(157e) Extracellular Carotenoid Production from Microalgae Under Increased CO2 Concentrations

Carbon dioxide fixation using fast-growing photoautotrophic microorganisms, in their natural habitats or in artificial cultivation systems, provides a very promising alternative for CO₂ mitigation. Besides, it is potentially cost-effective when CO₂ conversion is associated to the production of high value bioproducts (Borowitzka, 2013). Some of the bioactive compounds from microalgae are proteins, fatty acids, vitamins, pigments and carotenoids, some of them exhibiting antioxidant activity. Among the reported antioxidants produced by microalgae, astaxanthin is the highest-value carotenoid that has achieved commercial success. Only for astaxanthin, the worldwide market in 2010 was around US$230 million, synthetic and natural, and the consumers demand for natural products make the synthetic pigments much less desirable, providing an opportunity for the production of natural pigments. Other potential carotenoids are lutein and zeaxanthin, approved as food additives for preventing macular diseases. Currently, the main sources of lutein for commercial production are marigold flowers, and there is no commercial production of lutein from microalgae (Cezare-Gomes et al., 2019).

Parachlorella kessleri was pre-selected under 30% CO₂ from water and sediment samples, isolated using appropriate techniques, identified via molecular biology and evaluated with cultivations in shake flasks under CO₂ concentrations from 5 to 30%. Besides presenting relatively fast growth and high biomass production under all tested CO₂ conditions, the microalga produced an extracellular reddish pigment with antioxidant activity. The production of antioxidants in microalgae occurs, in some cases, when they are subjected to stress. During microalgae cultivation, CO₂ enrichment is a requirement to reach high productivity. At the same time, high CO₂ levels are normally stressful to microalgae, especially for the photosynthetic apparatus, which can induce carotenoid production. Likewise, high CO₂ levels can stimulate the production of fatty acids, with applications for biofuels or as nutraceuticals or pharmaceuticals.

This work evaluated extracellular carotenoid production from the isolated Parachlorella kessleri cultivated under 5, 15 and 30% CO₂ in stirred tank photobioreactors. In the 10^th day of cultivation, CO₂ supply was interrupted until the end of the cultivation (14^th day). It is believed that this causes a stress on microalgae cells and is an imperative condition to make them produce the antioxidant. To our knowledge, this method has not been reported before. Dissolved oxygen and pH sensors allowed the evaluation of both parameters in real time during the cultivations. Growth was monitored daily by optical density measures at 750 nm and cell dry weight concentrations. Chlorophyll-a concentrations, that relates to photosynthetic activity, were calculated using the equations in (Ritchie, 2006) after extraction of the samples with methanol and absorbance measures at 652, 665 and 750 nm. In the last day of cultivation, the biomass was centrifuged, lyophilized and destined to fatty acids composition and element analyses (C,N,H) for the calculus of CO₂ fixation rates. Total lipids were extracted by the modified Bligh&Dyer method, the final chloroform phase was evaporated and the lipids reacted with methanol in acid conditions to produce the fatty acid methyl esters (FAMEs), which were extracted with hexane and detected by GC-MS. Total carotenoids were spectrophotometrically quantified in the supernatant by absorbance measures at 450 nm and using molar extinction coefficient of 2500 (Britton, 1985).

The growth curves under all CO₂ concentrations were similar, although under 30% CO₂ there was a lag phase of one day, the exponential phase was shorter (3 days comparing to 4 days in the other conditions) and after exponential phase the growth was slower than the other conditions. The pH values varied differently among the cultivations. Under 5% CO₂, pH values increased from 5.7 on the first day to 10.5 on the 5^th day, decreased until the 10^th day and increased again after CO₂ supply was interrupted until the end of the cultivation. The same pattern was observed on the cultivations under 15% CO₂, but the highest pH values during exponential phase were around 6.5 and reached 10.5 on the last 2 days of cultivation because of interruption of CO₂ supply. Under 30% CO₂, the pH remained on 5 to 6 during the cultivation period, which might have contributed the lower growth after exponential phase under this condition. Chloropyll-a (Chl-a) profiles exhibited the highest values of Chl-a concentrations on days 4 and 5 of cultivation (highest photosynthetic activity), decreasing until the end of the cultivation, which is in accordance to the high pH values on these days.

