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.

Greenhouse gas balance due to the conversion of sugarcane areas from burned to green harvest, considering other conservationist management practices

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Flooding tolerance of Tabebuia cassinoides: Metabolic, morphological and growth responses

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

Zymomonas mobilis: um microrganismo promissor para a fermentação alcoólica

In many countries, fermentation studies regarding the use of bacteria instead of yeasts to reduce the period of alcoholic fermentation have been carried out. In Brazil, all the industrial alcohol production is carried out by yeasts as fermentation microorganisms and little is known about other microorganisms with potential to produce alcohol industrially. Brazil stands out in the energy sector worldwide and thus some institutions have been selecting microorganisms which are more efficient in the alcohol production process. Alcoholic bacteria from species Zymomonas mobilis present technological characteristics with potential to be used for alcoholic fermentation at industrial scale, since it exhibits promising abilities to transform sugars into alcohol and carbon dioxide, at conditions similar to the ones required by yeasts. Zymomonas mobilis is a unique bacteria among the microbial world, with peculiar growth, energy production and response to culture conditions, causing a great interest in scientific, biotechnological and industrial fields. The bacteria's ability to make possible energy production in favor of product formation, respond to physical and chemical environmental manipulation as well as its limited product formation make it an ideal microorganism for the study and development of microbial processes for ethanol production.

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.

Bioethanol - Brazil, 30 years of Proálcool

Brazil, which has always been in the forefront of sugarcane production, also occupies a prominent position as the first country to produce and use biofuel in its automobile fleet. This fact is a consequence of the introduction of a program which has already turned 30 years, the Próalcool (National Alcohol Program). The oil crisis in the seventies encouraged the government to develop an alternative way to replace gasoline. Bioethanol was then born as fuel obtained from fermentation of sugarcane juice, molasses or both. In the eighties, 85% of the cars ran exclusively on alcohol. Ethanol production in that decade exceeded sugarcane production by the mills. The installed units reached in that period the capacity to produce 18 billion liters of bioethanol per season, a volume equivalent to 100 million barrels of gasoline. The fermentation process, which so far had been restricted to manufacturing sugarcane liquor (aguardente) or ethanol as a byproduct of sugarcane, takes over the spotlight in the entrepreneurial scene. As a result, processes comprising engineering concepts came up and most of the biological phenomena involved in fermentation were understood. The knowledge gathered and the units installed have granted Brazil the hold of production technology and use of a clean fuel.

Development and Biotechnological Application of a Novel Endoxylanase Family GH10 Identified from Sugarcane Soil Metagenome

Metagenomics has been widely employed for discovery of new enzymes and pathways to conversion of lignocellulosic biomass to fuels and chemicals. In this context, the present study reports the isolation, recombinant expression, biochemical and structural characterization of a novel endoxylanase family GH10 (SCXyl) identified from sugarcane soil metagenome. The recombinant SCXyl was highly active against xylan from beechwood and showed optimal enzyme activity at pH 6,0 and 45°C. The crystal structure was solved at 2.75 Å resolution, revealing the classical (β/α)8-barrel fold with a conserved active-site pocket and an inherent flexibility of the Trp281-Arg291 loop that can adopt distinct conformational states depending on substrate binding. The capillary electrophoresis analysis of degradation products evidenced that the enzyme displays unusual capacity to degrade small xylooligosaccharides, such as xylotriose, which is consistent to the hydrophobic contacts at the +1 subsite and low-binding energies of subsites that are distant from the site of hydrolysis. The main reaction products from xylan polymers and phosphoric acid-pretreated sugarcane bagasse (PASB) were xylooligosaccharides, but, after a longer incubation time, xylobiose and xylose were also formed. Moreover, the use of SCXyl as pre-treatment step of PASB, prior to the addition of commercial cellulolytic cocktail, significantly enhanced the saccharification process. All these characteristics demonstrate the advantageous application of this enzyme in several biotechnological processes in food and feed industry and also in the enzymatic pretreatment of biomass for feedstock and ethanol production. © 2013 Alvarez et al.

Greenhouse gas mitigation potential from green harvested sugarcane scenarios in São Paulo State, Brazil

Brazil is a major sugarcane producer and São Paulo State cultivates 5.5 million hectares, close to 50% of Brazil's sugarcane area. The rapid increase in production has brought into question the sustainability of biofuels, especially considering the greenhouse gas (GHG) emissions associated to the agricultural sector. Despite the significant progress towards the green harvest practices, 1.67 million hectares were still burned in São Paulo State during the 2011 harvest season. Here an emissions inventory for the life cycle of sugarcane agricultural production is estimated using IPCC methodologies, according to the agriculture survey data and remote sensing database. Our hypothesis is that 1.67 million hectares shall be converted from burned to green harvest scenarios up to years 2021 (rate 1), 2014 (rate 2) or 2029 (rate 3). Those conversions would represent a significant GHG mitigation, ranging from 50.5 to 70.9 megatons of carbon dioxide equivalent (Mt CO2eq) up to 2050, depending on the conversion rate and the green harvest systems adopted: conventional (scenario S1) or conservationist management (scenario S2). We show that a green harvest scenario where crop rotation and reduced soil tillage are practiced has a higher mitigation potential (70.9 Mt CO2eq), which is already practiced in some of the sugarcane areas. Here we support the decision to not just stop burning prior to harvest, but also to consider other better practices in sugarcane areas to have a more sustainable sugarcane based ethanol production in the most dense cultivated sugarcane region in Brazil. © 2013 Elsevier Ltd. All rights reserved.

Produção de etanol a partir de mosto hidrolisado hemicelulósico de ponta e palha de cana-de-açúcar

Pós-graduação em Microbiologia Agropecuária - FCAV

Avaliação da tolerância de leveduras a um coquetel de inibidores que simula um hidrolisado de bagaço de cana quando adicionado a um meio sintético

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Análise por técnica de RDA (Representational Difference Analysis) da expressão gênica diferencial, para a identificação de fatores genéticos associados à produção de etanol em linhagem de Saccharomyces cerevisae

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

A utilização diferencial de glicose e frutose por leveduras vinícolas em meios contendo fontes de nittrogênio estruturalmente complexas

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Estudo da hidrólise do bagaço de cana-de-açúcar por fungos filamentosos

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)