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Optimization of bioprocess design for pharmaceutical metabolites and enzymes

by Parra, Roberto

Abstract (Summary)
This study examines the effect of ecophysiology on growth of cells and production of enzymes and secondary metabolites produced by the fungi Aspergillus niger (lysozyme) and a Phoma sp. (squalestatin S1). The effect of interactions of water activity (aw) (0.99-0.90), temperature (20, 30 and 35°C) and modifying aw solute (glycerol, NaCl) on growth and sporulation of a wild type strain of Aspergillus niger (W) and two genetically engineered lysozyme producing strains (L11, B1) was examined for the first time. Maximum growth rates were achieved for both strains (L11 and B1) under moderate aw levels. Optimum conditions for growth of strain L11 were estimated by means of contour plot surfaces and found to be 0.965 aw with glycerol as a solute at 35ºC (10.5 mm day-1). A model combining the effect of aw and temperature on growth of strains of Aspergillus niger, and comparison with data on food spoilage moulds in the literature was developed. The growth of two strains of A. niger, as a function of temperature (25-30oC) and aw (0.90-0.99) was developed. The estimation of the minimum aw (awmin) and optimal aw (awopt) levels were in accordance with data in the literature for a range of other Aspergillus and related species, regardless of the solutes used for aw modification. A central composition design was used to describe the effects of water activity (aw, 0.98, 0.97 and 0.96), inoculum size (2.7x105, 2.7x104 and 2.7x103 spores ml-1), and three autoclaving procedure (A = all components autoclaved together, B = medium autoclaved + maltose filtered and, C = medium autoclaved + maltose & soya milk filtered) on the production of lysozyme by two genetically-engineered strains of Aspergillus niger (B1 and L11) in a liquid culture fermentation. Although both strains produced similar lysozyme concentrations (15 mg l-1), different production patterns were found under the experimental conditions. However, strain B1 produced relatively higher amounts of lysozyme under water stress (0.96 aw) with all the substrates autoclaved together. Subsequently, a central composition design was used to investigate: different immobilized polymer types (alginate and pectate), polymer concentration (2 and 4% (w/v)), inoculum support ratios (1:2 and 1:4) and gel-inducing agent concentration (CaCl2, 2 and 3.5% (w/v)) on lysozyme production. Overall immobilization in Ca-pectate resulted in higher lysozyme production compared to immobilization in Ca-alginate. Similar effects were observed when the polymer concentration was reduced. A 13 fold higher lysozyme production was achieved with Ca-pectate in comparison to Ca-alginate (20-23 and 0.5-1.7 mg l-1 respectively). Polymer modifications also significantly affected the final pH and aw of the immobilized cell fermentation. The aw factor is a very significant parameter in the immobilization design. A combined statistical methodology of orthogonal design L27(313) and surface response methodology was applied to optimize the composition and concentration of a liquid fermentation medium for the production of squalestatin S1 by a Phoma species. Confirmatory experiments of the optimal medium composition produced average concentrations of 434 mg l 1 in five days fermentation at 25oC. This represented an improvement over 60% of the maximum concentration achieved in the initial experiment and a two-fold higher productivity in comparison with reported productivities of S1 in liquid fermentations with different fungal species. Different liquid height and column diameter (HL/Hr) ratios 3.7, 7.4 and 11.4 were studied in a bubble column (Dr=0.07 m) with a porous plate gas distributor, to find the effect on the gas hold up, power consumption (PG/VL) and volumetric mass transfer coefficient, kLa performance, under different superficial gas velocities calculated from the liquid properties and flow rates (2, 4, 6 and 8 l min-1) and temperatures (15, 25 and 30oC). Two kLa models were proposed based on the geometrical ratio (HL/Dr) and superficial gas velocity (m s-1) (R2=0.951), and power consumption (PG/VL) (R2=0.950). A free cell fermentation was performed in the bubble column, ratio (HL/Dr)= 3.7 and superficial gas velocity U= 0.120 m s-1, at 25oC. The S1 production reached a level of 420 mg l 1. The bioreactor scale up succeeded in maintaining the high S1 concentration obtained in our previous work 434 mg l 1 in Erlenmeyer flasks but in a shorter time. A Plackett-Burman design was used to improve the S1 produced by different immobilized designs. The immobilized cell fermentation design considered: polymerization with alginate and polygalacturonate and copolymerization, polymer concentration (alginate 3, 3.5 and 5 % w/v and pectate 4, 6 and 8 % w/v), 0.98, 0.96 and 0.94 aw levels, inoculum levels of 10, 20 and 30 % wt. v/v, gel-inducer (CaCl2) 3, 4 and 5 % w/v, gel-reinforce agent 0, 0.75 and 1.5 g l-1, air flow 4, 6 and 8 l min-1. Production of S1 reached levels of 883 mg l-1 which represent a 34 % improvement over the 660 mg l 1 produced in a stirred tank bioreactor (STR) with a free cell fermentation.
Bibliographical Information:

Advisor:Magan, Naresh (supervisor)

School:Cranfield University

School Location:United Kingdom

Source Type:Master's Thesis

Keywords:fungal growth

ISBN:

Date of Publication:08/01/2004

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