WO2025154009A1

WO2025154009A1 - Process for simultaneous production of sugars from biomass and of sugars and lipids from a fermentation broth

Info

Publication number: WO2025154009A1
Application number: PCT/IB2025/050514
Authority: WO
Inventors: Stefano Ramello; Mario BALDASSARRE; Riccardo PREDA; Valentina RODIGHIERO
Original assignee: Eni SpA; Versalis SpA
Current assignee: Eni SpA; Versalis SpA
Priority date: 2024-01-18
Filing date: 2025-01-17
Publication date: 2025-07-24
Anticipated expiration: 2026-07-18

Abstract

Process for simultaneous production of sugars from biomass including at least one polysaccharide and of sugars and lipids from a fermentation broth, comprising the following steps: (a) feeding at least one microorganism, preferably an oleaginous microorganism, to a fermentation device obtaining a fermentation broth comprising an aqueous suspension of cellular biomass comprising sugars and lipids; (b) optionally, at the end of the fermentation, subjecting the fermentation broth obtained in said step (a) to concentration obtaining a fermentation broth comprising an aqueous suspension of concentrated cellular biomass comprising sugars and lipids; (c) subjecting the fermentation broth obtained in said step (a) or the fermentation broth comprising an aqueous suspension of concentrated cellular biomass comprising sugars and lipids obtained in said step (b) and said biomass comprising at least one polysaccharide to hydrolysis in the presence of at least one organic acid having from 1 to 6 carbon atoms, preferably from 1 to 3 carbon atoms, obtaining a reaction mixture comprising a solid phase, an oily phase and an aqueous phase, said hydrolysis being carried out at a pH comprised between 0.6 and 1.6, preferably comprised between 0.9 and 1.3; (d) subjecting the reaction mixture obtained in said step (c) to separation obtaining a solid phase comprising lignin, cellulose and cellular debris, an oily phase comprising lipids and an aqueous phase comprising sugars. The sugars obtained from the aforesaid process can be advantageously used, for example, as carbon sources in the fermentation processes for the production of alcohols (e.g., ethanol, butanol), lipids, diols (e.g., 1,3 -propanediol, 1,3 -butanediol, 1,4-butanediol, 2,3-butanediol), bioplastics (e.g., polyhydroxyalkanoates), or in chemical synthesis processes for the production of other intermediates or chemicals. Said alcohols and lipids, can in turn be advantageously used in the production of biofuels (e.g., biodiesel or "Green Diesel"), which can be used as such, or in admixture with other automotive fuels, while said diols can be used in the production of products such as, for example, bio-butadiene which can in turn be used in the production of rubbers (e.g., polybutadiene or copolymers thereof). The lipids obtained from the aforesaid process can be advantageously used, for example, for the production of biofuels usable in diesel or aviation engines, or said lipids can be subjected to metathesis processes in order to obtain precursors of biofuels, waxes, plastics, cosmetics, personal care items. The aforesaid process is particularly useful in the case of a biorefinery.

Description

PROCESS FOR SIMULTANEOUS PRODUCTION OF SUGARS FROM BIOMASS AND OF SUGARS AND LIPIDS FROM A FERMENTATION BROTH

DESCRIPTION

The present invention relates to a process for simultaneous production of sugars from biomass and of sugars and lipids from a fermentation broth.

More particularly, the present invention relates to a process for simultaneous production of sugars from biomass including at least one polysaccharide and of sugars and lipids from a fermentation broth comprising subjecting said fermentation broth and said biomass comprising at least one polysaccharide to hydrolysis in the presence of at least one organic acid having from 1 to 6 carbon atoms, preferably from 1 to 3 carbon atoms.

The sugars obtained from the aforesaid process can be advantageously used, for example, as carbon sources in the fermentation processes for the production of alcohols (e.g., ethanol, butanol), lipids, diols (e.g., 1,3 -propanediol, 1,3 -butanediol, 1,4-butanediol, 2,3-butanediol), bioplastics (e.g., polyhydroxyalkanoates), or in chemical synthesis processes for the production of other intermediates or chemicals. Said alcohols and lipids, can in turn be advantageously used in the production of biofuels (e.g., biodiesel or “Green Diesel”), which can be used as such, or in admixture with other automotive fuels, while said diols can be used in the production of products such as, for example, bio-butadiene which can in turn be used in the production of rubbers (e.g., polybutadiene or copolymers thereof). The lipids obtained from the aforesaid process can be advantageously used, for example, for the production of biofuels usable in diesel or aviation engines, or said lipids can be subjected to metathesis processes in order to obtain precursors of biofuels, waxes, plastics, cosmetics, personal care items. The aforesaid process is particularly useful in the case of a biorefinery.

The production of sugars from biomass, in particular from 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 depending on the type of lignocellulosic biomass used. For example, in the case of plants, these amounts vary depending on the species and age of the plant.

Cellulose is the largest constituent of lignocellulosic biomass and is generally present in an amount comprised between 30% by weight and 60% by weight with respect to the total weight of lignocellulosic biomass. Cellulose consists of glucose molecules (from about 500 to 10,000 units) joined together by a P-l,4-glucosidic bond. The establishment of hydrogen bonds between the chains results in the formation of crystalline domains that impart strength and elasticity to the plant fibres. In nature it is found in its pure state only in annual plants such as cotton and flax, while in woody plants it is always accompanied by hemicellulose and lignin.

Hemicellulose, which is generally present in an amount comprised between 10% by weight and 40% by weight with respect to the total weight of the lignocellulosic biomass, appears as a relatively short (from 10 to 200 molecules) and branched mixed polymer formed by both sugars with six carbon atoms (glucose, mannose, galactose) and sugars with five carbon atoms (xylose, arabinose). The presence of hemicellulose is due to some important properties of plant fibres, the main one being that it promotes imbibition when water is present, causing them to swell. Hemicellulose also has adhesive properties and, therefore, tends to cement or become homy in consistency, with the consequence that said plant fibres become rigid and imbibe more slowly.

Lignin is generally present in an amount comprised between 10% by weight and 30% by weight with respect to the total weight of the lignocellulosic biomass. Its main function consists in binding and cementing the various plant fibres together so as to confer firmness and resistance to the plant and it also constitutes a protection against insects, pathogens, injuries and ultraviolet light. It is mainly used as a fuel but, currently, it is also widely used in industry as a dispersant, hardener, emulsifier, for plastic laminates, cardboards and rubber products. In addition, it can be chemically treated to produce aromatic compounds, such as vanillin, syringaldehyde, /?-hydroxybenzaldehyde, which can be used in pharmaceutical chemistry, or in the cosmetic and food industry.

In order to optimize the transformation of lignocellulosic biomass into products for energy use or usable as intermediates in the chemical industry, it is known to subject said biomass to a preliminary treatment so as to separate lignin and hydrolyse cellulose and hemicellulose to sugars such as, for example, glucose and xylose, which can then be subjected to fermentation processes.

The process usually used for the above purpose is acid hydrolysis, which can be carried out in the presence of dilute or concentrated strong acids.

For example, American patent No. 6,423,145 describes a process for hydrolysing a lignocellulosic biomass so as to obtain a high amount of fermentable sugars comprising: impregnating the lignocellulosic material with a mixture comprising a dilute acid catalyst (e.g., sulphuric acid, hydrochloric acid, nitric acid, sulphur dioxide, or any other strong acid capable of giving pH values of less than about 3) and a metal salt based catalyst (e.g., ferrous sulphate, ferric sulphate, ferric chloride, aluminium sulphate, aluminium chloride, magnesium sulphate), in an amount such as to provide a higher yield of fermentable sugars than that obtained in the presence of the diluted acid alone; feeding the impregnated lignocellulosic material to a reactor and heating (for example, to a temperature comprised between 120 C and 240°C) for a sufficient time (for example, for a time comprised between 1 minute and 30 minutes) to hydrolyse substantially all of the hemicellulose and more than 45% of the cellulose to water-soluble sugars; recovering the water soluble sugars.

International patent application WO 2010/102060 describes a process for the pre-treatment of biomass to be used in a biorefinery for the purpose of producing a fermentation product, comprising the following steps: subjecting the biomass to treatments (for example, removal of unwanted materials, grinding) before sending it to a pre-treatment; subjecting the biomass to pre-treatment by application of a dilute acid (for example, sulphuric acid) having a concentration comprised between about 0.8% by weight and about 1.1% by weight, at a temperature comprised between about 130°C and about 170°C, for a time comprised between about 8 minutes and about 12 minutes; wherein the fermentation product can be obtained by separating the pre-treated biomass into a liquid component comprising xylose and into a solid component from which glucose can be made available and recovering xylose for fermentation; wherein the biomass comprises lignocellulosic material; wherein the lignocellulosic material comprises corn cobs, corn plant husks, com plant leaves, and corn plant stalks.

International patent application WO 2010/071805 describes a process for pretreating lignocellulosic material comprising: exposing the lignocellulosic material to a first pre-treatment carried out under low severity operating conditions obtaining a first product; contacting said first product with an acid diluted in aqueous solution (for example, sulphuric acid, sulphurous acid, sulphur dioxide, phosphoric acid, carbonic acid) obtaining a second product. Said two-step process can provide products useful for the production of bioethanol.

American patent application US 2010/0227369 describes a method for producing a fermentation product in a fermentation system from biomass that has been pre-treated and separated into a first component and into a second component comprising the following steps: feeding the first component to a fermentation system; providing an ethanol-producing organism (“ethanologen”) to the fermentation system; maintaining the first component and the ethanol-producing organism (“ethanologen”) in the fermentation system at a temperature comprised between about 26 C and about 37°C and at a pH comprised between about 4.5 and about 6.0, for a time of not less than 18 hours; recovering the fermentation product from the fermentation system; wherein the ethanol-producing organism (“ethanologen”) is fed to the fermentation system in an amount of less than 150 grams of ethanol-producing organism (“ethanologen”) (dry weight) per litre of first component; wherein the biomass comprises lignocellulosic material; wherein the lignocellulosic material comprises at least one of corn cobs, corn plant husks, corn plant leaves and com plant stalks; wherein the first component comprises a pentose; wherein the pentose comprises xylose; wherein the ethanol-producing organism is capable of fermenting xylose into ethanol. Preferably, the pre-treatment of the biomass is done by contacting said biomass with an acid such as, for example, sulphuric acid, hydrochloric acid, nitric acid, phosphoric acid, acetic acid, or mixtures thereof.

American patent application US 2008/0274509 describes a process for preparing a hydrolysate from a lignocellulosic material comprising: a) pre-treating said lignocellulosic material with a compound selected from the group consisting of: sulphuric acid, alkalis, peroxydisulphates, potassium peroxide, and mixtures thereof, in the presence of water, obtaining an aqueous phase; and b) after removal of the aqueous phase and washing of the product obtained, treating said product with an enzyme useful for hydrolysis, in the presence of water, obtaining a hydrolysate, said hydrolysate being usable as a carbon source for fermentation.

Tsoutsos T. et al. in “Energies” (2011), Vol. 4, pp. 1601-1623, describe the optimization of the production of fermentable sugar solutions for the production of bioethanol from lignocellulosic biomass. In this regard, the lignocellulosic biomass is subjected to a two-step hydrolysis process, in the presence of a dilute acid. In particular, tests were carried out in the presence of acids (for example, hydrochloric acid, sulphuric acid, phosphoric acid, nitric acid) diluted to a concentration of up to 3% - 4% and at temperatures comprised between 100°C and 240 C. At temperatures comprised between 110°C and 140°C, hemicellulose hydrolysis occurs, while crystalline cellulose remains practically as it is up to 170°C and is hydrolysed at 240 C.

