AU618277B2 - Production of organic acid esters - Google Patents

Description

1 1_ COMMO N W E A L T H OF A U T A I PATENTS ACT 1952 COMPLETP SPECIFICATION6 1 8 2 (Original) FOR OFFICE USE Class Int. .Class Application Number: Lodged: Complete Specification Lodged: Accepted: Published: Priority: Related Art: .i Name of Applicant: Address of Applicant: ROYAL MELBOURNE INSTITUTE OF TECHNOLOGY LIMITED.

124 La Trobe Street, Melbourne, Victoria, 3000, Australia.

Felicity Anne RODDICK.

Margaret L. BRITZ.

a Actual Inventor(s): Address for Service: DAVIES COLLISON, Patent Attorneys, 1 Little Collins Street, Melbourne, 3000.

Complete specification for the invention entitled: "PRODUCTION OF ORGANIC ACID ESTERS" The following statement is a full description of this invention, including the best method of performing it known to US -1la S* 0 I PRODUCTION OF ORGANIC ACID ESTERS to the production of esters of organic acids produced 0 5 by microbial fermentation.

The high efficiency of energy conservation involved in chemical transformation reactions of anaerobic bacteria make them attractive candidates for developing processes involving bioconversions.

10 Strictly anaerobic bacteria have, indeed, been employed in a number of traditional biotechnological processes involving either mixed or pure culture fermentations; examples include anaerobic digestion with accompanying methane generation and acetone-butanol production. Although the economic feasibilitof anaerobic processes for synthesis of chemicals and fuels is governed by a fluctuating petrochemical economy, there has been an upsurge in interest in improving traditional processes, and in developing novel systems. These include intensifying the acetone-butanol fermentation (Ennis et al. 1986), converting C 1 compounds into C 2 to C 6 volatile 1 2 fatty acids (VFAs) (primarily butyric acid) for industrial uses (Pacaud et.al. 1986) or carbohydrates into VFAs as a feed for protein production (Mehta and Callihan 1984), and using anaerobic thermophilic bacteria for ethanol production from cellulose (see Zeikus 1980).

Many of the products of anaerobic fermentation have direct application as solvents and industrial chemicals or they may serve as feedstocks for other synthetic processes. For example, Millis (1984) has pointed out I 9. 10 that methyl and ethyl esters of C 2 to C 6 acids are useful as additives to gasoline (improving octane ratings), and the acids can serve as feedstocks for conversion to alkenes, ketones, ethers and, with ammonia, to nitriles. All of these derivatives are currently and variously used in plastics, coatings or resins, or as flavours and fragrances.

There are several problems in making chemicals through fermentation routes as alternatives to chemical synthesis or extraction: yields (product made/substrate consumed) are often low, the product is often in dilute aqueous solution (requiring concentration and purification, adding cost to the process) and the organism is frequently sensitive to toxic effects of the product, which limits accumulation of high product concentrations (Herrero 1983). One approach to solving these problems has been to intensify the processes by using continuous fermentation systems coupled with product removal, mainly aiming at concentrating the product and alleviating product inhibition hence improving productivity (Millis 1984; Mulder and Smolders 1986; Roffler et al. 1984). Suggested separation procedures include adsorption onto charcoal, membrane separation, and extraction into non-aqueous solvents with subsequent derivatisation occurring in this phase (Millis 1984; Playne 1986). A similar approach has been adopted to production of C 2 to C 6 volatile fatty acids by the anaerobic rumen bacterium, Megasphaera elsdenii (Roddick and Britz 1986). This organism ferments glucose to acetic, butyric and hexanoic acids and lactate to propionic and valeric acids as major end-products. The latter conversion occurs via acrylic acid.

In the production of organic acids by microbial fermentation of a carbonaceous substrate, it is found 10 that once the acid has reached a critical concentration, further production of acid is inhibited. This inhibition can be overcome by the adsorption of the acid onto an anion exchange resin added to or contacted with the fermentation medium. The removal of the acid from the fermentation broth results in an increase in the yield of acid, as well as extraction and purification of the 9. acid. The present inventors have now found that the acid can be conveniently and economically converted to an S* 0* ester while still adsorbed to the resin by reaction with an appropriate alcohol under suitable conditions.

