Solid state bioconversion of lignocellulosic residues by Inonotus obliquus for production of cellulolytic enzymes and saccharification

Accepted Manuscript Solid state bioconversion of lignocellulosic residues by Inonotus obliquus for production of cellulolytic enzymes and saccharification Xiangqun Xu, Mengmeng Lin, Qiang Zang, Song Shi PII: DOI: Reference:

S0960-8524(17)31506-7 http://dx.doi.org/10.1016/j.biortech.2017.08.192 BITE 18801

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Bioresource Technology

Received Date: Revised Date: Accepted Date:

12 July 2017 26 August 2017 29 August 2017

Please cite this article as: Xu, X., Lin, M., Zang, Q., Shi, S., Solid state bioconversion of lignocellulosic residues by Inonotus obliquus for production of cellulolytic enzymes and saccharification, Bioresource Technology (2017), doi: http://dx.doi.org/10.1016/j.biortech.2017.08.192

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Solid state bioconversion of lignocellulosic residues by Inonotus obliquus for production of cellulolytic enzymes and saccharification

Xiangqun Xu*, Mengmeng Lin, Qiang Zang, Song Shi College of Life Sciences, Zhejiang Sci-Tech University Email: [email protected]

Abstract White rot fungi have been usually considered for lignin degradation and ligninolytic enzyme production. To understand whether the white rot fungus Inonotus obliquus was able to produce highly efficient cellulase system, the production of cellulolytic enzyme cocktails was optimized under solid state fermentation. The activities of CMCase, FPase, and β-glucosidase reached their maximum of 27.15 IU/g, 3.16 IU/g and 2.53 IU/g using wheat bran at 40 (v/w) inoculum level, initial pH of 6.0 and substrate-moisture ratio of 1:2.5, respectively. The enzyme cocktail exhibited promising properties in terms of high catalytic activity at 40-60 °C and at pH 3.0 - 4.5, indicating that the cellulolytic enzymes represent thermophilic and acidophilic characteristics. Saccharification of raw wheat straw and rice straw by the cellulolytic enzyme cocktail sampled on Day 12 resulted in the release of reducing sugar of 130.24 mg/g and 125.36 mg/g of substrate after 48 h of hydrolysis, respectively. Keywords: Inonotus obliquus; cellulolytic enzymes; ligninolytic enzyme; solid state fermentation; optimal conditions; saccharification

1. Introduction Lignocellulosic biomass has been portrayed as potential low cost raw materials for the production of chemicals and biofuels by the degradation and conversion of the three major chemical components: lignin, hemicellulose and cellulose. In light of the high efficiency and environment protection, the degradation catalyzed by cellulases is a very helpful way to hydrolyze cellulose thoroughly (Yadav, 2017). Use of cellulolytic enzyme cocktails of fungal origin is one of the most promising ways to convert the cellulose and hemicellulose into the reducing sugars such as glucose and xylose for industrial utilizations, due to the fungi’s capability to synthesize the essential components of highly efficient cellulase system (Yadav, 2017). Currently, expensive cellulase production process, poor stability and low efficiency of cellulolytic enzymes still present a major obstacle in commercial and industrial applications. Thus, explorations of highly efficient cellulases or reducing the cost of large-scale production of cellulases is extremely pivotal for the overall process economics for bioconversion of lignocellulosic biomass into value-added products. Recently, a large number of microorganisms such as bacteria, actinomycetes and fungi have been recognized for their ability to hydrolyze lignocellulosic materials. The most commonly used microbes for the production of hydrolytic enzymes are Pseudomonas, Clostridium, Bacillus, Aspergillus, Trichoderma and Penicillium (Yadav, 2017). Among fungi, soft rot fungi, namely Trichoderma reesei and Aspergillus niger (Xue et al., 2017), and white rot fungi Phanerochaete chrysosporium (Manavalan et al.,

2015) have been extensively investigated for cellulase production. According to the previous studies, white rot fungi is one kind of several promising fungi in regards to biomass deconstruction and delignification, due to their capability to synthesize cellulolytic, hemicellulolytic and ligninolytic enzymes. The oxidative ligninolytic enzymes include lignin peroxidase (LiP), manganese peroxidase (MnP) and laccase (Lac) (Manavalan, et al. 2015). Recently, the authors reported that the white rot basidiomycete Inonotus obliquus was able to efficiently degrade both the lignin and crystalline cellulose of wheat straw, rice straw, and corn stover under submerged fermentation (Xu et al., 2017). However, no studies have been reported on the design of an optimized cocktail of cellulases isolated from I. obliquus. No information is available about saccharification using the cellulolytic enzyme cocktails from the white rot fungus either. Thus, the objective of the present study was to determine various process parameters for the production of cellulolytic enzyme cocktails using I. obliquus under solid state fermentation (SSF). The enzyme cocktail obtained under the optimal conditions was characterized in terms of its optimum pH and temperature, and thermal stability. The application of the enzyme cocktails in hydrolysis of raw wheat straw and rice straw was conducted. The cellulolytic [carboxymethyl cellulase (CMCase), filter paper cellulase (FPase) and β-glucosidase] and ligninolytic (MnP, LiP and Lac) enzymes on different time points during fermentation were investigated. The enzyme activity - dependent saccharification efficiency was discussed.

