Ethanol Inhibition Research Articles

A multi reaction kinetic model to describe the enzymatic transesterification reaction of jatropha oil using a fermented solid containing lipases

The advancement of more precise tools for sustainable process design in enzymatic biodiesel synthesis from renewable sources is crucial. Kinetics of solvent-free transesterification reactions were conducted across a temperature spectrum from 30 °C to 60 °C, utilizing Jatropha curcas oil (TG) and ethanol as substrates, alongside a fermented solid by Rhizopus homothallicus as the biocatalyst. The dynamics of chemical species concentrations were monitored through High-performance Thin-Layer Chromatography. Maximum productivities were achieved at 35 °C and 60 °C for biodiesel (293.24 and 299.02 g kg biocat−1 h−1, respectively), at 40 °C for diglycerides (1018.36 g kg biocat−1 h−1), and at 35 °C for monoglycerides (560.75 g kg biocat−1 h−1). Maximum yields were determined at 30 °C for fatty acid ethyl esters (0.56 g gTG−1), and at 40 °C for diglycerides (0.53 g gTG−1) and monoglycerides (0.30 g gTG−1). Based on the experimental findings, a kinetic model was formulated encompassing three reversible transesterification reactions. Individual reactions were structured following classical biochemical kinetics, inclusive of ethanol inhibition. Model fitting was executed through non-linear multivariable regression techniques, with the minimum of the average coefficient of variation of the residuals (ACVR) serving as the objective function. The resulting fit of the kinetic model to the experimental data proved satisfactory, with an ACVR of less than 5 % across all instances. Notably, the maximum biodiesel productivity, obtained in this work, represented the highest value, compared to other related studies, using a fermented solid as a biocatalyst.

Metabolic flux analysis of hydrogen production network by Ethanoligenens harbinense B49: effect of product inhibition

ABSTRACT 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.

Ethanol fumigation combined with modified atmosphere packaging delays potato greening under light

Potato greening under light causes post-harvest loss and compromises progress being made in the potato industry. In this study, we investigated the inhibiting effects of ethanol fumigation treatment on potato greening and water loss under light. This paper also examined the different treatments on the relationship between starch granule size and potato greening. Our results suggested that ethanol fumigation combined with modified atmosphere packaging have an effective in improving the overall visual quality and maintained a higher a* value and lower chlorophyll concentration. Ethanol fumigation combined with modified atmosphere packaging treatment deterred greening and water loss compared to the alone treatment. This was due to the larger starch granule size and fewer grana lamellae around the amyloplasts. Our results provide an effectively strategy that treating potatoes with 600 μL L−1 ethanol combined with modified atmosphere packaging to delay potato greening and explain the underlying mechanism of ethanol inhibition of potato greening.

Dietary soy prevents fetal demise, intrauterine growth restriction, craniofacial dysmorphic features, and impairments in placentation linked to gestational alcohol exposure: Pivotal role of insulin and insulin-like growth factor signaling networks.

Prenatal alcohol exposure can impair placentation and cause intrauterine growth restriction (IUGR), fetal demise, and fetal alcohol spectrum disorder (FASD). Previous studies showed that ethanol's inhibition of placental insulin and insulin-like growth factor, type 1 (IGF-1) signaling compromises trophoblastic cell motility and maternal vascular transformation at the implantation site. Since soy isolate supports insulin responsiveness, we hypothesized that dietary soy could be used to normalize placentation and fetal growth in an experimental model of FASD. Pregnant Long-Evans rat dams were fed with isocaloric liquid diets containing 0% or 8.2% ethanol (v/v) from gestation day (GD) 6. Dietary protein sources were either 100% soy isolate or 100% casein (standard). Gestational sacs were harvested on GD19 to evaluate fetal resorption, fetal growth parameters, and placental morphology. Placental insulin/IGF-1 signaling through Akt pathways was assessed using commercial bead-based multiplex enzyme-linked immunosorbent assays. Dietary soy markedly reduced or prevented the ethanol-associated fetal loss, IUGR, FASD dysmorphic features, and impairments in placentation/maturation. Furthermore, ethanol's inhibitory effects on the placental glycogen cell population at the junctional zone, invasive trophoblast populations at the implantation site, maternal vascular transformation, and signaling through the insulin and IGF1 receptors, Akt and PRAS40 were largely abrogated by co-administration of soy. Dietary soy may provide an economically feasible and accessible means of reducing adverse pregnancy outcomes linked to gestational ethanol exposure.

