Electroactive microorganisms synthesizing iron sulfide nanoparticles for enhanced hexavalent chromium removal in microbial fuel cells.

Microbial fuel cells (MFCs) have been considered a promising technology for Cr6+ removal, but they are limited by Cr6+-reducing biocathodes with low extracellular electron transfer (EET) and poor microbial activity. In this study, three kinds of nano-FeS hybridized electrode biofilms, obtained through synchronous biosynthesis (Sy-FeS), sequential biosynthesis (Se-FeS) and cathode biosynthesis (Ca-FeS), were applied as biocathodes for Cr6+ removal in MFCs. The Ca-FeS biocathode exhibited the best performance due to the superior properties of biogenic nano-FeS (e.g., more synthetic amount, smaller particle size, better dispersion). The MFC with the Ca-FeS biocathode achieved the highest power density (42.08 ± 1.42 mW/m2) and Cr6+ removal efficiency (99.18 ± 0.1 %), which were 1.42 and 2.08 times as high as those of the MFC with the normal biocathode, respectively. The synergistic effects of nano-FeS and microorganisms enhanced the bioelectrochemical reduction of Cr6+, first realizing deep reduction of Cr6+ to Cr0 in biocathode MFCs. This significantly alleviated the cathode passivation caused by Cr3+ deposition. In addition, the hybridized nano-FeS as "armor" layers protected the microbes from toxic attack by Cr6+, improving the biofilm physiological activity and extracellular polymeric substances (EPS) secretion. The hybridized nano-FeS as "electron bridges" facilitated the microbial community to form a balanced, stable and syntrophic ecological structure. This study proposes a novel strategy through the cathode in-situ biosynthesis of nanomaterials to fabricate hybridized electrode biofilms with enhanced EET and microbial activity for toxic pollutant treatment in bioelectrochemical systems.

High temperature shock threatens methane production via disturbing microbial interactions in anaerobic digestion.

Operational strategies shape microbial interactions determining anaerobic digesting process, but it is unclear whether and how the microbial network properties impact gas generation, especially in the transitional stage after operations. This research examined how the high temperature shock affected microbial diversity and network traits connected with the biogas production in a swine manure-fed anaerobic digester. Rising temperature (from 35 °C to 50 °C) significantly reduced biogas and methane production (p < 0.001) in the transitional stage due to the syntrophic loss of Methanomicrobiaceae and Firmicutes affiliated families. The high temperature shock reduced network modularity and thus caused the system functioning loss. Furthermore, the methanogenic stability was disrupted by high temperature shock (reduced the abundance of Methanosphaera but increased the abundance of Methanoculleus), which may result in the subsequent dysbiosis with other syntrophic communities. These findings suggest that the increased temperature-induced high network complexity and stability, but microbial communities need more time to restore the microenvironment via establishing the interactions of keystone species.

Using nanomaterials to increase the efficiency of chemical production in microbial cell factories: A comprehensive review.

Microbes have proven to be robust workhorses for the large-scale production of many chemicals. Especially, high-value biochemicals (e.g., natural pigments, unsaturated fatty acids) that cannot be derived from fossil fuels, can be produced by engineered microbes. There is a growing interest in both academia and industry to find new technologies that can enhance the efficiencies of microbial cell factories and boost the circular bioeconomy. Rapid technological innovations, such as microbial genome editing and synthetic biology, have greatly advanced the production of chemicals in engineered microbes. Nanomaterial-based technologies that exploit the unique physiochemical properties of nano-scale materials (e.g., large surface area, excellent catalytic activity, tunable optical and electrical performance) have demonstrated great potential and attracted increasing attention. There are many studies showing that nanomaterials can assist microbes in the synthesis of chemicals by providing micronutrients, inducing anti-ROS responses, promoting gas-liquid mass transfer, immobilizing microbial cells and promoting electron transfer in electrosynthesis. Furthermore, the latest studies demonstrate that nanomaterials can be used to construct photocatalyst-microbe hybrids and achieve solar driven chemical production. In this review, we comprehensively summarize these advances and discuss the current gaps as well as future perspectives. With the rapid development of synthetic biology and nanotechnology, we believe more nanomaterial-based technologies will be developed and used to improve the productivity of microbial cell factories.

N-acyl-homoserine lactones in extracellular polymeric substances from sludge for enhanced chloramphenicol-degrading anode biofilm formation in microbial fuel cells.

