This article appeared in a journal published by Elsevier The attached copy is furnished to the author for internal non-commercial research and education use, including for instruction at the authors institution and sharing with colleagues Other uses, including reproduction and distribution, or selling or licensing copies, or posting to personal, institutional or third party websites are prohibited In most cases authors are permitted to post their version of the article (e.g in Word or Tex form) to their personal website or institutional repository Authors requiring further information regarding Elsevier’s archiving and manuscript policies are encouraged to visit: http://www.elsevier.com/authorsrights Author's personal copy Bioresource Technology 163 (2014) 26–32 Contents lists available at ScienceDirect Bioresource Technology journal homepage: www.elsevier.com/locate/biortech The effective photoinduction of Haematococcus pluvialis for accumulating astaxanthin with attached cultivation Minxi Wan a,1, Dongmei Hou a,1, Yuanguang Li a,⇑, Jianhua Fan a, Jianke Huang a, Songtao Liang a, Weiliang Wang a, Ronghua Pan b, Jun Wang b, Shulan Li c a b c State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, PR China Jiaxing Zeyuan Bio-products Co., Ltd., Jiaxing 314007, PR China Shanghai Zeyuan Marine Bio-products Co., Ltd., Shanghai 200237, PR China h i g h l i g h t s H pluvialis can accumulate effectively astaxanthin with this attached cultivation Astaxanthin productivity of the attached system is 2.4-fold of that suspended Attached cells can accumulate astaxanthin under low light and temperature up to 35 °C This attached cultivation can strongly resist protozoan contamination This attached cultivation is superior on saving water, ease to harvest a r t i c l e i n f o Article history: Received 24 February 2014 Received in revised form April 2014 Accepted April 2014 Available online 16 April 2014 Keywords: Haematococcus pluvialis Astaxanthin Biomass Attached cultivation Photoinduction a b s t r a c t As the optimal source of astaxanthin, Haematococcus pluvialis was cultured for commercial production of astaxanthin through two continuous phases: cell growth and astaxanthin induction In this study, the efficiency of an attached system for producing astaxanthin from H pluvialis was investigated and compared to that of the suspended system (bubble column bioreactor) under various conditions Results showed that this attached system is more suitable for photoinduction of H pluvialis than the suspended bioreactor Under the optimal conditions, the astaxanthin productivity of the attached system was 65.8 mg mÀ2 dÀ1 and 2.4-fold of that in the suspended system This attached approach also offers other advantages over suspended systems, such as, producing astaxanthin under a wide range of light intensities and temperatures, saving water, ease to harvest cells, resisting contamination Therefore, the attached approach can be considered an economical, environmentally friendly and highly-efficient technology for producing astaxanthin from H pluvialis Ó 2014 Elsevier Ltd All rights reserved Introduction Astaxanthin (3,30 -dihydroxy-b,b-carotene-4,40 -dione) is a red ketocarotenoid with extraordinary antioxidant capability It has widespread applications in aquaculture and dietary supplements (Guerin et al., 2003; Higuera-Ciapara et al., 2006; Lorenz and Cysewski, 2000) Until now, the synthetic astaxanthin dominates current commercial astaxanthin market, of which the total value is more than $200 M per year (Li et al., 2011; Milledge, 2011) However, natural astaxanthin is more favorable in the market than ⇑ Corresponding author Address: Mail box 301, Meilong Road 130, Shanghai 200237, PR China Tel./fax: +86 21 64250964 E-mail addresses: wanminxi@gmail.com (M Wan), ygli@ecust.edu.cn (Y Li) These authors contributed equally to this work http://dx.doi.org/10.1016/j.biortech.2014.04.017 0960-8524/Ó 2014 Elsevier Ltd All rights reserved synthetic astaxanthin, while the market demand for natural astaxanthin is not being met Natural astaxanthin can be synthesized by some plants, bacteria, fungi, and green algae As astaxanthin content up to 1–5% of cell dry weight (He et al., 2007; Lorenz and Cysewski, 2000), the unicellular green algae Haematococcus pluvialis is recognized as the best biological source for natural astaxanthin, and far surpasses any other reported sources (Lorenz and Cysewski, 2000) Although the research and development of astaxanthin production from H pluvialis have been committed in these years, the successful strategy for the commercial production is established base on the two-step culture (Aflalo et