WO2022069725A1 - Process for cultivation of diatoms - Google Patents

Process for cultivation of diatoms Download PDF

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Publication number
WO2022069725A1
WO2022069725A1 PCT/EP2021/077142 EP2021077142W WO2022069725A1 WO 2022069725 A1 WO2022069725 A1 WO 2022069725A1 EP 2021077142 W EP2021077142 W EP 2021077142W WO 2022069725 A1 WO2022069725 A1 WO 2022069725A1
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WIPO (PCT)
Prior art keywords
diatoms
microsilica
aqueous composition
range
present
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PCT/EP2021/077142
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French (fr)
Inventor
Hans Christian EILERTSEN
Gunilla Kristina ERIKSEN
Richard Andre INGEBRIGTSEN
Jo STRØMHOLT
John-Steinar BERGUM
Original Assignee
Finnfjord As
Universitetet I Tromsø - Norges Arktiske Universitet
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Publication of WO2022069725A1 publication Critical patent/WO2022069725A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/12—Unicellular algae; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C01—INORGANIC CHEMISTRY
    • C01B—NON-METALLIC ELEMENTS; COMPOUNDS THEREOF; METALLOIDS OR COMPOUNDS THEREOF NOT COVERED BY SUBCLASS C01C
    • C01B32/00—Carbon; Compounds thereof
    • C01B32/90—Carbides
    • C01B32/914—Carbides of single elements
    • C01B32/956—Silicon carbide
    • C—CHEMISTRY; METALLURGY
    • C01—INORGANIC CHEMISTRY
    • C01B—NON-METALLIC ELEMENTS; COMPOUNDS THEREOF; METALLOIDS OR COMPOUNDS THEREOF NOT COVERED BY SUBCLASS C01C
    • C01B32/00—Carbon; Compounds thereof
    • C01B32/90—Carbides
    • C01B32/914—Carbides of single elements
    • C01B32/991—Boron carbide
    • C—CHEMISTRY; METALLURGY
    • C01—INORGANIC CHEMISTRY
    • C01B—NON-METALLIC ELEMENTS; COMPOUNDS THEREOF; METALLOIDS OR COMPOUNDS THEREOF NOT COVERED BY SUBCLASS C01C
    • C01B33/00—Silicon; Compounds thereof
    • C—CHEMISTRY; METALLURGY
    • C01—INORGANIC CHEMISTRY
    • C01B—NON-METALLIC ELEMENTS; COMPOUNDS THEREOF; METALLOIDS OR COMPOUNDS THEREOF NOT COVERED BY SUBCLASS C01C
    • C01B33/00—Silicon; Compounds thereof
    • C01B33/113—Silicon oxides; Hydrates thereof
    • C—CHEMISTRY; METALLURGY
    • C01—INORGANIC CHEMISTRY
    • C01B—NON-METALLIC ELEMENTS; COMPOUNDS THEREOF; METALLOIDS OR COMPOUNDS THEREOF NOT COVERED BY SUBCLASS C01C
    • C01B33/00—Silicon; Compounds thereof
    • C01B33/20—Silicates
    • C01B33/36—Silicates having base-exchange properties but not having molecular sieve properties
    • C01B33/46—Amorphous silicates, e.g. so-called "amorphous zeolites"
    • C—CHEMISTRY; METALLURGY
    • C01—INORGANIC CHEMISTRY
    • C01B—NON-METALLIC ELEMENTS; COMPOUNDS THEREOF; METALLOIDS OR COMPOUNDS THEREOF NOT COVERED BY SUBCLASS C01C
    • C01B35/00—Boron; Compounds thereof
    • C01B35/02—Boron; Borides
    • C01B35/04—Metal borides

Definitions

  • the present invention relates generally to algaculture, and in particular to cultivation of diatoms.
  • the present invention provides a growth medium for cultivation of diatoms in large volume vertical column bioreactors. Further, the invention also provides use of the growth medium for cultivation of diatoms as well as methods for cultivation of diatoms.
  • omega-3 fatty acids are associated with beneficial health effects in human nutrition.
  • Fat fish, including salmonids are good sources for the essential omega-3 long-chain polyunsaturated fatty acids EP A and DHA. Due to extensive fish farming the global human consumption of salmonids has increased substantially the last decades, where Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss) are the dominant species.
  • Algae is a large diverse group of photosynthetic eukaryotic organisms that are not necessarily closely related, and is thus polyphyletic. Algae includes organisms ranging from unicellular microalgae genera, such as Chlorella species and diatoms, to multicellular forms, such as the giant kelp, a large brown alga which may grow up to 50 m in length.
  • Diatoms are rich in lipids, and these algae, if cultivated economically, are therefore of particular interest for solving the need for high omega-3 formulated fish feed in the fish farming industry.
  • Diatoms are unicellular microalgae enclosed within a silica cell wall. The diatoms are microalgae with a size ranging from about 2 gm to about 500 gm (0.5 mm). They are living in the worlds’ oceans but are also found in fresh water. More than 8000 species of diatoms are recorded worldwide, and it is estimated that there are 20.000-200.000 extant diatoms species in the world.
  • Diatoms belongs to the group of photosynthesizing algae and it has been estimated that diatoms account for as much as 25 % of global photosynthetic fixation of carbon.
  • the major carbon storage compound in diatoms is lipids, among which triglycerides and fatty acids can make up 15-25% of the dry biomass.
  • the diatoms have also been demonstrated to be very effective in converting inorganic carbon to organic compounds.
  • the production of diatom biomass can aid to reduce local carbon footprints.
  • cultivation of diatoms represents a sustainable and environmentally friendly way of meeting the demand for omega-3 formulated fish feed in the fish farming industry.
  • the present inventors have been able to provide sustainable and cost-effective methodologies for cultivation of diatoms in large volume vertical column bioreactors by including a specific form of silica in the algae growth medium.
  • This specific form of silica has been demonstrated to result in an increased growth rate, and it has also been discovered that the increased growth rate may be increased even further by introducing factory smoke from the production of silicon and/or ferrosilicon into the growth medium.
  • factory smoke improves growth of the algae but also reduces the carbon footprint of the silicon and/or ferrosilicon factory; a win-win situation.
  • W02004048553 discloses a liquid for culturing diatom comprising nitrogen, silicon and phosphorus with a specific silicon : nitrogen ratio.
  • KR101888798 discloses a nano-silica solution for growing diatoms, the solution being prepared by including nano-silica, distilled water, iron chloride, ferric chloride, manganese chloride, zinc chloride, cobalt chloride, and copper chloride.
  • W02005121313 discloses a composition for a copious bloom of diatom algae comprising macro and or micronutrients adsorbed on metalate modified silica sol.
  • WO91 14427 discloses a method for producing single cell edible oils containing EPA from heterotrophic diatoms by cultivating diatoms in a nutrient solution containing nitrogen and silicate.
  • WO201 5041531 discloses culturing diatoms under non-limiting bioavailable silicon concentrations wherein the diatoms are subjected to a cycle of alternating dark phases and light phases and providing limitation of availability of at least one essential growth nutrient in one or more of said light phases.
  • WO201 5016720 relates to methods of producing diatom biomass using a continuous culture to produce a volumetric production rate of biomass of at least 0,2g dry weight/L/day, wherein the culture medium is designed to provide the essential nutrients to maintain the diatom in log phase growth.
  • Bioresource technology vol. 314, 2020, 123747 reports a novel solution-based method to trigger the growth of diatoms for enhanced biomass production, including use of inductively coupled plasma (ICP) synthesized nanosilika.
  • ICP inductively coupled plasma
  • a first aspect of the present invention relates to a method of preparing an aqueous composition for cultivation of diatoms, the method comprising the following step(s):
  • the amorphous SiCh is added in the form of microsilica.
  • the diatoms are marine diatoms.
  • the diatoms are photoautotrophic diatoms.
  • the diatoms are centric diatoms.
  • the mean diameter of the diatoms is > 25 pm.
  • the diatoms are a diatom monoculture.
  • the diatoms are a culture of diatoms wherein the majority of the diatoms are of the same species. In one embodiment more than 80% of the diatoms are of the same species, such as more than 85%, more than 90%, more than 95%, more than 96%, more than 97%, more than 98% or more than 99% of the diatoms are of the same species.
  • the diatoms are a culture of diatoms wherein a substantial number of the diatoms is of the same species.
  • the aqueous liquid is water, such as freshwater or seawater.
  • the aqueous liquid has a salt content in the range 20 to 50 g/L, preferably 20 to 45 g/L, more preferably a salt content in the range 25 to 45 g/L, even more preferably in the range 25 to 40 g/L and most preferably a salt content in the range 25 to 35 g/L, such as about 30 g/L.
  • the microsilica comprises > 70 % by weight amorphous silicon dioxide, preferably > 80 % by weight amorphous silicon dioxide, even more preferably > 85 % by weight amorphous silicon dioxide and most preferably > 90 % by weight amorphous silicon dioxide.
  • the microsilica is dissolved in the aqueous liquid and optionally any non-dissolved microsilica is removed from the aqueous liquid. In one embodiment, the microsilica is dissolved in the aqueous liquid and any nondissolved microsilica is removed from the aqueous liquid.
  • the microsilica is dispersed in the aqueous liquid.
  • the microsilica is a byproduct from the production of silicon and/or ferrosilicon.
  • the microsilica comprises a plurality of spherical microsilica particles.
  • the microsilica comprises a plurality of microsilica particles, wherein at least 10 % of particle volume is more than 100 nm. In another embodiment at least 20 % of particle volume is more than 100 nm. In another embodiment at least 30 % of particle volume is more than 100 nm. In another embodiment at least 40 % of particle volume is more than 100 nm. In another embodiment at least 50 % of particle volume is more than 100 nm. In another embodiment at least 55 % of particle volume is more than 100 nm. In yet another embodiment at least 60 % of particle volume is more than 100 nm. In yet another embodiment at least 65 % of particle volume is more than 100 nm.
  • At least 70 % of particle volume is more than 100 nm. In yet another embodiment at least 75 % of particle volume is more than 100 nm. In yet another embodiment at least 80 % of particle volume is more than 100 nm. In yet another embodiment at least 85 % of particle volume is more than 100 nm. In yet another embodiment at least 90 % of particle volume is more than 100 nm.
  • 55-95 % of particle volume is more than 100 nm, such as i) 60-90 % of particle volume is more than 100 nm, ii) 65-85 % of particle volume is more than 100 nm, iii) 70-80 % of particle volume is more than 100 nm or iv) 75% of particle volume is more than 100 nm.
  • the microsilica comprises a plurality of microsilica particles, wherein at least 20 % of particle volume is more than 150 nm. In another embodiment at least 25 % of particle volume is more than 150 nm. In another embodiment at least 30 % of particle volume more than 150 nm. In another embodiment at least 35 % of particle volume more than 150 nm. In another embodiment at least 40 % of particle volume more than 150 nm. In another embodiment at least 45 % of particle volume more than 150 nm. In yet another embodiment at least 50 % of particle volume more than 150 nm. In yet another embodiment at least 55 % of particle volume more than 150 nm. In yet another embodiment at least 60 % of particle volume more than 150 nm.
  • the microsilica comprises a plurality of microsilica particles, wherein 25-75 % of particle volume is less than 150 nm, such as i) 30-70 % of particle volume is less than 150 nm, ii) 35- 65 % of particle volume is less than 150 nm, iii) 40-60 % of particle volume is less than 150 nm or iv) 50% of particle volume is less than 150 nm.
  • the microsilica comprises a plurality of microsilica particles, wherein 5-45 % of particle volume is less than 100 nm, such as i) 10-40 % of particle volume is less than 100 nm, ii) 15- 35 % of particle volume is less than 100 nm, iii) 20-30 % of particle volume is less than 100 nm or iv) 25 % of particle volume is less than 100 nm.
  • the microsilica comprises a plurality of microsilica particles; the microsilica particles having an average particle diameter in the range 50-500 nm, more preferably in the range 50- 300 nm, even more preferably in the range 50-200 nm and most preferably in the range 100-200 nm.
  • the microsilica comprises a plurality of microsilica particles; the microsilica particles having an average particle diameter of more than 100 nm, such as more than 120 nm, more than 150 nm, more than 170 nm or more than 200 nm.
  • the microsilica has a bulk density in the range 70 to 800 kg/m 3 , such as in the range 200 to 800 kg/m 3 , in the range 300 to 800 kg/m 3 or in the range 400 to 700 kg/m 3 .
  • the microsilica has a bulk density in the range 400 to 800 kg/m 3 , such as 450 to 750 kg/m 3 , 500 to 700 kg/m 3 or 550 to 650 kg/m 3 .
  • the microsilica has a bulk density in the range 100 to 450 kg/m 3 , such as 150 to 400 kg/m 3 or 200 to 350 kg/m 3 .
  • the microsilica has a specific gravity in the range 1.5 to 3.0, more preferably in the range 2.0 to 2.5 and even more preferably in the range 2.2 to 2.3.
  • the microsilica has a specific surface area in the range 10 to 50 m 2 /g, more preferably in the range 12 to 40 m 2 /g and even more preferably in the range 15 to 30 m 2 /g.
  • the microsilica has a melting point in the range 1500 to 1600 °C, more preferably in the range 1540 to 1580 °C and even more preferably in the range 1550 to 1570 °C.
  • the microsilica comprises an iron source.
  • the microsilica comprises an iron source, the iron source being one or more iron oxide(s).
  • the one or more iron oxide(s) is Fe2O3.
  • the microsilica comprises 0.5 to 10 % iron oxide(s) by weight of the microsilica, preferably 0.5 to 5 % iron oxide(s) by weight of the microsilica, more preferably 1 to 5 % iron oxide(s) by weight of the microsilica and even more preferably 1 to 3 % iron oxide(s) by weight of the microsilica.
  • the iron oxide(s) is Fe2O3.
  • all nutrients essential to maintain growth of the diatoms are added to the aqueous liquid.
  • all nutrients essential to maintain the diatoms in exponential growth phase are added to the aqueous liquid.
  • the salt content of the aqueous composition is adjusted to a salt content in the range 20 to 50 g/L, preferably in the range 20 to 45 g/L, more preferably a salt content in the range 25 to 45 g/L, even more preferably in the range 25 to 40 g/L and most preferably a salt content in the range 25 to 35 g/L, such as a salt content of about 30 g/L.
  • the pH of the aqueous composition is adjusted to a pH in the range 5.5 to 9.0, preferably a pH in the range 6.0 to 9.0, more preferably a pH in the range 7 to 9 and even more preferably in the range 7 to 8.
  • At least 0,1 mg/L amorphous SiCL is added to the aqueous liquid to obtain the aqueous composition.
  • at least 0,14 mg/L amorphous SiCh is added to the aqueous liquid to obtain the aqueous composition.
  • at least 0,17 mg/L amorphous SiCh is added to the aqueous liquid to obtain the aqueous composition.
  • CO2(g) is introduced into the aqueous liquid; and/or NCL', NO2- or any mixture thereof is introduced into the aqueous liquid.
  • a phosphorus source such as fertilizer, is added to the aqueous liquid.
  • Substral® provided by Scotts Company A/S (see example 1, table 2) and YaraTera Kristalon® Purple provided by Yara are two examples of fertilizers suitable to be added to the aqueous composition.
  • factory smoke from production of silicon and/or ferrosilicon is introduced into the aqueous liquid.
  • the pH of the aqueous composition should preferably be >7, more preferably >7.5 and most preferably >7.8.
  • the pH of the aqueous composition is ⁇ 7, such as ⁇ 6.9 or ⁇ 6.8 it is preferred not to introduce any further factory smoke from production of silicon and/or ferrosilicon.
  • a second aspect of the present invention relates to an aqueous composition for cultivation of diatoms prepared by the method according to the first aspect of the present invention.
  • a third aspect of the present invention relates to an aqueous composition for cultivation of diatoms comprising an aqueous liquid and SiCh provided in the form of microsilica, wherein
  • the amount of SiCh provided in the form of microsilica in the aqueous composition is at least 0,07 mg/L.
  • the diatoms are marine diatoms.
  • the diatoms are photoautotrophic diatoms.
  • the diatoms are centric diatoms.
  • the mean diameter of the diatoms is > 25 pm.
  • the diatoms are a diatom monoculture.
  • the diatoms are a culture of diatoms wherein the majority of the diatoms are of the same species. In one embodiment more than 80% of the diatoms are of the same species, such as more than 85%, more than 90%, more than 95%, more than 96%, more than 97%, more than 98% or more than 99% of the diatoms are of the same species.
  • the diatoms are a culture of diatoms wherein a substantial number of the diatoms is of the same species.
  • the aqueous liquid is water, such as freshwater or seawater.
  • the aqueous liquid is water and has a salt content in the range 20 to 50 g/L, preferably 20 to 45 g/L, more preferably a salt content in the range 25 to 45 g/L, even more preferably in the range 25 to 40 g/L and most preferably a salt content in the range 25 to 35 g/L, such as about 30 g/L.
  • a salt content in the range 20 to 50 g/L, preferably 20 to 45 g/L, more preferably a salt content in the range 25 to 45 g/L, even more preferably in the range 25 to 40 g/L and most preferably a salt content in the range 25 to 35 g/L, such as about 30 g/L.
  • the microsilica comprises > 70 % by weight amorphous silicon dioxide, preferably > 80 % by weight amorphous silicon dioxide, even more preferably > 85 % by weight amorphous silicon dioxide and most preferably > 90 % by weight amorphous silicon dioxide.
  • the microsilica is dissolved in the aqueous liquid and optionally any non-dissolved microsilica is removed from the aqueous liquid. In one embodiment, the microsilica is dissolved in the aqueous liquid and any nondissolved microsilica is removed from the aqueous liquid.
  • the microsilica is dispersed in the aqueous liquid.
  • the microsilica is a byproduct from the production of silicon and/or ferrosilicon.
