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Concentration and desalting by membrane processes of a natural pigment produced by the marine diatom Haslea ostrearia Simonsen
Journal of Biotechnology 70 (1999) 393 – 402
Concentration and desalting by membrane processes of a natural pigment produced by the marine diatom Haslea ostrearia Simonsen L. Vandanjon a,*, P. Jaouen b, N. Rossignol b, F. Que´me´neur b, J.-M. Robert c b
a Laboratoire Polyme`res et Proce´de´s (L2P), Uni6ersite´ de Bretagne Sud, 4 rue Jean Zay, F-56325 Lorient ce´dex, France Laboratoire de Ge´nie des Proce´de´s (LGP), Institut des Substances et Organismes de la Mer (ISOMer), Boule6ard de l’Uni6ersite´, BP 406, F-44602 Saint-Nazaire ce´dex, France c Laboratoire de Biologie Marine (LBM), Institut des Substances et Organismes de la Mer (ISOMer), Rue de la Houssinie`re-BP 92208, F-44322 Nantes ce´dex 3, France
Received 9 October 1998; received in revised form 24 November 1998; accepted 22 December 1998
Abstract The marine microalga Haslea ostrearia, also called blue navicula , presents the unique peculiar property among the diatoms, to produce at its extremities a blue hydrosoluble pigment called marennine . It is presented the concentration and the desalting of the exocellular pigment by membrane processes (ultrafiltration, nanofiltration, reverse osmosis). Nanofiltration is particularly developed given the potential of this type of application both for the concentration of molecules and for desalting. It is shown the effect of velocity and pressure on performances of nanofiltration membranes. Permeation flux superior to 100 l h − 1 m − 2 (at 14.105 Pa) are obtained with the Kiryat Weizmann membrane MP 20 (polyester coated with a polyacrylonitrile layer, cut-off 450 Da). For the desalting of the blue pigment solution, nanofiltration membranes present a few advantages: a low salt rejection (less than 10% at 14.105 Pa) and a high pigment rejection (the nanofiltration membrane MP 20 retains more than 95% of the pigment). This membrane used in diafiltration mode allows an acceptable speed of desalting (700 g of salt eliminated per hour and per m2 at 25.105 Pa for a concentration of 18 g of salt per litre of solution). © 1999 Elsevier Science B.V. All rights reserved. Keywords: Concentration; Desalting; Haslea ostrearia; Nanofiltration; Pigment
1. Introduction * Corresponding author. Tel.: +33-2-97874531; fax: + 332-97874588. E-mail addresses: [email protected] (L. Vandanjon), [email protected] (P. Jaouen), [email protected] (J.-M. Robert)
The aim of the study is to develop the industrial application of the marine diatom Haslea ostrearia Simonsen, also called blue navicula , responsible for the greening of the oysters in France. This microalga presents the unique peculiar prop-
0168-1656/99/$ - see front matter © 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 8 - 1 6 5 6 ( 9 9 ) 0 0 0 9 2 - 9
394
L. Vandanjon et al. / Journal of Biotechnology 70 (1999) 393–402
erty among the diatoms, to produce at its extremities a blue hydrosoluble pigment called marennine (Robert, 1983). Ultrastructurally, the diatom shows marked changes depending on the stage of cell blueing by accumulation of marennine (Nassiri et al., 1998). The interest of this pigment is notably linked to: its colouring properties: the pigment might be used as a new blue natural dye in the food and agriculture fields, its ability for the greening of oysters: the only current industrial application is at the SOPROMA Company in Bouin (Vende´e, Atlantic coast, France), where the production of the diatom is performed in batch cultures, using tanks of 6 m3 in volume under carefully-controlled incubation conditions, its antiproliferative properties since aqueous extracts of the pigment show inhibitory effects both in vitro and in vivo against solid carcinoma lines (Belt et al., 1997) In this study, it is presented the concentration and the desalting of the exocellular pigment by membrane processes: performances of ultrafiltration, nanofiltration, reverse osmosis membranes are compared. Nanofiltration is particularly developed given the potential of this type of application both for the concentration of molecules and for desalting.
250 ml erlenmeyer flasks filled with 150 ml of ES 1/3 medium (first preculture). The temperature of incubation was 16°C and the light intensity was 3 × 1016 quanta cm − 2 s − 1 with a 14/10 h light/ dark cycle. The erlenmeyer flask contents were then inoculated into 25 l flasks containing 20 l of ES 1/3 medium (second preculture). After incubation of these larger flasks for ca 7 to 10 days in identical temperature and light conditions, their contents were transfered into 500 l tanks filled with 400 l underground sea-water from the Bouin district (Rouillard, 1996). In these natural incubation conditions, cell suspensions with concentration ranging between 60 and 100× 103 cells ml − 1 were obtained after incubation for 5–12 days. The algal mass collected by centrifugation, with a continuous centrifuger CEPA-PATBERG, LE (clarification cylinder type K, inox), was then collected and the blue coloured medium containing the exocellular marennine was recuperated for concentration and desalting by membrane processes.
2.2. Experimental set-up and membranes The experiments are carried out on two pilote plants: Millipore (Prolab Bench Top) and Gamma Filtration (Microlab 80S) built on the same principle (Fig. 1). The both pilote plants are constituted from a centrifugal pump allowing liquid recirculation and
2. Materials and methods
2.1. Biological material The study was performed using an axenic strain of H. ostrearia isolated in the Marine Biology Laboratory from the oyster-pond waters of the Bouin district (Vende´e, France). The clone cells were characterized by an average model length of 65 mm. The algal cultures were maintained by weekly transfer to fresh ES 1/3 medium (Robert, 1983; Lebeau et al., 1999). Algal precultures were precultivated by applying a two steps procedure, before to be used for mass production of the diatom. Cells from the clone pool were first precultured for ca 7 days in
Fig. 1. Schematic representation of the pilote plant.
