WO2018169402A1 - Procédé de production de microalgues - Google Patents

Procédé de production de microalgues Download PDF

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Publication number
WO2018169402A1
WO2018169402A1 PCT/NL2018/050166 NL2018050166W WO2018169402A1 WO 2018169402 A1 WO2018169402 A1 WO 2018169402A1 NL 2018050166 W NL2018050166 W NL 2018050166W WO 2018169402 A1 WO2018169402 A1 WO 2018169402A1
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WO
WIPO (PCT)
Prior art keywords
organic
microalgae
source
phosphorus
nitrogen
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PCT/NL2018/050166
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English (en)
Inventor
Marcel Hendrikus Oogink
Antonie Martinus Verschoor
Nick KOSTERINK
Original Assignee
Duplaco Holding B.V.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Duplaco Holding B.V. filed Critical Duplaco Holding B.V.
Priority to EP18716381.1A priority Critical patent/EP3596198A1/fr
Publication of WO2018169402A1 publication Critical patent/WO2018169402A1/fr

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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
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
    • A01G33/00—Cultivation of seaweed or algae
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management

Definitions

  • the present invention relates to a method for the production of microalgae, the invention further relates to the use of the microalgae in organic food and feed products, as nutrients or in other consumer products.
  • This invention relates to a method for the production of micro-algae.
  • a method for the production of biological micro-algae consisting of the use of substrates of organic and/or biological origin, in particular organic and/or biological substrates that can provide the nitrogen and phosphorus.
  • the method as described herein makes it possible that organic and/or biological algae extracts are produced and comply with regulations of the European Union for organic production of food for human consumption and feed for animals, such as the Regulation of the European Union (EC) 834/2007.
  • Organic production is an overall system of farm management and food production using natural substances and processes that combines best environmental practices, a high level of biodiversity and preservation of natural resources.
  • Organic production leads to the protection of the environment and animal welfare, as well as to rural development.
  • Organic production preferably relies on the use of renewable resources, such as, waste streams and by-products of plant and animal origin.
  • renewable resources such as, waste streams and by-products of plant and animal origin.
  • the preparation of processed organic food the product shall be mainly from ingredients of agricultural origin.
  • Genetically modified organisms (GMOs) and products thereof are incompatible with the concept of organic production and are therefore not used in the processing of organic products.
  • microalgae have become more important as food or feed ingredients.
  • Microalgae in particular green algae such as Chlorella, are used in animal feed and human nutrition.
  • the most important microalgae products for the food and feed sector are dried micro-algae (such as Spirulina and Chlorella).
  • Specific high-value components, such as pigments and fatty acids of microalgae are extracted for use as dietary supplements.
  • microalgae based molecules have specific advantages over their conventional alternatives, microalgae- based products and molecules are still far less (cost) competitive than comparable synthetic and traditional alternatives on the market.
  • the technology for the production of microalgae is still very much in development and much R&D has been done on cultivation systems of microalgae, aiming for benefits of scale.
  • microalgae grow photoautotrophically, that is, they convert inorganic carbon (C0 2 ) into organic molecules, making use of water, inorganic salts, and light energy.
  • Photoautotrophic microalgae are usually limited by the amount of light that can be brought into the system and the efficiency with which this light energy can be converted into chemical energy, typically giving low biomass yields.
  • the availability and intensity of light as the source of energy is mostly the limiting or inhibiting factor in the production process.
  • microalgae are chemoheterotrophic, meaning that they can use organic carbon compounds as source of energy and as building block for biomass synthesis.