2. Materials and Methods 2.1. Lignocellulosic residues Different lignocellulosic residues were screened as carbon sources for the fungus growth. Wheat bran (WB), wheat straw (WS), rice straw (RS), peanut shell (PS), sugarcane bagasse (SB), cassava peel (CP), birch branch (BI) and beech branch (BE) with a diameter of 0.4-0.6 cm were obtained from local farms of Shandong (WB, WS), Jiangxi (RS), Zhejiang (PS), Heilongjiang (BI, BE) provinces and Guanxi Zhuang autonomous region (SB, CP), China. The residues were first dried and, except for wheat bran, chopped into small pieces by a chopper ground to smaller particles in a hammer mill (FW135, TianJin, China), and then separated through a 20 mesh sieve. 2.2. Microorganism and culture conditions I. obliquus purchased from Centraalbureau voor Schimmelcultures, the Netherlands was grown and maintained on malt extract agar (MEA) composed of (g/L): malt extract 30.0, peptone 3 and agar 15.0 (pH 5.5) at 28 °C. The fungal cultures were maintained by periodical subculturing on MEA at 28 °C and stored at 4 °C. 2.3. Production of cellulases under SSF SSF was carried out in 250-mL Erlenmeyer flasks, each containing 5.0 g wheat bran or the other lignocellulosic residues moistened with 5 mL mineral salt solution containing (g/L): (NH4)2SO4 1.7, K2HPO4 2.0, CaCl2 0.3, peptone 1.0, MgSO4 0.3, FeSO4 0.005, MnSO4 0.002, ZnSO4.7H2O 0.0016, and CoCl2 0.0014, and 1 mL Tween-80 to attain the final substrate-to-moisture ratio of 1:1 (Maceno et al., 2016). The

flasks were sterilized by autoclaving at 121 °C (20 psi), and thereafter cooled to room temperature and inoculated with 2 mL spore suspension (106–107 spores/mL) from the 3-day-old fungus and cultured for 7 day at 28 °C with shaking at 150 rpm on a rotary shaker. After 7-day fermentation, 50 mL sodium citrate buffer (50 mM, pH 5.0) was added in each flask and the contents were mixed properly by shaking at 28 °C for 1 h at 150 rpm to let enzymes release completely, and then filtered through Whatman No.1 paper. The liquid fraction was assayed for enzyme activity (Soni et al., 2010). 2.4. Optimization of cellulase production under SSF Various process variables are aspects to be reckoned with in the fermentation, affecting cellulase production from I. obliquus under SSF. Therefore, different carbon sources, initial pH (3.5 - 9.0), substrate to moisture ratio (1:1 - 1:4) and inoculum size (v/w) were investigated. Finally, an experiment with optimal conditions of cellulase production was planned to observe the production of enzymes over time. 2.5. Characterization of cellulolytic enzyme cocktail The activities of CMCase, FPase, and β-glucosidase (using supernatants produced under the optimal SSF production conditions) were studied by varying the pH of reaction mixtures using different reaction buffers including acetate buffer (pH 3.0 - 7.0) and phosphate buffer (pH 7.5 - 8.0) at 50 mM concentration. The effects of temperature on the enzyme activities were obtained by assaying the activities at different reaction temperatures ranging from 30 to 80 °C. The thermostability profiles were studied by incubating the enzyme cocktail in 50 mM acetate buffer (pH 5.0) at 50 °C and 55 °C for

96 h and the residual activities were determined at regular intervals of time. The half lives of the enzyme cocktail were calculated by using the following equation (Singhal et al., 2012): X = X0 (1/2) t/g logX = logX0 – [(log 2)/g]t where X is residual activity, X0 is initial activity, t is time and g is half-life. 2.6. Enzymatic saccharification of raw wheat straw and rice straw Enzymatic saccharification was performed in 100-mL Erlenmeyer flasks. Each reaction mixture contained 1 g of raw wheat straw or rice straw in size of less than 3 mm and 10 mL of acetate buffer (50 mM, pH 5.0), containing 0.005% (w/v) sodium azide and the enzyme cocktail obtained on Day 6, 8, 10 and 12 was loaded for 5 FPU per gram of raw biomass. The reaction condition was at 50 °C and 150 rpm for 48 h. Samples were taken at 12, 24, 36 and 48 h, centrifuged for 10 min and the supernatant was analyzed for total reducing sugars released. Yield of reducing sugars was calculated as follows (Xue et al., 2017; Raghuwanshi et al., 2014): u ing sug

i



u ing sug

s

2.7. Analysis of chemical compositions of wheat straw and rice straw The cellulose, hemicellulose and lignin contents of raw wheat straw and rice straw were determined by the method of (van Soest, 1963). The loss of various components after enzymatic saccharification was calculated as follow:

2.8. Determination of cellulolytic and ligninolytic enzyme activity CMCase activity was assayed by measuring the release of reducing sugars in a reaction mixture containing 1 mL of enzyme cocktail and 1 mL of 2% (w/v) of CMC solution in 50 mM acetate buffer (pH 5.0) incubated at 50 °C for a period of 30 min. Released reducing sugar was estimated by the 3,5- dinitrosalicylic acid (DNS) method as glucose equivalent (Miller, 1959). FPase activity was assayed by measuring the release of reducing sugars in a reaction mixture containing Whatman No.1 filter paper (1× 6 cm ≈ 50.0 mg ) as the substrate in 50 mM acetate buffer (pH 5.0) incubated at 50 °C for 60 min and the reducing sugar was determined by the DNS method at 540 nm as glucose equivalent. One unit of cellulase activity was defined by the formation 1 μm

fgu s

quiv

ns

s

p

minu un

ss

n i i ns (Kovács et

al., 2009). β-Glucosidase assay was carried out in the reaction mixture (1 mL) containing 5 mM 4-nitrophenyl β-D-glucopyranoside (pNPG) in 50 mM acetate buffer pH 5

