Inhibition Of Anaerobic Digestion Research Articles

Inhibition of anaerobic digestion by various ammonia sources resulted in subtle differences in metabolite dynamics

The impact of ammonia on anaerobic digestion performance and microbial dynamics has been extensively studied, but the concurrent effect of anions brought by ammonium salt should not be neglected. This paper studied this effect using metabolomics and a time-course statistical framework. Metabolomics provides novel perspectives to study microbial processes and facilitates a more profound understanding at the metabolic level. The advanced statistical framework enables deciphering the complexity of large metabolomics data sets. More specifically, a series of lab-scale batch reactors were set up with different ammonia sources added. Samples of nine time points over the degradation were analyzed with liquid chromatography-mass spectrometry. A filtering procedure was applied to select the promising metabolomic peaks from 1262 peaks, followed by modeling their intensities across time. The metabolomic peaks with similar time profiles were clustered, evidencing the correlation of different biological processes. Differential analysis was performed to seek the differences in metabolite dynamics caused by different anions. Finally, tandem mass spectrometry and metabolite annotation provided further information on the molecular structure and possible metabolic pathways. For example, the consumption of 5-aminovaleric acid, a short-chain fatty acid obtained from l-lysine degradation, was slowed down by phosphates. Overall, by investigating the effect of anions on anaerobic digestion, our study demonstrated the effectiveness of metabolomics in providing detailed information in a set of samples from different experimental conditions. With the statistical framework, the approach enables capturing subtle differences in metabolite dynamics between samples while accounting for the differences caused by time variations.

Inhibitory Effects of Ammonia on Archaeal 16S rRNA Transcripts in Thermophilic Anaerobic Digester Sludge

High temperatures exacerbate the ammonia inhibition of anaerobic digestion coupled with methanogenesis. The inhibition of methane production by ammonia has been observed in other studies. However, the underlying mechanism is not well understood and requires further investigation. This study explored the effect of ammonia stress on archaeal 16S rRNA transcripts in thermophilic anaerobic digester sludge. Different ammonium concentrations were checked for their influence on the methanogenic rate and hydrogen accumulation. Quantitative PCR was used to compare the changes in total archaeal 16S rRNA expression. A Monte Carlo permutation test within redundancy analysis (RDA) was adopted for exploring the relationship between environmental variables and archaeal 16S rRNA and their transcripts. The results showed that with the increase in ammonium concentration, the methanogenic rate decreased and hydrogen accumulation occurred. The total archaeal 16S rRNA genes and transcripts copy numbers decreased significantly in treatments with higher ammonium concentrations (7 and 10 g NH4+-N/L), but did not change much at lower ammonia concentrations (3 g NH4+-N/L) compared with the 0 g NH4+-N/L treatment. The RDA analysis further revealed that most environmental variables, including ammonia and methane, except for formate, were significantly correlated with the community structure activity of archaeal 16S rRNA transcripts rather than the community structure of their genes. The composition of archaeal 16S rRNA transcripts showed that the hydrogenotrophic methanogen Methanothermobacter dominated the methanogenic community activity in all incubations. It exhibited sensitivity to ammonia stress and should be responsible for the methanogenic inhibition under thermophilic conditions. Our findings suggested that archaeal 16S rRNA transcripts, rather than 16S rRNA genes, are key indicators of ammonia stress and methanogenic activity.

Novel lanthanum-iron oxide nanoparticles alleviate the inhibition of anaerobic digestion by carbamazepine through adsorption and bioaugmentation

