WO2010046051A2

WO2010046051A2 - Process for the production of lipids from biomass

Info

Publication number: WO2010046051A2
Application number: PCT/EP2009/007388
Authority: WO
Inventors: Daniele Bianchi; Giuliana Franzoci; Anna Maria Romano
Original assignee: Eni S.P.A.
Priority date: 2008-10-21
Filing date: 2009-10-14
Publication date: 2010-04-29
Also published as: EA019318B1; EA201100551A1; ITMI20081863A1; BRPI0919588A2; EP2350300A2; EP2350300B1; PL2350300T3; UA103205C2; IT1392910B1; ES2618578T3; MX2011004250A; WO2010046051A8; EA019318B9; WO2010046051A3

Abstract

A process for the production of lipids from biomass including at least one polysaccharide comprising: subjecting said biomass to acid hydrolysis in the presence of an aqueous solution of at least one organic acid selected from alkyl- or aryl-sulfonic acids having from C₇ to C₂₀ carbon atoms, preferably from C₉ to C₁₅ carbon atoms, or from halogenated carboxylic acids, at a temperature ranging from 80°C to 160°C, preferably from 100°C to 150°C, obtaining a first mixture comprising a first solid phase and a first aqueous phase; - subjecting said first mixture to enzymatic hydrolysis obtaining a second mixture comprising a second solid phase and a second aqueous phase; subjecting said second aqueous phase to fermentation in the presence of at least one oleaginous yeast obtaining an oleaginous cellular biomass comprising lipids. The lipids thus obtained can be advantageously used in the production of biodiesel or green diesel which can be used as such, or in a mixture with other fuels for motor vehicles.

Description

PROCESS FOR THE PRODUCTION OF LIPIDS FROM BIOMASS

The present invention relates to a process for the production of lipids from biomass, including at least one polysaccharide . More specifically, the present invention relates to a process for the production of lipids from biomass including at least one polysaccharide which comprises subjecting said biomass to acid hydrolysis, subjecting the mixture obtained from said acid hydrolysis to enzymatic hy- drolysis, and subjecting the sugars obtained from said hydrolysis to fermentation in the presence of at least one oleaginous yeast .

The lipids thus obtained can be advantageously used in the production of biodiesel or green diesel which can be used as such, or in a mixture with other fuels for motor vehicles .

Generally speaking, a biomass is any substance with an organic, vegetable or animal matrix, which can be destined for energy purposes, for example as raw material for the production of biofuels, or of components which can be added to fuels. Biomass can therefore form a renewable energy source as an alternative to traditional raw materials of a fossil origin normally used in the production of fuels. For this purpose, lignocellulosic biomass is particularly useful. The production of sugars from biomass, in particular lignocellulosic biomass, is known in the art.

Lignocellulosic biomass is a complex structure comprising three main components: cellulose, hemicellulose and lignin. Their relative amounts vary according to the type of lignocellulosic biomass used. In the case of plants, for example, said amounts vary according to the species and age of the plant.

Cellulose is the greatest constituent of lignocellu- losic biomass and is generally present in an amount ranging from 30% by weight to 60% by weight with respect to the total weight of the lignocellulosic biomass. Cellulose consists of glucose molecules (from about 500 to 10,000 units) bound to each other through a β-1,4 gluco- side bond. The establishment of hydrogen bonds between the chains causes the formation of crystalline domains which give resistance and elasticity to vegetal fibres. In nature, it can only be found in its pure state in annual plants such as cotton and flax, whereas in ligneous plants it is always accompanied by hemicellulose and lignin.

Hemicellulose which is generally present in an amount ranging from 10% by weight to 40% by weight with respect to the total weight of the lignocellulosic bio- mass, appears as a mixed polymer, relatively short (from 10 to 200 molecules) and branched, made up of both sugars with six carbon atoms (glucose, mannose, galactose) and also sugars with five carbon atoms (xylose, arabinose) . Some important properties of vegetal fibres are due to the presence of hemicellulose, of which the main property- is that of favouring the imbibition of said vegetal fibres, when water is present, causing their swelling. Hemicellulose also has adhesive properties and therefore tends to cement or develop a horny consistency, with the consequence that said vegetal fibres become rigid and are imbibed more slowly.

Lignin is generally present in an amount ranging from 10% by weight to 30% by weight with respect to the total weight of the lignocellulosic biomass. Its main function consists in binding and cementing the various vegetal fibres with each other giving the plant compactness and resistance and also provides protection against insects, pathogen agents, lesions and ultraviolet light. It is mainly used as fuel but is also currently widely used in industry as a disperser, hardener, emulsifying agent, for plastic laminates, cartons and manufactured rubber articles . It can also be chemically treated to produce aromatic compounds, of the vanillin, syringalde- hyde, p-hydroxybenzaldehyde type, which can be used in pharmaceutical chemistry, or in the cosmetic and food in- dustry.

In order to optimize the transformation of lignocel- lulosic biomass into products for energy use, subjecting said biomass to a preliminary treatment to separate the lignin and hydrolyze the cellulose and hemicellulose to simple sugars, such as, for example, glucose and xylose, is known. These sugars can be used as carbon sources in fermentation processes in the presence of microorganisms for the production of alcohols and/or lipids. Patent application US 2008/0102176, for example, describes a method for the extraction of vegetable fats comprising: pulverizing the raw material containing cellulose in order to obtain particles with a diameter of 1- 2 mm,- immerging the particles in sulfuric acid at a con- centration equal to 1-2% to acidify said particles in order to increase the hydrolysis of the cellulose and to adjust the pH to a value of 4.5+0.5; removing the particles acidified by the sulfuric acid and adding, in sequence, cellulase and an oleaginous yeast to the acidi- fied particles and subjecting to fermentation for 8-9 days at a temperature of 25-30⁰C and a humidity of 85- 90%; adding an aliphatic hydrocarbon, as solvent, to the fermentation products in order to extract the fats, obtaining an extraction blend; removing the acidified par- tides remaining in the extraction blend and separating the fats from the solvent by distillation, obtaining raw oil. The cellulase is preferably Trichoderma viride and the oleaginous yeast is Rhodotorula glutinis. The fats obtained can be converted to biodiesel after esterifica- tion.

