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Enhancement of oxygen transfer rates in fermentation using oxygen-vectors
B]otech. Adv. Vol. 7, pp. 1-14, 1989
0734-9750/89 $0.00 + .50
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Copyright © 1989 Pergamon Press plc
E N H A N C E M E N T OF OXYGEN TRANSFER RATES IN F E R M E N T A T I O N USING OXYGEN-VECTORS J. L. ROLS and G. GOMA D~partement de Gdnie Bioehimique et Alimentaire, UA CNRS 544, Centre de Transfert en Biotechnologie Microbiologie, Institut National des Sciences Appliqudes, Avenue de Rangueil, 31077 Toulouse Cddex, France
ABSTRACT
Oxygen transfer is one of the bottlenecks in conventional fermentation technology and it has so far been almost totally overlooked with regards to high call densities and immobilized cells. This review presents some new concepts to improve oxygen supply in aerobic fermentations, especially the use of oxygen-vectors. The oxygen-vectors generally used are liquids which are insoluble in the fermentation media. Their utilization in an emulsified form can significantly increase the oxygen transfer coefficient between gas and aqueous phases. It seems that the vector acts as an active intermediate in the oxygen transport from gas bubbles to aqueous phase, but the mechanisms involved in this unconventional technique of aeration are not yet known. KEY WORDS
Oxygen-vector, enhanced oxygen transfer coefficient, hemoglobin, hydrocarbon, perfluorocarbon, emulsion, spreading coefficient, transfer way. INTRODUCTION
Although aeration is one of the most studied areas of biochemical engineering and increasing the active callconcentration in its reactor is a crucial goal, surprisingly little work has been done to attain high call concentrations in aerobic fermentations by developing new methods to supply oxygen. In many aerobic fermentations, the oxygen supply rate to the aqueous phase is the limiting factor because of its low solubility in water. Umitations occur
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when the oxygen demand rate exceeds the oxygen supply rate causing the dissolved oxygen level in the aqueous phase to fall below the critical oxygen concentration for the organism's metabolism. In conventional fermentation technology, oxygen is often supplied by bubbling air, or occasionally pure oxygen, through the fermentor. Vigorous stirring of the medium is necessary to get good oxygen transfer. Various novel methods have been reported by researchers to achieve good oxygen supply when high biocatalyst densities are used, such as in immobilized preparations (15,52). One approach has been to generate oxygen chemically in the aqueous phase by using a hydrogen-peroxide/catalase system (21,22,47), or by adding hydrogen-peroxide to the medium and using an organism with a high natural catalase activity (20). A biological approach to in situ oxygen generation would be to co-immobilize oxygen-consuming organisms with oxygen-producing organisms such as algae, but the flux of oxygen production is limited (1,2). Still another approach is to modify the medium in such a way that it dissolves more oxygen. The water solutions normally used are replaced by organic solvents, with the intention of increasing the solubility of both organic substrates and oxygen (6,13,23,28,51). Finally, another way of increasing the oxygen supply would be to modify the medium so that it could carry more oxygen. The interest in the production of single cell protein by growing microorganisms on various water-insoluble hydrocarbon substrates has resulted in several investigations of the nature of oxygen transfer in aerated systems with two liquid phases. The presence of a non-aqueous liquid phase may have a significant effect on the rate of oxygen transfer from the gas phase to the organisms. This new approach is the strategy of oxygen-vectors and consists of the addition to the growth medium of a compound or a liquid phase in which oxygen has a high solubility. The compounds generally used in biotechnology are hemoglobin (3), hydrocarbons (38) and perfluorocarbons (33). This present review covers the state of the oxygan-vector technology and presents a discussion of oxygen transfer in multiphase systems via the oxygan-vector. OXYGEN SUPPLY BY HEMOGLOBIN
In nature, several substances capable of transporting oxygen have been found. The most well-know are a series of iron and copper proteins that reversibly bind and transport oxygen via the blood streams of animals for the controlled catalysis of the transformation of biological substrates to useful products and thermal energy (18,46). In mammals, these are the heme proteins hemoglobin and myoglobin; in marine crustaceans the equivalent system is hemocyanin, a copper-containing protein; and in marine worms oxygen transport is via hemerythrin, a non-heme iron protein. WAKABAYASHI et al. (54) described a bacterial heme protein from V'~reoscilla that combines with molecular oxygen and has an amino-acid sequence with features characteristic of the globins. The hemoglobin content of V'~reoscilla increases almost 50-fold when the oxygen concentration of the growth medium falls below 10 per cent of atmoaphedc
ENHANCEMENT OF OXYGEN TRANSFER RATES
3
(5). Apparently, it may function to enable the organism to survive in oxygen-limited environments by acting as an oxygen storage-trap or to facilitate oxygen diffusion (42). Cloning of the hemoglobin gane from the bacterium Vitreoscilla has enabled expression of functional hemoglobin in Escherichia coli (4). Cells containing the Vitreoscilla hemoglobin exhibit increased respiration rates and grow more rapidly than hemoglobin-free calls under oxygen-limited conditions characteristic of high cell density growth, in both shake flask and fed-batch controlled fermentations. In addition, the cloned Vitreoscilla fragment also confers oxygen-mediated regulation of the expression of bacterial hemoglobin. In a project of MAI-I"IASSON and BORREBAECH (34) where glucose oxidase was used for analytical purposes, glucose oxidase and red blood calls containing hemoglobin were co-immobilized to concavalin A-Sepharose. On introducing a pulse of substrate, the partial pressure of oxygen within the matrix decreased concomitantly with the mobilization of the pool of oxygen stored in the hemeglobin packets of the calls. After the pulse had passed, Po2 increased and the hemoglobin was re,oxygenated. ADLERCREUTZ and MA'I-I'IASSON (3) supplemented the substrate solution with hemoglobin to increase oxygen supply to immobilized calls of Gluconobacter oxvdans for the conversion of glycerol to dihydroxyacatone. The dihydroxyacatone production varied linearly with the hemoglobin concentration. In this case, the role of hemoglobin is not unlike its normal role in animals. However this method has some limits: * Hemoglobin is highly soluble in water, but working with solutions more concantrated than 85 g/I does not seem realistic. * Hemoglobin has a high affinity for oxygen and therefore it is necessary for the medium to have a long residence time in the reactor if the oxygen supplied by the hemoglobin is to be effectively utilized. * If hemoglobin is used as an oxygen carrier in biotechnical processes, it is necessary that the protein can be used several times. It is know that hemoglobin is slowly oxidized by oxygen to methemoglobin. There are several reports concerning the reduction of mathemoglobin (38,49) but this reaction could not be driven to completness in this type of biotechnical processes. In an effort to exploit the advantages obtained with oxygan-vectors such as hemoglobin but avoid the disadvantages, synthetic carriers such as perfluorocarbons have been used instead. OXYGEN SUPPLY BY HYDROCARBON
