Volatile Fatty Acids Inhibition Research Articles

Microbiome re-assembly boosts anaerobic digestion under volatile fatty acid inhibition: focusing on reactive oxygen species metabolism

The accumulation of volatile fatty acids (VFAs) in anaerobic digestion (AD) systems resulting from food waste overload poses a risk of system collapse. However, limited understanding exists regarding the inhibitory mechanisms and effective strategies to address VFAs-induced stress. This study found that accumulated VFAs exert reactive oxygen species (ROS) stress on indigenous microbiota, particularly impacting methanogens due to their lower antioxidant capability compared to bacteria, which is supposed to be the primary reason for methanogenesis failure. To enhance the VFAs-stressed AD process, microbiome re-assembly using customized propionate-degrading consortia and bioaugmentation with concentrated digestate were implemented. Microbiome re-assembly demonstrated superior efficiency, yielding an average methane yield of 563.6±159.8 mL/L·d and reducing VFAs to undetectable levels for a minimum of 80 days. This strategy improved the abundance of Syntrophomonas, Syntrophobacter and Methanothrix, alleviating ROS stress. Conversely, microbial community in reactor with other strategy experienced an escalating intracellular damage, as indicated by the increase of ROS generation-related genes. This study fills knowledge gaps in stress-related metabolic mechanisms of anaerobic microbiomes exposed to VFAs and microbiome re-assembly to boost methanogenesis process.

Layer Inoculation as a New Technology to Resist Volatile Fatty Acid Inhibition during Solid-State Anaerobic Digestion: Methane Yield Performance and Microbial Responses

Solid-state anaerobic digestion is easily inhibited by high volatile fatty acid induced by high total solids, although it is a promising technology. Previous studies on volatile fatty acid inhibition mainly focused on total solid content, co-digestion substrates, and external additives. The present study proposed a new inoculation method named layer inoculation and compared it to premixing inoculation in the solid-state anaerobic digestion of pig manure and maize straw. The results showed that the cumulative CH4 yields from layer inoculation (211.5 mL/g-VS) were 5.64 times more than premixing inoculation (37.5 mL/g-VS) under a low inoculation ratio (25%), with the values of total volatile fatty acid being greater than 30.0 mg/g. The concentrations of total VFAs and acetic acid from layer inoculation decreased dramatically during days 18–43. Layer inoculation also showed wider specific methane yield peaks and shorter startup times than premixing inoculation. Methanosphaerula and Methanothrix were the most dominant genera, while the genus Methanosphaerula did not correlate with volatile fatty acids, pH, or total ammonia nitrogen. The hydrogenotrophic methanogen pathway was predominant during solid-state anaerobic digestion; the shift from hydrogenotrophic to acetoclastic occurred in premixing inoculation, and it was stable in layer inoculation (61.20–68.88%). Overall, layer inoculation can effectively enhance methane production under high volatile fatty acid concentrations compared with premixing inoculation.

Magnetite-based nanoparticles and nanocomposites for recovery of overloaded anaerobic digesters

The effect of magnetite nanoparticles and nanocomposites (magnetite nanoparticles impregnated into graphene oxide) supplement on the recovery of overloaded laboratory batch anaerobic reactors was assessed using two types of starting inoculum: anaerobic granular sludge (GS) and flocculent sludge (FS). Both nanomaterials recovered methane production at a dose of 0.27 g/L within 40 days in GS. Four doses of magnetite nanoparticles from 0.075 to 1 g/L recovered the process in FS systems between 30 and 50 days relaying on the dose. The presence of nanomaterials helped to reverse the effect of volatile fatty acids inhibition and enabled microbial communities to recover but also favoured the development of certain microorganisms over others. In GS reactors, the methanogenic population changed from being mostly acetoclastic (Methanothrix soehngenii) to being dominated by hydrogenotrophic species (Methanobacterium beijingense). Nanomaterial amendment may serve as a preventative measure or provide an effective remedial solution for system recovery following overloading.

