Effects of acetate on facultative autotrophy in Chlamydomonas reinhardtii assessed by photosynthetic measurements and stable isotope analyses

Abstract

The green alga Chlamydomonas reinhardtii can grow photoautotrophically utilizing CO(2), heterotrophically utilizing acetate, and mixotrophically utilizing both carbon sources. Growth of cells in increasing concentrations of acetate plus 5% CO(2) in liquid culture progressively reduced photosynthetic CO(2) fixation and net O(2) evolution without effects on respiration, photosystem II efficiency (as measured by chlorophyll fluorescence), or growth. Using the technique of on-line oxygen isotope ratio mass spectrometry, we found that mixotrophic growth in acetate is not associated with activation of the cyanide-insensitive alternative oxidase pathway. The fraction of carbon biomass resulting from photosynthesis, determined by stable carbon isotope ratio mass spectrometry, declined dramatically (about 50%) in cells grown in acetate with saturating light and CO(2). Under these conditions, photosynthetic CO(2) fixation and O(2) evolution were also reduced by about 50%. Some growth conditions (e.g. limiting light, high acetate, solid medium in air) virtually abolished photosynthetic carbon gain. These effects of acetate were exacerbated in mutants with slowed electron transfer through the D1 reaction center protein of photosystem II or impaired chloroplast protein synthesis. Therefore, in mixotrophically grown cells of C. reinhardtii, interpretations of the effects of environmental or genetic manipulations of photosynthesis are likely to be confounded by acetate in the medium.

Figure 1

Carbon isotope composition (δ¹³C) of cell biomass during photoautotrophic and heterotrophic growth of C. reinhardtii. A, Heterotrophic growth of C. reinhardtii on HSHA containing 29.4 mm acetate of varying δ¹³C. Cells of the non-photosynthetic mutant strain CC-744 were grown in dim light (5–25 μmol m⁻² s⁻¹) on HSHA plates (●) or 250-mL HSHA shake flask cultures (▪) formulated with sodium acetates differing in natural abundance of ¹³C. The stable carbon isotope compositions of the source acetates and lyophilized biomass were determined as described in the text. B, Photoautotrophic growth of wild-type C. reinhardtii in 250-mL liquid cultures bubbled with 5% CO₂ under moderate light (350 μmol m⁻² s⁻¹) at 25°C. Samples were harvested at the indicated times for hemocytometer counts, spectrophotometric determination of biomass concentration (A₇₅₀), and biomass δ¹³C measurement. Each value and data point are the means ± se (in parentheses) of two independent measurements. Data points were fitted to a logistic growth equation. Cell concentration at the beginning of the experiments was 2 × 10⁵ cells mL⁻¹ (A₇₅₀ = 0.01).

Figure 2

Photosynthetic fraction of carbon biomass from wild-type and mutant C. reinhardtii cells grown on ¹³C-TAP (17.5 mm acetate) in liquid culture. Cells of wild type (circles), spr/sr (diamonds), and dr (squares) were grown autotrophically to the early-/mid-exponential phase in HS bubbled with 5% CO₂ (δ¹³C = +2.9‰), heterotrophically in the dark on TAP supplemented with ¹³C (δ¹³C = +99‰; ▴), and mixotrophically on ¹³C TAP bubbled with 5% CO₂ at two irradiance levels, 350 μmol m⁻² s⁻¹ (closed symbols) and 600 μmol m⁻² s⁻¹ (open symbols). The fraction of carbon biomass resulting from photosynthetic carbon reduction was calculated as described in the text from the average photoautotrophic, heterotrophic, and mixotrophic biomass δ¹³C values.