Accepted Manuscript Enhancement in multiple lignolytic enzymes production for optimized lignin degradation and selectivity in fungal pretreatment of sweet sorghum bagasse Vartika Mishra, Asim K. Jana, Mithu Maiti Jana, Antriksh Gupta PII: DOI: Reference:
S0960-8524(17)30430-3 http://dx.doi.org/10.1016/j.biortech.2017.03.148 BITE 17852
To appear in:
Bioresource Technology
Received Date: Revised Date: Accepted Date:
26 January 2017 23 March 2017 24 March 2017
Please cite this article as: Mishra, V., Jana, A.K., Jana, M.M., Gupta, A., Enhancement in multiple lignolytic enzymes production for optimized lignin degradation and selectivity in fungal pretreatment of sweet sorghum bagasse, Bioresource Technology (2017), doi: http://dx.doi.org/10.1016/j.biortech.2017.03.148
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Enhancement in multiple lignolytic enzymes production for optimized lignin degradation and selectivity in fungal pretreatment of sweet sorghum bagasse Vartika Mishra1, Asim K. Jana1*, Mithu Maiti Jana2, Antriksh Gupta1 1
Department of Biotechnology, Dr B R A National Institute of Technology, Jalandhar - 144011,
Punjab, India 2
Department of Chemistry, Dr B R A National Institute of Technology, Jalandhar - 144011,
Punjab, India
* Corresponding author Dr Asim K Jana Department of Biotechnology, Dr B R A National Institute of Technology, G T Road Bye Pass, Jalandhar - 144011, Punjab, India Email:
[email protected] Phone: +91-9417710388 Fax: +91-181-2690320
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Abstract The objective of this work was to study the increase in multiple lignolytic enzyme productions through the use of supplements in combination in pretreatment of sweet sorghum bagasse (SSB) by Coriolus versicolor such that enzymes act synergistically to maximize the lignin degradation and selectivity. Enzyme activities were enhanced by metallic salts and phenolic compound supplements in SSF. Supplement of syringic acid increased the activities of LiP, AAO and laccase; gallic acid increased MnP; CuSO4 increased laccase and PPO to improve the lignin degradations and selectivity individually, higher than control. Combination of supplements optimized by RSM increased the production of laccase, LiP, MnP, PPO and AAO by 17.2, 45.5, 3.5, 2.4 and 3.6 folds respectively for synergistic action leading to highest lignin degradation (2.3 folds) and selectivity (7.1 folds). Enzymatic hydrolysis of pretreated SSB yielded ~2.43 times fermentable sugar. This technique could be widely applied for pretreatment and enzyme productions.
Keywords: Lignocellulosic biomass; fungal pretreatment; supplements; lignocellulolytic enzymes; lignin degradation; optimization.
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1. Introduction Agro-residues as substrates in fermentative processes have been explored for production of value added products enzymes, ethanol, amino acids etc. because of their abundance, carbon contents and disposal problems. Sorghum (Sorghum sp.) has higher biomass yields and sweet sorghum baggase (SSB) is a promising candidate for production of bioproducts and biofuels. The hindrance in the utilization of lignocellulosic wastes is the transformation of its structural polysaccharides into fermentable sugars due to recalcitrant lignin in which cellulosic fibrils are embedded. This can be achieved by chemical / enzymatic hydrolysis of the biomass after pretreatment that enhance the efficiency of hydrolysis by removal of lignin. In recent literature, physical and chemical pretreatment using organoslav (Tang et al., 2017), acid-alkali (MaitanAlfenas et al., 2015) and alkaline peroxide (Hideno, 2017) have been reported to degrade lignin in lignocellulosic biomass in few hours. But these processes have drawbacks of higher energy requirement, difficult operating conditions (such as high pressure, temperature and pH), requirements of environmentally sensitive chemicals and generation of inhibitors that required to be removed (Da Silva Machado & Ferraz, 2017; Ghorbani et al., 2015; Sindhu et al., 2016). Biological pretreatment of Eucalyptus globulus wood using Pringsheimia smilnergy acis with 33.1% lignin degradation in 28 days (Martín-Sampedro et al., 2015), miscanthus using Ceriporiopsis subvermispora with 30% lignin degradation after 28 days (Vasco-Correa et al., 2016), sugarcane bagasse using C. subvermispora with 48% lignin degradation after 60 days (Da Silva Machado & Ferraz, 2017), rice straw using Pleurotus ostreatus with 33.4% lignin degradation after 30 days (Mustafa et al., 2016) and using P. ostreatus in combination with milling (physical pretreatment) with 34.8% lignin degradation after 30 days (Mustafa et al., 2017) have been reported. Although WRF are the best lignin degraders, because of their ability
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to produce lignolytic enzymes laccase (phenol oxidase), lignin peroxidase (LiP), manganese peroxidase (MnP), arylalcohol oxidase (AAO) and polyphenol oxidase (PPO); but non-selective lignin degradation (Zhang et al., 2007a), long pretreatment time (Sindhu et al., 2016; Wan & Li, 2010) and cellulolytic enzyme expression are major concerns. Screening of fungal strains for obtaining high Selectivity value (SV) defined as a ratio of lignin degradation to cellulose consumption i.e. low cellulose consumption is an important parameter (Chang et al., 2012; Yu et al., 2009; Zhang et al., 2007a). Release of lignolytic enzymes could affect the SV and reduce the pretreatment time (Meehnian & Jana, 2016). The production of lignolytic enzymes including both oxidase and peroxidases can be controlled by inorganic compounds, phenolic and aromatic compounds (Levin et al., 2008; Mishra & Jana, 2017; Mishra et al., 2017; Singh & Chen, 2008; Wan & Li, 2011). Addition of phenolic supplements were reported to enhance the laccase (Liu et al., 2013) and LiP production (Christian et al., 2005). Very few studies of fungal pretreatment have reported towards processes development by production of lignolytic enzymes during pretreatment. The lignolytic enzyme (laccase, LiP, MnP) profiles during lignin degradation in pretreatment has been analyzed in recent studies (Ghorbani et al., 2015; Liu et al., 2015; Meehnian & Jana, 2016).
The degradation of lignin in biomass occurs by specific sequence of reactions by synergistic action / cooperative actions of the several lignolytic and cellulolytic enzymes produced by the fungi (Meehnian et al., 2017). It has been observed that simple increase in the lignolytic enzyme activities did not result into proportionate increase in lignin degradation (Karp et al., 2015; Sharma & Arora, 2010). Optimal combination of supplements used for enhancement of multiple lignolytic enzyme productions that act synergistically could give maximum lignin degradation and SV. No study has been reported in this direction till date. The objective of this work was to 4
study the increase in the multiple lignolytic enzyme (Laccase, LiP, MnP, PPO and AAO) productions through the use of supplements in combination in pretreatment of SSB such that enzymes act synergistically to maximize the lignin degradation and selectivity. Suitable fungal strain with high selectivity and three supplements were screened initially in preliminary study of pretreatment. This technique could be applied for improvement of lignin degradation in any fungal pretreatment in general irrespective of the fungal strain or biomass used.
2. Materials and methods 2.1.Materials
2,2'azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) was purchased from MP Biomedical (Mumbai, India). Cellulase (SaccariSEB EG) and β-glucosidase (SaccariSEB BG) were gifted by Advanced Enzyme Technologies Ltd (Thane, India). p-nitrophenyl-β-Dglucopyranoside, birchwood xylan, lignin sulfonic acid, catechol, vanillin and verataryl alcohol were purchased from Himedia Laboratory Pvt Ltd (Mumbai, India). Cellulose, gallic acid and syringic acid were purchased from Sigma Aldrich (Mumbai, India). Water was purified and deionized (DI) by Milli-Q purification system (minimum resistivity 18 MΩ cm; Millipore, Billerica, MA, USA). Rest of chemicals used were analytical grade chemicals made by S.D. fine chem. Ltd (Mumbai, India).