Several reports have shown that pharmaceuticals and personal care products (PPCPs) have some negative effects on anaerobic digestion (AD), yet there are no convenient and efficient strategies for mitigating the adverse influences. The typical PPCPs of carbamazepine have a strong negative effect on lactic acid AD process. Therefore, in this work, novel lanthanum-iron oxide (LaFeO3) nanoparticles (NPs) were used for adsorption and bioaugmentation to weak the negative effects of carbamazepine. The adsorption removal of carbamazepine increased from 0 to 44.30% as the dosage of LaFeO3 NPs was increased from 0 to 200 mg/L, providing the necessary prerequisites for bioaugmentation. Adsorption reduced the probability of direct contact between carbamazepine and anaerobes, partly alleviating the inhibition of carbamazepine on microbes. The highest methane (CH4) yield induced by LaFeO3 NPs (25 mg/L) was 226.09 mL/g lactic acid, increasing by 30.06% compared to the control yield with a recovery to 89.09% of the normal CH4 yield. Despite the ability of LaFeO3 NPs to restore normal AD performance, the biodegradation rate of carbamazepine remained below 10% due to its anti-biodegradability. Bioaugmentation was primarily reflected in the enhanced bioavailability of dissolved organic matter, while the intracellular LaFeO3 NPs promoted coenzyme F420 activity through binding to humic substances. Under the mediation of LaFeO3, a direct interspecies electron transfer system with Longilinea and Methanosaeta as functional bacteria was successfully constructed and the corresponding electron transfer rate was accelerated from 0.021 s−1 to 0.033 s−1. LaFeO3 NPs eventually recovered AD performance under carbamazepine stress in an adsorption and bioaugmentation manner.

Exploration of browning reactions during alkaline thermal hydrolysis of sludge: Maillard reaction, caramelization and humic acid desorption

The browning reaction produces melanoidins, 5-hydroxymethylfurfural (HMF) and humic acids which influence subsequent anaerobic digestion and protein recovery. This paper systematically evaluates the variation of organics that make sludge browning with heating temperature and reaction time, the effect of browning organics on protein recovery and anaerobic digestion, and finally proposes a pathway for the occurrence of the Maillard reaction (MR) in the sludge environment. The results show that the browning of sludge hydrolysate is related to the comprehensive influence of the MR, caramelization and humic acid desorption. The increase of temperature (80 °C–150 °C) and pH (9–13) will promote the extent of browning of sludge hydrolysate, and the sludge browning reaction basically stabilizes at the reaction time of 1 h. Humic acid and melanoidin could co-precipitate with the protein, thereby reducing the purity of the recovered protein. The inhibition of anaerobic digestion starts when the melanoidin concentration is 8.01 mmol/L. The three-dimensional fluorescence, GC-MS and FT-IR analysis show that melanoidins have the same functional groups and fluorescence properties as humic acid does, and the humic acid in the supernatant of the sludge treated with ATH was not only converted at its adsorbed state, but also possibly generated by the reaction of the dissolved proteins with polysaccharides. Finally, LC-MS/MS was used to identify the intermediate products of the MR and the possible structural formula of melanoidin. This study further clarifies the browning reaction in hydrothermal sludge treatment and provides help for the accuracy of subsequent studies.

Microbial shifts in anaerobic digestion towards phenol inhibition with and without hydrochar as revealed by metagenomic binning

The inhibition of anaerobic digestion (AD) by phenolic compounds is an obstacle to the efficient treatment of organic wastes. Besides, hydrochar produced from hydrothermal liquefaction of biomass has been previously reported to enhance AD. The present study aimed to provide deep insights into the microbial shifts at the species level to phenol (0–1.5 g/L) inhibition in AD of glucose with and without hydrochar by metagenomic analysis. Phenol higher than 1 g/L had severe inhibition on both the amount and rate of methane production in control experiments, while hydrochar significantly enhanced methane production, especially at phenol 1 g/L and 1.5 g/L. From metagenomic analysis, 78 High-quality metagenome-assembled genomes (MAGs) were obtained. Principal components analysis showed that the microbial communities were shifted when phenol concentration was increased to 0.25 g/L in control experiments and 1 g/L in hydrochar experiments. In control experiments, no MAGs involved in acetogenesis were found at phenol 1.5 g/L and Methanothrix sp.FDU243 was also inhibited. However, hydrochar resulted in the maintenance of several MAGs involved in acetogenesis and Methanothrix sp.FDU243 even at phenol 1.5 g/L, ensuring a persistent methane production. Furthermore, 6 phenol-degrading MAGs were identified, shifting dependent on the concentrations of phenol and the presence of hydrochar.

