Production of propionic acid - Le Lait

Lait (1995) 75, 453-461© Elsevier/INRA

453

Review

Production of propionic acid

P Boyaval, C Corre

Laboratoire de recherches de technologie laitière, INRA, 65, rue de Saint-Brieuc,35042 Rennes cedex, France

Summary - Propionic acid and its salts are widely used in industry and especially in the food indus-try as antifungal agents. The estimated world production in 1992 was about 100 000 tonnes. A largepart of this production is by petrochemical routes. Nevertheless, fermentation processes have beendescribed since 1923. The increasing consumer demand for biological products and the more effi-cient performance of new fermentation processes have revived researcher and industrial interest fora new investigation of biological propionic acid production. From the low productivity of batch pro-cesses (0.03 9 1-1 h-1), performance has rapidly increased to 2 to 14 9 1-1 h-1. This increase results fromthe emergence of high density cell bioreactor technology. These bioreactors make it possible to con-centrate large amounts of cells within the system during continuous fermentation and to reduce the cellinhibition effect of organic acid accumulation in the medium. Moreover, the use of glycerol as the prin-cipal carbon source enables propionic acid to be produced without acetic acid. The increased effi-ciency of membrane processes, such as electro-electrodialysis and electrodialysis with bipolar mem-brane, has considerably facilitated the recovery and purification steps from fermented media. Theincreased fermentation volumetrie productivity and downstream processing performance has led to aneconomic industrial biological propionic acid production.

propionic acid 1 bioreactor 1 purification 1glyceroll Propionibacterium

Résumé - La production d'acide propionique. L'acide propionique et les sels d'acide propioniquesont très largement utilisés dans l'industrie, et notamment dans les industries agro-alimentaires, pourleur rôle antifongique. La production mondiale estimée en 1992 était de 100000 tonnes. Une trèslarge proportion de cet acide est fabriquée par voie chimique, à l'aide de différents procédés issus del'industrie pétrolière. Néanmoins, des procédés de fermentations sont décrits depuis 1923. L'intérêt crois-sant des consommateurs pour des produits naturels et l'émergence de techniques de fermentation deplus en plus petiormantes ont renouvelé l'intérêt des chercheurs et des industriels pour une produc-tion biologique d'acide propionique. De très faibles productivités volumiques (0,03 g t ! tr ')pour lesprocédés batch, les résultats ont rapidement atteint de 2 à 14 g rI tri. Cette augmentation est prin-cipalement le fruit de l'introduction de la technologie des bioréacteurs à recyclage cellulaire qui permettentd'augmenter la biomasse au sein du système et de lever l'inhibition provoquée par l'accumulationd'acides dans le milieu de fermentation. L'introduction du glycérol en tant que principale source carbonéea conduit à l'obtention d'acide propionique sans la présence d'acide acétique. De plus, l'introductionde techniques de séparation petiormantes (électro-électrodialyse et électrodialyse à membranes bipo-

454 P Boyaval, C Corre

laires) a considérablement facilité la récupération et la purification de l'acide propionique à partir de moûtsde fermentation. La combinaison de ces différentes améliorations conduit actuellement à une indus-trie de production d'acide propionique par fermentation économiquement performante.

acide propionique / bioréacteur / purification / glycerol /Propionibacterium

INTRODUCTION

Propionic acid and its salts are used innumerous processes such as in the pro-duction of cellulose plastics (used in tex-tiles, filters, reverse osmosis membranes,lacquer formulations and moulding plastics),herbicides, in the manufacture of ester sol-vents, fruit flavours (citronellyl propionateand geranyl propionate), perfume basesand butyl rubber to improve processabilityand scorching resistance. In animal ther-apy, sodium propionate has been used indermatoses, wound infections, anti-arthriticdrugs and conjunctivitis. In the food industry,propionic acid (E 280) and its sodium (E281), calcium (E 282) and potassium salts(E 283) are incorporated to suppress thegrowth of mou Id and rope in breads andcakes, on the surface of cheeses, meats,fruits, vegetables, and tobacco, grain andsilage preservation, and to prevent the blow-ing of canned frankfurters without affectingtheir f1avour. Dipping containers, caps andwrappers in solutions of these salts is alsoeffective. Moreover, the association of pro-pionic acid with lactic and acetic acids hasbeen recommended for the preservation offoods. This recommendation was reinforcedby the works which demonstrated the syn-ergistic effect of these acids on the inhibitionof Listeria monocytogenes growth in foods.The Food and Drug Administration (FDA)lists the acid, the Na-, Ca++ and K+ salts aspreservatives in their summary of Gener-ally Recognized As Safe (GRAS) additivesand no upper limits are imposed, except forbread, rolls and cheeses (0.30 to 0.38%).Propionate is metabolized like other fattyacids in the mammalian body.

