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

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

Enhancement in multiple lignolytic enzymes production for optimized lignin degradation and selectivity in fungal pretreatment of sweet sorghum bagasse.

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.

Improvement of selective lignin degradation in fungal pretreatment of sweet sorghum bagasse using synergistic CuSO4-syringic acid supplements.

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 µmol g-1 SSB) and CuSO4 (4.4 µmol g-1 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 w-1) (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.

Fungal Pretreatment of Sweet Sorghum Bagasse with Combined CuSO4-Gallic Acid Supplement for Improvement in Lignin Degradation, Selectivity, and Enzymatic Saccharification.

Sweet sorghum (Sorghum sp.) has high biomass yield. Hydrolysis of lignocellulosic sweet sorghum bagasse (SSB) to fermentable sugar could be useful for manufacture of biofuel or other fermentation products. Pretreatment of lignocellulosic biomass to degrade lignin before enzymatic hydrolysis is a key step. Fungal pretreatment of SSB with combined CuSO4-gallic acid supplements in solid-state fermentation (SSF) to achieve higher lignin degradation, selectivity value (SV), and enzymatic hydrolysis to sugar was studied. Coriolus versicolor was selected due to high activities of ligninolytic enzymes laccase, lignin peroxidase (LiP), manganese peroxidase (MnP), polyphenol oxidase (PPO), and arylalcohol oxidase (AAO) and low activities of cellulolytic enzymes CMCase, FPase, and ß-glucosidase with high lignin degradation and SV in 20 days. CuSO4/gallic acid increased the activities of ligninolytic enzymes resulting in enhanced lignin degradations and SVs. Cumulative/synergistic effect of combined supplements further increased the activities of laccase, LiP, MnP, PPO, and AAO by 7.6, 14.6, 2.67, 2.06, and 2.15-folds, respectively (than control), resulting in highest lignin degradation 31.1 ± 1.4% w/w (1.56-fold) and SV 2.33 (3.58-fold). Enzymatic hydrolysis of pretreated SSB yielded higher (~2.2 times) fermentable sugar. The study showed combined supplements can improve fungal pretreatment of lignocellulosic biomass. XRD, SEM, FTIR, and TGA/DTG of SSB confirmed the results.

Cotton Stalk Pretreatment Using Daedalea flavida, Phlebia radiata, and Flavodon flavus: Lignin Degradation, Cellulose Recovery, and Enzymatic Saccharification.

Lignocellulolytic enzyme activities of selective fungi Daedalea flavida MTCC 145 (DF-2), Phlebia radiata MTCC 2791 (PR), and non-selective fungus Flavodon flavus MTCC 168 (FF) were studied for pretreatment of cotton stalks. Simultaneous productions of high LiP and laccase activities by DF-2 during early phase of growth were effective for lignin degradation 27.83 ± 1.25 % (w/w of lignin) in 20-day pretreatment. Production of high MnP activity without laccase in the early growth phase of PR was ineffective and delayed lignin degradation 24.93 ± 1.53 % in 25 days due to laccase production at later phase. With no LiP activity, low activities of MnP and laccase by FF yielded poor lignin degradation 15.09 ± 0.6 % in 20 days. Xylanase was predominant cellulolytic enzyme produced by DF-2, resulting hemicellulose as main carbon and energy source with 83 % of cellulose recovery after 40 days of pretreatment. The glucose yield improved more than two fold from 20-day DF-2 pretreated cotton stalks after enzymatic saccharification.

Effect of particle size, moisture content, and supplements on selective pretreatment of cotton stalks by Daedalea flavida and enzymatic saccharification.