Ethanol inhibition of undifferentiated rat neural progenitor cell replication can be prevented by chlorogenic acid via the NFATc4/CSE signaling pathway.

Prenatal ethanol exposure hinders oxidative stress-mediated neuroblast/neural progenitor cell proliferation by inhibiting G1-S transition, a process vital to neocortical development. We previously showed that ethanol elicits this redox imbalance by repressing cystathionine γ-lyase (CSE), the rate-limiting enzyme in the transsulfuration pathway in fetal brain and cultured cerebral cortical neurons. However, the mechanism by which ethanol impacts the CSE pathway in proliferating neuroblasts is not known. We conducted experiments to define the effects of ethanol on CSE regulation and the molecular signaling events that control this vital pathway. This enabled us to develop an intervention to prevent the ethanol-associated cytostasis. Spontaneously immortalized undifferentiated E18 rat neuroblasts from brain cerebral cortex were exposed to ethanol to mimic an acute consumption pattern in humans. We performed loss- and gain-of-function studies to evaluate whether NFATc4 is a transcriptional regulator of CSE. The neuroprotective effects of chlorogenic acid (CGA) against the effects of ethanol were assessed using ROS and GSH/GSSG assays as measures of oxidative stress, transcriptional activation of NFATc4, and expression of NFATc4 and CSE by qRT-PCR and immunoblotting. Ethanol treatment of E18-neuroblast cells elicited oxidative stress and significantly reduced CSE expression with a concomitant decrease in NFATc4 transcriptional activation and expression. In parallel, inhibition of the calcineurin/NFAT pathway by FK506 exaggerated ethanol-induced CSE loss. In contrast, NFATc4 overexpression prevented loss of ethanol-induced CSE. CGA increased and activated NFATc4, amplified CSE expression, rescued ethanol-induced oxidative stress, and averted the cytostasis of neuroblasts by rescuing cyclin D1 expression. These findings demonstrate that ethanol can perturb CSE-dependent redox homeostasis by impairing the NFATc4 signaling pathway in neuroblasts. Notably, ethanol-associated impairments were rescued by genetic or pharmacological activation of NFATc4. Furthermore, we found a potential role for CGA in mitigating the ethanol-related neuroblast toxicity with a compelling connection to the NFATc4/CSE pathway.

A Facile and Eco-Friendly Hydrothermal Synthesis of High Tetragonal Barium Titanate with Uniform and Controllable Particle Size.

The preparation of tetragonal barium titanate (BT) powders with uniform and suitable particle sizes is a significant prerequisite for ultra-thin and highly integrated multilayer ceramic capacitors (MLCCs). However, the balance of high tetragonality and controllable particle size remains a challenge, which limits the practical application of BT powders. Herein, the effects of different proportions of hydrothermal medium composition on the hydroxylation process are explored to obtain high tetragonality. The high tetragonality of BT powders under the optimal solvent condition of water:ethanol:ammonia solution of 2:2:1 is around 1.009 and increases with the particle size. Meanwhile, the good uniformity and dispersion of BT powders with particle sizes of 160, 190, 220, and 250 nm benefit from the inhibition of ethanol on the interfacial activity of BT particles (BTPs). The core-shell structure of BTPs is revealed by different lattice fringe spacings of the core and edge and the crystal structure by reconstructed atomic arrangement, which reasonably explains the trend between tetragonality and average particle size. These findings are instructive for the related research on the hydrothermal process of BT powders.

Effects of alcohol and PARP inhibition on RNA ribosomal engagement in cortical excitatory neurons.

