Optimization of temperature, sugar concentration, and inoculum size to maximize ethanol production without significant decrease in yeast cell viability

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Enrichment of a continuous culture of Saccharomyces cerevisiae with the yeast Issatchenkia orientalis in the production of ethanol at increasing temperatures

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Fermentation and epiphytic microflora dynamics in sugar cane silage

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Spittlebug infestation in sugarcane affects ethanolic fermentation

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Fermentation, losses, and aerobic stability of sugarcane silages treated with chemical or bacterial additives

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Development of rapidly fermenting strains of saccharomyces-diastaticus for direct conversion of starch and dextrins to ethanol

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

CURRENT ASPECTS OF FUEL ETHANOL-PRODUCTION IN BRAZIL

Brazil has become a great producer of bioethanol using sugarcane as the basic raw material. Fed-batch process and continuous process are used. Biogas generation from vinasse, production of dry yeast, and autolyzed bagasse for animal feed are making the ethanol production less polluting and more profitable. Bagasse surplus has also been converted into electrical energy. Another alternative use for bioethanol is its conversion to petrochemical derivatives. Up to the present, however, this conversion has been carried out on only a small scale by the industry.

THERMOTOLERANCE BEHAVIOR IN SUGAR-CANE SYRUP FERMENTATIONS OF WILD-TYPE YEAST STRAINS SELECTED UNDER PRESSURES OF TEMPERATURE, HIGH SUGAR AND ADDED ETHANOL

The selected yeast strains were examined for their ability lo grow, to retain cell viability and to ferment diluted sugar cane juice (15% total sugar, w/v) to ethanol at 40-degrees-C. The degree of agitation (aeration) affects the thermotolerance while the method used for isolation of the strains appears to have no significant effect. The yeast isolated are aerobically fermentative with increased levels of fermentation and growth resulting from agitation (aeration), the exact level of these increases being dependent on the strain used.

Altered patterns of maltose and glucose fermentation by brewing and wine yeasts influenced by the complexity of nitrogen source

Maltose and glucose fermentations by industrial brewing and wine yeasts strains were strongly affected by the structural complexity of the nitrogen source. In this study, four Saccharomyces cerevisiae strains, two brewing and two wine yeasts, were grown in a medium containing maltose or glucose supplemented with a nitrogen source varying from a single ammonium salt (ammonium sulfate) to free amino acids (casamino acids) and peptides (peptone). Diauxie was observed at low sugar concentration for brewing and wine strains, independent of nitrogen supplementation, and the type of sugar. At high sugar concentrations altered patterns of sugar fermentation were observed, and biomass accumulation and ethanol production depended on the nature of the nitrogen source and were different for brewing and wine strains. In maltose, high biomass production was observed under peptone and casamino acids for the brewing and wine strains, however efficient maltose utilization and high ethanol production was only observed in the presence of casamino acids for one brewing and one wine strain studied. Conversely, peptone and casamino acids induced higher biomass and ethanol production for the two other brewing and wine strains studied. With glucose, in general, peptone induced higher fermentation performance for all strains, and one brewing and wine strain produced the same amount of ethanol with peptone and casamino acids supplementation. Ammonium salts always induced poor yeast performance. The results described in this paper suggest that the complex nitrogen composition of the cultivation medium may create conditions resembling those responsible for inducing sluggish/stuck fermentation, and indicate that the kind and concentration of sugar, the complexity of nitrogen source and the yeast genetic background influence optimal industrial yeast fermentation performance.

GROWTH AND FERMENTATION CHARACTERISTICS OF NEW SELECTED STRAINS OF SACCHAROMYCES AT HIGH-TEMPERATURES AND HIGH CELL DENSITIES

Maintenance of high cell viability was the main characteristic of our new strains of thermotolerant Saccharomyces. Total sugar conversion to ethanol was observed for sugarcane juice fermentation at 38-40-degrees-C in less than 10 h and without continuous aeration of the culture. Invertase activity differed among the selected strains and increased during fermentation but was not dependent on cell viability. Invertase activity of the cells and optimum temperature for growth, as well as velocity of ethanol formation, were dependent on medium composition and the type of strain used. At high sugarcane syrup concentrations, the best temperature for ethanol formation by strain 781 was 35-degrees-C. Distinct differences among the velocities of ethanol production using selected strains were also observed in sugarcane syrup at 35-38-degrees-C.

