Carbon chain elongation characterizations of electrode-biofilm microbes in electro-fermentation.

The higher efficiency of electro-fermentation in synthesizing medium-chain fatty acids (MCFAs) compared to traditional fermentation has been acknowledged. However, the functional mechanisms of electrode-biofilm enhancing MCFAs synthesis remain research gaps. To address this, this study proposed a continuous flow electrode-biofilm reactor for chain elongation (CE). After 225 days of operation, stable electrode-biofilms formed and notably improved caproate yield by more than 38 %. The electrode-biofilm was enriched with more CE microorganisms and electroactive bacteria compared to the suspended sludge microorganisms, including Caproicibacterium, Oscillibacter and Pseudoramibacter. Besides, the upregulated CE pathways were evaluated by metagenomic analysis, and the results indicated that the pathways such as acetyl-CoA and malonyl-[acp] formation, reverse beta-oxidation, and fatty acid biosynthesis pathway were all markedly enhanced in cathodic biofilm, more than anodic biofilm and suspended microorganisms. Moreover, microbial community regulated processes like bacterial chemotaxis, flagellar assembly and quorum sensing, crucial for electrode-biofilm formation. Electron transfer, energy metabolism, and microbial interactions were found to be prominently upregulated in the cathodic biofilm, surpassing levels observed in anodic biofilm and suspended sludge microorganisms, which further enhanced CE efficiency. In addition, the statistical analyses further highlighted key microbial functions and interactions within the cathodic biofilm. Oscillospiraceae_bacterium was identified to be the most active microbe, alongside pivotal roles played by Caproiciproducens_sp._NJN-50, Clostridiales_bacterium, Prevotella_sp. and Pseudoclavibacter_caeni. Eventually, the proposed microbial collaboration mechanisms of cathodic biofilm were ascertained. Overall, this study uncovered the biological effects of the electrode-biofilm on MCFAs electrosynthesis, thereby advancing biochemicals production and filling the knowledge gaps in CE electroactive biofilm reactors.

13C-labelled Glucose Reveals Shifts In Fermentation Pathway During Cathodic Electro-Fermentation With Mixed Microbial Culture.

Cathodic Electro Fermentation (CEF) is an innovative approach to manage the spectrum of products deriving from anaerobic fermentation. Herein, mixed microbial culture fermentation using a ternary mixture containing labelled 13C glucose and non-labelled acetate and ethanol was studied to identify the role of polarization on the metabolic pathways of glucose fermentation. CEF at an applied potential of -700 mV (vs. SHE, Standard Hydrogen Electrode) enhanced the production yield of acetate, propionate, and butyrate (0.90 ± 0.10, 0.22 ± 0.03, and 0.34 ± 0.05 mol/mol; respectively) compared to control tests performed at open circuit potential (OCP) (0.54 ± 0.09, 0.15 ± 0.04 and, 0.21 ± 0.001 mol/mol, respectively). Results indicate that CEF affected the 13C labelled fermented product levels and their fractional 13C enrichments, allowing to establish metabolic pathway models. This work demonstrates that, under cathodic polarization, the abundance of both fully 13C labelled propionate and butyrate isotopomers increased compared to control tests. The effect of CEF is mainly due to intermediates initially produced from the glucose metabolic transformation in the presence of non-labelled acetate and ethanol as external substrates. These findings represent a significant advancement in current knowledge of CEF, which offers a promising tool to control mixed cultures bioprocesses.

Enhanced medium-chain fatty acid production from sewage sludge by combined electro-fermentation and anaerobic fermentation.