Dai et al . describe the production of biodiesel from oleaginous yeasts in the article: ""Biodiesel generation from oleaginous yeast J?hodotorula glutinis with xylose assimilating capacity" , published in "African Journal of Biotechnology" (2007) , Vol. 6 (18), pages 2130-2134. In said article, the lignocellulosic biomass is ground and subjected to acid hydrolysis in the presence of sulfuric acid. The sugars thus obtained are used as carbon sources in a fermentation process in the presence of a previously selected strain of Rhodotorula glutinis, capable of also using pentoses, xylose in particular, with the purpose of obtaining oils which are subsequently extracted by Sox- hlet extraction and subjected to transesterification in order to obtain biodiesel . The above processes however can have various drawbacks .

In order to hydrolyze both polysaccharide components of the biomass (e.g., hemicellulose and cellulose), for example, in particular in the presence of diluted sulfu- ric acid (e.g., at concentrations ranging from 0-5% to 11%) it is necessary to operate at high temperatures (e.g., over 160⁰C) . At said temperatures, there is the formation of by-products deriving from the dehydration of the sugars and from the partial depolymerization of the lignin. These by-products, in particular furfural, hy- droxyl methyl furfural, phenolic compounds, can act as growth inhibitors of the microorganisms normally used in the subsequent sugar fermentation processes. Furthermore, in the presence of diluted sulfuric acid, the sugar yield, glucose in particular, is normally low (e.g., lower than 50%) .

Furthermore, before subjecting the sugars to fermentation, it may be necessary to neutralize the sulfuric acid in order to adjust the pH to values acceptable for the subsequent fermentation. Neutralization is generally obtained by adding oxides and/or hydroxides such as, for example, calcium oxide, calcium hydroxide, or barium hydroxide, with the consequent formation of salts (e.g., calcium sulphate, calcium sulfate dihydrate ("gypsum")/ barium sulfate) which precipitate and must therefore be separated from the sugars before the latter are subjected to fermentation. In addition, these salts must be subsequently disposed of by means of suitable treatment with a consequent increase in the production cost. Furthermore, it is impossible to recover and reuse said inorganic acid.

The Applicant has now found that the production of lipids from biomass, in particular from biomass including at least one polysaccharide, can be advantageously ef- fected by means of a process which comprises subjecting said biomass to acid hydrolysis in the presence of an aqueous solution of at least one organic acid, at a temperature lower than or equal to 16O⁰C, subjecting the mixture obtained from said acid hydrolysis to enzymatic hydrolysis, and subjecting the sugars obtained from these hydrolyses to fermentation in the presence of at least one oleaginous yeast .

Numerous advantages are obtained by means of this process. Said process allows, for example, a high yield of pentose and hexose sugars to be obtained, deriving from the acid hydrolysis of said biomass, which can be subsequently used as carbon sources in fermentation processes for the production of lipids. Said lipids can be advantageously used in the production of biodiesel or green diesel which can be used as such, or mixed with other fuels for motor vehicles .

Furthermore, the possibility of operating at temperature which are not high, i.e. lower than or equal to 16O⁰C, allows the formation of by-products to be reduced, such as, for example, furfural, hydroxy1 methyl furfural, phenolic compounds, which can act as growth inhibitor of the microorganisms normally used in the subsequent fermentation processes of sugars.

Another relevant advantage, lies in the fact that said process allows the organic acid to be recovered, which can then be recycled to the above process, in particular, to the acid hydrolysis of the biomass. Said recovery also allows the neutralization step to be avoided and, therefore, the production of salts and their subse- quent disposal.

A further relevant advantage, is that the wet solid phase comprising cellular debris, in particular proteins and polysaccharides contained in the cell membrane of the oleaginous yeasts used, which is obtained at the end of the fermentation, can be recovered and used in the above- mentioned process, in particular it can be used in the acid hydrolysis. The acid hydrolysis of this wet solid phase allows to obtain amino acids from proteins and sugars from polysaccharides, which are subsequently sent to the fermentation step, allowing, in this way, to reduce the amount of nitrogen which must normally be added to the culture medium in which the fermentation takes place and to provide additional sugars for the fermentation.

An object of the present invention therefore relates to a process for the production of lipids from biomass including at least one polysaccharide comprising: - subjecting said biomass to acid hydrolysis in the presence of an aqueous solution of at least one organic acid selected from alkyl- or aryl-sulfonic acids hav- ing from C₇ to C₂o carbon atoms, preferably from C₉ to C₁₅ carbon atoms, or from halogenated carboxylic acids, at a temperature ranging from 80⁰C to 160⁰C, preferably from 100⁰C to 150⁰C, obtaining a first mixture comprising a first solid phase and a first aque- ous phase; subjecting said first mixture to enzymatic hydrolysis obtaining a second mixture comprising a second solid phase and a second aqueous phase; subjecting said second aqueous phase to fermentation in the presence of at least one oleaginous yeast obtaining an oleaginous cellular biomass comprising lipids.

For the purposes of the present description and of the following claims, the definition of the numerical ranges always include extremes, unless otherwise specified.

