Effect of Different Pretreatment Conditions on Saccharum spontaneum for Cellulase Production by B. subtilis K-18 Through Box–Bhenken Design Maria Ghazanfar, Muhammad Irfan, Fouzia Tabssum, Hafiz Abbdullah Shakir & Javed Iqbal Qazi Iranian Journal of Science and Technology, Transactions A: Science ISSN 1028-6276 Volume 42 Number 2 Iran J Sci Technol Trans Sci (2018) 42:313-320 DOI 10.1007/s40995-017-0463-y
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RESEARCH PAPER
Effect of Different Pretreatment Conditions on Saccharum spontaneum for Cellulase Production by B. subtilis K-18 Through Box–Bhenken Design Maria Ghazanfar1 • Muhammad Irfan2 • Fouzia Tabssum1 • Hafiz Abbdullah Shakir1 • Javed Iqbal Qazi1 Received: 12 February 2016 / Accepted: 13 December 2017 / Published online: 21 December 2017 Ó Shiraz University 2017
Abstract In the present investigation, dilute sulphuric acid pretreatment conditions were optimized for Saccharum spontaneum through Box–Bhenken design of response surface methodology for cellulase production. Cellulase production was carried out in 250 ml capacity Erlenmeyer flask using pretreated S. spontaneum as substrate by Bacillus subtilis K-18 incubated at 50 °C for 24 h of fermentation period. Results revealed that sulphuric acid followed by steam favored cellulase production as compared to sulphuric acid treatment. Maximum FPase production (1.389 IU/ml/min) was achieved under pretreatment condition of 1% H2SO4 concentration, 10% substrate concentration with residence time of 4 h at room temperature followed by steam at 121 °C for 15 min and 15 psi. The proposed model was found significant as revealed by F value, P value and coefficient of determination. The cellulase produced could effectively hydrolyze pretreated substrate releasing total sugars of 12.71 mg/ml after 20 h of incubation at 50 °C. These results suggested that the enzyme could effectively be used in industrial process especially in biofuel production from lignocellulosic biomass. Keywords Cellulase RSM Pretreatment Bacillus subtilis Submerged fermentation
1 Introduction Cellulases are the enzymes that hydrolyze b-1,4 relations in cellulose series which are produced by plants, animals, bacteria, fungi and protozoans. The catalytic components of cellulases have been categorized into several families on the basis of their amino acid strings and crystal formation (Henrissat 1991). It is a compound enzyme composed of three catalytic subunits that work in synergy to bring about the exchange of cellulose to a monomeric element, glucose that can later be agitated for bioethanol creation. These cellulase factors are endo-1,4-b-glucanase, exo-1,4-b-glucanase, and 1,4-b-D-glucosidase (Bayer et al. 1998; Henrissat 1994). Endo-1,4-b-glucanase (EG), also called endoglucanase, slice erratically intermolecular b-1,4& Muhammad Irfan
[email protected];
[email protected] 1
Microbial Biotechnology Laboratory, Department of Zoology, University of the Punjab, New Campus, Lahore 54590, Pakistan
2
Department of Biotechnology, University of Sargodha, Sargodha, Pakistan
glucosidic relations within the cellulose sequence. The endoglucanases are usually examined by thickness cuts in carboxymethyl cellulose (CMC) answer. The mode of action of endoglucanases and exoglucanases is different. Endoglucanases reduce the explicit viscosity of CMC appreciably with little hydrolysis due to intramolecular cleavages, while exoglucanases hydrolyze long series from the ends in a development process (Teeri et al. 1998; Zhang and Lynd 2004). The majority ordinary cellulolytic bacteria are Acetivibrio cellulolyticus, Bacillus spp., Cellulomonas spp., Clostridium spp., Erwinia chrysanthemi, Thermobispora bispara, Ruminococcus albus, Streptomyces spp., Thermonospora spp. and Thermobifida fusca (Sadhu and Maiti 2013). Scientifically important enzymes have conventionally been obtained from submerged fermentation (SmF) due to effortlessness of conducting and larger management of ecological issues like temperature and pH. Moreover, solid state fermentation (SSF) method can develop the yield and decrease the price of enzyme fabrication (Ghildyal et al. 1985; Hui et al. 2010). Response surface methodology (RSM) is a mathematical and geometric examination, helpful for representing
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and studying crisis where the reaction of attention is prejudiced by several unpredictable. RSM has been used widely for optimizing unusual steps in biotechnological processes and to optimize enzymatic hydrolysis. Box– Behnken design for three changeable at three stages was selected (Ahmed et al. 2012; Li et al. 2007). This study was aimed to optimize the pretreatment conditions for maximum cellulase production from pretreated Saccharum spontaneum under submerged fermentation by Bacillus subtilis K-18 and its application in maximum production of sugars during enzymatic hydrolysis of pretreated substrate.
