On-line analysis and in situ pH monitoring of mixed acid fermentation by Escherichia coli using combined FTIR and Raman techniques
On-line analysis and in situ pH monitoring of mixed acid fermentation by Escherichia coli using combined FTIR and Raman techniques
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DOI：
10.1007/s00216-020-02865-5
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发表时间：
2020-08-14
影响因子：
4.3
通讯作者：
Hippler, Michael
中科院分区：
文献类型：
--
作者：
Metcalfe, George D.;Smith, Thomas W.;Hippler, Michael
我们介绍了一个实验装置，允许连续监测细菌发酵过程中，同时光密度（OD）测量，长路径FTIR顶空监测CO2，乙醛和乙醇，和液体拉曼光谱的乙酸盐，甲酸盐和磷酸盐阴离子，没有采样。我们讨论了哪些光谱特征最适合用于检测，以及如何通过光谱特征的积分和最小二乘拟合来获得分压和浓度。噪声等效检测限为约2.6 mM的乙酸和3.6 mM的甲酸在5分钟的积分时间，提高到0.75 mM的乙酸和1.0 mM的甲酸在1小时的积分。分析范围扩展到至少1 M，误差百分比的标准偏差约为8%。磷酸盐缓冲液的阴离子的测量允许通过在6-8 pH范围内的改进的Henderson-Hasselbalch方程以优于0.1的准确度光谱原位测定细菌悬浮液的pH。使用亨利定律，4 m白色池FTIR测量提供了气相中乙醛的0.21 μ bar和乙醇的0.26 μ bar的噪声等效检测限，对应于溶液中的3.2 μ M乙醛和22 μ M乙醇。分析动态范围超过1 mbar乙醇，相当于溶液中的85 mM。作为应用实例，对大肠杆菌的混酸发酵进行了研究。CO2，乙醇，乙醛，酸，如甲酸和乙酸的生产，以及pH值的变化进行了讨论的背景下，混合酸发酵途径。研究发现，甲酸盐分解为CO（2）和H（2）受零级动力学速率定律支配，这表明向含有大肠杆菌的生物反应器中添加外源性甲酸盐预计对甲酸盐分解速率和生物产氢没有有益影响。
We introduce an experimental setup allowing continuous monitoring of bacterial fermentation processes by simultaneous optical density (OD) measurements, long-path FTIR headspace monitoring of CO2, acetaldehyde and ethanol, and liquid Raman spectroscopy of acetate, formate, and phosphate anions, without sampling. We discuss which spectral features are best suited for detection, and how to obtain partial pressures and concentrations by integrations and least squares fitting of spectral features. Noise equivalent detection limits are about 2.6 mM for acetate and 3.6 mM for formate at 5 min integration time, improving to 0.75 mM for acetate and 1.0 mM for formate at 1 h integration. The analytical range extends to at least 1 M with a standard deviation of percentage error of about 8%. The measurement of the anions of the phosphate buffer allows the spectroscopic, in situ determination of the pH of the bacterial suspension via a modified Henderson-Hasselbalch equation in the 6-8 pH range with an accuracy better than 0.1. The 4 m White cell FTIR measurements provide noise equivalent detection limits of 0.21 mu bar for acetaldehyde and 0.26 mu bar for ethanol in the gas phase, corresponding to 3.2 mu M acetaldehyde and 22 mu M ethanol in solution, using Henry's law. The analytical dynamic range exceeds 1 mbar ethanol corresponding to 85 mM in solution. As an application example, the mixed acid fermentation ofEscherichia coliis studied. The production of CO2, ethanol, acetaldehyde, acids such as formate and acetate, and the changes in pH are discussed in the context of the mixed acid fermentation pathways. Formate decomposition into CO(2)and H(2)is found to be governed by a zeroth-order kinetic rate law, showing that adding exogenous formate to a bioreactor withE.coliis expected to have no beneficial effect on the rate of formate decomposition and biohydrogen production.