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Bioresource Technology Reports
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Sequential production of ligninolytic, xylanolytic, and cellulolytic enzymes by Trametes hirsuta AA-017 under different biomass of Indonesian sorghum accessions-induced cultures
Ade Andriani
a,⁎, Alika Maharani
b, Dede Heri Yuli Yanto
c, Hartinah Pratiwi
a, Dwi Astuti
a, Isa Nuryana
a, Eva Agustriana
a, Sita Heris Anita
c, A.B. Juanssilfero
a, Urip Perwitasari
a, Carla Frieda Pantouw
a, Ade Nena Nurhasanah
a, Vincentia Esti Windiastri
a, Satya Nugroho
a, Dwi Widyajayantie
a, Jajang Sutiawan
d, Yuli Sulistyowati
a, Nanik Rahmani
a,
Ratih Asmana Ningrum
a, Yopi
ea Research Center for Biotechnology, Indonesian Institute of Sciences, Jl. Raya Bogor, Km. 46, Cibinong, Bogor 16911, Indonesia
b Department of Biology, Faculty of Sciences, Institut Teknologi Sepuluh November, Surabaya, Indonesia
c Research Center for Biomaterials, Indonesian Institute of Sciences (LIPI), Jl. Raya Bogor, Km. 46, Cibinong, Bogor 16911, Indonesia
d Department of Forest Product, Faculty of Forestry, Bogor Agricultural University, Bogor, Indonesia
e Research and Human Resource Development Centre, National Standardization Agency, Tangerang, Indonesia
A R T I C L E I N F O Keywords:
Sorghum White rot fungi Ligninolytic enzymes Cellulase
Xylanase
Sequential enzyme production
A B S T R A C T
Biorefinery concept encourages agricultural sector for developing zero waste technology. Utilization of agri- cultural residues for producing valuable products has become an important strategy to be investigated. Sorghum (Sorghum bicolor L) biomass, as one of agricultural residues, is potentially of great significance as a lignocellulosic material for production of lignocellulolytic enzymessuch as Laccase (Lac), Manganese Peroxidase (MnP), and Lignin Peroxidase (LiP), Cellulases (Cel), and Xylanases (Xyl). In the present study, 13 Indonesian sorghum accessions (with different lignocellulosic characteristics) were investigated due to their potency as potential substrate for production ligninolytic, hemicellulolytic, and cellulolytic enzymes by Trametes hirsuta AA-017 which has ability to sequentially produce the enzymes. Three accessions (Samurai, 4183, Kawali) were found as most suitable materials for enzymes production by the fungus with highest enhancement were more than 8000 and 71-fold, respectively for Lac and MnP. Total lignin in the biomass has shown a positive correlation with Lac (Pearson coefficient = 0.630; P < 0.01) and MnP produced by the fungus (Pearson coefficient = 0.595;
P < 0.01). Optimized fermentation (pH, substrate concentration, incubation times, and mineral inducers) has been also conducted with the maximum Lac, MnP, LiP, Cel, and Xyl were 25.7 × 103, 46.7 × 103, 91, 540, and 670 U/L. This is the first report of sequential production of ligninolytic, cellulolytic, hemicellulolytic enzymes by Trametes hirsuta AA-017 using Indonesian sorghum biomass and its selective behaviour of the fungus on the biomass degradation. Sequential production of the enzymes have important economic benefits for production of biorefinery related enzymes.
1. Introduction
Lignocellulosic biorefinery is a renewable technology for production of bioproducts such as bioenergy and biochemicals from lignocellulosic materials. These materials are abundant in nature. Every year more than 40 million tons of inedible plant material, including agricultural residues, are produced and much of them are thrown away. Biorefinery concept encourages agricultural sector for developing zero waste
technology. Utilization of agricultural residues for producing valuable products has become an important strategy to be investigated (Sanderson, 2011; Kumar et al., 2020).
Among the agricultural plants, Sorghum bicolor L is one potential agricultural products to support the development of biorefinery.
Nowadays, sorghum application has broadened from food source to a potential renewable materials for bioproducts such as bioenergy and biochemicals primarily due to its high biomass yield and abundant
https://doi.org/10.1016/j.biteb.2020.100562
Received 18 July 2020; Received in revised form 10 September 2020; Accepted 11 September 2020
⁎Corresponding author at: Research Centre for Biotechnology, Indonesian Institute of Sciences (LIPI), Jl. Raya Bogor, Km. 46, Cibinong, Bogor 16911, Indonesia.
E-mail addresses: [email protected] (A. Andriani), [email protected] (D.H.Y. Yanto).
Available online 16 September 2020
2589-014X/ © 2020 Elsevier Ltd. All rights reserved.
T
availability in environment as agricultural residues (Whitfield et al., 2012; Diallo et al., 2019; Wahyuni et al., 2019; Hassan et al., 2020).
Sorghum can be classified as sweet sorghum (for sugar production), grain sorghum (for food and feed), forage sorghum, fibre sorghum, and energy sorghum (sweet sorghum in stems and biomass sorghum). Sweet sorghum has been used widely as sugars source for bioethanol pro- duction (Dar et al., 2018; Diallo et al., 2019). Some countries, such as USA, China, India and Belgium, have developed bioethanol from sap stems of sorghum in industrial scale (Suryaningsih and Irhas, 2014).