  • the microsilica comprises a plurality of spherical microsilica particles.
  • the microsilica comprises a plurality of microsilica particles, wherein at least 10 % of particle volume is more than 100 nm. In another embodiment at least 20 % of particle volume is more than 100 nm. In another embodiment at least 30 % of particle volume is more than 100 nm. In another embodiment at least 40 % of particle volume is more than 100 nm. In another embodiment at least 50 % of particle volume is more than 100 nm. In another embodiment at least 55 % of particle volume is more than 100 nm. In yet another embodiment at least 60 % of particle volume is more than 100 nm. In yet another embodiment at least 65 % of particle volume is more than 100 nm.
  • At least 70 % of particle volume is more than 100 nm. In yet another embodiment at least 75 % of particle volume is more than 100 nm. In yet another embodiment at least 80 % of particle volume is more than 100 nm. In yet another embodiment at least 85 % of particle volume is more than 100 nm. In yet another embodiment at least 90 % of particle volume is more than 100 nm.
  • the microsilica comprises a plurality of microsilica particles, wherein at least 20 % of particle volume is more than 150 nm. In another embodiment at least 25 % of particle volume is more than 150 nm. In another embodiment at least 30 % of particle volume is more than 150 nm.
  • At least 35 % of particle volume is more than 150 nm. In another embodiment at least 40 % of particle volume is more than 150 nm. In another embodiment at least 45 % of particle volume is more than 150 nm. In yet another embodiment at least 50 % of particle volume is more than 150 nm. In yet another embodiment at least 55 % of particle volume is more than 150 nm. In yet another embodiment at least 60 % of particle volume is more than 150 nm. In yet another embodiment at least 65 % of particle volume is more than 150 nm. In yet another embodiment at least 70 % of particle volume is more than 150 nm. In yet another embodiment at least 75 % of particle volume is more than 150 nm.
  • At least 80 % of particle volume is more than 150 nm. It is preferred that 25-75 % of particle volume is more than 150 nm, such as i) 30-70 % of particle volume is more than 150 nm, ii) 35-65 % of particle volume is more than 150 nm, iii) 40-60 % of particle volume is more than 150 nm or iv) 50% of particle volume is more than 150 nm.
  • the microsilica comprises a plurality of microsilica particles, wherein 25-75 % of particle volume is less than 150 nm, such as i) 30-70 % of particle volume is less than 150 nm, ii) 35- 65 % of particle volume is less than 150 nm, iii) 40-60 % of particle volume is less than 150 nm or iv) 50% of particle volume is less than 150 nm.
  • the microsilica comprises a plurality of microsilica particles, wherein 5-45 % of particle volume is less than 100 nm, such as i) 10-40 % of particle volume is less than 100 nm, ii) 15- 35 % of particle volume is less than 100 nm, iii) 20-30 % of particle volume is less than 100 nm or iv) 25 % of particle volume is less than 100 nm.
  • the microsilica comprises a plurality of microsilica particles; the microsilica particles having an average particle diameter in the range 50-500 nm, more preferably in the range 50- 300 nm, even more preferably in the range 50-200 nm and most preferably in the range 100-200 nm.
  • the microsilica comprises a plurality of microsilica particles; the microsilica particles having an average particle diameter of more than 100 nm, such as more than 120 nm, more than 150 nm, more than 170 nm or more than 200 nm.
  • the microsilica has a bulk density in the range 70 to 800 kg/m 3 , such as in the range 200 to 800 kg/m 3 , in the range 300 to 800 kg/m 3 or in the range 400 to 700 kg/m 3 .
  • the microsilica has a bulk density in the range 400 to 800 kg/m 3 , such as 450 to 750 kg/m 3 , 500 to 700 kg/m 3 or 550 to 650 kg/m 3 .
  • the microsilica has a bulk density in the range 100 to 450 kg/m 3 , such as 150 to 400 kg/m 3 or 200 to 350 kg/m 3 .
  • the microsilica has a specific gravity in the range 1.5 to 3.0, more preferably in the range 2.0 to 2.5 and even more preferably in the range 2.2 to 2.3.
  • the microsilica has a specific surface area in the range 10 to 50 m 2 /g, more preferably in the range 12 to 40 m 2 /g and even more preferably in the range 15 to 30 m 2 /g.
  • the microsilica has a melting point in the range 1500 to 1600 °C, more preferably in the range 1540 to 1580 °C and even more preferably in the range 1550 to 1570 °C.
  • the microsilica comprises an iron source.
  • the microsilica comprises an iron source, the iron source being one or more iron oxide(s).
  • the one or more iron oxide(s) is Fe2O3.
  • the microsilica comprises 0.5 to 10 % iron oxide(s) by weight of the microsilica, preferably 0.5 to 5 % iron oxide(s) by weight of the microsilica, more preferably 1 to 5 % iron oxide(s) by weight of the microsilica and even more preferably 1 to 3 % iron oxide(s) by weight of the microsilica.
  • the iron oxide(s) is Fe2O3.
  • the aqueous composition comprises all nutrients essential to maintain growth of the diatoms. In one preferred embodiment according to the present invention the aqueous composition comprises all nutrients essential to maintain the diatoms in exponential growth.
  • the salt content of the aqueous composition is in the range 20 to 50 g/L, preferably in the range 20 to 45 g/L, more preferably a salt content in the range 25 to 45 g/L, even more preferably in the range 25 to 40 g/L and most preferably a salt content in the range 25 to 35 g/L, such as a salt content of about 30 g/L.
  • the pH of the aqueous composition is in the range 5.5 to 9.0, preferably in the range 6.0 to 9.0, more preferably in the range 7 to 9 and even more preferably in the range 7 to 8.
  • the amount of SiCh provided in the form of microsilica in the aqueous composition is at least 0,1 mg/L. In one embodiment according to the present invention, the amount of SiCh provided in the form of microsilica in the aqueous composition is at least 0,14 mg/L. In one embodiment according to the present invention, the amount of SiCh provided in the form of microsilica in the aqueous composition is at least 0,17 mg/L.
  • the aqueous composition comprises a carbon source and/or a nitrogen source, the carbon source being obtained by introducing CO2(g) into the aqueous composition and the nitrogen source being obtained by introducing NCL', NO2- or any mixture thereof into the aqueous composition.
  • the aqueous composition comprises a phosphorus source, such as fertilizer.
  • Substral® provided by Scotts Company A/S see example 1, table 2)
  • YaraTera Kristalon® Purple provided by Yara are two examples of fertilizers suitable to be added to the aqueous composition.
  • factory smoke from production of silicon and/or ferrosilicon is introduced into the aqueous composition.
  • the pH of the aqueous composition should preferably be >7, more preferably >7.5 and most preferably >7.8.
  • the pH of the aqueous composition is ⁇ 7, such as ⁇ 6.9 or ⁇ 6.8 it is preferred not to introduce any further factory smoke from production of silicon and/or ferrosilicon.
  • a fourth aspect of the present invention relates to a use of the aqueous composition according to the second or third aspect of the present invention for cultivation of diatoms.
  • the diatoms are marine diatoms.
  • the diatoms are photoautotrophic diatoms.
  • the diatoms are centric diatoms.
  • the mean diameter of the diatoms is > 25 pm.
  • the diatoms are a diatom monoculture.
  • the diatoms are a culture of diatoms wherein the majority of the diatoms are of the same species. In one embodiment more than 80% of the diatoms are of the same species, such as more than 85%, more than 90%, more than 95%, more than 96%, more than 97%, more than 98% or more than 99% of the diatoms are of the same species.
  • the diatoms are a culture of diatoms wherein a substantial number of the diatoms is of the same species.
  • a fifth aspect of the present invention relates to a method of producing a diatom biomass, the method comprising a step of culturing diatoms in the aqueous composition according to the second or third aspect of the present invention.
  • the diatoms are marine diatoms.
  • the diatoms are photoautotrophic diatoms.
  • the diatoms are centric diatoms.
  • the mean diameter of the diatoms is > 25 pm.
  • the diatoms are a diatom monoculture.
  • the diatoms are a culture of diatoms wherein the majority of the diatoms are of the same species. In one embodiment more than 80% of the diatoms are of the same species, such as more than 85%, more than 90%, more than 95%, more than 96%, more than 97%, more than 98% or more than 99% of the diatoms are of the same species.
  • the diatoms are a culture of diatoms wherein a substantial number of the diatoms is of the same species.
  • a first alternative aspect of the present invention relates to an aqueous composition for cultivation of diatoms, the aqueous composition comprising an aqueous liquid and microsilica.
  • the diatoms are marine diatoms.
  • the diatoms are photoautotrophic diatoms.
  • the diatoms are centric diatoms.
  • the mean diameter of the diatoms is > 25 pm.
  • the diatoms are a diatom monoculture.
  • the diatoms are a culture of diatoms wherein the majority of the diatoms are of the same species. In one embodiment more than 80% of the diatoms are of the same species, such as more than 85%, more than 90%, more than 95%, more than 96%, more than 97%, more than 98% or more than 99% of the diatoms are of the same species. In yet a further embodiment according to the present invention the diatoms are a culture of diatoms wherein a substantial number of the diatoms is of the same species.
  • the aqueous liquid is water, such as freshwater or seawater.
  • the aqueous liquid has a salt content in the range 20 to 50 g/L, preferably 20 to 45 g/L, more preferably a salt content in the range 25 to 45 g/L, even more preferably in the range 25 to 40 g/L and most preferably a salt content in the range 25 to 35 g/L, such as about 30 g/L.
  • the microsilica is amorphous silicon dioxide.
  • the microsilica contains > 60 % by weight amorphous silicon dioxide, > 70 % by weight amorphous silicon dioxide, preferably > 80 % by weight amorphous silicon dioxide, even more preferably > 85 % by weight amorphous silicon dioxide and most preferably > 90 % by weight amorphous silicon dioxide.
  • the microsilica is dissolved in the aqueous liquid and optionally any non-dissolved microsilica is removed from the aqueous liquid. In one embodiment, the microsilica is dissolved in the aqueous liquid and any nondissolved microsilica is removed from the aqueous liquid.
  • the microsilica is dispersed in the aqueous liquid.
  • the microsilica is a byproduct from the production of silicon and/or ferrosilicon.
  • the microsilica comprises a plurality of spherical microsilica particles.
  • the microsilica comprises a plurality of microsilica particles; and at least 80% of the microsilica particles have a diameter less than 1 pm, more preferably at least 90% of the microsilica particles have a diameter less than 1 pm and even more preferably all the microsilica particles have a diameter less than 1 pm.
  • the microsilica comprises a plurality of microsilica particles; and the microsilica particles have an average particle diameter in the range 50-500 nm, more preferably in the range 50- 300 nm, even more preferably in the range 50-200 nm and most preferably in the range 100-200 nm.
  • the microsilica comprises an iron source.
  • the microsilica comprises an iron source, the iron source being Fe2O3.
  • the microsilica comprises 0.5 to 10 % of one or more iron oxides(s) by weight of the microsilica, preferably 0.5 to 5 % of one or more iron oxides(s) by weight of the microsilica, more preferably 1 to 5 % of one or more iron oxide(s) by weight of the microsilica and even more preferably 1 to 3 % of one or more iron oxide(s) by weight of the microsilica.
  • the one or more iron oxides is Fe2O3.
  • the microsilica comprises 0.5 to 10 % Fe2C>3 by weight of the microsilica, preferably 0.5 to 5 % Fe2C>3 by weight of the microsilica, more preferably 1 to 5 % Fe2C>3 by weight of the microsilica and even more preferably 1 to 3 % Fe2C>3 by weight of the microsilica.
  • the microsilica has a bulk density in the range 70 to 800 kg/m 3 , preferably in the range 200 to 800 kg/m 3 , even more preferably in the range 300 to 800 kg/m 3 and most preferably in the range 400 to 700 kg/m 3 .
  • the microsilica has a specific gravity in the range 1.5 to 3.0, more preferably in the range 2.0 to 2.5 and even more preferably in the range 2.2 to 2.3.
  • the microsilica has a specific surface area in the range 10 to 50 m 2 /g, more preferably in the range 12 to 40 m 2 /g and even more preferably in the range 15 to 30 m 2 /g.
  • the microsilica has a melting point in the range 1500 to 1600 °C, more preferably in the range 1540 to 1580 °C and even more preferably in the range 1550 to 1570 °C.
  • the aqueous composition has a salt content in the range 20 to 50 g/L, preferably in the range 20 to 45 g/L, more preferably a salt content in the range 25 to 45 g/L, even more preferably in the range 25 to 40 g/L and most preferably a salt content in the range 25 to 35 g/L, such as a salt content of about 30 g/L.
  • the aqueous composition has a pH in the range 5.5 to 9.0, preferably a pH in the range 6.0 to 9.0, more preferably a pH in the range 7 to 9 and even more preferably in the range 7 to 8.
  • the amount of microsilica in the aqueous composition is > 0.00175 mg/L.
  • the amount of microsilica in the aqueous composition is at least 0.002625 mg/L; more preferably at least 0.0035 mg/L; even more preferably at least 0,005 mg/L and most preferably at least 0.006 mg/L; such as at least 0.01 mg/L, at least 0.015 mg/L, at least 0.02 mg/L, at least 0.025 mg/L, at least 0.03 mg/L, at least 0.035 mg/L, at least 0.04 mg/L, at least 0.045 mg/L, at least 0.05 mg/L, at least 0.06 mg/L, at least 0.07 mg/L, at least 0.08 mg/L, at least 0.09 mg/L, at least 0.1 mg/L, at least 0.15 mg/L, at least 0.2 mg/L, at least 0.25 mg/L, at least 0.3 mg/L, at least 0.35 mg/L,
  • the amount of microsilica in the aqueous composition is at least 0.1 mg/L, at least 0.15 mg/L, at least 0.2 mg/L, at least 0.25 mg/L, at least 0.3 mg/L, at least 0.35 mg/L, at least 0.4 mg/L, at least 0.45 mg/L, at least 0.50 mg/L, at least 0.6 mg/L, at least 0.7 mg/L, at least 0.8 mg/L, at least 0.9 mg/L, at least 1 mg/L, at least 5 mg/L or at least 10 mg/L.
  • the amount of microsilica in the aqueous composition is in the range 0.1 mg/L to 100 mg/L, such as 0.1 mg/L to 80 mg/L, 0.1 mg/L to 60 mg/L, 0.1 mg/L to 40 mg/L or 0.1 mg/L to 20 mg/L.
  • the amount of microsilica in the aqueous composition is in the range 0.2 mg/L to 100 mg/L, such as 0.2 mg/L to 80 mg/L, 0.2 mg/L to 60 mg/L, 0.2 mg/L to 40 mg/L or 0.2 mg/L to 20 mg/L.
  • the amount of microsilica in the aqueous composition is in the range 0.5 mg/L to 100 mg/L, such as 0.5 mg/L to 80 mg/L, 0.5 mg/L to 60 mg/L, 0.5 mg/L to 40 mg/L or 0.5 mg/L to 20 mg/L.
  • the amount of microsilica in the aqueous composition is in the range 1 mg/L to 100 mg/L, such as 1 mg/L to 80 mg/L, 1 mg/L to 60 mg/L, 1 mg/L to 40 mg/L or 1 mg/L to 20 mg/L.
  • the amount of microsilica in the aqueous composition is in the range 5 mg/L to 100 mg/L, such as 5 mg/L to 80 mg/L, 5 mg/L to 60 mg/L, 5 mg/L to 40 mg/L or 5 mg/L to 20 mg/L.
  • the amount of microsilica in the aqueous composition is in the range 0.002 mg/L to 1000 mg/L, more preferably in the range 0.004 mg/L to 1000 mg/L, even more preferably in the range 0.006 mg/L to 1000 mg/L and most preferably in the range 0.008 mg/L to 1000 mg/L, such as in the range 0.01 mg/L to 1000 mg/L, in the range 0.015 mg/L to 1000 mg/L, in the range 0.02 mg/L to 1000 mg/L, in the range 0.03 mg/L to 1000 mg/L, in the range 0.04 mg/L to 1000 mg/L, in the range 0.05 mg/L to 1000 mg/L, in the range 0.06 mg/L to 1000 mg/L, in the range 0.07 mg/L to 1000 mg/L, in the range 0.08 mg/L to 1000 mg/L, in the range 0.09 mg/L to 1000 mg/L or in the range 0.1 mg/L to 1000 mg/L.
  • the amount of microsilica in the aqueous composition is in the range 0.002 mg/L to 100 mg/L, more preferably in the range 0.004 mg/L to 100 mg/L, even more preferably in the range 0.006 mg/L to 100 mg/L and most preferably in the range 0.008 mg/L to 100 mg/L, such as in the range 0.01 mg/L to 100 mg/L, in the range 0.015 mg/L to 100 mg/L, in the range 0.02 mg/L to 100 mg/L, in the range 0.03 mg/L to 100 mg/L, in the range 0.04 mg/L to 100 mg/L, in the range 0.05 mg/L to 100 mg/L, in the range 0.06 mg/L to 100 mg/L, in the range 0.07 mg/L to 100 mg/L, in the range 0.08 mg/L to 100 mg/L, in the range 0.09 mg/L to 100 mg/L or in the range 0.1 mg/L to 100 mg/L.
  • the amount of microsilica in the aqueous composition is in the range 0.002 mg/L to 10 mg/L, more preferably in the range 0.004 mg/L to 10 mg/L, even more preferably in the range 0.006 mg/L to 10 mg/L and most preferably in the range 0.008 mg/L to 10 mg/L, such as in the range 0.01 mg/L to 10 mg/L, in the range 0.015 mg/L to 10 mg/L, in the range 0.02 mg/L to 10 mg/L, in the range 0.03 mg/L to 10 mg/L, in the range 0.04 mg/L to 10 mg/L, in the range 0.05 mg/L to 10 mg/L, in the range 0.06 mg/L to 10 mg/L, in the range 0.07 mg/L to 10 mg/L, in the range 0.08 mg/L to 10 mg/L, in the range 0.09 mg/L to 10 mg/L or in the range 0.1 mg/L to 10 mg/L.
