F
Green technology for paper industry: Cocktail of lignolytic and hemicellulolytic enzymes from Bacillus tequilensis LXM 55 for pulp biobleaching .
Steffy* Ph.D Research Scholar Dr. Naveen Gupta Department of Microbiology, BMS Block-I, Panjab University, Chandigarh 160014, India E-mail: steffyangural91@gmail.com •:
INTRODUCTION Environmental pollution has been a major challenge for the development of industry. Chemical used in various industrial processes are released into the environment. Pulp and Paper Industry is the 15th largest paper industry in the world. One of the most polluting industries worldwide. Bioprocessing of pulps using microbial enzymes for biobleaching is one of the most suitable biological applications for the pulp and paper
Raw Material Washing
Chipping at Chippers
Cooking in Digester
Single Stage Hot Stock Screening
Central composite design matrix values for all enzymes
RESULTS
Brown Stock washing
Pulp Bleaching
Std
Soil/sludge samples were collected from sites of effluent water from pulp and paper industry. Samples were enriched in M162 medium containing 2mM guaiacol as substrate, A total of 60 isolates showing reddish brown color colonies with different colony morphology were obtained. Further screened on the basis of production of extracellular laccase.
15 16 14 13 7 9 11 4 8 6 3 1 18 17 10 2 12 19 20 5
4 isolates showed the extracellular laccase activity Screening of Mannanase producing isolate/s Out of these, all the 4 isolates showed the activity of mannanase by DNSA method using locust bean gum as a substrate . Screening of Xylanase producing isolate/s Shortlisted isolate/s were screened for xylanase activity and two isolates (Isolate no. 54 and 55) showed xylanase activity by DNSA method using xylan as a substrate.
Run
A:Tryptone
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
grams 0.4 0.4 0.4 0.4 0.3 0.231821 0.4 0.5 0.5 0.5 0.3 0.3 0.4 0.4 0.568179 0.5 0.4 0.4 0.4 0.3
B:Inoculum percentage percentage 1.5 1.5 1.5 1.5 2 1.5 0.659104 2 2 1 2 1 1.5 1.5 1.5 1 2.3409 1.5 1.5 1
C:Moisture ratio percentage 3 3 4.68179 1.31821 4 3 3 2 4 4 2 2 3 3 3 2 3 3 3 4
Laccase activity Xylanase activity
250.62 200.4 33.78 435.5 130.03 250.78 400.28 195.4 30 43.75 400.68 410.62 150.28 266.09 145.46 436.87 165.31 190.18 190.4 61.09
Mannanase activity
90.9 82.19 30.69 170.97 40.8 34.59 108.4 174.2 170.8 23.48 11.09 181.6 105.34 182.79 170.4 180.19 100.09 100.5 100.89 10.8
19.59 19.9 1.91 30.9 1.29 15.7 22.35 32.1 12.29 19.7 32.62 29.65 20.51 19.44 24.88 31.2 17.14 18.66 31.3 4.48
Three Dimensional Graph for Laccase , Xylanase and Mannanase enzyme
Optimization of Laccase+Xylanase+Mannanase in SSF using OVAT Selection of solid substrate Incubation time Effect of moistening solutions Effect of different moisture level Incubation temperature Effect of different concentration of (CuSO4.5H2O) Effect of different concentration of inoculum
Bioprocessing of pulps using microbial enzymes for biobleaching is one of the most suitable biological applications for Pulp and Paper Industry Lignolytic Enzymes Hemicellulolytic Enzymes Laccase Xylanase, Mannanase Helps directly in the removal of lignin Depolymerizes hemicelluloses precipitated from the pulp on the surface of the fiber
: Wheat Bran : 96h : MS3 : 1:1 : 37ºC : 0.4 (µM) : 1%
Three Dimensional graphs for the effect of various parameters for laccase production A) Tryptone and Inoculum percentage
Important criteria for applying enzymes for Biobleaching
Statistical Optimization
Active at high temperature (60-80°C) Extracellular production High Yield A number of lignolytic and hemicellulolytic enzymes satisfying above criteria have been reported from different microorganisms and their application in pulp biobleaching has been explored.
