WO2010017610A1 - Microbial lipid production process and composition containing said lipids - Google Patents

Microbial lipid production process and composition containing said lipids Download PDF

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Publication number
WO2010017610A1
WO2010017610A1 PCT/BR2009/000250 BR2009000250W WO2010017610A1 WO 2010017610 A1 WO2010017610 A1 WO 2010017610A1 BR 2009000250 W BR2009000250 W BR 2009000250W WO 2010017610 A1 WO2010017610 A1 WO 2010017610A1
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process according
lipids
conducted
medium
carbon
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PCT/BR2009/000250
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French (fr)
Portuguese (pt)
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Telma Teixeira Franco
Saartje Hernalsteens
Camilo Sixto López GARZÓN
Carlos Eduardo Vaz Rossel
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Universidade Estadual De Campinas - Unicamp
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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
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11B—PRODUCING, e.g. BY PRESSING RAW MATERIALS OR BY EXTRACTION FROM WASTE MATERIALS, REFINING OR PRESERVING FATS, FATTY SUBSTANCES, e.g. LANOLIN, FATTY OILS OR WAXES; ESSENTIAL OILS; PERFUMES
    • C11B1/00—Production of fats or fatty oils from raw materials
    • C11B1/02—Pretreatment
    • C11B1/025—Pretreatment by enzymes or microorganisms, living or dead
    • 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
    • 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

  • the present invention is a process for obtaining lipids from hexoses and pentoses from various sources using oilseed microorganisms.
  • the lipids obtained are similar to vegetable oils and can be used as substitutes for them, including in the production of biodiesel.
  • the present invention is primarily in the fields of industrial biology, chemistry and biotechnology.
  • lipids produced by microorganisms are triglycerides, also having phospholipids, hydrocarbons, carotenoids and sterols (Ratledge, 1991).
  • Yeast-produced lipids are most similar to vegetable oils, they contain almost exclusively 16- and 18-carbon fatty acid chains with similar unsaturation and triglyceride structure.
  • Cryptococcus curvatus also known as Candida curvata or Apiotrichum curvatum
  • Cryptococcus curvatus also known as Candida curvata or Apiotrichum curvatum
  • Rhodotorula gracilis also known as Rhodotorula glutinis
  • Rhodosporidium toruloides due to high lipid productivity, good stability and relative ease in cultivation.
  • yeasts have common metabolic characteristics, which translates into similar operating conditions for the production of lipids, in particular as regards the nitrogen content in the culture medium, since at limiting nitrogen concentrations, cell growth is suppressed and all carbon consumed is transformed and stored as lipid inclusions (Johnson and Singh, 1995). ).
  • Rhodosporidium torufoides was used in batch crops reaching a maximum yield of 18.2 gL-1 biomass with a lipid percentage of 76% (w / w) using glucose as carbon source.
  • Cryptococcus curvatus has shown superior growth and yield characteristics, obtaining values of 0.03 g lipids. g "1. h " biomass (Gill et al., 1977), compared to 0.012 g lipids. g "1. h " 1 biomass, obtained for Rhodotorula gracilis under similar cultivation conditions (Choi and Dewey, 1982).
  • Yeast screening for the conversion of xylan and xylose to triglycerides resulted in the selection of some species with potential use.
  • strains belonging to the genera Candida, Cryptococcus, Lipomyces, Rhodosporidium, Rhodotorula and Trichosporon were tested, resulting in the production of 32 to 63% of lipids in relation to dry mass, which were 72 to 86% of triglycerides (Fali et al. al, 1984).
  • Sugarcane bagasse is the most favorable biomass to work in Brazil, due to the high availability and possibility of its use "on the spot" by the alcohol-producing plant itself, which has already transported and pretreated it ( crushing).
  • Granger LM. Periot P., et al., Efficiency of fatty acid synthesis by oleaginous yeasts: Prediction of yield and fatty acid cell content from consumed C / N ratio by a simple method, Biotechnology and Bioengineering, Vol. 42, No. 10 , Pag 1151-1156, 1983.
  • JP 05/070324 describes a skin affinity cosmetic formulation containing an oil extracted from yeast.
  • JP 05030981 describes the production of fatty acids and oil preparation from baking yeasts.
  • the present invention differs from such documents in that it is not about polyunsaturated fatty acids.
  • EP 0 624 202 describes a method for obtaining microbial oil comprising gamma linolenic acid from microorganisms grown in a medium comprising sugar free starch and containing monocarboxylic acid (acetic acid) as a carbon source.
  • the present invention differs from that document in that it does not use carboxylic acids as a carbon source and additionally comprises alternative carbon sources.
  • EP 1 138 759 describes the use of a Labyrinthula microorganism which may be used for the production of polyunsaturated fatty acids comprising culture media containing various oils (soybean and palm oil, among others) and fatty acids.
  • the present invention differs from that document in that it does not involve marine fungi and does not use oils and fatty acids as a carbon source, but a lipid forming process comprising the use of microorganisms, further comprising carbon and nitrogen sources in certain proportions, temperature, agitation, among others.
  • EP 0 568 608 describes a composition comprising arachidonic acid and the method for obtaining it comprising culturing Pythium insidiosum in a culture medium containing hexoses as carbon and nitrogen sources.
  • the present invention differs from that document in that it comprises only oleaginous microorganisms, comprising neither Pythium insidiosum nor the production of polyunsaturated fatty acids, and by restricting the Carbon: Nitrogen ratio range.
  • WO 93/12242 discloses a method for microbial production of gamma linolenic acid containing oil.
  • the microorganisms used are of the order Mucorales preferably of the genus Mortierella, Actinomucor, Mucor, Rhizomucor or Rhizopus. Said microorganism is grown in culture medium substantially free of starch and sugars, containing as carbon source at least one moncarboxylic acid of 2 to 5 carbon atoms, preferably acetic acid, and recovering the oil from the formed biome.
  • Patent application PI 0406347-3 discloses the production of microbial biomass for lipid extraction by submerged aerated or un agitated or agitated culture from sugarcane derivatives (juice, molasses, refined sugar, brown sugar , invert sugar or even a mixture of these raw materials in different proportions and / or concentrations) for biodiesel production applications or for direct use as fuel in diesel engines and / or in simple power and steam generation as a substitute for vegetable oils / lipids.
  • sugarcane derivatives juice, molasses, refined sugar, brown sugar , invert sugar or even a mixture of these raw materials in different proportions and / or concentrations
  • WO 06/033723 describes the production of gamma linolenic acid using recombinant oleaginous yeasts such as Yarrowia lipolytica for the purpose of expressing desaturases (delta-6 and delta-12).
  • the present invention differs from that document in that it does not require recombinant oleaginous yeasts.
  • US 2005/0266537 discloses a process for obtaining new fatty acids for various uses, said process conducted from the cultivation of recombinant oleaginous yeasts.
  • the present invention differs from that document in that it does not require recombinant oleaginous yeasts.
  • WO 08/042338 describes the production of carotenoids in recombinant yeast and oleaginous fungi that allow lipid accumulation and carotenoid production.
  • the present invention differs from that document in that it does not comprise carotenoids.
  • WO 04/101757 discloses a process for obtaining omega-3 or omega-6 fatty acids from the cultivation of oil yeasts containing genes encoding the biosynthetic pathway of such fatty acids in the presence of fermentable carbon sources, followed by recovery of said fatty acids.
  • EP 1 219 704 discloses a process for obtaining a prenyl alcohol (geranylgeraniol, farnesol and nerolidol) from the cultivation of a wide variety of microorganisms, including those of the genus Lypomyces.
  • the culture medium is high in sugar and contains surfactant, oil, fat or terpenes, thereby providing the accumulation of prenyl alcohol in the cells, which subsequently secrete it into the medium. .
  • the process of the present invention comprises the use of bioreactors.
  • the oleaginous microorganism used is a yeast of the genus Lypomyces.
  • the carbon source is obtained from lignocellulosic material or any other source, such as raw and refined sugar.
  • the process of the present invention is conducted at temperatures between 25 ° C and 40 ° C, preferably 30 ° C.
  • the process of the present invention is conducted at a pH of 3.0 to 7.0, preferably 4.5.
  • the process of the present invention is conducted with agitation between 100 and 900 rpm, preferably 300 rpm.
  • the process of the present invention is conducted at 1 to 4 vvm aeration, preferably 2 vvm.
  • the lipids obtained by this process mainly comprise triglycerides composed of 12 to 20 C chain fatty acids.
  • the process of the present invention further comprises a second phase of lipid accumulation when a medium preferably composed solely of carbon sources is added to the bioreactor in order to keep the carbohydrate concentration constant.
  • composition comprising the lipids obtained by this process.
  • Figure 1 shows a schematic representation of the pretreatment and hydrolysis process of lignocellulosic material (biorefinery), where: ML - lignocellulosic material; ** pre-treatment; Cel - cellulose; CoG - cogeneration, heat and strength extracted; HL - hemicellulose, lignin; *** hydrolysis; Hex - hexoses; CQPM - fuels, chemicals, polymers, materials; Pt - pentoses.
  • FIG. 2 shows a schematic representation of the batch (A), fed batch (B) and continuous (C) processes, the feed (*) and product outlet (P) points being indicated.
