BROTH

PRIORITY ^' CLAIM

The present Application claims benefit of priority from U.S. Provisional Application No. 61 /739,769, filed December 20. 20 2, the contents of which arc herein incorporated.

FIELD OF INVENTION

The present invention relates to a process for the production of carboxylic acids. In particular, the invention pertains to a method for recovering carboxylic acids from a fermentation broth.

BACKGROUND

Carboxylic acids, such as the four carbon succinic, malic, maieic and turn ark acids, as well as their derivatives play an. important role as precursor molecules for a variety of other chemicals, including the biodegradable polyester resins, dyesiibTs, and pharmaceuticals and as additives in the food Industry, Currently, for example, succinic acid Is largely produced commercially from crude oi! by catalytic hydrogenation of maieic anhydride to succinic anhydride and subsequent hydration or by direct catalytic hydrogenation of maieic acid. This traditional way of producing succinic acid from petrochemicals is costly and causes pollution problems. In recent years, many have sought to develop a more cost competitive and environmentally-friendly way of producing succinic acid by means of a biological -based fermentative process. The fermentative production of an important dioarboxylle acid Is advantageous not only because renewable substrates are used, but also because the greenhouse gas CO? Is incorporated into succinic acid during fermentation.

For instance, these biologically-derived succinic acid (BDSAs processes seek to produce succinic acid by fermenting glucose from biomass. separating and purifying the acid, and then caialyticaliy processing it s a platform chemical to produce, for example, 1 ,4 - huianedio) (BOO) and related products, tetrahydrofuran and γ-butyrolactone; -methyl pyrtoiidinone (N ^' MP). 2-pyrrohditxme or other chemicals that are used to make a wide assortment of products. Existing domestic markets for suc chemicals total almost 1 billion pounds, or more than $1.3 Billion, each year. The BDSA processes also promise to reduce reliance on imported oil and to expand markets for domestic agriculture to more than food sources. Ordinarily, however, the recovery of dicarboxylic acids from a fomen at on broth involves forming insoluble salts of the diacids, typically, insoluble calcium salts. In the case of fermentation by fungi such as Rhizopus oryzae or AsperigiU oryz , which preferentially make fumarie and malic acid, respectively, the calcium is typically introduced into the broth in the form of CaCC , which forms€a(H€<¾)2 in solution. The bicarbonate is effective to maintain the pH of the broth as the diacid being produced tends to lower the p The diacid is recovered as the calcium salt form. The calcium salts of such C diacids have a very low solubility in aqueous solutions (typically less than 3 g/liter at room temperature), and are not- suitable for many applications for which the free acid is needed, such as chemical conversion to derivative products like hutanediol and the like. Therefore, 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 tew commercial applications, and accordingly is typically discarded as a solid waste in landfills or other solid waste disposal sites.

In an alternative process described for example in WO2010/147920, instead of using calcium carbonate, the pH of the medium for fungi growth was maintained using a magnesium oxygen containing compound, such as MgO, Mg(OH) ₂ , MgCCo, or Mg{HCi ,) _. i all of which form the bicarbonate salt in aqueous solution _. The use of magnesium rather than calcium was found to enhance production of the acid by fermentation. The

fermentation was conducted at a pH of 5-8 and more preferably 6.0-7.0. The pH was maintained by the addition of the magnesium oxygen compound, and CO? was introduced into the medii n. in combination with the magnesium oxygen compound to maintain a molar fraction of bicarbonate (HCO ₃ ^* ) of at least 0.1 and most preferably about 0.3 based on the total moles of HCOf . C(¾ ^" and Cf in the medium. At the end of the fermentation, the liquid portion of the medium contained a majority of diacid as a soluble magnesium salt, which was separated from a solids portion of the medium containing precipitated, salts and other insoluble material. The dissolved acid salt was converted into the free acid form by- reducing the pH to below the isoelectric point of the diacid using a. mineral acid such as sulfuric acid, and lowering the temperature of the medium to (most preferably) not greater than 5°C, which precipitated the free acid from the solution.

While useful for producing a free acid, the techniques described for using the magnesium, salts results are expensive, first because the magnesium oxygen compounds cost considerably more than the analogous calcium compounds but also because the bulk of the magnesium remains in the fermentation medium in. the form of the .magnesium salt of the inorganic acid, and. is not useful for further fermeniaiion or other purposes, further, the need to lower the temperature of the recovered soluble salts to precipitate the free acid adds additional energy costs.

Although the fermentative production of carhoxylic acids, such as malic or succinic acid, has several, advantages over petrochemical-based processes, the generation of carboxyiic acid salts as just discussed carries significant processing costs because of the difficulties associated with the downstream processing and separation of the acids and their salts. When salts are generated io 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. All of these individual operational units contribute to the overall costs of the process.

Recovery of carboxyiic acids as salts has a number of associated problems and requires several different steps in post-fermentation, downstream processing to isolate free acids and to prepare the carboxyiic 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 byproducts. For instance, calcium salts of Q diacids have a very low solubility in aqueous broth solutions (t pically less than 3 g/iiter at room temperature), and are not suitable for many applications for which a free acid species is needed, such as chemical conversion to derivative products. Therefore, the calcium salt is typically dissolved in sulfuric acid, forming insoluble calcium sulfate, which can readily he separated f om the free diacid.