2.2.Biomass
Sweet Sorghum Bagasse (SSB) was collected from the local agricultural farms in Jalandhar, Punjab, India. The collected SSB was dried at 40 ºC for 72 h and grounded to pass through 5 mm
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sieve using a wiley mill. The ground SSB (about 3% moisture) was stored in air tight container at room temperature.
2.3.Fungal strain and maintenance
Coriolus versicolor (MTCC 138), Daedalea flavida (MTCC 145), Ganoderma lucidum (MTCC 1039), Phlebia radiata (MTCC 2791), Pleurotus ostreatus (MTCC 1801), Stereum hirsutum (MTCC 1099) were procured from Microbial Type Culture Collection (MTCC) Chandigarh, India and Pycnoporus cinnabarinus (NCIM 1181) from the National Collection of Industrial Microorganisms (NCIM) Pune, India. Coriolus versicolor, Ganoderma lucidum, Phlebia radiata and Pycnoporus cinnabarinus were grown and maintained in 2% (w v-1) malt extract agar media in petri plates at pH 4.5, 25 ºC. Pleurotus ostreatus and Stereum hirsutum were grown and maintained in potato dextrose agar media (potato infusion 20 %, dextrose 2 % and agar 1.5% w v-1) in petri plates at pH 5.5, 25 ºC. All strains were sub-cultured periodically after every 15 days.
2.4. Pretreatment of SSB and selection of supplements
Ten discs (using sterile cock borer with 10 mm in diameter) from the actively growing 7 days old culture in plates, were inoculated into 50 mL of 2% (w v-1) malt extract medium after sterilization by autoclave in 500 mL cotton plugged Erlenmeyer flask. After 7 day incubation at 27.5 ºC under static conditions, the liquid culture was aseptically homogenized in a sterilized blender for 15 sec × 3 cycles. Two mL (~2.4 x106 spore mL-1) of blended culture was used as inoculum for 5 g of SSB for pretreatment by solid state fermentation (SSF).
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5 g ground SSB (~3% moisture) was taken in 250 mL Erlenmeyer flask, 11.5 mL of distilled water was added with it to make 75% moisture initially and cotton plugged. Moistened SSB in flasks were autoclaved at 121 °C for 15 min. Sterilized flasks were inoculated with 2 mL inoculum and incubated stationary at 27.5 °C for 20 days. Samples were taken every 4th day for compositional analysis of treated biomass, lignocellulolytic enzyme assays and enzymatic hydrolysis of biomass.
Different phenolic supplements (veratryl alcohol, syringic acid, catechol, vanillin, guaiacol and gallic acid) were studied for improvement in lignin degradation and SV. Supplements were dissolved in DI water to make 1 mM supplement solution. 5 g of SSB (~3% moisture) was taken in 250 mL Erlenmeyer flasks and 11.5 mL of 1 mM supplement solution was mixed with it to make 2.2 µmol supplement g-1 SSB in SSF. Supplements with SSB in cotton plugged flasks were autoclaved and inoculated as mentioned above for the pretreatment. Supplements, significantly influencing the lignin degradation were studied individually and in combination to enhance the lignolytic enzyme production and lignin degradation. All the cultures were incubated statically at 27.5 ºC for 20 days. Culture without supplement was taken as control. All experiments were performed in triplicate.
2.5. Optimization of lignin degradation and SV by combination of supplements: Design of experiments Initial set of experiments were performed to identify the concentration range of the three selected supplements (CuSO4, gallic acid, syringic acid) for improvement of lignin degradation and SV. Effect of combination of three supplements on lignocellulolytic enzyme productions and their synergistic actions resulting in maximum degradations of lignin and SV in the pretreatment was studied by response surface methodology (RSM). The Central Composite Designs (CCD) used in 7
the RSM was a first-order (2N) design having additional centre and axial points to allow estimation of the parameters of a second-order model. The impact of three independent variables syringic acid (X1), gallic acid (X2) and CuSO4 (X3) on lignin degradation, cellulose loss and SV; their optimal concentrations were evaluated by CCD. The effect of each variable was studied at five different levels, namely -α, 1, 0, +1, +α. The results of the RSM were fitted to a secondorder polynomial Eq. representing behavior of the system Y = β0 + β1X1+ β2X2 + β3X3 + β1β1X12 + β2β2X22 + β3β3X32 + β1β2X1X2 + β1β3X1X3 + β2β3X2X3 where, Y is the response variable representing lignin degradation, cellulose loss and SV; β0 intercept; β1, β2, β3 are the linear coefficients; β1β1, β2β2, β3β3 are the squared coefficients; β1β2, β1β3, β2β3 are the interaction coefficients; X1, X2, X3, X12, X22, X32, X1X2, X1X3, X2X3 are the levels of the independent variables. Interaction was represented by combination of factors (such as X1X2) in the respective term. The data analysis and generation of the response surface graphs were done by the statistical software Design Expert (Design-Expert 7.0). The standard deviation (SD) was less than 5%.
2.6.Enzymatic hydrolysis of pretreated SSB
Enzymatic hydrolysis of cellulose, SSB were carried out following protocol described in the procedure NREL/TP-510-42630 (Dowe & McMillan, 2001). One gram of biomass (3% moisture) was taken in a 250 mL Erlenmeyer flask and 100 mL of 50 mM citrate buffer (pH 4.8) was added. Cellulase (SaccariSEB EG, 10 FPU dry g-1 solid) and β-glucosidase (SaccariSEB BG, 20 CBU dry g-1 solid) enzymes were added for hydrolysis of biomass. The flask was incubated in a shaking incubator at 165 rpm, 45 ºC at for 108 h and analysed for release of
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fermentable sugar. Sugar content was estimated using Agilent Hi-Plex-H column in HPLC (Agilent-1200 infinity series).
2.7.Analytical methods 2.7.1. Lignocellulolytic enzymes assays 50 mL acetate buffer (pH 4.5, 50 mM) was added to the each sample and shaked at 180 rpm at 27.5 °C for 1 h, and filtered under vacuum to recover most of the water-soluble components and enzymes in filtrate. The filtrate were stored at 4 °C and used for lignocellulolytic enzyme activity assays. The solid fractions from pretreated SSB were dried in hot air oven at 65 °C and used for composition analysis and enzymatic hydrolysis. 2.7.1.1.Lignolytic activity Lignin peroxidase (LiP) activity was determined by H2O2 dependent oxidation of azure-B at 651 nm (Ԑ651 = 48800 M-1 cm-1) (Archibald, 1992). Laccase activity was determined by oxidation of ABTS to ABTS-azine at 420 nm (Ԑ420 = 36000 M-1 cm-1) (Bourbonnais & Paice, 1990). Manganese peroxidase (MnP) activity was determined by phenol red oxidation (Ԑ610 = 22000 M-1 cm-1) (Glenn & Gold, 1985). Polyphenol oxidase (PPO) was assayed by oxidation of catechol to quinone at 30 ºC (Ԑ400 = 3450 M-1 cm-1) (Wong et al., 1971). Aryl alcohol oxidase (AAO) activity was monitored using the oxidation of veratryl alcohol to veratrylaldehyde (Ԑ310 = 9300 M-1 cm-1) (Guillen et al., 1992). One unit of enzyme activity was defined as the amount of enzyme required to oxidize 1 µmol of substrate per minute. All the enzymes were expressed as units per gram dry matter (U g-1).
