PRIORITY CLAIM

The present Application claims benefit of priority from U.S. Provisional Application No. 61/739.790, f led December 20.. 2012, die contents of which are herein incorporated.

F ELD OF IN VEN TION

The present inventio relates to a process for the production of certain biofueis. I particular, the invention pertains to a method or producing biofuei products from bio-derived esters of carboxylic acids by means of either hydrogenstson or hydrogenolysis,

BACKGROUND

Development of alternative fuels has gained increasing importance with increased awareness of environmental concerns and the wake of economic pressures and fuel costs that have resulted from supply volatility of petroleum and other fossil-based resources. Policy makers have recognized this importance and have encouraged production and increased use of biofueis. As interest has grown i moving away from petrochemical or natural gas-derived hydrocarbon sources, some have concentrated, on finding renewable and sustainable "green ^" carbon resources. Early so-called first generation biofueis are made by fermenting food products (e.g., starch-containing and sugary plants, such as corn, wheat, or sugar cane) into short-chain alcohols (e.g. ethanol, bntaaol) or tamsesterifymg fatty acids liberated from plant oils by giyeerolysis (e.g., rape seed, soybeans) to make fatty acid methyl esters used as biodiese!. (See e.g.. Cesar B, Granda et i., "Sustainable Liquid Biofueis and Their Environmental Impact," ENV IRONMENTAL PROGRESS. Vol. 26, No. 3 , pp. 233- 250 (Oct. 2007), incorporated herein by reference.)

Carboxylic acids such as used to make biodiesei can also be made by .fermentation from carbohydrate sources, in addition to being derived from plant oils. Although the fermentative production of carboxylic acids, such as malic or succinic acid, has several advantages over petrochemical-based fuels, the production costs for the bio-based carboxylic acids have been too high for bio-based production to be cost-competitive with petrochemical production regimes. (See e.g.. James McK inlay ef a!., "P os ects for a Bio-based Succinate indust ;' Ann ^; . Mk -ROi L.

ΒΚΧΓΕΓΗ ΌΙ... (200?) 76: 72? -740; incorporated herein by reference.) n fermentation of starches and sugars, carboxylic acids are generated using microorganisms. Microorganisms require certain conditions for their functions, for example, with most commercially viable succinate producing, microorganisms described in the literature, one needs to neutralize the fermentation broth to maintain an appropriate pH for maximum growth, conversion and productivity.

Currently, the carboxylic acids are recovered from fermentation broths as salts instead of as free acids. Typically, the pi t of the fermentation broth is maintained at. or near a pH of 7 by introduction of ammonium hydroxide or other base hue- the broth, thereby converting the carboxvlic acid into the corresponding acid salt. Generation of carboxvlic acid s lts versus the tree organic acid torn? brings about significant processing costs. About 60% of the total production costs are generated by downstream processing, because of the difficulties associated with the separation and purification of the acids and their salts in the fermentation broth.

Recovery of carbox lic acids as salts has number of associated problems and requires several different steps m post- fermentation, downstream processing to isolate free acids and to prepare the carboxvlic acids for chemical transformation and to convert the raw acids to useful compounds. When salts are generated in conventional fermentation processes, an equivalent of base is required for every equivalent of acid to neutralize. The amount of reagent used can increase costs. Further, one needs to remove the counter ions of the salts so as to yield free acids, and one needs to remove and dispose of any resulting waste and by-products. For instance, calcium salts of diacids have a very low solubility in aqueous broth solutions (typically loss than 3 g liter at room

temperature), and are not suitable for many applications for which a f ee acid species is needed, such as chemical conversion to derivative products like butanedioi and b fueis. T herefore, the calcium salt is typically dissolved in sulfuric acid, forming insoluble calcium sulfate, which can readily be separated from the free diacid. Calcium sulfate is a product having lew commercial applications, and accordingly is typically discarded as a solid waste in landfills or other solid waste disposal sites. All of these individual operational units contribute to the overall costs of the process,

To reduce waste and costs associated with generating free car boxy lie acids and to improve the recovery yield., a need exists for a better, more direct method of recovering a variety of carboxvlic acids, such as malic or succinic acid, and which can provide a successful route to combine a biologically-derived hydrocarbon feedstock with the production of various biofuei products, such as ethane, propane, propaooi or butanoi by means of hydrogen at ion or hydrogenolysis. Such a streamlined, green process would be a welcome innovation.

SU MARY OF THE INVENTION

The present invention concerns a process for producing biofuels. The process involves; a s obtaining a fermentation broth containing at least one free carboxvlic acid, or a mixture of carboxvlic acids, or a mi.xh.tre of carboxvlic acids of at least one carboxvlic acid and associated alkali or alkaline earth metal salts thereof; b) drying said fermentation broth containing free carboxvlic acids into a powder; and c) reacting said carboxvlic acids in said powder with an alcohol solvent or mixture of at least one alcohol under a CO atmosphere in substantial absence of any other acid catalyst at a reaction temperature and pressure that, corresponds to supercritical, critical or near critical conditions for the alcohol or CO; to synthesi e the corresponding ester or esters: d) subjecting the esters formed to either hydrogenolysis or hydrogenation to form one or more biofuels; and e) recovering the one or more biofuels. The ester can be a mono-ester, di-ester, or fri-ester. Preferably, the ester is a di-ester or tri~esk ^; r. Monoesters can convert to mono-alcohols., diesters to diols, and t iesters to triols. The biofuet products can be either an alkane or alcohol.

The esterifkation reaction temperature is between about i 50°C and about 250°C, and the operational reaction pressure is between about 400 psi and about 3,000 psi. Depending on the desired results, the ester ification reaction can be run for at least about 4 hours.

Additional features and advantages of the present methods will be disclosed in the following detailed description. It is understood that both the foregoing summary and the following detailed description and examples are merely representative of the Invention, and are intended to provide an overview for understanding the invention as claimed.

BRIEF DESCRIPTION OF FIGURES

f 10. i is a schematic diagram illustrating an iteration of the present process for es erifying an organic carboxyhc acid derived from fermentation broth, and further downstream processes that can Isolate the resulting esters and/or generate other compounds from such esters.

