Rates Of Ethanol Uptake Research Articles

Robotic workflows for automated long-term adaptive laboratory evolution: improving ethanol utilization by Corynebacterium glutamicum

BackgroundAdaptive laboratory evolution (ALE) is known as a powerful tool for untargeted engineering of microbial strains and genomics research. It is particularly well suited for the adaptation of microorganisms to new environmental conditions, such as alternative substrate sources. Since the probability of generating beneficial mutations increases with the frequency of DNA replication, ALE experiments are ideally free of constraints on the required duration of cell proliferation.ResultsHere, we present an extended robotic workflow for performing long-term evolution experiments based on fully automated repetitive batch cultures (rbALE) in a well-controlled microbioreactor environment. Using a microtiter plate recycling approach, the number of batches and thus cell generations is technically unlimited. By applying the validated workflow in three parallel rbALE runs, ethanol utilization by Corynebacterium glutamicum ATCC 13032 (WT) was significantly improved. The evolved mutant strain WT_EtOH-Evo showed a specific ethanol uptake rate of 8.45 ± 0.12 mmolEtOH gCDW−1 h−1 and a growth rate of 0.15 ± 0.01 h−1 in lab-scale bioreactors. Genome sequencing of this strain revealed a striking single nucleotide variation (SNV) upstream of the ald gene (NCgl2698, cg3096) encoding acetaldehyde dehydrogenase (ALDH). The mutated basepair was previously predicted to be part of the binding site for the global transcriptional regulator GlxR, and re-engineering demonstrated that the identified SNV is key for enhanced ethanol assimilation. Decreased binding of GlxR leads to increased synthesis of the rate-limiting enzyme ALDH, which was confirmed by proteomics measurements.ConclusionsThe established rbALE technology is generally applicable to any microbial strain and selection pressure that fits the small-scale cultivation format. In addition, our specific results will enable improved production processes with C. glutamicum from ethanol, which is of particular interest for acetyl-CoA-derived products.

Dynamics of ethanol and its metabolites in fruit: The impact of the temperature and fruit species

The objectives of this study were to: i) determine the effect of temperature on the ethanol vaporization rate, ii) determine the effect of temperature on the uptake rate of fruit species and cultivars, iii) understand the relationship between the temperature that ethanol is applied, ethylene metabolism, and respiration rate of fruit, and iv) evaluate the interaction between temperature that fruit are submitted to ethanol treatment on the metabolites produced by them as a response to ethanol treatment. The ethanol vaporization rate was performed in an empty container experiment at 2, 10, and 20 °C. Four experiments were conducted to identify the ethanol uptake rate in ‘Maxi Gala’ and ‘Cripps Pink’ apples, ‘BRS Isis’ table grapes, and ‘Rojo Brillante’ persimmons at 2, 10, and 20 °C. The metabolites produced by the fruit as a response to the ethanol treatment were determined using HS-SPME-GC/MS analysis. Our findings showed that the ethanol vaporization rate increases exponentially with the temperature and has a linear relationship with time. Moreover, the temperature and fruit species/cultivar affected the ethanol uptake rate, with the highest uptake rate at 20 °C. Furthermore, ethanol vapor uptake rate was positively correlated to respiration rate when all fruit species/cultivars were evaluated together, showing that fruit metabolism affects ethanol uptake rate. The temperature impacted the metabolite produced by fruit from ethanol application, evidencing the possibility of managing the volatile compounds produced from ethanol based on the ethanol treatment temperature. The fruit species has a decisive impact if ethanol is esterified or transformed to acetaldehyde, being predominantly esterified in apples and grapes, and converted to acetaldehyde in persimmons. Ethanol vapor treatment reduced the ethylene metabolism in apples at 20 °C, although ethanol increased ethylene in persimmons at 2 °C. Moreover, the respiration rate was generally unaffected or reduced in ethanol-treated fruit compared to untreated fruit of the same species/cultivar.

