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

Abstract

Process for simultaneous production 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) 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, said hydrolysis being carried out at a pH comprised between 0.6 and 1.6, preferably comprised between 0.9 and 1.3, obtaining a reaction mixture comprising cellular debris, an aqueous phase and an oily phase; (d) subjecting the reaction mixture obtained in said step (c) to separation obtaining a solid residue comprising cellular debris, an aqueous phase comprising sugars and an oily phase comprising lipids. The sugars obtained from the aforesaid process can be advantageously used, for example, 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), or in chemical synthesis processes for the production of other intermediates or chemical products. Said alcohols and lipids can in turn be advantageously used in the production of biofuels (e.g., biodiesel or 30 "Green Diesel"), which can be used as such or in a mixtures with other automotive 33 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 its copolymers). The lipids obtained from the aforesaid process can be advantageously used, for instance, for the production of biofuels that can be used in diesel or aviation engines, or these lipids can be subjected to metathesis processes in order to obtain biofuel precursors, waxes, plastics, cosmetics, and personal care articles. The aforesaid process is particularly useful in the case of a biorefinery.

Description

[0001] PROCESS FOR SIMULTANEOUS PRODUCTION OF SUGARS AND LIPIDS FROM A FERMENTATION BROTH

[0002] DESCRIPTION

[0003] The present invention relates to a process for simultaneous production of sugars and lipids from a fermentation broth.

[0004] More particularly, the present invention relates to a process for simultaneous production of sugars and lipids from a fermentation broth comprising subjecting said fermentation broth 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.

[0005] The sugars obtained from the aforesaid process can be advantageously used, for example, 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), or in chemical synthesis processes for the production of other intermediates or chemical products. 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 a mixtures 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 its copolymers). The lipids obtained from the aforesaid process can be advantageously used, for example, for the production of biofuels that can be used in diesel or aviation engines, or these lipids can be subjected to metathesis processes in order to obtain biofuel precursors, waxes, plastics, cosmetics, and personal care articles. The aforesaid process is particularly useful in the case of a biorefinery.

[0006] Processes for the recovery, quantification and characterisation of sugars in microorganisms are known in the art.

[0007] For example, Kanetsuna F. et al., in “Journal of Bacteriology" (1969), Vol. 97, No. (3), pp. 1036-1041, describe the cellular wall composition of the yeast Paracoccidioides brasiliensis. For this purpose, yeast was frozen and subjected to a process of mechanical lysis of the cellular walls by means of a French Press until it was almost completely crushed. After further treatment, the crushed cells were recovered by centrifugation and hydrolysed in a 1 N hydrochloric acid solution at 110°C, for a time comprised between 5 and 7 hours. The sugars in the obtained aqueous phase were analysed by liquid chromatography. No yield was reported and no other process conditions for sugar recovery were described. Moreover, the production of undesirable by-products deriving from secondary sugar-degrading reactions has not been addressed. These undesirable by-products, such as hydroxymethylfurfural (HMF), are in fact known in the art as inhibitors for possible downstream processes for the valorisation of sugars.

[0008] 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, p. 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 resulting aqueous phase. Also in this case, no yield was reported, no other process conditions for sugar recovery were described, and the production of undesirable by-products from secondary sugar-degrading reactions was not addressed.

[0009] The same approach was also adopted for the yeast Mucor rouxii 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 obtained aqueous phase. Also in this case, no yield was reported, no other process conditions for sugar recovery were described, and the production of undesirable by-products from secondary sugar-degrading reactions was not addressed.

[0010] Similarly, the nature of the sugars constituting the cellular wall of the yeast Aspergillius niger was studied as reported by Johnston I. R., in "Biochemical Journal" (1965), Vol. 96(3), p. 651-658, DOI: 10.1042 / bj0960651. Sugar hydrolysis was carried out in the presence of sulphuric acid (H2SO4) after yeast lysis and cellular wall recovery. Thus, glucose, mannose and galactose were identified and quantified in the obtained aqueous phase. In the experimental, neither alternative methods to hydrolysis with sulphuric acid (H2SO4), nor alternative methods for optimising sugar recovery were proposed, and it is assumed that sugar recovery is complete. Also in this case, the production of undesirable by-products from secondary sugar-degrading reactions was not addressed.

[0011] The same approach, in which it is assumed that sugar recovery is complete, was followed in a research aimed at the taxonomic evaluation of certain yeast species, in particular Rhodosporidium, Leucosporidium and Rhodotorula, as reported by Sugiyama J. et al. in "The Journal of General and Applied Microbiology" (1985), Vol. 31(6), p. 519-550, DOI: 10.2323 / jgam.31.519). The authors report a total recovery of the sugars of these yeasts, both those constituting the cellular wall and the endocellular sugars [i.e. sugars contained within the cell (for example, glycogen)], by treatment of the fermented product, 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 undesirable by-products from secondary sugar-degrading reactions was not addressed.

