Lignocellulose degradation and production of lignin modifying enzymes by Schizophyllum commune IBL-06 in solid-state fermentation

Biocatalysis and Agricultural Biotechnology 6 (2016) 195–201

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Lignocellulose degradation and production of lignin modifying enzymes by Schizophyllum commune IBL-06 in solid-state fermentation Muhammad Asgher, Abdul Wahab, Muhammad Bilal n, Hafiz Muhammad Nasir Iqbal Industrial Biotechnology Laboratory, Department of Biochemistry, University of Agriculture, Faisalabad 38040, Pakistan

art ic l e i nf o

a b s t r a c t

Article history: Received 7 March 2016 Received in revised form 9 April 2016 Accepted 11 April 2016 Available online 12 April 2016

The modification of lignin is recognized as an important aspect of the successful refining of lignocellulosic biomass. Schizophyllum commune, a white rot basidiomycete was studied for ligninolytic enzymes (manganese peroxidase, lignin peroxidase and laccase) production in solid-state fermentation (SSF) of rice straw. Various physiological factors such as incubation time, culture pH, incubation temperature, C:N ratio and addition of mediators were optimized to enhance enzymes productivity. Maximum enzyme recoveries were obtained at pH, 5.0; temperature, 35 °C; C:N ratio, 20:1; mediator, MnSO4; inoculum size, 4 mL after incubation time of 144 h. The crude ligninolytic extract thus produced was used for delignification of various agro-industrial residues. The enzyme extract caused 61.7%, 47.5%, 72.3% and 67.2% lignin removal from banana stalk, corn cobs, sugarcane bagasse, and wheat straw, respectively. The optimally delignified substrate was enzymatically digested by crude cellulase extract from Trichoderma harzaianum that resulted 47.3% and 69.4% cellulose hydrolysis from the native and pretreated bagasse, respectively. The results suggested that lignocellulosic waste could be utilized as lowcost substrate for the production of enzymes which play significant role in many industrial and biotechnological sectors. & 2016 Published by Elsevier Ltd.

Keywords: Schizophyllum commune Lignocellulosic materials Ligninases Process optimization Delignification Enzymatic saccharification

1. Introduction The microbial decomposition of lignin and plant cell-wall polysaccharides has become crucial for the development of innovative biotechnological processes in various industries such as pulp and paper, textile and chemical synthesis (El-Shishtawy et al., 2015; Garcia-Torreiro et al., 2016). The removal of encrusted lignin from the lignocellulosic biomass is quite challenging to accomplish and obviously a specific feature of filamentous fungi belonging to phylum Basidiomycota (Liers et al., 2011; Munir et al., 2015). In nature, lignocellulose transformation is predominantly attributed to the synergistic action of several oxidoreductases actively secreted by wood-rotting fungi accompanying with low-molecular-mass mediators (Hatakka and Hammel, 2010). Among them are the ligninmodifying peroxidases, including manganese-dependent peroxidase (MnP, EC 1.11.1.13), lignin peroxidase (LiP, EC 1.11.1.14) and versatile peroxidase (VP, EC 1.11.1.16) (Hofrichter et al., 2010). Laccases (phenol oxidases, EC 1.10.3.2) are another group of multi-copper oxidative biocatalysts which take part in biomass de-polymerization by oxidizing a variety of phenolic compounds and aromatic diamines (lignin, melanin and humic substances) (Baldrian, 2006). n

Corresponding author. E-mail address: [email protected] (M. Bilal).

http://dx.doi.org/10.1016/j.bcab.2016.04.003 1878-8181/& 2016 Published by Elsevier Ltd.

Agricultural wastes, forestry wastes and agro-industrial residues generally accumulated in the environment have ecological disadvantages (Asgher et al., 2014). However, these lignocellulosic wastes could be harnessed as potential raw materials for economic production of high added value products such as biocatalysts, fuel ethanol, single-cell protein, organic acids, secondary metabolites and other fine chemicals that currently remains the subject of considerable attention (Iqbal and Kamal, 2012). Delignification of biomass is regarded as the most important step in this regard (Asgher et al., 2016a, 2016b). Significant efforts were being made to convert this lignocellulose either as bio-fuel or as a valuable starting material for commodity chemical synthesis (Howard et al., 2003; Garcia-Torreiro et al., 2016). Since the cost of lignocellulosic substrates play central role in determining the economy of delignification process, lot of research focus had been given to the usage of low-price substrates and, therefore screening of agricultural wastes for release of sugars. Various agro-wastes have been delignified by other researchers employing individual and co-cultures of white-rots (Asgher et al., 2013a, 2013b). The solid-state fermentation (SSF) processes have been appeared particularly suitable route for the production of enzymes by filamentous fungi since they offer natural habitats on which fungus grows better (Bilal et al., 2015). In alternative to traditional submerged fermentation (SmF), SSF present advantages of improved yields, cost competitive, easier products recovery, and lack

