Bioprospecting of marine phytoplankton from large scale cultivation - Effect of culture conditions on bioactivity and biochemistry of the diatom Porosira glacialis

The diatoms make up the largest and most important group of marine microalgae. They are responsible for a quarter of all inorganic carbon fixation in the ocean and are thus of immense importance for global CO2 sequestration. This makes them good candidates for biological carbon capture and utilization (CCU) by mass cultivation using CO2 emissions from industrial point sources. Large scale cultivation of marine diatoms will produce valuable biomass containing marine lipids and proteins with possible commercial application. Commercial production of biomass requires extensive knowledge on the species to be cultivated in terms of optimal cultivation conditions, but also on the bioactivity, cytotoxicity and how the species responds to changing cultivation conditions. Most studies on the bioactivity and biochemistry of marine diatoms have been performed on species cultivated in the laboratory. Cultivation conditions are known to affect the biochemistry of diatoms, and large-scale cultivation will inevitably lead to a change in the environmental conditions. The present study was therefore designed to investigate how Porosira glacialis cultivated in large scale responds to changes in abiotic and biotic cultivation conditions. The first study was designed to investigate whether the direct addition of factory smoke as a source of CO2 would trigger production of bioactive or cytotoxic compounds in P. glacialis. Bioactivity testing revealed activity in anticancer, antibacterial and antibiofilm assays, in addition to cytotoxicity against human lung fibroblasts and effect on the development of sea urchin larvae. Nonetheless, the addition of factory smoke did not increase the cytotoxicity of P. glacialis, nor did it change the beneficial antibacterial and antibiofilm activity. These results are favourable for further development of large-scale production of diatom biomass. P. glacialis was co-cultivated with zooplankton to investigate whether the bioactivity and metabolic expression of the diatom would change when exposed to grazing pressure. The bioactivity testing revealed that the presence of zooplankton increased the cytotoxicity of P. glacialis towards human normal lung fibroblasts. Investigation of active compounds and metabolomic analysis showed that the grazing pressure possibly influence the carotenoid concentration in P. glacialis. It was also found that the bioactivity might be traced to primary metabolites such as chlorophyll derived compounds. The study also showed that the use of OSMAC (one strain many compounds) could be a useful method for further investigation of the bioactivity of diatoms. Investigation of the antibiofilm activity of P. glacialis resulted in isolation of two compounds showing inhibition of biofilm formation by Staphylococcus epidermidis. The compounds isolated were methyl 3-hydroxyoctadecanoate and a pheophorbide-like compound. This is the first time compounds with antibiofilm activity have been isolated from P. glacialis as well as the first evidence of such activity from both the isolated compounds.Kiselalgene utgjør den største og viktigste gruppen av marine mikroalger. De er ansvarlige for en fjerdedel av all uorganisk karbonfiksering i havet og er dermed av enorm betydning for det globale CO2 opptaket. Dette gjør dem til gode kandidater for biologisk karbonfangst og -utnyttelse (CCU) i massedyrking ved bruk av CO2 fra industrielle punktutslipp. Storskala dyrking av marine kiselalger vil produsere verdifull biomasse som inneholder marine lipider og proteiner med mulig kommersiell anvendelse. Kommersiell produksjon av biomasse krever stor kunnskap om arten som skal dyrkes med tanke på best mulige dyrkingsforhold, men også om bioaktivitet, toksisitet og hvordan arten reagerer på endrede dyrkingsforhold. De fleste studier på bioaktivitet og biokjemi av marine kiselalger er utført på arter dyrket i laboratoriet. Det er kjent at dyrkingsforhold påvirker biokjemien til kiselalger, og dyrking i stor skala vil uunngåelig føre til en endring i miljøforholdene. Denne studien ble derfor designet for å undersøke hvordan Porosira glacialis dyrket i stor skala reagerer på endringer i abiotiske og biotiske dyrkingsforhold. Den første studien undersøkte om direkte tilsetning av fabrikkrøyk som kilde til CO2 ville endre produksjon av bioaktive eller toksiske forbindelser i P. glacialis. Bioaktivitetstesting avdekket aktivitet i alle tester: anticancer, antibakteriell og anti-biofilm. I tillegg viste resultatene toksisitet som hemmet utvikling av kråkebolle-larver og humane lungefibroblaster. Likevel økte ikke tilsetting av fabrikkrøyk toksisiteten til P. glacialis, og den hemmet heller ikke den gunstige antibakterielle og anti-biofilm-aktiviteten til algene. Disse resultatene er svært positive for videre utvikling av storskala produksjon av kiselalger. P. glacialis ble dyrket sammen med dyreplankton for å undersøke om bioaktiviteten og utrykket av metabolitter ville endre seg når de ble utsatt for stress i form av beitetrykk. Testing av bioaktivitet avslørte at tilstedeværelsen av zooplankton økte toksisiteten til P. glacialis mot humane normale lungefibroblaster. Undersøkelse av aktive forbindelser og metabolittprofiler viste at beitetrykket muligens påvirker konsentrasjonen av karotenoider i P. glacialis, og at bioaktiviteten muligens kan spores til primære metabolitter som klorofyllderivater. Studien viste også at bruk av OSMAC (one strain many compounds) kunne være en nyttig metode for videre undersøkelse av bioaktiviteten til kiselalger. Undersøkelse av anti-biofilm-aktiviteten til P. glacialis resulterte i isolasjon av to forbindelser som viser inhibering av biofilmdannelse av bakterien Staphylococcus epidermidis. De isolerte forbindelsene var metyl-3-hydroksyoktadekanoat og en pheophorbid-lignende struktur. Dette er første gang en forbindelse med anti-biofilm-aktivitet er blitt isolert fra P. glacialis, og det første beviset på slik aktivitet fra begge de isolerte forbindelsene