In accordance with a preferred embodiment of the present invention, said polysaccharide can be selected from cellulose, hemicellulose or mixtures thereof. Cellulose, or mixtures of hemicellulose and cellulose, are particu- larly preferred.

In accordance with a further preferred embodiment of the present invention, said biomass is a lignocellulosic biomass. As mentioned above, the lignocellulosic biomass includes three components: hemicellulose, cellulose and lignin.

Said lignocellulosic biomass is preferably selected from:

- products of cultures expressly cultivated for energy use (for example, miscanthus, millet, common cane), including by-products, residues and wastes of said cultures or of their processing;

- products of agricultural cultivations, forestation and silviculture, comprising wood, plants, residues and by-products of agricultural processing, forestation and silviculture;

- agri-foodstuffs by-products intended for human feeding or zootechnics;

- residues, not chemically treated, of the paper in- dustry;

- waste products coming from the differentiated collection of solid urban waste (e.g., urban waste of a vegetable origin, paper) .

In accordance with a preferred embodiment of the present invention, said biomass can be subjected to a preliminary grinding process before being subjected to acid hydrolysis. Said biomass is preferably ground until particles having a diameter ranging from 0.1 mm to 10 mm, more preferably ranging from 0.5 mm to 4 mm, are ob- tained. Particles having a diameter of less than 1 mm are particularly preferred.

In accordance with a preferred embodiment of the present invention, said biomass is present in the reaction mixture in an amount ranging from 5% by weight to 40% by weight, preferably from 20% by weight to 35% by weight, with respect to the total weight of the reaction mixture .

For the purposes of the present description and of the following claims, the term "reaction mixture" means the mixture comprising the biomass and the aqueous solution of said at least one organic acid.

In accordance with a preferred embodiment of the present invention, said at least one organic acid is soluble in water and extractable with an organic solvent insolu- ble in water.

For the purposes of the present invention and of the following claims, the term "organic acid soluble in water" means an organic acid having a solubility in distilled water, at 25°C, of at least 0.5 g/100 ml of dis- tilled water, preferably of at least 2 g/100 ml of distilled water.

For the purposes of the present invention and of the following claims, the term "organic acid extractable with an organic solvent insoluble in water" means an organic acid which can be extracted with an organic solvent insoluble in water with a yield of at least 80%, preferably of at least 90%, said yield being calculated with respect to the total amount of organic acid present in the aque- ous solution.

For the purposes of the present invention and of the following claims, the term "organic solvent insoluble in water" means an organic solvent which has a solubility in distilled water, at 25⁰C, lower than 4% by volume, pref- erably lower than 2% by volume.

In accordance with a preferred embodiment of the present invention, said alkyl- or aryl sulfonic acids can be selected from: dodecyl-sulfonic acid, para-toluene- sulfonic acid, 1-naphthalene-sulfonic acid, 2- naphthalene-sulfonic acid, 1, 5-naphthalene-disulfonic acid, or mixtures thereof. Para-toluene-sulfonic acid, 2- naphthalene- sulfonic acid, 1, 5-naphthalene-disulfonic acid, or mixtures thereof, are particularly preferred.

In accordance with a preferred embodiment of the present invention, said halogenated carboxylic acids can be selected from those having a number of carbon atoms not higher than 20, preferably ranging from 2 to 15, such as, for example, trifluoroacetic acid, dichloroacetic acid, trichloroacetic acid, perfluoro-octanoic acid, or mixtures thereof .

In accordance with a preferred embodiment of the present invention, said at least one organic acid is present in the aqueous solution at a concentration ranging from 0.1% by weight to 5% by weight, preferably from 0.5% by weight to 2.5% by weight, with respect to the total weight of the aqueous solution.

Said organic acid acts as catalyst for the acid hydrolysis of said biomass. In particular, when the starting biomass is a lignocellulosic biomass, said organic acid specifically acts as catalyst for the acid hydrolysis of the hemicellulose . It should be noted that the process object of the present invention, when the starting biomass is a lignocellulosic biomass, not only allows the acid hydrolysis of the hemicellulose to be obtained, but also improves the disposition of the cellulose, said cellulose remaining substantially non-hydrolyzed, for the subsequent enzymatic hydrolysis, thanks to an improved destructuring of the starting biomass.

In accordance with a preferred embodiment of the present invention, said acid hydrolysis can be carried out for a time ranging from 20 minutes to 6 hours, preferably from 30 minutes to 3 hours.

Said acid hydrolysis can be carried out in reactors known in the art, such as, for example, autoclaves, or extruders .

In accordance with a preferred embodiment of the present invention, said first solid phase comprises lig- nin and cellulose and said first aqueous phase comprises at least one sugar having from C₅ to C₆ carbon atoms and said at least one organic acid. Said sugar is preferably xylose. Said xylose preferably derives from the acid hydrolysis of the hemicellulose .

Said acid hydrolysis allows at least one sugar having from C₅ to C₆ carbon atoms to be obtained, in particular xylose deriving from the acid hydrolysis of hemicellulose, with a high yield. More specifically, said acid hydrolysis allows a yield of xylose higher than or equal to 80% to be obtained, said yield being calculated with respect to the total amount of xylose present in the starting biomass .

Said acid hydrolysis also allows high yields of cellulose and lignin to be obtained.

In order to recover said at least one organic acid, said first mixture can be subjected to extraction with an organic solvent soluble in water. In accordance with a preferred embodiment of the present invention, said process also comprises subjecting said first mixture to extraction with at least one organic solvent insoluble in water. Preferably, said or- ganic solvent insoluble in water can be selected from: halogenated hydrocarbons such as, for example, methylene chloride, monochlorobenzene, dichlorobenzene, or mixtures thereof; aromatic hydrocarbons such as, for example, toluene, xylene, or mixtures thereof; aliphatic alcohols having from C₄ to C₆ carbon atoms such as, for example, n-butanol, n-pentanol, or mixtures thereof; or mixtures thereof . Mixtures of toluene and n-butanol are particularly preferred.

