Bioresource Technology 149 (2013) 575–578
Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Short Communication
Magnetophoretic harvesting of oleaginous Chlorella sp. by using biocompatible chitosan/magnetic nanoparticle composites Kyubock Lee a, So Yeun Lee a,b, Jeong-Geol Na a, Sang Goo Jeon a, Ramasamy Praveenkumar a, Dong-Myung Kim b, Won-Seok Chang c, You-Kwan Oh a,⇑ a b c
Clean Fuel Department, Korea Institute of Energy Research, 152 Gajeong-ro, Yuseong-gu, Daejeon 305-343, Republic of Korea Department of Fine Chemical Engineering and Applied Chemistry, Chungnam National University, Daejeon 305-764, Republic of Korea Korea District Heating Corp., 186 Bundang-dong, Bundang-gu, Seongnam-si, Gyoenggi-do 463-908, Republic of Korea
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Biocompatible and rapid
magnetophoretic harvesting process of oleaginous microalgae. Over 99% harvesting efficiency achieved by using chitosan–Fe3O4 composites. After harvesting, the used medium shows no adverse effect on microalgal growth.
a r t i c l e
i n f o
Article history: Received 14 June 2013 Received in revised form 16 September 2013 Accepted 18 September 2013 Available online 27 September 2013 Keywords: Chitosan Magnetic nanoparticle Microalgae Harvesting Medium recycling
a b s t r a c t The consumption of energy and resources such as water in the cultivation and harvesting steps should be minimized to reduce the overall cost of biodiesel production from microalgae. Here we present a biocompatible and rapid magnetophoretic harvesting process of oleaginous microalgae by using chitosan–Fe3O4 nanoparticle composites. Over 99% of microalgae was harvested by using the composites and the external magnetic field without changing the pH of culture medium so that it may be reused for microalgal culture without adverse effect on the cell growth. Depending on the working volume (20–500 mL) and the strength of surface magnetic-field (3400–9200 G), the process of harvesting microalgae took only 2– 5 min. The method presented here not only utilizes permanent magnets without additional energy for fast harvesting but also recycles the medium effectively for further cultivation of microalgae, looking ahead to a large scale economic microalgae-based biorefinement. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction With increasing petroleum oil price and growing emission of greenhouse gas, biomass-based biofuel production has been receiving much attention, especially since microalgae biomass has many advantages over the grain- and/or tree-based biomass in producing biodiesel (Lam and Lee, 2012; Mata et al., 2010). The microalgae have higher photosynthetic efficiency and high
⇑ Corresponding author. Tel.: +82 42 860 3697; fax: +82 42 860 3495. E-mail address: [email protected] (Y.-K. Oh). 0960-8524/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2013.09.074
lipid content of 15–77% of cell mass (Chisti, 2007), and they can be cultivated using carbon dioxide directly from power plants. In spite of the many advantages of microalgae-based biorefinery, it involves many steps from cultivation to harvesting, from lipid extraction to oil-to-biodiesel transition, which increase the cost of microalgal biodiesel (Lam and Lee, 2012). In the various steps in producing microalgal biodiesel, it is necessary to improve the technique of harvesting, which takes up 20–30% of the total cost (Mata et al., 2010). Since microalgae are cultivated in diluted culture medium, normally under 1 g/L, and have a negatively charged surface of a few microns in size, the dispersion of microalgae in the solution is very stable, which
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K. Lee et al. / Bioresource Technology 149 (2013) 575–578
makes harvesting a task worth challenging. Various harvesting methods have been applied till now. Conventional methods such as centrifugation, filtration and ultrasound have been widely used for microalgal harvesting (Bosma et al., 2003; Zhang et al., 2010). Electrolysis-base technologies have been shown to be powerful methods for harvesting microalgae even under the continuous process (Gao et al., 2010; Kim et al., 2012). However, those methods are not easy to scale-up and also require high energy consumption, which is not desirable for low cost production of microalgal biodiesel. Chemical flocculants such as polyvalent cations and cationic polymers or inorganics have been intensively investigated for large-scale applications (Lee et al., 2013; Sirin et al., 2012). To improve some drawbacks of chemical flocculants such as contaminations of final products and the toxic effects on microalgae (Kim et al., 2013), biocompatible flocculants such as chitosan and starch have been attempted (Beach et al., 2012; Farid et al., 2013). Recently, magnetic microalgal harvesting has been getting much attention. Microalgae are attached with magnetic particles coated with cationic substances by electrostatic interaction and then separated from the culture medium by external magnetic field (Lim et al., 2012; Xu et al., 2011). Lim et al. (2012) who applied the iron oxide magnetic nanoparticles and the cationic polyelectrolyte have reported that the harvesting efficiency of magnetic separation is as high as 99% and the harvesting completed within a few minutes. However, they did not report on the biocompatibility of the flocculent with polyelectrolyte as a functional group and the resulting recycling of medium. Chlorella sp. KR-1 is a newly isolated microalga that accumulates triacylglycerol approximately 37–41% (w/w) of cell mass (Lee et al., 2013; Na et al., 2011). In spite of the high lipid content, the small size of Chlorella sp. KR-1, around 3 lm in diameter (Lee et al., 2013), makes the harvesting by conventional methods more challenging. In this study, we synthesized chitosan–Fe3O4 magnetic nanoparticle (CS/MNP) composites and investigated the composites as biocompatible flocculants for the magnetophoretic harvesting of Chlorella sp. KR-1. The efficiency of microalgal harvesting was studied using the dosage and the ratio of chitosan to magnetic nanoparticles of the synthesized composites. Furthermore, the biocompatibility of the composites was tested by reusing both the used culture medium left over from microalgae harvesting and the microalgae-attached composites as inoculums.