2 Materials and Methods 2.1 Microbial Strain The bacterium B. subtilis K-18 was obtained from Microbial Biotechnology Laboratory, Department of Zoology, University of the Punjab, New campus Lahore Pakistan. The culture was maintained on nutrient agar slants and was used for production of cellulase in submerged fermentation.
Table 1 Coded and actual levels of the factors for three factor Box– Behnken design Independent variables
Symbols
Coded and actual values -1
0
?1
H2SO4 concentration (%)
X1
0.6
Substrate concentration (%)
X2
5
10
0.8
15
1
Time (h)
X3
4
6
8
medium pH of 5 which was autoclaved at 121 °C, for 15 min and 15 psi pressure. After sterilization, the flasks were allowed to cool at room temperature and 2% (v/v) of the vegetative cell culture was transferred aseptically to each of the fermentation flasks. After inoculation, the flasks were incubated at 50 °C with agitation speed of 120 rpm for 24 h of fermentation period. After completion of the fermentation period, the fermented broth was filtered through muslin cloth followed by centrifugation (Kokusan H-1500ER) for 10 min at 10,0009g and 4 °C for the removal of cell mass and unwanted particles. The clear filtrate obtained after centrifugation was used as a crude source of enzyme. Triplicate readings were taken for each of the experiment.
2.2 Pretreatment of Saccharum spontaneum 2.4 Cellulase Assay Pretreatment of S. spontaneum was done as described in our earlier reports (Arooj et al. 2017).
2.3 Enzyme Production Enzyme production was done in 250 ml Erlenmeyer flask capacity having 25 ml of fermentation medium containing 2% pretreated substrate and 1% yeast extract with initial Table 2 Cellulase production by H2SO4-treated Saccharum spontaneum using Box– Bhenken design
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Run #
X1
X2
X3
CMCase and FPase activity was determined as described in our earlier reports (Irfan et al. 2011). One unit of CMCase or FPase activity is defined as the amount of enzyme required to liberate one micromole of glucose from substrate per milliliter per minute under standard assay conditions.
CMCase activity (IU) Observed
Predicted
FPase activity (IU) Residual
Observed
Predicted
Residual
1
0.8
10
6
0.081391
0.081
0.000
0.012
0.012
2
1.0
10
8
0.059319
0.088
- 0.028
0.020
0.001
0.000 0.019
3
1.0
15
6
0.075873
0.157
- 0.081
0.014
0.047
- 0.032
4
1.0
10
4
0.404194
0.330
0.073
0.180
0.158
0.022 - 0.009
5
1.0
5
6
0.147607
0.110
0.036
0.037
0.046
6
0.6
15
6
0.091047
0.127
- 0.036
0.010
0.001
0.009
7
0.8
5
4
0.231756
0.341
- 0.110
0.164
0.177
- 0.012
8
0.6
10
8
0.052421
0.125
- 0.073
0.004
0.026
- 0.022
9
0.8
15
8
0.102083
- 0.008
0.110
0.022
0.009
0.012
10
0.6
10
4
0.484205
0.455
0.028
0.1037
0.123
- 0.019
11 12
0.6 0.8
5 5
6 8
0.383501 0.051042
0.302 0.058
0.081 - 0.007
0.116 0.033
0.083 0.043
0.032 - 0.010
13
0.8
15
4
0.288316
0.280
0.007
0.139
0.129
0.010
