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2,3-Butanediol (2,3-BD) is a highly valued building block, and optimizing its production by fermentation, particularly with crude glycerol, is crucial. Enterobacter aerogenes is a key microorganism for this process; however, there are limited studies addressing the inhibition effects of products and by-products on 2,3-BD production. This study investigates these inhibition effects to maximize 2,3-BD production. Final concentrations of 2,3-BD plus acetoin reached 89.3, 92.7, and 71.1 g.L-1 with productivities of 1.22, 1.69, and 0.99 g.L-1.h-1 in pure glycerol, glucose, and crude glycerol media, respectively. Acetic acid was the main by-product, with concentrations ranging from 10 to 15 g.L-1. The reinoculation of E. aerogenes cells highlighted the strong effect of 2,3-BD and acetic acid on microbial growth and metabolism, with the cultivation environment exerting selective pressure. Notably, cells reuse enhanced performance in crude glycerol media, achieving a specific productivity in relation to biomass (YP/X) of 9.18 g.g-1; about 25% higher than in fed-batch without cells reuse. By combining results from two fed-batch cycles, the total final concentration of 2,3-BD plus acetoin reached 99.4 g.L-1, alongside a 33% reduction in total acetic acid production with reused cells.

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S-Adenosylmethionine (SAM) is a crucial metabolic intermediate playing irreplaceable roles in organismal activities. However, the synthesis of SAM by methionine adenosyltransferase (MAT) is hindered by low conversion due to severe product inhibition. Herein structure-guided semirational engineering was conducted on MAT from Escherichia coli (EcMAT) to mitigate the product inhibitory effect. Compared with the wild-type EcMAT, the best variant E56Q/Q105R exhibited an 8.13-fold increase in half maximal inhibitory concentration and a 4.46-fold increase in conversion (150 mM ATP and l-methionine), leading to a SAM titer of 47.02 g/L. Another variant, E56N/Q105R, showed superior thermostability with an impressive 85.30-fold increase in half-life (50 °C) value. Furthermore, molecular dynamics (MD) simulation results demonstrate that the alleviation in product inhibitory effect could be attributed to facilitated product release. This study offers molecular insights into the mitigated product inhibition, and provides valuable guidance for engineering MAT toward enhanced catalytic performance.

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Lactic acid (LA) production from microbial fermentation using low-cost renewable sources has emerged as an attractive alternative to the use of petroleum-based products. This approach not only offers sustainable solutions for waste management but also enables the production of value-added products in an eco-friendly manner. However, to make this approach economically viable, optimizing the production process for high yield, productivity, and purity while minimizing costs is crucial. To address these challenges, various approaches have been proposed, including the use of neutralizing agents, high cell density cultures, co-cultures, fed-batch fermentation, and product removal strategies. Overall, this review underscores the potential of microbial fermentation for LA production as a sustainable and cost-effective solution to meet the growing demand for eco-friendly products. Further optimization of fermentation processes and the development of new microbial strains and fermentation techniques are key to advancing this approach. The production of LA through microbial fermentation presents a sustainable and eco-friendly solution to the increasing demand for eco-friendly products. With continued innovation, we can expect to see a significant reduction in the environmental impact of industrial processes, coupled with a more cost-effective and high-purity source of lactic acid for various industries.

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Blood coagulation is a network of biochemical reactions wherein dozens of proteins act collectively to initiate a rapid clotting response. Coagulation reactions are lipid-surface dependent, and this dependence is thought to help localize coagulation to the site of injury and enhance the association between reactants. Current mathematical models of coagulation either do not consider lipid as a variable or do not agree with experiments where lipid concentrations were varied. Since there is no analytic rate law that depends on lipid, only apparent rate constants can be derived from enzyme kinetic experiments. We developed a new mathematical framework for modeling enzymes reactions in the presence of lipid vesicles. Here the concentrations are such that only a fraction of the vesicles harbor bound enzymes and the rest remain empty. We call the lipid vesicles with and without enzyme TF:VIIa+ and TF:VIIa- lipid, respectively. Since substrate binds to both TF:VIIa+ and TF:VIIa- lipid, our model shows that excess empty lipid acts as a strong sink for substrate. We used our framework to derive an analytic rate equation and performed constrained optimization to estimate a single, global set of intrinsic rates for the enzyme-substrate pair. Results agree with experiments and reveal a critical lipid concentration where the conversion rate of the substrate is maximized, a phenomenon known as the template effect. Next, we included product inhibition of the enzyme and derived the corresponding rate equations, which enables kinetic studies of more complex reactions. Our combined experimental and mathematical study provides a general framework for uncovering the mechanisms by which lipid mediated reactions impact coagulation processes.

