History of Modern Genetics in Germany

5

Process Consideration

Selectivity higher than those attainable by current separation methods, saving on energy cost for final concentration of separated products,higher fluxes,compact installation, and low capital and operation costs are the common attractive feature of liquid membrane separation processes. The adsorption chromatography proposed as a final purification step by Fuji et al. [76] on filtered broth of cephalosporins has the limitation of generating dilute solutions necessitating energy intensive concentration operations and thereby increasing the price per kg of the material by several hundreds of dollars. Up to 50% of the total cost of a chromatographic process is eluent related. It is necessary to purify and recycle the spent solvent. A proportion (typically 3–5%) of solvent is usually lost and, considering the huge volume handled, this amount can be substantial. The liquid membrane process can be competitive to adsorption chromatography, but the following issues should be addressed for process design and practical application.

5.1

Chemical Driving Force

In liquid membrane extraction, if external driving force is not introduced, then driving force for the transport of cephalosporin anions is the chemical potential and dilution is inevitable. Phase ratio, use of counter-current flow, number of transfer stages, and reversibility of the extractant determine the degree to which the concentration of solute in the product solution approaches that in the feed phase [77]. Extractant design faces the choice between higher recovery and higher loading (strong extractant) on the one hand and higher product concentration (weaker extractant of higher reversibility) on the other. External driving force enables the concentration and higher recovery with product concentration to above its level in the feed phase (uphill transport). Product dilution through water co-transport should be avoided, e.g., by addition of salts to the feed phase, design of an acid/water selective membrane, or application of pressure on the stripping solution (in the case of HFM). Chemical and thermal energy can be used as driving forces in analogy to process in liquid-liquid extraction (electric energy in an electrodialysis resembling arrangement should also be considered).

Neutralization of the extracted molecule provides chemical energy. Thus, a NaOH-containing stripping phase was used by Li to provide the driving force for phenol separation and concentration through an ELM system [78]. Similar results were obtained by Friensen et al. [79] and by Basu and Sirkar [61], who showed up to three times higher transport rate and higher concentrations in the stripping phase. This type of chemical driving force can be applied for LM separation of cephalosporin antibiotics. A less prohibitive use of chemical energy for product concentration is the dilution of another compound. Such a process will be economic if the compound to be diluted is of relatively low cost and if there is a sink for its solution. Some examples of uphill pumping of the solute through dilution of another compound is as follows:

1.Water transport from product solution into a concentrated electrolyte solution (the membrane should transfer water and block the electrolyte and the solute).

2.Applying the common ion effect or salting out effect, the solute can be concentrated. The membrane should be designed to transfer the solute and block the additives and the impurities.

3.Simultaneous transport of solute with a transport of another solute in the same direction (syntrop) or in the opposite direction (antitrop).

5.2

Stability Problems

Membrane instability results in partial mixing of feed and stripping phases, which deteriorates the selectivity. In addition, raffinate and product are contaminated by the extractant, leading also to extractant losses. Economy of separation and hence industrial application of LM for separation of cephalosporins are strongly dependent on membrane stabilization.

Factors affecting ELM stability have been studied extensively [47, 80–83]. Usually the internal phase of ELM is much more concentrated than the external phase.Osmotic pressure driven water transport into the internal phase causes its swelling, sometimes to breaking point. The degree of swelling is affected by viscosity of the W/O emulsion, W/O/W dispersion, extractant composition, and surfactant and internal reagent compositions. Use of viscous paraffinic oils in the membrane phase and increasing surfactant concentration were proposed for decreasing ELM breakage. A tracer technique has been used to quantify emulsion break-up which is a time-dependent phenomena and can be expressed in terms of a measurable rate constant. However, in case of beta-lactam extraction in ELM, the emulsion swelling and break-up under specific experimental condition were found to be negligible [25, 26].

Although the emulsions were expected to be stabilized with increasing surfactant concentration, the degree of stability beyond a critical surfactant concentration becomes approximately constant due to the saturation of the surfactants at the oil-water interface. Again, higher concentration of surfactant will hinder demulsification after extraction. So a critical surfactant concentration value should be used for practical purposes.