FIG. 2 is a schematic diagram showing an example of ester production using succinic acid derived from fermentation, and a downstream process in which Na and Mg salts are recycled back into the fermentation reactor, in accordance with a part of art embodiment of the present process.

FIG. 3 is a diagram that illustrates CO _? -assisted esterifleation of free succinic acid in various alcohols that are converted to corresponding dimethyl, diethyl, or dibntyl esters, according to the present invention.

FIG. 4 is a diagram that illustrates COr-assisted esterifleation of other polycarboxyl ie acids. FIG, 5 shows a series of reaction diagrams that summarize variations in temperature tor CO assisted esienfioarkm of free succinic acid deri ved from fermentation broth.

FIG. 6 shows a series of reaction diagrams that, summarize variations in initial operational pressure for COj-assisted esterifleation of tree c&rboxylic acid according to the invention.

FIG. 7 shows a series of reaction diagrams that summarize variations in temperature, and reaction times for CO; assisted esterifkation of free oarboxyiie acid according to the invention.

FIG. 8 shows a series of reaction diagrams that summarize the results of reaction succinic acids and their Mg'" and Ca ^?"' salts.

DETAILED DESCRIPTION OF THE INVENTION

Section. 1. - Description

A.

The present disclosure describes, in part, a. process tor making various bioinei products by .means of either hydrogenatioo or ydrogeoolysis from a biologically-derived carbon source, such as sugar or other plant-based carbohydrates. The process joins an ability to recover a carboxyiic acid from a fennentation broth, with an ability to use the acid as a feedstock for biofael generating reactions in a streamlined procedure. The present process includes a method of converting the carhoxylic acid to its corresponding ester (e.g., mono-ester, di-ester, or tri-ester) in a relatively efficient and cost effective manner.

As used herein the term hiofueis" refers to a gaseous, liquid, or solid substance that is used as a. fuel, which is produced from renewable biological resources such as plant, cei!u!osic, or agricultural biomass or derivatives thereof. In particular, a biofuel refers to a materia! that can be used in or as a transportation fuel in interna! combustion engines, to power certain machinery, or energy generation applications. For instance, propanoi and butanol can be a gasoline additive much the same as efbanol. Butane and propane in liquefied petroleum gas (LPG) and ethane in natural gas can he adapted as fuels in certain transportation systems. Other biologically-derived hydrocarbons,, like oetaooi/octane. or alfcanes heavier than C _s or€ _<¾ may also be biofuels.

In particular, the present process involves obtaining a fermentation broth, from which cell mass and insoluble compounds have been either removed or not, containing a mixture of free carhoxylic acid of interest, optionally with associated alkali or alkaline earth metal salts (e.g., sodium, potassium, or magnesium salts); drying the ra or clarified fermentation broth containing free carhoxylic acid into a powder; reacting the carboxy!ic acid in the powder with an alcohol under a COrContaining atmosphere in substantial absence of any other acid catalyst a t a reaction temperature and pressure corresponding to supercritical, critical or near critical conditions for the alcohol and/or CO, ^, to synthesize an ester; and subjecting the ester to either hydrogenation or hydrogenolysis to form a. hiofuel product. As used herein the term ^vi free carhoxylic acid ^" refers to a carhoxylic acid compound that is at least 50% in its protonated state when in solution, at or below its pXa value. As used herein, the term "substantial absence ^" refers to a condition in which an acid catalyst is either largely or completely absent, or is present in minimis or trace amount of less than catalytic efficacy. In other words, no other acid catalyst is present, or is present at a level less than 10%, 5¾, 3%, or 1 % weight/weight relative to the carhoxylic acid in the reaction.

In another aspect the present invention involves the discovery of a simple but effective way of producing esters from carhoxylic acids that are otherwise costly and difficult to isolate. An advantage of the present invention is that, one can use free carhoxylic acids directly from a

.fermentation broth and generate corresponding esters therefrom without, the .need to isolate or purify the acids from the fermentation broth, as is necessar in conventional extractions from broth.

In comparison to certain fermentation processes that neutralize or convert the organic acids to their salts, the present process provides an easier way to isolate and extract organic acids iron ⁾ a fermentation broth. The present process eliminates a need for titration and neutralization of the fermentation broth that can precipitate metal salts, and certain purification steps to produce stock platform chemical The free organic acids are converted into esters, which are simpler to process and extract by distillation or other purification techniques without the use of expensive and complicated chromatographic separation columns or resins. For instance in a conventional process, one would need to use ion exchange chromatography to isolate the acids. A small amount of salts may unavoidably carry-over after the ion exchange. Hence, one may require mul iple units of operation to purify the acid to an acceptable quality level. With each added operational unit, the costs of the overall process increases. In contrast, with the present process la synthesizing the ester of the acid, one can .recover the salt as a carbonate or hydroxide, which can be used to regenerate the fermentation broth, and minimi?* waste, Rattier, an advantage of the present process is that one may further recycle- the synthesis by-products directly back into the fermentation broth. By converting the carboxyllc acids to their corresponding esters, we can avoid, such issues,

B.

Conventionally, esters are produced when carboxyllc acids are heated with alcohols in the presence of an acid catalyst. The mechanism for the formation of an ester from an acid and an alcohol are the reverse of the steps for the acid-catalyzed hydrolysis of an ester. The reaction can go in either direction depending on the conditions used. In a typical esterification process, a carboxyllc acid noes not. react vvith an alcohol unless a strong acid is used as a catalyst The catalyst is usually

concentrated sulfuric acid or hydrogen chloride. Protonation makes the carbonyl group more electrophilic and enables it to react with the alcohol, which is a weak nucieophile.

in general terms, the present esterification method involves a reaction of fermentation- derived, free organic carboxyllc acid with an alcohol in a carbon dioxide (CO; -oontaining atmosphere in substantial absence of any other acid catalyst to produce esters. T he esterification reaction is performed in solution under conditions that are either at supercritical, critical or near critical temperatures and/or pressures for either the alcohol and/or CX¾. Under such conditions, we believe that CO.- self-generates or functions in situ as an acid catalyst, and regenerates back alter the esterification reaction is completed. It is believed that a reactive intermediate nonoalkyloarhonic acid) is being made situ in large enough quantities to drive esterification and affect ester production. This intermediate, having a. similar pKa {e.g.,-4-5} as the f ee carboxyllc acid, functions as a carbonic acid, which is much weaker than the usual strong acids. The observed trend of greater ester conversion at higher temperatures adduces a relatively large energy of activation tor this process.

