摘要
2,3-丁二醇(2,3-butanediol)是一种非常重要的化工原料,在化工、食品、燃料等多个领域都有广泛的应用。本研究从自然界的土壤中筛选出一株能够以葡萄糖为底物发酵产2,3-丁二醇的枯草芽孢杆菌,进而对该菌株产2,3-丁二醇的摇瓶发酵培养基和发酵条件进行了研究,并在5 L发酵罐上进行了初步放大实验。主要内容和结果如下:
(1)从自然界的土壤中筛选出了能够以葡萄糖为底物发酵产2,3-丁二醇的微生物菌株42株,从中选择了一株2,3-丁二醇产量较高的菌株6-7作为本论文研究的对象。经初步发酵,2,3-丁二醇的产量达到48.7 g/L。经16S rDNA序列分析并结合常规生理生化实验鉴定,确定菌株6-7为枯草芽孢杆菌,命名为Bacillus subtilis 6-7。
(2)在摇瓶水平上考察了碳源、氮源、无机盐和金属离子对Bacillus subtilis 6-7发酵产2,3-丁二醇的影响。经单因素实验筛选,葡萄糖、玉米浆、(NH_4)_2SO_4、K_2HPO_4对菌株发酵产2,3-丁二醇影响较大,通过四因素三水平的正交实验优化了摇瓶发酵培养基,优化后培养基配方为(g/L):葡萄糖150,玉米浆12,(NH_4)_2SO_4 6,K_2HPO_4 10,乙酸钠3,KH_2PO_4 6,柠檬酸钠6,MgSO_4 0.05,ZnSO_4 0.01,FeSO_4 0.05,CaCl_2 0.05,NiCl_2 0.05。
(3)从pH、温度、摇床转速、装液量、接种量几个方面对摇床发酵产2,3-丁二醇的条件进行了研究,得到了摇床条件下最适的发酵条件:初始pH为6.5,温度37℃,摇床转速为160 r/min,装液量为60 mL/250 mL,接种量为5%(V/V),在此条件下发酵,2,3-丁二醇的产量可达到55.2 g/L,较初始产量提高了13.3%。
(4)在5 L发酵罐上对Bacillus subtilis 6-7发酵产2,3-丁二醇进行了初步研究,考察了搅拌速度、通气量和pH值对发酵罐分批发酵的影响,确定了发酵罐的最适发酵条件,搅拌转速为300 r/min、通气量为1 L/min、控制pH为6.5时,2,3-丁二醇产量最大可达56.3 g/L。为降低底物葡萄糖的抑制和提高2,3-丁二醇产量,对5 L发酵罐补料分批发酵进行了初步研究,最终测得2,3-丁二醇产量为72.6 g/L。
2,3-butanediol (2,3-BDO) is one of the important chemical intermediate, which can be used in many fields such as food, chemistry, fuel. A Bacillus sublitis strain producing 2,3-BDO from glucose as substrate was isolated from different samples. The fermentation medium and fermentation conditions were investigated. On the baisis of medium and fermentation conditons optimization, preliminary test was performed in 5 L fermentor. The main research contents and results are follows:
(1) 42 strains capable of producing 2,3-BDO from glucose were isolated from different natural environment samples. The strain 6-7 which could grow on medium containing glucose had a higher 2,3-BDO converting capability. After preliminary fermentation test, the concentration of 2,3-BDO reached 48.7 g/L. In addition to general morphaological and biochemical characteristics, the strain 6-7 was identified by 16S rDNA sequence and systematic analysis. The results showed that 16S rDNA sequence of strain 6-7 had similarity of 99% with Bacillus subtilis strain BIHB332, suggesting that the strain 6-7 is one of subspecied of Bacillus subtilis.
(2) Effect of carbon source, nitrogen source, mineral salt and metal ion on 2,3-BDO production of Bacillus subtilis 6-7 were investigated in the shaking-flask. Glucose, corn steep liquor, (NH_4)_2SO_4, K_2HPO_4 were the siginificant factors based on the results of single factor experiments. The optimum fermentation medium compositions were determined as follows(g/L): glucose 150; corn steap liquor 10; (NH_4)_2SO_4 6; CH3COONa 3; KH_2PO_4 6; K_2HPO_4 12; citrate sodium 6.0; MgSO_4 0.05; ZnSO_4 0.01; FeSO_4 0.05; CaCl_2 0.05; NiCl_2 0.05.
(3) Fermentation conditons of Bacillus subtilis 6-7 were optimized in the shaking-flask from following aspects: pH, temperature, rotation speed and so on. The best fermentation conditions were described as: initial pH 6.5, temperature37℃, the amount of inoculation 5%(V/V), the liquid volume 60 mL(in 250 mL, flask), rotation speed 160 rpm. Under optimum conditions, afer the strain 6-7 was cultured 72 hours, it produced 55.2 g/L 2,3-BDO, which increased about 13.3% more than before.
(4) In order to increase the 2,3-BDO production in 5 L fermentor, effect of pH, stirrer speed and air flow on the batch fermentation were studied. The results showed that high 2,3-BDO accumulation was acheieved at the condition follow as: pH 6.5, stirrer speed 300 r/min, air flow 1 L/min. To reduce substrate inhibition and increase 2,3-butanediol yield, the fed-batch fermentation in 5 L fermentor was studied, the final 2,3-butanediol production was 72.6 g/L.
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