Electro-fermentation (EF) was combined with anaerobic fermentation (AF) to promote medium-chain fatty acid (MCFA) from sewage sludge. Results showed that EF at acidification process significantly increased short-chain fatty acid (SCFA) production of by 0.5 times (82.4 mmol C/L). AF facilitated the chain elongation (CE) process by enhancing the SCFA conversion. Combined EF at acidification and AF at CE (EF-AF) achieved the highest MCFA production of 27.9 mmol C/L, which was 20 %-866 % higher than the other groups. Electrochemical analyses showed that enhanced SCFA and MCFA production was accompanied with good electrochemical performance at acidification and CE. Microbial analyses showed that EF-AF promoted MCFA production by enriching electrochemically active bacteria (EAB, Bacillus sp.). Enzyme analyses indicated that EF-AF promoted MCFA production by enriching the functional enzymes involved in Acetyl-CoA formation and the fatty acid biosynthesis (FAB) pathway. This study provided new insights into the production of MCFA from enhanced sewage sludge.

Efficient phosphate and hydrogen recovery from sludge fermentation liquid by sacrificial iron anode in electro-fermentation system.

Electro-fermentation (EF) has been extensively studied for recovering hydrogen and phosphorus from waste activated sludge (WAS), while was limited for the further application due to the low hydrogen yield and phosphorus recovery efficiency. This study proposed an efficient strategy for hydrogen and vivianite recovery from the simulated sludge fermentation liquid by sacrificial iron anode in EF. The optimum hydrogen productivity and the utilization efficiency of short chain fatty acids (SCFAs) reached 45.2 mmol/g COD and 77.6% at 5 d in pH 8. Phosphate removal efficiency achieved at 90.8% at 2 d and the high crystallinity and weight percentage of vivianite (84.8%) was obtained. The functional microbes, i.e., anaerobic fermentative bacteria, electrochemical active bacteria, homo-acetogens and iron-reducing bacteria were highly enriched and the inherent interaction between the microbial consortia and environmental variables was thoroughly explored. This work may provide a theoretical basis for energy/resource recovery from WAS in the further implementation.

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.

Sustainable Production of Biofuels and Biochemicals via Electro-Fermentation Technology.

The energy crisis and climate change are two of the most concerning issues for human beings nowadays. For that reason, the scientific community is focused on the search for alternative biofuels to conventional fossil fuels as well as the development of sustainable processes to develop a circular economy. Bioelectrochemical processes have been demonstrated to be useful for producing bioenergy and value-added products from several types of waste. Electro-fermentation has gained great attention in the last few years due to its potential contribution to biofuel and biochemical production, e.g., hydrogen, methane, biopolymers, etc. Conventional fermentation processes pose several limitations in terms of their practical and economic feasibility. The introduction of two electrodes in a bioreactor allows the regulation of redox instabilities that occur in conventional fermentation, boosting the overall process towards a high biomass yield and enhanced product formation. In this regard, key parameters such as the type of culture, the nature of the electrodes as well as the operating conditions are crucial in order to maximize the production of biofuels and biochemicals via electro-fermentation technology. This article comprises a critical overview of the benefits and limitations of this emerging bio-electrochemical technology and its contribution to the circular economy.

Effect of pH on metabolic pathway shift in fermentation and electro-fermentation of xylose by Clostridium autoethanogenum.

Clostridium autoethanogenum can to convert waste gases (CO2, CO, H2) and xylose from hydrolyzed biomass into acetate, lactate, formate, ethanol and 2,3-butanediol, being a candidate for the transformation of waste streams of lignocellulosic biorefineries. Electro-fermentation (EF) modify the pattern of traditional fermentations resulting in improved product yields as has been shown when using Clostridium strains. The aim of this work was to evaluate the influence of pH on microbial growth and product distribution during fermentation and EF of xylose by C. autoethanogenum DSM10061. Fermentation and EF were carried out in a H-type reactor at three controlled pH: 5.0, 5.5 and 5.8, and at a fixed potential of -600 mV (versus Ag/AgCl) in the EF. The experiments showed that maximum biomass concentration increased as the pH increased in fermentation and EF. In accordance with maximum biomass reached, the highest substrate conversion was observed at pH 5.8 for both systems, with 76.80 % in fermentation and 96.18 % in EF. Moreover, the highest concentrations of acetic acid (1.41 ± 0.07 g L-1) and ethanol (1.45 ± 0.15 g L-1) were obtained at the end of cultures in the EF at pH 5.8. The production of lactic and formic acid decreased by the application of the external potential regardless of the pH value, reaching the lowest productivity at pH 5.8. In contrast, the specific productivity of acetic acid and ethanol was lower in both fermentation and EF at the lowest pH. Furthermore, the presence of 0.06 g L-1 of 2,3-butanediol was only detected in EF at pH 5.8. The results revealed that EF modulated microbial metabolism, which can be explained by a possible increased generation of NADP+/NADPH cofactors, which would redirect the metabolic pathway to more reduced products.

