Switching industrial production processes from complex to defined media: method development and case study using the example of Penicillium chrysogenum

Abstract

Background: Filamentous fungi are versatile cell factories and widely used for the production of antibiotics, organic acids, enzymes and other industrially relevant compounds at large scale. As a fact, industrial production processes employing filamentous fungi are commonly based on complex raw materials. However, considerable lot-to-lot variability of complex media ingredients not only demands for exhaustive incoming components inspection and quality control, but unavoidably affects process stability and performance. Thus, switching bioprocesses from complex to defined media is highly desirable.

Results: This study presents a strategy for strain characterization of filamentous fungi on partly complex media using redundant mass balancing techniques. Applying the suggested method, interdependencies between specific biomass and side-product formation rates, production of fructooligosaccharides, specific complex media component uptake rates and fungal strains were revealed. A 2-fold increase of the overall penicillin space time yield and a 3-fold increase in the maximum specific penicillin formation rate were reached in defined media compared to complex media.

Conclusions: The newly developed methodology enabled fast characterization of two different industrial Penicillium chrysogenum candidate strains on complex media based on specific complex media component uptake kinetics and identification of the most promising strain for switching the process from complex to defined conditions. Characterization at different complex/defined media ratios using only a limited number of analytical methods allowed maximizing the overall industrial objectives of increasing both, method throughput and the generation of scientific process understanding.

Figure 1

Nitrogen metabolism for cultivations containing complex and defined nitrogen sources. Uptake rates for defined nitrogen components can be determined by simple enzymatic methods, uptake rates for complex nitrogen constituents can be inferred from stoichiometric balancing using carbon, degree of reduction and nitrogen balances.

Figure 2

Process performance charts for BCB1 batch cultivation on fully complex media.a) measured raw data. (Dots), Biomass formed; (Stars), Initial substrate consumed; (Squares), Gluconic acid formed; (Open triangles down) Mannitol. b) statistically verified measured raw data and calculated process variables. (Dots), Biomass formed; (Stars), Initial substrate consumed; (Squares), Gluconic acid formed; (Open triangles down), Mannitol formed; (Open hexagons), GF₂ formed; (Diamonds), Complex nitrogen consumed. c) physiological dynamics including cumulated yields and RQ. (Solid line), RQ; (Dots), Yield Biomass·Substrate^-1; (Open hexagons), Yield GF₂·Substrate^-1; (Squares), Yield Gluconic acid·Substrate^-1.d) specific formation rates for biomass and gluconic acid. (Dots), specific growth rate; (Squares), q_p Gluconic acid.

Figure 3

Process performance charts for BCB1 batch cultivation on 25% complex media.a) measured raw data. (Dots), Biomass formed; (Stars), Initial substrate consumed; (Squares), Gluconic acid formed; (Open triangles down), Mannitol formed; (Open Diamonds), NH₃-N consumed. b) statistically verified measured raw data and calculated process variables. (Dots), Biomass formed; (Stars), Initial substrate consumed; (Squares), Gluconic acid formed; (Open triangles down), Mannitol formed; (Diamonds), Complex nitrogen consumed, (Open Diamonds), NH₃-N consumed. c) physiological dynamics including cumulated yields and RQ. (Solid line), RQ; (Dots), Yield Biomass·Substrate^-1; (Squares), Yield Gluconic acid·Substrate^-1; (Crosses), Cumulated Percentage of Complex Nitrogen. d) specific formation rates for biomass and gluconic acid. (Dots), specific growth rate; (Squares), q_p Gluconic acid; (Diamonds), q_s complex nitrogen sources; (Open Diamonds), q_s NH₃-N.

Figure 4

Process performance charts for BCB1 batch cultivation on fully defined media.a) measured raw data. (Dots), Biomass formed; (Stars), Initial substrate consumed; (Open triangles down), Mannitol formed; (Open Diamonds), NH₃-N consumed. b) statistically verified measured raw data and calculated process variables. (Dots), Biomass formed; (Stars), Initial substrate consumed; (Open triangles down), Mannitol formed; (Open Diamonds), NH₃-N consumed. c) physiological dynamics including cumulated yields and RQ. (Dots), Yield Biomass·Substrate^-1; (Open dots), Yield O₂·Substrate^-1; (Triangles down), Yield CO₂·Substrate^-1. d) specific formation rate for biomass. (Dots), specific growth rate.

Figure 5

Key performance parameters for a 2-stage industrial penicillin production process at defined and complex batch cultivation conditions. (Dots), Normalized product yield using complex media; (Open dots), Normalized product yield using defined media; (Triangles down), Normalized specific productivity using standard complex media; (Open Triangles down), Normalized specific productivity using defined media.