Exploring an efficient acclimation strategy to obtain robust bioanodes is of practical significance for antibiotic wastewater treatment by bioelectrochemical systems (BESs). This study investigated the effects of two acclimation conditions on chloramphenicol (CAP)-degrading anode biofilm formation in microbial fuel cells (MFCs). The one was continuously added the extracellular polymeric substances (EPS) extracted from anaerobic sludge and increasing concentrations of CAP after the first start-up phase, while the other was added the EPS-1 (N-acyl-homoserine lactones, namely AHLs were extracted from the EPS) at the same conditions. The results demonstrated that AHLs in the sludge EPS played a crucial role for enhanced CAP-degrading anode biofilm formation in MFCs. The AHL-regulation could not only maintain stable voltage outputs but also significantly accelerate CAP removal in the EPS MFC. The maximum voltage of 653.83 mV and CAP removal rate of 1.21 ± 0.05 mg/L·h were attained from the EPS MFC at 30 mg/L of CAP, which were 0.84 and 1.57 times higher than those from the EPS-1 MFC, respectively. These improvements were largely caused by the thick and 3D structured biofilm, strong and homogeneous cell viability throughout the biofilm, and high protein/polysaccharide ratio along with more conductive contents in the biofilm EPS. Additionally, AHLs facilitated the formation of a biofilm with rich biodiversity and balanced bacterial proportions, leading to more beneficial mutualism among different functional bacteria. More bi-functional bacteria (for electricity generation and antibiotic resistance/degradation) were specifically enriched by AHLs as well. These findings provide quorum sensing theoretical knowledge and practical instruction for rapid antibiotic-degrading electrode biofilm acclimation in BESs.

The fate of anaerobic syntrophy in anaerobic digestion facing propionate and acetate accumulation.

How the acetate and propionate accumulation impact anaerobic syntrophy during methane formation is not well understood. To investigate such effect, continuous acetate (35 g/L), propionate (11.25 g/L) and bicarbonate (30 g/L) supplementation were used during mesophilic anaerobic digestion. The high throughput sequencing (16S rRNA and mcrA), Real-Time quantitative PCR, and stable carbon isotope fingerprinting were applied to investigate the structure and activity of microbial community members. The results demonstrated that the abundance of syntrophic acetate oxidizing bacteria exhibited a gradual decrease coupled with heavier stable carbon isotopic signature of methane (δ 13CH4) in the three reagents impacted reactors. The increased acetate and propionate concentrations exerted negative influence on biogas production but the relatively stable hydrogenotrophic methanogens together with syntrophic acetate/propionate oxidizing bacteria kept the stable methane formation facing acetate and propionate accumulation. The functional genes copy number of the hydrogenotrophic Methanocellaceae and Methanomicrobiaceae correlated significantly with δ 13CH4 (R2 > 0.74), but only the abundance of Methanocellaceae fitted well with δ 13CH4 (p < 0.05). The δ 13CH4 signatures can predict methanogenesis, as it directly reflects the main methanogenic pathway; yet, further investigation of isotope fractionation in acetate/propionate coupled with δ 13CH4 is needed. Collectively, these results provide deep insight into anaerobic syntrophy and reveal changes of synergistic relationships, both of which may contribute to the stability of biogas reactors.

A QSPR model for estimating Henry's law constant of H2S in ionic liquids by ELM algorithm.

Ionic liquids (ILs) as green solvents have been studied in the application of gas sweetening. However, it is a huge challenge to obtain all the experimental values because of the high costs and generated chemical wastes. This study pioneered a quantitative structure-property relationship (QSPR) model for estimating Henry's law constant (HLC) of H2S in ILs. A dataset consisting of the HLC data of H2S for 22 ILs within a wide range of temperature (298.15-363.15 K) were collected from published reports. The electrostatic potential surface area (SEP) and molecular volume of these ILs were calculated and used as input descriptors together with temperature. The extreme learning machine (ELM) algorithm was employed for nonlinear modelling. Results showed that the determination coefficient (R2) of the training set, test set and total set were 0.9996, 0.9989,0.9994, respectively, while the average absolute relative deviation (AARD%) of them were 1.3383, 2,4820 and 1.5820, respectively. The statistical parameters for the measurement of the model exhibited the great reliability, stability, and predictive power of the ELM model. The Applicability Domain (AD) of the ELM model is also investigated. It proves that the majority of ILs in the training and test sets are located in the model's AD and verifies the reliability of the model. The proposed model is potentially applicable to guide the application of ILs for gas sweetening.

