Magnetite particles accelerate methanogenic degradation of highly concentrated acetic acid in anaerobic digestion process.

The anaerobic digestion (AD) process has become significant for its capability to convert organic wastewater into biogas, a valuable energy source. Excessive acetic acid accumulation in the anaerobic digester can inhibit methanogens, ultimately leading to the deterioration of process performance. Herein, the effect of magnetite particles (MP) as an enhancer on the methanogenic degradation of highly-concentrated acetate (6 g COD/L) was examined through long-term sequential AD batch tests. Bioreactors with (AM) and without (AO) MP were compared. AO experienced inhibition and its methane production rate (qm) converged to 0.45 L CH4/g VSS/d after 10 sequential batches (AO10, the 10th batch in a series of the sequential batch tests conducted using bioreactors without MP addition). In contrast, AM achieved 3-425% higher qm through the sequential batches, indicating that MP could counteract the inhibition caused by the highly-concentrated acetate. MP addition to inhibited bioreactors (AO10) successfully restored them, achieving qm of 1.53 L CH4/g VSS/d, 3.4 times increase from AO10 after 8 days lag time, validating its potential as a recovery strategy for inhibited digesters with acetate accumulation. AM exhibited higher microbial populations (1.8-3.8 times) and intracellular activity (9.3 times) compared to AO. MP enriched Methanosaeta, Peptoclostridium, Paraclostridium, OPB41, and genes related to direct interspecies electron transfer and acetate oxidation, potentially driving the improvement of qm through MP-mediated methanogenesis. These findings demonstrated the potential of MP supplementation as an effective strategy to accelerate acetate-utilizing methanogenesis and restore an inhibited anaerobic digester with high acetate accumulation.

Key players in syntrophic propionate oxidation revealed by metagenome-assembled genomes from anaerobic digesters bioaugmented with propionic acid enriched microbial consortia.

Propionic acid (HPr) is frequently accumulated in anaerobic digesters due to its thermodynamically unfavorable degradation reaction. Here, we identify key players in HPr oxidation and organic overloading recovery from metagenome-assembled genomes (MAGs) recovered from anaerobic digesters inoculated with HPr-enriched microbial consortia before initiating organic overloading. Two independent HPr-enrichment cultures commonly selected two uncultured microorganisms represented with high relative abundance: Methanoculleus sp002497965 and JABUEY01 sp013314815 (a member of the Syntrophobacteraceae family). The relative abundance of JABUEY01 sp013314815 was 60 times higher in bioaugmented bioreactors compared to their unaugmented counterparts after recovery from organic overloading. Genomic analysis of JABUEY01 sp013314815 revealed its metabolic potential for syntrophic propionate degradation when partnered with hydrogenotrophic methanogens (e.g., Methanoculleus sp002497965) via the methylmalonyl-CoA pathway. Our results identified at least two key species that are responsible for efficient propionate removal and demonstrate their potential applications as microbial cocktails for stable AD operation.

Effect of magnetite supplementation on mesophilic anaerobic digestion of phenol and benzoate: Methane production rate and microbial communities.

Anaerobic sequential batch tests treating phenol and benzoate were conducted to evaluate the potential of magnetite supplementation to improve methanogenic degradation of phenol and benzoate, and to identify active microbial communities under each condition. Specific CH4 production rates during anaerobic digestion were 218.5 mL CH4/g VSS/d on phenol and 517.6 mL CH4/g VSS/d on benzoate. Magnetite supplementation significantly increased methanogenic degradation of phenol by 9.0-68.0% in CH4 production rate, and decreased lag time by 7.9-48.0%, with no significant reduction in CH4 yield. Syntrophorhabdus, Sporotomaculum, Syntrophus, Syntrophomonas, Peptoclostridium, Soehngenia, Mesotoga, Geobacter, Methanosaeta, Methanoculleus, and Methanospirillum were revealed as active microbial communities involved in anaerobic digestion of phenol and benzoate. Magnetite-mediated direct interspecies electron transfer between Geobacter, Peptoclostridium, and Methanosaeta harundinacea could contribute to this improvement.

