Jul 16, 2025
Co-cultivation Protocol for Porosira glacialis Diatom and Bacterial Isolates
- William Arnli1,
- Nerea Johanna Aalto2,3,
- Stina Krsmanovic2,3,
- Hans Christopher Bernstein2,3,4
- 1University of Tromsø;
- 2The Norwegian College of Fishery Science, Faculty of Biosciences, Fisheries and Economics, UiT - The Arctic University of Norway, Tromsø, Norway;
- 3Microalgae & Microbiomes Research Group (M2RG), UiT - The Arctic University of Norway, Tromsø, Norway;
- 4The Arctic Centre for Sustainable Energy (ARC), UiT - The Arctic University of Norway, Tromsø, Norway
Protocol Citation: William Arnli, Nerea Johanna Aalto, Stina Krsmanovic, Hans Christopher Bernstein 2025. Co-cultivation Protocol for Porosira glacialis Diatom and Bacterial Isolates . protocols.io https://dx.doi.org/10.17504/protocols.io.8epv5ow14g1b/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: June 22, 2025
Last Modified: July 16, 2025
Protocol Integer ID: 220677
Keywords: Phycosphere , Growth dynamics, Diatom cultivation , Synthetic Consortia, Characterization, Diatom-Bacteria Growth Dynamics, IAA, Indole-3-Acetic Acid, chlorophyll a extraction, Cell quantification , Cold-adapted diatom, Porosira glacialis , Sulfitobacter, marinus, Growth analysis, Bacterial associates, Synthetic consortia, Axenic cultivation, Paired cultivation, Chlorophyll a extraction, Marine growth media, Utermöhl settling method, Microalgae, Orbital shaker, Research protocol, Sustainable energy, Cocultivation , Co-cultivation, adapted marine diatom porosira, porosira glacialis diatom, marine diatom porosira, various algal strain, bacteria growth dynamic, bacterial isolate, uit201 diatom, microbiome, bacterial associate, microalgae, growth curves from measured cell density, paired cultivation
Disclaimer
This protocol was developed as part of a master's thesis affiliated with Microalgae & Microbiomes - The M2 Research Group at Norges fiskerihøgskole, University of Tromsø, Norway
Abstract
The protocol provides a platform for controlled experimentation on axenic and paired cultivation of the cold-adapted marine diatom Porosira glacialis UiT201 diatom with its bacterial associates in a synthetic consortia under variable environmental conditions, allowing the characterization of diatom-bacteria growth dynamics. Treatments include UIT201-Sulfitobacter marinus co-culture and UIT201 treated with Indole-3-Acetic Acid (IAA); however, this protocol can be adapted to various algal strains, bacterial isolates, and conditions to study diatom-bacteria growth dynamics. Expected results include growth curves from measured cell density and chlorophyll a extraction.
This protocol was developed as part of a master's thesis affiliated with Microalgae & Microbiomes - The M2 Research Group at Norges fiskerihøgskole, University of Tromsø, Norway
Guidelines
Suggested experimental workflow
Materials
MEADIA
FMAP (1 L)
15 g Marine Broth BD Difco™ Dehydrated Culture Media: Marine Broth 2216Thermo Fisher Scientific
5 g Peptone
Peptone from casein (Tryptone)Merck MilliporeSigma (Sigma-Aldrich)
300 mL Filtered seawater
700 mL MilliQ water
Protocol materials
Sodium metasilicateMerck MilliporeSigma (Sigma-Aldrich)Catalog #NA
1H-Indole-3-acetic acid, 98%Thermo Scientific
96-100% ethanol
Guillard′s (F/2) Marine Water Enrichment Solution 50 xMerck MilliporeSigma (Sigma-Aldrich)
Peptone from casein (Tryptone)Merck MilliporeSigma (Sigma-Aldrich)
BD Difco™ Dehydrated Culture Media: Marine Broth 2216Thermo Fisher Scientific
Troubleshooting
Safety warnings
Refer to the Safety Data Sheets (SDS) for information on health and environmental hazards. Be sure to follow your institution's Health and Safety (HMS) guidelines, wear appropriate personal protective equipment (PPE), and adhere to all established safety protocols to minimize risks
Before start
This protocol outlines the axenic and paired cultivation of Porosira glacialis UiT201 and Sulfitobacter marinus, including IAA treatment; however, it can easily be adapted to other algal strains, bacteria isolates, and variable conditions to investigate diatom-bacteria growth dynamics. FMAP media was utilized as an all-purpose marine growth media in the creation of this protocol (see Materials for media composition), however it is highly recommended to establish optimal growth media for each bacterial strain. Results are assessed through cell density and chlorophyll a extraction.
