Seokhwan 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.

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.

Selective optimization in thermophilic acidogenesis of cheese-whey wastewater to acetic and butyric acids : partial acidification and methanation

For partial acidogenesis of cheese-whey wastewater, a set of experiments were carried out to produce short-chain volatile fatty acids (VFA) in laboratory-scale continuously stirred tank reactors (CSTR). The maximum rate of acetic and butyric acid production associated with simultaneous changes in hydraulic retention time (HRT), pH, and temperature was investigated, in which the degree of acidification of the whey to the short-chain VFAs was less than 20% of the influent chemical oxygen demand (COD) concentration. Response surface methodology was successfully applied to determine the optimum physiological conditions where the maximum rates of acetic and butyric acid production occurred. These were 0.40-day HRT, pH 6.0 at 54.1 degrees C and 0.22-day HRT, pH 6.5 at 51.9 degrees C, respectively. The optimum conditions for acetic acid production were selected for partial acidification of cheese-whey wastewater because of a higher rate in combined productions of acetic and butyric acids than that at optimum conditions for butyric acid production. A thermophilic two-phase process with the partial acidification followed by a methanation step was operated. Performance of the two-phase process was compared to the single-phase anaerobic system. The two-phase process clearly showed a better performance in management of cheese-whey wastewater over the single-phase system. Maximum rate of COD removal and the rate of methane production in the two-phase process were, respectively, 116% and 43% higher than those of the single-phase system.

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 output power, target temperature, and solid concentration on the solubilization of waste activated sludge using microwave irradiation.

In this paper, we quantify the effect of heating pretreatment on the degree of solubilization of waste activated sludge. The pretreatment process was carried out using a lab-scale industrial microwave unit (2450 MHz frequency). Response surface analysis was applied to determine the combination of output power (400-1600 W), target temperature (60-120 degrees C), and total solid concentration (1-3% total solid (TS)). The power, temperature, and TS concentration significantly affected the solubilization degree of sludge. Within the design boundaries, the conditions predicted to maximize the solubilization degree of 17.9% were determined to be 400 W, 102 degrees C, and 2.3% TS.

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.