Sorghum biomass, which is categorized as agricultural residues, is promising industrial feedstock for bioproducts since it contains complex mixture of carbon sources such as lignin, hemicelluloses, and cellulose.
Indonesia has a huge diversity of sorghum plants including biomass sorghum cultivar. Wahyuni et al. (2019) reported that Indonesia has more than 30 accessions of sorghum plants with different character- istics of lignocelluloses content for various applications. Therefore, it is a chance to explore bioprospect of Indonesian sorghums for various applications. Cellulose content in the high biomass sorghum cultivar varying from 27 to 52%, while the hemicelluloses (17–23%) and lignin content ranges from 6.2 to 8.1% (Prakasham et al., 2014).The potential contents of sorghum lignocelluloses can be used as carbon sources and inducers for microorganism to produce various products such bior- efinery related enzymes.
Lignocellulosic degrading enzymes (LDEs) such as ligninolytic, cel- lulosic, and hemicellulosic enzymes have significant role for the pro- duction of second-generation biofuels. They can involve on the pro- cessing of lignocellulosic materials into sugar monomer such as glucose and xylose. Ligninolytic enzymes (LEs) such as Laccase (Lac), Manganese Peroxidase (MnP), and Lignin Peroxidase (LiP) have been widely used for delignification the materials on pretreatment process.
The enzymes are environmental friendly substitute for chemical pre- treatment which usually use strong acid or base solution in high tem- perature and pressure. Some benefits of this pretreatment is a low-cost and eco-friendly method for facilitating enzymatic hydrolysis (Mester and Tien, 2000; Plácido and Capareda, 2015; Masran et al., 2016; Su et al., 2018; Baruah et al., 2018). LEs are extracellular oxidative en- zymes which has broad range of substrates to degrade and commonly found in white rot fungi (WRF). In the present study, we used one potential WRF from our previous research, called Trametes hirsuta AA- 017 (Andriani et al., 2019). The fungus was found as the most potential producer of LEs among screened WRF, especially for Lac.
In this study, we also found the ability of T. hirsuta AA-017 to se- quentially produce cellulase and xylanase by using Indonesian sorghum accessions. Cellulases can convert the cellulose materials into sugar monomer such as β-glucose, shorter polysaccharides and/or oligo- saccharides while xylanase break down xylan materials into mono- saccharides such as β-xylose, shorter polysaccharides and/or oligo- saccharides (Takahashi et al., 2013; Obeng et al., 2017). The enzymes were produced by the fungus after the most optimum LEs were secreted (sequential production). Both enzymes, LEs and non-LEs (cellulase and
xylanase), were not produced simultaneously but in different incuba- tion times. Sequential production of those three enzyme groups in 1 batch culture has important economic benefits for production of bior- efinery related enzymes. The ability of WRF on lignocellulolytic en- zymes (LEs, cellulase, xylanase) production have been mentioned by some researchers (Cilerdzic et al., 2011; Vasina et al., 2017; Andriani et al., 2019). Irbe et al. (2014) have been reported that genus Trametes could secrete simultaneously cellulase, xylanase, Lac and MnP activities in different level of activity under wheat bran supported-solid state ferementation. Bhaumik et al. (2014) also reported that T. hirsuta is an efficient cellulase producer under wheat straw-supported solid state fermentation. In previous study, Vasina et al. (2016) also reported that Trametes hirsuta 072 could secrete various proteins with different phy- siological role such as for lignocellulolytic production. However, the study have not described the activity level of each enzyme such as cellulase and xylanase. Therefore the ability of this fungus on sequential production of LEs, cellulase, and xylanase and its selective behaviour on sorghum biomass degradation under one running batch have not been reported to date.
Considering the biotechnological importance of the enzymes, this study aims to observe the potency of 13 Indonesian sorghum accessions as potential lignocellulosic materials for sequentially production of biorefinery related enzymes: Lac, MnP, LiP, cellulase, and xylanase. We also investigated the optimized condition (potential sorghum accession, pH, substrate concentration, incubation days, and purification method) for fermentation by T. hirsuta AA-017 to produce high activity of these enzymes. This is the first report of sequential production of ligninolytic, cellulolytic, xylanolytic enzymes by the fungus using Indonesian sor- ghum biomass. Sequential production of these enzymes have important economic benefits for production of biorefinery related enzymes in 1 step culture. The enzymes have significant role for processing of lig- nocellulosic materials into sugar monomer which can be used for var- ious applications such as bioenergy and food industry.
2. Materials and methods 2.1. Microorganisms
The WRF strain used in this study was T. hirsuta AA-017. The fungus was cultivated at 25 °C on 2% malt extract agar (MEA) for 7 days in a disposable plastic Petri dish and maintained at 4 °C prior to use.
Nucleotide sequences of T. hirsuta AA-017 can be seen in Table 1. To obtain the sequences, DNA was extracted using genomic DNA extrac- tion with quick DNATM fungal/bacterial miniprep kit (Zymo Research, D6005). The extracted DNA was used as a template for PCR to amplify the ITS1-F (specific for higher fungi) and ITS4-B (specific for basidio- mycetefungi) regions. Products were then sequenced using PCR primers and the results of sequencing were compared with the National Center for Biotechnology Information (NCBI) GenBank database.
Table 1
Result of sequences of ITS1-F and ITS4-B regions for Trametes hirsuta AA-017.