  • concentration of the organic substrate and/or oxygen concentration are usually the limiting factor for growth. This means that reactor configuration is independent of light, and higher densities and yields can be achieved as compared to phototrophic growth. Therefore heterotrophic growth is increasingly applied for industrial production of microalgae.
  • Heterotrophic bioreactors dark
  • photobioreactors photobioreactors
  • microalgae technology Another important issue when using microalgae technology is food safely.
  • Cultivation and production needs particular consideration when algae are produced in open-air systems such as ponds, since they proved to be easily subject to contamination. This is much less / not an issue in the closed reactor systems.
  • closed system stainless steel fermenters are used for the large-scale cultivation and production of heterotrophic algae. Optimisation of culture conditions is an important issue in algae research and the main challenges in the cultivation of microalgae for food and feed are the reduction of production costs and improvement of the safety of food products during production.
  • Micro-algae comprise, on a dry matter basis, 45-72% carbon, 6-11% nitrogen and 1-2% phosphorus. Therefore, the most important nutrients for the cultivation of micro-algae are carbon, nitrogen and phosphorus, supplemented by minerals and trace elements.
  • Traditional heterotrophic culture of microalgae makes use of a carbon source (e.g. glucose, acetate or molasses) supplemented with nitrogen-containing minerals such as urea, ammonium or nitrate and mineral phosphorus, i.e. phosphate.
  • the organic sources being used are rich in carbon
  • the above object is met, according to a first aspect, by the present invention by a method for the production of microalgae, wherein the microalgae derive their nutritional requirement from organic substrate only, wherein the microalgae are cultivated in a bioreactor in the absence of light on organic substrate, wherein the organic substrate comprises at least one organic nitrogen (N) source of non-animal origin and at least one organic phosphorus (P) source of non-animal origin.
  • N organic nitrogen
  • P organic phosphorus
  • the microalgae are preferably cultured on an aqueous growth medium consisting of organic, non-animal substrate, for heterotrophic culture of microalgae.
  • the medium is comprised of one or more organic nitrogen and phosphorus sources.
  • the organic nitrogen and phosphorus sources of nutrients for the microalgae can be prepared by means of extraction, fermentation or hydrolysis from raw materials derived from plants, yeasts or fungi, preferably plant materials such as vegetables.
  • the growth medium further consists of one or more organic carbon source(s) or substrates, such as glucose, acetate, acetic acid or glycerol, derived from non-animal biotechnological or agricultural products such as molasses.
  • organic carbon source(s) or substrates such as glucose, acetate, acetic acid or glycerol
  • the present invention relates to the method, wherein the organic substrate further comprises at least one organic C-source of non-animal origin.
  • the present invention relates to the method, wherein the at least one organic C-source of non-animal origin is selected from the group consisting of glucose, sucrose acetate, acetic acid, glycerol, molasses and mixtures thereof.
  • the nitrogen and phosphorus sources or substrates comprise about 10 g to 500 g nitrogen and/or about 2 g to 40 g phosphorus per kg of raw material.
  • nitrogen and phosphorus sources are yeast extract, fermented wheat bran or hydrolysed plant material, raw biomass or waste streams thereof, wheat bran, peptone, tryptone, mushrooms, yeast, fungi, corn, legumes, rice.
  • the present invention relates to the method, wherein the at least one organic nitrogen and/or at least one phosphorus source is selected from the group consisting of hydrolysed, fermented and/or extracted protein rich materials, such as plants, yeast, fungi, preferably a yeast extract or a plant based hydrolysate.
  • the present invention relates to the method, wherein the at least one organic nitrogen and/or at least one phosphorus source is selected from the group consisting of yeast extract, hydrolysed wheat bran, hydrolysed beans, rice protein, tryptone, peptone, preferably yeast extract, hydrolysed beans or hydrolysed wheat bran.
