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Ploidy variants can be utilized to increase yield, introduce sterility, and modify specific traits with an economic impact. Despite economic importance of Saccharina species, their nuclear DNA content in different cell types and life stages remain unclear. The present research was initiated to determine the nuclear DNA content and intraindividual variation at different life cycle stages of the Laminarialean kelp Saccharina latissima. Nuclear DNA content in embryonic and mature sporophytes, released and unreleased zoospores, female, and male gametophytes from Sør-Trøndelag county in Norway were estimated by image analysis using the DNA-localizing fluorochrome DAPI and chicken’s red blood cells as a standard. DNA content of a total of 6905 DAPI-stained nuclei was estimated. This is the first study of nuclear DNA content which covered the life cycle of kelp. The lowest level of DNA content (1C) was observed in zoospores with an average of 0.76 pg. Male and female single spore gametophyte cultures presented higher average DNA content, more than double that of zoospores, suggesting the presence of polyteny. Female gametophyte nuclei were slightly larger and more variable in size than those of male gametophytes. The DNA content observed in embryonic sporophytes and in meristoderm cells from older sporophytes (1.51 pg) was 2C as expected and in the range of previously published studies of sporophytes of S. latissima. Mature sporophytes showed intra-plant variation with DNA content values ranging from 2-16C. The main difference was between meristoderm cells (mostly 2C) and cortical and medullary cells (2-16C).

Macroalgae aquaculture is thriving, and kelps such as Saccharina latissima, are now farmed over different production sites across Europe. Macroalgae production in integrated multi-trophic aquaculture setups is gaining relevance due to increased biomass yield and bioremediation potential. Nevertheless, there is a substantiated need to understand how this type of production shapes biomass composition. The present study evaluated the influence of time of harvesting and proximity to nutrient source (fish farm sea cages) on the elemental and biochemical composition of S. latissima, with special emphasis on its lipid content. Overall, the differences recorded for elemental, biochemical and lipid composition of S. latissima occurred consistently with harvesting period rather than with the distance to fish farm sea cages, evidencing that farming kelp in integrated multi-trophic aquaculture setups does not compromise nutritional quality while promoting increased biomass yield. Elemental and biochemical composition differed between kelp biomass harvested in April and those harvested in May and June, with lower C, H and carbohydrates, and higher ash contents in the later. Fatty acid profile analysis revealed an increase in monounsaturated fatty acids along the harvesting period, as well as a decrease in n-3 fatty acids with a concomitant increase of the n-6/n-3 ratio from April to May. Statistical analysis of the polar lipidome identified by liquid chromatography-mass spectrometry revealed differences in specific lipid signatures, displaying a perfect discrimination between harvesting periods. The discrimination between samples from reference and integrated multi-trophic aquaculture sites was only observed for kelp biomass harvested in June. Contributing particularly to these differences are betaine lipids, more abundant in the two later time points, and some lysolipid species, especially abundant in June. These findings will allow a more integrated look at macroalgae production under integrated multi-trophic aquaculture framework, thus promoting the systematization of farming practices that may enhance yield with biochemical quality to match the increasing demands by the food industry and sustainable biorefinary pipeline.

Micro- and macroalgae are a great and important source of raw material for manufacturing of bioactives and ingredients for food, feed, cosmetics, or pharmaceuticals. Macroalgae (or seaweeds) have been harvested locally from wild stocks in smaller volumes for a long time, and a production chain based on cultivated seaweed for the harvest of considerably larger amounts is in progress for several species. Microalgae and cyanobacteria such as Spirulina have been produced in "backyard ponds" for use in food and feed also for a long time, and now we see the establishment of large production plants to control the cultivation process and increase the production yields. There is also a shift from harvesting or cultivation centered in warmer, sunnier areas to increasing exploitation of natural resources in temperate to boreal regions. In locations with strong seasonal variations in solar irradiance and temperatures, we need to develop procedures to maximize the biomass production in the productive seasons and ensure efficient stabilization of the biomass for year-round processing and product manufacturing. Industrialized biomass production and large-scale manufacturing of bioactives also mean that we must employ sustainable, cost-effective, and environmentally friendly processing methods, including stabilization and extraction methods such as ensiling and subcritical water extraction (SWE) and advanced analytic tools to characterize the products. These topics are focus areas of the Nordic Centre of Excellence (NCoE) NordAqua, and here we present a review of current activities in the field of micro- and macroalgae biomass production sectors illustrated with some of our experiences from the NordAqua consortium.

NordAqua is a multidisciplinary Nordic Center of Excellence funded by NordForsk Bioeconomy program (2017–2022). The research center promotes Blue Bioeconomy and endeavours to reform the use of natural resources in a environmentally sustainable way. In this short communication, we summarize particular outcomes of the consortium. The key research progress of NordAqua includes (1) improving of photosynthetisis, (2) developing novel photosynthetic cell factories that function in a “solar-driven direct CO2 capture to target bioproducts” mode, (3) promoting the diversity of Nordic cyanobacteria and algae as an abundant and resilient alternative for less sustainable forest biomass and for innovative production of biochemicals, and (4) improving the bio-based wastewater purification and nutrient recycling technologies to provide new tools for integrative circular economy platforms.

