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Vol. 8: 637–646, 2016 AQUACULTURE ENVIRONMENT INTERACTIONS Published November 14 doi: 10.3354/aei00204 Aquacult Environ Interact OPENPEN ACCESSCCESS Bacterial community composition of flocculent matter under a salmonid aquaculture site in Newfoundland, Canada Joost T. P. Verhoeven1, Flora Salvo2, Dounia Hamoutene2, Suzanne C. Dufour1,* 1Department of Biology, Memorial University of Newfoundland, St. John’s, NL A1B 3X9, Canada 2Science Branch, Fisheries and Oceans Canada, PO Box 5667, St. John’s, NL A1C 5X1, Canada ABSTRACT: Aquaculture has become a rapidly growing industry: over the past 3 decades, com- mercial production has steadily increased, and further expansion seems likely. However, the rise of aquaculture has been accompanied by concerns, especially regarding environmental sustainability. Substrates located under aquaculture sites receive large influxes of organic matter that can subsequently create anoxic conditions and thereby impact existing benthic communities. Shifts in the relative abundance of specific groups of bacteria could prove to be important indicators of impact and remediation. Here, we investigated bacterial community composition via 16S rRNA gene sequencing on isolated DNA from flocculent matter samples and associated bacterial mats under a hard-bottom aquaculture site in Newfoundland, Canada. We describe the heterogeneous community present in the flocculent matter, characterized by high relative abundances of the genera Spirochaeta (12%), Prolixibacter (5.6%) and Marinifilum (4.6%). Bacterial mats were not composed of Beggiatoa as often hypothesized, but instead were dominated by the genera Spirochaeta (15%), Prevotella (21%), Meniscus (11%) and Odoribacter (20%). Our findings provide insights into the bacterial composition of flocculent matter deposited on hard substrates and undergoing de - gradation, and point to 3 unexpected bacterial genera as potential indicators of organic enrichment. KEY WORDS: Microbiome · Organic enrichment · Bacterial mats · Spirochaeta · Prolixibacter · Marinifilum INTRODUCTION and sustainability have arisen (Jusup et al. 2009). One major environmental concern is the increased In recent years, the aquaculture industry has expe- deposition of organic matter in the benthic environ- rienced rapid growth worldwide (Asche et al. 2008), ment, which usually occurs in the form of a complex substantiating the hypothesis that aquaculture could mixture known as flocculent matter composed of soon produce more than half of the seafood con- decomposing fish-food pellets, microbes, fish faeces sumed globally (Costa-Pierce 2010). Marine aqua - and other organic matter (Salvo et al. 2015). The culture operations in coastal Newfoundland (NL), accumulation of organic matter on the seafloor can Canada, notably the cage culture of salmonids and drive an increase in oxygen consumption by environ- long-line mussel culture, have seen significant growth mental or fish faeces-associated micro-organisms in the last decade (Anderson et al. 2005, Fisheries (Tett 2008). The resulting hypoxic conditions can and Oceans Canada 2016). negatively, and potentially permanently, affect the Along with the increase in finfish aquaculture sites benthic in- and epifauna (Karakassis et al. 2000, (or ‘farms’), concerns regarding ecological impacts Jusup et al. 2009, Pochon et al. 2015). © Fisheries and Oceans Canada and J. T. P. Verhoeven, S. C. Dufour 2016. Open Access under Creative Commons by Attribu- *Corresponding author: [email protected] tion Licence. Use, distribution and reproduction are unre- stricted. Authors and original publication must be credited. Publisher: Inter-Research · www.int-res.com 638 Aquacult Environ Interact 8: 637–646, 2016 Monitoring the ecological impact of fish farm oper- The presence of bacterial mats and flocculent mat ter ations on the environment is key to assess, under- are used as indicators in regulatory frameworks for stand and develop sustainable models for aquacul- aquaculture (Hamoutene et al. 2014). Further investi- ture. To this end, sediment sulphides and redox gating the microbial community composition of floc - potential measurements are often used as indicators culent matter and identifying key bacterial species to extrapolate the biological response to organic involved is a crucial step in developing our under- enrichment (Hargrave et al. 2008). In NL, many standing of how aquaculture operations initiate bacterial aquaculture operations occur in bays with water community changes and whether these shifts can be depths over 30 m and hard substrates comprised of used to measure impact and, potentially, remediation. rock and cobble, where grab sampling cannot be In this study, we investigated the bacterial commu- conducted, and therefore completing valid