Extracellular electron uptake from a cathode by the lactic acid bacterium Lactiplantibacillus plantarum.

A subset of microorganisms that perform respiration can endogenously utilize insoluble electron donors, such as Fe(II) or a cathode, in a process called extracellular electron transfer (EET). However, it is unknown whether similar endogenous EET can be performed by primarily fermentative species like lactic acid bacteria. We report for the first time electron uptake from a cathode by Lactiplantibacillus plantarum, a primarily fermentative bacteria found in the gut of mammals and in fermented foods. L. plantarum consumed electrons from a cathode and coupled this oxidation to the reduction of both an endogenous organic (pyruvate) and an exogenous inorganic electron acceptor (nitrate). This electron uptake from a cathode reroutes glucose fermentation toward lactate degradation and provides cells with a higher viability upon sugar exhaustion. Moreover, the associated genes and cofactors indicate that this activity is mechanistically different from that one employed by lactic acid bacteria to reduce an anode and to perform respiration. Our results expand our knowledge of the diversity of electroactive species and of the metabolic and bioenergetic strategies used by lactic acid bacteria.

Redox mediators stimulated chain elongation process in fluidized cathode electro-fermentation systems for caproate production.

Medium chain fatty acids (MCFAs), the secondary products of traditional anaerobic fermentation, can be produced via chain elongation (CE), a process often retarded due to the difficulty during interspecies electron transfer (IET). This study employed redox mediators, neutral red (NR), methyl viologen (MV), and methylene blue (MB) as electron shuttles to expedite the electro-fermentation for caproate production by improving IET. Results showed that MV increased the MCFAs production by promoting acetate to ethanol conversion, leading to the highest MCFAs selectivity of 68.73%. While NR was indicated to improve CE by encouraging H2 production, and the biocathode had the highest electrical activity due to the smallest internal resistance and largest capacitance increase of 96% than the control. A higher proportion of Sutterella, Prevotella, and Hydrogenophaga, linked with the H2 mediated interspecies electron transfer (MIET) during CE process, was observed across redox mediators supplied groups compared to the control. The presence of mediators led to an elevated abundance of key enzymes for enhanced CE process and electron transfer. This study provided the perspective of the stimulated electron transfer for improved MCFAs production in electro-fermentation systems.

Superior anodic electro-fermentation by enhancing capacity for extracellular electron transfer.

Anodic electro-fermentation (AEF), where an anode replaces the terminal electron acceptor, shows great promise. Recently a Lactococcus lactis strain blocked in NAD+ regeneration was demonstrated to use ferricyanide as an alternative electron acceptor to support fast growth, but the need for high concentrations of this non-regenerated electron acceptor limits practical applications. To address this, growth of this L. lactis strain, and an adaptively evolved (ALE) mutant with enhanced ferricyanide respiration capacity were investigated using an anode as electron acceptor in a bioelectrochemical system (BES) setup. Both strains grew well, however, the ALE mutant significantly faster. The ALE mutant almost exclusively generated 2,3-butanediol, whereas its parent strain mainly produced acetoin. The ALE mutant interacted efficiently with the anode, achieving a record high current density of 0.81 ± 0.05 mA/cm2. It is surprising that a Lactic Acid Bacterium, with fermentative metabolism, interacts so well with an anode, which demonstrates the potential of AEF.

