Kwanghyun Hwang

Quantitative analysis of methanogenic community dynamics in three anaerobic batch digesters treating different wastewaters.

Quantitative changes in methanogenic community structures, associated with performance data, were investigated in three anaerobic batch digesters treating synthetic glucose medium, whey permeate, and liquefied sewage sludge. All digesters were initially seeded with anaerobic sludge obtained from a local municipal wastewater treatment plant. Dynamics of methanogenic populations were monitored, at order and family levels, using real-time PCR based on the 16S rRNA gene. The molecular monitoring revealed that, in each digester, the quantitative structure of methanogenic community varied continuously over treatment time and the variation corresponded well to the changes in chemical profiles. Biphasic production of methane, associated with successive increases in aceticlastic (mainly Methanosarcinaceae) and hydrogenotrophic (mainly Methanomicrobiales) methanogenic groups, was observed in each digester. This corresponded to the diauxic utilization of acetate and longer-chain volatile fatty acids (C(3)-C(6)), mainly propionate. Additionally, the non-metric multidimensional scaling (NMDS) analysis of the quantification results demonstrated that the community shift patterns in three digesters were totally different from each other. Considering that the operating conditions in all trials were identical except substrates, the differences in quantitative shift profiles were suggested to be due to the different substrate compositions. This implied that the composition of wastewater could affect the evolution of quantitative methanogenic community structure in an anaerobic process. Overall, our results suggested that more attention to quantitative as well as qualitative approaches on microbial communities is needed for fundamental understanding of anaerobic processes, particularly under dynamic or transitional conditions.

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.

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.

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.

Effects of prolonged starvation on methanogenic population dynamics in anaerobic digestion of swine wastewater

This study investigated the relationship between the processes and microbial populations induced by long-term starvation. To demonstrate the effects of starvation, a laboratory-scale anaerobic reactor was operated in three phases (first reaction, starvation, second reaction) for 316 days. During the first reaction, the chemical oxygen demand (COD) concentration decreased by about 70% of the input swine wastewater and 64L of methane gas was produced; during the second reaction, there was a 63% COD reduction and 36L of methane was produced. The methanogenic diversity, qualitatively monitored with denaturing gradient gel electrophoresis using archaeal 16S rRNA gene primers, was not different between two reactions. However, DNA copy numbers of Methanosarcinales, quantitatively monitored with quantitative real-time polymerase chain reaction using order-level 16S rRNA gene primers, showed the changed results. Cell numbers of Methanosarcinales and methanogenic activity were important factors determining the different efficiencies of the process.

Use of order-specific primers to investigate the methanogenic diversity in acetate enrichment system

The applicability of order-specific primers in minimizing the possible underestimation of microbial diversity was evaluated via denaturing gradient gel electrophoresis (DGGE) analysis of a lab-scale anaerobic digester. Initially, a population analysis with real-time quantitative PCR demonstrated the existence of three methanogenic orders—Methanobacteriales, Methanomicrobiales, and Methanosarcinales—throughout the reaction period. DGGE analyses with three pairs of order-specific primers yielded eight operational taxonomic units (OTUs), whereas DGGE analysis with two independent Archaea-specific primers identified only five. Moreover, the order-specific primers amplified at least one OTU affiliated with each order, whereas no members of Methanobacteriales or Methanomicrobiales were identified with Archaea-specific primers in most samples. These findings provide evidence that order-specific analysis can detect the diversity of methanogens in greater detail than conventional Archaea-specific analysis.