al., 2007; Fábregas et al., 2001; Sarada et al., 2002) The first stage, green vegetative growth phase, is performed to obtain a large quantity of green vegetative cells under the favorable culture condition in tubular, bubble Author's personal copy M Wan et al / Bioresource Technology 163 (2014) 26–32 column, airlift photobioreactors, or the raceway ponds And the second stage, reddish inductive production phase, is the induction of cells to accumulate astaxanthin with the transition of green vegetative cells to reddish cyst cells in various stress conditions, such as nitrogen limitation, excess acetate addition, strong light intensity, salt stress, phosphate deficiency, or the addition of specific cell division inhibitors (Hata et al., 2001; Hu et al., 2008; Li et al., 2010; Sarada et al., 2002; Wang et al., 2003) Therefore, induction systems have a direct correlation with the astaxanthin content of cells and total productivity of astaxanthin Attached systems have been successfully used for culturing algae to remove nutrients from wastewater (Kebede-Westhead et al., 2006; Wilkie and Mulbry, 2002), and are developing to grow microalgae with the purpose of producing biofuel feedstock due to low consumptions of water and energy using attached systems (Johnson and Wen, 2010; Liu et al., 2013; Ozkan et al., 2012) Here, an attached technology was used in the astaxanthin accumulation of H pluvialis and compared with the traditional suspended technology, and the efficiency of attached technology for astaxanthin productivity was evaluated under various conditions Methods 2.1 Microalgal strain and culture conditions H pluvialis NIES-144 was obtained from the National Institute for Environmental Studies, Tsukuba, Japan The medium was NIES-C medium with 10 mM sodium acetate as the organic carbon source (Hata et al., 2001) The green cells were cultured under continuous illumination of 25 lmol mÀ2 sÀ1 at 25 °C, and were subsequently induced with continuous illumination of 150 lmol mÀ2 sÀ1 at 25 °C unless otherwise stated The peak, dominant, centroid and central wavelengths of illumination from the cold fluorescence lamp were 445, 504, 437 and 437 nm, respectively Furthermore, the color temperature was 6199 k Other photoinduction conditions for each experiment were described in the section of experimental design 2.2 Photobioreactor and experimental design for the attached induction 2.2.1 Photobioreactor A simple bioreactor was used to investigate the induction feasibility of attached algae cells (Fig 1) A gauze supported by wire mesh was vertically placed in a medium reservoir Filtered with a 0.2 lm microfiltration membrane (the diameter of 33 ± 0.5 cm2), suspended cells formed an algal film on the membrane Then the membrane with algal film was placed onto the gauze In order to Sparger B Algal “disk” 2.2.2 Experimental design for the attached induction All experiments were repeated three times The induction medium was NIES-N medium (Kang et al., 2005) for all induction experiments in this study This medium without nitrogen led to cells under nitrogen deficiency Except special requirement, H pluvialis was induced under continuous illumination of 150 lmol mÀ2 sÀ1 at 25 °C Temperatures and initial cell amount were set respectively to 15, 25, 35 °C and 10, 20, 40 g mÀ2 to investigate their effects on induction Illumination was in the horizontal direction, thus the vertical cross section of column bioreactor was considered light-receiving area in order to compare performances between both attached and suspended induction systems The biomass of suspended cells was transformed to that of algal disk according to the following formula: mdisk ¼ msuspend à V=S where the mdisk (g/m2) is the biomass concentration of algal disk, the msuspend (g LÀ1) is the biomass concentration in the column bioreactor, V (L) is the volume of the column bioreactor, S (m2) was the light-receiving area of cells Therefore, the starting cell densities per liter in the column bioreactor were 0.18, 0.36 and 0.65 g LÀ1 corresponding to the 10, 20 and 40 g mÀ2 in the attached bioreactor The induction was exposed to continuous illumination of 50, 70, 90, 120, 160, and 230 lmol photons mÀ2 sÀ1, respectively, to study the effect of light density on induction Furthermore, the flow rate of the induction medium was adjusted in order to investigate the effect of moisture on induction 2.3 Analytical methods 2.3.1 Dry weight For the column bioreactor, the algal cells were collected by centrifuging the induction fluid at 4000g for 10 min, washed with distilled water and dried at 80 °C for 24 h While the cell of algal disk was washed down and re-suspended