Gonzales-Hernandez J. C. et al., in “Journal of the Mexican Chemical Society” (2011), Vol. 56 (4), pp. 395-401, describe the hydrolysis of polysaccharides from tamarind seeds. In particular, the tamarind seeds were subjected to hydrolysis operating under different operating conditions: i.e. at a temperature comprised between 86 C and 130.2 C, at a concentration of nitric acid or sulphuric acid comprised between 0.32% and 3.68% (v/v), and at a contact time comprised between 13.2 minutes and 40 minutes. It has been seen that temperature and time are the factors that most influence sugar hydrolysis: in particular, the best operating conditions, for both acids, were: temperature equal to 130.2 C, concentration equal to 2% (v/v), contact time equal to 30 minutes, with a sugar yield equal to about 110 g/L.

Shatalov A. A. et al., in “Chemical Engineering & Process Technology” (2011), Vol. 2, Issue 5, pp. 1-8, describe the production of xylose, by hydrolysis in the presence of dilute sulphuric acid, at low temperature, in a single step, from thistle (Cynara cardunculus L.). In particular, operating under optimal conditions, i.e. temperature equal to 138.5 C, time equal to 51.7 minutes, acid concentration equal to 1.28%, there is a xylose recovery equal to 86%, with a low degradation of cellulose and a low production of furfurals (glucose = 2.3 g and furfural (F) 1.04 g per 100 g of thistle).

International patent application WO 2010/046051 relates to a process for the production of lipids from biomass including at least one polysaccharide which comprises: subjecting said biomass to acid hydrolysis in the presence of an aqueous solution of at least one organic acid having from C7 to C20 carbon atoms, preferably from C9 to C15 carbon atoms, at a temperature comprised between 80 C and 160 C, preferably comprised between 100 C and 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.

Lipids are therefore produced by fermentation of sugars obtained from the hydrolysis of biomass.

International patent application WO 2012/052368 relates to a process for the production of lipids from biomass including at least one polysaccharide which comprises: subjecting said biomass including at least one polysaccharide to acid hydrolysis obtaining a first mixture comprising a first solid phase and a first aqueous phase; feeding said first aqueous phase to a fermentation device in the presence of at least one oleaginous yeast obtaining a first fermentation broth comprising a first oleaginous cellular biomass; subjecting said first solid phase to acid hydrolysis or to enzymatic hydrolysis obtaining a second mixture comprising a second solid phase and a second aqueous phase; feeding said second aqueous phase to said fermentation device in the presence of said first fermentation broth obtaining a second fermentation broth comprising a second oleaginous cellular biomass including lipids; subjecting at least a part of said second fermentation broth to microfiltration obtaining a retentate and a permeate; feeding said retentate to said fermentation device.

Also in this case, lipids are produced by fermentation of sugars obtained from the hydrolysis of biomass.

However, the above processes may have some drawbacks.

For example, if the acid hydrolysis is carried out at high temperatures, for example above 140°C, reaction by-products deriving from the dehydration of sugars and from the partial depolymerization of lignin such as, for example, furfural (F), hydroxymethylfurfural (HMF), phenolic compounds, which act as inhibitors of the growth of microorganisms usually used in subsequent sugar fermentation processes, may be formed, resulting in a substantial decrease in the efficiency and productivity of said processes.

If, on the other hand, the acid hydrolysis is carried out at low temperatures, for example below 140 C, poor destructurization of the lignocellulosic biomass can be obtained, which destructurization is made necessary for the cellulose fibres to be released from the lignin network that covers them so that they can be advantageously used in the subsequent step of enzymatic hydrolysis. In fact, the cellulose fibres covered by lignin are difficult to reach by the enzymes usually used (for example, cellulase) in enzymatic hydrolysis.

Efforts were therefore made in the art in order to overcome the aforesaid drawbacks.

For example, the international patent application WO 2010/069583 describes a process for the production of one or more sugars from biomass including at least one polysaccharide which comprises contacting a biomass with an aqueous solution of at least one organic acid, preferably /?-toluene-sulfonic acid, 2-naphthalene- sulfonic acid, 1,5-naphthalene-disulfonic acid, at a temperature greater than or equal to 160°C, preferably comprised between 160°C and 230°C. In said patent application, alkyl sulfonic acids having from 4 to 16 carbon atoms, preferably from 8 to 12 carbon atoms, even more preferably octyl sulfonic acid and dodecyl sulfonic acid are also mentioned. However, the only examples of hydrolysis reported relate to the use of 2-naphthalene sulfonic acid. International patent application WO 2010/015404 describes a process for the production of sugars from biomass including at least one polysaccharide which comprises contacting a biomass with an aqueous solution of at least one organic acid having from 7 to 20 carbon atoms, preferably from 9 to 15 carbon atoms, more preferably /?-toluenesulfonic acid, 2-naphthalenesulfonic acid, 1,5- naphthalenesulfonic acid, at a temperature comprised between 80°C and 140 °C, preferably comprised between 100 C and 125°C.

International patent application WO 2015/087254, in the name of the Applicant, describes a process for the production of sugars from biomass including at least one polysaccharide which comprises contacting said biomass with an aqueous solution of at least one organic acid having from 1 to 6 carbon atoms, preferably from 1 to 3 carbon atoms, the pH of said aqueous solution being comprised between 0.6 and 1.6, preferably comprised between 0.9 and 1.3. Preferably, said at least one organic acid can be selected from alkyl sulfonic acids having general formula (I):

R-SO3H (I) wherein R represents a linear or branched C1-C6 alkyl group.

The American patent application US 2017/218094 in the name of the Applicant, relates to an integrated process for the transformation and valorisation of each part of the guayule plant, including the following sequential steps: separating the stem and branches from the leaves of said plant with a mechanical treatment; treating the leaves to produce waxes and essential oils, and a fraction comprising cellulose, hemicellulose and to a minor extent salts, organic compounds and lignin; extracting a liquid phase from the stem and branches, thus forming a first solid woody residue, referred to as bagasse; treating said first solid woody residue to form sugars, cellulose, hemicellulose and lignin.

The aforesaid integrated process is said to be able to further valorise the guayule plant by combining the production of latex, rubber, resin and bagasse with the production of fermentable sugars: said valorisation is particularly important in the case of biorefineries designed to produce organic intermediates other than ethanol, for example, to produce 1,3-butanediol which can be transformed, after the double catalytic dehydration thereof, into bio-butadiene. The production of fermentable sugars is carried out by means of a two-step saccharification treatment: in the first step an acid hydrolysis is carried out to transform lignin into monomeric sugars having 5 carbon atoms (C5), while in the second step an enzymatic, chemical or thermochemical hydrolysis is carried out in order to obtain monomeric sugars having 6 carbon atoms (C6).

International patent application WO 2019/145865, in the name of the Applicant, relates to a process for the production of sugars from biomass deriving from guayule plants comprising contacting an amount of said biomass (G2) (g) with an amount of water (Gi) (g) and with at least one organic acid, and optionally at least one inorganic acid, obtaining a mixture, said at least one organic acid and said at least one inorganic acid optionally present being used in amounts such that the total moles of said at least one organic acid and of said at least one inorganic acid optionally present (m TOT) contained in said mixture are calculated according to the following equation (1): mTOT = mi + m2 (1) wherein mi and m2 are defined according to the following equations (2) and (3), respectively: m1 = R1 • G1 (2) m2 = R2 G2 (3) wherein: R1 (mmol/g) is the ratio between a first portion of said at least one organic acid (mmol) and a first portion of said at least one inorganic acid (mmol) optionally present and the amount of water Gi (g) used, R1 being comprised between 0.06 mmol/g and 0.25 mmol/g, preferably comprised between 0.09 mmol/g and 0.18 mmol/g, said first portion of said at least one organic acid (mmol) and said first portion of said at least one inorganic acid (mmol) optionally present being referred to the amount of water Gi (g);

R2 (mmol/g) is: in the absence of said at least one inorganic acid, the ratio between a second portion of said at least one organic acid (mmol) and the amount of biomass G2 (g) used; or in the presence of said at least one inorganic acid, the ratio between the sum of said second portion of said at least one organic acid (mmol) and of a second portion of said at least one inorganic acid (mmol) and the amount of biomass G2 (g) used; or in the presence of said second portion of said at least one inorganic acid (mmol) and in the absence of said second portion of said at least one organic acid (mmol), the ratio between said second portion of said at least one inorganic acid (mmol) and the amount of biomass G2 (g) used; said second portion of said at least one organic acid (mmol) and said second portion of said at least one inorganic acid (mmol) being referred to the amount of biomass G2 (g);

R2 being comprised between 0.90 R (mmol/g) and 1.10 R (mmol/g), preferably comprised between 0.95 R (mmol/g) and 1.05 R (mmol/g), R being determined by the following algorithm (4), said algorithm (4) being obtained by the following elementary operations:

(i) preparing a volume V (1) of an aqueous solution of said at least one organic acid and of said at least a first portion of said at least one inorganic acid (mmol) optionally present, said aqueous solution having a pH(i) lower than 7, preferably comprised between 0.7 and 3;

(ii) adding to the aqueous solution obtained in (i) an amount of biomass Q (g), dried at 120 C for 15 h, said amount of biomass being preferably less than or equal to 60% by weight, more preferably comprised between 2% by weight and 40% by weight, with respect to the total weight of the mixture obtained;

(iii) measuring the pH of the mixture obtained in (ii), said pH hereinafter indicated as pH(2);

(iv) determine R according to the following algorithm (4): wherein pH(i), pH(2), V and Q have the same meanings reported above, the aforesaid elementary operations being carried out at room temperature; provided that, said at least one organic acid is present in such an amount that the ratio RMINIMUM (mmol/g) defined according to the following equation (5):

RMINIMUM = moRGANIC ACID/G2 (5) wherein IUORGANIC ACID are the mmol of organic acid present and G2 has the same meaning reported above, is greater than or equal to 0.20 mmol/g, preferably greater than or equal to 0.25 mmol/g and, in the case wherein said at least one inorganic acid is present, said mmol of organic acid (IUORGANIC ACID) are present in an amount less than the sum of the two portions of acid, i.e. the sum of the portion of inorganic acid (mmol) and the portion of organic acid (mmol), said sum corresponding to the total moles IHTOT (mmol) as defined in equation (1) reported above.

Processes for the recovery, quantification and characterization of sugars present in microorganisms are also known in the art.

For example, Kanetsuna F. et al., in “Journal of Bacteriology" (1969), Vol. 97, No. (3), pp. 1036-1041, describe the composition ofthe cellular wall ofthe yeast Paracoccidioides brasiliensis. For this purpose, the yeast was frozen and subjected to a process of mechanical lysis of the cellular walls by means of a French Press until almost complete crushing. The crushed cells, after further treatments, were recovered by centrifugation and hydrolysed in a 1 N hydrochloric acid solution, at 110 C, for a time comprised between 5 hours and 7 hours. The sugars obtained were analysed by liquid chromatography. The yield was not reported and no other process conditions for sugar recovery were described. Moreover, the production of undesired by-products deriving from secondary sugar-degrading reactions has not been addressed. Said undesired by-products, for example, hydroxymethylfurfural (HMF), are in fact known in the art as inhibitors for any downstream processes for valorisation of sugars.

