| As a multi-functional polyol salt,sodium gluconate(SG)has been widely used in food,pharmaceuticals,detergents,construction and other industries.In this thesis,submerged fermentation process for SG production using Aspergillus niger(A.niger)was studied.Based on the physiological metabolism of A.niger and the characteristics of the extracellular enzyme reaction for SG synthesis,SG production process was greatly improved and the effect of oxygen transfer on SG production was analysed using hybrid technologies of high-throughput screening,computational fluid dynamics(CFD)simulation,multi-scale parameters association analysis,metabolic flux analysis and so on.Then,an automatic cell-recycle continuous fermentation(CRCF)with a feedback control of feed rate for SG production was prposed and established by integrative analysis of the on-line physiological parameters(oxygen uptake rate;OUR,carbon dioxide evolution rate;CER,dissolved oxygen;DO and respiratory quotient;RQ)to regulate feed rate for the first time and was successfully applied to industrial SG production.The detailed research results were listed as follows:(1)In this study,a novel high-throughput screening method was established using NaNO3 and CaCO3 as selective pressure in agar plate and bromocresol green pH indicator as a fast screening indicating reagent in the 48-well microplate liquid cultivation.And a high-yield strain of SG(A.niger IV-7-C6)was screened out sucessfully from the original strain A.niger FY149.Compared to the original strain,production rate and yield of SG were increased by 19.5%and 5.8%,respectively.Improvement of the oxygen affinity of the A.niger mutant might be the reason for the higher productivity and yield.In order to better undstand the relationship between physiological metabolism and the product synthesis of the high-producing strain,kinetic models of cell growth,glucose consumption and SG production were constructed.Finally,effects of different osmolality caused by the initial glucose concentration on cell grwth and SG production were studied and the initial glucose concentration of 250.0 g L-1 was proved to be suitable in seed cultivation.Cell growth had no siginificant change under this glucose concentration,while,more glucose oxidase was induced to the synthesis for SG production.(2)In order to realize the online optimization of SG fermentation process,equations which can calculate real-time key-parameters during SG fermentation were established for the first time by on-line physiological data(OUR,CER and RQ)(Eq.1 and 2).Briefly,three rules can be established for SG production i.e.CER can be used to characterize cell respiration because carbon dioxide is not generated during the enzymatic production of GA;higher OUR2 meaning higher SG generation rate;Lower RQ meaning higher yield coefficient.Based on these results,industrial SG production was scaled-down to 50-L fermentor to figure out the limiting factors(fermentation medium recipe and oxygen transfer rate)in the initial batch fermentation process for SG production.Then,fermentation medium formula and agitation mode in batch fermentation were improved.After that,fermentation time of the improved batch fermentation was shortened from 34.0 h to 19.0 h and the overall yield of SG(0.943±0.012 mol mol-1)was increased by 16.0%compared to the initial batch fermentation.It was found that modification of fermentation medium recipe greatly shortened the lag-phase of batch fermentation and reduced the cell concentration to an optimal concentration(reduced from original 5.0 g L-1 to the present 1.5 g L-1).Improvement of agitation improved the oxygen transfer rate(OTR)level during the fermentation process.Actually,OTR in bioreactor is always the limiting factor for the catalytic reaction in presence of sufficient concentrations of glucose in the medium;therefore,any method that improves the OTR can enhance SG production.Compared to improved fermentation,higher metabolic flux of EMP and TCA cycle were occurred in the initial fermentation,indicating more glucose was used for cell growth and maintenance in the initial fermentation.Besides,lowest flux for citric acid(main by-product)biosynthesis was also obtained in the improved fermentation.Less glucose used for cell growth and maintenance and less formed by-product were the main reasons of the higher yield in the improved fermentation.Where rp(g L-1 h-1)and Yp(mol mol-1)are the real-time rate of SG production and yield of SG,respectively.(3)Viscosity of fermentation broth will be significantly increased when the mycelial morphology changes from pellet to dispersed form under high biomass concentration and this in turn negatively affects nutrient transport in the fermentation broth.Therefore,to avoid high viscosity,the pelleted mycelia are preferred in the SG fermentations.Although rheological problems can be alleviated by using mycelial pellets,the center of large pellets sometimes lacks enough oxygen supply,leading to autolysis of the fungus,which further negatively affects fermentation.While,the low biomass which had been achieved in the improved batch fermentation and high demand of oxygen in submerged fermentation of SG using A.niger may generate an optimum morphology of mycelia.Thus,SG fermentation with dispersed mycelia morphology was studied in this study.The fermentation results showed that conversion of mycelial morphology from pellets to dispersed state had negligible impact on viscosity of fermentation broth at low biomass concentration(1.7 g L-1),while coefficient of volumetric oxygen transfer(kLa)improved more than 13%,suggesting that the promotion of oxygen transfer rate(OTR)between liquid medium and mycelia greatly increased SG production.Fermentation time was shortened from 19.0 h to 15.0 h with a higher yield.(4)Based on the batch fermentation,a system of cell-recycle membrane continuous fermentor(CRMF)was established for continuous production of SG for the first time in this study using a ceramic membrane module to separate microbial cells from fermentation broth.Then,initial cell-recylce continuous fermentation(CRCF)with a constant feed rate was conducted.In the initial continuous fermentation,varieties of OTR in the bioreactor were found out using CFD technology.Additional power input due to the pump of the membrane module and increase of foam improved OTR level,while,addition of defomers significantly decreased OTR in the bioreactor.Feed rate of fresh medium should be regulated based on the OTR level during continuous fermentation to reach the highest reaction rate for SG production with the lowest residual concentration of glucose.Therefore,an automatic control scheme of feed rate by the on-line key parameters(OUR and DO)was proposed in the improved continuous fermentation by this CRMF system.Overall productivity and yiled of SG(0.984 mol mol-1),which was closed to the theoretical yield(1.0 mol mol-1),in the improved CRCF(240.0 h)using this automatic control method of feed rate were increased more than three-fold and 5.4%,respectively,compared to the improved batch fermentation.Additional power input and more dispersed mycelia morphology improved the OTR level in CRCF compared to batch fermentation.Less glucose used for cell growth due to the cell-ecyle process and less by-product(citric acid)formed were the main reasons of the higher yield in the optimized process.Finally,SG production using the commercial GOD solution was studied and a novel approach of stepwise addition of GOD solution for SG production by on-line physiological parameters association analysis was proposed in this research.Fermentation time of improved enzyme reaction(GOD and CAT)for SG production had a 5.0 h decrease compared the the initial reaction,and the total GOD solution amount used in the reaction was also decreasd by 19.4%.After the systematic optimization of fermentation process and establishment of cell-recycle continuous production system for SG production,more than 3.0-fold increase of overall productivity of SG was obtained compared to initial batch fermentation.Furthermore,the yield of SG also reached 0.98 mol mol-1,which is the highest yield reported in the literatures up to now.Production cost reduced about 50.0%after adopting these optimization procedure and the competitiveness of SG production enterprise was greatly improved. |
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