Influence of fumarate on interspecies electron transfer and the metabolic shift induced in Clostridium pasteurianum by Geobacter sulfurreducens.

AIMS: In previous studies, it was demonstrated that co-culturing Clostridium pasteurianum and Geobacter sulfurreducens triggers a metabolic shift in the former during glycerol fermentation. This shift, attributed to interspecies electron transfer and the exchange of other molecules, enhances the production of 1,3-propanediol at the expense of the butanol pathway. The aim of this investigation is to examine the impact of fumarate, a soluble compound usually used as an electron acceptor for G. sulfurreducens, in the metabolic shift previously described in C. pasteurianum. METHODS AND RESULTS: Experiments were conducted by adding along with glycerol, acetate and different quantities of fumarate in co-cultures of G. sulfurreducens and C. pasteurianum. Ametabolic shift was exhibited in all the co-culture conditions. This shift was more pronounced at higher fumarate concentrations. Additionally, we observed G. sulfurreducens growing even in the absence of fumarate and utilizing small amounts of this compound as an electron donor rather than an electron acceptor in the co-cultures with high fumarate addition. CONCLUSIONS: This study provided evidence that interspecies electron transfer continues to occur in the presence of a soluble electron acceptor, and the metabolic shift can be enhanced by promoting the growth of G. sulfurreducens.

Sequential dark fermentation and microbial electrolysis cells for hydrogen production: Volatile fatty acids influence and energy considerations.

Hydrogen production from food waste by coupling dark fermentation (DF) and microbial electrolysis cells (MEC) was studied. Metabolic patterns in DF, their effects on MECs efficiency, and the energy output of the coupling were investigated. Mesophilic temperature and acidic pH 5.5 resulted in 72 ± 20 mL H2/g CODin and a butyrate-enriched profile (C2/C4, 0.5-0.6) contrasting with an acetate-enriched profile (C2/C4, 1.8-1.9) and 36 ± 10 mL H2/g CODin at pH 7. Assessment in series of the DF effluents in MECs resulted in a higher hydrogen yield (566-733 mL H2/g CODin) and volatile fatty acids (VFAs) removal (84-95%) obtained from pH 7 effluents compared to pH 5.5 effluents (173-186 mL H2/g CODin and 29-59%). Finally, the output energy was lower in DF at pH 7, however, these effluents retrieved the highest energy in the MEC, showing the importance of process pH and VFAs profile to balance the coupling.

Lactic acid fermentation of food waste as storage method prior to biohydrogen production: Effect of storage temperature on biohydrogen potential and microbial communities.

This study aims to investigate the impact of utilizing lactic acid fermentation (LAF) as storage method of food waste (FW) prior to dark fermentation (DF). LAF of FW was carried out in batches at six temperatures (4 °C, 10 °C, 23 °C, 35 °C, 45 °C, and 55 °C) for 15 days followed by biological hydrogen potential (BHP) tests. Different storage temperatures resulted in different metabolites distribution, with either lactate or ethanol being dominant (159.2 ± 20.6 mM and 234.4 ± 38.2 mM respectively), but no negative impact on BHP (averaging at 94.6 ± 25.1 mL/gVS). Maximum hydrogen production rate for stored FW improved by at least 57%. Microbial analysis showed dominance of lactic acid bacteria (LAB) namely Lactobacillus sp., Lactococcus sp., Weisella sp., Streptococcus sp. and Bacillus sp. after LAF. Clostridium sp. emerged after DF, co-existing with LAB. Coupling LAF as a storage method was demonstrated as a novel strategy of FW management for DF, for a wide range of temperatures.

Dark fermentation and microalgae cultivation coupled systems: Outlook and challenges.

