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Production of propionic acid P Boyaval, C Corre

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P Boyaval, C Corre. Production of propionic acid. Le Lait, INRA Editions, 1995, 75 (4_5), pp.453- 461. ￿hal-00929451￿

HAL Id: hal-00929451 https://hal.archives-ouvertes.fr/hal-00929451 Submitted on 1 Jan 1995

HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Lait (1995) 75, 453-461 453 © Elsevier/INRA

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 large part of this production is by petrochemical routes. Nevertheless, processes have been described 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 for a 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 from the emergence of high 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 cell inhibition effect of organic acid accumulation in the medium. Moreover, the use of glycerol as the prin- cipal source enables propionic acid to be produced without . 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. The increased fermentation volumetrie productivity and downstream processing performance has led to an economic industrial biological propionic acid production. propionic acid 1 bioreactor 1 purification 1glyceroll

Résumé - La production d'acide propionique. L'acide propionique et les sels d'acide propionique sont très largement utilisés dans l'industrie, et notamment dans les industries agro-alimentaires, pour leur rôle antifongique. La production mondiale estimée en 1992 était de 100000 tonnes. Une très large proportion de cet acide est fabriquée par voie chimique, à l'aide de différents procédés issus de l'industrie pétrolière. Néanmoins, des procédés de sont décrits depuis 1923. L'intérêt crois- sant des consommateurs pour des produits naturels et l'émergence de techniques de fermentation de plus 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 les procédés 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 permettent d'augmenter la biomasse au sein du système et de lever l'inhibition provoquée par l'accumulation d'acides dans le milieu de fermentation. L'introduction du glycérol en tant que principale source carbonée a conduit à l'obtention d'acide propionique sans la présence d'acide acétique. De plus, l'introduction de 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 moûts de 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 MARKET SIZE AND COST

Propionic acid and its salts are used in Reliable statistics on production of the acid numerous processes such as in the pro- plant capacities and priee trends are not duction of plastics (used in tex- available for many countries which export tiles, filters, reverse osmosis membranes, propionic acid (China, etc). The main pro- lacquer formulations and moulding plastics), ducers are American (Union Carbide, East- herbicides, in the manufacture of sol- man and Celanese). In 1982, their produc- vents, fruit flavours (citronellyl propionate tion was 50 000 tonnes annually for a plant and geranyl propionate), perfume bases capacity of 107 000 tonnes. The current and butyl rubber to improve processability estimations are 60 000 tonnes per year in and scorching resistance. In animal ther- the United States and 40 000 tonnes in apy, propionate has been used in Western Europe. The cost has increased dermatoses, wound infections, anti-arthritic from US $0.73 kg-1 (1982) to US $1 kg-1 drugs and conjunctivitis. In the food industry, (1992). propionic acid (E 280) and its sodium (E 281), (E 282) and salts (E 283) are incorporated to suppress the PROPIONIC ACID PRODUCTION growth of mou Id and rope in breads and PROCESSES cakes, on the surface of cheeses, meats, fruits, vegetables, and tobacco, grain and preservation, and to prevent the blow- Chemical processes ing of canned frankfurters without affecting their f1avour. Dipping containers, caps and Several processes exist for the production of wrappers in solutions of these salts is also propionic acid. These include the Reppe effective. Moreover, the association of pro- process from , pionic acid with lactic and acetic acids has and steam, and the Larson process from been recommended for the preservation of foods. This recommendation was reinforced and carbon mono xide using boron by the works which demonstrated the syn- trifluoride as a catalyst. The acid is also ergistic effect of these acids on the inhibition obtained by oxidation of , as of Listeria monocytogenes growth in foods. a by-product in the Fischer-Tropsch pro- The Food and Drug Administration (FDA) cess for the synthesis of fuel and in wood lists the acid, the Na-, Ca++ and K+ salts as distillation as a by-product of the pyrolysis. in their summary of Gener- Very pure propionic acid can be obtained ally Recognized As Safe (GRAS) additives from propionitrile. and no upper limits are imposed, except for Fewer supplies and the higher cost of bread, rolls and cheeses (0.30 to 0.38%). oil, the opportunity to use by-products of the Propionate is metabolized like other fatty food industry as inexpensive media, the acids in the mammalian body. increasing consumer demand for organic Propionic acid production 455

