Using a non-precious metal catalyst for long-term enhancement of methane production in a zero-gap microbial electrosynthesis cell.

Microbial electrosynthesis (MES) cells exploit the ability of microbes to convert CO2 into valuable chemical products such as methane and acetate, but high rates of chemical production may need to be mediated by hydrogen and thus require a catalyst for the hydrogen evolution reaction (HER). To avoid the usage of precious metal catalysts and examine the impact of the catalyst on the rate of methane generation by microbes on the electrode, we used a carbon felt cathode coated with NiMo/C and compared performance to a bare carbon felt or a Pt/C-deposited cathode. A zero-gap configuration containing a cation exchange membrane was developed to produce a low internal resistance, limit pH changes, and enhance direct transport of H2 to microorganisms on the biocathode. At a fixed cathode potential of -1 V vs Ag/AgCl, the NiMo/C biocathode enabled a current density of 23 ± 4 A/m2 and a high methane production rate of 4.7 ± 1.0 L/L-d. This performance was comparable to that using a precious metal catalyst (Pt/C, 23 ± 6 A/m2, 5.4 ± 2.8 L/L-d), and 3-5 times higher than plain carbon cathodes (8 ± 3 A/m2, 1.0 ± 0.4 L/L-d). The NiMo/C biocathode was operated for over 120 days without observable decay or severe cathode catalyst leaching, reaching an average columbic efficiency of 53 ± 9 % based on methane production under steady state conditions. Analysis of microbial community on the biocathode revealed the dominance of the hydrogenotrophic genus Methanobacterium (∼40 %), with no significant difference found for biocathodes with different materials. These results demonstrated that HER catalysts improved rates of methane generation through facilitating hydrogen gas evolution to an attached biofilm, and that the long-term enhancement of methane production in MES was feasible using a non-precious metal catalyst and a zero-gap cell design.

Lactate-mediated medium-chain fatty acid production from expired dairy and beverage waste.

Fruits, vegetables, and dairy products are typically the primary sources of household food waste. Currently, anaerobic digestion is the most used bioprocess for the treatment of food waste with concomitant generation of biogas. However, to achieve a circular carbon economy, the organics in food waste should be converted to new chemicals with higher value than energy. Here we demonstrate the feasibility of medium-chain carboxylic acid (MCCA) production from expired dairy and beverage waste via a chain elongation platform mediated by lactate. In a two-stage fermentation process, the first stage with optimized operational conditions, including varying temperatures and organic loading rates, transformed expired dairy and beverage waste into lactate at a concentration higher than 900 mM C at 43 °C. This lactate was then used to produce >500 mM C caproate and >300 mM C butyrate via microbial chain elongation. Predominantly, lactate-producing microbes such as Lactobacillus and Lacticaseibacillus were regulated by temperature and could be highly enriched under mesophilic conditions in the first-stage reactor. In the second-stage chain elongation reactor, the dominating microbes were primarily from the genera Megasphaera and Caproiciproducens, shaped by varying feed and inoculum sources. Co-occurrence network analysis revealed positive correlations among species from the genera Caproiciproducens, Ruminococcus, and CAG-352, as well as Megasphaera, Bacteroides, and Solobacterium, indicating strong microbial interactions that enhance caproate production. These findings suggest that producing MCCAs from expired dairy and beverage waste via lactate-mediated chain elongation is a viable method for sustainable waste management and could serve as a chemical production platform in the context of building a circular bioeconomy.

Deciphering the role of granular activated carbon (GAC) in anammox: Effects on microbial succession and communication.

Anaerobic ammonium oxidation (anammox) offered an energy-efficient option for nitrogen removal from wastewater. Granular activated carbon (GAC) addition has been reported that improved biomass immobilization, but the role of GAC in anammox reactors has not been sufficiently revealed. In this study, it was observed that GAC addition in an upflow anaerobic sludge blanket (UASB) reactor led to the significantly shortened anammox enrichment time (shortened by 45 days) than the reactor without GAC addition. The nitrogen removal rate was 0.83 kg N/m3/day versus 0.76 kg N/m3/day in GAC and non-GAC reactors, respectively after 255 days' operation. Acyl-homoserine lactone (AHL) quorum sensing signal molecule C8-HSL had comparable concentrations in both anammox reactors, whereas the signal molecule C12-HSL was more pervasive in the reactor containing GAC than the reactor without GAC. Microbial analysis revealed distinct anammox development in both reactors, with Candidatus Brocadia predominant in the reactor that did not contain GAC, and Candidatus Kuenenia predominant in the reactor that contained GAC. Denitrification bacteria likely supported anammox metabolism in both reactors. The analyses of microbial functions suggested that AHL-dependent quorum sensing was enhanced with the addition of GAC, and that GAC possibly augmented the extracellular electron transfer (EET)-dependent anammox reaction.

