J. Chem. T echnol. Biotechnol. 1997, 70, 294È298

Multigram-Scale Production of AliphaticCarboxylic Acids by Oxidation of Alcohols withAcetobacter pasteurianus NCIMB 11664Francesco Molinari,1 Raþaella Villa,1 Fabrizio Aragozzini,1* Paolo Cabella,2Massimo Barbeni2 & Francesco Squarcia21 Dipartimento di Scienze e Tecnologie Alimentari e Microbiologiche. Sezione MicrobiologiaIndustriale-Universita degli Studi di Milano. Via Celoria 2, 20133 Milan, Italy2 San Giorgio Flavors S.p.a., via Fossato 114, 10147 Turin, Italy

(Received 3 October 1996 ; revised version received 10 April 1997 ; accepted 5 June 1997)

Abstract : Acetobacter pasteurianus NCIMB 11664 was selected for multigram-scale production of di†erent aliphatic carboxylic acids through oxidation of thecorresponding alcohols after screening di†erent acetic acid bacteria. Continuousproduction was carried out using an air-lift reactor, with overall yields of 1-propionic, 1-butyric, 2-methyl-1-butyric and 3-methyl-1-butyric acids rangingfrom 45 to 61 g dm~3.

J. Chem. T echnol. Biotechnol. 70, 294È298 (1997)No. of Figures : 2. No. of Tables : 1. No. of Refs : 7

Key words : microbial biotransformation, acetic acid bacteria, Acetobacter pas-teurianus, Ñavour, carboxylic acid

INTRODUCTION

The microbial production of aliphatic acids is playingan increasing role in the manufacture of natural Ña-vours. 1-Propionic, 1-butyric and branched valeric acids(2- and 3-methyl butyric) Ðnd application as Ñavourcomponents and precursors for ester synthesis.1 Amethod for their microbial production is from the oxi-dation of primary alcohols. Although it has been statedthat no microbial ability to oxidize isoamyl alcohol wasknown,1 the oxidation of this alcohol and of otherprimary alcohols by di†erent acetic acid bacteria hasbeen previously reviewed by Asai.2 Acetic acid bacteriapossess a number of dehydrogenases which enable themto oxidize di†erent alcohols with very high efficiencyand these highly productive enzyme systems have beenexploited for several industrial applications.3 Thispotential was exploited for the oxidation of various ali-phatic alcohols to their corresponding acids with astrain of Acetobacter roseus.4 Recently, n-propanol con-

* To whom correspondence should be addressed.

version to propionic acid by Gluconobacter oxydans in afed-batch conversion with high yield (37 g dm~3) hasbeen described.5 We have found that this ability is wide-spread among acetic acid bacteria and decided todevelop this promising method to achieve multigram-scale production of aliphatic carboxylic acids.

2 MATERIALS AND METHODS

2.1 Strains, media and growth conditions

The strains, which were obtained from an official collec-tion (NCIMB: National Collection of Industrial andMarine Bacteria, Aberdeen, UK) and from our collec-tions (MIM: Microbiologia Industriale, Milan) andMAAEM (Microbiologia Alimentare, Agraria ed Eco-logica, Milan), were maintained on GYC slants (glucose50 g dm~3, yeast extract 10 g dm~3, CaCO330 g dm~3, agar 15 g dm~3, pH 6É3) at 28¡C.

2941997 SCI. J. Chem. T echnol. Biotechnol. 0268-2575/97/$17.50. Printed in Great Britain(

Multigram-scale production of aliphatic carboxylic acids 295

The submerged cultures were carried out using fourdi†erent cultural media (GluY: glucose 25 g dm~3,yeast extract 10 g dm~3, pH 5; GlyY: glycerol25 g dm~3, yeast extract 10 g dm~3, pH 5; SorY: sorbi-tol 25 g dm~3, yeast extract 10 g dm~3, pH 5; ManY:mannitol 25 g dm~3, yeast extract 10 g dm~3, pH 5).The strains, grown on GYC slants for 24 h at 28¡C,were inoculated into 500 cm3 Erlenmeyer Ñasks con-taining 50 cm3 of each medium and incubated on areciprocal shaker (100 strokes per minute, spm) at 28¡C.For the determination of dry weight, 100 cm3 of cul-tures were centrifuged, washed with distilled water anddried at 110¡C for 24 h.

