Athanasios A. Koutinas

High-temperature alcoholic fermentation of whey using Kluyveromyces marxianus IMB3 yeast immobilized on delignified cellulosic material.

A novel system for high-temperature alcoholic fermentation of whey is described. This system consists of Kluyveromyces marxianus yeast immobilized on delignified cellulosic material (DCM). The effect of pH, initial lactose concentration and temperature on the fermentation of a synthetic medium containing lactose was studied. Batch fermentations of whey were also carried out and the formation of volatile by-products was examined. The concentrations of higher alcohols were found to be in very low levels leading to a product of improved quality. The fermented whey had an improved characteristic aroma compared to unfermented whey. The possibility to use fermented whey as raw material for the production of a novel, low alcohol content drink was also investigated.

Wine fermentations by immobilized and free cells at different temperatures. Effect of immobilization and temperature on volatile by-products

A biocatalyst was prepared by immobilization of Saccharomyces cerevisiae, strain AXAZ-1, on delignified cellulosic material (DCM) and gluten pellets (GP). Repeated batch fermentations were conducted using these biocatalysts and free cells, separately, at different temperatures. The volatile constituents were extracted with dichloromethane and the extracts were subsequently analyzed by HRGC/MS. Wines produced by DCM contained higher amounts of esters, at every temperature studied, whereas those produced by GP biocatalyst contained higher amounts of alcohols. Free cells and DCM biocatalyst gave wines with similar contents of alcohols. Wines produced by DCM biocatalyst gave better ratios of esters to alcohols and had dominating fruity aromas. GC/MS analysis proved that cell immobilization did not create serious changes in qualitative composition of the wine aroma. As regards the quantitative profile, the combined effect of temperature and immobilization resulted in the production of wines with more fruity character because of the higher ratio of esters to alcohols.

Solid state fermentation of food waste mixtures for single cell protein, aroma volatiles and fat production

Growth of selected microorganisms of industrial interest (Saccharomyces cerevisiae, Kluyveromyces marxianus and kefir) by solid state fermentation (SSF) of various food industry waste mixtures was studied. The fermented products were analysed for protein, and nutrient minerals content, as well as for aroma volatile compounds by GC/MS. The substrate fermented by K. marxianus contained the highest sum of fat and protein concentration (59.2% w/w dm) and therefore it could be considered for utilisation of its fat content and for livestock feed enrichment. Regarding volatiles, the formation of high amounts of ε-pinene was observed only in the SSF product of kefir at a yield estimated to be 4 kg/tn of SSF product. A preliminary design of a biorefinery-type process flow sheet and its economic analysis, indicated potential production of products (enriched livestock feed, fat and ε-pinene) of significant added value.

Grape skins as a natural support for yeast immobilization

Grape skins were used to immobilize Saccharomyces cerevisiae. In repeated batch fermentations of grape by immobilized and free cells, the maximum specific rate of alcohol production on glucose decreased from 7.98 h−1 at 25 °C to 0.7 h−1 at 5 °C. The rate was approximately twice as high as that on fructose. The rates for free cells were very low. The maximum alcohol yield (0.45 g g−1) was obtained at 5 °C when the immobilized biocatalyst was used.

Scale-up of extremely low temperature fermentations of grape must by wheat supported yeast cells.

A new biocatalyst was prepared by immobilization of Saccharomyces cerevisiae AXAZ-1 yeast cells on whole wheat grains. This biocatalyst was used for 30 repeated batch fermentations of glucose and grape must at various temperatures. The biocatalyst retained its operational stability for a long period and it was proved capable to produce dry wines of fine clarity even at extremely low temperatures (5 degrees C). After the completion of these fermentations the new biocatalyst was used in a scale-up system of 80 L for wine making at ambient (20 degrees C) and extremely low temperatures (2 degrees C). The scale-up process did not affect the fermentative ability of biocatalyst, even at low temperatures, while the produced wines had almost the same improved aromatic profile compare to free cells as revealed by GC and GC-MS analyses. More specifically the results showed that both systems with immobilized cells (laboratory scale and 80 L bioreactor) increased the formation of esters and produced wines with improved aromatic profile compared to those with free cells. Finally an increase in the percentages of total esters and a decrease in those of higher alcohols was observed in lower fermentation temperatures.