2. Methods 2.1. Synthesis of CS/MNP composites Fe3O4 magnetic particles of 10–30 nm in size were synthesized by the previously reported method (Liu et al., 2004). Briefly, two aqueous solutions of FeCl24H2O (P99.0%, Sigma–Aldrich, USA) and FeCl34H2O (97%, Sigma–Aldrich, USA) were mixed and heated at 85 °C for 30 min under N2 atmosphere followed by addition of NH4OH. The precipitated nanoparticles were washed several times with ethanol and subsequently with distilled water until pH of suspension reaches to 7. The prepared Fe3O4 nanoparticles (0.15 g) were dispersed in 12 mL of distilled water with 11 mL of 2 wt% Pluronic F-127 (Sigma, USA) solution using ultrasonication. Chitosan solution was prepared by dissolving 0.2 wt% of chitosan flakes (low molecular weight, Fluka, USA) in 0.5% acetic acid. The Fe3O4 dispersion and chitosan solutions were mixed for 30 min using a magnetic bar. For cross-linking of chitosan, 140 mL sodium tripolyphosphate solution (0.15 wt%, pH 6; Sigma–Aldrich, USA) was added by a peristaltic pump at 10 mL/min. The solution was aged overnight at 4 °C and then washed with distilled water several times. The total volume of each Fe3O4-chitosan dispersion solution was set to 50 mL in order to quantify Fe3O4-chitosan by volume.
The final products of CS/MNP composites were named ‘CS/MNP a’ of which a indicates the calculated weight ratio of CS to MNP (a: 0.13, 0.40 and 0.54). 2.2. Cultivation of microalgae Chlorella sp. KR-1, an indigenous freshwater microalga (Na et al., 2011) was used in this study. The microalgae were cultivated in a modified N8 medium in a 7 L Pyrex bubble-column photobioreactor supplied with 10% (v/v) CO2 in air at a rate of 0.75 L/min under illumination of 12 fluorescent lamps (light intensity: 80 lmol photons/m2 s). The medium contained 3 mM KNO3, 5.44 mM KH2PO4, 1.83 mM Na2HPO4, 0.20 mM MgSO47H2O, 0.12 mM CaCl2, 0.03 mM FeNaEDTA, 0.01 mM ZnSO47H2O, 0.07 mM MnCl24H2O, 0.07 mM CuSO4 and 0.01 mM Al2(SO4)318H2O with pH = 6.5. For the cultivation of microalgae on agar plate, the agarose gel was prepared by dissolving 1.5 wt% of agar (Bacto™ Agar, BD Bacto™, France) in a modified N8 medium in an autoclave at 121 °C for 15 min. The magnetophoretically harvested microalgae-CS/MNP flocs were plated on the agar plate, which was incubated for 3 days in the growth chamber (temperature, 25 °C; humidity, 60%; light intensity, 15 lmol photons/m2 s). 2.3. Harvesting of microalgae with CS/MNP The harvesting experiment was carried out using microalgae freshly taken from 7 L Pyrex bubble-column after 4 d incubation. The concentration of microalgae was 1.0 g/L (Optical density at 660 nm, OD660nm = 4.5). The harvesting efficiency, in this study, indicates the percentage of microalgae attached on CS/MNP which are separated by the magnetic field subsequently. After injection of the calculated amount of CS/MNP dispersion into 1 mL of microalgal solution, external magnetic field was applied to the mixture. The surface magnetic-field strength of a permanent NdFeB magnet chip (length, 20 mm; width, 9 mm; thickness 4 mm) was measured to be 3400 G by a Gauss meter (TM-701, KANETEC Co., Japan). The supernatant was taken for OD measurement after 3 min of magnetophoretic harvesting, thereby ensuring the measurement of the only concentration of unattached microalgae on CS/MNP. The harvesting efficiency was calculated by following equation (Lee et al., 2013).
