- Email: [email protected]
Production of exocellular pigment by the marine diatom Haslea ostrearia Simonsen in a photobioreactor equipped with immersed ultrafiltration membranes
Bioresource Technology 73 (2000) 197±200
Short communication
Production of exocellular pigment by the marine diatom Haslea ostrearia Simonsen in a photobioreactor equipped with immersed ultra®ltration membranes Nathalie Rossignol a, Pascal Jaouen a,*, Jean-Michel Robert b, Francis Quemeneur a a
b
Cnt. de Rech. et de Trans. de Tech., ISOMer ± Institut des Substances et Organismes de la Mer, Laboratorie de G enie des Proc ed es, Boulevard de l'Universit e, BP 406, F-44602 Saint-Nazaire Cedex, France Laboratoire de Biologie Marine, ISOMer ± Institut des Substances et Organismes de la Mer, Universit e de Nantes ± Facult e des Sciences et des Techniques, 2 rue de la Houssini ere, BP 92208, F-44322 Nantes Cedex 3, France Received 27 August 1999; received in revised form 24 October 1999; accepted 24 October 1999
Abstract A new photobioreactor coupled with an ultra®ltration system (immersed membranes) was investigated for the continuous culture of the microalga Haslea ostrearia in order to improve pigment (marennine) production and recovery. The system presents a commercial interest, because energetic costs were minimized, and the cells were not submitted to any shear stress due to pumping or circulation. To obtain this, since the photobioreactor was of cylindrical type, a membrane module was placed at the bottom of the reactor and the hydrostatic pressure (the height of the water column) used as driving force both for the permeation and periodical back¯ushing steps. The production of biomass and marennine was stable for a three-week period, with marennine speci®c productivity 30±35 mg 10ÿ9 cell dayÿ1 , marennine concentration 3 times higher than in a conventional batch photobioreactor. The permeation ¯ux obtained was acceptable (3±10 l hÿ1 mÿ2 , 3 kPa, 15°C), but for such applications, this type of integrated process needs further improvements. Owing to its simple design, the concept ``photobioreactor ± ultra®ltration with immersed membranes'' has good possibilities in biotechnology and aquaculture for continuous extraction of exocellular metabolites. Ó 2000 Elsevier Science Ltd. All rights reserved. Keywords: Photobioreactor; Ultra®ltration; Immersed membranes; Back¯ushing; Continuous production; Pigment
1. Introduction There is a growing interest in microalgae, and the development of biotechnology has allowed their commercial exploitation. In addition to aquaculture and the health food market, the uses of microalgae currently focus on the production of various high value metabolites (Gudin and Thepenier, 1986; Apt and Behrens, 1999) and on wastewater and euent treatments (Kaya et al., 1995). The use of a closed membrane photobioreactor is a means of improving the production of biomass and metabolites, since cultures are conducted under carefully controlled conditions. The biomass is then recycled, while a part of the exocellular products is extracted in the membrane permeate. Membrane separation (micro- or ultra®ltration) also allows inhibitors to be eliminated in the permeate and the metabolites to be *
Corresponding author. Fax: +33-2-4017-2618. E-mail address: [email protected] (P. Jaouen).
recovered without any particles (cells or debris); this can ease the subsequent steps of concentration and puri®cation. In the classical system (membrane module located in an external loop outside the bioreactor), circulation speeds are relatively high in order to limit the fouling of the membrane. This leads to high pumping costs and shear stress harmful to the cell viability (Gudin and Chaumont, 1991; Jaouen et al., 1999; Vandanjon et al., 1999a). An alternate solution to these problems is to integrate the membranes inside the bioreactor. This con®guration of membrane photobioreactor has been recently and successfully used in wastewater treatment (Shimizu et al., 1996; Praderie et al., 1998) and can also be designed to optimize fermentations (Suzuki et al., 1994). The goal of the present work was to study this concept for continuous culture of a marine diatom with the aim of increasing the production of pigment while trying to reduce operating costs.