Author's personal copy Iran J Sci Technol Trans Sci (2018) 42:313–320 Table 3 Cellulase production by H2SO4 followed by steamtreated Saccharum spontaneum using Box–Bhenken design
Run #
315
X1
X2
X3
CMCase activity (IU) Observed
Predicted
Residual
Observed
Predicted
Residual
1
0.8
10
6
0.543
0.543
0.000
0.331
0.331
2
1.0
10
8
0.855
0.809
0.045
0.954
0.883
0.070
3
1.0
15
6
0.433
0.592
- 0.159
0.767
0.902
- 0.135
4
1.0
10
4
0.882
0.715
0.167
1.389
1.281
0.108
0.000
- 0.042
5
1.0
5
6
0.802
0.856
- 0.053
1.080
1.123
6
0.6
15
6
0.938
0.884
0.053
1.080
1.037
0.042
7
0.8
5
4
0.769
0.883
- 0.113
0.850
0.915
- 0.065 - 0.108
8
0.6
10
8
0.573
0.740
- 0.167
0.790
0.898
9
0.8
15
8
1.007
0.893
0.113
0.962
0.897
0.065
10
0.6
10
4
0.783
0.828
- 0.045
0.746
0.816
- 0.070
11 12
0.6 0.8
5 5
6 8
0.767 1.009
0.607 1.001
0.159 0.007
0.674 0.788
0.538 0.815
0.135 - 0.027
13
0.8
15
4
0.997
1.005
- 0.007
1.140
1.113
0.027
2.5 Experimental Design To optimize different pretreatment conditions for cellulase production, Box–Bhenken design (BBD) was used for optimization study. The independent variables used were H2SO4 concentration (X1), substrate concentration, (X2) and residence time (X3) and their levels are mentioned in Table 1. This design is most suitable for quadratic response surface and generates second-order polynomial regression model. The relation between actual and coded values was described by the following equation; xi ¼
FPase activity (IU)
Xi Xo ; DXi
ð1Þ
where xi and Xi are the coded and actual values of the independent variable, Xo is the actual value of the independent variable at the center point and DXi is the change of Xi. The response is calculated from the following equation using STATISTICA software (99th edition). Y ¼ b0 þ b1 X1 þ b2 X2 þ b3 X3 þ b11 X12 þ b22 X22 þ b33 X32 þ b12 X1 X2 þ b13 X1 X3 þ b23 X2 X3 ;
ð2Þ
Y is the response, X1, X2 and X3 are the independent variables, b0 is the intercept, b1, b2 and b3 are linear coefficient, b11, b22 and b33 are square coefficients, b12, b13 and b23 are interaction coefficients.
3 Results and Discussion In this study, different pretreatment conditions were applied for maximum cellulase production in submerged fermentation by B. subtilis K-18 using S. spontaneum as substrate. For pretreatment, various concentrations of H2SO4, substrate
concentration and residence time were used in two methods, employing steam and without steam. The enzyme production was calculated by second-order polynomial equations (Eqs. 3–6). Two kinds of pretreatment were performed and highest filter paper activity (1.389 IU/ml/min) was observed under pretreatment condition of 1% H2SO4 concentration, 10% substrate concentration with residence time of 4 h at room temperature followed by steam at 121 °C for 15 min and 15 psi. The results of cellulase production using Box– Bhenken design under two different conditions were mentioned in Tables 2 and 3. Only sulphuric acid treatment yield lower enzyme production as compared to sulphuric acid followed by steam treatment. Equations for CMCase and FPase production from acidtreated substrate CMCase activity ðIUÞ ¼ 3:58 4:58X1 0:0543 X2 0:357X3 þ 2:19X12 þ 0:00023X22 þ 0:0203X32 þ 0:0552X1 X2 þ 0:054X1 X3 0:00014X2 X3 ; ð3Þ FPase activity ðIUÞ ¼ 0:983 0:354X1 0:0409X2 0:1710X3 þ 0:240X12 þ 0:000902X22 þ 0:01381X32 þ 0:0207X1 X2 0:0376 X1 X3 þ 0:00036X2 X3 : ð4Þ Equations for CMCase and FPase production from acid followed by steam-treated substrate