n

μL appropriately diluted enzyme solution. Incubation was carried out

at 50 °C for 10 min. Reaction was terminated by the addition of 2 mL Na 2CO3 (1 mol/L). After cooling, the sample was diluted with 10 mL distilled water and the liberated p-nitrophenol (pNP) was measured at 405 nm. Unit of enzyme activity was expressed by the enzyme that produced 1 mol of pNP per minute under the assay conditions (Kovács et al., 2009). LiP activity was measured by determining the oxidation rate of veratryl alcohol to veratraldehyde at 30 °C, and 1 mmol veratraldehyde formed per minute was defined as

the enzyme unit. The standard reaction mixture consisted of 2.7 mL of 0.2 M sodium tartrate buffer (pH 5.0),

mL f

mM v

,

μL f 2 mM

g n

peroxide solution and 0.1 mL of undiluted supernatant. The reaction was initiated by adding hydrogen peroxide and the change in absorbance was monitored at 310 nm (Pinto et al., 2012). MnP activity was determined based on the oxidation of Mn2+ to Mn3+ and the enzyme unit was defined as the amount of enzyme that oxidized 1 mmol MnSO4 per minute. MnSO4 (0.1 mL, 40 mmol/L) was added into 3.4 mL of sodium tartrate solution (50 mmol/L, pH 5.0) and 0.4 mL of appropriately diluted supernatant. The reaction was initiated by adding 0.1 mL of H2O2 (1.6 mmol/L) at 30 °C. The reaction was initiated by adding hydrogen peroxide and the change in absorbance was monitored at 240 nm (Xu et al., 2017). Lac activity was determined by oxidation of ABTS [2, 2-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid)] at 420 nm. The reaction mixture (3 mL) was comprised of 0.1 mL of sample, 0.2 mL of 0.5 mmol ABTS and 2.7 mL acetic acid buffer (50 mmol/L, pH 4.5) at 30 °C (Kumari and Das, 2016). One unit of activity is defined as the amount of enzyme which leads to the transformation of 1 μm

su s

p

minu

The experiments were performed using three replicates.

The data presented in the figures correspond to mean values with standard errors. 3. Results and Discussion 3.1. Optimization of medium conditions for cellulase production by I. obliquus 3.1.1. Screening of lignocellulosic substrates Selecting a suitable carbon source for I. obliquus growth is important for producing

its enzymes. The fungus was cultured under SSF using birch branch (BI), beech branch (BE), rice straw (RS), wheat straw (WS), wheat bran (WB), sugarcane bagasse (SB), cassava peel (CP) and peanut shell (PS) as lignocellulosic substrates, respectively. Fig. 1 shows the production profiles of CMCase, FPase and β-glucosidase activities sampled on Day 7. Of all the lignocellulosic residues evaluated, WB caused the maximum production of CMCase (17.66 ± 0.36 IU/g) and β-glucosidase (1.71 ± 0.01 IU/g), and SB resulted in the maximum FPase (5.55 ± 0.01 IU/g). A comparison of cellulase production by I. obliquus with past studies suggested that I. obliquus is a novel promising candidate for the production of cellulolytic enzyme systems. The CMCase activity in the cultures of WB was 1.72-fold higher than that with T. asperellum RCK2011 (Raghuwansh et al., 2014) using WB as a substrate and almost 1.35-fold higher than that produced in WB with fungus T. asperellum SR7 (Raghuwansh et al., 2014). The FPase activity in the culture of WB was 1.11-fold higher than that with T.asperellum RCK2011 on WB (Raghuwansh et al., 2014). 3.1.2. Determination of inoculum level The production of cellulosic enzymes in SSF on Day 7 using WB as a carbon source with different inoculum level was investigated to improve enzyme production. The maximum activities of CMCase (19.83 ± 0.18 IU/g), FPase (2.62 ± 0.08 IU/g) and β-glucosidase (2.53 ± 0.01 IU/g) achieved at 40 (v/w) inoculum level (Fig. 2a). These data are in accordance with the maximum CMCase and FPase productivity from a mutant of Trichoderma asperellum RCK2011 at 40% inoculum size (Raghuwanshi et

al., 2014). Inoculum size presented both benefits and challenges to cellulase production. Low amount of inoculum adversely affected the production of cellulases by I. obliquus due to low mycelia biomass. However, larger quantity of inoculum attributed to the enormous consumption of nutrients for the growth of the cells in a short time, thus the fungus suffered from malnutrition and influenced the enzyme production during late-stage fermentation. It was particularly evident that proper inoculum size sharply promoted the enzyme synthesis. 3.1.3. Determination of initial pH Initial pH is a principal process factor that affects cellulolytic enzyme production performance (Raghuwanshi et al., 2014). The cellulase production by I. obliquus in SSF on Day 7 using WB as a carbon source at 40 (v/w) inoculum level was tested at different pH ranging from 3.5 to 9.0 (Fig. 2b). The results demonstrated that I. obliquus was able to grow and produce the cellulases in a wide range of pHs. The fungus secreted maximum CMCase (20.72 ± 0.12 IU/g) and β-glucosidase (2.08 ±0.13 IU/g) at pH 6.0, whereas maximum FPase (3.21 ± 0.11 IU/g) production was observed at initial pH 4.0. The optimum pH in a range of 4.0 - 6.0 has been also reported for cellulase production in various fungi. In a study by Falkoski et al. (2013), a pH range starting from 2.0 to 7.0 was investigated and among these, pH 4.0 was optimized for the cellluase production of Chrysoporthe cubensis LPF-1. Trivedi et al., (2015) reported that a maximum cellulase production was also achieved at pH 4.0 using Cladosporium sphaerospermum. Deswal et al., (2011) reported that Fomitopsis sp.RCK2010 cellulase

production was the highest at pH 5.5. The selection of the optimal pH (6) was on the sis f