In fermentations using hydrocarbons as substrate, microbial oxygen demand is much higher that when carbohydrates are used, as reported by many authors (14,37,39). Also growth media containing hydrocarbons have been well studied (17,38). MIMURA eta/. (38) were the first to point out the effect of hydrocarbon phase on oxygen transport in air-waterhydrocarbon systems. They studied the oxygen transfer in dosed shaking flasks containing air, sodium sulfite solution, and kerosene by following the change in the gas phase oxygen
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J.L. ROLS a n d G. GOMA
partial pressure. They found that the oxygen transfer rate coefficient kLa/H increased exponentially with the volume fraction of kerosene ~ , when the total volume of the liquid mixture was held constant, for 0 < ~ < 0.85 (H: Henry's law constant). YOSHIDA et aL (56) determined values of kLa in both an agitated vessel and in a bubble column for aqueous dispersions of kerosene, a liquid paraffin and toluene. They pointed out that the spreading coefficient: Sp = ~T-WG - (g'OG + g--WO) , with 9"~ the interfacial tension between i and j phases, is negative for the kerosene and paraffin-water mixtures and for these systems kLa decreases linearly with increasing oil fraction. When Sp < 0, oil will simply form floating lenslike drops. For the toluene-water mixture, Sp > 0; kLa initially decreases and then increases with increasing oil fraction. The oil seems to spread as a thin film on the gas-liquid interface where it will act as a surface active agent to lower the surface tension and increase the specific interfacial area of the gaseous phase. COTY et al. (11) studied the oxygen uptake rate of mixtures containing hexadecane in which the oil phase was continuous. They found that the oxygen transfer rates from air to mixtures containing 50% and 66% oil in air lift reactors were much larger than for the medium without oil. MATSUMURA et aL (32), with appropriate modelling, determined volumetric oxygen transfer coefficients for transport between gas and water phases and water and hydrocarbon phases in an air-water-hydrocarbon (mainly n-pentedecane) system. They measured the gas phase oxygen partial pressure change in a closed agitated vessel with a draft tube, as oxygen was absorbed by the liquid mixture. They assumed the pathway of oxygen transport was from air to water to oil, and that the oil phase was in equilibrium with the water phase oxygen concentration present at the oil-water interface. The correlations they obtained are: (kLa)G w = (1.75.10-2.e 0.115.~. 0.8.10-3.e-46.9.,~).N.Vs1/3 and
(kLa)O w =
1 . (kLa)GW 1 + HOW (1/,~'-1)
with: : volume fraction of oil N: rotational impeller speed VS: linear velocity of aeration based on the cross sectional area of the stirred tank HOW: partition coefficient of oxygen in oil-water system. Both the volumetric absorption coefficients increased with an increase in the oil volume fraction. YAMANE and YOSHIDA (55) have shown that small oil droplets will very rapidly come into oxygen equilibrium when placed in an aqueous liquid. They also concluded that the main resistance for the oxygen absorption into O/3N emulsion in an agitated gas-liquid contactor exists in the water film around gas bubbles.
ENHANCEMENT OF OXYGEN TRANSFER RATES
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A dynamic method for estimating volumetric oxygen transfer coefficients between the gas and aqueous phases (kLa)GW, the gas and oil phases (kLa)GO and the oil and aqueous phases (kLa)Ow, and oxygen uptake rate from the aqueous and oil phases during cultivation was developed by YOSHIDA et al. (57). This method involves recording the oxygen concentration in the liquid following a step change in the gas phase oxygen concentration and a numerical analyse of the response. More recently, TORRIJOS (50) observed the effect of hydrocarbon emulsions on the growth of Aerobacter aeroaenas cells, which are highly sensitive to the flow of transfered oxygen (45).The analysis of cultures containing various concentrations of hydrocarbon (mixture of n-tridecane to n-heptadecane) indicated an increase of the oxygen transfer coefficient (up to 4.6-fold for 20%v/v organic phase), directly related to the addition of organic phase, resulting in an improvement of both production and growth kinetics. Furthermore, it has been demonstrated that it was possible to increase the kLa of a fermenter by the addition of n-alkanas in the aqueous phase, without changing the conditions of agitation-aeration and thus without supplying more energy. The n-alkane phase could be easily separated from the aqueous phase by decantation and centrifugation of the upper phase. The usa of these vectors is cost-effective since there are readily available, inexpensive and can be reused. O X Y G E N SUPPLY BY P E R F L U O R O C A R B O N
1- Properties of oerfluorocarl;~n~: Perfluorocarbons (PFCs) are cyclic or straight chain hydrocarbons in which hydrogen atoms have been replaced with fluorine. Due to the strength of the carbon-fluorine bond, PFCs are considered as being chemically inert (43). Many gases, for example oxygen and carbon dioxide, have a high solubility in PFC liquids; a solubility which varies linearly with the gas partial pressure, according to Henry's law (26). The gas-dissoMng capacity decreases in the order: CO 2 > > 0 2 > CO > N 2 > H 2 > He, apparently following the decrease in molecular volume of the solute. The high dissoMng capacity is related to the ease of formation of cavities capable of accomodating the small solute molecules within the solvent, rather than to any specific interaction. These properties make PFCs suitable as blood substitutes. They are used in emulsified form in the presence of a surfactant (Pluronic F68, a polyoxyethylene polyoxypropylene block polymer), by either ultrasonic treatment or high pressure homoganisation. Emulsions of PFCs dispersed in physiologically-acceptable electrolyte solutions have been widely tested as oxygen-carrying resuscitation fluids in several mammalian species (29). Moreover, one commercial emulsion (FluosoI-DA, Green Cros Corporation, Japan), which contains parfluorodecalin and perfluorotripropylamine emulsified with a poloxamer surfactant, is the only PFC preparation to have been tested in man (35). Another application is the use of oxygen-carrying PFC emulsion as an adjuvant to radiation therapy (48).
