Journal of Molecular Biology, Biochemistry and Genetics

Enzyme Technology for Lignocellulosic Biomass Conversion and Recycling to Valuable Paper and other Products: Challenges Ahead

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Published Date: October 26, 2017


Enzyme Technology for Lignocellulosic Biomass Conversion and Recycling to Valuable Paper and other Products: Challenges Ahead

Gursharan Singh1*, Shailendra K. Arya1, Vijaya Gupta2 and Prince Sharma2

1Department of Biotechnology, UIET, Panjab University, India

2Department of Microbiology, Panjab University, India


*Corresponding author: Gursharan Singh, Department of Biotechnology, UIET, Panjab University, India, E-mail:

Citation: Singh G, Arya SK, Gupta V, Sharma P (2017) Enzyme Technology for Lignocellulosic Biomass Conversion and Recycling to Valuable Paper and other Products: Challenges Ahead. J Mole Biol Tech 2(1): 105.




Enzymes have the potential to replace conventional, environment-polluting chemicals-based process for the transformation of lignocellulosic materials to valuable paper with other products. There are several challenges, however, for biocatalysts before their successful implementation in pulp and paper industry, like variant nature of lignocellulosic biomass from different sources and resistance to enzymatic attack, pH, temperature, stability and reductive yield of pulp by the use of xylanases. Low redox potential and need of Mediator System (LMS) for laccases and cost-intensive production of these enzymes at larger scale constitute an even greater hitch. Lack of technology for reuse of enzymes in biobleaching is also adding to the repeated investment cost. The demand of paper is continuously burgeoning at a pace much faster than the availability of fibers from the natural sources like wood biomass or agro-based lignocellulosic materials. Some of the lignocellulosic materials are present in excess like rice straw but are rigid to enzymatic attack thus showing the lack of potential to produce a quality paper. To cope with these situations, research efforts inclined towards the recycling of waste paper after its intended use are needed. Several biocatalysts like cellulase, lipase, xylanase and now laccase have been evaluated for their potential for deinking of different sort of waste papers for their recycling. Enzymes improve paper properties such as brightness and resistance as compared to the paper deinked by chemical methods. Interestingly, pulp and paper industry effluents have suddenly gained a great interest in the past few years for the production of valuable products like vanillin from the lignin present in the black liquor effluent. Current synthesis of such valuable products is achieved by cumbersome chemical methods due to the lack of eco-friendly enzymatic process development. This article emphasizes more profound knowledge on challenges that influence enzyme based bioconversion of lignocellulosic biomass and recycling of secondary pulp to valuable paper products.

Keywords:  Lignocellulose; Enzyme technology; Recycling; Paper; Xylanase; Laccase; Vanillin




In the 21st century, utilization of paper has increased four times more rapid than the human population. India is one of the emerging paper industries nations with 10% of growth rate which in all probability will expand more in future. Several pulp and paper industries still rely on chlorine dioxide (ClO2) as the Elemental Chlorine Free (ECF) agent for delignification of pulp for the production of white paper.  Biocatalysts give a simpler and remunerative way to reduce the consumption of ClO2, and other chlorine-based bleaching chemicals [1]. Viikari L, et al. [2] first reported in 1986 that the use of endoxylanases decreases the chemical consumption needed for bleaching of lignin-containing pulp. The applications of xylanase as a pre-bleaching agent have been evaluated in many laboratories and have also been commercially exploited in developed countries and in some developing nations. Besides xylanase, laccase is also being explored widely for lignocellulosic biomass conversion to pulp and paper in the presence of appropriate Laccase Mediator System (LMS). Laccase has the ability to delignify pulp without reduction of final yield in comparison to xylanases [3,4].  In last few years, recycled fiber has acquired importance in the paper industry. In Europe, the main sources of used paper recovery are trade and industry (50%), households (40%) and offices (10%). These are low-cost fibers for the manufacturing of paper after deinking, and through their use, forest resources can be preserved [5]. In the conventional deinking procedure, huge quantities of chemical products are utilized, which make the process environmentally harmful. To overcome the demerits of deinking by toxic chemicals, the enzymatic approach has drawn enough attention because it can detach ink particles from fibers without additional acquittal of pollutants [6,7]. Several organic composites present in the pulp and paper industrial effluent have been classified as mutagenic or carcinogenic. Chemical and physical techniques like incineration and coagulation of black liquor lignin have been reported as the common methods to reduce the toxicity and color of the pulp and paper industrial effluent. However, these methods are cost intensive as compared to biological methods of effluent treatment containing lignin. Several microbial species like wood rotting white-rot fungi have been frequently reported for the degradation of lignin as well as treatment of pulp and paper mill effluent because of their extracellular ligninases.  On the other hand, bacteria, which grow at a wide range of acidic to alkaline pH conditions, can play an influential role in stabilization of pulp and paper waste effluent without any need for change in pH. Some bacterial species have been recognized that can transform lignin monomers in the effluent to important aromatic compounds, like vanillic acid, ferulic acid and vanillin [8]. Although the enzyme technology for lignocellulosic biomass conversion to valuable paper and for deinking of waste paper is largely getting recognition for implication in paper mills, but several challenges are lying ahead like high production cost of enzymes, less stability of biocatalysts against high pH and temperatures and mandatory requirement of mediators by laccases due to their lower redox potential than lignin and non-lignin components of woody and agro-based pulps.


