Environmental Biotechnology Institute, University of Idaho, PO Box 441052, Moscow, ID 83844-1052, USA.
Biodegradation. 2011 Nov;22(6):1045-59. doi: 10.1007/s10532-011-9462-4. Epub 2011 Mar 1.
The Test Area North (TAN) site at the Idaho National Laboratory near Idaho Falls, ID, USA, sits over a trichloroethylene (TCE) contaminant plume in the Snake River Plain fractured basalt aquifer. Past observations have provided evidence that TCE at TAN is being transformed by biological natural attenuation that may be primarily due to co-metabolism in aerobic portions of the plume by methanotrophs. TCE co-metabolism by methanotrophs is the result of the broad substrate specificity of microbial methane monooxygenase which permits non-specific oxidation of TCE in addition to the primary substrate, methane. Arrays of experimental approaches have been utilized to understand the biogeochemical processes driving intrinsic TCE co-metabolism at TAN. In this study, aerobic methanotrophs were enumerated by qPCR using primers targeting conserved regions of the genes pmoA and mmoX encoding subunits of the particulate MMO (pMMO) and soluble MMO (sMMO) enzymes, respectively, as well as the gene mxa encoding the downstream enzyme methanol dehydrogenase. Identification of proteins in planktonic and biofilm samples from TAN was determined using reverse phase ultra-performance liquid chromatography (UPLC) coupled with a quadrupole-time-of-flight (QToF) mass spectrometer to separate and sequence peptides from trypsin digests of the protein extracts. Detection of MMO in unenriched water samples from TAN provides direct evidence of intrinsic methane oxidation and TCE co-metabolic potential of the indigenous microbial population. Mass spectrometry is also well suited for distinguishing which form of MMO is expressed in situ either soluble or particulate. Using this method, pMMO proteins were found to be abundant in samples collected from wells within and adjacent to the TCE plume at TAN.
美国爱达荷州爱达荷瀑布市附近爱达荷国家实验室的测试区北（TAN）场址位于斯内克河平原断裂玄武岩含水层中的三氯乙烯（TCE）污染物羽流上方。过去的观察结果提供了证据，表明 TAN 中的 TCE 正在通过生物自然衰减而转化，这种衰减可能主要是由于甲烷营养菌在羽流的需氧部分的共代谢作用。甲烷营养菌的 TCE 共代谢是微生物甲烷单加氧酶广泛的底物特异性的结果，这使得 TCE 可以除主要底物甲烷之外被非特异性氧化。已经利用一系列实验方法来理解驱动 TAN 中内在 TCE 共代谢的生物地球化学过程。在这项研究中，使用针对编码颗粒状 MMO（pMMO）和可溶性 MMO（sMMO）酶亚单位的基因 pmoA 和 mmoX 以及编码下游酶甲醇脱氢酶的基因 mxa 的保守区域的引物通过 qPCR 对好氧甲烷营养菌进行了计数。使用反相超高效液相色谱（UPLC）与四极杆-飞行时间（QToF）质谱联用仪从 TAN 的浮游生物和生物膜样品中鉴定蛋白质，以分离和测序来自蛋白质提取物的胰蛋白酶消化物中的肽。从 TAN 未富集中的水样中检测到 MMO 提供了直接证据，证明了土著微生物种群的内在甲烷氧化和 TCE 共代谢潜力。质谱法也非常适合区分原位表达的 MMO 是可溶性还是颗粒状。使用这种方法，在 TAN 的 TCE 羽流内和附近的井中采集的样品中发现了大量的 pMMO 蛋白。
