Chemical-free pretreatment of unwashed oil palm empty fruit bunch by using locally isolated fungus (Schizophyllum commune ENN1) for delignification

Food and Bioproducts Processing 1 1 8 ( 2 0 1 9 ) 207–216

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Chemical-free pretreatment of unwashed oil palm empty fruit bunch by using locally isolated fungus (Schizophyllum commune ENN1) for delignification Enis Natasha Noor Arbaain a , Ezyana Kamal Bahrin a,b,∗ , Nurshakinah Mohd Noor a , Mohamad Faizal Ibrahim a,b , Norhayati Ramli a , Suraini Abd-Aziz a a

Department of Bioprocess Technology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia b Institute of Tropical Forestry and Forest Product (INTROP), Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia

a r t i c l e

i n f o

a b s t r a c t

Article history:

Biological pretreatment of unwashed oil palm empty fruit bunch (OPEFB) by Schizopyllum com-

Received 30 March 2019

mune ENN1 was carried out to obtain optimum conditions of delignification. Locally isolated

Received in revised form 2

fungus, S. commune ENN1, was grown on oily OPEFB and the biomass was simultaneously

September 2019

delignified. Hence, the incubation time (7 d–28 d), temperature (25 ◦ C–40 ◦ C), and amount of

Accepted 9 September 2019

substrate (3 g–9 g) were investigated to improve the efficiency of lignin removal during the

Available online 16 September 2019

biological pretreatment. Maximum lignin peroxidase and manganese peroxidase were produced at each optimal parameter, and correlated with maximum lignin removal. A similar

Keywords:

pattern of maximum cellulase was produced by S. commune ENN1 at optimal parameters

Biological pretreatment

with only minimal activity of around 5 U/g (CMCase). A maximum lignin removal of 67.9%

Lignin

was achieved at optimum condition at a temperature of 30 ◦ C by using 5 g of OPEFB after

Ligninolytic enzyme

14 d of incubation time. The cellulose content was increased by 31.7% of the biologically

OPEFB

pretreated OPEFB at optimum condition. These findings showed that S. commune ENN1 was

Schizophyllum commune

feasible to remove the lignin and increase the cellulose content of unwashed OPEFB through biological pretreatment for enzymatic hydrolysis. © 2019 Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved.

1.

Introduction

The Malaysian palm oil industry has shown a continuous growth based on the upturn of palm oil production from 14.3 million tonnes in 1994 to 20 million tonnes in 2015 (Kamil and Omar, 2016). Through the intensive growth of the palm oil industry, the national economy is significantly boosted, socio-economic is developed, and the poverty rate in rural

˜ ond largest biomass produced from a palm oil mill (Garcia Nunez et al., 2010). To sustain the palm oil industry, good practice in managing OPEFB should be applied and OPEFB should be converted into valueadded products, such as biocompost, biogas, organic acid, and biofuel by using various pretreatment methods. The commonly used pretreatment in the industry is governed by chemical pretreatment by using chemicals or organic agent to break

areas is successfully reduced (Awalludin et al., 2015). However, it is expected that by the year 2020, about 100 million tonnes of dry oil palm biomass will be generated from this industry (Rozario, 2013). Approxi-

down the lignocellulosic structure of biomass. There are several types of applied chemical pretreatment in the industry, such as acid pretreatment, alkaline pretreatment, hydrothermal pretreatment, ozonolysis,

mately 23% oil palm empty fruit bunch (OPEFB) is produced per tonne of processed fresh fruit bunch (FFB) per day, which makes it the sec-

steam explosion and ammonia fibre expansion (Wang et al., 2017). Currently, biological pretreatment has gained great interest among

∗ Corresponding author at: Department of Bioprocess Technology, Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia. E-mail address: [email protected] (E.K. Bahrin). https://doi.org/10.1016/j.fbp.2019.09.001 0960-3085/© 2019 Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved.

