Department of Civil and Environmental Engineering, Michigan State University, East Lansing, MI, USA.
Department of Civil and Environmental Engineering, Michigan State University, East Lansing, MI, USA.
J Microbiol Methods. 2024 Apr;219:106908. doi: 10.1016/j.mimet.2024.106908. Epub 2024 Feb 23.
1,4-Dioxane, a likely human carcinogen, is a co-contaminant at many chlorinated solvent contaminated sites. Conventional treatment technologies, such as carbon sorption or air stripping, are largely ineffective, and so many researchers have explored bioremediation for site clean-up. An important step towards this involves examining the occurrence of the functional genes associated with 1,4-dioxane biodegradation. The current research explored potential biomarkers for 1,4-dioxane in three mixed microbial communities (wetland sediment, agricultural soil, impacted site sediment) using monooxygenase targeted amplicon sequencing, followed by quantitative PCR (qPCR). A BLAST analysis of the sequencing data detected only two of the genes previously associated with 1,4-dioxane metabolism or co-metabolism, namely propane monooxygenase (prmA) from Rhodococcus jostii RHA1 and Rhodococcus sp. RR1. To investigate this further, qPCR primers and probes were designed, and the assays were used to enumerate prmA gene copies in the three communities. Gene copies of Rhodococcus RR1 prmA were detected in all three, while gene copies of Rhodococcus jostii RHA1 prmA were detected in two of the three sample types (except impacted site sediment). Further, there was a statistically significant increase in RR1 prmA gene copies in the microcosms inoculated with impacted site sediment following 1,4-dioxane biodegradation compared to the control microcosms (no 1,4-dioxane) or to the initial copy numbers before incubation. Overall, the results indicate the importance of Rhodococcus associated prmA, compared to other 1,4-dioxane degrading associated biomarkers, in three different microbial communities. Also, the newly designed qPCR assays provide a platform for others to investigate 1,4-dioxane biodegradation potential in mixed communities and should be of particular interest to those considering bioremediation as a potential 1,4-dioxane remediation approach.
1,4-二恶烷是一种可能的人类致癌物，是许多氯化溶剂污染场地的共污染物。传统的处理技术，如碳吸附或空气汽提，效果不大，因此许多研究人员探索了生物修复来进行现场清理。这方面的一个重要步骤涉及检查与 1,4-二恶烷生物降解相关的功能基因的存在。目前的研究使用单加氧酶靶向扩增子测序，随后进行定量 PCR(qPCR)，探讨了三种混合微生物群落（湿地沉积物、农业土壤、受污染场地沉积物）中 1,4-二恶烷的潜在生物标志物。BLAST 分析测序数据仅检测到先前与 1,4-二恶烷代谢或共代谢相关的两个基因，即 Rhodococcus jostii RHA1 中的丙烷单加氧酶（prmA）和 Rhodococcus sp. RR1。为了进一步研究，设计了 qPCR 引物和探针，并在三个群落中使用这些测定方法来计数 prmA 基因拷贝数。在所有三个群落中都检测到 Rhodococcus RR1 prmA 的基因拷贝数，而在三个样本类型中的两个（除了受污染场地沉积物）中都检测到 Rhodococcus jostii RHA1 prmA 的基因拷贝数。此外，与对照微宇宙（无 1,4-二恶烷）或孵育前的初始拷贝数相比，在接种受污染场地沉积物的微宇宙中 1,4-二恶烷生物降解后，RR1 prmA 基因拷贝数有统计学意义的增加。总体而言，与其他 1,4-二恶烷降解相关生物标志物相比，结果表明在三个不同微生物群落中 Rhodococcus 相关 prmA 的重要性。此外，新设计的 qPCR 测定为其他研究人员在混合群落中研究 1,4-二恶烷生物降解潜力提供了一个平台，对于那些考虑生物修复作为潜在 1,4-二恶烷修复方法的人来说，应该特别感兴趣。
