WO2015076689A1 - Process for mixtrophic cultivation of algae - Google Patents

Process for mixtrophic cultivation of algae Download PDF

Info

Publication number
WO2015076689A1
WO2015076689A1 PCT/RO2013/000025 RO2013000025W WO2015076689A1 WO 2015076689 A1 WO2015076689 A1 WO 2015076689A1 RO 2013000025 W RO2013000025 W RO 2013000025W WO 2015076689 A1 WO2015076689 A1 WO 2015076689A1
Authority
WO
WIPO (PCT)
Prior art keywords
protein
algae
parts
mixture
biomass
Prior art date
Application number
PCT/RO2013/000025
Other languages
French (fr)
Inventor
Florin Oancea
Sandra VELEA
Emil Stepan
Lucia ILIE
Original Assignee
Institutul National De Cercetare-Dezvoltare Pentru Chimie Si Petrochimie - Icechim,
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Institutul National De Cercetare-Dezvoltare Pentru Chimie Si Petrochimie - Icechim, filed Critical Institutul National De Cercetare-Dezvoltare Pentru Chimie Si Petrochimie - Icechim,
Publication of WO2015076689A1 publication Critical patent/WO2015076689A1/en

Links

Classifications

    • 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
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/02—Oxygen as only ring hetero atoms
    • C12P17/04—Oxygen as only ring hetero atoms containing a five-membered hetero ring, e.g. griseofulvin, vitamin C
    • 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
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • 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
    • 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
    • C12P7/6458—Glycerides by transesterification, e.g. interesterification, ester interchange, alcoholysis or acidolysis
    • 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
    • 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