Sample of fungus Sequences
Isolate T. hirsuta AA-017 Assembly of 2 sequence 623 bp
1 CCCTTCCGTA GGGGAACCTG CGGAAGGATC ATTAACGAGT TTTGAAATGG GTTGTTGCTG 61 GCCTTCCGAG GCATGTGCAC GCCCTGCTCA TCCACTCTAC ACCTGTGCAC TTACTGTAGG 121 TTGGCGTGGG TTTCTAGCCT CCGGGCTGGG AGCATTCTGC CGGCCTATGT ACACTACAAA 181 CTCTAAAGTA TCAGAATGTA AACGCGTCTA ACGCATCTTA ATACAACTTT CAGCAACGGA 241 TCTCTTGGCT CTCGCATCGA TGAAGAACGC AGCGAAATGC GATAAGTAAT GTGAATTGCA 301 GAATTCAGTG AATCATCGAA TCTTTGAACG CACCTTGCGC TCCTTGGTAT TCCGAGGAGC 361 ATGCCTGTTT GAGTGTCATG AAATTCTCAA CCCATAAGTC CTTGTGATCT ATGGGCTTGG 421 ATTTGGAGGC TTGCTGGCCC TAGCGGTCGG CTCCTCTTGA ATGCATTAGC TTGATTCCGT 481 GCGGATCGGC TCTCAGTGTG ATAATTGTCT ACGCTGTGAC CGTGAAGCGT TTTGGCAAGC 541 TTCTAACCGT CCATTAGGAC AATCTTTCAA CATCTGACCT CAAATCAGGT AGGACTACCC 601 GCTGAACTTA AGCATATCAA TA
2.2. Biomass sorghum preparation
Thirteen sorghum (Sorghum bicolor) accessions were obtained from the Indonesia Cereals Research Institute, Indonesia. The sorghum ac- cessions were cultivated in the experimental field located in the Cibinong Science Center (Bogor, Indonesia) based on previous research (Wahyuni et al., 2019). The sorghum accessions used in this study were Buleleng, JP, WR, Super 1, KS, Kawali, 181, Samurai KLR, Numbu, 4183, and SMM.
For assay, only sorghum straws were used. Sorghum biomass (SB) were harvested, removed their leaves, cut using disk mill and ring flaker to obtain smaller pieces of biomass, dried, and in final step, grinded using willey mill to obtain biomass powder (60 mesh).
2.3. Selection of most potential sorghum biomass for ligninolytic enzyme production
To investigate the most potential sorghum biomass(SB) for T. hirsuta AA-017 in the enzyme production, 13 local accessions were used for medium culture. GYP medium was used as culture medium for the fungus. GYP contains glucose (20 g/L), yeast extract (5 g/L), and peptone casein (5 g/L) and MgSO4.7H2O (1 g/L). The medium were added by SB with concentration 2.5% (g/L) and then adjusted to pH 5 using HCl solution. Sterilization of the medium culture was conducted using autoclave at temperature 121 °C for 15 min. Three fungal disks (d = ± 0.5 cm) were added into medium culture and incubated for 12 days. The culture was conducted at a 300 mL-Erlenmeyer flask containing 150 mL liquid culture (50% of volume) with agitation 150 rpm at room temperature. Sampling was conducted at 0, 2, 4, 6, 8, 10, and 12 incubation days for LEs analysis.
2.4. Effect of medium, ligninolytic inducers, pH, and concentration of substrate for LEs production
To investigate a suitable medium, ligninolytic inducer, pH and concentration of substrate for the production of LEs by the fungus, several conditions were conducted. For medium optimization, 4 types of culture medium were used: PDB (potato dextrose broth), MEM (malt extract medium), Natural Nutrition/NN (commercialized mushroom medium), and GYP (glucose yeast peptone). MEM contains malt extract (20 g/L), glucose (20 g/L), peptone (1 g/L), and sterile water. NN contains C organic (8.37%), N (5.24%), Mg (0.24%), Ca (0.53%), Fe (34 ppm), Zn (273 ppm), hormone-HA (39.04 ppm), hormone-zeatin (35.28 ppm), and hormone-GA7 (88.23 ppm). Each 30 mL was diluted by sterile water (17 L). GYP contains glucose (20 g/L), yeast extract (5 g/L), and peptone casein (5 g/L) dan and MgSO4.7H2O (1 g/L). PDB medium contains PDB broth with concentration 24 g/L in purified water.
Ligninolytic inducers used in this study were veratryl alcohol 0.1 mM (VA) as LiP inducer, CuSO4 0.1 mM, as Lac inducer, and MnSO4
0.1 mM as MnP inducer. Addition of the inducers was conducted at 5 days incubation of preculture to minimize inhibition effect for the fungus. The treatments were conducted under SB-induced GYP culture medium. Control was also conducted using same medium with no in- ducers added.
pH is an important role factor for microorganisms to grow and se- crete their metabolites such as enzymes. In the present study, various pH (3–9) were applied to the culture medium to investigate the most pH condition for the fungus on ligninolytic production. GYP was selected for culture media. HCl or NaOH solution (1 M) was used for adjusting the medium culture to obtain a certain pH.
Percentage or concentration of sorghum biomass (SB)for the culture was also conducted. Various concentrations of SB were used: 2.5, 5, 7.5, and 10% (w/v). For optimization, all cultures were harvested for 8 days incubation for LEs analysis.
