THEREOF

The present invention pertains to the technical field of extracting PHA from biomass , in particular by means of solvents , and treating the extracted PHA to give a desired chemical intermediate . The present invention further relates to using such intermediate as an additive for fuel . For any of these purposes , the applied solvent is a key factor to improve both process efficiency and sustainability, especially on an industrial scale . The solvents must be chosen carefully with regard to costs , health and safety considerations as well as environmental impact .

In the past , solvents of considerable polarity have been used to treat PHA, in particular chlorinated hydrocarbons ( chloroform, dichloromethane , 1 , 2-dichloroethane ) . Halogenated carbons are powerful solvents due to their polarity . However, they are toxic and have a high environmental impact . Ef forts have been made to identi fy alternative solvents , which have comparatively low use of ressources of production, low associated hazards , low energy consumption upon distillation, good degradability, while still exhibiting desirable properties as a solvent . Classic solvents such as acetone , acetonitrile , alcohols , acetates , ethers , alkanes , cycloalkanes , aromatic hydrocarbons , aldehydes , etc . have been subj ect to comparative studies to optimi ze the above- mentioned aspects .

However, none of the classic solvents has satis factory biodegradability . In particular, when extracting polyhydroxalkanoates ( PHA) from biomasses , oil-derived solvents are generally used deteriorating the otherwise „green" attempt to produce bioplastics . It would be highly desirable to use an environmentally friendly and economically interesting extraction technology . Likewise , when processing extracted PHA to give the desired intermediates , suitable solvents are needed . Especially PHA having a short chain length, such as poly ( 3-hydroxybutyrate ) PHB, are di f ficult to dissolve . Such reactions were traditionally performed by using halogenated solvents - undermining the otherwise green approach for improved sustainability, especially on an industrial scale .

In US2003/ 0158441 is described a method for making intermediates from polyhydroxyalkanoates . The PHA are extracted from biomass and converted into an intermediate , e . g . , an ester, by treatment with a solvent/reagent , such as ethanol . However, reaction times are long and/or reaction set-ups laborious ; or they lead to unsatis factory yields . There remains a need for a method of treating biomass containing PHA for extracting/reacting the PHA by using suitable solvents which facilitate treatment steps while being less of an environmental burden .

Furthermore , PHA and their intermediates have been envisaged for use as a biofuel . In particular, after having been used as bioplastics , PHA can be methyl esteri fied to become a biofuel . Such an application extends the added value of PHA and, accordingly, improves the green footprint of such material . There is a need in the art to provide ef ficient biofuels , methods for providing biofuels , and methods of trans forming PHA into a biofuel .

This problem is solved by a method and a use having the features of the independent claims .

Speci fically, the invention relates to a method of reacting polyhydroxyalkanoates ( PHA) to form an intermediate , comprising the steps of a) providing a reaction mixture comprising

- polyhydroxyalkanoates (PHA) ;

- a reagent, in particular an alcohol or an amine;

- optionally : a catalyst; in a solvent comprising butyl 3-hydroxybutyrate; b) exposing the reaction mixture to elevated temperatures and/or elevated pressure, preferably temperatures of at least 90°C.

It is preferred that the PHA provided in step a) have been extracted from biomass ; particularly preferably have the PHA been extracted from biomass as is described hereinafter as a further aspect of the invention.

Another aspect of the invention relates to a method for extracting polyhydroxyalkanoates (PHA) from biomass, comprising the steps of a) combining a biomass containing PHA with a solvent to form a biomass liquor ; b) optionally : heating the biomass liquor to at least partially solubilize the PHA to form a PHA liquor ; c) recovering PHA from the PHA liquor; characterized in that the solvent used in step a) comprises butyl 3-hydroxybutyrate.

