An in-silico analysis of hydrodynamics and gas mass transfer characteristics in scale-down models for mammalian cell cultures.

Bioprocess scale-up and technology transfer can be challenging due to multiple variables that need to be optimized during process development from laboratory scale to commercial manufacturing. Cell cultures are highly sensitive to key factors during process transfer across scales, including geometric variability in bioreactors, shear stress from impeller and sparging activity, and nutrient gradients that occur due to increasing blend times. To improve the scale-up and scale-down of these processes, it is important to fully characterize bioreactors to better understand the differences that will occur within the culture environment, especially the hydrodynamic profiles that will vary in vessel designs across scales. In this study, a comprehensive hydrodynamic characterization of the Ambr® 250 mammalian single-use bioreactor was performed using time-accurate computational fluid dynamics simulations conducted with M-Star computational fluid dynamics software, which employs lattice-Boltzmann techniques to solve the Navier-Stokes transport equations at a mesoscopic scale. The single-phase and two-phase fluid properties within this small-scale vessel were analyzed in the context of agitation hydrodynamics and mass transfer (both within the bulk fluid and the free surface) to effectively characterize and understand the differences that scale-down models possess when compared to their large-scale counterparts. The model results validate the use of computational fluid dynamics as an in-silico tool to characterize bioreactor hydrodynamics and additionally identify important free-surface transfer mechanics that need to be considered during the qualification of a scale-down model in the development of mammalian bioprocesses.

Enhanced protein and amino acids of corn-ethanol co-product by Mucor indicus and Rhizopus oryzae.

Upcycle of co-products from corn-ethanol plant into protein-rich animal feed with balanced key amino acids via solid-state fermentation is a promising approach to economically support both biofuel and animal feed industries. However, there are multiple types of solid-state fermentation microorganisms and growth conditions that have not been tested. In this study, Mucor indicus and Rhizopus oryzae were used to ferment corn-based wet distiller's grains with solubles (WDGS). The effects of fermentation conditions (temperature, agitation, and moisture) and supplementations (extraneous carbon and nitrogen sources) were evaluated on protein production and amino acids profiles before and after fermentation. The study established best fermentation conditions (23 °C, static incubation for 4 days at 70% initial moisture content) to improve protein content for both R. oryzae and M. indicus. Moreover, urea supplied to R. oryzae and M. indicus improved protein concentration by 35 and 38%, and total amino acids content by 28 and 18%, respectively. The amount of 693.1 and 451.8 mg of additional total amino acids including 262.8 and 227.7 mg of key amino acids (lysine, methionine, tryptophan, and arginine) was synthesized by R. oryzae and M. indicus, respectively, per supply of 536 mg urea in 25 g of WDGS. This study demonstrated the feasibility of urea as a low-cost nitrogen source for amino acid biosynthesis in fungal fermentation of WDGS, which could contribute to the increasing demand for high-value monogastric animal feed.

Critical applications of Mucor circinelloides within a biorefinery context.

The establishment of an efficient and feasible biorefinery model depends on, among other factors, particularly the selection of the most appropriate microorganism. Mucor circinelloides is a dimorphic fungus species able to produce a wide variety of hydrolytic enzymes, lipids prone to biodiesel production, carotenoids, ethanol, and biomass with significant nutritional value. M. circinelloides also has been selected as a model species for genetic modification by being the first filamentous oleaginous species to have its genome fully characterized, as well as being a species characterized as a potential bioremediation agent. Considering the potential of replacing several nonrenewable feedstocks is widely dependent on fossil fuels, the exploitation of microbial processes and products is a desirable solution for promoting a green and sustainable future. Here, we introduce and thoroughly describe the recent and critical applications of this remarkable fungus within the context of developing a fungal-based biorefinery.

Mycoalgae biofilm: development of a novel platform technology using algae and fungal cultures.

BACKGROUND: Microalgae is considered a promising source for biofuel and bioenergy production, bio-remediation and production of high-value bioactive compounds, but harvesting microalgae is a major bottleneck in the algae based processes. The objective of this research is to mimic the growth of natural lichen and develop a novel biofilm platform technology using filamentous fungi and microalgae to form a lichen type of biofilm "mycoalgae" in a supporting polymer matrix. RESULTS: The possibility of co-existence of Chlorella vulgaris with various fungal cultures was tested to identify the best strain combination for high algae harvest efficiency. The effect of different matrices for cell attachment and biofilm formation, cell surface characterization of mycoalgae biofilm, kinetics of the process with respect to the algae-fungi cell distribution and total biomass production was studied. Mycoalgae biofilm with algae attachment efficiency of 99.0 % and above was achieved in a polymer-cotton composite matrix with glucose concentration of 2 g/L in the growth medium and agitation intensity of 150 rpm at 27 °C. The total biomass in the co-culture with the selected strain combination (Mucor sp. and Chlorella sp.) was higher than the axenic cultures of fungi and algae at the conditions tested. CONCLUSIONS: The results show that algae can be grown with complete attachment to a bio-augmenting fungal surface and can be harvested readily as a biofilm for product extraction from biomass. Even though, interaction between heterotrophic fungi and phototrophic algae was investigated in solid media after prolonged contact in a report, this research is the first of its kind in developing an artificial lichen type biofilm called "mycoalgae" biofilm completely attached on a matrix in liquid cultures. The mycoalgae biofilm based processes, propounds the scope for exploring new avenues in the bio-production industry and bioremediation.

Microbial production of virus-like particle vaccine protein at gram-per-litre levels.