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Table 4 Analysis of variance of cellulase from sulphuric acid-treated S. spontaneum
Table 5 Analysis of variance of cellulase from sulphuric acid followed by steam-treated S. spontaneum
Sources
Sources
df
Adj. SS
Adj. MS
F value
P value
CMCase (IU/ml/min)
df
Adj. SS
Adj. MS
F value
P value
CMCase (IU/ml/min)
Model
9
0.247904
0.027545
2.61
0.152
Model
9
0.377746
0.041972
1.47
0.349
Linear
3
0.184846
0.061615
5.84
0.043
Linear
3
0.001079
0.000360
0.01
0.998
X1
1
0.013137
0.013137
1.24
0.315
X1
1
0.000974
0.000974
0.03
0.860
X2
1
0.008230
0.008230
0.78
0.418
X2
1
0.000086
0.000086
0.00
0.958
X3
1
0.163479
0.163479
15.49
0.011
X3
1
0.000019
0.000019
0.00
0.980
Square X21
3 1
0.048983 0.028221
0.016328 0.028221
1.55 2.67
0.312 0.163
Square X21
3 1
0.282003 0.000357
0.094001 0.000357
3.30 0.01
0.116 0.915
X22
1
0.000120
0.000120
0.01
0.919
X22
1
0.122199
0.122199
4.29
0.093
X23
1
0.024356
0.024356
2.31
0.189
X23
1
0.179598
0.179598
6.31
0.054
2 way interaction
3
0.014075
0.004692
0.44
0.732
2-way interaction
3
0.094664
0.031555
1.11
0.428
X1 9 X2
1
0.012179
0.012179
1.15
0.332
X1 9 X2
1
0.073106
0.073106
2.57
0.170
X1 9 X3
1
0.001888
0.001888
0.18
0.690
X1 9 X3
1
0.008290
0.008290
0.29
0.613
X2 9 X3
1
0.000008
0.000008
0.00
0.980
X2 9 X3
1
0.013268
0.013268
0.47
0.525
Error
5
0.052765
0.010553
Error
5
0.142325
0.028465
Lack of fit
3
0.052765
0.017588
Lack of fit
3
0.142325
0.047442
Pure error
2
0.000000
0.000000
Pure error
2
0.000000
0.000000
14
0.300669
14
0.520072
Total
Total
FPase (IU/ml/min)
FPase (IU/ml/min)
Model
9
0.050916
0.005657
6.11
0.030
Model
9
1.26430
0.140478
8.39
0.015
Linear
3
0.035674
0.011891
12. 48
0.009
Linear
3
0.19020
0.063401
3.79
0.093
X1 X2
1 1
0.000044 0.003356
0.000044 0.003356
0.05 3.62
0.837 0.115
X1 X2
1 1
0.10128 0.03891
0.101276 0.038907
6.05 2.32
0.057 0.188
X3
1
0.032275
0.032275
34.85
0.002
X3
1
0.05002
0.050018
2.99
0.144
Square
3
0.012565
0.004188
4.52
0.069
Square
3
0.88386
0.294621
17.60
0.004
X21
1
0.000340
0.000340
0.37
0.571
X21
1
0.33623
0.336234
20.08
0.007
X22
1
0.001878
0.001878
2.03
0.214
X22
1
0.26328
0.263275
15.72
0.011
X23
1
0.011259
0.011259
12.16
0.018
X23
1
0.41810
0.418104
24.97
0.004
2-way interaction
3
0.002678
0.000893
0.96
0.478
2-way interaction
3
0.19024
0.063413
3.79
0.093
X1 9 X2
1
0.001721
0.001721
1.86
0.231
X1 9 X2
1
0.12948
0.129484
7.73
0.039
X1 9 X3
1
0.000904
0.000904
0.98
0.368
X1 9 X3
1
0.05738
0.057383
3.43
0.123
X2 9 X3
1
0.000053
0.000053
0.06
0.821
X2 9 X3
1
0.00337
0.003372
0.20
0.672
Error