  • the aqueous composition is saturated in respect of microsilica.
  • the amount of microsilica in the aqueous composition is in the range 0.00175 mg/L to saturated in respect of microsilica.
  • the amount of microsilica in the aqueous composition is in the range 0.002625 mg/L to saturated in respect of microsilica; more preferably in the range 0.0035 mg/L to saturated in respect of microsilica; even more preferably in the range 0,005 mg/L to saturated in respect of microsilica; and most preferably in the range 0.006 mg/L to saturated in respect of microsilica; such as in the range 0.01 mg/L to saturated in respect of microsilica, in the range 0.015 mg/L to saturated in respect of microsilica, in the range 0.02 mg/L to saturated in respect of microsilica, in the range 0.025 mg/L to saturated in respect of microsilica, in the range 0.03 mg/L to saturated in respect of microsilica, in the range 0.035 mg/L to saturated in respect of microsilica, in the range 0.
  • the aqueous composition comprises an aqueous liquid and microsilica, wherein the amount of microsilica in the aqueous composition is in the range 0.005 mg/L to saturated in respect of microsilica. In another embodiment, the amount of microsilica in the aqueous composition is in the range 0.01 mg/L to saturated in respect of microsilica. In yet another embodiment according to the present invention the amount of microsilica in the aqueous composition is in the range 0.05 mg/L to saturated in respect of microsilica.
  • the aqueous composition further comprises essential nutrients to maintain growth of the diatoms, preferably to maintain the diatoms in exponential growth phase.
  • the aqueous composition further comprises a carbon source and/or a nitrogen source and/or a phosphorus source or any mixture thereof.
  • the carbon source is obtained by introducing CO2( g ) into the aqueous composition.
  • the nitrogen source is obtained by introducing NO?’, NO2- or any mixture thereof into the aqueous composition.
  • the carbon source and the nitrogen source are obtained by introducing factory smoke from production of silicon and/or ferrosilicon into the aqueous composition.
  • factory smoke from production of silicon and/or ferrosilicon is to be introduced into the aqueous composition suitable for cultivating diatoms
  • the pH of the aqueous composition should preferably be >7, more preferably >7.5 and most preferably >7.8.
  • the pH of the aqueous composition is ⁇ 7, such as ⁇ 6.9 or ⁇ 6.8 it is preferred not to introduce any further factory smoke from production of silicon and/or ferrosilicon.
  • the phosphorus source is obtained by adding fertilizer into the aqueous composition.
  • Substral® provided by Scotts Company A/S (see example 1, table 2) and YaraTera Kristalon® Purple provided by Yara are two examples of fertilizers suitable to be added to the aqueous composition.
  • a second alternative aspect of the present invention relates to a use of the aqueous composition according to the first alternative aspect of the present invention, for cultivation of diatoms.
  • the diatoms are marine diatoms.
  • the diatoms are photoautotrophic diatoms.
  • the diatoms are centric diatoms.
  • the mean diameter of the diatoms is > 25 pm.
  • the diatoms are a diatom monoculture.
  • the diatoms are a culture of diatoms wherein the majority of the diatoms are of the same species. In one embodiment more than 80% of the diatoms are of the same species, such as more than 85%, more than 90%, more than 95%, more than 96%, more than 97%, more than 98% or more than 99% of the diatoms are of the same species.
  • the diatoms are a culture of diatoms wherein a substantial number of the diatoms is of the same species.
  • a third alternative aspect of the present invention relates to a method of producing a diatom biomass, the method comprising a step of culturing diatoms in the aqueous composition according to the first alternative aspect of the present invention.
  • the diatoms are marine diatoms.
  • the diatoms are photoautotrophic diatoms.
  • the diatoms are centric diatoms.
  • the mean diameter of the diatoms is > 25 pm.
  • the diatoms are a diatom monoculture.
  • the diatoms are a culture of diatoms wherein the majority of the diatoms are of the same species. In one embodiment more than 80% of the diatoms are of the same species, such as more than 85%, more than 90%, more than 95%, more than 96%, more than 97%, more than 98% or more than 99% of the diatoms are of the same species.
  • the diatoms are a culture of diatoms wherein a substantial number of the diatoms is of the same species.
  • Figure la illustrates production of diatom biomass applying the following five growth conditions:
  • Substral® (0.25 mL/ L) Substral® (0.25 mL/ L) + Silicate (3.5 g/L Na2O3Si°9H2O) Substral® (0.25 mL/ L) + microsilica (maximum dissolvable amount) Substral® (0.25 mL/ L) + introduction of factory smoke for 60 minutes per day
  • Horizontal axis Number of days after start of cultivation.
  • Figure lb illustrates production of diatom biomass applying the following five growth conditions: es per day
  • Horizontal axis Number of days after start of cultivation.
  • FIG. 1c illustrates the pH during production of biomass applying the following five growth conditions:
  • Figure 2 represents a flowchart to determine if a species is autotroph, heterotroph, or a subtype.
  • Figure 3a illustrates production of diatom biomass applying different amounts of microsilica in the growth medium, wherein Pl, P2 and P3 have high, intermediate, and low amounts of microsilica in the growth medium (see example 3) respectively.
  • P4 represents control with no added microsilica to the growth medium.
  • Horizontal axis Day number, wherein experiment starts at day 1.
  • Figure 3b shows the concentration of SiCh in a growth medium during cultivation of diatoms at different growth medium conditions at start.
  • Pl, P2 and P3 have high, intermediate and low amounts of microsilica in the growth medium (see example 3).
  • P4 represents control with no added microsilica to the growth medium.
  • Horizontal axis Day number, wherein experiment starts at day 1.
  • Figure 4 shows typical particle size distribution of microsilica, wherein about 25% of the particle volume is less than 0,1 pm and about 50% of the particle volume is less than 0,15 pm.
  • omega-3 fatty acids EPA (eicosapentaenoic acid) and DHA (docosahexaenoic acid), which are associated with beneficial health effects in human nutrition, are found in relatively high levels in diatoms which therefore represent a promising source of omega-3 fatty acids.
  • Diatoms are unicellular microalgae enclosed within a silica cell wall.
  • the silica cell wall is a unique property of this algae, and it is well known in the art that silicon availability is a key factor in the regulation of diatom growth.
  • Silicon is typically supplied to the ocean both dissolved as monomeric or oligomeric form or as solid particulates in variable size and crystallinity. Dissolved silicon can be loaded by river runoff as well as by the dissolution of particulate silicon present in the lithogenic and biogenic systems. In the sea the biogeochemical dissolution of this mineral is controlled by temperature, zooplankton grazing, diatom sinking and bacterial activity.
  • Silicon transport inside the cell seems to be carrier- mediated, and in marine diatoms the ion-carrier can be Na + dependent with metabolic energy supply.
  • the carrier function of silicon transport inside the cell can be mediated by the action of a protein: the silicon transporter (SIT), present in marine and freshwater diatoms.
  • SIT silicon transporter
  • Silica deposition and morphogenesis of the cell wall seems to occur in a specialized compartment: the Silica Deposition Vesicle, known as SDV.
  • the acidic environment contributes to the silicic acid polymerization into amorphous hydrated silica onto an organic template (proteins, polysaccharides, lipids), that drives the frustule silicification and morphogenesis.
  • Diatoms cultivated for 11 days in filtered seawater with 0.25 mL/ L substral® (example 1, table 2) were able to reach a biomass that was 2.5-fold higher than the biomass at start of cultivation (experimental setup A). If 3.5 g/L Na2O3Si°9H2O was added to the culture medium during the 11 days period, the culture reached a biomass that was about 4.9-fold higher than the biomass at start of cultivation (experimental setup B). The pH in the culture medium during the 11 days period was not significantly different between experimental setup A and experimental setup B.
  • experimental setup C diatoms were cultivated for 11 days in filtered seawater with 0.25 mL/ L substral® and dissolved amorphous silicon dioxide (microsilica).
  • the culture reached a biomass that was about 7-fold higher than the biomass at start of cultivation.
  • the pH in the culture medium during the 11 days period was not significantly different between experimental setup A, experimental setup B and experimental setup C. This surprising finding is a clear indication that diatoms have a preference for amorphous silicon dioxide as compared to silicate.
  • the amorphous silicon dioxide that was added to the culture medium in experimental setup C is a by-product of the production of silicon/ferrosilicon and was collected at a silicon/ferrosilicon factory in Finnsnes owned by Finnfjord AS. Another by-product of that process is the factory smoke which is known to contain inter alia carbon dioxide and nitrogen oxides (NO X ).
  • diatoms need a nitrogen source to grow and that they are very effective in converting carbon dioxide into organic compounds.
  • the inventors decided to investigate the effect on diatom growth of introducing factory smoke into the growth medium.
  • experimental setup D diatoms were cultivated for 11 days in filtered seawater with 0.25 mL/ L substral® wherein factory smoke, derived from a silicon/ferrosilicon factory in Finnsnes owned by Finnfjord AS, was introduced into the growth medium for a period of 60 minutes per day (bubbles of factory smoke introduced into the growth medium).
  • table 5 the culture reached a biomass that was about 3.9-fold higher than the biomass at start of cultivation.
  • experimental setup A without factory smoke
  • the culture reached a biomass that was about 2.5-fold higher than the biomass at start of cultivation.
  • the pH in the culture medium during the 11 days period was somewhat lower in experimental setup D as compared to experimental setup A, but the pH did not reach a critically low value.
  • the present inventors have demonstrated that both microsilica and factory smoke derived from a silicon/ferrosilicon factory are able to improve the growth rate of diatoms in large volume closed bioreactors.
  • the present inventors have been able to provide sustainable and cost-effective methodologies for cultivation of diatoms in large volume closed bioreactors.
  • a first aspect of the present invention relates to a method of preparing an aqueous composition for cultivation of diatoms, the method comprising the following step(s):
  • the amorphous SiCL is added in the form of microsilica.
  • a second aspect of the present invention relates to an aqueous composition for cultivation of diatoms prepared by the method according to the first aspect of the present invention.
  • a third aspect of the present invention relates to an aqueous composition for cultivation of diatoms comprising an aqueous liquid and SiCL provided in the form of microsilica, wherein - the amount of SiCh provided in the form of microsilica in the aqueous composition is at least 0,07 mg/L.
  • Amorphous silicon dioxide is the main ingredient in microsilica, typically constituting > 85 % by weight of the microsilica. Adding microsilica to an aqueous liquid will therefore increase the level of SiCh in the aqueous liquid significantly.
  • the amount of SiCh provided in the form of microsilica in the aqueous composition refers to the process of adding microsilica to the aqueous liquid and the resulting increase in the amount of SiCh in the aqueous liquid. Said in other words: the amount of SiCh in the aqueous liquid that originates from microsilica or the amount of SiCh in the aqueous liquid that is derived from microsilica.
  • aqueous compositions suitable for cultivation of diatoms there are several well-known aqueous compositions that are suitable for cultivation of diatoms, and the present invention lies in modifying those well-known products by adding silicon dioxide in the form of microsilica.
  • the first, second and third aspects of the present invention clearly identifies the product to be modified (aqueous compositions suitable for cultivation of diatoms) and specifies what is modified and in what way.
  • the other ingredients necessary for cultivation of diatoms is thus implied by the generic reference to an aqueous composition suitable for cultivation of diatoms.
  • microalgae require macro-, micronutrients and vitamins for growth (Scientific Reports volume 9, Article number: 1479 (2019)).
  • Macronutrients correspond to nitrogen (N) and phosphorus (P), while micronutrients correspond to trace metals such as iron, manganese, cobalt, etc.
  • vitamins such as thiamine, biotin and cobalamin (vitamins Bl, B7 and B12, respectively) are also often needed since some microalgal species are not able to synthesize them.
  • diatoms require silicon (Si), which is involved in building the outer cell wall, or frustule.
  • Both Substral® and Guillard’s f/2 marine nutrient enrichment solution referred to in table 1 below, provide required amounts of macronutrients, micronutrients and vitamins to facilitate growth of Diatoms. However, for optimal growth it may be required to add silicon, such as silicate or microsilica, and most preferably silicon in the form of microsilica. If silicate is to be used, it is preferred to use metasilicate pentahydrate.
  • the aqueous composition for cultivation of diatoms comprises macro-, micronutrients and optionally vitamins in sufficient amounts to facilitate growth of diatoms.
  • Said macronutrients comprising a source of nitrogen (N) and a source of phosphorus (P).
  • the source of nitrogen is selected from the group consisting of nitrate, ammonium nitrogen, ammonia and any mixture thereof.
  • the source of phosphorus is phosphate, such as sodium phosphate.
  • Said micronutrients comprising trace metals selected from the group consisting of iron, manganese, cobalt, zinc, magnesium, boron, potassium, sulphur and any mixture thereof.
  • Said vitamins being selected from the group consisting of thiamine, biotin, cobalamin (vitamins Bl, B7 and Bl 2, respectively) and any mixture thereof.
  • aqueous composition is a composition in which the solvent is water.
  • the water may be seawater, freshwater or any combination thereof.
  • the water has a salt content in the range 20 to 50 g/L, preferably in the range 20 to 45 g/L, more preferably a salt content in the range 25 to 45 g/L, even more preferably in the range 25 to 40 g/L and most preferably a salt content in the range 25 to 35 g/L, such as a salt content of about 30 g/L.
  • diatoms refers to a major group of microalgae found in the oceans, waterways and soils of the world.
  • the diatoms are marine diatoms.
  • marine diatoms referring to the diatoms that naturally live in the oceans, i.e. seawater.
  • Diatoms are unicellular: they occur either as solitary cells or in colonies, which can take the shape of e.g. ribbons, curls, waves, fans, zigzags, balls or stars. Individual cells typically range in size from 2 to 200 micrometers. In one embodiment according to the present invention, the mean diameter of the diatoms is > 25 pm.
  • Diatoms have two distinct shapes: a few (centric diatoms) are radially symmetric, while most (pennate diatoms) are broadly bilaterally symmetric.
  • the diatoms are centric diatoms, such as marine centric diatoms.
  • diatom anatomy A unique feature of diatom anatomy is that they are surrounded by a cell wall made of silica (hydrated silicon dioxide), called a frustule. These frustules have structural coloration due to their photonic nanostructure, prompting them to be described as “jewels of the sea” and "living opals”. Similar to plants, diatoms convert light energy to chemical energy by photosynthesis, although this shared autotrophy evolved independently in both lineages.
  • An autotroph or primary producer is an organism that produces complex organic compounds (such as carbohydrates, fats, and proteins) from simple substances present in its surroundings, generally using energy from light (photosynthesis) or inorganic chemical reactions (chemosynthesis).
  • Autotrophs are the producers in a food chain, such as plants on land or algae in water (in contrast to heterotrophs as consumers of autotrophs). They do not need a living source of energy or organic carbon. Autotrophs can reduce carbon dioxide to make organic compounds for biosynthesis and also create a store of chemical energy. Most autotrophs use water as the reducing agent, but some can use other hydrogen compounds such as hydrogen sulfide. Some autotrophs, such as green plants and algae, are phototrophs, meaning that they convert electromagnetic energy from sunlight into chemical energy in the form of reduced carbon.
  • Autotrophs can be photoautotrophs or chemoautotrophs.
  • Phototrophs use light as an energy source, while chemotrophs use electron donors as a source of energy, whether from organic or inorganic sources. However, in the case of autotrophs, these electron donors come from inorganic chemical sources.
  • Such chemotrophs are lithotrophs.
  • Lithotrophs use inorganic compounds, such as hydrogen sulfide, elemental sulfur, ammonium and ferrous iron, as reducing agents for biosynthesis and chemical energy storage.
  • Photoautotrophs and lithoautotrophs use a portion of the ATP produced during photosynthesis or the oxidation of inorganic compounds to reduce NADP+ to NADPH to form organic compounds.
  • the diatoms are photoautotrophic diatoms, such as marine photoautotrophic diatoms.
  • the diatoms are centric photoautotrophic diatoms, such as marine centric photoautotrophic diatoms.
  • aqueous liquid is a liquid in which the solvent is water.
  • the aqueous liquid is water.
  • the water may be seawater, freshwater or any combination thereof.
  • the water has a salt content in the range 20 to 50 g/L, preferably in the range 20 to 45 g/L, more preferably a salt content in the range 25 to 45 g/L, even more preferably in the range 25 to 40 g/L and most preferably a salt content in the range 25 to 35 g/L, such as a salt content of about 30 g/L.
  • the aqueous liquid has a salt content in the range 20 to 50 g/L, preferably a salt content in the range 20 to 45 g/L, more preferably in the range 25 to 45 g/L, even more preferably in the range 25 to 40 g/L and most preferably a salt content in the range 25 to 35 g/L, such as a salt content of about 30 g/L.
  • the aqueous composition according to the second or third aspect of the present invention comprises an aqueous liquid to which amorphous SiCh in the form of microsilica has been added. Further, the aqueous composition according to the first alternative aspect of the present invention relates to an aqueous composition comprising an aqueous liquid and microsilica.
  • Silica fume also known as microsilica, (CAS number 69012-64-2, EINECS number 273-761-1) is an amorphous (non-crystalline) polymorph of silicon dioxide, silica.
  • Microsilica is typically an ultrafine powder which may consist of spherical particles, non- spherical particles or a mixture thereof.
  • the microsilica comprises a plurality of spherical microsilica particles.
  • Microsilica may be collected as a by-product of the silicon and ferrosilicon alloy production, and such microsilica will typically have the following properties: about 25% of the particle volume less than 100 nm; about 50% of particle volume less than 150 nm; specific surface (BET) typically 20 m 2 /g (range 15-30);
  • amorphous SiCh 85 % by weight amorphous SiCh; some aluminum oxide, iron oxide and alkalis.