Said organic solvent insoluble in water is subse- quently evaporated obtaining a further solid phase comprising said at least one organic acid.

As specified above, the process object of the present invention allows said at least one organic acid to be recovered with a high yield, in particular, with a yield of at least 80%, preferably of at least 90%, said yield being calculated with respect to the total amount of organic acid present in said aqueous solution. Said at least one organic acid can therefore be subsequently reused according to the process object of the present in- vention. It should be noted that, when the recovery of said at least one organic acid is equal to 100%, said first aqueous phase will be free of said at least one organic acid.

In accordance with a preferred embodiment of the present invention, said process also comprises re-using said at least one organic acid in said acid hydrolysis.

For the purposes of the process object of the present invention, said enzymatic hydrolysis can be carried out according to techniques known in the art as de- scribed, for example, in United States Patents US 5,628,830, US 5,916,780 e US 6,090,595, using commercial enzymes such as, for example, Celluclast 1.5L (No- vozyraes) , Econase CE (Rohm Enzymes) , Spezyme (Genecor) , Novozym 188 (Novozymes) , used individually or mixed with each other.

In order to adjust the pH to values ranging from 4 to 6, preferably from 4.5 to 5.5, glacial acetic acid can be added to said first mixture, before the enzymatic hydrolysis, in an amount such as to obtain the desired pH. In accordance with a preferred embodiment of the present invention, said second solid phase comprises lig- nin and said second aqueous phase comprises at least one sugar having from C₅ to C₆ carbon atoms, preferably xylose, glucose, cellobiose, or mixtures thereof. In par- ticular, said second aqueous phase comprises xylose de- riving from the acid hydrolysis of hemicellulose, glucose and cellobiose deriving from the enzymatic hydrolysis of cellulose .

In particular, this enzymatic hydrolysis allows a high glucose yield to be obtained, more specifically a glucose yield higher than or equal to 90%, said yield being calculated with respect to the total amount of glucose present in the staring biomass .

The amounts of sugars contained in the starting bio- mass as also the amounts of sugars obtained after hydrolysis (acid and/or enzymatic hydrolysis) , can be determined by means of known techniques in the art such as, for example, High Performance Liquid Chromatography - HPLC. Said second solid phase and said second aqueous phase can be separated by techniques known in the art such as, for example, filtration, centrifugation. Said phases are preferably separated by filtration.

Said second solid phase, comprising lignin, can be upgraded as fuel, for example as a fuel for producing the energy necessary for sustaining the treatment processes of the biomass.

In accordance with a preferred embodiment of the present invention, said fermentation can be carried out at a temperature ranging from 20⁰C to 40⁰C, preferably from 25°C to 35°C.

In accordance with a preferred embodiment of the present invention, said fermentation can be carried out for a time ranging from 3 days to 10 days, preferably from 4 days to 8 days .

In accordance with a preferred embodiment of the present invention, said fermentation can be carried out at a pH ranging from 4.5 to 6.5, preferably from 5 to 6. In order to maintain the pH within the desired ranges, an aqueous solution of at least one inorganic base such as, for example, sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide, or mixtures thereof, can be added to the culture medium used for the fermentation, in an amount which is such as to obtain the desired pH.

In accordance with a preferred embodiment of the present invention, said oleaginous yeast can be selected from: Rhodotorula glutinis, Rhσdotorula gracilis, Rhodotorula graminis, Lypomices starkeyi, Lypomices Ii- pofer, Trigonopsis variabilis, Candida kefyr, Candida curvata, Candida lipolytica, Pichia stipitis, Torulopsis sp..

In accordance with a preferred embodiment of the present invention, said fermentation is a feed-batch fer- mentation. Before being used in said fermentation, said oleaginous yeast is preferably grown in a culture medium containing xylose, cellobiose, glucose, or mixtures thereof, at a concentration preferably ranging from 1% by weight to 2% by weight with respect to the total weight of said culture medium.

Said fermentation can be advantageously carried out in fermentors known in the art, in the presence of culture mediums normally used for the purpose comprising nu- trients such as, for example, nitrogen, potassium phosphate, magnesium, salts, vitamins.

In accordance with a preferred embodiment of the present invention, said process also comprises subjecting said oleaginous cellular biomass to mechanical treatment. Said mechanical treatment can be effected using equipment and techniques which are known in the art .

Said mechanical treatment can be advantageously effected, for example, using high-pressure homogenizers, preferably at a pressure ranging from 300 bar to 500 bar (e.g., Polytron homogenizer) , or using a French press cell.

A mechanical treatment is preferably effected using a French press cell, operating at a pressure ranging from 20 Kpsi to 50 Kpsi, preferably from 35 Kpsi to 45 Kpsi. In order to concentrate the oleaginous cellular bio- mass, before being subjected to mechanical treatment, said biomass can be subjected to centrifugation. Said centrifugation can be effected for a time ranging from 5 minutes to 30 minutes, preferably from 15 minutes to 25 minutes, at a rotation rate ranging from 3,000 rpm to 9,000 rpm, preferably from 4,000 to 8,000 rpm.

In order to deactivate the lipolytic enzymes (e.g., lipase), said oleaginous cellular biomass, before being subjected to mechanical treatment, can be subjected to thermal treatment. Said thermal treatment can be carried out at a temperature ranging from 70⁰C to 90⁰C, preferably from 75°C to 85⁰C, for a time ranging from 30 minutes to 3 hours, preferably from 1 to 2 hours.

