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1 Bioresource Technology 133 (2013) 9 15 Contents lists available at SciVerse ScienceDirect Bioresource Technology journal homepage: Optimal conditions of different flocculation methods for harvesting Scenedesmus sp. cultivated in an open-pond system Lu Chen a, Cunwen Wang a,, Weiguo Wang a, Jiang Wei b, a Key Laboratory for Green Chemical Process of Ministry of Education, Wuhan Institute of Technology, Xiongchu Road 693, Wuhan , China b Alfa Laval Nakskov A/S, Stavangervej 10, DK-4900 Nakskov, Denmark highlights " The Scenedesmus sp. discussed in this study was cultivated in an open-pond system. " Optimal conditions of six flocculants for harvesting Scenedesmus sp. were discussed. " Optimal ph to each flocculant was found for achieving high flocculation efficiency. " Increasing algal biomass concentration will increase the flocculant dosage needed. article info abstract Article history: Received 13 October 2012 Received in revised form 11 January 2013 Accepted 13 January 2013 Available online 24 January 2013 Keywords: Flocculation Scenedesmus sp. Open-pond cultivation Inorganic flocculants Organic flocculants The effects of culture medium ph, flocculant type (FeCl 3,Al 2 (SO 4 ) 3, Alum, Ca(OH) 2, chitosan, polyacrylamide), dosage and sedimental time on flocculation efficiency of harvesting Scenedesmus sp. cultivated in an open-pond system were investigated. Meanwhile, the relation between initial biomass concentration and the flocculant dosage needed was also investigated. The results from this work indicated that the flocculation efficiency achieved 97.4% after 10 min of sedimentation when the ph was adjusted to be 11.5, without adding flocculants. FeCl 3 and chitosan showed a good flocculation efficiency at dosage of 0.15 and 0.08 g/l, respectively without ph adjustment. The flocculation efficiency increased from 49.74% to 90.63% when the final medium ph was adjusted to 6 after adding 0.1 g/l Alum. An increment from 68.18% to 92.84% was observed after adding 0.1 g/l Al 2 (SO 4 ) 3 followed by ph adjustment. Finally, the most suitable flocculation method was discussed in this paper. Ó 2013 Elsevier Ltd. All rights reserved. 1. Introduction Corresponding authors. Tel./fax: (C. Wang), tel./fax: (J. Wei). addresses: wangcw118@hotmail.com (C. Wang), jiang.wei@alfalaval.com (J. Wei). As the depletion of fossil fuels, governments and research institutions all over the world are making a great effort to search for new fuels. Biodiesel is a non-toxic, biodegradable and renewable fuel that makes no net carbon dioxide or sulfur contribution to the atmosphere and emits less gaseous pollutants than conventional diesel fuels (Hu et al., 2006, 2008; Kim et al., 2011). Now biodiesel production from vegetable oils is a proven technology and is widely available in the world. However, plantation oil crops, waste vegetable oil and animal fat are only available in limited amounts (Ahmann and Dorgan, 2007; Lee et al., 2009; Wu et al., 2012). Moreover, using common food crops such as maize, sugarcane, soybean or oilseed rape for biodiesel production will lead to a decrease in food production (Schlesinger et al., 2012). Microalgae, as a potential source of biodiesel, has attracted worldwide attention as it has a high areal productivity and a relatively high lipid oil and protein content compared with traditional crops (Chisti, 2007; Halim et al., 2011; Mata et al., 2010). It has been reported that the average biodiesel yield produced by microalgae is nearly times higher than that from oleaginous seeds and/ or vegetable oils (Chisti, 2007; Tickell and Tickell, 2000). Nonetheless, due to the small size (3 30 lm) and low concentration (0.5 5 g/l) of microalgae, and the stable suspended state in the culture medium caused by their negative surface charge, the separation and recovery of microalgae from culture medium have been seen as a critical step in the microalgae biomass production process, which accounts for about 20 30% of the total production cost (Gudin and Thepenier, 1986; Wu et al., 2012). Thus, it is necessary for developing an efficient and low cost downstream process to harvest the microalgae cells from culture medium as well as to preserve their viability and bioactivity prior to use in the appropriate fields (Harith et al., 2009). Until now, several methods have been applied to harvest microalgae (Chen et al., 2011): centrifugation (Heasman et al., /$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.

