Bioethanol is a biofuel produced mainly from the fermentation of carbohydrates derived from agricultural feedstocks by the yeast Saccharomyces cerevisiae. One of the most widely adopted strains is PE-2, a heterothallic diploid naturally adapted to the sugar cane fermentation process used in Brazil. Here we report the molecular genetic analysis of a PE-2 derived diploid (JAY270), and the complete genome sequence of a haploid derivative (JAY291). The JAY270 genome is highly heterozygous (;2 SNPs/kb) and has several structural polymorphisms between homologous chromosomes. These chromosomal rearrangements are confined to the peripheral regions of the chromosomes, with breakpoints within repetitive DNA sequences. Despite its complex karyotype, this diploid, when sporulated, had a high frequency of viable spores. Hybrid diploids formed by outcrossing with the laboratory strain S288c also displayed good spore viability. Thus, the rearrangements that exist near the ends of chromosomes do not impair meiosis, as they do not span regions that contain essential genes. This observation is consistent with a model in which the peripheral regions of chromosomes represent plastic domains of the genome that are free to recombine ectopically and experiment with alternative structures. We also explored features of the JAY270 and JAY291 genomes that help explain their high adaptation to industrial environments, exhibiting desirable phenotypes such as high ethanol and cell mass production and high temperature and oxidative stress tolerance. The genomic manipulation of such strains could enable the creation of a new generation of industrial organisms, ideally suited for use as delivery vehicles for future bioenergy technologies.

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Sugar cane silages are characterized by extensive yeast activity, alcohol production and great dry matter -DM -losses. Better knowledge of the fermentation process is fundamental to the development of efficient ensilage techniques for this forage. This study evaluates temporal changes in chemical composition, DM losses and epiphytic microflora in sugar cane silage. Mature sugar cane, variety RB835486 (12 months of vegetative growth), was hand harvested, processed in a stationary chopper and ensiled in 20-L plastic buckets provided with valves for gas release and a device for effluent collection. Laboratory silos were kept at ambient temperature and sampled after ½, 1, 2, 3, 7, 15, 45, 90, 120 and 180 days. Ethanol concentration reached 6.4% in DM after 15 days of ensilage, followed by 71% water soluble carbohydrates -WSCs -disappearance. Gas and total DM losses reached a plateau on day 45 (16% and 29% of DM, respectively). Yeast count was higher on the second day (5.05 log cfu g -1 ). Silage pH declined to below 4.0 on the third day. Effluent yield was negligible (20 kg t -1 ). DM content in the forage decreased (35% to 26%) from day 0 to day 45. The increase in ethanol concentration showed an opposite trend to WSCs and true in vitro dry matter digestibility reductions in the silage. Developing methods to control yeasts, most probably through the use of additives, will enable more efficient production of sugar cane silage by farmers. Key words: ethanol, water soluble carbohydrates, losses, yeasts, bacteria DINÂMICA DA FERMENTAÇÃO E DA MICROFLORA EPÍFITA EM SILAGEM DE CANA-DE-AÇÚCARRESUMO: Silagens de cana-de-açúcar caracterizam-se pela extensa atividade de leveduras, alto teor de álcool e grandes perdas de matéria seca -MS. Conhecer melhor o processo fermentativo é fundamental para o desenvolvimento de técnicas eficientes de ensilagem da cana. Este trabalho avalia a mudança temporal na composição química, nas perdas de MS e na microflora epífita nestas silagens. Cana-de-açúcar (RB835486) foi colhida manualmente (12 meses de crescimento), picada em picadora estacionária e ensilada em baldes de plástico de 20 L com válvulas para gases e aparato para colheita de efluentes. Os silos laboratoriais foram mantidos sob temperatura ambiente e amostrados após ½, 1, 2, 3, 7, 15, 45, 90, 120 e 180 dias. Etanol atingiu 6,4% na MS no 15 o dia após ensilagem, seguido pelo desaparecimento de 71% dos carboidratos solúveis -CHOs. As perdas gasosas e a perda total de MS estabilizaram-se após 45 dias (16% e 29% da MS). A contagem de leveduras foi máxima no segundo dia (5,05 log ufc g -1 ). O pH atingiu nível abaixo de 4,0 no terceiro dia. A produção de efluentes foi insignificante (20,1 kg t -1 ). O teor de MS da forragem decresceu (35% para 26%) do dia 0 ao 45 o dia. O padrão de variação na concentração de etanol foi inverso à concentração de CHOs e à redução da digestibilidade da silagem. O desenvolvimento de métodos de controle das leveduras, provavelmente com o uso de aditivos, melhorará a eficiência no uso de silagen...