Different incubation techniques for yolk sac larvae of Atlantic halibut (Hippoglossus hippoglossus L.) were compared and evaluated on the basis of survival and amount of normally developed larvae. The three investigated incubator concepts were; 3-litre glass-bowls maintained with stagnant water, 100-litre flat bottom PVC-tanks run with slow water exchange, and 350–1450 litre flow through silos with up-streaming water. The survival at the end of the yolk sac stage in 3-litre bowls was 30–90%, and increased reproducibility was obtained by addition of 25 ppm antibiotics to the water at start of the incubation. In the PVC tanks the survival was lower (20–55%). The flow through silo-incubators with up-wards water flow gave survival of 30–70%, and exhibited the highest reproducibility in survival for replicates. The fraction of normally developed larvae ready for first feeding varied both with larval batches and incubator-type. For all batches of eggs, lowest amount of normal larvae were obtained in the 100 litre PVC-tanks. In the bowls and the silos the fraction of normal larvae were 55–75% in the investigated batches. The most dominating deformity was jaw deformity (gaping), and lowest relative fraction of gaping was obtained in the bowls and in silos in larval batch 2. An overall evaluation of the incubator potential of efficient large scale production of yolk sac larvae ready for first feeding suggests that the silo-incubators have the highest potential.

The Norwegian coastline covers more than 10° in latitude and provides a range in abiotic and biotic conditions for seaweed farming. In this study, we compared the effects of cultivation depth and season on the increase in biomass (frond length and biomass yield), chemical composition (protein, tissue nitrogen, intracellular nitrate and ash content) and biofouling (total cover and species composition) of cultivated Saccharina latissima at nine locations along a latitudinal gradient from 58 to 69° N. The effects of light and temperature on frond length and biofouling were evaluated along with their relevance for selecting optimal cultivation sites. Growth was greater at 1–2 m than at 8–9 m depth and showed large differences among locations, mainly in relation to local salinity levels. Maximum frond lengths varied between 15 and 100 cm, and maximum biomass yields between 0.2 and 14 kg m−2. Timing of maximum frond length and biomass yield varied with latitude, peaking 5 and 8 weeks later in the northern location (69° N) than in the central (63° N) and southern (58° N) locations, respectively. The nitrogen-to-protein conversion factor (averaged across all locations and depths) was 3.8, while protein content varied from 22 to 109 mg g−1 DW, with seasonality and latitude having the largest effect. The onset of biofouling also followed a latitudinal pattern, with a delayed onset in northern locations and at freshwater-influenced sites. The dominant epibiont was the bryozoan Membranipora membranacea. Our results demonstrate the feasibility of S. latissima cultivation along a wide latitudinal gradient in North Atlantic waters and underscore the importance of careful site selection for seaweed aquaculture.

The aquaculture of macroalgae for human consumption and other high-end applications is experiencing unprecedented development in European countries, with the brown algae Saccharina latissima being the flag species. However, environmental conditions in open sea culture sites are often unique, which may impact the biochemical composition of cultured macroalgae. The present study compared the elemental compositions (CHNS), fatty acid profiles, and lipidomes of S. latissima originating from three distinct locations (France, Norway, and the United Kingdom). Significant differences were found in the elemental composition, with Norwegian samples displaying twice the lipid content of the others, and significantly less protein (2.6%, while French and UK samples contained 6.3% and 9.1%, respectively). The fatty acid profiles also differed considerably, with UK samples displaying a lower content of n-3 fatty acids (21.6%), resulting in a higher n-6/n-3 ratio. Regarding the lipidomic profile, samples from France were enriched in lyso lipids, while those from Norway displayed a particular signature of phosphatidylglycerol, phosphatidylinositol, and phosphatidylcholine. Samples from the UK featured higher levels of phosphatidylethanolamine and, in general, a lower content of galactolipids. These differences highlight the influence of site-specific environmental conditions in the shaping of macroalgae biochemical phenotypes and nutritional value. It is also important to highlight that differences recorded in the lipidome of S. latissima make it possible to pinpoint specific lipid species that are likely to represent origin biomarkers. This finding is relevant for future applications in the field of geographic origin traceability and food control.

To reach the goal of an industrialised macroalgae industry in Norway and other high-cost countries in the near future, a standardised seedling production method to improve quality control and predictability of cultivated biomass is essential. A total of 11 different treatments for seeding twine or rope with meiospores, gametophytes or juvenile sporophytes from the kelp Saccharina latissima were measured for growth (frond length, frond area, biomass yield and density) and protein content after 80 and 120 days at sea. Meiospore- and gametophyte-seeded twines were pre-cultivated in the hatchery for 14–42 days prior to deployment, while juvenile sporophytes of different ages were seeded on ropes directly on the day of deployment using a commercial binder to attach the seedlings. The results showed that seeding with meiospores pre-cultivated in the hatchery for 42 days (S42) before deployment gave significantly longer fronds (77.0 ± 6.7 cm) and a higher biomass yield (7.2 ± 0.1 kg m−1) at sea compared to other treatments. The poorest growth was measured for the direct-seeded sporophytes pre-cultivated in free-floating cultures for 35 days prior to deployment (D35; 34.4 ± 2.4 cm frond length and 1.6 ± 0.4 kg m−1). Image analysis was used to measure the coverage of the twine substrate before deployment, and a correlation was found between substrate coverage and frond length at sea, indicating that this can be used as a tool for quantity and quality control during the hatchery phase and before deployment. The protein content did not reveal any large differences between the treatments after 120 days of cultivation. 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 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/.

