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An economic assessment of astaxanthin production by large scale cultivation of Haematococcus pluvialis
Biotechnology Advances 29 (2011) 568–574
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Biotechnology Advances j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / b i o t e c h a d v
Research review paper
An economic assessment of astaxanthin production by large scale cultivation of Haematococcus pluvialis Jian Li a, Daling Zhu b, Jianfeng Niu c, Songdong Shen d, Guangce Wang c,⁎ a
College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, P.R. China College of Marine Science and Engineering, Tianjin University of Science and Technology, Tianjin 300457, P.R. China Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, P.R. China d College of Life Sciences, SooChow University, Suzhou 215123, P.R. China b c
a r t i c l e
i n f o
Article history: Received 5 January 2011 Received in revised form 2 April 2011 Accepted 3 April 2011 Available online 9 April 2011 Keywords: Microalgae Astaxanthin Haematococcus pluvialis Process economics Photobioreactor Biofuels
a b s t r a c t Although natural sources have long been exploited for astaxanthin production, it is still uncertain if natural astaxanthin can be produced at lower cost than that of synthetic astaxanthin or not. In order to give a comprehensive cost analysis of astaxanthin production from Haematococcus, a pilot plant with two large scale outdoor photobioreactors and a raceway pond was established and operated for 2 years to develop processes for astaxanthin production from Haematococcus. The developed processes were scaled up to a hypothetical plant with a production capacity about 900 kg astaxanthin per year, and the process economics was preliminarily assessed. Based on the analysis, the production cost of astaxanthin and microalgae biomass can be as low as $718/kg and $18/kg respectively. The results are very encouraging because the estimated cost might be lower than that of chemically synthesized astaxanthin. © 2011 Elsevier Inc. All rights reserved.
Contents 1. 2.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Description of the production processes at pilot scale . . . . . . . . . . 2.1. Strains and media . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Methods of parameter measurements . . . . . . . . . . . . . . 2.3. The outdoor cell culture systems and downstream equipments . . 2.4. Production processes at pilot scale . . . . . . . . . . . . . . . . 2.5. Process parameter optimization and estimation . . . . . . . . . . 3. Assessment of process economics . . . . . . . . . . . . . . . . . . . 3.1. Process scale up . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Analysis of fixed capital investment . . . . . . . . . . . . . . . 3.3. Analysis of annual operational cost of production . . . . . . . . . 4. Results and discussions . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Production cost of natural astaxanthin . . . . . . . . . . . . . . 4.2. The reasons for the low cost production of natural astaxanthin . . 4.3. Raceway ponds versus photobioreactors . . . . . . . . . . . . . 4.4. The two step approach versus one step approach . . . . . . . . . 4.5. The chances to further decrease production cost of designed facility 5. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
⁎ Corresponding author. Tel.: + 86 532 82898574; fax: + 86 532 82898612. E-mail address: [email protected] (G. Wang). 0734-9750/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.biotechadv.2011.04.001
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1. Introduction Astaxanthin is a carotenoid naturally synthesized in some plants, algae and bacteria, and is amassed in some fishes, crustaceans and birds through food chains in nature. The primary applications of astaxanthin are in aquaculture and dietary supplements (HigueraCiapara et al., 2006). As an aquacultural feed additive, it is essential for salmon and trout farming (Lorenz and Cysewski, 2000). As a dietary supplement, it has effects of anti-aging, anti-inflammatory, sun proofing, and immune system boosting et al. (Guerin et al., 2003; Hussein et al., 2006; Lorenz and Cysewski, 2000). Astaxanthin is commercially available either from chemical synthesis or natural resources such as microalgae, yeast and crustacean byproducts (Higuera-Ciapara et al., 2006). The commercial astaxanthin is dominated by synthetic astaxanthin with applications in aquaculture. The total market value of synthetic astaxanthin is more than $200M per year, and the major manufacturers are DSM in Netherland, BASF in France and NHU in China. The estimated cost of production is about $1000/kg, and the market price is above $2000/kg (Milledge, 2010; Olaizola, 2003). Although chemical synthesis can provide a steady source of astaxanthin at large quantities, there are still concerns about its biological functions and food safety (Newsome, 1986). Synthetic astaxanthin is different in isomerism and chemical structure with natural astaxanthin. Synthetic astaxanthin is a mixture of three isomers, namely (3S, 3′S), (3R, 3′S), and (3R, 3′R) with a proportion ratio of 1:2:1 respectively, and is not esterified on its hydroxyl groups, but astaxanthin from microalgae is exclusively (3S, 3′S) or (3R, 3′R) isomers and mostly esterified by fatty acids (Higuera-Ciapara et al., 2006; Yuan and Chen, 1997). Moreover, the fact that synthetic astaxanthin is derived from petrochemicals raises the issues of food safety, pollution, and sustainability. Therefore the chemical astaxanthin is only allowed to be used in aquaculture, not allowed for human consumption and animal feed other than aquacultural applications. Due to the high production cost of synthetic astaxanthin and the market demand for natural astaxanthin, the biological sources of astaxanthin have long been widely exploited (Johnson and An, 1991). Among organisms such as microalgae, yeast, and crustaceans exploited, Haematococcus pluvialis, a freshwater microalga, was found out containing the highest level of astaxanthin, resulting in the belief that Haematococcus might provide a natural and inexpensive source of astaxanthin (Ausich, 1997; Bubrick, 1991). Extensive researches have been done to develop bioprocesses to produce astaxanthin from Haematococcus for the last three decades, and even several plants have been established (Del Campo et al., 2007; Milledge, 2010). But until today, it is still uncertain if natural astaxanthin can be produced at lower cost than synthetic astaxanthin or not although some researchers suggest that natural astaxanthin from Haematococcus be able to compete against synthetic astaxanthin on prices, based on a two step process employing both photobioreactors and raceway ponds (Guerin et al., 2003; Olaizola, 2003), and there is even not a report available analyzing the production cost of natural astaxanthin from Haematococcus. This paper is to give a preliminary economic assessment of production cost of natural astaxanthin from Haematococcus, trying to confirm the prediction that natural astaxanthin can be less expensively produced than synthetic astaxanthin with currently available technologies at low cost locales such as China. In addition, this paper would also be informative for those who develop processes for biofuels production by large scale cultivation of microalgae (Stephens et al., 2010; Wijffels and Barbosa, 2010). 2. Description of the production processes at pilot scale 2.1. Strains and media Six strains of algae Haematococcus pluvialis were obtained from Freshwater Algae Culture Collection at Institute of Hydrobiology, The
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Chinese Academy of Sciences, and one of them was screened out for outdoor pilot trials. The liquid medium for use in laboratory and outdoor photobioreactors contains 10 mM KNO3, 2 mM Na2HPO4, 0.5 mM CaCl2, 0.5 mM MgSO4, 2 mM NaHCO3, supplemented with a mixture of micronutrients containing, in final concentration, 50 μM H3BO3, 50 μM EDTA, 10 μM MnCl2, 5 μM FeCl3, 2 μM Na2MnO4, 1.5 μM NaVO3, 0.8 μM ZnSO4, 0.4 μM CuSO4, 0.2 μM CoCl2, 10 μg/L biotin, 1 μg/ L vitamin B12 and CO2 as carbon source. The medium recipe was referenced from the recent research reports (Gong and Chen, 1998; Hagen et al., 2001; Orosa et al., 2005) and modified by our studies (unpublished dada). The medium was sterilized with a 1 μm membrane filter before use in laboratory and with a 2 μm membrane filter before use in outdoor photobioreactors. The liquid medium for pond culture contains 2 mM NaHCO3 with automatic pH controlled addition of CO2, and the medium was subject to 5 μm bag filtration before pumping into ponds. 2.2. Methods of parameter measurements The cell number concentrations were determined by cell counting using a hematocytometer under a microscope. The biomass dry weight concentrations were determined by filtering culture through a dried 2 μm filter membrane and drying the filter with the algae cells in an oven until constant weight. The astaxanthin content was analyzed by extraction procedures and spectrophotometric readings at 477 nm according to the methods reported by Nobre et al. (2006). 2.3. The outdoor cell culture systems and downstream equipments Two large air-lifting tubular photobioreactors made of plastics were designed and constructed outdoors, one with capacity of 1000 L and another 8000 L. The small reactor had a downcomer and a riser, and the big bioreactor had two downcomers and two risers. Helical titanium heat exchangers were installed inside of the risers to control the temperature of the bioreactors. The temperature of both reactors could be controlled below 25 C by coolers. A CO2 injector controlled by a pH controller was installed on the bottom of the downcomers. The aeration was provided by high pressure air pumps through the sparger at the bottom of the riser. Both the air and CO2 were sterilized with 0.2 μm membrane filter before being injected into reactors. Each bioreactor was installed with 2 μm membrane filters for medium sterilization. A raceway pond of typical design with an area of 100 m2 was built to cultivate cells for astaxanthin accumulation (Goldman, 1979). The pH of culture in the pond was kept constant by controlled injection of CO2 into pond culture, and the cells were kept in suspension by the turbulence provided by the paddlewheel powered by a 400 W motor. A centrifuge (SCS600, China) was used to dehydrate the cell slurry from sedimentation tank, and the resulting algae paste was dried into powder by a spray dryer (LPG-5, China). The dried cells of Haematococcus were cracked by a fluidized bed airflow pulverizer (JZL100, China). 2.4. Production processes at pilot scale The pilot facility was established in Shenzhen, China, a city located at 22 32′ 0″ N/114 8′ 0″ E with an annual average temperature of 22 C and about 2200 h per year sunshine time. A typical two step approach was employed to cultivate Haematococcus to produce astaxanthin. Firstly, vegetative cells were scaled up and cultivated in 8000 L reactors semi-continuously to provide the inocula for the pond, and secondly in the pond, the encystment of cells and accumulation of astaxanthin took place. The axenic culture of Haematococcus was started in laboratory and scaled up to 10 plastic carboys each with 20 L culture. The 1000 L bioreactor was sterilized thoroughly by hydrochloride and ozone and filled with 800 L filtered media, and then the