2 10 L. Chen et al. / Bioresource Technology 133 (2013) ; Price et al., 1978), foam fractionation (Csordas and Wang, 2004; Lockwood et al., 1997), filtration (Turker et al., 2003), flocculation (Avinmelech et al., 1982) and gravity sedimentation. Most existing commercial systems choose centrifugation for harvesting microalgae, but there exists a non-negligible problem that it consumes a great deal of electric power (Divakaran and Sivasankara Pillai, 2002). Normally, rpm were used to separate the microalgae from culture medium by centrifugation, but sometimes the high-speed ( rpm) centrifugation will rupture the cells which lead to the contents inside cells flow into the medium (Das et al., 2012; González et al., 2003; Liu et al., 2012). Some microalgae can be harvested using filtration, but membranes will be rapidly fouled by the extracellular organic matter if the medium was filtered directly (Babel and Takizawa, 2010). Therefore, considering the economic and technological feasibility, flocculation can be an effective and convenient method to harvest microalgae from large quantities of microalgae cultures (Wu et al., 2012). Flocculation is the coalescence of separating suspended microalgal cells into large but loose particles. Through the interaction between the flocculant and the surface charge of microalgal cell, cells aggregate into large flocs and then settle out from the suspension subsequently (Knuckey et al., 2006). Lots of chemicals have been investigated as the flocculants for many types of microalgae. As it is reported, Scenedesmus sp. was considered as one of the most promising microalgae for biodiesel production because it has relatively high lipid content and productivity and it is relatively easy to be cultivated (Jena et al., 2012). The lipid content and productivity of Scenedesmus sp. were % dry weight biomass and mg/l/day, respectively (Mata et al., 2010). Many researchers have discussed the method for harvesting Scenedesmus sp. Different methods were investigated to reduce the membrane fouling for harvesting Scenedesmus sp. with polyvinylidene fluoride (PVDF) microfiltration membrane (Chen et al., 2012). It shows a potential of industrial micro-organisms harvesting by membrane. Consecutive treatment with CaCl 2 and FeCl 3, and a bioflocculant were used to be the flocculants to harvest Scenedesmus sp. with a high-density culture (Kim et al., 2011). However, the production process of bioflocculant is complex and the cost is relatively high. The ph increase of culture medium could induce the flocculation of microalgae. The flocculation efficiencies of several freshwater microalgae (Chlorella vulgaris, Scenedesmus sp., Chlorococcum sp.) and marine microalgae (Nannochloropsis oculata, Phaeodactylum tricomutum) have been discussed by increasing the ph value of culture medium (Halim et al., 2011). The flocculation potential of different microalgae depends on their properties, such as the cell wall compositions, the extent and type of excretions, physiological conditions, age and other factors (Avinmelech et al., 1982). Therefore, the suitable flocculation method for microalgae harvesting should be determined based on the properties of microalgae. In this study, the flocculation efficiency of different types of flocculants on harvesting Scenedesmus sp. cultivated in an openpond cultivation system was investigated. The effects of ph, sedimental time and flocculant dosage and ph adjustment after adding flocculants on flocculation efficiency were also discussed. 