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J, L. ROLS and G. GOMA
The effect of emulsified PFCs on microbial cells was studied by CHANDLER et aL (9) on the growth of E. ¢01i cells. The toxicity of these emulsions is thought to be at least partly caused by the surfactants and result in cytotoxic effects. At present, a new formulation of oxygen-carriers consists of selecting fluorinated compounds with a hydrogenated part, compatible with non-toxic hydrogenated surfactants (7). 2- Oxvaen SUDDIVto microoraanisms: In situ aeneration:
Several patents recommend the use of PFCs as oxygen-carriers to stimulate the growth of aerobic microorganisms. Although stated otherwise by ADLERCREUTZ and MA'I-I"IASSON (3), PFCs have previously been used to help improve the productivity of aerobic fermentations. CHIBATA et al. (10) described a new method whereby PFCs or silicone oils were added to batch aerobic fermentations which were carried out in shake flasks or gas-sparged stirred tank fermentors. The conversion of sorbitol to sorbose increased as more perfluorotributylamine was added. Bis(F-hexyl)athene has for example been used to supply oxygen to lymphoid cell cultures of the Navalma strain destined for the production of interferon (31). It is believed that microorganisms do not obtain oxygen directly from the PFC phase. In the fermenter experiments described above, air was passed through the water phase, delivering oxygen to it. When PFC was also present, oxygen dissolved in this phase and could subsequently be transported to the water phase (33). This apparently caused an increase in the total transport of oxygen from the gas phase to the water phase. Sometimes, the oxygen dissolved in the PFC is sufficient for the entire cultivation so that reoxygenation is not necessary. HERTL and RAMSAY (19) used PFC and silicone oil greases or gels as oxygen reservoirs, to stimulate the growth of various microorganisms in liquid or solid media. More recently, TORRIJOS (50) studied the effect of varying amounts of Forane F66E on the growth of A. aeroaenes cells in a 20 I. fermenter. The emulsion was obtained by ultrasonic treatment in the presence of Pluronic F68. The results indicated an increase of the oxygen transfer coefficient up to 4-fold for 4.5%v/v organic phase. External oxvaenation: Sometimes, it is beneficial to withdraw the liquid and oxygenate it outside the reactor in which case it is an advantage if the solubility of oxygen in the liquid is high. Emulsions of PFC (FC-72) were used in this way to increase oxygen supply to immobilized G. oxidans cells in a packed bed reactor (3). The reaction mixture containing FC-72 in an emulsified form was saturated with air and passed through the column. The use of this mixture improved the productivity of the cells 5 to 6 times (with 32.4%v/v) over that of the control. Another approach has been developed by DAMANIO and WANG (12) with perfluoromethyldecalin in cultivations of E. coil cells in a spray column fermentor. The PFC was continuously removed from the fermentor, reaeratad, and returned whereby oxygen
ENHANCEMENT OF OXYGEN TRANSFER RATES
7
was supplied to the free cell bacterial culture by means of liquid-liquid contacting. Little energy was needed to form the droplets and the oxygen transfer from the droplets to the aqueous phase was rapid. In some instances, other compounds may be better than PFCs for increasing the oxygen supply to microorganisms. LEONHARDT eta/. (27) did not find PFCs suitable as oxygen-carriers for Providencia cells immobilized in calcium alginate. Better cell growth was obtained with polydimethylsiloxane emulsified with a copolymer of polydimethylsiloxane and polyethylene oxide. PFC emulsions when used in medical applications, transport carbon dioxide as well as oxygen. The same is true for bacterial cultivations. CESCHIN et al. (8) improved growth of anaerobic bacteria such as Clostridium oerffinaens by supplying carbon dioxide-saturated perfluorineted oil. In addition it is probable that PFCs can be used to remove inhibitory gases from the reactor, e.g. CO2 in aerobic bioprocessas. There is a little chance of finding new PFC oxygan-vectors having significantly higher oxygen-dissolving capacity. With the prospect of large-scale commercialisation coming soon, the problem of the industrial feasability of both the preparation and the purification of the PFCs, which was often neglected in the screening stages, becomes more acute. At present, PFCs are relatively expensive compounds (FC-72 costs for example about 80 US $.kg -1) and effective recovery will be necessary. ROLE OF OXYGEN-VECTORS
The growth of microorganisms on carbohydrate substrate in presence of oxygenvector, is realised by dispersing organic and gas phases in a continuous phase (water in which salts and sugar are dissolved). The studied system is then composed of four phases: -the continuous phase which is generally the aqueous phase, -the dispersed phases: *solid phase: microorganisms for which the size varies between 0.5 and 5)Jm. *gaseous phase: air bubbles for which the diameter varies between 0.5 and 5 mm. *liquid phase: vector droplets for which the diameter varies between 0.5 and 40 IJm. The three dispersed phases are placed in a turbulent environment, provoking a permanent renewal of gas and vector surfaces. The liquid-liquid system should have much more surface area avaible for mass transfer (and thus higher oxygen transfer retas) than gas-aqueous system for the following reasons: 1) at equal work inputs, drops formed will be smaller than bubble due to surface tension differences; 2) bubbles generally coalesce into larger bubbles much more rapidly than do droplets; and 3) liquid hold-ups can be fixed at levels much higher than typical gas hold-ups. According to MAC LEAN (30), the dispersed liquid phase can influence oxygen transfer by absorbing oxygen or supplying it to other phases, or by influencing the fluid mechanics of the continuous phase, thereby influencing the mass transfer coefficients and
8
J.L. ROLS a n d G. GOMA
interfacial areas of the continuous phase. The various way that oxygen can flow to reach the bacterial cells are pointed out on the following scheme (fig.l).
O
CELL
2
VECTOR Q
GAS
WATER
3 4 Fioure 1- Possible wavs for oxvaen transfer from oas bubbles to cells. 1) Gas-liouid-solid transfer: It is the main way used by oxygen molecules in absence of oxygen-vector. The oxygen molecules are dissolved in the aqueous phase and used in the fermentation media by the microorganisms. The vector can act as an emulsifier of air bubbles and water particles (38). Thus with the vector-water system, it will be possible to increase the interfacial area between the gas and the emulsion. 2) Gas-solid tran#Qr: The cells use the oxygen directly at the interface of the gas bubbles. Microbial cells have a tendency to crowd themselves into the liquid film of the gas-liquid interface, due to surface adsorption. The cell concentration at the bubble surface is usually even higher than that in the liquid bulk. Since the cells consume the oxygen molecules being absorbed right at the gas-liquid interface, TSAO eta/. (53) believe that the cells will affect and enhance the oxygen absorption rate. The increase of the gas-liquid interface area by the emulsifier vector favours this second way.
ENHANCEMENT OF OXYGEN TRANSFER RATES
3) Gas-liQuid-liauid-solidtransfer: The organic phase dissolves' the oxygen which is then transfered to the aqueous phase where it is used by the cells. From the comparison of the estimated values of the volumetric coefficient for oxygen transfer through the water film around gas bubbles and the volumetric coefficient for overall oxygen transfer from water into oil drops, YAMANE and YOSHIDA (55) concluded that the main resistance for oxygen absorption into O/W emulsion in an agitated gas-liquid contactor exists in the water film around gas bubbles. MIMURA et a/. (38) assumed that the oxygen transfer from the oil to water is much faster than from air to oil. In this way, a direct oxygen transfer from air to water is accomplished. The type of interaction between air bubbles and vector droplets is not well established. YOSHIDA at a/. 's correlation (56) between the evolution of kLa with oil fraction and the value of spreading coefficient is very attractive. When the spreading coefficient is positive, the oil seems to spread as a thin film at the gas-liquid interface. A mechanism explaining interaction between air bubble-oil drop in water has been proposed by ROQUES et aL (44) in a study of water-hydrocarbon separations by flotation. For Sp > 0, the system goes through a recovery of the bubble by the drop. When Sp < 0, the oil simply forms a lenslike droplet floating on the bubble surface. Thus, the specific interracial area of gas bubble for oxygen transfer may decrease due to partial blanketing of the bubble surface with lanslike oil droplets and to slower diffusion of oxygen through oil droplets. It is also conceivable that the rate of surface renewal or eddy diffusion around gas bubbles decreases with presence of oil droplets. The dissolved oxygen profile present in the third way can be illustrated by the following scheme (fig.2). The oxygen vector is an active intermediate to carry oxygen from bubble to water. 4) Gas-liouid-soiid transfer: The cells are at the surface of the vector droplets and use the oxygen directly. This type of transfer would depend on the affinity of the cells for oxygen-vectors and on the size of the emulsified droplets in the aqueous phase. For microorganisms growing on carbohydrate, MIMURA et aL (40) and TORRIJOS (50) obtained uniformly dispersed bacteria in the aqueous phase when oxygen-vectors were respectively hydrocarbon and PFC. For hydrocarbon-assimilating bacteria, cells can adsorb onto the surface of oil droplets, forming dense flocs. The flocs tend to attach to the surface of the air bubbles (41). The cells take up oxygen from both organic phase and in the water film around the bubbles. YOSHIDA eta/. (57) proposed that cells utilize a considerable amount of oxygen from oil which spreads on the surface of air bubbles. The biological effect of the synthetic surface active agents which are used to emulsify,hydrocarbons, is to separate cells from oil droplets (40).
