| Pullulan,an important microbial exopolysaccharide,is usually produced under aerobic conditions by a yeast-like fungi of Aureobasidium pullulans.Owing to its unique chemical structure,this kind of water-soluble polymer has good plasticity,film formation ability and chemical stability,hence,pullulan can be safely used in food,medicine,environmental protection,light industry and other areas.Up to date,microbial fermentation using A.pullulans as the producer is the main approach for pullulan production.Many researches had focused on how to improve the titer and molecular weight(Mw)of pullulan,however,the study on the physiological and biochemical mechanisms within the batch pullulan production is just at the beginning.In this dissertation,a non-pigment producing strain of A.pullulans CCTCC M 2012259 was used as the starting strain for pullulan production.In order to obtaining a high titer and/or high Mw of pullulan,as well as decreasing the cost of material,a series of optimization strategies were applied during batch pullulan fermentation.The nutrimental and environmental conditions which had significant influence on batch pullulan fermentation were investigated,the physiological and biochemical mechanisms of efficient pullulan production were explored,and the economic analysis of the batch process for pullulan production was also carried out.The main results of this study are as follows:1.The effects of different concentrations of ammonium sulfate and yeast extract on pullulan production were investigated,and limited concentration of either or both organic nitrogen sources were found to be favorable to the improvement of pullulan production.When the concentration of ammonium sulfate and yeast extract were 0.3 g/L and 1.5 g/L,respectively,the maximum pullulan concentration of 25.3 g/L was obtained at 66 h,which was increase by 14.0%as compared to that with sufficient nitrogen source.The physiological and biochemical data showed that nitrogen limitation increased the activities of phosphoglucomutase(PGM)and glucosyltransferase(FKS),up-regulated the transcriptional levels of pgml and fks genes,improved the intracellular ATP content and ATP/ADP ratio,all of which provided sufficient material and energy for the overproduction of pullulan.Moreover,the strategy of nitrogen limitation and carbon supplement was applied in batch pullulan production,that is,glucose was fed at 36 h to a total concentration of 70 g/L.As a result,pullulan production of 39.8 g/L was achieved at 72 h,which was increased by 16.0%compared to that of the control with glucose feeding but without nitrogen limitation.The economic analysis of the batch process for pullulan fermentation indicated that the cost of raw material for pullulan production under nitrogen-limiting conditions was significantly decreased.2.Batch culture of A.pullulans CCTCC M 2012259 for pullulan production was carried out at various agitation speeds ranging from 200 to 600 rpm in a 5-L stirred tank fermentor,it was shown that the dissolved oxygen(DO)level at 500 rpm was beneficial to cell growth and maximized the Mw of pullulan,while the DO at 400 rpm favored the yield and final Mw of pullulan.Based on kinetic analysis of batch processes,activity assays of the enzymes associated with pullulan biosynthesis(FKS)and biodegradation,and intracellular ATP level as well as ATP/ADP ratio under different constant agitation speeds,a two-stage control strategy that shifted the agitation speed from 500 to 400 rpm at 18 h was proposed to simultaneously achieve high yield and high Mw of pullulan.A maximum pullulan concentration of 27.4 g/L and a final Mw of 3.09x 106 Da were obtained using the two-stage agitation speed control strategy,which were both greatly improved compared to those obtained at constant agitation speeds.The titer of pullulan under the two-stage strategy was increased by 13.7%and 17.6%compared to that obtained at constant agitation speeds of 400 rpm and 500 rpm,respectively.3.The influence of NaCl on the batch culture of A.pullulans CCTCC M 2012259 and pullulan production was investigated,it was illustrated that the addition of NaCl to the fermentation medium improved the titer of pullulan but decreased the Mw of pullulan.When NaCl concentration was 3 g/L,the maximum pullulan production was 28.9 g/L,which was increased by 26.7%compared to the control without NaCl supplementation,but the Mw of pullulan obtained at 72 h was only 47.0%that of the control.The assay of key enzymes related to pullulan biosynthesis and biodegradation were carried out,it was found that NaCl increased FKS activity during the early stage of batch fermentation,as well as improved pullulan degradation activity during the late phase of batch process.Furthermore,the transcriptome samples for cells of A.pullulans CCTCC M 2012259 derived from 0 g/L and 3 g/L NaCl conditions were sequenced and analyzed.The results illustrated that when NaCl concentration was 3 g/L,the transcriptional levels of 77 genes are up-regulated(including pgml and fks genes),while only 2 genes are down-regulated.The GO functional annotation and KEGG pathway annotation of these differentially expressed genes were analyzed using bioinformatics approaches,and NaCl addition was found to increased the expression of related genes of pullulan biosynthesis,secretion,hydrolysis and energy generation.All the above analysis partly explain the internal reason why NaCl improved pullulan biosynthesis but decreased the Mw of pullulan.4.Pullulan production by A.pullulans CCTCC M 2012259 using rice hull hydrolysate as the carbon source was conducted in flasks,the titer of pullulan was significantly lower than that with the sole carbon source of glucose or xylose.Based on the HPLC analysis of hydrolysate of rice hull with different pretreatment methods,as well as the verification experiment in flasks,acetic acid in the hydrolysate was demonstrated to exert a negative effect on pullulan production and proven to be the major factor to inhibit pullulan biosynthesis.In order to reduce the sensitivity of enzymes related to pullulan biosynthesis to acetic acid,the adaptive laboratory evolution of the parental strain on medium containing increasing concentration of hydrolysate of untreated rice hull was conducted.Instead of employing expensive methods to remove acetic acid from the hydrolysate,a mutant A.pullulans ARH-1 was isolated following 20 cycles of adaptive evolution.The maximum pullulan production achieved by the adapted mutant at 48 h using the hydrolysate of untreated rice hull was 22.2 g/L,while that obtained by the parental strain at 60 h was 15.6 g/L,which indicated that the mutant had a higher pullulan productivity than the parental strain.The assay of key enzymes associated with pullulan biosynthesis revealed that the inhibition of acetic acid was relieved in the mutant.5.Pullulan production using corn steep liquor(CSL)and soybean meal hydrolysate as organic nitrogen source was carried out in flasks.Based on the feasibility of both food wastes for pullulan production,response surface analysis was used to optimize the proportion of ammonium sulfate to CSL and soybean meal hydrolysate,respectively.For the sake of obtaining a high pullulan yield,the optimal combination of nitrogen sources was obtained as 1.28 g/L of ammonium sulfate and 4.68 g/L of CSL,as well as 1.25 g/L of ammonium sulfate and 12.41 mL/L of soybean meal hydrolysate,respectively.In addition,batch culture of A.pullulans CCTCC M 2012259 using the optimized combination of nitrogen sources was performed in a 5 L stirred fermentor,and the batch process using the complex nitrogen source of 0.6 g/L of ammonium sulfate and 3.0 g/L of yeast extract was designated as the control.It was shown that the Mw of pullulan derived from CSL was almost the same as that from yeast extract,while the titer of pullulan decreased by 14%;the production of pullulan derived from soybean meal hydrolysate was similar to that from yeast extract,but the Mw was much lower than that of the control.The economic analysis of the raw materials revealed that the cost of nitrogen sources in the fermentation medium for pullulan production greatly decreased when using the food wastes.The nitrogen cost of pullulan production using CSL and soybean meal hydrolysate is only 18.5%and 56.1%of that using yeast extract,respectively.The chemical structure characterization of the microbial polysaccharide produced by A.pullulans CCTCC M 2012259 with both alternative organic nitrogen sources was conducted,the peaks in FTIR spectra indicated that the polysaccharide is pullulan. |
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