隨著科技與產業的發展，在製造各式產品的過程中常伴隨大量有機或是具有毒性的污染物產生。石化產業屬於能源密集且高污染的工業，因此其廢水處理單元的穩定性非常重要，避免了許多污染物直接排放到大自然中影響生態或人體健康。傳統的廢水處理單元中，生物處理法中的活性污泥法是最被廣泛使用的方法。然而在傳統的控制方法上仍以操作導向並依賴經驗方法為主而非透過了解活性污泥內微生物的組成及特性進行控制。近年隨著分子生物技術的發展，對於活性污泥法中的微生物能有更深入的了解。本研究針對某石化產業之活性污泥系統進行水質監測，針對生物處理系統進出流的污染物進行特性分析，並利用批次試驗試圖釐清出流COD的可能來源與特性。另外利用分子生物技術如次世代定序(NGS)監測微生物族群變化;即時聚合酶連鎖反應(qPCR)對優勢菌群做定量分析。最後透過相關性分析找出水質與微生物間的關係，作為改善或控制活性污泥的參考。透過水質監測可以發現系統有COD去除效能受限的問題。廢水特性結果可得知進流廢水所含之短碳鏈有機物與揮發性物質都能被去除，另外根據分子量分佈分析，進流含分子量約900 Da的高分子物質也可被生物處理單元去除。從FEEM的結果分析出流溶解性有機物特性可能為類黃酸與類腐質酸物質。各股廢水批次測試與情境分析指出VCM製程廢水最為可能是造成出流COD無法完全降解的主要來源。NGS與PCA分析結果指出Saprospiraceae和Hyphomicrobiaceae (合佔36%)經過歲修後在活性污泥中仍保持生長優勢，進一步針對此兩種菌進行定量分析。定量結果與水質參數的相關性分析結果顯示進流COD和硝酸與一個月後的Hyphomicrobiaceae分別呈負相關與正相關。透過FISH確認屬於絲狀菌的Saprospiraceae則與F/M、溶氧有直接相關。Saprospiraceae較高的時候( > 4 * 107 copy/mL sludge)發生在F/M > 0.3 (kg COD/kg VSS-day) 與溶氧較低( < 1.9 mg/L)的操作條件下。從此結果可回饋實廠透過適當的調整F/M與溶氧能避免過度成長的絲狀菌造成的膨化問題。

Nowadays, there are many processes used in wastewater treatment including physical process, chemical process and biological process. The purpose is to remove pollutants that are harmful to human or ecosystem and make the effluent clean. Compared to other processes, biological treatment process, especially the activated sludge process (ASP) is widely used because it requires lower cost and is well established. While the performance of biological process sometimes could be affected by the fluctuation of influent in practice. Traditionally, the ASP is controlled by operational parameters while performance was directly evaluated by the water quality of influent and effluent, considering the ASP as a “black box” without understanding who is working inside. As the development of molecular biology tools, deeply understanding the microorganism in “black box” becomes possible. In this study, the activated sludge process in full-scale petrochemical industry was studied by monitoring the microbial communities and the wastewater characteristic in long-term period. Trying to link these two together and provide some useful information from the aspect of microorganism which could be used to improve or predict the ASP system. The standard methods and relative instruments were first used to analyze the water quality and characteristics including chemical oxygen demand (COD), nitrate, chloride ion, short-chain fatty acids, molecular weight distribution, volatile organic carbons (VOCs) and dissolved organic substances. The microbial information including community change and quantification were analyzed by molecular biology tools like next-generation sequencing (NGS) and quantitative polymerase chain reaction (qPCR) respectively. From the results of wastewater characteristics, the residual COD in the effluent may originate from the wastewater of VCM manufacturing process and their dissolved organic substance type could be classified into humic acid-like and fulvic acid-like region. In NGS results, Saprospiraceae and Hyphomicrobiaceae were observed as the dominant bacteria in family level (about 36% of total abundance) in the system. Saprospiraceae has been reported as filamentous or epifloral bacteria which cause bulking while Hyphomicrobiaceae was described as the methylotrophs and denitrifier. On the other hand, principal component analysis (PCA) was used and revealed that there was a changing point of microbial community due to the annual repair event. After quantifying dominant bacteria, the possible relationships were provided. The increase of influent COD would somehow decrease the amount of Hyphomicrobium after one month, while the increase of influent nitrate one month ago would increase the amount of Hyphomicrobium. Besides, there is a direct relationship between Saprospiraceae and food to microorganism ratio (F/M). The result indicated the tendency that higher amount of Saprospiraceae mostly occurred under the condition F/M higher than 0.3 kg COD/kg VSS-day and dissolved oxygen lower than 1.9 mg/L. These results could be served as a reference for bulking control.