By the term polyhydroxyalkanoates (PHA) is understood hereinafter a structure comprising randomly repeating monomer units of the structure I, wherein n is zero or an integer. Each of Ri, R2, R3, R4, R5, and Re are independently a hydrogen atom, a halogen atom or a hydrocarbon radical. A hydrocarbon radical contains at least one carbon atom. A hydrocarbon radical can be saturated or unsaturated, substituted or unsubstituted, branched or straight chained, and/or cyclic or acyclic. Examples of substituted hydrocarbon radicals include halo-substituted hydrocarbon radicals, hydroxysubstituted hydrocarbon radicals, nitrogen-substituted hydrocarbon radicals, and oxygen-substituted hydrocarbon radicals. Examples of hydrocarbon radicals include methyl, ethyl, propyl, butyl, pentyl, hexyl, octyl, and decyl. The units may be the same, as in a homopolymer, or be selected from two or more different units, as in a copolymer or terpolymer. A block copolymer is also encompassed by the term. The PHA can include one or more units, for example 10-100'000 units, preferably 100-30'000 units of the above Formula I. The polymers typically have a molecular weight above 270 Dalton, for example 10'000 to 3'000'000 Dalton.

By the term „biomass" is understood hereinafter plant biomass and/or microbial biomass (e.g., bacterial biomass, yeast biomass, fungal biomass, microalgal biomass, archeal biomass) . Bio- mass-derived PHA can be formed, for example, via enzymatic polymerization of the monomer units. The biomass can be formed of one or more of a variety of entities. Such entities include, for example, microbial strains for producing PHAs (e.g., Alcali- genes eutrophus/Cuprlavldus necator, Alcaligenes la- tus/ Azohydromonas lata, Rhodospirillum rubrum, Halomonas bluephagenesis , Azetobacter , Aeromonas , Comamonas , Pseudomonas') , Archaea (e.g., species of Bacillus, Halobacterium, Natronococ- cus, Natronobacterium, Halorubrum, Haloquadratum, Halococcus , Haloterrigena , Natrialba , Haloarcula , and Halo f erax) , and recombinant strains, genetically engineered organisms, preferably containing recombinant plasmids or introduced gene sequences on the chromosome, for producing PHAs (e.g., Escherichia coli species of Pseudomonas _r Ralstonia Klebsiella) , yeasts for producing PHAs, and plant systems for producing PHAs. Preferably, the PHA is isolated from plant biomass derived from plants such as sugar cane, switchgrass, soybean, cotton, coconuts, groundnuts, rapeseed, sunflower seed, olive, palm, sesame seed, linseed, castor, safflower seed, tobacco and potato. Most preferably, transgenic plants are used as a source for PHA. Transgenic plant derived PHA polymers or their derivatives can be processed and separated from plant biomass in commercially useful forms.

With regard to the method of reacting PHA to form an intermediate as claimed in claim 1, it is preferred that the formed intermediate is an ester, preferably an alkanoic acid ester. Examples of alkanoic esters include methyl 3- hydroxybutyrate, ethyl 3-hydroxybutyrate, methyl 3- hydroxypropionate, ethyl 3-hydroxypropionate, methyl 4- hydroxybutyrate, ethyl 4-hydroxybutyrate, propyl 3- hydroxybutyrate, propyl 4-hydroxybutyrate, isopropyl 3- hydroxybutyrate, isopropyl 4-hydroxybutyrate, butyl 3- hydroxybutyrate, butyl 4-hydroxyvalerate, derivatives of poly (3- hydroxypropionate) , etc. Treating a PHA to form an ester includes combining the PHA with an alcohol and - optionally - a catalyst, and exposing the PHA to elevated temperature and/or elevated pressure. Preferable alcohols include methanol, ethanol, propanol, isopropanol, and/or butanol, preferably n- butanol .

In an alternative embodiment, the formed intermediate may be an amide, preferably an alkenoic acid amide. Examples of alkenoic acid amides include N-methyl 3-hydroxybutyramide, N-ethyl 3- hydroxybutyramide, N-methyl 4-hydroxybutyramide, N-ethyl 4- hydroxybutyramide, N-hydroxyethyl 4-hydroxybutyramide, N-methyl 4-hydroxyvaleramide, derivatives of poly ( 3-hydroxypropionate, etc. Treating a PHA to form an amide includes combining the PHA with an amine (e.g. a primary, a secondary amine, such as ammonia, methyl amine, ethyl amine, pyrrolidone) and using elevated temperature and/or pressure.