L. Vandanjon et al. / Journal of Biotechnology 70 (1999) 393–402
395
Table 1 Characteristics of membranes used Trade Mark
Manufacturer
Geometry
Technique
Cut-off
Area (m2)
Material
Iris 3028
Tech-Sep, Ore´lis Millipore Kiryat Weizmann Kiryat Weizmann
Flat
UF
3 kDa
6.9×10−3
Polyethersulfone
Spiral Tubular
RO NF
B200 Da 450 Da
0.3 47×10−3
Tubular
NF
450 Da
47×10−3
Polyamide Polyester coated with a polyacrylonitrile layer Polysulfone on polypropylene
R 45P MPT 20 MPT 31
a volumetric pump assuming the feeding of the recirculation loop. In this way, pressure and tangential velocity adjustments are independent. Transmembrane pressure is measured upstream and downstream of the permeation module by manometers. A pressure of 50×105 Pa can be reached in the Millipore installation and 45× 105 Pa in the Gamma Filtration installation. A freezing group Mouvex RFA-30 allows to maintain a constant temperature of 25°C. The filtration module is either flat or tubular. Four membranes are tested: one ultrafiltration (UF) membrane, one loose reverse osmosis (RO) membrane and two nanofiltration (NF) membranes. These membranes and their characteristics are presented in the Table 1. The UF and NF experiments are carried out by total recycling of the retentate and the permeate. Steady flux and retention rates of the membranes are measured after 120 min. Concentration of the blue pigment solution is carried out with a loose RO membrane. Working pressure is 41×105 Pa and temperature is kept constant at 25°C. The concentrated solution is desalted by diafiltration using the same loose RO membrane in a continuous mode (Fig. 2).
The concentration of pigment in solution is determinated by spectrophotometry (Secomam SG 1000). Optical density is measured at 663 nm, which corresponds to the maximum absorbance of the marennine (Robert and Hallet, 1981). Salinity is determinated by measuring resistivity (Tacussel CD 60) of the marennine solution.
3. Results—discussion The pigment marennine seems to be composed of a mix of macromolecules of different sizes. Size repartition of pigment molecules has been estimated by using six UF membranes (bearing negligible adsorption rate) of different molecular cut-off comprised between 1 and 300 kDa (Vandanjon, 1997). By relying on the model proposed by Ferry (1936), we have shown that a large part of the pigment has a molecular weight comprised between 3 and 7 kDa (Fig. 3). So the ultrafiltration (low cut-off) could be convenient a technique for the concentration and the desalting of marennine solution. High purity molecules can be obtained by using multistage diafiltration (Muller, 1996). However, the existence of a small fraction of pigment whose molecular weight is close to hundreds Dalton involves the use of nanofiltration or reverse osmosis membranes for the recovery of the totality of the marennine.
3.1. Performance of ultrafiltration membrane (low cut-off ) Fig. 2. Diafiltration in a continuous mode.
The operating conditions are as following:
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Fig. 3. Molecular weight repartition of marennine molecules.
Pressure: 4×105 Pa Tangential velocity: 1 m s − 1 Results are presented on Fig. 4. The ultrafiltration membrane 3028 3 kDa presents a high rejection percentage for the pigment (90%) and a low rejection percentage for the salts. This membrane could be convenient for the desalting of pigment solution by diafiltration.
3.2. Performance of loose re6erse osmosis membrane 3.2.1. Concentration of the pigment solution In order to obtain a high volumic reduction factor (VRF), a volume (V0) of 350 l of culture medium containing the hydrosoluble pigment is concentrated. The blue water (culture medium) presents the following characteristics: Optical density (at 663 nm): 0.024 (length of the optical path=1 cm) Salt concentration: 30 g l − 1 pH 8 Concentration is achieved with the loose reverse osmosis membrane Millipore R45P. Fig. 5 presents the evolution of the permeate flux and the concentration factor (CF) of pigment and salts.
3.2.2. Comments Steady flux of 34 l m − 2 h − 1 is obtained after 26 h. Pigment is highly concentrated while salt concentration is constant. There is a gap between the VRF (VRF= 70) and the CF of pigment (CF=50). It can be explained by the loose of pigment in the permeate and the fixation of pigment on the membrane separators.
Fig. 4. Evolution of permeate flow and rejection percentage for an ultrafiltration membrane (cut-off 3 kDa).
L. Vandanjon et al. / Journal of Biotechnology 70 (1999) 393–402
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Fig. 5. Concentration of marennine with a loose reverse osmosis membrane.