Bartnicki, E.W., Castro, C.E., Biodehalogenation. The Pathway for Transhalogenation and the Stereochemistry of Epoxide Formation from Halohydrins. Biochemistry 8, 4677–4680, 1969.
Blanco, L., Hermosilla, D., Blanco, Á., Swinnen, N., Prieto, D., Negro, C., Assessment of the Performance of Membrane Bioreactors Applied to the Treatment of Industrial Effluents Containing Poly(vinyl alcohol). Ind. Eng. Chem. Res. 54, 5442–5449, 2015.
Bossert, I.D., Häggblom, M.M., Young, L.Y., Microbial Ecology of Dehalogenation, in: Dehalogenation. Kluwer Academic Publishers, pp. 33–52, 2005.
Cabezas, A., de Araujo, J.C., Callejas, C., Galès, A., Hamelin, J., Marone, A., Sousa, D.Z., Trably, E., Etchebehere, C., How to use molecular biology tools for the study of the anaerobic digestion process? Rev. Environ. Sci. Biotechnol. 2015.
Chaignaud, P., Maucourt, B., Weiman, M., Alberti, A., Kolb, S., Cruveiller, S., Vuilleumier, S., Bringel, F., Genomic and transcriptomic analysis of growth-supporting dehalogenation of chlorinated methanes in Methylobacterium. Front. Microbiol. 8, 2017.
Chen, C., Ming, J., Yoza, B.A., Liang, J., Li, Q.X., Guo, H., Liu, Z., Deng, J., Wang, Q., Characterization of aerobic granular sludge used for the treatment of petroleum wastewater. Bioresour. Technol. 271, 353–359, 2019.
Chen, W., Westerhoff, P., Leenheer, J.A., Booksh, K., Fluorescence Excitation-Emission Matrix Regional Integration to Quantify Spectra for Dissolved Organic Matter. Environ. Sci. Technol. 37, 5701–5710, 2003.
Davis, B.J.K., Jacobs, J.M., Zaitchik, B., DePaola, A., Curriero, F.C., Vibrio parahaemolyticus in the Chesapeake Bay: Operational in situ prediction and forecast models can benefit from inclusion of lagged water quality measurements. Appl. Environ. Microbiol. 85, 2019.
Díaz, M.P., Boyd, K.G., Grigson, S.J.W., Burgess, J.G., Biodegradation of crude oil across a wide range of salinities by an extremely halotolerant bacterial consortium MPD-M, immobilized onto polypropylene fibers. Biotechnol. Bioeng. 79, 145–153, 2002.
Fesefeldt, A., Holm, N.C., Gliesche, C.G., Genetic diversity and population dynamics of Hyphomicrobium spp. in a sewage treatment plant and its receiving lake. Water Sci. Technol. 37, 113–116, 1998.
Fetzner, S., Muller, R., Lingens1, F., Purification and Some Properties of 2-Halobenzoate 1,2-Dioxygenase, a Two-Component Enzyme System from Pseudomonas cepacia 2CBS, JOURNAL OF BACTERIOLOGY 1992.
Fierer, N., Jackson, J.A., Vilgalys, R., Jackson, R.B., Assessment of soil microbial community structure by use of taxon-specific quantitative PCR assays. Appl. Environ. Microbiol. 71, 4117–4120, 2005.
Gauthier, M.J., Flatau, G.N., Breittmayer, V.A., Protective effect of glycine betaine on survival of Escherichia coli cells in marine environments, in: Water Science and Technology 1991.
Gribble, G.W., A recent survey of naturally occurring organohalogen compounds. Environ. Chem. 12, 396, 2015.
Guo, F., Zhang, T., Profiling bulking and foaming bacteria in activated sludge by high throughput sequencing. Water Res. 46, 2772–2782, 2012.
Hong, J., Li, W., Lin, B., Zhan, M., Liu, C., Chen, B.Y., Deciphering the effect of salinity on the performance of submerged membrane bioreactor for aquaculture of bacterial community. Desalination 2013.
Huang, Z., Gedalanga, P.B., Asvapathanagul, P., Olson, B.H., Influence of physicochemical and operational parameters on Nitrobacter and Nitrospira communities in an aerobic activated sludge bioreactor. Water Res. 44, 4351–4358, 2010.
