WO2014060973A1 - Souches améliorées de microalgues et leur utilisation - Google Patents

Souches améliorées de microalgues et leur utilisation Download PDF

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WO2014060973A1
WO2014060973A1 PCT/IB2013/059407 IB2013059407W WO2014060973A1 WO 2014060973 A1 WO2014060973 A1 WO 2014060973A1 IB 2013059407 W IB2013059407 W IB 2013059407W WO 2014060973 A1 WO2014060973 A1 WO 2014060973A1
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Prior art keywords
microalgae
range
ioc
strains
biomass
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PCT/IB2013/059407
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English (en)
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Manoj Kumar
Mahendra Pratap Singh
Dheer SINGH
Anju CHOPRA
Deepak Kumar Tuli
Ravinder Kumar Malhotra
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Indian Oil Corporation Limited
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Priority to BR112015008824-4A priority Critical patent/BR112015008824B1/pt
Publication of WO2014060973A1 publication Critical patent/WO2014060973A1/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
    • 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
    • C12N1/125—Unicellular algae isolates
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/89—Algae ; Processes using algae

Definitions

  • the present invention relates to improved strains of microalgae, process for the same and use thereof
  • Microalgae are unicellular organism having ability to adapt to various environmental conditions. Microalgae are considered as promising source for biofuels due to their potential for attaining high yields per unit area without affecting land use pattern for food crops.
  • microalgae can convert solar energy into lipids at higher photosynthetic efficiencies.
  • Microalgae can be grown round the year and fix CQ2 efficiently from different sources, including industrial exhaust gases, and can thrive in waste water streams. They also have the potential for the production of various biofuels such as biodiesel, bio-oil, bio- syngas, bio-methane, bio-ethanol and bio-hydrogen. But for commercial viability modifications of certain traits are required. Multiple cycle of protoplast fusion and genome shuffling are a known art for improving the traits or to acquiring the a desired property
  • EP1707641 A2 provides methods employing iterative cycles of recombination and selection/screening for evolution of whole cells and organisms toward acquisition of desired properties. Examples of such properties include enhanced recombmogenieity, genome copy number, and capacity for expression and/ or secretion of proteins and secondary metabolites.
  • the technique of genome shuffling combines the advantage of multiparental crossing allowed by DNA shuffling together with the recombination of entire genomes normally associated with conventional breeding, or through protoplast fusion that increases the recombination process.
  • US6531646 relate to method for the genetic modification and improvement of Porphyra species utilizing protoplast fusion is disclosed.
  • the method of the invention features the use of conchoporangial branch conchoceiis for at least one of the sources of protoplasts for protoplast fusion.
  • Protoplasts fusion method involves either a chemical fusing agent Hive polyethylene glycol (PEG) or electrofusion.
  • US2010Q162620 provides systems and processes for optimizing each type of algal-based production of bioproducts (such as oil) separately and independently, thereby improving overall production of oil, lipids and other useful products. This process is advantageous because it allows the optimization of the individual steps and growth phases in the production of oil from biomass. This also allows the use of different feedstocks and growth conditions for the different process steps.
  • bioproducts such as oil
  • US20120028338 to mixed algal compositions able to proliferate on industrial waste water, and to methods of obtaining an algal biomass from such cultures for use in generating a biofuel.
  • the invention further encompass methods of cultivating mixed populations of freshwater and marine alga comprising a plurality of genera and species to provide a biomass from Which may be extracted lipids, or converted into biodiesel by such procedures as pyrolysis.
  • Deng et al. (201 1), African J. Agri.Res. Vol.6(16), pp. 3768-3774 is a scientific publication which relates to effects of selective medium on lipid accumulation of chlorellas and screening of high lipid mutants through ultraviolet mutagenesis
  • pp- 1 109- 1 i i 7 is also a scientific publication which provides an overview on the possibility of using mixed microalgae existing in ecological water-bodies for harnessing biodiesel.