3.2.3. Continuous diafiltration Diafiltration with the membrane R45P is achieved at a constant volume (5 l) and a constant pressure (10× 105 Pa). Fig. 6 presents the evolution of the salt concentration and the rejection rates for salts and pigment in function of time and added pure water volume. It can be noticed that the salts are not totally eliminated after 13 h of diafiltration. Experimental curves are compared to the theoritical model developed in diafiltration (Mulder, 1991). After integrating the overall mass balance sheet: dC −V0 · =Qv · Cp dt
(1)
the following equation is obtained: C =C0 · exp[− Qv · (1 −TR/100) · t/V0]
(2)
with: t, filtration time (s); C, salt concentration in the coloured water at the time t (kg m − 3); C0, salt concentration in the coloured water at the initial time (kg m − 3); Cp, salt concentration in the permeate (kg m − 3); Qv, volumetric flow of permeate
(m3 s − 1); TR(%) = 100 · [1–(Cp/C0)], salt rejection rate; and V0, initial volume of blue water (m3). If the salt rejection rate is constant with time, diafiltration experimental curves are close to the theoritical model. In the present case, the gap between theoritical curve and experimental curve is due to a salt rejection rate which is not perfectly constant (evolution of the osmotic pressure during diafiltration, adsorption phenomenom). However, it is possible to estimate the number of diavolumes (DV) necessary for desalting the solution with the relation (Tutunjian, 1985): C ln f C0 DV= TR −1 100
(3)
with Cf, salt concentration in the coloured water at the end time (kg m − 3). With the loose reverse osmosis membrane (salt rejection rate : 60%), it is possible to know the number of diavolumes for desalting from 15 to 1 g l − 1:
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DV =ln(1/15)/(60 − 100) · 100 =6.8 On the experimental curve (Fig. 6), it is necessary to add 35 l of pure water to 5 l of blue water for desalting the solution, i.e: DV= 35/ 5 = 7. These results confirm that the model is correct whenever the salt rejection rate is constant.
3.3. Performance of nanofiltration membranes 3.3.1. Permeation flux A few sets of experiments by total recycling of the retentate and the permeate are carried out at different pressures and velocities. For each couple pressure-velocity, the stabilization of the flux is reached after about 2 h. These points allow to draw steady flux versus pressure at different tangential velocities. Results
are presented on Fig. 7 (membranes MPT 20 and MPT 31). With the low pressures (DP B 105 Pa), filtrate flux increases linearly and does not depend from the recirculation velocity (Darcy’s law). Then flux increases slowly (primary polarization), but it is noticeable that it is strongly influenced by recirculation velocity (Brun, 1989). For these nanofiltration membranes, it can be considered that beyond 20×105 –25×105 Pa, increase of the pressure has no more significant influence on the filtrate flux. However, fluxes superior to 50 l m − 2 h − 1 at 30× 105 Pa can be considered as acceptable performances. The ultrafiltrate fluxes versus tangential velocity are often represented according to the relation (Que´me´neur and Schlumpf, 1980): J= a · u n
Fig. 6. Diafiltration of marennine solution with a loose reverse osmosis membrane.
(4)
L. Vandanjon et al. / Journal of Biotechnology 70 (1999) 393–402
399
Fig. 7. Steady flux versus pressure for nanofiltration membranes.
with: J, ultrafiltrate flux (m3 m − 2 s − 1); a, n, constants; and u, fluid velocity (m s − 1). In order to verify the validity of the Eq. (4) in nanofiltration, it is drawn on Fig. 8 the logarithmic permeate flux versus the logarithmic tangential velocity under a pressure of 25× 105 Pa. For each membrane, a straight line is obtained
Fig. 8. Experimental validation of the relation J = a · u n in nanofiltration. Graphic determination of the exponent n.
Fig. 9. Evolution of salt and pigment rejections with the pressure.
whose equation follows: ln J=ln a+n · ln u
(5)
In turbulent flow, the following values of the slope n can be determined graphically: Membrane MPT 20: n= 0.13 Membrane MPT 31: n= 0.18 These values are inferior to the most common values of the literature: n= 0.69–0.91 (Blatt et al., 1970; Zaitoun, 1979; Aimar, 1992). Generally, the coefficient n depends on hydrodynamic characteristics of the module geometry (flat, tubular, spiral etc.) and of the couple membrane-foulant (Gekas and Hallstro¨m, 1987; Nabetani et al., 1990). But the very low concentration of natural pigment and the high salinity of the marennine solution may be responsible for these results.
3.3.2. Selecti6ity of nanofiltration membranes From a practical or economical point of view, it is interesting to carry out desalting by diafiltration after concentration without any changing of material (pilot-plant and membrane). The ideal membrane should retain the maximum of pigment and be as permeable as possible for the salts. Fig. 9 describes performance, in term of salt and pigment rejection, of two nanofiltration membranes MPT 20 and MPT 31.
L. Vandanjon et al. / Journal of Biotechnology 70 (1999) 393–402
400
The membrane MPT 20 retains the fast totality of the pigment and its permeability for the salts remains high at high pressure. Rejection rate (TR) seems to be independent of the pressure, it would correspond to a mass transfer mechanism intermediate between capillar type mechanism (TR decreases with pressure) and diffusional type mechanism (TR increases with pressure). On the contrary, the nanofiltration membrane MPT 31 presents a rejection rate that increases with the applied pressure. This behaviour is characteristic of a diffusional type mechanism: B TR(%)=100 · 1− (6) A · (DP − Dp) with: A, solvent permeability of the membrane; and B, solute (salts) permeability of the membrane. With this type of nanofiltration membrane, close to reverse osmosis membranes, an increase of the pressure induces an increase of the permeate flux and the salt rejection rate; concurrently, it is observed a light decrease of the pigment rejection. That means that high permeate flows during concentration will induce loss of pigment and will penalize the next step of desalting. So it will be necessary to find a compromise in the aim to optimize the overall process of concentrationdesalting.
The most efficient membrane for desalting is the ultrafiltration membrane 3028 3 kDa. Salts can easily pass through the membrane but the low pigment retention makes the membrane unusable for a diafiltration. At the opposite, the membrane R45P retains perfectly the pigment but its high salt rejection induces a long and difficult desalting. The nanofiltration membrane MPT 20 constitutes the best compromise in term of desalting and pigment rejection and it may be used efficiently in diafiltration mode.