Similarly, the sugars constituting the cellular wall of the fungus Aspergillus nidulans were also studied as reported by Bull A.T., in “Journal of General Microbiology" (1970), Vol. 63, pp. 75-94, DOI: 10.1099/00221287-63-1-75. After lysis and separation, the cellular walls were subjected to a process of acid hydrolysis in the presence of sulphuric acid (H2SO4), after which glucose, mannose and galactose were identified in the aqueous phase. Also in this case, no yield was reported, no other process conditions for sugar recovery were described, and the production of undesired by-products deriving from secondary sugar-degrading reactions was not addressed.

The same approach was also adopted for Mucor rouxii yeast as reported by Dow J. M. et al., in “Microbiology” (1977), Vol. 99, Issue 1, 29-41 DOI: 10.1099/00221287-99-1-29. In this case, after lysis and separation, the cellular walls were subjected to a process of acid hydrolysis in the presence of sulphuric acid (H2SO4), after which mannose and galactose were identified in the aqueous phase. Also in this case, no yield was reported, no other process conditions for sugar recovery were described, and the production of undesired by-products deriving from secondary sugar-degrading reactions was not addressed.

In a similar way, the nature of the constituent sugars of the cellular wall of the yeast Aspergillius niger was also studied as reported by Johnston I. R., in “Biochemical Journal” (1965), Vol. 96(3), pp. 651-658, DOI: 10.1042/bj 0960651. Sugar hydrolysis was carried out in the presence of sulphuric acid (H2SO4) after yeast lysis and cellular wall recovery. Glucose, mannose and galactose were identified and quantified in this way. In the experimental part, neither alternative methods to hydrolysis with sulphuric acid (H2SO4), nor alternative methods for optimizing sugar recovery were proposed and it is assumed that sugar recovery is complete. Also in this case, the production of undesired by-products deriving from secondary sugar-degrading reactions has not been addressed.

The same approach, in which it is assumed that the recovery of sugars is complete, has been followed in a research aimed at the taxonomic evaluation of some species of yeasts, in particular Rhodosporidium, Leucosporidium and Rhodoiorula. as reported by Sugiyama J. et al., in “The Journal of General and Applied Microbiology” (1985), Vol. 31(6), pp. 519-550, DOI: 10.2323/jgam.31.519. The authors report a total recovery of the sugars of said yeasts, both those constituting the cellular wall and the endocellular ones (i.e. the sugars contained within the cell, such as glycogen), by treatment of the fermentate, dried, with trifluoroacetic acid. Again, the approach is uncritical with regard to both the amount and the type of sugars recovered. Also in this case, the production of undesired by-products deriving from secondary sugar-degrading reactions has not been addressed. Other authors, for analytical purposes, have faced the problem of identifying a useful acid in order to optimize the lysis of yeasts, in particular of the yeast Saccharomyces cerevisiae. with the simultaneous enhanced recovery of the sugars constituting the cellular walls as reported by Dallies N. et al., in “Yeast” (1998), Vol. 14, Issue 14, pp. 1297-1306, DOI: 10.1002/(SICI)1097- 0061(1998100)14: 14<1297:AID-YEA310>3.0.CO;2-L. The authors found that there were no standard methods for carrying out the desired analyses, so they relied in the first instance on a method developed for the determination of sugars in plant fibres, based on a treatment with sulphuric acid (H2SO4) reported by Selvendran R. 96, Issue 2, pp. 282-292, DOI: 10.1016/0003-2697(79) 90583-9) and subsequently they used other acids, including hydrochloric acid and trifluoroacetic acid. From the results of the tests carried out, the highest sugar yield was obtained using sulphuric acid (H2SO4). Also in this case, the production of undesired by-products deriving from secondary sugar-degrading reactions has not been addressed.

Other authors have also addressed the problem of identifying the optimal methodology for yeast lysis and its influence on the subsequent determination of sugars as reported, for example, by Bzducha- Wrobel A. et al., in “Molecules’" (2014), Vol. 19, Issue (12), pp. 20941-20961, DOI: 10.3390/moleculesl91220941. More particularly, the authors identified several methods for the lysis of yeast cells to establish their suitability for cellular wall preparation in the process of determining the sugars constituting the cellular wall. The methods studied included: autoclave sterilisation, thermally induced autolysis, homogenisation in a ball mill, sonication and combinations thereof. The highest degree of sugar release of the cellular wall of the yeast Saccharomyces cerevisiae was obtained following homogenization of the cells in a mill with zirconium glass beads. Similar results were obtained after autolysis combined with yeast grinding and sonication, but the time required for these processes was longer than 24 hours. The authors conclude that homogenization in a mill could be the ideal method for the cellular wall recovery process because it allows to eliminate further downstream treatments, such as sonication. Also in this case, the production of undesired by-products deriving from secondary sugar-degrading reactions has not been addressed. Other authors have addressed the problem of determining the content and composition of all sugars present (“whole cell approach”) in a series of yeasts, in particular in Ascomycetes and Basidiomycetes, as reported, for example, by Prillinger H. et al., in “TTze Journal of General and Applied Microbiology" (1993), Vol. 39, Issue 1, pp. 1-34, DOI: 10.2323/jgam.39.1. Despite the greater complexity of the problem addressed, i.e. the determination of not only cellular wall sugars but also endocellular sugars, the authors assumed that the method developed for the determination of the sugars in plant fibres, based on a treatment with sulphuric acid (H2SO4) reported in the article by Selvendran R. R. et al., in “Analytical Biochemistry" (1979), Vol. 96, Issue 2, pp. 282-292, DOI: 10.1016/0003- 2697(79)90583-9 mentioned above, was the most suitable and they followed the methodology without variations.

Some authors have studied the variation in the content and type of free sugars contained in yeast cells (endocellular sugars) during fermentation processes. Indeed, the commercial exploitation of various microorganisms to convert sugars into higher value-added chemicals is very important for several sectors such as agriculture, in the pharmaceutical and in the food industries. Yeasts are the most widely used microorganisms in the industry and are particularly important in the production of beer and wine. Therefore, the analysis of the endocellular sugar content of cells is of great scientific interest in the field of quality control of industrial processes. The variation in carbohydrate content in yeasts suggests that sugars play an important role during the yeast life cycle. The control and quantification of sugars is important because their concentrations are correlated to the metabolism of yeasts and their performance in the aforesaid industrial processes. Normally, the analysis involves lysing yeasts by acid treatment and analysing, in this case, not the cellular walls, but the sugars released in the aqueous phase obtained after cell lysis.

Recently, new methods for the characterization and quantification of sugars in yeasts have been proposed. For example, a quick and simple method to monitor variations in the carbohydrate composition of Saccharomyces cerevisiae. the yeast used for producing bread and similar products during fermentation, has been developed using spectroscopic analysis at mid-infrared (“mid-IR”) wavelengths as reported by Plata M. R. et al., in “ Analytical and Bioanalytical Chemistry” (2013), Vol. 405, pp. 8241-8250, DOI: 10.1007/s00216-013-7239-9). However, the method is limited by the incomplete type of sugars it allows to quantify: some are part of the cellular wall, others are endocellular sugars. In particular, the authors studied mannan, which is a cellular wall polysaccharide composed of mannose units, and intracellular carbohydrates, in particular trehalose, which is a glucose dimer, and glycogen, which is a polymer composed of glucose units. Spectroscopic analysis at mid-infrared (“mid-IR”) wavelengths was validated by determining sugars via ionexchange liquid chromatography, preceded by acid hydrolysis of the sugars (mannan, glycogen, trehalose) mentioned above. Furthermore, in order to obtain reference results for the aforesaid spectroscopic analysis, it was necessary to subject the yeast suspension to a lysis process using a bead mill. The set-up for the hydrolysis of the above reported sugars was particularly complex: mannan was hydrolysed in a hydrochloric acid (HC1) solution, while glycogen was hydrolysed enzymatically. To summarise, the method described is not attractive for industrial applications (neither at an analytical level nor as a teaching for industrial sugar production) due to the limited number of sugars it allows to quantify and to the need to periodically repeat the calibration by traditional methods, i.e. cell lysis, subsequent hydrolysis of mannose and glycogen by acid or enzymatic hydrolysis and subsequent analysis via ion exchange liquid chromatography.

Processes for obtaining and extracting lipids present in microorganisms are also known in the art.

For example, International Patent Application WO 2015/193547 relates to a method for the recovery of microbial lipids from oleaginous yeast biomass, said method comprising the steps of:

(i) providing an aqueous suspension comprising a fermentation broth containing oleaginous yeast biomass;

(ii) subjecting said aqueous suspension to hydrothermal treatment at a temperature of at least 160°C, for a time comprised between 1 second and 360 minutes, at a pressure of more than 5 bar;

(iii) subjecting the hydrothermally treated aqueous suspension to a separation step to obtain a liquid fraction and a yeast biomass fraction; (iv) subjecting the yeast biomass fraction to a drying step to obtain a dried yeast biomass fraction;

(v) subjecting the dried biomass fraction to a liquid solvent extraction step to produce a liquid fraction comprising microbial lipids and a solid fraction comprising the residual yeast biomass fraction;

(vi) recovering microbial lipids from the liquid fraction of step (v);

(vii) optionally, isolating the residual fraction of yeast biomass from the product of step (v).

The obtained lipids are said to contain low amounts of phosphorus and metals, a feature which is essential for their use in fuel production and which reduces the need for further purification steps.

International Patent Application WO 2001/053512 relates to a process for obtaining lipids from microorganisms comprising:

(a) lysing the cells of the microorganisms to produce a mixture of lysed cells;

(b) treating said mixture of lysed cells to produce a two-phase mixture comprising a heavy phase and a light phase, wherein said heavy phase comprises an aqueous solution and said light phase comprises lipids;

(c) separating said heavy phase from said light phase; and

(d) obtaining lipids from said light phase.

Lysis is carried out by heating the cells of the microorganism to at least 50 C in the presence of a base and/or chelating agent, and lipid extraction is carried out in the absence of solvents.

Patent application EP 2450425 relates to a method for the recovery of lipids from a microbial biomass comprising the steps of:

(i) providing a moist microbial biomass containing lipids to be extracted without breaking the cellular walls of the biomass;

(ii) subjecting said moist microbial biomass to extraction in the presence of a liquid extracting agent, at a high temperature of at least 170°C and at high pressure, where the combination of temperature and pressure is such that said lipids come into contact with said extracting agent; and

(iii) subsequently recovering the extracted lipids from or with said extracting agent. Said extracting agent is a non-polar organic solvent that is essentially, preferably totally, immiscible with water.

Dai C.-C. et al., in ''African Journal of Biotechnology" (2007), Vol. 6(18), pp. 2130-2134, describe the production of biodiesel from the oleaginous yeast Rhodotorula glutinis having the ability to assimilate xylose. In particular, a lignocellulosic biomass is ground and subjected to acid hydrolysis in the presence of sulphuric acid and the sugars thus obtained are used as carbon sources in a fermentation process in the presence of a Rhodotorula glutinis strain, previously selected, able to also use pentose, in particular xylose, in order to obtain oils which are subsequently extracted by Soxhlet extraction and subjected to transesterification in order to obtain biodiesel.

In the prior art mentioned above it is highlighted that the production of sugars from biomass including at least one polysaccharide and the production of sugars and lipids from microorganisms are distinct processes.

The Applicant has therefore faced the problem of finding a process for simultaneous production of sugars from biomass including at least one polysaccharide and sugars and lipids from microorganisms.

The Applicant has now surprisingly found that the simultaneous production of sugars from biomass including at least one polysaccharide and of sugars and lipids from microorganisms, can be obtained through a process comprising subjecting the fermentation broth deriving from the fermentation of microorganisms and said biomass comprising at least one polysaccharide to hydrolysis in the presence of at least one organic acid having from 1 to 6 carbon atoms, preferably from 1 to 3 carbon atoms.