Spatial distributions of granular activated carbon in up-flow anaerobic sludge blanket reactors influence methane production treating low and high solid-content wastewater.

The impacts of granular activated carbon (GAC) spatial distributions in up-flow anaerobic sludge blanket (UASB) reactors treating different solid-content wastewater were evaluated in the present study. When treating high solid-content wastewater, the highest methane yield was observed for UASB supplemented with self-floating GAC (74.2 ± 3.7 %), which was followed by settled + self-floating GAC reactor (65.1 ± 3.8 %), then settled GAC reactor (58.3 ± 1.4 %). When treating low solid-content wastewater, all UASBs achieved improved methane yield, and settled + self-floating GAC reactor achieved the highest methane yield (83.4 ± 3.3 %). Self-floating GAC amended reactor showed the best performance for treating high solid-content wastewater, while settled + self-floating GAC amended reactor was optimal for treating medium and low solid-content wastewater. The spatial distributions of microbial communities differed in the reactors with settled GAC and floating GAC. This study underlines the importance of considering feedwater characteristics when adopting GAC-based UASB processes.

Conductive biofilms in up-flow anaerobic sludge blanket enhanced biomethane recovery from municipal sewage under ambient temperatures.

The feasibility of municipal sewage treatment in laboratory-scale up-flow anaerobic sludge blankets was investigated in this work. Unlike previous studies, granular activated carbon (conductive) or sponge (non-conductive) was introduced to hollow plastic balls as carriers and suspended in the middle and upper layers of the reactors. The two bioreactors were operated at four different hydraulic retention times (stepwise reduced from 24 h to 8 h) for 100 days at ∼18 °C. The conductive-amended treatment was more effective than the non-conductive treatment in enhancing reactor performance. Interestingly, in the reactor containing conductive carriers, microorganisms enriched in the conductive biofilm were also dominant in the suspended sludge. In the reactor containing sponge carriers, the dominant microorganisms differed between the non-conductive biofilm and the suspended sludge. This study underlines that the enrichment of functional microbial communities and the positive impacts of biofilm on suspended sludge are the keys to improving biomethane recovery.

Enhancing the resistance to H2S toxicity during anaerobic digestion of low-strength wastewater through granular activated carbon (GAC) addition.

Low-strength wastewater was treated using two laboratory-scale up-flow anaerobic sludge blankets (UASB) for 130 days under sulfate-reducing conditions. Granular activated carbon (GAC) was added to one of the reactors. The GAC addition increased the total chemical oxygen demand removal by 21-28% and total methane production by 32-78%. The sludge from the GAC-amended UASB showed higher specific methanogenic activities (SMA) and higher activities in the presence of H2S, indicating that the GAC addition enhanced the resistance of methanogens to H2S toxicity. Further, the microbial communities showed that the GAC addition shifted microbial communities. A robust syntrophic partnership between bacteria (i.e., Bacteroidetes_vadinHA17 and Trichococcus) and methanogens was established in the GAC-amended UASB. Sulfate-reducing bacteria (SRB) were enriched in the GAC biofilm, indicating the coexistence of competition and cooperation between SRB and methanogens. These findings provide significant insights regarding microbial community dynamics, especially SRB and methanogens, in a GAC-amended anaerobic digestion process under sulfate-reducing conditions.

Anaerobic digestion of thickened waste activated sludge under calcium hypochlorite stress: Performance stability and microbial communities.

