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The M13 bacteriophage, a nature‐inspired environmentally friendly biomaterial, is used as a perovskite crystal growth template and a grain boundary passivator in perovskite solar cells. The amino groups and carboxyl groups of amino acids on the M13 bacteriophage surface function as Lewis bases, interacting with the perovskite materials. The M13 bacteriophage‐added perovskite films show a larger grain size and reduced trap‐sites compared with the reference perovskite films. In addition, the existence of the M13 bacteriophage induces light scattering effect, which enhances the light absorption particularly in the long‐wavelength region around 825 nm. Both the passivation effect of the M13 bacteriophage coordinating to the perovskite defect sites and the light scattering effect intensify when the M13 virus‐added perovskite precursor solution is heated at 90 °C prior to the film formation. Heating the solution denatures the M13 bacteriophage by breaking their inter‐ and intra‐molecular bondings. The denatured M13 bacteriophage‐added perovskite solar cells exhibit an efficiency of 20.1% while the reference devices give an efficiency of 17.8%. The great improvement in efficiency comes from all of the three photovoltaic parameters, namely short‐circuit current, open‐circuit voltage, and fill factor, which correspond to the perovskite grain size, trap‐site passivation, and charge transport, respectively., The M13 bacteriophage functions as an effective perovskite growth template and a passivator in perovskite solar cells. This is owing to its filamentous and uniform dimension, as well as the amino acids on its surface. These effects enhance when the M13 viruses are denatured at high temperature. The efficiency increases from 17.8% to 20.1% upon addition of the denatured viruses.

Methanogenesis and sulfidogenesis, the major microbial reduction reactions occurring in the anaerobic digestion (AD) process, compete for common substrates. Therefore, the balance between methanogenic and sulfidogenic activities is important for efficient biogas production. In this study, changes in methanogenic and sulfidogenic performances in response to changes in organic loading rate (OLR) were examined in two digesters treating sulfur-rich macroalgal waste under mesophilic and thermophilic conditions, respectively. Both methanogenesis and sulfidogenesis were largely suppressed under thermophilic relative to mesophilic conditions, regardless of OLR. However, the suppressive effect was even more significant for sulfidogenesis, which may suggest an option for H2S control. The reactor microbial communities developed totally differently according to reactor temperature, with the abundance of both methanogens and sulfate-reducing bacteria being significantly higher under mesophilic conditions. In both reactors, sulfidogenic activity increased with increasing OLR. The findings of this study help to understand how temperature affects sulfidogenesis and methanogenesis during AD.

This study compared single- versus two-phase systems for semi-continuous anaerobic digestion of food waste without pH control at varying organic loading rates (OLRs). The methanogenic reactors of both systems required trace element supplementation for stable operation at 3.0 g VS (volatile solids)/L∙d or higher OLRs. Under trace-element supplemented conditions, both systems achieved stable and efficient performance at OLRs up to 4.0 g VS/L∙d. The two-phase system outperformed the single-phase system at 1.0–4.0 g VS/L∙d OLRs, but it failed at an OLR of 5.0 g VS/L∙d. Meanwhile, the single-phase system maintained the stable performance and reached its maximum methane production at this OLR. These results suggest that a single-phase configuration is more advantageous for robust treatment of food waste without pH control at high organic and hydraulic loads. Hydrogenotrophic methanogens dominated the methanogen community throughout the experiment in both systems. Microbial community structure shifts correlated with reactor operation and performance characteristics.

Microbial community structure in a farm-scale anaerobic digester treating swine manure was investigated during three process events: 1) prolonged starvation, and changes of 2) operating temperature (between meso- and thermophilic) and 3) hydraulic retention time (HRT). Except during the initial period, the digester was dominated by hydrogenotrophic methanogens (HMs). The bacterial community structure significantly shifted with operating temperature and HRT but not with long-term starvation. Clostridiales (26.5–54.4%) and Bacteroidales (2.5–13.7%) became dominant orders in the digester during the period of HM dominance. Abundance of diverse meso- and thermophilic bacteria increased during the same period; many of these species may be H 2 producers, and/or syntrophic acetate oxidizers. Some of these species showed positive correlations with [NH 4 + -N] ( p

This study investigated the anaerobic digestion of Ulva biomass as an approach to diversifying energy sources and managing seaweed waste in a cost-effective manner. Ulva species are often identified as the main culprit of serious macroalgal blooms around the world, and their sulfur-rich nature causes difficulties in handling the biomass. Two reactors in continuous (Rc) and sequencing batch (Rs) modes were operated with decreasing hydraulic retention time in a stepwise manner from 20 to 6 days. Rs allowed significantly higher methane productivity (0.19–0.22 L/g chemical oxygen demand [COD] fed) and biomass retention capacity than Rc (0–0.16 L/g COD fed) throughout the experiment. Interestingly, sulfide production was also higher in Rs than in Rc. These findings, together with microbial quantification results, suggested that Rs operation enhanced biomass retention and the activity of both methanogens and sulfate-reducing bacteria. The Rc microbial community was less diverse and more variable than the Rs community. Accordingly, the performance of Rc was more significantly affected by changes in hydraulic and thus organic loads. Aceticlastic Methanosaetaceae dominated the methanogen community in both reactors, with the abundance of methanogens being significantly higher in Rs than in Rc. This may explain the more efficient and stable methane production despite the greater sulfidogenic activity in Rs, particularly at high hydraulic loads. Together, the results suggest that sequencing batch operation is advantageous over conventional continuous flow operation for the biomethanation of Ulva biomass and potentially other sulfur-rich feedstocks.