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culture in 10 carboys was sterilely inoculated into the 1000 L reactor. After several days of cultivation, the culture in 1000 L reactor was transferred to sterilized 8000 L reactor as inoculum. Again after several days of cultivation, the 8000 L reactor began to be operated semi-continuously to provide cell culture for pond cultivation. The cells were cultivated in pond for several days for cell encystment and astaxanthin accumulation. The encysted cells were subject to sedimentation for several hours in the pond by stopping the paddlewheel rotating. The supernatant was released to sump, and the deposited cell slurry was collected and pumped to a tank for further sedimentation. The concentrated cell slurry was further concentrated by centrifugation, and the resulted algae paste was then transferred to a spray dryer and dried into powder. If necessary, the cells of Haematococcus powder could be broken by the airflow pulverizer. The process flow diagram of astaxanthin production from Haematococcus is illustrated in Fig. 1. 2.5. Process parameter optimization and estimation The pilot plant had been operated for 2 years, and the process parameters were preliminarily optimized and estimated. The culture temperature in photobioreactors was controlled bellow 25 C by a 5 kW cooler for the 1000 L bioreactor and two 15 kW coolers for the 8000 L bioreactor respectively. The power consumption varied with weather conditions and ranged from 0 to 30 kWh and 0 to 200 kWh for small and big bioreactors respectively. The average values were about 10 kWh and 50 kWh for the whole three seasons of spring, summer and fall. The aeration rate was controlled at 0.05 v/v/min by air pumps at 0.03 MPa. Cell cultures in bioreactors were usually started at a cell concentration of 5–8 × 104 cells/ml, and cell concentration usually increased to above 5 × 105 cells/ml in 4– 5 days. The highest concentration of cell concentration in bioreactors reached to about 1 × 106 cells/ml. The small bioreactor served to provide seed culture for the large bioreactor. After the cell concentration reached 5 × 105 cells/ml, the 8000 L bioreactor was operated semi-continuously with a daily medium renewal rate of 25%. Except for extremely bad weather conditions such as rainfalls or in winters, the bioreactor could maintain a cell concentration level above 5 × 105 cells/ml. The pH of bioreactor cell culture was controlled at 7.5,
and the CO2 consumption was about 1.5 kg and 4.5 kg per day for 1000 L and 8000 L bioreactors respectively. The data of last 9 batches of the pond operations were used to estimate pond process parameters. The pond medium was prepared at a total volume of 13 tons, and was inoculated with 2 tons of cell culture from the 8000 L bioreactor. The pond pH was maintained at 8.0 by controlled addition of CO2, and the flow velocity of pond culture was controlled at 0.3 m/s during the full course of pond culture. The average CO2 consumption was about 2 kg per day for the 100 m2 pond, and the power supply of the paddle wheel was 0.4 kW. It took averagely about 8 to 9 days for the pond culture to reach the highest biomass and astaxanthin level at about 0.4 g/L and 12 mg/L respectively. The average maximal level of astaxanthin content in the pond was about 2.8% on dry biomass basis. The sedimentation of cells in the pond took about 6–8 h, and the volume of collected cell slurry was about 1500 L. The tank sedimentation took about 12 to 24 h, and the slurry for centrifugation was reduced to about 300 L. The slurry was further dehydrated by centrifugation to about 40 kg algae paste, and the paste was dried into about 6 kg powder to be pulverized. The average power consumption of the pulverizer to process 1 kg cell powder was about 3.5 kWh, and the average astaxanthin content of the final biomass was about 2.5%. All process parameters were summarized in Table 1. 3. Assessment of process economics 3.1. Process scale up Based on the estimated process parameters, the pilot operation was scaled up, and a 900 kg astaxanthin (about 36 ton biomass) per year facility was conceptually designed to be located in Kunming, China, a place with more sunshine time and less temperature variations than Shenzhen, China. The major components of the facility would include a laboratory, 1000 L reactors, 8000 L reactors, raceway ponds, tanks, centrifuges, a spray dryer, a pulverizer, downstream process buildings, storage rooms, laboratory rooms, bioreactor media station, pressured air supply, CO2 tank station and compressed air station et al. Assuming biomass could be harvested 30 times per year for each pond, 20 raceway ponds each with an area of 1000 m2
Fig. 1. Process flow diagram of astaxanthin production from Haematococcus.
J. Li et al. / Biotechnology Advances 29 (2011) 568–574
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Table 1 Summarization of process parameters. Photobioreactor process control parameters
Values
Ranges
Bioreactor culture temperature Bioreactor aeration rate Starting cell concentration of the 1000 L bioreactor Final cell concentration for the 1000 L bioreactor Starting cell concentration of the 8000 L bioreactor Daily renewal rate of the 8000 L bioreactor
Below 25 C 0.05 v/v/min 6 × 104 cells/ml 5 × 105 cells/ml 6 × 104 cells/ml 0.25 per day
10–25 C
Photobioreactor process performance parameters
Estimated values
Ranges
Power consumption for cooling the 1000 L bioreactor Power consumption for cooling the 8000 L bioreactor Aeration power input for the 1000 L bioreactor Aeration power input for the 8000 L bioreactor Days for 1000 L bioreactor cell culture to be ready Days for 8000 L bioreactor cell culture to be ready Working cell concentration for the 8000 L bioreactor Semi-continuous operation of the 8000 L bioreactor CO2 consumption of the 1000 L bioreactor CO2 consumption of the 8000 L bioreactor
10 kWh per day 50 kWh per day 0.1 kW 0.4 kW 5 days 5 days 5 × 105 cells/ml 40 days 1.5 kg per day 4.5 kg per day
0–30 kWh per day 0–200 kWh per day
4–6 days 4–6 days 4.2–6.0 × 105 cells/ml 30–60 days 1.0–2.0 kg per day 3.5–5.0 kg per day
Pond process control Parameters
Values
Ranges
Pond culture depth Pond water flow speed Pond inoculation cell concentration
15 cm 30 cm/s 6 × 104 cells/ml
5–7 × 104 cells/ml
5–8 × 104 cells/ml 5–7 × 105 cells/ml 5–7 × 104 cells/ml
Pond process performance parameters
Estimated values
Ranges
Pond reddening days Final pond culture concentration in biomass dry weight Pond CO2 consumption Pond power input
9 days 0.4 g/L 2 kg per day 400 W
8–9 days 0.27–0.55 g/L 1.5–2.5 kg per day
Downstream process parameters
Estimated values
Ranges
Dry biomass in cell slurry Dry biomass in cell paste Astaxanthin content in dry biomass Power consumption for centrifugation Oil consumption for spray drying Power consumption of cell pulverization
1.4% 13.5% 2.5% 3.0 kWh/kg biomass 1.5 kg oil/kg biomass 3.5 kWh/kg biomass
1.2%–1.5% 10%–15.5% 2.1%–3.2% 2.5–3.5 kWh/kg 1.2–1.5 kg/kg 3.2–3.6 kWh/kg
would give yearly biomass yield of about 36 tons. 20 big bioreactors would be needed to provide 40 tons of inoculum culture for two ponds each day. Assuming that 8000 L bioreactor could be operated at semi-continuous mode for about one month, another 6 small and big bioreactors would be required to prepare fresh 8000 L bioreactors and maintain the simultaneous operation of 20 big bioreactors at semicontinuous state. Each day one 1000 L and one 8000 L reactor would be initiated, and 10 carboys of seed culture from laboratory would be used as inoculum for the small bioreactor. Each day, two ponds, each with 150 ton culture, would be harvested, and each pond would yield about 15 tons of cell slurry. The slurry would be pumped to two 15 ton tanks for 24 h sedimentation. Each day about 6000 L concentrated slurry would be transferred to centrifuges. 4 centrifuges each with 200 L/h capacity would be employed for further dehydration, and the produced 800 kg algae paste would be dried by a dryer with hourly evaporation rate of about 100 kg water. The dried powder would be finally pulverized into broken pieces by a fluidized bed airflow pulverizer with an hourly capacity of 20 kg powder biomass. The total land occupation of the facility was assumed to be 25,000 m2, and the construction area for laboratory, offices, downstream process and product storage was assumed to be 500 m2. The media station would be constructed with 5 stock solution tanks each with a volume of 4000 L and a set of 5 μm filter system. The media would be mixed and filtered by 5 μm filter system then pumped to bioreactors individually equipped with a 2 μm filter. The pond media would be mixed and delivered to pond via plastic media pipes. The aeration of bioreactors would be collectively provided by two roots blowers each with a
capacity of 6 m3/min air. The total CO2 consumption for bioreactors and ponds was estimated at about 550 kg per day, and 20 ton CO2 storage tank would be constructed. A 5 kW air compressor would be installed to provide high pressure air for control purposes. 3.2. Analysis of fixed capital investment All the cost data are based on design and operational parameters of conceptually designed facility and the actual quotes or transacted prices. The land required is about 25,000 m2, and the cost for land acquisition and improvements is estimated at about $12/m2 and totally about $300,000 at Kunming, China. The building area unit cost is about $300/m2, and the total cost is $150,000. The cost of the laboratory instruments for seed cell culture and process parameter analysis is about $35,000. The costs of photobioreactors with individual medium and air filters are about $12,000 and $8000 for 8000 L and 1000 L bioreactors respectively. The total capital cost of all photobioreactors is about $360,000. The media station with 5 stock tanks and a set of 5 μm filter system for photobioreactors costs about $12,000. The low pressure air system including two roots blowers each with 5 kW power costs $6000. The pond cost including paddlewheels and CO2 diffusers averages about $12/m2, and thus the total 20 ponds costs about $240,000. The tank of 15 m3 costs $4000 each, and four tanks are to be installed. A 20 ton high pressure tank is to be used to store CO2, and together with CO2 tubing costs about $40,000. The centrifuge costs $8000 each, and 4 centrifuges installed to process about 6000 L concentrated cell slurry totals
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$32,000. The spray dryer costs about $30,000, and the pulverizer costs about $60,000. The compressor and tubing system costs about $2500. The pumps, valves, piping and control units for culture transfer are estimated to cost about $10,000. The electrical supply including a transformer and power cables costs $25,000. The project engineering and supervision are estimated at about $50,000, and the construction expenses are estimated to be $100,000. The total fixed capital investment is about $1.47M and is itemized in Table 2. Comparing with a Spirulina facility with the similar area, the designed Haematococcus facility costs about $0.5M more. The extra cost can be contributed to the expenses of photobioreactors, centrifuges, and the pulverizer which are not necessary for Spirulina production, and the other expenses would be almost equivalent (Vonshak, 1992).