Hypochlorite pretreatment has been proven effective in enhancing waste activated sludge (WAS) anaerobic digestion performances recently. In this study, two semi-continuous anaerobic sequencing batch reactors (ASBRs), one fed with Ca(ClO)2 pretreated thickened WAS (TWAS) and one with raw TWAS, were operated at mesophilic conditions (35 °C) for 145 days. Three loading shocks were introduced to each reactor to compare the performance stability and resilience between the digestion of Ca(ClO)2 pretreated TWAS and untreated TWAS. Microbial community shifts were quantified to reveal the microbiome responses to disturbances. The results suggested that 1% Ca(ClO)2 enhanced the digestion of TWAS by inactivating and transforming the biomass to more easily digested substrates. Co-occurrence network analysis revealed that the strongest interactions in the microbial community occurred in the steady state of TWAS anaerobic digestion.

Metagenomic insights into direct interspecies electron transfer and quorum sensing in blackwater anaerobic digestion reactors supplemented with granular activated carbon.

The addition of granular activated carbon (GAC) enhanced the performance of up-flow anaerobic sludge blanket (UASB) reactor treating blackwater at 35 °C. DNA were extracted from the sludge and biofilms attached to GAC and submitted for shotgun sequencing. In addition, the acyl-homoserine lactones (AHLs) were quantified. Diverse partners for direct interspecies electron transfer (DIET) were enriched in the sludge or biofilm (GAC-biofilm) of GAC amended UASB. Pedosphaera parvula, Syntrophus aciditrophicus and Syntrophorhabus aromaticivorans were dominant syntrophs. The analysis for type IV pilus assembly genes further suggested DIET may be functioned through GAC for GAC-biofilm, while through conductive pili for sludge aggregates. AHLs quantification and the analysis for quorum sensing (QS) related genes indicated higher QS activity at the population level was induced by GAC. Overall, the work illustrated the different DIET patterns, and suggested that QS played an important role in controlling the performance in GAC amended USAB.

Microbial co-occurrence network topological properties link with reactor parameters and reveal importance of low-abundance genera.

Operational factors and microbial interactions affect the ecology in anaerobic digestion systems. From 12 lab-scale reactors operated under distinct engineering conditions, bacterial communities were found driven by temperature, while archaeal communities by both temperature and substrate properties. Combining the bacterial and archaeal community clustering patterns led to five sample groups (ambient, mesophilic low-solid-substrate, mesophilic, mesophilic co-digestion and thermophilic) for co-occurrence network analysis. Network topological properties were associated with substrate characteristics and hydrolysis-methanogenesis balance. The hydrolysis efficiency correlated (p < 0.05) with clustering coefficient positively and with normalized betweenness negatively. The influent particulate COD ratio and the relative differential hydrolysis-methanogenesis efficiency (Defficiency) correlated negatively with the average path length (p < 0.05). Individual genera's topological properties showed more connector genera in thermophilic network, representing stronger inter-module communication. Individual genera's normalized degree and betweenness revealed that lower-abundance genera (as low as 0.1%) could perform central hub roles and communication roles, maintaining the stability and functionality of the microbial community.

Roles of granular activated carbon (GAC) and operational factors on active microbiome development in anaerobic reactors.

Ambient temperature municipal sewage was treated using two laboratory-scale up-flow anaerobic sludge blanket reactors for 225 days. Granular activated carbon (GAC) was added to one reactor to facilitate the development of direct interspecies electron transfer (DIET). The GAC addition increased total chemical oxygen demand removal by 5% - 18%. In addition to assessing the relative abundance of active amplicon sequence variants (ASVs), the mass balance model, the Mantel test, and the generalized linear models were applied to evaluate the dynamics of the active ASVs and the key operational factors controlling the bioreactor microbial community. These results demonstrated that, in addition to the GAC addition, extrinsic engineering operational factors played important roles in controlling (active) microbial communities. This study underlines the importance of taking a wholistic approach to assess microbial population dynamics. Reactor design and performance prediction should consider key engineering parameters when using DIET-based AD reactors in the future.

Calcium hypochlorite enhances the digestibility of and the phosphorus recovery from waste activated sludge.

