Concentration of amino acids by a liquid emulsion membrane with a cationic extractant

225

Journal of Membrane Science, 70 (1992) 225-235 Elsevler Science Publishers B V , Amsterdam

Concentration of amino acids by a liquid emulsion membrane with a cationic extractant Seong-Ahn

Hong, Hyung-Joon

Chow and Suk Woo Nam

Dwrszon of Process Engcneercng, Korea Institute of Sczence and Technology, Cheongryang, P 0 Box 131, Seoul (South Korea) (Received October 30,1991, accepted m revised form February 6,1992)

Abstract The concentration of L-phenylalanme m a hquld emulsion membrane system was studied usmg a cation complexmg agent, dl-2-ethylhexyl phosphonc acid (D2EHPA), as a earner The membrane formulation, the pH m the internal phase and the acid concentration m the external phase were optlmlzed with respect to concentration performance and membrane stability It was found that a hquld emulsion membrane obtained by demulslficatlon of the emulsion by an electrostatic coalescer could be reused Based on the laboratory tests, a contmuous multistage process for the concentration of ammo acids was proposed (with the development of a commercial process m mind) and its technical feaslblhty was discussed Keywords hquld emulsion membrane stability

membrane,

ammo acid concentration,

Introduction The separation and concentration of ammo acids using liquid emulsion membranes have been reported m several pubhcations [l-4] Among those, Thien et al. have examined the separation and concentration of L-phenylalarune m its amomc form using Ahquart 336 as a carrier [ 1 ] Although they confirmed the technical feaslbihty of using a liquid emulsion membrane for the concentration of ammo acids, one of several problems was not recogmzed with

Correspondence to Seong-Ahn Hong, Dlvlslon of Process Engineering, Korea Institute of Science and Technology, Cheongryang, P 0 BOX 131, Seoul (South Korea)

Q376-1388/92/$05

catlomc extractant,

demulslficatlon,

respect to the development of a commercial process. When an ammo acid is concentrated through a liquid emulsion membrane m its amomc form, counter ions, which are usually chloride ions, are transferred from the internal phase to the external phase across the membrane. If the residual amino acid concentration of the processed fermentation broth is not within acceptable hmits to be discarded, a contmuous multistage process such as either mixer-settler type or counter current contactor, may be required. In this situation, the accumulation of chloride ions m the external phase to be further treated will impair the separation efficiency because the drivmg force for the ammo acid extraction is usually provided by a concentration difference m counter-ion concentration be-

00 0 1992 Elsevler Science Publishers B V All rights reserved

S -A Hong et al/J

226

tween the external phase and the internal phase. In this article, the concentration of ammo acids in a liquid emulsion membrane system is examined using a cation complexmg agent, di2-ethyl hexyl phosphoric acid (D2EHPA) as a carrier. L-Phenylalanme is selected as a model solute because of its commercial significance. Currently, the downstream processing of a phenylalanme broth consists of cell removal, ion-exchange, evaporation and crystallization However, this process generates a lot of waste liquor for regeneration and much energy is wasted m evaporation. It has been shown previously that a liquid membrane process can be an alternative process for the separation and concentration of an amino acid even in its cationic form [ 31. The objective of this study is to clarify some potential problems, such as emulsion stability and membrane reuse, associated with the use of the liquid membrane process for commercial applications, and more specifically to develop a continuous process for ammo acid concentration by this technique. Experimental The membrane phase was prepared by blending all necessary components hsted in Table 1. Of the three organic solvents, kerosene, S-6ONR and S-31, the properties of which TABLE

