3.2.5. Integrated Technologies Based on Hydrolysis for Biofuel (Ethanol) Production

The quest to achieve a higher yield of biofuel per unit of biomass has led to the integration of different phases of the process; it reduces the cost of capital and makes biofuels more efficient and economically viable. Given that bioethanol is the most produced secondary biofuel today, the search for more efficient configurations for its production has been sought.

## Simultaneous Saccharification and Fermentation (SSnF)

Conventionally, ethanol production is carried out in separate phases, first the hydrolysis and later the fermentation. Due to this separation, each one is carried out under optimal conditions [89]. However, the excessive accumulation of sugars during the hydrolysis inhibits the enzymatic activity, reducing the process [63,69,90]. This problem led to the development of simultaneous saccharification and fermentation (SSnF). In this process, hydrolysis and fermentation occur together in the same reactor, reducing enzymatic inhibition due to sugars' presence since the monosaccharides produced are immediately consumed by fermenting microorganisms [65,70,90]. Compared to the conventional process, SSnF achieved a higher hydrolysis rate, and it conducted higher ethanol concentration. This strategy requires less equipment and a more straightforward operation, and the presence of ethanol in the wort makes it less susceptible to contamination by unwanted microorganisms [61,70,90].

However, this method's disadvantage is that the optimal operating conditions for hydrolysis and fermentation are different, implying difficulty for its control and optimization. Enzymatic hydrolysis is performed optimally at temperatures above 50 ◦C. For most fermenting microorganisms, the optimum temperature for their performance is between 28 and 37 ◦C; likewise, the optimum pH for the hydrolysis and fermentation stages is different [65,70,90].

Work performed on the search and selection of suitable enzymes and microorganisms for this strategy, and a promising option to overcome this difficulty, is the use of thermotolerant yeasts such as *Kluyveromyces marxianus*, which is a promising species since some of its strains grow at temperatures between 45 and 52 ◦C [91].

### Simultaneous Saccharification and Co-Fermentation (SSCF)

This configuration aims to complete all the sugars released during the pretreatment and hydrolysis of the biomass through mixed yeast cultures that can assimilate both the pentoses and the hexoses produced in the same reactor. This strategy offers this advantage. By continuously removing the final hydrolysis products, causing cellulase and glucosidase activity inhibition can lead to the high productivity of ethanol, giving greater yield compared to the SFS process. One drawback of this method is that the organisms that use hexoses grow faster than those that use pentoses, leading to inhibition by the high ethanol concentration. Genetically modified yeast or bacteria can be used to achieve efficient ethanol production. To carry out the fermentation of both, pentoses and hexoses are required [65,70,92,93].
