Methanol as a co-substrate with CO2 enhances butyrate production in microbial electrosynthesis.

Methanol is a promising feedstock for the bio-based economy as it can be derived from organic waste streams or produced electrochemically from CO2. Acetate production from CO2 in microbial electrosynthesis (MES) has been widely studied, while more valuable compounds such as butyrate are currently attracting attention. In this study, methanol was used as a co-substrate with CO2 to enhance butyrate production in MES. Feeding with CO2 and methanol resulted in the highest butyrate production rates and titres of 0.36 ± 0.01 g L-1 d-1 and 8.6 ± 0.2 g L-1, respectively, outperforming reactors with only CO2 feeding (0.20 ± 0.03 g L-1 d-1 and 5.2 ± 0.1 g L-1, respectively). Methanol acted as electron donor and as carbon source, both of which contributed ca. 50% of the carbon in the products. Eubacterium was the dominant genus with 52.6 ± 2.5% relative abundance. Thus, we demonstrate attractive route for the use of the C1 substrates, CO2 and methanol, to produce mainly butyrate. KEY POINTS: • Butyrate was the main product from methanol and CO2 in MES • Methanol acted as both carbon and electron source in MES • Eubacterium dominating microbial culture was enriched in MES.

Comparing the performance of fluidized and fixed granular activated carbon beds as cathodes for microbial electrosynthesis of carboxylates from CO2.

Microbial electrosynthesis (MES) can use renewable electricity to power microbial conversion of carbon dioxide (CO2) into carboxylates. To ensure high productivities in MES, good mass transfer must be ensured, which could be accomplished with fluidization of granular activated carbon (GAC). In this study, fluidized and fixed GAC bed cathodes were compared. Acetate production rate and current density were 42 % and 47 % lower, respectively, in fluidized than fixed bed reactors. Although similar microbial consortium dominated by Eubacterium and Proteiniphilum was observed, lowest biomass quantity was measured with fixed GAC bed indicating higher specific acetate production rates compared to fluidized GAC bed. Furthermore, charge efficiency was the highest and charge recovery in carboxylates the lowest in fixed GAC beds indicating enhanced hydrogen evolution and need for enhancing CO2 feeding to enable higher production rates of acetate. Overall, fixed GAC beds have higher efficiency for acetate production in MES than fluidized GAC beds.

Anode-assisted electro-fermentation with Bacillus subtilis under oxygen-limited conditions.

BACKGROUND: Bacillus subtilis is generally regarded as a ubiquitous facultative anaerobe. Oxygen is the major electron acceptor of B. subtilis, and when oxygen is absent, B. subtilis can donate electrons to nitrate or perform fermentation. An anode electrode can also be used by microorganisms as the electron sink in systems called anodic electro-fermentation. The facultative anaerobic character of B. subtilis makes it an excellent candidate to explore with different electron acceptors, such as an anode. This study aimed to optimise industrial aerobic bioprocesses using alternative electron acceptors. In particular, different end product spectrum of B. subtilis with various electron acceptors, including anode from the electro-fermentation system, was investigated. RESULTS: B. subtilis was grown using three electron acceptors, i.e. oxygen, nitrate and anode (poised at a potential of 0.7 V vs. standard hydrogen electrode). The results showed oxygen had a crucial role for cells to remain metabolically active. When nitrate or anode was applied as the sole electron acceptor anaerobically, immediate cell lysis and limited glucose consumption were observed. In anode-assisted electro-fermentation with a limited aeration rate, acetoin, as the main end product showed the highest yield of 0.78 ± 0.04 molproduct/molglucose, two-fold higher than without poised potential (0.39 ± 0.08 molproduct/molglucose). CONCLUSIONS: Oxygen controls B. subtilis biomass growth, alternative electron acceptors utilisation and metabolites formation. Limited oxygen/air supply enabled the bacteria to donate excess electrons to nitrate or anode, leading to steered product spectrum. The anode-assisted electro-fermentation showed its potential to boost acetoin production for future industrial biotechnology applications.

Modelling and techno-economic assessment of (bio)electrochemical nitrogen removal and recovery from reject water at full WWTP scale.