By the solvent comprising butyl 3-hydroxybutyrate, is understood a solvent, such as an alcohol, mixed with an amount of cosolvent of the Formula II

By applying the inventive method and paricularly by adding a share of butyl 3-hydroxybutyrate as a co-solvent, the reaction temperature can be chosen in a moderate range, such as 90° to 220°C, more preferably 100° to 150°C, even more preferably 115° to 135°C. When an elevated pressure is applied (e.g., 2 to 14 bar, preferably 5 to 10 bar) , the temperature can be raised correspondingly, for example to temperatures above 135°C, preferably 140-160°C. However, it is an advantage of the present invention that the reaction can be performed even under atmospheric pressure. The yield obtainable under such conditions is well above 60%, preferably above 70%, typically even above 80%, with a good degree of purity. Experiments have shown that without the inventive co-solvent, comparable yields can usually be achieved only under difficult reaction conditions, i.e. higher temperatures and/or pressures, or after long reaction times.

The elevated temperatures for forming an amide are typically lower, e.g., between 50 to 90°C. It is preferred that the PHA provided in step a) are extracted from biomass, preferably extracted by a method as described hereinafter. It is particularly preferred that the PHA in step a) is provided as a pretreated substance, having a purity of > 90%, measured by gas chromatography of methanolised PHA or by weight loss determination after dissolution/precipitation cycle. Most preferably, the PHA in step a) is provided as pure poly (3- hydroxybutyrate ) .

By the term "poly ( 3-hydroxybutyrate ) " (PHB) is understood herein a structure comprising repeating monomer units of the Formula III, preferably, a homopolymer comprising repeating monomer units of the above Formula III. When the PHB is obtained from biomass, the monomer typically has a stereocenter ( A-conf iguration) , giving the following Formula IV:

However, the skilled person is aware that R- and S- conf igurations (racemic or pure) may be obtained if the polymer is obtained by way of chemical synthesis. It is possible to directly react a biomass comprising a relevant PHB content with an alcohol or an amine in order to form an intermediate. But the method as described above has turned out to give particularly high yields if purified PHA or preferably PHB is provided as a starting material.

In a preferred embodiment of the above method, the catalyst used in step a) is a Broensted acidic ionic liquid, preferably 1- methyl-3- ( 3-sulfopropyl ) -imidazolium hydrogen sulphate ( [HSO3- pmimJHSO . In principle, suitable catalysts include sulfuric acid, para-toluene sulfonic acid, hydrochloric acid, and phosphoric acid (protic catalysts) , but also aprotic catalysts including certain transesterification catalysts (e.g., metal-containing transesterification catalysts such as tin compounds, but also titanium, zinc compounds and clays are possible) . Alternatively, enzymes (lipases such as Candida antarctica lipase B) can be used as catalysts for transesterification. However, Broensted acidic ionic liquids are suitable due to their thermal stability, reusability, and miscibility with organic compounds. Methyl-!^ 3-sulfopropyl ) -imidazolium hydrogen sulphate ( [HSO3- pmimJHSO of the Formula V has turned out to be particularly suitable for the ease of recycling for subsequent reactions.

In an alternative preferred embodiment, FeCls or FeC12 can be used as a catalyst. FeCl ₃ or FeC12 has turned out to give partic- ularly high yields in a method as described above. The catalyst can, however, be advantageously used also on a more general level in an method of reacting polyhydroxyalkanoates (PHA) to form an intermediate comprising the steps of a) providing a reaction mixture comprising

- PHA, preferably purified PHA;

- a reagent, in particular an alcohol or an amine;

- FeCl ₃ or FeC12 as a catalyst; in a solvent; b) exposing the reaction mixture to elevated temperature and/or elevated pressure, preferably temperatures of at least 90°C.

The catalyst has shown to deliver particularly high yields well above 70%, e.g., of butyl 3-hydroxybutyrate, even when used under moderate reaction conditions (atmospheric pressure, temperatures between 90 and 140°C) .

It is preferred that the solvent used for the reaction comprises at least 5 wt-% of butyl 3-hydroxybutyrate, preferably 8 to 50 wt-% of butyl 3-hydroxybutyrate, more preferably 10 to 40 wt-% of butyl 3-hydroxybutyrate. The skilled person will appreciate that amounts > 40 wt-% are likewise possible. However, such amounts are not preferable from an economical point of view.