Ethanol Production by Saccharomyces cerevisiae Immobilized on Mineral Kissiris

Abstract The attachment of Saccharomyces cerevisiae cells on the cheap and abundant ore Kissiris was investigated. Attached cells produced about 115 g/ l /d ethanol when inoculated in media containing glucose and 96 g/ l /d ethanol in raisin extract. No reduction in ethanol productivity and yield was observed for up to 29 repeated batch fermentations.

Nano-tubular cellulose for bioprocess technology development.

Delignified cellulosic material has shown a significant promotional effect on the alcoholic fermentation as yeast immobilization support. However, its potential for further biotechnological development is unexploited. This study reports the characterization of this tubular/porous cellulosic material, which was done by SEM, porosimetry and X-ray powder diffractometry. The results showed that the structure of nano-tubular cellulose (NC) justifies its suitability for use in “cold pasteurization” processes and its promoting activity in bioprocessing (fermentation). The last was explained by a glucose pump theory. Also, it was demonstrated that crystallization of viscous invert sugar solutions during freeze drying could not be otherwise achieved unless NC was present. This effect as well as the feasibility of extremely low temperature fermentation are due to reduction of the activation energy, and have facilitated the development of technologies such as wine fermentations at home scale (in a domestic refrigerator). Moreover, NC may lead to new perspectives in research such as the development of new composites, templates for cylindrical nano-particles, etc.

Progress in bacterial cellulose matrices for biotechnological applications

Bacterial cellulose (BC) is an extracellular polymer produced by many microorganisms. The Komagataeibacter genus is the best producer using semi-synthetic media and agricultural wastes. The main advantages of BC are the nanoporous structure, high water content and free hydroxyl groups. Modification of BC can be made by two strategies: in-situ, during the BC production, and ex-situ after BC purification. In bioprocesses, multilayer BC nanocomposites can contain biocatalysts designed to be suitable for outside to inside cell activities. These nanocomposites biocatalysts can (i) increase productivity in bioreactors and bioprocessing, (ii) provide cell activities does not possess without DNA cloning and (iii) provide novel nano-carriers for cell inside activity and bioprocessing. In nanomedicine, BC matrices containing therapeutic molecules can be used for pathologies like skin burns, and implantable therapeutic devices. In nanoelectronics, semiconductors BC-based using salts and synthetic polymers brings novel films showing excellent optical and photochemical properties.

Corn starch gel for yeast cell entrapment. a view for catalysis of wine fermentation.

A new biocatalyst was prepared by immobilization of Saccharomyces cerevisiae AXAZ-1 yeast cells in the matrix of corn starch gel. This biocatalyst was used for repeated batch fermentations of glucose and grape must at various sugar concentrations (110-280 g/L) and low-temperature winemaking (5 degrees C). The biocatalyst retained its operational stability for a long period, and it was proved to be capable of producing dry and semisweet wines. The produced wines were analyzed for volatile byproducts by GC and GC-MS, and the results showed an increase in the number and amount of esters by immobilized cells. In addition, an increase in the percentages of esters and a decrease in those of alcohols with the drop of fermentation temperature were reported. The activation energy (E(a)) was lower (approximately 36%) and the reaction rate constant (k) was higher (approximately 78% at 30 degrees C and approximately 265% at 15 degrees C) in the case of immobilized cells compared to free cells, especially at low temperatures. These results show that corn starch gel may act as a promoter for the enzymes that are involved in the process or as a catalyst of the alcoholic fermentation and can explain the capability of immobilized cells for extremely low-temperature winemaking. Therefore, these results open a new way for research to find new catalysts in biotechnological processes.

Oenococcus oeni cells immobilized on delignified cellulosic material for malolactic fermentation of wine

Oenococcus oeni ATCC 23279 cells immobilized on delignified cellulosic material (DCM) were used for malolactic fermentation (MLF). In first, eleven repeated alcoholic fermentation batches of white must of 11-12 degrees Be initial density were performed by Saccharomyces cerevisiae cells immobilized on delignified cellulosic material at 20 degrees C. Subsequently, the induction of MLF in the eleven taken wine batches by O. oeni cells immobilized on DCM took place at 27 degrees C. From the 3rd MLF batch up to 10th, the malic acid degradation was 53.1 up to 67.4% and the cfu of the immobilized cells/g of biocatalyst remained stable. The produced lactic acid was less than the stoichiometric yield and acetic acid content was significantly reduced after MLF not contributing in an important increase of the volatile acidity of wine. Ethanol, higher alcohols acetaldehyde and diacetyl contents in wines after MLF were in acceptable levels.