0960-8524/00/$ - see front matter Ó 2000 Elsevier Science Ltd. All rights reserved. PII: S 0 9 6 0 - 8 5 2 4 ( 9 9 ) 0 0 1 7 1 - 6
198
N. Rossignol et al. / Bioresource Technology 73 (2000) 197±200
The microalga Haslea ostrearia is commonly found in the littoral waters of the Atlantic coast in France. It has the unique property of synthesizing at its extremities a blue-green hydrosoluble pigment named ``marennine'', which is released into the medium (Robert, 1983; Nassiri et al., 1998) and known to be responsible for the greening of oysters. The interest in this pigment is notably linked to its colouring properties. Furthermore, antiproliferative properties (against human solid tumors) of this pigment have also been observed with aqueous extracts (Carbonnelle et al., 1999). 2. Methods 2.1. Membrane photobioreactor system The experimental set-up is shown in Fig. 1. The photobioreactor consisted of a 40 cm high glass cylinder with an inner diameter of 15 cm. The total volume was 7 l and the working volume 6 l. The internal membrane module consisted of two plane polyacrylonitrile ultra®ltration membranes (IRIS 3038 Orelis, Miribel-France) with molecular weight cut-o of 40 kDa and 2 ´ 45 cm2 area. Previous study selected the most ecient membrane for this application (Rossignol et al., 1999). The module was immersed in the reactor and hydrostatic pressure (the height of the water column) acted as driving force for the permeation. The culture medium was renewed by removing a part of the medium containing released pigment (permeate) and an equivalent volume of nutrient medium was added. To reduce concentration polarisation and fouling of membranes, growth medium was injected in the opposite direction to the deposit formed on the external membrane surface (back¯ushing) under a hydrostatic gradient (6 kPa). The renewal rate of culture medium was 10% of the total volume per day (600 ml). To improve nutrient transfer, the culture was mixed by a magnetic stirrer for 5 min after nutritive medium addition. 2.2. Microalgal strain and growth conditions The study was performed using an axenic strain of H. ostrearia isolated from oyster-ponds in the Bay of Bourgneuf (Vendee, France). Cultures were maintained in the modi®ed Provasoli medium described by Robert (1983) in 250 ml or 2-l ¯asks. Algal inoculum was collected by centrifugation (4000 ´ g, 6 min, 15°C) from such cultures in the exponential growth phase and introduced into the photobioreactor ®lled with the initial medium: sterile medium F/20 (Guillard, 1982). The salinity was adjusted to 28 g lÿ1 NaC1 and 80 mg lÿ1 NaHCO3 (source of carbon) was added. Marennine is synthesized under unfavourable nutritive conditions (Neuville and Daste, 1978) so, no nitrogen compounds
Fig. 1. Experimental set-up of photobioreactor with immersed membranes.
were added in the initial medium in order to starve and stress H. ostrearia from the beginning of the run. The renewal medium (F/20) was enriched with the minimal concentration of nitrates (88 lmol lÿ1 ) needed for cell metabolism. The pH was maintained at a value between 7.8 and 8.2 through daily acid addition (HCl, 0.1 N, 2 ml). Before incubation, photobioreactor, tubes and nutrient medium were autoclaved, while membranes and ultra®ltration module were ¯ushed with ethanol (95%) and rinsed with sterile deionized water. The photobioreactor was placed in an air-conditioned room with temperature adjusted to 15°C and the culture was submitted to 14 h light/10 h dark periodicity with a light intensity of 250 lmol photons mÿ2 sÿ1 measured by a spherical probe inside the culture (LI-1000 Data Logger quantameter). 2.3. Analytical procedures Marennine concentrations in the cell-free permeate were determined by measuring the optical density (OD)
N. Rossignol et al. / Bioresource Technology 73 (2000) 197±200
at 663 nm (Robert and Hallet, 1981) referring the OD values to a calibration curve. The algal population was assessed by two methods. Using the direct method, cell concentration (expressed as number of cells per litre of culture medium) was estimated using a Nageotte cell with an optical microscope. The indirect method consisted in evaluating the number of viable cells by chlorophyll a determination. According to the method of Lorenzen (1967), chlorophyll a concentration was determined by absorbance measurement at 665 nm after extraction from a culture sample by 90% acetone. The marennine volumetric and speci®c productivities were calculated from dilution rate and daily marennine, biomass and chlorophyll a concentration determinations, according to the following equations: Marennine production rate r (mg lÿ1 dayÿ1 ), i.e. volumetric productivity of marennine r
C ti ÿ C tiÿ1 DCm ti ÿ tiÿ1
with i P 1:
1
Speci®c productivity q (mg cellÿ1 dayÿ1 or mg mgÿ1 Chla dayÿ1 ) of marennine r ; 2 q Nm where D is the dilution rate (dayÿ1 ), ti the time after incubation (day), C(ti ) and C tiÿ1 the marennine concentrations (mg lÿ1 ) at time ti and tiÿ1 , Cm the average marennine concentration between times ti and tiÿ1 (mg lÿ1 ), and Nm is the average cell or chlorophyll a concentrations (cell lÿ1 or mg Chla lÿ1 ) between times ti and tiÿ1 : The ®rst part of Eq. (1) C ti ÿ C tiÿ1 = ti ÿ tiÿ1 represents the variation of marennine concentration per unit of time in the reactor. The second member of Eq. (1) DCm corresponds to the quantity of marennine extracted daily in the permeate with reference to reactional volume.
