Mukund Adsul

Development of biocatalysts for production of commodity chemicals from lignocellulosic biomass.

Lignocellulosic biomass is recognized as potential sustainable source for production of power, biofuels and variety of commodity chemicals which would potentially add economic value to biomass. Recalcitrance nature of biomass is largely responsible for the high cost of its conversion. Therefore, it is necessary to introduce some cost effective pretreatment processes to make the biomass polysaccharides easily amenable to enzymatic attack to release mixed fermentable sugars. Advancement in systemic biology can provide new tools for the development of such biocatalysts for sustainable production of commodity chemicals from biomass. Integration of functional genomics and system biology approaches may generate efficient microbial systems with new metabolic routes for production of commodity chemicals. This paper provides an overview of the challenges that are faced by the processes converting lignocellulosic biomass to commodity chemicals. The critical factors involved in engineering new microbial biocatalysts are also discussed with more emphasis on commodity chemicals.

Utilization of molasses sugar for lactic acid production by Lactobacillus delbrueckii subsp. delbrueckii mutant Uc-3 in batch fermentation.

ABSTRACT Efficient lactic acid production from cane sugar molasses by Lactobacillus delbrueckii mutant Uc-3 in batch fermentation process is demonstrated. Lactic acid fermentation using molasses was not significantly affected by yeast extract concentrations. The final lactic acid concentration increased with increases of molasses sugar concentrations up to 190 g/liter. The maximum lactic acid concentration of 166 g/liter was obtained at a molasses sugar concentration of 190 g/liter with a productivity of 4.15 g/liter/h. Such a high concentration of lactic acid with high productivity from molasses has not been reported previously, and hence mutant Uc-3 could be a potential candidate for economical production of lactic acid from molasses at a commercial scale.

Lactic acid production from waste sugarcane bagasse derived cellulose

Production of L(+)lactic acid from sugarcane bagasse cellulose, one of the abundant biomass materials available in India, was studied. The bagasse was chemically treated to obtain a purified bagasse cellulose sample, which is much more amenable to cellulase enzyme attack than bagasse itself. This sample, at high concentration (10%), was hydrolyzed by cellulase enzyme preparations (10 FPU g–1 cellulose) derived from mutants generated in our own laboratory. We obtained maximum hydrolysis (72%), yielding glucose and cellobiose as the main end products. Lactic acid was produced from this bagasse cellulose sample by simultaneous saccharification and fermentation (SSF) in a media containing a cellulase enzyme preparation derived from Penicillium janthinellum mutant EU1 and cellobiose utilizing Lactobacillus delbrueckii mutant Uc-3. A maximum lactic acid concentration of 67 g l–1 was produced from a concentration of 80 g l–1 of bagasse cellulose, the highest productivity and yield being 0.93 g l–1 h–1 and 0.83 g g–1, respectively. The mutant Uc-3 was found to utilize high concentrations of cellobiose (50 g l–1) and convert it into lactic acid in a homo-fermentative way. Considering that bagasse is a waste material available in abundance, we propose to valorize this biomass to produce cellulose and then sugars, which can be fermented to products such as ethanol and lactic acid.

D-(−)-Lactic acid production from cellobiose and cellulose by Lactobacillus lactis mutant RM2-24

Lactobacillus lactis mutant RM2-24 utilizes cellobiose efficiently, converting it into D-(−)-lactic acid. Cellobiose-degrading enzyme activities were determined for whole cells, cell extracts and disrupted cells. Aryl-β-glucosidase activity was detected in whole cells and disrupted cells, suggesting that these activities are confined to the cells. The mutant produced 80 g l−1 of lactic acid from 100 g l−1 of cellobiose with 1.66 g l−1 h−1 productivity. Production of D-lactic acid from different cellulose samples was also studied. The cellulose samples at high concentration (10%) were hydrolyzed by cellulase enzyme preparation (10 FPU g−1 cellulose) derived from Penicillium janthinellum mutant EU1 generated in our own laboratory. We obtained a maximum 72% hydrolysis, yielding glucose and cellobiose as the main end products. Lactic acid was produced from these cellulose samples by simultaneous saccharification and fermentation (SSF) in a media containing a cellulase enzyme preparation derived from Penicillium janthinellum mutant EU1 and cellobiose utilizing Lactobacillus lactis mutant RM2-24. A maximum lactic acid concentration of 73 g l−1 was produced from a concentration of 100 g l−1 of bagasse-derived cellulose, the highest productivity and yield being 1.52 g l−1 h−1 and 0.73 g g−1, respectively. Considering that bagasse is a waste material available in abundance, we propose to use this biomass to produce cellulose and then sugars, which can be fermented to valuable products such as ethanol and lactic acid.

Differential induction, purification and characterization of cold active lipase from Yarrowia lipolytica NCIM 3639

The production, purification and characterization of cold active lipases by Yarrowia lipolytica NCIM 3639 is described. The study presents a new finding of production of cell bound and extracellular lipase activities depending upon the substrate used for growth. The strain produced cell bound and extracellular lipase activity when grown on olive oil and Tween 80, respectively. The organism grew profusely at 20 °C and at initial pH of 5.5, producing maximum extracellular lipase. The purified lipase has a molecular mass of 400 kDa having 20 subunits forming a multimeric native protein. Further the enzyme displayed an optimum pH of 5.0 and optimum temperature of 25 °C. Peptide mass finger printing reveled that some peptides showed homologues sequence (42%) to Yarrowia lipolytica LIP8p. The studies on hydrolysis of racemic lavandulyl acetate revealed that extracellular and cell bound lipases show preference over the opposite antipodes of irregular monoterpene, lavandulyl acetate.

