산발효과정 최적화를 통한 돈분뇨의 이상혐기소화공정의 미생물 거동 및 주요 잔존 단백질 규명

Abstract

In this study, we investigated the microbial community dynamics in thermal acidogenesis using mesophilic sludge. From the result of optimization with a response surface methodology, the acidogenic optimum conditions predicted were a hydraulic retention time of 2.0 days and 51 ？C. Denaturing gradient gel electrophoresis (DGGE) profiles shows that the monitored bacterial community present consists of Pseudomonas mendocina, Bacillus halodurans, Clostridium hastiforme, Gracilibacter thermotolerans, and Thermomonas haemolytica. Among these, B. halodurans, G. thermotolerans, and T. haemolytica are reported to ferment carbohydrates thermotolerantly. In contrast, P. mendocina disappeared in the acidogenesis process because of its mesophilicity. In addition, C. hastiforme, G. thermotolerans originating from mesophilic anaeronic sludge were detected in the thermal acidogenesis. Based on this finding, we inferred that most thermophiles detected as DGGE bands could grow catalyzing carbohydrates metabolism in swine wastewater to produce volatile fatty acids thermotolerantly.The present study investigated the shifts in the chemical profiles of a two-phase anaerobic digestion system in methanogenic and acidogenic reactors for the treatment of swine wastewater. Acidogenic and methanogenic digesters were used with overall HRTs ranging from 27 to 6 d. In the optimized thermophilic / acidogenic phase throughout the entire experimental period, VS was reduced by 13.8% (1.6%)

however, COD hardly decreased because of the thermophilic hydrolysis of major organic materials, such as carbohydrates, proteins, and lipids, without any significant consumption of volatile fatty acids (VFAs). In the methanogenic/mesophilic phase, COD was reduced by 65.8 (1.1) % compared to a 47.4 (2.9)% reduction in VS reduction efficiency with the gradual increase in methane production during a methanogenic HRT between 25 and 10 d. This was probably due to the fact that methanogens consume a substantial amount of the VFAs produced as a result of the degradation of major organic materials by bacteria. A high protein degradation rate was observed in the optimized acidogenic phase, which is assumed to be due to the low content of carbohydrates in raw swine wastewater as well as the readily thermophilic hydrolysis of proteins. Additionally, the relatively high lipid degradation rate in the methanogenic phase is probably due to the fact that lipids were not involved in the formation of putative persistent organic materials during anaerobic digestion. Two-phase systems of anaerobic digestion consisting of optimized thermophilic and mesophilic methanogenic digesters exhibit comparative advantages with respect to VS reduction efficiency with OLRs less than 4 g VS/L&#8729

d. Qualitative and quantitative molecular analysis techniques were used to determine associations between differences in methanogenic microbial communities and the efficiency of batch anaerobic digesters. Two bioreactors were initially seeded with anaerobic sludge originating from a local municipal wastewater treatment plant and then supplemented with swine wastewater. Differences were observed in the total amount of methane produced in the two bioreactors (7.9 L/L, and 4.5 L/L, respectively). To explain these differences, efforts were taken to characterize the microbial populations present using a PCR-based DGGE analysis with methanogenic primer and probe sets. The groups Methanomicrobiales (MMB), Methanobacteriales (MBT), and Methanosarcinales (MSL) were detected, but Methanococcales (MCC) was not detected. Following this qualitative assay, real-time PCR was used to investigate quantitative differences in the populations of these methanogenic orders. MMB was found to be the dominant order present and its abundance patterns were different in the two digesters. The population profiles of the other methanogenic groups also differed. Through redundancy analysis, correlations between the concentrations of the different microbes and chemical properties such as volatile fatty acids were calculated. Correlations between MBT and MSL populations and chemical properties were found to be consistent in both digesters, however, differences were observed in the correlations between MMB and propionate. These results suggest that interactions between populations of MMB and other methanogens affected the final methane yield, despite MMB remaining the dominant group overall. The exact details of why changes in the MMB community caused different profiles of methane production could not be ascertained. However, this research provides evidence that microbial behavior is important for regulating the performance of anaerobic processes. In a two-stage anaerobic digestion process treating swine wastewater, the population dynamics of methanogens in the methanogenic stage were monitored at order level using real-time PCR at hydraulic retention times (HRTs) ranging from 25 to 5 d. Decreasing HRTs increased the loading rates of acetic, propionic, and butyric acids and ammonia. The relationship between the loading rate variation and methanogen community dynamics, and the relationship of these dynamics on daily methane production (DMP) and methane yield was statistically investigated using redundancy analysis. Methanomicrobiales (MMB) was dominant at longer than 12.5 d HRT, Methanosarcinales (MSL) was dominant at 8 and 10 d HRT, and Methanobacteriales (MBT) was dominant at shorter than 6 d HRT. Increased loading rates of volatile fatty acids (VFAs) and ammonia increased MBT, decreased MMB, and had no significant effect on MSL. DMP was positively correlated with populations of aceticlastic MSL. Methane yield increased slightly as MMB and MSL became predominant but decreased as MBT became predominant. The loading rates investigated seemed to be a selective pressure on the composition of methanogens

the methane production, in turn, was correlated with the methanogen community structure. These results suggest that multivariate analysis including quantitative methanogen populations can provide an insight into the understanding on the operation of anaerobic processes.Methanogenic plants at pilot scale were operated for 70 days at 20 days of HRT. To monitor the process, physicochemical parameters such as COD, sCOD, and VS, organics such as carbohydrates, proteins, and lipids, biogas production was investigated. Also, anaerobic digesters at serum bottle scale in the batch mode were operated to analyze persisteint proteins through SDS-PAGE and mass spectrometry. In the pilot plants, COD and VS reduction efficiencies were 65.3% (3.2), and 51.0% (5.7%), respectively. In particular, VS reduction efficiency in the pilot scale was lower than that in the lab scale, which might be due to the poor activity of acidogens in the pilot plants. Also, reductuion efficiencies of carbohydrates, proteins, and lipids were 57.3% (3.2%), 44.7% (4.6%), 60.7% (4.8%), respectively. In particular, carbohydrates reduction efficiency in the pilot scale was higher than in the lab scale, which was assumed to be due to the difference of proteins and carbohydrates compositions between pilot and lab scale plants. In order to analyze proteins from raw swine wastewater and methanogenic effluent in the pilot plants and serum bottle digesters, SDS-PAGE was carried out. As a result of it, five bands was detected as major bands, referring to kind of persistent proteins. To determine these bands, mass spectrometry was also accomplished. Through this spectrometry, three other proteins groups were determined as lipoproteins, Coenzyme-B sulfoethylthiotransferase, and N-acetyl-L-glutamate kinase (NAG kinase) as main remaining proteins. Since each protein was an essential enzyme for maintaining lives of microorganisms such as methanogens or acidogens in the anaerobic digesters, an additional subsequent reactor was considered to be necessary in order to remove them efficiently.