||Bioremediation of Soil Contaminated with Polychlorinated Dioxins under Hypoxic Conditions:Effects of Redox Potential Controls
||Department of Environmental Engineering
多氯戴奧辛物質是持久性環境毒物，由自然或人為的燃燒程序所產生，也是許多工業製程產生的不純物，累積在土壤環境造成污染，嚴重影響生態與健康。多氯戴奧辛化合物的生物分解通常需要厭氧菌先進行還原脫氯反應，再接續好氧菌的羥基化反應後進入中心代謝路徑。然而，多氯戴奧辛化合物厭氧還原脫氯反應非常緩慢，而限制兩階段生物復育戴奧辛污染的實際應用。最近發展出缺氧條件的單槽式生物處理系統，使八氯戴奧辛 (OCDD/F)快速分解，但是關於缺氧條件下降解OCDD/F的微生物種類、數量、動態以及其分解戴奧辛的生化代謝機制的了解仍然相當有限。因此，本研究運轉兩套缺氧生物處理系統，控制在不同變動方式的氧化還原電位條件，分別是「穩定型缺氧條件」 (-100 mV)的反應槽 A以及「變動型缺氧條件」 (-250 mV to +30 mV)的反應槽 B。「穩定型缺氧條件」的 A槽控制在14天即有顯著的 OCDD/F降解，而在127天的批次試程中，OCDD/F的去除率分別約有 68%以及62%，顯示若控制在穩定缺氧條件下，反應槽會有較佳的生物分解功能表現。不同批次的重覆測試與代謝產物分析可以驗證 OCDD/F的消失為生物分解貢獻，且添加堆肥可促進OCDD/F的分解速率。根據氣相層析高解析質譜鑑定代謝產物的結果，多氯二苯醚 (Polychlorinated Diphenyl Ethers)與烴基多氯聯苯 (Octachlorinated Hydroxylated biphenyls)可能是八氯戴奧辛在缺氧反應槽中相當重要的中間產物。兩個反應槽在堆肥添加期間，菌群結構在門階層變化趨勢相似，主要以Proteobacteria與Bacteroidetes為主，功能預測分析顯示這些菌群具有脫鹵、破環以及共代謝的潛力。最後，以A槽作為植種源的缺氧OCDD/F降解的批次實驗中，添加堆肥組約有55%的OCDD/F去除率，再次支持OCDD/F的缺氧生物分解。這些研究成果有助於未來發展類戴奧辛汙染物生物復育技術。
In this study, the effects of two oxidation-redox potential (ORP) control strategies were evaluated for the biodegradation of octachlorinated dibenzodioxin and dibenzofuran (OCDD/F) in a hypoxic reactor. Two sets of bioprocessing systems were successfully operated to control the ORP under various hypoxic conditions—CSTR-A for “stable hypoxic conditions” (−100 mV) and CSTR-B for “fluctuating hypoxic conditions” (−250 to +30 mV). After conducting treatment on multiple batches, the results demonstrate that CSTR-A (single-point hypoxic conditions) degrades significantly (~33%, p < 0.05) within 14 days, and the OCDD degradation efficiency was nearly 70% after a 127-day batch test. The degradation kinetic constant of OCDD/F in CSTR-A was approximately 0.011, which was 1.3 times that of CSTR-B. The GC/ECD profile indicated that CSTR-A and CSTR-B were not the same. Therefore, different conditions of hypoxic control may result in different metabolic paths, leading to different product compositions. The main reaction involved is hydrolysis and cometabolism, and PCDEs and OH-PCBs may be among the most crucial metabolites in the hypoxic biodegradation of OCDD/F. Microbiome analysis demonstrated that the community structures of various bacteria were similar at the phylum level. However, the structure of the contaminated soil added to the reactor in this study varied. Overall, this study provides further information for the biodegradation of PCDD/F-contaminated soils under hypoxic conditions.
During the 1980s, the production of pentachlorophenol (PCP) was as high as 50,000 to 60,000 metric tons per year globally (Borysiewicz et al. 2002). Taiwan was one of the major manufacturers of PCP in Southeast Asia at that time, with an annual output of up to 1,500 metric tons. PCP can result in production of approximately 1% highly toxic impurities such as polychlorinated-p-dibenzodioxin or polychlorinated dibenzofuran (PCDD/Fs) during normal manufacturing processes (Organization 1987).