199
Table 1 Marennine production by culture of Haslea ostrearia in a photobioreactor with immersed membranes (mean values between 10th and 21st days) Marennine concentration (mg lÿ1 ) Volumetric productivity (mg lÿ1 dayÿ1 )
20±30 2.5±3.5
Speci®c productivity with reference to Total biomass (mg 10ÿ9 cell dayÿ1 ) Viable cells (mg mgÿ1 Chla dayÿ1 )
30±35 5.5±6.5
vation, in which cells produced compensated decaying cells. The concentration of chlorophyll a, which better represents the viability of biomass, showed a stabilization of the active population from the second week of cultivation. The volumetric productivity of released marennine remained stable from the sixth day (3 mg lÿ1 dayÿ1 ). The simultaneous presence of active and degenerated cells as well as cell fragments in the photobioreactor did not aect the productivity of biomass. A stationary level of pigment production was reached without biomass purge. Parameters of marennine production are given in Table 1. The concentrations of exocellular marennine were three times higher than those usually reached with large volume (500 l) batch cultivations after 6±9 days of growth (Rouillard, 1996). 3.2. Permeation ¯ux The two main advantages of this system are the minimization of energy needs and shear stress eects on cells. The periodical back¯ushing improved the permeation ¯ux in comparison with continuous mode (without back¯ushing, direct nutrient addition into the reactor)
3. Results and discussion 3.1. Cell and marennine productivities Continuous culture of H. ostrearia in the immersed membrane photobioreactor was performed over a single period of three weeks and was deliberately stopped after this time. Every day, ultra®ltration was continuous for about 20 h and back¯ushing continuous for 4 h through injection of nutrient medium. This cycle was decided by the respective permeation ¯uxes: normal and reversed ¯ows. The cell concentration increased and, indeed, since the retention of the biomass was total, cells at dierent stages of growth were accumulated, including dead cells. A steady level was then reached after 16 days of culti-
Fig. 2. In¯uence of back¯ushing on permeation ¯ux of immersed ultra®ltration membranes (3 kPa, 15°C).
200
N. Rossignol et al. / Bioresource Technology 73 (2000) 197±200
as shown in Fig. 2. The average permeation ¯ux with back¯ushing procedure is about twice higher (4±5 l hÿ1 mÿ2 ) than without reversed ¯ow. These results are encouraging considering the low applied transmembrane pressure (3 kPa). However, back¯ushing does not entirely prevent ¯ux decline with time. Thus it is necessary to provide a means to improve the ultra®ltration system by increasing the membrane area or by optimizing the backwashing procedure, depending on the following main parameters: frequency, duration and applied differential pressure. 4. Conclusion Continuous culture of microalgae in a photobioreactor with immersed membrane is a suitable and promising technique for the production of marennine by H. ostrearia. For a three-week experiment duration, the viable biomass and marennine productivities reached a plateau phase at the end of the ®rst week. Then, during the last two weeks, marennine productivity remained stable. This economical system of cultivation could be used at the ®rst pigment production/extraction step, which could then be followed by concentration and puri®cation phases with another, specially designed membrane process such as nano®ltration or reverse osmosis (Vandanjon et al., 1999b). Acknowledgements The authors wish to thank P. Gaudin, Laboratoire de Biologie Marine, ISOMer-Nantes, for his technical contribution. References Apt, K.E., Behrens, P.W., 1999. Commercial developments in microalgal biotechnology. J. Phycol. 35, 215±226. Carbonnelle, D., Pondaven, P., Morancßais, M., Masse, G., Bosch, S., Jacquot, C., Briand, G., Robert, J.M., Roussakis, C., 1999. Antitumor and antiproliferative eects of an aqueous extract from the marine diatom H. ostrearia (Simonsen) against solid tumors lung carcinoma (NSCLC-N6), kidney carcinoma (E39) and melanoma (M96) cell lines. Anticancer Res. 19, 621±624. Gudin, C., Chaumont, D., 1991. Cell fragility ± the key problem of microalgae mass production in closed photobioreactors. Bioresource Technol. 38, 145±151.