An advantageous feature of the inventive process is that activation of the free carboxylic acid as an acyi halide (e.g., fluoride, chloride, bromide) or by using strong mineral acids is unnecessary unlike with some other techniques. Acyi halides are inconvenient to use because these species are inherently reactive, have issues with stability, waste treatment, and can be cumbersome and costly to snake.

in the present process, carbon dioxide functioning as a catalyst instead of the usual strong acids removes the need to introduce a strong acid into the esterification reaction. This feature can circumvent the usual need to adjust pH values in order to remove the catalyzing acid, enabling a simpler and cleaner synthesis. One can simply proceed to filter the resultant product to remove alkali or alkaline earth metal carbonate or othet salts. A cleaner product wis! save co ts in purification and downstream processing tor conversion to other chemical feedstock.

The process described herein is a snore environmentally benign way of producing esters. As it. is believed that the carbon dioxide can self-generate an acid catalyst in situ in the presence of the alcohol during the estenffcation reaction, the present method does not - equire the use or addition of another acid catalyst species, In other words, the reaction kinetics with 0 alone can drive the esieniicia ^' ion I the substantial absence of any other acid catalyst. To reiterate, the present process does not require activation of free acids as, for example, an acyl chloride or by strong acids (i.e., Fischer esterificaiion).

In general, the esterifieation is conducted at an operational or reaction temperature between about I SOX to about 2S0X, at a reaction pressure of between about 400 psi and 2,500 psi, for an extended period, such as 4 hours, up to about 12 hours. Typically, the temperature can be in a range between about 170 ^C C or 1 0°C to about 230%: or 245°C (e.g., 175 X, i 87°C\ 195 or 2 15X). and die operational pressure is between about 900 psi or 950 psi and about 2,200 psi or 2,400 psi (e.g., 960 psi, 989 psi, 1020 psi or 1050 psi). Alternatively, the temperature can be in a range between about 1 SO to about 2 5X (e.g., about 1 S5X or 200X or 210X to about 220X or 235X or

240X) and the operational pressure is between about 1090 psi and 2,350 psi (e.g., 1 , 100 psi, 1 ,200 psi, ! ,550 psi, 1 ,750 psi, 1 ,810 psi, or 1 ,900 psi). Other temperatures .may be within a. range, for example, from about 1 OOX or 185X to about 1 OX or 225X, and other operational pressures may be within a range, for example, from about 1 , 150 psi or ! ,500 psi to about ] ,800 si or 2.000 psi.

These reaction temperatures arid pressures correspond to supercritical, critical or near critical conditions for the alcohol(s} or€0>, Table I lists, for purpose of illustration, critical parameters for .some common solvents (i.e., methanol, ethanoi, 1 -propanoi. b-buianol, water, and€C½).

Table .1. Critics; Data for Select Substances {Yaws, C. L, Chemical Ρ?ο ·?οΐ½ Handbook, in McGraw-Hill: 199$; pp Χ-Ά)

Substance Nsm« Mo!eeulaf Weight Cfitica! lump, f }/T Critical Pressure {bsri pss Critical Density {g/em }

Methanol 32.042 512.58 / 239.43 S0.967 U74.22S5 0.2720

Ethanoi 46.069 516.25 / 243.10 63.84 / 925.9209 0.7.760

1-Propanol 60.095 537.4 / 264.25 51.02 / 739.9839 0.2754

t'-But no! 74..I S.?. $63.0 ± 0.3/ 2SS.8S 45.0 ± 4.0 / 652.671 0.3710

Water 18.015 647.13 / 373.98 220.53 / 313S.S071 0.3220

Carbon dioxide 44.010 304,19 / 31,04 73.82 / 1070.6885 0.4682

At conditions above the critical point li e,, critical temperature and/or pressure), the .fluid exists in a super critical phase where it exhibits properties that are in between those of a liquid and a gas, specifically, supercritical fluids have a liquid-like density and gas-l ike transport properties (i.e., diffusivity and viscosity }. This can be seen in Table 2, wherein the typical values of these properties are compared between the three fluid types - conventional liquids, supercritical fluids, and gases. Viscosity iPa-s}

Likewise, "near critical" refers to the conditions at which either the temperature or pressure of at least the alcohol species or CO? gas is below but within 1.5 OK (e.g., within 50- i 00K), or 220 psi (e.g.. within 30- 150 psi) of their respective critical points. It is believed that as temperatures and pressures reach near critical, critical or supercritical conditions, the solubility of the reagents are enhanced. which promotes the esterification reaction., in other words, the€<¾ gas, alcohol, and acid species are better able to interact under near critical, critical or supercritical conditions than under less rigorous conditions. The reaction does not require that both the alcohol species and C ^' Ov gas be at near-critical, critical or supercritical conditions; rather, the reaction is operative as long as either one of the species satisfies such a condition.

If the present esterification reactions are operated at. higher temperatures and greater pressures, up to about 2S0°C and 2,500 psi or 3,000 psi, respectively, for reaction tim.es of up to about 10 or 12 hours, one can produce significant amounts of ester product at relati vely high selectivity and level of purity within a shorter reaction time than before, which was about 18-20 hours. At lower operational temperatures (< 90°€), formation of monoester molecules of polycarboxylic acids is more prevalent, while reactions at temperatures > 1 0 ^':' € or I95€, will convert preferentially the polycarboxylic acids to diesters. By selecting a temperature in a higher range from about 1 0''C or 195% ^' or 200%? to about 245X or 2S0X\ one can preferentially drive the reaction to a greater rate of diester conversion. The esterification can yield a minimum of about 50%, desirably at least 65% or 70%, of a diester of the carbox lk acid. Reactions that are performed at. or near supercritical operating conditions tend to produce better results. When operated at or near critical conditions of about 230X or about ?A °C for methanol and about 3 i°C/1000 psi for€<¼, one is able to achieve conversions rates of about 90% or better, typically about 93% or 95%. One can achieve high yields by adjusting the permutations of different combinations of temperature and reaction times (e.g., higher temperatures and shorter reaction times (e.g., less than 10 or 12 hours, between 4 and 8 hours) or vice versa), which can be an advantage over current approaches. With optimization, esterification conducted at 250%; under either the same or greater CO; pressure, the yield would be nearly quantitative (i.e., 95% yield), for example, up to about 98%, 99%, or 99.9% conversion.