2.5. Ligninolytic enzymes (LEs) assays
Prior to analysis, all samples were centrifuged with agitation 10,000 rpm for 20 min to obtain clear supernatant. Lac activity was determined using syringaldazine as the substrate and extracellular en- zymes in sodium acetate buffer (Zavarzina and Zavarzin, 2006). MnP activity was assessed in a reaction with 20 mM 2,6-dimethoxyphenol as the substrate, 50 mM malonate buffer (pH 4.5), 20 mM MnSO4, and 2 mM H2O2 (Takano et al., 2004). LiP activity was analyzed by mixing 2 mM H2O2 and veratryl alcohol as the substrate (Collins et al., 1997).
Enzyme activities were expressed in units per liter (U/L) for the liquid treatment, defined as the amount of enzyme required to oxidize 1 μmol of substrate in one minute.
2.6. Screening for cellulase and xylanase activities
Screening using Congo Red (CR) two layer-agar medium was con- ducted to investigate the potency of the fungus on cellulase and xyla- nase production. Malt extract agar medium was used as first layer or bottom layer in petridish. Carboxymethyl cellulose (CMC) was used as substrate for cellulase screening while commercial xylan was used as substrate for xylanase screening in agar medium with concentration 0.5% (w/v). The medium were used as second layer or upper layer in petridish. Four μL enzymes solution (with various incubation days) were added into agar medium and incubated for 3 days at 30 °C. CR solution (0.25% w/v) was added into medium agar for twice. Sodium chloride (1% w/v) was used as washing solution to make clear zone (Rahmani et al., 2019).
2.7. Scanning electron microscope (SEM) analysis
The surface morphology of untreated and treated biomass samples was examined using SEM (Hitachi TM3030 with ion sputter Hitachi MC1000). Both untreated and treated samples were gold coating prior to analysis. The treated samples used for the assay were fungal treated SB for 8 incubation days. The untreated sample for control was only SB with no fungal treatment. The samples were analyzed with 2000×
magnifications to obtain clear structures of SB.
2.8. Cellulase and xylanase activity
For cellulase and xylanase assays were analyzed using DNS method as described by Rahmani et al. (2019). For xylanase, xilan beechwood (0.5% w/v) was used as substrate. Reducing sugar was determined using dinitro salicylic acid (DNS) solution. Xylanase activity (XA) was determined in a reaction substrate xylan solution (250 μL) and enzyme solution (250 μL) for 15 min at 40 °C. Blank, control, and standard of xylose were also prepared for XA calculation.Enzyme activities were expressed in units per liter (U/L) defined as the amount of 1 μmol re- ducing sugar formed (measure as xylose) in one minute under assay condition.
Cellulase activity (CA) was also determined using DNS method as mentioned above. CMC solution (0.5% w/v) was used as substrate for the assay. Enzyme activities were expressed in units per liter (U/L) defined as the amount of 1 μmol reducing sugar formed (measure as glucose) in one minute under assay condition. As a control, 250 μL of enzyme solution was inactivated (T = 100 °C for 15 min), then mixed with 250 μL of substrate solution and 750 μL of DNS reagent to correct for the reducing sugars in the substrate and the enzyme solution.
2.9. Lignin analysis of SB
Lignin content of SB was determined by the thioglycolic acid lignin (TGAL) assay as previously reported by Wahyuni et al. (2019). Sorghum biomass remained in the medium were separated from the fungal my- celia and suspension. Suspension then filtered and the biomass
remained in the filtration were oven vacuum for 24 h. Fungal mycelia was dried and the biomass attached in the dried mycelia was separated manually. All the biomass were collected for further lignin content.
2.10. Statistical analysis
The F-test was conducted on data to assess the significance of dif- ferences. Differences between means at a confidence level of 5%
(P < 0.05) were considered to be significant. For correlation analysis of lignin content in SB and LEs production, Pearson correlation coeffi- cient was measured.
3. Results and discussion
3.1. Selection of most potential accession for ligninolytic enzymes (LEs) production
Thirteen of SB (Fig. 1) used in the present study have different characteristics of lignocellulose, including lignin content, lignin che- mical structures, polysaccharide composition, wall-bound phenolic contents, and enzymatic saccharification efficiency (Wahyuni et al., 2019). The 13 SB have been categorized into three groups due to their lignin content: high lignin (19.7–22.9%), moderate lignin
(17.9–18.4%), and low lignin (14.4–16.7%). Lignin is a natural sub- strate for WRF to secrete and induce their ligninolytic system. Different lignocelluloses content of biomass has been reported to have significant difference of LEs production by microorganisms such as WRF (Thompson et al., 1998; Andriani and Tachibana, 2016). Therefore, lignin content in SB might be has a correlation and important role with LEs production. In the present study, T. hirsuta, an indigenous WRF, was used as LEs producer under different accessions of SB.
Based on analysis of LEs production by T. hirsuta under 13 acces- sions of SB (Fig. 1), we found that the fungus produced different activity in different SB. T. hirsuta could produce 2 predominant enzymes, Lac (778–12,874 U/L) and MnP (755–8657 U/L). LiP was also produced, but in low activity (< 200 U/L). We also found that all 13 SB-treated cultures resulted higher LEs compared with control treatment-SB un- treated- (Lac = 1.56 U/L; MnP = 97.42 U/L, and LiP = 28.91 U/L).