In accordance with a preferred embodiment of the present invention, said process also comprises subjecting said oleaginous cellular biomass to centrifugation, after mechanical treatment .

In accordance with a preferred embodiment of the present invention, said centrifugation can be carried out for a time ranging from 5 minutes to 30 minutes, preferably from 10 minutes to 25 minutes, at a rotation rate ranging from 3,000 rpm to 9,000 rpm, preferably from 4,000 rpm to 8,000 rpm.

At the end of the centrifugation, the following phases are obtained: (i) an oily phase comprising lipids; (ii) an aqueous phase comprising traces of non- separated lipids and non- fermented sugars; (iii) a wet solid phase comprising cellular debris and traces of non-separated lipids.

The process object of the present invention allows the lipids to be recovered with a yield ranging from 50% to 90%, preferably from 55% to 80%, said yield being calculated with respect to the total amount of lipids pre- sent in the oleaginous cellular biomass obtained after fermentation.

The lipids included in said oily phase (i) are preferably triglycerides, more preferably esters of glycerol with fatty acids having from Ci₄ to C₂o carbon atoms such as, for example, palmitic acid, stearic acid, oleic acid, α-linoleic acid, in an amount higher than or equal to 80% by weight, preferably higher than or equal to 90% by weight, with respect to the total weight of the lipids. Other lipids which can be present in said oily phase are: phospholipids, monoglycerides, diglycerides, or mixtures thereof .

The amount of lipids contained in the oleaginous cellular biomass obtained after fermentation, as also the amount of lipids contained in said oily phase, can be de- termined by means of techniques known in the art such as, for example, the colorimetric method which is based on the reaction of lipids with phosphoric acid and phospho- vanillin: further details relating to this method can be found, for example, in the following article: "Chemical Basis of the Sulpho-phospho-vanillin Reaction for Estimating Total Serum Lipids", J. A. Knight et al . , published in "Clinical Chemistry" (1972), Vol. 18, No. 3, pages 199-202.

As already specified above, the wet solid phase (iii) comprising cellular debris, in particular proteins and polysaccharides contained in the cell membrane of the oleaginous yeast used, can be recovered and used in the process object of the present invention, in particular it can be sent to acid hydrolysis. The proteins and polysac- charides of the membrane are then hydrolyzed to amino acids and simple sugars (for example, glucose, mannose) which can then be used as nitrogen and carbon source in the subsequent fermentation. It should be outlined the fact that the recovery and use of said wet solid phase (iii) allows nitrogen to be supplied to the culture medium used for the fermentation, consequently reducing the amount of nitrogen normally added to the culture medium in which the fermentation takes place and providing additional sugars for the fermentation. In accordance with a preferred embodiment of the present invention, said process also comprises sending said wet solid phase (iii) to acid hydrolysis. In this case, said first aqueous phase, in addition to said at least one sugar having from C₅ to C₆ carbon atoms and said at least one organic acid, wil comprise amino acids and simple sugars (for example, glucose and mannose) deriving from the acid hydrolysis of the proteins and of the polysaccharides of the membrane; whereas said first solid phase will comprise, in addition to cellulose and lignin, non-hydrolyzed cellular debris.

In order to obtain a higher yield of lipids, i.e. a yield higher than 90%, said aqueous phase (ii) and/or said wet solid phase (iii) can be subjected to extraction with a solvent, or with an alcohol/solvent mixture. In accordance with a preferred embodiment of the present invention, said process also comprises subjecting said aqueous phase (ii) and/or said wet solid phase (iii) to extraction with at least one solvent which can be selected from aliphatic hydrocarbons having from C₃ to Cio carbon atoms such as, for example, pentane, n-hexane, octane, or mixtures thereof; or with a mixture comprising at least one aliphatic alcohol having from C₃ to C₅ carbon atoms which can be selected from, for example, iso- propanol, n-butanol, or mixtures thereof, and at least one aliphatic hydrocarbon having from C₃ to C₁₀ carbon at- oms selected from those described above. Said organic solvent and/or said mixture, are subsequently evaporated obtaining a further oily phase comprising lipids, a further aqueous phase comprising non- fermented sugars and a further wet solid phase comprising cellular debris.

As described above for the wet solid phase (iii) , said further wet solid phase obtained after said extraction, can also be recovered and sent to acid hydrolysis. The lipids obtained according to the process of the present invention can be subjected to esterification in the presence of an alcohol having from 1 to 4 carbon atoms, preferably methanol, ethanol, and of an acid or base catalyst, in order to produce glycerol and alkyl esters, in particular methyl esters or ethyl esters (biodiesel) . Alternatively, said lipids can be subjected to hy- drogenation/deoxygenation in the presence of hydrogen and a catalyst in order to produce green diesel. Hydrogena- tion/deoxygenation processes are known in the art and are described, for example, in European patent application EP 1,728,844.

The present invention will now be described through an illustrative form with reference to Figure 1 provided hereunder .

According to a typical embodiment of the process ob- ject of the present invention, the biomass (e.g., previ- ously ground lignocellulosic biomass) is subjected to acid hydrolysis in the presence of an organic acid (e.g., 2-naphthalene -sulfonic acid) , obtaining a first mixture comprising a first aqueous phase including xylose deriv- ing from the hydrolysis of hemicellulose and said organic acid, and a first solid phase comprising lignin and cellulose .

As represented in Figure 1, said first mixture is subjected to extraction with an organic solvent insoluble in water (e.g., a mixtures of toluene and n-butanol) in order to recover said organic acid and recycle it, after evaporation of the solvent, to said acid hydrolysis.

