Exploring the impact of magnetic fields on biomass production efficiency under aerobic and anaerobic batch fermentation of Saccharomyces cerevisiae.

In this work, the effect of moderate electromagnetic fields (2.5, 10, and 15 mT) was studied using an immersed coil inserted directly into a bioreactor on batch cultivation of yeast under both aerobic and anaerobic conditions. Throughout the cultivation, parameters, including CO2 levels, O2 saturation, nitrogen consumption, glucose uptake, ethanol production, and yeast growth (using OD 600 measurements at 1-h intervals), were analysed. The results showed that 10 and 15 mT magnetic fields not only statistically significantly boosted and sped up biomass production (by 38-70%), but also accelerated overall metabolism, accelerating glucose, oxygen, and nitrogen consumption, by 1-2 h. The carbon balance analysis revealed an acceleration in ethanol and glycerol production, albeit with final concentrations by 22-28% lower, with a more pronounced effect in aerobic cultivation. These findings suggest that magnetic fields shift the metabolic balance toward biomass formation rather than ethanol production, showcasing their potential to modulate yeast metabolism. Considering coil heating, opting for the 10 mT magnetic field is preferable due to its lower heat generation. In these terms, we propose that magnetic field can be used as novel tool to increase biomass yield and accelerate yeast metabolism.

Ethanologenic fermentation by Parageobacillus thermoglucosidasius with continuous hot microbubble gas-stripping.

The increased use of biofuels in place of fossil fuels is one strategy to support the transition to net-zero carbon emissions, particularly in transport applications. However, expansion of the use of 1st generation crops as feedstocks is unsustainable due to the conflict with food use. The use of the lignocellulosic fractions from plants and/or co-products from food production including food wastes could satisfy the demand for biofuels without affecting the use of land and the availability of food, but organisms which can readily ferment all the carbohydrates present in these feedstocks often suffer from more severe bioethanol inhibition effects than yeast. This paper demonstrates the potential of hot gas microbubbles to strip ethanol from a thermophilic fermentation process using Parageobacillus thermoglucosidasius TM333, thereby reducing product inhibition and allowing production to continue beyond the nominal toxic ethanol concentrations of ≤ 2% v/v. Using an experimental rig in which cells were grown in fed-batch cultures on sugars derived from waste bread, and the broth continuously cycled through a purpose-built microbubble stripping unit, it was shown that non/low-inhibitory dissolved ethanol concentrations could be maintained throughout, despite reaching productivities equivalent to 4.7% v/v dissolved ethanol. Ethanol recovered in the condensate was at a concentration appropriate for dewatering to be cost effective and not prohibitively energy intensive. This suggests that hot microbubble stripping could be a valuable technology for the continuous production of bioethanol from fermentation processes which suffer from product inhibition before reaching economically viable titres, which is typical of most thermophilic ethanologenic bacteria.

Eupransor demetentiae gen. nov., sp. nov., a novel fructophilic lactic acid bacterium from bumble bees.

Strain LMG 33000T was isolated from a Bombus lapidarius gut sample. It shared the highest percentage 16S rRNA sequence identity, average amino acid identity, and amino acid identity of conserved genes with Convivina intestini LMG 28291T (95.86 %, 69.9 and 76.2 %, respectively), and the highest percentage OrthoANIu value with Fructobacillus fructosus DSM 20349T (71.4 %). Phylogenomic analyses by means of 107 or 120 conserved genes consistently revealed Convivina as nearest neighbour genus. The draft genome of strain LMG 33000T was 1.44 Mbp in size and had a DNA G+C content of 46.1 mol%. Genomic and physiological analyses revealed that strain LMG 33000T was a typical obligately fructophilic lactic acid bacterium that lacked the adhE and aldh genes and that did not produce ethanol during glucose or fructose metabolism. In contrast, Convivina species have the adhE and aldh genes in their genomes and produced ethanol from glucose and fructose metabolism, which is typical for heterofermentative lactic acid bacteria. Moreover, strain LMG 33000T exhibited catalase activity, an unusual characteristic among lactic acid bacteria, that is not shared with Convivina species. Given its position in the phylogenomic trees, and the difference in genomic percentage G+C content and in physiological and metabolic characteristics between strain LMG 33000T and Convivina species, we considered it most appropriate to classify strain LMG 33000T into a novel genus and species within the Lactobacillaceae family for which we propose the name Eupransor demetentiae gen. nov., sp. nov., with LMG 33000T (=CECT 30958T) as the type strain.

