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.

Elucidation of microbial interactions, dynamics, and keystone microbes in high pressure anaerobic digestion.

High-pressure anaerobic digestion (HPAD) is a promising technology for producing biogas enriched with high methane content in a single-step process. To enhance HPAD performance, a comprehensive understanding of microbial community dynamics and their interactions is essential. For this, mesophilic batch high-pressurized anaerobic reactors were operated under 3 bars (H3) and 6 bars (H6). The experimental results showed that the effect of high-pressure (up to 6 bar) on acidification was negligible while methanogenesis was significantly delayed. Microbial analysis showed the predominance of Defluviitoga affiliated with the phylum Thermotogae and the reduction of Thiopseudomonas under high-pressure conditions. In addition, the microbial cluster pattern in H3 and H6 was significantly different compared to the CR, indicating a clear shift in microbial community structure. Moreover, Methanobacterium, Methanomicrobiaceae, Alkaliphilus, and Petrimonas were strongly correlated in network analysis, and they could be identified as keystone microbes in the HPAD reactor.

Variations in antibiotic resistance genes and microbial community in sludges passing through biological nutrient removal and anaerobic digestion processes in municipal wastewater treatment plants.

Antimicrobial resistance (AMR) represents a relentless, silent pandemic. Contributing to this are wastewater treatment plants (WWTPs), a potential source of antibiotic resistance genes' (ARGs) transmission to the environment, threatening public health. The presence of ARGs in pathogenic bacteria and their release into the environment by WWTPs threatens the public health. The current study investigated changes in ARGs' abundance in biological nutrient removal (BNR) processes and anaerobic digestion (AD) reactors of two WWTPs. Also, microbial community structure, which is known to shape the distribution and abundance of ARGs, was also analyzed. The relative abundance of eight ARGs (tetX, tetA, tetM, TEM, sul1, sul2, ermB and qnrD) was quantified as ARGs' copies/16 S rRNA gene copies using quantitative polymerase chain reaction (qPCR). Microbial community composition was assessed by 16 S rRNA microbiome sequencing analysis. TetX was prevalent among the eight ARGs, followed by TEM and sul1. However, its abundance was decreased in the AD sludges compared to BNR sludges. Proteobacteria was the major bacterial phylum found in all the sludge samples, while Arcobacter, 12up and Acidovorax were the predominant genera. Acinetobacter and Flavobacterium were significantly more abundant in the BNR sludges, while 12up and Aeromonas were predominant in AD sludges. Principal component analysis (PCA) revealed a clear difference in dominant ARGs and bacteria between the sludges in the processes of BNR and AD of the two WWTPs. Clinically relevant bacterial genera, Klebsiella and Enterococcus, found in both the BNR and AD sludges, were significantly correlated with the tetX gene. Throughout this study, the relationship between microbial communities and specific ARGs was revealed, illustrating that the composition of the microbial community could play a vital role in the abundance of ARGs. These results will better inform future studies aimed at controlling the spread of ARGs and their potential hosts from WWTPs.

Tailoring a highly conductive and super-hydrophilic electrode for biocatalytic performance of microbial electrolysis cells.

Bioelectrochemical hydrogen production via microbial electrolysis cells (MECs) has attracted attention as the next generation of technology for the hydrogen economy. MECs work by electrochemically active bacteria reducing organic compounds at the anode. However, the hydrophobic nature of carbon-based anodes suppresses the release of the produced gas and water penetration, which significantly reduces the possibility of microbial attachment. Consequently, a limited surface area of the anode is used, which decreases hydrogen production efficiency. In this study, the bifunctional material poly(3,4-ethylenedioxythiophene):polystyrene sulfonate (PEDOT:PSS) was applied to the surface of a three-dimensional carbon felt anode to enhance the hydrogen production efficiency of an MEC owing to the high conductivity of PEDOT and super-hydrophilicity of PSS. In experiments, the PEDOT:PSS-modified anode almost doubled the hydrogen production efficiency of the MEC compared with the control anode owing to the increased capacitance current (239.3 %) and biofilm formation (220.7 %). The modified anode reduced the time required for the MEC to reach a steady state of hydrogen production by 14 days compared to the control anode. Microbial community profiles demonstrated that the modified anode had a greater abundance of electrochemically active bacteria than the control anode. This simple method could be widely applied to various bioelectrochemical systems (e.g., microbial fuel cells and solar cells) and to scaling up MECs.

