7. Fermentation organisms

There are few yeast species which assimilate lactose to produce ethanol [32]. K. marxianus is the microorganism widely used in industrial lactose to ethanol conversion. Other microorganisms used in industrial food and beverage manufacture have been examined at an experimental scale for their suitability for lactose to bioethanol conversion. These organisms include K. lactis, S. cerevisiae and Escherichia coli. Use of genetically engineered organisms for alcoholic beverage manufacture is currently not a common commercial practice. This is likely due to perceived consumer concerns about the consumption of genetically modified organisms.

### 7.1 K. marxianus and considerations for lactose to ethanol conversion

K. marxianus ability to convert lactose to ethanol is widely reported in scientific studies concerning bioethanol production. K. marxianus is the fermentative organism used for large scale manufacture of potable spirits and bioethanol produced from whey/whey permeate [8, 9]. Scientific studies often reference K. fragilis as a lactosefermenter; however, it is currently synonymous with K. marxianus [33]. Several studies have investigated the use of Candida pseudotropicalis as the fermentative organism for lactose to ethanol conversion [10]. C. pseudotropicalis, also referred to as Candida kefir, is the anamorph (asexual reproductive stage) of K. marxianus [33]. The species K. marxianus have a high degree of genetic variation and each strain's ability to produce ethanol can vary widely [34–36]. This is likely due to the species being present in a wide range of habitats [35]. K. marxianus is widely considered to be a Crabtree-negative organism, meaning the organism will preferentially respire instead of ferment when oxygen and glucose are abundant [37]. K. marxianus carries the genes necessary for fermentation and strains have been reported as Crabtree-positive (preferentially ferments in presence of oxygen and an abundance of glucose) [37, 38]. The ethanol tolerance of K. marxianus is lower than S. cerevisiae and can limit ethanol production [39]. Inhibition of ethanol production can occur at ethanol concentrations as low as 45–52 g/l or approximately 5.5–6.5% v/v [40]. Supplementation or concentration lactose within whey or whey permeate can cause substrate inhibition and limit ethanol production. This trait appears to be strain specific with reports varying of ethanol production inhibition at lactose concentration of 108–200 g/l [41, 42]. This wide variation in reported ranges highlights the importance of purchasing the proper fermentative strain of K. marxianus to meet each lactose to ethanol producer's requirements.

K. marxianus is generally recognized as safe (designated GRAS), which is advantageous for potable spirit producers because the yeast biomass can be further processed for livestock or human consumption. It has been reported that the fermentation process can reduce the biological oxygen demand of whey or whey permeate by 75% [43] and aerobic cultivation has reduced BOD by 90–95% [35].