Among 13 screened SB, we found that three accessions of SB (Samurai, 4183, and Kawali) showed the highest Lac (> 11,000 U/L) and MnP (6000 U/L). Highest Lac produced by T. hirsuta under supporting of 3 accessions were 12,874, 11,520, and 11,174 U/L for Samurai, 4183, and Kawali, respectively in 8 days incubation (Fig. 1a). Lac was en- hanced > 8000-fold for Samurai. Highest MnP was produced by the fungus under supporting of the 3 accessions were 6989, 6410, and 6146 U/L for Kawali, 4183, and Samurai, respectively (Fig. 1b). MnP Fig. 1. Production of Lac (a), MnP (b), LiP (c) in various sorghum biomass for 0, 4, 8, and 12 days (
= high lignin content SB = moderate lignin SB
= low lignin SB).
was enhanced > 71-fold for Kawali. Both Lac and MnP declined in 12 days incubation. LiP in all SB treatments was only produced in low activity with the highest was 172 U/L (Fig. 1c). Vasina et al. (2017) also reported similar result that LiP is less frequent in T. hirsuta compared to other studied basidiomycetes. However, in the present study, it could express high Lac and MnP.
Two of 3 potential accessions have high lignin content, 19.8 and 20.5%, respectively for 4183 and Samurai (both are high lignin SB group), while Kawali has lignin content > 18.4% (moderate lignin SB group). Fig. 1(a, b) shows the pattern Lac and MnP produced for low lignin SB group (KLR, Numbu, Buleleng, JP, Pahat) were lower com- pared other 2 groups. The highest lignin accession among screened SB (23%), KS, was not produced highest Les. Enzyme Lac and MnP for KS were 8123 and 4359 U/L, respectively in the optimum incubation day.
Lowest LEs produced under KS-induced culture might be due to its low enzymatic saccharification efficiency (ESE). Wahyuni et al. (2019) re- ported that among 30 Indonesian sorghum accessions, KS resulted the lowest ESE value with only 8.13%. A complex structure of biomass such as lignin content, and other cell wall components may affect their bioavaibility to biological attack (Reid, 2011). However, based on correlation study, we found that total lignin has positive correlation with Lac (Pearson coefficient = 0.630; P < 0.01) and MnP produced by the fungus (Pearson coefficient = 0.595; P < 0.01). The trend of this phenomena can be seen directly in Fig. 1.
Lignocellulosic characterics have important effects on the produc- tion of LEs by microorganisms such as WRF. In the present study, lignin content of SB was found to have positive correlation with LEs
production. Some studies also have reported various factors affected the ability of WRF on LEs production. Linares et al. (2018) stated that phenolic compounds released during lignocelluloses degradation could be toxic to cells and to inhibit enzyme activity produced by Phaner- ochaete chrysosporium. However, in tolerable concentration, phenolic compound could enhance the production of LEs by WRF (Cañas et al., 2007). Xie et al. (2016) reported that water holding capacity of lig- nocellulosic biomass (moisture content) and aeration were known to markedly affect the performance of fermentation. They reported that medium containing ramie stalks and kenaf stalks were found to best suitable cultivation medium for Pleurotus eryngii with biological effi- ciency achieved at 55% and 57%, respectively. Other factors such as nutrient (carbohydrate and protein), high degree porosity of biomass, inorganics content, C/N ratio, pH and other physical characteristics of lignocellulosic materials can affect LEs production by WRF (Andriani and Tachibana, 2016; Kumar and Chandra, 2020). In the present study, we used a kind of lignocellulosic material, SB, but in different acces- sions which contains different lignin content. We found that lignin content was a major cause for fluctuation of Les production by T. hir- suta. Fig. 1 shows for a correlation pattern for LEs production and lignin content in SB. High lignin SB group tends to have high LEs production, while low lignin SB group tends to have lower LEs production. There- fore, the use of high lignin SB group (especially Samurai, 4183, and Kawali) may be suitable accessions for LEs production in industrial scale.
This finding (high Lac and MnP produced by T. hirsuta under SB induction) were higher compared with several previous studies related to LEs production under induction of lignocellulosic biomass by this WRF. Cilerdzic et al. (2011) have been investigated the ability of T.
hirsuta to produce LEs using several agricultural residues such as wheat straw, cornstalks, mandarin orange, and orange peels. The highest ac- tivity of Lac (3827.0 ± 219.0 U/L) was noted in mandarin orange peels medium, while wheat straw was the optimum for MnP (1971.5 ± 23.0 U/L), respectively. Vasina et al. (2016) also reported that this fungus could produce Lac (1800 U/L), MnP (300 U/L) under CuSO4 and oat straw induction. Knežević et al. (2016) have reported that T. hirsuta BEOFB 301 under wheat straw induced-culture was 181.8 U/L. Our previous report (Andriani et al., 2019) also reported that this strain has highest activity > 2034 U/L when using sugar palm fruit cake as carbon source. Comparing all this findings, production of Lac and MnP under 3 potential accessions of SB showed excellent re- sults (Samurai, 4183, and Kawali) with very high activities even in unpurified forms/crude enzymes (> 11.000 U/L and > 6000 U/L for Lac and MnP, respectively in 8 days). Therefore, we can conclude that high lignin accessions of SB have potential advantages as high expres- sion LEs inducer for T. hirsuta.