  • Microalgal cultures from five water-bodies are cultivated in domestic wastewater in open-ponds and the harvested algal-biomass was processed through acid-catalyzed transesteri fication.
  • the prior art discloses random mutagenesis of algal strain for lipid and biomass productivity. Still, the major drawback in the microalgae for use in biofuel is the non-availability of suitable strains and cost effective method for cultivation and harvesting. Hence, there is need to develop cheaper methods for cultivation and fast growing strains with tolerance to adverse environmental conditions and ability to utilize high concentrations of C02 and lipid productivity with composition suitable for making fuels.
  • the main embodiment of the present invention provides strains of microalgae belonging to green algae and blue green algae selected from the group comprising of Nannochloropsis, Chlorella, Scenedesmus and Synechococcus.
  • Another embodiment of the present invention provides the strains which are Nannochloropsis IOC- 105, Chlorella vulgaris IOC- 106, Chlorella pro totheco ides IOC- 107, Chlorella emersonii IOC- 108, CMorella p renoidosa IOC- 109, Scenedesmits sp. IOC- 1 10, Synechococcus sp- IOC- 1 1 1.
  • Another embodiment of the present invention provide for a process of preparing strains of microalgae. said method comprising the steps of :
  • step (d) shuffling the protoplast obtained step (d) for fusion using 60% PEG-6000 ;
  • Another embodiment of the present invention provides for a process of enhancing the biomass and lipid content of microalgae, said process comprising the steps of:
  • step(b) Exposing microalgae of step(b) to UV for 2 hours in log phase;
  • step (d) Incubating the microalgae of step (d) atl 0°C for 6 hours;
  • step (e) Adding hot water extract from different plants to microalgae of step (e); and g) Obtaining microalgae with high lipid content and biomass.
  • Low quality water or “Poor quality water” or “Water containing heavy metals”
  • water containing heavy metals when used in the context of the present invention refers water which cannot be used directly for drinking, agriculture, human or animal consumption or other purpose. Such water is a was waste from industrial effluents, water containing heavy metals, hydrocarbons, water with high salinity, sewage water, reject water of reverse osmosis (RO) plant, river water with higher COD and BOD, water with coloring agent and other industry effluent etc.
  • RO reverse osmosis
  • Low quality water or “Poor quality water” also includes water, which is or is found to be undesirable and harmful to human, anima or aquatic life in resepct of drinking, living or for n ⁇ ' other purpose related an organism's survival or need.
  • High Value Products when used in the context of the present invention refers to vitamins, pigments, anti-oxidants, omega-3 & omega-6 polyunsaturated fatty acids, DFIA or EPA.
  • the term "Mutagenizing Agent/ s or Mutagenic Agent/s or Mutagens" when used in the context of the present invention refers to agent/s a chemical, ultraviolet light, or a radioactive element, that can induce or increase the frequency of mutation in an organism.
  • strain/s or Novel Strains when used in the context of the present invention refers to novel/new variants/strains of the microalgae produced or developed by the process of the present invention. These variants are genetically different in their control or parent or original forms. These variants are artificially developed and survive and perform better at extreme environmental conditions.
  • COD Ceral oxygen Demand
  • COD chemical oxygen Demand
  • Biological Oxygen Demand or BOD when used in the context of the present invention refers to amount of dissolved needed by aerobic biological organism in a body of water to break down organic material present in a given water sample at certain temperature over a specific period.
  • Biofuel/s when used in the context of the present invention refers to a fuel that uses energy from a carbon fixation produced from microalgae. These fuels are made from a microalgae biomass conversion.
  • Morphogenesis or mutagenized when used in the context of the present invention refers to a process by which the genetic information of an organism is changed in a stable manner, resulting in a mutation. In context of the present invention it achieved experimentally using laboratory procedure by exposing the microalgae to various mutagens.
  • Protoplast fusion or Somatic Fusion when used in context of the present invention refers to genetic modification of microalgae from same species by fusing their protoplasts (for e.g. pooled samples of C. vulgaris fused with another pooled samples of C. vulgaris) to form a new hybrid plant with the characteristics of both, a somatic hybrid.
