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.

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.

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.

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.

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.

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.

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.

Semi-continuous mono-digestion of OFMSW and Co-digestion of OFMSW with beech sawdust: Assessment of the maximum operational total solid content.

In this study, mono-digestion of the organic fraction of municipal solid waste (OFMSW) and co-digestion of OFMSW with beech sawdust, simulating green waste, were used to investigate the maximum operational total solid (TS) content in semi-continuous high-solids anaerobic digestion (HS-AD). To alleviate substrate overloading in HS-AD, the effluent mass was relatively reduced compared to the influent mass, extending the mass retention time. To this aim, the reactor mass was daily evaluated, permitting to assess the reactor content removal by biogas production. During mono-digestion of OFMSW, the NH3 inhibition and the rapid TS removal prevented to maintain HS-AD conditions (i.e. TS ≥ 10%), without exacerbating the risk of reactor acidification. In contrast, the inclusion of sawdust in OFMSW permitted to operate HS-AD up to 30% TS, before acidification occurred. Therefore, including a lignocellulosic substrate in OFMSW can prevent acidification and stabilize HS-AD at very high TS contents (i.e. 20-30%).

A comprehensive review on two-stage integrative schemes for the valorization of dark fermentative effluents.

This review provides the alternative routes towards the valorization of dark H2 fermentation effluents that are mainly rich in volatile fatty acids such as acetate and butyrate. Various enhancement and alternative routes such as photo fermentation, anaerobic digestion, utilization of microbial electrochemical systems, and algal system towards the generation of bioenergy and electricity and also for efficient organic matter utilization are highlighted. What is more, various integration schemes and two-stage fermentation for the possible scale up are reviewed. Moreover, recent progress for enhanced performance towards waste stabilization and overall utilization of useful and higher COD present in the organic source into value-added products are extensively discussed.

Hydrogen metabolic patterns driven by Clostridium-Streptococcus community shifts in a continuous stirred tank reactor.

The hydrogen (H2) production efficiency in dark fermentation systems is strongly dependent on the occurrence of metabolic pathways derived from the selection of microbial species that either consume molecular H2 or outcompete hydrogenogenic bacteria for the organic substrate. In this study, the effect of organic loading rate (OLR) on the H2 production performance, the metabolic pathways, and the microbial community composition in a continuous system was evaluated. Two bacterial genera, Clostridium and Streptococcus, were dominant in the microbial community depending on the OLR applied. At low OLR (14.7-44.1 gLactose/L-d), Clostridium sp. was dominant and directed the system towards the acetate-butyrate fermentation pathway, with a maximum H2 yield of 2.14 molH2/molHexose obtained at 29.4 gLactose/L-d. Under such conditions, the volumetric hydrogen production rate (VHPR) was between 3.2 and 11.6 LH2/L-d. In contrast, relatively high OLR (58.8 and 88.2 gLactose/L-d) favored the dominance of Streptococcus sp. as co-dominant microorganism leading to lactate production. Under these conditions, the formate production was also stimulated serving as a strategy to dispose the surplus of reduced molecules (e.g., NADH2+), which theoretically consumed up to 5.72 LH2/L-d. In such scenario, the VHPR was enhanced (13.7-14.5 LH2/L-d) but the H2 yield dropped to a minimum of 0.74 molH2/molHexose at OLR = 58.8 gLactose/L-d. Overall, this research brings clear evidence of the intrinsic occurrence of metabolic pathways detrimental for biohydrogen production, i.e., lactic acid fermentation and formate production, suggesting the use of low OLR as a strategy to control them.

Two-stage alkaline-enzymatic pretreatments to enhance biohydrogen production from sunflower stalks.