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researchers as an alternative for lignocellulosic biomass pretreatment as it offers an environmentally friendly approach, low pretreatment cost and low energy requirement (Sindhu et al., 2015). The biological pretreatment of biomass manipulates the capability of microorganisms, which are mainly fungi or enzymes, for delignification without utilising any chemical agents. The fungi used in biological pretreatment must be able to produce lignin modifying enzymes and concurrently produce a low amount of cellulolytic enzymes. Ligninolytic enzymes are capable to modify or selectively degrade the natural aromatic polymers structure of lignin in the lignocellulosic biomass (Zanirun et al., 2015). There are three most prominent ligninolytic enzymes expressed during the fungal catabolism of lignin, namely manganese peroxidase (MnP), lignin peroxidase (LiP) and laccase (Janusz et al., 2013). Fresh OPEFB have a significant amount of residual oil of approximately 3.35% after the milling process (Gomez et al., 2015). Nonetheless, most of the fungi cannot be cultivated on the oily substrate because of the oil residue presence on the surface of untreated OPEFB which inhibits the growth of non-indigenous fungi. Interestingly, indigenous fungi found on a pile of OPEFB are able to grow naturally on the oily biomass surface. Biological pretreatment by using indigenous fungus is a new strategy to pretreat the oily OPEFB without washing the biomass (Arbaain et al., 2019). Thus, the biological pretreatment using this fungus renders an advantage to pretreat the OPEFB by omitting the washing step and nutrient supplied throughout the biological pretreatment. Different types of fungi and biomass used in biological pretreatment may have different delignification performance under favourable conditions. Incubation time, temperature and amount of substrate are the crucial parameters in biological pretreatment that will influence the efficiency of lignin removal during pretreatment. Therefore, the objective of this study is to investigate the effect of different parameters on biological pretreatment by a locally isolated fungus, which is Schizophyllum commune ENN1, by using unwashed OPEFB for delignification.

2.

Materials and methods

2.1.

Oil palm empty fruit bunch (OPEFB)

OPEFB was obtained from Seri Ulu Langat palm oil mill, at Dengkil, Selangor, Malaysia with coordinates at 2◦ 85 14.50 N 101◦ 65 07.59 E. The shredded form of unwashed OPEFB with an average length of 10 cm–15 cm was stored in an airtight plastic container and kept in the freezer at −40 ◦ C to keep the freshness of the OPEFB and avoid any growth of fungi before pretreatment.

2.2.

Microorganism

Locally isolated fungus used in this study was isolated from an OPEFB pile at Biorefinery Complex, Universiti Putra Malaysia, Selangor, Malaysia with the coordinates at 2◦ 98 47.48 N 101◦ 71 29.98 E. The isolation of fungus was carried out by scraping a small number of fungal colonies on the OPEFB pile and cultured at the centre of a fresh M2 agar plate as the inoculum point. The composition of the M2 agar medium was as follows: glucose (10 g), glycerine (10 g), yeast extract (5 g), KH2 PO4 (0.3 g), MgSO4 ·7H2 O (0.1 g), agar (20 g) (NCIM, 2014). The pH value of the M2 medium was adjusted to pH 6.8. The fungus was cultivated and subcultured on the M2 agar at 30 ◦ C for 7 d for the mycelia to develop. Tween 80 (0.1% v/v) was used to harvest the fungal mycelia from the agar plate. The fungal mycelia suspension (0.5 mL) was mixed with 20% of glycerol (1 mL) in 1.5 mL tube and kept in −80 ◦ C freezer for long-term fungal preservation.

2.3.

Morphological observation

The microscopic observation was conducted by scraping seven-day old fungal mycelia culture from the plate and it was placed onto a clean glass slide. Then, a few drops of 0.5% (w/v) methylene blue was added onto the fungal mycelia for staining. The slide was examined under a light microscope (CX23, Olympus, Japan) at 40× and 100× magnification to get a clearer view of the fungal mycelia.

2.4.

Molecular identification of the isolated fungus

The isolated fungus was cultured on M2 agar for 7 d and matured mycelia (0.5 g) was scraped with a sterile loop and put into a tube containing bead solution. DNA of the fungal mycelia was extracted according to the MO BIO’s Powersoil® DNA Isolation Kit (MO BIO Laboratories Inc., USA) method. Next, the fungal internal transcribed spacer (ITS) region was PCR amplified using universal primer. DNA sequences obtained from ITS region were aligned by using BioEdit programme (version 7.2). The identity was then confirmed by using a BLAST search supported in National Centre for Biotechnology Identification (NCBI) database. The phylogenetic tree was constructed via the Neighbour-Joining method with 500 bootstrap numbers by using the MEGA 6.0 software.

2.5.

