Lignocellulolytic characterization and comparative secretome analysis of a Trichoderma erinaceum strain isolated from decaying sugarcane straw.

The fungus Trichoderma reesei is employed in the production of most enzyme cocktails used by the lignocellulosic biofuels industry today. Despite significant improvements, the cost of the required enzyme preparations remains high, representing a major obstacle for the industrial production of these alternative fuels. In this study, a new Trichoderma erinaceum strain was isolated from decaying sugarcane straw. The enzyme cocktail secreted by the new isolate during growth in pretreated sugarcane straw-containing medium presented higher specific activities of ß-glucosidase, endoxylanase, ß-xylosidase and α-galactosidase than the cocktail of a wild T. reesei strain and yielded more glucose in the hydrolysis of pretreated sugarcane straw. A proteomic analysis of the two strains' secretomes identified a total of 86 proteins, of which 48 were exclusive to T. erinaceum, 35 were exclusive to T. reesei and only 3 were common to both strains. The secretome of T. erinaceum also displayed a higher number of carbohydrate-active enzymes than that of T. reesei (37 and 27 enzymes, respectively). Altogether, these results reveal the significant potential of the T. erinaceum species for the production of lignocellulases, both as a possible source of enzymes for the supplementation of industrial cocktails and as a candidate chassis for enzyme production.

Genome structure of a Saccharomyces cerevisiae strain widely used in bioethanol production.

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 (approximately 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.

Contamination levels and preliminary assessment of the technical feasibility of employing natural attenuation in 5 priority areas of Presidente Bernardes Refinery in Cubatão, São Paulo, Brazil.

Five priority areas of potential impact by contaminants (API) were investigated at the Presidente Bernardes Refinery in Cubatão, São Paulo, Brazil with the following aims: (i) to identify both organic and inorganic contaminants present in soil and groundwater; (ii) to define the environmental conditions relevant for microbial activity at the site and (iii) to evaluate the feasibility of employing natural attenuation for treatment of the hydrocarbon contamination. One area (API 1) was an uncontrolled landfill, where waste materials from the refinery were deposited between 1954 and 1986, and four areas (API 4, 5, 7 and 11) were located in the operational section of the refinery. Soil contamination by regulated BTEX compounds (benzene, toluene, total xylenes) was restricted to two samples from API 1. Nonregulated ethylbenzene was detected in one soil sample from API 4, one from API 5 and two from API 1. No soil contained regulated PAH above threshold levels. Several nonregulated PAHs were found in 6 soil samples from API 1, 3 soil samples from API 4 and 1 soil sample from API 5. Site soils contained very high aluminium concentrations, but metal contamination was restricted to one soil sample from API 1, which contained nickel above threshold limits. BTEX contamination of groundwater was due mostly to benzene. Of the 17 PAH molecules tested, only naphthalene and 2-methylnaphthalene occurred in groundwater. The sum of total BTEX and total PAH exceeded 200 microg/L in only a few monitoring wells in API 4, 5 and 11 and was always below 2.640 microg/L. Be, Cd, Cr, Cu, Hg, Ni, Se, Ag, Tl and Zn were not detected in groundwater, which was contaminated in a few locations by aluminium (mostly below 1 mg/L), lead (<0.066 mg/L) and arsenic (<0.056 mg/L). S, K, Ca, Mg and Fe were present in groundwater in excess of physiological requirements for microbial growth, but low concentrations of N and P could become growth limiting. However, BTEX were efficiently degraded in saturated and unsaturated zone microcosms and nutrient amendments did not stimulate biodegradation rates measurably. The inorganic carbon pool in groundwater was up to one order of magnitude larger than the organic carbon pool. Total inorganic carbon (TIC) in API groundwater exceeded TIC of clean groundwater by factors of 2 (API 4), 6 (API 5, 7 and 11) or 10 (API 1). Most of the inorganic carbon incorporated into groundwater beneath the refinery originated from biodegradation in the unsaturated soil, which contained a microbiota (10(6) cells/g on average) capable of growth with most of the pure (benzene, toluene, ethylbenzene and xylene) and mixed hydrocarbons tested (diesel oil, gasoline, naphtha, condensate, aromatic residue and fuel oil). A viscous hydrocarbon paste uncovered in API 1 was insoluble in water but dissolved in dichloromethane. Many organic components of this paste were biodegradable as evidenced by weight reduction of the hydrocarbon paste and by the growth of suspended and attached biomass in saturated zone microcosms, where the paste was the only carbon source. This study indicates that monitored natural attenuation may be a technically feasible and efficient means for plume control in API 1, 4 and 5, provided the plumes in API 4 and 5 are not expanding. This technique is not suitable for contaminant reduction in API 11.

Control by sugar of Saccharomyces cerevisiae flocculation for industrial ethanol production.

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.