A comparison of high cell density fed-batch fermentations involving both induced and non-induced recombinantEscherichia coli under well-mixed small-scale and simulated poorly mixed large-scale conditions

Paraburkholderia sp. SOS3 is a plant growth-promoting bacterium (PGPB) that displays pleiotropic effects and has the potential to be applied at a large scale across several agronomically important crops. The use of SOS3 is a suitable option to reduce the use of chemical fertilisers. While the benefits of SOS3 have been demonstrated in vitro, its potential applications at large scale are limited due to low biomass yield in current batch culture systems. Here, we developed a strategy for high-cell density cultivation of SOS3 in instrumented bioreactors, moving from low-biomass yield in a complex medium to high-biomass yield in a semi-defined medium. We achieved a 40-fold increase in biomass production, achieving cell densities of up to 11 g/L (OD600 = 40). This result was achieved when SOS3 was cultivated using a fed-batch strategy. Biomass productivity, initially 0.02 g/L/h in batch cultures, was improved 12-fold, reaching 0.24 g/L/h during fed-batch cultures. The biomass yield was also improved 10-fold from 0.07 to 0.71 gbiomass/gsolids. Analysis of the fermentation profile of SOS3 indicated minimal production of by-products and accumulation of polyhydroxybutyrate (PHB) during the exponential growth phase associated with nitrogen limitation in the medium. By implementing proteomics analysis in fed-batch cultures, we identified the expression of four metabolic pathways associated with growth-promoting effects, which may be used as a qualitative parameter to guarantee the efficacy of SOS3 when used as a bioinoculant. Ultimately, we confirmed that the high-cell density cultures maintained their plant growth-promoting capacity when tested in sorghum and maize under glasshouse conditions.

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High Cell Density Cultivation of A Recombinant Bacillus Subtilis for Nattokinase Production

10.21203/rs.3.rs-91180/v1 ◽

2020 ◽

Author(s):

Jianhua Zhang ◽

Qing Cui ◽

Bingjun Qian ◽

Xiangjun Sun

Keyword(s):

Enzyme Activity ◽

Bacillus Subtilis ◽

Cell Density ◽

Large Scale ◽

High Cell Density ◽

Specific Activity ◽

Feeding Strategy ◽

Batch Process ◽

High Cell ◽

Fed Batch

Abstract Background: Nattokinase (NK), a fibrinolytic enzyme, can be produced by culturing recombinant Bacillus subtilis in Luria-Bertani broth in a shaking flask. For use as a nutraceutical, however, a large-scale preparation and a simple purification process are required.Results: The present study utilized a fed-batch process to cultivate a B. subtilis strain carrying a pHT01 plasmid with an NK-encoding gene (B. subtilis/pHT01-aprN1). For batch A (FB A), with a pH-stat two-stage fermentation strategy, we achieved an activity of 2910.5 ± 21.6 U mL-1 and a specific activity of 30.32 U ml-1 OD600-1. Then, we changed the strategy with a later induction and lower feeding rate to pursue higher cell density and thus higher enzyme activity, a 11.9-fold activity of 4521.8 ± 23.8 U mL-1 was acquired, however, the specific activity was lower than FB A. For the third batch, low-glycerol-level-maintain feeding strategy was followed, and finally, a NK activity of 7778 ±17.28 U mL-1 was obtained, according to our knowledge, it was the highest activity assayed by the fibrin plate method ever reported. Furthermore, fermentation supernatant was successively purified by ammonium sulfate precipitation and nickel column affinity chromatography with a total NK recovery rate of 65.2%.Conclusions: Our results indicate that there is a balance between the cell growth rate and NK expression when recombinant Bacillus subtilis is cultured with a fed-batch process. The equilibrium state can be attained by optimizing the induction and feeding strategy, and thus a high cell density and enzyme activity can be achieved.

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