Plackett-Burman design for screening of significant parameters affecting all the enzyme production using Bacillus sp. LXM 55
Limitation : For effective biobleaching, removal of both lignin and different hemicelluloses present in wood is required. No single enzyme is not able to remove lignin as well as hemicelluloses. So, cocktail of enzymes is required for achieving effective biobleaching of pulp making process eco-friendly. Strategies for making cocktail of lignolytic and hemicellulolytic enzymes Preparing a mixture of enzymes by growing different microorganisms separately; However it makes the process economically unviable Co-culturing of microorganisms producing different enzymes; But it is often difficult to standardize the conditions of co-culture fermentation moreover most of the researchers have reported the production of not more than two enzymes at one time by co-culturing Isolating a bacteria producing a cocktail of hemicellulolytic and lignolytic enzymes; Very few reports have shown the production of more than one enzyme (Maximum 2) from a single bacteria having application in pulp and paper industry
Exp
Facto
Facto
Facto
Facto
Factor
Factor
Factor
Factor
Factor
Factor
Factor
Respons
Respons
Response
r1
r2
r3
r4
5
6
7
8
9
10
11
e1
e2
3
A: B: C: CuSo4 MnSo MgSO
D: FeSO
Laccase Activity
4
F: TritonX 100
L: Yeast
4
E: Inoculum percentage
4
G: H: J: K: Moisture Peptone Kh2 PO Tryptone ratio 4
Xylanase Mannanas Activity e Activity
IUg-1
IUg-1
0.3
35
15.29
5.08
0
0
366.56
15.69
10.17
0.3
0.3
0
227.81
17.69
8.36
0
0.3
0.3
0.3
684.37
88
19.25
1
0
0
0.3
0
348.75
32.19
8.22
1
2
0
0
0
0.3
315.93
33.6
18.91
1
0.1
1
0
0.3
0
0.3
621.09
28.04
22.41
500
0.1
0.1
1
0.3
0
0.3
0.3
644.53
19.89
5.28
500
0
0.1
0.1
2
0
0.3
0.3
0
107.81
130.48
44.93
500
0
500
1
0.1
2
0.3
0.3
0
0
535
159.48
14.66
500
0
0
0
1
0.1
2
0.3
0
0.3
0.3
897.65
28.39
30.96
0
0
0
0
0.1
0.1
1
0
0
0
0
803.9
27.39
4.196
micro
micro
micro
mole
mole
mole
mole
1
500
500
0
0
2
500
0
500
3
0
0
4
0
5
%
Three Dimensional graphs for the effect of various parameters for laccase production A) Tryptone and Inoculum percentage
B) Moisture ratio and Tryptone C) Moisture ratio and Inoculum percentage
extract IUg-1
micro
B) Moisture ratio and Tryptone C) Moisture ratio and Inoculum percentage
%
%
%
%
%
%
(W/V)
(W/V)
(W/V)
(W/V)
(W/V)
(W/V)
0.1
1
1
0.3
0.3
0
500
0.1
1
2
0.3
0
500
0
1
1
1
0.3
0
0
500
0.1
1
2
500
500
0
500
1
1
6
0
500
500
0
1
7
500
0
500
500
8
0
500
500
9
500
500
10
0
11 12
Three Dimensional graphs for the effect of various parameters for laccase production A) Tryptone and Inoculum percentage
B) Moisture ratio and Tryptone C) Moisture ratio and Inoculum percentage
Preliminary characterization of the enzyme
Plackett Burman for Laccase enzyme
Application of cocktail of enzyme (L+X+M) for biobleaching of mixedwood kraft pulp
There is no report, where production of thermo alkali stable hemicellulases and lignolytic enzymes has been reported from a single bacterial strain.
Isolate a bacterial strain producing a cocktail of hemicellulolytic and lignolytic enzymes and standardizing a procedure for their application in pulp and paper industry to make the whole process of paper making eco friendly
Plackett Burman for Mannanase enzyme
METHODOLOGY Isolation and selection of novel bacterial strain which is capable of producing a cocktail of hemicellulolytic and lignolytic enzymes.
Control (Without enzyme treated) Paper
Reduction in chlorine consumption by cocktail of laccase + xylanase + mannanase (L+X+M) treatment of mixedwood kraft pulp
Identification of the isolate by microscopic, biochemical tests and 16S rDNA sequence analysis. Evaluation of various cultural and environmental factors for designing an economically viable and simple medium leading to the hyper production of all the enzyme components in solid state fermentation was done by using: (i) By classical methods using OVAT (One variable at a time method) (ii) By statistical modelling using (PB and RSM)
Parameters
Plackett Burman for Xylanase enzyme
U*
(L+X+M)
Kappa Number
2.05
0.45
Brightness (%)
89.23
97.78
Whiteness (%)
72.26
84.48
Conclusion: Significant reduction in chlorine use was achieved by pretreating the pulp with cocktail of enzymes. Therefore this cocktail of enzymes is highly suitable candidate for developing a cleaner and economical process for Pulp bleaching.
Characterization of the enzyme with respect to temperature optima/stability, pH optima/stability was done. Application of cocktail of enzymes in pulp and paper industry for pulp biobleaching was done by optimizing the important process parameters for optimal biobleaching.
Test (With enzyme treated) Paper
Out of 11 factors, three factors viz. tryptone, inoculum percentage, Moisture ratio having high positive influence on laccase, mannanase and xylanase production. So they are further selected for RSM
References Bajpai P, Anand A, Sharma N, Mishra SP, Bajpai PK and Lachenal D (2006) Enzymes improve ECF bleaching of pulp. Bioresources 1: 34-44. Chauhan PS, Soni SK, Sharma P, Saini A and Gupta N (2014) A Mannanase from Bacillus nealsonii PN-11: Statistical optimization of production and Application in Biobleaching of pulp in combination with xyalanse. International Journal of Pharma and Bio Sciences 5: 237-251. Gupta V, Garg S, Capalash N, Gupta N and Sharma P (2015) Production of thermo-alkali-stable laccase and xylanase by co-culturing of Bacillus sp. and B. halodurans for biobleaching of kraft pulp and deinking of waste paper. Bioprocess and Biosystem Engineering 38: 947-956.