ODf 100 Harvesting efficiency ½% ¼ 1 ODi where ODi and ODf were the initial OD and the OD of the supernatant after magnetophoretic harvesting, respectively. 2.4. Reuse of medium solution and microalgae-attached CS/MNP as an inoculum Microalgae were harvested by using CS/MNP composite and a magnet rod (surface magnetic-field strength, 9200 G; diameter, 22 mm; length, 500 mm) from 0.5 L of microalgal solution (see Graphical Abstract). After adjusting the concentration of nitrate of used medium to 3 mM, new cells were inoculated and cultivated in 1 L bubble columns for 7 d under the same conditions mentioned previously. For comparison, the medium solution was reused for the next cultivation after harvesting microalgae with a widely using flocculant, Alum (Kim et al., 2011). In addition, the magnetophoretically harvested black slurry containing microalgae-attached CS/MNP (the 4th image of Graphical Abstract, Fig. S3a and S4b) was directly used as an inoculum in the new N8 medium to investigate the biocompatibility CS/MNP composite versus microalgae.
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2.5. Analytical methods
3.3. Reuse of culture medium after harvesting of microalgae
The OD measurement was performed using UV–VIS spectrophotometer (Optizen 2120 UV, Mecasys Co., Korea). The optical microscopic images were obtained with an oil immersion objective lens at 100 magnification (Microscope Axio Imager.A2, Carl Zeiss Microscopy GmbH, Jena, Germany). After harvesting, microalgaeattached CS/MNP composites were freeze-dried at 55 °C for field emission scanning electron microscope (FE-SEM) and energy-dispersive spectroscopy (EDS) analysis (S-4800, HITACHI, Japan).
The medium was reused for microalgal culture after harvesting (Fig. 1b). As shown in Graphical Abstract, over 97% of microalgae was recovered within 5 min from 1 L bubble column containing 0.5 L microalgal solution by using CS/MNP 0.40 and a permanent magnetic rod with a maximum surface magnetic field strength of 9200 G. Cell growth in the reused medium that had been used once in CS/MNP harvesting was similar to that of the new medium (Fig. 1b). This implies that the use of CS/MNP composites as flocculants has no inhibitory effect on algal cells. For comparison, Alum which has been used widely as a chemical flocculant was tested for magnetophoretic harvesting of oleaginous microalgae and for the reuse of the medium after harvesting. It took one hour to settle 90.6% and 97.3% microalgae in 1 L bubble column with 0.9 and 5 mM of Alum, respectively. Microalgal cell growth was inhibited in both reused media of Alum. This is consistent with the report by Kim et al. (2011). The Lewis acidity of Al3+ caused a pH drop of microalgal solution down to 3 when Alum was used. This seems to have caused the inhibition of cell growth. The pH levels were kept at 6–7 during the cell growth in the new N8 medium or the reused medium after harvesting with CS/MNP (data not shown). Chitosan is known to be a widely used biocompatible material (Kumar, 2000). The biocompatibility of the CS/MNP composites was tested by plating the harvested microalgae-CS/MNP flocs on an agar plate (Fig. S3a). After three days of incubation, microalgal growth was evident as visible colonies on the agar surface (Fig. S3b). When the revival of microalgae was verified microscopically, the development of actively dividing colonies from the microalgae-CS/MNP flocs was observed (Fig. S3b). This test showed
3. Results and discussion 3.1. CS/MNP composites and magnetophoretic harvesting of microalgae Fig. S1a shows the snapshots of magnetophoretic harvesting of microalgae at different time points after injecting CS/MNP suspension into 20 mL of 1 g/L microalgal solution and applying a NdFeB magnet chip. It took less than 2 min for the complete harvesting of microalgae. The kinetics of magnetophoretic harvesting is not comparable to that of gravitational harvesting. It has been reported that microalgal harvesting by settling only with chitosan as flocculants required one hour to harvest 97% of microalgae (Farid et al., 2013). Flocculation was observed when CS/MNP was mixed with microalgal solution. As soon as the magnet was externally applied to the vial containing the mixture, the magnet pulled flocs of CS/MNP and microalgae toward the magnet (Fig. S1a). Fig. S1b shows the differential interference contrast (DIC) microscopic image of CS/MNP composite synthesized with brownish aggregated Fe3O4 nanoparticles embedded in the chitosan matrix. The transparent chitosan matrix could be observed in DIC mode, which enhances the contrast of chitosan by the principle of interferometry. After mixing CS/MNP with microalgal solution, it was observed that microalgal cells were attached onto CS/MNP forming flocs (Fig. S1a and c). SEM image clearly shows microalgal cells attached onto CS/MNP as indicated by arrows in Fig. S1d. The protonated amino groups of chitosan in water are positively charged to present NH3+ ions and, therefore, induce a charge neutralization, which means attachment of negatively charged microalgal cells (Renault et al., 2009; Sirin et al., 2012). Nanosized Fe3O4 particles were also observed, and they were confirmed to be a Fe-rich region by energy-dispersive X-ray spectroscopy (EDAX) analysis and mapping (Figs. S1d and S2).