natural products and the emergence of more and propionic acids; and (iii) separation and efficient fermentation processes have led concentration of the acid is expensive (Iow to a new opportunity for microbial production concentration, small difference of volatility to be economically attractive. between acid and , and presence of other acids, especially acetic acid). Nevertheless, a lot of work has been Fermentation processes done to establish the most important char- acteristics of the production of propionic Microbiology of the fermentations acid by fermentation with propionic acid bac- teria. Playne (1985) reported more than 40 The first works on propionic acid fermenta- patents resulting in increased productivity, tion resulted in the formulation of the Fitz inexpensive media (wood pulp waste liquor, equation: steep water, maize gluten, whey, starch hydrolysates, sulphite waste liquors, ligno- 3 -> 2 propionic acid + 1 acetic cellulose, etc) or more efficient recovery. acid + 1 CO + 1 H 0 2 2 Propionibacterium were also grown in mixed or cultures with lactic acid . They have 1.5 -> 2 propionic acid + 1 acetic been immobilized in calcium-alginate beads, acid + 1 CO2 + 1 H20 in polyacrylamide gels and in membrane Theoretical maximum yields are 54.8% bioreactors. (w/w) as propionic acid and 77% as total The recent improvements in fermenta- acids. Formation of propionic acid is accom- tion technology, with the introduction of high panied by the formation of acetate for stoi- cell density bioreactors, the enhancement chiometric reasons and to maintain hydro- of recovery efficiency of membrane pro- gen and balances. The dicarboxylic cesses and the increasing demand of con- acid pathway is the most common pathway sumers for biological products, have led the for the formation of propionic acid. The Laboratory of Dairy Technology Research of acrylic pathway, restricted to a few species INRA (Rennes, France) to a systematic of bacteria ( propionicum, investigation of the improvement of propi- Megasphaera elsdenii, Bacteroides rumini- onic acid fermentation. cola), also leads to propionic acid forma- tion. Enhancement of propionic acid produc- The processes: state of the art tivity of fermentation processes

Today, the industrial production of propi- Process improvement onic acid is almost entirely by petrochemical Propionibacterium acidi propionici was used routes, although numerous fermentation to ferment lactose from whey permeate to processes have been patented since 1923, propionic acid in a continuous stirred tank mainly with strains of the Propionibacterium reactor with cell recycle (fig 1). The cells genus. They have never passed the pilot were separated from the medium and recy- plant level because: (i) propionic acid fer- c1ed back to the bioreactor using an ultrafil- mentation is a very fastidious task (1 to 2 tration unit. This fermentation system makes weeks to be completed in batch); (ii) the it possible to reduce the propionic acid production of organic acids by Proploni- inhibitory effect on the cells, to increase the bacterium is end-product-inhibited by acetic concentration of viable microbes in the biore- 456 P Boyaval, C Corre actor and consequently to obtain higher vol- cess was converted to a sequential one umetrie productivity than the conventional (Colomban et al, 1993). method. If batch fermentation realized volu- The sequential mode of operation 1 1 metrie productivity of 0.03 9 1- h- , this offered several advantages. The technical membrane bioreactor attained a productivity operations were very similar to batch fer- 1 1 of 14.3 9 t- h- : more than 480 times greater mentation, therefore the process was than the traditional process (Boyaval and rapidly assimilated by the staff. This pro- Corre, 1987). As already observed during gramme allows utilization of only 1 mem- lactic acid production, the specifie acid pro- brane device for several bioreactors, which ductivity decreased when the biomass is very useful from an economic point of increased in the bioreactor: from 0.4 9 of view. The time for filtration was shorter com- propionic acid per gram of cells per hour pared to the continuous use of a membrane (g 1-1h") at 10 9 1-1to 0.17 9 g-1 h-1 at 80 9 bioreactor. There was less clogging of the 1-1. This decreased cell specifie productiv- membrane, so the permeate was higher ity at high cell concentration was probably and the cleaning procedures were less the result of physiological perturbations (inhi- involved. In addition, this membrane filter bition, substrate limitation or variations in the can be washed during fermentation, a step intracellular water content of the cells, etc). impossible to achieve in continuous runs The acid concentration was 25 9 1-1and except if the bioreactor is coupled to 2 units, the biomass reached 100 9 1-1 (dry weight 1 being used for filtration while the other is [DW]). A periodic bleeding of this concen- being washed (however, 2 pumps are used trated biomass, used to keep a high cell in this case). Finally, the filtration device specifie productivity, could be an interest- can be employed to sterilize the medium ing source of propionic acid bacteria starters, before fermentation between normal oper- which play an important role in the ripening ating steps. of Swiss-type cheeses. Table 1 presents the Nevertheless, only a few studies have productivity of the principal types of fer- dealt with that type of operation. Semicon- mentation processes for propionic acid pro- tinuous processes have been used to duction. These results, obtained with a improve the yield of propionic acid in a con- CSTR with ultrafiltration recycle, led to a ventional bioreactor. Repeated recycling pilot plant and an industrial plant (37 m3) culture has been tested previously by Lee cou pied with 32 m2 of ceramic membranes and Chang (1990) to alleviate the acetic (Boyaval et al, 1991). The continuous pro- acid inhibition in E coli and by Denis and