We have evaluated the cultivation potential of sugar kelp (Saccharina latissima) as a function of latitude and position (near- and offshore) along the Norwegian coast using a coupled 3D hydrodynamic-biogeochemical-kelp model system (SINMOD) run for four growth seasons (2012-2016). The results are spatially explicit and may be used to compare the suitability of different regions for kelp cultivation, both inshore and offshore. The simulation results were compared with growth data from kelp cultivation experiments and in situ observations on coverage of naturally growing kelp. The model demonstrated a higher production potential offshore than in inshore regions, which is mainly due to the limitations in nutrient availability caused by the stratification found along the coast. However, suitable locationsareas for kelp cultivation were also identified in areas with high vertical mixing close to the shore. The results indicate a latitudinal effect on the timing of the optimal period of growth, with the prime growth period being up to 2 months earlier in the south (58 °N) than in the north (71 °N). Although the maximum cultivation potential was similar in the six marine ecoregions in Norway (150-200 tons per unit area per year) in the six marine ecoregions in Norway, the deployment time of the cultures seems to matter significantly in the south, but less so in the north. The relevance of the results is discussed with a view towards use in decision support tools and a management context. The results are discussed, focusing on their potential significance for optimized cultivation and to support decision making towards sustainable management. The kelp cultivation potential in coastal and offshore regions of Norway

The article Effect of seeding methods and hatchery periods on sea cultivation of Saccharina latissima (Phaeophyceae): a Norwegian case study, written by Silje Forbord, Kristine B. Steinhovden, Torfinn Solvang, Aleksander Handa, Jorunn Skjermo.

This study represents a large-scale investigation into iodine contents in three commercially important and edible seaweed species from the North Atlantic: the brown algae Saccharina latissima and Alaria esculenta, and the red alga Palmaria palmata. Variability among and within species were explored in terms of temporal and spatial variations in addition to biomass source. Mean iodine concentration in bulk seaweed biomass was species-specific: Saccharina > Alaria > Palmaria. Iodine contents of Saccharina biomass were similar between years and seasons, but varied significantly between sampling locations and biomass sources. In Alaria and Palmaria, none of the independent variables examined contributed significantly to the small variations observed. Our data suggest that all three species are rich sources of iodine, and only 32, 283, or 2149 mg dry weight of unprocessed dry biomass of Saccharina, Alaria, or Palmaria, respectively, meets the recommended daily intake levels for most healthy humans.

New consumer trends, market demands, and opportunities for multiple uses of macroalgae such as food, bioactive components for functional food and feed ingredients, fertilizers, and biofuels have strengthened the motivation for industrial macroalgae cultivation in Europe. The cultivation of kelp in sea requires production of seedlings on substrate to be deployed at sea farms. There are three main strategies for seedling production of kelp, aiming for seeding of the growth substrate with spores, gametophytes, or small sporophytes, whereof seeding with spores and gametophytes so far is the most incorporated techniques. Seeding with spores requires fertile sporophytes and this is seasonally dependent if these are collected in natural habitats, but fertility can also be induced by artificial day rhythm, and thus enables access to spores independent of natural seasons. Gametophytes can be kept in continuous cultures and be available for year-through seeding or production of microscopic sporophytes for direct seeding. This is advantageous as the use of incubation facilities on land can be shortened by several weeks or, in the case of direct seeding, omitted completely. The presented protocol focuses on Saccharina latissima seedlings production from spores and gametophytes and is adapted to the infrastructure and equipment that are available at typical aquaculture research laboratories, although some adjustments may be necessary to meet the needs of the different life stages. Keywords: Saccharina latissima, cultivation, seeding, laboratory protocol, gametophyte cultures, contamination control, deployment

Seaweeds are increasingly used in European cuisines due to their nutritional value. Many algal constituents, such as polyphenols, are important antioxidants and thus considered beneficial to humans. However, many seaweed species can accumulate heavy metals and exhibit potential health risks upon ingestion. We investigated temporal and spatial variations in polyphenol and heavy metal (As, Cd, Hg, Pb) concentrations of three edible seaweed species. The brown algae Saccharina latissima and Alaria esculenta, and the red alga Palmaria palmata were sourced from natural populations and aquaculture in the NE Atlantic and processed as bulk biomass mimicking industrial scales. The mean polyphenol content was species-specific (Alaria > Saccharina > Palmaria), and highest in winter (for Alaria and Saccharina) and spring (for Palmaria); inter-annual and spatial variations were marginal. Heavy metal concentrations varied between species and depended on collection site, but seasonal variations were minimal. Our data suggest that all three species are good sources of antioxidants, and the heavy metal concentrations are below the upper limits set by the French recommendation and the EU Commission Regulation on contaminants in foodstuffs. A health risk assessment indicated that consumption of these seaweed species poses a low risk for humans with regard to heavy metals. However, an EU-wide regulation on maximal concentration of heavy metals in seaweeds should be established.