Use of Hemicellulases for the Bioconversion of Lignocellulosic Biomass to Pulp and Paper


Among the hemicellulases, xylanases are considered as the preferable option to enhance the brightness of pulp through delignification that can reduce or replace the toxic chlorine compounds used in the chemical bleaching methods [9,10]. Xylanases have been widely evaluated for their potential applicability in the pulp and paper mills since they proved delignification of pulp by depolymerizing xylan closely placed with lignin in the plant cell wall. Lignin removal by xylanases can reduce 5.0–7.0 kg of ClO2 per ton of pulp and there is an estimated reduction of 2.0–4.0 units in the kappa number of pulp [12]. Xylanase from B. megaterium was reported for bringing 8.1 and 1.1% more brightness and viscosity respectively, 13% reduction in kappa number and 31% subtraction in chlorine utilization [13]. Xylanase from B. stearothermophilus SDX reduced the chlorine consumption up to 15% while its combination with pectinase resulted in 20% reduction [14]. Xylanase from Thermomyces lanuginosus SK was reported for delignification potential of the non-purified enzyme for bagasse pulp where brightness was increased by 3.6 units than untreated pulp. Sugars and lignin-derived compounds were released in large amounts in xylanase treated pulp [9]. Thermo tolerant and alkali stable xylanase from B. pumilus SV-85S showed (at pH 9.0, 55°C for 2 h) subtraction in kappa units by 1.6 and increased brightness by 1.9 points. The prebleaching of pulp with xylanase resulted in 29% subtraction in chlorine utilization while maintaining the similar brightness as in control [15]. Thermo-alkali stable mannanase of Streptomyces sp. PG-08-3 was evaluated for delignification of wheat straw-rich pulp at pH 8.5 and 55°C that enhanced the brightness by 7.3% and decreased the kappa number by 24% after 4h. Mannanase based prebleaching of pulp decreased the utilization of hypochlorite by 16% to bring the brighter of pulp sheets same as produced through chemical processing of pulp [16].


How Xylanases Improves Delignification?


Extensive information gathered by Senior et al. [17], exhibited how xylanases hydrolyze the xylan present in the pulp fibers. Xylan is linked to the cellulose and lignin, thus it follows that destruction of the xylan backbone affects their separation during bleaching. Xylanase was also shown to increase fiber wall swelling and in turn enhanced the speed of diffusion through the walls [18]. Another hypothesis that came from the research was that xylanase enzymes catalyze the hydrolysis of xylan that has reprecipitated on the fibers during alkaline pulping. Xylanase acts as a bleaching aid rather than as a true delignification agent since the enzyme does not directly degrade lignin [19].


Application of Laccases for the Bioconversion of Lignocellulosic Biomass to Pulp and Paper


Laccase can catalyze the four electron reduction of O2 to water with simultaneously single-electron oxidation of several phenolic substrates. The laccase is widely reported from fungi, plants, and bacteria [20]. Lignin oxidation by fungal laccases has been frequently studied as compared to prokaryotic laccases. Lignin is a complex polymer in which only phenolic units are invaded by laccase by giving oxygen-centered radicals which react further through nonenzymatic ways [11]. There is no report in the literature that laccase can deteriorate lignin in pulp or wood without a mediator. Lignin’s of wood and pulp are considered as poor substrates for laccases as this lignin contains a low quantity of free phenolic hydroxyl groups. Moreover, generally, this biocatalyst is a large molecule (~60–120Kd). As a result, the laccase cannot enter the cell wall to access lignin directly [21].

It is a well-known fact that the range of laccase substrates can be extended to non-phenolic subunits of lignin in the presence of an appropriate mediator or co-substrate in the reaction mixture. Laccase was used in the presence of ABTS as a mediator for the delignification of kraft pulp [22].  Figure 1 shows the mechanism of lignin degradation by laccase and mediator system. Laccases have an advantage over other lignin oxidizing enzymes, like lignin peroxidases (Lip) in that the redox potential of Lip increases with a decrease in pH of reaction environment.  For laccases, such data is not available, as logically alkalitolerant-laccases will be the best choice for pulp bleaching where alkaline conditions are demanded at large [23,24].


