Growth data

In order to simplify basic culture maintenance and experimentation, growth rates of eukaryotic algal stocks C. vulgaris, C. emersonii, S. vacuolatus, P. ellipsoidea (‘obi’ and ‘ni’) were determined in BBM and ‘Denso media’ (recommended medium for P. ellipsoidea by Denso) under otherwise ‘standard’ growth conditions outlined in Chapter 2.1.4. Both cell count (Figure 3.3) and optical density (OD) (Figure 3.4) were measured. OD is an excellent indicator of biomass, yet this requires careful calibration for each species. Cell count is more accurate to monitor growth, as absorbance can change as health or metabolism of the culture changes (i.e. cell flocculation, (data not shown)).

Overall, all strains have faster cell doublings (growth rates) in BBM than ‘Denso media’, with the exception of P. ellipsoidea ‘ni’ where growth rates in both media were comparable (Figure 3.3). Cell doublings took place approximately every 72h for all strains. The large difference in cell number between C. vulgaris and all other strains is due to its significantly smaller cell size (Figure 3.3) ~8times smaller than C. emersonii and S. vacuolatus (data not shown). Absorbance readings showed that in terms of biomass, C. vulgaris accumulated a similar quantity to the other strains (Figure 3.4). Up to day 12 growth rates of all strains under the different media were comparable. After day 12 however, high cell doubling rates continued for strains in BBM, yet were slowed in ‘Denso medium’ suggesting depletion of a particular nutrient. These results were also reflected in the OD readings (Figure 3.4).

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Figure 3.3: Microalgal growth of stock eukaryotic strains (C. vulgaris, C. emersonii, S. vacuolatus and P. ellipsoidea ‘obi’ and ‘ni’) in both BBM and ‘Denso media’ under ‘standard conditions’ measured by cell count using a haemocytometer. Three repeats of measurements were carried out for each measurement, error bars = S.D.

Figure 3.4: Microalgal growth of stock eukaryotic strains (C. vulgaris, C. emersonii, S. vacuolatus and P. ellipsoidea ‘obi’ and ‘ni’) in both BBM and ‘Denso media’ under ‘standard conditions’ measured by optical density(OD) at 550nm using a spectrophotometer. Three repeats of measurements were carried out for each measurement, error bars = S.D.

83 Growth measurements were repeated in BBM for our 3 stocks C. vulgaris, C. emersonii and S. vacuolatus, measuring growth by cell count, absorbance and dry weight (Figure 3.5). These results were highly comparable to those obtained previously (Figures 3.3 and 3.4). Under standard growth conditions in BBM, C. vulgaris, C. emersonii and S. vacuolatus had cell doubling times of ~3d (Figure 3.5a) and a comparable pace of ‘approximated’ biomass accumulation (Figure 3.5b). So as not to greatly reduce the volume in our standard 100ml culture flasks (as this could affect growth characteristics) and to ensure repetition, determination of biomass (Figure 3.5c) was carried out using very small sample sizes. Both C. vulgaris and C. emersonii were difficult to pellet as cells adhered to the outer walls of Eppendorf tubes and upon removal of supernatant, would be drawn off the walls as a result of liquid surface tension. Due to this and the small sample sizes there was a large error margin, too large to accurately calibrate the absorbance curve to ascertain biomass with accuracy (Figure 3.5b).

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Figure 3.5: C. vulgaris, C. emersonii and S. vacuolatus cultured in BBM under ‘standard conditions’.

Growth measured by (a) cell count, (b) absorbance 550nm and (c) dry weight. Three repeats of measurements were carried out for each measurement, error bars = S.D.

85 3.2.2 Visualising the cell wall using staining

Stains were screened on all strains to determine their behaviour, in particular cell wall stains, with the vision of using staining as a means for detecting changes in the cell wall after enzyme treatment. Firstly the common cell wall stain calcofluor (which stains β1,4 and β1,3-glucans) was investigated on our 5 stock strains (Figure 3.6).

Figure 3.6: Microalgal stock strains (C. vulgaris, C. emersonii, S. vacuolatus and P. ellipsoidea ‘obi’ and

‘ni’) stained with calcofluor and viewed under the light microscope and confocal (ex488nm).

Cv (C. vulgaris) is the only strain to show strong staining with calcofluor. Bar = 20µm.

It is accepted that all Chlorophyta contain some -polysaccharides in their cell wall yet C. emersonii, S. vacuolatus did not appear to stain at all and P. ellipsoidea ‘obi’ and ‘ni’

stained very weakly. These 4 strains did however, show strong staining when the cell wall was broken or damaged, as if calcofluor could leak through an impenetrable outer layer of the wall. The variation in strength of staining was further investigated with C. vulgaris, by looking at cell cycle stages to determine if stain intensity changed with stages of the cell cycle (Figure 3.7).

Figure 3.7: Different growth stages of C. vulgaris stained with calcofluor under the light microscope and confocal.

Cell size and cycle stage appear to have no discernable differences in staining, with percieved increases in brightness due to layers of cell wall material. Confocal excitation 488nm, bar = 20µm.

86 Cell walls of C. vulgaris showed that there was no change in level of staining during different growth stages. Dividing cells inside the mother cell appear slightly brighter due to layers of stained material (Yamamoto et al., 2004). It appeared that the ability of algae to stain with calcofluor was species specific. As calcofluor could not be used as a means of monitoring cell wall change in algae treated with enzyme mixtures, it was decided to screen other common stains, if only to observe any other unusual staining behaviour. Chapter 2.2.5 outlines the target material or chemical of the commonly used stains tested. Included in the screening of stains were isolated algae from the Roman Baths (discussed in Chapters 5 and 6).

Table 3.2: Summarised stain screening results for all strains.

Aceto

-no staining, + stains target weakly, ++ stains target strongly *also stains the cell wall

Inability to stain with cell wall stains was found for C. emersonii, S. vacuolatus, C.saipanenensis, P. ellipsoidea ‘obi’ and ‘ni’ and the cyanobacteria O. sancta, M. chthonoplastes and C. thermalis. As far back as 1985 (Rahat and Reich) it was noted that C. emersonii contained a tough biopolymer in its outer wall. The inability to stain cell walls of microalgae is often linked to the presence of this biopolymer (Rodriguez and Cerezo, 1996).

This biopolymer is observed in many species and has since been termed algaenan (Burczyk et al., 1999). Algaenan can be visualised using TEM (Chapter 3.2.5).