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2.1.1.1.Cellulolytic activity
Carboxymethyl cellulase activity (CMCase / endo-β-1-4-glucanase), filter paper activity (FPase) and xylanase activities were estimated based on dinitrosalicylic acid (DNS) method. Filter Paper cellulase (FPase) and Carboxymethyl cellulase (CMCase) activity was determined using whatman No. 1 filter paper strip and carboxymethyl cellulose as substrate respectively (Ghose, 1987). One unit of activity was defined as the amount of enzyme required to produce 1 µmol reducing sugars per min. β-Glucosidase activity was assayed using p-nitrophenyl-β-Dglucopyranoside as substrate (Wood & Bhat, 1988). One unit of activity was defined as the amount of enzyme required to produce 1 µmol p-nitrophenol per min. Xylanase activity was assayed using 1% w v-1 solution of birchwood xylan in 50 mM citrate buffer (pH 5.0) as the substrate and measured at 520 nm using xylose as standard (Bailey et al., 1992). One unit of activity was defined as the amount of enzyme required to produce 1 µmol xylose per min under standard condition of pH and temperature. All the enzymes were expressed as units per gram dry matter (U g-1).
2.7.2. Fungal growth estimation Fungal hyphal growth was estimated in terms of N-acetyl glucosamine content. (Aidoo et al., 1981). The glucosamine content of the fungal cell wall was used to monitor fungal biomass. At the beginning, glucosamine standard curve was obtained using N-acetyl glucosamine (NAG). Glucosamine content of fungi (biomass vs. glucosamine) was determined through liquid culture cultivation. The fungal cell wall chitins were converted to NAG using 2N HCl and measured colorimetrically using Erhlick’s reagent at 530 nm by UV-vis spectrophotometer (Eppendorf Ltd, USA). 10
2.7.3. Compositional analysis of SSB
Compositions of SSB were determined before and after solid state fermentation. Lignin, cellulose and hemicelluloses of the SSB samples were determined by the protocol described in NREL report (NREL/TP-510-42618) (Sluiter et al., 2010) using two-stage acid hydrolysis. The first hydrolysis step used 72% v v-1 sulfuric acid at 30 ºC for 1 hr. The samples were diluted immediately to 4% v v-1and autoclaved for 1 hr. Autoclaved hydrolyzed solution was vacuum filtered through previously weighed filtering crucibles. Crucible and acid insoluble residue was dried at 105 ºC until a constant weight was achieved. Aliquot sample collected from vacuum filtration was used to determine acid soluble lignin, cellulose and hemicellulose. The acidsoluble lignin was measured by UV–Vis spectrophotometer (Kinetic, Biospectrometer, Eppendorf, Germany) using lignin sulphonic acid as standard. Cellulose and hemicellulose in samples were acid-hydrolyzed into monomeric sugars. Monomeric sugars were measured by HPLC (Agilent 1200 series, MN, USA) equipped with Agilent Hi-Plex-H column and refractive index detector (RID) that were maintained at 80 º and 55 ºC, respectively. HPLC grade water was used as the mobile phase, at flow rate of 0.6 mL min-1. The content of cellulose in the sample was calculated from glucose and galactose obtained, while hemicelluloses was calculated from the sum of xylose and arabinose obtained, and using an anhydro correction of 0.90 for C-6 sugars and 0.88 for C-5 sugars, respectively. The cellulose and hemicellulose contents were calculated as Cellulose% = (GR × 0.9)/w × 100 and Hemicellulose% = (XGA × 0.88)/w × 100 respectively, where GR is the glucose and galactose released (g); w is the sample dry weight (g) and XGA is the xylose and arabinose released (g). The percentage of lignin degradation, which is defined as the percentage of total lignin reduced after pretreatment, was calculated using equation Lignin degradation% = (1 – w(α + β)/(wo (αo + βo))) × 100, where w is the sample dry 11
weight (g) of the pretreated SSB; wo is the initial dry weight (g) of the untreated SSB; α, β are the % of acid-soluble and acid-insoluble lignin in the pretreated SSB, respectively; αo, βo are the % of acid-soluble and acid-insoluble lignin in the untreated SSB, respectively. Dry mass loss was calculated as the percentage of total solids loss after pretreatment and after removing the fungal biomass content.
2.8.Characterization of SSB
Morphological, structural and chemical changes in SSB were characterized by scanning electron microscopy (SEM), X-ray diffraction (XRD), Fourier transformed infrared spectroscopy (FTIR), and Thermogravimetric/Differential Thermogravimetric (TGA/DTG). For SEM, samples were dried in an oven at 45 °C for 48 h and then gradually dehydrated using acetone–water mixtures, with the acetone concentration increasing up to 100%. Samples were sputter-coat with Au–Pd and examined in SEM (EVO 40 EP-CARL ZEISS, Germany) at 1500X and 3000X magnification using an accelerating voltage of 20 kV. Sample was analyzed using FTIR (AGILENT CARY-630, USA) spectrometer in the attenuated total reflectance mode to compare the changes in the functional group of the SSB samples after lignin degradation during solid state fermentations. The samples were prepared by blending and grinding 1% (w w-1) SSB sample in KBr. Scans were taken for each sample from 4000 to 400 cm-1 at a resolution of 2 cm-1. To analyze the degradation of biomass, thermogravimetric (TG) analysis was conducted using TGA/DTG (SII EXSTAR 6000 [TG/DTA 6300], Japan) from 30 ºC to 750 ºC. Weight loss experiments were performed on the SSB samples weighing 5 mg. The heating rate was 10 ºC min-1 with inert atmosphere (nitrogen flow of 20 mL min-1). To determine the crystallinity of the biomass, the XRD patterns of the SSB samples were collected by X-ray Diffractometer
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(PANALYTICAL [XPERT-PROMPT, Netherland) using copper X-ray source (k= 0.15418 nm). The X-ray generator was operated at 45 kV and 40 mA. All samples were ground and sieved with a 60 mesh to make homogenous mixtures. Homogenous powders were dispersed in a thin film (~ 1 mm thickness) in a flat sample holder. The holder was filled with freeze dried coffee powder and pressed into a compact material for the analysis. Scans were taken at 1 ºminute from 5 to 50 º 2θ. The percentage of crystallinity (%Cr) and crystallinity index (CrI) was calculated by (Segal et al., 1959) equation %Cr = Icrys/(Icrys + Iamor) × 100 and CrI = (Icrys - Iamor)/ Icrys, where Icrys is the overall intensity of the peak at 2θ about 22 º and Iamor is the intensity at 2θ about 18 º.
3. Results and discussion 3.1.Screening of selective strain and supplements Maximum selective lignin degrading fungal strain was screened from the fungal strains reported in literature (Aguiar et al., 2014; Chang et al., 2012; Knezevic et al., 2013; Taniguchi et al., 2005; Zhang et al., 2007b) based on their high lignolytic and low cellulolytic abilities and selectivity value (SV) (lignin degradation/cellulose loss) during pretreatment of SSB.