We report on the effects of ethanol (EtOH) and Poly (ADP-ribose) polymerase (PARP) inhibition on RNA ribosomal engagement, as a proxy for protein translation, in prefrontal cortical (PFC) pyramidal neurons. We hypothesized that EtOH induces a shift in RNA ribosomal-engagement (RE) in PFC pyramidal neurons, and that many of these changes can be reversed using a PARP inhibitor. We utilized the translating ribosome affinity purification (TRAP) technique to isolate cell type-specific RNA. Transgenic mice with EGFP-tagged Rpl10a ribosomal protein expressed only in CaMKIIα-expressing pyramidal cells were administered EtOH or normal saline (CTL) i.p. twice a day, for four consecutive days. On the fourth day, a sub-group of mice that received EtOH in the previous three days received a combination of EtOH and the PARP inhibitor ABT-888 (EtOH + ABT-888). PFC tissue was processed to isolate both, CaMKIIα pyramidal cell-type specific ribosomal-engaged RNA (TRAP-RNA), as well as genomically expressed total-RNA from whole tissue, which were submitted for RNA-seq. We observed EtOH effects on RE transcripts in pyramidal cells and furthermore treatment with a PARP inhibitor "reversed" these effects. The PARP inhibitor ABT-888 reversed 82% of the EtOH-induced changes in RE (TRAP-RNA), and similarly 83% in the total-RNA transcripts. We identified Insulin Receptor Signaling as highly enriched in the ethanol-regulated and PARP-reverted RE pool and validated five participating genes from this pathway. To our knowledge, this is the first description of the effects of EtOH on excitatory neuron RE transcripts from total-RNA and provides insights into PARP-mediated regulation of EtOH effects.

Role of α6-Nicotinic Receptors in Alcohol-Induced GABAergic Synaptic Transmission and Plasticity to Cholinergic Interneurons in the Nucleus Accumbens.

The prevailing view is that enhancement of dopamine (DA) transmission in the mesolimbic system, consisting of DA neurons in the ventral tegmental area (VTA) that project to the nucleus accumbens (NAc), underlies the reward properties of ethanol (EtOH) and nicotine (NIC). We have shown previously that EtOH and NIC modulation of DA release in the NAc is mediated by α6-containing nicotinic acetylcholine receptors (α6*-nAChRs), that α6*-nAChRs mediate low-dose EtOH effects on VTA GABA neurons and EtOH preference, and that α6*-nAChRs may be a molecular target for low-dose EtOH. However, the most sensitive target for reward-relevant EtOH modulation of mesolimbic DA transmission and the involvement of α6*-nAChRs in the mesolimbic DA reward system remains to be elucidated. The aim of this study was to evaluate EtOH effects on GABAergic modulation of VTA GABA neurons and VTA GABAergic input to cholinergic interneurons (CINs) in the NAc. Low-dose EtOH enhanced GABAergic input to VTA GABA neurons that was blocked by knockdown of α6*-nAChRs. Knockdown was achieved either by α6-miRNA injected into the VTA of VGAT-Cre/GAD67-GFP mice or by superfusion of the α-conotoxin MII[H9A;L15A] (MII). Superfusion of MII blocked EtOH inhibition of mIPSCs in NAc CINs. Concomitantly, EtOH enhanced CIN firing rate, which was blocked by knockdown of α6*-nAChRs with α6-miRNA injected into the VTA of VGAT-Cre/GAD67-GFP mice. The firing rate of CINs was not enhanced by EtOH in EtOH-dependent mice, and low-frequency stimulation (LFS; 1Hz, 240 pulses) caused inhibitory long-term depression at this synapse (VTA-NAc CIN-iLTD) which was blocked by knockdown of α6*-nAChR and MII. Ethanol inhibition of CIN-mediated evoked DA release in the NAc was blocked by MII. Taken together, these findings suggest that α6*-nAChRs in the VTA-NAc pathway are sensitive to low-dose EtOH and play a role in plasticity associated with chronic EtOH.

Zone of Inhibition of Ethanol and Ethyl Acetate Extracts of Forest Betel Leaves against Escherichia Coli

The betel plant is one of the medicinal plants known by the common people. This plant consists of more than 1000 species. One of the species is the betel forest. The part of the betel plant that is commonly used as medicine is the leaves. This study aims to determine the phytochemical content and inhibition of Escherichia coli in the ethanol and ethyl acetate extracts of forest betel leaves (Piper aduncum L.). The method used in the antibacterial test is the good diffusion method. The research showed that ethanol extract of forest betel leaves (Piper aduncum L.) leaves contains secondary metabolites, namely flavonoids, saponins, terpenoids, and tannins, while the ethyl acetate extract contains secondary metabolites, namely flavonoids, terpenoids, and tannins. The ethanol and ethyl acetate extracts of forest betel leaves (Piper aduncum L.) can inhibit the growth of Escherichia coli in the strong category