As the accompanying Examples will show, variation in reaction conditions suggests that one can generate more diester product with higher temperatures and/or protracted reaction times. As stated before, however, different permutations in temperature can influence the duration of the esterification reactions to produce the same amount of ester product. The reactions according to the present method are not conducive to a significant degree of side product formation; hence one can avoid eyclixatioo of the carbox lie acids and other starting reagents. Potential dangers of

decarb xylatio at high temperatures (i.e., 145%: or >150X) are not observed in the present ^, method. Using an amount of the alcohol solvent in excess of the carboxylic acid, one can produce a very clean esterification. The present synthesis process produces very clean ester products at about ?0 -?2% initial purity, without generation of .significant amounts of side products such as low molecular weight acids - acetic or formic acid - molecular rearrangements or cyclic products, which one could normally .find in standard acid catalyzed esterification at high temperatures. The esters can be reoned to achieve about 90-98% purity. The purification car; be accomplished, for instance, by means of crystallization, chromatography, or distillation.

Typically, the resulting ester products can he either monoesfers or d testers, or form a mixture of both. One can control the reaction to drive the esterification toward either one ester form or another. For instance, one may select an operational temperature and pressure that preferentially drives the esterification reaction towards formation of diester molecules. Likewise, one can control whether esters are formed from either a single carboxylic acid species (e.g., succinic acid) or a .mixture of multiple different kinds carboxylic acids (e.g., acetic, citric, lactic, malic, maleic, succinic acids) that may be present and derivable from fermentation broth, in other words, one can use a variety of different kinds of carboxylic acids in accord with the present esterification reaction to produce a variety of different esters. These esters, in turn, can be isolated, further modified in downstream chemical processes and converted, in certain embodiments, into useful compounds such as for pharmaceutical cosmetic, food, feed, polymer materials or biofuel . For instance, succinic esters can be converted into a polymer, such as polybutylene succinate (PBS).

l the present esterification process, both the catalyst (COj) and the esterification reagent

(alcohol ? are present in large excess relative to the amount of free carboxylic acid. CO should be in the gas phase during the reaction phase, regardless of its origi (e.g., gas tank or dry ice), as the reaction is conducted at high temperatures. Addition of solid CO _? is strategic in the ease where sealed pressure reactors are used, in that it allows for slow sublimation of gaseous C<¾ formation as the reaction apparatus is being assembled. This can minimize CO; loss. I.n a€€¾ (i.e., COrContaining) atmosphere, the concentration of CO-;, in the reaction atmosphere can be at least 10% or 15% by volume, favorably about 25% or 30%, preferably greater than 50%, For better reaction results, the concentration of CO _? , should be maximized. Desirable concentrations of€0 ₂ are from about 75% or 80% to about 99.9% by volume, typically between about 85% and about <>S%. Nitrogen (>½} gas or air is perm issible in the reactor, but preferably the concentration of gases other than CO - is kept, at either a minor percentage (< 50%) or de minimis amount.

Any liquid alcohol with an R-group of C :-€;«, can serve as the solvent reagent or first alcohol species. The -aroup cats be saturated, unsaturated, or aromatic, A mixture of different kinds of alcohols (e.g.., C _: -<.¾) can also be used in the reaction, but will produce a corresponding mixture of different esters depending on the particular R-group. Certain lower alcohol species with C _r Q alkyi groups are preferred as the reagent in the fir esterification with C<¼ in view of their common availability, inexpensiveness, and mechanistic simplicity in the esterification reaction. Further, alcohols such as methanol, sthanoi, propanol, or butanol arc preferred because of parameters such as their comparatively simple structure and that the reactions are more easily controlled with respect to the supercritical critical or near critical temperatures and pressures of these alcohol species.

Alternatively, in some embodiments, the alcohol can also be a C ^' yCVdiol. Esterifieatiou with a d»ol can generate monomers or low molecular weight oligomers that, can foe readily polymerized.

One can use a variety of different carboxylic acids, for example, selected from the group consisting of: a) monocarboxylic acids: formic acid, acetic acid, propionic acid , lactic acid, butyric acid, isobutyric acid, valeric acid, hexanoic acid, heptanoic acid, decanoic acid, laurio acid, myristle acid, and€;.··€;* fatty acids; b) dicarbox iic acids: fumaric acid, naconic acid, malic acid, succinic acid, maleic acid, rnalonic acid, glutaric acid, glucaric acid, oxalic acid, adipic acid, pimelic acid, suberic acid, azelaic acid, sebacic acid, dodecanedioic acid, giutaconic acid, ortho-ph hallc acid, isophihaiie acid, terephthalic acid; or c) tricarboxylic acids: citric acid, isoci ric acid, aeonhie acid, tricarbailyiifc acid, and trimesic acid. The carboxylic acids can include a mix of associated alkali or alkaline earth metal (e.g., sodium, potassium, or magnesium) salts of these carboxylic acids.

Desirably, the carboxylic acid is a poiyearboxylic acid, such as a dicarboyx!k or tricarboxylic acid.

The process can reduce the amount of waste by means of recycling of by-products back into the fermentation broth, either in a continuous or batch process. We have also found that in the present esterification process, when, free carboxylic acid is reacted with an alcohol and CX¾ absent any other acid catalyst, the free protonated form of the carboxylic acids has greater solubility in the alcohol solvent than their corresponding salts. Performed under similar reaction conditions, the esterification reaction using the free carboxylic acid as a reagent will yield about 2-3 times greater amount of ester product tlian the reaction that uses the salt species as a reagent. {This result can be seen when one- compares the reaction of accompanying Figure 38 (free acid) with that of Figure 5.A. (acid salt), and in Table 4. Examples 2 and 3 (acid salt), with Examples 5 and 6 (free acid}, respectively.) It is believed that solubility is a factor for the difference. For instance, since the solubility of magnesium salts in methanol and ethanol are significantly better than that of calcium salts, product yield from a reaction of a calcium salt is much lower than that produced from a starting reagent of a corresponding magnesium salt.

