Walter Vieira Guimarães

Genetic diversity of the ectomycorrhizal fungus Pisolithus tinctorius based on RAPD-PCR analysis

Abstract Twenty Pisolithus tinctorius isolates from different geographic locations and different hosts were characterized by the random amplified polymorphic DNA technique. Thirteen arbitrary primers generated 87 DNA fragments, all of them polymorphic. These data were used to calculate genetic distances among the isolates. The pairwise genetic distances ranged from 1 to 100%, with an average of 58.7%. Cluster analysis based on the amplified fragments grouped the isolates according to their host and geographical origins. Group I contained isolates collected in Brazil and group II those collected in the Northern Hemisphere. In addition to the diversity seen at the molecular level, the isolates also showed host specificity. Greenhouse experiments demonstrated that isolates from the Northern Hemisphere colonized mainly Pinus whereas isolates from Brazil colonized only Eucalyptus. The molecular data suggest that the Pisolithus tinctorius isolates analyzed belong to two distinct groups. The data also suggest new guidelines for future investigations on the taxonomy and systematic of this important fungus species. Furthermore, these results support future experiments aimed at the selection and development of improved isolates of P. tinctorius.

Ethanolic fermentation of sucrose, sugarcane juice and molasses by Escherichia coli strain ko11 and Klebsiella oxytoca strain P2

Escherichia coli KO11 and Klebsiella oxytoca P2 recombinants fermented sucrose to ethanol. In minimal medium with 2% or 12% added sucrose KO11 produced 75% and 41%, respectively, of the maximum theoretical yield (0.54g ethanol/g sucrose). In Luria-Bertani (LB) broth with up to 8% sucrose, KO11 presented a 94-96% yield and with 12% sucrose, KO11 presented about 69% yield (44.5g ethanol/L). P2 presented 55% of the theoretical maximum yield in minimal medium supplemented with 2% sucrose and 47% of the maximum in 12% sucrose. In LB broth with 2 or 4% sucrose, P2 presented 94-95% of the theoretical maximum yield, which fell to 73% with 8% added sucrose (31.4g ethanol/L) and 58% with 12% sucrose (37.5 g/L). Volumetric productivity in LB broth containing 2% sucrose was 0.41 g/L/h for KO11 and 1.1 g/L/h for P2, while in LB broth with 12% added sucrose, productivity was 0.96 g/L/h (KO11) and 1.4 g/L/h (P2). During fermentation of sugar cane juice by E. coli KO11 and K. oxytoca P2 produced 39.4 g/L and 42.1 g/L ethanol when supplemented with 0.5% yeast extract, micronutrients and thiamine. In sugar cane juice supplemented with LB broth ingredients, KO11 ethanol fermentation was inhibited with production of only 23.0 g ethanol/L, while P2 produced 44.2 g/L. Ethanol production by KO11 and P2, respectively, in sugarcane juice was a) 25.3 and 30.2 g/L with 0.2% ammonium sulfate, b) 24.9 and 31.6 g/L with ammonium sulfate and micronutrients, c) 25.6 and 37.5 g/L with ammonium sulfate, micronutrients and thiamine. During molasses fermentation E. coli KO11 presented low ethanol production and high lactic acid production. K. oxytoca P2 produced 25 g ethanol/L in molasses diluted 10-fold in water, with or without addition of 0.5% yeast extract, and 27.8 g/L with addition of LB broth ingredients after 96h. P2 produced 24.5, 26.9, and 28.0 g ethanol/L in molasses diluted 10-fold in vinasse, vinasse with 0.5% added yeast extract and with LB broth ingredients, respectively.

FERMENTATION OF SWEET WHEY BY RECOMBINANT ESCHERICHIA COLI KO11

A producao de etanol a partir de soro de leite empregando a cepa Escherichia coli KO11 recombinante, em fermentacao de batelada, foi testada. O rendimento maximo de etanol foi obtido em 96h, representando apenas 38% do rendimento teorico. A suplementacao do soro com os componentes do caldo LB aumentou o rendimento para 96% em 72h. A adicao de 0,5% de extrato de levedura ao soro resultou em um rendimento maximo de 74% em 36h que aumentou para acima de 100% quando se adicionou extrato de levedura e uma solucao de metais traco (Fe++, Mn++ e Zn++).

Production and regeneration of protoplasts from the mycorrhizal fungus Suillus granulatus

Experiments were performed with the mycorrhizal fungus Suillus granulatus to define the parameters for production and regeneration of protoplasts. Protoplasts were released at frequencies between 1 and 3×107/ml from mycelium 3 to 7 days old. The best osmotic stabilizer for protoplast release was MgSO4 (0.7 m). To optimize protoplast release and regeneration an enzyme (Novozym 234) concentration 1.7 mg/ml was chosen, with a digestion time of 1 to 2 h. Regenerated colonies formed mycorrhizae within 60 days after inoculation in Pinus caribaea var. hondurensis seedlings.

Ethanol production from starch by recombinant Escherichia coli containing integrated genes for ethanol production and plasmid genes for saccharification

Ethanologenic strains ofEscherichia coli have been developed which can express thermostable enzymes for starch saccharification as intracellular products. These enzymes can be harvested within cells at the end of fermentation and liberated by heating to the temperature at which they exhibit maximal activity (60°C to 70°C). Organisms such as these could be used to supply enzymes for yeast-based fermentations while producing ethanol as a co-product.

Fermentation of maltose and starch by Klebsiella oxytoca P2

Klebsiella oxytoca P2, which has genes from Zymomonas mobilis encoding the alcohol dehydrogenase and pyruvate decarboxylase integrated in its chromosome, fermented 50 g maltose/l to 25.4 g of ethanol/l. It also fermented 10, 20 and 40 g starch/l yielding 4, 8.4, and 17.7 g ethanol/l, respectively, representing 72, 75 and 78% of the theoretical yield.