Effort to Mitigate Volatile Fatty Acid Inhibition by Using Mixed Inoculum and Compost for the Degradation of Food Waste and the Production of Biogas

Food waste is a rich organic matter that can potentially be converted into biogas as a source of renewable energy. The limitation in energy production lies in the presence of volatile fatty acid (VFA) during the anaerobic digestion of food waste due to the high degradation rate. The accumulation of VFA leads to a decrease in pH that exceeds the optimal pH range of 6.8–7.6 for methanogens, thus inhibiting methanogenesis and affecting biogas production. In the present study, a symbiotic culture of bacteria and yeast (SCOBY) and kombucha mixed inoculum and compost was applied as an alternative treatment method to alleviate inhibition. The digestion efficiency was evaluated on pH, total alkalinity (TA), total volatile fatty acid (TVFA), total solid (TS), and volatile solid (VS) throughout the digestion period of 80 days to analyse the stability of the system. The results revealed that SCOBY and kombucha mixed inoculum caused system instability, inducing inhibition at TVFA of 12,874.1 mg/L, while the pH dropped to 5.23. The inhibition in the digestion system with only the SCOBY inoculum occurred at TVFA of 11,908.3 mg/L, and the pH dropped to 5.67. The biogas and methane yield quantified from the mixed inoculum is 8.792E−4 L/L d, comparatively lower than the ethanol pre-fermentation treatment method. These findings indicate that the addition of compost improved the pH, VS, and TVFA.

Effects of initial volatile fatty acid concentrations on process characteristics, microbial communities, and metabolic pathways on solid-state anaerobic digestion

Solid-state anaerobic digestion (SSAD) is vulnerable to excess volatile fatty acids (VFA), mainly acetate and propionate. The co-effects of VFAs and microbial dynamics under VFA accumulation were investigated in SSAD of pig manure and corn straw. Adding 2 and 4 mg/g acetate or propionate caused initial increases in total VFAs, followed by decreases after day 6, resulting in ‘mild’ VFA accumulation, while adding 6 mg/g caused similarly increased VFAs, but with no subsequent decrease, causing ‘severe’ VFA accumulation and poor methanation performance. Mild propionate accumulation promoted acetate consumption, whereas acetate accumulation inhibited propionate degradation by affecting crucial redox reactions. Under severe VFA accumulation, hydrolysis and acidification mainly conducted by acid-tolerant Clostridium sp. exacerbated VFA inhibition, causing a competition between Methanosarcina and Methanosaeta, and impairments of acetoclastic and hydrogenotrophic methanogenesis and interspecies formate transfer. This study provides new insights into mechanisms of VFA accumulation in SSAD, and its effects on methanogenesis.

Effect of bioaugmentation combined with activated charcoal on the mitigation of volatile fatty acids inhibition during anaerobic digestion

Volatile fatty acids (VFAs) accumulation is a common problem in anaerobic digestion, which is constraining the process. In this study, bioaugmentation was initially adopted as a measure to tackle the acidification problem in a continuous anaerobic digester. However, after bioaugmentation stopped, the promotive effect of bioaugmentation was hard to sustain. In comparison, the combination of bioaugmentation and Activated charcoal (AC) enabled a better reactor performance. The positive impact of AC on recovery from acidification in sequential batch reactors was thus investigated by feeding either propionate or acetate. The results implied that the addition of AC significantly increased the degradation rate of propionate, thus accelerating the biogas production rate (104 ~ 371%). Moreover, the role of AC is mainly in the immobilization of microorganisms to form bio-AC owing to AC’s physical characteristics (high-surface-area and large number of pores enables adsorption of microorganisms). The biofilms on the AC decreased the distance between the microbes and delayed the washout effect of microorganisms, enabling a longer-lasting effect of bioaugmentation. The 16S-rRNA analysis showed that the addition of AC led to the enrichment of syntrophic microbial guilds (syntrophic VFAs oxidizing bacteria and hydrogenotrophic methanogens). The potential function analysis (PICRUSt2) indicated the significant enhancement of formylmethanofuran dehydrogenase (EC 1.2.7.12) and coenzyme F420 hydrogenase (EC 1.12.98.1) by AC. The results suggested that the addition of AC promoted interspecies electron transfer.