Lignolytic ability i.e. activities of laccase, lignin peroxidase (LiP), polyphenol oxidase (PPO), and cellulolytic and hemicelluloytic ability i.e. activities of carboxymethyl cellulase (CMCase) and xylanase, were analyzed by measuring the diameter of characterstic zones formed in petri plates containing the substrates lignin sulfonate salt, 2,2'azino-bis (3-ethylbenzothiazoline-6sulfonic acid) (ABTS), guaiacol, azure-B, tannic acid, CMC (congo red test) and xylan respectively (Table S1). Zone diameter ≥ 30 mm indicated high activity of enzyme, while diameter ≤ 20 mm indicated low activity of enzyme. Strains exhibited the varying lignolytic,
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cellulolytic and hemicellulolytic abilities; but C. versicolor showed highest lignolytic and hemicellulolytic ability with low cellulolytic ability.
Strains showed significant differences in lignocellulolytic enzymes activities, when grown on SSB in SSF (Table S2). C. versicolor showed the highest laccase, LiP, MnP and PPO activity. C. versicolor also produced enzyme AAO, but less than G. lucidium and P. radiata. D. flavida, P. ostreatus and S. hirusita didn’t show any AAO activity. Only C. versicolor, G. lucidium and P. radiata produced all lignolytic enzymes i.e. laccase, LiP, MnP, PPO and AAO. P. radiata showed the high activities celluloytic enzymes i.e CMCase, FPase and ß-glucosidase; while C. versicolor showed the lowest CMCase activity, and low FPase and ß-glucosidase activities. C. versicolor produced highest xylanase activity i.e. capability to utilize hemicelluloses as carbon source; produced low levels of cellulolytic enzymes and high lignolytic enzymes with high degradation of lignin in SSB.
Fungal strains showed different SVs due to the varied lignin degradation, cellulose loss and hemicellulose loss depending upon their lignolytic and cellulolytic abilities. C. versicolor showed highest SV 0.65 after 20 days, 0.59 after 30 days of SSF (Table S3) and degradation of hemicellulose was high. Strain showing ability to degrade xylan can be advantage for pretreatment, because dependency of strains on hemicelluloses can reduce the loss of cellulose. C. versicolor was selected for the pretreatment of SSB with supplements. In a preliminary study, SSB was pretreated with individual supplements (2.2 µmol g-1 SSB) of veratryl alcohol, syringic acid, catechol, vanillin, guaiacol, gallic acid, MnSO4 and CuSO4 to observe the effect on lignin degradation and SVs (Fig. S1). Addition of CuSO4, gallic acid, syringic acid resulted maximum degradation in lignin 24.8±2.3%, 22.8 ± 1.0% and 22.5 ± 1.0% 14
w w-1 respectively, while lower for other supplements 18.5 to 22.0% w w-1. Presence of CuSO4, gallic acid and syringic acid resulted low cellulose losses in biomass and achieved SV values 1.15, 0.75 and 0.89 respectively, higher than the control (without supplements). Three supplements were selected for further study of the effects of individual and combined supplements on lignocellulolytic enzyme activities and lignin degradations in pretreatment.
Pretreatment of SSB was studied with the three above selected individual supplements at 0.0 11.0 µmol g-1 SSB (Table S4). SSB was composed (w w-1) of lignin 25.14%, cellulose 38.02% and hemicellulose 25.01%. Maximum lignin degradation 24.8 ± 2.3% w w-1 with cellulose loss 21.6 ± 1.01% w w-1 and SV 1.15 was obtained with CuSO4 2.2 µmol g-1 SSB. With the gallic acid 4.4 µmol g-1 SSB, maximum SV was 1.13 with 22.8 ± 2.1% w w-1 of lignin degradation and 20.1 ± 1.8% w w-1 of cellulose loss. Syringic acid 8.8 µmol g-1 SSB, resulted in highest lignin degradation 28.7 ± 1.7% w w-1 with minimum cellulose consumption 16.6 ± 1.0% w w-1 and highest SV 1.77. The addition of higher concentration of supplements decreased the lignin degradation and SV, which might be due to the inhibition of fungal growth and lignolytic enzyme activities.
3.3. Optimization of lignin degradation and SV by combination of supplements CCD was used to find the values of the three independent variables i.e. concentrations of supplements syringic acid, gallic acid, CuSO4 in combinations for high lignin degradation, low cellulose loss and high SV in pretreatment. The sets of experiments with supplement concentrations upto 11.00 µmol g-1 SSB conducted in pretreatment was suggested by the software. The sets of experiments and their results comprised the data for CCD (Table 1). Data fitted to the quadratic equation with good agreement between experimental results and the
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predicted response values of lignin degradations, cellulose loss and SVs. All the coefficients i.e. linear, quadratic and interaction coefficients of the model (Table 2) were significant (p <0.05). The quality of the model was evaluated by the coefficient R2, its statistical significance determined by F-test and annova value (Table 2). High R2 (> 0.75) signified the acceptance of the model in the range of variables for the response and R2 > 0.92 in all cases indicated that model fitted well with the experimental results. The analysis of variance indicated that syringic acid with CuSO4 had significant positive effect on degradation of lignin and SV as well (Table 2). CuSO4 showed a positive effect with gallic acid and with syringic acid, on lignin degradation and SV. Syringic acid showed positive effect with gallic acid and with CuSO4 on cellulose loss, while combination of gallic acid with CuSO4 had negative effect on cellulose loss. Presence of the supplements resulted in decrease in the cellulose loss.
Analysis of contour plots depicted the effect of two variables on lignin degradation, cellulose loss and SV by keeping the other variable constant at zero level (Fig. 1). Combination of high concentration of syringic acid and low concentration of gallic acid with CuSO4 at zero level (5.5 µ mol g-1) resulted in high degradation of lignin (Fig. 1A), justified by the negative coefficients in anova analysis (Table 2). There was strong cooperative action between syringic acid and CuSO4 for effect on lignin degradation (Fig. 1B); cooperative action between gallic acid and syringic acid (Fig. 1G), CuSO4 and syringic acid (Fig. 1H), and CuSO4 and gallic acid (Fig. 1I) for the effect on SVs. High concentration of supplements resulted in decrease in the consumption of cellulose (Fig. 1D-F). Lignin degradation reached to maximum (Fig. 1B, Table 1) due to increased cooperative action between syringic acid and CuSO4 with their concentrations. Low concentration of gallic acid with high concentration of syringic acid resulted in high SV, while 16
higher gallic acid resulted in decline in the SVs (Fig. 1G). SV increased with an increase in concentration of CuSO4 and syringic acid (Fig. 1H), while low gallic acid with high CuSO4 showed high SV due to low cellulose loss (Fig. 1I). Syringic acid was a significant factor for the improvement of lignin degradation and SV by individual as well as combined effect of supplements. The maximum lignin degradation 45.8% w w-1, highest SV 4.67 and 9.81% w w-1 of cellulose loss was observed at the combination of supplements (µmol g-1 SSB) syringic acid 8.8, gallic acid 2.2 and CuSO4 8.8. But, the minimum cellulose loss 8.32 % w w-1 was obtained at (µmol g-1 SSB) syringic acid 8.8, gallic acid 2.2 and CuSO4 2.2. The increase in lignin degradation was 2.3 folds and SV 7.1 folds higher at the optimal combination of supplements in pretreatment than control. 3.4.Validation of the model and degradation of SSB during pretreatment The validity of model equation and regression was tested by carrying out pretreatments of SSB with ten different combinations of supplements suggested by the software (Table 4). The experimental results of lignin degradations, cellulose loss and SVs showed good agreement with the predicted values (Table 4). Pretreatment of SSB with optimal combination of supplements showed significant improvement in lignin degradation than pretreatment with individual supplements or control (without supplement) (Fig. 2A), might be due to increased productions of multiple lignolytic enzymes during pretreatment in SSF. With combined supplements, rate of cellulose loss during initial days was high, but after 4 to 8 days it became slower (Fig. 2B). This was due to the consumption of small amount of easily accessible cellulose for primary growth of fungi during early days. Due to low cellulose loss in pretreatment with the combined supplements, highest SV 4.67 (7.1 fold higher than control) was achieved (Fig. 2C). 17
Maximum hemicellulose consumption (35.0 ± 1.3% w w-1) was obtained in the case of pretreatment with combined supplements (Fig. 2D). Presence of supplements in culture stimulated C. versicolor to consume hemicelluloses. Ratio of loss of hemicellulose to cellulose loss (H/C) (Fig. 2E) was similar to pattern of change in SVs. Higher H/C ratio indicated that combination of supplements stimulated fungi to feed more on hemicellulose for its growth rather than to feed on cellulose. C. versicolor grew well in SSF of SSB with supplements (Fig. 2F) and prolific colonization was observed in 20 days. The fungal growth was slow during first four days of pretreatment due to fungi adapted to the new environment in lag phase. The fungal biomass were high (474.0 ± 28.2 mg gdm-1) with the combined supplement than with syringic acid (442.2 ± 26.3 mg gdm-1), gallic acid (413.8 ± 38.0 mg gdm-1), control (384.5 ± 31.2 mg gdm-1) or with CuSO4 (328.3 ± 18.4 mg gdm-1). It was interesting to note that supplements caused reduction in the exponential phase of fungal growth in SSF and stationary phase achieved earlier. It seemed that detail study of the production of lignolytic and cellulolytic enzymes in presence of supplements would be useful to understand the results of degradation characteristics of SSB during pretreatment.