Gaining insights into the responses of individual yeast cells to ethanol fermentation using Raman tweezers and chemometrics.

Saccharomyces cerevisiae is the most common microbe used for the industrial production of bioethanol, and it encounters various stresses that inhibit cell growth and metabolism during fermentation. However, little is currently known about the physiological changes that occur in individual yeast cells during ethanol fermentation. Therefore, in this work, Raman spectroscopy and chemometric techniques were employed to monitor the metabolic changes of individual yeast cells at distinct stages during high gravity ethanol fermentation. Raman tweezers was used to acquire the Raman spectra of individual yeast cells. Multivariate curve resolution-alternating least squares (MCR-ALS) and principal component analysis were employed to analyze the Raman spectra dataset. MCR-ALS extracted the spectra of proteins, phospholipids, and triacylglycerols and their relative contents in individual cells. Changes in intracellular biomolecules showed that yeast cells undergo three distinct physiological stages during fermentation. In addition, heterogeneity among yeast cells significantly increased in the late fermentation period, and different yeast cells may respond to ethanol stress via different mechanisms. Our findings suggest that the combination of Raman tweezers and chemometrics approaches allows for characterizing the dynamics of molecular components within individual cells. This approach can serve as a valuable tool in investigating the resistance mechanism and metabolic heterogeneity of yeast cells during ethanol fermentation.

Transcriptomics to evaluate the influence mechanisms of ethanol on the ester production of Wickerhamomyces anomalus with the induction of lactic acid.

Wickerhamomyces anomalus is one of the most important ester-producing strains in Chinese baijiu brewing. Ethanol and lactic acid are the main metabolites produced during baijiu brewing, but their synergistic influence on the growth and ester production of W. anomalus is unclear. Therefore, in this paper, based on the contents of ethanol and lactic acid during Te-flavor baijiu brewing, the effects of different ethanol concentrations (3, 6, and 9% (v/v)) combined with 1% lactic acid on the growth and ester production of W. anomalus NCUF307.1 were studied and their influence mechanisms were analyzed by transcriptomics. The results showed that the growth of W. anomalus NCUF307.1 under the induction of lactic acid was inhibited by ethanol. Although self-repair mechanism of W. anomalus NCUF307.1 induced by lactic acid was initiated at all concentrations of ethanol, resulting in significant up-regulation of genes related to the Genetic Information Processing pathway, such as cell cycle-yeast, meiosis-yeast, DNA replication and other pathways. However, the accumulation of reactive oxygen species and the inhibition of pathways associated with carbohydrate and amino acid metabolism may be the main reason for the inhibition of growth in W. anomalus NCUF307.1. In addition, 3% and 6% ethanol combined with 1% lactic acid could promote the ester production of W. anomalus NCUF307.1, which may be related to the up-regulation of EAT1, ADH5 and TGL5 genes, while the inhibition in 9% ethanol may be related to down-regulation of ATF2, EAT1, ADH2, ADH5, and TGL3 genes.

Comparative Metabolomics and Microbiome Analysis of Ethanol versus OMNImet/gene•GUT Fecal Stabilization.

