Comprehensive studies on optimization of ligno-hemicellulolytic enzymes by indigenous white rot hymenomycetes under solid-state cultivation using agro-industrial wastes

Journal of Environmental Management 259 (2020) 110056

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Research article

Comprehensive studies on optimization of ligno-hemicellulolytic enzymes by indigenous white rot hymenomycetes under solid-state cultivation using agro-industrial wastes Yuvarani Naidu a, *, Yasmeen Siddiqui b, **, Abu Seman Idris a a b

Biology Division, Malaysian Palm Oil Board, 6, Persiaran Institusi, Bandar Baru Bangi, 43000, Kajang, Selangor, Malaysia Laboratory of Plantation Science and Technology, Institute of Plantation Studies, Universiti Putra Malaysia, Serdang, 43400, Selangor, Malaysia

A R T I C L E I N F O

A B S T R A C T

Keywords: Waste management Agro-industrial waste Lignocellulosic biomass Oxidative enzymes Hydrolytic enzymes PCA Biplot

The disposal of oil palm biomass is a huge challenge in Malaysian oil palm plantations. The aim of this study was to develop efficient solid-state cultivated (SSC) ligno-hemicellulolytic bio-degrader formulations of indigenous white-rot hymenomycetes (Trametes lactinea FBW and Pycnoporus sanguineus FBR) utilizing oil palm empty fruit bunches (EFB), rubber wood sawdust (SD) and vermiculite (V) either alone or in combination as substrates. Based on significant laccase (849.40 U mg 1 protein), xylanase (42.26 U g 1 protein) and amylase (157.49 U g 1 protein) production, SDþV (T5) and V (T3) were the optimum substrates for SSC of T. lactinea FBW. Whereas, utilizing EFB (T1) substrate for SSC of P. sanguineus FBR enhanced the production of MnP (42.51 U mg 1 pro­ tein), LiP (103.20 U mg 1 protein) and CMCase (34.39 U g 1 protein), enzymes. Apparently, this is the first study reporting on the protein profiles by T. lactinea FBW, producing two isoforms of un-purified laccase (~55 and 70 kDa) and MnP (~40 and 60 kDa) and a CMCase band (~60 kDa) during SSC on SDþV (T5) substrate. Inter­ estingly, this is also the first report to document a single isoform of un-purified laccase (~50 kDa), MnP (~45 kDa), CMCase (~60 kDa) and xylanase (~55 kDa) by P. sanguineus FBR during SSC on empty fruit bunches substrate. The computed Principal Component Analysis (PCA) Biplot analysis elucidated the relationship be­ tween the solid substrate compositions, the hymenomycete strain, ligno-hemicellulolytic enzyme profiles, and cultivation time. Therefore, it is suggested to use PCA as a tool for multivariate analysis method for compre­ hensive selection and optimization of ligno-hemicellulolytic enzyme cocktails by the indigenous white rot hymenomycetes. These non-toxic (acute oral toxicity) formulations are safe to be used in field applications to efficiently degrade oil palm trunks and root mass that had been felled, chipped or pulverized under zero burning waste management program. This study could also serve as an alternative method for efficient utilization of agroindustrial waste as substrates for the development of cost-effective bio-degraders formulations for agro-waste management.

1. Introduction The palm oil and rubber industry play an important role in the economic development of Malaysia in enhancing the economic welfare of the population. Despite the obvious benefits of this industrial devel­ opment, it also contributes to environmental pollution. Inevitably, there are more than 80 millions of tonnes of green waste/lignocellulosic biomass in Malaysia which is produced annually from the oil palm in­ dustries (Loh, 2017), including oil palm trunks (OPT), oil palm fronds (OPF), oil palm empty fruit bunch (OPEFB), oil palm pressed fibres

(OPPF), oil palm kernel shell (OPKS) and palm oil mill effluent (POME). Disposal of these wastes are subjected to various regulations under the Malaysian Environmental Quality Act 1974. Since 1993, the practice of land clearing using the clean burn method has been replaced by enforcement of Zero Burning Policy in Malaysian oil palm plantations. Generally, ageing trees, trunks and root mass of diseased oil palm stands are felled, chipped, or pulverized and left in situ for natural degradation. This biomass takes approximately two years to completely degrade due to high levels of lignin and polysaccharides (Murai et al., 2009). In addition, the biomass provides a substrate for

* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (Y. Naidu), [email protected] (Y. Siddiqui). https://doi.org/10.1016/j.jenvman.2019.110056 Received 12 July 2019; Received in revised form 11 December 2019; Accepted 31 December 2019 Available online 9 January 2020 0301-4797/© 2020 Elsevier Ltd. All rights reserved.

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Journal of Environmental Management 259 (2020) 110056

breeding of pest such as rhinoceros beetle (Oryctes rhinoceros) and serves as a source of Ganoderma sp. inoculum, to initiate basal stem rot infec­ tion, the most devastating disease of oil palm. Currently, disposal of these oil palm generated wastes remains a huge challenge under zero burning policy. Similarly, in the rubber plantation timber and stumps left after latex extracting age of the rubber trees have not been fully utilized and is conventionally removed by burning, which is only allowed for specific cases after obtaining special permission from Department of Environ­ ment Malaysia (DOE) (Yiew, 1998). Though, open burning is regulated but still contributes to environmental pollution. Therefore, fundamental principles of waste management are deemed necessary to minimize and recycle the lignocellulosic biomass, recover the energy and finally dispose the waste (Rizal et al., 2018). Studies on utilizing the agricul­ tural biomass, including the rubber trunk, branches, twigs and leaves for many downstream processes have been reported (Ratnasingam and Scholz, 2009; Srinivasakannan and Zailani, 2004). For instance, ligno­ cellulosic residues from agricultural and forest industries are known to stimulate the production of ligno-hemicellulolytic enzymes (Winquist et al., 2008; Camarero et al., 1997). These residues can be used as non-inert materials in solid-state fermentation (SSF) or cultivation (SSC) owing to their easy availability, high biodegradability and rich source of nutrients such as carbon (Gassara et al., 2010), thereby reducing the production costs and the environmental impact of their disposal (Sadh et al., 2018). In addition to existing sanitation techniques, an alternative approach of biodegradation of agro-waste by utilizing white-rot hymenomycetes could be opted. Abiding with the law, this practice could aid in reducing environmental pollution, allow complete restoration of soil organic matter and contribute positively towards efforts in minimizing global warming. The study reported here is part of an ongoing project dealing with oil palm-generated waste management in the plantations. To date, there are no scientific reports that elucidate the use of white-rot hymenomycetes as a bio-degrader in the waste management program in the oil palm plantations. In addition, there is no proper delivery technologies of the bio-degrader have been discovered to make this approach a success. Therefore, this study was carried with the objective to mass-produce indigenous white-rot hymenomycetes, namely Pycno­ porus sanguineus FBR and Trametes lactinea FBW using different agroindustrial wastes as carrier substrates. Besides that, factors affecting the quality of the developed bio-degrader formulations were also eval­ uated. The optimized bio-degrader formulations will be further used as a green and cleaner technology approach under the zero burning waste management program.

