Throughout the world’s oceans in global nutrient cycles, food chains, and climate, as well as increasingly in human-made industrial processes, a diverse set of planktonic microbes, such as algae, play an integral role. For nearly all of these planktonic microbes, however, little is known about them genetically beyond a few marker sequences, while their morphology, biological interactions, metabolism, and ecological significance remain a mystery.

Algae produce half of the oxygen in earth’s atmosphere and some forms of algae are used in industrial applications — such as producing high omega-3 fatty acids for baby formula or being used for biofuels — so there are many reasons a better understanding of algae could be beneficial. There is another side to algae, however, as some species can create harmful algal blooms (HABs), and those have been the focus of the research of University of Delaware’s Kathryn Coyne.

Advancing the study of microbes

To help advance the understanding of the cellular instructions that underpin microbial life in the sea, Coyne joined more than 100 scientists from institutions around the globe to publish a compilation of methods, or protocols, for laboratory experiments that will help scientists gain a better understanding of the genetic underpinnings of marine algae as a resource article in the journal Nature Methods.

The work was funded by the Gordon and Betty Moore Foundation’s Marine Microbiology Initiative. For her contribution to the collaboration, Coyne worked specifically with Heterosigma akashiwo, a species of algae that can produce HABs.

One of the mysteries about H. akashiwo is that while some strains produce toxins that can kill fish, other strains are non-toxic.

“We don’t have a clear understanding of what kind of toxin they produce. We just know that when there are blooms of this algae in some areas of the world, they are associated with massive fish kills,” said Coyne, an associate professor of marine biosciences in UD’s College of Earth, Ocean, and Environment (CEOE), and director of the Delaware Sea Grant program. “We also don’t know why some strains produce toxins, or what stimulates this toxin production.”

Scientists often use genome manipulation to better understand how microbes respond to the environment or to identify genes that may be involved in a specific response, like production of toxins. Unlike other algal species that serve as models for genome manipulations, however, H. akashiwo doesn’t have a cell wall, instead having only a thin membrane that holds the cell shape. Coyne explained that having a cell wall can be an impediment to genome manipulations and that these kinds of experiments usually entail some effort initially just to remove the cell wall or make it more porous.

Because H. akashiwo lacked a cell wall, Coyne and her research team proposed that genome manipulation might be more straightforward with this species, and were able to demonstrate that using a couple of gene manipulation methods that have been successful on other model species.

“We created a piece of genetic material that could be introduced into Heterosigma cells that would make them resistant to a specific antibiotic,” said Coyne. “If we were successful, we would be able to grow them in this antibiotic and cells that had incorporated the resistance gene would survive.”

Coyne worked with Deepak Nanjappa, a postdoctoral researcher in her lab who is also an author on the paper, as well as Pam Green and her lab members, Vinay Nagarajan, a postdoctoral researcher, and Monica Accerbi, a research associate in Green’s lab at the Delaware Biotechnology Institute (DBI).

Together, they tried a handful of methods and optimized those that were successful for Heterosigma. One method in particular was replicated successfully several times, showing that they were able to produce a genetically modified strain of Heterosigma. Using this approach, scientists can now probe the genome of Heterosigma akashiwo to gain a better understanding of how this species responds to environmental cues, or what genes are responsible for its toxicity.

One of the aims of the project was to make all of the methods developed freely available so that scientists can take that information and use it in their own research.

“The Moore Foundation funded this project with the expectation that all of the methods developed during this research would be published,” said Coyne. “Nothing is proprietary for this project, so we can share any of the protocols that we developed for Heterosigma.”

Immobilizing algicidal bacteria

In addition, Coyne had another paper published in the scientific journal, Harmful Algae, that detailed her work with Yanfei Wang, a doctoral student in CEOE, studying the algicidal bacterium Shewanella and how it could be used to remediate HABs.

Shewanella, which is an algicidal bacterium that has been isolated from the Delaware Inland Bays, is being developed as a biological control for HABs. It secretes water-soluble compounds that inhibit the growth of dinoflagellates, single-celled organisms that often produce toxins and contribute to HABs. Other research on this species of Shewanella shows that it has no negative effects on the growth of other species of algae, or on fish or shellfish. Since it was isolated from local waters, it may be considered an “environmentally neutral” solution to controlling HABs.

In order to use Shewanella in the natural environment to control HABs, there first needs to be a method to safely deploy the bacterium in areas that are at risk for HABs.

To move this HAB control solution closer to reality, Coyne and Wang immobilized Shewanella into several porous materials. Funded by Delaware Sea Grant, this research determined how well each material retained the bacteria over time, and whether the immobilized form of Shewanella was effective at controlling the growth of dinoflagellates.

Unlike other HAB control approaches, such as application of toxic chemicals like copper sulfate, the advantage of using immobilized algicidal bacteria is the potential for continuous control of HABs without the need for frequent reapplication. The immobilized bacteria can also be removed when it is no longer needed.

This research found that an alginate hydrogel was the most successful of the porous materials used in the study, and had the best retention of Shewanella cells.

This research also showed that Shewanella cells immobilized in alginate beads were as effective as free bacteria in controlling the growth of the harmful species while at the same time having no negative impacts on a non-harmful control species.

Overall, the study suggests that immobilized Shewanella may be used as an environmentally friendly approach to prevent or mitigate the blooms of harmful dinoflagellates and provides insight and directions for future studies.