ADVANCED BIOTECHNOLOGY FOR INTENSIVE FRESHWATER AQUACULTURE WASTEWATER REUSE

ABAWARE's aim is to develop and implement innovative technologies for better usage of water in aquaculture and minimize its negative impact on the environment and human health. At present, RASs (recirculating aquaculture systems) use significant amounts of fresh water due to inability to remove "waste" material such as suspended solids and fish metabolites, as well as low microbial quality of the recirculated water. Hence, developing technologies for efficient removal and/or utilization of such material as well as improving and maintaining a stable microbial quality within the RAS is important for the efficiency of production and fish health. A large amount of the suspended solids in RAS stems from undigested plant fiber used as matrix for fish pellets. In accordance with the activities outlined in WP1 we have collected larvae from wood feeding insects capable of utilizing complex carbohydrates such as cellulose. The microbial populations from the guts of three larvae were enriched in liquid microbiological medium and characterized for their ability to produce cellulolytic enzymes. Enrichment cultures displayed strong cellulolytic activities in enzymatic assays. The secreted enzymes were identified by LC-MS. The enrichment cultures were analyzed by using shotgun metagenomics approach (Illumina). The stability of the microbial communities was studied over time using 16S rDNA metabarcoding (Illumina). The screening of single bacterial isolates for cellulolytic activity and their identification by 16S rDNA sequencing is ongoing. Three candidates have also undergone whole-genome sequencing using SMRT technology (PacBio). The latter provided us with complete high quality genome assemblies for selected isolates and gave insights into their cellulose degrading potential. Detailed analyses of the full-genome sequencing data is currently ongoing. In addition to the search for cellulose degrading microorganisms we have performed detailed analysis of the dynamics of microbial populations in the cold water RAS. The analysis was performed with the intention of using these microorganisms in RAS wastewater treatment as well as tracking potential spread of antibiotic resistance and release of pathogens from RAS into the environment. The samples were collected monthly from a newly established RAS during a 12-month period. The biodiversity and dynamics of the microbial communities in cold water RAS were studied by 16S rDNA metabarcoding (Illumina) as well as traditional cultivation approaches. In total, we were able to collect forty microbial isolates. Genomes from six isolates were sequenced by using SMRT technology (PacBio) and further subjected to analysis to study their potential as vectors of antibiotic resistance, virulence factors or tools for improved wastewater treatment. In accordance with the objectives of WP4 linked to microbial water quality in warm water RAS we have performed sampling and analysis of microbial communities in a RAS facility, AQUATERRA (Romania). The analysis was performed using 16S rDNA metabarcoding on an Illumina platform. The obtained data provides a starting point for information that will be used to evaluate efficacy of the implemented wastewater treatment technology developed by DRF systems (Romania) and installed at the Aquaterra facility. In line with the ABAWARE project plan the biofilters and the biofilm of the same Norwegian cold water RAS facility for salmonids that was used as sampling site in WP1 and in addition a flow-through facility for commercial smolt production in Northern Norway was sampled and analyzed for odour related geosmin compounds in WP1 of ABAWARE. The results verify that there are odour and taste related geosmins in biofilters and biofilm in freshwater flow-through facilities for salmonid smolt production while not existing as a relevant phenomenon in the sampled flow-through facility.

The ABAWARE project is linking research in fish farming, sewage handling and development of antibiotic resistance with no tradition in co-operation. Commercial fish farms based on recirculation technologies (RAS) has created a biological waste product of uneaten feed and fish feces that potentially may harm the farmed fish, impact the environment negatively and that potentially could be harvested as a by-product with a higher commercial and biological value if prepared by microbial processing. The processes employed in the human sewage plants for decades will be developed to handle water and biological sediments in the fish farms with RAS. The innovative outcome and potential impact of ABAWARE will help the aquaculture industry in developing a side-product from the feed put into the RAS farms and will in develop a market for producers of infrastructure needed to perform the protocols suggested in ABAWARE.

Aquaculture is currently estimated to be the fastest-growing area of food production in the world. Although aquaculture can provide an important food source, these types of farms must be developed in a responsible and sustainable way. The rapid growth of intensive aquaculture systems has already caused important damage affecting both the environment and human health. The surrounding waters are affected by the chaotic algae growth generated by the high quantity of discharged pollutants (organic matters, P and N compounds etc.). This water pollution in some cases can prove deadly for certain aquatic species and indirectly constitute a danger to human population, who end up eating contaminated fishes and uses an inadequate quality water. When an ecosystem becomes too compromised, the fish farm is simply moved elsewhere and the environmental problems remain. Members of this consortium have identified the role of aquaculture as reservoirs of specific antibiotic resistance of importance to human health. In this context, the ABAWARE's main objectives are to develop and implement innovative technologies for the monitoring of surface and groundwater bodies for effective integrated water and waste management in freshwater aquaculture sectors by developing an advanced biotechnology for intensive recirculated aquaculture systems with minimum costs and footprint. In order to measure the effects of such an innovative system we will assess the nitrate and phosphorus loads from freshwater aquaculture, understand and decrease the environmental risks from freshwater aquaculture to human health (environmental exposure from water uses and food) and the reduce these loads and risks by the implementation of the technology. Thus, providing ways to avoid the risks of eutrophication of rivers and lakes, and propose management approaches for reducing impacts on ecosystem biodiversity and economic sectors.