Gudin, C., Thepenier, C., 1986. Bioconversion of solar energy into organic chemicals by microalgae. Adv. Biotechnol. Processes 6, 73± 110. Guillard, R.R.L., 1982. Culture of phytoplancton for feeding marine invertebrates. In: Smith, W.L., Charley, M.H (Eds.), Culture of Marine Invertebrates Animals. Plenum Press, New York, pp. 108± 132. Jaouen, P., Vandanjon, L., Quemeneur, F., 1999. The shear stress of microalgal cell suspensions (Tetraselmis suecica) in tangential ¯ow ®ltration systems: the role of pumps. Bioresource Technol. 68 (2), 149±154. Kaya, V.M., de la No ue, J., Picard G., 1995. A comparative study of four systems for tertiary wastewater treatment by Scenedesmus bicellularis: new technology for immobilization. J. Appl. Phycol. 7, 85±95. Lorenzen, C.J., 1967. Determination of chlorophylles and phaeopigments: spectrophotometric equations. Limnol. Oceanogr. 12, 343± 346. Nassiri, Y., Robert, J.M., Rince, Y., GinsburgerVogel, T., 1998. The cytoplasmic ®ne structure of the diatom H. ostrearia (Bacillariophyceae) in relation to marennine production. Phycologia 37 (2), 84±91. Neuville, D., Daste, P., 1978. Recherche sur le determinisme de la production de marennine par la diatomee marine Navicula ostrearia (Gaillon) Bory en culture in vitro. Revue Generale de Botanique 85, 255±303. Praderie, M., Buisson, H., Paillard, H., Vouillon, T., 1998. Traitement des euents industriels par boues activees a membranes immergees. Surfaces 276, 20±26. Robert, J.M., 1983. Fertilite des claires ostreicoles et verdissement: utilisation de l'azote par les diatomees dominantes. Ph.D. thesis, Nantes, France. Robert, J.M., Hallet, J.N., 1981. Absorption spectrum in vivo of the blue pigment marennine of the pennate diatom Navicula ostrearia Bory. J. Exp. Bot. 32, 341±345. Rossignol, N., Vandanjon, L., Jaouen, P., Quemeneur, F., 1999. Membrane technology for the continuous separation microalgae/ culture medium: compared performances of cross-¯ow micro®ltration and ultra®ltration. Aquacult. Eng. 20 (3), 191±208. Rouillard, I., 1996. Optimisation de la production en masse de Haslea ostrearia Simonsen sur eau souterraine salee, importance de la souche et des conditions de culture, comparaison avec Skeletonema costatum (Grev.) Cleve. Ph.D. thesis, Nantes, France. Shimizu, Y., Uryu, K., Okuno, Y., Watanabe, A., 1996. Cross-¯ow micro®ltration of activated sludge using submerged membrane with air-bubbling. J. Ferment. Bioeng. 81 (1), 55±60. Suzuki, T., Sato, T., Kominami, M., 1994. A dense cell retention culture system using a stirred ceramic membrane reactor. Biotechnol. Bioeng. 44, 1186±1192. Vandanjon, L., Rossignol, N., Jaouen, P., Robert, J.M., Quemeneur, F., 1999a. Eects of shear on two microalgae species. Contribution of pumps and valves in tangential ¯ow ®ltration systems. Biotechnol. Bioeng. 63 (1), 1±9. Vandanjon, L., Jaouen, P., Rossignol, N., Quemeneur, F., Robert, J.M., 1999b. Concentration and desalting by membrane processes of a natural pigment produced by the marine diatom H. ostrearia Simonsen. J. Biotechnol. 70, 393±402.