  • This invention refer to a process of mixotrophic cultivation of unicellular algae, in a mineral medium supplemented with glycerol and hydrolysates of proteins and nucleic acids, intended to be used for the production of algal biomass, which could be further processed into biofuels, including aviation bio-based fuel.
  • Patent application WO2010/123848 reveals a process that involves the formation of algal cultures, by combining the populations of algae that have the ability to grow on industrial waste water, with an growing media with industrial waste water, supplemented optionally with a carbon source, represented by glucose, sucrose, fructose, glycerol, acetate, methanol or lignocellulose hydrolysates, as well as any other combination thereof.
  • a carbon source represented by glucose, sucrose, fructose, glycerol, acetate, methanol or lignocellulose hydrolysates, as well as any other combination thereof.
  • Patent application WO2012/035262 describes a mixotrophic process of cultivation of algae in the presence of a discontinuous light source, in form of flashes.
  • Cultivation medium is supplemented with various carbon sources: 5 mM acetate, 5 g/l glucose, 10 g/l lactose, 10 g/l sucrose and 5 g/l glycerol.
  • Flashes of light which are between 20 and 30 per hour, have amplitude equal or superior to 10 pEm "2 s "1 , and last between 20 seconds to 10 minutes, preferably between 10 seconds and 2 minutes, and most preferably between 20 seconds and 1 minute.
  • cultivated algae accumulate large amounts of biomass, ranging between 100 and 150 g/l, with a fat content of more than 30% higher than the same autotroph cultivated algae, and cultivation time is reduced to less than 40 hours.
  • Patent application US 2013/0217084 describes a process of mixotrophic cultivation of algae on media supplemented with 30 g/l of raw glycerol from biodiesel production and 10 g/l yeast extract, wherein unicellular algae biomass obtained have a high content of omega-3 fatty acids.
  • Hydrolysis of protein and nucleic acids from defatted algal biomass with a mixture of hydrolases initially with a mixture consisting of 0.25 parts of protein, with specific endonuclease activity protein of 1 x 10 6 endonuclease units/mg of protein, and 0.25 parts of protein, with specific protein phosphatase, PP, activity of 300 units PP/mg of protein, to 1000 parts of defatted algal biomass, expressed as dry matter, for 12 hours at 45°C and pH 6.5, followed by a treatment with 0.1 parts protein, with specific protease activity of 2.4 units Anson (AU) per gram, and 0.1 parts of protein, with a aminopeptidase activity of 500 LAPU/g, to 1000 parts defatted algal biomass, expressed as dry matter, at pH 6.5 and temperature of 60°C, for 16 hours.
  • AU Anson
  • the process according to the invention presents the following advantages: Superior accumulation of unicellular biomass and of lipids in algal biomass, due to the supplementation of cultivation media with organic sources of carbon, nitrogen and phosphorus, with high bio-availability;
  • Example 1 Algal biomass is harvested after cultivation in a photobioreactor Biostat PBR 2S (Sartorius Stedim Biotech, Goettingen, Germany), by centrifugation on a continuous laboratory centrifuge Westfalia Laboratory Separator, model SA 1 - 02-175 (GEA Westfalia Separator Group, Germany, Oelde), which is operated at a speed of the centrifugal disks of 10,000 rpm, equivalent of 8,500 x g, and at a feeding rate of 0.3 liters/min, with continuous separation of clarified culturing media and a discontinuous one for algae concentrated, when this reach the density of 1 100 kg/m 3 .
  • Algal biomass concentrate is homogenized on a high pressure piston homogenizer GEA Niro Soavi Arriete NS2006 (GEA Niro Soavi, Parma, Italy) with a knife edge valve, three cycle at 150 MPa, 0.3 liters / min.
  • High pressure homogenization causes disruption of the algal cells by lysis induced by pressure variations and forced passing through the knife edge valve, with the expression of cell content and cell wall exposure.
  • lipids are extracted with a mixture of chloroform : methanol 2 : 1 , in a ratio of 5 parts solvent mixture to 1 part homogenate.
  • Lipids are trans-esterified using a basic catalyst, potassium alkoxide. 100 parts of algal oil, react in autoclave, under protective atmosphere of nitrogen, and at 40°C for 8 hours with a solution obtained from 0.8 g sodium hydroxide 99.1 % and 1 1.3 g methanol 99.9%, with the recovery of the raw glycerol.
  • Raw glycerol pH is corrected to neutral with phosphoric acid, and this is used as a source of carbon for supplementation of the cultivation media of algae.
  • Transesterification can be performed by any other known process, with the recovery of the raw glycerol, which pH is corrected to neutral either with phosphoric acid, either with potassium hydroxide, and this raw glycerol is used as source of carbon to algae cultivation media.
  • Nucleic acids and proteins from the defatted algal biomass are hydrolyzed with a mixture of hydrolases.