Membrane engineering of Escherichia coli based on "Building bridges" and "Digging tunnels" to improve electro-fermentation of succinate.

Succinate is the end product of anaerobic metabolism of Escherichia coli, and its over-production needs abundant reducing force. Electro-fermentation (EF) is a novel biotechnology to steer and control fermentative processes by supplying extra electrons. However, E.coli is a non-electroactive strain which needs the support of electron shuttle in EF. Here, membrane engineering strategies of "Building bridges" via screening direct electron transport pathway and "Digging tunnels" via screening membrane porins were developed to improve the transmembrane transport of electron during the cathodic electro-fermentation (CEF). As a result, the total electron quantity during electro-fermentation was increased from 1.21 mmol to 7.90 mmol, and succinate yield was increased by 23.3% when these strategies simultaneously were applied to the succinate candidate E. coli Suc260. Hence, this study provides a reference mode for designing and constructing non-electroactive bacteria for electro-fermentation of reductive metabolites.

Electricity-Driven Microbial Metabolism of Carbon and Nitrogen: A Waste-to-Resource Solution.

Electricity-driven microbial metabolism relies on the extracellular electron transfer (EET) process between microbes and electrodes and provides promise for resource recovery from wastewater and industrial discharges. Over the past decades, tremendous efforts have been dedicated to designing electrocatalysts and microbes, as well as hybrid systems to push this approach toward industrial adoption. This paper summarizes these advances in order to facilitate a better understanding of electricity-driven microbial metabolism as a sustainable waste-to-resource solution. Quantitative comparisons of microbial electrosynthesis and abiotic electrosynthesis are made, and the strategy of electrocatalyst-assisted microbial electrosynthesis is critically discussed. Nitrogen recovery processes including microbial electrochemical N2 fixation, electrocatalytic N2 reduction, dissimilatory nitrate reduction to ammonium (DNRA), and abiotic electrochemical nitrate reduction to ammonia (Abio-NRA) are systematically reviewed. Furthermore, the synchronous metabolism of carbon and nitrogen using hybrid inorganic-biological systems is discussed, including advanced physicochemical, microbial, and electrochemical characterizations involved in this field. Finally, perspectives for future trends are presented. The paper provides valuable insights on the potential contribution of electricity-driven microbial valorization of waste carbon and nitrogen toward a green and sustainable society.

Production of acetone, butanol, and ethanol by electro-fermentation with Clostridium saccharoperbutylacetonicum N1-4.

This manuscript describes the effect of altering the extracellular redox potential during the production of acetone, butanol, and ethanol on a dual chamber H-type microbial fuel cell by fermenting glucose with Clostridium saccharoperbutylacetonicum N1-4. Extracellular redox potential modification was achieved by either supplementing the microbial broth with the redox agent NADH or by poising the cathode potential at -600 mV vs. Ag/AgCl. The addition of NADH was found to foment the production of acetone via fermentation of glucose. The addition of 200 mM of NADH to the catholyte rendered the highest production of acetone (2.4 g L-1), thus outperforming the production of acetone by conventional fermentation means (control treatment) by a factor of 2.2. The experimental evidence gathered here, indicates that cathodic electro-fermentation of glucose favors the production of butanol. When poising the cathode potential at -600 mV vs Ag/AgCl (electro-fermentation), the largest production of butanol was achieved (5.8 g L-1), outperforming the control treatment by a factor of 1.5. The production of ABE solvents and the electrochemical measurements demonstrate the electroactive properties of C. saccharoperbutylacetonicum N1-4 and illustrates the usefulness of bio-electrochemical systems to improve conventional fermentative processes.

Efficient butyrate production from rice straw in an optimized cathodic electro-fermentation process.