  • Microsilica may be provided in undensified form, which typically has a density of 200-350 kg/m 3 , or in densified form which typically has a density of 550-650 kg/m 3 .
  • microsilica particles are polydisperse, which means that the particles in an ensemble have different sizes. I addition to size differences, the particles in an ensemble will also typically have different geometrical shape.
  • the size is an indirect measure, obtained by a model that transforms, in abstract way, the real particle shape into a simple and standardized shape, like a sphere (the most usual) or a cuboid (when minimum bounding box is used), where the size parameter (ex. diameter of sphere) makes sense.
  • the size parameter ex. diameter of sphere
  • volume-based particle size model One way of expressing particle size is the so called “volume-based particle size model”.
  • volume-based particle size model the volume-based particle size equals the diameter of the sphere that has the same volume as a given particle.
  • This model is typically used in sieve analysis, as shape hypothesis (sieve's mesh size as the sphere diameter).
  • Amorphous silicon dioxide is the main ingredient in microsilica, typically constituting > 85 % by weight of the microsilica. Adding microsilica to an aqueous liquid will therefore increase the level of SiCh in the aqueous liquid significantly. Reference is made to the following examples illustrating the amount of microsilica required to obtain a desired amount of SiCh.
  • Calculation X adding 0,1 mg microsilica to IL of an aqueous liquid will provide an aqueous liquid containing 0,07 mg (amorphous) SiCh provided that 70 % by weight of the microsilica is amorphous SiCh.
  • Calculation Y adding 0,07 mg amorphous SiCh, in the form of microsilica, to an aqueous liquid will require that you add 0,1 mg microsilica to IL of an aqueous liquid provided that 70 % by weight of the microsilica is amorphous SiCL.
  • microsilica collected as a by-product of the silicon and ferrosilicon alloy production typically comprises i) an iron source, such as one or more iron oxide(s), e.g. Fe2O3; ii) an aluminum source, such as one or more aluminum oxide(s), e.g. AI2O3; and iii) a magnesium source, such as one or more magnesium oxide(s), e.g. MgO.
  • an iron source such as one or more iron oxide(s), e.g. Fe2O3
  • an aluminum source such as one or more aluminum oxide(s), e.g. AI2O3
  • a magnesium source such as one or more magnesium oxide(s), e.g. MgO.
  • the microsilica comprises an iron source, such as one or more iron oxide(s), the iron source preferably being Fe2C>3.
  • the microsilica comprises 0.5 to 10 % by weight of one or more iron oxide(s), such as 0.5 to 10 % by weight Fe2C>3.
  • the microsilica comprises 1 to 5 % by weight of one or more iron oxide(s), such as 1 to 5 % by weight Fe2O3.
  • the microsilica comprises 1 to 3 % by weight of one or more iron oxide(s), such as 1 to 3 % by weight Fe2O3.
  • the microsilica comprises an aluminum source, such as one or more aluminum oxide(s), the aluminum source preferably being AI2O3.
  • the microsilica comprises 0.1 to 10 % by weight of one or more aluminum oxide(s), such as 0.1 to 10 % by weight AI2O3.
  • the microsilica comprises 0,3 to 5 % by weight of one or more aluminum oxide(s), such as 0,3 to 5 % by weight AI2O3.
  • the microsilica comprises 0,5 to 1,5 % by weight of one or more aluminum oxide(s), such as 0,5 to 1,5 % by weight AI2O3.
  • the microsilica comprises a magnesium source, such as one or more magnesium oxide(s), the magnesium source preferably being MgO.
  • the microsilica comprises 0.1 to 10 % by weight of one or more magnesium oxide(s), such as 0.1 to 10 % by weight MgO.
  • the microsilica comprises 0,3 to 5 % by weight of one or more magnesium oxide(s), such as 0,3 to 5 % by weight MgO.
  • the microsilica comprises 0,5 to 2 % by weight of one or more magnesium oxide(s), such as 0,5 to 2 % by weight MgO; and most preferably the microsilica comprises 0,5 to 1,5 % by weight of one or more magnesium oxide(s), such as 0,5 to 1,5 % by weight MgO.
  • Non-axenic clonal culture of a planktonic marine diatom was established from a single cell from a sample collected in the Barents Sea. Isolation method applied was a combination of microscope aided manual capillary pipetting and serial dilution transfer (i.e. transferring a single cell to a well in a 4-well chamber (Nunc) filled with autoclaved seawater and serial transferring it 5-10 times to rinse it). Although non-axenic, bacterial numbers were very low.
  • the culture collection and innocula for mass cultures were cultivated in pasteurized seawater from 10m depth in Tromsoysund with 4 mL L -1 of Guillard’s f/2 x 50 marine water enrichment solution (Sigma-Aldrich, table 1). Table 1
  • the diatoms were cultivated in 150L transparent plexiglas columns in temperature and irradiance-controlled rooms (temperature of about 7 °C and scalar irradiance inside the columns of about 30 pmol photons m -2 s -1 ).
  • the cultivation took place in Millipore filtered seawater (0.22-pm pore size) with nutrients as indicated in table 2.
  • Each treatment was represented by triplicate cultures, hence the given cell numbers are means of the triplicates. All given C.V. were below 4%, hence data conclusions are based on did not overlap.
  • Photosynthetical active radiation (PAR) was measured with a QSL-100 scalar irradiance meter (Biosperical Instruments Inc.). All cultures were mixed/aerated at a rate of ca. 2.5 L min -1 .
  • microsilica is a by-product from the production of silicon/ferrosilicon.
  • the microsilica was collected at a silicon/ferrosilicon factory in Finnsnes owned by Microsilica Finnfjord AS. An analysis of the microsilica is provided in table 6.
  • Factory smoke Factory smoke was derived from a silicon/ferrosilicon factory in Finnsnes owned by Finnfjord AS.
  • the algae cultures were sampled at day 0, 2, 4, 7, 9 and 11 after start of cultivation. Each sample was analyzed with respect to number of cells, counted in inverted microscopes after minimum 4 hrs. sedimentation in Nunclon 4 well 2 mL chambers (table 3) and pH (table 4). Table 3
  • the number of cells at start of cultivation varies considerably between experimental setups. In order to be able to compare the biomass growth between experimental setups, it was decided to set the number of cells at start of cultivation equal to 100 %. The number of cells at day 2, 4, 7, 9 and 11 after start of cultivation was calculated relatively to the number of cells at day 0.
  • microsilica product utilized in this experiment is a by-product from the production of silicon and ferrosilicon and contains more than 85 % by weight amorphous silicon dioxide (analysis of the microsilica product utilized in this experiment is presented in table 6). Thus, it is believed that it is amorphous silicon dioxide that is the factor being responsible for the observed increase in growth rate. However, the microsilica product utilized in this experiment also contains other elements that may contribute to the increased growth rate. Iron in the form of Fe2O3 being an interesting candidate in this respect.
  • factory smoke may contain microsilica and that the content of microsilica will vary to some extent.
  • the factory smoke referred to in said master thesis had a regular content of microsilica, the factory smoke should have added about 0.0105 g microsilica to 6000 L culture media during the experiment.
  • the culturing media referred to in said master thesis is assumed to contain about 0.00175 mg/L microsilica.
  • example 1 of the present application provides comparative growth rate data using either a culturing media to which microsilica has been added or culturing media to which factory smoke has been introduced for 60 minutes.
  • example 1 of the present application provides comparative growth rate data using either a culturing media to which microsilica has been added or culturing media to which 3.5 g/L Na2O3Si°9H2O has been added.
  • Non-axenic clonal culture of a planktonic centric marine diatom was retrieved from stock monoculture samples (kept at UiT The arctic university of Norway) originally collected in the Arctic Barents Sea. The culturing was performed using conventional Guillards f/2 growth medium without Si (control, Treatment P4) , while Si was added as microsilica dissolved in water and added to different concentrations (Treatments Pl, P2, P3) and Si was measured underway in the experiment by standard colorimetric methods (Strickland and Parsons, 1972) using a Flow Solution IV analyzer from O.I. Analytical, USA (see Table 7 and figure 3b). The analyzer was calibrated using reference seawater from Ocean Scientific International Ltd. UK.
  • Bioresource technology vol. 314, 2020, 123747 reports a novel solution-based method to trigger the growth of diatoms for enhanced biomass production, including use of inductively coupled plasma (ICP) synthesized nanosilika.
  • ICP inductively coupled plasma
  • microsilica provides higher growth rates at lower light intensities at SiCh concentrations from 175 000 to 350 000 times lower than what was being used in Bioresource technology, vol. 314, 2020, 123747.

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Abstract

The present invention relates generally to algaculture, and in particular to cultivation of diatoms. The present invention provides a growth medium for cultivation of diatoms in large volume vertical column bioreactors. Further, the invention also provides use of the growth medium for cultivation of diatoms as well as methods for cultivation of diatoms.

Description

PROCESS FOR CULTIVATION OF DIATOMS
Field of the invention
The present invention relates generally to algaculture, and in particular to cultivation of diatoms. The present invention provides a growth medium for cultivation of diatoms in large volume vertical column bioreactors. Further, the invention also provides use of the growth medium for cultivation of diatoms as well as methods for cultivation of diatoms.
Background of the invention
The omega-3 fatty acids are associated with beneficial health effects in human nutrition. Fat fish, including salmonids, are good sources for the essential omega-3 long-chain polyunsaturated fatty acids EP A and DHA. Due to extensive fish farming the global human consumption of salmonids has increased substantially the last decades, where Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss) are the dominant species.
Originally, salmonid feeds were mainly based on marine resources and therefore naturally rich in the omega-3 fatty acids. However, through the increase in global fish farming (including non-salmonid species), and increased direct human consumption of marine oils, the marine resources that traditionally has been used in feeds can no longer meet the demand of the fish farming industry.
Over the years the feed used in the fish farming industry has therefore changed from being primarily marine based to be more terrestrial based, e.g. on soy, maize and rapeseed. Coinciding with the shift in composition of salmonid feed a decrease in omega-3 content of farmed salmon has been observed. It is believed that the decrease in omega-3 content of farmed salmon is caused by replacing the marine oils with vegetable oils less rich in the omega-3 fatty acids. Thus, there is an urgent need for increased amounts of high omega-3 formulated fish feed in the fish farming industry.
All omega-3 fatty acids are originally synthesized by microalgae, and they hence represent the original source of omega-3 fatty acids. Algae is a large diverse group of photosynthetic eukaryotic organisms that are not necessarily closely related, and is thus polyphyletic. Algae includes organisms ranging from unicellular microalgae genera, such as Chlorella species and diatoms, to multicellular forms, such as the giant kelp, a large brown alga which may grow up to 50 m in length.
Diatoms are rich in lipids, and these algae, if cultivated economically, are therefore of particular interest for solving the need for high omega-3 formulated fish feed in the fish farming industry. Diatoms are unicellular microalgae enclosed within a silica cell wall. The diatoms are microalgae with a size ranging from about 2 gm to about 500 gm (0.5 mm). They are living in the worlds’ oceans but are also found in fresh water. More than 8000 species of diatoms are recorded worldwide, and it is estimated that there are 20.000-200.000 extant diatoms species in the world.
Diatoms belongs to the group of photosynthesizing algae and it has been estimated that diatoms account for as much as 25 % of global photosynthetic fixation of carbon. The major carbon storage compound in diatoms is lipids, among which triglycerides and fatty acids can make up 15-25% of the dry biomass. The omega-3 fatty acids EPA (eicosapentaenoic acid) and DHA (docosahexaenoic acid), which are associated with beneficial health effects in human nutrition, are found in relatively high levels in diatoms. Despite these attractive attributes, the diatoms have until now been underrepresented in production projects.
In addition to representing a promising source of omega-3 fatty acids, the diatoms have also been demonstrated to be very effective in converting inorganic carbon to organic compounds. By being able to convert carbon dioxide into organic compounds, such as sugars, the production of diatom biomass can aid to reduce local carbon footprints. Thus, cultivation of diatoms represents a sustainable and environmentally friendly way of meeting the demand for omega-3 formulated fish feed in the fish farming industry.
In order to be able to industrialize production of omega-3 fatty acids derived from diatoms to meet the demand of the fish farming industry, it is important to establish sustainable and cost-effective methodologies for cultivation.
The present inventors have been able to provide sustainable and cost-effective methodologies for cultivation of diatoms in large volume vertical column bioreactors by including a specific form of silica in the algae growth medium. This specific form of silica has been demonstrated to result in an increased growth rate, and it has also been discovered that the increased growth rate may be increased even further by introducing factory smoke from the production of silicon and/or ferrosilicon into the growth medium. The introduction of factory smoke improves growth of the algae but also reduces the carbon footprint of the silicon and/or ferrosilicon factory; a win-win situation.
W02004048553 discloses a liquid for culturing diatom comprising nitrogen, silicon and phosphorus with a specific silicon : nitrogen ratio. KR101888798 discloses a nano-silica solution for growing diatoms, the solution being prepared by including nano-silica, distilled water, iron chloride, ferric chloride, manganese chloride, zinc chloride, cobalt chloride, and copper chloride.
W02005121313 discloses a composition for a copious bloom of diatom algae comprising macro and or micronutrients adsorbed on metalate modified silica sol.
WO91 14427 discloses a method for producing single cell edible oils containing EPA from heterotrophic diatoms by cultivating diatoms in a nutrient solution containing nitrogen and silicate.
WO201 5041531 discloses culturing diatoms under non-limiting bioavailable silicon concentrations wherein the diatoms are subjected to a cycle of alternating dark phases and light phases and providing limitation of availability of at least one essential growth nutrient in one or more of said light phases.
WO201 5016720 relates to methods of producing diatom biomass using a continuous culture to produce a volumetric production rate of biomass of at least 0,2g dry weight/L/day, wherein the culture medium is designed to provide the essential nutrients to maintain the diatom in log phase growth.
Master Thesis in Marine Biology entitled Bioactivity Potential of an Arctic Marine Diatom Species Cultivated at Different Conditions”, 2018 discloses a method for the cultivation of diatoms (centric marine). The diatoms are grown in seawater wherein factory smoke is introduced, said smoke being a source of CO2, NOX and microsilica.
Algal Culturing Techniques (R, A. Andersen, eds), 1 January 2005, pages 429-538 discloses f/2 Medium for growing diatoms containing sodium silicate. However, no disclosure of any microsilica in the medium is being made.
Bioresource technology, vol. 314, 2020, 123747 reports a novel solution-based method to trigger the growth of diatoms for enhanced biomass production, including use of inductively coupled plasma (ICP) synthesized nanosilika. However, no disclosure of any microsilica in the medium is being made.
Summary of the invention
A first aspect of the present invention relates to a method of preparing an aqueous composition for cultivation of diatoms, the method comprising the following step(s):
- adding at least 0,07 mg/L amorphous SiCh to an aqueous liquid to obtain the aqueous composition; wherein
- the amorphous SiCh is added in the form of microsilica.
Diatom
In a first embodiment according to the present invention, the diatoms are marine diatoms.
In another embodiment according to the present invention the diatoms are photoautotrophic diatoms.
In a further embodiment according to the present invention the diatoms are centric diatoms.
In yet a further embodiment according to the present invention the mean diameter of the diatoms is > 25 pm.
In yet a further embodiment according to the present invention the diatoms are a diatom monoculture.
In yet a further embodiment according to the present invention the diatoms are a culture of diatoms wherein the majority of the diatoms are of the same species. In one embodiment more than 80% of the diatoms are of the same species, such as more than 85%, more than 90%, more than 95%, more than 96%, more than 97%, more than 98% or more than 99% of the diatoms are of the same species.
In yet a further embodiment according to the present invention the diatoms are a culture of diatoms wherein a substantial number of the diatoms is of the same species.
Aqueous liquid
In one embodiment according to the present invention, the aqueous liquid is water, such as freshwater or seawater.
In a further embodiment according to the present invention, the aqueous liquid has a salt content in the range 20 to 50 g/L, preferably 20 to 45 g/L, more preferably a salt content in the range 25 to 45 g/L, even more preferably in the range 25 to 40 g/L and most preferably a salt content in the range 25 to 35 g/L, such as about 30 g/L.
Microsilica
In another embodiment according to the present invention, the microsilica comprises > 70 % by weight amorphous silicon dioxide, preferably > 80 % by weight amorphous silicon dioxide, even more preferably > 85 % by weight amorphous silicon dioxide and most preferably > 90 % by weight amorphous silicon dioxide.
In yet a further embodiment, the microsilica is dissolved in the aqueous liquid and optionally any non-dissolved microsilica is removed from the aqueous liquid. In one embodiment, the microsilica is dissolved in the aqueous liquid and any nondissolved microsilica is removed from the aqueous liquid.
In another embodiment according to the present invention, the microsilica is dispersed in the aqueous liquid.
In a further embodiment according to the present invention, the microsilica is a byproduct from the production of silicon and/or ferrosilicon.
In one embodiment according to the present invention, the microsilica comprises a plurality of spherical microsilica particles.
In a further embodiment according to the present invention, the microsilica comprises a plurality of microsilica particles, wherein at least 10 % of particle volume is more than 100 nm. In another embodiment at least 20 % of particle volume is more than 100 nm. In another embodiment at least 30 % of particle volume is more than 100 nm. In another embodiment at least 40 % of particle volume is more than 100 nm. In another embodiment at least 50 % of particle volume is more than 100 nm. In another embodiment at least 55 % of particle volume is more than 100 nm. In yet another embodiment at least 60 % of particle volume is more than 100 nm. In yet another embodiment at least 65 % of particle volume is more than 100 nm. In yet another embodiment at least 70 % of particle volume is more than 100 nm. In yet another embodiment at least 75 % of particle volume is more than 100 nm. In yet another embodiment at least 80 % of particle volume is more than 100 nm. In yet another embodiment at least 85 % of particle volume is more than 100 nm. In yet another embodiment at least 90 % of particle volume is more than 100 nm. It is preferred that 55-95 % of particle volume is more than 100 nm, such as i) 60-90 % of particle volume is more than 100 nm, ii) 65-85 % of particle volume is more than 100 nm, iii) 70-80 % of particle volume is more than 100 nm or iv) 75% of particle volume is more than 100 nm.