Waste activated sludge (WAS) can be treated using anaerobic digestion (AD) for biogas recovery and volume reduction. However, the poor digestibility and hydrolysis of WAS limit AD applications. The current study investigated the feasibility of applying calcium hypochlorite as a WAS pretreatment strategy to improve AD treatment efficiency using laboratory reactors. The results showed that pretreatment with 5 - 20% Ca(ClO)2 (total suspended solids basis) significantly enhanced WAS anaerobic digestibility, and led to significantly enhanced methane production rate and biomethane yield comparing to the AD of raw WAS (P < 0.05). Low Ca(ClO)2 pretreatment (5 - 10%) significantly enhanced digestion efficiency, which can be attributed to the development of fermentative and syntrophic bacteria. However, high Ca(ClO)2 doses (>20%) reduced microbial activities, leading to slow release of dissolved organic compounds and prolonged methane production lag phase. In addition, high Ca(ClO)2 removed 82.7% of the initial phosphate by calcium-phosphate binding, reducing the phosphorus in liquid digestate.

Cometabolism accelerated simultaneous ammoxidation and organics mineralization in an oxygen-based membrane biofilm reactor treating greywater under low dissolved oxygen conditions.

Carbon/nitrogen ratio is an important parameter during the biological wastewater treatment. Our study emphasizes revealing the mechanisms of chemical oxygen demand/total nitrogen (COD/TN) ratio dependent improved greywater (GW) treatment in an oxygen based membrane biofilm reactor (O2-MBfR). Results showed that reducing COD/TN ratio from 40 to 20 g COD/g N by supplementing NH4Cl to GW improved the relative abundance of genera related to LAS-biodegradation (from 8.39% to 35.7%), nitrification (from 0.20% to 0.62%) and denitrification (from 3.01% to 7.59%). Reducing COD/TN ratio also led to an increase in the ammonia monooxygenase (AMO) activity (from 7.56 to 10.2 mg N/g VSS-h), as well as improved ammoxidation and linear alkylbenzene sulfonate (LAS) mineralization although the dissolved oxygen (DO) concentration and pH decreased. Much higher NH4+ - N at lower COD/TN ratio (10 units) led to lower DO (0.13 ± 0.01 mg/L) and pH (6.72 ± 0.02), but the continuously increased AMO activity (up to 12.9 mg N/g VSS-h) enabled the cometabolism of ammoxidation and LAS mineralization, leading to the efficient removal of organics and nitrogen under the low DO condition.

Effects of micro-aeration on microbial niches and antimicrobial resistances in blackwater anaerobic digesters.

Anaerobic digestion (AD) of source-diverted blackwater (toilet flush) at ambient room temperature presents challenges for fast hydrolysis of particulate matters. This study investigated the effect of different micro-aeration dosages for blackwater AD. Sequencing batch reactors were operated at ambient room temperature (22 ± 1°C) with micro-aeration (0, 5, 10, 50, and 150 mg O2 g-1 CODfeed per cycle) and gradually reduced hydraulic retention times from 5 d to 2 d. The methanogenesis efficiencies were greater at low oxygen dosages (i.e., 0, 5, 10) while the volatile fatty acids (VFAs) accumulated more at high oxygen dosages (i.e., 50, 150). Microbial communities were significantly different under different oxygen dosages (p<0.05), with segregation of microbial ecological niches in low and high oxygen dosage communities. The low-oxygen-dosage niche (0, 5, and 10 mg g-1 CODfeed) was inhabited by fermenting and syntrophic bacteria (e.g., Cytophaga, Syntrophomonas) and methanogens (e.g., Methanobacterium, Methanolinea, Methanosaeta). The high-oxygen-dosage niche (50 and 150 mg g-1 CODfeed) had significantly (p<0.05) more facultative anaerobic bacteria (Ignavibacteriales and Cloacamonales), and aerobic bacteria (Rhodocyclales). Moreover, blackwater can be a source of antimicrobial resistance genes (ARGs), which are affected by different oxygen dosages. The ARG variation correlated with the microbial community composition (p<0.05). Low-oxygen-dosage communities contained a higher prevalence of mobile gene elements (intI1 and korB) and tetM, ermB, sul1, sul2, and blaCTX-M than the high-oxygen-dosage communities, indicating that oxygen dosage influenced the prevalence of populations carrying ARGs. These findings suggest that application of micro-aeration to AD can be used to control ARG profiles.

Shaping biofilm microbiomes by changing GAC location during wastewater anaerobic digestion.