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Sophorolipid (SL) production by Candida catenulata from sunflower fatty acids was studied in a bubble column reactor (BCR). The specific oxygen uptake rate was 0.021 mg gcell-1 min-1 which indicates the importance of aeration in SL biosynthesis. The measurement of oxygen transfer rate (OTR) in the BCR showed a satisfactory OTR value of about 0.093 min-1 in the system. However, further SL production was stopped after 30 h in the BCR mainly due to the product accumulation in the culture and its inhibitory effects on cell growth and SL synthesis. Since an extensive foam was generated in the BCR under the absence of an antifoam agent, the development of an in situ foam recovery system provided the integration of production and separation of SL to handle the problem. The application of the foam recovery system enhanced biomass and titer SL concentration by 38.5 and 28.2% in comparison with the conventional BCR, respectively. Further studies in the system were performed by monitoring the size of bubbles and their effects on the biomass and SL enrichment in the foam stream at different aeration rates where the SL enrichment varied from 900 to 100% at 12 and 50 h of the fermentation.

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Protocatechuic acid (3, 4-dihydroxybenzoic acid, PCA) is widely used in the pharmaceuticals, health food, and cosmetics industries owing to its diverse biological activities. However, the inhibition of 3-dehydroshikimate dehydratase (AroZ) by PCA and its toxicity to cells limit the efficient production of PCA in Escherichia coli. In this study, a high-level strain of 3-dehydroshikimate, E. coli DHS01, was developed by blocking the carbon flow from the shikimate-overproducing strain E. coli SA09. Additionally, the PCA biosynthetic pathway was established in DHS01 by introducing the high-activity ApAroZ. Subsequently, the protein structure and catalytic mechanism of 3-dehydroshikimate dehydratase from Acinetobacter pittii PHEA-2 (ApAroZ) were clarified. The variant ApAroZR363A, achieved by modulating the conformational dynamics of ApAroZ, effectively relieved product inhibition. Additionally, the tolerance of the strain E. coli PCA04 to PCA was enhanced by adaptive laboratory evolution, and a biosensor-assisted high-throughput screening method was designed and implemented to expedite the identification of high-performance PCA-producing strains. Finally, in a 5 L bioreactor, the final strain PCA05 achieved the highest PCA titer of 46.65 g/L, a yield of 0.23 g/g, and a productivity of 1.46 g/L/h for PCA synthesis from glucose using normal fed-batch fermentation. The strategies described herein serve as valuable guidelines for the production of other high-value and toxic products.

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The first and committed step in proline synthesis from glutamate is catalyzed by δ1 -pyrroline-5-carboxylate synthetase (P5CS). Two P5CS genes have been found in most angiosperms, one constitutively expressed to satisfy proline demand for protein synthesis, the other stress-induced. Despite the number of papers to investigate regulation at the transcriptional level, to date, the properties of the enzymes have been subjected to limited study. The isolation of Arabidopsis thaliana P5CS isoenzymes was achieved through heterologous expression and affinity purification. The two proteins were characterized with respect to kinetic and biochemical properties. AtP5CS2 showed KM values in the micro- to millimolar range, and its activity was inhibited by NADP+ , ADP and proline, and by glutamine and arginine at high levels. Mg2+ ions were required for activity, which was further stimulated by K+ and other cations. AtP5CS1 displayed positive cooperativity with glutamate and was almost insensitive to inhibition by proline. In the presence of physiological, nonsaturating concentrations of glutamate, proline was slightly stimulatory, and glutamine strongly increased the catalytic rate. Data suggest that the activity of AtP5CS isoenzymes is differentially regulated by a complex array of factors including the concentrations of proline, glutamate, glutamine, monovalent cations and pyridine dinucleotides.

