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

最近的调查已经确定了地表以下2800米（mbls）的各种地壳环境中的微生物群落。然而，由于取心成本高昂，只有一小把大陆地壳深处的微生物样本（500毫升）存在。然而，南非2.9 Ga Witwatersrand Supergroup的金矿提供了一个独特的机会，可以在2000至3500 mb的深度研究微生物群落。最终达到5000毫升。从3200米的开采深度收集了富铀、含金、碳质岩石和水的侦察样本，用于微生物分析。那里的岩石和水的温度在50到55摄氏度之间。在开采和取样过程中采取了避免污染的措施。样品装在无菌的、冰上的厌氧罐中运到美国，在无菌厌氧条件下进行处理，然后分发到其他微生物实验室。显微镜观察表明，两种类型的样品中都存在完整的细胞。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，岩石样品中含有蓝藻和硫酸盐还原菌（SRB）。在岩石样品的有氧和厌氧富集中检测到生长。水样产生了一种热菌菌株，这是第一个报道的热菌来还原铁（III）和第一个兼性，嗜热铁（III）还原细菌（IRB）。请拨资金返回南非，在现场设立一个样品处理实验室，并进行为期三个月的实地研究，以解决这次样品勘测所提出的下列问题：(采矿活动对岩石和水样的污染程度如何，必须制订何种取样战略以减少和量化这种污染？具体来说，蓝藻代表采矿污染吗？(从富含铀的碳质层发出的辐射是否为本地微生物群落提供了重要的能量来源？特别是，与水的辐射分解反应是否为IRB-SA等兼性微生物的生长产生足够的氧气？(如果这些微生物群落自2.0 Ga的最后一次热事件以来就存在于岩石中，那么SRB和IRB的原位活动是否可以解释草莓状黄铁矿和丝状金聚集体的出现？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，在隧洞开挖过程中，采用无菌法在三维网格（3x3x12米）中采集碳质层和宿主岩的岩石样品。为了回答第二个和第三个问题，将在现场检查岩石中的矿物学和细菌之间的关系，并检查培养的微生物在原位条件下沉淀矿物质的能力。为了限制微生物群落的居住时间，将使用钻孔温度和热时计提供的热约束来模拟现代和古水文。所有样品的孵化将在现场进行。随后21个月的场外分析将包括：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)吖啶橙直接计数（AODC），原位探针和场发射枪扫描电子显微镜（FEG-SEM） （Princeton-Rutgers）；4)化学和同位素分析（普林斯顿-印第安纳大学）；裂变径迹磷灰石分析（普林斯顿大学）佩恩的)。SRB和铀还原活性的磷成像（PNNL-Princeton）。

9714215 White 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, carbo naceous 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).