As described earlier, the invention includes a method for extracting polyhydroxyalkanoates (PHA) from biomass, comprising the steps of a) combining a biomass containing PHA with a solvent to form a biomass liquor; b) optionally : heating the biomass liquor to at least partially solubilize the PHA to form a PHA liquor ; c) recovering PHA from the PHA liquor; characterized in that the solvent used in step a) comprises butyl 3-hydroxybutyrate.

In the extraction method described above, steps a) to c) can be perfomed simultaneously or sequentially.

The above described method is particularly suitable for the extraction of short chain length PHA (scl PHA) . By the term «short chain length PHA» is understood hereinafter PHA of the above Formula I, wherein n is zero, one, two, three or four. It is particularly preferred that the biomass provided in step a) comprises a high amount of poly ( 3-hydroxybutyrate ) (PHB) , preferably a PHB content of > 80%. Most particularly preferred is the method for the extraction of poly ( A-3-hydroxybutyrate) .

Short chain length PHA (scl PHA) , and most typically poly(A-3- hydroxybutyrate ) , are often isolated from plant biomass derived from, e.g., sugar cane, switchgrass, soybean, cotton, coconuts, groundnuts, rapeseed, sunflower seed, olive, palm, sesame seed, linseed, castor, safflower seed, tobacco, potato, and algae. Such plants may be transgenic or wild type. Extraction of short chain length PHA places high demands on the performance of extraction solvent, due to the relatively high polarity of the polymer. The superior performance of butyl 3-hydroxybutyrate is more pronounced when used for extraction of short chain length PHA. For example, a quantitative extraction can be performed in comparatively short time under mild reaction conditions.

With regard to the method for extracting polyhydroxyalkanoates (PHA) from biomass, it is particularly preferred that the PHA in step c) is recovered from the PHA liquor by filtration. For example, the biomass residues (e.g. cell debris) may be filtered off the PHA solution. The filter cake may be dissolved once more in solvent, e.g., a different solvent, and filtered for increasing the PHA yield. Alternatively, the PHA may be recovered by precipitating the PHA from the solution using an anti-solvent such as methanol or ethanol or through other methods such as sedimentation, re-suspension, centrifugation, cooling, solvent evaporation and combinations thereof.

The use of butyl 3-hydroxybutyrate of the Formula IT has turned out to be particularly suitable for the purposes of extraction. Butyl 3-hydroxybutyrate can be used as an extraction solvent in steps a) and subsequently step b) . For example, the biomass can be suspended in the solvent in a ratio of 1-2 g per 100 ml, and the extraction can be performed at temperatures of 50° to 140°C, preferably 55° to 90°C, even at atmospheric pressure, to give a quantitative yield.

It is preferred that the solvent used in step a) comprises at least 5 wt-% of butyl 3-hydroxybutyrate, preferably 8 to 50 wt-% of butyl 3-hydroxybutyrate, more preferably 10 to 40 wt-% of butyl 3-hydroxybutyrate. The skilled person will appreciate that amounts > 40 wt-% are likewise possible. However, such amounts are not preferable from a commercial point of view.

It is preferred that in the method as described above, in step b) the biomass liquor is heated to temperatures of 50 to 140°C, preferably 55° to 90°C, at atmospheric pressure. High yields are thus obtainable even after short extraction periods. It is preferred that the biomass containing PHA is provided as a dry biomass . The PHA or preferably PHB may have been prewashed .

In a preferred embodiment the method as described above is for producing a biofuel and comprises the steps of a ) providing a reaction mixture comprising polyhydroxyalkanoates ( PHA) ; an alcohol , in particular methanol , ethanol , ( iso- ) propanol , butanol or a mixture thereof ; optionally : a catalyst ; in a solvent comprising butyl 3-hydroxybutyrate ; b ) exposing the reaction mixture to elevated temperatures and/or elevated pressure , preferably temperatures of at least 90 ° C ; and c ) combining the substance resulting in step b ) with a chemical fuel .