  • 1000 parts of defatted algal biomass, which has a residual humidity of over 80% is treated with a mixture consisting of 0.25 parts protein, with specific endonuclease activity of 1 x 10 6 endonuclease units / mg protein (about 0.28 part Benzonase, purity degree II > 90%, Merck Millipore, Darmstad, Germany, mixture of endonuclease from Serratia marcensens, obtained by expression of specific genes in E.
  • Benzonase unit is defined as the amount of enzyme that causes, in standard conditions, an increase in absorbance A260 with 1.0 in 30 min, corresponding to a complete digestion of 37 ⁇ g DNA.
  • the standard reaction conditions are 1 mg/ml sonicated DNA substrate, i.e.
  • alkaline phosphatase is defined as the amount of enzyme that causes, in standard conditions, release of 1 ⁇ p-nitrophenol per min. Standard Conditions are: 0.1 ml enzyme sample added over 1.9 ml solution of p-nitro-phenyl phosphate, disodium salt (2 mg per ml in buffer Tris-HCI 1 M, pH 10.0) and incubation mixture at 50°C. Any combination of endonuclease and alkaline phosphatase may be used, with the condition to provide a similar enzyme activity in the mixture.
  • Anson unit is defined as the amount of enzyme which, in standard conditions, at pH 7.5, 25°C and 10 min reaction time, digests hemoglobin with an initial rate which produced within a minute a quantity of trichloroacetic acid soluble compounds that give the same color with the Folin-Ciocalteu reagent as 1 miliechivalent of tyrosine.
  • a LAPU unit, unit leucine aminopeptidase is the amount of enzyme that hydrolyzes 1 pmol of leucine-p-nitroanilide / min, in standard conditions, 26 mM of L leucine-p-nitroanilide as substrate, buffer Tris-HCI 0.1 M, pH 8.0, 40 ° C).
  • the supernatant from centrifugation is tangentially ultra-filtrated on a system of tangential ultrafiltration Prostak (Merck Millipore, Billerica, MA, USA), provided with a membrane Ultracel PLAC (Merck Millipore), and from regenerated cellulose with a cut-off of 5 KDa.
  • the concentration in the permeate of phosphorus, hydrolyzed protein, amino acids and/or peptides, is continued till reaching a 5% concentrations of total nitrogen and 1.5% of total phosphorus in the permeate, controlled by the determination of total nitrogen and phosphorus.
  • the permeate is sampled periodically analyzes the contents of total nitrogen and phosphorus, using Kjeldahl method ( 3342 NE-2001 ) and after extracting in Aqua Regis (EN 13346-2000).
  • Cultivation of unicellular algae is done on mineral medium Z, Zarouk, supplemented with 16 g/l NaHC0 3 , and mixotrophic media, Zarouk supplemented by 16 g/l NaHC03, raw glycerol in concentration between 2 and 5 g/l, and 12.5 g/l hydrolyzed proteins and nucleic acids, with 5% N and 1.5% P, whose formulas are presented in table 1 below.
  • Tab. 1 The composition of the Zarouk media and Zarouk media supplemented with glycerol and hydrolyzed algal protein and nucleic acid (Zarouk mixotrophic).
  • the growth conditions in photobioreactor are: working volume - 3 I; working temperature: 28°C; illumination 250 ⁇ ⁇ "2 3 "1 , with photoperiod / alternation of light / dark cycles of 12 : 12 hours; the aeration with the following synthetic gas mixture: 7% CO 2, 14% O 2 and 79% N 2 , at a rate of 30 ml/min, corresponding to an aeration of 1 liter of gas mixture with 7% CO2 per min, per 100 liters; the speed for recirculation of peristaltic pump 70%, i.e.
  • a recirculating debit of 3500 ml/min programming from photobioreactor software for measurement and registration of the working parameters: pH, turbidity (OD), temperature, light, recirculation flow, debit of CO 2 / synthetic gas mixture used for aeration.
  • the growing medium is inoculated with 300 ml of inoculum form a culture of Nannochloris sp. 424-1 strain, min. 10 9 ufc/ml. It is grown for 5 days, until reaching the total concentration of 100 g/l algal biomass, followed by harvesting algal biomass.
  • the average results obtained are shown in table 2 below.
  • Example 2 Proceed as in example 1 , with the difference that it is used, for micro-algae cultivation, an integrated photosynthesis system, described in detail in patent RO 0123480.
  • this photosynthesis system is composed from a central body in the form of U shape tank, open at the top, wherein are placed a variable number of photosynthesis cells, linked each other with parallel pipeline for feeding nutrient solutions (or nutrient solutions + algal mass or mass algal brought till the stage of exponential growth) and, respectively with pipes supplying the system with gases with variable content of carbon dioxide. Over the U shape tanks is placed the lighting system.
  • This integrated photosynthesis system combines the advantages of the open systems, of pond types, with those of the close system with flat plates, and could be included in the hybrid photobioreactor class.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • Genetics & Genomics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Oil, Petroleum & Natural Gas (AREA)
  • Botany (AREA)
  • Cell Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Virology (AREA)
  • Biomedical Technology (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