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Fermentation of Theobroma cacao (cacao) seeds is an absolute requirement for the full development of chocolate flavor precursors. An adequate aeration of the fermenting cacao seed mass is a fundamental prerequisite for a satisfactory fermentation. Here, we evaluated whether a controlled inoculation of cacao seed fermentation using a Kluyveromyces marxianus hybrid yeast strain, with an increased pectinolytic activity, would improve an earlier liquid drainage ('sweatings') from the fermentation mass, developing a superior final product quality. Inoculation with K. marxianus increased by one third the volume of drained liquid and affected the microorganism population structure during fermentation, which was detectable up to the end of the process. Introduction of the hybrid yeast affected the profile of total seed protein degradation evaluated by polyacrylamide gel electrophoresis, with improved seed protein degradation, and reduction of titrable acidity. Sensorial evaluation of the chocolate obtained from beans fermented with the K. marxianus inoculation was more accepted by analysts in comparison with the one from cocoa obtained through natural fermentation. The increase in mass aeration during the first 24 h seemed to be fundamental for the improvement of fermentation quality, demonstrating the potential application of this improved hybrid yeast strain with superior exogenous pectinolytic activity.

The goal of this study was to develop conditional flocculant yeasts for use in the alcohol fermentation industry. Promoters were constructed to completely repress gene transcription in the presence of sugar and to support strong expression after the exhaustion of this compound. A fusion of regulatory regions of the ADH2 promoter with the FLO5 core promoter was constructed to regulate the FLO5 gene. This construct was inserted into multicopy plasmids and transformed into laboratory strains of Saccharomyces cerevisiae, whereby the transformed cells were selected by sedimentation from the bulk medium after sugar exhaustion, without decreasing ethanol production. The ADH2-FLO5 region was converted into an integrative cassette to disrupt the CAN1 gene in industrial yeast strains. Transformed cells became resistant to canavanine and demonstrated conditional flocculation. Although ethanol production was significantly decreased in the industrial transformants, this development reveals a promising technology for the substitution of centrifugation in industrial ethanol production.

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Anthracnose is a crop disease usually caused by fungi in the genus Colletotrichum or Gloeosporium. These are considered one of the main pathogens, causing significant economic losses, such as in peppers and guarana. The current forms of control include the use of resistant cultivars, sanitary pruning and fungicides. However, even with the use of some methods of controlling these cultures, the crops are not free of anthracnose. Additionally, excessive application of fungicides increases the resistance of pathogens to agrochemicals and cause harm to human health and the environment. In order to find natural antifungal agents against guarana anthracnose, endophytic fungi were isolated from Amazon guarana. The compounds piliformic acid and cytochalasin D were isolated by chromatographic techniques from two Xylaria spp., guided by assays with Colletotrichum gloeosporioides. The isolated compounds were identified by spectrometric techniques, as NMR and mass spectrometry. This is the first report that piliformic acid and cytochalasin D have antifungal activity against C. gloeosporioides with MIC 2.92 and 2.46 μmol mL−1 respectively. Captan and difenoconazole were included as positive controls (MIC 16.63 and 0.02 μmol mL−1, respectively). Thus, Xylaria species presented a biotechnological potential and production of different active compounds which might be promising against anthracnose disease.