2.2. Solid substrate preparation

2. Materials and methods

2.4.2. Production of lignocellulolytic enzymes during solid-state cultivation The oxidative enzymes, laccase, manganese peroxidase (MnP), and lignin peroxidase (LiP) and the hydrolytic enzymes, carboxymethyl cellulase (CMCase), xylanase and amylase secreted by P. sanguineus FBR and T. lactinea FBW in each of the solid-substrate cultures were quan­ tified and analyzed according to Naidu et al. (2017). Briefly, at each sampling time, the substrate and mycelium from each bottle was transferred to a 250-mL Erlenmeyer flask containing 100 mL of cold 50 mM sodium acetate buffer. The pH of the extraction buffer was adjusted depending on the type of enzyme being extracted. The flasks were then placed on an incubator shaker set at 200 rpm at 4 � C for 18 h (Kumaran et al., 1997). After shaking, the mixtures were filtered using a sterilized muslin cloth and centrifuged at 4 � C (9000 rpm) for 30 min. The crude culture filtrates were analyzed to determine the initial and final pH of the filtrates, and the activity of the oxidative and hydrolytic enzymes. The crude filtrates were stored at 20 � C for 24 h prior to performing enzyme assays and sodium dodecyl sulfate-polyacrylamide gel electro­ phoresis (SDS-PAGE).

Two agro-industrial wastes, ground oil palm empty fruit bunches (EFB) and rubber wood sawdust (SD), together with vermiculite (V), were selected as solid substrates. The EFB, SD, and V particles ranged in size from 0.50 to 0.85 mm. The fresh EFB was collected from MPOBUKM Research Station, Bangi, Malaysia. Finely ground SD was collected from a rubber-milling factory, Seberang Perak, Malaysia, whereas the rice bran (RB) which is used as an additive was purchased from MARDI, Serdang, Selangor, Malaysia. The vermiculite was ob­ tained from a commercial agricultural company, Serdang, Malaysia. The lignocellulosic composition of the agro-industrial wastes is listed in Supplementary 1. 2.3. Solid-state cultivation Solid-state cultivation (SSC) was used to mass-produce cultures of the white-rot hymenomycetes according to a modified protocol by Chawachart et al. (2004). The fungi were cultivated in tissue-culture bottles (350 mL capacity) containing 16 g of either a single or a com­ bination of the solid substrate. The substrate ratios and nutrient amendments are listed in Supplementary 2. The moisture content of 75–85%, w/w was adjusted by adding distilled water to each bottle. The prepared substrate mixtures were sterilized for 30 min at 121 � C for three consecutive days. The sterilized substrates were inoculated with 20 g of P. sanguineus FBR or T. lactinea FBW spawn (Section 2.1), fol­ lowed by incubation in the dark at 28 � 2 � C under static conditions for 21 days. Quantitative enzyme assays were performed in quadruplets for each treatment after 7 and 21 days of incubation. Substrate formulations giving the better performance based on the mycelial growth extension rate and secretion of lignocellulolytic enzymes were selected for further studies. 2.4. Solid substrate selection 2.4.1. Mycelial growth during solid-state cultivation Sterilized Petri dishes measuring 14 cm in diameter were aseptically filled with 20 g of a sterilized formulation (Supplementary 2). The centre of each Petri dish was inoculated with spawn grains (2 g) of either P. sanguineus FBR or T. lactinea FBW and then incubated in the dark at 28 � 2 � C, with six replicates per treatment. The average mycelial diameter was measured as described in section 2.1. The average reading was plotted against time (day) to obtain the mycelial extension rate in mm/day.

2.1. Spawn preparation Barley, sorghum and wheat grains obtained from a commercial grocery shop (Bangi, Selangor, Malaysia) were evaluated to determine their suitability as spawn substrates for two white-rot hymenomycetes, Pycnoporus sanguineus FBR and Trametes lactinea FBW. One-week-old pure fungal cultures were obtained from culture stocks of P. sanguineus FBR and T. lactinea FBW, which were previously isolated and identified (Naidu et al., 2015). The substrates for spawn production were culti­ vated in tissue-culture bottles (350 mL capacity) using a modified method of Narh et al. (2011). The length of the mycelia from the edge of the inoculum plug to the edge of the radial growth was measured at two different angles with a mechanical ruler (millimetres) and recorded as the mycelium radial extension (Narh et al., 2011). After 14 days of in­ cubation, the mycelial fresh weight was determined. The most suitable grain substrate was selected for use as the spawn substrate in the solid-state cultivation studies.

2.4.3. Protein infiltration and storage The crude culture filtrates (section 2.4.2) were concentrated using 2

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Journal of Environmental Management 259 (2020) 110056

ultrafiltration (Amicon Ultra-15 Centrifugal Filter Unit with an Ultracel10 kDa MWCO membrane). The unpurified crude protein was stored at 20 � C for 24 h prior to performing the SDS-PAGE and native-PAGE (for zymogram analyses).

