Chapter 6 Figure 6.3.1
4.3 Results and Discussion
4.3.4 Comparative assessment of results with published literature
The conclusion of this study is also supported by the outcome of the study by Deka et al. (2013), where overall optimization of medium composition and fermentation parameters resulted in eight-fold enhancement in CMCase activity from Bacillus sp. and the contribution of medium optimization alone was six fold. A comparative account of optimization of medium as well as optimization of fermentation parameters is given in Table 4.3.9A and B. The enzyme activity reported by Lee et al. (2008) and Lee et al. (2010) for Bacillus sp. is significantly higher (i.e., 137 and 153 U/mL) than that reported in this study as well as several other studies cited in Table 4.3.9A. However, it should be noted that the Lee et al.
(2008) and Lee et al. (2010) determined the enzyme activity at bioreactor scale as compared with shake flask scale in present study, which has better controlled system in terms of operating parameters. Moreover, the carbon source for all species reported in Table 4.3.9A is different from each other, which influences the activity of the enzyme. For example, Sethi et al. (2013) used glucose as the sole carbon source, while Shabeb et al. (2010) used dual carbon source in the form of molasses and cellulose. It was observed that microorganisms, producing cellulase activity within a similar range as B. amyloliquefaciens SS35, were able to hydrolyze several lignocellulosic biomasses significantly. For example: (1) Sago pith waste hydrolysis
by B. amyloliquefaciens UMAS 1002 with CMCase activity 0.63 U/mL (Apun et al.
2000); (2) enzymatic hydrolysis of sugarcane bagasse for bioethanol production using cellulase with 0.112 and 0.902 U/mL produced by Trichoderma longibrachiatum PTCC 5140 and Aspergillus niger, respectively (Shaibani et al.
2011); (3) Wild grass (Achnatherum hymenoides) hydrolysis by B. subtilis AS3 with 0.57 U/mL CMCase activity (Deka et al. 2013).
It was proven with rigorous study of numerous commercial enzyme preparations (from suppliers such as Novozymes, Dyadic, Genencor, Rhodia- Danisco, Lyven) comprising cellulase, cellobiase and xylanases that the enzyme activities predicted by standard assays using commercially available substrates do not really reflect the actual activity of the enzyme on plant (lignocellulosic) materials (Kabel et al. 2006). The enzyme showing lower activity with commercial substrates can do efficient hydrolysis with a lignocellulosic substrate. In the study by Kabel et al. (2006), xylanase activity analyzed in the standard xylanase assay did not present a high correlation with the degradation of xylan-rich fractions of wheat bran and grass.
A remarkable example is the comparison of the standard activity of Cellubrix (107 U/mL) and Cellulase 2000L (568 U/mL) with the degradation of xylan to xylose of Cellubrix (only 50-60%) and Cellulase 2000L (only 18-20%). The enzyme activity towards the cellulose- and xylan-enriched fractions from grass and wheat bran was revealed to be markedly different than that found with standard assays. Therefore, the actual degradation of the xylan- and cellulose-rich fractions from wheat bran and grass could not be correlated with the (relatively low) activity of enzymes as indicated by standard assays. Therefore, in general, the choice of most suitable
enzyme preparation is dependent on the substrate characteristics also other than on standard enzyme activities measured.
Table 4.3.9 Comparison of various optima reported in literature for CMCase production by Bacillus spp.
(A) Representative literature review on optimization of medium components.
Bacillus sp. Source Medium
Component Method
Optimum Conc.
(g/L)
Scale
Cellulase activity (U/mL)
Reference
Bacillus sp. VG1
Hot spring
soil
CMC
OVAT
10.0
Shake
flask 0.63 Singh et al. 2001
Tryptone 5.0
B. pumilus EB3
Oil palm empty
fruit bunch
CMC
OVAT
10.0
Shake
flask 0.076 Ariffin et al. 2008
Yeast extract 2.5
(NH4)2SO4 2.5
B. amyloliquefaciens
DL3 Soil
Rice hull
OVAT
20.0
7 L
Bioreactor 153.0 Lee et al.
2008
Peptone 2.5
(NH4)2SO4 0.6
B. subtilis subsp.
subtilis A-53
Sea water
Rice bran
OVAT 20.0 7 L
Bioreactor 137.0 Lee et al.
2010
Yeast extract 2.5
B. subtilis KO
Sugar factory product
Molasses +
Cellulose OVAT 1.0 Shake
flask 35.0 Shabeb et al. 2010 (NH4)2PO4/tryptone 2.0
B. subtilis AS3 Cow
dung
CMC Plackett-
Burman and CCD
18.0
Shake
flask 0.43 Deka et al. 2011
Yeast extract 4.79
Peptone 8.0
B. subtilis Soil Glucose
OVAT 50.0 Shake
flask 1.0 Sethi et al. 2013
(NH4)2SO4 5.0
B. amyloliquefaciens SS35
Rhino- ceros dung
CMC Plackett-
Burman and CCD
19.05
Shake
flask 0.55
This study (Singh et al. 2014)
Yeast extract 8.0
Peptone 2.0
(B) Representative literature review for optimization of fermentation parameters.
Bacillus sp. Source Optimization
Parameter Method Optimum
value Scale
Cellulase activity (U/mL)
Reference
B. amyloliquefaciens UMAS1002
Sago pith waste
pH
OVAT
6.0
Shake flask
9.38 Khan and Hussaini 2006
Temperature (oC) 40
Inoculum size (%, v/v) 4
Shaking speed (rpm) 100
Bacillus sp.
Coir retting effluent
pH
OVAT
7.0
Shake flask
0.02 Immanuel et al. 2006
Temperature (oC) 40
Inoculum size (%, v/v) ND
Shaking speed (rpm) ND
B. amyloliquefaciens
DL3 Soil
pH
OVAT
6.8
7 L
Bioreactor 367 Lee et al.
2008
Temperature (oC) 37
Inoculum size (%, v/v) ND
Shaking speed (rpm) ND
B. subtilis subsp.
subtilis A-53
Sea water
pH
OVAT
6.8
7 L
Bioreactor 137 Lee et al.
2010
Temperature (oC) 30
Inoculum size (%, v/v) ND
Shaking speed (rpm) ND
B. amyloliquefaciens UNPDV-22
Hot spring
pH
CCD
5.25
Shake
flask 13
Vasudeo and Lew 2011
Temperature (oC) 42.24
Inoculum size (%, v/v) 4.95
Shaking speed (rpm) ND
B. subtilis AS3 Cow
dung
pH
CCD
7.2
Shake
flask 0.56 Deka et al. 2013
Temperature (oC) 39
Inoculum size (%, v/v) ND
Shaking speed (rpm) 121
B. subtilis Soil
pH
OVAT
10.0
Shake
flask 0.9 Sethi et al. 2013
Temperature (oC) 40
Inoculum size (%, v/v) ND
Shaking speed (rpm) ND
B. amyloliquefaciens SS35
Rhino- ceros dung
pH
CCD
5.65
Shake
flask 0.69
This study (Singh et al. 2014)
Temperature (oC) 40.4
Inoculum size (%, v/v) 6.96
Shaking speed (rpm) 120
ND - not determined