Key information

Title: PROCESS FOR CULTIVATION OF DIATOMS
Application number: 20201076
Case type: National
Status: 01.10.2020 Andre og senere realitetsskriv foreligger
Filed: 01.10.2020
Effective date: 01.10.2020
Publicly available: 04.04.2022
Next annual fee due: 31.10.2027
Applicant: Universitetet i Tromsø - Norges Arktiske Universitet (NO) et al.
Inventor: Hans Christian Eilertsen (NO) et al.
Agent: ACAPO ONSAGERS AS (NO)

Abstract and drawing

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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.

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Date	Doc. No.	Process	Case number	In/Out	Journal description	To/from
20.01.2026	19-06	Saksbehandling	20201076	INN	20260120 Response to Office Action 1294269	ACAPO ONSAGERS AS
20.01.2026	19-05	Saksbehandling	20201076	INN	20260105 ARIV 1290195	ACAPO ONSAGERS AS
20.01.2026	19-04	Saksbehandling	20201076	INN	20260105 ARIII 1290194	ACAPO ONSAGERS AS
20.01.2026	19-03	Saksbehandling	20201076	INN	20260105 ARII 1290193	ACAPO ONSAGERS AS
20.01.2026	19-02	Saksbehandling	20201076	INN	20260105 ARI 1290192	ACAPO ONSAGERS AS
20.01.2026	19-01	Saksbehandling	20201076	INN	Korrespondanse (Hovedbrev inn)	ACAPO ONSAGERS AS
03.11.2025	18-01	Saksbehandling	20201076	UT	Realitet patent	ACAPO ONSAGERS AS
23.06.2023	17-10	Saksbehandling	20201076	INN	P27730NO00 Besvarelse av OA NIPO	ONSAGERS AS
23.06.2023	17-09	Saksbehandling	20201076	INN	ARIV MarkUp 1119015	ONSAGERS AS
23.06.2023	17-08	Saksbehandling	20201076	INN	ARIV Clean 1119016	ONSAGERS AS
23.06.2023	17-07	Saksbehandling	20201076	INN	ARIII MarkUp 1119018	ONSAGERS AS
23.06.2023	17-06	Saksbehandling	20201076	INN	ARIII Clean 1119017	ONSAGERS AS
23.06.2023	17-05	Saksbehandling	20201076	INN	ARII MarkUp 1119020	ONSAGERS AS
23.06.2023	17-04	Saksbehandling	20201076	INN	ARII Clean 1119019	ONSAGERS AS
23.06.2023	17-03	Saksbehandling	20201076	INN	ARI MarkUp 1119022	ONSAGERS AS
23.06.2023	17-02	Saksbehandling	20201076	INN	ARI Clean 1119021	ONSAGERS AS
23.06.2023	17-01	Saksbehandling	20201076	INN	Korrespondanse (Hovedbrev inn)	ONSAGERS AS
19.04.2023	16-02	Saksbehandling	20201076	INN	P27730NO00 Response to OA	ONSAGERS AS
19.04.2023	16-01	Saksbehandling	20201076	INN	Korrespondanse (Hovedbrev inn)	ONSAGERS AS
10.02.2023	15-01	Saksbehandling	20201076	UT	20201076_1672812_PT 08	ONSAGERS AS
