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Semi-continuous Cultivation of Haematococcus pluvialis for Commercial Production
Esra Imamoglu & Meltem Conk Dalay & Fazilet Vardar Sukan
Received: 20 October 2008 / Accepted: 25 March 2009 # Humana Press 2009
Abstract The objectives of the present study on the growth of Haematococcus pluvialis were to indicate the effects of a long-term semi-continuous cultivation, sterilization, carbon dioxide, and different culture media by using artesian well water. This investigation was an enterprise in order to commercialize the production economically. When the effect of CO2 was investigated in basal culture medium, the influence of sterilization was also researched in Rudic’s culture medium in vertical panel-type photobioreactors for 31 days of semi-continuous cultivation. The maximum cell concentration of 10.55×105 cells ml−1, which corresponds to the growth rate of 0.271 day−1 with the areal productivity of 3.531 g m−2 day−1, was found in non-sterilized RM medium on the 24th day of the third run of semi-continuous cultivation at a renewal rate of 50% in a vertical panel-type photobioreactor. Keywords Carbon dioxide . Haematococcus pluvialis . Nutrient medium . Photobioreactor . Semi-continuous cultivation . Sterilization Abbreviations Cb Biomass concentration (g l−1) Cc Cell count (cells ml−1) DT Doubling time (day) Pba Areal productivity (g m−2 day−1) Pbv Volumetric productivity (g l−1 day−1) µ Specific growth rate (day−1) Introduction The major market for astaxanthin is as a pigmentation source in aquaculture, primarily salmon, trout, and sea beam. Astaxanthin sells for approximately $2,500 kg−1 with the annual worldwide aquaculture market estimated at $200 million. Although over 95% of this
E. Imamoglu (*) : M. C. Dalay : F. V. Sukan Department of Bioengineering, Faculty of Engineering, Ege University, Izmir 35100, Turkey e-mail: firstname.lastname@example.org
Izmir. (c) CO2. allowing to change species and to remedy defects in the system rapidly. protect against the damaging effects of ultraviolet radiation. Thus. only high cell densities are considered to be economically justified . the semi-continuous method yields more algae than the batch method for a given tank size . Semi-continuous cultures may be indoors or outdoors. The very cheap method of growing Haematococcus pluvialis in open ponds will not materialize because of contamination problems . 6]. 10] at 25 °C under continuous illumination (100 µmol photons m−2 s−1) in a 10-l flask. Turkey. Continuous aeration was provided by bubbling air. Growth Conditions The temperature was measured in the center of the reactor with a thermocouple (Dixell-XT115). 2 min) and the supernatant was removed. For the preparation of the inoculum.Appl Biochem Biotechnol market consumes synthetically derived astaxanthin. The objectives of the present study on growth of H. and vitamin E. controlled by an air conditioner. Although often considered as the most reliable method.000-ml Erlenmeyer flask containing 800 ml of fresh BG11 medium under continuous illumination (75 µmol photons m−2 s−1). and also enhance the immune system . using a blower (Nitto Kohki Co. zeaxanthin. Additionally. and chemically induced cancers.160×g. but usually their duration is unpredictable. Batch culture systems are widely applied because of their simplicity and flexibility. pluvialis was grown photoautotrophically in BG11 medium [9. and (d) different culture media. Since the culture is not harvested completely. Turkey) were used to provide . at 25°C for 4 days. batch culture is not necessarily the most efficient method. A 4-day-old culture was used as inoculum at 10% volume for all experiments. This investigation was an enterprise in order to commercialize the production economically. (b) sterilization. In bioreactor cultures of Haematococcus algae. The collected cells were transferred and incubated aseptically in a 1. Air was supplied to the culture at a flow rate of 1 l min−1 (1. Materials and Methods Algal Strain and Inoculum Preparation H.25 vvm). Two types of bioreactor systems are suitable for cultivation of Haematococcus lacustris at high cell densities. Rotameters (Özgül-air. Irradiance was measured in the center of the reactor with a quantum meter (Lambda L1-185). Illumination was provided by standard cool white fluorescent lamps (18 W) from one side of the reactor. the cells from the stock culture were collected and concentrated by centrifugation (1. pluvialis Flotow EGE MACC-35 was obtained from the Culture Collection of Microalgae at the University of Ege. Izmir. the pharmaceutical and cosmetic industries are increasingly interested in astaxanthin since it can. Stock culture of H. Ltd). for instance. pluvialis were to indicate the effects of (a) a long-term semi-continuous cultivation. These include photobioreactors  and conventional heterotrophic fermenters [5. the red ketocarotenoid astaxanthin surpasses the antioxidant activity of other carotenoids like β-carotene. due to the high cost of capital investment and the requirements for medium sterilization and aseptic operation. consumer demand for natural products makes the synthetic pigments much less desirable and provides opportunity for the production of natural astaxanthin by Haematococcus .