In some cases,swelling due to water transport caused by surfactant,e.g.,Span80 destabilized the emulsion droplets. Use of a surfactant with low value of hy- drophilic-lipophilic balance is essential for reducing the swelling. The surfactant should carry virtually no water during operation so as to alleviate osmotic swelling [83]. Span-80 as an emulsifier exhibits poor stability due to hydrolysis, especially when NaOH is incorporated into the internal phase. In such cases, PX100 and ECA4360,polyamine surfactants may be used or Span-80 may be used by replacing NaOH with Na2CO3. Membrane with ECA4360 alone exhibits low osmotic swelling but high resistance to mass transfer. In contrast, membrane with Span-80 shows high swelling but low mass transfer resistance. By adding 2% Span-80 to the membrane with ECA4360, the mass transfer resistance was substantially lowered while the swelling was still kept to a low tolerable level [82].

Extractant leakage from the pores of the polymeric membrane in SLM is due to osmotic flow of massive quantities of water through the membrane. Membrane stability decreases with increasing osmotic pressure gradient and depends upon composition of the SLM system.A high tendency to solubilize water,low extractant/aqueous interfacial tension,and high wettability of polymeric membrane leads to less stable SLMs. The following measures have been proposed for improvement of stability:

–Maintaining an interface immobilizing pressure difference across the porous membrane in a direction and of a magnitude effective to oppose the tendency of leakage [83, 84].

–Modification of extractant and the aqueous phases for achieving lower surface activity and lower miscibility [85].

–Modification of polymeric membrane for better adhesion of the extractant or designing it so that interface between the phases is immobilized at the porous membrane [84, 85].

–Blocking leakage by applying a thin gel layer, preferably by a gel network with a low mesh size built with chemically stable crosslinking. [86].

–Various degrees of binding the extractant to the polymeric membrane [87].

5.3

Membrane Recoverability and Reuse

Generally, an emulsion prepared with a high energy density input (such as by an electrostatic method) will have very small droplets. This will enhance membrane stability if the surfactant concentration is high enough. Meanwhile, the small droplet size gives a very large interfacial area for mass transfer, but an ultrastable emulsion should be avoided because of possible difficulties later during the demulsification step. Usually, a membrane leakage rate of about 0.1% is allowable for a practical process [47].

Two principal approaches for the demulsification of the loaded emulsion are chemical and physical treatments. Chemical treatment involves the addition of a demulsifier to the emulsion. This method seems to be very effective. However, the added demulsifier will change the properties of the membrane phase and thus inhibits its reuse. In addition, the recovery of the demulsifier by distillation is rather expensive. Therefore, chemical treatment is usually not suitable for breaking emulsion liquid membrane, although few examples of chemical demulsification have been reported for certain liquid membrane systems [88].

Physical treatment methods including heating, centrifugation, ultrasonics, solvent dissolution, high shear, and use of high voltage electrostatic fields. Liquid membrane emulsion (both W/O and O/W type) can be effectively broken by the use of a specially formulated solvent mixture and high shear. The solvent mixture breaks the emulsion without damaging the surfactant. The solvents used are low boiling compound and they can be easily recovered later by evaporation. The method of demulsification by high shear includes the use of centrifugation as the first step, followed by pumping the half-broken emulsion through a high shear device [89].

because of reduced equipment volume and the low cost required for a particular duty. In an LM process, the external driving force allows high product recovery with product concentration well above its level in the feed (uphill pumping). Extractor design can be based on the choice between high recovery and high loading (strong extractant) on the one hand and high product concentration (weaker extractant of higher reversibility) on the other.

Except for the demulsification step, the process design principles of ELM process is fairly well understood. In the case of cephalosporins, membrane formulations as regards the extractant, stripping agent, surfactant, etc., can be based on thermodynamic and kinetic considerations identical to those applied for penicillins. Modeling and simulation of the reaction diffusion phenomena and validation from experimental results with independently measured model parameters may be required to understand the process design principles of the LM extraction of cephalosporins. For large scale application, the column extractor used for a conventional solvent extraction process can be effective for ELM processes. The well-understood design principle available for conventional extractors can be easily extended to ELM extractors [29].

The process design principles of SLM, non-dispersive extraction, and hybrid liquid membrane systems need to be understood through bench scale experiments using feed solution of practical relevance.While the economic analysis of an ELM process can be performed from small scale experiments, such an analysis is difficult for other LM systems. In particular, availability and cost of hollow fiber membranes for commercial application are not known apriori. A simple rule of thumb for cost scale-up may not be applicable in the case of an HF membrane. Yet we feel that the pilot plant tests would be adequate to make realistic cost benefit analysis of a liquid membrane process, since the volume of production in b-lactam antibiotic industries is usually low.