2. Methods 2.1. Microalgae and culture condition The microalgae used in this study were Scenedesmus sp. cultivated in an open-pond system, which was provided by Green Center Algae Innovation Center Lolland, Denmark. It was cultivated by using Bold s medium (BBM) in some big basins with volume around 2.5 m 3. Cells were harvested in the late logarithmic growth phase, which usually takes days to reach that phase, and stored under darkness at 4 C for the subsequent use in the flocculation experiments Flocculation experiments Effect of ph The effect of ph on flocculation efficiency was carried out by using 5 M sodium hydroxide and 1 N hydrochloric acid for adjusting the culture medium (1 L) ph ranging from 7.5 to The ph was measured by a ph meter (ph M83 Autocal ph meter, TTT 80 titrator, ABU80 autoburette, Radiometer Copenhagen Co., Copenhagen, Denmark). The medium was mixed rapidly (800 rpm) until the required ph was achieved and then slowly (250 rpm) for 1 min using a magnetic bar stirrer. After sedimentation under gravity for different sedimentation times (10, 30, 60, 120 min), an aliquot of medium was withdrawn for measuring the optical density (OD) at the height of two-thirds from the bottom. The OD of the aliquot was measured by a UV-spectrophotometer (Hach Lange DR5000) at a wavelength of 665 nm to evaluate the flocculation efficiency, which was calculated using the following equation (Kim et al., 2011; Wu et al., 2012): Flocculationefficiencyð%Þ ¼ð1 B=AÞ100 where A is the OD of the initial culture medium at 665 nm and B is the OD of the sample at 665 nm Effect of flocculants with different dosages Six flocculants (chitosan, polyacrylamide (PAM), Alum, Al 2 (- SO 4 ) 3, Ca(OH) 2 and FeCl 3 ), which were purchased from Sigma (Denmark), were used for harvesting Scenedesmus sp. from the culture medium. All of them were common chemicals that have been proved to be efficient flocculants to many types of microalgae and widely used in many flocculation processes (Bajza and Hitrec, 2004; Harith et al., 2009; Schlesinger et al., 2012). Several dosages of these flocculants were added into the culture medium (1 L). The medium was mixed rapidly (800 rpm) for 1 min and then slowly (250 rpm) for 1 min using a magnetic bar stirrer. The dosages for each flocculant were: FeCl 3 (0.06, 0.08, 0.1, 0.15 and 0.2 g/l), Al 2 (SO 4 ) 3 and Alum (0.02, 0.03, 0.05, 0.1 and 0.3 g/l), Chitosan (0.03, 0.06, 0.08, 0.1 and 0.12 g/l), PAM (0.02, 0.04, 0.05, 0.06 and 0.08 g/l) and Ca(OH) 2 (0.2, 0.3, 0.4, 0.5 and 0.6 g/l). Thereafter, an aliquot of medium was taken for measuring the flocculation efficiency at the height of two-thirds from the bottom after sedimentation under gravity for different sedimentation times (2, 5, 10, 30, 60 and 120 min) Effect of flocculation with ph adjustment The effect of medium ph after adding flocculants on flocculation efficiency was carried out by adjusting the ph using 1 M sodium hydroxide and 1 N hydrochloric acid. The base or acid were added into the medium with vigorous magnetic stirring (800 rpm) for 1 min to achieve homogeneity in ph of the whole medium solution. After the required ph value was reached, the medium was agitated at 250 rpm for 1 min. After 10 min of sedimentation, an aliquot of medium was taken for measuring the flocculation efficiency at the height of two-thirds from the bottom Effect of flocculant dosage with different algal concentrations Four initial algal biomass concentrations (0.23, 0.41, 0.53 and 0.66 g/l) were investigated to test the effect of flocculant dosage with different algal concentrations. The experimental method was the same as previously mentioned in Section The sedimentation time was 10 min.