1

Typical condltlon of experiments Internal phase Membrane phase

H,SO, 15 M S-6ONR 75 wt % Paranox 100 5 wt % DBEHPA 20 wt %

External

L-phe 12-35 g/l pH = 2 5 (with H2S04) 7110

phase

W/O ratio (W/0)/W ratio Temperature Stirring speed

l/4 25°C 300 rpm

Membrane SCL 70 (1992) 225-235

differ somewhat from each other (Table 2)) S60NR was mostly used. Paranox 100 was used as a surfactant and D2EHPA, a kmd of metal extractant, was used as a carrier. S-6ONR and S-31, precursors of lubricants, were obtained from SSang Yong Oil Refinery Company (Korea) A reagent-grade r.,-phenylalanme was purchased from Sigma and was mostly used. The fermentation broth used as a feed source for the results summarized by Fig. 11 was supplied by Miwon Co. (Korea). The broth was autoclavecd at 120 oC and then filtered through &atomaceous earth as a pretreatment before use Concentration of amino acids by means of liquid emulsion membranes was performed in a batch operation A stable water-m-oil emulsion was made by slow addition of the aqueous internal phase to the membrane phase under high shear provided by a homogenizer. The agitation speed was so high that the temperature of the emulsion was increased during the emulsification step Thus, a water-jacketted beaker which was kept at 10” C was used to keep the temperature of the emulsion below 25°C The size of the water droplets m the emulsion was measured by using a centrifugal automatic particle size analyzer, the average diameter was in the range of 0 4-1.5 pm. The W/O emulsion was then dispersed using mild agitation mto a baffled vessel contaunng 400 cm3 of contmuous external phase to give a dispersion of emulsion globules in an aqueous solution, 1.e a W/O/W liquid emulsion system. TABLE 2 The physical properties of solvents Composrtlon (vol % )

S-6ONR s-31 Kerosene

Aromatics

Olefins Paraffins

10 10 19 9

500 33 0 05

49 0 66 0 79 6

vlscoslty (cP, 40°C)

Spec grav1tY

71 25 4 17

0 87 0 87 080

227

S -A Hong et al ,LJ membrane Scz 70 (1992) 225-235

Samples were taken at a given interval and the external phase was separated from the emulsion phase for analysis Concentrations of Lphenylalanme were analysed by high performance liquid chromatography using a reverse phase p-Bondapak Cl8 column Typical experimental conditions are summarized m Table 1, unless otherwise mentioned The (W/0)/W multiple emulsion was separated by density difference, and the separated (W/O) emulsion was demulsified by an electrostatic coalescer &picted in Fig. 1 The volumes of internal phase and membrane phase were measured and the internal phase was analyzed for L-phenylalanme concentration,

Results and discussion

Figure 2 shows the solute concentration profiles versus time m the external phase and m the internal phase for the standard experimental condltlons, listed m Table 1. Characterlstlc of this result 1sthe rapId in&al decrease m the solute concentration of the external phase due to the drlvmg force of the hydrogen ion concentration gradient This 1s followed by a decreased flux as the dnvmg force decreases. The detailed transport mechanism was discussed elsewhere [ 3,5] (see also Fig 3). The slight m-

1OKv 30KHz

.

1

‘0 ‘

io ~io

’

’ ’

40

Contact tlme(mln)

5’ 8

Fig 2 Typical resuk of L-phe concentration

HA Phe+ Emulsion

Phe+

V

1

Water

Fig 1 Schematic diagram of demulslfier

Phe+ A-

Exterior

Phase

Membrane

Phase

L-phe 31y/l

S-60NR

pH = 2 5

PxiOO

5%

UIEHPA

20%

Fig 3 Phenylalanme

75%

Interior Phase H2Slr4

transport as a catlomc form

I 5F1

S -A Hong et al/J

228

crease m the solute concentration of the external phase after 20 min contact may be due to membrane breakage However, m a complementary experiment using a tracer only 1.7% of the solute in the internal phase was found m the external phase as a result of membrane breakage after a 40 mm period. Meanwhile, the concentration profile of the internal phase indicates a rapid increase during the first 10 min. The slight decrease in the concentration of the internal phase after 30 mm contact should be attributed to swelling of the internal phase. The driving force for this swellmg is the osmotic pressure gradient across the membrane between the internal phase (1.5 M H2S04) and the external phase (0.2 M L-phenylalanme) A time course of membrane swellmg for this experiment is shown m Fig. 4, where a dramatic swellmg behavior is observed after 30 mm contact. One surprismg observation here is that the swelling gradient mcreases with time, this contradicts most literature studies [l] which report a decreasing swelling gra&ent with time The reason behind this observation has yet to be explained, but entrainment of the external phase to the emulsion phase and emulsion stability change with time may produce this effect Considering the concentration efficiency and emulsion stability, the optimum contact time for this system is lo-20 min dur-