Metabolites from feces provide important insights into the functionality of the gut microbiome. As immediate freezing is not always feasible in gut microbiome studies, there is a need for sampling protocols that provide the stability of the fecal metabolome and microbiome at room temperature (RT). Here, we investigated the stability of various metabolites and the microbiome (16S rRNA) in feces collected in 95% ethanol (EtOH) and commercially available sample collection kits with specific preservatives OMNImet•GUT/OMNIgene•GUT. To simulate field-collection scenarios, the samples were stored at different temperatures at varying durations (24 h + 4 °C, 24 h RT, 36 h RT, 48 h RT, and 7 days RT) and compared to aliquots immediately frozen at -80 °C. We applied several targeted and untargeted metabolomics platforms to measure lipids, polar metabolites, endocannabinoids, short-chain fatty acids (SCFAs), and bile acids (BAs). We found that SCFAs in the nonstabilized samples increased over time, while a stable profile was recorded in sample aliquots stored in 95% EtOH and OMNImet•GUT. When comparing the metabolite levels between aliquots stored at room temperature and at +4 °C, we detected several changes in microbial metabolites, including multiple BAs and SCFAs. Taken together, we found that storing samples at RT and stabilizing them in 95% EtOH yielded metabolomic results comparable to those from flash freezing. We also found that the overall composition of the microbiome did not vary significantly between different storage types. However, notable differences were observed in the α diversity. Altogether, the stability of the metabolome and microbiome in 95% EtOH provided results similar to those of the validated commercial collection kits OMNImet•GUT and OMNIgene•GUT, respectively.

Design of a sustainable system for wastewater treatment and generation of biofuels based on the biomass of the aquatic plant Eichhornia Crassipes.

Colombia's continuous contamination of water resources and the low alternatives to produce biofuels have affected the fulfillment of the objectives of sustainable development, deteriorating the environment and affecting the economic productivity of this country. Due to this reality, projects on environmental and economic sustainability, phytoremediation, and the production of biofuels such as ethanol and hydrogen were combined. The objective of this article was to design and develop a sustainable system for wastewater treatment and the generation of biofuels based on the biomass of the aquatic plant Eichhornia crassipes. A system that simulates an artificial wetland with live E. crassipes plants was designed and developed, removing organic matter contaminants; subsequently, and continuing the sustainability project, bioreactors were designed, adapted, and started up to produce bioethanol and biohydrogen with the hydrolyzed biomass used in the phytoremediation process, generating around 12 g/L of bioethanol and around 81 ml H2/g. The proposed research strategy suggests combining two sustainable methods, bioremediation and biofuel production, to preserve the natural beauty of water systems and their surroundings.

ALDH2 polymorphism and myocardial infarction: From alcohol metabolism to redox regulation.

Acute myocardial infarction (AMI) remains a leading cause of death worldwide. Increased formation of reactive oxygen species (ROS) during the early reperfusion phase is thought to trigger lipid peroxidation and disrupt redox homeostasis, leading to myocardial injury. Whilst the mitochondrial enzyme aldehyde dehydrogenase 2 (ALDH2) is chiefly recognised for its central role in ethanol metabolism, substantial experimental evidence suggests an additional cardioprotective role for ALDH2 independent of alcohol intake, which mitigates myocardial injury by detoxifying breakdown products of lipid peroxidation including the reactive aldehydes, malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE). Epidemiological evidence suggests that an ALDH2 mutant variant with reduced activity that is highly prevalent in the East Asian population increases AMI risk. Additional studies have uncovered a strong association between coronary heart disease and this ALDH2 mutant variant. It appears this enzyme polymorphism (in particular, in ALDH2*2/2 carriers) has the potential to have wide-ranging effects on thiol reactivity, redox tone and therefore numerous redox-related signaling processes, resilience of the heart to cope with lifestyle-related and environmental stressors, and the ability of the whole body to achieve redox balance. In this review, we summarize the journey of ALDH2 from a mitochondrial reductase linked to alcohol metabolism, via pre-clinical studies aimed at stimulating ALDH2 activity to reduce myocardial injury to clinical evidence for its protective role in the heart.

Up-regulation of Retrograde Response in yeast increases glycerol and reduces ethanol during wine fermentation.

