乳乳乳乳乳乳乳乳乳乳乳乳乳乳乳乳乳 * 乳乳 1,2 乳乳 1 乳乳乳 1** 乳乳乳 1,2 乳乳乳 1,2 乳乳乳 1 1 乳乳乳乳乳乳乳乳乳乳乳乳乳乳 610041 2 乳乳乳乳乳乳乳乳乳 100049 乳 乳 乳乳乳乳乳乳乳乳 一,体,, 。统 乳乳 。,pH 乳乳乳乳 5.5-7.5 乳 乳 ,20g L -1 -40g L -1 乳乳 , 1.5g L -1 -3.5g L -1 乳 。乳乳乳 ,一乳 pH 乳 乳 、 Box-Behnken 乳乳乳乳乳乳乳乳乳乳乳 , 乳 乳乳乳 ,。 乳乳 :pH 乳乳 6.72 乳 , 27.83 g L -1 乳 , 2.79 g L -1 乳 乳乳乳乳乳 , 2.47 g L -1 D -1 乳 。 2.43 g L -1 D -1 乳乳 ,, 47.27% 乳乳 。,( Miseq 乳乳乳 乳 ), Clostridium sensustricto、Lactobacillus 乳 Clostridium IV 乳 , 69.35%,15.41%乳 10.05% 乳 。 8 乳 3 乳 34 乳乳乳乳 乳乳 乳乳乳乳乳 ;;;; CLC Construction of the process of butyrate production from lactate and the optimization of fermentation parameters * LIANG Cheng 1,2 ,TAO Yong 1 , ZHU Xiaoyu 1** ,ZHANG Yanyan 1,2 ,XIANG Yuanying 1,2 &HE Xiaohong 1 1 Chengdu Institute of Biology, Chinese Academy of Sciences,Chengdu 610041,China 2 University of Chinese Academy of Sciences,Beijing 100049,China Abstract Objectives: This paper reported a new process that can produce butyrate from lactate using mixed culture. In order to reduce the cost of butyrate fermentation as well as improve the yield of butyrate, optimization of fermentation conditions were investigated. Methods: First, functional mixed culture was acclimated by fed-batch fermentation in a designated reactor; Second, the optimal range of various factors were determined by conducting single factor experiments; Third, optimal conditions were obtained by using response surface methodology about Box-Behnken design; Finally, microbial community structure was analyzed by using high-throughput sequencing technologies. Results: The single factor experiments firstly showed that the optimal range of pH was between 5.5 and 7.5, lactate concentration was between 20g L -1 and 40g L -1 , and additional acetate concentration was between 1.5g L -1 and 3.5gL - 1 . Based on response surface methodology, it was found that the highest production rate of butyrate (2.47 g L -1 D -1 ) can be achieved at pH 6.72 with lactate concentration of 27.83 g L -1 and acetate concentration of 2.79 g L -1 . 1 乳乳乳乳 Received 乳乳乳乳 : Accepted: *乳乳乳乳乳乳乳乳乳乳 ( No.31300116 , No.31270531 , No.31470020 乳乳乳乳乳乳 乳乳乳乳乳 乳乳乳乳乳乳乳乳乳乳 ) 、西西( Y4C5011100 乳乳乳乳乳乳乳乳乳 )、 乳 2016JZ0010 ) Supported by the National Natural Science Foundation of China (No.31300116 , No.31270531 , No.31470020) 、 The Western Light Talent Culture Project乳 Y4C5011100)、Sichuan Science and Technology Support Program乳 2016JZ0010) ** 乳乳乳乳 Corresponding author (E-mail: [email protected]) 1 2 3 4 5 6 7 8 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 1 2 3 4 5 6
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达 1.65 g L-1 D-1,乳酸平均消耗速率为 4.33 g L-1 D-1;在 pH4.5 和 8.5 条件下,丁酸的生成和乳酸的消耗明显受到抑制,经 7天时间发酵,丁酸没有明显的增加,乳酸的消耗也很缓慢。因此确定 pH5.5-7.5 为丁酸发酵的适宜范围。
图 2 在不同初始 pH条件下丁酸的生成及乳酸的消耗情况,误差线为均值的标准方差。Fig.2 Effects of different initial pH on the rate of butyrate-producing and lactate-utilizing.Error bars
indicate standard deviation from the mean.
2.2 乳酸浓度对丁酸产率的影响试验设置乳酸初始浓度分别为 10 g L-1、20 g L-1、30 g L-1、40 g L-1 和 50 g L-1共 5 个梯度,控制 pH6.5
±0.1,发酵温度 30±1℃,外加乙酸浓度为 2.0 g L-1。由图 3 可知,随着乳酸初始浓度的增加,丁酸的产率先增大后减少,在 30 g L-1 浓度条件下,丁酸产率最大,平均可达 1.80 g L-1 D-1,乳酸平均消耗速率为 4.88
g L-1 D-1;在乳酸初始浓度为 50 g L-1 时,反应明显受到抑制,说明此发酵过程存在底物抑制作用。结果表明乳酸初始浓度 20 g L-1-40 g L-1 为丁酸发酵的适宜范围。图3
在不同初始乳酸浓度条件下丁酸的生成和乳酸的消耗情况,误差线为均值的标准方差。Fig3. Effects of different initial lactate concentrations on the rate ofbutyrate-producing and lactate-utilizing.
Error bars indicate standard deviation from the mean.
2.3 外加乙酸浓度对丁酸产率的影响试验对外加乙酸浓度分别为 0.5 g L-1、1.5 g L-1、2.5 g L-1、3.5 g L-1 和 4.5 g L-1 条件下丁酸产率变化进
行研究,发酵过程中控制 pH6.5 ±0.1,发酵温度 30±1℃,乳酸浓度为 30 g L-1。试验结果如图 4 所示,随外加乙酸浓度增大,丁酸产率先增大后减小,乙酸浓度为 2.5 g L-1 时,丁酸平均产率最大可达 1.71 g L-1 D-
1,乳酸平均消耗速率达 4.31 g L-1 D-1。因此得出外加乳酸浓度在 1.5 g L-1-3.5 g L-1 为适宜范围。
时放出能量供给其自身生命活动,乳酸杆菌属在自然界分布极为广泛,除少数外,其中绝大部分都是人体内必不可少的且具有重要生理功能的菌群 [34]。因此,在本工艺中,该菌种的作用与丁酸合成并没有直接关系。综上所述,在本试验工艺中,Clostridium sensustricto 可能是丁酸合成的关键功能菌群。参考文献[References]1 Agler MT, Werner JJ, Iten LB, Dekker A, Cotta MA, Dien BS, Angenent LT. Shaping Reactor Microbiomes to Produce the Fuel
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