Biokinetics of protein degrading Clostridium cadaveris and Clostridium sporogenes in batch and continuous mode of operations.

A quantitative real-time polymerase chain reaction (QPCR) was applied to estimate biokinetic coefficients of Clostridium cadaveris and Clostridium sporogenes, which utilize protein as carbon source. Experimental data of changes in peptone concentration and 16S rRNA gene copy numbers of C. cadaveris and C. sporogenes were fitted to model. The fourth-order Runge-Kutta approximation with non-linear least squared analysis was employed to solve the ordinary differential equations to estimate biokinetic coefficients. The maximum specific growth rate (µmax), half saturation concentration (Ks), growth yield (Y), and decay coefficient (Kd) of C. cadaveris and C.sporogenes were 0.73 ± 0.05 and 1.35 ± 0.32 h-1, 6.07 ± 1.52 and 5.67 ± 1.53 g/L, 2.25 ± 0.75 × 1010 and 7.92 ± 3.71 × 109 copies/g, 0.002 ± 0.003 and 0.002 ± 0.001 h-1, respectively. The theoretical specific growth rate of C. sporogenes always exceeded than that of C. cadaveris at peptone concentration higher than 3.62 g/L. When the influent peptone concentration was 5.0 g/L, the concentration of C.cadaveris gradually decreased to the steady value of 2.9 × 1010 copies/mL at 4 hours HRT, which indicates 67.1% of the initial population reduction, but the wash out occurred at 1.9 and 3.2 hours HRTs. The 16S rRNA gene copy numbers of C. sporogenes gradually decreased to steady values ranging from 1.1 × 1010 to 2.9 × 1010 copies/mL. C. sporogenes species was predicted to wash out at an HRT of 1.6 h.

Magnetite as an enhancer in methanogenic degradation of volatile fatty acids under ammonia-stressed condition.

Anaerobic batch tests with a 22 full-factorial design of ammonia (1.5, 6.5 g N/L) and magnetite concentrations (0, 20 mmol/L) were conducted separately for methanogenic degradation of acetate, propionate, and butyrate (volatile fatty acids (VFAs)) to 1) quantify the effect of magnetite as an enhancer in methanogenic degradation of each of the VFAs in conditions without ammonia stress (1.5 g N/L) and with ammonia stress (6.5 g N/L), and 2) identify methanogenic consortia that are related to such enhancement. Among the three VFAs, methanogenic degradation of propionate was the least feasible (57% lower specific methanogenic activity RCH4 and three times longer lag time λ than acetate degradation). At low ammonia concentration, only propionate showed improvement in RCH4 (46%) with supplementation of magnetite. In the ammonia-stressed condition without magnetite, RCH4 decreased by 38-58% and λ increased 2.2-8.8 times for all VFAs; magnetite supplementation significantly alleviated these effects. These results demonstrate that magnetite supplementation effectively increases methanogenic degradation of the VFAs even under ammonia-stressed conditions. 16S metagenomic sequencing revealed that distinctive methanogenic consortia were active in the different combinations of substrate, ammonia and magnetite. Alkaliphilus, Hyphomonadaceae SWB02 and Clostridia DTU014, Clostridia D8A-2, Christensenellaceae R-7 group and Rikenellaceae DMER64 were identified as potential syntrophic bacteria that can establish magnetite-mediated direct electron transfer with methanogens (Methanosaeta concilii, Methanosaeta harundinacea, Methanolinea tarda, Methanoculleus bourgensis and Methanosarcina spp.) during methanogenic degradation of VFAs. The results may be useful as a reference to develop effective strategies using magnetite supplementation to remediate anaerobic digestion processes that have been afflicted by VFA accumulation and ammonia inhibition.

Microbial community structure in full scale anaerobic mono-and co-digesters treating food waste and animal waste.

Five mesophilic full-scale anaerobic digesters treating food waste (FW-digester), animal waste (AW-digester), and co-substrate of food waste and animal waste (CO-digesters) were monitored identify bacterial and archaeal communities and to quantify the effect of substrate characteristics on them, and to identify 'core' microorganism. The substrate characteristics and microbial communities of the FW-digester, AW-digester, and CO-digesters were statistically different. Organic concentration and [Na+] were identified as major variations that effect microbial community. Methanogen community was more diverse in AW-digester than in FW-digester. Methanogen community in CO-digester was as diverse as in AW-digester, and the most dominant species was Methanoculleus bourgensis same as in FW-digester. Twenty-one bacterial genera and four methanogen species were found in all digesters as a consequence of their metabolic versatility to degrade organic and inhibitor compounds. The results implied that these core microorganisms may contribute to maintaining a stable microbial ecosystem.

Development of an interspecies interaction model: An experiment on Clostridium cadaveris and Clostridium sporogenes under anaerobic condition.