sulphide nity composition of multiple flocculent matter samples and redox potential measurements is difficult under a salmonid farm in the Hermitage Bay area, on (Hamoutene et al. 2014, 2015). the south coast of NL. Our analysis was performed by More recently, visual monitoring has documented sequencing the V6/V8/V9 region of the 16S rRNA the close association of flocculent matter and white gene. Multiple samples were analysed to detect within- Beggiatoa-like bacterial mats with the elevated sul- site variation and determine the homogeneity of the phide levels typically found near farms in NL flocculent matter observed. We also collected multiple (Hamoutene 2014). When the collection of sulphides samples of bacterial mats and attempted to identify and redox potential measurements is not feasible, the bacterial community driving mat formation. drop-camera based observation of flocculent matter and bacterial mats is a valid alternative indicator of organic enrichment, as these features are generally MATERIALS AND METHODS not observed in areas where no active aquaculture operations are conducted (Crawford et al. 2001, Collection of flocculent matter Hamoutene 2014, Hamoutene et al. 2015). The formation of bacterial mats in response to aquaculture Samples of flocculent matter were collected on 27 operations is indicative of a shift in bacterial commu- August 2015 at a salmonid aquaculture site in ‘Little nity composition in the farm vicinity. Indeed, previ- Passage’, located between Bay D’Espoir and Her- ous studies have shown fish farm-dependent organic mitage Bay on the south coast of NL. This site, part of enrichment and anoxia to be closely associated with a larger ongoing study of benthic remediation pro- large-scale shifts in bacterial communities (Asami et cesses at aquaculture sites, had been out of production al. 2005, Dowle et al. 2015). Such shifts are usually (in fallow) for 3 mo at the time of sampling. Drop- characterized by an increase in abundance of sul- camera visualization was performed to detect stations phur, nitrogen and methane cycle-associated bacte- (locations within the site) presenting flocculent matter ria in the direct environment (Asami et al. 2005, and bacterial mats. A total of 7 stations, located ap- Dowle et al. 2015). proximately 10 m from each other, were investigated, The apparent plasticity of the microbial community and an average of 7 grabs (Eckman or box corer), di- in response to aquaculture operations suggests that rectly adjacent to cage edges, were completed at each these communities could have intrinsic indicator po- station, at an average depth of 72 m. Four stations tential for indirectly assessing ecological impacts and were selected (based on grab success and retrieved remediation. However, shifts in microbial community flocculent matter) for molecular analysis, and a total of composition are not fully understood, and many ques- 15 samples (approximately 4 per station) were collected tions remain as to what drives these changes. For ex- from grab material using sterile spoons or cut pipette ample, the white bacterial mats observed near farms, tips. Samples were subsequently stored in sterile con- until recently considered to be Beggiatoa, have not tainers and frozen at −20°C until arrival at the labora- yet been identified (Dowle et al. 2015). Furthermore, tory, where they were transferred to a −80°C freezer. bacterial changes in one region might not be compa- rable to those observed elsewhere due to differences in geographical location, bathymetry and other envi- DNA extraction and high-throughput sequencing ronmental factors. In NL, for example, there is often (HTS) very little natural sediment, and flocculent matter build-up represents a drastic change of substrate for Flocculent matter samples were thawed on ice and benthic organisms (Hamoutene et al. 2016). transferred aseptically to bead beating tubes con- Verhoeven et al.: Bacterial community composition of flocculent matter 639 taining 0.1 mm glass beads (MO Bio Laboratories). (utilizing USEARCH global implementation, percent- Total microbial DNA was subsequently isolated age identity = 97%). using the MO BIO PowerViral DNA/RNA isolation Taxonomy was inferred for each OTU through an kit (MO Bio Laboratories) according to the manufac- internal SPONS wrapper function which invokes 4 turer’s specifications. Extracted nucleic acids were taxonomic classifiers through the assign_taxonomy. py measured for quality and concentration using a Nano - command within the QIIME framework: UCLUST Drop 1000 (Thermo Scientific) and outsourced for (maximum number of hits to consider = 1, minimum Illumina MiSeq 2x300-bp paired-end sequencing of percent similarity = 0.8, minimum consensus fraction = the 16S rRNA (V6/V7/V8 region)

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