4. Conclusion Nanofiltration seems to be the best performing technique for desalting the blue coloured solution. The model used in diafiltration with a loose reverse osmosis membrane may be used with nanofiltration membranes, whether salt rejection rate is about constant. In these conditions, it is possible to calculate, from laboratory scale experiments, performances of pilot-plant or industrial process. In this study, we have shown the effect of velocity and pressure on performance of nanofiltration membranes. Flows superior to 50 l m − 2 h − 1 (at 30× 105 Pa) are obtained with the Weizmann membrane MPT 20 (in polyacryloni-
3.4. Comparison of membranes for diafiltration Diafiltration is carried out at a constant volume: V0 =5 l. Three membranes are tested functionning in diafiltration mode: an ultrafiltration membrane Ore´lis – Iris 3028 3 kDa at DP =4×105 Pa, a nanofiltration membrane Kyriat Weizmann MPT 20 at DP = 25 ×105 Pa, a loose reverse osmosis membrane Millipore R45P at DP = 10 ×105 Pa. With the aim to evaluate the efficiency of the three membranes (ultrafiltration, nanofiltration and reverse osmosis) to carry out a diafiltration, it has been drawn on Fig. 10 the graphic of desalting speed versus salts concentration in the retentate.
Fig. 10. Compared efficiency of the membranes for the elimination of the salts.
L. Vandanjon et al. / Journal of Biotechnology 70 (1999) 393–402
trile coated on polyester, cut-off 450 Da). Higher fluxes could be obtained by optimizing hydrodynamic conditions or the couple tangential velocity/pressure. For the desalting of the blue – green pigment solution, nanofiltration membranes present a few advantages over loose reverse osmosis and ultrafiltration: a low salt rejection rate (less than 10% at 14 bars) and a high pigment rejection (the nanofiltration membrane MPT 20 retains the fast totality of the pigment). This membrane used in diafiltration mode allows to obtain an acceptable speed of desalting (for example, 700 g of salt eliminated per hour and per m2 at 25 × 105 Pa for a concentration of 18 g of salt by litre of solution). So the nanofiltration membrane MPT 20 is interesting in the aim of a simultaneous concentration-desalting (without changing of processunit or membrane) of the marennine solution produced by H. ostrearia. The diafiltration does not allow to eliminate entirely the salts, but it does not constitute an obstacle in the aim of food enhancement of the marennine. Indeed, legislation makes difficult the commercialization of new natural dyestuffs (Labatut and In, 1990). In these conditions, non purified marennine should preferently be enhanced as a food ingredient. However, a high degree of purity is necessary for the use of marennine for therapeutical application in cancerology for example (Riou et al., 1993). The dialysis technique is then well suited for the final elimination of the salts when using small volumes of concentrated pigment solution. One further development of this study is the membrane photobioreactor which combines biological reaction and separation. Biological reaction: the research work consists in optimizing culture conditions of H. ostrearia for the highest pigment production: choice of the clones, study of the shear effects caused by pumps and valves in a recirculating loop (Vandanjon et al., 1999), addition of nutriments. Separation: for the extraction of pigment, two stages of membranes are necessary. Ultrafiltration (cut-off 40 kDa) can be used for the extraction of the exometabolite from the culture in a continuous mode (Rossignol et al., 1999). Then after-
401
wards, the marennine solution is concentrated and desalted by nanofiltration. References Aimar, P., 1992. Limiting flux in membrane separations: a model based on the viscosity dependency of the mass transfer coefficient. Chem. Eng. Sci. 47 (3), 579 – 586. Belt S.T., Robert J.-M., Roussakis C., Rowland S., 1997. Chemotherapeutic compounds from microalgae. Patent n°GB97-08936-6, ISOMer (University of Nantes, Nantes, France) and University of Plymouth, Plymouth, UK. Blatt, W.F., Dravid, A., Michaels, A.S., Nelsen, L., 1970. Solute polarization and cake formation in membrane ultrafiltration. In: Membrane Science and Technology, vol. 47. Plenum Press, New York. Brun J.-P., 1989. Proce´de´s de se´paration par membranes. Masson, Paris, 270 p, ISBN 2-225-81573-9. Ferry, J.D., 1936. Ultrafilter membranes and ultrafiltration. Chem. Rev. 18, 373 – 455. Gekas, V., Hallstro¨m, B., 1987. Critical literature review and adaptation of existing Sherwood correlations to membrane operations. J. Membr. Sci. 30, 153 – 170. Labatut, M.-L., In, T., 1990. La couleur au naturel. Biofutur November, 37 – 42. Lebeau, T., Junter, G.-A., Jouenne, T., Robert, J.-M., 1999. Marennine production by agar-entrapped Haslea ostrearia Simonsen. Biores. Technol. 67 (1), 13 – 17. Mulder, M., 1991. Basic Principles of Membrane Technology. Kluwer, Dordrecht. Muller A., 1996. Proce´de´ d’obtention d%a-lactalbumine de haute purete´: e´tapes e´le´mentaires du fractionnement des prote´ines de lactose´rum et mise en cascade. Ph.D. thesis, ENSAR Rennes, France. Nabetani, H., Nakajima, M., Watanabe, A., Nakao, S.I., Kimura, S., 1990. Effect of osmotic pressure ans adsorption on ultrafiltration of ovalbumine. AIChE J. 36 (6), 907 – 915. Nassiri, Y., Robert, J.-M., Rince´, Y., Ginsburger-Vogel, T., 1998. The cytoplasmic fine structure of the diatom Haslea ostrearia (Bacillariophyceae) in relation to marennine production. Phycologia 37 (2), 84 – 91. Que´me´neur, F., Schlumpf, J.-P., 1980. Traitement des huiles solubles par ultrafiltration. Entropie 93, 22 – 29. Riou, D., Roussakis, C., Biard, J.-F., Verbist, J.-F., 1993. Comparative study of the antitumour activity of bistramides A, D and K against a non-small cell broncho-pulmonary carcinoma. Anticancer Res. 13, 2331 – 2334. Robert J.-M., 1983. Fertilite´ des eaux de claires ostre´icoles et verdissement: utilisation de l’azote par les diatome´es dominantes. Ph.D. thesis, Nantes University, Nantes, France, 281 p. Robert, J.-M., Hallet, J.-N., 1981. Absorption spectrum in vivo of the blue pigment marennine of the pennate diatom Na6icula ostrearia Bory. J. Exp. Botany 32 (127), 341 – 345.