[0012] Other authors, for analytical purposes, have faced the problem of identifying a useful acid to optimise the lysis of yeasts, in particular 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, p. 1297-1306, DOI: 10.1002 / (SICI)1097-0061(1998100)14: 14<1297:AID- YEA3 10>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 determining sugars in plant fibres, based on a sulphuric acid (H2SO4) treatment reported by Selvendran R. R. et al. in "Analytical Bioche mistry" (1979), Vol. 96, Issue 2, p. 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 undesirable byproducts from secondary sugar-degrading reactions was not addressed.

[0013] 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 in order to establish their suitability in 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 homogenisation of the cells in a mill with zirconium glass beads. Similar results were obtained after autolysis combined with yeast milling and sonication, but the time required for these processes was longer than 24 hours. The authors conclude that homogenisation in a mill might be the ideal method for the cellular wall recovery process because it eliminates further downstream treatments, such as sonication. Also in this case, the production of undesirable by-products from secondary sugar-degrading reactions was not addressed.

[0014] Other authors have addressed the problem of determining the content and composition of all sugars present (“whole cell approach”) in a range of yeasts, particularly in Ascomycetes and Basidiomycetes, as reported, for example, by Prillinger H. et al. in “TTze Journal of General and Applied Microbiology" (1993), Vol. 39, Issuel, p. 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 determining sugars in plant fibres, based on a sulphuric acid (H2SO4) treatment reported in the article by Selvendran R. R. et al. in "Analytical Biochemistry" (1979), Vol. 96, Issue 2, p. 282-292, DOI: 10.1016 / 0003-2697(79)90583-9) cited above, was the most suitable and followed the method without variation.

[0015] 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 (for example, the agriculture sector), in the pharmaceutical industry and in the food industry. Yeasts are the most widely used microorganisms in the industry and are particularly important in the production of beer, wine, and in distillation processes. Therefore, the analysis of the endocellular sugar content of yeast 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 related to the metabolism of yeasts and their performance in the above-mentioned 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.

[0016] Recently, new methods for the characterisation and quantification of sugars in yeasts have been proposed. For example, a quick and simple method to monitor changes 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, p. 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, particularly 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) reported 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 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 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.

[0017] Processes for obtaining and extracting lipids present in microorganisms are also known in the art.

[0018] 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:

[0019] (i) providing an aqueous suspension comprising a fermentation broth containing oleaginous yeast biomass;

[0020] (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;

[0021] (iii) subjecting the hydrothermally treated aqueous suspension to a separation step to obtain a liquid fraction and a yeast biomass fraction;

[0022] (iv) subjecting the yeast biomass fraction to a drying step to obtain a dried yeast biomass fraction,

[0023] (v) subjecting the dried yeast 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,

[0024] (vi) recovering microbial lipids from the liquid fraction of step (v),

[0025] (vii) optionally, isolating the residual fraction of yeast biomass from the product of step (v).

[0026] The resulting 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.

[0027] International Patent Application WO 2001 / 053512 relates to a process for obtaining lipids from microorganisms comprising:

[0028] (a) lysis of microorganism cells to produce a mixture of lysed cells; (b) processing 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;

[0029] (c) separating said heavy phase from said light phase; and

[0030] (d) obtaining lipids from said light phase.

[0031] Lysis is performed by heating the cells of the microorganism to at least 50°C in the presence of a base and / or a chelating agent, and lipid extraction is carried out in the absence of solvents.

[0032] European Patent Application EP 2450425 relates to a method for the recovery of lipids from a microbial biomass comprising the steps of

[0033] (i) providing a moist microbial biomass containing lipids to be extracted without breaking the cell walls of the biomass;

[0034] (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 high pressure, where the combination of temperature and pressure is such that said lipids come into contact with said extracting agent; and

[0035] (iii) subsequently recovering the extracted lipids from or with said extracting agent.

[0036] Said extracting agent is a non-polar organic solvent that is essentially, preferably totally, immiscible with water.

[0037] The above-mentioned prior art highlights that the production of sugars and lipids from microorganisms are two distinct processes.

[0038] The Applicant therefore faced the problem of finding a process for simultaneous production of sugars and lipids from microorganisms.

[0039] The Applicant has now surprisingly found that the simultaneous production of sugars and lipids from microorganisms, preferably oleaginous microorganisms, can be obtained by a process comprising subjecting the fermentation broth obtained from the fermentation of said microorganisms 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.