Numerous advantages are obtained by said process.

For example, said process allows to obtain a high conversion of the hemicellulosic component and, consequently, a high yield of monomeric sugars having 5 carbon atoms (C5), such as xylose, arabinose (i.e. a yield of monomeric sugars having 5 carbon atoms (C5) greater than or equal to 95%, said yield being calculated as reported below), deriving from the acid hydrolysis of said biomass. Furthermore, the possibility of obtaining a high conversion of the hemicellulosic component, and consequently a high yield of monomeric sugars having 5 carbon atoms (C5), such as xylose, arabinose, and simultaneously obtaining a high recovery of monomeric sugars having 6 carbon atoms (C6) from the fermentation broth deriving from the fermentation of microorganisms, allows sending to subsequent fermentations solutions of sugars particularly rich in monomeric sugars having 5 carbon atoms (C5) and monomeric sugars having 6 carbon atoms (C6), thus avoiding the addition of monomeric sugars having 6 carbon atoms (C6) from streams deriving from different bioprocesses (for example, solutions of monomeric sugars having 6 carbon atoms (C6) deriving from the enzymatic hydrolysis of cellulose) and, consequently, to optimize said fermentation processes. It is known, in fact, that the microorganisms used in fermentation give a fermented biomass having different characteristics in terms, for example, of accumulation of intermediate products, accumulation of undesired metabolic products, depending on the sugars supplied in the feed. It is also known that the microorganisms used in fermentation processes are susceptible to feeding: for example, some strains of microorganisms do not metabolize an excessive amount of monomeric sugars having 5 carbon atoms (C5). It is therefore extremely advantageous to have a sugar solution particularly rich in both monomeric sugars having 5 carbon atoms (C5) and monomeric sugars having 6 carbon atoms (C6), so as to be able to destine said sugar solution to numerous and different fermentation processes and, consequently, optimize said fermentation processes thanks to greater flexibility with respect to the dietary needs of the different strains of microorganisms.

Furthermore, said process allows to obtain a simultaneous high recovery of monomeric sugars having 5 carbon atoms (C5) and monomeric sugars having 6 carbon atoms (C6) in aqueous phase, recovered respectively from the hemicellulose of the biomass and from the fermentation broth deriving from the fermentation of microorganisms, with a simultaneous reduced production of undesired by-products [e.g., furfural (F), hydroxymethylfurfural (HMF)] deriving from degradation reactions of the sugars which, as mentioned above, act as inhibitors in the processes in which the sugars can be conveniently recycled (for example, the fermentation itself). Said process makes it possible in fact to obtain an aqueous phase comprising sugars that can be recycled, for example to subsequent fermentations, directly without the need for further purification and/or concentration steps with advantages both from the point of view of process times and from the economic point of view. Consequently, the sugars obtained from the aforesaid process can be advantageously used as carbon sources in fermentation processes for the production of alcohols (e.g., ethanol, butanol), lipids, diols (e.g., 1,3 -propanediol, 1,3- butanediol, 1,4-butanediol, 2, 3 -butanediol), bioplastics (e.g., polyhydroxyalkanoates), or in chemical synthesis processes for the production of other intermediates or chemicals. Said alcohols and lipids, can in turn be advantageously used in the production of biofuels (e.g., biodiesel or “Green Diesel”), which can be used as such, or in admixture with other automotive fuels, while said diols can be used in the production of products such as, for example, biobutadiene which can in turn be used in the production of rubbers (e.g., polybutadiene or copolymers thereof).

Moreover, said process makes it possible to obtain, thanks to the simultaneous lysis of the cellular walls of the microorganisms used, high-quality lipids with a high yield, and an amount of Free Fatty Acids (FFA) of less than 2 per cent, a fundamental characteristic that determines the commercial value of oils and fats. In particular, in case of using oleaginous microorganisms, a lipid yield greater than or equal to 95% is obtained, said lipid yield being calculated as reported below. The lipids obtained can be advantageously used, for example, for the production of biofuels that can be used in diesel or aviation engines, or said lipids can be subjected to metathesis processes in order to obtain biofuel precursors, waxes, plastics, cosmetics, and personal care items. The aforesaid process, thanks to the integration of two processes, the production of sugars from biomass including at least one polysaccharide and the production of lipids from the fermentation broth deriving from the fermentation of microorganisms, is particularly useful in the case of a biorefinery. In fact, the union of two unitary operations normally carried out separately, i.e. the simultaneous production of sugars from biomass and the production of sugars and lipids from the fermentation broth deriving from the fermentation of microorganisms, in a single reactor facilitates and makes the process more economical, without negatively affecting the yields of said sugars and lipids.

Furthermore, the aforesaid process can be carried out in the presence of high biomass loads: for example, it is possible to load in said reactor an amount of total biomass comprised between 30% by weight and 40% by weight, with respect to the aqueous component (for example 13% by weight of biomass including at least one polysaccharide and 20% by weight of cellular biomass deriving from the fermentation of microorganisms) while maintaining in any case an aqueous suspension capable of being treated with a generic stirred reactor, thus minimizing the energy required for stirring.

It is therefore an object of the present invention a process for simultaneous production of sugars from biomass including at least one polysaccharide and of sugars and lipids from a fermentation broth, comprising the following steps:

(a) feeding at least one microorganism, preferably an oleaginous microorganism, to a fermentation device obtaining a fermentation broth comprising an aqueous suspension of cellular biomass comprising sugars and lipids;

(b) optionally, at the end of the fermentation, subjecting the fermentation broth obtained in said step (a) to concentration obtaining a fermentation broth comprising an aqueous suspension of concentrated cellular biomass comprising sugars and lipids;

(c) subjecting the fermentation broth obtained in said step (a) or the fermentation broth comprising an aqueous suspension of concentrated cellular biomass comprising sugars and lipids obtained in said step (b) and said biomass comprising at least one polysaccharide to hydrolysis in the presence of at least one organic acid having from 1 to 6 carbon atoms, preferably from 1 to 3 carbon atoms, obtaining a reaction mixture comprising a solid phase, an oily phase and an aqueous phase, said hydrolysis being carried out at a pH comprised between 0.6 and 1.6, preferably comprised between 0.9 and 1.3;

(d) subjecting the reaction mixture obtained in said step (c) to separation obtaining a solid phase comprising lignin, cellulose and cellular debris, an oily phase comprising lipids and an aqueous phase comprising sugars.

For the purpose of the present description and of the following claims, the definitions of the numerical ranges always comprise the extreme values unless otherwise specified.

For the purpose of the present description and of the following claims, the term “comprising” also includes the terms “essentially consisting of’ or “consisting of’.

For the purpose of the present description and of the following claims, the term “monomeric sugar having 5 carbon atoms (C5)” means a pentose sugar, or more simply a pentose, which is a monosaccharide glucide composed of five carbon atoms having the chemical formula C5H10O5.

For the purpose of the present description and of the following claims, the term “monomeric sugar having 6 carbon atoms (C6)” means a hexose sugar, or more simply a hexose, which is a monosaccharide glucide composed of six carbon atoms having the chemical formula CeHnOe.

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

In accordance with a preferred embodiment of the present invention, said biomass including at least one polysaccharide is a lignocellulosic biomass. As already mentioned above, lignocellulosic biomass comprises three components: hemicellulose, cellulose and lignin.

In accordance with a preferred embodiment of the present invention, said biomass including at least one polysaccharide can be selected, for example, from: scraps, residues and waste of products deriving from crops expressly cultivated for energy use such as, for example, miscanthus, panicum (Panicum virgatum), common cane (Arundo donctx): scraps, residues and waste of products deriving from agriculture such as, for example, guayule (Parthenium argentatum), corn, soybean, cotton, linseed, rapeseed, sugar cane, palm oil, poplar, alder, birch, residues deriving from the oil palm tree [for example, palm leaf, trunks, leaf midribs, empty palm oil fruits (EFB - Empty Fruit Bunches) ], wheat straw, rice straw, corn stalks, cotton stalks, sorghum, bagasse [for example, sugar cane bagasse, guayule (Parthenium argentatum) bagasse]; scraps, residues and waste of products deriving from forestation or forestry comprising scraps, residues and waste resulting from such products or their processing; scraps from agri-food products intended for human consumption or animal husbandry; residues, not chemically treated, from the paper industry; waste coming from the separate collection of solid urban waste (for example urban waste of vegetal origin, paper); algae such as, for example, microalgae or macroalgae, in particular macroalgae.

In accordance with a particularly preferred embodiment of the present invention, said biomass including at least one polysaccharide can be selected, for example, from scraps, residues and waste deriving from miscanthus, panicum (Panicum virgatum), common cane (Arundo donctx). guayule (Parthenium argentatum), poplar, alder, birch, residues deriving from the oil palm tree [such as, for example, leaf mibrids, empty palm oil fruits (EFB - “Empty Fruit Bunches”)], wheat straw, rice straw, com stalks, cotton stalks, sorghum, sugar cane bagasse, guayule (Parthenium argentatum) bagasse; preferably from scraps, residues and wastes deriving from guayule (Parthenium argentatum), more preferably is guayule (Parthenium argentatum) bagasse.

For the purpose of the present description and of the following claims, the term “bagasse” means the residual portion of plant material deriving from the extraction processes to which sugarcane plants and guayule plants can be subjected. Bagasse can include, in addition to lignin and polysaccharides (e.g., cellulose and hemicellulose), even small amounts of non-plant material (e.g., soil, sand, etc.) typically associated with plant roots and deriving from plant growing soil.

Preferably, said biomass including at least one polysaccharide can be subjected to a preliminary grinding process before being used in the hydrolysis step (c). Preferably, said lignocellulosic biomass can be ground up to particles having a diameter comprised between 0.1 mm and 10 mm, more preferably comprised between 0.5 mm and 4 mm. Particles having a diameter of less than 1 mm are particularly preferred.

In accordance with a further embodiment of the present invention, said biomass including at least one polysaccharide can be selected, for example, from sugar processing scraps, in particular from sugar beet or sugar cane, such as molasses.

For the purpose of the present invention said step (a) can be carried out according to methods known in the art.

Preferably, said microorganism, before being fed to the fermentation device in step (a), can be grown in a suitable culture medium obtaining an inoculum. For this purpose, said microorganism can be fed to a fermentation device obtaining a fermentation broth (inoculum). Preferably, in said fermentation device, fermentation can be carried out: at a temperature comprised between 20°C and 40 C, preferably comprised between 25 C and 35 C; and/or for a time comprised between 10 hours and 36 hours, preferably comprised between 12 hours and 26 hours; and/or at a pH comprised between 4.5 and 7.0, preferably comprised between 5.0 and 6.5.

In order to maintain the pH in the desired ranges, an aqueous solution of at least one inorganic base such as, for example, sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide, ammonium hydroxide, preferably sodium hydroxide, ammonium hydroxide, or mixtures thereof, or of at least one inorganic acid such as, for example, phosphoric acid, sulphuric acid, preferably sulphuric acid, or mixtures thereof, can be added to the culture medium used for the fermentation in such an amount as to obtain the desired pH.

When the microorganism has reached a concentration higher than or equal to 8 g/L, preferably comprised between 10 g/L and 20 g/L, said fermentation broth (inoculum) is fed to the fermentation device used in step (a).

In accordance with a preferred embodiment of the present invention, in said step (a), in said fermentation device, fermentation can be carried out: at a temperature comprised between 20°C and 40°C, preferably comprised between 25 C and 35 C; and/or for a time comprised between 2 days and 10 days, preferably comprised between 3 days and 8 days; and/or at a pH comprised between 4.5 and 7.0, preferably comprised between 5.0 and 6.5.