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J.L. ROLS a n d G. GOMA
OXYGEN
CONCENTRATION
Cv*
LEVEL
Cv
Cv.Cw*•. Cv"
i
I I GAS
VECTOR
[
Cw
I I
DISTANCE CELL
WATER
TRANSFER WAY
I=,
Fiaure 2- Oxvoen transfer oroflle in a fermentation media with an insoluble vector The total oxygen transfer rate measured in a fermentation media including an oxygenvector, will be the sum of the oxygen transfer rates from the four ways described above. It seems difficult to estimate the part of each way on the total transfer. However, some authors have proposed some methods for the determination of kLa at the different interfaces dating from mass transfer balances (32,57). More recently, KREYSA and WOEBCKEN (24,25) and FURIET et al. (16) have developed a transitory electrochemical technique measuring kLa between gas and liquid phases or between two liquid phases. These different methods will perhaps set in evidency the role of the oxygen-vectors.
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ENHANCEMENT OF OXYGEN TRANSFER RATES
11
3-P. ADLERCREUTZ and B. MA]-rlASSON, Oxygen supply to immobilized cells: 3- oxygen supply by hemoglobin or emulsions of perfluorochemicals, Eur. J. Appl. Microbiol. Biotechnol., 16, 165-170 (1982). 4-J.E. BAILEY, Enhancement of cell growth characteristics using genetic engineering, Proceedings VIII Internatl. Biotechnol. Sym., Pads (1988). 5-S.J. BOERMAN and D.A. WEBSTER, Control of heme content in V'~reoscilla by oxygen, J. Gen. Appl. Microbiol., 28, 35-43 (1982). 6-B.C. BUCKLAND, P. DUNNILL and M.D. ULLY, The enzymatic transformation of waterinsoluble reactants in nonaqueous solvents- Conversion of cholesterol to cholest-4-ene3-one by a Nocardia sp, Biotechnol. Bioeng., 17, 815-826 (1975). 7-M.C. CECUTrl, Formulation de milieux transporteurs d'oxyg~ne: optimisetion de nouvelles micro6mulsions par modulation des composants fluor6s, Th~Dse Docteur I.N.P., Toulouse, France (1987). 8-C. CESCHIN, M.C. MALET-MARTINO, G. MICHEL and A. LATTES, C. R. Acad. Sc. Paris, Ser.lll, 18, 669-672 (1985). 9-D. CHANDLER, M.R. DAVEY, K.Co LOWE and B.J. MULLIGAN, Effect of emulsified perfluorochemicals on growth and ultrastructure of microbial cells in culture, Biotechnol. Latt., 9, 195-200 (1987). 10-1. CHIBATA, S. YAMADA, M. WADA, N. IZUO and T. YAMAGUCHI, US Patent 3 850 753 (1974). 11-V.F. COTY, R.L GORRING, I.J. HEILWEIL, R.I. LEAVITT and S. SRINIVASAN, Growth of microbes in an oil-continuous environment, Biotechnol. Bioeng., 13. 825-842 (1971). 12-D. DAMIANO and S.S. WANG, Novel use of a perfluorocarbon for supplying oxygen to aerobic submerged cultures, Biotechnol. Lett., L 81-86 (1985). 13-J.M.C. DUARTE and M.D. LILLY, The use of free and immobilized cells in the presence of organic solvents: the oxidation of cholesterol by Nocardia rhodochrous. Enz. Eng., 5, 363 (1980). 14-A. EINSELE, H. SCHNEIDER and A. FIECHTER, Characterization of microemulsions in a hydrocarbon fermentation by electronmicroscopy, J. Ferment. Technol., 53, 241-243 (1975). 15-SOO. ENFORS and B. MATTIASSON, Oxygenation of processes involving immobilized cells, in Immobilized cells and orgenelles, Mattiesson ed., CRC Press, i~, 41-60 (1983). 16-C. FURIET, A. STORCK and F. LAPICQUE, Mdthode 61ectrochimique transitoire de d~ermination des conductances de transfart liquide-liquide, in R6cents progr6s en g6nie des procdd6s, Lavoisier, Paris, vol. 1,386-391 (1987). 17-G. GOMA, Contribution & I'(~ude des fermentations sur hydrocarbures: transfeft de mati&re, lois de croissance, Th(~se Docteur d'Etat Universit6 Paul Sabatier, Toulouse, France (1975). 18-D. HAYAISHI, Molecular mechanisms of oxygen activation, Academic press, New-York (1974). 19-W. HERTL and W.S. RAMSAY, US Patent 4 166 006 (1979).
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20-O. HOLST, S.O. ENFORS and B. MA'I-FIASSON, Oxygenation of immobilized cells using hydrogen-peroxide; a model study of Gluconobacter oxydans converting glycerol to dihydroxyacetone, Eur. J. Appl. Microbiol. Biotechnol., 14, 64-68 (1982). 21-M. IBRAHIM and H.G. SCHLEGEL, Oxygen supply to bacterial suspensions of high cell densities by hydrogen-peroxide, Biotechnol. Bioeng., 22, 1877-1894 (1980). 22-M. IBRAHIM and H.G. SCHLEGEL, Efficiency of bovine liver catalase as a catalyst to cleave H20 2 added continually to buffer solutions, Biotechnol. Bioeng., 22, 1895-1906 (1980). 23-A.M. KUBANOV,
Enzymes that work in organic solvents, Chemtech, June, 354-359
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37-A. MIMURA, Recent problems in petroleum fermentation: the problems on the biochemical engineering, J. Ferment. Technol., 48, 449-460 (1970). 38-A. MIMURA, T. KAWANO and R. KODAIRA, Biochemical engineering analysis of hydrocarbon fermentation: 1- oxygen transfer in the oil-water system, J. Ferment. Technol., 47, 229-236 (1989). 39-A. MIMURA, M. SUGENO, T. OOKA and I. TAKEDA, Biochemical engineering analysis of hydrocarbon fermentation: 2- oxygen demand of hydrocarbon-assimilating yeasts, J. Ferment. TechnoL, 49, 245-254 (1971). 40-A. MIMURA, S. WATANABE and I. TAKEDA, Biochemical engineering analysis of hydrocarbon fermentation : 3- analysis of emulsification phenomena, J. Ferment. Technol., 49, 255-271 (1971). 41-A. MIMURA, I. TAKEDA and R. WAKASA, Some characteristic phenomena of oxygen transfer in hydrocarbon fermentation, Biotechnol. Bioeng. Symp., 4, 467-484 (1973). 42-M.F. PERUTZ, A bacterial haemoglobin, Nature, 322, 405 (1986). 43-J.G. RIESS and M. LE BLANC, Solubility and transport phenomena in perfluorochemicals relevant to blood substitution and other biomedical applications, Pure Appl. Chem., 54, 2383-2406 (1982). 44-H. ROQUES, Y. AURELLE, M. AOUDJEHANE, N. SIEM and J. RABAT, Etude exp6rimentale des ph6nom6nes de coalescence dans les syst~mes "bulles-gouttas', Revue de rlnstitut Fran(~ais du P6trole, 42, 163-177 (1987). 45-J.M. SABLAYROLLES and G. GOMA, Butanediol production by A0robacter aerooenes NRRL B-119: effects of initial substrate concentration and aeration-agitation, Biotechnol. Bioeng., 26, 148-155 (1984). 46-D.T. SAWYER, Chemtech, june, 369-375 (1988). 47-H.G. SCHLEGEL, Aeration without air: oxygen supply by hydrogen-peroxide, Biotechnol. Bioeng., 19, 413-424 (1977). 48-B.A. TEICHER and C.M. ROSE, Oxygen-carrying perfluorochemical emulsion as an adjuvant to radiation therapy in mice, Cancer Res., 44, 4285-4288 (1984). 49-A. TOMADA, S. MATSUKAWA, M. TAKESHITA and Y. YONEYAMA, Effect of organic phosphates on methemoglobin reduction by ascorbic acid, J. Biol. Chem., 251, 74947498 (1976). 50-M. TORRIJOS, Evaluation des techniques non conventionnelles d'intensification des transferts d'oxyg~ne en fermentation, Th6se Docteur Ing6nieur I.N.S.A., Toulouse, France (1987). 51-J. TRAMPER, Immobilizing biocatalists for use in syntheses, Trends in Biotechnol.,3, 45-50 (1985). 52-J. TRAMPER and W.J.J. VAN DEN TWEEL, Continuous production of gluconic acid by an immobilized Gluconobacter oxvdans system, Abstr. Comm. Sec. Eur. Congr. Biotechnol. Eastbourne, 161 (1981).
