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Improvement of selective lignin degradation in fungal pretreatment of sweet sorghum bagasse using synergistic CuSO4-syringic acid supplements
Journal of Environmental Management xxx (2017) 1e9
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Research article
Improvement of selective lignin degradation in fungal pretreatment of sweet sorghum bagasse using synergistic CuSO4-syringic acid supplements Vartika Mishra a, Asim K. Jana a, *, Mithu Maiti Jana b, Antriksh Gupta a a b
Department of Biotechnology, Dr B R A National Institute of Technology, Jalandhar, 144011, Punjab, India Department of Chemistry, Dr B R A National Institute of Technology, Jalandhar, 144011, Punjab, India
a r t i c l e i n f o
a b s t r a c t
Article history: Received 2 November 2016 Received in revised form 23 January 2017 Accepted 21 February 2017 Available online xxx
Sweet sorghum bagasse (SSB) generated in large quantities could be hydrolyzed to sugar and then fermented to green fuels. The hydrolysis of SSB polysaccharides interlocked in recalcitrant lignin network is the major problem. Pretreatment of SSB in SSF by using Coriolus versicolor with CuSO4-syringic acid supplements for effects on production of ligninocellulolytic enzymes, lignin degradation and selectivity values (SV) were studied. C. versicolor was selected based on high ligninolytic and low cellulolytic abilily. Individually, CuSO4 increased the activities of laccase (4.9 folds) and PPO (1.9 folds); syringic acid increased LiP (13 folds), AAO (2.8 folds) and laccase (5.6 folds) resulting in increased lignin degradation and SVs. Combined syringic acid (4.4 mmol g1 SSB) and CuSO4 (4.4 mmol g1 SSB) increased the activities of laccase, LiP, MnP, PPO and AAO by 11.2, 17.6, 2.8, 2.4 and 2.3 folds respectively due to synergistic effect, resulting in maximum lignin degradation 35.9 ± 1.3% (w w1) (1.86 fold) and highest SV 3.07 (4.7 fold). Enzymatic hydrolysis of pretreated SSB yielded higher (~2.2 times) fermentable sugar. Pretreated SSB was characterized by XRD, SEM, FTIR and TGA/DTG analysis to confirm results. It is possible to improve fungal pretreatment of agricultural waste by combination of supplements. © 2017 Elsevier Ltd. All rights reserved.
Keywords: Ligninocellulosic waste Fungal pretreatment Ligninolytic enzymes Supplements Delignification Enzymatic hydrolysis
1. Introduction Biofuels produced from ligninocellulosic biomass/agricultural residues showed the potential as an alternative fuel to overcome the need of fossil fuels and benefits with respect to environmental management. Sweet sorghum (Sorghum sp.) bagasse (SSB) is promising for use in biofuel production due to its higher yield of waste biomass. This ligninocellulosic waste mainly consists of lignin (cross-linked phenol polymers), cellulose (homopolymer of glucose) and hemicellulose (heteropolymer of pentoses and hexoses) (Ratnavathi et al., 2011). The polysaccharides i.e. cellulose and hemicellulose (55e75% on dry weight basis) could be hydrolyzed to sugars and then fermented to alcohols and biofuels. But, these polysaccharides are interlocked in the polymeric network structure of recalcitrant lignin. To overcome the hindrance of lignin, pretreatment of ligninocellulosic biomass is necessary step. Biological
* Corresponding author. Department of Biotechnology, Dr B R A National Institute of Technology, G T Road Bye Pass, Jalandhar, 144011, Punjab, India. E-mail address: [email protected] (A.K. Jana).
pretreatment comprises the application of microorganisms such as white rot fungi (WRF) for selective degradation of lignin and hemicellulose (Alexandropoulou et al., 2016). WRF vary in abilities to degrade lignin due to their ligninolytic abilities because of activities of enzymes laccase (phenol oxidase), lignin peroxidase (LiP), manganese peroxidase (MnP), arylalcohol oxidase (AAO) and polyphenol oxidase (PPO) which breakdown the lignin polymer into soluble compounds that are further mineralize (completely) into CO2 & H2O (Hatakka, 1994). WRF Phanerochaete chrysosporium, Abortiporus biennis and C. versicolor have been studied for their delignification abilities during pretreatment of rice straw (Chang et al., 2012), sawdust (Alexandropoulou et al., 2016) and bamboo residue (Zhang et al., 2007a) respectively. Although WRF are the best lignin degraders; non-selective lignin degradation (Sitarz et al., 2013), long pretreatment time (Shi et al., 2009) and high cellulolytic activities are the problems during pretreatment. To screen the fungal strain for high lignin degradation with low cellulose consumption, selectivity value (SV) is an important parameter (Hakala et al., 2004; Shi et al., 2009; Zhang et al., 2007b). SV is defined as a ratio of amount of lignin degraded to amount of cellulose consumed by fungal strain
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Please cite this article in press as: Mishra, V., et al., Improvement of selective lignin degradation in fungal pretreatment of sweet sorghum bagasse using synergistic CuSO4-syringic acid supplements, Journal of Environmental Management (2017), http://dx.doi.org/10.1016/ j.jenvman.2017.02.057
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during pretreatment. Few WRF such as Coriolus versicolor (Aguiar et al., 2014), Pleurotus ostreatus (Taniguchi et al., 2005), Trametes hirusuta (Knezevic et al., 2013) and Phanerochaete chrysosporium (Chang et al., 2012) showed high SV. It depended on lignocellulolytic ability of the fungal strains, substrates used and culture conditions (Chang et al., 2012; Salvachua et al., 2013). Addition of supplements for enhancement in production of certain ligninolytic enzymes by the fungal strains have been studied (Nousiainen et al., 2014; Zerva et al., 2016). It was possible to control the productions of ligninolytic enzymes oxidases and peroxidases by using metallic salts, phenolic and aromatic compound supplements. An addition of phenolic and aromatic supplements (ferulic acid, xylidine, veratric acid, vanillic acid, cinnamic acid and guaiacol) reported to enhance the laccase production (Elisashvili et al., 2010; Liu et al., 2013). Metallic salt MnSO4 enhanced the MnP activity during biodegradation of Eucalyptus grandis (Vicentim and Ferraz, 2007), while CuSO4 and MnSO4 inhibited cellulolytic enzymes (Geiger et al., 1998; Tejirian and Xu, 2010). The degradation of lignin in pretreatment process occurs by complex reactions involving multiple ligninolytic and cellulolytic enzymes produced by the fungal strains. The achievement of high lignin degradation and SV in pretreatment could be possible only through the enhancement in production of multiple lignocellullytic enzymes and their interactions. Although, effect of individual supplements on specific enzyme production have been reported as above, combination of more than one supplements to enhance multiple ligninolytic enzymes, lignin degradation and SV has not been studied yet. The objective of the present study was to enhance the degradation of lignin with high SV in a pretreatment of SSB by fungal strain using combined effect of an inorganic metallic salt and a phenolic supplement. Fungal strain having high selectivity was screened initially out of different strains used in the pretreatment (Aguiar et al., 2014; Chang et al., 2012; Knezevic et al., 2013; Taniguchi et al., 2005; Zhang et al., 2007a). The effect of the combination of CuSO4 and syringic acid on production of ligninolytic enzymes, cellulolytic enzymes and selective lignin degradation compared to the effect of the individual supplements have been reported for the first time to the best of our knowledge. 2. Materials and methods 2.1. Materials 2,2'azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) was purchased from MP Biomedical (Mumbai, India). p-nitrophenyl-b-D-glucopyranoside (p-NPG), birchwood xylan, lignin sulfonic acid, catechol, vanillin and verataryl alcohol were purchased from Himedia Laboratory Pvt Ltd (Mumbai, India). Azur-B and bovine serum albumin (BSA) were purchased from S.D. fine chem. Ltd (Mumbai, India). Cellulose and syringic acid were purchased from Sigma Aldrich (Mumbai, India). Water was purified and deionized (DI) by Milli-Q purification system (minimum resistivity 18 MU cm; Millipore, Billerica, MA, USA). Rest of chemicals used were analytical grade chemicals made by S.D. fine chem. Ltd (Mumbai, India). Cellulase (SaccariSEB EG) and b-glucosidase (SaccariSEB BG) were gifted from Advanced Enzyme Technologies Ltd (Thane, India). 2.2. Biomass Sweet Sorghum Bagasse (SSB) was collected from the local agricultural farms in Jalandhar, Punjab, India. SSB was dried at 40 C for 72 h and ground to pass through 5 mm 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 eryngii (MTCC 1798), 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. All strains were grown and maintained in 2% (w v1) malt extract agar media in petri plates at pH 4.5, 25 C, and sub-cultured periodically after every 15 days. 2.4. Ligninocellulolytic abilities 2.4.1. Ligninolytic ability Basal medium (BM) was prepared containing (g L1) ammonium tartrate 5.0, yeast extract 0.1, potassium phosphate monobasic 1.0, calcium chloride 0.001, magnesium sulfate 0.5 and 1.6% (w v1) agar (pH 4.5). Substrates i.e. 0.1% (w v1) ABTS, 0.1% (v v1) guaiacol, 0.01% (w v1) tannic acid, 0.25% (w v1) lignin sulfonic acid and 0.01% (w v1) azure-B were used to determine the extent of ligninolytic ability (Pointing, 1999). Single disc taken from actively growing 7 days old fungal culture plate using sterile cock borer (10 mm diameter), was used as inoculums. Sterilized petri plates containing BM and supplemented with substrates were inoculated with test strains and incubated (Innova 42R, Eppendorf ltd, USA) at 27.5 C for 5 days. The colourless agar medium turned green due to the oxidation of ABTS to ABTS-azine due to laccase produced by fungus. Colorless medium containing guaiacol turned to brick red due to laccase produced by fungus. The appearance of a brown oxidation zone around colonies in petri plates containing tannic acid signified the polyphenol oxidase production. In lignin sulfonic acid plate, 1% (w v1) aqueous solution of ferric chloride and 1% (w v1) potassium ferricyanide was flooded, washed with DI water after 10 min, and observed for clear zones of degraded lignin around colonies against blue green colour of undegraded lignin. Clearance of blue colour of agar in the petri plate containing azure B showed the ability of lignin peroxidases production by fungal strain. Diameter of these colour zones around the colonies were taken as the ligninolytic ability of the strains. 2.4.2. Cellulolytic and hemicellulolytic (xylanolytic) ability The cellulolytic ability of fungal strains was detected by the Congo red test (Teather and Wood, 1982). Petri plates containing BM with carboxy methyl cellulose (1.1% (w v1)) as substrate were sterilized. Single disc taken from actively growing 7 days old fungal culture plate using sterile cock borer (10 mm diameter) was inoculated and incubated at 27.5 C for 5 days. Cultivated plates were flooded with aqueous Congo red solution (1 mg mL1). After 15 min of exposure, dye was drained and plates were washed three times with 1 M NaCl and observed for production of yellow zones around the colonies. For xylanolytic ability, xylan (4% (w v1)) was supplemented with BM in petri plates (Pointing, 1999). Sterile plates were inoculated with test strains and incubated at 27.5 C for 5 days. Plates were flooded with iodine stain (0.25% (w v1) aqueous I2 and 0.25% (w v1) KI). After 5 min of exposure, dye was drained and plates were washed with DI water, and observed for appearance of a yellow-opaque area (signified the release of xylanase) against a blue/reddish purple colour for undegraded xylan. Diameter of coloure zones around fungal discs were taken as cellulolytic and hemicellulolytic ability of strains. 2.5. Pretreatment of SSB Ten discs (using sterile cock borer with 10 mm in diameter) from
