Rajesh P. Singh

Enhanced production of itaconic acid from corn starch and market refuse fruits by genetically manipulated Aspergillus terreus SKR10

A potent itaconic acid producing strain, Aspergillus terreus SKR10, was isolated from horticulture waste. Market refuse, apple and banana, were explored as novel substrates for itaconic acid production with yields of 20+/-2.0 and 20.0+/-1.0 g l(-1), respectively. Itaconic acid yields of 28.5+/-2.2 and 31.0+/-1.7 g l(-1) were obtained with acid and alpha-amylase hydrolyzed corn starch. The efficiency of itaconic acid production by this wild type strain was improved by ultraviolet, chemical and mixed mutagenic treatments. Two high itaconic acid yielding mutants, N45 and UNCS1 were obtained by gradient plating. These two mutants were capable of producing twice the yield of itaconic acid as the parent strain.

Biodiesel production from non-edible lignocellulosic biomass of Cassia fistula L. fruit pulp using oleaginous yeast Rhodosporidium kratochvilovae HIMPA1

This study explored biodiesel production from a low cost, abundant, non-edible lignocellulosic biomass from aqueous extract of Cassia fistula L. (CAE) fruit pulp. The CAE was utilized as substrate for cultivating novel oleaginous yeast Rhodosporidium kratochvilovae HIMPA1. This oleaginous yeast accumulates high amount of triacylglycerides as large intracellular lipid droplets (4.35±0.54μm) using CAE as sole nutritional source. Total lipids (4.86±0.54g/l) with lipid content of 53.18% (w/w) were produced by R. kratochvilovae HIMPA1 on CAE. The FAME profile obtained revealed palmitic acid (C16:0) 43.06%, stearic acid (C18:0) 28.74%, and oleic acid (C18:1) 17.34% as major fatty acids. High saturated fatty acids content (72.58%) can be blended with high PUFA feedstocks to make it an industrially viable renewable energy product.

Boosting accumulation of neutral lipids in Rhodosporidium kratochvilovae HIMPA1 grown on hemp (Cannabis sativa Linn) seed aqueous extract as feedstock for biodiesel production

Hemp seeds aqueous extract (HSAE) was used as cheap renewable feedstocks to grow novel oleaginous yeast Rhodosporidium kratochvilovae HIMPA1 isolated from Himalayan permafrost soil. The yeast showed boosted triglyceride (TAG) accumulation in the lipid droplets (LDs) which were transesterified to biodiesel. The sonicated HSAE prepared lacked toxic inhibitors and showed enhanced total lipid content and lipid yield 55.56%, 8.39±0.57g/l in comparison to 41.92%, 6.2±0.8g/l from industrially used glucose synthetic medium, respectively. Supersized LDs (5.95±1.02μm) accumulated maximum TAG in sonicated HSAE grown cells were visualized by fluorescent BODIPY (505/515nm) stain. GC-MS analysis revealed unique longer carbon chain FAME profile containing Arachidic acid (C20:0) 5%, Behenic acid (C22:0) 9.7%, Heptacosanoic acid (C27:0) 14.98%, for the first time in this yeast when grown on industrially competent sonicated HSAE, showing more similarity to algal oils.

Co-cultivation of mutant Penicillium oxalicum SAUE-3.510 and Pleurotus ostreatus for simultaneous biosynthesis of xylanase and laccase under solid-state fermentation

