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The microbiota of recirculating aquaculture systems (RAS) is of major importance for optimal fish health. However, the microbial communities in commercial RAS are highly complex and more knowledge is needed to potentially control and maintain beneficial microbial communities for good fish production. In this study we monitored microbial communities in a commercial RAS producing Atlantic salmon fry (Salmo salar) during seven consecutive production batches. The water of rearing tanks and the water sump downstream of the biofilter/upstream of the UV, as well as biofilm of the wall of the rearing tanks and the fixed bed biofilter were analysed using 16S rRNA gene amplicon sequencing to elucidate the spatial-temporal microbial dynamics. The results showed that the microbiota composition of water and biofilm varied within and between the production batches, and that the fallowing periods had a substantial effect on the microbial communities. The correlation of the water and biofilm microbiota to fish presence in the system was confirmed by supervised machine learning. Shifts in the composition of the microbiota were identified in conjunction with variations in organic matter loading both during production and fallowing. In addition, variables like oxygen saturation, biomass, and feed type, showed good correlation with variations in the water microbiota composition. Although microbiota changed at fallowing, the microbiota returned to similar compositions during the production phases and was especially evident for the water microbiota. This indicates that the development of microbiota composition is strongly dictated by the similar selection pressure in the system. Nitrifying communities were dominated by Nitrospira, and the third most abundant Nitrospira OTUs were related to the comammox Nitrospira nitrificans. The microbial communities in the biofilter biofilm and water were significantly different but shared abundant taxa and followed the same temporal microbial dynamics and indicates an interaction between the biofilter biofilm and the suspended bacteria. CFU analysis showed that the fraction of rapid-growing bacteria was significantly higher in the rearing water than in the water sump upstream the UV disinfection, indicating that disinfection upstream the rearing tanks allowed for growth of opportunistic bacteria. A community with considerable potential for opportunistic regrowth can have consequences for the microbial water quality and the resistance against pathogen invasion The absence of an in-line disinfection step or placing the disinfection unit upstream the biofilter might provide better microbial water quality and a more resilient system against pathogen proliferation.

Recirculating aquaculture systems (RAS) harbour complex microbial communities which can have an impact on the growth and development of the reared fish. This study aimed to improve our understanding of microbial community dynamics in a RAS involving three production batches of Atlantic salmon fry and parr during a period of 20 months. Water for analysis of microbiota was sampled at different positions in the RAS, and we also examined the effect of UV treatment on the water microbiota. Microbial communities were characterized by 16S rDNA amplicon sequencing of water samples taken directly upstream and downstream of the UV treatment unit and from three of the rearing tanks. In total 6 sampling events were made during a 20-month period. The study showed that: 1) Two of the production batches had a highly similar water microbiota despite disinfection of the system between the batches and rearing fish of different stages. In contrast, the first production batch showed a different water microbiota with variable composition through the system and over time. A more immature biofilter in the first batch may explain these differences. 2) The full-flow UV treatment directly upstream the rearing tanks had no observable effect on the community composition of the water microbiota in the different sampling positions in the RAS. This was likely a consequence of the low hydraulic retention time (HRT) (23 min) in rearing tanks, low bacterial regrowth in the fish tanks and community changes throughout the RAS loop. 3) The disinfection effect on viable bacterial densities in the water directly downstream of the UV treatment was around 89%, when the water was clear. Regrowth of bacteria following disinfection was low compared to those reported for marine RAS with UV disinfection and long HRT in fish tanks. The study shows that UV disinfection can be used to efficiently reduce bacterial density without compromising the microbial water quality in the fish tanks in RAS with low HRT.

Understanding how periodical disturbances affect the community assembly processes is vital for predicting temporal dynamics in microbial communities. However, the effect of dilutions as disturbances are poorly understood. We used a marine bacterial community to investigate the effect of disturbance (+/−) and carrying capacity (high/low) over 50 days in a dispersal-limited 2 × 2 factorial study in triplicates, with a crossover in the disturbance regime between microcosms halfway in the experiment. We modelled the rate of change in community composition between replicates and used this rate to quantify selection and ecological drift. The disturbed communities increased in Bray–Curtis similarity with 0.011 ± 0.0045 (Period 1) and 0.0092 ± 0.0080 day−1 (Period 2), indicating that selection dominated community assembly. The undisturbed communities decreased in similarity at a rate of −0.015 ± 0.0038 day−1 in Period 1 and were stable in Period 2 at 0.00050 ± 0.0040 day−1, suggesting drift structured community assembly. Interestingly, carrying capacity had minor effects on community dynamics. This study is the first to show that stochastic effects are suppressed by periodical disturbances resulting in exponential growth periods due to density-independent biomass loss and resource input. The increased contribution of selection as a response to disturbances implies that ecosystem prediction is achievable.

Background Fish skin represents an ancient vertebrate mucosal surface, sharing characteristics with other mucosal surfaces including those of the intestine. The skin mucosa is continuously exposed to microbes in the surrounding water and is therefore important in the first line defense against environmental pathogens by preventing bacteria from accessing the underlying surfaces. Understanding the microbe-host interactions at the fish skin mucosa is highly relevant in order to understand and control infection, commensalism, colonization, persistence, infection, and disease. Here we investigate the interactions between the pathogenic bacteria Aeromonas salmonicida (A. salmonicida) and Yersinia ruckeri (Y. ruckeri), respectively, and the skin mucosal surface of Atlantic salmon fry using AFM force spectroscopy. Results The results obtained revealed that when retracting probes functionalized with bacteria from surfaces coated with immobilized mucins, isolated from salmon mucosal surfaces, rupture events reflecting the disruption of adhesive interactions were observed, with rupture strengths centered around 200 pN. However, when retracting probes functionalized with bacteria from the intact mucosal surface of salmon fish fry no adhesive interactions could be detected. Furthermore, rheological measurements revealed a near fluid-like behavior for the fish fry skin mucus. Taken together, the experimental data indicate that the adhesion between the mucin molecules within the mucous layer may be significantly weaker than the interaction between the bacteria and the mucin molecules. The bacteria, immobilized on the AFM probe, do bind to individual mucins in the mucosal layer, but are released from the near fluid mucus with little resistance upon retraction of the AFM probe, to which they are immobilized. Conclusion The data provided in the current paper reveal that A. salmonicida and Y. ruckeri do bind to the immobilized mucins. However, when retracting the bacteria from intact mucosal surfaces, no adhesive interactions are detected. These observations suggest a mechanism underlying the protective function of the mucosal surface based on the clearing of potential threats by adhering them to loosely attached mucus that is subsequently released from the fish skin.

