Recovery of erythromycin by a liquid membrane

journal of MEMBRANE SCIENCE

ELSEVIER

Journal of Membrane Science 112 (1996) 209-217

Recovery of erythromycin by a liquid membrane J. Kawasaki a, R. Egashira a, T. Kawai a, H. Hara a, L. Boyadzhiev b,, a Department of Chemical Engineering, Tokyo Institute of Technology, Tokyo 152, Japan b Institute of Chemical Engineering, Bulgarian Academy of Sciences, Sofia 1113, Bulgaria Received 11 May 1995; revised 27 October 1995; accepted 27 October 1995

Abstract A supported liquid membrane technique was applied for extraction of erythromycin A from its dilute slightly alkaline aqueous solutions, l-decanol was used as an intermediate (membrane) phase and a buffered acidic aqueous solution to strip the organic membrane. The antibiotic distribution coefficient between the membrane used and the feed was found to be 122, providing relatively high solute fluxes across the membrane, although the transfer resistance remains in the filled membrane pores. It was shown that due to existing equilibrium between the protonised and non-protonised forms of the solute, the antibiotic can be completely transferred and concentrated in the receiving, low pH solution. Keywords: Liquid membrane; Pertraction; Erythromycin extraction; Antibiotic recovery; Erythromycin; Antibiotic extraction

1. Introduction Erythromycin A is a macrolide antibiotic, produced by Streptomyces erythreus microorganisms. According to the technological scheme adopted, after the fermentation step is completed it is extracted from the filtered broth [1,2]. A large number of solvents were proposed for its extraction [3,4] and some of them (butyl acetate, iso-amyl acetate or pentyl ethanoate) are applied on an industrial scale [2]. Russin and co-workers [5,6] studied the effects of temperature, solution pH-value and some additives on erythromycin distribution in a w a t e r - b u t y l acetate system. Bosnjakovic extracted the antibiotic with methyl-iso-butyl ketone and concentrated it 15 times in the extract solution [7], while for the same purpose Lu proposed to use tri-iso-butyl phosphate [8]. For in-situ extraction of erythromycin Mueller et al. [9] used a non-ionic alkyl phenol ethoxylate. Maydanov et al. [10] reported for important synergetic effects if couples of extractants such as pentanol-chloroform or b u t a n o l - c h l o roform are used. A comparison of the distribution coefficients for various prospective erythromycin extractants was published by Charykov et al. [11]. They found that these coefficients increase in the following order: n-hexane > n-heptane > decaline > toluene > carbon tetrachloride > octanol > pentanol > chloroform and that they depend strongly on erythromycin concentration and the pH-value of the aqueous solution.

* Corresponding author. 0376-7388/96/$15.00 © 1996 Elsevier Science B.V. All rights reserved SSDI 0376-7388(95)00282-0

J. Kawasaki et aL /Journal of Membrane Science 112 (1996) 209-217

210

Although the number of the studied extractants is very large, most of them should be avoided because of their high solubility in the aqueous solutions or due to their toxic properties. Convenient solvents with respect to the latter, as C7-C10 alkanes or slightly soluble in water C8-C1~ aliphatic alcohols provide lower distribution coefficients and therefore they are considered not suitable for practical use. However, if the classical extraction operation is replaced by a liquid membrane separation, these extractants might be successfully used for erythromycin recovery. In the recent years membrane separation processes are becoming increasingly attractive for recovery and separation of various substances. Among the numerous membrane processes special interests represent the so-called liquid membrane or pertraction separations, where an intermediate, immiscible liquid plays the role of a membrane, separating the feed and the stripping solutions. Combining the extraction and the stripping steps in one operation, these separation processes provide maximum driving force and allow the use of non-conventional, harmless extractants [ 12]. The aim of the present work is to reveal the possibilities for erythromycin recovery and concentration from its dilute aqueous solutions by means of a supported liquid membrane of 1-decanol.

2. Transport mechanism Dissolved in water, erythromycin exists in two forms: molecular and protonised. The equilibrium between these forms can be conveniently represented by the expression: EH+~ E + H ÷

(1)

with an equilibrium constant [4]: K = [E][H + ] / [ E H + ] = 10 -8.8 ( m o l / m 3)

(2)

Therefore, erythromycin is a weak organic base, slightly soluble in water (2.1 g/1 at 28°C [4]), existing in acidic water solutions (pH < 6) predominantly as a cation EH ÷ and in alkaline medium (pH > 10) as a neutral molecule E, only. Since, in contrast to the latter, the cationic form EH ÷ is completely insoluble in the organic solvent, applying the scheme shown in Fig. 1, erythromycin can be almost completely extracted from the feed F and concentrated into the stripping solution R.