At conventional wastewater treatment plants (WWTPs), reject waters originating from the dewatering of anaerobically digested sludge contain the highest nitrogen concentrations within the plant and thereby have potential for realising nitrogen recovery in a reusable form. At the same time, nitrogen removal from reject waters has potential to reduce the energetic and chemical demands of the WWTP due to a reduced nutrient load to the activated sludge process. In recent years, (bio)electrochemical methods have been extensively studied for nitrogen recovery from reject waters in laboratory-scale but not yet implemented in real WWTP environments, particularly due to concerns about the need for large capital investments. This study assessed the techno-economic feasibility of retrofitting a (bio)electrochemical nitrogen removal and recovery (NRR) unit into the reject water circulation line of a full-scale WWTP through modelling. Data from laboratory-scale (bio)electroconcentration ((B)EC) experiments was used to construct a simple, semi-empirical model block integrated into the Benchmark Simulation Model No. 2 (BSM2) simulating a generalised WWTP. The effects of nitrogen removal from the reject water on both the effluent quality and operational costs of the WWTP were assessed and compared to the BSM2 performance without an NRR unit. In all studied scenarios, the effluent quality index was improved by 4-11%, while both the aeration (7-19% decrease) and carbon (24-71%) requirements were reduced. The additional energy consumed by the NRR unit increased the total operational cost index by >18%, but the revenue assumed for the generated nutrient product (20 EUR kgN-1) was enough to make the BEC-NRR scenarios at realistically low current densities (1 and 5 A m-2) economically attractive compared to the control. A sensitivity analysis revealed that electricity price and nutrient product value had the most notable effects on the feasibility of the NRR unit. The results suggest a key factor in making (bio)electrochemical NRR economically viable is to reduce its electricity consumption further, while the anticipated increases in nitrogen fertiliser prices can help accelerate the adoption of these methods in larger scale.

The effects of weathering-induced degradation of polymers in the microplastic study involving reduction of organic matter.

The analysis of microplastics in complex environmental samples requires the use of chemicals to reduce the organic matrix. This procedure should be evaluated in terms of the preservation of the microplastic's integrity, typically done with pristine reference microplastics. However, real microplastics are most likely degraded due to weathering, so pristine reference microplastics might not depict the appropriateness of the process. This study performed a purification process using sodium dodecyl sulfate and hydrogen peroxide on sewage sludge containing LLDPE, HDPE, PP, PS, PET, PA66 and SBR samples exposed to simulated environmental weathering. The degradation of the polymers was assessed by analyzing surface morphology, mass variation, and mechanical, thermal and chemical properties. Comparison with pristine polymers revealed that the purification process can lead to more detrimental effects if the polymers are weathered. After the purification process, some important observations were: 1) LLDPE, PP and SBR surfaces had cracks in the weathered samples that were not observed in the pristine samples, 2) weathered LLDPE, PP and PA66 experienced greater mass loss than pristine, 3) the fragmentation propensity of weathered LLDPE, HDPE, PP, PS and SBR increased compared to pristine samples and 4) the main characteristic peaks in FTIR spectrum could be identified and used for chemical identification of most polymers for pristine and weathered samples. Based on the findings of this study, when analyzing the efficiency and adequacy of a purification process with methods based on surface morphology, mass variation and particle counting indicators, it is recommended to consider the differences that potentially arise between pristine and weathered microplastics, especially for polyolefins (PEs and PP).

The effects of digestate pyrolysis liquid on the thermophilic anaerobic digestion of sewage sludge - Perspective for a centralized biogas plant using thermal hydrolysis pretreatment.

The use of pyrolysis process to valorize digestate from anaerobic digestion (AD) of municipal sewage sludge for biochar production was piloted in a central biogas plant. The pyrolysis also generates pyrolysis liquid with high organics and nutrient contents that currently has no value and requires treatment, which could potentially be done in AD. As the pyrolysis liquid may contain inhibitory compounds, we investigated the effects of adding the pyrolysis liquid on AD of sewage sludge and thermal hydrolysis pretreated sewage sludge (THSS) simulating the full-scale centralized biogas plant conditions. In batch assays, the pyrolysis liquid as such did not produce any methane, and the 1% and 5% (v/w) shares suppressed the methane production from THSS by 14-19%, while a smaller decrease in methane production was observed with sewage sludge. However, in the semi-continuous reactor experiments, pyrolysis liquid at a 1% (v/w) share was added in sewage sludge or THSS feed without affecting the methane yields or digestate characteristics. The laboratory results indicated that pyrolysis liquid can be treated in AD, while extrapolating the results to the centralized biogas plant indicated minor increase in the overall methane production and an increased potential for ammonium recovery.

Hydrothermal carbonization of pulp and paper industry wastewater treatment sludges - characterization and potential use of hydrochars and filtrates.

