Efeito do tamanho do inóculo na fermentaçäo alcoólica por Zymomonas mobilis ZAP / Effects of inoculum size on alcoholic fermentation by Zymomonas mobilis ZAP

The effects of inoculon size, centrifuged or not, on the batch alcoholit fermentation by Zymomonas mobilis (strain ZAP) were studied. The results show that the ethanol yeld factor, the "economical" and the biochemical" efficiencies and the productivity increase with the elevation of inoculum independently of the inoculation methodology (centrifuged or not). The specific rates of cells growth (micronx), glucose uptake (microngs) and ethanol production (microngp) decrease with the elevation of inoculum level. It was observed an apparente limitation of growth with the increase above 20% (0,50 g/l) of the uncentrifuged inoculum

The effect of temperature on the formation of ethanol by Candida albicans in blood.

The effect of temperature on microbial fermentation in blood was studied. Specimens of human blood from a blood bank were inoculated with Candida albicans, an organism capable of causing fermentation. A preservative was added to a portion of the inoculated specimens. These inoculated specimens, as well as uninoculated blood, were stored under various temperature conditions. Production of ethyl alcohol was monitored over a period of six months. Fermentation was found to be highly temperature dependent, with refrigeration proving to be most effective at inhibiting ethanol formation.

Influence of substrate carbon on the metabolism of Clostridium thermohydrosulfuricum.

The concentration of carbon sources has a significant influence on the growth, carbohydrate uptake and metabolite distribution in Clostridium thermohydrosulfuricum. The growing concentrations of glucose or starch increase the production of ethanol and lactate, the intracellular fructose-1,6-diphosphate (FDP) and the specific activity of lactate dehydrogenase (LDH), but decrease the ethanol/lactate ratio.

Nutrition evaluation and mutagenicity testing of freeze-dried distiller's grains with solubles.

Distiller's grains with solubles (DGS) was obtained from a commercial fermentation of 90% corn and 10% wheat for ethanol production. The DGS was freeze-dried and proximate analysis resulted in 17.6% protein, 5.5% fat, 3.0% ash, 17.3% moisture, 21.6% neutral detergent fiber, and 35% carbohydrate by difference. Fatty acid analysis and amino acid analysis were similar to those of corn. Tryptophan was the limiting amino acid with a chemical score of 47. Protein Efficiency Ratio (PER) was 1.49. Mutagenicity testing of lipid and aqueous extracts of DGS in the Ames Salmonella mammalian microsome assay were negative.

Metabolic responses of Saccharomyces cerevisiae CBS 8066 and Candida utilis CBS 621 upon transition from glucose limitation to glucose excess.

When chemostat cultures of Saccharomyces cerevisiae CBS 8066 and Candida utilis CBS 621, grown under glucose limitation, were pulsed with excess glucose, both organisms initially exhibited similar rates of glucose and oxygen consumption. However, striking differences were apparent between the two yeasts with respect to the production of cell mass in the culture and metabolite excretion. Upon transition from glucose limitation to glucose excess, S. cerevisiae produced much ethanol but the growth rate remained close to that under glucose limitation. C. utilis, on the other hand, produced little ethanol and immediately started to accumulate cell mass at a high rate. This high production rate of cell mass was probably due to synthesis of reserve material and not caused by a high rate of protein synthesis. Upon a glucose pulse both yeasts excreted pyruvate. In contrast to C. utilis, S. cerevisiae also excreted various tricarboxylic acid cycle intermediates, both under steady-state conditions and after exposure to glucose excess. These results and those of theoretical calculations on ATP flows support the hypothesis that the ethanol production as a consequence of pyruvate accumulation in S. cerevisiae, occurring upon transition from glucose limitation to glucose excess, is caused by a limited capacity of assimilatory pathways.

Effects of elevated temperature on growth, respiratory-deficient mutation, respiratory activity, and ethanol production in yeast.

Some mesophilic yeasts and a thermotolerant strain of Saccharomyces cerevisiae were found to grow at 40 degrees C in complex media containing 1% yeast extract when an inoculum of 10(6) or more cells.mL-1 was used. Yeast extract (6%) permitted Saccharomyces cerevisiae to grow at 40 degrees C even with a smaller inoculum size (10(5) cells.mL-1). The fraction of respiratory-deficient (petite) mutants in 40 degrees C grown culture was less than 10% except for the thermotolerant strain, which showed greatly increased levels depending on culture conditions. Seven of eight yeast strains exhibited extremely reduced cytochrome oxidase activity when grown at 40 degrees C irrespective of the frequency of the petite mutation. In contrast, the accumulation of ethanol in the medium and the ethanol-producing activity of the cells were not affected by growth at 40 degrees C.

