Propionate is a valuable platform chemical, in particular as a precursor for the production of sustainable jet fuel. However, its biological production remains a challenge, particularly in mixed-culture waste fermentation, where a mixture of volatile fatty acids is typically generated with a low propionate selectivity. This study investigates a two-stage fermentation process to convert food waste into propionate using two different inocula. The strategy relies on driving microbial metabolism toward lactate production in the first-stage fermentation, which will serve as the intermediate for propionate production during the second step of the fermentation. With both inocula, lactate was effectively consumed and preferentially converted into propionate and acetate, with propionate concentrations reaching up to 17.90 +/- 0.35 g L-1, representing 45.51 +/- 1.81% of total COD metabolites. This production was associated with the growth of Anaerosporobacter, Tyzzerella and some Clostridium genera, in both inocula, suggesting their pivotal role in the lactate-propionate pathway. Overall, these results demonstrate that a two-stage fermentation strategy can efficiently target high propionate concentrations and reasonable selectivity using mixed cultures, by specifically targeting the lactate-propionate pathway, regardless of the initial inoculum.

The production of biofuels from lignocellulosic biomass is one of the most promising alternatives for generating clean energy. However, various challenges arise when using this recalcitrant biomass in biotechnological processes for the production of biofuels (ethanol, biogas, biohydrogen, and methane), such as low yields due to the structural complexity of lignocellulose. In this sense, this study evaluated a biological pretreatment of a lignocellulosic waste mixture consisting of fruit peels (FRP) and brewer's spent grain (BSG), through solid-state fermentation (SSF) using the white-rot fungi Trametes versicolor and Pleurotus ostreatus. Different pretreatment conditions were assessed, including duration (15, 25, and 35 days), temperature (28 and 32 degrees C), and fungal strain (T. versicolor and P. ostreatus), to determine their impact on reducing sugars concentration and delignification and improving the bioaccesibility of the biomass in further bioenergy obtention process. The most suitable pretreatment condition was to carry out the SSF with T. versicolor at 28 degrees C for 25 days, obtaining a delignification of 15 +/- 0.14 % and a reducing sugars consumption of 24 +/- 0 %. The substrate pretreated under these conditions (TV-H) resulted in a 2.06-fold improvement in hydrogen production (mLH2 L-1) compared to the raw substrate as well as a shift in microbial communities and metabolic pathways involved in comparison to the raw substrate in dark fermentation, demonstrating an improvement in the process when biomass is pretreated with Trametes versicolor.

Biogas upgrading through in-situ biomethanation offers a sustainable alternative to traditional technologies, yet the impact of key factors such as substrate biochemical composition remains inadequately explored. This study investigates how the substrate composition, such as protein-rich, lipid-rich, and carbohydrate-rich substrates, affects batch in-situ biomethanation performance. Using inoculum pre-adapted through parent continuous anaerobic digestion reactors, experiments revealed distinct methane yields and microbial dynamics influenced by substrate composition. Protein-rich substrates resulted in elevated ammonia levels (2 g total ammonia nitrogen-N/L), leading to a higher abundance of hydrogenotrophic methanogens. In contrast, carbohydrate-rich substrates demonstrated stable methane production and low ammonium accumulation, facilitating balanced microbial activity. Reactors processing lipid-rich and carbohydrate-rich substrates maintained consistent microbial communities, contrasting with the higher abundance of hydrogenotrophic methanogens in protein-rich waste reactors. Despite these variations, hydrogen consumption rates were comparable across all reactors, regardless of inoculum source (37 ± 1.5, 38 ± 2.0, and 34 ± 4.0 mg COD/L/h for protein-rich (COD being Chemical Oxygen Demand), lipid-rich and carbohydrate-rich substrates, respectively). These findings underscore that substrate biochemical composition does not significantly affect hydrogen consumption kinetics during the start-up period of batch in-situ biomethanation., providing valuable insights for advancing biogas upgrading technologies.