Ethanol tolerance of Clostridium thermocellum: the role of chaotropicity, temperature and pathway thermodynamics on growth and fermentative capacity

BackgroundClostridium thermocellum is a promising candidate for consolidated bioprocessing of lignocellulosic biomass to ethanol. The low ethanol tolerance of this microorganism is one of the remaining obstacles to industrial implementation. Ethanol inhibition can be caused by end-product inhibition and/or chaotropic-induced stress resulting in increased membrane fluidization and disruption of macromolecules. The highly reversible glycolysis of C. thermocellum might be especially sensitive to end-product inhibition. The chaotropic effect of ethanol is known to increase with temperature. This study explores the relative contributions of these two aspects to investigate and possibly mitigate ethanol-induced stress in growing and non-growing C. thermocellum cultures.ResultsTo separate chaotropic from thermodynamic effects of ethanol toxicity, a non-ethanol producing strain AVM062 (Pclo1313_2638::ldh* ∆adhE) was constructed by deleting the bifunctional acetaldehyde/alcohol dehydrogenase gene, adhE, in a lactate-overproducing strain. Exogenously added ethanol lowered the growth rate of both wild-type and the non-ethanol producing mutant. The mutant strain grew quicker than the wild-type at 50 and 55 °C for ethanol concentrations ≥ 10 g L−1 and was able to reach higher maximum OD600 at all ethanol concentrations and temperatures. For the wild-type, the maximum OD600 and relative growth rates were higher at 45 and 50 °C, compared to 55 °C, for ethanol concentrations ≥ 15 g L−1. For the mutant strain, no positive effect on growth was observed at lower temperatures. Growth-arrested cells of the wild-type demonstrated improved fermentative capacity over time in the presence of ethanol concentrations up to 40 g L−1 at 45 and 50 °C compared to 55 °C.ConclusionPositive effects of temperature on ethanol tolerance were limited to wild-type C. thermocellum and are likely related to mechanisms involved in the ethanol-formation pathway and redox cofactor balancing. Lowering the cultivation temperature provides an attractive strategy to improve growth and fermentative capacity at high ethanol titres in high-cellulose loading batch cultivations. Finally, non-ethanol producing strains are useful platform strains to study the effects of chaotropicity and thermodynamics related to ethanol toxicity and allow for deeper understanding of growth and/or fermentation cessation under industrially relevant conditions.

Inhibition of Candida albicans and Staphylococcus epidermidis mixed biofilm formation in a catheter disk model system treated with EtOH-EDTA solution.

Microbial colonization and the formation of biofilms on catheter surfaces pose a great risk for medical-related infections. We aimed (a) to evaluate polymicrobial biofilm formation of Candida albicans and Staphylococcus epidermidis and (b) to investigate the inhibition and effects of ethanol (EtOH) and EtOH-EDTA solutions on biofilms. Catheter disks were made and used as a substrate for biofilm formation. Varying concentrations of EtOH and EtOH-EDTA solutions were compared in deterring biofilm formation. The EtOH-EDTA solutions were further tested to remove mature and preformed biofilms. Compared to their monospecies counterparts, biofilm concentration significantly increases when C. albicans is co-cultured with S. epidermidis. Moreover, all treatments with EtOH-EDTA solution significantly lowered biofilm formation compared to EtOH alone (P≤0.05). Lastly, biofilm was dramatically reduced when treated with 20%, 30%, 40%, and 50% EtOH-EDTA solutions (P≤0.05). Our findings suggest that biofilms become more resilient to treatment when formed by multiple organisms. Nonetheless, treatment with EtOH-EDTA is effective against these polymicrobial biofilms.

Molecular mechanism of enhanced ethanol tolerance associated with hfq overexpression in Zymomonas mobilis.