6

Research Needs of Pragmatic Importance

The liquid membrane (LM) technique for extraction of cephalosporin antibiotics appears to be the future generation technology of promise. The LM can perhaps function best for natural cephalosporins, which are usually present at considerably low concentration in the fermentation broth. The capital investment of the LM process is likely to be low because of reduced equipment volume required for a particular duty.In an LM process,the external driving force allows a high product recovery with product concentration well above its level in feed (uphill pumping). The importance of the LM process for cephalosporin separation will be more pronounced if the same is applied for direct extraction from culture broth. Since enzyme/microbial cell activity is likely to be effected under the reactive extraction condition, it is necessary to address this aspect from experiments using culture broth. Toxic effects of the extractants should be related to the membrane stability and enzyme/cell activity.As far as process application is concerned, it is not clear where an LM should belong in the downstream processing train. If LM is to be used early in the downstream processing for crude separation and concentration, it must be demonstrated that LM application can

compete commercially with well proven commercial processes like adsorption chromatography. If LM is to be used later in the downstream processing train, it must be shown that adequate, specific separation can be achieved, and product loss would be acceptable. It is apparent that scale-up studies on LM extraction of cephalosporin antibiotics will be highly useful as the economic incentives for such a process are expected to be very attractive.

7 Conclusion

Developments in liquid membrane separation of cephalosporin antibiotics are rather meager, but will be quite rewarding. In particular, ELM has great potential to be practically utilized for separation and concentration from dilute solution such as that encountered in fermentation broth. The most probable application of ELM appears to be the recovery and concentration of CPC, 7-ACA, and cephalhexin in downstream operation of biochemical processes. The integrated product formation and recovery by liquid membrane extraction will gain impetus in the future. Reactive extraction in liquid membrane and non-dispersive reactive extraction in hollow fiber membrane need extensive investigation leading to generation of process design information for practical exploitation in technological perspectives.

References

1.Martin JF, Liras P (1989) Enzymes involved in penicillin, cephalosporin and cephamycin biosynthesis.In: Fiechter A (ed) Adv in Biochem Eng Biotech.Springer,Berlin Heidelberg New York

2.Newton GGF, Abraham EP (1955) Nature 24:443

3.Lancini G, Lorenzetti R (1993) Biotechnology of antibiotics and other bioactive microbial metabolites, 2nd edn. Plenum Press, New York

4.Vandamme EJ (1990) Recent advances in the biotechnology of b-lactam and microbial bioactive peptides. In: Klein Kauf H, Von Dohren H (eds) Biochemistry of peptides antibiotics. Watter De Gruyter, Berlin

5.Hyun CK, Kim JK, Ruy DDY (1993) Biotech Bioeng 42:800

6.Schewale JG, Sivaraman H (1998) Proc Biochem 8X:148

7.Leinn JJ (1988) Eur Pat 0,293,218

8.Aramori I, Fukugawa M, Tsumara M, Iwami M, Ono H, Ishitani Y, Kojo H, Kohsaka M, Ueda Y, Iomanak H (1992) J Ferment Bioeng 73:185

9.Abraham EP, Loder HP (1972) In: Flynn HE (ed) Cephalosporins and penicillins: chemistry and biology. Academic Press, New York

10.Binder R, Brown J, Romanick G (1994) Appl Environ Microbiol 60:1805

11.Parmer A, Kumar H, Marwaha SS, Kenedy JF (1998) Crit Rev Biotech 18:1

12.Ghosh AC, Mathur RK, Dutta NN (1997) Extraction and purification of cephalosporin antibiotics. In: Scheper T (ed) Adv Biochem Eng Biotech 56:111–145

13.Li NN (1971) Ind Eng Chem (Proc Des Devel) 10:215

14.Wald AS, Leysez LJ, Matson LS (1989) Paper presented at the 3rd Annual meeting of the North American Membrane Society, 19 May, Texas, USA

15.Schugerl K (1994) Solvent extraction in biotechnology: recovery of primary and secondary metabolites, 1st edn. Springer, Berlin Heidelberg New York