with a volume of distilled water, and then the dry weight of algal disk was measured as same as above Thus the biomass of algal disk was calculated as: where the mt was the dry weight of algal disk after induction, and S was the light-receiving area of cells Net biomass productivity : ðMt À M Þ=t Medium flow Gauze Induction medium Pump reservoir Circulation pipe fix the algal film, the small magnets were put on the edge of membrane, and brought magnetic force with wire mesh support During the induction, the induction medium flowed down from a sparger on the top brim of gauze so that medium permeated the gauze and microfiltration membrane to wet algal film The flow rate for each algal disk in the attached system is ml/min, and can be controlled at the ranges of 0.05–190 ml/min to change moisture of attached cells For normal experiment, The control experiment was performed in L column bioreactor (height: 26 cm, radius: 3.5 cm, the vertical cross section was 182 cm2) under the same condition Aeration and mixing were done by sparging air with the rate of 0.5 vvm Mt ¼ mt =S Wire mesh support Light 27 Algal film Microfiltration membrane A Fig The attached bioreactor for the induction of H pluvialis (A) The overview of the attached induction (B) The details of the algae ‘disk’ where Mt was dry weight after induced, and M0 was the initial dry weight 2.3.2 Moisture of algal disk The flow rate of medium was adjusted to change moisture of algal disk which was calculated by the ratio of dry cell weight to wet cell weight Firstly, wet cells were scraped from algal disk, and then were divided into two groups and weighed separately Group was directly dried at 80 °C for 24 h to measure the total Author's personal copy 28 M Wan et al / Bioresource Technology 163 (2014) 26–32 dry weight of cells and salts inside induction medium Group was washed with distilled water, and then cells were collected by centrifuging the fluid at 4000g for 10 min, and dried at 80 °C for 24 h to measure dry cell weight Thus the moisture of the algal disk was calculated as: -2 (a) 2.0 1.8 where md1 was dry weight of Group (including cells and salts), and Mw1 was the wet weight of Group (including cells, water, and salts), md2 was dry weight of Group (only including cells), and Mw1 was the wet weight of Group (including cells, water, and salts) 1.6 where the Va was the volume of extracts, the Vb was the volume of culture sample (For the attached culture, the Vb was the volume of distilled water for the algal cells washed down and re-suspended.), and A490 was the absorbance of extracts at 490 nm Astaxanthin content ð%Þ : ct =mt where ct was the astaxanthin concentration after induced and mt was the dry weight (For the attached culture, the mt was the dry weight in distilled water for the algal cells washed down and resuspended.) after induced 1.4 1.2 1.0 0.8 0.6 0.4 0.2 10 12 Time (d) -2 (b) Attached induction with initial cell amount of 10 g m -2 Attached induction with initial cell amount of 20 g m -2 Attached induction with initial cell amount of 40 g m -2 Suspended induction with initial cell amount of 10 g m -2 Suspended induction with initial cell amount of 20 g m -2 Suspended induction with initial cell amount of 40 g m 100 90 80 70 -2 Biomass (g m ) c ¼ 4:5  A490  V a =V b Astaxanthin (%, w/w) Moisture ¼ ð1 À md1 =mw1 Þ=ð1 À md1 =mw1 À md2 =mw2 Þ 2.3.3 Determination of astaxanthin The algal cells in the attached system were washed down and re-suspended with distilled water for the astaxanthin determination The astaxanthin content was measured with modified Boussiba method (Boussiba and Vonshak, 1991) For astaxanthin determination, mL culture sample was centrifuged for 10 at 4000g, and the pellet was first saponified by using a solution of 5% KOH in 30% (v/v) methanol at 65 °C for 15 to destroy the chlorophyll And then the supernatant was discarded, and the pellet was then washed three times with de-ionized water to remove the residual lye The remaining pellet was extracted with mL DMSO (Labkim A.R., 99.5%) using an ultrasonic processor for 10 to recover the astaxanthin The extraction procedure was repeated at least three times until the cell debris was almost colorless The absorbance of the combined extracts was measured at 490 nm Per unit volume astaxanthin concentration (c, mg/L) calculated as: Attached induction with initial cell amount of 10 g m -2 Attached induction with initial cell amount of 20 g m -2 Attached induction with initial cell amount of 40 g m -2 Suspended induction with initial cell amount of 10 g m -2 Suspended induction with initial cell amount of 20 g m -2 Suspended induction with initial cell amount of 