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WO2017033188A1 - Huile et biocarburant d'origine algale et leurs procédés de production - Google Patents

Huile et biocarburant d'origine algale et leurs procédés de production Download PDF

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Publication number
WO2017033188A1
WO2017033188A1 PCT/IL2016/050921 IL2016050921W WO2017033188A1 WO 2017033188 A1 WO2017033188 A1 WO 2017033188A1 IL 2016050921 W IL2016050921 W IL 2016050921W WO 2017033188 A1 WO2017033188 A1 WO 2017033188A1
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Prior art keywords
tag
microalgae
oil
tags
virus
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Inventor
Assaf Vardi
Asaph Aharoni
Sergey Malitsky
Shilo ROSENWASSER
Carmit ZIV
Daniella Schatz
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Yeda Research and Development Co Ltd
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Yeda Research and Development Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6436—Fatty acid esters
    • C12P7/649—Biodiesel, i.e. fatty acid alkyl esters
    • 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
    • C12N1/06—Lysis of microorganisms
    • 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
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6436—Fatty acid esters
    • C12P7/6445—Glycerides
    • C12P7/6463—Glycerides obtained from glyceride producing microorganisms, e.g. single cell oil
    • 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
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel

Definitions

  • the present invention in some embodiments thereof, relates to algal oil and biofuel and method of producing same.
  • Biofuel such as biodiesel
  • TAG triacylglycerides
  • Microalgae are groups of photosynthetic microorganisms found in freshwater and marine biosystems that can utilize inorganic nutrients (e.g., carbon, nitrogen, phosphorus) from the environment to produce organic compounds, such as protein, pigment, and oil.
  • Algal blooms are regulated by both abiotic factors, such as nutrient supply, temperature and light, and biotic interactions with grazers and viruses.
  • Viral infection of microaiagae is typically accompanied by coordinated modulation of flux through host metabolic pathways to supply building blocks such as fatty acids, amino acids and nucleotides to facilitate replication and assembly of the virus.
  • viruses can expand the metabolic capabilities of their host by expressing metabolic genes encoded by their own genomes with unique biochemical features.
  • viral infection may redefine the chemical composition and the metabolic profile of the infected microalgae and release the synthesized metabolites to the microenvironment during the lytic phase of infection [see e.g., Fulton et al., (2014) Environ Microbiol 16: 1 137-1 149; and Rosenwasser et al., (2014) The Plant cell 26, 2689-2707].
  • Microalgae are recognized as an excellent source for the production of economically viable biofuel given their fast growth rate, the fact that they can be cultured in sea water, in salty or in waste water in areas not suitable for agriculture and can also serve as biofilters for flue gas emitted by power plants and other carbon dioxide emitting industries.
  • byproducts of microalgae following oil extraction can be utilized as a source of protein for animal and fish feed, as fertilizers in agriculture and as sources for high-value pigments and vitamins.
  • Lipid composition and content vary between different microalgal species and depends on the different stages of the life cycle and environmental and culture conditions. However, many microalgae have the ability to produce substantial amounts of TAGs under adverse environmental conditions. Indeed, the most common strategies to induce TAGs accumulation include stress induction e.g., nitrogen, sulfate or phosphate limitation, silicon limitation, low temperature or high light intensities. Thus, high levels of TAGs are accumulated only under growth limiting conditions which maximize lipid productivity. These conditions, however, inhibit cell division and biomass production, resulting in overall limited lipid productivity per liter culture (Hu Q. et al., 2008. Plant J 54:621 -639). As a result, the overall lipid productivity of most algal species per liter culture decreases at growth limiting conditions, due to the large decrease in biomass productivity.
  • stress induction e.g., nitrogen, sulfate or phosphate limitation, silicon limitation, low temperature or high light intensities.
  • stress induction e.g., nitrogen, sulf
  • Additional background art includes International Patent Application Publication No. WO 2008083352; U.S. Patent Application Publication No. U.S. 20120165490; and U.S. Patent Nos. 8557514, 8636815 and 8450090.
  • a method of producing oil comprising:
  • a method of producing an algal lysate comprising:
  • the agent is a pathogen.
  • the pathogen is a virus.
  • the virus is a lytic virus.
  • the lytic virus is a Coccolithovirus.
  • the Coccolithovirus virus is N-(2-aminoethovirus virus
  • the pathogen comprise bacteria.
  • the bacteria comprise Roseobacter bacteria.
  • the method further comprises fractionating the lysate so as to obtain a fraction which is able to increase TAGs in the algae.
  • the fraction is pathogen-free.
  • the lysate consists of 100 kDa molecules or lower.
  • composition of matter comprising a pathogen-free algal lysate capable of increasing TAGs content in an algal culture.
  • the pathogen-free algal lysate consists of molecules of 100 kDa or lower.
  • the composition further comprises a preservative.
  • the preservative is selected from the group consisting of antioxidants or protease inhibitors.
  • the algal culture comprises Emiliania huxleyi microalgae.
  • the extracting is effected following virion release.
  • the extracting is effected prior to virion release.
  • the method comprising extracting oil from virions released from the microalgae following the infecting.
  • the infecting is effected at a multiplicity of infection (MOI) of about 1 viral particle per 1 cell.
  • the infecting is effected in a microalgae bioreactor.
  • an oil produced according to the method there is provided an oil produced according to the method.
  • TAGs triacylglycerols
  • the TAGs comprise saturated and/or mono-unsaturated TAGs.
  • a method of producing biofuel comprising:
  • the reaction is selected from the group consisting of transesterification, hydrocracking and hydrogenation.
  • the extracting is effected 24- 72 hours following infection with the virus.
  • the extracting is effected following virion release.
  • the extracting is effected prior virion release.
  • the method comprising extracting oil from virions released from the microalgae following infection with the virus.
  • the microalgae were infected with the virus at a multiplicity of infection (MOI) of about 1 viral particle per 1 cell. According to some embodiments of the invention, the microalgae were infected with the virus in a microalgae bioreactor.
  • MOI multiplicity of infection
  • the method comprising purifying the biofuel following the processing.
  • the method comprising isolating a glycerol following the processing.
  • a biofuel from Emiliania huxley i microalgae having an increased content of saturated and/or mono-unsaturated fatty acids; as compared to biofuel from Emiliania huxleyi microalgae not infected with a virus and/or biofuel from Emiliania huxleyi microalgae following nitrogen deprivation.
  • the biofuel is biodiesel.
  • a method of producing an algal cake comprising:
  • the removing is effected 24-
  • an algal cake produced according to the method.
  • an algal cake comprising virus infected Emiliania huxleyi microalgae.
  • the virus is a lytic virus. According to some embodiments of the invention, the virus is a Coccolithovirus.
  • the Coccolithovirus is EhV201.
