L-酪氨酸连续发酵工艺研究

Study on Continuous Fermentation Technology of L-Tyrosine

当前工业上用微生物发酵法生产L-酪氨酸的工艺中,基本都是前期向发酵罐内加入基础培养基,中后期再流加各类营养物质,虽然整个发酵过程中减少了取料和放料的操作,保证发酵中物料不被浪费,但是,随着流加物质的不断增加,发酵液体系不断扩大,该过程在发酵后期需要不断地人为调控发酵参数,而人为调控中,难以避免调控参数波动,并最终影响发酵结果。因此,实验采用高效连续发酵,该工艺可以大大提高菌体活力,并且有效延长产酸高峰期,为了优化L-酪氨酸的高密度连续发酵生产工艺,通过对大肠杆菌TYR-05发酵培养,考察了L-酪氨酸发酵过程并且分析了菌体量、产酸量、产酸效率、糖酸转化率的情况,确定L-酪氨酸高密度连续发酵过程中接种量、糖速率、糖耗量、底糖浓度等关键条件,以达到高密度连续发酵工艺控制条件优化。实验结果表明在5 L发酵罐中,选择30%的种子接种量,发酵起始时,底物氯化胆碱浓度为1 g/L并每4 h向罐内流加0.2 g/L的氯化胆碱,发酵至12 h时,底糖耗尽,开始以12 g/(L·h)的补糖速率向罐内提供葡萄糖,并在此时开始放液,放液速率为0.13 L/h,使得装液量恒定在20%左右,发酵25 h时开始流加复合营养液,发酵35 h时最高菌体OD_(600)达到65,产酸量为55.8 g/L,糖酸转化率为25.4%,为L-酪氨酸连续发酵工业化生产提供了重要参考。

In the current industrial process of producing L-tyrosine by microbial fermentation method,the basic culture medium is generally added into the fermentation tank in the early stage,and then various nutrients are fed in the middle and later stages.Although in the entire fermentation process,the operations of taking and releasing materials are reduced,ensuring that materials are not wasted during the fermentation,with the continuous increase of fed-in materials,the fermentation liquid system continues to expand.The fermentation parameters need to be continuously regulated and controlled artificially in the later stage of fermentation.However,it is difficult to avoid the fluctuation of the regulation parameters in the artificial regulation and control,and eventually affecting the fermentation results.Therefore,efficient continuous fermentation is adopted in this experiment,which can greatly improve the cell vitality and effectively prolong the peak period of acid production.In order to optimize the high-density continuous fermentation production process of L-tyrosine,the fermentation process of L-tyrosine is investigated through the fermentation culture of E.coli TYR-05,and the biomass,acid production amount,acid production efficiency and sugar-acid conversion rate are analyzed.Key conditions such as inoculation amount,sugar rate,sugar consumption amount and base sugar concentration are determined during the high-density continuous fermentation process of L-tyrosine,in order to achieve the optimization of the control conditions for the high-density continuous fermentation process.The experimental results show that in a 5 L fermentation tank,30%seed inoculation amount is selected.At the beginning of fermentation,the substrate choline chloride concentration is 1 g/L,and 0.2 g/L choline chloride is fed into the tank every 4 h.After 12 h of fermentation,the base sugar is depleted,and glucose is supplied into the tank at a sugar supplement rate of 12 g/(L·h).At this time,the liquid is released at a rate of 0.13 L/h to make the liquid volume constant at about 20%.After 25 h of fermentation,compound nutrient liquid is fed in.After 35 h of fermentation,the highest cell OD_(600) reaches 65,the acid production amount is 55.8 g/L,and the sugar-acid conversion rate is 25.4%,which has provided important references for the industrial production of L-tyrosine continuous fermentation.