10.01.2023	14-02	Saksbehandling	20201076	INN	Reminder til NIPO 20201076	ONSAGERS AS
10.01.2023	14-01	Saksbehandling	20201076	INN	Korrespondanse (Hovedbrev inn)	ONSAGERS AS
05.12.2022	13-02	Saksbehandling	20201076	INN	P27730NO00 Påminnelse	ONSAGERS AS
05.12.2022	13-01	Saksbehandling	20201076	INN	Korrespondanse (Hovedbrev inn)	ONSAGERS AS
27.09.2022	12-01	Saksbehandling	20201076	UT	Generelt Brev Patent	ONSAGERS AS
21.09.2022	11-02	Saksbehandling	20201076	INN	P27730NO00 Påminnelse til Patentstyret	ONSAGERS AS
21.09.2022	11-01	Saksbehandling	20201076	INN	Korrespondanse (Hovedbrev inn)	ONSAGERS AS
01.09.2022	10-01	Saksbehandling	20201076	UT	PT Varsel om betaling av første årsavgift (3317) (PT20201076)
09.11.2021	09-02	Saksbehandling	20201076	INN	Besvarelse av Office Action NO20201076	ONSAGERS AS
09.11.2021	09-01	Saksbehandling	20201076	INN	Korrespondanse (Hovedbrev inn)	ONSAGERS AS
04.11.2021	08-01	Saksbehandling	20201076	UT	Realitet patent	ONSAGERS AS
20.09.2021	07-01	Saksbehandling	20201076	INN	Korrespondanse (Hovedbrev inn)	ONSAGERS AS
01.09.2021	06-04	Saksbehandling	20201076	INN	P27730NO00 Tilsvar Patentstyret	ONSAGERS AS
01.09.2021	06-03	Saksbehandling	20201076	INN	P27730NO00 Amended Claims MarkUp	ONSAGERS AS
01.09.2021	06-02	Saksbehandling	20201076	INN	P27730NO00 Amended Claims Clean	ONSAGERS AS
01.09.2021	06-01	Saksbehandling	20201076	INN	Korrespondanse (Hovedbrev inn)	ONSAGERS AS
26.04.2021	05-02	Saksbehandling	20201076	UT	PT report 11:02:17
26.04.2021	05-01	Saksbehandling	20201076	UT	Realitet patent	ONSAGERS AS
28.12.2020	04-04	Saksbehandling	20201076	INN	Universitetet i Tromsø	ONSAGERS AS
28.12.2020	04-03	Saksbehandling	20201076	INN	P27730NO00 Signed Declaring rights to invention	ONSAGERS AS
28.12.2020	04-02	Saksbehandling	20201076	INN	Finnfjord AS	ONSAGERS AS
28.12.2020	04-01	Saksbehandling	20201076	INN	Korrespondanse (Hovedbrev inn)	ONSAGERS AS
02.10.2020	03-01	Saksbehandling	20201076	UT	Infobrev til oppfinner	ONSAGERS AS
02.10.2020	02-01	Saksbehandling	20201076	UT	Formalia 1	ONSAGERS AS
01.10.2020	01-03	Saksbehandling	20201076	INN	Specification as filed 962575	ONSAGERS AS
01.10.2020	01-02	Saksbehandling	20201076	INN	Figures as filed 962576	ONSAGERS AS
01.10.2020	01-01	Saksbehandling	20201076	INN	Søknadsskjema Patent	ONSAGERS AS