  • FIGs. 1 A-B show graphs demonstrating the effect of viral infection on E. hiLxleyi cell survival.
  • Host cell numbers ( Figure 1 A) and extracellular viral numbers (Figure IB) are presented at the indicated time points following infection (hours post infection, denoted hereinafter hpi) with lytic or non-lytic virus as compared to non- infected control cells.
  • FIG. 2 shows lipidome chromatograms illustrating total lipidome analysis derived from E. htixleyi cultures 48 hpi with lytic or non-lytic virus as compared to non-infected control cells.
  • the retention time area of the major lipid classes (described in Table 1 ) is marked and the number of species identified for each class is specified in brackets.
  • FIG. 3 shows principal component analysis (PCA) graphs illustrating distinct segregation in the lipid composition derived from E. huxleyi cultures infected with lytic or non-lytic virus as compared to non-infected control cells at the indicated time points following infection (hpi).
  • PCA principal component analysis
  • FIG. 4 is a clustergram representation of the alterations in the contents of 200 lipids derived from E. huxleyi cultures infected with lytic or non-lytic virus as compared to non-infected control cells at the indicated time points following infection (hpi).
  • the right panel represents the association of the detected lipids to the five major lipid classes: phospholipids (PL), sphingolipids (SPL), betaine lipids (BL), glycolipids (GL) and triacylglycerols (TAG).
  • PL phospholipids
  • SPL sphingolipids
  • BL betaine lipids
  • GL glycolipids
  • TAG triacylglycerols
  • the change in the abundance of viral- derived sphingolipid (vGSL) is marked with an arrow.
  • FIGs. 5A-B are pie charts demonstrating that TAGs are over produced during lytic viral infection and accumulated in the isolated virions.
  • Figure 5A illustrates the relative abundance of lipids associated with the major lipid classes derived from E. hiixleyi cultures infected with lytic or non-lytic virus as compared to non-infected control cells at the indicated time points (hpi).
  • Figure 5B illustrates the relative abundance of lipids associated with the major lipid classes derived from the isolated virions of the lytic (EhV201) virus 72 hpi.
  • FIG. 5C illustrates a transmission electron micrographs (TEM) of the sample of EhV201 virions after purification by filtration and concentration (see Methods). Scale of inset 200nm.
  • FIGs. 6A-D demonstrate that neutral lipids are accumulated in lipid droplets during lytic viral infection.
  • Figure 6 A shows fluorescent microscopy photomicrographs illustrating the accumulation of lipids in E. hiixleyi cultures 24 and 48 hpi with lytic virus, as compared to non-infected control. The images are composites of bright field (BF) and BODIPY fluorescence (green) alone or combined with chlorophyll auto-fluorescence (red).
  • Figure 6B is a graph illustrating neutral lipids levels in E. huxleyi cultures infected with lytic virus, as compared to non- infected control in the indicated time points following infection, as determined by flow cytometry with BODIPY staining.
  • Figure 6C is a graph illustrating the area of high intensity BODIPY staining in control cells (blue) and cells infected with the lytic virus (red) plotted against the bright detail intensity of the BODIPY staining, as assessed by imaging flow cytometry. The two treatments were found to be significantly different (MANOVA, p ⁇ 0.001 ).
  • Figure 6D shows fluorescent microscopy photomicrographs acquired during imaging flow cytometry analysis of control cells or cells infected with the lytic virus for 48 hpi. Images are composites of bright field (BF), BODIPY fluorescence (green), chlorophyll auto- fluorescence (red) and a merged image.
  • FIG. 7 illustrates the abundance of TAGs with various saturation levels in E. huxleyi cultures 72 hpi with lytic virus (left panel) and in the isolated virions (right panel).
  • FIG. 8 illustrates the expression profile of genes encoding for TAGs biosynthesis proteins, 1 and 24 hpi with lytic virus as compared to non-lytic virus; indicating de-novo TAG biosynthesis during lytic viral infection.
  • FIGs. 9A-F are graphs demonstrating that lytic viral infection resulted in higher TAGs amounts and saturation levels compared to nitrogen starvation.
  • E. hiixleyi cells were subjected to lytic viral infection or nitrogen starvation for 96 hours and total lipidome was analyzed using LC-MC.
  • Total lipid content Figures 9A-B
  • TAGs levels Figures 9C-D
  • saturation level Figures 9E-F
  • FIG. 10 shows calibration curves of TAG standards generated to calculate the data presented in Table 7, presenting the area under peak of each TAG versus the concentration.
  • FIGs. 11A-B are bar graphs showing that virus-free lysate (VFL) can mimic viral infection and induce TAGs formation without induction of cell death.
  • VFL virus-free lysate
  • 1 1 A depicts neutral lipids levels in E. huxleyi cultures, 24 hours after supplemented with either VFL or control lysate, as compared to untreated control culture or E. huxleyi cultures that were infected with lytic virus for 24h (24hpi), as determined by
  • Figure 1 1 B depicts cell death levels in E. huxleyi cultures, 24 hours after supplemented with either VFL or control lysate, as compared to untreated control culture or E. huxleyi cultures that were infected with lytic virus for 24h (24hpi), as assessed by Sytox green staining in the flow cytometry.
  • FIG. 12 is a bar graph showing that VFL can mimic viral infection and induce specific TAGs formation in E. huxleyi.
  • the graph illustrating the level of 9 TAGs in E. huxleyi cultures, 72 hours after supplemented with VFL, as compared to untreated control culture or E. huxleyi cultures that were infected with lytic virus for 72h
  • FIG. 13 is a bar graph showing that VFL can mimic viral infection and induce
  • the present invention in some embodiments thereof, relates to algal oil and biofuel and method of producing same.
  • Microalgae are recognized as an excellent source for the production of economically viable biofuel, such as biodiesel, that can serve as a possible alternative to petroleum-based fuels.
  • byproducts of microalgae following oil extraction can be utilized as a source of protein for animal and fish feed, as fertilizers in agriculture and as sources for high-value pigments and vitamins.
  • high levels of triacylglycerides (TAGs) are accumulated only under growth limiting conditions such as nitrogen, sulfate or phosphate limitation, low temperature or high light intensities which maximize lipid productivity but on the other hand inhibit cell division and biomass production, resulting in overall limited lipid productivity per liter culture.
  • TAGs composed of saturated and/or mono-unsaturated fatty acyl chains
  • the TAG content and the high proportion of saturated and monounsaturated fatty acids, is considered optimal for e.g., high quality biofuel and more specifically for biodiesel production.
  • the present teachings suggest the use of viral infection in obtaining microalgae oil that can further be used for production of biofuel or in the food, feed and agricultural industries.
  • EhV201 viral infection of E. huxleyi cultures induces growth arrest 24 hours post infection, accumulation of viral particles in the medium and subsequent lysis 72 hours post infection (Example 1 , Figures 1 A-B).