A sodium percarbonate/ultraviolet system generated free radicals for degrading capsaicin to alleviate inhibition of methane production during anaerobic digestion of lipids and food waste

To alleviate inhibition of anaerobic digestion caused by capsaicin, which is easily soluble in the lipid components of food waste (FW), an advanced oxidation process with sodium percarbonate/ultraviolet (SPC/UV) was used to generate free radicals for degrading capsaicin and recovering methane production. Free radical sweeping showed that the free radicals OH, O2— and CO3— worked together to degrade capsaicin. Gas chromatography-mass spectrometry showed that capsaicin likely had four degradation pathways via conversion into benzoquinone, and finally into carbon dioxide and water. The degradation rate of capsaicin in lipids increased from 62.2% to 96.0% when the SPC concentration increased from 2 mmol/L to 32 mmol/L (UV intensity = 20.66 mW/cm2). The degradation rate increased from 70.9% to 94.6% when the UV intensity increased from 20.66 mW/cm2 to 46.86 mW/cm2 (SPC concentration = 4 mmol/L). The subsequent products after capsaicin degradation were subjected to anaerobic digestion either directly or by adding FW. The reduced intracellular oxidative kinases of anaerobic digestion microorganisms recovered the CH4 yield from 27.2 mL/g-total volatile solids (TVS) with capsaicin to 311.2 mL/g-TVS with degraded capsaicin, which was 40.7% that of the control group (765.3 mL/g-TVS without capsaicin). After adding FW, the CH4 yield of SPC/UV degradation effluent was 504.1 mL/g-TVS, which was 82.6% that of the control group (610.4 mL/g-TVS).

Integrating independent microbial studies to build predictive models of anaerobic digestion inhibition by ammonia and phenol

Anaerobic digestion (AD) is a process that can efficiently degrade organic waste into renewable energies. AD failure is however common as the underpinning microbial mechanisms are highly vulnerable to a wide range of inhibitory compounds. Sequencing technologies enable the identification of microbial indicators of digesters inhibition, but existing studies are limited. They used different inocula, substrates, sites and types of reactors and reported different or contradictory indicators. Our aim was to identify a robust signature of microbial indicators of phenol and ammonia inhibitions across four independent AD microbial studies. To identify such signature, we applied an original multivariate integrative method on two in-house studies, then validated our approach by predicting the inhibitory status of samples from two other studies with more than 90% accuracy. Our approach shows how we can efficiently leverage on existing studies to extract reproducible microbial community patterns and predict AD inhibition to improve AD microbial management.

Overcome inhibition of anaerobic digestion of chicken manure under ammonia-stressed condition by lowering the organic loading rate

The synergetic inhibition of fatty acids accumulation and ammonia on the metabolic capability of methanogens limits the efficiency of nitrogen-rich substrates anaerobic digestion (AD). In this study, AD of chicken manure under different experimental conditions (namely, organic loading rate (OLR) of 1.25, 2.5 and 5 g.TS L−1d−1) with ammonia-stressed system (⁓6000 mg-NH4+-N/L) were investigated. The results demonstrated an inhibited-steady state of methane production at OLR of 5.0 g.TS L−1d−1 which resulted in up to 30%–37% methane yield reduction. The highest methane yield (356 mL g VSin−1) and organics removal efficiency (66%) were achieved at OLR of 2.5 g.TS L−1d−1 by extending the hydraulic retention time to 40 days. Methanosarcina was found more tolerant to ammonia stress but Methanobacterium and Methanoculleus dominated in the higher methane yield digester. The results indicated that the proper trade-off between high OLR and ammonia inhibition may develop a high efficiency AD for nitrogen-rich materials.