In order to maintain the pH in the desired ranges, an aqueous solution of at least one inorganic base such as, for example, sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide, ammonium hydroxide, preferably sodium hydroxide, ammonium hydroxide, or mixtures thereof, or of at least one inorganic acid such as, for example, phosphoric acid, sulphuric acid, preferably sulphuric acid, or mixtures thereof, may be added to the culture medium used for the fermentation in such an amount as to obtain the desired pH.

For the purpose of the present invention, said fermentation can be carried out in one or more steps, in discontinuous mode (“feed-batch fermentation”), repeated discontinuous mode (“repeated feed-batch fermentation”), semi-continuous, or continuous. In the case of semi-continuous or continuous culture fermentation, there is a continuous addition of nutrients and a possible recirculation of cellular biomass after elimination of the exhausted fermentation broth by, for example, microfiltration.

Said fermentation can be advantageously carried out in fermentation devices known in the art, in the presence of culture media usually used for the purpose comprising, in addition to sugars, various nutrients such as, for example, nitrogen, potassium phosphate, magnesium, salts, vitamins, trace elements.

For the purpose of the present invention, said fermentation devices can be selected from reactors known in the art such as, for example, autoclaves, continuous biomass feed “slurry” reactors (CSTR - “Continuous Stirred-Tank Reactor”), extruders. Preferably, said reactor is selected from among continuous biomass feed “slurry” reactors (CSTR - “Continuous Stirred-Tank Reactor”).

At the end of fermentation, in order to deactivate the lipolytic enzymes (e.g., lipase), said fermentation broth can be subjected to heat treatment. Said heat treatment can be carried out at a temperature comprised between 70°C and 120 C, preferably comprised between 75°C and 110°C, for a time comprised between 5 minutes and 3 hours, preferably comprised between 30 minutes and 2.5 hours.

In accordance with a preferred embodiment of the present invention, in said step (a), said oleaginous microorganism can be selected, for example, from oleaginous yeasts such as, for example: Rhodotorula glutinis, Rhodotorula gracilis, Rhodotorula graminis, Lypomices starkeyi, Lypomices lipofer, Trigonopsis variabilis, Candida kefyr, Candida curvala. Candida lipolytica, Torulopsis sp., Pichia stipitis, Trichosporon cacaoliposimilis, Rhodosporidium sp. , Cryptococcus ciirvalus. Trichosporon oleaginosus.

In accordance with a particularly preferred embodiment of the present invention, in said step (a), said oleaginous microorganism can be selected from Rhodosporidium sp., more preferably is Rhodosporidium azoricum DSM 29495 (mutant described in international patent application WO 2016/108185).

In accordance with a preferred embodiment of the present invention, said step (b) can be carried out by centrifugation, filtration, tangential microfiltration, preferably by tangential microfiltration.

In accordance with a preferred embodiment of the present invention, in said step (c), said at least one organic acid can be selected, for example, from alkyl sulfonic acids having general formula (I):

R-SO3H (I) wherein R represents a Ci-Ce alkyl group, preferably C1-C3, linear or branched.

In accordance with a preferred embodiment of the present invention, in said step (c), said at least one organic acid is methanesulphonic acid (CH3-SO3H).

In order to achieve the desired pH value said at least one organic acid can be used in a mixture with at least one inorganic acid.

In accordance with a preferred embodiment of the present invention, in said step (c), said at least one organic acid is used in a mixture with at least one inorganic acid.

In accordance with a preferred embodiment of the present invention, in said step (c), said at least one inorganic acid can be selected, for example, from strong inorganic acids such as, for example, hydrochloric acid (HC1), nitric acid (HNO3), sulphuric acid (H2SO4), or mixtures thereof.

In accordance with a preferred embodiment of the present invention, in said mixture said at least one organic acid may be present in an amount comprised between 10% by weight and 90% by weight, preferably comprised between 15% by weight and 85% by weight, even more preferably comprised between 20% by weight and 80% by weight, with respect to the total weight of said mixture. In accordance with a preferred embodiment of the present invention, said step (c) can be carried out at a temperature comprised between 100 C and 180 C, preferably comprised between 130 C and 150°C.

In accordance with a preferred embodiment of the present invention, said step (c) can be carried out for a time comprised between 10 minutes and 2 hours, preferably comprised between 20 minutes and 1 hour.

Before being subjected to step (d), the reaction mixture comprising a solid phase, an aqueous phase and an oily phase, obtained at the end of said step (c), is cooled to room temperature (25°C).

In accordance with a preferred embodiment of the present invention, said separation step (d) can comprise the following steps:

(di) subjecting the reaction mixture comprising a solid phase, an oily phase and an aqueous phase, obtained at the end of said step (c), to extraction in the presence of at least one non-polar organic solvent, obtaining a first mixture comprising:

(i) an organic phase comprising lipids dissolved in solvent (extract);

(ii) an aqueous phase comprising monomeric sugars having 5 carbon atoms (C5), monomeric sugars having 6 carbon atoms (C6), organic acid or a mixture thereof with at least one inorganic acid and traces of unseparated lipids and part of the non-polar organic solvent;

(iii) a solid phase comprising lignin, cellulose and cellular debris;

(d2) subjecting said first mixture to filtration obtaining an organic phase comprising lipids dissolved in solvent (extract) and a second mixture comprising an aqueous phase comprising monomeric sugars having 5 carbon atoms (C5), monomeric sugars having 6 carbon atoms (C6), organic acid or a mixture thereof with at least one inorganic acid, traces of non-separated lipids and part of the non-polar organic solvent and a solid phase comprising lignin, cellulose and cellular debris;

(ds) subjecting said second mixture to filtration or centrifugation, preferably to filtration, obtaining an aqueous phase comprising monomeric sugars having 5 carbon atoms (C5), monomeric sugars having 6 carbon atoms (C6), organic acid or a mixture thereof with at least one inorganic acid, traces of unseparated lipids and part of the non-polar organic solvent (refined) and a solid phase comprising lignin, cellulose and cellular debris.

The solid phase comprising lignin, cellulose and cellular debris obtained at the end of step (ds) can be dehumidified and valorised as fuel, or subjected to enzymatic treatments.

For example, said solid phase comprising lignin, cellulose and cellular debris, can be used in an enzymatic hydrolysis process, in order to hydrolyse the cellulose to glucose. The enzymatic hydrolysis process can be carried out according to techniques known in the art as described, for example, in the American patents US 5,628,830, US 5,916,780 and US 6,090,595, using commercial enzymes such as, for example, Celluclast 1.5L (Novozymes), Econase CE (Rohm Enzymes), Spezyme (Genecor), Novozym 188 (Novozymes), used singly or mixed together. A further solid phase comprising lignin and cellular debris and a further aqueous phase comprising glucose deriving from the hydrolysis of cellulose are obtained from the enzymatic hydrolysis of said solid phase. Said further solid phase and said further aqueous phase can be separated by techniques known in the art such as, for example, filtration, centrifugation. Preferably, said phases are separated by filtration. Said further aqueous phase comprising glucose, can be used as such, or in a mixture with solutions particularly rich of sugars having 5 carbon atoms (C5), as raw material in the fermentation processes. Said further solid phase, comprising lignin and cellular debris, can be exploited as a fuel, for example as a fuel to produce the energy necessary to support biomass treatment processes.

In accordance with a preferred embodiment of the present invention, in said step (di) said non-polar organic solvent can be selected, for example, from cyclohexane, //-hexane, //-heptane, //-octane, /.w-octane, or mixtures thereof; preferably cyclohexane.

In accordance with a preferred embodiment of the present invention, said step (di) can be carried out at a temperature comprised between 20°C and 200 C, preferably at the boiling temperature of the solvent used.

In accordance with a preferred embodiment of the present invention, said step (di) can be carried out in the presence of an amount of a non-polar organic solvent comprised between 1 times and 4 times, preferably comprised between 1 times and 2 times, the volume of said first mixture.

Said first mixture is cooled to room temperature (25°C).

In said step (d2), filtration in order to obtain said organic phase (extract) and said second mixture, can be carried out by filtration, preferably by filtration through a filter with a dip tube.

In said step (ds), filtration in order to obtain said aqueous phase (refined) and said solid phase can be carried out via a Buchner filter.

In order to recover the lipids, said organic phase (i) (extract) is subjected to evaporation, obtaining as a residue an oil comprising lipids and a liquid phase comprising the solvent that can be recycled to the aforesaid extraction [i.e. to said step (di)].

Preferably, the lipids comprised in said organic phase (i) (extract) are triglycerides, more preferably esters of glycerol with fatty acids having from 14 to 24 carbon atoms such as, for example, palmitic acid, stearic acid, oleic acid, a- linoleic acid, in an amount greater than or equal to 80% by weight, preferably greater than or equal to 90% by weight, with respect to the total weight of the lipids. Other lipids that may be present in said organic phase (i) are: phospholipids, monoglycerides, diglycerides, free fatty acids (FFAs) in amounts of less than 2%, or mixtures thereof.

In order to recover the sugars, said aqueous phase (ii) (refined), is subjected to a process comprising:

(d4) subjecting said aqueous phase (ii) (refined) to distillation or evaporation obtaining a second aqueous phase (second refined) comprising monomeric sugars having 6 carbon atoms (C6), organic acid or a mixture thereof with at least one inorganic acid and traces of unseparated lipids, and a liquid phase consisting of two phases:

(iii) a phase consisting of the remaining non-polar organic solvent mixed with the aqueous solution;

(m) a phase consisting of water;

(ds) separating said phase (iii) and (m) by known techniques, for example, decantation

(de) recycling the phase (iii) to the aforesaid extraction [step (di)]; (d7) sending the phase (H2) to water treatment or subsequent valorisations, for example, to fermentation processes of oleaginous microorganisms;

(d8) optionally, subjecting said second aqueous phase (second refined) to separation, preferably by means of ionic resins, in order to recover said at least one organic acid or a mixture thereof with at least one inorganic acid, and a third aqueous phase (third refined) comprising monomeric sugars having 6 carbon atoms (C6) and water;

(d9) recycling said organic acid or a mixture thereof with at least one inorganic acid to step (c).

In accordance with a further embodiment of the present invention, said separation step (d) can comprise the following steps:

(d10) subjecting the reaction mixture comprising a solid phase, an oily phase and an aqueous phase, obtained at the end of said step (c), to filtration obtaining a solid phase comprising lignin, cellulose and cellular debris, and a first mixture comprising an oily phase and an aqueous phase;

(d11) subjecting said first mixture to extraction in the presence of at least one nonpolar organic solvent, obtaining a second mixture comprising:

(i) an organic phase comprising lipids dissolved in solvent (extract);

(ii) an aqueous phase comprising monomeric sugars having 5 carbon atoms (C5), monomeric sugars having 6 carbon atoms (C6), organic acid or a mixture thereof with at least one inorganic acid, traces of unseparated lipids and part of the non-polar organic solvent (refined).

Said step (du) can be carried out as reported above for step (ds).

Said step (dio) can be carried out as reported above for step (di).

Said organic phase (i) (extract) and said aqueous phase (ii) (refined) can be separated by processes known in the art such as, for example, decantation, centrifugation, preferably decantation.

The recovery of lipids from said organic phase (i) (extract) and the recovery of sugars from said aqueous phase (ii) (refined) are carried out as described above.

The present invention will now be illustrated in greater detail by an embodiment with reference to Figure 1 reported below.