Short communication
Production of exocellular pigment by the marine diatom Haslea ostrearia Simonsen in a photobioreactor equipped with immersed ultra®ltration membranes Nathalie Rossignol a, Pascal Jaouen a,*, Jean-Michel Robert b, Francis Quemeneur a a
b
Cnt. de Rech. et de Trans. de Tech., ISOMer ± Institut des Substances et Organismes de la Mer, Laboratorie de G enie des Proc ed es, Boulevard de l'Universit e, BP 406, F-44602 Saint-Nazaire Cedex, France Laboratoire de Biologie Marine, ISOMer ± Institut des Substances et Organismes de la Mer, Universit e de Nantes ± Facult e des Sciences et des Techniques, 2 rue de la Houssini ere, BP 92208, F-44322 Nantes Cedex 3, France Received 27 August 1999; received in revised form 24 October 1999; accepted 24 October 1999
Abstract A new photobioreactor coupled with an ultra®ltration system (immersed membranes) was investigated for the continuous culture of the microalga Haslea ostrearia in order to improve pigment (marennine) production and recovery. The system presents a commercial interest, because energetic costs were minimized, and the cells were not submitted to any shear stress due to pumping or circulation. To obtain this, since the photobioreactor was of cylindrical type, a membrane module was placed at the bottom of the reactor and the hydrostatic pressure (the height of the water column) used as driving force both for the permeation and periodical back¯ushing steps. The production of biomass and marennine was stable for a three-week period, with marennine speci®c productivity 30±35 mg 10ÿ9 cell dayÿ1 , marennine concentration 3 times higher than in a conventional batch photobioreactor. The permeation ¯ux obtained was acceptable (3±10 l hÿ1 mÿ2 , 3 kPa, 15°C), but for such applications, this type of integrated process needs further improvements. Owing to its simple design, the concept ``photobioreactor ± ultra®ltration with immersed membranes'' has good possibilities in biotechnology and aquaculture for continuous extraction of exocellular metabolites. Ó 2000 Elsevier Science Ltd. All rights reserved. Keywords: Photobioreactor; Ultra®ltration; Immersed membranes; Back¯ushing; Continuous production; Pigment
1. Introduction There is a growing interest in microalgae, and the development of biotechnology has allowed their commercial exploitation. In addition to aquaculture and the health food market, the uses of microalgae currently focus on the production of various high value metabolites (Gudin and Thepenier, 1986; Apt and Behrens, 1999) and on wastewater and euent treatments (Kaya et al., 1995). The use of a closed membrane photobioreactor is a means of improving the production of biomass and metabolites, since cultures are conducted under carefully controlled conditions. The biomass is then recycled, while a part of the exocellular products is extracted in the membrane permeate. Membrane separation (micro- or ultra®ltration) also allows inhibitors to be eliminated in the permeate and the metabolites to be *
Corresponding author. Fax: +33-2-4017-2618. E-mail address: [email protected] (P. Jaouen).
recovered without any particles (cells or debris); this can ease the subsequent steps of concentration and puri®cation. In the classical system (membrane module located in an external loop outside the bioreactor), circulation speeds are relatively high in order to limit the fouling of the membrane. This leads to high pumping costs and shear stress harmful to the cell viability (Gudin and Chaumont, 1991; Jaouen et al., 1999; Vandanjon et al., 1999a). An alternate solution to these problems is to integrate the membranes inside the bioreactor. This con®guration of membrane photobioreactor has been recently and successfully used in wastewater treatment (Shimizu et al., 1996; Praderie et al., 1998) and can also be designed to optimize fermentations (Suzuki et al., 1994). The goal of the present work was to study this concept for continuous culture of a marine diatom with the aim of increasing the production of pigment while trying to reduce operating costs.