  • EhV201 viral infection induced accumulation of TAGs, phospholipids (PL) and viral specific GSLs (Example 1 , Figures 2-4 and Tables 2-3).
  • TAGs were found to account for 20 % of the total detected lipids in the Eh V201 -infected cells, in contrary to only 2 % in the non- infected and the EhV163-infected cells (Example 1, Figure 5A).
  • Lipidome analysis of the purified released virions demonstrated that the viral particles were enriched with TAGs as well (Example 1, Figures 5B-5C and Table 4).
  • the present inventors have shown that TAGs accumulated in the EhV201 -infected cells and in the purified virions were significantly enriched with saturated and mono- un saturated TAGs (Example 1 , Figure 7).
  • the present inventors have shown that TAGs abundance and saturation level were higher during EhV201 viral infection as compared to nitrogen starvation which is commonly used to enhance TAGs production for biofuel (Example 2, Figures 9A-F, 10 and Tables 6 and 7).
  • the method of the present invention is advantageous over commonly used method for inducing TAGs accumulation by nutrient starvation such as nitrogen starvation.
  • the present inventors further shown that the induction of TAGs formation can also be done by supplementing an algal culture with a virus-free lysate (VFL) prepared by isolating a 100 kDa fraction of algal culture infected with the virus ( Figures 1 1 - 13). This is of significance value as the induction of TAGs without free virions substantially reduces cell death ( Figure I IB).
  • VFL virus-free lysate
  • a method of producing oil comprising:
  • the term "Emiliana hiixleyi (E. hiixleyi) microalgae” refers to a species within the Coccolithophores which are a class of unicellular eukaryotic microalgae belonging to the phylum haptophytes, and may possess calcium carbonate plates (or scales) called coccoliths.
  • the E. huxleyi is of a strain selected from the group consisting of CCMP2090, 374, 92 F, 1516, CCMP 1 516, or RCC 1216 [Allen et al., Environmental Microbiology, 9(4) 971 -982 (2007)].
  • the E. huxleyi is CCMP2090.
  • the E. huxleyi strain is infectable by EhV201.
  • Coccolithovirus E. huxleyi virus 201 refers to an EhV201 strain of a dsDNA lytic virus within the monophyletic Phycodnaviridae capable of infecting E. huxleyi. Following infection the EhV201 can replicate, produce virions, induce lysis of the infected E. huxleyi cell membrane and release the progeny virions which can infect other E. huxleyi cells. According to specific embodiments the infection cycle ranges between 4- 120 hours, 4-96 hours, 4-72 hours, 20-96 hours, 20-72 hours, 48-120 hours or 48-96 hours.
  • the terms "infecting”, “infection” and “infected”, which are used interchangeably, refer to the step of incubating E. huxleyi microalgae with a virus under conditions which allow the virus to invade the microalgae.
  • the conditions include ratios of the number of virus particles to the number of target algal cells [i.e. multiplicity of infection (MOI)] used for infection which include but are not limited to at least 1 viral particle per 100 cells, at least 1 viral particle per 10 cells, at least 1 viral particle per 1 cell, at least 5 viral particles per 1 cell, at least 10 viral particles per 1 cell or at least 100 viral particles for 1 cell.
  • MOI multiplicity of infection
  • the infection is effected at a multiplicity of infection ( MOI) of 0.5- 10 viral particles per 1 cell.
  • MOI multiplicity of infection
  • the infection is effected at a multiplicity of infection (MOI) of about 1 viral particle per 1 cell.
  • MOI multiplicity of infection
  • Methods of evaluating viral infection include determining viral adsorption by e.g., counting free viral particles immediately after viral addition and at early time points (e.g., 30 minutes) following viral addition; determining viral replication activity by e.g., DNA sequencing and Southern blot analysis; determining viral lytic activity by e.g., optical density and dye indicators for viability.
  • the infection is effected in a microalgae bioreactor.
  • the term "bioreactor” refers to a structure that supports the growth of E. huxleyi microalgae.
  • the bioreactor may a sterile or a non-sterile bioreactor.
  • the bioreactor is a non-sterile bioreactor.
  • E. huxleyi microalgae cultivation requires water, carbon dioxide, light and minerals (also refers to herein as growth medium).
  • the bioreactor contains at least 100 liters, at least 1 cube, at least 100 cubes or at least 1000 cubes of growth medium comprising E. huxleyi microalgae.
  • any algal bioreactor known in the art can be used including open or closed bioreactor operated in a batch, semi-batch or a continuous mode (see e.g., U.S. Department of Energy, 2009 National Algal Biofuels Technology Roadmap. Washington, D.C.: U.S. Department of Energy; and Sheehan, J. et al., ( 1998). A Look Back at the U.S. Department of Energy 's Aquatic Species Program - Biodiesel from Algae. Golden: National Renewable Energy Laboratory; and Benemann, J. R. (2008). Open Ponds and Closed Photobioreactors - Comparative Economics. 5th Annual World Congress on Industrial Biotechnology & Bioprocessing. Chicago).
  • the bioreactor is an open bioreactor.
  • open bioreactor refers to an uncovered pond, in which the surface of the growth media is directly exposed to the surrounding environment.
  • a raceway pond or high-rate pond growth media is circulated to provide mixing.
  • the bioreactor is a closed bioreactor.
  • closed bioreactor refers to a closed system which partially isolates the microalgae by circulating growth media through a system of tubes or other containers.
  • batch mode refers to a process in which a reactor is filled with a growth medium, inoculated with microalgae, and then left to grow.
  • the term "semi-batch mode" refers to a process in which a reactor is filled with a growth medium, inoculated with microalgae, and then a fraction of the reactor volume is replaced during each reactor cycle.
  • continuous mode refers to a process in which a reactor is inoculated with microalgae and then fresh media is added multiple times following predetermined time points while bioreactor fluid containing waste products is constantly removed following predetermined time points, while leaving at least an inoculum.
  • the bioreactor contains growth medium which comprises nutrients required for the growth of the E. huxleyi microalgae.
  • the growth medium is a liquid medium.
  • the growth medium used by the present invention can be a water-based medium which includes a combination of substances such as salts, nutrients, minerals, vitamins, amino acids, nucleic acids, proteins and small molecules all of which are needed for microalgae proliferation and survival.
  • the growth medium is based on seawater, such as disclosed for example in Keller et al., Journal of Phycology ( 1987) 23, 633-638.
  • the growth medium is based on municipal wastewater.
  • a culture medium can be a synthetic growth medium such as K/2 medium or F/2 medium, as described by Keller et al., Journal of Phycology (1987) 23, 633-638 and can be purchased from e.g., Florida-Aqua-Farms.
  • oil is extracted.