[EH÷]s IE]F

~ DF

High pH

Low pH

F [EH÷]F

S - [E]~ [E] =0

Fig. 1. Diagram of erythromycin transport between two aqueous solutions with different pH-values, separated by a l-decanol supported liquid membrane.

J. Kawasaki et al. / Journal of Membrane Science 112 (1996) 209-217

211

The distribution coefficient of the erythromycin between organic (membrane) liquid and the aqueous solution is:

O = [ E ] / [ E w ],

(3)

but it should be noticed that most of the analytical procedures do not distinguish the two forms and give as a result the total ([E] + [EH+]) erythromycin concentration. Therefore, the apparent distribution coefficient Da measured is always smaller than the real one and it decreases rapidly with the increase of the proton concentration. The relation between the two coefficients is given by: D =D~(1 + [ H + ] / K )

(4)

The concentration profiles of both erythromycin species in the considered pertraction system are shown in Fig. 1. It is presumed that the protolytic equilibria are established instantaneously and that only non-associated molecules [E] are transported across the membrane. The rates of erythromycin transfer are represented by the local fluxes: JF = kF([E]F -- [E]MF/DF [m°l/m2 s]

(5)

JM = (-@rM/T6)([E]MF -- [E]Ms) [mol/m2 s]

(6)

Js = ks([E]Ms/Ds - [E]s) [mol/m2 s]

(7)

and

where k F and k s are the local mass transfer coefficients, -@'M the erythromycin diffusivity in the organic membrane, r and 6 the membrane tortuosity and thickness, respectively. Assuming that the erythromycin amount in the membrane is negligible regarding its contents in the aqueous solutions, one can write: J = J F = J M =Js [mol/m2 s]

(8)

Combining Eqs. (5) to (8) one can derive the rate of the feed exhaustion: d[E]F dt

"AF J Vv

AF

DF[E]F -- Ds[E]s

[mol/m 3 s]

(9)

V F 7"6/..@ M "t- D F / k F q- D s / k s

It should be emphasised that the concentrations [E]v and [E]s are those of the non protonised erythromycin in the appropriate solutions, but not of the total antibiotic contents. Since the analysis, as mentioned, give the total concentrations [E]F.T = ([E] F + [EH+]F) and [E]s,v = ([E] s + [EH+]s), it is more convenient from practical point of view to use the modified rate equation: d[E]F dt

A F K D F [ E ] F ' T / ( K + [H+]F) - D s [ E ] s ' T / ( K + VF r3/~ M + DF/k v + Ds/k s

[H+]s) [mol/m3 s]

(10)

When the pH value of the stripping solution is lower than 5.5 and relatively constant, and the two distribution coefficients D F and D s are very close, Eq. (10) is simplified to: d[E]F dt

AF

K[E]F,T

-- V F ( K + [H+ ]F ) ( ~ 6 / D F ~ M + I / k v + l / k s )

[m°l/m3s]

(11)

The rate equations (10) and (11) can be solved analytically with the condition that the distribution coefficients and the pH-values do not depend on the solute concentration; otherwise they should be solved numerically.

2 t2

J. Kawasaki et a l . / J o u r n a l o f Membrane Science 112 (1996) 209-217

3. Experimental A 0.003 M buffered solution of erythromycin A, was used as a feed phase. The buffering solution contained 0.025 mol/1 citric acid, 0.10 tool/1 boric acid and 0.05 mol/1 sodium phosphate, all p.a. grade products. To prevent the pH variation of the stripping solution caused by the protonisation of the transferred erythromycin molecules it was buffered also. The membrane liquid used for impregnation the porous Teflon ~ support was, as mentioned, 1-decanol purum grade. The support, supplied by Goretex ~ was 60 /xm thick with an average pore size of 0.02 /zm and 48% porosity. The total erythromycin concentrations [E]v.v and [E]s, T in the aqueous solutions were measured with a HPLC (Hitachi). All experiments were carried out at 25°C. The erythromycin distribution coefficients were obtained using thermostated separation funnels. Samples of 20 ml aqueous solution, containing initially 0.03 M erythromycin were contacted for 20 min with 2 ml of 1-decanol. The pertraction (mass transfer) experiments were performed in the two-compartment mass transfer cell, made of Pyrex ~ glass shown in Fig. 2. Both compartments of 125 ml each, were stirred independently by means of four blade agitators, rotating with constant velocity. The membrane, soaked with decanol, was clamped between the two compartments, providing 13.02 cm 2 exposed area. Each compartment had openings for periodic liquid sampling using micro syringes and for temperature control. The whole device was thermostated.