2.2 Air-lift reactor

An air-lift reactor (Fig. 1) was designed to carry out bio-transformations on 1 dm3 scale. The air was suppliedthrough a porous sparger after sterilization through a

0É2 km microÐltration membrane ; the air Ñow rate was1É0 dm3 dm~3 min~1. A condenser, with cold water(6¡C) circulating, was placed at the gas outlet of thereactor to reduce the stripping of volatile compounds. Acold trap (liquid nitrogen) at the outlet of the condenserwas able to recover substrates and products stripped bythe air stream.

2.3 Biotransformations

The bench-scale experiments were carried out with 24 hsubmerged cultures. Undiluted substrates were directlyadded to cultures and the Ñasks shaken on a reciprocalshaker (100 spm).

The air-lift reactor experiments were accomplishedusing bacteria grown directly inside the reaction vessel.Continuous addition of aqueous NaOH (34% v/v) andisoamyl alcohol was via a multichannel Watson-Marlow 503 U/R peristaltic pump connected to a pH

Fig. 1. Air-lift reactor used for production of aliphatic carboxylic acids.

296 F. Molinari, R. V illa, F. Aragozzini, P. Cabella, M. Barbeni, F. Squarcia

controller (pH/ORP Controller 3675, JencoElectronics).

2.4 Analytical methods

Substrate and product concentrations were determinedby gas chromatographic analysis on a Carlo Erba Frac-tovap G1 instrument equipped with a hydrogen Ñameionization detector. The column (3] 2000 mm) waspacked with Carbopack B-DA (4% CW 20M) and thecolumn temperature maintained at 200¡C. Samples(0É5 cm3) were taken at intervals and added to an equalvolume of an internal standard (1-butanol, except for1-butanol oxidation where 3-methyl-1-butanol wasused) as a methanol solution. The results are theaverage of three replicates.

3 RESULTS AND DISCUSSION

3.1 Selection of the strain

The screening of acetic acid bacteria able to oxidize 3-methyl-1-butanol (isoamyl alcohol) has been describedpreviously where the production of aliphatic aldehydeswas investigated.6 Most of the strains tested convertedthe alcohol directly to acid with high molar conversion.Other aliphatic alcohols, namely 1-propanol, 1-butanoland 2-methyl-1-butanol, were tested as substrates(2É5 g dm~3) for the present biotransformation studies(Table 1).

The results obtained show that the ability of aceticacid bacteria to oxidize primary aliphatic alcohols totheir corresponding carboxyl acids in quite widespread.Complete conversions of all the alcohols were achievedwith three strains (Acetobacter pasteurianus NCIMB8618, A. pasteurianus NCIMB 11664 and Gluconobacteroxydans NCIMB 8035). Among them, A. pasteurianusNCIMB 11664 exhibited the highest reaction rates, con-verting the four alcohols completely within 2È3 h. A.pasteurianus NCIMB 11664 was, therefore, chosen asthe biocatalyst for subsequent experiments.

The evaluation of various carbon sources (glucose,glycerol, mannitol, sortibol) for microbial growth indi-cated that glycerol was particularly good at inducingthis speciÐc oxidative activity in A. pasteurianusNCIMB 11664. Negligible oxidation of primary alcoholcould be detected using glucose. Therefore, bacteriawere grown for 24 h on glycerol, giving an average dryweight of 5É0 g dm~3 and a Ðnal pH of 7É0.

3.2 Biotransformation

The toxicity of the substrate toward A. pasteurianusNCIMB 11664 was analysed by performing reactionswith di†erent alcohol concentrations. Experiments werecarried out in Erlenmeyer Ñasks using the four primaryalcohols and indicated that the highest initial rates wereobtained with alcohol concentrations in the range 2É5È5 g dm~3, whilst higher concentrations were severelytoxic for the microorganism activity.

The biotransformation was then studied in the air-liftreactor with bacteria grown inside the vessel with an airÑow rate of 1É0 dm3 dm~3 min~1, giving growthsimilar to that observed in the Ñasks. The oxidation of

TABLE 1Molar Conversions (%) of the Oxidation of 1-Butanol, 2-Butanol, 2-Methyl-1-butanol and3-Methyl-1-butanol (substrate concentration 2É5 g dm~3) with Di†erent Acetic Acid Bacteria

after 24 h

Microorganism 1-Propanol 1-Butanol 2-Methyl-1- 3-Methyl-1-butanol butanol6

A. aceti MAAEM 100 100 32 44A. lovaniensis MAAEM 74 75 2 45A. mesoxydans MAAEM 74 84 96 95A. pasteurianus NCIMB 8618 100 100 100 100A. pasteurianus NCIMB 11664 100 100 100 100A. rancens MAAEM 26 36 6 44G. oxydans NCIMB 8035 100 100 100 100A. C1 MIM 95 92 100 100A. sp. CA MIM 95 88 95 97A. sp. CB MIM 95 90 95 100A. sp. CH MIM 97 95 100 100A. sp. GYCE MIM 97 100 100 100A. sp. GYCF MIM 95 100 100 100

