Janzen Nils H, Schmidt Michael, Krause Christian, Weuster-Botz Dirk
Lehrstuhl für Bioverfahrenstechnik, Technische Universität München, Boltzmannstr. 15, 85748, Garching, Germany,
Bioprocess Biosyst Eng. 2015 Sep;38(9):1685-92. doi: 10.1007/s00449-015-1409-4. Epub 2015 May 13.
Optical chemical sensors are the standard for pH monitoring in small-scale bioreactors such as microtiter plates, shaking flasks or other single-use bioreactors. The dynamic pH range of the so far commercially available fluorescent pH sensors applied in small-scale bioreactors is restricted to pH monitoring around neutral pH, although many fermentation processes are performed at pH < 6 on industrial scale. Thus, two new prototype acidic fluorescence pH sensors immobilized in single-use stirred-tank bioreactors, one with excitation at 470 nm and emission at 550 nm (sensor 470/550) and the other with excitation at 505 nm and emission at 600 nm (sensor 505/600), were characterized with respect to dynamic ranges and operational stability in representative fermentation media. Best resolution and dynamic range was observed with pH sensor 505/600 in mineral medium (dynamic range of 3.9 < pH < 7.2). Applying the same pH sensors to complex medium results in a drastic reduction of resolution and dynamic ranges. Yeast extract in complex medium was found to cause background fluorescence at the sensors' operating wavelength combinations. Optical isolation of the sensor by adding a black colored polymer layer above the sensor spot and fixing an aperture made of adhesive photoresistant foil between the fluorescence reader and the transparent bottom of the polystyrene reactors enabled full re-establishment of the sensor's characteristics. Reliability and operational stability of sensor 505/600 was shown by online pH monitoring (4.5 < pH < 5.8) of parallel anaerobic batch fermentations of Clostridium acetobutylicum for the production of acetone, butanol and ethanol (ABE) with offline pH measurements with a standard glass electrode as reference.
光学化学传感器是小型生物反应器（如微孔板、摇瓶或其他一次性生物反应器）中pH监测的标准方法。尽管许多工业规模的发酵过程是在pH < 6的条件下进行的，但目前应用于小型生物反应器的市售荧光pH传感器的动态pH范围仅限于中性pH附近的pH监测。因此，我们对两种固定在一次性搅拌罐生物反应器中的新型酸性荧光pH传感器进行了表征，一种在470 nm激发、550 nm发射（传感器470/550），另一种在505 nm激发、600 nm发射（传感器505/600），研究了它们在代表性发酵培养基中的动态范围和操作稳定性。在矿物培养基中，pH传感器505/600表现出最佳的分辨率和动态范围（动态范围为3.9 < pH < 7.2）。将相同的pH传感器应用于复杂培养基会导致分辨率和动态范围大幅降低。发现复杂培养基中的酵母提取物会在传感器的工作波长组合下产生背景荧光。通过在传感器点上方添加黑色聚合物层对传感器进行光学隔离，并在荧光读取器和聚苯乙烯反应器的透明底部之间固定由粘性光刻胶箔制成的孔径，能够完全恢复传感器的特性。通过在线pH监测（4.5 < pH < 5.8）丙酮丁醇梭菌平行厌氧分批发酵生产丙酮、丁醇和乙醇（ABE），并以标准玻璃电极的离线pH测量作为参考，展示了传感器505/600的可靠性和操作稳定性。