The implementation of a sustainable bio-based economy is considered a top priority today. There is no doubt about the necessity to produce renewable bioenergy and bio-sourced chemicals to replace fossil-derived compounds. Under this scenario, strong efforts have been devoted to efficiently use organic waste as feedstock for biohydrogen production via dark fermentation. However, the technoeconomic viability of this process needs to be enhanced by the valorization of the residual streams generated. The use of dark fermentation effluents as low-cost carbon source for microalgae cultivation arises as an innovative approach for bioproducts generation (e.g., biodiesel, bioactive compounds, pigments) that maximizes the carbon recovery. In a biorefinery context, after value-added product extraction, the spent microalgae biomass can be further valorised as feedstock for biohydrogen production. This integrated process would play a key role in the transition towards a circular economy. This review covers recent advances in microalgal cultivation on dark fermentation effluents (DFE). BioH2 via dark fermentation processes and the involved metabolic pathways are detailed with a special focus on the main aspects affecting the effluent composition. Interesting traits of microalgae and current approaches to solve the challenges associated to the integration of dark fermentation and microalgae cultivation are also discussed.

Effect of organic loading rate and effluent recirculation on biogas production of desulfated skim latex serum using up-flow anaerobic sludge blanket reactor.

High sulfate contents in skim latex serum (SLS) can be reduced by rubber wood ash (RWA). Subsequently, the desulfated skim latex serum (DSLS) can be further anaerobically treated more effectively with the accompanying generated biomethane. In this study, DSLS was treated using an up-flow anaerobic sludge blanket (UASB) reactor operated at 10-day HRT and under mesophilic (37 °C) conditions. The effect of organic loading rates (OLR) at 0.89, 1.79 and 3.57 g-COD/L-reactor∙d on DSLS biodegradability was investigated in Phase I-IV using NaHCO3 as an external buffering agent. Maximum methane production yield of 226.35 mL-CH4/g-CODadded corresponding to 403.25 mL-CH4/L reactor·d was achieved at the suitable OLR of 1.79 g-COD/L-reactor∙d. UASB effluent recirculation which was then applied to replace the NaHCO3. It was found that with 53% effluent recirculation similar to an OLR of 2.01 g-COD/L-reactor∙d, an average of 185.70 mL-CH4/g-CODadded corresponding to 371.40 mL/L reactor·d of methane production was reached. The dominant bacteria in UASB reactor were members of Proteobacteria, Bacteroidota, Firmicutes, and Desulfobacterota phyla. Meanwhile, the archaeal community was majorly dominated by the genera Methanosaeta sp. and Methanomethylovorans sp. The study clearly indicates the capabilities of UASB reactor with effluent recirculation to treat DSLS anaerobically.

Lactic acid production from food waste using a microbial consortium: Focus on key parameters for process upscaling and fermentation residues valorization.

In this study, the production of lactic acid from food waste in industrially relevant conditions was investigated. Laboratory assays were first performed in batch conditions to determine the suitable operational parameters for an efficient lactic acid production. The use of compost as inoculum, the regulation of temperature at 35 °C and pH at 5 enhanced the development of Lactobacillus sp. resulting in the production of 70 g/L of lactic acid with a selectivity of 89% over the other carboxylic acids. Those parameters were then applied at pilot scale in successive fed-batch fermentations. The subsequent high concentration (68 g/L), yield (0.38 g/gTS) and selectivity (77%) in lactic acid demonstrated the applicability of the process. To integrate the process into a complete value chain, fermentation residues were then converted into biogas through anaerobic digestion. Lastly, the experiment was successfully replicated using commercial and municipal waste collected in France.

Mechanisms underlying Clostridium pasteurianum's metabolic shift when grown with Geobacter sulfurreducens.