Calcium sulfate is product having few 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.

The production costs for the bio-based carboxyiic acids ha ve as a result been too high for bio-based production io be cost-competitive with petrochemical production regimes. ⁽ See e.g., James McKmlay et al, "'Prospects for a Bio-based Succinate industry," APPL.

iCROBiOL. BK:>TECHNOL., (2007) 76:727-740; incorporated herein by reference.) 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. Typically, the pH of the fermentation broth is maintai ned at or near a pH of 7 by introduction of ammonium hydroxide or other base into the broth, thereby converting the di-acid into the corresponding di-acid salt. About 60% of the total production costs are generated by downstream processing, e.g. , the isolation and purification of the product in the fermentation broth.

Over the years, various other approaches have been proposed to isolate the di-acids.

These techniques have involved using ultra-filtration, precipitation with calcium hydroxide or ammonia, eieetrodialysis, liquid-liquid extraction, sorption and ion exchange

chromatography. (See, T ^' anja urzroek ei aL "Recovery of Succinic Acid from Fermentation Br th " Review, BIOTECHNOLOGY LETTER, (2010) 32:331-339; incorporated herein by reference.) Alternative approaches that some nave proposed include operating a iermentaiion reactor at low pH„ which functionally would, be similar to operating the .fermentation with minimum level of salts, (See, e.g., Carol A. Roa Engel et aL, "Development of Low-pH Fermentation Strategy ^? for Fumaric Acid Production by Rhizopus oryzaef ^' ENZYME AND MICROBIAL TECHNOLOGY, Vol . 48. Issue 1 , pp. 39-47, 5 January 201 1 , incorporated herein by reference.)

For example. Figure 1 shows a schematic diagram of a known process for extracting organic acids from a fermentation broth. Glucose, corn steep liquor, or other sugars, and CaCO* are introduced into a .fermentation reactor / and subjected to microbial fermentation 2. A .fermentation broth liquid containing a mixture of organic acids and other by-products J is extracted and filtered 4. The broth, is neutralized 5 with a strong acid, such as H- SO.<.. which generates CaSCY*. The reaction mixture is then filtered 6 to remove cell mass and the CaSi>4 7, which is waste that cannot be recycled; hence, it is disposed of in landfill or employed, for gypsum-using applications. The remaining organic acids, glycerol, and. other by-products $ can he recovered and fed back into the fermentation reactor as a carbon source, such as described in U.S. Patent No. 8,183,022, the content of which is incorporated herein by reference. The products can he separated by various techniques, such as crystallization or ion exchange 9. The organic acids can be purified 10, for example, over a carbo bed .

An alternative approach some have described involves the synthesis of alky] monoesters by direct esterification of alkali metal salts of earboxyiie acids, such, as calcium lactate, sodium acetate, sodium benzoate, and sodium salicylate, using carbon dioxide and an alcohol as a way of making bio-based chemicals in an. environmentall friendly manner (see, Prashant P. Barve, ef aL, "Preparation of Pure Methyl Esters From Corresponding Alkali Metal Sail of Carboxylic Acids Using Carbon Dioxide and Methanol" I >, ENG. CHEM. RES., 15 Sept. 2011 ,), The esterification process, however, has a limited application and do not describe the recovery of polycarboxy!ic acids.

Although these techniques have had some success, they are not able to provide a direct route by which fennentatlon-derived dicarbox lic or poiyearboxyll acids can be recovered in a simple, cost-efficient process from a fermentation broth. Rather, these fermentation techniques oilers involve the need to go through several different steps to prepare the carboxyiic acids in fermentation broth for chemical transformation and to convert the raw acids to useful compounds.

To reduce waste and costs associated with generating free carboxyiic acids and to improve the recovery yield, a need exists for a better, more direct method of recovering a variety of ca.rbox l.ic acids, such as malic or succinic acid, and which can provide a successful route to simplify downstream chemical conversions .from, a biological ly-derived feedstock. Such a streamlined, green process would be a welcome innovation. SUMMARY OF THE IN VENTION

The present invention describes, in part, a process for recovering and using carbox iic acids from a fermentation broth by converting a carboxyiic acid to one or more of its corresponding esters (i.e. , rnortoester, diester, or triester) in a relatively efficient and cost effective manner. In particular, the present process involves obtaining a fermentation broth. from which cell mass and insoluble compounds have been either removed or not, containing at least one ree carboxyiic aeid, or a mixture of carboxyiic acids, or at least one free carboxyiic acid and an associated alkali or alkaline earth metal salts of the carboxyiic acid (e.g., sodium, potassium, or magnesium salts): drying the raw or clarified fermentation broth containing free carboxyiic acid, into a powder; and reacting the carboxyiic acid in the powder with an alcohol under a C ¾ atmosphere in the substantial absence of any other acid catalyst, at a reaction temperature or pressure corresponding to supercritical, critical or near critical conditions lor at least the alcohol or€(¼, to synthesize the corresponding ester or esters from the carboxyiic acid in the powder. In subsequent steps, the esters can be converted back to their corresponding tree acid form. One may recycle the synthesis by-products directly back into the original or a new fermentation broth.