[0040] Numerous advantages are obtained by said process. For example, said process allows to obtain a high recovery of sugars from microorganisms, particularly monomeric sugars with 6 carbon atoms (C6), and minimises the formation of undesirable by-products, particularly hydroxymethylfurfural (HMF), resulting from undesirable secondary sugardegrading processes which, as mentioned above, act as inhibitors in processes where sugars can be conveniently recycled, such as fermentation itself. Said process makes it possible to obtain an aqueous phase comprising sugars, in particular monomeric sugars with 6 carbon atoms (C6), at a concentration greater than or equal to 50 g / 1, which can be recycled, for example at fermentation, directly, with no need for further purification and / or concentration steps, with advantages in terms of both process time and economy. The sugars obtained from the above process can advantageously be 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), 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 a mixture with other automotive fuels, while these 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 its copolymers). In addition, said process allows to obtain a high lipid yield. In particular, in the case of using oleaginous microorganisms, said process allows to obtain a lipid yield greater than or equal to 95%, said lipid yield being calculated as grams of lipids obtained per gram of lipids present in the starting microorganism. The lipids obtained can be advantageously used, for instance, for the production of biofuels that can be used in diesel or aviation engines, or these lipids can be subjected to metathesis processes in order to obtain biofuel precursors, waxes, plastics, cosmetics, and personal care articles. The aforesaid process is particularly useful in the case of a biorefinery.

[0041] It is therefore an object of the present invention to provide a process for simultaneous production 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;

[0042] (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;

[0043] (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) 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, said hydrolysis being carried out at a pH comprised between 0.6 and 1.6, preferably comprised between 0.9 and 1.3, obtaining a reaction mixture comprising cellular debris, an aqueous phase and an oily phase;

[0044] (d) subjecting the reaction mixture obtained in said step (c) to separation obtaining a solid residue comprising cellular debris, an aqueous phase comprising sugars and an oily phase comprising lipids.

[0045] 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.

[0046] 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.

[0047] For the purpose of the present description and of the following claims, the term "monomeric sugar with 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.

[0048] For the purpose of the present invention, said step (a) can be carried out according to methods known in the art.

[0049] Preferably, said microorganism, before being fed to the fermentation device in step (a), can be grown in a suitable culture medium by obtaining an inoculum. For this purpose, this 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.

[0050] In order to maintain the pH in the desired range, 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 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 fermentation, in such an amount as to achieve the desired pH.

[0051] When the microorganism has reached a concentration higher than or equal to 8 g / 1, preferably comprised between 10 g / 1 and 20 g / 1, said fermentation broth (inoculum) is fed to the fermentation device used in step (a).

[0052] According to 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.

[0053] In order to maintain the pH in the desired range, 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 fermentation, in such an amount as to achieve the desired pH.

[0054] 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 removal of the exhausted fermentation broth by, for example, microfiltration.

[0055] For the purpose of the present invention, in said step (a), said fermentation can advantageously be carried out in fermentation devices known in the art, in the presence of culture media usually used for this purpose, comprising, in addition to sugars, various nutrients such as, for example, nitrogen, potassium phosphate, magnesium, salts, vitamins, trace elements.

[0056] For the purpose of the present invention, said fermentation devices may 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 among continuous biomass feed “slurry” reactors (CSTR- “Continuous Stirred-Tank Reactor”).

[0057] 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.

[0058] According to a preferred embodiment of the present invention, in said step (a), said oleaginous microorganism can be selected from oleaginous yeasts such as, for example: Rhodotorula gl minis. Rhodotorula gracilis, Rhodotorula graminis, Lypomices siarkeyi, Lypomices lipofer, Trigonopsis variabilis, Candida kefyr, Candida curvaia, Candida lipolytica, Torulopsis sp. , Pichia siipiiis, Trichosporon cacaoliposimilis, Rhodosporidium sp. , Cryptococcus curvatus, Trichosporon oleaginosus.

[0059] According to a particularly preferred embodiment of the present invention, in said step (a), said oleaginous microorganism can be selected from Rhodosporidium sp., most preferably it is Rhodosporidium azoricum DSM 29495 (mutant described in International Patent Application WO 2016 / 108185).

[0060] According to a preferred embodiment of the present invention, said step (b) can be carried out by centrifugation, filtration, tangential microfiltration, preferably by tangential microfiltration.

[0061] According to a preferred embodiment of the present invention, in said step (c), said at least one organic acid can be selected from alkyl sulphonic acids having general formula (I):

[0062] R-SO3H (I) wherein R represents a Ci-Ce alkyl group, preferably C1-C3, linear or branched.

[0063] According to a preferred embodiment of the present invention, in said step (c), said at least one organic acid is methanesulphonic acid (CH3-SO3H).

[0064] In order to achieve the desired pH value said at least one organic acid may be used in a mixture with at least one inorganic acid.