Fermentation and metabolic characteristics of Gluconacetobacter oboediens for different carbon sources

The metabolism of Gluconacetobacter oboediens was investigated in relation to different carbon sources for the continuous cultures at the dilution rate of 0.05 h(-1). The 13C-flux result implies the formation of metabolic recycles for the case of using glucose and acetate as carbon sources. When glucose and ethanol were used as carbon sources, the specific ethanol uptake rate and the specific acetate production rate increased as the feed ethanol concentration was increased from 40 to 60 g/l, while the specific CO2 production rate and the biomass concentration decreased, where the 13C-metabolic flux result indicates that the glycolysis, oxidative PP pathway, and the tricarboxylic acid (TCA) cycle were less active, resulting in less biomass concentration. The flux result also implies that oxaloacetate decarboxylase flux became negative, so that oxaloacetate is backed up by this pathway, resulting in less activity of glyoxylate pathway. When gluconate was added for the case of using glucose and ethanol as carbon sources, the acetate and cell concentrations as well as gluconate concentrations increased. The glucose and ethanol concentrations decreased concomitantly with the increased feed gluconate concentration. In accordance with these fermentation characteristics, the enzyme activity result indicates that glucose dehydrogenase and glucose-6-phosphate dehydrogenase pathways became less active, while the glycolysis and the TCA cycle was activated as the feed gluconate concentration was increased.

Bio-ethanol production to be blended with gasoline: Improvements in energy use by adsorption

Batch adsorption experiments were conducted to examine the liquid-phase adsorption of ethanol from ethanol-water solutions. Experiments performed established the kinetic and equilibrium behaviour of the various adsorbents in solution. The experiments with the ZSM-5 adsorbents indicate that the silica to alumina ratio had little effect on the ethanol-water separation at low ethanol concentrations. In general, ZSM-5 adsorbents were outperformed by the activated carbon adsorbents, which showed higher adsorption capacities. The capacity of activated carbon adsorbents correlated strongly with cumulative pore volume and Brunauer, Emmet and Teller (BET) surface area. Particle size was found to be the most influential factor in the ethanol uptake rate. The large pellets showed sluggish kinetics when compared to their powdered counterparts. When considering kinetic performance and adsorption capacity XTRUSORB A754 and M-30 activated carbon show the most potential for the selective adsorption of ethanol. The adsorbent screening performed herein applies to the energy efficient production of bio-ethanol via adsorption.

The role of radiation produced peroxides and superoxide in the radiation response of A549 cells

Rates of ethanol oxidation by perfused livers from fasted female rats were decreased from 82 ± 8 to 11 ± 7 μmol/g/hr by 4-methylpyrazole, an inhibitor of alcohol dehydrogenase. The subsequent addition of fatty acids of various chain lengths in the presence of 4-methylpyrazole increased rates of ethanol uptake markedly. Palmitate (1 mM) increased rates of ethanol oxidation to 95 ± 8 μmol/g/hr, while octanoate and oleate increased rates to 58 ± 11 and 68 ± 15 μmol/g/hr, respectively. Hexanoate, a short-chain fatty acid oxidized predominantly in the mitochondria, had no effect. Addition of oleate also increased the steady-state level of catalase-H2O2. Pretreatment of rats for 1.5 hours with 3-amino-1,2,4-triazole (1.0 g/kg), an inhibitor of catalase, prevented the ethanol-dependent decrease in the steady-state level of catalase-H2O2 completely. Under these conditions, aminotriazole decreased rates of ethanol oxidation by about 50% and blocked the stimulation of ethanol oxidation by fatty acids. Oleate decreased rates of aniline hydroxylation by about 50%, indicating that cytochrome P450 is not involved in the stimulation of ethanol uptake by fatty acids. Furthermore, oleate stimulated ethanol uptake in livers from ADH-negative deermice indicating that fatty acids do not simply displace 4-methylpyrazole from alcohol dehydrogenase. It is concluded that the stimulation of ethanol oxidation by fatty acids is due to increased H2O2 supplied by the peroxisomal β-oxidation of fatty acids for the catalase-H2O2 peroxidation pathway.

Effect of external pH on ethanol oxidation byCandida utilis

The external pH affects both ethanol and oxygen uptake rates by nongrowing cells of Candida utilis suspended either in distilled water or in phthalate buffer. The buffering properties of organic acids control the maximum rates of exogenous respiration and ethanol uptake. The substrate limitation of ethanol uptake rate and endogenous respiration rate increase proportionally with increasing hydrogen ion concentration in the medium.

Acute and chronic ethanol treatment in vivo increases malate-aspartate shuttle capacity in perfused rat liver.

