Effects of glycerol on the thermal dependence of the stability of human erythrocytes

Incubation of human blood in saline solution of 0–36% (v/v) ethanol for 30 min produces lysis or stabilization of erythrocytes depending on the ethanol concentration. Under less elevated concentrations of ethanol, erythrocytes are present in expanded shapes (R state) that present lower stability and suffer lysis with increase in the ethanol concentration. Under more elevated concentrations of ethanol, erythrocytes are present in contracted shapes (T state) that have higher stability and suffer lysis at even more elevated ethanol concentrations. This work evaluated the effects of glycerol (0 to 2.0 M) and temperature (7 to 47°C) on the stability of the R erythrocytes, characterized by the ethanol concentration at the mid-transition point (D _50R ) of the hemolysis curve (D _50R ). D _50R declined sigmoidally with increase in the glycerol concentration or temperature, due to transition of the R to the T state erythrocytes. In 1.5 M glycerol, the erythrocytes stability decreased below 32 but increased above 37°C. The combination of temperature, glycerol and ethanol actions generates a critical value of osmotic pressure below which the R state predominates and above which the T state predominates. At 7°C 1.5 M glycerol decreased the erythrocytes stability against ethanol but increased the erythrocytes stability against hypotonic shock. Those conditions favor the R state, which has a lower stability against ethanol; however, in the absence of ethanol, glycerol determines less water entrance in the erythrocytes, making more difficult its lysis by hypotonicity.     

Temperature profiles of growth and ethanol tolerance of the xylose-fermenting yeasts Candida shehatae and Pichia stipitis

Summary The effect of different ethanol concentrations on the growth of Candida shehatae and Pichia stipitis with xylose as substrate was evaluated in a temperature gradient incubator. The upper limit of the temperature profiles of ethanol tolerance of both yeast strains were similar, although P. stipitis appeared to have a slightly higher ethanol tolerance in the higher temperature range. An increase in the ethanol concentration severely depressed the maximum growth temperature, and also increased the minimum growth temperature slightly. The ethanol tolerance limit of 46–48 g·l^-1 occurred within a narrow temperature plateau of 11 to 22° C. The low ethanol tolerance of these pentose fermenting yeasts is detrimental for commercial ethanol production from hemicellulose hydrolysates.     

Ethanol disrupts the formation of hypochord and dorsal aorta during the development of embryonic zebrafish

Exposure to ethanol during human embryonic period has severe teratogenic effects on the cardiovascular system. In our study, we demonstrated that ethanol of gradient concentrations can interfere with the establishment of circulatory system in embryonic zebrafish. The effective concentration to cause 50% malformations (EC₅₀) was 182.5 mmol/L. The ethanol pulse exposure experiment displayed that dome stage during embryogenesis is the sensitive time window to ethanol. It is found that 400 mmol/L ethanol pulse exposure can induce circulatory defects in 43% treated embryos. We ruled out the possibility that ethanol can interfere with the process of hematopoiesis in zebrafish. By employing in situ hybridization with endothelial biomarker (Flk-1), we revealed that ethanol disrupts the establishment of trunk axial vasculature, but has no effect on cranial vessels. Combined with the results of semi-thin histological sections, the in situ hybridization experiments with arterial and venous biomarkers (ephrinB2, ephB4) suggested that ethanol mainly interrupts the development of dorsal aorta while has little effect on axial vein. Further study indicated the negative influence of ethanol on the development of hypochord in zebrafish. The consequent lack of vasculogenic factors including Radar and Ang- 1 partly explains the defects in formation and integrity of dorsal aorta. These results provide important clues to the study of adverse effects of ethanol on the cardiovascular development in human fetus.     

Effects of temperature on microbial ethanol production I. The temperature profile curve of ethanol production of the yeast strain saccharomyces cerevisiae Sc 5

The temperature-profile curve of ethanol production of the yeast Saccharomyces cerevisiae Sc 5 is shown. Accordingly the biokinetic sphere of ethanol formation consists of 5 ranges. The maximum specific ethanol formation rate v₀ is reached within the temperature limits of 32°C ≦ T ≦ 36°C, and the maximum temperature for ethanol formation amounts to T″_max = 57°C. Within the first suboptimum temperature range ethanol formation is not retarded thermally. Using a modified ARRHENIUS equation the activation energy of ethanol formation was calculated to be ΔH_ÊtOH = (78.5 ± 2.2) kJ/mol.     