Please cite this article in press as: Mishra, V., et al., Improvement of selective lignin degradation in fungal pretreatment of sweet sorghum bagasse using synergistic CuSO4-syringic acid supplements, Journal of Environmental Management (2017), http://dx.doi.org/10.1016/ j.jenvman.2017.02.057
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actively growing 7 days old fungal culture plates were inoculated into 500 mL cotton plugged Erlenmeyer flask containing 50 mL of 2% (w v1) malt extract medium. After 7 days incubation at 27.5 C under static condition, the liquid culture was aseptically homogenized by a sterilized blender for 15 s 3 cycles. Two mL (~2.4 106 spore mL1) of blended culture was used as inoculum for 5 g of SSB. 5 g ground SSB (~3% moisture) was taken in 250 mL Erlenmeyer flasks, 11.5 mL of distilled water was added with it (75% moisture) and cotton plugged. Moistened SSB in flask was autoclaved at 121 C for 15 min. Sterilized flask was inoculated with 2 mL inoculum and incubated stationary at 27.5 C for 20 days solid state fermentation (SSF). Samples were taken every 4th day for compositional analysis of treated biomass, lignocellulolytic enzyme assays and enzymatic hydrolysis of biomass. Different supplements (veratryl alcohol, syringic acid, catechol, vanillin, guaiacol, CuSO4 and MnSO4) were studied to enhance the selectivity value and lignin degradation. All the supplements were dissolved in DI water to make 1 mM supplement solution. 5 g of SSB (~3% moisture) were taken in 250 mL Erlenmeyer flasks, mixed with 11.5 mL of supplement solution to make concentration of 2.2 mmol g1 SSB in culture. Pretreatment of SSB with supplements were carried out by SSF as described above. Supplements, significantly influencing the lignin degradation were studied individually and in combination to enhance the production of ligninolytic enzymes and lignin degradations. All the cultures were incubated statically at 27.5 C for 20 days. The flask culture without supplements was served as control. All experiments were performed in triplicate. 2.6. Ligninocellulolytic enzymes assays 50 mL acetate buffer (pH 4.5, 50 mM) was added to the each sample, shaked at 180 rpm and 27.5 C for 1 h, filtered under vacuum to recover 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 oven at 65 C and used for compositional analysis and enzymatic hydrolysis. 2.6.1. Ligninolytic activity Lignin peroxidase (LiP) activity was determined by H2O2 dependent oxidation of azure-B at 651 nm (ε651 ¼ 48800 M1 cm1) (Archibald, 1992). Laccase activity was determined by oxidation of ABTS to ABTS-azine at 420 nm (ε420 ¼ 36000 M1 cm1) (Bourbonnais and Paice, 1990). Manganese peroxidase (MnP) activity was determined by phenol red oxidation (ε610 ¼ 22000 M1 cm1) (Glenn and Gold, 1985). Polyphenol oxidase (PPO) was assayed by oxidation of catechol to quinone at 30 C (ε400 ¼ 3450 M1 cm1) (Wong et al., 1971). Aryl alcohol oxidase (AAO) activity was monitored by using the oxidation of veratryl alcohol to veratrylaldehyde (ε310 ¼ 9300 M1 cm1) (Guillen et al., 1992). One unit of enzyme activity was defined as the amount of enzyme required to oxidize 1 mmol of substrate per minute. Activities of all enzymes were expressed as units per gram dry matter (U gdm1). 2.6.2. Cellulolytic activity Carboxymethyl cellulase activity (CMCase/endo-b-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) activities were determined using Whatman No. 1 filter paper strip and carboxymethyl cellulose respectively as substrates (Ghose, 1987). One unit of enzyme activity was defined as the amount of enzyme required to produce 1 mmol reducing sugar per min. b-Glucosidase
3
activity was assayed by using p-NPG as substrate (Wood and Bhat, 1988). One unit of enzyme activity was defined as the amount of enzyme required to produce 1 mmol p-nitrophenol per min. Xylanase activity was assayed using 1% (w v1) 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 enzyme activity was defined as the amount of enzyme required to produce 1 mmol xylose per min under standard condition of pH and temperature. Activities all enzymes were expressed as units per gram dry matter (U gdm1). 2.7. Fungal growth estimation N-acetyl glucosamine content of the fungal cell wall was used to monitor fungal biomass (Aidoo et al., 1981). 2.8. Compositional analysis of SSB Compositions of SSB were determined before and after pretreatment by solid state fermentation. Lignin, cellulose and hemicellulose 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 v1) sulfuric acid at 30 C for 1 h. The samples were diluted immediately to 4% (v v1) and autoclaved for 1 h. Autoclaved hydrolyzed solution was vacuum filtered through previously weighed filtering crucibles. Crucible and acid insoluble residue was dried at 105 C until a constant weight was achieved. Aliquot sample collected from vacuum filtration was used to determine acid soluble lignin, cellulose and hemicellulose. The acid-soluble lignin was measured by UVeVis spectrophotometer (Kinetic, Biospectrometer, Eppendorf, Germany) using lignin sulfonic 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 HiPlex-H column and refractive index detector (RID) that were maintained at 80 and 55 C, respectively. HPLC grade water was used as the mobile phase, at flow rate of 0.6 mL min1. The content of cellulose in the sample was calculated from glucose and galactose obtained while hemicellulose 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 using equations %Cellulose ¼ (GR 0.9)/w 100 and %Hemicellulose ¼ (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, defined as the percentage of total lignin reduced after pretreatment, was calculated using equation %Lignin degradation ¼ (1 w(a þ b)/(wo (ao þ bo))) 100; where w is the sample dry weight (g) of the pretreated SSB; wo is the initial dry weight (g) of the untreated SSB; a, b and ao, bo are the % of acid-soluble and acid-insoluble lignin in the pretreated and untreated SSB, respectively. Dry mass loss was calculated as the percentage of total solids loss after pretreatment after removing fungal biomass content. 2.9. Enzymatic hydrolysis of pretreated SSB Enzymatic hydrolysis of cellulose (avicel), untreated and treated SSB were carried out following protocol described in NREL/TP-51042630 (Dowe and McMillan, 2001). One gram of biomass (3% moisture) was taken in 250 mL Erlenmeyer flask and 100 mL of 50 mM citrate buffer (pH 4.8) was added. Cellulase (SaccariSEB EG, 10 filter paper unit g1 solid) and b-glucosidase (SaccariSEB BG, 20
Please cite this article in press as: Mishra, V., et al., Improvement of selective lignin degradation in fungal pretreatment of sweet sorghum bagasse using synergistic CuSO4-syringic acid supplements, Journal of Environmental Management (2017), http://dx.doi.org/10.1016/ j.jenvman.2017.02.057
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cellobiose unit g1 solid) enzymes were added for hydrolysis of biomass. The flask was incubated in a shaking incubator at 165 rpm, 45 C at for 108 h and analyzed for release of fermentable sugar. Sugar content was estimated using Agilent Hi-Plex-H column in HPLC (Agilent-1200 infinity series) as discussed above in Section 2.8.
the lowest CMCase activity, and low FPase and ß-glucosidase activity. In overall, C. versicolor produced highest xylanase activity i.e. capability to utilize hemicellulose as carbon source, low cellulolytic enzymes and high ligninolytic enzymes with high degradation of lignin in SSB. Fungal strains showed different SVs, due to the varied lignin degradation, cellulose and hemicellulose loss depending on their lignocellulolytic abilities. C. versicolor showed maximum SV of 0.65 after 20 days and 0.59 after 30 days of SSF (Table S3) with high degradation of hemicellulose. Strain showing ability to degrade xylan is advantage for pretreatment as dependency on hemicellulose help to preserve cellulose. SV of pretreatment by C. versicolor decreased at latter phase of SSF (0.65 at 20th day to 0.59 at 30th day). This was due to shielded structure of lignin acted as barrier against consumption of cellulose at initial stage. C. versicolor was selected for pretreatment of SSB with supplements. SSB was pretreated with supplements (2.2 mmol g1 SSB initial) veratryl alcohol, syringic acid, catechol, vanillin, guaiacol, CuSO4 and MnSO4 for effect on lignin degradation and SVs (Fig. S1). CuSO4 resulted in maximum degradation of lignin (24.8 ± 2.3% (w w1)) followed by syringic acid (22.5 ± 1.0% (w w1)), while lower with other supplements (18.5e22.0% (w w1)). CuSO4 caused minimum consumption of cellulose (21.6 ± 1.01% (w w1)). Presence of CuSO4 and syringic acid resulted in SV values 1.15 and 0.89 respectively, higher than the control (without supplements). CuSO4 and syringic acid supplements were selected for study of the effects on lignocellulolytic enzyme activities, lignin degradations and SVs in pretreatment.
2.10. Characterization of SSB Changes in morphological, structural, chemical and thermal characteristics of SSB were characterized by the techniques of scanning electron microscopy (SEM) (EVO 40 EP-CARL ZEISS, Germany) (Zhang et al., 2007b), X-ray diffraction (XRD) (PANALYTICAL [XPERT-PROMPT, Netherland) (Zeng et al., 2011), Fourier transformed infrared spectroscopy (FTIR) (AGILENT CARY-630, USA) (Zhang et al., 2007b) and Thermogravimetric/Differential Thermogravimetric analysis (TGA/DTG) (SII EXSTAR 6000 [TG/DTA 6300], Japan) (Zeng et al., 2011). The percentage of crystallinity (% Cr) and crystallinity index (CrI) was calculated by equation (Segal et al., 1959),
%Cr ¼ Icrys
Icrys þ Iamor 100 and CrI ¼ Icrys Iamor Icrys
where Icrys is the overall intensity of the peak at 2q about 22 and Iamor is the intensity at 2q about 18 . 3. Results and discussion 3.1. Screening of lignin selective strain and supplements
Table 2 Degradation of lignin, cellulose, hemicellulose and variation of selectivity value (SV) in the presence of syringic acid and CuSO4 in combination.