Antimikrobielle forbindelser i marine diatomeer (kiselalger)

Bioprospektering er definert som leting etter og karakterisering av nye bioaktive forbindelser fra naturens ressurser med den hensikt å benytte denne kunnskapen kommersielt til utvikling av medisiner, helsekost eller stoffer med annen anvendelse. Blant moderne medisiner er hele 40% basert kunnskap fra naturstoffer, og blant kreftmedisiner og medisiner mot infeksiøse sykdommer er andelen enda høyere. Verdenshavene dekker 70% av jordens overflate, og innehar en enorm biodiversitet som enda er lite utforsket, og derfor antas ressurser herfra å ha et potensiale som opphav til nye bioaktive forbindelser. I denne sammenheng antas de marine diatomeene å være viktige, både fordi de er de mest tallrike av alle mikroalger i det marine økosystemet, men også fordi de utgjør en enorm biodiversitet både i artsmangfold og biokjemi. Dette studiet besto av massedyrking av fire ulike marine diatomeer i monokulturer (Attheya longicornis, Cylindrotheca closterium, Porosira glacialis og Thalassiosira nordenskioeldii), samt en blandingskultur av P. glacialis og A. longicornis. Naturstoffer fra disse artene ble ekstrahert, videre separert ved hjelp av fast-fase ekstraksjon (SPE) og testet for antimikrobiell aktivitet mot fire ulike bakteriestammer; Eschericia coli, Pseudomonas aeruginosa, Staphylococcus Aureus og Corynebacterium glutamicum. Det ble registret antimikrobiell aktivitet i SPE-eluater fra alle de ulike artene dyrket i renkultur, samt i blandingskulturen. Noen av eluatene viste antimikrobiell effekt mot alle de fire bakteriestammene. Aktive SPE-eluater ble fraksjonert ved hjelp av høytrykks-væskekromatografi (HPLC), og analysert med massespektrometri LC-MS. Det ble oppdaget antimikrobielt riboflavin og flavin mononukleotid i 30% SPE-eluatene av P. glacialis, T. nordenskioeldii og blandingskulturen. Det ble i tillegg funnet et antimikrobielt protein med molekylvekt på ca. 11 kDa i 40% SPE-eluatet av T. nordenskioeldii. Én fraksjon fra 30% SPE-eluatet av T. nordenskioeldii inneholdt en lavmolekylær forbindelse som ble karakterisert til å være tryptofan eller tryptofan-lignende derivater

Bioactivity of a Marine Diatom (Porosira glacialis [Grunow] Jorgensen 1905) Cultivated With and Without Factory Smoke CO2

Using industrial emissions as a strategy for CO2 sequestration through carbon capture and utilization (CCU) in cultivation of microalgae can potentially change cultivation factors such as pH, nutrient availability and presence of trace metals, which could alter the growth and metabolism of the microalgae. It is therefore important to investigate whether such changes in culturing conditions can lead to changes in the diatoms metabolism, such as production of unwanted toxic compounds or by reduction of the diatoms' natural ability to control the growth of competing microorganisms (e.g., by decreasing the production of antibacterial compounds). The cold-water marine diatom Porosira glacialis was cultivated in two, 6,000-L photobioreactors in an industrial setting; one culture had the direct addition of factory smoke, and to the other fresh air was added. The biomass was extracted and screened for toxicity in viability assays against human cells (cancer and normal lung fibroblasts) and development of sea urchin larvae (Paracentrotus lividus). Bioactivity was tested in two bacterial assays: growth inhibition assay and anti-biofilm assay. The results confirm earlier reports on the presence of toxic compounds against human cell lines and P. lividus larvae, but no elevated toxicity could be detected using factory smoke. Anti-biofilm activity was present in both cultures. This indicates that the natural toxic properties of the microalgae do not increase by adding factory smoke, and that we keep the beneficial ability of the microalga to suppress growth of bacteria. These are key elements in a successful, industrial-scale cultivation as the product is safe and at the same time the monocultures are not being contaminated by competing organisms

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

Bacterial diversity of aMasi, a South African fermented milk product, determined by clone library and denaturing gradient gel electrophoresis analysis

In the present study, we investigated the bacterial diversity of aMasi, a traditional South African fermented milk product, by 16S rRNA clone library and Denaturing Gradient Gel Electrophoresis (DGGE) analysis. Two hundred and eighty two clones from clone library were isolated and identified from aMasi, prepared from the milk of four cows from one herd in the EkuPindiseni Community, North West of Hluhluwe-iMfolozi Park in KwaZulu-Natal Province. The majority of the identified sequences corresponded to lactic acid bacteria (LAB), with the genus Lactococcus as major representative. The species Lactococcus lactis accounted for 179 of the identified clones. In addition, several species of Lactobacillus, Leuconostoc and Enterococcus were detected. Furthermore, several clones belonging to Acinetobacter, Aeromonas and genera within the Enterobacteriaceae were detected. It is important to note that human pathogens such as Klebsiella pneumoniae were identified in aMasi in the present study. Conversely, zoonotic bacteria such as Brucella abortus and Mycobacterium bovis were not detected in aMasi, although, they are present in the cattle population in the study area. Thirty (30) clones were identified as uncultured bacterial clones. Nine DGGE bands were successfully sequenced, of which four were classified as L. lactis with other bands belonging to lactobacilli, Clostridium acidurici, Enterobacter sp., Acinetobacter baumannii and an un-culturable bacterium. Even though there was some discrepancy between the two culture independent methods used to study the bacteriological community in aMasi, a general conclusion can be drawn, L. lactis may be considered as the dominant bacterium within a diverse bacterial community in this locally-produced dairy product

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