In this study, nitrification before and after abrupt cross-transfer in salinity was investigated in two moving bed biofilm reactors inoculated with nitrifying cultures that had adaptation to freshwater (FR) and seawater salinities (SR). FR and SR MBRRs were exposed to short and long term cross-transfer in salinity, and the functional capacity of nitrifying microbial communities was quantified by the estimation of ammonia and nitrite oxidation rates. Salinity induced successions were evaluated before and after salinity change by deep sequencing of 16S rRNA gene amplicons and statistical analysis. The bacterial community structure was characterized and Venn diagrams were included. The results indicated that after salinity cross-transfer, the FR was not significantly recovered at seawater salinity whereas SR showed high resistance to stress caused by low-salt. Succession and physiological plasticity were the main mechanisms of the long-term adaption of the nitrifying communities exposed to abrupt salinity changes. Independently of salinity, some nitrifiers presented high physiological plasticity towards salinity and were very successful at both zero and full seawater salinity. SR culture is robust and suitable inoculum for ammonium removal from recirculating aquaculture systems and industrial wastewaters with variable and fast salinity changes. Our findings contradict the current perspective of the significance of salinity on the structure of nitrifying communities. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

Oil spilled in the Arctic may drift into ice-covered areas and become trapped until the ice melts. To determine if exposure to oil during freezing may have a priming effect on degradation of the oil, weathered dispersed oil (2-3 mg/L) was frozen into solid ice for 200 days at -10 °C, then melted and incubated for 64 days at 4 °C. No degradation was measured in oil frozen into ice prior to melting. Both total amount of oil and target compounds were biotransformed by the microbial community from the melted ice. However, oil released from melted ice was degraded at a slower rate than oil incubated in fresh seawater at the same temperature (4 °C), and by a different microbial community. These data suggest negligible biodegradation of oil frozen in sea ice, while oil-degrading bacteria surviving in the ice may contribute to biodegradation when the ice melts.

Backgound: The present study investigated the effect of different lipid source in the feed on the colonization and the bacterial succession in early life stages (fertilized eggs until 93 days post first feeding) of S. salar. The two diets used in this study, FD (fish oil based diet) and VD (vegetable oil based diet), were formulated to cover the fish nutritional requirements and except the lipid source the components were identical between them.Hindgut samples collected at 0, 35, 65 and 93 days post first feeding (dpff). Moreover, fertilized eggs, yolk sac larvae, rearing water and feed were also sampled in order to assess a possible contribution of their microbiota to the colonization of the gut. To analyze the composition of the bacterial communities, the Illumina MiSeq platform was used. Results: S. salar growth variables (mean wet weight and total length) did not differ significantly during the experiment (p> 0.05) across replicate tanks and between dietary treatments. The analysis of the 16S rDNA sequencing data revealed a total of 4548 unique OTUs, affiliated in 21 bacterial phyla. Proteobacteria, Firmicutes, Actinobacteria and Bacteroidetes were the dominant bacterial phyla. 13 OTUs were shared among all S. salar samples independent of life stage and diet treatment. Similarity percentages analysis (SIMPER) based on Bray–Curtis distance, showed that the average dissimilarity among the groups of the same life stages was 76.0%, whereas the average dissimilarity within groups of the same dietary treatment was 78.5% (FD) and 83.6% (VD). Conclusion: Feeding on either fish oil or vegetable oil-based diets, did not result in significant differences in the intestinal microbiota. The composition of gut microbiota did not differ significantly between the two dietary treatments, but changed with age, and each stage was characterized by different dominant bacteria. These OTUs are related to species that provide different functions and have been isolated from a variety of environments. Finally, this study revealed the occurrence of a core microbiota independent of the studied life stages and diet during the early life stages of Atlantic salmon.

An important step in water treatment in recirculating aquaculture systems (RAS) is the removal of particulate organic matter. While commonly applied treatment technologies remove most of the particles, fine particulate organic matter typically accumulate in RAS, and serve as substrate for heterotrophic bacteria in the system. Membrane ultrafiltration has been shown to reduce the concentrations of total organic carbon and to counteract blooms of opportunistic bacteria in periods with high organic loading. The potential influence of such opportunistic bacteria on the fish microbiomes and fish welfare is poorly understood. In this study, we investigated the effects of high organic loading on the water and hindgut microbiomes in RAS with Atlantic salmon parr. Two pilot-scale RAS, one with membrane ultrafiltration of a 10–15% side-stream (mRAS), and one without (cRAS), were operated in periods with low and high organic loadings, and the water and hindgut microbiomes were characterized by Illumina sequencing of 16S rDNA amplicons. At the end of the experiment, after a period of high organic loading, two OTUs representing Mycobacterium and Spartobacteria dominated the water microbiomes in cRAS, with relative abundances as high as 24 and 17%, respectively. In the mRAS water they were rare (relative abundances of 1.9 and 0.1%). The membrane ultrafiltration stabilized the water microbiota and efficiently prevented the growth of these bacterial populations in RAS. The parr hindgut microbiomes were generally highly distinct from the RAS water microbiomes and were dominated by the bacterial phyla Bacilli and Firmicutes. Furthermore, an OTU probably representing Carnobacterium inhibens, originally isolated from the gut of adult salmon, was highly abundant in the gut microbiomes, with relative abundances reaching over 30%. However, for the cRAS fish after a period with high organic loading, the relative abundance of this Carnobacterium OTU was reduced, and an increased similarity between the gut and the water microbiomes was observed. The Mycobacterium and Spartobacteria OTUs that were abundant in the cRAS water were also abundant in the cRAS hindgut samples. This indicates a direct influence of the water microbiomes on the gut microbiomes, and that high organic loading in the cRAS resulted in growth of opportunistic bacteria that were able to colonize the fish gut. The present study shows that membrane filtration is an applicable strategy to counteract growth of opportunistic bacteria in RAS and to promote positive fish – microbe interactions.