The present invention includes a method for estcrifying a poiyearboxylic acid. The esterification method involves: providing and reacting a solution of one or more free carboxylic acids with an alcohol in CO; atmosphere without the presence of any other acid catalyst; and selecting an operational reaction temperature or reaction pressure corresponding to supercritical, critical or near critical conditions for the alcohol and/or CO? to yield an ester. The reaction temperature and pressure conditions preferentially drive the reaction towards the formation of at least diester molecules over monoester molecules when the carboxylic acid is a poiyearboxylic acid. As with the recovery- process, the operational reaction temperature is between about 1 5Q°C and about 50°C, and the operational reaction pressure is between about 400 psi and about 2,500 psi. Depending on the desired results, the reaction can be run for up to about 1.2 hours,

C.

The estenfieatioo process described above can be integrated into fem-entation-based production of carbon chain feedstocks and to provide a snore convenient method of generating esters from earboxyiie acids derived ftom a renewable source. The present invention provides a direct route by which biologically-derived earboxyiie acids can be recovered in a simple, cost-efficient process from a fermentation broth, converted into esters, and then subjected to hydrogenation or

hyd ogenoiysis to snake biefaels. he accompanying figures illustrate how the esters are integrated into further downstream processes.

Figure 1 is a schematic representation showing a general process of extracting carboxylic acids from fermentation broth that includes a version of the present esterification reaction integrated with further processes that can utilize the resulting esters. As shown, fermentation broth 10 from a reactor is filtered (ultra) 12 to remove biomateriais such as cell mass, and yield earboxyiie acids including their salts, by products and other compounds. All of these materials are then dried 13 to make an unrefined mixture 14. This dried mixture of -materials is then reacted 16 in a liquid system with an alcohol (R-C)H; R ^::: aikyi .-C- ) and€<¾ at an elevated operational reaction temperature and pressure to yield either monoesters or diesters, or a mixture of both. Only the earboxyiie acids react in solution. The resulting mixture 18 is filtered 20 to separate the esters 22 and other by-products 24. The esters are soluble while other by-product compounds are insoluble. The by-products include carbonate salts of calcium, magnesium, or sodium, which can he recovered and recycled 26 back into a fermentation broth 28 in the reactor JO. This recycling can lead to significant cost savings and improves the efficiency of the overall fermentation and extraction process. The esters can be processed subsequently either by distillation SO, hydrogenation 32, or hydrogenioysis treatment 34, respectively, to separate the different esters, produce C.< platform compounds and biofue!s ⁽ e.g., ethane. ethanoL butane, butanol, etc.)

Through the distillation process one can concentrate the esters by driving off the alcohol, and then filter the by-products resultant from ester synthesis. Further distillation of a mixed-acid ester product mixture according to the boiling points of the different ester species., permits one to separate the various individual esters. For instance, Table 3 provides boiling points for a am l of common esters that may be present in an ester product mixture according to the present invention.

$ 0 inmi;;:hyS"di:r«te 176 (16 mm Hg) fr ^j ethyf-ct ^' ttate 3SJ1SG mm Kg)

After recovering the esters in the retra ning solution, the materials are in a readily usable form and one can either distill the ester mixture to separate the different ester species and any remaining alcohol. Once the esters are recovered, one can use the monoesters as precursors tor conversion into chelating agents, and the testers as solvents.

An advantage of recovering the carboxyiie acids from fermentation in the form of their corresponding esters is that downstream processing of the esters is less energy intensive than the h rogenation of the free acids. Another advantage of the present esters fkation process is that, one will l½d the present process simpler and easier, as compared to other approaches, to refine carboxyiie acids for€< chetnicai platforms from fermentation, it simplifies efforts to separate esters from the other insoluble materials, as well as minimizes the amount of salt that one needs to separate, in an integrated process enables one to directly esterify a combination of free acid and salts that is produced in a iow~pH fermentation, in which the fermentation is operated at a pi i of less than the pKa of the carboxyiie acids.

Figure 2 shows a schematic diagram of a downstream processing that incorporates an iteration of the present esterificatien process, in particular, figure 2 depicts an example of using succinic acid or any other kind of carboxyiie acid derived from a fermentation broth is extracted and reacted with an alcohol in the presence of excess C0 ₂ to generate esters. According to this iteration of the process, glucose, corn steep liquor, or other sugats, and Mg(OI-l);. / NaOH are introduced into a fermentation reactor 2 and fermented 23 to produce carboxyiie acids. A fermentation broth liquid 25 containing a mixture of carboxyiie acids, salts (e.g., succinic acid and its sodium or magnesium salt), and other byproducts is filtered 27 to remove cell .mass and other insoluble matter 27a. The fermentation is performed at a low p-1-ϊ value, in which one starts at a higher pK (e.g., pH ~? or 8} and during the course of the fermentation, the pH value drops to about. 2-3 , One will produce a mixture of salts and free acid present, for example, in a ratio range of about 9: i w ^' w to 7:3 w/w of sail to acid. The fermentation broth is retrieved from a fermentation reactor at a pH value of less than the p La of the carboxyiie acids, (e.g., pH 5}.. Typically, the fermentation broth is at a pH value in a range between about \ .5 and about 4.5.

The broth extract is then dried 29 to a powder. When drying the mixed acid filtrate should remove as much water as possible. The drying step can be accomplished, for instance, by means of spray drying, drum drying, or cryodesiccation. As with esterification in general, relatively low water content is desired, otherwise the reversible reaction will tend to hydroiyze back to the dicarbox iic acid. In the present process, a maximum residual moisture content of about 5% by weigh t should be maintained. One would expect an increase in ester yield of up to about 98 or 99% with samples that contain less than 3% wt. of water. The dried powder (average moisture content between about l wt.% and 5wt.%, desirably 3 wt.%) is then reacted i/ with an alcohol X? which serves as an alkylating agent, in excess CO; at a temperature between about 1$0*€ to about 250°C for a duration of about 4 hours or more to esterify the earboxyiie acids. In this example, succinic acid is reacted in methanol and CO. _? to generate dimethyl succinate. Along with the free e?irboxy he acid, any remaining free amino acids which were in the fermentation broth are also esters ned.

