Luiz Carlos Basso

Fuel ethanol after 25 years

After 25 years, Brazil and North America are still the only two regions that produce large quantities of fuel ethanol, from sugar cane and maize, respectively. The efficiency of ethanol production has steadily increased and valuable co-products are produced, but only tax credits make fuel ethanol commercially viable because oil prices are at an all-time low. The original motivation for fuel-ethanol production was to become more independent of oil imports; now, the emphasis is on its use as an oxygenated gasoline additive. There will only be sufficient, low-cost ethanol if lignocellulose feedstock is also used.

Ethanol Production in Brazil: The Industrial Process and Its Impact on Yeast Fermentation

Luiz Carlos Basso1, Thiago Olitta Basso2,3 and Saul Nitsche Rocha4 1University of Sao Paulo, Escola Superior de Agricultura Luiz de Queiroz, Departamento de Ciencias Biologicas, Piracicaba (SP) 2University of Sao Paulo, Escola Politecnica, Departamento de Engenharia Quimica, Sao Paulo (SP) 3University of Sao Paulo Programa de Pos-Graduacao Inter-unidades em Biotecnologia, Sao Paulo (SP) 4Positivo University, Programa de Pos-Graduacao em Biotecnologia Industrial Curitiba (PR) Brazil

What do we know about the yeast strains from the Brazilian fuel ethanol industry

The production of fuel ethanol from sugarcane-based raw materials in Brazil is a successful example of a large-scale bioprocess that delivers an advanced biofuel at competitive prices and low environmental impact. Two to three fed-batch fermentations per day, with acid treatment of the yeast cream between consecutive cycles, during 6–8xa0months of uninterrupted production in a nonaseptic environment are some of the features that make the Brazilian process quite peculiar. Along the past decades, some wild Saccharomyces cerevisiae strains were isolated, identified, characterized, and eventually, reintroduced into the process, enabling us to build up knowledge on these organisms. This information, combined with physiological studies in the laboratory and, more recently, genome sequencing data, has allowed us to start clarifying why and how these strains behave differently from the better known laboratory, wine, beer, and bakers strains. All these issues are covered in this minireview, which also presents a brief discussion on future directions in the field and on the perspectives of introducing genetically modified strains in this industrial process.

Homo- and heterofermentative lactobacilli differently affect sugarcane-based fuel ethanol fermentation.

Bacterial contamination during industrial yeast fermentation has serious economic consequences for fuel ethanol producers. In addition to deviating carbon away from ethanol formation, bacterial cells and their metabolites often have a detrimental effect on yeast fermentative performance. The bacterial contaminants are commonly lactic acid bacteria (LAB), comprising both homo- and heterofermentative strains. We have studied the effects of these two different types of bacteria upon yeast fermentative performance, particularly in connection with sugarcane-based fuel ethanol fermentation process. Homofermentative Lactobacillus plantarum was found to be more detrimental to an industrial yeast strain (Saccharomyces cerevisiae CAT-1), when compared with heterofermentative Lactobacillus fermentum, in terms of reduced yeast viability and ethanol formation, presumably due to the higher titres of lactic acid in the growth medium. These effects were only noticed when bacteria and yeast were inoculated in equal cell numbers. However, when simulating industrial fuel ethanol conditions, as conducted in Brazil where high yeast cell densities and short fermentation time prevail, the heterofermentative strain was more deleterious than the homofermentative type, causing lower ethanol yield and out competing yeast cells during cell recycle. Yeast overproduction of glycerol was noticed only in the presence of the heterofermentative bacterium. Since the heterofermentative bacterium was shown to be more deleterious to yeast cells than the homofermentative strain, we believe our findings could stimulate the search for more strain-specific antimicrobial agents to treat bacterial contaminations during industrial ethanol fermentation.

Produções de ácido acético, etanol e dos isômeros óticos do ácido lático por linhagens de Lactobacillus isoladas de fermentações alcoólicas industriais