4. Results and discussions
3.3. Analysis of annual operational cost of production The operational cost of production includes raw materials, utilities, and labor et al. The major chemicals for culture media are potassium nitrate, sodium phosphate and sodium bicarbonate, and the total annual consumption is about 15 tons, 3 tons and 150 tons respectively. The total cost is about $80,000. The consumption of other nutrients is totally estimated as 3 tons per year at $2000 per ton. The consumption of CO2 is predicted to be 150 tons per year, and the total cost is about $22,500. The total water consumption is about 90,000 tons, and the total cost is about $27,000 per year. According to our experiences, the medium filter for each photobioreactor needs to be replaced every two months, and the air filter needs to be replaced every three months. For about 32 bioreactors, about 170 units of medium filters and 130 units of air filters are to be consumed per year. The medium filter and air filter cost about $100 and $60 respectively, and the total costs are $17, 000 and $7800 per year. The other consumables both for laboratory and bioreactor application are estimated to cost $12,000 per year. The power consumption arises from bioreactor cooling, low pressure air supply, compressed air supply, pond paddle wheeling, cell pulverization, pumping and control et al. The estimated consumption is about 270,000 kWh, 72,000 kWh, 7500 kWh, 288,000 kWh, 36,000 kWh, and 43,200 kWh for cooling, paddling, pulverization, low pressure air, compressed air, and pumping respectively, and the costs are about $36,000, $9600, $1000, $38,400, $4800 and $5760 respectively. It takes 48 tons of oil to
Table 2 Summarization of fixed capital cost for the conceptually designed facility. Items 1. Land acquisition and improvements 2. Building area 3. Laboratory instruments 4. 8000 L bioreactor systems 5. 1000 L bioreactor systems 6. Medium supply station for bioreactors 7. Low pressure air supply 8. Raceway ponds 9. Harvest cell broth storage tank 10. CO2 storage tank 11. Centrifuge 12. Dryer 13. Pulverizer 14. Air compressor and tubing 15. Pumps, valves, piping and control 16. Electrical 17. Engineering and supervision 18. Construction expenses Total fixed cost
Unit costs
Quantities
Units
$12
25,000
m2
$300,000
$300 $35,000 $12,000 $8,000 $12,000
500 1 26 6 1
m2 Set Set Set Set
$150,000 $35,000 $312,000 $48,000 $12,000
$3,000 $12 $4,000 $40,000 $8,000 $30,000 $60,000 $2,500 $10,000
2 20,000 4 1 4 1 1 1 1
Set m2 Units Unit Units Unit Unit Set Set
$6,000 $240,000 $16,000 $40,000 $32,000 $30,000 $60,000 $2,500 $10,000
$25,000 $50,000 $100,000
1 1 1
Set
process 36 tons dry biomass, and 48 tons of oil cost about $48,000. About 19 workers and 4 supervisors or chemists are required to operate the production, and the total estimated expenses are $95,000 and $32,000 respectively. The maintenance including all the replacing machinery parts and outsourcing repairing work is estimated to be about $20,000 per year. The general plant overhead is estimated to be $50,000 per year. The total annual operational cost of production is about $0.5M and is summarized in Table 3. Comparing with a Spirulina facility with the similar area, the Haematococcus facility would cost much more. The labor, supervision, maintenance and electricity expenses would be substantially reduced because the photobioreactors, the centrifuges and the pulverizer would not be operated in a Spirulina facility (Vonshak, 1992).
Total cost ($)
$25,000 $50,000 $100,000 US$1,468,500
4.1. Production cost of natural astaxanthin For the conceptually designed facility, the total fixed capital investment and the total direct cost of production per year are estimated to be $1,468,500 and $499,705 respectively. The direct production of cost of biomass and astaxanthin would be about $14/kg and $555/kg. If a 10 year depreciation of fixed capital cost is adopted, the unit production cost of biomass and astaxanthin would be $18/kg and $718/kg respectively. If a 5 year depreciation of fixed capital cost is adopted, the unit production cost of biomass and astaxanthin would be $22/kg and $882/kg respectively. The production cost of synthetic astaxanthin by DSM, BASF, and NHU is estimated to be about $1000/ kg astaxanthin, and therefore the conceptually designed facility would be able to produce natural astaxanthin at a cost lower than that of synthetic astaxanthin.
4.2. The reasons for the low cost production of natural astaxanthin There are several established facilities, Cyanotech Inc., Algatechnologies, Ltd., Mera Pharmaceuticals Inc., and Biogenic Inc., et al.(Del Campo et al., 2007; Milledge, 2010), producing astaxanthin from Haematococcus world widely, and their production costs are estimated to be above $3000/kg astaxanthin, which is much higher than the estimated level in this research, namely $718/kg. There might be several reasons accounted for the low cost of our predicted production. Firstly photobioreactors developed and employed in this research are proved to be very efficient and inexpensive. The big photobioreactor with a culture carrying capacity of 8000 L can be operated at 5 × 105 cells/ml cell concentration at semi-continuous state for a period of two months and can be built at a cost of $12,000. This cost level is less than one third of current industrial level with the same total culture volume and similar performance (Kaewpintong et al., 2007; Kunjapur and Eldridge, 2010; Molina Grima et al., 2003). Secondly raceway ponds instead of photobioreactors are used as the primary microalgae cell cultivation tools, and photobioreactors are only used to provide vegetative cells for raceway ponds. The cost of building raceways is only about one fifteenth of photobioreactors with the same culture carrying capacity in our situations. At the same time, the maintenance and consumables cost of raceway ponds is only about 5% of that of photobioreactors according to our analysis. In fact, some of current operations of astaxanthin production from Haematococcus employ only photobioreactors for both vegetative cell proliferation and astaxanthin accumulation. Thirdly the low cost labor, land and utilities in China substantially reduce the production cost of natural astaxanthin. For example, if the same production facility were established in USA, the labor would approximately cost $600 per kg astaxanthin while only about $120 per kg in China according to analysis.