3.2. Harvesting efficiency and kinetics of magnetophoretic separation of microalgae This section investigates the relationships between the harvesting efficiency of microalgae and the concentration of the composites as well as the ratio of chitosan to magnetic nanoparticles. The harvesting efficiencies were improved to almost 100% by increasing the dosage of CS/MNP (Fig. 1a). Just as Rashid et al. (2013) reported, the zeta-potential of Chlorella vulgaris increased from 21 to +1.9 mV with the chitosan dosage. The charge neutralization seems to have occurred when the harvesting efficiency reached 100%. When the composite with a higher ratio of CS to MNP was used, the higher harvesting efficiency was obtained with same dosage. In the case of CS/MNP 0.54, a 99% harvesting efficiency was obtained by the 1.4 g/L dosage. On the other hand, only 95% of microalgae were harvested with 3.4 g/L of CS/MNP 0.13 because chitosan provides adsorption sites for microalgae and, therefore, the composite with a higher ratio of CS to MNP has more adsorption sites.
Fig. 1. (a) Plots of harvesting efficiency of CS/MNP with various ratio of CS to MNP as a function of the concentration of CS/MNP in 1 g/L of microalgal solution. (b) Microagal growth in the reused medium after harvesting microalgae by using CS/MNP 0.40 ( ), 0.9 mM ( ) and 5 mM of Alum ( ) as flocculants after adjusting the concentration of nitrate to the level of new N8 medium (d).
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that the composites do not affect the growth of microalgae, thus they could be considered as biocompatible materials. In addition, microalgae were successfully cultured by using microalgae-attached CS/MNP as an inoculum (black slurry, the 4th image of Graphical Abstract, Figs. S3a and S4b). The cell growth showed a typical pattern of microalgal growth in 1 L bubblecolumn with exponential growth followed by the stationary pattern whose OD reached 9.15 in 7 d (Supplementary materials, Fig. S4a). The optical microscope image of microalgae-attached CS/MNP after re-culture of 7 d shows that green microalgal cells are still alive and attached onto the composites (Supplementary materials, Fig. S4c). These results imply that CS/MNP composites are biocompatible with Chlorella sp. It is worth noting that a large portion of the cost in microagal culture is attributed to water, since the concentration of microalgae in culture medium is very low, less than 1 g/L (Mata et al., 2010). It is important, therefore, to reuse the water effectively to decrease the cost of microalgal biodiesel. It has been reported that the harvesting of microalgae by using chitosan as flocculants is most effective when pH is around 10 (Beach et al., 2012; Sirin et al., 2012). In our study, harvesting efficiency reached over 99% without changing the pH of the culture medium. The averaged pH of the culture medium was 6.90 ± 0.16 after harvesting with various ranges of dosage of CS/MNP. In addition, the effect of pH on the harvesting performance was not significant and the efficiency was kept over 95% in the ranges of pH 2–12 (data not shown). This is a favorable condition for medium recycling and cost saving cultivation. Farid et al. (2013) have estimated the cost for harvesting microalgae by using nano-chitosan to be about $0.0246/(kg dry microalgal biomass), which is promising based on the consideration of the economic viability of algal biodiesel and the minimum cost for harvesting microalgae ($0.08/kg dry mass). Even though we composite chitosan with additionally costing magnetic nanoparticles and need more dosage of chitosan than that of nano-chitosan for the flocculation, the economic viability of the present microalgal harvesting method is expected with taking account of rapid harvesting as well as recycling the composites and the culture medium. For future research, we are exploring the sustainability of a long-term medium recycling and the possibility of separating microalgae from CS/MNP composites. 4. Conclusions Chitosan–Fe3O4 nanoparticle composites were successfully applied to a biocompatible and rapid magnetophoretic harvesting of oleaginous microalgae. Over 99% harvesting efficiency of Chlorella sp. KR-1 was obtained without changing the pH of the culture medium by using the composites. Furthermore, the composites had no adverse effects on microalgal growth to be reused as culture medium after harvesting. This means that medium recycling is now made possible in microalgal culture. The present results show potential cost saving in microalgal diesel production by using the magnetophoretic harvesting process and biocompatible flocculants. Acknowledgements This work was supported by the Advanced Biomass R&D Center (ABC) of Global Frontier Project funded by the Ministry of Education, Science and Technology’’ (ABC-2012-053880), the Korea
Institute of Energy Technology Evaluation and Planning (KETEP) and Ministry of Knowledge Economy (MKE) as a parts of the Project of ‘‘Process demonstration for bioconversion of CO2 to highvalued biomaterials using microalgae’’ (2012-T-100201516) in ‘‘Energy Efficiency and Resources R&D project’’, and the Development of Biohydrogen Production Technology using the Hyperthermophilic Archaea program of the Ministry of Oceans and Fisheries.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.biortech.2013. 09.074.