After extraction with the solvent, said first mixtures is subjected to enzymatic hydrolysis, in the pres- ence of an enzyme (e.g., an aqueous solution of Cellu- clast 1,5L and Novozym 188) obtaining a second mixture comprising a second aqueous phase including xylose deriving from the acid hydrolysis of hemicellulose, glucose and cellobiose deriving from the enzymatic hydrolysis of cellulose, and a second solid phase including lignin.

Before being subjected to enzymatic hydrolysis, glacial acetic acid can be added to said first mixture in order to adjust the pH to values ranging from 4 to 6 (not represented in Figure 1) . Said second aqueous phase and said second solid phase are separated by filtration (not represented in Figure 1) .

Said second aqueous phase is subjected to fermentation in the presence of an oleaginous yeast (e.g., Lypo- myces starkey NRRL 1389) .

At the end of the fermentation, an oleaginous cellular biomass is obtained, which is subjected to mechanical treatment (e.g., by means of a French press cell) , and to subsequent centrifugation obtaining the following three phases:

(i) an oily phase comprising lipids; (ii) an aqueous phase comprising traces of non- separated lipids and non- fermented sugars; (iii) a wet solid phase comprising cellular debris and traces of non-separated lipids.

In order to concentrate the oleaginous cellular biomass, before being subjected to mechanical treatment, said biomass can be subjected to centrifugation (not represented in Figure 1) . In order to deactivate the lipolytic enzymes (e.g., lipase) , before being subjected to mechanical treatment, said oleaginous cellular biomass can be subjected to thermal treatment (not represented in Figure 1) .

As represented in Figure 1, said wet solid phase (iii) is sent to acid hydrolysis. In order to obtain a higher yield of lipids, said aqueous phase (ii) and said wet solid phase (iii) can be subjected to extraction in the presence of a mixture comprising at least one aliphatic alcohol and at least one aliphatic hydrocarbon (e.g., a mixture of iso-propanol and n-hexane) (not represented in Figure 1) .

Some illustrative and non- limiting examples are provided for a better understanding of the present invention and for its embodiment. EXAMPLE 1

Acid hydrolysis of the biomass

300 g of coniferous wood previously ground (particle diameter < 1 mm) were added to a solution of 20 g of 2- naphthalenesulfonic acid in 1 1 of water. The composition of the starting biomass was the following: 50% by weight of cellulose, 25% by weight of hemicellulose, 25% by weight of lignin, with respect to the total weight of the starting biomass.

The reaction mixture thus obtained, was maintained, under stirring, in an autoclave, at 140⁰C, for 1 hour, obtaining a first mixture comprising 225 g (dry weight) of a first solid phase (comprising cellulose and lignin) and 1,095 g of a first aqueous phase (comprising xylose deriving from the hydrolysis of hemicellulose and 2- naphthalene-sulfonic acid) . After cooling to 25°C, said first mixture was subjected to extraction with 1 1 of a mixture of toluene and n-butanol (3/1 by volume) .

The mixture of toluene and n-butanol was subse- quently separated and evaporated, at reduced pressure, obtaining a further solid phase comprising 19.6 g (dry weight) of 2-naphthalenesulfonic acid (recovery yield 98% calculated with respect to the total amount of the acid present in the aqueous solution) . After separation of the acid, glacial acetic acid was added to said first mixture until a pH of said first aqueous phase equal to 5 was obtained.

An aqueous solution of the enzyme Celluclast 1,5L (Novozymes) corresponding to 1485 FPU (Filter Paper Units) and the enzyme Novozym 188 (from Novozymes) corresponding to 18000 BGU (Beta Glucanase Units) was subsequently added. The suspension thus obtained was maintained under stirring, for 72 hours, at 45°C.

At the end of the enzymatic hydrolysis, 90 g (dry weight) of a second solid phase comprising lignin (75 g - dry weight) and non-hydrolyzed cellulose (15 g - dry weight) and 1,200 g of a second aqueous phase comprising glucose (152 g - dry weight) and xylose (72 g - dry weight) , were separated by filtration. Fermentation 250 ml of said second aqueous phase obtained as described above, containing 200 g/1 of sugars were suitably- diluted with distilled water until a solution having a final concentration of sugars equal to 50 g/1 was ob- tained.

The following nutrients were added to this solution: ammonium sulfate 0,1%, KH₂PO₄ 0,1%, MgSO₄ 7H₂O- 0,005%, NaCl 0,001%, CaCl₂ 0,001%, yeast extract 0,1%. The fermentation broth (culture medium) thus obtained was inocu- lated in a fermentor with yeast cells {Lypomices starkey NRRL 1389) (inoculum equal to 2,5 g/1 - dry weight) for a fermentation volume equal to 1 litre and the pH was maintained at 5.5 by the addition of an aqueous solution of NaOH 0.1 M . The yeast cells were grown, under stirring at 500 rpm, at 30⁰C, aeration 0.5 1/min of sterile air.

A further 250 ml of said second aqueous phase obtained from the hydrolysis of the biomass, containing 200 g/1 of sugars, suitably diluted with distilled water to obtain a solution having a final concentration of sugars equal to 50 g/1, were added to the fermentor.

After 150 hours, the yeast cells were collected and centrifuged at 7,000 rpm, for 20 minutes, obtaining 52 g (dry weight) of oleaginous cellular biomass. Recovery of the lipids The oleaginous cellular biomass thus obtained was maintained at 8O⁰C, for two hours, in order to inhibit the lipolytic enzymes and was then resuspended in 200 ml of water . The suspension thus obtained was subjected to mechanical treatment by means of a French press cell, operating at a pressure of 39 Kpsi obtaining a cellular lys- ate.

