Production Of Ligninolytic Enzymes Research Articles

Scale‐up and economic analysis of the production of ligninolytic enzymes from a side‐stream of the organosolv process

AbstractBACKGROUNDOrganosolv pretreatment produces a hemicellulose‐rich stream with limited applications due to its toxicity. In this study, beech wood organosolv liquor was valorized through the production of ligninolytic enzymes, the extraction of ganoderic acid and the detoxification of this process stream.RESULTSLaccase and manganese peroxidase (MnP) were produced in 30 L bioreactors by the white‐rot fungi Ganoderma lucidum and Irpex lacteus, respectively, using the hemicellulose‐rich liquor as carbon source. Both fungi caused a significant reduction in the hemicellulose stream toxicity (between 6.1 and 8.3 times lower). The extraction of ganoderic acid (24.3 mg g−1 of G. lucidum biomass) as a fungal metabolite with high pharmacological activity greatly increased the profitability of the process. Finally, the economic viability of the proposed valorization strategy was analyzed on the basis of the operational costs estimated for the process scale‐up. The specific costs of MnP and laccase production were €2.27 kU−1and €1.08 kU−1, respectively, which are 1109 and 78 times lower than the average price of commercially available enzymes.CONCLUSIONIt was found that the integration of the proposed valorization strategy into an organosolv‐based biorefinery would help to increase the economic feasibility of this pretreatment technology. © 2018 Society of Chemical Industry

Application of a combined fungal and diluted acid pretreatment on olive tree biomass

A biological pretreatment of olive tree biomass (OTB) was carried out. First, seven white-rot fungi (WRF) were screened on solid-state fermentations by analyzing the substrate composition, ligninolytic enzymes production and enzymatic hydrolysis yields at three different pretreatment times (15, 30 and 45 days). Glucose released by enzymatic hydrolysis of OTB pretreated with Irpex lacteus for 45 days doubled that obtained with the control (non-inoculated). In addition, to enhance this yield, the combination of fungal pretreatment with a chemical pretreatment was studied. It was also found that the order of the pretreatment combination has a relevant effect on the glucose yield. Thereby, the best option determined, fungal pretreatment with I. lacteus after 28 days of culture followed by diluted acid pretreatment (2% w/v H2SO4, 130 °C and 90 min), enhanced 34% the enzymatic hydrolysis yield compared with the acid pretreatment alone. Applying the best pretreatment combination, the overall sugar yield of the whole process (sequential pretreatment plus enzymatic hydrolysis) was 51% of the theoretical yield.

High level production of laccases and peroxidases from the newly isolated white-rot basidiomycete Marasmiellus palmivorus VE111 in a stirred-tank bioreactor in response to different carbon and nitrogen sources

Abstract The present work was carried out to determine the best sources and concentrations of carbon and nitrogen in the production of laccases, total peroxidases and manganese peroxidase by Marasmiellus palmivorus VE111. Among the carbon and nitrogen sources used, the combination of glucose and casein led to higher production of laccases (5134 U/mL), total peroxidases (187 U/mL) and manganese peroxidase (57 U/mL). Central composite rotatable design (CCRD) was used to determine the optimum concentrations of glucose and casein to produce enzymes. The experiments with better results were scaled up in a 5 L bioreactor. It was verified that lower concentrations of glucose (5 and 10 g/L) stimulated higher activity of total peroxidases (1285 U/mL) and laccases (3420 U/mL), respectively, while a higher concentration of glucose (30 g/L) favoured the production of manganese peroxidase (59 U/mL). These data indicate high performance of M. palmivorus VE111 in the production of ligninolytic enzymes.

Biofuels: Production of fungal-mediated ligninolytic enzymes and the modes of bioprocesses utilizing agro-based residues

Abstract The depletion of fossil fuels and rise in their global energy consumption and demand have had a major impact on the stability of the ecosystem and highlighted the need for efficient, sustainable, and renewable alternative sources of energy. Lignocellulosic biomass-based biofuels are highly advantageous due to their enormous supply in nature. Enzymatic bioconversion of lignocellulose polysaccharides into monomeric sugars has a higher efficiency than traditional chemical modes of action. Fungal-mediated ligninolytic enzymes offer even greater advantages in the bioconversion of lignocelluloses into simple sugars due to their thermostability, activity across a wide range of pH values, high specificity, and minimal by-products. This review, based on recent developments in the field of fungal-derived ligninolytic enzymes, discusses their mechanisms of action along with their production and the modes of bioprocesses involved as well as different techniques, such as heterologous gene expression, mutagenesis, and co-culturing, that enhance production and improve catalytic and stability properties.