According to related research, the use of reductive dichlorination of anaerobic microorganisms is preferable for the biological remediation of highly chlorined DD/Fs such as octachlorinated dibenzodioxin and dibenzofuran (OCDD/F). Anaerobic microorganisms convert highly chlorined DD/Fs into lower-chlorine-content homologues, after which aerobic microorganisms are applied. Due to the different redox environments, two-stage biological procedures are required to detoxify highly chlorined DD/Fs (Long et al. 2015). A recent study specified that OCDD/F can be rapidly decomposed and detoxified in a single-tank system that controls hypoxic conditions, with redox levels constantly fluctuating between −400 and +80 mV (Chen et al. 2016). Moreover, biodegradation of highly toxic chlorine dioxins was found to be more effective under hypoxic conditions.
However, controlling hypoxic conditions is not as easy as controlling aerobic or anaerobic conditions. Moreover, the mechanism of biodegrading OCDD/F under hypoxic conditions has still not been comprehensively researched because the type, amount, and dynamics of microorganisms differ. Therefore, the purpose of this study was to control the conditions in a hypoxic environment. The hypoxic conditions were further explored by determining whether the environment should be controlled at a stable point or changes in the gray area should be allowed.
A novel bioremediation method was developed for reducing PCDD/Fs in contaminated soil under hypoxic conditions. Because oxidation–reduction potential (ORP) is considerably sensitive to the presence of oxygen in solution, ORP controllers are used to regulate the reactor in a hypoxic environment. ORP has frequently been employed to control hypoxic environments (Duangmanee 2009; Nghiem et al. 2014a; Takahashi et al. 2011). In this study, two sets of bioprocessing systems were successfully operated to control the ORP conditions under two hypoxic conditions: “stable hypoxic conditions” (−100 mV) and “fluctuating hypoxic conditions” (−250 to +30 mV). Nutrients such as compost and basal medium were added to the reactor to vary the ORP conditions (single-point vs. two-point control). Steady and long-term operation of the hypoxic reactor was established to optimize the OCDD/F biodegradation. In addition, next-generation sequencing technology was employed to determine, whether it is different from the traditional anaerobic and aerobic flora, as well as special microbiome to participate for an absence of oxygen in the environment.
MATERIALS AND METHODS
The soil used in this study was from the site of a PCP plant in An-Shun, northwestern Tainan, Taiwan. The soil in this area contains high concentrations of PCDD/Fs, of which OCDDs and OCDF congeners contribute approximately 90% of the total amount of toxic substances. Moisture content, total organic carbon, total nitrogen, and available phosphorus were approximately 9.8%, 1.2%, 0.1%, and 2.23 mg/kg, respectively.
Slurry bioreactor setup and operation
The reactor was constructed using a steel drum of approximately 40 cm in length and 21 cm in inner diameter. Figure 1 displays the setup of the mixing equipment and aeration device. The working volume of the reactor was approximately 9 L, and the soil to water ratio was 1:2. At the beginning of each batch operation, contaminated soil (600 g), cow dung compost (150 g), and basal medium (1.5 L) was added. Moreover, 20% (w:w) slurry was drained after the batch operations. ORP control was conducted using an ORP sensor system (WTW, Germany) and aerator to control changes in the hypoxic conditions. CSTR-A was controlled at −100 mV for single-point control, and CSTR-B was controlled in the range of −250 to +30 mV for two-point control.
Chemical analysis (OCDD/F and metabolite analysis TOC)
OCDD/F was extracted through ultrasonic extraction (NIEA M167.01C, (Taiwan 2013). Dioxin analysis was performed using the standards of the American Society for Testing and Materials (ASTM STP1075) (Draper et al. 1991) hrough gas chromatography (GC; HP 6890N Series) with an electron capture detector (ECD) employing a DB-17 GC column (0.32 mm × 30 m i.d., 0.25-µm-thick film, Agilent J&W, USA). The metabolites analysis used in this study were obtained from National Chiao Tung University. High-resolution (HR) GC (Agilent 7890 CB) and HR mass spectrometry (MS) (AccuTOF GCX, JEOL) were utilized.
16S rRNA gene miseq sequencing and data processing
In this study, soil DNA extraction was performed using a commercial DNA extraction kit (MO BIO Laboratories, USA) by referring to the standard procedures specified by the manufacturer. This extraction was followed by using a rapid tissue homogenizer (FastPrep-24 5G, MP Biomedicals, USA). The extracted DNA was dissolved in polymerase chain reaction (PCR)-grade sterile water and stored at −20 °C for later use. In this study, Welgene Biotech Co., Ltd. was entrusted to conduct the next-generation sequencing. The extracted DNA was amplified by using the Illumina MiSeq high-throughput sequencing platform, and the sequence of 16S rRNA V3-V4 was analyzed. The resulting OTUs (Operational Taxonomic Units) were annotated using QIIME, and their biodiversity indices were estimated to analyze the distribution of microbial flora at different levels.
