Continuous flocculation-sedimentation for harvesting Nannochloropsis salina biomass

Journal of Biotechnology 222 (2016) 94–103

Contents lists available at ScienceDirect

Journal of Biotechnology journal homepage: www.elsevier.com/locate/jbiotec

Continuous flocculation-sedimentation for harvesting Nannochloropsis salina biomass Tawan Chatsungnoen, Yusuf Chisti ∗ School of Engineering, Massey University, Private Bag 11 222, Palmerston North, New Zealand

a r t i c l e

i n f o

Article history: Received 5 December 2015 Received in revised form 3 February 2016 Accepted 11 February 2016 Available online 12 February 2016 Keywords: Flocculation–sedimentation microalgae Nannochloropsis salina Biofuels

a b s t r a c t A continuous flow process is developed for recovery of the biomass of the marine microalga Nannochloropsis salina. Flocculation–sedimentation is used to recover the biomass from an algal suspension with an initial dry biomass concentration of 0.5 g L−1 , as would be typical of a raceway-based biomass production system. More than 85% of the biomass initially in suspension could be settled by gravity in a flocculation–sedimentation device with a total residence time of ∼148 min. Aluminum sulfate was used as an inexpensive, readily available and safe flocculant. The optimal flocculant dosage (as Al2 (SO4 )3 ) was 229 mg L−1 . Relative to a highly effective 62-min batch flocculation–sedimentation process for the same alga and flocculant, the continuous flow operation took longer and required nearly double the flocculant dose. The design of the flocculation–sedimentation system is explained. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Nannochloropsis salina is a marine microalga that has attracted much attention as a potential source of oils for making biodiesel and other fuels (Chatsungnoen, 2015; Doan et al., 2011; Kilian et al., 2011; Mirsiaghi and Reardon, 2015; Rodolfi et al., 2009). Typically, the alga is grown in shallow, open ponds to a peak attainable biomass concentration of about 0.5 g L−1 on a dry basis (Chisti, 2010; Sompech et al., 2012). For oil recovery, the biomass must be separated from the water. This dewatering is expensive (Chisti and Yan, 2011; Uduman et al., 2010). Dewatering methods include various types of filtrations (Danquah et al., 2009a; Gerardo et al., 2015; Mo et al., 2015), centrifugation (Gerardo et al., 2015; Molina Grima et al., 2003), electrocoagulation (Lee et al., 2013) and forward osmosis (Mazzuca Sobczuk et al., 2015). Some of these methods are used in commercial dewatering processes, but are too expensive for harvesting the biomass for cheap products such as algal fuel oils. Chemically induced flocculation, or agglomeration, of the biomass followed by gravity sedimentation of the flocs is the least expensive option for partial dewatering (Lee et al., 2009; Molina Grima et al., 2003; Vandamme et al., 2013). Cheap, readily available and inexpensive metal salts such as aluminum sulfate are effective flocculants for colloidal particles and have a long history of use in commercial water treatment processes (Bratby, 2006).

∗ Corresponding author. E-mail address: [email protected] (Y. Chisti). http://dx.doi.org/10.1016/j.jbiotec.2016.02.020 0168-1656/© 2016 Elsevier B.V. All rights reserved.

Flocculation–sedimentation can remove nearly two-thirds, or more of the water (Chatsungnoen, 2015; Chatsungnoen and Chisti, 2016). The metal cations in aluminum sulfate and other inorganic salts neutralize the negative surface charge on the algal cells (Gerardo et al., 2015). This reduces the mutual repulsion of the cells and allows them to come together and attach by van der Waals forces (Ndikubwimana et al., 2015). Other mechanisms also contribute to flocculation (Duan and Gregory, 2003). The flocculated cells, or flocs, are much larger than the individual cells and readily settle under gravity. Other effective flocculants include cationic polymers, but these tend to be much more expensive than the metal salts such as aluminum sulfate. The flocculation–sedimentation process involves three steps: (1) a rapid mixing step for uniformly dispersing the flocculant in the algal slurry; (2) a step of gentle mixing to facilitate cell–cell encounters while minimizing the breakup of agglomerates; and (3) a quiescent step to allow the flocs to settle by gravity. Formation and growth of flocs, or flocculation, actually occurs mostly in step 2. This work reports on a continuous flow process for flocculation–sedimentation of N. salina. Aluminum sulfate is used as an effective, cheap and safe flocculant. Floculation–sedimentation is demonstrated for an algal broth with a relatively low but commercially relevant biomass concentration of 0.5 g L−1 . The engineering design of the flocculation–sedimentation system is discussed. Prior studies of flocculation of microalgae all used batch flocculation–sedimentation processes (Chen et al., 2013; Danquah et al., 2009b; Farid et al., 2013; Garzon-Sanabria et al., 2012;

T. Chatsungnoen, Y. Chisti / Journal of Biotechnology 222 (2016) 94–103

95

2. Materials and methods Nomenclature A A680 a b Cb Cc CF Ci D Db Dc d db dc Hb Hc Hh Hr Hs L Mb N Pb Pr Ps Q Sb T tR V Vb Vh Vs vo

vs W Wb Wc

Total surface area of raceway ponds Spectrophotometric absorbance at 680 nm The hopper dimension identified in Fig. 4 The hopper dimension identified in Fig. 4 Dry biomass concentration Impeller clearance from the bottom of the continuous mixing tank, or flocculation tank Flocculant dosage Impeller clearance from the bottom of the batch mixing vessel, or batch flocculation vessel Dilution factor Internal diameter of batch mixing vessel Internal diameter of the continuous flow mixing vessel, or flocculation vessel Impeller diameter Impeller diameter of batch mixer Impeller diameter of the continuous flow mixer, or flocculation mixer Broth depth in batch mixing vessel Broth depth in continuous flow mixing vessel, or flocculation vessel The hopper dimension identified in Fig. 4 Sedimentation depth at the inlet of the settling tank Broth depth at the bottom of the slope in the settling tank Length of the settling tank Floc production rate Impeller rotational speed Areal productivity of dry algal biomass Percentage of the microalgal biomass removed from the broth by settling Percentage by weight of solids in the harvested biomass Volume flow rate of feed, or broth Specific gravity of the biomass Impeller tip speed Residence time Volume of the algal broth in vessel, or the working volume Volume production rate of the flocculated biomass Volume of the biomass hopper Required volume of large scale flocculation–sedimentation system Overflow velocity in sedimentation tank Settling velocity of floc Width of the sedimentation tank Impeller blade height in the batch mixer Impeller blade height in continuous flow mixing vessel, or flocculating vessel