4. Results and discussion

4.1. Equilibrium distribution Fig. 3 shows the variation of the apparent distribution coefficient D a versus the concentration of solution hydrogen ions. The pronounced decrease of this coefficient with the increase of hydrogen ion concentration from 3.10 - t ° to 10 -7 m o l / l is due to the increase of erythromycin protonisation with the solution acidity. However, when these experimental results are recalculated according to Eq. (4), one can obtain an approximately constant value for the real distribution coefficient D, which average was found to be D = 122. This

4

5

4

Fig. 2. Scheme of the two-compartment mass transfer cell: 1, phase compartment; 2, four-blade agitators; 3, membrane; 4, temperature probes; 5, sampling probes; 6, membrane support; 7, membrane liquid.

J. Kawasaki et al. / Journal of Membrane Science 112 (1996) 209-217 D.Do

,

,

213

,

[-1 i0z ~

-D--'O- .....

100

e ' ~

I

llJ~

I

169

k

llj 8

llJT[H+]rnot/t

Fig. 3. Effect of hydrogen ion concentrationon real (D) and apparent(Da) distribution coefficients.

relatively high value makes the 1-decanol an attractive membrane liquid for erythromycin recovery from dilute production solutions or from waste effluents, provided that the mass transfer coefficients and the other process parameters are favourable, also. 4.2. Transfer kinetics

As mentioned, erythromycin transfer includes three consecutive transport steps, assuming the protonisation and deprotonisation reactions to be instantaneous. Since the distribution coefficient D is relatively high, a priori it was presumed that the three mass transfer resistances, in the right-hand side denominator of Eq. (10) have comparable values. To check this hypothesis we carried out experiments at various intensities of agitation of the feed and the stripping solutions. However, the data in Fig. 4, obtained at 150 and 250 rpm, show that even a modest phase homogenisation eliminates completely the mass transfer resistances in both aqueous boundary

(

,

,

,

•

i

.

.

.

.

i

[E]~rxl03 [mot/L]

2

[]

[]

/ 1

I

0

I i

i

~

I

i

,

i

5

i

I

10 time [h]

Fig. 4. Effect of aqueous phase stirring on the rate of erythromycinremovalfrom the feed.

214

J. Kawasaki et al. / Journal of Membrane Science 112 (1996) 209-217

3(

. . . .

'

. . . .

'

[El'x~03 [mot/t]

o-pH =8,5 I ~ " ~ , ' ~

O-pH=9.51 1

.

.

.

"%~.-'0.

pH =10.5 I

a.

I

0

i

i

5

,

i

time [h]

I

10

Fig. 5. Influence of feed pH value on the rate of erythromycin removal from the feed. Stripping solution pH = 5.5.

layers. Therefore, the erythromycin transport in the stagnant liquid membrane is the only rate controlling step and hence, for stirring velocities over 150 rpm Eq. (10) can be reduced to: - d [E]F = A---LvDF[E]F'T/( K + [H + ]F) -- Ds[E]s,T/( K + [H + ]s) [ m o l / m 3 s] dt VF 7' 8//.,~r M K

(12)

Since in these experimental runs the pH value of the strip solution was kept constant at pH s = 5.5, the negative term in the right-hand side of Eq. (12) can also be omitted: d[E]F dt

AF2MKDF[E]F'T[mol/m3 s] VF~'6(K +

(13)

[H+ ]F)

Hydrogen ion concentrations in the two aqueous solutions are important factors for erythromycin recovery. The difference between their concentrations in the feed and strip solutions defines the process driving force on the condition that the solute in the former solution is not protonised.

K

.

.

.

.

l

.

.

.

.

i

[E],rxl03t "NTk

oNL 2

" t ~

v

v - pHs = 8,5 I 0

5

time [h]

10

Fig. 6. Influence of stripping phase pH-value on the rate of erythromycin removal from the feed. Feed phase pH = 9.5.

J. Kawasaki et aL / Journal of Membrane Science 112 (1996) 209-217 3~

.

0

5

I

.

..

.

10

.

215

"r=

1'5

210

I

time[hi

Fig. 7. Extraction of erythromycin from the feed and its accumulation in the stripping solution vs. time. Feed phase pH = 9.5. Stripping phase pH = 5.5.