The pulp and paper industry's mixed sludge represents waste streams with few other means of disposal than incineration. Hydrothermal carbonization (HTC) could be advantageous for the sludge refinement into value-added products, thus complementing the concept of pulp and paper mills as biorefineries. Laboratory HTC was performed on mixed sludge (at 32% and 15% total solids) at temperatures of 210-250 °C for 30 or 120 min, and the characteristics of the HTC products were evaluated for their potential for energy, carbon, and nutrient recovery. The energy content increased from 14.9 MJ/kg in the mixed sludge up to 20.5 MJ/kg in the hydrochars. The produced filtrates had 12-15-fold higher COD and 3-5-fold higher volumetric methane production than untreated sludge filtrates, even though the methane yield against g-COD was lower. The increased value of the hydrochars in terms of energy content and carbon sequestration potential promote HTC deployment in sludge treatment and upgrading.

Optimised reduction of total solids and organic matter of sewage sludge matrix for an improved extraction of microplastics.

The extraction of microplastics from complex environmental matrices, such as sewage sludge, has proven challenging because of their high organic content. A common procedure for the extraction of microplastics from sludge involves conducting a chemical digestion to reduce the amount of organic matter in the sample, followed by a density separation of microplastics. In order to increase the reliability of the density-based separation, an optimisation of the chemical digestion is needed. The aim of this study was to maximise the total solids and carbon content reduction of sludge by optimising the sodium dodecyl sulphate (SDS) pretreatment and the duration of H2O2 digestion. A reduction in total solids by 95.6% and in carbon content by 98.1% were achieved with the optimised digestion method, which involved an application of 1% SDS and a 2-day H2O2 treatment in the first digestion step. The inclusion of the SDS pretreatment significantly increased the reduction of total solids and carbon content. The optimised digestion process had no significant visible effects on tested reference microplastics and provided an extraction efficiency of 84% for 150 µm reference microspheres and 72% for 650 µm long microfibres. To enable the application of the optimised digestion process to other types of sludges, the consumption of SDS and H2O2 were also presented as per grams of organic matter in the untreated sludge.

An online flow-imaging particle counter and conventional water quality sensors detect drinking water contamination in the presence of normal water quality fluctuations.

Contamination detection in drinking water is crucial for water utilities in terms of public health; however, current online water quality sensors can be unresponsive to various possible contaminants consisting of particulate and dissolved content or require a constant supply of reagents and sample preparation. We used a two-line test environment connected to a drinking water distribution system with flow-imaging particle counters and conventional sensors to assess their responses to the injection of contaminants into one line, including stormwater, treated wastewater, wastewater, well water, and Escherichia coli, while simultaneously measuring responses to normal water quality fluctuations in the other line. These water quality fluctuations were detected with all of the conventional sensors (except conductivity) and with 3 out of 5 of the size- and shape-derived particle classes of the flow-imaging particle counter. The flow-imaging particle counter was able to detect all of the studied contaminants, e.g. municipal wastewater at 0.001% (v/v), while the oxidation-reduction potential sensor outperformed other conventional sensors, detecting the same wastewater at 0.03% (v/v). The presence of particles less than 1 µm in size was shown to be a generic parameter for the detection of particulates present in the studied contaminants; however, they manifested a considerable response to fluctuations which led to lower relative response to contaminants in comparison to larger particles. The particle size and class distributions of contaminants were different from those of drinking water, and thus monitoring particles larger than 1 µm or specific particle classes of flow-imaging particle counter, which are substantially more abundant in contaminated water than in pure drinking water, can improve the detection of contamination events. Water utilities could optimize contamination detection by selecting water quality parameters with a minimal response to quality fluctuations and/or a high relative response to contaminants.

Cathodic biofilms - A prerequisite for microbial electrosynthesis.

Cathodic biofilms have an important role in CO2 bio-reduction to carboxylic acids and biofuels in microbial electrosynthesis (MES) cells. However, robust and resilient electroactive biofilms for an efficient CO2 conversion are difficult to achieve. In this review, the fundamentals of cathodic biofilm formation, including energy conservation, electron transfer and development of catalytic biofilms, are presented. In addition, strategies for improving cathodic biofilm formation, such as the selection of electrode and carrier materials, cell design and operational conditions, are described. The knowledge gaps are individuated, and possible solutions are proposed to achieve stable and productive biofilms in MES cathodes.

Microbial community assembly and dynamics in Granular, Fixed-Biofilm and planktonic microbiomes valorizing Long-Chain fatty acids at 20 °C.