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of foam formation. Furthermore, due to low water contents, contamination risks were significantly eliminated and, therefore the volume of residual wastes also decreases (Yasmeen et al., 2013). The industrial scale enzyme synthesis through medium optimization and selection of appropriate growth promoting substrate plays a noteworthy role in the design of an efficient biotechnology (Moldes et al., 2004: Bilal and Asgher, 2015a, 2015b). Schizophyllum commune is a ubiquitous white-rot fungus with a worldwide distribution that can degrade complex plant biomass, including the recalcitrant lignin (Irshad and Asgher, 2011; Horisawa et al., 2015). Since the genome of Schizophyllum commune encodes an extensive catalog of genes implicated in lignocellulose degradation, its lignocellulolytic enzyme pool is expected to provide a prospective enzyme source for biotechnological applications (Zhu et al., 2016). In fact, S. commune has the most complete polysaccharide breakdown machinery of all basidiomycetes examined. This complete machinery is consistent with the wide variety of substrates that support growth of S. commune (Ohm et al., 2010). The current study was aimed to optimize the culturing conditions for enhanced lignin-modifying enzymes (LMEs) production in SSF by Schizophyllum commune IBL-06. The potential of enzyme extract for the delignification of different agricultural wastes followed by saccharification using cellulases extract from T. harzianum was also investigated.

2. Materials and methods 2.1. Agricultural waste Rice straw collected from students Research Farms, University of Agriculture, Faisalabad was chosen as the nutrient source for production of LMEs through SSF. Before use, the substrate was oven dried at 60 °C, crushed in a commercial mill (Ashraf Herbal Laboratories limited, Faisalabad) and sieved to 40 mesh particle size. 2.2. Fungal strain, medium and inoculum development S. commune IBL-06 was obtained from culture stock of the Industrial Biotechnology Laboratory, Department of Biochemistry, University of Agriculture; Faisalabad. The fungal culture was maintained on potato dextrose agar (PDA) slants for 3–5 days at pH 4.5 and 28 °C and preserved at 4 °C. Inoculum was prepared by growing the strain on a rotary shaker (150 rpm and 30 °C) in 250mL sterilized culture flask filled with 100-mL of Kirk′s basal medium with additionally 1.0% (w/v) glucose solution (sterilized through filtration). The basal medium contained; ammonium tartrate (0.22 g L 1), KH2PO4 (0.21 g L 1), MgSO4
7H2O (0.05 g L 1), CaCl2 (0.01 g L 1), Thiamine (0.001 g L 1), 10 mL Tween 80 (10%), 10 mL 100 mM veratryl alcohol and 10 mL trace-metal solution. The traced element solution was prepared by mixing the salts of CuSO4, (0.08 g L 1); NaMoO4, (0.05 g L 1), MnSO4. H20, (0.07 g L 1), ZnSO4
7H2O (0.043 g L 1) and FeSO4, (0.05 g L 1). Prior to sterilization, pH of the medium was adjusted to pH 4.5 (WTW pH-meter; InoLab pH 730). After 5–7 days of cultivation, homogeneous spore suspension (1  106–1  108 spores/mL) was attained and used as inoculum (Yasmeen et al., 2013).