0960-8524/00/$ - see front matter Ó 2000 Elsevier Science Ltd. All rights reserved. PII: S 0 9 6 0 - 8 5 2 4 ( 9 9 ) 0 0 1 7 1 - 6
198
N. Rossignol et al. / Bioresource Technology 73 (2000) 197±200
The microalga Haslea ostrearia is commonly found in the littoral waters of the Atlantic coast in France. It has the unique property of synthesizing at its extremities a blue-green hydrosoluble pigment named ``marennine'', which is released into the medium (Robert, 1983; Nassiri et al., 1998) and known to be responsible for the greening of oysters. The interest in this pigment is notably linked to its colouring properties. Furthermore, antiproliferative properties (against human solid tumors) of this pigment have also been observed with aqueous extracts (Carbonnelle et al., 1999). 2. Methods 2.1. Membrane photobioreactor system The experimental set-up is shown in Fig. 1. The photobioreactor consisted of a 40 cm high glass cylinder with an inner diameter of 15 cm. The total volume was 7 l and the working volume 6 l. The internal membrane module consisted of two plane polyacrylonitrile ultra®ltration membranes (IRIS 3038 Orelis, Miribel-France) with molecular weight cut-o of 40 kDa and 2 ´ 45 cm2 area. Previous study selected the most ecient membrane for this application (Rossignol et al., 1999). The module was immersed in the reactor and hydrostatic pressure (the height of the water column) acted as driving force for the permeation. The culture medium was renewed by removing a part of the medium containing released pigment (permeate) and an equivalent volume of nutrient medium was added. To reduce concentration polarisation and fouling of membranes, growth medium was injected in the opposite direction to the deposit formed on the external membrane surface (back¯ushing) under a hydrostatic gradient (6 kPa). The renewal rate of culture medium was 10% of the total volume per day (600 ml). To improve nutrient transfer, the culture was mixed by a magnetic stirrer for 5 min after nutritive medium addition. 2.2. Microalgal strain and growth conditions The study was performed using an axenic strain of H. ostrearia isolated from oyster-ponds in the Bay of Bourgneuf (Vendee, France). Cultures were maintained in the modi®ed Provasoli medium described by Robert (1983) in 250 ml or 2-l ¯asks. Algal inoculum was collected by centrifugation (4000 ´ g, 6 min, 15°C) from such cultures in the exponential growth phase and introduced into the photobioreactor ®lled with the initial medium: sterile medium F/20 (Guillard, 1982). The salinity was adjusted to 28 g lÿ1 NaC1 and 80 mg lÿ1 NaHCO3 (source of carbon) was added. Marennine is synthesized under unfavourable nutritive conditions (Neuville and Daste, 1978) so, no nitrogen compounds
Fig. 1. Experimental set-up of photobioreactor with immersed membranes.
were added in the initial medium in order to starve and stress H. ostrearia from the beginning of the run. The renewal medium (F/20) was enriched with the minimal concentration of nitrates (88 lmol lÿ1 ) needed for cell metabolism. The pH was maintained at a value between 7.8 and 8.2 through daily acid addition (HCl, 0.1 N, 2 ml). Before incubation, photobioreactor, tubes and nutrient medium were autoclaved, while membranes and ultra®ltration module were ¯ushed with ethanol (95%) and rinsed with sterile deionized water. The photobioreactor was placed in an air-conditioned room with temperature adjusted to 15°C and the culture was submitted to 14 h light/10 h dark periodicity with a light intensity of 250 lmol photons mÿ2 sÿ1 measured by a spherical probe inside the culture (LI-1000 Data Logger quantameter). 2.3. Analytical procedures Marennine concentrations in the cell-free permeate were determined by measuring the optical density (OD)
N. Rossignol et al. / Bioresource Technology 73 (2000) 197±200
at 663 nm (Robert and Hallet, 1981) referring the OD values to a calibration curve. The algal population was assessed by two methods. Using the direct method, cell concentration (expressed as number of cells per litre of culture medium) was estimated using a Nageotte cell with an optical microscope. The indirect method consisted in evaluating the number of viable cells by chlorophyll a determination. According to the method of Lorenzen (1967), chlorophyll a concentration was determined by absorbance measurement at 665 nm after extraction from a culture sample by 90% acetone. The marennine volumetric and speci®c productivities were calculated from dilution rate and daily marennine, biomass and chlorophyll a concentration determinations, according to the following equations: Marennine production rate r (mg lÿ1 dayÿ1 ), i.e. volumetric productivity of marennine r
C ti ÿ C tiÿ1 DCm ti ÿ tiÿ1
with i P 1:
1
Speci®c productivity q (mg cellÿ1 dayÿ1 or mg mgÿ1 Chla dayÿ1 ) of marennine r ; 2 q Nm where D is the dilution rate (dayÿ1 ), ti the time after incubation (day), C(ti ) and C tiÿ1 the marennine concentrations (mg lÿ1 ) at time ti and tiÿ1 , Cm the average marennine concentration between times ti and tiÿ1 (mg lÿ1 ), and Nm is the average cell or chlorophyll a concentrations (cell lÿ1 or mg Chla lÿ1 ) between times ti and tiÿ1 : The ®rst part of Eq. (1) C ti ÿ C tiÿ1 = ti ÿ tiÿ1 represents the variation of marennine concentration per unit of time in the reactor. The second member of Eq. (1) DCm corresponds to the quantity of marennine extracted daily in the permeate with reference to reactional volume.