  • Figure 3 shows lipid accumulation at different C / N ratios by Rhodotorula glutinis (in gray) and Lipomyces starkey (single batch).
  • Figure 4 shows the composition of the biodiesel produced (methyl fatty acid esters).
  • suitable "carbon sources” are chosen from the group comprising: glucose; xylose; sucrose and / or combinations thereof.
  • these carbon sources are derived from materials such as: pretreated and / or hydrolyzed lignocellulosic material, molasses and juice from sugarcane, corn, straw and bagasse from various sources.
  • the process of the present invention utilizes 20-100 g / l carbohydrate from lignocellulosic material.
  • suitable nitrogen sources are chosen from the group comprising: urea, ammonium salts, yeast extract or hydrolysates, peptone, caseinates, agrochemical industry residues. food and soy residues, pure and / or combinations thereof.
  • ammonium salts are used.
  • the oleaginous microorganisms comprise fungi and / or yeast and / or oleaginous bacteria selected from the group comprising Candida sp, Rhodotorula sp, Cryptococcus sp, Lypomyces sp, Rhodococcus sp. and / or combinations thereof. Combinations thereof are understood to be pooled co-cultivation and / or the use of genetically modified organisms comprising genes of the genera Candida sp, Rhodotorula sp, Cryptococcus sp, Lypomyces sp., Rhodococcus sp.
  • these genes may be selected from the list of genes cited in the Candida Species Genome Projects such as Candida albicans and Cryptococcus such as Cryptococcus neoformans found on the website of the US National Biotechnological Information Center (NCBI - www. ncbi.nlm.nih.gov)
  • the present invention utilizes a strain of Lipomyces starkeyi.
  • the microorganisms used are registered in the DSMZ (German Collection of Microorganisms and Cell Cultures Collection) Biological Material Banks, the ARS Culture Collection (Agricultural Research Service, also known as the NRRL Collection), INQS (National Institute for Quality Control in Health) and CCT (respectively as Rhodotorula glutinis CCT 0783, Rhodotorula glutinis DSM 10134, Rhodotorula glutinis DSM 70398, Rhodotorula mucilaginous INCQS 40157, Rhodosporidium toruloides DSM 4444, Cryptococcus albidus Crystococcus albidus Crystococcus albidus Cryptococcus albidus 70022, Lipomyces starkeyi NRRL 1 1557, Lipomyces starkeyi NRRL 27493, Lipomyces starkeyi DSM 70296, Candida lipolytica DSM 8218, Candida lipolytica DSM 1345, Lipomy
  • suitable culture medium is chosen from the group comprising single batch, fed batch and / or continuous process techniques.
  • the cultivation further comprises specific temperature, agitation, specific pH, aeration, carbon sources and nitrogen sources.
  • the culture medium contains some salts which give the process greater yield, KH 2 P0 4 (0 to 7 g / L), Na 2 HPO 4 (0 to 7 g / L), MgS0 4 of ( 0 to 4 g / l), plus traces (from 0 to 0.1 g / l) of CaCl 2 , ZnSO 4 , CuSO 4 and MnSO 4 .
  • the process of the present invention is conducted at temperatures between 25 ° C and 40 ° C, pH 3.0 to 7.0, agitation between 100 and 900 rpm, aeration 1 to 4 vvm from a medium containing from 20 to 100 gL- 1 of a carbon source and a Carbon / Nitrogen (w / w) ratio of 10 to 100.
  • the process of the present invention is performed at 30 ° C, pH of 4.5, 300 ppm agitation and 2vvm aeration.
  • Example 1 Process for obtaining lipids
  • the present invention provides a process for obtaining lipids in bioreactors using oleaginous microorganisms, preferably yeasts belonging to the genera Candida sp., Rhodotorula sp., Cryptococcus sp., Lypomyces sp., And bacteria belonging to the genus Rhodococcus sp. Lipomyces starkey. Both synthetic and industrial media containing carbon and nitrogen sources from the agri-food industry can be used (molasses, lignocellulosic material hydrolyzate, maize maceration water, cheese whey, pie and soya leaves, among others).
  • Both synthetic and industrial media should contain between 20 and 100 g / l carbohydrate (m / v) and sufficient nitrogen source to maintain the C / N ratio (m / m) between 10 and 100.
  • m / v g / l carbohydrate
  • nitrogen source to maintain the C / N ratio (m / m) between 10 and 100.
  • hydrolysis products from lignocellulosic material using the concept of biorefinery ( Figure 1).
  • Example 1 Preferred Process for Obtaining Lipids
  • the process can be performed in one or two steps, for the accumulation of lipids simultaneous to cell growth should preferably maintain 30 g / L carbohydrate and C / N ratio of 50 ( Figure 3).
  • the first phase is for cell development, which medium should preferably contain 70g / L carbohydrate and C / N ratio of 15 and the second phase of lipid accumulation when a medium is preferably composed Carbohydrate-only is added to the bioreactor to keep the carbohydrate concentration constant, preferably at 20 g / L ( Figure 2).
  • the culture medium should contain some salts which give the process greater yield, KH 2 P0 4 (0 to 7 g / L), Na 2 HPO 4 (0 to 7 g / L), MgSO 4 of (0 to 4 g / L), plus traces (0 to 0.1 g / L) of CaCl 2 , ZnSO 4 , CuSO 4 and MnSO 4 .
  • the processes are preferably performed in bioreactors, the pH being adjusted between pH 3.0 and pH 7.0, and to which 5 to 20% (v / v) inoculum is added.
  • the inoculum is preferably obtained by incubating the stored cell mass at 25 ° C to 30 ° C in flasks shaken at 150 to 200 rpm for 12 to 24 hours in synthetic medium containing (per liter): 30 g carbohydrate, 1 g (NH 4 ) 2 SO, 3 g KH 2 PO 4 , 1 g Na 2 HPO 4 , 1.5 g MgSO 4 .7H 2 O, 2 g CaCl 2 .6H 2 O and 2 g yeast extract ( pH between 4.5 and 5.0).
  • certain preferred parameters are thus maintained: temperature between 25 and 40 ° C, preferably 30 ° C; agitation between 100 and 900 rpm, preferably 300 rpm, aeration between 0.5 vvm and 4 vvm, preferably 2 vvm.
  • the process pH is controlled and maintained between pH 3.5 and 7.0, preferably pH 4.5; with the addition of acidic or basic solution, preferably 1 M HCl and 1 M NaOH.
  • the fed or continuous batch process is fed with a solution containing from 100 to 700 g / l carbohydrate, preferably 200 g / l carbohydrate, and whether or not it contains a nitrogen source.
  • the feed should contain enough nitrogen to maintain a C / N ratio of 50 to 200.
  • the addition of this solution should be such that the carbohydrate concentration in the culture medium is between 10 and 100 g / l, preferably 20%. g / L, and the C / N ratio of 60.
  • the fermented broth is clarified by any usual method, preferably by centrifugation and the lipids extracted also using any usual method.
  • any usual method preferably by centrifugation and the lipids extracted also using any usual method.
  • biodiesel without the need for prior extraction by direct transesterification.
  • lipid production is performed by the batch-fed Lypomyces starkey yeast, using an initial medium containing 50 g / l carbohydrate and C / N ratio of 15 (1 g / l yeast hydrolyzate or yeast extract). yeast and the remainder of ammonium sulphate). After 36 to 48 hours of reaction at 30 ° C, 2vvm and 300 rpm, and pH 4.5 (controlled with the addition of 1 M NaOH and 1 M HCl), a solution containing 300 g / L of carbohydrate to maintain approximately 20 g / l of carbohydrate in the culture medium until it is observed that there is no increase in lipid content and carbohydrate consumption.

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Abstract

The present invention provides a process for producing lipids from hexoses and pentoses of various origins, using oleaginous micro-organisms. The resultant lipids are similar to vegetable oils and can be used instead of the latter, including in the production of biodiesel.

Description

Relatório Descritivo de Patente de Invenção  Patent Invention Descriptive Report
PROCESSO DE PRODUÇÃO MICROBIANA DE LIPÍDEOS E COMPOSIÇÃO COMPREENDENDO TAIS LIPÍDEOS  MICROBIAN LIPID PRODUCTION PROCESS AND COMPOSITION UNDERSTANDING SUCH LIPIDS
Campo da Invenção  Field of the Invention
A presente invenção trata de um processo de obtenção de lipídeos a partir de hexoses e pentoses, provenientes de diversas fontes, utilizando microrganismos oleaginosos. Os lipídeos obtidos são semelhantes aos óleos vegetais, podendo ser utilizados como substitutos dos mesmos, inclusive na produção de biodiesel. A presente invenção situa-se principalmente nos campos da biologia, química e biotecnologia industrial.  The present invention is a process for obtaining lipids from hexoses and pentoses from various sources using oilseed microorganisms. The lipids obtained are similar to vegetable oils and can be used as substitutes for them, including in the production of biodiesel. The present invention is primarily in the fields of industrial biology, chemistry and biotechnology.