  • the growth medium is further supplemented with one or more organic or mineral trace elements to obtain a well balanced medium for heterotrophic growth of the microaigae.
  • Preferred concentrations of these elements are: copper (5 nM- 10 ⁇ ), molybdenum (2 nM- 20uM). cobalt (1 nM -20 nM), boron (0.5 ⁇ -200 ⁇ ), manganese (0.5 ⁇ -100 ⁇ ), zinc (0.5 ⁇ -100 ⁇ ), iron ( 10 ⁇ -25 ⁇ ), magnesium (30 ⁇ -3 mM) and calcium (5 ⁇ -200 ⁇ ).
  • the present invention relates to the method, wherein the at least one organic nitrogen source comprises between 10 g to 500 g, preferably between 30 g to 300 g of nitrogen per kg raw material.
  • the present invention relates to the method, wherein the at least one phosphorus source comprises between 2 g to 40 g.
  • the at least one phosphorous source comprises between 10 g to 25 g of phosphorus per kg raw material.
  • the present invention relates to the method, wherein the at least one organic nitrogen source and the at least one organic phosphorus source are from one organic substrate.
  • the present invention relates to the method, wherein the cultivation of microaigae is carried out in a bioreactor at a temperature between about 15° C to 45 ° C, preferably between about 20° C to 37° C and a pH between about 5 to 8, preferably between about 6 to 7.
  • the cultivation of microaigae is preferably carried out in a cylindrical stainless steel or glass bioreactor, wherein the reactor preferably has a size of between 0.001 m 3 and 100 m '1 . Production of algae on industrial scale further depends on factors that are of importance for optimal growth like pH, temperature, nutrients and aeration.
  • the stirring speed is typically between 250 and 5000 rpm; the oxygen content in the reactor is preferably above 10% air saturation, and the glucose concentration is preferably between 20 and 200 g L "1 .
  • the micro-algae are grown in a batch, fed batch or continuous process, with or without the use of biomass retention by perfusion, recycling in the reactor, encapsulation, flocculation or biofilm formation.
  • the preferred culture method is by fed-batch culture.
  • the present invention relates to the method, wherein the microalgae is selected from the group consisting of Chlorophyceae (green microalgae), Traustochytriaceae (traustochytrids), Cyanophyceae, Bacillariophyceae,
  • Eustigmatophyceae preferably, Raphidophyceae, Chrysophyceae and Rhodophyceae, preferably
  • Chlorophyceae or Traustochytriaceae are Chlorophyceae or Traustochytriaceae.
  • the present invention relates to the method, wherein the green microalgae is selected from the group of Chlorella ( C.) soroklniana, C. vulgaris, C. pyrenoidosa, C. minutissima, C. protothecoid.es. C. zofingiensis, C. fusca, C. kessleri, C. regularis, C. eUipsoidea, C.
  • Chlorella ( C.) soroklniana C. vulgaris, C. pyrenoidosa, C. minutissima, C. protothecoid.es. C. zofingiensis, C. fusca, C. kessleri, C. regularis, C. eUipsoidea, C.
  • Chlorella sorokiniana is a fresh water microalga with a high growth rate. This species can double approximately 5 times a day under optimal growth conditions. The optimum growth temperature is about 37 ° C and the optimum pH is about pH 6.7. Experiments have shown that Chlorella strains reach higher densities grown heterotrophically (using an organic carbon and energy source) as compared to
  • Chlorella strains have been cultivated heterotrophically to densities of approximately 100 g/1.
  • the present invention relates to the method, wherein the Traustochytriaceae is selected from the group consisting of Aurantiochytrium sp., Schizochytrium sp., Thraustochytrium sp., and Ulkenia sp..
  • the method of present invention may also be used for the heterotrophic cultivation of microorganisms other than microalgae, such as bacteria, archaea, yeasts, fungi, protozoa, etc. These microorganisms could subsequently be applied in certified organic foodstuffs and animal feed.
  • the present invention relates to a use of microalgae, parts of microalgae or extract thereof obtained according to the method of present invention as ingredient or semi-finished product in food and feed products, including animal feed and plant feed, biofuels and biochemicals.
  • Processed food products may be labelled as organic or biological when produced via the method of present invention, since most sources/ingredients are of organic origin, provided that all non-organic sources/ingredients comply with legislation on organic food- and feedstuffs.