Nutrient signaling pathways play a pivotal role in regulating the balance among metabolism, growth and stress response depending on the available food supply. They are key factors for the biotechnological success of the yeast Saccharomyces cerevisiae during food-producing fermentations. One such pathway is Retrograde Response, which controls the alpha-ketoglutarate supply required for the synthesis of amino acids like glutamate and lysine. Repressor MKS1 is linked with the TORC1 complex and negatively regulates this pathway. Deleting MKS1 from a variety of industrial strains causes glycerol to increase during winemaking, brewing and baking. This increase is accompanied by a reduction in ethanol production during grape juice fermentation in four commercial wine strains. Interestingly, this does not lead volatile acidity to increase because acetic acid levels actually lower. Aeration during winemaking usually increases acetic acid levels, but this effect reduces in the MKS1 mutant. Despite the improvement in the metabolites of oenological interest, it comes at a cost given that the mutant shows slower fermentation kinetics when grown in grape juice, malt and laboratory media and using glucose, sucrose and maltose as carbon sources. The deletion of RTG2, an activator of Retrograde Response that acts as an antagonist of MKS1, also results in a defect in wine fermentation speed. These findings suggest that the deregulation of this pathway causes a fitness defect. Therefore, manipulating repressor MKS1 is a promising approach to modulate yeast metabolism and to produce low-ethanol drinks.

Effectiveness of genetic feedback on alcohol metabolism to reduce alcohol consumption in young adults: an open-label randomized controlled trial.

BACKGROUND: It is unclear whether brief interventions using the combined classification of alcohol-metabolizing enzymes aldehyde dehydrogenase 2 (ALDH2) and alcohol dehydrogenase 1B (ADH1B) together with behavioral changes in alcohol use can reduce excessive alcohol consumption. This study aimed to examine the effects of a brief intervention based on the screening of ALDH2 and ADH1B gene polymorphisms on alcohol consumption in Japanese young adults. METHODS: In this open-label randomized controlled trial, we enrolled adults aged 20-30 years who had excessive drinking behavior (average amount of alcohol consumed: men, ≥ 4 drinks/per day and women, ≥ 2 drinks/per day; 1 drink = 10 g of pure alcohol equivalent). Participants were randomized into intervention or control group using a simple random number table. The intervention group underwent saliva-based genotyping of alcohol-metabolizing enzymes (ALDH2 and ADH1B), which were classified into five types. A 30-min in-person or online educational counseling was conducted approximately 1 month later based on genotyping test results and their own drinking records. The control group received traditional alcohol education. Average daily alcohol consumption was calculated based on the drinking diary, which was recorded at baseline and at 3 and 6 months of follow-up. The primary endpoint was average daily alcohol consumption, and the secondary endpoints were the alcohol-use disorder identification test for consumption (AUDIT-C) score and behavioral modification stages assessed using a transtheoretical model. RESULTS: Participants were allocated to the intervention (n = 100) and control (n = 96) groups using simple randomization. Overall, 28 (29.2%) participants in the control group and 21 (21.0%) in the intervention group did not complete the follow-up. Average alcohol consumption decreased significantly from baseline to 3 and 6 months in the intervention group but not in the control group. The reduction from baseline alcohol consumption values and AUDIT-C score at 3 months were greater in the intervention group than in the control group (p < 0.001). In addition, the behavioral modification stages were significantly changed by the intervention (p < 0.001). CONCLUSIONS: Genetic testing for alcohol-metabolizing enzymes and health guidance on type-specific excessive drinking may be useful for reducing sustained average alcohol consumption associated with behavioral modification. TRIAL REGISTRATION: R000050379, UMIN000044148, Registered on June 1, 2021.

Key role of K+ and Ca2+ in high-yield ethanol production by S. Cerevisiae from concentrated sugarcane molasses.