For the purposes of this invention, by the term „chemical fuel" is understood a fuel selected from the group of gasoline , diesel , and alcohol fuel . In particular, the alcohol fuel is selected from the group consisting of ethanol , n-propanol and n- butanol .

The inventors have found that PHA which is esteri fied by the method as described above can be reacted in a particularly ef ficient way into an additive for fuel , due to the excellent dissolvability of PHA in butyl 3-hydroxybutyrate . In particular, after having been used as a bioplastic, PHA can be turned into an additive for a fuel , whereby the application value of PHA is further increased . Alternatively, the PHA provided in the reac- tion mixture of step a) may be contained in biomass and/or may have been extracted from biomass, preferably by a method as described above.

It is therefore particularly preferred that the PHA provided in step a) are contained in biomass and/or in an object made from PHA-based plastic. For example, the biomass may be an active sludge from waste water treatment. It can be directly dissolved in butyl 3-hydroxybutyrate ; however, one or more optional iso- lation/purif ication steps may proceed the esterification reaction. In the case of a PHA plastic, the PHA may e.g., be present as a waste object made from PHA plastic, hence having an even better environmental impact due to the chain of applications.

It is particularly preferred that PHB, and more preferably poly ( .R-3-hydroxybutyrate ) , is provided in step a) of the method. Most preferably the PHB are contained in biomass and/or in an object made from PHA plastic. PHB is preferred not only due to their abundancy as a product of biomass synthesis. Also, PHB provides good combustion heat when admixed to a chemical fuel, and offers interesting commercial prospects.

The invention further relates to the use of butyl 3- hydroxybutyrate as an additive for a chemical fuel, preferably in a ratio of 5 to 40 wt-%, preferably 10 to 30 wt-%, of the total resulting fuel.

Experiments have shown that butyl 3-hydroxybutyrate is readily miscible with chemical fuels, in particular gasoline or diesel.

The following examples further illustrate the invention. They are not meant to limit the scope of the claims. The Figures show :

Fig 1 Diagram of measured torque over timing advance in engine tests for pure gasoline (SP98) , 20% Butyl 3-hydroxybutyrate blend, 20% Methyl 3- hydroxybutyrate blend and 20% n-Butanol blend, respectively;

Fig 2 Diagram of measured power over engine speed in engine tests for pure gasoline (SP98) , 20% butyl 3- hydroxybutyrate blend, 20% methyl 3- hydroxybutyrate blend and 20% n-butanol blend, respectively;

Fig 3 Diagram of measured torque over fuel-air equivalence ratio in engine tests for pure gasoline (SP98) , 20% butyl 3-hydroxybutyrate blend, 20% Methyl 3-hydroxybutyrate blend and 20% n-Butanol blend, respectively;

Fig 4 Diagram of measured engine efficiency relative to pure gasoline (SP98) over engine speed in engine tests for 20% butyl 3-hydroxybutyrate blend, 20% methyl 3-hydroxybutyrate blend and 20% n-butanol blend, respectively.

EXAMPLES

Example 1: Butyl 3-hydroxybutyrate from pure PHB at high pressure

In a 300 ml stainless steel autoclave, PHB (17.39 g, 0.20 mol, 1.00 eq) and the catalyst [HSO4] [HSCh-pmim] (3.02 g, 0.01 mol, 0.05 eq) in n-butanol (92.5 mL, 1.00 mol, 5.0 eq) were introduced. The apparatus was flushed three times with nitrogen (8 bar) and a leak test was performed to ensure the system integrity (8 bar for 10 min) . The reaction mixture was heated up to 140°C for 24 hours. Then, the device was cooled down to room temperature. The mixture was filtered to remove the unreacted PHB . The filtrate was concentrated under vacuum. The crude oil was distilled under reduced pressure (0.3 mbar, 60-70°C) to afford a colourless liquid (26.1 g, 0.163 mol, 82%, purity = 95 ± 3%) .

The distillation residue was reused as catalyst without further treatment for the next synthesis.

