Digambar Gokhale

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.

Purification and characterization of acidic lipase from Aspergillus niger NCIM 1207

An extracellular lipase from Aspergillus niger NCIM 1207 has been purified to homogeneity using ammonium sulfate precipitation followed by phenyl sepharose and Sephacryl-100 gel chromatography. This protocol resulted in 149 fold purification with 54% final recovery. The purified enzyme showed a prominent single band on SDS-PAGE. The purified enzyme is a monomeric protein of 32.2 kDa molecular weight and exhibits optimal activity at 50 degrees C. One interesting feature of this enzyme is its highly acidic pH optimum. The isoelectric point (pI) of lipase was 8.5. The purified lipase appears to be unique since it cleaved triolein at only 3-position releasing 1,2-diolein. Chemical modification studies revealed that His, Ser, Carboxylate and Trp are involved in catalysis.

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.

Lignocellulose processing: a current challenge

Lignocellulosic biomass, of which inedible crops are a renewable source, is expected to become one of the key renewable energy resources in the near future, to deal with global warming and the depletion of conventional fossil fuel resources. It also holds the key to supplying societys basic needs for the sustainable production of chemicals and fuels without impacting the human food supply. Despite this, the production of 2nd generation biofuels and chemicals has not yet been commercialized. Therefore, the challenges involved in the production of lignocellulosic biomass derived fuels and chemicals must be addressed. The search for economic pretreatment methods has been recognized as one of the main hurdles for the processing of biomass to biofuels and chemicals. The conversion of all biomass components, lignin in particular, would greatly contribute to the economic viability of biomass based processes for 2nd generation biofuels and chemicals. The highly organized crystalline structure of cellulose presents an obstacle to its hydrolysis. Hydrolysis of lignocellulose carbohydrates into fermentable sugars requires a number of different biomass degrading enzymes such as cellulases and hemicellulases. Still, a number of technical and scientific issues within pretreatment and hydrolysis remain to be solved. Depending on the raw material and pretreatment technology, the enzyme mixtures must be designed to degrade biomass carbohydrates. Rapid advances in enzyme, microbial and plant engineering would provide the necessary breakthroughs for the successful commercialization of biomass derived fuels and chemicals.

Biomass to biodegradable polymer (PLA)

Lignocellulosic biomass is renewable and cheap, and it has the potential to displace fossil fuels for the production of fuels and chemicals. Biomass derived lactic acid is an important compound that can be used as a chemical platform for the production of a variety of important chemicals on a large scale. The quality of the monomers of lactic acid and lactide, as well as the chemical changes induced during polymerization and processing, are crucial parameters for controlling the properties of the resulting polylactic acid (PLA) products. In this review, we outline the process of exploiting biomass for the production of polylactic acid, a biodegradable polymer which is well-known as a sustainable bioplastic material.

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.

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.

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.