Optimal conditions of different flocculation methods for harvesting Scenedesmus sp. cultivated in an open-pond system

Lu Chen1, Cunwen Wang1*, Weiguo Wang1, Jiang Wei2* 1

Key Laboratory for Green Chemical Process of Ministry of Education, Wuhan Institute of Technology, Xiongchu road 693, Wuhan 430073, China

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Alfa Laval Nakskov A/S, Stavangervej 10 , DK-4900 Nakskov, Denmark

*Corresponding author: Cunwen Wang: Key Laboratory for Green Chemical Process of Ministry of Education, Wuhan Institute of Technology, Xiongchu road 693, Wuhan 430073, P.R.China Email: [email protected] Tel./fax: +8627 87195639 Jiang Wei: Alfa Laval Nakskov A/S, Stavangervej 10 , DK-4900 Nakskov, Denmark Email: [email protected] Tel./fax: +45 54971771

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Abstract

The effects of flocculation efficiency on harvesting Scenedesmus sp. cultivated in an open-pond system were investigated by changing culture medium pH value, flocculants (FeCl3, Al2(SO4)3, Alum, Ca(OH)2, chitosan, polyacrylamide), different dosages and sedimental times. 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 minutes sedimentation when the pH was adjusted to be 11.5, without adding flocculants. FeCl3 and chitosan showed a good flocculation efficiency at 0.15 g/l 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 Al2(SO4)3 followed by pH adjustment. Finally, the most suitable flocculation method is discussed in this paper.

Keywords: flocculation; Scenedesmus sp.; open-pond cultivation; inorganic flocculants; organic flocculants

1. Introduction 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., 2008; Hu et al., 2006; Kim et al., 2011). Now biodiesel production from vegetable oils

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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 will entail a decrease in food production(Schlesinger et al., 2012). Microalgae, as a potential source, have been attracting the worldwide attention as it has a high areal productivity, 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 yield of biodiesel produced by microalgae is nearly 10-20 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 µm), low concentration (0.5-5 g/L) of microalgae and the stable suspended state in the culture medium due to 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’s 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., 2000; Price et al., 1978), foam fractionation(Csordas and Wang, 2004; Lockwood et al., 1997)，filtration(Turker et al.,

2003)，flocculation(Avinmelech Yoram et al., 1982) and gravity sedimentation. Most

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existing commercial systems choose centrifugation, the traditional method, for harvesting microalgae, but there exists a non-negligible problem that it consumes a great deal of electric power. And high-speed centrifugation usually ruptures the cells which leads to the contents inside cells flow into the medium(Divakaran and Sivasankara Pillai, 2002). Some microalgae can be harvested using filtration, but membranes will rapidly fouled by extracellular organic matter if filtrating the medium directly(Babel and Takizawa, 2010). Therefore, considering the feasibility in terms of economy and technology, flocculation can be an effective and convenient method to harvest microalgae from large quantities of microalgae cultures rapidly(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 of the suspension subsequently(Knuckey et al., 2006). Lots of chemicals have been investigated as the flocculants for many types of microalgae. As it’s reported, Scenedesmus sp. was considered as one of the most promising microalgae for biodiesel because it has relatively high lipid productivity and it is relatively easy to be cultivated(Jena et al., 2012). Until now, 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 CaCl2 and FeCl3, and a bioflocculant were used to be the flocculants to harvest Scenedesmus sp. with a high density(Kim et al., 2011). However, the production process of bioflocculant is complex and the cost is relative expensive. The pH increase

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of culture medium could induce the flocculation of microalgae. The flocculation efficiency 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 various flocculation potential of different microalgae depends on their different properties, such as the cell wall compositions, the extent and type of excretions, physiological conditions, age and other factors (Avinmelech Yoram et al., 1982). Therefore, the optimal flocculation method for the microalgae should be chosen according to their own situation. In this study, the flocculation efficiency of different types of flocculants on harvesting Scenedesmus sp. cultivated in an open-pond 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. Materials and methods 2.1 Microalgae and culture condition The microalgae used in this study were Scenedesmus sp. which was provided by Algae Innovation Center of Denmark. It was cultivated using Bold’s Basal medium (BBM) in an open-pond cultivation system. Cells were harvested at late logarithmic growth phase and stored under darkness at 4°C for subsequent use in the flocculation experiments.

2.2 Flocculation experiments 2.2.1 Effect of pH

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The effect of pH value on flocculation efficiency was carried out by adjusting the pH of 1 L culture medium ranging from pH 7.5 to pH 12.5 using 5M sodium hydroxide and 1N hydrochloric acid. The medium was mixed rapidly (800rpm) until the required pH value was achieved and then slowly (250rpm) for 1 minute using magnetic bar stirrer. After sedimentation under gravity for different sedimentation times, an aliquot of medium was withdrawn for measuring the optical density at the height of two-thirds from the bottom. The optical density (OD) of the aliquot was measured by UVspectrophotometer (Hach Lange DR5000) at a wavelength of 665 nm to evaluate the flocculation efficiency. The flocculation efficiency was calculated using the following equation: Flocculation efficiency (%) = (1-B/A)*100 Where A is the optical density of the initial culture medium at 665 nm and B is the optical density of the sample at 665 nm.

