7.2 Environmental considerations and fermentation parameters for K. marxianus

Several adjustable factors can influence the rate and quality of fermentation by K. marxianus. These factors include the presence of oxygen, nutrient supplementation, substrate, pH and fermentation temperature. In general, hypoxic and anoxic environments favor K. marxianus's fermentative metabolism, and ethanol yields are greater than in an aerobic environment [10, 43]. Aerobic conditions favor the building of cell density and are commercially applied for cell propagation in vessels called "Donas" [9]. The doubling time of K. marxianus is approximately 70 min, and it has one of the fastest growth rates of any eukaryote [37].

Nutrients are not added to the whey/whey permeate during commercial fermentations [9]. Additional supplementation of nitrogen and phosphorus to whey/ whey permeate was shown not to affect ethanol production during fermentation [44]. It has been illustrated experimentally that supplementation of concentrated whey (200 g/l lactose) with bacto-peptone, ergosterol and linoleic acid reduced fermentation time from over 90 to less 60 h [10]. This is a substantial decrease in fermentation time, however large-scale commercial lactose to ethanol fermentations range from 12 to 24 h [8, 9].

K. marxianus is a thermotolerant yeast with reported maximum growth temperatures ranging from 47 to 52°C [10, 35]. Ethanol production has been reported at temperatures as high as 45°C [45] and, other studies indicate that the optimum fermentation temperature is lower. Studies indicate the optimal fermentation temperature range to be 30–40°C [36, 39, 41, 42, 46–48]. This wide range of reported temperatures can likely be attributed to the genetic diversity of K. marxianus strains and differences in experimental design. A pH of approximately 5 is widely reported as the optimum fermentation pH value [36, 39, 42, 46–48]. Agitation of fermenting whey/whey permeate occurs in industrial lactose to ethanol conversion and has been incorporated experimentally [8, 9, 47, 49]. To the authors' knowledge, the effects of the rate of agitation on fermentation efficiency of K. marxianus have not been examined.

Large scale lactose to ethanol production facilities will adjust fermentation time, temperature, tank pressure, and agitation rate to meet production goals [8, 9]. K. marxianus strain UFV-3 may have potential for potable ethanol production. K. marxianus strain UFV-3 was able to produce ethanol at yields 90% of the theoretical maximum with fermentation temperatures between 33.3 and 38.5°C, pH 4.7–5.7 and lactose concentrations between 50 and 108 g/l [42].

#### 7.3 Other fermentation organisms

K. lactis is used to produce lactase and recombinant bovine chymosin on an industrial scale. It is the sister organism to K. marxianus that is more widely studied. K. lactis synthesizes β-galactosidase much like K. marxianus and most strains of K. lactis are considered Crabtree-negative [50]. A small number of isolate have been used by researchers working with K. lactis and it is ubiquitous to fewer environments than K. marxianus [10, 32]. This has led to less genetic variation than within in the species than K. marxianus. Some strains of K. lactis exhibit Crabtree-positive

### Whey to Vodka DOI: http://dx.doi.org/10.5772/intechopen.81679

metabolic characteristics [51, 52] and have been genetically engineered for lactose to bioethanol conversion [53]; however, they have not been adopted on a commercial level for lactose to ethanol conversion.

S. cerevisiae is the microorganism widely used in alcoholic beverage and bioethanol production. S. cerevisiae is used for traditional potable spirit production for several reasons including its fermentative capacity and ethanol tolerance, being considered Crabtree-positive (preferentially ferments in presence of oxygen and an abundance of glucose), and it's GRAS designation [10]. S. cerevisiae is ill-suited for the conversion of lactose to potable ethanol because wild S. cerevisiae does not express the genes necessary to produce β-galactosidase. This requires the lactose within whey/whey permeate to be pre-hydrolyzed or S. cerevisiae to be genetically engineered to produce β-galactosidase. While pre-hydrolysis of lactose has been explored on an experimental scale for bioethanol production, it would require an additional input (enzymes) and/or additional processing equipment. S. cerevisiae preferentially uptakes glucose after lactose hydrolysis and the presence of glucose causes the catabolic repression of enzymes necessary to uptake galactose [54]. The enzymes necessary to uptake galactose will only be synthesized after the glucose has been depleted. This repression causes an increase in fermentation time due to a diauxic lag [10, 55]. While S. cerevisiae has been genetically engineered to synthesize β-galactosidase and to reduce catabolic repression, genetically engineered yeast are not commonly used for beverage production [19, 21, 56].

E. coli has been genetically altered to produce ethanol since 1987 [57]. In 2010, E. coli was genetically modified to express the Vitreoscilla hemoglobin for direct fermentation of sugar to ethanol [58]. This technology has been experimentally developed for the efficient fermentation of whey and other organic by-products. Recently, microbial immobilization has been experimentally applied to E. coli expressing Vitreoscilla hemoglobin and has shown an increase in lactose to bioethanol production efficiency without producing the microbial biomass associated with the traditional fermentation process [59, 60].

The use of genetically modified organisms for the conversion of whey to potable spirit has the potential to increase production efficiency and reduce operating costs. The use of these organisms will require consumer acceptance of potable spirits produced from this technology.