Best lignin degrading fungal strain was screened out of eight fungal strains reported in literature (Aguiar et al., 2014; Chang et al., 2012; Knezevic et al., 2013; Taniguchi et al., 2005; Zhang et al., 2007a) based on high ligninolytic, low cellulolytic abilities and SV (lignin degradation/cellulose loss) during pretreatment of SSB. All strains exhibited the different ligninolytic, cellulolytic and hemicellulolytic abilities; but Coriolus versicolor showed highest ligninolytic and hemicellulolytic ability with low cellulolytic ability (Table S1). C. versicolor showed the highest laccase, LiP, MnP and PPO activity when grown on SSB in SSF (Table S2). C. versicolor also produced AAO enzyme, but it was less than Ganoderma lucidium and Phlebia radiata. Daedalea flavida, Pleurotus ostreatus and Stereum hirusita didn't show any AAO activity. Only C. versicolor, G. lucidium and P. radiata produced all ligninolytic enzymes (laccase, LiP, MnP, PPO and AAO). P. radiata showed the high celluloytic activity i.e CMCase, FPase and ß-glucosidase. C. versicolor showed
Supplement (mmol g1 SSB)
Degradation (% (w w1))
Syringic acid
CuSO4
Lignin
0.0 0.0 8.8 8.8 8.8 8.8 4.4 4.4 4.4 2.2 2.2 2.2
0.0 2.2 0.0 2.2 4.4 8.8 2.2 4.4 8.8 2.2 4.4 8.8
19.9 24.8 28.7 33.7 33.2 29.9 32.6 35.9 30.8 32.1 32.9 30.4
± ± ± ± ± ± ± ± ± ± ± ±
Cellulose 1.6 2.3 1.7 1.2 1.1 1.4 1.8 1.3 1.2 1.6 1.5 1.3
30.4 21.6 16.6 15.5 12.1 26.1 15.1 11.7 25.1 14.9 12.2 24.4
± ± ± ± ± ± ± ± ± ± ± ±
2.2 1.0 1.0 0.7 0.9 1.1 0.5 0.4 1.1 0.5 0.6 1.0
SV Hemicellulose 19.7 21.7 28.2 27.3 25.7 26.9 27.2 27.8 26.4 26.1 27.1 25.7
± ± ± ± ± ± ± ± ± ± ± ±
1.5 1.9 1.7 1.1 1.2 1.2 1.1 1.3 1.4 1.4 1.3 1.2
0.65 1.15 1.77 2.17 2.74 1.15 2.16 3.07 1.23 2.15 2.70 1.25
Table 1 Degradation of lignin, cellulose, hemicellulose and variation of selectivity value (SV) in the presence of supplements CuSO4 and syringic acid individually after 20 days of pretreatment by C. versicolor. Supplement
Concentration (mmol g1 SSB)
Degradation (% (w w1)) Lignin
None CuSO4
Syringic acid
0.0 0.11 1.1 2.2 4.4 8.8 22.0 0.11 1.1 2.2 4.4 8.8 22.0
19.9 22.3 23.6 24.8 23.9 23.4 19.3 23.5 23.7 22.4 29.4 28.7 20.7
± ± ± ± ± ± ± ± ± ± ± ± ±
SV Cellulose
1.6 1.0 1.1 2.3 2.2 1.4 1.3 1.2 1.0 1.0 2.7 1.7 1.4
30.4 32.8 31.7 21.6 22.1 23.8 28.3 35.5 27.9 25.8 25.0 16.6 33.7
± ± ± ± ± ± ± ± ± ± ± ± ±
2.2 1.9 1.8 1.0 1.6 1.5 1.7 1.7 2.3 2.3 2.3 1.0 2.3
Hemicellulose 19.7 21.4 19.0 21.7 20.1 19.2 17.6 22.5 23.7 24.9 26.0 28.2 25.8
± ± ± ± ± ± ± ± ± ± ± ± ±
1.5 1.0 1.0 1.9 1.6 1.1 1.2 1.1 1.4 1.3 2.3 1.7 1.8
0.65 0.68 0.74 1.15 1.08 0.98 0.68 0.66 0.84 0.86 1.17 1.77 0.61
Please cite this article in press as: Mishra, V., et al., Improvement of selective lignin degradation in fungal pretreatment of sweet sorghum bagasse using synergistic CuSO4-syringic acid supplements, Journal of Environmental Management (2017), http://dx.doi.org/10.1016/ j.jenvman.2017.02.057
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3.2. Effects of selected individual supplement and their combination 3.2.1. Biomass degradation and fungal growth Effects of CuSO4, syringic acid (0.0e22.0 mmol g1 SSB) on degradation of SSB individually (Table 1) and their combination (Table 2) were studied. The SSB was composed (w w1) of lignin
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(25.14%), cellulose (38.02%) and hemicellulose (25.01%). At CuSO4 2.2 mmol g1 SSB, maximum lignin degradation was 24.8 ± 2.3% (w w1) with cellulose loss 21.6 ± 1.01% (w w1) and SV 1.15. Addition of higher concentration of supplement decreased in the lignin degradation and SV, might be due to inhibition of fungal growth and ligninolytic enzyme activity. 8.8 mmol g1 SSB syringic acid
Fig. 1. Effect of supplements on degradation of (A) lignin; (B) cellulose; (C) selectivity value; (D) degradation of hemicellulose; (E) hemicellulose loss to cellulose loss ratio; (F) growth of C. versicolor during pretreatment of SSB.
Please cite this article in press as: Mishra, V., et al., Improvement of selective lignin degradation in fungal pretreatment of sweet sorghum bagasse using synergistic CuSO4-syringic acid supplements, Journal of Environmental Management (2017), http://dx.doi.org/10.1016/ j.jenvman.2017.02.057
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resulted in high lignin degradation (28.7 ± 1.7% (w w1)) with minimum cellulose consumption (16.6 ± 1.0% (w w1)) and high SV 1.77. As maximum lignin degradation and SV value was obtained at 8.8 mmol g1 SSB syringic acid, its combinations with CuSO4 was studied for further enhancement of lignin degradation. Syringic acid 4.4 mmol g1 SSB and CuSO4 4.4 mmol g1 SSB combination gave the maximum lignin degradation (35.9 ± 1.3% (w w1)) and SV 3.07 (Table 2). Pretreatment of SSB supplemented with syringic acid and CuSO4 in combination showed the significant improvement in lignin degradation than supplements with syringic acid, CuSO4 or control (Fig. 1A), probably due to increased activities of ligninolytic enzymes in culture. During first 4 days of pretreatment, lignin degradation was slow, while rate of cellulose loss was high (Fig. 1B) due to the consumption of easily accessible small amount of cellulose for primary growth of fungi. The cellulose content in SSB pretreated with supplement in combination was higher due to minimum cellulose loss during pretreatment. The availability and vulnerability of cellulose to subsequent hydrolysis is an important criterion for evaluating the performance of fungal pretreatment, because higher cellulose content in substrates, eventually provide higher accessibility of carbohydrates for enzymatic saccharification and sugar yield (Salvachua et al., 2013). Due to low cellulose loss with supplement in combination, highest SV 3.07 was achieved (Fig. 1C) (4.7 fold higher than the control). Presence of supplements in culture stimulated C. versicolor to consume hemicellulose in SSB. Maximum hemicellulose consumption (27.8 ± 1.3% (w w1)) was obtained with supplements in combination (Fig. 1D). Ratio of loss of hemicellulose to cellulose loss (H/C) (Fig. 1E) was similar to change of SV. Although difference of hemicellulose loss with syringic acid (28.2 ± 1.7% (w w1)) and with supplements in combination was small, but H/C with combined supplements was higher due to low cellulose consumption during pretreatment in latter case. Higher H/C ratio signified that combined supplements stimulated the fungi to feed on hemicellulose rather than cellulose for its primary growth. C. versicolor grew well on SSB in the presence of supplements in SSF (Fig. 1F) and prolific colonization observed in 20 days. The fungal growth was slow during first 4 days of pretreatment due to the lag phase. The fungal biomass were much higher (442.2 ± 26.3 mg gdm1) with phenolic supplement syringic acid compared to control (384.5 ± 31.2 mg gdm1) or with CuSO4 (328.3 ± 18.4 mg gdm1). When CuSO4 was added in combination with syringic acid, fungal biomass (431.3 ± 21.0 mg gdm1) was slightly lower than syringic acid alone might be due to oxidative stress by CuSO4 on fungi. Metallic salts have been reported to result in low fungal biomass (Baldrian, 2003; Geiger et al., 1998), while the phenolic supplements such as syringic acid and gallic acid have reported to increase in fungal growth and hyphae till a maximum concentration in the medium (D'souza et al., 1999; Srivastava et al., 2013). This could be a possible reason that syringic acid resulted in
higher fungal biomass when present alone and in combination with copper. It was interesting to note that exponential phase the culture reduced and stationary phase achieved earlier with the supplements. 3.2.2. Ligninolytic and cellulolytic enzyme productions Peak activities (U g1) of ligninolytic enzymes of 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. Presence of supplements increased ligninolytic enzyme productions during pretreatment (Table 3). CuSO4 (2.2 mmol g1 SSB) increased peak activities of laccase and PPO enzyme, while LiP and AAO was little affected, might be due to induction effect of copper on laccase and related oxidative enzyme (PPO) at transcription level (Tinoco et al., 2011). Unexpectedly, significant increase in MnP activity was also observed. In the presence of CuSO4, all ligninolytic enzymes showed peak activity on 12th day of fermentation except of LiP which peaked on 16th day. The stability of laccase activity was observed, might be due to the inhibition of extracellular proteolytic enzymes in the presence of Cu2þ, resulting in inhibition of laccase degradation (Kaur and Sudhakara, 2011). Syringic acid (8.8 mmol g1 SSB) enhanced the LiP activity to 1.7 ± 0.08 U g1, 13 folds higher than control. This might be due to 3 folds increase in AAO activity by syringic acid had produced H2O2 to increase the LiP activity significantly. Additionally, laccase activity (67.4 ± 7.7 U g1) increased to 7 folds and MnP activity (19.0 ± 1.1 U g1) increased to 3 folds with respect to control. All ligninolytic enzymes showed their peak activities on 16 - 20th day of fermentation. The significant increase in enzyme activities was might be due to structural similarity of syringic acid with syringyl unit present in the lignin. Difference between syringic acid and syringyl unit is functional group at para site of benzyl ring. Structural similarity can result in more inducing effect for release of ligninolytic enzymes (D'souza et al., 1999). This increase in ligninolytic enzymes had supported the significant increase in lignin degradation. CuSO4 4.4 mmol g1 SSB and syringic acid 4.4 mmol g1 SSB in combination, increased the activities of laccase (133.2 ± 7.1 U g1) by 11.2 folds, LiP (2.3 ± 0.1 U g1) by 17.6 folds, MnP (22.7 ± 1.0 U g1) by 2.8 folds, PPO (11.8 ± 0.6 U g1) by 2.4 folds and AAO (3.3 ± 0.1 U g1) by 2.3 folds compared to control. Although fungal biomass with syringic acid and CuSO4 together was lower than culture with syringic acid supplement alone, higher ligninolytic enzymes activities were detected. This clearly indicated the synergistic effect of supplements in combination on over expression of multiple ligninolytic enzyme activities that was responsible for high lignin degradation and SV. Presence of CuSO4 enhanced the laccase and related enzyme (PPO) and syringic acid enhanced laccase, LiP and AAO activity; which resulted in cumulative increase in multiple ligninolytic enzymes activities with combined supplements. Laccase showed peak activity on 12th day with combined
Table 3 Peak ligninolytic, cellulolytic and hemicellulolytic enzyme activity during pretreatment. Supplement (mmol g1 SSB)
Ligninolytic enzyme (U g1) Laccase
without supplement 11.9 ± 0.3 (control) (16th day) 58.2 ± 4.3 CuSO4 (2.2) (12th day) Syringic acid (8.8) 67.4 ± 7.7 (20th day) 133.2 ± 7.1 Syringic acid (4.4) þ CuSO4 (4.4) (12th day)
Cellulolytic enzyme (U g1)
Hemicellulolytic enzyme (U g1)
LiP
MnP
PPO
AAO
CMCase
FPase
ß-glucosidase
Xylanase
0.13 ± 0.0 (12th day) 0.17 ± 0.02 (16th day) 1.7 ± 0.08 (16th day) 2.3 ± 0.1 (8th day)
8.1 ± 0.2 (12th day) 15.6 ± 2.1 (12th day) 19.0 ± 1.1 (20th day) 22.7 ± 1.0 (12th day)
4.9 ± 0.07 (16th day) 9.4 ± 1.2 (12th day) 7.5 ± 0.8 (16th day) 11.8 ± 0.6 (16th day)
1.39 ± 0.04 (12th day) 2.2 ± 0.3 (12th day) 3.9 ± 0.1 (16th day) 3.3 ± 0.1 (8th day)
6.4 ± 0.06 (20th day) 3.0 ± 0.4 (16th day) 2.2 ± 0.1 (16th day) 1.9 ± 0.08 (20th day)
12.7 ± 0.42 (20th day) 10.0 ± 1.3 (16th day) 6.7 ± 0.9 (16th day) 4.3 ± 0.1 (16th day)
12.6 ± 0.44 (20th day) 6.1 ± 0.8 (12th day) 4.8 ± 0.2 (16th day) 2.9 ± 0.1 (20th day)
7.9 ± 0.06 (20th day) 7.5 ± 0.67 (20th day) 8.9 ± 0.82 (20th day) 9.2 ± 0.4 (16th day)
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supplements and CuSO4 alone, while it peaked on 20th day with syringic acid. This indicated the presence of syringic acid resulted in an over expression of laccase activity on 12th day with CuSO4. LiP
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and AAO showed an early and over expression (peak activity on 8th day) with combined supplements. MnP activity also increased with combined supplements and it overlapped with MnP activity with
Fig. 2. Characterization of untreated and pretreated SSBs. (A) Scanning electron microscopy (SEM) (untreated), (B) SEM (pretreated with supplements syringic acid and CuSO4), (C) Fourier transformed infrared spectroscopy (FTIR), (D) X-ray diffraction (XRD), (E) Thermogravimetric (TGA)/Differential Thermogravimetric Analysis (DTG).
