Oct 25, 2024
Version 2
Labelling kelps with 13C and 15N for isotope tracing or enrichment experiments V.2
- Anton Kuech1,
- Ursula Witte1,
- Inka Bartsch2
- 1School of Biological Sciences, University of Aberdeen, Aberdeen AB24 3FX, United Kingdom;
- 2Alfred Wegener Institute, Helmholtz Centre for Polar and Marine Research, Am Handelshafen 12, D-27515 Bremerhaven, Germany
Protocol Citation: Anton Kuech, Ursula Witte, Inka Bartsch 2024. Labelling kelps with 13C and 15N for isotope tracing or enrichment experiments. protocols.io https://dx.doi.org/10.17504/protocols.io.8epv597rdg1b/v2Version created by Anton Kuech
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: November 29, 2021
Last Modified: October 25, 2024
Protocol Integer ID: 110094
Keywords: Macroalgae, Kelp, Seaweed, Cultivation, Isotopic enrichment, Isotope tracing experiments, Enrichment experiments
Funders Acknowledgements:
Natural Environment Research Council
Grant ID: NE/S007377/1
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Abstract
Isotope tracing experiments can be used to trace organic material flow through the ecosystem by artificially adding labelled biomass into a system. The advantage of this process is the direct control of carbon and nitrogen addition to the system for measuring uptake rates by consumers which can substantially reduce the uncertainties associated with food web models. This protocol details the steps involved in successfully culturing and isotopically enriching (13C & 15N) juvenile sporophytes of two common North Atlantic kelp species (Laminariales): Saccharina latissima and Laminaria digitata. A first successful isotopic enrichment study of S. latissima, as well as the first inclusion of 15N enrichment for L. digitata, is detailed. This protocol provides a comprehensive description of the stable isotope enrichment process in two kelp species, potentially serving as a foundation for its application in other macroalgal taxa.
Materials
Equipment:
- Juvenile kelp sporophytes
- 5 L sterile culture bottles (glass or plexiglass)
- 2 L Kautex bottles with lids or similar
- Dry air and tubes
- Plant/algae grow-lights
- Clean and pasteurised seawater
- Dissecting scissors
- Rotating or tilting shakers
- Deionized (DI) water
- Freeze-dryer
- Aluminium foil
- Tweezers
- Zip-lock bags
- Combusted 14 mL glass vials
- Ball mill
- Aluminium cups
- Analytical laboratory scale
- 96-well plate
Growth media chemicals:
- Von Stosch (VS) medium chemicals
- Provasoli Enriched Seawater (PES) medium chemicals
- Sodium nitrate-15N (CAS# 31432-45-8)
- Sodium bicarbonate-13C (CAS# 87081-58-1)
Protocol materials
Sodium nitrateP212121
Step 3
Liquid nitrogen
Step 14
Sodium nitrate-15NMerck MilliporeSigma (Sigma-Aldrich)Catalog #364606-5G
Step 7
Sodium glycerophosphate hydrate
Step 7
Sodium bicarbonate-13CMerck MilliporeSigma (Sigma-Aldrich)Catalog #372382-5G
Step 7
Sodium 2-glycerophosphate pentahydrate
Step 3
Before start
The generation of juvenile sporophyte material for isotopic labelling can be achieved through various methods, such as sampling from the field or growing sporophytes from spores or vegetative stock gametophytes. For details of our experiment see PLOS ONE article. Juvenile kelp sporophytes for labelling purposes have to be cultured in a way, which ensures good growth to produce actively growing healthy material. The following steps are tailored to the two species cultivated for this experiment and may require adaptation based on the available equipment and sporophyte physiology.
Culture set-up (pre-labelling)
Culture set-up (pre-labelling)
Sterile culture bottles (glass or plexiglass) of sufficient size are used for cultivation. In our case, sporophytes of Laminaria digitata and Saccharina latissima were cultured in 5 L DURAN glass bottles with the nutrient addition of 100 mL (half concentration) Provasoli Enriched Seawater (PES) in 10 L fresh seawater stored in temperature-controlled laboratories at 10 °C . Medium was changed weekly and continuously bubbled with dry air via tubes. Irradiance should be set to around 40-50 µmol photons m-2 s-1 in long-day conditions (16 h light : 8 h dark; 16:8 LD). For cultivation of sporophytes, clean and pasteurised seawater is needed, either from natural fully marine sources or artificial seawater at approximately 30-33 PSU. Here, seawater was filtered through a 5" Polypropylene Yarn Water Filter and was pasteurised for 04:00:00 at 99 °C with a combi steamer (PALUX, Germany).
Protocol
NAME
Provasoli Enriched Seawater (PES) medium solutionCREATED BY
Anton Kuech
Note
During the culturing process full PES (200 mL PES in 10 L seawater) can also be used to induce higher growth rates (Fortes & Lüning, 1980). For optimal results, it is recommended to add the full concentration a few days prior to the labelling start.