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In order to further understand the effect of product inhibition on the metabolism of hydrogen production bacteria, and to seek an effective way to increase the hydrogen yield in fermentation, a simplified metabolic model of Ethanoligenens harbinense B49 was constructed to analyse the metabolic flux under acetate and ethanol inhibition separately and to analyse the flux changes of the nodes. Based on the changes in metabolic flux distribution, Glucose 6-phosphate (G6P), Pyruvate (PYR), and Acetyl-CoA (AcCoA) were identified as key nodes of hydrogen production in the metabolic network. Robustness analysis showed that G6P was flexible, while AcCoA and PYR were weakly rigid, indicating that acetate flux could be increased by adding inhibitors or using genetic manipulation. Furthermore, releasing inhibition of acetate could effectively increase hydrogen production. These findings suggested that the addition of acetate separation in ethanol-type fermentation process is expected to improve hydrogen production, which might be a promising way to full-scale produce biohydrogen in industrial applications. Further, for the first time, we report the effect of product inhibition on key nodes in the E. harbinense B49 hydrogen production metabolism network.

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Product inhibition caused by organic acids is a serious issue in establishing economical biochemical production systems. Herein, for enhanced production of glutaric acid by overcoming product inhibition triggered by glutaric acid, a whole-cell bioconversion system equipped with biocatalyst recycling process and in situ product recovery by adsorption was developed successfully. From the whole-cell bioconversion reaction, we found that both dissociated and undissociated forms of glutaric acid acted as an inhibitor in the whole-cell bioconversion reaction, wherein bioconversion was hindered beyond 200 mM glutaric acid regardless of reaction pH. Therefore, as the promising solution for the inhibition issue by glutaric acid, the biocatalyst-recycled bioconversion process integrated with in situ product recovery by adsorption was introduced in the whole-cell bioconversion. As a result, 592 mM glutaric acid was produced from 1000 mM 5-aminovaleric acid with 59.2% conversion. We believe that our system will be a promising candidate for economically producing organic acids with high titer.

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This chapter describes methods for cultivation and characterization of the growth of Mycolicibacterium spp. mutants in a microbioreactor system in the presence of steroids and/or phytosterols followed by high-throughput mass spectrometry analysis to describe their ability to convert phytosterols into the target steroid androstenedione (AD). We focus on Mycolicibacterium neoaurum NRRL B-3805 ΔkstD which can convert phytosterol into androstenedione (AD) as one of its major steroid products, and mutants thereof with increased tolerance towards this end-product. By using BioLector 48-well plates with optodes at the bottom of each well, bacterial growth can be monitored online despite the turbidity of the growth medium resulting from non-dissolved phytosterol and steroid particles. To cope with the large number of samples that accumulate during growth experiments in microbioreactors and similar formats (e.g., microtiter plates), protocols for extraction and subsequent RapidFire-MS analysis are presented. This reduces the analysis time per sample to 10 s from 10 min required for regular LC-MS analysis.

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Glucose mother liquor (GML) is a byproduct of the glucose (G1) crystallization process. However, the presence of maltooligosaccharides and isomaltooligosaccharides within GML imposes limitations on its reutilization. Furthermore, the high concentration of G1 in GML leads to product inhibition of G1-producing enzymes. To overcome these challenges, a variant enzyme called V219A was developed through genetic mutation. The V219A exhibits the ability to hydrolyze both maltooligosaccharides and isomaltooligosaccharides. Product inhibition kinetics showed that the IC50 value of V219A was 7 times higher than that of the wild type. Upon subjecting primary, secondary, and tertiary GML to treatment with V219A, the G1 content exhibited notable increases, reaching 96.88, 95.70, and 90.46%, respectively. These significant findings not only establish an innovative and environmentally conscious approach for G1 production from GML but also provide a promising strategy for enzyme construction that caters to the demands of industrial-scale production.