References Beach, E.S., Eckelman, M.J., Cui, Z., Brentner, L., Zimmerman, J.B., 2012. Preferential technological and life cycle environmental performance of chitosan flocculation for harvesting of the green algae Neochloris oleoabundans. Bioresour. Technol. 121, 445–449. Bosma, R., van Spronsen, W.A., Tramper, J., Wijffels, R.H., 2003. Ultrasound, a new separation technique to harvest microalgae. J. Appl. Phycol. 15, 143– 153. Chisti, Y., 2007. Biodiesel from microalgae. Biotechnol. Adv. 25, 294–306. Farid, M.S., Shariati, A., Badakhshan, A., Anvaripour, B., 2013. Using nanochitosan for harvesting microalga Nannochloropsis sp. Bioresour. Technol. 131, 555–559. Gao, S.S., Yang, J.X., Tian, J.Y., Ma, F., Tu, G., Du, M.A., 2010. Electro-coagulationflotation process for algae removal. J. Hazard. Mater. 177, 336–343. Kim, D.-G., La, H.-J., Ahn, C.-Y., Park, Y.-H., Oh, H.-M., 2011. Harvest of Scenedesmus sp. with bioflocculant and reuse of culture medium for subsequent high-density cultures. Bioresour. Technol. 102, 3163–3168. Kim, J., Ryu, B.G., Kim, K., Kim, B.K., Han, J.I., Yang, J.W., 2012. Continuous microalgae recovery using electrolysis: Effect of different electrode pairs and timing of polarity exchange. Bioresour. Technol. 123, 164–170. Kim, J., Yoo, G., Lee, H., Lim, J., Kim, K., Kim, C.W., Park, M.S., Yang, J.-W., 2013. Methods of downstream processing for the production of biodiesel from microalgae. Biotechnol. Adv. 31, 862–876. Kumar, M., 2000. A review of chitin and chitosan applications. React. Funct. Polym. 46, 1–27. Lam, M.K., Lee, K.T., 2012. Microalgae biofuels: a critical review of issues, problems and the way forward. Biotechnol. Adv. 30, 673–690. Lee, Y.-C., Kim, B., Farooq, W., Chung, J., Han, J.-I., Shin, H.-J., Jeong, S.H., Park, J.-Y., Lee, J.-S., Oh, Y.-K., 2013. Harvesting of oleaginous Chlorella sp. by organoclays. Bioresour. Technol. 132, 440–445. Lim, J.K., Chieh, D.C.J., Jalak, S.A., Toh, P.Y., Yasin, N.H.M., Ng, B.W., Ahmad, A.L., 2012. Rapid magnetophoretic separation of microalgae. Small 8, 1683–1692. Liu, X.Q., Ma, Z.Y., Xing, J.M., Liu, H.Z., 2004. Preparation and characterization of amino-silane modified superparamagnetic silica nanospheres. J. Magnetism Magnet. MatER. 270, 1–6. Mata, T.M., Martins, A.A., Caetano, N.S., 2010. Microalgae for biodiesel production and other applications: A review. Renew. Sustain. Energy Rev. 14, 217–232. Na, J.-G., Lee, H., Oh, Y.-K., Park, J.-Y., Ko, C., Lee, S.-H., Yi, K., Chung, S., Jeon, S., 2011. Rapid estimation of triacylglycerol content of Chlorella sp. by thermogravimetric analysis. Biotechnol. Lett. 33, 957–960. Rashid, N., Rehman, M.S.U., Han, J.-I., 2013. Use of chitosan acid solutions to improve separation efficiency for harvesting of the microalga Chlorella vulgaris. Chem. Eng. J. 226, 238–242. Renault, F., Sancey, B., Badot, P.M., Crini, G., 2009. Chitosan for coagulation/ flocculation processes – an eco-friendly approach. Eur. Poly. J. 45, 1337– 1348. Sirin, S., Trobajo, R., Ibanez, C., Salvado, J., 2012. Harvesting the microalgae Phaeodactylum tricornutum with polyaluminum chloride, aluminium sulphate, chitosan and alkalinity-induced flocculation. J. Appl. Phycol. 24, 1067–1080. Xu, L., Guo, C., Wang, F., Zheng, S., Liu, C.Z., 2011. A simple and rapid harvesting method for microalgae by in situ magnetic separation. Bioresour. Technol. 102, 10047–10051. Zhang, X.Z., Hu, Q., Sommerfeld, M., Puruhito, E., Chen, Y.S., 2010. Harvesting algal biomass for biofuels using ultrafiltration membranes. Bioresour. Technol. 101, 5297–5304.
Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Short Communication
Magnetophoretic harvesting of oleaginous Chlorella sp. by using biocompatible chitosan/magnetic nanoparticle composites Kyubock Lee a, So Yeun Lee a,b, Jeong-Geol Na a, Sang Goo Jeon a, Ramasamy Praveenkumar a, Dong-Myung Kim b, Won-Seok Chang c, You-Kwan Oh a,⇑ a b c
Clean Fuel Department, Korea Institute of Energy Research, 152 Gajeong-ro, Yuseong-gu, Daejeon 305-343, Republic of Korea Department of Fine Chemical Engineering and Applied Chemistry, Chungnam National University, Daejeon 305-764, Republic of Korea Korea District Heating Corp., 186 Bundang-dong, Bundang-gu, Seongnam-si, Gyoenggi-do 463-908, Republic of Korea
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Biocompatible and rapid
magnetophoretic harvesting process of oleaginous microalgae. Over 99% harvesting efficiency achieved by using chitosan–Fe3O4 composites. After harvesting, the used medium shows no adverse effect on microalgal growth.
a r t i c l e
i n f o
Article history: Received 14 June 2013 Received in revised form 16 September 2013 Accepted 18 September 2013 Available online 27 September 2013 Keywords: Chitosan Magnetic nanoparticle Microalgae Harvesting Medium recycling
a b s t r a c t The consumption of energy and resources such as water in the cultivation and harvesting steps should be minimized to reduce the overall cost of biodiesel production from microalgae. Here we present a biocompatible and rapid magnetophoretic harvesting process of oleaginous microalgae by using chitosan–Fe3O4 nanoparticle composites. Over 99% of microalgae was harvested by using the composites and the external magnetic field without changing the pH of culture medium so that it may be reused for microalgal culture without adverse effect on the cell growth. Depending on the working volume (20–500 mL) and the strength of surface magnetic-field (3400–9200 G), the process of harvesting microalgae took only 2– 5 min. The method presented here not only utilizes permanent magnets without additional energy for fast harvesting but also recycles the medium effectively for further cultivation of microalgae, looking ahead to a large scale economic microalgae-based biorefinement. Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction With increasing petroleum oil price and growing emission of greenhouse gas, biomass-based biofuel production has been receiving much attention, especially since microalgae biomass has many advantages over the grain- and/or tree-based biomass in producing biodiesel (Lam and Lee, 2012; Mata et al., 2010). The microalgae have higher photosynthetic efficiency and high
⇑ Corresponding author. Tel.: +82 42 860 3697; fax: +82 42 860 3495. E-mail address: [email protected] (Y.-K. Oh). 0960-8524/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2013.09.074
lipid content of 15–77% of cell mass (Chisti, 2007), and they can be cultivated using carbon dioxide directly from power plants. In spite of the many advantages of microalgae-based biorefinery, it involves many steps from cultivation to harvesting, from lipid extraction to oil-to-biodiesel transition, which increase the cost of microalgal biodiesel (Lam and Lee, 2012). In the various steps in producing microalgal biodiesel, it is necessary to improve the technique of harvesting, which takes up 20–30% of the total cost (Mata et al., 2010). Since microalgae are cultivated in diluted culture medium, normally under 1 g/L, and have a negatively charged surface of a few microns in size, the dispersion of microalgae in the solution is very stable, which
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K. Lee et al. / Bioresource Technology 149 (2013) 575–578
makes harvesting a task worth challenging. Various harvesting methods have been applied till now. Conventional methods such as centrifugation, filtration and ultrasound have been widely used for microalgal harvesting (Bosma et al., 2003; Zhang et al., 2010). Electrolysis-base technologies have been shown to be powerful methods for harvesting microalgae even under the continuous process (Gao et al., 2010; Kim et al., 2012). However, those methods are not easy to scale-up and also require high energy consumption, which is not desirable for low cost production of microalgal biodiesel. Chemical flocculants such as polyvalent cations and cationic polymers or inorganics have been intensively investigated for large-scale applications (Lee et al., 2013; Sirin et al., 2012). To improve some drawbacks of chemical flocculants such as contaminations of final products and the toxic effects on microalgae (Kim et al., 2013), biocompatible flocculants such as chitosan and starch have been attempted (Beach et al., 2012; Farid et al., 2013). Recently, magnetic microalgal harvesting has been getting much attention. Microalgae are attached with magnetic particles