This study demonstrates the feasibility of large-scale production of murine polyomavirus VP1 protein in recombinant Escherichia coli as pentamers which are able to subsequently self-assemble in vitro into virus-like particles (VLPs). High-cell-density pH-stat fed-batch cultivation was employed to produce glutathione-S-transferase (GST)-VP1 fusion protein in soluble form. The expression of recombinant VP1 was induced with IPTG at different cell optical densities (OD at 600 nm of 20, 60 or 100). GST-VP1 production was highest when the culture was induced at a cell density of OD 60, with volumetric yield reaching 4.38 gL⁻¹ in 31h, which we believe is the highest volumetric productivity for viral capsid protein reported to date. The induction cell density is shown to have a significant effect on the overall volumetric yield of recombinant VP1 and on final cell density, but not on VLP quality. VP1 yield was enhanced 15-fold by scaling-up from shake flask to pH-stat fed-batch cultivation in a bioreactor. Although numerous studies have expressed structural viral protein in E. coli, we believe this is the first report of translation to bioreactors yielding gram-per-litre levels. This VLP production technology overcomes major drawbacks associated with eukaryotic cell-based vaccine production technologies, and propounds the scope for large-scale commercially viable E. coli based VLP production by significantly reducing vaccine production time and cost.

Lipase catalyzed ester synthesis for food processing industries

Lipases are one of the most important industrial biocatalyst which catalyzes the hydrolysis of lipids. It can also reverse the reaction at minimum water activity. Because of this pliable nature, it is widely exploited to catalyze the diverse bioconversion reactions, such as hydrolysis, esterification, interesterification, alcoholysis, acidolysis and aminolysis. The property to synthesize the esters from the fatty acids and glycerol promotes its use in various ester synthesis. The esters synthesized by lipase finds applications in numerous fields such as biodiesel production, resolution of the recemic drugs, fat and lipid modification, flavour synthesis, synthesis of enantiopure pharmaceuticals and nutraceuticals. It plays a crucial role in the food processing industries since the process is unaffected by the unwanted side products. Lipase modifications such as the surfactant coating, molecular imprinting to suit for the non-aqueous ester synthesis have also been reported. This review deals with lipase catalyzed ester synthesis, esterification strategies, optimum conditions and their applications in food processing industries.

Lipases são catalizadores industriais dos mais importantes, os quais catalizam a hidrólise de lipídeos. Também podem reverter a reação a um mínimo de atividade de água. Devido sua natureza flexível, é amplamente explorada para catalizar uma diversidade de reações de bioconversão como hidrólise, esterificação, interesterificação, alcoólise, acidólise e aminólise. A propriedade de síntese de esteres a partir de ácidos graxos e glicerol promoveu seu uso em várias sínteses de esteres. Os esteres sintetizados por lipases encontram aplicação em numerosos campos como a produção de biodiesel, resolução de drogas racêmicas, modificação de gorduras e lipídios, sintese de aromas, síntese de produtos farmacêuticos enantiopuro e nutracêuticos. As lipases possuem um papel crucial nas indústrias de processamento de alimentos, pois os processos não são afetados por subprodutos indesejáveis. Modificações nas lipases como revestimento tensoativo, impressão molecular, para permitir a síntese de esteres não aquosos também são reportados. Esta revisão trata da síntese de éster catalizada por lipase, estratégia de esterificação, condições ótimas e suas aplicações em indústrias de processamento de alimento.

Evaluation of medium components by Plackett-Burman statistical design for lipase production by Candida rugosa and kinetic modeling.

Lipase production by Candida rugosa was carried out in submerged fermentation. Plackett-Burman statistical experimental design was applied to evaluate the fermentation medium components. The effect of twelve medium components was studied in sixteen experimental trials. Glucose, olive oil, peptone and FeCl3.6H2O were found to have more significance on lipase production by Candida rugosa. Maximum lipase activity of 3.8 u mL(-1) was obtained at 50 h of fermentation period. The fermentation was carried out at optimized temperature of 30 degrees C, initial pH of 6.8 and shaking speed of 120 r/min. Unstructured kinetic models were used to simulate the experimental data. Logistic model, Luedeking-Piret model and modified Luedeking-Piret model were found suitable to efficiently predict the cell mass, lipase production and glucose consumption respectively with high determination coefficient(R2). From the estimated values of the Luedeking-Piret kinetic model parameters, alpha and beta, it was found that the lipase production by Candida rugosa is growth associated.

Optimization of medium composition for lipase production by Candida rugosa NCIM 3462 using response surface methodology.

A sequential optimization approach using statistical design of experiments was employed to enhance the lipase production by Candida rugosa in submerged batch fermentation. Twelve medium components were evaluated initially using the Plackett-Burman 2-level factorial design. The significant variables affecting lipase production were found to be glucose, olive oil, peptone, (NH4)2SO4, and FeCl3.6H2O. Various vegetable oils were tested in the second step, and among them, groundnut oil was found to be the best inducer for lipase production by C. rugosa. The third step was to identify the optimal values of the significant medium components with groundnut oil as the inducer using response surface methodology. The regression equation obtained from the experimental data designed using a central composite design was solved, and analyzing the response surface contour plots, the optimal concentrations of the significant variables were determined. A maximum lipase activity of 5.95 U.mL-1, which is 1.64 times the maximum activity obtained in the Plackett-Burman experimental trials, was observed. The optimum combination of medium constituents contained 19.604 g.L-1 glucose, 13.065 mL.L-1 groundnut oil, 7.473 g.L-1 peptone, 0.962 g.L-1 (NH4)2SO4, 0.0019 g.L-1 FeCl3.6H2O, and other insignificant components at the fixed level. A predictive model of the combined effects of the independent variables using response surface methodology and an artificial neural network was proposed. The unstructured kinetic models, logistic model, and Luedeking-Piret model were used to describe cell mass and lipase production. The parameters of the models were evaluated and the lipase production by C. rugosa was found to be growth associated.