(5:1, 10:1, 15:1, 20:1, 25:1) and effect of mediators (MnSO4, oxalate, ABTS, veratryl alcohol and H2O2) were optimized to achieve maximum ligninolytic enzyme production. Classical optimization strategy i.e., by varying one variable at a time and keeping the previously optimized factors at optimum level was employed for optimization study. 2.4. Production and extraction of ligninolytic enzyme The SSF was carried out in triplicate Erlenmeyer flasks (250mL) containing 5 g of rice straw pre-moistened with sterilized Kirk's basal salts medium (66% w/v) without glucose. After maintaining the medium pH to 5.0, the fermentation flasks were inoculated with 4 mL homogeneous mycelium suspension and subjected to fermentation at 35 °C for 144 h, using a C:N ratio of 20:1 (molasse:ammonium sulphate) and MnSO4 as mediator. After designated time, the fermented biomass was harvested for extracellular enzymes with 100 mL of distilled water. The extracts in the flasks were shaken (120 rpm for 30 min) followed by centrifugation at 4000 rpm for 10 min. The clear filtrate was used as crude enzyme extract for activity assays (Bilal and Asgher, 2015a, 2015b). 2.5. Ligninolytic enzyme assays Activities of all the oxidoreductases were measured spectrophotometrically (HALO-DB 20). MnP activity was specifically assayed as described previously by monitoring the formation of manganic-malonate complexes at 270 nm (Wariishi et al., 1992). A 2.6-mL of assay mixture comprised MnSO4 (1 mL; 1 mM), 1 mL of 50 mM Na-malonate buffer pH 4.5, 500 mL of H2O2 and 100 mL of crude enzyme solution. Activity of LiP was measured by the method of Tien and Kirk (1988) following the H2O2 dependent oxidation of veratryl alcohol to veratraldehyde at 25 °C. Reactive mixture (2.6 mL) contained 1 mL tartrate buffer (100 mM) of pH 3, 1 mL of 4 mM veratryl alcohol, 500 mL of H2O2 and 100 mL of enzyme aliquots. Laccase was assayed by monitoring 2, 2 azinobis (3ethylbenzthiazoline) 6 sulphonate (ABTS) oxidation in Na-malonate buffer at 436 nm (Wolfenden and Willson, 1982). A 2.1-mL of reaction mixture containing 1 mL of 50 mM Na-malonate buffer (pH 4.0), 1 mL of ABTS and 100 mL of enzyme solution. Blank contained 100 mL of distilled water instead of enzyme solution. Enzyme activities were represented in units per gram of dry substrate (U/gds), with one unit of enzyme activity defined as the amount of enzyme that catalyzed the formation of 1 μmol of corresponding products in one min under the given assay conditions. 2.6. Pretreatment with ligninolytic enzymes extract Varying volumes of ligninoytic enzymes extract containing LiP, MnP and laccase was applied at different doze levels (5, 10, 15, 20, and 25 mL) for 24 and 48 h at 35 °C. The volume of each flask containing 30 g substrate was made to 200 mL mark with 50 mM Na-malonate buffer of pH 5.0. The percentage of lignin was calculated before and after ligninolytic enzymes treatment in order to determine the enzyme efficiency and percentage lignin removal from the substrate. 2.7. Determination of lignin and percent delignification

2.3. Optimization of culture conditions for enzyme production The effect of various physicochemical parameters including incubation time (48, 96, 144, 192, 240 h), incubation pH (3, 4, 5, 6, 7), temperature (20, 25, 30, 35, 40 °C), carbon (molasses, glucose, fructose, starch) and nitrogen sources (ammonium sulphate, peptone, urea, and ammonium chloride), carbon/nitrogen ratio

Untreated and ligninolytic enzymes treated substrates were analyzed for lignin content and percent delignification according to Johnson et al. (1961), with slight modifications. The sample was dissolved in 10 mL 25% (w/v) acetyl bromide in re-distilled glacial acetic acid (GAA Z99%) by heating at 70 °C7 2 °C in a water bath for 30 min. The sample was stored in a special digester tube with a

M. Asgher et al. / Biocatalysis and Agricultural Biotechnology 6 (2016) 195–201

notched glass stopper. After 30 min, the dissolved sample was transferred into a 200 mL volumetric flask containing 5 mL mixture of acetic acid and caustic soda in 1:1 ratio. Interfering substances were removed by adding 0.2 g hydroxylamine hydrochloride. The sample was diluted to 15 mL volume with 99% acetic acid, and absorbance was read at 280 nm.

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pH 4.8). After 72 h of incubation at 37 °C in orbital shaker (150 rpm), the saccharified materials were centrifuged (at 10,000 rpm for 10 min) and aqueous solution was analyzed for cellulose (David, 1969) and glucose (Gadjil et al., 1995) determination. 2.9. Statistical analysis

2.8. Enzymatic hydrolysis (Saccharification) of delignified residues The enzymatic hydrolysis of de-lignified substrates was performed with crude cellulase extract obtained from T. harzianum under optimized growth conditions. Triplicate Erlenmeyer flasks (500 mL) containing 5% solids loading (5 g dry weight per 100 mL) were incubated with 10 mL cellulase extract and volume was maintained up to 100 mL with citrate phosphate buffer (100 mM;

All the treatments and enzyme assays were performed in triplicate, and the obtained data were subjected to analysis of variance (ANOVA) under Completely Randomized Design (CRD). Comparisons between treatment means were made by Duncan's Multiple Range Test (DMR) (Steel et al., 1997). The data was presented as mean 7S.E. (standard error) and S.E. values have been shown as Y error bars in figures.