approximately 1×106 cells ml−1) was inoculated into 1. (b) basal culture medium without the addition of CO2 under non-aseptic conditions. The gas spargers were placed from both opposite corners of the photobioreactor so as to supply enriched air to the culture. 9 light source . Turkey). The photobioreactor was made of Plexiglas (0. Chemical sterilization was applied by using 1.4 cm (single nozzle). for 10 s every 10 min to the air stream (1. (c) Rudic’s (RM) culture medium  with the addition of CO2 under non-aseptic conditions. The reactors were incubated at 25 °C under the light intensity of 75 µmol photons m−2 s−1.5% (v/v) hypochlorite for the reactors. The sparger diameter is 0. 5 harvesting outlet. The multifactorial experimental design was achieved by two different culture media for 31 days of semi-continuous cultivation with the following conditions: (a) basal culture medium  with the addition of CO2 under non-aseptic conditions.Appl Biochem Biotechnol the desired air flow rate. 1 A schematic diagram of the vertical panel-type photobioreactor: 1 air and CO2 injection. 2 rotameter.5 cm W×25 cm H). 8 control valve. The total volume of the photobioreactor was 1.000 ml fresh media in 1. Vertical Panel-Type Photobioreactor A schematic diagram of the vertical panel-type photobioreactor is shown in Fig. and (d) RM culture medium with the addition of CO2 under aseptic conditions. 6 baffle. Styrofoam was used to avoid light penetration between experiments. pure CO2 was added intermittently. 4 electrodes and sensors.5% v/v) in order to provide inorganic carbon to the culture and keep the pH value below 8. 7 culture medium vessel.5 l (25 cm L×2. Each culture medium was prepared and added to Fig. A 4-day-old culture (100 ml. using a timer (Akboru BND-50/G1. 1. 3 sparger. If necessary. Izmir.5 cm wall thickness). Air was supplied to the culture at a flow rate of 1 l min−1 (1 vvm).500 ml panel reactors.
The Rudic’s culture medium contained (per liter): 300 mg NaNO3. Cultures were harvested by draining the culture media into a plankton collector equipped with 50 to 150 µm mesh. second. The basal culture medium contained (per 100 ml): 15 mg Ca (NO3)2⋅4H2O. Measurements and Analytical Methods Samples were taken at indicated times and growth parameters were measured immediately. a percentage of the volume of the culture (renewal rate) was harvested when a deceleration of the growth velocity was observed.2 mg Co(NO3)2⋅H2O. and 60% were applied at the end of the first. one of the media was autoclaved.Appl Biochem Biotechnol artesian well water.). 0. the aeration of the photobioreactors was turned off and the cells were allowed to settle. 80 mg K2HPO4.08 mg CuSO4⋅5H2O. All reagents used were of analytical grade (Merck & Co. as m ¼ ðln Cc2 À ln Cc1 Þ=delta t . Before harvesting. 0. respectively.3 mg (NH4) 6Mo7O24⋅4H2O. 20 mg KH2PO4. For semi-continuous cultivation. Furthermore.25 mg Na2MoO4⋅2H2O. and third runs of semicontinuous cultivation. The value of linear regression coefficient is 0. Dry weight was determined in duplicate by filtering a 5-ml culture sample through preweighed Whatman GF/C filters and drying the cell mass at 105 °C for 2 h. 20 mg NaCl. the cell concentration was determined by counting triplicate samples in a Neubauer hemocytometer.3 mg H3BO3.01 μg biotin. 10 mg MgSO4⋅7H2O.4 mg CoCl2⋅6H2O. 10 mg KNO3. 5 mg β-Na2 glycerophosphate.6 mg FeCl3⋅6H2O. and delta t is the time required for the increase in concentration from Cc1 to Cc2. 2. Doubling time (DT) was also calculated as DT ¼ ln 2=m: The volumetric productivity of the biomass is calculated : Pbv ¼ mCb . ð2Þ where Cc2 is the final cell concentration. 0. The sediments were stirred up manually following harvest. as follows : Pba A ¼ Pbv V . The PIV metals contained (per 100 ml): 19. 0.6 mg MnCl2⋅4H2O. 0. ð5Þ .1 mg ZnSO4⋅7H2O. 3. 0. 1. The areal productivity Pba is related with volumetric productivity.01 μg vitamin B12.2 mg ZnSO4⋅7H2O. 0. to re-suspend cells. 0. 50%. The renewal rates of 40%. Cc1 is the initial cell concentration. The cell capacity to flocculate made harvesting easier even in the vegetative stage. The specific growth rate (µ) of the cells was calculated from the initial logarithmic phase of growth for at least 48 h. and 100 mg EDTA-Na2. and 50 mg trisaminomethane. 0. 0. thoroughly mixing the cultures. 1 μg thiamine–HCl.5 mg MnSO4⋅H2O. 4 mg MgSO4⋅7H2O.5 mg EDTA. 47 mg CaCl2. where V is the volume of the reactor and A is the land area occupied by it.99 for Eq. ð1Þ where Cb is the dry weight biomass concentration. The correlation between the cell count (Cc) and the biomass concentration (Cb) was: À Á À Á Cb glÀ1 ¼ 0:0668 Cc 105 cell=ml þ 0:1304. 17 mg FeCl3⋅6H2O. and 7. ð4Þ ð3Þ where Pbv is the volumetric productivity and Cb is the concentration of the biomass. and the same volume of freshly prepared culture medium was added for each experiment. 1.3 ml PIV metals.