Antecedentes da Invenção Background of the Invention
Produção de Lipídios  Lipid Production
Normalmente, 80 a 90% dos lipídeos produzidos por microrganismos são triglicerídeos, apresentando também fosfolipídeos, hidrocarbonetos, carotenóides e esteróis (Ratledge, 1991 ). Lipídeos produzidos por leveduras são os mais semelhantes aos óleos vegetais, eles contêm quase que exclusivamente cadeias de ácidos graxos de 16 e 18 carbonos com insaturação e estrutura trigíicerídica semelhantes.  Typically, 80 to 90% of the lipids produced by microorganisms are triglycerides, also having phospholipids, hydrocarbons, carotenoids and sterols (Ratledge, 1991). Yeast-produced lipids are most similar to vegetable oils, they contain almost exclusively 16- and 18-carbon fatty acid chains with similar unsaturation and triglyceride structure.
Uma revisão dos estudos referentes a esta tecnologia mostra que os microorganismos mais importantes na produção de óleos microbianos são as leveduras oleaginosas, entre estas destacam-se: Cryptococcus curvatus (conhecido também como Cândida curvata ou Apiotrichum curvatum) (Gill et al., 1977; Johnson e Singh, 1995; Li e Lio, 2006; Pinkart et aí., 1998; Kaar et al., 1998), Rhodotorula gracilis (também conhecida como Rhodotorula glutinis) (Choi e Dewey, 1982; Granger e Periot, 1983; Martin et al., 2006; Meesters e Wal, 1996; Ykema e Verbree, 1986) e Rhodosporidium toruloides (Lee, 2005; Turcotte e Kosaric, 1988); devido à alta produtividade de lipídeos, boa estabilidade e a relativa facilidade no cultivo.  A review of studies on this technology shows that the most important microorganisms in the production of microbial oils are oilseeds, among them the following: Cryptococcus curvatus (also known as Candida curvata or Apiotrichum curvatum) (Gill et al., 1977; Johnson and Singh, 1995; Li and Lio, 2006; Pinkart et al., 1998; Kaar et al., 1998), Rhodotorula gracilis (also known as Rhodotorula glutinis) (Choi and Dewey, 1982; Granger and Periot, 1983; Martin et al., 2006; Meesters and Wal, 1996; Ykema and Verbree, 1986) and Rhodosporidium toruloides (Lee, 2005; Turcotte and Kosaric, 1988); due to high lipid productivity, good stability and relative ease in cultivation.
Estas leveduras possuem características metabólicas comuns, o que se traduz em condições de operação similares para a produção dos lipídeos, em particular no que se refere ao teor de nitrogénio no meio de cultivo, já que em concentrações limitantes de nitrogénio, o crescimento celular é reprimido e todo o carbono consumido é transformado e armazenado na forma de inclusões lipídicas (Johnson e Singh, 1995). These yeasts have common metabolic characteristics, which translates into similar operating conditions for the production of lipids, in particular as regards the nitrogen content in the culture medium, since at limiting nitrogen concentrations, cell growth is suppressed and all carbon consumed is transformed and stored as lipid inclusions (Johnson and Singh, 1995). ).
A produção de lipídeos por estas leveduras oleaginosas foi estudada em diferentes processos: batelada, batelada alimentada (Kaar et al., 1998) e processo contínuo (Gill et al., 977; Carvalho et al., 2005), sendo os dois últimos os mais apropriados para maximizar a produção de lipídeos já que é possível ajustar a relação Carbono: Nitrogénio para obtenção inicial de altas concentrações celulares e depois para estimular a produção de lipídeos.  The lipid production by these oilseeds was studied in different processes: batch, fed batch (Kaar et al., 1998) and continuous process (Gill et al., 977; Carvalho et al., 2005). more suitable for maximizing lipid production since it is possible to adjust the Carbon: Nitrogen ratio to initially obtain high cellular concentrations and then to stimulate lipid production.
Rhodosporidium torufoides foi utilizado em cultivos batelada atingindo produção máxima de 18,2 g.L-1 de biomassa com uma porcentagem de lipídeos de 76% (p/p) utilizando glicose como fonte de carbono. Nos três modos de operação, especialmente no cultivo contínuo, Cryptococcus curvatus tem mostrado características superiores de crescimento e de produtividade, obtendo valores de 0,03 g lipídeos. g biomassa"1. h" (Gill et al., 1977), em comparação com os 0,012 g lipídeos. g biomassa"1. h"1, obtidos para Rhodotorula gracilis em condições similares de cultivo(Choi e Dewey, 1982). Estudos envolvendo modelagem matemática (Granger e Periot, 1983; Carvalho et al., 2005) e desenvolvimento de metodologias de determinação e recuperação de lipídeos (Yamauvhi et al., 1983; Misra et al., 1984) demonstram o potencial tecnológico desta alternativa. Rhodosporidium torufoides was used in batch crops reaching a maximum yield of 18.2 gL-1 biomass with a lipid percentage of 76% (w / w) using glucose as carbon source. In all three modes of operation, especially in continuous cultivation, Cryptococcus curvatus has shown superior growth and yield characteristics, obtaining values of 0.03 g lipids. g "1. h " biomass (Gill et al., 1977), compared to 0.012 g lipids. g "1. h " 1 biomass, obtained for Rhodotorula gracilis under similar cultivation conditions (Choi and Dewey, 1982). Studies involving mathematical modeling (Granger and Periot, 1983; Carvalho et al., 2005) and the development of lipid determination and recovery methodologies (Yamauvhi et al., 1983; Misra et al., 1984) demonstrate the technological potential of this alternative.
O screening de leveduras para a conversão de xilana e xilose em triglicerídeos resultou na seleção de algumas espécies com potencial de utilização. Neste estudo foram testadas linhagens pertencentes aos géneros Cândida, Cryptococcus, Lipomyces, Rhodosporidium, Rhodotorula e Trichosporon, resultando na produção de 32 a 63% de lipídeos em relação à massa seca, sendo estes, compostos de 72 a 86% de triglicerídeos (Fali et al, 1984).  Yeast screening for the conversion of xylan and xylose to triglycerides resulted in the selection of some species with potential use. In this study strains belonging to the genera Candida, Cryptococcus, Lipomyces, Rhodosporidium, Rhodotorula and Trichosporon were tested, resulting in the production of 32 to 63% of lipids in relation to dry mass, which were 72 to 86% of triglycerides (Fali et al. al, 1984).
Materiais Lignocelulósicos O interesse nos materiais lignocelulósicos vem crescendo paralelamente ao aumento do preço do petróleo e seus derivados. Estes materiais não são ainda utilizados e sua aplicação representa incremento significativo da produção de energia, sem exigir aumento da área de plantio, favorecendo a redução de rejeitos e o aproveitamento integral da biomassa. O bagaço de cana-de-açúcar é a biomassa mais favorável para se trabalhar no Brasil, devido à elevada disponibilidade e possibilidade de sua utilização "in loco" pela própria usina produtora de álcool, a qual já o transportou e o pré-tratou (esmagamento). Lignocellulosic Materials Interest in lignocellulosic materials has been growing in parallel with the rising price of oil and its derivatives. These materials are not yet used and their application represents a significant increase in energy production, without requiring an increase in the planting area, favoring the reduction of waste and the full use of biomass. Sugarcane bagasse is the most favorable biomass to work in Brazil, due to the high availability and possibility of its use "on the spot" by the alcohol-producing plant itself, which has already transported and pretreated it ( crushing).
Para utilização do bagaço e da palha da cana para produção de biocombustíveis, é preciso transformar as cadeias poliméricas de carboidratos em açúcares, essencialmente glicose e xilose (e outros açúcares encontrados em menores concentrações no hidrolisado). Este processo de transformação requer pré-tratamento na biomassa (físico, químico ou físico-químico), seguido de hidrólise (ácida ou enzimática), e posteriormente a fermentação dos açúcares, convertendo-os em combustíveis, sendo que este processo deve ser o mais simples possível para obtenção de um produto com viabilidade comercial.  To use sugarcane bagasse and straw for biofuel production, it is necessary to transform the polymeric chains of carbohydrates into sugars, essentially glucose and xylose (and other sugars found in lower concentrations in the hydrolyzate). This transformation process requires pre-treatment in biomass (physical, chemical or physicochemical), followed by hydrolysis (acid or enzymatic), and then the fermentation of sugars, converting them into fuels, and this process should be the most possible to obtain a commercially viable product.
A literatura científica contempla alguns documentos relacionados ao objeto da invenção, sem porém antecipá-la ou sequer sugeri-la, sendo listados a seguir os principais para referência:  The scientific literature contemplates some documents related to the object of the invention, without anticipating or even suggesting it, being listed below the main ones for reference:
• Carvalho, W.; Santos, J.C.; Canilha, L; Silva, S.S.; Perego, P.;  • Oak, W .; Santos, J.C .; Canilla, L; Silva, S.S .; Perego, P .;
Converti, A. Xilitol production from sugarcane bagasse hydrolysate. Metabolic behavior of Cândida guilliermondii cells entrapped in Ca- alginate Biochemical Enginnering Journal, v. 25, p.25-31 , 2005.  Converti, A. Xylitol production from sugarcane bagasse hydrolysate. Metabolic behavior of Candida guilliermondii cells entrapped in Calcinate Biochemical Enginnering Journal, v. 25, pp. 25-31, 2005.
