杨木酶水解液培养枯草芽孢杆菌试验  

The production of Bacillus subtilis by fermentation from enzymatic hydrolysate of poplar

作　　者：游佳欣 应文俊 杨倩倩 廖红 徐勇[1] 张军华[1,2] YOU Jiaxin;YING Wenjun;YANG Qianqian;LIAO Hong;XU Yong;ZHANG Junhua(College of Chemical Engineering,Nanjing Forestry University,Nanjing 210037,China;College of Forestry,Northwest A&F University,Yangling 712100,Shaanxi,China)

机构地区：[1]南京林业大学化学工程学院,南京210037 [2]西北农林科技大学林学院,陕西杨凌712100

出　　处：《林业工程学报》2021年第4期94-100,共7页Journal of Forestry Engineering

基　　金：国家重点研发计划(2017YFD0601001);南京林业大学科研启动基金(163030103)。

摘　　要：为了更好地利用林业废弃物杨木屑,将杨木屑进行预处理和酶水解制备了富含单糖的酶水解液,并将杨木酶水解液作为培养基中的碳源培养枯草芽孢杆菌。首先通过单因素试验优化了培养基组成,研究了培养基中氮源、无机盐和微量元素以及各组分添加量对枯草芽孢杆菌生长的影响。采用软件Design-Expert 12设计响应面优化试验,对培养基组成进一步优化。根据预测的最佳条件进行5次重复试验,确定最佳培养基组成为:3.20%(体积分数)杨木酶水解液、2.30%(质量分数)鱼粉蛋白胨、0.06%(质量分数)K_(2)HPO_(4)、0.04%(质量分数)MgSO_(4)。通过单因素试验优化了杨木酶水解液作为碳源培养枯草芽孢杆菌的培养条件,确定最佳培养条件为:温度35℃、pH 7.0、转速190 r/min、初始接种量2.0%(体积分数)、装液量10 mL(相对于50 mL)。杨木屑经过乙酸、过氧化氢-乙酸预处理和纤维素酶水解后得到了含有101.7 g/L葡萄糖和20.7 g/L木糖的酶水解液。在最佳培养基组成和培养条件下,酶水解液经过发酵24 h后,培养基中26.8 g/L的葡萄糖和5.6 g/L的木糖分别被枯草芽孢杆菌消耗至0.4和0 g/L,培养得到的枯草芽孢杆菌活菌数可达到6.46×10^(8) CFU/mL,最终单糖利用率可达98.7%。试验结果表明,杨木酶水解液可应用于枯草芽孢杆菌培养,可为杨木高值化利用提供参考。Poplar tree species including hybrid varieties are considered as promising biomass feedstock for biofuels and biochemicals production due to their fast growing,short vegetative cycle,and widely distribution.Bacillus subtilis is a gram-positive bacterium with fast growth and simple nutritional requirements,and has a good fermentation basis,which is one of the state-permitted feed additives,and widely used in the animal husbandry and feed processing industry,with broad development prospects.In this paper,we used hydrogen peroxide-acetic acid pretreatment to remove the lignin of poplar and the enzymatic hydrolysate was used as carbon source for the fermentation of B.subtilis.The aim of this work is to optimize the cultivation medium and conditions of B.subtilis using poplar hydrolysate as carbon source.In this work,the effects of nitrogen source,inorganic salts,trace elements,temperature,pH value,shaking speed,bacterial loading,and liquid volume on the growth of B.subtilis were investigated.The single factor experiments with response surface optimization were used to optimize culture conditions and medium compositions.The optimal culture conditions were 35℃,pH of 7.0,190 r/min,2% bacterial loading,and 10/50 mL,and the optimized concentrations of poplar hydrolysate,fishmeal peptone and K_(2)HPO_(4) were 3.20%,2.30% and 0.06%,respectively.The concentrations of glucose and xylose in poplar hydrolysate were 101.7 and 20.7 g/L,respectively.The growth process of B.subtilis was studied.The slow phase of B.subtilis was 0-3 h,the logarithmic growth phase was 3-24 h and the stable growth phase was 24 h.After the fermentation by B.subtilis under optimized conditions and medium for 24 h,the concentrations of glucose and xylose in medium decreased from 26.8 and 5.6 g/L to 0.4 and 0 g/L,respectively,with a total monosaccharide utilization rate of 98.7%,and the highest viable count reached 6.34×10^(8) CFU/mL.The enzymatic hydrolysate of poplar contains glucose,xylose and cellobiose.According to the growth process of B.subtilis

关 键 词：酶水解液 杨木 枯草芽孢杆菌 发酵培养基 响应面法 

分 类 号：TQ35[化学工程]