Recently, a study showed that glycerol fermentation by Clostridium pasteurianum could be metabolically redirected when the electroactive bacterium Geobacter sulfurreducens was added in the culture. It was assumed that this metabolic shift of the fermentative species resulted from an interspecies electron transfer. The aim of this study was to find out the mechanisms used for this interaction and how they affect the metabolism of C. pasteurianum. To get insights into the mechanisms involved, several coculture setups and RNA sequencing with differential expression analysis were performed. As a result, a putative interaction model was proposed: G. sulfurreducens produces cobamide molecules that possibly modify C. pasteurianum metabolic pathway at the key enzyme glycerol dehydratase, and affect its vanadium nitrogenase expression. In addition, the results suggested that G. sulfurreducens' electrons could enter C. pasteurianum through its transmembrane flavin-bound polyferredoxin and cellular cytochrome b5-rubredoxin interplay, putatively reinforcing the metabolic shift. Unravelling the mechanisms behind the interaction between fermentative and electroactive bacteria helps to better understand the role of bacterial interactions in fermentation setups. KEY POINTS: • C. pasteurianum-G. sulfurreducens interaction inducing a metabolic shift is mediated • C. pasteurianum's metabolic shift in coculture might be induced by cobamides • Electrons possibly enter C. pasteurianum through a multiflavin polyferredoxin.

Mixotrophic Growth of Chlorella sorokiniana on Acetate and Butyrate: Interplay Between Substrate, C:N Ratio and pH.

Microalgae can be cultivated on waste dark fermentation effluents containing volatile fatty acids (VFA) such as acetate or butyrate. These VFA can however inhibit microalgae growth at concentrations above 0.5-1 gC.L-1. This study used the model strain Chlorella sorokiniana to investigate the effects of acetate or butyrate concentration on biomass growth rates and yields alongside C:N:P ratios and pH control. Decreasing undissociated acid levels by raising the initial pH to 8.0 allowed growth without inhibition up to 5 gC.L-1 VFAs. However, VFA concentration strongly affected biomass yields irrespective of pH control or C:N:P ratios. Biomass yields on 1.0 gC.L-1 acetate were around 1.3-1.5 gC.gC -1 but decreased by 26-48% when increasing initial acetate to 2.0 gC.L-1. This was also observed for butyrate with yields decreasing up to 25%. This decrease in yield in suggested to be due to the prevalence of heterotrophic metabolism at high organic acid concentration, which reduced the amount of carbon fixed by autotrophy. Finally, the effects of C:N:P on biomass, lipids and carbohydrates production dynamics were assessed using a mixture of both substrates. In nutrient replete conditions, C. sorokiniana accumulated up to 20.5% carbohydrates and 16.4% lipids while nutrient limitation triggered carbohydrates accumulation up to 45.3%.

Robust operation through effluent recycling for hydrogen production from the organic fraction of municipal solid waste.

The stability of fermentative hydrogen production from the organic fraction of municipal solid waste (OFMSW) was evaluated in this work using a strategy of effluent recycling. Three pretreatment conditions were applied on the recycled effluent: a) no heat shock treatment, b) one initial heat shock treatment (90 °C, 30 min) and c) systematic heat shock treatment at the beginning of each fermentation. When a systematic heat shock was applied, a maximal hydrogen yield of 17.2 ± 3.8 mLH2/gVS was attained. The hydrogen productivity was improved by 331% reaching a stable value of 1.51 ± 0.29 mLH2/gVS/h, after 8 cycles of effluent recycling. This strategy caused a sharp decrease of diversity with stable co-dominance of hydrogen- and lactate-producing bacteria, ie. Clostridiales and Lactobacillales, respectively. For the other conditions, a sharp decrease of the hydrogen yields was observed showing the importance of applying a heat shock treatment for optimal hydrogen production with effluent recycling.

Temperature and Inoculum Origin Influence the Performance of Ex-Situ Biological Hydrogen Methanation.

The conversion of H2 into methane can be carried out by microorganisms in a process so-called biomethanation. In ex-situ biomethanation H2 and CO2 gas are exogenous to the system. One of the main limitations of the biomethanation process is the low gas-liquid transfer rate and solubility of H2 which are strongly influenced by the temperature. Hydrogenotrophic methanogens that are responsible for the biomethanation reaction are also very sensitive to temperature variations. The aim of this work was to evaluate the impact of temperature on batch biomethanation process in mixed culture. The performances of mesophilic and thermophilic inocula were assessed at 4 temperatures (24, 35, 55 and 65 °C). A negative impact of the low temperature (24 °C) was observed on microbial kinetics. Although methane production rate was higher at 55 and 65 °C (respectively 290 ± 55 and 309 ± 109 mL CH4/L.day for the mesophilic inoculum) than at 24 and 35 °C (respectively 156 ± 41 and 253 ± 51 mL CH4/L.day), the instability of the system substantially increased, likely because of a strong dominance of only Methanothermobacter species. Considering the maximal methane production rates and their stability all along the experiments, an optimal temperature range of 35 °C or 55 °C is recommended to operate ex-situ biomethanation process.