  • the biomass is harvested.
  • the microalgae are concentrated for example by sedimentation, centrifugation or filtration, and optionally followed by a rinsing step to remove any pollutants or left over media.
  • the recovered and concentrated algae are preferably kept refrigerated until further processing. Further processing of the algae may comprise the direct processing of the fresh concentrate into food or feed products, the isolation of specific high value ingredients such as proteins, pigments or fatty acids, or drying of the microalgae, further processing of the microalgae, or fractions into products or semi-finished products for the benefit of food, animal feed, fertilizer, microbial growth media, fuels or chemicals.
  • Figure 1 shows the maximum optical density (OD 750 , lllax ) of Chlorella sorokiniana obtained during a 6-day growth assay. The maximum densities were determined after growing the microalgae on six different media containing various yeast extracts
  • Figure 1 shows the initial phosphorus content of these media.
  • Figure 2 shows the maximum optical density (maximum OD 75 o, max ) of Chlorella
  • sorokiniana obtained during a 6-day growth assay.
  • the maximum densities were determined after growing the microalgae on eight different media, including several types of hydrolysed plant material (see Table 2).
  • Example 1 heterotrophic growth of Chlorella sorokiniana on yeast extract
  • yeast extract is a dissolved product which is obtained by enzymatic hydrolysis of yeast.
  • Table 1 shows the phosphorus (P) content of a number of commonly available organic yeast extracts (GE1 to GE4), dried yeast (DG), and a control medium (CM) containing mineral phosphorus.
  • CM mineral control medium
  • CM 5.18 mM KH 2 P0 4 + 0.63 mM KH 2 P0 4 ) - 179.74
  • Media containing the yeast extracts of Table 1 were used to determine the heterotrophic growth of the green microalgae Chlorella sorokiniana.
  • the results are plotted in FIGURE 1 wherein the maximum density (OD750max) obtained of Chlorella sorokiniana during a 6-day growth assay is shown, per yeast product used.
  • the medium comprised 0.75 g L 1 of the relevant yeast product, 5 g L "1 glucose, and trace elements such as naturally present in the tap water used to prepare the medium.
  • the results include a control medium (CM), having the same amounts of glucose and trace elements, but supplemented by mineral phosphate (180 mg P0 4 -P L '), and mineral nitrate (255 mg of N0 3 -N L "1 ) instead of organic nitrogen and organic phosphorus.
  • CM control medium
  • Example 2 heterotrophic growth of Chlorella sorokiniana on hydrolysed plant material
  • the hydrolysed plant materials as indicated in table 2 have been used to determine the heterotrophic growth of the green microalgae Chlorella sorokiniana.
  • the tests were performed using 8 different growth media, in which only the control medium (1 and 2) contained mineral nitrogen (255 mg of NO 3 -N L ⁇ l ), and only one formulation (1) contained mineral phosphorus (180 mg P0 4 -P L "! ):
  • Control medium with mineral phosphorus (CM in Table 1);
  • Rice protein with organic phosphorus (wheat bran hydrolysate);
  • Soybean hydrolysate with organic phosphorus (wheat bran hydrolysate);
  • FIGURE 2 The results are plotted in FIGURE 2 wherein the maximum optical density (OD750) of Chlorella sorokiniana is shown, per substrate type.
  • the highest density was achieved on the mineral control medium (1 , 2). Within this control medium, the highest densities were obtained when growing on an organic phosphorus source (hydrolysed wheat bran). This difference in density was also observed when within the black bean hydrolysates with and without organic phosphorus source (compare 7 and 8).
  • Figure 1 and Figure 2 much higher maximum densities (approximately 3 to 4 times higher) were achieved on a plant based source as compared to using yeast extract as organic nutrient source.
  • Example 3 substrates used for organic cultivation
  • Substrates that can be used are hydrolysates of organic compounds.
  • hydrolysates are mixed with glucose and demi water and autoclaved. 0.195 g of hydrolysates and 1 ,25 g of glucose are added. 2,5 mL of preculture is added to the mixture. The medium is transferred in cultivation flasks (50 mL). The microalgae are cultivated in an incubator under dark conditions. Sampling was performed, at a time interval of 3 hours 5 mL sample is taken to determine the OD (680 and 750 run) and pH.

