Extraction of ‘algaenan’

The following method is adapted from Allard et al., (1998). A large quantity of lyophilised algal biomass (over 10g) was put under reflux in a 500ml round bottom flask with CHCl₃:MeOH (2:1 v/v) for 3h, the solid filtered and rinsed with CHCl₃ (to extract lipids). The solid was then hydrolysed in 2N TFA (100ºC, 3h), then further hydrolysed in 4N TFA (100ºC, 18h) before final hydrolysis in 6N TFA (100ºC, 18h) (to extract polysaccharides). Solid residue was thoroughly washed with dH2O before putting under reflux in dH2O for 1h. Solid then refluxed in CHCl3:MeOH (2:1 v/v) for 3h, saponified by reflux in 5% KOH in 2-methoxyethanol:H2O (88:12 v/v) for 1h. Solid then put under reflux in 6N HCl 100ºC for 24h.

Solid residue filtered and washed with H2O before refluxing in H2O for 1h and finally refluxing CHCl3:MeOH (2:1 v/v) for 3h. Final residue was filtered out and rinsed with H2O before allowing to dry at room temperature overnight.

56 2.2.10 Cultivation of Fusarium oxysporum f.sp elaeidis and exposure to algaenan

Fusarium oxysporum f.sp. elaeidis, an oil palm pathogen, was donated by Hefni Rusli (University of Bath) for investigating the digestion of algaenan with a live fungus. Czapek-Dox media with different concentrations of sucrose (carbon source) were prepared (Table 2.9). Minimal carbon media (Cmin) was tested alongside carbon deficient media (C-) as some fungi require ‘metabolic momentum’ in order to synthesise enzymes for the digestion of tough materials (Cooper, 2012).

Table 2.9: Recipe for Czapek-Dox media for the liquid culture of F.oxysporum f.sp. elaeidis including media modification for the reduction of carbon content.

Adapted from Rusli, (2012).

Magnesium sulphate MgSO₄ 0.500 0.500 0.500

Potassium chloride KCl 0.500 0.500 0.500

Ferrous sulphate FeSO₄ 0.010 0.010 0.010

Liquid media were adjusted to pH 7.3 using HCl prior to autoclaving, as ~pH7 is optimal for cutinase production in F.oxysporum (Pio et al., 2008). Due to the formation of a cloudy residue after autoclaving, 15ml of media was filtered using a 25ml NORM-JEX ® syringe and sterile MILLEX GS 0.22µm filter into sterile 100ml conical flasks covered with foil. Flasks were incubated at 28°C at 150xg in a Stuart S150 orbital incubator for 5 days. For the investigation into the digestion of algaenan or algae (autoclaved, frozen and live) by F.oxysporum .sp. elaeidis flasks were inoculated to give a final concentration of algal cells of

~1.5 x10⁶ cells ml^-1. For samples containing algaenan a visible quantity was added (not weighable <1mg). The growth of F.oxysporum f.sp. elaeidis was measured over 5 days using a haemocytometer. In samples where some hyphal growth was observed, ‘cell counts’ for hyphae were estimated based on the length of an average spore (¼ of the length of the side of the smallest haemocytometer well ~ 0.625µm).

Where F.oxysporum sp. elaeidis was cultured on agar plates, plates were prepared by adding 1% (w/v) of agar to the media. After autoclaving molten agar-media was poured into 90mm Petri dishes (Sterilin). Plates were inoculated centrally with F.oxysporum f.sp. elaeidis using 10µl of culture and allowed to dry for 5 min in a laminar flowhood. For the investigation into the digestion of the algal cell wall, 10μl algal samples (autoclaved, frozen and live) were

57 dropped approximately 1cm from the edge of the plates. In the case of algaenan, small visible samples (~5mm in diameter) were transferred 1cm from the edge of the plate. Plates were incubated at 28°C for 5 days.

After 5 days plates were photographed and viewed under the inverted microscope. Liquid samples containing algae (autoclaved, frozen and live) were transferred to 15ml greiner falcon tubes and pelleted at 3000xg for 5 min. Pellets were then resuspended in 5ml sodium phosphate buffer. 1ml of this sample was then transferred to an Eppendorf tube containing 0.05% calcofluor in 25mM sodium phosphate buffer, inverted to mix, and incubated for 5min.

Samples were then transferred to a microscope slide and viewed under the confocal microscope.

Liquid cultures of F.oxysporum f.sp. elaeidis in ‘Cmin’ media were transferred to 15ml greiner falcon tube and pelleted (13,000xg, 5min). The supernatant was transferred to a new 15ml greiner falcon tube and a visible quantity of C. emersonii algaenan added (<1mg). The sample was then incubated at 28°C, 100xg. After 24hr the sample was filtered and supernatant retained for further processing and GC-MS analysis.

Liquid samples in Cmin media containing algaenan; the control (with no fungus), fungus cultivated in the presence of algaenan and Cmin fungal culture supernatant was subsequently filtered and used for the digestion of algaenan (described above), were all filtered and then subjected to transesterification, washing and GC-MS analysis as follows. To ~15ml filtrate samples 20ml CHCl3:MeOH (2:1) and 1 drop concentrated H2SO4 were added into a 50ml round bottomed flask and shaken well. Samples were then transesterified at 80°C and stirred at ~300xg for 3h. Samples were then washed with dH2O, dried and dissolved in 200μl of remaining solution was located directly under a UV biocidal radiation lamp (BioTechne Hepa UV laminar flow hood) for 5 min (~53cm from bulb). After 5 min the solution was agitated, 1ml sample was then taken.This procedure was repeated for samples taken at 10, 15, 20, 25,

58 30min (this was changed to sampling every 2min after the first UV mutation curves were generated). These samples were then diluted 1 in 100 (10µl samples to 990µl BBM), then further diluted 1 (40µl) in 5 (200µl), to give a final 1 in 500 dilution.

30µl of each sample (and replicates) was then plated onto 90mm Sterilin plates (BBM 1%

(w/v) agar) and appropriately labelled. The plates were then sealed with Parafilm and stored in the algal Fitotron ® growth room until colonies appeared. Suitable exposure time for UV mutagenesis was determined by plotting number of survivors against UV exposure.

C. emersonii underwent further mutagenesis with UV. Mutagenesis was carried out as described above at the ‘10% survival UV mutagenic dose’ (for C. emersonii this was 11min), omitting the final 1 in 5 dilution step prior to plating. Approximately 100 plates were prepared from this sample in order to screen for mutants.