The effects of acute and chronic treatment with ethanol on transport of reducing equivalents into mitochondria via the malate-aspartate shuttle were studied in perfused rat liver. The shuttle capacity was estimated from the decrease in rates of glucose production from the reduced substrate sorbitol caused by an increase in the NADH/NAD+ ratio in the cytosol due to metabolism of ethanol. The greater the capacity of the malate-aspartate shuttle, the smaller the inhibition of glucose synthesis by ethanol. Glucose synthesis was decreased about 2-fold less in livers from fasted rats treated acutely 2.5 h earlier with ethanol than in untreated controls. Chronic treatment with ethanol for 3-5 weeks prevented completely the decrease in glucose synthesis from sorbitol due to ethanol oxidation. Rates of ethanol uptake were elevated significantly from 69 +/- 7 mumols/g/h in livers from control rats up to 92 +/- 7 mumols/g/h in livers from SIAM rats. Similarly, rates of ethanol uptake were stimulated by chronic ethanol treatment from 71 +/- 6 to 222 +/- 15 mumols/g/h; this increase was largely sensitive to aminooxyacetate. Taken together, these data indicate that flux of reducing equivalents over the malate-aspartate shuttle is increased by both acute and chronic treatment with ethanol and that movement of reducing equivalents from the cytosol into the mitochondria via the malate-aspartate shuttle is an important rate determinant in hepatic ethanol oxidation.

Oscillatory behavior of Saccharomyces cerevisiae in continuous culture: II. Analysis of cell synchronization and metabolism

Sustained oscillations of biomass, ethanol, and ammonium concentrations, specific growth rate, and specific uptake rates of ethanol, ammonium, and oxygen were found in continuous cultures of Saccharomyces cerevisiae under controlled dissolved oxygen (DO), pH, and temperature conditions. The period of oscillations was approximately 2.5-3 h at a pH of 5.5 and 2-2.5 h at a pH of 6.5. Oscillations were observed only under conditions of low carbon (glucose below the minimum detectable level), nitrogen nutrient (ammonium concentration varied between 0.00001 and 0.0015M), and ethanol concentration (0.002-0.085 g/L) in the bioreactor.The oscillatory behavior at pH 5.5 was also characterized by partially synchronized cell growth and reproduction. Not only did the total percentage of budding cells oscillate with the same period as observed for the global biomass and nutrient concentrations, but the peaks in the individual subpopulations of initial budding, middle budding, and late budding cells appeared sequentially during the oscillation period. This provides strong evidence of the hypothesis that variations in metabolism during different periods in the cell cycle of a partially synchronized cell population are responsible for the observed oscillatory bioreactor behavior.The specific nutrient uptake rates for ammonium and oxygen as well as the net specific ethanol uptake rate oscillated with the same period as the biomass oscillations. These results show a dramatic increase in the ammonium and oxygen consumption rates prior to the initial budding of the synchronized subpopulation and a decrease in these rates during the late budding phase. At a pH of 5.5, the late budding phase is characterized by high specific ethanol productivity; however, the ethanol productivity lags the late budding phase at a pH pf 6.5. The observed time-varying metabolism in the oscillatory operating regime appears to be the result of the metabolic changes which occur during the cell cycle. Models which can predict the oscillatory biomass concentration and nutrient levels in this regime must be capable of predicting the concentrations and metabolic rates of the subpopulations as well.

Catalase-dependent ethanol oxidation in perfused rat liver. Requirement for fatty-acid-stimulated H2O2 production by peroxisomes.

