The raw and pretreated pine sawdust samples were hydrolyzed using enzymes to produce sugar for biofermentation. Effects of reaction time and enzyme dose on the glucose yield, total sugar yield, and weight loss of the raw and pretreated pine sawdust were systematically studied. The results of enzymatic hydrolysis are presented as follows.

Figure 19.2 shows the effect of reaction time on the glucose yield under two differ­ent enzyme doses. The general trends observed in Fig. 19.2a (7.67 FPU) are similar to those in Fig. 19.2b (11.76 FPU). In the first 12 h of operation, the glucose yield increased significantly with time for all samples. After that, the glucose yield leveled off for 12-48 h of reaction. This could be explained by the fact that the enzymatic hydrolysis rate, especially the initial hydrolysis rate, strongly depends on the initial extent of enzyme adsorption and the effectiveness of the adsorbed enzymes [54]. At the beginning of the hydrolysis reaction, there were ideally a maximum number of ac­tive binding sites on the surface of the substrate, and enzymes could be fully absorbed onto the area. At this time, the hydrolysis rate could be considered the fastest. After a certain period of time, the process of enzymes adsorption and desorption reached a saturation point. Additionally, the production of cellobiose and glucose, which have been considered inhibitory to enzymatic hydrolysis, might be accumulated and inhibited enzymatic hydrolysis. As shown in Fig. 19.2, there are obvious differences in the glucose yield among pine sawdust samples pretreated using different methods. From Fig. 19.2, it is clear that the glucose yield increased with an increase in the enzyme dose (Fig. 19.2a vs. b). The raw pine sawdust (untreated) only had 5-6 % glucose yield while pine sawdust treated with (organosolv + ultrasound + NaOH) contributed to nearly 16-18 % glucose yield (Fig. 19.2b), which is more than three times the yield obtained with the raw pine sawdust sample. This can be explained by that the pretreatment removed lignin, loosened the cellulose crystalline structures and increased its accessibility to enzyme. The order of glucose yield positively cor­related to the order of PE and DE, as shown in Fig. 19.4. In other words, a higher PE and DE led to a higher glucose yield. The results suggest that pretreatment did play an important role in enzymatic hydrolysis [50, 55].

Organosolv+ultrasound*NaOH

Organosolv* NaOH

Reaction time (h)

Organosolv* ultrasound

Reaction time (h)

Fig. 19.2 Glucose yield from various pretreated pine sawdust samples at 50 °C in sodium citrate solution, pH 4.8 and 100 rpm shaking speed: a 7.67 FPU; b 11.76 FPU

Admittedly, the maximum glucose yield from this work was lower than those reported in some other studies, for example by Sannigrahi et al. [50] and Palonen et al. [55] using different pretreatment methods. Sannigrahi et al. [50] obtained a nearly 70 % of sugar yield when 65 % ethanol/water solution containing 1.1 % sulfuric acid was used as the pretreatment reagent. The addition of acid could lead to

Fig. 19.3 Total sugar yield from various pretreated pine sawdust samples at 50 °C in sodium citrate buffer solution, pH 4.8, and 100 rpm shaking speed: a 7.67 FPU; b 11.76 FPU

a higher pretreatment efficiency not only for lignin but also for hemicellulose. The higher lignin and hemicellulose removal efficiency led to the higher glucose yield.

The glucose yields achieved in this study for Jack pine sawdust pretreated with the combination of (organosolv + ultrasound + NaOH) methods are lower than those

reported in the work of Zhu et al. [4-6] where very high percentage of cellulose conversion (up to 90 %) was reported for hardwood and softwood samples with combined pretreatment methods of dilute acid and sulfite and disk milling [4-6]. However, compared with the disk milling pretreatment, a mechanical pretreatment approach, the organosolv-based pretreatment approaches may be advantageous in terms of pretreatment energy costs and the overall economics of the process. An acid-free organosolv process can overcome the problems caused by acid-catalyzed pretreatment. It shall be noted that the effect of the organosolv-based pretreatment on cellulose conversion can be greatly improved by combining it with dilute acid pretreatment, as demonstrated by Sannigrahi et al. [50]. Moreover, one of the striking advantages of the organosolv-based pretreatment approaches is that lignin of high purity is generated as a byproduct from the organosolv-based pretreatment processes, which can be utilized as a highly valuable feedstock for the production of phenolic resins and adhesives and biophenols [56].

Figure 19.3a and 19.3b shows the total sugar yield as the function of hydrolysis reaction time. Similar to the results obtained for glucose yield as dicussed above, for the sample pretreated with organo + ultrasound + NaOH, the total sugar yield increased to approximately 27 % in the initial 24 h and then reached the maximum yield of about 30 %. In the next 24 h, there was no significant increase in total sugar yield. The total sugar yields of other pretreated pine sawdust samples were lower but followed the same trend. Interestingly, in contrast to the glucose yield, a higher PE and DE led to a lower total sugar yield (Fig. 19.4). This was probably due to the fact that the higher removal efficiency of hemicellulose at a higher PE resulted in a lower content of hemicellulose in the solid residues, which decreased the formation of non-glucose carbohydrates (such as xylose). From the Fig. 19.4, the effects of enzyme dose on the total sugar yield are less significant (with approximately 5 % differecne in the yield).

19.4 Conclusions

1. Various pretreatment methods (organosolv extraction, followed by ultrasonic and/or NaOH treatment) resulted in a significant removal of lignin and hemi — cellulose.

2. The observations from SEM, FTIR, and XRD clearly demonstrate that the pre­treatment led to disordered cell wall, twisted and exposed inner structure, reduced lignin and hemicelluloses contents, accompanied by increased cellulose content in the solid residues after treatment.

3. Pretreatment of pine sawdust samples had a significant impact on the glucose yield and total sugar yield. The treatment with different methods produced two — tothree-fold increase in the glucose and total sugar yields. The maximum glu­cose and total sugar yields were 5.8 % and 7.1 %, respectively for the raw pine sample, but they were increased to 9.6 % and 30.1 % with organosolv pretreat­ment, 10.7 % and 24.1 % with organosolv + ultrasound pretreatment, 13.6 %

Fig. 19.4 Effects of PE and DE on glucose yield (a) and total sugar yield (b): • organosolv ex­traction; 4 organosolv + ultrasound; ■ organosolv + NaOH; A organosolv + ultrasound + NaOH; * untreated sawdust

and 26.8 % with organosolv + NaOH pretreatment, and 19.3 % and 22.4 % with organosolv + ultrasound + NaOH pretreatment.

4. In enzymatic hydrolysis of the pretreated pine sawdust, increasing PE and DE led to an increase in glucose yield, but a decrease in total sugar yield.

Acknowledgments The authors are grateful for the financial support from the Ontario Ministry of Energy and Ontario Centers of Excellence (OCE) through the Atikokan Bioenergy Research Center (ABRC) program. The authors would also like to acknowledge Natural Sciences and Engineering Research Council of Canada (NSERC) for the Discovery Grants.

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