5
0.004630
0.000926
Error
5
0.08372
0.016744
Lack of fit
3
0.004630
0.001543
Lack of fit
3
0.08372
0.027907
Pure error
2
0.00000
0.000000
Pure error
2
0.00000
0.000000
14
0.055546
14
1.34802
Total
Total
CMCase activity ðIUÞ ¼ 2:57 þ 0:22X1 0:002 X2 0:694X3 þ 0:25X12 þ 0:00728X22 þ 0:0551X32 0:1352X1 X2 þ 0:114X1 X3 0:00576X2 X3 ;
FPase activity ðIUÞ ¼ 5:85 7:91X1 0:0383X2 0:780X3 þ 7:54X12 þ 0:01068X22 þ 0:0841X32 0:1799X1 X2 0:299X1 X3 0:00290X2 X3 : ð6Þ ð5Þ
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All the data were statistically analyzed by analysis of variance and the proposed model was found significant for FPase production in both treatments while the model for CMCase production was not significant. The Fisher’s F test
Author's personal copy Iran J Sci Technol Trans Sci (2018) 42:313–320
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Fig. 1 Contour plots for CMCase (IU/ml/min) and FPase (IU/ml/min) production from sulphuric acid-treated Saccharum spontaneum by Bacillus subtilis K-18 in submerged fermentation
value of FPase model was 6.11 and 8.39 with P value of 0.030 and 0.015 (Tables 4, 5) in acid-treated and acid followed by steam-treated substrates, respectively. The
model fitness was further checked by coefficient of determination (R2 value) which showed that the predicted model was 91.66 and 93.79% accurately explained the predicted
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Fig. 2 Contour plots for CMCase (IU/ml/min) and FPase (IU/ml/min) production from sulphuric acid followed by steam-treated Saccharum spontaneum by Bacillus subtilis K-18 in submerged fermentation
response in acid-treated and acid followed by steam-treated substrates, respectively. Furthermore, the adjusted R2 value supported the model with values of 76.66 and 82.61% in
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acid-treated and acid followed by steam-treated substrates, respectively.
Author's personal copy Iran J Sci Technol Trans Sci (2018) 42:313–320 Acid
319
Acid steam
Total Sugars (mg/ml)
(a) 16 14 12 10 8 6 4 2 0
5
16
20
23
Time (h)
Reducing sugars (mg/ml)
(b)
Acid
substrate for cellulase production due to its high cellulose content as compared to other substrates (Ilyas et al. 2012). The crude cellulase enzyme produced from B. subtilis K-18 under submerged fermentation was applied to hydrolyze the pretreated S. spontaneum. The experiment was conducted for different time intervals to check the maximum production of fermentable sugars. Results (Fig. 3) revealed that maximum total sugars of 12.71 mg/ml were released at 20 h of incubation at 50 °C; after that the sugar production was reduced. Acid steam-treated S. spontaneum yield higher reducing sugar production (0.60 mg/ml) as compared to acid-treated (0.53 mg/ml) S. spontaneum.
Acid steam
0.7
Acknowledgements Technical staff of microbial biotechnology Lab of department of zoology, university of the Punjab Lahore Pakistan is highly acknowledged.