Effects of inhibitions by sodium ion and ammonia and different inocula on acetate-utilizing methanogenesis: Methanogenic activity and succession of methanogens.

Acetate-fed anaerobic sequential batch experiments with four different inhibitory conditions (non-inhibitory (Lo), sodium-ion inhibitory (Na), ammonia inhibitory (Am), combined inhibitory (Hi)) were conducted using thirteen different inocula to investigate the inhibition effects by sodium-ion and ammonia and different inocula on acetate-utilizing methanogenesis and succession of methanogens. Sodium-ion and ammonia significantly extended lag-time λ and reduced specific-methanogenic-activity RCH4, and caused synergistic inhibition. The inhibition differed according to the initial methanogen community structures: the inhibition effects on λ and RCH4 were strongest ininocula with Methanosaeta concilii dominant and weakest in inocula with Methanoculleus bourgensis dominant. These inhibitory conditions determined the succession of methanogens: the most competitive methanogens were Methanosaeta concilii in Lo, Methanosarcina sp. in Na, Methanosarcina sp. and Methanoculleus bourgensis in Am, Methanoculleus bourgensis in Hi. This study provides valuable information for microbial management and optimization for AD processes treating wastewater that is rich in protein and/or salt.

A snapshot of microbial community structures in 20 different field-scale anaerobic bioreactors treating food waste.

This study aimed to identify significant factors shaping the microbial populations in biogas plants treating food waste (FW). Twenty full-scale anaerobic acidogenic/methanogenic bioreactors, located at 11 FW treatment facilities, were compared to find patterns in their microbial community structures and potential interactions with the process parameters. Temperature, hydraulic retention time, and organic loading rate were design parameters that systematically influenced the microbial communities. The latter two clearly separated the acidogenic and methanogenic bioreactors. Lactobacillus was the dominant (69.7 ±â€¯19.8%) bacteria in the acidogenic reactors, while hydrogen-utilizing methanogens, such as Methanoculleus (65.1 ±â€¯33.5%), were the dominant archaea in most methanogenic digesters. Defluiviitoga was the dominant (82.7 ±â€¯1.4%) bacteria in the thermophilic digesters, but was also the most abundant (33.1-33.6%) bacteria in dry mesophilic digesters. The two bioreactor categories had lower bacterial diversities, and also higher propionate concentrations (>5 g/L in 4 out of 5 cases), which may impose potential risks for the management of such digesters. The current 'snapshot' of the microbial communities suggests several bacterial and archaeal taxa as potential indicators of bioreactor categories and/or process variables.

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.

Single and combined inhibition of Methanosaeta concilii by ammonia, sodium ion and hydrogen sulfide.

Single and combined inhibition of lag time λ and specific methanogenic activity RCH4 of Methanosaeta concilii by NH3, Na+ and H2S were investigated using inhibition tests with a single inhibitor and a 33 full-factorial experiment of NH3, Na+ and H2S concentrations (1.5 ≤ total ammonia nitrogen (TAN)/L ≤ 4.5 g, 1 ≤ Na+/L ≤ 4.3 g, 14.2 ≤ total hydrogen sulfide sulfur (THSS)/L ≤ 836 mg). All three inhibitors significantly increased λ and reduced RCH4 of M. concilii. The half-maximal inhibitory concentrations of NH3, Na+ and H2S for M. concilii were 6.4 g TAN/L, 5.2 g Na+/L and 1.6 g THSS/L. Partial cubic models adequately approximated the corresponding response surfaces of λ and RCH4 from the 33 full-factorial experiment. The inhibitors inhibited RCH4 synergistically, but inhibited λ in a complex manner. The combination of NH3 and Na+ showed the strongest synergistic inhibition of both λ and RCH4.

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.

Temporal variation in bacterial and methanogenic communities of three full-scale anaerobic digesters treating swine wastewater.