Synergistic Effect of Hemicellulases and Laccases for Lignocellulosic Biomass Conversion to Pulp and Paper


The combination of laccase and xylanase for prebleaching of pulps can result in increased brightness by removing the lignin from pulps [25]. A blend of laccase and xylanase was evaluated for the biobleaching of mixed wood pulp. The co-cultivation method was used for the production of enzymes by Penicillium oxalicum and Pleurotus ostreatus grown by using solid substrate fermentation. Biobleaching of pulp with a cocktail of enzymes brought a reduction in kappa units and enhanced brightness as compared to the use of the only xylanase. Review of bio-bleaching sequences demonstrated the enzyme cocktail (laccase: xylanase 1:22) led to increased biobleaching during an experiment at 10% consistency of pulp (55°C, pH 9.0) for 3.0 h [26]. Laccase and xylanase were produced through cost-effective strategy for enzyme production up to 10 kg of the substrate and checked for elemental chlorine free bleaching of kraft pulp. Compared to the pulp pre-bleached with xylanase (15%) or laccase (25%) individually, the ClO2 savings were higher with sequential treatment with xylanase followed by laccase (35%) at laboratory scale. The same bio-catalytic treatment when applied at pilot scale (50 kg pulp), resulted in improved pulp properties (50% reduction of post color number, 15.71% increased tear index) and reduced (34%) organo-chlorine compounds (measured as AOX) levels in bleach effluents [27].  Recently, Gupta et al. [28] reported the simultaneous production of laccase {L} and xylanase {X} from Bacillus sp. and B. halodurans by solid-state fermentation. Both biocatalysts showed similar pH and temperature optima of 9.0 and 70°C and were applied for biobleaching of pulp and deinking of Old Newsprint (ONP). For pulp biobleaching, XL showed the significant enhancement in brightness by 13%, whiteness by 106% and subtraction in kappa number by 15% after the alkali extraction step and peroxide stage. This increase in properties of pulp showed the combined effect of bleaching enzymes brought in a single step.


Deinking of Old Newsprint and Waste Paper by Enzyme Treatment


Enzymes have accelerated and remodeled the process of deinking for getting the higher brightness of secondary pulp in comparison to traditional chemical deinking methods. There are several advantages of recycling of waste paper by deinking, like decreasing the requirement of wood as a conventional raw material, lower energy inputs, reliable and easy availability of pulp for the production of commercial white paper and low price of deinked pulp in comparison to a wood-based pulp ( Deinking is a crucial step for the recycling of fiber. Nevertheless, with each successive deinking cycle the firmness of new paper is reduced which further decreases the papermaking potential of recycled fiber. A comparison of the traditional chemical deinking method with enzymatic one got the attention owing to capital investment and environmental issues [29]. Xu et al. [6] reported the old newsprint (ONP) deinked by Laccase and Violuric acid System (LVS). Experimental outcomes showed that the molecular mass of lignin from LVS-deinked pulp was decreased than lignin from the control pulp. The process starts with the oxidation of violuric acid by laccase in an oxygenated environment and converted to VIO complex which contained potent oxidation potential this complex easily diffused into the fibers of pulp and extract lignin through the series of oxidative degradation reactions. As the lignin removes, bondings between fibers and ink particles became loosen. Further with the aid of shear forces giving the deinking equipment, ink particles detached from the pulp fibers. The probable mechanism of laccase based deinking by using mediator system is shown in Figure 2. Hemicellulase and cellulase alone or in combination are regularly utilized in several paper mills [30]. On the other hand, laccase is now considered worth for the deinking of ONP [7]. ONP deinked by laccase and violuric acid system (LVS) improved the tensile and tear strength by 20 and 13% respectively. The brightness was also increased by 4.2% after bleaching with H2O2 [31]. Recently, Virk et al. [7] reported a novel laccase that does not require any mediator involvement for deinking of ONP pulp and a blend of xylanase and laccase was used for effective deinking.


Production of Aromatic Compounds from Pulp and Paper Industrial Waste


Annual utilization of approximately 15,000 tons of vanillin (4-hydroxy-3-methoxybenzaldehyde) is considered as the most widely-used flavor component in baked food, chocolates, and dairy products. Alone 0.2% of the total requirement is achieved from vanilla beads, but the rest is generated by the chemical process and ferulic acid based process. The natural flavor of vanilla is a mixture of several constituents, although the aroma is majorly contributed by vanillin. Due to the less availability and higher cost of native vanilla juice, there has been a persisting interest for its synthesis biotechnologically. Although ferulic acid is placed excessively in plant cell wall and is considered as the preferred precursor for vanillin synthesis, the high cost of ferulic acid has diverted the search for alternative natural precursor substrates for the synthesis of natural vanillin [32]. Lignin is the major and easily attainable byproduct of biofuel and paper industry and one of the wonderful naturally available sources of aromatic compounds. Transformation of lignin as a complicated bio-polymer to less complex organic compounds has attracted great attention. Three bacterial strains viz. Aneurinibacillus aneurinilyticus, Bacillus sp. and Paenibacillus sp. showed the potential for effluent treatment of pulp and paper industry. The aromatic compounds identified in extracts of treated effluent were identified as p-cinnamic acid, ferulic acid, 3-hydroxy-4-methoxyphenol, vanillic acid, vanillin and gallic acid that indicating the transformation of lignin present in the effluent [8].