3.5. Lignolytic and cellulolytic enzymes production Peak activities of lignolytic enzymes (U g-1); laccase 11.9 ± 0.3 (16th day), LiP 0.13 ± 0.01 (12th day), MnP 8.1 ± 0.2 (12th day), PPO 4.9 ± 0.7 (16th day) and AAO 1.39 ± 0.04 (12th day) were detected in control culture (without supplement). Presence of supplements increased the production of lignolytic enzymes during pretreatment (Table 3). With supplement of 2.2 µmol CuSO4 g-1 SSB, significant increase in peak activity of laccase and PPO enzyme was observed,
18
while LiP and AAO was little affected, might be due to copper has induction effect on laccase and related oxidative enzyme (PPO) at transcription level (Kaur & Sudhakara, 2011). Unexpectedly, significant increase in MnP activity was also observed. All lignolytic enzymes showed peak activity on 12th day of fermentation except of LiP which peaked on 16th day. Gallic acid at 4.4 µmol g-1 SSB resulted in high activities of laccase, MnP and AAO with optimum LiP activity. Positive effect of gallic acid on lignolytic enzymes was might be due to its structural similarity with lignin components. MnP, AAO activity peaked on 12th and 20th day respectively, while laccase activity peaked on 16th day. Syringic acid 8.8 µmol g-1 SSB increased the LiP activity to 1.7 ± 0.08 U g-1, 13 folds higher than activity in control. This might be due to 3 fold increase in AAO activity by syringic acid produced H2O2 in the culture, which significantly increased the LiP activity. Additionally, laccase activity (67.4 ± 7.7 U g-1) increased upto 7 fold and MnP activity (19.0 ± 1.1 U g-1) increased upto 2.5 times. All lignolytic enzymes showed significant increase in peak activities on 16 - 20th day, might be due to structural similarity of syringic acid with syringyl unit present in the lignin. Only difference in syringic acid and syringyl unit is the functional group at para site of benzyl ring. Structural similarity could result in high inducing effect for release of lignolytic enzyme (D’souza et al., 1999). These increase in lignolytic enzymes activities by supplements, supported the increase in lignin degradation.
Lignins in SSB are predominantly composed of β-O-4′ aryl ether linkages, minor amounts of ββ′, β-5′, β-1′ and α,β-diaryl ether linkages (Sun et al., 2013). Cleavage of Cα - Cβ bonds and Cα oxidation by lignolytic enzymes breaks these linkages. All lignolytic enzymes posses ability to break these linkages, but LiP has highest ability due to high redox potential (E’o ~ 1.2 V). The depolymerization of lignin was initiated by AAO (produces H2O2 and initiate LiP) and LiP after breaking the inter-unit β-O-4’ and β-β’ linkages by cleaving of Cα - Cβ bonds present in lignin 19
structure. This resulted in production of partially degraded lignin and different types of aromatic monomers of lower molecular weight and further degraded by laccase, PPO and MnP (Bourbonnais & Paice, 1990; Hatakka & Hammel, 2010). With combination of supplements optimized at (µmol g-1 SSB) syringic acid 8.8, gallic acid 2.2 and CuSO4 8.8; the peak activities (U g-1) of laccase 205.01 ± 10.1 (16th day), LiP 5.94 ± 0.2 (12th day), MnP 28.7 ± 1.1 (12th day), PPO 12.20 ± 0.5 (16th day) and AAO 5.1 ± 0.2 (8th day) were observed. Combination of supplements resulted in cumulative increase in the productions of multiple lignolytic enzymes in culture. Activities of laccase, LiP, MnP, PPO and AAO increased by 17.2, 45.5, 3.5, 2.4 and 3.6 fold respectively. AAO activity peaked earlier (8th day) in culture having optimized combination of supplements than with the individual supplements. Early expression of AAO resulted in early release of H2O2 in the culture, which enhanced the early expression of LiP enzyme (peak activity on 12th day) with optimized combination of supplements. Laccase showed peak activity on 16th day with optimized combination of supplements similar to gallic acid alone, later than with CuSO4 alone, while it peaked on 20th day with syringic acid. This indicated the presence of syringic acid and CuSO4 resulted in an over expression of laccase activity on 16th day with gallic acid. Early expression of AAO on 8th day and LiP on 12th day, and an over expression of laccase on 16th day due to cumulative effect of three supplements might be responsible for higher lignin degradation in the culture. Results showed the synergistic effect of multiple lignolytic enzymes activities were responsible for higher lignin degradation and SV compared to control and supplements individually. With the addition of CuSO4, CMCase activity (3.0 ± 0.21 U g-1) reduced upto half of control, FPase (10.0 ± 1.3 U g-1) and β-glucosidase activity (6.1 ± 0.8 U g-1) was also low. Syringic acid resulted in minimum cellulolytic activities i.e. CMCase, FPase and β-glucosidase 2.2 ± 0.1, 6.7 ± 20
0.9,4.8 ± 0.2 U g-1 respectively (Table 3). CMCase, FPase and β-glucosidase activities were lowest 1.3 ± 0.09, 3.1 ± 0.3 and 2.0 ± 0.1 U g-1 respectively with the combined supplements, while xylanase activity increased to 11.9 ± 1.13 U g-1 (Table 3). Decrease in cellulolytic enzymes production with supplements individually and in combination is due to structural similarity of gallic acid and syringic acid with lignin. Availability of the supplements on the surface of SSB forced the early release of lignolytic enzymes rather than cellulolytic enzymes. Reduced cellulolytic activities resulted in low consumption of cellulose which improved the SV. Wan and Li (Wan & Li, 2011) studied the effect of inorganic metallic salts supplements on delignification of wheat straw by C. subvermispora and targeted only laccase production during pretreatment. Delignification increased to 9% from 4% w w-1 after 21 days of fermentation. Fungal delignification of wheat straw using Irpex lacteus by SSF in the presence of inducer (MnSO4) was studied by Salvachua and coworkers (Salvachúa et al., 2013). Lignin degradation was 28.2% and 31.5% w w-1 in the absence and presence of inducer respectively in 21 days. Increase in delignification was not significant, might be due to limited positive effect of MnP enzymes by MnSO4. Pretreatment of rice straw using Pleurotus ostreatus resulted in 33.4% lignin degradation after 30 days (Mustafa et al., 2016), but increased marginally in combination with milling (physical pretreatment) to 34.8% (Mustafa et al., 2017), showed the importance of lignolytic enzymes. In case of pretreatment sugarcane bagasse with corn steep liquor supplement using C. subvermispora, lignin degradation was 48% in long 60 days and selectivity value was low due to high cellulose loss (Da Silva Machado & Ferraz, 2017). In our study, upto 46% of lignin degradation was obtained in 20 days pretreatment process with SV4.67 is much better than studies reported in literature.
