Proteomics Reveals an Increase in the Abundance of Glycolytic and Ethanolic Fermentation Enzymes in Developing Sugarcane Culms During Sucrose Accumulation.

Sugarcane is an economically important crop contributing to the sugar and ethanol production of the world with 80 and 40%, respectively. Despite its importance as the main crop for sugar production, the mechanisms involved in the regulation of sucrose accumulation in sugarcane culms are still poorly understood. The aim of this work was to compare the quantitative changes of proteins in juvenile and maturing internodes at three stages of plant development. Label-free shotgun proteomics was used for protein profiling and quantification in internodes 5 (I5) and 9 (I9) of 4-, 7-, and 10-month-old-plants (4M, 7M, and 10M, respectively). The I9/I5 ratio was used to assess the differences in the abundance of common proteins at each stage of internode development. I9 of 4M plants showed statistically significant increases in the abundance of several enzymes of the glycolytic pathway and proteoforms of alcohol dehydrogenase (ADH) and pyruvate decarboxylase (PDC). The changes in content of the enzymes were followed by major increases of proteins related to O2 transport like hemoglobin 2, ROS scavenging enzymes, and enzymes involved in the ascorbate/glutatione system. Besides, intermediates from tricarboxylic acid cycle (TCA) were reduced in I9-4M, indicating that the increase in abundance of several enzymes involved in glycolysis, pentose phosphate cycle, and TCA, might be responsible for higher metabolic flux, reducing its metabolites content. The results observed in I9-4M indicate that hypoxia might be the main cause of the increased flux of glycolysis and ethanolic fermentation to supply ATP and reducing power for plant growth, mitigating the reduction in mitochondrial respiration due to the low oxygen availability inside the culm. As the plant matured and sucrose accumulated to high levels in the culms, the proteins involved in glycolysis, ethanolic fermentation, and primary carbon metabolism were significantly reduced.

Dynamics of microbial contaminants is driven by selection during ethanol production.

Brazil is the second largest ethanol producer in the world and largest using sugarcane feedstock. Bacteria contamination is one of the most important issues faced by ethanol producers that seek to increase production efficiency. Each step of production is a selection event due to the environmental and biological changes that occur. Therefore, we evaluated the influence of the selection arising from the ethanol production process on diversity and composition of bacteria. Our objectives were to test two hypotheses, (1) that species richness will decrease during the production process and (2) that lactic acid bacteria will become dominant with the advance of ethanol production. Bacterial community assemblage was accessed using 16S rRNA gene sequencing from 19 sequential samples. Temperature is of great importance in shaping microbial communities. Species richness increased between the decanter and must steps of the process. Low Simpson index values were recorded at the fermentation step, indicating a high dominance of Lactobacillus. Interactions between Lactobacillus and yeast may be impairing the efficiency of industrial ethanol production.

Seasonal Variation of Carbon Metabolism in the Cambial Zone of Eucalyptus grandis.

Eucalyptus species are the most widely hardwood planted in the world. It is one of the successful examples of commercial forestry plantation in Brazil and other tropical and subtropical countries. The tree is valued for its rapid growth, adaptability and wood quality. Wood formation is the result of cumulative annual activity of the vascular cambium. This cambial activity is generally related to the alternation of cold and warm, and/or dry and rainy seasons. Efforts have focused on analysis of cambial zone in response to seasonal variations in trees from temperate zones. However, little is known about the molecular changes triggered by seasonal variations in trees from tropical countries. In this work we attempted to establish a global view of seasonal alterations in the cambial zone of Eucalyptus grandis Hill ex Maiden, emphasizing changes occurring in the carbon metabolism. Using transcripts, proteomics and metabolomics we analyzed the tissues harvested in summer-wet and winter-dry seasons. Based on proteomics analysis, 70 proteins that changed in abundance were successfully identified. Transcripts for some of these proteins were analyzed and similar expression patterns were observed. We identified 19 metabolites differentially abundant. Our results suggest a differential reconfiguration of carbon partioning in E. grandis cambial zone. During summer, pyruvate is primarily metabolized via ethanolic fermentation, possibly to regenerate NAD(+) for glycolytic ATP production and cellular maintenance. However, in winter there seems to be a metabolic change and we found that some sugars were highly abundant. Our results revealed a dynamic change in E. grandis cambial zone due to seasonality and highlight the importance of glycolysis and ethanolic fermentation for energy generation and maintenance in Eucalyptus, a fast growing tree.