A large amount of cotton stalk waste biomass with high cellulose content are incinerated by the farmers causing air pollution. The high cellulose content of cotton stalks can be converted to fermentable sugars by fungal delignification pretreatment of lignocellulosic biomass and enzymatic saccharification. The effect of particle size, moisture content, and media supplements was studied for delignification of cotton stalks by Daedalea flavida MTCC 145 (DF-2) in solid-state fermentation. The highest lignolytic enzyme activities, optimal lignin degradation 29.88 ± 0.97% (w/w) with cellulose loss 11.70 ± 1.30% (w/w), were observed in cotton stalks at particle size 5 mm with 75% moisture content after 20 days. Cellulolytic enzyme activity increased with decrease in particle size and increased moisture content. The addition of Cu2+, gallic acid, and veratryl alcohol enhanced the lignolytic enzyme production and the lignin degradation. In addition to increased laccase activity, Cu2+ inhibited the cellulolytic activity. Supplements Cu2+ at 0.5 mM/g gave the best results of lignin degradation 33.74 ± 1.17% (w/w) and highest selectivity value (SV) 3.15 after pretreatment. The glucose yield increased to 127.44 ± 4.56 mg/g from 20 day pretreated cotton stalks with Cu2+ supplements, ~threefolds higher than untreated cotton stalks. The study is important for the production of fermentable sugars from cotton stalks residues which can further be utilized in production of bioethanol and other applications.

Chitosan-assisted immobilization of serratiopeptidase on magnetic nanoparticles, characterization and its target delivery.

BACKGROUND: Magnetic nanoparticles (MNPs) gained attentions as universal carrier for drug delivery and for enzyme immobilization. PURPOSE: Target delivery of serratiopeptidase for treatment using this enzyme and applications in drug delivery. METHOD: Serratiopeptidase was immobilized on chitosan amino-functionalized MNPs by covalent bonding through glutaraldehyde. Targeting of MNPs with immobilized enzyme (EMNPs) was carried out in vitro in modified diffusion cell and in vivo in rats. RESULTS AND DISCUSSION: MNPs and EMNPs were 15.43 ± 5.22 and 18.43 ± 3.23 nm (transmission electron microscopy), crystallite size 16.89 and 21.05 nm (X-ray diffraction) and saturation magnetization 62 and 35.2 emug(-1), respectively. Maximum protein and enzyme loading on EMNPs were 264 mg g(-1) and 325 U g(-1), respectively. In the molecular level, maximum 52 enzyme molecules could bind to each particle showing residual activity 68%, little effect on KM and Vmax, good storage stability. Magnetic targeting of EMNPs increased the delivery (permeation) of drug through membrane in vitro and the enhanced anti-inflammatory effect on carrageenan-induced paw oedema in rats in vivo at much lower doses of enzyme than the doses required for treatment with free enzyme. CONCLUSIONS: The enzymatic preparation of MNPs showed enhanced effects (permeation enhancement and anti-inflammatory activity) at lower concentration with magnetic targeting.

Preparation, characterization and targeted delivery of serratiopeptidase immobilized on amino-functionalized magnetic nanoparticles.

Targeted delivery of serratiopeptidase enzyme immobilized on magnetic nanoparticles (MNPs) of Fe3O4 has been reported for the treatment using this enzyme. The enzyme was immobilized by covalent bonding through glutaraldehyde after amino functionalization of MNPs and parameters was studied. The enzyme bound MNPs (EMNPs) were characterized for size, crystallographic identity, phase purity, zeta potential and magnetic properties along with elemental and thermal analysis. The binding of enzyme had little effect on sizes (~10-17 nm) and on magnetic properties, but the zeta potential increased from -25 mV to +14.5 mV with surface amino groups up to 350 µmoles g(-1) MNPs, to stabilize its suspensions. In the molecular level, maximum of 17 molecules of enzyme could bind to each particle of MNPs that showed residual activity 67%, decreased KM and Vmax, good storage stability. Magnetic targeting of EMNPs increased the delivery (permeation) of drug through the membrane in in vitro study and enhanced the anti-inflammatory effect on carrageenan induced paw oedema in rats in in vivo study at much lower doses of enzyme than the doses required for treatment with free enzyme.

Effect of citric acid on the biosynthesis and composition of xanthan.

The effect of citric acid metabolism by Xanthomonas campestris on composition of xanthan has been studied. Citric acid consumption in fed-batch and continuous fermentation increased the pyruvic acid content of xanthan. An increase in pyruvic acid content in xanthan has been explained with the help of energy balance in xanthan biosynthesis.