Biological pretreatment

Biological pretreatment by using an identified local isolate fungus was carried out in 250 mL Erlenmeyer flasks. The unwashed OPEFB with an initial moisture content of 55.2% was placed in the flasks. The flasks were sterilised at 121 ◦ C for 20 min by using an autoclave. Five agar plugs of sevenday old fungal mycelia (average size of 1 cm) were aseptically inoculated onto the OPEFB by using a sterile cork-borer. The biological pretreatment was carried out without any supplies of moistening agents or nutrients. Parameters such as incubation time, temperature and amount of substrate of biological pretreatment were investigated by employing the one-factorat-a-time (OFAT) method. Incubation time for the biological pretreatment was 7 d, 14 d, 21 d and 28 d at constant conditions of temperature (30 ◦ C) and amount of substrate (5 g). The temperature effect was studied at 25 ◦ C, 30 ◦ C, 35 ◦ C and 40 ◦ C with the same amount of substrate (5 g) for 14 d. Meanwhile, the effect on the amount of substrate was conducted by using a different amount of substrate, which was 3 g, 5 g, 7 g and 9 g at constant temperature (30 ◦ C) and incubation time (14 d). The biological pretreatment by using identified fungus was carried out by using the optimum conditions in order to validate the findings for each investigated parameter. The chemical composition of biologically pretreated OPEFB samples was analysed after the pretreatment to identify the changes of lignocellulosic components.

2.6.

Ligninolytic enzyme assays

Manganese peroxidase (MnP) and lignin peroxidase (LiP) extractions were done according to Kong et al. (2016). Manganese peroxidase (MnP) activity was assayed by oxidation of guaiacol to the coloured product according to Li et al. (2009). Lignin peroxidase (LiP) activity was determined based on the oxidation of veratryl alcohol to veratraldehyde according to Tien and Kirk (1988), with some modifications. The

Food and Bioproducts Processing 1 1 8 ( 2 0 1 9 ) 207–216

gradient of the kinetic reaction was calculated and used to measure the MnP and LiP activities. Ligninolytic enzyme activities were determined by using the UV–vis spectrophotometer (UV-1800 Shimadzu, Japan). The ligninolytic enzyme activity was expressed as U/g (unit per gram of dry OPEFB).

2.7.

Cellulase assays

Extraction of cellulase enzyme was carried out according to Ang et al. (2013), with a slight modification. Pretreated OPEFB was suspended in 0.05 M sodium acetate buffer (pH 4.8) with substrate to buffer ratio 2:1 (w/v). The mixture was incubated at 24 ◦ C for 30 min with 150 rpm agitation. OPEFB and fungi cell debris was separated by centrifugation at 5000 rpm, 4 ◦ C for 5 min to obtain the supernatant which contained the crude enzyme. The crude enzyme was temporarily stored at 4 ◦ C prior to enzyme assays. Determination of FPase, CMCase and ␤-glucosidase activities was assayed as described by Wood and Bhat (1988) by using a UV–vis spectrophotometer (UV-1800 Shimadzu, Japan). All enzyme activities were expressed as U/g (unit per gram of dry OPEFB).

Macromorphology and micromorphology are the primary method to identify the fungi before confirmation through fungal sequencing (Sasso et al., 2017). Fig. 1b shows the hyphae structure of S. commune ENN1 under microscopy observation. Based on the figure, it was clearly shown the marginal hyaline hyphae with 2.5 ␮m–4 ␮m in diameter, thin-walled, nodose septate and sparsely to moderately branched. The microscopic examination of S. commune ENN1 hyphae was comparable with other studies. The morphology of S. commune isolate by Hanafusa et al. (2016) showed hyaline, septate, and nondichotomously branching hyphae. Based on the phylogenetic tree in Fig. 2, isolated fungus in this study showed 97% homology in genomic alignment with the nucleotide sequence of a strain isolated from a medicinal plant in North India and was identified as S. commune (Mishra and Singh, 2015). This homology value was the highest as compared to other species. From the results of morphological observation and phylogenetic tree, the isolated fungus was identified as S. commune and the sequence was deposited to GenBank as S. commune ENN1 (Accession no MH539647.1).

3.2. 2.8.

Analytical methods

The moisture content and ash content analyses of the OPEFB samples were carried out according to the standardised method of the National Renewable Energy Laboratory (NREL, USA) (Sluiter et al., 2008). Approximately 1.0 g of OPEFB sample was weighed and the moisture content was recorded based on the measured values by using a digital moisture analyser (A&D, MX-50, Japan). The determination of water and solvent extractive components was carried out according to the NREL procedure reported by Sluiter et al. (2012). The lignocellulosic compositions (lignin, cellulose and hemicellulose) of OPEFB before and after pretreatment were determined according to the method by Iwamoto et al. (2008). All analyses were carried out in triplicate.

2.9.

