Seung Gu Shin

A comprehensive microbial insight into two-stage anaerobic digestion of food waste-recycling wastewater

Microbial community structures were assessed in a two-stage anaerobic digestion system treating food waste-recycling wastewater. The reactors were operated for 390 d at 10 different hydraulic retention times (HRTs) ranging from 25 to 4 d. Stable operation was achieved with the overall chemical oxygen demand (COD) removal efficiency of 73.0-85.9% at organic loading rate of up to 35.6 g COD/L·d. Performance of the acidogenic reactors, however, changed significantly during operation. This change coincided with transition of the bacterial community from one dominated by Aeriscardovia- and Lactobacillus amylovorus-related species to one dominated by Lactobacillus acetotolerans- and Lactobacillus kefiri-like organisms. In methanogenic reactors, the microbial community structures also changed at this stage along with the shift from Methanoculleus- to Methanosarcina-like organisms. This trend was confirmed by the non-metric multidimensional scaling joint plot of microbial shifts along with performance parameters. These results indicated that the overall process performance was relatively stable compared to the dynamic changes in the microbial structures and the acidogenic performance.

Qualitative and quantitative assessment of microbial community in batch anaerobic digestion of secondary sludge

Microbial community shifts were determined by denaturing gradient gel electrophoresis (DGGE) and real-time PCR for an anaerobic batch digester treating secondary sludge. The batch process was successfully operated with an organic removal efficiency of 35% associated with a 91% decrease in the bacterial 16S rRNA gene concentration. The microbial community structures showed continuous shifts within four bacterial phyla and three archaeal orders. Several bacterial species, such as Fusibacter-related, Clostridium-like, and Syntrophus-like organisms, appeared to be responsible for acidogenesis or syntrophic acid degradation. Both hydrogenotrophic and aceticlastic methanogens appear to have been involved in the methanogenesis with the acidogenic products. The quantitative structure of the methanogenic populations varied continuously, with the growth of Methanomicrobiales and Methanosarcinales in series, to result in a Methanomicrobiales-dominant population. The ordination of microbial community structures demonstrated that the quantitative methanogenic structure converged to the seed inoculum while the bacterial and archaeal DGGE band patterns diverged. These results provide an insight into the microbial behavior in the transitional phase (e.g., a start-up period) of anaerobic sludge digestion.

Real-time PCR determination of rRNA gene copy number: absolute and relative quantification assays with Escherichia coli

Real-time polymerase chain reaction (PCR)-based methodology for the determination of rRNA gene (rrn) copy number was introduced and demonstrated. Both absolute and relative quantifications were tested with Escherichia coli. The separate detection of rRNA gene and chromosomal DNA was achieved using two primer sets, specific for 16S rRNA gene and for D-1-deoxyxylulose 5-phosphate synthase gene (dxs), respectively. As dxs is a single-copy gene of E. coli chromosomal DNA, the rrn copy number can be determined as the copy ratio of rrn to dxs. This methodology was successfully applied to determine the rrn copy number in E. coli cells. The results from absolute and relative quantifications were identical and highly reproducible with coefficient of variation (CV) values of 1.8–4.6%. The estimated rrn copy numbers also corresponded to the previously reported value in E. coli (i.e., 7), indicating that the results were reliable. The methodology introduced in this study is faster and cost-effective without safety problems compared to the traditionally used Southern blot analysis. The fundamentals in our methodology would be applicable to any microorganism, as long as having the sequence information of the rRNA gene and another chromosomal gene with a known copy number.

Monitoring bacterial and archaeal community shifts in a mesophilic anaerobic batch reactor treating a high-strength organic wastewater

Shifts in bacterial and archaeal communities, associated with changes in chemical profiles, were investigated in an anaerobic batch reactor treating dairy-processing wastewater prepared with whey permeate powder. The dynamics of bacterial and archaeal populations were monitored by quantitative real-time PCR and showed good agreement with the process data. A rapid increase in bacterial populations and a high rate of substrate fermentation were observed during the initial period. Growth and regrowth of archaeal populations occurred with biphasic production of methane, corresponding to the diauxic consumption of acetate and propionate. Bacterial community structure was examined by denaturing gel gradient electrophoresis (DGGE) targeting 16S rRNA genes. An Aeromonas-like organism was suggested to be mainly responsible for the rapid fermentation of carbohydrate during the initial period. Several band sequences closely related to the Clostridium species, capable of carbohydrate fermentation, lactate or ethanol fermentation, and/or homoacetogenesis, were also detected. Statistical analyses of the DGGE profiles showed that the bacterial community structure, as well as the process performance, varied with the incubation time. Our results demonstrated that the bacterial community shifted, reflecting the performance changes and, particularly, that a significant community shift corresponded to a considerable process event. This suggested that the diagnosis of an anaerobic digestion process could be possible by monitoring bacterial community shifts.

