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Abstract One of the most important biotechnological challenges is to develop environment friendly technologies to produce new sources of energy. Microbial production of biohydrogen through dark fermentation, by conversion of residual biomass, is an attractive solution for short-term development of bioH2 producing processes. Efficient biohydrogen production relies on complex mixed communities working in tight interaction. Species composition and functional traits are of crucial importance to maintain the ecosystem service. The analysis of microbial community revealed a wide phylogenetic diversity that contributes in different—and still mostly unclear—ways to hydrogen production. Bridging this gap of knowledge between microbial ecology features and ecosystem functionality is essential to optimize the bioprocess and develop strategies toward a maximization of the efficiency and stability of substrate conversion. The aim of this review is to provide a comprehensive overview of the most up-to-date biodata available and discuss the main microbial community features of biohydrogen engineered ecosystems, with a special emphasis on the crucial role of interactions and the relationships between species composition and ecosystem service. The elucidation of intricate relationships between community structure and ecosystem function would make possible to drive ecosystems toward an improved functionality on the basis of microbial ecology principles. This review establishes basic knowledge about the taxonomic and metabolic diversity of hydrogen producers in fermentation bioprocesses, and emphasizes the crucial role of interactions between effective producers, negative effectors inhibiting or outcompeting hydrogen production, and positive contributors facilitating the ecosystem function, with the final objective to propose bioengineering strategies and microbial resource management to enhance bioprocess function.

Sugarcane is a lignocellulosic crop which is used to produce sugar in sugarcane processing industries. Globally, sugarcane processing industries generate solid and liquid wastes amounting to more than 279 million tons per annum and by-products; namely, trash, bagasse, mill mud, and molasses. The valorisation of waste and by-products has recently increased and is playing a significant role in achieving policies and goals associated with circular bioeconomy and sustainable development. For the valorisation of sugarcane processing industry waste and by-products, a number of technologies are well established and in use, while other innovative technologies are still ongoing through research and development with promising futures. These by-products obtained from sugarcane processing industries can be converted into biofuels like hydrogen and methane via anaerobic digestion. Molasses belongs to the first-generation (1G) waste, while trash, bagasse, and mill mud belong to second-generation (2G) waste. Various studies have been carried out in converting both first- and second-generation sugarcane processing industry wastes into renewable energy, exploiting anaerobic digestion (AD) and dark fermentation (DF). This review emphasises the various factors affecting the AD and DF of 1G and 2G sugarcane processing industry wastes. It also critically addresses the feasibility and challenges of operating a two-stage anaerobic digestion process for hydrogen and methane production from these wastes.

Knowledge of the behaviour of bacterial communities is crucial for understanding biogeochemical cycles and developing environmental biotechnology. Here we demonstrate the formation of an artificial consortium between two anaerobic bacteria, Clostridium acetobutylicum (Gram-positive) and Desulfovibrio vulgaris Hildenborough (Gram-negative, sulfate-reducing) in which physical interactions between the two partners induce emergent properties. Molecular and cellular approaches show that tight cell–cell interactions are associated with an exchange of molecules, including proteins, which allows the growth of one partner ( D. vulgaris ) in spite of the shortage of nutrients. This physical interaction induces changes in expression of two genes encoding enzymes at the pyruvate crossroads, with concomitant changes in the distribution of metabolic fluxes, and allows a substantial increase in hydrogen production without requiring genetic engineering. The stress induced by the shortage of nutrients of D. vulgaris appears to trigger the interaction. Bacterial communities adapt to changing environments by modulating patterns of nutrient flow between species. Benomar et al . show that under nutrient stress, the sulfate-reducing bacterium Desulfovibrio vulgaris can exchange cytoplasmic material with Clostridium acetobutylicum , altering metabolic flux.

End-product accumulation during dark fermentation leads to process instability and hydrogen production inhibition. To overcome this constraint, microbial community adaptation to butyric acid can induce acid tolerance and thus enhance the hydrogen yields; however, adaptation and selection of appropriate microbial communities remains uncertain when dealing with complex substrates in a continuous fermentation mode. To address this question, a reactor fed in continuous mode with food waste (organic loading rate of 60 gVS·L·d−1; 12 h hydraulic retention time) was first stressed for 48 h with increasing concentrations of butyric acid (up to 8.7 g·L−1). Performances were compared with a control reactor (unstressed) for 13 days. During 6 days in a steady-state, the pre-stressed reactor produced 2.2 ± 0.2 LH2·L·d−1, which was 48% higher than in the control reactor (1.5 ± 0.2 LH2·L·d−1). The pretreatment also affected the metabolites’ distribution. The pre-stressed reactor presented a higher production of butyric acid (+44%) achieving up to 3.8 ± 0.3 g·L−1, a lower production of lactic acid (−56%), and an enhancement of substrate conversion (+9%). The performance improvement was attributed to the promotion of Clostridium guangxiense, a hydrogen -producer, with a relative abundance increasing from 22% in the unstressed reactor to 52% in the stressed reactor.