Endogenous bacterial communities present on biomass exhibit hydrolytic and fermentative activities, producing liquid and gaseous metabolites during fermentation. Several pretreatment methods are employed to hydrolyse complex biomass into simpler forms before conversion by fermentation. However, the impact of these pretreatments on endogenous microbial communities, particularly in the absence of external inoculum, is not well understood. This study investigates the effects of thermal and thermo-acid pretreatments on the selection of endogenous bacteria in sugar beet pulp and their subsequent production of volatile fatty acids (VFAs) and hydrogen through acidogenic fermentation. Pretreatment increased soluble sugar availability, leading to a metabolic shift toward butyrate production and enhanced hydrogen production up to 91 +/- 14 NmL/gVS under thermo-acid conditions. In contrast, the highest VFA yields were achieved without pretreatment (0.72 +/- 0.16 gCOD/gVS with inoculum and 0.59 +/- 0.05 gCOD/gVS without inoculum). While pretreatment initially reduced microbial population density and metabolite yield in non-inoculated fermentations at day 8, extending fermentation to 14 days allowed the endogenous community to recover, achieving comparable yields with inoculated fermentation at day 8. These results demonstrate that pretreatment can selectively drive metabolic pathways to target products, while fermentation duration and inoculation strongly influence overall metabolite production.

Palm oil mill effluent (POME) is a high-strength wastewater generated in large quantities by the palm oil industry and represents a promising substrate for bioenergy recovery. However, the mechanisms governing biohydrogen production from POME during dark fermentation remain unclear, particularly whether hydrogen yield is primarily controlled by metabolite accumulation or by microbial selection driven by operating conditions. This study investigates the effects of inoculum heat treatment and fermentation temperature on biohydrogen and short-chain fatty acid (SCFA) production from POME via dark fermentation. Batch experiments were conducted under mesophilic (37 degrees C) and thermophilic (55 degrees C) conditions using heat-treated and non-heat-treated inocula. Under thermophilic fermentation (55 degrees C) with heat-treated inoculum, acetic and butyric acids were the dominant SCFAs, contributing 54.1% and 42.3% of the total SCFA concentration, respectively, while propionic acid remained low (3.6%), indicating the predominance of acetate- and butyrate-type fermentation pathways. The highest biohydrogen yield of 25 mL H2/g VS was achieved at 55 degrees C with heat-treated inoculum, representing a 1.14-fold increase compared with mesophilic operation under identical conditions. Microbial community analysis revealed a temperature-dependent shift in dominant hydrogen-producing bacteria, with Clostridium prevailing at 37 degrees C and Thermoanaerobacterium dominating at 55 degrees C. Despite comparable SCFA distributions across conditions, hydrogen productivity differed substantially, indicating that biohydrogen production was primarily governed by temperature-mediated microbial selection rather than SCFA accumulation. These findings highlight the critical role of fermentation temperature in controlling microbial ecology and hydrogenogenic metabolism, supporting thermophilic dark fermentation as an effective strategy for POME valorisation into renewable hydrogen.

Intermittent electro-anaerobic digestion (EAD) combines electrochemical regulation with anaerobic digestion, potentially improving the efficiency of biomethane production and achieving efficient storage of renewable electricity. In this process, the electrodes periodically act as electron donors or acceptors, and by regulating the charging and discharging rhythm, the metabolic activity of microorganisms, the electron transfer rate and the community synergy are significantly enhanced. This work critically reviews emerging progress in our understanding of the electron transfer mechanism, the evolutionary patterns of the composite microbial community, electrochemical performance, and biofilm morphology under various intermittent polarization EAD strategies. The potential for low-carbon energy conversion processes within circular bioeconomy systems with industrial applications is emphasized, while perspectives for future engineering optimization are proposed.

Microbial electrolysis cells (MECs) are conventionally used for renewable biohydrogen production, yet their potential to steer carbon fluxes toward value-added molecules remains underexplored. Here, continuous dark fermentation (DF) of food waste (FW) operated at increasing organic loading rates (30–60 g VS/L·d) at neutral pH. The resulting effluents were enriched in acetate (26–31%), ethanol (3–15%), propionate (10–14%) and succinate (8–22%) on a chemical oxygen demand (COD) basis, while butyrate decreased from 25% to 8%. The effluent produced at 60 g VS/L·d was selected for MEC operation because of its high content of readily degradable substrates (acetate and ethanol), low butyrate level, and increased succinate concentration (a known propionate precursor), making it suitable for propionate enrichment and hydrogen production. MECs developed stable electroactive biofilms achieving H2 productions of 0.54–0.59 L H2/g COD, cathodic H2 recoveries up to 90%, and Coulombic efficiencies of 86 ± 2%, with current densities up to 11.5 A/m². Propionate selectivity increased from 12 ± 3% in the DF effluent to 59 ± 5% after MEC operation, as a result of preferential consumption of readily degradable metabolites, particularly acetate, together with succinate conversion into propionate. Microbial community analyses revealed enrichment of hydrolytic and acidogenic taxa during DF, and dominance of Geobacter-like electroactive bacteria (up to 66 ± 5%) alongside fermentative populations during MEC operation, consistent with the observed metabolite transformations. These findings highlight MECs as selective polishing units for simultaneous energy recovery and targeted carbon valorization, advancing the DF–MEC concept toward sustainable bio-based chemical production.