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Table 3 Summarization of operational costs for the conceptually designed facility. Items
Unit costs
Quantities
Units
Total costs
1. Potassium nitrate 2. Disodium hydrogen phosphate 3. Sodium bicarbonate 4. Carbon dioxide 5. Other nutrients 6. Water and waste water treatment 7. Medium filters 8. Air filters 9. Other consumables 10. Power for bioreactor cooling 11. Power for low pressure air 12. Power for compressed air 13. Power for pond paddling 14. Power for cell pulverization 15. Power for pumping 16. Oil consumption for the dryer 17. Labor 18. Supervision 19. Maintenance 20. General plant overheads Total direct cost of production Direct production cost of biomass Direct production cost of astaxanthin Depreciation of fixed capital cost (10 years) Unit production cost of biomass Unit production cost of astaxanthin Depreciation of fixed capital cost (5 years) Unit production cost of biomass Unit production cost of astaxanthin
$500 $800 $250 $200 $2,000 $0.3 $100 $60 $8,000 $0.15 $0.15 $0.15 $0.15 $0.15 $0.15 $1,000 $5,000 $8,000 $20,000 $50,000
15 3 150 170 3 90,000 170 130 1 270,000 72,000 7500 288,000 36,000 43,200 48 19 4
Tons Tons Tons Tons Tons Tons Pieces Pieces
$7,500 $2,400 $37,500 $34,000 $6,000 $27,000 $17,000 $7,800 $8,000 $40,500 $10,800 $1,125 $43,200 $5,400 $6,480 $48,000 $95,000 $32,000 $20,000 $50,000 US$499,705 US$14 US$555 US$146,850 US$18 US$718 US$293,700 US$22 US$882
4.3. Raceway ponds versus photobioreactors Raceways ponds have long been used in commercial cultivation of microalgae, but because raceway ponds are open to air and subject to contamination, only several species, such as Spirulina, Chlorella, and Dunaliella, could have been successfully cultivated in raceway ponds (Milledge, 2010). To overcome the problem of contamination, a bunch of photobioreactors have been developed to cultivate microalgae at large scales. Though developed photobioreactors provide a closed and controlled cultivation environment for microalgae and are able to cultivate most microalgae species at large scale, the building and maintenance expenses of photobioreactors are much higher than raceways ponds. For example, the building cost of photobioreactors reported in this paper are fifteen times more expensive than raceway ponds with the same culture volume, and the maintenance fees are even 20 times higher. If only our developed photobioreactors and no raceway ponds are employed to cultivate Haematococcus for astaxanthin production at 900 kg per year capacity, the production cost of astaxanthin would be more than $3600/kg according to our analysis, which would be about 4.5 times higher than the estimated production cost of conceptually designed facility and might be the cases of some current commercial operation of astaxanthin production from Haematococcus. In this research and design, photobioreactors serve to provide vegetative cell culture for raceway ponds, and raceway ponds are operated at batch mode for a short period of time to avoid contamination. This approach was suggested for large scale cultivation of microalgae to produce biofuels (Huntley and Redalje, 2007), and the data reported in this paper could serve as an evidence to support for the suggestion, which was especially suitable for algae species undergoing different stages with different nutrient requirements for lipid accumulation (Liu et al., 2008; Qiao et al., 2009). 4.4. The two step approach versus one step approach The typical two step approach has been employed to cultivate Haematococcus for astaxanthin production, which is characterized by
kWh kWh kWh kWh kWh kWh Tons People People
a quick dilution of cell culture to reduce the concentration levels of nutrients after vegetative growth of cells (Fábregas et al., 2001; Olaizola, 2000). Recent researches of astaxanthin production from Haematococcus suggest a one step approach which either controls the supply of nutrients to the cell culture or reduce the level of nutrients by growth of Haematococcus cells (Del Río et al., 2008; Esperanza Del Río et al., 2005; García-Malea et al., 2006, 2009; Zhang et al., 2009). In the former situation, cells can be cultivated and harvested continuously, but both the cell growth rate and astaxanthin content in dry biomass are much lower than those of two step approaches. In addition, to cultivate microalgae continuously, closed photobioreactor systems might need to be used. Therefore if substantial improvements are not achieved, the cost of production would be much higher than the current two step approach. In the latter situation, although a batch production is proposed in raceway ponds, the prolonged cell growth period in open environments would endanger cell culture to serious contamination problems if selective medium for Haematococcus is not successfully developed. A research work reported by Aflalo et al. (2007) also argues that two step approach is much more efficient than one step approach in terms of astaxanthin production. 4.5. The chances to further decrease production cost of designed facility The conceptually designed facility is based on preliminary researches at pilot scale in Shenzhen, China, and our experiences, industrial data and research reports do support the belief that the cost would be able to be further decreased substantially. The pilot scale experiments have been done in Shenzhen, China, and the conceptually designed facility would be located at a place with more sunshine and less temperature variations such as Kunming, China. The processes would be able to be further optimized, and the production cost should be able to be decreased substantially. The average content of astaxanthin in biomass of designed facility is only 2.5%, and the biomass product of 3.5% astaxanthin is commercially available from Biogenic Co., Ltd. at Kunming, China, and even higher level of astaxanthin has been reported in literatures (Aflalo et al., 2007; Bubrick, 1991; Harker et al., 1996;
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Margalith, 1999; Ranjbar et al., 2008). Recent researches on genomics and proteomics of Haematococcus might lead to genetic modification of Haematococcus to enhance astaxanthin accumulation in cells (Jin et al., 2006; Kathiresan and Sarada, 2009; Sandesh Kamath et al., 2008; Steiger and Sandmann, 2004). Apparently, the production cost would be proportionally reduced with the increase of average astaxanthin content in final biomass products assuming the same processes being used. 5. Conclusions Inexpensive production of astaxanthin by cultivation of Haematococcus can be realized with current available technologies. The production cost of astaxanthin by our conceptually designed facility is estimated to be $718/kg astaxanthin or about $18/kg biomass with 2.5% astaxanthin. The cost is much lower than that the current industrial operations and is even lower than that of synthetic astaxanthin. The reasons of the low cost predictions can be contributed to low cost photobioreactors employed, two step approach using raceways ponds, and low labor and other costs in China. The cost might be able to be further reduced with the advances of technologies and optimization of processes. Acknowledgments This work was supported by National Innovation Fund of China with the funding number 06C26214421651, by Science and Technology Planning Project of Shenzhen Municipality, China, with the funding number SZKJ0531, by Main Direction Program of Knowledge Innovation of Chinese Academy of Sciences with the funding number KGCX2-YW-374-3, and by the open fund of Tianjin Key Laboratory of Marine Resources and Chemistry with the funding number 200903. We appreciate SCW Medicath Ltd. for providing its building rooftop at Shenzhen, China, on which the pilot plant of this research was established. References Aflalo C, Meshulam Y, Zarka A, Boussiba S. On the relative efficiency of two-vs. onestage production of astaxanthin by the green alga Haematococcus pluvialis. Biotechnol Bioeng 2007;98:300–5. Ausich R. Commercial opportunities for carotenoid production by biotechnology. Pure Appl Chem 1997;69:2169–74. Bubrick P. Production of astaxanthin from Haematococcus. Bioresour Technol 1991;38: 237–9. Del Campo J, García-Gonzáez M, Guerrero M. Outdoor cultivation of microalgae for carotenoid production: current state and perspectives. Appl Microbiol Biotechnol 2007;74:1163–74. Del Río E, Acién F, Garcia Malea M, Rivas J, Molina Grima E, Guerrero M. Efficiency assessment of the one-step production of astaxanthin by the microalga Haematococcus pluvialis. Biotechnol Bioeng 2008;100:397–402. Esperanza Del Río F, García-Malea M, Rivas J, Molina-Grima E, Guerrero M. Efficient one-step production of astaxanthin by the microalga Haematococcus pluvialis in continuous culture. Biotechnol Bioeng 2005;91:808–15. Fábregas J, Otero A, Maseda A, Domínguez A. Two-stage cultures for the production of astaxanthin from Haematococcus pluvialis. J Biotechnol 2001;89:65–71. García-Malea M, Acién F, Fernández J, Cerón M, Molina E. Continuous production of green cells of Haematococcus pluvialis: modeling of the irradiance effect. Enzyme Microb Technol 2006;38:981–9. García-Malea M, Acién F, Del Río E, Fernández J, Cerón M, Guerrero M, et al. Production of astaxanthin by Haematococcus pluvialis: taking the one-step system outdoors. Biotechnol Bioeng 2009;102:651–7.
