Valorization of Biomass to Value-Added Commodities, 2020
Depleting fossil fuel reserves and increased climate changes have led the research community to l... more Depleting fossil fuel reserves and increased climate changes have led the research community to look for sustainable and environment-friendly energy sources. Agricultural/forest feedstock has a tremendous potential to contribute for the production of biofuels and to reduce carbon dioxide emission. However, the biological transformation of lignocellulosic biomass is complicated due to their complex structure. Therefore, pretreatment, enzymatic hydrolysis, and fermentation technologies were being investigated for the conversion of lignocellulosic biomass into biofuels. Various factors that adversely affect ethanol production include presence of various fermentation inhibitors (furfurals, formic acid, and acetic acid) and presence of hexoses (glucose and mannose) that compete with or inhibit xylose utilization. This study compares and scrutinizes the various advanced biotechnological strategies applied on microbes such as strain adaptation, mutagenesis, genome shuffling, and metabolic engineering to enhance sugars and biofuel (bioethanol/biodiesel) production. Further, evaluation of BOLT-ON technology was carried out to study the impact of co-fermentation of sugars derived from molasses and lignocellulosics on ethanol production. Thus, it was concluded that co-utilization of hexoses and pentoses streams can be carried out by using advanced microbial strains that must be adapted to high sugars and ethanol concentration as well as fermentation inhibitors in future.
The LC/MS-based exo-proteome analysis of thermophilic fungus Thermomyces lanuginosus showed 22.59... more The LC/MS-based exo-proteome analysis of thermophilic fungus Thermomyces lanuginosus showed 22.59% (40 proteins) of the total identified proteins (177) as CAZymes (carbohydrate-active enzymes). The CAZymes were primarily represented by glycosyl hydrolases (72.5%) belonging to 21 different GH families. Xylanase (GH11) was found to be the major protein (24.3%) in addition to the β-glucanase and another complex polysaccharide-degrading (carbohydrate-active enzymes) CAZymes in the secretome. FPLC-based fractionation of secretome was employed to identify proteins responsible for enhancing the catalytic efficacy of Cellic Ctec2 during hydrolysis of acid pre-treated lignocellulosics and eventually xylanase (GH11) and β-glucanases (GH64 and GH81) were identified and purified. The purified xylanase when supplemented with Cellic Ctec2 resulted in 2.05-, 1.79-, and 1.60-fold increase in the release of glucose from acid-treated bagasse at 10, 15, and 20% substrate loading rate, respectively when compared with the control. Similarly, enhanced hydrolysis of acid pre-treated corn residue and rice straw was observed upon supplementation with xylanase. Spiking benchmark cellulase with purified GH64 and GH81 also resulted in 1.18- and 1.23-fold enhanced hydrolysis of acid pre-treated sugarcane bagasse. The supplementation of xylanase (1000 units/g substrate) was found to reduce the Cellic Ctec2 loading rate from 36 mg/g substrate by 2.70-, 2.88-, and 2.57-fold required for hydrolysis of acid treated bagasse, corn residue, and rice straw, respectively, at high substrate loading rate (20%) and thus, it is an important candidate for economizing the 2G ethanol process.
Two Lytic polysaccharide Mono-Oxygenases (LPMOs), non-modular (PMO_08942) and modular (PMO_07920)... more Two Lytic polysaccharide Mono-Oxygenases (LPMOs), non-modular (PMO_08942) and modular (PMO_07920), from thermotolerant fungus Aspergillus terreus 9DR cloned and expressed in Pichia pastoris X33 and purified to homogeneity using ion-exchange chromatography were found to be of~29 and~40 kDa, respectively. Both LPMOs were optimally active at 50°C; PMO_08942 was active under acidic condition (pH 5.0) and PMO_07920 at pH 7.0. Modular LPMO (PMO_07920) tethered to CBM-1 was found to be versatile as it showed appreciable activity on complex polysaccharide (both cellulose and xylans) as compared to non-modular (PMO_08942). The t 1/2 of PMO_08942 (~192 h, pH 5.0) and PMO_0792 (~192 h, pH 7.0) at 50°C, suggests highly stable nature of these LPMOs. Fluorescently tagged modular AA9 was studied microscopically to understand interaction with pretreated biomass. Priming of biomass for up to 6 h with LPMOs prior to initiating hydrolysis with core cellulase enzyme resulted in significantly higher saccharification.