Lumpfish (Cyclopterus lumpus L.) in Norway is currently produced in traditional flow-through systems (FTS). Hatcheries frequently show signs of bacterial infections, unstable microbial communities in the rearing water and varying mortality. Recirculating aquaculture systems (RAS) is proposed to create stable and healthy microbial environments, with less probabilities for blooming of opportunistic microbes. Studies have also shown that RAS increases the survival of marine fish. The aim with this study was to investigate the effect of various RAS water treatment designs on water and biofilm microbiota, survival, growth and gill health of lumpfish. An experiment with lumpfish was conducted, from 2 months post hatch to the transfer into sea cages. Five different water treatment regimens were compared: 1. RAS with no additional water treatment, 2. RAS with a filtration unit for removal of small particles, 3. RAS with filtration and disinfection with UV-irradiation, 4. RAS with filtration and disinfection with UV-irradiation and ozone and 5. FTS as a reference. The microbiota of the rearing water and tank wall biofilm were sampled and characterized by Illumina sequencing of 16S rDNA amplicons. Lumpfish juveniles reared in the RAS treatments were exposed to a more stable and diverse rearing water microbiota, with a lower share of opportunistic bacteria, a probable reason for the higher survival and better gill health of the fish compared to siblings reared in the FTS. Lumpfish reared in RAS without disinfection were exposed to a more diverse and stable water microbiota, with a lower share of opportunistic and potential harmful bacteria, compared to the lumpfish reared in RAS with disinfection and FTS. This resulted in better gill health. Fish in RAS with filtration, but no disinfection, had a better gill health than the fish in the RAS without filtration, possibly due to the reduction of small particles. The lumpfish were exposed to different microbial communities of both water and biofilm, due to the different treatments of the incoming tank water. In conclusion, our results indicate that implementation of RAS in the production of lumpfish has a potential to increase both survival, growth and gill health of the fish and that RAS with filtration of small particles, but without disinfection, result in the best fish health and performance among the investigated treatments. © 2020 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

As ice extent in the Arctic is declining, oil and gas activities will increase, with higher risk of oil spills to the marine environment. To determine biotransformation of dispersed weathered oil in newly formed ice, oil dispersions (2–3 ppm) were incubated in a mixture of natural seawater and frazil ice for 125 days at −2 °C. Dispersed oil in seawater without frazil ice were included in the experimental setup. Presence or absence of frazil ice was a strong driver for microbial community structures and affected the rate of oil degradation. n-alkanes were degraded faster in the presence of frazil ice, the opposite was the case for naphthalenes and 2–3 ring PAHs. No degradation of 4–6 ring PAHs was observed in any of the treatments. The total petroleum oil was not degraded to any significant degree, suggesting that oil will freeze into the ice matrix and persist throughout the icy season. © 2020 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Many studies demonstrate the importance of the commensal microbiomes to animal health and development. However, the initial community assembly process is poorly understood. It is unclear to what extent the hosts select for their commensal microbiota, whether stochastic processes contribute, and how environmental conditions affect the community assembly. We investigated community assembly in Atlantic cod larvae exposed to distinct microbial metacommunities. We aimed to quantify ecological processes influencing community assembly in cod larvae and to elucidate the complex relationship between the bacteria of the environment and the fish. Selection within the fish was the major determinant for community assembly, but drift resulted in inter-individual variation. The environmental bacterial communities were highly dissimilar from those associated with the fish. Still, differences in the environmental bacterial communities strongly influenced the fish communities. The most striking difference was an excessive dominance of a single OTU (Arcobacter) for larvae reared in two of the three systems. These larvae were exposed to environments with higher fractions of opportunistic bacteria, and we hypothesise that detrimental host–microbe interactions might have made the fish susceptible to Arcobacter colonisation. Despite strong selection within the host, this points to a possibility to steer the metacommunity towards mutualistic host–microbe interactions and improved fish health and survival., Bacterial community assembly in Atlantic cod larvae is driven by deterministic and stochastic processes and influenced by the bacterial metacommunity structure.

A key challenge in recirculating aquaculture systems (RAS) is the accumulation of particulate organic matter, especially the fine and colloidal fraction due to low removal efficiency of today's technology. The supply of organic matter is typically the limiting resource determining the carrying capacity (CC) of heterotrophic bacteria in the system. An appropriate and stable CC is proposed as a strategy for an optimal microbial environment in RAS with less blooms of opportunistic bacteria and more stable community dynamics. In this study, we investigated the effects of including a membrane for ultrafiltration in the RAS water treatment loop (treating 10–15% of the total water flow) to reduce the amount of fine and colloidal organic matter. Atlantic salmon parr (Salmo salar) were reared in two pilot-scale RAS (mRAS: membrane, cRAS: conventional). To evaluate the bacterial dynamics with and without membrane filtration at different organic loadings, the water exchange rates of the systems were manipulated equally to create periods with high and low loading of organic matter. The results showed that in the mRAS water, the level of organic matter was more stable throughout the experiment for the changing organic matter loadings. As a consequence, water in mRAS had higher microbial diversity, lower and shorter bacterial blooms and generally lower bacterial densities than in cRAS. All variables indicate a better microbial environment in the water of the system with membrane filtration. Also, the physicochemical water quality was better in mRAS in terms of lower turbidity and particulate organic matter (POC), and slightly lower concentrations of total ammonia nitrogen (TAN). The composition of the microbial communities was significantly different between the two systems, and temporal variations in the community dynamics were observed in both systems during the periods with different organic loadings. At high organic loading, the genus Mycobacterium had high relative abundance in the cRAS water (up to 0.25) compared to mRAS (0.01–0.03). The fish in mRAS were significantly bigger (14%) than fish in cRAS at the end of the experiment, however it is hard to conclude whether the better growth in mRAS was due to higher temperatures (caused by membrane operation) or better water quality, as it was probably a combination of both. We can conclude that membrane filtration gave more stable and better physicochemical and microbial water quality, which will reduce the probability for microbially related accidents in RAS. © 2020 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