Statistical analysis

Statistical analysis was conducted to analyse the significant effect from each parameter on the pretreatment process. All calculations were performed by using analysis of variance (ANOVA) by Statistical Analysis Software (SAS) Version 9.4. The statistical significance was verified by considering p <0.05.

3.

Results and discussion

3.1.

Identification of isolated fungus

As fungi are morphologically and phylogenetically diverse, the morphology of fungal growth and molecular identification to species level are paramount steps in fungal identification. Based on morphological observation of seven days old fungal culture, the fungal mycelia had a hairy texture with light orange colour (Fig. 1a). These morphological features corresponded with the fungal genus of Schizophyllum sp. and were complementary with other studies, which showed white coloured mycelial mat spread throughout the surface of the agar medium and changed to orange coloured mycelia under controlled condition (Ujor et al., 2012). The raised marginal hyphae and non-sporulating (only vegetative growth) fungal colony were other characteristics of the isolated fungus.

209

Preliminary run of biological pretreatment

The relation between mycelia density of S. commune ENN1 grown on OPEFB throughout the biological pretreatment was essential in understanding the delignification process. Fig. 3 shows the growth of S. commune ENN1 throughout 21 d of biological pretreatment. Based on the observation throughout the biological pretreatment, the density of fungal mycelia increased from Day 0 to Day 14. From the bottom view of the flask, the OPEFB were completely covered with the fungal mycelia of S. commune ENN1 and remain attached to the biomass on Day 14. This suggested that the S. commune ENN1 had the highest growth up to 14 d of incubation, fully adapted with the environment and efficiently catabolised the carbon source contained in the OPEFB (Teoh and Don, 2012). According to Manan and Webb (2016), mycelia density and colour changes of the pretreated substrate during biological pretreatment provided a good indicator for fungal growth estimation. On the other hand, the density of S. commune ENN1 mycelia depleted on Day 21 of biological pretreatment probably due to the depletion of carbon source.

3.3.

Effect of incubation time

The ability of white-rot fungi to modify the lignocellulosic structure of biomass may differ for a different period of time. In this study, the biological pretreatment was carried out at four different incubation times, which were 7 d, 14 d, 21 d and 28 d. The effect of incubation time on the lignocellulosic composition of OPEFB, before and after biologically pretreated with S. commune ENN1, was investigated (Table 1). The highest lignin removal was recorded on Day 14 with 55.2% of lignin removal and closely followed by 49.5% and 50.9% of lignin removal on Day 21 and Day 28. The results were in agreement with Asgher et al. (2012) who demonstrated that the ability of ligninolytic enzymes to modify the lignocellulosic structure increased with increase in incubation time. However, prolonging the pretreatment time for more than 28 days showed no significant difference in terms of lignin removal of OPEFB due to the low activity of ligninolytic enzymes. By comparing with the untreated OPEFB, the cellulose content in pretreated samples increased up to 32.5% on Day 21 and decreased to 7.3% on Day 28. The cellulose content of the

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Fig. 1 – Morphological structure of Schizophyllum commune ENN1 (a) culture on the agar plate (b) microscopic image of fungal mycelia (arrows show cross wall septa of the fungal hyphae).

Fig. 2 – Phylogenetic tree constructed using the Neighbor-Joining method based on based on the amplified ITS region of the isolated fungus and related fungi. Table 1 – Composition of biologically pretreated oil palm empty fruit bunch (OPEFB) by Schizophyllum commune ENN1 at different incubation times. The temperature and amount of substrate were kept constant at 30 ◦ C and 5 g, respectively during biological pretreatment. OPEFB samples

*Composition (%) Lignocellulosic components

Untreated 7 days 14 days 21 days 28 days

**Total extractives

Lignin

Hemicellulose

Cellulose

21.2 ± 0.1b 12.1 ± 0.4a 9.5 ± 0.1a 10.7 ± 0.8a,b 10.4 ± 0.3a

30.2 ± 0.5c 36.1 ± 0.5b,c 46.8 ± 0.4a,b 45.4 ± 2.2a 54.3 ± 0.4a

32.8 ± 0.5b 48.5 ± 0.7a 41.1 ± 0.5b 48.6 ± 0.4b 35.4 ± 0.4b

8.2 ± 0.4a 17.5 ± 0.3b 21.0 ± 1.1b 18.4 ± 0.4b 17.3 ± 0.2b

Ash

1.2 ± 0.2b 1.5 ± 0.0a 2.6 ± 0.4b 2.5 ± 0.0b 2.3 ± 0.0a

Moisture

55.2 ± 0.2a 50.0 ± 0.3a 50.6 ± 0.0a 34.6 ± 0.9b 10.7 ± 0.0c

*Values reported represent average values ± standard deviation of triplicate samples. Different letters in the same column indicate significant differences between incubation time at p < 0.05 level. *Total extractive = water-soluble extractive + solvent soluble extractive.