BACKGROUND: Saccharomyces cerevisiae is an important microorganism in ethanol synthesis, and with sugarcane molasses as the feedstock, ethanol is being synthesized sustainably to meet growing demands. However, high-concentration ethanol fermentation based on high-concentration sugarcane molasses-which is needed for reduced energy consumption of ethanol distillation at industrial scale-is yet to be achieved. RESULTS: In the present study, to identify the main limiting factors of this process, adaptive laboratory evolution and high-throughput screening (Py-Fe3+) based on ARTP (atmospheric and room-temperature plasma) mutagenesis were applied. We identified high osmotic pressure, high temperature, high alcohol levels, and high concentrations of K+, Ca2+, K+ and Ca2+ (K+&Ca2+), and sugarcane molasses as the main limiting factors. The robust S. cerevisiae strains of NGT-F1, NGW-F1, NGC-F1, NGK+, NGCa2+ NGK+&Ca2+-F1, and NGTM-F1 exhibited high tolerance to the respective limiting factor and exhibited increased yield. Subsequently, ethanol synthesis, cell morphology, comparative genomics, and gene ontology (GO) enrichment analysis were performed in a molasses broth containing 250 g/L total fermentable sugars (TFS). Additionally, S. cerevisiae NGTM-F1 was used with 250 g/L (TFS) sugarcane molasses to synthesize ethanol in a 5-L fermenter, giving a yield of 111.65 g/L, the conversion of sugar to alcohol reached 95.53%. It is the highest level of physical mutagenesis yield at present. CONCLUSION: Our results showed that K+ and Ca2+ ions primarily limited the efficient production of ethanol. Then, subsequent comparative transcriptomic GO and pathway analyses showed that the co-presence of K+ and Ca2+ exerted the most prominent limitation on efficient ethanol production. The results of this study might prove useful by promoting the development and utilization of green fuel bio-manufactured from molasses.

Isolation, identification, and tolerance analysis of yeast during the natural fermentation process of Sidamo coffee beans.

Yeast, which plays a pivotal role in the brewing, food, and medical industries, exhibits a close relationship with human beings. In this study, we isolated and purified 60 yeast strains from the natural fermentation broth of Sidamo coffee beans to screen for indigenous beneficial yeasts. Among them, 25 strains were obtained through morphological characterization on nutritional agar medium from Wallerstein Laboratory (WL), with molecular biology identifying Saccharomyces cerevisiae strain YBB-47 and the remaining 24 yeast strains identified as Pichia kudriavzevii. We investigated the fermentation performance, alcohol tolerance, SO2 tolerance, pH tolerance, sugar tolerance, temperature tolerance, ester production capacity, ethanol production capacity, H2S production capacity, and other brewing characteristics of YBB-33 and YBB-47. The results demonstrated that both strains could tolerate up to 3% alcohol by volume at a high sucrose mass concentration (400 g/L) under elevated temperature conditions (40 ℃), while also exhibiting a remarkable ability to withstand an SO2 mass concentration of 300 g/L at pH 3.2. Moreover, S. cerevisiae YBB-47 displayed a rapid gas production rate and strong ethanol productivity. whereas P. kudriavzevii YBB-33 exhibited excellent alcohol tolerance. Furthermore, this systematic classification and characterization of coffee bean yeast strains from the Sidamo region can potentially uncover additional yeasts that offer high-quality resources for industrial-scale coffee bean production.

Hexanoic acid production and microbial community in anaerobic fermentation: Effects of inorganic carbon addition.

Carbon dioxide (CO2) plays a crucial role in carbon chain elongation with ethanol serving as an electron donor. In this study, the impacts of various carbonates on CO2 concentration, hexanoic acid production, and microbial communities during ethanol-butyric acid fermentation were explored. The results showed that the addition of MgCO3 provided sustained inorganic carbon and facilitated interspecific electron transfer, thereby increasing hexanoic acid yield by 58%. MgCO3 and NH4HCO3 inhibited the excessive ethanol oxidation and decreased the yield of acetic acid by 51% and 42%, respectively. The yields of hexanoic acid and acetic acid in the CaCO3 group increased by 19% and 15%, respectively. The NaHCO3 group exhibited high headspace CO2 concentration, promoting acetogenic bacteria enrichment while reducing the abundance of Clostridium_sensu_stricto_12. The batch addition of NaHCO3 accelerated the synthesis of hexanoic acid and increased its production by 26%. The relative abundance of Clostridium_sensus_stricto_12 was positively correlated with hexanoic acid production.

Cofactor engineering in Thermoanaerobacterium aotearoense SCUT27 for maximizing ethanol yield and revealing an enzyme complex with high ferredoxin-NAD+ reductase activity.

