Monitoring Microbial Water Quality in Near Real-Time

BactiQuant’s Contribution to an Optimal and Stable Production Environment

By Stavros Panoutsopoulos, Sales Engineer, BactiQuant, Denmark

 Aquaculture is a growing industry worldwide, with farmed fish production increasing steadily in recent years. The most crucial element influencing fish performance and health is water quality.

To provide the best conditions for the fish being raised, the fish farmer must be able to monitor water quality parameters. Most operations seek to keep these water quality variables within ranges that promote maximum growth while utilizing less water and reducing waste. Nutrient inputs stimulate microbes and phytoplankton, which may lead to poor water quality. The amount of nutrients in the effluent is directly correlated with water retention time and hydraulic turnover rate (Tucker et al. 2005). As a result, the intensity and type of aquaculture systems being used directly affect the water quality.

Modern aquaculture production facilities like RAS operate at or close to maximum carrying capacity and have relatively complex circulation, aeration, and biofilter systems. Because of the high biological risk factors in these systems, vigilance in monitoring and prompt action are required to reduce mortality rates (Helfrich and Libey, 1991).

On the one hand, in RAS systems, a biological risk element is the continuous, high concentration of bacteria that surrounds the fish. Some of them are slow-growing bacteria that commonly inhabit the intestines, gills, and skin’s mucus (A.L. Aam, 2015), while detrimental and pathogenic species can be found among the fast-growing opportunistic bacteria (Allen et al., 2004). According to Rojas and Tirado (2019), changes in water quality may lead to increased bacterial abundance and activity, reduce biological stability (Attramadal et al., 2012), and cause stress in the fish population (Harmin, T.D., 2009). Stressed individuals are more vulnerable to infection by dangerous, fast-growing pathogens (Raman et al., 2013). Yet, in aquafarm systems like RAS, monitoring microbiological water quality indicators has been impeded by time-consuming, ineffective, and/or complex tests (Rojas-Tirado, 2018). Today, there is a growing demand for operational technologies that may be applied to RAS management to quickly monitor microbiological water quality and improve process effect understanding (L. Pedersen and P.B. Pedersen, 2016). On the other hand, hatcheries of catfish, salmon, and trout species face challenges from various fungi responsible for devastating infections on fish in aquaculture, fish farms, and hobby fish tanks. The most significant cause of economic losses in aquaculture is diseased fish, and oomycete (water molds) infections are second only to bacterial diseases in their impact (Meyer 1991).

Bactiquant-water – A new Microbial Water Quality Parameter:

The Danish company BactiQuant has created and patented a new microbiological water quality parameter called Bactiquant-water (BQW). The BQW method is a quick field test for fluorometric detection of a specific bacterial hydrolase enzyme activity. Gram-positive and gram-negative bacteria, representing all of the major bacterial taxonomic groups, exhibit enzyme activity. The process is easy, rapid, and highly reproducible (McKernan et al., 2012). The measurement can be performed on the spot, even under rugged conditions, and requires no extraction procedures. The US-EPA verified the BactiQuant method in 2012 in collaboration with Batelle (McKernan et al., 2012), and the technology was awarded the seal of excellence by the EU in 2018. The technology has been used in research studies on RAS to monitor bacterial dynamics in the water phase, the impact of feed loading on microbial water quality, and bacterial activity on particles (Rojas-Tirado et al., 2019). Additionally, it has been used to monitor bacterial activity and conduct studies in the wetlands of a German trout farm (C. Naas, 2014).

Measurement principle

BactiQuant is based on a very sensitive fluorescence technology. The assay consists of three simple steps:

1. The bacteria in a water sample are concentrated using a Millex syringe-driven filter unit (PES, 0,22µm, express, Millipore).
2. A fluorophore-labelled enzyme substrate targeting a specific bacterial hydrolase activity is transferred to the filter unit using a syringe. The filter is saturated with the enzyme substrate, and the enzyme reaction is initiated.
3. Following the reaction step, the fluorophore produced from the reaction between the bacterial hydrolase activity and the enzyme substrate is flushed out of the filter into a cuvette. The fluorophore concentration is determined using a handheld fluorometer.

The fluorescence output from a water sample is linear with the time and volume of the sample. The result of a Bactiquant analysis is calculated based on standard conditions: Fluorescence output (FLU) per 250 ml of the water sample, reacted at 23^oC for 30 minutes. This is defined as the BactiQuant-Water value. The BQW value is a proxy for the total bacterial presence in the analyzed water sample. The technology can detect particle-associated bacteria (PAB´s), slough of biofilm, and planktonic bacteria, providing a relevant and comprehensive measure of the total bacterial exposure in RAS, compared to the limited capabilities of the traditional methods. The sensitivity to bacterial concentration can be readily adjusted by changing the water volume filtrated and/or the reaction time. A typical protocol for an aquaculture water analysis is the filtration of a 50 ml water sample reacted for five minutes at ambient temperature.  The enzyme activity is calculated according to the following formula:

Reading blank*(sample volume/250)*reaction time* e3.33440.0522*temperature​

Why rapid microbial biomass assessment is a necessity and not a luxury

One of the key factors in reducing microbial risk in RAS systems is providing stable water microbiology. Opportunistic microorganisms have a lower risk of taking over a system in which all parameters are maintained at steady levels, including water quality. Microbial water quality is essential not only for larvae and juvenile performance in hatcheries, which are the most vulnerable stages of fish developmentbut also for identifying problems in the later stages of the water cycle in a RAS system, such as the impact and optimization of sanitizing agents, the impact of feed on water quality, the effect of biofilter maintenance, etc. With the help of BactiQuant-water (for bacterial measurement) and FungiCount (for fungal measurement – able to measure micro fragments, spores, and hyphae), producers can generate their unique baseline of data and compare it to operational practices or altered assumptions (such as a new feed, feed spill, changes in operating practices for sludge drainage or recycling biofilters).

The baseline data will change throughout the course of production. They will represent various fish species, growth phases (hatching, start feed), fish size, densities, feeding schedules, seasonal variations in source water quality, and operational routines like cleaning schedules for biofilters and tanks.

In RAS systems, all components are equally crucial in ensuring the efficiency and safety of the system and the cultured species, like gears in a mechanical watch. BactiQuant allows the user to monitor each gear potentially and decide if changes need to be made since each gear contributes to fish welfare. Providing stable microbial water quality is an essential starting point that significantly contributes to better performance, increased growth, and reduced mortality. By optimizing processes, adjusting routines, and constantly monitoring microbial water quality, the user can increase proactivity and verify stable water quality.

Applications in Aquaculture systems

systems and, thus, assist operators in selecting and develop better production system designs. Last but not least, as bigger RAS systems have multiple hatcheries and grow-out tanks, the technology can be used to benchmark production out of control. monitoring the microbial activity in Critical Control Points (CCP’s), such as before and after the water treatment chain. This will result in a deeper understanding of the efficiency of the installed water treatment technologies and allow for adjustments of the treatment steps in near real-time. Thirdly, BactiQuant’s products can be utilised to evaluate the impact different operating and handling processes have on the microbiome. Unsanitary handling of fish (i.e., during vaccination), movement of fish in grow-out tanks, changing of equipment, feed type, feed frequency, etc., are critical examples of operational routines that can significantly disrupt the balance of the microbiome around the fish and allow for opportunistic microbes to proliferateby processes treatment watera stable production environment. Secondly, BactiQuant enables users to optimise their While microbial biomass is a critical process parameter for water quality, the ability to do measurements in near real-time allows early detection of deviations in water quality. This enables the operator to quickly adjust production processes and operational routines to maintain