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.

Complex network analysis of slaughterhouse waste anaerobic digestion: From failure to success of long-term operation.

The study explored slaughterhouse waste (SHW) as prime feedstock associated with and without supplement of an external slowly degradable lignocellulosic carbon source to overcome the synergistic co-inhibitions of ammonia and fatty acids. Long-term solid-state digestion (SSD) and liquid-state digestion (LSD) were investigated using a mixture of pork liver and fat. At 2.0 g volatile solids (VS) L-1 d-1 of organic loading rate (OLR), the two reactors of SSD experienced operational instability due to ammonia inhibition and volatile fatty acid (VFA) accumulation while LSD successfully produced 0.725 CH4 L CH4 g-1VS during 197 d of working days under unfavorable condition with high total ammonia nitrogen (>4.7 g/L) and VFAs concentration (>1.9 g/L). The network analysis between complex microflora and operational parameters provided an insight for sustainable biogas production using SHW. Among all, hydrogenotrophic methanogens have shown better resistance than acetoclastic methanogens.

Startup of Demo-Scale Anaerobic Digestion Plant Treating Food Waste Leachate: Process Instability and Recovery.

A demo-scale (600 m3 working volume) anaerobic digester treating food waste leachate was monitored during its startup period. The operation strategy was adjusted twice (i.e., three distinct phases) during the operation to recover the process from instability. During the first phase, the organic loading rate (OLR) > 2.7 kg chemical oxygen demand (COD)/m3∙day corresponded to volatile fatty acid (VFA) accumulation along with a decreasing pH, resulting in the drop in biogas yield to 0.43 ± 0.9 m3/kg CODin. During phase 2, fast recovery of this process was aimed at using a sequencing batch operation. One batch cycle (5 to 2 days) consisted of the combined drawing and feeding step (5 h), the reacting step (91 to 17 h), and the settling step (24 h). The duration of the reacting step was determined for each cycle such that (1) the biogas production ceased before the cycle end and (2) the residual VFA concentration was < 1 g/L. In total, 11 cycles were operated with a gradual increase in biogas yield to 0.55 m3/kg CODin with the absence of any sign of system disturbance. After phase 2, the digester was fed at the designed OLR of 4.1 ± 0.3 kg COD/m3∙day. The biogas yield was elevated to 0.58 ± 0.2 m3/kg CODin during phase 3 with the residual VFA concentration maintained at 2.2 ± 0.6 g/L. Methanogen populations, as determined by real-time PCR, did not change significantly throughout the period. These results imply that the adaptation of this process to the OLR of ca. 4 kg COD/m3∙day was not due to the increase in methanogen population but due to the elevation of its activity. Overall, this study suggests that the sequencing batch operation with adjustable cycle duration can be one successful recovery strategy for biogas plants under system instability.

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.

Identification of factors affecting removal of antibiotic resistance genes in full-scale anaerobic digesters treating organic solid wastes.

Efficiencies of removing antibiotic resistance genes (ARGs) and intI1 were explored using eight full-scale anaerobic digesters. The digesters demonstrated different characteristics on the basis of substrate types (food waste, manure or sludge); configuration (single or two-stage); temperature (psychrophilic, mesophilic or thermophilic); hydraulic retention time (HRT) (9.7-44 days); and operation mode (continuous stirred tank reactor or plug flow reactor). Digesters' configuration or operating parameters showed a greater effect on abundance of ARGs than the type of input substrate. Redundancy analysis (RDA) accounted for 85.2% of the total variances and digesters with the same configuration and operational conditions showed similar performance for removal of ARGs. The highest efficiencies of removing ARGs (99.99%) were observed in two-stage thermophilic digesters with relatively long HRTs (32 days). The lowest removal efficiency (97.93%) was observed in single-stage mesophilic with relatively short HRTs (9.7 days), likely due to vertical and horizontal gene transfer.

Effects of Different pH Control Strategies on Microalgae Cultivation and Nutrient Removal from Anaerobic Digestion Effluent.