CMC n β-glucosidase activities, which play a key role in lignocellulose

saccharification (Yadav, 2017). The pH was a determining factor of enzyme activity. It directly affected the charge of the cell membrane, thus affecting the permeability of the cell membrane, and ultimately affected the secretion of cellulases from cell into extracellular space (Chen & Liang, 2015). The results demonstrated that the optimal pH (6) was the best condition for the fungus to produce these enzymes. 3.1.4. Determination of substrate to moisture ratio Cellulolytic enzyme production depends greatly on the substrate to moisture ratio. The moisture requirements vary with microorganisms and substrates, affecting both the microbial growth and secondary metabolism (Deswal et al., 2011). The production of cellulases by I. obliquus in SSF on Day 7 using WB at 40 (v/w) inoculum level and at initial pH of 6.0 was tested in a substrate-moisture ratio ranging from 1:1 to 1:4 (Fig. 2c). The substrate-moisture ratio of 1:2.5 was the best one suited for the production of CMCase and FPase revealing the yields of 25.91 ± 0.42 IU/g and 3.30 ± 0.10 IU/g. β-glucosidase of 1.85 ± 0.28 IU/g was observed at a substrate-moisture ratio of 1:1.5. These results are in accordance with cellulase produced through SSF from T. asperellum RCK2011, where the best suited substrate-moisture ratio was 1:2.5, with activity of 1.56–7.08 IU/g (Raghuwanshi et al., 2014). The substrate-moisture ratio of 1:1.5 was reported for the maximum activity in the case of Aspergillus sp. S4B2F (Soni et al., 2010). The substrate with high moisture contents presented a barrier to oxygen

transfer that in turn created an unfavorable environment for the cell growth and the enzyme production. 3.2. Time courses of cellulolytic and ligninolytic enzyme activity under the optimal conditions Fig. 3 shows

im

us s f

u

i

CMC s , FP s

n β-glucosidase) (Fig.

3a) and ligninolytic (MnP, LiP and Lac) (Fig. 3b) enzyme activities during fermentation under the optimal conditions. T

ivi i s f CMC s , FP s

n β-glucosidase

increased with the fermentation time with some fluctuations. The highest activities of CMCase and FPase (27.15 ± 1.17 IU/g and 3.16 ± 0.06 IU/g, respectively) achieved on Day 10, and the m ximum β-glucosidase activity (2.53 ± 0.01 IU/g) was observed on Day 12. However, under un-optimized conditions [wheat bran 5.0 g, initial pH 5.5, inoculum size 40% (v/w) and substrate-moisture ratio of 1:1], the maximum CMCase activity of 16.16 ± 1.29 IU/g, FPase activity of 2.16 ± 0.24 IU/g and β-glucosidase activity of 1.77 ± 0.13 IU/g were obtained on Day 10, Day 9 and Day 12, respectively. Therefore, optimization of process parameters resulted in 1.68-fold, 1.46-fold and 1.43-f

in CMC s , FP s

n β-Glucosidase production in SSF using I. obliquus,

respectively (Fig. 3a). It is noted that the maximum FPase production was not same as the result in the section 3.1.4. It could be explained by the relative complex process of cellulase production. The process parameters affect cellulase production including pH, incubation time, inoculums concentration, and the substrate-moisture ratio. The difference in FPase activity between the two series experiments may be caused by the

alteration in biological activity of the fungus in a small range and measurement error, which could be considered within the allowed error range. The phenomenon occurred in a previous study as well (Salgado et al., 2015). A comparison of cellulase production by I. obliquus with that by the other fungi showed that I. obliquus is a novel promising candidate for production of cellulolytic nz m s s ms T

p

u i n f β-glucosidase by Aspergillus uvarum in solid-state

cultivation using exhausted grape marc with olive pomace at ratio of 3:1 was studied with an initial water content of 75% (w/w) at 29 °C. The maximum β-glucosidase activity of 1.12 ± 0.09 U/g was obtained after 8 days of cultivation (Salgado et al., 2015). The m ximum p

u i n f CMC s , FP s

n β-Glucosidase (10.25, 1.60

and 6.32 IU/g, respectively) was obtained in SSF of wheat bran by wild strain T. asperellum RCK2011 under the optimized conditions (Raghuwanshi et al., 2014). T. reesei Rut C-30 that was engineered from wild strain T. reesei QM 9414 produced the maximum CMCase, FPase n β-Glucosidase (68.57 ± 3.56, 22.89 ± 0.69 and 13.58 ± 0.56 IU/g, respectively) when it was cultured on wheat bran after 4 days (Dhillon et al., 2011). It is possible to further improve the ability of cellulase production by the I. obliquus strain by genetic engineering means. T

m ximum FP s

n β-Glucosidase

activities (13.57 IU/g and 21.69IU/g, respectively) were obtained with A. niger BC-1 using wheat bran as a substrate after 4 days, and the highest CMCase activity of 48.22 IU/g was observed on Day 5 (Dhillon et al., 2011). Although working with other microorganisms (A. niger, T. reseei) the levels of cellulase enzymes produced were

