TITLE: Extraction of Cyanobacterial Slime from Community Samples and Subsequent Analysis via GC-MS
AUTHORS: Kelsey Cremin, Jerko Rosko, Sarah J.N. Duxbury, Mary Coates, Lijiang Song, Orkun Soyer

[DESCRIPTION]
This protocol details the extraction of cyanobacterial slime from community samples and subsequent analysis via GC-MS.

[GUIDELINES]
NOTE: 

This protocol is for extraction of slime (a.k.a exopolysaccharides, EPS) from a filamentous cyanobacterial microbial community (from a freshwater environment) that forms extensive biofilms and granular structures.1 For other cyanobacterial samples, the protocol can be adapted / simplified.

This protocol is based on the slime extraction method described in Plude et al. 19912, and the EPS extraction method described in Olenska et al. 20213 (therefore this protocol uses these original papers’ definitions of EPS and slime). Both papers perform cyanobacterial slime/EPS extraction and perform GC-MS based analytical studies - via hydrolysis - on the extracts, however, details of some of the preparation and analysis steps are limited. Here, elements of hydrolysis method descriptions from the above two papers have been combined with a more rigorous and hopefully total hydrolysis method4, so to further optimise this process. 

The protocol also contains steps for preparation of a selection of monosaccharide standards for analysis, to be used as comparison against the samples and treated using the same GC-MS based analytical methods. 

Associated protocols / media sheets:
DSMZ_Medium_BG11+_1593 (omitting Vitamin B12 and replacing with full vitamin mix supplement as described below in Table 1)

OSP_35: This protocol. 

 
References 

(1) Duxbury, S. J. N.; Raguideau, S.; Rosko, J.; Cremin, K.; Coates, M.; Quince, C.; Soyer, O. S. Reproducible spatial structure formation and stable community composition in the cyanosphere predicts metabolic interactions. bioRxiv 2022. 

(2) Plude, J. L.; Parker, D. L.; Schommer, O. J.; Timmerman, R. J.; Hagstrom, S. A.; Joers, J. M.; Hnasko, R. Chemical Characterisation of Polysaccharide from the Slime Layer of the Cyanobacterium Microcystis flos-aquae C3-40. Applied and Environmental Microbiology 1991, 57 (6).

(3) Olenska, E.; Malek, W.; Kotowska, U.; Wydrych, J.; Polinska, W.; Swiecicka, I.; Thijs, S.; Vangronsveld, J. Exopolysaccharide Carbohydrate Structure and Biofilm Formation by Rhizobium leguminosarum bv. trifolii Strains Inhabiting Nodules of Trifoliumrepens Growing on an Old Zn-Pb-Cd-Polluted Waste Heap Area. Int J Mol Sci 2021, 22 (6).

(4) Becker, M.; Ahn, K.; Bacher, M.; Xu, C.; Sundberg, A.; Willfor, S.; Rosenau, T.; Potthast, A. Comparative hydrolysis analysis of cellulose samples and aspects of its application in conservation science. Cellulose (Lond) 2021, 28 (13), 8719-8734.

(5) Nakagawa, M.; Takamura, Y.; Yagi, O. Isolation and Characterization of the Slime from a Cyanobacterium,Microcystis aeruginosaK-3A. Agricultural and Biological Chemistry 2014, 51 (2), 329-337.

(6) Zhu, R.; Lin, Y. S.; Lipp, J. S.; Meador, T. B.; Hinrichs, K. U. Optimizing sample pretreatment for compound-specific stable carbon isotopic analysis of amino sugars in marine sediment. Biogeosciences Discuss 2014, 11, 593–623.

[STEPS]
SECTION: PART A: Slime/EPS extraction
1. 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
     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   
 
SECTION: PART A: Slime/EPS extraction
2. Once grown, centrifuge the cultures at 4000 x g, 20 min, 4 °C to pellet the cells. 
 


SECTION: PART A: Slime/EPS extraction
2.1. 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. 
 

SECTION: PART A: Slime/EPS extraction
3. Next, extract any slime/EPS from the remaining cell pellet, using an adapted method inspired by both Plude et al. (1991)2 and Nakagawa et al. (1987)5. 
SECTION: PART A: Slime/EPS extraction
3.1. 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 1 mL of the tube, then add 30 mL of ddH2O. Store the suspension    at 4 °C. The slime should separate from the cell pellet and enter the ddH2O. 

SECTION: PART A: Slime/EPS extraction
3.2. Next morning, centrifuge the cell pellet solution again 4000 x g, 20 min, 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.
 