Damages of spittlebug on sugarcane quality and fermentation process

The quality of the raw material defines the industrial potential production. The harvest of raw sugarcane promoted a favorable environment for the increase of Mahanarva fimbriolata (Stål, 1854) pest that affects the plant development. This research was undertaken to evaluate the effects of M. fimbriolata on sugarcane quality and fermentation process. For the technological analysis, four damage levels and two evaluation periods were evaluated, while four fermentation cycles and two evaluation periods were tested for microbiological analysis, with three replications. The determined parameters were Brix (soluble solids), Pol (apparent sucrose), Purity, reducing sugars (RS), total reducing sugars (TRS), Total Acidity, pH and Total Phenolic Compounds in juice. In the fermentation process cellular viability analysis, yeast sprout and bacterial concentration were carried out. The wine was submitted to alcohol content, TRRS (total residual reducing sugars) and fermentation efficiency. The treatment with 60% of damage steams exhibited the lowest averages of Brix, Pol, Purity and TRS of the juice. An increase of the RS% of the juice and content of total phenolic compounds was observed. The cellular and sprouts viability were reduced with the increase in the damages caused by M. fimbriolata. The fermentation process exhibited larger quantities of contaminators. With the damages, the wine TRRS quantity was superior and, consequently there was a decrease of the alcoholic content and fermentation efficiency. The damages caused by M. fimbriolata affected the quality of the raw material, compromising the fermentative process, and increase the production of phenolic compounds and contaminators.

A qualidade da matéria-prima define o potencial de produção da indústria. A colheita de cana sem queima proporcionou um ambiente favorável ao aumento da infestação da Mahanarva fimbriolata (Stål, 1854), que suga a planta e afeta seu desenvolvimento. Avaliaram-se os danos promovidos pela M. fimbriolata na qualidade da cana e no processo fermentativo. Os tratamentos utilizados foram quatro níveis de danos e duas épocas de avaliações para as análises tecnológicas, e quatro níveis de danos, quatro ciclos fermentativos e duas épocas de avaliações para as análises microbiológicas com três repetições. Foram determinados Brix (sólidos solúveis), Pol (sacarose aparente), Pureza, açúcar redutor total (ART), açúcar redutor (AR), Acidez Total, pH, e teor de compostos fenólicos totais no caldo. No processo fermentativo foram realizadas análises de viabilidade celular, brotos de leveduras e concentração de bactérias. Determinaram-se açúcar redutor residual Total (ARRT), teor alcoólico nos vinhos e a eficiência da fermentação. Os tratamentos com 60% dos colmos danificados apresentaram menor média de Brix, Pol, Pureza e ART do caldo. Verificou-se aumento do AR% caldo e dos teores de compostos fenólicos totais. A viabilidade celular e de brotos foram reduzidas com o aumento dos danos provocados pela M. fimbriolata. O processo fermentativo apresentou maiores quantidades de contaminantes. Com os danos a quantidade de ARRT dos vinhos foi maior, houve redução do teor alcoólico, assim como na eficiência da fermentação. Os danos causados pela M. fimbriolata afetaram a qualidade da matéria-prima, comprometendo o processo fermentativo, além de aumentar a produção de compostos fenólicos e contaminantes.