Goldman J. Outdoor algal mass cultures—I. Applications. Water Res 1979;13:1–19. Gong X, Chen F. Influence of medium components on astaxanthin content and production of Haematococcus pluvialis. Process Biochem 1998;33:385–91. Guerin M, Huntley M, Olaizola M. Haematococcus astaxanthin: applications for human health and nutrition. Trends Biotechnol 2003;21:210–6. Hagen C, Grünewald K, Xyländer M, Rothe E. Effect of cultivation parameters on growth and pigment biosynthesis in flagellated cells of Haematococcus pluvialis. J Appl Phycol 2001;13:79–87. Harker M, Tsavalos A, Young A. Autotrophic growth and carotenoid production of Haematococcus pluvialis in a 30 liter air-lift photobioreactor. J Ferment Bioeng 1996;82:113–8. Higuera-Ciapara I, Felix-Valenzuela L, Goycoolea F. Astaxanthin: a review of its chemistry and applications. Crit Rev Food Sci Nutr 2006;46:185–96. Huntley M, Redalje D. CO 2 mitigation and renewable oil from photosynthetic microbes: a new appraisal. Mitig Adapt Strat Glob Change 2007;12:573–608. Hussein G, Sankawa U, Goto H, Matsumoto K, Watanabe H. Astaxanthin, a carotenoid with potential in human health and nutrition. J Nat Prod 2006;69:443–9. Jin E, Lee C, Polle J. Secondary carotenoid accumulation in Haematococcus (Chlorophyceae): biosynthesis, regulation, and biotechnology. J Microbiol Biotechnol 2006;16:821. Johnson E, An G. Astaxanthin from microbial sources. Crit Rev Biotechnol 1991;11: 297–326. Kaewpintong K, Shotipruk A, Powtongsook S, Pavasant P. Photoautotrophic highdensity cultivation of vegetative cells of Haematococcus pluvialis in airlift bioreactor. Bioresour Technol 2007;98:288–95. Kathiresan S, Sarada R. Towards genetic improvement of commercially important microalga Haematococcus pluvialis for biotech applications. J Appl Phycol 2009;21: 553–8. Kunjapur A, Eldridge R. Photobioreactor design for commercial biofuel production from microalgae. Ind Eng Chem Res 2010;49:3516–26. Liu Z, Wang G, Zhou B. Effect of iron on growth and lipid accumulation in Chlorella vulgaris. Bioresour Technol 2008;99:4717–22. Lorenz R, Cysewski G. Commercial potential for Haematococcus microalgae as a natural source of astaxanthin. Trends Biotechnol 2000;18:160–7. Margalith P. Production of ketocarotenoids by microalgae. Appl Microbiol Biotechnol 1999;51:431–8. Milledge J. Commercial application of microalgae other than as biofuels: a brief review. Rev Environ Sci Biotechnol 2010:1-11. Molina Grima E, Belarbi E, Acien Fernandez F, Robles Medina A, Chisti Y. Recovery of microalgal biomass and metabolites: process options and economics. Biotechnol Adv 2003;20:491–515. Newsome R. Food colors. Food Technol-Chicago 1986;40:49–56. Nobre B, Marcelo F, Passos R, Beir o L, Palavra A, Gouveia L, et al. Supercritical carbon dioxide extraction of astaxanthin and other carotenoids from the microalga Haematococcus pluvialis. Eur Food Res Technol 2006;223:787–90. Olaizola M. Commercial production of astaxanthin from Haematococcus pluvialis using 25,000-liter outdoor photobioreactors. J Appl Phycol 2000;12:499–506. Olaizola M. Commercial development of microalgal biotechnology: from the test tube to the marketplace. Biomol Eng 2003;20:459–66. Orosa M, Franqueira D, Cid A, Abalde J. Analysis and enhancement of astaxanthin accumulation in Haematococcus pluvialis. Bioresour Technol 2005;96:373–8. Qiao H, Wang G, Zhang X. Isolation and characterization of Chlorella sorokiniana GXNN01 (Chlorophyta) with the properties of heterotrophic and microaerobic growth. J Phycol 2009;45:1153–62. Ranjbar R, Inoue R, Shiraishi H, Katsuda T, Katoh S. High efficiency production of astaxanthin by autotrophic cultivation of Haematococcus pluvialis in a bubble column photobioreactor. Biochem Eng J 2008;39:575–80. Sandesh Kamath B, Vidhyavathi R, Sarada R, Ravishankar G. Enhancement of carotenoids by mutation and stress induced carotenogenic genes in Haematococcus pluvialis mutants. Bioresour Technol 2008;99:8667–73. Steiger S, Sandmann G. Cloning of two carotenoid ketolase genes from Nostoc punctiforme for the heterologous production of canthaxanthin and astaxanthin. Biotechnol Lett 2004;26:813–7. Stephens E, Ross I, King Z, Mussgnug J, Kruse O, Posten C, et al. An economic and technical evaluation of microalgal biofuels. Nat Biotechnol 2010;28:126–8. Vonshak A. Microalgal biotechnology: is it an economic success? Biotechnology: Economic and Social Aspects: Issues for Developing Countries; 1992. p. 70. Wijffels RH, Barbosa MJ. An outlook on microalgal biofuels. Science 2010;329:796. Yuan JP, Chen F. Identification of astaxanthin isomers in Haematococcus lacustris by HPLC-photodiode array detection. Biotechnol Tech 1997;11:455–9. Zhang B, Geng Y, Li Z, Hu H, Li Y. Production of astaxanthin from Haematococcus in open pond by two-stage growth one-step process. Aquaculture 2009;295:275–81.
Contents lists available at ScienceDirect
Biotechnology Advances j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / b i o t e c h a d v
Research review paper
An economic assessment of astaxanthin production by large scale cultivation of Haematococcus pluvialis Jian Li a, Daling Zhu b, Jianfeng Niu c, Songdong Shen d, Guangce Wang c,⁎ a
College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457, P.R. China College of Marine Science and Engineering, Tianjin University of Science and Technology, Tianjin 300457, P.R. China Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, P.R. China d College of Life Sciences, SooChow University, Suzhou 215123, P.R. China b c
a r t i c l e
i n f o
Article history: Received 5 January 2011 Received in revised form 2 April 2011 Accepted 3 April 2011 Available online 9 April 2011 Keywords: Microalgae Astaxanthin Haematococcus pluvialis Process economics Photobioreactor Biofuels
a b s t r a c t Although natural sources have long been exploited for astaxanthin production, it is still uncertain if natural astaxanthin can be produced at lower cost than that of synthetic astaxanthin or not. In order to give a comprehensive cost analysis of astaxanthin production from Haematococcus, a pilot plant with two large scale outdoor photobioreactors and a raceway pond was established and operated for 2 years to develop processes for astaxanthin production from Haematococcus. The developed processes were scaled up to a hypothetical plant with a production capacity about 900 kg astaxanthin per year, and the process economics was preliminarily assessed. Based on the analysis, the production cost of astaxanthin and microalgae biomass can be as low as $718/kg and $18/kg respectively. The results are very encouraging because the estimated cost might be lower than that of chemically synthesized astaxanthin. © 2011 Elsevier Inc. All rights reserved.
Contents 1. 2.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Description of the production processes at pilot scale . . . . . . . . . . 2.1. Strains and media . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Methods of parameter measurements . . . . . . . . . . . . . . 2.3. The outdoor cell culture systems and downstream equipments . . 2.4. Production processes at pilot scale . . . . . . . . . . . . . . . . 2.5. Process parameter optimization and estimation . . . . . . . . . . 3. Assessment of process economics . . . . . . . . . . . . . . . . . . . 3.1. Process scale up . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Analysis of fixed capital investment . . . . . . . . . . . . . . . 3.3. Analysis of annual operational cost of production . . . . . . . . . 4. Results and discussions . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Production cost of natural astaxanthin . . . . . . . . . . . . . . 4.2. The reasons for the low cost production of natural astaxanthin . . 4.3. Raceway ponds versus photobioreactors . . . . . . . . . . . . . 4.4. The two step approach versus one step approach . . . . . . . . . 4.5. The chances to further decrease production cost of designed facility 5. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
⁎ Corresponding author. Tel.: + 86 532 82898574; fax: + 86 532 82898612. E-mail address: [email protected] (G. Wang). 0734-9750/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.biotechadv.2011.04.001
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1. Introduction Astaxanthin is a carotenoid naturally synthesized in some plants, algae and bacteria, and is amassed in some fishes, crustaceans and birds through food chains in nature. The primary applications of astaxanthin are in aquaculture and dietary supplements (HigueraCiapara et al., 2006). As an aquacultural feed additive, it is essential for salmon and trout farming (Lorenz and Cysewski, 2000). As a dietary supplement, it has effects of anti-aging, anti-inflammatory, sun proofing, and immune system boosting et al. (Guerin et al., 2003; Hussein et al., 2006; Lorenz and Cysewski, 2000). Astaxanthin is commercially available either from chemical synthesis or natural resources such as microalgae, yeast and crustacean byproducts (Higuera-Ciapara et al., 2006). The commercial astaxanthin is dominated by synthetic astaxanthin with applications in aquaculture. The total market value of synthetic astaxanthin is more than $200M per year, and the major manufacturers are DSM in Netherland, BASF in France and NHU in China. The estimated cost of production is about $1000/kg, and the market price is above $2000/kg (Milledge, 2010; Olaizola, 2003). Although chemical synthesis can provide a steady source of astaxanthin at large quantities, there are still concerns about its biological functions and food safety (Newsome, 1986). Synthetic astaxanthin is different in isomerism and chemical structure with natural astaxanthin. Synthetic astaxanthin is a mixture of three isomers, namely (3S, 3′S), (3R, 3′S), and (3R, 3′R) with a proportion ratio of 1:2:1 respectively, and is not esterified on its hydroxyl groups, but astaxanthin from microalgae is exclusively (3S, 3′S) or (3R, 3′R) isomers and mostly esterified by fatty acids (Higuera-Ciapara et al., 2006; Yuan and Chen, 1997). Moreover, the fact that synthetic astaxanthin is derived from petrochemicals raises the issues of food safety, pollution, and sustainability. Therefore the chemical astaxanthin is only allowed to be used in aquaculture, not allowed for human consumption and animal feed other than aquacultural applications. Due to the high production cost of synthetic astaxanthin and the market demand for natural astaxanthin, the