Effect of copper ions on the ligninolytic enzyme complex and the antioxidant enzyme activity in the white-rot fungus Trametes trogii 46

White-rot fungi of the Phylum Basidiomycota are quite promising in ligninolytic enzyme production and the optimization of their synthesis is of particular significance. The aim of this study was to investigate the effect of enhanced concentration of copper (Cu) ions (25–1000 μg/ml) on the activity of the ligninolytic enzyme complex (laccase, Lac; lignin peroxidase, LiP; Mn-peroxidase, MnP) in Trametes trogii 46, as well as the changes in the antioxidant cell response. All concentrations tested reduced significantly in growth and glucose consumption. Cu ions affected the ligninolytic enzyme activity in a dose dependent manner. Concentrations in the range of 25–100 μg/ml strongly stimulated Lac production (a 5–6-fold increase compared to the control). LiP activity was also induced by Cu, with the peak value being recorded following exposure to 50 μg/ml metal ions. In contrast, the addition of Cu ions had a positive effect on MnP activity at a concentration higher than 100 μg/ml. The maximum enzyme level was achieved at 1000 μg/ml. The results obtained on superoxide dismutase and catalase activities indicated that exposure of T. trogii 46 mycelia to Cu ions promoted oxidative stress. Both enzyme activities were co-ordinately produced with Lac and LiP but not co-ordinately with MnP.

Effect of Domestic Wastewater as Co-Substrate on Biological Stain Wastewater Treatment Using Fungal/Bacterial Consortia in Pilot Plant and Greenhouse Reuse

In this study, a pilot wastewater treatment plant was used to evaluate the co-treatment of biological-staining residues and domestic wastewater under non-sterile conditions. A novel microbial consortia formed by Trametes versicolor, Trametes sp, Pleurotus ostreatus, Pseudomonas fluorescens, Pseudomonas azotoformans, Pseudomonas sp, Enterobacter xianfangensis and Bacillus subtillis was inoculated in an extended aeration type bio-reactor. The treatment units were operated during three consecutive cycles during a period of 147 h. After the last operating cycle, the concentrations of Chemical Oxygen Demand, Biochemical Oxygen Demand, Color Units, Total suspended solids, and the pH value were 1695 mg/L, 105 mg/L, 106 CU, 5), 1367 (CU), 566 mg/L (TSS) and 7.0 (pH) respectively. The reduction of pollutants load was related with the ratio of the two types of wastewater (3.5:0.5) combined to increase biodegradability, the concentration of fungi and bacteria used in the consortia (30 × 103 - 55 × 106 CUF/mL Total Fungi and 70 × 107 - 83 × 108 CFU/mL of Total Bacteria) and ligninolytic enzymes production, Laccase (13 - 96 U/L), MnP (9.8 - 39 U/L) and LiP (0.3 - 5.3 U/L). The post-treated effluent was used as irrigation water. Lolium perenne plants were watered during 60 days with post-treated effluent. The results of root weight showed that there are significant differences between the initial water and the effluent obtained after the operational cycles (p = 0.00470). The highest root weights (1 - 1.12 g) were found in plants irrigated with water obtained from the last treatment cycle.

Enhanced Delignification of Lignocellulosic Biomass by Recombinant Fungus Phanerochaete chrysosporium Overexpressing Laccases and Peroxidases

Ligninolytic enzyme production and lignin degradation are typically the rate-limiting steps in the biofuel industry. To improve the efficiency of simultaneous bio-delignification and enzyme production, Phanerochaete chrysosporium was transformed by shock wave-induced acoustic cavitation to co-overexpress 3 peroxidases and 1 laccase and test it on the degradation of sugarcane bagasse and wheat bran. Lignin depolymerization was enhanced by up to 25% in the presence of recombinant fungi in comparison with the wild-type strain. Sugar release on lignocellulose was 2- to 6-fold higher by recombinant fungi as compared with the control. Wheat bran ostensibly stimulated the production of ligninolytic enzymes. The highest peroxidase activity from the recombinant strains was 2.6-fold higher, whereas the increase in laccase activity was 4-fold higher in comparison to the control. The improvement of lignin degradation was directly proportional to the highest peroxidase and laccase activity. Because various phenolic compounds released during lignocellulose degradation have proven to be toxic to cells and to inhibit enzyme activity, a significant reduction (over 40%) of the total phenolic content in the samples treated with recombinant strains was observed. To our knowledge, this is the first report that engineering P. chrysosporium enhances biodegradation of lignocellulosic biomass.

