Flow cytometry pulse width data enables rapid and sensitive estimation of biomass dry weight in the microalgae Chlamydomonas reinhardtii and Chlorella vulgaris

Abstract

Dry weight biomass is an important parameter in algaculture. Direct measurement requires weighing milligram quantities of dried biomass, which is problematic for small volume systems containing few cells, such as laboratory studies and high throughput assays in microwell plates. In these cases indirect methods must be used, inducing measurement artefacts which vary in severity with the cell type and conditions employed. Here, we utilise flow cytometry pulse width data for the estimation of cell density and biomass, using Chlorella vulgaris and Chlamydomonas reinhardtii as model algae and compare it to optical density methods. Measurement of cell concentration by flow cytometry was shown to be more sensitive than optical density at 750 nm (OD750) for monitoring culture growth. However, neither cell concentration nor optical density correlates well to biomass when growth conditions vary. Compared to the growth of C. vulgaris in TAP (tris-acetate-phosphate) medium, cells grown in TAP + glucose displayed a slowed cell division rate and a 2-fold increased dry biomass accumulation compared to growth without glucose. This was accompanied by increased cellular volume. Laser scattering characteristics during flow cytometry were used to estimate cell diameters and it was shown that an empirical but nonlinear relationship could be shown between flow cytometric pulse width and dry weight biomass per cell. This relationship could be linearised by the use of hypertonic conditions (1 M NaCl) to dehydrate the cells, as shown by density gradient centrifugation. Flow cytometry for biomass estimation is easy to perform, sensitive and offers more comprehensive information than optical density measurements. In addition, periodic flow cytometry measurements can be used to calibrate OD750 measurements for both convenience and accuracy. This approach is particularly useful for small samples and where cellular characteristics, especially cell size, are expected to vary during growth.

Figure 1. Cell size affects OD₇₅₀ measurements.

(a) Cells of Chlorella vulgaris grown photoautotrophically in unsupplemented TP have a smaller average diameter than (b) cells grown to stationary phase in Tris-Phosphate (TP) medium containing 100 mM glucose. (c) Changes in cell morphology affect the relationship between OD₇₅₀ and biomass dry weight, yielding different calibration factors (line slope) for C. vulgaris in TP (○) and in TP + 100 mM glucose (•). Error bars are standard error of the mean (SEM; n = 3).

Figure 2. Growth curves monitored in 96 well plates by OD₇₅₀ in a plate reader (effective path length ∼0.2 cm) at different initial cell densities.

(a) Chlamydomonas reinhardtii in TAP at initial cell concentration of 10⁶ cells/mL (○) and at initial cell concentration of 2.5×10⁵ cells/mL (•). (b) Chlorella vulgaris in TAP at initial cell concentration of 10⁶ cells/mL (□) and at initial cell concentration of 2.5×10⁵ cells/mL (▪). Error bars are SEM (n = 3).

Figure 3. Growth curves monitored in 96 well plates, measuring cell concentration with flow cytometry.

Cell concentration (cells/µL) was estimated from flow cytometry events, calibrated using CountBright Absolute Counting Beads (Invitrogen). Error bars are SEM (n = 3). (a) Chlamydomonas reinhardtii strain 137c at initial cell concentration of 10⁶ cells/mL (○). (b) C. reinhardtii strain 137c at initial cell concentration of 2.5×10⁵ cells/mL (•). (c) Chlorella vulgaris at initial cell concentration of 10⁶ cells/mL (□). (d) C. vulgaris at initial cell concentration of 2.5×10⁵ cells/mL (▪).

Figure 4. Biomass determination by dry weight, compared to OD₇₅₀ and cell concentration (by flow cytometry and direct counting) at three points in the growth curve.

