**1.4 Simultaneous saccharification and fermentation (SSF) process and key variables**

Simultaneous saccharification and fermentation SSF is a process in which sugars from the liquefied substrates are saccharified and fermented in a single fermenter using enzyme and yeast. The drawback of SSF of cellulose using enzymes is

*Simultaneous Saccharification and Fermentation and Factors Influencing Ethanol Production… DOI: http://dx.doi.org/10.5772/intechopen.86480*

feedback inhibition by the product. Separate Hydrolysis and Fermentation uses different temperature for hydrolysis and fermentation but the main disadvantage is the end product inhibition of glucose that accumulates in the hydrolysis step [31]. SSF process overcomes this difficulty of accumulation of sugars inside the fermenter by simultaneous fermentation of sugar by suitable yeast [32, 34]. The flow sheet of the SSF process using corn starch is shown in **Figure 2**.

Verma et al. [35] studied the conversion of starch to ethanol in a SSF process using co culture of amylolytic yeast and *S. cerevisiae*. The optimum temperature was reported as 30°C. Banerjee et al. [36] reported an optimum temperature of 37°C for *S. diastaticus* using soluble starch as a substrate. Saha and Ueda et al. [37] reported that 38°C gave a maximum ethanol yield by *S. cerevisiae* in a fermentation of glucoamylase treated starch. Bandaru et al. [38] had optimized the operating variables of fermentation for the production of ethanol using sago starch using coimmobilized glucoamylase and *Z. mobilis* and he reported an optimum temperature of 32.4°C and desirable pH at 4.93.

Amutha et al. [39] studied the ethanol from pretreated cassava starch by coimmobilized cells of *Z. mobilis* and *S. diastaticus* in batch and continuous fermentation. Pretreatment of substrate was carried out using BAB at 75°C for 1 hour. The

**Figure 1.** *EMP pathway for glucose to ethanol.*

#### **Figure 2.**

*Flow sheet for simultaneous saccharification and fermentation.*

batch fermentation was carried out at a temperature of 30°C and at an initial pH of 6.0. 37.5 gL<sup>−</sup><sup>1</sup> of ethanol production was reported using free cells in mixed culture fermentation and 46.7 gL<sup>−</sup><sup>1</sup> using co-immobilized cells in batch fermentation.

Neves et al. [40] studied the ethanol production from wheat flour by SSF process. SSF process was conducted at 5°C and a controlled pH of 4.5 using glucoamylase 200 U/g of flour and *S. cerevisiae* in a batch fermenter. The fermentation time was 72 hour. 38.76 gL<sup>−</sup><sup>1</sup> of ethanol production was reported.

Davis et al. [41] studied the production of ethanol using waste starch stream by SSF process using *Z. mobilis* and *S. cerevisiae*. The operating conditions for SSF process were a controlled pH of 5.0 and temperature at 30°C. A maximum ethanol production of 39 gL<sup>−</sup><sup>1</sup> was reported.

Nakumara et al 1997 [42] studied the production from raw wheat flour using glucoamylase and *S. cerevisiae*. The pre-treatment of starch was carried out by adding 0.02 g of Termamyl/kg of starch and at a temperature of 95°C for 2 hour. Ethanol concentration of 67 gL<sup>−</sup><sup>1</sup> was reported using SSF process at a controlled temperature of 35°C and controlled pH of 4.5. The alcoholic fermentation of whey using K. marxianus yeast immobilized on delignified cellulose material. The optimum pH value was reported as 4.5. The optimum temperature for fermentation was reported as 37°C.

Pavla et al. [43] had studied the SSF process using wheat bran as substrate. Wheat bran was pre-treated with FAA followed by saccharification using glucoamylase. Pre-treatment temperature for FAA was 55°C and pH 6.0 for 4 hour and saccharification at 55°C for 48 hour to ensure the total hydrolysis of starch. The fermentation of filtrates resulting from pre-treatment using *S. cerevisiae* was carried out with initial pH of 5.5 and 30°C. The ethanol yield reported was 0.41 g/g of glucose fermented.

Reddy et al. [44] had studied the direct fermentation of potato starch to ethanol by co culture of *A. niger* and *S. cerevisiae*. The optimum pH for maximum ethanol production was reported as 5 to 6. The temperature of the fermentation medium was controlled at 30°C.

*Simultaneous Saccharification and Fermentation and Factors Influencing Ethanol Production… DOI: http://dx.doi.org/10.5772/intechopen.86480*

SSF process using maize starch as substrate by glucoamylase and *S. cerevisiae* at 35°C with the initial pH 5.5 was carried out. A maximum ethanol productivity of 1.23 gL<sup>−</sup><sup>1</sup> h<sup>−</sup><sup>1</sup> was reported.

Kadam and Newman [33] evaluated several industrially available nutrient sources for their effectiveness in the SSF of pretreated starch with *Saccharomyces cerevisiae* D5A. Ethanol production was found to increase for a combination of 0.3% CSL and 2.5 mM MgSO4.7H2O Hence, it is more industrially relevant medium than the medium containing rich nutrients.

The pH and temperature of the medium plays a vital role in all types of fermentation processes. As temperature increases the rate of biological reactions also increases upto a certain temperature and further increase in temperature may result in lesser product formation. That temperature was always chosen as the optimum temperature for the fermentation. This characteristic is similar to chemical reaction. This increase in rate of biological reaction may be due to more production of required enzymes at the faster rate. The ethanol producing microorganisms such as *S. cerevisiae, K. marxianus, S. diastaticus* prefer to grow best at 30°C [47]. Most of the microorganisms prefer to grow at neutral pH and hence we have more contamination at that pH. Ethanol producing yeast prefer to grow and metabolize in the pH


#### **Table 5.**

*Production of ethanol from starch sources using SSF process.*

5–6 and a controlled pH environment is always preferred for maximum ethanol production. Very low pH is also not preferred as the rate of growth was very less. Hence an optimum pH of 5–6 must be maintained in the medium. In addition to that the medium should have optimum mineral concentration which provides more biomass and in turn more ethanol yield **Table 5**.