Please note that your experimental design may vary. For this protocol, samples are measured and Indole-3-acetic acid (IAA) is supplemented every other day. Chlorophyll a and cell counts are recorded continuously throughout the experiment, which lasts until two repeated measurements are taken during the stationary phase. A suggested workflow is provided in the Guidelines section.
Cultivation set-up
10m
Position a large orbital shaker in a 7 °C cold-room and arrange flexible 200 × 50W RGB LED strips along the shaker to provide a sufficient cultivation light source
Equipment
PSU-20i Orbital Shaking Platform
NAME
Orbital Shaking Platform
TYPE
Grant bio
BRAND
NA
SKU
LINK
PSU-20i Orbital Shaking Platform with RGB light fixture
5m
Day 1 (T= -2) - Treatment preparations
3h 20m
Inoculate bacteria isolates in5 mL FMAP media in 15 mL culture
tubes and shake Overnight
10m
Prepare a stock solution of 0.25 millimolar (mM) 1H-Indole-3-acetic acid, 98%Thermo Scientific dissolved in Milli-Q water. Wrap the stock solution in tinfoil to prevent degradation, and place in a refrigerator for short term storage. For long term storage, consider storing at -20 °C .
10m
The Utermöhl settling method (Edler, L., and Elbrächter, M., 2010) is utilized to count the stock algae culture: Sample 4 mL of stock diatom culture and fix 2 mL diatoms in duplicates with 7 µL Lugol's iodine solution in 2 mL cell culture wells for 1-2 hours in a 4 °C refrigerator.
2h
Once the samples have settled, observe it under a microscope. Diatom cells are identified and counted in several randomly selected fields of view to ensure a representative sample. If the diatom concentration is too large for an accurate count (>100), determine a dilution factor and proceed to dilute the culture. For each duplicate sample, count the diatom cells first horizontally, then vertically along an axis. For the horizontal count, exclude cells on the bottom edge of the microscopic view, and for the vertical count, exclude cells on the right edge. Repeat the process for the second duplicate sample. This results in four values which are averaged to calculate the concentration of cells per unit volume of the original water sample.
Counting diatom cells using the Utermöhl method. Cells are counted horizontally and vertically, excluding those on the bottom and right edges (outlined in red boxes). This process is repeated for the second duplicate sample.
1h
Aiming for a starting culture of 1-2 mill cells/L, dilute with Milli-Q to 1:3 of the starting algae concentration.
Note
This step decreases lag-phase, ensuring that a quick transition to log phase. Skipping this step could result in an extended lag-phase.
Day 2 (T= -1) - Treatment preparations
15m
Move the overnight bacteria inoculation tubes to a 7-degree cold room to acclimate. Leave Overnight for additional inoculation.
5m
Prepare a stock of 3.5 g/L Sodium metasilicateMerck MilliporeSigma (Sigma-Aldrich)Catalog #NA in Milli-Q
10m
Day 3 (T=0) - Initiating cultivation
4h 35m
For determining stock diatom culture sample, utilize the Utermöhl settling method: 4 mL of stock diatom culture and fix 2 mL diatoms in duplicates with 7 µL Lugol's iodine solution in 2 mL cell culture wells for 1-2 hours.
2h
Prepare fresh media by adding 8 mg/mL of Guillard′s (F/2) Marine Water Enrichment Solution 50 xMerck MilliporeSigma (Sigma-Aldrich) and 8 mg/mL of the stock sodium metasilicate to 5 L of filtered seawater*
* 5 L is sufficient for 9 (3 treatments * 3 biological replicates) replicates
5m
Filter the media through 0.22 µm sterile filters and fill 9500 mL tissue culture flasks
Note
Follow and do not exceed the recommended capacity of the filters
Equipment
Sterivex™ Pressure Filter
NAME
0.22 μm filter
TYPE
Millipore®
BRAND
00-00-0
SKU
LINK
Equipment
Masterflex L/S
NAME
Peristaltic Pump
TYPE
Masterflex
BRAND
NA
SKU
LINK
30m
After 1-2 hours of fixation, utilize the Utermöhl method in step 5 to count the stock algae culture.
