Measuring Cell Number
Various techniques are available to determine the concentration of biomass in liquid cultures. The most accurate but also laborious method is the determination of the dry weight content which requires washing, drying, and weighing of a sample. Due to the many necessary steps, this method is not readily automated and can thus not be used to control the cultivation process on-line. Alternative approaches which also allow on-line measurements are based on either spectroscopic, electrochemical, or optical priciples. The most commonly employed method is the absorption spectroscopic measurement using off-line photospectrometers or specific on-line probes. The turbidity is determined measuring the light transmission through the sample. The resulting ratio between the intensity of the light irradiating the sample to the intensity of the emerging light is usually interpreted with the help of the Lambert-Beer law:
A = log(I0/I) = ε(λ) * c * d
It relates the concentration of the light absorbing substance (c) to the ratio of incoming (I0) to received light (I) under consideration of the length of the optical path (d), i.e. here the diameter of the reactor vessel, and the wavelength-dependent extinction coefficient (ε). Importantly, this formulation considers only the reduction of light due to absorption by dissolved substances. However, suspensions containing particles also experience attenuation of transmitted light due to scattering. For a nice explanation of scattering and absorption in cell quantificaiton see [Myers2013]. Despite this inconsistency, it is well established that OD600 is proportional to the cellular dry weight (CDW) and can thus be used as a substitute readout [Geppert1984]. This can not be directly tranferred to cyanobacteria, which feature a range of pigments which absorb light in the 600nm range (see absorption spectrum of two popular cyanobacterial strains below). Accordingly, measuring the turbidity at that wavelength would provide a convolution of the cell number with the pigment content of each cell. This can be avoided by using a longer wavelength such as 850nm at which light does not interact significantly with pigments or chlorophyll.
The setup for the turbidity measurement is simple (schema shown below) featuring a light source placed on one side of the reactor vessel and a light detector on the other. Due to the application of a water bath to reduce refraction at the round bottle wall, both the source and detector are placed inside a protective housing. Cuvettes used in commercial photospectrometers are used due to their high optical quality, low price and availability.
The next step shows that the NinjaPBR setup does essentially what a commercial photospectrometer does. Therefore, a geometric dilution series of a concentrated liquid culture of Thermosynechococcus sp. PCC 7002 was prepared and measured. For direct comparability, OD values were measured withthe spectrophotometer Spekol from Carl Zeiss Jena in parallel to the NinjaPBR. Both devices yield curves which are typical for the setup geometry. The sensitivity of the turbidity signal to changes in the actual particle concentration decreases with increasing particle concentration.
In addition to the Spekol, the handheld Buglab OD scanner was used. In contrast to the former devices, the OD scanner is based on the measurement of laser light being scattered back from the sample. This approach provides a wider range in which the detected light intensity correlates linearly with the particle concentration. Accordingly, the obtained particle concentration, here measured in nephelometric turbidity unsits (NTU), show a linear increase until a dilution factor of 0.5 as opposed to the Spekol, which offered a linear correlation up to a dilution of 0.0625.
The direct comparison of measured OD with NTU data emphasizes the accuracy of the NinjaPBR setup at low particle concentrations. It is important to note, that linear correlation of cell number and measured parameter is not necessary. Every parameter must be calibrated to the actual cell number, permitting the application of linear as well as non-lininear functions. However, the accuracy of the obtained cell number estimate decreases with the smaller slope.
Due to the wider informative range of scattering-based setups, the NinjaPBR is prepared for a second housing to add a side-scatter sensor in later iterations.
-- [Geppert, G., & Thielemann, H. (1984). Streulichtphotometer zur kontinuierlichen Bestimmung der Biomassekonzentration in Fermentationsmedien. Acta Biotechnologica, 4(4), 361–367. doi:10.1002/abio.370040411]
[Myers, J. A., Curtis, B. S., & Curtis, W. R. (2013). Improving accuracy of cell and chromophore concentration measurements using optical density. BMC Biophysics, 6(1), 4. doi:10.1186/2046-1682-6-4]