Said cellular lysate was centrifuged at 7,000 rpm, for 20 minutes obtaining three phases: an oily phase insoluble in water, an aqueous phase and a wet solid phase. The oily phase insoluble in water was separated and dried in an oven at 60⁰C, obtaining 19 g (dry weight) of an oily residue comprising lipids. The aqueous phase comprising traces of non-separated lipids and non- fermented sugars and the wet solid phase comprising cellular debris and traces of non- separated lipids, were extracted with 50 ml of a mixture comprising n-hexane and isopropanol in a proportion of 3:1. The mix- ture of solvents was separated and evaporated, at reduced pressure, obtaining 12.2 g (dry weight) of a further oily residue comprising lipids, a further wet solid phase and a further aqueous phase .

The total amount of lipids obtained is therefore equal to 31.2 g (dry weight) (recovery yield of 60% cal- culated with respect to the total amount of the oleaginous cellular biomass obtained after fermentation) .

The lipids were determined by means of the colori- metric method which is based on the reaction of the lip- ids with phosphoric acid and phospho-vanillin.

The further wet solid phase, obtained after extraction of the lipids, comprising water and cellular debris, including 42% by weight (dry weight) of polysaccharides and 45% by weight of proteins (dry weight) , was separated by centrifugation from the further aqueous phase comprising non- fermented sugars. EXAMPLE 2

The further wet solid phase, obtained after extraction of the lipids as described in Example 1, was sent to acid hydrolysis, operating under the conditions indicated below.

42 g (dry weight) of said further wet solid phase and 260 g of coniferous wood previously ground (particle diameter < 1 mm) were added to a solution of 20 g of 2- naphthalenesulfonic acid in 1 1 of water.

The composition of the starting biomass was the following: 50% by weight of cellulose, 25% by weight of hemicellulose, 25% by weight of lignin, with respect to the total weight of the starting biomass. The reaction mixture thus obtained, was maintained, under stirring, in an autoclave, at 14O⁰C, for 1 hour, obtaining a first mixture comprising 198 g (dry weight) of a first solid phase (comprising cellulose, lignin and cellular debris) and 1,100 g of a first aqueous phase (comprising xylose deriving from the hydrolysis of hemi- cellulose, glucose, mannose and amino acids deriving from the hydrolysis of the polysaccharides and of the membrane proteins included in the cellular debris and 2- naphthalenesulfonic acid) . After cooling to 25°C, said first mixture was subjected to extraction with 1 1 of a mixture of toluene and n-butanol (3/1 by volume) .

The mixture of toluene and n-butanol was subsequently separated and evaporated, at reduced pressure, obtaining a further solid phase comprising 19.5 g (dry weight) of 2-naphthalenesulfonic acid (recovery yield 98% calculated with respect to the total amount of the acid present in the aqueous solution) .

After separation of the acid, glacial acetic acid was added to said first mixture until a pH of said first aqueous phase equal to 5.0 was obtained.

An aqueous solution of the enzyme Celluclast 1,5L

(Novozymes) corresponding to 1400 FPU (Filter Paper

Units) and the enzyme Novozym 188 (from Novozymes) corre- sponding to 18000 BGU (Beta Glucanase Units) was subse- quently added. The suspension thus obtained was maintained under stirring, for 72 hours, at 45⁰C.

At the end of the enzymatic hydrolysis, 80 g (dry weight) of a second solid phase comprising lignin (65 g - dry weight) , non-hydrolyzed cellulose (13 g - dry weight) and cellular debris (2 g - dry weight) and 1,220 g of a second aqueous phase comprising glucose (139 g - dry weight) , mannose (9 g - dry weight) , xylose (67 g - dry weight) and amino acids (20 g - dry weight) , were sepa- rated by filtration.

Said second aqueous phase can be subjected to fermentation operating as described in Example 1.

Claims

1. A process for the production of lipids from biomass including at least one polysaccharide comprising: subjecting said biomass to acid hydrolysis in the presence of an aqueous solution of at least one organic acid selected from alkyl- or aryl-sulfonic acids having from C₇ to C₂o carbon atoms, or from halogenated carboxylic acids, at a temperature ranging from 80⁰C to 160⁰C, obtaining a first mixture comprising a first solid phase and a first aqueous phase,- subjecting said first mixture to enzymatic hydrolysis obtaining a second mixture comprising a second solid phase and a second aqueous phase,- subjecting said second aqueous phase to fermentation in the presence of at least one oleaginous yeast obtaining an oleaginous cellular biomass comprising lipids .

2. The process according to claim 1, wherein said alkyl- or aryl-sulfonic acids have from C₉ to Ci₅ carbon at- oms .

3. The process according to claim 1 or 2, wherein said temperature ranges from 100⁰C to 150°C.

4. The process according to any one of the preceding claims, wherein said polysaccharide is selected from cellulose, hemicellulose, or their mixtures.

5. The process according to any one of the preceding claims, wherein said biomass is a lignocellulosic bio- mass .

6. The process according to claim 5, wherein said ligno- cellulosic biomass is selected from: products of cultures expressly cultivated for energy use (for example, miscanthus, millet, common cane), including by-products, residues and wastes of said cultures or of their processing; - products of agricultural cultivations, forestation and silviculture, comprising wood, plants, residues and by-products of agricultural processing, forestation and silviculture; agri-foodstuffs by-products intended for human feeding or zootechnics; residues, not chemically treated, of the paper industry; waste products coming from the differentiated collection of solid urban waste.

7. The process according to any one of the preceding claims, wherein said biomass is subjected to a preliminary grinding process before being subjected to acid hydrolysis.

8. The process according to claim 7, wherein said biomass is ground until particles having a diameter ranging from 0.1 mm to 10 mm are obtained.

9. The process according to claim 8, wherein said biomass is ground until particles having a diameter lower than 1 mm are obtained.