• Choi S.Y., Dewey D.Y., et al, Production of microbial lipid: Effects of growth rate and oxygen on lipid synthesis and fatty acid composition of Rhodotorula gracilis, Biotechnology and Bioengineering, Vol 24, N° 5, pag 1 165-1 172, 1982. Fali, R.; Phelps, P.; Spindler, D. Bioconversion of xylan to triglycerides by oil-rich yeasts Applied and environmental microbiology, v.47:1130-1134, 1984. Choi SY, Dewey DY, et al, Production of Microbial Lipid: Effects of Growth Rate and Oxygen on Lipid Synthesis and Fatty Acid Composition of Rhodotorula gracilis, Biotechnology and Bioengineering, Vol 24, No. 5, page 1 165-1 172, 1982. Fali, R .; Phelps, P .; Spindler, D. Bioconversion of xylan to triglycerides by oil-rich yeasts Applied and environmental microbiology, v.47: 1130-1134, 1984.
Gill C.O., Hall M.J., Ratledge C, Lipid accumulation in an oleaginous yeast (Cândida 107) growing on glucose in single-stage continuous culture, Applied and Environmental Microbiology, Vol. 33, N° 2, pag 231-239, 1977.  Gill C.O., Hall M.J., Ratledge C, Lipid accumulation in an oleaginous yeast (Candida 107) growing on glucose in single-stage continuous culture, Applied and Environmental Microbiology, Vol. 33, No. 2, page 231-239, 1977.
Granger L-M., Periot P., et ali, Efficiency of fatty acid synthesis by oleaginous yeasts: Prediction of yield and fatty acid cell content from consumed C/N ratio by a simple method, Biotechnology and Bioengineering, Vol. 42, N° 10, Pag 1151-1156, 1983.  Granger LM., Periot P., et al., Efficiency of fatty acid synthesis by oleaginous yeasts: Prediction of yield and fatty acid cell content from consumed C / N ratio by a simple method, Biotechnology and Bioengineering, Vol. 42, No. 10 , Pag 1151-1156, 1983.
Johnson V.W., Singh M., et ali, Utilization of molasses for the production of fat by an oleaginous yeast, Rhodotorula glutinis IIP-30, Journal of Industrial Microbiology and Biotechnology, Vol. 14, N° 1 , pag 1-4, 1995. Johnson VW, Singh M., et al., Utilization of molasses for the production of fat by an oleaginous yeast, Rhodotorula glutinis IIP-30, Journal of Industrial Microbiology and Biotechnology, Vol. 14, No. 1, page 1-4, 1995 .
Kaar, W.E., Gutierres, C.V., Kinoshita, C.M., Steam explosion of sugarcane bagasse as a pretreatment for conversion for ethanol. Biomass and Bioenergy, v. 14, n° 3, p. 277-287, 1998.  Kaar, W.E., Gutierres, C.V., Kinoshita, C.M., Steam explosion of sugarcane bagasse as a pretreatment for conversion to ethanol. Biomass and Bioenergy, v. 14, no. 3, p. 277-287, 1998.
Lee, Y., Oxidation of sugarcane bagasse using a combination of hypochlorite and peroxide. Louisiana, 2005. 65p. Tese (Mestrado) - Faculty of the Louisiana State University and Agricultural and Mechanical College. Lee, Y., Oxidation of sugarcane bagasse using a combination of hypochlorite and peroxide. Louisiana, 2005. 65p. Thesis (Faculty) - Faculty of the Louisiana State University and Agricultural and Mechanical College.
Li Y-H, Lio B., et ali, Optimization of culture conditions for lipid production by Rhodosporidium toruloides, Chinese Journal of Biotechnology, Vol. 22, N° 4, pag 650-656, 2006  Li Y-H, Lio B., et al., Optimization of culture conditions for lipid production by Rhodosporidium toruloides, Chinese Journal of Biotechnology, Vol. 22, No. 4, page 650-656, 2006
Martin, C, González.Y., Fernández, T., Thomsen, A., Investigation of cellulosic convertibility and ethanol fermentation of sugarcane bagasse pretreated by wet oxidation and steam explosion. Journal of Chemical Technology and Biotechnology, v. 81 , p. 1669-1677, 2006 • Meesters P.A., Wal H., et all, Cultivation of t e oleaginous yeast Cryptococcus curvatus in a new reactor with improved mixing and mass transfer characteristics (Surer®), Biotechnology Techniques, Vol. 10, N° 4r pag 277-282, 1996. Martin, C. González.Y., Fernández, T., Thomsen, A., Investigation of cellulosic convertibility and ethanol fermentation of sugarcane bagasse pretreated by wet oxidation and steam explosion. Journal of Chemical Technology and Biotechnology, v. 81, p. 1669-1677, 2006 • Meesters PA, Wal H., et al., Cultivation of the oleaginous yeast Cryptococcus curvatus in a new reactor with improved mixing and mass transfer characteristics (Surer®), Biotechnology Techniques, Vol. 10, No. 4 r page 277-282, 1996
· Misra, S.; Ghosh, A.; Dutta, J. Production and composition of microbial fat from Rhodotorula glutinis Journal of Sei. Food Agric. 35: 59-65, 1984.  · Misra, S .; Ghosh, A .; Dutta, J. Production and composition of microbial fat from Rhodotorula glutinis Journal of Sci. Food Agric. 35: 59-65, 1984.
• Pinkart H.C., Devereux R., Chapman P.J., Rapid separation of microbial lipids using solid phase extraction columns, Journal of Microbiological Methods, Vol 43, N° 1 , pag 9-15, 1998.  • Pinkart H.C., Devereux R., Chapman P.J., Rapid separation of microbial lipids using solid phase extraction columns, Journal of Microbiological Methods, Vol 43, No. 1, page 9-15, 1998.
• Ratledge C, Microorganisms for lipids, Acta Biotechnologica, Vol. 1 1 , N° 5, pag 429-438, 1991.  • Ratledge C, Microorganisms for lipids, Acta Biotechnologica, Vol. 11, No. 5, page 429-438, 1991.
• Turcotte G., Kosaric N., Biosynthesis of lipids by Rhodosporidium toruloides ATCC 10788, Journal of Biotechnology, Vol 8, N° 3, pag 221-237, 1988.  • Turcotte G., Kosaric N., Biosynthesis of Lipids by Rhodosporidium toruloides ATCC 10788, Journal of Biotechnology, Vol 8, No. 3, page 221-237, 1988.
• Yamauchi, H.; Mori, H.; Kobayashi, T.; SHAMIZU, S. Mass production of lipids of Lipomyces starkeyi in microcomputer-aided fed- batch culture. Journal of Fermentation technology 61 :275-280, 1983. • Yamauchi, H .; Mori, H .; Kobayashi, T .; SHAMIZU, S. Mass production of lipids of Lipomyces starkeyi in microcomputer-aided fed batch culture. Journal of Fermentation Technology 61: 275-280, 1983.
• Ykema A., Verbree E.C., et all, Mathematical modeling of lipid production by oleaginous yeasts in continuous cultures, Antonie van• Ykema A., Verbree E.C., et al., Mathematical modeling of lipid production by oleaginous yeasts in continuous cultures, Antonie van
Leeuwenhoek, Vol. 52, N° 6, pag 491-506, 1986. Leeuwenhoek, Vol. 52, No. 6, pages 491-506, 1986.
No âmbito patentário, poucos documentos versam sobre produção microbiana de lipídios por diferentes microorganismos.  In the patent area, few documents deal with microbial lipid production by different microorganisms.
O documento JP 05/070324 descreve uma formulação cosmética com afinidade pela pele contendo um óleo extraído de leveduras. Já o documento JP 05030981 descreve a obtenção de ácidos graxos e preparação de óleos, a partir de leveduras de panificação. A presente invenção difere de tais documentos por não tratar de ácidos graxos poliinsaturados. O documento EP 0 624 202 descreve um método para a obtenção de óleo microbiano compreendendo ácido gama-linolênico a partir de microorganismos cultivados em um meio compreendendo amido isento de açúcar e que contém ácido monocarboxílico (ácido acético) como fonte de carbono. A presente invenção difere desse documento por não utilizar ácidos carboxílicos como fonte de carbono e por adicionalmente compreender fontes alternativas de carbono. JP 05/070324 describes a skin affinity cosmetic formulation containing an oil extracted from yeast. JP 05030981 describes the production of fatty acids and oil preparation from baking yeasts. The present invention differs from such documents in that it is not about polyunsaturated fatty acids. EP 0 624 202 describes a method for obtaining microbial oil comprising gamma linolenic acid from microorganisms grown in a medium comprising sugar free starch and containing monocarboxylic acid (acetic acid) as a carbon source. The present invention differs from that document in that it does not use carboxylic acids as a carbon source and additionally comprises alternative carbon sources.
O documento EP 1 138 759 descreve a utilização de um microorganismo Labyrinthula, que pode ser utilizado para a produção de ácidos graxos poliinsaturados compreendendo meios de cultura contendo diversos óleos (óleo de soja e de palma, entre outros) e ácidos graxos. A presente invenção difere desse documento por não envolver fungos marinhos e não utilizar óleos e ácidos graxos como fonte de carbono, mas de um processo de obtenção de lipídeos compreendendo o uso de microorganismos, compreendendo adicionalmente fontes de carbono e nitrogénio em determinadas proporções, temperatura, agitação, entre outros.  EP 1 138 759 describes the use of a Labyrinthula microorganism which may be used for the production of polyunsaturated fatty acids comprising culture media containing various oils (soybean and palm oil, among others) and fatty acids. The present invention differs from that document in that it does not involve marine fungi and does not use oils and fatty acids as a carbon source, but a lipid forming process comprising the use of microorganisms, further comprising carbon and nitrogen sources in certain proportions, temperature, agitation, among others.