OCDD/F degradation batch experiment
In the OCDD/F degradation batch experiments, three control groups, a compost group, and substrate groups were designed by using various substrates to promote rapid biodegradation of OCDD/F. Slurry was obtained from CSTR-A and centrifuged in a 250-mL polypropylene vial at 8000 g for 20 min using a high-capacity centrifuge to remove the supernatant. Moreover, 10 g of wet soil (moisture content of approximately 40%) was added to a 160-mL serum bottle. According to the group required, pretreatment prepared material was added. Subsequently, 1 mL of OCDD/F stock solution (200 mg/L in Toluene) and 30 mL of medium were added. The mixture was aerated with 3 L/min of nitrogen for 1 min by capping with a stopper and aluminum cap. All groups were established in triplicate and incubated at room temperature and sampled over a 14-days period (Dat 0 sample were taken after 12 hours from onset) for both total dissolved organic carbon (DOC) and OCDD /F concentrations analysis.
RESULTS AND DISCUSSION
Figure 2 displays the real-time ORP state and frequency distribution at different stages of operation. The ORP was maintained at −111 ± 56 mV for CSTR-A, and the frequency distribution of CSTR-A in the second and third phases was approximately −100 mV. CSTR-B controlled the ORP in a fluctuating environment of 1 ± 114 mV. Moreover, the figure shows that the ORP demonstrated a bimodal trend with time.
Table 1 lists the degradation efficiencies and reaction kinetic constants of the two reactors for different batches. CSTR-A had a 25% to 34% OCDD/F degradation efficiency (p < 0.05) for a 14-day batch test; however, no significant degradation was observed in CSTR-B. For the 28-day batch test, CSTR-A and CSTR-B had a significant degradation efficiency of approximately 33% and 37%, respectively, at this stage. In batches 8 and 9, a continuous analysis of length up to 127 days indicated that CSTR-A had an OCDD/F degradation efficiency of 68%. However, CSTR-B had an OCDD/F degradation efficiency of less than half that of CSTR-A, as shown in Figure 3. The total DOC results displayed in Figure 4 for batch 8 suggested that the DOC accumulated to approximately 4667 mg/L in CSTR-A. However, the accumulation was not as considerable in CSTR-B (2900 mg/L).
The GC/ECD profile was analyzed using nonmetric multidimensional scaling analysis, as shown in Figure 5. The results for CSTR-A and CSTR-B are not the same. Therefore, different conditions of hypoxic control may result in different metabolic paths, leading to different product compositions. The metabolite results obtained using HRGC-MS are provided in Table 2. Octachlorinated diphenyl ethers or octachlorinated hydroxylated biphenyls were present in the hypoxic reactor.
The diversity index results obtained from next-generation sequencing analysis are shown in Table 3. The contaminated soil (Soil-G) had higher diversity than other samples. The quantitative PCR data indicated that the total amount of bacteria was approximately 105 copies/g- dry slurry less than that in the reactor (109 copies/g dry slurry). Figure 6 presents a Venn diagram of the OTU level. The microbiome of the bacteria in the contaminated soil was different from that in the reactor. The metagenomics function prediction heat map (Figure 7) shows that the function of CSTR-A was more stable than that of CSTR-B. This implies that by exerting single-point control on the hypoxic conditions, the microbiome gradually adapts to the environmental pressure and then displays stable performance.
OCDD/F degradation batch experiment
Finally, an OCDD/F biodegradation batch experiment was conducted to compare the cometabolism substrates provided to the microbiome under hypoxic conditions. Figure 8, shows that positive control has a degradation efficiency of approximately 55%. Moreover, the water-washed group (Ws, Wis) has a similary degradation efficiency.
In this study, the effects of two ORP control strategies on the biodegradation of OCDD/F were evaluated in a hypoxic reactor. After treating multiple batches, the results obtained indicated that under the single-point hypoxic conditions, CSTR-A was considerably degraded after 14 days and its function was stable, compared to CSTR-B. The primary reactions were hydrolysis and dichlorination. PCDEs and OH-PCBs may be among the most crucial metabolites in the reaction tank. This fact provides information for conducting further studies on the biodegradation of PCDD/Fs under hypoxic conditions. In the OCDD/F degradation batch experiment, compost could be added through a rapid provision of DOC as a substrate for microbial cometabolism, and the hydrolysis of the compost by microorganisms could also contribute to the biodegradation of OCDD/F.