199
Table 1 Marennine production by culture of Haslea ostrearia in a photobioreactor with immersed membranes (mean values between 10th and 21st days) Marennine concentration (mg lÿ1 ) Volumetric productivity (mg lÿ1 dayÿ1 )
20±30 2.5±3.5
Speci®c productivity with reference to Total biomass (mg 10ÿ9 cell dayÿ1 ) Viable cells (mg mgÿ1 Chla dayÿ1 )
30±35 5.5±6.5
vation, in which cells produced compensated decaying cells. The concentration of chlorophyll a, which better represents the viability of biomass, showed a stabilization of the active population from the second week of cultivation. The volumetric productivity of released marennine remained stable from the sixth day (3 mg lÿ1 dayÿ1 ). The simultaneous presence of active and degenerated cells as well as cell fragments in the photobioreactor did not aect the productivity of biomass. A stationary level of pigment production was reached without biomass purge. Parameters of marennine production are given in Table 1. The concentrations of exocellular marennine were three times higher than those usually reached with large volume (500 l) batch cultivations after 6±9 days of growth (Rouillard, 1996). 3.2. Permeation ¯ux The two main advantages of this system are the minimization of energy needs and shear stress eects on cells. The periodical back¯ushing improved the permeation ¯ux in comparison with continuous mode (without back¯ushing, direct nutrient addition into the reactor)
3. Results and discussion 3.1. Cell and marennine productivities Continuous culture of H. ostrearia in the immersed membrane photobioreactor was performed over a single period of three weeks and was deliberately stopped after this time. Every day, ultra®ltration was continuous for about 20 h and back¯ushing continuous for 4 h through injection of nutrient medium. This cycle was decided by the respective permeation ¯uxes: normal and reversed ¯ows. The cell concentration increased and, indeed, since the retention of the biomass was total, cells at dierent stages of growth were accumulated, including dead cells. A steady level was then reached after 16 days of culti-
Fig. 2. In¯uence of back¯ushing on permeation ¯ux of immersed ultra®ltration membranes (3 kPa, 15°C).
200
N. Rossignol et al. / Bioresource Technology 73 (2000) 197±200
as shown in Fig. 2. The average permeation ¯ux with back¯ushing procedure is about twice higher (4±5 l hÿ1 mÿ2 ) than without reversed ¯ow. These results are encouraging considering the low applied transmembrane pressure (3 kPa). However, back¯ushing does not entirely prevent ¯ux decline with time. Thus it is necessary to provide a means to improve the ultra®ltration system by increasing the membrane area or by optimizing the backwashing procedure, depending on the following main parameters: frequency, duration and applied differential pressure. 4. Conclusion Continuous culture of microalgae in a photobioreactor with immersed membrane is a suitable and promising technique for the production of marennine by H. ostrearia. For a three-week experiment duration, the viable biomass and marennine productivities reached a plateau phase at the end of the ®rst week. Then, during the last two weeks, marennine productivity remained stable. This economical system of cultivation could be used at the ®rst pigment production/extraction step, which could then be followed by concentration and puri®cation phases with another, specially designed membrane process such as nano®ltration or reverse osmosis (Vandanjon et al., 1999b). Acknowledgements The authors wish to thank P. Gaudin, Laboratoire de Biologie Marine, ISOMer-Nantes, for his technical contribution. References Apt, K.E., Behrens, P.W., 1999. Commercial developments in microalgal biotechnology. J. Phycol. 35, 215±226. Carbonnelle, D., Pondaven, P., Morancßais, M., Masse, G., Bosch, S., Jacquot, C., Briand, G., Robert, J.M., Roussakis, C., 1999. Antitumor and antiproliferative eects of an aqueous extract from the marine diatom H. ostrearia (Simonsen) against solid tumors lung carcinoma (NSCLC-N6), kidney carcinoma (E39) and melanoma (M96) cell lines. Anticancer Res. 19, 621±624. Gudin, C., Chaumont, D., 1991. Cell fragility ± the key problem of microalgae mass production in closed photobioreactors. Bioresource Technol. 38, 145±151.
