Apr 14, 2026

Enriched Seawater for Cyanobacterial Culture

DOI
https://dx.doi.org/10.17504/protocols.io.5qpvo183xg4o/v1
  • 1Federal University of Rio de Janeiro, Brazil.
  • LAABio-IPPN-UFRJ
Rômulo Pereira de Jesus
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DOI: https://dx.doi.org/10.17504/protocols.io.5qpvo183xg4o/v1
Protocol Citation: Rômulo Pereira de Jesus, Gabriel da Silva Ferreira de Lima, Paulo Iiboshi Hargreaves, Ricardo Moreira Borges 2026. Enriched Seawater for Cyanobacterial Culture. protocols.io https://dx.doi.org/10.17504/protocols.io.5qpvo183xg4o/v1
Manuscript citation:
Borges, R. M., Ferreira, G. A., Chagas, F., Pauloihc, & Chagas, F. F. O. C. (2023, September 18). Cyanobacteria growth (Version 2). protocols.io. https://doi.org/10.17504/protocols.io.4r3l22o8xl1y/v2
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: February 04, 2026
Last Modified: April 14, 2026
Protocol  Integer ID: 242640
Keywords: Cyanobacteria, Seawater, Biomass production, Large-scale cultivation, enriched seawater for cyanobacterial cultivation, enriched seawater for cyanobacterial culture, cyanobacterial cultivation, cyanobacterial culture, preparing enriched seawater, improved growth performance in seawater, enriched seawater, cyanobacteria, seawater due to the high cost, commercial marine salt, seawater, scale cultivation, future harvesting, large amounts of biomass
Abstract
This work describes a standardized protocol for preparing enriched seawater for cyanobacterial cultivation. We adopted seawater due to the high cost of commercial marine salts when scaling up cultures. In addition to economic advantages, cyanobacteria showed improved growth performance in seawater compared to other culture media. Because the primary goal was to produce large amounts of biomass for future harvesting, seawater proved to be an efficient, sustainable, and cost-effective alternative for large-scale cultivation.
Pre-collection considerations
Seawater should not be collected following rainfall events, as precipitation promotes sediment resuspension, increases the load of organic matter and suspended particles, and introduces continental contaminants such as sewage, soil, and excess nutrients. These factors significantly alter seawater quality and may compromise the stability of the culture medium.

Recommendation:
Seawater collection should be carried out only after at least 1–2 consecutive weeks of dry and sunny weather, ensuring greater physicochemical stability of the seawater.

  • Select flasks ranging from 1 to 5 liters according to the number of cultures required. The flasks must be cleaned with Extran and rinsed thoroughly with distilled water.
  • Label each flask with the internal code used to identify the cyanobacteria culture. It is recommended to use thermoresistant labels; otherwise, they may be damaged during autoclaving.
  • Avoid selecting any non-thermoresistant material for flasks.




Seawater collection
Seawater collection should be carried out near the shoreline, using surface water, while avoiding areas with strong sediment resuspension or visible accumulation of organic matter.

Collection procedure:
  • Use clean containers suitable for water sampling (glass or appropriate plastic).
  • Before the final sampling, rinse the container with the seawater itself, repeating the procedure five consecutive times (5×).
  • After rinsing, collect the required volume of surface water, keeping the container facing against the current at all times to prevent the entry of sand particles.
  • Seal the container immediately after collection.
Filtration
Upon arrival at the laboratory, the seawater should be immediately subjected to vacuum filtration.

Materials used:
  • Kitasato flask
  • Büchner funnel
  • Standard laboratory filter paper

Procedure:
  • Vacuum filtration aims to remove coarse particles such as sand, debris, and suspended particulate matter, ensuring a cleaner solution for subsequent medium preparation.
  • Filter the water carefully, pouring small amounts at a time. Remember to change the filters after every few filtrations, as retained sand slows the filtration rate.
  • When transferring the water to the cleaned culture flasks, make sure to measure the volume in each flask. It is recommended to use a 1-liter graduated cylinder.
Supplementation
After filtration, the seawater must be supplemented to enhance the culture medium.
For each 1 L of filtered salt water add: 1 mL of NaNO3 1 mL of NaH2PO4•H2O 0.5 mL of trace metals

  • Mix carefully after the addition of the supplements. For further details on stock solutions preparation, refer to Cyanobacteria Growth V.2, DOI: dx.doi.org/10.17504/protocols.io.4r3l22o8xl1y/v2.

Sterilization
After supplementation, the medium must be sterilized by autoclaving.

‱ ATTENTION: The autoclaving time depends on the volume of solution in each container and may vary according to the amount being autoclaved.
Cooling
After autoclaving, the containers should be:
  • Removed from the autoclave
  • Allowed to cool naturally on the laboratory bench, without external interference
InoculationUntitled section
Once the medium has cooled:
  • Perform the inoculation of the cyanobacteria.
  • Do not forget to add the vitamin MIX.
Final considerations
The vitamin MIX must be added only after the medium has been autoclaved and allowed to cool, at a ratio of 1 mL per 1 L of medium, since some of its components are heat-sensitive and may degrade during sterilization.
Protocol references
Borges RM, Ferreira GA, Chagas F, pauloihc, Chagas FOC. Cyanobacteria growth. protocols.io; 2023. DOI: 10.17504/protocols.io.4r3l22o8xl1y/v2.