significantly higher than those reported in this work, white rot fungus I. obliquus was able to produce high-activity-level ligninolytic enzymes as well (Fig. 3b), while soft rot fungus T. reesei and A. niger could not achieve. Lignin removal by ligninolytic enzymes is crucial for biological pretreatment and saccharification efficiency of raw lignocellullosic wastes (Liguori & Faraco, 2016). The activities of MnP and LiP had two peaks during fermentation (Fig. 3b). The first one for MnP appeared on Day 5 with 1603±7.76 IU/g, reached another peak of 1379.92±20.35 IU/g on Day 10, after which activity decreased to 833.33±14.21 IU/g on Day 12. The maximum LiP activity appeared on Day 6 with 1500±21.44 IU/g, the second on Day 10 with 930.11±17.81 IU/g and then decreased to 220.43±10.24 IU/g on Day 12. Different from MnP and LiP production, Lac activity was detectable after 5 days of fermentation and reached maximum activity of 81.94±7.55 IU/g on Day 9, after which activity decreased to15.28±3.59 IU/g on Day 12 (Fig. 3b). The past studies demonstrated that a few white rot fungi produced all these enzymes, while most produced only one or two of them (Manavalan et al., 2015). This study first found that I. obliquus secreted all the three components of ligninolytic enzymes under SSF, and the ligninolytic enzymes represented relatively high catalytic capability among various MnP, LiP and Lac sources. The MnP activity from I. obliquus was 1001.9-fold higher than that from Pleurotus pulmonarium MTCC 1805 (Kumari and Das, 2016) using sugarcane top as a substrate and 419.6-fold higher than that produced on cotton stalks by the fungus Phlebia radiata MTCC 2791(Meehnian et al., 2017). Meehnian et al.,

(2017) reported that Daedalea flavida MTCC 145 produced both LiP and Lac in the fermentation of cotton stalk. However, the activity of LiP and Lac of 1.75 ± 0.11 IU/g and 4.26 ± 0.38 IU/g after 10-15 days was much lower than the values reported in this study. The Lac activity from I. obliquus was almost 2.85 times higher than that from Pandoraea sp. ISTKB using sugarcane bagasse (Kumar et al, 2016) and was equivalent to that from Daedalea flavida MTCC 145 on cotton stalk sampled on Day10 (Meehnian et al, 2017). Lac is the currently preferred ligninase enzyme. Nevertheless, it can only directly oxidise phenolic lignin units, which usually comprise less than 10% of the total polymer content of natural lignin (Manavalan et al., 2015). By contrast, LiP is the most effective oxidizer ligninase known to date and is capable of catalyzing the oxidation of phenolic or non-phenolic compounds, aromatic amines, aromatic ethers, and polycyclic aromatic hydrocarbons (Manavalan et al., 2015). The catalytic mechanism of MnP is mostly similar to that of LiP but differs in utilizing Mn2+ as the electron donor (Manavalan et al., 2015). Thus, MnP-LiP complexes from I. obliquus with earlier MnP and LiP production peaks on Days 5-6 and second peaks on Day 10 (Fig. 3b) can be expected to be efficient at lignin degradation. These results signified a great leap forward in terms of the production of highly effective enzymes. This study demonstrated that I. obliquus has advantage over soft rot fungi in terms of ligninolytic enzyme production and over other white rot fungi in terms of cellulase production.

3.3. Characterization of cellulolytic enzyme cocktail Being considered both the cellulolytic and ligninolytic enzyme activity (Fig. 3) in one enzyme cocktail, the cellulase cocktail produced on Day 10 by I. obliquus under the optimal conditions was characterized. 3.3.1. Effect of pH on enzyme activity The influence of pH ranging from 3.0 to 8.0 on the activity of the enzymes revealed that the highest activities were obtained at pH between 3.0 and 4.5 (Fig. 4a). CMCase exhibited its maximum activity at pH 3.0-3.5, dropping slightly at pH 4.0. The optimum catalytic activity of CMCase from the commercial strain T. reesei occurs in a narrower pH range between 4.0 and 5.0 than that from I. obliquus in this study (Kogo et al., 2009) The highest FPase activity was observed in the pH range 3.5 - 4.5, with maximum activity at pH 4.0 (Fig. 4a). Bansal et al. (2014) reported a greater FPase activity in the pH range from 3.0 to 9.0, with the maximum activity at pH 4.0 for enzyme extracts produced by the fungus A. niger NS-2 T

β-glucosidase component gave the maximal

activity at pH 3.5 with significant decreases at pH > 5.5 in this study. da Silva Delabona et al. (2013) obtained the m ximum β- glucosidase activity at pH 4.0 as well. Jung et al. 2

2

p

m ximum

ivi

f β-glucosidase produced by mutant

Trichoderma reesei MT-2 at pH 4.8. In comparison to the intolerance of the cellulases from T. reesei, the cellulases from I. obliquus presented great tolerance toward acidic pH, showing acidophilic characteristics. As seen, the expensive cellulase production process and poor stability of cellulases are major concerns in industrial applications,

obviously, it is advisable to use extremozyme in the industrial processes (Yadav, 2017). Therefore, the valorization of these results in this study was significant. 3.3.2. Effect of temperature on the activity and stability of enzyme cocktail Fig. 4b presents the cellulase activity profiles at different temperatures between 30 and 80 °C. The optimal temperature for the enzyme activity of CMCase, FPase and β-glucosidase was 55, 40 and 60 °C, respectively. The enzymatic activity decreased slowly as the temperature was higher than the optimal temperature. At 70°C, the activity f FP s

n β-glucosidase maintained 71% and 72% of the optimal activity, whereas

the CMC activity significantly decreased to about 47% of the maximum activity. These results demonstrated that the cellulases from I. obliquus worked well under thermophilic condition, to some extent, this type of the enzymes is favored in the commercial and industrial processes. The results were comparable to those reported in the literature. Optimum temperature for the CMCase produced by I. obliquus was similar to that produced by Petriella setifera LH (Zhao et al., 2013) but lower than that produced by T. reesei (60 °C) (Kogo et al., 2009). Bansal et al. (2014) obtained the maximum FPase activity at 40 °C, using A. fumigatus AR1. The past studies also reported 60 °C to be the optimum temperature fo β-glucosidase from A. niger NS-2 (Bansal et al., 2014) and Trichoderma harzianum PPDDN10 NFCCI-2925 (Pathak et al., 2014). The CMCase component of the cocktail produced by I. obliquus revealed a half-life of 12 h at 50 °C, and 2 h at 55 °C (Fig. 5), better than that from Aspergillus oryzae