O documento EP 0 568 608 descreve uma composição compreendendo ácido araquidônico e o método de obtenção da mesma compreendendo o cultivo de Pythium insidiosum em um meio de cultivo contendo hexoses como fontes de carbono e nitrogénio. A presente invenção difere desse documento por compreender somente microorganismos oleaginosos, não compreendendo Pythium insidiosum e nem a produção de ácidos graxos poliinsaturados, e por restringir a faixa de proporção Carbono: Nitrogénio.  EP 0 568 608 describes a composition comprising arachidonic acid and the method for obtaining it comprising culturing Pythium insidiosum in a culture medium containing hexoses as carbon and nitrogen sources. The present invention differs from that document in that it comprises only oleaginous microorganisms, comprising neither Pythium insidiosum nor the production of polyunsaturated fatty acids, and by restricting the Carbon: Nitrogen ratio range.
O documento WO 93/12242 revela um método para a produção microbiana de óleo contendo ácido gama-linolênico. No referido documento, os microrganismos utilizados são da ordem Mucorales preferencialmente do género Mortierella, Actinomucor, Mucor, Rhizomucor ou Rhizopus. Referidos microrganismo são cultivados em meio de cultura substancialmente livre de amido e açúcares, contendo, como fonte de carbono, pelo menos um ácido moncarboxílico de 2 a 5 átomos de carbono, preferencialmente ácido acético, e a recuperando o óleo da biomoassa formada. WO 93/12242 discloses a method for microbial production of gamma linolenic acid containing oil. In said document, the microorganisms used are of the order Mucorales preferably of the genus Mortierella, Actinomucor, Mucor, Rhizomucor or Rhizopus. Said microorganism is grown in culture medium substantially free of starch and sugars, containing as carbon source at least one moncarboxylic acid of 2 to 5 carbon atoms, preferably acetic acid, and recovering the oil from the formed biome.
Recentemente, com o crescimento do interesse na produção de lipídeos visando à obtenção de biodiesel, alguns grupos alcançaram resultados interessantes utilizando algas como, por exemplo, o processo revelado no documento US 2007/0048848.  Recently, with the growing interest in the production of lipids to obtain biodiesel, some groups have achieved interesting results using algae such as the process disclosed in US 2007/0048848.
O pedido de patente PI 0406347-3 revela a produção de biomassa microbiana para extração de lipídios por cultura submersa, aerada ou não, agitada ou não, a partir dos derivados da cana-de-açúcar (caldo, melaço, açúcar refinado, açúcar mascavo, açúcar invertido ou ainda mistura dessas matérias-primas em diferentes proporções e/ou concentrações) para aplicações na produção de biodiesel ou para uso direto como combustível em motores do tipo Diesel e/ou na geração simples de energia e vapor, como um substitutivo aos óleos/lipídios vegetais.  Patent application PI 0406347-3 discloses the production of microbial biomass for lipid extraction by submerged aerated or un agitated or agitated culture from sugarcane derivatives (juice, molasses, refined sugar, brown sugar , invert sugar or even a mixture of these raw materials in different proportions and / or concentrations) for biodiesel production applications or for direct use as fuel in diesel engines and / or in simple power and steam generation as a substitute for vegetable oils / lipids.
O documento WO 06/033723 descreve a produção de ácido gama- linolênico utilizando leveduras oleaginosas recombinantes, como Yarrowia lipolytica, com a finalidade de expressar desaturases (delta-6 e delta-12). A presente invenção difere desse documento por não necessitar de leveduras oleaginosas recombinantes.  WO 06/033723 describes the production of gamma linolenic acid using recombinant oleaginous yeasts such as Yarrowia lipolytica for the purpose of expressing desaturases (delta-6 and delta-12). The present invention differs from that document in that it does not require recombinant oleaginous yeasts.
O documento US 2005/0266537 revela um processo para a obtenção de novos ácidos graxos para diversos usos, referido processo conduzido a partir do cultivo de leveduras recombinantes oleaginosas. A presente invenção difere desse documento por não necessitar de leveduras oleaginosas recombinantes.  US 2005/0266537 discloses a process for obtaining new fatty acids for various uses, said process conducted from the cultivation of recombinant oleaginous yeasts. The present invention differs from that document in that it does not require recombinant oleaginous yeasts.
O documento WO 08/042338 descreve a produção de carotenóides em leveduras e fungos oleaginosos recombinantes que permitem o acúmulo de lipídeos e a produção de carotenóides. A presente invenção difere desse documento por não compreender carotenóides. O documento WO 04/101757 revela um processo para a obtenção dos ácidos graxos omega-3 ou omega-6, a partir do cultivo de leveduras oleaginosas contendo genes codificantes da via biosintética de tais ácidos graxos, na presença de fontes de carbono fermentescíveis, seguida de recuperação dos referidos ácidos graxos. WO 08/042338 describes the production of carotenoids in recombinant yeast and oleaginous fungi that allow lipid accumulation and carotenoid production. The present invention differs from that document in that it does not comprise carotenoids. WO 04/101757 discloses a process for obtaining omega-3 or omega-6 fatty acids from the cultivation of oil yeasts containing genes encoding the biosynthetic pathway of such fatty acids in the presence of fermentable carbon sources, followed by recovery of said fatty acids.
O documento EP 1 219 704 revela um processo para a obtenção de um álcool prenílico (geranilgeraniol, farnesol e nerolidol) a partir do cultivo de uma ampla variedade de microrganismos, incluindo os do género Lypomyces. No processo descrito no referido documento, ao contrário da presente invenção, o meio de cultivo tem elevado teor de açúcar e contém surfactante, óleo, gordura ou terpenos, proporcionando desta forma o acúmulo de álcool prenílico nas células, que posteriormente o secretam para o meio.  EP 1 219 704 discloses a process for obtaining a prenyl alcohol (geranylgeraniol, farnesol and nerolidol) from the cultivation of a wide variety of microorganisms, including those of the genus Lypomyces. In the process described in said document, unlike the present invention, the culture medium is high in sugar and contains surfactant, oil, fat or terpenes, thereby providing the accumulation of prenyl alcohol in the cells, which subsequently secrete it into the medium. .
Do que se depreende da literatura pesquisada, não foram encontrados documentos antecipando e/ou sugerindo a presente invenção, que contorna diversas dificuldades da técnica anterior e/ou proporciona vantagens técnicas em relação às mesmas.  From the research literature, no documents were found anticipating and / or suggesting the present invention, which circumvent various prior art difficulties and / or provide technical advantages over them.
Sumário da Invenção Summary of the Invention
É um objeto da presente invenção proporcionar um processo de obtenção de lipídeos a partir do cultivo de microorganismos.  It is an object of the present invention to provide a process for obtaining lipids from the culture of microorganisms.
Em um aspecto, sendo, portanto, outro dos objetos da invenção, é revelado um processo de obtenção de lipídeos compreendendo as etapas de:  In one aspect, therefore, being another object of the invention, a process for obtaining lipids comprising the steps of:
a) cultivo dè microorganismos oleaginosos em um meio compreendendo:  a) cultivating oilseed microorganisms in a medium comprising:
a1 ) 20-100 g/L de fonte de carbono; e  a1) 20-100 g / l carbon source; and
a2) fonte de nitrogénio (q.s.),  a2) nitrogen source (q.s.),
de maneira que a relação C/N seja mantida entre 10 e 100 g/g. Em uma realização preferencial, o processo da presente invenção compreende o uso de biorreatores. such that the C / N ratio is maintained between 10 and 100 g / g. In a preferred embodiment, the process of the present invention comprises the use of bioreactors.
Em uma realização preferencial, o microorganismo oleaginoso utilizado é uma levedura do género Lypomyces.  In a preferred embodiment, the oleaginous microorganism used is a yeast of the genus Lypomyces.
Em uma realização preferencial, a fonte de carbono é obtida de material lignocelulósico ou outra fonte qualquer, como açúcar bruto e refinado.  In a preferred embodiment, the carbon source is obtained from lignocellulosic material or any other source, such as raw and refined sugar.
Em uma realização preferencial, o processo da presente invenção é conduzido em temperaturas entre 25°C e 40°C, preferencialmente 30°C.  In a preferred embodiment, the process of the present invention is conducted at temperatures between 25 ° C and 40 ° C, preferably 30 ° C.
Em uma realização preferencial, o processo da presente invenção é conduzido em pH de 3,0 a 7,0, preferencialmente 4,5.  In a preferred embodiment, the process of the present invention is conducted at a pH of 3.0 to 7.0, preferably 4.5.
Em uma realização preferencial, o processo da presente invenção é conduzido em agitação entre 100 e 900 rpm, preferencialmente 300 rpm.  In a preferred embodiment, the process of the present invention is conducted with agitation between 100 and 900 rpm, preferably 300 rpm.