Distinct microbial assemblages evolve in anaerobic digestion (AD) reactors to drive sequential conversions of organics to methane. The spatio-temporal development of three such assemblages (granules, biofilms, planktonic) derived from the same inoculum was studied in replicated bioreactors treating long-chain fatty acids (LCFA)-rich wastewater at 20 °C at hydraulic retention times (HRTs) of 12-72 h. We found granular, biofilm and planktonic assemblages differentiated by diversity, structure, and assembly mechanisms; demonstrating a spatial compartmentalisation of the microbiomes from the initial community reservoir. Our analysis linked abundant Methanosaeta and Syntrophaceae-affiliated taxa (Syntrophus and uncultured) to their putative, active roles in syntrophic LCFA bioconversion. LCFA loading rates (stearate, palmitate), and HRT, were significant drivers shaping microbial community dynamics and assembly. This study of the archaea and syntrophic bacteria actively valorising LCFAs at short HRTs and 20 °C will help uncover the microbiology underpinning anaerobic bioconversions of fats, oil and grease.

Hydrothermal carbonisation of mechanically dewatered digested sewage sludge-Energy and nutrient recovery in centralised biogas plant.

This study aimed to assess the role of hydrothermal carbonisation (HTC) in digestate processing in centralised biogas plants receiving dewatered sludge from regional wastewater treatment plants and producing biomethane and fertilisers. Chemically conditioned and mechanically dewatered sludge was used as such (total solids (TS) 25%) or as diluted (15% TS) with reject water in 30 min or 120 min HTC treatments at 210 °C, 230 °C or 250 °C, and the produced slurry was filtered to produce hydrochars and filtrates. The different hydrochars contributed to 20-55% of the original mass, 72-88% of the TS, 74-87% of the energy content, 71-92% of the carbon, above 86% of phosphorous and 38-64% of the nitrogen present in the original digestates. The hydrochars' energy content (higher heating values were 11.3-12.2 MJ/kg-TS) were similar to that of the digestates, while the ash contents increased (from 43% up to 57%). HTC treatments produced filtrates in volumes of 42-76% of the dewatered digestate, having a soluble chemical oxygen demand (SCOD) of 28-44 g/L, of which volatile fatty acids (VFAs) contributed 10-34%, and methane potentials of 182-206 mL-CH4/g-SCOD without any major indication of inhibition. All 32 pharmaceuticals detected in the digestates were below the detection limit in hydrochars and filtrates, save for ibuprofen and benzotriazole in filtrate, while heavy metals were concentrated in the hydrochars but below the national limits for fertiliser use, save for mercury. The integration of HTC to a centralised biogas plant was extrapolated to enhance the annual biogas production by 5% and ammonium recovery by 25%, and the hydrochar was estimated to produce 83 GJ upon combustion or to direct 350 t phosphorous to agriculture annually.

Anodic electro-fermentation: Empowering anaerobic production processes via anodic respiration.

In nature as well as in industrial microbiology, all microorganisms need to achieve redox balance. Their redox state and energy conservation highly depend on the availability of a terminal electron acceptor, for example oxygen in aerobic production processes. Under anaerobic conditions in the absence of an electron acceptor, redox balance is achieved via the production of reduced carbon-compounds (fermentation). An alternative strategy to artificially stabilize microbial redox and energy state is the use of anodic electro-fermentation (AEF). This emerging biotechnology empowers respiration under anaerobic conditions using the anode of a bioelectrochemical system as an undepletable terminal electron acceptor. Electrochemical control of redox metabolism and energy conservation via AEF can steer the carbon metabolism towards a product of interest and avoid the need for continuous and cost-inefficient supply of oxygen as well as the production of mixed reduced by-products, as is the case in aerobic production and fermentation processes, respectively. The great challenge for AEF is to establish efficient extracellular electron transfer (EET) from the microbe to the anode and link it to central carbon metabolism to enhance the synthesis of a target product. This article reviews the advantages and challenges of AEF, EET mechanisms, microbial energy gain, and discusses the rational choice of substrate-product couple as well as the choice of microbial catalyst. Besides, it discusses the potential of the industrial model-organism Bacillus subtilis as a promising candidate for AEF, which has not been yet considered for such an application. This prospective review contributes to a better understanding of how industrial microbiology can benefit from AEF and analyses key-factors required to successfully implement AEF processes. Overall, this work aims to advance the young research field especially by critically revisiting the fundamental aspects of AEF.