Ethanol production by thermophilic bacteria: biochemical basis for ethanol and hydrogen tolerance in Clostridium thermohydrosulfuricum.

The metabolic and enzymatic bases for growth tolerance to ethanol (4%) and H2 (2 atm [1 atm = 101.29 kPa]) fermentation products in Clostridium thermohydrosulfuricum were compared in a sensitive wild-type strain and an insensitive alcohol-adapted strain. In the wild-type strain, ethanol (4%) and H2 (2 atm) inhibited glucose but not pyruvate fermentation parameters (growth and end product formation). Inhibition of glucose fermentation by ethanol (4%) in the wild-type strain was reversed by addition of acetone (1%), which lowered H2 and ethanol production while increasing isopropanol and acetate production. Pulsing cells grown in continuous culture on glucose with 5% ethanol or 1 atm of H2 significantly raised the NADH/NAD ratio in the wild-type strain but not in the alcohol-adapted strain. Analysis of key oxidoreductases demonstrated that the alcohol-adapted strain lacked detectable levels of reduced ferredoxin-linked NAD reductase and NAD-linked alcohol dehydrogenase activities which were present in the wild-type strain. Differences in the glucose fermentation product ratios of the two strains were related to differences in lactate dehydrogenase and hydrogenase levels and sensitivity of glyceraldehyde 3-phosphate dehydrogenase activity to NADH inhibition. A biochemical model is proposed which describes a common enzymatic mechanism for growth tolerance of thermoanaerobes to moderate concentrations of both ethanol and hydrogen.

The control of glycogen metabolism in yeast. 1. Interconversion in vivo of glycogen synthase and glycogen phosphorylase induced by glucose, a nitrogen source or uncouplers.

The addition of glucose to a suspension of yeast initiated glycogen synthesis and ethanol formation. Other effects of the glucose addition were a transient rise in the concentration of cyclic AMP and a more prolonged increase in the concentration of hexose 6-monophosphate and of fructose 2,6-bisphosphate. The activity of glycogen synthase increased about 4-fold and that of glycogen phosphorylase decreased 3-5-fold. These changes could be reversed by the removal of glucose from the medium and induced again by a new addition of the sugar. These effects of glucose were also obtained with glucose derivatives known to form the corresponding 6-phosphoester. Similar changes in glycogen synthase and glycogen phosphorylase activity were induced by glucose in a thermosensitive mutant deficient in adenylate cyclase (cdc35) when incubated at the permissive temperature of 26 degrees C, but were much more pronounced at the nonpermissive temperature of 35 degrees C. Under the latter condition, glycogen synthase was nearly fully activated and glycogen phosphorylase fully inactivated. Such large effects of glucose were, however, not seen in another adenylate-cyclase-deficient mutant (cyr1), able to incorporate exogenous cyclic AMP. When a nitrogen source or uncouplers were added to the incubation medium after glucose, they had effects on glycogen metabolism and on the activity of glycogen synthase and glycogen phosphorylase which were directly opposite to those of glucose. By contrast, like glucose, these agents also caused, under most experimental conditions, a detectable rise in cyclic AMP concentration and a series of cyclic-AMP-dependent effects such as an activation of phosphofructokinase 2 and of trehalase and an increase in the concentration of fructose 2,6-bisphosphate and in the rate of glycolysis. Under all experimental conditions, the rate of glycolysis was proportional to the concentration of fructose 2,6-bisphosphate. Uncouplers, but not a nitrogen source, also induced an activation of glycogen phosphorylase and an inactivation of glycogen synthase when added to the cdc35 mutant incubated at the restrictive temperature of 35 degrees C without affecting cyclic AMP concentration.

Ethanol tolerance and the induction of stress proteins by ethanol in Candida albicans.

Ethanol is one of the products of the metabolism of glucose by Candida albicans. The amount produced is directly related to the concentration of glucose in the medium. The fungus utilizes ethanol as a sole source of carbon but is relatively intolerant of ethanol in its environment. Ethanol induces germ tube formation by blastoconidia of C. albicans. Germination was not seen under fermentation conditions even though the amount of ethanol produced was in the range form stress proteins that are similar to heat shock proteins. The possibility that stress proteins may regulate germ tube formation by C. albicans is discussed.

Influence of gamma radiation on ethanol production from yeast.

The effect of up to 6,000 Gray (Gy; 1 Gy = 1 J/k) 60Co gamma irradiation on the fermentative capacity of two strains of yeast cells is reported. Ethanol production by the irradiated cells was unchanged for both strains at 3,000 Gy and reduced 43% for only one strain at 6,000 Gy in spite of a marked decrease in viability at higher doses (2-8% at 3,000 Gy and 0.01% at 6,000 Gy). These results suggest that the yeast fermentation system for converting sugar to alcohol is a relatively radioresistant process and not inhibited by the stable by-products produced during irradiation. Furthermore, these data indicate that radiation polymerization for immobilizing these cells should not interfere with their fermentation capacity.