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53-G.T. TSAO, A. MUKERJEE and Y.Y. LEE, Gas-liquid-cell oxygen absorption in fermentation, in Fermentation Technology Today, Proceedings IV Internatl. Ferment. Sym., Kyoto, 65-71 (1972). 54-S. WAKABAYASHI, H. MATSUBURA and D.A. WEBSTER, Primary sequence of a dimeric bacterial haemoglobin from Vitreoscilla, Nature, 322, 481-483 (1986). 55-T. YAMANE and F. YOSHIDA, Comments on oxygen absorption into oil-water emulsions, J. Ferment. Technol., 52, 445-450 (1974). 56-F. YOSHIDA, T. YAMANE and Y. MIYAMOTO, Oxygen absorption into oil-in-water emulsions: a study on hydrocarbon fermentors, Ind. Eng. Chem. Process Des. Develop., 9, 570-577 (1970). 57-T. YOSHIDA, K. YOKOYAMA, K.C. CHEN, T. SUNOUCHI and H. TAGUCHI, Oxygen transfer in hydrocarbon fermentation by Candida rugosa. J. Ferment. Technol., 55, 7683 (1977).
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E N H A N C E M E N T OF OXYGEN TRANSFER RATES IN F E R M E N T A T I O N USING OXYGEN-VECTORS J. L. ROLS and G. GOMA D~partement de Gdnie Bioehimique et Alimentaire, UA CNRS 544, Centre de Transfert en Biotechnologie Microbiologie, Institut National des Sciences Appliqudes, Avenue de Rangueil, 31077 Toulouse Cddex, France
ABSTRACT
Oxygen transfer is one of the bottlenecks in conventional fermentation technology and it has so far been almost totally overlooked with regards to high call densities and immobilized cells. This review presents some new concepts to improve oxygen supply in aerobic fermentations, especially the use of oxygen-vectors. The oxygen-vectors generally used are liquids which are insoluble in the fermentation media. Their utilization in an emulsified form can significantly increase the oxygen transfer coefficient between gas and aqueous phases. It seems that the vector acts as an active intermediate in the oxygen transport from gas bubbles to aqueous phase, but the mechanisms involved in this unconventional technique of aeration are not yet known. KEY WORDS
Oxygen-vector, enhanced oxygen transfer coefficient, hemoglobin, hydrocarbon, perfluorocarbon, emulsion, spreading coefficient, transfer way. INTRODUCTION
Although aeration is one of the most studied areas of biochemical engineering and increasing the active callconcentration in its reactor is a crucial goal, surprisingly little work has been done to attain high call concentrations in aerobic fermentations by developing new methods to supply oxygen. In many aerobic fermentations, the oxygen supply rate to the aqueous phase is the limiting factor because of its low solubility in water. Umitations occur
2
1. L, ROLS a n d G. GOMA
when the oxygen demand rate exceeds the oxygen supply rate causing the dissolved oxygen level in the aqueous phase to fall below the critical oxygen concentration for the organism's metabolism. In conventional fermentation technology, oxygen is often supplied by bubbling air, or occasionally pure oxygen, through the fermentor. Vigorous stirring of the medium is necessary to get good oxygen transfer. Various novel methods have been reported by researchers to achieve good oxygen supply when high biocatalyst densities are used, such as in immobilized preparations (15,52). One approach has been to generate oxygen chemically in the aqueous phase by using a hydrogen-peroxide/catalase system (21,22,47), or by adding hydrogen-peroxide to the medium and using an organism with a high natural catalase activity (20). A biological approach to in situ oxygen generation would be to co-immobilize oxygen-consuming organisms with oxygen-producing organisms such as algae, but the flux of oxygen production is limited (1,2). Still another approach is to modify the medium in such a way that it dissolves more oxygen. The water solutions normally used are replaced by organic solvents, with the intention of increasing the solubility of both organic substrates and oxygen (6,13,23,28,51). Finally, another way of increasing the oxygen supply would be to modify the medium so that it could carry more oxygen. The interest in the production of single cell protein by growing microorganisms on various water-insoluble hydrocarbon substrates has resulted in several investigations of the nature of oxygen transfer in aerated systems with two liquid phases. The presence of a non-aqueous liquid phase may have a significant effect on the rate of oxygen transfer from the gas phase to the organisms. This new approach is the strategy of oxygen-vectors and consists of the addition to the growth medium of a compound or a liquid phase in which oxygen has a high solubility. The compounds generally used in biotechnology are hemoglobin (3), hydrocarbons (38) and perfluorocarbons (33). This present review covers the state of the oxygan-vector technology and presents a discussion of oxygen transfer in multiphase systems via the oxygan-vector. OXYGEN SUPPLY BY HEMOGLOBIN
In nature, several substances capable of transporting oxygen have been found. The most well-know are a series of iron and copper proteins that reversibly bind and transport oxygen via the blood streams of animals for the controlled catalysis of the transformation of biological substrates to useful products and thermal energy (18,46). In mammals, these are the heme proteins hemoglobin and myoglobin; in marine crustaceans the equivalent system is hemocyanin, a copper-containing protein; and in marine worms oxygen transport is via hemerythrin, a non-heme iron protein. WAKABAYASHI et al. (54) described a bacterial heme protein from V'~reoscilla that combines with molecular oxygen and has an amino-acid sequence with features characteristic of the globins. The hemoglobin content of V'~reoscilla increases almost 50-fold when the oxygen concentration of the growth medium falls below 10 per cent of atmoaphedc
ENHANCEMENT OF OXYGEN TRANSFER RATES
3
(5). Apparently, it may function to enable the organism to survive in oxygen-limited environments by acting as an oxygen storage-trap or to facilitate oxygen diffusion (42). Cloning of the hemoglobin gane from the bacterium Vitreoscilla has enabled expression of functional hemoglobin in Escherichia coli (4). Cells containing the Vitreoscilla hemoglobin exhibit increased respiration rates and grow more rapidly than hemoglobin-free calls under oxygen-limited conditions characteristic of high cell density growth, in both shake flask and fed-batch controlled fermentations. In addition, the cloned Vitreoscilla fragment also confers oxygen-mediated regulation of the expression of bacterial hemoglobin. In a project of MAI-I"IASSON and BORREBAECH (34) where glucose oxidase was used for analytical purposes, glucose oxidase and red blood calls containing hemoglobin were co-immobilized to concavalin A-Sepharose. On introducing a pulse of substrate, the partial pressure of oxygen within the matrix decreased concomitantly with the mobilization of the pool of oxygen stored in the hemeglobin packets of the calls. After the pulse had passed, Po2 increased and the hemoglobin was re,oxygenated. ADLERCREUTZ and MA'I-I'IASSON (3) supplemented the substrate solution with hemoglobin to increase oxygen supply to immobilized calls of Gluconobacter oxvdans for the conversion of glycerol to dihydroxyacatone. The dihydroxyacatone production varied linearly with the hemoglobin concentration. In this case, the role of hemoglobin is not unlike its normal role in animals. However this method has some limits: * Hemoglobin is highly soluble in water, but working with solutions more concantrated than 85 g/I does not seem realistic. * Hemoglobin has a high affinity for oxygen and therefore it is necessary for the medium to have a long residence time in the reactor if the oxygen supplied by the hemoglobin is to be effectively utilized. * If hemoglobin is used as an oxygen carrier in biotechnical processes, it is necessary that the protein can be used several times. It is know that hemoglobin is slowly oxidized by oxygen to methemoglobin. There are several reports concerning the reduction of mathemoglobin (38,49) but this reaction could not be driven to completness in this type of biotechnical processes. In an effort to exploit the advantages obtained with oxygan-vectors such as hemoglobin but avoid the disadvantages, synthetic carriers such as perfluorocarbons have been used instead. OXYGEN SUPPLY BY HYDROCARBON
