運用多維體學策略來解析環境微生物與有機污染物如阻燃劑之動態降解機制
Study environmental microbiology by using multi-OMICS approaches: characterization of microbial degradation mechanisms toward organic contaminants such as flame retardants
This project focused on the microbial degradation of the novel environmental organic pollutants, such as brominated flame retardants (BFR). BFRs are prevailing used as anti-flame additives, and among all, hexabromocyclododecane (HBCD) is one of the most frequently used. Because of the extensive use of HBCD as an addictive FR in textile and electronic products, and the fact that it had been declared as one of the persistent organic pollutants (POP) by Stockholm Convention. The aim of this study is to search for other microorganisms capable of degrading BFRs and further expand the scale of selecting other native environmental microcosms with the ability of degrading or transforming BFRs by multi-omics approaches, and compare the metagenomics, metatranscriptomics, and proteomics to decipher the optimal conditions, molecular mechanisms, metabolic pathways for degrading and transforming BFRs in the environment.
High-throughput sequencing innovations, for example, the Illumina sequencing platform, give a novel intends to examine the elements of transcriptional variety related to metabolic pathways. This methodology is fundamental for explaining the confounded molecular system hidden behind the biotransformation pathway in R. palustris. Time-course transcriptome analysis revealed that the mechanism by which cells degrade HBCD degradation in this study involved unique expression characteristics, i.e. both hydroxylation and debromination pathways were used for HBCD degradation. This was different from other microbial modes of HBCD degradation, i.e. aerobic hydroxylation and anaerobic debromination, which demonstrate that there are fewer restrictions on oxygen demand for both reactions to occur. Specifically, the regulators for HBCD degradation enzymes were confirmed which include transmembrane proteins of class II cytochromes P450 and the TCS; higher levels of gene expression precisely controlled by the LysR-Type transcriptional regulators including GST and HAHD during longer HBCD treatment condition reveal the appropriate transitioning of HBCD tolerance state to positive degradation.
Two soil samples, Chiang Chun Soil and River Bank Soil, were examined with the HBCD degradation capacity. In Chiang Chun Soil, 40 % HBCD removal was reached after 4 days of incubation and in River Bank Soil, the same level of HBCD degradation was observed after 7 days of incubation. The HBCD debromination metabolites, PBCDE, Tri-BCD, CDT, and epoxide oxidation product ECDD were identified in Chiang Chun Soil. The composition of Chiang Chun Soil was analyzed with the metagenomics approach. The metagenomics network was built according to the correlation between microbes, and separate modules contained microbes with similar expression trends. To find which module correlated to HBCD degradation, we examine the correlation between the eigenvalue of modules and sample traits. We chose the blue module owing to the highest positive correlation to HBCD_treatment and HBCD_degradation. The top 10 bacteria were selected according to the correlation to the trait, including Brevundimonas spp., Chondrocystis spp., Microvirga spp., Phenylobacterium spp., Vulgatibacter spp., Cupriavidus spp., Caulobacter spp., Tessaracoccus spp., Bosea spp., and Polaromonas spp.. These better understandings provide more biodegradation knowledge for the remediation for POPs such as HBCD and the new insights to use bioinformation tools in the study of environmental microbiology.
對於微生物降解新興環境有機污染物例如溴化阻燃劑，在其中六溴環十二烷(hexabromocyclododecane, HBCD）是最被常使用的其中一種，且HBCD被斯德哥爾摩公約列為持久性有機污染物之一。藉由高通量定序技術與分析和轉錄體表現差異相關的代謝途徑，了解Rhodopseudomonas palustris（沼澤紅假單孢菌）生物轉化HBCD的分子機制有重要的貢獻。時程轉錄基因體分析顯示出在沼澤紅假單孢菌中獨特的生物轉化HBCD機制，同時包括羥化作用、脫溴作用等代謝途徑。這和其他能夠降解利用HBCD的微生物不同，例如好氧性的羥化作用或是厭氧性的脫溴機制，表示沼澤紅假單孢菌對於降解HBCD的養氣需求限制較低，以達到同時具有羥化作用以及脫溴作用途徑。具體來說，調節HBCD生物轉化降解的調控者，包含跨膜蛋白第二型細胞色素P450以及雙分子調控系統調節者 (Two component system regulator) ，而在長時間HBCD處理下被LysR轉錄因子調控，且具有更高基因表現量的谷胱甘肽S-轉移酶(Glutathione S-transferase)以及鹵酸脫鹵酶 (Haloacid dehalogenase)，則是顯示在沼澤紅假單孢菌中受到HBCD刺激後從忍受狀態轉換成主動降解的過程。另外，在兩種環境土壤的微生物族群有不同忍受以及降解HBCD的能力。在將軍系土壤中，40%的HBCD在4天處理後被土壤微生物群降解，而在新店溪底泥土中同樣40%的HBCD則是需要7天來達到。HBCD的脫溴代謝產物包含pentabromocyclododecane、tribromocyclododecane、cyclododecane 以及環氧化作用產物epoxy-cyclodecadiene 均有在處理過的將軍系土壤中有偵測到。並進行總體基因體學分析，表現趨勢相同的微生物分類至同一模組中，檢測每一微生物模組的特徵值與HBCD降解表現趨勢之間的關聯性。最後我們選擇的模組與HBCD_treatment和HBCD_degradation等樣本特性之間是高度正相關，其中前10名的細菌屬別包括Brevundimonas spp.、 Chondrocystis spp., Microvirga spp.、 Phenylobacterium spp.、 Vulgatibacter spp., Cupriavidus spp.、 Caulobacter spp.、 Tessaracoccus spp.、 Bosea spp.以及 Polaromonas spp.。這些對HBCD微生物降解更好的了解與知識提供了整治復育持久性有機污染物如HBCD的處理與使用生物資訊於環境微生物學的新見解與應用。