In a further embodiment according to the present invention, the microsilica comprises a plurality of microsilica particles, wherein at least 20 % of particle volume is more than 150 nm. In another embodiment at least 25 % of particle volume is more than 150 nm. In another embodiment at least 30 % of particle volume
Figure imgf000008_0001
more than 150 nm. In another embodiment at least 35 % of particle volume
Figure imgf000008_0002
more than 150 nm. In another embodiment at least 40 % of particle volume
Figure imgf000008_0003
more than 150 nm. In another embodiment at least 45 % of particle volume
Figure imgf000008_0004
more than 150 nm. In yet another embodiment at least 50 % of particle volume
Figure imgf000008_0005
more than 150 nm. In yet another embodiment at least 55 % of particle volume
Figure imgf000008_0006
more than 150 nm. In yet another embodiment at least 60 % of particle volume
Figure imgf000008_0007
more than 150 nm. In yet another embodiment at least 65 % of particle volume
Figure imgf000008_0008
more than 150 nm. In yet another embodiment at least 70 % of particle volume
Figure imgf000008_0009
more than 150 nm. In yet another embodiment at least 75 % of particle volume
Figure imgf000008_0010
more than 150 nm. In yet another embodiment at least 80 % of particle volume is more than 150 nm. It is preferred that 25-75 % of particle volume is more than 150 nm, such as i) 30-70 % of particle volume is more than 150 nm, ii) 35-65 % of particle volume is more than 150 nm, iii) 40-60 % of particle volume is more than 150 nm or iv) 50% of particle volume is more than 150 nm.
In a further embodiment according to the present invention, the microsilica comprises a plurality of microsilica particles, wherein 25-75 % of particle volume is less than 150 nm, such as i) 30-70 % of particle volume is less than 150 nm, ii) 35- 65 % of particle volume is less than 150 nm, iii) 40-60 % of particle volume is less than 150 nm or iv) 50% of particle volume is less than 150 nm.
In a further embodiment according to the present invention, the microsilica comprises a plurality of microsilica particles, wherein 5-45 % of particle volume is less than 100 nm, such as i) 10-40 % of particle volume is less than 100 nm, ii) 15- 35 % of particle volume is less than 100 nm, iii) 20-30 % of particle volume is less than 100 nm or iv) 25 % of particle volume is less than 100 nm.
In another embodiment according to the present invention, the microsilica comprises a plurality of microsilica particles; the microsilica particles having an average particle diameter in the range 50-500 nm, more preferably in the range 50- 300 nm, even more preferably in the range 50-200 nm and most preferably in the range 100-200 nm.
In another embodiment according to the present invention, the microsilica comprises a plurality of microsilica particles; the microsilica particles having an average particle diameter of more than 100 nm, such as more than 120 nm, more than 150 nm, more than 170 nm or more than 200 nm. In another embodiment according to the present invention, the microsilica has a bulk density in the range 70 to 800 kg/m3, such as in the range 200 to 800 kg/m3, in the range 300 to 800 kg/m3 or in the range 400 to 700 kg/m3.
In another embodiment according to the present invention, the microsilica has a bulk density in the range 400 to 800 kg/m3, such as 450 to 750 kg/m3, 500 to 700 kg/m3 or 550 to 650 kg/m3.
In another embodiment according to the present invention, the microsilica has a bulk density in the range 100 to 450 kg/m3, such as 150 to 400 kg/m3 or 200 to 350 kg/m3.
In a further embodiment, the microsilica has a specific gravity in the range 1.5 to 3.0, more preferably in the range 2.0 to 2.5 and even more preferably in the range 2.2 to 2.3.
In yet a further embodiment, the microsilica has a specific surface area in the range 10 to 50 m2/g, more preferably in the range 12 to 40 m2/g and even more preferably in the range 15 to 30 m2/g.
In yet another embodiment, the microsilica has a melting point in the range 1500 to 1600 °C, more preferably in the range 1540 to 1580 °C and even more preferably in the range 1550 to 1570 °C.
In another embodiment according to the present invention, the microsilica comprises an iron source.
In a further embodiment, the microsilica comprises an iron source, the iron source being one or more iron oxide(s). Preferably the one or more iron oxide(s) is Fe2O3.
In yet a further embodiment, the microsilica comprises 0.5 to 10 % iron oxide(s) by weight of the microsilica, preferably 0.5 to 5 % iron oxide(s) by weight of the microsilica, more preferably 1 to 5 % iron oxide(s) by weight of the microsilica and even more preferably 1 to 3 % iron oxide(s) by weight of the microsilica. Preferably the iron oxide(s) is Fe2O3.
Aqueous composition
In one embodiment according to the present invention, all nutrients essential to maintain growth of the diatoms are added to the aqueous liquid. In one preferred embodiment according to the present invention, all nutrients essential to maintain the diatoms in exponential growth phase are added to the aqueous liquid. In one embodiment according to the present invention, wherein the salt content of the aqueous composition is adjusted to a salt content in the range 20 to 50 g/L, preferably in the range 20 to 45 g/L, more preferably a salt content in the range 25 to 45 g/L, even more preferably in the range 25 to 40 g/L and most preferably a salt content in the range 25 to 35 g/L, such as a salt content of about 30 g/L.
In another embodiment according to the present invention, the pH of the aqueous composition is adjusted to a pH in the range 5.5 to 9.0, preferably a pH in the range 6.0 to 9.0, more preferably a pH in the range 7 to 9 and even more preferably in the range 7 to 8.
General
In one embodiment according to the present invention, at least 0,1 mg/L amorphous SiCL is added to the aqueous liquid to obtain the aqueous composition. In one embodiment according to the present invention, at least 0,14 mg/L amorphous SiCh is added to the aqueous liquid to obtain the aqueous composition. In one embodiment according to the present invention, at least 0,17 mg/L amorphous SiCh is added to the aqueous liquid to obtain the aqueous composition.
In one embodiment according to the present invention, CO2(g) is introduced into the aqueous liquid; and/or NCL', NO2- or any mixture thereof is introduced into the aqueous liquid. In one embodiment according to the present invention, a phosphorus source, such as fertilizer, is added to the aqueous liquid. Substral® provided by Scotts Company A/S (see example 1, table 2) and YaraTera Kristalon® Purple provided by Yara are two examples of fertilizers suitable to be added to the aqueous composition.
In one embodiment according to the present invention, factory smoke from production of silicon and/or ferrosilicon is introduced into the aqueous liquid. If factory smoke from production of silicon and/or ferrosilicon is to be introduced into the aqueous composition suitable for cultivating diatoms, the pH of the aqueous composition should preferably be >7, more preferably >7.5 and most preferably >7.8. However, if the pH of the aqueous composition is < 7, such as < 6.9 or <6.8 it is preferred not to introduce any further factory smoke from production of silicon and/or ferrosilicon.
A second aspect of the present invention relates to an aqueous composition for cultivation of diatoms prepared by the method according to the first aspect of the present invention. A third aspect of the present invention relates to an aqueous composition for cultivation of diatoms comprising an aqueous liquid and SiCh provided in the form of microsilica, wherein
- the amount of SiCh provided in the form of microsilica in the aqueous composition is at least 0,07 mg/L.
Diatom
In a first embodiment according to the present invention, the diatoms are marine diatoms.
In another embodiment according to the present invention the diatoms are photoautotrophic diatoms.
In a further embodiment according to the present invention the diatoms are centric diatoms.
In yet a further embodiment according to the present invention the mean diameter of the diatoms is > 25 pm.
In yet a further embodiment according to the present invention the diatoms are a diatom monoculture.
In yet a further embodiment according to the present invention the diatoms are a culture of diatoms wherein the majority of the diatoms are of the same species. In one embodiment more than 80% of the diatoms are of the same species, such as more than 85%, more than 90%, more than 95%, more than 96%, more than 97%, more than 98% or more than 99% of the diatoms are of the same species.
In yet a further embodiment according to the present invention the diatoms are a culture of diatoms wherein a substantial number of the diatoms is of the same species.
Aqueous liquid
In one embodiment according to the present invention, the aqueous liquid is water, such as freshwater or seawater.
In a further embodiment according to the present invention, the aqueous liquid is water and has a salt content in the range 20 to 50 g/L, preferably 20 to 45 g/L, more preferably a salt content in the range 25 to 45 g/L, even more preferably in the range 25 to 40 g/L and most preferably a salt content in the range 25 to 35 g/L, such as about 30 g/L. Microsilica
In another embodiment according to the present invention, the microsilica comprises > 70 % by weight amorphous silicon dioxide, preferably > 80 % by weight amorphous silicon dioxide, even more preferably > 85 % by weight amorphous silicon dioxide and most preferably > 90 % by weight amorphous silicon dioxide.
In yet a further embodiment, the microsilica is dissolved in the aqueous liquid and optionally any non-dissolved microsilica is removed from the aqueous liquid. In one embodiment, the microsilica is dissolved in the aqueous liquid and any nondissolved microsilica is removed from the aqueous liquid.
In another embodiment according to the present invention, the microsilica is dispersed in the aqueous liquid.
In a further embodiment according to the present invention, the microsilica is a byproduct from the production of silicon and/or ferrosilicon.
In one embodiment according to the present invention, the microsilica comprises a plurality of spherical microsilica particles.
In a further embodiment according to the present invention, the microsilica comprises a plurality of microsilica particles, wherein at least 10 % of particle volume is more than 100 nm. In another embodiment at least 20 % of particle volume is more than 100 nm. In another embodiment at least 30 % of particle volume is more than 100 nm. In another embodiment at least 40 % of particle volume is more than 100 nm. In another embodiment at least 50 % of particle volume is more than 100 nm. In another embodiment at least 55 % of particle volume is more than 100 nm. In yet another embodiment at least 60 % of particle volume is more than 100 nm. In yet another embodiment at least 65 % of particle volume is more than 100 nm. In yet another embodiment at least 70 % of particle volume is more than 100 nm. In yet another embodiment at least 75 % of particle volume is more than 100 nm. In yet another embodiment at least 80 % of particle volume is more than 100 nm. In yet another embodiment at least 85 % of particle volume is more than 100 nm. In yet another embodiment at least 90 % of particle volume is more than 100 nm. It is preferred that 55-95 % of particle volume is more than 100 nm, such as i) 60-90 % of particle volume is more than 100 nm, ii) 65-85 % of particle volume is more than 100 nm, iii) 70-80 % of particle volume is more than 100 nm or iv) 75% of particle volume is more than 100 nm. In a further embodiment according to the present invention, the microsilica comprises a plurality of microsilica particles, wherein at least 20 % of particle volume is more than 150 nm. In another embodiment at least 25 % of particle volume is more than 150 nm. In another embodiment at least 30 % of particle volume is more than 150 nm. In another embodiment at least 35 % of particle volume is more than 150 nm. In another embodiment at least 40 % of particle volume is more than 150 nm. In another embodiment at least 45 % of particle volume is more than 150 nm. In yet another embodiment at least 50 % of particle volume is more than 150 nm. In yet another embodiment at least 55 % of particle volume is more than 150 nm. In yet another embodiment at least 60 % of particle volume is more than 150 nm. In yet another embodiment at least 65 % of particle volume is more than 150 nm. In yet another embodiment at least 70 % of particle volume is more than 150 nm. In yet another embodiment at least 75 % of particle volume is more than 150 nm. In yet another embodiment at least 80 % of particle volume is more than 150 nm. It is preferred that 25-75 % of particle volume is more than 150 nm, such as i) 30-70 % of particle volume is more than 150 nm, ii) 35-65 % of particle volume is more than 150 nm, iii) 40-60 % of particle volume is more than 150 nm or iv) 50% of particle volume is more than 150 nm.
In a further embodiment according to the present invention, the microsilica comprises a plurality of microsilica particles, wherein 25-75 % of particle volume is less than 150 nm, such as i) 30-70 % of particle volume is less than 150 nm, ii) 35- 65 % of particle volume is less than 150 nm, iii) 40-60 % of particle volume is less than 150 nm or iv) 50% of particle volume is less than 150 nm.
In a further embodiment according to the present invention, the microsilica comprises a plurality of microsilica particles, wherein 5-45 % of particle volume is less than 100 nm, such as i) 10-40 % of particle volume is less than 100 nm, ii) 15- 35 % of particle volume is less than 100 nm, iii) 20-30 % of particle volume is less than 100 nm or iv) 25 % of particle volume is less than 100 nm.
In another embodiment according to the present invention, the microsilica comprises a plurality of microsilica particles; the microsilica particles having an average particle diameter in the range 50-500 nm, more preferably in the range 50- 300 nm, even more preferably in the range 50-200 nm and most preferably in the range 100-200 nm.
In another embodiment according to the present invention, the microsilica comprises a plurality of microsilica particles; the microsilica particles having an average particle diameter of more than 100 nm, such as more than 120 nm, more than 150 nm, more than 170 nm or more than 200 nm.
In another embodiment according to the present invention, the microsilica has a bulk density in the range 70 to 800 kg/m3, such as in the range 200 to 800 kg/m3, in the range 300 to 800 kg/m3 or in the range 400 to 700 kg/m3.
In another embodiment according to the present invention, the microsilica has a bulk density in the range 400 to 800 kg/m3, such as 450 to 750 kg/m3, 500 to 700 kg/m3 or 550 to 650 kg/m3.
In another embodiment according to the present invention, the microsilica has a bulk density in the range 100 to 450 kg/m3, such as 150 to 400 kg/m3 or 200 to 350 kg/m3.
In a further embodiment, the microsilica has a specific gravity in the range 1.5 to 3.0, more preferably in the range 2.0 to 2.5 and even more preferably in the range 2.2 to 2.3.
In yet a further embodiment, the microsilica has a specific surface area in the range 10 to 50 m2/g, more preferably in the range 12 to 40 m2/g and even more preferably in the range 15 to 30 m2/g.
In yet another embodiment, the microsilica has a melting point in the range 1500 to 1600 °C, more preferably in the range 1540 to 1580 °C and even more preferably in the range 1550 to 1570 °C.
In another embodiment according to the present invention, the microsilica comprises an iron source.
In a further embodiment, the microsilica comprises an iron source, the iron source being one or more iron oxide(s). Preferably the one or more iron oxide(s) is Fe2O3.
In yet a further embodiment, the microsilica comprises 0.5 to 10 % iron oxide(s) by weight of the microsilica, preferably 0.5 to 5 % iron oxide(s) by weight of the microsilica, more preferably 1 to 5 % iron oxide(s) by weight of the microsilica and even more preferably 1 to 3 % iron oxide(s) by weight of the microsilica. Preferably the iron oxide(s) is Fe2O3.
Aqueous composition
In one embodiment according to the present invention, the aqueous composition comprises all nutrients essential to maintain growth of the diatoms. In one preferred embodiment according to the present invention the aqueous composition comprises all nutrients essential to maintain the diatoms in exponential growth.
In one embodiment according to the present invention the salt content of the aqueous composition is in the range 20 to 50 g/L, preferably in the range 20 to 45 g/L, more preferably a salt content in the range 25 to 45 g/L, even more preferably in the range 25 to 40 g/L and most preferably a salt content in the range 25 to 35 g/L, such as a salt content of about 30 g/L.
In another embodiment according to the present invention, the pH of the aqueous composition is in the range 5.5 to 9.0, preferably in the range 6.0 to 9.0, more preferably in the range 7 to 9 and even more preferably in the range 7 to 8.
General
In one embodiment according to the present invention, the amount of SiCh provided in the form of microsilica in the aqueous composition is at least 0,1 mg/L. In one embodiment according to the present invention, the amount of SiCh provided in the form of microsilica in the aqueous composition is at least 0,14 mg/L. In one embodiment according to the present invention, the amount of SiCh provided in the form of microsilica in the aqueous composition is at least 0,17 mg/L.
In one embodiment according to the present invention, the aqueous composition comprises a carbon source and/or a nitrogen source, the carbon source being obtained by introducing CO2(g) into the aqueous composition and the nitrogen source being obtained by introducing NCL', NO2- or any mixture thereof into the aqueous composition. In yet a further embodiment, the aqueous composition comprises a phosphorus source, such as fertilizer. Substral® provided by Scotts Company A/S (see example 1, table 2) and YaraTera Kristalon® Purple provided by Yara are two examples of fertilizers suitable to be added to the aqueous composition.
In one embodiment according to the present invention, factory smoke from production of silicon and/or ferrosilicon is introduced into the aqueous composition. If factory smoke from production of silicon and/or ferrosilicon is to be introduced into the aqueous composition suitable for cultivating diatoms, the pH of the aqueous composition should preferably be >7, more preferably >7.5 and most preferably >7.8. However, if the pH of the aqueous composition is < 7, such as < 6.9 or <6.8 it is preferred not to introduce any further factory smoke from production of silicon and/or ferrosilicon. A fourth aspect of the present invention relates to a use of the aqueous composition according to the second or third aspect of the present invention for cultivation of diatoms.
Diatom
In a first embodiment according to the present invention, the diatoms are marine diatoms.
In another embodiment according to the present invention the diatoms are photoautotrophic diatoms.
In a further embodiment according to the present invention the diatoms are centric diatoms.
In yet a further embodiment according to the present invention the mean diameter of the diatoms is > 25 pm.
In yet a further embodiment according to the present invention the diatoms are a diatom monoculture.
In yet a further embodiment according to the present invention the diatoms are a culture of diatoms wherein the majority of the diatoms are of the same species. In one embodiment more than 80% of the diatoms are of the same species, such as more than 85%, more than 90%, more than 95%, more than 96%, more than 97%, more than 98% or more than 99% of the diatoms are of the same species.