In fermentations using hydrocarbons as substrate, microbial oxygen demand is much higher that when carbohydrates are used, as reported by many authors (14,37,39). Also growth media containing hydrocarbons have been well studied (17,38). MIMURA eta/. (38) were the first to point out the effect of hydrocarbon phase on oxygen transport in air-waterhydrocarbon systems. They studied the oxygen transfer in dosed shaking flasks containing air, sodium sulfite solution, and kerosene by following the change in the gas phase oxygen
4
J.L. ROLS a n d G. GOMA
partial pressure. They found that the oxygen transfer rate coefficient kLa/H increased exponentially with the volume fraction of kerosene ~ , when the total volume of the liquid mixture was held constant, for 0 < ~ < 0.85 (H: Henry's law constant). YOSHIDA et aL (56) determined values of kLa in both an agitated vessel and in a bubble column for aqueous dispersions of kerosene, a liquid paraffin and toluene. They pointed out that the spreading coefficient: Sp = ~T-WG - (g'OG + g--WO) , with 9"~ the interfacial tension between i and j phases, is negative for the kerosene and paraffin-water mixtures and for these systems kLa decreases linearly with increasing oil fraction. When Sp < 0, oil will simply form floating lenslike drops. For the toluene-water mixture, Sp > 0; kLa initially decreases and then increases with increasing oil fraction. The oil seems to spread as a thin film on the gas-liquid interface where it will act as a surface active agent to lower the surface tension and increase the specific interfacial area of the gaseous phase. COTY et al. (11) studied the oxygen uptake rate of mixtures containing hexadecane in which the oil phase was continuous. They found that the oxygen transfer rates from air to mixtures containing 50% and 66% oil in air lift reactors were much larger than for the medium without oil. MATSUMURA et aL (32), with appropriate modelling, determined volumetric oxygen transfer coefficients for transport between gas and water phases and water and hydrocarbon phases in an air-water-hydrocarbon (mainly n-pentedecane) system. They measured the gas phase oxygen partial pressure change in a closed agitated vessel with a draft tube, as oxygen was absorbed by the liquid mixture. They assumed the pathway of oxygen transport was from air to water to oil, and that the oil phase was in equilibrium with the water phase oxygen concentration present at the oil-water interface. The correlations they obtained are: (kLa)G w = (1.75.10-2.e 0.115.~. 0.8.10-3.e-46.9.,~).N.Vs1/3 and
(kLa)O w =
1 . (kLa)GW 1 + HOW (1/,~'-1)
with: : volume fraction of oil N: rotational impeller speed VS: linear velocity of aeration based on the cross sectional area of the stirred tank HOW: partition coefficient of oxygen in oil-water system. Both the volumetric absorption coefficients increased with an increase in the oil volume fraction. YAMANE and YOSHIDA (55) have shown that small oil droplets will very rapidly come into oxygen equilibrium when placed in an aqueous liquid. They also concluded that the main resistance for the oxygen absorption into O/3N emulsion in an agitated gas-liquid contactor exists in the water film around gas bubbles.
ENHANCEMENT OF OXYGEN TRANSFER RATES
5
A dynamic method for estimating volumetric oxygen transfer coefficients between the gas and aqueous phases (kLa)GW, the gas and oil phases (kLa)GO and the oil and aqueous phases (kLa)Ow, and oxygen uptake rate from the aqueous and oil phases during cultivation was developed by YOSHIDA et al. (57). This method involves recording the oxygen concentration in the liquid following a step change in the gas phase oxygen concentration and a numerical analyse of the response. More recently, TORRIJOS (50) observed the effect of hydrocarbon emulsions on the growth of Aerobacter aeroaenas cells, which are highly sensitive to the flow of transfered oxygen (45).The analysis of cultures containing various concentrations of hydrocarbon (mixture of n-tridecane to n-heptadecane) indicated an increase of the oxygen transfer coefficient (up to 4.6-fold for 20%v/v organic phase), directly related to the addition of organic phase, resulting in an improvement of both production and growth kinetics. Furthermore, it has been demonstrated that it was possible to increase the kLa of a fermenter by the addition of n-alkanas in the aqueous phase, without changing the conditions of agitation-aeration and thus without supplying more energy. The n-alkane phase could be easily separated from the aqueous phase by decantation and centrifugation of the upper phase. The usa of these vectors is cost-effective since there are readily available, inexpensive and can be reused. O X Y G E N SUPPLY BY P E R F L U O R O C A R B O N
1- Properties of oerfluorocarl;~n~: Perfluorocarbons (PFCs) are cyclic or straight chain hydrocarbons in which hydrogen atoms have been replaced with fluorine. Due to the strength of the carbon-fluorine bond, PFCs are considered as being chemically inert (43). Many gases, for example oxygen and carbon dioxide, have a high solubility in PFC liquids; a solubility which varies linearly with the gas partial pressure, according to Henry's law (26). The gas-dissoMng capacity decreases in the order: CO 2 > > 0 2 > CO > N 2 > H 2 > He, apparently following the decrease in molecular volume of the solute. The high dissoMng capacity is related to the ease of formation of cavities capable of accomodating the small solute molecules within the solvent, rather than to any specific interaction. These properties make PFCs suitable as blood substitutes. They are used in emulsified form in the presence of a surfactant (Pluronic F68, a polyoxyethylene polyoxypropylene block polymer), by either ultrasonic treatment or high pressure homoganisation. Emulsions of PFCs dispersed in physiologically-acceptable electrolyte solutions have been widely tested as oxygen-carrying resuscitation fluids in several mammalian species (29). Moreover, one commercial emulsion (FluosoI-DA, Green Cros Corporation, Japan), which contains parfluorodecalin and perfluorotripropylamine emulsified with a poloxamer surfactant, is the only PFC preparation to have been tested in man (35). Another application is the use of oxygen-carrying PFC emulsion as an adjuvant to radiation therapy (48).
