現地相反轉與生物還原脫氯法衍生之微生物群系再造技術研發

Development of Microbiome Reengineering from In-Situ Phase Inversion Emulsification and Biological Reductive Dechlorination for Environmental Remediation

蔡家昇(Chia-Sheng Tsai) ； 張書奇(Shu-Chi Chang) ； 葉家瑋(Chia-Wei Yeh) ； 李思寬(Szu-Kuang Lee) ； 詹艾霖(Ai-Lin Chan) ； 陳姿文(Tzu-Wen Chen) ； 蔡利局(Li-Chu Tsai)

《土壤及地下水污染整治》 6卷1期 (2021/06) Pp. 1-28

https://doi.org/10.6499/JSGR.202106_6(1).0001

摘要

環境污染整治為過去40餘年來國內外環境工程界之重要課題，諸如河川底泥中持久性有機污染物（persistent organic pollutants, POPs）的嚴重污染與地下水中氯化有機溶劑之污染。這些有機物之污染因其毒性與持久性，不僅造成環境生態系長遠負面影響，也可經由食物鏈進入人體，導致人類健康風險增加。目前部分污染物在實驗室中已經可以有效分解或是處理，但在大型實場整治上仍有相當多難題。本文藉由介紹一項新穎之跨環境介質平台整治技術（cross-matrices platform remediation technology）與目前所獲得之部分宏觀基因體學（metagenomics, MG）資料，嘗試提供未來生物整治技術研發方向，提供各界環境工程先進參考，或許可以有效解決國際間環境污染整治所面臨之困境。本團隊所研發之整合現地相反轉法與生物降解法（in-situ phase inversion emulsification and biological reductive dechlorination, ISPIE/BiRD），目前在底泥與土壤污染整治已應用成功，歸結為微生物群系再造（microbiome reengineering, MBRE）為最可能之原因，並於模擬地下水中有機污染物生物降解研究中證實的確可以有效提升生物降解效率，MBRE也成為歷來生物整治界以生物刺激與生物擴增技術為主流技術外之第三種可能未來主流技術。本文將介紹MBRE應用於底泥中典型POPs：多氯聯苯Aroclor 1254及六氯苯（hexachlorobenzene, HCB）的快速整治、土壤中靈丹（γ-hexachlorocyclohexane, lindane）與滴滴涕（dichlorodiphenyl tri-chloroethane, DDT）之生物降解及模擬地下水中三氯乙烯（trichloroethylene, TCE）之快速分解等成果。在底泥整治模場試驗中，經實驗證實單次ISPIE回收後，可分別去除約62%風化之Aroclor 1254及31%風化之HCB，再經由70天BiRD，可將風化的Aroclor 1254及HCB有效去除達98%及約100%：土壤整治研究顯示可於28天內去除DDT達99.94%；TCE污染之地下水菌群經過熱篩後，可在1天內將3.5 mg/L之TCE完全降解完畢，為歷來TCE生物降解速率最佳者。本研究結果顯示MBRE應用於大規模之現地底泥、土壤及地下水整治均具有高度可行性；盱衡國際綠色永續整治與人工智慧（artificial intelligence, AI）應用兩大浪潮，如何應用AI與生物資訊學（bioinformatics, BI）由MG之大數據中解開高效生物整治之機制成為當務之急。

