| Poly malic acid(PMA)produced by yeast-like fungus Aureobasidium pullulans is a polyester polymer that is polymerized by L-malic acid as the sole monomer,which has a lot of characteristics,such as water-soluble,biodegradable,easily-modified,and non-immunogenic.PMA can be chemically modified,and has a wide range of applications including biodegradable material,drug carrier,tissue repair etc.Currently,the synthesis of PMA is mainly composed of two methods: chemical synthesis and biological fermentation.Chemical synthesis has many disadvantages,such as the low molecular weight of PMA,separation and purification is difficult,the reaction conditions are harsh.However,PMA was synthesized by microbial fermentation are pure,large molecular weight,and no environmental pollution.Therefore,it has more advantages to obtain PMA by microbial fermentation.Currently,the production and the yield of PMA can not meet the needs of industrial production.So we use Aureobasidium pullulans CCTCC M2012223 as the starting strain,to carry out research on high throughput screening and fermentation characteristics of strains.The main contents are as follows:1.An easily synthesizable rhodamine-based fluorescent probe R was designed and characterized.Fluorescence intensity showed a good linearity(Y=?1555.6X+6837.2 R2 = 0.9744),with a pH value ranging from 2.4 to 4.4,covering the change of pH values during the PMA fermentation process.It was applied to a novel strategy integrating the probe R and ARTP for high-throughput screening of high-titer PMA mutants.Among them,we found that the mutant AH-21,with a fluorescence intensity that reached 181.14±17.82,28.57% higher than the one of the wild type strain,has a maximal capacity to produce PMA.This finding was further confirmed with shake-flask fermentation.Compared with the wild type,the growth rate of AH-21 cells was faster,but there was no significant difference in glucose consumption rate in a 3-L fermentor;the titer of PMA reached 41.9g/L at 76 h,and the titer increased by 13.8% under the same culture condition.Fed-batch fermentation was performed in a 30-L fermentor,the titer and production rate of PMA were 128.2 g/L and 0.71g/L h,and the yield was 0.51 g/g glucose.2.The growth of A.pullulans in different culture conditions was studied.With the increase of NH4NO3 concentration,the number of viable cells(capacitance)increased from 2.3pF/cm to 11.9pF/cm,and the difference was significant when seeds were cultured 36 h.4g/L NH4NO3 group had the fastest growth rate,from the initial 8.0pF/cm to 11.9 pF/cm.There was no significant difference in production of PMA under high nitrogen(4 g/L and 8g/L NH4NO3),but the production was increased by compared with that of medium nitrogen(2g/L NH4NO3).Therefore,the best seed culture conditions is that incubation time and concentration of NH4NO3 were 72 h and 4g/L.Further investigated in a 5L fermentor,the results are as follows.Compared to control group(2 g/L NH4NO3),the titer of PMA in optimized group(4 g/L NH4NO3)was increased by 8.9%;conductivity increased by 10.2%.On line detection of the fermentation process of 5L fermentor by biomass sensor.The fc value which is inversely proportional to the cell radius decreases rapidly at the cell growth stage,which indicates that the cell radius increases gradually;This prediction was confirmed by microscopic observation of cell morphology.At the late stage of fermentation(25-66h),PMA and conductivity were fitted linearly.The results showed that there was a good linear relationship between PMA content and conductivity,y=0.0649x+3.5799,R2=0.9895.The optimized seeds were inoculated in the 30 L fermentor for fed-batch fermentation.PMA production reached 147.5 g/L at 156 h,which was 13.8% higher than the original process conditions.The yield was 0.58 g/g glucose,which was 13.7% higher than control.The above results show that AH-21 has a good PMA production capacity and potential for industrial production... |
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