Effect of Moisture Content and Inoculum Size on Cell Wall Composition and Ethanol Yield from Switchgrass after Solid-State Pleurotus ostreatus Treatment

Abstract. The white-rot fungus is capable of selectively degrading lignin over polymeric sugars. Solid-state cultivation and subsequent simultaneous saccharification and fermentation for ethanol production were performed. Effects of moisture content (MC) and fungus inoculum on biomass degradation, ligninolytic enzyme, and ethanol production were evaluated. First, fungal pretreatment was performed with varied MC and inoculum levels and sampled every 20 days. The highest xylose yield observed was 15.6% for samples with 75% MC and 5 mL inoculum at fungal pretreatment of 40 days. The highest lignin degradation of 52% and highest ethanol yield of 31% (based on the glucan present in the raw switchgrass) were achieved for 80-day fungal-treated samples with 75% MC and 5 mL inoculum. Keywords: KLywords. Bioenergy, Fungal pretreatment, Oyster mushroom, Perennial grass.

Effects of Different Induction Media as Inducers on Laccase Activities of Pleurotus ostreatus Strains in Submerged Fermentation

Sequential submerged cultivation with different induction media as inducers of ligninolytic enzyme production by Pleurotus ostreatus strains was assessed by measuring laccase activities. An unconventional material, alkali lignin, was used for the first time as an inducer for different strains to enhance laccase activity. The P. ostreatus strains secreted similar but relatively high levels of laccase activity when the induction media contained alkali lignin with or without glucose. The laccase enzyme of different P. ostreatus strains in the different media exhibited large differences, and the wild strain YAASM 0568 exhibited enhanced production of laccase compared to other cultivated strains. The laccase activities of wild strain YAASM 0568 were nearly 3.4-, 3.3-, and 5.4-fold higher than that for cultivated strains CCMSSC 00322, CCMSSC 00406, and CCMSSC 00336, respectively, when the induction media contained alkali lignin, inorganic salt, and vitamin B1. In general, induction media containing alkali lignin with or without glucose were favorable for laccase secretion. The results revealed that the type of induction material and the nature of the fungus play important roles in the expression of ligninolytic enzymes. These findings would be helpful for selection of the appropriate type of strain and for optimization of integrated industrial ligninolytic enzyme production.

Simultaneous valorization and detoxification of the hemicellulose rich liquor from the organosolv fractionation

Fractionation of lignocellulosic biomass with solvents (organosolv process) generates a hemicellulose-rich liquor with a high content of phenolics which is particularly toxic. This work addresses the utilization of this stream as a potential carbon source for the production of ligninolytic enzymes (LE). Among six basidiomycetes species, Irpex lacteus and Ganoderma lucidum presented the highest activities of manganese peroxidase (646 ± 122 U L−1) and laccase (1497 ± 161 U L−1), respectively, growing on a medium composed mainly of cellulose fibers, lignin and hemicellulose. The influence of each lignocellulosic fraction on the LE production mechanisms was studied in more detail. The high concentration of phenolic compounds in the hemicellulose-rich stream acted as inducer of LE production, with levels even greater than those of xylose. Acute toxicity tests on Vibrio fischeri revealed a substantial reduction of the toxicity after the fungal treatment (by ca. 12–21 times). The proposed valorization and detoxification of this currently non-exploited and abundant by-product stream is a promising strategy to enhance the industrial feasibility of the organosolv fractionation process.