Co-cultivation of mutant Penicillium oxalicum SAU(E)-3.510 and Pleurotus ostreatus MTCC 1804 was evaluated for the production of xylanase-laccase mixture under solid-state fermentation (SSF) condition. Growth compatibility between mutant P. oxalicum SAU(E)-3.510 and white rot fungi (P. ostreatus MTCC 1804, Trametes hirsuta MTCC 136 and Pycnoporus sp. MTCC 137) was analyzed by growing them on potato dextrose agar plate. Extracellular enzyme activities were determined spectrophotometrically. Under derived conditions, paired culturing of mutant P. oxalicum SAU(E)-3.510 and P. ostreatus MTCC 1804 resulted in 58% and 33% higher levels of xylanase and laccase production, respectively. A combination of sugarcane bagasse and black gram husk in a ratio of 3:1 was found to be the most ideal solid substrate and support for fungal colonization and enzyme production during co-cultivation. Maximum levels of xylanase (8205.31 ± 168.31 IU g(-1)) and laccase (375.53 ± 34.17 IU g(-1)) during SSF were obtained by using 4 g of solid support with 80% of moisture content. Furthermore, expressions of both xylanase and laccase were characterized during mixed culture by zymogram analysis. Improved levels of xylanase and laccase biosynthesis were achieved by co-culturing the mutant P. oxalicum SAU(E)-3.510 and P. ostreatus MTCC 1804. This may be because of efficient substrate utilization as compared to their respective monocultures in the presence of lignin degradation compounds because of synergistic action of xylanase and laccase. Understanding and developing the process of co-cultivation appears productive for the development of mixed enzyme preparation with tremendous potential for biobleaching.

Kinetics of chlorophenol degradation by benzoate-induced culture of Rhodococcus erythropolis M1

A pure culture of Rhodococcus erythropolis was isolated with the ability to degrade 2-chlorophenol, 4-chlorophenol and 2,4-dichlorophenol. Degradation of 2-chlorophenol by the uninduced culture of Rhodococcus erythropolis began after a prolonged lag period and complete mineralization of the substrates took 45 days. With the aim of reducing the lag period and subsequently improving the rate of degradation, the cells of the isolate were induced with benzoate, phenol, toluene and catechol individually. Benzoate-induced cells showed the highest rate of degradation and were thus used for the study of the degradation kinetics of 2-chlorophenol, 4-chlorophenol and 2,4-dichlorophenol. Complete mineralization of these substrates was achieved up to a concentration of 300, 100 and 50 mg l−1 respectively. Degradation of the chlorophenols was initiated without any significant lag and took the remarkably short time periods of 84, 64 and 144 h for the highest concentrations of the substrate. Evaluation of kinetic parameters showed chlorophenol degradation to follow substrate inhibition kinetics. This is evident from the decrease in specific growth rate, growth yield and substrate uptake rate with increase in the initial substrate concentrations. Toxicity of the chlorophenols was observed to depend on the position of chlorine on the benzene ring and the degree of chlorination.

Antibiofilm activity of quercetin- encapsulated cytocompatible nanofibers against Candida albicans

In this study, nanofibers against pro dimorphic fungal sessile growth were developed. Quercetin was successfully encapsulated within poly(d,l-lactide-co-glycolide)–poly(ε-caprolactone) nanofibers using an electrospinning technique. Field emission scanning electron microscopy, fluorescent microscopy, and Fourier-transformed infrared spectrometer were used to confirm the formation as well as encapsulation of quercetin within the nanofibers. These fabricated nanofibers were further evaluated to determine the effectiveness of the antibiofilm activity against Candida albicans. The cytocompatibility of quercetin-encapsulated nanofibers was found to be similar to control and pure polymeric nanofibers based on 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay against human embryonic kidney (HEK-293) cell lines. These fabricated nanofibers potentially could be used as coatings on biomedical devices to inhibit microbial contaminations.

Process optimization for fabrication of gellan based electrospun nanofibers.

In this investigation, the nanofiber formation ability of gellan, a FDA approved low cost natural polysaccharide, has been achieved for the first time using electrospinning technique. The gellan based ultrafine nanofibers were fabricated by using a blend mixture of gellan with another biodegradable polymer polyvinyl alcohol (PVA). The morphology of resulting gellan-PVA nanofibers was analyzed using field emission scanning electron microscopy (FESEM). The mass ratio of 50:50 for gellan:PVA was recorded as an optimum solution ratio to obtain uniform bead free nanofibers with an average diameter of 40 ± 15.8 nm. Data depicted that among different parameters evaluated, viscosity and the mass ratio of gellan:PVA were the key parameters that influence the nanofiber morphology and diameter.