3 L. Chen et al. / Bioresource Technology 133 (2013) Determination of cell growth A calibration curve of known OD values and corresponding algal biomass was obtained by measuring the dry cell weight (DCW) of microalgal culture. The dry weight was determined gravimetrically after centrifugation at 4000 rpm for 15 min and then drying the algal cells at 60 C in the oven until constant weight was reached. There was a direct correlation between OD 665 and dry weight expressed by a function: Drycellweightðg=LÞ ¼0:7323 OD 665 ðr 2 ¼ 0:9983Þ The relationship between DCW and OD 665 was described by a power regression with a R 2 close to 1 with an OD ranging from 0.15 to 1.8. Based on this relation, all the OD values were converted to concentration of biomass (g/l). The results presented in this paper are based on the average of the three replicates. 3. Results and discussion 3.1. Autoflocculation by ph adjustment Fig. 1 shows the effect of ph ranging from 7.5 to 12.5 on the flocculation efficiency for harvesting Scenedesmus sp. at an algal biomass concentration of 0.54 g/l. The original ph of culture medium was Around this ph, 50% of algae cells were settled down after 120 min. The efficiency increased to 97.4% after only 10 min of sedimentation when the ph was adjusted up to After sedimentation, some algal cells may resuspend. The stability of the flocs was monitored with time. After 120 min of sedimentation, the flocculation efficiencies could still keep higher than 96% when the ph was adjusted to 11.5 or The pictures of flocculation at different ph after 10 min of sedimentation were shown in Fig. 2. The changes of flocculation efficiency with ph could be seen clearly in the figure. Therefore, the results demonstrated that effective flocculation for harvesting Scenedesmus sp. could be attained by increasing ph value of the medium. Various groups have realized that microalgae could be flocculated at high ph. They proposed that the reason for autoflocculation could be due to the metal cations in the medium such as calcium and magnesium ions that could form hydroxide precipitates with a positive superficial charge as ph increased. These positively charged precipitates would absorb the negatively charged algal cells, causing the Fig. 1. Effect of the ph adjustment on the flocculation efficiency of Scenedesmus sp. at 0.54 g/l. compression of the electrical double-layer and then the cells become destabilized and hence to flocculate (Lavoie and de la Noüe, 1987; Schlesinger et al., 2012; Semerjian and Ayoub, 2003). However, some algae cells in the medium at ph 12.5 died after 24 h. Hence, the medium with ph 11.5 was more suitable for effective flocculation. Wu et al. also determined the effects of the ph increase on the flocculation efficiency for harvesting Scenedesmus sp. (Wu et al., 2012). They found the efficiency was greatly raised when the ph was increased to The result was similar to the conclusion in this work. Nevertheless, the microalgae were cultivated under different conditions so that the suitable ph for flocculation in the two cases (this case versus Wu s case) was not the same Effect of different types and dosages of flocculants on flocculation efficiency As the flocculant dosage will influence both the extent and the rate of flocculation reaction, it has been recognized as a critical parameter in flocculation processes. Therefore, preliminary experiments were undertaken to determine the optimal flocculant dosage and sedimental time for flocculating the algal cells. Six flocculants were evaluated for algal cell flocculation in this study. Among them, FeCl 3, Alum, Al 2 (SO 4 ) 3 and Ca(OH) 2 belong to inorganic flocculants, Chitosan is an organic cationic polymer that could only be dissolved in dilute acid, and PAM is a polymer flocculant with a high molecular weight. The reason for adding cationic flocculants is that the positive charge carried by them could neutralize the negatively charged microalgal cells. All of them have been used as efficient flocculants for many algae, such as Chlorella and Thalassiosira pseudonana (Divakaran and Sivasankara Pillai, 2002; Knuckey et al., 2006). Fig. 3 shows the flocculation efficiencies of harvesting Scenedesmus sp. from the culture medium by different types of flocculants. As Fig. 3 shows, FeCl 3,Al 2 (SO 4 ) 3, Alum and Chitosan exhibited a high flocculation efficiency over 95% at a relatively short time while the other two, especially PAM, showed a low flocculation efficiency. The flocculation efficiency of FeCl 3 sharply increased from 53.31% to 