PROPIONIC Boyaval (1991) to produce an extracellular ~--+--,----. ACID microbial enzyme in a membrane bioreactor. This mode of bioreactor operation will prob- ably be more appreciated by professionals for industrial fermentation plants th an the continuous fermentation because it is eas- ier to set up in the factory. The long-term stability of the process has been proved with the runs presented here (1 000 h) and with numerous others. We have observed no loss of catalytic activity Fig 1. Schematic flow sheet diagram of the pro- of our strains. Moreover, it seems possible cess. (UF: ultrafiltration unit). to increase the resistance of P acidi-propi- Schéma simplifié du procédé. 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 Reference source concentration acid productivity (g r') concentration (gr1h-1) (g rI)

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

P acidi-propionici CSTR Glucose 1.51099.51011 3.74 0.19 (ATCC 25562) Xylose ce Ils ml-1 0.18 Clausen & Gaddy Plug Ilow Glycose 0.49 (1984) tubular Xylose 0.40 reactor

Propionibacterium sp Calcium Na-lactate 4109 cells 5 2 Cavin etai alginate (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 & Corre cell recycle (1987)

odic cultures with medium enriched in pro- tic, succinic, pyruvic, malic, fumaric, iso- pionic and acetic acids. valerie and formic acids. These "by-prod- At present, trials are performed with 30% ucts" must be carefully examined because dry matter (220 9 1-1 lactose) in order to variations in the process and especially in achieve higher propionic acid contents and the growth medium and agitation speed lead to increase the propionic/acetic acid ratio. to important variations in the concentration These runs are conducted with a higher of these products. A decreased yield of pro- biomass content (>80 9 1-1). The aim of pionic acid production and a modification of these experiments is to find a balance the final product of the fermentation are between an increased propionic acid con- then observed. centration and the decreased specifie pro- Moreover, the recent work of Hsu and ductivity due to the higher biomass and acid Yang (1991) shows that even if neutral pH is contents of the medium, without neglecting optimum for the growth of Propionibacterium a total lactose consumption. acidi-propionici, the propionic acid yield is Even though propionic and acetic acids low. On the other hand, in the acidic pH are the 2 major end products of lactose fer- range, the growth rate is low but the yield mentation by propionic acid bacteria, other is double. With our particular mode of oper- acids could be produced in the medium: lac- ation, it will be easy to multiply the cells, in 458 P Boyaval, C Corre the first cycles, at a neutral pH and to allow Glycerol has now become an inexpen- an acidification for the next ones to enhance sive carbon source. The new processes the yield. A safer process will be obtained at used to extract fuel from vegetable oils pro- this lower pH. This process makes it pos- duce a large quantity of glycerol as a by- sible to produce concentrated cells (200 9 1-1 product which needs new uses. The increas- DW) which can be used as cheese starters ing interest in a natural source of propionic in the factory. acid highlights the great demand of this biosynthesis for industrial processes that Propionic acid production with glycerol need high-quality acid. as carbon source