Please cite this article in press as: Mishra, V., et al., Improvement of selective lignin degradation in fungal pretreatment of sweet sorghum bagasse using synergistic CuSO4-syringic acid supplements, Journal of Environmental Management (2017), http://dx.doi.org/10.1016/ j.jenvman.2017.02.057
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CuSO4 alone, indicating slight over expression due to syringic acid. Lignins in SSB is predominantly composed of b-O-40 aryl ether linkages, together with minor amounts of b-b0 , b-50 , b-10 and a,bdiaryl ether linkages (Sun et al., 2013). Although all ligninolytic enzymes possess ability to break these linkages by cleaving Ca - Cb bonds in lignin structure and by Ca oxidation, but LiP has highest and laccase has lowest redox potential. The depolymerization of lignin was initiated by AAO (AAO produces H2O2; initiator for LiP) and LiP, broke the inter-unit b-O-40 and b-b’ linkages by cleaving of Ca - Cb bonds. This resulted in production of partially degraded lignin and different types of aromatic monomers of lower molecular weights; further degraded by laccase, PPO and MnP (Aguiar et al., 2014; Bourbonnais and Paice, 1990; Machuca and Ferraz, 2001). Early and over expression of LiP and AAO on 8th day, and laccase on 12th day due to synergistic effect of two supplements in the culture were responsible for higher lignin degradation and SV. CuSO4 supplement reduced CMCase activity (3.0 ± 0.21 U g1) to half of control and FPase (10.0 ± 1.3 U g1) and b-glucosidase activity (6.1 ± 0.8 U g1) was low. Syringic acid also resulted in low cellulolytic activities 2.2 ± 0.1, 6.7 ± 0.9 and 4.8 ± 0.2 U g1 of CMCase, FPase and b-glucosidase, respectively. Syringic acid and CuSO4 in combination showed maximum inhibitory effect on cellulolytic enzyme (Table 3). b-glucosidase, CMCase and FPase activities were lowest 2.9 ± 0.1, 1.9 ± 0.08 and 4.3 ± 0.1 U g1 respectively with combined supplements, while xylanase activity was 9.2 ± 0.4 U g1. Decreased cellulolytic enzyme activities in the presence of syringic acid were due to its structural similarity with lignin. This forced an early release and over expressions of ligninolytic enzymes than the cellulolytic enzymes. High degradation of lignin and low consumption of cellulose improved the SV. Wan and Li (2011) studied the effect of metallic salt supplements on delignification of wheat straw by C. subvermispora and targeted only laccase production during pretreatment. Delignification increased to 9% from 4% (w w1) after 21 day. Fungal delignification of wheat straw using Irpex lacteus by SSF in presence of inducer MnSO4 was studied by Salvachua et al. (2013). Lignin degradation was 28.2% and 31.5% (w w1) 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 MnSO4 only on MnP enzyme. In present study, synergistic effect of CuSO4 and syringic acid increased multiple ligninolytic enzymes and delignification reached 35.9 ± 1.3% (w w1) with SV 3.07 after 20 days. 3.3. Characterization of SSB SSB were brownish in presence of the lignin, but colour changed to light yellow after pretreatment, could be due to exposure of cellulose at the surface or the change to phenolic structure. Structure of single strand of SSB was covered with small particles or globules and lignocelluloses had an intact surface structure initially (Fig. 2A). Removal of lignin resulted in a rugged and partially broken surface (Fig. 2B). A visible destruction in cell wall structure, alteration in intact morphology, formation of holes and crevices on the biomass surfaces was observed in pretreated SSB. Functional groups of untreated SSB showed broad peaks at 3418 cm1 (hydrogen bonded OeH stretching), 2930 cm1 (CeH stretching), 1638 cm1 (C]C stretching of aromatic ring), 1435 cm1 (CeH stretching) and 1130 cm1 (CeO stretching) (Zeng et al., 2011) by FTIR spectroscopy (Fig. 2C), similar to lignin (Fig. S2). Pretreatment of SSB resulted in decrease in the respective peaks and intensities especially at 1638 cm1 (C]C stretching for aromatic ring, mainly originating from lignin in particular) (Fig. 2C). The largest decrease in the intensities of lignin was observed in SSB pretreated with CuSO4 and syringic acid combination.
Fig. 3. Enzymatic hydrolysis of pretreated SSB to sugar.
X-ray diffraction analysis displayed the change in percentage crystallinity (%Cr) and crystallinity index (CrI) of pretreated SSB with reference to avicel (Fig. 2D and Table S4). The decrease in %Cr and CrI indicated the lowering of the crystallinity of pretreated SSB due to degradation of lignin. Thermal degradation characteristics, TGA and DTG curves of SSB showed three stages (Fig. 2E and Table S5); (i) dehydration and volatization at <180 C, (ii) degradation between 190 and 500 C corresponding to depolymerization of hemicellulose and cellulose components, and (iii) charcoal formation at >500 C by slow decomposition of the solid residues of lignin. Shifting of DTG curves towards the lower temperature, higher percentage of mass loss and increased DTG peaks intensities indicated the higher thermal degradation rate of pretreated samples, due to degradation of lignin and reduction in cellulose crystallinity. 3.4. Enzymatic hydrolysis Significant differences in glucose yields from the untreated and pretreated SSB were observed (Fig. 3). The addition of supplements during pretreatment affected the enzymatic hydrolysis of SSB. The glucose yield from SSB pretreated with CuSO4 (4.4 mmol g1 SSB) þ syringic acid (4.4 mmol g1 SSB) supplements was maximum (362.8 ± 11.1 mg g1). Syringic acid þ CuSO4 resulted in maximum lignin degradation and porosity of SSB after pretreatment that enabled the penetration of hydrolytic enzymes to increase the glucose yield. The sugar yield was ~2.2 and 1.91 times than the untreated SSB and control (pretreatment without supplement), respectively. 4. Conclusion Pretreatment of SSB by using C. versicolor yielded high lignin degradation and SV. Combination of supplements CuSO4 and syringic acid interacted synergistically to alter the activities of several ligninocellulolytic enzymes during pretreatment. Activities of laccase, LiP, MnP, PPO, AAO enhanced by 11.2, 17.6, 2.8, 2.4, 2.3 folds respectively to increase the lignin degradation (1.86 folds) and SV (4.7 folds). Enzymatic hydrolysis of pretreated SSB yielded higher (~2.2 times) fermentable sugar. It is possible to improve fungal pretreatment of agricultural waste by combination of supplements and convert usable cellulose to fermentable sugar for green fuel industry.