Degradation of common polymers in sewage sludge purification process developed for microplastic analysis.

To enable and/or facilitate analysis of microplastics from environmental samples, a purification process is required to reduce the organic matter content. The development of such process has as one main concern, besides achieving efficient organic matter reduction, the preservation of the microplastics. In this study, a three-step method for sewage sludge purification was proposed employing sodium dodecyl sulfate and hydrogen peroxide. The effects of the purification method on seven polymers (LLDPE, HDPE, PP, PS, PET, PA66 and SBR) were evaluated in terms of mass change, surface characteristics, mechanical properties, thermal properties and functional groups change. It was also assessed how the polymers were affected by the purification chemicals without the presence of sewage sludge. The purification process led to changes in all tested plastics, but in different intensities. LLDPE, HDPE, PP, PS and PET did not suffer considerable degradation. PET was more affected by hydrolysis than oxidation. On the other hand, the integrities of PA66 and SBR were noticeably affected. The effects of the purification process were considered to be due to the plasticizer behavior of water and oxidation on PA66 and loss of filler and oxidation on SBR. For both polymers there was a reduction on the tensile strength of around 50-60% after the purification, indicating they could be prone to fragmentate into smaller pieces along the process. After purification, PA66 also started to decompose at a temperature around 10 °C lower comparing to virgin samples. Except for SBR, the presence of sewage sludge and its oxidation was more harmful to the polymers than the purification chemicals without the presence of sewage sludge. This study serves as an evaluation of the effects of the purification process on the degradation of microplastics and a methodology for such assessment when designing a purification process.

Volatile fatty acid production from Kraft mill foul condensate in upflow anaerobic sludge blanket reactors.

The utilization of foul condensate (FC) collected from a Kraft pulp mill for the anaerobic production of volatile fatty acids (VFA) was tested in upflow anaerobic sludge blanket (UASB) reactors operated at 22, 37 and 55°C at a hydraulic retention time (HRT) of ∼75 h. The FC consisted mainly of 11370, 500 and 592 mg/L methanol, ethanol and acetone, respectively. 42-46% of the organic carbon (methanol, ethanol and acetone) was utilized in the UASB reactors operated at an organic loading of ∼8.6 gCOD/L.d and 52-70% of the utilized organic carbon was converted into VFA. Along with acetate, also propionate, isobutyrate, butyrate, isovalerate and valerate were produced from the FC. Prior to acetogenesis of FC, enrichment of the acetogenic biomass was carried out in the UASB reactors for 113 d by applying operational parameters that inhibit methanogenesis and induce acetogenesis. Activity tests after 158 d of reactor operation showed that the biomass from the 55°C UASB reactor exhibited the highest activity after the FC feed compared to the biomass from the reactors at 22 and 37°C. Activity tests at 37°C to compare FC utilization for CH4 versus VFA production showed that an organic carbon utilization >98% for CH4 production occurred in batch bottles, whereas the VFA production batch bottles showed 51% organic carbon utilization. Furthermore, higher concentrations of C3-C5 VFA were produced when FC was the substrate compared to synthetic methanol rich wastewater.

The effect of start-up on energy recovery and compositional changes in brewery wastewater in bioelectrochemical systems.

Start-up of bioelectrochemical systems (BESs) fed with brewery wastewater was compared at different adjusted anode potentials (-200 and 0 mV vs. Ag/AgCl) and external resistances (50 and 1000 Ω). Current generation stabilized faster with the external resistances (9 ± 3 and 1.70 ± 0.04 A/m3 with 50 and 1000 Ω, respectively), whilst significantly higher current densities of 76 ± 39 and 44 ± 9 A/m3 were obtained with the adjusted anode potentials of -200 and 0 mV vs. Ag/AgCl, respectively. After start-up, when operated using 47 Ω external resistance, the current densities and Coulombic efficiencies of all BESs stabilized to 9.5 ± 2.9 A/m3 and 12 ± 2%, respectively, demonstrating that the start-up protocols were not critical for long-term BES operation in microbial fuel cell mode. With adjusted anode potentials, two times more biofilm biomass (measured as protein) was formed by the end of the experiment as compared to start-up with the fixed external resistances. After start-up, the organics in the brewery wastewater, mainly sugars and alcohols, were transformed to acetate (1360 ± 250 mg/L) and propionate (610 ± 190 mg/L). Optimized start-up is required for prompt BES recovery, for example, after process disturbances. Based on the results of this study, adjustment of anode potential to -200 mV vs. Ag/AgCl is recommended for fast BES start-up.

