LEXEN: Collaborative Research: A Window into the Extreme Environment of Deep Subsurface Microbial Communities: Witwatersrand Deep Microbiology Project

LEXEN：合作研究：了解深层地下微生物群落极端环境的窗口：Witwatersrand 深层微生物学项目

• 批准号: 9714214
• 负责人: Tullis Onstott
• 金额: $ 19.25万
• 依托单位: Princeton University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-10-01 至 1999-09-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9714214&HistoricalAwards=false
• 关键词: LEXEN; Collaborative; Research; Window; into

9714214 Onstott Recent investigations have identified microbial communities in various crustal environments down to 2800 meters below the surface (mbls). Only a hand full of deep microbial samples from continental crust (500 mbls.) exists, however, because coring is expensive. The gold mines of the 2.9 Ga Witwatersrand Supergroup in South Africa, however, provide a unique opportunity to study microbial communities at depths ranging from 2000 to 3500 mbls. And eventually up to 5000 mbls. Reconnaissance samples of a uranium-rich, gold-bearing, carbonaceous rock and of water from a gallery borehole were collected for microbial analyses from mined depth of 3200 mbls., where the rock and water temperatures were 50 to 55(C. Measures were taken to avoid contamination during mining and sampling. Samples were shipped to the U.S.A. in sterile, anaerobic canisters on ice, processed under sterile anaerobic conditions and distributed to other microbiology labs. Microscopic observations indicated the presence of intact cells in both types of samples. Electron microscopy reveals filamentous microorganisms in the rock samples. Phospholipid fatty acid and DNA analyses indicate that the rock samples contain Cyanobacteria and sulfate-reducing bacteria (SRB). Growth was detected in aerobic and anaerobic enrichments of the rock samples. The water sample yielded a strain of Thermus that is the first reported Thermus to reduce Fe (III) and the first facultative, thermophilic Fe (III) reducing bacteria (IRB). Funds are requested to return to South Africa, set up a sample-processing laboratory on site, and carry out a three-month field study to address the following issues raised by this sample reconnaissance: ( To what extent are rock and water samples contaminated by mining activity and what sampling strategies must be developed to reduce and quantify this contamination? Specifically, do the Cyanobacteria represent mining contamination? ( Does the radiation emanating from the uranium-rich, car bonaceous layer provide a significant source of energy for indigenous microbial communities? In particular, does the radiolytic reaction with water generate sufficient oxygen for the growth of facultative microorganisms like IRB-SA? ( If these microbial communities have been present in the rock since the time of the last thermal episode at 2.0 Ga, then does the in situ activity of the SRB and IRB explain the occurrence of framboidal pyrite and filamentous aggregates of gold? To address the first question, samples of mining water, borehole water, and filtered air samples will be collected and analyzed. For the second question, rock samples of the carbonaceous layer and host rock will be collected with aseptic methods in a three dimensional grid (3x3x12 meter) during excavation of an access tunnel. To answer the second and third question, the relationships between the mineralogy and the bacteria in the rocks will be examined in situ and the capability of the cultured microorganisms to precipitate minerals at in situ conditions will be examined. To constrain the habitation time of the microbial community, the present day and paleo hydrology will be modeled using thermal constraints provided by borehole temperatures and thermochronometry. Incubations of all samples will be initiated on site. Off site analyses during the subsequent 21 months will include 1) phospholipid, glycolipid, ether lipid analyses (Univ. of Tenn.); 2) DNA extraction, polymerase chain reaction (PCR) amplification, cloning, and sequencing (Pacific Northwest National Laboratories-PNNL); 3) acridine orange direct counts (AODC), in situ probe, and field emission gun scanning electron microscopy (FEG-SEM) (Princeton-Rutgers); 4) chemical and isotopic analyses (Princeton-Indiana University); 5) fission track apatite analyses (Princeton-Univ. of Penn.). 6) Phosphorimaging of SRB and uranium reducing activity (PNNL-Princeton).

小行星9714214 最近的调查已经确定了各种地壳环境中的微生物群落，深度可达地表以下2800米（mbls）。 只有一只手从大陆地壳深层微生物样本（500 mbls）。然而，由于取芯是昂贵的，所以存在。 然而，南非2.9 Ga Witwatersrand超群的金矿为研究2000至3500 MBLs深度的微生物群落提供了独特的机会。最终达到5000 mbl。 从3200 mbls的开采深度采集了富铀、含金、碳质岩石和坑道钻孔中的水的勘察样品，用于微生物分析，岩石和水的温度为50到55摄氏度。 已采取措施，避免在采矿和取样过程中受到污染。 将样品置于置于冰上的无菌厌氧罐中运输至美国，在无菌厌氧条件下处理，并分发至其他微生物学实验室。 显微镜观察结果表明，两种类型的样品中均存在完整细胞。 电子显微镜显示岩石样品中的丝状微生物。 磷脂脂肪酸和DNA分析表明，岩石样品含有蓝藻和硫酸盐还原菌（SRB）。 在岩石样品的好氧和厌氧富集中检测到生长。 该水样产生了一株栖热菌，这是第一个报道的还原Fe（III）的栖热菌和第一个兼性嗜热Fe（III）还原菌（IRB）。 请求提供资金，以便返回南非，在现场建立一个样本处理实验室，并进行为期三个月的实地研究，以解决这次样本勘察提出的下列问题：（岩石和水样本受到采矿活动污染的程度，必须制定何种取样战略来减少和量化这种污染？ 具体来说，蓝细菌是否代表采矿污染？ （富含铀的碳质层发出的辐射是否为本土微生物群落提供了重要的能量来源？ 特别是，与水的辐解反应是否产生足够的氧气供兼性微生物（如IRB-SA）生长？ （如果这些微生物群落自2.0 Ga的最后一次热事件以来一直存在于岩石中，那么SRB和IRB的原位活动是否可以解释framboidal黄铁矿和丝状金聚集体的发生？ 为了解决第一个问题，将收集和分析采矿水、钻孔水和过滤空气样品。 对于第二个问题，在开挖交通隧道期间，将采用无菌方法在三维网格（3x 3x 12米）中采集碳质层和主岩的岩石样本。 为了回答第二个和第三个问题，将在原地检查矿物学和岩石中的细菌之间的关系，并检查培养的微生物在原地条件下沉淀矿物的能力。 为了限制微生物群落的居住时间，将使用钻孔温度和热计时法提供的热约束来模拟现今和古水文。 所有样本的孵育将在研究中心开始。 在随后的21个月期间的场外分析将包括1）磷脂、糖脂、醚脂分析（田纳西大学）; 2)DNA提取、聚合酶链反应（PCR）扩增、克隆和测序（太平洋西北国家实验室-PNNL）; 3）吖啶橙子直接计数（AODC）、原位探针和场发射枪扫描电子显微镜（FEG-SEM）（莱顿-罗格斯大学）; 4）化学和同位素分析（莱顿-印第安纳大学）; 5）裂变径迹磷灰石分析（莱顿-宾夕法尼亚大学）。6)SRB和铀还原活性的磷光成像（PNNL-Princeton）。