Enzyme studies

C. vulgaris, C. emersonii, S. vacuolatus responded similarly to enzyme treatments (with the former being most affected and subjected to further testing). All enzymes tested singly and in mixtures (including Nicotiana spp. or Lilium spp. bud extracts) had little effect on the cell

87 wall when observed with calcofluor staining and with subsequent addition of SDS to rupture sensitive cells. The discovery of ‘algaenan’ and its effect on staining and enzymatic resistance its resistance to enzymes, led to one final set of experiments using enzymes on C. vulgaris (as an algaenan negative species), P. ellipsoidea ‘obi’ and ‘ni’ using sensitivity to SDS and a haemocytometer as a means to measure changes or weakening of the cell wall.

Figure 3.8: C. vulgaris cell sensitivity to lysis after exposure to various enzymes (12%w/v, over 3h).

Enzymes were tested singly and as a mixture (‘All’). Samples were analysed using a hemocytometer and results expressed as a percentage of intact cells compared to the control. Three repeats were performed for each measurement, error bars = S.D.

Figure 3.9: P. ellipsoidea ‘obi’ and ‘ni’ cell sensitivity to lysis after exposure to various enzymes (12%w/v, over 3h).

Enzymes were tested singly and as a mixture (‘All’). Samples were analysed using a hemocytometer and expressed as a percentage of intact cells compared to the control. Three repeats were performed for each measurement, error bars = S.D.

C. vulgaris was most sensitive to single enzymes β-glucanase, cellulase, pectinase and natural mixtures ‘Driselase’ and ‘Macerozyme’ (Figure 3.8). Affected cells however did not represent

88 more than 20% of the population, which is perhaps likely due to the affected cells representing a proportion, either undergoing cell division or cell death. The most effective were enzyme mixtures Driselase and ‘All’ (all listed enzymes). This suggests that despite the lack of algaenan C. vulgaris cell walls remain strong and resistant to enzymatic attack. The cell wall of P. ellipsoidea ‘obi’ was marginally more weakened by enzyme exposure than P. ellipsoidea ‘ni’. Both show similar behaviour when exposed to enzymes, with the exception of Macerozyme which strongly affected ‘obi’ but not ‘ni’ (Figure 3.9). Driselase appeared most effective for ‘ni’. However, like C. vulgaris only a small fraction of cells was ever affected.

3.2.4 Scanning electron microscopy of C. vulgaris and S. vacuolatus

SEM was used to try and identify why in a concentrated culture, cells of S. vacuolatus began to autoflocculate (unlike our other strains). Autoflocculation is a desirable trait, as dewatering of algal cultures adds a large cost to its production. It was found that S. vacuolatus cells were encased in a loose ‘membrane’, creating furrows which may have helped cells stick together (Figure 3.10). In contrast C. vulgaris cells were smooth. This could be caused by a difference in osmotic requirements between the species, despite both having been cultured under identical conditions. In addition pH can also affect flocculation of algal cultures due to surface charges (Kaloudis, 2012)

Figure 3.10: SEM images of (a) C. vulgaris and (b) S. vacuolatus.

(a) cell of C. vulgaris showing a smooth outer cell surface. (b) cells of S. vacuolatus showing a wrinkly cell surface. Cells of S. vacuolatus showed loose outer membranous material.

89 3.2.5 Transmission electron microscopy for visualisation of the cell wall

TEM sections were made of all stock species and Roman Bath isolates (Chapters 5 and 6) to better visualise the cell wall due to the odd staining behaviour exhibited. For C. vulgaris and P. ellipsoidea samples were prepared and viewed at the University of Bath, with voltage settings on the TEM at 120kV, for good contrast of biological samples with minimal sample damage. C. vulgaris had a thicker cell wall than P. ellipsoidea and there was no presence of a TLS (Figure 3.11). There was a visible TLS in P. ellipsoidea, which appeared to be the only visible constituent of the cell wall, making it remarkably thin for the overall cell size (Potter, 2009). The TLS is likely to be a biopolymer such as algaenan. In P. ellipsoidea the algaenan cell wall not only accumulated in the medium but strongly associated with cells, sticking dividing cells together forming clumps.

Figure 3.11: TEM images comparing the cell walls of C. vulgaris and P. ellipsoidea.

(a) C. vulgaris with a thick cell wall lacking a TLS and (b) P. ellipsoidea with a very thin cell wall comprised exclusively of a TLS likely algaenan. pm = plasma membrane, TLS = trilaminar structure, bar = 0.1μm.

Image quality was not always good for the Roman Bath isolates as moisture was present in the resin curing oven, which had rendered them brittle and difficult to section. In order to save on project budget, Roman Bath isolates were sectioned and viewed under TEM at the Johnson Matthey site in Sonning Common. The settings of their TEM were at a much higher voltage due to the nature of their chemical samples (voltage 200kV). As protection against the higher voltage used for viewing samples were carbon coated prior to viewing. The carbon coating and higher voltage resulted in poorer contrast and added granularity to the images. Despite

90 carbon coating sections also began to show ‘bleaching’ after prolonged periods of viewing,

‘burning’ samples under the electron beam. This accounted for differences in image quality shown in the Figures.

Figure 3.12: TEM images of C. emersonii, S. vacuolatus, P. ellipsoidea ‘ni’ and C. saipanensis.

All these are green eukaryotic algae which exhibit a TLS structure in the outer wall, likely algaenan. Inserts are to show the accumulation of algaenan-containing cell walls in the media which are strongly associated with cells.

Bar = 0.2μm.

TEM images of green eukaryotic algae which showed poor staining with cell wall stains such as crystal violet and calcofluor, confirmed the presence of a TLS (Figure 3.12). This suggested the presence of the biopolymer algaenan. Inserts show accumulation of old cell walls, which remained closely associated with cells. All the algaenan containing species showed differences in the cell wall structure. C. emersonii showed multiple layers of electron dense and electron translucent layers (TLS) closely packed at the surface of the cell in addition to a thick cell wall underneath. S. vacuolatus had a single layer of what appeared to be an amorphous ‘unraveling’ TLS on top of a thick cell wall; this is also observed with C. saipanensis. P. ellipsoidea only possessed a single thin layer of TLS and apparent absence of a cell wall.

91 TEM images of the remaining Roman Bath strains revealed diverse cell wall morphologies (Figure 3.13). Species which did stain such as Klebsormidium sp., M. chthonoplastes and M. laminosus appeared to have thin walls with no apparent biopolymer. Those which showed unusual or poor staining O. sancta, M. chthonoplastes and C. thermalis all had thicker walls comprising of two or more layers and or a mucilaginous sheath. In some of these layers, fibres could be seen.

Figure 3.13: TEM images of K. sp., M. chthonoplastes , M. laminosus, O. sancta and C. thermalis.

These species do not exhibit a TLS structure in the outer wall. Some cells have thick walls comprising two or more layers. Bar = 1μm (with the exception of Os where bar = 0.2 μm).