The invention refers to a process of mixotrophic cultivation of unicellular algae. Through this process the compounds resulted from preparation of algal biomass processed for biofuels production are used for supplementation of the autotrophic cultivation media, respectively raw glycerol, resulting from the transesterification of algal lipids, as source of carbon, and hydrolysates of proteins and nucleic acids from unicellular algae defatted biomass, as source of nitrogen and phosphorus. Raw glycerol is added in concentrations between 2 and 5 g/l mixotrophic media and hydrolysates of proteins and nucleic acids from algal biomass, normalized to 5% toal nitrogen and 1.5% total phosphorus, are used in the concentration of 12.5 g/l.

Description

PROCESS FOR MIXTROPHIC CULTIVATION OF ALGAE
This invention refer to a process of mixotrophic cultivation of unicellular algae, in a mineral medium supplemented with glycerol and hydrolysates of proteins and nucleic acids, intended to be used for the production of algal biomass, which could be further processed into biofuels, including aviation bio-based fuel.
There are known several patents related to different mixotrophic cultivation processes of unicellular algae, which involves supplementation of mineral growing media with various organic compounds. Patent application WO2010/123848 reveals a process that involves the formation of algal cultures, by combining the populations of algae that have the ability to grow on industrial waste water, with an growing media with industrial waste water, supplemented optionally with a carbon source, represented by glucose, sucrose, fructose, glycerol, acetate, methanol or lignocellulose hydrolysates, as well as any other combination thereof.
Patent application WO2012/035262 describes a mixotrophic process of cultivation of algae in the presence of a discontinuous light source, in form of flashes. Cultivation medium is supplemented with various carbon sources: 5 mM acetate, 5 g/l glucose, 10 g/l lactose, 10 g/l sucrose and 5 g/l glycerol. Flashes of light, which are between 20 and 30 per hour, have amplitude equal or superior to 10 pEm"2s"1, and last between 20 seconds to 10 minutes, preferably between 10 seconds and 2 minutes, and most preferably between 20 seconds and 1 minute. Through this process, cultivated algae accumulate large amounts of biomass, ranging between 100 and 150 g/l, with a fat content of more than 30% higher than the same autotroph cultivated algae, and cultivation time is reduced to less than 40 hours.
Patent application US 2013/0217084 describes a process of mixotrophic cultivation of algae on media supplemented with 30 g/l of raw glycerol from biodiesel production and 10 g/l yeast extract, wherein unicellular algae biomass obtained have a high content of omega-3 fatty acids.
A common disadvantage of processes described till now is the related to the fact that supplementation of mineral autotroph growing media is done by adding compounds that originate from terrestrial organisms photosynthesis, thus this supplementation could be done in very good conditions through the re-use of compounds derived from algal biomass processing, harvested also from mixotrophic media supplemented with carbon, nitrogen and phosphorus sources derived from unicellular algae biomass. Another disadvantage is that the predominant supplementation is done with sources of carbon, the main deficit in the commercial production of biofuels from micro-algae being that of nitrogen and phosphorus sources - see e.g. the review Chisti, 2013, J. Biotech., 167: 2012-214.
It is an object of this invention to describe a process of mixotrophic cultivation of unicellular algae, wherein are used, for supplementation of the mineral autotroph cultivation media, compounds derived from preparation of algal biomass processed for biofuel production, respectively raw glycerol, resulted from algal lipids transesterification, as a source of carbon, and protein and nucleic acids hydrolysates from spent unicellular algae biomass, as source of nitrogen and phosphorus. It is another object of this invention to describe a process by which the proteins and nucleic acids hydrolysates from unicellular algae biomass, used to supplement the autotrophic media, are obtained.
The process according to the invention consists of the following steps:
Harvesting of the biomass of algae grown on mixotrophic medium and disruption of the cells of unicellular algae by high pressure homogenization;
V Extraction of algal lipids from micro-algae homogenate, using a solvent mixture, followed by the recovery of solvents by distillation and separation of lipids;
Transesterification of lipid through known processes, with recovery of raw glycerol, which is used, after correction to neutral pH with phosphoric acid, as the carbon source to supplement the micro-algae cultivation mixotrophic medium, in concentrations between 2 and 5 g/l;
Enzymatic hydrolysis of extracted biomass with a mixture of hydrolases, phosphatases, nucleases and proteases, followed by separation by centrifugation of the algal material resistant to enzymatic hydrolysis, which consists predominantly of recalcitrant (ligno)cellulose, and further processing of this material to furans and levulinic acid, usable for bio-fuels production;
V Normalization of proteins and nucleic acids hydrolysates by tangential ultrafiltration on a membrane with a cut-off of 5 kDa, till 5% total nitrogen and .5% total phosphorus, and use of these as sources of nitrogen and phosphorous, to supplement micro-algae cultivation medium, in a concentration of 12.5 g/l;
Cultivation of unicellular algae on mineral medium, supplemented with 16 g/l NaHC03, raw glycerol in concentration between 2 and 5 g/l and 12.5 g/l hydrolysates of proteins and nucleic acids, with 5% N and 1.5% P, on an aerated medium, with 1 ...10 liters of gaseous mixture, with 7% C02, per min per 100 I of medium, at a temperature of 28°C and at illumination intensities between 250 and 1000 μΕπϊ , for 2 to 5 days, until the total concentration of 100 g/l algal biomass is reached, followed by harvesting algal biomass.
The favorite aspects of the process described above are:
Harvesting by centrifugation at 8000 x g , and disruption of unicellular algae cells through repeated passing on a high pressure piston homogenizer with knife edge valve, 3 cycles at 150 MPa;
Extraction of lipids from unicellular algae homogenate, with a mixture of chloroform : methanol 2: 1 , in a ratio of 5 parts solvent mixture at 1 part homogenate;