Damages of spittlebug on sugarcane quality and fermentation process

The quality of the raw material defines the industrial potential production. The harvest of raw sugarcane promoted a favorable environment for the increase of Mahanarva fimbriolata (Stål, 1854) pest that affects the plant development. This research was undertaken to evaluate the effects of M. fimbriolata on sugarcane quality and fermentation process. For the technological analysis, four damage levels and two evaluation periods were evaluated, while four fermentation cycles and two evaluation periods were tested for microbiological analysis, with three replications. The determined parameters were Brix (soluble solids), Pol (apparent sucrose), Purity, reducing sugars (RS), total reducing sugars (TRS), Total Acidity, pH and Total Phenolic Compounds in juice. In the fermentation process cellular viability analysis, yeast sprout and bacterial concentration were carried out. The wine was submitted to alcohol content, TRRS (total residual reducing sugars) and fermentation efficiency. The treatment with 60% of damage steams exhibited the lowest averages of Brix, Pol, Purity and TRS of the juice. An increase of the RS% of the juice and content of total phenolic compounds was observed. The cellular and sprouts viability were reduced with the increase in the damages caused by M. fimbriolata. The fermentation process exhibited larger quantities of contaminators. With the damages, the wine TRRS quantity was superior and, consequently there was a decrease of the alcoholic content and fermentation efficiency. The damages caused by M. fimbriolata affected the quality of the raw material, compromising the fermentative process, and increase the production of phenolic compounds and contaminators.

A qualidade da matéria-prima define o potencial de produção da indústria. A colheita de cana sem queima proporcionou um ambiente favorável ao aumento da infestação da Mahanarva fimbriolata (Stål, 1854), que suga a planta e afeta seu desenvolvimento. Avaliaram-se os danos promovidos pela M. fimbriolata na qualidade da cana e no processo fermentativo. Os tratamentos utilizados foram quatro níveis de danos e duas épocas de avaliações para as análises tecnológicas, e quatro níveis de danos, quatro ciclos fermentativos e duas épocas de avaliações para as análises microbiológicas com três repetições. Foram determinados Brix (sólidos solúveis), Pol (sacarose aparente), Pureza, açúcar redutor total (ART), açúcar redutor (AR), Acidez Total, pH, e teor de compostos fenólicos totais no caldo. No processo fermentativo foram realizadas análises de viabilidade celular, brotos de leveduras e concentração de bactérias. Determinaram-se açúcar redutor residual Total (ARRT), teor alcoólico nos vinhos e a eficiência da fermentação. Os tratamentos com 60% dos colmos danificados apresentaram menor média de Brix, Pol, Pureza e ART do caldo. Verificou-se aumento do AR% caldo e dos teores de compostos fenólicos totais. A viabilidade celular e de brotos foram reduzidas com o aumento dos danos provocados pela M. fimbriolata. O processo fermentativo apresentou maiores quantidades de contaminantes. Com os danos a quantidade de ARRT dos vinhos foi maior, houve redução do teor alcoólico, assim como na eficiência da fermentação. Os danos causados pela M. fimbriolata afetaram a qualidade da matéria-prima, comprometendo o processo fermentativo, além de aumentar a produção de compostos fenólicos e contaminantes.

Effects of corn meal and sulphuric acid on the production of cachaça / Efeitos do fubá e ácido sulfúrico sobre a produção de cachaça

This work was carried out to evaluate the effects of using corn meal and treating yeasts with sulfuric acid on fermentation microorganisms, wine acidity, ethanol content and cachaça yield and composition. The experiment was arranged in randomized block design, in a 2x3 factorial with five replications. The methods applied in this study are recommended by distilleries. Results showed that the yeast sulfuric acid treatment transferred acidity to the fermenting juice, without any influence on yeast viability, ethanol content and cachaça yield. On the other hand, the acid treatment controlled lactic bacteria in the inoculum. Addition of corn meal increased the concentration of lactic bacteria in the end of the fermentation and increased the levels of higher alcohols in cachaça, especially propyl and isobutyl alcohol.

Avaliou-se o efeito da adição do fubá de milho no mosto de xarope de cana e o tratamento ácido do pé-de-cuba sobre a microbiota do processo fermentativo, acidez do vinho, grau alcoólico, rendimento e composição da cachaça. O delineamento experimental utilizado foi o de blocos casualizados, no esquema fatorial 2x3 e cinco repetições. A metodologia empregada e as análises foram as recomendadas pelo setor aguardenteiro. Os resultados permitiram concluir que a adição do ácido sulfúrico no pé-de-cuba transferiu a acidez para o vinho, não influenciando na viabilidade das leveduras, rendimento e composição da cachaça. Por outro lado, a acidificação do meio controlou as bactérias láticas no pé-de-cuba. A adição do fubá aumentou a concentração de bactérias lácticas ao final do processo fermentativo e dos álcoois homólogos superiores na cachaça, particularmente, os álcoois propílico e isobutílico.