Integrated multi-trophic aquaculture (IMTA) has the potential of reducing open-cage fish farming impacts on the environment while also introducing new value chains. The aim of this study was to investigate the growth and composition of the kelp Saccharina latissima in salmon-driven IMTA, and to assess the spatial extent of the influence of salmon derived nitrogen in order to evaluate the upscaling potential for IMTA. S. latissima was cultivated 100, 200, and 1,000 m east and 1,000 m west of a 5,000 tons salmon farm in Western Norway from February to September 2013. The proportion of salmon derived nitrogen available for the kelp showed a clear decline with distance from the farm. Accordingly, the kelp cultivated near the salmon cages grew faster during the spring season, and growth rate decreased with increasing distance from the farm. A spatially explicit numerical model system (SINMOD), including compartments for dissolved nutrients and kelp growth, was tuned to the field data and used to investigate the potential for upscaling IMTA production. The model was used to introduce a new metric—the impacted area IA—for the areal effects of IMTA in terms of the increase in production by IMTA. The model showed that a 25 hectare kelp farm in the vicinity of the studied salmon farm could take up 1.6 of the 13.5 tons of dissolved inorganic nitrogen released during kelp cultivation, amounting to almost 12% of the ammonia released during the cultivation period from February to June. The 25 hectare kelp farm would have a production yield of 1,125 tons fresh weight (FW), being 60% more than that of a non-IMTA kelp farm, while a 20% increase of kelp FW could be obtained over a 110 hectar area in salmon-driven IMTA. To achieve an even mass balance, an area of approximately 220 ha−1 would be needed to cultivate enough kelp to fix an equivalent of the nitrogen released by the fish. Copyright © 2018 Fossberg, Forbord, Broch, Malzahn, Jansen, Handå, Førde, Bergvik, Fleddum, Skjermo and Olsen. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY).

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.

Economic and environmental developments in the last decades call for the displacement of fossil fuels to alternative energy sources. Biofuels are considered as a part of the solution for this challenge. Sustainable raw materials for the production of transportation biofuels such as biodiesel, biobutanol and bioethanol, can be obtained from algal biomass. In particular, marine macroalgal biomass is a promising feedstock for transportation biofuels because of (the) its fast growth and its potential cultivation on seawater, avoiding competition of resources with conventional agriculture of terrestrial plants used for food. In addition, dissolved inorganic nutrients like nitrogen, phosphorous and carbon are taken up by macroalgae, helping to alleviate eutrophication in seas and oceans. Using biological, chemical and engineering advances in the last decades, technologies to provide cost efficient cultivation, harvesting, extraction and processing of sustainable biofuels have to be elaborated. This paper provides a review of macroalgae based biorefineries with offshore cultivation and consequent biomass conversion into transportation liquid biofuels. We discuss the methods for offshore cultivation, harvesting, and conversion of macroalgae biomass into liquid transportation biofuels. Based on the current information and global experience, we present local perspectives specific for France, Germany, Norway, the Netherlands and Israel on the potential use of Exclusive Economic Zone for transportation biofuels production. Marketable suggestions for future research need to include all stakeholders of a given site for offshore biorefinery development.

article i nfo Hydrothermal liquefaction of sugar kelp Laminaria saccharina, a brown macro-alga harvested in Trondheim bay (Norway), was experimentally studied by a high-throughput screening technique using sealed quartz capillary reactors. Very high heating rates were achieved by the experiment technique, which significantly intensified the liquefaction and resulted in much higher bio-oil yields. The highest bio-oil yield of 79% w (dry and ash free) was obtained from the test at the highest heating rate of 585 °C/min (temperature = 350 °C, holding time = 15 min, and kelp/water ratio = 1/10 w/w). The HHV of the bio-oil was also significantly improved, being as high as of 35.97 MJ/kg (dry and ash free). In addition, a numerical prediction and modelling supported by regression analyses showed a good agreement between the present study and the literature, with respect to the effect of heating rate on the bio-oil yield.

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.

This paper investigated the assimilation of dissolved inorganic nitrogen (DIN) in Saccharina latissima in proximity to salmon cages in coastal waters. The bioassays were performed on plants from three stations located in the vicinity of a salmon farm (Salmo salar) in exposed waters at Tristein (63° 52′ N, 9° 37′ E) in Central Norway. The growth, the C and N content, and the nitrogen isotope ratios (δ15N) of S. latissima were monitored over 1 year. The DIN concentrations in seawater were higher at the salmon farm stations than at the reference station during the winter, and the N/P ratio at the salmon farm stations was higher from September to January and in June. S. latissima at the salmon farm stations grew faster than at the reference station. The length of S. latissima increased by 50 % when integrated with the salmon farm compared to the reference station. The N content of S. latissima was positively correlated to the DIN concentration in seawater (p

The interest to develop an industrialized cultivation of several macroalgae in Europe is growing rapidly. Here, we demonstrate cultivation of the sugar kelp Saccharina latissima in integration with salmon (Salmo salar) aquaculture in a Norwegian coastal area. Sporophytes of S. latissima were deployed at 2, 5 and 8 m depths at a salmon farm and at a reference station 4 km away in August, November, February and June (2010–2011). The growth was good in late autumn and in spring, with peak lengths of the sporophytes in June, while being poor in winter and summer. As a result of a faster initial growth at the fish farm than at the reference station from August to November, the August-sporophytes reached a significantly longer length than those at the reference station in 5 out of 10 sampling months at 2 m depth, and in 9 out of 10 months at 5 m depth (p

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.