In accordance with the present invention, there i is provided a method for the production of an ester of an organic acid produced by microbial fermentation of a carbonaceous substrate, which comprises the steps of: recovering the organic acid produced by said microbial fermentation from the culture medium by adsorption of the acid onto an anion exchange resin; and (ii) reacting said organic acid while adsorbed on said anion exchange resin with an alcohol to esterify said acid.

As pointed out above, adsorption of the acid onto the anion exchange resin provides a simple and efficient means of extraction and purification of the 4 organic acid, whilst simultaneously increasing the yield of acid. Esterification of the recovered acid in situ on the resin similarly provides an effective method of producing the desired ester without the need for eluting the acid as an additional step prior to esterification.

It will, of course, be appreciated that where two or more organic acids are produced simultaneously by microbial fermentation, they can be adsorbed together onto the anion exchange resin to remove them from the culture 10 medium. Subsequently, they may either be simultaneously esterified in situ to form a mixture of esters, or alternatively they may be selectively eluted to leave only a single adsorbed acid prior to the esterification step.

The anion exchange resin used may be any suitable such resin, for example a resin having S. trimethylammonium groups such as Amberlite IRA 400 or Amberlite IRA 900.

The in situ esterification of the organic acid may be effected by any standard esterification procedure known to persons skilled in the art.

Preferably, the water content of the resin with the acid adsorbed thereon is reduced prior to the esterification step to improve the yield and conversion rate. Suitable methods of reducing the water content include drying, for example at a vacuum pump, and/or washing with ethanol.

The method of the present invention is applicable to the production of a wide variety of esters of organic acids produced in well known processes by a variety of microorganisms. The production of esters of hexanoic acid and other volatile fatty acids produced by Megasphaera eldenii is particularly described herein to exemplify the method of this invention, and in this particular embodiment, acidic eri nroducts including hexanoic acid, have been shown tc ju absorbed onto the anion exchange resin and to remain bound during the elution procedure. It has been found that most of the butyric acid can be eluted while hexanoic acid remains bound, where it can be subsequently chemically converted into esters by procedures such as acidic-ethanol S. treatment. It is to be understood, however, that tis *invention is not limited to the production of these 10 particular esters, or the use of this particular microorganism.

In general terms, a variety of carbonaceous substrates, for example glucose, sucrose, fructose, maltose, lactose and/or starch, may be used in accordance with this invention, and these can be fermented by any suitable microorganism to produce organic acids, for example acetic, propionic, butyric, valeric, hexanoic, and acrylic acids. In addition, the production of isoand n-acids by microbial fermentation may be utilised in accordance with this invention in the production of specific high value esters. The yield of acid is increased by removal from the fermentation medium by adsorption onto an anion exchange resin, e.g. one with trimethylammonium groups, and the adsorption of the acid onto the resin also provides a means of extraction and purification of the acid from the fermentation medium.

Whilst the acid is still adsorbed to the resin, an in situ conversion of the acid to an ester by the addition of an appropriate alcohol preferably under acidic conditions, e.g. ethanol, iso- and n-propanol, iso-, sec- and tert-butyl alcohol, pentanol, or vinyl alcohol is performed. Examples of esters which may be produced by this process include butyl acetate, ethyl acrylate, ethyl propionate, ethyl butyrate, methyl hexanoate, ethyl valerate, and ethyl hexanoate.

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Whilst the anion exchange resin may be added to the fermentation broth to adsorb the organic acid from the broth, in order to more efficiently remove the acid the fermentation broth may be continuously or periodically passed through a column of the resin. In one particularly efficient embodiment, a system of two or more columns may be provided whereby broth from a fermenter is continuously or periodically circulated o. through a first column until the resin in that column is 10 saturated with acid, and the broth is then switched to another column whilst the drying, esterification and regeneration procedures are applied to the first column.

The first column is then available for re-use.