This study investigated nutrient removal from anaerobic digestion effluent by cultivating mixed-culture microalgae enriched from anaerobic sludge under different pH conditions: RUC (uncontrolled), R7-8 (maintained at 7-8), and R<8 (maintained below 8). Significant amounts of NH4+-N were lost by volatilization in RUC cultures due to increased pH values (≤8.6) during the early period of cultivation. The pH control strategies significantly affected the biological NH4+-N removal (highest in R7-8), microalgal growth (highest in R7-8), biomass settleability (highest in R<8), and microalgal growth relative to bacteria (highest in R<8) in the cultures. Parachlorella completely dominated the microalgal communities in the inoculum and all of the cultures, and grew well at highly acidic pH (<3) induced by culture acidification with microalgal growth. Microalgae-associated bacterial community structure developed very differently among the cultures. The findings call for more attention to the influence and control of pH changes during cultivation in microalgal treatment of anaerobic digestion effluent.

Responses of Coagulant Type, Dosage and Process Conditions to Phosphate Removal Efficiency from Anaerobic Sludge.

Phosphorus, a crucial component of life, may cause eutrophication if it is discharged untreated into the aquatic ecosystem. Phosphate (PO43-) may exist at an elevated level in anaerobic digestion (AD) effluents and can lead to the clogging of pipes by forming struvite crystals. This study was conducted to assess the responses of coagulant type, dosage and process conditions to phosphate removal efficiency from anaerobic sludge. The experiments were performed in two steps. First, a sensitivity test was conducted to compare five coagulant types (alum, poly-aluminum chloride (PAC), FeCl2, FeCl3 and PAC + FeCl3) at standard coagulation conditions. The results showed that PAC would be the best coagulant among the tested, while a combination of PAC and FeCl3 may be beneficial under circumstances. Second, an optimization study was performed for PAC using response surface methodology employing central composite design. Among the three independent variables (coagulant dosage, slow mixing duration and agitation speed), the dosage was the sole significant variable for phosphate removal efficiency, while the other two had limited effects. A future study to optimize the rapid mixing conditions would give additional insights into the process. The results of this study may be useful to design a process to counteract phosphate discharges from AD plants, as well as to reduce the risks of pipe clogging and maintenance problems due to crystalline struvite formation in the later stage of AD.

Long-term effectiveness of bioaugmentation with rumen culture in continuous anaerobic digestion of food and vegetable wastes under feed composition fluctuations.

Biogas plants treating food waste (FW) often experience feed load and composition fluctuations. In Korea, vegetable waste from the preparation of kimchi comprises over 20% of the total FW production during the Kimjang season. The large production of Kimjang waste (KW) can cause mechanical and operational problems in FW digesters. This study investigated the long-term effectiveness of bioaugmentation with rumen culture (38 months) in an anaerobic reactor co-digesting FW with varying amounts of KW. The bioaugmented reactor maintained better and stabler performance under recurrent fluctuations in feed characteristics than a non-bioaugmented control reactor, particularly under high ammonia conditions. Bioaugmentation increased microbial diversity, thereby improving the resilience of the microbial community. Some augmented microorganisms, especially Methanosarcina, likely played an important role in it. The results suggest that the proposed bioaugmentation strategy may provide a means to effectively treat and valorize KW-and potentially other seasonal lignocellulosic wastes-by co-digestion with FW.

Tracking microbial community shifts during recovery process in overloaded anaerobic digesters under biological and non-biological supplementation strategies.

Anaerobic digestion encounters operational instability due to fluctuations in organic loading. Propionic acid (HPr) is frequently accumulated due to its unfavorable reaction thermodynamics. Here, 'specific' bioaugmentation using HPr enrichment cultures (three different injection regimes of quantity and frequency) was compared with 'non-specific' bioaugmentation using anaerobic sludge, and with non-biological supplementation of magnetite or coenzyme M. The specific bioaugmentation treatments showed superior recovery responses during continuous feeding after a peak overload. A 'one-shot' bioaugmentation with enrichment showed the best remediation, with ~25% recovery time and >10% CH4 conversion efficiency compared to the control. Consecutive bioaugmentation showed evidence of increased stability of the introduced community. Families Synergistaceae, Syntrophobacteraceae, and Kosmotogaceae were likely responsible for HPr-oxidation, in potential syntrophy with Methanoculleus and Methanobacterium. The different supplementation strategies can be considered to reduce the effect of start-up or overload in anaerobic digesters based on the availability of supplementation resources.