2.8. Statistical analysis All the experiments were conducted independently in duplicates, and the data presented here are the mean values with standard error (�SE). To achieve normality, homoscedasticity, and linearity of the data, some of the response variables were arcsine transformed. The data were analyzed using SAS statistical software (PC-SAS software V8.2, SAS Institute, Cary, NC, USA) for analysis of variance (ANOVA). Means were separated by Fisher’s Protected Least Significant Difference (LSD) test at 5% levels of probability. In addition, a principal component analysis (PCA) was performed to assess the correlation between the SSC, the two hymenomycetes, oxidative and hydrolytic enzymes production, biomass yield, and biomass loss. The data matrix consisted of six substrates (factor A), five parameters [three oxidative enzymes or three hydrolytic enzymes þ biomass yield and biomass loss] (factor B) and two hymenomycetes (factor C). The PCA Biplot was visualized by plotting the first column of the corresponding component matrices vs. the second column. The PCA analysis was constructed using XLSTAT Software (https://www.xlstat. com/en/).

2.4.4. Molecular weight determination Prior to zymography, SDS-PAGE was carried out according to the method described in Naidu et al. (2017). The zymogram analyses of CMCase, xylanase, and amylase were performed on native-PAGE as described by Manavalan et al. (2012), with slight modifications. The native-PAGE analyses were prepared using 10% separating gels that were incorporated with 1% carboxymethyl cellulose (20 μL CMC; Fluka, Switzerland), birchwood xylan (40 μL; Sigma-Aldrich 223), or starch (40 μL; Sigma-Aldrich 223) and a 4% stacking gel. For SDS-PAGE, total protein (10–15 μg) and loading buffer (ratio 1:4) were heated in a water bath at 98 � C for 5 min before loading in a sample well, whereas for zymography the total protein and the loading buffer were not heated (Adav et al., 2012; Manavalan et al., 2012). Electrophoresis was per­ formed at a constant 80 V for 2 h using a Mini-PROTEAN III (Bio-Rad), as described by Sarnthima et al. (2009) with slight modifications. To determine the activity of the hydrolytic enzymes, the native gel was removed from the glass plates, rinsed twice with distilled water and then incubated in sodium acetate buffer (50 mM, pH 7.0 and containing 40% isopropanol) for 15 min on an orbital shaker, followed by incu­ bation at 50 � C for another 30 min. CMCase and xylanase bands were developed after staining with 0.1% Congo red for 30 min followed by destaining with 1 mM NaCl for another 15 min. The reaction was fixed with 5% acetic acid solution for 10 min. Bands were visualized as clear zones under light and photographed using a NIKON DSLR D3100. To visualize the amylase band, the gel was incubated at 39 � C in phosphatecitrate-NaCl buffer (0.1 M phosphate citrate and 0.05 M NaCl buffer, pH 6.0) as described by Zaferanloo et al. (2013)). By contrast, for zymogram analyses of laccase and MnP activities, the native gels (without substrate) were rinsed twice with distilled water and stained as reported by Manavalan et al. (2012) and Naidu et al. (2017). Protein bands exhibiting laccase and MnP activities were stained orange under light and photographed using a NIKON DSLR D3100. The mo­ lecular weights of the proteins were determined by comparing them with standard protein markers ranging in size from 10 kDa to 250 kDa (Kaleidoscope™ Prestained Protein Standards, New England Biolabs, Ipswich, MA).

3. Results and discussion 3.1. Selection of grains as spawn stock Nwanze et al. (2008) have previously shown that the spawn grain and growth medium can have a significant effect on carpophore pro­ duction by basidiomycetes or hymenomycete fungi. Therefore, to select a suitable grain for use as spawn, the growth and multiplication of P. sanguineus FBR and T. lactinea FBW on barley, sorghum, and wheat grains was assessed. The mycelial grain weight of T. lactinea FBW was significantly (P � 0.05) higher when grown on wheat (5.55) than on sorghum (4.19) grains, whereas P. sanguineus FBR showed similar levels of growth on wheat (5.31 g) and sorghum (4.98 g), after 14 days of incubation. No fungal growth was observed on barley, which indicates that barley grains were not suitable for the growth of these hymenomycetes (Sup­ plementary 3). Both T. lactinea FBW and P. sanguineus FBR colonized sorghum more rapidly (within 6 and 7 days, respectively) than wheat grain (7 and 8 days, respectively); however, mycelial radial extension rates on wheat and sorghum grains were not significantly different (Supplementary 3). Based on these results, both wheat and sorghum appeared to be suitable grains for spawn production; however, wheat was selected as the spawn grain for inoculation in the SSC formulation because of the ready availability of wheat.

2.5. Estimation of protein biomass and substrate utilization After centrifugation of the culture filtrate (section 2.4.2), the fungal biomass (with substrate) of each of the solid-substrate cultures was measured using pre-weighed Whatman No. 4 filter papers to obtain the initial weight (WI). The biomass was then dried at 60 � C � 3 � C until a constant mass was reached to obtain the final dry mass (WF). Substrate utilization in terms of biomass loss was calculated using the following formula:

3.2. Solid substrate selection 3.2.1. Mycelial growth Six formulations comprising different agro-industrial wastes and their combinations were assessed to determine the most suitable solid substrate. The optimum SSC formulation was selected based on the minimum time required to colonize the entire substrate. T. lactinea FBW completely colonized the entire SDþV (T5) substrate in 7 days and demonstrated a significantly higher growth rate (18 � 1 mm/day) compared with that on other substrates (Supplementary 4a). By contrast, P. sanguineus FBR attained optimum growth rate (17 � 1 mm/ day) when grown on EFB alone (T1) (Supplementary 4b) and required 8 days to fully colonize the EFB substrate formulation. Lignin in lignocellulosic materials acts as a barrier for any solutions, enzymes or microorganisms, preventing them from penetrating the interior lignocellulosic structure or uses as a source of carbon and energy (Dashtban et al., 2010). White-rot fungi are able to degrade lignin, making other carbon sources (such as cellulose, hemicellulose, and amylase, which are more readily assimilated sources of carbon) avail­ able to microbial degradation (Dashtban et al., 2010; Saritha et al.,