  • extracting oil refers to the process in which oil is removed from the infected microalgae and/or the virions released from same.
  • Exemplary time ranges for extracting the oil following infection include but are not limited to 12-72 hours post infection, 20-72 hours post infection, 24-72 hours post infection, 24-48 hours post infection, 48-72 hours post infection, 24-96 hours post infection, or 48-96 hours post infection.
  • the oil is extracted from the infected microalgae 24-72 hours following infection.
  • the oil is extracted from the infected microalgae following virion release. According to specific embodiments the oil is extracted from the infected microalgae prior to virion release.
  • Oil or crude oil refers to the hydrophobic liquid containing TAGs which can be extracted from virus infected E. huxleyi microalgae (e.g., lytic Coccolithovirus such as EhV201) and/or from virions (e.g., Coccolithovirus virions such as EhV201 virions) released from E. huxleyi microalgae.
  • E. huxleyi microalgae e.g., lytic Coccolithovirus such as EhV201
  • virions e.g., Coccolithovirus virions such as EhV201 virions
  • oil refers to crude oil (e.g., emulsions) obtained by extraction without further steps of enrichment for specific lipids or refinement or manipulation (e.g., by splitting).
  • oil includes derivatives thereof, including racemic mixtures, enantiomers, diastereomers, hydrates, salts, solvates, metabolites, analogs, and homologs.
  • TAG triacylglyceride
  • HPLC high performance liquid chromatography
  • MS Mass spectrometry
  • fatty acid refers to a carboxylic acid having an aliphatic tail which is either saturated or unsaturated.
  • the fatty acid may be in a free stated (i.e. non-esterified) or in an esterified form such as part of a TAG.
  • saturated fatty acid refers to a fatty acid with no double bonds along the carbon chain.
  • the term "mono-unsaturated fatty acid” refers to a fatty acid with one carbon-carbon double bond along the carbon chain.
  • polyunsaturated fatty acid refers to a fatty acid with more than one carbon-carbon double bond along the carbon chain.
  • glycol and “glycerin”, which are interchangeably used, refer to an organic trihedral alcohol with the formula C3H 5 (OH)3.
  • the TAGs comprise saturated and/or mono-unsaturated fatty acids, i.e. saturated and/or mono-unsaturated TAGs.
  • the mono-unsaturated TAGs comprise TAG46:1, TAG48:1 and TAG50:1.
  • Methods of extracting oil from microalgae are known in the art such as disclosed in e.g., Nagle, N. and Lemke, P. (1989) Microalgal Fuel Production Processes: Analysis of Lipid Extraction and Conversion Methods, Aquatic Species Program Annual Report 1989, SER I/SP-231 -3579. Golden, CO: Solar Energy Research Institute; US Patent No. US 8,313,648; and International Application Publication No. WO 2013005209.
  • the microalgae biomass is concentrated prior to oil extraction.
  • Concentration can be accomplished by e.g., filtration, screening, centrifugation, flotation or sedimentation which can be combined with coagulation and flocculation (see e.g., Benemann, et al., Algae Biomass (1980) 457- 495; and Metcalf and Eddy. (2003). Wastewater Engineering: Treatment and Reuse. New York: McGraw-Hill).
  • Exemplary oil extraction methods involve cell lysis by mechanical, thermal, enzymatic or chemical methods. These methods result in emulsions, requiring an expensive cleanup process.
  • the emulsion is a complex mixture, containing neutral lipids, polar lipids, proteins, and other algal products, necessitating refining processes to isolate the neutral lipids (e.g., TAGs).
  • mechanical cell disruption may be effected by bead milling, homogenizing or sonication (see e.g., Doucha, J., & Livansky, K. (2008). Biotechnological Products and Process Engineering , 431 -440; and Shuler, M. L. (2002). Bioprocess Engineering: Basic Concepts. Upper saddle River, NJ: Prentice- Hall, Inc.).
  • exemplary oil extraction methods include, but not limited to, the use of solvents or chemicals to extract lipids from a growing algal culture.
  • solvents or chemicals to extract lipids from a growing algal culture.
  • the Bligh and Dyer method or a variation thereof of lipid extraction uses a ch loroform-methanol solvent system (Bligh & Dyer, 1959; and Van Mooy et al., (2006), Proc. Natl. Acad. Sci. USA 103, 8607).
  • the Soxhlet extraction uses hexane as the standard extraction solvent.
  • various mixtures of short-chain alcohols and alkanes are more commonly used for extractions, for example, methylene chloride.
  • the solvent can be removed by e.g., a reduced pressure distillation.
  • the TAG fraction or a specific TAG population may be separated from the oil by e.g., preparative chromatography, such as high performance liquid chromatography (HPLC) or with solid phase extraction methods (SPE).
  • preparative chromatography such as high performance liquid chromatography (HPLC) or with solid phase extraction methods (SPE).
  • At least 70 %, at least 80 %, at least 90 %, at least 95 % or at least 99 % of the purified fraction is TAGs.
  • EhV201 virions released from the E. huxleyi microalgae are characterized by a modified oil composition (i.e. enriched with TAGs), the present teachings suggest extracting the oil from the released virions.
  • the method comprising extracting oil from virions released from the microalgae following viral infection.
  • the virions are purified from the algal cells before oil extraction.
  • Methods of isolating virions include density gradient separation (e.g., OptiPrep gradient) such as disclosed e.g., in Lawrence JE, Steward GF. Purification of viruses by centrifugation. In: Wilhelm SW, Weinbauer MG, Suttle C A, editors. Manual of aquatic viral ecology. College Station, TX, USA: ASLO; 2010. pp. 166 181 .
  • density gradient separation e.g., OptiPrep gradient
  • the virions are present in a preparation which is devoid of intact algal cell, e.g., less than 30 %, less than 20 %, less than 15 %, less that 10 %, less than 5 % or less than 1 % algal cells.
  • the algal cells are present in a preparation which is devoid of intact virions, e.g., less than 30 %, less than 20 %, less than 15 %, less that 10 %, less than 5 % or less than 1 % virions.
  • microalgae oil generated according to the above teachings is characterized by a modified lipid composition.
  • oil produced by the method of some embodiments of the invention is provided.
  • an Emiliania huxleyi microalgae oil having an increased content of TAGs as compared to an oil of Emiliania huxleyi microalgae not infected with a Coccolithovirus EhV201 and/or an oil of Emiliania huxleyi microalgae following nitrogen deprivation.
  • the increased content of TAGs in the oil is of at least 5 %, at least 10 %, at least 20 %, at least 30 %, at least 40 %, at least 50 %, at least 60 %, at least 70 %, at least 80 %, at least 90 % or more than 100 % higher than that found in an oil of Emiliania huxleyi microalgae of the same strain not infected with a Coccolithovirus EhV201 and/or an oil of Emiliania huxleyi microalgae of the same strain following nitrogen deprivation, as determined by e.g., HPLC and/or MS.
