Process Control And Optimization Of Cephalosporin C Fermentation Based On Metabolic Analysis And Computational Fluid Dynamics Simulation

Cephalosporin C （CPC） is an important intermediate for producing the widely used β-lactamantibiotics, and Cephalosporins are fermentatively produced by several Streptomyces spp. andfilamentous fungi. The semi-synthetic derivatives of CPC are less toxic, high stability onβ-lactamase and broad-spectrum antibiotics. Even though the history of CPC fermentativeproduction in China is relatively long, a lot of practical problems still remained unsolved inindustrial CPC productions, such as fermentation performance/stability dependence onknowledge/experience of expert/skilled operators, high accumulation of the majorby-metabolite of deacetoxycephalosporin （DAOC）, difficulty in controlling dissolved oxygenconcentration （DO） control due to the extensively high oxygen consumption, high costs ofraw materials and operations, etc. Focusing on CPC fermentations by Cephalosporinsacremonium HC-3, this thesis investigated the optimal substrates feeding strategies, theoptimal impeller combinations in a stirred bioreactor for CPC production, and CPCfermentation techniques of co-feeding mixed carbon sources. The effects of various automaticfeeding strategies of carbon/nitrogen sources and impeller combinations on CPC fermentationperformance were explored and compared. Metabolic analysis under different feedingmanners of soybean and mixed carbon sources was also conducted to find out the reasons ofenhanced CPC fermentation performance achieved under the optimal feeding conditions. Themajor results of this dissertation were summarized as follows:（1） A novel and automatic （NH₄）₂SO₄feeding strategy which coupled （NH₄）₂SO₄supplementwith soybean oil addition was proposed. The results indicated that the （NH₄）₂SO₄-soybeanoil coupled feeding strategy （mass ratio of soybean oil versus （NH₄）₂SO₄about1:0.17）could control NH4+-N concentration in a range of3-5g/L throughout the fermentations,which satisfied the requirements of cell growth/CPC synthesis on N-/S-sources andaccelerated mycelia differentiation. This strategy laid a platform or prerequisite forefficient CPC production during its major production phase.（2） On the basis of the proposed （NH₄）₂SO₄-soybean oil coupled feeding strategy, CPCfermentations during the major CPC production phase using different soybean oil feedingmethods including intermittent, constant rate and DO-Stat based feeding were conductedand their performance was compared. The method of adopting DO-Stat for soybean oilfeeding with oxygen-enriched-air for aeration could conduct CPC fermentations in a wayof higher CPC but lower DAOC accumulation. In this case, final CPC concentration andCPC yield reached the levels of35.77g/L and13.3%, respectively. DAOC accumulationand DAOC/CPC ratio ended at lower levels of0.178g/L and0.5%, respectively.（3） CFD calculation technique was used for simulation and optimization of the suitableimpeller numbers and combinations for CPC production. CPC fermentation is anextremely high oxygen consuming process, the impeller numbers/combinations directlyaffects oxygen mass transfer/mixng effects in the broth. C.acremonium is sensitive toshear rate. Extrassively high shear rate causes irreservable damage on cells and mycelia breakage at improper/early stage, and severely deteriorates cell growth/CPC synthesis.The optimal impeller combination/numbers/mounting positions for CPC fermentationwere determined on the basis of CFD simulation/calculation results, which were impellernumber of2; impeller combination of a bottem six-bladed turbine plus a upperfour-pitched-blade turbine; mounting distance of the bottom turbine and tank bottom of57mm; and mounting distance of the two turbines of97mm. The optimal impellers wereapplied for CPC fermentation experiments for verification. The experimental resultsindicated that the entire fermentation performance was largely enhanced when adoptingthe optimized impellers, CPC concentration increased by52%and DAOC accumulationdecreased by71%as compared with those of using the standard impellers.（4） When automatically feeding mixed carbon sources consisting of soybean oil and glucose,glucose concentration could be controlled at very low level and CPC synthesis repressiondue to excessive glucosed did not occur. When using glucose as an assistant carbon source,cellular activity could be maintained or even enhanced as glucose was quickly utilized bythe cells; soybean oil feeding and CPC synthesis rates were increased as O2mass transferrate in broth was indirectly improved and “platform” effect in DO control was eliminated.Under the conditions of adopting the novel mixed carbon sources feeding strategy andstandard impellers, aerating air for oxygen supply throughout the fermentation, maximalCPC concentration reached a higher level of36.99g/L; CPC yield on expensive soybeanoil increased up to25.14%; DAOC/CPC ratio stayed at very low level of0.28%qualifying industrial standard. With the new mixed carbon sources feeding strategy,carbon fluxes towards to CPC synthesis route was improved; CPC yield was significantlyenhanced; accumulation of by-metabolite was largely relieved; and quality of the CPCproduct was ensured, while CPC productivity was maintained at compabably high level.（5） During CPC major production phase, metabolic analysis under various soybean oilfeeding conditions was conducted. The results revealed that, when using DO-Stat basedfeeding method for supplementing soybean oil, more carbon fluxes could be directed intothe formation of CPC synthesis precursors; extracellular methionine could be directlyutilized to run the entire CPC biosynthesis in a more efficient way; carbon fluxesdistribution in TCA could be slightly weakened. Under this condition, CO2releasedecreased and CPC yield increased.（6） A novel soybean oil feeding strategy, namely, a combined strategy of constant rate feeding+DO-Stat feeding which absorbed the advantages of both was proposed and conducted in50L scaled-up fermentors. This strategy increased rates of soybean oil feeding and CPCsynthesis, and stably controlled DO in a range of20%-40%. In this case, CPC yieldsignificantly increased, DAOC accumulation decreased, and DAOC/CPC ratio reduced toa lower level of0.46%qualifying the industrial standard, as compared with the results ofusing the original feeding method.