Biomethanation processes: new insights on the effect of a high H2 partial pressure on microbial communities.

BACKGROUND: Biomethanation is a promising solution to upgrade the CH4 content in biogas. This process consists in the injection of H2 into an anaerobic digester, using the capacity of indigenous hydrogenotrophic methanogens for converting the injected H2 and the CO2 generated from the anaerobic digestion process into CH4. However, the injection of H2 could cause process disturbances by impacting the microbial communities of the anaerobic digester. Better understanding on how the indigenous microbial community can adapt to high H2 partial pressures is therefore required. RESULTS: Seven microbial inocula issued from industrial bioprocesses treating different types of waste were exposed to a high H2 partial pressure in semi-continuous reactors. After 12 days of operation, even though both CH4 and volatile fatty acids (VFA) were produced as end products, one of them was the main product. Acetate was the most abundant VFA, representing up to 94% of the total VFA production. VFA accumulation strongly anti-correlated with CH4 production according to the source of inoculum. Three clusters of inocula were distinguished: (1) inocula leading to CH4 production, (2) inocula leading to the production of methane and VFA in a low proportion, and (3) inocula leading to the accumulation of mostly VFA, mainly acetate. Interestingly, VFA accumulation was highly correlated to a low proportion of archaea in the inocula, a higher amount of homoacetogens than hydrogenotrophic methanogens and, the absence or the very low abundance in members from the Methanosarcinales order. The best methanogenic performances were obtained when hydrogenotrophic methanogens and Methanosarcina sp. co-dominated all along the operation. CONCLUSIONS: New insights on the microbial community response to high H2 partial pressure are provided in this work. H2 injection in semi-continuous reactors showed a significant impact on microbial communities and their associated metabolic patterns. Hydrogenotrophic methanogens, Methanobacterium sp. or Methanoculleus sp. were highly selected in the reactors, but the presence of co-dominant Methanosarcinales related species were required to produce higher amounts of CH4 than VFA.

Novel Outlook in Microbial Ecology: Nonmutualistic Interspecies Electron Transfer.

Recent advances in microbial electrochemical technologies have revealed the existence of numerous and highly diverse microorganisms able to exchange electrons with electrodes. This diversity could reflect the capacity of microorganisms to release and/or retrieve electrons with each other in natural environments. So far, this interspecies electron transfer has been studied with a special focus on syntrophy and was successfully demonstrated for several couples of species. In this article we argue that electron exchange between microbes exists beyond syntrophy or mutualism and could also promote competitive and even parasitic behaviour. Based on three interesting case studies identified from the literature, we also highlight that such nonmutualistic interactions could be widespread and of particular significance for the survival of pathogens or the shaping of complex microbial communities.

Standardized protocol for determination of biohydrogen potential.

Biohydrogen production potential (BHP) depends on several factors like inoculum source, substrate, pH, among many others. Batch assays are the most common strategy to evaluate such parameters, where the comparison is a challenging task due to the different procedures used. The present method introduces the first internationally validated protocol, evaluated by 8 independent laboratories from 5 different countries, to assess the biohydrogen potential. As quality criteria, a coefficient of variation of the cumulative hydrogen production (H max) was defined to be <15 %. Two options to run BHP batch tests were proposed; a manual protocol with periodic measurements of biogas production, needing conventional laboratory materials and analytical equipment for biogas characterization; and an automatic protocol, which is run in a device developed for online measurements of low biogas production. The detailed procedures for both protocol options are presented, as well as data validating them. The validation showed acceptable repeatability and reproducibility, measured as intra- and inter-laboratory coefficient of variation, which can be reduced up to 9 %.

