Mutants of Pichia stipitis NRRL Y-7124 able to tolerate and produce ethanol from hardwood spent sulfite liquor (HW SSL) were obtained by UV mutagenesis. P. stipitis cells were subjected to three successive rounds of UV mutagenesis, each followed by screening first on HW SSL gradient plates and then in diluted liquid HW SSL. Six third generation mutants with greater tolerance to HW SSL as compared to the wild type (WT) were isolated. The WT strain could not grow in HW SSL unless it was diluted to 65% (v/v). In contrast, the third generation mutants were able to grow in HW SSL diluted to 75% (v/v). Mutants PS301 and PS302 survived even in 80% (v/v) HW SSL, although there was no increase in cell number. All the third generation mutants exhibited higher growth rates but significantly lower growth yields on xylose or glucose compared to the WT. The mutants fermented 4% (w/v) glucose as efficiently as the WT and fermented 4% (w/v) xylose more efficiently with a higher ethanol yield than the WT. In a medium containing 4% (w/v) each of xylose and glucose, all the third generation mutants utilized glucose as efficiently and xylose more efficiently than the WT. This resulted in higher ethanol yield by the mutants. The mutants retained the ability to utilize galactose and mannose and ferment them to ethanol. Arabinose was consumed slowly by both the mutants and WT with no ethanol production. In 60% (v/v) HW SSL, the mutants utilized and fermented glucose, mannose, galactose and xylose while the WT could not ferment any of these sugars.

Transformation of pBR322 DNA into Shigella occurred at a low frequency. The efficiency of transformation was highest in S. dysenteriae 1 and lowest in S. flexneri. Treatment of cells with CaCl2 for a prolonged period (24h) increased the efficiency of transformation in all strains, except in S. flexneri, where transformation efficiency could not be improved by a variety of manipulations. Transformation efficiency did not increase in any of the strains when transformation was carried out with plasmid DNA obtained from a transformant (homologous transformation), suggesting the absence of a strong restriction-modification system. Extracellular deoxyribonuclease (DNase) levels were low in all the strains tested, but the levels of endogenous DNAse, released after CaCl2 treatment or sonication of the cells, were high. Washing the cells with a solution of CaCl2 did not enhance transformation, suggesting that endogenous DNase could be a significant factor affecting transformation efficiency in species of Shigella.