Figure 1 schematically shows an embodiment of the process object of the present invention. For this purpose, at least one microorganism, preferably an oleaginous microorganism, is fed to a fermentation device (1), obtaining a fermentation broth (2) comprising an aqueous suspension of cellular biomass comprising sugars and lipids. A biomass including at least one polysaccharide (3) is added to said fermentation broth (2) and the whole is subjected to hydrolysis (4) in the presence of at least one organic acid, preferably methanesulfonic acid (CHa- SO3H), obtaining a reaction mixture comprising a solid phase, an aqueous phase and an oily phase (not shown in Figure 1). Said reaction mixture is subjected to extraction (5) obtaining a first mixture (6) comprising:

(i) an organic phase comprising lipids dissolved in solvent (extract);

(ii) an aqueous phase comprising monomeric sugars having 5 carbon atoms (C5), monomeric sugars having 6 carbon atoms (C6), organic acid or a mixture thereof with at least one inorganic acid, traces of unseparated lipids and part of the non-polar organic solvent;

(iii) a solid phase comprising lignin, cellulose and cellular debris.

Said first mixture (6) is subjected to filtration (7), preferably through a filter with a dip tube, obtaining an organic phase (8) comprising lipids dissolved in solvent (extract) and a second mixture (9) comprising an aqueous phase comprising monomeric sugars having 5 carbon atoms (C5), monomeric sugars having 6 carbon atoms (C6), organic acid or a mixture thereof with at least one inorganic acid, traces of non-separated lipids and part of the non-polar organic solvent and a solid phase comprising lignin, cellulose and cellular debris.

Said second mixture (9) is subjected to filtration (10), preferably through Buchner filter, obtaining an aqueous phase (11) comprising monomeric sugars having 5 carbon atoms (C5), monomeric sugars having 6 carbon atoms (C6), organic acid or a mixture thereof with at least one inorganic acid, traces of unseparated lipids and part of the non-polar organic solvent (refined) and a solid phase (12) comprising lignin, cellulose and cellular debris.

Said solid phase (12) comprising lignin, cellulose and cellular debris can be dehumidified and valorised as fuel, or subjected to enzymatic treatments as reported above (not shown in Figure 1).

Said organic phase (8) comprising lipids dissolved in solvent (extract) is subjected to evaporation (13), obtaining as a residue an oil comprising lipids (14) and a liquid phase (15) comprising the solvent that can be recycled to the aforesaid extraction (5).

In order to recover the sugars, said aqueous phase (ii) (refined) (11) is subjected to a process comprising:

(d4) subjecting said aqueous phase (ii) (refined) (11) to evaporation (16) obtaining a second aqueous phase (second refined) (17) comprising monomeric sugars having 6 carbon atoms (C6), organic acid or a mixture thereof with at least one inorganic acid and traces of unseparated lipids and a liquid phase (18) consisting of two phases:

(iii) a phase consisting of water.

Said phase (iii) and (H2) can be separated by known techniques, for example, decantation, obtaining a phase (iii) that can be recycled to the aforesaid extraction and a phase (H2) that can be sent to water treatment or subsequent valorisations, for example to fermentation processes of oleaginous microorganisms (not shown in Figure 1).

In order to better understand the present invention and to put it into practice, some illustrative and non-limiting examples thereof are reported below. Methodologies of analysis and characterization

The analysis and characterization methodologies reported below were used. pH determination

The pH was determined by a Metrohm 781 pH meter/Ion meter. Analysis of the starting biomass

The starting biomass (i.e. guayule bagasse used in the examples below reported) was analysed using the Van Soest fibrous fractions system for the quantification of the constituents of the cellular walls, in particular hemicellulose, cellulose and lignin, operating as described in Van Soest P.J. et al., in “ Journal of Association of Official Analytical Chemistry", 1967, Vol. 50, Issue 1, pp. 50-55 DOI: 10.1093/jaoac/50.1.50.

Lipid analysis The quantitative analysis of the obtained lipids was carried out using a colorimetric method (Total lipids - sulpho-phospho vanillin kit marketed by Spinreact S.S.U., Ctra. Santa Colma, 7 E-17176 St. Steve d’en Bas (GI), Spain) operating as described by Mishra S. K.et al. in “Bioresource Technology" (2014), Vol. 155, pp. 330-333, DOI: 10.1016/j.biortech.2013.12.077.

The aforesaid analysis was also confirmed by gravimetric method operating as described by Sperry W. M. et al. in “The Journal of Biological Chemistry" (1955), Vol. 123, Issue 1, pp. 69-76, DOI: 10.1016/80021-9258(18)71045-4.

Three complementary gas chromatographic methods have also been developed to characterize and quantify the lipids and the C12-C24 acids: one involves quantifying the lipids after treatment with a silanizing agent [BSTFA (N,O-bis(trimethylsilyl)trifluoroacetamide)], the second involves evaluating only the free fatty acids (in particular the FFA fraction = “Free Fatty Acids”, i.e. the sum of C16 and Cl8 free acids) after subjecting the sample to acid esterification, the third method involves using a polar column particularly suitable for better separation of unsaturated homologues. In all cases, the analysis is based on gas chromatographic separation combined with a flame ionization detector (FID). The quantification method was carried out by internal standardization operating as reported below.

Method A

The samples were prepared as follows: about 10 mg -15 mg of the sample to be analysed, weighed accurately, were dissolved in 10 ml of methylene chloride (Merck). Subsequently, to 1 ml of the solution obtained, after evaporation under nitrogen flow, were added: 0.2 ml of pyridine (Merck), 0.1 ml of N,O- bis(trimethylsilyl)trifluoroacetamide (BSTFA) (Merck), 50 pl of an internal standard comprising n-tetradecane [i.e. of a stock solution previously prepared by dissolving 40.77 mg of n-tetradecane (Merck) in 10 ml of hexane (Merck)] and 25 pl (microlitres) of an internal standard comprising tricaprin [i.e. of a stock solution containing 8000 ppm of tricaprin (Merck) in pyridine (Merck)]: the whole was heated to 70°C, for 40 minutes, and finally diluted to about 1 ml with methylene chloride (Merck) for subsequent analysis.

The chromatographic analysis was carried out using the following operating conditions: instrument: gas chromatograph GC 7890B Agilent, SupelcoPetrocol EX2887 capillary column (5 m x 0.53 mm); temperature program: initial temperature: 50 C, isothermal for 2 minutes, 10°C increase/min up to the temperature of 350°C; transport gas: helium 0.68 psi (40 cm/sec at constant flow); detector: flame ionization detector (FID), temperature 350 C, air: 400 ml/min, hydrogen: 35 ml/min; injector: cool on column track oven; injection volume: 1 μl.

Method B

The samples, before analysis, were treated with methanol, in an acidic environment, so as to esterify the acids.

To this end, 10 ml of toluene (Merck) and 20 ml of a 1% solution of sulphuric acid (Merck) in methanol (Merck) were added to about 50 mg of sample: the whole was heated to reflux for 4 hours. Subsequently, 50 ml of a 5% sodium chloride solution (Merck) in water was added and two extractions were carried out with 50 ml of hexane (Merck) each. The extracts obtained were combined and added with 40 ml of 2% sodium bicarbonate (Merck) in water obtaining an aqueous phase and an organic phase: the organic phase was separated by decantation through separating funnel. 50 pl of an internal standard comprising n-tetradecane [i.e. of a stock solution previously prepared by dissolving 40.77 mg of n-tetradecane (Merck) in 10 ml of hexane (Merck)] were added to 1 ml of the organic phase obtained: the sample obtained was subjected to gas chromatography analysis.

The chromatographic analysis was carried out using the following operating conditions: instrument: gas chromatograph GC 7890B Agilent, MDN column (30 m x 0.25 mm); temperature program: initial temperature: 50 C, isothermal for 2 minutes, 10°C increase/min up to the temperature of 350°C, isothermal for 20 minutes; transport gas: helium 16.53 psi (40 cm/sec at constant flow); detector: flame ionization detector (FID), temperature 280°C, air: 400 ml/min, hydrogen: 35 ml/min; injector: “splitless” (1 minute), temperature 320 C; injection volume: 1 ml.

Method C

The samples to be analysed were prepared as described above in Method B.

The chromatographic analysis was carried out using the following operating conditions: instrument: gas chromatograph GC 7890B Agilent, Supelcowax column (30 m x 0.32 mm); temperature program: initial temperature: 50 C, isothermal for 2 minutes, 10°C increase/min up to the temperature of 275 C, isothermal for 5 minutes; transport gas: helium 9.54 psi (40 cm/sec at constant flow); detector: flame ionization detector (FID), temperature 280 C, air: 400 ml/min, hydrogen: 35 ml/min; injector: “splitless” (1 minute), temperature 280 C; injection volume: 1 ml.

Calculation of lipid recovery yield

The yield was expressed, based on the analytical results (i.e. the lipid analysis carried out as described above), as the percentage ratio of the recovered oil with respect to the total amount of lipids in the starting fermentation broth, according to the following formula:

Yield = oil/lipids* 100 wherein: oil = oil recovered at the end of the process (g); lipids = lipids present in the starting fermentation broth (g).

Analysis of the compounds present in the aqueous phase

Analyses of the sugars and by-products, i.e. hydroxymethylfurfural (HMF), were carried out on the second refined, by liquid chromatography using two complementary methods. The instrumentation used and the operating conditions are reported below.

Method I: instrument: HPLC (“High Performance Liquid Chromatography”) consisting of pump, autosampler, oven, refractive index and diode array detector (RI);

Agilent Metacarb 67H 300x6.5 mm column, with similar pre-column; pump flow: 0.8 mL/min (5 rnM sulphuric acid); injection volume: 20 pL; oven temperature and column: 45 C; diode array detectors (RI) temperature: 35°C; diode array detectors (RI) wavelengths: 210 nm and 280 nm; analysis time: 35 minutes.

Method II: instrument: HPLC (“High Performance Liquid Chromatography”) consisting of pump, autosampler, oven, refractive index and diode array detector (RI);

Biorad Aminex HPX 87P 300x7.8 mm column, with cation/anion exchange pre-columns; pump flow: 0.6 mL/min (ultrapure water); injection volume: 10 μL; oven temperature and column: 65°C; diode array detectors (RI) temperature: 35°C; diode array detectors (RI) wavelengths: 210 nm and 280 nm; analysis time: 50 minutes.

Calculation of the degradation of monomeric sugars having 6 carbon atoms (C6) and monomeric sugars having 5 carbon atoms (C5) to undesired products

The carbohydrates contained in the microorganisms used are present as polymers or oligomers consisting of monomeric sugars having 6 carbon atoms (C6). Glucose, mannose and galactose have the molecular formula CeHnCL with a molecular weight of 180. However, during the undesired degradation processes of said sugars, hydroxymethylfurfural (HMF) is formed. The molecular weight of hydroxymethylfurfural (HMF), equal to 126, was therefore used to calculate the degradation ratio of the monomeric sugars having 6 carbon atoms (C6), defined below. Thus, from 180 g of monomeric sugars having 6 carbon atoms (C6), it is possible to obtain 126 g of hydroxymethylfurfural (HMF). The degradation ratio was then calculated accordingly, as in the formula reported below, using the calculation expedients reported by Dhepe P. L. et al., in “Green Chemistry" (2010), Vol. 12, pp. 2153-2156, DOI: 10.1039/C004128A.