[0065] According to a preferred embodiment of the present invention, in said stage (c), said at least one organic acid can be used in a mixture with at least one inorganic acid.

[0066] According to a preferred embodiment of the present invention, in said stage (c), said at least one inorganic acid can be selected from strong inorganic acids such as, for example, hydrochloric acid (HC1), nitric acid (HNO3), sulphuric acid (H2SO4), or mixtures thereof.

[0067] According to a preferred embodiment of the present invention, in said mixture said at least one organic acid can be present in an amount comprised betweenl0% 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.

[0068] According to 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.

[0069] According to 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.

[0070] Before being subjected to step (d), the reaction mixture comprising cellular debris, an aqueous phase and an oily phase, obtained in said step (c), is cooled to room temperature (25°C).

[0071] According to a preferred embodiment of the present invention, said step (d) can comprise the following steps:

[0072] (di) subjecting the reaction mixture comprising cellular debris, an aqueous phase and an oily phase, obtained in said step (c), to filtration obtaining cellular debris and a first mixture comprising an aqueous phase and an oily phase;

[0073] (dz) subjecting said first mixture to extraction in the presence of at least one nonpolar organic solvent, obtaining a second mixture comprising:

[0074] (i) an organic phase comprising lipids dissolved in solvent (extract);

[0075] (ii) an aqueous phase comprising monomeric sugars with 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).

[0076] The cellular debris obtained at the end of the step (di) can be dehumidified and used as fuel.

[0077] According to a preferred embodiment of the present invention, in said step (dz) said non-polar organic solvent can be selected, for example, from cyclohexane, / / -hexane, / / -heptane, / / -octane, Ao-octane, or mixtures thereof; preferably cyclohexane.

[0078] According to a preferred embodiment of the present invention, said step (dz) can be carried out at a temperature comprised between 20°C and 200°C, preferably at the boiling temperature of the solvent used.

[0079] According to a preferred embodiment of the present invention, said step (dz) can be carried out in the presence of an amount of non-polar organic solvent comprised between 1 time and 4 times, preferably comprised between 1 time and 2 times, the volume of said first mixture.

[0080] Before being subjected to separation, said second mixture is cooled to room temperature (25°C).

[0081] 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.

[0082] 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 containing the non-polar organic solvent that can be recycled to the aforesaid extraction [i.e. to said stage (d2)].

[0083] 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 present in said organic phase (i) (extract). Other lipids that can be present in said organic phase (i) are: phospholipids, monoglycerides, di glycerides, free fatty acids, or mixtures thereof.

[0084] In order to recover the sugars, said aqueous phase (ii) (refined) is subjected to a process comprising:

[0085] (ds) subjecting said aqueous phase (ii) (refined) to distillation or evaporation obtaining a second aqueous phase (second refined) comprising monomeric sugars with 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:

[0086] (111) a phase consisting of the remaining non-polar organic solvent mixed with the aqueous solution;

[0087] (112) a phase consisting of water;

[0088] (d4) separating said phase (iii) and ( ) by known techniques, for example, decantation; (ds) recycling phase (iii) to the aforesaid extraction [step (d2)];

[0089] (de) sending the phase (H2) to water treatment or subsequent valorisation, for example to fermentation processes of oleaginous microorganisms;

[0090] (d?) 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 with 6 carbon atoms (C6) and water;

[0091] (d?) recycling said organic acid or a mixture thereof with at least one inorganic acid to stage (c).

[0092] In accordance with a further embodiment of the present invention, said separation step (d) can comprise the following steps:

[0093] (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 extraction in the presence of at least one non-polar organic solvent, obtaining a first mixture comprising:

[0094] (i) an organic phase comprising lipids dissolved in solvent (extract);

[0095] (ii) an aqueous phase comprising monomeric sugars with 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;

[0096] (iii) a solid phase comprising cellular debris;

[0097] (du) 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 with 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 and a solid phase comprising cellular debris;

[0098] (d 12) subjecting said second mixture to filtration or centrifugation, preferably to filtration, obtaining an aqueous phase comprising monomeric sugars with 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 (refined) and a solid phase including cellular debris.

[0099] Said step (dio) can be carried out as above-mentioned for step (d2).

[0100] In said step (du), filtration in order to obtain said organic phase (extract) and said second mixture, can be carried out by filtration, preferably by suction filtration on a porous septum.

[0101] In said step (dn), filtration in order to obtain said aqueous phase (refined) and said solid phase can be carried out via a Buchner filter.

[0102] 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.

[0103] The present invention will now be illustrated in greater detail by an embodiment with reference to Figure 1 reported below.