  • the TAGs comprise saturated and/or mono-unsaturated TAGs
  • the increased content of saturated and/or mono-unsaturated TAGs in the oil is of at least 5 %, at least 10 %, at least 20 %, at least 30 %, at least 40 %, at least 50 %, at least 60 %, at least 70 %, at least 80 %, at least 90 % or more than 100 % higher than that found in an oil of Emiliania huxleyi microalgae of the same strain not infected with a Coccolithovirus EhV201 and/or an oil of Emiliania huxleyi microalgae of the same strain following nitrogen deprivation, as determined by e.g., HPLC and/or MS.
  • the increased content of TAGs and/or the increased content of saturated and/or mono-unsaturated TAGs is determined in the crude oil (e.g., emulsions) obtained by extraction without further steps of enrichment for specific lipids or refinement or manipulation (e.g., by splitting).
  • nitrogen deprivation refers to nitrogen conditions which are less than optimal, in the case of E. huxleyi below 300 ⁇ .
  • Exemplary time ranges for nitrogen deprivation include, but are not limited to, 96 hours, 72 hours, 48 hours and 24 hours.
  • the nitrogen deprivation is effected in the absence of nitrogen for 72 hours.
  • nitrogen refers to nitrogen that can be assimilated by E. huxleyi microalgae to produce proteins.
  • the remaining mass of algal cells following oil removal, which comprise proteins and carbohydrates, can be dried and pressed into E. huxleyi microalgae cake.
  • a lysate of an algal culture free of viruses is sufficient to increase TAGs formation in an algal culture treated therewith.
  • agents that can be used to increase the TAGs content in the microalgae include, but are not limited to, a pathogenic agent and or a growth condition.
  • pathogenic agents include a viral infection or a bacterial infection.
  • the agent is a pathogen.
  • the pathogen is a virus.
  • the virus is a lytic virus.
  • the lytic virus is a Coccolithovirus.
  • the Coccolithovirus virus is EhV201 or
  • the pathogen is bacteria.
  • the bacteria comprise Roseobacter bacteria.
  • the bacteria is Sulfitobacter sp. or Phaeobacter sp.
  • growth conditions include, but are not limited to nitrogen limitation, stationary phase and other stress conditions.
  • Oleaginous algae produce TAGs during Stationary phase (i.e aging culture) or when placed under stress conditions imposed by chemical or physical environmental stimuli like nutrient starvation (e.g. nitrogen limited medium).
  • Additional factors that may affect TAG formation include salinity and growth-medium pH and also temperature and light intensity (Hu et al. 2008 Microalgal triacylglycerols as feedstocks for biofuel production: perspectives and advances. Plant J 54, 621 -639).
  • the treatment may cause the lysis and thus lysate formation.
  • Infection may be effected according to the teachings provided herein or as known to those of skills in the art.
  • lysate refers to a preparation that does not include intact organisms or viable or replicative organisms such as virions, bacteria and/or algae.
  • virus/bacteria- free algal lysate or "VFL"/ "BFL” refers to an algal free, virus free, bacterial free preparation.
  • the lysate may be a result of a lytic virus or pathogenic bacteria or a result of an artificial (man-made) induced lysis of algal cells.
  • free refers to below 1 -10, 10- 100, 100- 1000 virions per mL, or below 1 -10, 10-100, 100-1000 intact algal cells per mL or below 1 -10, 10-100, 100-
  • the lysate may be further subjected to fractionation so as to obtain a fraction which is able to increase TAGs in said algae.
  • the virus-free algal lysate consists of 100 kDa molecules or lower. It may be supplemented with other macromolecules (above 100 kDa) from a heterologous source (i.e., not the virus of the algae-infected therewith).
  • composition of matter comprising a virus-free algal lysate (e.g., belonging to Coccolithophores, to Diatoms, to Nannochloropsis, or to other microalgae) capable of increasing TAGs content in an algal culture.
  • a virus-free algal lysate e.g., belonging to Coccolithophores, to Diatoms, to Nannochloropsis, or to other microalgae
  • the composition comprises a virus-free algal lysate (VFL) or bacteria-free algal lysate (BFL) consisting of molecules of 100 kDa or lower.
  • VFL virus-free algal lysate
  • BFL bacteria-free algal lysate
  • molecules of higher molecular weight may also be present in the composition.
  • the composition may further comprise an agent such as a preservative.
  • composition may be used fresh or stored e.g., cryo-preserved or kept in the dark.
  • the preservative is selected from the group consisting of antioxidants or protease inhibitors.
  • compositions described herein can be used to increase TAGs content in an algal culture, negating the need for infection and reducing algal death (as a result of pathogen infection) significantly.
  • reduced cell death refers to less than 10%, 20%, 30%, dying cells out of the total cells, as determined by as assessed by methods well known in the art e.g., Sytox green staining in flow cytometry
  • a method of increasing content of triacylglycerols (TAGs) in algae comprising treating an algal culture (belonging to Coccolithophores, Diatoms, Nannochloropsis, or to other microalgae) with the composition comprising the VFL, as described herein, thereby increasing content of triacylglycerols (TAGs) in algae.
  • VFL may be of an algal source identical to the algal culture treated therewith (i.e., same algal species).
  • VFL may be of an algal source different than the algal culture treated therewith (i.e., different algal species).
  • any algal culture stage can be treated with the composition described herein.
  • the algal culture is at the exponential stage.
  • the algal culture is at the stationary stage.
  • algal cake refers to a microalgae product obtained as the residue from oil extraction.
  • the cake can be used for e.g., animal feed, fish feed, nutritional supplements (e.g., vitamins and antioxidants), fertilizer, a dry fuel (i.e. "green coal"), or in the generation of bioethanol via carbohydrate fermentation.
  • Oil removal is effected by extracting the oil from the microalgae as disclosed in details hereinabove.
  • oil removal is effected 24-72 hours following infection.
  • the virus is a lytic virus.
  • lytic virus refers to a virus capable of infecting E. huxleyi and follows the lytic pathway. Following infection the lytic virus can replicate, produce virions, induce lysis of the infected E. huxleyi cell membrane and release the progeny virions which can infect other E. huxleyi cells.
  • the virus is a Coccolithovirus.
  • Coccolithovirus refers to a dsDNA virus within the monophyletic Phycodnaviridae capable of infecting E. huxleyi. According to specific embodiments, the Coccolithovirus is a Coccolithovirus E. huxleyi virus (EhV). According to specific embodiments the Coccolithovirus is a lytic Coccolithovirus.