Some studies of optimization for LEs production by other WRFs also showed that T. hirsuta under SB-supported GYP medium resulted higher LEs than several assayed WRF. Zhou et al. (2007) reported that under optimized condition (glucose feeding), MnP produced by P. chrysos- porium has only activity 200 U/L. Wattanakitjanukul et al. (2019) have investigated that maximum Lac and MnP activity produced by Xylaria sp under palm empty fruit bunch (PEFB) induced fermentation were of 16.3 and 24.8 U/g PEFB. However, in the present study, we found that T. hirsuta could produce both Lac and MnP in very high activity in the present of SB, up to 25.7 × 103 and 46.7 × 103 U/L.
3.2. Optimization production for ligninolytic enzymes by T. hirsuta AA-017 3.2.1. Optimization of incubation days for LEs production
To obtain optimum condition for T. hirsuta on LEs production under SB-supported culture, several optimization treatments such as incuba- tion days, pH, nutrient medium, LEs inducer, and SB concentration were conducted. We cultivated T. hirsuta using 3 potential SB accessions (Samurai, 4183, and Kawali) as LEs inducer in several incubation days (0–10 days). The highest Lac for Samurai, 4183, and Kawali were found Fig. 2. Production of Lac (a), MnP (b), and LiP (c) by 3 selected sorghum
biomass in various incubation times (0–12 days).
at 8 days incubation with Lac activity 12,874, 11,520, and 11,174 U/L, respectively (Fig. 2a). While the highest MnP for Kawali and 4183 were found at 6 days incubation with activity 46,796 and 36,544 U/L, re- spectively (Fig. 2b). For Samurai, the highest MnP was found at 7 days incubation with lower activity (6230 U/L). The highest LiP were found at 7 days for Kawali and 4183 and 8 days for samurai with low activity
(< 100 U/L) (Fig. 2c). Optimum LEs produced by WRF varies depend on fungal strain and their treatment. Optimum days for LEs production has been reported in the range 4–14 days. The incubation times using submerged culture commonly have shorter period compared with solid state fermentation/SSF (up to 60 days) (Widiastuti and Wulaningtyas, 2008; Babič et al., 2012). However, SSF has been reported can produce Fig. 3. SEM analysis of sorghum biomass before treatment: 4183 (a), Samurai (b), Kawali (c) and after treatment: 4183 (d), Samurai (e), and Kawali (f).
higher LEs than submerged culture.
In Fig. 3, the comparison of fungal treated SB (for 3 potential ac- cessions) and control (only biomass) using SEM analysis shows a sig- nificant structure changes of SB. The compact structures in untreated SBs have disminished after fungal treatment. These images also de- monstrate that the fungal treated SB exposed some internal areas in the biomass compared with the images for untreated samples. It also be- came cracked, perforated, and porous. The morphological alteration
indicated the disruption of linkages in treated-SB and the remarkable reduction of lignin content after fungal treatment. During the incuba- tion in early stage, the fungus has used lignin as its natural substrate and inducer for LEs production (Gai et al., 2014; Plácido and Capareda, 2015; Dong et al., 2019).
3.2.2. Optimization of pH and SB concentration for LEs production The pH was very crucial to production of LEs (Kumar and Chandra, 2020). In the present study, the pH of the culture medium (Samurai) was varied from 3 to 10. However, the optimal activity in 8 days in- cubation is mainly obtained in the pH range of 3.0–9.0. The highest Lac production (19,073 U/L) was obtained at pH 5.0. While the highest MnP was found at pH 6.0 (5062 U/L). Further rise in pH showed no increase in the production of enzyme. Alkaline (pH 7) might inhibit the LEs production by the fungus. This may be attributed to the poor my- celial growth at an elevated pH which may restrict the LEs production.
Several studies reported that WRF tends to grow better under acidic condition than basic condition (Andriani et al., 2016; Asgher et al., 2011). Similar result has been reported by Patel and Gupte (2016) with maximum enzyme production at pH 5.0 (12 × 104 U/g substrate). In the highest pH (pH 9), T. hirsuta also produced highly both Lac (> 5000 U/L) and MnP (> 1900 U/L). The WRF have ability to pro- duce various organic acids that can change the pH medium from basic Table 2
Effect of various ligninolytic inducer and nutrition on enzyme production using concentration sorghum 2.5% (5 day preculture and 3 day addition).
Treatment Enzyme activity (U/L)
Lac MnP LiP
Inducer VA 0.1 mM 12,276.3 2020 n.da
CuSO4 0.1 mM 19,758.5 3315 0.6
MnSO4 0.1 mM 10,215.6 1230 0.8
Control (No addition) 16,751.5 4394 2.4
Nutrition PDB 927.8 282 16.1
ME 881.1 312 0.3
Nutrisi Natural 50.2 49 2.6
GYP 16,751.5 4394 2.4
a n.d = not detected.
Fig. 4. Screening assay for xilanase and cellulase of produced enzyme in congo red medium (various incubation medium 1–10 days). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
pH (> 7) to acidic pH (< 7) (Silva et al., 2004).
Analysis of various concentration of SB was also conducted for ef- ficiency purpose. We found that 7.5% (w/v) was the most optimum condition for the fungus on LEs production. The highest Lac and MnP were > 25,000 and 14,000 U/L, respectively for 8 incubation days. LiP was detected in a very lower activity (< 100 U/L) compared with Lac and MnP. SB concentration above 10% resulted solid state for the fungus.
