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Genetic Engineering & Biotechnology News | GENengnews.com | SEPTEMBER 1, 2016 | 31 sensor. Three source-detector pairs are incor- porated into a single sensor, and are automati- cally arbitrated between in order to maintain linear response to biomass over three orders of magnitude cell biomass. 1 This approach has been successfully employed in vessels hav- ing volumes of 500 mL and larger. To accommodate the trend toward smaller vessels, a more recent approach (BE3000 probe), uses a small diameter (e.g. 3 mm) fiber optic probe measuring back-reflec- tance at 1310 nm. At this wavelength, water absorbance limits optical penetration into the medium to 3 cm or less. 2,3 At shorter wave- lengths (i.e. towards the visible region of the spectrum) the penetration depth increases due to weaker water absorbance. For example, at 850 nm the penetration depth into the me- dium can be more than 10 cm. Particularly in small vessels, this can make it difficult to avoid interference from other probes and ob- jects such as impellers. At longer wavelengths, the penetration depth into water rapidly diminishes due to increasing absorbance of light by water. For example, at 1450 nm, the penetration depth is less than 1 mm. This has the effect of re- ducing the effective measurement volume to such an extent that the sensitivity to cell bio- mass is substantially diminished. At 1310 nm the measurement volume is small enough so that measurements can be made in small vessels (e.g. as small as 50 mL in a 250 mL vessel), while at the same time maintaining a very wide linear range of sensi- tivity to biomass. 3 The optical reflectance of Saccharomyces cerevisiae in a 250 mL ves- sel is shown in Figure 1, measured at 1310 nm as a function of more than four orders of magnitude of yeast dry cell weight. Mitigating the Effects of Bubbles One of the most well-known sources of interference with on-line optical measure- ments of cell biomass is bubbles. For micro- bial cells grown in bioreactors, high gassing and stirring rates are often employed. By using a small fiber optic probe and a source wavelength having limited penetration depth, the BE3000 measurement volume is limited to approximately 200 µL or less. The number of microbial cells in this vol- ume will be in the thousand to millions at the lowest concentrations of interest, and will range up into the millions to billions at high concentrations. As a result, the individual microbial cells that are inside the optically sampled volume will change over time as the cells move through the medium, but the mean number of cells will be nearly constant. Bubbles are generally larger and less numerous than the cells, so the number of bubbles within the optically sampled volume will vary widely as a function of time. By cre- ating a 2D map of biomass as a function of the reflectance distribution and central value, the effects of changing bubbles and biomass can be effectively separated. 3 By applying this map to new measurements, accurate biomass prediction is achieved over four or- ders of biomass magnitude, despite widely varying agitation and sparging conditions. 3 The bulk scattering effects measured by transmission and reflectance probes is affect- ed by the size of the scattering particles. For this reason, calibration is required in order to report biomass in absolute units such as dry cell weight or cell counts. The BE3000 instrument comes pre-cal- ibrated for dry cell weight of Saccharomy- ces cerevisiae, E. coli, and the micro-algae Chlorella vulgaris. Calibration to other unicellular organisms or off-line methods is a straight-forward process. References 1. U.S. Patent 8,603,772 2. U.S. Patent 8,405,033 3. U.S. Patent Application 20150300938 and International Patent Application PCT/ US2015/026702 Save the date BIOPROCESSING Martin P. Debreczeny, Ph.D. (martin@ buglab.com), is the co-founder of BugLab. Website: www.buglab.com. Tech Note