Comparing pulse amplitude modulated (PAM) fluorometry with radiocarbon technique for determination of inorganic carbon fixation in Chlorella vulgaris (Trebouxiophyceae, Chlorophyta)

ABSTRACT

Integration of pulse amplitude modulated (PAM) fluorometry and conventional methods for estimating carbon assimilation in microalgae is important for physiological, ecological and economic purposes. In this study, we compared PAM fluorometry and carbon-14 (¹⁴C) uptake techniques to estimate the carbon fixation rate in Chlorella vulgaris under controlled laboratory conditions. The key parameter for this comparison was the electron yield for carbon fixation (Ф_e), commonly assumed when converting electron transport rate (ETR) values into the chlorophyll-specific carbon fixation rate (P^B). Additional analyses of maximum (Φ_M) and effective (Φ’_M) quantum efficiency of photosystem II, photochemical (qP) and non-photochemical (NPQ) quenching, and photosynthesis-irradiance response curves demonstrated that the photophysiology of C. vulgaris did not change after a 2-h incubation with NaH¹⁴CO₃ and Na₂CO₃ (control). The association of P^B obtained through the ¹⁴C method (151 ± 8.77 µmol C [mg chl a]^–1 h^–1) with ETR (411 ± 3.91 µmol e⁻ [mg chl a]^–1 h^–1) resulted in an average Ф_e of 0.37 ± 0.02 µmol C [µmol e⁻]^–1, which is higher than theoretical Ф_e values usually reported in the literature (e.g. 0.20 and 0.25). We attributed this discrepancy to a possible inaccuracy in ETR due to underestimated values of chlorophyll-specific absorption cross-section (a*) and the common assumption that only 50% of total light is absorbed by photosystem II. We here demonstrate the importance of associating chlorophyll fluorescence with other primary production techniques, so that adjustments to calculation procedures can be made in accordance to species-specific physiological traits and particularities regarding culturing conditions.

KEYWORDS:

• Carbon-14
• chlorophyll fluorescence
• electron transport rate
• microalgae
• photophysiology
• photosynthesis
• primary production

Acknowledgements

The authors are grateful to the National Council for Scientific and Technological Development (CNPq; grant number 302175/2015-6) and the São Paulo Research Foundation (FAPESP; grant number 2018/07988-5) for financial support. OP was supported by the Grant Agency of the Czech Republic (GACR; project 18-07822S).

Disclosure statement

No potential conflict of interest was reported by the authors.

Supplementary information

The following supplementary material is accessible via the Supplementary Content tab on the article’s online page at https://doi.org/10.1080/09670262.2021.1885065

Supplementary fig. S1. In vivo absorption spectrum of C. vulgaris cells (dashed line) and emission spectra of the white LED used during ¹⁴C incubation (orange line) and Phyto-PAM red actinic light (red line). The emission spectra were adjusted to fit with the measured overall PAR intensities (~150 µmol photons m^–2 s^–1).

Supplementary fig. S2. Emission spectra for the saturating flash (Phyto-PAM, Walz, Germany) used to induce F_m and F’_m.

Supplementary fig. S3. Maximum (Φ_M) and effective (Φ’_M) quantum efficiency of C. vulgaris after 2 h incubation with Na₂CO₃ (control – light grey bars) and NaH¹⁴CO₃ (dark grey bars) as carbon sources. Error bars represent standard deviation of the average (n = 3).

Supplementary fig. S4. Photochemical (qP) and non-photochemical (NPQ) quenching in C. vulgaris after 2 h incubation with Na₂CO₃ (control – light grey bars) and NaH¹⁴CO₃ (dark grey bars) as carbon sources. Error bars represent standard deviation of the average (n = 3).

Author contributions

E.C. Camargo: drafting and editing manuscript; R.A. Rossi: experimental set-up and culture experiments; J.C. Silva: culture experiments. A.C.P. Miwa: ¹⁴C analysis; M.C. Calijuri: ¹⁴C analysis supervision and manuscript review; A.T. Lombardi and O. Prášil: original concept and manuscript review.

Additional information

Funding

This work was supported by the Conselho Nacional de Desenvolvimento Científico e Tecnológico [302175/2015-6]; Fundação de Amparo à Pesquisa do Estado de São Paulo [2018/07988-5]; Grantová Agentura České Republiky [18-07822S].