Thermoanaerobacterium aotearoense SCUT27 is a prominent producer of biofuels from lignocellulosic materials. To provide sufficient NAD(P)H for ethanol production, redox-related genes, including lactate dehydrogenase (ldh), redox-sensing transcriptional repressor (rex), and hydrogenase (hfsB), were knocked out. However, the growth of strain PRH (Δldh/Δrex/ΔhfsB) was suppressed due to the intracellular redox state imbalance with the increased NADH concentration. Coincidentally, when the Bcd-EtfAB (BCD) complex was overexpressed, the resulting strain PRH-B3 (Δldh/Δrex/ΔhfsB::BCD) grew rapidly and produced ethanol with a high yield. With lignocellulosic hydrolysates, PRH-BA (Δldh/Δrex/ΔhfsB::BCD::adhE) demonstrated high ethanol productivity and yield, reaching levels of 0.45-0.51 g/L/h and 0.46-0.53 g/g sugars, respectively. The study results shed light on the cofactor balance for cell stability and the high ferredoxin-NAD+ reductase activity of the BCD complex under an intracellular low redox state. They also provide an essential reference for developing strains for improved biofuel production.

Agricultural Waste Management by Production of Second-Generation Bioethanol from Sugarcane Bagasse Using Indigenous Yeast Strain.

In the wake of rapid industrialization and burgeoning transportation networks, the escalating demand for fossil fuels has accelerated the depletion of finite energy reservoirs, necessitating urgent exploration of sustainable alternatives. To address this, current research is focusing on renewable fuels like second-generation bioethanol from agricultural waste such as sugarcane bagasse. This approach not only circumvents the contentious issue of food-fuel conflicts associated with biofuels but also tackles agricultural waste management. In the present study indigenous yeast strain, Clavispora lusitaniae QG1 (MN592676), was isolated from rotten grapes to ferment xylose sugars present in the hemicellulose content of sugarcane bagasse. To liberate the xylose sugars, dilute acid pretreatment was performed. The highest reducing sugars yield was 1.2% obtained at a temperature of 121 °C for 15 min, a solid-to-liquid ratio of 1:25 (% w/v), and an acid concentration of 1% dilute acid H2SO4 that was significantly higher (P < 0.001) yield obtained under similar conditions at 100 °C for 1 h. The isolated strain was statistically optimized for fermentation process by Plackett-Burman design to achieve the highest ethanol yield. Liberated xylose sugars were completely utilized by Clavispora lusitaniae QG1 (MN592676) and gave 100% ethanol yield. This study optimizes both fermentation process and pretreatment of sugarcane bagasse to maximize bioethanol yield and demonstrates the ability of isolated strain to effectively utilize xylose as a carbon source. The desirable characteristics depicted by strain Clavispora lusitaniae shows its promising utilization in management of industrial waste like sugarcane bagasse by its conversion into renewable biofuels like bioethanol.

A new Zymomonas mobilis platform strain for the efficient production of chemicals.

BACKGROUND: Zymomonas mobilis is well known for its outstanding ability to produce ethanol with both high specific productivity and with high yield close to the theoretical maximum. The key enzyme in the ethanol production pathway is the pyruvate decarboxylase (PDC) which is converting pyruvate to acetaldehyde. Since it is widely considered that its gene pdc is essential, metabolic engineering strategies aiming to produce other compounds derived from pyruvate need to find ways to reduce PDC activity. RESULTS: Here, we present a new platform strain (sGB027) of Z. mobilis in which the native promoter of pdc was replaced with the IPTG-inducible PT7A1, allowing for a controllable expression of pdc. Expression of lactate dehydrogenase from E. coli in sGB027 allowed the production of D-lactate with, to the best of our knowledge, the highest reported specific productivity of any microbial lactate producer as well as with the highest reported lactate yield for Z. mobilis so far. Additionally, by expressing the L-alanine dehydrogenase of Geobacillus stearothermophilus in sGB027 we produced L-alanine, further demonstrating the potential of sGB027 as a base for the production of compounds other than ethanol. CONCLUSION: We demonstrated that our new platform strain can be an excellent starting point for the efficient production of various compounds derived from pyruvate with Z. mobilis and can thus enhance the establishment of this organism as a workhorse for biotechnological production processes.

Wine fermentation process evaluation through NMR analysis: Polysaccharides, ethanol quantification and biological activity.