(Javed et al., 2009) but less thermal stable than that from Alkalilimnicola sp. NM-DCM1; the CMCase lost 50% activity when kept at 55 °C for 169 min (Mesbah et al., 2017). The FPase from I. obliquus showed half-lives of 1.5 h and 48 h at 50 °C and 55 °C, respectively (Fig. 5), better than the FPase produced by Trichoderma sp. B-8; it retained less than 50% activities when kept at 55 °C for 3 h (Fang et al., 2011). The β-glucosidase from I. obliquus retained 50% of its original activities after incubation for 0.5 h at 55 °C, and the enzyme activity was completely lost after 5 h. Half-life of 1 h was estimated at 50 °C (Fig. 5). Our results indicated that the enzymes produced by I. obliquus exhibited relatively excellent thermal stability when compared with those studies. 3.4. Enzymatic saccharification of lignocellulosic substrates Enzymatic hydrolysis of raw lignocellulosic substrates was carried out with the cellulolytic enzyme cocktail from I. obliquus at pH 5.0 and its optimum conditions of 50 °C. Supplementary Fig. 1 shows that 50 °C was the best temperature in the ranges of 40 - 60 °C for the saccharification of wheat straw and rice straw. The enzymatic saccharification is not only reliant on the characteristics of lignocellulosic substrates, but also the effect of the proportion of individual enzyme components. The crude cellulase cocktails collected from different time points exihibited different enzymatic components (Fig. 3). To elucidate the cellulolytic performances of cellulases obtained at different time intervals, the cellulase cocktails obtained on Day 6, 8, 10 and 12 were used for the hydrolysis of raw rice straw (RS) and wheat straw (WS). The time course

of enzymatic saccharification revealed that the enzyme cocktail sampled on Day 12 achieved the highest release of reducing sugar from the raw RS (125.36 mg/g of substrate) and WS (130.24 mg/g of substrate) after 48 h of hydrolysis (Fig. 6). These results were probably caused by the action of high-activity-level MnP-LiP and β-glucosidase (see the time courses of cellulolytic and ligninolytic enzyme activities in Figs. 3a, b). The MnP-LiP played an important part in the process of biodegradation of lignin. The enzymes removed or disrupted lignin and liberated cellulose from lignin, and reduced the cellulase adsorption onto lignin, which facilitated the process of saccharification for the reducing sugar production (Yamagishi et al. 2011). The β-glucosidase is the rate-limiting enzyme of the final step of enzymatic saccharification, sufficiently converting cellobiose into glucose (Cripwell et al., 2015). Interestingly, hydrolysis efficiency of the cellulase cocktail sampled on Day 6 was higher than the enzyme cocktails sampled on Day 8 and 10, possibly also because of ligninolytic enzymes that played key roles in the degradation of lignin (see the time courses of ligninolytic enzyme activities in Fig. 3b). To put it another way, ligninolytic enzymes secreted by I. obliquus in the early stage of fermentation selectively removed lignin and then cellulose was prone to being effectively degraded by the cellulases. Through the saccharification processing after 48 h of hydrolysis using the cellulase cocktail sampled on Day 6, the compositions of raw RS and WS significantly changed. The enzymatic hydrolysis of WS was accompanied by 23.5 % cellulose, 11.7% hemicellulose and 22.4% lignin degradation, respectively. The RS achieved 18.9% cellulose loss, 11.2%

hemicelluloses loss and 14.8% lignin loss, respectively. The conversion yields in the hydrolysis of WS and RS by the cellulolytic enzyme cocktails from I. obliquus were higher than the commercial cellulase enzymes. Tiwari et al., (2013) reported that the amount of total reducing sugars increased to 50.89 mg/g with raw paddy straw after 48 h of hydrolysis using the commercial cellulase enzyme Accellerase ® 1500. The higher yield of the reducing sugar (98.31 mg/g) was obtained by enzymatic hydrolysis of unautoclaved and uninoculated paddy straw using Accellerase ® 1500 (Saritha et al., 2013). The reducing sugar yield obtained after 72 h of reaction using Novozyme SP188 and Fusarium verticillioides secretome was 65.4 mg/g of wheat straw (Ravalason et al., 2012). The reducing sugars generation by the cellulase cocktail from I. obliquus was much higher than that of parent strain T. asperellum RCK2011 with raw wheat straw and equivalent to that of mutant strain SR1-7. The good performance of the cellulase cocktail from I. obliquus as a powerful white rot fungus could be attributed to the production levers of both the ligninolytic and hydrolytic enzymes. Overall, it turned out that the white rot fungus I. obliquus showed potential application value in the reutilization of cellulosic waste and conversion of biomass to lignocellulose-derived bioenergy. 4. Conclusion This study, for the first time, demonstrated that the white rot fungus I. obliquus was an efficient and effective producer in terms of high-activity-level cellulolytic and ligninolytic enzyme production under solid state fermentation. The cellulase cocktail