Methanogenic population dynamics assessed by real-time quantitative PCR in sludge granule in upflow anaerobic sludge blanket treating swine wastewater

A pilot-scale upflow anaerobic sludge blanket (UASB) treating swine wastewater was operated for 382 days to evaluate the process performance and methanogenic population dynamics. A real-time quantitative PCR (QPCR) was used to detect and quantify the 16S rRNA gene concentrations of the domain Archaea, the four methanogenic orders, and the two aceticlastic families. Extended intervals of consistently stable and efficient wastewater treatment with a final hydraulic retention time (HRT) of 3.5 days were sustained. A high abundance of hydrogenotrophic methanogens was observed, with Methanobacteriales as the major group, suggesting that hydrogenotrophic methanogenesis, with the syntrophic oxidation of volatile fatty acids (VFAs), was a major route of methane formation. This phenomenon was mainly attributed to the high ammonium concentration in swine wastewater, which has a severe inhibitory effect mainly on aceticlastic methanogens. Although there was no significant growth of Methanosaetaceae, its abundance contributed to the formation and maintenance of granule.

Effect of high temperature on bacterial community dynamics in anaerobic acidogenesis using mesophilic sludge inoculum

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 degrees 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 anaerobic 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.

Quantitative and qualitative transitions of methanogen community structure during the batch anaerobic digestion of cheese-processing wastewater.

Qualitative and quantitative shifts in methanogen community structure, associated with process performance data, were investigated during the batch anaerobic digestion of a cheese-processing wastewater, whey permeate. Denaturing gradient gel electrophoresis (DGGE) and real-time PCR techniques were applied to obtain qualitative and quantitative microbial data sets, respectively, based on methanogen 16S rRNA genes. Throughout the operation, dynamic variations in both qualitative and quantitative community structures were observed, with repeated shifts in dominance between the aceticlastic Methanosarcinaceae (suggested mainly by the detection of a Methanosarcina-like population) and the hydrogenotrophic Methanomicrobiales (suggested mainly by the detection of a Methanofollis-like population). This trend corresponded well to the diauxic utilization of acetate and longer-chain fatty acids (C3–C6), mainly propionate. Joint-plot non-metric multidimensional scaling (NMS) analysis demonstrated that the qualitative and quantitative community shifts had significant correlations with the composition of residual organic acids and the methane production rate, respectively. This suggests the potential use of microbial community shift analysis as an indicative tool for diagnosing anaerobic digestion processes. The results suggest that more attention should be directed to quantitative, as well as qualitative, approaches for a better understanding of anaerobic digestion, particularly in terms of biogas production efficiency, under dynamic and transitional conditions.

Methanogenic community shift in anaerobic batch digesters treating swine wastewater

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 (7.9L/L, and 4.5L/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.

Continuous fermentation of food waste leachate for the production of volatile fatty acids and potential as a denitrification carbon source

This study investigated the simultaneous effects of hydraulic retention time (HRT) and pH on the continuous production of VFAs from food waste leachate using response surface analysis. The response surface approximations (R(2)=0.895, p<0.05) revealed that pH has a dominant effect on the specific VFA production (PTVFA) within the explored space (1-4-day HRT, pH 4.5-6.5). The estimated maximum PTVFA was 0.26g total VFAs/g CODf at 2.14-day HRT and pH 6.44, and the approximation was experimentally validated by running triplicate reactors under the estimated optimum conditions. The mixture of the filtrates recovered from these reactors was tested as a denitrification carbon source and demonstrated superior performance in terms of reaction rate and lag length relative to other chemicals, including acetate and methanol. The overall results provide helpful information for better design and control of continuous fermentation for producing waste-derived VFAs, an alternative carbon source for denitrification.

Common key acidogen populations in anaerobic reactors treating different wastewaters: Molecular identification and quantitative monitoring

Bacterial population dynamics during the start-up of three lab-scale anaerobic reactors treating different wastewaters, i.e., synthetic glucose wastewater, whey permeate, and liquefied sewage sludge, were assessed using a combination of denaturing gradient gel electrophoresis (DGGE) and real-time PCR techniques. The DGGE results showed that bacterial populations related to Aeromonas spp. and Clostridium sticklandii emerged as common and prominent acidogens in all reactors. Two real-time PCR primer/probe sets targeting Aeromonas or C. sticklandii were developed, and successfully applied to quantitatively investigate their dynamics in relation to changes in reactor performance. Quantitative analysis demonstrated that both Aeromonas- and C. sticklandii-related populations were highly abundant for acidogenic period in all reactors. Aeromonas populations accounted for up to 86.6-95.3% of total bacterial 16S rRNA genes during start-up, suggesting that, given its capability of utilizing carbohydrate, Aeromonas is likely the major acidogen group responsible for the rapid initial fermentation of carbohydrate. C. sticklandii, able to utilize specific amino acids only, occupied up to 8.5-55.2% of total bacterial 16S rRNA genes in the reactors tested. Growth of this population is inferred to be supported, at least in part, by non-substrate amino acid sources like cell debris or extracellular excretions, particularly in the reactor fed on synthetic glucose wastewater with no amino acid source. The quantitative dynamics of the two acidogen groups of interest, together with their putative functions, suggest that Aeromonas and C. sticklandii populations were numerically as well as functionally important in all reactors tested, regardless of the differences in substrate composition. Particularly, the members of Aeromonas supposedly play vital roles in anaerobic digesters treating various substrates under acidogenic, fermentative start-up conditions.