CITATION
Picture of pre-labelling setup depicting kelp sporophytes in culture bottles bubbled with dry air via tubes.
Equipment
DURAN™ Original Laboratory Bottle, Clear, with DIN 168-1 Thread, Graduated
NAME
DURAN glass bottle
TYPE
DWK Life Sciences
BRAND
Z232122-1EA
SKU
5000 mL (narrow neck)
SPECIFICATIONS
Equipment
5" Polypropylene Yarn Water Filter
NAME
Water Filter
TYPE
Vyair
BRAND
n.a.
SKU
LINK
Equipment
PALUX Touch 'n' Steam 611QL
NAME
Combi Steamer
TYPE
PALUX
BRAND
E611 QBRN 000000
SKU
LINK
Determining growth rates
Determining growth rates
Macroalgal growth rates should be determined in simulated labelling conditions prior to labelling start to ensure the material is growing well. Otherwise, successful incorporation of labelled compounds is not guaranteed. Wet weight (mg) and surface area (cm2) are the main variables that should be measured at regular intervals (e.g. every 4 days), but blade length can also be used as a variable. In addition, pH measurements can be taken to monitor the acidity of the media.
Note
To ensure uptake of enriched chemicals, the algal biomass should ideally double during the labelling period.
Labelling medium
The simulated medium for labelling consists of von Stosch (VS) medium (in contrast to PES, which is used during early cultivation – see Note below) modified to exclude phosphate and nitrate. Phosphate (0.01546 millimolar (mM) Sodium 2-glycerophosphate pentahydrate C3H17Na2O11P), nitrate (0.54908 millimolar (mM) Sodium nitrateP212121 NaNO3) and bicarbonate (2.32545 millimolar (mM) Sodium bicarbonateMerck MilliporeSigma (Sigma-Aldrich) NaHCO₃) were added individually in the same concentrations as in PES (table below). The full concentration is 200 mL VS for 10 L fresh seawater.
| A | B | C | D | |
| Chemical compound | CAS number | Concentration (μmol/L) [as in full PES] | Concentration in millimolar (mM) | |
| Sodium bicarbonate | 144-55-8 | 2325.45 μmol/L | 2.32545 | |
| Sodium nitrate | 7631-99-4 | 549.08 μmol/L | 2.32545 | |
| Sodium glycerophosphate – hydrate | 55073-41-1 | 15.46 μmol/L | 0.01546 |
Chemical concentrations added individually to von Stosch (VS) medium used for cultures (pre-labelling) with CAS Registry Number of chemical substances.
Note
Best growth conditions of macroalgae are generally achieved with Provasoli Enriched Seawater (PES), however, given the complexity of the preparation of this medium and the difficulty in leaving out all nitrate sources, the preferred medium for isotopic labelling is von Stosch (VS) (according to Guiry & Cunningham, 1984). Phosphate was added separately because of ongoing separate experiments, however, for the sole purpose of isotopical labelling, phosphate can be left in the VS medium.
CITATION
Protocol
NAME
Von Stosch (VS) enriched seawater medium solutionCREATED BY
Anton Kuech
Sporophyte preparation for labelling
Separate sporophytes of the two species by cutting them at the stipe just above the holdfast using dissecting scissors. This enables single unidirectional growth as compared to an unequal distribution of growth between blade and holdfast. Select sporophytes of a good growth stage (e.g. widening of blade for S. latissima) and avoid individuals with white spots or other noticeable damaged areas. The blade should have an even brown colour.
Picture of Saccharina latissima and Laminaria digitata including the cut-off point marked by the red line.
Equipment
Fisherbrand™ Dissecting Scissors
NAME
Scissors
TYPE
Fisherbrand
BRAND
15277168
SKU
LINK
Example incubation of sporophytes for determining growth rates
Either two sporophytes of Saccharina latissima or four sporophytes of Laminaria digitata were placed into a single 2 L Kautex bottle (ensure sufficient replication). Wet weight (mg), surface area (cm2) and pH were measured in 4-day intervals for a 14-day period.
Note
The number of sporophytes placed into the 2 L Kautex bottles will depend on (a) the amount of enriched organic matter needed for the incubations (see PLOS ONE article for example), (b) the size of the pre-labelling sporophytes and (c) the ratio between sporophyte size and bottles. For this experiment, we used sporophytes of approximately 11 cm and 4 cm length of a single S. latissima and L. digitata thallus respectively. The average wet weight of a single sporophyte was approximately 104 mg and 24 mg for S. latissima and L. digitata respectively. Thus, two sporophytes of S. latissima and four sporophytes of L. digitata were placed into a Kautex bottle each. In case that you need more material or sporophytes are bigger, you should also increase the size of the incubation bottles to ensure good growth.