  • the Coccolithovirus is selected from the group consisting of EhVl , EhV84, EhV86, EI1V88, EhV163, EhV201 , EhV2G2, EhV203, EhV204, EhV205, EhV206, EhV207, EhV2G8, EhV209, EhV-V2 and EhVice.
  • the Coccolithovirus is EhV201.
  • microalgae cake generated according to the above teachings is characterized by the presence of viral (e.g., lytic Coccolithovirus e.g., EhV201) DNA.
  • viral e.g., lytic Coccolithovirus e.g., EhV201
  • an algal cake produced by the method of some embodiments of the invention.
  • an algal cake comprising virus infected Emiliania huxleyi microalgae.
  • Methods of determining the presence of viral DNA (e.g., EhV201 ) in the cake are well known in the art and include e.g., PCR, DNA sequencing and Southern blot.
  • processed products of the microalgae oil of some embodiments of the invention including but not limited to biofuel, oil for food, food additive, feed, nutritional supplement, cosmetics, soap, detergent, candle, paint, a personal care product, a medicinal product and a veterinary product.
  • a method of producing biofuel comprising:
  • a method of producing biofuel comprising:
  • biofuel refers to an organic fuel derived from E. huxleyi microalgae.
  • the biofuel is biodiesel.
  • biodiesel refers to monoalkyl (methyl, propyl or ethyl) esters of long chain fatty acids derived from lipids (e.g., TAGs) of E. huxleyi microalgae, which can be used in diesel-engine vehicles.
  • TAGs lipids
  • the properties of biodiesel are largely determined by the structure of its component fatty acid esters. For example, saturated fats produce a biodiesel with superior oxidative stability and a higher cetane number, but rather poor low-temperature properties. Biodiesels produced using these saturated fats are more likely to gel at ambient temperatures.
  • TAG triacylglycerol
  • Such reactions are well known in the art and are disclosed e.g., in Gardner: Chapter 8: Oil Seed and Algal Oils as Biofuel Feedstocks 121 - 143; US Patent No. US 8313648; and US Application Publication No US 20100081835, the contents of which are incorporated herein by reference in their entirety.
  • reaction is selected from the group consisting of transesterification, hydrocracking and hydrogenation.
  • the term "transesterification” refers to the process of reacting a TAG molecule with an excess of alcohol, typically methanol, in the presence of a catalyst to produce glycerin and alkyl esters (i.e. biodiesel).
  • glycerin and alkyl esters i.e. biodiesel.
  • Tran se steri ficati on can be accomplished by using traditional chemical processes such as acid or base catalyzed reactions [such as sulfuric acid (H 2 S0 4 ) and hydrochloric acid (HCl)J or by using enzyme-catalyzed reactions (such as lipase). See e.g., Nagle, N. and Lemke, P.
  • hydrocracking refers to a catalytic cracking process assisted by the presence of an elevated partial pressure of hydrogen gas.
  • the process employs high pressure, high temperature, a catalyst, and hydrogen.
  • Hydrocracking is normally facilitated by a bifunctional catalyst that is capable of rearranging and breaking hydrocarbon chains as well as adding hydrogen to aromatics and olefins to produce naphthenes and alkanes.
  • the products of this process are saturated hydrocarbons that can be used as e.g, jet fuel. See e.g., US Patent No. US 83 13648, the contents of which are incorporated herein by reference in their entirety.
  • the saturated fatty acid chains can undergo dehydration, decarbonylation or decarboxylation reactions to produce normal alkanes that can be used as renewable diesel or renewable jet fuel, sometimes branded "green diesel” or “green jet”.
  • the glycerol backbone is hydrogenated to propane so there is substantially no glycerol produced as a byproduct. See e.g., Ruber (2007) Applied Catalysis A: General. 329: 120-129; and US Patent No. US 8636815, the contents of which are incorporated herein by reference in their entirety.
  • the biofuel may be isolated and purified.
  • the method comprising purifying the biofuel following the processing.
  • Methods of purifying the biofuel e.g., biodiesel
  • the glycerol is isolated from the biofuel following the processing. Glycerol is denser than biodiesel and can be drained out of a reactor. Other impurities can be removed by washing the product with water, using ion exchange resins or solid adsorbents. Residual methanol can be removed by distillation.
  • the glycerol obtained according to the methods of some embodiments of the present invention can further be used in the production of e.g., food, food additive, feed, nutritional supplement, cosmetic, soap, detergent, toothpaste, explosives, candle, paint, tobacco, emulsifiers, a personal care product, a medicinal product and a veterinary product.
  • the biofuel generated according to the above teachings is characterized by a modified fatty acid composition.
  • a biofuel produced by the method of some embodiments of the invention.
  • a biofuel from Emiliania huxleyi microalgae having an increased content of saturated and/or mono-unsaturated fatty acids; as compared to biofuel from Emiliania huxleyi microalgae not infected with a virus and/or biofuel from Emiliania huxleyi microalgae following nitrogen deprivation.
  • the increased content of saturated and/or mono-unsaturated fatty acids in the biofuel is of at least 5 %, at least 10 %, at least 20 %, at least 30 %, at least 40 %, at least 50 %, at least 60 %, at least 70 %, at least 80 %, at least 90 % or more than 100 % higher than that found in biofuel from Emiliania huxleyi microalgae of the same strain not infected with a virus (e.g., lytic Coccolithovirus e.g., EhV201) and/or biofuel from Emiliania huxleyi microalgae of the same strain following nitrogen deprivation, as determined by e.g., HPLC and/or MS.
  • a virus e.g., lytic Coccolithovirus e.g., EhV201
  • biofuel from Emiliania huxleyi microalgae of the same strain following nitrogen deprivation as determined by e
  • the properties of biodiesel are largely determined by the structure of its component fatty acid esters.
  • the biofuel (i.e. biodiesel) of the present invention present a better quality biofuel demonstrating e.g., higher oxidative stability and higher cetane number as compared to biofuel from Emiliania huxleyi microalgae not infected with a virus (e.g., lytic Coccolithovirus e.g., EhV2Ql) and/or biofuel from Emiliania huxleyi microalgae following nitrogen deprivation.
  • a virus e.g., lytic Coccolithovirus e.g., EhV2Ql
  • compositions, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.
  • a compound or “at least one compound” may include a plurality of compounds, including mixtures thereof.
  • range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1 , 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.
  • method refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.
  • sequences that substantially correspond to its complementary sequence as including minor sequence variations, resulting from, e.g., sequencing errors, cloning errors, or other alterations resulting in base substitution, base deletion or base addition, provided that the frequency of such variations is less than 1 in 50 nucleotides, alternatively, less than 1 in 100 nucleotides, alternatively, less than 1 in 200 nucleotides, alternatively, less than 1 in 500 nucleotides, alternatively, less than 1 in 1000 nucleotides, alternatively, less than 1 in 5,000 nucleotides, alternatively, less than 1 in 10,000 nucleotides.