40 g m 60 50 40 30 20 10 0 10 12 Time(d) Astaxanthin yield Cị : C ẳ c V=S where the c was the astaxanthin concentration of algal disk after induction, V was the volume of distilled water for the algal cells washed down and re-suspended, and S was the light-receiving area of cells Net astaxanthin productivity : ðC t À C Þ=t where Ct was the astaxanthin yield after induced, and C0 was the initial astaxanthin yield Results and discussion 3.1 The effect of initial cell amount on the cell growth and astaxanthin content In order to assess whether the attached cultivation approach would be suitable for H pluvialis induction, and how much of the initial cell amount would be better for the induction, H pluvialis was induced with different initial cell amount in the attached bioreactor under continuous illumination of 150 lmol mÀ2 sÀ1 at 25 °C As indicated in Fig 2, the lower the initial cell amount was, the higher the astaxanthin content was The maximal Fig The astaxanthin contents (a) and biomass concentrations (b) of H pluvialis with three different initial cell amounts in the attached bioreactor and the column bioreactor astaxanthin content was achieved at the initial cell amount of 10 g mÀ2 But the growth rates at initial cell amount of 20 and 40 g mÀ2 were similar, and were higher than that of 10 g mÀ2 The induction effect of the attached bioreactor was compared with that of the column bioreactor As the results, the variation trend of the astaxanthin in the column bioreactor was similar to that in the attached induction, namely, the astaxanthin contents increased with reducing initial cell amounts At the initial cell amount of 10 and 20 g mÀ2, the astaxanthin content in the attached induction was higher than that in the column bioreactor An interesting phenomenon in the suspended induction system has been observed widely that cells are easy to attach on the walls of bioreactors, and these attached cells redden much faster than those suspended in liquid H pluvialis with high astaxanthin content is usually found in the bottom of dried out rock pools and bird baths (Pocock, 1960), which provide a semiarid niche and are similar with the attached system, implying the niche in the attached bioreactor may be is Author's personal copy 29 M Wan et al / Bioresource Technology 163 (2014) 26–32 Attached induction at 15 oC Attached induction at 25 oC Attached induction at 35 oC Traditional induction at 15 oC Traditional induction at 25 oC Traditional induction at 35 oC (a) 2.0 1.8 Astaxanthin (%, w/w) 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 10 12 14 10 12 14 Time (d) Attached induction at 15 oC Attached induction at 25 oC Attached induction at 35 oC Suspended induction at 15 oC Suspended induction at 25 oC Suspended induction at 35 oC (b) 80 70 60 Biomass (g m-2) consistent with natural environment where cells accumulate astaxanthin Therefore, the attached induction strategy may be more suitable to astaxanthin accumulation for H pluvialis in the term of bioecology compared to another induction technology with suspended liquid In the early period of induction, biosynthetic rate of astaxanthin in the attached bioreactor was significantly higher than that in the suspended bioreactor, which is well matched with the above phenomenon The cell growth in the column bioreactor was slightly higher than that in the attached induction before the days induction, then the cell growth in the column bioreactor slowed down but that in the attached induction was still kept at a rapid rate Besides, both the astaxanthin productivity and biomass productivity were reached the maximum at the initial cell amount of 20 g mÀ2 (Table 1) The astaxanthin productivity and the biomass productivity of attached induction were about 2.4-fold and 2.8-fold of that in the suspended induction, respectively At the initial cell amount of 20 and 40 g mÀ2, the astaxanthin productivity and the biomass productivity of attached induction were higher than those of the column bioreactor as well For the same algae species and induction medium, the astaxanthin content was 1% under a temperature of 23 °C and light intensity of 200 lmol mÀ2 sÀ1 in the other report (Kang et al., 2005), and was lower than that in this attached induction Also, biomass and astaxanthin productivities in this attached induction were similar or higher than those in other reports (Domı´nguez-Bocanegra et al., 2004; Hata et al., 2001; Kang et al., 2010, 2007) Although attached cultivation system for microalgae have made a great progress on feedstock production for biofuel (Gross et al., 2013; Johnson and Wen, 2010; Liu et al., 2013; Ozkan et al., 2012), this study showed