Membrane Scl 70 (1992) 225-235

mg which 33 g/l of L-phenylalanine m the external phase can be concentrated to 170 g/l of L-phenylalanine in the internal phase Membrane formulatton The membrane composition is one of the most important variables m the liquid emulsion membrane process It determines not only the stability of the emulsion but also influences a lot of other parameters, like osmosis and water solubihty The membrane usually consists of a solvent, a surfactant to stabilize the primary emulsion and a carrier. An optimized membrane formulation in this study is listed m Table 1. The properties of the organic solvent constituting a liquid membrane play an important role m determmmg the stability of the liquid membrane and the transport rate of the solute. Three organic solvents were used to test the effect of membrane viscosity and membrane composition on the liquid emulsion membrane system. The result is illustrated m Fig. 5. Experimental observations indicate that both the emulsion stability and concentration performance were greatly affected by organic solvents Emulsion instability IS mamly caused by membrane swelhng and membrane breakage. When kerosene was used as the organic solvent, the membrane phase begins to expand by entrammg the

o Kerosene

00

Fig

4

Time course of membrane swellmg

Fig

10 20 Contact 5

30 40 tlme(min)

C

Effect of solvents on separation performance

229

S -A Hong et al /J Membrane Scz 70 (1992) 225-235

external aqueous phase and eventually all the external phase is transported into the membrane phase Figure 5 also shows that a high membrane break-up occurred after 10 min contact. When more viscous solvents, e.g. S-6ONR and S31, which have lower aromatic content than kerosene (see Table 2 ) , were used, however, the stability of a liquid membrane was much improved. This result is quite consistent with the previous findmgs [6] that a liquid emulsion membrane consisting of ahphatic hydrocarbons rather than aromatics is more stable and its stability increases with increasing carbon number. It is also shown m Fig. 5 that the transport rate of L-phenylalanme strongly depends on the viscosity of the orgamc solvent. The less viscous solvent, S-GONR,enhanced the permeation of the solute across the membrane more than S-31. The next important things in the membrane formulation are the selection of surfactant and the determmation of the carrier concentration With few exceptions, only two kmds of surfactant have been usually used in hquid emulsion membrane process, SPAN 80 and a polyamine surfactant. It is well known [ 71, however, that SPAN 80, sorbitan mono-oleate, favors swellmg of the emulsion by osmosis Futhermore, it has a high solubility m water. Meanwhile, Paranox 100, a polyamme, does not suffer from these shortcomings and thus makes emulsions more stable. Based on our preliminary screening experiments, a Paranox 100 concentration of 5% and a DBEHPA concentration of 20% (see Fig 6) were found to be optimal for this system with respect to the emulsion stability and concentration performance Effect of pH m external phase It follows from the results of experiments on the liquid-liquid extraction of metals with D2EHPA that the pH of the aqueous solution substantially governs the process of metal sep-

-06

00

10 20 30 40 Contact tlme(mln)

Fig 6 Effect of carrier concentration performance

on separation

-06 %

00

10 20 30 40 Contact ttme(mln)

1D

Fig 7 Effect of pH m the external phase on separation performance

aration [ 81. Since the separation using liquid membranes is based on extraction, a similar dependence of the separation preference on pH in the external phase should be expected. Figure 7 shows the effect of the pH of the external phase The increase of pH leads to a higher separation of L-phenylalanine m a shorter time. For pH 1.0, the separation of Lphenylalanme is about 25%, while for pH 2.5, it is about 70%. At the lower pH values, the reverse reaction is higher than the forward reaction m the followmg ion-exchange reaction Phe+ +HA*Phe+A-

+H’

However, if the pH is too high, the separation efficiency decreases because less L-phenylala-

S -A Hong et al/J

230

rune is present m a cationic form. Based on the result of Fig. 7, an initial pH of 2.5-3 is optimal for this system. This pH dependency explains why the separation efficiency reaches a limit of approx 70% at optimal conditions: because Phe+ is exchanged for H+, as shown in Fig. 3, the pH m the external phase gradually decreases as Lphenylalanme is transferred into the internal phase For example, the final pH after 40 min contact time turned out to be about 17 for an initial pH of 2 5 m Fig 7. If the pH of the external phase is kept unchanged during the experiment, either by addition of a strong NaOH solution or by using buffer solution, an enhancement of separation performance can be expected. This effect is shown m Fig. 8 The separation of L-phenylalanine was improved to 80% after 10 mm contact time using a formic acid-NaOH buffer solution, while it is 64% without pH adJustment Effect of acid concentratton m the mternal phase Figure 9 shows how the transport rate varies with the concentration of sulfuric acid in the internal phase. The rate increases with mcreasing sulfuric acid concentration because the driving force by the hydrogen ion gradient m-