Improving fermentative methane production of glycerol trioleate and food waste pretreated with ozone through two-stage dark hydrogen fermentation and anaerobic digestion

In order to alleviate substantial inhibition of anaerobic digestion by undegraded lipids that coat microorganisms in food wastes, an ozonation pretreatment was employed to degrade the lipids and promote methane production through two-stage dark hydrogen fermentation and anaerobic digestion. GC–MS analysis showed that degradation rate of glycerol trioleate into hexadecanoic acid and tetradecanoic acid increased to 78.6% when ozone concentration increased to 0.8 g-O3/g-TVS in the pretreatment. SEM and FTIR analysis indicated that coating of methanogens by lipids during the anaerobic digestion process was substantially relieved after ozonation pretreatment. The methane yield from glycerol trioleate after dark hydrogen fermentation increased by 81.9% to 946.5 mL/g-TVS after 0.8 g-O3/g-TVS pretreatment. When food waste (carbohydrate 45.7%, protein 21.4%, lipid 28.1%) and glycerol trioleate were mixed with a total volatile solids (TVS) ratio of 1:1, an ozonation pretreatment of 0.02 g-O3/g-TVS gave the highest energy conversion efficiency of 78.7% through two-stage dark hydrogen fermentation and anaerobic digestion. However, excessive ozone (≥0.05 g-O3/g-TVS) pretreatment decreased not only hydrogen yield but also methane yield, because most small-molecular carbohydrates and a part of proteins were destroyed by ozone oxidization.

Co-digestion of tannery waste activated sludge with slaughterhouse sludge to improve organic biodegradability and biomethane generation

A novel approach was used to overcome inhibition of anaerobic digestion of tannery waste activated sludge by co-digesting with slaughterhouse sludge. The seasonal characteristics of the tannery sludge varied significantly. Macronutrient ratios were typically not ideal for anaerobic digestion, mainly due to an over-abundance of nitrogen. In addition, potentially inhibitory concentrations of metals were present. Optimal co-digestion with 50% (vol./vol.) slaughterhouse sludge achieved a CH4 yield of 215mLCH4/gVS, and biodegradability of 55.7% VS, 45.5% TS and 48.2% COD. This resulted in a synergistic increase of ≥59.4% in biodegradability and 156% in CH4 yield compared to mono-digestion of tannery waste activated sludge. Furthermore, the regression of the cumulative CH4 yield using the Logistic, Cone and modified Gompertz model (adj R2≥0.998) showed an increase of 20.4–21.9%, 58.6–252%, and 164% in the ultimate CH4 yield, maximum CH4 production rate and the specific rate constant respectively, and 59.3% reduction of the lag phase. An alternative cleaner production technique that vertically integrates the value chain improves solid waste management and provides an income stream was demonstrated through this co-digestion strategy.

Effect of COD/SO42- Ratio on Anaerobic Digestion of Penicillin Bacterial Residues

In order to assess the sulfate-induced inhibition of anaerobic digestion of antibiotic manufacturing bio-waste. The effect of COD/SO42- ratio on biogas production potential and substrate utilization characteristics during the acidogenic phase of anaerobic digestion of penicillin bacterial residues were investigated through batch experiments. The results obtained indicated that biogas production was gradually enhanced after 10 days of anaerobic digestion. However, the maximum cumulative methane production probably exceeded about 208 mL·g-1(on TS basis) since COD/SO42-≥3. Because adaptive acclimation, more than 71% COD removal, which may have been converted to methane, was achieved. Consequently, at COD/SO42-≤1.5, methane production was suppressed by 49% and 100% when the organics and SO42- removal rates were less than 17% and 5%, respectively. This indicated that methanogens and sulfate-reducing bacteria were inhibited at high sulfate loading rates. In addition, the COD balance analysis revealed that less than 9.1% of the COD was converted to methane. However, 5.0%-9.0% of the COD was used for sulfate reduction. This means that methanogens are more susceptible than sulfate-reducing bacteria to sulfide-induced inhibition. The S balance showed that the reduced sulfate was mainly present as sulfide in the digester. A small fraction of it was present as hydrogen sulfide in the biogas. The analysis of substrate utilization characteristics during the acidogenic phase revealed that methanation of soluble protein was initiated after methanation of soluble carbohydrate.