Zymomonas mobilis is a promising microorganism for industrial bioethanol production. However, ethanol produced during fermentation is toxic to Z. mobilis and affects its growth and bioethanol production. Although several reports demonstrated that the RNA-binding protein Hfq in Z. mobilis contributes to the tolerance against multiple lignocellulosic hydrolysate inhibitors, the role of Hfq on ethanol tolerance has not been investigated. In this study, hfq in Z. mobilis was either deleted or overexpressed and their effects on cell growth and ethanol tolerance were examined. Our results demonstrated that hfq overexpression improved ethanol tolerance of Z. mobilis, which is probably due to energy saving by downregulating flagellar biosynthesis and heat stress response proteins, as well as reducing the reactive oxygen species induced by ethanol stress via upregulating the sulfate assimilation and cysteine biosynthesis. To explore proteins potentially interacted with Hfq, the TEV protease mediated Yeast Endoplasmic Reticulum Sequestration Screening system (YESS) was established in Z. mobilis. YESS results suggested that Hfq may modulate the cytoplasmic heat shock response by interacting with the heat shock proteins DnaK and DnaJ to deal with the ethanol inhibition. This study thus not only revealed the underlying mechanism of enhanced ethanol tolerance by hfq overexpression, but also provided an alternative approach to investigate protein-protein interactions in Z. mobilis.

Effects of ethanol on GluN1/GluN2A and GluN1/GluN2B NMDA receptor-ion channel gating kinetics.

The N-methyl-D-aspartate receptor (NMDAR) is a major molecular target of alcohol action in the central nervous system, yet many aspects of alcohol's modulation of the activity of this ion channel remain unclear. We and others have shown that ethanol inhibition of NMDAR involves alterations in gating, especially a reduction in mean open time. However, a full description of ethanol's effects on NMDAR kinetics, including fitting them to a kinetic model, has not been reported. To determine ethanol's effects on NMDAR kinetics, we used steady-state single-channel recording in outside-out patches from HEK-293 cells transfected with recombinant GluN1/GluN2A or GluN1/GluN2B NMDAR subunits. Very low glutamate concentrations were used to isolate individual activations of the receptor. In both subunit types, ethanol, at approximate whole-cell IC50 values (156 mM, GluN2A; 150 mM, GluN2B), reduced open probability (po ) by approximately 50% and decreased mean open time without changing the frequency of opening. Open and shut time distributions exhibited two and five components, respectively; ethanol selectively decreased the time constant and relative proportion of the longer open time component. In the GluN2A subunit, ethanol increased the time constants of all but the longest shut time components, whereas in the GluN2B subunit, shut times were unchanged by ethanol. Fitting of bursts of openings (representing individual activations of the receptor) to the gating portion of a kinetic model revealed that ethanol altered two rates: the rate associated with activation of the GluN2A or GluN2B subunit, and the rate associated with the closing of the longer of the two open states. These results demonstrate that ethanol selectively alters individual kinetic rates and thus appears to selectively affect distinct conformational transitions involved in NMDAR gating.

Analyze and control on the membrane ethanol fermentation process with periodic exogenous signals

Bio-ethanol production by continuous fermentation reactor would generate self-oscillatory dynamics at high substrate concentrations, which exacerbates the control problem. Andronov-Hopf bifurcation analysis of the process model indicates that the self-oscillatory dynamics is caused by ethanol inhibition, and an ethanol selective membrane is assembled to the fermentation reactor for in-situ product removal (ISPR). The simulation results show that the membrane reactor eliminates the self-oscillatory dynamics effectively, but considering the operational cost for the membrane reactor is substantial, periodic operation and on/off control of the sweeping stream in the membrane separator is adopted, and it is interesting to find that when the ethanol content follow fine designed periodic trajectories, the corresponding control actions become regular and square waveform is formulated, which is beneficial from real-time operational perspective. This result could aid periodic operation and control of the fermentation reactor at high ethanol concentrations.

The ethanol inhibition of basolateral amygdala neuron spiking is mediated by a γ-aminobutyric acid type A-mediated tonic current.