Em uma realização preferencial, o processo da presente invenção é conduzido em aeração de 1 a 4 vvm, preferencialmente 2 vvm.  In a preferred embodiment, the process of the present invention is conducted at 1 to 4 vvm aeration, preferably 2 vvm.
Em uma realização preferencial, os lipídeos obtidos por esse processo compreendem principalmente triglicerídeos compostos de ácidos graxos de cadeia de 12 a 20 C.  In a preferred embodiment, the lipids obtained by this process mainly comprise triglycerides composed of 12 to 20 C chain fatty acids.
Em ainda outra realização, o processo da presente invenção adicionalmente compreende uma segunda fase de acúmulo de lipídeos, quando um meio preferencialmente composto somente de fontes de carbono é adicionado ao biorreator, a fim de manter a concentração de carboidratos constante.  In yet another embodiment, the process of the present invention further comprises a second phase of lipid accumulation when a medium preferably composed solely of carbon sources is added to the bioreactor in order to keep the carbohydrate concentration constant.
É um adicional objeto da presente invenção uma composição compreendendo os lipídeos obtidos por esse processo.  It is a further object of the present invention a composition comprising the lipids obtained by this process.
Esses e outros objetos da invenção serão valorizados e ficarão mais aparentes a partir da descrição detalhada a seguir.  These and other objects of the invention will be appreciated and will become more apparent from the following detailed description.
Breve Descrição das Figuras Brief Description of the Figures
A figura 1 mostra uma representação esquemática do processo de pré-tratamento e hidrólise de material lignocelulósico (biorrefinaria), onde: ML - material lignocelulósico; ** pré-tratamento; Cel - celulose; CoG - cogeração, calor e força extraídas; HL - hemicelulose, lignina; *** hidrólise; Hex - hexoses; CQPM - combustíveis, químicos, polímeros, materiais; Pt - pentoses. Figure 1 shows a schematic representation of the pretreatment and hydrolysis process of lignocellulosic material (biorefinery), where: ML - lignocellulosic material; ** pre-treatment; Cel - cellulose; CoG - cogeneration, heat and strength extracted; HL - hemicellulose, lignin; *** hydrolysis; Hex - hexoses; CQPM - fuels, chemicals, polymers, materials; Pt - pentoses.
A figura 2 mostra uma representação esquemática dos processos de batelada (A), batelada alimentada (B) e contínuo (C), sendo indicados os pontos de alimentação (*) e de saída de produto (P).  Figure 2 shows a schematic representation of the batch (A), fed batch (B) and continuous (C) processes, the feed (*) and product outlet (P) points being indicated.
A figura 3 mostra o acúmulo de lipídeos em diferentes relações C/N por Rhodotorula glutinis (em cinza) e Lipomyces starkey (batelada simples).  Figure 3 shows lipid accumulation at different C / N ratios by Rhodotorula glutinis (in gray) and Lipomyces starkey (single batch).
A figura 4 mostra a composição do biodiesel produzido (metil ésteres de ácidos graxos).  Figure 4 shows the composition of the biodiesel produced (methyl fatty acid esters).
Descrição Detalhada da Invenção Detailed Description of the Invention
Os exemplos a seguir não têm a intenção de limitar a invenção, mas somente de exemplificar uma das inúmeras maneiras de concretizá-la. Diversas variantes podem ser preparadas a partir dos ensinamentos da presente invenção.  The following examples are not intended to limit the invention, but merely to exemplify one of the numerous ways of carrying it out. Several variants may be prepared from the teachings of the present invention.
Fonte de carbono Carbon source
Na presente invenção, as "fontes de carbono" adequadas são escolhidas do grupo que compreende: glicose; xilose; sacarose e/ou a combinações dos mesmos. Em uma realização preferencial, essas fontes de carbono são originadas de materiais como: material lignocelulósico pré- tratado e/ou hidrolisado, melaço e suco de cana de açúcar, milho, palha e bagaço de diversas fontes. Em uma realização preferencial, o processo da presente invenção utiliza de 20-100 g/L de carboidrato proveniente de material lignocelulósico.  In the present invention, suitable "carbon sources" are chosen from the group comprising: glucose; xylose; sucrose and / or combinations thereof. In a preferred embodiment, these carbon sources are derived from materials such as: pretreated and / or hydrolyzed lignocellulosic material, molasses and juice from sugarcane, corn, straw and bagasse from various sources. In a preferred embodiment, the process of the present invention utilizes 20-100 g / l carbohydrate from lignocellulosic material.
Fonte de nitrogénio Nitrogen source
Na presente invenção, as fontes de nitrogénio adequadas são escolhidas do grupo que compreende: uréia, sais de amónia, extrato ou hidrolisados de levedura, peptona, caseinatos, resíduos da indústria agro- alimentar e resíduos de soja, puros e/ou combinações dos mesmos. Em uma realização preferencial, utiliza-se sais de amónia. In the present invention suitable nitrogen sources are chosen from the group comprising: urea, ammonium salts, yeast extract or hydrolysates, peptone, caseinates, agrochemical industry residues. food and soy residues, pure and / or combinations thereof. In a preferred embodiment, ammonium salts are used.
Microorganismos oleaginosos Oilseed microorganisms
Na presente invenção, os microorganismos oleaginosos compreendem fungos e/ou leveduras e/ou bactérias oleaginosas escolhidas do grupo que compreende os géneros Cândida sp, Rhodotorula sp, Cryptococcus sp, Lypomyces sp, Rhodococcus sp. e/ou combinações dos mesmos. Entende-se por combinações do mesmo o co-cultivo em pool e/ou o uso de organismos modificados geneticamente compreendendo genes dos géneros Cândida sp, Rhodotorula sp, Cryptococcus sp, Lypomyces sp., Rhodococcus sp. Em uma realização preferencial, esses genes podem ser selecionados da lista de genes citados nos Projetos Genoma de espécies de Cândida, como Cândida albicans e de Cryptococcus, como Cryptococcus neoformans, encontrados no site do Centro Nacional de Informação Biotecnológica dos EUA (NCBI - www.ncbi.nlm.nih.gov) Em uma realização preferencial, a presente invenção utiliza uma cepa de Lipomyces starkeyi.  In the present invention, the oleaginous microorganisms comprise fungi and / or yeast and / or oleaginous bacteria selected from the group comprising Candida sp, Rhodotorula sp, Cryptococcus sp, Lypomyces sp, Rhodococcus sp. and / or combinations thereof. Combinations thereof are understood to be pooled co-cultivation and / or the use of genetically modified organisms comprising genes of the genera Candida sp, Rhodotorula sp, Cryptococcus sp, Lypomyces sp., Rhodococcus sp. In a preferred embodiment, these genes may be selected from the list of genes cited in the Candida Species Genome Projects such as Candida albicans and Cryptococcus such as Cryptococcus neoformans found on the website of the US National Biotechnological Information Center (NCBI - www. ncbi.nlm.nih.gov) In a preferred embodiment, the present invention utilizes a strain of Lipomyces starkeyi.
Os microorganismos utilizados estão registrados nos bancos de material biológico DSMZ (Coleção Alemã de microorganismos e culturas de células), na ARS Culture Collection (Agricultural Research Service, conhecido também como coleção NRRL), INQS (Instituto Nacional de Controle de Qualidade em Saúde) e CCT (, respectivamente como Rhodotorula glutinis CCT 0783, Rhodotorula glutinis DSM 10134, Rhodotorula glutinis DSM 70398, Rhodotorula mucilaginosa INCQS 40157, Rhodosporidium toruloides DSM 4444, Cryptococcus albidus INCQS 40077, Cryptococcus albidus INCQS 40190, Cryptococcus, Cryptococcus laurentii DSM 70766, Cryptococcus curvatus DSM 70022, Lipomyces starkeyi NRRL 1 1557, Lipomyces starkeyi NRRL 27493, Lipomyces starkeyi DSM 70296, Cândida lipolytica DSM 8218, Cândida lipolytica DSM 1345, Lipomyces lipofer NRRL 1 555, Yarrowia lipolytica DSM 70561 . Meio de cultivo The microorganisms used are registered in the DSMZ (German Collection of Microorganisms and Cell Cultures Collection) Biological Material Banks, the ARS Culture Collection (Agricultural Research Service, also known as the NRRL Collection), INQS (National Institute for Quality Control in Health) and CCT (respectively as Rhodotorula glutinis CCT 0783, Rhodotorula glutinis DSM 10134, Rhodotorula glutinis DSM 70398, Rhodotorula mucilaginous INCQS 40157, Rhodosporidium toruloides DSM 4444, Cryptococcus albidus Crystococcus albidus Crystococcus albidus Cryptococcus albidus 70022, Lipomyces starkeyi NRRL 1 1557, Lipomyces starkeyi NRRL 27493, Lipomyces starkeyi DSM 70296, Candida lipolytica DSM 8218, Candida lipolytica DSM 1345, Lipomyces lipofer NRRL 1 555, Yarrowia lipolytica DSM 70561. Cultivation medium
Na presente invenção, o meio de cultivo adequado é escolhido do grupo que compreende as técnicas de batelada simples, batelada alimentada e/ou processo contínuo. O cultivo adicionalmente compreende temperatura específica, agitação, pH específico, aeração, fontes de carbono e fontes de nitrogénio. O meio de cultura contém, preferencialmente, alguns sais que proporcionam um maior rendimento ao processo, KH2P04 (de 0 a 7 g/L), Na2HP04 (de 0 a 7 g/L), MgS04 de (0 a 4 g/L), alem de traços (de 0 a 0, 1 g/L) de CaCI2, ZnS04, CuS04 e MnS04. In the present invention, suitable culture medium is chosen from the group comprising single batch, fed batch and / or continuous process techniques. The cultivation further comprises specific temperature, agitation, specific pH, aeration, carbon sources and nitrogen sources. Preferably, the culture medium contains some salts which give the process greater yield, KH 2 P0 4 (0 to 7 g / L), Na 2 HPO 4 (0 to 7 g / L), MgS0 4 of ( 0 to 4 g / l), plus traces (from 0 to 0.1 g / l) of CaCl 2 , ZnSO 4 , CuSO 4 and MnSO 4 .
