Ujjval Trivedi

Production of poly(β-hydroxybutyrate) by Comamonas testosteroni during growth on naphthalene

Comamonas testosteroni has been found to produce poly(β-hydroxybutyrate) (PHB) during its growth on naphthalene. Fourier transform infrared spectroscopy (FTIR) and 13C nuclear magnetic resonance (NMR) analysis confirmed it as a homopolymer of 3-hydroxybutyrate. Oxygen and essential nutrient limitation other than carbon source play a major role in maximum PHB production. Nitrogen limitation was found to have a profound effect, with 0.2 g ammonium nitrate/l optimum for PHB production. Both aeration and iron were found to be essential for growth and PHB accumulation. Ferric chloride at 0.04 g/l concentration was found to be optimum for PHB accumulation. Phosphate source variation showed no significant effect. Using naphthalene as a sole carbon source in optimized Bushnell Haas medium, 85% of the dry cell mass was extracted as chloroform-soluble PHB.

Optimization and characterization of PHA from isolate Pannonibacter phragmitetus ERC8 using glycerol waste

Polyhydroxyalkanoates (PHAs) have been considered as a good alternative for petrochemical based polymers due to its biodegradability. However, a high production cost limits their acceptance in industries. In present work, efforts have been made to optimize the production of PHA by Pannonibacter phragmitetus ERC8 using glycerol waste as a sole carbon source, with enhanced polymer production in a cost effective way. To check the possibility of growth and polymer accumulation potential of P. phragmitetus ERC8, various low cost substrates such as food waste, mutton tallow, whey, sugarcane bagasse, corn steep liquor and glycerol waste were used. Optimum concentration of selected factors obtained as response of statistical experimental design were 0.8% (v/v) glycerol waste, 0.26% (w/v) BHM and 1.25%OD as an inoculum for the maximum PHA production. The suggested model was validated and maximum 1.36 g/L of PHA production was obtained after 96 h. PHA production of 1.87 g/L was achieved in 5L (working volume 3 L) lab scale bioreactor with the suggested media components by RSM (Response Surface Methodology). Characterization of the PHA by NMR spectroscopy revealed that the polymer was a hetromonomer of (R)-3-hydroxybutyrate and medium chain length 3HA[(R)-3-hydroxyalkanoate] monomers.

Optimization of glucoamylase production by Colletotrichum sp. KCP1 using statistical methodology

Glucoamylase is a key enzyme used in the food processing as well as in commercial production of glucose from starch. A natural fungal strain identified as Colletotrichum sp. KCP1 using 18S rDNA partial genome sequencing has been studied for optimization of glucoamylase production. Media components were screened and optimized through the statistical approach for the synthesis of glucoamylase in solid state fermentation using wheat bran as the substrate. The medium components influencing the enzyme production were identified using Plackett-Burman design. Among various variables screened along with wheat bran as major growth substrate, starch, whey, and casein acid hydrolyasate were found to be most significant. The optimum concentrations of these significant parameters were determined employing the response surface central composite design, revealing starch concentration (1.5 g), whey (0.1 mL), and casein acid hydrolysate (0.1 g) per 5 g of wheat bran for highest enzyme production.

Chapter 10:Biodegradation of Poly (3-hydroxyalkanoates)

Polyhydroxyalkanoates (PHAs) are polyesters of hydroxyalkanoates synthesized by numerous bacteria as an intracellular carbon and energy storage compounds. PHAs have attracted much attention as substitute for non-degradable plastics derived from petrochemicals because of their similar material properties to conventional plastics along with complete biodegradability under natural environmental conditions upon disposal. In this chapter, extracellular as well as intracellular biodegradation of PHAs by diverse bacteria and fungi is reviewed. Several topics namely, factors affecting biodegradation of plastics, methods used for isolation of PHA degrading organisms, biodegradation of PHAs, biochemical properties of several PHA depolymerases produced by variety of organisms are discussed in detail in this chapter. It also includes mechanisms of action of intracellular as well as extracellular PHB depolymerases with major emphasis on mode of action of extracellular PHB depolymerase from Pseudomonas lemoignei and Penicillium funiculosum as well as intracellular PHB depolymerase of Cupriavidus necator.