Isidorov, V.A., Zenkevich, I.G., Ioffe, B. V, Volatile Organic Compounds in Solfataric Gases, Journal of Atmospheric Chemistry 1990.
Ju, F., Xia, Y., Guo, F., Wang, Z., Zhang, T., Taxonomic relatedness shapes bacterial assembly in activated sludge of globally distributed wastewater treatment plants. Environ. Microbiol. 16, 2421–2432, 2014.
Kaetzke, A., Jentzsch, D., Eschrich, K., Quantification of Microthrix parvicella in activated sludge bacterial communities by real-time PCR. Lett. Appl. Microbiol. 40, 207–211, 2005.
Kim, E., Yulisa, A., Kim, S., Hwang, S., Monitoring microbial community structure and variations in a full-scale petroleum refinery wastewater treatment plant. Bioresour. Technol. 306, 123178, 2020.
Kim, J., Lim, J., Lee, C., Quantitative real-time PCR approaches for microbial community studies in wastewater treatment systems: Applications and considerations. Biotechnol. Adv. 2013.
Kincannon, D.F., Gaudy, A.F., Response of biological waste treatment systems to changes in salt concentrations. Biotechnol. Bioeng. 10, 483–496, 1968.
Kiragosyan, K., van Veelen, P., Gupta, S., Tomaszewska-Porada, A., Roman, P., Timmers, P.H.A., Development of quantitative PCR for the detection of Alkalilimnicola ehrlichii, Thioalkalivibrio sulfidiphilus and Thioalkalibacter halophilus in gas biodesulfurization processes. AMB Express 9, 1–8, 2019.
Kohler-Staub, D., Frank, S., Leisinger, T., Dichloromethane as the sole carbon source for Hyphomicrobium sp. strain DM2 under denitrification conditions. Biodegradation 6, 229–235, 1995.
Kohler-Staub, D., Leisinger, T., Dichloromethane Dehalogenase of Hyphomicrobium sp. Strain DM2 1985.
Kong, Y., Xia, Y., Nielsen, J.L., Nielsen, P.H., Structure and function of the microbial community in a full-scale enhanced biological phosphorus removal plant. Microbiology 153, 4061–4073, 2007.
Kumar, G., Cho, S.K., Sivagurunathan, P., Anburajan, P., Mahapatra, D.M., Park, J.H., Pugazhendhi, A., Insights into evolutionary trends in molecular biology tools in microbial screening for biohydrogen production through dark fermentation. Int. J. Hydrogen Energy 2018.
Kylefors, K., Ecke, H., Lagerkvist, A., Accuracy of cod test for landfill leachates. Water. Air. Soil Pollut. 146, 153–169, 2003.
Lai, C.-H., Optimization of Biological treatment for petrochemical wastewater and development of molecular tools for monitoring functional microorganisms 2020.
Lefebvre, O., Habouzit, F., Bru, V., Delgenes, J.P., Godon, J.J., Moletta, R., Treatment of hypersaline industrial wastewater by a microbial consortium in a sequencing batch reactor. Environ. Technol. 25, 543–553, 2004.
Lešinský, D., Fritz, J., Braun, R., Biological degradation of PVA/CH blends in terrestrial and aquatic conditions. Bioresour. Technol. 96, 197–201, 2005.
Levantesi, C., Beimfohr, C., Geurkink, B., Rossetti, S., Thelen, K., Krooneman, J., Snaidr, J., Van Der Waarde, J., Tandoi, V., Filamentous Alphaproteobacteria associated with bulking in industrial wastewater treatment plants. Syst. Appl. Microbiol. 27, 716–727, 2004.
Li, B., Wei, Z., Huang, Z., Xiao, X., Ming, S., Jiao, H., Cheng, X., Removal of Toluene from Synthetic Waste Gas through Aerobic Denitrification in Biotrickling Reactor. Environ. Eng. Sci. 37, 769–781, 2020.
Li, W.H., Sheng, G.P., Liu, X.W., Yu, H.Q., Characterizing the extracellular and intracellular fluorescent products of activated sludge in a sequencing batch reactor. Water Res. 42, 3173–3181, 2008.
Löffler, F., Müller, R., Lingens, F., Dehalogenation of 4-chlorobenzoate by 4-chlorobenzoate dehalogenase from Pseudomonas sp. CBS3: An ATP/coenzyme A dependent reaction. Biochem. Biophys. Res. Commun. 176, 1106–1111, 1991.