Lactic acid fermentation has recently been shown to be a robust storage strategy for food waste prior to conversion to biohydrogen through dark fermentation. However, the importance of initial microbial communities and, more particularly, exogenous microorganisms on the conversion of lactic acid-rich stored substrate is not yet fully elucidated. This study investigates the impact of introducing exogenous inoculum to lactic acid-rich stored food waste prior to biohydrogen production in dark fermentation. Results showed exogenous inoculation produced a statistically significant increase in biohydrogen production rate (Rm) by 199%, 250%, 137%, 130%, 19%, and 10% compared to non-inoculated stored food waste after food waste storage at 4 °C, 10 °C, 23 °C, 35 °C, 45 °C, and 55 °C, respectively. Interestingly, no impact on the maximum production yield (Pm) was observed, but exogenous inoculation increased the accumulation of acetate, up to 160% more compared to endogenous inoculum. The main hydrogen-producing bacteria (HPB) were affiliated with Clostridium sp., while Prevotella_9 sp., another known HPB, was found after the fermentation of the food waste stored at 23 °C. In this study, the interest of exogenous inoculation to convert food waste stored by lactic acid fermentation was demonstrated through an increase in production rate along with higher accumulation of co-products, e.g., acetate. Such findings are promising for further development of process coupling, combining storage and conversion by fermentation of complex food waste.

The recently reported electro-anaerobic digestion with intermittent electrical stimulation (IEAD) offers new opportunities for renewable energy storage. However, the mechanistic insights and resilience of long-term continuous and stable IEAD are still yet to be better understood. This study lasted up to 155 days of continuous operation of IEAD in up-flow anaerobic sludge blanket bioreactors to evaluate the long-term effects of different intermittent power periods on IEAD performance. The optimal electrical stimulation period was obtained for 12 h:12 h intermittence, resulting in a 15.9 % improvement of the specific methane yield while achieving an organic removal efficiency higher than 98 %. Intermittent electrical stimulation favored biofilm growth, electroactive substance secretion, and enhanced electrochemical activity and cellular repair. Genomic analysis demonstrated that IEAD microbial community was dominated by Methanothrix, whereas continuous energization promoted Methanobacteriaceae enrichment. Proteomic analysis suggested that intermittent power supply preserved functional proteins and upregulated dominant enzymes such as acetyl-CoA carboxylase biotin carboxyl carrier protein and enoyl-CoA hydratase, while conferring flexible carbon source allocation and electron transfer capacity to the suspension zone. Life cycle assessment results showed that the IEAD system could achieve an energy conversion rate of 20.6 and low carbon emissions of 7.3 g CO2-eq/MJ.

Selective butyrate production via anaerobic fermentation holds promise for sustainable biochemicals. Despite butyrate frequently accumulate in anaerobic systems, its mechanisms and optimal conditions remain unclear. To address this gap, batch experiments with glucose were conducted to explore the effect of pH (4.5-7.0 and 8.5-11.0), temperature (37 °C and 55 °C) and inoculum-to-substrate ratio (ISR; 3:1, 2:1, 1:1, 1:2, and 1:3) on butyrate production. The results indicated that the optimal conditions were pH 5.5, 37 °C, and an ISR of 1:3, refining previously inconsistent reports. Metagenomic analysis demonstrated that temperature and ISR significantly influenced microbial communities, identifying key butyrate producers: Clostridium, Caproicibacter, Caproicibacterium, Sporolactobacillus, and Ethanoligenens. The functional gene analysis revealed enriched reverse β-oxidation genes under optimal conditions, highlighting the key role of carbon chain elongation in butyrate production. These findings provide mechanistic insights into butyrate biosynthesis and practical strategies for improving selective butyrate production in anaerobic fermentation.