exhibited excellent thermal stability and great tolerance toward acidic pH. Interestingly, saccharification efficiency of raw rice straw and wheat straw was dependent on both the cellulolytic and ligninolytic enzyme components and activities in the cellulase cocktails. This study provided an approach to solve the problem of highly efficient both lignolytic and cellulase production for bioconversion of lignocellulosic biomass into biofeuls. Acknowledgements The authors thank the financial support for the study from Zhejiang Provincial Natural Science Foundation, China under grant LY16B020013. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version. References Bansal, N., Janveja, C., Tewari, R., Soni, R., Soni , S.K., 2014. Highly thermostable and pH-stable cellulases from Aspergillus niger NS-2: properties and application for cellulose hydrolysis. Appl. Biochem. Biotechnol. 172 (1), 141-156. Chen, L., Liang, J.F., 2015. The Potential Roles of Cell Surface pHs in Bioactive Peptide Activation. Chem. Biol. Drug Des. 85(2), 208-215. Cripwell, R., Favaro, L., Rose, S.H., Basaglia, M., Cagnin, L., Casella, S., van Zyl, W., 2015. Utilisation of wheat bran as a substrate for bioethanol production using recombinant cellulases and amylolytic yeast. Appl. Energy.160, 610-617. da Silva Delabona, P., Pirota, R.D.P.B., Codima, C.A., Tremacoldi, C.R., Rodrigues, A., Farinas, C.S., 2013. Effect of initial moisture content on two Amazon rainforest

Aspergillus strains cultivated on agro-industrial residues: Biomass-degrading enzymes production and characterization. Ind. Crops and Prod. 42, 236-242. Deswal, D., Khasa, Y.P., Kuhad, R.C., 2011. Optimization of cellulase production by a brown rot fungus Fomitopsis sp. RCK2010 under solid state fermentation. Bioresour. Technol. 102 (10), 6065-6072. Dhillon, G.S., Oberoi, H.S., Kaur, S., Bansal, S., Brar, S.K. 2011. Value-addition of agricultural wastes for augmented cellulase and xylanase production through solid-state tray fermentation employing mixed-culture of fungi. Ind. Crops and Prod. 34 (1), 1160-1167. Falkoski, D.L., Guimarães, V.M., de Almeida, M.N., Alfenas, A.C., Colodette, J.L., de Rezende, S.T., 2013. Chrysoporthe cubensis: a new source of cellulases and hemicellulases to application in biomass saccharification processes. Bioresour. Technol. 130, 296-305. Fang, X., Shi, H., Ma, J., Jiang, Z., Dai, G., Wu, J., Qin, X., Lin, S., Liu, W., 2011. Studies on the cellulase production using corncob residue from xylose manufacture by Trichoderma sp. B-8 and the characterization of the cellulase. In New Technology of Agricultural Engineering (ICAE), 2011 International Conference on (pp. 684-687). IEEE. Javed, M.R., Rashid, M.H., Nadeem, H., Riaz, M., Perveen, R., 2009. Catalytic and thermodynamic characterization of endoglucanase (CMCase) from Aspergillus oryzae cmc-1. Appl. Biochem. Biotechnol. 157 (3), 483-497.

Jung, Y.R., Shin, H.Y., Yoo, H.Y., Um, Y., Kim, S.W., 2012. Production of cellulases n β-glucosidase in Trichoderma reesei mutated by proton beam irradiation. Korean J. Chem. Eng. 29(7), 925-930. Kogo, T., Yoshida, Y., Koganei, K., Matsumoto, H., Watanabe, T., Ogihara, J., Kasumi, T., 2017. Production of rice straw hydrolysis enzymes by the fungi Trichoderma reesei and Humicola insolens using rice straw as a carbon source. Bioresour. Technol. 233, 67-73. Kovács, K., Szakacs, G., Zacchi, G., 2009. Comparative enzymatic hydrolysis of pretreated spruce by supernatants, whole fermentation broths and washed mycelia of Trichoderma reesei and Trichoderma atroviride. Bioresour. Technol. 100(3), 1350-1357. Kumar, M., Singhal, A., Thakur, I.S., 2016. Comparison of submerged and solid state pretreatment of sugarcane bagasse by Pandoraea sp. ISTKB: Enzymatic and structural analysis. Bioresour. Technol. 203, 18-25. Kumari, S., Das, D., 2016. Biologically pretreated sugarcane top as a potential raw material for the enhancement of gaseous energy recovery by two stage biohythane process. Bioresour. Technol. 218, 1090-1097. Liguori, R., Faraco, V., 2016. Biological processes for advancing lignocellulosic waste biorefinery by advocating circular economy. Bioresour. Technol. 215, 13-20. Maceno, M.A.C., de Souza Vandenberghe, L.P., Woiciechowski, A.L., Soccol, C.R., Spier, M.R., 2016. Production of Cellulases by Phanerochaete sp. Using Empty

Fruit Bunches of Palm (EFB) as Substrate: Optimization and Scale-Up of Process in Bubble Column and Stirred Tank Bioreactors (STR). Waste. Biomass. Valor. 7 (6), 1327-1337. Manavalan, T., Manavalan, A., Heese, K., 2015. Characterization of lignocellulolytic enzymes from white-rot fungi. Curr. Microbiol. 70, 485–498. Meehnian, H., Jana, A.K., Jana, M.M., 2017. Pretreatment of cotton stalks by synergistic interaction of Daedalea flavida and Phlebia radiata in co-culture for improvement in delignification and saccharification. Int. Biodeterior. Biodegrad. 117, 68-77. Mesbah, N.M., Wiegel, J., 2017. A halophilic, alkalithermostable, ionic liquid-tolerant cellulase and its application in in situ saccharification of rice straw. Bioenerg. Res.10(2), 583-591. Miller, G.L., 1959. Use of dinitrosalicylic acid reagent for determination of reducing sugar. Anal. Chem. 31(3), 426-428. Mishra, V., Jana, A.K., Jana, M.M., Gupta, A., 2017. Enhancement in multiple lignolytic enzymes production for optimized lignin degradation and selectivity in fungal pretreatment of sweet sorghum bagasse. Bioresour. Technol. 236, 49-59. Pathak, P., Bhardwaj, N.K., Singh, A.K., 2014. Production of crude cellulase and xylanase from Trichoderma harzianum PPDDN10 NFCCI-2925 and its application in photocopier waste paper recycling. Appl. Biochem. Biotechnol. 172 (8), 3776-3797.