  • mutant strains when used in context of the present invention refers to modified microalgae by a mutagen and protoplast fusion, wherein the fusion has been carried out in the microalgae of same species or pool of microalgae of same species.
  • the mutant strains of the present invention do not in any manner or meant to refer to transgenic mutants or transgenic microalgae or transgenics or transgenic material.
  • the strains do not comprise genes of any unrelated higher life-form's or organism/s or unrelated microorganism/s. In the present invention even the microalgae from different genus have not be crossed or nor n ⁇ ' genetical material from different microalgae genus have been fused.
  • the present invention relates to improved strains microalgae.
  • the microalga species in the present invention were collected from various locations in India.
  • the algal species used to develop the strains of the present invention includes fresh water green microalgae like Nannochloropsis spp., Chlorella species such as Chlorella vulgaris, Chlorella salina, Chloretta protothecoides, Chlorella ellipsoidea, Chlorella emersonii, Chlorella miniitissima, Chlorella pyrenoidosa, Chlorella sorokiniarta, Chroomonas slairta; Cyclotella sp., Dimaliella sp., Botryococciis sp., Haematococcus sp., Nartnochloris sp., Neochloris, Onoraphidium sp., Scenedesmus sp., Spirulina platensis, Chlamydomonas
  • Nannochloropsis sp. and Scenedesmus sp. were collected from IOCL refinery Panipat, Haryana, India.
  • Chlorella vulgaris, Chlorella pyrenoidosa and Synechococcus sp. were collected from soil of IOCL of R&D Centre, Faridabad, Haryana, India.
  • Chlorella protothecoides and Chlorella emersonii were collected from Yamuna River Bank, New Delhi, India.
  • the preferred strains of microalgae that were developed are:
  • Nannochloropsis sp. (referred to herein as IOC- 105) which was deposited with Culture Collection of Algae and Protoza (CC AP), UK under Budapest Treaty on , 2013 and given accession number ; Chlorella vulgaris ( referred to herein as IOC- 106) which was deposited with (Culture (Collection of Algae and Protoza (CCAP), UK under Budapest
  • Chlorella protothecoides (referred to herein as IOC- 107) which was deposited with Culture Collection of Algae and Protoza (CCAP), UK under Budapest Treaty on , 2013 and given accession number :, Chlorella emersonii (referred to herein as IOC- 108) which was deposited with Culture Collection of Algae and Protoza (CCAP), U K under Budapest Treaty on
  • Chlorella pyrenoidosa Chlorella pyrenoidosa
  • IOC- 109 which was deposited with Culture Collection of Algae and Protoza (CCAP), UK under Budapest Treaty on , 2013 and given accession number
  • IOC-1 11 which was deposited with Culture Collection of Algae and Protoza (CCAP), UK under Budapest Treaty on , 2013 and given accession number
  • CCAP Culture Collection of Algae and Protoza
  • the present invention also relates to a process for improving the mutant strains of microalgae obtained by mutagenic and/or chemostat mediated adaptation.
  • the microalgae strains were improved by recursive mutagenesis and protoplast fusion.
  • the microalgae were mutagenised by chemical and radiation mutagen.
  • the pooled mutant population is shuffled by homologous recombination using protoplast fusion followed by selecting improved progenies and subjecting the same to next round of selection. This process was carried out for six cycles
  • the microalgae were mutagenized by chemical (EMS, mitomycin C, N-methyl-N'-nitro-N-nitrosoguanidine, benzo(a)pyrene and 4-nitroqumoline 1- oxide) and radiation mutagen (IJV, gamma-rays) or their combination.
  • EMS mitomycin C
  • N-methyl-N'-nitro-N-nitrosoguanidine benzo(a)pyrene
  • 4-nitroqumoline 1- oxide radiation mutagen
  • IJV gamma-rays
  • microalgae strains thus developed by the process of the invention have the ability to grow in presence of salt up to 3%, at a temperature range from 10-45°C, high light intensity, pH 4.5-10 and heavy metal up to 100-1000 ppm or more (TABLE-l).
