Microbial Community Dynamics during a Harmful Chrysochromulina leadbeateri Bloom in Northern Norway

A harmful algal bloom occurred in late spring 2019 across multiple, interconnected fjords and bays in northern Norway. The event was caused by the haptophyte Chrysochromulina leadbeateri and led to severe fish mortality at several salmon aquaculture facilities. This study reports on the spatial and temporal succession dynamics of the holistic marine microbiome associated with this bloom by relating all detectable 18S and 16S rRNA gene amplicon sequence variants to the relative abundance of the C. leadbeateri focal taxon. A k-medoid clustering enabled inferences on how the causative focal taxon cobloomed with diverse groups of bacteria and microeukaryotes. These coblooming patterns showed high temporal variability and were distinct between two geographically separated time series stations during the regional harmful algal bloom. The distinct blooming patterns observed with respect to each station were poorly connected to environmental conditions, suggesting that other factors, such as biological interactions, may be at least as important in shaping the dynamics of this type of harmful algal bloom. A deeper understanding of microbiome succession patterns during these rare but destructive events will help guide future efforts to forecast deviations from the natural bloom cycles of the northern Norwegian coastal marine ecosystems that are home to intensive aquaculture activities

Marine diatoms are a rich source of bioactivity

As part of the MabCent-SFI drug-discovery initiative organized by the University of Tromsø, the coastal areas of northern Norway and the Barents Sea have been investigated (2006-2014). We saw that bioac- tive compounds from diatoms are very rarely reported (1-4). There- fore we desided that during the initial MabCent research cruises, di- atom strains should be isolated and kept in a culture collection and subsequently mass-cultivated in order to investigate their bioactivity by screening the diatom extracts using the Marbio (UiT) assay line

Light and temperature effects on bioactivity in diatoms

Isolates of five pelagic North Atlantic marine diatoms (Bacillariophyceae): Attheya longicornis, Chaetoceros socialis, Chaetoceros furcellatus, Skeletonema marinoi and Porosira glacialis were cultivated in large photobioreactors at two light and two temperature regimes to test if this affected bioactivity. We screened for bioactivity in assays representing five different therapeutic areas: diabetes II (PTP1b), cancer (melanoma cells, A2058), anti-oxidants (FRAP), immunomodulation (TNFa) and anti-infection (MRSA, Enterococcus faecalis, Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa). All the diatom strains showed activity in two or more assays. We detected differences in bioactivity both between species and within species cultivated with different light and temperature regimes. Our results demonstrate the potential for a more exhaustive exploitation of diatom metabolites that can be obtained by manipulation of the cultivation conditions

Field sampling marine plankton for biodiscovery

Microalgae and plankton can be a rich source of bioactivity. However, induction of secondary metabolite production in lab conditions can be difficult. One simple way of bypassing this issue is to collect biomass in the field and screen for bioactivity. Therefore, bulk net samples from three areas along the coast of northern Norway and Spitsbergen were collected, extracted and fractionated. Biomass samples from a strain of a mass-cultivated diatom Porosira glacialis were used as a reference for comparison to field samples. Screening for bioactivity was performed with 13 assays within four therapeutic areas: antibacterial, anticancer, antidiabetes and antioxidation. We analysed the metabolic profiles of the samples using high resolution - mass spectroscopy (HR-MS). Principal component analysis showed a marked difference in metabolite profiles between the field samples and the photobioreactor culture; furthermore, the number of active fractions and extent of bioactivity was different in the field compared to the photobioreactor samples. We found varying levels of bioactivity in all samples, indicating that complex marine field samples could be used to investigate bioactivities from otherwise inaccessible sources. Furthermore, we hypothesize that metabolic pathways that would otherwise been silent under controlled growth in monocultures, might have been activated in the field samples

The Effect of Freeze/Thaw Cycles on Reproducibility of Metabolic Profiling of Marine Microalgal Extracts Using Direct Infusion High-Resolution Mass Spectrometry (HR-MS)

During normal sample preparation, storage in freezers and subsequent freeze/thaw cycles are commonly introduced. The effect of freeze/thaw cycles on the metabolic profiling of microalgal extracts using HR-MS was investigated. Methanolic extracts of monocultures of Arctic marine diatoms were analyzed immediately after extraction, after seven days of storage at −78 °C (one freeze/thaw cycle), and after additional seven days at −20 °C (two freeze/thaw cycles). Repeated direct infusion high-resolution mass spectrometry analysis of microalgae extracts of the same sample showed that reproducibility was ca. 90% when a fresh (unfrozen) sample was analyzed. The overall reproducibility decreased further by ca. 10% after the first freeze/thaw-cycle, and after one more freeze/thaw cycle the reproducibility decreased further by ca. 7%. The decrease in reproducibility after freeze-thaw cycles could be attributed to sample degradation and not to instrument variability

Adding zooplankton to the OSMAC toolkit: Effect of grazing stress on the metabolic profile and bioactivity of a diatom