關鍵字

生物整治 ； 多氯聯苯 ； 滴滴涕 ； 三氯乙烯 ； 微生物群系再造

並列摘要

1. Introduction. Over the past 40 years, environmental remediation has gained an important place in environmental engineering and aims to tackle persistent organic pollutants (POPs) in river sediments and chlorinated solvents in groundwater. The toxicity of these pollutants result not only in adverse impacts on ecosystems, but also on human health through food chains. Though some contaminants have been effectively degraded in the laboratory, widespread remediation remains difficult. This study presents a cross-matrix platform remediation technology using metagenomic data to guide future research in bioremediation. Our team developed a new method by integrating in-situ phase inversion emulsification and biological reductive dechlorination (ISPIE/BiRD) for efficient remediation of chlorinated POPs in river sediments and soils. ISPIE/BiRD has been successfully tested in two laboratory-based studies and a pilot study. The key to this success is largely attributed to microbiome reengineering (MBRE) through heat selection. The MBRE approach was also successful in its application to the biodegradation of trichloroethylene (TCE) in simulated groundwater. In the following sections, MBRE for remediation of sediment contaminated by polychlorinated biphenyls (PCBs) and hexachlorobenzene (HCB), clean-up of soil contaminated by γ-hexachlorocyclohexane (lindane) and dichlorodiphenyl tri-chloroethane (DDT), and TCE biodegradation in simulated groundwater will be discussed. Finally, future research potential is discussed. 2. MBRE in Sediment Remediation. 2.1 Theory and Technology Development: MBRE is developed from ISPIE/BiRD and the background and development of ISPIE/BiRD are presented below. There are three obstacles to the rapid bioremediation of sediment sites contaminated by Arochlor 1254 (a commercial mixture of PCBs) and HCB: slow desorption, low water solubility, and slow biodegradation rate. Slow desorption and low water solubility can be partially solved by increasing the temperature, but this may cause adverse impacts on the microbial ecosystem. However, it has been reported that the hydrogen production can be effectively altered by heating, and the dechlorinating bacteria grow well in the 28-37°C temperature range. Coincidentally, heating an emulsion made of nonionic surfactants can cause phase inversion, forming a water-in-oil (W/O) emulsion from an oil-in-water (O/W) emulsion. Because the heated W/O emulsion exhibits hydrophobic behavior in the sediment matrix, such as sorption onto the surface of sediment organic matter and exhibition of non-wetting tendency toward the surface of sediment particles, they are likely to interact with the highly hydrophobic POPs in the sediments. Higher temperatures can also increase the desorption rate, rapid repartitioning into the oil phase, and the water solubility of POPs (Figure 1). Please refer to published papers for a detailed discussion of ISPIE/BiRD (Chang et al., 2019a; Chang et al., 2019b). 2.2 Lab Study: The test groups and the results are shown in Table 1, Figure 3, Figure 4, and Table 2 in the full-length paper in Chinese. Among all groups, test number 7 displayed the most effective removal of both Aroclor 1254 and HCB. For both contaminants, the optimum conditions for removal were: high temperature (30°C), high emulsion concentration (10%), high organic matter (1.0%), and a pH range of 7.0-8.5. The ratios of the first order biodegradation rate constants of HCB to Aroclor 1254 (k_(HCB)/k_(PCB)) under nine different conditions were ranging from 2.14-3.04, with a mean of 2.59, and a 13.6% coefficient of variance. This indicates that HCB can be removed at a higher rate than PCBs. This ratio is consistent under all tested environmental conditions. The Arrhenius equation showed that the activation energy of Aroclor 1254 was approximately 3.3 times higher than that of HCB, and HCB degradation was largely dependent on temperature. These results also showed that acclimated cultures were effective. 2.3 Pilot Study: The pilot study results are shown in Table 3, Figure 5, Figure 6, and Figure 7 in the full-length paper in Chinese. There were eight different groups (Table 3), and the ISPIE operation contributed to about 50% removal of the PCBs; the removal rates of fresh PCBs were higher than those of weathered PCBs. The variation of HCB was large, and the freshly added samples produced consistent results (Figure 5). For the BiRD stage, it is evident that weathered samples showed much higher removals than fresh samples for both PCBs and HCB (Figure 6). For the overall ISPIE/BiRD process, the removal rates for weathered and natural recovery (WNR) and weathered and bioaugmentation (WBA) were over 98.0% in the weathered PCBs. Similarly, the removal of weathered HCB in WNR, weathered and biostimulation (WBS), and WBA was 100%. However, the removal rates in the fresh samples were significantly lower. As evidenced in the benchmarking, this study is the first to be conducted in the field using ISPIE/BiRD with very high percentages of POP removal, in a relatively short period of time. 2.4 Microbial Community Profiles: The microbial communities of the upper and lower sections of WNR and WBK were sequenced using next-generation sequencing (NGS), and the results are shown in Figure 8 and Table 5. The tag numbers and operational taxonomic