Hydrolysis of protein and nucleic acids from defatted algal biomass with a mixture of hydrolases, initially with a mixture consisting of 0.25 parts of protein, with specific endonuclease activity protein of 1 x 106 endonuclease units/mg of protein, and 0.25 parts of protein, with specific protein phosphatase, PP, activity of 300 units PP/mg of protein, to 1000 parts of defatted algal biomass, expressed as dry matter, for 12 hours at 45°C and pH 6.5, followed by a treatment with 0.1 parts protein, with specific protease activity of 2.4 units Anson (AU) per gram, and 0.1 parts of protein, with a aminopeptidase activity of 500 LAPU/g, to 1000 parts defatted algal biomass, expressed as dry matter, at pH 6.5 and temperature of 60°C, for 16 hours.
The process according to the invention presents the following advantages: Superior accumulation of unicellular biomass and of lipids in algal biomass, due to the supplementation of cultivation media with organic sources of carbon, nitrogen and phosphorus, with high bio-availability;
> Stimulation of the growth of algae due to accumulation of algal phytohormones and of amino acids precursor of phytohormones, on the enzymatic hydrolysates of defatted algal biomass;
> Promotion of the adaptation to adverse culturing conditions, represented by high intensities light and high concentrations of C02 in the gases used for aeration of the culturing media, due to the accumulation of osmoprotectants compounds, in particular proline, in enzymatic hydrolysates of defatted algal biomass;
> Use by recycling within the process of mixotrophic cultivation of raw glycerol and of fractions of protein and nucleic acids from algal biomass, by-products of the processing of lipids and carbohydrates from algal biomass into biofuels. Below are examples of invention embodiments, which illustrate the invention without limit it.
Example 1. Algal biomass is harvested after cultivation in a photobioreactor Biostat PBR 2S (Sartorius Stedim Biotech, Goettingen, Germany), by centrifugation on a continuous laboratory centrifuge Westfalia Laboratory Separator, model SA 1 - 02-175 (GEA Westfalia Separator Group, Germany, Oelde), which is operated at a speed of the centrifugal disks of 10,000 rpm, equivalent of 8,500 x g, and at a feeding rate of 0.3 liters/min, with continuous separation of clarified culturing media and a discontinuous one for algae concentrated, when this reach the density of 1 100 kg/m3. Algal biomass concentrate is homogenized on a high pressure piston homogenizer GEA Niro Soavi Arriete NS2006 (GEA Niro Soavi, Parma, Italy) with a knife edge valve, three cycle at 150 MPa, 0.3 liters / min. High pressure homogenization causes disruption of the algal cells by lysis induced by pressure variations and forced passing through the knife edge valve, with the expression of cell content and cell wall exposure.
From algae homogenate the lipids are extracted with a mixture of chloroform : methanol 2 : 1 , in a ratio of 5 parts solvent mixture to 1 part homogenate. Lipids are trans-esterified using a basic catalyst, potassium alkoxide. 100 parts of algal oil, react in autoclave, under protective atmosphere of nitrogen, and at 40°C for 8 hours with a solution obtained from 0.8 g sodium hydroxide 99.1 % and 1 1.3 g methanol 99.9%, with the recovery of the raw glycerol. Raw glycerol pH is corrected to neutral with phosphoric acid, and this is used as a source of carbon for supplementation of the cultivation media of algae.
Transesterification can be performed by any other known process, with the recovery of the raw glycerol, which pH is corrected to neutral either with phosphoric acid, either with potassium hydroxide, and this raw glycerol is used as source of carbon to algae cultivation media.
Nucleic acids and proteins from the defatted algal biomass are hydrolyzed with a mixture of hydrolases. Initially 1000 parts of defatted algal biomass, which has a residual humidity of over 80%, is treated with a mixture consisting of 0.25 parts protein, with specific endonuclease activity of 1 x 106 endonuclease units / mg protein (about 0.28 part Benzonase, purity degree II > 90%, Merck Millipore, Darmstad, Germany, mixture of endonuclease from Serratia marcensens, obtained by expression of specific genes in E. coli) and 0.25 parts protein with specific activity of protein phosphatase, PP, of 300 units PP / mg (0.5 parts of alkaline phosphatase purified from culture of Bacillus licheniformis MTCC 1483, in accordance with the procedure described by Pandey and Banik, 2011 , Biores. Technol., 102: 4226-4231), for 12 hours at 45°C and pH 6.5. Benzonase unit is defined as the amount of enzyme that causes, in standard conditions, an increase in absorbance A260 with 1.0 in 30 min, corresponding to a complete digestion of 37 μg DNA. The standard reaction conditions are 1 mg/ml sonicated DNA substrate, i.e. salmon sperm, in 50 mM Tris- HCI buffer, pH 8.0, 0.1 mg/ml BSA, 1 mM MgCI2( incubation at 37°C. A unit of alkaline phosphatase is defined as the amount of enzyme that causes, in standard conditions, release of 1 μηιοΐβε p-nitrophenol per min. Standard Conditions are: 0.1 ml enzyme sample added over 1.9 ml solution of p-nitro-phenyl phosphate, disodium salt (2 mg per ml in buffer Tris-HCI 1 M, pH 10.0) and incubation mixture at 50°C. Any combination of endonuclease and alkaline phosphatase may be used, with the condition to provide a similar enzyme activity in the mixture.
After the enzymatic hydrolysis of nucleic acids, with mixture of endonucleases and alkaline phosphatase, from defatted algal biomass proteins are hydrolyzed. 1000 parts of defatted algal biomass, with a residual humidity of over 80%, is treated with a mixture consisting of 0.1 parts protein, having a specific protease activity of 2.4 Anson units (AU) per gram (0.1 parts Alcalase AF 2.4 L, Novozyme, Novozyme A/S, Bagvaerd, Denmark, bacterial endopeptidase from Bacillus licheniformis, with subtilisin / serin endo-peptidase as the main component, the specific enzymatic activity being of 2.4 Anson units (AU) per gram) and 0.5 g parts of protein, with a aminopeptidase specific activity of 500 LAPU/g (0,5 parts Flavourzyme 500 MG, Novozyme, a complex of amidopeptidase / exopeptidaze and endo-proteases, obtained from Aspergillus oryzae, with a 500 LAPU/g) at pH 6.5 and temperature of 60°C for 16 hours. Anson unit is defined as the amount of enzyme which, in standard conditions, at pH 7.5, 25°C and 10 min reaction time, digests hemoglobin with an initial rate which produced within a minute a quantity of trichloroacetic acid soluble compounds that give the same color with the Folin-Ciocalteu reagent as 1 miliechivalent of tyrosine. A LAPU unit, unit leucine aminopeptidase, is the amount of enzyme that hydrolyzes 1 pmol of leucine-p-nitroanilide / min, in standard conditions, 26 mM of L leucine-p-nitroanilide as substrate, buffer Tris-HCI 0.1 M, pH 8.0, 40 ° C). Any kind of combination of endoproteases and amidopeptidases / exo-proteases could be used, with the condition to provide a similar enzyme activity in the mixture. The algal material resistant to enzymatic hydrolysis, which consists predominantly of recalcitrant (ligno)cellulose is separated by centrifugation, at 8,000 x g, and is subsequently processed to furans and levulinic acid, which are usable for bio-fuels production.
