的大型海洋永久缺氧盆地。它也是国际CARIACO（有色海洋中的碳保留）时间序列项目的所在地，该项目正在研究海洋生产力和沉积的长期变化。该项目的主要长期目标是在Cariaco时间序列站点发现和表征Cariaco盆地氧化还原过渡带中尚未描述的微生物，并为了解它们在碳，硫和金属循环中的意义奠定基础。正在追求下列实验目标：使用16S rRNA文库/T-RFLP或DGGE技术描述氧化还原过渡区内具有最高垂直分辨率的原核生物群落；2. 以16S rDNA文库为基础，设计针对三种微生物领域（好氧、嗜微氧和厌氧）中特定群原核生物的寡核苷酸探针。利用这些探针的荧光原位杂交（FISH）和项目头两年收集的样品来测试在16S rDNA文库/DGGE凝胶中发现的原核生物在各自的行会中是否具有数字优势；3. 利用14C放射自显影和原位杂交相结合的方法鉴定三个行会的自养成分；4. 通过核酮糖-1,5-二磷酸羧化酶/加氧酶和柠檬酸连接酶基因的多样性来表征这三个行业；5. 生成硫化物、硫代硫酸盐、亚硫酸盐、单质硫、MnII和MnO2、溶解铁和颗粒铁以及FeS的浓度分布曲线，以解释这些Cariaco行会可能的生物地球化学性质；6. 分离具有新型代谢的原核生物——化学自养生物氧化硫化物和硫代硫酸盐，使用氧化锰或氧化铁/氢氧化物作为电子受体，并创建其他代谢的富集培养物：(a)好氧硫化物氧化剂，(b)（微）嗜氧金属氧化剂，(c)厌氧反硝化硫化物氧化剂，(d)厌氧MnO2/S0还原剂（Geobacteraceae和生理上相似的细菌）和(e) S0和亚硫酸盐/硫代硫酸盐歧化细菌；7. 用FISH用16S rDNA文库设计的探针鉴定富集（或纯）培养物。将这些分离物的16S rDNA和CO2固定基因与各自行会的分子信息（16S rDNA， CO2固定基因，FISH）进行比较，以验证其在自然界中的重要性。分离细菌的生理特性正在研究它们在潜在的电子供体、受体和碳源上生长的能力。该项目包括一个重要的教育组成部分，与委内瑞拉科学家（西蒙玻利瓦尔大学和拉萨尔自然科学基金会）的国际合作，以及与由美国国家科学基金会资助的正在进行的CARIACO项目的密切合作。至少有两名研究生和两名本科生在项目过程中接受培训。与拉丁美洲一所主要院校的合作，不断为招收委内瑞拉籍学生提供机会，并为形成正式合作提供机会。

Project summaryThe interface between oxic and anoxic environments plays an important role in cycling of many elements in the ocean, particularly those with several redox states, including carbon, sulfur, nitrogen manganese and iron. The interface lies within a redox transition zone where reduced (sulfide, reduced metals, organic) and oxidized (oxygen, nitrate, metal oxides) chemical species coexist in opposing concentration gradients. Many important reactions in the transition zone are biologically mediated (e.g., sulfide oxidation and MnO2 reduction) and these reactions support microbial populations unable to exist anywhere else. The Cariaco Basin is the world's second largest pelagic anoxic systems and is the only large, truly marine, permanently anoxic basin. It is also the site of the international CARIACO (CArbon Retention In A Colored Ocean) time series program, which is examining long-term variability in ocean productivity and sedimentation. The major long term goal of this project is discovering and characterizing yet undescribed microorganisms in the redox transition zone of the Cariaco Basin at the CARIACO time series site, and laying groundwork for understanding their significance in carbon, sulfur and metal cycling. The following experimental objectives are being pursued: 1. Describing the prokaryotic community with the highest feasible vertical resolution within the redox transition zone using 16S rRNA libraries/T-RFLP or DGGE techniques; 2. Designing oligonucleotide probes targeting specific groups of prokaryotes in each of the three microbial guilds (aerobic, microaerophilic and anaerobic) on the basis of 16S rDNA libraries. Using fluorescent in situ hybridization with these probes (FISH) and samples collected during first two years of the project to test whether prokaryotes found in 16S rDNA libraries/DGGE gels are numerically dominant within their respective guilds; 3. Identifying autotrophic components of the three guilds using a combination of 14C autoradiography with in situ hybridization; 4. Characterizing the three guilds by the diversity of ribulose-1,5-bisphosphate carboxylase/oxygenase and citrate liase genes; 5. Producing concentration profiles of sulfide, thiosulfate, sulfite, elemental sulfur, MnII and MnO2, dissolved and particulate Fe, and FeS to interpret possible biogeochemical properties of these Cariaco guilds; 6. Isolating prokaryotes with novel types of metabolism--chemoautotrophs oxidizing sulfide and thiosulfate using manganese oxide or iron oxide/hydroxide as electron acceptors and creating enrichment cultures of other metabolisms: (a) aerobic sulfide oxidizers, (b) (micro)aerophilic metal oxidizers, (c) anaerobic denitrifying sulfide oxidizers, (d) anaerobic MnO2/S0 reducers (Geobacteraceae and physiologically similar bacteria) and (e) S0 and sulfite/thiosulfate disproportionating bacteria; 7. Identifying enrichment (or pure) cultures by FISH with probes designed from 16S rDNA libraries. 16S rDNA and CO2 fixing genes from these isolates are being compared to the molecular information (16S rDNA, CO2 fixing genes, FISH) from their respective guilds to validate their importance in nature. Physiological properties of isolated bacteria are being studied for their ability to grow on potential electron donors, acceptors, and sources of carbon. This project includes a substantial educational component, an international collaboration with Venezuelan scientists (Simon Bolivar University and Fundacion La Salle de Ciencias Naturales) and close collaboration with the ongoing CARIACO project funded by NSF. At least two graduate students and two undergraduates are being trained during the course of the project. Collaboration with one of the leading Latin American institutions is offering continuing opportunities to recruit students of Venezuelan origin and opportunities for forming formal collaborations.