¹ HNMR (300 MHz, chlorof orm-d) 5 4.33 - 3.97 (m, 3H) , 3.21 (s, 1H) , 2.62 - 2.33 (m, 2H) , 1.63 (quint, 2H) , 1.39 (sext, J = 14.4, 7.3 Hz, 2H) , 1.23 (d, J = 6.3 Hz, 3H) , 0.94 (t, J = 7.3 Hz, 3H) .

¹³ CNMR (300 MHz, chlorof orm-d) 5 173.05, 64.57, 64.25, 42.77, 30.55, 22.41, 19.07, 13.63.

Scale-up experiments have been performed successfully with comparable yields (data not shown) .

Example 2: Butyl 3 -hydroxybutyrate from pure PHB at atmospheric pressure with 20% B3HB as co-solvent

In a 100 mL round bottom flask equipped with an air condenser, the catalyst [HSO4] [HSCh-pmim] (1.85 g, 6.12 mmol, 0.05 eq) and PHB (10.67 g, 122.5 mmol, 1.0 eq) in n-butanol (45 mL, 490 mmol, 4.0 eq) and butyl 3-hydroxybutyrate co-solvent (9.39 mL, 56.86 mmol, 20% of the solvent/co-solvent solution) were introduced. The apparatus was flushed three times with argon. The reactive mixture was mechanically mixed with a half-moon shaped PTFE stirrer. The jacket was heated in order to reach a T _r of 115°C (solvent reflux) for 24 h. Then, the reaction mixture was cooled down to room temperature, filtered in order to eliminate the PHB residues and transferred in a 100 mL single neck round bottom flask connected to a distillation apparatus. N-Butanol was evap- orated in a vacuum ( T j 60°C, 30 mbar) . The product was distilled under high vacuum ( T j = 110°C, T _vap = 35°C, p= 3.7*10~ ³ mbar) to give 0.78 g crude butyl 3-hydroxybutyrate (80% yield by NMR assay using 1 , 4-dimethoxybenzene as internal standard and corrected considering the initial addition of butyl 3-hydroxybutyrate as co-solvent) .

Scale-up experiments have been performed successfully with comparable yields (data not shown) .

Example 3: Butyl 3-hydroxybutyrate from pure PHB at atmospheric pressure by using FeCla as a catalyst

In a 2 L glass reactor equipped with an air condenser, the catalyst FeCls (35.9 g, 0.218 mol, 0.1 eq) and PHB (190.4 g, 2.18 mol, 1.0 eq) in n-butanol (1 L, 10.98 mol, 5.0 eq) were introduced. The apparatus was flushed three times with argon. The reactive mixture is mechanically mixed with a half-moon shaped PTFE stirrer. The jacket was heated in order to reach a Tr of 115°C (solvent reflux) for 24 h. Then, the reaction mixture was cooled down to room temperature, filtered in order to eliminate the PHB residues and transferred in a 2 L single neck round bottom flask connected to a distillation apparatus. N-Butanol was evaporated in a vacuum (Tj 60°C, 30 mbar) . The product was distilled under high vacuum (Tj = 110°C, Tvap = 35°C, p= 3.7*10-3 mbar) to give 319.5 g crude butyl 3-hydroxybutyrate (92% yield by NMR assay using 1 , 4-dimethoxybenzene as internal standard) .

Example 4 : Butyl 3-hydroxybutyrate from PHB biomass with 6% B3HB as co-solvent

4.0 g (corresponding to 3.44 g PHB, 0.040 mol, 1.00 eq, ) of biomass with a PHB content of 86.2% were washed with 40 mL of acetone during 1 h, filtered and dried in an oven (40°C, 3 h) . This pre-washed biomass was introduced in a 100 mL round bottom flask equipped with an air condenser. The catalyst FeC12 (520.1 mg, 0.0040 mol, 0.1 eq) in n-butanol (54.5 mL, 0.60 mol, 15.0 eq) and 3-hydroxybutyrate co-solvent (3.2 mL, 0.019 mol, 6% of the solvent/co-solvent solution) were introduced as well. The apparatus was flushed three times with argon. The reactive mixture is magnetically stirred. The jacket was heated in order to reach a T _r of 115°C (solvent reflux) for 24 h. The reaction mixture was cooled down to room temperature, filtered in order to eliminate the catalyst and biomass residues, then transferred in a 50 mL single neck round bottom. N-butanol was evaporated in a vacuum (Tj 60°C, 30 mbar) at the rotatory evaporator to give 4.02 g crude butyl 3-hydroxybutyrate (63% yield by NMR assay using 1,4- dimethoxybenzene as internal standard and corrected considering the initial addition of butyl 3-hydroxybutyrate as co-solvent) .