Stimulation of the non-specific defence enhances the disease resistance and growth, and has good potentials as a measure for increased microbial control in juvenile production of marine fish and shellfish. So far, the most commonly used immunostimulants are β-(1 → 3, 1 → 6)-glucans, and in this study the stimulatory potential of a β-(1 → 3, 1 → 6)-glucan of marine origin, the storage polysaccharide from the marine diatom Chaetoceros mülleri, was examined. The glucan (chrysolaminaran) was extracted from cultures of C. mülleri, and used as a dietary supplement in two first feeding experiments with larvae of Atlantic cod Gadus morhua L. In one experiment the microalgal glucan was compared to the commercial yeast-glucan product MacroGard®, and in the other to an alginate with a high content of mannuronic acid (High-M alginate) isolated from Durvillaea antarctica. The stimulants were given via rotifers, and weaning to formulated feed was initiated at day 17 or 18 after hatching. The survival ± SEM at day 27 after hatching was 24.5 ± 2.0%, 14.8 ± 4.5% and 13.1 ± 1.4% for the groups fed C. mülleri-glucan, yeast glucan and for the control, respectively, in the first experiment. The group fed C. mülleri-glucan group had higher survival compared to the control (P < 0.05) group, whereas the yeast glucan had no positive effect on the survival (p > 0.05). The dry weights of the groups at day 27 were low, with 203.2 ± 52.2, 165.2 ± 43.4 and 198.5 ± 58.1 μg per larva for the C. mülleri-glucan, yeast glucan and control groups, respectively. In the second experiment the survival in the period of feeding formulated feed (days 18-30) were 44.6 ± 4.3%, 44.7 ± 1.3%, and 33.8 ± 4.1% survival for the C. mülleri-glucan, High-M alginate and control group, respectively. The cod larvae fed C. mülleri-glucan reached an average weight of 531.6 ± 17.2 μg at day 30, which was significantly higher (p < 0.05) than the control group that had an average of 473.6 ± 3.5 μg. The larvae fed High-M alginate had an average weight of 470.3 ± 31.6 μg per larva at day 30, and not significantly different from the control (p > 0.05). The early weaning to formulated diet had detrimental effect on the growth of the larvae. In both experiments the C. mülleri-glucan group was the only group showing a positive growth rate in the period of weaning to dry feed. The microbial conditions in larval gut and water were monitored with respect to total colony forming units on Marine agar, and Vibrio- and Pseudomonas-like species on selective agars (TCBS and marine Pseudomonas Agar with CFC-supplement). The larvae were rapidly colonised after hatching, but no or weak effects of the stimulants were observed on the colonisation rates or the composition. The total CFU varied from 10 1 to 10 2 CFU per μg larva after initiation of the first feeding. The percentages of Pseudomonas-like bacteria increased throughout the period, whereas the levels of Vibrio-like bacteria were low and stable. The chrysolaminaran from the diatom C. mülleri was shown to be a promising candidate for use as an immunostimulatory feed additive, and which should be further explored. © 2006 Elsevier B.V. All rights reserved.

The diversity of the microbiota in 15 Atlantic cod (Gadus morhua L.) larvae from three different hatcheries with variable food type and water treatment was characterized with culture dependent and independent approaches. The culture-dependent approach was phenotypic characterization of bacterial isolates based on 16 tests, and the culture-independent approach was terminal restriction fragment length polymorphism (T-RFLP) of PCR amplified 16S rRNA. Both approaches revealed large inter-individual differences in the microbiota of cod larvae. Data from real time PCR indicate substantially lower variation in bacterial number larva � 1 both within and between hatcheries than the data on colony forming units, resulting in an estimated four orders of magnitude variation in culturability between larvae. Considerably higher richness and diversity were observed with T-RFLP than with phenotypic characterization, and no correlation between the two approaches was found for diversity indexes calculated at the individual level. Dissimilarity in the composition of the microbiota between individuals from the same rearing environment was comparable to the dissimilarities between hatcheries. The large inter-individual variation is important to consider when designing experiments, as pooling of individuals will completely mask this variability. We conclude that colonization of individual cod larvae by bacteria seems to be a stochastic and not a deterministic process, which is not well understood.

Two primary selection criteria were used to collect a pool of nearly 500 candidate probiotic bacteria from Atlantic cod (Gadus morhua L.) larvae, i.e. the dominant intestinal bacterial flora and isolates with antagonistic activity against Vibrio anguillarum. Bacteria were isolated from cod larvae from five rearing groups with variable rearing technologies. The bacteria were brought to pure culture and characterized phenotypically. Based on properties such as uniqueness, dominance and fermentative ability, a selection of approximately 10% of the isolates were chosen from the initial pool of bacteria to reduce the number of candidates. These 55 isolates were characterized further in vitro regarding antagonism, adhesion to mucus, growth in mucus, production of extracellular enzymes, fish bile resistance and haemolytic properties. Based on the results of the in vitro tests, the number of probiotic candidates was further reduced to seven isolates. To evaluate the probiotic potential and to assure that the seven isolates were not harmful to the host, yolk sac larvae of cod were exposed to the isolates in a small-scale in vivo experiment. The in vivo experiment excluded two of the candidate bacteria due to increased mortality of cod larvae, whereas three isolates from the dominant (Vibrio and two different strains of Microbacterium) and two from the antagonistic (Ruegeria and Pseudoalteromonas) group improved the survival of larvae compared to the positive control. Thus, a combination of the two screening methods was suited for making multistrain probiotics with complementary modes of action.