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L. Vandanjon et al. / Journal of Biotechnology 70 (1999) 393–402 Vandanjon L., 1997. Etude d’un proce´de´ de valorisation d’une microalgue marine: concentration et purification par techniques a` membranes d’un pigment naturel produit par la diatome´e Haslea ostrearia. Ph.D. thesis, Nantes University, Nantes, France, 257 p. Vandanjon, L., Rossignol, N., Jaouen, P., Robert, J.-M., Que´me´neur, F., 1999. Effects of shear on two microalgae species. Contribution of pumps and valves in tangential flow filtration systems. Biotechnol. Bioeng. 63 (1), 1 – 9. Zaitoun A., 1979. Osmose inverse et ultrafiltration en milieu organique. Equations de transport. Application a` l’ultrafiltration des huiles moteur. Ph.D. thesis, INP Lorraine, France.
Rossignol N., Vandanjon L., Jaouen P., Que´me´neur F., 1999. Membrane technology for microalgae harvesting: compared performances of cross-flow microfiltration and ultrafiltration. Aquacult. Eng. (submitted for publication). Rouillard I., 1996. Optimisation de la production en masse de Haslea ostrearia Simonsen sur une eau souterraine sale´e: importance de la souche et des conditions de culture; comparaison avec Skeletonema costatum (Grev) Cleve. Ph.D. thesis, Nantes University, Nantes, France, 270 p. Tutunjian, R.S., 1985. Scale-up considerations for membrane processes. Biotechnology 3, 615–626.
.
Concentration and desalting by membrane processes of a natural pigment produced by the marine diatom Haslea ostrearia Simonsen L. Vandanjon a,*, P. Jaouen b, N. Rossignol b, F. Que´me´neur b, J.-M. Robert c b
a Laboratoire Polyme`res et Proce´de´s (L2P), Uni6ersite´ de Bretagne Sud, 4 rue Jean Zay, F-56325 Lorient ce´dex, France Laboratoire de Ge´nie des Proce´de´s (LGP), Institut des Substances et Organismes de la Mer (ISOMer), Boule6ard de l’Uni6ersite´, BP 406, F-44602 Saint-Nazaire ce´dex, France c Laboratoire de Biologie Marine (LBM), Institut des Substances et Organismes de la Mer (ISOMer), Rue de la Houssinie`re-BP 92208, F-44322 Nantes ce´dex 3, France
Received 9 October 1998; received in revised form 24 November 1998; accepted 22 December 1998
Abstract The marine microalga Haslea ostrearia, also called blue navicula , presents the unique peculiar property among the diatoms, to produce at its extremities a blue hydrosoluble pigment called marennine . It is presented the concentration and the desalting of the exocellular pigment by membrane processes (ultrafiltration, nanofiltration, reverse osmosis). Nanofiltration is particularly developed given the potential of this type of application both for the concentration of molecules and for desalting. It is shown the effect of velocity and pressure on performances of nanofiltration membranes. Permeation flux superior to 100 l h − 1 m − 2 (at 14.105 Pa) are obtained with the Kiryat Weizmann membrane MP 20 (polyester coated with a polyacrylonitrile layer, cut-off 450 Da). For the desalting of the blue pigment solution, nanofiltration membranes present a few advantages: a low salt rejection (less than 10% at 14.105 Pa) and a high pigment rejection (the nanofiltration membrane MP 20 retains more than 95% of the pigment). This membrane used in diafiltration mode allows an acceptable speed of desalting (700 g of salt eliminated per hour and per m2 at 25.105 Pa for a concentration of 18 g of salt per litre of solution). © 1999 Elsevier Science B.V. All rights reserved. Keywords: Concentration; Desalting; Haslea ostrearia; Nanofiltration; Pigment
1. Introduction * Corresponding author. Tel.: +33-2-97874531; fax: + 332-97874588. E-mail addresses: [email protected] (L. Vandanjon), [email protected] (P. Jaouen), [email protected] (J.-M. Robert)
The aim of the study is to develop the industrial application of the marine diatom Haslea ostrearia Simonsen, also called blue navicula , responsible for the greening of the oysters in France. This microalga presents the unique peculiar prop-
0168-1656/99/$ - see front matter © 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 8 - 1 6 5 6 ( 9 9 ) 0 0 0 9 2 - 9
394
L. Vandanjon et al. / Journal of Biotechnology 70 (1999) 393–402
erty among the diatoms, to produce at its extremities a blue hydrosoluble pigment called marennine (Robert, 1983). Ultrastructurally, the diatom shows marked changes depending on the stage of cell blueing by accumulation of marennine (Nassiri et al., 1998). The interest of this pigment is notably linked to: its colouring properties: the pigment might be used as a new blue natural dye in the food and agriculture fields, its ability for the greening of oysters: the only current industrial application is at the SOPROMA Company in Bouin (Vende´e, Atlantic coast, France), where the production of the diatom is performed in batch cultures, using tanks of 6 m3 in volume under carefully-controlled incubation conditions, its antiproliferative properties since aqueous extracts of the pigment show inhibitory effects both in vitro and in vivo against solid carcinoma lines (Belt et al., 1997) In this study, it is presented the concentration and the desalting of the exocellular pigment by membrane processes: performances of ultrafiltration, nanofiltration, reverse osmosis membranes are compared. Nanofiltration is particularly developed given the potential of this type of application both for the concentration of molecules and for desalting.