16.Likidis Z, Schugerl K (1987) J Biotechnol 5:293

17.Reschke N, Schugerl K (1986) Chem Eng Biochem Eng J 32:B1–B5

18.Muller B, Schlichting E, Schugerl K (1988) Appl Microbiol. Biotechnol, 27:484

19.Likidis Z, Schlichting E, Bishoff E, Schugerl K (1989) Biotech Bioeng 33:1385

20.Lee KH, Lee WK (1994) Sep Sci. Technol 29:602

21.Harris TAJ, Khan S, Reuban GB, Shokoya T (1990) In: Pyle DL (ed) Separations for biotechnology. Elsevier, London, pp 172–180

22.Hano T, Matsumoto M, Ohtake T, Hori F (1992) J Chem Eng Jpn 25:293

23.Sahoo GC, Ghosh AC, Dutta NN, Mathur RK (1996) J Membr Sci 112:147

24.Sahoo GC, Ghosh AC, Dutta NN (1997) Process Biochem 32:265

25.Sahoo GC, Dutta NN (1998) J Membr Sci 145:15

26.Sahoo GC, Dass NN, Dutta NN (1999) J Membr Sci 157:251

27.Sahoo GC, Borthakur S, Dass NN, Dutta NN (1999) Bioprocess Eng 20:117

28.Li NN (1968) US Pat 3,410,794

29.Ho WSW, Li NN (1992) In: Ho WSW, Sirkar KK (eds) Membrane handbook, 1st edn. Van Nostrand Reinhold, New York

30.Mutihac L, Mutihac RZ, Nocolace LC, Constantinescu T (1992) Rev Roum Chim 37:91

31.Mutihac L, Luca C (1991) Rev Roum Chim 36:85

32.Mutihac L, Mutihac R (1992) Acta Chim Hung 129:573

33.Lazarova Z, Peeva L (1994) Biotechnol Bioeng 43:907

34.Roberts J, Caserio M (1964) Basic principles of organic chemistry, 1st edn. Benjamin Inc, New York, 2:247

35.Scheper T, Likidis Z, Makryaleas K, Nowottry C, Schugerl K (1987) Enz Microb Technol 9:625

36.Boayadzhiev L, Attanassova I (1991) Biotech Bioeng 38:1059

37.Smith BD (1996) In: Bartsch RA, Way JD (eds) Chemical separation with liquid membrane. ACS Symposium Series: 642. American Chemical Society, Washington, DC, pp 194–205

38.Smith BD (1996) Supramol Chem 7:55

39.Smith BD, Gardiner SJ (1998) Adv Supramol Chem 5:157

40.Cascaval D, Oniscu C, Cascaval C, Romanic M (1997) Roum Biotechnol Lett 2:407

41.Sahoo GC, Dutta NN, Dass NN (2000) Chem Eng Comm (in press)

42.Habaki H, Isobe S, Ega-Shiva R, Kawasaki J (1998) J Chem Eng Jpn 31:47

43.Tsikas D, Scheper T, Schugerl K (1989) Chem Ing Tech 61:418

44.Chan CC,Yang JF (1995) Sep Sci Technol 30(15):3001

45.Thien MP, Hatton TA, Wang DIC (1988) Biotechnol Bioeng 32:604

46.Reisinger H, Marr R (1993) J Membr Sci 80:85

47.Hong S-A, Choi H-J, Nam SW (1992) J Membr Sci 70:225

48.Chaudhuri JB, Pyle DL (1992) Chem Eng Sci 47:49

49.Wildfenur EM (1985) In: Leroith O, Schiloach J, Leaby JT (eds) ACS Symposium Series, ACS, Washington DC, 77:155–174

50.Hano T, Ohtake T, Matsumoto M, Ogawa SI, Hari F (1990) J Chem Eng Jpn 23:772

51.Bora MM, Dutta NN, Bhattacharya KG (1998) J Chem Eng Data 43:318

52.Bora MM, Ghosh AC, Dutta NN, Mathur RK (1997) Canad J Chem Eng 75:520

53.Lee SC, Lee WK (1992) J Chem Tech Biotechnol 53:251

54.Lee KH, Lee SC, Lee WK (1994) J Chem Tech Biotech 59:365

55.Lee KH, Lee SC, Lee WK (1994) J Chem Tech Biotech 59:371

56.Ghosh AC, Borthakur S, Roy MK, Dutta NN (1995) Sep Technol 5:121

57.Marchese J, Lopez L, Quin JA (1989) J Chem Tech Biotech 46:149

58.Lee JC,Yeh JH,Yang WJ, Kan RC (1993) Biotech Bioeng 42:527

59.Lee JC,Yeh JH, Kan RC (1994) Biotech Bioeng 43:309

60.Tsikas D, Kaltsidon SE, Brunner G (1992) Chem Ing Tech 64:545

61.Basu R, Sirkar KK (1991) AIChE J 37:383

62.Yang FZ, Rindfleisch D, Scheper T, Schuregl K (1993) 3rd International Conference on Effective Membrane Processes, New Perspectives, Bath, UK, 12–14 May