Because of their rich composition in carbohydrates, lignocellulosic residues represent an interesting source of biomass to produce biohydrogen by dark fermentation. Nevertheless, pretreatments should be applied to enhance the solubilization of holocelluloses and increase their further conversion into biohydrogen. The aim of this study was to investigate the effect of thermo-alkaline pretreatment alone and combined with enzymatic hydrolysis to enhance biohydrogen production from sunflower stalks. A low increase of hydrogen potentials from 2.3 ± 0.9 to 4.4 ± 2.6 and 20.6 ± 5.6 mL of H2 g(-1) of volatile solids (VS) was observed with raw sunflower stalks and after thermo-alkaline pretreatment at 55 °C, 24 h, and 4% NaOH and 170 °C, 1 h, and 4% NaOH, respectively. Enzymatic pretreatment alone showed an enhancement of the biohydrogen yields to 30.4 mL of H2 g(-1) of initial VS, whereas it led to 49 and 59.5 mL of H2 g(-1) of initial VS when combined with alkaline pretreatment at 55 and 170 °C, respectively. Interestingly, a diauxic effect was observed with sequential consumption of sugars by the mixed cultures during dark fermentation. Glucose was first consumed, and once glucose was completely exhausted, xylose was used by the microorganisms, mainly related to Clostridium species.

High current density via direct electron transfer by the halophilic anode respiring bacterium Geoalkalibacter subterraneus.

In this study the characterization of Geoalkalibacter subterraneus is presented, which is a novel halophilic anode respiring bacterium (ARB) previously selected and identified in a potentiostatically controlled bioelectrochemical system (BES) inoculated with sediments from a salt plant. Pure culture electroactive biofilms of Glk. subterraneus were grown during chronoamperometric batch experiments at a graphite electrode poised at +200 mV (vs. SCE) with 10 mM acetate as the electron donor. These biofilms exhibited the highest current density (4.68 ± 0.54 A m(-2)) reported on a planar material with a pure culture under saline conditions (3.5% NaCl). To investigate possible anodic electron transfer (ET) mechanisms, cyclic voltammetry (CV) of mature visible apparent reddish biofilms was performed under bioelectrocatalytic substrate consumption (turnover) and in the absence of the substrate (non-turnover). CV evidenced a well defined typical sigmoidal shape and a pair of clear redox couples under turnover and non-turnover conditions, respectively. Moreover, the calculation of their formal potentials indicated the presence of a common ET mechanism present in both CV conditions between -427.6 ± 0.5 (Ef,2) and -364.8 ± 4.5 mV (Ef,3). Confocal laser scanning microscopy inspection showed a biofilm structure composed of several layers of metabolically active bacteria that spread all over the electrode material within a biofilm up to 76 ± 7 µm thick. Such high value compared to the thickness values normally reported in the literature for pure culture electroactive bacteria justifies further investigations. Taken together, these results suggest that Glk. subterraneus performs a direct ET mechanism in contact with the electrode material. Furthermore, direct current generation from saline wastewater significantly expands the application of BESs.

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.

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.

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.

Predictive models of biohydrogen and biomethane production based on the compositional and structural features of lignocellulosic materials.

In an integrated biorefinery concept, biological hydrogen and methane production from lignocellulosic substrates appears to be one of the most promising alternatives to produce energy from renewable sources. However, lignocellulosic substrates present compositional and structural features that can limit their conversion into biohydrogen and methane. In this study, biohydrogen and methane potentials of 20 lignocellulosic residues were evaluated. Compositional (lignin, cellulose, hemicelluloses, total uronic acids, proteins, and soluble sugars) as well as structural features (crystallinity) were determined for each substrate. Two predictive partial least square (PLS) models were built to determine which compositional and structural parameters affected biohydrogen or methane production from lignocellulosic substrates, among proteins, total uronic acids, soluble sugars, crystalline cellulose, amorphous holocelluloses, and lignin. Only soluble sugars had a significant positive effect on biohydrogen production. Besides, methane potentials correlated negatively to the lignin contents and, to a lower extent, crystalline cellulose showed also a negative impact, whereas soluble sugars, proteins, and amorphous hemicelluloses showed a positive impact. These findings will help to develop further pretreatment strategies for enhancing both biohydrogen and methane production.

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.

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.

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%.

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.