6
J, L. ROLS and G. GOMA
The effect of emulsified PFCs on microbial cells was studied by CHANDLER et aL (9) on the growth of E. ¢01i cells. The toxicity of these emulsions is thought to be at least partly caused by the surfactants and result in cytotoxic effects. At present, a new formulation of oxygen-carriers consists of selecting fluorinated compounds with a hydrogenated part, compatible with non-toxic hydrogenated surfactants (7). 2- Oxvaen SUDDIVto microoraanisms: In situ aeneration:
Several patents recommend the use of PFCs as oxygen-carriers to stimulate the growth of aerobic microorganisms. Although stated otherwise by ADLERCREUTZ and MA'I-I"IASSON (3), PFCs have previously been used to help improve the productivity of aerobic fermentations. CHIBATA et al. (10) described a new method whereby PFCs or silicone oils were added to batch aerobic fermentations which were carried out in shake flasks or gas-sparged stirred tank fermentors. The conversion of sorbitol to sorbose increased as more perfluorotributylamine was added. Bis(F-hexyl)athene has for example been used to supply oxygen to lymphoid cell cultures of the Navalma strain destined for the production of interferon (31). It is believed that microorganisms do not obtain oxygen directly from the PFC phase. In the fermenter experiments described above, air was passed through the water phase, delivering oxygen to it. When PFC was also present, oxygen dissolved in this phase and could subsequently be transported to the water phase (33). This apparently caused an increase in the total transport of oxygen from the gas phase to the water phase. Sometimes, the oxygen dissolved in the PFC is sufficient for the entire cultivation so that reoxygenation is not necessary. HERTL and RAMSAY (19) used PFC and silicone oil greases or gels as oxygen reservoirs, to stimulate the growth of various microorganisms in liquid or solid media. More recently, TORRIJOS (50) studied the effect of varying amounts of Forane F66E on the growth of A. aeroaenes cells in a 20 I. fermenter. The emulsion was obtained by ultrasonic treatment in the presence of Pluronic F68. The results indicated an increase of the oxygen transfer coefficient up to 4-fold for 4.5%v/v organic phase. External oxvaenation: Sometimes, it is beneficial to withdraw the liquid and oxygenate it outside the reactor in which case it is an advantage if the solubility of oxygen in the liquid is high. Emulsions of PFC (FC-72) were used in this way to increase oxygen supply to immobilized G. oxidans cells in a packed bed reactor (3). The reaction mixture containing FC-72 in an emulsified form was saturated with air and passed through the column. The use of this mixture improved the productivity of the cells 5 to 6 times (with 32.4%v/v) over that of the control. Another approach has been developed by DAMANIO and WANG (12) with perfluoromethyldecalin in cultivations of E. coil cells in a spray column fermentor. The PFC was continuously removed from the fermentor, reaeratad, and returned whereby oxygen
ENHANCEMENT OF OXYGEN TRANSFER RATES
7
was supplied to the free cell bacterial culture by means of liquid-liquid contacting. Little energy was needed to form the droplets and the oxygen transfer from the droplets to the aqueous phase was rapid. In some instances, other compounds may be better than PFCs for increasing the oxygen supply to microorganisms. LEONHARDT eta/. (27) did not find PFCs suitable as oxygen-carriers for Providencia cells immobilized in calcium alginate. Better cell growth was obtained with polydimethylsiloxane emulsified with a copolymer of polydimethylsiloxane and polyethylene oxide. PFC emulsions when used in medical applications, transport carbon dioxide as well as oxygen. The same is true for bacterial cultivations. CESCHIN et al. (8) improved growth of anaerobic bacteria such as Clostridium oerffinaens by supplying carbon dioxide-saturated perfluorineted oil. In addition it is probable that PFCs can be used to remove inhibitory gases from the reactor, e.g. CO2 in aerobic bioprocessas. There is a little chance of finding new PFC oxygan-vectors having significantly higher oxygen-dissolving capacity. With the prospect of large-scale commercialisation coming soon, the problem of the industrial feasability of both the preparation and the purification of the PFCs, which was often neglected in the screening stages, becomes more acute. At present, PFCs are relatively expensive compounds (FC-72 costs for example about 80 US $.kg -1) and effective recovery will be necessary. ROLE OF OXYGEN-VECTORS
The growth of microorganisms on carbohydrate substrate in presence of oxygenvector, is realised by dispersing organic and gas phases in a continuous phase (water in which salts and sugar are dissolved). The studied system is then composed of four phases: -the continuous phase which is generally the aqueous phase, -the dispersed phases: *solid phase: microorganisms for which the size varies between 0.5 and 5)Jm. *gaseous phase: air bubbles for which the diameter varies between 0.5 and 5 mm. *liquid phase: vector droplets for which the diameter varies between 0.5 and 40 IJm. The three dispersed phases are placed in a turbulent environment, provoking a permanent renewal of gas and vector surfaces. The liquid-liquid system should have much more surface area avaible for mass transfer (and thus higher oxygen transfer retas) than gas-aqueous system for the following reasons: 1) at equal work inputs, drops formed will be smaller than bubble due to surface tension differences; 2) bubbles generally coalesce into larger bubbles much more rapidly than do droplets; and 3) liquid hold-ups can be fixed at levels much higher than typical gas hold-ups. According to MAC LEAN (30), the dispersed liquid phase can influence oxygen transfer by absorbing oxygen or supplying it to other phases, or by influencing the fluid mechanics of the continuous phase, thereby influencing the mass transfer coefficients and
8
J.L. ROLS a n d G. GOMA
interfacial areas of the continuous phase. The various way that oxygen can flow to reach the bacterial cells are pointed out on the following scheme (fig.l).
O
CELL
2
VECTOR Q
GAS
WATER
3 4 Fioure 1- Possible wavs for oxvaen transfer from oas bubbles to cells. 1) Gas-liouid-solid transfer: It is the main way used by oxygen molecules in absence of oxygen-vector. The oxygen molecules are dissolved in the aqueous phase and used in the fermentation media by the microorganisms. The vector can act as an emulsifier of air bubbles and water particles (38). Thus with the vector-water system, it will be possible to increase the interfacial area between the gas and the emulsion. 2) Gas-solid tran#Qr: The cells use the oxygen directly at the interface of the gas bubbles. Microbial cells have a tendency to crowd themselves into the liquid film of the gas-liquid interface, due to surface adsorption. The cell concentration at the bubble surface is usually even higher than that in the liquid bulk. Since the cells consume the oxygen molecules being absorbed right at the gas-liquid interface, TSAO eta/. (53) believe that the cells will affect and enhance the oxygen absorption rate. The increase of the gas-liquid interface area by the emulsifier vector favours this second way.