The intestinal microbiota in 27 Atlantic cod larvae (Gadus morhua L.) from five rearing groups was screened for antagonistic activity against Listonella anguillarum. A total of 98 antagonistic isolates were phenotypically characterized and belonged to 12 clusters at the 80% similarity level. The 16S rDNA of representative isolates was partially sequenced and compared to sequences in the GenBank database. The majority of antagonistic bacterial isolates had high sequence similarity to the γ-Proteobacteria, and the rest were members of the α-Proteobacteria. Antagonistic isolates with sequence similarity to Vibrionaceae were found in larvae from all five rearing groups, and this was the most numerous group of antagonistic isolates. The other antagonistic bacteria had sequence similarities to Hyphomicrobium, Marinomonas, Pseudoalteromonas, Roseobacter and Shewanella. The composition of the antagonistic bacterial flora varied both between individual cod larvae from the same rearing group and between larvae from the five different rearing groups. Also, the average percentage of antagonistic isolates was significantly different in one of the five rearing groups.

In light of the Marine Strategy Framework Directive (MSFD) and the EU Thematic Strategy on the Sustainable Use of Natural Resources, environmental biotechnology could make significant contributions in the exploitation of marine resources and addressing key marine environmental problems. In this paper 14 propositions are presented focusing on (i) the contamination of the marine environment, and more particularly how to optimize the use of biotechnology-related tools and strategies for predicting and monitoring contamination and developing mitigation measures; (ii) the exploitation of the marine biological and genetic resources to progress with the sustainable, eco-compatible use of the maritime space (issues are very diversified and include, for example, waste treatment and recycling, anti-biofouling agents; bio-plastics); (iii) environmental/marine biotechnology as a driver for a sustainable economic growth.

The chrysolaminaran from the marine diatom Chaetoceros debilis was isolated and characterized by NMR spectroscopy. Cells were harvested in the stationary phase of growth after the medium had been depleted of nitrate when the chrysolaminaran content was expected to be at its highest. The chrysolaminaran was isolated with an yield of 17.5 mg/L, which corresponds to 15.8 pg/cell. 1H NMR indicated that the structure was similar to that of a beta-(1-->3) main chain with beta-(1-->6)-linked side chains. The degree of polymerization was found to be 30, corresponding to a molecular weight of approximately 4900. Thirty-three percent of the residues were found to be beta-(1-->6)-linked branches. The characteristics of the beta-(1-->6) branching were examined by NOESY NMR, which suggested pustulan-like branches, being beta-(1-->6) linked chains connected to the main chain with few branch points. Confirmation of the 1H NMR data was done by 13C-DEPT, TOCSY, COSY and HMQC NMR spectroscopy. The assignment of the resonances of the main beta-(1-->3) and beta-(1-->6) chains is presented. The structure proposed from our analyses is compared against other chrysolaminaran structures.

A strategy for microbial management in marine juvenile production should include nonspecific immunostimulation to reduce losses caused by infectious diseases. Halibut (Hippoglossus hippoglossus L.) larvae (250 day degrees) were distributed into first-feeding tanks of 10.000 l, at a Norwegian halibut juvenile farm. Immunomodulatory high-M alginate was bioencapsulated in Artemia by use of alginate microbeads (2–30 μm), and administered to halibut larvae at three stages during the first feeding period (days 7–9, 20–22 and 41–43). A fourth treatment was run after weaning, 5–3 days prior to a challenge test with two different doses of Vibrio anguillarum O2 that was started at day 90 after first feeding. After 15 days, the mortality at the highest dose was 45±1% in the control and 28±8% in the stimulated group, corresponding to 38% reduction in mortality, whereas no significant differences were measured at the low dose. It is concluded that feeding immunostimulants to halibut larvae for four short periods during 3 months enhanced resistance against vibriosis, and that high-M alginates are suitable as immunostimulants for marine larvae during first feeding and weaning.

High densities of marine larvae are often associated with microbial problems, mostly caused by opportunistic pathogens, resulting in poor growth and mass mortalities. The early life stages of fish have a very limited immune defence system. Therefore stimulation of non-specific immune mechanisms in fish larvae might be a very interesting tool. The present study evaluates the effects of a known immunostimulant on protein turnover in larval turbot (Scophthalmus maximus). Protein turnover and food intake was determined at day 13 using a time course after feeding the larvae with 15N-labelled rotifers. Turbot larvae fed with rotifers enriched with the immunostimulant FMI at first feeding had significantly higher fractional rates of protein synthesis when compared to a control group. This resulted in three-fold higher protein turnover in the larvae given the immunostimulant compared to the control group. Food intake, larval size and survival at the end of the experiment were similar in the two treatments. The effect of FMI in increasing protein turnover probably imply a higher larval viability and survival in case of environmental/disease stress. However, protein turnover is costly, and this may cause a trade-off between fast growth and stress-resistance/survival in fish larvae.

Nauplii of Artemia franciscana were incubated in two different concentrations (undiluted and 1:9 in autoclaved sea water) of a divalent bacterin composed of two different serovars of Vibrio anguillarum. In order to investigate uptake and further processing of a bacterin in the live feed organism A. franciscana, immunohistochemistry was applied, visualising the presence of whole bacterial cells and antigens from the bacterin in individual nauplii. By using ELISA, it was shown that approximately 1.5-2-5 x 10(5) cells were incorporated into each Artemia under the conditions used. Maximum incorporation of cells was measured after 30 min, whereas after 60 min there was a decline to levels of 0.9-1.6 x 10(5) cells per Artemia. Immediately after incubation in the bacterin solution, the nauplii were transferred to a culture of the alga Isochrysis galbana, in order to simulate transfer of the nauplii to rearing tanks for fish larvae. From the ELISA, it could be concluded that the incorporated bacterial cells were excreted from the Artemia nauplii rapidly, however a large variation among different nauplii could be visualised by immunohistochemistry.