In nitrifying biofilms, the organic carbon to ammonia nitrogen (C/N) supply ratio can influence resource competition between heterotrophic and nitrifying bacteria for oxygen and space. We investigated the impact of acute and chronic changes in carbon supply on inter-guild competition in two moving bed biofilm reactors (MBBR), operated with (R1) and without (R0) external organic carbon supply. The microbial and nitrifying community composition of the reactors differed significantly. Interestingly, acute increases in the dissolved organic carbon inhibited nitrification in R1 ten times more than in R0. A sustained increase in the carbon supply decreased nitrification efficiency and increased denitrification activity to a greater extent in R1, and also increased the proportion of potential denitrifiers in both bioreactors. The findings suggest that autotrophic biofilms subjected to increases in carbon supply show higher nitrification and lower denitrification activity than carbon-fed biofilms. This has significant implications for the design of nitrifying bioreactors. Specifically, efficient removal of organic matter before the nitrification unit can improve the robustness of the bioreactor to varying influent quality. Thus, maintaining a low C/N ratio is important in nitrifying biofilters when acute carbon stress is expected or when anoxic activity (e.g. denitrification or H2S production) is undesirable, such as in recirculating aquaculture systems (RAS). © The Author(s) 2020. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Te images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

The rearing environment is important for a stable production of good quality lobster juveniles. By providing an environment excluding pathogens and dominated by mutualistic bacteria, the probability of developing healthy host-microbe relationships and produce healthy juveniles is increased. Disinfection of water and sudden increase in the supply of organic matter in culture tanks are processes that open for uncontrolled microbial regrowth in the rearing water. This increase the variability in the development of the microbiota between replicate rearing tanks and promotes selection for potentially harmful opportunistic bacteria. In two start feeding experiments with European lobster (Homarus gammarus) we compared the bacterial environment in three types of rearing systems: a recirculating aquaculture system (RAS) with UV treatment directly in front of the rearing raceways, a RAS without disinfection, and a conventional flow through system (FTS). The RAS with no disinfection was hypothesised to stabilise the microbiota of the rearing water, select against opportunistic bacteria, and reduce variability in production outcome between replicate tanks compared to the other systems. As predicted, the three different systems developed significantly different compositions of the microbiota in the rearing water and the larvae. On average, the survival of larvae in RAS without disinfection increased with 43 and 275 %, in the first experiment, and 64 and 18 % in the second experiment, compared to RAS with UV and FTS, respectively. Also, the RAS without disinfection showed less variability in the survival of larvae between replicate tanks and batches compared to the other treatments. The results are promising for controlling the microbiota of the rearing water to improve, increase and stabilise the production of marine larvae by competent use of water treatment and selection regimes. Based on the presented and previous work, RAS is recommended over FTS, and in RAS it is recommended to avoid point-disinfection of the recirculating water, to provide a stable and beneficial microbial environment in the cultivation of marine larvae. https://doi.org/10.1016/j.aquaeng.2021.102176 © 2021 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

Fish live in close contact with microbes in their surrounding water. For cultivated marine fish larvae, stable microbial environments, characterized by slow-growing, K-selected bacteria, have been found to improve growth and survival. Compared to flow-through systems, recirculating aquaculture systems (RAS) offer a better possibility for obtaining microbial stability in the rearing water. However, the microbial ecology in RAS is complex and little studied, and to optimize the microbial water quality more knowledge is needed on microbial community dynamics and interactions between microbes and fish. In the present study we investigated microbial community dynamics in three large-scale RAS, operated with different salinities (12, 22 and 32 ppt), for production of Atlantic salmon post-smolts. We focused on the stability of microbial communities over time in the bioreactors and the fish tanks, and compared the microbial communities within and between the three RAS. The study showed that: 1) The microbial community of the water was similar between the compartments of each RAS at the same time point, 2) The microbial communities of the water (and to a lesser extent the biofilm) within each system underwent large changes over time, 3) The microbiota of biofilm and water differed significantly within each system, 4) Biofilm community profiles were more similar between RAS than the water community profiles, and 5) Salinity structured the microbial community composition. The present study provides new knowledge on how the microbiota in RAS varies with time and how it is influenced by environmental factors.

The anammox activity of a freshwater anammox consortium was strongly inhibited at low-salinity level. Stepwise adaptation from 0 to 3 g NaCl L−1 took 153 days. Further adaptation to high-salinity concentration (from 3 to 30 g L−1) took only 40 days, and no inhibition was observed. A comprehensive insight into the salinity-induced successions of the total and the anammox communities was obtained by 454 pyrosequencing of 16S rRNA gene amplicons and statistical analysis. A major succession in the anammox community was observed at 3 g L−1 where the dominating population shifted from Candidatus Brocadia fulgida to Ca. Kuenenia stuttgartiensis. The latter dominated at high salinity and seemed to be essential for the high (˃96%) ammonium and nitrite removal efficiencies achieved. SIMPER analysis indicated that these two dominating anammox species explained most to the differences in community structure among samples and helped in identifying other important members at different salinities.

