Microbial water quality within Recirculating Aquaculture Systems

Empirical observations suggest causality between water quality and fish performance. Direct monitoring of microbial water quality (MWQ) is presently not part of recirculating aquaculture systems (RAS) management. Current standard methods to assess microbial water quality in RAS under operational conditions are based on direct predictive tools such as turbidity or visual observation on fish performance.

Factors affecting MWQ in RAS include but are not limited to organic matter input and accumulation, environmental conditions, biological processes, RAS treatment units and management. As RAS development takes place at a high pace, there is an urgent need to understand the link between operational factors and microbial dynamic.

In order to achieve this, rapid and reliable monitoring tools are required. The aim of this PhD thesis was to assess selected aspects of microbial water quality submitted in freshwater RAS experimentally controlled under different conditions. Microbial water quality changes were measured through a set of new, rapid, culture-independent and reliable methods such as Bactiquant®, hydrogen peroxide (HP) degradation assay and flow cytometry.

The present thesis encloses three scientific articles and unpublished data collected during the last three years. The three articles are related to: 1) bacterial activity dynamic in the water phase during start-up of RAS; 2) detection of changes in RAS MWQ associated to changes in feed loading; 3) monitoring of abrupt changes in MWQ within RAS water.

The first manuscript (Paper I) evaluated bacterial activity development in six identical , pilot scale freshwater RAS stocked with rainbow trout (Oncorhynchus mykiss) during a three months period from start-up. The systems were operated under constant conditions in terms of feed and water exchange. Bacterial activity was assessed with a new method called Bactiquant®, which measures bacterial activity indirectly by targeting a specific enzyme from the hydrolase class.

The results showed that during start-up, bacterial activity increased (quantified by Bactiquant®) with substantial fluctuations (variation) between RAS. After a three weeks period, the bacterial activity stabilized, which was correlated to both particulate and dissolved fractions of organic matter measured as chemical oxygen demand (COD).

The second study (Paper II) investigated changes in RAS microbial water quality associated with changes in the feed loading. After an experimental stabilization period, under constant conditions, the RAS were divided into three treatment groups according to the feed loading. The effect of feed loading was evaluated based on duplicate RAS: i) unchanged (3.13 kg/m3); ii) stopped feeding (0 kg/m3); and iii) doubled feeding (6.25 kg/m3).

Microbial water quality was assessed in terms of bacterial activity with Bactiquant® and HP degradation assay and bacterial abundance with flow cytometry. The overall results showed that microbial water quality responded (directly) positively to feed loading changes. Bacterial activity was highly related to the accumulation of particulate organic matter whereas abundance of free-living bacteria was associated with the dissolved organic matter content.

A delay in bacterial activity and abundance response in the water phase was observed and it was suggested that the biofilter buffered transient and prolonged changes by biofilm/water phase interactions. The third study (Paper III) was performed in an extensive experimental setup including twelve separate and identical freshwater RAS.

The 1.7 m3 pilot scale RAS with rainbow trout were kept under constant conditions and managed after predefined protocols for five months. The aim of this study was to evaluate the effects of different levels of easy biodegradable substance on MQW in RAS subjected to acetate spiking trials. For three consecutive days, acetate was spiked (daily pulse addition of 40 mg/l acetate) into the fish tanks and associated bacterial activity was investigated before, during and after addition in both batch and full scale experiments .

Microbial water quality was assessed in terms of activity (Bactiquant® and HP degradation assay) and abundance (flow cytometry). The bacterial dynamics and potential influence be the biofilter, was also evaluated within the full scale spike experiments. The experimental setup group consisted on four treatments: i) control RAS with biofilter, ii) control RAS without biofilter, iii) RAS with biofilter, spiked with acetate; and iv) RAS without biofilter, spiked with acetate.

Each treatment was evaluated in triplicated RAS. The main results showed that bacteria were substrate limited but as soon as the systems were spiked with the readily, easy degradable carbon source, bacterial increased in terms of activity and number. The biofilter related communities was found to be the main source of consumption of the additional carbon source, suppressing the growth of bacteria in the water column.

In conclusion, both of the rapid and simple methods - Bactiquant® and HP degradation assay - proved to provide a reliable, broad picture regarding microbial activity, by taking into consideration both free-living bacteria and particles-associated bacteria. Bacterial activity was related to the presence of particulate organic matter.

Flow cytometry quantified the numbers of free-living cells in the water phase, which was highly associated with the dissolved fraction of the readily available organic matter. Furthermore, this PhD study elucidated that when monitoring bacteria in the water phase there are two main interactions that has to be taken into consideration in future studies: i) biofilter bacterial population vs suspended bacterial population (both free-living and particle-associated bacteria); and ii) free-living bacterial population vs particle associated bacterial population