Pulsed feeding during fed‐batch fungal fermentation leads to reduced viscosity without detrimentally affecting protein expression

Abstract

The goal in this study was to determine if pulsed addition of substrate could be used to alter filamentous fungal morphology during fermentation, to result in reduced broth viscosity. In all experiments, an industrially relevant strain of Aspergillus oryzae was grown in 20-liter fermentors. As a control, cultures were fed limiting substrate (glucose) continuously. Tests were performed by altering the feeding strategy so that the same total amount of glucose was fed in repeated 300-s cycles, with the feed pump on for either 30 or 150 s during each cycle. Variables indicative of cellular metabolic activity (biomass concentration, oxygen uptake rate, base consumed for pH control) showed no significant difference between continuous and pulse-fed fermentations. In addition, there was no significant difference between total extracellular protein expression or the apparent distribution of these proteins. In contrast, fungal mycelia during the second half of pulse-fed fermentations were approximately half the size (average projected area) of fungi during fermentations with continuous addition of glucose. As a result, broth viscosity during the second half of pulse-fed fermentations was approximately half that during the second half of continuous fermentations. If these results prove to be applicable for other fungal strains and processes, then this method will represent a simple and inexpensive means to reduce viscosity during filamentous fungal fermentation.

Enzymatic resolution of racemic phenyloxirane by a novel epoxide hydrolase from Aspergillus niger SQ-6 and its fed-batch fermentation

“…Pulsed addition of a limiting carbon source to reduce broth viscosity during heterologous protein expression has been studied thoroughly in fed-batch fermentation in bacteria, yeast, and in one filamentous fungus (A. oryzae [7][8][9][10]). In this paper, this technique was successfully applied in an A. niger fed-batch fermentation with excellent results.…”

“…Unfortunately, submerged cultivation of filamentous fungi is often characterized by high broth viscosity, which can result in a number of problems (e.g., insufficient oxygen mass transfer, high power requirement, formation of nutrient concentration zones) that have the potential to reduce productivity [31]. Bhargava et al [7,8] recently proposed a possible solution: ''pulsed'' addition of the limiting carbon source in an Aspergillus oryzae fed-batch fermentation led to significantly reduced broth viscosity and improved oxygen mass transfer efficiency without detrimentally affecting cellular metabolic activity or total secreted protein. The effect of total cycle time for substrate pulsing was also studied in detail, revealing that high values of cycle time (more than 15 min) showed significantly reduced recombinant enzyme production [10].…”

Pilot-scale process development and scale up for antifungal production

“…Two principle causes are high biomass concentration and a dispersed filamentous culture morphology [50][51][52]. Although biomass was high at 130 g/L dcw, broth consisted of loosely aggregated mucoid clumps of mycelia, suspended in a black liquid containing unicellular bodies suspected to be conidia.…”

“…Besides broth dilution, two alternative methods were found, neither proven for secondary metabolite production: (1) reduction of cultivation temperature from 30 to 25°C (pigment production by Monascus sp. ; [59], and (2) pulse-feeding of a limiting carbon source (enzyme production by Aspergillus; [50,60,61]). …”

Aspergillus sojae

“…This can be achieved either by changing the process parameters, which could cause a difference in the growth characteristics of the fungus (Bhargava et al 2003), or by changing the morphology of the fungus itself. Consequently, the latter option -to isolate morphological A. sojae mutants of reduced viscosity -was chosen.…”