The addition of granular activated carbon (GAC) to up-flow anaerobic sludge blanket (UASB) reactors treating synthetic wastewater enhanced methane production by stimulating direct interspecies electron transfer (DIET). A modified UASB reactor with GAC packed in plastic carriers that allowed the GAC to float in the upper reactor zone achieved enhanced performance compared to a UASB reactor with GAC settled at the bottom of the reactor. Microbial communities in the biofilms developed on settled or floated GAC were compared. Methanosarcina (56.3-73.3%) dominated the floated-GAC biofilm whereas Methanobacterium (84.9-85.1%) was greatly enriched in the settled-GAC biofilm. Methanospirillum and Methanocorpusculum were enriched in the floated-GAC biofilm (8.8-19.8% and 5.1-9.5%, respectively), but only existed in low abundances in the settled-GAC biofilm (3.4-3.6% and 0-0.4%, respectively). The floated GAC developed bacterial communities with higher diversity and more syntrophic bacteria enrichments on its surface, including Geobacter, Smithella, and Syntrophomonas, than the settled-GAC biofilm. Common hydrogen-donating syntrophs and hydrogenotrophic archaea, Methanospirillum and Methanoregula, were identified as potential electro-active microorganisms related to DIET.

Lumen air pressure (LAP) affecting greywater treatment in an oxygen-based membrane biofilm reactor (O2-MBfR).

Several technologies have been employed to treat greywater (GW) for domestic use. Aerobic biological treatment has achieved high efficiency, the main cost being the necessary source of oxygen (O2). This study explores the effects of lumen air pressure (LAP) on reactor performance and microbial community succession in an O2-based membrane biofilm reactor (O2-MBfR) treating GW. At high LAP (≥0.8 psi), the dissolved oxygen (DO) concentration inside the reactor was higher than 0.38 ± 0.02 mg/L, leading to removal efficiencies of 90%, 98%, and 80%, of total chemical oxygen demand, total linear alkylbenzene sulfonate (LAS), and total nitrogen, respectively. Lower LAP (<0.8 psi) led to a decrease in DO inside the system, and a less effective GW treatment. Low O2 pressure decreased organic biodegradation and ammoniation, and caused LAS accumulation in the biofilm, leading to the solubilization of extracellular polymeric substances and cell lysis. Comprehensive consideration of reactor performance and energy input, DO inside the MBfR at 0.38 ± 0.02 mg/L could be selected as the optimized condition for GW treatment. Microbial community analyses results also revealed that improved LAP was favorable for the enrichment of LAS-biodegradation related genus (Pseudomonas, Parvibaculum, Magnetospirillum, Clostridium, Zoogloea, Dechloromonas and Mycobacterium), nitrifiers (Nitrosomonas and Sphingomonas) and facultative microorganisms (Dechloromonas, Flavobacterium, Pseudomonas, Aeromonas and Zoogloea) that can carry out denitrification under relatively high DO conditions (>0.38 mg/L), but led to the reduction of the relative abundance of heterotrophs (Acidovorax, Thermomonas, Brevundimonas and Enterobacter) that are more sensitive towards high DO conditions.

Impacts of conductive materials on microbial community during syntrophic propionate oxidization for biomethane recovery.