To investigate the effects of temporal variations of process parameters on microbial community structures in the two types of full-scale anaerobic digester treating swine wastewater, three full-scale anaerobic digesters were monitored. An anaerobic filter (AF)-type digester located in Gong-Ju (GJ) showed the highest COD removal among three digesters and maintained stable efficiency. A digester in Hong-Seong (HS) was of the same type as it GJ and showed improved efficiency over the sampling period. A continuously stirred tank reactor (CSTR)-type digester in Soon-Cheon (SC) showed decreasing efficiency due to a high residual concentration of VFAs and NH4+. These process efficiencies were closely correlated to the Simpson indices of the methanogenic communities. Genera Bacillus, Methanosaeta, and Methanospirillum that have filamentous morphology were dominant in both AF-type digesters, but genera Acholeplasma, Methanosarcina, and Methanoculleus that have spherical or coccoid morphology were dominantly abundant in the CSTR-type digester. Correlation between populations suggests a possible syntrophic relationship between genera Desulfobulbus and Methanosaeta in digesters GJ and HS.

Comprehensive analysis of microbial communities in full-scale mesophilic and thermophilic anaerobic digesters treating food waste-recycling wastewater.

Microbes were sampled for a year in a full-scale mesophilic anaerobic digester (MD) and a thermophilic anaerobic digester (TD) treating food waste-recycling wastewater (FRW), then microbial community structure, dynamics and diversity were quantified. In the MD, Fastidiosipila, Petrimonas, vadinBC27, Syntrophomonas, and Proteiniphilum were dominant bacterial genera; they may contribute to hydrolysis and fermentation. In the TD, Defluviitoga, Gelria and Tepidimicrobium were dominant bacteria; they may be responsible for hydrolysis and acid production. In the MD, dominant methanogens changed from Methanobacterium (17.1 ±â€¯16.9%) to Methanoculleus (67.7 ±â€¯17.8%) due to the increase in ammonium concentration. In the TD, dominant methanogens changed from Methanoculleus (42.8 ±â€¯13.6%) to Methanothermobacter (49.6 ±â€¯11.0%) due to the increase of pH. Bacteria and archaea were more diverse in the MD than in the TD. These results will guide development of microbial management methods to improve the process stability of MD and TD treating FRW.

Microbial communities underpinning mesophilic anaerobic digesters treating food wastewater or sewage sludge: A full-scale study.

Ten mesophilic full-scale anaerobic digesters treating food wastewater (FW-digesters) or sewage sludge (SL-digesters) were monitored for 1 year to investigate: (1) microbial communities underpinning FW-digesters and SL-digesters, (2) the effects of total ammonia-nitrogen concentration [TAN] and Na+ concentration [Na+] on variations of these communities. [TAN] and [Na+] in the digester varied among digesters: 1.7-6.5 g TAN/L and 1.0-3.6 g Na+/L for the FW-digesters, and 0.1-2.2 g TAN/L and 0.1-1.2 g Na+/L for the SL-digesters; [TAN] negatively correlated with the process efficiency of the FW-digesters. Microbial communities were less diverse in the FW-digesters than in the SL-digesters. The FW- and SL-digesters formed very distinct microbial community structures; [TAN] and [Na+] in the digester were the critical factors shaping these structures. Immigrant bacteria from influent sludge significantly influence the bacterial communities of the SL-digesters. Methanoculleus might be tolerant to high ammonia in AD of such organic wastewater.

A comparative study on the process efficiencies and microbial community structures of six full-scale wet and semi-dry anaerobic digesters treating food wastes.

