Simultaneous effect of cathode potentials and magnetite concentrations on methanogenesis of acetic acid under different ammonia conditions

Electromethanogenesis (EM) is a system that facilitates direct interspecies electron transfer (DIET) in anaerobic digestion (AD) by providing an external power supply to favor desired reactions. Substrates of AD commonly contain ammonia (NH3) as biodegradation product of nitrogen-rich compounds that can deteriorate the stability of AD process. Optimized cathode potential (VCAT) and magnetite (Mag) concentration ([Mag]) are expected to improve AD efficiency in the presence of NH3. Response surface analysis with central composite face-centered design was used in this study to investigate the effect of VCAT and [Mag] under different total ammonia nitrogen concentration ([TAN]). Highest cumulative methane production was achieved at VCAT = -737.4 mV, [Mag] = 18.2 mM, and [TAN] = 1.5 g/L; highest acetate degradation rate was achieved at VCAT = 757.6 mV, [Mag] = 21.4 mM, and [TAN] = 1.5 g/L. The study demonstrated that VCAT promotes either microbial growth or electrochemical NH3 removal. A Shift from acetoclastic to hydrogenotrophic pathway was also observed by the increase of hydrogenotrophic methanogen populations at the end of experiment. This study is beneficial for process control of AD under different NH3 conditions.

Chasing the metabolism of novel syntrophic acetate-oxidizing bacteria in thermophilic methanogenic chemostats

Background: Acetate is the major intermediate of anaerobic digestion of organic waste to CH4. In anaerobic methanogenic systems, acetate degradation is carried out by either acetoclastic methanogenesis or a syntrophic degradation by a syntrophy of acetate oxidizers and hydrogenotrophic methanogens. Due to challenges in isolation of syntrophic acetate-oxidizing bacteria (SAOB), the diversity and metabolism of SAOB, as well as the mechanisms of their interactions with methanogenic partners remain poorly understood. Results: In this study, we successfully enriched previously unknown SAOB by operating continuous thermophilic anaerobic chemostats fed with acetate, propionate, butyrate, or isovalerate as the sole carbon and energy source. They represent novel clades belonging to Clostridia, Thermoanaerobacteraceae, Anaerolineae, and Gemmatimonadetes. In these SAOB, acetate is degraded through reverse Wood-Ljungdahl pathway or an alternative pathway mediated by the glycine cleavage system, while the SAOB possessing the latter pathway dominated the bacterial community. Moreover, H2 is the major product of the acetate degradation by these SAOB, which is mediated by [FeFe]-type electron-confurcating hydrogenases, formate dehydrogenases, and NADPH reoxidation complexes. We also identified the methanogen partner of these SAOB in acetate-fed chemostat, Methanosarcina thermophila, which highly expressed genes for CO2-reducing methanogenesis and hydrogenases to supportively consuming H2 at transcriptional level. Finally, our bioinformatical analyses further suggested that these previously unknown syntrophic lineages were prevalent and might play critical roles in thermophilic methanogenic reactors. Conclusion: This study expands our understanding on the phylogenetic diversity and in situ biological functions of uncultured syntrophic acetate degraders, and presents novel insights on how they interact with their methanogens partner. These knowledges strengthen our awareness on the important role of SAO in thermophilic methanogenesis and may be applied to manage microbial community to improve the performance and efficiency of anaerobic digestion. Keywords: Thermophilic anaerobic digestion, Microbial community, Syntrophic acetate oxidation, Glycine cleavage, Energy conservation

Endogenous esterases of Clostridium thermocellum are identified and disrupted for enhanced isobutyl acetate production from cellulose