Prediction of ammonia absorption in ionic liquids based on extreme learning machine modelling and a novel molecular descriptor SEP.

The large amounts of ammonia emissions generated from industrial production have caused serious environmental pollution problems, such as soil acidification, eutrophication, the formation of fine particles and changes in the global greenhouse balance, and also greatly endanger human health. At present, effectively reducing ammonia emissions or recovering ammonia is still a huge challenge. Ionic liquids (ILs) as a new class of green solvent have been introduced for ammonia absorption with great potential, but a huge number on combination systems of ILs lead to the difficulty of measuring the ammonia solubility in all ILs by experiments (e.g., danger and cost). Hereby, this study proposed a novel approach for estimating the ammonia solubility in different ILs. A predictive model was developed based on the novel Algorithm - extreme learning machine (ELM) and the molecular descriptors of electrostatic potential surface areas (SEP) as input parameters. Besides, 502 data points of ammonia solubility in 17 ILs were gathered with a wide range of pressure and temperature. For the total set, the determination coefficient (R2) and the average absolute relative deviation (AARD) of the developed model were 0.9937 and 2.95%, respectively. The regression plots revealed good consistency between predictive and experimental data points. Results show the good performance and reliability of the developed model, indicating that the proposed approach can be potentially applied for screening reasonable ILs to absorb ammonia from chemical industry processes.

Effects of loading rates and plant species on sludge characteristics in earthworm assistant sludge treatment wetlands.

Sludge treatment wetlands (STWs) are widely used to treat surplus sludge in recent years. However, the effects of plant species and loading rates on sludge characteristics in earthworm assistant STWs remain unclear. In the current study, six STWs planted with two plant species (Phragmites australis, Typha angustifolia) were investigated under four loading rates (60, 80, 90 and 120 kg DS/m2/yr) regarding the influence on sludge characteristics. Furthermore, earthworms were added in three STWs to evaluate their role on sludge stabilization during resting period. Results showed that the best sludge dewatering (dry solids (DS) of 45.0%) and stabilization (volatile solids to total solids (VS/TS) of 40.5%) were determined in the P. australis STWs at the loading rate of 80 kg DS/m2/yr. Furthermore, VS/TS and Escherichia coli contents in earthworm STWs were 5.5-11.2% and 12-39% lower than that in the control without earthworm addition. Meanwhile, earthworm also decreased the nutrient contents in STWs. However, earthworms had insignificant effects on heavy metal contents in STWs. Nevertheless, the bioavailability of Cd and Cr in STWs were decreased by earthworm addition, with an acid-soluble fraction of Cd and Cr reduced by 11.2-18% and 2.5-7.5%, respectively. In conclusion, sludge characteristics can be improved by earthworm addition in P. australis STWs.

Effects of copper salts on performance, antibiotic resistance genes, and microbial community during thermophilic anaerobic digestion of swine manure.

This study investigated methane production and ARGs reduction during thermophilic AD of swine manure with the addition of different Cu salts (cupric sulfate, cupric glycinate, and the 1:1 mixture of these two salts). Results showed methane production was increased by 28.78% through adding mixed Cu salts. The mixed Cu group effectively reduced total ARGs abundance by 26.94%, suggesting mixed Cu salts did not promote the potential ARGs risk. The positive effects of mixed Cu salts on AD performance and ARGs removal might be ascribed to the low bioavailability. Microbial community analysis indicated the highest abundances of Clostridia_MBA03 and Methanobacterium in the mixed Cu group might cause the increased methane production. Spearman's rank correlation analysis elucidated the succession in microbial community induced by environmental factors was the main driver for shaping ARGs profiles. Thus, mixed Cu salts could be an alternative to replace the inorganic Cu salt in animal feed additives.

Temperature regulations impose positive influence on the biomethane potential versus digesting modes treating agricultural residues.