Membrane Set 70 (1992) 225-235

t

I

OOo

10 Conta26

5(3 tlmT(mrn;

Fig 9 Effect of acid concentration m the internal phase

00

10

ContaS

Fig

10

3 40 tume?(mln)

:

Effect of carrier type on separation performance

creases However, the degree of the mcrease becomes smaller when the sulfuric acid concentration is higher than 2 M. This is because increase of the sulfuric acid concentration increases the membrane swellmg which is caused by the water transfer from the external phase to the internal phase. Effect of carrier type

-06

Contact

tlme(mln)

Fig 8 Time course of L-phe concentration m the formic acid-NaOH buffer

Figure 10 illustrates the separation performance using two different types of carrier: DBEHPA when L-phenylalanme is transported as a cation and Adogen 464 when it is transported as an anion. For the given experimental conditions - which were presumed to be optlmum - DBEHPA led to much faster extraction

S -A Hong et al /J Membrane SCL 70 (1992) 225-235

than Adogen 464. In addition, with the use of DBEHPA there 1s room to further mcrease the transport rate by adJusting the pH of the external phase. While the accumulation of chloride ions makes the continuous multistage process impossible for the Adogen 464 system, the increase m hydrogen ion concentration resulting from the transfer from the internal phase may be easily adJusted for the DPEHPA system Fermentation broths often contain slgnificant quantities of inorganic anions as well as inorganic cations These morgamc ions are required by the microorganisms during fermentation and represent potential competitors for the carriers Although the effect of competing ions on the separation performance was not mvestlgated systematically in this study, the technical feasibihty of this liquid emulsion membrane system to be applied to real fermentation broths was studled One of the results 1s illustrated m Fig. 11 The fermentation broth used in this experiment has a L-phenylalanme concentration of 34 g/l and a sulfate ion concentration of 0.05% In the case where L-phenylalanme was transported as an amon using Adogen 464, the separation performance for the fermentation broth decreased markedly compared with that for the reagent grade L-phenylalamne This decrease m extraction performance 1s mainly attributed to the presence of

231

inorganic anions, especially sulfate ions, m the fermentation broth. However, there was not much difference of extraction performance for the reagent-grade phenylalanme and the fermentation broth when the L-phenylalanme was transported as a cation using DBEHPA Emu&on splttfng Once the solute has been concentrated into the internal phase, the product must be recovered and the liquid membrane must be reused for the liquid membrane process to be commercialized. Many methods to split W/O emulsions effectively with low cost have been tried. thermal and chemical splitting, centrifugation, ultrasonics and electrostatic coalescer [ 91 Among them, the well-known electrostatic splitting of water from crude 011 has been well adopted m the liquid emulsion membrane technique Many devices have been suggested [lo131 for the apphcatlon of a direct and alternatmg current The mechanism of the sphttmg is not very clear yet; not only the applied voltage, but also the frequency 1s an important parameter In Fig. 12, these effects on demulslficatlon of a W/O emulsion are illustrated using a high voltage, A C coalescer shown m Fig 1 Based

Voltage

{/-Gk-%+

0

(30KHz) 6

e (2OKv

, 0

Contact

tlme(mln)

Fig 11 Effect of competmg Ions on separation performance

I

,

I

,

10 20 Frequency(KHz)

I

30

’

Fig 12 Effects of A C voltage and frequency on emulsion sphttmg

S -A Hong et al /J Membrane Scz 70 (1992) 225-235

232

on these results, a high voltage of 20 kV with 20 kHz was chosen as the operating condition. A time course of demulsification under this condition 1s shown in Fig. 13, where 100% demulsification was obtained within 10 min. Membrane reuse Figure 14 shows the possiblhty of membrane reuse Here, demulslfication of the emulsion was conducted using the apparatus shown in Fig 1 and the membrane phase was reused for L-phenylalanine concentration without any chemical or physical treatment. This result clearly indicates that the liquid membrane can be reused without efficiency decline.