  • huxleyi virus EhV201 (lytic) and Eh V 163 (non-lytic) (Schroeder et al., 2002) were used to infect E. huxleyi cultures using a 1 : 100 volumetric ratio of viral lysate to culture (multiplicity of infection of -4 : 1 viral particles per cell). All experiments were performed with exponential phase cultures (5xl0 5 to 10 6 cells / ml).
  • Enumeration of Cell and Virus Abundance - Cells were monitored and quantified using an Eclipse (iCyt) flow cytometer, equipped with 405- and 488- ran solid state-air cooled lasers (both 25 mW on the flowCell) and standard filter set-up. Algae were identified by plotting chlorophyll fluorescence in the red channel (737- 663 nm) versus green fluorescence (500-550 nm) or side scatter. For extracellular viral production, samples were fixed with glutaraldehyde at a final concentration of 0.5 % for 30 min at 4 °C, followed by freezing in liquid nitrogen and storage at -80 °C until analysis.
  • Microscopy and Flow cytometry - Fluorescence microscopy images were obtained using 1X71 SI F-3-5 motorized inverted Olympus microscope, equipped with a 60x objective, with the following filter systems: BODIPY (ex:470/40nm, em:525/50nm), chlorophyll auto-fluorescence (ex:500/2()nm, em:650nm LP). Images were captures using an EXi BlueTM (Q Imaging).
  • Multispectral imaging flow-cytometry (ImageStreamX) analysis, cells were stained for BODIPY as described above and imaged using multispectral Imaging Flow Cytometry (ImageStream mark 11 flow cytometer; Amnis Corp, part of EMD millipore, Seattle, WA) using a 60X lens.
  • the dye was excited by the 488nm laser (l OOmW) and imaged on channel 2 (480-560nm), and the chlorophyll was excited by the 4()5nm laser and measured on channel 10 (595-640nm).
  • Approximately 5X 10 ' ' cells were collected from each sample and data were analyzed using image analysis software (IDEAS 6. 1 ; Amnis Corp). Images were compensated for fluorescent dye overlap by using single-stain controls.
  • Lipid extraction for LC-MS analysis Lipids were extracted from non- infected E. huxleyi cells and E. huxleyi cells infected with either EhV201 (lytic) or Eh V 163 (non-lytic) 1 , 4, 24, 32, 48 and 72 hours post infection (hpi). 50 ml of cultures in three biological replicates were collected on filters (Vardi et al., 2009).
  • the dried lipid extracts were re- suspended in 300 ⁇ mobile phage buffer B (acetonitrile: isopropanol in a ratio of 7 : 3, with 1 % 1 M NH4Ac and 0.1 % acetic acid) and centrifuged again at 10,000 g at 4 °C for 5 min. The supernatant was transferred to an autosampler vial and an aliquot of 3 ⁇ was subjected to UPLC- MS (Ultra performance liquid chromatography - mass spectrometry) analysis.
  • 300 ⁇ mobile phage buffer B acetonitrile: isopropanol in a ratio of 7 : 3, with 1 % 1 M NH4Ac and 0.1 % acetic acid
  • Lipids extraction from virions was performed by Bligh and Dyer method (Bligh and Dyer 1959).
  • the mobile phases consisted of water (UPLC grade) with 1 % 1 M NH4Ac, 0.1 % acetic acid (mobile phase A), or acetonitrile: isopropanol (7 : 3) with 1 % 1 M NH4Ac, 0.1 % acetic acid (mobile phase B).
  • the column was maintained at 40 °C with mobile phase flow rate of 0.4 ml / min.
  • MS parameters were as follows: the source and de-solvation temperatures were maintained at 120 °C and 450 °C, respectively.
  • the capillary voltage and cone voltage were set to 1.0 kV and 40 V, respectively.
  • itrogen was used as the de-solvation gas and cone gas at a flow rate of 800 1 / h and 20 1 / h, respectively.
  • the mass spectrometer was operated in full scan MS 1* positive resolution mode over a mass range of 50 Da- 1500 Da.
  • TAGs triacylglycerols
  • RNA extraction for RNAseq transcriptome analysis - RNA was extracted from host E. huxleyi 1 and 24 hours following infection with the lytic virus (EhV201) or with the non-lytic virus (Eh VI 63) as described previously by (Schatz et al., 2014).
  • Viral infection of algae triggers a global remodeling of the host lipidome
  • Remodeling of host primary metabolism toward lipid biosynthesis was found to be critical for successful infection of E. huxleyi cell by EhV (Rosenwasser et al., 2014). To determine the consequences of this metabolic remodeling, profiling the lipidome of infected host and purified virions was effected by LC/MS-based global lipidomics approach.
  • a comparative host-virus system was utilized in which a single host E. huxleyi (strain CCMP2090) was exposed to either the lytic virus (EhV201) or the non-lytic virus (EhV 163) and growth dynamics was followed over the infection time course.
  • Cultures of E. huxleyi not infected with virus served as control.
  • Figure 1 A while cultures infected with the lytic virus exhibited growth arrest 24 hours post infection (denoted hereinafter as hpi) and subsequent lysis 72 hpi, the control cultures as well as the cultures infected with the non-lytic virus exhibited exponential growth throughout the experiment.
  • accumulation of viral particles in the medium was observed only during infection with the lytic virus, reaching a maximal extracellular level 48 hpi (see Figure I B).
  • lipidome profiling In order to characterize a specific pattern of lipids associated with distinct phases of the infection, cells from the different cultures were sampled for lipidome profiling at various time points along the experiment (i.e., 1 , 4, 24, 32, 48 and 72 hpi). In addition, the viral particles fraction of the culture infected by the lytic virus was sampled and purified 72 hpi in order to characterize the lipids contents of the virions. The lipids extracted from the different samples were separated by liquid chromatography and analyzed by mass-spectroscopy using a targeted approach, by which the identification of pre-selected lipids was validated according to retention time, accurate mass and expected fragmentation.
  • PC A Principal component analysis of the identified lipids
  • Figure 3 showed a clear separation between the samples of cells infected with the lytic virus as compared with cells of either the control or cells infected with the non-lytic virus. More specifically, while samples derived from earlier stages of infection ( 1 -4 hpi) were not significantly different in their lipid composition, a clear separation between the samples was evident 24 hpi, and this separation was even more pronounced towards later stages of infection (i.e. 48 hpi). These results indicated a substantial modulation of the lipidome during the mid-late phase of lytic viral infection and towards maximal viral release (Tables 2 and 3 below provide detailed description of the identified lipids and their relative abundance during infection).
  • Table 2 Lipids distribution in E. huxleyi following infection and in the virus virions.
  • Table 3 Lipids distribution in E. huxleyi following infection and in the virus virions.