The specific primer and probe sets for quantifying Clostridium cadaveris and Clostridium sporogenes using a quantitative real-time PCR were designed. Each primer and probe set detected only the target species very specifically. The two species were cultivated in pure and mixed culture in batch mode with glucose as the only carbon source. The designed QPCR sets were used successfully to estimate the biokinetic parameters of each species in pure culture: i.e., maximum specific growth rate µmax, half saturation concentration Ks, growth yield Y, and decay coefficient Kd. of C. cadaveris and C. sporogenes were 0.311 ±â€¯0.020 and 0.360 ±â€¯0.019 h-1, 4.241 ±â€¯1.653 and 5.171 ±â€¯1.097 g/L, 0.301 ±â€¯0.065 and 0.199 ±â€¯0.037 1011 copies/g, 0.005 ±â€¯0.043 and 0.009 ±â€¯0.025 h-1, respectively. The effect of interspecific interaction of on substrate consumption rate and microbial growth was evaluated using mixed culture; curve fitting and comparison of coefficients detected increase in substrate consumption rate but decrease in microbial growth rate; these results imply interspecific interaction effect. A new model was of the interspecific interaction was developed, with focus on accuracy, realism, simplicity and biological significance. This interspecific interaction model may be extended to more-complex bioprocesses such as biological wastewater treatment systems and anaerobic digestion.

Bacteria and archaea communities in full-scale thermophilic and mesophilic anaerobic digesters treating food wastewater: Key process parameters and microbial indicators of process instability.

In this study, four different mesophilic and thermophilic full-scale anaerobic digesters treating food wastewater (FWW) were monitored for 1-2years in order to investigate: 1) microbial communities underpinning anaerobic digestion of FWW, 2) significant factors shaping microbial community structures, and 3) potential microbial indicators of process instability. Twenty-seven bacterial genera were identified as abundant bacteria underpinning the anaerobic digestion of FWW. Methanosaeta harundinacea, M. concilii, Methanoculleus bourgensis, M. thermophilus, and Methanobacterium beijingense were revealed as dominant methanogens. Bacterial community structures were clearly differentiated by digesters; archaeal community structures of each digester were dominated by one or two methanogen species. Temperature, ammonia, propionate, Na+, and acetate in the digester were significant factors shaping microbial community structures. The total microbial populations, microbial diversity, and specific bacteria genera showed potential as indicators of process instability in the anaerobic digestion of FWW.

Identifying methanogen community structures and their correlations with performance parameters in four full-scale anaerobic sludge digesters.

Four full-scale mesophilic anaerobic digesters treating waste sludge were monitored to characterize methanogen communities and their relationship with process parameters. The performance of the four digesters were dissimilar with the average chemical oxygen demand removal efficiencies between 24 and 45% and differing pH. Real-time quantitative PCR showed that archaeal 16S rRNA gene concentration ([ARC]) and, more pronouncedly, its ratio to bacterial counterpart ([ARC]/[BAC]) correlated positively with the performance parameters, including the lipid removal efficiency. Pyrosequencing identified 12 methanogen genera, of which Methanolinea, Methansaeta, and Methanospirillum collectively accounted for 79.2% of total archaeal reads. However, Methanoculleus, a numerically minor (1.9±2.6%) taxa, was the most promising biomarker for positive performance, while Methanoregula was abundant in samples with poor performance. These results could be useful for the control and management of anaerobic sludge digestion.

Correlations between bacterial populations and process parameters in four full-scale anaerobic digesters treating sewage sludge.

Process parameters and bacterial populations were investigated in four full-scale anaerobic digesters treating sewage sludge. Although the four digesters were operated under similar conditions, digesters A and B had higher pH (7.2-7.4) and lipid removal efficiencies (>50%) than C and D (pH 6.1-6.4; average lipid removal <16%). Bacterial richness, diversity, and evenness were higher in digesters C and D. Among the top-populated genera, ten (group I) were more abundant in digesters A and/or B; they were putative syntrophic fatty acid or protein/amino acid-utilizers. In contrast, fifteen others (group II) were less abundant in A and/or B and included potentially dormant/dead cells originated from activated sludge. Despite the overall richness trend, the presence of the 25 genera in groups I/II was greater in digesters A and B (24) than in C and D (17); this observation suggests that group I bacteria might be essential in AD of sewage sludge.

Nutrient Recovery of Starch Processing Waste to Cordyceps militaris: Solid State Cultivation and Submerged Liquid Cultivation.