In order to effectively express the production of by-products from monomeric sugars having 6 carbon atoms (C6), i.e. hydroxymethylfurfural (HMF), the degradation ratio was calculated according to the following formula:

C6 sugar degradation ratio (%) = [(HMF/126*180)/(C6 sugars + HMF/126*180)]*100 wherein:

C6 sugars = monomeric sugars having 6 carbon atoms (C6) (g/1) present in the second refined;

HMF = hydroxymethylfurfural (g/1) present in the second refined.

Hemicellulose is a polymer consisting mainly of monomeric sugars having 5 carbon atoms, in particular xylose and arabinose. Xylose and arabinose have the molecular formula C5H10O5 with a molecular weight of 150. However, during the formation of hemicellulose from the xylose and arabinose units there is loss of water (H2O) with molecular weight equal to 18 (like in the hydrolysis reaction of hemicellulose water is added and xylose and arabinose are formed as products). For this reason, 18 was subtracted from 150 and 132 was obtained as the molecular weight of hemicellulose (CsHsO^n for the purpose of yield calculation. The molecular weight of furfural (F), equal to 96, was used for the calculation of the degradation to furfural (F). Then, from 132 g of hemicellulose, it is possible to obtain 150 g of xylose and arabinose and 96 g of furfural (F). The yield was then calculated accordingly, as in the formulas reported below.

C5 yield (%) = (xylose + arabinose)/(hemicellulose*150/132)*100 wherein: xylose = xylose (g) obtained from the process; arabinose = arabinose (g) obtained from the process; hemicellulose = hemicellulose (g) contained in the starting biomass.

In order to effectively express the degradation of C5 sugars to furfural (F), an undesired by-product deriving from consecutive reactions of xylose and arabinose, the degradation ratio was calculated according to the following formula:

C5 degradation ratio (%) = (F*150/96)/(xylose + arabinose + F*150/96)*100 wherein: xylose = xylose (g) present in the aqueous phase at the end of the process; arabinose = arabinose (g) present in the aqueous phase at the end of the process;

F = furfural (g) present in the aqueous phase at the end of the process.

EXAMPLE 1

Preparation oi Rhodosporidium azoricum DSM 29495 inoculum

The inoculum (i.e. first fermentation broth) was prepared using 5 litres of an aqueous solution containing glucose 50 g/L, yeast extract 2 g/L, ammonium sulphate 5 g/L, KH₂PO₄ 1 g/L, MgSO₄-7H₂O 0.05 g/L, NaCl 0.01 g/L, CaCl₂-2H₂O 0.01 g/L, placed in a 7-litre fermenter, equipped with a stirrer, the pH of the mixture obtained was brought to 5 by adding a few drops of potassium hydroxide (KOH) 2.5 M. The mixture obtained was sterilized in an autoclave at 120 C, for 20 minutes. At the end of sterilization, the mixture obtained was brought to room temperature (25°C) and inoculated with Rhodosporidium azoricum DSM 29495 cells, which were allowed to grow, for 24 hours, at 30°C, under stirring (600 rpm), insufflating 1 wm of air (normal volume over culture volume per minute), until an initial fermentation broth with a concentration of oleaginous cellular biomass equal to 2% by weight (dry weight) was obtained.

EXAMPLE 2

Fermentation oi Rhodosporidium azoricum DSM 29495 [feed-batch]

The fermentation test with Rhodosporidium azoricum DSM 29495 cells was carried out in feed-batch mode in a 200-litre fermenter, operating under the following conditions:

100 g/L of glucose;

2 g/1 of yeast extract;

5 g/1 of com steep solid;

5 g/L of (NH₄)₂SO₄;

6 g/L of KH₂PO₃;

0.3 g/L MgSO₄-7H₂O;

0.06 g/L NaCl;

0.06 g/L CaCl₂-2H₂O; fed air: flow equal to 1 vvm; operating pH of 5 maintained by adding a few drops of a 5 M potassium hydroxide (KOH) solution when necessary; stirring equal to 200 rpm - 900 rpm, modulated with the air flow so as to maintain the oxygen concentration (O2) above 30%; initial volume: 80 litres; inoculum of Rhodosporidium azoricum DSM 29495 (i.e. first fermentation broth) obtained as described in Example 1, diluted at 10% (v/v) with the culture medium used for fermentation, so that fermentation starts with an oleaginous cellular biomass concentration equal to 0.06% by weight (dry weight).

Fermentation was carried out in batch mode for the first 24 hours, obtaining a fermentation broth with an oleaginous cellular biomass concentration equal to 3.5% by weight (dry weight) and a residual sugar concentration of 30 g/L.

Subsequently, the initial concentrations of yeast extract and ammonium sulphate (2 g/L and 5 g/L respectively) were restored to the fermentation broth and the administration of glucose was operated in a feed-batch mode in order to maintain a constant concentration at 30 g/L in the fermentation reactor.

At the end of the fermentation, after 165 hours, a second fermentation broth was obtained with an oleaginous cellular biomass concentration equal to 8.4% by weight (dry weight) with respect to the total amount of oleaginous cellular biomass obtained. Said second fermentation broth was subjected to heat treatment (pasteurization), at 80°C, for 2 hours, in order to inhibit lipase activity.

Subsequently, said second fermentation broth was concentrated by tangential microfiltration in a P19 HAR plant on P1960 ceramic membrane with 0.2 micron porosity. The 1020 mm-long filter membrane consisted of a module of 19 channels with 6 mm diameter for a total filter surface area of 0.36 m². For this purpose, 135.7 kg of said second fermentation broth were loaded into said system and the following operating conditions were adopted: average recirculation flow rate: 8000 L/h, average temperature: 60°C; average cross flow (CF) speed: 4.1 m/s transmembrane pressure (TMP): 1-2 bar; time: 600 minutes.

At the end of the tangential microfiltration, the concentration of oleaginous cellular biomass was found to be equal to 21% by weight (dry weight), with respect to a starting dry weight of the solution equal to 8.4% by weight, thus obtaining a final concentration ratio equal to 2.5. The residual glucose content in the concentrated fermentation broth, determined by liquid chromatography operating as reported above (Method I and Method II), was found to be equal to 14.5 g/L, while the final total lipid content in the concentrated fermentation broth, determined by colorimetric method and confirmed by gravimetric method operating as reported above, was found to be equal to 129.6 g/L of said cellular biomass (the data are reported in Table 1).

EXAMPLE 3 (invention)

Hydrolysis of guayule bagasse dispersed in the fermentation broth

For this purpose, 1 kg of fermentation broth obtained as described in Example 2 was used, consisting of 790 g of aqueous phase and 210 g of Rhodosporidium azoricum DSM 2949 cells to which 150 g of previously ground guayule (Parthenium argentatum) bagasse (particle diameter less than 2 mm) was added, so as to have a bagasse concentration equal to 13% by weight with respect to the total weight of said fermentation broth: 70% methanesulfonic acid (MSA) (Aldrich) was added to the suspension obtained to bring the pH to 1.0.

The composition of guayule bagasse, determined as described above, was as follows: 47.0% of cellulose, 20.5% of hemicellulose, 26.8% of lignin, with respect to the total weight of the starting guayule bagasse. The remaining part was found to consist of organic acids, protein and non-protein nitrogenous substances, lipids, mineral salts.

The reaction mixture obtained was poured into a 2-litre Buchi autoclave equipped with refrigerant jacket, kept under stirring (600 rpm) and heated until reaching the temperature of 140 C (in about 46 minutes): the temperature was maintained for 26 minutes, then the autoclave was abruptly cooled to room temperature (25 C) with water through the refrigerant jacket.

Lipid extraction

At the end of hydrolysis, the reaction mixture thus obtained was poured into a continuous stirred tank reactor (CSTR) made of Pyrex glass, stirred and thermostated, to which cyclohexane (Aldrich, code 227048) was added so as to have a reaction mixture: solvent volume ratio equal to 1 :2: the temperature was raised to 71 C and the mixture was left at said temperature, for 2 hours, under stirring (200 rpm). At the end of the 2 hours, the reactor was allowed to cool to room temperature (25 C) obtaining a first mixture comprising an organic phase (extract), an aqueous phase and a solid phase. Said first mixture was discharged from the bottom of the reactor and subjected to filtration by means of a Pirex filter with a dip-tube with porous septum and peristaltic pump, obtaining an organic phase (extract) and a second mixture comprising an aqueous phase and a solid phase. The organic phase (extract) was placed in a rotary evaporator, at 60° C, 250 mbar, for the evaporation of the cyclohexane and recovery thereof. The residue remaining in the evaporator was again treated at 100 mbar and 60 C in order to completely eliminate the cyclohexane obtaining 127.4 g of an oil comprising lipids, which correspond to 99.1% by weight of the total lipids present in the starting fermentation broth determined by colorimetric method and confirmed by gravimetric method operating as reported above.

The recovered oil was also analysed by operating as reported above and the data obtained are reported in Table 2.

Solvent recovery from the aqueous phase (refined) and quantification of sugars in the refined

For this purpose, said second mixture comprising an aqueous phase and a solid phase was filtered on a Buchner G5 2-5 pm filter, obtaining an aqueous phase (refined) and a solid phase. Said aqueous phase (refined) was placed in a rotary evaporator, at 60 C, 300 mbar for evaporation and recovery of the residual solvent (i.e. cyclohexane). During evaporation, the pressure was gradually brought to 150 mbar and continued until the light phase, i.e. the solvent, was no longer present in the condensate. At the end of the evaporation process, a liquid phase equal to 4.9% by weight of the total weight of the aqueous phase (refined) was obtained, said liquid phase consisting of 70% by weight of cyclohexane, 30% by weight of water, with respect to the total weight of said liquid phase and a second aqueous phase (second refined). The second aqueous phase (second refined) was analysed by operating as reported above, obtaining the following results (reported in Table 3):

C5 sugar yield: 98.5% (determined as described above);

C5 sugar recovery: 38.4 g/L (determined as described above);

C6 sugar recovery: 70.5 g/L (determined as described above);

C6 degradation ratio (HMF degradation): 1.1% (calculated as described above),

C5 degradation ratio (F degradation): 0.2% (calculated as described above). EXAMPLE 4 (comparative)

Hydrolysis of the fermentation broth

For this purpose, 1 kg of fermentation broth, obtained as described in Example 2, was used consisting of 790 g of aqueous phase and 210 g of Rhodosporidium azoricum DSM 29495 cells, containing 129.6 g of lipids, based on the quantitative determination of lipids carried out as described above, i.e. by colorimetric method and confirmed by gravimetric method.

70% methanesulfonic acid (MSA) (Aldrich, code 471348) was added to said fermentation broth to bring the pH to 1 : the resulting suspension was poured into a 2-litre Buchi autoclave equipped with refrigerant jacket, kept under stirring (600 rpm) and heated until reaching the temperature of 140 C (in about 46 minutes): the temperature was maintained for 20 minutes, then the autoclave was abruptly cooled to room temperature (25°C) with water through the refrigerant jacket.

Lipid extraction

At the end of hydrolysis, the reaction mixture obtained was subjected to lipid extraction operating as described in Example 3, obtaining 127.4 g of an oil comprising lipids, which corresponds to 98.3% by weight of the total lipids present in the starting fermentation broth determined by the colorimetric method and confirmed by the gravimetric method operating as described above.

For this purpose, the aqueous phase (refined) was treated as described in Example 2, obtaining the following results (reported in Table 3):

C6 sugars: 70.0 g/L (determined as described above);

C6 degradation ratio (HMF degradation): 1.1 (calculated as described above). EXAMPLE 5 (comparative)

Hydrolysis of guayule bagasse dispersed in water

For this purpose, 1 kg of water and 150 g of previously ground guayule (Parthenium argentatum) bagasse (particle diameter less than 2 mm) were loaded to a 2-litre Brignole autoclave, equipped with refrigerant jacket, so as to have a bagasse concentration equal to 13% by weight with respect to the total weight of said fermentation broth: 70% methanesulfonic acid (MSA) (Aldrich) was added to the suspension obtained to bring the pH to 1.0.