Description, claims and drawings

Disclaimer: This text has been machine-scanned and may contain errors – please refer to "Publications" for legally binding content.

Description

PROCESS FOR CULTIVATION OF DIATOMS

Field of the invention

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 EPA 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 µm to about 500 µm (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 represent 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.

WO2004048553 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.

WO2005121313 discloses a composition for a copious bloom of diatom algae comprising macro and or micronutrients adsorbed on metalate modified silica sol.

WO9114427 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.

WO2015041531 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.

WO2015016720 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.

Summary of the invention

A first 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 µm.

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 ≥ 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 µm, more preferably at least 90% of the microsilica particles have a diameter less than 1 µm and even more preferably all the microsilica particles have a diameter less than 1 µm.

In yet a further embodiment according to the present invention, the microsilica comprises a plurality of microsilica particles; and less than 1 % of the microsilica particles have a diameter ≥ 45 µm, preferably less than 0.5 % of the microsilica particles have a diameter ≥ 45 µm, more preferably less than 0.2 % of the microsilica particles have a diameter ≥ 45 µm and most preferably less than 0.1 % of the microsilica particles have a diameter ≥ 45 µm.

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 % Fe2O3 by weight of the microsilica, preferably 0.5 to 5 % Fe2O3 by weight of the microsilica, more preferably 1 to 5 % Fe2O3 by weight of the microsilica and even more preferably 1 to 3 % Fe2O3 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 or at least 0.1 mg/L.

In one embodiment the aqueous composition comprises an aqueous liquid and microsilica, wherein the amount of microsilica in the aqueous composition is at least 0.005 mg/L. In another embodiment, the amount of microsilica in the aqueous composition is at least 0.01 mg/L. In yet another embodiment according to the present invention the amount of microsilica in the aqueous composition is at least 0.05 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 NO3-, 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. In yet another embodiment, the phosphorus source is obtained by adding fertilizer into the aqueous composition.

A second aspect of the present invention relates to use of the aqueous composition according to the first 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 µm.

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 a substantial number of the diatoms is of the same species.

A third 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 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 µm.

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 a substantial number of the diatoms is of the same species.

Brief description of drawings

Figure 1a 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

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 1a, 1b 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 1b 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

Vertical axis: Biomass growth (%).

Horizontal axis: Number of days after start of cultivation.

The data presented in figure 1a, 1b 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)

b Substral® (0.25 mL/ L) Silicate (3.5 g/L Na2O3Si°9H2O)

c Substral® (0.25 mL/ L) microsilica (maximum dissolvable amount)

d 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 1a, 1b 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.

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 carriermediated, 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 to the best of our knowledge not been studied so far. 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 1b.

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.

Thus, a first aspect of the present invention relates to an aqueous composition for cultivation of diatoms, the aqueous composition comprising an aqueous liquid and microsilica.

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 in the form of microsilica. The first aspect 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 B1, 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:

YaraTera Kristalon® Purple should be added to an aqueous solution, such as water, according to the following formula: Amount of YaraTera Kristalon® = number of algae cells (million) / 10 0.5. Round down to whole closest number. Thus, if there are 25 million algae cells /L aqueous solution, the amount of YaraTera Kristalon® Purple to be added per liter aqueous solution is 0.01g/L.

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 B1, B7 and B12, respectively) and any mixture thereof.

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.

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 µm.

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.

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 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 first aspect of the present invention relates to an aqueous composition for cultivation of diatoms, the aqueous composition comprising an aqueous liquid and microsilica.

Experimental setup C of example 1 refers to an aqueous composition comprising filtered seawater with substral® to which microsilica has been added. The microsilica was dissolved in the aqueous composition and any non-dissolved material was removed. Thus, the aqueous composition referred to in experimental setup C was saturated with respect to the amount of dissolved microsilica. Thus, in one embodiment according to the present invention, the aqueous composition is saturated with respect to the amount of dissolved microsilica.

In yet another embodiment according to the present invention, the amount of microsilica in the aqueous composition is > 0.00175 mg/L, i.e. the aqueous composition comprises > 0.00175 mg microsilica per liter aqueous composition. 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 or at least 0.1 mg/L.

In one embodiment according to the present invention, the microsilica is dispersed in the aqueous liquid. In a preferred embodiment, the microsilica is dissolved in the aqueous liquid and any non-dissolved microsilica is being removed, i.e. by filtration. In an alternative embodiment according to the present invention, the microsilica is dissolved in the aqueous liquid and any non-dissolved microsilica is not being removed. In the most preferred embodiment according to the present invention, the microsilica is dissolved in the aqueous liquid and any non-dissolved microsilica is dispersed in the aqueous liquid.

In an alternative embodiment according to the present invention, the aqueous composition comprises dissolved microsilica. Preferably, the aqueous composition comprises dissolved microsilica and non-dissolved microsilica, the non-dissolved microsilica preferably being dispersed in the aqueous composition. Non-dissolved microsilica is preferably particulate microsilica.