97.32% when its dosage increased from 0.1 to 0.15 g/l after 2 min of sedimentation. Fig. 3e shows that it has no significant change of flocculation efficiency with different dosages of PAM. The mechanism of flocculation by PAM is bridging. The flocculation efficiency will be affected strongly by the solution properties of the polymer. The PAM used in this study was a common one whose chain is not expanded enough for bridging the cells. Modified PAM has better flocculation efficiency than the common one because of the influence of chain end group (Qian et al., 2004). For Ca(OH) 2, the flocculation efficiency reached 90% after 120 min of sedimentation when the dosage of Ca(OH) 2 was 0.4 g/ L and did not further increase with the increment of flocculant dosage. Almost similar results were observed for flocculation using Al 2 (SO 4 ) 3 and Alum. High flocculation efficiency, 97.88% and 94.93%, respectively, was obtained after 10 min of sedimentation when the dosage of them was 0.3 g/l. However, the flocs produced from this dosage were not very dense and showed a tendency to float and the high dosage of flocculants was also harmful to the algal cells. After adding high dosage of flocculants, the color of the culture medium changed. It turned to be light white when the Al 2 (- SO 4 ) 3 and Alum dosage reached 0.3 g/l and turned to be orange when the FeCl 3 dosage reached 0.2 g/l. This phenomenon might be caused by the excess flocculants. Part of flocculants reacted with the algal cells, the excess flocculants stayed in the medium as ionic compounds. The color of ferric chloride solution was orange and the Al(OH) 3 precipitate was white. After 24 h, most of cells were dead and floated on the surface with adding high dosages of

4 12 L. Chen et al. / Bioresource Technology 133 (2013) 9 15 Fig. 2. Pictures of flocculation at different ph, (a) 7.5, (b) 8.5, (c) 9.5, (d) 10.5, (e) 11.5, (f) 12.5, after 10 minutes of sedimentation. Fig. 3. Effect of the different dosages of six flocculants, (a) FeCl 3, (b) Al 2 (SO 4 ) 3, (c) alum, (d) chitosan, (e) PAM and (f) Ca(OH) 2, at different sedimental time on the flocculation efficiency. The biomass concentration of the Scenedesmus sp. culture medium was 0.54 g/l. Al 2 (SO 4 ) 3, Alum and FeCl 3. Based on these results, the optimal flocculant dosage producing stable flocs and high flocculation efficiency at a shorter time should be chosen to flocculate the algae according to the conditions of the original algae culture medium. If the supernatant after flocculation is reused for cultivating the algae, organic cationic polymer like chitosan will be a suitable choice because it has no toxic effects and does not contaminate growth medium (Wu et al., 2012). Otherwise, if the purpose of flocculation process focuses on harvesting the algae economically and conveniently, the inorganic flocculants such as FeCl 3,Al 2 (SO 4 ) 3 and Alum can be chosen as they are cheaper and easier to get Effect of flocculation efficiency with ph adjustment after adding flocculant The changes in flocculation efficiency at different ph adjusted with 1 M sodium hydroxide and 1 N hydrochloric acid followed by addition of several flocculants are given in Fig. 4. The importance of ph on flocculation process has been reported by many researchers (Harith et al., 2009; Bajza and Hitrec, 2004). As ph affects the zeta potential of charged particles, it may interfere with flocculation after adding flocculants. A slight change in flocculation efficiency was shown in Fig. 4a for chitosan at different ph be-

5 L. Chen et al. / Bioresource Technology 133 (2013) Fig. 4. Changes in flocculation efficiency at different ph adjustment followed by the addition of different flocculants (a) 0.08 g/l chitosan, (b) 0.1 g/l alum, (c) 0.1 g/l Al 2 (SO 4 ) 3 and (d) 0.05 g/l PAM. The biomass concentration of Scenedesmus sp. culture medium was 0.54 g/l. Fig. 5. Relation between the initial algal biomass concentration and the flocculant dosage required (line) to achieve the high flocculation efficiency (column) (a) FeCl 3, (b) Al 2 (SO 4 ) 3, (c) alum, (d) chitosan. tween 5 and 10. Due to the acidic characteristic of chitosan solution, the ph of the culture medium reduced from10 to 7 after the addition of the flocculant. The highest flocculation efficiency over 95% was observed at ph 9 when the dosage of chitosan was 0.08 g/l. Chitosan s molecular structure can be influenced by ph. The positive charge gradually disappeared and chitosan tended to form coli structure and precipitate when the ph was alkaline (Chen and Hwa, 1996). The algal cells had the highest negative charge