attached system has a good performance for astaxanthin production from H pluvialis Furthermore, all standard deviations of experiments in this study are less than 10%, indicating all results were statistically significant 50 40 30 20 10 3.2 The effect of temperature on the cell growth and astaxanthin content The large-scale commercial production of astaxanthin from H pluvialis under outdoor condition was affected by the season, mainly in the light intensity and temperature Most studies have been reported the suitable temperature for the astaxanthin accumulation of H pluvialis was between the 20 °C and 28 °C (Kang et al., 2010, 2006, 2005; Lababpour et al., 2005; Yoo et al., 2012) Furthermore, it was reported that H pluvialis can accumulate astaxanthin at 35 °C (Tjahjono et al., 1994) This may be because of the difference of H pluvialis species and induction condition In order to assess the scope of temperature for the attached induction, the induction temperatures were set at 15 °C, 25 °C and 35 °C, respectively According to our results (Fig 3), the astaxanthin content at 15 °C was the highest, followed by those at 25 °C and 35 °C after 12 days induction, while the biomass was achieved the maximum at the 25 °C, followed by that at 35 °C, and the growth Table The net astaxanthin and biomass productivities of H pluvialis with different initial cell amount Induction bioreactor Initial cell amount (g mÀ2) Astaxanthin productivity (mg mÀ2 dÀ1) Biomass productivity (g mÀ2 dÀ1) Attached 10 20 40 10 20 40 44.1 ± 1.7 65.8 ± 1.7 58.7 ± 2.3 22.4 ± 0.8 27.3 ± 1.7 33.9 ± 3.1 2.1 ± 0.2 3.7 ± 0.3 3.3 ± 0.3 0.9 ± 0.2 1.3 ± 0.2 1.1 ± 0.2 Suspended 0 Time (d) Fig The astaxanthin contents (a) and biomass concentrations (b) of H pluvialis at temperatures of 15 °C, 25 °C and 33 °C in the attached bioreactor and the column bioreactor was slowest at 15 °C The astaxanthin productivity was 32.3, 65.8, 43.6 mg mÀ2 dÀ1 and the biomass productivity was 1.1, 3.7, 2.6 g mÀ2 dÀ1, respectively Thus, the best temperature for the attached induction was about 25 °C and similar to other reports (Domı´nguez-Bocanegra et al., 2004; Kang et al., 2010, 2006, 2005; Yoo et al., 2012) In this study, the induction in the column reactor was carried out to verify the performance of attached induction under different temperature In the column photobioreactor, the astaxanthin concentration was similar at 15 °C and 25 °C, but biomass at 25 °C was slightly higher than that at 15 °C But, the astaxanthin accumulation in the attached photobioreactor was still faster than that in the column photobioreactor The cell growth in the column bioreactor was slightly higher than the attached induction before the days induction, then the growth rate in the column bioreactor slowed down, but those in the attached induction still increased rapidly At 35 °C, the astaxanthin accumulation was similar between column bioreactor and attached bioreactor before the 6th day Then, cells in column bioreactor were death, but those in attached photobioreactor were growing, suggesting astaxanthin induction of H pluvialis in the attached system can be conducted at a higher Author's personal copy M Wan et al / Bioresource Technology 163 (2014) 26–32 3.3 The relationships of light intensity with cell growth and astaxanthin accumulation in the attached bioreactor Light is a key factor for inducing rapidly the astaxanthin biosynthesis Differed with suspended system, thick algal film in the attached system may cause insufficient illumination for the innermost cells Thus initial cell amount was set to a high level of 40 g mÀ2 to study the relationships of light intensity with cell growth and astaxanthin accumulation As the light intensity increased until 120 lmol mÀ2 sÀ1, the astaxanthin content of cells in attached induction increased (Fig 4) And the astaxanthin content reached the maximum under light intensity of 120 lmol mÀ2 sÀ1, and decreased slightly with the increasing light intensity The biomass productivities were similar under light intensities below 90 lmol mÀ2 sÀ1 In the range of light intensities from 90 to 160 lmol mÀ2 sÀ1, the biomass increased with light intensity However, the increase rate of biomass slowed down when the light intensity exceeded 160 lmol mÀ2 sÀ1 These results indicated that the 160 lmol mÀ2 sÀ1 of light density could be considered as the light saturation point (LSP) for this attached induction system in our investigated conditions This result was different than those in suspended induction that the astaxanthin content was reached the maximum at the light intensity 300 lmol mÀ2 sÀ1 (Li et al., 2010; Liang, 2009) Therefore, the attached induction