2000

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0 MnSO4

Oxalate

ABTS

V. Alcohol

H2O2

Mediators Fig. 1. Effect of A) incubation time, B) pH C) temperature D) C:N ratios and E) mediators on ligninase production by S. commune IBL-06; MnP (

), LiP (

), Lac (

).

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3. Results and discussion Low yields of ligninolytic enzymes in most of the white-rot basidiomycetes are the leading issues delaying the implementation of these enzymes in various environmental and biotechnological applications. Generally extracellular enzymes are only produced in small quantities; therefore all the attempts being devoted to improve the production of enzymes through optimization strategies could be regarded as advantageous (Yasmeen et al., 2013). In present study, S. commune IBL-06 was used as fermentative organism for the production of ligninolytic enzymes in SSF and different experiments were carried out to optimize different parameters such as incubation time, pH, temperature, and supplementation of carbon and nitrogen sources, C:N ratio, and effect of mediators. 3.1. Optimization studies 3.1.1. Enzyme secretion over the incubation period The time course of ligninolytic enzyme production by S. commune in SSF using rice straw as a cheap substrate was investigated and results are shown in Fig. 1A. It was observed that enzyme yield was significantly (P o0.05) affected at different incubation time. Apparently, maximum enzyme titers (MnP, 1846.7 U/gds; LiP, 1347.2 U/gds and Lac, 316.28 U/gds) were achieved after 144 h. Beyond this optimal cultivation period, the enzyme activity started to decline that might be attributed to the medium nutrients depletion or enzyme deactivation and/or denaturation caused by the interaction with other components in the medium (Munir et al., 2015). The results were in agreement with Irshad and Asgher (2011) who recorded maximum enzyme activities from S. commune after 192 h using banana stalk. While, Silva et al. (2014) encountered optimum enzyme production by Tremates villosa after 15 days. Generally, the optimum growth time for most of the WRF strains varies between 4 and 10 days (Giardina et al., 2000). The genetic variations among microbial strains as well as nature and composition of the substrates have been correlated with varying expression of LMEs after different fermentation durations (Giardina et al., 2000; Yasmeen et al., 2013). 3.1.2. Effect of pH The pH is an important factor that influences the microbial growth and enzyme production during SSF. Filamentous fungi possess a unique optimum pH, as well as a pH range for its activity and growth. These are believed to thrive over a broader pH range in solid-state culture since the solid substrate offer a better buffering capacity (Sun and Xu, 2008). To determine the effect of pH on ligninases production, rice straw was moistened with kirks nutrient medium of varying pH (3.0–7.0). The data in Fig. 1B reveal that S. commune showed capability of producing enzyme in all the tested pHs with optimum enzyme activity at pH 5.0. Ligninases produced by S. commune NI-07 in submerged fermentation recorded an optimal pH from 4.43 to 4.46, and further rise in pH deactivated enzyme protein (Kumar et al., 2015). The optimal pH of purified laccase produced by S. commune IBL-06 strain in solid culture on banana stalks was reported as 6.0 for ABTS oxidation (Irshad et al., 2011). The maximum ligninolytic activities by T. versicolor were detected at pH 4.0 (Wang et al., 2014; Carabajal et al., 2013), and marked reduction in enzyme activity by P. ostreatus was observed at extreme pH values of 3.0 and 8.0 (Fernandez-Fueyo et al., 2014). 3.1.3. Effect of incubation temperature Temperature is another very imperative parameter for extracellular enzyme production. Different temperatures ranging from 20 to 40 °C were maintained in SSF system to decipher the

