Extraction of Cyanobacterial Slime from Community Samples and Subsequent Analysis via GC-MS

Use mature cultures (at least 35 days old, grown in BG11+ with full vitamin mix – see Table 1) and record culture origin and date of initiation.

Table 1. Full vitamin mix, prepared as a one thousand times concentrated stock, as referenced in Duxbury et al. (2023)1 Table 1. Full vitamin mix, prepared as a one thousand times concentrated stock, as referenced in Duxbury et al. (2023) ¹

A	B	C	D	E
Vitamins	g/L (stock)	g/L (medium)	g/mol	Mol/l (medium)
Biotin	0.020	0.00002	244.31	8.2E-08
Folic Acid	0.020	0.00002	441.40	4.5E-08
Pyridoxin HCl	0.100	0.0001	205.63	4.9E-07
Thiamine HCl	0.050	0.00005	337.26	1.5E-07
Riboflavin	0.050	0.00005	376.26	1.3E-07
Nicotinic Acid	0.050	0.00005	123.11	4.1E-07
D-Ca-Panthotenate	0.050	0.00005	238.27	2.1E-07
p-Aminobenzoic Acid	0.050	0.00005	137.14	3.6E-07
Vitamin B12	0.001	0.000001	1355.37	7.4E-10
Lipoic Acid	0.050	0.00005	206.33	2.4E-07

Once grown, centrifuge the cultures at 4000x g,4°C to pellet the cells.

Culture Supernatant slime/EPS. Following centrifugation – in step 2 - remove the supernatant carefully into a separate container. Filter the supernatant through a Nuclepore 5-mm pore size filter, then through a Gelman 0.45 mm membrane filter and store frozen at -4°C until lyophilised, and then use for analysis .

Next, extract any slime/EPS from the remaining cell pellet, using an adapted method inspired by both Plude et al. (1991)²and Nakagawa et al. (1987)⁵.

After centrifugation – in step 2 - approximately measure the culture pellet volume and resuspend in 30 times the pellet volume of ddH₂O (vortex vigorously). For this dilution step, we use a centrifuge tube which has volume markings, so if the pellet occupies 1mL of the tube, then add 30mL of ddH₂O. Store the suspension at 4°C. The slime should separate from the cell pellet and enter the ddH₂O.

Next morning, centrifuge the cell pellet solution again 4000x g,4°C, to pellet the cells. Remove this supernatant and pass into sterile glassware, store this at 4°C whilst we extract the rest of the slime.

Suspend the cell pellet (which can measure approx. 5mL for a 200 mL culture) in 25mL of ddH₂O (or more, scale accordingly with the initial culture size).

1. Vortex the samples for 2 minutes max, split between twelve 2 mL centrifuge tubes and centrifuge at 15000x g in a benchtop centrifuge. This sheds a portion of the slime and the fragments enter the supernatant. Extract the supernatant from each tube, pool it together, and resuspend each pellet in fresh 1.5mL of ddH₂O. (1/5)
2. Vortex the samples for 2 minutes max, split between twelve 2 mL centrifuge tubes and centrifuge at 15000x g in a benchtop centrifuge. This sheds a portion of the slime and the fragments enter the supernatant. Extract the supernatant from each tube, pool it together, and resuspend each pellet in fresh 1.5mL of ddH₂O. (2/5)
3. Vortex the samples for 2 minutes max, split between twelve 2 mL centrifuge tubes and centrifuge at 15000x g in a benchtop centrifuge. This sheds a portion of the slime and the fragments enter the supernatant. Extract the supernatant from each tube, pool it together, and resuspend each pellet in fresh 1.5mL of ddH₂O. (3/5)
4. Vortex the samples for 2 minutes max, split between twelve 2 mL centrifuge tubes and centrifuge at 15000x g in a benchtop centrifuge. This sheds a portion of the slime and the fragments enter the supernatant. Extract the supernatant from each tube, pool it together, and resuspend each pellet in fresh 1.5mL of ddH₂O. (4/5)
5. Vortex the samples for 2 minutes max, split between twelve 2 mL centrifuge tubes and centrifuge at 15000x g in a benchtop centrifuge. This sheds a portion of the slime and the fragments enter the supernatant. Extract the supernatant from each tube, pool it together, and resuspend each pellet in fresh 1.5mL of ddH₂O. (5/5)
6. Collect the supernatant with each iteration.
   Note

   By the third iteration, the supernatant – for our samples - had a slight blue colour, possibly from cellular photopigments being extracted as well.

Further slime extraction from pellet . Following on from the last cycle of step 3.3 collect the remaining pellets – using ddH₂O - into one falcon tube, and add 10mL of ddH₂O to further loosen the pellet.

• Shear the cyano pellet solution, by pipetting up and down into a 10-mL syringe through a 18G needle 20 times, follow this by repeating the shearing steps with a 25G needle, also 20 times. Hope this removes the final remaining slime sheaths from cyanobacteria filaments.
• Centrifuge the cyano pellet again, and collect the supernatant and add to the other supernatant solutions collected in the previous steps 3.2 and 3.3.
• Keep the remaining cyano pellet for further analysis.
  Note

  => this sample is called “Culture cyano pellet” below.