In yet a further embodiment according to the present invention the diatoms are a culture of diatoms wherein a substantial number of the diatoms is of the same species.
A fifth aspect of the present invention relates to a method of producing a diatom biomass, the method comprising a step of culturing diatoms in the aqueous composition according to the second or third aspect of the present invention.
Diatom
In a first embodiment according to the present invention, the diatoms are marine diatoms.
In another embodiment according to the present invention the diatoms are photoautotrophic diatoms.
In a further embodiment according to the present invention the diatoms are centric diatoms. In yet a further embodiment according to the present invention the mean diameter of the diatoms is > 25 pm.
In yet a further embodiment according to the present invention the diatoms are a diatom monoculture.
In yet a further embodiment according to the present invention the diatoms are a culture of diatoms wherein the majority of the diatoms are of the same species. In one embodiment more than 80% of the diatoms are of the same species, such as more than 85%, more than 90%, more than 95%, more than 96%, more than 97%, more than 98% or more than 99% of the diatoms are of the same species.
In yet a further embodiment according to the present invention the diatoms are a culture of diatoms wherein a substantial number of the diatoms is of the same species.
A first alternative aspect of the present invention relates to an aqueous composition for cultivation of diatoms, the aqueous composition comprising an aqueous liquid and microsilica.
In a first embodiment according to the present invention, the diatoms are marine diatoms.
In another embodiment according to the present invention the diatoms are photoautotrophic diatoms.
In a further embodiment according to the present invention the diatoms are centric diatoms.
In yet a further embodiment according to the present invention the mean diameter of the diatoms is > 25 pm.
In yet a further embodiment according to the present invention the diatoms are a diatom monoculture.
In yet a further embodiment according to the present invention the diatoms are a culture of diatoms wherein the majority of the diatoms are of the same species. In one embodiment more than 80% of the diatoms are of the same species, such as more than 85%, more than 90%, more than 95%, more than 96%, more than 97%, more than 98% or more than 99% of the diatoms are of the same species. In yet a further embodiment according to the present invention the diatoms are a culture of diatoms wherein a substantial number of the diatoms is of the same species.
In one embodiment according to the present invention, the aqueous liquid is water, such as freshwater or seawater.
In a further embodiment according to the present invention, the aqueous liquid has a salt content in the range 20 to 50 g/L, preferably 20 to 45 g/L, more preferably a salt content in the range 25 to 45 g/L, even more preferably in the range 25 to 40 g/L and most preferably a salt content in the range 25 to 35 g/L, such as about 30 g/L.
In one embodiment according to the present invention, the microsilica is amorphous silicon dioxide.
In another embodiment according to the present invention, the microsilica contains > 60 % by weight amorphous silicon dioxide, > 70 % by weight amorphous silicon dioxide, preferably > 80 % by weight amorphous silicon dioxide, even more preferably > 85 % by weight amorphous silicon dioxide and most preferably > 90 % by weight amorphous silicon dioxide.
In yet a further embodiment, the microsilica is dissolved in the aqueous liquid and optionally any non-dissolved microsilica is removed from the aqueous liquid. In one embodiment, the microsilica is dissolved in the aqueous liquid and any nondissolved microsilica is removed from the aqueous liquid.
In another embodiment according to the present invention, the microsilica is dispersed in the aqueous liquid.
In a further embodiment according to the present invention, the microsilica is a byproduct from the production of silicon and/or ferrosilicon.
In one embodiment according to the present invention, the microsilica comprises a plurality of spherical microsilica particles.
In a further embodiment according to the present invention, the microsilica comprises a plurality of microsilica particles; and at least 80% of the microsilica particles have a diameter less than 1 pm, more preferably at least 90% of the microsilica particles have a diameter less than 1 pm and even more preferably all the microsilica particles have a diameter less than 1 pm. In another embodiment according to the present invention, the microsilica comprises a plurality of microsilica particles; and the microsilica particles have an average particle diameter in the range 50-500 nm, more preferably in the range 50- 300 nm, even more preferably in the range 50-200 nm and most preferably in the range 100-200 nm.
In another embodiment according to the present invention, the microsilica comprises an iron source.
In a further embodiment, the microsilica comprises an iron source, the iron source being Fe2O3.
In yet a further embodiment, the microsilica comprises 0.5 to 10 % of one or more iron oxides(s) by weight of the microsilica, preferably 0.5 to 5 % of one or more iron oxides(s) by weight of the microsilica, more preferably 1 to 5 % of one or more iron oxide(s) by weight of the microsilica and even more preferably 1 to 3 % of one or more iron oxide(s) by weight of the microsilica. Preferably, the one or more iron oxides is Fe2O3.
In yet a further embodiment, the microsilica comprises 0.5 to 10 % Fe2C>3 by weight of the microsilica, preferably 0.5 to 5 % Fe2C>3 by weight of the microsilica, more preferably 1 to 5 % Fe2C>3 by weight of the microsilica and even more preferably 1 to 3 % Fe2C>3 by weight of the microsilica.
In another embodiment according to the present invention, the microsilica has a bulk density in the range 70 to 800 kg/m3, preferably in the range 200 to 800 kg/m3, even more preferably in the range 300 to 800 kg/m3 and most preferably in the range 400 to 700 kg/m3.
In a further embodiment, the microsilica has a specific gravity in the range 1.5 to 3.0, more preferably in the range 2.0 to 2.5 and even more preferably in the range 2.2 to 2.3.
In yet a further embodiment, the microsilica has a specific surface area in the range 10 to 50 m2/g, more preferably in the range 12 to 40 m2/g and even more preferably in the range 15 to 30 m2/g.
In yet another embodiment, the microsilica has a melting point in the range 1500 to 1600 °C, more preferably in the range 1540 to 1580 °C and even more preferably in the range 1550 to 1570 °C. In one embodiment according to the present invention, the aqueous composition has a salt content in the range 20 to 50 g/L, preferably in the range 20 to 45 g/L, more preferably a salt content in the range 25 to 45 g/L, even more preferably in the range 25 to 40 g/L and most preferably a salt content in the range 25 to 35 g/L, such as a salt content of about 30 g/L.
In another embodiment according to the present invention, the aqueous composition has a pH in the range 5.5 to 9.0, preferably a pH in the range 6.0 to 9.0, more preferably a pH in the range 7 to 9 and even more preferably in the range 7 to 8.
In yet another embodiment according to the present invention, the amount of microsilica in the aqueous composition is > 0.00175 mg/L. In another embodiment the amount of microsilica in the aqueous composition is at least 0.002625 mg/L; more preferably at least 0.0035 mg/L; even more preferably at least 0,005 mg/L and most preferably at least 0.006 mg/L; such as at least 0.01 mg/L, at least 0.015 mg/L, at least 0.02 mg/L, at least 0.025 mg/L, at least 0.03 mg/L, at least 0.035 mg/L, at least 0.04 mg/L, at least 0.045 mg/L, at least 0.05 mg/L, at least 0.06 mg/L, at least 0.07 mg/L, at least 0.08 mg/L, at least 0.09 mg/L, at least 0.1 mg/L, at least 0.15 mg/L, at least 0.2 mg/L, at least 0.25 mg/L, at least 0.3 mg/L, at least 0.35 mg/L, at least 0.4 mg/L, at least 0.45 mg/L, at least 0.50 mg/L, at least 0.6 mg/L, at least 0.7 mg/L, at least 0.8 mg/L, at least 0.9 mg/L or at least 1 mg/L.
In a preferred embodiment, the amount of microsilica in the aqueous composition is at least 0.1 mg/L, at least 0.15 mg/L, at least 0.2 mg/L, at least 0.25 mg/L, at least 0.3 mg/L, at least 0.35 mg/L, at least 0.4 mg/L, at least 0.45 mg/L, at least 0.50 mg/L, at least 0.6 mg/L, at least 0.7 mg/L, at least 0.8 mg/L, at least 0.9 mg/L, at least 1 mg/L, at least 5 mg/L or at least 10 mg/L.
In a preferred embodiment according to the present invention, the amount of microsilica in the aqueous composition is in the range 0.1 mg/L to 100 mg/L, such as 0.1 mg/L to 80 mg/L, 0.1 mg/L to 60 mg/L, 0.1 mg/L to 40 mg/L or 0.1 mg/L to 20 mg/L.
In a preferred embodiment according to the present invention, the amount of microsilica in the aqueous composition is in the range 0.2 mg/L to 100 mg/L, such as 0.2 mg/L to 80 mg/L, 0.2 mg/L to 60 mg/L, 0.2 mg/L to 40 mg/L or 0.2 mg/L to 20 mg/L.
In a preferred embodiment according to the present invention, the amount of microsilica in the aqueous composition is in the range 0.5 mg/L to 100 mg/L, such as 0.5 mg/L to 80 mg/L, 0.5 mg/L to 60 mg/L, 0.5 mg/L to 40 mg/L or 0.5 mg/L to 20 mg/L.
In a preferred embodiment according to the present invention, the amount of microsilica in the aqueous composition is in the range 1 mg/L to 100 mg/L, such as 1 mg/L to 80 mg/L, 1 mg/L to 60 mg/L, 1 mg/L to 40 mg/L or 1 mg/L to 20 mg/L.
In a preferred embodiment according to the present invention, the amount of microsilica in the aqueous composition is in the range 5 mg/L to 100 mg/L, such as 5 mg/L to 80 mg/L, 5 mg/L to 60 mg/L, 5 mg/L to 40 mg/L or 5 mg/L to 20 mg/L.
In another embodiment according to the present invention, the amount of microsilica in the aqueous composition is in the range 0.002 mg/L to 1000 mg/L, more preferably in the range 0.004 mg/L to 1000 mg/L, even more preferably in the range 0.006 mg/L to 1000 mg/L and most preferably in the range 0.008 mg/L to 1000 mg/L, such as in the range 0.01 mg/L to 1000 mg/L, in the range 0.015 mg/L to 1000 mg/L, in the range 0.02 mg/L to 1000 mg/L, in the range 0.03 mg/L to 1000 mg/L, in the range 0.04 mg/L to 1000 mg/L, in the range 0.05 mg/L to 1000 mg/L, in the range 0.06 mg/L to 1000 mg/L, in the range 0.07 mg/L to 1000 mg/L, in the range 0.08 mg/L to 1000 mg/L, in the range 0.09 mg/L to 1000 mg/L or in the range 0.1 mg/L to 1000 mg/L.
In another embodiment according to the present invention, the amount of microsilica in the aqueous composition is in the range 0.002 mg/L to 100 mg/L, more preferably in the range 0.004 mg/L to 100 mg/L, even more preferably in the range 0.006 mg/L to 100 mg/L and most preferably in the range 0.008 mg/L to 100 mg/L, such as in the range 0.01 mg/L to 100 mg/L, in the range 0.015 mg/L to 100 mg/L, in the range 0.02 mg/L to 100 mg/L, in the range 0.03 mg/L to 100 mg/L, in the range 0.04 mg/L to 100 mg/L, in the range 0.05 mg/L to 100 mg/L, in the range 0.06 mg/L to 100 mg/L, in the range 0.07 mg/L to 100 mg/L, in the range 0.08 mg/L to 100 mg/L, in the range 0.09 mg/L to 100 mg/L or in the range 0.1 mg/L to 100 mg/L.
In another embodiment according to the present invention, the amount of microsilica in the aqueous composition is in the range 0.002 mg/L to 10 mg/L, more preferably in the range 0.004 mg/L to 10 mg/L, even more preferably in the range 0.006 mg/L to 10 mg/L and most preferably in the range 0.008 mg/L to 10 mg/L, such as in the range 0.01 mg/L to 10 mg/L, in the range 0.015 mg/L to 10 mg/L, in the range 0.02 mg/L to 10 mg/L, in the range 0.03 mg/L to 10 mg/L, in the range 0.04 mg/L to 10 mg/L, in the range 0.05 mg/L to 10 mg/L, in the range 0.06 mg/L to 10 mg/L, in the range 0.07 mg/L to 10 mg/L, in the range 0.08 mg/L to 10 mg/L, in the range 0.09 mg/L to 10 mg/L or in the range 0.1 mg/L to 10 mg/L.
In another embodiment according to the present invention, the aqueous composition is saturated in respect of microsilica.
In yet another embodiment according to the present invention, the amount of microsilica in the aqueous composition is in the range 0.00175 mg/L to saturated in respect of microsilica. In another embodiment the amount of microsilica in the aqueous composition is in the range 0.002625 mg/L to saturated in respect of microsilica; more preferably in the range 0.0035 mg/L to saturated in respect of microsilica; even more preferably in the range 0,005 mg/L to saturated in respect of microsilica; and most preferably in the range 0.006 mg/L to saturated in respect of microsilica; such as in the range 0.01 mg/L to saturated in respect of microsilica, in the range 0.015 mg/L to saturated in respect of microsilica, in the range 0.02 mg/L to saturated in respect of microsilica, in the range 0.025 mg/L to saturated in respect of microsilica, in the range 0.03 mg/L to saturated in respect of microsilica, in the range 0.035 mg/L to saturated in respect of microsilica, in the range 0.04 mg/L to saturated in respect of microsilica, in the range 0.045 mg/L to saturated in respect of microsilica, in the range 0.05 mg/L to saturated in respect of microsilica, in the range 0.06 mg/L to saturated in respect of microsilica, in the range 0.07 mg/L to saturated in respect of microsilica, in the range 0.08 mg/L to saturated in respect of microsilica, in the range 0.09 mg/L to saturated in respect of microsilica or in the range 0.1 mg/L to saturated in respect of microsilica.
In one embodiment the aqueous composition comprises an aqueous liquid and microsilica, wherein the amount of microsilica in the aqueous composition is in the range 0.005 mg/L to saturated in respect of microsilica. In another embodiment, the amount of microsilica in the aqueous composition is in the range 0.01 mg/L to saturated in respect of microsilica. In yet another embodiment according to the present invention the amount of microsilica in the aqueous composition is in the range 0.05 mg/L to saturated in respect of microsilica.
The person skilled in the art will understand that if the amount of microsilica in the aqueous composition exceeds the amount of microsilica that is dissolvable in the aqueous composition, the non-dissolved microsilica may still be contained in the aqueous composition in the form of non-dissolved matter or may be removed from the aqueous composition by e.g. precipitation. Precipitation may be facilitated e.g. by centrifugation. In one embodiment according to the present invention, the aqueous composition further comprises essential nutrients to maintain growth of the diatoms, preferably to maintain the diatoms in exponential growth phase.
In another embodiment according to the present invention, the aqueous composition further comprises a carbon source and/or a nitrogen source and/or a phosphorus source or any mixture thereof. In one embodiment, the carbon source is obtained by introducing CO2(g) into the aqueous composition. In another embodiment, the nitrogen source is obtained by introducing NO?’, NO2- or any mixture thereof into the aqueous composition.
In yet a further embodiment, the carbon source and the nitrogen source are obtained by introducing factory smoke from production of silicon and/or ferrosilicon into the aqueous composition. If factory smoke from production of silicon and/or ferrosilicon is to be introduced into the aqueous composition suitable for cultivating diatoms, the pH of the aqueous composition should preferably be >7, more preferably >7.5 and most preferably >7.8. However, if the pH of the aqueous composition is < 7, such as < 6.9 or <6.8 it is preferred not to introduce any further factory smoke from production of silicon and/or ferrosilicon.
In yet another embodiment, the phosphorus source is obtained by adding fertilizer into the aqueous composition. Substral® provided by Scotts Company A/S (see example 1, table 2) and YaraTera Kristalon® Purple provided by Yara are two examples of fertilizers suitable to be added to the aqueous composition.
A second alternative aspect of the present invention relates to a use of the aqueous composition according to the first alternative aspect of the present invention, for cultivation of diatoms.
In one embodiment the diatoms are marine diatoms.
In another embodiment, the diatoms are photoautotrophic diatoms.
In yet another embodiment the diatoms are centric diatoms.
In yet a further embodiment the mean diameter of the diatoms is > 25 pm.
In yet a further embodiment according to the present invention the diatoms are a diatom monoculture.
In yet a further embodiment according to the present invention the diatoms are a culture of diatoms wherein the majority of the diatoms are of the same species. In one embodiment more than 80% of the diatoms are of the same species, such as more than 85%, more than 90%, more than 95%, more than 96%, more than 97%, more than 98% or more than 99% of the diatoms are of the same species.
In yet a further embodiment according to the present invention the diatoms are a culture of diatoms wherein a substantial number of the diatoms is of the same species.
A third alternative aspect of the present invention relates to a method of producing a diatom biomass, the method comprising a step of culturing diatoms in the aqueous composition according to the first alternative aspect of the present invention.
In one embodiment the diatoms are marine diatoms.
In another embodiment, the diatoms are photoautotrophic diatoms.
In yet another embodiment the diatoms are centric diatoms.
In yet a further embodiment the mean diameter of the diatoms is > 25 pm.
In yet a further embodiment according to the present invention the diatoms are a diatom monoculture.
In yet a further embodiment according to the present invention the diatoms are a culture of diatoms wherein the majority of the diatoms are of the same species. In one embodiment more than 80% of the diatoms are of the same species, such as more than 85%, more than 90%, more than 95%, more than 96%, more than 97%, more than 98% or more than 99% of the diatoms are of the same species.
In yet a further embodiment according to the present invention the diatoms are a culture of diatoms wherein a substantial number of the diatoms is of the same species.
Brief description of drawings
Figure la illustrates production of diatom biomass applying the following five growth conditions:
Substral® (0.25 mL/ L)
Figure imgf000024_0001
Substral® (0.25 mL/ L) + Silicate (3.5 g/L Na2O3Si°9H2O) Substral® (0.25 mL/ L) + microsilica (maximum dissolvable amount)
Figure imgf000025_0001
Substral® (0.25 mL/ L) + introduction of factory smoke for 60 minutes per day
Vertical axis: Number of cells/L.