[0104] 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. Said fermentation broth (2) is subjected to hydrolysis (3) in the presence of at least one organic acid, preferably methanesulphonic acid (CH3-SO3H), obtaining a reaction mixture comprising cellular debris, an aqueous phase and an oily phase (not shown in Figure 1). Said reaction mixture is subjected to filtration (4) obtaining cellular debris (5) that can be dehumidified and valorised into fuel (not shown in Figure 1) and a first mixture comprising an aqueous phase and an oily phase (6). Said first mixture (6) is subjected to extraction (7) in the presence of at least one non-polar organic solvent, obtaining a second mixture (8) comprising:

[0105] (i) an organic phase comprising lipids dissolved in solvent (extract);

[0106] (ii) an aqueous phase comprising monomeric sugars with 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).

[0107] Said organic phase (i) (extract) and said aqueous phase (ii) (refined) are separated by decantation (9), obtaining an organic phase (extract) (10) and an aqueous phase (refined) (11).

[0108] In order to recover the lipids, said organic phase (extract) (10) is subjected to evaporation (12), obtaining as a residue an oil comprising lipids (13) that can be used for the production of biofuels, and a liquid phase containing the organic nonpolar solvent (14) that is recycled to the aforesaid extraction (7).

[0109] In order to recover the sugars, said aqueous phase (refined) (11) is subjected to evaporation (15), obtaining a second aqueous phase (second refined) (16) comprising monomeric sugars with 6 carbon atoms (C6), organic acid or a mixture thereof with at least one inorganic acid and traces of unseparated lipids (refined) that can be used as such in fermentation processes, and a liquid phase (17) consisting of two phases:

[0110] (iii) a phase consisting of the remaining non-polar organic solvent mixed with the aqueous solution;

[0111] (iii) a phase consisting of water.

[0112] Alternatively, said second aqueous phase (second refined) can be subjected 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, which can be recycled to hydrolysis (3) and a third aqueous phase (third refined) comprising monomeric sugars with 6 carbon atoms (C6) and water (not shown in Figure 1) which can be used in fermentation processes.

[0113] 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 valorisation, for example to fermentation processes of oleaginous microorganisms (not shown in Figure 1).

[0114] In order to better understand the present invention and to put it into practice, some illustrative and non-limiting examples thereof are reported below.

[0115] Analysis and characterization methods

[0116] The analysis and characterization methods reported below were used. pH determination pH was determined using a pH meter Metrohm 781 pH / Ion meter. analysis

[0117] 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, p. 330-333, DOI: 10.1016 / j.biortech.2013.12.077.

[0118] 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, p. 69-76, DOI: 10.1016 / 80021-9258(18)71045-4.

[0119] Calculation of lipid recovery yield

[0120] 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:

[0121] Yield = oil / lipids* 100 wherein: oil = oil recovered at the end of the process (g); lipids = lipids present in the starting fermentation broth (g).

[0122] Analysis of compounds in the aqueous phase

[0123] Analyses of 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 shown below.

[0124] Method I: instrument: HPLC (“High Performance Liquid Chromatography”) consisting of pump, autosampler, oven, refractive index and diode array detector (RI);

[0125] Agilent Metacarb 67H 300x6.5 mm column, with similar pre-column; pump flow: 0.8 mL / min (sulphuric acid 5 mM); injection volume: 20 pL; oven and column temperature: 45°C; diode array detectors (RI) temperature: 35°C; diode array detectors (RI) wavelengths: 210 nm and 280 nm; analysis time: 35 minutes.

[0126] 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 pL; oven and column temperature: 65°C; diode array detectors (RI) temperature: 35°C; diode array detectors (RI) wavelengths: 210 nm and 280 nm; analysis time: 50 minutes.

[0127] Calculation of the degradation of monomeric sugars with 6 carbon atoms (C6) to undesirable products

[0128] The carbohydrates contained in the microorganisms used are present as polymers or oligomers consisting of monomeric sugars with 6 carbon atoms (C6). Glucose, mannose and galactose have the molecular formula CeHnCL with a molecular weight of 180. However, during the undesirable degradation processes of these sugars, hydroxymethylfurfural (HMF) is formed. The molecular weight of hydroxymethylfurfural (HMF), which is 126, was therefore used to calculate the degradation ratio of monomeric sugars with 6 carbon atoms (C6), defined below. Thus, from 180 g of monomeric sugars with 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, p. 2153-2156, DOI: 10.1039 / C004128A.

[0129] In order to effectively express the production of by-products from monomeric sugars with 6 carbon atoms (C6), i.e. hydroxymethylfurfural (HMF), the degradation ratio was calculated according to the following formula:

[0130] C6 sugar degradation ratio (%) = [(HMF / 126*180) / (C6 sugars + HMF / 126*180)]*100 wherein:

[0131] C6 sugars = monomeric sugars with 6 carbon atoms (C6) (g / 1) present in the second refined;

[0132] HMF = hydroxymethylfurfural (g / 1) present in the second refined.