The effect of Fe2NiO4 and Fe4NiO4Zn magnetic nanoparticles on anaerobic digestion activity

Two types of magnetic nanoparticles (MNPs), i.e. Ni ferrite nanoparticles (Fe2NiO4) and Ni Zn ferrite nanoparticles (Fe4NiO4Zn) containing the trace metals Ni and Fe, were added to the anaerobic digestion of synthetic municipal wastewater at concentrations between 1 and 100 mg Ni L−1 in order to compare their effects on biogas (methane) production and sludge activity. Using the production of methane over time as a measure, the assays revealed that anaerobic digestion was stimulated by the addition of 100 mg Ni L−1 in Fe2NiO4 NPs, while it was inhibited by the addition of 1–100 mg Ni L−1 in Fe4NiO4Zn NPs. Especially at 100 mg Ni L−1, Fe4NiO4Zn NPs resulted in a total inhibition of anaerobic digestion. The metabolic activity of the anaerobic sludge was tested using the resazurin reduction assay, and the assay clearly revealed the negative effect of Fe4NiO4Zn NPs and the positive effect of Fe2NiO4 NPs. Re-feeding fresh synthetic medium reactivated the NPs added to the anaerobic sludge, except for the experiment with 100 mg Ni L−1 addition of Fe4NiO4Zn NPs. The findings in this present study indicate a possible new strategy for NPs design to enhance anaerobic digestion.

Increasing the tolerance to polyphenols of the anaerobic digestion of olive wastewater through microbial adaptation

The valorisation of olive oil mill wastewater (OMW) through anaerobic digestion requires identifying the concentration of polyphenols (PP) that causes failure of the process of digestion. In addition, the advantages of the possible microbial adaptation, in terms of increased methane production, to significant concentrations of PP as well as the kinetics of OMW anaerobic degradation requires evaluation. To fill these knowledge gaps, anaerobic digestion batch tests were carried out on three blends of OMW and inoculum (digestate from a biogas plant fed with agro-wastes) at a PP concentration of 0.5, 1.0 and 2.0 g l−1 in mesophilic conditions. Total inhibition of anaerobic digestion was found at a PP concentration of 2.0 g l−1 (non-adapted inoculum group). A positive effect of the adaptation to the substrate was, instead, observed for the blends with adapted inoculum at a PP concentration of 1.0 and 2.0 g l−1. Methane yields increased by 70% (PP = 1.0 g l−1) and 300% (2.0 g l−1) in the group with adapted inoculum compared to the group with non-adapted inoculum. The results suggest that OMW should not be subject to anaerobic digestion at high PP concentrations (i.e. higher than about 1 g l−1) due to the microbial inhibition detected. Moreover, given the benefits of the adaptation of the microbial population that was more evident at the highest PP concentration tested, it is advisable to allow the progressive adaptation of the digestion to OMW feeding. Thanks to the increased methane yield, because of the improved microbial tolerance to inhibiting compounds, the anaerobic digestion of OMW could be a viable and environmentally sound solution for the treatment of agro-industrial wastewater.

The inhibition of anaerobic digestion by model phenolic compounds representative of those from Sargassum muticum

Practical yields of biogas from the anaerobic digestion of macroalgae and, Sargassum muticum in particular, are substantially below the theoretical maximum. There is considerable conjecture about the reasons for the relatively low practical methane yields from seaweed, and polyphenols are suggested as one of the elements in the low yield of methane from brown seaweeds. However, there appears to be little information on the effect of specific phenolics on defined substrates. This paper examines the effect of some simple phenolic compounds, representative of those reported in S. muticum on methane production from a range of model substrates. Three simple phenolics were selected, gallic acid, epicatechin and phloroglucinol; at four addition levels, 0, 0.5, 3.5 and 7.5% w/w of substrate; for four substrates, a readily digested simple organic substance, glycerol, and three polymers found in seaweed, cellulose, alginic acid and the sodium salt of alginic acid. Alginic acid and its sodium salt were found to be recalcitrant with average methane yields of equivalent to only 23–28% of their theoretical methane potential. Methane yield was further reduced by the presence of high concentrations (7% of substrate equivalent to 17.5 mg L−1) of phloroglucinol and epicatechin. None of the phenolic compounds studied appeared to inhibit the breakdown of the simple and readily digested compound, glycerol. Low methane yield in seaweed may be due to the recalcitrance of complex hydrocolloids and phenolic inhibition of the breakdown of more complex molecules in the initial hydrolysis stage of anaerobic digestion, but further research is required.