Interspecies electron transfer (IET) between microbial populations with different functions is critical to stable anaerobic digestion. This study, in an attempt to facilitate IET, investigated the effect of magnetite supplementation on the biomethanation of dairy effluent in continuous mode. The magnetite-added reactor (RM) was significantly more resistant and resilient to process imbalance than the reactor run without magnetite addition (RC). RC showed unstable performance with repeated process upsets, but its performance improved to be comparable to that of RM after applying magnetite supplementation. Magnetite was particularly effective in stabilizing a build-up of propionic acid and therefore improving the process robustness and reliability. The enhanced biomethanation in terms of productivity and stability was attributed to the facilitated direct IET (DIET) between exoelectrogens and methanogens via magnetite particles. Methanosaeta was the predominant methanogen group in the experimental reactors and likely played a key role in both DIET-mediated carbon dioxide-reducing and aceticlastic methanogenesis.

The feasibility of co-digestion of Ulva with whey was investigated at varying substrate mixing ratios in two continuous reactors run with increasing and decreasing proportions of Ulva , respectively. Co-digestion with whey proved beneficial to the biomethanation of Ulva , with the methane yield being greater by up to 1.6-fold in co-digestion phases than in the Ulva mono-digestion phases. The experimental reactors responded differently, in terms of process performance and community structure, to the changes in the substrate mixing ratio. This can be attributed to the different operating regimes between two reactors, which may have caused the microbial communities to develop in different ways to acclimate. Methanosaeta -related populations were the predominant methanogens responsible for the production of methane regardless of different substrate mixing ratios in both reactors. Considering the methane recovery and the Ulva treatment capacity, the optimal fraction of Ulva in the substrate mixture is suggested to be 50–75%.

Potential of microalgal cultivation as an alternative approach to the treatment of anaerobic digestion (AD) effluents was examined using two representative Chlorella species, Chlorella vulgaris (CV) and Chlorella protothecoides (CP). Both species effectively removed NH4+-N from the AD effluents from four digesters treating different wastes under different operating conditions. In all experimental cultures on the AD effluents, NH4+-N (initial concentration, 40 mg/L) was completely removed within 10 days without residual NO3−-N or NO2−-N in batch mode. Compared to CP, CV showed greater biomass and lipid yields (advantageous for biodiesel production), regardless of the media used. Prolonged nitrogen starvation significantly increased the lipid accumulation in all cultures on the AD effluents, and the effect was more pronounced in the CV than in the CP cultures. On the other hand, compared to CV, CP showed significantly faster settling (advantageous for biomass harvesting) in all media. Our results suggest that the Chlorella cultivation on AD effluents under non-sterile, mixed-culture conditions may provide a viable way to manage and valorize the problematic effluents. Diverse bacteria derived from the AD effluents co-existed and presumably interacted with the Chlorella species in the cultures.

Anaerobic digestion (AD) of Ulva biomass, a promising next-generation feedstock for energy production, was investigated in sequencing batch mode. Over five cycles of operation, the methane yield decreased more than twofold (from 0.15 to 0.07 L/g CODfed), while the organic treatment efficiency (i.e., chemical oxygen demand (COD) removal) remained fairly constant (53.7–61.1%). Such changes in reactor performance were related with structural variations in the microbial community, particularly the bacterial community, with repeated cycles. Methanosaeta- and Methanolinea-related populations were most likely the main aceticlastic and hydrogenotrophic methanogens, respectively, in the reactor. The emergence and prevalence of sulfate-reducing bacteria (SRBs), primarily a Solitalea-related population, most likely resulted in increased consumption of organic substrates for sulfate reduction, rather than methane production, in later cycles. Our observations suggest that the metabolic properties of the reactor changed with the transition of the bacterial community structure over cycles, and the metabolic shift had a negative effect on methanogenesis. The sequencing batch operation strategy applied in this study was not suitable for maximizing methane production from Ulva biomass, although the treatment efficiency was fairly stable. Robust control of SRB activity is necessary for more stable and efficient biomethanation of Ulva biomass in sequencing batch mode.