Facile Approach for the Dispersion of Regenerated Cellulose in Aqueous System in the Form of Nanoparticles

This study reports a facile method to disperse cellulose in deionized water, wherein a critical condition of regenerated cellulose is discovered, where it completely disperses up to a maximum of 5 g L(-1) concentration in deionized water with the help of ultrasonication. The dispersed cellulose is characterized by TEM and DLS, the latter among which shows 200 nm hydrodynamic radii of cellulose nanoparticles dispersed in deionized water. FTIR analysis of dispersed cellulose reveals that dispersed cellulose losses its crystallinity during regeneration and dispersion step employed in this study. The dispersed cellulose reported in this study is able to form free-standing, transparent films, which were characterized by SEM, XRD, TGA, EDX, and FTIR spectroscopy and show resistance against dissolution in water. Additionally, the dispersed cellulose is able to undergo at least three times faster enzymatic hydrolysis in comparison to pristine microcrystalline cellulose under similar reaction conditions. The dispersed cellulose reported here could be a better material for reinforcement, preparation of hydrogels, and drug delivery applications under physiological environment.

Enhanced cellulase production by Penicillium oxalicum for bio-ethanol application

Present study was focused on cellulase production from an indigenously isolated filamentous fungal strain, identified as Penicillium oxalicum. Initially, cellulase production under submerged fermentation in shake flasks resulted in cellulase activity of 0.7 FPU/mL. Optimization of process parameters enhanced cellulase production by 1.7-fold and resulted in maximum cellulase activity of 1.2 FPU/mL in 8 days. Cellulase production was successfully scaled-up to 7 L fermenter under controlled conditions and incubation time was reduced from 8 days to 4 days for achieving similar cellulase titer. Optimum pH and temperature for activity of the crude enzyme were pH 5 and 50 °C, respectively. At 50 °C the produced cellulase retained approximately 50% and 26% of its activity at 48 h and 72 h, respectively. Hydrolytic efficiency of P. oxalicum was comparable to commercial cellulase preparations which indicate its great potential for application in the lignocellulose hydrolysis.

Biochemical characterization of two xylanases from yeast Pseudozyma hubeiensis producing only xylooligosaccharides

Two distinct xylanases from Pseudozyma hubeiensis NCIM 3574 were purified to homogeneity. The molecular masses of two native xylanases were 33.3 kDa (PhX33) and 20.1 kDa (PhX20). PhX33 is predominant with alpha-helix and PhX20 contained predominantly beta-sheets. Xylanase, PhX33, possesses three tryptophan and one carboxyl residues at the active site. The active site of PhX20 comprises one residue each of tryptophan, carboxyl and histidine. Carboxyl residue is mainly involved in catalysis and tryptophane residues are solely involved in substrate binding. Histidine residue present at the active site of PhX20 appeared to have a role in substrate binding. Both the xylanases produced only xylooligosaccharides (XOS) with degree of polymerization (DP) 3-7 without formation of xylose and xylobiose. These XOS could be used in functional foods or as prebiotics. Lc ms-ms ion search of tryptic digestion of these xylanases revealed that there is no significant homology of peptides with known fungal xylanase sequences which indicate that these xylanases appear to be new.

Bioethanol production from wheat straw via enzymatic route employing Penicillium janthinellum cellulases.

This study concerns in-house development of cellulases from a mutant Penicillium janthinellum EMS-UV-8 and its application in separate hydrolysis and fermentation (SHF) and simultaneous saccharification and fermentation (SSF) processes for bioethanol production from pre-treated wheat straw. In a 5L fermentor, the above strain could produce cellulases having activity of 3.1 FPU/mL and a specific activity of 0.83 FPU/mg of protein. In-house developed cellulase worked more efficiently in case of SSF as ethanol concentration of 21.6g/L and yield of 54.4% were obtained which were higher in comparison to SHF (ethanol concentration 12 g/L and 30.2% yield). This enzyme preparation when compared with commercial cellulase for hydrolysis of pre-treated wheat straw was found competitive. This study demonstrates that P. janthinellum EMS-UV-8 is a potential fungus for future large-scale production of cellulases.

Combined strategy for the dispersion/dissolution of single walled carbon nanotubes and cellulose in water

Co-dispersion of native cellulose and single walled carbon nanotubes in water is demonstrated. The pH of the water should be between 6 and 10 for better dispersion. Raman spectra confirm debundling of nanotubes in water and FTIR spectra reveal that the co-solubility is likely caused through disruption of intramolecular hydrogen bonds in the cellulose by hydroxyl groups present on nanotubes surface and the creation of intermolecular hydrogen bonds between cellulose and carbon nanotubes. This is a very simple method for co-dispersion/dissolution of nanotubes and cellulose in water without covalent modifications and expands the repertoire of nanotube modification strategies that are amenable to biological applications.