pretreated OPEFB exhibited an increment instead of a gradual reduction over time. This phenomenon suggested that the cellulose fibrils were exposed during the pretreatment and the cellulose accessibility had increased. There was an increment pattern in hemicellulose content starting from Day 7 to Day 28. The result was in agreement with the previous report by Ishola et al. (2014), which showed an increase in the hemicellulose composition of OPEFB after biological pretreatment by using Pleurotus floridanus LIPIMC996. The highest amount of total extractive (21%) was recorded on Day 14 of biological pretreatment and in line with the highest percentage of lignin removal. The total extractives of biologically pretreated OPEB were not significantly (p < 0.05) affected at different incubation times. Besides, the biological pretreatment of OPEFB by S. commune ENN1 could maintain the initial moisture content up to Day 14 for efficient delig-

nification. The sudden drop in moisture content after 14 d was caused by several factors, such as evaporation and low growth rate of fungi during pretreatment. This was supported by Alam et al. (2010) who stated that a complete life cycle for basidiomycete S. commune was approximately 10 d. The highest LiP and MnP activities were produced at Day 14 with 0.93 ± 0.09 U/g and 0.05 ± 0.02 U/g, respectively (Fig. 4a). In contrast, Asgher et al. (2012) obtained maximum enzyme activities from S. commune after 8 d of incubation time by using a banana stalk. On the other hand, the LiP and MnP activities were found much lower at other incubation times of biological pretreatment. The results obtained showed that the activity of ligninolytic enzymes was significantly affected at different incubation times. Apparently, laccase was one of the enzymes in the lignin degradation mechanism. However, laccase was not detected in the enzyme cocktail of S. commune

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Fig. 3 – Front and bottom view of biologically pretreated OPEFB by Schizophyllum commune ENN1 (a) day 0 (b) day 7 (c) day 14 and (d) day 21. ENN1; hence, only LiP and MnP were determined throughout this study. Similarly, Zhu et al. (2016) reported that laccase was not detected when S. commune was grown on Jerusalem artichoke stalk. Cellulase activity of S. commune ENN1 in this pretreatment was measured to determine the cellulase action during the delignification process. Fig. 4b shows the activities of the cellulolytic enzymes of pretreated OPEFB at different incubation times. Day 14 showed the highest cellulase activities with 4.889 ± 0.002 U/g, 0.417 ± 0.003 U/g, and 0.719 ± 0.002 U/g for CMCase, FPase and ␤-glucosidase, respectively. Sornlake et al. (2017) reported that the cellulase activity increased gradually over time and remained constant after 20 d. The high

amount of CMCase activity by S. commune was detected after lignin was removed and the cellulose available in the substrate (Tsujiyama and Ueno, 2011). Although S. commune ENN1 produced cellulase, the activity of the enzyme was considered low and insufficient to degrade the cellulose in OPEFB. This result correlated with the cellulose content obtained from the pretreated OPEFB.

3.4.

Effect of temperature

The efficiency of fungi performance in delignification of biomass varies at different temperatures. Hence, the most favourable temperature for delignification of OPEFB by S. com-

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Fig. 4 – Enzymatic activities of Schizophyllum commune ENN1 during biological pretreatment of OPEFB at different incubation times (a) ligninolytic enzyme activities ( LiP; MnP), (b) cellulase activities ( CMCase; ␤-glucosidase, FPase). Error bars represent the standard deviation of triplicate samples. Different letters on error bar indicate significant differences between incubation time at p < 0.05 level.

Table 2 – Composition of biologically pretreated oil palm empty fruit bunch (OPEFB) at different temperatures. The incubation time and amounts of substrate were kept constant at 14 d and 5 g, respectively during biological pretreatment. OPEFB samples