Screening and Selection of Medium Components for Cyclodextrin Glucanotransferase Production by New Alkaliphile Microbacterium terrae KNR 9 Using Plackett-Burman Design.

Cyclodextrin glucanotransferase (CGTase, EC 2.4.1.19) production using new alkaliphile Microbacterium terrae KNR 9 was investigated by submerged fermentation. Statistical screening for components belonging to different categories, namely, soluble and raw starches as carbon sources, complex organic and inorganic nitrogen sources, minerals, a buffering agent, and a surfactant, has been carried out for CGTase production using Plackett-Burman factorial design. To screen out k (19), number of variables, k + 1 (20), number of experiments, were performed. Among the fourteen components screened, four components, namely, soluble starch, corn flour, yeast extract, and K2HPO4, were identified as significant with reference to their concentration effect and corresponding p value. Although soluble starch showed highest significance, comparable significance was also observed with corn flour and hence it was selected as a sole carbon source along with yeast extract and K2HPO4 for further media optimization studies. Using screened components, CGTase production was increased to 45% and 87% at shake flask level and laboratory scale fermenter, respectively, as compared to basal media.

Kinetic and thermodynamic characterization of lipase produced by Cellulomonas flavigena UNP3

Lipase of Cellulomonas flavigena UNP3 was purified by two‐step purification process comprising ammonium sulfate precipitation followed by gel permeation chromatography (GPC). The recovery of lipase after GPC was found to be 1.70% with 20.98‐fold increase in specific activity. The molecular weight of lipase protein was found to be 45.2 kDa by SDS–PAGE. Activation energy for p‐nitrophenol palmitate (pNPP) hydrolysis was 26.45 kJ mol−1, while temperature quotient (Q10) was found to be 1.64. The enzyme was found to be stable over wide pH range and thermally stable at 30–40 °C up to 60 min of incubation while exhibited maximum activity at 30 °C with pH 7.0. Vmax, Km, and Kcat for pNPP were found to be 666.71 U ml−1, 1.33 mM (pNPP) and 433 min−1, respectively. Activation energy for irreversible inactivation Ea(d) of lipase was 64.32 kJ mol−1. Thermodynamic parameters of irreversible inactivation of lipase and pNPP hydrolysis were also determined.

β-Cyclodextrin Production by Cyclodextrin Glucanotransferase from an Alkaliphile Microbacterium terrae KNR 9 Using Different Starch Substrates

Cyclodextrin glucanotransferase (CGTase, EC 2.4.1.19) is an important member of α-amylase family which can degrade the starch and produce cyclodextrins (CDs) as a result of intramolecular transglycosylation (cyclization). β-Cyclodextrin production was carried out using the purified CGTase enzyme from an alkaliphile Microbacterium terrae KNR 9 with different starches in raw as well as gelatinized form. Cyclodextrin production was confirmed using thin layer chromatography. Six different starch substrates, namely, soluble starch, potato starch, sago starch, corn starch, corn flour, and rice flour, were tested for CD production. Raw potato starch granules were found to be the best substrate giving 13.46 gm/L of cyclodextrins after 1 h of incubation at 60°C. Raw sago starch gave 12.96 gm/L of cyclodextrins as the second best substrate. To achieve the maximum cyclodextrin production, statistical optimization using Central Composite Design (CCD) was carried out with three parameters, namely, potato starch concentration, CGTase enzyme concentration, and incubation temperature. Cyclodextrin production of 28.22 (gm/L) was achieved with the optimized parameters suggested by the model which are CGTase 4.8 U/L, starch 150 gm/L, and temperature 55.6°C. The suggested optimized conditions showed about 15% increase in β-cyclodextrin production (28.22 gm/L) at 55.6°C as compared to 24.48 gm/L at 60°C. The degradation of raw potato starch granules by purified CGTase was also confirmed by microscopic observations.