2.6. Percentage biomass loss (%) ¼ {(WI–WF)}/(WI) � 100 Total nitrogen was analyzed according to the Kjeldahl method. True protein was calculated as the total nitrogen multiplied by 4.38, as illustrated by Kachlishvili et al. (2005). 2.7. Effect of incubation temperature on mycelial growth SSC of P. sanguineus FBR and T. lactinea FBW were subjected to different incubation temperatures (4 � C, 15 � C, 30 � C and 45 � 2 � C) to determine its stability at different temperature range. This study was carried out in Petri dishes in a similar manner as described in Section 2.4.1, with six replicates per treatment. The average reading was plotted against time (day) to obtain the mycelial extension rate in mm/day. 3

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2012). Thus, the presence of a ligninase system is necessary for the complete degradation of lignocellulosic materials (Lechner and Papi­ nutti, 2006). In this study, both T. lactinea FBW and P. sanguineus FBR degraded lignocellulose (substrates) competently. Besides the amount of nutrients added to substrates during fermentation, whether the substrate anchors the fungi during mycelial growth is another important consid­ eration when choosing a substrate for SSC (Hong et al., 2011).

Table 2 Correlation matrix [Pearson (n)] for combined principle component analysis of the formulations comprised of Pycnoporus sanguineus FBR and Trametes lactinea FBW – oxidative enzymes.

3.2.2. Biomass yield and substrate utilization by white-rot hymenomycetes under solid-state cultivation The growth of the two hymenomycetes on the agro-industrial wastes was confirmed by the increase in biomass yield content in the final biomass. After 7 days of incubation, the biomass yield of T. lactinea FBW was greatest when grown on the SDþV (17) substrate (76 mg 1 bottle, see Table 1 for an explanation of codes) and lowest on EFBþV (18) (34 mg 1 bottle). However, after 21 days of incubation, T. lactinea FBW growing on SDþV (23) showed a two-fold decline in biomass (37 mg 1 bottle) (Supplementary 5). By contrast, P. sanguineus FBR produced the greatest biomass yield (57 mg 1 bottle) after 21 days of incubation on EFB (7). The accumulation of true protein was generally observed on day 7 for T. lactinea FBW and on day 21 for P. sanguineus FBR, which cor­ responded to the production of oxidative and hydrolytic enzymes by these hymenomycetes. The pH of the different formulations tested during the SSC of P. sanguineus FBR and T. lactinea FBW ranged between 4.3 and 4.7 and pH 4.3 and 6.0, respectively (Supplementary 6). The pH ranges fall within the expected range reported previously (Levin et al., 2008; Eggert et al., 1996). Although, Levin et al. (2008) reported that enzyme pro­ duction by Trametes trogii was not affected by pH variations ranging between pH 3 and 6, they observed that an increase in pH triggered cellulolytic and hemicellulolytic enzyme activity. Furthermore, Eggert et al. (1996) observed that Pycnoporus cinnabarinus showed laccase ac­ tivity between pH 4.5 and 5.0 and that the oxidative enzymes seemed to be more enhanced under acidic conditions than on alkaline medium (pH 7 and 8). Suitable substrate utilization during SSC was clearly evident in terms of biomass loss (%). P. sanguineus FBR showed a significantly (P � 0.05) greater percentage of biomass loss during SSC on EFB on days 7 and 21 (codes 1 and 7, respectively, see Table 1 for an explanation of codes), on EFBþSD on days 7 and 21 (codes 4 and 10, respectively), and on SDþV on day 21 (11) than when grown on the other formulations. T. lactinea FBW showed significantly greater biomass loss when cultivated on SDþV for 7 days or 21 days (17 and 23, respectively), EFB for 7 or 21 days (13 and 19, respectively), and EFBþSD for 21 days (22) than when grown on the other formulations (Supplementary 7). Biomass yield positively correlated with biomass loss (Tables 2 and 3). Generally, the optimal substrates that contributed to significant (P � 0.01) biomass loss were EFB (T1) and EFBþSD (T4). Thus, the best substrate utilization

Code

Formulation Trametes lactinea FBW

Code

T1-EFBD7 T2-SDD7 T3-VD7 T4-EFBþSDD7 T5-SDþVD7 T6-EFBþVD7 T1-EFBD21 T2-SDD21 T3-VD21 T4-EFBþSDD21 T5-SDþVD21 T6-EFBþVD21

1 2 3 4 5 6 7 8 9 10 11 12

T1-EFBD7 T2-SDD7 T3-VD7 T4-EFBþSDD7 T5-SDþVD7 T6-EFBþVD7 T1-EFBD21 T2-SDD21 T3-VD21 T4-EFBþSDD21 T5-SDþVD21 T6-EFBþVD21

13 14 15 16 17 18 19 20 21 22 23 24

Laccase

MnP

LiP

Biomass loss (%)

Biomass yield

Laccase MnP LiP Biomass loss (%) Biomass yield

1

0.094 1 0.308 0.393

0.009 0.308 1 0.547

0.257 0.393 0.547 1

0.544 0.176 0.411 0.436

0.176

0.411

0.436

1

0.094 0.009 0.257

0.544

Values in bold are different from 0 with a significance level of alpha¼0.05. Abbreviations: LiP - lignin peroxidase; MnP - manganese peroxidase. Table 3 Correlation matrix [Pearson (n)] for combined principle component analysis of the formulations comprised of Pycnoporus sanguineus FBR and Trametes lactinea FBW – hydrolytic enzymes. Variables

CMCase

Xylanase

Amylase

Biomass loss (%)