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ω-transaminase has attracted growing attention for chiral amine synthesis, although it commonly suffers from severe by-product inhibition. ω-transaminase CrmG is critical for the biosynthesis of Caerulomycin A, a natural product that possesses broad bioactivity, including immunosuppressive and anti-cancer. Compared to L-Arg, amino donor L-Glu, L-Gln or L-Ala is more preferred by CrmG. In this study, we determined the crystal structure of CrmG in complex with amino donor L-Arg, unveiling the detailed binding mode. Specifically, L-Arg exhibits an extensive contact with aromatic residues F207 and W223 on the roof of CrmG active site via cation-π network. This interaction may render the deamination by-product of L-Arg to be an inhibitor against PMP-bound CrmG by stabilizing its flexible roof, thus reducing the reactivity of L-Arg as an amino donor for CrmG. These data provide further evidence to support our previous proposal that CrmG can overcome inhibition from those by-products that are not able to stabilize the flexible roof of active site in PMP-bound CrmG. Thus, our result can not only facilitate the biosynthesis of CRM A but also be beneficial for the rational design of ω-transaminase to bypass by-product inhibition.

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Almost half of the people who die from sudden cardiac arrest have no detectable heart disease. Among children and young adults, the cause of approximately one-third of deaths from sudden cardiac arrest remains unexplained after thorough examination. Sudden cardiac arrest and related sudden cardiac death are attributed to dysfunctional cardiac ion-channels. The present perspective paper proposes a pathophysiological mechanism by which phosphate toxicity from cellular accumulation of dysregulated inorganic phosphate interferes with normal calcium handling in the heart, leading to sudden cardiac arrest. During cardiac muscle relaxation following contraction, SERCA2a pumps actively transport calcium ions into the sarcoplasmic reticulum, powered by ATP hydrolysis that produces ADP and inorganic phosphate end products. Reviewed evidence supports the proposal that end-product inhibition of SERCA2a occurs as increasing levels of inorganic phosphate drive up phosphate toxicity and bring cardiac function to a sudden and unexpected halt. The paper concludes that end-product inhibition from ATP hydrolysis is the mediating factor in the association of sudden cardiac arrest with phosphate toxicity. However, current technology lacks the ability to directly measure this pathophysiological mechanism in active myocardium, and further research is needed to confirm phosphate toxicity as a risk factor in individuals with sudden cardiac arrest. Moreover, phosphate toxicity may be reduced through modification of dietary phosphate intake, with potential for employing low-phosphate dietary interventions to reduce the risk of sudden cardiac arrest.

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PERIOD (PER) and Casein Kinase 1δ regulate circadian rhythms through a phosphoswitch that controls PER stability and repressive activity in the molecular clock. CK1δ phosphorylation of the familial advanced sleep phase (FASP) serine cluster embedded within the Casein Kinase 1 binding domain (CK1BD) of mammalian PER1/2 inhibits its activity on phosphodegrons to stabilize PER and extend circadian period. Here, we show that the phosphorylated FASP region (pFASP) of PER2 directly interacts with and inhibits CK1δ. Co-crystal structures in conjunction with molecular dynamics simulations reveal how pFASP phosphoserines dock into conserved anion binding sites near the active site of CK1δ. Limiting phosphorylation of the FASP serine cluster reduces product inhibition, decreasing PER2 stability and shortening circadian period in human cells. We found that Drosophila PER also regulates CK1δ via feedback inhibition through the phosphorylated PER-Short domain, revealing a conserved mechanism by which PER phosphorylation near the CK1BD regulates CK1 kinase activity.