optimum temperature for enhanced ligninases production. Variations in incubation temperature had significant (P o0.05) effect on enzyme yield, and peaked ligninolytic activities by S. commune were noted when fermentation was processed at 35 °C (Fig. 1C). Earlier reports verified 30 °C as best temperature for ligninases production by T. versicolor using rice straw (Iqbal et al., 2012). S. commune NI-07 laccase showed optimal activity at a temperature of 30 °C, losing over 50% activity with a variation of 75 °C (Kumar et al., 2015). Fernandez-Fueyo et al. (2014) emphasized that the activities of different peroxidases, particularly MnP by P. ostreatus were significantly dropped at extreme temperature (above 37 °C). Enzyme deactivation might be the contributing factor that led to the decreased enzyme activity at higher temperatures. However, expression of ligninolytic enzymes by white-rot fungi (WRF) is supposed to be species and strain dependent (Cupul et al., 2014). 3.1.4. Effect of carbon and nitrogen sources Supplementation of carbon (1.0%) and nitrogen (0.2%) sources in different combinations (Table 1) in the SSF medium of rice straw was studied to appraise their stimulatory and/or inhibitory effect on LMEs production. It was observed that addition of carbon and nitrogen sources contributed stimulatory effect on enzyme activities and optimum enzyme yield was achieved with combination of molasses (C source) and ammonium sulphate (N source). At optimized carbon and nitrogen combination, the enzyme extract contained 1964.32, 1487.28 and 431.17 U/gds of MnP, LiP and Lac, respectively. In order to produce higher LMEs, Irshad and Asgher (2011) suggested glucose and yeast extract as best carbon and nitrogen additives for WRF strains, including P. ostreatus, S. commune and T. versicolor. In comparison to urea, ammonium sulphate, and ammonium tartrate, Hou et al., (2004) achieved 1.99 and 1.79 fold increased activity of laccase by P. ostreatus using peptone and yeast extract as nitrogen sources, respectively. Similarly, Mishra and Kumar, (2007) also verified that yeast extract was preferred inorganic nitrogen sources for enhancement of ligninolytic enzyme production by P. ostreatus MTCC1804. Among different nitrogen sources tested, peptone in medium greatly promoted the production of MnP and laccase production by Thalictrum pubescens and Bjerkandera sp. strain BOS 55, respectively (Galhaup et al., 2002). Table 1 Activities of ligninases produced by S. commune IBL-06 with varying carbon and nitrogen sources. Enzyme activities (U/gds) Nitrogen sources (0.2%)

Carbon sources (1%) Molasses (C1) Glucose (C2) Fructose (C3) Starch (C4)

Ammonium sulphate (N1)

MnP 1964.32 LiP 1487.28 Lac 431.17

1106.31 1200.09 315.28

1633.53 1317.63 293.33

1399.31 1188.77 234.72

Urea (N3)

MnP 1690.75 LiP 1068.82 Lac 260.07

1210.02 1237.63 263.39

1530.68 918.28 311.39

1555.75 1054.54 247.78

Urea (N3)

MnP LiP Lac

1106.31 1104.3 256.39

1777.87 1106.03 222.24

1826.27 996.24 315.27

1337.08 1048.39 257.83

Ammonium chloride (N4)