Please cite this article in press as: Mishra, V., et al., Improvement of selective lignin degradation in fungal pretreatment of sweet sorghum bagasse using synergistic CuSO4-syringic acid supplements, Journal of Environmental Management (2017), http://dx.doi.org/10.1016/ j.jenvman.2017.02.057
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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. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.jenvman.2017.02.057. References Aguiar, A., Gavioli, D., Ferraz, A., 2014. Metabolite secretion, Fe3þ-reducing activity and wood degradation by the white-rot fungus Trametes versicolor ATCC 20869. Fungal Biol. 118, 935e942. Aidoo, K.E., Hendry, R., Wood, B.J.B., 1981. Estimation of fungal growth in a solidstate fermentation system. Eur. J. Appl. Microbiol. Biotechnol. 12, 6e9. Alexandropoulou, M., Antonopoulou, G., Fragkou, E., Ntaikou, I., Lyberatos, G., 2016. Fungal pretreatment of willow sawdust and its combination with alkaline treatment for enhancing biogas production. J. Environ. Manag. http:// dx.doi.org/10.1016/j.jenvman.2016.04.006 (In press). Archibald, F.S., 1992. A new assay for lignin-type peroxidases employing the dye azure B. Appl. Environ. Microbiol. 58, 3110e3116. Bailey, M.J., Biely, P., Poutanen, K., 1992. Interlaboratory testing of methods for assay of xylanase activity. J. Biotechnol. 23, 257e270. Baldrian, P., 2003. Interactions of heavy metals with white-rot fungi. Enzyme Microb. Technol. 32, 78e91. Bourbonnais, R., Paice, M.G., 1990. Oxidation of non-phenolic substrates: an expanded role for laccase in lignin biodegradation. FEBS Lett. 267, 99e102. Chang, A.J., Fan, J., Wen, X., 2012. Screening of fungi capable of highly selective degradation of lignin in rice straw. Int. Biodeterior. Biodegr. 72, 26e30. 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, 5307e5313. Dowe, N., McMillan, J., 2001. SSF Experimental Protocols: Lignocellulosic Biomass Hydrolysis and Fermentation. Laboratory Analytical Procedure (LAP), National Renewable Energy Laboratory (NREL) Analytical Procedures. Golden, Co: National Renewable Energy Laboratory. NREL/TP 510 42630. Elisashvili, V., Kachlishvili, E., Khardziani, T., Agathos, S.N., 2010. Effect of aromatic compounds on the production of laccase and manganese peroxidase by whiterot basidiomycetes. J. Ind. Microbiol. Biotechnol. 37, 1091e1096. Geiger, G., Brandl, H., Furrer, G., Schulin, R., 1998. The effect of copper on the activity of cellulase and b-glucosidase in the presence of montmorillonite or Almontmorillonite. Soil Biol. Biochem. 30, 1537e1544. Ghose, T., 1987. Measurement of cellulase activities. Pure Appl. Chem. 59, 257e268. Glenn, J.K., Gold, M.H., 1985. Purification and characterization of an extracellular Mn (II)-dependent peroxidase from the lignin-degrading basidiomycete, Phanerochaete chrysosporium. Arch. Biochem. Biophys. 242, 329e341. Guillen, F., Martinez, A.T., Martinez, M.J., 1992. Substrate specificity and properties of the aryl-alcohol oxidase from the ligninolytic fungus Pleurotus eryngii. Eur. J. Biochem. 209, 603e611. Hakala, T.K., Maijala, P., Konn, J., Hatakka, A., 2004. Evaluation of novel wood-rotting polypores and corticioid fungi for the decay and biopulping of Norway spruce (Picea abies) wood. Enzyme Microb. Technol. 34, 255e263. Hatakka, A., 1994. Lignin modifying enzymes from selected white rot fungi: Production and role in lignin degradation. FEMS Microbiol. Rev. 13, 125e135. Kaur, H., Sudhakara, K.M., 2011. Effect of carbon, nitrogen sources and inducers on ligninolytic enzyme production by Morchella crassipes. World J. Microbiol. Biotechnol. 27, 687e691. Knezevic, A., Milovanovic, I., Stajic, M., Vukojevic, J., 2013. Potential of Trametes species to degrade lignin. Int. Biodeterior. Biodegrad. 85, 52e56. Liu, Y.T., Luo, Z., Su, Y.J., 2013. Effect of supplements Mn2þ, Cu2þ, and aromatic compounds and Penicillium decumbens on lignocellulosic enzyme activity and
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Please cite this article in press as: Mishra, V., et al., Improvement of selective lignin degradation in fungal pretreatment of sweet sorghum bagasse using synergistic CuSO4-syringic acid supplements, Journal of Environmental Management (2017), http://dx.doi.org/10.1016/ j.jenvman.2017.02.057
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Research article
Improvement of selective lignin degradation in fungal pretreatment of sweet sorghum bagasse using synergistic CuSO4-syringic acid supplements Vartika Mishra a, Asim K. Jana a, *, Mithu Maiti Jana b, Antriksh Gupta a a b
Department of Biotechnology, Dr B R A National Institute of Technology, Jalandhar, 144011, Punjab, India Department of Chemistry, Dr B R A National Institute of Technology, Jalandhar, 144011, Punjab, India
a r t i c l e i n f o
a b s t r a c t
Article history: Received 2 November 2016 Received in revised form 23 January 2017 Accepted 21 February 2017 Available online xxx
Sweet sorghum bagasse (SSB) generated in large quantities could be hydrolyzed to sugar and then fermented to green fuels. The hydrolysis of SSB polysaccharides interlocked in recalcitrant lignin network is the major problem. Pretreatment of SSB in SSF by using Coriolus versicolor with CuSO4-syringic acid supplements for effects on production of ligninocellulolytic enzymes, lignin degradation and selectivity values (SV) were studied. C. versicolor was selected based on high ligninolytic and low cellulolytic abilily. Individually, CuSO4 increased the activities of laccase (4.9 folds) and PPO (1.9 folds); syringic acid increased LiP (13 folds), AAO (2.8 folds) and laccase (5.6 folds) resulting in increased lignin degradation and SVs. Combined syringic acid (4.4 mmol g1 SSB) and CuSO4 (4.4 mmol g1 SSB) increased the activities of laccase, LiP, MnP, PPO and AAO by 11.2, 17.6, 2.8, 2.4 and 2.3 folds respectively due to synergistic effect, resulting in maximum lignin degradation 35.9 ± 1.3% (w w1) (1.86 fold) and highest SV 3.07 (4.7 fold). Enzymatic hydrolysis of pretreated SSB yielded higher (~2.2 times) fermentable sugar. Pretreated SSB was characterized by XRD, SEM, FTIR and TGA/DTG analysis to confirm results. It is possible to improve fungal pretreatment of agricultural waste by combination of supplements. © 2017 Elsevier Ltd. All rights reserved.
Keywords: Ligninocellulosic waste Fungal pretreatment Ligninolytic enzymes Supplements Delignification Enzymatic hydrolysis
1. Introduction Biofuels produced from ligninocellulosic biomass/agricultural residues showed the potential as an alternative fuel to overcome the need of fossil fuels and benefits with respect to environmental management. Sweet sorghum (Sorghum sp.) bagasse (SSB) is promising for use in biofuel production due to its higher yield of waste biomass. This ligninocellulosic waste mainly consists of lignin (cross-linked phenol polymers), cellulose (homopolymer of glucose) and hemicellulose (heteropolymer of pentoses and hexoses) (Ratnavathi et al., 2011). The polysaccharides i.e. cellulose and hemicellulose (55e75% on dry weight basis) could be hydrolyzed to sugars and then fermented to alcohols and biofuels. But, these polysaccharides are interlocked in the polymeric network structure of recalcitrant lignin. To overcome the hindrance of lignin, pretreatment of ligninocellulosic biomass is necessary step. Biological
* Corresponding author. Department of Biotechnology, Dr B R A National Institute of Technology, G T Road Bye Pass, Jalandhar, 144011, Punjab, India. E-mail address: [email protected] (A.K. Jana).
pretreatment comprises the application of microorganisms such as white rot fungi (WRF) for selective degradation of lignin and hemicellulose (Alexandropoulou et al., 2016). WRF vary in abilities to degrade lignin due to their ligninolytic abilities because of activities of enzymes laccase (phenol oxidase), lignin peroxidase (LiP), manganese peroxidase (MnP), arylalcohol oxidase (AAO) and polyphenol oxidase (PPO) which breakdown the lignin polymer into soluble compounds that are further mineralize (completely) into CO2 & H2O (Hatakka, 1994). WRF Phanerochaete chrysosporium, Abortiporus biennis and C. versicolor have been studied for their delignification abilities during pretreatment of rice straw (Chang et al., 2012), sawdust (Alexandropoulou et al., 2016) and bamboo residue (Zhang et al., 2007a) respectively. Although WRF are the best lignin degraders; non-selective lignin degradation (Sitarz et al., 2013), long pretreatment time (Shi et al., 2009) and high cellulolytic activities are the problems during pretreatment. To screen the fungal strain for high lignin degradation with low cellulose consumption, selectivity value (SV) is an important parameter (Hakala et al., 2004; Shi et al., 2009; Zhang et al., 2007b). SV is defined as a ratio of amount of lignin degraded to amount of cellulose consumed by fungal strain
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during pretreatment. Few WRF such as Coriolus versicolor (Aguiar et al., 2014), Pleurotus ostreatus (Taniguchi et al., 2005), Trametes hirusuta (Knezevic et al., 2013) and Phanerochaete chrysosporium (Chang et al., 2012) showed high SV. It depended on lignocellulolytic ability of the fungal strains, substrates used and culture conditions (Chang et al., 2012; Salvachua et al., 2013). Addition of supplements for enhancement in production of certain ligninolytic enzymes by the fungal strains have been studied (Nousiainen et al., 2014; Zerva et al., 2016). It was possible to control the productions of ligninolytic enzymes oxidases and peroxidases by using metallic salts, phenolic and aromatic compound supplements. An addition of phenolic and aromatic supplements (ferulic acid, xylidine, veratric acid, vanillic acid, cinnamic acid and guaiacol) reported to enhance the laccase production (Elisashvili et al., 2010; Liu et al., 2013). Metallic salt MnSO4 enhanced the MnP activity during biodegradation of Eucalyptus grandis (Vicentim and Ferraz, 2007), while CuSO4 and MnSO4 inhibited cellulolytic enzymes (Geiger et al., 1998; Tejirian and Xu, 2010). The degradation of lignin in pretreatment process occurs by complex reactions involving multiple ligninolytic and cellulolytic enzymes produced by the fungal strains. The achievement of high lignin degradation and SV in pretreatment could be possible only through the enhancement in production of multiple lignocellullytic enzymes and their interactions. Although, effect of individual supplements on specific enzyme production have been reported as above, combination of more than one supplements to enhance multiple ligninolytic enzymes, lignin degradation and SV has not been studied yet. The objective of the present study was to enhance the degradation of lignin with high SV in a pretreatment of SSB by fungal strain using combined effect of an inorganic metallic salt and a phenolic supplement. Fungal strain having high selectivity was screened initially out of different strains used in the pretreatment (Aguiar et al., 2014; Chang et al., 2012; Knezevic et al., 2013; Taniguchi et al., 2005; Zhang et al., 2007a). The effect of the combination of CuSO4 and syringic acid on production of ligninolytic enzymes, cellulolytic enzymes and selective lignin degradation compared to the effect of the individual supplements have been reported for the first time to the best of our knowledge. 2. Materials and methods 2.1. Materials 2,2'azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) was purchased from MP Biomedical (Mumbai, India). p-nitrophenyl-b-D-glucopyranoside (p-NPG), birchwood xylan, lignin sulfonic acid, catechol, vanillin and verataryl alcohol were purchased from Himedia Laboratory Pvt Ltd (Mumbai, India). Azur-B and bovine serum albumin (BSA) were purchased from S.D. fine chem. Ltd (Mumbai, India). Cellulose and syringic acid were purchased from Sigma Aldrich (Mumbai, India). Water was purified and deionized (DI) by Milli-Q purification system (minimum resistivity 18 MU cm; Millipore, Billerica, MA, USA). Rest of chemicals used were analytical grade chemicals made by S.D. fine chem. Ltd (Mumbai, India). Cellulase (SaccariSEB EG) and b-glucosidase (SaccariSEB BG) were gifted from Advanced Enzyme Technologies Ltd (Thane, India). 