introduction: Prepping Water for Grow Out

Treating raw water is essential to ensure the incoming water doesn’t have potential pathogens or pests that could cause problems during grow out. However, the techniques that effectively reduce pathogens (UV, ozone, bleach, etc.) are also good at eliminating beneficial microbes in the microbiome, which form the foundation of health in all water environments.

There is an inherent balance between removing pathogens and preserving beneficial microbes that are required to support the health of stock during grow out. To determine ozone’s effect on the microbiome and optimize the use of ozone, we compared untreated raw seawater with ozone-treated seawater.

methodology: Time Series

We utilized our patent-pending auto sampling equipment for this time series to standardize the samples and eliminate what’s known as the batch effect. 

Batch effects occur when outside factors, such as hand sampling, influence the data produced and can lead to inaccurate conclusions. Once biases are introduced, it’s impossible to determine if the observed changes are due to actual microbiome shifts or differences due to introduced errors from sample collection techniques.

Using our autosampler, we took a water baseline for two days leading up to the ozone treatment and three days after treatment. We then extracted the DNA of all five samples, performed Next-Gen sequencing, and analyzed the data using our AI-driven advanced algorithms.

results: Deep Instability

The microbiome in the raw seawater was stable leading up to the ozone treatment and showed good biodiversity. Biodiversity is a crucial driver of healthy water systems, and the presence of diversity means the tank or pond water has resilience and is less prone to future disease events.

Day one post-ozone shows a complete crash in diversity and the rise of Ruegeria, a gram-negative bacteria found in the Phylum Proteobacteria. Reugeria promptly crashed out the following day, leading to the increase of Altermonas and Brevundimonas, which has antioxidant properties and are widely used as feed supplements due to their vital functions for fish.

But any benefits that Brevundimonas might have conferred to the surrounding water were lost when the group crashed out on day three. After the treatment, we found that the overall number of species had dropped significantly.

We then saw the rise of yet another genus Limibaculum. Aside from these swings, a lack of diversity was reflected in our raw data.

WIld swings in the microbiome communitry structure post ozone indicates deep instability.
Effects on Algae

Cyanobacteria, otherwise called blue-green algae, can cause significant issues in aquaculture. Certain species are responsible for releasing neurotoxins into the water and toxic algal blooms.

However, all are not bad. Some species do confer benefits, which is why it’s vital to understand the community on a species-level rather than looking at the group as a whole.

The phytoplankton Synechococcus, for example, can remove ammonium from brackish aquaculture wastewater and has been targeted for potential use as a biofuel feedstock.

In this case study, we found a tiny cohort of Cyanobacteria in the fresh seawater that did not survive the ozone treatment, specifically: CalothrixGeitlerinema, and Dulcicalothrix.

However, there were cyano survivors after treatment, specifically Synechcococcus and Stanieria. This could indicate a better adaption mechanism for surviving ozone as these can withstand intense UV radiation from the sun and should be taken into consideration for future water treatments. 

Since both survived an extinction event, it would be wise to watch their numbers during growout for both benefits conferred and overgrowth.

Blue-green algae can be both good and bad. It's best to evaluate them on a species-level.
Diversity

Diversity on the Bray-Curtis chart allows us to see how similar or dissimilar the microbiome community is from sample to sample.

We can see that the two days before ozone resemble each other. And the two days post ozone are similar. However, taken as a group, they are entirely different, as they are on opposite sides of the MDS2 graph.

It’s interesting to note that ozone pushed the community structure in a polar opposite direction, showing how powerful ozone can be in shifting community dynamics.

Conclusion

Microbiome communities have a certain elasticity, but the dynamics are highly complex. In this case study, we observed ozone’s powerful effect on community structure. There were some benefits conferred but also some disadvantages.

Overall, the application of ozone severely disrupted the microbiome, and the wild swings in species oscillating daily is a hazardous point in which to start grow-out, especially if hitchhiker parasites or pathogens are accidentally introduced with newly arrived stock.

Contact us today to find out if your water is grow out ready.

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