Bioethanol is an alcohol made by fermenting the rich sugar components of biomass which is seen as a good fuel alternative. The use of bioethanol as a biofuel has very important advantage — it is generally CO2 neutral. This is achieved because in the growing phase of the biomass plants, CO2 is absorbed and then released in the same volume during combustion of the fuel (Stephenson et al., 2010). This creates an obvious advantage over fossil fuels which only emit CO2 as well as other poisonous gasses. Bioethanol can be used as a fuel for transport in its pure form, but it is usually used as a gasoline additive to increase its octane rating and improve vehicle efficiency (Balat & Balat, 2009).

Nowadays, the bioethanol market has continued to grow rapidly, for example, from about 46 billion L of ethanol produced worldwide in 2007 to the expected value of 100 billion L in 2015 (Balat & Balat, 2009; Sarkar et al., 2012). The USA is the world leader in the production of bioethanol with 48 billion L in 2009 (Muthaiyan & Ricke, 2010), followed by Brazil with 27,0 billion L in 2009 (Soccol et al., 2010) which determined 62% of the worldwide production (Sarkar et al., 2012). In the USA, bioethanol is mainly used as a 10% petrol additive (E10 is the standard petrol fuel, in 2011 introduced E15). In Brazil, it is offered both as a pure fuel (E100) and is blended with conventional petrol with a content of 20 to 25% (E20, E25). In Europe, with the adoption of the Biofuel Directive 2003/30/EC in 2003, the framework conditions were especially created for European bioethanol production. Today France is a leading producer of bioethanol, then Germany, Spain, Sweden and Dutch are the significant producers in Europe (Gnansounou, 2010). Current large scale production of fuel ethanol is mainly based on sugarcane (Brasil), corn (the USA), sugar beet and wheat (Europe), (Balat & Balat, 2009). The recent rise in the prices of food ethanol biomass has shifted in focus towards a possibility of deriving fuel ethanol from any type of biomass, especially cellulosic biomass (corn or wheat straw, sugarcane bagasse, wood, grass) and food waste biomass (organic waste and wastewater from food processing industries) (Sarkar et al., 2012; Soccol et al., 2010).

According to the literature, cheese whey could be a suitable substrate for bioethanol production (Kourkoutas et al., 2002; Zafar & Owais, 2006). Lewandowska & Kujawski (2007) used a solution of dried UF whey permeate as a substrate for semi-continuous ethanol fermentation. Silveira et al. (2005) fermented the solution of UF whey permeate in batch cultures. Ghaly & El-Taweel (1997) developed a kinetic model for continuous ethanol fermentation from lactose. Moreover, in 2008 there were two industrial scale whey-ethanol plants in the United States which produced 8 million gallons of fuel ethanol per year (Ling, 2008). In New Zealand there were whey-ethanol plants with an annual production of about 5 million gallons of ethanol (Ling, 2008). Industrial-scale plants producing bioethanol form whey permeate are operated in Ireland (de Glutz, 2009).

There are many reports of potential applications of yeast strains in ethanol production from UF whey permeate streams, but most of them focused on Kluyveromyces sp. due to its ability to directly ferment lactose (Kourkoutas et al., 2005; Ozmihci & Kargi, 2008; Silveira et al., 2005; ). These yeasts generally suffer from low conversion yields (0.4 kg ethanol kg-1 lactose) and are very sensitive to product (ethanol) inhibition at concentrations as low as 20 g L-1 (de Glutz, 2009). An alternative is to employ indirect fermentation yeasts, such as Saccharomyces cerevisiae, which show considerably better ethanol fermentation performance (0.520 kg ethanol kg-1 lactose) and much higher alcohol tolerance (100 — 120 g L-1) (Cote et al., 2004; de Glutz, 2009). The disadvantage of using S. cerevisiae is the inability to directly ferment lactose. It can be solved by genetic manipulation of yeasts or facilitate the process with a simultaneous lactose hydrolysis, for example by co-immobilization of yeast cells with the enzyme (Cote et al., 2004; Guimaraes et al., 2008). Moreover, higher ethanol production could be achieved by application of different stimulation processes, improving biological activity of yeasts. Many researchers have found that ultrasonic stimulation has the function of promoting the activity of enzyme, cell growth and cell membrane permeability (Chisti, 2003; Liu et al., 2003a; Liu et al., 2007; Schlafer et al., 2000). However, application of ultrasonic irradiation at improper intensity or period has destructive impact on cells by disrupting the cell membranes and deactivating biological molecules such as enzymes or DNA (Liu et al., 2007).

The objectives of the studies were: (1) to investigate bioethanol production from UF whey permeate in continuous fermentation in UASB reactors by K. marxianus 499, (2) to evaluate the effects of low intensity ultrasound (20 kHz, 1 W L-1) for ethanol production from UF whey permeate by S. cerevisiae B4.