The method of the present invention is further exemplified by the following Examples and drawings. In *the accompanying drawings: Figure 1 shows growth, acid synthesis and glucose utilisation for batch cultures performed with or without manual pH control. Symbols: pH, no pH control pH, adjusted to 7 butyric acid concentration hexanoic acid concentration Figure 2 shows effects of the presence of an 00 anionic exchange resin on growth and glucose consumption by M.elsdenii in batch culture. Symbols: no resin; resin in dialysis bags; Q loose resin.

Figure 3 shows growth, acid synthesis and glucose utilisation for cultures of free cells cells immobilised in K-carrageenan and immobilised cells cultured in the presence of resin butyric acid concentration: hexanoic acid concentration.

Figure 4 shows fed-batch culture of immobilised cells grown in the presence of resin. Symbols: (0) 7 absorbance for cells in gel; absorbance of culture supernatant fluids; glucose concentration; (L) hexanoic and butyric acid concentrations.

EXAMPLE 1 PRODUCTION AND RECOVERY OF ORGANIC ACIDS.

Materials and methods Strains, media and growth conditions.

M.elsdenii strain ATCC25940 was maintained as a 10 freeze-dried culture and by sub-culturing on THBAG plates (Todd-Hewitt broth, Oxoid, solidified with 2% agar after supplementing with 1% glucose, 0.05% cysteine-HCl and 6% ii horse blood, CSL). Liquid cultures were performed in PY medium (Holdeman and Moore 1972) supplemented with 4% glucose (PYG), unless otherwise noted. When used, S C(U)]-glucose (Amersham) was added to give approximately 105 d.p.m./mL. Plates and starter cultures(100 mL. inoculated from colonies on THBAG plates) were incubated in anaerobic jars under an atmosphere of hydrogen plus carbon dioxide (GasPak, Oxoid) after flushing jars with oxygen-free nitrogen. A palladium catalyst was used.

(ii) Batch fermentation conditions.

Inocula were grown for 48 to 72 h before being transferred under nitrogen flow into 500 mL of PYG in a 1 L-capacity glass vessel with bottom stirring and temperature controlled at 37 0 C by a waterbath. Nitrogen flow was stopped once growth was sufficient to maintain a positive gas pressure. Fermentations were normally performed with manual pH control, where the pH was measured periodically then adjusted to 7 using 2M NaOH.

Samples were also removed periodically under nitrogen flow and analysed for VFAs, glucose concentration, 8 absorbance at 550 nm and, when appropriate, radioactivity. Batch cultures performed in parallel with immobilised cell cultures were inoculated with cells concentrated into 0.9% saline (see below) and 500 mL of medium was used.

(iii) Immobilisation of cells.

Typically, cells from 100 mL starter cultures were harvested by centrifugation after 48 to 72 h growth 10 then resuspended in 5 mL of 0.9% sterile saline, After warming to 45 0 C, the cells were added to 100 mL of 4% K-carrageenan held at 45°C following autoclaving, mixed .o rapidly then the suspension poured into Petri dishes.

When set, the gel was cut into cubes of 1.5-2 mm edge then hardened by suspension in 0.3M KC1 for 45 min. When monitoring growth, samples were withdrawn periodically S. and absorbances of the culture supernatant fluids measured after removing the gel. The gel was then o. homogenised and absorbances for cells released from the gel were measured against an homogenised gel blank. pH was recorded and manually adjusted to 7. In fed-batch j mode for both immobilised cells and batch fermentation controls, additional glucose final concentration) was added at times indicated on graphs and samples again removed for measuring total glucose concentrations.

(iv) Use of the resin.

The resin, Amberlite IRA 400 (trimethylammonium on polystyrene, BDH), was activated immediately prior to use by washing with distilled water then 2M NaOH followed by extensive washing and distilled water to remove excess NaOH. When the resin was used loose in the fermentation broth, it was sterilised prior to activation by autoclaving at 121°C for 10 min. When used in dialysis Al 9 bags (Union Carbide type 20, 20 mm diameter), the resin was suspended in PYG before transfer into sterile tubing. Early batch experiments were performed using wet weight of resin in 600 mL fermentations. This was increased to 150-200g wet weight of resin in 500 mL cultures to avoid exceeding the binding capacity of the resin. The resin was collected at the end of the fermentations, washed over glass wool with PYG to remove cells, then eluted by serial washing in 3X160 mL 2M NaOH, 10 3X160 mL distilled water and 3X160 mL HC1 (for every 100 g of resin) then the washings pooled for measurement of VFAs. The binding capacity of the resin was determined by stirring 10g wet weight of resin in 5X20 mL lots of PYG containing 50 mM each of acetic, butyric and hexanoic acids (pH followed by elution with 3X15 mL aliquots each of 2M NaOH, d.H 0 and 2M HC1. The 10 g bound 3.4 *2 mmoles (0.3 g) butyric and 5.0 mmoles (0.6 g) of hexanoic acids; the recovery was 92% and 31% respectively. All calculations of VFA concentrations were corrected for 20 acid remaining bound to the resin.

