Ultrafiltration to Produce Pathogen Free Water in Shellfish Farms

Ultrafiltration process was tested to treat inlet water of shellfish hatcheries and nurseries farms. The aim of the study was to protect animals from pathogens linked to mass mortalities in shellfish culture. A semi industrial process was confronted to two qualities of water: natural pre-treated seawater and an effluent from an oyster breeding. The control of total load bacteria and elimination of vibrio was confirmed in both cases. Indeed, whatever the quality of inlet water, a removal of bacteria was obtained. Moreover, the ultrafiltration sustainability confronted to these two water qualities was validated. Chemical cleanings carried out every 12 hours minimum on hardest conditions of water qualities, led to a performance recover on the period of the tests. Finally, ultrafiltered seawater and effluent were used for fecundation and oyster breedings application. The results obtained, in comparison to prefiltered (1 μm) - UV treated seawater, showed that the ultrafiltration could be applied to shellfish culture.

Introduction

Shellfish culture is imperilled by the degradation of coastal water quality. Indeed, the profession must face pollution from different origins, variable with season and weather, with different impacts depending on the shellfish species. Since 2008, mass mortality episodes strike oyster cultures at different steps of their lives. Two pathogens were shown as partially responsible of these deleterious phenomena for the profession: Vibrio aestuarianus bacteria and an herpes virus, OsHv-1 (Cochennec-Laureau and Baud, 2011; Renault, 2011). In-land shellfish structures, hatcheries and nurseries may be exposed to these contaminants and then need to control and treat inlet waters to protect animals. Nowadays, physical disinfection using UV treatment is commonly used and more generally in aquaculture, chemical oxidation with chlorine or ozone is also employed (Summerfelt, 2003). These processes were proved efficient against the targeted pathogens, but they present disadvantages such as a treatment efficiency linked to the water quality (UV treatment) or, in the case of chemical oxidation, the generation of disinfection by products which could potentially be harmful for the animals (Gullian et al., 2012; Powell and Scolding, 2016). The consequence is the need of a pre-treatment when the disinfection is carried out with UV, to ensure the satisfying eliminating of contaminants or a post treatment to eliminate byproducts created with chemical oxidation (Lekang, 2013; Ozawa et al., 1991). Therefore, in shellfish culture, there is a need of a disinfection process able to deliver pathogen free water with a quality adapted for the breedings.

The aim of the study is to evaluate the performances of the ultrafiltration process to treat inlet waters in shellfish hatcheries or nurseries. This new process must (i) remove troublesome pathogens, (ii) deliver water with a quality adapted to the different stages of development of the Crassostrea gigas oyster and (iii) demonstrate its ability to face different water qualities and remain stable over time.

Material and Methods

The first part of the study was realised with a semi industrial unit able to treat 20 m. 3 .d -1 as presented in Figure 1. Membranes were PES hollow fibres (Aquasource) with a Molecular Weight Cut Off (MWCO) of 0.02 μm and a filtration surface of 8 m². Frontal filtration was carried out. Backwashes, air backwashes (backwash with a pre-injection of air in membranes) were performed at a chosen frequency and chemical cleaning was operated when the permeability decreased lower than 300 L.h -1 .m -2 .bar -1 . This pilot was confronted to two qualities of feed water during this study: natural seawater pre-treated with sand filtration (25 – 30 μm) and an effluent of an adult oyster breeding. In the case of natural seawater filtration, the aim was to validate the retention of Vibrio bacteria and total bacteria load. Moreover, the ultrafiltered water was used to realise fecundation of oysters. Fecundation rates were calculated and compared to the ones obtained with a control water treated with a filtration at 1 μm and UV. In the case of the treatment of the effluent of oyster breeding, the retention of Vibrio and bacteria was controlled with harder quality of water to treat. The ultrafiltered effluent was used for oyster spat breedings: the growth of shells in this water quality was compared to a control spat supplied with seawater filtered 1 μm and UV treated. Water quality parameters (physico chemicals and bacteriologic) and the evolution of spats were followed for a period superior to one month. In both studies, bacterial measurements were realised by petri dish culture in marine agar for total bacteria load and TCBS for Vibrio . These Vibrio measurements concerned the whole family of Vibrio and not only the targeted one, Vibrio aestuarianus . Hydraulic performances (transmembrane pressure and permeability) of the filtration unit were continuously monitored, taking account of the temperature, to validate the stability of the process.

. The second part of the study was focused on the retention of specific pathogens Vibrio aesturianus and herpes virus OsHv-1 with a lab scale pilot. The same membranes were used and the working surface of 0.14 m² was determined in order to work with the same volumetric concentration factor as the semi industrial unit but with a higher pathogen and virus concentrations. The retention of pathogens was controlled by (i) dipping oyster in permeate water and (ii) injecting permeate in oyster. The mortality of the animals was followed and compared with positive and negative controls.

Results and Discussion

Filtration of Natural Seawater

The conditions of filtration were based on previous studies and literature (Cordier et al., 2018, 2019; Guilbaud et al., 2013). A flux of 60 L.h -1 .m -2 and a time of 60 min between two backwashes were then imposed. The evolution of permeability in these conditions and the turbidity on the same period are presented in Figure 2. The turbidity of seawater feeding was not constant because dependant on the environment (weather, tides). Moreover, twice a day, spikes of turbidity generated by the cleaning of the sand filtered that could reach 20 NTU were monitored. Under these conditions of filtration and quality of water, a chemical cleaning of the membranes was carried out about every 40 h and the hydraulic performances remained stable.