Marine fish larvae are characterized by immature immune and digestive systems at hatching. Therefore, they are particularly vulnerable to detrimental interactions with microbes. In this chapter, we review studies performed in our group on the microbiota of cod larvae. The overall aim of these studies was to clarify which factors affected the composition of the larval microbiota in the early developmental stages and thereby be able to identify possible rearing strategies to promote positive microbe–larvae interactions in aquaculture rearing systems. Through careful experimental designs, we managed to separate the effects of the microbiota in rearing water and diet and were surprised to find that the water microbiota seemed to be a more important determinant for the composition of the larval microbiota than the feed. The larval microbiota changed over time, and our results indicated that this was due to developmental changes in the gastrointestinal system and that selection in the digestive tract of the host structured the larval microbiota. We further tested the potential for manipulating the microbiota of larvae through introduction of probiotic candidates. This was not successful, as the probionts were only transiently present in the larval microbiota. This observation was independent of the developmental stage of the cod larvae and despite the fact that the strains had been isolated from cod larvae. We suggest that the best strategy for promoting beneficial microbe–larvae interactions in aquaculture systems is to manage the microbial water quality. To obtain this, microbial ecology must be taken into consideration. With proper management and system design, it is possible to maintain stable, K-selected microbial environments in both flow-through and recirculating aquaculture systems (RAS). This will promote beneficial larvae–microbe interactions and improve the viability of the larvae.

Aquaculture has the potential to become a major food supplier in a world with an increasing human population, and increased consumption of fish will likely have positive health implications. For marine aquaculture, the production of high quality juveniles is a bottleneck. Survival until the juvenile stage is typically as low as 10–15% for many species, which indicates suboptimal rearing conditions. Substantial evidence indicates that the poor performance and viability of larvae is largely due to detrimental larvae-microbiota interactions. This emphasises the need for microbial management strategies in the cultivation of marine fish larvae. Disinfection and probiotics are the most studied microbial management methods so far. However, most studies on these methods overlooked the role of mutualistic relationships between microbes and hosts, and have not proposed or examined methods steering toward such relationships. Based on ecological theory and a number of experiments, we find support for the hypothesis that current practise in aquaculture generally selects for r-strategic, opportunistic microbes, which results in detrimental host–microbiota interactions. Thus, the challenge is to develop technology and methods for microbial management at the ecosystem level that creates a K-selected microbial community, and by this mean select against r-strategic opportunists. Here we summarise experiments done during 25 years and with marine larvae of five different species showing that: (1) K-selection strategies result in different water microbiota with less opportunists, (2) this influences the microbiota of the fish larvae, and (3) the larvae cultivated in water inhabited by a K-selected microbiota perform better. Improved performance of larvae includes improved appetite, earlier onset of and faster growth, increased survival, and increased robustness to stress. K-selection as a method for management of the microbial community is a robust approach that allows steering of host–microbiota interactions in larviculture toward mutualism. It could also be applicable for young stages of other domesticated animals. Our review illustrates that a change from a “beat-them” to a “join-them” strategy for microbial management in larval rearing can lead to a more sustainable aquaculture industry. Copyright © 2018 Vadstein, Attramadal, Bakke and Olsen. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY)

This paper describes an experimental study of microbial communities of three moving bed biofilm reactors (MBBR) inoculated with nitrifying cultures originated from environments with different salinity; freshwater, brackish (20‰) and seawater. All reactors were run until they operated at a conversion efficiency of >96%. The microbial communities were profiled using 454-pyrosequencing of 16S rRNA gene amplicons. Statistical analysis was used to investigate the differences in microbial community structure and distribution of the nitrifying populations with different salinity environments. Nonmetric multidimensional scaling analysis (NMDS) and the PERMANOVA test based on Bray-Curtis similarities revealed significantly different community structure in the three reactors. The brackish reactor showed lower diversity index than fresh and seawater reactors. Venn diagram showed that 60 and 78% of the total operational taxonomic units (OTUs) in the ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) guild, respectively, were unique OTUs for a given reactor. Similarity Percentages (SIMPER) analysis showed that two-thirds of the total difference in community structure between the reactors was explained by 10 OTUs, indicating that only a small number of OTUs play a numerically dominant role in the nitrification process. Acute toxicity of salt stress on ammonium and nitrite oxidizing activities showed distinctly different patterns, reaching 97% inhibition of the freshwater reactor for ammonium oxidation rate. In the brackish culture, inhibition was only observed at maximal level of salinity, 32‰. In the fully adapted seawater culture, higher activities were observed at 32‰ than at any of the lower salinities.

The selection pressure can be used to reduce the opportunities for proliferation of potentially harmful bacteria. Hypothetically the chance of proliferation of opportunistic bacteria in the fish tank can be minimized by microbial maturation of the incoming water at a microbial carrying capacity similar to that in the rearing tanks. In a start feeding experiment with Ballan wrasse (Labrus bergylta) we compared the bacterial environment in two flow through systems: a microbially matured system (MMS), where the water presented to the fish was matured in a biofilter, and a fed microbially matured system (F-MMS), where the biofilter was fed organic matter to increase the microbial carrying capacity. As predicted, the MMS showed a more variable and often high microbial growth potential in the tank water. The microbial community composition of the tank water was more stable, diverse and species rich in the F-MMS than in the MMS. The results are promising for controlling the microbiota of the rearing water by competent use of water treatment and selection regimes. Statement of relevance The experiment shows that small changes in management (organic load and maturation of water) of water treatment give significant different microbiota in fish tanks. The experiment also shows that by increasing the microbial carrying capacity in a maturation unit to the level of the tank microbial carrying capacity, the microbial community in the fish tank becomes more stable and less open for opportunistic proliferation. This work reveals promising possibilities for controlling the microbiota of the rearing water in land based aquaculture by competent use of water treatment and selection regimes. Improved control and understanding of microbial control is very relevant for the aquaculture industry.