Theaim of this study was to determine whether it is possible to influence thespecies composition of the gut microflora in 70-days old halibut larvae(Hippoglossus hippoglossus L.) by addition of bacteriabioencapsulated in Artemia franciscana in two shortpulses.Two Vibrio strains, PB 1-11 and PB 6-1 were used. Twotreatments received each strain alone, a third treatment received the twobacterial strains in mixture, whereas in a control treatment no bacteria wereadded. Five fish tanks were included in each treatment. The bacteria werebioencapsulated in Artemia franciscana metanauplii duringashort-term incubation in bacterial suspensions of the specific strains, andwereadded in two pulses, on day 1 and day 10 of the experiment. Addition ofbacteriadid not increase the total number of colony-forming units (CFU) in the larvalgut. The total CFU in the water was lower in tanks added bioencapsulatedbacteria than in the control treatment (p 0.05). The addition of bioencapsulated bacteria intwo pulses within a ten days period was therefore not sufficient to influencethe species composition of the microflora of the halibut larvae.

The accumulation of bacteria in Brachionus plicatilis and Artemia franciscana during a short-term incubation was quantified using immunocolony blot (ICB) and an enzyme-linked immunosorbent assay (ELISA). Four bacterial strains, isolated from turbot and halibut, were grazed effectively by both species when given at high concentrations (≥5×10 7 bacteria ml −1 ). B. plicatilis accumulated 21–63×10 3 bacteria per rotifer and A. franciscana up to 45×10 3 bacteria per metanauplius after 20–60 min of grazing. The composition of the bacterial microflora of the live food organisms changed drastically, as the bioencapsulated strains comprised up to 100% of the total count of colony-forming units. After incubation in the bacterial suspensions, B. plicatilis and A. franciscana were transferred to seawater with added microalgae ( Tetraselmis sp., 2 mg C l −1 ), to evaluate the persistence of the changed bacterial composition in conditions similar to those present in a first feeding tank. The bioencapsulated bacteria decreased in numbers, but in most cases remained present in both live food organisms after 24 h. It is possible, after a short-term incubation, to replace opportunistic (r-selected) bacteria present in the live food cultures with other bacteria, which persist as a dominant part of the bacterial flora of the live food for a relatively long period of time (4–24 h).

Two bacterial strains, 4:44 and PB52, isolated from turbot(Scophthalmus maximus L.) were used during a first feedingexperiment with turbot larvae. Bacteria were either added directly tothe water on the day of hatching of the larvae (day 0), orbioencapsulated in rotifers (Brachionus plicatilis) distributedon day 2 after hatching. The two bacterial strains were found to bepresent in the water of the rearing tanks throughout the experiment. Theaddition of bacteria influenced the species-composition of themicroflora associated with intensively produced rotifers added to thetanks, and resulted in colonization of the gut of the larvae by theadded bacterial strains. The strain 4:44 showed a peak on day 9 afterhatching, and reached a mean(sem) of 2.5(1.4) × 104bacteria per larva, when added in a mixture with PB52, and 0.4(0.1)× 104 bacteria per larva, when added alone. The strainPB52, on day 12 after hatching, reached 5.2(1.5) × 104bacteria per larva when added in a mixture with strain 4:44, and12.5(0.7) × 104 bacteria per larva, when added alone.The added bacteria colonized the gut turbot larvae, whereas thebacterial land, the survival and growth of the larvae were in most casesnot influenced in a negative way by the addition of bacteria.

The impacts of varying algal growth rates on the bacterial load in the cultures were studied for six marine microalgae: Skeletonema costatum ,Chaetoceros mulleri (Bacillariophyceae), Nannochloropsis oculata (Eustigmatophyceae), Isochrysis galbana, Pavlova lutheri (Haptophyceae), and Tetraselmis sp. (Prasinophyceae). Samples were taken in batch cultures during early exponential phase and in stationary phase when the growth rate was below 5% of maximum growth rate (µmax). Except for C. mulleri and N. oculata, the microalgae were thereafter grown semi-continuously at dilution rates of 20% and 60% of µmax and samples taken during a period of 10 days. Higher bacterial levels were associated with slow-growing microalgae on a per volume basis. Per algal cell, variations in the bacterial load were more related to species. Relatively high bacterial densities (5.7–16.4 CFU algal cell−1) with large proportions of opportunistic and haemolytic species were associated with the Bacillariophyceae. For the other species, the bacterial density was 0.2–4.3 CFU algal cell−1. Bacterial levels were in general lower and not affected to the same extent by algal growth conditions in Tetraselmis sp. and P. lutheri. No or very low levels of presumptive Vibrio spp. were observed, except in semi-continuous cultures of P. lutheri. Cultures of Tetraselmis sp. had low levels of opportunistic bacteria at all growth conditions.