Gudin, C., Thepenier, C., 1986. Bioconversion of solar energy into organic chemicals by microalgae. Adv. Biotechnol. Processes 6, 73± 110. Guillard, R.R.L., 1982. Culture of phytoplancton for feeding marine invertebrates. In: Smith, W.L., Charley, M.H (Eds.), Culture of Marine Invertebrates Animals. Plenum Press, New York, pp. 108± 132. Jaouen, P., Vandanjon, L., Quemeneur, F., 1999. The shear stress of microalgal cell suspensions (Tetraselmis suecica) in tangential ¯ow ®ltration systems: the role of pumps. Bioresource Technol. 68 (2), 149±154. Kaya, V.M., de la No ue, J., Picard G., 1995. A comparative study of four systems for tertiary wastewater treatment by Scenedesmus bicellularis: new technology for immobilization. J. Appl. Phycol. 7, 85±95. Lorenzen, C.J., 1967. Determination of chlorophylles and phaeopigments: spectrophotometric equations. Limnol. Oceanogr. 12, 343± 346. Nassiri, Y., Robert, J.M., Rince, Y., GinsburgerVogel, T., 1998. The cytoplasmic ®ne structure of the diatom H. ostrearia (Bacillariophyceae) in relation to marennine production. Phycologia 37 (2), 84±91. Neuville, D., Daste, P., 1978. Recherche sur le determinisme de la production de marennine par la diatomee marine Navicula ostrearia (Gaillon) Bory en culture in vitro. Revue Generale de Botanique 85, 255±303. Praderie, M., Buisson, H., Paillard, H., Vouillon, T., 1998. Traitement des euents industriels par boues activees a membranes immergees. Surfaces 276, 20±26. Robert, J.M., 1983. Fertilite des claires ostreicoles et verdissement: utilisation de l'azote par les diatomees dominantes. Ph.D. thesis, Nantes, France. Robert, J.M., Hallet, J.N., 1981. Absorption spectrum in vivo of the blue pigment marennine of the pennate diatom Navicula ostrearia Bory. J. Exp. Bot. 32, 341±345. Rossignol, N., Vandanjon, L., Jaouen, P., Quemeneur, F., 1999. Membrane technology for the continuous separation microalgae/ culture medium: compared performances of cross-¯ow micro®ltration and ultra®ltration. Aquacult. Eng. 20 (3), 191±208. Rouillard, I., 1996. Optimisation de la production en masse de Haslea ostrearia Simonsen sur eau souterraine salee, importance de la souche et des conditions de culture, comparaison avec Skeletonema costatum (Grev.) Cleve. Ph.D. thesis, Nantes, France. Shimizu, Y., Uryu, K., Okuno, Y., Watanabe, A., 1996. Cross-¯ow micro®ltration of activated sludge using submerged membrane with air-bubbling. J. Ferment. Bioeng. 81 (1), 55±60. Suzuki, T., Sato, T., Kominami, M., 1994. A dense cell retention culture system using a stirred ceramic membrane reactor. Biotechnol. Bioeng. 44, 1186±1192. Vandanjon, L., Rossignol, N., Jaouen, P., Robert, J.M., Quemeneur, F., 1999a. Eects of shear on two microalgae species. Contribution of pumps and valves in tangential ¯ow ®ltration systems. Biotechnol. Bioeng. 63 (1), 1±9. Vandanjon, L., Jaouen, P., Rossignol, N., Quemeneur, F., Robert, J.M., 1999b. Concentration and desalting by membrane processes of a natural pigment produced by the marine diatom H. ostrearia Simonsen. J. Biotechnol. 70, 393±402.