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Abstract

La présente invention concerne un procédé de production de microalgues. Elle concerne en particulier un procédé de production de microalgues organiques, comprenant l'utilisation de substrats d'origine organique, en particulier de substrats organiques qui rend possible l'enrichissement en algues en termes d'azote et de phosphore selon les exigences nutritionnelles. Le procédé de la présente invention rend possible l'obtention de microalgues organiques et les utilise dans les produits alimentaires et de nutrition organique selon les réglementations de l'Union Européenne.
PCT/NL2018/050166 2017-03-17 2018-03-16 Procédé de production de microalgues WO2018169402A1 (fr)

Priority Applications (1)

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EP18716381.1A EP3596198A1 (fr) 2017-03-17 2018-03-16 Procédé de production de microalgues

Applications Claiming Priority (2)

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NL2018539A NL2018539B1 (en) 2017-03-17 2017-03-17 Method for the production of microalgae
NL2018539 2017-03-17

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WO2018169402A1 true WO2018169402A1 (fr) 2018-09-20

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112430161A (zh) * 2020-12-01 2021-03-02 鹤山市新的生物制品有限公司 一种有机发酵膏肥及其制备方法

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US3320693A (en) * 1964-09-11 1967-05-23 Kk Method of industral cultivation of unicellular green algae such as chlorella
EP1724357A1 (fr) * 2004-03-04 2006-11-22 Suntory Limited Procede pour la fabrication des lipides contenant de l'astaxanthine
WO2010104922A1 (fr) * 2009-03-10 2010-09-16 Srs Energy Fractionnement d'une biomasse d'algues
US20160068799A1 (en) 2014-09-05 2016-03-10 City University Of Hong Kong Medium, method and system for cultivation of chlorella pyrenoidosa or organisms derived from chlorella pyrenoidosa
US20160298149A1 (en) * 2013-12-19 2016-10-13 Roquette Freres Method for enriching the biomass of thraustochytrium genus microalgae with dha

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US3320693A (en) * 1964-09-11 1967-05-23 Kk Method of industral cultivation of unicellular green algae such as chlorella
GB1109659A (en) 1964-09-11 1968-04-10 Yakult Honsha Kk Method of industrial cultivation of unicellular green algae such as chlorella
EP1724357A1 (fr) * 2004-03-04 2006-11-22 Suntory Limited Procede pour la fabrication des lipides contenant de l'astaxanthine
WO2010104922A1 (fr) * 2009-03-10 2010-09-16 Srs Energy Fractionnement d'une biomasse d'algues
US20160298149A1 (en) * 2013-12-19 2016-10-13 Roquette Freres Method for enriching the biomass of thraustochytrium genus microalgae with dha
US20160068799A1 (en) 2014-09-05 2016-03-10 City University Of Hong Kong Medium, method and system for cultivation of chlorella pyrenoidosa or organisms derived from chlorella pyrenoidosa

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PEREZ-GARCIA O ET AL: "Heterotrophic cultures of microalgae: Metabolism and potential products", WATER RESEARCH, ELSEVIER, AMSTERDAM, NL, vol. 45, no. 1, 1 January 2011 (2011-01-01), pages 11 - 36, XP027536678, ISSN: 0043-1354, [retrieved on 20100827] *
SANSAWA H ET AL: "Production of intracellular phytochemicals in Chlorella under heterotrophic conditions", JOURNAL OF BIOSCIENCE AND BIOENGINEE, ELSEVIER, AMSTERDAM, NL, vol. 98, no. 6, 1 January 2004 (2004-01-01), pages 437 - 444, XP004727088, ISSN: 1389-1723 *
SUNJIN KIM ET AL: "Growth rate, organic carbon and nutrient removal rates of Chlorella sorokiniana in autotrophic, heterotrophic and mixotrophic conditions", BIORESOURCE TECHNOLOGY., vol. 144, 1 September 2013 (2013-09-01), GB, pages 8 - 13, XP055404798, ISSN: 0960-8524, DOI: 10.1016/j.biortech.2013.06.068 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112430161A (zh) * 2020-12-01 2021-03-02 鹤山市新的生物制品有限公司 一种有机发酵膏肥及其制备方法

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EP3596198A1 (fr) 2020-01-22

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