Silica fume, also known as microsilica, (CAS number 69012-64-2, EINECS number 273-761-1) is typically an amorphous (non-crystalline) polymorph of silicon dioxide, silica. Thus, in one embodiment according to the present invention, the microsilica is amorphous silicon dioxide.

Further, microsilica is typically an ultrafine powder collected as a by-product of the silicon and ferrosilicon alloy production and may consist of spherical particles with an average particle diameter of 150 nm.

In one embodiment according to the present invention, the microsilica contains ≥ 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 another embodiment according to the present invention, the microsilica is a byproduct from the production of silicon and/or ferrosilicon.

In another embodiment according to the present invention, the microsilica comprises a plurality of spherical microsilica particles. Preferably, the microsilica consists of spherical particles of amorphous silicon dioxide.

In an alternative 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 µm, more preferably at least 90% of the microsilica particles have a diameter less than 1 µm and even more preferably all the microsilica particles have a diameter less than 1 µm.

In yet a further alternative embodiment according to the present invention, the microsilica comprises a plurality of microsilica particles; and less than 1 % of the microsilica particles have a diameter ≥ 45 µm, preferably less than 0.5 % of the microsilica particles have a diameter ≥ 45 µm, more preferably less than 0.2 % of the microsilica particles have a diameter ≥ 45 µm and most preferably less than 0.1 % of the microsilica particles have a diameter ≥ 45 µm.

Microsilica collected as a by-product of the silicon and ferrosilicon alloy production typically comprises an iron source, such as Fe2O3. Thus, in one embodiment according to the present invention, the microsilica comprises an iron source, the iron source preferably being Fe2O3.

In another embodiment according to the present invention, the microsilica comprises an aluminum source, the aluminum source preferably being Al2O3.

In yet a further embodiment, the microsilica comprises 0.1 to 10 % Al2O3 by weight of the microsilica, preferably 0.3 to 5 % Al2O3 by weight of the microsilica, more preferably 0.5 to 2 % Al2O3 by weight of the microsilica and even more preferably 0.5 to 1.5 % Al2O3 by weight of the microsilica.

In another embodiment according to the present invention, the microsilica comprises a magnesium source, the magnesium source preferably being MgO.

In yet a further embodiment, the microsilica comprises 0.1 to 10 % MgO by weight of the microsilica, preferably 0.3 to 5 % MgO by weight of the microsilica, more preferably 0.5 to 2 % MgO by weight of the microsilica and even more preferably 0.5 to 1.5 % MgO by weight of the microsilica.

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.

Thus, in one embodiment according to the present invention, the aqueous composition further comprises a carbon source, a nitrogen source, 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 NO3-, 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 aspect of the present invention relates to use of the aqueous composition according to the first aspect of the present invention for cultivation of diatoms.

An alternative aspect of the present invention relates to a method of cultivating diatoms, the method comprising a step of culturing diatoms in the aqueous composition according to the first aspect of the present invention.

In one embodiment according to the present invention, the diatom(s) are marine diatom(s).

In one embodiment according to the present invention, the mean diameter of the diatoms is ≥ 25 µm.

In one embodiment according to the present invention, the diatoms are centric diatoms, such as marine centric diatoms.

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.

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 Tromsøysund with 4 mL L−1 of Guillard’s f/2 × 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 μmol photons m−2 s−1). The cultivation took place in Millipore filtered seawater (0.22-μm 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

Substral® Substral® (Scotts Company (Nordics) A/S)), see table 1

Silicate Silicate, Na2O3Si·9H2O, Sigma–Aldrich

Microsilica 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 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

Experimental setup A, B, C and D are presented in table 2. A graphical presentation of the results is presented in figure 1a.

Table 4

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

Experimental setup A, B, C and D are presented in table 2. A graphical presentation of the results is presented in figure 1b.

The results presented in figure 1b 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.

Table 6

The 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 1a and 1b, 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.