*Composition (%) Lignocellulosic components Lignin

Untreated 25 ◦ C 30 ◦ C 35 ◦ C 40 ◦ C

21.2 ± 0.1 9.8 ± 0.2a 7.2 ± 0.5a 11.7 ± 2.2a 14 ± 0.9a,b

b

**Total extractives

Hemicellulose

Cellulose

30.2 ± 0.5 29.1 ± 0.0a 33.5 ± 1.2a 29.6 ± 2.0a 28.8 ± 0.2a

32.8 ± 0.5c 58.6 ± 0.1a 52.6 ± 1.5b 48.6 ± 0.4b 47.0 ± 0.3b

a

8.2 ± 0.4a 20.6 ± 0.2c 24.7 ± 0.7c 21.5 ± 0.3b,c 18.8 ± 0.4b

Ash

1.2 ± 0.2c 1.3 ± 0.0a 1.5 ± 0.1b 1.5 ± 0.0b 1.3 ± 0.0a

Moisture

55.2 ± 0.2a 53.4 ± 0.4a 51.5 ± 0.4a 46.5 ± 0.3b 35.2 ± 0.1c

*Values reported represent average values ± standard deviation of triplicate samples. Different letters in the same column indicate significant differences between the temperature at p < 0.05 level. *Total extractive = water-soluble extractive + solvent soluble extractive.

mune ENN1 had to be determined. The biological pretreatment was carried out at four different temperatures, which were 25 ◦ C, 30 ◦ C, 35 ◦ C and 40 ◦ C. Based on Table 2, the maximum removal of lignin by S. commune ENN1 was recorded at temperature of 30 ◦ C with 66% of lignin removal. Meanwhile, the removal of lignin decreased to 34% at a temperature of more than 30 ◦ C. The hemicellulose content was inclined at an optimal temperature of 30 ◦ C with 33.5% of hemicellulose content

after the biological pretreatment. There was no significant difference in the hemicellulose content between untreated (30.2%) and other pretreated OPEFB. However, the hemicellulose content was slightly degraded by 3.6%, 1.9% and 4.6% at the temperatures of 25 ◦ C, 35 ◦ C and 40 ◦ C, respectively. Meanwhile, the maximum cellulose content of the pretreated OPEFB was achieved at 25 ◦ C and 30 ◦ C and increased by 1.8-fold and 1.6-fold, respectively. This positive result showed that cellu-

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Fig. 5 – Enzymatic activities of Schizophyllum commune ENN1 during biological pretreatment of OPEFB at different MnP), (b) cellulase activities ( CMCase; ␤-glucosidase, temperatures (a) ligninolytic enzyme activities ( LiP; FPase). Error bars represent the standard deviation of triplicate samples. Different letters on error bar indicate significant differences between the temperature at p < 0.05 level. Table 3 – Composition of biologically pretreated oil palm empty fruit bunch (OPEFB) at different amounts of substrate. The temperature and incubation time was kept constant at 30 ◦ C and 14 d, respectively during biological pretreatment. OPEFB samples

*Composition (%) Lignocellulosic components

Untreated 3g 5g 7g 9g

**Total extractives

Lignin

Hemicellulose

Cellulose

21.2 ± 0.1c 5.6 ± 0.4a 6.0 ± 0.2a 12.8 ± 0.1b 13.6 ± 0.4b

30.2 ± 0.5a 27.3 ± 0.4a 28.5 ± 0.9a 29.2 ± 0.3a 29.9 ± 0.1a

32.8 ± 0.5d 51.6 ± 0.4a 51.0 ± 0.4a 47.2 ± 0.0b 45.5 ± 0.2c

8.2 ± 0.4a 19.2 ± 0.4b 19.4 ± 0.3b 21.8 ± 0.0b,c 26.8 ± 0.3c

Ash

1.2 ± 0.2a 1.3 ± 0.0a 1.3 ± 0.0a 1.4 ± 0.0a,b 1.5 ± 0.1b

Moisture

55.2 ± 0.2a 45.5 ± 0.2a 51.5 ± 0.3a 53.7 ± 0.2a 54.6 ± 0.3a

*Values reported represent average values ± standard deviation of triplicate samples. Different letters in the same column indicate significant differences between the amount of substrate at p < 0.05 level. *Total extractive = water-soluble extractive + solvent soluble extractive.

lose was exposed after the removal of lignin, and thus caused the increase in cellulose contents (Shirkavand et al., 2017). The total extractives contents of all the pretreated OPEFB were higher than untreated OPEFB (8.2%). This was in agreement with Lee et al. (2007) who reported that the extractive contents of softwood, Pinus densiflora, was increased after biologically pretreated by three white rot fungi, namely Ceriporia lacerata, Stereum hirsutum, and Polyporus brumalis. The researchers deduced that low molecular weight of carbohydrates and lignin degradation products were generated during the biological pretreatment. The highest amount of total extractives (24.7%) was recorded at a temperature of 30 ◦ C in