The supernatant from centrifugation is tangentially ultra-filtrated on a system of tangential ultrafiltration Prostak (Merck Millipore, Billerica, MA, USA), provided with a membrane Ultracel PLAC (Merck Millipore), and from regenerated cellulose with a cut-off of 5 KDa. The concentration in the permeate of phosphorus, hydrolyzed protein, amino acids and/or peptides, is continued till reaching a 5% concentrations of total nitrogen and 1.5% of total phosphorus in the permeate, controlled by the determination of total nitrogen and phosphorus. The permeate is sampled periodically analyzes the contents of total nitrogen and phosphorus, using Kjeldahl method ( 3342 NE-2001 ) and after extracting in Aqua Regis (EN 13346-2000).
Cultivation of unicellular algae is done on mineral medium Z, Zarouk, supplemented with 16 g/l NaHC03, and mixotrophic media, Zarouk supplemented by 16 g/l NaHC03, raw glycerol in concentration between 2 and 5 g/l, and 12.5 g/l hydrolyzed proteins and nucleic acids, with 5% N and 1.5% P, whose formulas are presented in table 1 below.
Tab. 1. The composition of the Zarouk media and Zarouk media supplemented with glycerol and hydrolyzed algal protein and nucleic acid (Zarouk mixotrophic).
Figure imgf000007_0001
*Micronutrients stock solution (g/l): H3B03, 2.860; MnS04-4H20, 2.030; ZnS04-7H20 0.222; Mo03 (85%) 0,018; CuS04-5H20 0.079; Co(N03)2-6H20 0.494.
* For preparation of chelated Fe stock solution are dissolved 0.69 g FeS04-7H20 and 0.93 g Na2EDTA in 80 ml of distilled water. After boiling for a short duration of time and cooling to the room temperature the final solution is brought in to a volume of 100 ml in volumetric flask. In the photobioreactor Biostat PBR 2S (Sartorius) are introduced 2.7 liters of Zarouk medium or Zarouk medium supplemented with 2 g glycerol and 12.5 g/l of hydrolysates of algal protein and nucleic acids (Zarouk mixotrophic).
The growth conditions in photobioreactor are: working volume - 3 I; working temperature: 28°C; illumination 250 μΙ μΕιτι"23 "1, with photoperiod / alternation of light / dark cycles of 12 : 12 hours; the aeration with the following synthetic gas mixture: 7% CO 2, 14% O2and 79% N2, at a rate of 30 ml/min, corresponding to an aeration of 1 liter of gas mixture with 7% CO2 per min, per 100 liters; the speed for recirculation of peristaltic pump 70%, i.e. a recirculating debit of 3500 ml/min; programming from photobioreactor software for measurement and registration of the working parameters: pH, turbidity (OD), temperature, light, recirculation flow, debit of CO2 / synthetic gas mixture used for aeration.
The growing medium is inoculated with 300 ml of inoculum form a culture of Nannochloris sp. 424-1 strain, min. 109ufc/ml. It is grown for 5 days, until reaching the total concentration of 100 g/l algal biomass, followed by harvesting algal biomass. The average results obtained are shown in table 2 below.
Tab 2. Autotrophic and mixotrophic growth of Nannochloris sp 424-1 photobioreactor Biostat PBR 2S, 250 μΙ Em"2s"\ with 12 hours photoperiod.
Figure imgf000008_0001
Example 2. Proceed as in example 1 , with the difference that it is used, for micro-algae cultivation, an integrated photosynthesis system, described in detail in patent RO 0123480. In essence, this photosynthesis system is composed from a central body in the form of U shape tank, open at the top, wherein are placed a variable number of photosynthesis cells, linked each other with parallel pipeline for feeding nutrient solutions (or nutrient solutions + algal mass or mass algal brought till the stage of exponential growth) and, respectively with pipes supplying the system with gases with variable content of carbon dioxide. Over the U shape tanks is placed the lighting system. This integrated photosynthesis system combines the advantages of the open systems, of pond types, with those of the close system with flat plates, and could be included in the hybrid photobioreactor class. In order to test the growth of 424-1 strain, firstly are loaded the photosynthesis cells with Zarouk medium with 16.8 g/l NaHCO3, or with Zarouk supplemented with 5 g/l glycerol and 12.5 g/l of hydrolysates from nucleic acids and protein from algae (mixotrophic media). Freshly prepared inoculum, in a volumetric ratio 9: 1 , is added. The loading is done to fill the photosynthesis cells, till the feeding fluid overflow into the photobioreactor central tank, through the spillways. The lighting system is coupled and then the gas mixture with carbon dioxide is introduced through the feeding pipe and re-circulated by the blower, with a pre-established flow, which bubbling on the media and maintain a good agitation on the photosynthesis cells.
Experiments were performed in the greenhouse conditions, at 22±2°C during the day and 17±2°C during the night, with 12 hours photoperiod. In laboratory conditions, the lighting was done with halogen lamps to 250 μΕιτ 1. In greenhouse conditions solar light was used, with intensities that reach 1100 mg E m'V during the afternoon, supplemented with light intensity of 160 μΕπϊ 1, derived from halogen lamps, when the light intensity decreased below 500 μΕηΥν. Determinations of the exponential growth rate and doubling time (Wood et al., 2005. Algal culturing techniques, 269-285), of the lipid content (Bligh and Dyer, 1959, Can. J. Biochem. Physiology 37: 911-917) and carotenoids (Wright and Jeffrey, 1997, in Jeffrey SW, Mantoura RFC, Wright SW (eds), Phytoplankton Pigments in Oceanography, Unesco Publishing, Paris, pp. 327-341) were done. The average of the results obtained is presented in table 3 below.
Tab 3. Autotrophic and mixotrophic growth of Nannochloris sp. 424-1 on the integrated photosynthetic system, at an illumination between 500-1100 μΕιη"25"1, with 12 hours photoperiod.
Figure imgf000009_0001
These results support the effectiveness of the proposed mixotrophic process for micro-algae cultivation; the process increased the yields of unicellular algae cultivation, recycling the nitrogen and phosphorus from proteins and nucleic acids of algal biomass, which are not used for biofuels production.