  • the strains so developed have lipid composition suitable for biofuel (TABLE-2).
  • mutant algal strains were cultured in various bio-chemical-physical factors like growth hormones, specific bacteria, protein synthesis inhibiting chemicals- sodium thio-sulphate, ultra-violet rays; high and low temperature to improve biomass, lipid content and/or its composition (TABLE-3).
  • microalgae strains of the present, invention are used to sequester carbon dioxide from various sources like flue gas, bio-gas plant exhaust and other source of concentrated C0 2 having C0 2 in the range of 0.05-50%, thereby helping in abating pollution (TABLE-4).
  • the main embodiment of the present invention provides strains of microalgae belonging to green algae and blue green algae selected from genus comprising of Nannochloropsis, Chioreila, Scenedesmus and Synechococcus.
  • strains which are Nannochloropsis IOC- 105, Chioreila vulgaris IOC- 106, Chioreila protothecoides IOC- 107, Chioreila emersonii IOC- 108, Chioreila pyrenoidos IOC- 109, Scenedesmus sp. IOC- 1 10, Synechococcus sp- IOC- 1 1 1.
  • Yet another embodiment of the present invention provides strains having high lipid content, higher growth rate, C0 2 utilization ability and biomass desired lipid composition.
  • strains wherein the strains are useful as biofueis and for value added products selected from group comprising of Vitarnms, pigments, 20 antioxidants, omega-3 and omega-6 polyunsaturated fatty acids, Docosahexaenoic acid (DHA) and Eicosapentaenoic acid (EPA).
  • DHA Docosahexaenoic acid
  • EPA Eicosapentaenoic acid
  • Another embodiment of the present invention provides for strains wherein the strains tolerates high salinity, pH, heavy metal contamination, temperature and light intensity.
  • strains wherein the strains have the ability to grow under salinity conditions in range of 0.5-3%.
  • One more embodiment of the present invention provides for strains, wherein the strains can grow 30 in the temperature range from 10-45°C.
  • strains wherein the strains can grow in wide range of pH from 6.5-10.
  • strains can have heavy metal tolerance in the range of 100-1000 ppm.
  • microalgae strain wherein the microalgae strain have saturates in the range of 26-85%.
  • microalgae strain wherein the microalgae strain have saturates in the range of 32-70%
  • Yet another embodiment of the present invention provides microalgae strain wherein the microalgae strain has lipid content in the range of 20-35%.
  • microalgae strain wherein the microalgae strain has lipid content in the range of 26-33%
  • microalgae strain wherein the microalgae strain has biomass in the range of 2-7g/L
  • microalgae strain wherein the microalgae strain has biomass in the range of 2-6g/l.
  • microalgae strain wherein the microalgae strain has biomass in the range of 2.82-5.1 g/1.
  • Another embodiment of the present invention provides microalgae strain wherem the microalgae strain has saturates in the range of 26-70%.
  • microalgae strain wherein the microalgae strain has lipid content in the range of 21.4-30.45%.
  • Another embodiment of the present invention provide for a process of preparin niicroalgae, said method comprising the steps of :
  • micro algae by culturing in medium comprising of algal isolation medium, low quality water containing heavy metals in range of 100-1000 ppm, hydrocarbons in the range of 0.001%-2% and high concentration of salt of opto 3%;
  • step (d) for fusion using 60% PEG-6000 ; culturing the microalgae of the fused protoplast in AIM media containing 0,5 M osmotic agent ;
  • Yet another embodiment of the present invention provides for a process, wherem the microalgae is selected from the group comprising of green algae and blue-green aigae.
  • One more embodiment of the present invention provides for a process, wherein the microalgae belong to genus selected from the group comprising of Nannochloropsis, Chiorella, Sceriedesmus and Synechococcus.