Density profile modeling for real-time estimation of liquid level in anaerobic digester using multiple pressure meters.

The liquid level of a bioreactor is an important operating parameter governing the hydraulic retention time. In this study, a novel method is proposed to estimate the liquid level of anaerobic digesters. The proposed method has an advantage over typical differential pressure measurement as it considers the heterogeneity of the digestate along the level using multiple pressure meters. The real-time measurement generates a model to fit the densities at different liquid columns, predicts the density of the surface layer and determines the overall liquid level. A pilot-scale (0.33 m3 working volume; 1.2 m liquid level) digester, equipped with seven pressure meters, was operated to test the methodology. The performance of the digester was confirmed stable during a long-term (175 d) operation. A set of density-pressure models was developed and were validated using the long-term experimental data. The new method employing cubic model showed significantly better estimation of the reactor level (mean error rate of 1.31%) with improved CDF, as compared with the traditional differential pressure method (mean error rate of 5.71%). The methodology proposed in this study is simple, robust, and cost-effective and can be used to provide additional insights into the operation of an anaerobic digester such as assessing the mixing efficiency.

Effects of Microbes from Coal-Related Commercial Humic Substances on Hydroponic Crop Cultivation: A Microbiological View for Agronomical Use of Humic Substances.

Here, coal-related humic substances (HSs) were examined to confirm whether sterilization treatments induce their inferior ability to stimulate lettuce in hydroponic cultivations. Interestingly, a drastic reduction in both lettuce biomass and microbial colony-forming units of the crop culture solutions was observed when the autoclaved HSs were treated. Some microbial genera (i.e., Bacillus and Aspergillus) identifiable in the bare HS-treated hydroponic systems were able to be isolated by direct inoculation of bare HS powders on conventional microbial nutrients, supporting that flourishing microbes in the hydroponic cultivations derive from bare HSs-treated. Moreover, coincubation of some isolated bacterial and fungal strains (i.e., Bacillus and Aspergillus genera) from HSs with lettuce resulted in a significant increase in plant biomass and enhanced resistance to NaCl-related abiotic stresses. Microbial volatile organic compounds renowned for plant stimulation were detected by using solid-phase microextraction coupled with gas chromatography-mass spectrometry. It was finally confirmed that the isolates are capable of utilizing carbon substrates such as pectin and tween 20 or 40, which are relevant to those of microbes isolated from peat and leonardite (i.e., HS extraction sources). Overall, our results suggest that microbiological factors could be considered when commercial coal-related HSs are applied in hydroponic crop cultivations.

Long-term enrichment of anaerobic propionate-oxidizing consortia: Syntrophic culture development and growth optimization.

Propionate is a quantitatively important methanogenic intermediate in anaerobic digesters and only limited number of microbes can utilize it under syntrophic association with methanogens. The syntrophic propionate oxidizing bacterias are known to be slow growers due to the low energy yield. Thus, propionate get accumulated frequently in anaerobic digesters and it negatively affect digester performance. In this study, propionate degrading consortia from four different seeding sources were enriched in sequential bath mode in two phases; first adaption phase with 1 g/L of propionate concentration and later, high-strength phase with 3 g/L. From 16s rRNA gene based metagenomics analysis of the former phase, four syntrophic microbial groups, Syntrophaceae, Syntrophomonadaceae, Methanobacterium and Methanosaeta were found to be dominant with complete degradation of propionate. The substrate accelerated microbial shifts were observed at high-strength phase with significant decrease of Syntrophaceae up to 26.9 %. Using Response Surface Methodology, pH 6.8-6.9 and temperature 34.5-34.9 °C were found to be optimum growth conditions for the propionate degradation culture. Observed results could be useful to improve degradation efficiencies and obtained enriched culture can be used to recover propionate-accumulated digesters by bio-augmentation.