Vinasse degradation using Pleurotus sajor-caju in a combined biological – Electrochemical oxidation treatment

Abstract Industrial wastewater treatment, by combined processes, has recently been seen as a novel and economically attractive strategy. The use of Pleurotus sajor-caju , followed by electrochemical oxidation (EO) for sugarcane vinasse degradation was investigated. Vinasse degradation treatment with P. sajor-caju yielded efficient color removal (97%), turbidity (99%), chemical oxygen demand (COD) (50.6%), and total organic carbon (TOC) (57.3%). Ligninolytic enzyme production was seen, and the highest laccase activity were obtained after 12th day of fermentation (varying between 341–400 UI L –1 ) and for peroxidase manganese the highest activity was seen between the 9th and 12th day of fermentation (varying between 1400–1494 UI L –1 ). Concomitantly, the mycelial biomass presented a high concentration (13.63 g L –1 ) on the 15th day of growth. In optimized conditions, the EO using as anode Ti/(RuO 2 ) 0.7 (IrO 2 ) 0.1 (Sb 2 O 3 ) 0.2 further degraded vinasse, reaching a final color removal of 100%, COD of 66% and TOC of 61%. This additional procedure allowed the elimination of vinasse toxicity reaching 0% lethality for Lactuca sativa and Raphidocelis subcapitata.

Characterization of ligninolytic enzyme production in white-rot wild fungal strains suitable for kraft pulp bleaching

Fungal strains identified by phylogenetic analysis of the ITS rDNA region as Trametes versicolor (CMU-TA01), Irpex lacteus (CMU-84/13), and Phlebiopsis sp. (CMU-47/13) are able to grow on and bleach kraft pulp (KP) in a simple solid-state fermentation (SSF) assay conducted in Petri dishes. Kappa number reductions obtained with Phlebiopsis sp. (48.3%), T. versicolor (43%), and I. lacteus (39.3%), evidence their capability for lignin breakdown. Scanning electron microscopy images of KP fibers from SSF assays demonstrated increased roughness and striation, evidencing significant cell wall modification. T. versicolor produces laccase (Lac), manganese peroxidase (MnP), and lignin peroxidase (LiP) in potato dextrose broth (PDB), PDB+CuSO4, and PDB+KP, whereas Phlebiopsis sp. and I. lacteus showed no Lac and low LiP activities in all media. Compared to PDB, the highest increase in Lac (7.25-fold) and MnP (2.37-fold) activities in PDB+CuSO4 occur in T. versicolor; for LiP, the greatest changes (6.95-fold) were observed in I. lacteus. Incubation in PDB+KP shows significant increases in Lac and MnP for T. versicolor, MnP and LiP for Phlebiopsis sp., and none for I. lacteus. SSF assays in Petri plates are a valuable tool to select fungi that are able to delignify KP. Here, delignification by Phlebiopsis sp. of this substrate is reported for the first time, and MnP activity was strongly associated with the delignification ability of the studied strains. The obtained results suggest that the studied fungal strains have biotechnological potential for use in the paper industry.

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

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

Production of ligninolytic enzymes and some diffusible antifungal compounds by white-rot fungi using potato solid wastes as the sole nutrient source.

The aim of this study was to evaluate the synthesis of ligninolytic enzymes and some diffusible antifungal compounds by white-rot fungi (WRF) using peels or discarded potato as the sole nutrient source. The strain Trametes hirsuta Ru-513 highlighted for its laccase activity (595±33Ul-1 ), which is able to decolourize 87% of an anthraquinone dye using potato peels as the sole nutritional support. A native polyacrylamide gel of laccase proteins showed the presence of two isoenzymes, corresponding to proteins of 56 and 67kDa, which were detected by SDS-PAGE. The antifungal activity of ethyl acetate extracts was evaluated by the agar diffusion method, where Anthracophyllum discolor Sp4 and Inonotus sp. Sp2 showed the highest inhibition zones of Mucor miehei. The fungal extracts also inhibited Fusarium oxysporum and Botrytis cinerea growth, with inhibition zones of up to 18mm. The extract with the highest antifungal activity, from A. discolor Sp4 grown in discarded potato medium, was analysed using a gas chromatograph coupled to a mass spectrometer. Among the identified compounds, chlorinated aromatic compounds and veratryl alcohol were the most abundant compounds. The results revealed the relevance of potato waste valorization for the sustainable production of ligninolytic enzymes and antifungal compounds. This study reports the synthesis of ligninolytic enzymes and diffusible antifungal compounds by WRF using potato wastes as the sole nutrient source and suggests a relationship between the enzymatic activity and the synthesis of antifungal compounds. These compounds and the synthesis of halogen compounds by WRF using agro-industrial wastes have been poorly studied before.