  • Another embodiment of the present invention provides for a process, wherein the novel strains comprise of Nannochloropsis IOC- 105, Chiorella vulgaris IOC- 106, Chiorella protothecoides IOC- 107, Chiorella emersonii IOC- 108, Chiorella pyrenoidosa IOC- 109, Sceriedesmus sp. IOC- 1 10, Synechococcus sp- IOC-11 1.
  • step (e) provides for a process, wherein the mutagenizing agents in step (e) are selected from the group comprising of EMS, mitomycin C, N- methyl- '-nitro- -nitrosoguanidine, benzo(9)pyrene and 4-nitroquinoline 1 -oxide, UV rays, gamma- rays or their combination therof, preferably EMS,
  • the osmotic agent in step (i) is sucrose.
  • microalgae is useful for the production of biofuels and value added products selected from group comprising of Vitamins, pigments, antioxidants, ornega-3 and omega-6 polyunsaturated fatty acids, DHA or EPA.
  • One more embodiment of the present invention provides for a process, wherein the microalgae has high salinity tolerate upto 3%, pH tolerance in the range of 4.5-10, heavy metal contamination tolerance in the range of 300- 1 OOOppm, temperature tolerance in the range of 10- 45°C and light intensity tolerance.
  • Another embodiment of the present invention provides for a process, wherein the microalgae has higher growth rate, lipid productivity, C0 2 utilization ability and biomass desired lipid composition.
  • Another embodiment of the present invention provides for process wherein the microalgae strain have saturates in the range of 26-85%.
  • Another embodiment of the present invention provides for a process wherein the microalgae strain have saturates in the range of 32-70%.
  • One more embodiment of the present invention provides for a process wherein the microalgae strain has biomass in the range of 2-7g/L
  • Yet another embodiment of the present invention provides for a process wherein the microalgae strain has biomass in the range of 2-6g/l.
  • Another embodiment of the present invention provides for a process wherein the microalgae strain has biomass in the range of 2.82-5.1 g/L
  • Another embodiment of the present invention provides for a process wherein the microalgae strain has saturates in the range of 26-70%.
  • Another embodiment of the present invention provides for a process wherem the microalgae strain has lipid content in the range of 21.4-30.45%.
  • Another embodiment of the present invention provides for a novel process for enhancing the biomass and lipid content of microalgae, said process comprising the steps of:
  • step(b) Exposing microalgae of step(b) to UV for 2 hours in log phase;
  • step (c) Adding sodium thiosulfate in the range of 0.05-2 % in the late log phase for microalgae of step (c);
  • step (d) Incubating the microalgae of step (d) at 10°C for 6 hours;
  • microalgae of step (e) Adding hot water extract from different plants to microalgae of step (e); and g) Obtaining microalgae with high lipid content and biomass.
  • Yet another embodiment of the present invention provides for a novel process for enhancing the biomass and lipid content of microalgae, wherein the microalgae are a green algae or blue-green algae.
  • microalgae belong to genus selected from the group comprising of Nannochloropsis, Chlorella, Scenedesmus and Synechococcus.
  • One embodiment of the present invention provides for a novel process for enhancing the biomass and lipid content of microalgae, wherein the micro algae strains comprise of Nannochioropsis IOC- 105, Chlorella vulgaris IOC- 106, Chlorella protothecoides IOC- 107, Chlorella emersonii IOC- 108, Chlorella pyrenoidosa IOC- 109, Scenedesmus sp. IOC- 110, Synechococcus sp- IOC- 1 11.
  • Another embodiment of the present invention provides for a novel process for enhancing the biomass and lipid content of microalgae, wherein the steps (b) to (f) is to be performed in a said sequential manner to obtain the strains of microalgae with high biomass and lipid content.
  • Another embodiment of the present invention provides for a novel process for enhancing the biomass and lipid content of microalgae, wherein the novel strains are useful for the production of biofuels and value added products selected from group comprising of Vitamins, pigments, antioxidants, omega-3 and omega-6 polyunsaturated fatty acids, DHA or EPA,
  • Yet another embodiment of the present invention provides for a novel process for enhancing the biomass and lipid content of microalgae, wherein the novel strains tolerate high salinity upto 3%, pH in the range of 4.5-10, heavy metal contamination in the range of 100- 1 OOOppm, temperature in the range of 1() ⁇ 45°C and light intensity.