The effect of feed pH value is shown in Fig. 5. As expected, the higher is the feed pH value, the faster is the erythromycin pertraction, but beyond the level of pH = 10.5 the transfer rate does not change because all solute content in the feed exists practically in a transferable, non-ionic form. In these experiments the pH of the stripping solution was 5.5, providing complete protonisation of the accumulated erythromycin. The solid lines in Fig. 5 represent the changes of erythromycin feed concentrations, calculated according Eq. (11). An explanation of the noticeable discrepancy between the experimental and calculated data for the case of pH = 8.5 might be the association of the erythromycin molecules in the feed, modifying and complicating the protolytic equilibrium represented by Eq. (1). Indications for such a behaviour were reported earlier [10] and this phenomenon deserves a special, more detailed study. When the pH of the stripping solution is higher than 5.5, the solute flux decreases due to growing effect of the negative term in the right-hand side nominator in Eq. (12). The experimental results, shown in Fig. 6, carried out at constant feed pH = 9.5, confirm this statement: There is no remarkable difference between the pertraction rates at pH = 5.5 (99.95% protonisation), pH = 6.0 (99.84% protonisation) and pH -= 7.0 (98.4% protonisation), while for pH = 8.5 (50% protonisation) the transfer rate is clearly slower. As mentioned, the existence of erythromycin in an aqueous solution in two different forms, whose ratio depends on the solution pH value, provides an opportunity for its nearly complete and supposedly selective extraction from fermentation broths or waste solutions. This "uphill" transport across the liquid membrane and concentration of erythromycin in the stripping solution is illustrated in Fig. 7. It should be emphasised that this result should be considered merely as an illustrative example, but not as a recommended procedure for practical application: The fluxes in these experiments are very small due to very low surface-to-volume ratio ( A/V - 10 -5 m 2 / m 3) of the experimental cell. They are at least hundred times lower than the fluxes, provided by the conventional ELM, SLM or RFP liquid membrane techniques [12]. The relatively thick porous supports used retained enough carried to provide a stable operation at least for 200 h. However, the real lifetime limit of the membrane was not measured in this study.

5. Conclusion

Erythromycin A can be recovered from its dilute alkaline solutions applying a supported liquid membrane technique, using 1-decanol as a carrier. Due to the existing equilibrium between protonised and non-protonised

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J. Kawasaki et al. / Journal of Membrane Science 112 (1996) 209-217

erythromycin molecules, easily controlled by solution pH values it is possible to extract the antibiotic almost completely and to concentrate it in the stripping solution. The carrier used is non-toxic, it has very low solubility in water (less than 0.01%) and provides high distribution coefficients. When a supported liquid membrane technique is applied, the mass transfer resistance of the membrane is the rate controlling factor, on the condition that the feed and the stripping solutions are well stirred.

6. Symbols used A D E EH F j K M S V _~

interfacial area solute distribution coefficient erythromycin protonised erythromycin feed phase solute flux equilibrium constant membrane phase stripping phase phase volume diffusivity membrane thickness ~" tortuosity factor Subscripts a denotes apparent values F refers to the feed M the membrane MF membrane/feed interface Ms membrane/stripping solution interface s stripping phase v denotes total water W

References [1] A.K. Malams, Macrolide antibiotics, in Kirk-Othmer's Encyclopaedia of Chemical Technology, Vol. 2, Wiley, New York, 1978, p. 941. [2] D.M. Trachtenberg, Antibiotic Producfon, Khimia, Moscow, 1970. [3] J.M. McGuire, R.L. Bunch, R.G. Anderson, H.E.Boaz, E.H. Flinn, H.M. Powell and J.W. Smith, Ilotycin-a new antibiotic, Antibiot. Chemotherapy, 6 (1952) 281. [4] P.J. Weiss, M.L. Andrew and W.W. Wright, Solubilities of antibiotics in 24 solvents - use in analysis, Antibiot. Chemotherapy, 11 (1957) 374. [5] V.V. Russin, S.A. Zhukovskaya and V.L. Pebalk, Equilibrium conditions and effect of solution pH on erythromycin purification by extraction, Antibiotiki, 20 (1975) 222. [6] V.V. Russin, A.L. Solodov, S.A. Zhukovskaya and V.L. Pebalk, Use of ETSD-type extractor for extraction of antibiotics, Khim. Farm. Zh., l0 (1976) 114. [7] A. Bosnjakovic A., Extraction of erythromycin from fermentation broth, Kern. Ind., 29 (1984) 173. [8] Z. Lu, Extraction of antibiotics from fermentation liquors with organo-phosphorous compounds, Chinese. Pat., 1,037,343/22 November 1989.

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[9] V. Mueller, U. Merrettig, M. Traeger and U. Onken, In-situ extraction of secondary metabolites, Proc. Dechema Int. Biotechnol. Conf., part 3, 1989, p. 175. [10] N.N. Maydanov, N.L. Egutkin, V.V. Maydanov and Yu. Nikitin, Khim. Farm. Zh., 18 (1984) 336. [11] A.K. Charykov and L.I.Shirokova, Estimation of the efficiency of the extraction of erythromycin by different organic solvents by means of pH potentiometric method, Vestn. Len. Univ., Ser. 4, Fiz. Khim., 99 (1987). [12] L. Boyadzhiev and Z. Lazarova, Liquid membranes, in R. Noble and A. Stem (Eds.), Membrane Separation Technology. Principles and applications, Elsevier, Amsterdam, 1995, pp. 283-352.