Temperature is a crucial factor that significantly influences the microbial activity and so the methanation performance of an anaerobic digestion (AD) process. Therefore, how to control the operating temperature for optimal activity of the microbes involved is a key to stable AD. This study examined the response of a continuous anaerobic reactor to a series of temperature shifts over a wide range of 35-65 °C using a dairy-processing byproduct as model wastewater. During the long-term experiment for approximately 16 months, the reactor was subjected to stepwise temperature increases by 5 °C at a fixed HRT of 15 days. The reactor showed stable performance within the temperature range of 35-45 °C, with the methane production rate and yield being maximum at 45 °C (18% and 26% greater, respectively, than at 35 °C). However, the subsequent increase to 50 °C induced a sudden performance deterioration with a complete cessation of methane recovery, indicating that the temperature range between 45 °C and 50 °C had a critical impact on the transition of the reactor's methanogenic activity from mesophilic to thermophilic. This serious process perturbation was associated with a severe restructuring of the reactor microbial community structure, particularly of methanogens, quantitatively as well as qualitatively. Once restored by interrupted feeding for about two months, the reactor maintained fairly stable performance under thermophilic conditions until it was upset again at 65 °C. Interestingly, in contrast to most previous reports, hydrogenotrophs largely dominated the methanogen community at mesophilic temperatures while acetotrophs emerged as a major group at thermophilic temperature. This implies that the primary methanogenesis route of the reactor shifted from hydrogen- to acetate-utilizing pathways with the temperature shifts from mesophilic to thermophilic temperatures. Our observations suggest that a mesophilic digester may not need to be cooled at up to 45 °C in case of undesired temperature rise, for example, by excessive self-heating, which offers a possibility to reduce operating costs.

The effects of mild-temperature thermochemical pretreatments with HCl or NaOH on the solubilization and biomethanation of Ulva biomass were assessed. Within the explored region (0-0.2M HCl/NaOH, 60-90°C), both methods were effective for solubilization (about 2-fold increase in the proportion of soluble organics), particularly under high-temperature and high-chemical-dose conditions. However, increased solubilization was not translated into enhanced biogas production for both methods. Response surface analysis statistically revealed that HCl or NaOH addition enhances the solubilization degree while adversely affects the methanation. The thermal-only treatment at the upper-limit temperature (90°C) was estimated to maximize the biogas production for both methods, suggesting limited potential of HCl/NaOH treatment for enhanced Ulva biomethanation. Compared to HCl, NaOH had much stronger positive and negative effects on the solubilization and methanation, respectively. Methanosaeta was likely the dominant methanogen group in all trials. Bacterial community structure varied among the trials according primarily to HCl/NaOH addition.

The inoculation of conventional activated sludge is a prospective option against transferring anaerobic ammonium oxidation (ANAMMOX) biomass from a currently existing ANAMMOX process in places where large amounts of ANAMMOX biomass are not available. In this study, poly(vinyl alcohol)/sodium alginate gel beads were utilized as an immobilization system to entrap activated sludge. Despite the longer start-up periods than those in previous examples using the ANAMMOX inoculum, the acclimation of ANAMMOX bacteria in the gel beads was successful. The maximum nitrogen removal rate of 1.12 kg N/m3-day was established at a nitrogen loading rate of 1.26 ± 0.04 kg N/m3-day with a total nitrogen removal efficiency of 88.9%. The exponential growth rate in the initial phase was enhanced by a higher nitrogen loading rate. The area-to-volume ratio of the gel beads was the significant control factor for the lag period, showing an inverse relationship due to the internal mass transport limitation. Based on the 16S rRNA gene, a sharp increase in the amount of ANAMMOX bacteria was identified along with an increase in the nitrogen removal rate. The responsible ANAMMOX bacteria were the species related to Candidatus ‘Brocadia sinica’.

In article number 2000782, Il Jeon, Yutaka Matsuo, Jin‐Woo Oh, and co‐workers demonstrate the application of the M13 bacteriophages to perovskite solar cells. The M13 bacteriophage is a nature‐inspired material which has an aspect ratio of 880 nm to 6.6 nm. Along with the eco‐friendly merits, their uniform size and many Lewis base functional groups on the surface qualify them for crystal growth templates and passivators. The device efficiency improves from 17.8% to 20.1% upon the addition of the M13 bacteriophage. The effectiveness can be maximized by denaturing the M13 bacteriophage at 90 °C before the perovskite film fabrication. [Image: see text]

Biogas upgrading through chemical absorption is an energy-intensive and costly process, but it is essential for the higher-value utilization of biogas as biomethane. The loss of absorbents and the energy consumption for their regeneration lower the economic viability of the process. This study investigated the potential of human urine as an economical and sustainable source of solvent for biogas upgrading. Hydrolyzed urine (HU) is rich in ammonia (>4.4 g N/L) and alkaline (approximately pH 9), which makes it attractive as an absorbent to capture CO2. The hydrolysis characteristics of urine were assessed with and without urease addition at different temperatures (4, 25, and 35 °C). Urease induced immediate urine hydrolysis, and temperature had a more significant effect on the hydrolysis rate when urease was not added. Conductivity, but not pH, proved to be a reliable indicator for monitoring urine hydrolysis. The CO2 capture capacity observed in the biogas upgrading experiments with simulated biogas (60% CH4 and 40% CO2, v/v) was 0.41–0.53 mol CO2/mol total ammonia nitrogen (TAN). The experiments using real biogas (71% CH4) showed that CO2 was effectively captured by HU with complete removal of H2S (2440 ppmv). This study is the first to demonstrate the direct use of HU as a sole solvent for biogas upgrading and suggest the possibility of recovering fertilizer material (e.g., ammonium bicarbonate, NH4HCO3) from the spent HU. The results suggest interesting opportunities to improve the economic feasibility of biogas upgrading and to valorize human urine.