  • One more embodiment of the present invention for a novel process for enhancing the biomass and lipid content of microalgae, wherein the stains have higher growth rate, lipid productivity, C0 2 utilization ability and biomass desired lipid composition.
  • Another embodiment of the present invention provides for a novel process for enhancing the biomass and lipid content of microalgae wherein the microalgae strain have saturates in the range of 26-85%.
  • Another embodiment of the present invention provides for a novel process for enhancing the biomass and lipid content of microalgae wherein the microalgae strain have saturates in the range of 32-70%.
  • Yet another embodiment of the present invention provides for a novel process for enhancing the biomass and lipid content of microalgae wherein the microalgae strain has lipid content in the range of 20-35%.
  • Yet another embodiment of the present invention provides for a novel process for enhancing the biomass and lipid content of microalgae wherein the microalgae strain has lipid content in the range of 26-33%
  • One more embodiment of the present invention provides for a novel process for enhancing the biomass and lipid content of microalgae wherein the microalgae strain has biomass in the range of 2-7g/l.
  • Yet another embodiment of the present invention provides for a novel process for enhancing the biomass and lipid content of microalgae wherein the microalgae strain has biomass in the range of 2-6g/i.
  • Another embodiment of the present invention provides for a novel process for enhancing the biomass and lipid content of microalgae wherein the microalgae strain has biomass in the range of 2.82-5.1 g/1.
  • Another embodiment of the present invention provides for a novel process for enhancing the biomass and lipid content of microalgae wherein the microalgae strain has saturates in the range of 26-70%,
  • Another embodiment of the present invention provides for a novel process for enhancing the biomass and lipid content of microalgae wherein the microalgae strain has lipid content in the range of 21.4-30.45%.
  • cost effective media components mainly nitrogen, phosphorus and micronutrient and their concentration ranging from 5 ppm to 5% for high growth and lipid content have been used.
  • These sources include fertilizers, biogas plant residue extract, corn steep liquor, cow dung extract, plants extract, poultry dropping extract and so forth.
  • antibacterial and/or fungicidal compounds of plant origin are added to inhibit growth of bacteria/fungi in cultivation media of microalgae.
  • hot water extract of plant was to inhibit growth of bacteria/fungi in cultivation media of algae.
  • the hot water extract of plant origin not only inhibits the growth of undesired microbes in open pond but also improves the growth of algae.
  • the present invention further discloses a method for cultivation in open pond/photo bioreactor/ ' poiybags.
  • the algal biomass cultivated in open pond/photo bioreactor/'poiybags or in other cultivation medias is harvested followed by cell disruption by mechanical method, chemical exposure, salinity changes or pH changes and subsequently extracting the oil using combination of polar and nonpolar solvents at a suitable temperature, wherein the temperature may range between 30-80 degree C.
  • Algal isolation media containing (g/L) Na2 C03 (0.5-5), NaHC03 (1-5) KH 2 P0 4 (0.5-4.8), K 2 HP0 4 (0.5-5), MgS0 4 (0.01-1.0), (NH4) 2 S0 4 (0.25-0.50), KN0 3 (0.15-4.75), Urea (0.15-4.75), Di-ammonium phosphate (0.15-4.75), ZnS04 (0.2-2.1), NaCl (0.2-10) Trace element (2ml to 10 ml of solution).
  • AIM Algal isolation media
  • the trace element, solution (gram per liter) comprises Nitrilotriacetic acid (0.1-1,0), FeS0 4 .7H 2 0 (0.01-0.15), MnCl 2 .4H 2 0 (0.001- 0.005), CoCl 2 .6H 2 0 (0.005-0.02), CaCl 2 .2H 2 0 (0.01-0.5), ZnCl 2 (0.01 -0.15), CuCl 2 .H 2 0 (0.01-0.03), H 3 B0 3 (0.002-0.02), Na 2 Mo0 4 (0.001 -0.02), Na 2 Se() 3 (0.005-0.02), NiS0 4 (0.01-0.03), SnCl 2 (0.01 -0.03).
