Fluorescence sensor for the monitoring and control of microalgal cultivations

By Luis Porras Reyes

Microalgae are well known photosynthetic microbes used as cell factories for the production of relevant biotechnological compounds (e.g., pigments, unsaturated fatty acids) and environmental applications (e.g., wastewater treatment, lipids for biofuels). Despite these outstanding properties, the industrial scale production of microalgae is still at an early stage of development compared to other cell-based bioprocesses. An important bottleneck in this support is the lack of suitable online sensors for reliable monitoring of biological parameters in microalgae bioprocesses.

Fluorescence spectroscopy offers several advantages as a powerful analytical technique in the monitoring of different processes. Its high sensitivity, selectivity, fast measurements, versatility, and non-invasiveness make it a widely used tool for research, quality control, process monitoring, and diagnostic applications in fields such as biochemistry, bioprocess engineering, pharmacology, environmental science, and materials sciences. But first, what do we call fluorescence phenomena? When referring to this concept we have to consider that a molecule, called fluorophore, absorbs light energy which promotes electrons into a temporary excited state (fluorescence lifetime), followed by a return to a basal state in which the excess energy is dissipated through the emission of photons (emitted fluorescence light), i.e., energy whose wavelength is larger than that which generated the excited state, due to energy loss by, e.g., vibration. In order to show fluorescence, fluorophores must contain an aromatic system in their chemical structure.

In biotechnology, fluorescence measurements are commonly used for the detection of biological molecules such as proteins fractions (e.g., tryptophan, tyrosine, and phenylalanine), vitamins, and cofactors (e.g., FAD and NADH) which have been used in the estimation of critical process parameters (CPP) (e.g. biomass, metabolites, substrate consumption, product formation). In microalgae bioprocesses, pigments like chlorophyll and phycobiliproteins should also be considered as candidate fluorophores for the monitoring and control of these microorganisms.

Researchers have reported using sensor software and fluorescence spectroscopy to assess CPPs in microalgae cultures (e.g., biomass, pigment, and lipid content). However, the fluorescence data is still collected through offline measurements. This presents an opportunity for the development of online fluorescence sensors that can provide real-time, non-invasive, and reliable information on the status of microalgae cultures.

Our recent studies focused on developing an online prototype fluorescence sensor to monitor microalgae cultures. We began by screening Chlorella vulgaris cultures to identify the most important intracellular fluorophores (refer to Figure 1). With this basic information, it was possible to design a simple and low-cost sensor prototype that has demonstrated promising results. The fluorescence signals obtained with riboflavin (chemical reagent) at different concentrations showed a linear trend with both the on-line prototype and the reference method: fluorescence spectrophotometer (refer to Figure 2). This trend was also observed in the online and offline measurements with NADH (results not shown). Finally, by using dilutions of C. vulgaris cultures, it was able to obtain online fluorescence signals for both riboflavin and chlorophyll. The fluorescence signal intensity decreases significantly when measuring riboflavin, while the measurements associated with chlorophyll are found throughout the sensor intensity detection range (refer to Figure 3). The behaviour of the evaluated dilutions shows a parabolic trend from the most diluted samples to the undiluted samples. These results are consistent in both fluorophores and could be possibly explained due to a signal saturation caused by the accumulation of the sidescatter light.

The results are promising for developing an online fluorescence sensor for microalgae cultures. However, future adjustments to the electrical current that feeds the light source for each fluorophore’s excitation should be considered. Online fluorescence sensors, when coupled with software sensors, can be used to estimate CPPs relevant in the monitoring and control of microalgae cultures.

Figure 2. Online (left) and offline (right) measurements of different concentrations of riboflavin (0,005, 0,05, 0,5, 1, 2,5 and 5 mg/L in PBS pH 7,5).

 

Figure 3. Online riboflavin measurements (left) and online chlorophyll measurements (right) using C.vulgaris cultivated cells at different dilutions (1:50, 1:10, 1:5, 1:3, 1 using NaCl 0,9%).