2.2.2 Effect of different flocculants with different dosages Six flocculants (chitosan, polyacrylamide (PAM), Alum, Al2(SO4)3, Ca(OH)2 and FeCl3), which were purchased from Sigma (Denmark), were used for harvesting Secendesmus sp. from culture medium. All of them were common chemicals that have been proved to be efficient flocculants to many types of microalgae and widely used on many flocculation processes (Bajza and Hitrec, 2004; Harith et al., 2009; Schlesinger et al., 2012). Several dosages of these flocculants were added to 1L culture medium and mixed rapidly (800rpm) for 1 minute and then slowly (250rpm) for 1 minute using magnetic bar stirrer. Thereafter, an aliquot of medium was taken for measuring the

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flocculation efficiency at the height of two-thirds from the bottom after sedimentation under gravity for different sedimentation times.

2.2.3 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 1M sodium hydroxide and 1N hydrochloric acid. The base or acid were added to the medium at high mixing rate (800rpm) provided by agitation using magnetic bar stirrer. After the required pH value was reached, the medium was agitated at 250 rpm for 1 minute to achieve homogeneity in pH in whole medium solution. After 10 minutes sedimentation, an aliquot of medium was taken for measuring the flocculation efficiency at the height of two-thirds from the bottom.

2.2.4 Effect of flocculant dosage with different algal concentrations Four initial algal biomass concentrations (0.23 g/l, 0.41 g/l, 0.53 g/l 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 2.2.2. The sedimentation time was 10 minutes.

2.3 Determination of cell growth A calibration curve of known OD values and corresponding dry weights was calculated by measuring the dry cell weight (DCW) of microalgal culture. The dry weight was determined gravimetrically after centrifugation at 4000 rpm for 15 minutes and then drying the algal cells at 60 ◦C in the oven until constant weight was reached. There was a direct correlation between OD665 and dry weight expressed by a function:

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Dry weight (g/l) =0.7323*OD665 (R2=0.9983) The relationship between DCW and OD665 was described by a power regression with a R2 close to 1 within an OD range from 0.15-1.8. Based on this relation, all the OD values were converted to 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

Figure 1. Effect of pH adjustment on flocculation efficiency of Scenedesmus sp. at 0.54 g/l Figure 1 shows the effect of pH ranging from 7.5 to 12.5 on flocculation efficiency for harvesting Scenedesmus sp. at an algal biomass concentration of 0.54 g/l. The original pH value of culture medium was 10.3. Around this pH, 50% algae cells were settled down after 120 minutes. The efficiency increased to 97.4% after only 10 minutes

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sedimentation when the pH was adjusted up to 11.5. In addition, after 120 minutes sedimentation, the flocculation efficiencies could keep higher than 96% when the pH was adjusted to 11.5 or 12.5. Therefore, the results demonstrated that effective flocculation for harvesting Scenedesmus sp. could be attained by pH increase. Various groups have realized that microalgae could be flocculated by high pH value. 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 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, the algal cells in the medium at pH 12.5 were dead partially after 24 h and the color turned to be yellow. Hence, the flocculation efficiency could be the highest at the shortest time when the pH was adjusted to 11.5.

3.2 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 all flocculation processes. Therefore, preliminary experiments were undertaken to determine the optimum flocculant dosage and sedimental time on the flocculation of algal cells. Six flocculants were chosen to be discussed in this study. Among them FeCl3, Alum, Al2(SO4)3 and Ca(OH)2 belong to inorganic flocculants, Chitosan is organic cationic polymer which only dissolved in dilute acid and PAM is an polymer flocculant with high molecular weight. The reason for adding cationic flocculants is the positive charge carried by them

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could neutralize the negative charged microalgal cells. All of them have been used as efficient flocculants on many algae, such as Chlorella, Thalassiosira pseudonana.

Figure 2. Effect of different dosages of six flocculants, (a) FeCl3, (b) Al2(SO4)3, (c) Alum, (d) Chitosan, (e) PAM, (f) Ca(OH)2, at different sedimental time on flocculation efficiency. The biomass concentration of Scenedesmus sp. culture medium was 0.54 g/l.

Figure 2 shows the flocculation efficiencies of harvesting Scenedesmus sp. culture medium by different types of flocculants. As Figure 2 shown, FeCl3, Al2(SO4)3, Alum and Chitosan could lead to a high flocculation efficiency over 95% at a relatively short