Mild-temperature thermo-alkaline pretreatment of spent coffee grounds (SCG) was studied to improve its solubilization and methanation. The simultaneous effects of NaOH concentration (0–0.2 M) and temperature (60–90 °C) were investigated. Significant solubilization of SCG was achieved by the pretreatment, particularly under high-NaOH-concentration and high-temperature conditions. However, adding NaOH above a certain concentration adversely affected the methane production. Therefore, the degree of solubilization (SD) correlated poorly with methane yield (Ym). Response surface models of SD and Ym were successfully generated. The maximum response of SD (36.4%) was obtained at 0.18 M NaOH and 90.0 °C, while that of Ym (263.31 mL CH4/g COD added) was obtained at 0.13 M NaOH and 70.5 °C. Hydrogenotrophic Methanospirillum species were the dominant methanogens in all the SCG digestion tests. It is likely that NaOH concentration had a more significant influence on the development of microbial community structure, particularly of methanogens than temperature.

Characterizing microbial community structure using molecular techniques is becoming a popular approach in studies of waste/wastewater treatment processes. A rapid and robust tool to analyze microbial communities is required for efficient process monitoring and control. In this study, a new community fingerprinting method based on high-resolution melting (HRM) analysis was developed and applied to compare methanogenic community structures of five different anaerobic digesters. The new method produced robust community clustering and ordination results comparable to the results from the commonly used denaturing gradient gel electrophoresis (DGGE) performed in parallel. This method transforms melting peak plots (MPs) of community DNA samples generated by HRM analysis to molecular fingerprints and estimates the relationships between the communities based on the fingerprints. The MP-based fingerprinting would provide a good alternative to monitor variations in microbial community structure especially when handling large sample numbers due to its high-throughput capacity and short analysis time.

Ulva biomass was evaluated as a co-substrate for anaerobic digestion of spent coffee grounds at varying organic loads (0.7–1.6 g chemical oxygen demand (COD)/L d) and substrate compositions. Co-digestion with Ulva (25%, COD basis) proved beneficial for SCG biomethanation in both terms of process performance and stability. The beneficial effect is much more pronounced at higher organic and hydraulic loads, with the highest COD removal and methane yield being 51.8% and 0.19 L/g COD fed, respectively. The reactor microbial community structure changed dynamically during the experiment, and a dominance shift from hydrogenotrophic to aceticlastic methanogens occurred with increase in organic loading rate. Network analysis provides a comprehensive view of the microbial interactions involved in the system and confirms a direct positive correlation between Ulva input and methane productivity. A group of populations, including Methanobacterium - and Methanoculleus -related methanogens, was identified as a possible indicator for monitoring the biomethanation performance.

Functioning biocathodes are essential for electromethanogenesis. This study investigated the development of a biocathode from non-acclimated anaerobic sludge in an electromethanogenesis cell at a cathode potential of -0.7V (vs. standard hydrogen electrode) over four cycles of repeated batch operations. The CO2-to-CH4 conversion rate increased (to 97.7%) while the length of the lag phase decreased as the number of cycles increased, suggesting that a functioning biocathode developed during the repeated subculturing cycles. CO2-resupply test results suggested that the biocathode catalyzed the formation of CH4 via both direct and indirect (H2-mediated) electron transfer mechanisms. The biocathode archaeal community was dominated by the genus Methanobacterium, and most archaeal sequences (>89%) were affiliated with Methanobacterium palustre. The bacterial community was dominated by putative electroactive bacteria, with Arcobacter, which is rarely observed in biocathodes, forming the largest population. These electroactive bacteria were likely involved in electron transfer between the cathode and the methanogens.

Promotion of direct interspecies electron transfer (DIET) between exoelectrogenic bacteria and electron-utilizing methanogens has recently been discussed as a new method for enhanced biomethanation. This study evaluated the effect of magnetite-promoted DIET in continuous anaerobic digestion of dairy effluent and tested the magnetic separation and recycling of magnetite to avoid continuous magnetite addition. The applied magnetite recycling method effectively supported enhanced DIET activity and biomethanation performance over a long period (>250days) without adding extra magnetite. DIET via magnetite particles as electrical conduits was likely the main mechanism for the enhanced biomethanation. Magnetite formed complex aggregate structures with microbes, and magnetite recycling also helped retain more biomass in the process. Methanosaeta was likely the major methanogen group responsible for DIET-based methanogenesis, in association with Proteobacteria and Chloroflexi populations as syntrophic partners. The recycling approach proved robust and effective, highlighting the potential of magnetite recycling for high-rate biomethanation.

