Study On 2-keto-l-gulonic Acid Fermentation Process Based On The Analysis Of Physiological Characteristics

This thesis aimed to further enhance 2-keto-L-gulonic acid (2-KLG) production efficiency using the mixed culture of Ketogulonicigenium vulgaer and Bacillus megaterium. For this, the cell growth and metabolic characteristics of the mixed strains under different environmental (hyperosmotic stress) and nutritional (the addition of amino acids) conditions were carefully investigated, and those optimum fermentation process parameters were industrial developed in 1 m3 and 200 m3 fermentor. The main results were described as follows:(1) Based on the time course of osmolality during 2-KLG industrial scale fermentation and effects of osmolality on cells growth and 2-KLG production, a novel strategy for enhancing K. vulgare growth and 2-KLG production by improving B. megaterium growth with sucrose was developed. Results showed that the accumulation of 2-KLG and the feeding of alkaline matter led to an osmolality rise of 832 mOsmol/kg in the culture broth. High osmotic stress (1250 mOsmol/kg) made the growth of B. megaterium and K. vulgare decreased 15.4% and 31.7%, respectively, and consequently the titer and productivity of 2-KLG reduced 67.5% and 69.3%, respectively. When supplemented sucrose under high osmotic condition (1 250 mOsmol/kg), B. megaterium growth was improved significantly, with the result that 2-KLG production was increased 87%. Furthermore, by applying this sucrose addition strategy further to batch fermentation in 31 fermentor, the productivity of 2-KLG increased 10.4%, and the duration of fermentation declined 10.8%.(2) By combination of systems biology strategies (the complete genome sequence analysis and the proteomics) and biochemical engineering strategy (effects of CSL compositions on 2-KLG production), the key amino acids that affected 2-KLG production mostly were determined. The results shown that proteins (enzymes) contributed to K. vulgare de novo biosynthesis of L-serine, L-threonine, L-tryptophan, L-methionine, L-isoleucine, L-valine, L-phenylalanine and L-glutamine were up-regulated when co-cultured with the lysate of B. megaterium. Genome analysis shown that K. vulgare lacked of the complete biosynthesis pathways for L-histidine, L-glycine, L-lysine, L-threonine, L-methionine, L-leucine and L-isoleucine. When supplementation of CSL with the key amino acids L-glycine, L-proline and L-threonine, the fermentation time decreased to 58,62 and 60 h, respectively, as consequently,2-KLG productivity reached to 1.12±0.02 (20.4% increase), 1.04+0.01 (11.8% increase) and 1.09±0.02 g/l/h (17.2% increase), respectively.(3) A strategy for enhancing 2-KLG production efficiency with gelatin addition was proposed based on the fact that gelatin was rich in the two key amino acids (L-glycine and L-proline). Composition analysis of gelatin shown that the total content of L-glycine and L-proline was accounted for 34.21% of dry weight. When supplemented with 0.8 g/1 gelatin to the fermentation broth in 1 m3 fermentor, the fermentation period decreased to 43 h (decreased 15.6%), and 2-KLG productivity reached at 1.89 g/l/h (increased 25.2%). This result was expand to 200 m3 airlift fermentor, and 2-KLG titer achieved to 102.28 g/1, the fermentation time was shortened by 8%(5 h), and 2-KLG productivity increased from 1.70 g/l/h to 1.86g/l/h.