i Analytical procedures.

Glucose was measured using the DNS method (Sumner and Somers 1944). Concentrations of VFAs (mM) were measured by GLC (FID) on Chromosorb 101 (Carlsson 1973) after acidifying 1 mL samples with 0.5 mL formic acid containing 0.005% isovaleric acid as internal standard. Total mmoles of acids found in culture fluids and eluted from resin were calculated and the "effective concentration" (mM or g/L) defined as the concentration of acid produced in the original culture volume.

Radioactivity was measured as described previously (Britz and Lowther 1981).

Results Batch fermentation, with and without PH adjustment.

Earlier experiments using test-tube cultures (Britz and Wilkinson 1984) indicated that the major product of glucose fermentation by M. elsdenii was hexanoic acid, which was made at concentration of 10-11 mM Butyric acid was produced at a 10 concentration of 4.5 mM (0.4 g/L) and acetic acid was a minor product. In 600mL stirred-batch fermentations (Fig. total acids increased to 3.2 g/L (2.6 g/L hexanoic acid). Although levels of acids continued to increase for up to 200 h fermentation, most product was formed in the first 50 hours of culture; the pH decreased to 5.2 within 20 h of culture but did not decrease further. In contrast, when the pH was manually adjusted to 7, the period of acid synthesis was increased, final concentrations of acids were higher (hexanoic,7.3 g/L; butyric, 2.1 g/L) and more glucose was consumed, indicating that low pH was deleterious to metabolism. In both cultures, absorbance peaked within 30 h of culture then declined, presumably corresponding to cell lysis.

Productivity over the first 50 h of culture where pH was uncontrolled was 0.052 g/L/h, whereas when the pH was adjusted, this was 0.093 g/L/h for the same period (average productivity over 120 h, the period in which acid concentrations continued to increase, was 0.060 Yields appeared to be higher (see summary in Table 2) in the pH uncontrolled culture, but this was difficult to measure because of the small amount of glucose consumed. The yield for the pH adjusted culture was 0.3 g hexanoic acid/g glucose. In all subsequent cultures, the pH was adjusted to 7 periodically (Fig. 1 shows typical increments).

11 (ii) Effects of including an anionic exchange resin in batch cultures.

The resin was present either free in the culture or contained within dialysis bags. Free resin increased both the rate and the degree of glucose consumption and prevented the decrease in absorbance normally seen in ageing cultures (Fig. maximum absorbances achieved I '"did not increase. When D-[ 14 C(U)l]-glucose was i included during culture, counts detectable in cultures I 10 grown in the presence of resin decreased during the fermentation and 46% of the radioactivity supplied was S i absorbed onto the resin. Only 20% of the bound radioactivity was removed by the elution procedure, indicating that acidic end-products, including hexanoic 15 acid, remained bound. Radioactivity in the control fermentation lacking resin did not decrease during the course of the experiment.

Although there was some variation between experiments, the presence of resin (loose or in bags) always increased the productivity and effective concentrations of acids produced (Table Resin in dialysis bags was not as effective as free resin and its presence did not alter growth or glucose consumption profiles markedly, probably due to poor accessibility of binding sites.

(iii) Production of acids by immobilised cells and effects of resin.

Preliminary results indicated that cells immobilised in K-carrageenan could convert glucose into butyric and hexanoic acids but that rates of synthesis and final concentrations achieved were not as high as those for free cells (Roddick and Britz 1986). However, 12 when inocula were increased so that the initial absorbances for immobilised cells was about twice that for free cells, the glucose consumption profiles for immobilised cells mimicked those for free cells (see Fig.