. Figure 2: Evolution of permeability vs. time [filtration of seawater; J = 60 L.h -1 .m -2 , t filtration = 60 min]

. The bacterial water quality of the feed and the initial and final permeate was followed under these filtration conditions. The results obtained are presented in Figure 3 a. and b. Ultrafiltration led to a removal of the total load of bacteria and in the case of Vibrio , the graph puts in light a total retention of these microorganisms potentially harmful for oysters and thus, whatever the concentration in feed water.

. The pilot was able to control the bacterial water quality with the removal of Vibrio from seawater and stable conditions of filtration. To be used in shellfish culture, it is also necessary to validate that the quality of the water produced is adapted for the application. Therefore, ultrafiltered seawater was used to realise fecundations of Crassostrea gigas and the results were compared to the one obtained using a seawater with classical treatments, 1 μm filtration and UV treatment, as a control. Triplicates of fecundations were performed and fecundation rates were calculated from the number of oyster oocytes put in contact with spermatozoa and the number of larvae obtained 24 h after the fecundation. Results are presented in Figure 4.

. Fecundation rates obtained in both water qualities are similar, around 60 % reflecting that ultrafiltration produces water quality adapted for the fecundation of larvae, a sensible step in the oyster larvae life. This part of the study highlighted the efficiency of ultrafiltration process to protect oyster farms even in the case of sensible breedings. The removal of Vibrio bacteria from seawater was demonstrated and the sustainability of the process to treat seawater was validated.

Feeding with an effluent from oyster breeding

The pilot was supplied with a real effluent containing faeces, pseudo faeces and uneaten microalgae cells by the oysters. Two filtration conditions were tested: J = 60 L.h -1 .m -2 and t filtration = 30 min with an air-backwash every 5 backwashes and J =60 L.h -1 .m -2 and tfiltration = 60 min with an air-backwash every 3 backwashes. Evolution of permeability versus time (Figure 5a. and b.) highlight the stability of the process facing this effluent with a turbidity of an average of 2.7 NTU with daily picks reaching 30 NTU due to the cleaning of breeding tanks. A chemical cleaning was carried out every 12 hours in both cases leading to the conclusions that the process was sustainable in those conditions.

With the objective to prove that the process was able to protect the breedings from pathogens, bacterial water quality of the treated water was followed in order to validate the retention of total load bacteria and especially Vibrio bacteria. Figure 6.a. presents the concentrations measured in effluent before treatment, in ultrafiltered effluent and in control water used to feed the breedings. These graphs put in light the retention of Vibrio by the ultrafiltration process. Indeed, except at the beginning of the test, no Vibrio was detected in waters feeding oyster spats. A protection of the oysters towards these microoganisms was obtained even in the case of a loaded effluent. To validate that the ultrafiltered effluent was adapted to breeding applications, it was used to supply an oyster spat for 2 months. The evolution of height and weight of oysters compared to the control ones, supplied with control seawater filtered 1 μm and UV treated is presented Figure 6.b. The growth evolution being similar for both spats, we concluded hat besides protecting oysters from Vibrio , ultrafiltration produces a water with a quality adapted to breeding applications.

. Figure 5: Evolution of permeability vs. time for the treatment of an oyster breeding effluent – a. [J = 60 L.h -1 .m -2 and t filtration = 30 min] and b. . [J = 60 L.h -1 .m -2 and t filtration = 60 min] Conclusion

The study demonstrated that ultrafiltration process was a solution to treat inlet waters in shellfish farms. Indeed, this process was able to control the total load bacteria brought to animals and remove Vibrio bacteria, microorganisms potentially pathogens for the shells. This result was confirmed for two qualities of water. Moreover, the stability of the pilot was validated facing natural seawater and an effluent. Ultrafiltration remained sustainable for periods superior to three months in both cases. The chemical cleanings proceed by the filtration unit (every 12 hours) led to a recover of the initial performances. The quality of the water produced was tested for the targeted applications. Ultrafiltered seawater and effluent showed rearing efficiency similar to the one obtained with UV treated seawater, validating the efficiency of the process in fecundation or spat breedings applications. Finally, following the presented tests, the efficiency of the process to remove specific pathogens, Vibrio aestuarianus and virus OsHv-1, which are pathogens for oysters, is still studied using the lab scale ultrafiltration pilot.

. Conclusion

The study demonstrated that ultrafiltration process was a solution to treat inlet waters in shellfish farms. Indeed, this process was able to control the total load bacteria brought to animals and remove Vibrio bacteria, microorganisms potentially pathogens for the shells. This result was confirmed for two qualities of water. Moreover, the stability of the pilot was validated facing natural seawater and an effluent. Ultrafiltration remained sustainable for periods superior to three months in both cases. The chemical cleanings proceed by the filtration unit (every 12 hours) led to a recover of the initial performances. The quality of the water produced was tested for the targeted applications. Ultrafiltered seawater and effluent showed rearing efficiency similar to the one obtained with UV treated seawater, validating the efficiency of the process in fecundation or spat breedings applications. Finally, following the presented tests, the efficiency of the process to remove specific pathogens, Vibrio aestuarianus and virus OsHv-1, which are pathogens for oysters, is still studied using the lab scale ultrafiltration pilot.

To conclude, this study demonstrates that, whatever the quality of the seawater, the ultrafiltration process delivers a disinfected water with a quality adapted to shellfish culture, and thus, even in the delicate case of early life stages of oysters and with no pre or post treatment needed.