| Currently, the researches on microbial fermentation process are getting more and more profound, which has been developed from the extracellular to intracellular analysis, from macrocosmic to microcosmic metabolism of the microbes. Therefore, an industrial systems bioprocess engineering by integrating the multi scale physiological metabolism information is more essential for elevating the power of the domestic industrial fermentation. In the present work, the methods for determining and analyzing the macro-and micro-metabolic information were established in the industrial vitamin B12 fermentation by Pseudomonas denitrificans. Vitamin B12 has become one of the most important industrial materials for its well utilization in medicine and nutrition; more and more researches were focused on the improvement of the productivity and the optimization of the fermentation process controlling strategy. Integrated with the systematical analysis of the macro and micro metabolism information, the effects and the controlling strategies of the betaine, oxygen supply, and CO2 on the vitamin B12 fermentation and scale-up were investigated in this thesis.The structurally conserved and ubiquitous pathways of central carbon metabolism were identified and quantified in vitamin B12 producing strain P. denitrificans. The protocol for fast sampling and the principles underlying the calculation of metabolic flux ratios from the mass spectra of amino acids were developed. Furthermore, a mini-scale sensor reactor system was constructed for the 13C-labeling experiments, which has only 200 ml volume and all the online parameters reflecting the physiological characters (like pH, dissolved oxygen, OUR, CER, RQ, etc.) can be monitored and controlled. And also the labeled 45CO2 could be well determined. The central carbon metabolism in vitamin B12 producing strain P. denitrificans were identified and quantified with labeling experiments based on the [1-13C] and [U-13C] glucose. The results demonstrated that glucose was mainly consumed along the Entner-Doudoroff pathway, the pentose phosphate pathway, and the EMP pathway was inactive. Although the main result was identical to other reported Pseudomonas, but there was a high pentose phosphate pathway and no malic enzyme activity in this strain. The C1 metabolism associated with the serine, glycine and betaine was also illustrated in detail. The mechanism and the optimal controlling strategy of betaine were constructed in vitamin B12 production based on the [U-13C] glucose experiments. The fate of betaine and the metabolic fluxes analysis were illustrated under 13C-constrained flux balancing analysis, complemented with stoichiometric relations and measured extracellular rates. Fluxes in central metabolism of exponentially growing P. denitrificans revealed that most of the betaine was disassembled for cell growth, only small amount of it was served as methyl group donor for vitamin B12 biosynthesis. Meanwhile the betaine also had the function for enhancement of the respiration and maintaining the constant pH in the fermentation process. Furthermore, the feeding strategy of the betaine was optimized combined with OUR. The optimal betaine supplementation time began just at the moment of the decreasing point of OUR, which would not only accelerate vitamin B12 production rate and decrease the consumption of the betaine significantly (about 58.7% lower than that of control), but also avoid the inhibition of high betaine concentration on cell growth, and thus a cost-effective industrial vitamin B12 fermentation technology was achieved.Integrated with the macro and micro metabolic parameters analysis, a novel oxygen supply controlling strategy based on oxygen uptake rates (OUR) was achieved for cost effective vitamin B12 production. The physiological metabolic characters of P. denitrificans on vitamin B12 fermentation were investigated under fed-batch and continuous cultivations, a high affinity to oxygen was demonstrated in the strain, and oxygen supply played an important role on vitamin B12 production. Therefore, the effects of different oxygen transfer rates (OTR) on the cell growth and vitamin B12 biosynthesis of P. denitrificans were investigated under dissolved oxygen limiting conditions. The results demonstrated that high OTR accelerated cell growth and initial vitamin B12 biosynthesis rate, while lower OTR was critical for higher productivity in the late fermentation process. The OUR corresponded well with OTR. Based on the metabolic intermediates analysis and the metabolic flux analysis, a step-wise OUR control strategy was proposed. The strategy was successfully implemented in scale-up to an industrial fermenter (120 m3). A stable maximum vitamin B12 production of 208±2.5 mg/1 was achieved, which was increased by 17.3 percent compared with the control. Furthermore, the glucose consumption coefficient to vitamin B12 was 34.4 percent lower than that of the control. An efficient and economical fermentation process based on OUR criterion was established for industrial vitamin B12 fermentation by P. denitrificans.Metabolic flux analysis using 13C-labeled substrates is a well-developed method for investigating cellular behavior in steady state culture condition. To extend its application, in particular to typical industrial conditions, such as batch and fed-batch cultivations, a novel method of 13C metabolic flux analysis based on intracellular free amino acids was proposed. Metabolic flux distributions were determined in both exponential and stationary phases. Using this new approach, a culture phase-dependent metabolic shift on anaplerotic reaction catalyzed by pyruvate carboxylase was detected in the industrial fed-batch fermentation. The approach presented here has great potential for investigating cellular behavior in industrial processes. Considering the importance of the CO2 on the anaplerotic reaction, the effect of increased levels of dissolved CO2 on fed-batch cultures of P. denitrificans was investigated by means of continuous gassing with inlet gas mixtures containing various ranges of CO2 concentration. Combined with the systematical analysis of the macro and micro metabolism information, an optimal CO2 controlling strategy through adjustment of the agitation and aeration strategy was proposed, with which the highest vitamin B12production of 235.4 mg/1 was obtained, which was 32.9% higher than that of the control (177.1 mg/1) before the optimization.Overall, detecting and integrating the macro and micro metabolism information of industrial microorganisms are feasible approach for the optimization of fermentation process. This strategy could also be well utilized for better understanding the mechanism behind the fermentation phenomenon and revealing the synergistic effects of genetic modifications and environment parameters on cell metabolism, thus the better industrial fermentation process optimization could be achieved.
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