2.2. Biomass Sweet Sorghum Bagasse (SSB) was collected from the local agricultural farms in Jalandhar, Punjab, India. SSB was dried at 40 C for 72 h and ground to pass through 5 mm 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 eryngii (MTCC 1798), 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. All strains were grown and maintained in 2% (w v1) malt extract agar media in petri plates at pH 4.5, 25 C, and sub-cultured periodically after every 15 days. 2.4. Ligninocellulolytic abilities 2.4.1. Ligninolytic ability Basal medium (BM) was prepared containing (g L1) ammonium tartrate 5.0, yeast extract 0.1, potassium phosphate monobasic 1.0, calcium chloride 0.001, magnesium sulfate 0.5 and 1.6% (w v1) agar (pH 4.5). Substrates i.e. 0.1% (w v1) ABTS, 0.1% (v v1) guaiacol, 0.01% (w v1) tannic acid, 0.25% (w v1) lignin sulfonic acid and 0.01% (w v1) azure-B were used to determine the extent of ligninolytic ability (Pointing, 1999). Single disc taken from actively growing 7 days old fungal culture plate using sterile cock borer (10 mm diameter), was used as inoculums. Sterilized petri plates containing BM and supplemented with substrates were inoculated with test strains and incubated (Innova 42R, Eppendorf ltd, USA) at 27.5 C for 5 days. The colourless agar medium turned green due to the oxidation of ABTS to ABTS-azine due to laccase produced by fungus. Colorless medium containing guaiacol turned to brick red due to laccase produced by fungus. The appearance of a brown oxidation zone around colonies in petri plates containing tannic acid signified the polyphenol oxidase production. In lignin sulfonic acid plate, 1% (w v1) aqueous solution of ferric chloride and 1% (w v1) potassium ferricyanide was flooded, washed with DI water after 10 min, and observed for clear zones of degraded lignin around colonies against blue green colour of undegraded lignin. Clearance of blue colour of agar in the petri plate containing azure B showed the ability of lignin peroxidases production by fungal strain. Diameter of these colour zones around the colonies were taken as the ligninolytic ability of the strains. 2.4.2. Cellulolytic and hemicellulolytic (xylanolytic) ability The cellulolytic ability of fungal strains was detected by the Congo red test (Teather and Wood, 1982). Petri plates containing BM with carboxy methyl cellulose (1.1% (w v1)) as substrate were sterilized. Single disc taken from actively growing 7 days old fungal culture plate using sterile cock borer (10 mm diameter) was inoculated and incubated at 27.5 C for 5 days. Cultivated plates were flooded with aqueous Congo red solution (1 mg mL1). After 15 min of exposure, dye was drained and plates were washed three times with 1 M NaCl and observed for production of yellow zones around the colonies. For xylanolytic ability, xylan (4% (w v1)) was supplemented with BM in petri plates (Pointing, 1999). Sterile plates were inoculated with test strains and incubated at 27.5 C for 5 days. Plates were flooded with iodine stain (0.25% (w v1) aqueous I2 and 0.25% (w v1) KI). After 5 min of exposure, dye was drained and plates were washed with DI water, and observed for appearance of a yellow-opaque area (signified the release of xylanase) against a blue/reddish purple colour for undegraded xylan. Diameter of coloure zones around fungal discs were taken as cellulolytic and hemicellulolytic ability of strains. 2.5. Pretreatment of SSB Ten discs (using sterile cock borer with 10 mm in diameter) from
Please cite this article in press as: Mishra, V., et al., Improvement of selective lignin degradation in fungal pretreatment of sweet sorghum bagasse using synergistic CuSO4-syringic acid supplements, Journal of Environmental Management (2017), http://dx.doi.org/10.1016/ j.jenvman.2017.02.057
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actively growing 7 days old fungal culture plates were inoculated into 500 mL cotton plugged Erlenmeyer flask containing 50 mL of 2% (w v1) malt extract medium. After 7 days incubation at 27.5 C under static condition, the liquid culture was aseptically homogenized by a sterilized blender for 15 s 3 cycles. Two mL (~2.4 106 spore mL1) of blended culture was used as inoculum for 5 g of SSB. 5 g ground SSB (~3% moisture) was taken in 250 mL Erlenmeyer flasks, 11.5 mL of distilled water was added with it (75% moisture) and cotton plugged. Moistened SSB in flask was autoclaved at 121 C for 15 min. Sterilized flask was inoculated with 2 mL inoculum and incubated stationary at 27.5 C for 20 days solid state fermentation (SSF). Samples were taken every 4th day for compositional analysis of treated biomass, lignocellulolytic enzyme assays and enzymatic hydrolysis of biomass. Different supplements (veratryl alcohol, syringic acid, catechol, vanillin, guaiacol, CuSO4 and MnSO4) were studied to enhance the selectivity value and lignin degradation. All the supplements were dissolved in DI water to make 1 mM supplement solution. 5 g of SSB (~3% moisture) were taken in 250 mL Erlenmeyer flasks, mixed with 11.5 mL of supplement solution to make concentration of 2.2 mmol g1 SSB in culture. Pretreatment of SSB with supplements were carried out by SSF as described above. Supplements, significantly influencing the lignin degradation were studied individually and in combination to enhance the production of ligninolytic enzymes and lignin degradations. All the cultures were incubated statically at 27.5 C for 20 days. The flask culture without supplements was served as control. All experiments were performed in triplicate. 2.6. Ligninocellulolytic enzymes assays 50 mL acetate buffer (pH 4.5, 50 mM) was added to the each sample, shaked at 180 rpm and 27.5 C for 1 h, filtered under vacuum to recover 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 oven at 65 C and used for compositional analysis and enzymatic hydrolysis. 2.6.1. Ligninolytic activity Lignin peroxidase (LiP) activity was determined by H2O2 dependent oxidation of azure-B at 651 nm (ε651 ¼ 48800 M1 cm1) (Archibald, 1992). Laccase activity was determined by oxidation of ABTS to ABTS-azine at 420 nm (ε420 ¼ 36000 M1 cm1) (Bourbonnais and Paice, 1990). Manganese peroxidase (MnP) activity was determined by phenol red oxidation (ε610 ¼ 22000 M1 cm1) (Glenn and Gold, 1985). Polyphenol oxidase (PPO) was assayed by oxidation of catechol to quinone at 30 C (ε400 ¼ 3450 M1 cm1) (Wong et al., 1971). Aryl alcohol oxidase (AAO) activity was monitored by using the oxidation of veratryl alcohol to veratrylaldehyde (ε310 ¼ 9300 M1 cm1) (Guillen et al., 1992). One unit of enzyme activity was defined as the amount of enzyme required to oxidize 1 mmol of substrate per minute. Activities of all enzymes were expressed as units per gram dry matter (U gdm1). 2.6.2. Cellulolytic activity Carboxymethyl cellulase activity (CMCase/endo-b-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) activities were determined using Whatman No. 1 filter paper strip and carboxymethyl cellulose respectively as substrates (Ghose, 1987). One unit of enzyme activity was defined as the amount of enzyme required to produce 1 mmol reducing sugar per min. b-Glucosidase
3
activity was assayed by using p-NPG as substrate (Wood and Bhat, 1988). One unit of enzyme activity was defined as the amount of enzyme required to produce 1 mmol p-nitrophenol per min. Xylanase activity was assayed using 1% (w v1) 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 enzyme activity was defined as the amount of enzyme required to produce 1 mmol xylose per min under standard condition of pH and temperature. Activities all enzymes were expressed as units per gram dry matter (U gdm1). 2.7. Fungal growth estimation N-acetyl glucosamine content of the fungal cell wall was used to monitor fungal biomass (Aidoo et al., 1981). 2.8. Compositional analysis of SSB Compositions of SSB were determined before and after pretreatment by solid state fermentation. Lignin, cellulose and hemicellulose 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 v1) sulfuric acid at 30 C for 1 h. The samples were diluted immediately to 4% (v v1) and autoclaved for 1 h. Autoclaved hydrolyzed solution was vacuum filtered through previously weighed filtering crucibles. Crucible and acid insoluble residue was dried at 105 C until a constant weight was achieved. Aliquot sample collected from vacuum filtration was used to determine acid soluble lignin, cellulose and hemicellulose. The acid-soluble lignin was measured by UVeVis spectrophotometer (Kinetic, Biospectrometer, Eppendorf, Germany) using lignin sulfonic 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 HiPlex-H column and refractive index detector (RID) that were maintained at 80 and 55 C, respectively. HPLC grade water was used as the mobile phase, at flow rate of 0.6 mL min1. The content of cellulose in the sample was calculated from glucose and galactose obtained while hemicellulose 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 using equations %Cellulose ¼ (GR 0.9)/w 100 and %Hemicellulose ¼ (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, defined as the percentage of total lignin reduced after pretreatment, was calculated using equation %Lignin degradation ¼ (1 w(a þ b)/(wo (ao þ bo))) 100; where w is the sample dry weight (g) of the pretreated SSB; wo is the initial dry weight (g) of the untreated SSB; a, b and ao, bo are the % of acid-soluble and acid-insoluble lignin in the pretreated and untreated SSB, respectively. Dry mass loss was calculated as the percentage of total solids loss after pretreatment after removing fungal biomass content. 2.9. Enzymatic hydrolysis of pretreated SSB Enzymatic hydrolysis of cellulose (avicel), untreated and treated SSB were carried out following protocol described in NREL/TP-51042630 (Dowe and McMillan, 2001). One gram of biomass (3% moisture) was taken in 250 mL Erlenmeyer flask and 100 mL of 50 mM citrate buffer (pH 4.8) was added. Cellulase (SaccariSEB EG, 10 filter paper unit g1 solid) and b-glucosidase (SaccariSEB BG, 20
Please cite this article in press as: Mishra, V., et al., Improvement of selective lignin degradation in fungal pretreatment of sweet sorghum bagasse using synergistic CuSO4-syringic acid supplements, Journal of Environmental Management (2017), http://dx.doi.org/10.1016/ j.jenvman.2017.02.057
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cellobiose unit g1 solid) enzymes were added for hydrolysis of biomass. The flask was incubated in a shaking incubator at 165 rpm, 45 C at for 108 h and analyzed for release of fermentable sugar. Sugar content was estimated using Agilent Hi-Plex-H column in HPLC (Agilent-1200 infinity series) as discussed above in Section 2.8.
the lowest CMCase activity, and low FPase and ß-glucosidase activity. In overall, C. versicolor produced highest xylanase activity i.e. capability to utilize hemicellulose as carbon source, low cellulolytic enzymes and high ligninolytic enzymes with high degradation of lignin in SSB. Fungal strains showed different SVs, due to the varied lignin degradation, cellulose and hemicellulose loss depending on their lignocellulolytic abilities. C. versicolor showed maximum SV of 0.65 after 20 days and 0.59 after 30 days of SSF (Table S3) with high degradation of hemicellulose. Strain showing ability to degrade xylan is advantage for pretreatment as dependency on hemicellulose help to preserve cellulose. SV of pretreatment by C. versicolor decreased at latter phase of SSF (0.65 at 20th day to 0.59 at 30th day). This was due to shielded structure of lignin acted as barrier against consumption of cellulose at initial stage. C. versicolor was selected for pretreatment of SSB with supplements. SSB was pretreated with supplements (2.2 mmol g1 SSB initial) veratryl alcohol, syringic acid, catechol, vanillin, guaiacol, CuSO4 and MnSO4 for effect on lignin degradation and SVs (Fig. S1). CuSO4 resulted in maximum degradation of lignin (24.8 ± 2.3% (w w1)) followed by syringic acid (22.5 ± 1.0% (w w1)), while lower with other supplements (18.5e22.0% (w w1)). CuSO4 caused minimum consumption of cellulose (21.6 ± 1.01% (w w1)). Presence of CuSO4 and syringic acid resulted in SV values 1.15 and 0.89 respectively, higher than the control (without supplements). CuSO4 and syringic acid supplements were selected for study of the effects on lignocellulolytic enzyme activities, lignin degradations and SVs in pretreatment.
