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. A metabolic 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.

Screening and Application of Ligninolytic Microbial Consortia to Enhance Aerobic Degradation of Solid Digestate.

Recirculation of solid digestate through digesters has been demonstrated to be a potential simple strategy to increase continuous stirred-tank reactor biogas plant efficiency. This study extended this earlier work and investigated solid digestate post-treatment using liquid isolated ligninolytic aerobic consortia in order to increase methane recovery during the recirculation. Based on sampling in several natural environments, an enrichment and selection method was implemented using a Lab-scale Automated and Multiplexed (an)Aerobic Chemostat system to generate ligninolytic aerobic consortia. Then, obtained consortia were further cultivated under liquid form in bottles. Chitinophagia bacteria and Sordariomycetes fungi were the two dominant classes of microorganisms enriched through these steps. Finally, these consortia where mixed with the solid digestate before a short-term aerobic post-treatment. However, consortia addition did not increase the efficiency of aerobic post-treatment of solid digestate and lower methane yields were obtained in comparison to the untreated control. The main reason identified is the respiration of easily degradable fractions (e.g., sugars, proteins, amorphous cellulose) by the selected consortia. Thus, this paper highlights the difficulties of constraining microbial consortia to sole ligninolytic activities on complex feedstock, such as solid digestate, that does not only contain lignocellulosic structures.

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%.

Modeling of interspecies electron transfer in anaerobic microbial communities.

Interspecies electron transfer (IET) is a key phenomenon in anaerobic ecosystems, which is traditionally modeled as hydrogen transfer. Recently discovered alternative mediated IET (MIET) or direct IET (DIET) offer exciting alternative mechanisms of microbial partnerships that could lead to new strategies for the improvement of biotechnologies. Here, we analyze mathematical modeling of DIET and MIET in anaerobic ecosystems. Bioenergetics approaches already enable the evaluation of different energy sharing scenarios between microorganisms and give interesting clues on redox mediators and on possible ways of driving microbial communities relying on IET. The modeling of DIET kinetics however is currently only in its infancy. Recent concepts introduced for the modeling of electroactive biofilms should be further exploited. Recent modeling examples confirms the potential of DIET to increase the IET rates compared to H2-MIET, but also point out the need for additional characterizations of biological components supporting IET to improve predictions.

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.

The impact of biogas digestate typology on nutrient recovery for plant growth: Accessibility indicators for first fertilization prediction.

In recent years, anaerobic digestion of organic waste (OW) is rapidly appearing as a winning waste management strategy by producing energy and anaerobic digestates that can be used as fertilizers in agricultural soils. In this context, the management of the OW treatment process to maximize agro-system sustainability satisfying the crop nutrient demands represents the main goal. To investigate these traits, two protocols to assess the plant availability of digestate nitrogen (N) and phosphorus (P) were evaluated. With this aim, the N and P availability was determined on 8 digestates and 2 types of digestate-based compost from different OW via sequential chemical extractions (SCE). In addition, the digestates were tested in soil incubations and in plant pot tests with Italian ryegrass and compared with chemical fertilizer and a non-amended control soil. The N extracted from digestates via SCE was related to soil N mineralization and plant N recovery. The C: N ratio had negative impact on mineralized N and its recovery in shoots (ShootsN = -0.0085.(C/N)+0.172, r2 = 0.67), whereas water extractable mineral N was positevely related to the root N apparent recovery fraction (N-ARF) with (RootsN = 5E-5.Nsolublemin+0.0138, r2 = 0.53). The shoot P-ARF was positively correlated with the inorganic water extractable fraction of P (ShootsP =0.1153.H2O-Pi-0.2777.H2O-Po+0.0249, r2 = 0.71) whereas the root P-ARF was positively correlated with the less accessible fractions (RootsP = (b)   0.0955.NaHCO3-Po+0.0955.NaOH-Po-0.0584NaHCO3-Pi+0.0128, r2 = 0.8641). Feedstock digestate typology impacted the N and P recovery results leading to a better description of the typology properties and a first nutrients ARF prediction.

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.

Soft Microwave Pretreatment to Extract P-Hydroxycinnamic Acids from Grass Stalks.

The aim of this article is to provide an analysis of microwave effects on ferulic and coumaric acids (FA and CA, respectively) extraction from grass biomass (corn stalks and miscanthus). Microwave pretreatment using various solvents was first compared to conventional heating on corn stalks. Then, microwave operational conditions were extended in terms of incident power and treatment duration. Optimal conditions were chosen to increase p-hydroxycinnamic acids release. Finally, these optimal conditions determined on corn stalks were tested on miscanthus stalks to underlie the substrate incidence on p-hydroxycinnamic acids release yields. The optimal conditions-a treatment duration of 405 s under 1000 W-allowed extracting 1.38% FA and 1.97% CA in corn stalks and 0.58% FA and 3.89% CA in miscanthus stalks. The different bioaccessibility of these two molecules can explain the higher or lower yields between corn and miscanthus stalks.