Responses of anaerobic digestion of food waste to coupling effects of inoculum origins, organic loads and pH control under high load: Process performance and microbial characteristics

This study investigated responses of anaerobic digestion (AD) of food waste (FW) with different inocula to varying organic loads and to pH control under high load in terms of process performance and microbial characteristics. Without pH control, digester inoculated by thickened sludge obtained high methane yield of 547.8 ± 27.8 mL/g VS under organic load of 7.5 g VS/L but was inhibited by volatile fatty acids (VFAs) under higher loads (15 and 30 g VS/L). However, digesters inoculated by anaerobic sludge obtained high methane yields of 575.9 ± 34.2, 569.3 ± 24.8 and 531.9 ± 26.2 mL/g VS under organic loads of 7.5, 15 and 30 g VS/L and VFAs inhibition only appeared under extremely high load of 45 g VS/L. Digesters under VFA inhibition with high load were significantly enhanced by controlling single ecological factor pH at 6.5, 7.0 and 7.5, as indicated by shorter lag phases, higher peak values of methane production rate, greater methane yields and fast VFAs degradation. Maximum methane recovery was obtained with pH control at 7.5 under high load. VFA inhibition was accompanied by the degeneration of ecological functions of Syntrophomonadaceae and unidentified Bacteroidales and the dominant growth of unidentified Clostridiales. Under high load and pH control, high stability was strongly associated with obvious growth of Methanosarcina, which enriched methanogenic pathways thus improved system robustness and tolerance to VFAs. Moreover, pH control stimulated the growth of syntrophic Bacteria Syntrophomonadaceae while maintaining the high activity of hydrogenotrophic methanogens therefore sustained efficient syntrophic communities of Bacteria and methanogens and avoided over accumulation of VFAs. pH control promoted adaptive selection of methanogens, leading to obvious decline of archaeal community diversity. This study provided practical guidance on digester configurations of high-load AD of FW and expanded the understanding of responses to coupling effects of inoculum origins, organic loads and pH control under high load concerning process performance and microbial community dynamics.

Comparison of the microbial communities in anaerobic digesters treating high alkalinity synthetic wastewater at atmospheric and high-pressure (11 bar)

High-pressure anaerobic digestion is an appealing concept since it can upgrade biogas directly within the reactor. However, the decline of pH caused by the dissolution of CO2 is the main barrier that prevents a good operating high-pressure anaerobic digestion process. Therefore, in this study, a high-pressure anaerobic digestion was studied to treat high alkalinity synthetic wastewater, which could not be treated in a normal-pressure anaerobic digester. In the high-pressure reactor, the pH value was 7.5 ~ 7.8, and the CH4 content reached 88% at 11 bar. Unlike its normal-pressure counterpart (2285 mg/L acetic acid), the high-pressure reactor ran steadily (without volatile fatty acids inhibition). Furthermore, the microbial community changed in the high-pressure reactor. Specifically, key microbial guilds (Syntrophus (11.2%), Methanosaeta concilii (50.9%), and Methanobrevibacter (26.8%)) were dominant in the high-pressure reactor at 11 bar, indicating their fundamental roles under high-pressure treating high alkalinity synthetic wastewater.

Role of trace elements in anaerobic digestion of food waste: Process stability, recovery from volatile fatty acid inhibition and microbial community dynamics

Role of trace elements (TEs) in long-term anaerobic digestion of food waste (FW) under fixed and stepwise increasing loads and under early and medium volatile fatty acid (VFA) inhibition was investigated. Digesters under high load suffered VFA inhibition. Mismatch between scarce TEs in FW and essential TEs for sustainable methanogenesis suppressed Methanosaeta causing blocked aceticlastic methanogenesis and shift to CO2 reduction pathway, as indicated by decreased Methanosaeta from above 70.0% to below 42.0% and enriched hydrogenotrophic methanogens (Methanospirillum, Methanoculleus, Methanobacterium) from below 15.0% to above 53.6%. Dual stresses of VFA inhibition and TEs deficiency resulted in recession of syntrophic Bacteria Syntrophomonadaceae. Conversely, digesters with TEs supplementation maintained high activity of Syntrophomonadaceae and ensured predominant aceticlastic methanogenesis and powerful methanogenic community functions. Early and medium VFA inhibition were reversed by TEs supplementation or coupling with pH adjustment by stimulating VFAs degradation via syntrophic metabolism and unclogging acetate conversion via aceticlastic methanogenesis.

