Ashish A. Prabhu

Turbulent flux events in a nearly neutral atmospheric boundary layer

We propose here a novel method of analysing turbulent momentum flux signals. The data for the analysis come from a nearly neutral atmospheric boundary layer and are taken at a height of 4 m above ground corresponding to 1.1×105 wall units, within the log layer for the mean velocity. The method of analysis involves examining the instantaneous flux profiles that exceed a given threshold, for which an optimum value is found to be 1 s.d. of the flux signal. It is found feasible to identify normalized flux variation signatures separately for positive and negative ‘flux events’—the sign being determined by that of the flux itself. Using these signatures, the flux signal is transformed to one of events characterized by the time of occurrence, duration and intensity. It is also found that both the average duration and the average time-interval between successive events are of order 1 s, about four orders of magnitude higher than a wall unit in time. This episodic description of the turbulence flux in the time domain enables us to identify separately productive, counter-productive and idle periods (accounting, respectively, for 36, 15 and 49% of the time), taking as criterion the generation of the momentum flux. A ‘burstiness’ index of 0.72 is found for the data. Comparison with laboratory data indicates higher (/lower) ejection (/sweep) quadrant occupancy but lower (/higher) contributions to flux from the ejection (/sweep) quadrant at the high Reynolds numbers of the atmospheric boundary layer. Possible connections with the concept of active and passive motion in a turbulent boundary layer are briefly discussed.

Reverse micellar extraction of papain with cationic detergent based system: An optimization approach

ABSTRACT In this study, reverse micellar extraction of papain model system was performed using cetyltrimethylammonium bromide (CTAB)/iso-octane/hexanol/butanol system to optimize the forward and back extraction efficiency (BEE). A maximum forward extraction efficiency of 55.0, 61.0, and 54% was achieved with an aqueous phase pH of 11.0, 150 mM CTAB/iso-octane and 0.1 M NaCl, respectively. Taguchi’s orthogonal array was applied to optimize the pH of stripping phase, concentration of isopropyl alcohol (IPA) and potassium chloride (KCl) for maximizing BEE. The optimal levels of stripping phase pH, concentration of IPA and KCl were found to be 6, 20% (v/v), and 0.8 M, respectively. Under these optimal levels, the BEE was found to be 88% after which enzyme activity was recovered with 2.5-fold purification. Further optimization was performed using artificial neural network-linked genetic algorithm, where the BEE was improved to 90.52% with pH 6, IPA (%) = 19.938, and KCl (M) = 0.729.

Gene and process level modulation to overcome the bottlenecks of recombinant proteins expression in Pichia pastoris

BACKGROUND Process development involving system metabolic engineering and bioprocess engineering has become one of the major thrust for the development of therapeutic proteins or enzymes. Pichia pastoris has emerged as a prominent host for the production of therapeutic protein or enzymes. Regardless of producing high protein titers, various cellular and process level bottlenecks restrict the expression of recombinant proteins in P. pastoris. RESULT AND CONCLUSION In the present review, we have summarized the recent developments in the expression of foreign proteins in P. pastoris. Further, we have discussed various cellular engineering strategies which include codon optimization, pathway engineering, signal peptide processing, development of protease deficient strain and glyco-engineered strains for the high yield protein secretion of recombinant protein. Bioprocess development of recombinant proteins in large-scale bioreactor including medium optimization, optimum feeding strategy and co-substrate feeding in fed-batch as well as continuous cultivation have been described. The recent advances in system and synthetic biology studies including metabolic flux analysis in understanding the phenotypic characteristics of recombinant Pichia and genome editing with CRISPR-CAS system have also been summarized.

Dual-substrate inhibition kinetic studies for recombinant human interferon gamma producing Pichia pastoris

ABSTRACT Pichia pastoris is considered as one of the prominent host extensively used as a platform for heterologous protein production. In the present study, the growth inhibition kinetics of recombinant P. pastoris expressing human interferon gamma was studied under different initial substrate concentrations of gluconate (10–100 g L−1) and methanol (2–50 g L−1) in modified FM22 medium. The highest specific growth rate of 0.0206 and 0.019 hr−1 was observed at 60 g L−1 of gluconate and 10 g L−1 of methanol, respectively. Various three- and four-parametric Monod-variant models were chosen to analyze the inhibition kinetics. The model parameters as well as goodness of fit were estimated using nonlinear regression analysis. The three-parameter Haldane model was found to be best fit for both gluconate (R2 = 0.95) and methanol substrate (R2 = 0.96). The parameter sensitivity analysis revealed that µmax, Ki, and Ks are the most sensitive parameters for both methanol and gluconate. Different substrate inhibition models were fitted to the growth kinetic data and the additive form of double Webb model was found to be the best to explain the growth kinetics of recombinant P. pastoris.