unit (OTU) numbers in the four samples are provided in Figure 8(a), and the relative abundance of the top 10 phyla are shown in Figure 8(b). In Figure 8(c), the top 35 genera are presented in a heatmap format clustered in vertical directions, showing the similarity of trends between different genera. From Figure 8(c), it is evident that the patterns are very different. The top 12 most abundant OTUs are listed in Table 5 and it can be seen that the related dechlorinating bacteria and archaea behaved very differently. For example, samples 2, 6, and 7 under the phylum Chloroflexi were often found in dechlorination consortia but displayed a negative correlation with the removal rates. Methanosaeta spp. (samples 1 and 10) showed a highly positive correlation with removal rates. These archaea were classed as the best dechlorinating bacteria. 3. ISPIE/BiRD in Soil Remediation. 3.1 Batch Study: As shown in Table 6 in the Chinese version, under nine different experimental conditions for BiRD test, lindane displayed quite significant degradation (median = 79.8%, 74.8% and 83.6% as inter-percentile 25% and 75%), but DDT showed various levels of removal (median = 33.5%, 8.6% and 73.5% as inter-percentile 25% and 75%). The best combination of the parameters was: high water content (25%), medium soil organic matter (0.1%), medium emulsion level (1.0%), and medium to high pH (pH 7.0-8.5). The half-life was as short as 3.2 days for lindane. This result showed that the Er-Ren River (ERR) consortium is feasible for lindane biodegradation in soils. 3.2 Column Study: To test the performance ISPIE in soil, a column study was conducted. The results showed that the removal of DDT was significantly higher than that of lindane (Figure 9(a)). The most probable reason is the difference of log K_(ow) (Figure 9(b)). 3.3 Sandbox Study: In the sandbox test, the test conditions were based on the best environmental parameter combinations obtained from the batch study. Four combinations of the presence of acclimated microorganisms and emulsion were tested. The results were very different from batch study. The removal of DDT achieved as high as 99.94 ± 0.10% for the samples with the presence of both acclimated microorganisms and emulsion (Table 8). The best removal for lindane was 92.26 ± 0.33% of the samples with neither acclimated microorganisms nor emulsion. Further statistical analysis showed that emulsion addition can cause significant difference in DDT removal. 4. MBRE in TCE Biodegradation. Due to the success of the MBRE in sediment remediation, a mixed culture for a TCE-contaminated site was used for the heat selection test. Currently, this is pending a patent approval and hence the details are not provided here. However, it has been confirmed that heat selection increases the biodegradation rate. The half-life of TCE biodegradation was the shortest in the literature, and the first-order kinetic rate constant increased more than 70 times as compared to the original untreated culture. According to the model, the predicted rate constant can be more than 90 times higher than that of the untreated sample under optimal conditions. Thus, MBRE possesses high potential in the application to field remediation. 5. Future Research Perspectives. Considering the wave of green sustainable remediation and its cost-effectiveness, the application of bioremediation has increased in the past decade. It has been estimated that the compound annual growth rate of the bioremediation market is as high as 7.72%. This MBRE technology has the potential to become a mainstream bioremediation technology, in addition to biostimulation and bioaugmentation, as shown in Figure 10. It is recommended that MBRE be tested in various environmental matrices, contaminants, and cultures. It is also crucial to form cross-disciplinary teams for the testing of new artificial intelligence algorithms, bioinformatic exploration in massive metagenomic data, while conducting continuous MBRE studies to identify new key species, effective mixed cultures, enzymes, and functional genes. 6. Conclusions. MBRE was chanced upon during ISPIE/BiRD research, and it has now been established as a cross-matrix platform technology. Some common dechlorinating bacteria were negatively correlated with removal of the tested POPs and it has also been confirmed that these are useful in changing the microbial structure while biodegrading TCE in simulated groundwater. The improved rate constant was 70 times higher than that of the untreated culture. It is highly recommended to form cross-disciplinary teams for the testing of new artificial intelligence algorithms and bioinformatic exploration in massive metagenomic data, while conducting continuous MBRE studies to identify new key species, effective mixed cultures, enzymes, and functional genes that supplement effective bioremediation. Acknowledgement. This study was funded by the research project supported by the Taiwan Environmental Protection Administration (EPA). The views or opinions expressed in this article are those of the writers and should not be construed as opinions of the Taiwan EPA. Mention of trade names, vendor names, or commercial products does not constitute endorsement or recommendation by Taiwan EPA.

並列關鍵字

bioremediation ； polychlorinated biphenyls ； dichlorodiphenyl tri-chloroethane ； trichloroethylene ； microbiome reengineering