The reaction mixture obtained was kept under stirring (600 rpm) and heated until reaching the temperature of 140 C (in about 46 minutes): the temperature was maintained for 26 minutes, then the autoclave was abruptly cooled to room temperature (25°C) with water through the refrigerant jacket obtaining a reaction mixture comprising a solid phase comprising lignin and cellulose and an aqueous phase comprising the sugars deriving from hemicellulose: said phases were separated by filtration.

The aqueous phase was analysed as described above, obtaining the following results (reported in Table 3):

C5 sugar yield: 96.4% (determined as described above);

C5 sugar recovery: 29.7 g/L (determined as described above);

C5 degradation ratio (F degradation): 2.6% (calculated as described above).

Table 1 ⁽¹⁾: hydroxymethylfurfural;

⁽²⁾: furfural.

Table 2

(1): free fatty acids

5 Table 3

⁽¹⁾: hydroxymethylfurfural;

⁽²⁾: furfural.

In Table 3, from the comparison between the results of Example 3 (invention) and Example 5 (comparative), it is observed that the amount of monomeric sugars having 5 carbon atoms (C5) present in the aqueous phase (second refined) equal to 38.4 g/L in Example 3 (invention), is higher than the amount of monomeric sugars having 5 carbon atoms (C5) present in the aqueous phase equal to 29.7 g/L in Example 5 (comparative). From said comparison it can also be seen how the yield of monomeric sugars having 5 carbon atoms (C5) equal to 98.5% obtained in Example 3 (invention) is higher than the yield of monomeric sugars having 5 carbon atoms (C5) equal to 96.4% obtained in Example 5 (comparative) supporting the fact that the presence of the fermentation broth does not negatively affect said yield but even improves it.

Furthermore, based on the data reported in Table 3, from the comparison between the results of Example 3 (invention) and Example 5 (comparative), it is surprising the drastic decrease in the degradation of the monomeric sugars having 5 carbon atoms (C5) (F degradation ) that goes from 2.6% to 0.2%, based on the only difference of the dispersing medium of guayule bagasse: fermentation broth in Example 3 (invention) with respect to water of Example 5 (comparative).

Furthermore, on the basis of the data reported in Table 3, it can be seen that both Example 3 (invention) and Example 4 (comparative) show the same degradation ratio of monomeric sugars having 6 carbon atoms (C6) (HMF degradation), equal to 1.1%: therefore, the presence of guayule bagasse in Example 3 (invention) does not entail any increase in undesired products deriving from the degradation of the monomeric sugars having 6 carbon atoms (C6).

Also based on the data reported in Table 3, it can be seen how Example 3 (invention) allows to obtain a higher concentration of monomeric sugars having 5 carbon atoms and having 6 carbon atoms (C5 + C6) in the aqueous phase (second refined) (equal to 108.9 g/L) compared to Example 5 (comparative) (equal to 29.7 g/L) and Example 4 (comparative) (equal to 70 g/L).

Regarding the lipids obtained from the fermentation broth deriving from the fermentation of microorganisms, based on the data reported in Table 2, an almost quantitative yield (99.1%) in Example 3 (invention) is surprising, which is improving compared to the yield obtained in Example 4 (comparative) (98.3%), supporting the fact that the presence of guayule bagasse does not negatively affect said recovery but, even improves it.

Furthermore, on the basis of the data reported in Table 2, from the comparison between the compositions of the oil obtained in Example 3 (invention) and the oil obtained in Example 4 (comparative), a surprisingly better quality of the recovered oil is found, thanks to the presence of a lower amount of free fatty acids (FFA), a fundamental characteristic linked to the quality and commercial value of oils and fats, which goes from 1.2% of Example 3 (invention) to 2.3% of Example 4 (comparative).

Claims

1. Process for simultaneous production of sugars from biomass including at least one polysaccharide and of sugars and lipids from a fermentation broth, comprising the following steps:

2. Process for simultaneous production of sugars from biomass including at least one polysaccharide and of sugars and lipids from a fermentation broth according to claim 1, wherein said polysaccharide is selected from cellulose, hemicellulose, or mixtures thereof, preferably from cellulose, or mixtures of hemicellulose and cellulose.

3. Process for simultaneous production of sugars from biomass including at least one polysaccharide and of sugars and lipids from a fermentation broth according to claim 1 or 2, wherein said biomass including at least one polysaccharide is a lignocellulosic biomass.

4. Process for simultaneous production of sugars from biomass including at least one polysaccharide and of sugars and lipids from a fermentation broth according to claim 3, wherein said biomass including at least one polysaccharide is selected from: scraps, residues and waste of products deriving from crops expressly cultivated for energy use such as miscanthus, panicum (Panicum virgatum), common cane (Arundo donax): scraps, residues and waste of products deriving from agriculture such as, for example, guayule (Parthenium argentatum), corn, soybean, cotton, linseed, rapeseed, sugar cane, palm oil, poplar, alder, birch, residues deriving from the oil palm tree [such as palm leaf, trunks, leaf midribs, empty palm oil fruits (EFB - Empty Fruit Bunches) ], wheat straw, rice straw, com stalks, cotton stalks, sorghum, bagasse [such as sugar cane bagasse, guayule (Parthenium argentatum) bagasse]; scraps, residues and waste of products deriving from forestation or forestry comprising scraps, residues and waste resulting from such products or their processing; scraps from agri-food products intended for human consumption or animal husbandry; residues, not chemically treated, from the paper industry; waste coming from the separate collection of solid urban waste (such as urban waste of vegetal origin, paper); algae such as microalgae or macroalgae, in particular macroalgae. preferably from scraps, residues and waste deriving from miscanthus, panicum (Panicum virgatum), common cane (Arundo donax), guayule (Parthenium argentatum), poplar, alder, birch, residues deriving from the oil palm tree [such as, for example, leaf mibrids, empty palm oil fruits (EFB - “Empty Fruit Bunches”)], wheat straw, rice straw, com stalks, cotton stalks, sorghum, sugar cane bagasse, guayule (Parthenium argentatum) bagasse; more preferably from scraps, residues and wastes deriving from guayule (Parthenium argentatum), even more preferably is guayule (Parthenium argentatum) bagasse.

5. Process for simultaneous production of sugars from biomass including at least one polysaccharide and of sugars and lipids from a fermentation broth according to claim 1 or 2, wherein said biomass including at least one polysaccharide is selected from the sugar processing scraps, in particular from sugar beet or sugar cane, such as molasses.

6. Process for simultaneous production of sugars from biomass including at least one polysaccharide and of sugars and lipids from a fermentation broth according to any one of the preceding claims, wherein in said step (a), in said fermentation device, the fermentation is carried out: at a temperature comprised between 20°C and 40 C, preferably comprised between 25°C and 35 C; and/or for a time comprised between 2 days and 10 days, preferably comprised between 3 days and 8 days; and/or at a pH comprised between 4.5 and 7.0, preferably between 5.0 and 6.5.

7. Process for simultaneous production of sugars from biomass including at least one polysaccharide and sugars and lipids from a fermentation broth according to any one of the preceding claims, wherein in said step (a), said oleaginous microorganism is selected from oleaginous yeasts such as: Rhodotorula glutinis, Rhodotorula gracilis, Rhodotorula graminis, Lypomices slarkeyi, Lypomices lipofer, Trigonopsis variabilis, Candida kefyr, Candida curvata, Candida lipolytica, Torulopsis sp. , Pichia stipitis, Trichosporon cacaoliposimilis, Rhodosporidium sp. , Cryptococcus curvatus, Trichosporon oleaginosus: preferably from Rhodosporidium sp., more preferably is Rhodosporidium azoricum DSM 29495.

8. Process for simultaneous production of sugars from biomass including at least one polysaccharide and of sugars and lipids from a fermentation broth according to any one of the preceding claims, wherein said step (b) is carried out by centrifugation, filtration, tangential microfiltration, preferably by tangential microfiltration.

9. Process for simultaneous production of sugars from biomass including at least one polysaccharide and of sugars and lipids from a fermentation broth according to any one of the preceding claims, wherein in said step (c), said at least one organic acid is selected from alkyl sulfonic acids having general formula (I):

R-SO3H (I) wherein R represents a Ci-Ce, preferably C1-C3, alkyl group, linear or branched, preferably is methanesulfonic acid (CH3-SO3H).

10. Process for simultaneous production of sugars from biomass including at least one polysaccharide and of sugars and lipids from a fermentation broth according to any one of the preceding claims, wherein in said step (c), said at least one organic acid is used in a mixture with at least one inorganic acid, said inorganic acid being selected from strong inorganic acids such as hydrochloric acid (HC1), nitric acid (HNO3), sulphuric acid (H2SO4), or mixtures thereof.

11. Process for simultaneous production of sugars from biomass including at least one polysaccharide and sugars and lipids from a fermentation broth according to claim 10, wherein in said mixture said at least one organic acid is present in an amount comprised between 10% by weight and 90% by weight, preferably comprised between 15% by weight and 85% by weight, even more preferably comprised between 20% by weight and 80% by weight, with respect to the total weight of said mixture.

12. Process for simultaneous production of sugars from biomass including at least one polysaccharide and of sugars and lipids from a fermentation broth according to any one of the preceding claims, wherein said step (c) is carried out: at a temperature comprised between 100 C and 180 C, preferably comprised between 130°C and 150°C; and/or for a time comprised between 10 minutes and 2 hours, preferably comprised between 20 minutes and 1 hour.

13. Process for simultaneous production of sugars from biomass including at least one polysaccharide and of sugars and lipids from a fermentation broth according to any one of the preceding claims, wherein said separation step (d) comprises the following steps:

(i) an organic phase comprising lipids dissolved in solvent (extract);

(iii) a solid phase comprising lignin, cellulose and cellular debris,

14. Process for simultaneous production of sugars from biomass including at least one polysaccharide and of sugars and lipids from a fermentation broth according to claim 13, wherein: in said step (di) said non-polar organic solvent is selected from cyclohexane, //-hexane, //-heptane, //-octane, /.w-octane, or mixtures thereof; preferably cyclohexane; and/or said step (di) is carried out at a temperature comprised between 20°C and 200°C, preferably at the boiling temperature of the solvent used; and/or said step (di) is carried out in the presence of an amount of solvent comprised between 1 and 4 times, preferably comprised between 1 and 2 times, the volume of said first mixture.

15. Process for simultaneous production of sugars from biomass including at least one polysaccharide and of sugars and lipids from a fermentation broth according to any one of claims 1 to 12, wherein said separation step (d) comprises the following steps: (dio) subjecting the reaction mixture comprising a solid phase, an oily phase and an aqueous phase, obtained at the end of said step (c), to filtration obtaining a solid phase comprising lignin, cellulose and cellular debris, and a first mixture comprising an oily phase and an aqueous phase; (du) subjecting said first mixture to extraction in the presence of at least one nonpolar organic solvent, obtaining a second mixture comprising:

(i) an organic phase comprising lipids dissolved in solvent (extract);

(ii) an aqueous phase comprising monomeric sugars having 5 carbon atoms (C5), monomeric sugars having 6 carbon atoms (C6), organic acid or a mixture thereof with at least one inorganic acid and traces of unseparated lipids (refined), and part of the non-polar organic solvent.