This study demonstrated the potential for managing starch processing waste (SPW) by bioconversion to Cordyceps militaris mycelia using solid state cultivation (SSC) and submerged liquid cultivation (SLC). The growth characteristics of C. militaris mycelium were accessed and compared for SSC and SLC systems on SPW under various conditions of initial SPW concentration, pH, and operating temperature. To quantify the mycelial biomass in SLC, original primer sets targeting the 18S rRNA gene of C. militaris were developed. In SSC, a maximum mycelial growth rate (543.1 mm(2)/day) was predicted to occur at 25.6 g SPW/L, pH 5.5, and 23.8 °C. In SLC, a maximum mycelial growth rate (1918.6 mg/L/day) was predicted to occur at 35.5 g SPW/L, pH 5.5, and 22.0 °C. Temperature was suggested as the most significant factor in both systems. The higher optimum substrate concentration observed for SLC than for SSC was likely due to difference in mycelial morphology and mixing effect.

Nitrification resilience and community dynamics of ammonia-oxidizing bacteria with respect to ammonia loading shock in a nitrification reactor treating steel wastewater.

The aim of this study was to investigate the nitrification resilience pattern and examine the key ammonia-oxidizing bacteria (AOB) with respect to ammonia loading shocks (ALSs) in a nitrification bioreactor treating steel wastewater. The perturbation experiments were conducted in a 4-L bioreactor operated in continuous mode with a hydraulic retention time of 10 d. Three sequential ALSs were given to the bioreactor (120, 180 and 180 mg total ammonia nitrogen (TAN)/L. When the first shock was given, the nitrification process completely recovered after 14 d of further operation. However, the resilience duration was significantly reduced to ∼1 d after the second and third ALSs. In the bioreactor, Nitrosomonas aestuarii dominated the other AOB species, Nitrosomonas europaea and N. nitrosa, throughout the process. In addition, the population of N. aestuarii increased with ammonia utilization following each ALS; i.e., this species responded to acute ammonia overloadings by contributing to ammonia oxidation. This finding suggests that N. aestuarii could be exploited to achieve stable nitrification in industrial wastewaters that contain high concentrations of ammonia.

Anaerobic digestion of cattle offal: protein and lipid-rich substrate degradation and population dynamics of acidogens and methanogens.

Anaerobic digestion of cattle offal was investigated in batch reactors at 35 °C to determine the feasibility of using cattle offal as a feedstock. The organic content [i.e., volatile solids (VS)] of the cattle offal was mainly composed of protein (33.9%) and lipids (46.1%). Hydrolysis along with acidogenesis was monitored to investigate the substrate degradation and generation of intermediate products (e.g., volatile fatty acids, ammonia). Acetate (2.03 g/L), propionate (0.60 g/L), n-butyrate (0.39 g/L), and iso-valerate (0.37 g/L) were major acidogenesis products (91% of total volatile fatty acid concentration). Overall protein and lipid degradation were 82.9 and 81.8%, respectively. Protein degraded first, and four times faster (0.28 day(-1)) than lipid (0.07 day(-1)). Methane yields were 0.52 L CH4/g VSadded and 0.65 L CH4/g VSremoved, indicating that anaerobic digestion of the offal was feasible. A quantitative QPCR assay was conducted to understand the microbial dynamics. The variation patt erns in the gene concentrations successfully indicated the population dynamics of proteolytic and lipolytic acidogens. A fourth-order Runge-Kutta approximation was used to determine the kinetics of the acidogens. The molecular biotechnology approach was appropriate for the evaluation of the acidogenic biokinetics. The maximum growth rate, µ m, halfsaturation coefficients, K s, microbial yield coefficient, Y, cell mass decay rate coefficient, k d, of the proteolytic acidogens were 9.9 day(-1), 37.8 g protein/L, 1.1 × 10(10) copies/g protein, and 3.8 × 10(-1), respectively. Those for the lipolytic acidogens were 1.2 × 10(-1) day(-1), 8.3 g lipid/L, 1.5 × 10(9) copies/g lipid, and 9.9 × 10(-3) day(-1), respectively.

Temporal variation in methanogen communities of four different full-scale anaerobic digesters treating food waste-recycling wastewater.

Methanogen communities were investigated using 454 pyrosequencing in four different full-scale anaerobic digesters treating food waste-recycling wastewater. Seasonal samples were collected for 2 years, and 24 samples were available for microbial analysis from a plug flow thermophilic (PT) digester, a continuously-stirred tank thermophilic (CT) digester, an upflow anerobic sludge blanket mesophilic (UM) digester, and a continuously-stirred tank mesophilic (CM) digester. Methanoculleus, Methanobacterium, Methanothermobacter, and Methanosaeta were revealed to be key methanogens in full-scale anaerobic digestion process treating food waste-recycling wastewater. In the PT digester, Methanoculleus was dominant (96.8%). In the CT digester, Methanoculleus was dominant (95.4%) during the first year of operation, but the dominant genus was shifted to Methanothermobacter (98.5%) due to pH increase. In the UM digester, Methanosaeta was dominant (87.2%). In the CM digester, Methanoculleus was constantly dominant (74.8%) except during CM5 when Methanosaeta was dominant (62.6%) due to the low residual acetate concentration (0.1 g/L).