Monitoring for wild yeast contaminants is an essential component of the management of the industrial fuel ethanol manufacturing process. Here we describe the isolation and molecular identification of 24 yeast species present in bioethanol distilleries in northeast Brazil that use sugar cane juice or cane molasses as feeding substrate. Most of the yeast species could be identified readily from their unique amplification-specific polymerase chain reaction (PCR) fingerprint. Yeast of the species Dekkera bruxellensis, Candida tropicalis, Pichia galeiformis, as well as a species of Candida that belongs to the C. intermedia clade, were found to be involved in acute contamination episodes; the remaining 20 species were classified as adventitious. Additional physiologic data confirmed that the presence of these major contaminants cause decreased bioethanol yield. We conclude that PCR fingerprinting can be used in an industrial setting to monitor yeast population dynamics to early identify the presence of the most important contaminant yeasts.

Aims: The present work focuses on the possibility to use conserved primers that amplify yeast ITS1-5AE8S-ITS2 ribosomal DNA locus (rDNA) to detect the presence of non-Saccharomyces cerevisiae yeast in fermentation must of bioethanol fermentation process. Methods and Results: Total DNA was extracted from pure or mixed yeast cultures containing different cell concentrations and different contaminant/fermenting yeast concentrations and submitted to PCR. Upon improvement of detection limits and DNA extraction protocol, must samples of distillery were checked for the presence of contaminant yeast. Contaminant rDNA bands were detected only in industrial samples during contamination episodes, but not in noncontaminated must. Conclusions: The method described here could detect the presence of contaminant yeast from industrial must in eight hours after sampling. Significance and Impact of the Study: The improved procedure may help to avoid severe contamination episodes at fermentation industries by decreasing the detection time from 5 days to 8 h and possible quantification of contaminant yeasts that can impose economical loss to the process.

Fuel ethanol fermentation process is a complex environment with an intensive succession of yeast strains. The population stability depends on the use of a well-adapted strain that can fit to a particular industrial plant. This stability helps to keep high level of ethanol yield and it is absolutely required when intending to use recombinant strains. Yeast strains have been previously isolated from different distilleries in Northeast Brazil and clustered in genetic strains by PCR-fingerprinting. In this report we present the isolation and selection of a novel Saccharomyces cerevisiae strain by its high dominance in the yeast population. The new strain, JP1 strain, presented practically the same fermentative capacity and stress tolerance like the most used commercial strains, with advantages of being highly adapted to different industrial units in Northeast Brazil that used sugar cane juice as substrate. Moreover, it presented higher transformation efficiency that pointed out its potential for genetic manipulations. The importance of this strain selection programme for ethanol production is discussed.

Article Info Fungus/Allergen Size (kD) Biological Activity Sequence access number Penicillium brevicompactum Pen b 13 33 Alkaline serine protease * Pen b 26 11 Ribosomal acid protein 60S AY786077 Penicillium chrysogenum Pen ch 13 34 Alkaline serine protease AF193420 Pen ch 18 32 Vacuolar serine protease AF264027 Pen ch 20 68 N-acetyl-glucosaminidase S77837 Pen ch 31 Calreticulin AY850367 Pen ch 33 16 unknown EF206657.1 Pen ch 35 36.5 Transaldolase GQ925430 Penicillium citrinum Pen c 3 18 Peroxisomal membrane protein AF144753 Pen c 13 33 Alkaline serine protease * Pen c 19 70 Heat shock protein P70 U64207 Pen c 22 46 Enolase AF254643 Pen c 24 Elongation factor 1β AY363911 Pen c 30 97 Catalase DQ288844 Pen c 32 40 Pectate lyase EF159713 Penicillium crustosum Pen cr 26 11 Acid ribosomal phosphoprotein P1 60s JN791438 Penicillium oxalicum

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