[0133] EXAMPLE 1

[0134] Preparation of Rhodosporidium azoricum DSM 29495 inoculum

[0135] The inoculum (i.e. first fermentation broth) was prepared using 5 litres of an aqueous solution containing glucose 50 g / 1, yeast extract 2 g / 1, ammonium sulphate 5 g / 1, KH2PO4 1 g / 1, MgSO4-7H2O 0,05 g / 1, NaCl 0.01 g / 1, CaCl2-2H2O 0.01 g / 1, placed in a 7-litre fermenter, equipped with a stirrer, the pH of the obtained mixture was raised to 5 by adding a few drops of 2.5 M potassium hydroxide (KOH). The mixture obtained was sterilised in an autoclave at 120°C, for 20 minutes. After sterilisation, 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 h, 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 of 2% by weight (dry weight) was obtained.

[0136] EXAMPLE 2

[0137] Fermentation of Rhodosporidium azoricum DSM 29495 [discontinuous mode ("feed-batch")]

[0138] The fermentation test with Rhodosporidium azoricum DSM 29495 cells was carried out in discontinuous mode (“feed batch”) in a 200-litre fermenter, operating under the following conditions:

[0139] 100 g / L glucose;

[0140] 2 g / 1 yeast extract;

[0141] 5 g / 1 corn steep solid;

[0142] 5 g / 1 (NH4)2SO4;

[0143] 6 g / 1 KH2PO3;

[0144] 0.3 g / 1 MgSO4-7H2O; 0.06 g / 1 NaCl;

[0145] 0.06 g / 1 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 of 200 rpm - 900 rpm, modulated with the air flow so as to keep 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 of 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 of 3.5 % by weight (dry weight) and a residual sugar concentration of 30 g / 1.

[0146] Subsequently, the initial concentrations of yeast extract and ammonium sulphate (2 g / 1 and 5 g / 1, respectively) were restored to the fermentation broth and the glucose feed was operated in feed batch mode in order to maintain a constant concentration at 30 g / 1 in the fermentation reactor.

[0147] At the end of the fermentation, after 165 hours, a second fermentation broth was obtained with an oleaginous cellular biomass concentration of 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 (pasteurisation), at 80°C, for 2 hours, in order to inhibit lipase activity.

[0148] Subsequently, this second fermentation broth was concentrated by tangential microfiltration in a HAR Pl 9 system on a P 1960 ceramic membrane with a porosity of 0.2 microns. 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 was loaded into said system< and the following operating conditions were adopted: average recirculation flow rate: 8000 1 / h, average temperature: 60°C; average Cross Flow (CF) speed: 4.1 m / s transmembrane pressure (TMP): 1-2 bar; time: 600 minutes.

[0149] At the end of the tangential microfiltration, the concentration of oleaginous cellular biomass was equal to 21% by weight (dry weight), with respect to a starting dry weight of the solution of 8.4% by weight, thus obtaining a final concentration ratio of 2.5. The residual glucose content in the concentrated fermentation broth is of 14.5 g / 1, determined by liquid chromatography operating as above (Method I and Method II). The final total lipid content was 12.9 % of said cellular biomass, determined by colorimetric method and confirmed by gravimetric method operating as reported above.

[0150] EXAMPLE 3 (invention)

[0151] Hydrolysis of the fermentation broth

[0152] 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 caried out as described above, i.e. by colorimetric method and confirmed by gravimetric method.

[0153] 70% methanesulphonic acid (MSA) (Aldrich, code 471348) was added to said fermentation broth, to bring the pH to 1.0: the resulting suspension was poured into a 2-litre Buchi autoclave equipped with a refrigerant jacket, kept stirring (600 rpm) and heated to a 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.

[0154] Lipid extraction

[0155] At the end of hydrolysis, the reaction mixture thus obtained was filtered through a porous septum, then 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 2 hours, the reactor was allowed to cool to room temperature (25°C) and the organic phase (extract) and aqueous phase (refined) were allowed to separate by decantation. The organic phase (extract) was recovered from the reactor by means of a peristaltic pump, while the aqueous phase (refined) was discharged from the bottom of the reactor. 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, resulting in 127.4 g of a high-boiling oil consisting of 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 reported above.

[0156] Solvent recovery from the aqueous phase (refined) and quantification of sugars in the refined

[0157] For this purpose, the 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 is gradually increased to 150 mbar and continues until the light phase, i.e. the solvent, is 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) is 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).

[0158] The second aqueous phase (second refined) was analysed by operating as above, obtaining the following results (shown in Table 1):

[0159] C6 sugars: 70.0 g / 1 (determined as described above); degradation ratio C6: 1.1% (calculated as described above).