Biomass yield

CMCase Xylanase Amylase Biomass loss (%) Biomass yield

1 0.177 0.044 0.344

0.177 1 0.637 0.280

0.044 0.637 1 0.073

0.344 0.280 0.073 1

0.029 0.074 0.090 0.436

0.074

0.090

0.436

1

0.029

Values in bold are different from 0 with a significance level of alpha¼0.05. Abbreviation: CMCase - carboxymethyl cellulase.

by the two hymenomycetes was in following descending order: EFB (T1) > EFBþSD (T4) > SDþV (T5) > SD (T2) > V (T3) > EFBþV (T6). 3.2.3. Production of lignocellulolytic enzymes during solid-state cultivation 3.2.3.1. Oxidative enzymes. Generally, oxidative enzymes in T. lactinea FBW had been triggered by day 7 of the incubation period, with maximum laccase production on day 7. Laccase production was signif­ icantly (P � 0.05) higher in SDþV (T5) (849.40 U mg 1 protein) and V (T3) (717.77 U mg 1 protein) (Fig. 1a). However, by day 21, laccase production on these two substrates had declined 11-fold and 19-fold, respectively. By contrast, P. sanguineus FBR produced little laccase on day 7, regardless of the SSC medium (<20.0 U mg 1 protein) (Fig. 1a). How­ ever, by day 21 laccase activity had increased significantly on all media, with a 4.3 fold increase on V (T3), followed by 4.2-fold increase on SDþV (T5), 4.0-fold increase on both EFBþSD (T4) and SD (T2) and 3.7fold increase on EFB (T1) (Fig. 1a). Therefore, the optimum substrates for significant laccase production were SDþV (T5) and V (T3) and the best laccase producer was T. lactinea FBW. The results presented in this study suggest that only a short incu­ bation period is needed to produce high levels of ligninolytic enzyme, which is in line with findings reported elsewhere (Akpinar and Urek, 2014; Goh et al., 2017; Kne�zevi�c et al., 2017; Reddy et al., 2003). These authors reported that maximum specific activities of oxidative enzymes were obtained for Pleurotus ostreatus, Pleurotus sajor-caju, Pleurotus eryngii and Trametes sp., respectively when cultured between 9 and 20 days. However, in the case of P. sanguineus FBR cultivated on EFB (T1), the optimum production of oxidative enzymes was obtained during the exponential fungal growth phase (21 days), in accordance with the findings of Mansur et al. (2003). Conversely, the current findings are not in agreement with those of Vikineswary et al. (2006), who reported that the maximum production of laccase enzymes was obtained on day 11 of SSF of P. sanguineus using sago hampas and rubber wood sawdust as substrates, followed by a decline in the production on day 21. These

Table 1 Number codes referring to the different treatments under this study. Formulation Pycnoporus sanguineus FBR

Variables

Abbreviations: EFB - oil palm empty fruit bunches; SD - rubber wood sawdust; V Vermiculite; D7 - Day 7; D21- Day 21. 4

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Fig. 1. Activity of oxidative enzymes secreted by Trametes lactinea FBW and Pycnoporus sanguineus FBR growing on different agro-industrial wastes under solid state cultivation (SSC) at 7 days (D7) and 21 days (D21). (a) Laccase, (b) manganese peroxidase (MnP), and (c) lignin peroxidase (LiP) production levels. Each value represents the mean of six replicates. Vertical bars indicate the standard error.

variations in maximum laccase production times might be due to utili­ zation of the different substrates (EFB vs. sago hampas and rubber wood sawdust) by the fungus, as suggested by Winquist et al. (2008). The findings are in corroboration with previous findings stating that the type of hymenomycete and substrate used in SSC influence the time at which oxidative enzymes are produced, regardless of the particular oxidative enzyme. Significantly higher MnP activity was recorded on day 7 by P. sanguineus FBR (42.51 U mg 1 protein) and (32.25 U mg 1 protein) SSC on EFB (T1) and SDþV (T5), respectively than on other substrates (Fig. 1b). High level was also obtained for the SSC of T. lactinea FBW on SDþV (T5) (23.96 U mg 1 protein) on day 7. By contrast, by day 21 the three biggest declines in MnP activity were P. sanguineus FBR on EFB (T1) (3.4-fold decline) and P. sanguineus FBR and T. lactinea FBW on SDþV (T5), which showed a 1.7-fold and 1.6-fold decline, respectively (Fig. 1b). Therefore, the best substrates for MnP production were SDþV (T5) and EFB (T1) and the best MnP producer was P. sanguineus FBR.

LiP activity showed a similar trend. Significantly (P � 0.05) higher LiP activity was attained on day 7 during SSC of P. sanguineus FBR (73.20 U mg 1 protein) and T. lactinea FBW (57.26 U mg 1 protein) on an EFB substrate (T1) than for the other treatments (Fig. 1c). The second highest LiP levels were produced on day 7 during SSC of T. lactinea FBW (45.38 U mg 1 protein) and P. sanguineus FBR (44.79 U mg 1 protein) on SDþV substrate (T5) (Fig. 1c). However, by day 21, LiP activity during SSC of T. lactinea FBW on EFB (T1) and SDþV (T5) substrates had declined 1.7fold and 1.3-fold, respectively. By contrast, LiP activity during SSC of P. sanguineus FBR on EFB (T1) had increased 1.4-fold by day 21 (Fig. 1c). Significantly more LiP activity was produced when using EFB (T1) as a substrate, followed by SDþV (T5) and EFBþSD (T4) and P. sanguineus FBR produced higher level of LiP than T. lactinea FBW. The addition of available carbon and nitrogen sources could be essential for the production of enzymes during SSC. The increase in oxidative enzyme production during SSC could be due to the addition of rice bran together with other carbon (sucrose) and nitrogen (yeast 5