Mesophilic biomethanation of Ulva biomass was performed in a batch bioreactor, and a high organic removal of 77% was obtained on the basis of chemical oxygen demand (COD) after a month of operation. The estimated methane yield was 0.43 ± 0.02 L CH4/g COD(removed) which is close to the theoretical methane potential. Transitions of bacterial and archaeal community structures, associated with process performance data, were investigated using a combination of molecular fingerprinting and biostatistical tools. During the operation, archaeal community structure had no significant changes while bacterial community structure shifted continuously and dynamically. The reactor completely stabilized volatile fatty acids (primarily acetate and propionate) accumulated from the acidogenesis phase, with Methanosaeta- and Methanolinea-related microbes respectively being the main aceticlastic and hydrogenotrophic methanogens. Methanolinea- and Syntrophobacter-related populations were likely the key members to form a syntrophic propionate-degrading consortium. A Methanolinea-related population was likely the dominant methane producer in the experimental reactor.

Quantitative real-time PCR (qPCR) has been widely used in recent environmental microbial ecology studies as a tool for detecting and quantifying microorganisms of interest, which aids in better understandings of the complexity of wastewater microbial communities. Although qPCR can be used to provide more specific and accurate quantification than other molecular techniques, it does have limitations that must be considered when applying it in practice. This article reviews the principle of qPCR quantification and its applications to microbial ecology studies in various wastewater treatment environments. Here we also address several limitations of qPCR-based approaches that can affect the validity of quantification data: template nucleic acid quality, nucleic acid extraction efficiency, specificity of group-specific primers and probes, amplification of nonviable DNA, gene copy number variation, and limited number of sequences in the database. Even with such limitations, qPCR is reportedly among the best methods for quantitatively investigating environmental microbial communities. The application of qPCR is and will continue to be increasingly common in studies of wastewater treatment systems. To obtain reliable analyses, however, the limitations that have often been overlooked must be carefully considered when interpreting the results.

This study investigated changes in microbial community structure, associated with changes in process performance, in an anaerobic bioreactor treating cheese-processing wastewater in repeated-batch mode. During the operation of three batch cycles, the overall reaction rate increased with the increase in starting microbial population size over cycles, indicating a positive effect of biomass accumulation on process performance. Bacterial community structure varied significantly over cycles in the reactor. In contrast, methanogen community structure was maintained almost unchanged (>90% similarity) during the experiment. This indicates that changes in bacterial community structure interestingly had little influence on the formation of methanogen community structure in the reactor. Another notable point is that the reactor methanogen community was greatly dominated by hydrogenotrophic methanogens (up to 90% of the total population), likely suggesting H2-utilizing pathway as the major methanogenesis route in the reactor. This is contrary to the conventional wisdom that aceticlastic methanogens are the main drivers of methanogenesis in anaerobic digesters under normal conditions.

Mixed-culture fermentation that does not require an energy-intensive sterilization process is a viable approach for the economically feasible production of lactic acid (LA) due to the potential use of organic waste as feedstock. This study investigated mixed-culture LA fermentation of whey, a high-strength organic wastewater, in continuous mode. Variations in the hydraulic retention time (HRT) from 120 to 8 h under different pH regimes in two thermophilic reactors (55 °C) were compared for their fermentation performance. One reactor was maintained at a low pH (pH 3.0) during operation at HRTs of 120 to 24 h and then adjusted to pH 5.5 in the later phases of fermentation at HRTs of 24 to 8 h (R1), while the second reactor was maintained at pH 5.5 throughout the experiment (R2). Although the LA production in R1 was negligible at low pH, it increased dramatically after the pH was raised to 5.5 and exceeded that in R2 when stabilized at HRTs of 8 and 12 h. The maximum yield (0.62 g LA/g substrate fed as the chemical oxygen demand (COD) equivalent), the production rate (11.5 g/L day), and the selectivity (95 %) of LA were all determined at a 12-h HRT in R1. Additionally, molecular and statistical analyses revealed that changes in the HRT and the pH significantly affected the bacterial community structure and thus the fermentation characteristics of the experimental reactors. Bacillus coagulans was likely the predominant LA producer in both reactors. The overall results suggest that low pH start-up has a positive effect on yield and selectivity in mixed-culture LA fermentation.

Purpose Oxidative stress has been implicated in the pathogenesis of various diseases. Catalase is one of the main defense mechanisms against oxidative stress. To examine the possible relationship between oxidative stress, and gastric and hepatocellular carcinomas, HinfI restriction length polymorphism (RFLP) in the human catalase gene was assessed. Materials and methods The genotype and allele frequencies in the promoter region of the catalase gene were studied by PCR-RFLP in 108 Korean controls, 80 Korean gastric carcinoma (GC) and 106 Korean hepatocellular carcinoma (HCC) patients. Results No statistically significant differences were found in the genotypic distribution and allelic frequencies between the controls and both types of carcinoma patient. Conclusion To address the possible contribution of oxidative stresses to the pathogenesis of gastric and hepatocellular carcinomas, the associations between the catalase gene polymorphism and GC and HCC susceptibilities were studied. As a result, the catalase gene polymorphism was found not to be determinant of GC and HCC susceptibilities. Further studies are required on various other oxidative stress related genes to elucidate the mechanisms of GC and HCC.

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.

