Rodolfo J. Ertola

Permeabilization of yeast cells (Kluyveromyces lactis) with organic solvents

The permeabilization of Kluyveromyces lactis (CBS 683) cells in relation to β-galactosidase (β-d-galactoside galactohydrolase E.C. 3.2.1.23) activity was studied using chloroform, toluene, and ethanol. The performance of these solvents was dependent on the incubation time, the incubation temperature, and the concentration of both cells and solvents. Maximum enzyme activity was achieved with chloroform or with a mixture of chloroform and 10% ethanol at temperatures between 5 and 37°C. At 37°C, the process was very quick and permeabilization occurred in 5 min or less. Similar results were obtained with toluene and a mixture of toluene and 10% ethanol, except at 5°C where permeabilization did not occur. The minimum amount of chloroform and toluene needed to obtain the maximum enzyme activity increases the higher the cell concentration and the lower the temperature of incubation. When working with a cell concentration between 3 and 15.109 cells ml-1 at 37°C, the critical amount of both solvents lies between 1.5 and 3%. The presence of ethanol in the incubation mixture reduces these values to 0.75 and 2.5%. Ethanol (40%) was an effective permeabilizing agent at 5 and 30°C, but not at 37°C due to the inactivation of the enzyme. Solvent-permeabilized cells showed the same β-galactosidase activity as a suspension of disrupted cells. The relative enzyme activity was correlated with the fraction of cells that were actually permeabilized, as evidenced by the relationship observed between enzyme activity and the fraction of methylene blue-stainable cells.

Some factors affecting pectinase production from apple pomace in solid-state cultures

Abstract Some factors affecting pectinase production from apple pomace in solid-state culture (medium composition, inoculum size, temperature and type of apple pomace used) were investigated. To obtain high enzymatic activities, apple pomace requires the addition of an adequate level of organic nitrogen components like those present in peptone, yeast extract or corn-steep. Inoculum size is not critical and optimum temperature for the fermentation process is 30°C. In connection with apple-pomace quality, the sugar concentration is the most important parameter which controls pectinase yields. By using Granny Smith pomace mixtures, containing 4% of sugars, production as high as 1300 U g−1 was attained in 36 h of culture. Pectinase levels ranging from 1000 to 2000 U litre−1 of juice for 1 to 3 h were necessary to achieve clarification in three different apple juices.

Butanol production from apple pomace

SummaryThe use of apple pomace for butanol production was studied by employing strains ofClostridium acetobutylicum andbutylicum. Yields of butanol between 1.9 and 2.2 % of fresh apple pomace are reported.

High level of sporulation of Metarhizium anisopliae in a medium containing by-products

SummarySolid-state fermentation of rice bran or rice-bran-husk mixtures byMetarhizium anisopliae proved to be highly successful in spore yield. Optimum sporulation response on bran medium occurred when the initial water activity values ranged from 0.982 to 0.999. When bran was supplemented with 50% rice husk, the spore yield almost doubled, but a narrower initial water activity range (0.997–0.999) was optimum. Under these conditions, spore yields were 5–15 times higher (depending on the strain) than those currently obtained on the basis of rice grain fermentations.

Effect of high concentration of nutrients onBacillus thuringiensis cultures

SummaryBacterial insecticide production using a strain ofBacillus thuringiensis var. kurstaki was studied in batch culture considering the influence of increasing concentration of components of a glucose — yeast extract — mineral salts medium.It was found that spore counts were increased from 1.08×1012 spores. 1−1 to 7.36×1012 spores. 1−1 and toxin level from 1.05 mg.ml−1 to 6.85 mg.ml−1, when the concentration of glucose was increased from 8 to 56 (g 1−1), with the corresponding increase in the rest of medium components. Higher concentration of nutrients inhibit either spore count or toxin production.Preliminary experiments of fed-batch cultures which allows the use of high amounts of nutrients were also carried out. In this case spore counts of 1.2×1013 spores.1−1 were achieved.

Apple pomace as raw material for pectinases production in solid state culture

Abstract Apple pomace was used for pectinases production on a small scale by solid state cultures. Viscosimetric enzyme activities as high as 1062 U g−1 were attained in 48 h. Characterization of the enzymatic pool showed that it is adequate for practical purposes.

Characterization of growth and sporulation of Metarhizium anisopliae in solid-substrate fermentation

The entomopathogenic fungus Metarhizium anisopliae was cultivated by solid-substrate fermentation both with and without forced aeration using a medium based on a mixture of rice bran and rice husk; the water activity was fixed at an initial value of 0.998. An equation to describe the growth and sporulation processes was derived from stoichiometric considerations. By means of elemental mass balances and using the concept of c-mole, the stoichiometric coefficients of the growth equation were determined. It was concluded that under the culture conditions tested, M. anisopliae can grow at a maximum specific growth rate of 0.08 h−1. After 90–100 h of culture when growth has stopped, a spore yield of 2.3 × 1010 spores g−1 rice bran was obtained. This is as high as 80.4% of the maximum theoretical value. The heat generated during the culture did not affect growth and sporulation since, even when high initial amounts of solids were used, no desiccation was observed. Nevertheless, the yields obtained under these conditions suggested the appearance of another type of growth limitation not related to substrate consumption.

Water balance in solid-state fermentation without forced aeration

Abstract Water balances, based on the evaluation of water released from the fermentor, were estimated in a solid-state fermentation process performed without forced aeration. It was proved that the net gain or loss of water during the course of the process depends on the dry weight of the solid substrate at the beginning of the process and also on the initial water activity. A linear relationship was found between the wet weight losses of cultures recorded after the end of the growth period and the initial dry weight of the substrate which enables the evaluation of the water released from the vessels. These values were included into water balances from which it was possible to calculate the water produced by metabolism of carbohydrates, which was estimated at 0.40 mol of H 2 O per C-mol of glucose consumed.

Effect of nutritional factors on the culture of Nostoc sp. as a source of phycobiliproteins

SummaryThe effect of nutritional factors in a new culture medium (BW3) is described for the cyanobacterium Nostoc sp. The growth of Nostoc sp. was higher in BW3 than in control media currently used for cyanobacteria. With medium BW3 the content of the pigment c-phycocyanin depended on the culture conditions employed, particularly the nature of the nitrogen and carbon sources. Higher amounts of c-phycocyanin amounting to 20.1% on a dry-weight basis were accumulated when both sources were supplied in the gas phase of the culture. The phycobiliproteins of Nostoc sp. were resolved into two components: c-phycocyanin (λmax = 614 nm) and allophycocyanin (λmax = 652 nm). The phycobiliprotein composition was 30% allophycocyanin and 70% c-phycocyanin. The culture of Nostoc sp. in BW3 medium seems promising as a source of biomass for the production of natural dyes.

A new medium for growth and delta-endotoxin production by Bacillus thuringiensis var. kurstaki

SummaryThe influence of composition of media used for growth and delta-endotoxin production by B. thuringiensis var. kurstaki was studied with the idea of finding a cheap medium for attaining high yields of spore-crystal preparations. A new medium, based on malt sprouts is proposed. Data on growth and bioinsecticidal activity are given. A concentration of 2.1 × 109 spores.ml-1 was attained in a 48 h process. The spore-crystal preparations obtained present a LC50 of 2.18 × 108 spores.g-1 against larvae of Galleria mellonella.