MARKET SIZE AND COST

Reliable statistics on production of the acidplant capacities and priee trends are notavailable for many countries which exportpropionic acid (China, etc). The main pro-ducers are American (Union Carbide, East-man and Celanese). In 1982, their produc-tion was 50 000 tonnes annually for a plantcapacity of 107 000 tonnes. The currentestimations are 60 000 tonnes per year inthe United States and 40 000 tonnes inWestern Europe. The cost has increasedfrom US $0.73 kg-1 (1982) to US $1 kg-1

(1992).

PROPIONIC ACID PRODUCTIONPROCESSES

Chemical processes

Several processes exist for the production ofpropionic acid. These include the Reppeprocess from ethylene, carbon monoxideand steam, and the Larson process fromethanol and carbon mono xide using borontrifluoride as a catalyst. The acid is alsoobtained by oxidation of propionaldehyde, asa by-product in the Fischer-Tropsch pro-cess for the synthesis of fuel and in wooddistillation as a by-product of the pyrolysis.Very pure propionic acid can be obtainedfrom propionitrile.

Fewer supplies and the higher cost ofoil, the opportunity to use by-products of thefood industry as inexpensive media, theincreasing consumer demand for organic

Propionic acid production

natural products and the emergence of moreefficient fermentation processes have ledto a new opportunity for microbial productionto be economically attractive.

Fermentation processes

Microbiology of the fermentations

The first works on propionic acid fermenta-tion resulted in the formulation of the Fitzequation:

3 lactic acid -> 2 propionic acid + 1 aceticacid + 1 CO2 + 1 H20or1.5 glucose -> 2 propionic acid + 1 aceticacid + 1 CO2 + 1 H20

Theoretical maximum yields are 54.8%(w/w) as propionic acid and 77% as totalacids. Formation of propionic acid is accom-panied by the formation of acetate for stoi-chiometric reasons and to maintain hydro-gen and redox balances. The dicarboxylicacid pathway is the most common pathwayfor the formation of propionic acid. Theacrylic pathway, restricted to a few speciesof bacteria (Clostridium propionicum,Megasphaera elsdenii, Bacteroides rumini-cola), also leads to propionic acid forma-tion.

The processes: state of the art

Today, the industrial production of propi-onic acid is almost entirely by petrochemicalroutes, although numerous fermentationprocesses have been patented since 1923,mainly with strains of the Propionibacteriumgenus. They have never passed the pilotplant level because: (i) propionic acid fer-mentation is a very fastidious task (1 to 2weeks to be completed in batch); (ii) theproduction of organic acids by Proploni-bacterium is end-product-inhibited by acetic

455

and propionic acids; and (iii) separation andconcentration of the acid is expensive (Iowconcentration, small difference of volatilitybetween acid and water, and presence ofother acids, especially acetic acid).

Nevertheless, a lot of work has beendone to establish the most important char-acteristics of the production of propionicacid by fermentation with propionic acid bac-teria. Playne (1985) reported more than 40patents resulting in increased productivity,inexpensive media (wood pulp waste liquor,steep water, maize gluten, whey, starchhydrolysates, sulphite waste liquors, ligno-cellulose, etc) or more efficient recovery.Propionibacterium were also grown in mixedcultures with lactic acid bacteria. They havebeen immobilized in calcium-alginate beads,in polyacrylamide gels and in membranebioreactors.

The recent improvements in fermenta-tion technology, with the introduction of highcell density bioreactors, the enhancementof recovery efficiency of membrane pro-cesses and the increasing demand of con-sumers for biological products, have led theLaboratory of Dairy Technology Research ofINRA (Rennes, France) to a systematicinvestigation of the improvement of propi-onic acid fermentation.

