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Prosjektnummer

901470

Prosjektinformasjon

Prosjektnummer: 901470
Status: Avsluttet
Startdato: 05.01.2018
Sluttdato: 30.05.2019

Microbial colonization in recirculation aquaculture systems (RAS) / Mikrobiell kolonisering i resirkuleringsanlegg for havbruk

​Ny kunnskap som bidrar til bedret fiskehelse og redusert dødelighet i resirkuleringsanlegg
​• En mikrobiell suksesjon ble observert både i vannet og biofilteret i løpet av den første produksjonssyklusen, og de mikrobielle populasjonene i biofilterets biofilm er stabile over tid, selv ved varierende vannkvalitet.
• Biofilteret knyttet til kar med lav biomasse hadde høyest andel av nitrifiserende bakterier, og en større andel saktevoksende bakterier som omdanner nitritt til nitrat.
• Den nitrifiserende populasjonen i kommersielt inokulum vokser saktere og har lavest effektivitet av ammonium-oksidasjon, sammenlignet med et modnet RAS biofilter. Dette betyr at det tar lang tid før filteret er operativt med tanke på nitrifiseringsprosessene.  
• Overføring av biofilterbærere fra et modnet RAS biofilter gav den tidligste og høyeste produksjonen av nitrat og nitritt i veksteksperimentene, sammenlignet med kommersielt inokulum.
• I kulturer tilsatt biofilterbærere fra et modnet RAS ble det påvist bakterier som kunne utføre hele oksidasjonsprosessen fra ammonium til nitrat, mens kulturer tilsatt kommersielt inokulum inneholdt bakterier og arker som kunne utføre oksidasjon av ammonium til nitritt.
• Gitt at mikroorganismene i biofiltermateriale er nøye kartlagt og analysert for å unngå smitterisiko, vil overføring av biofilmbærere fra biofilteret i et modnet, velfungerende RAS til et nytt RAS være den mest effektive måten å inokulere det nye biofilteret med nitrifiserende bakterier.​
Sammendrag av resultater fra prosjektets sluttrapport (summary in English below)
I lukkede resirkulerende akvakultur system (RAS) er det viktig med god vannkvalitet for vellykket vekst og utvikling av post-smolt laks. De nitrifiserende mikroorganismene i biofilteret bidrar til god vannkvalitet ved å omdanne ammonium til nitrat, og redusere mengden organisk materiale i vannet. En av de største utfordringene ved oppstart av nytt RAS er å få etablert en populasjon med nitrifiserende bakterier i biofilteret slik at filteret er operativt når fisken blir satt inn. I dette prosjektet har man fulgt etableringen av nitrifiserende bakterier i to nye RAS biofiltre, og sammenlignet effektiviteten av to ulike inokulum for RAS biofilter i veksteksperimenter. Resultatene viste at de nitrifiserende populasjonene i de to nye RAS biofiltre utviklet seg ulikt. Dette ble sannsynligvis forårsaket av store variasjoner i vannkvaliteten de første fire månedene av driften, og ulik biomasse tilknyttet hvert biofilter. Biofilteret tilknyttet kar med lite fisk hadde best modning i form av lavere nitritt-konsentrasjoner og høyere andel nitrifiserende bakterier etter fire måneder. Pågående korrelasjonsanalyser vil muligens vise faktorer som er gunstige for biofilteraktivering. Vekstforsøkene viste at overført biofiltermateriale fra et modnet RAS oppnådde nitritt og nitrat produksjon tidligst sammenlignet med kommersielt inokulum, og nådde de høyeste konsentrasjonene over tid. Sekvensering av 16S rRNA gener viste også at kulturene med overførte biofilter materiale hadde størst andel nitrifiserende bakterier etter 39 dagers inkubasjon. 

Vitenskapelig publisering
– Irene Roalkvam, Karine Drønen, Håkon Dahle and Heidrun Inger Wergeland, ‘Comparison of active biofilm carriers and commercially available inoculum for activation of biofilters in marine recirculating aquaculture systems (RAS)’, Aquaculture, 514 (2020). https://doi.org/10.1016/j.aquaculture.2019.734480.

Summary of results from the project’s final report
In recirculating aquaculture systems (RAS) good water quality is essential for successful breeding of post-smolt Atlantic salmon. The nitrifying microorganisms in the biofilter play an impor​tant role in water improvement; by converting ammonium to nitrite and nitrate, and removing some of the organic matter. One of the greatest challenges when starting a new RAS is to establish the nitrifying population in the biofilter so that the biofilter is fully operational before the fish is installed. This project has monitored the establishment of a nitrifying population in two new RAS biofilters, and compared the nitrifying performances of two different biofilter inoculums in growth experiments. The results showed great differences in the development of nitrifying population in the new biofilters. This is probably caused changing water quality during the first four months of production, and differences in stocki​ng densities in tanks associated with each biofilter. The biofilter associated with low biomass had the most successful activation progress, due to lower concentrations of nitrite during production and high relative abundance of nitrifying bacteria after four months. Statistical analyses are ongoing, and might identify correlations between factors that can influence the biofilter activation process. The growth experiments showed that transferred biofilter material from an established RAS generated nitrite and nitrate earliest, compared to commercial inoculum, and also achieved the highest concentrations of nitrite and nitrate. The 16S rRNA gene sequencing showed that growth cultures containing transferred biofilter material had the highest proportion of nitrifying bacteria in the total population after 39 days of incubation.

