An on-line high cell density measuring device was developed in this study. It has similar principle as spectrophotomtry in measurement, but it can regulate light path length and measure cell concentration on-line which are impossible in a spectrophotometer.
The cell measuring device is small enough to operate in a spectrophotometer HP-8453, and is able to use deuterium lamp which is originally prepared for the spectrophotometer as a light source. A sealed space is made between two metal plates with a thin rubber supported by a metal ring. Within the space, two quartz-crystal glasses are attatched seperately at each of the plates to pass light. The path length of light can be varied precisely by moving one of the plates using a micrometer. Two small holes are made to circulate fermentation broths using a peristaltic pump and tube.
Two strains were used to measure cell concentrations by this device, S.cerevisiae and Lactobacillus. In case of S.cerevisiae, linear relationship between cell mass calculated by OD value of spectrophotometer and absorbance measured by the device is obtained up to 70 g/L cell concentration. Higher cell densities also have linearity, but large errors can be obtained during calculations. The change of slope in high cell concentrations is due to mechaical deviation from Beer's law, not morphological change of cells.
In case of Lactobacillus measurements, cell concentration shows linearity of up to 40 g/L against absorbance. Over 40 g/L, there's some deviation from the slope obtained at lower concentratioin. Such deviation can be explained by aggregation of cells. When the aggregation occurs in fermentation broth, there are some cells which are surrounded tightly by other cells and give no effect to light scattering. Therefore, the apparent absorbance gets lower. During 5 repeated experiments at each concentration, the device shows good reproducibility with standard deviations less than 0.09.
In Lactobacillus continuous culture using total cell recycle, dry cell weight and cell mass calculated from the absorbance obtained by the device were compared. The relative errors are about 10%, and it is reliable to use this device to measure cell mass during fermentation.
This device has advantages compared to previous high cell density measurements with its simple deisgn, durability, and reduction of cost.