The basolateral nucleus of the amygdala (BLA) plays an important role in the development of fear and anxiety-related behaviors. The BLA receives inputs from all sensory stimuli. After processing those stimuli, BLA neurons signal neurons within the central amygdala and other brain regions, including the ventral and dorsal striatum and frontal cortex. Studies suggest that the BLA is involved in drug dependence and in the reinforcing actions of ethanol. For example, acute exposure to ethanol reduces anxiety, while withdrawal from chronic ethanol exposure alters BLA synaptic transmission, which increases anxiety, a common underlying cause of relapse. Exposure to and withdrawal from chronic alcohol also disrupts many brain areas that connect with the BLA. Despite these important findings, the acute actions of alcohol on the intrinsic excitability of BLA neurons have not been fully characterized. Brain slices containing the BLA were prepared from adult C57BL/6J male mice. Whole-cell and sharp electrode electrophysiological recordings were performed to characterize the effects of acute ethanol on BLA neuronal and astrocyte function, respectively. Ethanol inhibited action potential (AP) firing of BLA neurons but had no effect on BLA astrocyte resting membrane potential. The ethanol-induced inhibition of firing was concentration-dependent (11 to 66 mM) and accompanied by a reduction in the input resistance and an increase in the rheobase of BLA neurons. The inhibitory effect of ethanol was suppressed by picrotoxin, which blocks both γ-aminobutyric acid type A (GABAA ) and glycine receptors, but not by the selective glycine receptor antagonist strychnine, which suggests an involvement of GABAA receptors. Ethanol did not affect spontaneous inhibitory postsynaptic currents suggesting that the inhibition of BLA neuronal excitability by ethanol was not due to an increase in GABAA -mediated synaptic transmission. However, acute ethanol enhanced the amplitude of the holding current of BLA neurons, an effect that was prevented by picrotoxin, which by itself reduced the holding current. These results suggest that BLA neurons express a GABA-mediated tonic current that is enhanced by acute ethanol, which leads to reduced excitability of BLA neurons.

Cellulosic Ethanol Production Using a Dual Functional Novel Yeast.

Reducing the cost of cellulosic ethanol production, especially for cellulose hydrolytic enzymes, is vital to growing a sustainable and efficient cellulosic ethanol industry and bio-based economy. Using an ethanologenic yeast able to produce hydrolytic enzymes, such as Clavispora NRRL Y-50464, is one solution. NRRL Y-50464 is fast-growing and robust, and tolerates inhibitory compounds 2-furaldehyde (furfural) and 5-hydroxymethyl-2-furaldehyde (HMF) associated with lignocellulose-to-fuel conversion. It produces three forms of β-glucosidase isozymes, BGL1, BGL2, and BGL3, and ferment cellobiose as the sole carbon source. These β-glucosidases exhibited desirable enzyme kinetic parameters and high levels of enzyme-specific activity toward cellobiose and many oligosaccharide substrates. They tolerate the product inhibition of glucose and ethanol, and are stable to temperature and pH conditions. These characteristics are desirable for more efficient cellulosic ethanol production by simultaneous saccharification and fermentation. NRRL Y-50464 provided the highest cellulosic ethanol titers and conversion rates at lower cellulase loadings, using either pure cellulose or agricultural residues, as so far reported in the literature. This review summarizes NRRL Y-50464 performance on cellulosic ethanol production from refined cellulose, rice straw, and corn stover processed in various ways, in the presence or absence of furfural and HMF. This dual functional yeast has potential to serve as a prototype for the development of next-generation biocatalysts. Perspectives on continued strain development and process engineering improvements for more efficient cellulosic ethanol production from lignocellulosic materials are also discussed.

Kinetic model of ethanol inhibition for Kluyveromyces marxianus CCT 7735 (UFV-3) based on the modified Monod model by Ghose & Tyagi

Kinetic model of ethanol inhibition for Kluyveromyces marxianus CCT 7735 (UFV-3) based on the modified Monod model by Ghose & Tyagi

Ethanol treatment suppresses the yellowing of fresh-cut yam by repressing the phenylpropanoid pathway and bisdemethoxycurcumin biosynthesis

An ethanol treatment (10 % v/v) inhibited the yellowing of fresh-cut yam during storage. The ethanol treatment also prevented bisdemethoxycurcumin formation, and reduced the contents of metabolites in fresh-cut yam during storage at 25 °C. Ethanol treatment also inhibited the activities of key enzymes in the phenylpropanoid pathway, including phenylalanine ammonia lyase (PAL), cinnamic acid-4-hydroxylase (C4H), and 4-coumarate-CoA ligase (4CL), and decreased the transcription level expressions of PAL, C4H, and 4CL compared to the control. The expressions of diketide-CoA synthase, curcumin synthase 3, and curcumin synthase were also reduced by ethanol treatment compared to the control, in agreement with the absence of bisdemethoxycurcumin in fresh-cut yam treated with ethanol. These findings suggest that ethanol can be used to attenuate the yellowing of fresh-cut yam stored at 25 °C, which may be related to ethanol inhibition of the phenylpropanoid pathway and bisdemethoxycurcumin biosynthesis.