correlation with the highest lignin removal. Incubation temperature of more than 30 ◦ C caused a gradual reduction of moisture content in the biological pretreatment system. High lignin removal of OPEFB was achieved with moderate moisture content (51%–53%) in the temperature range of 25 ◦ C–30 ◦ C. Therefore, the moisture content of pretreated OPEFB at 25 ◦ C and 30 ◦ C promoted better conditions for the S. commune ENN1 growth and consequently enhance its ability to degrade lignin. The highest LiP and MnP activities of S. commune in this biological pretreatment were produced at 30 ◦ C with 2.083 ± 0.047 U/g and 1.752 ± 0.033 U/g, respectively (Fig. 5a). LiP and MnP activities were found very low at other tempera-

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Fig. 6 – Enzymatic activities of Schizophyllum commune ENN1 during biological pretreatment of OPEFB at different amount of substrates (a) lignin peroxidase (LiP) and manganese peroxidase (MnP) activities (

LiP;

MnP), (b) cellulase activities (

CMCase; ␤-glucosidase, FPase). Error bars represent the standard deviation of triplicate samples. Different letters on error bar indicate significant differences between the amount of substrate at p < 0.05 level. Table 4 – Chemical composition of untreated and biologically pretreated OPEFB by Schizophyllum commune ENN1 at optimum condition. *Composition (%)

Untreated OPEFB

Lignocellulosic components 21.2 ± 0.1b Lignin Cellulose 32.8 ± 0.5b Hemicellulose 30.2 ± 0.5b **Total extractives 10.5 ± 0.2a Ash 1.7 ± 0.1a Moisture content 49.6 ± 0.0a

produced the highest cellulase as compared to Phanerochaete chrysosporium and Ceriporiopsis subvermispora.

Pretreated OPEFB

3.5.

6.8 ± 0.4a 48.0 ± 0.1a 38.5 ± 0.0a 15.8 ± 0.5b 1.5 ± 0.2a 51.4 ± 0.2a

The amount of unwashed OPEFB (3 g, 5 g, 7 g and 9 g) used in this biological pretreatment might influence the ability of S. commune ENN1 to modify the lignocellulosic structure. High lignin removal of 73.6% and 71.7% were obtained by using 3 g and 5 g of OPEFB, respectively (Table 3). An insignificant difference was statistically recorded in lignin removal between the two amounts (3 g and 5 g) by using SAS. However, 5 g of OPEFB was considered as the optimum amount of substrate in this pretreatment. Several studies reported that 4 g–5 g was the optimum amount of substrate range for biological pretreatment in 250 mL Erlenmeyer flask (Asgher et al., 2016). Both the amount of substrates, 3 g and 5 g, recorded an increment in the percentage of cellulose content by 36.4% and 35.7%, respectively. Yao and Nokes (2014) also discovered that enhancement of lignin removal was accompanied by an increase in cellulose content because of more accessible cellulose for fungal metabolism. In contrast, a minimal reduction in the hemicellulose content (9.6%) was recorded at 3 g of OPEFB after biological pretreatment. There was no significant difference in the percentage of hemicellulose content as the amount of OPEFB

*Values reported represent average values ± standard deviation of triplicate samples. Different letters in the same column indicate significant differences between samples of OPEFB at p < 0.05 level. *Total extractive = water-soluble extractive + solvent soluble extractive.

tures of biological pretreatment. A study by Mishra and Singh (2015) reported that the optimal ligninolytic enzymes activity of S. commune was 30 ◦ C and lost over 50% activity with a variation of ±2 ◦ C. Fernández-Fueyo et al. (2014) claimed that the activity of the ligninolytic enzyme by Pleurotus ostreatus was significantly dropped above 37 ◦ C. Fig. 5b shows the cellulase activities of biologically pretreated OPEFB and 30 ◦ C displayed the highest CMCase activity at 4.972 ± 0.056 U/g. This was supported by Zhu et al. (2016) who claimed that S. commune

Effect of amount of substrate

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Table 5 – Effects of different parameters in biological pretreatment on lignin removal from various lignocellulosic biomass. Fungus

Stereum hirsutum, Trametes versicolor Daedalea flavida Phanerochaete chrysosporium Pleurotus floridanus LIPIMC996 Trametes versicolor Schizophyllum commune ENN1

Biomass

Parameters

Initial lignin Lignin content (%) removal (%)

References

Duration Temperature (day) (◦ C)

Amount of substrate (g)

Radiata pine

49

25

10

34.0

22.0

Shirkavand et al. (2017)