A new strategy to recover from volatile fatty acid inhibition in anaerobic digestion by photosynthetic bacteria

The accumulation of volatile fatty acids (VFAs) can decrease reactor pH and inhibit methane-producing process. For the first time, photosynthetic bacteria (PSB) were used to recover from VFAs inhibition (pH 6.0) of an anaerobic digestion system. After adding PSB for 12 days with and without light condition, the methane content recovered from 33.3% to 60.5% and from 32.1% to 59.3%, respectively; the pH increased to 7.1 and 6.8, respectively, the system alkalinity rapidly increased to 2238 and 1921 mg/L, respectively; the sCOD decreased from 5600 to 995 mg/L and from 5575 to 2025 mg/L, respectively; and the contents of formic acid, acetic acid, propionic acid and total VFA were greatly reduced. Microbial analysis found that PSB bioaugmentation could maintain microbial diversity of the system. PSB bioaugmentation could effectively relieve acids accumulation and stimulate methane production especially under light condition. It is also found that light could accelerate recovery with or without bioaugmentation.

Strong pH dependence of hydrogen production from glucose by Rhodobacter sphaeroides

Purple non-sulfur bacteria (PNSB) are well known for converting short-chain organic acids to H2, however, a decrease in pH caused by metabolic acids production limited H2 production during the photo-fermentation from glucose. Here we address why volatile fatty acids (VFA) excreted as fermentation products cannot be further degraded by R. sphaeroides that readily use them. We found that the photo-fermentation with pH controlled at 6.9 ± 0.1 resulted in a 90% increase of H2 yield and a 107.6% increase in volume H2 production relative to the pH-uncontrolled culture. Comparative fermentations on glucose at pH 5.8 and pH 7.1 using culture medium supplemented with 50% spent fermentation broth demonstrated that low pH alone is not the limiting factor and compounds present in the supernatants along with pH decrease were the most inhibitory to H2 production. The impact of byproducts VFA on phototrophic H2 production was dependent on both the pH and VFA concentrations; even 7 mM VFA addition totally inhibited H2 production from glucose at pH 5.4. H2 production with pH control for the Δhup strain was not discernibly different from the parent strain, which are all significantly higher than high-performance strains by metabolic engineering. These results demonstrate that pH dependent VFA inhibition can be turned into a driving force for enhanced H2 production from glucose by pH regulation.

Biogas production from high solids anaerobic co-digestion of food waste, yard waste and waste activated sludge

High-solids anaerobic co-digestion (HS-AcD) has the potential to recover energy and reduce environmental impacts of the organic fraction of municipal solid waste and waste activated sludge. We investigated the impact of substrate to inoculum (S/I) ratios, alkalinity sources (sodium bicarbonate and oyster shells), co-substrate mixing ratios and inoculum acclimation on HS-AcD of food waste, yard waste, and waste activated sludge using batch studies. Long-term HS-AcD performance was evaluated under the optimal conditions through a semi-continuous biodigester study with leachate recirculation. The digester with S/I = 1 using a mixture of crushed oyster shells and sodium bicarbonate as alkalinity sources had the highest methane yields (183 mL CH4/g VS). Addition of waste activated sludge to food waste and yard waste alleviated acidification (pH 6.86 ± 0.12) during the start-up period, which and improved digester stability. Mixtures with FW/YW/WAS = 0.8:1.7:0.5 had higher methane yields (134 ± 15 mL CH4/gVS) than mixtures with FW/YW/WAS = 1:1:1, but required a longer time (10 days) for self-recovery from volatile fatty acid inhibition. The use of an acclimated inoculum eliminated the lag time during start-up and produced 38% higher methane yield. In the semi-continuous biodigester, an average volatile solids reduction of 38% and methane yield of 186 mL/gVS was achieved. Improved performance in the semi-continuous biodigester compared with batch reactors was likely due to leachate recirculation, which can improve mass transfer of substrates to the microbial community. Digestate produced from HS-AcD of waste organics had a 1.7–2.3 fold higher nitrogen, similar phosphorous and 0.2–0.3 fold lower potassium content than commercially available bioorganic fertilizer.