Bioaugmentation enhances dark fermentative hydrogen production in cultures exposed to short-term temperature fluctuations.

Hydrogen-producing mixed cultures were subjected to a 48-h downward or upward temperature fluctuation from 55 to 35 or 75 °C. Hydrogen production was monitored during the fluctuations and for three consecutive batch cultivations at 55 °C to evaluate the impact of temperature fluctuations and bioaugmentation with synthetic mixed culture of known H2 producers either during or after the fluctuation. Without augmentation, H2 production was significantly reduced during the downward temperature fluctuation and no H2 was produced during the upward fluctuation. H2 production improved significantly during temperature fluctuation when bioaugmentation was applied to cultures exposed to downward or upward temperatures. However, when bioaugmentation was applied after the fluctuation, i.e., when the cultures were returned to 55 °C, the H2 yields obtained were between 1.6 and 5% higher than when bioaugmentation was applied during the fluctuation. Thus, the results indicate the usefulness of bioaugmentation in process recovery, especially if bioaugmentation time is optimised.

Assessing practical identifiability during calibration and cross-validation of a structured model for high-solids anaerobic digestion.

High-solids anaerobic digestion (HS-AD) of the organic fraction of municipal solid waste (OFMSW) is operated at a total solid (TS) content ≥ 10% to enhance the waste treatment economy, though it might be associated to free ammonia (NH3) inhibition. This study aimed to calibrate and cross-validate a HS-AD model for homogenized reactors in order to assess the effects of high NH3 levels in HS-AD of OFMSW, but also to evaluate the suitability of the reversible non-competitive inhibition function to reproduce the effect of NH3 on the main acetogenic and methanogenic populations. The practical identifiability of structural/biochemical parameters (i.e. 35) and initial conditions (i.e. 32) was evaluated using batch experiments at different TS and/or inoculum-to-substrate ratios. Variance-based global sensitivity analysis and approximate Bayesian computation were used for parameter optimization. The experimental data in this study permitted to estimate up to 8 biochemical parameters, whereas the rest of parameters and biomass contents were poorly identifiable. The study also showed the relatively high levels of NH3 (i.e. up to 2.3 g N/L) and ionic strength (i.e. up to 0.9 M) when increasing TS in HS-AD of OFMSW. However, the NH3 non-competitive function was unable to capture the acetogenic/methanogenic inhibition. Therefore, the calibration emphasized the need for target-oriented experimental data to enhance the practical identifiability and the predictive capabilities of structured HS-AD models, but also the need for further testing the NH3 inhibition function used in these simulations.

Modelling non-ideal bio-physical-chemical effects on high-solids anaerobic digestion of the organic fraction of municipal solid waste.

This study evaluates the main effects of including 'non-ideal' bio-physical-chemical corrections in high-solids anaerobic digestion (HS-AD) of the organic fraction of municipal solid waste (OFMSW), at total solid (TS) between 10 and 40%. As a novel approach, a simple 'non-ideal' module, accounting for the effects of ionic strength (I) on the main acid-base equilibriums, was coupled to a HS-AD model, to jointly evaluate the effects of 'non-ideality' and the TS content dynamics on the HS-AD bio-physical-chemistry. 'Non-ideality' influenced the pH, concentration of inhibitors (i.e. NH3), and liquid-gas transfer (i.e. CO2), particularly at higher TS (i.e. ≥ 20%). Meanwhile, fitting the experimental data for batch assays at 15% TS showed that HS-AD of OFMSW might be operated at I ≥ 0.5 M. Therefore, all HS-AD simulations should account for 'non-ideal' corrections, when assessing the main inhibitory mechanisms (i.e. NH3 buildup and acidification) potentially occurring in HS-AD of OFMSW.

Behavior of two-chamber microbial electrochemical systems started-up with different ion-exchange membrane separators.