Hydrometeorological Influence on Antibiotic-Resistance Genes (ARGs) and Bacterial Community at a Recreational Beach in Korea.

We investigated the occurrence and distribution of antibiotic-resistance genes (ARGs) and the composition of a bacterial community under conditions of rainfall on a recreational beach in Korea. Seawater samples, collected every 1‒5 hours in June 2018 and May 2019, were analyzed using quantitative real-time polymerase chain reaction and next-generation sequencing. We found a substantial influence of rainfall and tidal levels on the relative abundance of total ARGs and bacterial operational taxonomic units (OTUs), which showed 1.9 × 103 and 1.1 × 101 fold increases, respectively. In particular, the elevated levels of ARGs were maintained for up to 32 hours after rainfall. An increased abundance of sewage-related ARGs and bacterial OTUs suggested that combined sewer overflow (CSO) may be the major factor contributing to the increase in the number and diversity of ARGs and related bacterial communities. Network analysis of ARGs and OTUs indicated that, at the genus level, Acinetobacter, Pseudomonas, and Prevotella were the main potential pathogens carrying the observed ARGs in the recreational seawater. Overall, these findings highlight the potential threat to public health on beaches, and indicate the requirement for more adequate monitoring, with greater efforts to mitigate the propagation of ARGs arising from CSOs.

Fungal mycelia functionalization with halloysite nanotubes for hyphal spreading and sorption behavior regulation: A new bio-ceramic hybrid for enhanced water treatment.

Filamentous fungi are believed to remove a wide range of environmental xenobiotics due to their characteristically non-specific catabolic metabolisms. Nonetheless, irregular hyphal spreading can lead to clogging problems in treatment facilities and the dependence of pollutant bioavailability on hyphal surface features severely limits their applicability in water treatment. Here, we propose a scalable and facile methodology to structurally modify fungal hyphae, allowing for both the maximization of pollutant sorption and fungal pellet morphology self-regulation. Halloysite-doped mycelium architectures were efficiently constructed by dipping Aspergillus fumigatus pellets in halloysite nanotube-dispersed water. Ultrastructure analyses using scanning electron microscopy equipped with energy-dispersive X-ray spectroscopy revealed that the nanotubes were mainly attached to the outer surface of the pellets. Fungal viability and exoenzyme production were hardly affected by the halloysites. Notably, nanotube doping appeared to be extremely robust given that detachments rarely occurred even in high concentrations of organic solvents and salt. It was also demonstrated that the doped halloysites weakened hyphal growth-driven gelation, thus maintaining sphere-like pellet structures. The water treatment potential of the hybrid fungal mycelia was assessed through both cationic toxic organic/inorganic-contaminated water and real dye industry wastewater clean-ups. Aided by the mesoporous halloysite sites on their surface, the removal abilities of the hybrid structures were significantly enhanced. Moreover, inherent low sorption ability of HNT for heavy metals was found to be overcome by the aid of fungal mycelia. Finally, universal feature of the dipping-based doping way was confirmed by using different filamentous fungi. Given that traditional approaches to effectively implement fungus-based water treatment are based mostly on polymer-based immobilization techniques, our proposed approach provides a novel and effective alternative via simple doping of living fungi with environmentally-benign clays such as halloysite nanotubes.

Direct inoculation of a biotrickling filter for hydrogenotrophic methanogenesis.

Hydrogenotrophic biomethanation in a biotrickling filter has been reported to be a proven technology for biological biogas upgrading in recent studies. However, the preparation of enriched hydrogenotrophic methanogens in a separate reactor prior to biomethanation in a trickled bed is a lengthy procedure and therefore hard to apply on an industrial scale. This study explored the direct inoculation of anaerobic biogas digestate for simultaneous enrichment of hydrogenotrophic methanogens and biofilm immobilisation in a trickled bed system. The direct inoculation and formation of hydrogenotrophic biofilm was successful and resulted in a stable H2 loading rate of 11  [Formula: see text] , with the highest specific methane productivity recorded at 3.03 Nm3mR-3d-1 and a purity of 98% CH4 in thermophilic conditions. The DNA analysis confirmed that hydrogenotrophic methanogens dominated the archaeal consortia.