Temperature regulations (mesophilic/thermophilic) and digesting modes (mono-/co-digestion) play key roles in the biomethane potential of anaerobic digestion, but limited research focus on the synergetic effects on microbial interconnections of the biomethane process. In this study, the pineapple and maize residues under different operations were monitored by batch biogas assays and 16S high-throughput sequencing to explore: 1) biomethane potential regarding different operations, 2) microbial communities in different treated reactors, and 3) significant factors determine microbial distribution. Results showed that the co-digestion had higher methanogenic abundance and biomethane production (~3300 mLn) versus mono-digestion under mesophilic condition. To the thermophilic condition, the co-digestion had less methanogenic abundance but more biomethane production (~5000 mLn). Statistical evidence uncovered that the Clostridiaceae and Thermoanaerobacteraceae dominated pathways linked closely with methanogenesis which may contribute the more biomethane production in the thermophilic condition. This study demonstrated the temperature regulations drove rare taxa as major contributors for biomethane production.

Fermentation conditions optimization, purification, and antioxidant activity of exopolysaccharides obtained from the plant growth-promoting endophytic actinobacterium Glutamicibacter halophytocola KLBMP 5180.

In this study, an endophytic actinobacterium Glutamicibacter halophytocola KLBMP 5180, was investigated for the production and antioxidant activity of exopolysaccharides (EPSs). First, the suitable fermentation time, temperature, inoculation volume, pH value, and the carbon and nitrogen sources for EPSs production were obtained using the one variable at a time method (OVAT). Then, a central composition design was used for fermentation conditions optimization to obtain the maximum EPS yield. The optimal medium and condition were as follows: 100 mL broth in 250 mL Erlenmeyer flasks, including 3.65 g/L maltose, 9.88 g/L malt extract, 3.40 g/L yeast extract, 1.41 g/L MnCl2, pH 7.5, culture temperature 28 °C, and 200 rpm for 7 days, which increased the yield of EPSs to 2.89 g/L. Two purified EPSs, 5180EPS-1 (MW 58.9 kDa) and 5180EPS-2 (10.5 kDa), comprising rhamnose, galacturonic acid, glucose, glucuronic acid, xylose, and arabinose, were obtained for chemical analysis and antioxidant evaluation. The scavenging ability and reducing power of the superoxide anion and hydroxyl radicals demonstrated the moderate in vitro antioxidant activities of the two EPSs, thus indicating their potential to be a new source of natural antioxidants. However, further structure elucidation and functional studies need to be continued.

Positive effects of concomitant heavy metals and their reduzates on hexavalent chromium removal in microbial fuel cells.

Cr(vi) laden wastewaters generally comprise a range of multiple heavy metals such as Au(iii) and Cu(ii) with great toxicity. In the present study, cooperative cathode modification by biogenic Au nanoparticles (BioAu) reduced from aqueous Au(iii) and in situ Cu(ii) co-reduction were investigated for the first time to enhance Cr(vi) removal in microbial fuel cells (MFCs). With the co-existence of Cu(ii) in the catholyte, the MFC with carbon cloth modified with nanocomposites of multi-walled carbon nanotubes blended with BioAu (BioAu/MWCNT) obtained the highest Cr(vi) removal rate (4.07 ± 0.01 mg L-1 h-1) and power density (309.34 ± 17.65 mW m-2), which were 2.73 and 3.30 times as high as those for the control, respectively. The enhancements were caused by BioAu/MWCNT composites and deposited reduzates of Cu(ii) on the cathode surface, which increased the adsorption capacity, electronic conductivity and electrocatalytic activity of the cathode. This study provides an alternative approach for efficiently remediating co-contamination of multiple heavy metals and simultaneous bioenergy recovery.

Effect of one step temperature increment from mesophilic to thermophilic anaerobic digestion on the linked pattern between bacterial and methanogenic communities.

Process fluctuation caused by temperature modification of anaerobic digestion is routinely monitored via operational parameters, such as pH and gas production, but these parameters are lagging on microbial community performance. In this study, 13C isotope fractionation in CH4 and CO2 of biogas together with microbial community dynamics were applied to evaluate process stability in response to temperature increment. Results showed that the weakening correlated links between Firmicutes affiliated families and Methanomicrobiaceae were found regarding temperature increase. In contrast, Methanosarcinaceae and Methanobacteriaceae strengthened their links with multiple bacterial groups. This suggests that the 13C isotope fractionation in CH4 can predict the collapse of certain microbial interconnections and process instability, the new reinforced microbial links directly reflect the microbial community redundancy for maintaining function of syntrophic populations.

Assessment of the start-up process of anaerobic digestion utilizing swine manure: 13C fractionation of biogas and microbial dynamics.