This review covers recent advances in the fixing of dinitrogen in microbial bioelectrochemical systems (BES) where bacteria release or accept electron to/from electrodes for their respiratory metabolism, either directly or indirectly. We discuss how BES may be interesting platforms for producing ammonium or biomass from N2 fixation. The potential for N2-fixation in BES is first discussed with a focus on possible metabolism and different mechanism that may lead to an increase of fixed dinitrogen. We then review recent examples where dinitrogen is fixed at the cathodes of BES, either by pure cultures of hydrogenotrophic and/or diazotrophic bacteria using cathodic H2 or reduced redox mediators as the electron, or by mixed enriched consortia. A section is then devoted to the special case of nitrogen fixation at anodic microbial electrode where organic matter oxidation also occurs. Finally, a comparison of the reported current performance of nitrogen fixation in BES with other biotic (anerobic digestion) or abiotic (Haber-Bosch process, electrochemical N2 reduction) is provided together with a perspective on possible optimization and application of this emerging microbial electrochemical and technological process.

The impact of a two-stage anaerobic digestion (AD) system integrating a dark fermentation (DF) step prior to AD, referred to as DF-AD2, on the organic matter (OM) quality and the microbial communities in anaerobic digestates was investigated. Two treatment routes (one-stage AD (AD1) and DF-AD2) were compared by advanced characterization using the same feedstock and treatment duration. The DF-AD2 improved the percentage of CH4 during AD2 by 8.3 % and the volatile solids removal by 6.8 % compared to AD1. The DF step increased the dissolved OM and mineralized nitrogen after AD despite similar OM complexity and predicted carbon mineralization in soils. Moreover, respirometry tests related the enhanced bioaccessibility of DF effluent to greater biological activity (126.3 ± 5.8 mg O2) compared to the substrate (51.1 ± 5.8 mg O2). Nonetheless, DF-AD2 did not impact the biological stability of digestates (32.09 ± 1.1 and 30.2 ± 1.5 mg O2 for AD1 and DF-AD2, respectively). Low-stress operational conditions of the tests might smooth the DF-AD2 effect on digestate biological stability and microbial communities. Archaea varied after DF but homogenized during AD2, with the genus Methanosarcina comprising 71-79 % of the relative abundance. Concurrently, the orders Bacteroidales, Spirochaetales and Cloacimonadales dominated Bacteria in both AD1 and AD2. Overall, this study evidence that a DF-AD2 system is a feasible way to improve both OM removal and the nitrogen fertilizing value of digestates, without hindering digestate biological stability or microbial communities. However, optimizing operational parameters and pre-treatment processes may be necessary to enhance the system's energy output.

This study focuses on thermal and non-thermal effects of microwave pretreatment on horse dung as indigenous inoculum before xylose and wheat straw fermentation, emphasizing metabolite production and microbial community changes. Two horse dung microwave pretreatments (MW40°C and MW95°C) were compared with a conventional thermal pretreatment (Th95°C) and an unpretreated condition (Ctrl). Microwave heating pretreatment (MW95°C) leads to similar production as a conventional thermal pretreatment (Th95°C) for xylose and wheat straw fermentation while MW40°C treatment was similar to the control. The two thermal pretreatments (MW95°C and Th95°C) produced respectively 19 % and 45 % more than the Ctrl and MW40°C conditions. Nonetheless, for wheat straw, microwave irradiation without heating affected the microbial community by increasing the alpha diversity richness. The Ruminiclostridium genus, known for its hydrolytic activity, represented at least 25 % of the total relative abundance after fermentation with thermal shock pretreatment, potentially explaining the enhancement of the metabolite production from straw.

This study aimed to investigate the influence of microwave pretreatment and fermentation parameters on acidogenic fermentation of wheat straw and horse manure. Four different inoculation strategies were tested on wheat straw fermentation, including horse dung, cow dung, aerobic sludge, and wheat straw indigenous inoculum at two different pH (6 and 10). The best yield was achieved with horse dung, reaching 0.172 ± 0.005 gCOD/gVS after seven days at pH 6. However, for horse manure fermentation, lignocellulosic substrate suitable for fermentation without external inoculation, a pretreatment was needed to deactivate methanogenic archaea and solubilize sugars. Microwave parameters such as power (200 or 1000 W), liquid/solid (L/S) ratio (6 or 17) and energy (7 Wh/gVS or 12 Wh/gVS) were assessed on substrate solubilization and metabolic yield. The best pretreatment condition, at 200 W for 15 min with a L/S ratio of 17, led to a metabolic yield of 0.134 ± 0.029 g COD/gVS after seven days. This condition suppressed methanogenic activity while increasing soluble sugars and Chemical Oxygen Demand (COD) by 44 % and 58 %, respectively. Finally, increasing volatile solid (VS) concentration during batch fermentation from 14 to 28 gVS/L of horse manure led to a significant metabolic profile change. Butyrate production was quadrupled for the 200 W pretreatment while total metabolite yield was unchanged. Overall, this study demonstrates that microwave pretreatment is an effective strategy to inhibit methanogenesis and enhance substrate solubilization, without significantly promoting volatile fatty acid production. In addition, modifying VS concentration can induce a notable metabolic shift.

