Anaerobic syntrophic consortia degrading terephthalate, methylbenzoate and benzoate

• Main Authors: Wu Jer-Horng, 吳哲宏
• Other Authors: Cheng Sheng-Shung
• Format: Others
• Language: zh-TW
• Published: 2001
• Online Access: http://ndltd.ncl.edu.tw/handle/14544554109053602968

博士 === 國立成功大學 === 環境工程學系 === 89 === Chemical production of Purified Terephthalic Acid (PTA) as the raw material was one of the important petrochemical and related industry in Taiwan. In addition to acetate, wastewater generated from the PTA manufacturing process mainly contained aromatic compounds including terephthalate (isomers), 4-methylbenzoate and benzoate. In the preliminary studies of biodegradability assays, the results revealed that the aromatic compounds were relatively slowly mineralized by the methanogenic consortia with the acclimation periods of more than 3 to 16 months. The thesis was thus aimed for providing the ecological insight into the microbial diversity associated with the degradation of terephthalate, 4-methylbenzoate and benzoate by the methanogenic consortia. Energetics analysis suggested that the carboxylated aromatic compounds had to be degraded syntrophically by fermenting bacteria at the conditions with the low levels of acetate and hydrogen which were manipulated by the methanogens in the methanogenic consortia. The syntrophic degradation was evidently supported by the observation that the primary intermediates such as acetate and hydrogen were identified in the degradation of aromatic compounds. With built-up of the acetate and/or hydrogen by adding the selective inhibitors (2-bromoethanesulfonic acid or sodium molybdate), the anaerobic degradation of terephthalate and 4-methylbenzoate reached the thresholds with the actual Gibbs free energy change in the range from —72 to —82 kJ/mol, equivalent to the energy quantum needed for the synthesis of one ATP unit, approximately. The energy conserved in the bacteria responsible for degrading terephthalate and 4-methylbenzoate was substantially higher than that for degrading benzoate and fatty acids. It was likely that the higher energy was conserved from the decarboxylation and demethylation processes. In addition, anaerobic degradation of terephthalate and 4-methylbenzoate was shown to be rate-limited in the fermentation step. Morphological characterization of the mature biofilm and the granulated sludge individually obtained from the anaerobic fluidized bed (AnFB) reactor and the upflow anaerobic sludge bed (UASB) reactors treating PTA wastewater was conducted by using the scanning and the transmission electron microcopies. The micrographs clearly demonstrated the architectures of the biofilm and the granule with the distribution of microbial clusters that were composed of the unidentified bacteria in syntrophic association with the hydrogenotrophic methanogen-like (Methanobrevibacter-like) and/or the acetoclastic methanogen-like (Methanosaeta-like and Methanosarcina-like) bacteria. The comparative results further revealed the morphotypic similarity of the overall microbial composition but the distribution discrepancy of the Methanosaeta-like and Methanosarcina-like populations between the biofilm and the granule degrading the mixed substrates of PTA wastewater. In the methanogenic consortia individually enriched with terephthalate, 4-methylbenaoate and benzoate, the methanogen-like populations were quite similar but the predominant bacteria with the rod morphotypes were obviously different. The rod bacteria with the distinctive traits were also frequently observed in the biofilm and granule from the AnFB and UASB reactors treating PTA wastewater, suggesting the role in the anaerobic degradation of aromatic compounds. Using 16S rDNA-based molecular techniques, the phylogenetic diversity of the members within the methanogenic consortia degrading terephthalate, 4-methylbenzoate and benzoate was investigated. 16S rDNA sequence fragments were amplified by polymerase chain reaction (PCR) with genomic DNA directly extracted from the methanogenic consortia and retrieved by the cloning and sequencing approach. Most of the retrieved 16S rDNA sequences were related to the populations without the known isolates, suggesting the unique community structures. Comparative analysis of the identified clonal sequences and closely related references from the public database indicated that the bacterial members, which might be involved in the aromatic degradation were phylogenetically affiliated with the prevalent divisions including Proteobacteria (delta subdivision), green non-sulfur bacteria (subdivision I), candidate novel MBA1 and many others, specific to the individual community. It was suggested that the members represented by the delta-proteobacterial sequences were the predominant populations in the three communities. More importantly, that the identified delta-proteobacterial sequences were individually classified into three clusters in the phylogenetic tree seemed to depend on the aromatic substrates, suggesting that the populations degrading terephthalate, 4-methylbenzoate and benzoate were probably different. Furthermore, the Archaea members within the terephthalate-enriched methanogenic consortium were found to closely affiliate with the Methanosaeta- and the Methanospirillum-related populations. Fluorescence in situ hybridization (FISH) with the group-specific, 16S rRNA-targeted oligonucleotide probes was used to localize the members in the granules. The fluorescent signals correctly hybridized with the targets revealed that the random or mixed distribution of the microbial populations was elucidated for the non-layered topology of the granule. The FISH technique with the 16S rRNA-targeted probe detected the presence of the Methanobacteriaceae, which the corresponding sequences were not retrieved in the clone library, further suggesting that the microbial diversity of the community could not completely unveiled by only one method. Based on the phenotypic and phylotypic database established in this thesis, it can provide the microbial indicators or information for the further application in the anaerobic processes treating PTA wastewater and in the isolation of the novel anaerobic microorganisms.