Greek letters w Density of water

2.1. The microalga and culture conditions The marine microalga N. salina (CCAP849/3) was used in view of its high oil content and productivity compared to many other algae (Chatsungnoen, 2015; Chatsungnoen and Chisti, 2016). The alga was purchased from the Culture Collection of Algae and Protozoa (CCAP), Argyll, United Kingdom. The alga was maintained and cultured aseptically in the BG11 medium (Andersen et al., 2005) made with artificial seawater (Chatsungnoen, 2015; Chatsungnoen and Chisti, 2016). For inoculum preparation, a stock culture from a liquid or solid medium was transferred to 40 mL of the fresh medium in a 250 mL Erlenmeyer flask. The flask was held in an incubator shaker (25 ◦ C, 130–140 rpm) under fluorescent light (∼30 ␮mol photons m−2 s−1 ), for around 20–30 days. This culture (40 mL) was used to inoculate 360 mL of BG11 in a 1 L Duran bottle (borosilicate glass 3.3, LabServ, Biolab, Auckland, New Zealand). This bottle was incubated at room temperature (∼25 ◦ C) for approximately 7–14 days. This preculture (400 mL) was used to inoculate 1.6 L of the fresh medium in a 2 L Duran bottle for further growth for around 7–14 days. The resulting inoculum was split into 400 mL lots and transferred to 5 fresh 2-L Duran bottles, each with 1.6 L of BG11. The Duran bottles (1 L, 2 L) were maintained at room temperature (24–26 ◦ C) under continuous light (∼219 ␮mol photons m−2 s−1 ) from a bank of six fluorescent lamps (Philips TLD 58w/840, cool white, Thailand). All Duran bottle cultures were continuously bubbled (0.375 L min−1 ) with humidified air mixed with 5% (vol/vol) carbon dioxide. The inlet and exhaust gases were sterile filtered by passing through 0.2 ␮m Teflon membrane filters (Midisart® 2000; Sartorius AG, Goettingen, Germany). The culture was harvested in the stationary phase (day 55) and kept at 4 ◦ C in the dark. This broth was used in flocculation studies within 7-days of harvest.

2.2. Biomass concentration in algal broth A 20 mL sample of the algal broth was vacuum filtered using a pre-weighed microfiber disc filter (Whatman GF-C, 0.45 ␮m, 90 mm). The biomass was washed with 0.5 M ammonium formate (2 × 20 mL) and dried overnight at 80 ◦ C. Subsequently, the biomass was cooled to room temperature in a desiccator and weighed to calculate the dry biomass in 20 mL of the algal broth (Lee and Shen, 2004). An algal broth sample with a precisely known concentration (dry weight) of the biomass was serially diluted with the fresh medium and the optical density of the samples was measured at 680 nm in a spectrophotometer (Ultrospec 2000 spectrophotometer, Pharmacia Biotech, Model 80-21062106-00, England). The blank was the fresh medium. At least six dilutions were measured such that the maximum measured optical density did not exceed 0.6. The measured absorbance was plotted against the calculated dry weight to obtain the following linear calibration equation:

Cb = Papazi et al., 2010; Poelman et al., 1997; Rwehumbiza et al., 2012; Schlesinger et al., 2012; S¸irin et al., 2012; Sukenik et al., 1988; Vandamme et al., 2013). Batch processes are difficult to implement in continuously operated large-scale commercial biomass production operations. Therefore, there is need for in-depth studies of continuous flow flocculation–sedimentation for concentration of algal biomass.

A680 × D 5.6773

(1)

where Cb (g L−1 ) is the dry biomass concentration of the sample, A680 is the measured absorbance and D is the dilution factor. Subsequently, the biomass concentration of an unknown sample was calculated using the measured absorbance of an appropriately diluted sample.

96

T. Chatsungnoen, Y. Chisti / Journal of Biotechnology 222 (2016) 94–103

2.3. Continuous flocculation–sedimentation process Continuous flocculation–sedimentation studies were carried out using aluminum sulfate (Al2 (SO4 )3 ·18H2 O; Riedel-de Haen, Hanover, Germany) as the flocculant as it had been earlier shown to be highly effective (Chatsungnoen, 2015; Chatsungnoen and Chisti, 2016) for batch flocculation of exactly the same alga as used in the present study. The initial dosage of the flocculant (CF , mg L−1 ) for an algal broth with a given dry biomass concentration Cb (g L−1 ) was calculated using the following equation: CF (mg L−1 ) = 229.57Cb (g L−1 )

(2)

The above equation was developed for batch flocculation (Chatsungnoen, 2015) and it provided CF in terms of the Al2 (SO4 )3 , not its hydrated form. The calculated dosage was for 95% removal of the biomass from an algal broth with an initial biomass concentration Cb . The flocculant solution (885 mg L−1 calculated as Al2 (SO4 )3 ) was prepared by dissolving the salt in deionized water. The solution was kept at room temperature (24–26 ◦ C) and used within 14-days. 2.3.1. The flocculation–sedimentation equipment The continuous flocculation–sedimentation system used in this study is shown in Fig. 1. The algal broth was continuously pumped from a 20 L reservoir (1, Fig. 1) via the pump 2 (Fig. 1) to the rapid mixing tank (5, Fig. 1). Simultaneously, the flocculant solution (885 mg L−1 as Al2 (SO4 )3 ) from the 5-L reservoir 3 (Fig. 1) was continuously pumped (pump 4, Fig. 1) into the rapid mixing tank 5 (Fig. 1). In this tank, the algal broth and the flocculant solution were mixed rapidly (high-speed motor 8, Fig. 1) and the mixed slurry continuously overflowed by gravity into the flocculation tank (6, Fig. 1). Here the suspension was gently mixed using the low-speed agitator (9, Fig. 1). The flow from the flocculation tank (9, Fig. 1) was by gravity into the sedimentation tank (7, Fig. 1). The clarified broth left the sedimentation tank by overflow (10, Fig. 1) while the algal solids remained in the sedimentation tank (7, Fig. 1). The flow rates of the algal broth and the flocculant solution could be adjusted as desired using the variable speed peristaltic pumps 2 and 4 (Fig. 1), respectively. As the volumes of the rapid mixing tank (∼113 mL) and the flocculation tank (∼1065 mL) were fixed, the residence time of the fluid in these vessels depended on the combined flow rate of the algal broth and the flocculant solution. 2.3.2. The design of the mixing vessel, the flocculation tank and the sedimentation tank 2.3.2.1. The mixing vessel. Rapid or flash mixing is required to mix the flocculant with the algal broth to initiate the particle aggregation process (Letterman and Yiacoumi, 2011). Rapid mixing is usually carried out with a high-speed impeller in a small mixing vessel. A cylindrical mixing tank with a 2-bladed impeller was used (5, Fig. 1). The mixing systems was a scaled up version of the mixing tank that had been previously successfully used in our batch flocculation studies (Chatsungnoen, 2015; Chatsungnoen and Chisti, 2016). The dimensions of the batch mixing tank described in earlier publications (Chatsungnoen, 2015; Chatsungnoen and Chisti, 2016) are as shown in Fig. 2. In a batch flocculation–sedimentation operation, the same vessel is used sequentially for mixing, flocculation and sedimentation steps, but under different conditions of agitation (Chatsungnoen and Chisti, 2016). The batch mixing vessel (Fig. 2) had an internal diameter of 6.3 cm (Db ); a broth depth (Hb ) of 6.4 cm; and an impeller diameter (db ) of 5 cm. A 2-bladed impeller was used. The impeller clearance (Ci ) from the bottom of the vessel was 2.5 cm. The impeller blade width (Wb ) was 1.1 cm and the blades were inclined at a 25◦ angle from the horizontal. For rapid mixing of the broth and the flocculant in the previously described batch operation (Chatsungnoen, 2015; Chatsungnoen and Chisti,