Scn4b regulates the hypnotic effects of ethanol and other sedative drugs

The voltage‐gated sodium channel subunit β4 (SCN4B) regulates neuronal activity by modulating channel gating and has been implicated in ethanol consumption in rodent models and human alcoholics. However, the functional role for Scn4b in ethanol‐mediated behaviors is unknown. We determined if genetic global knockout (KO) or targeted knockdown of Scn4b in the central nucleus of the amygdala (CeA) altered ethanol drinking or related behaviors. We used four different ethanol consumption procedures (continuous and intermittent two‐bottle choice (2BC), drinking‐in‐the dark and chronic intermittent ethanol vapor) and found that male and female Scn4b KO mice did not differ from their wild‐type (WT) littermates in ethanol consumption in any of the tests. Knockdown of Scn4b mRNA in the CeA also did not alter 2BC ethanol drinking. However, Scn4b KO mice showed longer duration of the loss of righting reflex induced by ethanol, gaboxadol, pentobarbital and ketamine. KO mice showed slower recovery to basal levels of handling‐induced convulsions after ethanol injection, which is consistent with the increased sedative effects observed in these mice. However, Scn4b KO mice did not differ in the severity of acute ethanol withdrawal. Acoustic startle responses, ethanol‐induced hypothermia and clearance of blood ethanol also did not differ between the genotypes. There were also no functional differences in the membrane properties or excitability of CeA neurons from Scn4b KO and WT mice. Although we found no evidence that Scn4b regulates ethanol consumption in mice, it was involved in the acute hypnotic effects of ethanol and other sedatives.     

Inhibition of ethanol-producing yeast and bacteria by degradation products produced during pre-treatment of biomass

An overview of the different inhibitors formed by pre-treatment of lignocellulosic materials and their inhibition of ethanol production in yeast and bacteria is given. Different high temperature physical pre-treatment methods are available to render the carbohydrates in lignocellulose accessible for ethanol fermentation. The resulting hydrolyzsates contain substances inhibitory to fermentation—depending on both the raw material (biomass) and the pre-treatment applied. An overview of the inhibitory effect on ethanol production by yeast and bacteria is presented. Apart from furans formed by sugar degradation, phenol monomers from lignin degradation are important co-factors in hydrolysate inhibition, and inhibitory effects of these aromatic compounds on different ethanol producing microorganisms is reviewed. The furans and phenols generally inhibited growth and ethanol production rate (Q_EtOH) but not the ethanol yields (Y_EtOH) in Saccharomyces cerevisiae. Within the same phenol functional group (aldehyde, ketone, and acid) the inhibition of volumetric ethanol productivity was found to depend on the amount of methoxyl substituents and hence hydrophobicity (log P). Many pentose-utilizing strains Escherichia coli, Pichia stipititis, and Zymomonas mobilis produce ethanol in concentrated hemicellulose liquors but detoxification by overliming is needed. Thermoanaerobacter mathranii A3M3 can grow on pentoses and produce ethanol in hydrolysate without any need for detoxification.     

Influence of the biogenic amine tyramine on ethanol‐induced behaviors in Drosophila

The biogenic amine tyramine has been implicated in drug‐induced behavior. The Drosophila inactive mutant is characterized by reduced tyramine and octopamine levels and is defective in cocaine sensitization. To test whether there is an overlap in the use of the amine neurotransmitter system in ethanol‐ and cocaine‐induced behaviors, mutant analyses were extended to the phenotypic characterization of inactive and other mutants effecting the tyramine and octopamine neurotransmitter system. The inactive mutant displays increased ethanol sensitivity and is impaired in the initial startle response upon ethanol application. Furthermore, this mutant fails to regulate its alcohol‐induced hyperactivity properly. In contrast to the defects seen after cocaine application, inactive mutants develop normal ethanol tolerance and sensitize to the locomotor activating effect of ethanol. The tyramine‐β‐hydroxylase mutant (TβH) with increased tyramine and depleted octopamine levels displays normal ethanol sensitivity, a startle repression, and hyperactivates more in response to ethanol. In addition, TβH mutants fail to develop a tolerance to the hyperactivating effect of ethanol. Ethanol‐induced sensitization does not seem to be impaired in either mutant, suggesting that tyramine is not required for this process. The comparative analysis of the phenotypes associated with inactive and TβH mutants suggests that the fine tuning of ethanol‐induced hyperactivity can be correlated with different tyramine levels. Defects in other aspects of ethanol‐induced behaviors might be due to different molecules or mechanisms. © 2005 Wiley Periodicals, Inc. J Neurobiol, 2005     

The effect of aeration on xylose fermentation by Candida shehatae and Pachysolen tannophilus