The purpose of this study was to investigate the effect of different types of food wastes on the process efficiency and microbial community structures in full-scale anaerobic digesters and to identify parameters that affect these criteria. Six full-scale anaerobic digesters were investigated; three were operated under "wet" condition (total solids TS≤10%), and three were run under "semi-dry" condition (10%≤TS≤20%). Removal efficiency of volatile solids was much higher in the wet digesters (75.2±3.8%) than in the semi-dry digesters (42.6±5.5%). The bacterial and archaeal communities were distinctly characterized by families Porphyromonadaceae, Sphingobacteriaceae, Syntrophomonadaceae, and Methanobacteriaceae in the wet digesters; and of Clostridiaceae, Patulibacteraceae, Pseudonocardiaceae, Lachnospiraceae, Rikenellaceae, and Methanomicrobiaceae in the semi-dry digesters. The discriminant parameters identified were TS content of influent, concentration of total ammonia nitrogen and the ratio of soluble chemical oxygen demand (COD) to COD in the digester.

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.

Microbial community shifts in a farm-scale anaerobic digester treating swine waste: Correlations between bacteria communities associated with hydrogenotrophic methanogens and environmental conditions.

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 H2 producers, and/or syntrophic acetate oxidizers. Some of these species showed positive correlations with [NH4+-N] (p<0.1); this relationship suggests that ammonia was a significant parameter for bacterial selection. The bacterial niche information reported in this study can be useful to understand the ecophysiology of anaerobic digesters treating swine manure that contains high ammonia content.

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.

Characterization of antibiotic resistance genes in representative organic solid wastes: Food waste-recycling wastewater, manure, and sewage sludge.

In this research, the distribution of antibiotic resistance genes (ARGs) was characterized in representative organic solid waste (OSW) in Korea: food waste-recycling wastewater (FRW), manure, and sewage sludge. The amounts of total ARG (gene copies/16S rRNA gene copies) was greatest in manure followed by sewage sludge and FRW. Interestingly, there were significantly different patterns in the diversity and mechanisms of ARGs. For example, a significant proportion of ARGs were tetracycline resistant genes in all the OSW (40.4-78.2%). ß-lactam antibiotics resistant genes were higher in the FRW samples than in other types of OSW but sulfonamides resistant genes represented the greatest proportion in sludge. Regarding the characteristics of antibiotic resistance mechanisms, there was a relatively higher proportion of the ribosomal protection mechanism to tetracycline observed in the FRW and manure samples. However, tetracycline resistant genes with direct interaction were relatively higher in the sewage sludge samples. sul1 was the dominant subtype in all the OSW types and detection of ermB was observed although there was no ermC detected in sewage sludge. There were significant correlations between the occurrences of ARG subtypes: tetB and tetG in all OSW (P<0.01); tetE and tetQ only in sludge (P<0.01). The Class 1 integron-integrase gene (intI1) was significantly correlated with total ARGs only in manure and sludge (P<0.05), revealing potential horizontal gene transfer in these OSW.

Mesophilic Acidogenesis of Food Waste-Recycling Wastewater: Effects of Hydraulic Retention Time, pH, and Temperature.

The effects of hydraulic retention time (HRT), pH, and operating temperature (T OP) on the degradation of food waste-recycling wastewater (FRW) were investigated in laboratory-scale hydrolysis/acidogenesis reactors. Response surface analysis was used to approximate the production of volatile organic acids and degradation of volatile suspended solids (VSS), carbohydrate, protein, and lipid with regard to the independent variables (1 ≤ HRT ≤ 3 days, 4 ≤ pH ≤ 6, 25 ≤ T OP ≤ 45 °C). Partial cubic models adequately approximated the corresponding response surfaces at α < 5 %. The physiological conditions for maximum acidification (0.4 g TVFA + EtOH/g VSadded) and the maximal degradation of VSS (47.5 %), carbohydrate (92.0 %), protein (17.7 %), and lipid (73.7 %) were different. Analysis of variance suggested that pH had a great effect on the responses in most cases, while T OP and HRT, and their interaction, were significant in some cases. Denaturing gradient gel electrophoresis analysis revealed that Sporanaerobacter acetigenes, Lactobacillus sp., and Eubacterium pyruvivorans-like microorganisms might be main contributors to the hydrolysis and acidogenesis of FRW. Biochemical methane potential test confirmed higher methane yield (538.2 mL CH4/g VSadded) from an acidogenic effluent than from raw FRW.

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.

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.

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.