Multigram-scale production of aliphatic carboxylic acids 297

Fig. 2. Production of di†erent aliphatic carboxylic acids propionic, butyric, 2-methyl-1-butyric, 3-methyl-1-butyric) by(= + L KAcetobacter pasteurianus NCIMB 11664 with continuous addition of the substrates.

the alcohols was performed at di†erent pH values, tem-peratures and air Ñow rates. The highest rates wereachieved at pH 6È7, 28¡C and at an air Ñow rate of1É0 dm3 dm~3 min~1. A. pasteurianus NCIMB 11664converted all the cited alcohols with an average rate of1É1 g dm~3 h~1.

3.3 Continuous production of acids

The overall yield of these bioconversions could beincreased by performing continuous production of theacid in the presence of a constant alcohol concentrationat which the biocatalyst shows the highest transform-ation rates. This can be achieved by adding the sub-strate at the same rate as consumption occurs. pHcontrol is also important since acid production causesa pH reduction, with negative e†ects on microbialactivity.

Adjustment of the pH to a constant value of 6É5 wasattained using a pH controller. The controller was con-nected to a multichannel peristaltic pump so that thesolution of NaOH was added together with an equi-molar amount of substrate to maintain the alcohol con-centration close to 4È5 g dm~3. The time courses of1-propanol, 1-butanol, 2-methyl-1-butanol and 3-methyl-1-butanol oxidation are reported in Fig. 2.

The conversions were performed over 90 h and ageneral decrease of microbial activity was observed onlyat the end of this period when the alcohol concentrationrose above 5 g dm~3. The decline of the oxidation ratewas somewhat reduced for shorter chain acids (1-propi-onic and 1-butyric) which showed similar proÐles givinga Ðnal yield of about 60 g dm~3. This corresponded toan overall productivity of 0É7 g dm~3 h~1 and to pro-ductivity numbers (PN)7 of 1É6 and 1É4 for propionic

and butyric acid, respectively. The production of thetwo-branched valeric acid was lower, with a 45 g dm~3yield (PN\ 0É9).

4 CONCLUSION

Acetic acid bacteria have long been utilized to catalysethe two-step transformation of primary alcohols intoacids2 but their potential for the preparation of di†erentaliphatic carboxylic acids is still scarcely exploited. Theoxidation of amyl alcohols to produce the correspond-ing acids has previously been described as very difficult1and, more recently, their batch production has beenclaimed in a patent, with only limited yields (10È12 g dm~3).4 The use of Acetobacter pasteurianusNCIMB 11664 as biocatalyst for the continuous pro-duction of various acids in an appropriate air-liftreactor furnished interesting yields of carboxylic acidswith minimal e†ort in process optimization. The pro-cedure is very simple since no immobilization or specialtreatment of the microorganism are needed. Work is inprogress to improve the productivity of the bioprocessby selective and in-situ removal of the products.

REFERENCES

1. Sharpell, F. H., Microbial Ñavors and fragrances. In Com-prehensive Biotechnology, ed. M. Moo-Young. PergamonPress, Oxford, 1985, Vol. 3, pp. 965È81.

2. Asai, T., Oxidation of other aliphatic alcohols. In AceticAcid Bacteria. University of Tokyo Press, Japan, 1968, pp.139È41.

298 F. Molinari, R. V illa, F. Aragozzini, P. Cabella, M. Barbeni, F. Squarcia

3. Kulhanek, M., Microbial dehydrogenations of monosac-charides. Adv. Appl. Microbiol., 34 (1989) 141È82.

4. GatÐeld, I. & Sand, T., European Patent Application289822, 1988.

5. Svitel, J. & Sturdik, E., n-Propanol conversion to propionicacid by Gluconobacter oxydans. Enzyme Microb. T echnol.,17 (1995) 546È50.

6. Molinari, F., Villa, R., Manzoni, M. & Aragozzini, F., Alde-hyde production by alcohol oxidation with Gluconobacteroxydans. Appl. Microbiol. Biotechnol., 43 (1995) 989È94.

7. Simon, H., Bader, J., Gunther, H., Neumann, S. & Thanos,J., Chiral compounds synthesized by biocatalytic reactions.Angew. Chem. Int. Ed. Engl., 24 (1985) 539È53.