Fig 13 Time course of demulslficatlon

No of

000

10

Contact Fig 14 Posslbhty

cycle

20 tlme(mln)

SO

of membrane reuse

Contrnuous mubstage process Process flowsheets for the recovery of Lphenylalanme using hquld emulsion membranes are not much different from those proposed for the separation of heavy metals. Based on the results obtained from the laboratory batch operations, a continuous multistage process 1s proposed as shown m Fig. 15 The flow &agram uses a counter-current mixer-settler type cascade followed by electrostatic coalescence of the emulsion and recycle of the hquld membrane Crystalhzatlon of L-phenylalanine from the internal phase takes advantage of muumum solublhty at isoelectric pH’s. One of the most important steps determmmg the process shown m Fig. 15 1s the selectron of the type of mass transfer apparatus Although many types of mass transfer apparatus have been used m the past, the mixer-settler type seemed to be most suitable for this system A counter-current extraction column might also be used, but special consideration should be given. As discussed earlier, the pH of the external phase 1s very critical m determining the separation performance, it decreases to a nonoptimal value during the mass transfer. This means that one counter-current column can never be sufticlent to achieve the required separation. This unwanted sltuatlon might be resolved if pH adlustment could be made along the column However, it was found m our preliminary experiments with a counter-current extraction column that pH adlustment was not possible by ad&tlon of a strong NaOH solution because this caused excessive membrane breakup and emulsion mstablhty Based on these observations, the mixer-settler type was chosen where pH adjustment could be made at the outlet of each stage as shown m Fig 15 One maJor economical and technical question m the process design 1s how many stages of mixer-settler units are needed to achieve the required separation. This, of course, depends

233

S -A Hong et al /J Membrane Scr 70 (1992) 225-235 Membrane phase Internal phase

Broth

1 I

I

Homogemzer

I

1

-Demulslfler

c

J I

Contact0

7-

T NaOH

NaOl NabH Waste -

Recycle of

mternal

phase

L-phenylalanme Crystal

Fig 15 Schematic

diagram of L-phe recovery process by mixer-settler

very much on the process parameters, the required concentration in the effluent and on the results of the pilot plant. These questions will be discussed in detail in a subsequent paper, but a simulated experiment using a batch operation was conducted to check the technical feasibility of the multistage process proposed m Fig 15. The result is shown in Fig 16, where the initial L-phenylalanme concentration of 34 5 g/l was reduced to 0 4 g/l with four serial extractions. In this particular experiment, a fresh emulsion was used for each extraction step and the pH in the external phase was adJusted to 2.5 after each extraction step This result m-

dicates that a contmuous multistage extraction process is possible without efficiency declme when L-phenylalanme is transported across the membrane as a cation Another thing to be noted in the process proposed in Fig. 15 is the recycle of mternal phase. The internal phase separated in the demulsitier is first neutralized by a strong NaOH solution and then crystallized. This mternal phase is recycled and mixed with pretreated fermentation broth, not with the fresh internal phase. This point is different from the process proposed by Thien and Hatton [ 21. Because the solubility of L-phenylalanme at its isoelectric

S -A Hong et al /J Membrane Set 70 (1992) 225-235

234

60,

No

c” \

230 r” 8

A 0 l

1st 2nd

q .

3rd 4th

of extractlon

nl;

NaISOI

Fig 16 Posslblhty of the senal extraction of L-phe solution

00

10

20

Contact

tlme(rnlnf 30

0

0

Fig 17 Effect of the Na,SO, m the external phase on separation performance

point is about 30 g/l, it should be noted that the concentration of recycled mternal phase is close to the concentration of fermentation broth Also, the recycled internal phase contains the sodium sulfate which is formed by the reaction of NaOH with H&SO, Therefore, the effect of the presence of Na$SO, m the external phase was examined The result is shown m Fig. 17, where the rate was remarkably enhanced by the add&ion of sodnim sulfate m the external phase In this figure, the pH m the parenthesis mdicates the final pH of the external phase after 40 mm contact Without the addition of Na,SO,, the pH of the external phase was shifted from the mltial nH of 2.5 to 1 7 because