In modelling of complex organic substrates like sugarcane vinasse, glucose is assumed as the sole sugar source, despite the fact that it contains glucose and sucrose. While both substrates produce similar byproducts, the literature reports glucose fermentation as more stable. Sucrose fermentation is known for its instability, leading to a decline in volumetric hydrogen productivity, often attributable to the occurrence of the Wood-Ljungdahl metabolic pathway. This study evaluated the feasibility of modelling dark fermentation (DF) of mono- and disaccharide in an anaerobic fixed-bed reactor (AFBR) using a unified dynamic model. A 3.5 L AFBR was operated for 60 days, under mesophilic conditions (25 degrees C) with an initial concentration of 2 kg COD & sdot;m- 3. The proposed model was calibrated using glucose and validated using sucrose. To represent the retention of microorganisms, a fixation term (alpha) was added to simulate it as a homogenous bioreactor. Statistical analysis showed that the model accurately predicted the fermentation of both carbohydrates. Consequently, the model was applied to simulate the DF of sugarcane vinasse. This confirms that glucose can be treated as the primary carbohydrate in mathematical models for sugar-rich substrates, without compromising the model's accuracy. Differences in hydrogen production from both substrates were attributed to mass transfer limitations.

Food waste fermentation offers a promising approach for the production of volatile fatty acids (VFAs). However, controlling the VFA profile, particularly enriching propionic acid, remains a challenge due to the complexity of microbial communities involved. This study investigates the influence of key operating parameters-initial pH, temperature, and substrate concentration-on VFA production and microbial community dynamics. Batch fermentation experiments using reconstituted food waste were conducted under varying conditions. The highest propionic acid selectivity was achieved at an initial pH of 9, a temperature of 35 degrees C, and a substrate concentration of 7.8 g VS/L, with statistically significant improvement over other conditions. Microbial community analysis based on 16S rDNA sequencing revealed distinct profiles shaped by the operational settings. Notably, increased relative abundances of Enterobacteriaceae, Lachnospiraceae, and Exiguobacterium spp. were associated with higher propionic acid production. These results highlight the strong interplay between fermentation conditions, microbial ecology, and metabolite profiles, providing insights for optimising food waste valorisation towards selective VFA production.

Horse manure is a lignocellulosic biomass found in significant quantities with a vast indigenous flora, not yet fully valorized apart from anaerobic digestion. Its use in the fermentation process can lead to the production of higher-value metabolites. This study investigates three inoculation strategies coupled with five pretreatment conditions for horse manure fermentation. Two microwave pretreatments (200W and 1000W) were compared with a conventional thermal pretreatment, a thermo-acid pretreatment, and an unpretreated condition. The sole horse manure indigenous microorganisms were used in fermentation and compared with two inoculation strategies using external inoculum, which was i) thermally treated or ii) pretreated simultaneously with manure. A statistically similar total metabolite production (0.088 ± 0.010 gCOD/gVS) was observed, with more than 50 % of acetate produced for all the pretreated conditions. When no pretreatment was performed (Ctrl), methane was produced as a major metabolite. The metabolic profile of the thermo-acid pretreatment condition using solely indigenous microorganisms was different from the other conditions, with ethanol (0.015 ± 0.004 gCOD/gVS) and hydrogen (0.009 ± 0.002 gCOD/gVS) production. This was related to the Klebsiella genus abundance increase recorded for this condition. Both microwave pretreatments shared similar metabolite results and microbial composition with the conventional thermal pretreatment. However, a heat shock is needed to inhibit methane production from archaea and can be performed by microwave or conventional thermal pretreatment. To conclude, indigenous horse manure microorganisms are suitable for fermentation with equivalent yields compared to an external inoculum from a wastewater treatment plant whatever the pretreatment applied. However, a heat shock is needed to inhibit methane production from archaea and can be performed via microwave or conventional thermal pretreatment.