respectively (Table 1). The best growth was obtained in the third run of semi-continuous cultivation. When the second run of semi-continuous cultivation was started with a renewal rate of 40%.243 and 0. pluvialis at a light intensity of 75 µmol photons m−2 s−1 for 31 days of semi-continuous cultivation period. RM culture medium with the addition of CO2 under aseptic conditions . It was recorded that cell division was inhibited in the fourth run of semi-continuous 12 10 -1 8 6 4 2 0 0 4 8 12 16 20 24 28 32 10 cells ml 5 Time (days) Fig. 9.Appl Biochem Biotechnol Results The effects of four different conditions on the growth of H.271 day−1 with the areal productivity of 3.55×105 cells ml−1. was obtained in non-sterilized RM medium and the lowest in non-sterilized basal medium without the addition of CO2 (7.531 g m−2 day−1. all the cells were still in the vegetative form. the cultures in basal and RM media were harvested at about 60% of the volume of the culture. At the end of the third run. It should be noted here that when the volumetric productivity decreased due to the dilute culture broth. 2 Effect of different conditions on the growth of H.25×105 cells ml−1) at a renewal rate of 40% on the 17th day. No significant increase in cell concentration was noted at each experimental parameter with a renewal rate of 60% on the 31st day of the fourth run of semi-continuous cultivation. These were equivalent to the growth rates of 0. basal culture medium with the addition of CO2 under non-aseptic conditions. corresponding to the growth rate of 0. empty triangle. It should be noted here that the optimum carrying capacity of the system was reached in the third run.55×105 cells ml−1. empty square. the maximum cell concentration of 8. was found in nonsterilized RM medium on the 24th day of the third run of semi-continuous cultivation with a renewal rate of 50%. which corresponds to the volumetric productivity of 0. Filled triangle. RM culture medium with the addition of CO2 under nonaseptic conditions. which indicated that cells could adjust well to the growth conditions. After that.135 g l−1 day−1 (2. As shown in Fig. The maximum cell concentration of 10. this could have caused an increase in the cost of recovering the biomass. was obtained in non-sterilized RM medium on the 10th day of the first run of semi-continuous cultivation. pluvialis were simultaneously investigated for 31 days of semi-continuous cultivation period. 2.25× 105 cells ml−1. The maximum cell concentration. This was to control the cell concentration for the next run. basal culture medium without the addition of CO2 under non-aseptic conditions.109 g m−2 day−1) . filled square.157 day−1. the cultures in basal and RM media were harvested at about 50% of the volume of the culture on the 17th day of cultivation.