2016), the tip seed of the impeller was 20.95 cm s−1 (80 rpm) and the duration of the mixing was 2 min. rapid mixing vessel for the continuous The flocculation–sedimentation process (5, Fig. 1) was a scaled up version of the small batch mixing vessel shown in Fig. 2. In scaling up mixing systems, the geometric ratios of the small system are kept the same at the larger scale. In this case, the larger scale was the continuous mixing tank (5, Fig. 1). The suggested geometric proportions for a circular mixing tank are a broth depth (Hb ) to vessel internal diameter (Db ) ratio of 1 (Hemrajani and Tatterson, 2004; Oldshue, 1983). Based on this, a suitable vessel which was available in the laboratory was used. It had a 5.2 cm internal diameter and could accommodate a 5.3 cm broth depth. The broth depth-to-vessel internal diameter ratio was 1.02, or close to the recommended value and nearly the same as for the batch mixing vessel (Fig. 2). The volume of the microalgal broth in the vessel (V, mL) could be calculated as follows: V=

Db2 4

Hb

(3)

The calculated volume was 112.57 mL, or ∼113 mL. The flow rate (Q, mL min−1 ), required for a given residence time (tR ) in the vessel was estimated as follows: Q =

V tR

(4)

The value of the residence time was set at 2 min to correspond to the 2 min mixing time used in the batch process (Chatsungnoen, 2015; Chatsungnoen and Chisti, 2016). Thus, the broth flow rate through the continuous rapid mixing vessel was 56.5 mL min−1 . For geometric similarity with the batch mixing impeller (Fig. 2), the diameter of the impeller for the continuous flow mixer was calculated as follows: db dc = Db Dc

(5)

where db is impeller diameter of the batch vessel (5 cm); Db is the internal diameter of the batch vessel (6.3 cm); dc is impeller diameter of the continuous rapid mixing vessel (cm); Dc is internal diameter of the continuous rapid mixing vessel (5.2 cm). Based on this, the impeller diameter for the continuous flow mixing vessel was 4.13 cm. The vertical blade height (Wc , cm), of the continuous flow mixing impeller was calculated using the following relationship: Wb Wc = db dc

(6)

where Wb is vertical blade height of the batch impeller (1.10 cm; Fig. 2); db is impeller diameter in the batch mixer (5.0 cm, Fig. 2); Wc (cm) is vertical blade height of the impeller in the continuous flow mixer; and dc is diameter of the continuous flow impeller (4.13 cm). Thus, the vertical blade height of the impeller in the continuous flow vessel was 0.91 cm. The impeller clearance (Cc , cm), from the bottom of the continuous mixing tank had to satisfy the following relationship for geometric similarity: Db Dc = Ci Cc

(7)

where Ci is the clearance in the batch mixing tank (Fig. 2) and Cc is the clearance in the continuous mixing tank. As the internal diameter of the batch vessel (Db ) was 6.3 cm and Ci was 2.5 cm, a Cc value of 2.1 cm was calculated. For similar levels of mixing at the two scales in geometrically similar mixers, the tip speed of the impeller at the two scales needs

T. Chatsungnoen, Y. Chisti / Journal of Biotechnology 222 (2016) 94–103

Fig. 1. The continuous flocculation–sedimentation setup.

Fig. 2. Geometric details of the batch mixer: mixing tank (A) and the 2-bladed impeller (B). Dimensions in cm.

Fig. 3. The sedimentation tank. Dimensions in mm.

97

98

T. Chatsungnoen, Y. Chisti / Journal of Biotechnology 222 (2016) 94–103

Table 1 Design summary of continuous flow mixing and flocculation vessels. Description

Mixing tank

Flocculation tank

Internal diameter of vessel (Dc , cm) Broth depth in vessel (Hc , cm) Vessel aspect ratio (Hc /Dc ) Working volume of tank (V, mL) Impeller diameter (dc , cm) Impeller vertical blade height (Wc , cm) Impeller clearance from bottom (Cc , cm) Impeller tip speed (T, cm s−1 ) Broth flow rate (Q, mL min−1 ) Retention time (tR , min)

5.2 5.3 1.02 113 4.13 0.91 2.1 20.95 56.5 2

11 11.2 1.02 1065 8.73 1.92 4.37 5.24 56.5 19

o =

to be identical. The impeller tip speed (T) at any scale was calculated as follows: T=

Nd 60

velocity (vo ) in the tank will settle out (Hendricks, 2006). Therefore the maximum overflow velocity (vo ) had to be 0.256 cm min−1 . As the broth flows upwards through the cross-section of the tank, the tank surface area for the maximum overflow velocity was calculated using the following equation:

(8)

where T (cm s−1 ) is the tip speed; N is impeller rotational speed (rpm); and d is impeller diameter. In the batch mixing vessel the tip speed was 20.95 cm s−1 (80 rpm). Thus, the impeller rotational speed in the continuous flow mixing vessel needed to be 96.87 rpm (97 rpm). 2.3.2.2. The continuous flocculation vessel. In batch flocculation studies, the mixing vessel (Fig. 2) was also used for the flocculation step, but at a reduced agitation speed (Chatsungnoen, 2015; Chatsungnoen and Chisti, 2016). Therefore, the mixing tank and flocculation tank of the batch operation were identical. The flocculation time used in batch studies was 30 min (Chatsungnoen, 2015). The continuous flocculation vessel and impeller were geometrically similar to the system used in the batch operations (Fig. 2). The continuous flocculation was performed in a vessel with an internal diameter of 11 cm and a broth depth of 11.2 cm. The broth volume in the vessel was 1064.51 mL, or ∼1065 mL. Based on the previously calculated feed flow rate (Q) of 56.5 mL min−1 (Eq. (4)), the retention time in the continuous flocculation vessel was 19 min. This was deemed sufficient based on prior experience, although it was less than the 30 min used in the batch operations (Chatsungnoen, 2015; Chatsungnoen and Chisti, 2016). In view of the earlier mentioned requirement of geometric similarity, the impeller diameter for the continuous flocculation vessel was calculated to be 8.73 cm. The calculated vertical blade height was 1.92 cm. The calculated impeller clearance from the bottom was 4.36 cm, or 4.4 cm. The impeller tip speed in the continuous flocculation tank needed to be the same as the tip of 5.24 cm s−1 used in the batch flocculation operation (Chatsungnoen, 2015). In the continuous flocculation tank, this tip speed was equivalent to an impeller rotational speed of 11.46 rpm, or 11.5 rpm. The results from the above calculations are summarized in Table 1. 2.3.2.3. The sedimentation tank. Although both rectangular and circular sedimentation tanks are commonly used in continuous large scale water treatment processes (Joint Task Force of the Water Environment Federation and the American Society of Civil Engineers, 1992; Lin, 2007), rectangular tanks have certain advantages compared to circular tanks (Liu and Lipták, 1999). A rectangular sedimentation tank (7, Figs. 1 and 3) was designed for this work. The relevant calculations (Hendricks, 2006; Kaira and Christian, 2006) and operation are described below. Based on batch sedimentation experiments, at least 25 min were required by the flocs to settle in a 6.4 cm deep tank (Chatsungnoen, 2015). Therefore, the settling velocity of the flocs was 0.256 cm min−1 . In concept, in a sedimentation tank a particle with a settling velocity (vs ) greater than or equal to the overflow

Q W ×L

(9)

where vo is the overflow velocity (0.256 cm min−1 ), Q is the volume flow rate of the microalgal broth through the sedimentation tank (the same flow rate as in the mixing and flocculation tanks, or 56.5 mL min−1 ), W is the width of the tank (cm) and L is the length of the settling tank (cm). The required surface area of the tank was 220.7 cm2 , or 221 cm2 . For rectangular sedimentation tanks, an economically acceptable length (L) to width (W) ratio (L/W) of 4 to 66 has been recommended (Gregory and Edzwald, 2011; Hendricks, 2006; Riffat, 2013). A ratio of 4 was used in this work. For a tank with a surface area of L × W (= 221 cm2 ) and L/W value of 4, L = 4W and the surface area is 4W × W = 221 cm2 . Therefore, W = 7.43 cm, or 7.5 cm, and L = 30 cm. The sedimentation depth Hr at the inlet of the rectangular tank was kept identical to the sedimentation depth in the batch settling vessel (Chatsungnoen, 2015). Therefore, Hr = 6.4 cm. As a result the volume (= W × L × Hr ) of the rectangular tank was calculated to be 1440 mL. The dimensions of the biomass collection hopper at the end of the sedimentation tank (Fig. 3) were then calculated using an estimate of the generation rate of the flocs. The floc production rate (Mb , g min−1 ) was estimated for 95% removal of the biomass from the broth on a dry weight basis. Thus, Mb = Pr × Cb × Q

(10)

where Pr (= 95%) is the percentage of the microalgal biomass removed from the broth; Cb (= 1 g L−1 ) is the biomass concentration in the broth; and Q is flow rate of the broth through the sedimentation tank (= 56.5 mL min−1 , or 56.5 × 10−3 L min−1 ). The production rate of the flocs was therefore 0.054 g min−1 . The volume production rate of the flocculated biomass (Vb , mL min−1 ) was estimated as follows: Vb =

Mb w Sb Ps

(11)

where w is the density of water (= 0.997 g mL−1 at 25 ◦ C); Sb is the specific gravity of the biomass (= 1.10; Fogg (1975)); and Ps is the percentage of solids in the harvested biomass expressed as a decimal fraction (= 0.20, as the moisture content of the biomass was ∼80% w/w based on preliminary experiments). Therefore, the volume production rate of the flocs was 0.25 mL min−1 .The capacity of the biomass collection hopper (Fig. 3) was then calculated assuming that the biomass was removed from the hopper every 4-h by pumping. Therefore, the hopper had to have biomass storage capacity for 4-h, or 240 min. Hence, the capacity of the hopper needed to be 240 × Vb , or 60 mL. Generally, the hopper bottoms are designed with a trapezoidal shape (Kaira and Christian, 2006) as shown in Fig. 4. The volume (Vh ) of such a shape with a channel width W is given by the following equation: Vh =

Hh (a + b) W 2

(12)

where the dimensions Hh , a and b are identified in Fig. 4. For the specified volume of 60 mL and a tank width W of 7.5 cm (Fig. 3), suitable values of Hh , a and b were 2 cm, 5 cm and 3 cm, respectively. The sedimentation tank was designed with a slope of 20% based on the typical values used in the literature (Kaira and Christian,

T. Chatsungnoen, Y. Chisti / Journal of Biotechnology 222 (2016) 94–103

99

Table 2 The operational conditions of the continuous flocculation–sedimentation process. Description

Value

Residence time in mixing tank (min) Residence time in flocculation tank (min) Residence time in sedimentation tank (min) Total residence time (min) Flow rate of algal broth (mL min−1 ) (90% of the total flow rate) Flow rate of aluminum sulfate solution (mL min−1 ) (10% of the total flow rate) Total flow rate (mL min−1 ) Rapid mixing impeller speed (rpm) Flocculation impeller speed (rpm)

2 18.85 38.16 59.01 50.9 5.6 56.5 97 11.5

Fig. 4. The biomass collection hopper of the sedimentation tank.

start of flow. Similarly, the agitation speed in the flocculation tank had been set to 11.5 rpm. Samples (5 mL) were taken from the outlet zone of the sedimentation tank (Fig. 3) and the biomass concentration (g L−1 ) was determined spectrophotometrically as previously explained. The samples were taken every 30 min until the flocculation–sedimentation process reached a steady state (i.e. after the passage of 4-residence times, based on total volume of the equipment, from the instance of a complete filling of the equipment).