[0160] EXAMPLE 4 (comparative)

[0161] Hydrolysis of the fermentation broth

[0162] 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.

[0163] 70% methanesulphonic acid (MSA) (Aldrich, code 471348) was added to this fermentation broth, to bring the pH to 2.3 : the resulting suspension was poured into a 2-litre Buchi autoclave equipped with a refrigerant jacket, kept stirring (600 rpm) and heated to a temperature of 140°C (in about 46 minutes): the temperature was maintained for 240 minutes, then the autoclave was abruptly cooled to room temperature (25°C) with water by means of the cooling jacket.

[0164] Lipid extraction

[0165] At the end of hydrolysis, the reaction mixture obtained was subjected to lipid extraction operating as described in Example 3, obtaining 127.1 g of an oil consisting of lipids, which corresponds to 98.1 % 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.

[0166] Solvent recovery from the aqueous phase (refined) and quantification of sugars in the refined

[0167] For this purpose, the aqueous phase (refined) was treated as described in Example 2, obtaining the following results (shown in Table 1):

[0168] C6 sugars: 25.3 g / 1 (determined as described above); degradation ratio C6: 27.1% (calculated as described above).

[0169] EXAMPLE 5 (comparative)

[0170] Hydrolysis of the fermentation broth

[0171] 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.

[0172] 96% sulphuric acid (H2SO4) (Aldrich, code 258105) was added to said fermentation broth, to bring the pH to 1.0: the resulting suspension was poured into a 2-litre Buchi autoclave equipped with a refrigerating jacket, kept stirring (600 rpm) and heated to a 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 by means of the cooling jacket.

[0173] Lipid extraction

[0174] At the end of hydrolysis, the reaction mixture obtained was subjected to lipid extraction operating as described in Example 3, obtaining 125.7 g of an oil consisting of lipids, which corresponds to 97.0 % 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.

[0175] Solvent recovery from the aqueous phase (refined) and quantification of sugars in the refined

[0176] For this purpose, the aqueous phase (refined) was treated as described in Example 2, obtaining the following results (shown in Table 1):

[0177] C6 sugars: 62.7 g / 1 (determined as described above); degradation ratio C6: 3.0% (calculated as described above).

[0178] EXAMPLE 6 (comparative)

[0179] Hydrolysis of the fermentation broth

[0180] 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, containing 129.6 g of lipids, based on the quantitative determination of lipids performed as described above, i.e. by colorimetric method and confirmed by gravimetric method.

[0181] 96% sulphuric acid (H2SO4) (Aldrich, code 258105) was added to this fermentation broth, to bring the pH to 2.3: the resulting suspension was poured into a 2-litre Buchi autoclave equipped with a refrigerating jacket, kept stirring (600 rpm) and heated to a temperature of 140°C (in about 46 minutes): the temperature was maintained for 4 hours, then the autoclave was abruptly cooled to room temperature (25°C) with water by means of the cooling jacket.

[0182] Lipid extraction

[0183] At the end of hydrolysis, the reaction mixture obtained was subjected to lipid extraction operating as described in Example 3, obtaining 129.6 g of an oil consisting of 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.

[0184] Solvent recovery from the aqueous phase (refined) and quantification of sugars in the refined

[0185] For this purpose, the aqueous phase (refined) was treated as described in Example 2, obtaining the following results (shown in Table 1):

[0186] C6 sugars: 22.3 g / 1 (determined as described above); degradation ratio C6: 30.0% (calculated as described above).

[0187] Table 1

[0188] By comparing Example 3 (invention), Example 4 (comparative), Example 5 (comparative) and Example 6 (comparative), it can be observed that the lipid recovery yield is quite indifferent under the conditions used for yeast lysis (pH, temperature, time) and the type of acid [methanesulphonic acid (MSA) or sulphuric acid (H2SO4)]. In particular, the lipid recovery yield is in all cases above 95%.

[0189] Surprising differences are, however, observed in relation to sugars where, depending on the type of acid used, i.e. methanesulphonic acid (MSA) versus sulphuric acid (H2SO4), the organic acid allows to obtain better sugar production and less sugar degradation under the same conditions. In particular, the quality of monomeric sugars with 6 carbon atoms (C6) is better because of a lower presence of degradation products due to undesirable secondary reactions [in particular hydroxymethylfurfural HMF, a degradation product of monomeric sugars with 6 carbon atoms (C6)]. Furthermore, from a comparison between the analysis results of Example 3 (invention), in which methanesulphonic acid (MSA) (pH 1.0, 140°C, 20 minutes) was used and Example 5 (comparative), in which sulphuric acid (H2SO4) (pH 1,1, 140°C, 20 minutes) was used, a higher sugar production (70.0 g / 1) and, at the same time, a lower degradation ratio to hydroxymethylfurfural (HMF) (1.1%) can be observed when, all other process conditions being equal, methanesulphonic acid (MSA) was used.