Final concentrations of hexanoic acid were similar (9.1 g/L control culture, 8.2 g/L immobilised) with yields about 36%. The final absorbance for the immobilised cells was equivalent to that of the maximum for free cells (10.3) although there was some leakage of 10 cells from the gel (absorbance in culture fluids was 2.8 S* at the end of culture): as samples were not taken during the period of fermentation to determine absorbances of cells growing in the gel, it is not known if this corresponds to maximum growth. When resin was included in the culture of immobilised cells, all of the glucose .supplied was consumed within 70 h of culture and the **effective concentration of hexanoic acid was 13 g/L.

There was little leakage of immobilised cells from the gel and final absorbance was approximately 14, hence growth was improved or lysis was impaired relative to the control culture.

(iv) Effects of fed-batch mode on immobilised cells cultured in the presence of resin.

The above results using immobilised cells cultured with resin suggested that there was a possibility that final amounts of hexanoic acid synthesised could be increased if more glucose was supplied during culture. Using fed-batch operation for free immobilised cells did not improve final concentrations, yields or productivity (see summary in Table However, feeding glucose during culture in the presence of resin (Fig. 4) resulted in 71 g/L of glucose being consumed and an effective hexanoic acid concentration of 24.1 g/L (34% yield) (butyric acid, 3.4 Growth within the gel peaked after 60-80 h culture, with a small decline in absorbance seen with prolonged culture; again, little leakage from the gel occurred (maximum absorbances of culture supernatant were about 0.05).

Discussion I. 10 It has previously been shown (Roddick and Britz 1986) that M. elsdenii is unable to initiate growth in high concentrations of neutralized C 2 to C 6 acids, 0: 2 6 where the degree of inhibition increases with chain length. Further, the present results have now shown that when pH was adjusted to neutral during culture, final concentrations of products increase and more glucose was consumed. However, growth was not stimulated significantly and decreases in absorbances of ageing cultures were still seen, merely indicating that the metabolic activity of the cells was maintained for a longer period with higher productivity if pH did not remain at 5.2 for prolonged periods. Employing an anionic exchange resin during culture for in situ product removal improved glucose consumption and hexanoic acid production, although butyric acid synthesis remained largely unchanged. Table 2 presents a summary of results. Immobilising cells in K-carrageenan and using fed-batch mode, coupled with removal of acid products onto resin, resulted in the greatest amount of hexanoic acid being synthesised with a productivity level equivalent to free cells cultured in the presence of resin. Yields did not increase despite the mode of growth, remaining at an average of 0.34 g hexanoic acid/g glucose used.

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S. S 14 TABLE 1 Effects on VFA production of addition of Trimethylammonium-resin to batch cultures.

Time of Control Dialysis Bags Loose Resin harvest Acid g/L/ha g/Lb g/L/h g/L g/L/h g/L Expt. No.1 But. 0.027 1.8 NTc 0.029 1.9 Hex. 0.126 8.2 0.228 14.8 Expt. No.2 122 But. 0.017 2.1 0.015 1.8 0.017 2.1 Hex. 0.029 3.5 0.063 7.7 0.107 13.0 Ext. No.3 96 But. 0.023 2.2 0.023 2.2 0.023 2.2 Hex. 0.077 7.4 0.114 10.9 0.137 13.1 Expt. N0.4 95 But. 0.021 2.0 0.023 2.2 NT Hex. 0.073 6.9 0.100 Expt. 96 But. 0.016 1.5 0.032 3.0 NT Hex. 0.049 4.7 0.066 6.4 As resin was collected only at the end of the fermentation, productivity figures were calculated at the point of termination.

Effective concentration of acid (total in culture fluids and resin-bound).

Not tested.

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r 21 TABLE 2 Hexanoic acid concentrations, productivity and yields for different growth conditions.