Once the carboxylic acid esters are generated and recovered from the fermentation broth they can be further processed as feed stock in either hydrogenation or hydrogenoiysis systems,

Hydrogenation or hydrogenoiysis can be conducted according to various different methods, systems, and their permutations, such as described in U.S. Patent os. 7,498.45002 (relating to homogenous hydrogenation of dicarbocilic acids and/or anhydrides), 6,Τί3, 193B, or 5,969,164, (relating to a process for production of tetra ydrofut n and γ-hntyrolactone by hydrogenation of rna!eic anhydride); U.S. Patent No. 4,584,4 (relating to process for the production of butane - 1 , -dioi involving the hydrogenation of a difCj to O* aikyi) ester of a C dicarboxyiic acid); UK Patent Application No. GB2207914A (relating to a process for production of a mixture of butane 1 ,4-diol γ-butyroiactone. a d tetrahydrofuran from maleate and tiunerate ; International Patent Application os. WOSS00937A (relating to a process for the co-production of butane- 1 ,4-diol and γ-butyrolactone by means of hydrogenation of dialkyi maleate) or WO 82/03854 (relating to a process for hydrogeuo lysis of a carboxylic acid ester), or an article by S. Varadarajan e( L, "Catalytic Upgrading of Fermentation- Derived Organic Acids," ΒίθΊΈθΗΝθΐ. PROG. 1 99, 15, 845-854, the content of each of the preceding disclosures is incorporated herein in Its entirety by reference.

As the example illustrates in Figure 2, when reacted with methanol in accord with She reaction temperatures and pressure parameters defined above, succinic acid esteriiled to prod*. ice dimethyl succinate (as predominant product), aPICO _? , MgCO _:! / Mg(HCO _:i )?. and excess methanol 35. The dimethyl succinate and methanol 37 are separated from NaHCO _? and gCO? 39. The carbonates, unlike CaSG , can be recycled 41 back into the reactor 2L either for a continuous process or in a fresh batch process. The dimethyl succinate and methanol are further separated 43 from each other with the methanol 33 being recycled 45. Subsequently, the dimethyl succinate 47 is subjected to either hydrogenation or hydrogenolysis 49 and transformed into a variety of biofuei products 51, including alkanes and/or alcohols, for instance: ethane, ethanol, propane, propane!, butane, or butanoi.

Another advantage of the present process is that it can simplify the transport and processing of crops for fermentation products. For instance, with a dried fermentation broth powder one is freed front issues associated with working with wet ot liquid stock. A dried fermentation broth powder can be more economically shipped to a location different from where the fermentation broth is made or obtained. This will enable the reaction for ester synthesis to be performed at a remote location different from where the fermentation broth is obtained, and expand the geography of where the Final processing facilities can be situated. Section Π. ·- Examples

A.

Examples prepared according to the present esterification method are integrated into a process for isolating free carboxylic acid from fermentation broth. The method involves generally the following steps: a} filtering a crude fermentation broth to remove ceil mass and other biological debris from fermentation, broth: b) desiccating the fermentation broth; c) reacting the dried fermentation broth with an excess of metha ol <CM _;i OH) or ethane; (CT-fjOH) and carbon dioxide (CO;) at a temperature about 150°C up to the near critical or critical temperature and under near critical or critical pressure of the alcohol and/or CO _¾ reagents, to produce a mixture of monoesters and diesters and carbonate (NaH ^' €03/ MgC0 ₅ ): d) filtering the reaction product to remove by-products; and e) purifying by distilling die esters.

The fermentation broth filtrate was dried to remove all or nearly all of the water to produce a powder of mixed organics. Using a spray dryer or drum dryer, one aerosolizes the raw solution containing mixed carboxylic acids to desiccate into a powder. The desiccated powder is suspended in an alcohol solvent. The powder reacts with the alcohol according to the conditions described herein to essenfy into either monoesters or diesters.

Each of the following examples was performed according to the following general protocol, except for variations in reaction temperature, pressure, time, and/or acid species as indicated, mutatis mutandis. Ten grams of ireeze-dried succinic acid fermentation broth (off-white powder) and 300 g of met H noi were charged to a 1 1 stainless steel vessel, jacketed, and fixed to a Parr reactor. While stirring mechanically at .1 .100 rpni. the internal headspace of the reactor vessel was purged with K and then pressurised initially to 400 psi with CO _? and .heated to 180 C for 5 hours. The internal pressure was observed to be - 1 50 psi at ISO C. After the reaction time, the reactor body was cooled in a water bath until reaching room temperature and pressure released. The heterogeneous mixture was then filtered and solids were dried overnight under vacuum. Samples of the solid material and the solution were analysis quantitatively using gas-chfomatography/tnass spectrometry (GC MS). The yield of dimethy l succinate was determined to be 31 .9% with more than 95% of the available magnesium succinate consumed in the reaction. The remaining balance of product included the corresponding monoesters as the greater part, and was in a range of about 60% to about 65%.

As the reactions depicted in the accompanying figures and tables show, modification and selection of certain temperature and pressure parameters causes reactions to yield preferentially more of the diester compounds. In certain examples of the present process, the esterifkafion reactions yielded more thart 50%, typically more than 70% or 80% di-aky! succinate or malate. As stated before, the unieacted materials and the undesired products are recycled into the fermentation reactor. Subsequent separation of ^' the mono-esters and di-esters w achieved by crystallization. Figure 3 shows a series of esterifjcation reactions which sumrca ize CO ssisted estenilcation of tree succinic acid in various alcohols. Figure 3 A. shows succinic acid reacted w ith methanol in 400 psi C0 ₂ gas, at 150°C for 5 hours, which achieved a yield of about 3?% dimethyl succinate. When the operational temperature was increased to I WC in the reaction of Figure 3B and all other parameters kept the same as in Figure 3 A, the amount of dimethyl succinate yield increases more than twofold to about 8 1 .2%.

Figure 3C represents free succinic acid reaction at I ¾*°C under present operational conditions in ethano!. which generates diethyl succinate in good yield of about 60.8%. In Figure 3D, free succinic acid was reacted at .! SCFC under operational conditions in n-b tanol, which generates dibutyl succinate at about 52.2% yield. These examples demonstrate the versatility of the present

esterification reaction in view of different kinds of alcohols.