Butyrate production from renewable biomass shows great potential against climate change and over-consumption of fossil fuels. Herein, key operational parameters of a cathodic electro-fermentation (CEF) process were optimized for efficient butyrate production from rice straw by mixed culture. The cathode potential, controlled pH and initial substrate dosage were optimized at -1.0 V (vs Ag/AgCl), 7.0 and 30 g/L, respectively. Under the optimal conditions, 12.50 g/L butyrate with yield of 0.51 g/g-rice straw were obtained in batch-operated CEF system. In fed-batch mode, butyrate production significantly increased to 19.66 g/L with the yield of 0.33 g/g-rice straw, but 45.99% butyrate selectivity still needs to be improved in future. Enriched butyrate producing bacteria (Clostridium cluster XIVa and IV) with proportion of 58.75% on the 21st day of the fed-batch fermentation, contributed to the high-level butyrate production. The study provides a promising approach for efficient butyrate production from lignocellulosic biomass.

Highly selective butanol production by manipulating electron flow via cathodic electro-fermentation.

Butanol production by solventogenic Clostridia shows great potential to combat the energy crisis, but is still challenged by low butanol selectivity and high downstream cost. In this study, a novel cathodic electro-fermentation (CEF) system mediated by methyl viologen (MV) was proposed and sequentially optimized to obtain highly selective butanol production. Under the optimal conditions (-0.60 V cathode potential, 0.50 mM MV, 30 g/L glucose), 7.17 ± 0.55 g/L butanol production were achieved with the yield of 0.32 ± 0.02 g/g. With the supplement of 4 g/L butyric acid as co-substrate, butanol production further improved to 13.14 ± 1.14 g/L with butanol yield and selectivity as high as 0.43 ± 0.01 g/g and 90.44 ± 1.66%, respectively. The polarized electrode enabled the unbalanced fermentation towards butanol formation and MV further inhibited hydrogen production, both of which contributed to the high-level butanol production and selectivity. The MV-mediated CEF system is a promising approach for cost-effective bio-butanol production.

Electrochemically mediated bioconversion and integrated purification greatly enhanced co-production of 1,3-propanediol and organic acids from glycerol in an industrial bioprocess.

In this study, we show how electrochemically mediated bioconversion can greatly increase the co-production of 1,3-propanediol and organic acids from glycerol in an industrial bioprocess using a Clostridum pasteurianum mutant. Remarkably, an enhanced butyrate formation was observed due to a weakened butanol pathway of the mutant. This allowed the strain to have a higher ATP generation for an enhanced growth, higher glycerol consumption and PDO production. The PDO titer reached as high as 120.67 g/L at a cathodic current of -400 mA, which is 33% higher than that without electricity, with a concurrent increase of butyric acid by 80%. To fully recover the increased PDO and organic acids, a novel downstream process combining thin film evaporation of PDO and esterification of organic acids with ethanol was developed. This enables the efficient co-production of PDO, ethyl acetate and ethyl butyrate with a high overall carbon use of 87%.

Anode-assisted electro-fermentation with Bacillus subtilis under oxygen-limited conditions.

BACKGROUND: Bacillus subtilis is generally regarded as a ubiquitous facultative anaerobe. Oxygen is the major electron acceptor of B. subtilis, and when oxygen is absent, B. subtilis can donate electrons to nitrate or perform fermentation. An anode electrode can also be used by microorganisms as the electron sink in systems called anodic electro-fermentation. The facultative anaerobic character of B. subtilis makes it an excellent candidate to explore with different electron acceptors, such as an anode. This study aimed to optimise industrial aerobic bioprocesses using alternative electron acceptors. In particular, different end product spectrum of B. subtilis with various electron acceptors, including anode from the electro-fermentation system, was investigated. RESULTS: B. subtilis was grown using three electron acceptors, i.e. oxygen, nitrate and anode (poised at a potential of 0.7 V vs. standard hydrogen electrode). The results showed oxygen had a crucial role for cells to remain metabolically active. When nitrate or anode was applied as the sole electron acceptor anaerobically, immediate cell lysis and limited glucose consumption were observed. In anode-assisted electro-fermentation with a limited aeration rate, acetoin, as the main end product showed the highest yield of 0.78 ± 0.04 molproduct/molglucose, two-fold higher than without poised potential (0.39 ± 0.08 molproduct/molglucose). CONCLUSIONS: Oxygen controls B. subtilis biomass growth, alternative electron acceptors utilisation and metabolites formation. Limited oxygen/air supply enabled the bacteria to donate excess electrons to nitrate or anode, leading to steered product spectrum. The anode-assisted electro-fermentation showed its potential to boost acetoin production for future industrial biotechnology applications.