Granular biofilms underpin the operation of several categories of anaerobic wastewater treatment bioreactors. Recent studies have demonstrated the feasibility of treating both industrial and domestic wastewaters at their discharge temperatures (usually

A medicinal mushroom, Phellinus linteus, was successfully cultivated using a cheese-processing waste, whey, and the optimal bioconversion conditions for the maximum mycelial growth rate was also estimated through solid-state cultivation experiments. Response surface analysis with a face-centered design (center point replication = 5) was applied to statistically approximate the simultaneous effects of the three variables, i.e., substrate concentration (10–30 g lactose l−1), temperature (20–30°C), and pH (4–6), on the mycelial growth rate of P. linteus. The following is a partial cubic model where η is the mycelial growth rate (K r ) and x k is the corresponding variable term (k = substrate concentration, temperature, and pH in order): η = −23.8 + 8.67 × 10−2 x 1 + 1.48x 2 + 1.77x 3 + 8.00 × 10−4 x 1 x 2 + 7.25 × 10−2 x 1 x 3 + 5.13 × 10−2 x 2 x 3 −1.28 × 10−2 x 1 2 –3.18 × 10−2 x 2 2 . −2.64 × 10−1 x 3 2 −3.28 × 10−3 x 1 x 2 x 3 + 4.68 × 10−4 x 1 2 x 2. The produced response surface model proved to be significant (r 2 > 0.99, P-value

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 (C(3)-C(6)), 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.

Anaerobic granulation describes the self-immobilisation of methanogenic consortia into dense, particulate biofilms. This procedure underpins the operation of several categories of high-rate anaerobic wastewater treatment system. Full-scale anaerobic granular sludge plants have been generally operated in the mesophilic (20-45 degrees C) or thermophilic (45-65 degrees C) temperature range. On the other hand, recent studies highlighted the economic advantages of treating wastewaters at their discharge temperatures (mostly under 18 degrees C), removing a costly heating process and increasing net biogas yield. However, as yet, relatively little information is available about the microbial behaviour and interactions in anaerobic granular sludge formed under psychrophilic conditions. To this end, and in order to provide a microbial insight into low-temperature anaerobic granulation, we monitored the changes in methanogenic community structure, associated with the changes in process performance. Three, laboratory-scale, expanded granular sludge bed (EGSB) bioreactors treating a synthetic glucose wastewater were tested at two temperatures of 37+/-1 degrees C (R1) and 15+/-1 degrees C (R2 and 3). Quantitative real-time PCR and specific methanogenic activity assays highlighted a community shift towards hydrogenotrophic methanogens, particularly the order Methanomicrobiales in the low-temperature bioreactors. Corresponding to this, denaturing gradient gel electrophoresis (DGGE) analysis identified the emergence and maintenance of a Methanocorpusculum-like organism. Our results indicate that hydrogenotrophic methanogens, particularly the Methanomicrobiales-related populations, are likely to play important roles in low-temperature anaerobic granular sludge systems. This suggests that the process efficiency could be improved by facilitating the growth and retention of this group.

This study investigated the bacterial community of a full-scale anaerobic digester, which suffers from intermittent seawater contaminations, using 16S rRNA gene clone analysis over different seasons. Bacteroidetes, Proteobacteria, and unclassifiable bacteria were the three major bacterial groups within the clone library (a total of 290 clones). A significant portion of the total clones (29.3%) was not affiliated to any previously reported phylum, and 55.3% of the unclassifiable clones (16.9% of the total clones) showed potential relations to the species of Thermotogae, rarely present under normal mesophilic anaerobic conditions. These results suggested that the novel populations may have the potential to play an important role in anaerobic processes, particularly under abnormal environmental conditions. Additionally, statistical analysis supported that seasonal variations in influent characteristics, and potential competitions among different populations, may be related to the unusual bacterial diversity and community dynamics observed over the study period.

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.

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.

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.

Real-time QPCR based methods for determination of plasmid copy number in recombinant Escherichia coli cultures are presented. Two compatible methods based on absolute and relative analyses were tested with recombinant E. coli DH5α harboring pBR322, which is a common bacterial cloning vector. The separate detection of the plasmid and the host chromosomal DNA was achieved using two separate primer sets, specific for the plasmid β-lactamase gene ( bla ) and for the chromosomal d -1-deoxyxylulose 5-phosphate synthase gene ( dxs ), respectively. Since both bla and dxs are single-copy genes of pBR322 and E. coli chromosomal DNA, respectively, the plasmid copy number can be determined as the copy ratio of bla to dxs . These methods were successfully applied to determine the plasmid copy number of pBR322 of E. coli host cells. The results of the absolute and relative analyses were identical and highly reproducible with coefficient of variation (CV) values of 2.8–3.9% and 4.7–5.4%, respectively. The results corresponded to the previously reported values of pBR322 copy number within E. coli host cells, 15–20. The methods introduced in this study are convenient to perform and cost-effective compared to the traditionally used Southern blot method. The primer sets designed in this study can be used to determine plasmid copy number of any recombinant E. coli with a plasmid vector having bla gene.