Dynamic behaviors of batch anaerobic systems of food waste for methane production under different organic loads, substrate to inoculum ratios and initial pH

This study characterized dynamic behaviors of batch anaerobic digesters treating food waste in terms of methane production, organics destruction and process stability under different organic loads (OLs), substrate to inoculum (S/I) ratios [on volatile solid (VS) basis] and initial pH. The results showed that OL, S/I ratio and initial pH significantly affected batch anaerobic process. Methane yield was proved to be inversely proportional to OL and S/I ratio. Digester with lowest OL (5g VS/L) obtained greatest methane yield (551.4mL/g VS), highest organics removal (94.1%) and good stability. Enhancing OL to 10g VS/L was recommended for satisfactory stability and higher volumetric methane productivity. When OL was designated as 10g VS/L, digester with low S/I ratio (1/2) achieved satisfactory methane yield (539.3mL/g VS), high organics removal (92.3%) and stable performance. When OL was relatively high (20g VS/L), adjusting initial pH to 7.5 contributed to stable performance via enhancing buffering capacity against volatile fatty acids (VFA) disturbance. Strong VFA inhibition occurred under high OL (40g VS/L) or great S/I ratio (2/1) or acidic initial pH (6.5). In this case, acetate was dominant VFA, followed by butyrate. However, when digester was stable, acetate was main VFA, followed by propionate. This study provided practical guidance on process configurations for batch digesters of food waste needed to achieve satisfactory performance and stability.

Inhibition of volatile fatty acids on methane production kinetics during dry co-digestion of food waste and pig manure

Compared with wet digestion, dry digestion of organic wastes reduces reactor volume and requires less energy for heating, but it is easily inhibited by high volatile fatty acid (VFA) or ammonia concentration. The inhibition on methane production kinetics during dry co-digestion of food waste and pig manure is rarely reported. The aim of this study was to explore the inhibition mechanisms and the microbial interactions in food waste and pig manure dry co-digestion systems at different inoculum rates (25% and 50% based on volatile solids) and food waste/pig manure ratios (0:100, 25:75, 50:50, 75:25 and 100:0 based on volatile solids). The results showed that the preferable operation conditions were obtained at the inoculum rate of 50% and food waste/pig manure ratio of 50:50, with a specific methane yield of 263 mL/g VSadded. High VFA concentration was the main inhibition factor on methane production, and the threshold VFA inhibition concentrations ranged 16.5–18.0 g/L. Syntrophic oxidation with hydrogenotrophic methanogenesis might be the main methane production pathway in dry co-digestion systems due to the dominance of hydrogenotrophic methanogens in the archaeal community. In conclusion, dry co-digestion of food waste and pig manure is feasible for methane production without pH adjustment and can be operated stably by choosing proper operation conditions.

Enhanced PHA Production with Mixed Cultures Using a Robust and Simple Controller

This work proposes the use of feedback control tools to enhance the production of bacterial polyhydroxyalkanoates (PHA) in an aerobic bioreactor fed with volatile fatty acids (VFA), by regulating the dissolved oxygen (DO) concentration at an optimal reference value and using the airflow signal behaviour to decide when to stop the reaction. The proposed method uses a standard proportional-integral (PI) controller to regulate the DO at a reference value by modifying the airflow, and this latter signal as an indicator to stop the reaction. Experiments were carried out in a batch aerobic bioreactor inoculated with enriched PHA-producing biomass from another bioreactor to determine the optimal DO reference value for PHA production and to compare the proposed operational scheme with the usual strategy that uses a constant airflow. The proposed method allows operation at a DO set-point that is optimal for PHA production and is able to identify the total degradation of substrate by a simple algorithm. It can cope with high VFA influent concentrations and inhibition because of PHA accumulation, by adapting the reaction time on-line. The PHA productivity rate was enhanced and the air consumption was decreased when compared with the usual operation strategy for this type of system. The method is able to identify the end of the reaction using the online measured airflow signal, enhances PHA production, improves the substrate rate and saves air consumption.

Real-time recovery strategies for volatile fatty acid-inhibited anaerobic digestion of food waste for methane production

This study investigated effects of real-time recovery strategies on VFA (volatile fatty acid)-inhibited anaerobic system of FW (food waste) and identified key driver of process recovery. The long-term anaerobic system of FW encountered serious VFA (mainly propionate) inhibition. The pH adjustment (PA) strategy could not reverse process imbalance but only delayed the process failure. The short-term effect of reinoculation (RI) strategy was greatly effective, but its long-term effect was non-sustainable. Trace elements were key drivers of process recovery owing to their indispensable roles in activating methanogenesis and therefore stimulating propionate conversion. From the viewpoint of economic feasibility, the single strategy of trace elements supplementation (TE) and the combinational strategy of PA + TE were respectively recommended in the initial and medium VFA-inhibition stages. The three-in-one strategy of PA + TE + RI was always effective but was costly. This study provided practical guidance on real-time recovery of VFA-inhibited anaerobic system of FW.