of H pluvialis, even at initial cell amount of 40 g mÀ2, can work well under lower light intensity than suspended approach Furthermore, the attached induction is easy to avoid harmful high light intensity by tilting attached bioreactor toward the light direction, which means more induction bioreactors in the same illumination area can be operated 3.4 The relationships of moisture with cell growth and astaxanthin accumulation in the attached bioreactor In the attached photobioreactor, attached cells must keep a certain moisture to avoid death caused by lacks of water However, high moisture means more electronic power cost for enhancing the flow rate of induction medium In this study, the effects of moisture on cell growth and astaxanthin accumulation of H pluvialis were investigated Due to water in cells, the lowest moisture of H pluvialis cells in the attached photobioreactor was 75% when Biomass Astaxanthin (%, w/w) 1.8 1.6 (a) 1.8 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0 70 50 30 10 12 14 Time (d) 60 1.0 Moisture 95% Moisture 85% Moisture 75% 1.6 60 40 1.2 For a large scale production of H pluvialis, 1000–1500 tons of fresh water (without water recycling) will be required to produce ton of H pluvialis dry mass in suspended induction systems (Aflalo et al., 2007; García-Malea et al., 2009; Lopez et al., 2006; Torzillo et al., 2003; Zhang et al., 2009) In particular, drainage without treatment after cell collection will bring a serious environment Such huge amount of water consumption and effluent treatment are two of main reasons caused high cost for producing astaxanthin For the attached induction approach, however, only a little water with the nutrients was required to keep the algal cell 70 50 1.4 3.5 The water saving potential and other merits for attached induction approach (b) 80 Astaxanthin Biomass (g m-2) 2.0 keeping cells living The curves of biomass and astaxanthin content during the induction of H pluvialis with different moisture were shown in Fig In the first days, the accumulation rate of growth and astaxanthin content under the 95% moisture is faster than others Then the growth rates with 85% and 75% moisture increased, and the astaxanthin accumulation rate under three moistures slowed down At the 12 day, astaxanthin content and biomass were close among three moistures Hence, the wet of the algal disk could not cause the great differences in the growth and the astaxanthin accumulation, suggesting H pluvialis can to grow and synthesize astaxanthin in little water, and is very suitable to the induction in attached bioreactors Astaxanthin (%, w/w) temperature than that in conventional bioreactors Therefore, the attached system of H pluvialis may be able to reduce the cost of temperature control, compared to the conventional approach Biomass (g m -2) 30 Moisture 95% Moisture 85% Moisture 75% 40 30 20 20 10 10 0.8 0.6 40 60 80 100 120 140 160 180 200 220 240 Light intensity (µmol m-2 s-1) Fig The astaxanthin contents and biomass concentrations of H pluvialis in the attached bioreactor under different light intensities for 12 days of photoinduction 10 12 Time (d) Fig The astaxanthin contents (a) and biomass concentrations (b) of H pluvialis with different moistures in the attached bioreactor Author's personal copy M Wan et al / Bioresource Technology 163 (2014) 26–32 Table The amount of paramecium in the attached bioreactor and the column bioreactor Induction bioreactor Attached induction Suspended induction The total amount of paramecium (cells per dry algal cell weight, Â107 cells/g) days 12 days 7±1 51 ± 35 ± 103 ± 12 wet As been in our experiments, the water consumption of the attached induce was less than 30% of that in the open pond Therefore, the attached induction approach can dramatically reduce the water requirement and the amount of wastewater The contamination by protozoan does great harm to microalgae mass cultivation (Tang et al., 2011) The contamination control for conventional open aqueous-suspended cultivation was quite difficult due to the huge water body As shown in Table 2, the contamination by protozoan, mainly be paramecium, in the attached system was less than in the open column reactors Furthermore, small dose of pesticides or antibiotics would be enough to readily control the contamination in the attached system due to the small water body Thus, the effect of contamination on cell cultivation could be minimized Other merits of attached induction include: (1) power costeffective Mixture of medium is required and energy-intensive in suspended induction systems, but is not needed anymore in the attached induction Furthermore, algal biomass