Ludzack, F.J., Noran, D.K., Tolerance of High Salinities by Conventional Wastewater Treatment Processes. J. (Water Pollut. Control Fed. 37, 1404–1416, 1965.
Madoni, P., Davoli, D., Gibin, G., Survey of filamentous microorganisms from bulking and foaming activated- sludge plants in Italy. Water Res. 34, 1767–1772, 2000.
Martínez, J.P., Garay, E., Alcaide, E., Hernández, E., The genus thiobacillus: Physiology and industrial applications. Acta Biotechnol. 3, 99–124, 1983.
Maymó-Gatell, X., Chien, Y.T., Gossett, J.M., Zinder, S.H., Isolation of a bacterium that reductively dechlorinates tetrachloroethene to ethene. Science (80-. ). 276, 1568–1571, 1997.
Meßmer, M., Reinhardt, S., Wohlfarth, G., Diekert, G., Studies on methyl chloride dehalogenase and O-demethylase in cell extracts of the homoacetogen strain MC based on a newly developed coupled enzyme assay. Arch. Microbiol. 165, 18–25, 1996.
Miao, Y., Wang, Z., Liao, R., Shi, P., Li, A., Assessment of phenol effect on microbial community structure and function in an anaerobic denitrifying process treating high concentration nitrate wastewater 2017.
Miyoshi, T., Tsuyuhara, T., Ogyu, R., Kimura, K., Watanabe, Y., Seasonal variation in membrane fouling in membrane bioreactors (MBRs) treating municipal wastewater. Water Res. 43, 5109–5118, 2009.
Muneron Mello, J.M., Brandão, H.L., Valério, A., de Souza, A.A.U., de Oliveira, D., da Silva, A., de Souza, S.M.A.G.U., Biodegradation of BTEX compounds from petrochemical wastewater: Kinetic and toxicity. J. Water Process Eng. 32, 2019.
Munz, G., Gori, R., Mori, G., Lubello, C., Powdered activated carbon and membrane bioreactors (MBRPAC) for tannery wastewater treatment: long term effect on biological and filtration process performances. Desalination 207, 349–360, 2007.
Nagata,’, Y., Nariya,’, T., Ohtomo,’, R., Fukuda, M., Yano, K., Takagi’, M., Cloning and Sequencing of a Dehalogenase Gene Encoding an Enzyme with Hydrolase Activity Involved in the Degradation of y-Hexachlorocyclohexane in Pseudomonas paucimobilis, JOURNAL OF BACTERIOLOGY 1993.
Olson, T. Le, Van De Meene, A.M.L., Francis, J.N., Pierson, B.K., Blankenship, R.E., Pigment analysis of “Candidatus Chlorothrix halophila,” a green filamentous anoxygenic phototrophic bacterium. J. Bacteriol. 189, 4187–4195, 2007.
Ozalp, G., Gomec, C.Y., Ozturk, I., Gonuldinc, S., Altinbas, M., Effect of high salinity on anerobic treatment of low strength effluents, in: Water Science and Technology 2004.
Palm, J.C., Jenkins, D., Parker, D.S., Relationship between organic oxygen sludge dissolved loading , and concentration in the completely settleability mixed activated process sludge. J. WPCF 52, 2484–06, 1980.
Prieto, D., Swinnen, N., Blanco, L., Hermosilla, D., Cauwenberg, P., Blanco, Á., Negro, C., Drivers and economic aspects for the implementation of advanced wastewater treatment and water reuse in a PVC plant. Water Resour. Ind. 14, 26–30, 2016.
Putri, D.W., 國立成功大學 環境工程研究所 碩士論文 探討光電產業廢水生物處理程序中 dimethyl sulfoxide 與 dimethyl sulfide 對微生物族群之影響 2011.
Qi, W., Taherzadeh, M.J., Ruan, Y., Deng, Y., Chen, J.S., Lu, H.F., Xu, X.Y., Denitrification performance and microbial communities of solid-phase denitrifying reactors using poly (butylene succinate)/bamboo powder composite. Bioresour. Technol. 305, 123033, 2020.
RENE, E., KIM, S., PARK, H., Effect of COD/N ratio and salinity on the performance of sequencing batch reactors. Bioresour. Technol. 99, 839–846, 2008.
Rodríguez, E., García-Encina, P.A., Stams, A.J.M., Maphosa, F., Sousa, D.Z., Meta-omics approaches to understand and improve wastewater treatment systems. Rev. Environ. Sci. Biotechnol. 14, 385–406, 2015.