Growth conditions Yield Effective conc. Productivity coefficient of hexanoic (Yp)s acid (g hexanoic/ g glucose).

p .*n pp p.

p* batch, no pH control 2 pH adjusted to 7 b 6 batch, pH adjusted to 7: resin in dialysis bagsc 8 loose resind 13 fed-batch 8 .6 .1 .6 .6 .2 0.026 a 0.060 0.086 0.157 0.085 0.073 0.115 0.062 0.134 0.52 0.30 0.37 0.46 0.32 0.33 0.37 0.21 0.34 immobilised cells, pH batch loose resin fed-batch fed-batch, loose resin adjusted to 7: 8.2 13.0 7.3 24.1 calculated for 120 h culture; productivity in first 50 h was 0.052 g/L/h.

average of 5 experiments.

average of 4 experiments.

average of 3 experiments.

0e p p 16 EXAMPLE 2 ESTERIFICATION OF RECOVERED ORGANIC ACIDS.

Adsorption and Desorption of VFAs from Anionic Resins.

Anionic resins tested were Amberlite IRA400 and IRA900. The moisture content of each resin preparation was measured, and 10.0g dry weight of each resin was tested for adsorption of butyric and hexanoic acids each) from fermentation broth (PYG broth see Example 1).

Immediately prior to adsorption, the resins had 10 been washed and activated as per the following regime: d. HO 2 x 100mL 2M NaOH 1 x 100mL d. H20 2 x lOOmL 2.3 M HC 1 x 100mL d. H20 2 x 100mL **2M NaOH 1 x 100mL d. H 2 0 until wash water neutral pH of d. H 2 0).

Adsorption was conducted using 50mL aliquots of I 20 fresh medium which was changed every 20min total of 9 x 50mL lots of medium, each for 20min; so as to simulate the fermentative production of the organic acids at semi-controlled pH). Adsorption was performed in sealed conical flasks in a shaking water bath (140 opm) at 37 0

C.

The results are set out in Table 3: TABLE 3 Acid adsorbed after 9 x 20min exposures in aliquots broth (mmoles/lOg.d.wt.) Acid IRA 400 IRA 900 butyric 5.4 4.2 hexanoic 10.1 6.4 17 In order to test the resins for desorption of VFAs, the fermentation broth was removed by filtration and the VFAs eluted by the following washing regime, each wash being conducted with shaking for 2M NaOH 2 x d. HO2 2 x 2.3M HC1 3 x *of* The results are set out in Table 4: TABLE 4 Total desorbed acid (mmole) and percentage recovery g 1.

i j

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1 S S

S

Acid IRA 400 IRA 900 butyric 3.8 [71] 4.2 [100] hexanoic 3.0 [30] 6.0 [94] 20 Hence Amberlite IRA 400 has a greater capacity for adsorption of hexanoic acid, however a much better recovery of both butyric and hexanoic acids was obtained from Amberlite IRA 900.

Amberlite IRA 400 was used in the subsequent experiments described below because of its greater capacity for hexanoic acid.

Esterification of Hexanoic Acid in the absence of resin.

Hexanoic acid (~10mmoles) was added to 25mL AR absolute (99.89%) ethanol and the degree of esterification in the presence of varying concentrations of conc.H 2

SO

4 (catalyst) established. The reaction was conducted at 55 0 C with shaking (140opm) over a period of 80 min, and was sampled at 20 min. intervals.

Degree of conversion of hexanoic acid into ethyl hexanoate initial mmoles hex.acid final mmoles hex.acid x 100% initial mmoles hex.acid The results are set out in Table TABLE 10 Concentration of H2SO 4 Conversion wt. H SO4/wt.ethanol) too 0060 0 S 6 S@ 60

SS

0 O S S 0 56 6 S S .5 S.,r 0 6* *0 *0 0

SS

5O 2 98.7 3 98.9 5 98.9 10 99.2 The above experiment was repeated using no 20 H 2

SO

4 after 6h the degree of conversion was 3.8%.

All reactions reached equilibrium within the initial reaction rate being directly dependent on the concentration of H2SO4 within the reaction mixture.

Esterification of Resin bound hexanoic acid.

Resin (5 lots of 10g dry weight) was washed and activated according to the regime described in above.

Resin aliquots were added to individual flasks containing fermentation broth at pH7 (PYG-broth see Example supplemented with hexanoic acid (=200mM).