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time while the flocculation efficiencies of Ca(OH)2 and PAM were lower especially PAM. The flocculation efficiency of FeCl3 sharply increased from 53.31% to 97.32% when the dosage increased from 0.1 g/l to 0.15 g/l after 2 minutes sedimentation. Figure 2e shows that it has no significant change of flocculation efficiency with different dosages of PAM. The mechanism of flocculation by PAM was 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 highest flocculation efficiency was 90% after 120 minutes sedimentation with adding 0.4 g/l solution to the medium for flocculating the Scenedesmus sp. and the efficiency did not further increase with the increment of flocculant dosage. Almost similar results were observed for flocculation using Al2(SO4)3 and Alum. High flocculation efficiency, 97.88% and 94.93% respectively, were obtained after 10 minutes sedimentation when the dosage of them was 0.3 g/l. However, the flocs produced from that dosage of Al2(SO4)3 and Alum were not very dense and showed a tendency to float and the high dosage of flocculants was also harmful to the algal cells. The culture medium turned to be light white when the Al2(SO4)3 and Alum dosage reached 0.3 g/l and turned to be orange when the FeCl3 dosage reached 0.2 g/l after adding the flocculants. This phenomenon might be caused by the excess flocculants. Part of flocculants reacted with the algal cells, the excess flocculants stayed in the medium in ionic state. Ferric chloride solution was orange and the Al(OH)3 precipitate was white. And after 24 hours, most of cells were dead and floated on the surface with adding high dosages of Al2(SO4)3, Alum and FeCl3. Based on these results, the optimum

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flocculant dosage produces stability 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 FeCl3, Al2(SO4)3 and Alum can be chosen as they are cheaper and easier to get.

3.3 Effect of flocculation efficiency with flocculant and pH adjustment

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Figure 3. Changes in flocculation efficiency at different pH adjustment followed by addition of different flocculants (a)0.08 g/l chitosan, (b)0,1 g/l Alum, (c)0.1 g/l Al2(SO4)3 and (d)0.05 g/l PAM. The biomass concentration of Scenedesmus sp. culture medium was 0.54 g/l. The changes in flocculation efficiency at different pH adjustments with 1M sodium hydroxide and 1N hydrochloric acid followed by addition of several flocculants are given in Figure 3. The importance of pH on flocculation process has been reported by many researchers. As pH affects the zeta potential of charged particles, it may interfere with flocculation after adding flocculant. A slight change in flocculation efficiency was shown in Figure 3a for chitosan at different pH values between 5 and 10. Due to acidic characteristic of chitosan solution, the pH of the culture medium reduced from10 to7 after the addition the flocculant. The highest flocculation efficiency over 95% was observed at pH 9 with 0.08 g/l chitosan solution. 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 (Rong Huei and Hwa, 1996). The algal cells had the highest negative charge at the pH (neutralization point), thus the flocculation efficiency was enhanced when the pH increased to that point. Because of chitosan and the algal cells interact with each other through the electrostatic interaction. Bridges were formed more than once as the polymer chain had sufficient length to bind the cells (Harith et al., 2009). As it shown in Figure 3b and 3c, the flocculation efficiency was higher than 90% with addition of 0.1 g/l Alum and Al2(SO4)3 followed by adjusting the pH to 6 and 5 respectively. Aluminum salts released hydrogen ions and consequently lower the medium pH value after adding into the culture medium. The existing form of Al3+ was affected by pH value. Al(OH)3 was

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the predominant aluminum species around pH 5 and 6. Around this pH, the initially formed colloidal precipitate was colloidally stable, and it was positively charged. The flocs stability decreased when the pH increased further because of the soluble anionic form Me(OH)4 becomes dominant in the solution(Bajza and Hitrec, 2004). It shows that the flocculation efficiency has no substantially changes from pH 7 to 11 after the addition of 0.05 g/l PAM in Figure 3d. A sharp increment was shown 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.

3. 4 Effect of flocculant dosages with different algal biomass concentrations

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Figure 4. Relation between initial algal biomass concentration and the flocculant dosage required (line) to achieve high flocculation efficiency (column) (a) FeCl3, (b) Al2(SO4)3, (c) Alum, (d) Chitosan Different initial algal biomass concentrations might influence the efficiency during the flocculation process. Therefore, the relation of algal biomass concentration and flocculant dosage was investigated and the results are shown in Figure 4. 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 Figure 4. 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. As figures shown, when the initial biomass concentration was 0.66 g/l, the optimal dosage of these four flocculants (FeCl3, Al2(SO4)3, Alum and Chitosan) was 0.2 g/l, 0.4 g/l , 0.4 g/l and 0.1 g/l respectively. Compared with Al2(SO4)3 and Alum, the consumption of FeCl3 and chitosan was smaller. The higher the flocculant dosage is, the higher the residual ions concentration may be. The residual ions will contaminate the medium and 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 flocculant will lower the cost of flocculate process.

4. Conclusions

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Six flocculants and their suitable conditions were investigated for harvesting the Scenedesmus sp. cultivated in an open-pond cultivation system. The flocculant needed to obtain high flocculation efficiency depends on the conditions of algae and further downstream process. The pH adjustment and nontoxic flocculant like chitosan can be chosen when the supernatant need to be reused after flocculation. Inorganic flocculants will be a good choice if there is no strict demand for the rest supernatant or a system coupled with filtration. A liner relation between the dosage needed and the initial algal biomass concentration was observed for different flocculants.

Acknowledgements This work was financed by National Natural Science Foundation of China (Grant No.20976140). 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 Algae Innovation Center of Denmark for providing algae suspensions and helpful discussions.

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