250 ml erlenmeyer flasks filled with 150 ml of ES 1/3 medium (first preculture). The temperature of incubation was 16°C and the light intensity was 3 × 1016 quanta cm − 2 s − 1 with a 14/10 h light/ dark cycle. The erlenmeyer flask contents were then inoculated into 25 l flasks containing 20 l of ES 1/3 medium (second preculture). After incubation of these larger flasks for ca 7 to 10 days in identical temperature and light conditions, their contents were transfered into 500 l tanks filled with 400 l underground sea-water from the Bouin district (Rouillard, 1996). In these natural incubation conditions, cell suspensions with concentration ranging between 60 and 100× 103 cells ml − 1 were obtained after incubation for 5–12 days. The algal mass collected by centrifugation, with a continuous centrifuger CEPA-PATBERG, LE (clarification cylinder type K, inox), was then collected and the blue coloured medium containing the exocellular marennine was recuperated for concentration and desalting by membrane processes.
2.2. Experimental set-up and membranes The experiments are carried out on two pilote plants: Millipore (Prolab Bench Top) and Gamma Filtration (Microlab 80S) built on the same principle (Fig. 1). The both pilote plants are constituted from a centrifugal pump allowing liquid recirculation and
2. Materials and methods
2.1. Biological material The study was performed using an axenic strain of H. ostrearia isolated in the Marine Biology Laboratory from the oyster-pond waters of the Bouin district (Vende´e, France). The clone cells were characterized by an average model length of 65 mm. The algal cultures were maintained by weekly transfer to fresh ES 1/3 medium (Robert, 1983; Lebeau et al., 1999). Algal precultures were precultivated by applying a two steps procedure, before to be used for mass production of the diatom. Cells from the clone pool were first precultured for ca 7 days in
Fig. 1. Schematic representation of the pilote plant.
L. Vandanjon et al. / Journal of Biotechnology 70 (1999) 393–402
395
Table 1 Characteristics of membranes used Trade Mark
Manufacturer
Geometry
Technique
Cut-off
Area (m2)
Material
Iris 3028
Tech-Sep, Ore´lis Millipore Kiryat Weizmann Kiryat Weizmann
Flat
UF
3 kDa
6.9×10−3
Polyethersulfone
Spiral Tubular
RO NF
B200 Da 450 Da
0.3 47×10−3
Tubular
NF
450 Da
47×10−3
Polyamide Polyester coated with a polyacrylonitrile layer Polysulfone on polypropylene
R 45P MPT 20 MPT 31
a volumetric pump assuming the feeding of the recirculation loop. In this way, pressure and tangential velocity adjustments are independent. Transmembrane pressure is measured upstream and downstream of the permeation module by manometers. A pressure of 50×105 Pa can be reached in the Millipore installation and 45× 105 Pa in the Gamma Filtration installation. A freezing group Mouvex RFA-30 allows to maintain a constant temperature of 25°C. The filtration module is either flat or tubular. Four membranes are tested: one ultrafiltration (UF) membrane, one loose reverse osmosis (RO) membrane and two nanofiltration (NF) membranes. These membranes and their characteristics are presented in the Table 1. The UF and NF experiments are carried out by total recycling of the retentate and the permeate. Steady flux and retention rates of the membranes are measured after 120 min. Concentration of the blue pigment solution is carried out with a loose RO membrane. Working pressure is 41×105 Pa and temperature is kept constant at 25°C. The concentrated solution is desalted by diafiltration using the same loose RO membrane in a continuous mode (Fig. 2).
The concentration of pigment in solution is determinated by spectrophotometry (Secomam SG 1000). Optical density is measured at 663 nm, which corresponds to the maximum absorbance of the marennine (Robert and Hallet, 1981). Salinity is determinated by measuring resistivity (Tacussel CD 60) of the marennine solution.
3. Results—discussion The pigment marennine seems to be composed of a mix of macromolecules of different sizes. Size repartition of pigment molecules has been estimated by using six UF membranes (bearing negligible adsorption rate) of different molecular cut-off comprised between 1 and 300 kDa (Vandanjon, 1997). By relying on the model proposed by Ferry (1936), we have shown that a large part of the pigment has a molecular weight comprised between 3 and 7 kDa (Fig. 3). So the ultrafiltration (low cut-off) could be convenient a technique for the concentration and the desalting of marennine solution. High purity molecules can be obtained by using multistage diafiltration (Muller, 1996). However, the existence of a small fraction of pigment whose molecular weight is close to hundreds Dalton involves the use of nanofiltration or reverse osmosis membranes for the recovery of the totality of the marennine.