The calculated maximum specific velocities were 0.74 ± 0.09, 0.74 ± 0.08 and 0.77 ± 0.11 under 5, 15 and 30% CO₂, respectively, and the differences were not statistically significant (p>0.05). Cell dry weight concentrations on the 14^th day of cultivation were 1.09 ± 0.12, 1.24 ± 0.15 and 1.09 ± 0.25 g L^-1, total lipids were 241 ± 11, 208 ± 5 e 183 ± 14 mg g^-1 (d.w.) and CO₂ fixation rates were 158 ± 7, 165 ± 5 and 141 ± 3 mg L^-1d^-1, under 5, 15 and 30% CO₂, respectively. Minhas and coworkers (2016) reported 21.42% (d.w) of lipid content on P. kessleri , and de Morais and Costa (2007) reported 163 mg L^-1d^-1 of CO₂ fixation rate under 18% CO₂. Both values corroborate the results obtained in this work. The reddish carotenoid production was observed in all cultivations, with concentrations of approximately 0.030 ± 0.005, 0.026 ± 0.006 and 0.011 ± 0.002 µg mL^-1 under 5, 15 and 30% CO₂, and the differences were statistically significant (p<0.05).

The fatty acids analysis of the biomass presented the major fatty acids: palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18:1), linoleic acid (C18:2) and α-linolenic acid (C18:3). Their relative concentrations were different depending on the CO₂ concentration. For instance, C16:0 yields were higher under 30% CO₂ (around 60%) compared to the other conditions (around 30%). In contrast, polyunsaturated fatty acids (PUFAs) were higher under 5% CO₂ (around 50%) and decreased to 28% and 22% under 15 and 30% CO₂, respectively. Another significant difference was on C18:1 production, which increased from 14% to 30%, under 5 and 15% CO₂, and decreased to 11% under 30% CO₂. The increase in oleic acid production has been previously reported as an indication of high cell density and accumulation of lipids, normally promoted because of nutrient limitations (van der Ha et al., 2012). This indicates that high CO₂ levels up to 15% can induce lipid production on this microalga.

The carotenoids produced were extracted with methanol and the methanolic extracts were characterized by UV-Vis spectrophotometry and by High Performance Liquid Chromatography (HPLC). Two fractions were separated in the HPLC, yellow and red, which presented λ_max of 405 and 427 nm, for the yellowish pigment, and 455, 485, 524 nm, for the reddish one. Adjacent absorbance peaks on these regions of the visible light spectrum are characteristic of carotenoids and indicate multiple chromophore groups. LC-MS/MS (Liquid Chromatography coupled to Mass Spectrometry) analysis of the reddish carotenoid fraction pointed to a carotenoid with molecular weight of 569 g mol^-1, which may correspond to zeaxanthin or lutein. The MS/MS fragmentation of the molecular ion m/z 569 presented the normally reported mass losses for carotenoids but was not conclusive on the complete molecular elucidation.

To our knowledge, this is the first report of extracellular carotenoid production by the microalga Parachlorella kessleri. Botryococcus braunii has been reported to produce the extracellular carotenoid equinenone (Cheng et al., 2019). Extracellular production is particularly interesting considering the costs and the environmental impact of using organic solvents and harsh conditions to extract intracellular carotenoids. This could reduce the costs of the final product and increase the sustainability of the process. This work revealed that CO₂ concentrations up to 15% can be used to improve carotenoid production, also favoring lipid production, with particular interest on the PUFAs, which are high value bioproducts.