biological sources of astaxanthin have long been widely exploited (Johnson and An, 1991). Among organisms such as microalgae, yeast, and crustaceans exploited, Haematococcus pluvialis, a freshwater microalga, was found out containing the highest level of astaxanthin, resulting in the belief that Haematococcus might provide a natural and inexpensive source of astaxanthin (Ausich, 1997; Bubrick, 1991). Extensive researches have been done to develop bioprocesses to produce astaxanthin from Haematococcus for the last three decades, and even several plants have been established (Del Campo et al., 2007; Milledge, 2010). But until today, it is still uncertain if natural astaxanthin can be produced at lower cost than synthetic astaxanthin or not although some researchers suggest that natural astaxanthin from Haematococcus be able to compete against synthetic astaxanthin on prices, based on a two step process employing both photobioreactors and raceway ponds (Guerin et al., 2003; Olaizola, 2003), and there is even not a report available analyzing the production cost of natural astaxanthin from Haematococcus. This paper is to give a preliminary economic assessment of production cost of natural astaxanthin from Haematococcus, trying to confirm the prediction that natural astaxanthin can be less expensively produced than synthetic astaxanthin with currently available technologies at low cost locales such as China. In addition, this paper would also be informative for those who develop processes for biofuels production by large scale cultivation of microalgae (Stephens et al., 2010; Wijffels and Barbosa, 2010). 2. Description of the production processes at pilot scale 2.1. Strains and media Six strains of algae Haematococcus pluvialis were obtained from Freshwater Algae Culture Collection at Institute of Hydrobiology, The
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Chinese Academy of Sciences, and one of them was screened out for outdoor pilot trials. The liquid medium for use in laboratory and outdoor photobioreactors contains 10 mM KNO3, 2 mM Na2HPO4, 0.5 mM CaCl2, 0.5 mM MgSO4, 2 mM NaHCO3, supplemented with a mixture of micronutrients containing, in final concentration, 50 μM H3BO3, 50 μM EDTA, 10 μM MnCl2, 5 μM FeCl3, 2 μM Na2MnO4, 1.5 μM NaVO3, 0.8 μM ZnSO4, 0.4 μM CuSO4, 0.2 μM CoCl2, 10 μg/L biotin, 1 μg/ L vitamin B12 and CO2 as carbon source. The medium recipe was referenced from the recent research reports (Gong and Chen, 1998; Hagen et al., 2001; Orosa et al., 2005) and modified by our studies (unpublished dada). The medium was sterilized with a 1 μm membrane filter before use in laboratory and with a 2 μm membrane filter before use in outdoor photobioreactors. The liquid medium for pond culture contains 2 mM NaHCO3 with automatic pH controlled addition of CO2, and the medium was subject to 5 μm bag filtration before pumping into ponds. 2.2. Methods of parameter measurements The cell number concentrations were determined by cell counting using a hematocytometer under a microscope. The biomass dry weight concentrations were determined by filtering culture through a dried 2 μm filter membrane and drying the filter with the algae cells in an oven until constant weight. The astaxanthin content was analyzed by extraction procedures and spectrophotometric readings at 477 nm according to the methods reported by Nobre et al. (2006). 2.3. The outdoor cell culture systems and downstream equipments Two large air-lifting tubular photobioreactors made of plastics were designed and constructed outdoors, one with capacity of 1000 L and another 8000 L. The small reactor had a downcomer and a riser, and the big bioreactor had two downcomers and two risers. Helical titanium heat exchangers were installed inside of the risers to control the temperature of the bioreactors. The temperature of both reactors could be controlled below 25 C by coolers. A CO2 injector controlled by a pH controller was installed on the bottom of the downcomers. The aeration was provided by high pressure air pumps through the sparger at the bottom of the riser. Both the air and CO2 were sterilized with 0.2 μm membrane filter before being injected into reactors. Each bioreactor was installed with 2 μm membrane filters for medium sterilization. A raceway pond of typical design with an area of 100 m2 was built to cultivate cells for astaxanthin accumulation (Goldman, 1979). The pH of culture in the pond was kept constant by controlled injection of CO2 into pond culture, and the cells were kept in suspension by the turbulence provided by the paddlewheel powered by a 400 W motor. A centrifuge (SCS600, China) was used to dehydrate the cell slurry from sedimentation tank, and the resulting algae paste was dried into powder by a spray dryer (LPG-5, China). The dried cells of Haematococcus were cracked by a fluidized bed airflow pulverizer (JZL100, China). 2.4. Production processes at pilot scale The pilot facility was established in Shenzhen, China, a city located at 22 32′ 0″ N/114 8′ 0″ E with an annual average temperature of 22 C and about 2200 h per year sunshine time. A typical two step approach was employed to cultivate Haematococcus to produce astaxanthin. Firstly, vegetative cells were scaled up and cultivated in 8000 L reactors semi-continuously to provide the inocula for the pond, and secondly in the pond, the encystment of cells and accumulation of astaxanthin took place. The axenic culture of Haematococcus was started in laboratory and scaled up to 10 plastic carboys each with 20 L culture. The 1000 L bioreactor was sterilized thoroughly by hydrochloride and ozone and filled with 800 L filtered media, and then the
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culture in 10 carboys was sterilely inoculated into the 1000 L reactor. After several days of cultivation, the culture in 1000 L reactor was transferred to sterilized 8000 L reactor as inoculum. Again after several days of cultivation, the 8000 L reactor began to be operated semi-continuously to provide cell culture for pond cultivation. The cells were cultivated in pond for several days for cell encystment and astaxanthin accumulation. The encysted cells were subject to sedimentation for several hours in the pond by stopping the paddlewheel rotating. The supernatant was released to sump, and the deposited cell slurry was collected and pumped to a tank for further sedimentation. The concentrated cell slurry was further concentrated by centrifugation, and the resulted algae paste was then transferred to a spray dryer and dried into powder. If necessary, the cells of Haematococcus powder could be broken by the airflow pulverizer. The process flow diagram of astaxanthin production from Haematococcus is illustrated in Fig. 1. 2.5. Process parameter optimization and estimation The pilot plant had been operated for 2 years, and the process parameters were preliminarily optimized and estimated. The culture temperature in photobioreactors was controlled bellow 25 C by a 5 kW cooler for the 1000 L bioreactor and two 15 kW coolers for the 8000 L bioreactor respectively. The power consumption varied with weather conditions and ranged from 0 to 30 kWh and 0 to 200 kWh for small and big bioreactors respectively. The average values were about 10 kWh and 50 kWh for the whole three seasons of spring, summer and fall. The aeration rate was controlled at 0.05 v/v/min by air pumps at 0.03 MPa. Cell cultures in bioreactors were usually started at a cell concentration of 5–8 × 104 cells/ml, and cell concentration usually increased to above 5 × 105 cells/ml in 4– 5 days. The highest concentration of cell concentration in bioreactors reached to about 1 × 106 cells/ml. The small bioreactor served to provide seed culture for the large bioreactor. After the cell concentration reached 5 × 105 cells/ml, the 8000 L bioreactor was operated semi-continuously with a daily medium renewal rate of 25%. Except for extremely bad weather conditions such as rainfalls or in winters, the bioreactor could maintain a cell concentration level above 5 × 105 cells/ml. The pH of bioreactor cell culture was controlled at 7.5,
and the CO2 consumption was about 1.5 kg and 4.5 kg per day for 1000 L and 8000 L bioreactors respectively. The data of last 9 batches of the pond operations were used to estimate pond process parameters. The pond medium was prepared at a total volume of 13 tons, and was inoculated with 2 tons of cell culture from the 8000 L bioreactor. The pond pH was maintained at 8.0 by controlled addition of CO2, and the flow velocity of pond culture was controlled at 0.3 m/s during the full course of pond culture. The average CO2 consumption was about 2 kg per day for the 100 m2 pond, and the power supply of the paddle wheel was 0.4 kW. It took averagely about 8 to 9 days for the pond culture to reach the highest biomass and astaxanthin level at about 0.4 g/L and 12 mg/L respectively. The average maximal level of astaxanthin content in the pond was about 2.8% on dry biomass basis. The sedimentation of cells in the pond took about 6–8 h, and the volume of collected cell slurry was about 1500 L. The tank sedimentation took about 12 to 24 h, and the slurry for centrifugation was reduced to about 300 L. The slurry was further dehydrated by centrifugation to about 40 kg algae paste, and the paste was dried into about 6 kg powder to be pulverized. The average power consumption of the pulverizer to process 1 kg cell powder was about 3.5 kWh, and the average astaxanthin content of the final biomass was about 2.5%. All process parameters were summarized in Table 1. 3. Assessment of process economics 3.1. Process scale up Based on the estimated process parameters, the pilot operation was scaled up, and a 900 kg astaxanthin (about 36 ton biomass) per year facility was conceptually designed to be located in Kunming, China, a place with more sunshine time and less temperature variations than Shenzhen, China. The major components of the facility would include a laboratory, 1000 L reactors, 8000 L reactors, raceway ponds, tanks, centrifuges, a spray dryer, a pulverizer, downstream process buildings, storage rooms, laboratory rooms, bioreactor media station, pressured air supply, CO2 tank station and compressed air station et al. Assuming biomass could be harvested 30 times per year for each pond, 20 raceway ponds each with an area of 1000 m2
Fig. 1. Process flow diagram of astaxanthin production from Haematococcus.