Em uma realização preferencial, o processo da presente invenção é conduzido em temperaturas entre 25°C e 40°C, pH de 3,0 a 7,0, agitação entre 100 e 900 rpm, aeração de 1 a 4 vvm, a partir de um meio contendo de 20 a 100 g.L"1 de uma fonte de carbono e uma relação Carbono/Nitrogénio (m/m) de 10 a 100. Em uma realização preferencial, o processo da presente invenção é realizado a 30° C, pH de 4,5, agitação de 300 ppm e aeração de 2vvm. In a preferred embodiment, the process of the present invention is conducted at temperatures between 25 ° C and 40 ° C, pH 3.0 to 7.0, agitation between 100 and 900 rpm, aeration 1 to 4 vvm from a medium containing from 20 to 100 gL- 1 of a carbon source and a Carbon / Nitrogen (w / w) ratio of 10 to 100. In a preferred embodiment, the process of the present invention is performed at 30 ° C, pH of 4.5, 300 ppm agitation and 2vvm aeration.
Exemplo 1. Processo de obtenção de lipídeos Example 1. Process for obtaining lipids
A presente invenção proporciona um processo de obtenção de lipídeos em biorreatores utilizando microrganismos oleaginosos, preferencialmente leveduras pertencentes aos géneros Cândida sp., Rhodotorula sp., Cryptococcus sp., Lypomyces sp., e bactéria pertencente ao género Rhodococcus sp., preferencialmente uma cepa de Lipomyces starkey. Podem ser utilizados tanto meios sintéticos quanto meios industriais, contendo fontes de carbono e nitrogénio provenientes da indústria agro-alimentar (melaço, hidrolisado de material lignocelulósico, água de maceração de milho, soro de queijo, torta e folhas de soja, entre outros). Tanto o meio sintético quanto o meio industrial deve conter entre 20 e 100 g/L de carboidrato (m/v) e fonte de nitrogénio o suficiente para manter a relação C/N (m/m) entre 10 e 100. Como fonte de carboidratos pode-se utilizar também produtos da hidrólise de material lignocelulósico, utilizando- se o conceito de biorrefinaria (Figura 1 ). The present invention provides a process for obtaining lipids in bioreactors using oleaginous microorganisms, preferably yeasts belonging to the genera Candida sp., Rhodotorula sp., Cryptococcus sp., Lypomyces sp., And bacteria belonging to the genus Rhodococcus sp. Lipomyces starkey. Both synthetic and industrial media containing carbon and nitrogen sources from the agri-food industry can be used (molasses, lignocellulosic material hydrolyzate, maize maceration water, cheese whey, pie and soya leaves, among others). Both synthetic and industrial media should contain between 20 and 100 g / l carbohydrate (m / v) and sufficient nitrogen source to maintain the C / N ratio (m / m) between 10 and 100. As a source of carbohydrates one can Also use hydrolysis products from lignocellulosic material, using the concept of biorefinery (Figure 1).
Exemplo 1. 1 Processo preferencial de obtenção de lipídeos  Example 1. 1 Preferred Process for Obtaining Lipids
O processo pode ser realizado em uma ou duas etapas, para o acúmulo de lipídeos simultâneo ao crescimento celular deve-se manter preferencialmente 30 g/L de carboidrato e relação C/N de 50 (Figura 3). No caso de um processo em batelada alimentada ou contínuo, a primeira fase visa o desenvolvimento celular, cujo meio deve conter preferencialmente 70g/L de carboidrato e relação C/N de 15 e a segunda fase de acúmulo de lipídeos, quando um meio preferencialmente composto somente de carboidrato é adicionado ao biorreator, para manter a concentração de carboidratos constante, preferencialmente em 20 g/L (Figura 2).  The process can be performed in one or two steps, for the accumulation of lipids simultaneous to cell growth should preferably maintain 30 g / L carbohydrate and C / N ratio of 50 (Figure 3). In the case of a fed or continuous batch process, the first phase is for cell development, which medium should preferably contain 70g / L carbohydrate and C / N ratio of 15 and the second phase of lipid accumulation when a medium is preferably composed Carbohydrate-only is added to the bioreactor to keep the carbohydrate concentration constant, preferably at 20 g / L (Figure 2).
O meio de cultura deve conter, preferencialmente, alguns sais que proporcionam um maior rendimento ao processo, KH2P04 (de 0 a 7 g/L), Na2HP04 (de 0 a 7 g/L), MgS04 de (0 a 4 g/L), alem de traços (de 0 a 0,1 g/L) de CaCI2, ZnS04, CuS04 e MnS04. Preferably, the culture medium should contain some salts which give the process greater yield, KH 2 P0 4 (0 to 7 g / L), Na 2 HPO 4 (0 to 7 g / L), MgSO 4 of (0 to 4 g / L), plus traces (0 to 0.1 g / L) of CaCl 2 , ZnSO 4 , CuSO 4 and MnSO 4 .
Os processos são preferencialmente realizados em biorreatores, sendo o pH ajustado entre pH 3,0 e pH 7,0, e ao qual são adicionados de 5 a 20% (v/v) de inoculo. O inoculo é obtido preferencialmente por incubação da massa celular estocada a temperatura de 25°C a 30°C, em frascos agitados entre 150 e 200 rpm, por 12 a 24 horas em meio sintético contendo (por litro): 30 g de carboidrato, 1 g (NH4)2SO , 3 g KH2PO4, 1 g Na2HPO4, 1 ,5 g de MgSO4.7H2O, 2 g CaCI2.6H2O e 2 g extrato de levedura (pH entre 4,5 e 5,0). The processes are preferably performed in bioreactors, the pH being adjusted between pH 3.0 and pH 7.0, and to which 5 to 20% (v / v) inoculum is added. The inoculum is preferably obtained by incubating the stored cell mass at 25 ° C to 30 ° C in flasks shaken at 150 to 200 rpm for 12 to 24 hours in synthetic medium containing (per liter): 30 g carbohydrate, 1 g (NH 4 ) 2 SO, 3 g KH 2 PO 4 , 1 g Na 2 HPO 4 , 1.5 g MgSO 4 .7H 2 O, 2 g CaCl 2 .6H 2 O and 2 g yeast extract ( pH between 4.5 and 5.0).
Durante o processo, certos parâmetros preferenciais são assim mantidos: temperatura entre 25 e 40°C, preferencialmente 30°C; agitação entre 100 e 900 rpm, preferencialmente 300 rpm, aeração entre 0,5 vvm e 4 vvm, preferencialmente 2 vvm. O pH do processo é controlado e mantido entre pH 3,5 e 7,0, preferencialmente pH 4,5; com a adição de solução ácida ou básica, preferencialmente HCI 1 M e NaOH 1 M. O processo em batelada alimentada, ou contínuo, é alimentado com uma solução contendo de 100 a 700 g/L de carboidrato, preferencialmente 200 g/L de carboidrato, e conter ou não uma fonte de nitrogénio. Preferencialmente, a alimentação deve conter nitrogénio o suficiente para manter uma relação C/N de 50 a 200. A adição desta solução deve ser realizada de forma que a concentração de carboidrato no meio de cultura seja entre 10 e 100 g/L, preferencialmente 20 g/L, e a relação C/N de 60. During the process certain preferred parameters are thus maintained: temperature between 25 and 40 ° C, preferably 30 ° C; agitation between 100 and 900 rpm, preferably 300 rpm, aeration between 0.5 vvm and 4 vvm, preferably 2 vvm. The process pH is controlled and maintained between pH 3.5 and 7.0, preferably pH 4.5; with the addition of acidic or basic solution, preferably 1 M HCl and 1 M NaOH. The fed or continuous batch process is fed with a solution containing from 100 to 700 g / l carbohydrate, preferably 200 g / l carbohydrate, and whether or not it contains a nitrogen source. Preferably, the feed should contain enough nitrogen to maintain a C / N ratio of 50 to 200. The addition of this solution should be such that the carbohydrate concentration in the culture medium is between 10 and 100 g / l, preferably 20%. g / L, and the C / N ratio of 60.
O caldo fermentado é clarificado por qualquer método usual, preferencialmente por centrifugação e os lipídeos são extraídos também utilizando qualquer método usual. Além disso, há a possibilidade de se obter biodiesel sem a necessidade de extração prévia, por trans-esterificação direta.  The fermented broth is clarified by any usual method, preferably by centrifugation and the lipids extracted also using any usual method. In addition, there is the possibility of obtaining biodiesel without the need for prior extraction by direct transesterification.