  • Each 1000 ml of flask contained 500 mi of above media was autoclaved. It was inoculated with 5-10% of soil or water sample. The flasks were incubated at 45°C for 2-10 days in presence of light and continuously C02 was sparged. After completion of incubation the 1 ml culture was centrifuged at 3000 rpm for 5 minutes. The ceils which are floating were re-inoculated in above media where N03 was omitted from AIM. This was repeated for total twelve cycles. After 5 la cycle the solvent used for media preparation was low quality water i.e., water containing heavy metals (100-500 ppm), hydrocarbons (0.001%-2%), & high salinity (3%).
  • the resulting 18S rRNA gene sequences were aligned and compared to the nucleotide sequences of some known microalgae in GenBank database of the National Center for Biotechnology Information by using Basic Local Alignment Search Tool (BLAST ® ) .
  • Protoplast was prepared according to method known in prior art. For shuffling, protoplasts were fused by suspension in buffer (0.5 M sucrose, lOmM Tris -HCL 20mM MgCl 2 ) containing 15% dimethyl sulphoxide and 60% PEG-6000. The resulting suspension was incubated at 25°C for 50 min.
  • the fused protoplast preparation was diluted with regeneration media (AIM media containing 0.5M sucrose) and protoplasts were harvested by centrifugation at 3500 rpm for 10 min at 25°C.
  • the protoplast cells floating was collected and were re-suspended in regeneration media and shaken at 200 rpm for 12 h before plating on agar plates higher salt concentration 2%, heavy metal concentration (250 ppm), pH ( 1 1 ) .
  • the plated were scraped to generate a pooled fusion library.
  • the formation of protoplasts, their fusion and their subsequent regeneration was repeated six times with pooled regenerated cells from one fusion being the inoculum for the subsequent protoplast culture.
  • Non-shuffled controls were prepared by 5 the recursive formation and regeneration of protoplasts without exposure to PEG. This process was carried out for six cycles. This accelerates directed evolution through recursive recombination of improved progeny, thereby improving multiple traits.
  • the niicroalgae strains thus developed by the process of the invention have the ability to grow in 10 presence of salt up to 3%, at a temperature range from 10-45 degree C, high light intensity, pH 4.5- 10 and heavy metal 100-1000 ppm (Table- 1).
  • the strains so developed have lipid composition suitable for biofuel (Table-2).
  • the above given factors are added in a sequential manner in suitable concentrations.
  • the novel feature of the process relates to the sequence in which the various ingredients of the composition are added during the process.
  • the adding and/or mixing the various compositions of the ingredients in the sequential manner as described as herein, description of the specification and the claims allows or provides a maximum beneficial mode to enhance the biomass, lipid and saturate contents of the algal strains. It has been found that this sequential mode is not only useful for enhancing the biomass, lipid and saturate contents of control or unmodified microalgae but also of the novel strains.
  • Table 3 Modulation of growth conditions leading to improved biomass of improved strain (Strain: Chlorella vulgaris IOC- 106). Sequence of addition, timing and concentration various bio-chemical-physical factors like growth hormones, specific bacteria, protein synthesis inhibiting chemicals- sodium thio-sulphate, ultra-violet rays; high and low r temperature are used to improve biomass, lipid content and/or its composition.
  • the composition of the invention includes extract of different part of the plants such as lantana, tobacco, neem, ma endi, vegetative and/or fruit plant material and/or mixtures thereof
  • Plant material includes the stem, leaves and fruit of the plant and any part of the plant.
  • the plant material is dried, powdered and extracted with water, sequentially and/or simultaneously at different temperature, pressure to remove the compounds having ability inhibit the growth of undesired bacteria and stimulate growth of algae.
  • the temperature ranges from 40-100 °C, preferably 55-80 °C and the pressure ranges from atmospheric to 15 lbs.