Three changes of fermentation broth were used, each exposure being for 1.5h in a shaking water bath at 140 opm and 37 0

C.

r i 19 Following acid adsorption, the resin aliquots were collected by vacuum filtration on a sintered glass funnel and the adsorbed hexanoic acid subjected-to esterification after various resin treatments: resin used immediately after filtration; immediately after filtration resin washed with 100mL abs.ethanol with agitation for 5min, then used immediately after filtration; resin dried at vacuum pump on filter for S 10 resin dried at vacuum pump on filter for resin dried at vacuum pump on filter for washed with 50mL abs.ethanol and then dried at vacuum pump for S i* The esterification conditions used were as follows: and 25mL ethanol; 50mL ethanol; all contained 10% H 2

SO

4 (10% wt.conc.acid/wt.ethanol).

The reactions were conducted at 5° 0 C with shaking (140 opm) and were sampled at 30 minute intervals.

.I 00 li~C- The results are set out in Table 6: TABLE 6: 0 0*00 0* 0 00 0 @0 00 0 0 00 S 0 Initial Hex. Hex.Acid Conversiona Yieldb Recoveryc Acid conc. Adsorbed (Hex as (mM) (mmoles) acid ester) immediately after filtration -212 23.4 3 2 84 ethanol wash after filtration 212 25.3 22 15 dried at vacuum pump 185 16.7 85 89 104 dried at vacuum pump 185 15.7 87 97 105 dried at vacuum pump 50mL ethanol wash, dried at vacuum pump, 5 min, 185 15.0 95 88 Conversion mmole hex.ester x 100% 0000 0 0 0 00* °o (po o oo mmole hex.ester mmole hex.acid Yield mmole hex.ester x 100% mmole adsorbed hex.acid Recovery mmole hex.ester mmole hex.acid x 100% mmole adsorbed hex acid Resin treatment i.e 90min drying at vacuum pump, plus ethanol wash, plus 5min drying at vacuum pump, gave the fastest rate of reaction with equilibrium being reached after 60min. Clearly the water content of reaction mixture (as contained in resin) was critical to the reaction rate.

21 Recyclability of resin used for in situ esterification of adsorbed hexanoic acid.

The resin samples, and used in the esterification tests above were tested for further hexanoic acid adsorption, and resin sample also for further hexanoic acid esterification.

The resin samples were washed with d.H 2 0 (3 x 250mL), activated with 2M NaOH (lxl00mL) and washed with distilled water until the pH of the washings was that of 10 d.H 2 0.

The resins were subjected to hexanoic acid adsorption, as described in above, and esterification of the acid adsorbed to resin sample was performed, after the pretreatment described below. The results are set out in Table 7: TABLE 7: Resin Initial Hexanoic Hexanoic Acid Treatment Acid conc.(mM) Adsorbed (mmoles) Se ariea at vacuum pump 5 min 195 17.6 dried at vacuum pump 90 min 195 16.2 dried at vacuum pump 90 min, ethanol wash 5 min, dried at vacuum pump min. 195 16.4 The treatment for resin sample after hexanoic acid adsorption and prior to esterification was as follows: dried at vacuum pump 5 min; ethanol wash 50mL -5 min; dried at vacuum pump 22 After subsequent esterification, as described in above, the results were: conversion 96% yield 77% recovery ease sees*: 99 0 so0 S0.0 23

REFERENCES

Britz ML, Lowther DA (1981) Some properties of neutral proteinases from lysosomes of rabbit polymorphonuclear leucocytes. AJEBAK 59:63-75 Britz ML, Wilkinson, RG (1984) Production of longer chained volatile fatty acids by anaerobic bacteria. Abstracts, VII International Biotechnology Symposium, New Delhi, p 404.

Carlsson J (1973) Simplified gas chromatographic procedure for identification of bacterial metabolic products. Appl Microbiol 25:287-289.

Ennis BM, Gutierrez NA, Maddox IS (1986) The acetone-butanol fermentation: a current assessment. Process Biochem 21:131-147.

Herrero AA (1983) End-product inhibition in anaerobic fermentations. Trends Biotechnol 1:49-53.