Real-time polymerase chain reaction (PCR) is a highly sensitive method that can be used for the detection and quantification of microbial populations without cultivating them in anaerobic processes and environmental samples. This work was conducted to design primer and probe sets for the detection of methanogens using a real-time PCR with the TaqMan system. Six group-specific methanogenic primer and probe sets were designed. These sets separately detect four orders (Methanococcales, Methanobacteriales, Methanomicrobiales, and Methanosarcinales) along with two families (Methanosarcinaceae and Methanosaetaceae) of the order Methanosarcinales. We also designed the universal primer and probe sets that specifically detect the 16S rDNA of prokaryotes and of the domain Bacteria and Archaea, and which are fully compatible with the TaqMan real-time PCR system. Target-group specificity of each primer and probe set was empirically verified by testing DNA isolated from 28 archaeal cultures and by analyzing potential false results. In general, each primer and probe set was very specific to the target group. The primer and probe sets designed in this study can be used to detect and quantify the order-level (family-level in the case of Methanosarcinales) methanogenic groups in anaerobic biological processes and various environments.

A mixed bacterial culture capable of growing in potassium-thiocyanate containing medium (200 mg KSCN) has been isolated from bacterial suspensions of soil samples collected near gold mines in Kumjung (Korea). The isolates were initially characterized by metabolic profile analysis and were identified as Bacillus thermoglucosidasius, Bacillus cereus, Bacillus licheniformis, Bacillus mycoides, Brevibacterium epidermidis, Brevibacterium otitidis ,a ndCorynebacterium nitrilophilus. One of the seven isolates was initially characterized as Brevibacterium epidermidis, which is not known to degrade thiocyanate. However, using 16S rDNA sequencing, this strain was identified as a member of Klebsiella. The strain shows high similarity values (95.8 to 96.4%) with Klebsiella species, and the closest known relative was found to be K. ornithinolytica ATCC 31898. The result indicates that species of the genus Klebsiella were the closest phylogenetic relatives of the investigated strain. This is the first known report of a member of Klebsiella that is capable of utilizing thiocyanate as sole source of carbon and nitrogen.

The organic loading rate (OLR) is a critical factor that controls the treatment efficiency and biogas production in anaerobic digestion (AD). Therefore, organic shock loads may cause significant process imbalances accompanied by a drop in pH and acid accumulation or even failure. This study investigated the response of a continuous mesophilic anaerobic bioreactor to a series of transient organic shock loads of the substrate whey permeate, a high-strength organic wastewater from cheese making. The reactor was subjected to organic shock loads of increasing magnitude (a one-day pulse of elevated feed organic concentration) under controlled (near 7) and uncontrolled pH conditions at a fixed HRT of 10 days. The reactor was resilient to up to a shock load of up to 8.0 g SCOD/L·d under controlled pH conditions but failed to recover from the serious imbalance caused by a 3.0-g SCOD/L·d shock load, thus indicating the critical effect of pH on system resilience. The acidified reactor was not restored by interrupted feeding under the acidic conditions that were formed (pH ≤ 4.5) but was successfully restored after pH adjustment to 7. The reactor subsequently reverted to continuous mode without pH control and showed a performance comparable to the stable performance at the design OLR of 1.0 g SCOD/L·d. The bacterial community structure shifted dynamically in association with disturbances in the reactor conditions, whereas the archaeal community structure remained simple and less variable during the shock loading experiments. The structural shifts of the bacterial community were well correlated with the process performance changes, and performance recovery was generally accompanied by recovery of the bacterial community structure. The overall results suggest that the reactor pH, rather than simply acting as an accumulation of organic acids, had a crucial effect on the resilience and robustness of the microbial community and thus on the reactor performance under organic shock loads.

Low-temperature thermo-alkaline pretreatment of waste activated sludge (WAS) was studied, within the region of 0-0.2 M NaOH and 60-90°C, for the effects of NaOH concentration and temperature on sludge degradability in anaerobic digestion (AD). Significant disintegration of sludge solids (up to 75.6%) and an increase in methane production (up to 70.6%) were observed in the pretreatment trials. Two quadratic models were successfully generated by response surface analysis (R(2)>0.9, p

Microbial community structure and dynamics were examined in two anaerobic reactors run in repeated-batch mode to treat whey permeate. Despite being started up using different seeding sources, the reactors showed generally similar reaction patterns and performances. During the repeated-batch operation for three cycles, the overall reaction rate increased with the increase in the initial population size of both bacteria and methanogens over cycles. Clostridium- and Methanospirillum-related microorganisms were likely the main acidogenic and methanogenic populations, respectively, in both reactors. Bacterial community structure shifted dynamically over cycles, while little change was observed in methanogen community structure throughout the operation. This means that the changes in bacterial community structure changes had little influence on the formation and evolution of methanogen community structure in the reactors. The increased methanogenesis rate with cycles seemed therefore more likely due to the effect of the increase in methanogen abundance rather than the alteration of community structure.