Biomass removal (% of initial)

100 90 80 70 60 50 40

Flocculant dose

30

Control (114.4 mg/L) 1.5 x control 2.0 x control 3.0 x control

20 10 0 0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

Time (h) Fig. 5. The biomass removal efficacy of the continuous flocculation–sedimentation process operated with various dosages of aluminum sulfate. All measurements were made from the instance of the sedimentation tank being filled to operational level (i.e. 59 min on the time axis is shown as 0 h).

2006) and the preliminary experiments. For this slope, the broth depth (Hs ) at the bottom of the slope was calculated as follows: Hs = 0.2 (L − a)

(13)

where L (= 30 cm) is the length of the settling tank and a is width of the hopper bottom (= 5 cm; Fig. 4). The slope depth was 5.0 cm. Therefore, the overall depth of the settling tank was 13.4 cm (= Hr + Hs + Hh ). For the calculated overall length of the settling tank, a 10% length was added for the inlet zone and the outlet zone. Therefore, the overall length of the settling tank was 36 cm. The overall retention time of the designed sedimentation tank for a broth flow rate of 56.5 mL min−1 was 38.2 min. 2.3.3. Continuous flocculation–sedimentation procedure The microalga was grown to the stationary phase, as previously explained. The broth was then diluted with the fresh medium to a biomass concentration of 0.5 g L−1 , as is typically found in algal broths produced in commercial raceway ponds (Borowitzka, 2005; Chisti, 2012, 2013; Molina Grima et al., 2003). The flocculant solution (885 mg L−1 as Al2 (SO4 )3 ) was prepared separately. The broth flow rate was set at the desired value. The flocculant flow rate was adjusted such that the flocculant dose corresponded to the optimal dosage calculated using Eq. (2). The start-up procedure consisted of establishing the flows of the algal broth and the flocculant solution at the desired rates. Allowing the mixing tank, the flocculation tank and the sedimentation tank (Fig. 1) to fill to the operating levels. The agitation speed in the mixing tank had been set to 97 rpm prior to the

3. Results and discussion The operational conditions of the continuous flow flocculation–sedimentation process are summarized in Table 2. N. salina was used in this study as it is known to have a high lipid productivity (Chatsungnoen, 2015; Doan et al., 2011; Kilian et al., 2011; Mirsiaghi and Reardon, 2015; Rodolfi et al., 2009). Furthermore, it has a small cell diameter (4.8 ± 0.4 ␮m; Chatsungnoen, 2015) and, therefore, its flocs are expected to be harder to settle compared to the flocs of many other oil producing microalgae (e.g. Neochloris sp., Chlorella vulgaris, Cylindrotheca fusiformis; Chatsungnoen, 2015). If a continuous flow flocculation–sedimentation process is effective for N. salina, it is likely to be effective for most other microalgae. The initial total flow rate of the microalgal broth and the aluminum sulfate solution were set at the calculated value 56.5 mL min−1 (Eq. (4)). This was the baseline, or the control flow rate. Mixing of the aluminum sulfate solution with the algal broth in rapid mixing tank (5, Fig. 1) caused an immediate destabilization of the microalgal suspension and the cells began to aggregate into small flocs. From the mixing tank, the algal suspension flowed into the gentle mixing environment of the flocculation tank (6, Fig. 1). Here the small flocs developed into larger flocs through further agglomeration. The residence time, or the flocculation time in tank 6 (Fig. 1) was 19 min at the control value of the feed (algal broth) flow rate. The flow containing the large flocs then entered the sedimentation tank (7, Fig. 1). Here the flocs settled by gravity and the clarified liquor exited the tank at the outflow (10, Fig. 1). Once the entire flocculation–sedimentation equipment (i.e. the tanks 5, 6 and 7; Fig. 1) had been filled to the required levels, samples (5 mL) were taken at the outlet zone of the rectangular sedimentation tank for measuring the flocculation efficiency. Samples were taken every 30 min until the process attained a steady state. That is, the biomass reading in the clarified liquor at the outlet (10, Fig. 1) had become steady. A steady state was considered to exist after the elapse of 4-residence times of the broth in the entire equipment, counting from the point of complete filling of the equipment. At steady state the biomass concentration readings in the outflow broth were steady.

100

T. Chatsungnoen, Y. Chisti / Journal of Biotechnology 222 (2016) 94–103

Fig. 6. Comparison of the continuous flocculation–sedimentation (A1, B1, C1, D1) with the batch process (A2, B2, C2, D2) at identical dosages of the flocculant. The residence time in the continuous flow tanks (A1, B1, C1, D1) was 4 h. The residence time in the batch tanks (A2, B2, C2, D2) was 30 min (i.e. the settling time). The flocculant dosages were as follows: control dosage of 114.4 mg L−1 (A1, A2); 1.5× control (B1, B2); 2× control (C1, C2); 3× control (D1, D2).

3.1. Effect of aluminum sulfate dosage on biomass removal As previously shown for batch flocculation work with a variety of microalgae including N. salina (Chatsungnoen, 2015; Chatsungnoen and Chisti, 2016), the flocculant dosage for a given concentration of the biomass in the broth is the most important factor influencing floc formation. The dosage influences both the rate of flocculation and its extent (Lee et al., 1998). Therefore, this experiment focused on identifying the optimal aluminum sulfate dosage for the recovery of the N. salina biomass in the continuous flocculation–sedimentation process. Several dosages of the aluminum sulfate flocculant were trialled. The control dosage was taken to be 114.4 mg L−1 (Eq. (2)), a value that had earlier been shown to the optimal in batch flocculation of the same alga grown under the same conditions (Chatsungnoen, 2015; Chatsungnoen and Chisti, 2016) as in the present study. As shown in Fig. 5,

with the control value of the dosage the steady state biomass removal was in the range of 40–50% of the initial biomass concentration. In batch flocculation studies, the same dosage had resulted in 95% biomass removal (Chatsungnoen, 2015; Chatsungnoen and Chisti, 2016). Therefore, the optimal dosage of the batch flocculation–sedimentation process was clearly insufficient for the continuous flow process. Therefore, flocculant dosages of 1.5× control (i.e. 171.6 mg L−1 ), 2× control (228.8 mg L−1 ) and 3× control (343.2 mg L−1 ) were trialled. Increasing dosage improved biomass removal (Fig. 5). A dosage of 2-fold the control value was the minimum for satisfactory biomass removal (Fig. 5). A further increase in dosage did not enhance the biomass removal (Fig. 5). At the minimum effective dosage of 2-times the control dose, the biomass removal was ∼80%, significantly lower than the 95% removal that could be achieved in the batch flocculation operation (Chatsungnoen, 2015; Chatsungnoen and Chisti, 2016). A

