Combinatorial Biobleaching of Mixedwood Pulp with Lignolytic and Hemicellulolytic Enzymes for Paper Making

Abstract

Microbial enzymes are the safe alternatives to chemical based bleaching of pulp in paper mills. For effective biobleaching, both hemicellulolytic and lignolytic enzymes are required. This study reports laccase (L) + xylanase (X) and laccase (L) + mannanase (M) enzyme concoctions for pulp biobleaching derived from Bacillus sp. LX and Bacillus sp. LM isolated from the decaying organic matter. All enzymes were thermo-alkali-stable, hence were suitable for their application in pulp biobleaching. When a mixture of L + X/L + M was used for mixedwood pulp biobleaching, 46.32/40.25% reduction in kappa number; 13.21/10.01% and 3.36/2.76% improvement in brightness and whiteness was achieved respectively. Moreover, no laccase mediator system was required in the current process. Significant changes in the structure of enzymatically treated pulp were also observed. All these properties make these concoctions of enzymes suitable for their application in pulp and paper mill.

Introduction

Pulp and paper mills uses chemical based processes such as ‘chlorine alkali-extraction’ for the removal of lignin from pulp, which leads to the release of highly toxic and bio-accumulating chlorinated organic substances in the effluent [1]. Therefore, newer approaches are being explored to reduce the use of chemicals; out of which most suitable is the use of enzymes for the bioprocessing of pulp [2].

Enzymes such as xylanase(s) and mannanase(s) help in the removal of hemicellulolytic components which facilitate the removal of lignin from the pulp with reduced chemical requirement [3, 4]. Lignolytic enzymes like laccase(s) directly act on lignin and degrade it by attacking the phenolic subunits [5]. For effective enzymatic treatment of pulp, both lignolytic and hemicellulolytic enzymes are required. Application of cocktail of enzymes has been explored in many industrial processes [6, 7]. There are a few reports showing the use of a combination of enzymes in pulp biobleaching but in all these studies, the enzymatic combination was prepared from two different microorganisms grown individually [5, 8] or by co-culturing [9]. Moreover, most of the times, enzymatic treatment was given sequentially [10]. There is no report to date which has shown the production of lignolytic enzymes (laccases) and hemicellulolytic enzymes (xylanases and/or mannanases) from a single bacterium and their application in pulp and paper Industry. This study was undertaken for the extensive screening of bacteria which can produce a combination of thermo alkali-stable extracellular laccase + xylanase and/or mannanase and their application for pulp biobleaching.

Soil samples were collected from the areas where organic matter was decaying. Although there are some reports of extracellular laccases [5] from bacteria but most of them are intracellular or spore bound [11]. With this viewpoint in mind, first screening of the samples was done for extracellular laccase producing bacteria. Enrichment of the samples was done in M162 basal medium plates having 2 mM guaiacol [5, 12]. The screening of plates showed 35 laccase positive isolates having different colony morphologies were screened for the presence of extracellular laccase [5]. Out of these, eleven isolates produced the enzyme extracellularly with varying yield (Table 1). Laccase positive isolates were screened for extracellular xylanase and mannanase by adding xylan and locust bean gum (LBG) in the medium by Congo red staining [3, 4] and enzyme assay in the liquid medium [4, 13]. Four isolates were positive for extracellular xylanase and three for extracellular mannanase. No strain was positive for all the three enzymes (Table 1). One International Unit was defined as micromoles of substrate converted into product per minute by one ml of the enzyme. There are numerous reports of bacteria producing xylanase(s) [13, 14] and mannanase(s) [15, 16] individually. Some bacteria producing xylanase + mannanase have also been reported [17]. However, there is no report till date for the production of laccase + xylanase or laccase + mannnase from any bacterium.

Table 1.

Screening of bacteria for Lignolytic + hemicellulolytic enzyme(s)

Isolate No.	Laccase (L)		Xylanase (X)		Mannanase (M)
	Screening on plate	Extracellular activity (IUml−1)	Screening on plate	Extracellular activity (IUml−1)	Screening on plate	Extracellular activity (IUml−1)
4	+ +	10.6 ± 0.05	+	1.2 ± 01.4	–	–
9	+	6.2 ± 0.15	+	0.8 ± 0.17	–	–
11	+ +	15.8 ± 0.14	–	–	–	–
15	+ +	13.3 ± 0.13	–	–	–	–
18	+ +	10.8 ± 0.11	–	–	+	0.8 ± 0.18
20	+	7.2 ± 0.18	–	–	–	–
23	+ +	15.4 ± 0.14	+	5.3 ± 0.11	–	–
24	+ +	16.8 ± .0.15	–	–	–	–
29	+ +	18.8 ± 0.14	–	–	+	6.4 ± 0.11
30	+	5.8 ± 0.19	–	–	+	0.6 ± 0.15
33	+ +	11.3 ± 0.11	+	4.6 ± 0.18	–	–

