introduction: Prepping Water for Grow Out

Disinfection is a standard water treatment that reduces the pathogen load before grow-out begins. Effective treatment options include UV, ozone, and chlorine. They all work by degrading DNA non-selectively, which means the treatment kills off both good and bad microbes. Ozone, in particular, is very powerful.

Beneficial microbes that deliver essential services for water quality, like ammonia and nitrite reduction, are reduced or eliminated during disinfection. Probiotics are also degraded, which means losing huge benefits like immune support, stress relief, higher growth rates, and lower mortality in stock. 

However, the most devastating aspect of water disinfection is the drastic reduction of the diversity and abundance of the overall microbial community, which forms the foundation of health in the aquatic setting. 

Once the microbiome destabilises, we lose the competitive pressure of other microbes that keep pathogens in check. And without any competition for space or resources, survivor pathogens are free to wreak havoc in a system, which they do. 

There is an inherent balance between removing pathogens and preserving beneficial microbes that are required to support fish and shrimp during growout. To determine ozone’s effect on water and to optimize the use of ozone, we looked at how the microbiome fared after a 12-hour ozone treatment. 

methodology: Time Series

To carry out this study, we took six samples overall; three consecutive days before the ozone treatment and three consecutive days after ozone. 

We collected samples with  our patent-pending auto sampling equipment for this time series to standardize the samples and eliminate batch effects. 

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 three days leading up to the ozone treatment and three days after treatment. We then extracted the DNA from all samples, performed Next Gen sequencing, and analyzed the data using our AI-driven advanced algorithms.

results: Deep Instability

Before ozone, we can see that the community changes very little, with only a small drop in the population of Pseudomonas (a good thing). Candidatus Pelagibacter shifts a bit, but overall we would label this kind of movement as statistically irrelevant.  The three samples taken at days -3, -2, and -1 before ozone showed a solid baseline in the water as nature intended it: diverse and stable. 

The three samples taken consecutive days after the ozone treatment shows a very different story. After the ozone application, we see dramatic shifts in the community due to the crash out of almost all the microbial species.

At this point, there is a race for equilibrium to fill the newly created void. Over the next three days, we see wild swings in the rise and crash of various species which indicates deep instability. Even three days after the ozone treatment, equilibrium had yet not been reached. 

Microbiome Baselines Three Days Before and After Ozone

Pre-ozone days K1, K2, and K3,  show stability and microbial diversity. Post-ozone days K4, K5, and K6 shows wild swings, voids, and a drop in diversity. Water with microbiome instability shown here is unlikely to be able to support high productivity. 

Blue-green algae can be good, bad, or neutral. Tracking survival post-disinfection at the species-level is critical.

The Algal Factor

While disinfection is mostly aimed at reducing the bacterial load in water, algal survivors and downstream management is a factor to consider when prepping water for growout. 

‘Algae’ is a broad terms that covers both complex multi-cellular organisms called Eukaryotes and simple-cell life forms belonging to the phylum Cyanobacteria. These Prokaryotes are also called called blue-green algae.

Both can deliver benefits or cause significant issues in aquaculture when certain toxic species are inadvertently allowed to grow. For example, some species of Aphanizomenon release endotoxins in the water while Aureococcus spp is responsible for brown tides

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

We’ve seen both species survive disinfection, which is why it’s important to understand what’s happening at the species-level rather than looking at the group as a whole. And track microbes after disinfection.

In this case study, we found a small cohort of Cyanobacteria  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 which indicates instability. 

Day One
K4 is one day post ozone. Red arrow indicates Day One when tanks are stocked and grow out typically begins. However, at this stage in the water recovery process we can see that microbiome, which forms the foundation of health, is compromised. 
Setting The Stage For Success

If pathogens survive this type of extinction event (which they do), they now face very favourable conditions: very little competition for resources, very little downward pressure from other microbes to prevent outbreaks, and no buffering forces from probiotics or algae to outcompete them. This is the worst place to start grow out. 

The first day after ozone treatment is when the microbiome becomes severely compromised. Water in this state cannot support high productivity or survivability rates. In fact, we’ve seen tanks promptly crash with a 100% mortality after adding water freshly treated with ozone.   

Some operations add probiotics and seeding products to the water at points during growout, which is a very prudent thing to do, but our research shows that adding positive counter-forces at this stage is far too late. 

To counter balance the destabilization of the microbiome, disinfection protocols should be optimised for both pathogen load reduction and the subsequent rehabilitation of the water before growout starts, especially in RAS systems.   

conclusion: Lay the Foundation for High Productivity

In an ideal outcome, all pathogens would be killed off during the disinfection protocol, so the only challenge a farmer faces is rebalancing the water with effective re-seeding protocols. Effective being the key word. But many pathogens, especially Vibrio, are unwelcome hitchhikers that are accidentally introduced with newly imported stock. 

To find out what happens to the microbiome when infected stock was added to pathogen-free water read our case study: Hitchhiker Pathogens: Testing Quarantined Stock

The microbiome has a certain amount of elasticity, but the dynamics are highly complex. In this case study, we observed ozone’s powerful effect on community structure. 

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

Water holds the keys to high productivity, specifically the microbes that live in it. Together, they have the power to deliver high survival rates, booming growth, and a very healthy bottom line. If neglected, or unintentionally eliminated, all the benefits they deliver will be drastically reduced, along with profit margins.

Disinfection is Ground Zero for establishing a pattern for success or failure for growout. Similarly, water used for water exchange should also be rehabilitated and prepped for growout.

Optimizing disinfection protocols with particular attention paid to how inputs and treatments affect the microbiome are a prudent step to take in the successful management of water, especially in RAS systems.

Contact us today to find out
if your disinfection protocol is optimised.