Until now, ail of the carbon sources used to produce propionic acid by fermentation PROPIONICi GLYCEROL have led to the cosynthesis of acetic acid. ACID g/L '.>' g/L Using Propionibacterium thoenii NCDO 40 . 1082, we demonstrated that fermentation of glycerol by this propionic acid bacteria led only to propionic acid with no acetic acid. Fed-batch experiments were per- formed (fig 2). The highest productivity was 30 0.3 9 1-1 h-1, and the maximum concentra- 30 tion was 40 ± 2 9 1-1. The specific acid pro- duction rate was equal to zero when the acid concentration reached this value. Con- tinuous fermentation with a membrane 20 bioreactor realized the production of high- 20 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 carbon source or with glycerol only during "pro- 10 10 duction steps", while a complete medium can 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 medium 100 300 TIME (HOURS) does not show any components issuing 200 from yeast extract or other corn- pounds. This greatly simplifies the down- Fig 2. Typical fed-batch experiment. Four addi- stream processing for that product. For tions of pure glycerol of indicated by arrows on the example, direct electro-electrodialysis figure 15, 30, 40 and 30 ml were done during this (EED) or electrodialysis (ED) with bipolar run. Initial volume 1.2 1; pH 6.8; temperature membranes will be successfully used to 30°C. (e) Propionic acid; (0) glycerol. concentrate the propionate salt and to con- Résultat d'une expérience de fed-batch. Quatre additions de glycérol pur de 15, 30, 40 et 30 ml vert it to the acid form. Clogging of the ont été réalisées au cours de cette fermentation. membranes, which is the main problem of Le volume initial était de 1,21, le pH 6,8 et la tem- this type of technology, will be greatly pérature 300C. (.) Acide propionique; (0) gly- reduced. cérol. Propionic acid production 459

PROPIONIC ACID RECOVERY comitant concentration of the product. ED AND PURIFICATION with bipolar membranes (BPM: water-split- ting at the junction of 2 homopolar mem- branes) and EED enable inorganic and State of the art organic anions to be extracted and com- bined with protons to produce acids (fig 3). Downstream processing of organic acids The evolution of time of propionic acid from fermentation is often the major financial concentration from the solution (fig 4) is drawback of the process. This is also true for characteristic of the extraction-reconcen- the recovery of propionic acid. This acid is tration curve obtained by ED. In the con- highly hydrophilic and the difference of centrate, the amount of the extracted elec- volatility between propionic acid and water trolyte first increases linearly before reaching is small. Moreover, the optimum pH range the reconcentration plateau. This recon- for that fermentation is, like many other aci- centration level is limited by the leak- dogenic fermentations, between 6.0 and age through the membrane and the water 7.0. This means that the acid is in the ion- transport. The results recorded here sug- ized state and hence is completely non- gest that the limiting process is the water volatile. The acid concentration is gener- transport through the anion exchange mem- ally, for fermentation processes, less than 50 brane. Moreover, in the presence of only 9 1-1. This low concentrated product, among propionic acid, the water transport through many other present in the fer- the BPM has the same effect as the water mentation broth, embarrasses the purifica- consumption taking place during the EED tion. The main purification processes experiment and resulting from the water employed today are: acidification of the fer- electrolysis at the anode. mentation media followed by recovery with The results show that the acetate and anion exchange resins; extraction lactate , which are present with propi- and distillation; salting out procedures; ester- onate ions in the fermentation broth, are ification of the acid with ethanol or butanol extracted at the same time, giving rise to (the ester is less soluble in water); mem- the production of acetic and lactic acid in brane techniques; and other techniques compartment 2. In the 2 processes, after such as supercritical fluid extraction and 25 h, the propionic acid concentration adsorption on zeolite (others have been increases again with time. This result may examined but, to our knowledge, are not be explained by the fact that the totality of largelyemployed). acetate and lactate ions having been extracted from the fermentation broth, only the propionate ion is carried through the Enhancementofdownsueam anion exchange membrane and the effi- processing efficiency ciency of the electrotransport of propionate becomes higher. Therefore, the amount of As for other organic acids, the pH must be propionic acid will most likely be increased, maintained during fermentation in order to leading to a more concentrated propionic increase the productivity. Then salts such acid, with longer experiments. as sodium or calcium propionate are Concentrated propionic acid was obtained. For numerous uses, however, the obtained by EED and ED with bipolar mem- acid form is required. Thus, it was interest- branes of the fermentation broth. The current ing to convert easily and economically pro- efficiency was high but decreased with time, pionate salts to propionic acid, with a con- especially with bipolar membranes. The 460 P Boyaval, C Corre

