Sustainable resource production for manufacturing bioactives from micro- and macroalgae: Examples from harvesting and cultivation in the Nordic region.

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, a Nordic Center of Excellence to develop an algae-based photosynthetic production platform.

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.

Nuclear DNA Content Variation in Different Life Cycle Stages of Sugar Kelp, Saccharina latissima.

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).

The Unique Lipidomic Signatures of Saccharina latissima Can Be Used to Pinpoint Their Geographic Origin.

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.

Iodine content in bulk biomass of wild-harvested and cultivated edible seaweeds: Inherent variations determine species-specific daily allowable consumption.

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.

Managing the Microbial Community of Marine Fish Larvae: A Holistic Perspective for Larviculture.

The availability of high-quality juveniles is a bottleneck in the farming of many marine fish species. Detrimental larvae-microbe interactions are a main reason for poor viability and quality in larval rearing. In this review, we explore the microbial community of fish larvae from an ecological and eco-physiological perspective, with the aim to develop the knowledge basis for microbial management. The larvae are exposed to a huge number of microbes from external and internal sources in intensive aquaculture, but their relative importance depend on the rearing technology used (especially flow-through vs. recirculating systems) and the retention time of the water in the fish tanks. Generally, focus has been on microbes entering the system, but microbes from growth within the system is normally a substantial part of the microbes encountered by larvae. Culture independent methods have revealed an unexpected high richness of bacterial species associated with larvae, with 100-250 operational taxonomic units associated with one individual. The microbiota of larvae changes rapidly until metamorphosis, most likely due to changes in the selection pressure in the digestive tract caused by changes in host-microbe and microbe-microbe interactions. Even though the microbiota of larvae is distinctly different from the microbiota of the water and the live food, the microbiota of the water strongly affects the microbiota of the larvae. We are in the early phase of understanding larvae-microbe interactions in vivo, but some studies with other animals than fish emphasize that we so far have underestimated the complexity of these interactions. We present examples demonstrating the diversity of these interactions. A large variety of microbial management methods exist, focusing on non-selective reduction of microbes, selective enhancement of microbes, and on improvement of the resistance of larvae against microbes. However, relatively few methods have been studied extensively. We believe that there is a lot to gain by increasing the diversity of approaches for microbial management. As many microbial management methods are perturbations of the microbial community, we argue that ecological theory is needed to foresee and test for longer term consequences in microbe-microbe and microbe-larvae interactions. We finally make some recommendations for future research and development.

A branched beta-D-(1-->3,1-->6)-glucan from the marine diatom Chaetoceros debilis (Bacillariophyceae) characterized by NMR.

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.

Structural characterization of beta-D-(1-->3)-glucans from different growth phases of the marine diatoms Chaetoceros mülleri and Thalassiosira weissflogii.

We have investigated the content and structure of the chrysolaminarans isolated from the two marine diatoms Chaetoceros mülleri and Thalassiosira weissflogii. Samples were taken from different phases of growth, and the structure of the chrysolaminaran was seen in relation to the specific growth rate of the diatoms. The structure determined for the glucan from C. mülleri was found not to vary with different specific growth rates. T. weissflogii showed some variance in the structure, both throughout the different stages of growth and between samples taken from the stationary phase. C. mülleri was found to have a chrysolaminaran with a degree of polymerization (DP) of 22-24 and a degree of beta-(1-->6) branching of 0.006-0.009. These results corresponded well with previous results obtained in our laboratories. The chrysolaminaran isolated from T. weissflogii was found to have a DP of 5-13 and no beta-(1-->6) branching. This is to our knowledge the first characterization of the chrysolaminaran from T. weissflogii.

The role of environmental biotechnology in exploring, exploiting, monitoring, preserving, protecting and decontaminating the marine environment.

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.

Characterization of a beta-D-(1-->3)-glucan from the marine diatom Chaetoceros mülleri by high-resolution magic-angle spinning NMR spectroscopy on whole algal cells.

High-resolution magic-angle spinning (hr-MAS) NMR spectroscopy was used to record NMR spectra of a cell paste from the marine diatom Chaetoceros mülleri. This gave information on a cellular storage polysaccharide identified as a beta-D-(1-->3)-linked glucan, using hr-MAS one-dimensional 1H and 13C, two-dimensional 1H,1H-COSY and 13C,1H-correlation spectroscopy. The same structural information was deduced from the liquid state NMR data on the glucan extracted from C. mülleri. The extracted glucan proved to be a beta-D-(1-->3)-linked glucan with a degree of polymerization of 19 and a degree of beta-D-(1-->6) branching of 0.005. The hr-MAS spectrum of the diatom showed several nonglucan resonances in the carbohydrate region of the NMR spectrum (60-103 ppm) that were shown to be noncarbohydrate resonances by means of two-dimensional 13C,1H- and 1H,1H-correlated NMR data.

Live feed is not a major determinant of the microbiota associated with cod larvae (Gadus morhua).

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.

Selection of candidate probionts by two different screening strategies from Atlantic cod (Gadus morhua L.) larvae.

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.