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extract) sources during SSC on SDþV (T5) and EFB (T1). Similar ob­ servations were reported by several authors during SSF of T. versicolor supplemented with glucose (carbon)/yeast extract (nitrogen), T. trogii supplemented with glucose (carbon)/asparagine (nitrogen) and Tra­ metes pubescens supplemented with glucose (carbon)/peptone (nitrogen) on the secretion of laccase and LiP activities, respectively (Asgher and Iqbal, 2011; Galhaup et al., 2002; Levin and Forchiassin, 2001). More­ over, Trametes versicolor produced a significant level of MnP activity when both high levels of carbon and nitrogen were present in the me­ dium (Joung et al., 2001). Albeit, higher nitrogen levels are required for laccase production by Trametes sp., white-rot fungi such as P. sanguineus or P. cinnabarinus require carbon and nitrogen-limited culture conditions to enhance lac­ case production (Janusz et al., 2013; Pointing et al., 2000). This could explain the relatively low amount of laccase secreted by P. sanguineus FBR (on most of the substrates tested) compared with T. lactinea FBW in the present study. The genus Trametes and Pycnoporus are promising

lignin degraders due to their well-developed ligninolytic enzyme sys­ tems comprised of Mn-oxidizing peroxidases and laccases (Kne�zevi�c et al., 2013; Ramírez-Cavazos et al., 2014). 3.2.3.2. Hydrolytic enzymes. The production of CMCase, xylanase and amylase enzymes was enhanced during SSC of T. lactinea FBW on SD (T2) and/or a combination of SDþV (T5) and during SSC of P. sanguineus FBR on EFB (T1) after 21 days (Fig. 2a, b and 2c). The best CMCase producer was P. sanguineus FBR and the maximum xylanase and amylase activities were registered in T. lactinea FBW. The substantial production of endoglucanase, β-glucosidase and xylanase by the genus Pycnoporus and Trametes have been reported on culture broth and wood-based solid mediums, respectively (Coniglio et al., 2017; Levin et al., 2008). Apparently, the amylolytic ability of the genus Trametes has been quantitatively determined in comparison to the Pycnoporus (Okamoto et al., 2011; Olguin-Maciel et al., 2019). In the current study, the hydrolytic enzymes produced during the

Fig. 2. Activity of hydrolytic enzymes secreted by Trametes lactinea FBW and Pycnoporus sanguineus FBR growing on different agro-industrial wastes under solid state cultivation (SSC) for 7 days (D7) and 21 days (21D). (a) Carboxymethyl cellulase (CMCase), (b) xylanase, and (c) amylase production levels. Each value represents the mean of six replicates. Vertical bars indicate the standard error. 6

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SSC of P. sanguineus FBR on EFB were generally enhanced during the fungal exponential growth phase. The enhanced production of hydro­ lytic enzymes detected on day 21 was possibly due to the actively growing fungal hyphae producing hydrolytic enzymes that metabolized large quantities of polysaccharides instead of lignin found in the EFB. EFB is suggested as the most appropriate substrate for the cultivation of P. sanguineus FBR because it contains about 25–32% lignin, 24–34% hemicellulose, 41–46% cellulose, and 3.5% ash. In addition, EFB also contain sugars in the form of galactose (25%) and glucose (33.1%) (Thambirajah et al., 1995). These could be an abundantly available source of substrate for hydrolytic enzyme production by the respective fungus. Pycnoporus sanguineus FBR has been reported to grow well on agroindustrial wastes such as sago hampas, rubber wood sawdust (Vikines­ wary et al., 2006), oil palm frond parenchyma tissue (Annuar et al., 2010; Vikineswary et al., 2006), and pine sawdust (Gambato et al., 2016); however, the use of oil palm EFB as a substrate has received little attention. EFB has been evaluated as an alternative growth substrate for the cultivation of other basidiomycete fungi such as P. ostreatus (Ali et al., 2013), Auricularia polytricha (Abd Razak et al., 2013), Lentinus sp., Phanerochaete sp., and Ganoderma sp. (Maceno et al., 2016).

(on SDþV, T5) and P. sanguineus FBR (on EFB, T1) were elucidated by SDS-PAGE. Multiple bands with molecular mass ranging from 15 to 100 kDa are shown in Fig. 3a and b. The laccase enzyme migrated as a single isoform on 10% (w/v) native-PAGE, with an estimated molecular mass of ~50 kDa secreted by P. sanguineus FBR (Fig. 3b). Two isoforms of laccase were visualized for T. lactinea FBW with molecular masses of ~55 and 70 kDa (Fig. 3a). The MnP enzyme in P. sanguineus FBR was detected after staining with DMP as the substrate, to reveal a single protein band with a mo­ lecular mass of ~45 kDa corresponding to the position of MnP activity. Two isoforms of the MnP enzyme in T. lactinea FBW were detected with molecular masses of ~40 and 60 kDa. At the end of the incubation period (day-21), an isoform of the MnP band (~40 kDa) was only apparent. Both laccase and MnP isoforms were visible throughout the incubation periods on the native-PAGE for both of the tested hymenomycetes. An endoglucanase (CMCase) band with a molecular mass of ~60 kDa that corresponded to the endoglucanase band detected in albumin bovine serum (control) was also secreted in the crude extracts obtained from both hymenomycetes (Fig. 3a and b). In addition, although a xylanase band with a molecular mass of ~55 kDa was detected in crude extracts secreted by P. sanguineus FBR (Fig. 3b), the specific band was not visualized in crude extracts secreted by T. lactinea FBW (Fig. 3a). A zymogram pattern for amylase activity was not detected in the present study. Therefore, the zymogram protocol needs to be further optimized

3.4. Estimation of protein profiles The protein profiles of the crude extracts secreted by T. lactinea FBW

Fig. 3. Zymogram analysis of the crude extract from (a) Trametes lactinea FBW and (b) Pycnoporus sanguineus FBR during SSC on different agro-industrial wastes using native-polyacrylamide gel electrophoresis (resolving gel 10%). Lane 1, Marker; lanes 2 and 3, laccase activity on day 7 and 21, respectively; lanes 4 and 5, manganese peroxidase (MnP) activity on day 7 and 21, respectively; lanes 6 and 7, carboxymethyl cellulase activity on day 7 and 21, respectively; lanes 8 and 9, xylanase activity on day 7 and 21, respectively; lane 10, albumin bovine serum (BSA) – control. Marker size: 10–230 kDa (Blue Prestained Protein Standards, Broad Range, New England Biolabs). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.) 7