1h
Aiming for a starting culture of 1-2 mill cells/L, transfer diatoms to the 500 mL tissue culture flasks
10m
Determine bacteria isolate concentrations by measuring OD600. Aiming for a 50:1 bacteria:diatom ratio, proceed to pipette the appropriate amount to each of the bacteria treatment culture flasks
30m
From the 0.25 millimolar (mM) 1H-Indole-3-acetic acid, 98%Thermo Scientific stock spike100 µL of IAA to each of the IAA treatment culture flasks to achieve a final concentration of 50 nanomolar (nM) . Immediately proceed to step 16.
5m
Turn over the culture flasks 10 times to homogenize the cultures and place the flasks on the orbital shaker in the 7 °C cold-room. Set the orbital shaker to 50 rpm, 7°C with 300 reciprocal motion for 12 seconds and vibration set to 2.
Note
Measure light intensity ensuring consistent light intensity across all flasks. It is recommended to alternate the positioning of the bottles.
Culture bottle placement on orbital shaker in the 7 °C cold-room
Orbital shaker settings
15m
Sampling for chlorophyll extraction and cell count
30m
Retrieve and extract 25 mL samples from the each of the culture bottles in 50 mL falcon tubes. To minimize incubation room downtime, immediately return the culture flasks to the orbital shaker.
Note
For each measurement 4 mL is needed for cell counting and 20 mL for chlorophyll extraction. Extracting 25 mL of sample allows for some margin. As the diatom culture enters log phase, the samples need to be diluted for measurements and less extracted sample volume is needed.
30m
Diatom cell count
2h
From each of the 25 mL samples, the utilize Utermöhl settling method is used to create duplicates count the cells (steps 4 and 5): pipette 2* 2 mL of culture to2 mL cell culture wells and fix with 7 µL Lugol's iodine solution for 1-2 hours. Proceed to count the cells according to the previously described method.
2h
Chlorophyll extraction
15h
Assemble a vacuum pump with a fitted filtration system. From the remaining 21 mL of each sample culture, create 5 technical replicates by depositing 5 *4 mL on 1.2 µm glass microfiber filter papers. Proceed to create a vacuum and filter the culture samples.
Equipment
Grade GF/C Glass Microfiber Filter Papers
NAME
Microfiber Filter Papers
TYPE
Whatman
BRAND
NA
SKU
LINK
1.2um, 25mm Dia., Binder-Free
SPECIFICATIONS
Equipment
Chemical Duty Pump
NAME
Vacuum pump
TYPE
Merck Millipore
BRAND
NA
SKU
LINK
220 V/50 Hz
SPECIFICATIONS
Equipment
1225 Sampling Manifold
NAME
Millipore
BRAND
NA
SKU
LINK
15 mL process volume
SPECIFICATIONS
1h
Transfer the glass microfiber filters to separate test tubes and add 5 mL 96-100% ethanol . For extraction, immediately cover the samples in tinfoil and place them in a 7 °C cold-room Overnight .
12h
After allowing overnight extraction, proceed to measure chlorophyll a on a fluorometer with excitation wavelength set to 430-450 nm (typically around 435 nm) and emission wavelength to 675-685 nm. A chlorophyll a factor of 0.00417 is used to convert absorbance readings into the concentration of chlorophyll a (Chla μg/L) in a sample.
Equipment
TD-700 Laboratory Fluorometer
NAME
Fluorometer
TYPE
Turner Designs
BRAND
00-00-0
SKU
LINK
2h
Protocol references
Edler, L., and Elbrächter, M. (2010). “The utermöhl method for quantitative phytoplankton analysis,” in Microscopic and Molecular Methods for Quantitative Phytoplankton Analysis, Vol. 110, eds B. Karlson, C. Cusack, and E. Bresnan (Paris: Intergovernmental Oceanographic Commission Manual and guides), 13–20