A novel reverse micellar purification strategy for histidine tagged human interferon gamma (hIFN-γ) protein from Pichia pastoris

In the present study, we have demonstrated the process development of human interferon gamma (hIFN-γ) (upstream to downstream). The codon optimized hIFN-γ gene was cloned in Pichia pastoris X-33 and the expression was evaluated in batch reactor study. The purification was carried out with modified nickel chelated reverse micellar system and compared with the existing Nickle- Nitrilotriacetic acid (NI-NTA) method. The parameter optimization for forward extraction demonstrated a significant enhancement of 72% in forward extraction efficiency (FEE). Furthermore, the factors governing back extraction efficiency (BEE) were also optimized with sequential optimization involving Taguchi orthogonal array and Artificial Neural Network linked Simulated Annealing Algorithm (ANN-SA). The optimization resulted in 91.2% back extraction efficiency of recombinant human interferon gamma (rhIFN-γ). The development of this purification system with optimized parameters led to an efficient recovery of 67.3% and improved purity of 79.54%. Alongside, the anti-proliferative activity in MCF-7 cell lines were also investigated and it demonstrated that at 60ngmL-1 concentration of rhIFN-γ more that 25%.

Batch and fed-batch bioreactor studies for the enhanced production of glutaminase-free L-asparaginase from Pectobacterium carotovorum MTCC 1428

ABSTRACT The effect of dissolved oxygen (DO) level and pH (controlled/uncontrolled) was first studied to enhance the production of novel glutaminase-free L-asparaginase by Pectobacterium carotovorum MTCC 1428 in a batch bioreactor. The optimum level of DO was found to be 20%. The production of L-asparaginase was found to be maximum when pH of the medium was maintained at 8.5 after 12 h of fermentation. Under these conditions, P. carotovorum produced 17.97 U/mL of L-asparaginase corresponding to the productivity of 1497.50 U/L/h. The production of L-asparaginase was studied in fed-batch bioreactor by feeding L-asparagine (essential substrate for production) and/or glucose (carbon source for growth) at the end of the reaction period of 12 h. The initial medium containing both L-asparagine and glucose in the batch mode and L-asparagine in the feeding stream was found to be the best combination for enhanced production of glutaminase-free L-asparaginase. Under this condition, the L-asparaginase production was increased to 38.8 U/mL, which corresponded to a productivity of 1615.8 U/L/h. The production and productivity were increased by 115.8% and 7.9%, respectively, both of which are higher than those obtained in the batch bioreactor experiments.

Rhizoremediation of Environmental Contaminants Using Microbial Communities

Over last few decades, the contamination of water and soil has become a major threat to ecosystem and human health. Bioremediation is an attractive tool to overcome the challenges posed by the traditional methods such as incineration and excavation. Recently, phytoremediation has been widely used to remediate the pollutants (such as organic and inorganic) from the environment, but certain compounds and heavy metals tend to inhibit the growth of the plants. In this chapter, we have emphasized on most accepted bioremediation process known as rhizoremediation, which involves the mutualism between microorganisms and plants that degrades the recalcitrant compounds present in the soil and makes eco-friendly environment. Furthermore, we discussed the important factors such as temperature, pH, and organic matter present in the soil, which affects the growth and metabolism of not only the organism but also the plants, interaction between plant and microorganisms, and role of endophytic and rhizobacteria in bioremediation of heavy metals and organic pollutants.

Application of medium optimization tools for improving recombinant human interferon gamma production from Kluyveromyces lactis

ABSTRACT The present study is focused upon improving biomass of Kluyveromyces lactis cells expressing recombinant human interferon gamma (hIFN-γ), with the aim of augmenting hIFN-γ concentration using statistical and artificial intelligence approach. Optimization of medium components viz., lactose, yeast extract, and trace elements were performed with Box–Behnken design (BBD) and artificial neural network linked genetic algorithm (ANN-GA) for maximizing biomass of recombinant K. lactis (objective function). The studies resulted over 1.5-fold improvement in the biomass concentration in a medium composed of 80 g/L lactose, 10.353 g/L yeast extract, and 15 mL/L trace elements as compared with initial biomass value. In the same study hIFN-γ concentration reached 881 µg/L which was 2.28-fold higher as compared with initial hIFN-γ concentration obtained in unoptimized medium. Further the batch fermentation study displayed mixed growth associated kinetics with the maximum hIFN-γ production rate of 1.1 mg/L. BBD and ANN-GA, both optimization techniques predicted a higher lactose concentration was clearly beneficial for augmenting K. lactis biomass which in turn increased hIFN-γ concentration.

Improvement of Phytonutrients and Antioxidant Properties of Wheat Bran by Yeast Fermentation

Introduction: Utilizing nonedible part of food crop as functional food components has received more attention in recent years. Wheat bran is a reservoir of several health beneficial components. But due to the presence of non-starch polysaccharides in the cell wall, the bioavailability of phytonutrients will be very low. Our main objective is to understand the effect of fermentation by Baker’s yeast (Saccharomyces cerevisiae) on Phytonutrients and antioxidant properties of wheat bran. Methods: The wheat bran is subjected to fermentation with Baker’s yeast (Saccharomyces cerevisiae) for 24h time period. Later the variations in the Phytonutrients and antioxidant properties of fermented wheat bran was analysed and compared with that of control. Results: It was found that the soluble, bound and total polyphenol content was increased by 48%, 21.5% and 34% respectively and flavonoid content was increased by 20%. Anti-nutritional compounds such as tannins were reduced by 35%. Due to enhancement of phytonutrients components such as polyphenols and flavonoids, the in-vitro total antioxidant capacity was also increased by 2 folds, whereas the free radical scavenging was enhanced by 56% compared to control samples. Conclusion: This findings shows that, with the aid of fermentation process modification in structural and functional properties of bran can be achieved and thus the bioprocessed bran can be used as the ingredient for functional food preparation.