Example 5: PHB extraction from biomass

1.6 g (corresponding to 1.0 g PHB) of biomass with a PHB content of 86.2% were washed with 20 mL acetone during Ih, filtered and dried in an oven (40°C, 3 h) . This pre-washed biomass was introduced in a 200 mL round bottom flask equipped with an air condenser. 100 mL of butyl 3-hydroxybutyrate were introduced as well. The suspension was magnetically stirred during 1 h at 60 °C. The obtained white suspension was filtered. The solid part was dissolved in 100 mL of 1 , 1 , 1-3 , 3 , 3-hexaf luoro-2-propanol and filtered again in order to separate the extracted PHB from the biomass residues. The two solutions containing PHB were evaporated (Tj = 110°C, Tvap = 35°C, p= 3.7*10-3 mbar for butyl 3- hydroxybutyrate solution and Tj = 40 °C, 200 mbar for 1,1,1- 3 , 3 , 3-hexaf luoro-2-propanol solution) . The obtained white solid was dried in an oven at 60 °C for 6 h (quantitative extraction, 1 g PHB) . Example 6: Engine performance using M3HB and B3HB as a fuel additive .

Butyl 3-hydroxybutyrate (B3HB) was added to gasoline (SP98) to give an amount of 20 vol-% and was measured to have a density at 20°C of 0.78 g/mL and a combustion heat of 38'664 J/g. This is well comparable to known conventional fuel additives: Methyl-3- hydroxybutyrate (M3HB) was added to gasoline (SP98) to an amount of 20 vol-% and the fuel was measured to have a density at 20°C of 0.79 g/mL and a combustion heat of 37'381 J/g. N-butanol was added to gasoline (SP98) to an amount of 20 vol-% and the fuel was measured to have a density at 20°C of 0.75 g/mL and a combustion heat of 40'416 J/g.

The fuels, each including an additive (M3HB, B3HB, n-butanol) , were tested in a test engine. Overall, the engine ran smoothly on all fuels. There were runs in a series of comparative studies for each blended fuel and commercial grade gasoline (SP98) as such :

Fig 1 shows a plot of corrected measured torque ( [Nm] ) over timing advance ( [°] ) for pure gasoline (SP98) , 20% butyl 3- hydroxybutyrate blend, 20% methyl 3-hydroxybutyrate blend and 20% n-butanol blend respectively. Almost identical torque values were achieved for all fuels at 4000 rpm during optimisation of ignition timing.

Fig 2 shows a plot of measured corrected power ( [ kW ] ) over engine speed ( [rpm] ) for pure gasoline (SP98) , 20% butyl 3- hydroxybutyrate blend, 20% methyl 3-hydroxybutyrate blend and 20% n-butanol blend respectively. Almost identical power values were achieved for all fuels at full load. Fig 3 shows a plot of corrected torque over fuel-air equivalence ratio for pure gasoline (SP98) , 20% butyl 3-hydroxybutyrate, 20 methyl 3-hydroxybutyrate and 20% n-butanol respectively. Similar torque values were achieved for all fuels at 4000 rpm. Nevertheless, it was found that on the test engine, the alternative fuel containing 20% B3HB had a higher minimum fuel-air equivalence ratio than the other fuel blends, i.e., it had to be slightly richer for the engine to maintain combustion.

Fig 4 shows a plot of engine efficiency (cylinder pressure) relative to pure gasoline (given in %) over variable engine speeds. The cylinder pressures for all fuels were similar to those achieved with SP98, with maximum variations ranging from -1.7% to +8.2% compared to SP98. In principle, the relative efficiency of the different fuel blends is slightly lower than that of standard SP98 fuel. This may be explained by the fact that the fuel blends have lower heating values (LHV/HHV) than pure commercial grade gasoline and the engine was designed specifically for such fuel.