  • TAG triacylglycerol
  • PL phospholipids
  • This accumulation of TAGs was coincided with a decrease in the relative abundance of MGDG, DGDG and SQDG in the lytic-infected cells:
  • the MGDG fraction was only 20 % in the lytic- infected cells 48 hpi, compared to 31 % in the control cells; the DGDG accounted for 16 % in the control while only 1 1 % in the lytic-infected cells; and SQDG was accounted for 8 % in the lytic-infected cells as compared with 13 % in the control cells.
  • the amount of the glycolipids per cell was not different in the lytic- infected cells in comparison to the control cells (Table 2 above).
  • LD lipid droplets
  • FFA free fatty acids
  • Lipids Lipids: Lipids: Lipids: Lipids: Lipids: Lipids:
  • PPDMS 44 12 4. ⁇ 7 ⁇ +5 TAG 52:2 3.13E+6 Lyso-DGDG 18:1 PE 32:1 (2)
  • the TAGS composition is enriched with saturated and monounsaturated TAGs
  • TAGs accumulated in the lytic-infected cells 72 hpi were significantly enriched with saturated and especially monounsaturated TAGs, which accounted for 57 % of the total TAGs detected ( Figure 7, left panel).
  • the most abundant TAGs in the lytic-infected cells were TAG46: 1 , TAG48: 1 and TAG50:1 that reached their maximum level at 72 hpi.
  • the profile of TAGs identified in the purified virion showed a similar, though more pronounced, preference towards the more saturated fatty acids in TAGs, as 80 % of the TAGs in the virions were either saturated or with 1 -2 double bonds (relative to 70 % in the lytic-infected algal cell). This may imply a physiological role for saturated TAGs in determining the infectivity and decay rates of EhV.
  • G3P glycerol-3-phosphate
  • GPAT glycerol-3- phosphate acyltransferase
  • the second acylation is catalyzed by acyl-CoA:lyso-phosphatidic acid acyltransferase (LP AT, EC 2.3.1.51 ).
  • DAG 7i-/,2-diacylglycerol
  • PAP phosphatide acid phosphatase
  • PA phosphatidic acid
  • acyl-CoA-dependent acylation is catalyzed by acyl-CoA:diacylglycerol acyltransferase (DGAT, EC 2.3.1.20).
  • acyl- CoA lyso-phosphatidic acid acyltransferase (LPAT, 4 fold increase), phosphatidic acid phosphatase (PAP2, 14.5 fold increase) and some of the acyl-CoA:diacylglycerol acyltransferase orthologues genes (DGAT2, 3.5-4 fold increase), supporting the induction of TAG biosynthesis during lytic infection.
  • Table 5 Expression profiles of genes encoding for TAG biosynthesis enzymes. Data is presented as fold change normalized to control.
  • DGAT2 462450 1.86 - 1.24 -1.03 -1.09 [Monodelphis domestica] (model%: 61, hii%: 59. score: 369. %id: 23) [no tax name]
  • DGAT2 213149 1.09 -1.71 3.96 -6.62 >gi
  • DGAT2 440315 1.16 -1.31 3.52 -3.32 >gi
  • Nitrogen (N) starvation is commonly used to induce TAGs accumulation in algae (Breuer et al., 2012, Razeghifard 2013, Yang et al., 2013) for e.g., biofuel production.
  • N Nitrogen starvation
  • the mon o-un saturated 48: 1 TAG was found to be the most abundant TAG in the viral-infected cells accumulating to 72.2 ⁇ 4.2 fg/cell, while the unsaturated 58: 10 TAG was the most abundant TAG in the -deprived cells accumulating to 53 ⁇ 5.8 fg/ cell (Table 7).
  • Table 6 Relative quantification (AU, normalized peak intensity per cell) of the identified TAGs in control compared to following lytic viral infection or N- starvations for 72 hours.
  • Table 7 Absolute quantification of several TAGs following lytic viral infection -starvation. Data is presents as fg/cell.
  • VIRUS-FREE LYSATE CAN INCREASE TAGS IN NON-INFECTED ALGAE Virus-Free Lysate was produced by filtering E. huxleyi 2090 culture infected with EhV201 for 72 hours.
  • the culture of 72 (hours post infection, hpi) was first filtered through a sterile 0.45 ⁇ PVDF filter (Stericaup 500ml Durapore, Milipore) and then through a 100k D filter device with Ultracel® low binding regenerated cellulose with 100,000NMWL cutoff (Amicon).
  • the flow through of the two sequential filtration steps resulted in VFL.
  • Control lysate was produced in a similar way from a non-infected E. huxleyi 2090 culture.
  • VFL or the control lysate were added to an exponential growing culture (1 -
  • E. huxleyi 2090 2*10 6 cells/ ml) of E. huxleyi 2090 in a 1 : 1 v/v ratio.
  • the E. huxleyi 2090 cultures that were supplemented with VFL or control lysate were monitored for TAGs formation using BODIPY staining quantified on the flow cytometry ( Figure 1 1 A) and for induction of death using Sytox Green staining quantified on the flow cytometry ( Figure 1 I B).
  • the E. huxleyi 2090 culture that was supplemented with VFL was also analyzed using LC-MC for the production of 9 most abundant TAGs ( alitsky et al., 2016) ( Figure 12).
  • TAGs determined were: TAG 44:0, TAG 46:0, TAG 46: 1 , TAG 48:0, TAG 48: 1 , TAG 50:1 , TAG 50:2, TAG 54:1, TAG 54:3.
  • the integration area under the peaks of the selected TAGs was normalized against DGCC 38:6 or DGTS 32:4, lipids shown to be consistently abundant during exponential growth and during viral infection (Malitsky et al., 2016). As both normalizations showed similar results, only the DGTS 32:4 normalized data is presented in Figures 12-13.

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Abstract

L'invention concerne des procédés de production d'huile algale et de biocarburant algal. Par conséquent, l'invention concerne un procédé de production d'huile, le procédé comprenant l'infection de microalgues Emiliania huxleyi avec un Coccolithovirus Eh V201 ; et l'extraction de l'huile à partir des microalgues 24 à 72 heures après l'infection. L'invention concerne également un procédé de production de biocarburant, le procédé comprenant l'extraction d'huile à partir de microalgues Emiliania huxleyi infectées par le virus ; et le traitement de l'huile par une réaction qui sépare les chaînes d'acides gras d'un triacylglycérol (TAG) contenu dans l'huile de son squelette glycérine. Une huile, un biocarburant et un tourteau à base de microalgues Emiliania huxleyi et leurs procédés de production sont en outre décrits.
PCT/IL2016/050921 2015-08-24 2016-08-24 Huile et biocarburant d'origine algale et leurs procédés de production Ceased WO2017033188A1 (fr)

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