Cyanobacteria are bacteria that are capable of photosynthesizing, just like plants—and that’s unusual in the world of microorganisms. In addition, some cyanobacteria can even fix nitrogen. Nurseries can be a unique setting, often with areas that provide optimal growing conditions (moisture, nutrients, light) for these microorganisms. But nurseries also contain another area where cyanobacteria can grow: recycling ponds. We have set out to study the biology of cyanobacteria that contribute to these unique aquatic ecosystems. We have established a robust isolating and culturing protocol and now have over 10 individual species in pure culture.
First, we had to collect water samples from which to isolate cyanobacteria. We collaborated with three nurseries on this task; our collaborators were happy to support us in providing access to their recycling ponds. Depending on the size of the nursery’s recycling pond, we collected between 5-10 water samples, varying the depth of collection. Water samples were collected in sterile containers; water temperature and pH were also recorded. One nursery was troubled with cyanobacteria growing in a production area experiencing overwatering. We collected water from this area as well.
Isolation and Growth on Solid Media
Isolation process and culture media: We first wanted to visualize the diversity of microalgae growth, both bacterial and eukaryotic, from the water samples. To accomplish this, we pipetted a small amount (between 1-2 mL) of each water sample onto solid BG-11 media (basic algal culture media). We used a sterile bacterial spreader to ensure the samples were distributed evenly around their respective plates. These plates were sealed and placed under LED light at room temperature (23℃) for 2 weeks to allow microalgae to grow.
Cleaning cultures on selective media: However, we weren’t certain if the green colonies were eukaryotic microalgae or cyanobacteria. We wanted to select only cyanobacteria and continue the purification process with these isolates. To accomplish this, we transferred colonies and grew them on a selective media. This selective media was made by adding 100 mg/L of cycloheximide to our basic BG-11 media. Cycloheximide inhibits protein synthesis in eukaryotic cells (e.g. algae) but allows for the growth of bacteria.
The purification of these colonies took several months; cyanobacteria can sometimes occur with fungal or bacterial partners. Purification involved multiple, sequential transfers of a very small amount of cell growth to fresh media. The picture on the right demonstrates the beautiful growing pattern of purified cyanobacteria cultures.
Increasing Cell Volume in Bioreactors
What is a bioreactor? Remember, cyanobacteria are bacteria that are capable of photosynthesis. A bioreactor uses light and carbon dioxide, two ingredients for photosynthesis, to increase the growth of cyanobacteria.
With this background, we purchased three UTEX Photobioreactors with the corresponding UTEX RGB-LED Lighting Platform (Culture Collection of Algae at the University of Texas, Austin). Throughout the remainder of our project, we maintained our cultures in these units.
Over the course of the project, we had a fabulous student worker who helped us with isolation and microscopic characterization. She found a unique isolate that has some special structures! Can you see cells with different shapes in the picture on the right? Among these structures are heterocysts, which are present in species that fix nitrogen, and akinetes, structures that act as energy reserves in some cyanobacteria species.
Pigment Extraction and Future Directions
We tested our cyanobacteria's ability to reduce phosphate and nitrate concentrations in solutions. We hope to repeat this experiment, as preliminary results are very promising! In addition, a couple of our isolates demonstrated the ability to grow in the presence of an herbicide containing glyphosate. One of our isolates produces a vibrant blue/purple pigment as you can see on the left! We look forward to exploring possibilities for pigment extraction and use. In addition, we will be exploring antagonistic abilities of cyanobacteria extract. The possibilities are endless with these marvelous microalgae!