Avaliaram-se no presente trabalho, as producoes de etanol e dos acidos acetico e latico, bem como das proporcoes dos isomeros oticos D(-) e L(+) desse ultimo, por 17 linhagens de Lactobacillus isoladas de fermentacoes industriais de producao de etanol. As linhagens foram crescidas a 32oC por 24 horas, em meio contendo 1% de glucose, 1% de frutose, 1% de extrato de levedura, sais nutrientes (K, Mg e Mn) e tampao fosfato. Foram estimados os teores de acido latico, acido acetico e etanol mediante cromatografia liquida de alta eficiencia, assim como dos isomeros oticos D(-) e L(+) do acido latico mediante espectrofotometria ao ultra-violeta, empregando desidrogenases laticas estereoespecificas. O crescimento bacteriano foi inferido pela absorvância a 600 nm. Os resultados obtidos mostraram, pelos perfis de excrecao dos metabolitos, a presenca de 8 linhagens homofermentativas obrigatorias (produzindo unicamente acido latico), 8 linhagens heterofermentativas obrigatorias (com producoes de acidos latico, acetico e etanol) e 1 linhagem supostamente heterofermentativa facultativa. Observou-se tambem, em relacao a formacao dos estereoisomeros, que 12 linhagens foram incluidas no grupo DL, 4 no grupo L e 1 no grupo D. Os resultados permitem concluir que os Lactobacillus que contaminam processos fermentativos industriais de producao de etanol, podem se apresentar nos 3 biotipos fermentativos e produzindo as mais variadas proporcoes dos dois estereoisomeros do acido latico, com relevantes implicacoes biotecnologicas. Este e o primeiro relato sobre as producoes dos isomeros oticos do acido latico por bacterias do genero Lactobacillus isoladas de fermentacoes industriais baseadas na cana-de-acucar.

Use of Sugar Cane Vinasse to Mitigate Aluminum Toxicity to Saccharomyces cerevisiae

Owing to its toxicity, aluminum (Al), which is one of the most abundant metals, inhibits the productivity of many cultures and affects the microbial metabolism. The aim of this work was to investigate the capacity of sugar cane vinasse to mitigate the adverse effects of Al on cell growth, viability, and budding, as the likely result of possible chelating action. For this purpose, Fleischmann’s yeast (Saccharomyces cerevisiae) was used in growth tests performed in 125-mL Erlenmeyer flasks containing 30xa0mL of YED medium (5.0xa0g/L yeast extract plus 20xa0g/L glucose) supplemented with the selected amounts of either vinasse or Al in the form of AlCl3xa0·xa0H2O. Without vinasse, the addition of increasing levels of Al up to 54xa0mg/L reduced the specific growth rate by 18%, whereas no significant reduction was observed in its presence. The toxic effect of Al on S. cerevisiae growth and the mitigating effect of sugar cane vinasse were quantified by the exponential model of Ciftci et al. (Biotechnol Bioeng 25:2007–2023, 1983). The cell viability decreased from 97.7% at the start to 84.0% at the end of runs without vinasse and to 92.3% with vinasse. On the other hand, the cell budding increased from 7.62% at the start to 8.84% at the end of runs without vinasse and to 17.8% with vinasse. These results demonstrate the ability of this raw material to stimulate cell growth and mitigate the toxic effect of Al.

The fermentation of sugarcane molasses by Dekkera bruxellensis and the mobilization of reserve carbohydrates.

The yeast Dekkera bruxellensis is considered to be very well adapted to industrial environments, in Brazil, USA, Canada and European Countries, when different substrates are used in alcoholic fermentations. Our previous study described its fermentative profile with a sugarcane juice substrate. In this study, we have extended its physiological evaluation to fermentation situations by using sugarcane molasses as a substrate to replicate industrial working conditions. The results have confirmed the previous reports of the low capacity of D. bruxellensis cells to assimilate sucrose, which seems to be the main factor that can cause a bottleneck in its use as fermentative yeast. Furthermore, the cells of D. bruxellensis showed a tendency to deviate most of sugar available for biomass and organic acids (lactic and acetic) compared with Saccharomyces cerevisiae, when calculated on the basis of their respective yields. As well as this, the acetate production from molasses medium by both yeasts was in marked contrast with the previous data on sugarcane juice. Glycerol and ethanol production by D. bruxellensis cells achieved levels of 33 and 53xa0% of the S. cerevisiae, respectively. However, the ethanol yield was similar for both yeasts. It is worth noting that this yeast did not accumulate trehalose when the intracellular glycogen content was 30xa0% lower than in S. cerevisiae. The lack of trehalose did not affect yeast viability under fermentation conditions. Thus, the adaptive success of D. bruxellensis under industrial fermentation conditions seems to be unrelated to the production of these reserve carbohydrates.