 
SECTION: PART A: Slime/EPS extraction
3.3. Suspend the cell pellet (which can measure approx. 5 mL for a 200 mL culture) in 25 mL of ddH2O (or more, scale accordingly with the initial culture size). 
Vortex the samples for 2 minutes max, split between twelve 2 mL centrifuge tubes and centrifuge at 15000 x g, 15 min 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 1.5 mL of ddH2O. (1/5)
Vortex the samples for 2 minutes max, split between twelve 2 mL centrifuge tubes and centrifuge at 15000 x g, 15 min 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 1.5 mL of ddH2O.  (2/5)
Vortex the samples for 2 minutes max, split between twelve 2 mL centrifuge tubes and centrifuge at 15000 x g, 15 min 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 1.5 mL of ddH2O. (3/5)
Vortex the samples for 2 minutes max, split between twelve 2 mL centrifuge tubes and centrifuge at 15000 x g, 15 min 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 1.5 mL of ddH2O. (4/5)
Vortex the samples for 2 minutes max, split between twelve 2 mL centrifuge tubes and centrifuge at 15000 x g, 15 min 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 1.5 mL of ddH2O. (5/5)
Collect the supernatant with each iteration. 
 

SECTION: PART A: Slime/EPS extraction
4. 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 10 mL 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. 
 

SECTION: PART A: Slime/EPS extraction
5. Following the iterations of slime extraction, which gives a final volume of 90 mL supernatant containing slime, to which add 45 mg of CaCl2 (final conc. of 500 mg/L CaCl2). Mix this  and leave it   in the fridge (4 °C).
SECTION: PART A: Slime/EPS extraction
6. On the next day, centrifuge the solution (from step 4) at 15000 x g, 30 min, 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. 
 


SECTION: PART A: Slime/EPS extraction
7. Also, store the “slime pellet” from step 5 with a final volume of less than 1 mL . 
 


SECTION: PART A: Slime/EPS extraction
8. Prior to GC, lyophilise the Culture supernatant, Culture cyano pellet, Culture slime supernatant and Culture slime pellet fractions.
SECTION: PART B: Preparation of samples (and known standards) for GC-MS
9. 
 

SECTION: PART B: Sample/control prep for GC
10. 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. 
 

SECTION: PART B: Sample/control prep for GC
10.1. Weigh 40 mg of each standard, control blank, and the lyophilised culture fraction samples are into separate clean glass vials. 
In the first stage, add 1.5 mL of 72% aq. H2SO4  to each sample. This is left stirring under Room temperature for 120 min. 

SECTION: PART B: Sample/control prep for GC
10.2. In the second stage, add 2 mL of H2O to each mixture and heat them in an oven at 80 °C for 60 min. Cool down the hydrolysis solution in an ice bath and stored at 4 °C 60 min. 
SECTION: PART B: Sample/control prep for GC
10.3. Neutralise the hydrolysed sample to pH 7, using Na2CO3 until CO2 evolution subsides. 
 

SECTION: PART B: Sample/control prep for GC
10.4. Filter the resulting pH 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.
 


SECTION: PART B: Sample/control prep for GC
11. Trimethylsilylation. 
 
Dissolve the dried hydrolysates in 1.5 mL of pyridine (Scientific Laboratory Supplies Ltd, W296600, 99+%), shake by hand for 30 s, and then incubate at Room temperature for 30 min. 
SECTION: PART B: Sample/control prep for GC
11.1. Then add 0.5 mL of 1-(Trimethylsilyl)imidazole (1-TMS-imidazole, Merck, A12512.06, 97%), and  incubate the samples in a shaking incubator at 60 °C. 
SECTION: PART B: Sample/control prep for GC
11.2. Cool at Room temperature. Remove the pyridine fully by drying with nitrogen gas, as described above. Drying will again take 30-60 minutes. 
 


SECTION: PART B: Sample/control prep for GC
11.3. To the dried compounds, add 2 mL 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. 
SECTION: PART B: Sample/control prep for GC
12. Concentrations for GC. Each sample contains 40 mg of the species of interest; therefore, dissolve each sample into 8 mL of hexane (>99%, LC grade, solvent used for GC), thereby creating 5 mg/mL master stocks. 
SECTION: PART B: Sample/control prep for GC
12.1. From this master stock, take 1 mL and dilute into hexane to give a total volume of 1 mL in brown glass GC vials – resulting in final concentration of 5 mg/L. 
 

SECTION: PART B: Sample/control prep for GC
12.2. This creates a set of 5 mg/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. 
 

SECTION: PART B: Sample/control prep for GC
13. 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 2 min, and increase at a rate of 8 °C/min to 250 °C, then hold for 17 min. Use Helium as a carrier gas (1.2 mL /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 1 mL , with an injection dispense speed of 6000 mL/min. The total run time is 32 minutes. Use electron ionisation, with a MS scan range 50-750 m/z.