A comparative study of aldehyde dehydrogenase and alcohol dehydrogenase activities in crucian carp and three other vertebrates: apparent adaptations to ethanol production.

In the final step of the pathway producing ethanol in anoxic crucian carp (Carassius carassius L.), acetaldehyde is reduced to ethanol by alcohol dehydrogenase. The presence of aldehyde dehydrogenase in the tissues responsible for ethanol production could cause an undesired oxidation of acetaldehyde to acetate coupled with a reduction of NAD+ to NADH. Moreover, acetaldehyde could competitively inhibit the oxidation of reactive biogenic aldehydes. In the present study, the distribution of aldehyde dehydrogenase (measured with a biogenic aldehyde) and alcohol dehydrogenase (measured with acetaldehyde) were studied in organs of crucian carp, common carp (Cyprinus carpio L.), rainbow trout (Salmo gairdneri Richardson), and Norwegian rat (Rattus norvegicus Berkenhout). The results showed that alcohol dehydrogenase and aldehyde dehydrogenase activities were almost completely spatially separated in the crucian carp. These enzymes occurred together in the other three vertebrates. In the crucian carp, alcohol dehydrogenase was only found in red and white skeletal muscle, while these tissues contained exceptionally low aldehyde dehydrogenase activities. Moreover, the low aldehyde dehydrogenase activity found in crucian carp red muscle was about 1000 times less sensitive to inhibition by acetaldehyde than that found in other tissues and other species. The results are interpreted as demonstrating adaptations to avoid a depletion of ethanol production, and possibly inhibition of biogenic aldehyde metabolism.

Metabolism of acetaldehyde in human and baboon renal cortex. Ethanol synthesis by isolated baboon kidney-cortex tubules.

Acetaldehyde (1-20 mM) was metabolized at high rates and in a dose-dependent manner in isolated human and baboon kidney-cortex tubules. Acetaldehyde removal was accompanied by a large accumulation of acetate in both human and baboon tubules. By contrast, a large synthesis of ethanol was observed only in baboon tubules. Consistent with the latter finding, ethanol was found to be metabolized at significant rates in baboon but not human tubules. In the tubules from both species, a significant fraction of the acetaldehyde removed was also completely oxidized to CO2 and H2O. These results suggest that, in both man and baboon, the kidneys participate in the in vivo metabolism of acetaldehyde; they also suggest that, in contrast with the human kidneys, the baboon kidneys contribute to the detoxication of circulating ethanol.

Alcoholic fermentation by 'non-fermentative' yeasts.

All type strains of 'non-fermentative' yeasts, available in the culture collection of the Centraalbureau voor Schimmelcultures, were reinvestigated for their capacity to ferment glucose in the classical Durham tube test. Although visible gas production was absent, nearly all strains produced significant amounts of ethanol under the test conditions. Under conditions of oxygen-limited growth, even strong alcoholic fermentation may occur in a number of yeasts hitherto considered as non-fermentative. Thus, shake-flask cultures of Hansenula nonfermentans and Candida silvae fermented more than half of the available sugar to ethanol. It is concluded that the taxonomic test for fermentation capacity, which relies on detection of gas formation in Durham tubes, is not reliable for a physiological classification of yeasts as fermentative and non-fermentative species.

Intergeneric hybrids of Saccharomyces cerevisiae and Zygosaccharomyces fermentati obtained by protoplast fusion.

To obtain strains that are able to efficiently produce ethanol from different carbohydrates, mainly cellulose hydrolysates, several species of the genus Candida and a Zygosaccharomyces fermentati strain were examined for their ability to utilize cellobiose and produce ethanol, as well as for their thermotolerance and the possibility of genetic manipulation. Candida obtusa and Zygosaccharomyces fermentati tolerated the maximal temperature for growth, possessed the highest cellobiase activity, and offered the possibility of genetic manipulation, although neither of them proved to be a good producer of ethanol. Intergeneric hybrids of Saccharomyces cerevisiae and Z. fermentati were obtained after protoplast fusion. They were selected as prototrophic strains, after isolation of auxotrophic mutants from Z. fermentati and fusion with an S. cerevisiae strain which was also auxotrophic. The hybrids, which appeared at a frequency of 2 X 10(-7), presented characteristics of both parents, such as resistance to certain drugs and the ability to grow with either cellobiose or lactic acid as the sole carbon source; they were very stable, even under nonselective conditions. These hybrids may have important industrial applications as good fermenting strains.