Horizontal axis: Number of days after start of cultivation.
Area under the curve: Represents the amount of biomass produced
The data presented in figure la, lb and 1c were collected from the same experiment. The cell count was measured at day 0, 2, 4, 7, 9 and 11 after start of cultivation while the cell count at day 1, 3, 5, 6, 8 and 10 are estimated values.
Figure lb illustrates production of diatom biomass applying the following five growth conditions:
Figure imgf000025_0002
es per day
Vertical axis: Biomass growth (%).
Horizontal axis: Number of days after start of cultivation.
The data presented in figure la, lb and 1c were collected from the same experiment. The number of cells was measured at day 0, 2, 4, 7, 9 and 11 after start of cultivation. The number of cells at day 0 was set equal to 100% and the values for days 2, 4, 7, 9 and 11 were calculated relatively to the value at day 0. The values count at day 1, 3, 5, 6, 8 and 10 are estimated values.
Figure 1c illustrates the pH during production of biomass applying the following five growth conditions:
■ a Substral® (0.25 mL/ L) Substral® (0.25 mL/ L) + Silicate (3.5 g/L Na2O3Si°9H2O)
Substral® (0.25 mL/ L) + microsilica (maximum dissolvable amount)
Substral® (0.25 mL/ L) + introduction of factory smoke for 60 minutes per day
Vertical axis: pH
Horizontal axis: Days after start of cultivation The data presented in figure la, lb and 1c were collected from the same experiment. The pH was measured at day 0, 2, 4, 7, 9 and 11 after start of cultivation.
Figure 2 represents a flowchart to determine if a species is autotroph, heterotroph, or a subtype.
Figure 3a illustrates production of diatom biomass applying different amounts of microsilica in the growth medium, wherein Pl, P2 and P3 have high, intermediate, and low amounts of microsilica in the growth medium (see example 3) respectively. P4 represents control with no added microsilica to the growth medium.
Vertical axis: Number of cells per liter.
Horizontal axis: Day number, wherein experiment starts at day 1.
Figure 3b shows the concentration of SiCh in a growth medium during cultivation of diatoms at different growth medium conditions at start. Pl, P2 and P3 have high, intermediate and low amounts of microsilica in the growth medium (see example 3). P4 represents control with no added microsilica to the growth medium.
Vertical axis: mg SiCh per liter
Horizontal axis: Day number, wherein experiment starts at day 1.
Figure 4 shows typical particle size distribution of microsilica, wherein about 25% of the particle volume is less than 0,1 pm and about 50% of the particle volume is less than 0,15 pm.
Vertical axis: percent
Horizontal axis: microns (d)
Detailed description of the invention
Unless specifically defined herein, all technical and scientific terms used have the same meaning as commonly understood by a skilled artisan in the fields of biochemistry and molecular biology.
All methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, with suitable methods and materials being described herein. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will prevail. Where a numeric limit or range is stated, the endpoints are included. Also, all values and subranges within a numerical limit or range are specifically included as if explicitly written out.
There is an urgent need for high omega-3 formulated fish feed in the fish farming industry. The omega-3 fatty acids EPA (eicosapentaenoic acid) and DHA (docosahexaenoic acid), which are associated with beneficial health effects in human nutrition, are found in relatively high levels in diatoms which therefore represent a promising source of omega-3 fatty acids.
However, in order to be able to industrialize production of omega-3 fatty acids from diatoms, it is important to establish sustainable and cost-effective methodologies for cultivation of such algae.
Diatoms are unicellular microalgae enclosed within a silica cell wall. The silica cell wall is a unique property of this algae, and it is well known in the art that silicon availability is a key factor in the regulation of diatom growth. Silicon is typically supplied to the ocean both dissolved as monomeric or oligomeric form or as solid particulates in variable size and crystallinity. Dissolved silicon can be loaded by river runoff as well as by the dissolution of particulate silicon present in the lithogenic and biogenic systems. In the sea the biogeochemical dissolution of this mineral is controlled by temperature, zooplankton grazing, diatom sinking and bacterial activity.
Studies on the uptake of dissolved silicon by diatom cells have demonstrated that active silicon transport and the specific Si-dependent cell- division rate follow Michaelis-Menten kinetics. Silicon transport inside the cell seems to be carrier- mediated, and in marine diatoms the ion-carrier can be Na+ dependent with metabolic energy supply. The carrier function of silicon transport inside the cell can be mediated by the action of a protein: the silicon transporter (SIT), present in marine and freshwater diatoms. Silica deposition and morphogenesis of the cell wall seems to occur in a specialized compartment: the Silica Deposition Vesicle, known as SDV. In the SDV, the acidic environment contributes to the silicic acid polymerization into amorphous hydrated silica onto an organic template (proteins, polysaccharides, lipids), that drives the frustule silicification and morphogenesis.
Generally, it is assumed that diatoms take up silicon as undissociated silicic acid, Si(OH)4, at pH 8.0 from the marine environment. This is the dominant form, constituting 97% of total dissolved Si (dSi); the remainder is essentially SiO(OH)3. Despite numerous studies focused on characterizing the chemistry of silicon uptake and deposition, the influence on diatom growth of different particulate silica forms as a silicon source has not been extensively studies. This issue is of high interest when establishing sustainable and cost-effective methodologies for cultivation of such algae. In the search for sustainable and cost-effective methodologies for cultivation of diatoms, the present inventors have studied the influence on diatom growth of different forms of silica. The results of that research are presented in example 1, and in particular table 5 and figure lb.
Diatoms cultivated for 11 days in filtered seawater with 0.25 mL/ L substral® (example 1, table 2) were able to reach a biomass that was 2.5-fold higher than the biomass at start of cultivation (experimental setup A). If 3.5 g/L Na2O3Si°9H2O was added to the culture medium during the 11 days period, the culture reached a biomass that was about 4.9-fold higher than the biomass at start of cultivation (experimental setup B). The pH in the culture medium during the 11 days period was not significantly different between experimental setup A and experimental setup B. These results give clear indication that silicon availability is a key factor in the regulation of diatom growth.
Further, in experimental setup C, diatoms were cultivated for 11 days in filtered seawater with 0.25 mL/ L substral® and dissolved amorphous silicon dioxide (microsilica). The culture reached a biomass that was about 7-fold higher than the biomass at start of cultivation. The pH in the culture medium during the 11 days period was not significantly different between experimental setup A, experimental setup B and experimental setup C. This surprising finding is a clear indication that diatoms have a preference for amorphous silicon dioxide as compared to silicate.
The amorphous silicon dioxide that was added to the culture medium in experimental setup C is a by-product of the production of silicon/ferrosilicon and was collected at a silicon/ferrosilicon factory in Finnsnes owned by Finnfjord AS. Another by-product of that process is the factory smoke which is known to contain inter alia carbon dioxide and nitrogen oxides (NOX).
It is well known that diatoms need a nitrogen source to grow and that they are very effective in converting carbon dioxide into organic compounds. In an effort to reduce the carbon footprint of the silicon/ferrosilicon factory and at the same time provide a cost-effective methodology for cultivating diatoms, the inventors decided to investigate the effect on diatom growth of introducing factory smoke into the growth medium.
In experimental setup D, diatoms were cultivated for 11 days in filtered seawater with 0.25 mL/ L substral® wherein factory smoke, derived from a silicon/ferrosilicon factory in Finnsnes owned by Finnfjord AS, was introduced into the growth medium for a period of 60 minutes per day (bubbles of factory smoke introduced into the growth medium). As shown in example 1, table 5 the culture reached a biomass that was about 3.9-fold higher than the biomass at start of cultivation. In experimental setup A (without factory smoke), the culture reached a biomass that was about 2.5-fold higher than the biomass at start of cultivation. The pH in the culture medium during the 11 days period was somewhat lower in experimental setup D as compared to experimental setup A, but the pH did not reach a critically low value. This surprising finding is a clear indication that diatoms are able to feed on the factory smoke with the result being improved growth rate and a reduced carbon footprint for the silicon/ferrosilicon factory; a win-win situation.
In summary, the present inventors have demonstrated that both microsilica and factory smoke derived from a silicon/ferrosilicon factory are able to improve the growth rate of diatoms in large volume closed bioreactors. Thus, the present inventors have been able to provide sustainable and cost-effective methodologies for cultivation of diatoms in large volume closed bioreactors.
In order to investigate the minimum amount of microsilica needed to have a positive effect on the growth rate of diatoms, the inventors designed a study where four different groups of diatoms (Example 3, Pl, P2, P3 and P4) were cultivated under different growth conditions. As shown in figure 3 a, the growth rates of Pl and P2 (0,171 and 0,142 mg/L SiCh) were substantially higher than P3 and P4 (0,066 and 0,003 mg/L SiCh) and P3 (0,066 mg/L SiCL) had a significantly better growth rate compared to P4 (0,003 mg/L SiCh). The final yield was also substantially higher in terms of final cell numbers (i.e. biomass) in that Pl and P2 were 6 934 632 cells/L and 5 697 180 cells/L respectively while P3 and P4 were 2 205 360 and 1 353 625 cells/L respectively.
In short, the present inventors have demonstrated that 0,066 mg/L SiCh (provided in the form of microsilica) is sufficient to have a significant effect on the growth rate of diatoms and that > 0,142 mg/L SiCh is superior to the effect of 0,066 mg/L SiCb.
Thus, a first aspect of the present invention relates to a method of preparing an aqueous composition for cultivation of diatoms, the method comprising the following step(s):
- adding at least 0,07 mg/L amorphous SiCh to an aqueous liquid to obtain the aqueous composition; wherein
- the amorphous SiCL is added in the form of microsilica.
A second aspect of the present invention relates to an aqueous composition for cultivation of diatoms prepared by the method according to the first aspect of the present invention.
A third aspect of the present invention relates to an aqueous composition for cultivation of diatoms comprising an aqueous liquid and SiCL provided in the form of microsilica, wherein - the amount of SiCh provided in the form of microsilica in the aqueous composition is at least 0,07 mg/L.
Amorphous silicon dioxide is the main ingredient in microsilica, typically constituting > 85 % by weight of the microsilica. Adding microsilica to an aqueous liquid will therefore increase the level of SiCh in the aqueous liquid significantly.
The expression “the amount of SiCh provided in the form of microsilica in the aqueous composition” refers to the process of adding microsilica to the aqueous liquid and the resulting increase in the amount of SiCh in the aqueous liquid. Said in other words: the amount of SiCh in the aqueous liquid that originates from microsilica or the amount of SiCh in the aqueous liquid that is derived from microsilica.
Nutritional needs
There are several well-known aqueous compositions that are suitable for cultivation of diatoms, and the present invention lies in modifying those well-known products by adding silicon dioxide in the form of microsilica. The first, second and third aspects of the present invention clearly identifies the product to be modified (aqueous compositions suitable for cultivation of diatoms) and specifies what is modified and in what way. The other ingredients necessary for cultivation of diatoms is thus implied by the generic reference to an aqueous composition suitable for cultivation of diatoms.
In general, it is well known that microalgae require macro-, micronutrients and vitamins for growth (Scientific Reports volume 9, Article number: 1479 (2019)). Macronutrients correspond to nitrogen (N) and phosphorus (P), while micronutrients correspond to trace metals such as iron, manganese, cobalt, etc. In addition, vitamins such as thiamine, biotin and cobalamin (vitamins Bl, B7 and B12, respectively) are also often needed since some microalgal species are not able to synthesize them. Further, diatoms require silicon (Si), which is involved in building the outer cell wall, or frustule.
Both Substral® and Guillard’s f/2 marine nutrient enrichment solution, referred to in table 1 below, provide required amounts of macronutrients, micronutrients and vitamins to facilitate growth of Diatoms. However, for optimal growth it may be required to add silicon, such as silicate or microsilica, and most preferably silicon in the form of microsilica. If silicate is to be used, it is preferred to use metasilicate pentahydrate.
Another well-known product which is able to provide the required amounts of macronutrients and micronutrients to facilitate growth of diatoms is YaraTera Kristalon® Purple. Said product is provided by Yara and has the following composition:
Figure imgf000031_0001
Figure imgf000031_0002
Thus, in one embodiment according to the present invention the aqueous composition for cultivation of diatoms comprises macro-, micronutrients and optionally vitamins in sufficient amounts to facilitate growth of diatoms.
Said macronutrients comprising a source of nitrogen (N) and a source of phosphorus (P). In one embodiment the source of nitrogen is selected from the group consisting of nitrate, ammonium nitrogen, ammonia and any mixture thereof. In one embodiment, the source of phosphorus is phosphate, such as sodium phosphate.
Said micronutrients comprising trace metals selected from the group consisting of iron, manganese, cobalt, zinc, magnesium, boron, potassium, sulphur and any mixture thereof.
Said vitamins being selected from the group consisting of thiamine, biotin, cobalamin (vitamins Bl, B7 and Bl 2, respectively) and any mixture thereof.
Aqueous composition
The term “aqueous composition” is a composition in which the solvent is water. The water may be seawater, freshwater or any combination thereof.
In one embodiment according to the present invention, the water has a salt content in the range 20 to 50 g/L, preferably in the range 20 to 45 g/L, more preferably a salt content in the range 25 to 45 g/L, even more preferably in the range 25 to 40 g/L and most preferably a salt content in the range 25 to 35 g/L, such as a salt content of about 30 g/L.
Diatoms
The term “diatoms” refers to a major group of microalgae found in the oceans, waterways and soils of the world. In one embodiment according to the present invention, the diatoms are marine diatoms. The term “marine diatoms” referring to the diatoms that naturally live in the oceans, i.e. seawater.
Diatoms are unicellular: they occur either as solitary cells or in colonies, which can take the shape of e.g. ribbons, curls, waves, fans, zigzags, balls or stars. Individual cells typically range in size from 2 to 200 micrometers. In one embodiment according to the present invention, the mean diameter of the diatoms is > 25 pm.
Diatoms have two distinct shapes: a few (centric diatoms) are radially symmetric, while most (pennate diatoms) are broadly bilaterally symmetric. In one embodiment according to the present invention, the diatoms are centric diatoms, such as marine centric diatoms.
A unique feature of diatom anatomy is that they are surrounded by a cell wall made of silica (hydrated silicon dioxide), called a frustule. These frustules have structural coloration due to their photonic nanostructure, prompting them to be described as "jewels of the sea" and "living opals". Similar to plants, diatoms convert light energy to chemical energy by photosynthesis, although this shared autotrophy evolved independently in both lineages. An autotroph or primary producer, is an organism that produces complex organic compounds (such as carbohydrates, fats, and proteins) from simple substances present in its surroundings, generally using energy from light (photosynthesis) or inorganic chemical reactions (chemosynthesis). They are the producers in a food chain, such as plants on land or algae in water (in contrast to heterotrophs as consumers of autotrophs). They do not need a living source of energy or organic carbon. Autotrophs can reduce carbon dioxide to make organic compounds for biosynthesis and also create a store of chemical energy. Most autotrophs use water as the reducing agent, but some can use other hydrogen compounds such as hydrogen sulfide. Some autotrophs, such as green plants and algae, are phototrophs, meaning that they convert electromagnetic energy from sunlight into chemical energy in the form of reduced carbon.
Autotrophs can be photoautotrophs or chemoautotrophs. Phototrophs use light as an energy source, while chemotrophs use electron donors as a source of energy, whether from organic or inorganic sources. However, in the case of autotrophs, these electron donors come from inorganic chemical sources. Such chemotrophs are lithotrophs. Lithotrophs use inorganic compounds, such as hydrogen sulfide, elemental sulfur, ammonium and ferrous iron, as reducing agents for biosynthesis and chemical energy storage. Photoautotrophs and lithoautotrophs use a portion of the ATP produced during photosynthesis or the oxidation of inorganic compounds to reduce NADP+ to NADPH to form organic compounds.
In one embodiment according to the present invention, the diatoms are photoautotrophic diatoms, such as marine photoautotrophic diatoms.
In a preferred embodiment according to the present invention, the diatoms are centric photoautotrophic diatoms, such as marine centric photoautotrophic diatoms.
Aqueous liquid
The term “aqueous liquid” is a liquid in which the solvent is water. In one embodiment according to the present invention the aqueous liquid is water. The water may be seawater, freshwater or any combination thereof.
In one embodiment according to the present invention, the water has a salt content in the range 20 to 50 g/L, preferably in the range 20 to 45 g/L, more preferably a salt content in the range 25 to 45 g/L, even more preferably in the range 25 to 40 g/L and most preferably a salt content in the range 25 to 35 g/L, such as a salt content of about 30 g/L.
In one embodiment according to the present invention, the aqueous liquid has a salt content in the range 20 to 50 g/L, preferably a salt content in the range 20 to 45 g/L, more preferably in the range 25 to 45 g/L, even more preferably in the range 25 to 40 g/L and most preferably a salt content in the range 25 to 35 g/L, such as a salt content of about 30 g/L.
Microsilica
The aqueous composition according to the second or third aspect of the present invention comprises an aqueous liquid to which amorphous SiCh in the form of microsilica has been added. Further, the aqueous composition according to the first alternative aspect of the present invention relates to an aqueous composition comprising an aqueous liquid and microsilica.
Silica fume, also known as microsilica, (CAS number 69012-64-2, EINECS number 273-761-1) is an amorphous (non-crystalline) polymorph of silicon dioxide, silica.
Microsilica is typically an ultrafine powder which may consist of spherical particles, non- spherical particles or a mixture thereof. In one embodiment according to the present invention, the microsilica comprises a plurality of spherical microsilica particles.
Microsilica may be collected as a by-product of the silicon and ferrosilicon alloy production, and such microsilica will typically have the following properties: about 25% of the particle volume less than 100 nm; about 50% of particle volume less than 150 nm; specific surface (BET) typically 20 m2/g (range 15-30);
- > 85 % by weight amorphous SiCh; some aluminum oxide, iron oxide and alkalis.