Enhancement of propionic acid produc-tivity of fermentation processes

Process improvement

Propionibacterium acidi propionici was usedto ferment lactose from whey permeate topropionic acid in a continuous stirred tankreactor with cell recycle (fig 1). The cellswere separated from the medium and recy-c1ed back to the bioreactor using an ultrafil-tration unit. This fermentation system makesit possible to reduce the propionic acidinhibitory effect on the cells, to increase theconcentration of viable microbes in the biore-


actor and consequently to obtain higher vol-umetrie productivity than the conventionalmethod. If batch fermentation realized volu-metrie productivity of 0.03 9 1-1 h-1, thismembrane bioreactor attained a productivityof 14.3 9 t-1 h-1: more than 480 times greaterthan the traditional process (Boyaval andCorre, 1987). As already observed duringlactic acid production, the specifie acid pro-ductivity decreased when the biomassincreased in the bioreactor: from 0.4 9 ofpropionic acid per gram of cells per hour(g 1-1h") at 10 9 1-1to 0.17 9 g-1 h-1 at 80 91-1. This decreased cell specifie productiv-ity at high cell concentration was probablythe result of physiological perturbations (inhi-bition, substrate limitation or variations in theintracellular water content of the cells, etc).

The acid concentration was 25 9 1-1andthe biomass reached 100 9 1-1 (dry weight[DW]). A periodic bleeding of this concen-trated biomass, used to keep a high cellspecifie productivity, could be an interest-ing source of propionic acid bacteria starters,which play an important role in the ripeningof Swiss-type cheeses. Table 1 presents theproductivity of the principal types of fer-mentation processes for propionic acid pro-duction. These results, obtained with aCSTR with ultrafiltration recycle, led to apilot plant and an industrial plant (37 m3)cou pied with 32 m2 of ceramic membranes(Boyaval et al, 1991). The continuous pro-

PROPIONIC~--+--,----.

ACID

Fig 1. Schematic flow sheet diagram of the pro-cess. (UF: ultrafiltration unit).Schéma simplifié du procédé.

cess was converted to a sequential one(Colomban et al, 1993).

The sequential mode of operationoffered several advantages. The technicaloperations were very similar to batch fer-mentation, therefore the process wasrapidly assimilated by the staff. This pro-gramme allows utilization of only 1 mem-brane device for several bioreactors, whichis very useful from an economic point ofview. The time for filtration was shorter com-pared to the continuous use of a membranebioreactor. There was less clogging of themembrane, so the permeate flux was higherand the cleaning procedures were lessinvolved. In addition, this membrane filtercan be washed during fermentation, a stepimpossible to achieve in continuous runsexcept if the bioreactor is coupled to 2 units,1 being used for filtration while the other isbeing washed (however, 2 pumps are usedin this case). Finally, the filtration devicecan be employed to sterilize the mediumbefore fermentation between normal oper-ating steps.

Nevertheless, only a few studies havedealt with that type of operation. Semicon-tinuous processes have been used toimprove the yield of propionic acid in a con-ventional bioreactor. Repeated recyclingculture has been tested previously by Leeand Chang (1990) to alleviate the aceticacid inhibition in E coli and by Denis andBoyaval (1991) to produce an extracellularmicrobial enzyme in a membrane bioreactor.This mode of bioreactor operation will prob-ably be more appreciated by professionalsfor industrial fermentation plants th an thecontinuous fermentation because it is eas-ier to set up in the factory.

The long-term stability of the process hasbeen proved with the runs presented here(1 000 h) and with numerous others. Wehave observed no loss of catalytic activityof our strains. Moreover, it seems possibleto increase the resistance of P acidi-propi-onicito organic acid inhibition by long, peri-

Propionic acid production 457

Table 1. Comparison of propionic acid fermentation processes.Comparaison des procédés de fermentation pour la production d'acide propionique.

Organism System Carbon Cell Propionic Volumetrie Referencesource concentration acid productivity

(g r') concentration (gr1h-1)(g rI)

P acidi-propionici Batch Lactose 2.37 0.033 Sherman & ShawGlucose (1923)

P acidi-propionici CSTR Glucose 1.51099.51011 3.74 0.19(ATCC 25562) Xylose ce Ils ml-1 0.18

Clausen & GaddyPlug Ilow Glycose 0.49 (1984)tubular Xylose 0.40reactor

Propionibacterium sp Calcium Na-lactate 4109 cells 5 2 Cavin etaialginate (Iree) ml-4 (1989)

gelbeads

P acidi-propionici CSTR + UF Xylose 95 18 2.2 Carrondo et al(ATCC 25562) cell recycle (1987)

P acidi-propionici CSTR + UF Lactose 100 25 14.3 Boyaval & Correcell recycle (1987)

odic cultures with medium enriched in pro-pionic and acetic acids.