In this study, microbial fuel cells (MFCs) - operated with novel cation- and anion-exchange membranes, in particular AN-VPA 60 (CEM) and PSEBS DABCO (AEM) - were assessed comparatively with Nafion proton exchange membrane (PEM). The process characterization involved versatile electrochemical (polarization, electrochemical impedance spectroscopy - EIS, cyclic voltammetry - CV) and biological (microbial structure analysis) methods in order to reveal the influence of membrane-type during start-up. In fact, the use of AEM led to 2-5 times higher energy yields than CEM and PEM and the lowest MFC internal resistance (148 ±â€¯17 Ω) by the end of start-up. Regardless of the membrane-type, Geobacter was dominantly enriched on all anodes. Besides, CV and EIS measurements implied higher anode surface coverage of redox compounds for MFCs and lower membrane resistance with AEM, respectively. As a result, AEM based on PSEBS DABCO could be found as a promising material to substitute Nafion.

Reversibility of hydrolysis inhibition at high hydrogen partial pressure in dry anaerobic digestion processes fed with wheat straw and inoculated with anaerobic granular sludge.

In dry anaerobic digestion (AD), methanogenic performances are lowered by high solid contents. Low performances are often caused by a decrease of the gas-liquid transfer kinetics leading to local accumulation of inhibitory by-products. Hydrogen was previously identified as an inhibitor of hydrolytic and acetogenic microbial activities in dry AD. CO2 is also generated but its impact on the microbial activity remains unknown. In this study, the reversibility of dry AD inhibition at high H2 partial pressure (PH2 of 1 bar) was investigated by adding CO2 (400 mbars) after 11 and 18 days of methanogenesis inhibition, in an AD process operated at 25% TS, using wheat straw as substrate and inoculated with anaerobic granular sludge. As soon as CO2 was added, the methanogenic activity rapidly recovered within 3 days, from 0.41 ±â€¯0.1 to 3.77 ±â€¯0.8 and then 2.25 ±â€¯0.3, likely through the hydrogenotrophic pathway followed by the acetoclastic pathway, respectively. This result was confirmed by the high abundance of Methanomicrobiales (83%) and the emergence of Methanosarcinales sp (up to 17%) within the methanogenic community. Furthermore, the recovery kinetics were impacted by the duration of the inhibition period suggesting a different impact of the high PH2 on hydrogenotrophic and acetoclastic methanogens.

Enhancement of corn stover conversion to carboxylates by extrusion and biotic triggers in solid-state fermentation.

Solid-state fermentation is a potential technology for developing lignocellulosic biomass-based biorefineries. This work dealt with solid-state fermentation for carboxylates production from corn stover, as building blocks for a lignocellulosic feedstock-based biorefinery. The effect of extrusion pretreatment, together with the action of a microbial consortia and hydrolytic enzymes as biotic triggers, was investigated on corn stover conversion, microbial metabolic pathways, and populations. The extrusion caused changes in the physical and morphological characteristics, without altering the biochemical composition of the corn stover. Extrusion also led to remarkable differences in the composition of the indigenous microbial population of the substrate. Consequently, it affected the structure of community developed after fermentation and the substrate conversion yield, which increased by 118% (from 23 ± 4 gCOD/kgVSi obtained with raw substrate to 51 ± 1 gCOD/kgVSi with extruded corn stover) with regard to self-fermentation experiments. The use of activated sludge as inoculum further increased the total substrate conversion into carboxylates, up to 60 ± 2 gCOD/kgVSi, and shaped the microbial communities (mainly composed of bacteria from the Clostridia and Bacteroidia classes) with subsequent homogenization of the fermentation pathways. The addition of hydrolytic enzymes into the reactors further increased the corn stover conversion, leading to a maximum yield of 142 ± 1 gCOD/kgVSi. Thus, extrusion pretreatment combined with the use of an inoculum and enzyme addition increased by 506% corn stover conversion into carboxylates. Beside biomass pretreatment, the results of this study indicated that biotic factor greatly impacted solid-state fermentation by shaping the microbial communities and related metabolic pathways.