Pinto, P.A., Dias, A.A., Fraga, I., Marques, G., Rodrigues, M.A., Colaço, J., Sampaio, A., Bezerra, R.M., 2012. Influence of ligninolytic enzymes on straw saccharification during fungal pretreatment. Bioresour. Technol. 111, 261-267. Raghuwanshi, S., Deswal, D., Karp, M., Kuhad, R.C., 2014. Bioprocessing of enhanced cellulase production from a mutant of Trichoderma asperellum RCK2011 and its application in hydrolysis of cellulose. Fuel. 124, 183-189. Ravalason, H., Grisel, S., Chevret, D., Favel, A., Berrin, J.G., Sigoillot, J.C., Herpoël-Gimbert, I., 2012. Fusarium verticillioides secretome as a source of auxiliary enzymes to enhance saccharification of wheat straw. Bioresour. Technol. 114, 589-596. Salgado, J.M.,

un s , L ,

n n i , A., Domínguez, J.M., Belo, I., 2015.

Enhancing the bioconversion of winery and olive mill waste mixtures into lignocellulolytic enzymes and animal feed by Aspergillus uvarum using a packed-bed bioreactor. J. Agric. Food. Chem. 63(42), 9306-9314. Saritha, M., Arora, A., Singh, S., Nain, L., 2013. Streptomyces griseorubens mediated delignification of paddy straw for improved enzymatic saccharification yields. Bioresour. Technol. 135, 12-17. Singhal, A., Choudhary, G., Thakur, I.S., 2012. Characterization of laccase activity produced by Cryptococcus albidus. Prep. Biochem. Biotechnol. 42 (2),113-124. Soni, R., Nazir, A., Chadha, B.S., 2010. Optimization of cellulase production by a versatile Aspergillus fumigatus fresenius strain (AMA) capable of efficient

deinking and enzymatic hydrolysis of Solka floc and bagasse. Ind. Crops and Prod. 31 (2), 277-283. Soni, S.K., Batra, N., Bansal, N., Soni, R., 2010. Bioconversion of sugarcane bagasse into second generation bioethanol after enzymatic hydrolysis with in-house produced cellulases from Aspergillus sp. S4B2F. BioResources.5 (2), 741-757. Tiwari, R., Rana, S., Singh, S., Arora, A., Kaushik, R., Agrawal, V.V., Saxena, A.A., Nain, L., 2013. Biological delignification of paddy straw and Parthenium sp. using a novel micromycete Myrothecium roridum LG7 for enhanced saccharification. Bioresour. Technol. 135, 7-11. Trivedi, N., Reddy, C.R.K., Radulovich, R., Jha, B., 2015. Solid state fermentation (SSF)-derived cellulase for saccharification of the green seaweed Ulva for bioethanol production. Algal Res. 9, 48-54. Van Soest, P.J., 1963. Use of detergents in the analysis of fibrous feeds. 2. A rapid method for the determination of fiber and lignin. J. Assoc. Off. Agric. Chem. 46, 829-835. Xu, X., Xu, Z., Shi, S., Lin, M., 2017. Lignocellulose degradation patterns, structural changes, and enzyme secretion by Inonotus obliquus on straw biomass under submerged fermentation. Bioresour. Technol. 241, 415-423. Xue, D.S., Liang, L.Y., Zheng, G., Lin, D.Q., Zhang, Q.L., Yao, S.J., 2017. Expression of Piromyces rhizinflata cellulase in marine Aspergillus niger to enhance halostable cellulase activity by adjusting enzyme-composition. Biochem. Eng. J.

117, 156-161. Yadav, S.K., 2017. Technological advances and applications of hydrolytic enzymes for valorization of lignocellulosic biomass. Bioresour. Technol. https://doi.org/10.1016/j.biortech.2017.05.066. Yamagishi, K., Kimura, T., Watanabe, T., 2011. Treatment of rice straw with selected Cyathus stercoreus strains to improve enzymatic saccharification. Bioresour. Technol. 102, 6937–6943. Zhao, X.H., Wang, W., Wei, D.Z., 2013. Identification of Petriella setifera LH and characterization of its crude carboxymethyl cellulase for application in denim biostoning. J. Microbiol. 51 (1), 82.

Figure caption Fig. 1 Effect of lignocellulosic substrates on CMC s , FP s

n β-glucosidase

production by I. obliquus under SSF. Fig. 2 Effect of (a) inoculum size, (b) pH, (c) moisture content on CMCase, FPase and β-glucosidase production by I. obliquus under SSF. Fig. 3 Time courses of (a) cellulolytic and (b) ligninolytic enzyme activity produced by I. obliquus under the optimal SSF. Fig. 4 Temperature and pH profiles of CMC s , FP s

n β-glucosidase activities.

Fig. 5 Thermal stability at (a) 50 °C and (b) 55 °C f CMC s , FP s

n β-glucosidase

produced by I. obliquus. Fig. 6 Enzymatic saccharification profiles of raw wheat straw (a) and rice straw (b) using cellulolytic enzyme cocktails from I. obliquus.

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Highlights I. obliquus is a novel promising source for lignocellulosic conversion. Optimization of process parameters promoted the cellulase production in SSF. The cellulase enzymes worked well under thermophilic and acidophilic conditions. The best performances of cellulases produced on Day 12 in straw saccharification. The saccharification efficiency was dependent on lignocellulolytic enzyme activities.

Graphical Abstract