2.10. Characterization of SSB Changes in morphological, structural, chemical and thermal characteristics of SSB were characterized by the techniques of scanning electron microscopy (SEM) (EVO 40 EP-CARL ZEISS, Germany) (Zhang et al., 2007b), X-ray diffraction (XRD) (PANALYTICAL [XPERT-PROMPT, Netherland) (Zeng et al., 2011), Fourier transformed infrared spectroscopy (FTIR) (AGILENT CARY-630, USA) (Zhang et al., 2007b) and Thermogravimetric/Differential Thermogravimetric analysis (TGA/DTG) (SII EXSTAR 6000 [TG/DTA 6300], Japan) (Zeng et al., 2011). The percentage of crystallinity (% Cr) and crystallinity index (CrI) was calculated by equation (Segal et al., 1959),
%Cr ¼ Icrys
Icrys þ Iamor 100 and CrI ¼ Icrys Iamor Icrys
where Icrys is the overall intensity of the peak at 2q about 22 and Iamor is the intensity at 2q about 18 . 3. Results and discussion 3.1. Screening of lignin selective strain and supplements
Table 2 Degradation of lignin, cellulose, hemicellulose and variation of selectivity value (SV) in the presence of syringic acid and CuSO4 in combination.
Best lignin degrading fungal strain was screened out of eight fungal strains reported in literature (Aguiar et al., 2014; Chang et al., 2012; Knezevic et al., 2013; Taniguchi et al., 2005; Zhang et al., 2007a) based on high ligninolytic, low cellulolytic abilities and SV (lignin degradation/cellulose loss) during pretreatment of SSB. All strains exhibited the different ligninolytic, cellulolytic and hemicellulolytic abilities; but Coriolus versicolor showed highest ligninolytic and hemicellulolytic ability with low cellulolytic ability (Table S1). C. versicolor showed the highest laccase, LiP, MnP and PPO activity when grown on SSB in SSF (Table S2). C. versicolor also produced AAO enzyme, but it was less than Ganoderma lucidium and Phlebia radiata. Daedalea flavida, Pleurotus ostreatus and Stereum hirusita didn't show any AAO activity. Only C. versicolor, G. lucidium and P. radiata produced all ligninolytic enzymes (laccase, LiP, MnP, PPO and AAO). P. radiata showed the high celluloytic activity i.e CMCase, FPase and ß-glucosidase. C. versicolor showed
Supplement (mmol g1 SSB)
Degradation (% (w w1))
Syringic acid
CuSO4
Lignin
0.0 0.0 8.8 8.8 8.8 8.8 4.4 4.4 4.4 2.2 2.2 2.2
0.0 2.2 0.0 2.2 4.4 8.8 2.2 4.4 8.8 2.2 4.4 8.8
19.9 24.8 28.7 33.7 33.2 29.9 32.6 35.9 30.8 32.1 32.9 30.4
± ± ± ± ± ± ± ± ± ± ± ±
Cellulose 1.6 2.3 1.7 1.2 1.1 1.4 1.8 1.3 1.2 1.6 1.5 1.3
30.4 21.6 16.6 15.5 12.1 26.1 15.1 11.7 25.1 14.9 12.2 24.4
± ± ± ± ± ± ± ± ± ± ± ±
2.2 1.0 1.0 0.7 0.9 1.1 0.5 0.4 1.1 0.5 0.6 1.0
SV Hemicellulose 19.7 21.7 28.2 27.3 25.7 26.9 27.2 27.8 26.4 26.1 27.1 25.7
± ± ± ± ± ± ± ± ± ± ± ±
1.5 1.9 1.7 1.1 1.2 1.2 1.1 1.3 1.4 1.4 1.3 1.2
0.65 1.15 1.77 2.17 2.74 1.15 2.16 3.07 1.23 2.15 2.70 1.25
Table 1 Degradation of lignin, cellulose, hemicellulose and variation of selectivity value (SV) in the presence of supplements CuSO4 and syringic acid individually after 20 days of pretreatment by C. versicolor. Supplement
Concentration (mmol g1 SSB)
Degradation (% (w w1)) Lignin
None CuSO4
Syringic acid
0.0 0.11 1.1 2.2 4.4 8.8 22.0 0.11 1.1 2.2 4.4 8.8 22.0
19.9 22.3 23.6 24.8 23.9 23.4 19.3 23.5 23.7 22.4 29.4 28.7 20.7
± ± ± ± ± ± ± ± ± ± ± ± ±
SV Cellulose
1.6 1.0 1.1 2.3 2.2 1.4 1.3 1.2 1.0 1.0 2.7 1.7 1.4
30.4 32.8 31.7 21.6 22.1 23.8 28.3 35.5 27.9 25.8 25.0 16.6 33.7
± ± ± ± ± ± ± ± ± ± ± ± ±
2.2 1.9 1.8 1.0 1.6 1.5 1.7 1.7 2.3 2.3 2.3 1.0 2.3
Hemicellulose 19.7 21.4 19.0 21.7 20.1 19.2 17.6 22.5 23.7 24.9 26.0 28.2 25.8
± ± ± ± ± ± ± ± ± ± ± ± ±
1.5 1.0 1.0 1.9 1.6 1.1 1.2 1.1 1.4 1.3 2.3 1.7 1.8
0.65 0.68 0.74 1.15 1.08 0.98 0.68 0.66 0.84 0.86 1.17 1.77 0.61
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3.2. Effects of selected individual supplement and their combination 3.2.1. Biomass degradation and fungal growth Effects of CuSO4, syringic acid (0.0e22.0 mmol g1 SSB) on degradation of SSB individually (Table 1) and their combination (Table 2) were studied. The SSB was composed (w w1) of lignin
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(25.14%), cellulose (38.02%) and hemicellulose (25.01%). At CuSO4 2.2 mmol g1 SSB, maximum lignin degradation was 24.8 ± 2.3% (w w1) with cellulose loss 21.6 ± 1.01% (w w1) and SV 1.15. Addition of higher concentration of supplement decreased in the lignin degradation and SV, might be due to inhibition of fungal growth and ligninolytic enzyme activity. 8.8 mmol g1 SSB syringic acid
Fig. 1. Effect of supplements on degradation of (A) lignin; (B) cellulose; (C) selectivity value; (D) degradation of hemicellulose; (E) hemicellulose loss to cellulose loss ratio; (F) growth of C. versicolor during pretreatment of SSB.
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resulted in high lignin degradation (28.7 ± 1.7% (w w1)) with minimum cellulose consumption (16.6 ± 1.0% (w w1)) and high SV 1.77. As maximum lignin degradation and SV value was obtained at 8.8 mmol g1 SSB syringic acid, its combinations with CuSO4 was studied for further enhancement of lignin degradation. Syringic acid 4.4 mmol g1 SSB and CuSO4 4.4 mmol g1 SSB combination gave the maximum lignin degradation (35.9 ± 1.3% (w w1)) and SV 3.07 (Table 2). Pretreatment of SSB supplemented with syringic acid and CuSO4 in combination showed the significant improvement in lignin degradation than supplements with syringic acid, CuSO4 or control (Fig. 1A), probably due to increased activities of ligninolytic enzymes in culture. During first 4 days of pretreatment, lignin degradation was slow, while rate of cellulose loss was high (Fig. 1B) due to the consumption of easily accessible small amount of cellulose for primary growth of fungi. The cellulose content in SSB pretreated with supplement in combination was higher due to minimum cellulose loss during pretreatment. The availability and vulnerability of cellulose to subsequent hydrolysis is an important criterion for evaluating the performance of fungal pretreatment, because higher cellulose content in substrates, eventually provide higher accessibility of carbohydrates for enzymatic saccharification and sugar yield (Salvachua et al., 2013). Due to low cellulose loss with supplement in combination, highest SV 3.07 was achieved (Fig. 1C) (4.7 fold higher than the control). Presence of supplements in culture stimulated C. versicolor to consume hemicellulose in SSB. Maximum hemicellulose consumption (27.8 ± 1.3% (w w1)) was obtained with supplements in combination (Fig. 1D). Ratio of loss of hemicellulose to cellulose loss (H/C) (Fig. 1E) was similar to change of SV. Although difference of hemicellulose loss with syringic acid (28.2 ± 1.7% (w w1)) and with supplements in combination was small, but H/C with combined supplements was higher due to low cellulose consumption during pretreatment in latter case. Higher H/C ratio signified that combined supplements stimulated the fungi to feed on hemicellulose rather than cellulose for its primary growth. C. versicolor grew well on SSB in the presence of supplements in SSF (Fig. 1F) and prolific colonization observed in 20 days. The fungal growth was slow during first 4 days of pretreatment due to the lag phase. The fungal biomass were much higher (442.2 ± 26.3 mg gdm1) with phenolic supplement syringic acid compared to control (384.5 ± 31.2 mg gdm1) or with CuSO4 (328.3 ± 18.4 mg gdm1). When CuSO4 was added in combination with syringic acid, fungal biomass (431.3 ± 21.0 mg gdm1) was slightly lower than syringic acid alone might be due to oxidative stress by CuSO4 on fungi. Metallic salts have been reported to result in low fungal biomass (Baldrian, 2003; Geiger et al., 1998), while the phenolic supplements such as syringic acid and gallic acid have reported to increase in fungal growth and hyphae till a maximum concentration in the medium (D'souza et al., 1999; Srivastava et al., 2013). This could be a possible reason that syringic acid resulted in
higher fungal biomass when present alone and in combination with copper. It was interesting to note that exponential phase the culture reduced and stationary phase achieved earlier with the supplements. 3.2.2. Ligninolytic and cellulolytic enzyme productions Peak activities (U g1) of ligninolytic enzymes of 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. Presence of supplements increased ligninolytic enzyme productions during pretreatment (Table 3). CuSO4 (2.2 mmol g1 SSB) increased peak activities of laccase and PPO enzyme, while LiP and AAO was little affected, might be due to induction effect of copper on laccase and related oxidative enzyme (PPO) at transcription level (Tinoco et al., 2011). Unexpectedly, significant increase in MnP activity was also observed. In the presence of CuSO4, all ligninolytic enzymes showed peak activity on 12th day of fermentation except of LiP which peaked on 16th day. The stability of laccase activity was observed, might be due to the inhibition of extracellular proteolytic enzymes in the presence of Cu2þ, resulting in inhibition of laccase degradation (Kaur and Sudhakara, 2011). Syringic acid (8.8 mmol g1 SSB) enhanced the LiP activity to 1.7 ± 0.08 U g1, 13 folds higher than control. This might be due to 3 folds increase in AAO activity by syringic acid had produced H2O2 to increase the LiP activity significantly. Additionally, laccase activity (67.4 ± 7.7 U g1) increased to 7 folds and MnP activity (19.0 ± 1.1 U g1) increased to 3 folds with respect to control. All ligninolytic enzymes showed their peak activities on 16 - 20th day of fermentation. The significant increase in enzyme activities was might be due to structural similarity of syringic acid with syringyl unit present in the lignin. Difference between syringic acid and syringyl unit is functional group at para site of benzyl ring. Structural similarity can result in more inducing effect for release of ligninolytic enzymes (D'souza et al., 1999). This increase in ligninolytic enzymes had supported the significant increase in lignin degradation. CuSO4 4.4 mmol g1 SSB and syringic acid 4.4 mmol g1 SSB in combination, increased the activities of laccase (133.2 ± 7.1 U g1) by 11.2 folds, LiP (2.3 ± 0.1 U g1) by 17.6 folds, MnP (22.7 ± 1.0 U g1) by 2.8 folds, PPO (11.8 ± 0.6 U g1) by 2.4 folds and AAO (3.3 ± 0.1 U g1) by 2.3 folds compared to control. Although fungal biomass with syringic acid and CuSO4 together was lower than culture with syringic acid supplement alone, higher ligninolytic enzymes activities were detected. This clearly indicated the synergistic effect of supplements in combination on over expression of multiple ligninolytic enzyme activities that was responsible for high lignin degradation and SV. Presence of CuSO4 enhanced the laccase and related enzyme (PPO) and syringic acid enhanced laccase, LiP and AAO activity; which resulted in cumulative increase in multiple ligninolytic enzymes activities with combined supplements. Laccase showed peak activity on 12th day with combined