10. The process according to any one of the preceding claims, wherein said biomass is present in the reaction mixture in an amount ranging from 5% by weight to 40% by weight with respect to the total weight of the reaction mixture.

11. The process according to any one of the preceding claims, wherein said at least one organic acid is soluble in water and extractable by means of an organic solvent insoluble in water.

12. The process according to any one of the preceding claims, wherein said alkyl- or aryl sulfonic acids are selected from: dodecyl- sulfonic acid, para-toluene- sulfonic acid, 1-naphthalene-sulfonic acid, 2- naphthalene-sulfonic acid, 1, 5-naphthalene-disulfonic acid, or their mixtures; halogenated carboxylic acids such as difluoroacetic acid, trifluoroacetic acid, trichloroacetic acid, perfluoro-octanoic acid, or their mixtures; or their mixtures.

13. The process according to claim 12, .wherein said at least one organic acid is selected from para-toluene- sulfonic acid, 2 -naphthalene- sulfonic acid, 1,5- naphthalene-disulfonic acid, or their mixtures.

14. The process according to any one of the previous claims, wherein said halogenated carboxylic acids are selected from those having a number of carbon atoms not higher than 20 such as trifluoroacetic acid, di- chloroacetic acid, trichloroacetic acid, perfluoro- octanoic acid, or mixtures thereof.

15. The process according to any one of the preceding claims, wherein said at least one organic acid is pre- sent in the aqueous solution at a concentration ranging from 0.1% by weight to 5% by weight with respect to the total weight of the aqueous solution.

16. The process according to any one of the preceding claims, wherein said acid hydrolysis is carried out for a time ranging from 20 minutes to 6 hours.

17. The process according to any one of the preceding claims, wherein said first solid phase comprises lig- nin and cellulose.

18. The process according to any one of the preceding claims, wherein said first aqueous phase comprises at least one sugar having from C₅ to C₆ carbon atoms and said at least one organic acid.

19. The process according to claim 18, wherein said at least one sugar is xylose.

20. The process according to any one of the preceding claims, wherein said first mixture is subjected to extraction with an organic solvent insoluble in water.

21. The process according to claim 20, wherein said organic solvent insoluble in water is selected from: halogenated hydrocarbons such as methylene chloride, monochlorobenzene, dichlorobenzene, or mixtures thereof; aromatic hydrocarbons such as toluene, xylene, or mixtures thereof; aliphatic alcohols having from C₄ to C₆ carbon atoms such as n-butanol, n- pentanol, or mixtures thereof; or mixtures thereof.

22. The process according to claim 21, wherein said organic solvent insoluble in water is selected from mixtures of toluene and n-butanol.

23. The process according to any one of the claims from 20 to 22, wherein said organic solvent insoluble in water is evaporated obtaining a further solid phase comprising said at least one organic acid.

24. The process according to any one of the preceding claims, wherein said process comprises reutilizing said at least one organic acid in said acid hydrolysis .

25. The process according to any one of the preceding claims, wherein said second solid phase comprises lig- nin.

26. The process according to any one of the preceding claims, wherein said second aqueous phase comprises at least one sugar having from C₅ to C₅ carbon atoms .

27. The process according to any one of the preceding claims, wherein said second aqueous phase comprises xylose, glucose and cellobiose.

28. The process according to any one of the preceding claims, wherein said fermentation is carried out at a temperature ranging from 20⁰C to 40⁰C.

29. The process according to any one of the preceding claims, wherein said fermentation is carried out for a time ranging from 3 days to 10 days .

30. The process according to any one of the preceding claims, wherein said fermentation is carried out at a pH ranging from 4.5 to 6.5.

31. The process according to any one of the preceding claims, wherein said oleaginous yeast is selected from: Rhodotorula glutinis, Rhodotorula gracilis, Rhodotorula graminis, Lypomices starkeyi, Lypomices lipofer, Trigonopsis variabilis, Candida kefyr, Candida curvata, Candida lipolytica, Pichia stipitis, To- rulopsis sp..

32. The process according to any one of the preceding claims, wherein said fermentation is a feed-batch fer- mentation.

33. The process according to any one of the preceding claims, wherein said oleaginous cellular biomass is subjected to mechanical treatment.

34. The process according to claim 33, wherein said me- chanical treatment is carried out using a "French press cell", operating at a pressure ranging from 20 Kpsi to 50 Kpsi.

35. The process according to claim 33 or 34, wherein said oleaginous cellular biomass is subjected to cen- trifugation after mechanical treatment.

36. The process according to claim 35, wherein said centrifugation is carried out for a period of time ranging from 5 minutes to 30 minutes.

37. The process according to claim 35 or 36, wherein said centrifugation is carried out at a rotational speed ranging from 3,000 rpm to 9,000 rpm.

38. The process according to any one of the claims from 34 to 36, wherein at the end of the centrifugation the following three phases are obtained: (i) an oily phase comprising lipids;

(ii) an aqueous phase comprising traces of non- separated lipids and non- fermented sugars,- (iii) a wet solid phase comprising cellular debris and traces of non-separated lipids.

39. The process according to any one of the preceding claims, wherein said wet solid phase (iii) is conveyed to the acid hydrolysis.

40. The process according to any one of the preceding claims, wherein said aqueous phase (ii) and/or said wet solid phase (iii) are subjected to extraction with at least one solvent which can be selected from aliphatic hydrocarbons having from C₃ to C₁₀ carbon atoms; or with a mixture comprising at least one aliphatic alcohol having from C₃ to C₅ carbon atoms and at least one aliphatic hydrocarbon having from C₃ to Ci₀ carbon atoms .

41. Biodiesel or green diesel obtained from lipids produced by means of a process according to any one of the preceding claims .