极端微生物的生物勘探以解决环境污染问题
1Department of Biology,University of Naples Federico II, 2Institute of Polymers, Composites and Biomaterials (IPCB),Consiglio Nazionale delle Ricerche CNR, 3Division of Biological Systems and Engineering,Lawrence Berkeley National Laboratory, 4BAT Center-Interuniversity Center for Studies on Bioinspired Agro-Environmental Technology,University of Napoli Federico II
Summary
从地热泉中分离出抗重金属微生物是生物修复和环境监测生物系统发展的热门话题。本研究为从温泉中分离和鉴定重金属耐受细菌提供了一种方法学方法。
Abstract
地热泉富含各种金属离子，这是由于深含水层中发生的岩石和水之间的相互作用。此外，由于pH和温度的季节性变化，在这些极端环境中定期观察到元素组成的波动，从而影响环境微生物群落。在火山热喷口中茁壮成长的极端微生物已经发展出抵抗机制来处理环境中存在的几种金属离子，从而参与复杂的金属生物地球化学循环。此外，极端微生物及其产品在市场上找到了广泛的立足点，这对于他们的酶尤其如此。在这种情况下，它们的表征对于开发用于环境监测和生物修复的生物系统和生物过程具有功能。迄今为止，在实验室条件下分离和培养极端微生物仍然是充分利用其生物技术潜力的瓶颈。这项工作描述了从温泉中分离嗜热微生物的简化方案，并通过以下步骤对其进行基因型和表型鉴定：（1）从地热场所（“Pisciarelli”，意大利那不勒斯Campi Flegrei的火山区）中取样微生物;（2）重金属抗微生物的分离;（3）微生物分离物的鉴定;（4）分离株的表型表征。本著作中描述的方法通常也适用于从其他极端环境中分离微生物。
Introduction
我们星球上的极端环境是能够忍受恶劣条件（即温度，pH值，盐度，压力和重金属）的微生物的极好来源1，2，包括冰岛，意大利，美国，新西兰，日本，中非和印度，这是最认可和研究的火山地区3，4，5，6，7，8，9.嗜热性在45°C至80°C10，11，12的温度范围内的恶劣环境中进化而来。嗜热微生物，无论是属于古菌还是细菌王国，都是研究生物多样性，系统发育和生产用于工业应用的独家生物分子的储存库13，14，15，16。事实上，在过去几十年中，全球市场的持续工业需求鼓励了极端微生物和热酶在几个生物技术领域的多样化应用的开发17，18，19。
温泉是生物体生活在联盟中的丰富来源，是生物多样性的丰富来源，因此代表了研究微生物生态学的有吸引力的栖息地20，21。此外，这些富含火山金属的地区通常被微生物定殖，这些微生物已经进化出耐受系统，以生存和适应重金属22，23 的存在，因此积极参与其生物地球化学循环。如今，重金属被认为是人类和环境的优先污染物。抗重金属微生物能够通过转化和重塑其生态系统来溶解和沉淀金属24，25。对重金属抗性分子机制的理解是开发新型绿色方法26，27，28的紧迫性的热门话题。在此背景下，发现新的耐受细菌是制定环境生物修复新战略的起点24，29。在通过微生物程序探索热液环境和增加对支撑重金属耐受性的基因的作用的认识的同时，在意大利Campi Flegrei温泉区进行了微生物筛查。这种富含重金属的环境显示出强大的热液活动，喷气孔和沸腾池，其pH值和温度随季节，降雨和地下地质运动而变化30。从这个角度来看，我们描述了一种易于应用且有效的方法来分离对重金属有抗药性的细菌，例如，来自Campi Flegrei的Pisciarelli地区的 Geobacillus stearothermophilus GF16 31 （命名为分离株1）和 Alicyclobacillus mali FL1832（命名为分离株2）。
Protocol
1. 地热场微生物采样 选择采样地点作为具有所需温度和pH值的标准地点。通过数字热电偶探头测量物理参数，将其插入选定的池或泥浆中。 收集20克土壤样本（在这种情况下，从Pisciarelli Solfatara热液现场的泥浆中），用无菌勺子将它们捡起。为每个选择的地点至少采集两个样本。 将样品放入50 mL无菌聚丙烯管中，立即关闭。 使用数字热电偶探头直接插入…
Representative Results
采样现场该协议说明了从温泉中分离重金属抗性细菌的方法。在这项研究中，将酸性硫化地热环境Pisciarelli地区用作采样点 （图1）。该生态系统的特点是来自火山活动的侵蚀性硫流体的流动。已经证明，酸性硫磺地热系统中的微生物群落受到由高浓度重金属存在产生的极端选择性压力。这些样本是在一年中的两个不同时期（4月和9月）从<sup class=…