Fungal pretreatment of sweet sorghum bagasse with supplements: improvement in lignin degradation, selectivity and enzymatic saccharification

Sweet sorghum bagasse (SSB) from food processing and agricultural industry has attracted the attention for uses in production of biofuel, enzymes and other products. The alteration in lignocellulolytic enzymes by use of supplements in fungal pretreatment of SSB to achieve higher lignin degradation, selectivity value and enzymatic hydrolysis to fermentable sugar was studied. Fungal strain Coriolus versicolor was selected for pretreatment due to high ligninolytic and low cellulolytic enzyme production resulting in high lignin degradation and selectivity value. SSB was pretreated with supplements of veratryl alcohol, syringic acid, catechol, gallic acid, vanillin, guaiacol, CuSO4 and MnSO4. The best results were obtained with CuSO4, gallic acid and syringic acid supplements. CuSO4 increased the activities of laccase (4.9-fold) and polyphenol oxidase (1.9-fold); gallic acid increased laccase (3.5-fold) and manganese peroxidase (2.5-fold); and syringic acid increased laccase (5.6-fold), lignin peroxidase (13-fold) and arylalcohol oxidase (2.8-fold) resulting in enhanced lignin degradations and selectivity values than the control. Reduced cellulolytic enzyme activities resulted in high cellulose recovery. Enzymatic hydrolysis of pretreated SSB yielded higher sugar due to degradation of lignin and reduced the crystallinity of cellulose. The study showed that supplements could be used to improve the pretreatment process. The results were confirmed by scanning electron microscopy, X-ray diffraction, Fourier transform infrared spectroscopy and thermogravimetric/differential thermogravimetric analysis of SSB.

Production of ligninolytic enzymes by solid state fermentation using Pleurotus ostreatus

Abstract Solid state fermentation (SSF) stands out in the production of lignocellulolytic and other industrially important enzymes. SSF, an alternative culture method, has several advantages over the conventional submerged ones, like higher yields of enzymes. The production of ligninolytic enzymes, such as laccase (Lac), manganese peroxidase (MnP), lignin peroxidase (LiP) and aryl alcohol oxidase (AAO) by Pleurotus ostreatus (Jacq.) Pleurotus Kumm. (MCC16) was investigated under SSF, which was performed using a support-substrate from potato peel waste (PPW). PPW as dry and fresh was pretreated with base to neutralize organic acids and distilled water. Chemical content of PPW was investigated. Ligninolytic enzyme production patterns were investigated during the growth of the organism for a period of 23 days at 25 °C in the stationary SSF using pretreated PPW. The highest Lac and MnP activities were determined in dry PPW, pretreated with distilled water as 6708.3 ± 75 U/L and 2503.6 ± 50 U/L on day 17, respectively. In addition, amylase and protease enzyme activities were detected under same conditions. According to the results, PPW has a potential as supports for ligninolytic enzyme production by P. ostreatus under SSF.

Producción de enzimas ligninolíticas durante la degradación del herbicida paraquat por hongos de la pudrición blanca

Paraquat is a widely used herbicide in agriculture. Its inappropriate use and wide distribution represents a serious pollution problem for soil and water. White rot fungi are capable of degrading pollutants having a similar structure to that of lignin, such as paraquat. This study evaluated the degradation effect of paraquat on the production of ligninolytic enzymes by white rot fungi isolated from the South of Mexico. Six fungal strains showed tolerance to the herbicide in solid culture. Three of the six evaluated strains showed levels of degradation of 32, 26 and 47% (Polyporus tricholoma, Cilindrobasidium laeve and Deconica citrispora, respectively) after twelve days of cultivation in the presence of the xenobiotic. An increase in laccase and manganese peroxidase (MnP) activities was detected in the strains showing the highest percentage of degradation. Experiments were done with enzyme extracts from the extracellular medium with the two strains showing more degradation potential and enzyme production. After 24hours of incubation, a degradation of 49% of the initial paraquat concentration was observed for D. citrispora. These results suggest that paraquat degradation can be attributed to the presence of extracellular enzymes from white rot fungi. In this work the first evidence of the biodegradation potential of D. citrispora and Cilindrobasidium leave is shown.