Negative H2S character and flocculation as yeast strain markers for inoculum recovery

Routine identification of yeast behavior is essential to measure the control of the alcohol production process and to maintain product quality standards. This work utilized the non-hydrogen sulfide production and flocculation traits as characteristic strain markers for the evaluation of cell recycling during the alcoholic fermentation process for production of sugarcane alcohol. This study evaluated the behavior of a recombinant yeast bank made by protoplast fusion, for strain screening purposes; strain fermentative kinetics in comparison to commercial baker yeast; viability and recovery of the selected strain on differential media, after five consecutive fermentation batches; and the recovery of the selected strain from fermentation with mixed strain cultures. The strain selected for the H2S negative character kept its viability during successive recyclings, with contamination levels not detected by the method of analysis. It also presented a kinetic behavior similar to that of baker yeast, either in single or mixed culture fermentations, opening new possibilities for further work on quality control of cell recycling in the alcoholic fermentation process.

A identificação rotineira do comportamento das leveduras é essencial à manutenção do controle e do padrão de qualidade dos processos fermentativos para produção de álcool. Em função da baixa freqüência de ocorrência de leveduras não produtoras de ácido sulfídrico e floculantes, pretendeu-se neste trabalho utilizar estas características como marcadores de linhagem na avaliação da reciclagem de células em processos de fermentação alcoólica do caldo de cana para a produção de aguardente. O trabalho foi elaborado com base no acompanhamento das características de um banco de leveduras recombinantes produzidas por fusão de protoplastos para seleção de linhagem; no acompanhamento da cinética fermentativa da linhagem selecionada em comparação ao fermento de panificação comercial, em função de seu emprego comum em processos fermentativos; na avaliação da viabilidade e da recuperação da linhagem selecionada em meio diferencial após cinco reciclos; na avaliação da recuperação desta linhagem em cultura mista com o fermento de panificação comercial. A levedura selecionada para o caráter H2S negativo manteve sua viabilidade durante os reciclos, com níveis de contaminação não detectados pela metodologia empregada, apresentando comportamento cinético semelhante ao da levedura de panificação tanto em cultura pura quanto em cultivo misto, apresentando uma nova possibilidade para estudos sobre o controle de qualidade de processos de fermentação alcoólica com reciclagem de células.

Negative H2S character and flocculation as yeast strain markers for inoculum recovery

Routine identification of yeast behavior is essential to measure the control of the alcohol production process and to maintain product quality standards. This work utilized the non-hydrogen sulfide production and flocculation traits as characteristic strain markers for the evaluation of cell recycling during the alcoholic fermentation process for production of sugarcane alcohol. This study evaluated the behavior of a recombinant yeast bank made by protoplast fusion, for strain screening purposes; strain fermentative kinetics in comparison to commercial baker yeast; viability and recovery of the selected strain on differential media, after five consecutive fermentation batches; and the recovery of the selected strain from fermentation with mixed strain cultures. The strain selected for the H2S negative character kept its viability during successive recyclings, with contamination levels not detected by the method of analysis. It also presented a kinetic behavior similar to that of baker yeast, either in single or mixed culture fermentations, opening new possibilities for further work on quality control of cell recycling in the alcoholic fermentation process.

A identificação rotineira do comportamento das leveduras é essencial à manutenção do controle e do padrão de qualidade dos processos fermentativos para produção de álcool. Em função da baixa freqüência de ocorrência de leveduras não produtoras de ácido sulfídrico e floculantes, pretendeu-se neste trabalho utilizar estas características como marcadores de linhagem na avaliação da reciclagem de células em processos de fermentação alcoólica do caldo de cana para a produção de aguardente. O trabalho foi elaborado com base no acompanhamento das características de um banco de leveduras recombinantes produzidas por fusão de protoplastos para seleção de linhagem; no acompanhamento da cinética fermentativa da linhagem selecionada em comparação ao fermento de panificação comercial, em função de seu emprego comum em processos fermentativos; na avaliação da viabilidade e da recuperação da linhagem selecionada em meio diferencial após cinco reciclos; na avaliação da recuperação desta linhagem em cultura mista com o fermento de panificação comercial. A levedura selecionada para o caráter H2S negativo manteve sua viabilidade durante os reciclos, com níveis de contaminação não detectados pela metodologia empregada, apresentando comportamento cinético semelhante ao da levedura de panificação tanto em cultura pura quanto em cultivo misto, apresentando uma nova possibilidade para estudos sobre o controle de qualidade de processos de fermentação alcoólica com reciclagem de células.