Effect of sugar catabolite repression in correlation with the structural complexity of the nitrogen source on yeast growth and fermentation

Biomass and ethanol production by industrial Saccharomyces cerevisiae strains were strongly affected by the structural complexity of the nitrogen source during fermentation in media containing galactose, and supplemented with a nitrogen source varying from a single ammonium salt (ammonium sulfate) to free amino acids (casamino acids) and peptides (peptone). Diauxie was observed at low galactose concentrations independent of nitrogen supplementation. At high sugar concentrations altered patterns of galactose utilisation were observed. Biomass accumulation and ethanol production depended on the nature of the nitrogen source and were different for baking and brewing ale and lager strains. Baking yeast showed improved galactose fermentation performance in the medium supplemented with casamino acids. High biomass production was observed with peptone and casamino acids for the ale brewing strain, however high ethanol production was observed only in the presence of casamino acids. Conversely, peptone was the nitrogen supplement that induced higher biomass and ethanol production for the lager brewing strain. Ammonium salts always induced poor yeast performance. The results with galactose differed from those obtained with glucose and maltose which indicated that supplementation with a nitrogen source in the peptide form (peptone) was more positive for yeast metabolism, suggesting that sugar catabolite repression has a central role in yeast performance in a medium containing nitrogen sources with differing levels of structural complexity.

Structural complexity of the nitrogen source and influence on yeast growth and fermentation

The structural complexity of the nitrogen source strongly affects both biomass and ethanol production by industrial strains of Saccharomyces cerevisiae, during fermentation in media containing glucose or maltose, and supplemented with a nitrogen source varying from a single ammonium salt (ammonium sulfate) to free amino acids (casamino acids) and peptides (peptone). Diauxie was observed at low glucose and maltose concentrations independent of nitrogen supplementation. At high sugar concentrations diauxie was not easily observed. and growth and ethanol production depended on the nature of the nitrogen source. This was different for baking and brewing ale and lager yeast strains. Sugar concentration had a strong effect on the shift from oxido-fermentative to oxidative metabolism. At low sugar concentrations, biomass production was similar under both peptone and casamino acid supplementation. Under casamino acid supplementation, the time for metabolic shift increased with the glucose concentration, together with a decrease in the biomass production. This drastic effect on glucose fermentation resulted in the extinction of the second growth phase, probably due to the loss of cell viability. Ammonium salts always induced poor yeast performance. In general, supplementation with a nitrogen source in the peptide form (peptone) was more positive for yeast metabolism, inducing higher biomass and ethanol production, and preserving yeast viability, in both glucose and maltose media, for baking and brewing ale and lager yeast strains. Determination of amino acid utilization showed that most free and peptide amino acids present, in peptone and casamino acids, were utilized by the yeast, suggesting that the results described in this work were not due to a nutritional status induced by nitrogen limitation.

Chlorine dioxide against bacteria and yeasts from the alcoholic fermentation

The ethanol production in Brazil is carried out by fed-batch or continuous process with cell recycle, in such way that bacterial contaminants are also recycled and may be troublesome due to the substrate competition. Addition of sulphuric acid when inoculum cells are washed can control the bacterial growth or alternatively biocides are used. This work aimed to verify the effect of chlorine dioxide, a well-known biocide for bacterial decontamination of water and equipments, against contaminant bacteria ( Bacillus subtilis, Lactobacillus plantarum, Lactobacillus fermentum and Leuconostoc mesenteroides) from alcoholic fermentation, through the method of minimum inhibitory concentration ( MIC), as well as its effect on the industrial yeast inoculum. Lower MIC was found for B. subtilis ( 10 ppm) and Leuconostoc mesenteroides ( 50 ppm) than for Lactobacillus fermentum ( 75 ppm) and Lactobacillus plantarum ( 125 ppm). Additionally, these concentrations of chlorine dioxide had similar effects on bacteria as 3 ppm of Kamoran (R) ( recommended dosage for fermentation tanks), exception for B. subtilis, which could not be controlled at this Kamoran (R) dosage. The growth of industrial yeasts was affected when the concentration of chlorine dioxide was higher than 50 ppm, but the effect was slightly dependent on the type of yeast strain. Smooth yeast colonies ( dispersed cells) seemed to be more sensitive than wrinkled yeast colonies ( clustered cells/pseudohyphal growth), both isolated from an alcohol-producing unit during the 2006/2007 sugar cane harvest. The main advantage in the usage of chlorine dioxide that it can replace antibiotics, avoiding the selection of resistant populations of microorganisms.