Table 3 Peak ligninolytic, cellulolytic and hemicellulolytic enzyme activity during pretreatment. Supplement (mmol g1 SSB)
Ligninolytic enzyme (U g1) Laccase
without supplement 11.9 ± 0.3 (control) (16th day) 58.2 ± 4.3 CuSO4 (2.2) (12th day) Syringic acid (8.8) 67.4 ± 7.7 (20th day) 133.2 ± 7.1 Syringic acid (4.4) þ CuSO4 (4.4) (12th day)
Cellulolytic enzyme (U g1)
Hemicellulolytic enzyme (U g1)
LiP
MnP
PPO
AAO
CMCase
FPase
ß-glucosidase
Xylanase
0.13 ± 0.0 (12th day) 0.17 ± 0.02 (16th day) 1.7 ± 0.08 (16th day) 2.3 ± 0.1 (8th day)
8.1 ± 0.2 (12th day) 15.6 ± 2.1 (12th day) 19.0 ± 1.1 (20th day) 22.7 ± 1.0 (12th day)
4.9 ± 0.07 (16th day) 9.4 ± 1.2 (12th day) 7.5 ± 0.8 (16th day) 11.8 ± 0.6 (16th day)
1.39 ± 0.04 (12th day) 2.2 ± 0.3 (12th day) 3.9 ± 0.1 (16th day) 3.3 ± 0.1 (8th day)
6.4 ± 0.06 (20th day) 3.0 ± 0.4 (16th day) 2.2 ± 0.1 (16th day) 1.9 ± 0.08 (20th day)
12.7 ± 0.42 (20th day) 10.0 ± 1.3 (16th day) 6.7 ± 0.9 (16th day) 4.3 ± 0.1 (16th day)
12.6 ± 0.44 (20th day) 6.1 ± 0.8 (12th day) 4.8 ± 0.2 (16th day) 2.9 ± 0.1 (20th day)
7.9 ± 0.06 (20th day) 7.5 ± 0.67 (20th day) 8.9 ± 0.82 (20th day) 9.2 ± 0.4 (16th day)
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supplements and CuSO4 alone, while it peaked on 20th day with syringic acid. This indicated the presence of syringic acid resulted in an over expression of laccase activity on 12th day with CuSO4. LiP
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and AAO showed an early and over expression (peak activity on 8th day) with combined supplements. MnP activity also increased with combined supplements and it overlapped with MnP activity with
Fig. 2. Characterization of untreated and pretreated SSBs. (A) Scanning electron microscopy (SEM) (untreated), (B) SEM (pretreated with supplements syringic acid and CuSO4), (C) Fourier transformed infrared spectroscopy (FTIR), (D) X-ray diffraction (XRD), (E) Thermogravimetric (TGA)/Differential Thermogravimetric Analysis (DTG).
Please cite this article in press as: Mishra, V., et al., Improvement of selective lignin degradation in fungal pretreatment of sweet sorghum bagasse using synergistic CuSO4-syringic acid supplements, Journal of Environmental Management (2017), http://dx.doi.org/10.1016/ j.jenvman.2017.02.057
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CuSO4 alone, indicating slight over expression due to syringic acid. Lignins in SSB is predominantly composed of b-O-40 aryl ether linkages, together with minor amounts of b-b0 , b-50 , b-10 and a,bdiaryl ether linkages (Sun et al., 2013). Although all ligninolytic enzymes possess ability to break these linkages by cleaving Ca - Cb bonds in lignin structure and by Ca oxidation, but LiP has highest and laccase has lowest redox potential. The depolymerization of lignin was initiated by AAO (AAO produces H2O2; initiator for LiP) and LiP, broke the inter-unit b-O-40 and b-b’ linkages by cleaving of Ca - Cb bonds. This resulted in production of partially degraded lignin and different types of aromatic monomers of lower molecular weights; further degraded by laccase, PPO and MnP (Aguiar et al., 2014; Bourbonnais and Paice, 1990; Machuca and Ferraz, 2001). Early and over expression of LiP and AAO on 8th day, and laccase on 12th day due to synergistic effect of two supplements in the culture were responsible for higher lignin degradation and SV. CuSO4 supplement reduced CMCase activity (3.0 ± 0.21 U g1) to half of control and FPase (10.0 ± 1.3 U g1) and b-glucosidase activity (6.1 ± 0.8 U g1) was low. Syringic acid also resulted in low cellulolytic activities 2.2 ± 0.1, 6.7 ± 0.9 and 4.8 ± 0.2 U g1 of CMCase, FPase and b-glucosidase, respectively. Syringic acid and CuSO4 in combination showed maximum inhibitory effect on cellulolytic enzyme (Table 3). b-glucosidase, CMCase and FPase activities were lowest 2.9 ± 0.1, 1.9 ± 0.08 and 4.3 ± 0.1 U g1 respectively with combined supplements, while xylanase activity was 9.2 ± 0.4 U g1. Decreased cellulolytic enzyme activities in the presence of syringic acid were due to its structural similarity with lignin. This forced an early release and over expressions of ligninolytic enzymes than the cellulolytic enzymes. High degradation of lignin and low consumption of cellulose improved the SV. Wan and Li (2011) studied the effect of metallic salt supplements on delignification of wheat straw by C. subvermispora and targeted only laccase production during pretreatment. Delignification increased to 9% from 4% (w w1) after 21 day. Fungal delignification of wheat straw using Irpex lacteus by SSF in presence of inducer MnSO4 was studied by Salvachua et al. (2013). Lignin degradation was 28.2% and 31.5% (w w1) 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 MnSO4 only on MnP enzyme. In present study, synergistic effect of CuSO4 and syringic acid increased multiple ligninolytic enzymes and delignification reached 35.9 ± 1.3% (w w1) with SV 3.07 after 20 days. 3.3. Characterization of SSB SSB were brownish in presence of the lignin, but colour changed to light yellow after pretreatment, could be due to exposure of cellulose at the surface or the change to phenolic structure. Structure of single strand of SSB was covered with small particles or globules and lignocelluloses had an intact surface structure initially (Fig. 2A). Removal of lignin resulted in a rugged and partially broken surface (Fig. 2B). A visible destruction in cell wall structure, alteration in intact morphology, formation of holes and crevices on the biomass surfaces was observed in pretreated SSB. Functional groups of untreated SSB showed broad peaks at 3418 cm1 (hydrogen bonded OeH stretching), 2930 cm1 (CeH stretching), 1638 cm1 (C]C stretching of aromatic ring), 1435 cm1 (CeH stretching) and 1130 cm1 (CeO stretching) (Zeng et al., 2011) by FTIR spectroscopy (Fig. 2C), similar to lignin (Fig. S2). Pretreatment of SSB resulted in decrease in the respective peaks and intensities especially at 1638 cm1 (C]C stretching for aromatic ring, mainly originating from lignin in particular) (Fig. 2C). The largest decrease in the intensities of lignin was observed in SSB pretreated with CuSO4 and syringic acid combination.
Fig. 3. Enzymatic hydrolysis of pretreated SSB to sugar.
X-ray diffraction analysis displayed the change in percentage crystallinity (%Cr) and crystallinity index (CrI) of pretreated SSB with reference to avicel (Fig. 2D and Table S4). The decrease in %Cr and CrI indicated the lowering of the crystallinity of pretreated SSB due to degradation of lignin. Thermal degradation characteristics, TGA and DTG curves of SSB showed three stages (Fig. 2E and Table S5); (i) dehydration and volatization at <180 C, (ii) degradation between 190 and 500 C corresponding to depolymerization of hemicellulose and cellulose components, and (iii) charcoal formation at >500 C by slow decomposition of the solid residues of lignin. Shifting of DTG curves towards the lower temperature, higher percentage of mass loss and increased DTG peaks intensities indicated the higher thermal degradation rate of pretreated samples, due to degradation of lignin and reduction in cellulose crystallinity. 3.4. Enzymatic hydrolysis Significant differences in glucose yields from the untreated and pretreated SSB were observed (Fig. 3). The addition of supplements during pretreatment affected the enzymatic hydrolysis of SSB. The glucose yield from SSB pretreated with CuSO4 (4.4 mmol g1 SSB) þ syringic acid (4.4 mmol g1 SSB) supplements was maximum (362.8 ± 11.1 mg g1). Syringic acid þ CuSO4 resulted in maximum lignin degradation and porosity of SSB after pretreatment that enabled the penetration of hydrolytic enzymes to increase the glucose yield. The sugar yield was ~2.2 and 1.91 times than the untreated SSB and control (pretreatment without supplement), respectively. 4. Conclusion Pretreatment of SSB by using C. versicolor yielded high lignin degradation and SV. Combination of supplements CuSO4 and syringic acid interacted synergistically to alter the activities of several ligninocellulolytic enzymes during pretreatment. Activities of laccase, LiP, MnP, PPO, AAO enhanced by 11.2, 17.6, 2.8, 2.4, 2.3 folds respectively to increase the lignin degradation (1.86 folds) and SV (4.7 folds). Enzymatic hydrolysis of pretreated SSB yielded higher (~2.2 times) fermentable sugar. It is possible to improve fungal pretreatment of agricultural waste by combination of supplements and convert usable cellulose to fermentable sugar for green fuel industry.
Please cite this article in press as: Mishra, V., et al., Improvement of selective lignin degradation in fungal pretreatment of sweet sorghum bagasse using synergistic CuSO4-syringic acid supplements, Journal of Environmental Management (2017), http://dx.doi.org/10.1016/ j.jenvman.2017.02.057
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Please cite this article in press as: Mishra, V., et al., Improvement of selective lignin degradation in fungal pretreatment of sweet sorghum bagasse using synergistic CuSO4-syringic acid supplements, Journal of Environmental Management (2017), http://dx.doi.org/10.1016/ j.jenvman.2017.02.057