a PROPlPcr1C PROPIONIC ACID (g.I-' ) 120

J rtO-lt- o BO

'l' 40 lT,1 N.t PROPIONATE

o proproruc 10 20 b acid TI ME (heurs) BPM t SPM ® © ® © @ © 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 time - H+ 1 of the concentration of propionic acid in the corn- OH OH- H+ G 20 partment 2: (0) EED, (.) ED with bipolar mem- IJI~branes (BPM). Current density 70 mA cm-2. G, +r.~;- Électro-électrodialyse (EED) et électrodialyse (ED) avec des membranes bipolaires d'une solu- ~1NaL tion d'acide propionique à 40 g rI. Évolution de la PROPIONATE concentration de l'acide propionique en fonction du temps dans le compartiment 2: (0) EED, (.) Fig 3a. Laboratory cell device for electro-elec- ED avec des membranes bipolaires. Densité de trodialysis. c = Cation exchange membrane; a = courant 70 mA ctrrê. anion exchange membrane. b. Laboratory cell device for electrodialyser with bipolar membranes (BPM). a. Équipement de laboratoire pour t'éiectro-étec- trodialyse (EED). c = membrane échangeuse de tor. Compared with solvent extraction, the cations; a = membrane échangeuse d'anions. membrane processes described assume b. Équipement de laboratoire pour l'électrodia- no trace of extractive solvent in the product lyse (ED) avec des membranes bipolaires. with the subsequent problems to food acceptability; no problem of pollution by chemical release and associated production which can be re-used presence of inorganic ions led to the for- in the fermentation process. Previous ultra- mation of other acids (Boyaval et al, 1993). filtration of the fermented whey UF perme- The influence of the water transport as ate achieved in continuous fermentations weil as the current density need to be anal- with membrane bioreactors permits in line ysed more thoroughly, taking into consider- use of this sophisticated technology with- ation the electrical energy consumption of out c10gging problems. these processes in order to obtain data which are necessary for economic estimations. ln an extractive fermentation process, CONCLUSION EED and ED with bipolar membranes can be used to decrease the acid level in the At present, continuous stirred tank reactor medium and then allow high cell multiplica- cou pied with ultrafiltration cell recycle may tion and fast acid production. Moreover, the prove to be the most appropriate process for sodium hydroxide produced can be easily propionic acid production. Nanofiltration mem- employed for the pH control of the bioreac- branes have been used to show the feasi- Propionic acid production 461

bility of the production of highly pure propionic Boyaval P, Corre C, Madec M-N (1994) Propionic acid acid with no residuallactose. In addition, the production in a membrane bioreactor. Enzyme Microb Techno/16, 883-886 biological propionic acid colour was greatly Colomban A, Roger L, Boyaval P (1993) Production of reduced. The performance obtained with a propionic acid from whey permeate by sequential bioreactor cou pied to nanofiltration mem- fermentation, uhrafiltration, and ceU recycling. Biotech- brane for lactic acid production is very promis- nol Bioeng42, 1091-1098 ing for the production of propionic acid. Carrondo MJT, Crespo JPSG, Moura MJ (1988) Pro- duction of propionic acid using a xylose utilizing Pro- The complete overview of the perfor- pionibacterium. Appl Biochem Biotechno/17, 295- mances of EED and the use of bipolar mem- 312 branes for the recovery and concentration of Cavin JF, Saint C, Divies C (1985) Continuous produc- propionic acid (or other organic acids tion of Emmental cheese flavours and propionic acid obtained by fermentation processes) need a starters by immobilized cells of a propionic acid bac- serious examination of different parameters terium. Biotechnol Lett7, 821-826 (membrane nature, current density) and an Clausen EC, Gaddy JL (1984) Organic acids from biomass by continuous fermentation. Chem Eng economic evaluation of the process is Prog 80, 59-63 essential. Denis S, Boyaval P (1991) Microbial enzyme produc- tion in a membrane bioreactor. Appl Microbiol Biotechno/34,608-612 REFERENCES Hsu ST, Yang ST (1991) Propionic acid fermentation of lactose by Propionibacterium acidipropionici: effects of pH. Biotechnol Bioeng38, 571-578 Boyaval P, Corre C (1987) Continuous fermentation of sweet whey permeate for propionic acid production in Lee YL, Chang HN (1990) High cell density culture of a CSTR with UF recycle. Biotechnol Lett9, 801-806 a recombinant producing penicillin acylase in a membrane cell recycle fermentor. Boyaval P, Colomban A, Roger L (1991) Semi-continu- Biotechnol Bioeng 36, 330-337 ous fermentation process for the production of bio- logical propionic acid based preparations. European Playne MJ (1985) Propionic and butyric acids. In: Com- Patent n° 9146002206 prehensive Biotechnology, Vol 3 (M Moo- Young, ed) Pergamon Press, Oxford, UK, 731-759 Boyaval P, Seta J, Gavach C (1993) Concentrated pro- pionic acid production by electrodialysis. Enzyme Sherman JM, Shaw RH (1943) The propionic acid fer- Microb Techno/15, 683-686 mentation of lactose. J Biol Chem 56,695-700