The purpose of this study was to measure rates of catalase-dependent ethanol uptake and rates of H2O2 generation in perfused rat livers in the presence of fatty acids of varying chain length. Rates of ethanol uptake in livers from fasted rats, perfused in a recirculating system, of about 80 mumol g-1 h-1 were decreased to about 10 mumol g-1 h-1 by the addition of an inhibitor of alcohol dehydrogenase (ADH), 4-methylpyrazole. The medium-chain-length fatty acid, laurate (12:0; 1 mM), increased rates of 4-methylpyrazole-insensitive ethanol uptake maximally to 80-85 mumol g-1 h-1. Rates of ethanol uptake diminished as the chain length of fatty acid was decreased [hexanoate (6:0) = 23 mumol g-1 h-1; octanoate (8:0) = 55 mumol g-1 h-1; decanoate (10:0) = 65 mumol g-1 h-1] or increased [myristate (14:0) = 77 mumol g-1 h-1; palmitate (16:0) = 80 mumol g-1 h-1; stearate (18:0) = 29 mumol g-1 h-1; oleate (18:1) = 60 mumol g-1 h-1; erucate (22:3) = 22 mumol g-1 h-1] from 12:0. Oleate did not increase rates of hydroxylation of p-nitrophenol, a substrate for the ethanol-inducible form of cytochrome P-450, indicating that the stimulation of ethanol uptake by fatty acids was not due to increased mixed-function oxidation. The increase of ethanol uptake was also not due to displacement of 4-methylpyrazole from ADH by fatty acids, since oleate stimulated ethanol uptake by about 50% in perfused livers from deermice genetically deficient in ADH. The increase in 4-methylpyrazole-insensitive ethanol uptake by fatty acids was blocked by the catalase inhibitor, aminotriazole, indicating the involvement of catalase. Rates of H2O2 generation by livers perfused in a non-recirculating system with 1.7% albumin were increased from 6 +/- 1 to 23 +/- 5 mumol g-1 h-1 by oleate (1 mM). Because of the discrepancy between rates of ethanol metabolism and H2O2 production, methods were developed to measure H2O2 production in a recirculating perfusion system. H2O2 generation was determined from the time necessary for steady-state level of catalase-H2O2, measured spectrophotometrically (660-640 nm) through a lobe of the liver, to return to basal values after the addition of a known quantity of methanol, which is not metabolized by ADH in the rat.(ABSTRACT TRUNCATED AT 400 WORDS)

Identification of P-450 ALC in microsomes from alcohol dehydrogenase-deficient deermice: Contribution to ethanol elimination in vivo

Isozyme 3a of rabbit hepatic cytochrome P-450, also termed P-450 ALC, was previously isolated and characterized and was shown to be induced 3- to 5-fold by exposure to ethanol. In the present study, antibody against rabbit P-450 ALC was used to identify a homologous protein in alcohol dehydrogenase-negative (ADH −) and -positive (ADH +) deermice, Peromyscus maniculatus. The antibody reacts with a single protein having an apparent molecular weight of 52,000 on immunoblots of hepatic microsomes from untreated and ethanol-treated deermice from both strains. The level of the homologous protein was about 2-fold greater in microsomes from naive ADH − than from naive ADH + animals. Ethanol treatment induced the protein about 3-fold in the ADH + strain and about 4-fold in the ADH − strain. The antibody to rabbit P-450 ALC inhibited the microsomal metabolism of ethanol and aniline. The homologous protein, termed deermouse P-450 ALC, catalyzed from 70 to 80% of the oxidation of ethanol and about 90% of the hydroxylation of aniline by microsomes from both strains after ethanol treatment. The antibody-inhibited portion of the microsomal activities, which are attributable to the P-450 ALC homolog, increased about 3-fold upon ethanol treatment in the ADH + strain and about 4-fold in the ADH − strain, in excellent agreement with the results from immunoblots. The total microsomal P-450 content and the rate of ethanol oxidation were induced 1.4-fold and 2.2-fold, respectively, by ethanol in the ADH + strain and 1.9-fold and 3.3-fold, respectively, in the ADH − strain. Thus, the total microsomal P-450 content and ethanol oxidation underestimate the induction of the P-450 ALC homolog in both strains. A comparison of the rates of microsomal ethanol oxidation in vitro with rates of ethanol elimination in vivo indicates that deermouse P-450 ALC could account optimally for 3 and 8% of total ethanol elimination in naive ADH + and ADH − strains, respectively. After chronic ethanol treatment, P-450 ALC could account maximally for 8% of the total ethanol elimination in the ADH + strain and 22% in the ADH − strain. Further, cytochrome P-450 ALC appears to be responsible for about one-half of the increase in the rate of ethanol elimination in vivo after chronic treatment with ethanol. These results indicate that the contribution of P-450 ALC to ethanol oxidation in the deermouse is relatively small. Desferrioxamine had no effect on rates of ethanol uptake by perfused livers from ADH-negative deermice, indicating that ethanol oxidation by a hydroxyl radical-mediated mechanism was not involved in ethanol metabolism in this mutant. Peroxisomal β-oxidation capacity was increased 40% over control values by ethanol treatment, consistent with the hypothesis that the increase in ethanol elimination in the ADH-negative deermouse is mediated predominantly via catalase-H 2O 2.