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571
Table 1 Summarization of process parameters. Photobioreactor process control parameters
Values
Ranges
Bioreactor culture temperature Bioreactor aeration rate Starting cell concentration of the 1000 L bioreactor Final cell concentration for the 1000 L bioreactor Starting cell concentration of the 8000 L bioreactor Daily renewal rate of the 8000 L bioreactor
Below 25 C 0.05 v/v/min 6 × 104 cells/ml 5 × 105 cells/ml 6 × 104 cells/ml 0.25 per day
10–25 C
Photobioreactor process performance parameters
Estimated values
Ranges
Power consumption for cooling the 1000 L bioreactor Power consumption for cooling the 8000 L bioreactor Aeration power input for the 1000 L bioreactor Aeration power input for the 8000 L bioreactor Days for 1000 L bioreactor cell culture to be ready Days for 8000 L bioreactor cell culture to be ready Working cell concentration for the 8000 L bioreactor Semi-continuous operation of the 8000 L bioreactor CO2 consumption of the 1000 L bioreactor CO2 consumption of the 8000 L bioreactor
10 kWh per day 50 kWh per day 0.1 kW 0.4 kW 5 days 5 days 5 × 105 cells/ml 40 days 1.5 kg per day 4.5 kg per day
0–30 kWh per day 0–200 kWh per day
4–6 days 4–6 days 4.2–6.0 × 105 cells/ml 30–60 days 1.0–2.0 kg per day 3.5–5.0 kg per day
Pond process control Parameters
Values
Ranges
Pond culture depth Pond water flow speed Pond inoculation cell concentration
15 cm 30 cm/s 6 × 104 cells/ml
5–7 × 104 cells/ml
5–8 × 104 cells/ml 5–7 × 105 cells/ml 5–7 × 104 cells/ml
Pond process performance parameters
Estimated values
Ranges
Pond reddening days Final pond culture concentration in biomass dry weight Pond CO2 consumption Pond power input
9 days 0.4 g/L 2 kg per day 400 W
8–9 days 0.27–0.55 g/L 1.5–2.5 kg per day
Downstream process parameters
Estimated values
Ranges
Dry biomass in cell slurry Dry biomass in cell paste Astaxanthin content in dry biomass Power consumption for centrifugation Oil consumption for spray drying Power consumption of cell pulverization
1.4% 13.5% 2.5% 3.0 kWh/kg biomass 1.5 kg oil/kg biomass 3.5 kWh/kg biomass
1.2%–1.5% 10%–15.5% 2.1%–3.2% 2.5–3.5 kWh/kg 1.2–1.5 kg/kg 3.2–3.6 kWh/kg
would give yearly biomass yield of about 36 tons. 20 big bioreactors would be needed to provide 40 tons of inoculum culture for two ponds each day. Assuming that 8000 L bioreactor could be operated at semi-continuous mode for about one month, another 6 small and big bioreactors would be required to prepare fresh 8000 L bioreactors and maintain the simultaneous operation of 20 big bioreactors at semicontinuous state. Each day one 1000 L and one 8000 L reactor would be initiated, and 10 carboys of seed culture from laboratory would be used as inoculum for the small bioreactor. Each day, two ponds, each with 150 ton culture, would be harvested, and each pond would yield about 15 tons of cell slurry. The slurry would be pumped to two 15 ton tanks for 24 h sedimentation. Each day about 6000 L concentrated slurry would be transferred to centrifuges. 4 centrifuges each with 200 L/h capacity would be employed for further dehydration, and the produced 800 kg algae paste would be dried by a dryer with hourly evaporation rate of about 100 kg water. The dried powder would be finally pulverized into broken pieces by a fluidized bed airflow pulverizer with an hourly capacity of 20 kg powder biomass. The total land occupation of the facility was assumed to be 25,000 m2, and the construction area for laboratory, offices, downstream process and product storage was assumed to be 500 m2. The media station would be constructed with 5 stock solution tanks each with a volume of 4000 L and a set of 5 μm filter system. The media would be mixed and filtered by 5 μm filter system then pumped to bioreactors individually equipped with a 2 μm filter. The pond media would be mixed and delivered to pond via plastic media pipes. The aeration of bioreactors would be collectively provided by two roots blowers each with a
capacity of 6 m3/min air. The total CO2 consumption for bioreactors and ponds was estimated at about 550 kg per day, and 20 ton CO2 storage tank would be constructed. A 5 kW air compressor would be installed to provide high pressure air for control purposes. 3.2. Analysis of fixed capital investment All the cost data are based on design and operational parameters of conceptually designed facility and the actual quotes or transacted prices. The land required is about 25,000 m2, and the cost for land acquisition and improvements is estimated at about $12/m2 and totally about $300,000 at Kunming, China. The building area unit cost is about $300/m2, and the total cost is $150,000. The cost of the laboratory instruments for seed cell culture and process parameter analysis is about $35,000. The costs of photobioreactors with individual medium and air filters are about $12,000 and $8000 for 8000 L and 1000 L bioreactors respectively. The total capital cost of all photobioreactors is about $360,000. The media station with 5 stock tanks and a set of 5 μm filter system for photobioreactors costs about $12,000. The low pressure air system including two roots blowers each with 5 kW power costs $6000. The pond cost including paddlewheels and CO2 diffusers averages about $12/m2, and thus the total 20 ponds costs about $240,000. The tank of 15 m3 costs $4000 each, and four tanks are to be installed. A 20 ton high pressure tank is to be used to store CO2, and together with CO2 tubing costs about $40,000. The centrifuge costs $8000 each, and 4 centrifuges installed to process about 6000 L concentrated cell slurry totals
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$32,000. The spray dryer costs about $30,000, and the pulverizer costs about $60,000. The compressor and tubing system costs about $2500. The pumps, valves, piping and control units for culture transfer are estimated to cost about $10,000. The electrical supply including a transformer and power cables costs $25,000. The project engineering and supervision are estimated at about $50,000, and the construction expenses are estimated to be $100,000. The total fixed capital investment is about $1.47M and is itemized in Table 2. Comparing with a Spirulina facility with the similar area, the designed Haematococcus facility costs about $0.5M more. The extra cost can be contributed to the expenses of photobioreactors, centrifuges, and the pulverizer which are not necessary for Spirulina production, and the other expenses would be almost equivalent (Vonshak, 1992).
4. Results and discussions
3.3. Analysis of annual operational cost of production The operational cost of production includes raw materials, utilities, and labor et al. The major chemicals for culture media are potassium nitrate, sodium phosphate and sodium bicarbonate, and the total annual consumption is about 15 tons, 3 tons and 150 tons respectively. The total cost is about $80,000. The consumption of other nutrients is totally estimated as 3 tons per year at $2000 per ton. The consumption of CO2 is predicted to be 150 tons per year, and the total cost is about $22,500. The total water consumption is about 90,000 tons, and the total cost is about $27,000 per year. According to our experiences, the medium filter for each photobioreactor needs to be replaced every two months, and the air filter needs to be replaced every three months. For about 32 bioreactors, about 170 units of medium filters and 130 units of air filters are to be consumed per year. The medium filter and air filter cost about $100 and $60 respectively, and the total costs are $17, 000 and $7800 per year. The other consumables both for laboratory and bioreactor application are estimated to cost $12,000 per year. The power consumption arises from bioreactor cooling, low pressure air supply, compressed air supply, pond paddle wheeling, cell pulverization, pumping and control et al. The estimated consumption is about 270,000 kWh, 72,000 kWh, 7500 kWh, 288,000 kWh, 36,000 kWh, and 43,200 kWh for cooling, paddling, pulverization, low pressure air, compressed air, and pumping respectively, and the costs are about $36,000, $9600, $1000, $38,400, $4800 and $5760 respectively. It takes 48 tons of oil to
Table 2 Summarization of fixed capital cost for the conceptually designed facility. Items 1. Land acquisition and improvements 2. Building area 3. Laboratory instruments 4. 8000 L bioreactor systems 5. 1000 L bioreactor systems 6. Medium supply station for bioreactors 7. Low pressure air supply 8. Raceway ponds 9. Harvest cell broth storage tank 10. CO2 storage tank 11. Centrifuge 12. Dryer 13. Pulverizer 14. Air compressor and tubing 15. Pumps, valves, piping and control 16. Electrical 17. Engineering and supervision 18. Construction expenses Total fixed cost
Unit costs
Quantities
Units
$12
25,000
m2
$300,000
$300 $35,000 $12,000 $8,000 $12,000
500 1 26 6 1
m2 Set Set Set Set
$150,000 $35,000 $312,000 $48,000 $12,000
$3,000 $12 $4,000 $40,000 $8,000 $30,000 $60,000 $2,500 $10,000
2 20,000 4 1 4 1 1 1 1
Set m2 Units Unit Units Unit Unit Set Set
$6,000 $240,000 $16,000 $40,000 $32,000 $30,000 $60,000 $2,500 $10,000
$25,000 $50,000 $100,000
1 1 1
Set
process 36 tons dry biomass, and 48 tons of oil cost about $48,000. About 19 workers and 4 supervisors or chemists are required to operate the production, and the total estimated expenses are $95,000 and $32,000 respectively. The maintenance including all the replacing machinery parts and outsourcing repairing work is estimated to be about $20,000 per year. The general plant overhead is estimated to be $50,000 per year. The total annual operational cost of production is about $0.5M and is summarized in Table 3. Comparing with a Spirulina facility with the similar area, the Haematococcus facility would cost much more. The labor, supervision, maintenance and electricity expenses would be substantially reduced because the photobioreactors, the centrifuges and the pulverizer would not be operated in a Spirulina facility (Vonshak, 1992).
Total cost ($)
$25,000 $50,000 $100,000 US$1,468,500
4.1. Production cost of natural astaxanthin For the conceptually designed facility, the total fixed capital investment and the total direct cost of production per year are estimated to be $1,468,500 and $499,705 respectively. The direct production of cost of biomass and astaxanthin would be about $14/kg and $555/kg. If a 10 year depreciation of fixed capital cost is adopted, the unit production cost of biomass and astaxanthin would be $18/kg and $718/kg respectively. If a 5 year depreciation of fixed capital cost is adopted, the unit production cost of biomass and astaxanthin would be $22/kg and $882/kg respectively. The production cost of synthetic astaxanthin by DSM, BASF, and NHU is estimated to be about $1000/ kg astaxanthin, and therefore the conceptually designed facility would be able to produce natural astaxanthin at a cost lower than that of synthetic astaxanthin.