We have previously shown that biological nitrogen removal by pre-denitrification as illustrated may be a feasible approach for treating wastes generated by amine based CO2 capture. In order to identify limiting factors for successful up-scaling, we first compared the nitrifying activity of moving bed biofilm reactors (MBBR) with or without chronic exposure to organic loading in the form of acetate while monitoring population dynamics in the biofilms by pyro-sequencing. Our results show that the long-term abundance of heterotrophic bacteria is an essential factor in inhibition of nitrification efficiency. Secondly, the inhibition potential of the commonly applied amines monoethanolamine (MEA), 2-amino-2-methyl-1-propanol (AMP), diethanolamine (DEA), methyldiethanolamine (MDEA), piperazine (Pip), as well as MEA based reclaimer waste (RW) were tested on separate nitrifying and denitrifying MBBRs. Results show that nitrification was inhibited by 50% at EC50 concentrations from 9 to 120 mM, whereas denitrification was stimulated by all compounds at concentrations up to 100 mM. Nitrifying biofilms long-term adapted to organic loadings were 5⿿20 times more sensitive towards inhibition than those maintained without organic feeding, by both MEA and by organic loading. The crucial factor for the total process is therefore maintaining nitrification by avoiding overloading of amines or other organics in the second reactor.

We have previously shown that K-selection and microbial stability in the rearing water increases survival and growth of Atlantic cod (Gadus morhua) larvae, and that recirculating aquaculture systems (RAS) are compatible with this. Here, we have assessed how water treatment influenced the larval microbiota and host responses at the gene expression level. Cod larvae were reared with two different rearing water systems: a RAS and a flow-through system (FTS). The water microbiota was examined using a 16S rDNA PCR/DGGE strategy. RNA extracted from larvae at 8, 13, and 17 days post hatching was used for microbiota and microarray gene expression analysis. Bacterial cDNA was synthesized and used for 16S rRNA amplicon 454 pyrosequencing of larval microbiota. Both water and larval microbiota differed significantly between the systems, and the larval microbiota appeared to become more dissimilar between systems with time. In total 4 phyla were identified for all larvae: Actinobacteria, Bacteroidetes, Firmicutes, and Proteobacteria. The most profound difference in larval microbiota was a high abundance of Arcobacter (Epsilonproteobacteria) in FTS larvae (34 ± 9% of total reads). Arcobacter includes several species that are known pathogens for humans and animals. Cod larval transcriptome responses were investigated using an oligonucleotide gene expression microarray covering approximately 24,000 genes. Interestingly, FTS larvae transcriptional profiles revealed an overrepresentation of upregulated transcripts associated with responses to pathogens and infections, such as c1ql3-like, pglyrp-2-like and zg16, compared to RAS larvae. In conclusion, distinct water treatment systems induced differences in the larval microbiota. FTS larvae showed up-regulation of transcripts associated with responses to microbial stress. These results are consistent with the hypothesis that RAS promotes K-selection and microbial stability by maintaining a microbial load close to the carrying capacity of the system, and ensuring long retention times for both bacteria and water in the system. Copyright © 2018 Vestrum, Attramadal, Winge, Li, Olsen, Bones, Vadstein and Bakke. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

All animals need a mutualistic interaction with their microbiota for proper development and functioning. Also for the fish-microbiota interaction considerable research has been done, and especially for reared fish larvae this interaction is crucial for their viability. However, during the 1980s and 1990s a number of findings revealed at that time current methods were not suitable for studying the total microbial community and that data on composition of microbiota was biased. Several recent methodological revolutions have boosted the possibilities for addressing questions related to fish larvae-microbiota interactions that previously lacked suitable tools for proper evaluation. These methodological achievements include the development of experimental rearing systems including gnotobiotic systems for fish, new visualization tools, and molecular “omics” tools for characterizing the response of the host on a variety of levels and for characterizing both composition and activity of fish microbiota. We present and review these tools and give examples on how they have been used to improve our understanding of fish larvae-microbiota interactions. With respect to understanding, this includes in particular how the microbiota is established and maintained, what the functionality of the microbiota is and how it affects fish health, and finally how we can apply this knowledge for management of a healthy and beneficial microbiota in aquaculture settings.

In upflow anaerobic sludge bed (UASB) reactors, biomass present as granules allows for long solids retention time. Here, granules from a process treating pulp and paper industrial wastewater were successfully applied as inoculum in UASB reactors treating pig manure supernatant, despite high particle content and high ammonium concentrations in the influent. We did a detailed characterization of archaeal and bacterial communities associated with the inoculum and with the aggregated and dispersed fractions of the influent and the reactors after one year of operation. The granular communities underwent major changes and adapted to the highly distinct conditions without disintegration of the granules. Although the granules persisted in the reactors, non-granular aggregates accumulated, and partly replaced the granules. Particles introduced to the reactors by the pig manure influent apparently contributed both as food and biofilm growth support. Archaeal communities in the dispersed reactor phase were similar to those dispersed in the influents, implying successful retention and little loss of archaeal biomass due to detachment or disintegration of granules and other aggregates. Unique bacterial communities developed in the dispersed fraction of the reactors despite of low hydraulic retention times. They probably consisted of fast growing organisms consuming readily degradable organic matter. © The Author(s) 2018 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

Marine fish larvae are immature upon hatching, and share their environment with high numbers of bacteria. The microbial communities associated with developing fish larvae might be structured by other factors than those important in developing terrestrial animals. Here, we analysed the beta (β)-diversity of the microbiota associated with developing cod larvae and compared it with the bacterial communities in water and live feed by applying pyrosequencing of bar coded v4 16S rDNA amplicons. A total of 15 phyla were observed in the cod larval microbiota. Proteobacteria was the most abundant, followed by Firmicutes, Bacteroidetes and Actinobacteria. The composition and diversity of the cod larval microbiota changed considerably with age. The temporal and spatial patterns of β-diversity could not be explained by stochastic processes, and did not coincide with changes in the rearing conditions. Furthermore, the larval microbiota was highly distinct from the water and the live feed microbiota, particularly at early developmental stages. However, the similarity between larval and water microbiota increased with age. This study suggests that strong selection in the host structures the cod larval microbiota. The changes in community structure observed with increasing age can be explained by altered selection pressure due to development of the intestinal system.