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Self-replication of nucleic acids in the absence of enzymes represents an important and poorly understood step in the origin of life as such reported systems are strongly hindered by product inhibition. Studying one of the few successful examples of enzymatic DNA self-replication based on a simple ligation chain reaction, lesion-induced DNA amplification (LIDA), can shed light on how this fundamental process may have originally evolved. To identify the unknown factors that lead LIDA to overcome product inhibition we have employed isothermal titration calorimetry and global fitting of time-dependent ligation data to characterize the individual steps of the amplification process. We find that incorporating the abasic lesion into one of the four primers substantially decreases the stability difference between the product and intermediate complexes compared with complexes without the abasic group. In the presence of T4 DNA ligase this stability gap is further reduced by two orders of magnitude revealing that the ligase also helps overcome product inhibition. Kinetic simulations reveal that the intermediate complex stability and the magnitude of the ligation rate constant significantly impact the rate of self-replication, suggesting that catalysts that both facilitate ligation and stabilize the intermediate complex might be a route to efficient nonenzymatic replication.

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Many biotechnology applications tend to be for low production volumes and relatively high-value products such as insulin and vaccines. More difficult to perfect at scale are bioprocesses for high-volume products with lower value, especially if the target product is a reduced chemical such as a solvent or a plastic. Historically, industrial microbiology succeeded under special circumstances when fossil feedstocks were either unavailable or expensive. Inevitably, as these circumstances relaxed, bioprocesses struggled to compete with petrochemistry. Why try to compete? Fossil resources will be phased out in the coming decades in the struggle with climate change. To reach net-zero carbon by 2050 will require all sectors to transition, not only energy and transportation. This may herald a new opportunity for industrial bioprocesses with much better tools.

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Efficient waste management is necessary to transition towards a more sustainable society. An emerging trend is to use mixed culture biotechnology to produce chemicals from organic waste. Insights into the metabolic interactions between community members and their growth characterization are needed to mediate knowledge-driven bioprocess development and optimization. Here, a granular sludge bioprocess for the production of caproic acid through sugar-based chain elongation metabolism was established. Lactic acid and chain-elongating bacteria were identified as the two main functional guilds in the granular community. The growth features of the main community representatives (isolate Limosilactobacillus musocae G03 for lactic acid bacteria and type strain Caproiciproducens lactatifermentans for chain-elongating bacteria) were characterized. The measured growth rates of lactic acid bacteria (0.051 ± 0.005 h-1) were two times higher than those of chain-elongating bacteria (0.026 ± 0.004 h-1), while the biomass yields of lactic acid bacteria (0.120 ± 0.005 g biomass/g glucose) were two times lower than that of chain-elongating bacteria (0.239 ± 0.007 g biomass/g glucose). This points towards differential growth strategies, with lactic acid bacteria resembling that of a r-strategist and chain-elongating bacteria resembling that of a K-strategist. Furthermore, the half-saturation constant of glucose for L. mucosae was determined to be 0.35 ± 0.05 g/L of glucose. A linear trend of caproic acid inhibition on the growth of L. mucosae was observed, and the growth inhibitory caproic acid concentration was predicted to be 13.6 ± 0.5 g/L, which is the highest reported so far. The pre-adjustment of L. mucosae to 4 g/L of caproic acid did not improve the overall resistance to it, but did restore the growth rates at low caproic acid concentrations (1-4 g/L) to the baseline values (i.e., growth rate at 0 g/L of caproic acid). High resistance to caproic acid enables lactic acid bacteria to persist and thrive in the systems intended for caproic acid production. Here, insights into the growth of two main functional guilds of sugar-based chain elongation systems are provided which allows for a better understanding of their interactions and promotes future bioprocess design and optimization.