3.1. Performance of ultrafiltration membrane (low cut-off ) Fig. 2. Diafiltration in a continuous mode.
The operating conditions are as following:
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Fig. 3. Molecular weight repartition of marennine molecules.
Pressure: 4×105 Pa Tangential velocity: 1 m s − 1 Results are presented on Fig. 4. The ultrafiltration membrane 3028 3 kDa presents a high rejection percentage for the pigment (90%) and a low rejection percentage for the salts. This membrane could be convenient for the desalting of pigment solution by diafiltration.
3.2. Performance of loose re6erse osmosis membrane 3.2.1. Concentration of the pigment solution In order to obtain a high volumic reduction factor (VRF), a volume (V0) of 350 l of culture medium containing the hydrosoluble pigment is concentrated. The blue water (culture medium) presents the following characteristics: Optical density (at 663 nm): 0.024 (length of the optical path=1 cm) Salt concentration: 30 g l − 1 pH 8 Concentration is achieved with the loose reverse osmosis membrane Millipore R45P. Fig. 5 presents the evolution of the permeate flux and the concentration factor (CF) of pigment and salts.
3.2.2. Comments Steady flux of 34 l m − 2 h − 1 is obtained after 26 h. Pigment is highly concentrated while salt concentration is constant. There is a gap between the VRF (VRF= 70) and the CF of pigment (CF=50). It can be explained by the loose of pigment in the permeate and the fixation of pigment on the membrane separators.
Fig. 4. Evolution of permeate flow and rejection percentage for an ultrafiltration membrane (cut-off 3 kDa).
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Fig. 5. Concentration of marennine with a loose reverse osmosis membrane.
3.2.3. Continuous diafiltration Diafiltration with the membrane R45P is achieved at a constant volume (5 l) and a constant pressure (10× 105 Pa). Fig. 6 presents the evolution of the salt concentration and the rejection rates for salts and pigment in function of time and added pure water volume. It can be noticed that the salts are not totally eliminated after 13 h of diafiltration. Experimental curves are compared to the theoritical model developed in diafiltration (Mulder, 1991). After integrating the overall mass balance sheet: dC −V0 · =Qv · Cp dt
(1)
the following equation is obtained: C =C0 · exp[− Qv · (1 −TR/100) · t/V0]
(2)
with: t, filtration time (s); C, salt concentration in the coloured water at the time t (kg m − 3); C0, salt concentration in the coloured water at the initial time (kg m − 3); Cp, salt concentration in the permeate (kg m − 3); Qv, volumetric flow of permeate
(m3 s − 1); TR(%) = 100 · [1–(Cp/C0)], salt rejection rate; and V0, initial volume of blue water (m3). If the salt rejection rate is constant with time, diafiltration experimental curves are close to the theoritical model. In the present case, the gap between theoritical curve and experimental curve is due to a salt rejection rate which is not perfectly constant (evolution of the osmotic pressure during diafiltration, adsorption phenomenom). However, it is possible to estimate the number of diavolumes (DV) necessary for desalting the solution with the relation (Tutunjian, 1985): C ln f C0 DV= TR −1 100
(3)
with Cf, salt concentration in the coloured water at the end time (kg m − 3). With the loose reverse osmosis membrane (salt rejection rate : 60%), it is possible to know the number of diavolumes for desalting from 15 to 1 g l − 1:
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DV =ln(1/15)/(60 − 100) · 100 =6.8 On the experimental curve (Fig. 6), it is necessary to add 35 l of pure water to 5 l of blue water for desalting the solution, i.e: DV= 35/ 5 = 7. These results confirm that the model is correct whenever the salt rejection rate is constant.
3.3. Performance of nanofiltration membranes 3.3.1. Permeation flux A few sets of experiments by total recycling of the retentate and the permeate are carried out at different pressures and velocities. For each couple pressure-velocity, the stabilization of the flux is reached after about 2 h. These points allow to draw steady flux versus pressure at different tangential velocities. Results
are presented on Fig. 7 (membranes MPT 20 and MPT 31). With the low pressures (DP B 105 Pa), filtrate flux increases linearly and does not depend from the recirculation velocity (Darcy’s law). Then flux increases slowly (primary polarization), but it is noticeable that it is strongly influenced by recirculation velocity (Brun, 1989). For these nanofiltration membranes, it can be considered that beyond 20×105 –25×105 Pa, increase of the pressure has no more significant influence on the filtrate flux. However, fluxes superior to 50 l m − 2 h − 1 at 30× 105 Pa can be considered as acceptable performances. The ultrafiltrate fluxes versus tangential velocity are often represented according to the relation (Que´me´neur and Schlumpf, 1980): J= a · u n
Fig. 6. Diafiltration of marennine solution with a loose reverse osmosis membrane.
(4)
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Fig. 7. Steady flux versus pressure for nanofiltration membranes.
with: J, ultrafiltrate flux (m3 m − 2 s − 1); a, n, constants; and u, fluid velocity (m s − 1). In order to verify the validity of the Eq. (4) in nanofiltration, it is drawn on Fig. 8 the logarithmic permeate flux versus the logarithmic tangential velocity under a pressure of 25× 105 Pa. For each membrane, a straight line is obtained
Fig. 8. Experimental validation of the relation J = a · u n in nanofiltration. Graphic determination of the exponent n.