'Killer' character of yeasts isolated from ethanolic fermentations

The number of killer, neutral and sensitive yeasts was determined from strains isolated from substrates related to alcoholic fermentations. From 113 isolates, 24 showed killer activity against NCYC 1006 (standard sensitive strain), while 30 were sensitive to NCYC 738 (standard killer strain), and 59 had no reaction in assays at 25-27°C. Two wild yeast strains of Saccharomyces cerevisiae and one of Candida colliculosa were tested against 10 standard killer strains and one standard sensitive strain in a cell x cell and well-test assays at four different pHs. None of the isolates displayed strong killer activity or were sensitive to the standard strains. All belonged to the neutral type. It was concluded that although the number of killer strains was high, this character cannot be used to protect ethanol fermentation processes against yeast contaminants like those which form cell clusters.

Avaliou-se o número de linhagens 'killer', sensíveis e neutras em leveduras isoladas de substratos relacionados à fermentação etanólica. Das 113 linhagens, 24 mostraram atividade 'Killer' contra o isolado NCYC 1006 (padrão de sensibilidade), 30 foram sensíveis ao isolado NCYC 738 (padrão 'Killer') e 59 apresentaram reação neutra para ambos os isolados. Três cepas de leveduras selvagens do processo (duas de Sacch. cerevisiae e uma de Candida colliculosa, que formam cachos de células não separáveis por tratamentos químicos ou físicos), foram testadas contra 10 isolados padrões do tipo 'Killer' e um isolado padrão de sensibilidade. Os ensaios células x células e células X toxinas foram realizados em diferentes pH e a 30ºC. As três cepas contaminantes mostraram reação neutra a todos os isolados padrões testados. Apesar do alto número de linhagens 'Killer' entre aquelas testadas, concluiu-se que este caráter não pode ser utilizado na proteção do processo de fermentação etanólica contra essas leveduras selvagens contaminantes.

'Killer' character of yeasts isolated from ethanolic fermentations

The number of killer, neutral and sensitive yeasts was determined from strains isolated from substrates related to alcoholic fermentations. From 113 isolates, 24 showed killer activity against NCYC 1006 (standard sensitive strain), while 30 were sensitive to NCYC 738 (standard killer strain), and 59 had no reaction in assays at 25-27°C. Two wild yeast strains of Saccharomyces cerevisiae and one of Candida colliculosa were tested against 10 standard killer strains and one standard sensitive strain in a cell x cell and well-test assays at four different pHs. None of the isolates displayed strong killer activity or were sensitive to the standard strains. All belonged to the neutral type. It was concluded that although the number of killer strains was high, this character cannot be used to protect ethanol fermentation processes against yeast contaminants like those which form cell clusters.

Avaliou-se o número de linhagens 'killer', sensíveis e neutras em leveduras isoladas de substratos relacionados à fermentação etanólica. Das 113 linhagens, 24 mostraram atividade 'Killer' contra o isolado NCYC 1006 (padrão de sensibilidade), 30 foram sensíveis ao isolado NCYC 738 (padrão 'Killer') e 59 apresentaram reação neutra para ambos os isolados. Três cepas de leveduras selvagens do processo (duas de Sacch. cerevisiae e uma de Candida colliculosa, que formam cachos de células não separáveis por tratamentos químicos ou físicos), foram testadas contra 10 isolados padrões do tipo 'Killer' e um isolado padrão de sensibilidade. Os ensaios células x células e células X toxinas foram realizados em diferentes pH e a 30ºC. As três cepas contaminantes mostraram reação neutra a todos os isolados padrões testados. Apesar do alto número de linhagens 'Killer' entre aquelas testadas, concluiu-se que este caráter não pode ser utilizado na proteção do processo de fermentação etanólica contra essas leveduras selvagens contaminantes.