ABSTRACTMedium chain esters are potential drop-in biofuels and versatile chemicals. Currently, these esters are largely produced by the conventional chemical process that uses harsh operating conditions and requires high energy input. Alternatively, the microbial conversion route has recently emerged as a promising platform for sustainable and renewable ester production. The ester biosynthesis pathways can utilize either esterases/lipases or alcohol acyltransferase (AAT), but the AAT-dependent pathway is more thermodynamically favorable in aqueous fermentation environment. Even though cellulolytic thermophiles such as Clostridium thermocellum harboring the engineered AAT-dependent pathway can directly convert lignocellulosic biomass into esters, the production is currently not efficient and requires optimization. One potential bottleneck is the ester degradation caused by the endogenous carbohydrate esterases (CEs) whose functional roles are poorly understood. In this study, we developed a simple, high-throughput colorimetric assay to screen the endogenous esterases of C. thermocellum responsible for ester hydrolysis. We identified, characterized, and disrupted two critical endogenous esterases that significantly contributes to isobutyl acetate degradation in C. thermocellum. We demonstrated that not only did the engineered esterase-deficient strain alleviate ester hydrolysis but also helped improve isobutyl acetate production while not affecting its robust metabolism for effective cellulose assimilation.IMPORTANCECarbohydrate esterases (CEs) are important enzymes in the deconstruction of lignocellulosic biomass by the cellulolytic thermophile C. thermocellum, yet some are potential ester degraders in a microbial ester production system. Currently, the functional roles of CEs for hydrolyzing medium chain esters and negatively affecting the ester microbial biosynthesis are not well understood. This study discovered novel CEs responsible for isobutyl acetate degradation in C. thermocellum and hence identified one of the critical bottlenecks for direct conversion of lignocellulosic biomass into esters.

Anaerobic Co-Digestion of Sludge and Organic Food Waste—Performance, Inhibition, and Impact on the Microbial Community

Anaerobic co-digestion allows for under-utilised digesters to increase biomethane production. The organic fraction of municipal solid waste (OFMSW), i.e., food waste, is an abundant substrate with high degradability and gas potential. This paper investigates the co-digestion of mixed sludge from wastewater treatment plants and OFMSW, through batch and continuous lab-scale experiments, modelling, and microbial population analysis. The results show a rapid adaptation of the process, and an increase of the biomethane production by 20% to 40%, when co-digesting mixed sludge with OFMSW at a ratio of 1:1, based on the volatile solids (VS) content. The introduction of OFMSW also has an impact on the microbial community. With 50% co-substrate and constant loading conditions (1 kg VS/m3/d) the methanogenic activity increases and adapts towards acetate degradation, while the community in the reference reactor, without a co-substrate, remains unaffected. An elevated load (2 kg VS/m3/d) increases the methanogenic activity in both reactors, but the composition of the methanogenic population remains constant for the reference reactor. The modelling shows that ammonium inhibition increases at elevated organic loads, and that intermittent feeding causes fluctuations in the digester performance, due to varying inhibition. The paper demonstrates how modelling can be used for designing feed strategies and experimental set-ups for anaerobic co-digestion.

The joint acute effect of tetracycline, erythromycin and sulfamethoxazole on acetoclastic methanogens

In this study, we aimed to develop an understanding of the triple effects of sulfamethoxazole–erythromycin–tetracycline (ETS) and the dual effects of sulfamethoxazole–tetracycline (ST), erythromycin–sulfamethoxazole (ES) and erythromycin–tetracycline (ET) on the anaerobic treatment of pharmaceutical industry wastewater throughout a year of operation. Concentrations of the antibiotics in the influent were gradually increased until the metabolic collapse of the anaerobic sequencing batch reactors (SBRs), which corresponded to ETS (40 + 3 + 3 mg/L) and ST (25 + 2.5 mg/L), ET (4 + 4 mg/L) and ES (3 + 40 mg/L). Acetate accumulation in the anaerobic SBRs, acetoclastic activity of the anaerobic sludge taken from different antibiotic feeding stages and also expression of acetyl-coA synthetase from the acetoclastic methanogenic pathway on the mRNA level were assessed. The results indicated that, while acetate accumulation and decrease of acetoclastic activity were observed after stage 3 in the ST and ES reactors, and stage 7 in the ETS and ET reactors, the expression of acetyl-coA synthetase was mostly decreased in the last stages in all SBRs, in which antibiotic mixture feeding was terminated. It might be speculated that acetoclastic methanogens have an important role in acetate degradation by expressing acetyl-coA synthetase.