The esterification reaction temperature is between about 150 ^C C and about 2S0°€, and the operational reaction pressure is between about 400 psi and about 3,000 psi (gauge).

Depending on the desired results, the reaction can be run for about 4 hours, up to about 12 hours.. in another aspect the present invention pertains to a method for este ifying a po!ycarhoxyiic acid derived from fermentation. The esierification method involves:

providing a solution of one or more free carboxylic acids from a fermentation broth and reacting the fee carboxylic acids with an alcohol in a C0 ₂ 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 at least the alcohol or CO?, to yield an ester corresponding to the free carboxylic acids. The reaction, temperature and pressure conditions preferentially drive the reaction towards the formation of diester molecules over monoester .molecules when the carboxylic acid is a pol carboxylic acid. The reaction temperature is between about 150°€ and about 250°C, and the reaction pressure is between about 400 psi and about 3,000 psi. Depending on the desired results, the reaction can be ru for up to about 12 hours.

In another aspect, the present invention pertains to a method of processing an agricultural product or biomass. The .method includes obtaining carbohydrates from the agricultural, product or biomass, fermenting the carbohydrates to produce a fermentation broth, drying the fermentation broth to produce a fennentation broth powder, and transporting the iemientation broth powder to a. second processing site. The second processing site can be located nearer to a source of demand for a product derivable from the fermentation broth powder, which can be processed or transformed at the second site to produce a product therefrom.

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 DESCRI PTION OF FIGURES FIG. 1 is a schematic diagram illustrating an extraction of organic acids from a .fermentation broth mixture and downstream processing as practiced conventionally.

FIG, 2 is a schematic diagram illustrating an iteration of the present process for esterifying an organic carboxylic acid derived from fermentation broth, and further downstream processes that can isolate the resulting esters and/or generate other compounds horn such esters,

FIG, 3 is a schematic diagram showing an example of ester production using succinic acid derived from fermentation, and a downstream process in which a and g salts are recycled back into the fermentation reactor, in accordance with another embodiment of the present process.

FIG. 4 is a diagram, that illustrates COrassisted esteriilcation of free succinic acid in various alcohols that are converted to corresponding dimethyl, diethyl, or dibutyi esters, according to the present invention.

FIG. 5 is a diagram that illustrates CO?-assisted esterification of other polycarboxylic acids,

FIG. 6 shows a series of reaction diagrams that summarize variations in temperature for CO assisted este ifieation of free succinic acid derived from fermentation broth.

FIG. 7 shows a series of reaction diagrams that summarize variations in initial operational pressure for C ⁽ ¾-assisted esterificaikm of tree carboxyiic acid accordin to the invention.

FIG. 8 shows a series of reaction diagrams that summarize variations in temperature, and reaction times for COa-assisted esteriflcation of fee carboxyiic acid according to the invention.

FiG, 9 shows a series of reaction diagrams that summarize the reaction results of succinic acids and the r Mg' ^{: '} and Ca ²⁺ salts,

DETAILED DESCRIPTION OF THE .INVENTION

Section I - Description

The present process modifies a conventional, extraction of carboxyiic acids-derived from fermentation. As compared to the process shown in Figure I, the present approach has several advantages; such as, contrary to convention, one can. avoid a need to neutralize the fermentation broth. In. another aspect of the present disclosure. Fig re 2 is a schematic representation showing a general process of extracting carboxyiic acids from fermentation broth that includes a version of the present esteriflcation reaction integrated wit further processes that can utilize the resulting esters. As shown, .fermentation broth i from a reactor is filtered (ultra) 3 to remove hiomaterials such as cell mass, and yield carboxyiic acids including their salts, by products and other compounds. All of these materials are then dried 3a to make an unrefined mixture 5. This dried mixture of materials is then reacted 7 in a liquid system with an alcohol s ^' -OH; R ^:::: alky! Cj-Co) ^a »d 0¾ at an elevated operational reaction temperature and pressure to yield either monoesters or diesters. or a mixture of both. Only the carboxyiic acids react in solution. The resulting mixture 9 is filtered 11 to separate the esters 13 and other by-products 15, The esters are soluble while other by-product compounds are insoluble. The by-products include carbonate salts of calcium, magnesium, or sodium, which can be recovered and recycled 17 back into the iennentation reactor 19. 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 21, hydrogen tion 23, or hydrogenolysis treatment 25, respectively, to separate the esters, produce <¾ platform compounds, such as the hydrogenation products (e.g., BDO, GBL, NMP.. etc.), and. biofoeis (e.g., ethane, ethanol.. butane., butanol, propane, propano!, etc.).

As used herein the term "biofuels" refers to a gaseous, liquid, or solid substance that is used as a fuel, which is produced from renewable biological resources such as plant, ceilnlosic, or agricultural bio-nass or derivatives thereof. In particular, a. biofuel refers to a material that can be used in or as a transportation fuel in internal, combustion engines, to power certain machinery, or energy generation applications. For instance, propanol and butano.1 can be a gasoline additive much the same as ethanol. Butane and propane in liquefied petroleum gas (LPG) and ethane in natural gas can be adapted as fuels in certai transportation systems. Other biologically-derived hydrocarbons, like octanol/ociane, or alkanes heavier than C$ or€<, may also be biofuels.

A.