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Fermentation technology is commonly used as a mature process to produce numerous products with the help of micro-organisms. However, these organisms are sometimes inhibited by the accumulation of these products or their by-products. One route to circumvent this is via extractive fermentation, which combines the fermentation process with extraction. To facilitate this, novel bioreactor designs are required, such as the semi-partition bioreactor (SPB) which has been recently proposed for in-situ extractive fermentation. The latter combines a fermentation and an extraction unit into a single vessel using a mixer-settler principle. Where the bioproduct is produced in the mixer and removed continuous in the settler. As the SPB functionality is a subject of interest, this study builds on demonstrating different process conditions in the production of a sample bioprocess (lactic acid (LA)) which is susceptible to product inhibition. The results showed a 34.5 g/L LA concentration was obtained in the pH-controlled condition. While LA production can suffer from product inhibition, neutralizing agents can be easily used to curb inhibitory problems, however, the LA fermentation is a simple (and well-studied) example, which can demonstrate an alternative route to avoiding product inhibition (for systems which cannot be rescued using pH control). Hence, to replicate a scenario of product inhibition, two different process conditions were investigated, no pH control with no extraction (non-integrated), and no pH control with integrated extractive fermentation. Key findings showed higher LA concentration in integrated (25.10 g/L) as compared to the non-integrated (14.94 g/L) case with improved yield (0.75 gg-1 (integrated) versus 0.60 gg-1 (non-integrated)) and overall productivity (0.35 gL-1h-1(integrated) versus 0.20 gL-1h-1(non-integrated)) likewise. This is the first demonstration of an SP bioreactor, and shows how the reactor can be applied to improve productivity. Based on these results, the SPB design can be applied to produce any product liable to product inhibition.

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BACKGROUND: Malic acid, a dicarboxylic acid mainly used in the food industry, is currently produced from fossil resources. The utilization of low-cost substrates derived from biomass could render microbial processes economic. Such feedstocks, like lignocellulosic hydrolysates or condensates of fast pyrolysis, can contain high concentrations of acetic acid. Acetate is a suitable substrate for L-malic acid production with the filamentous fungus Aspergillus oryzae DSM 1863, but concentrations obtained so far are low. An advantage of this carbon source is that it can be used for pH control and simultaneous substrate supply in the form of acetic acid. In this study, we therefore aimed to enhance L-malate production from acetate with A. oryzae by applying a pH-coupled feeding strategy. RESULTS: In 2.5-L bioreactor fermentations, several feeding strategies were evaluated. Using a pH-coupled feed consisting of 10 M acetic acid, the malic acid concentration was increased about 5.3-fold compared to the batch process without pH control, resulting in a maximum titer of 29.53 ± 1.82 g/L after 264 h. However, it was not possible to keep both the pH and the substrate concentration constant during this fermentation. By using 10 M acetic acid set to a pH of 4.5, or with the repeated addition of NaOH, the substrate concentration could be maintained within a constant range, but these strategies did not prove beneficial as lower maximum titers and yields were obtained. Since cessation of malic acid production was observed in later fermentation stages despite carbon availability, a possible product inhibition was evaluated in shake flask cultivations. In these experiments, malate and succinate, which is a major by-product during malic acid production, were added at concentrations of up to 50 g/L, and it was found that A. oryzae is capable of organic acid production even at high product concentrations. CONCLUSIONS: This study demonstrates that a suitable feeding strategy is necessary for efficient malic acid production from acetate. It illustrates the potential of acetate as carbon source for microbial production of the organic acid and provides useful insights which can serve as basis for further optimization.

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Microbial electrosynthesis (MES) of acetate is a process using electrical energy to reduce CO2 to acetic acid in an integrated bioelectrochemical system. MES powered by excess renewable electricity produces carbon-neutral acetate while benefitting from inexpensive but intermittent energy sources. Interruptions in electricity supply also cause energy limitation and starvation of the microbial cells performing MES. Here, we studied the effect of intermittent electricity supply on the performance of hydrogen-mediated MES of acetate. Thermoanaerobacter kivui produced acetic acid for more than 4 months from intermittent electricity supplied in 12 h on-off cycles in a semicontinuously-fed MES system. After current interruptions, hydrogen utilization and acetate synthesis rates were severely diminished. They did not recover to the steady-state rates of continuous MES within the 12 h current-on period under most conditions. Accumulating high product (acetate) concentration exacerbated this effect and prolonged recovery. However, supply of a low background current of 1-5% of the maximum current during "off-times" reduced the impact of current interruptions on subsequent MES performance. This study presents sustained MES at a rate of up to 2 mM h-1 acetate at an average concentration of 60-90 mM by a pure thermophilic microbial culture powered by intermittent electricity. We identified product inhibition of accumulating acetic acid as a key challenge to improving the efficiency of intermittently powered MES.

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