Thermophilic anaerobic digestion of pre-treated orange peel: Modelling of methane production

Abstract The valorisation of waste generated during orange juice manufacturing (OPW) by anaerobic digestion is gaining attention due to the several economic, social and environmental advantages of the process. Given that anaerobic digestion is a process that requires rigorous control to improve the biodegradability of the waste and methane production, this paper highlights the importance of modelling for adequate process monitoring. Specifically, this study fitted three sigmoidal models (Logistic, Gompertz, and Sigmoid models) to methane production from OPW. At laboratory scale, two thermophilic continuously stirred-tank reactors working in semi-continuous mode was used. d -limonene removal of 70% was previously reached by applying a pre-treatment on OPW. The results showed a biodegradability of the pre-treated waste of up to 96.7% in COD. The Logistic, Gompertz and Sigmoid models fitted adequately to cumulative methane production (R2 was 0.9691; 0.9492 and 0.9511, respectively). However, the Logistic model showed the best fit to the experimental data even under critical conditions. Specifically, the Logistic model predicted the maximum methane production rate within loads of 2.5–5.0 g COD/L. Therefore, this model might be very useful in predicting the maximum methane production rate and the maximum load that could be added without the inhibition of anaerobic digestion at real scale.

Efficient Anaerobic Digestion of Microalgae Biomass: Proteins as a Key Macromolecule.

Biogas generation is the least complex technology to transform microalgae biomass into bioenergy. Since hydrolysis has been pointed out as the rate limiting stage of anaerobic digestion, the main challenge for an efficient biogas production is the optimization of cell wall disruption/hydrolysis. Among all tested pretreatments, enzymatic treatments were demonstrated not only very effective in disruption/hydrolysis but they also revealed the impact of microalgae macromolecular composition in the anaerobic process. Although carbohydrates have been traditionally recognized as the polymers responsible for the low microalgae digestibility, protease addition resulted in the highest organic matter solubilization and the highest methane production. However, protein solubilization during the pretreatment can result in anaerobic digestion inhibition due to the release of large amounts of ammonium nitrogen. The possible solutions to overcome these negative effects include the reduction of protein biomass levels by culturing the microalgae in low nitrogen media and the use of ammonia tolerant anaerobic inocula. Overall, this review is intended to evidence the relevance of microalgae proteins in different stages of anaerobic digestion, namely hydrolysis and methanogenesis.

High-rate anaerobic digestion of thermally hydrolyzed wasted sludge (THWS) with high-strength ammonia

In this study, the anaerobic digestion of thermally hydrolyzed wasted sludge with high-strength ammonia was evaluated through ammonia-stripping and diverse types of anaerobic-digestion reactors. Although anaerobic digestion was conducted after ammonia-stripping and removal, anaerobic-digestion inhibition occurred due to the problem of the toxicity of sodium. Therefore, the sodium concentration should be maintained at lower than inhibitory concentration for effective anaerobic digestion. The results of the operating reactors, gas production amount, methane content, organic-matter removal efficiency, and methane yield were high in the order of the fluidized bed reactor (FBR), upflow anaerobic sludge blanket reactor, and continuously stirred tank reactor types. As the microorganism concentrations in the FBR type were more than twice higher than those in the other reactors, the FBR type was able to keep the microorganism concentrations high, and the microbial proximity between the hydrogen-producing microorganisms and the hydrogen-consuming microorganisms was closest in the FBR type. Therefore, the reactor type for the efficient anaerobic digestion of wastewater containing high-concentration ammonia nitrogen is the FBR type.