0.6 0.5 0.4 0.3
References
0.2 0.1 0
5
16
20
23
Time (h)
Fig. 3 Sugars released after enzymatic hydrolysis at various time periods
Figures 1 and 2 illustrated the contour plots for CMCase and FPase production from H2SO4-treated and H2SO4 followed by steam-treated S. spontaneum. Cellulase production in this study was higher as compared to previous reports. Patagundi et al. (2014) isolated and characterized cellulase producing bacteria from soil and reported that maximum cellulase production was observed by Bacillus cereus (0.440 and 0.410 IU/ml/min), followed by B. subtilis (0.357 IU/ml/min) and Bacillus thuringiensis (0.334 IU/ml/min) using Acacia arabica pod as a substrate in submerged fermentation. Arooj et al. (2017) obtained highest FPase activity using pretreatment conditions of 0.4 N H2SO4, 15% banana peduncle as substrate concentration with residence time of 6 h in chemical treatment using B. subtilis K-18 in submerged fermentation. The highest CMCase production of 1.009 IU/ml/ml was observed by 0.8% H2SO4 followed by steam-treated S. spontaneum and this activity was higher as compared to (Ladeira et al. 2015) who reported CMCase activity of 0.29 U/ml cultivated at 50 °C for 168 h or fermentation using sugarcane bagasse as substrate. Saccharum spontaneum is abundantly available substrate which is potential feed stock used for bioethanol production due to its high cellulose contents (Sharma et al. 2015). Sateesh et al. (2012) reported 0.8 IU/ml of cellulase from dilute acid hydrolyzates of S. spontaneum on fourth day of fermentation by Trichoderma reesei NCIM 992. Saccharum spontaneum was good
Ahmed FM, Rahman RS, Gomes DJ (2012) Saccharification of sugarcane bagasse by enzymatic treatment for bioethanol production. Malays J Microbiol 8(2):97–103 Arooj A, Irfan M, Tabbasum F, Shakir HA, Qazi JI (2017) Effect of dilute sulphuric acid pretreatment on cellulase production by Bacillus subtilis (K-18) through response surface methodology. Proc Pak Acad Sci B Life Environ Sci 54(1):11–20 Bayer E, Chanzy H, Lamed R, Shoham Y (1998) Cellulose, cellulase and cellulosomes. Curr Opin Struct Biol 8:548–557 Ghildyal NP, Lonsane BK, Sreekantiah KR, Sreenivasa MV (1985) Economics of submerged and solid state fermentation for the production of amyloglucosidase. J Food Sci Technol 22:171–176 Henrissat B (1991) A classification of glycosyl hydrolases based on amino acid sequence similarities. Biochem J 280(Pt 2):309–316 Henrissat B (1994) Cellulases and their interaction with cellulose. Cellulose 1:169–196 Hui L, Wan C, Hai-tao D, Xue-jiao C, Qi-fa Z, Yu-hua Z (2010) Direct microbial conversion of wheat straw into lipid by a cellulolytic fungus of Aspergillus oryzae A-4 in solid-state fermentation. Bioresour Technol 101:7556–7562 Ilyas U, Ahmed S, Majeed A, Nadeem M (2012) Exploration of indigenous agrowastes for cellulase production by Aspergillus niger. Afr J Biotechnol 11(38):9276–9279 Irfan M, Gulsher M, Abbas S, Syed Q, Nadeem M, Baig S (2011) Effect of various pretreatment conditions on enzymatic saccharification. Songklanakarin J Sci Technol 33(4):397–404 Ladeira SA, Cruz E, Delatorre AB, Barbosa JB, Martins MLL (2015) Cellulase production by thermophilic Bacillus sp. SMIA-2 and its detergent compatibility. Electron J Biotechnol 18:110–115 Li W, Du W, Liu DH (2007) Optimization of whole cell-catalyzed methanolysis of soybean oil for biodiesel production using response surface methodology. J Mol Catal B Enzym 45:12–127 Patagundi BI, Shivasharan CT, Kaliwal BB (2014) Isolation and characterization of cellulase producing bacteria from soil. Int J Curr Microbiol Appl Sci 3(5):59–69 Sadhu S, Maiti TK (2013) Cellulase production by bacteria: a review. British Microbiol Res J 3:235–258 Sateesh L, Naseeruddin S, Rodhe AV, Prasad Y, Yadav KS, Rao LV (2012) Simultaneous cellulase production, saccharification and detoxification using dilute acid hydrolysate of S. spontaneum with Trichoderma reesei NCIM 992 and Aspergillus niger. Indian J Microbiol 52(2):258–262
123
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Sharma D, Joshi B, Bhatt MJ, Joshi J, Malla R, Bhattarai T, Sreerama L (2015) Isolation of cellulolytic organisms from the gut contents of termites native to nepal and their utility in saccharification and fermentation of lignocellulosic biomass. J Biomass Biofuel 2:11–20 Teeri TT, Koivula A, Linder M, Wohlfahrt G, Divine C, Jones TA (1998) Trichoderma reesei cellobiohydrolases: why so efficient on crystalline cellulose? Biochem Soc Trans 26:173–178
123
Zhang YHP, Lynd LR (2004) Toward an aggregated understanding of enzymatic hydrolysis of cellulose: noncomplexed cellulase systems. Biotechnol Bioeng 88:797–824