At present, trials are performed with 30%dry matter (220 9 1-1 lactose) in order toachieve higher propionic acid contents andto increase the propionic/acetic acid ratio.These runs are conducted with a higherbiomass content (>80 9 1-1). The aim ofthese experiments is to find a balancebetween an increased propionic acid con-centration and the decreased specifie pro-ductivity due to the higher biomass and acidcontents of the medium, without neglectinga total lactose consumption.

Even though propionic and acetic acidsare the 2 major end products of lactose fer-mentation by propionic acid bacteria, otheracids could be produced in the medium: lac-

tic, succinic, pyruvic, malic, fumaric, iso-valerie and formic acids. These "by-prod-ucts" must be carefully examined becausevariations in the process and especially inthe growth medium and agitation speed leadto important variations in the concentrationof these products. A decreased yield of pro-pionic acid production and a modification ofthe final product of the fermentation arethen observed.

Moreover, the recent work of Hsu andYang (1991) shows that even if neutral pH isoptimum for the growth of Propionibacteriumacidi-propionici, the propionic acid yield islow. On the other hand, in the acidic pHrange, the growth rate is low but the yieldis double. With our particular mode of oper-ation, it will be easy to multiply the cells, in


the first cycles, at a neutral pH and to allowan acidification for the next ones to enhancethe yield. A safer process will be obtained atthis lower pH. This process makes it pos-sible to produce concentrated cells (200 9 1-1DW) which can be used as cheese startersin the factory.

Propionic acid production with glycerolas carbon source

Until now, ail of the carbon sources usedto produce propionic acid by fermentationhave led to the cosynthesis of acetic acid.Using Propionibacterium thoenii NCDO1082, we demonstrated that fermentationof glycerol by this propionic acid bacterialed only to propionic acid with no aceticacid. Fed-batch experiments were per-formed (fig 2). The highest productivity was0.3 9 1-1 h-1, and the maximum concentra-tion was 40 ± 2 9 1-1. The specific acid pro-duction rate was equal to zero when theacid concentration reached this value. Con-tinuous fermentation with a membranebioreactor realized the production of high-quality propionic acid with a volumetric pro-ductivity of 1 9 1-1 h-1 (Boyaval et al, 1994).The process can be conducted with a com-plete medium with glycerol as the carbonsource or with glycerol only during "pro-duction steps", while a complete mediumcan be used periodically to "regenerate"the production potential of the cells visu al-ized by a decreasing specifie acid produc-tivity. As a result, the fermented mediumdoes not show any components issuingfrom yeast extract or other nitrogen corn-pounds. This greatly simplifies the down-stream processing for that product. Forexample, direct electro-electrodialysis(EED) or electrodialysis (ED) with bipolarmembranes will be successfully used toconcentrate the propionate salt and to con-vert it to the acid form. Clogging of themembranes, which is the main problem ofthis type of technology, will be greatlyreduced.

Glycerol has now become an inexpen-sive carbon source. The new processesused to extract fuel from vegetable oils pro-duce a large quantity of glycerol as a by-product which needs new uses. The increas-ing interest in a natural source of propionicacid highlights the great demand of thisbiosynthesis for industrial processes thatneed high-quality acid.

PROPIONICiACID g/L

40 .

GLYCEROL'.>' g/L

30

10

30

20 20

10

100 300 TIME (HOURS)200

Fig 2. Typical fed-batch experiment. Four addi-tions of pure glycerol of indicated by arrows on thefigure 15, 30, 40 and 30 ml were done during thisrun. Initial volume 1.2 1; pH 6.8; temperature30°C. (e) Propionic acid; (0) glycerol.Résultat d'une expérience de fed-batch. Quatreadditions de glycérol pur de 15, 30, 40 et 30 mlont été réalisées au cours de cette fermentation.Le volume initial était de 1,21, le pH 6,8 et la tem-pérature 300C. (.) Acide propionique; (0) gly-cérol.


