Madan Kumar

Comparison of submerged and solid state pretreatment of sugarcane bagasse by Pandoraea sp. ISTKB: Enzymatic and structural analysis.

Pretreatment of sugarcane bagasse by Pandoraea sp. ISTKB was evaluated under submerged (SmF) and solid state (SSF) culture conditions. Number of bacteria was 2.7 times higher in SmF as compared with SSF. Enzymes produced under SmF were xylanase, LiP, MnP and laccase. In SSF xylanase and laccase were detected. CMCase, FPase and β-glucosidase were not detected. Delignification was highest in SmF with 19.94% and 10.43% removal of hemicelluloses and lignin, respectively. FTIR analysis suggested the degradation of lignin/hemicellulose component. SEM analysis showed pores were three times bigger in SmF as compared with raw bagasse. Maximum CR dye was absorbed by treated SmF bagasse. Enzymatic saccharification increased by 3.7 times after SmF treatment in comparison to raw bagasse. Pretreatment of bagasse by Pandoraea sp. ISTKB was more efficient under SmF than SSF. High negative correlation between saccharification vs lignin/hemicelluloses content justified the need for pretreatment of lignocellulosic waste before saccharification.

Immobilized lipase from Schizophyllum commune ISTL04 for the production of fatty acids methyl esters from cyanobacterial oil.

Novel lipase from model mushroom Schizophyllum commune strain ISTL04 produced by solid state fermentation of Leucaena leucocephala seeds, was immobilized onto Celite for enzymatic FAMEs production from cyanobacterial endolith Leptolyngbya ISTCY101. The isolate showed vigorous growth and produced remarkable lipase activity of 146.5 U g(-1) dry solid substrate, without any external lipase inducer. Single-factor experiments were carried out to study the effects of various reaction parameters on the FAMEs yield. The best conditions for enzymatic transesterification as revealed by the results were: 1:3 oil to methanol molar ratio, added at 3h intervals, 12% water content, 1581.5 U g(-1) immobilized lipase, temperature 45 °C, and time 24h. Under these conditions, the maximum FAMEs yield reached 94%. The immobilized lipase was able to produce >90% of the relative FAMEs yield after four repeated transesterification cycles. This immobilized lipase exhibited potential for application in biodiesel industry.

Proteomic characterization and schizophyllan production by Schizophyllum commune ISTL04 cultured on Leucaena leucocephala wood under submerged fermentation

In this study Schizophyllum commune ISTL04 was grown on Leucaena leucocephala wood (LLW) for secretome analysis and schizophyllan production. There is no report on extracellular protein profile and schizophyllan production on woody biomass by this fungus under submerged fermentation conditions. Leucaena leucocephala, a promising bioenergy crop having high holocellulose content was used as substrate without pretreatment. The maximum sugar, extracellular protein and exopolysaccharide (EPS) production during fermentation was found to be 8.53±0.07mgmL-1, 391±7.51mgL-1 and 4.2±0.1gL-1 or 0.21gg-1LLW on day 18 respectively. The secretome profile was dominated by glycoside hydrolases followed by carbohydrate esterase and other oxidative enzymes. EPS was further characterized by FTIR and GC-MS for functional group, monomer composition and linkage analysis and was identified as schizophyllan. The result indicated that LLW can be utilized as a low cost substrate for enzyme cocktail and schizophyllan production.

Production and Characterization of Polyhydroxyalkanoate from Lignin Derivatives by Pandoraea sp. ISTKB

The present study investigates polyhydroxyalkanoate (PHA) production from lignin and its derivatives by a previously reported lignin-degrading bacterial strain Pandoraea sp. ISTKB. PHA production was screened by fluorescence microscopy and flow cytometry using a Nile red stain. PHA and biomass accumulation, while screening, was found to be maximum on 4-hydroxybenzoic acid followed by p-coumaric acid, vanillic acid, 2,6-dimethoxyphenol, and kraft lignin after 96 h. Monomer composition was analyzed by gas chromatography–mass spectrometry (GC–MS) and was followed by Fourier transform infrared and 1H NMR analysis, indicating PHA to be a copolymer of P(hydroxybutyrate-co-hydroxyvalerate). Genomic analysis of Pandoraea sp. ISTKB also complemented the results of GC–MS and NMR, and the relevant genes responsible for the synthesis of small chain length PHA were discovered in the genome. Process parameters were optimized by response surface methodology for enhanced production of PHA and biomass on 4-hydroxybenzoate. Optimization results showed 30 and 66% increase in the biomass and PHA production, respectively. The results obtained were promising and indicated that if lignin is depolymerized into low-molecular-weight intermediates, then it can easily be utilized and converted into value-added products like PHA by microbes.

Recycling of carbon dioxide by free air CO2 enriched (FACE) Bacillus sp. SS105 for enhanced production and optimization of biosurfactant

Carbon dioxide utilizing bacterium Bacillus sp. SS105 was isolated from FACE (free air CO2 enriched) sample. The strain was grown in shake flask containing minimal salt medium with 50mM NaHCO3 as autotrophic carbon source and molasses as a low cost byproduct for mixotrophic growth. Carbon dioxide sequestration property of Bacillus sp. SS105 was determined by enzyme assay of carbonic anhydrase and ribulose-1, 5-bisphosphate carboxylase/oxygenase (RuBisCO). Along with CO2 sequestration this strain produced biosurfactant and its characterization by FTIR and 1H NMR indicated lipopeptide nature. Optimization of process parameter along with nutrient sources for higher biosurfactant production was done by Response Surface Methodology (RSM). Under optimized conditions, the yield of biosurfactant and biomass was 2.65 and 2.78gL-1 respectively. The study revealed simultaneous CO2 sequestration and biosurfactant production by Bacillus sp. SS105.