T. Chatsungnoen, Y. Chisti / Journal of Biotechnology 222 (2016) 94–103

3.2. Effect of the total flow rate on biomass removal in continuous flocculation–sedimentation process In principle, an increase in the fluid residence time in the flocculation tank should increase the average floc size. These larger flocs combined with a longer residence time in the sedimentation tank should result in an improved recovery of the biomass in the sedimentation vessel. Therefore, using a flocculant concentration of 229.0 mg L−1 that was most effective in the continuous flocculation process with a total residence time of 59 min, lower and higher flow rate than the control flow rate of 56.5 mL min−1 were tested. The results are shown in Table 3. A 20% greater flow rate than control, reduced the residence time of the slurry in the various zones of the flocculation–sedimentation system (Table 3) but barely affected the biomass removal efficiency. However, reducing the flow rate relative to control resulted in a progressive enhancement in the flocculation efficiency, or the biomass removal efficiency (Table 3). This is clearly seen in Figs. 7 and 8 where a reducing flow rate improves the clarification of the overflow stream and more biomass settles out of the broth. Whilst reducing the flow rate did improve sedimentation (Fig. 7), in principle the same effect can be achieved at a given flow rate by designing the sedimentation tank such that the residence time of the broth in it is increased. At a constant flow rate, a sedimentation tank of an increased size should allow more biomass to settle. Other options for improving the biomass recovery are a reduced agitation speed in the mixing vessel to reduce fragmentation of the flocs being formed and an increase in the fluid residence time in the flocculation vessel so that larger flocs are formed (Hogg, 2000; Sastry et al., 2000). Furthermore, polymeric flocculants may be used in conjunction with aluminum sulfate to increase the resistance of the flocs to disintegration during mixing and flow (Rebhun, 1990), although this may be impractical as extremely large scale operations are envisaged for biomass production for algal oil as a

100

Biomass removal (% of initial)

visual comparison of the biomass removal in the continuous flow flocculation–sedimentation and the batch process at various identical dosages is shown in Fig. 6. Clearly, the batch process was always more effective in biomass removal, but the efficacy of the continuous flow process improved with increasing dosage of the flocculant (Fig. 6). In the continuous flow process, the flocculation efficiency increased substantially from approximately 48% to nearly 70% when the flocculant dosage was increased from 114.5 mg L−1 to 171.7 mg L−1 (Fig. 5). As shown in Fig. 6, an increase in the flocculant dosage relative to control improved the sedimentation behavior. At 4 h, a greater quantity of the biomass could be sedimented with increasing dosage (Fig. 6). The optimal dosage of the flocculant in the continuous flocculation–sedimentation process was 229.0 mg L−1 , or twice the optimal dosage of the batch flocculation process. At this dosage 75% of the biomass was removed from the broth. This difference in performance relates to the differences in the flocculation conditions in the batch and continuous systems. The very high flocculation efficiency of the batch operation was because all the particles had the same exposure time in the mixing vessel (2 min), the flocculation vessel (30 min) and the sedimentation vessel (30 min). In the continuous flow operation, there was a distribution of residence times in the mixing tank, the flocculation tank as well as the sedimentation tank. The floc size in a batch flocculation system is known to increase progressively with time, but in the continuous flow system the size of the flocs has been observed to have a broader range (Hogg, 2000; Rattanakawin and Hogg, 2000). The smallest of the flocs tended to be washed out of the sedimentation tank with the overflow and only the larger flocs readily settled at the operating conditions used.

101

90

80

Feed flow rate 70

Control (56.5 mL/min) 20% higher (67.8 mL/min) 20% lower (45.2 mL/min) 40% lower (33.9 mL/min) 60% lower (22.6 mL/min)

20 10 0 0

1

2

3

4

5

6

7

8

9

10

11

Time (h) Fig. 7. The biomass removal efficacy of continuous flocculation–sedimentation process at various feed flow rates. All measurements were made from the instance of the sedimentation tank being filled to the operational level (59 min = 0 h for control; 49 min = 0 h for 20% higher flow rate; 74 min = 0 h for 20% lower flow rate; 99 min = 0 h for 40% lower flow rate; 147.5 min = 0 h for 60% lower flow rate). The higher and lower flow rates are relative to control. The flocculant dosage was 229.0 mg L−1 .

potential fuel. Polymeric flocculants are too expensive for use in such an operation. 3.3. Feasibility of continuous flocculation–sedimentation in a large-scale process The flocculation–sedimentation system examined in this study required a total residence time of ∼148 min to recover 86% of the biomass (Table 3). Here we assess if such a system would be technically feasible in terms of the required volume in a large-scale process for producing algal biomass. A large facility using raceways to produce algal biomass may have a total pond surface area (A) of 0.5 ha, or 5000 m2 . A realistic dry biomass productivity (Pb ) for such a facility would be 0.020 kg m−2 /day (Chisti, 2012) and a typical dry biomass concentration (Cb ) in the culture broth at harvest would be 0.5 kg m−3 (Chisti, 2012). For such a facility, the volumetric production rate (Q) of the broth can be estimated as follows: Q =

APb 5000 × 0.020 = 200 m3 /day = Cb 0.5

(14)

For processing this flow rate through a flocculation–sedimentation system with an average residence time tR , the required system volume (Vs ) can be calculated as follows: Vs = QtR

(15)

Therefore, for a residence time of 148 min, or 0.103 day, the system volume would be 20.6 m3 . If the culture depth in the raceway ponds was a typical 0.25 m (Chisti, 2012; Sompech et al., 2012), the total working volume of the ponds would be 1250 m3 . Therefore, the above estimated volume of the flocculation–sedimentation system is a reasonable 1.6% of the volume of the raceways. Such a flocculation–sedimentation system is clearly technically realistic. It would provide nearly 30 tons (= 0.5 × 200 × 0.86 × 365 × 0.95 × 10−3 ) of biomass annually if operated for 95% of the calendar year. 4. Concluding remarks A continuous flow flocculation–sedimentation of microalgal biomass is feasible as demonstrated here using N. salina, an alga