MnP 1452.03 LiP 1231.41 Lac 384.17

1246.33 1241.93 309.44

1143.47 1225.81 264.44

1406.22 1205.38 281.07

M. Asgher et al. / Biocatalysis and Agricultural Biotechnology 6 (2016) 195–201

3.1.6. Effect of mediators Effect of different mediators (MnSO4, Oxalate, ABTS, Veratryl alcohol, H2O2) in the pre-optimized fermentation medium of rice straw for maximum production of ligninases by S. commune was investigated, and responses are shown in Fig. 1E. Mediators increase the surface area for growth of microorganism and subsequently affect enzyme production (Camarero et al., 2004; Batool et al., 2013). All mediators exhibited stimulatory effect on the ligninase production by S. commune but their effects on different enzyme activities were highly variable. Noticeably, MnSO4 exerted most stimulatory effect on ligninolytic enzyme production. In support to our study, Urek and Pazarlioglu (2008) recorded the significant increase in MnP enzyme activity by adding MnSO4 in fermentation medium. 3.2. Delignification of plant residues The crude ligninolytic enzyme extract produced under optimized conditions of pH 5.0, temperature 35 °C, C:N ratio 20:1, mediator MnSO4, inoculum size 4 mL after 144 h of incubation time was used for enzymatic delignification of banana stalk (BS), corncobs (CC), sugarcane bagasse (SCB), and wheat straw (WS) for different time periods (24 and 48 h). The extent of lignin removal (%) for selected plant residues after the treatment with ligninolytic extract is presented in Fig. 2. A marked reduction in lignin contents of all the plant residues was recorded with a maximum delignification of 72.3 (%) in SCB with the dose level of 25 mL of ligninolytic extract after 48 h. The lignin degradation trend in other substrates was also promising. By increasing the time from 24 to 48 h, the enhancement in lignin removal was observed. Previously, various authors accomplished considerable lignin disruption by employing ligninolytic enzymes as shown in Table 2. Although all cited reports showed good results, the lignin removal achieved in this work using ligninolytic enzyme was comparatively higher. A huge variety of pretreatment methodologies have been previously evaluated in literature to delignify various agro-industrial residues for fuel ethanol production (Singh et al., 2014). Acid and/ or alkali pre-treatment of substrates have been regarded as the current leading pretreatment technologies; however, they substantially add to the overall production cost and also contributes to environmental issues. In contrast, the use of crude ligninolytic enzyme extracts could be a novel approach that can help developing a cost-effective and environmentally acceptable technology (Zhu et al., 2016). The practical exploitation of cheap crude enzyme extract can be more proficient than isolated enzymes in cases, where high substrate specificity is not required (Mai et al., 2004). Crude culture filtrates from WRF presents several additional advantages over the use of purified enzymes. The presence of proteins, mediators or other factors in the medium may stabilize crude enzymes and mediate the action of these enzymes (Asgher et al., 2013a, 2013b). The process of biomass delignification is very complicated,

24 h 48 h 24 h 48 h 24 h 48 h 24 h 48 h

70 60

Lignin removal (%)

3.1.5. Effect of carbon:nitrogen ratio After selection of best carbon and nitrogen sources, the effect of varying C:N ratios (5:1, 10:1, 15:1, 20:1, 25:1) on ligninase production by S. commune was investigated, and responses are shown in Fig. 1D. Maximum enzyme production was recovered in the medium with 20:1 as C: N ratio, showing activities of MnP (1979.4 U/gds), LiP (1498.1 U/gds) and laccase (446.39 U/gds). Previously, Xiaoping and Xin (2008) described the effect of different carbon to nitrogen (C/N) ratios and levels. At low C/N ratio, the fungus was carbon starved and did not produce extracellular enzymes, whereas under excess C/N ratio, relatively large amounts of enzyme activities were achieved from fermented culture.

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50 40 30 20 10 0 5

10

15

20

25

Ligninolytic extract (mL) Fig. 2. Lignin removal pattern of different agricultural wastes (black color linesbanana stalk, blue color lines-corncobs, red color lines-sugarcane bagasse, and olive color lines-wheat straw) by crude enzymatic extract of S. commune. The optimal conditions for ligninolytic enzyme production were pH 5.0, temperature 35 °C, C: N ratio 20:1, mediator MnSO4, inoculum size 4 mL and incubation time 144 h. Table 2 Comparison of enzymatic delignification of various fungal pretreated substrates by ligninolytic enzyme. Substrate

Microbial source

% lignin removal

Reference

Wheat straw

Tremetes versicolor Daedalea flavida Daedalea squalens Phanerochaete chrysosporium Phlebia fascicularia Phlebia floridensis Phlebia radiata Pleurotus ostreatus Tremetes species

12.3 18.8 16.4 17.2

Arora et al. (2002)

25.2 22.8 18.5 41 10.2–50.1

Pleurotus ostreatus

33.6

Taniguchi et al. (2005) Moniruzzaman and Ono (2012) Asgher et al. (2013a)

Ganoderma lucidum Pleurotus florida

39.6 7.91

Asgher et al. (2013b) Deswal et al. (2013)

Coriolopsis caperata Ganoderma sp. Trametes pubescens

5.84 5.58 5.9

Tremetes versicolor Tremetes villosa

34.7 35.05

Knezevic et al. (2013a, 2013b) Wang et al. (2014) Silva et al. (2014)

63.11 39.61 61.7

Present study

47.5 63.6

Present study Present study

58.6

Present study

Rice straw Wood biomass/ fiber Sugarcane bagasse Wheat straw Sugarcane bagasse

Oak sawdust Rice straw Sugarcane bagasse Sisal fiber Coconut shell Banana stalk Corncobs Sugarcane bagasse Wheat straw