21
3.6. Characterization of SSB Brownish SSB due to presence of lignin became light yellow after pretreatment indicating the exposed cellulose on surface. SEM images showed strands of SSB were covered with small globules and lignocellulose had an intact surface structure (Fig. S2A). The surface of pretreated SSB became rugged and partially broken (Fig. S2B) due to the removal of lignin and breaking of lignocellulose networks. A visible destruction in intact morphology, and formation of holes and crevices on the surfaces of biomass were observed after pretreatment. Maximum rugged surface and pores were visualized in SSB pretreated with combined syringic acid, gallic acid and CuSO4 supplements.
The functional groups observed by FTIR spectroscopy of lignin (Fig. S3) and SSB (Fig. S2C), showed broad peaks at 3418 cm-1 (hydrogen bonded –OH stretching), 2930 cm-1 (C–H stretching), 1638 cm-1 (C=C stretching of aromatic ring), 1435 cm-1 (C–H stretching) and 1130 cm-1 (C-O stretching) (Zeng et al., 2011). Pretreatment of SSB decreased in the respective peaks and intensities at 1638 cm-1 (C=C stretching for aromatic ring, mainly originating from lignin in particular) (Fig. S2C). The maximum decrease in the intensities observed in SSB pretreated with optimized combination of supplements. X-ray diffraction analysis displayed change in percentage crystallinity (%Cr) and crystallinity index (CrI) of pretreated SSB with reference to avicel (Fig. S2D & Table 5). The %Cr and CrI of avicel was 88.1% and 0.86. The %Cr and CrI of SSB for untreated, pretreated without supplement and pretreated with combination of supplements were 75.2, 73.9, 64.8% and 0.72, 0.65, 0.45 respectively. The decrease in %Cr and CrI indicated the lowering of the crystallinity of SSB due to degradation of lignin after pretreatment.
22
Thermal degradation (TGA) curves and the derivative of the TGA curves (DTG) of SSB showed three stages (Fig. S2E & Table S5): (i) dehydration and volatization at <180 °C, (ii) a degradation between 190 - 500 °C corresponding to depolymerization of hemicelluloses and cellulose components, and (iii) charcoal formation at >500 °C by slow decomposition of the solid residues of lignin. The percentage mass loss was more, DTG curves shifted towards the lower temperature range and the intensity of DTG peaks increased in pretreated samples, indicated the higher rate of thermal degradation. This was due to lower thermal stability of pretreated samples after the reduction in cellulose crystallinity and degradation of lignin.
3.7.Enzymatic hydrolysis
Enzymatic hydrolysis of avicel and SSB were carried out (Fig. 3). The addition of supplements during pretreatment affected the hydrolysis of SSB. The glucose yield on hydrolysis of avicel, SSB untreated, SSB pretreated without supplement and with combined supplements were 764.0 ± 37, 162.7 ± 6.6, 189.3 ± 9.2 and 395.8 ± 14.4 mg g-1 respectively. Supplements in combination increased the lignin degradation and porosity of SSB that enabled the higher penetration of hydrolytic enzymes to increase in the glucose yields 2.43 and 2.09 times than untreated SSB and control (pretreatment without supplement) respectively.
Release of sugar was higher in our study than from the sugarcane bagasse treated with 1% H2SO4 and 1% NaOH (66.6 mg g-1 ) (Maitan-Alfenas et al., 2015), Eucalyptus globulus chips pretreated with endophytic fungi Ulocladium sp (180.0 mg g-1) (Martín-Sampedro et al., 2015), rice straw pretreated with oxidases and hydrolytic enzymes (315 mg g-1) (Dhiman et al., 2015), sugarcane bagasse using C. subvermispora (210 mg g-1) (Da Silva Machado & Ferraz, 2017), short time
23
pretreatment of rice straw with alkaline peroxide (sugar yield increased 2.1 times) (Hideno, 2017).
In recent study, the cost of pretreatment process for different process were compared on the basis of Techno-economic models to observe that cost of sugar yield by biological pretreatment were high (Baral & Shah, 2017). In our study fermentable sugar increased 2.09 folds than control (pretreated without supplement) showed that production cost of sugar will come down to half. We used low cost supplements in small amounts without affecting the other process parameters of SSF i.e. without increasing normal cost for biological pretreatment. Decreased production cost of sugar will make our process to be competitive.
4. Conclusion C. versicolor showed high lignin degradation with minimum cellulose loss in pretreatment of SSB by SSF. Supplements of syringic acid increased the production of LiP, AAO and laccase; gallic acid increased MnP and CuSO4 increased laccase and PPO. Optimized combination of supplements increased the production of laccase, LiP, MnP, PPO and AAO to 17.2, 45.5, 3.5, 2.4 and 3.6 folds respectively resulting in maximum lignin degradation (2.3 folds) and SV (7.1 folds) by synergistic action of enzymes. Enzymatic hydrolysis of pretreated SSB yielded higher (~2.43 times) fermentable sugar. This technique could be applied for improvement in pretreatment and enzyme productions. Supplementary three figures and five tables submitted as background work of the article.