Mitigating inhibition of undissociated volatile fatty acids (VFAs) for enhanced sludge-rice bran composting with ferric nitrate amendment

This study investigated the effect of ferric nitrate on mitigating the inhibition of volatile fatty acids (VFAs) during the initial phase of sewage sludge composting amended with rice bran. During the 34-day lab-scale composting, the supplementation of ferric nitrate enhanced the degradation of VFAs by up to 3 times as compared to the control. The organic matters loss (OML) rate in the treatment reactor was almost doubled with supplementation of ferric nitrate as compared to the control reactor during the initial phase. Eventually the treatment reactor achieved a 39.0% OML by the end of composting, which was 22% higher than the control. Ferric nitration addition mitigated the inhibition of VFAs by stimulating denitrification which consumed protons and VFAs. Ferric nitrate addition also decreased the electrical conductivity by 23% in the final compost product, reducing the possibility of phytotoxicity issue upon soil application. In summary, the results demonstrated that ferric nitrate addition could be an effective strategy for enhanced sludge composting.

Synergism and effect of high initial volatile fatty acid concentrations during food waste and pig manure anaerobic co-digestion

Anaerobic co-digestion of food waste (FW) and pig manure (PM) was undertaken in batch mode at 37°C in order to identify and quantify the synergistic effects of co-digestion on the specific methane yield (SMY) and reaction kinetics. The effects of the high initial volatile fatty acid (VFA) concentrations in PM on synergy observed during co-digestion, and on kinetic modelling were investigated. PM to FW mixing ratios of 1/0, 4/1, 3/2, 2/3, 1/4 and 0/1 (VS basis) were examined. No VFA or ammonia inhibition was observed. The highest SMY of 521±29ml CH4/gVS was achieved at a PM/FW mixing ratio of 1/4. Synergy in terms of both reaction kinetics and SMY occurred at PM/FW mixing ratios of 3/2, 2/3 and 1/4. Initial VFA concentrations did not explain the synergy observed. Throughout the study the conversion of butyric acid was inhibited. Due to the high initial VFA content of PM, conventional first order and Gompertz models were inappropriate for determining reaction kinetics. A dual pooled first order model was found to provide the best fit for the data generated in this study. The optimal mixing ratio in terms of both reaction kinetics and SMY was found at a PM/FW mixing ratio of 1/4.

Solid state anaerobic co-digestion of tomato residues with dairy manure and corn stover for biogas production

Solid-state anaerobic co-digestion of tomato residues with dairy manure and corn stover was conducted at 20% total solids under 35°C for 45days. Results showed digestion of mixed tomato residues with dairy manure and corn stover improved methane yields. The highest VS reduction (46.2%) and methane yield (415.4L/kg VSfeed) were achieved with the ternary mixtures of 33% corn stover, 54% dairy manure, and 13% tomato residues, lead to a 0.5–10.2-fold higher than that of individual feedstocks. Inhibition of volatile fatty acids (VFAs) to biogas production occurred when more than 40% tomato residues were added. The results indicated that ternary mixtures diluted the inhibitors that would otherwise cause inhibition in the digestion of tomato residues as a mono-feedstock.

The positive effects of waste leachate addition on methane fermentation from food waste in batch trials.

The purposes of this study were to investigate the effect of waste leachate (WL) addition on batch anaerobic digestion of food waste (FW), and to examine the influence of mixture ratio on the co-digestion process. The results showed that anaerobic digestion of FW was greatly enhanced by WL addition, as indicated by the higher methane yield, higher methane content, more volatile solids (VS) destruction, and higher stability. Although WL was rich in volatile fatty acids (VFA), its addition did not cause VFA inhibition. It was found that WL addition was beneficial to accelerate the start-up and shorten the long reaction time of the batch anaerobic process. The time to reach the peak methane yield was reduced by 1, 2, and 4 days with WL addition. The optimum FW to WL ratio was 77.9:22.1 with the highest methane yield (416 mL/g VS), the highest methane content (64.3%), the greatest VS removal (77.6%), and stable performance. These results confirmed the positive effects of WL addition on methane fermentation from FW.