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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.

Decline in fish oil and fish meal availability has forced the aquaculture sector to investigate alternative and sustainable aquafeed ingredients. Despite that several studies have evaluated the effect of fish oil replacement in aquaculture fish species, there is a knowledge gap on the effects of alternative dietary lipid sources on the gut microbiota in early life stages of Salmo salar. The present study evaluated the influence of dietary administration of two different lipid sources (fish oil and vegetable oil) on the intestinal microbiota of first feeding Atlantic salmon (S. salar) up to 93 days post first feeding (dpff). The two diets used in this study, FD (fish oil diet) and VD (blend of rapeseed, linseed and palm oils diet), were formulated to cover the fish nutritional requirements. Apart from the lipid source, the rest of the feed components were identical in the two diets. Hindgut samples were collected at 0, 35, 65, and 93 dpff. Moreover, fertilized eggs, yolk sac larvae, rearing water and feed were also collected in order to assess a possible contribution of their microbiota to the colonization and bacterial succession of the fish intestines. To analyze the bacterial communities, amplicon sequencing was used targeting the V3–V4 region of the 16S rRNA gene. The findings indicate that feeding on either fish oil or vegetable oil-based diet, fish growth variables (mean wet weight and total length) did not differ significantly during the experiment (p > 0.05). No significant differences were also found between the two dietary groups, regarding their gut bacteria composition, after the analysis of the 16S rRNA sequencing data. Instead, gut microbiota changed with age, and each stage was characterized by different dominant bacteria. These operational taxonomic units (OTUs) were related to species that provide different functions and have been isolated from a variety of environments. The results also show little OTUs overlap between the host and rearing environment microbiota. Overall, this study revealed the occurrence of a core microbiota in early life of Atlantic salmon independent of the feed-contained oil origin. Copyright © 2021 Nikouli, Kormas, Jin, Olsen, Bakke and Vadstein. 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(s) 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.

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.

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.

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.

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.

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.

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.

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.

Aims To elucidate how granular sludge inoculum and particle-rich organic loading affect the structure of the microbial communities and process performance in upflow anaerobic sludge bed (UASB) reactors. Methods and Results We investigated four reactors run on dairy manure filtrate and four on pig manure supernatant for three months achieving similar methane yields. The reactors fed with less particle rich pig manure stabilized faster and had highest capacity. Microbial community dynamics analysed by a PCR/denaturing gradient gel electrophoresis approach showed that influent was a major determinant for the composition of the reactor communities. Comparisons of pre- and non-adapted inoculum in the reactors run on pig manure supernatant showed that the community structure of the nonadapted inoculum adapted in approximately two months. Microbiota variance partitioning analysis revealed that running time, organic loading rate and inoculum together explained 26 and 31% of the variance in bacterial and archaeal communities respectively. Conclusions The microbial communities of UASBs adapted to the reactor conditions in treatment of particle rich manure fractions, obtaining high capacity, especially on pig manure supernatant. Significance and Impact of the Study These findings provide relevant insight into the microbial community dynamics in startup and operation of sludge bed reactors for methane production from slurry fractions, a major potential source of biogas.

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.

Secondary structure features of mitochondrial ribosomal RNAs (mt-rRNAs) of bony fishes were investigated by a DNA sequence alignment approach. The small subunit (SSU) and large subunit (LSU) mt-rRNA genes were found to contain several additional variable regions compared to their mammalian counterparts. Fish mt-LSU rRNA genes were found to be longer than the mammalians due to increased length of some of the variable regions. The 5' and 3' ends of Atlantic cod mt-rRNAs were precisely mapped. The 3' ends of mt-SSU rRNAs were found to be homogenous and mono-adenylated, whereas that of the mt-LSU rRNAs were heterogenous and oligo-adenylated. The 5' ends of mt-SSU rRNAs appeared to be heterogenous, corresponding to the presumed first and second positions of the gene. Sequences of the central domain and the D-domain of the mt-SSU and mt-LSU rRNA genes, respectively, were determined and characterized for 11 gadiform species (representing the families Gadidae, Lotidae, Ranicipitidae, Merlucciidae, Phycidae, and Macrouridae) and one Lophiidae species. Detailed secondary structure models of the RNA regions are presented for the Atlantic cod (Gadus morhua) and Roundnose grenadier (Coryphaeonides rupestris). Saturation plots revealed that DNA nucleotide positions corresponding to unpaired RNA regions become saturated with transitions at sequence divergence levels about 0.15. Phylogenetic analyses revealed some aspects of gadiform relationships. Gadidae was identified as the most derived of the gadiform families. Lotidae was found to be the family closest related to Gadidae, and Ranicipitidae was also recognized as a derived gadiform taxon.