Propionate is one of the most important intermediates in anaerobic digestion, and its degradation requires a syntrophic partnership between propionate-oxidizing bacteria and hydrogenotrophic methanogens. Anaerobic digestion efficiency can be improved by direct interspecies electron transfer (DIET) through conductive materials. This study aimed to investigate the effects of DIET on syntrophic propionate oxidization under room temperature (20°C) and reveal the syntrophic partners. Firstly, conventional anaerobic consortium and conductive material-enriched consortium were tested for DIET under high H2 partial pressure. The latter supplemented with granular activated carbon (GAC) can mitigate H2 inhibition through DIET. Secondly, a DIET consortium was enriched for testing GAC and magnetite, both showed DIET facilitation. Microbial communities in GAC- and magnetite-supplemented reactors were similar. Syntrophic propionate-oxidizing bacteria, for example, Smithella (3.9%-9.9%) and a genus from the family Syntrophaceae (1.9%-3.6%) and methanogens Methanobacterium (30.3%-75.2%), Methanolinea (8.5%-25.2%), Methanosaeta (11.4%-36.7%), and Candidatus Methanofastidiosum (3.6%-6.6%), were predominant. Functional genes for cell mobility and membrane transport (3.3% and 9.5% in control reactor) increased with GAC (3.7% and 11.1%, respectively) and magnetite (3.7% and 10.9%, respectively) addition. Syntrophic propionate-oxidizing bacteria and methanogenesis partners were revealed by co-occurrence network, for example, Methanobacterium with Smithella, Syntrophobacter, Dechloromonas, and Trichococcus, signifying the importance of the syntrophic partnership in DIET environment. PRACTITIONER POINTS: DIET improved syntrophic propionate oxidization under room temperature condition (20°C). Microbial communities were similar for GAC- and magnetite-supplemented reactors, different with control reactor. Syntrophic propionate-oxidizing bacteria and methanogenesis partners were revealed by co-occurrence network. Methanobacterium and Smithella, Syntrophobacter, Dechloromonas, and Trichococcus were correlated.

Different micro-aeration rates facilitate production of different end-products from source-diverted blackwater.

The effects of micro-aeration on the performance of anaerobic sequencing batch reactors (ASBR) for blackwater treatment were investigated in this study. Different micro-aeration rates, 0, 5, 10, 50, and 150 mg O2/L-reactor/cycle, and their effect on the hydrolysis, acidogenesis, and methanogenesis of blackwater were evaluated and compared at ambient temperature. Source-diverted blackwater (toilet water) contains high organic contents which can be recovered as biogas. Previous studies have found that anaerobic digestion of blackwater without micro-aeration can only recover upwards of less than 40% of chemical oxygen demand (COD) to methane at room temperature due to the low hydrolysis rate of biomass content in blackwater. This study achieved increases in blackwater hydrolysis (from 34.7% to 48.7%) and methane production (from 39.6% to 50.7%) with controlled micro-aeration (5 mg O2/L-reactor/cycle). The microbial analysis results showed that hydrolytic/fermentative bacteria and acetoclastic methanogens (e.g. Methanosaeta) were in higher abundances in low-dose micro-aeration reactors (5 and 10 mg O2/L-reactor/cycle), which facilitated syntrophic interactions between microorganisms. The relative abundance of oxygen-tolerant methanogen such as Methanosarcina greatly increased (from 1.5% to 11.4%) after oxygen injection. High oxygen dosages (50 and 150 mg O2/L-reactor/cycle) led to reduced methane production and higher accumulation of volatile fatty acids, largely due to the oxygen inhibition on methanogens and degradation of organic matters by aerobic growth and respiration, as indicated by the predicted metagenome functions. By combining reactor performance results and microbial community analyses, this study demonstrated that low-dose micro-aeration improves blackwater biomethane recovery by enhancing hydrolysis efficiency and promoting the development of a functional microbial population, while medium to high-dose micro-aeration reduced the activities of certain anaerobes. It was also observed that medium-dose micro-aeration maximizes VFA accumulation, which may be used in two-stage anaerobic digesters.

Enhancement of the sludge disintegration and nutrients release by a treatment with potassium ferrate combined with an ultrasonic process.

Sludge disintegration by ultrasound is a promising sludge treatment method. In order to enhance the efficiency of the sludge reduction and hydrolysis, potassium ferrate (K2FeO4) (PF) was used. A novel method was developed to improve the sludge disintegration-sludge pretreatment by using PF in combination with an ultrasonic treatment (PF + ULT). After a short-term PF + ULT treatment, 17.23% of the volatile suspended solids (VSS) were reduced after a 900-min reaction time, which is 61.3% higher than the VSS reduction for the raw sludge. The supernatant soluble chemical oxygen demand (SCOD), total nitrogen (TN), volatile fatty acids (VFAs), soluble protein and polysaccharides increased by 522.5%, 1029.4%, 878.4%, 2996.6% and 801.9%, respectively. The constituent parts of the dissolved organic matter of the sludge products were released efficiently, which demonstrated the positive effect caused by the PF + ULT. The enhanced sludge disintegration process further alleviates environmental risk and offers a more efficient and convenient method for utilizing sludge.