Analysis and optimization of process parameters for production of polyhydroxyalkanoates along with wastewater treatment by Serratia sp. ISTVKR1

A previously reported biodegrading bacterial strain Serratia sp. ISTVKR1 was studied for polyhydroxyalkanoate (PHA) production along with wastewater contaminant removal. Nile red fluorescence, GC-MS, FT-IR, NMR and TEM confirmed the accumulation of homopolymer poly-3-hydroxyvalerate (PHV) within the bacterial cells. Analysis of culture after 72h of bacterial treatment showed maximum COD removal (8.4-fold), non-detection of organic contaminants such as 1H-Cyclopropa [a] naphthalene (R.T.=10.12) using GC-MS and increased proportion of elements like Cr, Mn, Fe, Ni, Cu, Cd and Pb in the bacterial cell pellets by SEM-EDX analysis. Optimization of process parameters for enhanced PHA production along with wastewater treatment done using Response Surface Methodology (RSM) showed 5% and 0.74% increase in the PHA production (0.3368±0.13gL-1) and % COD reduction (88.93±2.41) of wastewater, respectively. The study, thus established the production of PHA along with wastewater contaminant removal by Serratia sp. ISTVKR1.

Production, Process optimization and Molecular characterization of Polyhydroxyalkanoate (PHA) by CO 2 sequestering B. cereus SS105

Carbon dioxide sequestering bacterial strains were previously isolated from free air CO2 enriched (FACE) soil. In the present study, these strains were screened for PHA accumulation and Bacillus cereus SS105 was found to be the most prominent PHA accumulating strain on sodium bicarbonate and molasses as carbon source. This strain was further characterized by Spectrofluorometric method and Confocal microscopy after staining with Nile red. PHA granules in inclusion bodies were visualized by Transmission Electron Microscopy. The PHA and its monomer composition were characterized by GC-MS followed by FTIR and NMR. The genetic basis of PHA production was confirmed by the amplification, cloning and analysis of PHA biosynthesis genes phaR, phaB and phaC from B. cereus with the degenerate primers. The PHA production was further optimized by Response Surface Methodology and the percent increase observed after optimization was 55.16% (w/v).

Genome Sequence of Pandoraea sp. ISTKB, a Lignin-Degrading Betaproteobacterium, Isolated from Rhizospheric Soil

ABSTRACT We report here the genome sequence of Pandoraea sp. ISTKB, a betaproteobacterium isolated from rhizospheric soil in the backwaters of Alappuzha, Kerala, India. The strain is alkalotolerant and grows on medium containing lignin as a sole carbon source. Genes and pathways related to lignin degradation were complemented by genomic analysis.

Genome Sequence of Carbon Dioxide-Sequestering Serratia sp. Strain ISTD04 Isolated from Marble Mining Rocks

ABSTRACT The Serratia sp. strain ISTD04 has been identified as a carbon dioxide (CO2)-sequestering bacterium isolated from marble mining rocks in the Umra area, Rajasthan, India. This strain grows chemolithotrophically on media that contain sodium bicarbonate (NaHCO3) as the sole carbon source. Here, we report the genome sequence of 5.07 Mb Serratia sp. ISTD04.

Genomic and proteomic analysis of lignin degrading and polyhydroxyalkanoate accumulating β-proteobacterium Pandoraea sp. ISTKB

BackgroundLignin is a major component of plant biomass and is recalcitrant to degradation due to its complex and heterogeneous aromatic structure. The biomass-based research mainly focuses on polysaccharides component of biomass and lignin is discarded as waste with very limited usage. The sustainability and success of plant polysaccharide-based biorefinery can be possible if lignin is utilized in improved ways and with minimal waste generation. Discovering new microbial strains and understanding their enzyme system for lignin degradation are necessary for its conversion into fuel and chemicals. The Pandoraea sp. ISTKB was previously characterized for lignin degradation and successfully applied for pretreatment of sugarcane bagasse and polyhydroxyalkanoate (PHA) production. In this study, genomic analysis and proteomics on aromatic polymer kraft lignin and vanillic acid are performed to find the important enzymes for polymer utilization.ResultsGenomic analysis of Pandoraea sp. ISTKB revealed the presence of strong lignin degradation machinery and identified various candidate genes responsible for lignin degradation and PHA production. We also applied label-free quantitative proteomic approach to identify the expression profile on monoaromatic compound vanillic acid (VA) and polyaromatic kraft lignin (KL). Genomic and proteomic analysis simultaneously discovered Dyp-type peroxidase, peroxidases, glycolate oxidase, aldehyde oxidase, GMC oxidoreductase, laccases, quinone oxidoreductase, dioxygenases, monooxygenases, glutathione-dependent etherases, dehydrogenases, reductases, and methyltransferases and various other recently reported enzyme systems such as superoxide dismutases or catalase–peroxidase for lignin degradation. A strong stress response and detoxification mechanism was discovered. The two important gene clusters for lignin degradation and three PHA polymerase spanning gene clusters were identified and all the clusters were functionally active on KL–VA.ConclusionsThe unusual aerobic ‘-CoA’-mediated degradation pathway of phenylacetate and benzoate (reported only in 16 and 4–5% of total sequenced bacterial genomes), peroxidase-accessory enzyme system, and fenton chemistry based are the major pathways observed for lignin degradation. Both ortho and meta ring cleavage pathways for aromatic compound degradation were observed in expression profile. Genomic and proteomic approaches provided validation to this strain’s robust machinery for the metabolism of recalcitrant compounds and PHA production and provide an opportunity to target important enzymes for lignin valorization in future.