The nucleotide sequences of intergenic spacers located between the tRNA(Thr) and tRNA(Pro) genes in mitochondrial DNA of cod fishes (order Godiformes) were determined. Spacers from eight species representing two families of cod fishes were analyzed and found to vary in size from 25 to 99 bp. Each spacer sequence contains one or two copies of a conserved 17-bp motif. Four to five central nucleotides of this motif constitute a substitutional hot spot as observed from interspecific and intraspecific comparisons. The substitution rate of the spacer is approximately twice that of the variable part I of the mitochondrial DNA control region, making this sequence region interesting as a molecular marker in population studies or stock assessments of cod fishes. We propose that the spacer originated in a duplication event and evolved into a functional domain, perhaps by binding regulatory proteins.

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.

The minke whale (Balaenoptera acutorostrata) is subject to commercial whaling, but stock identification and assessment are still uncertain. Mitochondrial DNA (mtDNA) sequences were determined to examine the population structure of minke whales from the central and northeastern parts of the North Atlantic, as well as the Antarctic regions IV and V. The analyses include 345 nucleotide positions of the control region of 110 individuals, and 250 nucleotide positions of the NADH dehydrogenase subunit 2 gene for a representative selection of North Atlantic minke whales. Maximum parsimony analyses and sequence divergence calculations did not reveal any genetic differentiation between individuals from the central and northeastern parts of the North Atlantic. These results do not support the International Whaling Commission's separation of minke whales in this area into different management units, and they are in conflict with previously reported results from allozyme analyses. Comparison of minke whale control region sequences showed that the sequence diversity of North Atlantic minke whales is substantially lower (0.0065) than that of Antarctic minke whales (0.0166), and clearly demonstrated that individuals from these two areas represent genetically distinct populations.

Summary Secondary structures and the short Shine–Dalgarno sequence in the 5′‐untranslated region of bacterial mRNAs (UTR) are known to affect gene expression at the level of translation. Here we report the use of random combinatorial DNA sequence libraries to study UTR function, applying the strong, σ32/σ38‐dependent, and positively regulated Pm promoter as a model. All mutations in the libraries are located at least 8 bp downstream of the transcriptional start site. The libraries were screened using the ampicillin‐resistance gene (bla) as reporter, allowing easy identification of UTR mutants that display high levels of expression (up to 20‐fold increase relative to the wild‐type at the protein level). Studies of the two UTR mutants identified by a modified screening procedure showed that their expression is stimulated to a similar extent at both the transcript and protein product levels. For one such mutant a model analysis of the transcription kinetics showed significant evidence of a difference in the transcription rate (about 18‐fold higher than the wild type), while there was no evidence of a difference in transcript stability. The two UTR sequences also stimulated expression from a constitutive σ70‐dependent promoter (P1/Panti‐tet), demonstrating that the UTR at the DNA or RNA level has a hitherto unrecognized role in transcription.

PCR primers targeting conserved regions of the SSU rRNA gene are commonly used in bacterial community studies. For microbes associated with eukaryotes, co-amplification of eukaryotic DNA may preclude the analysis. We present a simple and efficient PCR strategy to obtain pure bacterial rDNA amplicons from samples predominated by eukaryotic DNA.

Mitochondrial DNA sequences of selected regions of the small subunit and large subunit ribosomal RNAs and cytochrome b genes were analyzed for 10 gadid species, representing 8 genera within Gadidae, and 10 species representing 5 other gadiform families. Phylogenetic analyses revealed that Gadiculus is the most basal gadid genus, and that Trisopterus and Micromesistius constitute a relatively basal clade. Lotidae was identified as the family most closely related to Gadidae. Estimation of divergence times indicated that the most ancient Gadidae split between Gadiculus and the remaining gadid genera occurred about 20 million years ago. The clade including the most recent species (Gadus, Boreogadus, Merlangius, Melanogrammus, and Pollachius) diverged from the Trisopterus/Micromesistius clade approximately 12 million years ago.

The primary structure of the Alca torda mitochondrial control region was determined and conserved structural features were identified based on sequence comparisons to other bird species. In a population survey using control region analysis, five individuals were found to possess heteroplasmic point mutations at the variable 5′ end of the control region. The pattern of variable nucleotide positions among individuals was compared to the distribution of heteroplasmic sites and the heteroplasmic condition was further characterised by a cloning procedure applied to two individuals which harboured one and two heteroplasmic point mutations, respectively. These results are in support of recent evidence that single site heteroplasmy may be more common than previously thought.

Pigeons learned either an easy or a difficult line angle discrimination (Experiment 1) or wavelength discrimination (Experiment 2), and then they were given a reacquisition test of retention after delays of 1 min, 1 day, or 1 week. Both percentage of responses to the S+ in the initial 10-trial block and number of blocks to criterion showed a progressive memory loss which was greater for the difficult problem. These results extend recent findings by using a free operant rather than a discrete trial task and by varying problem difficulty by altering the dimensional separation between training stimuli. In Experiment 3, pigeons were given variable interval training with either a wavelength or a line angle stimulus, and then they were tested for generalization in extinction after delays of 1 min, 1 day, and 1 week. With both dimensions, the relative gradients became progressively flatter with increasing delay intervals. This replicates earlier findings and extends them to the line angle dimension. The evidence of substantial forgetting in the first 24 h in all three experiments suggests that operant free-response procedures are more sensitive to forgetting effects than are discrete trial tasks.