Inhibition of catalase-dependent ethanol metabolism in alcohol dehydrogenase-deficient deermice by fructose.

Hepatic microsomal fractions from ADH (alcohol dehydrogenase)-negative deermice incubated with an NADPH-generating system metabolized butanol and ethanol at rates around 10 nmol/min per mg. In contrast, cytosolic catalase from ADH-negative deermouse liver oxidized ethanol, but not butanol, when incubated with an H2O2-generating system. Thus butanol is oxidized by cytochrome P-450 in microsomal fractions, but not by cytosolic catalase, in tissues from ADH-negative deermice. In perfused livers from ADH-negative deermice, rates of ethanol uptake at low concentrations of ethanol (1.5 mM) were about 60 mumol/h per g, yet butanol (1.5 mM) uptake was undetectable (less than 4 mumol/h per g). At higher concentrations of alcohol (25-30 mM), rates of ethanol uptake were about 80 mumol/h per g, whereas rates of butanol uptake were only about 9 mumol/h per g. Because rates of butanol metabolism via cytochrome P-450 in deermice were more than an order of magnitude lower than rates of ethanol uptake in livers from ADH-negative deermice, it is concluded that ethanol uptake by perfused livers from ADH-negative deermice is catalysed predominantly via catalase-H2O2. In support of this conclusion, rates of H2O2 generation, which are rate-limiting for the peroxidation of ethanol by catalase, were about 65 mumol/h per g in livers from ADH-negative deermice, values similar to rates of ethanol uptake of about 60 mumol/h per g measured under identical conditions. Rates of ethanol uptake by perfused livers from ADH-positive, but not from ADH-negative, deermice were increased by about 50% by infusion of fructose. Thus it is concluded that the stimulation of hepatic ethanol uptake by fructose is dependent on the presence of ADH. Unexpectedly, fructose decreased rates of ethanol metabolism and H2O2 generation by about 60% in perfused livers from ADH-negative deermice, probably by decreasing activation of fatty acids and thus diminishing rates of peroxisomal beta-oxidation.

Fatty acid-dependent ethanol metabolism

Rates of ethanol oxidation by perfused livers from fasted female rats were decreased from 82 ± 8 to 11 ± 7 μmol/g/hr by 4-methylpyrazole, an inhibitor of alcohol dehydrogenase. The subsequent addition of fatty acids of various chain lengths in the presence of 4-methylpyrazole increased rates of ethanol uptake markedly. Palmitate (1 mM) increased rates of ethanol oxidation to 95 ± 8 μmol/g/hr, while octanoate and oleate increased rates to 58 ± 11 and 68 ± 15 μmol/g/hr, respectively. Hexanoate, a short-chain fatty acid oxidized predominantly in the mitochondria, had no effect. Addition of oleate also increased the steady-state level of catalase-H 2O 2. Pretreatment of rats for 1.5 hours with 3-amino-1,2,4-triazole (1.0 g/kg), an inhibitor of catalase, prevented the ethanol-dependent decrease in the steady-state level of catalase-H 2O 2 completely. Under these conditions, aminotriazole decreased rates of ethanol oxidation by about 50% and blocked the stimulation of ethanol oxidation by fatty acids. Oleate decreased rates of aniline hydroxylation by about 50%, indicating that cytochrome P 450 is not involved in the stimulation of ethanol uptake by fatty acids. Furthermore, oleate stimulated ethanol uptake in livers from ADH-negative deermice indicating that fatty acids do not simply displace 4-methylpyrazole from alcohol dehydrogenase. It is concluded that the stimulation of ethanol oxidation by fatty acids is due to increased H 2O 2 supplied by the peroxisomal β-oxidation of fatty acids for the catalase-H 2O 2 peroxidation pathway.

The respirative breakdown of glucose by Saccharomyces cerevisiae: an assessment of a physiological state.