24
Acknowledgement Mrs. Vartika Mishra gratefully acknowledges Ministry of Human Resource Development (MHRD), Govt. of India for providing the fellowship during the study. All authors are highly thankful to National Institute of Technology (NIT), Jalandhar for providing grants and administrative supports for the study. References 1.Aguiar, A., Gavioli, D., Ferraz, A., 2014. Metabolite secretion, Fe3+-reducing activity and wood degradation by the white-rot fungus Trametes versicolor ATCC 20869. Fungal biology, 118(11), 935-942. 2.Aidoo, K.E., Hendry, R., Wood, B.J.B., 1981. Estimation of fungal growth in a Solid-State Fermentation system. Eur. J. Appl. Microbiol. Biotechnol., 12(1), 6-9. 3.Archibald, F.S., 1992. A new assay for lignin-type peroxidases employing the dye azure B. Appl. Environ. Microbiol., 58(9), 3110-3116. 4.Bailey, M.J., Biely, P., Poutanen, K., 1992. Interlaboratory testing of methods for assay of xylanase activity. J. Biotechnol., 23(3), 257-270. 5.Baral, N.R., Shah, A., 2017. Comparative techno-economic analysis of steam explosion, dilute sulfuric acid, ammonia fiber explosion and biological pretreatments of corn stover. Bioresour. Technol., 232, 331-343. 6.Bourbonnais, R., Paice, M.G., 1990. Oxidation of non-phenolic substrates: an expanded role for laccase in lignin biodegradation. FEBS letters, 267(1), 99-102. 7.Chang, A.J., Fan, J., Wen, X., 2012. Screening of fungi capable of highly selective degradation of lignin in rice straw. Int. Biodeterior. Biodegrad., 72, 26-30. 8.Christian, V., Shrivastava, R., Shukla, D., Modi, H., Vyas, B.R.M., 2005. Mediator role of veratryl alcohol in the lignin peroxidase-catalyzed oxidative decolorization of Remazol Brilliant Blue R. Enzyme Microb. Technol., 36(2), 327-332. 9.D’souza, T.M., Merritt, C.S., Reddy, C.A., 1999. Lignin-modifying enzymes of the white rot basidiomycete Ganoderma lucidum. Appl. Environ. Microbiol., 65(12), 5307-5313. 10. Da Silva Machado, A., Ferraz, A., 2017. Biological pretreatment of sugarcane bagasse with basidiomycetes producing varied patterns of biodegradation. Bioresour. Technol., 225, 17-22. 11. Dhiman, S.S., Haw, J.-R., Kalyani, D., Kalia, V.C., Kang, Y.C., Lee, J.-K., 2015. Simultaneous pretreatment and saccharification: Green technology for enhanced sugar yields from biomass using a fungal consortium. Bioresour. Technol., 179, 50-57. 12. Dowe, N., McMillan, J. 2001. SSF experimental protocols: lignocellulosic biomass hydrolysis and fermentation: Laboratory Analytical Procedure (LAP). Golden, Co: National Renewable Energy Laboratory. 13. Ghorbani, F., Karimi, M., Biria, D., Kariminia, H., Jeihanipour, A., 2015. Enhancement of fungal delignification of rice straw by Trichoderma viride sp. to improve its saccharification. Biochemical Engineering Journal, 101, 77-84. 25
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Figures and Tables (captions) Fig. 1 Contour plots showing the effect of (µmol g-1 SSB) CuSO4, gallic acid and syringic acid and their cooperative actions on lignin degradation (% w w-1), cellulose loss (% w w-1) and SV Fig. 2 Effect of supplements on degradation of (A) lignin, (B) cellulose; (C) selectivity value (SV); (D) degradation of hemicelluloses; (E) hemicellulose loss to cellulose loss ratio; (F) growth of C. versicolor during pretreatment (
) without supplement; (
g-1 SSB; (
) CuSO4 2.2 µmol g-1 SSB; (
) syringic acid 8.8 µmol g-1 SSB; (
) gallic acid 4.4 µ mol
) syringic acid 8.8 µmol g-1 SSB +
gallic acid 2.2 µmol g-1 SSB + CuSO4 8.8 µmol g-1 SSB Fig. 3 Enzymatic hydrolysis of SSB to fermentable sugar (
) SSB untreated; SSB pretreated (
supplements; (
) without supplement, (
) with combined
) cellulose
Table 1 CCD design for variables and their responses Table 2 Regression coefficients and identification of significant variables (p < 0.05) Table 3 Peak lignolytic, cellulolytic and hemicellulolytic enzyme activities during pretreatment of SSB Table 4 Solutions obtained from the CCD optimization of concentration of syringic acid, gallic acid and CuSO4 for lignin degradation and SV Table 5 Percentage crystallinity (%Cr) and crystallinity index (CrI) of avicel, untreated and treated SSB
29
List of supplementary tables and figures (captions) Table S1. Lignolytic and cellulolytic ability of white rot fungi measured by diameter of characteristic zones Table S2. Peak lignolytic, cellulolytic and hemicellulolytic enzyme activities during pretreatment by fungal strains Table S3. Degradation of lignin (LIG), cellulose (CEL), hemicellulose (HEM) and variation of selectivity value (SV) during pretreatment of SSB by fungal strains Table S4 Degradation of lignin, cellulose, hemicellulose and variation of selectivity value (SV) in the presence of supplements CuSO4, gallic acid and syringic acid individually after 20 days of pretreatment by C. versicolor Table S5 Thermal studies of untreated and treated SSB Figure S1 Effect of different metallic and phenolic compounds on degradation (% w w-1) of SSB and selectivity value after 20 days pretreatment by C. versicolor (
) Lignin, (
) cellulose, (
) hemicelluloses and (
) selectivity value
Figure S2 Characterization of SSB (A) SEM (untreated), (B) SEM (pretreated with combined supplements), (C) FTIR, (D) XRD, (E) TGA (
) avicel; (
) untreated; pretreated (
) without supplement, (
) with
combined supplements Figure S3 FTIR spectra of lignin
30
Lignin Degradation (% w/w)
10.00
Lignin Degradation (% w/w)
10.00
35.2831
B 7.50
6 5.00
35.2831
36.4521
38.0345
45.2726
CuSO4 (umol/g SSB)
Gallic Acid (umol/g SSB)
A
39.617 41.1994
42.7818
2.50
7.50
42.7818 41.1994
36.4521
39.617 38.0345
6
5.00 35.2831 33.3926
2.50 28.834
0.00
0.00
0.00
2.50
5.00
7.50
10.00
Lignin Degradation (% w/w)
10.00
0.00
D
39.1264
7.50
38.0345
6 5.00
36.4521 35.2831 33.3926
2.50 28.834
Gallic Acid (umol/g SSB)
39.617
CuSO4 (umol/g SSB)
C
5.00
7.50
10.00
cellulose loss (% w/w)
10.00
Syringic Acid (umol/g SSB)
2.50
Syringic Acid (umol/g SSB)