Summary The ability of a Candida shehatae and a Pachysolen tannophilus strain to ferment D-xylose to ethanol was evaluated in defined and complex media under different levels of aeration. Aeration enhanced the ethanol productivity of both yeasts considerably. C. shehatae maintained a higher fermentation rate and ethanol yield than P. tannophilus over a wide range of aeration levels. Ethanol production by C. shehatae commenced during the early stage of the fermentation, whereas with P. tannophilus there was a considerable lag between the initiation of growth and ethanol production. Both yeasts produced appreciable quantities of xylitol late in the fermentation. P. tannophilus failed to grow under anoxic conditions, producing a maximum of only 0.5 g · l^-1 ethanol. In comparison, C. shehatae exhibited limited growth in anoxic cultures, and produced ethanol much more rapidly. Under the condition of aeration where C. shehatae exhibited the highest ethanol productivity, the fermentation parameters were: maximum specific growth rate, 0.15 h^-1; maximum volumetric and specific rates of ethanol production, 0.7 g (l · h)^-1 and 0.34 g ethanol (g cells · h)^-1 respectively; ethanol yield, 0.36 g (g xylose)^-1. The best values obtained with P. tannophilus were: maximum specific growth rate, 0.14 h^-1; maximum volumetric and specific rates of ethanol production, 0.22 g (l · h)^-1 and 0.07 h^-1 respectively; ethanol yield coefficient, 0.28. Because of its higher ethanol productivity at various levels of aeration, C. shehatae has a greater potential for ethanol production from xylose than P. tannophilus.     

Inhibition effects of ethanol concentration history and ethanol concentration change rate onZymomonas mobilis

Summary The inhibition effects onZymomonas mobilis of ethanol concentration history (time-integrated exposure to ethanol) and ethanol concentration change rate have been investigated. It was found that the ethanol concentration history had no significant effect on the fermentative capability ofZ. mobilis, while the ethanol concentration change rate had a quite intense inhibitory effect.     

The role of ethanol oxidation during carboxydotrophic growth of Clostridium autoethanogenum

The Wood–Ljungdahl pathway is an ancient metabolic route used by acetogenic carboxydotrophs to convert CO into acetate, and some cases ethanol. When produced, ethanol is generally seen as an end product of acetogenic metabolism, but here we show that it acts as an important intermediate and co-substrate during carboxydotrophic growth of Clostridium autoethanogenum. Depending on CO availability, C. autoethanogenum is able to rapidly switch between ethanol production and utilization, hereby optimizing its carboxydotrophic growth. The importance of the aldehyde ferredoxin:oxidoreductase (AOR) route for ethanol production in carboxydotrophic acetogens is known; however, the role of the bifunctional alcohol dehydrogenase AdhE (Ald–Adh) route in ethanol metabolism remains largely unclear. We show that the mutant strain C. autoethanogenum ∆adhE1a, lacking the Ald subunit of the main bifunctional aldehyde/alcohol dehydrogenase (AdhE, CAETHG_3747), has poor ethanol oxidation capabilities, with a negative impact on biomass yield. This indicates that the Adh–Ald route plays a major role in ethanol oxidation during carboxydotrophic growth, enabling subsequent energy conservation via substrate-level phosphorylation using acetate kinase. Subsequent chemostat experiments with C. autoethanogenum show that the wild type, in contrast to ∆adhE1a, is more resilient to sudden changes in CO supply and utilizes ethanol as a temporary storage for reduction equivalents and energy during CO-abundant conditions, reserving these ‘stored assets’ for more CO-limited conditions. This shows that the direction of the ethanol metabolism is very dynamic during carboxydotrophic acetogenesis and opens new insights in the central metabolism of C. autoethanogenum and similar acetogens.     

Contributions of immobilized and free cells of salt-tolerantZygosaccharomyces rouxii andCandida versatilis to the production of ethanol and 4-ethylguaiacol

Summary The contribution of immobilized cells and free cells released from gel beads to ethanol production by the salt-tolerant yeastsZygosaccharomyces rouxii andCandida versatilis, and 4-ethylguaiacol (4-EG) production byC. versatilis were investigated using an airlift reactor. The amounts of ethanol produced by free cells were about 65% and about 90% of total ethanol in the reactor forZ. rouxii andC. versatilis, respectively. It was found that immobilized cells gave a much lower specific productivity of ethanol (ethanol production per hour per cell) than free cells of both yeasts, especially ofC. versatilis. 4-EG was produced mainly by immobilized cells ofC. versatilis; the amount of 4-EG produced by free cells was about 20% of the total 4-EG, in contrast to the results of ethanol production. However, the specific productivity of 4-EG (4-EG production per hour per cell) by immobilized and free cells was fairly similar.     