Shizophyllum commune

involving the synergistic action of laccases and peroxidases. Although oxidoreductases are directly involved in lignin modification, their production level is not essentially correlated to the extent of losses of wood components. Lignin bio-degradation could occur until the latest phases of fermentation, even if the highest enzymatic activity is achieved earlier (Knezevic et al., 2013a, 2013b). The effective degradation of phenolic/aromatic

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60

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Fig. 3. Saccharification and glucose yield of native and ligninolytic enzyme treated sugarcane bagasse by crude cellulase extract from T. harzianum.

compounds during enzymatic pre-treatment also reduces the toxicity of the medium for subsequent saccharification and fermentation process (Sitarz et al., 2013). Treatment of agricultural waste i.e., wheat straw with ligninolytic enzyme extracts, obtained from the WRF, T. versicolor, Bjerkandera adusta and Fomes fomentarius, versus the non-treated straw control showed an increase in digestibility of treated straw. Supplementation of different doses of a mixture of three exogenous ligninolytic enzymes, Lac, LiP and MnP failed to elicit any changes in either the cell wall components or the in-vitro digestibility of straw. However, treating the straw with the same enzymes for 24 h at a ratio of 2: 5 (v/ w) showed marked reduction in these cell wall components as well as in the in-vitro digestibility (Sridhar et al., 2014). The results indicated that the crude ligninolytic enzyme extract obtained under the optimized conditions by S. commune IBL-06 could be a possible alternative to promote lignin removal in plant biomass. The process of enzymatic delignification may lead to better results when using optimized reaction conditions, such as time, temperature, enzyme concentration, stirring, and type of residues, which can be determined in further studies. 3.3. Enzymatic saccharification Beside bio-delignification, the availability of the cellulose is also an important criterion for evaluating the efficiency of biological pretreatment; which in turn could provide higher amount of cellulose (carbohydrate) for enzymatic saccharification (McKee et al., 2016). After delignification of selected lignocellulsoic substrates, only the best pretreated biomass (SCB) was further subjected to enzymatic digestion by the crude cellulases (β 1, 4 endoglucanase, 53.5 71.24 U/mL; β 1, 4 exoglucanase, 41.371.31 U/mL; β 1, 4 glucosidase, 46.8 71.43 U/mL) from T. harzaianum. Incubation of both native and ligninolytic pretreated sugarcane bagasse with cellulase extract resulted in glucose recoveries during saccharification process. However, higher cellulose hydrolysis (69.4%) was obtained with ligninolytic pretreated substrate in comparison to untreated SCB (47.3%). Likewise, about 37.9% more glucose yields were achieved from enzymatic hydrolysis of SCB pretreated with ligninases in comparison to yields from untreated SCB (Fig. 3). The enhanced enzymatic saccharification in ligninolytic treated substrate may be attributed to the partial lignin degradation, which may be responsible for preventing the penetration of cellulases (Dias et al., 2010; Zhu et al., 2016). Moreover, the improvement in enzymatic hydrolysis could be due to the improvement in porosity of plant material because of biological pretreatment with fungal ligninolytic enzymes to an

increase in the initial adsorption of cellulase to cellulose (Horisawa et al., 2015). The cellulose hydrolysis and concomitant glucose liberation obtained after 72 h was in agreement with previous reports for other lignocellulosics (Asgher et al., 2013a, 2013b). Similarly, Kadarmoidheen et al. (2012) reported highest 51.59% saccharification by Trichoderma viride followed by 40.06% and 28.75% with Aspergillus niger and Fusarium oxysporum, respectively. The currently investigated ligninolytic approach resulted commendable conversion of cellulose to glucose under mild and eco-friendly processing conditions, which are safer and stable than various acidic, alkali or other pretreatment methods.

4. Conclusions In this work, lignin-modifying enzyme production by S. commune IBL-06 was successfully enhanced by media optimization, using inexpensive agro-industrial waste rice straw as substrate. The crude enzyme extract from S. commune was capable of reducing the lignin content by 61.7%, 47.5%, 72.3% and 67.2% of banana stalk, corncobs, sugarcane bagasse, and wheat straw, respectively, after 48 h. Further studies are currently underway to optimize the reaction conditions in order to improve the lignin degradation efficiency in these plant residues and others commonly found in Pakistan.

Acknowledgment The present study was a part of the research project focused on development of ligninolytic enzymes for industrial applications. The financial support for this project by Higher Education Commission (HEC) Islamabad, Pakistan (Grant No.: 20-1652/R&D/2011) is thankfully acknowledged.

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