利用味精废水培养枯草芽孢杆菌产γ-聚谷氨酸及初步表征

Cultivation of Bacillus Subtilis with Monosodium Glutamate Wastewater to Produce γ-Polyglutamic Acid and Preliminary Characterization

味精废水是一种高浓度有机废水,具有酸性强、高COD、高BOD、高硫酸根、高菌体含量和低温等特点,其中含有大量的L-谷氨酸、还原糖与氨氮,如果任其排放不仅浪费了宝贵资源,而且会造成严重的环境污染。实验利用味精废水作为培养基,培养枯草芽孢杆菌168得到产物γ-聚谷氨酸,对于探索味精废水的资源化利用途径和降低微生物法合成γ-聚谷氨酸的生产成本具有积极意义。单因素实验研究了培养条件对枯草芽孢杆菌168生长和γ-聚谷氨酸产量的影响,结果表明,味精废水浓度、初始pH、接种量和培养时间对枯草芽孢杆菌168生长和γ-PGA产量具有重要的影响。通过Box-Behnken响应面法进一步优化了枯草芽孢杆菌168的培养条件,优化后的培养条件为:味精废水稀释倍数3.88,培养初始pH 5.84,50 mL味精废水接种量10.55 mL,在37℃、180 r·min^(-1)条件下培养48 h,γ-聚谷氨酸的产量达到(53.51±0.92) g?L^(-1)。茚三酮比色法确定粗产品中γ-聚谷氨酸含量为78.24%(质量分数)。紫外扫描光谱分析显示检测样品中氨基和羧基的特点符合γ-PGA标准图谱特征,γ-聚谷氨酸的聚合键与蛋白质的肽键结构有明显区别,γ-聚谷氨酸是由γ-酰胺键聚合而成。傅立叶红外光谱和核磁共振分析显示,样品中含有酰胺基、羧基、羰基-CH、-CH_2等基团。对紫外光谱、红外光谱和核磁共振结果进行综合分析,可以初步确定样品中存在酰胺键连接的多聚体结构,即γ-聚谷氨酸的结构。研究结果显示,利用味精废水培养枯草芽孢杆菌168得到产物γ-聚谷氨酸在技术上是可行的,为味精废水资源化利用探索出一条可能的途径,且具有降低微生物法合成γ-聚谷氨酸成本的潜力。

As a kind of high-concentration organic wastewater,monosodium glutamate wastewater(MSGW)features strong acidity,high COD,high BOD,high sulfate radical,high bacterial content and low temperature,and it contains a large amount of L-glutamic acid,reducing sugar and ammonia nitrogen.If it is discharged without limit,not only valuable resources will be wasted,but also serious environmental pollution can be caused.This study focused on the cultivation of Bacillus subtilis 168 with MSGW to produceγ-polyglutamic acid(γ-PGA),which was of positive significance to explore the resource utilization channels of MSGW and reduce the production cost of synthesizingγ-PGA using the microbial method.The effects of cultivation conditions on Bacillus subtilis 168 growth andγ-PGA production were investigated by single-factor experiments,the results of which showed that MSGW concentration,initial pH of culture solution,inoculation concentration and cultivation time had an important influence on Bacillus subtilis 168 growth andγ-PGA production.The optimal cultivation conditions for the production ofγ-PGA by Bacillus subtilis 168 were obtained through Box-Behnken Design(BBD).Optimal fermentation conditions were determined based on results of experiments:Dilution fold of MSGW 3.88,initial culture pH 5.84,inoculation volume 10.55 mL for 50 mL diluted MSGW,and the cultivation time 48 h.Productivity ofγ-PGA reached up to(53.51±0.92)g·L^-1 when B.subtilis 168 was cultivated under the optimal conditions.The mass percent ofγ-PGA in crude product was measured 78.24%through ninhydrin colorimetry.According to the UV scanning spectrum analysis,the characteristics of the amino and carboxyl in the sample were in accordance with the characteristics of theγ-PGA standard map.The polymerization bonds ofγ-PGA and the peptide bond structure of the protein were significantly different,forγ-PGA was polymerized byγ-amide bon.Fourier infrared spectroscopy and nuclear magnetic resonance analysis showed that the sample contained amide,carboxyl,carbonyl-CH and carbonyl-CH2.As determined by the comprehensive analysis of the ultraviolet spectrum,infrared spectrum and nuclear magnetic resonance results,there was a polymer structure connected by amide bonds in the sample,i.e.theγ-PGA structure.The results show that it is technically feasible to cultivate B.subtilis 168 with MSGW to produceγ-PGA.As a possible approach explored for the utilization of MSGW,it has the potential to reduce the production cost of synthesizingγ-PGA using the microbial method.