The present disclosure describes, in part, a process for recovering and using an carboxylic acid from a fermentation broth. The process includes a method of esteri lying free earboxylie acids. As used herein the term "free carboxylic acid ^'5 refers to a carbox lic acid compound that is at least 50% in its protonated state when in solution, at or below its p a value. ^' The present invention involves the discovery of a simple but effective way of producing esters from organic acids that are otherwise costly and difficult to isolate.

The recovery process and esterifieation method can be applied to producing chemical feedstock molecules from free carboxylic acids derived from a fermentation broth. An advantage of the present invention is that one can use free carboxylic 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 necessary in conventional extractions from broth, in comparison to certain fermentation processes that neutralize or convert the carboxylic acids to their salts, the present process provides an easier way to isolate and extract carboxylic acids from a fermentation, broth. The present process eliminates a need tor titration and neutralization of the fermentation broth that can precipitate metal salts, and certain purification steps to produce a stock platform chemical The free carboxylic 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 multiple units of operation to purify the acid to n acceptable quality level With each added operational unit the costs of the overall process increases. Moreover, in 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. minimize waste. By converting the organic acids to their corresponding esters, we can void such issues.

Conventionally, esters are produced when carboxylic 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

esterii!cadon process, a carboxylic acid does not react with an alcohol unless a strong acid is used as a catalyst. The catalyst is usually concentrated sulfuric acid or hydrogen chloride. Protonaiiott makes the carbonyl group more eiectrophiiic and enables it to react with the alcohol, which, is a weak nuoleophile.

in general terms, the present esterifieation method involves a reaction of

fermentation-derived, tree organic carboxylic acid with an alcohol in a carbon dioxide (€€½>- predominant atmosphere in substantial absence of any other acid catalyst to produce esters. The esterifieation reaction is performed in solution under conditions that are either at supercritical _. , critical or near critical temperatures and/or pressures for at least one of the alcohol or€<¾. Under such conditions, we believe that€ ⁽ ¾ sell-generates or functions in situ as an acid catalyst and regenerates back after the esterifieation reaction is completed. It is believed that a reactive intermediate troonoalkylcarbonic acid) is being made in itu in large enough quantities to drive esterifieation and affect ester production. This intermediate, having a similar pKa (e.g..-4-5} as the bee carboxylic 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 for this process. 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 de minimis or trace amount of less than catalytic efficacy. In other words, no other acid catalyst is present, or is present at a

9

SUBSmiJTE SHEET (RULE 26) level less than 10%, 5%, 3%, or 1% weight/weight relative to the carboxylic acid in the reaction.

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, 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 make.

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 other salts. A cleaner product will save costs in purification and downstream processing lor conversion to other chemical feedstock,

The process described herein is a. more environmental ly benign way of producing esters. As it is believed that the carbon dioxide can self-generate an acid calalysl in situ in the presence of the alcohol during the esterification reaction, the present method does not require the use or addition of another acid catal st species. In other words, the reaction kinetics with CO? alone can drive the esterification in the substantial absence of any other acid catalyst. To reiterate, the present process does not require activation of tree acids as, for example, an acyi chloride or by strong acids (i.e., Fischer esterification}.

In general , the esterification is conducted at an operational or reaction temperature between about 150 C to about 250°C, at a reaction pressure of between about 400 psi and 2,500 psi or 3,000 psi (gauge), for an extended period, such as about 4 hours, up to about 12 hours. Typically, the temperature can be in a range between about I 70 ^'' C or I90°C to about 230°C or 245°C (e.g., 175°C. 187°C. I95°C or 2 I 5°C). and the operational pressure is between about 900 psi or 950 psi and about 2,200 psi or 2,400 psi (e.g., 960 psi. 980 psL 1020 psi or 1050 psi). Alternatively, the temperature can be in a range between about 18(P€ to about 245°C (e.g., about 185°C or 200"C or 210 ^C C to about 22C C or 235 or 240 ^¾ C), and the operational pressure is between about 1000 psi and 2,350 psi (e.g., 1 ,1.00 psi, 1 ,200 psi, 1,550 psi, 1 ,750 psi, 1 ,810 psi, or 1 ,900 psi). Other temperatures may be within a range, for example, from about 160°C or 1.85°C to about 2i0°C or 225°C, and other operational pressures may be within a range,, for example, from about 1 ,150 psi or 1 ,500 psi to about 1 ,800 psi or 2.000 psi. These reaction temperatures and pressures correspond to supercritical, critical or near critical conditions for the alcohol(s) or C(¾. Table 1 lists, for purpose of iliusi.rat.ion, critical parameters tor some common solvents (i.e... methanol, ethanol, l-propanol, 1 -butanoi, water, aod CO >.

Table i. Critical Data for Select Substances (Yaws,. C. L, Chemical Properties Handbook, In cGraw-HHI: 1999; pp 1-29.)