Claims

1. Process according to the invention characterized in that consist of the following steps: harvesting of the biomass of algae grown on mixotrophic medium and disruption of the cells of unicellular algae by high pressure homogenization; extraction of algal lipids from micro-algae homogenate, using a solvent mixture, followed by the recovery of solvents by distillation and separation of lipids; transesterification of lipid through known processes, with recovery of raw glycerol, which is used, after correction to neutral pH with phosphoric acid, as the carbon source to supplement the micro-algae cultivation mixotrophic medium, in concentrations between 2 and 5 g/l; enzymatic hydrolysis of extracted biomass with a mixture of hydrolases, phosphatases, nucleases and proteases, followed by separation by centrifugation of the algal material resistant to enzymatic hydrolysis, which consists predominantly of recalcitrant (ligno)cellulose, and further processing of this material to furans and levulinic acid, usable for bio-fuels production; normalization of proteins and nucleic acids hydrolysates by tangential ultrafiltration on a membrane with a cut-off of 5 kDa, till 5% total nitrogen and 1.5% total phosphorus, and use of these as sources of nitrogen and phosphorous, to supplement micro-algae cultivation medium, in a concentration of 12.5 g/l; cultivation of unicellular algae on mineral medium, supplemented with 16 g/l NaHC03, raw glycerol in concentration between 2 and 5 g/l and 12.5 g/l hydrolysates of proteins and nucleic acids, with 5% N and 1.5% P, on an aerated medium, with 1 ...10 liters of gaseous mixture, with 7% CO2, per min per 100 I of medium, at a temperature of 28°C and at illumination intensities between 250 and 1000 μΕηη' 1, for 2 to 5 days, until the total concentration of 100 g/l algal biomass is reached, followed by harvesting algal biomass.
2. Process according to claim 1 characterized in that harvesting by centrifugation is done at 8000 x g, and disruption of unicellular algae cells is done through repeated passing on a high pressure piston homogenizer with knife edge valve, 3 cycles at 150 MPa.
3. Process according to claim 1 characterized in that extraction of lipids from unicellular algae homogenate, with a mixture of chloroform : methanol 2: 1 , in a ratio of 5 parts solvent mixture at 1 part homogenate.
4. Process according to claim 1 characterized in that hydrolysis of protein and nucleic acids from defatted algal biomass is done with a mixture of hydrolases, initially with a mixture consisting of 0.25 parts of protein, with specific endonuclease activity protein of 1 x 106 endonuclease units/mg of protein, and 0.25 parts of protein, with specific protein phosphatase, PP, activity of 300 units PP/mg of protein, to 1000 parts of defatted algal biomass, expressed as dry matter, for 12 hours at 45°C and pH 6.5, followed by a treatment with 0.1 parts protein, with specific protease activity of 2.4 units Anson (AU) per gram, and 0.1 parts of protein, with a aminopeptidase activity of 500 LAPU/g, to 1000 parts defatted algal biomass, expressed as dry matter, at pH 6.5 and temperature of 60°C, for 16 hours.
PCT/RO2013/000025 2013-11-25 2013-11-27 Process for mixtrophic cultivation of algae WO2015076689A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
ROA201300896 2013-11-25
ROA201300896A RO130353B1 (en) 2013-11-25 2013-11-25 Process for mixotrophic growth of unicellular algae

Publications (1)

Publication Number Publication Date
WO2015076689A1 true WO2015076689A1 (en) 2015-05-28

Family

ID=53179857

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/RO2013/000025 WO2015076689A1 (en) 2013-11-25 2013-11-27 Process for mixtrophic cultivation of algae