Ethanol tolerance of thermotolerant yeasts cultivated on mixtures of sucrose and ethanol

The final levels of ethanol (levels of ethanol produced plus that added initially to the media) reached by the thermotolerant yeasts were highest (16.5-20.3%, v/v) at 8% initial ethanol. The thermotolerant yeasts were found to have the following characteristics: constant levels of ethanol formation (10.5-12.3%, v/v), fog additions of external ethanol within the range 2-8% (v/v) of initial ethanol; constant values of product coefficients when initial ethanol was in the range of 2-6%, which increased or decreased, depending on the strain, when initial ethanol exceeded 6%; growth activity was inhibited at different levels of addition of external ethanol when final biomass and specific rate of growth were compared; significant differences among the yeast strains in the amount of external ethanol capable of reducing biomass formation by one half. In addition, the viability of the strains (early stationary phase) varied with the amount of external ethanol, the lowest viabilities occurring at concentrations of initial ethanol ranging from 4 to 7% and the highest in the range of 7 to 8% (v/v). The relative levels of trehalose (with/without 7% ethanol added initially) in the yeast strains (the stationary phase) ranged from 1.03 to 1.75, suggesting that the effect of produced ethanol on trehalose accumulation was stronger than that of external ethanol. The levels of final ethanol shown by the yeast strains were also correlated with the cellular levels of glycerol-3-phosphate dehydrogenase (increase in enzyme levels with decrease in final ethanol) for cells harvested at the stationary phase.

During the initiation of fermentation overexpression of hexokinase PII in yeast transiently causes a similar deregulation of glycolysis as deletion of Tps1

In the yeast Saccharomyces cerevisiae a novel control exerted by TPS1 (=GGS1=FDP1=BYP1=CIF1=GLC6=TSS1)-encoded trehalose-6-phosphate synthase, is essential for restriction of glucose influx into glycolysis apparently by inhibiting hexokinase activity in vivo. We show that up to 50-fold overexpression of hexokinase does not noticeably affect growth on glucose or fructose in wild-type cells. However, it causes higher levels of glucose-6-phosphate, fructose-6-phosphate and also faster accumulation of fructose-1,6-bisphosphate during the initiation of fermentation. The levels of ATP and Pi correlated inversely with the higher sugar phosphate levels. In the first minutes after glucose addition, the metabolite pattern observed was intermediate between those of the tps1Δ mutant and tile wild-type strain. Apparently, during the start-up of fermentation hexokinase is more rate-limiting in the first section of glycolysis than phosphofructokinase. We have developed a method to measure the free intracellular glucose level which is based on the simultaneous addition of D-glucose and an equal concentration of radiolabelled L-glucose. Since the latter is not transported, the free intracellular glucose level can be calculated as the difference between the total B-glucose measured (intracellular + periplasmic/extracellular) and the total L-glucose measured (periplasmic/extracellular). The intracellular glucose level rose in 5 min after addition of 100 mM-glucose to 0.5-2 mM in the wild-type strain, ± 10 mm in a hxk1Δ hxk2Δ glk1Δ and 2-3 mM in a tps1Δ strain. In the strains overexpressing hexokinase PII the level of free intracellular glucose was not reduced. Overexpression of hexokinase PII never produced a strong effect on the rate of ethanol production and glucose consumption. Our results show that overexpression of hexokinase does not cause the same phenotype as deletion of Tps1. However, it mimics it transiently during the initiation of fermentation. Afterwards, the Tps1-dependent control system is apparently able to restrict Properly up to 50-fold higher hexokinase activity.