Substance Molecul r Critical Temp. Critical Pressure Critics! Density

Weight {bar)/psi !g/cm ³ )

Methanol 32.042 512.58 / 239,43 80.96 / 1174,226 0.2720

Ethanol 46.069 _h 516.25 / 243.10 63.84 / 925.920 0.2760 l~Propanol 60,095 537.4 / 264.25 51.02 / 739.983 0.2754

1-Butanol 74.122 563.0 ± 0.3/ 45.0 ± 4.0 / 652.671 0.3710

289.35

Water 18.015 647.13 / 373.98 220.55 / 3198.807 0.3220

Carbon 44.010 304.19 / 31.04 73.82 / 1070,669 0.4682 dioxide

At conditions above the critical point (i.e.. critical temperature and pressure), the fluid exists in a supercritical phase where it exhibits properties that are in between those of a liquid and a gas. More specifically, supercritical fluids (SCFs) have a liquid-like density and gas-like 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.

Table 2. Comparison of Typical Physical Property Values of Liquids, Supercritical Fluids,, and Gases.

Property Liquid SC Gas

Density (g ml) 1 0.3

Diffusivity {cm2/sj SxlO ^'6 0.1

Viscosity iPa-s) 10 " likewise, "'near critical ^" refers to the conditions at which cither the temperature or pressure of at least the alcohol species or CO? gas is below but within 1 5 OK (e.g., within 50-100K). 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 solubilit of the reagents are enhanced, which promotes the esteriilcation reaction, in other words, the€<¼ 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 thai both the alcohol species and€<¾ 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 250°C and about 3,000 psi (gauge), respectively, tor reaction times of up to about 10 or 1.2 hours, one can produce significant amounts of ester product at relatively greater selectivity and level of purity within a shorter reaction time than before, which was about. 1 S-20 hours. At lower operational temperatures { ^' < 190°C), fon.nat.ion of monoester molecules of po!yearhoxyik acids is more prevalent, while reactions at temperatures > 1 0°C or 195°C, wiii convert preferentially the poiyearboxyiic acids to diesters. By selecting a temperature in a higher range from about 190°C or 195°€ or 200°C to about 245*C or 250°C, one can preferentially drive the reaction to a greater rate of diester conversion. The esteri 11 cat on can yield a minimum of about 50%, desirably about. 65% or 70%, of a diester of the earboxylie 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.230*C or about 240°C for methanol and about 3 PC/I 000 psi .for C<¼, one is able to achieve conversions rates of about 90% or better, typically about 93% or 95%. One can achieve greater 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 1 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°C under either the same or greater CC pressure, the yield would be nearly quantitative (i.e., > 95% yield), tor example, up to about 98%., 99%, or 99.9% conversion.

As the accompanying Examples will snow, variation in react on conditions suggests tha 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 cyclization of the earboxylie acids and other starting reagents. Potential dangers of decarboxylation at high temperatures (i.e., >1.45°€ or > ^' { 50°C) are not observed in the present method.

Using an amount of the alcohol solvent in excess of the earboxylie acid, one can produce a very clean esterification. T he present synthesis process produces very clean osier products at about 70%-72% 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 rind standard acid cataly ed estcri ncation at high temperatures. The esters can be refined to achieve about 90-98% purity. The purification can be accomplished, for instance, by means of crystallization,

chromatography, or distillation.

Typically, the resulting ester products can be either mo.noest.ers or diesters, 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 arid pressure that preferentially dnves 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, made, nialeic. succinic acids) that may be present and derivable from fomie.ntati.on 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 or teed ingredient, polymer materials or hiomels. For instance, succinic esters can be converted into a polymer, such as poiybutyiene succinate (PBS).

In the present esterification process, both the catalyst (C<¼) 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 origin (e.g., gas tank or dry ice), as the reaction is conducted at high temperatures. Addition of solid CO? is strategic in. the case where sealed pressure reactors are used, in that it allows for slow sublimation of gaseous C0 ₂ formation as the reaction apparatus is being assembled. T his can minimize CO loss, in a CO; (i.e., CO2-co.ntaini.ng} atmosphere, the concentration of CO?, in the reaction atmosphere can be at least 10% or 1 5% 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 CO? are from about 75% or 80% to about 99.9% by volume, typically between about 85% and about 98%. Nitrogen (r½) gas or air is permissible in the reactor, but preferably the concentration of gases other than CO?, is kept at either a minor percentage (<■ 50%) or e minimis amount.

Any liquid alcohol with an R-gronp of Cj-Cso can serve as the solvent reagent or first alcohol species. The R-group can be saturated, unsaturated, or aromatic. A mixture of different, kinds of alcohols (e.g., C _\ -Cn) can also be used in the reaction, but will produce a corresponding mixture of different esters depending on the particular -group. Certain lower alcohol species with€ _r Q> alkyl groups are preferred as the reagent in the first esterification with€0.2 i view of their common availability,, inexpensiveness, and mechanistic simplicity in the esterification reaction. Further, alcohols such as methanol, ethanol. propane K or butanoi are 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 C2-C? _> ~dk>i. Esterification with a dio! can generate monomers or low molecular weight oligomers that can. be readily polymerized.

One can use a variety of different carboxylic acids, for example, selected from: a) monoearboxyiic acids: formic acid, acetic acid, propionic acid , lactic acid, butyric acid, isobutyrie acid, valeric acid, hexanok acid, heptanoie acid, decanoic acid, lauric acid, rnyristic acid, and€; €sx fatty acids; b) dkarboxyik acids: fa marie acid, itaeonic acid, malic acid, succinic acid, tnaieic acid, maionic acid, glutaric acid, glucaric acid, oxalic acid, adi ic acid, pimeiic acid, suberic acid, azel.aic acid, sebacic acid, dodecanedioic acid, glutaconk acid, otlho-phthalic acid, isop haiic acid, terephihalie acid; or c) tricarboxylic acids: citric acid, isocitric acid, aconitic acid, trkarballyhc 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 carboxy lic acids. Desirably, the acid is a dicarhoyxlk or tricarboxylic acid.