1 2

concskobon(rnol/l)

Fig 18 Effect of the Na,SO, m the external phase on membrane swelling

of counter ion transfer from the internal phase Meanwhile, the pH drop m the external phase was sigmficantly reduced by the addition of Na,SO,. The smaller change of pH m the external phase by the addition of Na$SO, is explained by the buffering effect of Na,SO,, and contributes to the enhancement of permeation rate by keeping the external pH around the optimum Another thing to be noted here is that the ad&tion of Na$O, to the external phase greatly reduces membrane swelling by reducing the osmotic pressure difference between the phases. This effect is shown dramatically m Fig 18. Conclusions (1) The contmuous process of ammo acid concentration with a liquid emulsion techmque was developed and the technical feasibility of this process was confirmed through the performance tests of its unit process (2) The concentration of ammo acid as a cation has several advantages over that as an anion (3 ) The membrane formulation, pH m the external phase and acid concentration m the internal phase were optimized with respect to the separation performance and membrane stabihty

235

S -A Hong et al /J Membrane Scr 70 (1992) 225-235

(4) The electrostatic coalescer for the contmuous demulsificatlon of emulsion was developed and its operation condition (frequency and A C voltage) was optimized. (5) Liquid emulsion membrane obtained from the demulsificatlon of the emulsion could be reused without any efficiency decline Based on these laboratory results, it is concluded that liquid emulsion membrane has a great potential to be practically utilized for the concentration of ammo acids Contmuous efforts to commercialize this developed process should be made Acknowledgement The authors wish to thank Mr. Chang-Ki Hur for his assistance m the experimental work. This work was supported by the Ministry of Science and Technology, South Korea. List of symbols C

CO H+ HA L-phe Phe+

L-phenylalanme concentration m the external phase (mol/l) Initial concentration of L-phenylalanine m the external phase (mol/l) hydrogen ion (di-2-ethylhexylphosDBEHPA phoric acid) L-phenylalanme L-phenylalanme cation

References 1

2

3

4

5

6

7

8

9

10 11 12

13

M P Thlen, T A Hatton and D I C Wang, Separatlon and concentration of ammo acids usmg liquid emulsion membranes, Blotechnol Bloeng ,32 (1988) 604 M P Thlen and T A Hatton, Liquid emulsion membrane and their apphcatlons m blochemlcal processmg, Sep Scl Technol ,23 (1988) 819 H Itoh, M P Thlen, T A Hatton and D I C Wang, A liquid emulsion membrane process for the separation of ammo acids, Blotechnol Bloeng , 35 (1990) 853 H Y Ha and S A Hong, A study on enzymatic reaction using a liquid emulsion membrane technique, Blotechnol Bloeng ,39 (1992) 125 R Marr and A Kopp, Liquid membrane technology A survey of phenomena, mechamsms, and models, Int Chem Eng ,22 (1982) 44 T Kmugawa, K Watanabe and H Takeuchl, Effect of organic solvents on stability of liquid surfactant membranes, J Chem Eng Jpn ,22 (1989) 593 J Draxler and R Marr, Emulsion liquid membranes Part I Phenomena and mdustnal apphcatlon, Chem Eng Progr ,20 (1986) 319 G M Rltcey and A W Ashbrook, Solvent Extraction Prmclples and Apphcatlons to Process Metallurgy Part I, Elsevler, Amsterdam, 1984 K J Llssant, Demulslflcantlon Industrial Apphcatlon, Surfactant Science Ser Vol 13, Marcel Dekker, New York, NY, 1983 E C Hsu and N N Ll, U S Patent 4,415,426,1983 R Marr, H J Bart and J Draxler, Liquid membrane permeation, Chem Eng Process, 27 (1990) 59 M Goto, J Ine, K Kondo and F Nakashlo, Electrical demulslflcatlon of W/O emulsion by contmuous tubular coalescer, J Chem Eng Jpn ,22 (1989) 401 K Fujimawa, M Morlshlta, M Hozawa, N Imalshl and H Ino, Demulslficatlon of W/O emulsion by use of high voltage of A C fields, J Chem Eng Jpn , 17 (1984) 632