Penicillium oxalicum SAEM-51: a mutagenised strain for enhanced production of chitin deacetylase for bioconversion to chitosan.

A novel chitin deacetylase (CDA) producing strain Penicillium oxalicum ITCC 6965 was isolated from residual materials of sea food processing industries. Strain following mutagenesis using ethidium bromide (EtBr) and microwave irradiation had resulted into a mutant P. oxalicum SAE(M)-51 having improved levels of chitin deacetylase (210.71 ± 1.65 Ul(-1)) as compared to the wild type strain (108.26 ± 1.98 Ul(-1)). Maximum enzyme production was achieved in submerged fermentation following 144 hours of incubation with notably improved productivity of 1.46 ± 0.82 Ul(-1) h(-1) as compared to the wild type strain (0.75 ± 0.53 Ul(-1)h(-1)). Scanning electron micrographs of mutant and wild type strains had revealed distinct morphological features. Evaluation of kinetic parameters viz. Q(s), Q(p), Y(p/x), Y(p/s), q(p), q(s) had denoted that strain P. oxalicum SAE(M)-51 is a hyper producer of chitin deacetylase. Glucose as compared to chitin or colloidal chitin had resulted in increased levels of enzyme production. However, replacement of glucose with chitinous substrates had prolonged the duration for enzyme production. The mutant strain had two pH optima that is 6.0 and 8.0 and had an optimum temperature of 30 °C for growth and enzyme production.

A novel gellan-PVA nanofibrous scaffold for skin tissue regeneration: Fabrication and characterization.

In this investigation, we have introduced novel electrospun gellan based nanofibers as a hydrophilic scaffolding material for skin tissue regeneration. These nanofibers were fabricated using a blend mixture of gellan with polyvinyl alcohol (PVA). PVA reduced the repulsive force of resulting solution and lead to formation of uniform fibers with improved nanostructure. Field emission scanning electron microscopy (FESEM) confirmed the average diameter of nanofibers down to 50 nm. The infrared spectra (IR), differential scanning calorimetry (DSC) and X-ray diffraction (XRD) analysis evaluated the crosslinking, thermal stability and highly crystalline nature of gellan-PVA nanofibers, respectively. Furthermore, the cell culture studies using human dermal fibroblast (3T3L1) cells established that these gellan based nanofibrous scaffold could induce improved cell adhesion and enhanced cell growth than conventionally proposed gellan based hydrogels and dry films. Importantly, the nanofibrous scaffold are biodegradable and could be potentially used as a temporary substrate/or biomedical graft to induce skin tissue regeneration.

Synergistic effect of fermentable and non-fermentable carbon sources enhances TAG accumulation in oleaginous yeast Rhodosporidium kratochvilovae HIMPA1

Novel strategy for enhancing TAG accumulation by simultaneous utilization of fermentable and non-fermentable carbon sources as substrate for cultivation of oleaginous yeast Rhodosporidium kratochvilovae HIMPA1 were undertaken in this investigation. The yeast strain showed direct correlation between the size of lipid bodies, visualized by BODIPY stain (493-515 nm) and TAG accumulation when examined on individual fermenting and non-fermenting carbon sources and their mixtures. Maximum TAG accumulation (μm) in glucose (2.38 ± 0.52), fructose (4.03 ± 0.38), sucrose (4.24 ± 0.45), glycerol (4.35 ± 0.54), xylulose (3.94 ± 0.12), and arabinose (2.98 ± 0.43) were observed. Synergistic effect of the above carbon sources (fermentable and non-fermentable) in equimolar concentration revealed maximum lipid droplet size of 5.35 ± 0.76 μm and cell size of 6.89 ± 0.97 μm. Total lipid content observed in mixed carbon sources was 9.26 g/l compared to glucose (6.2g/l). FAME profile revealed enhanced longer chain (C14:0-C24:0) fatty acids in mix carbon sources.