Relationship Between Effect of Ethanol on Proton Flux Across Plasma Membrane and Ethanol Tolerance, inPichia stipitis

Pichia stipitisefficiently converts glucose or xylose into ethanol but is inhibited by ethanol concentrations exceeding 30 g/L. InSaccharomyces cerevisiae, ethanol has been shown to alter the movement of protons into and out of the cell. InP. stipitisthe passive entry of protons into either glucose- or xylose-grown cells is unaffected at physiological ethanol concentrations. In contrast, active proton extrusion is affected differentially by ethanol, depending on the carbon source catabolized. In fact, in glucose-grown cells, the H⁺-extrusion rate is reduced by low ethanol concentrations, whereas, in xylose-grown cells, the H⁺-extrusion rate is reduced only at non-physiological ethanol concentrations. Thus, the ethanol inhibitory effect on growth and ethanol production, in glucose-grown cells, is probably caused by a reduction in H⁺-extrusion. Comparison of the rates of H⁺-flux with the relatedin vitroH⁺-ATPase activity suggests a new mechanism for the regulation of the proton pumping plasma membrane ATPase (EC 3.6.1.3) ofP. stipitis, by both glucose and ethanol. Glucose activates both the ATP hydrolysis and the proton-pumping activities of the H⁺-ATPase, whereas ethanol causes an uncoupling between the ATP hydrolysis and the proton-pumping activities. This uncoupling may well be the cause of ethanol induced growth inhibition of glucose grownP. stipitiscells.     

Isolation and characterization of ethanol-tolerant mutants of Escherichia coli KO11 for fuel ethanol production

Genetically engineered Escherichia coli KO11 is capable of efficiently producing ethanol from all sugar constituents of lignocellulose but lacks the high ethanol tolerance of yeasts currently used for commercial starch-based ethanol processes. Using an enrichment method which selects alternatively for ethanol tolerance during growth in broth and for ethanol production on solid medium, mutants of KO11 with increased ethanol tolerance were isolated which can produce more than 60 g ethanol L⁻¹ from xylose in 72 h. Ethanol concentrations and yields achieved by the LY01 mutant with xylose exceed those reported for recombinant strains of Saccharomyces and Zymomonas mobilis, both of which have a high native ethanol tolerance. Received 18 September 1997/ Accepted in revised form 07 January 1998     

Production of ethanol from sugar cane molasses by Zymomonas mobilis

Summary Zymomonas mobilis strains were compared with each other and with a Saacharomyces cerevisiae strain for the production of ethanol from sugar cane molasses in batch fermentations. The effect of pH and temperature on ethanol production by Zymomonas was studied. The ability of Z. mobilis to produce ethanol from molasses varied from one strain to another. At low sugar concentrations Zymomonas compared favourably with S. cerevisiae. However, at higher sugar concentrations the yeast produced considerably more ethanol than Zymomonas.     

Effect of temperature on ethanol tolerance of thermotolerant Isshatchenkia orientalis IPE100

Tolerance to high temperature and ethanol is a major factor in high‐temperature bio‐ethanol fermentation. The inhibitory effect of exogenously added ethanol (0–100 g L^?1) on the growth of the newly isolated thermotolerant Issatchenkia orientalis IPE100 was evaluated at a range of temperatures (30–45°C). A generalized Monod equation with product inhibition was used to quantify ethanol tolerance, and it correlated well with the experimental data on microbial growth inhibition of ethanol at the temperatures of 30–45°C. The maximum inhibitory concentration of ethanol for growth (P_m) and toxic power (n) at the optimal growth temperature of 42°C were estimated to be 96.7 g L^?1 and 1.23, respectively. The recently isolated thermotolerant I. orientalis IPE100 shows therefore a strong potential for the development of future high‐temperature bio‐ethanol fermentation technologies. This study provides useful insights into our understanding of the temperature‐dependent inhibitory effects of ethanol on yeast growth.     

Enzymatic responses of Drosophila melanogaster to long- and short-term exposures to ethanol

The effects of environmental ethanol on larva-to-pupa survival and on the activities of four enzymes were investigated in three Drosophila melanogaster strains. The strains had different allelic combinations at the Odh and Aldox loci on their third chromosomes, but they all carried the Adh ^S -Gpdh ^F allelic combination on the second chromosome. Replicates of each of the strains were exposed to three different ethanol treatments: (i) no ethanol in the medium (control); (ii) 5% ethanol for a single generation (short-term exposure); (iii) 5% ethanol for 20 generations (long-term exposure). In all experiments, the activities of four enzymes (ADH, ODH, GPDH and AOX) were measured in larvae, pupae and adults. The results showed that (i) the larval and adult metabolic responses to environmental ethanol were different; (ii) enzyme activity changes under short-term exposure differed from those measured under long-term exposure; (iii) the activities of the allozymes common to all strains (ADH-S and GPDH-F), differed depending on the genetic background. Changes in larva-to-pupa survival were seen when the larvae of control and exposed lines of the three strains were confronted with various concentrations of ethanol. In all three strains, the exposed lines had significantly higher initial survival rate and ethanol tolerance than the control lines. Strain-specific differences were observed in the ethanol tolerance of both types of line. Received: 26 November 1996 / Accepted: 14 February 1997