797 17 17 17 17 0.155 0. To evaluate the semi-continuous cultivation.844 2.851 3. as shown in Fig.781 1.179 2.652 3.415 2.226 0.685 0. 2.149 0.630 3.180 0. The result of semi-continuous cultivation means that the next run could be achieved successfully with higher specific growth rates by using correct renewal rates.672 0.658 0.221 0.182 0.702 0.086 0.601 0.850 4. In other words. It is possible that higher renewal rates could have caused an inhibition of cell division. the experiment was over.685 0. Semi-continuous culture is also useful for long-term cultivation. .745 0.191 0.109 2.175 4.748 0. green vegetative cells were produced in semi-continuous cultures . When a slowdown was observed in the fourth run compared to those grown in other runs.135 0.243 0.844 Cb (g l−1) µ (day−1) DT (day) Pbv (g l−1 day−1) Pba (g m−2 day−1) cultivation so the experiment was finished on the 31st day of semi-continuous cultivation.151 0.099 0.835 0. Several experiments have been carried out by different authors on the growth of Haematococcus sp.114 0.516 2.802 0.828 1.Appl Biochem Biotechnol Table 1 Results of obtaining kinetic parameters of H.177 0.181 0. the runs were compared.151 4. in order to obtain maximum biomass productivity.132 0.672 0.108 0.135 0.590 3.531 2.107 0.194 0. The second run was 8.547 2.109 1.217 4.852 3.558 3.920 2.688 0. pluvialis cultured semi-continuously in different conditions.062 3.672 1.820 0.104 0.143 0.591 3.748 0.615 0. It is also worth noting that the renewal rate is the main factor affecting the cell concentration in semi-continuous cultivation.118 1.695 0. As reported by Fàbregas et al.167 0.2% higher than the first run.136 4.961 0.193 0. The third run was also 24% higher whereas the fourth run was 3% lower than the first run. the value of the renewal rate had exceeded the saturation point such as the well-known phenomenon of photoinhibition for the fourth run of semi-continuous cultivation.097 0. Discussion Batch culture is widely used for commercial cultivation of algae for its ease of operation and simple culture system .271 0.344 1.625 2.131 0.157 0.281 10 10 10 10 0.223 3.146 1.472 4. Day Run 1 Basal with CO2 Basal without CO2 Non-sterilized RM Sterilized RM Run 2 Basal with CO2 Basal without CO2 Non-sterilized RM Sterilized RM Run 3 Basal with CO2 Basal without CO2 Non-sterilized RM Sterilized RM Run 4 Basal with CO2 Basal without CO2 Non-sterilized RM Sterilized RM 24 24 24 24 31 31 31 31 0.047 2.
In the bubble column photobioreactor. There were natural buffering agents in basal culture medium. 0. who successfully achieved the semicontinuous culture. 64×106 cells l−1 day−1 was obtained with a daily renewal rate of 20%. no significant effect of CO2 in non-sterilized basal medium was recorded due to its buffering agents. cement plants.55 g l−1 day. with an overall biomass productivity of 0. Sodium glycerophosphate also makes the trace metal salts less prone to precipitation in the basal medium. The maximum daily biomass productivity. but only in the small scale (in 500 ml Erlenmeyer flask) with a productivity of 6. pluvialis in the tubular photobioreactor. Product quality is varied and maintained by adjusting the sterilization and process control for various final products depending on the different industrial .Appl Biochem Biotechnol maintained with daily renewal rates between 10% and 40%. Ethanol production as the main agro-industrial fermentation process is considered as an ideal source of carbon dioxide for algal cultivation as the gas can be used without costly purification.52 cells ml−1 day−1. Carbon dioxide uptake is accompanied by changes in pH. pluvialis was cultivated under photoautotrophic conditions in a bubble column with fed-batch addition of nutrients. has served different industries as astaxanthin producer. in the present study. and a cell number above 5×106 cells mL−1 with the overall productivity of 0.) were reached after 16 days. biomass concentrations of 7. the location of algae ponds near such plants is an important consideration in reducing algae production costs.41 g l−1 day. have been tried. On the other hand.4 g l−1. The highest cell productivity.31 day−1 and 5. and it is the best means available to control and maintain specific pH levels that are optimal for mass-cultivated species .0 g l−1 (d. however. pluvialis was successfully implemented with the optimal airlift bioreactor design and under optimal conditions. For the outdoor culture of H. Haematococcus sp.8 cells ml−1 day−1 . This procedure is thus particularly efficient for growing large-scale cultures of Haematococcus. Since pure carbon dioxide is expensive. the price of pure CO2 in Turkey is approximately US $1 kg−1 which is 25% more expensive than in European countries.40 g l−1 day−1was attained after 300 h . Therefore. all these possibilities remain unrealized and have not gained any economical importance . Trisaminomethane plays an important role as a buffer. Contaminants have eventually built up in the culture under non-sterilized conditions for the long-term bio-productions. The CO2–H2CO3–HCO3−–CO32− system is the most important buffer generally present in freshwaters. or fermentation. such as waste gas from industrial combustion process. the maximum biomass concentration reached was also 1. Therefore. . while effectively preventing contamination by other microorganisms without the need for expensive and time-consuming steps such as careful sterilization of all apparatus and media and the use of antibiotics or any other antimicrobial agents . The large-scale production of Haematococcus cells was achieved by the use of a relatively high initial Haematococcus cell concentration which permitted the Haematococcus cells to predominate immediately after their inoculation into the photobioreactor. the effect of CO2 on the growth of H. The specific growth rate and productivity of the semicontinuous culture were 0. so far. The semi-continuous culture of H. diesel engines. Consequently. respectively. H. This result was comparable to that reported by Hata et al. Buffers are used to prevent precipitation in the media with minimal pH change. was reached in the tubular photobioreactor for an average irradiance inside the culture of 130 μE/m2 s. . thereby preventing any possible contaminating microorganisms to grow in the photobioreactor. the basal medium was selected to indicate the effect of CO2.06 g l−1 day. As reported in 2007 by Habas Firm. especially nitrate. with an overall biomass productivity of 0. However. alternative and cheaper sources of this gas.wt. pluvialis could change depending on the culture medium used.