Following the iterations of slime extraction, which gives a final volume of 90mL supernatant containing slime, to which add 45mg of CaCl₂ (final conc. of 500mg/L CaCl₂). Mix this and leave it in the fridge (4°C).

On the next day, centrifuge the solution (from step 4) at 15000x g, to pellet out the slime. Remove the supernatant - a green-tinged transparent gelatinous material in our case - and store in the fridge for further analysis if wished.

Also, store the “slime pellet” from step 5 with a final volume of less than 1mL .

Prior to GC, lyophilise the Culture supernatant, Culture cyano pellet, Culture slime supernatant and Culture slime pellet fractions.

*Hydrolysis (see also Hydrolysis NOTE). Use acid hydrolysis over acidic methanolysis as total hydrolysis is desired, and a more thorough hydrolysis can be achieved with the harsher acid hydrolysis method.

Weigh 40mg of each standard, control blank, and the lyophilised culture fraction samples are into separate clean glass vials.

• In the first stage, add 1.5mL of 72% aq . H₂SO₄ to each sample. This is left stirring under Room temperature for 2h 0m 0s.

In the second stage, add 2mL of H₂O to each mixture and heat them in an oven at 80°C for 1h 0m 0s. Cool down the hydrolysis solution in an ice bath and stored at 4°C 1h 0m 0s.

Neutralise the hydrolysed sample to 7, using Na₂CO₃until CO₂ evolution subsides.

Filter the resulting 7 solutions through Gelman 0.45 mm filter into new test tubes. Then dry the hydrolysates under flowing nitrogen gas.

• In this set up, place a single branching capillaries from a line splitter into each test tube where it sits about the liquid level, then connect the line to a nitrogen cannister, which is set to allow a steady flow of nitrogen across the sample. In this set up the samples dry to completeness in 30-60 minutes.
  Note

  Hydrolysis NOTE In our experience it is possible that some known monosaccharides fail to derivatise with the above hydrolysis method. Thus, an alternative hydrolysis method would be to use HCl instead of H₂SO₄. Such a method would be similar to the one used by Zhu et al. (2014) for amino sugar hydrolysis, where it was found to have high recovery rates.⁶Alternative hydrolysis methods. Alternative hydrolysis methods. Weigh 40mg samples and standards into glass test tubes, to which add 1mL of 6Molarity (M) HCl. Then heat this to 105°C for 8h 0m 0s on the heat block, inside the chemical fume hood. Adjust this solution to pH 6.5–7.0 with 1Molarity (M) sodium hydroxide (NaOH), dry by evaporating it under N₂ as above, re-dissolved in 2mL methanol (MeOH, 99+%). Samples collected in the supernatant after centrifugation, as above. The methanol step will remove the salt formed from neutralisation. Subsequent derivatisation steps after hydrolysis as the same as described from (step 11 onwards).

Trimethylsilylation Trimethylsilylation.

Dissolve the dried hydrolysates in 1.5mL of pyridine (Scientific Laboratory Supplies Ltd, W296600, 99+%), shake by hand for 0h 0m 30s, and then incubate at Room temperature for 0h 30m 0s.

Then add 0.5mL of 1-(Trimethylsilyl)imidazole (1-TMS-imidazole, Merck, A12512.06, 97%), and incubate the samples in a shaking incubator at 60°C.

Cool at Room temperature. Remove the pyridine fully by drying with nitrogen gas, as described above. Drying will again take 30-60 minutes.

To the dried compounds, add 2mL heptane (>99.3%, LC grade) and dry under nitrogen again. Repeat the addition and drying of heptane several times until the samples no longer smell of pyridine.

Concentrations for GC. Concentrations for GC . Each sample contains 40mg of the species of interest; therefore, dissolve each sample into 8mL of hexane (>99%, LC grade, solvent used for GC), thereby creating 5mg/mL master stocks.

From this master stock, take 1mL and dilute into hexane to give a total volume of 1mL in brown glass GC vials – resulting in final concentration of 5mg/L.

This creates a set of 5mg/L working stocks, which can be taken directly to the GC. Smaller sample concentrations than this are difficult to interpret as the signal would be too low and it is feared that some compounds will fall within the noise level.

GC-MS conditions. GC-MS conditions . Perform all GC-MS experiments on an Agilent 7890GC coupled with 5977B MSD detector. Use an Agilent HP-5MS with 5% Phenyl Methyl Silox column (30 m × 250 mm × 0.25 mm). Set the initial column temperature to 150°C, hold for 0h 2m 0s, and increase at a rate of 8°C/min to 250°C, then hold for 0h 17m 0s. Use Helium as a carrier gas (1.2mL /min). Front inlet temperature is 275°C, transfer line temperature is 280°C, MS source temperature is 230°C, and MS quad temperature is 150°C. Use an injection volume of 1mL , with an injection dispense speed of 6000mL/min. The total run time is 32 minutes. Use electron ionisation, with a MS scan range 50-750 m/z.