Note
In case you use new Kautex bottles, which are made from polyethylene terephthalate glycol (PETG), you have to take care to reduce the risk of contamination. To reduce the impact of the new PETG material and polyethylene (PE) foam insert of the closures on the macroalgae, the bottles were filled with tap water, closed and stored Overnight . This was followed by washing the closures and bottles in the dishwasher without detergent at 50 °C and dried in the oven at 40 °C .
Equipment
Wide Necked square container, PETG, 2000mL w/ screw closure, crystal clear
NAME
Wide neck container
TYPE
Kautex
BRAND
225274374
SKU
LINK
Labelling of kelps
Labelling of kelps
Pre-labelling start conditions
A few days prior to labelling start, full PES (200 mL PES in 10 L pasteurized seawater) should be added and irradiance should be set to 50 µmol photons m-2 s-1 to induce optimum growth rates (16 h light : 8 h dark per 24-hour period).
Note
Depending on species and developmental stage the conditions for inducing optimum growth rates may vary.
Labelling conditions
The concentrations of sodium nitrate and sodium bicarbonate used in von Stosch (VS) were adjusted to represent the same concentration that the macroalgae would receive in the full PES mixture (200 mL PES in 10 L pasteurized seawater). This concentration is considered the ideal medium for growth and is also in accordance with the concentrations used by Braeckman et al. (2019) and Rossi et al. (2013). Exact concentrations of chemicals added separately were 0.01546 millimolar (mM) Sodium glycerophosphate hydrate , 0.55457 millimolar (mM) Sodium nitrate-15NMerck MilliporeSigma (Sigma-Aldrich)Catalog #364606-5G and 2.35294 millimolar (mM) Sodium bicarbonate-13CMerck MilliporeSigma (Sigma-Aldrich)Catalog #372382-5G . Remaining nutrients and vitamins were added according to the von Stosch (VS) enrichment medium modified to exclude phosphate and nitratego to step #3 .
| A | B | C | D | |
| Chemical compound | CAS number | Concentration (μmol/L) [as in full PES] | Concentration in millimolar (mM) | |
| Sodium glycerophosphate – hydrate | 55073-41-1 | 15.46 μmol/L | 0.01546 | |
| Sodium nitrate-15N | 31432-45-8 | 554.57 μmol/L | 0.55457 | |
| Sodium bicarbonate-13C | 87081-58-1 | 2352.94 μmol/L | 2.35294 |
Concentrations of main chemicals used for labelling with CAS Registry Number of chemical substances.
Protocol
NAME
Von Stosch (VS) enriched seawater medium solutionCREATED BY
Anton Kuech
CITATION
CITATION
Separate sporophytes of the two species by cutting them at the stipe just above the holdfast using nail scissors as detailed in step #4. go to step #4
Incubation of sporophytes for labelling
Selected kelp sporophytes are stored in Kautex bottles, filled with the pasteurized seawater and labelling solution to the top, tightly closed and placed on rotating or tilting shakers for 9 days. Kautex bottles lie on their sides to allow full illumination. Make sure that there are no air bubbles in the bottle. We used Orbitron rotating shakers at 90 rpm, 10°C . Irradiance set to 50 µmol photons m-2 s-1 in a 16 : 8 LD cycle.
Picture of labelling setup depicting Kautex bottles containing kelp sporophytes placed on a rotating shaker.
Note
The optimal incubation period for sporophytes in the enriched medium may vary depending on the species selected and their respective growth rates. To ensure uptake of enriched chemicals, the algal biomass should ideally double during the labelling period.
Equipment
Wide Necked square container, PETG, 2000mL w/ screw closure, crystal clear
NAME
Wide neck container
TYPE
Kautex
BRAND
225274374
SKU
LINK
Harvesting biomass
Harvesting biomass
Remove sporophytes from a single Kautex bottle.
Take picture and wet weight of sporophytes using a macro photography camera or similar.
Separation of growing from non-growing blade parts
After the incubation period, newly formed blade parts of sporophytes normally become visible by a widening at the basis (see image below). We expected that the basal blade part that was formed during incubation would have a higher labelling result than the distal blade part. This assumption was supported by our results (see PLOS ONE article for details). Thus, if you cut sporophytes at the intersection between the estimated new growth (highly labelled) and prior biomass (slightly labelled), you separate material with a differential labelling result.
Examples of cut off points (red lines) for Laminaria digitata and Saccharina latissima, which approximately separate the newly formed basal blade area during labelling incubation (highly labelled area) from the distal blade part which had been formed during pre-cultivation (slightly labelled part).
Note
When dealing with other kelp species, their behaviours can vary. Separating the blades halfway along their length after termination of the labelling process is generally expected to generate highly and slightly labelled material. Kelps also distribute chemical compounds throughout the blades via translocation and thus, both the new biomass and ‘old’ biomass should be stored.