Em uma concretização preferida, a produção de lipídeos é realizada pela levedura Lypomyces starkey em batelada alimentada, utilizando um meio inicial contendo 50 g/L de carboidrato e relação C/N de 15 (sendo 1 g/L de hidrolisado de levedura ou extrato de levedura e o restante de sulfato de amónia). Após 36 a 48 horas de reação a 30°C, 2vvm e 300 rpm, e pH 4,5 (controlado coma adição de NaOH 1 M e HCI 1 M), inicia-se a alimentação de uma solução contendo 300 g/L de carboidrato de modo a se manter aproximadamente 20 g/L de carboidrato no meio de cultura até se observar que não há incremento no teor de lipídeos e consumo de carboidrato.  In a preferred embodiment, lipid production is performed by the batch-fed Lypomyces starkey yeast, using an initial medium containing 50 g / l carbohydrate and C / N ratio of 15 (1 g / l yeast hydrolyzate or yeast extract). yeast and the remainder of ammonium sulphate). After 36 to 48 hours of reaction at 30 ° C, 2vvm and 300 rpm, and pH 4.5 (controlled with the addition of 1 M NaOH and 1 M HCl), a solution containing 300 g / L of carbohydrate to maintain approximately 20 g / l of carbohydrate in the culture medium until it is observed that there is no increase in lipid content and carbohydrate consumption.
Nesta concretização é possível obter até 100 g/L de massa celular seca contendo de 30 à 50% de lipídeos, compostos principalmente de triglicerídeos, como mostrado na Figura 4.  In this embodiment it is possible to obtain up to 100 g / l dry cell mass containing from 30 to 50% lipids, composed mainly of triglycerides, as shown in Figure 4.
Os versados na arte valorizarão imediatamente os importantes benefícios decorrentes do uso da presente invenção. Pequenas variações na forma de concretizá-la devem ser compreendidas como dentro do espírito da invenção e das reivindicações anexas.  Those skilled in the art will immediately appreciate the important benefits arising from the use of the present invention. Minor variations in the embodiment thereof should be understood to be within the spirit of the invention and the appended claims.

Claims

Reivindicações Claims
1. Processo de obtenção de lipídeos caracterizado por compreender as etapas de:  1. A lipid production process comprising the steps of:
a) cultivo de microorganismos oleaginosos em um meio compreendendo:  a) cultivation of oilseed microorganisms in a medium comprising:
a1 ) 20-100 g/L de fonte de carbono; e  a1) 20-100 g / l carbon source; and
a2) fonte de nitrogénio (q.s.),  a2) nitrogen source (q.s.),
de maneira que a relação C/N (m/m) seja mantida entre 10 e 100 . so that the C / N ratio (w / w) is maintained between 10 and 100.
2. Processo, de acordo com a reivindicação 1 , caracterizado pelo microorganismo oleaginoso utilizado ser escolhido do grupo que compreende: Cândida sp, Rhodotorula sp., Cryptococcus sp., Lypomyces sp., Rhodococcus sp. ou combinações dos mesmos. Process according to Claim 1, characterized in that the oleaginous microorganism used is chosen from the group comprising: Candida sp, Rhodotorula sp., Cryptococcus sp., Lypomyces sp., Rhodococcus sp. or combinations thereof.
3. Processo, de acordo com a reivindicação 2, caracterizado pelo microorganismo oleaginoso ser uma levedura escolhida do género Lypomyces.  Process according to Claim 2, characterized in that the oleaginous microorganism is a chosen yeast of the genus Lypomyces.
4. Processo, de acordo com a reivindicação 1 , caracterizado pela fonte de carbono ser obtida do grupo que compreende açúcar bruto, melaços, refinado e/ou material lignocelulósico.  Process according to Claim 1, characterized in that the carbon source is obtained from the group comprising raw sugar, molasses, refined sugar and / or lignocellulosic material.
5. Processo, de acordo com a reivindicação 1 , caracterizado por compreender o uso de biorreatores.  Process according to Claim 1, characterized in that it comprises the use of bioreactors.
6. Processo, de acordo com a reivindicação 1 , caracterizado por ser conduzido em temperaturas entre 25°C e 40°C.  Process according to Claim 1, characterized in that it is conducted at temperatures between 25 ° C and 40 ° C.
7. Processo, de acordo com a reivindicação 6, caracterizado por ser preferencialmente conduzido a 30°C.  Process according to Claim 6, characterized in that it is preferably conducted at 30 ° C.
8. Processo, de acordo com a reivindicação 1 , caracterizado por ser conduzido em pH de 3,0 a 7,0.  Process according to Claim 1, characterized in that it is conducted at a pH of 3.0 to 7.0.
9. Processo, de acordo com a reivindicação 8, caracterizado por ser preferencialmente conduzido em pH de 4,5.  Process according to Claim 8, characterized in that it is preferably conducted at a pH of 4.5.
10. Processo, de acordo com a reivindicação 1 , caracterizado por ser conduzido sob agitação entre 100 e 900 rpm. Process according to Claim 1, characterized in that it is conducted under agitation between 100 and 900 rpm.
1 1. Processo, de acordo com a reivindicação 10, caracterizado por ser preferencialmente conduzido sob agitação de 300 rpm. Process according to Claim 10, characterized in that it is preferably conducted under agitation of 300 rpm.
12. Processo, de acordo com a reivindicação 1 , caracterizado por ser conduzido em aeração de 1 a 4 vvm.  Process according to Claim 1, characterized in that it is conducted at 1 to 4 vvm aeration.
13. Processo, de acordo com a reivindicação 12, caracterizado por ser preferencialmente conduzido em aeração de 2 vvm.  Process according to Claim 12, characterized in that it is preferably conducted in 2 vvm aeration.
14. Processo, de acordo com a reivindicação 1 , caracterizado pelo fato de que os lipídeos obtidos por esse processo compreendem principalmente triglicerídeos compostos de ácidos graxos de cadeia de 12 a 20 C.  Process according to Claim 1, characterized in that the lipids obtained by this process mainly comprise triglycerides composed of 12 to 20 C chain fatty acids.
15. Processo, de acordo com a reivindicação 1 , caracterizado por ser um processo em batelada simples, batelada alimentada ou contínuo.  Process according to Claim 1, characterized in that it is a single batch, fed batch or continuous batch process.
16. Processo, de acordo com a reivindicação 15, caracterizado pela alimentação de um meio contendo fontes de carbono ou qualquer outro meio que mantenha a concentração de carboidratos ser constante.  Process according to Claim 15, characterized in that a medium containing carbon sources or any other medium which maintains the carbohydrate concentration is constant.
17. Composição contendo lipídeos caracterizada por ser obtida por um processo compreendendo as etapas de:  17. Lipid-containing composition characterized in that it is obtained by a process comprising the steps of:
a) cultivo de microorganismos oleaginosos em um meio compreendendo:  a) cultivation of oilseed microorganisms in a medium comprising:
a1 ) 20-100 g/L de fonte de carbono; e  a1) 20-100 g / l carbon source; and
a2) fonte de nitrogénio (q.s.),  a2) nitrogen source (q.s.),
de maneira que a relação C/N (m/m) seja mantida entre 10 e 100. so that the C / N ratio (m / m) is maintained between 10 and 100.
18. Composição, de acordo com a reivindicação 17, caracterizada por compreender compostos de ácidos graxos de cadeia de 12 a 20 carbonos em sua cadeia. Composition according to Claim 17, characterized in that it comprises 12 to 20 carbon chain fatty acid compounds in its chain.
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021198895A1 (en) * 2020-03-31 2021-10-07 Pirelli Tyre S.P.A. Manufacture of elastomeric compounds comprising oils with a plasticising action obtained from oleaginous microbial cells

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008134836A2 (en) * 2007-05-02 2008-11-13 Ouro Fino Participações E Empreendimentos S.A. Process to produce biodiesel and/or fuel oil

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008134836A2 (en) * 2007-05-02 2008-11-13 Ouro Fino Participações E Empreendimentos S.A. Process to produce biodiesel and/or fuel oil

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
EVANS, C.T. ET AL.: "A comparison of the oleaginous yeast, Candida curvata, grown on different carbon sources in continuous and batch culture.", LIPIDS, vol. 18, no. 9, 1983, pages 623 - 629 *
RATLEDGE C. ET AL.: "The biochemistry and molecular biology of lipids accumulation in oleaginous microorganisms.", ADVANCES IN APPLIED MICROBIOLOGY, vol. 51, 2002, pages 1 - 44 *
ZHAO, X. ET AL.: "Medium optimization for lipid production through co-fermentation of glucose and xylose by oleaginous yeast Lipomyces starkeyi.", EUROPEAN JOURNAL OF LIPID SCIENCE AND TECHNOLOGY, vol. 110, no. 5, May 2008 (2008-05-01), pages 405 - 412 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021198895A1 (en) * 2020-03-31 2021-10-07 Pirelli Tyre S.P.A. Manufacture of elastomeric compounds comprising oils with a plasticising action obtained from oleaginous microbial cells

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