Challenges for the Enzyme Technology Appraised as the Right Substitute to Chemical Technology 


Lack of technology and interest for recycling of enzymes in biobleaching process are the major drawbacks and are unnoticed thrust areas of green technology which need dedicated efforts to find possible methods how to reuse the biocatalysts after biobleaching of pulps.  There are few reports on recycling of enzymes like cellulase for the production of ethanol from lignocellulosic biomass. Tu and Saddler [33,34] reported the concept of recycling of cellulase after organosolv-pretreatment of lignocellulose by the inclusion of surfactant like Tween 80. Cellulase can be recycled and used in succession for five hydrolysis cycles. The use of Tween 80 can save 60% of the total cellulase cost. There are several challenges before the enzyme technology is implemented to lignocellulosic materials conversion to paper products. Laccase has lower redox potential than lignin, so these biocatalysts need costly mediators for increasing their redox potential.


Mediator Requirement by Laccases for Delignification of Pulp is a Major Hurdle for Use at Industrial Scale


Veratryl alcohol and 1,2-dimethoxybenzene are nonphenolic units of lignin polymer with high (?1400 mV) redox potential. However, it has been noticed that laccase has the capability to oxidize some small compounds (redox mediators) having more redox potential than laccase itself, although this mechanism by which this happens is unclear. By the addition of such mediators, laccase shows the ability to oxidize nonphenolic lignin model components and decrease pulp kappa number to a great extent [35]. The standard redox potential range for laccase activity is usually between 500–800 mV versus normal hydrogen electrode (NHE) range which can attack only phenolic moieties comprising less than 10% of the total lignin [36]. A redox mediator could be the smallest molecule that acted as an ‘electron shuttle’ in between enzyme and substrate. Since the inclusion of ABTS as a redox mediator for pre-bleaching of pulp is cost intensive [2] at the large scale.  1-Hydroxybenzotriazol (HBT) has also been shown to inactivate laccases after some time and possesses high toxicity often at less concentration [37,38], therefore an extensive search is required to find out cost effective and nontoxic natural mediators [39].


Cost Intensive Production of Enzymes is a Setback for Recycling of Waste Paper and Delignification of Pulp


Niladevi et al. [40] applied Response Surface Methodology (RSM), a statistical method, for the enzyme production from Streptomyces psammoticus, but following this technology only 3.0 fold enzyme productions was increased in comparison to the conventional method of laccase optimization. Singh et al. [3] reported the pre-bleaching of wheat straw rich soda pulp by alkali-tolerant laccase of γ -proteobacterium. Production of this laccase was low (8 × 103 nkatL-1) with the conventional method of enzyme production (OVAT system). Later, RSM based optimization enhanced the laccase production by 9.3 fold [23]. There are several reports of laccase production enhancements available from fungi in literature, but still favorable results are not forthcoming according to the pulp and paper industries requirements. Recently, Sharma et al. [41] reported the method to avoid the step of xylanase production for delignification of pulp. Xylanase-producing Bacillus halodurans FNP 135 cells were directly tried for pre-bleaching of kraft pulp by using submerged fermentation (SmF) and Solid State Fermentation (SSF). This study concluded the potential application of B. halodurans cells for biobleaching of hardwood kraft pulp and eliminates the cost-intensive steps of enzyme production and extraction before their application in biobleaching. This method of biobleaching has a great advantage to avoid the cumbersome process of media optimization for the cost-effective production of xylanase.




After the discovery of the lignolytic enzymes, the pulp and paper industry, in particular, had great expectations with regard to implementing cell-free enzymatic delignification/ bleaching systems in its industrial processes in the foreseeable future. However, no cost-effective solution based on these inventions that are capable of fulfilling this requirement has emerged to date. All the mediated oxidoreductase systems like the LMS system (laccase-mediator system) and others have different but significant drawbacks that are related to either cost, environment, or performance and/or technical feasibility. Modern biotechnology can provide novel enzymes with improved properties under a diverse physiological condition such as broad working pH and temperature range and high redox potential laccases.




Authors are thankful to SERB/DST, Delhi, India, for providing the research funding under Fast Track Young Scientist Program (SB/FT/LS-315/2012).


Conflict of Interest


The authors declared there is no conflict of interest.




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