Russell, J.B., Cook, G.M., Energetics of Bacterial Growth: Balance of Anabolic and Catabolic Reactions, MICROBIOLOGICAL REVIEWS 1995.
Schäfer, H., Miller, L.G., Oremland, R.S., Murrell, J.C., Bacterial Cycling of Methyl Halides. Adv. Appl. Microbiol. 61, 307–346, 2007.
Schauer, M., Hahn, M.W., Diversity and phylogenetic affiliations of morphologically conspicuous large filamentous bacteria occurring in the pelagic zones of a broad spectrum of freshwater habitats. Appl. Environ. Microbiol. 71, 1931–1940, 2005.
Sezgin, M., Jenkins, D., Parker, D.S., A unified theory of filamentous activated sludge bulking. J. Water Pollut. Control Fed. 50, 362–381, 1978.
Shariati, S.R.P., Bonakdarpour, B., Zare, N., Ashtiani, F.Z., The effect of hydraulic retention time on the performance and fouling characteristics of membrane sequencing batch reactors used for the treatment of synthetic petroleum refinery wastewater. Bioresour. Technol. 102, 7692–7699, 2011.
Sheng, G.P., Yu, H.Q., Li, X.Y., Extracellular polymeric substances (EPS) of microbial aggregates in biological wastewater treatment systems: A review. Biotechnol. Adv. 2010.
Shi, K., Zhou, W., Zhao, H., Zhang, Y., Performance of halophilic marine bacteria inocula on nutrient removal from hypersaline wastewater in an intermittently aerated biological filter. Bioresour. Technol. 113, 280–287, 2012.
Stefanis, S.K., Livingston, A.G., Pistikopoulos, E.N., Minimizing the environmental impact of process Plants: A process systems methodology. Comput. Chem. Eng. 19, 39–44, 1995.
Suman Raj, D.S., Anjaneyulu, Y., Evaluation of biokinetic parameters for pharmaceutical wastewaters using aerobic oxidation integrated with chemical treatment. Process Biochem. 40, 165–175, 2005.
Tabassum, S., Ji, Q., Li, C., Chi, L., audia Silva, C.G., Ajlouni, A.F., Chu, C., Alnoman, R., Zhang, Z., Treatment of centrifugal mother liquid of polyvinyl chloride by internal circulation aerobic biofilm reactor: Lab to plant scale system 2018.
Tan, B.F., Ng, C., Nshimyimana, J.P., Loh, L.L., Gin, K.Y.H., Thompson, J.R., Next-generation sequencing (NGS) for assessment of microbial water quality: Current progress, challenges, and future opportunities. Front. Microbiol. 6, 2015.
Tobiszewski, M., Tsakovski, S., Simeonov, V., Namieśnik, J., Chlorinated solvents in a petrochemical wastewater treatment plant: An assessment of their removal using self-organising maps. Chemosphere 87, 962–968, 2012.
van Hylckama Vlieg, J.E., Janssen, D.B., Bacterial degradation of 3-chloroacrylic acid and the characterization of cis-and trans-specific dehalogenases, Biodegradatton 1992.
Voelkerding, K. V, Dames, S.A., Durtschi, J.D., Next-generation sequencing:from basic research to diagnostics. Clin. Chem. 2009.
Wu, C., Zhou, Y., Sun, Q., Fu, L., Xi, H., Yu, Y., Yu, R., Appling hydrolysis acidification-anoxic-oxic process in the treatment of petrochemical wastewater: From bench scale reactor to full scale wastewater treatment plant. J. Hazard. Mater. 309, 185–191, 2016a.
Wu, C., Zhou, Y., Sun, Q., Fu, L., Xi, H., Yu, Y., Yu, R., Appling hydrolysis acidification-anoxic-oxic process in the treatment of petrochemical wastewater: From bench scale reactor to full scale wastewater treatment plant. J. Hazard. Mater. 309, 185–191, 2016b.
Xia, Y., Kong, Y., Thomsen, T.R., Nielsen, P.H., Identification and ecophysiological characterization of epiphytic protein-hydrolyzing Saprospiraceae (“Candidatus epiflobacter” spp.) in activated sludge. Appl. Environ. Microbiol. 74, 2229–2238, 2008.
Yu, H.Q., Gu, G.W., Song, L.P., The effect of fill mode on the performance of sequencing-batch reactors treating various wastewaters. Bioresour. Technol. 58, 49–55, 1996.