GFP fluorescence detection via Native PAGE.

<p>To identify GFP expressing mutants 1(wt), mutant 1 (mut1) and mutant 2 (mut2) algae were loaded and intrinsic GFP fluorescence was used for detection. 10, 5, 2 and 1 ng of GFP protein were used as standard.</p

Cell size affects OD₇₅₀ measurements.

<p>(a) Cells of <i>Chlorella vulgaris</i> 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 <i>C. vulgaris</i> in TP (○) and in TP + 100 mM glucose (•). Error bars are standard error of the mean (SEM; n = 3).</p

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

<p>Biomass production in 100<i>C. vulgaris</i> 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).</p

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

<p>(a) OD₇₅₀ calibration constants (i.e. the slope of OD₇₅₀ vs dry weight biomass) were measured for a range of <i>C. vulgaris</i> 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.</p

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

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

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

<p>(a) <i>Chlamydomonas reinhardtii</i> in TAP at initial cell concentration of 10⁶ cells/mL (○) and at initial cell concentration of 2.5×10⁵ cells/mL (•). (b) <i>Chlorella vulgaris</i> 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).</p

Schematic illustration of the chloroplast vector system.

<p>Destination vector and entry vector are recombined through L-R reaction yielding the final chloroplast transformation vector. Destination vector: Chloroplast homologous sequences (plastome DNA) with ribosomal (<i>rrn5</i>) and <i>psbA</i> (<i>psbA</i>) genes are indicated. <i>attR</i> sites (<i>aatR1</i>, <i>attR2</i>) supporting L-R reaction are indicated. Entry vector: <i>attL</i> sites (<i>attL1</i>, <i>attL2</i>) participating in L-R reaction are indicated as well as the expression cassette of the selection marker <i>aadA</i> and the <i>gfp</i> expression cassette. Important restriction sites used for cloning are indicated as well as the recombination reaction performed between the <i>attR</i> and <i>attL</i> sites (<i>L-R reaction</i>). Transformation vector: Schematic illustration of final transformation vector resulting from L-R reaction between destination and entry vector.</p

Partial dehydration reduces density differences between cell populations.

<p><i>C. vulgaris</i> 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) <i>C. vulgaris</i> in TP. (2) <i>C. vulgaris</i> + 1 M NaCl. (3) <i>C. vulgaris</i> in TAP. (4) <i>C. vulgaris</i> in TAP + 1 M NaCl 1 M. (5) <i>C. vulgaris</i> in TP + glucose (100 mM). (6) <i>C. vulgaris</i> in TP + glucose (100 mM) + 1 M NaCl. (7) Standard density marker beads (g.mL⁻¹) with densities indicated.</p

Effect of sucrose, DMSO and sucrose/DMSO mixtures on cryopreservation recovery.

<p>Row 1 (black): Control lacking sucrose and DMSO. Row 2 (blue): 200 mM sucrose. Row 3 (orange): 10% DMSO. Row 4 (red), 200 mM sucrose and 10% DMSO. Numbers represent different algae isolates. The data values are an average of triplicates and error bars represent the standard deviation.</p

The effect of cell concentration on cryopreservation efficiency.

<p>Cell viability of strains 3 (red square), 12 (blue triangle) and 19 (black circle) expressed as % CFU / theoretically plated cells. Results of duplicate experiments are shown for 1, 5, 10 and 68 million cell mL⁻¹. Error is represented as the standard error of the mean and lines of best fit are spline fits.</p