Power production and microbial community composition in thermophilic acetate-fed up-flow and flow-through microbial fuel cells.

The microbial communities developed from a mixed-species culture in up-flow and flow-through configurations of thermophilic (55 °C) microbial fuel cells (MFCs), and their power production from acetate, were investigated. The up-flow MFC was operated for 202 days, obtaining an average power density of 0.13 W/m3, and Tepidiphilus was the dominant transcriptionally-active microorganisms. The planktonic community developed in the up-flow MFC was used to inoculate a flow-through MFC resulting in the proliferation of Ureibacillus, whose relative abundance increased from 1 to 61% after 45 days. Despite the differences between the up-flow and flow-through MFCs, including the anode electrode, hydrodynamic conditions, and the predominant microorganism, similar (p = 0.05) volumetric power (0.11-0.13 W/m3), coulombic efficiency (16-18%) and acetate consumption rates (55-69 mg/L/d) were obtained from both. This suggests that though MFC design can shape the active component of the thermophilic microbial community, the consortia are resilient and can maintain similar performance in different MFC configurations.

Anaerobic treatment of LCFA-containing synthetic dairy wastewater at 20 °C: Process performance and microbial community dynamics.

Facilitating anaerobic degradation of long-chain fatty acids (LCFA) is key for tapping the high methane production potential of the fats, oil and grease (FOG) content of dairy wastewaters. In this study, the feasibility of using high-rate granular sludge reactors for the treatment of mixed LCFA-containing synthetic dairy wastewater (SDW) was assessed at 20 °C. The effects of the LCFA concentration (33-45% of COD) and organic loading rates (2-3 gCOD/L·d) were determined using three parallel expanded granular sludge bed reactors. For the first time, long term anaerobic treatment of LCFA-containing feed at 20 °C was shown to be feasible and was linked to the microbial community dynamics in high-rate reactors. During a two-month operation, a soluble COD removal of 84-91% and COD to methane conversion of 44-51% was obtained. However, granular sludge flotation and washout occurred after two months in all reactors without volatile fatty acids (VFA) accumulation, emphasizing the need for sludge retention for long-term granular sludge reactor operation with LCFA-containing feed at low ambient temperatures. The temporal shifts in microbial community structure were studied in the high-rate treatment of SDW, and the process disturbances (elevated LCFA loading, LCFA accumulation, and batch operation) were found to decrease the microbial community diversity. The relative abundance of Methanosaeta increased with higher LCFA accumulation in the settled and flotation layer granules in the three reactors, therefore, acetoclastic methanogenesis was found to be crucial for the high-rate treatment of SDW at 20 °C. This study provides an initial understanding of the continuous anaerobic treatment of LCFA-containing industrial wastewaters at low ambient temperatures.

Acetotrophic Activity Facilitates Methanogenesis from LCFA at Low Temperatures: Screening from Mesophilic Inocula.

The inoculum source plays a crucial role in the anaerobic treatment of wastewaters. Lipids are present in various wastewaters and have a high methanogenic potential, but their hydrolysis results in the production of long chain fatty acids (LCFAs) that are inhibitory to anaerobic microorganisms. Screening of inoculum for the anaerobic treatment of LCFA-containing wastewaters has been performed at mesophilic and thermophilic conditions. However, an evaluation of inocula for producing methane from LCFA-containing wastewater has not yet been conducted at low temperatures and needs to be undertaken. In this study, three inocula (one granular sludge and two municipal digester sludges) were assessed for methane production from LCFA-containing synthetic dairy wastewater (SDW) at low temperatures (10 and 20°C). A methane yield (based on mL-CH4/g-CODadded) of 86-65% with acetate and 45-20% with SDW was achieved within 10 days using unacclimated granular sludge, whereas the municipal digester sludges produced methane only at 20°C but not at 10°C even after 200 days of incubation. The acetotrophic activity in the inoculum was found to be crucial for methane production from LCFA at low temperatures, highlighting the role of Methanosaeta (acetoclastic archaea) at low temperatures. The presence of bacterial taxa from the family Syntrophaceae (Syntrophus and uncultured taxa) in the inoculum was found to be important for methane production from SDW at 10°C. This study suggests the evaluation of acetotrophic activity and the initial microbial community characteristics by high-throughput amplicon sequencing for selecting the inoculum for producing methane at low temperatures (up to 10°C) from lipid-containing wastewaters.