Decolorization of complex dyes and textile effluent by extracellular enzymes of Cyathus bulleri cultivated on agro-residues/domestic wastes and proposed pathway of degradation of Kiton blue A and reactive orange 16.

In this study, the white-rot fungus Cyathus bulleri was cultivated on low-cost agro-residues, namely wheat bran (WB), wheat straw (WS), and domestic waste orange peel (OP) for production of ligninolytic enzymes. Of the three substrates, WB and OP served as good materials for the production of laccase with no requirement of additional carbon or nitrogen source. Specific laccase activity of 94.4Umg-1 extracellular protein and 21.01Umg-1 protein was obtained on WB and OP, respectively. Maximum decolorization rate of 13.6μmolh-1U-1 laccase for reactive black 5 and 22.68μmolh-1U-1 laccase for reactive orange 16 (RO) was obtained with the WB culture filtrate, and 11.7μmolh-1U-1 laccase for reactive violet 5 was observed with OP culture filtrate. Importantly, Kiton blue A (KB), reported not to be amenable to enzymatic degradation, was degraded by culture filtrate borne activities. Products of degradation of KB and RO were identified by mass spectrometry, and a pathway of degradation proposed. WB-grown culture filtrate decolorized and detoxified real and simulated textile effluents by about 40%. The study highlights the use of inexpensive materials for the production of enzymes effective on dyes and effluents.

Discoloration of the azo dye Congo Red by manganese-dependent peroxidase from Pleurotus sajor caju

This paper describes the production of ligninolytic enzymes by the white-rot fungus Pleurotus sajor caju under solid-state fermentation conditions using a cost-effective medium consisting of agro-industrial wastes. From the different agro-industrial wastes tested (i.e. orange, banana, mango and cantaloupe peels), banana peels led to the highest manganese-dependent peroxidase (MnP) activity (6.3 U/mL on the 10 day). MnP from banana peel cultures was purified and applied to the discoloration of the azo dye Congo Red (CR). The optimum temperature, pH and enzyme concentration for maximum discoloration (i.e. 95% in 1 h) were found to be 35°C, 4.0, and 1.4 U/mL, respectively. In addition, the phytotoxicity (with respect to Sorghum vulgare and Phaseolus radiatus seeds) of CR was considerably reduced after the treatment of plant material with MnP produced by P. sajor caju. The products obtained after discoloration of CR were characterized using GC/MS as 8-amino naphthol 3-sulfonic acid, 3-hydroperoxy 8-nitrosonaphthol, p-p'-dihydroxybiphenyl. Therefore, this approach holds promise for the production and application of MnP from P. sajor caju on a larger scale.

Synergistic effect of syringic acid and gallic acid supplements in fungal pretreatment of sweet sorghum bagasse for improved lignin degradation and enzymatic saccharification

Abstract Sweet Sorghum Bagasse (SSB) has potential for uses in production of ligninocellulosic biofuel and other fermentation products due to higher biomass yield. Pretreatment of SSB by Coriolus versicolor with synergistic syringic acid-gallic acid supplements for enhanced ligninolytic enzyme production, lignin degradation, selectivity value (SV) and enzymatic saccharification have been studied. C. versicolor was selected due to its high ligninolytic and low cellulolytic enzyme activities in SSF with 19.3 ± 0.8% w w −1 of lignin degradation in 20 days and SV 0.65. Supplement gallic acid increased the production of MnP; while syringic acid increased the production of LiP, AAO and laccase with enhanced lignin degradation and SV. Combined supplements increased the production of multiple ligninolytic enzymes further than the effect of individual supplements due to synergistic interactions. Activities of laccase, LiP, MnP, PPO and AAO increased by 9.1, 14.6, 2.6, 1.9 and 2.2 folds respectively, which resulted in highest lignin degradation (1.56 times) and SV (3.87 times). Enzymatic hydrolysis of SSB pretreated with combined supplements yielded higher fermentable sugar (∼2.18 times). Combined supplements could be used for improvement in pretreatment by SSF. XRD, SEM, FTIR and TGA/DTG analysis of SSB confirmed the results.