102

T. Chatsungnoen, Y. Chisti / Journal of Biotechnology 222 (2016) 94–103

Table 3 The operational conditions of the continuous flocculation–sedimentation process at various flow rates. Description

Total flow rate (mL min−1 ) Total residence time (min) Residence time in mixing tank (min) Residence time in flocculation tank (min) Residence time in sedimentation tank (min) Flocculation efficiency (%)a a

Feed flow rate Normal

20% higher

20% lower

40% lower

60% lower

56.5 59.0 2 18.85 38.16 74.8 ± 0.1

67.8 49.2 1.67 15.70 31.80 75.1 ± 0.3

45.2 73.8 2.5 23.56 47.70 80.0 ± 0.4

33.9 98.8 3.33 31.42 64 83.1 ± 0.2

20.4 147.5 5 47.12 95.4 86.1 ± 0.1

At steady state (i.e. at any time greater than the 4-residence times of the entire flocculation–sedimentation system).

biomass concentration of 0.5 g L−1 and a total residence time in the flocculation–sedimentation system of ∼148 min.

Acknowledgements This research was supported in part by Ministry of Science and Technology, Thailand, and Maejo University, Chiang Mai, Thailand. The authors gratefully acknowledge this support.

References

Fig. 8. The sedimentation of microalgal flocs in the rectangular sedimentation tank at different total flow rates: (A) a 20% higher total flow rate than control (56.5 mL min−1 ) at 3.5 h of operation (Fig. 6); (B) a 20% lower total flow rate relative to control and 5 h of operation (Fig. 6); (C) a 40% lower total flow rate relative to control and 6.5 h of operation at steady state; (D) a 60% lower total flow rate relative to control and at 10 h of operation. All photographs were at steady state. In all cases the flocculant dosage was 229.0 mg L−1 .

that can be relatively hard to settle because of the small size of its cells. A biomass recovery of at least 86% is feasible using an aluminium sulfate dosage of 229 mg L−1 for a broth with an initial dry

Andersen, R.A., Berges, J.A., Harrison, P.J., Watanabe, M.M., 2005. Recipes for freshwater and seawater media. In: Andersen, R.A. (Ed.), Algal Culturing Techniques. Elsevier, Burlington, MA, p. 429. Borowitzka, M.A., 2005. Culturing microalgae in outdoor ponds. In: Andersen, R.A. (Ed.), Algal Culturing Techniques. Elsevier, Burlington, MA, pp. 205–218. Bratby, J., 2006. Coagulation and Flocculation in Water and Wastewater Treatment, second ed. IWA Publishing, Seattle, WA. Chatsungnoen, T., Chisti, Y., 2016. Harvesting microalgae by flocculation–sedimentation. Algal Res. 13, 271–283. Chatsungnoen, T., 2015. An assessment of inexpensive methods for recovery of microalgal biomass and oils. In: PhD Thesis. Massey University, New Zealand. Chen, L., Wang, C., Wang, W., Wei, J., 2013. Optimal conditions of different flocculation methods for harvesting Scenedesmus sp.: cultivated in an open-pond system. Bioresou. Technol. 133, 9–15. Chisti, Y., Yan, J., 2011. Algal biofuels—a status report. Appl. Energy 88, 3277–3279. Chisti, Y., 2010. Fuels from microalgae. Biofuels 1, 233–235. Chisti, Y., 2012. Raceways-based production of algal crude oil. In: Posten, C., Walter, C. (Eds.), Microalgae Biotechnology: Potential and Production. Walter de Gruyter, Berlin, pp. 113–146. Chisti, Y., 2013. Constraints to commercialization of algal fuels. J. Biotechnol. 167, 201–214. Danquah, M.K., Gladman, B., Moheimani, N., Forde, G.M., 2009a. Microalgal growth characteristics and subsequent influence on dewatering efficiency. Chem. Eng. J. 151, 73–78. Danquah, M.K., Ang, L., Uduman, N., Moheimani, N., Forde, G.M., 2009b. Dewatering of microalgal culture for biodiesel production: exploring polymer flocculation and tangential flow filtration. J. Chem. Technol. Biotechnol. 84, 1078–1083. Doan, T.T.Y., Sivaloganathan, B., Obbard, J.P., 2011. Screening of marine microalgae for biodiesel feedstock. Biomass Bioenergy 35, 2534–2544. Duan, J., Gregory, J., 2003. Coagulation by hydrolysing metal salts. Adv. Colloid Interface Sci. 100–102, 475–502. Farid, M.S., Shariati, A., Badakhshan, A., Anvaripour, B., 2013. Using nano-chitosan for harvesting microalga Nannochloropsis sp. Bioresou. Technol. 131, 555–559. Fogg, G.E., 1975. Algal Cultures and Phytoplankton Ecology, second ed. University of Wisconsin Press, Madison, pp. 175. Garzon-Sanabria, A.J., Davis, R.T., Nikolov, Z.L., 2012. Harvesting Nannochloris oculata by inorganic electrolyte flocculation Effect of initial cell density, ionic strength, coagulant dosage, and media pH. Bioresou. Technol. 118, 418–424. Gerardo, M.L., Van Den Hende, S., Vervaeren, H., Coward, T., Skill, S.C., 2015. Harvesting of microalgae within a biorefinery approach: a review of the developments and case studies from pilot-plants. Algal Res. 11, 248–262. Gregory, G., Edzwald, J.K., 2011. Sedimentation and flotation. In: Edzwald, J.K. (Ed.), Water Quality and Treatment: A Handbook on Drinking Water/American Water Works Association. , sixth ed. McGraw-Hill, New York, pp. 9.1–9.92. Hemrajani, R.R., Tatterson, G.B., 2004. Mechanically stirred vessels. In: Paul, E.L., Atiemo-Obeng, V.A., Kresta, S.M. (Eds.), Handbook of Industrial Mixing: Science and Practice. Wiley, Hoboken, NJ, pp. 345–390. Hendricks, D.W., 2006. Water Treatment Unit Processes: Physical and Chemical. Taylor and Francis, Boca Raton, FL, pp. 139–197. Hogg, R., 2000. Flocculation and dewatering. Int. J. Miner. Process. 58, 223–236. Joint Task Force of the Water Environment Federation and the American Society of Civil Engineers, 1992. Design of municipal wastewater treatment plants. In: Water Environment Federation. American Society of Civil Engineers, Alexandria, VA.