F igure 4 shows examples of COj-assist ^' ed esterification of other kinds ofcarhoxyik poiyackls. in Figure 4 and 48. succinic acid was substituted respectively with citric acid, a tricarboxylic acid, and malic acid. The yield of trimeihylciirate was reasonable at about 20.1 %.

demonstrating that the Ci.Vassist.ed protocol can he applied to tricarboxylic acids. The yield of the dimethyl analogue of malic acid was good at about 84.3%. Hence, the new method of esteri ication is feasible for general use with other acids.

Table 4 summarizes results of several reactions thai were performed, according to the esterification method of the present disclosure as depicted in Figures 5, 6, and 7. Each set of examples is arranged in terms of a variation of an operational condition under which the reaction was performed: A s temperature, B) pressure, and C) reaction time, in each of the examples, succinic acid from a fermentation broth is used as the substrate. The filtered clarified broth containing free acid and. salts are dried and later reacted with methanol and C<¾ in solution. (As the reactions are heated, the actual operational temperatures and pressures within the reactor vessel will exceed the initial temperatures and pressures provided herein.)

i.o the three examples of Set A, we carried out the reaction for 5 hours at an initial CO* pressure of 400 psi. under different temperatures: Ex. A- i at 180 ^ft C, Ex. A -2 at 210°C, and Ex. A-3 at 230'"€·. The percent conversion of acid to its corresponding d tester increased with higher operational temperature. Figure < shows the effect of an-ing temperature in a series of esteriflcation reactions of succinic acid and its salt, m Figure 5 A, the esterifieation of succinic acid is performed at a temperature of about 180°€, over a period of 5 hours. The reaction produced about ^; 3.9% yield of dimethyl succinate. Figure 5B shows the same reaction as in. Figure 5 A, when the reaction time held constant, hut. with the temperature raised to about 2 I 0°C which yields about 42.9%. Figure SC shows a. reaction at 230%: and yields about 72.4%. This suggests that as the temperature increases, the reaction kinetics drives toward a more complete reaction of the acid and alkylating agent, and a greater yield of the dialkyoester. Reactions performed at or near critical temperature and/or pressure conditions can produce at least 95%. likely > 97% or 98%. conversion. In Set 8 nd Figure 6, sve performed the esterifkation r a i n i r 5 hours at an initial temperature of 180°C, and varied the initial€(¾ gas pressures: Ex. B-d at 400 psi, Ex. B-2 at S00 psL and Ex. B-3 at 600 psi. The percent conversion of acid to its corresponding diester was moderate, and the amount yield did not show significant difference statistically. The initial Ci¾ gas pressure in the reactor did not exert much effect in conversion of the acid to its dies e but the operational pressures in the reactor during the reaction suggest an effect on yields.

In Set C and Figure 7, we performed the esters fieat ion feaei n at a constant pressure and temperature but varied the duration of the reaction. Ex. C- i at 5 hours. Ex, C-2 at 2 hours, and E.x. C~ at 0.5 hours. T he examples shown in Figure 8 suggest that a greater amount of diester was converted from she acid with increased reaction time.

Figure 8 shows a first set of CCVassisted esterific&tion reactions using a concentration of succinate salts of about 4% w/w, which are presented as Examples I -3 in Table 5. In Examples I and 2, succinic acid and its magnesium (Mg ^~" > salt was reacted in methanol and ethanol at 21 *0 and 1 80 C respectively, for a reaction time of 5 hours. The reactions produced about 33% dimethyl succinate and about 1 % diethyl succinate, respectively. Methanol exhibits a greater capacity to dissol ve the succinate salt than ethanol. Magnesium succinate exhibits a reasonable level of solubility in methanol, while it. exhibits limited solubility in ethanol, even at high temperatures. Hence, the yield of dieth Succinate was negligible. Example 3 shows & reaction using calcium (Ca ^" > succinate, at i 80 ^c C, over 5 hours. The reactio yields only about 1 3% of the corresponding

dimelhylsnccinate. Relatively low conversion rates in ^' Examples 2 and 3. also highlights the solubility difference between corresponding alkali earth salts. The calcium succinate salt is insoluble in methanol, even at high temperatures. The methanol to salt molar ratio used n the CO.? experiments was approximately 1 10: 1 for methanol to magnesium succinate. Likewise, the ratio was about 100: 1 for methanol to the other earboxylic acids.

Ί able 5 lists results from other examples of esterifieation reactions according to the present ^■ method. Examples 1 , 2 and 3 demonstrate the Importance of substrate solubility of succinic ac id as compared to the salts of succinate. Examples 4-7 is a second set of reactions in which free succinic acid was reacted in methanol etbanol, and l-butanoi in similar .fashion. Examples 8 and 9 show thai reactions with other carboxylic acids, such as citric acid, and malic acid can achieve relatively good ields of about 20% and 86%, respectively.

Free succinic acid reacts readily with the alcohols, since it is completely soluble in methanol,. ethanoL butanol, and other alcohols up to and including octanol (C-8 alcohol). In Examples 6 and 7. succinic acid reacted in eihanol and I -butanol, yields 60.8% and 52.2% conversion, respectively.

The solubility of carboxylic salts in. a particular solvent, can have an influence on the esterification process. The greater solubility of free-acid permits a greater reactivity than the carboxykte. salt which lacks an acid functionality. Accordingly, the yields of the corresponding esters tend to be significantly greater than the control samples when comparing the two sets of reactions. The reactions of Examples 4-7 y ielded significantly greater amounts of corresponding d!e ters than that of Examples 1 -3. The carboxylic acid itself may be sufficient to catalyze the esterification reaction under the present operational temperature and pressure conditions. One can adjust the substrate solubility for successful esterification according to the present method.

B.

Example: Kydroge na ion

Using a process like one of those described in the references cited above, one can perform direct hydrogenation of the carboxylic acid esters collected from the esteri ication process described above. For examples, one can use metallic copper catalysts for the hydrogenation of dia!ky! succinate esters to BDO, CiBL, and THF. The following describes an illustration of the hydrogenation process.

