Apr 03, 2023
Extraction of Cyanobacterial Slime from Community Samples and Subsequent Analysis via GC-MS
- Kelsey Cremin1,
- Jerko Rosko1,
- Sarah J.N. Duxbury1,
- Mary Coates1,
- Lijiang Song1,
- Orkun Soyer1
- 1University of Warwick
Protocol Citation: Kelsey Cremin, Jerko Rosko, Sarah J.N. Duxbury, Mary Coates, Lijiang Song, Orkun Soyer 2023. Extraction of Cyanobacterial Slime from Community Samples and Subsequent Analysis via GC-MS. protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvorw8dv4o/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: March 04, 2023
Last Modified: April 03, 2023
Protocol Integer ID: 78088
Keywords: Slime/EPS extraction, Preparation of samples for GC-MS, Trimethylsilylation
Funders Acknowledgement:
Gordon and Betty Moore Foundation
Grant ID: 9200
Abstract
This protocol details the extraction of cyanobacterial slime from community samples and subsequent analysis via GC-MS.
Attachments
663-1393.docx
50KB
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.
Materials
Materials
- vitamin mix
- BG11+ growth media
- Nuclepore 5-μm pore size filter
- Gelman 0.45 μm membrane filter
- ddH2O
- 18G needle
- syringe
- 25G needle
- CaCl2
- H2SO4
- Na2CO3
- sodium hydroxide
- methanol
- pyridine
- 1-(Trimethylsilyl)imidazole
- heptane
- Phenyl Methyl Silox column
- Agilent 7890GC
PART A: Slime/EPS extraction
PART A: Slime/EPS extraction
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.
Note
In our experience, an approximate volume of 200 mL of culture produces dried slime with a mass of 20-30 mg , therefore greater initial volumes of culture are advised.
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 4000 x g, 4°C, 00:20:00 to pellet the cells.
Note
Plude et al. believes the pellet will contain the adherent slime, whilst Olenska et al. work on the principle that the EPS exists in the supernatant - therefore, we suggest that both should be kept (from the same cultures) and analysed concurrently.
20m
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.
Note
=> this sample is called “Culture Supernatant” below.
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.
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 Overnight at 4 °C . The slime should separate from the cell pellet and enter the ddH2O.
Next morning, centrifuge the cell pellet solution again 4000 x g, 4°C, 00:20:00 , 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.
Note
Note: The supernatant contains the slime according to Plude et al. (1991), while in our case we combine it with supernatants obtained from further treatment of the cell pellet, as explained next.
20m
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, 00:15:00 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, 00:15:00 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, 00:15:00 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, 00:15:00 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, 00:15:00 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.
Note
By the third iteration, the supernatant – for our samples - had a slight blue colour, possibly from cellular photopigments being extracted as well.
1h 15m
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.
Note
=> this sample is called “Culture cyano pellet” below.
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 Overnight in the fridge (4 °C ).
On the next day, centrifuge the solution (from step 4) at 15000 x g, 00:30:00 , 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.
Note
=> this sample is called “Culture slime supernatant” below.
30m
Also, store the “slime pellet” from step 5 with a final volume of less than 1 mL .
Note
=> this sample is called “Culture slime pellet” below.
Prior to GC, lyophilise the Culture supernatant, Culture cyano pellet, Culture slime supernatant and Culture slime pellet fractions.
PART B: Preparation of samples (and known standards) for GC-MS
PART B: Preparation of samples (and known standards) for GC-MS
Note
Standards for known monosaccharides can be prepared, so that their GC-MS spectra can be compared to that of actual sample. Monosaccharide standards will all be of HPLC grade and will be prepared to 1 mg/L sample, through a serial dilution. Two control samples (‘blanks’), one consisting of fresh BG11+ growth media, and one of ddH2O were included in the GC-MS analysis.
Choice of monosaccharide standards should be project specific, but here we focus on the following monosaccharides based on previous studies of cyanobacterial slime/EPS:
- Glucose,
- Xylose,
- Galactose,
- Mannose,
- Rhamnose,
- Galacturonic acid,
- Galactonate
- Gluconarate
- D-Fructose,
- L-Fucose,
- Adonitol,
- Fumaric Acid,
- L-Aspartic Acid,
- L-Arabinose
The samples to analyse come from the different fractions resulting from the extraction step – part A – listed above:
- Media control (BG11+ with vitamin mix),
- ddH2O water control,
- Culture Supernatant,
- Culture cyano cell pellet,
- Culture slime supernatant, and
- Culture slime pellet.
PART B: Sample/control prep for GC
PART B: Sample/control prep for GC
4h 49m 30s
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.
Note
The hydrolysis method is based on Becker et al. 2021.4 Hydrolysis consists of two stages.
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 02:00:00 .
2h
In the second stage, add 2 mL of H2O to each mixture and heat them in an oven at 80 °C for 01:00:00 . Cool down the hydrolysis solution in an ice bath and stored at 4 °C Overnight .
2h
Neutralise the hydrolysed sample to 7 , using Na2CO3 until CO2 evolution subsides.
Note
This will require a considerable amount of Na2CO3 and will involve salt formation.
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.6
Alternative hydrolysis methods. Weigh 40 mg samples and standards into glass test tubes, to which add 1 mL of 6 Molarity (M) HCl. Then heat this to 105 °C for 08:00:00 on the heat block, inside the chemical fume hood. Adjust this solution to pH 6.5–7.0 with 1 Molarity (M) sodium hydroxide (NaOH), dry by evaporating it under N2 as above, re-dissolved in 2 mL 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.
Note
This trimethylsilylation method has been originated in Becker et al. 2013.4
Dissolve the dried hydrolysates in 1.5 mL of pyridine (Scientific Laboratory Supplies Ltd, W296600, 99+%), shake by hand for 00:00:30 , and then incubate at Room temperature for 00:30:00 .
30m 30s
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 .
Cool at Room temperature . Remove the pyridine fully by drying with nitrogen gas, as described above. Drying will again take 30-60 minutes.
Note
Due to safety concerns and the unpleasant odour of pyridine, it is highly encouraged to perform this in a chemical fume hood with outside extraction.
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.
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.
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 .
Note
NOTE: There may be a precipitate left over from the silylation – step 11, above - at the bottom of the vials. This should be avoided and only the solution should be taken as the solution only contains the monosaccharides.
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.
Note
NOTE: these samples should be produced close to the actual GC-MS date, if not, store the trimethylsilyl (TMS) derivatives moisture-sealed and in a -20 °C freezer where they will remain stable for several months.
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 00:02:00 , and increase at a rate of 8 °C /min to 250 °C , then hold for 00:17:00 . 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.
19m