Clostridium kluyveri enhances caproate production by synergistically cooperating with acetogens in mixed microbial community of electro-fermentation system.

As a chain elongation (CE) model strain, Clostridium kluyveri has been used in the studies of bioaugmentation of caproate production. However, its application in the novel electro-fermentation CE system for bioaugmentation is still unclear. In this study, the CE performances, with or without bioaugmentation and in conventional or electro-fermentation systems were compared. And the mechanism of electrochemical-bioaugmentation by constructing a co-culture of Acetobacterium woodii and Clostridium kluyveri were further verified. Results demonstrated that the bioaugmentation treatments have better CE performance, especially in electro-fermentation system, with a highest caproate concentration of 4.68 g·L-1. Mechanism analysis revealed that C. kluyveri responded to the electric field and emerged synergy with the acetogens, which was proved by the increases of C. kluyveri colonization and the acetogens abundance in biofilm and supported by the co-culture experiment. This study provides a novel insight of microbial synergy mechanism of C. kluyveri during CE bioaugmentation in electro-fermentation system.

Product spectrum analysis and microbial insights of medium-chain fatty acids production from waste biomass during liquor fermentation process: Effects of substrate concentrations and fermentation modes.

Substrate toxicity would limit the upgrading of waste biomass to medium-chain fatty acids (MCFAs). In this work, two fermentation modes of electro-fermentation (EF) and traditional fermentation (TF) with different concentration of liquor fermentation waste (20%, 40%, 60%) were used for MCFAs production as well as mechanism investigation. The highest caproate (4.04 g/L) and butyrate (13.96 g/L) concentrations were obtained by EF at 40% substrate concentration. TF experiments showed that the substrate concentration above 40% severely inhibited ethanol oxidation and products formation. Compared with TF mode, the total substrates consumption and product yields under EF mode were significantly increased by 2.6%-43.5% and 54.0%-83.0%, respectively. Microbial analysis indicated that EF effectively alleviated substrate toxicity and enriched chain elongation bacteria, particularly Clostridium_sensu_stricto 12, thereby promoting ethanol oxidation and products formation. Caproiciproducens tolerated high-concentration substrates to ensure normal lactate metabolism. This study provides a new way to produce MCFAs from high concentration wastewater.

Recent advances in electro-fermentation technology: A novel approach towards balanced fermentation.

Biotransformation of organic substrates via acidogenic fermentation (AF) to high-value products such as C1-C6 carboxylic acids and alcohol serves as platform chemicals for various industrial applications. However, the AF technology suffers from low product titers due to thermodynamic constraints. Recent studies suggest that augmenting AF redox potential can regulate the metabolic pathway and provide seamless electron flow by lowering the activation energy barrier, thus positively influencing the substrate utilization rate, product yield, and speciation. Hence, the augmented AF system with an exogenous electricity supply is termed as electro-fermentation (EF), which has enormous potential to strengthen the fermentation technology domain. Therefore, this critical review systematically discusses the current understanding of EF with a special focus on the extracellular electron transfer mechanism of electroactive bacteria and provides perspectives and research gaps to further improve the technology for green chemical synthesis, sustainable waste management, and circular bio-economy.