Darin Ridgway

Increased heterologous protein production in Aspergillus niger fermentation through extracellular proteases inhibition by pelleted growth.

The dependence of filamentous fungal protease secretion on morphology was investigated by employing the recombinant Aspergillus niger strain AB4.1[pgpdAGLAGFP] which contains a gene for the glucoamylase‐GFP (green fluorescence protein) fusion protein. Different inoculum levels were used to obtain different sizes of pellet or free mycelia. The extracellular protease activity of the cultures varied with the pellet size and decreased dramatically when the morphology was changed from free mycelia to pellets. The culture with an optimal pellet size of 1.6 mm was obtained from an inoculum of 4 × 106 spores/mL. It resulted in a specific protease activity of 158 units/L, only one‐third of that in free mycelial growth, and a maximum specific GFP yield of 0.98 mg/g (cell mass) compared to 0.29 mg/g for free mycelial growth with an inoculum of 107 spores/mL. The results indicate that this bioprocessing strategy can be effectively used to inhibit protease activity in filamentous fungal fermentation and thereby to enhance heterologous protein production.

Enhanced heterologous protein production in Aspergillus niger through pH control of extracellular protease activity

The extracellular protease activity of Aspergillus niger AB4.1[pgpdAGLAGFP]#11, a recombinant strain producing a glucoamylase (GLA)–green fluorescent protein (GFP) fusion protein, was investigated in a 15 l stirred tank reactor and accordingly a pH control strategy was designed to minimize protease activity and increase recombinant yield. By maintaining pH at 6 recombinant protein production was enhanced over 10-fold to 21.0 mg/l compared to growth at acidic pH or without pH control. Protease activity was found to increase after 2 days of culture corresponding to the point where glucose in the culture medium had been completely utilized. When grown at pH 6, A. niger protease activity in the culture was decreased 6-fold to 560 U/l, compared to 3600 U/l under normal, acidic culture conditions. Protease activity at fermentation pH 6 was consistently lower than that at fermentation pH 3 regardless of assay pH, and results indicate that this decrease in activity was a combination of sub-optimal enzyme activity and variation in the spectrum of proteases secreted under the different pH conditions. A comparison of the concentrations of recombinant GLA and GFP demonstrated that high protease activity was responsible for GFP losses. More GFP was secreted in the pH 3 run, but less GFP remained in the broth because of the high protease activity. Although controlling pH at 6 did not completely inhibit the proteases, the GFP concentration in the fermentation broth was increased greatly.

Production of poly-β-hydroxybutyrate on molasses by recombinant Escherichia coli

Beet molasses successfully replaced glucose as sole carbon source to produce poly-β-hydroxybutyrate by a recombinant Escherichia coli strain (HMS174/pTZ18u-PHB). The fermentation with molasses was cheaper than with glucose. The final dry cell weight, PHB content and PHB productivity were 39.5 g/L, 80% (w/w) and 1 g/Lh, respectively, in a 5 L stirred tank fermenter after 31.5 h fed-batch fermentation with constant pH and dissolved O2 content.

Inhibition of extracellular protease secretion by Aspergillus niger using cell immobilization

A wild-type A. niger strain was employed as a model to investigate the effect of cell immobilization on extracellular protease secretion during fermentation. A metal-coated pad of polyester latex felt was used to immobilize the cells in shake flasks. Compared with free suspension culture, the maximum specific activity of the extracellular protease from immobilized cells was reduced from 129 units/g to 28 units/g.

A segregated model for heterologous amylase production by Bacillus subtilis

A segregated model was proposed to investigate the inherent relationships between growth, substrate consumption, cell differentiation and product formation in a Bacillus subtilis fermentation producing heterologous amylase in a 22-l bioreactor. The segregated model includes three distinguishable cell states and the transition from vegetative phase to sporangium and finally to mature spore. An age-based population balance model was applied to describe the maturity of sporangium toward the formation of spores. Parameters in the model were determined by fitting the model with experimental data. The model was able to predict the transient behavior of B. subtilis in both batch and fed-batch cultures.

Oxygen mass-transfer performance of low viscosity gas-liquid-solid system in a split-cylinder airlift bioreactor

The O2 mass-transfer coefficient, kLa, decreased by 20% when the viscosity of a simulated broth increased from 1.38 × 10−3 to 3.43 × 10−3 Pa s in a split-cylinder airlift bioreactor with a broth volume of 41 l. When the paper pulp concentration was below 10 g l−1, kLa hardly changed. While at 30 g l−1, kLa decreased by 56%. C2O42− and Na+ were found to have some effect on the kLa value.

KINETIC MODELING OF CELL GROWTH AND PRODUCT FORMATION IN SUBMERGED CULTURE OF RECOMBINANT ASPERGILLUS NIGER

The kinetics of cell growth and protein production for the recombinant Aspergillus niger strain AB4.1[pgpdAGLAGFP]#11 were investigated. An unstructured kinetic model was developed to describe the cell growth and product formation mathematically. The dynamics of glucose consumption and biomass production can be well-described by the model. The assumption that the degradation of GFP by proteases is a first-order reaction can express the trend of the GFP profile reasonably well. The dynamics of protease production is also described reasonably well by considering the inhibition effect of glucose on protease production. Because of the high initial glucose concentration and keeping the pH value of the broth at 6, the protease activity could be kept low most of the time during the fermentation. The increase of protease activity near the end of the culture might be caused by cell lysis because the intracellular protease activity was much higher than the extracellular activity.