B.

The present esterification process car- be integrated into fomentation-based production of carbon chain feedstocks and to provide a more convenient method of generating esters from carboxylic acids derived from a renewable source. The process cars 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€<¾ 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 than, the reaction that uses the salt species as a reagent. This result can be seen when one compares the reaction of accompanying Figure 4B (free acid) with that of Figure 6A (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 e hanol 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.

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 sample of common, esters thai may be present in an ester product mixture according to the present invention.

Table 3. Boiling Points for Some Common Esters

£$ft r Species Boiling Point (X) Ester Species Bailing Point (T) methyl-acetate 56.9 ethyl-acetate 77.1

methyl-formate 32 ethyl-formate " 54.0

methyldactate 145 ethyl-lactate 151-155

dsmethyi-malate 104-108 (1 mm Hgj diethyl-ma!a e 281.6

dimethyl- 200 diethyl-succinate 217-2.18

succinate

tn lhy --c.i†.rate 176 (IS mm Hg) triethyi-citrate 2.35 {150 mm Hg)

After recovering the esters in the remaining 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 for conversion into chelating agents, and the diesters as solvents.

An advantage of recovering the carboxyiie acids from iennentation in the form, of flick corresponding esters is that downstream processing of the esters is less energy intensive than the hydrogenation of the tree acids. Another advantage of the present esterification process is that, one w ll find the present process simpler and easier, as compared to other approaches, to refine carboxyiie acids for C _? chemical platforms from iennentation. It simplifies efforts to separate esters from the other insoluble materials, as well as minimizes the amount of salt thai one needs to separate. In an integrated process enables one to directly esterif a combination of free acid and salts that is produced in a low-pH fermentation, in which, the fermentation s operated at a pH of less than, the p a of the carboxylic acids, ^' foe process can be less energy intensive that current recovery approaches.

We iO now expound in .more detail the concepts of the general process depicted in Figure 2. Figure 3 shows a schematic diagram of a downstream processing that incorporates an. iteration of the present esterificadon process, in particular. Figure 3 depicts an example of using succinic acid or any other kind of carboxylic acid derived from a fermentation, broth is extracted and reacted with an alcohol in the presence of excess€<¾. to generate esters.

According to this iteration of the process, glucose, com sleep liquor, or other sugars, and !Vig(< ^} f ¾ / NaOH are introduced into a fermentation reactor i and fermented 2 to produce succinic acid and. its sodium, or magnesium salt. A fermentation broth liquid containing a mixture of carboxylic acids, salts, and other by-products 4 is filtered 6 to remove eel! mass S and other insoluble matter. The fermentation is performed at a low pB value, in which one starts at a higher pB (e.g., pH --7 or 8) and during the course of the ierrnen.tati.on, 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: 1 w/w to 7:3 w/w of salt to acid. The fermentation broth is retrieved from a fermentation reactor at a pE value of less than the pKa of the carboxylic acids, (e.g., pH 5). Typically, the fermentation broth is at a pH value in a range between about 1 .5 and about 4.5.

The broth extract is then dried S 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 esierification in general, relatively low water content is desired, otherwise the reversible reaction will tend to hydrolyze back to the dicarboxylic acid, in the present process, a .maximum residual moisture content of about 5% by weight 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% i. of water.

The dried powder (average moisture content between about 1 t.% and 5wt.%, desirably < 3 wt.%) is then reacted 12 with an alcohol 14 which serves as an alkylating agent, in excess C(¾ at a temperature between about 180°C to about. 250°C for a duration of about 4 hours or more to esterify the carboxylic acids. In this example, succinic acid is reacted in methanol and€<¾ to generate dimethyl succinate. Along with the free carboxylic acid, an remaining free amino acids which were in the fermentation broth are also esteri bed.

One can also produce various precursor chemicals by subjecting the ester mixture to hy drogenafion. One can produce a variety of compounds, including for example: 1 ,4~buiane~ diol (BOO), tetrahydrofuran (THF), y-butyrolactone (GBL), or N-Methy!-2-pyrrolido.ne (NMPh which in turn can be further modified to other useful compounds, by means of hydrogenation processes such as described in U.S. Patent No. 4,584,419A (relating to process for the production of 1 .4~butane-dto! involving the hydrogenation of a di(C, to Cs alkyl) ester of a Ci dicarboxylic acid); UK Patent Application No. G.B2207914A. (relating to a process for production of a mixture of butane 1 ,4~diol, γ-butyro lactone, and tetrahydrofuran from maleate and fumerate): International Patent Application Nos. WO8800937A (relating to a process for the co-production of butane- 1 ,4-dio.l and v-btnyrolactone by means of hydrogenation of dialkyl maleate) or WO 82/03854 (relating to a process for ^' hydrogenoiysis of a carboxy!ie acid ester), the content of each, of the preceding patent disclosures is i ncorporated herein in its entirety by reference.