​Prosjektet har fremskaffet viktig kunnskap om hvordan optimalisere og forbedre mikrobiologiske parametere i resirkuleringsanlegg.

Background
Recirculation aquaculture systems (RAS) presently offer the best supporting technology for microbial control in fish farming. This requires, however, the use of biofilters with a defined microbial community that can perform the processes needed for good water quality. The main function of these bacteria is to remove ammonium by oxidizing it to nitrite and then to nitrate.

Furthermore, microbes in the biofilter are efficient in processes like organic matter flocculation and utilization, which remove some of the organic matter in the water. However, the slow growing ammonium oxidizing population can be out-competed or displaced by the fast growing organotrophic population. In general, this is a main problem in aquaculture RAS operation and other wastewater treatment technology.
 
This project will follow the development of microbial communities from the very start-up of two new biofilters at Erko Settefisk AS, using the same methodology as applied in the project ‘Adapting monitoring tools for bacterial load detection in closed marine fish farms: for improved fish health and reduced mortalities (MONITOR)’ (Norwegian Research Council’s project no. 267545).

Based on the results from this comparative study, information and recommendations regarding biofilter start-up will be available for RAS fish farms on a national scale. The project will be coordinated with the ongoing MONITOR project.
Objectives
Objectives: Study A
To reduce risk of mortality and mortalities in RAS tanks and thus improve fish health and production volume.

Sub-objectives
• to provide data on microbial composition microorganisms in the start-up period of two biofilters;
• to compare different biofilter start-up strategies.
 
Objectives: Study B
To identify the optimal salinity for microbial inoculum establishment in biofilters.

Sub-objectives
• to determine microbial composition;
• to determine the optimal salinity for maximum ammonia oxidation rates in the start-up period.
Expected project outcome
Outcomes of study A
Reduce risk of mortality in RAS tanks and thus improve fish health and production volume, by:
• transferable knowledge on microbial composition to RAS farms;
• generate highly relevant data as commercial microbial inoculum is used;
• help to plan RAS biofilter start-up and to promote fish Health;
• reduce risk of fish mortalities, by optimising water quality and microbial composition;
• lower insurance tax for the first fish batch.

Outcomes study B
Identify the optimal salinity for successful biofilter start-up, including reduced incubation time and increased ammonium oxidation rates.

This will:
• give in-depth information on microflora and possible changes in turnover rates caused by changing salinities;
• predict effect on addition of freshwater and, if positive, make recommendations to fish farmers on which salinity gives the highest ammonium oxidation rates;

The test model can be applied to other RAS farms to test the actual inoculum at their sites.
Project design and implementation
Study A: Comparative study of microbial colonization and establishment in two marine RAS biofilters

The establishment of microbial communities in biofilters will follow two strategies:
1. Supply biofilter inoculum with NH4+ for three months, as in accordance with the SINTEF / Norwegian Institute for Water Research (NIVA) recommendations, prior to small smolt addition;
2. Supply biofilter inoculum with NH4+ for one month before introducing water and large post smolt from a currently running unit (see below). After two months the robust fish is replaced by the small post-smolt.
 
Regime 2 ensures first colonization by the original inoculum, thus, the introduced water and fish from regime 1 will probably only complement the established microbes.

Sampling of biofilter carriers and water will follow the routines implemented in the MONITOR project. The microbial community development over time in each regime will be revealed using molecular methods for microbial ecology studies, i.e. 16S rRNA amplicon libraries, high throughput sequencing and bioinformatics tools.

This extensive sampling will provide data with high standard and high resolution, which allow for comparisons between the different start-up regimes. In addition, comparisons can also be made between these new biofilters and the established RAS biofilter already included in the MONITOR project.
 
Study B: Testing optimal salinity for microbial oxidation of ammonium to nitrate in biofilters
We will compare two biofilter inoculums (new and maturated) at 9 different salinities between 16 and 32‰ (with three parallels for each), and controls without inoculum. Filtered fresh and seawater from Erko Settefisk AS will be mixed adequately for desired salinities. The samples will be enriched with NH4+ and organic matter (only one series). Samples will be incubated for three weeks at 14°C in incubators at UiB. The microorganisms in the cultures will be quantified using CASY cell counter, and the microbial community analyses will be performed on samples from all salinities using the same methods as described above and used in the MONITOR project. The NO3-, NO2- and NH4+ concentrations will be measured in order to monitor the efficiency of ammonia oxidation performed by the different microbial communities. The test model can later be applied to other RAS farms to test the actual inoculum at their sites.
Formidling er planlagt gjennom:
• seminarer og konferanser (både nasjonale og internasjonale)
• fagblad som Norsk Fiskeoppdrett
• vitenskapelige artikler, vil bli koordinert med resultater fra MONITOR-prosjektet
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