3.2.3. Optimization of nutrient medium and LEs inducers for LEs production Four medium culture (PDB, ME, NN, and GYP) and LEs inducers (VA 0.1 mM, CuSO4 0.1 mM, and MnSO4 0.1 mM) were investigated to obtain a suitable media culture for T. hirsuta under SB induction.
Among medium tested, GYP and CuSO4 0.1 mM resulted highest both Lac and MnP (Table 2), 16,751.5 and 4394 U/L, respectively. LiP ac- tivity were detected in very low activity in all medium (< 20 U/L). GYP might be provide a suitable nutrient content and ratio (C/N) for T.
hirsuta to grow well and produce certain metabolism products such LEs (Iranzo et al., 2004; Kumar and Chandra, 2020). The complexity or completeness of nutrient in the media seems has no significant effect for the fungus on LEs production. Media NN, as a commercial media for
mushroom with complex nutrient (as mentioned in method), had no effect for boosting LEs production. Lac, MnP, and LiP in NN med- iumwere only 50.2, 49.0, and 2.6 U/L, respectively. PDB and ME both resulted < 1000 U/L for lac and < 500 U/L for MnP, respectively.
While GYP could result > 16-fold for Lac and 10-fold for MnP com- pared with these two medium. GYP, with only contain glucose as an initial carbon source, yeast extract and peptone as protein sources, and essential mineral of MgSO4.7H2O could be a complementary for SB on high production of LEs by T. hirsuta. Microorganisms, such as fungi, have preferences for consuming nutrient media to produce certain metabolites (Tramontano et al., 2018). GYP–supported SB might be a suitable growing media for T. hirsuta on LEs production.
CuSO4 0.1 mM, as Lac inducer, could increase Lac activity ap- proximately 20% compared with control treatment (no CuSO4). Zhu et al. (2016) reported that among the tested substances (carbon and nitrogen source, phenolic compounds and metal ions), yeast extract and copper showed the strongest effect on laccase activity by Pleurotus sp.
Collins and Dobson (1997) also stated that the copper increased laccase transcript levels were detected within 15 min. However, in the present study the addition of copper ion to culture could only enhance not >
20% of Lac activity and not stimulated other LEs such as MnP or LiP activity. The effect of copper was not as high as addition of SB into the culture (> 8000-fold and 71-fold for Lac and MnP, respectively).
Therefore, SB (with specific accessions) can be used as a natural bioinducer for LEs for substituting the copper.
3.3. Sequential production for cellulase and xylanase by T. hirsuta AA-017 Since SB contains complex mixture of other natural carbohydrates such as cellulose and hemicelluloses, we investigated the ability of T.
hirsuta on non-LEs production such as cellulolytic and hemicellulolytic group such as cellulase and xylanase. Both are important enzymes for biprocessing of lignocellulosic materials into sugar monomer such as glucose and xylose. Furthermore the sugar monomer can be converted into various biochemicals and biofuels via fermentation (Farinas et al., 2010; Song et al., 2016). Together with LEs, cellulase and xylanase are categorized as important enzymes for biorefinery purpose.
As preliminary study, qualitative study using double layer agar medium containing specific substrate were conducted to detect the presence of cellulase and xylanase in SB containing culture medium (CMC for cellulase and xylan for xylanase). The presence of both en- zymes were analyzed from 1 to 10 days incubation with clear zone detection in the congo red containing agar medium. Based on the re- sults (Fig. 4), cellulase were detected initially at 8 incubation days (in 2 accessions of SB, Samurai and Kawali). The clear zone showed bigger as the increasing incubation days (8–10 days). Xylanase was detected in earlier incubation days than cellulase (started at 7 incubation days) and the clear zone also became bigger by the days (up to 10 days). Cellulase was found in only in 2 SB accessions, while xylanase was detected in all three potential SB (4183, Samurai, and Kawali). Congo red assay is usually used for qualitative analysis of polysaccharides degrading en- zymes such as cellulase and hemicellulase. Congo red is capable of forming complexes with polysaccharides in a helical conformation and causing a red zone. Clear zone in the agar medium (as shown in Fig. 4) indicates the hydrolysis of polysaccharides into sugar monomer such as glucose and xylose (Baharuddin et al., 2010; Gupta et al., 2012).
Quantitative analysis for cellulase and xylanase using DNS method to measure the activity (Fig. 5). Highest cellulase and xylanase were found after 12 and 10 incubation days with activity 540 and 670/L, respec- tively.
In the present study, the effect of glucose in SB (Samurai)-supported culture was analyzed (Table 3). We have found that the presence of glucose could boost the production of Lac and MnP > 2-fold and 5-fold, for 8 and 4 days respectively. However, the presence of glucose in the culture inhibited the production of cellulase and xylanase in the early stage of incubation. No cellulase and xylanase were detected in the Fig. 5. Xilanase (a) and cellulase (b) produced by Trametes hirsuta AA-017.
Table 3
Effect of glucose in the production of LDEs in the SB-supported culture (Samurai Kultivar).
