The response of steady state chemostat cultures of a recombinant Aspergillus niger (B1-D), secreting both a heterologous enzyme (Hen Egg White Lysozyme [HEWL]) and a native enzyme (Glucoamylase), to varying levels of O2 enrichment of the process gas was evaluated. Formation of both the native and the foreign enzyme increased with increasing O2 supply. Conversely, biomass levels and total extracellular protein levels were generally not increased under O2 enriched conditions. Two distinct micromorphologies were apparent in these cultures, one, typically seen under O2 limiting conditions (i. e. at 0 and 10% enrichment levels), tended to be represented by long, sparsely branched hyphal elements, with low percentages of "active" length (i. e. how much of the hypha is cytoplasm filled); whilst, a second micromorphology, typical of O2 enriched cultures at 30 and 50% O2 enrichment, was represented by shorter hyphal elements, with more branching and a higher % "active" length. At these higher O2 levels, formation of a yellow pigment occurred, and signs of culture autolysis were noted. At 50% enrichment, a "stranded" aggregate morphology was apparent, possibly as a response to a hyperoxidant state. Production of both the native enzyme and HEWL correlated well with a simple morphological measure (tip number) or, with % "active" length. It is proposed the morphological changes noted in the cultures were associated with the increased production of both HEWL and glucoamylase.

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The in¯uence of bioreactor operational conditions on the micromorphology of batch cultures of Aspergillus niger B1-D, containing a hen egg white lysozome (HEWL) marker protein, was examined using computerised image analysis. Signi®cant differences in micromorphology were observed with increased stirrer speed, with shorter organisms with shorter hyphal elements occurring as agitation speed increased, even though mean tip numbers were similar. This may explain the observed increase in the total extracellular protein, since the ratio of synthetic (tip) to non-synthetic zones became increasingly favourable. HEWL concentrations fell above 500 rpm, probably due to the effects of DOT (dissolved oxygen tension) on the glucoamylase HEWL fusion. HEWL was susceptible to proteolytic degradation, by native proteases, during the autolytic phase. Such insights may indicate why a gene from one mould expressed in a close relative can give production levels equivalent to levels of native enzymes, while secretion of a gene from a`distant' source, eg a higher eukaryotic gene, occurs at much lower levels.

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