Cells of Saccharomyces cerevisiae exhibiting respirative glucose metabolism in continuous culture were able to use ethanol as a co-substrate. The ethanol uptake rate was dependent on the residual respirative capacity of the cells. The activities of gluconeogenic enzymes and of malate dehydrogenase were higher in cells degrading glucose respiratively than in cells metabolizing glucose respiro-fermentatively, but were lower than in cells growing on ethanol only. The pattern of distribution of the mitochondrial cytochromes was similar but the differences were less distinct. In synchronously growing cells, the activities of gluconeogenic enzymes and of malate dehydrogenase oscillated, with activities increasing during the budding phase. The increase was preceded by the appearance of ethanol in the culture medium.

Effect of dietary fat upon ethanol metabolism in rats

The effect of dietary fat upon ethanol metabolism was studied in rats. Wistar strain male rats were divided into four groups according to diet, namely alcohol-high fat, alcohol-low fat, control-high fat, and control-low fat. After 4 weeks of feeding, blood ethanol levels following an intraperitoneal injection of 0.2 g ethanol/100 g of body weight were measured. The disappearance rate of blood ethanol was faster and the metabolic rate of ethanol was significantly greater in the alcohol-high fat group compared to the alcohol-low fat or non-alcoholic groups. Microsomal enzymes, such as the microsomal ethanol-oxidizing system, aniline hydroxylase. and glucose-6-phosphatase, were significantly higher in the alcohol-high fat group than in the alcohol-low fat or non-alcoholic groups. The ethanol uptake rate of the isolated perfused liver was increased significantly in the alcoholic groups. In the alcoholic rats, the high fat group showed significantly higher uptake than the low fat group. Although the ethanol uptake rate after 4-methylpyrazole treatment was not significantly different among the four groups, its fraction of the total ethanol uptake was increased significantly in the alcohol-high fat group. These results suggest that high fat diets accelerate ethanol metabolism through the microsomal ethanoloxidizing system.

Characteristics of Ethanol and Acetaldehyde Oxidation on Flavin and Pyridine Nucleotide Fluorescence Changes in Perfused Rat Liver

Abstract Data presented in this paper describe how the percentage decomposition of the catalase-H2O2 intermediate by methanol and ethanol as determined from absorbance changes (660 to 640 nm) through a lobe of the perfused rat liver can be used to calculate the intracellular ethanol concentration. Values obtained were similar to those measured in the effluent fluid from the liver. Using this technique, a value of 0.09 mm for the apparent Km of alcohol dehydrogenase in situ for ethanol and a Vmax value of 1.25 µmol per min per g, wet weight of liver were calculated. The hepatic ethanol concentration giving half-maximal reduction of the pyridine nucleotides as determined from pyridine nucleotide fluorescence changes (366-nm excitation, 460-nm emission) was 0.07 mm. When arterial fluid ethanol concentrations were used, the apparent Km for ethanol was about 0.4 mm. The apparent Km values for ethanol measured in situ are 5- to 20-fold lower than values measured with isolated rat liver alcohol dehydrogenase, indicating that ethanol metabolism by the liver is not directly limited by alcohol dehydrogenase activity but rather by the rate of reoxidation of cytosolic NADH. At the low rates of hydrogen peroxide production observed in substrate-free perfused livers, rates of ethanol oxidation via alcohol dehydrogenase calculated from the different effectiveness of ethanol and methanol in decomposing the catalase-H2O2 intermediate were similar to rates of ethanol uptake measured directly from arteriovenous concentration differences. Changes of flavin and pyridine nucleotide fluorescence as measured by surface fluorometry were compared with changes in the ratios of lactate to pyruvate and β-hydroxybutyrate to acetoacetate in the effluent fluid perfusing the liver. Titrations with octanoate to reduce the pyridine nucleotides in the mitochondrial compartment showed that the oxidation-reduction state of the fluorescent flavoproteins was in rapid equilibrium with the ratio of β-hydroxybutyrate to acetoacetate. A comparison of pyridine nucleotide oxidation-reduction changes during ethanol infusion, and titrations with acetaldehyde in the absence and presence of 4-methylpyrazole (an inhibitor of alcohol dehydrogenase), showed that at acetaldehyde concentrations above 0.2 to 0.4 mm a dismutation between alcohol and acetaldehyde dehydrogenase occurred in the cytosolic compartment. At lower acetaldehyde concentrations commensurate with those occurring during ethanol oxidation, the evidence indicates that acetaldehyde is normally oxidized in the mitochondrial compartment.