PROPIONIC ACID RECOVERYAND PURIFICATION

State of the art

Downstream processing of organic acidsfrom fermentation is often the major financialdrawback of the process. This is also true forthe recovery of propionic acid. This acid ishighly hydrophilic and the difference ofvolatility between propionic acid and wateris small. Moreover, the optimum pH rangefor that fermentation is, like many other aci-dogenic fermentations, between 6.0 and7.0. This means that the acid is in the ion-ized state and hence is completely non-volatile. The acid concentration is gener-ally, for fermentation processes, less than 509 1-1. This low concentrated product, amongmany other molecules present in the fer-mentation broth, embarrasses the purifica-tion. The main purification processesemployed today are: acidification of the fer-mentation media followed by recovery withanion exchange resins; solvent extractionand distillation; salting out procedures; ester-ification of the acid with ethanol or butanol(the ester is less soluble in water); mem-brane techniques; and other techniquessuch as supercritical fluid extraction andadsorption on zeolite (others have beenexamined but, to our knowledge, are notlargelyemployed).

Enhancementofdownsueamprocessing efficiency

As for other organic acids, the pH must bemaintained during fermentation in order toincrease the productivity. Then salts suchas sodium or calcium propionate areobtained. For numerous uses, however, theacid form is required. Thus, it was interest-ing to convert easily and economically pro-pionate salts to propionic acid, with a con-

459

comitant concentration of the product. EDwith bipolar membranes (BPM: water-split-ting at the junction of 2 homopolar mem-branes) and EED enable inorganic andorganic anions to be extracted and com-bined with protons to produce acids (fig 3).

The evolution of time of propionic acidconcentration from the solution (fig 4) ischaracteristic of the extraction-reconcen-tration curve obtained by ED. In the con-centrate, the amount of the extracted elec-trolyte first increases linearly before reachingthe reconcentration plateau. This recon-centration level is limited by the ion leak-age through the membrane and the watertransport. The results recorded here sug-gest that the limiting process is the watertransport through the anion exchange mem-brane. Moreover, in the presence of onlypropionic acid, the water transport throughthe BPM has the same effect as the waterconsumption taking place during the EEDexperiment and resulting from the waterelectrolysis at the anode.

The results show that the acetate andlactate ions, which are present with propi-onate ions in the fermentation broth, areextracted at the same time, giving rise tothe production of acetic and lactic acid incompartment 2. In the 2 processes, after25 h, the propionic acid concentrationincreases again with time. This result maybe explained by the fact that the totality ofacetate and lactate ions having beenextracted from the fermentation broth, onlythe propionate ion is carried through theanion exchange membrane and the effi-ciency of the electrotransport of propionatebecomes higher. Therefore, the amount ofpropionic acid will most likely be increased,leading to a more concentrated propionicacid, with longer experiments.

Concentrated propionic acid wasobtained by EED and ED with bipolar mem-branes of the fermentation broth. The currentefficiency was high but decreased with time,especially with bipolar membranes. The


aPROPlPcr1C

J rtO-lt- olT, 'l'1 N.t

PROPIONATE

b proprorucacid

BPM t SPM

® © ® © @ ©

~120IJI~- H+ 1OH OH- H+ G

G, +r.~;-LNa

PROPIONATE

Fig 3a. Laboratory cell device for electro-elec-trodialysis. c = Cation exchange membrane; a =anion exchange membrane. b. Laboratory celldevice for electrodialyser with bipolar membranes(BPM).a. Équipement de laboratoire pour t'éiectro-étec-trodialyse (EED). c = membrane échangeuse decations; a = membrane échangeuse d'anions.b. Équipement de laboratoire pour l'électrodia-lyse (ED) avec des membranes bipolaires.

presence of inorganic ions led to the for-mation of other acids (Boyaval et al, 1993).