(2003). F. Journal of General Microbiology. Hagen. B. J. O. J. (2004). Bioresource Technology. 51. 19–24. 2. Conclusion In this study.. obtained in non-sterilized RM medium. 13.).. (1997). 62. The maximum areal productivity of 3. Chen. 111. Xylander. E. 281–288. (1991). Herdman. the cells grown in RM medium were more effective than the basal medium. & Palsson. (2001). 10. & Gong. H. Journal of Applied Phycology. 5.. 43–46. Handbook of microalgal culture: Biotechnology and applied phycology (2nd ed. 1: An introduction to microalgal biotechnology and role of photobioreactors.109 and 2. Torzillo. R. Rippka. Trends in Biotechnology.. Hence. T. Taroda. 1–61. K. Dalay. R. P. whereas on 31st day of the forth run of semi-continuous cultivation. Acknowledgment We wish to thank Ege University Scientific Research Fund for financial support. Applied Microbiology Biotechnology. was found in non-sterilized RM medium on the 24th day of the third run of semi-continuous cultivation at a renewal rate of 50% under the light intensity of 75 µmol photons m−2 s−1 in the vertical panel-type photobioreactor. respectively. In the fourth run of semi-continuous cultivation. C. no significant effect of CO2 in the basal medium was recorded due to its natural buffering agents. 395–402. 7.Appl Biochem Biotechnol applications. 7. pluvialis must be cultivated under sterilized conditions for the long-term bio-production. Journal of Applied Phycology.55×105 cells ml−1. Y. (2001). & Masojidek. 11. pp. 421–426. G. Faraloni. Journal of Applied Phycology. which corresponds to the growth rate of 0. & Tanaka.. long-term effects of sterilization must be tested especially for large-scale production which the control of the growth conditions is much more difficult during longer-term cultivation... 6. R.. Kocky. The results have demonstrated that the maximum cell concentration of 10.. H. vol. 8.. Hata.. was much higher than the 2. pluvialis was considered as a pigment producer for the aquaculture industry. growth increased by 21% in sterilized RM medium with respect to non-sterilized RM medium with a renewal rate of 60% on the 31st day. 38.. References 1. (1995). Javanmardian. International workshop and training course on photobioreactor. (1996). F.. Furthermore. (1999). RM culture medium was chosen to also demonstrate the effect of sterilization on the growth of H. by comparing the runs of semi-continuous cultivation. C. H. pp. N. C. 79–87.062 g m−2 day−1 were obtained on the 24th day of the third run of semi-continuous cultivation in the basal medium with the addition of CO2 and basal medium without the addition of CO2. 431–438. . 3. M. USA: Iowa State Press. 9. & Stanier. Richmond.. Izmir. Hasegawa. pluvialis. & Rothe. 1182–1189. 13.. Iowa. (1979). X.531 g m−2 day−1. J. Margalith. Dalay (Ed. Chen. the effect of CO2 was investigated in the basal medium. (2003).271 day−1 with the areal productivity of 3. Journal of Applied Phycology.. 6–7. In M..). the areal productivity decreased by only 37% in non-sterilized RM medium in comparison with sterilized RM medium. 14. Considering each run of the semi-continuous cultivation. In this study. Chen. M.828 g m−2 day−1 obtained in sterilized RM medium on the 24th day of the third run of semi-continuous cultivation. Ogbonna. M. Goksan. F. Y. A. H.. J. B. M. Turkey: Ebiltem. 127–136.531 g m−2 day−1. Deruelles. M. C. Grünewald. Hence. 4. 15. H. Chen. Biotechnology and Bioengineering. and similar areal productivities of 2. & Johns.
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