Inhibition of anaerobic digestion by dissolved lignin derived from alkaline pre-treatment of an aquatic macrophyte

Delignification by chemical pre-treatment improves the anaerobic digestibility of lignocellulosic biomass, but the by-products can potentially inhibit the digestion process. The present study investigates the inhibitory effect of dissolved lignin on three steps of anaerobic digestion: methanogenesis, acidogenesis, and hydrolysis. Dissolved lignin was extracted from a lignin-rich aquatic macrophyte, and anaerobic toxicity tests were performed in batch mode at dissolved lignin concentrations of 0, 0.5, 1.0, 2.5, and 5.0gL−1 under mesophilic conditions. Minor reductions of up to 15% and 10% against the control were observed in methanogenic and acidogenic activities, respectively, at a dissolved lignin concentration of 5.0gL−1. By contrast, hydrolysis efficiency dropped by 25% against the control at a dissolved lignin concentration of 1.0gL−1 and dropped by 35% against the control at a dissolved lignin concentration of 5.0gL−1. These results suggest that enzymatic hydrolysis of cellulose is the step that is most susceptible to inhibition by dissolved lignin in the anaerobic digestion process.

Stimulation and Inhibition of Anaerobic Digestion by Nickel and Cobalt: A Rapid Assessment Using the Resazurin Reduction Assay

Stimulation of anaerobic digestion by essential trace metals is beneficial from a practical point of view to enhance the biodegradability and degradation rate of wastes. Hence, a quick method to determine which metal species, and at what concentration, can optimize anaerobic digestion is of great interest to both researchers and operators. In this present study, we investigated the effect of nickel(II), cobalt(II), and their mixture, on the anaerobic digestion of synthetic municipal wastewater. Using a volumetric method, that is, measuring methane production over time, revealed that anaerobic digestion was stimulated by the addition of 5 mg L-1 nickel(II), and cobalt(II), and their mixture in day(s). However, using a novel resazurin reduction assay, and based on its change in rate over time, we evaluated both inhibition at 250 mg L-1 nickel(II) and cobalt(II), and also the stimulatory effect of 5 mg L-1 nickel(II), and cobalt(II), and their mixture, in just 6 h. By investigating the dynamic distribution of these metals in the liquid phase of the anaerobic system and kinetics of resazurin reduction by nickel spiked anaerobic sludge, the concentration of nickel(II) on anaerobic digestion performance was profiled. Three critical concentrations were determined; stimulation starting (around 1 mg L-1), stimulation ending (around 100 mg L-1) and stimulation maximizing (around 10 mg L-1). Hence, we propose that the resazurin reduction assay is a novel and quick protocol for studying the stimulation of anaerobic bioprocesses by bioavailable essential trace metals.

Identification of syntrophic acetate-oxidizing bacteria in anaerobic digesters by combined protein-based stable isotope probing and metagenomics.

Inhibition of anaerobic digestion through accumulation of volatile fatty acids occasionally occurs as the result of unbalanced growth between acidogenic bacteria and methanogens. A fast recovery is a prerequisite for establishing an economical production of biogas. However, very little is known about the microorganisms facilitating this recovery. In this study, we investigated the organisms involved by a novel approach of mapping protein-stable isotope probing (protein-SIP) onto a binned metagenome. Under simulation of acetate accumulation conditions, formations of (13)C-labeled CO2 and CH4 were detected immediately following incubation with [U-(13)C]acetate, indicating high turnover rate of acetate. The identified (13)C-labeled peptides were mapped onto a binned metagenome for improved identification of the organisms involved. The results revealed that Methanosarcina and Methanoculleus were actively involved in acetate turnover, as were five subspecies of Clostridia. The acetate-consuming organisms affiliating with Clostridia all contained the FTFHS gene for formyltetrahydrofolate synthetase, a key enzyme for reductive acetogenesis, indicating that these organisms are possible syntrophic acetate-oxidizing (SAO) bacteria that can facilitate acetate consumption via SAO, coupled with hydrogenotrophic methanogenesis (SAO-HM). This study represents the first study applying protein-SIP for analysis of complex biogas samples, a promising method for identifying key microorganisms utilizing specific pathways.