Microsilica may be provided in undensified form, which typically has a density of 200-350 kg/m3, or in densified form which typically has a density of 550-650 kg/m3.
Based on the above data it is clear that microsilica particles are polydisperse, which means that the particles in an ensemble have different sizes. I addition to size differences, the particles in an ensemble will also typically have different geometrical shape.
There are a number of different techniques in the art for analyzing particle size in such complex systems. In most of these methods the size is an indirect measure, obtained by a model that transforms, in abstract way, the real particle shape into a simple and standardized shape, like a sphere (the most usual) or a cuboid (when minimum bounding box is used), where the size parameter (ex. diameter of sphere) makes sense. Definition of the particle size for an ensemble (collection) of particles of different size presents another problem. The notion of particle size distribution reflects this polydispersity.
One way of expressing particle size is the so called “volume-based particle size model”. In this model, the volume-based particle size equals the diameter of the sphere that has the same volume as a given particle. This model is typically used in sieve analysis, as shape hypothesis (sieve's mesh size as the sphere diameter).
Figure imgf000035_0001
D diameter of representative sphere
V: volume of particle
Amorphous silicon dioxide is the main ingredient in microsilica, typically constituting > 85 % by weight of the microsilica. Adding microsilica to an aqueous liquid will therefore increase the level of SiCh in the aqueous liquid significantly. Reference is made to the following examples illustrating the amount of microsilica required to obtain a desired amount of SiCh.
Calculation X: adding 0,1 mg microsilica to IL of an aqueous liquid will provide an aqueous liquid containing 0,07 mg (amorphous) SiCh provided that 70 % by weight of the microsilica is amorphous SiCh.
Calculation Y: adding 0,07 mg amorphous SiCh, in the form of microsilica, to an aqueous liquid will require that you add 0,1 mg microsilica to IL of an aqueous liquid provided that 70 % by weight of the microsilica is amorphous SiCL.
Further, microsilica collected as a by-product of the silicon and ferrosilicon alloy production typically comprises i) an iron source, such as one or more iron oxide(s), e.g. Fe2O3; ii) an aluminum source, such as one or more aluminum oxide(s), e.g. AI2O3; and iii) a magnesium source, such as one or more magnesium oxide(s), e.g. MgO.
Thus, in one embodiment according to the present invention, the microsilica comprises an iron source, such as one or more iron oxide(s), the iron source preferably being Fe2C>3. In yet a further embodiment, the microsilica comprises 0.5 to 10 % by weight of one or more iron oxide(s), such as 0.5 to 10 % by weight Fe2C>3. In a preferred embodiment, the microsilica comprises 1 to 5 % by weight of one or more iron oxide(s), such as 1 to 5 % by weight Fe2O3. Even more preferably the microsilica comprises 1 to 3 % by weight of one or more iron oxide(s), such as 1 to 3 % by weight Fe2O3. In another embodiment according to the present invention, the microsilica comprises an aluminum source, such as one or more aluminum oxide(s), the aluminum source preferably being AI2O3. In yet a further embodiment, the microsilica comprises 0.1 to 10 % by weight of one or more aluminum oxide(s), such as 0.1 to 10 % by weight AI2O3. In a preferred embodiment, the microsilica comprises 0,3 to 5 % by weight of one or more aluminum oxide(s), such as 0,3 to 5 % by weight AI2O3. Even more preferably the microsilica comprises 0,5 to 1,5 % by weight of one or more aluminum oxide(s), such as 0,5 to 1,5 % by weight AI2O3.
In another embodiment according to the present invention, the microsilica comprises a magnesium source, such as one or more magnesium oxide(s), the magnesium source preferably being MgO. In yet a further embodiment, the microsilica comprises 0.1 to 10 % by weight of one or more magnesium oxide(s), such as 0.1 to 10 % by weight MgO. In a preferred embodiment, the microsilica comprises 0,3 to 5 % by weight of one or more magnesium oxide(s), such as 0,3 to 5 % by weight MgO. Even more preferably the microsilica comprises 0,5 to 2 % by weight of one or more magnesium oxide(s), such as 0,5 to 2 % by weight MgO; and most preferably the microsilica comprises 0,5 to 1,5 % by weight of one or more magnesium oxide(s), such as 0,5 to 1,5 % by weight MgO.
Having generally described this invention, a further understanding can be obtained by reference to the following example, which is provided herein for purposes of illustration only, and are not intended to be limiting unless otherwise specified.
Examples
Example 1: Cultivation of diatoms
Non-axenic clonal culture of a planktonic marine diatom was established from a single cell from a sample collected in the Barents Sea. Isolation method applied was a combination of microscope aided manual capillary pipetting and serial dilution transfer (i.e. transferring a single cell to a well in a 4-well chamber (Nunc) filled with autoclaved seawater and serial transferring it 5-10 times to rinse it). Although non-axenic, bacterial numbers were very low. The culture collection and innocula for mass cultures were cultivated in pasteurized seawater from 10m depth in Tromsoysund with 4 mL L-1 of Guillard’s f/2 x 50 marine water enrichment solution (Sigma-Aldrich, table 1). Table 1
Figure imgf000037_0001
The diatoms were cultivated in 150L transparent plexiglas columns in temperature and irradiance-controlled rooms (temperature of about 7 °C and scalar irradiance inside the columns of about 30 pmol photons m-2 s-1). The cultivation took place in Millipore filtered seawater (0.22-pm pore size) with nutrients as indicated in table 2. Each treatment was represented by triplicate cultures, hence the given cell numbers are means of the triplicates. All given C.V. were below 4%, hence data conclusions are based on did not overlap. Osram L 58W/954 Daylight tubes placed around the columns provided illumination, and the photoperiod was 14: 10 (light/dark). Photosynthetical active radiation (PAR) was measured with a QSL-100 scalar irradiance meter (Biosperical Instruments Inc.). All cultures were mixed/aerated at a rate of ca. 2.5 L min-1.
Table 2
Figure imgf000037_0002
Substral Substral (Scotts Company (Nordics) A/S)), see table 1
Silicate Silicate, Na2O3Si-9H2O, Sigma-Aldrich
The microsilica is a by-product from the production of silicon/ferrosilicon. The microsilica was collected at a silicon/ferrosilicon factory in Finnsnes owned by Microsilica Finnfjord AS. An analysis of the microsilica is provided in table 6.
Factory smoke Factory smoke was derived from a silicon/ferrosilicon factory in Finnsnes owned by Finnfjord AS.
The algae cultures were sampled at day 0, 2, 4, 7, 9 and 11 after start of cultivation. Each sample was analyzed with respect to number of cells, counted in inverted microscopes after minimum 4 hrs. sedimentation in Nunclon 4 well 2 mL chambers (table 3) and pH (table 4). Table 3
Figure imgf000038_0001
Experimental setup A, B, C and D are presented in table 2. A graphical presentation of the results is presented in figure la.
Table 4
Figure imgf000038_0002
Experimental setup A, B, C and D are presented in table 2. A graphical presentation of the results is presented in figure 1c.
As can be seen from table 3, the number of cells at start of cultivation varies considerably between experimental setups. In order to be able to compare the biomass growth between experimental setups, it was decided to set the number of cells at start of cultivation equal to 100 %. The number of cells at day 2, 4, 7, 9 and 11 after start of cultivation was calculated relatively to the number of cells at day 0.
The results are presented in table 5 below. Table 5
Figure imgf000039_0001
Experimental setup A, B, C and D are presented in table 2. A graphical presentation of the results is presented in figure lb.
The results presented in figure lb clearly show that: diatoms grow significantly faster when factory smoke is introduced into the growth medium as compared to diatoms grown with no introduction of factory smoke; and
- diatoms grow significantly faster in the presence of microsilica as compared to diatoms grown in the presence of silicate. The microsilica product utilized in this experiment is a by-product from the production of silicon and ferrosilicon and contains more than 85 % by weight amorphous silicon dioxide (analysis of the microsilica product utilized in this experiment is presented in table 6). Thus, it is believed that it is amorphous silicon dioxide that is the factor being responsible for the observed increase in growth rate. However, the microsilica product utilized in this experiment also contains other elements that may contribute to the increased growth rate. Iron in the form of Fe2O3 being an interesting candidate in this respect.
able 6
Figure imgf000040_0001
he bulk density being kg/m3 while the other values representing % by weight of the microsilica powder.
Reference example 2a: Prior art cultivation of diatoms I
Master Thesis in Marine Biology entitled Bioactivity Potential of an Arctic Marine Diatom Species Cultivated at Different Conditions”, 2018, section 4, pages 21-22 refers to a diatom monoculture that was cultivated in a photobioreactor with the addition of factory smoke. The factory smoke is said to contain, amongst others, CO2, NOX and microsilica. However, the amount of microsilica in said culture media is not being disclosed.
It is well known that factory smoke may contain microsilica and that the content of microsilica will vary to some extent. Provided that the factory smoke referred to in said master thesis had a regular content of microsilica, the factory smoke should have added about 0.0105 g microsilica to 6000 L culture media during the experiment. Thus, the culturing media referred to in said master thesis is assumed to contain about 0.00175 mg/L microsilica.
Reference is made to example 1 of the present application, and in particular the results presented in figure la and lb, which provides comparative growth rate data using either a culturing media to which microsilica has been added or culturing media to which factory smoke has been introduced for 60 minutes.
Reference example 2b: Prior art cultivation of diatoms II
Algal Culturing Techniques (R, A. Andersen, eds), 1 January 2005, pages 429-538 discloses f/2 Medium for growing diatoms containing sodium silicate.
Reference is made to example 1 of the present application, and in particular the results presented in figure la and lb, which provides comparative growth rate data using either a culturing media to which microsilica has been added or culturing media to which 3.5 g/L Na2O3Si°9H2O has been added.
Example 3: Cultivation of diatoms
Non-axenic clonal culture of a planktonic centric marine diatom was retrieved from stock monoculture samples (kept at UiT The arctic university of Norway) originally collected in the Arctic Barents Sea. The culturing was performed using conventional Guillards f/2 growth medium without Si (control, Treatment P4) , while Si was added as microsilica dissolved in water and added to different concentrations (Treatments Pl, P2, P3) and Si was measured underway in the experiment by standard colorimetric methods (Strickland and Parsons, 1972) using a Flow Solution IV analyzer from O.I. Analytical, USA (see Table 7 and figure 3b). The analyzer was calibrated using reference seawater from Ocean Scientific International Ltd. UK. The cultivation took place in 2L PAH 98% light transmittent flasks in temperature and irradiance-controlled rooms. Temperature was 7 - 8°C, light period was 16:8 (L:D) and scalar irradiance in the middle of the flasks was 70-30 pmol photons m-2 s’1 (self-shading occurs when cultures grow). Photosynthetical active radiation (PAR) was measured with a QSL-100 scalar irradiance meter (Biosperical Instruments Inc.).
All cultures were mixed/aerated twice daily. Each treatment was represented by triplicate cultures and the given cell and Si concentrations are means of the triplicates. All separate values were statistically significant, i.e. there was no overlaps between data sets. Biomass and growth rates were monitored with cell numbers that was achieved by counting Lugol fixated samples from the culture flasks. A minimum of 100 cells (in transects) was counted three times in each treatment triplicate (using 4 well 1.9 mL Nunc counting chambers), see Figure 3a. Counting took place in inverted microscopes after minimum 4 hours sedimenting time.
Table 7
Figure imgf000042_0001
The results of this experiment shows that growth rates are substantially higher (Fig. 3a) with initial SiCh concentrations of 0,171 and 0,142 mg/L (treatment Pl and P2) compared to 0,066 and 0,003 mg/L (treatment P3 and P4), see Fig. 3b and Table 7, i.e. growth increases with increased SiCh concentrations. The final yield was also substantially higher in terms of final cell numbers (i.e. biomass) in that Pl and P2 were 6 934 632 cells/L and 5 697 180 cells/L respectively while P3 and P4 were 2 205 360 and 1 353 625 cells/L respectively.
Reference example 4: Prior art cultivation of diatoms III
Bioresource technology, vol. 314, 2020, 123747 reports a novel solution-based method to trigger the growth of diatoms for enhanced biomass production, including use of inductively coupled plasma (ICP) synthesized nanosilika. The test was performed using either 0,3g IPC nanosilika/lOOml or 0,6g IPC nanosilika/lOOml and growth rates at different points in time is disclosed in table 1.
Based on the data presented in table 1 of Bioresource technology, vol. 314, 2020, 123747, the growth rate (as doublings per day) has been calculated (see table 8, second column) and compared with the growth rate (as doublings per day) obtained in example 3 (see table 8, third column).
Table 8
Figure imgf000043_0001
The above comparison shows that microsilica provides higher growth rates at lower light intensities at SiCh concentrations from 175 000 to 350 000 times lower than what was being used in Bioresource technology, vol. 314, 2020, 123747.

Claims

42 CLAIMS
1.
A method of preparing an aqueous composition for cultivation of diatoms, the method comprising the following step(s):
- adding at least 0,07 mg/L amorphous SiCh to an aqueous liquid to obtain the aqueous composition; wherein
- the amorphous SiCh is added in the form of microsilica.
2.
The method according to claim 1, wherein all nutrients essential to maintain growth of the diatoms are added to the aqueous liquid.
3.
The method according to any one of the preceding claims, wherein pH of the aqueous composition is adjusted to a pH in the range 5.5 to 9.0.
4.
The method according to any one of the preceding claims, wherein at least 0,1 mg/L amorphous SiCh is added to the aqueous liquid.
5.
The method according to any one of the preceding claims, wherein at least 0,14 mg/L amorphous SiCh is added to the aqueous liquid.
6.
The method according to any one of the preceding claims, wherein at least 0,17 mg/L amorphous SiCh is added to the aqueous liquid.
7.
The method according to any one of the preceding claims, wherein the diatoms are marine photoautotrophic diatoms.
8.
The method according to any one of the preceding claims, wherein the aqueous liquid is water and the salt content of the water is adjusted to a salt content in the range 20 to 50 g/L. 43
9.
The method according to any one of the preceding claims, wherein the aqueous liquid is seawater.
10.
The method according to any one of the preceding claims, wherein the microsilica comprises > 70 % by weight amorphous SiCh, such as e.g. > 80 % by weight amorphous SiCh or > 85 % by weight amorphous SiCh.
11.
The method according to any one of the preceding claims, wherein the microsilica comprises 0.5 to 10 % by weight iron oxide(s), such as e.g. 1 to 5 % by weight iron oxide(s); wherein the iron oxide(s) preferably is Fe2O3.
12.
The method according to any one of the preceding claims, wherein i) CO2(g) is introduced into the aqueous liquid; and/or ii) NCh’, NO2- or any mixture thereof is introduced into the aqueous liquid.
13.
The method according to any one of the preceding claims, wherein factory smoke from production of silicon and/or ferrosilicon is introduced into the aqueous liquid.
14.
An aqueous composition for cultivation of diatoms prepared by the method according to any one of claims 1-13.
15.
An aqueous composition for cultivation of diatoms comprising an aqueous liquid and SiO2 provided in the form of microsilica, wherein the amount of SiCh provided in the form of microsilica in the aqueous composition is at least 0,07 mg/L.
16.
The aqueous composition according to claim 15, wherein the aqueous composition comprises all nutrients essential to maintain growth of the diatoms.
17.
The aqueous composition according to any one of claims 15-16, wherein pH of the aqueous composition is in the range 5.5 to 9.0. 44
18.
The aqueous composition according to any one of claims 15-17, wherein the amount of SiCh provided in the form of microsilica in the aqueous composition is at least 0,1 mg/L.
19.
The aqueous composition according to any one of claims 15-18, wherein the amount of SiCh provided in the form of microsilica in the aqueous composition is at least 0,14 mg/L.
20.
The aqueous composition according to any one of claims 15-19, wherein the amount of SiCh provided in the form of microsilica in the aqueous composition is at least 0,17 mg/L.
21.
The aqueous composition according to any one of claims 15-20, wherein the diatoms are marine photoautotrophic diatoms.
22.
The aqueous composition according to any one of claims 15-21, wherein the aqueous liquid is water and the salt content of the water is in the range 20 to 50 g/L.
23.
The aqueous composition according to any one of claims 15-22, wherein the aqueous liquid is seawater.
24.
The aqueous composition according to any one of claims 15-23, wherein the microsilica comprises > 70 % by weight amorphous SiCh, such as e.g. > 80 % by weight amorphous SiCh or > 85 % by weight amorphous SiCL.
25.
The aqueous composition according to any one of claims 15-24, wherein the microsilica comprises 0.5 to 10 % by weight iron oxide(s), such as e.g. 1 to 5 % by weight iron oxides(s), wherein the iron oxide(s) preferably is Fe2O3.
26.
The aqueous composition according to any one of claims 15-25, wherein the aqueous composition comprises a carbon source and a nitrogen source, the carbon source being obtained by introducing CO2(g) into the aqueous composition and the nitrogen source being obtained by introducing NCh’, NO2- or any mixture thereof into the aqueous composition.
27.
The aqueous composition according to any one of claims 15-26, wherein factory smoke from production of silicon and/or ferrosilicon is introduced into the aqueous composition.
28.
Use of the aqueous composition according to any one of claims 14-27, for cultivation of diatoms.
29.
The use according to claim 28, wherein the diatoms are marine photoautotrophic diatoms.
30.
The use according to any one of claims 28-29, wherein the diatoms is a monoculture of diatoms.
31.
A method of producing a diatom biomass, the method comprising a step of culturing diatoms in the aqueous composition according to any one of claims 14-27.
32.
The method according to claim 31, wherein the diatoms are marine photoautotrophic diatoms.
33.
The method according to any one of claims 31-32, wherein the diatoms are a monoculture of diatoms.
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Citations (7)

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