The enzymatic mixture containing Novozyme Cellic CTec2 spiked with xylanase derived from Thermomy... more The enzymatic mixture containing Novozyme Cellic CTec2 spiked with xylanase derived from Thermomyces lanuginosus (1000 U xylanase per g of substrate) resulted in enhanced saccharification of native, hydrothermally and autohydrolytically pretreated sugarcane bagasse when compared to the benchmark Cellic CTec2. Hydrothermally pretreated bagasse upon hydrolysis with the enzymatic mixture yielded considerably higher levels of sugars as compared to benchmark Cellic CTec2, resulting in saccharification efficiencies of 85.7, 77.8 and 78.9% at 10, 15 and 20% (weight per volume) substrate loadings, respectively. Autohydrolysed and native sugarcane bagasse samples had lower saccharification yields, although xylanase addition to the benchmark cocktail still was beneficial. Chemical compositional analysis and detailed morphological examination of the substrates after pretreatments as well as the enzymatic hydrolysis reactions employing confocal laser scanning microscopy (CLSM), field emission scanning electron microscopy (FESEM), X-Ray diffraction (XRD) and solid state nuclear magnetic resonance (ssNMR) provided fundamental insights into the structural changes during deconstruction and shed light on how such changes affected the biomass enzyme hydrolysis.
Evaluating novel fungal secretomes for efficient saccharification and fermentation of composite s... more Evaluating novel fungal secretomes for efficient saccharification and fermentation of composite sugars derived from hydrolysate and molasses into ethanol,
The following study reports bioconversion of corncob into ethanol using hybrid approach for couti... more The following study reports bioconversion of corncob into ethanol using hybrid approach for coutilization of dilute acid hydrolysate (pentose rich stream) and hexose rich stream obtained by enzymatic saccharification employing commercial cellulase Cellic CTec2 as well as in-house cellulase preparations derived from Malbranchea cinnamomea, Scytalidium thermophilium and a recombinant Aspergillus strain. Acid hydrolysis (1% H 2 SO 4) of corncob at 1:15 solid liquid ratio led to removal of 80.5% of hemicellulosic fraction. The solid glucan rich fraction (63.5% glucan, 8.3% pentosans and 27.9% lignin) was hydrolysed at 10% substrate loading rate with different enzymes for 72 h at 50 C resulting in release of 732 and 535 (mg/g substrate) total sugars by Cellic CTec2 and M. cinnamomea derived enzymes, respectively. The fermentation of enzyme hydrolysate with co-culture of Saccharomyces cerevisiae and Pichia stipitis added in sequential manner resulted in 3.42 and 2.50% (v/v) ethanol in hydrolysate obtained from commercial Cellic CTec2 and M. cinnamomea, respectively. Employing a hybrid approach, where dilute acid hydrolysate stream was added to solid residue along with enzyme Cellic CTec2 during staggered simultaneous saccharification and fermentation at substrate loading rate of 15% resulted in 252 g ethanol/kg corncob.
This study reports production of microbial oil from a yeast strain Trichosporon sp., (RW) isolate... more This study reports production of microbial oil from a yeast strain Trichosporon sp., (RW) isolated from decayed wood. Preliminary analysis based on fluorescence microscopy and spectroscopy of Nile red stained yeast cells showed accumulation of lipid globules. The potential of the yeast to produce lipids was evaluated on glucose, glycerol and acid hydrolysate of sugarcane bagasse, where Trichosporon sp. (RW) was found to accumulate 21.45 (59.6%), 18.41 (56%) and 10.25g/l (40.5%) of the lipids after 120h of fermentation at 30°C. FAME analysis of lipids by GC-FID and NMR revealed oleic acid (18:1) as the major constituent, corresponding to 50.05, 46.48 and 54.66% of the accumulated lipids in glucose, glycerol and hydrolysate grown cultures, respectively. Other accumulated lipids included palmitic (16:0), linoleic (18:2) and stearic acids (18:0) in that order. The cetane number of the lipids ranged from 52.39 to 59.57 indicating suitability for biodiesel production.