Cotton stalks OPEFB OPEFB OPEFB OPEFB

20 14 35 14 14

28 30 30 28 30

5 5 25 4 5

30.2 21.0 NA 19.0 21.4

33.7 38.6 27.2 15.6 67.9

Meehnian et al. (2016) Arbaain et al. (2019) Harmini et al. (2013) Kamcharoen et al. (2014) This study

NA: not available.

increased (more than 5 g). Meanwhile, total extractives contents of all different amounts of OPEFB samples after the pretreatment were higher than the untreated OPEFB possibly because protein and enzymes were secreted during the delignification. The ligninolytic enzymes activity corresponded with the lignocellulosic composition analysis which showed high lignin removal at 3 g of OPEFB. The maximum LiP (1.219 ± 0.000 U/g) and MnP activities (0.055 ± 0.008 U/g) were achieved by using 3 g of OPEFB (Fig. 6a). Less amount of substrate that occupied the vessel had a great advantage of efficient gas exchange and heat transfer. Thus, give better oxygen supply for the growth of fungi, easier removal of carbon dioxide and heat generated (Manan and Webb, 2016). The activities of ligninolytic enzymes were reduced when the amount of substrate was more than 3 g of OPEFB. This occurrence was mainly due to the catabolic repression of enzyme expression caused by excess substrates and oxygen deficiency (Mallek-Fakhfakh et al., 2017). This eventually inhibited the growth of microorganism which subsequently declined the production of the enzyme (Qian et al., 2012). Meanwhile, the amount of substrate at 5 g recorded a higher activity of CMCase and FPase, which were 3.532 ± 0.020 U/g and 0.404 ± 0.001 U/g, respectively (Fig. 6b), as compared to 3 g substrate. Essentially, the cellulase activity correlated very well with the moisture content in this studied parameter. In fact, cellulase is a type of hydrolase enzyme that requires a good level of moisture content to hydrolyse its substrate.

3.6. Biological pretreatment of OPEFB at optimum conditions Biological pretreatment of OPEFB was carried out at optimum conditions by using 5 g of OPEFB and incubated at 30 ◦ C for 14 days. Lignin removal of the pretreated OPEFB was 67.9% and this significant result proved that the S. commune ENN1 was feasible to enhance the lignin removal (Table 4). The degradation of lignin during the biological pretreatment increased the pore size in the biomass and provided a more available surface area of the cellulose (Nazarpour et al., 2013) while simultaneously conserving the cellulose polymer (Yao and Nokes, 2014). The cellulose content was increased by 31.7% in the pretreated OPEFB at the optimum condition, which was comparable with the increment obtained during optimisation at different variables (incubation time, temperature and amount of substrate). Different parameters gave diverse effects on the removal of lignin during biological pretreatment of lignocellulosic biomass (Table 5). In this study, 67.9% of lignin removal was achieved at the optimum condition of 14 days, 30 ◦ C and 5 g

of OPEFB from 21.4% of initial lignin content. Kamcharoen et al. (2014) used Trametes versicolor to biologically pretreat 4 g of OPEFB for 14 d at 28 ◦ C, which resulted in 15.6% of lignin removal from 19.0% of initial lignin content. The longest incubation time was the pretreatment of Radiata pine that was carried out up to 49 days by using two different types of fungus, which were S. hirsutum and T. versicolor (Shirkavand et al., 2017). Typically, most of the biological pretreatments were carried out in the range of 25 ◦ C–35 ◦ C, similar to the favourable temperature for most white-rot fungi. The amount of substrate can be varied between 1 g–10 g of biomass based on the type of biomass and the fungus ability to degrade the biomass.

4.

Conclusions

Above results indicated that S. commune ENN1 isolate successfully performed delignification of unwashed OPEFB through biological pretreatment. Optimisation of the pretreatment conditions, particularly incubation time, temperature, and amount of substrate greatly improved the lignin removal of OPEFB with satisfactory ligninolytic enzymes secretion during the biological pretreatment. A maximum lignin removal of 67.9% was achieved at optimum conditions by using 5 g of the substrate after 14 d of incubation time at a temperature of 30 ◦ C. This new S. commune ENN1 could be employed in the chemical-free pretreatment for the conversion of various bioproducts from lignocellulosic biomass.

Acknowledgements The authors would like to thank the MyBrain 15 scholarship from the Ministry of Higher Education Malaysia, UPM Graduate Research Fellowship (GRF), Putra Grant IPM from Universiti Putra Malaysia (Grant Scheme no. 9484600) for their financial support in this research.

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