  • the extracted material was further purified using known art like column chromatography and each fraction was evaluated for their ability to inhibit growth of undesired bacteria and stimulate growth of algae.
  • the plant extract was effective in the concentration ranging from 1 -10% (v/v) in media.

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Abstract

La présente invention concerne des souches améliorées de microalgues, des procédés associés et leur utilisation.
PCT/IB2013/059407 2012-10-19 2013-10-17 Souches améliorées de microalgues et leur utilisation WO2014060973A1 (fr)

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CN104480153A (zh) * 2014-11-25 2015-04-01 临沂大学 促进微拟球藻大量积累高不饱和脂肪酸的方法
CZ306149B6 (cs) * 2014-09-25 2016-08-24 Ecofuel Laboratories S.R.O. Produkční kmen Japonochytrium sp. AN4-10, jeho použití a způsob produkce kyseliny dokosahexaenové
WO2016160999A1 (fr) * 2015-03-31 2016-10-06 Solazyme, Inc. Micro-algues conçues pour des conditions de culture hétérotrophes
EP3109315A4 (fr) * 2014-10-15 2017-07-26 Universidade de Santiago de Compostela Procédé d'enrichissement de biomasse de microalgues en acides gras polyinsaturés
WO2018112177A1 (fr) 2016-12-14 2018-06-21 Heliae Development, Llc Procédés et compositions se rapportant à des microalgues enrichies en phytohormones
CN109576314A (zh) * 2017-09-28 2019-04-05 中国石油化工股份有限公司 一种混合培养制备微藻油脂的方法
CN110628759A (zh) * 2019-09-23 2019-12-31 西南科技大学 一种耐辐射野生型丛枝菌根真菌诱变筛选富集植物共生菌新种质的方法
CN114394670A (zh) * 2021-12-15 2022-04-26 海南大学 一种小球藻及其在重金属废水处理中的应用

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Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104084221A (zh) * 2014-06-23 2014-10-08 北京科技大学 一种生物吸附溶液中重金属离子制备光催化材料的方法
CZ306149B6 (cs) * 2014-09-25 2016-08-24 Ecofuel Laboratories S.R.O. Produkční kmen Japonochytrium sp. AN4-10, jeho použití a způsob produkce kyseliny dokosahexaenové
EP3109315A4 (fr) * 2014-10-15 2017-07-26 Universidade de Santiago de Compostela Procédé d'enrichissement de biomasse de microalgues en acides gras polyinsaturés
CN104480153A (zh) * 2014-11-25 2015-04-01 临沂大学 促进微拟球藻大量积累高不饱和脂肪酸的方法
US11352602B2 (en) 2015-03-31 2022-06-07 Corbion Biotech, Inc. Microalgae adapted for heterotrophic culture conditions
WO2016160999A1 (fr) * 2015-03-31 2016-10-06 Solazyme, Inc. Micro-algues conçues pour des conditions de culture hétérotrophes
CN107889502A (zh) * 2015-03-31 2018-04-06 柯碧恩生物技术公司 适应于异养培养条件的微藻
EP3447124A1 (fr) * 2015-03-31 2019-02-27 Corbion Biotech, Inc. Micro-algues conçues pour des conditions de culture hétérotrophes
WO2018112177A1 (fr) 2016-12-14 2018-06-21 Heliae Development, Llc Procédés et compositions se rapportant à des microalgues enrichies en phytohormones
CN109576314A (zh) * 2017-09-28 2019-04-05 中国石油化工股份有限公司 一种混合培养制备微藻油脂的方法
CN110628759A (zh) * 2019-09-23 2019-12-31 西南科技大学 一种耐辐射野生型丛枝菌根真菌诱变筛选富集植物共生菌新种质的方法
CN114394670A (zh) * 2021-12-15 2022-04-26 海南大学 一种小球藻及其在重金属废水处理中的应用
CN114394670B (zh) * 2021-12-15 2023-05-23 海南大学 一种小球藻及其在重金属废水处理中的应用

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