coated with cationic substances by electrostatic interaction and then separated from the culture medium by external magnetic field (Lim et al., 2012; Xu et al., 2011). Lim et al. (2012) who applied the iron oxide magnetic nanoparticles and the cationic polyelectrolyte have reported that the harvesting efficiency of magnetic separation is as high as 99% and the harvesting completed within a few minutes. However, they did not report on the biocompatibility of the flocculent with polyelectrolyte as a functional group and the resulting recycling of medium. Chlorella sp. KR-1 is a newly isolated microalga that accumulates triacylglycerol approximately 37–41% (w/w) of cell mass (Lee et al., 2013; Na et al., 2011). In spite of the high lipid content, the small size of Chlorella sp. KR-1, around 3 lm in diameter (Lee et al., 2013), makes the harvesting by conventional methods more challenging. In this study, we synthesized chitosan–Fe3O4 magnetic nanoparticle (CS/MNP) composites and investigated the composites as biocompatible flocculants for the magnetophoretic harvesting of Chlorella sp. KR-1. The efficiency of microalgal harvesting was studied using the dosage and the ratio of chitosan to magnetic nanoparticles of the synthesized composites. Furthermore, the biocompatibility of the composites was tested by reusing both the used culture medium left over from microalgae harvesting and the microalgae-attached composites as inoculums.
2. Methods 2.1. Synthesis of CS/MNP composites Fe3O4 magnetic particles of 10–30 nm in size were synthesized by the previously reported method (Liu et al., 2004). Briefly, two aqueous solutions of FeCl24H2O (P99.0%, Sigma–Aldrich, USA) and FeCl34H2O (97%, Sigma–Aldrich, USA) were mixed and heated at 85 °C for 30 min under N2 atmosphere followed by addition of NH4OH. The precipitated nanoparticles were washed several times with ethanol and subsequently with distilled water until pH of suspension reaches to 7. The prepared Fe3O4 nanoparticles (0.15 g) were dispersed in 12 mL of distilled water with 11 mL of 2 wt% Pluronic F-127 (Sigma, USA) solution using ultrasonication. Chitosan solution was prepared by dissolving 0.2 wt% of chitosan flakes (low molecular weight, Fluka, USA) in 0.5% acetic acid. The Fe3O4 dispersion and chitosan solutions were mixed for 30 min using a magnetic bar. For cross-linking of chitosan, 140 mL sodium tripolyphosphate solution (0.15 wt%, pH 6; Sigma–Aldrich, USA) was added by a peristaltic pump at 10 mL/min. The solution was aged overnight at 4 °C and then washed with distilled water several times. The total volume of each Fe3O4-chitosan dispersion solution was set to 50 mL in order to quantify Fe3O4-chitosan by volume.
The final products of CS/MNP composites were named ‘CS/MNP a’ of which a indicates the calculated weight ratio of CS to MNP (a: 0.13, 0.40 and 0.54). 2.2. Cultivation of microalgae Chlorella sp. KR-1, an indigenous freshwater microalga (Na et al., 2011) was used in this study. The microalgae were cultivated in a modified N8 medium in a 7 L Pyrex bubble-column photobioreactor supplied with 10% (v/v) CO2 in air at a rate of 0.75 L/min under illumination of 12 fluorescent lamps (light intensity: 80 lmol photons/m2 s). The medium contained 3 mM KNO3, 5.44 mM KH2PO4, 1.83 mM Na2HPO4, 0.20 mM MgSO47H2O, 0.12 mM CaCl2, 0.03 mM FeNaEDTA, 0.01 mM ZnSO47H2O, 0.07 mM MnCl24H2O, 0.07 mM CuSO4 and 0.01 mM Al2(SO4)318H2O with pH = 6.5. For the cultivation of microalgae on agar plate, the agarose gel was prepared by dissolving 1.5 wt% of agar (Bacto™ Agar, BD Bacto™, France) in a modified N8 medium in an autoclave at 121 °C for 15 min. The magnetophoretically harvested microalgae-CS/MNP flocs were plated on the agar plate, which was incubated for 3 days in the growth chamber (temperature, 25 °C; humidity, 60%; light intensity, 15 lmol photons/m2 s). 2.3. Harvesting of microalgae with CS/MNP The harvesting experiment was carried out using microalgae freshly taken from 7 L Pyrex bubble-column after 4 d incubation. The concentration of microalgae was 1.0 g/L (Optical density at 660 nm, OD660nm = 4.5). The harvesting efficiency, in this study, indicates the percentage of microalgae attached on CS/MNP which are separated by the magnetic field subsequently. After injection of the calculated amount of CS/MNP dispersion into 1 mL of microalgal solution, external magnetic field was applied to the mixture. The surface magnetic-field strength of a permanent NdFeB magnet chip (length, 20 mm; width, 9 mm; thickness 4 mm) was measured to be 3400 G by a Gauss meter (TM-701, KANETEC Co., Japan). The supernatant was taken for OD measurement after 3 min of magnetophoretic harvesting, thereby ensuring the measurement of the only concentration of unattached microalgae on CS/MNP. The harvesting efficiency was calculated by following equation (Lee et al., 2013).