pastes are collected easy and cost-effectively by scraping down the biomass directly without further dewatering (Gross et al., 2013; Liu et al., 2013; Ozkan et al., 2012) (2) wide induction condition Cells can be induced with the attached approach to synthesize astaxanthin under higher or lower temperature and light intensity resulting from cloudy day or the season, compared to the suspended approach It is one of noteworthy features of attached technology for the commercial application, because controlling cells under a small range of temperatures and light intensities is high-cost for outdoor culture (3) Easy to scale-up The scale-up of conventional cultivation devices may reduce the performance of bioreactor due to the changes of mixing characteristics For the attached induction system without mixing, the constraints in conventional bioreactor design and scale-up were released greatly The medium can be cycled with sprays instead of pumps to wet to cells, and the wire mesh support can be replaced by cheaper, thinner and water retentive materials Conclusions In the present study, an attached cultivation approach was successfully applied in the induction of Haematococcus pluvialis for astaxanthin production Under the optimal condition, biomass and astaxanthin productivities in the attached cultivation were 2.8-fold (3.7 g mÀ2 dÀ1) and 2.4-fold (65.8 mg mÀ2 dÀ1) of those in the suspended bioreactor, respectively Furthermore, the attached cultivation approach is superior to suspended induction approach in other aspects, such as, lower water consumption and smaller risk of contamination, indicating this approach provides a promising way to boost economic benefit and considerably reduce production cost of astaxanthin from H pluvialis Acknowledgements This research was funded by National Basic Research Program China (973 Program: 2011CB200903 & 2011CB200904), National Key Technologies R&D Program (2011BAD23B04), China Postdoctoral Science Foundation (2013M530183) 31 References Aflalo, C., Meshulam, Y., Zarka, A., Boussiba, S., 2007 On the relative efficiency of two- vs one-stage production of astaxanthin by the green alga Haematococcus pluvialis Biotechnol Bioeng 98, 300–305 Boussiba, S., Vonshak, A., 1991 Astaxanthin accumulation in the green alga Haematococcus pluvialis Plant Cell Physiol 32, 1077–1082 Domı´nguez-Bocanegra, A.R., Guerrero Legarreta, I., Martinez Jeronimo, F., Tomasini Campocosio, A., 2004 Influence of environmental and nutritional factors in the production of astaxanthin from Haematococcus pluvialis Bioresour Technol 92, 209–214 Fábregas, J., Otero, A., Maseda, A., Domínguez, A., 2001 Two-stage cultures for the production of Astaxanthin from Haematococcus pluvialis J Biotechnol 89, 65– 71 García-Malea, M.C., Acién, F.G., Del Río, E., Fernández, J.M., Cerón, M.C., Guerrero, M.G., Molina-Grima, E., 2009 Production of astaxanthin by Haematococcus pluvialis: taking the one-step system outdoors Biotechnol Bioeng 102, 651– 657 Gross, M., Henry, W., Michael, C., Wen, Z., 2013 Development of a rotating algal biofilm growth system for attached microalgae growth with in situ biomass harvest Biotechnol Bioeng 150, 195–201 Guerin, M., Huntley, M.E., Olaizola, M., 2003 Haematococcus astaxanthin: applications for human health and nutrition Trends Biotechnol 21, 210–216 Hata, N., Ogbonna, J.C., Hasegawa, Y., Taroda, H., Tanaka, H., 2001 Production of astaxanthin by Haematococcus pluvialis in a sequential heterotrophic– photoautotrophic culture J Appl Phycol 13, 395–402 He, P., Duncan, J., Barber, J., 2007 Astaxanthin accumulation in the green alga Haematococcus pluvialis: effects of cultivation parameters J Integr Plant Biol 49, 447–451 Higuera-Ciapara, I., Felix-Valenzuela, L., Goycoolea, F., 2006 Astaxanthin: a review of its chemistry and applications Crit Rev Food Sci Nutr 46, 185–196 Hu, Z., Li, Y., Sommerfeld, M., Chen, F., Hu, Q., 2008 Enhanced protection against oxidative stress in an astaxanthin-overproduction Haematococcus mutant (Chlorophyceae) Eur J Phycol 43, 365–376 Johnson, M.B., Wen, Z., 2010 Development of an attached microalgal growth system for biofuel production Appl Microbiol Biotechnol 85, 525–534 Kang, C.D., Lee, J.S., Park, T.H., Sim, S.J., 2005 Comparison of heterotrophic and photoautotrophic induction on astaxanthin production by Haematococcus pluvialis Appl Microbiol Biotechnol 68, 237–241 Kang, C.D., An, J.Y., Park, T.H., Sim, S.J., 2006 Astaxanthin biosynthesis from simultaneous N and P uptake by the green alga Haematococcus pluvialis in primary-treated wastewater Biochem Eng J 31, 234–238 Kang, C.D., Lee, J.S., Park, T.H., Sim, S.J., 2007 Complementary limiting factors