The aim of this study was to investigate how the microbial community structure adapts during the start-up phase and how the 13C fractionation of biogas reflects the microbial population dynamics in two parallel swine manure-fed anaerobic digesters. Two swine manure-fed reactors for the start-up of continuously stirred tank reactors at mesophilic condition were evaluated. Changes in community structure were monitored using 16S rRNA high-throughput sequencing to measure the abundance of fermenting bacteria and methanogens. Digesters with relatively stable Methanosarcinaceae started up successfully and contained high gas production and low levels of propionate. In contrast, the digester that experienced a difficult start-up period had reduced Methanosarcinaceae along with accumulated propionate and low gas production. Specific gas production, specific methane production, and 13C fractionation of biogas were influenced significantly by Methanosarcinaceae, Methanobacteriaceae, and Clostridiaceae, indicating that the 13C fractionation of biogas had significant potential to reflect microbial population changes and digester performance during the start-up period.

Purification of leachate from sludge treatment beds by subsurface flow constructed wetlands: effects of plants and hydraulic retention time.

Sludge treatment beds (STBs) have been used widely in many countries due to low energy consumption, low operating and maintenance costs, and better environmental compatibility. Penetration, evaporation, and transpiration are the main processes for sludge dewatering in STBs. However, the leachate quality from STBs usually cannot meet discharge limits. Moreover, such leachate has very low COD/N ratio, which makes it difficult to treat. In the present study, two subsurface flow (SSF) constructed wetlands (CWs) were investigated for the treatment of leachate from STBs under three different hydraulic retention time (HRT) (3 days, 4 days, 6 days), aiming for evaluating the effects of plants and HRT on treatment performance, as well as the potential of SSF CWs to treat sludge leachate with low COD/N ration. The results showed that plants play an important role in leachate treatment. The best treatment performance was achieved with HRT of 4 days. In this condition, the mean removal efficiencies of COD (chemical oxygen demand), NH4+-N, TN (total nitrogen), and TP (total phosphorus) in the planted and the unplanted CWs were 61.6% (unplanted - 3.7%), 76.6% (unplanted 43.5%), 70% (unplanted 41%), and 65.6% (unplanted 6%), respectively. Heavy metal concentrations were below the Chinese integrated wastewater discharge standard during the experimental period in the planted CW, and the removal efficiencies in the planted CW system were higher than in the unplanted CW system. In all, planted SSF CWs can be an effective approach in removing leachate from sludge treatment beds. Furthermore, considering to temperature and seasonal variation, the leachate from STBs needs to be further studied in pilot- and full-scale condition.

Effects of various feedstocks on isotope fractionation of biogas and microbial community structure during anaerobic digestion.

Feedstock type influences bacterial and methanogenic communities in anaerobic digestion. These two communities work tightly to maintain the stability of anaerobic digestion. How to quick report the changes of microbial community structure especially methanogenesis is the key issue for optimizing anaerobic digestion process. In this study, 13C isotope fractionations of CH4 and CO2 in biogas and microbial community composition were analyzed in 5 different feedstocks. Our results showed that grass silage, maize silage and swine manure fed reactors had similar δ 13C values and methanogenic community composition, dominated by Methanosarcinaceae. The lowest δ 13CH4 values were detected in straw and chicken manure fed reactors, reflecting reduced microbial degradation of material or the presence of toxic components in these feedstocks. The straw fed bioreactor lead to low δ 13CH4 values, probably reflecting relatively high levels of the syntrophic acetate oxidizing bacteria, Synergistaceae and Syntrophaceae, which might work collectively with hydrogenotrophic methanogens, resulting in the low δ 13CH4 values in this bioreactor. Significantly, all core microbes in the 5 different feedstock fed bioreactors were either Clostridia species or related to the Synergistaceae (syntrophic acetate oxidizing bacteria).

Microbial community shifts in biogas reactors upon complete or partial ammonia inhibition.