Fig. 9. Evolution of salt and pigment rejections with the pressure.
whose equation follows: ln J=ln a+n · ln u
(5)
In turbulent flow, the following values of the slope n can be determined graphically: Membrane MPT 20: n= 0.13 Membrane MPT 31: n= 0.18 These values are inferior to the most common values of the literature: n= 0.69–0.91 (Blatt et al., 1970; Zaitoun, 1979; Aimar, 1992). Generally, the coefficient n depends on hydrodynamic characteristics of the module geometry (flat, tubular, spiral etc.) and of the couple membrane-foulant (Gekas and Hallstro¨m, 1987; Nabetani et al., 1990). But the very low concentration of natural pigment and the high salinity of the marennine solution may be responsible for these results.
3.3.2. Selecti6ity of nanofiltration membranes From a practical or economical point of view, it is interesting to carry out desalting by diafiltration after concentration without any changing of material (pilot-plant and membrane). The ideal membrane should retain the maximum of pigment and be as permeable as possible for the salts. Fig. 9 describes performance, in term of salt and pigment rejection, of two nanofiltration membranes MPT 20 and MPT 31.
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The membrane MPT 20 retains the fast totality of the pigment and its permeability for the salts remains high at high pressure. Rejection rate (TR) seems to be independent of the pressure, it would correspond to a mass transfer mechanism intermediate between capillar type mechanism (TR decreases with pressure) and diffusional type mechanism (TR increases with pressure). On the contrary, the nanofiltration membrane MPT 31 presents a rejection rate that increases with the applied pressure. This behaviour is characteristic of a diffusional type mechanism: B TR(%)=100 · 1− (6) A · (DP − Dp) with: A, solvent permeability of the membrane; and B, solute (salts) permeability of the membrane. With this type of nanofiltration membrane, close to reverse osmosis membranes, an increase of the pressure induces an increase of the permeate flux and the salt rejection rate; concurrently, it is observed a light decrease of the pigment rejection. That means that high permeate flows during concentration will induce loss of pigment and will penalize the next step of desalting. So it will be necessary to find a compromise in the aim to optimize the overall process of concentrationdesalting.
The most efficient membrane for desalting is the ultrafiltration membrane 3028 3 kDa. Salts can easily pass through the membrane but the low pigment retention makes the membrane unusable for a diafiltration. At the opposite, the membrane R45P retains perfectly the pigment but its high salt rejection induces a long and difficult desalting. The nanofiltration membrane MPT 20 constitutes the best compromise in term of desalting and pigment rejection and it may be used efficiently in diafiltration mode.
4. Conclusion Nanofiltration seems to be the best performing technique for desalting the blue coloured solution. The model used in diafiltration with a loose reverse osmosis membrane may be used with nanofiltration membranes, whether salt rejection rate is about constant. In these conditions, it is possible to calculate, from laboratory scale experiments, performances of pilot-plant or industrial process. In this study, we have shown the effect of velocity and pressure on performance of nanofiltration membranes. Flows superior to 50 l m − 2 h − 1 (at 30× 105 Pa) are obtained with the Weizmann membrane MPT 20 (in polyacryloni-
3.4. Comparison of membranes for diafiltration Diafiltration is carried out at a constant volume: V0 =5 l. Three membranes are tested functionning in diafiltration mode: an ultrafiltration membrane Ore´lis – Iris 3028 3 kDa at DP =4×105 Pa, a nanofiltration membrane Kyriat Weizmann MPT 20 at DP = 25 ×105 Pa, a loose reverse osmosis membrane Millipore R45P at DP = 10 ×105 Pa. With the aim to evaluate the efficiency of the three membranes (ultrafiltration, nanofiltration and reverse osmosis) to carry out a diafiltration, it has been drawn on Fig. 10 the graphic of desalting speed versus salts concentration in the retentate.
Fig. 10. Compared efficiency of the membranes for the elimination of the salts.
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trile coated on polyester, cut-off 450 Da). Higher fluxes could be obtained by optimizing hydrodynamic conditions or the couple tangential velocity/pressure. For the desalting of the blue – green pigment solution, nanofiltration membranes present a few advantages over loose reverse osmosis and ultrafiltration: a low salt rejection rate (less than 10% at 14 bars) and a high pigment rejection (the nanofiltration membrane MPT 20 retains the fast totality of the pigment). This membrane used in diafiltration mode allows to obtain an acceptable speed of desalting (for example, 700 g of salt eliminated per hour and per m2 at 25 × 105 Pa for a concentration of 18 g of salt by litre of solution). So the nanofiltration membrane MPT 20 is interesting in the aim of a simultaneous concentration-desalting (without changing of processunit or membrane) of the marennine solution produced by H. ostrearia. The diafiltration does not allow to eliminate entirely the salts, but it does not constitute an obstacle in the aim of food enhancement of the marennine. Indeed, legislation makes difficult the commercialization of new natural dyestuffs (Labatut and In, 1990). In these conditions, non purified marennine should preferently be enhanced as a food ingredient. However, a high degree of purity is necessary for the use of marennine for therapeutical application in cancerology for example (Riou et al., 1993). The dialysis technique is then well suited for the final elimination of the salts when using small volumes of concentrated pigment solution. One further development of this study is the membrane photobioreactor which combines biological reaction and separation. Biological reaction: the research work consists in optimizing culture conditions of H. ostrearia for the highest pigment production: choice of the clones, study of the shear effects caused by pumps and valves in a recirculating loop (Vandanjon et al., 1999), addition of nutriments. Separation: for the extraction of pigment, two stages of membranes are necessary. Ultrafiltration (cut-off 40 kDa) can be used for the extraction of the exometabolite from the culture in a continuous mode (Rossignol et al., 1999). Then after-
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