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.

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 Graphical abstract

Over the past decades, biodiesel production has sharply increased worldwide and has led to an overproduction of glycerol, as by‐product. Therefore, glycerol is not only produced at low cost with a wide availability but is also a versatile precursor of useful value‐added chemicals such as1,3‐propanediol. At an industrial scale, glycerol conversion into 1,3‐propanediol is almost entirely carried out by fermentation processes as they have shown the best economic and environmental performances. The aim of this article is to provide an up‐to‐date state of the art on the fundamentals and fermentation process strategies for the microbial conversion of glycerol into 1,3‐propanediol. Glycerol fermentation metabolism is detailed and strategies concerning microbial inoculum (i.e., pure cultures of natural or genetically modified strains vs. mixed cultures or artificial consortia), process configuration (i.e., batch, fed‐batch and continuous reactors, biomass immobilisation) and related operational parameters (i.e., temperature, pH, oxido‐reduction potential) are discussed for the optimisation of 1,3‐propanediol production by fermentation. This review highlights recent advances in microbial fermentation strategies for converting glycerol, a biodiesel by‐product, into 1,3‐propanediol. It discusses metabolic pathways, microbial inocula and key operational parameters to optimise production, supporting sustainable chemical synthesis aligned with circular economy principles.

Biogas upgrading via biomethanation has been extensively studied recently, but the influence of organic loading rate on process performance remains to be fully understood. This is particularly significant because both organic loading rate and hydrogen injection can lead to volatile fatty acid accumulation during anaerobic digestion. This study investigated the impact of a wide range of organic loading rates (from 1.25 to 3.25 g VS/L/d) on hydrogen consumption rates, organic acid accumulation, and microbial communities during in situ biomethanation. It also provided kinetics data and metabolite production data for different control reactors, including anaerobic digestion, ex situ biomethanation, and endogenous control reactors. Hydrogen was injected into parallel batch reactors using digestate from a semi-continuous lab-scale reactor subjected to increasing organic loading rates (1.25–3.25 g VS/L/d) as an inoculum. The inoculum was well adapted to each tested organic loading rate. The batch experiments were replicated following a 12 h hydrogen starvation period to assess the stability of hydrogen consumption rates. High organic loading rate values resulted in increased hydrogen consumption rates, peaking at 68 mg COD/L/h at an organic loading rate of 3.25 g VS/L/d (maximum value tested), with no significant organic acid accumulation despite the high hydrogen partial pressures. The hydrogen consumption rates were maintained after the starvation period. Furthermore, the addition of an organic substrate did not impact the hydrogen consumption rate (i.e., the in situ and ex situ rates were similar). A higher organic loading rate resulted in higher relative abundances of hydrogenotrophic methanogens (i.e., Methanospirillum sp.). This study highlights that increasing the organic loading rate can accelerate the rate of hydrogen consumption during in situ biomethanation, consequently reducing both capital and operational costs.

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.

Extracellular electron transfer (EET) is a mechanism that allows energetic coupling between two microorganisms or between a microorganism and an electrode surface. EET is either supported by direct physical contacts or mediated by electron shuttles. So far, studies dealing with interspecies EET (so-called IET) have mainly focused on possible syntrophic interactions between microorganisms favoured by this mechanism. In this article, the case of fermentative bacteria receiving extracellular electrons while fermenting a substrate is considered. A thermodynamical analysis based on metabolic energy balances was applied to re-investigate experimental data from the literature. Results suggest that the observations of a decrease of cell biomass yields of fermentative electron-accepting species, as mostly reported, can be unravelled by EET energetics and correspond to parasitism in case of IET. As an illustration, the growth yield decrease of Propionibacterium freudenreichii (−14%) observed in electro-fermentation experiments was fully explained by EET energetics when electrons were used by this species at a potential of −0.12 ± 0.01 V vs SHE. Analysis of other cases showed that, in addition to EET energetics in Clostridium pasteurianum , biological regulations can also be involved in such biomass yield decrease (−33% to −38%). Interestingly, the diminution of bacterial biomass production is always concomitant with an increased production of reduced compounds making IET-mediated parasitism and electro-fermentation attractive ways to optimize carbon fluxes in fermentation processes.