Treatment Incubation days Enzyme activity (U/L)
Cellulase Xylanase Lac MnP LiP
No glucose 4 70 126 n.d 956 n.da
6 82 368 5258 7108 n.d
8 84 319 6341 5438 n.d
Glucose 4 n.d n.d 3473 1746 n.d
6 n.d n.d 6210 37,087 n.d
8 n.d n.d 12,875 6147 55
a n.d = not detected.
culture up to 8 incubation days. However, the activities of both en- zymes were detected in no glucose-treatment. Xylanase was produced 4-fold higher than cellulase in the treatment. Some previous studies (Xiao et al., 2004; Hsieh et al., 2014) have reported that the presence of monosaccharides such as glucose could inhibit the production of cel- lulase. In contrast, the presence of glucose stimulated both fungal biomass growth and the LEs production. Similar result has been also reported in other WRF, P. chrysosporium (Zhou et al., 2007). Since lignin is outer part of the biomass, high lignin production in the early in- cubation days is necessary. Therefore, glucose was used as initial carbon source to stimulate the fungal growth. We also analyzed lignin content before and after fungal treatment. Higher degradation of lignin of SB was found in glucose-supported culture. Lignin content after treatment was 11% (or 42% degradation of initial lignin) in addition of glucose and 13% (or 31% degradation of initial lignin) with no addition of glucose.
In lignocellulosic materials, lignin acts as a barrier to any solutions or enzymes and prevents penetration of LEs to the interior of lig- nocellulosic structure due to its recalcitrant characteristics (Leonowicz et al., 1999; Dashtban et al., 2010; Masran et al., 2016). Due to the mechanism of WRF on lignocellulose degradation, WRF can be divided in 2 groups. For the first, WRF which attack lignin, hemicellulose and cellulose simultaneously. Some WRF have been found to have this characteristics, such as T. versicolor (Tanaka et al., 1999), P.e chrysos- porium (Dashtban et al., 2010), and Irpex lacteus (Xu et al., 2009). For the second group, WRF which selectively attack lignin before degrade other components of cell wall (cellulose and hemicelluloses). Some examples are Ceriporiopsis subvermispora (Guerra et al., 2004), Physis- porinus rivulosus (Hilden et al., 2007) and Phlebia spp (Arora and Sharma, 2009). These type of WRF degrade the lignocellulosic materials in a sequential manner. In the present study, we also found that T.
hirsuta degrade SB in the selective manner (Fig. 6). Under SB supported- GYP, after LEs were produced (2–12 days), the fungus produced se- quentially cellulase and xylanase after 8 days incubation. Both enzymes were produced after highest peak for LEs was obtained. It indicates a
selectively manner of T. hirsuta on lignocellulosic degradation. Some studies have reported the ability of this fungus on cellulase and xyla- nase production (Cilerdzic et al., 2011; Vasina et al., 2016), however the mechanism of T. hirsuta on lignocelllulosic biomass degradation via sequentially production of LEs, xylanase, and cellulase, have not been reported to date. Sequential production of the enzymes have important economic benefits for production of biorefinery related enzymes. The enzymes have significant role for the processing of lignocellulosic ma- terials into sugar monomer.
4. Conclusions
Three accessions (Samurai, 4183, Kawali) were found as most sui- table materials for enzymes production by the fungus. Total lignin in the biomass has shown a positive correlation with Lac (Pearson coef- ficient = 0.630; P < 0.01) and MnP produced by the fungus (Pearson coefficient = 0.595; P < 0.01). T. hirsuta could also sequentially produce cellulase and xylanase after LEs completely produced. The maximum Lac, MnP, LiP, Cellulase, and Xylanase were 25.7 × 103, 46.7 × 103, 91, 540, and 670 U/L, respectively for 8, 5, 8, 12, and 10 incubation days. The use of high lignin SB may be convenient for the industrial production of lignocellulosic degrading enzymes.
CRediT authorship contribution statement
Ade Andriani: Conceptualization, Methodology, Investigation, Writing - original draft. Alika Maharani: Investigation, Formal ana- lysis. Dede Heri Yuli Yanto: Supervision, Writing - review & editing, Data curation. Hartinah Pratiwi: Investigation. Dwi Astuti: Resources.
Isa Nuryana: Investigation. Eva Agustriana: Investigation, Software.
Sita Heris Anita: Investigation. A.B. Juanssilfero: Validation. Urip Perwitasari: Writing - original draft. Carla Frieda Pantouw:
Resources. Ade Nena Nurhasanah: Resources. Vincentia Esti Windiastri: Resources. Satya Nugroho: Supervision. Dwi Widyajayantie: Investigation, Resources. Jajang Sutiawan:
Fig. 6. Mechanism degradation of sorghum biomass by Trametes hirsuta AA-017.
Investigation, Resources. Yuli Sulistyowati: Resources. Nanik Rahmani: Validation. Ratih Asmana Ningrum: Project administra- tion. Yopi: Funding acquisition, Supervision.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influ- ence the work reported in this paper.
Acknowledgments
This research was supported by Program of National Priority 2019
“DIPA 052-bioprocess technology to enhance the production of bioca- talyst and bioenergy” (Research Center for Biotechnology-LIPI). The authors also acknowledge the facilities, the scientific and technical assistance of the Integrated Laboratory of Bioproducts at the Indonesian Institute of Science. This research was also supported by Center of Excellence “Integrated Biorefinery”, Research Center of Biotechnology LIPI.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.biteb.2020.100562.
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