The influence of the water transport asweil as the current density need to be anal-ysed more thoroughly, taking into consider-ation the electrical energy consumption ofthese processes in order to obtain data whichare necessary for economic estimations.

ln an extractive fermentation process,EED and ED with bipolar membranes canbe used to decrease the acid level in themedium and then allow high cell multiplica-tion and fast acid production. Moreover, thesodium hydroxide produced can be easilyemployed for the pH control of the bioreac-

PROPIONICACID

(g.I-' )120

BO

40

o10 20

TI ME (heurs)

Fig 4. Electro-electrodialysis (EED) and electro-dialysis (ED) with bipolar membrane of a solu-tion of propionic acid (40 9 1-1).Variation with timeof the concentration of propionic acid in the corn-partment 2: (0) EED, (.) ED with bipolar mem-branes (BPM). Current density 70 mA cm-2.Électro-électrodialyse (EED) et électrodialyse(ED) avec des membranes bipolaires d'une solu-tion d'acide propionique à 40 g rI. Évolution de laconcentration de l'acide propionique en fonctiondu temps dans le compartiment 2: (0) EED, (.)ED avec des membranes bipolaires. Densité decourant 70 mA ctrrê.

tor. Compared with solvent extraction, themembrane processes described assumeno trace of extractive solvent in the productwith the subsequent problems to foodacceptability; no problem of pollution bychemical release and associated sodiumhydroxide production which can be re-usedin the fermentation process. Previous ultra-filtration of the fermented whey UF perme-ate achieved in continuous fermentationswith membrane bioreactors permits in lineuse of this sophisticated technology with-out c10gging problems.

CONCLUSION

At present, continuous stirred tank reactorcou pied with ultrafiltration cell recycle mayprove to be the most appropriate process forpropionic acid production. Nanofiltration mem-branes have been used to show the feasi-


bility of the production of highly pure propionicacid with no residuallactose. In addition, thebiological propionic acid colour was greatlyreduced. The performance obtained with abioreactor cou pied to nanofiltration mem-brane for lactic acid production is very promis-ing for the production of propionic acid.

The complete overview of the perfor-mances of EED and the use of bipolar mem-branes for the recovery and concentration ofpropionic acid (or other organic acidsobtained by fermentation processes) need aserious examination of different parameters(membrane nature, current density) and aneconomic evaluation of the process isessential.

REFERENCES

Boyaval P, Corre C (1987) Continuous fermentation ofsweet whey permeate for propionic acid production ina CSTR with UF recycle. Biotechnol Lett9, 801-806

Boyaval P, Colomban A, Roger L (1991) Semi-continu-ous fermentation process for the production of bio-logical propionic acid based preparations. EuropeanPatent n° 9146002206

Boyaval P, Seta J, Gavach C (1993) Concentrated pro-pionic acid production by electrodialysis. EnzymeMicrob Techno/15, 683-686

461

Boyaval P, Corre C, Madec M-N (1994) Propionic acidproduction in a membrane bioreactor. EnzymeMicrob Techno/16, 883-886

Colomban A, Roger L, Boyaval P (1993) Production ofpropionic acid from whey permeate by sequentialfermentation, uhrafiltration, and ceU recycling. Biotech-nol Bioeng42, 1091-1098

Carrondo MJT, Crespo JPSG, Moura MJ (1988) Pro-duction of propionic acid using a xylose utilizing Pro-pionibacterium. Appl Biochem Biotechno/17, 295-312

Cavin JF, Saint C, Divies C (1985) Continuous produc-tion of Emmental cheese flavours and propionic acidstarters by immobilized cells of a propionic acid bac-terium. Biotechnol Lett7, 821-826

Clausen EC, Gaddy JL (1984) Organic acids frombiomass by continuous fermentation. Chem EngProg 80, 59-63

Denis S, Boyaval P (1991) Microbial enzyme produc-tion in a membrane bioreactor. Appl MicrobiolBiotechno/34,608-612

Hsu ST, Yang ST (1991) Propionic acid fermentation oflactose by Propionibacterium acidipropionici: effectsof pH. Biotechnol Bioeng38, 571-578

Lee YL, Chang HN (1990) High cell density culture ofa recombinant Escherichia coli producing penicillinacylase in a membrane cell recycle fermentor.Biotechnol Bioeng 36, 330-337

Playne MJ (1985) Propionic and butyric acids. In: Com-prehensive Biotechnology, Vol 3 (M Moo- Young, ed)Pergamon Press, Oxford, UK, 731-759

Sherman JM, Shaw RH (1943) The propionic acid fer-mentation of lactose. J Biol Chem 56,695-700

Production of propionic acid - Le Lait

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