Evaluation of On-Site Produced Enzymes by Enzymatic Hydrolysis Experiment

The objective of this study was to investigate the cultivation of mesophilic fungi for cellulase production and evaluate the hydrolytic efficiency of the resulting enzyme preparations toward wet-exploded lignocellulosic biomass. In brief, the substrates for the enzymatic hydrolysis experiments were wet-exploded corn stover and loblolly pine and were hydrolyzed at two solid loadings of 5 and 10 % using two enzyme loadings of 5 and 15 FPU/g glucan. Enzymatic hydrolysis was performed at comparatively low substrate concentration (50 and 100 g/l) to avoid the end-product inhibition on cellulase activity.

Comparison of On-Site and Commercial Cellulolytic Enzyme Efficiency on Corn Stover

After 72 h hydrolysis of wet-exploded corn stover (WECS) with 5 FPU/g glucan enzyme loading at 5 % (w/w) substrate concentration, commercial cellulase converted 10 % more glucose, 4 % more xylose, and 18 % more arabinose compared to on-site cellulase. With 5 FPU/g glucan enzyme loading at 10 % (w/w) substrate concentration, xylose and arabinose yield differences remain the same; however, cellulose conversion increased to 19 %. As can be observed in Fig. 3.3, after 72 h of enzymatic hydrolysis using 5 FPU/g glucan enzyme loading of commercial and on-site enzymes, 51 and 46 % glucose yields were obtained at 5 % substrate concentration and 42 and 34 % at 10 % (w/w) substrate concentration.

Monomeric sugar yield obtained from the hydrolysis of wet-exploded corn stover with

Fig. 3.3 Monomeric sugar yield obtained from the hydrolysis of wet-exploded corn stover with

on-site enzymes (T. reesei and A. saccharolyticus) and commercial enzymes (Celluclast 1.5L and

Novozym 188) at 5 and 10 % substrate concentrations and 5 FPU and 10 CBU loadings

At a higher enzyme loading, 15 FPU/g glucan after 72 h hydrolysis, at 5 % (w/w) substrate concentration, commercial cellulase converted 5 % more glucose and 12 % more arabinose; however, xylose conversion remained the same for samples hydrolyzed with on-site cellulase. With 15 FPU/g glucan enzyme loading at 10 % (w/w) substrate concentration, the sugar yields obtained were comparable with very small differences (see Fig. 3.4). At 5 % substrate concentration, the enzymatic hydrolysis of WECS with commercial and on-site enzymes resulted in a final glucose concentration of 88 and 81 %, respectively, and at 10 % substrate concentration, the enzymatic hydrolysis of WECS with commercial and on-site enzymes yielded final glucose concentrations of 85 and 80 %, respectively. High glucose yields demonstrated that the cellulolytic activities of enzymes produced on-site were enhanced when the carbon source and substrate for hydrolysis were the same. These results indicate that it might be advantageous to use the same carbon source and substrate for the hydrolysis as fungi produce enzymes to degrade the material on which it was grown as previously shown (Juhasz et al. 2005).

High yields of xylose (above 90 %) could be explained by the fact that Novozym 188 has high amount of xylanase, 107.3 nkat/mL, and that improved xylose as well as glucose yields during hydrolysis by decreasing the xylooligomers accumulation which are known to inhibit cellulase activity (Kumar and Wyman 2009).

We found similarity in results with past studies performed on comparison of commercial and on-site enzymes for the hydrolysis of lignocellulosic biomass. Previously a comparison between hydrolysis of Celluclast 1.5 L and Novozym 188 mixture and on-site Trichoderma atroviride TUB F-1663 enzyme using steam-pretreated sugarcane bagasse at 2 % substrate concentration had shown glucose yield, 8.3 and 6.7 g/l, respectively (Kovacs et al. 2009). Another study demonstrated comparison of hydrolysis between on-site enzymes produced from T. reesei RUT-C30 on steam-pretreated corn stover, spruce, and willow and had shown glucose yields 46, 31, and 40 %, respectively, whereas with commercial enzyme (Celluclast 1.5 L and Econase), yield was 52 % (Juhasz et al. 2005). Kurabi et al. have reported difference in glucose yields when comparing commercial enzymes (Trichoderma spp.) with novel complex obtained from mutagenesis of Trichoderma strain on organosolv-pretreated Douglas fir as 56.1 % and

51.1 %, respectively, at 5 % substrate concentration (Kurabi et al. 2005).

Comparable sugar yield obtained from on-site and commercial enzymes might be due to the fact that besides p-glucosidase from A. saccharolyticus, other enzyme components present in cell wall of fungi such as endoglucanase, exoglucanases, and xylanase can increase the hydrolytic potential of the on-site cellulase cocktail. As reported previously, no measurable amount of FPU, CMCase, or xylanase activities was found in Novozym 188 (Kovacs et al. 2009) and that explains comparable yields from on-site produced enzymes. Furthermore, comparable conversion of cellulose in both corn stover and loblolly pine may also be related to the fact that on-site enzyme cocktail had higher p-glucosidase-to-FPA ratio than the commercial mixture.

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Fig. 3.4 Monomeric sugar yield obtained from the hydrolysis of wet-exploded corn stover with on-site enzymes (T. reesei and A. saccharolyticus) and commercial enzymes (Celluclast 1.5L and Novozym 188) at 5 and 10 % substrate concentrations and 15 FPU and 30 CBU loadings

 
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