+ (< 10U); + + (>10U); – negative

High yield is also an important criterion for the commercial application of any enzyme [6]. Therefore, final selection of the isolates was done on the basis of yield of enzymes. Isolates number 23 and 29 were selected for L + X and L + M respectively on the basis of high enzyme yield. Morphological, biochemical and 16S rRNA sequence (GenBank accession number MT020109 and MT020110) analysis showed that both the isolates belonged to genus Bacillus and were designated as Bacillus sp. LX and Bacillus sp. LM respectively. For large scale application it is required that an enzyme should be active/stable at the process conditions used in the Industry [5]. In a paper mill, pulp is processed at high temperature and pH [10, 14] therefore; temperature and pH profiles of the enzymes were studied. Optimum temperature and pH of the enzymes were determined by carrying out the enzyme assays at different temperatures (40–80 °C) and pH (5.5–10.0). Stability profiles of the enzymes were determined by incubating the enzymes at different temperatures/pH for different time intervals and measuring the enzyme activity under standard assay conditions. Optimum temperature/pH of laccase and xylanase from Bacillus sp. LX was found to be 75 °C/7.0 and 70 °C/9.0 respectively (Fig. 1) and 70 °C/7.0 and 60 °C/8.5 for laccase and mannanase respectively from Bacillus sp. LM (Fig. 2). Results indicated that all the enzymes were thermo-alkali stable. Although optimum pH of laccase from both the isolates was 7.0 when assayed using syringaldazine as substrate however, pH profile of laccase(s) is known to be substrate dependent [18].

Fig. 1.

Activity profiles of Laccase (▲) and Xylanase (●) from Bacillus sp. LX with respect to a Temperature b pH

Fig. 2.

Activity profiles of Laccase (▲) and Mannanase (●) from Bacillus sp. LM with respect to a Temperature b pH

Combination of enzymes L + X and L + M were applied for biobleaching of pulp. Mixed wood pulp was obtained from Ballarpur Industries Limited (BILT), Yamunanagar, Haryana, India and was washed with distilled water and dried at 55 °C for overnight in an oven. Enzymatic treatment was given at 5% pulp consistency (20 ml reaction volume containing 1 g of pulp), at 70 °C, pH 8.5 for 4 h using 1 ml of L + X (3:1) and L + M (3:1) mixtures respectively in a water bath shaker. Pulp treated under the same conditions but without enzyme was taken as control. Standard methods of TAPPI (Technical Associations of Pulp and Paper Industry) were used for analysis of kappa number, brightness and whiteness [19]. A significant reduction of 46.32% in kappa number; 13.21% of improvement in brightness; and 3.36% in whiteness was achieved after enzymatic treatment with L + X. Similar results were obtained when pulp was treated with L + M enzymes that showed 40.25% reduction in kappa number and 10.01% and 2.76% improvement in brightness and whiteness respectively (Table 2). Some studies have shown the use of L + X for pulp biobleaching but improvement in the pulp properties was lesser; moreover enzymes were obtained from two different organisms and have been used sequentially for pulp biobleaching [10].

Table 2.

Biobleaching of pulp using a mixture of Laccase + Xylanase from Bacillus sp. LX and Laccase Mannanase from Bacillus sp. LM

Effect of enzymatic treatment
Parameters	U	Change in pulp properties				% change
		L + X	LMS + X	L + M	LMS + M	L + X	LMS + X	L + M	LMS + M
Kappa number	15.80	8.48 ± 0.12	8.25 ± 0.14	9.44 ± 0.11	8.71 ± 0.18	46.32 ↓	47.78 ↓	40.25 ↓	44.87 ↓
Brightness (%)	27.46	31.09 ± 0.14	31.4 ± 0.18	30.21 ± 0.18	30.98 ± 0.11	13.21 ↑	14.34 ↑	10.01 ↑	12.81 ↑
Whiteness (%)	− 43.38	− 41.92 ± 0.15	− 41.63 ± 0.14	− 42.18 ± 0.11	− 41.87 ± 0.18	3.36 ↑	4.03 ↑	2.76 ↑	3.48 ↑

U: Untreated pulp; L_MS: Laccase mediator system

A number of mediators have been reported to increase the redox potential of laccase(s) [20] and most of the studies carried out in the past has reported the requirement of a mediator(s) for the application of laccase in pulp processing [10]. However, because of the high costs involved, requirement of a mediator makes the application of laccase(s) for pulp biobleaching non-economical [6, 10]. Therefore another set of enzymatic treatment of pulp was done using 2, 2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) as laccase mediator system (L_MS + X; L_MS + M). ABTS is the best mediator used for these processes and is known not to effect the activity of other enzymes [10]. However, the results obtained were comparable to those when no mediator system was used. The non requirement of a mediator for the enzymes reported in the present study further increases their feasibility for industrial application by reducing the cost of the process. Sondhi et al., 2015 is the only study which has reported a laccase which does not require a mediator for pulp biobleaching [5].

Samples of control and untreated pulp were prepared [5] and examined under Scanning Electron Microscope (SEM) to analyze structural changes in the enzyme treated pulp. Control pulp showed integral structures whereas visible changes in the properties of enzymatic treated pulp were observed with dissociation of fibers leading to increased roughness, cracks and pores (Fig. 3). This can be because of degradation of hemicellulose by xylanase/mannanase and dissolution of lignin by laccase. These changes indicated that lesser amount of chemicals will be required in the subsequent chemical based bleaching of pulp. A number of other reports have also shown similar changes in the enzymatically treated pulp [5, 10].

Fig. 3.

Scanning electron micrographs a Control- untreated pulp b Laccase + Xylanase treated pulp c Laccase + Mannanase treated pulp

Production of lignolytic and hemicellulolytic enzymes from a single bacterium and non requirement of laccase mediator system make these enzymatic concoctions highly suitable for the development of an environment friendly process for pulp and paper industry.

Compliance with Ethical Standards

Conflict of interest

The authors declare that they have no conflict of interest.