Acid phosphatase activity and leaf phosphorus content in soybean cultivars

A adubacao fosfatada corresponde a fracao mais onerosa do custo de producao da cultura da soja. A obtencao de cultivares de soja eficientes na absorcao e utilizacao de fosforo (P) em condicao de media disponibilidade deste nutriente pode contribuir para aumentar o potencial produtivo da cultura. Trinta e dois cultivares de soja [Glycine max (L.) Merr.], de ciclo precoce, semiprecoce, semitardio e tardio, recomendados para o cerrado, foram cultivados em Latossolo Vermelho-Amarelo distrofico tipico, do cerrado, objetivando avaliar a atividade da fosfatase acida, concentracao de P na folha diagnostico e biomassa da parte aerea. Ocorreram diferencas entre os cultivares dentro de todos os ciclos de maturacao na atividade da fosfatase acida e na biomassa da parte aerea. Cultivares de ciclos semitardio e tardio apresentaram diferencas significativas quanto a concentracao de fosforo na folha diagnostico. A atividade da fosfatase acida correlacionou-se positivamente com a biomassa da parte aerea nos cultivares dos ciclos semiprecoce (r = 0,46) e tardio (r = 0,47) e, negativamente (r = -0,40), com a concentracao de P na folha-diagnostico, nos cultivares do ciclo tardio. A ocorrencia de cultivares de soja com alta e baixa atividade da fosfatase acida dentro do mesmo ciclo de maturacao sinalizam a existencia de diferentes mecanismos envolvidos na mobilizacao de P no solo e remobilizacao interna deste nutriente na planta entre os grupos de cultivares.

Efeitos do cádmio sobre o crescimento das leveduras Saccharomyces cerevisiae PE-2 e Saccharomyces cerevisiae IZ-1904, e a capacidade da vinhaça em atenuar a toxicidade

The present study was carried out in order to evaluate the capability of different cadmium concentration (0; 0,05, 0,10 and 0,50mM) to affect the growth of two S. cerevisiae strains (PE-2 and IZ-1904) in YED (yeast extract 1% and dextrose 2%) medium, and to evaluate the three vinasse concentration capability (0,15 and 30%) to attenuate the two cadmium concentration toxicity (0,1 and 0,5mM), using S. cerevisiae PE-2 strain in YED medium. In the first assay, the medium was inoculated in aseptic conditions with 1mL of 1% yeast suspension (PE-2 or IZ-1904) and incubated at 30oC, 70 RPM for 18 hours. During anaerobic growth (0, 2, 4, 6, 8, 10, 12, 14, 16 and 18 hours), portions of cell suspension were taken out and biomass concentration was determined. At the end of fermentation, yeast viability, budding rate and bacterial contamination were determined. Both, initial and final trehalose, was measured. In the second assay, the medium was inoculated in aseptic conditions with 2mL of 1% PE-2 suspension and incubated at 30oC, 120 rpm for 18 hours. During the anaerobic growth (0, 2, 4, 6, 8, 10, 12,14, 16 and 18 hours) portions of cell suspension were taken out and biomass concentration was determined. At the end of fermentation, alcohol production, yeast viability, budding rate and bacterial contamination were determined. Both, initial and final trehalose, was measured. The increase of cadmium levels showed a reduction on yeast growth and cell viability. Vinasse showed low toxicity, but protected yeast cells very effectively against the toxic effects of cadmium.

Fermentation of endogenous trehalose and glycogen by Saccharomyces cerevisiae

As linhagens PE-2 e VR-1 de Saccharomyces cerevisiae foram submetidas a fermentacao das reservas endogenas na temperatura de 40oC. No intervalo de 0 a 24 horas foram recolhidas as amostras para a determinacao de etanol, nitrogenio no fermento e no vinho, bem como os carboidratos de reserva (trealose e glicogenio) e a viabilidade celular. A trealose foi esgotada durante 24 horas. Os teores de glicogenio sofreram muitas oscilacoes ao longo do tempo, entre a mobilizacao e a sintese e embora nao esgotado, deve ter contribuido significativamente para a formacao de alcool na suspensao. Foi observada a relacao proporcional entre a mobilizacao de trealose e a queda da viabilidade celular. No transcorrer da fermentacao das reservas de carboidratos houve aumento nos teores de nitrogenio no fermento ate 6 e 8 horas, sendo tal incremento afetado pela linhagem de levedura. No prosseguimento da fermentacao ocorreu a autolise celular, que foi percebida pelo aumento brusco de nitrogenio no vinho (de 200 para 1500mg/L) e pela queda da viabilidade celular. O ganho alcancado com a fermentacao endogena foi de 40 e 68 litros de alcool por tonelada de levedura seca com incremento de 25 e 27% de proteina no fermento para as linhagens PE-2 e VR-1, respectivamente. Este resultado tem reflexos positivos quando da comercializacao da levedura seca como proteina microbiana.