of astaxanthin synthesis during photoautotrophic induction of Haematococcus pluvialis: C/N ratio and light intensity Appl Microbiol Biotechnol 74, 987–994 Kang, C., Han, S., Choi, S., Sim, S., 2010 Fed-batch culture of astaxanthin-rich Haematococcus pluvialis by exponential nutrient feeding and stepwise light supplementation Bioproc Biosyst Eng 33, 133–139 Kebede-Westhead, E., Pizarro, C., Mulbry, W.W., 2006 Treatment of swine manure effluent using freshwater algae: production, nutrient recovery, and elemental composition of algal biomass at four effluent loading rates J Appl Phycol 18, 41–46 Lababpour, A., Shimahara, K., Hada, K., Kyoui, Y., Katsuda, T., Katoh, S., 2005 Fedbatch culture under illumination with blue light emitting diodes (LEDs) for astaxanthin production by Haematococcus pluvialis J Biosci Bioeng 100, 339– 342 Li, Y., Sommerfeld, M., Chen, F., Hu, Q., 2010 Effect of photon flux densities on regulation of carotenogenesis and cell viability of Haematococcus pluvialis (Chlorophyceae) J Appl Phycol 22, 253–263 Li, J., Zhu, D., Niu, J., Shen, S., Wang, G., 2011 An economic assessment of astaxanthin production by large scale cultivation of Haematococcus pluvialis Biotechnol Adv 29, 568–574 Liang, S.C.a.Y., 2009 Effects of illumination on the chlorophyll fluorescence parameters and astaxanthin content of Haematococcus pluvialis South China Fisheries Sci 5, 1–8 Liu, T., Wang, J., Hu, Q., Cheng, P., Ji, B., Liu, J., Chen, Y., Zhang, W., Chen, X., Chen, L., Gao, L., Ji, C., Wang, H., 2013 Attached cultivation technology of microalgae for efficient biomass feedstock production Bioresour Technol 127, 216–222 Lopez, M.C., Sanchez Edel, R., Lopez, J.L., Fernandez, F.G., Sevilla, J.M., Rivas, J., Guerrero, M.G., Grima, E.M., 2006 Comparative analysis of the outdoor culture of Haematococcus pluvialis in tubular and bubble column photobioreactors J Biotechnol 123, 329–342 Lorenz, R.T., Cysewski, G.R., 2000 Commercial potential for Haematococcus microalgae as a natural source of astaxanthin Trends Biotechnol 18, 160–167 Milledge, J.J., 2011 Commercial application of microalgae other than as biofuels: a brief review Rev Environ Sci Bio/Technol 10, 31–41 Ozkan, A., Kinney, K., Katz, L., Berberoglu, H., 2012 Reduction of water and energy requirement of algae cultivation using an algae biofilm photobioreactor Bioresour Technol 114, 542–548 Pocock, M., 1960 Haematococcus in southern Africa Trans R Soc South Afr 36, 5– 55 Sarada, R., Tripathi, U., Ravishankar, G.A., 2002 Influence of stress on astaxanthin production in Haematococcus pluvialis grown under different culture conditions Process Biochem 37, 623–627 Author's personal copy 32 M Wan et al / Bioresource Technology 163 (2014) 26–32 Tang, H., Chen, M., Garcia, M., Abunasser, N., Ng, K., Salley, S.O., 2011 Culture of microalgae Chlorella minutissima for biodiesel feedstock production Biotechnol Bioeng 108, 2280–2287 Tjahjono, A.E., Hayama, Y., Kakizono, T., Terada, Y., Nishio, N., Nagai, S., 1994 Hyperaccumulation of astaxanthin in a green alga Haematococcus pluvialis at elevated temperatures Biotechnol Lett 16, 133–138 Torzillo, G., Goksan, T., Faraloni, C., Kopecky, J., Masojídek, J., 2003 Interplay between photochemical activities and pigment composition in an outdoor culture of Haematococcus pluvialis during the shift from the green to red stage J Appl Phycol 15, 127–136 Wang, B., Zarka, A., Trebst, A., Boussiba, S., 2003 Astaxanthin accumulation in Haematococcus pluvialis (chlorophyceae) as an active photoprotective process under high irradiance J Phycol 39, 1116–1124 Wilkie, A.C., Mulbry, W.W., 2002 Recovery of dairy manure nutrients by benthic freshwater algae Bioresour Technol 84, 81–91 Yoo, J., Choi, S., Kim, B., Sim, S., 2012 Optimal design of scalable photo-bioreactor for phototropic culturing of Haematococcus pluvialis Bioproc Biosyst Eng 35, 309–315 Zhang, B.Y., Geng, Y.H., Li, Z.K., Hu, H.J., Li, Y.G., 2009 Production of astaxanthin from Haematococcus in open pond by two-stage growth one-step process Aquaculture 295, 275–281 ... higher than that of 10 g mÀ2 The induction effect of the attached bioreactor was compared with that of the column bioreactor As the results, the variation trend of the astaxanthin in the column bioreactor... Fig The attached bioreactor for the induction of H pluvialis (A) The overview of the attached induction (B) The details of the algae ‘disk’ where Mt was dry weight after induced, and M0 was the. .. concentration in the column bioreactor, V (L) is the volume of the column bioreactor, S (m2) was the light-receiving area of cells Therefore, the starting cell densities per liter in the column bioreactor