Valorization of Biomass to Value-Added Commodities, 2020
Depleting fossil fuel reserves and increased climate changes have led the research community to l... more Depleting fossil fuel reserves and increased climate changes have led the research community to look for sustainable and environment-friendly energy sources. Agricultural/forest feedstock has a tremendous potential to contribute for the production of biofuels and to reduce carbon dioxide emission. However, the biological transformation of lignocellulosic biomass is complicated due to their complex structure. Therefore, pretreatment, enzymatic hydrolysis, and fermentation technologies were being investigated for the conversion of lignocellulosic biomass into biofuels. Various factors that adversely affect ethanol production include presence of various fermentation inhibitors (furfurals, formic acid, and acetic acid) and presence of hexoses (glucose and mannose) that compete with or inhibit xylose utilization. This study compares and scrutinizes the various advanced biotechnological strategies applied on microbes such as strain adaptation, mutagenesis, genome shuffling, and metabolic engineering to enhance sugars and biofuel (bioethanol/biodiesel) production. Further, evaluation of BOLT-ON technology was carried out to study the impact of co-fermentation of sugars derived from molasses and lignocellulosics on ethanol production. Thus, it was concluded that co-utilization of hexoses and pentoses streams can be carried out by using advanced microbial strains that must be adapted to high sugars and ethanol concentration as well as fermentation inhibitors in future.
The LC/MS-based exo-proteome analysis of thermophilic fungus Thermomyces lanuginosus showed 22.59... more The LC/MS-based exo-proteome analysis of thermophilic fungus Thermomyces lanuginosus showed 22.59% (40 proteins) of the total identified proteins (177) as CAZymes (carbohydrate-active enzymes). The CAZymes were primarily represented by glycosyl hydrolases (72.5%) belonging to 21 different GH families. Xylanase (GH11) was found to be the major protein (24.3%) in addition to the β-glucanase and another complex polysaccharide-degrading (carbohydrate-active enzymes) CAZymes in the secretome. FPLC-based fractionation of secretome was employed to identify proteins responsible for enhancing the catalytic efficacy of Cellic Ctec2 during hydrolysis of acid pre-treated lignocellulosics and eventually xylanase (GH11) and β-glucanases (GH64 and GH81) were identified and purified. The purified xylanase when supplemented with Cellic Ctec2 resulted in 2.05-, 1.79-, and 1.60-fold increase in the release of glucose from acid-treated bagasse at 10, 15, and 20% substrate loading rate, respectively when compared with the control. Similarly, enhanced hydrolysis of acid pre-treated corn residue and rice straw was observed upon supplementation with xylanase. Spiking benchmark cellulase with purified GH64 and GH81 also resulted in 1.18- and 1.23-fold enhanced hydrolysis of acid pre-treated sugarcane bagasse. The supplementation of xylanase (1000 units/g substrate) was found to reduce the Cellic Ctec2 loading rate from 36 mg/g substrate by 2.70-, 2.88-, and 2.57-fold required for hydrolysis of acid treated bagasse, corn residue, and rice straw, respectively, at high substrate loading rate (20%) and thus, it is an important candidate for economizing the 2G ethanol process.
Two Lytic polysaccharide Mono-Oxygenases (LPMOs), non-modular (PMO_08942) and modular (PMO_07920)... more Two Lytic polysaccharide Mono-Oxygenases (LPMOs), non-modular (PMO_08942) and modular (PMO_07920), from thermotolerant fungus Aspergillus terreus 9DR cloned and expressed in Pichia pastoris X33 and purified to homogeneity using ion-exchange chromatography were found to be of~29 and~40 kDa, respectively. Both LPMOs were optimally active at 50°C; PMO_08942 was active under acidic condition (pH 5.0) and PMO_07920 at pH 7.0. Modular LPMO (PMO_07920) tethered to CBM-1 was found to be versatile as it showed appreciable activity on complex polysaccharide (both cellulose and xylans) as compared to non-modular (PMO_08942). The t 1/2 of PMO_08942 (~192 h, pH 5.0) and PMO_0792 (~192 h, pH 7.0) at 50°C, suggests highly stable nature of these LPMOs. Fluorescently tagged modular AA9 was studied microscopically to understand interaction with pretreated biomass. Priming of biomass for up to 6 h with LPMOs prior to initiating hydrolysis with core cellulase enzyme resulted in significantly higher saccharification.
