Effect of Novozym 188 Supplementation on In-House Produced Enzymes for WELP Hydrolysis

Figure 4.6 shows glucose, xylose, and mannose yields after enzymatic hydrolysis of WELP supplementation assay with Novozym 188. Novozym 188 contains high amount of p-glucosidase and helps in cellobiose conversion to glucose and also prevents end-product inhibition of cellulase due to accumulation of cellobiose.

After 72 h, glucose yield increased by 6 %, 13 %, 14 %, 16 %, and 20 %; xylose yield increased by 5 %, 6 %, 9 %, 13 %, and 17 %; and mannose yield increased by 2 %, 3 %, 6 %, 10 %, and 14 % with 1, 10, 30, 50, and 100 % supplementation, respectively, compared to non-supplemented control. Glucose increase was supported statistically at 95 % confidence level. Less difference was observed in xylose and mannose production. Novozym 188 supplementation resulted in slightly higher concentration of xylose and mannose compared to non-supplemented control; however, these differences were not significant at 95 % confidence level of statistical analysis.

A parallel study of enzymatic hydrolysis with only commercial cellulases, Cellic®Ctec2 (control-2), was conducted. Enzyme loading of 45 FPU/mL was used for hydrolysis. Glucose, xylose, and mannose concentrations after 72 h of hydrolysis were 79.1, 18.5, and 16.0 mg/mL corresponding to 99 %, 96 %, and 89 % yields, respectively. Comparing the yields control-1 (in-house enzymes without any supplementation) and control-2 (completely commercial enzyme), we found that supplementation of in-house enzymes supplemented with commercial enzymes resulted in higher conversion of polysaccharides after wet explosion pretreatment of loblolly pine. When 15 FPU/mL of in-house enzymes were boosted with 15 FPU/ mL of commercial cellulase, Cellic®Ctec2 resulted in 99 %, 93 %, and 93 % of glucose, xylose, and mannose yields, respectively. When 15 FPU/mL of in-house enzymes were boosted with 15 FPU/mL of commercial hemicellulase, Cellic®Htec2, glucose, xylose, and mannose yields reached to 99 %, 99 %, and 94 %, respectively. Similar cellulose conversion was achieved with Cellic®Ctec and Cellic®Htec2 as can be explained by similar FPA activities (100 and 94.3 FPU/mL, respectively), but higher yields of xylose and mannose was found with hemicellulase, Cellic®Htec2.

Table 4.2 summarizes cellulose conversions from hydrolysis of WELP. Cellulose conversion was increased with increasing supplementation of all three commercial enzymes. Probably due to insufficient hemicellulolytic activity, higher accumulation of hemicellulosic oligomers was observed with non-supplemented enzyme preparation.

The best enzymatic hydrolysis yields were obtained when the in-house cellulase cocktail was supplemented with Cellic®Htec2. When 15 FPU/mL of in-house cellulase was boosted with 7.5 FPU/mL of commercial hemicellulase, Cellic®Htec2 (50 % supplementation), 85 % glucose, 92 % xylose, and 86 % mannose were produced. Contrary to some previous studies (Wu et al. 1999; Cullis et al. 2004; Monavari et al. 2009), higher yields (>70 %) of glucose can be obtained with high loadings (30-80 FPU/g cellulose) of commercial enzymes. We obtained higher glucose yield (85 %) with lower FPU/g cellulose. Increasing the supplementation from 50 to 100 % resulted in a near-complete sugar conversion. The enzymes will remain functional during fermentation and therefore be able to convert remaining sugars to monomers along with the fermentation which means that any remaining cellulose can be converted to glucose during subsequent fermentation step.

With 1 % supplementation of in-house enzymes, Cellic®Ctec2, Cellic®Htec2, and Novozym 188 showed glucose yield of 37 %, 41 %, and 34 % corresponding to the increase in glucose yield from non-supplemented control-1 (in-house enzymes)

Enzyme

Code

Supplementation (%)

Enzyme loading (FPU)

Enzyme loading (CBU)

CC“

ccb

Total cellulose1-' conversion (g)

In-house

cellulase - control-1

100

No

15.0

30.0

0.76

0.38

11.44

Cellic®Ctec2

supplementation

1

1

15.2

30.0

2.41

0.44

13.14

2

10

16.5

30.0

2.82

0.56

16.72

3

30

19.5

30.0

3.02

0.71

21.17

4

50

22.5

30.0

3.27

0.88

26.46

5

100

30.0

30.0

3.32

1.19

35.79

Cellic®E[tec2

supplementation

11

1

15.2

30.0

2.71

0.49

14.76

12

10

16.5

30.0

3.43

0.68

20.35

13

30

19.5

30.0

3.24

0.76

22.70

14

50

22.5

30.0

3.79

1.02

30.71

15

100

30.0

30.0

3.30

1.19

35.60

Novozym 188 supplementation11

21

1

15.0

30.3

2.24

0.40

12.11

22

10

15.0

33.0

2.43

0.40

13.10

23

30

15.0

39.0

2.48

0.34

13.37

24

50

15.0

45.0

2.53

0.30

13.64

25

100

15.0

60.0

2.64

0.24

14.23

Commercial cellulase - control-2 (Cellic®Ctec2)

200

No

45.0

0.00

2.20

-

35.63

“Grams of cellulose converted per FPU (gFPU-1) •Grams of cellulose converted per CBU (gCBU-1) “Total cellulose present in loblolly pine. 35.97 g “•Supplementation is percentage of CBU as 13 %, 23 %, and 6 %, respectively. A 100 % supplementation of in-house enzymes with Cellic®Ctec2, Cellic®Htec2, and Novozym 188 resulted into glucose yield of 99 %, 99 %, and 40 %, respectively. At highest supplementation (100 %), Cellic®Ctec2 and Cellic®Htec2 resulted into similar yield of 99 % and that could be explained as Cellic®Ctec2 primarily focuses on cellulose degradation, while Cellic®Htec2 degrades hemicellulose and thus releases cellulose for cellulase attack. Also, higher enzyme/substrate ratio eliminates enzyme inhibition due to substrate characteristics. 50 % supplementation of in-house enzymes with Cellic®Ctec2, Cellic®Htec2, and Novozym 188 showed glucose yield of 74 %, 85 %, and 38 %.

Considering economics and yield, 50 % supplementation (7.5 FPU) with Cellic®Htec2 to in-house produced enzyme cocktail (15 FPU) was determined as optimal. Thus, it is imperative that in-house produced cellulases from mesophilic fungi and commercially available cellulase formulations be made to work synergis- tically as a lignocellulolytic enzyme system, providing better saccharification yields from lignocellulosic biomass for a lower cost than using commercial enzymes alone. It is important to note that although the focus in this study was on monomeric sugar release, enzymatic hydrolysis for longer period (more than 72 h) will likely result in higher sugar yields. Further studies are needed to evaluate the effect of other important factors such as pH, temperature, and hydrolysis period on the efficiency of mixed enzyme cocktail.

 
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