ORP control for boosting ethanol productivity in gas fermentation systems and dynamics of redox cofactor NADH/NAD+ under oxidative stress

Gas fermentation processes have attracted considerable attention in recent years as they hold high potential for capturing and converting C1 waste gases into a range of biofuels and commodity chemicals. The production of solvents in gas fermentation is typically achieved by exploiting the solventogenic metabolism of acetogenic cultures, which is generally triggered upon exposure to stressful conditions, e.g. low pH. Although the oxidoreduction potential (ORP) is a well-known trigger of the cellular stress response, it has been scarcely investigated as a process control parameter in gas fermentation. Thus, this study focused on evaluating the potential of ORP control strategies for boosting the productivity of ethanol by exploiting the metabolic response to oxidative stress of acetogenic cultures. The dynamics of the redox cofactor pool and ratio as a function of the extracellular ORP and other operational parameters were also studied by monitoring the intracellular levels of the redox cofactor NADH/NAD+. The results showed that increasing the ORP to oxidizing conditions using dilute H2O2 triggered a 3.7-fold increase in the specific ethanol productivity, from 0.63 ± 0.04 mmol∙gCDW−1 h−1 at an ORP of -210 mV to 2.32 ± 0.19 mmol∙gCDW−1 h−1 at 160 mV. Additionally, the concentration and product selectivity towards ethanol also increased considerably due to the partial inhibition of the chain elongation under oxidative stress. Boost in ethanol productivity and inhibition of the chain elongation were both found to be driven by the presence of H2O2 rather than by the ORP per se. Studying the profile of the redox cofactors revealed a highly dynamic nature in the pool and ratio of NADH/NAD+ as a function of the specific uptake rate and the ratio of acetate-to-ethanol, respectively. The latter was explained by analyzing the thermodynamics of the aldehyde:ferredoxin oxidoreductase (AOR) pathway, which showed that the intrinsic thermodynamic limitation of this pathway imposes a high Fdred/Fdox ratio (>88 % of reduced ferredoxin) while forcing a highly dynamic NADH/NAD+ ratio in order to maintain the thermodynamic drive in the forward direction. The dynamics of the NADH/NAD+ ratio were also found to be significantly affected by the oxidative stress triggered by dilute H2O2, which confirmed the involvement of the AOR pathway in the detoxification of reactive oxygen species.

Omics analysis reveals mechanism underlying metabolic oscillation during continuous very-high-gravity ethanol fermentation by Saccharomyces cerevisiae.

During continuous very-high-gravity (VHG) ethanol fermentation with Saccharomyces cerevisiae, the process exhibits sustained oscillation in residual glucose, ethanol, and biomass, raising a question: how do yeast cells respond to this phenomenon? In this study, the oscillatory behavior of yeast cells was characterized through transcriptome and metabolome analysis for one complete oscillatory period. By analyzing the accumulation of 26 intracellular metabolites and the expression of 90 genes related to central carbonmetabolism and stress response, we confirmed that the process oscillation was attributed to intracellular metabolic oscillation with phase difference, and the expression of HXK1, HXT1,2,4, and PFK1 was significantly different from other genes in the Embden-Meyerhof-Parnas pathway, indicating that glucose transport and phosphorylation could be key nodes for regulating the intracellular metabolism under oscillatory conditions. Moreover, the expression of stress response genes was triggered and affected predominately by ethanol inhibition in yeast cells. This progress not only contributes to the understanding of mechanisms underlying the process oscillation observed for continuous VHG ethanol fermentation, but also provides insights for understanding unsteady state that might develop in other continuous fermentation processes operated under VHG conditions to increase product titers for robust production.