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to detect the amylase enzyme produced by these two particular hymenomycetes. The intensity of the bands elucidated by SDS-PAGE from the crude extracts secreted by P. sanguineus FBR during SSC on EFB (T1) or T. lactinea FBW on SDþV (T5) corresponded with the activities of the oxidative and hydrolytic enzymes, unlike those elucidated for other five substrates tested. However, the protein profiles obtained from the crude extracts secreted by either P. sanguineus or T. lactinea when grown on the EFBþV substrate were not apparent. Typical fungal laccases are described as glycosylated multi-copper proteins, which are produced as extracellular monomeric forms of approximately 60–80 kDa (Gonzalez et al., 2013; Patel et al., 2014). The majority of laccase mass secreted by genus Pycnoporus and Trametes generally have molecular mass between 55 and 80 kDa (Gonzalez et al., 2013; Ramírez-Cavazos et al., 2014; Thurston, 1994; Xiao et al., 2003). T. pubescens and T. versicolor grown on agro-industrial wastes produced two laccase isoforms with molecular masses of 100–120 kDa (Lac1) and 60 kDa (Lac2) (Gonzalez et al., 2013; Martínez-Morales et al., 2015). Meanwhile, MnP enzymes secreted by white-rot fungi usually have molecular mass ranging between 32 and 75 kDa (de Oliveira et al., 2009;

Shin et al., 2005), and two isoforms of MnP’s have been detected secreted by Trametes polyzona with molecular mass of 44 kDa (MnP1) and 42 kDa (MnP2) (Lueangjaroenkit et al., 2019). Laccases and MnP’s are widely distributed in fungi and bacteria. Many laccases have been reported from genus Trametes including T. polyzona (Chairin et al., 2014; Lueangjaroenkit et al., 2019), T. trogii (Campos et al., 2016), T. versicolor (Asgher and Iqbal, 2011; Asgher et al., 2016), T. hirsuta (Patil and Yadav, 2018) and T. maxima (Suman et al., 2018), however, none were reported from the species, T. lactinea. This is the first time that rubber wood sawdust and vermiculite were investigated as possible substrates for enzyme production and secretion of two isoforms of un-purified laccase and MnP’s under solid-state cultivation with indigenous strain, T. lac­ tinea FBW. Apparently, an endoglucanase (CMCase) was also detected during the SSC by this similar strain. Laccase secretion by P. sanguineus was well studied albeit, none of the findings have been reported on the enhancement and secretion of protein profiles during SSC using empty fruit bunches of oil palm. Interestingly, this is the first report to document a single isoform of unpurified laccase, MnP, CMCase and xylanase by the indigenous strain, P. sanguineus FBR for future biotechnological approaches. Fig. 4. Principal Component Analysis (Biplot) of the solid substrate cultivation (SSC) of Trametes lactinea FBW and Pycnoporus sanguineus FBR oxidative en­ zymes (a) and hydrolytic enzymes (b). SSC formula­ tions are indicated by blue dots (observations). Red lines (variables) indicate the production of enzymes, biomass yield, and biomass loss. Distinct clusters are indicated by green circles, the blue circle, and orange circles. Abbreviations: CMCase, carboxymethyl cellu­ lase; LiP, lignin peroxidase; MnP, manganese peroxi­ dase. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

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3.5. Principal component analysis (PCA)

palm-generated waste treatment in plantations under zero burning policy. The production of oxidative and hydrolytic enzymes in this study, as illustrated by the PCA Biplot analyses, was significantly gov­ erned by the fungal species and cultivation time. The enzyme profiles correlated with the utilization of the respective substrates in terms of biomass yield or biomass loss.