第一章 前言 1
第二章 文獻回顧 3
2.1. 五氯酚 3
2.1.1. 五氯酚全球過去產量 3
2.1.2. 五氯酚工業製程之不純物 4
2.1.3. 五氯酚於環境中自發形成高氯數戴奧辛 5
2.2. 戴奧辛污染 6
2.2.1. 戴奧辛 6
2.2.2. 戴奧辛污染事件 7
2.3. 戴奧辛生物降解機制 8
2.3.1. 戴奧辛同源物好氧降解 8
2.3.2. 戴奧辛同源物厭氧分解 9
2.3.3. 真菌酵素分解戴奧辛 10
2.3.4. 兼性好氧/厭氧類戴奧辛生物分解 11
2.3.5. 高氯戴奧辛缺氧分解假說 12
2.4. 微生物脫氯反應機制 13
2.4.1. 氧化脫鹵作用(Oxidative Dehalogenation) 14
2.4.2. 脫氫氯反應 (Dehydrohalogenation) 15
2.4.3. 取代脫氯反應 (Substitutive Dehalogenation) 15
2.4.4. 經甲基轉換的脫氯作用 (Dechlorination via Methyl Transfer) 16
2.4.5. 還原脫氯作用 (Reductive dehalogenation) 17
2.5. 微生物共代謝作用 18
2.5.1. 共代謝定義 18
2.5.2. 含氯化合物共代謝作用 18
220.127.116.11. 含氯脂肪烴 19
18.104.22.168. 含氯單環化合物 19
22.214.171.124. 含氯多環化合物 20
2.6. 戴奧辛生物復育技術 23
2.6.1. 泥漿相生物反應器 23
126.96.36.199. 泥漿相生物反應器於生物復育之原理及應用 23
188.8.131.52. 影響泥漿相生物反應器之操作因子 24
2.6.2. 掩埋處理程序 25
184.108.40.206. 堆肥處理技術 26
220.127.116.11. 堆肥添加於生物復育之原理及應用 26
2.7. 氧化還原電位 28
2.7.1. 氧化還原電位定義 28
2.7.2. 氧化還原電位監測與控制應用 29
第三章 實驗材料與方法 31
3.1. 研究架構 31
3.2. 研究策略 32
3.3. 系統設計及操作條件 33
3.4. 氧化還原電位控制 34
3.5. 土壤樣本來源 35
3.6. 泥漿採樣與保存 35
3.7. 總溶解性有機碳分析 36
3.8. OCDD/F缺氧共代謝脫氯批次實驗 36
3.8.1. 堆肥組前處理製備流程 36
3.8.2. 定義基質組前處理製備流程 37
3.8.3. 植種流程 37
3.9. 土壤戴奧辛萃取與分析 37
3.9.1. 超音波震盪萃取 37
3.9.2. OCDD/F分析 38
3.10. 代謝產物分析 38
3.10.1. 高解析氣相層析儀分離條件 38
3.10.2. 高解析質譜儀分析條件 39
3.11. 土壤DNA萃取 39
3.12. 分子生物分析方法 39
3.12.1. 聚合酶鏈鎖反應（Polymerase chain reaction, PCR） 39
3.12.2. 瓊酯膠電泳 40
3.12.3. 即時定量聚合酶鏈鎖反應(Quantitative real-time Polymerase Chain Reactions, qPCR) 40
3.12.4. 次世代定序 (Next Generation Seguencing, NGS) 41
第四章 結果與討論 42
4.1. 反應槽不同控制之氧化還原電位變化 42
4.2. 反應槽功能表現 46
4.2.1. 反應槽 OCDD/F濃度變化結果 46
4.2.2. 反應槽OCDD/F降解反應動力探討 53
4.2.3. 總溶解性有機碳變化結果 58
4.2.4. OCDD與總溶解性有機碳(DOC)變化相關性分析 60
4.3. 反應槽不同操作時期代謝產物分析 66
4.3.1. GC/ECD圖譜分析結果 66
4.3.2. HRGC-HRMS分析未知代謝產物探討 69
4.4. 反應槽不同時期微生物體分析 78
4.4.1. 總細菌16S qPCR分析結果 78
4.4.2. 次世代高通量定序結果 79
4.4.3. 菌群結構分析 80
4.4.4. 宏基因體功能預測分析 85
4.5. 堆肥水解缺氧降解OCDD/F批次實驗 92
4.5.1. 溶解有機碳變化分析 94
4.5.2. OCDD/F分解效率探討 96
4.5.3. OCDD/F與溶解性有機碳(DOC)變化相關性分析 99
第五章 結論與建議 101
5.1. 結論 101
5.2. 建議 104
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