6 14 L. Chen et al. / Bioresource Technology 133 (2013) 9 15 when the ph reached the neutralization point. Thus the flocculation efficiency was enhanced when the ph increased to that point, because of the electrostatic interaction between the algal cells and chitosan. Bridges were formed more than once as the polymer chain had the sufficient length to bind the cells (Harith et al., 2009). As shown in Fig. 4b and c, the flocculation efficiency was higher than 90% after the ph was adjusted to 6 and 5, respectively, when the dosage of Alum and Al 2 (SO 4 ) 3 in the medium were 0.1 g/ L. The addition of aluminum salts could lower the medium ph due to the release of hydrogen ions from them. The existing form of Al 3+ was affected by ph. Al(OH) 3 was the predominant aluminum species around ph 5 and 6. Around this ph, the initially formed colloidal precipitate was colloidally stable and positively charged. The flocs stability decreased when the ph increased further because the soluble anionic form MeðOHÞ 4 becomes dominant in the solution (Bajza and Hitrec, 2004). As shown in Fig. 4d, the flocculation efficiency has no substantial change from ph 7 to 11 with dosage of PAM at 0.05 g/l. A sharp increment can be seen when the ph value increased to 12. However, this increment at the high ph was probably caused by the autoflocculation, which occurs at ph 11.5 or higher Effect of flocculant dosages with different algal biomass concentrations on flocculation efficiency Initial algal biomass concentrations might influence the efficiency during the flocculation process. Therefore, the relation between algal biomass concentration and flocculant dosage was investigated and the results are shown in Fig. 5. For each initial algal biomass concentration, different flocculant dosages were compared to get the optimal one which could result in the highest flocculation efficiency for harvesting algal cells. A linear relation between the dosage needed and the initial algal biomass concentration was shown in Fig. 5. The dosage needed increased with the increment of the initial algal biomass concentration. This phenomenon could be explained by the mechanism of flocculation. The amount of suspended algal cells increased with the increase of the biomass concentration. Thus higher flocculant dosages were needed to interact with the surface charges of algal cells. In the work by Kim et al., they tested three concentrations of FeCl 3 and Al 2 (SO 4 ) 3 with four different Scenedesmus sp. culture densities. The flocculation efficiencies decreased with the increment of culture densities when the concentrations of flocculants were the same (Kim et al., 2011). Their result shows the same tendency with this work. Higher dosage of flocculant was needed for high algal biomass. As Fig. 5 show, when the initial biomass concentration was 0.66 g/l, the optimal dosage of these four flocculants (FeCl 3, Al 2 (SO 4 ) 3, Alum and Chitosan) was 0.2, 0.4, 0.4 and 0.1 g/l, respectively. Compared to Al 2 (SO 4 ) 3 and Alum, the consumption of FeCl 3 and chitosan was smaller. It is known that the higher the flocculant dosage is, the higher the residual ions concentration may be. The residual ions may contaminate the medium and may be harmful to the cell vitality. Therefore, it is better to choose the suitable flocculants according to the biomass concentration. Furthermore, reducing the amount of flocculants will lower the cost of the flocculation process. 4. Conclusions Six flocculants were investigated for harvesting the Scenedesmus sp. cultivated in an open-pond cultivation system. The flocculant needed to obtain the high flocculation efficiency depends on the conditions of algae and the downstream process. The ph adjustment and nontoxic flocculants like chitosan can be chosen when the supernatant needs to be reused after the flocculation. Inorganic flocculants could be a good choice if there is no strict demand for the rest supernatant or the flocculation coupled with filtration. A liner relation between the dosage needed and the initial algal biomass concentration was observed for each flocculant. Acknowledgements This work was financed by National Natural Science Foundation of China (Grant No ). The authors wish to acknowledge Alfa Laval Nakskov A/S for the support of the work and would like to thank Jørgen Enggaard Boelsmand at Green Center Algae Innovation Center Lolland, Denmark for providing algae suspensions and helpful discussions. 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