CITATION
Dip sporophytes briefly in deionized (DI) water to remove residual saltwater and dry material with clean tissue paper.
Shock freezing of samples
Sporophytes wrapped in punctured aluminium foil (highly/slightly labelled separate) and either (a) dipped in Liquid nitrogen until bubbling of liquid stops or (b) stored for a minimum of 24 hours at -80 °C prior to freeze-drying.
Freeze-drying of samples for 48 hours.
Equipment
RVC Alpha 3-4 LSCbasic
NAME
Laboratory freeze-dryer
TYPE
CHRIST
BRAND
n.a.
SKU
LINK
Storage of freeze-dried biomass in zip lock bags in freezer at -20 °C (or -80 °C ). Material can also be stored in a desiccator filled with silica gel for shorter periods.
Determining labelling uptake
Determining labelling uptake
Freeze-dried material should ideally be made into powder, best with a ball mill. Remove sporophytes from foil and transfer into the metal tubes from the ball mill. Ball mill set to 00:03:00 at 25 Hz. Afterwards, store the powder in combusted glass vials (previously combusted at 500 °C overnight).
Safety information
Handle hot glass vials with care! After combustion glass vials should be left to stand for 02:00:00 to cool down. Use heat resistant gloves when handling.
Equipment
Mixer Mill MM 400
NAME
Retsch
BRAND
MM400
SKU
LINK
Equipment
Fisherbrand™ Snap Cap Vial, Clear Glass
NAME
Glass vial
TYPE
Fisherbrand
BRAND
10749644
SKU
LINK
14 mL volume
SPECIFICATIONS
Random sub-samples of the powder, such as 4 replicates per species and distal/basal blade part, are weighed into aluminium cups (approximately 1 mg per sample), encapsulated and added onto a 96 well plate (can also be stored in a desiccator filled with silica gel for shorter periods).
Equipment
Aluminium capsules/pans for solids
NAME
Aluminium capsules
TYPE
Elemental Microanalysis
BRAND
D3089
SKU
LINK
Aluminium Capsules Pressed 8.75 x 3.5mm pack of 100
SPECIFICATIONS
Equipment
Corning™ Clear Polystyrene 96-Well Microplates
NAME
96-well plate
TYPE
Corning
BRAND
10377601
SKU
LINK
Analysis of samples for 13C and 15N isotopes. For details of the results for this experiment see PLOS ONE article.
Note
UC Davis Stable Isotope Facility (SIF) performed 13C and 15N isotope analyses using an elemental analyser interfaced to a continuous flow isotope ratio mass spectrometer (IRMS). As per UC Davis SIF guidelines, small plant samples are analysed for 13C and 15N isotopes using a PDZ Europa ANCA-GSL elemental analyser interfaced to a PDZ Europa 20-20 isotope ratio mass spectrometer (Sercon Ltd., Cheshire, UK). Samples are combusted at 1000°C in a reactor packed with chromium oxide and silvered copper oxide. Following combustion, oxides are removed in a reduction reactor (reduced copper at 650°C). The helium carrier then flows through a water trap (magnesium perchlorate and phosphorous pentoxide). N2 and CO2 are separated from the samples via a Carbosieve GC column prior to entering the IRMS. Calibrated reference materials are added to the samples during analysis. During analysis, the samples are interspersed with multiple replicates of at least four distinct laboratory reference materials. These reference materials have undergone prior calibration against internationally recognized standards, including IAEA-600, USGS-40, USGS-41, USGS-42, USGS-43, USGS-61, USGS-64, and USGS-65. A sample's initial isotope ratio is assessed relative to a reference gas peak, which is analyzed alongside each sample. These initial values are then refined by adjusting them for the entire batch using the established values of the laboratory reference materials included in the analysis. Standard deviations are figured at 0.2 ‰ for 13C and 0.3 ‰ for 15N. The final delta values are expressed relative to international standards VPDB (Vienna Pee Dee Belemnite) and Air for carbon and nitrogen, respectively.
Citations
Step 1
M. D. Fortes & K. Lüning. Growth rates of North Sea macroalgae in relation to temperature, irradiance and photoperiod
https://doi.org/10.1007/BF01983538Step 7
Rossi F, Gribsholt B, Gazeau F, Di Santo V, Middelburg JJ. Complex Effects of Ecosystem Engineer Loss on Benthic Ecosystem Response to Detrital Macroalgae.
https://doi.org/10.1371/journal.pone.0066650Step 7
Braeckman U, Pasotti F, Vázquez S, Zacher K, Hoffmann R, Elvert M, Marchant H, Buckner C, Quartino ML, Mác Cormack W, Soetaert K, Wenzhöfer F, Vanreusel A. Degradation of macroalgal detritus in shallow coastal Antarctic sediments.
https://doi.org/10.1002/lno.11125