63.Prasad R, Sirkar KK (1989) J Membr Sci 47:235

64.Basu R, Sirkar KK (1992) Solvent Extr Ion Exch 10:119

65.Dahuron L, Cussler EL (1988) AIChE J 37:383

66.Dekker M, Riet K Van’t, Wijmans JMGM, Baltussen JWA (1987) 1st International Congress Membrane and Membrane Processes, Tokyo

67.Prasad R, Sirkar KK (1990) J Membr Sci 50:153

68.Casamatta G, Boyadzhiev L, Angelio H (1994) Chem Eng Sci 29:2005

69.Teramoto M, Matsuyama H, Nakai K, Uesaka T, Ohnishi N (1994) J Membr Sci 91:203

70.Chaudhuri JB, Pyle DL (1992) Chem Eng Sci 47:41

71.Mok YS, Lee WK (1995) Membrane J 5:44

72.Calderback PH, Moo-Young MB (1961) Chem Eng Sci 16:39

73.Wilke CR, Chang P (1955) AIChEJ 264–266

74.Jefferson TB, Witzell OW, Libbit WL (1958) Ind Eng Chem 50:1518

75.Gu ZM, Wasan DT, Li NN (1985) Sep Sci Tech 20:599

76.Fuji T, Matsumoto K, Watanabe T (1976) Proc Biochem 11:12

77.Eyal AM, Bressler E (1993) Biotech Bioeng 41:287

78.Li NN (1973) US Pat. 3,779,907

79.Friensen DI, Babcock WC, Brose DJ, Chambers AR (1991) J Membr Sci 56:127

80.Bhakta A, Ruckenstein E (1995) Langmuir 11:4642

81.Szabo C (1992) Biotechnol Bioeng 4:247

82.Draxler J, Marr R (1986) Chem Eng Process 20:319

83.Li W, Dai X, Shi Y (1992) Proc Int. Solvent Extraction Conf., Kyoto, Japan, Part B, p 1655

84.Sirkar KK (1991) US Pat. 4,921,612

85.Sirkar KK (1991) US Pat. 4,997,569

86.Kemperman AJB, Bergeman D, Van Den BT, Strathmann H (1996) Sep Sci Technol 31(20):2733

87.Neplenbrock AM, Bergeman D, Smolders CA (1992) J Membr Sci 67:149

88.Zhang X-J, Huang P-Y, Chen X-J (1988) Proc First Sino-Jap Symp Liq Membr 24–27 Sept, Chona, pp 83–85

89.Kato S,Kawasaki J (1988) Proc First Sino-Jap Symp Liq Membr 24–27 September,Chona, pp 86–88

90.Lu G, Lu Q, Li P (1997) J Membr Sci 128:1

91.Sun D, Duan X,Yanling X, Zhang LT, Zhou D (1996) Harbin Gongye Daxue Xuebao 28:68

92.Ghosh AC, Bora MM, Dutta NN (1996) Bioseparation 6:91

93.Behr JP (1973) J Am Chem Soc 95:6108

94.Lehn JM (1975) J Am Chem Soc 97:2532

95.Itosh H, Thien MP, Hatton TA, Wang DIC (1990) Biotech Bioeng 35:853

96.Boydzhiev L, Atannossova I (1994) Proc Biochem 29:237

97.Hano T, Matsumoto M, Kawozu T, Ohtake T (1995) J Chem Tech Biotech 62:66

98.Yamaguchi T, Nishimura K, Shinbo T, Seigura M (1985) Chem Lett 10:1552

99.Bryjak M, Weiczorek P, Kafarski P, Leiczark B (1988) J Membr Sci 37:287

100.Wieczorek P, Kocorek A, Bryjak M, Kafarski P, Lejczak B (1993) J Membr Sci 78:83

Received: March 2000