The first two principal components, PC1 and PC2, are shown in Fig. 4a, accounting for 72.59% of the variability [(43.29% (F1) and 29.29% (F2)]. Two distinct clusters were computed based on oxidative enzymes production by the tested hymenomycetes. SDþV (T5) (code 17) (see upper right of the PCA Biplot) was characterized as the main contributor for the variables laccase and biomass yield (refer to treatment codes in Table 1). The variables that ordinated on the lower right and left of the PCA Biplot produced low levels of laccase during SSC on SDþV (T5) (code 5) and EFBþV (T6) (codes 6 and 12), and low biomass yield on SDþV (T5) (code 5). The variables laccase and biomass yield were positively correlated (Fig. 4a; Table 2). On the lower right of the PCA Biplot, a second cluster was observed, in which EFB (T1) (code 7) was characterized as the main contributor for the variables LiP and biomass loss. These variables were low in EFBþV treatment (T6) (codes 6 and 18). Interestingly, the variable LiP was positively correlated with both biomass yield and biomass loss (Table 2). A subset of the second cluster was evident in which the EFB treatment (T1) (code 1) was characterized as the main contributor for the variable MnP. The variable MnP ordinated on the opposite side of the PCA Biplot for treatments EFBþV (T6) (code 18), EFB (T1) (code 19), EFBþSD (T4) (code 22) and EFBþV (T6) (code 24), which produced low levels of MnP during SSC (Fig. 4a). In the second PCA Biplot (Fig. 4b), the first two principal compo­ nents, PC1 and PC2, accounted for 64.87% of the variability [(34.59% (F1) and 30.28% (F2)]. Two distinct groups were computed based on the hydrolytic enzymes produced by the test hymenomycetes. On the upper left of the PCA Biplot, the EFB treatment (T1) (code 7) was characterized as the main contributor for the variables CMCase and biomass loss. Variable CMCase ordinated low for treatments V (T3) (code 21), EFBþSD (T4) (code 22), and EFBþV (T6) (code 24), which produced low levels of CMCase during SSC. Variable biomass loss ordinated low for treatments EFBþV (T6) (code 6), V (T3) (code 15), and EFBþV (T6) (code 18). Another subset of this cluster was formed by the SDþV treatment (T5) (code 17), which was characterized as the main contributor for the variable biomass yield, albeit biomass yield was low in treatments SDþV (T5) (code 5), EFBþV (T6) (code 6), and EFBþV (T6) (code 12). A second cluster on the upper right side of the PCA Biplot charac­ terized treatments SDþV (T5) (code 23) and SD (T2) (code 20) as the main contributors for the variable amylase (Fig. 4b). However, this variable ordinated low for treatments SD (T2) (code 8), V (T3) (code 9), EFBþSD (T4) (code 10), SDþV (T5) (code 11), and EFBþV (T6) (code 12). In another subset of the second cluster, treatments V (T3) (code 15), SDþV (T5) (code 23), and SD (T2) (code 20) were characterized as the main contributors for the variable xylanase. The variable ordinated on the opposite side of the PCA Biplot and was noted to be low for treat­ ments that showed similar xylanase production trend as those seen for amylase. Interestingly, the variable amylase correlated positively with xylanase (Table 3). The increase in biomass yield can be attributed to the secretion of extracellular enzymes (Chen et al., 2016). The present findings confirmed that the production of the oxidative enzymes laccase and LiP and the hydrolytic enzymes amylase and xylanase were significantly influenced by the biomass yield. By contrast, the production of these enzymes was ordinated on the opposite side to biomass loss, supporting the idea of a gradual decrease in biomass loss as the enzyme production increased. Interestingly, only the production of LiP (an oxidative enzyme) was influenced by biomass loss. This was the first attempt where strains of P. sanguineus and T. lactinea were mass cultivated using EFB of oil palm supplemented with rice bran and other C and N sources. The main reason for using EFB as a substrate was to provide a reservoir of lignin, cellulose, and hemi­ cellulose that could be consumed during the growth and multiplication of the white-rot hymenomycetes as part of the development of an oil

3.6. Effect of incubation temperature on the mycelial growth of white-rot hymenomycetes on the solid-state cultivation formulations To determine the temperature range suitable for SSC of P. sanguineus FBR and T. lactinea FBW, both were subjected to a range of tempera­ tures. Both fungi failed to grow at 4 � C and 45 � C, but grew well at 15 � C

Fig. 5. Growth rate (mm/day) of (a) Trametes lactinea FBW during solid state cultivation (SSC) on rubber wood sawdust and vermiculite (T5) and (b) Pyc­ noporus sanguineus FBR during SSC on oil palm empty fruit bunches (T1) at different incubation temperatures: 4 � C, 15 � C, 34 � C, and 45 � 2 � C. Each value represents the mean of six replicates. Vertical bars indicate the standard error. 9

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and 34 � C, with the fastest growth rate recorded on days 8–9 (Fig. 5a and b). T. lactinea FBW showed the fastest growth rate (1.10 mm/day, day 8) when cultivated on a substrate containing SDþV (T5), followed by P. sanguineus FBR (0.98 mm/day, day 9) when cultivated on EFB (T1). � These findings agree with those of Snajdr and Baldrian (2007), who reported that P. ostreatus achieved the highest levels of laccase and MnP production at temperatures ranging between 25 � C and 30 � C and that enzyme production peaked for T. versicolor at 35 � C. Buswell et al. (1996) also reported that 30 � C is an ideal temperature for producing the cellulolytic enzymatic complex secreted by fungi of the genus Pycnoporus. Based on the findings presented here, SDþV (T5) was selected as a solid substrate for cultivating T. lactinea FBW and EFB (T1) was selected for cultivating P. sanguineus FBR in future studies. T. lactinea FBW (PI 2015702852) and P. sanguineus FBR (PI 2015702850) were patented under the Intellectual Property Corporation of Malaysia (MyIPO) prior to the nursery evaluation. In addition, an acute oral toxicity test was carried out to estimate the acute toxicity of the chemicals (SSC formu­ lations comprising T. lactinea FBW and P. sanguineus FBR) and to provide an estimation of the median lethal dose (LD50) and its confidence level. The toxicity test service was conducted by the Industrial Biotechnology Research Centre, SIRIM Berhad, Malaysia. The two formulations are classified as Category 5 according to the Globally Harmonised System. As a result, the respective formulations are safe and recommended for use in oil palm plantations and could be applied during oil palm replanting as a strategy to degrade oil palm generated waste and mini­ mize the pest and pathogen survival in an eco-friendly manner.

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4. Conclusions The preference of T. lactinea FBW for lignin, hemicellulose and starch degradation over cellulose in SDþV formulation, and a hightemperature tolerance and colonization rate make the SSC of T. lactinea on SDþV ideal for future field applications to degrade oil palm-generated biomass or other agro-wastes. Likewise, the preference of P. sanguineus FBR for degrading components of oil palm EFB (lignin, cellulose, hemicellulose and starch), efficient substrate utilization and an ability to colonize the substrate at high temperatures, make the SSC of P. sanguineus on EFB suitable for use at a larger scale in field appli­ cations. Despite of being a green and cleaner technology, the developed formulations will be further tested under field condition in degrading oil palm trunks and root mass that had been felled, chipped or pulverized under zero burning waste management program. Author contribution section Yuvarani naidu: Conceived and designed the analysis, Collected the data, Contributed data or analysis tools, Performed the analysis, Wrote the paper, Other contribution. Yasmeen siddiqui: Wrote the paper, Other contribution. Abu seman idris: Other contribution. Acknowledgements The authors thank the Malaysian Palm Oil Board (MPOB) for finan­ cial assistance, granting permission to publish, and all the staff of the Ganoderma and Diseases Research for Oil Palm (GanoDROP) unit, Biology Division, MPOB, for their assistance. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.jenvman.2019.110056.

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