The copper catalysts were prepared by wet impregnation of copper salts onto the following supports (all - 16+30 mesh): silica-alumina (93% silica. 7% alumina Sigma Chemicals) and two chromatographic silicas (Phase Separations, Inc.) (XOA-400 and XOB-030). Ten milliliters of solution containing copper nitrate to produce the desired loading was added to 10 g of support. The slurry was stirred at room temperature for 2 hrs. and then dried for 2 hrs. under vacuum at 60 ^' "€ - 70 °C. The dried solid was then calcined in air in a furnace at 500 ^": C for 1 1 - 12 h to gi e copper oxides. The catalyst material was then loaded into the .reactor and reduced in-situ in pure hydrogen at 200 °C and 200 psig for 3 hrs. Catalyst supports were characterized before and after reaction by nitrogen BET surface axea and mercury poroslroetry. Support acidify measurements were made using butylamine as a titrant i dry benzene with / -dimethyian -iazobenzene (pK» 3.9) as the indicator.

A cone closure reactor (Autoclave Engineers, Inc.) made of 3 16 Stainless Steel, 1 7 mm long

·'· 5 mm inner diameter (i.d.j, was charged with one gram of catalyst supported on a quartz frit. The reactor was surrounded by a clamshell furnace controlled by an Omega series CN -201

I S progrannnable temperature controller. Dimethyl succinate was fed to the top of the reactor as 30 wt % solution in methanol using a iorad H.PLC pump. Hydrogen gas was also fed to the top of the reactor from a standard tank and high-pressure regulator; a rotameter was used to monitor gas flow rate at the reactor outlet. The liquid feed rate was fixed at 0.05 mL/rnin, and the hydrogen rate was set at 400 mL of S P in to give a weight hourly space velocity (WHSV) of 0.9 f D S of cat/h and a H ucc ate ratio of 200: ! .

Condensable products were collected in single-ended 10 mL Whitey sample cylinders immersed in. ice baths. Three-way valves were used to divert reaction products to either of two sample cylinders; during reaction., condensable products were collected in the traps for a timed period, after which the trap was removed and weighed and the contents were removed for analysis. Gas exiting the collection cylinders passed through a. rupture disk assembly and was stepped down to atmospheric pressure using a back-pressure regulator. Gas products were collected in gas bags for analysis using gas chromatography to quantify non-condensable product formation.

Condensable products were weighed following collection and analyzed in a Varian 3 00 gas cinematograph equipped w ith a flame ionization detector and a Supeico SPB-i wide-bore (0.5 mm) capillary column (50-200 X 12 °C/snm, hold @ 200 X). Methyl lactate was used as an internal standard to facilitate the calculation of product concentrations.

Example: Hydrogenolysls

The esters resulting from the fermentation extraction described above is then hydrogeno!yzed over a catalyst (e.g., reduced CuO/ZnO). which should obtain high conversions ( 98%) and selec ivities (e.g., international Patent Application No, WG 82/03854),

Alternatively, one can proceed according to a process such as described in U.S. Patent No. 4. 8 , 19. A. stainless steel with an oil jacket maintained at 23 i. ^¾ C was used for this reaction.

Hydrogen was introduced by way of a pressure regulator and flow controller (not shown) through line to the bottom end of a vaporiser containing a number of steel bails. Ester was metered as a liquid to vaporiser through a line. The resultin vaporous mixture of ester and hydrogen was passed through preheating coil to reactor. This contained a layer of glass halls, on which rested the catalyst bed. The remainder of the reactor was illed with glass balls and the upper end of the reactor was fitted with an exit tube which led to a condenser (not shown j and then to a pressure let-down valve. The exit gas flow rate was measured downstream front the condenser using a wet gas meter.

A charge of 30 ml of a granulated copper chromite catalyst was placed in the reactor which was then purged with nitrogen at 42 bar. The oil bath was raised to a temperature of 2 1 .degree. C ^, A 2% H.sub.2 in N.sub.2 gaseous mixtu e at 42 bar was then passed over the catalyst tor 8 hours, followed by 10% H.sub.2 In Rsn.b.2 (still at 42 bar) for a further 1 hours, and then by pure H.sub.2 (also at 42 bar) tor an additional 12 hours. Diethyl succinate was then introduced into the vaporizer corresponding to & liquid hourly space velocity of 0.2/hr. The hydrogen gamester molar ratio in the vaporous mixture was 313: 1. The temperature of ^" the sand bath was maintained at. 231*0. The condensate was analyzed by gas chromatography using a i .82 meter long stainless steel column wuh an internal diameter of 3.18 mm containing % dieth iene glycol succinate on Chromosorb PAW, a helium gas flow rate of 30 ml/minute and a flame iom ^' sation detector. The instrument was fitted with a chart recorder having a peak integrator and was calibrated using a mixture of diethyl maleate. diatky! succinate,

butyrolaetone, butane- 1.4-diol, ietrahydrofuran and water of known composition. The exit gas was also sampled and analyzed b gas chromatography using the same technique. The .identity of the peaks was confirmed by comparison of the retention times observed with those of authentic specimens of the materials in. question and by mass spectroscopy. The following compounds were detected in the reaction mixture: diethyl succinate, butyrolaetone, butane- 1, -dioL tetrahydrofuran and water. Trace amounts of minor byproducts, including 2- ethoxy tetrahydrofuran and 2~ ethox butane- 1 , -dioi were also detected in the reaction mixture. From the results obtained it appeared that diethyl succinate had been smoothly converted to products with a selectivity to tenrahydrofuran of ^' 52.2mol %. a selectivity to n-butanol of 1 1.6 mo! %, a selectivity to gamma- hntyroiactooe of 26. i oi %, and a selectivity to butane- 1 ,4-dioi of 10.1 mol % ₅ the balance being minor byproducts.

The present invention has been described in general and in detail by way of examples.

Persons of skill in the art understand that the invention is not limited necessarily to the embodiments specifically disclosed, but that modi.ficat.ions and variations may be made without departing from the scope of the Invention as defined by the following claims or their equivalents, including other equivalent components presently known, or to be developed, which may be used within the scope of the present invention. Therefore, unless changes otherwise depart from the scope of the invention, the changes should be construed as being included herein.