ENHANCEMENT OF OXYGEN TRANSFER RATES
3) Gas-liQuid-liauid-solidtransfer: The organic phase dissolves' the oxygen which is then transfered to the aqueous phase where it is used by the cells. From the comparison of the estimated values of the volumetric coefficient for oxygen transfer through the water film around gas bubbles and the volumetric coefficient for overall oxygen transfer from water into oil drops, YAMANE and YOSHIDA (55) concluded that the main resistance for oxygen absorption into O/W emulsion in an agitated gas-liquid contactor exists in the water film around gas bubbles. MIMURA et a/. (38) assumed that the oxygen transfer from the oil to water is much faster than from air to oil. In this way, a direct oxygen transfer from air to water is accomplished. The type of interaction between air bubbles and vector droplets is not well established. YOSHIDA at a/. 's correlation (56) between the evolution of kLa with oil fraction and the value of spreading coefficient is very attractive. When the spreading coefficient is positive, the oil seems to spread as a thin film at the gas-liquid interface. A mechanism explaining interaction between air bubble-oil drop in water has been proposed by ROQUES et aL (44) in a study of water-hydrocarbon separations by flotation. For Sp > 0, the system goes through a recovery of the bubble by the drop. When Sp < 0, the oil simply forms a lenslike droplet floating on the bubble surface. Thus, the specific interracial area of gas bubble for oxygen transfer may decrease due to partial blanketing of the bubble surface with lanslike oil droplets and to slower diffusion of oxygen through oil droplets. It is also conceivable that the rate of surface renewal or eddy diffusion around gas bubbles decreases with presence of oil droplets. The dissolved oxygen profile present in the third way can be illustrated by the following scheme (fig.2). The oxygen vector is an active intermediate to carry oxygen from bubble to water. 4) Gas-liouid-soiid transfer: The cells are at the surface of the vector droplets and use the oxygen directly. This type of transfer would depend on the affinity of the cells for oxygen-vectors and on the size of the emulsified droplets in the aqueous phase. For microorganisms growing on carbohydrate, MIMURA et aL (40) and TORRIJOS (50) obtained uniformly dispersed bacteria in the aqueous phase when oxygen-vectors were respectively hydrocarbon and PFC. For hydrocarbon-assimilating bacteria, cells can adsorb onto the surface of oil droplets, forming dense flocs. The flocs tend to attach to the surface of the air bubbles (41). The cells take up oxygen from both organic phase and in the water film around the bubbles. YOSHIDA eta/. (57) proposed that cells utilize a considerable amount of oxygen from oil which spreads on the surface of air bubbles. The biological effect of the synthetic surface active agents which are used to emulsify,hydrocarbons, is to separate cells from oil droplets (40).
10
J.L. ROLS a n d G. GOMA
OXYGEN
CONCENTRATION
Cv*
LEVEL
Cv
Cv.Cw*•. Cv"
i
I I GAS
VECTOR
[
Cw
I I
DISTANCE CELL
WATER
TRANSFER WAY
I=,
Fiaure 2- Oxvoen transfer oroflle in a fermentation media with an insoluble vector The total oxygen transfer rate measured in a fermentation media including an oxygenvector, will be the sum of the oxygen transfer rates from the four ways described above. It seems difficult to estimate the part of each way on the total transfer. However, some authors have proposed some methods for the determination of kLa at the different interfaces dating from mass transfer balances (32,57). More recently, KREYSA and WOEBCKEN (24,25) and FURIET et al. (16) have developed a transitory electrochemical technique measuring kLa between gas and liquid phases or between two liquid phases. These different methods will perhaps set in evidency the role of the oxygen-vectors.
REFERENCES
1-P. ADLERCREUTZ and B. MA'I-rlASSON, Oxygen supply to immobilized cells:l- oxygen production by immobilized (~hlorella Dyrenoidosa, Enzyme Microb. Technol., 4. 332-336 (1982). 2-P. ADLERCREU'rZ, O. HOLST and B. MA'R'IASSON, Oxygen supply to immobilized cells: 2- studies on a co-immobilized algae-bactaria preparation with in situ oxygen generation, Enzyme Microb. Technoi., 4, 395-400 (1982).
ENHANCEMENT OF OXYGEN TRANSFER RATES
11
3-P. ADLERCREUTZ and B. MA]-rlASSON, Oxygen supply to immobilized cells: 3- oxygen supply by hemoglobin or emulsions of perfluorochemicals, Eur. J. Appl. Microbiol. Biotechnol., 16, 165-170 (1982). 4-J.E. BAILEY, Enhancement of cell growth characteristics using genetic engineering, Proceedings VIII Internatl. Biotechnol. Sym., Pads (1988). 5-S.J. BOERMAN and D.A. WEBSTER, Control of heme content in V'~reoscilla by oxygen, J. Gen. Appl. Microbiol., 28, 35-43 (1982). 6-B.C. BUCKLAND, P. DUNNILL and M.D. ULLY, The enzymatic transformation of waterinsoluble reactants in nonaqueous solvents- Conversion of cholesterol to cholest-4-ene3-one by a Nocardia sp, Biotechnol. Bioeng., 17, 815-826 (1975). 7-M.C. CECUTrl, Formulation de milieux transporteurs d'oxyg~ne: optimisetion de nouvelles micro6mulsions par modulation des composants fluor6s, Th~Dse Docteur I.N.P., Toulouse, France (1987). 8-C. CESCHIN, M.C. MALET-MARTINO, G. MICHEL and A. LATTES, C. R. Acad. Sc. Paris, Ser.lll, 18, 669-672 (1985). 9-D. CHANDLER, M.R. DAVEY, K.Co LOWE and B.J. MULLIGAN, Effect of emulsified perfluorochemicals on growth and ultrastructure of microbial cells in culture, Biotechnol. Latt., 9, 195-200 (1987). 10-1. CHIBATA, S. YAMADA, M. WADA, N. IZUO and T. YAMAGUCHI, US Patent 3 850 753 (1974). 11-V.F. COTY, R.L GORRING, I.J. HEILWEIL, R.I. LEAVITT and S. SRINIVASAN, Growth of microbes in an oil-continuous environment, Biotechnol. Bioeng., 13. 825-842 (1971). 12-D. DAMIANO and S.S. WANG, Novel use of a perfluorocarbon for supplying oxygen to aerobic submerged cultures, Biotechnol. Lett., L 81-86 (1985). 13-J.M.C. DUARTE and M.D. LILLY, The use of free and immobilized cells in the presence of organic solvents: the oxidation of cholesterol by Nocardia rhodochrous. Enz. Eng., 5, 363 (1980). 14-A. EINSELE, H. SCHNEIDER and A. FIECHTER, Characterization of microemulsions in a hydrocarbon fermentation by electronmicroscopy, J. Ferment. Technol., 53, 241-243 (1975). 15-SOO. ENFORS and B. MATTIASSON, Oxygenation of processes involving immobilized cells, in Immobilized cells and orgenelles, Mattiesson ed., CRC Press, i~, 41-60 (1983). 16-C. FURIET, A. STORCK and F. LAPICQUE, Mdthode 61ectrochimique transitoire de d~ermination des conductances de transfart liquide-liquide, in R6cents progr6s en g6nie des procdd6s, Lavoisier, Paris, vol. 1,386-391 (1987). 17-G. GOMA, Contribution & I'(~ude des fermentations sur hydrocarbures: transfeft de mati&re, lois de croissance, Th(~se Docteur d'Etat Universit6 Paul Sabatier, Toulouse, France (1975). 18-D. HAYAISHI, Molecular mechanisms of oxygen activation, Academic press, New-York (1974). 19-W. HERTL and W.S. RAMSAY, US Patent 4 166 006 (1979).
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J.L. ROLS and G. GOMA
20-O. HOLST, S.O. ENFORS and B. MA'I-FIASSON, Oxygenation of immobilized cells using hydrogen-peroxide; a model study of Gluconobacter oxydans converting glycerol to dihydroxyacetone, Eur. J. Appl. Microbiol. Biotechnol., 14, 64-68 (1982). 21-M. IBRAHIM and H.G. SCHLEGEL, Oxygen supply to bacterial suspensions of high cell densities by hydrogen-peroxide, Biotechnol. Bioeng., 22, 1877-1894 (1980). 22-M. IBRAHIM and H.G. SCHLEGEL, Efficiency of bovine liver catalase as a catalyst to cleave H20 2 added continually to buffer solutions, Biotechnol. Bioeng., 22, 1895-1906 (1980). 23-A.M. KUBANOV,
Enzymes that work in organic solvents, Chemtech, June, 354-359
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