Effect of Single and Sequential Cellulolytic Enzyme Cocktail on the Fermentable Sugar Yield from Pretreated Agricultural Residues of Cassava

Abstract

Cassava cultivation generates three types of lignocellulosic residues such as stems, leaves and peels. As a part of the investigations on their potential for second generation (2G) bioethanol production, the effect of two cellulolytic enzyme cocktails in enhancing fermentable sugar yield from pretreated (hydrothermal and microwave assisted diluted acid treated) residues was studied. Highest release of reducing sugars was observed from cassava peels, followed by stems, during saccharification for 120 h with Cellic, a multicellulolytic enzyme. Cassava leaves were highly recalcitrant to enzymatic hydrolysis and microwave pretreatment was ineffective. Standardization of enzyme levels showed that 500 mg enzyme protein was adequate for saccharification. Further, sequential saccharification of the pretreated residues using another cellulolytic enzyme, Accellerase for 72 h followed by Cellic for 48 h (at half the optimal dose of both the enzymes) was not advantageous in enhancing the fermentable sugar yield. Two stage process in which the pretreated residues were first saccharified with Accellerase, followed by further hydrolysis by Cellic was also inferior to the single enzyme (Cellic) saccharifiaction. Ultrastructural studies on Cellic treated residues using scanning electron microscopy gave evidence for the hydrolysis of starch almost completely, while fragmented sheaths of cellulose were evident in cassava stems and leaves. The study showed that whilst the carbohydrate in cassava peels could be almost totally saccharified to fermentable sugars, the yield from the other two substrates was not optimal. The presence of higher quantities of lignin in the latter two biomasses compared to peels could be attributed to the lower extent of saccharification.

Microwave-Assisted Alkali Delignification Coupled with Non-Ionic Surfactant Effect on the Fermentable Sugar Yield from Agricultural Residues of Cassava

“…38 and 73 g/L RS after 120 h saccharification, while ca. 27 g/L was only released from the leaves [26]. This showed that AHP treatment of steam-exposed slurry was superior to simple steam pretreatment in the case of leaves only.…”

“…Native (untreated) stem b. MW-assisted alkali pretreated stem c. Saccharified residue from MWassisted alkali pretreated stem d. Native (untreated) leaf e. MW-assisted alkali pretreated leaf f. Saccharified residue from MWassisted alkali pretreated leaf g. Native (untreated) peel h. MW-assisted alkali pretreated peel i. Saccharified residue from MWassisted alkali pretreated peel Fig. 4 (a-i) Pooja and Padmaja [26] reported the presence of alphaamylase existing as a co-activity along with cellulase and β-glucosidase in Cellic CTec 2. However, the levels may not be sufficient to bring about complete hydrolysis of starch.…”

“…The optimum temperature and pH of Cellic were standardized in our laboratory on these biomasses and were found to be 50 °C and 5.5 respectively [26]. 2.3 Pretreatment 2.3.1.…”

“…The equipment had MW radiation at power levels ranging from 100 W to 900 W. The flasks after exposure were treated with 2.5 ml and 5.0 ml 35% H2O2 and pH adjusted to 11.5 with 4M NaOH and volume raised to 100 ml. The microwave power regime was chosen based on our earlier studies [25,26]. The flasks were then incubated either for 4 h at 50 ºC or 24 h at room temperature (30±1 ºC) in a thermostatic water bath (M/s Julabo SW22).…”

“…Subsequent studies using another cellulolytic complex, Cellic CTec 2 also showed that very high yield of sugars was possible from peel. Nevertheless, optimum hydrolysis of polysaccharides could not be achieved for the other two biomasses [26]. Non-ionic surfactants such as Tween 20 (polyethylene glycol sorbitan monolaurate) and Tween 80 (polyethylene glycol sorbitan monooleate) have been reported to prevent the non-productive binding of lignin to cellulases [27].…”