*1.2.9 Enzyme combinations in saccharification process*

Single enzymes are rarely used for saccharification process. Enzymes may be combined successfully in mashing processes and fermentation. As reported by [24], different combinations of technical enzymes may exhibit either complementary or inhibitory effects. "OPTIMALT" is an industrially used enzyme combination off GAR GAA and FAA [28]. The concentration of fermentable sugars in mashes rises rapidly when enzyme combination is used in SSF process.

### **1.3 Microorganisms for ethanol production using SSF process**

The yeast species mainly *S. cerevisiae, S. uvarum, Schizosaccharomyces pombe, Kluyveromyces marxianus and Candida utilis* are used for industrial alcohol production using SSF process [29]. *Saccharomyces cerevisiae* is the common microbe used for industrial ethanol production owing to its use for long time food industry. *Kluyveromyces marxianus* yeast grows well even up to 40°C [30]. This species is mainly used for production of alcohol from cellulosic, starch and saccharine substrates using SSF process. The activity of the yeast is very high at high temperatures and results in high ethanol production in less fermentation time.

Yeasts can utilize a variety of substrates. In general, they are able to grow and efficiently ferment in a pH between 3.5–6.0 and temperature in the range of 28–35°C. The overall productivity of the fermentation was less due to ethanol product inhibition and substrate inhibition [26]. This drawback of substrate inhibition can be overcome in SSF process where simultaneous utilization of substrate by microbes and synthesis of glucose by enzymes at faster rates.

Yeast, under anaerobic conditions, converts glucose to ethanol by the Embden-Meyerhof pathway and is shown in **Figure 1**. 2 mol of ethanol, CO2, and ATP per mol of glucose fermented were synthesized in this pathway with a yield coefficient of 0.51 g alcohol [27].