Discussion
温泉含有未开发的微生物组多样性，具有同样多样化的代谢能力12.开发分离能够有效将重金属转化为毒性较小的化合物10 的微生物的策略代表了全世界越来越感兴趣的研究领域。本文旨在描述一种筛选和分离具有抗有毒化学物质能力的微生物的简化方法。所描述的方法可以很容易地修改，以从不同的环境来源（如水，食物，土壤或沉积物）中分离微生物。然而?…
Divulgations
The authors have nothing to disclose.
Acknowledgements
这项工作得到了ERA-NET Cofund Martera的支持：“FLAshMoB：用于海洋生物传感的功能性淀粉样蛋白嵌合体”，PRIN 2017-PANACEA CUP：E69E19000530001和GoodbyWaste：GetGOOD产品-利用副产品-减少浪费，MIUR 2017-JTNK78.006，意大利。我们感谢莫妮卡·皮奥奇博士和安吉拉·莫尔蒙博士（意大利那不勒斯维苏维亚诺大学国家地质研究所）对地热场址的鉴定和定性。
Materials
| Ampicillin | Sigma Aldrich | A9393 | |
| Aura Mini | bio air s.c.r.l. | Biological hood | |
| Bacitracin | Sigma Aldrich | B0125 | |
| Cadmium chloride | Sigma Aldrich | 202908 | |
| Chloramphenicol | Sigma Aldrich | C0378 | |
| Ciprofloxacin | Sigma Aldrich | 17850 | |
| Cobalt chloride | Sigma Aldrich | C8661 | |
| Copper chloride | Sigma Aldrich | 224332 | |
| Erythromycin | Sigma Aldrich | E5389 | |
| Exernal Service | DSMZ | Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures GmbH | |
| Genomic DNA Purification Kit | Thermo Scientific | #K0721 | |
| Kanamycin sulphate | Sigma Aldrich | 60615 | |
| MaxQTM 4000 Benchtop Orbital Shaker | Thermo Scientific | SHKE4000 | |
| Mercury chloride | Sigma Aldrich | 215465 | |
| NanoDrop 1000 Spectrophotometer | Thermo Scientific | ||
| Nickel chloride | Sigma Aldrich | 654507 | |
| Orion Star A221 Portable pH Meter | Thermo Scientific | STARA2218 | |
| Sodium (meta) arsenite | Sigma Aldrich | S7400 | |
| Sodium arsenate dibasic heptahydrate | Sigma Aldrich | A6756 | |
| Sodium chloride | Sigma Aldrich | S5886 | |
| Streptomycin | Sigma Aldrich | S6501 | |
| Tetracycline | Sigma Aldrich | 87128 | |
| Tryptone BioChemica | Applichem Panreac | A1553 | |
| Vancomycin | Sigma Aldrich | PHR1732 | |
| Yeast extract for molecular biology | Applichem Panreac | A3732 |
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Citer Cet Article
Gallo, G., Aulitto, M., Contursi, P., Limauro, D., Bartolucci, S., Fiorentino, G. Bioprospecting of Extremophilic Microorganisms to Address Environmental Pollution. J. Vis. Exp. (178), e63453, doi:10.3791/63453 (2021).