Mass Cultivation of Microalgae: II. A Large Species Pulsing Blue Light Concept.

If mass cultivation of photoautotrophic microalgae is to gain momentum and find its place in the new "green future", exceptional optimizations to reduce production costs must be implemented. Issues related to illumination should therefore constitute the main focus, since it is the availability of photons in time and space that drives synthesis of biomass. Further, artificial illumination (e.g., LEDs) is needed to transport enough photons into dense algae cultures contained in large photobioreactors. In the present research project, we employed short-term O2 production and 7-day batch cultivation experiments to evaluate the potential to reduce illumination light energy by applying blue flashing light to cultures of large and small diatoms. Our results show that large diatom cells allow more light penetration for growth compared to smaller cells. PAR (400-700 nm) scans yielded twice as much biovolume-specific absorbance for small biovolume (avg. 7070 µm3) than for large biovolume (avg. 18,703 µm3) cells. The dry weight (DW) to biovolume ratio was 17% lower for large than small cells, resulting in a DW specific absorbance that was 1.75 times higher for small cells compared to large cells. Blue 100 Hz square flashing light yielded the same biovolume production as blue linear light in both the O2 production and batch experiments at the same maximum light intensities. We therefore suggest that, in the future, more focus should be placed on researching optical issues in photobioreactors, and that cell size and flashing blue light should be central in this.

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.

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.

Chemical diversity as a function of temperature in six northern diatom species.

In this study, we investigate how metabolic fingerprints are related to temperature. Six common northern temperate diatoms (Attheya longicornis, Chaetoceros socialis, Chaetoceros furcellatus, Porosira glacialis, Skeletonema marinoi, and Thalassiosira gravida) were cultivated at two different temperatures, 0.5 and 8.5 °C. To exclude metabolic variations due to differences in growth rates, the growth rates were kept similar by performing the experiments under light limited conditions but in exponential growth phase. Growth rates and maximum quantum yield of photosynthesis were measured and interpreted as physiological variables, and metabolic fingerprints were acquired by high-resolution mass spectrometry. The chemical diversity varied substantially between the two temperatures for the tested species, ranging from 31% similarity for C. furcellatus and P. glacialis to 81% similarity for A. longicornis. The chemical diversity was generally highest at the lowest temperature.

Isolation and characterisation of a cytotoxic polyunsaturated aldehyde from the marine phytoplankter Phaeocystis pouchetii (Hariot) Lagerheim.

Several investigators have documented that the marine phytoplankter Phaeocystis pouchetii produce and excrete some compound that has adverse effects on its surroundings, but the chemical composition and structure of the active agent has so far been unknown. In the present study we used mass spectrometry to investigate the structural properties of the putative toxin. Colonial cells of P. pouchetii were collected along the coast of northern Norway and cultivated in the lab for a limited period of time prior to harvesting by filtration. Harvested cells and culture filtrate were extracted separately with organic solvents, and a yeast cell bioassay was used to track the toxic fractions during extraction and purification with HPLC. We found the organic extract from the culture filtrate to be toxic, and after purification with RP-HPLC the cytotoxic activity was recovered as one fraction. When the toxic fractions were pooled and analysed by GC-MS we were able to identify 2-trans-4-trans-decadienal by comparing retention time and fragmentation pattern to a commercial standard. This is the first report of a polyunsaturated aldehyde produced by a marine alga belonging to the class Haptophyceae, and this implies that production and release of these reactive compounds are not limited to diatoms.

Anti-mitotic activity towards sea urchin embryos in extracts from the marine haptophycean Phaeocystis pouchetii (Hariot) Lagerheim collected along the coast of northern Norway.

The marine bloom-forming alga Phaeocystis pouchetii is suspected to produce some toxic compound responsible for reduced growth, fecundity and survival of other marine organisms. Sea urchin early development was used as a model to investigate the degree and nature of toxicity. Colonial cells of P. pouchetii were collected during its spring-bloom along the coast of northern Norway and maintained in culture for a short period of time in order to evaluate the concentration of toxic compounds present inside the cells or excreted to the surrounding seawater medium. Cells were harvested by filtration and toxins were extracted separately from the collected cells and the filtrate using organic solvents. We found that extracts from the filtered seawater at a concentration corresponding to 9.0 x 10(5) cells ml(-1) completely blocked cell divisions in embryos of the sea urchin Sphaerechinus granularis, whereas extracts from intact algal cells were only mildly cytotoxic. When the extracts from seawater culture medium were purified by RP-HPLC, cytotoxic activity towards S. granularis embryos was recovered in three consecutive fractions. Moreover, unfertilised eggs incubated in the active HPLC fractions became unproductive, whereas incubation of sperm gave a reduced fertilisation rate. This anti-proliferative effect was further characterized by immunofluorescence staining of sea urchin embryos. DNA labelling revealed that incubating sea urchin embryos in the purified algal extracts inhibited both pronuclei migration and fusion. Incorporation and detection of the DNA-base analogue 5-bromo-2-deoxyuridine showed that DNA-replication was blocked. Furthermore, staining of alpha-tubulin subunits demonstrated that embryonic tubulin organisation was altered. We conclude that P. pouchetii produce some anti-mitotic compound, and that senescent colonial cells to a great extent excrete this compound to their surroundings.