[0190] Better performance, when methanesulphonic acid (MSA) was used compared to sulphuric acid (H2SO4), was observed even when operating under drastically different process conditions (in particular, pH). In fact, from a comparison between the analysis results of Example 4 (comparative), in which methanesulphonic acid (MSA) (pH 2.3, 140°C, 240 minutes) was used and Example 6 (comparative), in which sulphuric acid (H2SO4) (pH 2,3, 140°C, 240 minutes) was used, a higher sugar production (25.3 g / 1) and, at the same time, a lower degradation ratio to hydroxymethylfurfural (HMF) (27.1%) can be observed when, all other process conditions being equal, methanesulphonic acid (MSA) was used.

Claims

CLAIMS1. Process for simultaneous production 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) 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, said hydrolysis being carried out at a pH of comprised between 0.6 and 1.6, preferably comprised between 0.9 and 1.3, obtaining a reaction mixture comprising cellular debris, an aqueous phase and an oily phase;(d) subjecting the reaction mixture obtained in said step (c) to separation obtaining a solid residue comprising cellular debris, an aqueous phase comprising sugars and an oily phase comprising lipids.

2. Process for simultaneous production of sugars and lipids from a fermentation broth according to claim 1, 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 comprised between 5.0 and 6.5.

3. Process for simultaneous production of sugars and lipids from a fermentationbroth according to claim 1 or 2, 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.

4. Process for simultaneous production of sugars and lipids from a fermentation broth according to any one of the previous claims, wherein in said step (a), said oleaginous microorganism is selected from Rhodosporidium sp., more preferably it is Rhodosporidium azoricum DSM 29495.

5. Process for simultaneous production of sugars and lipids from a fermentation broth according to any one of the previous claims, wherein said step (b) is carried out by centrifugation, filtration, tangential microfiltration, preferably by tangential microfiltration.

6. Process for simultaneous production of sugars and lipids from a fermentation broth according to any one of the previous claims, wherein, in said step (c), said at least one organic acid is selected from alkyl-sulfonic acids having the general formula (I):R-SO3H (I) wherein R represents a Ci-Ce alkyl group, preferably C1-C3, linear or branched.

7. Process for simultaneous production of sugars and lipids from a fermentation broth according to any one of the previous claims, wherein, in said step (c), said at least one organic acid is methanesulfonic acid (CH3-SO3H).

8. Process for simultaneous production of sugars and lipids from a fermentation broth according to any one of the previous claims, wherein, in said step (c), said at least one organic acid is used in mixture with at least one inorganic acid.

9. Process for simultaneous production of sugars and lipids from a fermentation broth according to claim 8, wherein, in said step (c), said at least one inorganic acid is selected from strong inorganic acids such as hydrochloric acid (HC1), nitric acid (HNO3), sulfuric acid (H2SO4), or mixtures thereof.

10. Process for simultaneous production of sugars and lipids from a fermentationbroth according to claim 8 or 9, 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.

11. Process for simultaneous production of sugars and lipids from a fermentation broth according to any one of the previous 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.

12. Process for simultaneous production of sugars and lipids from a fermentation broth according to any one of the previous claims, wherein said step (d) comprises the following steps:(di) subjecting the reaction mixture comprising cellular debris, an aqueous phase and an oily phase, obtained at the end of said step (c), to filtration obtaining cellular debris and a first mixture comprising an aqueous phase and an oily phase;(d2) 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 with 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).

13. Process for simultaneous production of sugars and lipids from a fermentation broth according to any one of the previous claims, wherein in said step (d2) said non-polar organic solvent is selected from cyclohexane, w-hexane, ^-heptane, n- octane, / .w-octane, or mixtures thereof; preferably cyclohexane.

14. Process for simultaneous production of sugars and lipids from a fermentationbroth according to any one of the previous claims, wherein said step (d2) is carried out at a temperature comprised between 20°C and 200°C, preferably at the boiling temperature of the solvent used.

15. Process for simultaneous production of sugars and lipids from a fermentation broth according to any one of the previous claims, wherein said step (d2) is carried out in the presence of an amount of solvent comprised between 1 and 4 times, preferably comprised between 1 time and 2 times, the volume of said first mixture.

16. Process for simultaneous production 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 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 with 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 cellular debris;(du) 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 with 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 and a solid phase comprising cellular debris;(d 12) subjecting said second mixture to filtration or centrifugation, preferably to filtration, obtaining an aqueous phase comprising monomeric sugars with 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 (refined) and a solid phase including cellular debris.

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