Growth parameters and responses of green algae across a gradient of phototrophic, mixotrophic and heterotrophic conditions

Abstract

Many studies have shown that algal growth is enhanced by organic carbon and algal mixotrophy is relevant for physiology and commercial cultivation. Most studies have tested only a single organic carbon concentration and report different growth parameters which hampers comparisons and improvements to algal cultivation methodology. This study compared growth of green algae Chlorella vulgaris and Chlamydomonas reinhardtii across a gradient of photoautotrophic-mixotrophic-heterotrophic culture conditions, with five acetate concentrations. Culture growth rates and biomass achieved were compared using different methods of biomass estimation. Both species grew faster and produced the most biomass when supplied with moderate acetate concentrations (1-4 g L^-1), but light was required to optimize growth rates, biomass yield, cell size and cell chlorophyll content. Higher acetate concentration (10 g L^-1) inhibited algal production. The choice of growth parameter and method to estimate biomass (optical density (OD), chlorophyll a fluorescence, flow cytometry, cell counts) affected apparent responses to organic carbon, but use of OD at 600, 680 or 750 nm was consistent. There were apparent trade-offs among exponential growth rate, maximum biomass, and culture time spent in exponential phase. Different cell responses over 1-10 g L^-1 acetate highlight profound physiological acclimation across a gradient of mixotrophy. In both species, cell size vs cell chlorophyll relationships were more constrained in photoautotrophic and heterotrophic cultures, but under mixotrophy, and outside exponential growth phase, these relationships were more variable. This study provides insights into algal physiological responses to mixotrophy but also has practical implications for choosing parameters for monitoring commercial algal cultivation.

Keywords: Acetate; Algal cultures; Carrying capacity; Chlorophyll a fluorescence; Exponential growth; Flow cytometry; Optical density; Photosynthesis; Physiology.

Figure 1. Example growth curve showing growth parameters used for analysis.

Plot of growth of Chlorella vulgaris in a batch culture example to illustrate the growth parameters reported in Figs. 2–5. The period of exponential growth phase (grey box) was estimated from the linear portion of a plot of the natural log of biomass over time (top plot).

Figure 2. Algal growth rates based on chl a fluorescence and optical density measurements.

Growth rates of Chlorella vulgaris and Chlamydomonas reinhardtii, based on Chl a fluorescence (A and B) or OD (600, 680, 750 nm; C and D). Growth rates were calculated during exponential growth phase of batch cultures. Cultures were maintained in light with 0 g L⁻¹ acetate (photoautotrophic conditions), supplied with light and acetate (1, 2, 3, 4 or 10 g L⁻¹) (mixotrophic), or 2 g L⁻¹ acetate in the dark (heterotrophic). Bars are mean + standard deviation from four replicate cultures, and treatments with significant different values are indicated with different lowercase letters (1-way ANOVA). For OD, statistical differences between treatments are shown only for OD at 680 nm.

Figure 3. Maximum algal biomass achieved in cultures.

Maximum biomass achieved in batch cultures of green algae, based on Chl a fluorescence (A and B) or OD (600, 680, 750 nm; C and D). Values for Chl a fluorescence or OD were estimated during stationary phase of growth. Treatments are the same as Fig. 2. Bars are mean + standard deviation from four replicate cultures, and treatments with significant different values are indicated with different lowercase letters (one-way ANOVA). For OD, statistical differences are shown for OD at 680 nm.

Figure 4. (A–D) Culture time period spent in exponential phase of growth.

Time spent in exponential growth phase for batch cultures of green algae based on Chl a fluorescence or OD (at 600, 680 or 750 nm). Treatments are as in Fig. 2. Bars are mean + standard deviation from four replicate cultures, and treatments with significant different values are indicated with different lowercase letters (one-way ANOVA). For OD, statistical differences are shown for OD at 680 nm only.

Figure 5. Algal culture growth rates based on different estimates of biomass.

(A) Chlorella. (B) Chlamydomonas. Growth rates calculated for batch cultures of green algae based on cell counts from flow cytometry and haemocytometer counts, chl fluorescence and OD during exponential phase of growth. Three treatments were 0 g L⁻¹ acetate (photoautotrophic), 2 g L⁻¹ acetate plus light (mixotrophic), and 2 g L⁻¹ acetate in the dark (heterotrophic). Bars are mean + standard deviation from three replicate cultures. Statistically significant growth rates within each species and treatment are indicated with different lowercase letters (two-way ANOVA).

Figure 6. Cell size and chl fluorescence per cell in cultures.

Changes in cell size and chl fluorescence per cell estimates over the culture growth period in the two algal species in photoautotrophic (A–B), mixotrophic (2 g/L acetate; C–D) and heterotrophic (E–F) growth conditions, for Chlorella vulgaris (A, C, E) and Chlamydomonas reinhardtii (B, D, F). Cell size estimates are based on the mean FSC-H for each sample (filled symbols, left scales) and chl fluorescence per cell (open symbols, right scales) is based on mean FL-3 values for each sample, both using the same flow cytometry gates determined for each species. Plots of the two species for each growth condition treatment are on the same scales. Exponential growth period is indicated with grey bars at top of plots. All points represent a mean of mean FSC-H or FL-3 values for three replicate cultures ± standard deviation.

Figure 7. Relationships between cell size and cell chl fluorescence.

Relationships between flow-cytometry estimates of cell size (mean FSC) and chl fluorescence per cell (mean FL-3) in Chlorella vulgaris (filled symbols) and Chlamydomonas. reinhardtii (open symbols) during photoautotrophic, mixotrophic (2 g/L acetate) or heterotrophic culture conditions. Points are mean FSC and FL-3 values from samples from three replicate cultures (mean values over time are shown in Fig. 6). A, C, E plots show all points over culture experiment and B, D, and F plots show points only during exponential growth phase. Lines for each species are fitted by linear regression. R² values for linear regressions: All points Exponential Growth Photoautotrophic: Chlorella – 0.263, Chlamy – 0.317, Chlorella – 0.247, Chlamy – 0.284; Mixotrophic: Chlorella – 0.0174, Chlamy – 0.0776, Chlorella – 0.134, Chlamy – 0.364; Heterotrophic: Chlorella – 0.284, Chlamy – 0.671 Chlorella – 0.747, Chlamy – 0.168.