The enzymatic mixture containing Novozyme Cellic CTec2 spiked with xylanase derived from Thermomy... more The enzymatic mixture containing Novozyme Cellic CTec2 spiked with xylanase derived from Thermomyces lanuginosus (1000 U xylanase per g of substrate) resulted in enhanced saccharification of native, hydrothermally and autohydrolytically pretreated sugarcane bagasse when compared to the benchmark Cellic CTec2. Hydrothermally pretreated bagasse upon hydrolysis with the enzymatic mixture yielded considerably higher levels of sugars as compared to benchmark Cellic CTec2, resulting in saccharification efficiencies of 85.7, 77.8 and 78.9% at 10, 15 and 20% (weight per volume) substrate loadings, respectively. Autohydrolysed and native sugarcane bagasse samples had lower saccharification yields, although xylanase addition to the benchmark cocktail still was beneficial. Chemical compositional analysis and detailed morphological examination of the substrates after pretreatments as well as the enzymatic hydrolysis reactions employing confocal laser scanning microscopy (CLSM), field emission scanning electron microscopy (FESEM), X-Ray diffraction (XRD) and solid state nuclear magnetic resonance (ssNMR) provided fundamental insights into the structural changes during deconstruction and shed light on how such changes affected the biomass enzyme hydrolysis.
Evaluating novel fungal secretomes for efficient saccharification and fermentation of composite s... more Evaluating novel fungal secretomes for efficient saccharification and fermentation of composite sugars derived from hydrolysate and molasses into ethanol,
The following study reports bioconversion of corncob into ethanol using hybrid approach for couti... more The following study reports bioconversion of corncob into ethanol using hybrid approach for coutilization of dilute acid hydrolysate (pentose rich stream) and hexose rich stream obtained by enzymatic saccharification employing commercial cellulase Cellic CTec2 as well as in-house cellulase preparations derived from Malbranchea cinnamomea, Scytalidium thermophilium and a recombinant Aspergillus strain. Acid hydrolysis (1% H 2 SO 4) of corncob at 1:15 solid liquid ratio led to removal of 80.5% of hemicellulosic fraction. The solid glucan rich fraction (63.5% glucan, 8.3% pentosans and 27.9% lignin) was hydrolysed at 10% substrate loading rate with different enzymes for 72 h at 50 C resulting in release of 732 and 535 (mg/g substrate) total sugars by Cellic CTec2 and M. cinnamomea derived enzymes, respectively. The fermentation of enzyme hydrolysate with co-culture of Saccharomyces cerevisiae and Pichia stipitis added in sequential manner resulted in 3.42 and 2.50% (v/v) ethanol in hydrolysate obtained from commercial Cellic CTec2 and M. cinnamomea, respectively. Employing a hybrid approach, where dilute acid hydrolysate stream was added to solid residue along with enzyme Cellic CTec2 during staggered simultaneous saccharification and fermentation at substrate loading rate of 15% resulted in 252 g ethanol/kg corncob.
This study reports production of microbial oil from a yeast strain Trichosporon sp., (RW) isolate... more This study reports production of microbial oil from a yeast strain Trichosporon sp., (RW) isolated from decayed wood. Preliminary analysis based on fluorescence microscopy and spectroscopy of Nile red stained yeast cells showed accumulation of lipid globules. The potential of the yeast to produce lipids was evaluated on glucose, glycerol and acid hydrolysate of sugarcane bagasse, where Trichosporon sp. (RW) was found to accumulate 21.45 (59.6%), 18.41 (56%) and 10.25g/l (40.5%) of the lipids after 120h of fermentation at 30°C. FAME analysis of lipids by GC-FID and NMR revealed oleic acid (18:1) as the major constituent, corresponding to 50.05, 46.48 and 54.66% of the accumulated lipids in glucose, glycerol and hydrolysate grown cultures, respectively. Other accumulated lipids included palmitic (16:0), linoleic (18:2) and stearic acids (18:0) in that order. The cetane number of the lipids ranged from 52.39 to 59.57 indicating suitability for biodiesel production.
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