The study shows that high rate anaerobic digestion may be an efficient way to obtain sustainable energy recovery from slurries such as pig manure. High process capacity and robustness to 5 % daily load increases are observed in the 370 ml sludge bed AD reactors investigated. The supernatant from partly settled, stored pig manure was fed at rates giving hydraulic retention times, HRT, gradually decreased from 42 to 1.7 hours imposing a maximum organic load of 400 g COD L-1 reactor d-1. The reactors reached a biogas production rate of 97 g COD L-1 reactor d-1 at the highest load at which process stress signs were apparent. The yield was ∼0.47 g COD methane g-1 CODT feed at HRT above 17 h, gradually decreasing to 0.24 at the lowest HRT (0.166 NL CH4 g-1 CODT feed decreasing to 0.086). Reactor pH was innately stable at 8.0 ± 0.1 at all HRTs with alkalinity between 9-11 g L-1. The first stress symptom occurred as reduced methane yield when HRT dropped below 17 h. When HRT dropped below 4 h the propionate removal stopped. The yield from acetate removal was constant at 0.17 g COD acetate removed per g CODT substrate. This robust methanogenesis implies that pig manure supernatant, and probably other similar slurries, can be digested for methane production in compact and effective sludge bed reactors. Denaturing gradient gel electrophoresis (DGGE) analysis indicated a relatively fast adaptation of the microbial communities to manure and implies that non-adapted granular sludge can be used to start such sludge bed bioreactors. © 2015 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http:// creativecommons.org/licenses/by-nc-nd/4.0/).

An Atlantic cod larva (Gadus morhua) feeding experiment was carried out to compare three different live feed enrichment products for Brachionus ibericus and Artemia salina: 1) Nannochloropsis sp. microalgal concentrate (frozen) (N), 2) Pavlova viridis microalgal concentrate (frozen) (P), and 3) Larviva Multigain (R) (BioMar, Denmark)-formulated commercial product (CP). Over the experimental period of 42 days larvae of groups P and CP showed significantly higher instantaneous growth rates (G: 0.074 +/- 0.001 d(-1) and 0.079 +/- 0.009 d(-1)) than larvae of group N (G: 0.04 +/- 0.0 d(-1)). Survival at the end of the trial of group CP was highest (20 +/- 8%), whereas significantly lower survival was observed for groups P (7 +/- 2%) and N (

The use of probiotic bacteria is a strategy suggested to overcome microbial problems during early stages of farmed animals, including fish. However, few studies have investigated the efficiency in the establishment of probiotics in the fish and how lasting such treatments are. The objective of this study was to evaluate whether the microbiota of Atlantic cod larvae Gadus morhua L can be steered by introduction of selected probiotic candidates, and if there are particular developmental stages where this is more easily obtained. Cod larvae were given a mixture of 4 candidate probiotic strains (Microbacterium (ID3-10), Ruegeria (RA4-1), Pseudoalteromonas (RA7-14) and Vibrio (RD5-30)) through the live feed and the water during a treatment period of 24 h. The strains originated from reared cod larvae and are proven to have probiotic properties. The larvae were treated at seven different ages, namely days 0, 2, 4, 8, 16, 30 and 45 post-hatching, with one new experimental group started each time. A real-time PCR strategy was developed to quantify the relative amounts of the four added bacterial strains in live feed, and in water and cod larvae during 10 days after exposure. Only ID3-10 was measured to constitute considerable fractions of the larval microbiota for most of the treatments, despite the fact that all the probiotic candidates originated from cod larvae intestines. In most cases, the amounts of the added strains decreased to approximately background levels after a maximum of 4 days, indicating only a transient presence in the larvae. The results indicate that probiotic treatment aiming for colonization with new strains in the cod larvae is difficult, and challenges the probiotic concept for the larval stage unless continuous or repeated addition to the fish larvae is used.

We examined the effect of ammonium and temperature on methane production in high rate upflow anaerobic sludge bed reactors treating pig manure supernatant. We operated four reactors at two ammonium concentrations (‘low’ at 1.9, ‘high’ at 3.7 g L−1, termed LA and HA reactors, respectively) and at variable temperatures over 358 days. Archaeal and bacterial communities were characterized by Illumina sequencing of 16S rRNA amplicons. Ammonium was a major selective factor for bacterial and archaeal community structure. After ~200 days of adaptation to high ammonium levels, acetate and propionate removal and methane production improved substantially in HA reactors. Aceticlastic Methanosaeta was abundant and positively correlated to methane yield in the HA reactors, whereas Methanosarcina was more abundant in LA reactors. Furthermore, a group of monophyletic OTUs that was related to Thaumarchaeota in phylogenetic analysis was highly abundant in the archaeal communities, particularly in the HA reactors. The most abundant bacterial OTU in LA reactors, representing Syntrophomonadaceae, was also positively correlated to methane yield in the HA reactors, indicating its importance in methane production under ammonia stress. In conclusion, efficient methane production, involving aceticlastic methanogenesis by Methanosaeta took place in the reactors at free ammonia concentrations as high as 1 g L−1.

A high share of opportunistic and fast-growing microbes is generally negative for the performance of marine fish larvae. It is hypothesised that larvae that are reared in water dominated by K-strategists (mature microbial communities) will perform better, because they are less likely to encounter opportunistic (r-selected) microbes and develop detrimental host–microbe interactions. K-selection can be set up by securing competition for resources by maintaining a low nutrient supply per microbe in a maturing biofilter in a flow through system or in a recirculation system without disinfection. In an experiment with Atlantic cod larvae (Gadus morhua L.) the development of the microbial community in the inflowing water to tanks as well as in the tank water was studied in a conventional flow through system (FTS) and two hypothesised K-selective systems: a microbially matured flow through system (MMS) and a recirculation aquaculture system (RAS). As predicted, the K-selective treatments resulted in a significantly more mature and stable microbial community composition in the water going in to the fish tanks compared to the FTS. The larvae in the K-selective treatments showed 72% higher survival than the larvae in the FTS. In K-selective systems where the inflowing water to the fish tanks is matured at a microbial carrying capacity that is significantly lower than the carrying capacity in the rearing tank (i.e. MMS) there is hypothetically an opening for proliferation of opportunists to match the higher carrying capacity in the tank water. As predicted, the RAS showed K-selection of the incoming water at a carrying capacity similar to that of the tank water, which resulted in a significantly more stable microbial community composition in the tank water of the RAS than in the MMS and FTS. The results of this experiment showed a high potential for increasing fish survival by using K-selection of bacteria, which is a cheap and easy method that can be used in all kinds of new or existing aquaculture systems.