T. Chatsungnoen, Y. Chisti / Journal of Biotechnology 222 (2016) 94–103 Kaira, G.L., Christian, R.A., 2006. Wastewater Treatment: Concepts and Design Approach, 133–162. Prentice-Hall of India Private Limited, New Delhi, pp. 12–41. Kilian, O., Benemann, C.S.E., Niyogi, K.K., Vick, B., 2011. High-efficiency homologous recombination in the oil-producing alga Nannochloropsis sp. Proc. Nat. Acad. Sci. U.S.A. 108, 21265–21269. Lee, Y.K., Shen, H., 2004. Basic culturing techniques. In: Richmond, A. (Ed.), Handbook of Microalgal Culture: Biotechnology and Applied Phycology. Blackwell Science, Oxford, UK, pp. 40–56. Lee, S.J., Kim, S.B., Kim, J.E., Kwon, G.S., Yoon, B.D., Oh, H.M., 1998. Effects of harvesting method and growth stage on the flocculation of the green alga Botryococcus braunii. Lett. Appl. Microbiol. 27, 14–18. Lee, A.K., Lewis, D., Ashman, P., 2009. Microbial flocculation, a potentially low-cost harvesting technique for marine microalgae for the production of biodiesel. J. Appl. Phycol. 21, 559–567. Lee, A.K., Lewis, D.M., Ashman, P.J., 2013. Harvesting of marine microalgae by electroflocculation The energetics, plant design, and economics. Appl. Energy 108, 45–53. Letterman, R.D., Yiacoumi, S., 2011. Coagulation and flocculation. In: Edzwald, J.K. (Ed.), Water Quality and Treatment: A Handbook on Drinking Water/American Water Works Association. , sixth ed. McGraw-Hill, New York, pp. 8.1–8.72. Lin, S.D., 2007. Water and Wastewater Calculations Manual, 2nd ed. McGraw-Hill, New York, pp. 531–884. Liu, D.H.F., Lipták, B.G., 1999. Environmental Engineers’ Handbook, 17. CRC Press, Boca Raton, FL, pp. 7. ˜ González, M.J., Molina Grima, E., Chisti, Y., 2015. Mazzuca Sobczuk, T., Ibánez Forward osmosis with waste glycerol for concentrating microalgae slurries. Algal Res. 8, 168–173. Mirsiaghi, M., Reardon, K.F., 2015. Conversion of lipid-extracted Nannochloropsis salina biomass into fermentable sugars. Algal Res. 8, 145–152. Mo, W., Soh, L., Werber, J.R., Elimelech, M., Zimmerman, J.B., 2015. Application of membrane dewatering for algal biofuel. Algal Res. 11, 1–12. Molina Grima, E., Belarbi, E.H., Acién Fernández, F.G., Robles Medina, A., Chisti, Y., 2003. Recovery of microalgal biomass and metabolites: process options and economics. Biotechnol. Adv. 20, 491–515. Ndikubwimana, T., Zeng, X., He, N., Xiao, Z., Xie, Y., Chang, J.-S., Lin, L., Lu, Y., 2015. Microalgae biomass harvesting by bioflocculation-interpretation by classical DLVO theory. Biochem. Eng. J. 101, 160–167. Oldshue, J.Y., 1983. Fluid Mixing Technology. McGraw-Hill, New York, NY, pp. 574. Papazi, A., Makridis, P., Divanach, P., 2010. Harvesting Chlorella minutissima using cell coagulants. J. Appl. Phycol. 22, 349–355.

103

Poelman, E., De Pauw, N., Jeurissen, B., 1997. Potential of electrolytic flocculation for recovery of micro-algae. Resour. Conserv. Recycl. 19, 1–10. Rattanakawin, C., Hogg, R., 2000. Aggregate size distributions in flocculation. Colloids Surf. A 177, 87–98. Rebhun, M., 1990. Floc formation and breakup in continuous flow flocculation and in contact filtration. In: Hahn, H., Klute, R. (Eds.), Chemical Water and Wastewater Treatment. Springer, Berlin, pp. 117–126. Riffat, R., 2013. Fundamentals of Wastewater Treatment and Engineering. CRC Press, Boca Raton, FL, pp. 99–118. Rodolfi, L., Zittelli, G.C., Bassi, N., Padovani, G., Biondi, N., Bonini, G., Tredici, M.R., 2009. Microalgae for oil strain selection, induction of lipid synthesis and outdoor mass cultivation in a low-cost photobioreactor. Biotechnol. Bioeng. 102, 100–112. Rwehumbiza, V.M., Harrison, R., Thomsen, L., 2012. Alum-induced flocculation of preconcentrated Nannochloropsis salina residual aluminium in the biomass, FAMEs and its effects on microalgae growth upon media recycling. Chem. Eng. J. 200, 168–175. Sastry, K.V.S., Wilson, T., Moothedath, S.K., 2000. Influence of operating variables on continuous flocculation of mineral fines. In: Paper presented at the International Symposium on Processing of Fines, Jamshedpur, India, 2–3 November. S¸irin, S., Trobajo, R., Ibanez, C., Salvadó, J., 2012. Harvesting the microalgae Phaeodactylum tricornutum with polyaluminum chloride, aluminium sulphate, chitosan and alkalinity-induced flocculation. J. Appl. Phycol. 24, 1067–1080. Schlesinger, A., Eisenstadt, D., Bar-Gil, A., Carmely, C., Einbinder, S., Gressel, J., 2012. Inexpensive non-toxic flocculation of microalgae contradicts theories; overcoming a major hurdle to bulk algal production. Biotechnol. Adv. 30, 1023–1030. Sompech, K., Chisti, Y., Srinophakun, T., 2012. Design of raceway ponds for producing microalgae. Biofuels 3, 387–397. Sukenik, A., Bilanovic, D., Shelef, G., 1988. Flocculation of microalgae in brackish and sea waters. Biomass 15, 187–199. Uduman, N., Qi, Y., Danquah, M.K., Forde, G.M., Hoadley, A., 2010. Dewatering of microalgal cultures: a major bottleneck to algae-based fuels. J. Renewable Sustainable Energy 2, 1–15. Vandamme, D., Foubert, I., Muylaert, K., 2013. Flocculation as a low-cost method for harvesting microalgae for bulk biomass production. Trends Biotechnol. 31, 233–239.