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.

Effect of ammonium, electron donor and sulphate transient feeding conditions on sulphidogenesis in sequencing batch bioreactors.

This work aimed to study the effect of transient feeding conditions on sulphidogenesis in 8 sequencing batch bioreactors (SBR). SBR L1 and H1, operated under steady-state conditions were used as the control reactors, while four SBR were tested under transient feeding conditions using moderate (L2 and L3, feast and famine: 2.5 and 0 g SO42-·L-1) and high (H2 and H3, feast and famine: 15 and 0 g SO42-·L-1) loads. The sulphate removal efficiency (RE) was ≥90% in SBR L2, L3 and H1. The NH4+ famine conditions resulted in a higher sulphate RE (≥40% H3) compared to feast conditions (≤20% H2). Besides, the sulphidogenic first-order kinetic constant was 4% larger and the use of electron donor was 16.6% more efficient under NH4+ famine conditions. Sulphidogenesis is robust to transient feeding conditions, but not when applying high loading rates (SBR H2 and H3).

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.

Microbial anodic consortia fed with fermentable substrates in microbial electrolysis cells: Significance of microbial structures.

Microbial community structure of anodic biofilms plays a key role in bioelectrochemical systems (BESs). When ecosystems are used as inocula, many bacterial species having interconnected ecological interactions are present. The aim of the present study was to identify these interactions for the conversion of single substrates into electrical current. Dual-chamber reactors were inoculated with activated sludge and fed in batch mode with acetate, lactate, butyrate and propionate at 80 mMe- equivalents in quadruplicate. Analyses of biofilms and planktonic microbial communities showed that the anodic biofilms were mainly dominated by the Geobacter genus (62.4% of the total sequences). At the species level, Geobacter sulfurreducens was dominant in presence of lactate and acetate, while Geobacter toluenoxydans and Geobacter pelophilus were dominant with butyrate and propionate as substrates. These results indicate for the first time a specificity within the Geobacter genus towards the electron donor, suggesting a competitive process for electrode colonization and the implementations of syntrophic interactions for complete oxidation of substrates such as propionate and butyrate. All together, these results provide a new insight into the ecological relationships within electroactive biofilms and suggest eco-engineering perspectives to improve the performances of BESs.

Addition of granular activated carbon and trace elements to favor volatile fatty acid consumption during anaerobic digestion of food waste.

The effect of supplementing granular activated carbon and trace elements on the anaerobic digestion performance of consecutive batch reactors treating food waste was investigated. The results from the first batch suggest that addition of activated carbon favored biomass acclimation, improving acetic acid consumption and enhancing methane production. Adding trace elements allowed a faster consumption of propionic acid. A second batch proved that a synergy existed when activated carbon and trace elements were supplemented simultaneously. The degradation kinetics of propionate oxidation were particularly improved, reducing significantly the batch duration and improving the average methane productivities. Addition of activated carbon favored the growth of archaea and syntrophic bacteria, suggesting that interactions between these microorganisms were enhanced. Interestingly, microbial analyses showed that hydrogenotrophic methanogens were predominant. This study shows for the first time that addition of granular activated carbon and trace elements may be a feasible solution to stabilize food waste anaerobic digestion.

Methanosarcina plays a main role during methanogenesis of high-solids food waste and cardboard.

Anaerobic digestion of food waste is a complex process often hindered by high concentrations of volatile fatty acids and ammonia. Methanogenic archaea are more sensitive to these inhibitors than bacteria and thus the structure of their community is critical to avoid reactor acidification. In this study, the performances of three different inocula were compared using batch digestion tests of food waste and cardboard mixtures. Particular attention was paid to the archaeal communities in the inocula and after digestion. While the tests started with inocula rich in Methanosarcina led to efficient methane production, VFAs accumulated in the reactors where inocula initially were poor in this archaea and no methane was produced. In addition, higher substrate loads were tolerated when greater proportions of Methanosarcina were initially present in the inoculum. Independently of the inoculum origin, Methanosarcina were the dominant methanogens in the digestates from the experiments that efficiently produced methane. These results suggest that the initial archaeal composition of the inoculum is crucial during reactor start-up to achieve stable anaerobic digestion at high concentrations of ammonia and organic acids.