As the example illustrates in Figure 3, when reacted with methanol in accord with the reaction temperatures and pressure parameters defined above, succinic acid esterified to produce dimethyl succinate (as predominant product), NaHCiX Mg€<¾ / Mg(HC<¼)2 and excess methanol 16. The dimethyl succinate and methanol 18 are separated from NaHC<¾ and MgCOi 20. The carbonates, unlike CaSCV can be recycled 22 back into the reactor /, either for a continuous process or in a fresh batch process. The dimethyl succinate and methanol are further separated 24 from each other with the methanol 7 being recycled 26. Subsequently, the dimethyl, succinate 28 can be hydrogenated S9 into a variety of different chemical products 32, including for instance; .1 ,4-butane~ldiol (BDO), tetrahydrofuran (T.HF), γ-butyrolacione (GBLk or -methy 1 -2-pyrroi.idone (NMP).

Another advantage of the present process is that it can simplify the transport and processing of crops for fermentatio products. For instance, with a dried fermentation broth powder one is freed from issues associated with working with wet or liquid stock. A dried fermentation broth powder can be more economically shipped, to a location different from where the fermentation broth is made or sourced. This will enable the reactio for ester synthesis to be performed at a remote location different from where the fermentation broth is sourced, rid expand the geography of where the final processing facilities can be situated.

Hence, we also envision that the esterification process described herein can be integrated into a method for processing an agricultural product or biomass. The method involves obtaining carbohydrates from the agricultural product or biomass, fermenting the carbohydrates to produce a fermentation broth, drying the fermentation broth to produce a fermentation broth powder, and transporting the fermentation broth powder to a second processing site. This second site can be located closer to a source of demand for a product i ? derivable from the broth powder, which can be esteriiied and/or otherwise processed at the second site to produce a product therefrom.

Section II - Examples

Examples prepared, according to the present esterTOeation method are integrated into a process for isolating free carboxyiic acid from a fermentation broth. The method involves generally the following steps: a) filtering a crude fermentation broth to remove ceil mass and other biological debris from a fermentation broth; b) desiccating the fermentation broth; c) reacting the dried fermentation broth with an excess of methanol (€Η ΟΗ) or ethane! CQjHsOH) and carbon dioxide (C<¼) at a temperature about 150°€ up to the near critical or critical temperature and under near critical or critical pressure of the alcohol and/or C0 ₂ reagents, to produce a mixture of monoesters and diesters and carbonate i afl€03/ gC<¾): _> d) filtering the reaction product to remove by-products; and e) purifying by distilling the 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 carboxyiic 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 esterify 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 freeze- dried succinic acid fermentation broth (oft- white powder) and 300 g of methanol, were charged to a 1 L stainless steel vessel jacketed, and -fixed to a Parr reactor. While stirring mechanically at 1 1 0 rp.m, the internal headspace of the reactor vessel was purged with j and then pressurized initially to 400 psi with CO¾ and heated to 1.80 C for 5 hours. The internal pressure was observed to be -1650 psi at ! SO€. 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 tittered and solids were dried overnight under vacuum. Samples of the solid materia! and the solution. were analysis quantitatively using gas-chroniatography/mass spectrometry ⁽ GC/MS). The yield of dimethyl 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

I S the corcespo.udi.og raonoesters as the greater part, and was in a range of about 60% to about 65%.

As the reactions depicted in ⁽ he 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 esterilkation reactions yielded more than 50%, typically more than 70% or 80% di-akyi succinate or malate. As stated before, the unreacted materials and the undesired products are recycled into the fermentation reactor. Subsequent separation, of the mono-esters and di- esters w s achieved by crystallization,

Figure 4 shows a series of esterification reactions which summarize COa-assisted esterification of free succinic acid in various alcohols. Figure 4 A shows succinic acid reacted with .methanol in 400 psi CO? gas. at 150 - for 5 hours, which achieved a yield of about 37% dimethyl succinate. When the operational temperature was increased to 180°C in the reaction of Figure 4B and all other parameters kept the same as in Figure 4A, the amount of dimethyl succinate yield increases more than, two-fold to about 81.2%.

Figure 4C represents tree succinic acid reaction at 180°C under present operational conditions in. ethanoL which generates diethyl succinate in good yield of about 60.8%. In Figure 4D, free succinic acid was reacted at 180°C under operational conditions in n-butanol. which generates dibuty! succinate at about 52.2% yield. These examples demonstrate the versatility of the present esterification reaction in view of different kinds of alcohols.

Figure 5 shows examples of CO _? -assisted esterification of other kinds of carboxylic po!yacids. in Figure 5A and SB, succinic acid was substituted respectively with citric acid, a tricarboxylic acid, and .malic acid. The yield of trimethykitrate was reasonable at about 20.1 %, demonstrating that the CO^-asslsted protocol can be applied to tricarboxylic acids. The yield of the dimethyl analogue of malic acid was good at about 84.3%. Flenee, the new method of esterification is feasible for general use with other acids.

'fable 4 summarizes results of several reactions that were performed according to the esteriftcaiion method of the present disclosure as depicted in Figures 6. 7. and 8. Each set of examples is arranged in terms of a variation of an operational condition under which the reaction was performed: A) temperature, B) pressure, and€} reaction time. In each of the examples, succinic acid from a fermentation broth is used as the substrate. The filtered clarified broth containing tree acid and salts are dried and later reacted with methanol and Ci¾ 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.)