“One strain many compounds” (OSMAC) based approaches have been widely used in the search for bioactive compounds. Introducing stress factors like nutrient limitation, UV-light or cocultivation with competing organisms has successfully been used in prokaryote cultivation. It is known that diatom physiology is affected by changed cultivation conditions such as temperature, nutrient concentration and light conditions. Cocultivation, though, is less explored. Hence, we wanted to investigate whether grazing pressure can affect the metabolome of the marine diatom Porosira glacialis, and if the stress reaction could be detected as changes in bioactivity. P. glacialis cultures were mass cultivated in large volume bioreactor (6000 L), first as a monoculture and then as a coculture with live zooplankton. Extracts of the diatom biomass were screened in a selection of bioactivity assays: inhibition of biofilm formation, antibacterial and cell viability assay on human cells. Bioactivity was found in all bioassays performed. The viability assay towards normal lung fibroblasts revealed that P. glacialis had higher bioactivity when cocultivated with zooplankton than in monoculture. Cocultivation with diatoms had no noticeable effect on the activity against biofilm formation or bacterial growth. The metabolic profiles were analyzed showing the differences in diatom metabolomes between the two culture conditions. The experiment demonstrates that grazing stress affects the biochemistry of P. glacialis and thus represents a potential tool in the OSMAC toolkit

Inclusion of photoautotrophic cultivated diatom biomass in salmon feed can deter lice

The aim of this study was to evaluate the potential of diatom (microalgae) biomass as a lice-reducing ingredient in salmon feed. The original hypothesis was based on the fact that polyunsaturated aldehydes (PUAs), e.g. 2-trans, 4-trans decadenial (A3) produced by diatoms can function as grazing deterrents and harm copepod development. Salmon lice (Lepeophtheirus salmonis) is a copepod, and we intended to test if inclusion of diatom biomass in the feed could reduce the infestation of lice on salmon. We performed experiments where salmon kept in tanks were offered four different diets, i.e. basic feed with diatoms, fish oil, Calanus sp. oil or rapeseed oil added. After a feeding period of 67 days a statistically representative group of fishes, tagged with diet group origin, were pooled in a 4000L tank and exposed to salmon lice copepodites whereafter lice infestation was enumerated. Salmon from all four diet groups had good growth with SGR values from 1.29 to 1.44% day-1 (increase from ca. 130 g to 350 g). At the termination of the experiment the number of lice on salmon offered diatom feed were statistically significantly lower than on salmon fed the other diets. Mean lice infestation values increased from diatom feed through Calanus and fish oil to standard feed with terrestrial plant ingredients. Analysis of the chemical composition of the different diets (fatty acids, amino acids) failed to explain the differences in lice infestation. The only notable result was that diatom and Calanus feed contained more FFA (free fatty acids) than feed with fish oil and the control feed. None of the potential deleterious targeted polyunsaturated aldehydes could be detected in skin samples of the salmon. What was exclusive for salmon that experienced reduced lice was diatom inclusion in the feed. This therefore still indicates the presence of some lice deterring ingredient, either in the feed, or an ingredient can have triggered production of an deterrent in the fish. An obvious follow up of this will be to perform experiments with different degrees of diatom inclusion in the feeds, i.e. dose response experiments combined with targeted PUA analyses, as well as to perform large scale experiments under natural conditions in aquaculture pens

Mass Cultivation of Microalgae: I. Experiences with Vertical Column Airlift Photobioreactors, Diatoms and CO2 Sequestration

From 2015 to 2021, we optimized mass cultivation of diatoms in our own developed vertical column airlift photobioreactors using natural and artificial light (LEDs). The project took place at the ferrosilicon producer Finnfjord AS in North Norway as a joint venture with UiT—The Arctic University of Norway. Small (0.1–6–14 m3) reactors were used for initial experiments and to produce inoculum cultures while upscaling experiments took place in a 300 m3 reactor. We here argue that species cultivated in reactors should be large since biovolume specific self-shadowing of light can be lower for large vs. small cells. The highest production, 1.28 cm3 L-1 biovolume (0.09–0.31 g DW day-1), was obtained with continuous culture at ca. 19% light utilization efficiency and 34% CO2 uptake. We cultivated 4–6 months without microbial contamination or biofouling, and this we argue was due to a natural antifouling (anti-biofilm) agent in the algae. In terms of protein quality all essential amino acids were present, and the composition and digestibility of the fatty acids were as required for feed ingredients. Lipid content was ca. 20% of ash-free DW with high EPA levels, and omega-3 and amino acid content increased when factory fume was added. The content of heavy metals in algae cultivated with fume was well within the accepted safety limits. Organic pollutants (e.g., dioxins and PCBs) were below the limits required by the European Union food safety regulations, and bioprospecting revealed several promising findings

Mass Cultivation of Microalgae: I. Experiences with Vertical Column Airlift Photobioreactors, Diatoms and CO2 Sequestration

From 2015 to 2021, we optimized mass cultivation of diatoms in our own developed vertical column airlift photobioreactors using natural and artificial light (LEDs). The project took place at the ferrosilicon producer Finnfjord AS in North Norway as a joint venture with UiT—The Arctic University of Norway. Small (0.1–6–14 m3) reactors were used for initial experiments and to produce inoculum cultures while upscaling experiments took place in a 300 m3 reactor. We here argue that species cultivated in reactors should be large since biovolume specific self-shadowing of light can be lower for large vs. small cells. The highest production, 1.28 cm3 L-1 biovolume (0.09–0.31 g DW day-1), was obtained with continuous culture at ca. 19% light utilization efficiency and 34% CO2 uptake. We cultivated 4–6 months without microbial contamination or biofouling, and this we argue was due to a natural antifouling (anti-biofilm) agent in the algae. In terms of protein quality all essential amino acids were present, and the composition and digestibility of the fatty acids were as required for feed ingredients. Lipid content was ca. 20% of ash-free DW with high EPA levels, and omega-3 and amino acid content increased when factory fume was added. The content of heavy metals in algae cultivated with fume was well within the accepted safety limits. Organic pollutants (e.g., dioxins and PCBs) were below the limits required by the European Union food safety regulations, and bioprospecting revealed several promising findings