Biomass production in 100C. vulgaris in g dry weight (g_dw) L⁻² day⁻¹ was estimated by collecting 3×12 ml at each of 3 time points; “lag phase” (t = 0), “log phase” (after ∼10 hours of growth) and “stationary phase” (24–27 hours; immediately after growth stabilised). Filled bars signify cells grown in TAP, open bars represent cells grown in TP + 100 mM glucose. Measurements at each time point were (a) Flow cytometry (200 µL culture was diluted into 1.5 mL TP and cell concentration measured by flow cytometry) (b) OD₇₅₀ (estimated in triplicate using 100 µL aliquots of culture diluted with 900 µL TAP or TP + 100 mM glucose) (c) Dry weight determinations (performed as described in the Methods section and (d) Microscopy (cell concentration was estimated microscopically in triplicate using a haemocytometer, from 10 µL of culture). Error bars are SEM (n = 3).

Figure 5. Use of flow cytometry side scatter pulse width (SSC-W) to estimate bead diameter.

Mean SSC-W was estimated for a range of silica and polystyrene size marker beads. (a) Histogram plots of silica beads (Si2: 2 µm; Si8: 8 µm) and polystyrene beads (P2: 2 µm; P7: 7.4 µm) contrasted with the SSC-W profile of Chlorella vulgaris (Cv). (b) Calibration curves of SSC-W vs bead diameter for silica (•) and polystyrene (▴) beads.

Figure 6. Flow cytometry pulse width measurements correlate well with cell diameter.

Fluorescence activated cell sorting (FACS) of C. vulgaris cells was used to sort a mixture of cells and beads, according to SSC-W range. Cells and polystyrene marker beads were photographed after sorting (a) in the range SSC-W = 60000–65000 (cells grown in TP), and (b) in the range SSC-W = 70000–80000 (cells grown in TP+100 mM glucose). Representative beads (black arrows) and cells (grey arrows) marked are indicated. (c) Diameters were measured microscopically using IMARIS software for ∼100 cells or beads after sorting and the mean, standard deviation and range was plotted for each population of cells and the marker beads.

Figure 7. Partial dehydration reduces density differences between cell populations.

C. vulgaris biomass was measured during lag phase, log phase and stationary phase in TP with 100 mM glucose. (a) Flow cytometry was used to estimate average cell diameter using SSC-W measurements and plotted vs average dry weight per cell. Flow cytometry was performed on cells diluted into TP growth medium (▵) or hypertonic media (1 M NaCl in TP) (▵) and allowed to dehydrate for 10min. The use of NaCl improves the linearity of the relationship between biovolume and biomass dry weight. All measurements were conducted in triplicate; error bars represent the standard error of the mean. (b) Percoll density gradient centrifugation demonstrates a lower, more uniform range of cell densities when measured in medium containing 1 M NaCl. Tubes are labelled as follows: (1) C. vulgaris in TP. (2) C. vulgaris + 1 M NaCl. (3) C. vulgaris in TAP. (4) C. vulgaris in TAP + 1 M NaCl 1 M. (5) C. vulgaris in TP + glucose (100 mM). (6) C. vulgaris in TP + glucose (100 mM) + 1 M NaCl. (7) Standard density marker beads (g.mL⁻¹) with densities indicated.

Figure 8. OD₇₅₀ measurements can be corrected using flow cytometry to yield reliable biomass estimates.

(a) OD₇₅₀ calibration constants (i.e. the slope of OD₇₅₀ vs dry weight biomass) were measured for a range of C. vulgaris cultures at different growth stages (with different average cell diameter). These values were plotted against flow cytometry (peak SSC-W) measurements taken on the same cultures. (b) Using this calibration curve, biomass dry weight was then predicted for a different set of previous cultures for which both biomass and OD₇₅₀ were known. Comparison of the predicted biomass and measured biomass demonstrates a linear relationship (r² = 0.981), suggesting the use of FC measurements to correct OD₇₅₀ measurements enables a linear relationship to be established between OD₇₅₀ and biomass dry weight regardless of the cell morphology or culture stage.