Impacts on microbial communities in sediment by aquaculture farming during one salmon cycle

In recent years, the salmon farming industry has grown significantly worldwide, and in the Faroe Islands, it has become a major industry with an annual production of over 94,000 tonnes, yielding 24% of the GDP. According to environmental regulations, the ocean floor is monitored during every production cycle at all farming sites, involving macrofaunal, sensory, and chemical analyses. However, the impact of farming activity on microorganisms in the Faroe Islands remains unknown. This study aimed to assess the impact of Atlantic salmon farming on benthic microbial communities, giving a better understanding of the effects on the foundation of the benthic food web and to assess if these are more prone to environmental impact than traditional macrofaunal biomonitoring. Sediment cores were sampled along a transect from directly below the salmon cages to a background reference site. The sampling occurred prior to the release of salmon into the cages (‘before stocking’) and immediately before the salmon were harvested (‘peak biomass’). The 16S rRNA (V4-V5) gene was sequenced on an Illumina MiSeq from our sediment samples at the surface, 3 cm, and 10 cm depth. Significant shifts in microbial community composition were observed between ‘before stocking’ and ‘peak biomass’, as well as between different depth layers. Microbial diversity increased with increasing distance from the cages and was at its highest ‘before stocking’, indicating a significant impact of the salmon farming on the microbial community structure. In contrast to the regularly executed environmental monitoring, the results from this study showed an impact on the sediments by the salmon farming, underlining the powerful alternative of DNA-metabarcoding when biomonitoring an aquaculture area.

Methods
Sample collection and preparation

Sediment samples were collected on the 25th of September 2019 and 15th July 2020 at the fish farming area A-83 in Sørvágar, Faroe Islands (Figure 1). The first sampling effort was conducted after a three-month fallowing period, and the second sampling effort was carried out when the salmon biomass was at its highest.

Starting from the cage station RS20, six stations were sampled along a transect (Figure 1). The first station was collected directly at the periphery of the cage (0 m), and then approximately at 12 m, 40 m, 150 m, 190 m, and 600 m from RS20. The transect into the fjord follows the prevailing current direction, measured approximately 225 m west of the farm (Norði et al., 2023) (Figure 1). The sampling station at 600 meters was designated as a background sample, based on findings from a study indicating that the maximum impact range of organic enrichment from fish farms in Faroese fjords is 200 m (Mortensen et al., 2021). Our background site, 600 meters away, was based on a threshold set by the Aquaculture Stewardship Council (ASC) of >500 m distance between fish farm and reference station (Mortensen et al., 2021). However, the 600 meters sampling station is taken at a slightly shallower depth (≈ 35 m depth) than the other samples (≈ 45 m depth). Due to the small size of the fjord, we chose the best fitting reference point in a 500 m radius. During the ‘peak biomass’ sampling effort, two additional samples (1+ and 2+, illustrated in Figure 1) were taken due to the relocation of cages (RS20, RS22, RS24 and RS26) during farming cycles. To ensure consistent comparison with the ‘before stocking’ sampling effort, the additional samples were collected from RS28 (representing 0 m and 12 m distance), which was the outermost cage station on the same transect. Thus, samples were collected at stations 1, 2, 3, 4, 5, and 6 on both sampling events but only at stations 1+ and 2+ during ‘peak biomass’. This resulted in a total of 14 stations.

Samples were collected with a kayak corer, consisting of 47 cm plex tubes with a diameter of 44 mm (Supplementary File 1). Each of the 14 sediment cores was sliced into 1 cm intervals down to a depth of 10 cm. Three sediment layers were analyzed: the surface (scraping the surface), 3 cm depth, and 10 cm depth (Supplementary File 1). Three replicates were collected from each core and layer, resulting in a total of 126 samples. The 10 cm layer was the deepest layer that could be collected from all samples. The samples were stored at -18°C within 2-5 hours after collection and kept until DNA extraction, which was conducted 2-7 weeks later. Concurrently with our sample collection, an environmental condition assessment of the area was performed. This assessment is a statutory requirement before smolts are led into the cages and when the biomass is at its highest (i.e., before slaughter).

Samples for the legislated national environmental condition assessment, as described in ICES (2023), were collected by an impartial company. The environmental condition assessment was conducted in accordance with Norwegian Standard 9410:2016, which is also used by the Faroese Environmental Agency (Agency, 2018). The impact status is categorized as follows: ‘no impact’, ‘some impact’, ‘high impact’ and ‘very high impact’ (Agency, 2018).

DNA extraction and sequencing

The samples were thawed at room temperature and then homogenized to ensure uniform consistency. Each sample was divided into three laboratory analysis replicates and DNA was extracted using the Qiagen DNeasy PowerSoil kit according to the manufacturer’s instructions. The DNA yields from the extractions were quantified using a nanophotometer (3117, Implen). Average DNA yields for the samples were 41 ± 62 ng/mL, with an average A260/A280 ratios of 1.863 ± 0.304. A schematic of the sampling and laboratory protocol is shown in Supplementary File 2.

PCR and PCR clean-up

Sequencing was done of the V4-V5 region on the 16S rRNA gene using the primers 515F-Y and 926R according to Parada et al. (2015). PCR reactions were carried out in triplicate for each laboratory replicate, with a 1:10 dilution for all DNA extracts to reduce PCR inhibitors (Apprill et al., 2015; Djurhuus et al., 2017). In brief, 1 mL DNA template was added to the master mix, consisting of 12.5 mL 2X Phusion Master Mix (Thermo Fisher Scientific, USA), 10.50 mL nuclease-free water, and 0.5 mL of each primer (0.2 mM) (Parada et al., 2015), resulting in a 25 mL reaction. The PCR reaction occurred at 98°C for 30 s, followed by 27 cycles of 98°C for 10 s, 50°C for 30 s, and 72°C for 30 s, with a final extension at 72°C for 10 min. After PCR, the triplicates were pooled. To confirm the presence of targeted bands and to check for clean negative controls, the pooled amplicon products were run on 1.5% agarose gels using UView (Bio-Rad) for visualization. All non-template controls (NTCs) and blanks tested negative. The pooled amplicon products were purified using 1x magnetic Agencourt AMPure XP beads (Beckman Coulter, USA), following the manufacturer’s protocols. The concentration of all clean PCR products was quantified using a nanophotometer. A second PCR reaction was conducted to attach dual barcode indices and Illumina sequencing adapters with the Nextera XT Index Kit v2. In brief, 5 mL of each DNA amplicon was added to a master mix consisting of 25 mL 2X KAPA HiFi (Kapa Biosystems Inc., Woburn, U.S.) HotStart ReadyMix, 10 mL nuclease-free water, and 5 mL of primers Nextera XT Index 1 and Nextera XT Index 2, resulting in a total volume of 50 mL. PCR was performed with the following cycling conditions: 95°C for 3 min, 8 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 30 s, followed by a final extension at 72°C for 5 min. Subsequently, the products were purified using AMPure XP beads and quantified using the Qubit 2.0 Fluorometer and the Qubit dsDNA HS Assay Kit (Invitrogen, USA). Following quantification, library normalization was performed to get an even sequencing depth per sample. Sequencing libraries were verified using an Agilent Bioanalyzer 2100 (Agilent Technologies, USA) to check the sizes of PCR libraries. These were then sequenced on an Illumina MiSeq platform, generating 2 x 250 bp paired-end reads.