The intensive cultivation conditions for marine larvae may easily cause microbial problems, resulting in poor growth and mass mortality, and techniques for improvement by enhancing environmental and larval factors should be developed. Establishment of a beneficial, protective microflora of marine larvae can be obtained by use of microbial matured water and probiotics. Microbial maturation of the rearing water before use in the larval tanks, can be obtained by running the water through a maturation unit that selects for non-opportunistic microflora. A diverse bacterial flora established by non-opportunists is believed to inhibit proliferation of opportunistic pathogenic bacteria in the water and the larvae. When used in the earliest developmental stages of marine fish larvae, enhanced growth and survival can be obtained. Also, the introduction of probiotic bacteria may promote the defence of the gut flora against pathogenic bacteria. Probiotic bacteria can be added directly to the water or administered to the larvae via live food, such as rotifers and Artemia. Further developments in these techniques are needed for improved control of bacterial number, growth and colonisation in the larval gut, as well as the identification of bacteria with probiotic effect on the host. Disinfection of eggs is a positive factor for control of growth and transfer of bacteria in larviculture. For marine fish eggs glutaraldehyde has suitable properties as a disinfectant, which improves hatching, development and survival of larvae. Because marine fish larvae have no specific immune system at hatching, non-specific defence is very important during the first developmental stages, and suitable techniques for stimulation of the non-specific immune defence may become important in future marine larval rearing. Enhanced viability of marine fish larvae and juveniles, has been obtained by treatment with FMI, which is a mannuronic acid polymer. Routes of administration were either into the water or via live food. A technique for incorporating immunostimulants in rotifers and Artemia for controlled transfer of immunostimulants to marine larvae, is described.

A first feeding experiment with turbot was arranged as a two factorial design with filtered vs. microbially matured water as one factor, and with vs. without microalgae added to the tanks as the other. Application of microbially matured water had a highly positive effect on the growth of the larvae from day 5 after hatching and onwards. Algal addition gave the greatest effect on size from day 12, although both factors were highly significant. A positive interaction effect of combining matured water with the addition of microalgae was also observed. The positive effects may be related to the observed delay in colonization of the larvae in the early stages of first feeding. Evaluation of the water microflora by criteria based on the degree of maturation showed a lower proportion of opportunistic (i.e., r-selected) bacteria in tanks with matured water containing microalgae. It is suggested that rearing of turbot larvae in microbially matured water to which microalgae are added will lower the proliferation of opportunistic bacteria on the mucosal surfaces of the larvae, with more viable and fast-growing larvae as the result.

Before transfer to larval incubators, water was membrane filtered to remove >95% of the bacteria and then transiently maintained in a biofilter that promoted recolonization of the water by non-opportunistic bacteria. The process is termed microbial maturation of the water. Hypothetically the bacterial flora in the matured water should protect the marine larvae from colonization and proliferation by opportunistic bacteria. Testing of the hypothesis demonstrated 76% higher survival of yolk sac larvae of Atlantic halibut (Hippoglossus hippoglossus) in matured than in membrane filtered water. Proliferation of opportunistic bacteria was observed in the rearing water after hatching of turbot eggs (Scophthalmus maximus), but to a less extent in the microbially matured water. In the early phase of first feeding of turbot larvae, the matured water induced qualitative differences in the gut microflora. Significantly higher initial growth rate of the turbot larvae in the matured water affected 51% higher average weight of 13 days old larvae than in membrane filtered water. Algal addition to the matured water enhanced the larval growth further. The experiments conducted supported the proposed hypothesis that microbial maturation selects for non-opportunistic bacteria, which protects the marine larvae from proliferation of detrimental opportunistic bacteria.

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.

A 3-dimensional hydrodynamic−ecological model system (SINMOD) was used to estimate the full-scale cultivation potential of the brown alga Saccharina latissima in integrated multi-trophic aquaculture (IMTA) with Atlantic salmon Salmo salar. A previously developed model for the frond size and composition (carbon and nitrogen content) of S. latissima sporophytes was adjusted to data from an outdoor mesocosm growth experiment and then coupled and run online with the 3-dimensional model system. Results from simulations were compared with data from an IMTA field experiment, providing partial validation of the hydrodynamic–ecological– kelp model system. The model system was applied to study the large-scale cultivation potential of S. latissima in IMTA with salmon and to quantify its seasonal bioremediation potential. The results suggest a possible yield of 75 t fresh weight S. latissima ha−1 in 4 mo (February to June) and about 170 t fresh weight ha−1 in 10 mo (August to June). The results further suggest that the net nitrogen consumption of a 1 ha S. latissima installation in the vicinity of a fish farm producing approximately 5000 t salmon in a production cycle is about 0.36 (0.15) t NH4 +-N, or a removal of 0.34% (0.41%) of the dissolved inorganic nitrogen effluent with a cultivation period from August (February) to June. Due to the differing seasonal growth patterns of fish and kelp, there was a mismatch between the maximum effluent of NH4 +-N from the fish farm and the maximum uptake rates in S. latissima. © The authors 2013. Open Access under Creative Commons by Attribution Licence. Use, distribution and reproduction are un - restricted. Authors and original publication must be credited.

Saccharina latissima is attractive for industrial cultivation for different usages, such as biofuels, feed supplements, and derivation of chemicals. A continuous supply of kelp sporelings throughout the year may ensure a year-round production of kelp juveniles on ropes. In this study, induction of sporangial areas (sorus portions) on the blade of S. latissima was performed throughout the year at three locations: Trondheim (Norway), Grenaa (Denmark), and Sylt island (Germany). The results indicate that a year-round sorus induction in S. latissima is possible and that this induction is controlled by applying short-day treatment of adult sporophytes throughout the year and by the removal of the basal blade meristem. The artificially induced and released zoospores formed viable sporelings at all seasons, but cultivation in the sea in Norway was successful only during autumn, winter, and spring, while the growth conditions were poor during the summer. The results are important for industrial scale-up and continuous production of kelp biomass.