Winemaking production is old knowledge of the combination of saccharification and fermentation processes. During the fermentation process, ethanol concentration is one of the main key parameters that provides the quality of wine and is linked to the consumption of carbohydrates present in wine. In this work was determined the better fermentation time, where the wine retains its highest concentration of ethanol and a higher concentration of the polysaccharides of Bordo wine of Vitis labrusca by 1D and 2D NMR measurements. The study provides information on the polysaccharide content for improving features and quality control of winemaking. Moreover, following previous studies by our group (de Lacerda Bezerra et al., 2018, de Lacerda Bezerra, Caillot, de Oliveira, Santana-Filho, & Sassaki, 2019; Stipp et al., 2023) showed that the soluble polysaccharides also inhibited the production of inflammatory cytokines (TNF-α and IL-1ß) and mediator (NO) in macrophage cells stimulated with LPS, bringing some important health benefits of wine.

Substitution of both histidines in the active site of yeast alcohol dehydrogenase 1 exposes underlying pH dependencies.

Histidine residues 44 and 48 in yeast alcohol dehydrogenase (ADH) bind to the coenzymes NAD(H) and contribute to catalysis. The individual H44R and H48Q substitutions alter the kinetics and pH dependencies, and now the roles of other ionizable groups in the enzyme were studied in the doubly substituted H44R/H48Q ADH. The substitutions make the enzyme more resistant to inactivation by diethyl pyrocarbonate, modestly improve affinity for coenzymes, and substantially decrease catalytic efficiencies for ethanol oxidation and acetaldehyde reduction. The pH dependencies for several kinetic parameters are shifted from pK values for wild-type ADH of 7.3-8.1 to values for H44R/H48Q ADH of 8.0-9.6, and are assigned to the water or alcohol bound to the catalytic zinc. It appears that the rate of binding of NAD+ is electrostatically favored with zinc-hydroxide whereas binding of NADH is faster with neutral zinc-water. The pH dependencies of catalytic efficiencies (V/EtKm) for ethanol oxidation and acetaldehyde reduction are similarly controlled by deprotonation and protonation, respectively. The substitutions make an enzyme that resembles the homologous horse liver H51Q ADH, which has Arg-47 and Gln-51 and exhibits similar pK values. In the wild-type ADHs, it appears that His-48 (or His-51) in the proton relay systems linked to the catalytic zinc ligands modulate catalytic efficiencies.

Optimization of CO fermentation by Clostridium carboxidivorans in batch reactors: Effects of the medium composition.

OBJECTIVES: The objective of this study was to investigate the effects of medium composition on CO fermentation by Clostridium carboxidivorans. The focus was to reduce the medium cost preserving acceptable levels of solvent production. METHODS: Yeast extract (YE) concentration was set in the range of 0-3 g/L. Different reducing agents were investigated, including cysteine-HCl 0.6 g/L, pure cysteine 0.6 g/L, sodium sulphide (Na2S) 0.6 g/L, cysteine-sodium sulphide 0.6 g/L and cysteine-sodium sulphide 0.72 g/L. The concentration of the metal solution was decreased down to 25 % of the standard value. Fermentation tests were also carried out with and without tungsten or selenium. RESULTS: The results demonstrated that under optimized conditions, namely yeast extract (YE) concentration set at 1 g/L, pure cysteine as the reducing agent and trace metal concentration reduced to 75 % of the standard value, reasonable solvent production was achieved in less than 150 h. Under these operating conditions, the production levels were found to be 1.39 g/L of ethanol and 0.27 g/L of butanol. Furthermore, the study revealed that selenium was not necessary for C. carboxidivorans fermentation, whereas the presence of tungsten played a crucial role in both cell growth and solvent production. CONCLUSIONS: The optimization of the medium composition in CO fermentation by Clostridium carboxidivorans is crucial for cost-effective solvent production. Tuning the yeast extract (YE) concentration, using pure cysteine as the reducing agent and reducing trace metal concentration contribute to reasonable solvent production within a relatively short fermentation period. Tungsten is essential for cell growth and solvent production, while selenium is not required.