Journal of Energy Bioscience 2024, Vol.15, No.3, 135-146 http://bioscipublisher.com/index.php/jeb 138 sugars, which are then suitable for fermentation. This process has shown to reduce the treatment time to 2 hours and yield a reducing sugar level comparable to the conventional two-stage acid and enzymatic hydrolysis (Li, 2024). Novel enzymes such as Spezyme® Xtra and Stargen™ 001 have been optimized for the liquefaction and saccharification of cassava starch, with Spezyme optimally active at 90 ℃ and pH 5.5, and Stargen effective at room temperature. These advancements in the enzymatic conversion of cassava starch to sugars are crucial for efficient bioethanol production. 3.2 Fermentation efficiency The conversion of cassava biomass to bioethanol involves fermenting the hydrolyzed sugars using various microorganisms. Saccharomyces cerevisiae and Clostridium butyricum have been used to ferment the reducing sugars obtained from cassava pulp, yielding ethanol concentrations of up to 12.9 g/L when cassava starch wastewater is used (Virunanon et al., 2013). Another study demonstrated the use of a genetically engineered Thermoanaerobacterium aotearoense in a fibrous-bed bioreactor, achieving an ethanol yield of 0.364 g/g from glucose (Cai et al., 2012). Additionally, the fermentation of hydrolyzed cassava stem using Saccharomyces cerevisiae resulted in an ethanol concentration of 7.55 g/L (Han et al., 2011). These studies highlight the potential of various biochemical pathways in converting cassava biomass to bioethanol and other biofuels. 3.3 Genetic engineering Genetic engineering has been employed to increase the fermentable sugar yields from cassava. A genetically engineered strain of Thermoanaerobacterium aotearoense, for instance, has been used to ferment cassava pulp hydrolysate in a fibrous-bed bioreactor, resulting in high ethanol yields and productivity (Cai et al., 2012). Additionally, the use of a multi-enzyme activity from Aspergillus niger and simultaneous fermentation with Candida tropicalis has been shown to produce ethanol from cassava pulp efficiently, suggesting that genetic and enzymatic enhancements can significantly improve bioenergy production (Rattanachomsri et al., 2009). Furthermore, the use of a co-culture of Aspergillus sp. and Saccharomyces cerevisiae has been explored for the direct bioconversion of raw starch from wild cassava to bioethanol at low temperatures, achieving high yields and efficiencies (Moshi et al., 2016). These advancements in genetic engineering and enzyme technology are pivotal for optimizing cassava as a bioenergy crop. 4 Case Studies 4.1 Successful modifications Genetic and biochemical modifications have played a pivotal role in enhancing cassava's potential as a bioenergy crop. One of the landmark studies in this area demonstrated that genetic transformation could overcome the challenges of high heterozygosity and trait separation in traditional breeding, leading to rapid improvement in target traits such as pest and disease resistance, biofortification, and starch quality (Liu et al., 2011). Another significant advancement was the discovery of a single nucleotide polymorphism in the phytoene synthase gene, which led to a substantial increase in provitamin A accumulation in cassava roots. This modification not only has implications for bioenergy but also for combating vitamin A deficiency (Welsch et al., 2010). Furthermore, the development of transgenic technologies has enabled the production of cassava with enhanced resistance to diseases and pests, improved nutritional content, and modified starch metabolism, which are essential for bioenergy applications (Taylor et al., 2004). 4.2 Pilot projects Pilot projects have been instrumental in testing the scalability and practicality of cassava-based bioenergy production. The optimization of Agrobacterium-mediated transformation systems for the large-scale production of transgenic cassava plants is a notable example. This system has been successfully applied to elite cassava cultivars, resulting in the integration of genetic constructs for disease resistance and nutritional enhancement into a significant number of plants, which is a critical step towards commercial bioenergy production (Chauhan et al., 2015).
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