Evaluation of the effects of dye-mediated irradiation on metabolite production in Chlamydomonas reinhardtii
Authors:   , Ramanna Luveshan 
Published: June 2022

Microalgae have emerged as one of the most promising feedstocks for the production of high-value metabolites and bioactive compounds. However, the commercial success of largescale microalgal cultivation systems has been restricted due to the limited productivity of biomass and metabolites, which has been largely due to limited light availability. Light is one of the main factors that influence microalgal photosynthetic efficiency, carbon fixation capacity, cellular metabolism, and biomass production. Solar radiation is composed of a wide range of wavelengths such as ultraviolet (UV) (290 – 400 nm), visible (450 – 700 nm), and infrared rays (700 nm – 30 µm). Green microalgae only use the blue (400 – 500 nm) and red (600 – 700 nm) bands of the light spectrum for photosynthesis, referred to as photosynthetically active radiation (PAR). Spectral wavelengths that do not contribute to photosyntheticallydriven energy conversion pass through the microalgal cells and remain unused. Hence, the conversion of photons from the unused to the usable wavelengths can produce/supplement a spectrum amenable to potentially increase metabolite production in microalgae. This study aimed to enhance light availability to Chlamydomonas reinhardtii using organic dyes as incident light converters, thereby potentially improving photosynthetic efficiency and metabolite production. The first objective of this study dealt with determining the optimal solvent and dye concentration. Diphenylanthracene [DPA], Diphenyloxazole [DPO], Rhodamine 6G [R6G], Rhodamine 8G [R8G], Rhodamine 800 [R800], Fluorescein Isothiocyanate [FITC], Lumogen Yellow [LY], and Lumogen Red, [LR], were evaluated for their ability to absorb, convert, and emit photon flux densities (PFDs) in the PAR region. The DPA and DPO dyes absorbed light in the UV range (280 – 350 nm) and emitted in the violet/blue wavelengths (390 – 500 nm). Rhodamine 8G absorbed and emitted light between 200 – 300 and 450 – 500 nm, respectively, suggesting a blue-green region that could stimulate pigment and/or lipid production in algae. The fluorescent dye LY showed several sharp peaks at the green and blue light spectra (400 – 525 nm). The fluorescence intensity also extended to more than 600 nm, representing the green to yellow and red PAR portions. The LR dye demonstrated an excitation and emission range between 320 – 400 nm and 590 – 660 nm, respectively, while R6G, R800, and FITC did not exhibit irradiations in the PAR wavelengths. Most of the dyes displayed higher fluorescent intensities in methanol (10 mgL-1) when compared to dyes dissolved in ethanol and acetone at higher concentrations. Lumogen Yellow, R8G, and LR emitted the highest emissions in the PAR regions which were 161873, 113138, and 61824 a.u, respectively. The next objective focused on determining the effects of increased LY and R8Gmediated irradiance on the physiological responses in C. reinhardtii. Experiments were conducted in double-jacketed cylindrical photo-bioreactors. Outer jackets contained dye solutions while inner jackets contained microalgae. The dyes were dissolved in methanol (10 and 100 mgL-1) and were stimulated using daylight tubes. The initial increase in irradiance from LY and R8G induced light stress in Chlamydomonas, which was characterised by nonphotochemical quenching (NPQ). Increased NPQ from ~ 0.05 to 0.5 signified damage to the microalgal photosystem (PS). Accordingly, the quantum efficiencies of PSII decreased from approximately 0.6 to 0.2, resulting in transient growth until day 8, and low biomass production. The cultures grown under R8G irradiations showed increased carotenoid production in response to the light stress (~ 37%), while the LY-irradiated cultures showed the highest carotenoids throughout the experimental period (12 mgg-1). After the 4th day of experimentation, there was an inactivation in the NPQ process which was accompanied by a simultaneous increase in the quantum efficiencies in cultures grown under R8G and LY dye-irradiance (47 and 34% respectively). The findings indicated that the alga required an initial dye-acclimation period, after which it was able to regulate the excess energy. Toward the end of the growth cycle, decreases in biomass were ascribed to light attenuation in the culture medium. Moreover, the effects of increased dye-irradiance may have manifested indirectly through the production of stress relief metabolites (protein, carbohydrate, and lipid). The final objectives dealt with evaluating the dye spectral conversion ability and assessing light utilisation and metabolite production in C. reinhardtii. The emission intensities of the fluorescent dyes were increased using UV fluorescent tubes. The R8G dye pattern showed a sharp peak at about 498 nm. The LY dye demonstrated several sharp peaks at the green and blue light spectra; however, there was a drastic reduction in emissions indicative of photo-degradation. The LR dye exhibited an emission spectrum peak in the orange-red light region, providing effective light energy for microalgal growth. Cultures grown under LR irradiance exhibited increased photochemical energy utilisation [Y(II)] and decreased regulated heat dissipation [Y(NPQ)]. Additionally, the LR-irradiated cultures showed a 1.6- and 2.9-fold up-regulation in RuBisCo gene expression, verifying the increase in the photosynthetic process under increased red-light wavelengths. Carbohydrate and lipids were produced early in the growth phase, while biomass and protein concentrations were accumulated toward in later stages. Dye irradiations (LR and/or R8G) was associated with increased biomass productivity and metabolic content compared with the control (with no dye addition). The findings showed that R8G and LR can be applied to microalgal cultivation systems to improve light availability and enhance photosynthetic output. Hence, this study demonstrated the potential applicability of organic dyes as effective spectral converters of incident irradiation, supporting photosynthetic energy utilisation, and increasing metabolite yields with less input of energy in the algal systems.