7.50
11.114
6 5.00
10.847 10.571 10.296 10.021 9.746
2.50 8.675 8.675
0.00 0.00
5.00
7.50
0.00
10.00
0.00
cellulose loss (% w/w)
10.00
Gallic Acid (umol/g SSB)
F
7.50
6 5.00
10.847
10.296
10.571
10.021 9.746
2.50
10.00
2.50 5.00 7.50 cellulose loss (% w/w)
10.00
Syringic Acid (umol/g SSB)
CuSO4 (umol/g SSB)
CuSO4 (umol/g SSB)
E
2.50
7.50
10.296
10.571
6 5.00 10.296 10.021
10.847
9.746
2.50
8.675
8.675 8.675
0.00
0.00 0.00
2.50
5.00
7.50
0.00
10.00
SV
H
7.50 3.33701
6 5.00
3.63441 3.94949
2.50
7.50
10.00
SV
10.00
4.26456
3.63441
CuSO4 (umol/g SSB)
Gallic Acid (umol/g SSB)
G
5.00
Gallic Acid (umol/g SSB)
Syringic Acid (umol/g SSB) 10.00
2.50
4.26456
7.50 3.94949
6 5.00
3.33701
31
2.50
2.96908
4.57964 4.89472
4.26456
0.00 0.00
0.00 2.50
5.00
7.50
10.00
0.00
2.50
5.00
7.50
10.00
CuSO4 (umol/g SSB)
I
SV
10.00
7.50
4.26456
3.63441 3.94949
6
5.00 3.33701
2.50
2.96908
0.00 0.00
2.50
5.00
7.50
10.00
Gallic Acid (umol/g SSB)
32
35
B
A Cellulose loss (% w w -1 )
Lignin degradation(% w w -1 )
50
40
30
20
10
14
0 0
4
5
8
12
16
20
0
4
Pretreatment time (Day)
Hemicelluloss loss (% w w -1 )
4
3
2
1 4.0 0
E
8
12
16
20
Pretreatment time (Day)
3.2
12
16
20
16
20
16
20
D
36 30 24 18 12 6 0 500
4
2.4
1.6
0.8
Fungal biomass (mg gdm -1 )
0
8
Pretreatment time (Day)
C
SV
21
7
0
Hemicellulose loss / cellulose loss
28
0
F
4
8
12
Pretreatment time (Day) 400
300
200
100
0
0.0 0
4
8
12
Pretreatment time (Day)
16
20
0
4
8
12
Pretreatment time (Day)
33
Fermentable sugar (mg g-1 dry matter)
800 700 600 500 400 300 200 100 0 0
24
48
72
96
120
Hydrolysis time (hr)
34
Table 1 CCD design for variables and their responses
Variable
Run
Syringic acid (µmol g-1 SSB) X1
Responses
Gallic acid (µmol g SSB)
-1
CuSO4 (µmol g SSB)
X2
X3
-1
Lignin degradation
Cellulose loss
(% w w-1)
(% w w-1)
SV
1
5.5
0
5.5
33.13
8.95
3.82
2
0
5.5
5.5
35.06
9.13
3.84
3
8.8
8.8
2.2
33.35
10.19
3.27
4
5.5
5.5
5.5
37.68
11.19
3.37
5
2.2
8.8
2.2
29.58
9.85
3.00
6
8.8
2.2
2.2
33.92
8.32
4.08
7
5.5
5.5
5.5
38.05
11.03
3.45
8
5.5
5.5
11.0
37.22
10.19
3.65
9
11.0
5.5
5.5
43.86
10.15
4.32
10
2.2
2.2
2.2
29.87
8.84
3.38
35
11
5.5
11.0
5.5
37.05
10.62
3.49
12
2.2
2.2
8.8
36.59
9.22
3.97
13
5.5
5.5
5.5
37.51
10.89
3.44
14
8.8
2.2
8.8
45.80
9.81
4.67
15
8.8
8.8
8.8
44.06
10.29
4.28
16
2.2
8.8
8.8
36.48
9.78
3.73
17
5.5
5.5
5.5
36.99
10.88
3.40
18
5.5
5.5
5.5
37.51
10.58
3.55
19
5.5
5.5
0
23.66
8.83
2.68
20
5.5
5.5
5.5
37.22
11.09
3.36
36
Table 2 Regression coefficients and identification of significant variables (p < 0.05)
Coefficient
Lignin degradation (% w w-1)
Cellulose loss SV -1
(% w w )
37.48
10.94
3.43
X1 (syringic acid)
2.89 a
0.19 a
0.22a
X2 (gallic acid)
-0.33 a
0.49 a
-0.23 a
X3 (CuSO4)
4.32 a
0.31 a
0.33 a
X1 * X2
-0.24 b
0.09 b
0.072 a
X1 * X3
1.12 a
0.016 a
0.035 b
X2 * X3
0.007 a
-0.23 b
0.069 b
X1
0.80 a
-0.47 a
0.25 a
X2
0.14 a
-0.42 b
0.17 a
Constant Linear
Interactions
Quadratic
37
X3
-2.39 a
-0.51 a
-0.07 b
R2
0.98
0.92
0.94
Model significance
a
a
a
Lack of Fit
b
b
b
a – significant (p < 0.05); b - not significant
38
Table 3 Peak lignolytic, cellulolytic and hemicellulolytic enzyme activities during pretreatment of SSB
Lignolytic enzyme (U g-1)
Supplem ent (µmol g-1 SSB) without supplem ent (control)
CuSO4 2.2
Laccase
LiP
MnP
PPO
AAO
11.9±0.3
0.13±0 .0
8.1±0. 2
4.9±0. 07
1.39±0 .04
6.4±0. 06
12.7±0 .42
12.6±0.4 4
(12th day)
(12th day)
(16th day)
(12th day)
(20th day)
(20th day)
(20th day)
0.17±0 .02
15.6± 2.1
9.4±1. 2
2.2±0. 3
3.0±0. 4
10.0±1 .3
6.1±0.8
(16th day)
58.2±4.3
42.1±3.6 (16th day)
Syringic acid 8.8
FPase
ßglucosid ase
CMC ase
(12th day)
Gallic acid 4.4
Cellulolytic enzyme (U g-1)
67.4±7.7 (20th day)
th
th
(16 day)
(12 day)
0.23±0 .01
20.3± 1.1
(16th day)
(12th day)
1.7±0. 08
19.0± 1.1
(16th day)
(20th day)
th
(12 day)
th
th
th
(12 day)
(16 day)
(16 day)
2.5±0. 1
5.7±0. 03
10.6±1 .04
(20th day)
(16th day)
(16th day)
7.5±0. 8
3.9±0. 1
2.2±0. 1
6.7±0. 9
(16th day)
(16th day)
(16th day)
(16th day)
6.2±0. 9 (20th day)
(12th day)
7.3±0.3 (20th day)
4.8±0.2 (16th day)
Hemicellulo lytic enzyme (U g-1)
Xylanase
7.9±0.06 (20th day)
7.5±0.67 (20th day)
7.7±0.13 (16th day)
8.9±0.82 (20thday)
39
CuSO4 8.8 + Gallic acid 2.2 + Syringic acid 8.8
205.01± 10.1 (16th day)
5.94±0 .2 (12th day)
28.7± 1.1 (12th day)
12.20± 0.5 (16th day)
5.1±0. 2 (8th day)
1.3±0. 09 (20th day)
3.1±0. 3 (16th day)
2.0±0.1 (20th day)
11.9±1.13 (16th day)
40
Table 4 Solutions obtained from the CCD optimization of concentration of syringic acid, gallic acid and CuSO4 for lignin degradation and SV
Syringic
Gallic
Acid
Acid
(µmol g-1
(µmol g-
SSB)
1
SSB)
CuSO4
Lignin Degradation
(µmol g-1
(% w w-1 )
SV
SSB) Predicted
Experimental
Predicted
Experimental
value
value
value
value
8.8
2.2
8.8
45.04167
45.8 ± 1.1
4.588089
4.67 ± 0.13
8.91
2.42
8.25
45.017001
46.12 ± 0.6
4.54597
4.6 ± 0.15
8.8
2.35
8.8
44.99787
45.1 ± 1.0
4.562023
4.54 ± 0.11
8.75
2.2
8.49
44.86869
44.7 ± 1.3
4.562593
4.58 ± 0.11
8.8
2.2
8.23
44.83367
45.0 ± 0.9
4.56055
4.51 ± 0.09
8.8
2.2
8.17
44.80582
44.5 ± 1.2
4.557628
4.49 ± 0.17
8.8
2.2
8.1
44.77025
44.9 ± 1.3
4.554033
4.52 ± 0.10
8.8
2.2
7.78
44.57432
45.0 ± 1.0
4.536092
4.50 ± 0.13
41
8.8
2.2
7.61
44.44696
44.7 ± 1.1
4.52558
4.50 ± 0.10
8.8
2.59
8.16
44.69036
44.8 ± 1.3
4.489461
4.48 ± 0.09
42
Table 5 Percentage crystallinity (%Cr) and crystallinity index (CrI) of avicel, untreated and treated SSB 2θ scale Sample
%Cr
CrI
I22
I18
Avicel
1021
137
88.1
0.86
Untreated SSB
607
168
75.2
0.72
462
158
73.9
0.65
347
188
64.8
0.45
SSB (pretreated without supplement)
SSB pretreated with supplement (µ mol g-1 SSB) Syringic acid 8.8 + gallic acid 2.2 + CuSO4 8.8
43
•
Coriolus versicolor selected for pretreatment of sweet sorghum bagasse by SSF
•
Combined supplements enhanced multiple lignolytic enzymes production
•
CuSO4, gallic and syringic acid combination optimized
•
Synergistic action of enzymes yielded highest lignin degradation and selectivity
•
Sugar yield increased ~2.43 times after enzymatic hydrolysis
44