Anaerobic digestion of nitrogen-rich substrate often causes process inhibition due to the susceptibility of the microbial community facing ammonia accumulation. However, the precise response of the microbial community has remained largely unknown. To explore the reasons, bacterial communities in ammonia-stressed reactors and control reactors were studied by amplicon pyrosequencing of 16S rRNA genes and the active methanogens were followed by terminal restriction fragment length polymorphism (T-RFLP) analyses of mcrA/mrtA gene transcripts. The results showed that the diversity of bacterial communities decreased in two parallel ammonia-inhibited reactors compared with two control reactors, but different levels of inhibitions coinciding with different community shifts were observed. In one reactor, the process was completely inhibited, which was preceded by a decreasing relative abundance of the phylum Firmicutes. Despite the same operating conditions, the process was stabilized in the parallel, partially inhibited reactor, in which the relative abundance of Firmicutes greatly increased. In particular, both ammonia-inhibited reactors lacked taxa assumed to be syntrophic bacteria (Thermoanaerobacteraceae, Syntrophomonadaceae, and Synergistaceae). Besides the predominance of the hydrogenotrophic methanogens Methanoculleus and Methanobacterium, activity of Methanosarcina and even of the strictly aceticlastic genus Methanosaeta were found to contribute at very high ammonia levels (> 9 g NH4-N L-1) in the stabilized reactor (partial inhibition). In contrast, the lack of aceticlastic activity in the parallel reactor might have led to acetate accumulation and thus process failure (complete inhibition). Collectively, ammonia was found to be a general inhibitor while accumulating acetate and thus acidification might be the key factor of complete process failure.

Reduction of the hydraulic retention time at constant high organic loading rate to reach the microbial limits of anaerobic digestion in various reactor systems.

The effects of hydraulic retention time (HRT) reduction at constant high organic loading rate on the activity of hydrogen-producing bacteria and methanogens were investigated in reactors digesting thin stillage. Stable isotope fingerprinting was additionally applied to assess methanogenic pathways. Based on hydA gene transcripts, Clostridiales was the most active hydrogen-producing order in continuous stirred tank reactor (CSTR), fixed-bed reactor (FBR) and anaerobic sequencing batch reactor (ASBR), but shorter HRT stimulated the activity of Spirochaetales. Further decreasing HRT diminished Spirochaetales activity in systems with biomass retention. Based on mcrA gene transcripts, Methanoculleus and Methanosarcina were the predominantly active in CSTR and ASBR, whereas Methanosaeta and Methanospirillum activity was more significant in stably performing FBR. Isotope values indicated the predominance of aceticlastic pathway in FBR. Interestingly, an increased activity of Methanosaeta was observed during shortening HRT in CSTR and ASBR despite high organic acids concentrations, what was supported by stable isotope data.

Improved Monitoring of Semi-Continuous Anaerobic Digestion of Sugarcane Waste: Effects of Increasing Organic Loading Rate on Methanogenic Community Dynamics.

The anaerobic digestion of filter cake and its co-digestion with bagasse, and the effect of gradual increase of the organic loading rate (OLR) from start-up to overload were investigated. Understanding the influence of environmental and technical parameters on the development of particular methanogenic pathway in the biogas process was an important aim for the prediction and prevention of process failure. The rapid accumulation of volatile organic acids at high OLR of 3.0 to 4.0 gvs·L⁻¹·day⁻¹ indicated strong process inhibition. Methanogenic community dynamics of the reactors was monitored by stable isotope composition of biogas and molecular biological analysis. A potential shift toward the aceticlastic methanogenesis was observed along with the OLR increase under stable reactor operating conditions. Reactor overloading and process failure were indicated by the tendency to return to a predominance of hydrogenotrophic methanogenesis with rising abundances of the orders Methanobacteriales and Methanomicrobiales and drop of the genus Methanosarcina abundance.

Stable isotope composition of biogas allows early warning of complete process failure as a result of ammonia inhibition in anaerobic digesters.

Four 15-L lab-scale continuous stirred tank reactors were operated under mesophilic conditions to investigate the effect of ammonia inhibition. Stable isotope fingerprinting of biogas was applied as a process monitoring tool. Ammonia inhibition was initiated by amendment of chicken manure to maize silage fed reactors. During the accumulation of ammonia, the concentration of volatile fatty acids increased while the biogas production and pH decreased. However, in one reactor, an inhibited steady state with stable gas production even at high ammonia levels was achieved, while the other reactor proceeded to complete process failure. A depletion of the δ(13)CH4 and δ(13)CO2 values preceded the process inhibition. Moreover, the stable isotope composition of biogas also forecasted the complete process failure earlier than other standard parameters. The stable isotope analyses of biogas have a potential for mechanistic insights in anaerobic processes, and may be used to pre-warn process failure under stress conditions.