Particle accumulation in recirculating aquaculture systems (RAS) are generated from uneaten food and feces, and bacteria tends to proliferate. Small organic particles act as substrate for bacterial growth and may by direct interference with gills cause stress and reduce disease resistance for the cultivated species. Most diseases in aquaculture of marine fish larvae are caused by opportunistic microorganisms that become pathogenic when the fish is under stressed conditions. The present study investigated the potential of using a membrane bioreactor (MBR) as a part of the water treatment in a RAS for production of marine fish larvae, since the small pore size (50 nm) efficiently removes organic particles including the colloidal fraction in the system. The experiment was a 50 day start-feeding experiment with Atlantic cod ( Gadus morhua L.), comparing water quality and larval performance in a conventional RAS (cRAS) with a membrane filtrated RAS (mRAS). In mRAS, 8.5% of the water flow was at any time membrane filtrated. The present study showed that the MBR improved water quality significantly by reducing turbidity and by lowering the total number of bacteria in the rearing water (up to 80% reduction). Microbial communities in water samples were significantly different between cRAS and mRAS already on 5 days post-hatching (dph) and throughout the experiment. The microbial community composition of the rearing water was significantly different from the composition of the larval microbiota during the experiment, although differences between larval samples were different only on 50 dph. The present study showed the potential of using MBR to lower the bacterial carrying capacity (CC) by efficiently removing organic particles and bacteria. In average a 13% higher cod larval growth (weight, %) at 40 dph and a 3.0% higher survival rate at 50 dph were measured in the mRAS scheme.

The gastrointestinal (GI) tract of newly hatched fish is probably colonized by bacteria present in the water, but how environmental and internal factors affect the development of the GI microbiota is poorly understood. In this study, we investigated the effect of diet and of rearing in separate tanks on the cod larval microbiota. Cod larvae were fed three different live feed diets. For each diet, larvae were reared in three replicate tanks. The microbial communities were investigated for water, live feed and individual larvae using a PCR/DGGE (Denaturing Gradient Gel Electrophoresis) strategy. Statistical tests were applied to investigate differences in the larval microbiota between groups of individuals. We found no differences in the larval microbiota due to diet after 8 dph (days post hatching). Moreover, the larval microbiota was similar at 17 and 32 dph, despite a change in live feed at 18 dph. The larval microbiota was generally more similar to the water microbiota than to live feed microbiota. We further found that rearing of larvae in replicate tanks with identical diet could result in significant differences in larval microbiota. These findings indicate that diet does not entail major changes to the composition of cod larval microbiota.

Racemates of hydrophobic amino acids with linear side chains are known to undergo a unique series of solid-state phase transitions that involve sliding of molecular bilayers upon heating or cooling. Recently, this behaviour was shown to extend also to quasiracemates of two different amino acids with opposite handedness [Görbitz & Karen (2015).J. Phys. Chem. B,119, 4975–4984]. Previous investigations are here extended to an L-2-aminobutyric acid–D-methionine (1/1) co-crystal, C4H9NO2·C5H11NO2S. The significant difference in size between the –CH2CH3and –CH2CH2SCH3side chains leads to extensive disorder at room temperature, which is essentially resolved after a phase transition at 229 K to an unprecedented triclinic form where all four D-methionine molecules in the asymmetric unit have different side-chain conformations and all three side-chain rotamers are used for the four partner L-2-aminobutyric acid molecules.

The vertebrate gut is host to large communities of bacteria, and one of the beneficial contributions of this commensal gut microbiota is the increased nutritional gain from feed components that the host cannot degrade on its own. Fish larvae of similar age and under the same rearing conditions often diverge with regards to growth. The underlying reasons for this could be differences in genetic background, feeding behavior or digestive capacity. Both feeding behavior and digestion can be influenced by differences in the microbiota. To investigate possible correlations between the size of fish larvae and their gut microbiota, we analyzed the microbiota small and large genetically homogenous killifish and genetically heterogeneous cod larvae by Bray-Curtis Similarity measures of 16S DNA DGGE patterns. A significant difference in richness (p = 0.037) was observed in the gut microbiota of small and large killifish, but the overall gut microbiota was not found to be significantly different (p = 0.13), indicating strong genetic host selection on microbiota composition at the time of sampling. The microbiota of small and large cod larvae was significantly different with regards to evenness and diversity (p = 0.0001), and a strong correlation between microbiota and growth was observed. This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/

Directed Evolution of the Transcription Factor XylS for Development of Improved Expression Systems

Summary Seven polyene macrolides with alterations in the polyol region and exocyclic carboxy group were obtained via genetic engineering of the nystatin biosynthesis genes in Streptomyces noursei . In vitro analyses of the compounds for antifungal and hemolytic activities indicated that combinations of several mutations caused additive improvements in their activity-toxicity properties. The two best analogs selected on the basis of in vitro data were tested for acute toxicity and antifungal activity in a mouse model. Both analogs were shown to be effective against disseminated candidosis, while being considerably less toxic than amphotericin B. To our knowledge, this is the first report on polyene macrolides with improved in vivo pharmacological properties obtained by genetic engineering. These results indicate that the engineered nystatin analogs can be further developed into antifungal drugs for human use.