Claims

CLAIMS

1. An aqueous composition for cultivation of diatoms, the aqueous composition comprising an aqueous liquid and microsilica, wherein the amount of microsilica in the aqueous composition is at least 0.005 mg/L.

2. The aqueous composition according to claim 1, wherein the amount of microsilica in the aqueous composition is at least 0.01 mg/L.

3. The aqueous composition according to any one of the preceding claims, wherein the amount of microsilica in the aqueous composition is at least 0.05 mg/L.

4. The aqueous composition according to any one of the preceding claims, wherein the diatoms are marine photoautotrophic diatoms.

5. The aqueous composition according to any one of the preceding claims, wherein the aqueous liquid is water with a salt content in the range 20 to 50 g/L.

6. The aqueous composition according to any one of the preceding claims, wherein the microsilica is amorphous silicon dioxide.

7. The aqueous composition according to any one of the preceding claims, wherein the aqueous composition has a pH in the range 5.5 to 9.0.

8. The aqueous composition according to any one of the preceding claims, wherein the aqueous composition comprises essential nutrients to maintain growth of the diatoms.

9. The aqueous composition according to any one of the preceding claims, 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 NO3-, NO2-or any mixture thereof into the aqueous composition.

10. The aqueous composition according to any one of the preceding claims, wherein factory smoke from production of silicon and/or ferrosilicon is introduced into the aqueous composition.

11. Use of the aqueous composition according to any one of the preceding claims, for cultivation of diatoms.

12. The use according to claim 11, wherein the diatoms are marine photoautotrophic diatoms.

13. 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 1-10.

14. The method according to claim 13, wherein the diatoms are marine photoautotrophic diatoms.

15. The method according to any one of claims 13 or 14, wherein the diatoms is a monoculture of diatoms.

Drawings

Priority

No priority

Patent classes

IPC classes

C12N 1/12

C01B 33/00

C01B 33/113

C01B 33/46

CPC classes

C12N 1/12

C01B 33/00

C01B 33/113

C01B 33/46

Citations

ELVEDAL et al; Bioactivity Potential of an Arctic Marine Diatom Species Cultivated at Different Conditions; Masteroppgave ved Norges Fiskerihøgskole, UiT, 2018 ()

SAXENA et al; Inducible coupled plasma nanosilica based growth method for enhanced biomass production in marine diatom algae; Bioresource technology, vol. 314, 2020, s. 123747, ISSN 1873-2976. https://doi.org/10.1016/j.biortech.2020.123747 ()

f/2 medium for growing diatoms, recipe ()

KR 101888798 B1 (B1)

WO 2005121313 A2 (A2)

Applicants, owners and inventors

Applicants

Universitetet i Tromsø - Norges Arktiske Universitet

Postboks 6050 Langnes

9037 TROMSØ

NO (TROMSØ Municipality, Troms county)

Org. number: 970422528

Finnfjord AS

Ferroveien 1

9308 FINNSNES

NO (SENJA Municipality, Troms county)

Org. number: 934039254

Inventors

Hans Christian Eilertsen

Bjørnøygata 3

9007 TROMSØ

NO (TROMSØ Municipality, Troms county)

Richard Andre Ingebrigtsen

Svarthammervegen 6

9020 TROMSDALEN

NO (TROMSØ Municipality, Troms county)

Gunilla Kristina Eriksen

Selnesveien 150

9042 LAKSVATN

NO (BALSFJORD Municipality, Troms county)

Jo Strømholt

Sven Eidissens vei 2

9300 FINNSNES

NO (SENJA Municipality, Troms county)

John-Steinar Bergum

Skavikveien 30 A

9300 FINNSNES

NO (SENJA Municipality, Troms county)

Agent

ACAPO ONSAGERS AS

Edvard Griegs vei 1

5059 BERGEN

NO (BERGEN Municipality, Vestland county)

Org. number: 932214636

Reference: P27730NO00

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