4.2. The reasons for the low cost production of natural astaxanthin There are several established facilities, Cyanotech Inc., Algatechnologies, Ltd., Mera Pharmaceuticals Inc., and Biogenic Inc., et al.(Del Campo et al., 2007; Milledge, 2010), producing astaxanthin from Haematococcus world widely, and their production costs are estimated to be above $3000/kg astaxanthin, which is much higher than the estimated level in this research, namely $718/kg. There might be several reasons accounted for the low cost of our predicted production. Firstly photobioreactors developed and employed in this research are proved to be very efficient and inexpensive. The big photobioreactor with a culture carrying capacity of 8000 L can be operated at 5 × 105 cells/ml cell concentration at semi-continuous state for a period of two months and can be built at a cost of $12,000. This cost level is less than one third of current industrial level with the same total culture volume and similar performance (Kaewpintong et al., 2007; Kunjapur and Eldridge, 2010; Molina Grima et al., 2003). Secondly raceway ponds instead of photobioreactors are used as the primary microalgae cell cultivation tools, and photobioreactors are only used to provide vegetative cells for raceway ponds. The cost of building raceways is only about one fifteenth of photobioreactors with the same culture carrying capacity in our situations. At the same time, the maintenance and consumables cost of raceway ponds is only about 5% of that of photobioreactors according to our analysis. In fact, some of current operations of astaxanthin production from Haematococcus employ only photobioreactors for both vegetative cell proliferation and astaxanthin accumulation. Thirdly the low cost labor, land and utilities in China substantially reduce the production cost of natural astaxanthin. For example, if the same production facility were established in USA, the labor would approximately cost $600 per kg astaxanthin while only about $120 per kg in China according to analysis.
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573
Table 3 Summarization of operational costs for the conceptually designed facility. Items
Unit costs
Quantities
Units
Total costs
1. Potassium nitrate 2. Disodium hydrogen phosphate 3. Sodium bicarbonate 4. Carbon dioxide 5. Other nutrients 6. Water and waste water treatment 7. Medium filters 8. Air filters 9. Other consumables 10. Power for bioreactor cooling 11. Power for low pressure air 12. Power for compressed air 13. Power for pond paddling 14. Power for cell pulverization 15. Power for pumping 16. Oil consumption for the dryer 17. Labor 18. Supervision 19. Maintenance 20. General plant overheads Total direct cost of production Direct production cost of biomass Direct production cost of astaxanthin Depreciation of fixed capital cost (10 years) Unit production cost of biomass Unit production cost of astaxanthin Depreciation of fixed capital cost (5 years) Unit production cost of biomass Unit production cost of astaxanthin
$500 $800 $250 $200 $2,000 $0.3 $100 $60 $8,000 $0.15 $0.15 $0.15 $0.15 $0.15 $0.15 $1,000 $5,000 $8,000 $20,000 $50,000
15 3 150 170 3 90,000 170 130 1 270,000 72,000 7500 288,000 36,000 43,200 48 19 4
Tons Tons Tons Tons Tons Tons Pieces Pieces
$7,500 $2,400 $37,500 $34,000 $6,000 $27,000 $17,000 $7,800 $8,000 $40,500 $10,800 $1,125 $43,200 $5,400 $6,480 $48,000 $95,000 $32,000 $20,000 $50,000 US$499,705 US$14 US$555 US$146,850 US$18 US$718 US$293,700 US$22 US$882
4.3. Raceway ponds versus photobioreactors Raceways ponds have long been used in commercial cultivation of microalgae, but because raceway ponds are open to air and subject to contamination, only several species, such as Spirulina, Chlorella, and Dunaliella, could have been successfully cultivated in raceway ponds (Milledge, 2010). To overcome the problem of contamination, a bunch of photobioreactors have been developed to cultivate microalgae at large scales. Though developed photobioreactors provide a closed and controlled cultivation environment for microalgae and are able to cultivate most microalgae species at large scale, the building and maintenance expenses of photobioreactors are much higher than raceways ponds. For example, the building cost of photobioreactors reported in this paper are fifteen times more expensive than raceway ponds with the same culture volume, and the maintenance fees are even 20 times higher. If only our developed photobioreactors and no raceway ponds are employed to cultivate Haematococcus for astaxanthin production at 900 kg per year capacity, the production cost of astaxanthin would be more than $3600/kg according to our analysis, which would be about 4.5 times higher than the estimated production cost of conceptually designed facility and might be the cases of some current commercial operation of astaxanthin production from Haematococcus. In this research and design, photobioreactors serve to provide vegetative cell culture for raceway ponds, and raceway ponds are operated at batch mode for a short period of time to avoid contamination. This approach was suggested for large scale cultivation of microalgae to produce biofuels (Huntley and Redalje, 2007), and the data reported in this paper could serve as an evidence to support for the suggestion, which was especially suitable for algae species undergoing different stages with different nutrient requirements for lipid accumulation (Liu et al., 2008; Qiao et al., 2009). 4.4. The two step approach versus one step approach The typical two step approach has been employed to cultivate Haematococcus for astaxanthin production, which is characterized by
kWh kWh kWh kWh kWh kWh Tons People People
a quick dilution of cell culture to reduce the concentration levels of nutrients after vegetative growth of cells (Fábregas et al., 2001; Olaizola, 2000). Recent researches of astaxanthin production from Haematococcus suggest a one step approach which either controls the supply of nutrients to the cell culture or reduce the level of nutrients by growth of Haematococcus cells (Del Río et al., 2008; Esperanza Del Río et al., 2005; García-Malea et al., 2006, 2009; Zhang et al., 2009). In the former situation, cells can be cultivated and harvested continuously, but both the cell growth rate and astaxanthin content in dry biomass are much lower than those of two step approaches. In addition, to cultivate microalgae continuously, closed photobioreactor systems might need to be used. Therefore if substantial improvements are not achieved, the cost of production would be much higher than the current two step approach. In the latter situation, although a batch production is proposed in raceway ponds, the prolonged cell growth period in open environments would endanger cell culture to serious contamination problems if selective medium for Haematococcus is not successfully developed. A research work reported by Aflalo et al. (2007) also argues that two step approach is much more efficient than one step approach in terms of astaxanthin production. 4.5. The chances to further decrease production cost of designed facility The conceptually designed facility is based on preliminary researches at pilot scale in Shenzhen, China, and our experiences, industrial data and research reports do support the belief that the cost would be able to be further decreased substantially. The pilot scale experiments have been done in Shenzhen, China, and the conceptually designed facility would be located at a place with more sunshine and less temperature variations such as Kunming, China. The processes would be able to be further optimized, and the production cost should be able to be decreased substantially. The average content of astaxanthin in biomass of designed facility is only 2.5%, and the biomass product of 3.5% astaxanthin is commercially available from Biogenic Co., Ltd. at Kunming, China, and even higher level of astaxanthin has been reported in literatures (Aflalo et al., 2007; Bubrick, 1991; Harker et al., 1996;
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Margalith, 1999; Ranjbar et al., 2008). Recent researches on genomics and proteomics of Haematococcus might lead to genetic modification of Haematococcus to enhance astaxanthin accumulation in cells (Jin et al., 2006; Kathiresan and Sarada, 2009; Sandesh Kamath et al., 2008; Steiger and Sandmann, 2004). Apparently, the production cost would be proportionally reduced with the increase of average astaxanthin content in final biomass products assuming the same processes being used. 5. Conclusions Inexpensive production of astaxanthin by cultivation of Haematococcus can be realized with current available technologies. The production cost of astaxanthin by our conceptually designed facility is estimated to be $718/kg astaxanthin or about $18/kg biomass with 2.5% astaxanthin. The cost is much lower than that the current industrial operations and is even lower than that of synthetic astaxanthin. The reasons of the low cost predictions can be contributed to low cost photobioreactors employed, two step approach using raceways ponds, and low labor and other costs in China. The cost might be able to be further reduced with the advances of technologies and optimization of processes. Acknowledgments This work was supported by National Innovation Fund of China with the funding number 06C26214421651, by Science and Technology Planning Project of Shenzhen Municipality, China, with the funding number SZKJ0531, by Main Direction Program of Knowledge Innovation of Chinese Academy of Sciences with the funding number KGCX2-YW-374-3, and by the open fund of Tianjin Key Laboratory of Marine Resources and Chemistry with the funding number 200903. We appreciate SCW Medicath Ltd. for providing its building rooftop at Shenzhen, China, on which the pilot plant of this research was established. References Aflalo C, Meshulam Y, Zarka A, Boussiba S. On the relative efficiency of two-vs. onestage production of astaxanthin by the green alga Haematococcus pluvialis. Biotechnol Bioeng 2007;98:300–5. Ausich R. Commercial opportunities for carotenoid production by biotechnology. Pure Appl Chem 1997;69:2169–74. Bubrick P. Production of astaxanthin from Haematococcus. Bioresour Technol 1991;38: 237–9. Del Campo J, García-Gonzáez M, Guerrero M. Outdoor cultivation of microalgae for carotenoid production: current state and perspectives. Appl Microbiol Biotechnol 2007;74:1163–74. Del Río E, Acién F, Garcia Malea M, Rivas J, Molina Grima E, Guerrero M. Efficiency assessment of the one-step production of astaxanthin by the microalga Haematococcus pluvialis. Biotechnol Bioeng 2008;100:397–402. Esperanza Del Río F, García-Malea M, Rivas J, Molina-Grima E, Guerrero M. Efficient one-step production of astaxanthin by the microalga Haematococcus pluvialis in continuous culture. Biotechnol Bioeng 2005;91:808–15. Fábregas J, Otero A, Maseda A, Domínguez A. Two-stage cultures for the production of astaxanthin from Haematococcus pluvialis. J Biotechnol 2001;89:65–71. García-Malea M, Acién F, Fernández J, Cerón M, Molina E. Continuous production of green cells of Haematococcus pluvialis: modeling of the irradiance effect. Enzyme Microb Technol 2006;38:981–9. García-Malea M, Acién F, Del Río E, Fernández J, Cerón M, Guerrero M, et al. Production of astaxanthin by Haematococcus pluvialis: taking the one-step system outdoors. Biotechnol Bioeng 2009;102:651–7.
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