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 organic materials, such as carbohydrates, proteins, and lipids, without any significant consumption of volatile fatty acids. 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. 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. Two-phase systems of anaerobic digestion consisting of optimized thermophilic and mesophilic methanogenic digesters showed a stable performance with respect to VS reduction efficiency with OLRs less than 3 g VS/L·d, in other words, more than 10 days of methanogenic HRT in this study.

Methanogenic community structure and population dynamics were investigated in two anaerobic reactors treating a dairy wastewater, an Inverted Fluidized Bed (IFB) and Expanded Granular Sludge Bed (EGSB). A combination of real-time PCR, denaturing gradient gel electrophoresis and statistical techniques was employed. Distinct methanogenic communities developed in the IFB and EGSB reactors reflecting step-wise reductions in the applied hydraulic retention time from 72 to 12 h during the 200-day trial. The aceticlastic family Methanosarcinaceae was only detected in the IFB and the order Methanomicrobiales was also much more abundant in this reactor, while the aceticlastic family Methanosaetaceae was more abundant in the EGSB. The hydrogenotrophic order, Methanobacteriales, predominated in both reactors under all applied operational conditions. Non-metric multidimensional scaling (NMS) and moving-window analyses, based on absolute and relative abundance quantification data, demonstrated that the methanogenic communities developed in a different manner in the IFB, compared to the EGSB reactor. In our study, relative abundance-based quantification by NMS and moving-window analysis appeared to be a valuable molecular approach that was more applicable to reflect the changes in the anaerobic digestion process than approaches based either on qualitative analysis, or solely on absolute quantification of the various methanogenic groups. The overall results and findings provided a comparative, quantitative and qualitative insight into anaerobic digestion processes, which could be helpful for better future reactor design and process control.

Although Aeromonas caviae is pathogenic to a broad range of invertebrates including human, frequent in aquatic environments, and potentially vital for acidogenesis in anaerobic digestion, virtually no biokinetic information on its anaerobic growth is at hand. Therefore, this study focused on evaluating its anaerobic growth kinetics on glucose. To provide a set of relevant biokinetic coefficients for modeling, a combination of curve fitting and numerical modeling was used. Microcultivations were performed at eight different initial glucose concentrations of 0.1 to 2.5 g l(-1) to establish a function of specific growth rate versus substrate concentration. A batch anaerobic bioreactor was then operated to collect a data set for the numerical analysis. Kinetic coefficients were estimated from three different biomass growth profiles monitored by optical density, volatile suspended solids (VSS), or DNA measurement, and applied for simulating continuous operations at various hydraulic retention times (HRTs). Assuming the influent glucose concentration is 5,000 mg l(-1), the substrate utilization efficiency predicted to be 77.2% to 92.0% at 17 to 36 h HRTs. For the VSS-model-based simulation, the washout HRT was estimated to be 16.6 h, and similar for the other models. Overall, the anaerobic biokinetic coefficients of A. caviae grown on glucose were successfully estimated and found to follow a substrate inhibition model.

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.

Changes in microbial community structure, associated with changes in process performance, were investigated with respect to the sludge retention time (SRT) in bioreactors treating thiocyanate. Among the seven reactors operated at 0.8–3.0 d SRTs, respectively, the reactor at 2.0 d SRT displayed the maximal thiocyanate removal rate of 240.2 mg/L/d. However, the thiocyanate removal efficiency suddenly decreased from 96.1% to 43.1% when the SRT was reduced from 2.0 to 1.8 d, corresponding to a 50.1% drop in the removal rate. Microbial communities in the reactors operated at short SRTs, near washout, were analyzed by denaturing gradient gel electrophoresis (DGGE) based on bacterial 16S rRNA genes. All band sequences recovered were assigned to two phyla, Proteobacteria and Bacteriodetes . A Thiobacillus -like microorganism was commonly detected in all the reactors and is suggested to be the main organism responsible for thiocyanate decomposition. Several DGGE band sequences were closely related to the environmental clones detected in environments rich in sulfur and/or nitrogen compounds. Statistical analysis of the DGGE profiles demonstrated that the structure of thiocyanate-degrading communities, as well as the process performance, changed with change in SRT. The microbial community profiles were not always more closely related to those at similar SRT than those at less similar SRT on the non-metric multidimensional scaling (NMDS) map. This was also supported by clustering analysis. These results were contrary to the general notion that the community structures in continuous systems will be controlled by the washout of microbial populations. Our experimental results suggest that the structure of a microbial thiocyanate-degrading community at a given SRT would not be determined only by the washout effect.

The methanogenic community structures of four different anaerobic processes were characterized using a quantitative real-time PCR with group-specific primer and probe sets targeting the 16S rRNA gene (rDNA). The group specific primer and probe sets were developed and used to detect the orders Methanosarcinales, and the families Methanosarcinaceae and Methanosaetaceae. Two separate sets targeting the domains Archaea and Bacteria were also used. Each microbial population in different anaerobic processes was determined and the relative abundance in the system was compared with each other. Dominant methanogenic populations and the community structures in the processes were varied by hydraulic retention time and acetate concentration. This indicates that the real-time PCR method with the primer and probe sets is a promising tool to analyze community structures in anaerobic processes.