In the three examples of Set A, we carried out the reaction for 5 hours at an initial €C½ pressure of 400 psi, under different temperatures: Ex. A--1 at I 80°C Ex. A-2 at 2J0 ^o C, S and Ex. A- 3 at 230°C. The percent conversion of acid to its corresponding diester increased with higher operational temperature. Figure 6 shows the effect of varying temperature in a series of esterification reactions of succinic acid and its salt. m Figure 6 A, the esterification. of succinic acid is performed at a temperature of about 180°C, over a period of 5 hours. The reaction produced about 13.9% yield of dimethyl succinate. Figure 6B shows the same

0 reaction as in Figure 6A, when the reaction, time held constant, but with the temperature

raised to about 210°€, which yields about 42.9%. Figure 6C shows a reaction at 230°C 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 dialkyl-ester. Reactions performed at or near critical temperature and/or pressure3 conditions can produce about 95%, likely > 97% or 98%, conversion.

in Set B and Figure 7, we performed the esterification reaction for 5 hours at an initial temperature of 1 80°C. and varied the initial€<¾ gas pressures: Ex. B-i at 400 psi, Ex. B-2 at 500 psi, 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, The0 initial€■{¼ gas pressure in the reactor did not exert much effect in conversi n of the acid to its diester, but the operational, pressures in the reactor during the reaction suggest an effect on yields.

In Set€ and Figure 8, we performed the esterification reaction, at a constant pressure and temperature but varied the duration of the reaction. Ex. C-l at 5 hours, Ex. C~2 at 2S hours, and Ex. C-3 at 0.5 hours. The examples shown in Figure 8 suggest that a greater

amount of diester was converted from the acid with increased reaction time.

Figure 9 shows a first set oi ^' COj-assisted esterification reactions using a concentration of succinate salts of about 4% w/w, which are presented as Examples 1.-3 in Table 5. In Examples 1 and 2. succinic acid and its magnesium (Mg"'} salt was reacted in0 methanol and ethanol at 210°C and 18C ^' 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 dissolve 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 diethy!sucemate was negligible. Example 3 shows a reaction using calcium (Ca" ^'' ') succinate., a 180°C, over 5 hours. The reaction yields only about 1.33% of the corresponding dimethyisuccinate.

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 in the C(>> experiments was approximately 1. 10: 1. for methanol to magnesium succinate. Likewise, the ratio was about 100:1 for methanol to the other carboxyltc acids.

TABLE 4 - Variations in Reaction Conditions

Exam le Reaction Time Temperature Initial C0 ₂ % Conversion

Substrate Aicohof (h) pressure (psi) to Diester

A

1 Succinic acid

fermentation

broth, ²ⁱ salt Methanol 5 180 400 13.9

Succinic: -acid

fermentation Temperature broth, g ² * sait Methanol r 210 400 49.2 Variation i

3 Succinic acid

fermentation

broth.. Mg ²⁴ salt Methanol 5 230 400 72.4

e

1 Succinic acid

fermentation

broth, Mg ² ' sait Methanol 5 ISO 400 13.9

2 Succinic acid

fermentation Pressure broth, Mg^ sait Methanol S 180 500 11.4 Variation

3 Succinic acid

fermentation

broth, g ²⁺ salt Methanol 5 1.80 600 9.6

C

1 Succinic acid

fermentation

broth, Mg*- salt Methanol S 180 400 13.9

2 Succinic acid

fermentation Reaction Time \ broth, Mg ² * sait Methanol 2 180 400 5.4 Variation !

I O o O i O;

00 ; _" "~> Ϊ 00 00 00 : iXS 00! 00Ϊ

j

9i

- ! - _™ © o i Ό o

Oi ST ί O o C c

¾ ! c TO ΓΪ O

.sr. O i ^■ χ· .c ¾ Ϊ ¾

: Ί T

¾-> : : iii I e& S : < 1 *s Έ

Table 5 lists results from other examples of esterification reactions according to the present method, Examples I , 2 and 3 demonstrate the importance of substrate solubility of succinic acid 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, ethanoL and I. -butanol in similar fashion. Examples 8 and 9 show thai reactions with other carboxyiic acids, such as citric acid and malic acid can achieve relatively good yields of about 20% and H6%, respectively.

Free succinic acid reacts readi ly with the alcohols, since it is completely soluble in methanol. ethanoL butanol, and other alcohols up to and including octanot { ^' ۤ alcohol). In Examples and 7, succinic acid reacted in ethanoi and 1 -butanol, yields 60.8% and 52.2% conversion, respectively.

The solubility of carboxyiic salts in a particular solvent can have an influence on the es eri kaiion process. The greater solubility of free-acid permits a greater reactivity than the carboxylate 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 yielded significantly greater amounts of corresponding diesters than that of Examples 1 -3. The carboxyiic acid itself may be sufficient to catalyze the esterif cation reaction under the present operational temperature and pressure conditions. One can adjust the substrate solubility for successful esterification according to the present method.

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 necessari ly to the embodiments specifically disclosed, but that modifications 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.