Improving Determination of Pigment Contents in Microalgae Suspension with Absorption Spectroscopy: Light Scattering Effect and Bouguer–Lambert–Beer Law

Abstract

The Bouguer–Lambert–Beer (BLB) law serves as the fundamental basis for the spectrophotometric determination of pigment content in microalgae. Although it has been observed that the applicability of the BLB law is compromised by the light scattering effect in microalgae suspensions, in-depth research concerning the relationship between the light scattering effect and the accuracy of spectrophotometric pigment determination remains scarce. We hypothesized that (1) the precision of spectrophotometric pigment content determination using the BLB law would diminish with increasing nonlinearity of absorbance, and (2) employing the modified version of the BLB (mBLB) law would yield superior performance. To assess our hypotheses, we cultivated Phaeodactylum tricornutum under varying illumination conditions and nitrogen supplies in controlled indoor experiments, resulting in suspensions with diverse pigment contents. Subsequently, P. tricornutum samples were diluted into subsamples, and spectral measurements were conducted using different combinations of biomass concentrations and path lengths. This was carried out to assess the applicability of the BLB law and the nonlinearity of absorbance. The chlorophyll a and fucoxanthin contents in the samples were analyzed via high-performance liquid chromatography (HPLC) and subsequently used in our modeling. Our findings confirm our hypotheses, showing that the modified BLB law outperforms the original BLB law in terms of the normalized root mean square error (NRMSE): 6.3% for chlorophyll a and 5.8% for fucoxanthin, compared to 8.5% and 7.9%, respectively.

Comparative Analysis of Laboratory-Based and Spectroscopic Methods Used to Estimate the Algal Density of Chlorella vulgaris

“…This correlation can be explained by the phenomenon that, after a certain number of algae cells, there is a statistically high chance of covering each other, so the appearance of another cell will no longer cause as much of a drop in light intensity in a more concentrated suspension as it does in a dilute case. Therefore, for these parameters, we applied the Box–Lucas function (y = a·(1 − exp(−bx))) [ 132 ], which is a built-in factor of the Origin software and is equivalent to the well-known Bouguer–Lambert–Beer equation, which describes the light scattering effect during pigment determination [ 133 ]. As it is presented in Figure 4 , the recommended logistic function resulted in a much higher coefficient of determination (R 2 = 0.9989) than the linear model (R 2 = 0.9879).…”