Agitation effects on morphology and protein productive fractions of filamentous and pelleted growth forms of recombinant Aspergillus niger

Abstract

Recombinant Aspergillus niger genetically engineered to produce glucose oxidase using the constitutive gpdA promoter and the glucoamylase signal sequence for secretion was grown in batch cultures at agitation speeds of 200 -800 rpm covering the industrial relevant power input range of 0.1 -5 W kg -1 . The growth morphology ranged from large pellets with an average diameter of 1500 µm at low power input up to micropellets embedded in a filamentous network at high power input. A correlation of agitation intensity with growth morphology and glucose oxidase production revealed an increase of the protein production capability with the change of the growth morphology from pelleted to filamentous growth forms. However, the exposure to higher shear stress with increasing power input also resulted in lower biomass yields as well as increased transient formation of polyol (xylitol) and higher final concentrations of oxalic acid. The highest specific production rates were found in young filamentous growth forms at high power input. Although intermediate agitation intensity leading to small pellets became more favorable during prolonged cultivation. An acridine orange staining procedure discriminating between RNA rich (red) and RNA poor regions (green) of the fungal biomass proved that active protein production is restricted to filamentous growth forms and the outer layer of fungal pellets. A correlation between the RNA rich fraction of the biomass determined by image analysis and the productivity is shown.

Heterogeneity of Aspergillus niger Microcolonies in Liquid Shaken Cultures

“…This finding indicates that the RNA levels of these genes per unit mass of hypha are much higher in zone 4 than in zone 2. In agreement with this, acridine orange staining showed that the periphery of microcolonies of A. niger is rich in RNA, while the center is not (5). Acridine orange binds to single-and double-stranded nucleic acids, resulting in red and green fluorescence, respectively.…”

Impact of dissolved oxygen concentration on some key parameters and production of rhG-CSF in batch fermentation

“…Although the highest cell growth was obtained at an agitation speed of 750 rpm, the highest specific and volumetric yield was obtained at an agitation speed of 550 rpm. Our results coincided with the the results of others investigations [32,33]. But, no earlier reports have discussed about the impact of agitation speed on cell growth, plasmid stability, acetate accumulation and rhG-CSF expression [25,26] In the production of rhG-CSF by E. coli BL21 (DE3) PLysS, the relative viscosity of culture broth became steep declined slope (Fig.…”

Changes in morphology of Rhizopus chinensis in submerged fermentation and their effect on production of mycelium-bound lipase

“…Other researchers also reported that 100 lm is a limitation distance for mycelium which with biofilm [39]. Investigations have shown that pellet formation is necessary for the production of some enzymes from filamentous microorganisms, such as polygalacturonase [14,37], phytase [16], glucoamylase [8], fructosyltransferase [15], and other proteins [33]. Based on other researchers and their results, Papagianni concluded that such phenomena may be related to diffusional limitations in pellets, which either reduce the extent of catabolic repression in pellets or limit the oxygen supply, preventing oxidative inactivation of a specific set of enzymes [1,8].…”