Country Status (2)

Country Link
RO (1) RO130353B1 (en)
WO (1) WO2015076689A1 (en)

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030213745A1 (en) * 2002-05-07 2003-11-20 Haerther Daryl W. System and method for remediation of waste
WO2009094440A1 (en) * 2008-01-25 2009-07-30 Aquatic Energy Llc Algal culture production, harvesting, and processing
WO2010042842A2 (en) * 2008-10-09 2010-04-15 Eudes De Crecy A method of producing fatty acids for biofuel, biodiesel, and other valuable chemicals

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030213745A1 (en) * 2002-05-07 2003-11-20 Haerther Daryl W. System and method for remediation of waste
WO2009094440A1 (en) * 2008-01-25 2009-07-30 Aquatic Energy Llc Algal culture production, harvesting, and processing
WO2010042842A2 (en) * 2008-10-09 2010-04-15 Eudes De Crecy A method of producing fatty acids for biofuel, biodiesel, and other valuable chemicals

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
LANCU P. ET AL.: "Flue gas CO2 capture by microalgae in photobioreactor: a sustainable technology''.", CHEMICAL ENGINEERING, vol. 29, 2012, pages 799 - 804 *
LI XIN ET AL.: "«Effects of different nitrogen and phosphorus concentrations on the growth, nutrient uptake, and lipid accumulation of a freshwater microalga Scenedesmus sp.».", BIORESOURCE TECHNOLOGY, vol. 101, 2010, pages 5494 - 5500 *
PINCHUK GRIGORIY E. ET AL.: "Utilization of DNA as a Sole Source of Phosphorus, Carbon, and Energy by Shewanella spp.: Ecological and Physiological Implications for Dissimilatory Metal Reduction''.", APPLIED AND ENVIRONMENTAL MICROBIOLOGY, vol. 74, no. 4, 2008, pages 1198 - 1208 *
WETTSTEIN STEPHANIE G. ET AL.: "A roadmap for conversion of lignocellulosic biomass to chemicals and fuels'' .", CURRENT OPINION IN CHEMICAL ENGINEERING, vol. 1, 2012, pages 218 - 224 *

Also Published As

Publication number Publication date
RO130353A2 (en) 2015-06-30
RO130353B1 (en) 2017-12-29

Similar Documents

Publication Publication Date Title
Lowrey et al. Heterotrophic and mixotrophic cultivation of microalgae for biodiesel production in agricultural wastewaters and associated challenges—a critical review
García et al. Development of a process for the production of l-amino-acids concentrates from microalgae by enzymatic hydrolysis
Zhou et al. Microalgae cultivation and photobioreactor design
Thevarajah et al. Large-scale production of Spirulina-based proteins and c-phycocyanin: A biorefinery approach
Silva et al. Combining glucose and sodium acetate improves the growth of Neochloris oleoabundans under mixotrophic conditions
WO2023278317A1 (en) Microorganism-derived protein hydrolysates and methods of preparation and use thereof
Callejo-López et al. Versatile method to obtain protein-and/or amino acid-enriched extracts from fresh biomass of recalcitrant microalgae without mechanical pretreatment
CN107384844A (en) A kind of recombination bacillus coli for producing phospholipase D and its application
Li et al. Co-cultivation of Rhodotorula glutinis and Chlorella pyrenoidosa to improve nutrient removal and protein content by their synergistic relationship
Kumari et al. Cultivation of Spirulina platensis using NPK-10: 26: 26 complex fertilizer and simulated flue gas in sintered disk chromatographic glass bubble column
El-Sheekh et al. Influence of molasses on growth, biochemical composition and ethanol production of the green algae Chlorella vulgaris and Scenedesmus obliquus
Callejo-López et al. Main variables affecting a chemical-enzymatic method to obtain protein and amino acids from resistant microalgae
CN110846351B (en) Threonine fermentation medium prepared by using mycoprotein as raw material
JP2022519874A (en) Methods for Producing Microbial Lipids
CN108949617B (en) Bacillus subtilis, application thereof and enzyme preparation
CN112312771A (en) Method for producing cell-rich media using chemoautotrophic microorganisms
WO2015076689A1 (en) Process for mixtrophic cultivation of algae
TWI463012B (en) Duckweed hydrolysate and use thereof
RU2397247C1 (en) Lipase biosynthesis method
KR20140042566A (en) Method for producing succinic acid using spent microalgae as a nitrogen source
US20190218504A1 (en) Methods of culturing aurantiochytrium using acetate as an organic carbon source
AU2005254730A1 (en) Fermentation process using faba beans as nitrogen source
WO2018169402A1 (en) Method for the production of microalgae
CN110846369B (en) Process for jointly hydrolyzing mycoprotein and soybean protein
RO130238B1 (en) Process for continuously growing microalgae in autotrophic - mixotrophic cycle, with water and nutrient recycling

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 13897810

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 13897810

Country of ref document: EP

Kind code of ref document: A1