Holdeman LV, Moore WEC (eds) (1972) Anaerobics Laboratory Manual, Virginia Polytechnic Institute and State University, Blacksburg.

Mehta KI, Callihan CD (1984) Production of protein and fatty acids in anaerobic fermentation of molasses by E. ruminantium. JAOCS 61:1728-1734.

Millis NF (1984) Solvents and chemical feedstocks: can microbes help? In Dean I, Ellwood DC, Ivan CGT (eds) Continuous culture 8: Biotechnology, medicine and the environment. Ellis Horwood Ltd., Chichester, p 292 Mulder MHV, Smolders CA (1986) Continuous ethanol production controlled by membrane processes.

Process Biochem 20:35-39.

Pacaud S, Loubiere P, Goma G, Lindley ND (1986) Organic acid production during methylotrophic growth of Eubacterium limosum B2: displacement W~ i towards increased butyric acid yields by supplementing with acetate. Appl Microbiol Biotechnol 23:330-335 Playne MJ (1986) Propionic and butyric acids. In: Cooney CL, Humphrey, AE (eds) Comprehensive biotechnology: the principles, applications and regulations of biotechnology in industry, agriculture and medicine, vol 3. Pergamon Press, New York. p 731.

Roddick FA, Britz ML (1986) Influence of product removal on volatile fatty acid production by Megasphaera elsdenii. Proceedings, VIIth Australian Biotechnology Conference, Melbourne, p 386, Roffler SR, Blanch, HW, Wilke, CR (1984) In situ recovery of fermentation products. Trends Biotechnol 2:129-136.

Sumner J, Somers G (1944) Laboratory experiments in biological chemistry, Academic Press, New York.

Zeikus JG (1980) Chemical and fuel production by anaerobic bacteria. Ann Rev Microbiol 34:423-464

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Claims (9)

1. A method for the production of an ester of an organic acid produced by microbial fermentation of a carbonaceous substrate, which comprises the steps of: recovering the organic acid produced by said microbial fermentation from the culture medium by adsorption of the acid onto an anion exchange resin; and (ii) reacting said organic acid while adsorbed on j said anion exchange resin with an alcohol to esterify said acid.

2. A method according to claim 1, wherein water is S" removed from said anion exchange resin after adsorption of the organic acid from the culture medium and prior to said esterification step.

3. A method according to claim 1 or claim 2, wherein said resin is added to said culture medium and j• subsequently said resin having organic acid adsorbed thereon is removed from said culture medium.

4. A method according to claim 1 or claim 2, i e wherein said culture medium is continuously or j 'periodically passed through a column of said resin to enable organic acid in said culture medium to be adsorbed on said resin. A method according to claim 4, wherein at least two resin-containing columns are provided, and said culture medium is passed through a first column to load organic acid onto the resin in said first column, and subsequently said culture medium is passed through another column whilst the organic acid on said first column is esterified and the resin in said first column is subsequently regenerated.

MW-- 26

6. A method according to any one of claims 1 to wherein said anion exchange resin contains trimethyl- ammonium groups.

7. A method according to claim 6 wherein said anion ezchange resin is selected from Amberlite IRA 400 and Ainberlite IRA 900. i

8. A method according to any one of claims 1 to 7, wherein said esterification step comprises reaction of the organic acid with an alcohol under acidic conditions. .00

9. A method according to claim 8, wherein said esterification step comprises reaction of the organic acid with acidic ethanol to form an ethyl ester. e A method according to claim 1, wherein said organic acid is hexanoic acid produced by anaerobic fermentation of Megasphaera eldenii, and said hexanoic o- acid is reacted in situ on said anion exchange resin with acidic-ethanol to produce ethyl hexanoate. '.dl 0i.9- Thg t S, f n n n ier and nmpann l referred to or indicated in th ecification and/or claims of this applicati r individually or collectively, and any and a combinations of any two or more of said st oer features. Dated this 29th day of August, 1988. ROYAL MELBOURNE INSTITUTE OF TECHNOLOGY LIMITED, By its Patent Attorneys, DAVIES COLLISON. A. >OFC -s