ODf 100 Harvesting efficiency ½% ¼ 1 ODi where ODi and ODf were the initial OD and the OD of the supernatant after magnetophoretic harvesting, respectively. 2.4. Reuse of medium solution and microalgae-attached CS/MNP as an inoculum Microalgae were harvested by using CS/MNP composite and a magnet rod (surface magnetic-field strength, 9200 G; diameter, 22 mm; length, 500 mm) from 0.5 L of microalgal solution (see Graphical Abstract). After adjusting the concentration of nitrate of used medium to 3 mM, new cells were inoculated and cultivated in 1 L bubble columns for 7 d under the same conditions mentioned previously. For comparison, the medium solution was reused for the next cultivation after harvesting microalgae with a widely using flocculant, Alum (Kim et al., 2011). In addition, the magnetophoretically harvested black slurry containing microalgae-attached CS/MNP (the 4th image of Graphical Abstract, Fig. S3a and S4b) was directly used as an inoculum in the new N8 medium to investigate the biocompatibility CS/MNP composite versus microalgae.
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2.5. Analytical methods
3.3. Reuse of culture medium after harvesting of microalgae
The OD measurement was performed using UV–VIS spectrophotometer (Optizen 2120 UV, Mecasys Co., Korea). The optical microscopic images were obtained with an oil immersion objective lens at 100 magnification (Microscope Axio Imager.A2, Carl Zeiss Microscopy GmbH, Jena, Germany). After harvesting, microalgaeattached CS/MNP composites were freeze-dried at 55 °C for field emission scanning electron microscope (FE-SEM) and energy-dispersive spectroscopy (EDS) analysis (S-4800, HITACHI, Japan).
The medium was reused for microalgal culture after harvesting (Fig. 1b). As shown in Graphical Abstract, over 97% of microalgae was recovered within 5 min from 1 L bubble column containing 0.5 L microalgal solution by using CS/MNP 0.40 and a permanent magnetic rod with a maximum surface magnetic field strength of 9200 G. Cell growth in the reused medium that had been used once in CS/MNP harvesting was similar to that of the new medium (Fig. 1b). This implies that the use of CS/MNP composites as flocculants has no inhibitory effect on algal cells. For comparison, Alum which has been used widely as a chemical flocculant was tested for magnetophoretic harvesting of oleaginous microalgae and for the reuse of the medium after harvesting. It took one hour to settle 90.6% and 97.3% microalgae in 1 L bubble column with 0.9 and 5 mM of Alum, respectively. Microalgal cell growth was inhibited in both reused media of Alum. This is consistent with the report by Kim et al. (2011). The Lewis acidity of Al3+ caused a pH drop of microalgal solution down to 3 when Alum was used. This seems to have caused the inhibition of cell growth. The pH levels were kept at 6–7 during the cell growth in the new N8 medium or the reused medium after harvesting with CS/MNP (data not shown). Chitosan is known to be a widely used biocompatible material (Kumar, 2000). The biocompatibility of the CS/MNP composites was tested by plating the harvested microalgae-CS/MNP flocs on an agar plate (Fig. S3a). After three days of incubation, microalgal growth was evident as visible colonies on the agar surface (Fig. S3b). When the revival of microalgae was verified microscopically, the development of actively dividing colonies from the microalgae-CS/MNP flocs was observed (Fig. S3b). This test showed
3. Results and discussion 3.1. CS/MNP composites and magnetophoretic harvesting of microalgae Fig. S1a shows the snapshots of magnetophoretic harvesting of microalgae at different time points after injecting CS/MNP suspension into 20 mL of 1 g/L microalgal solution and applying a NdFeB magnet chip. It took less than 2 min for the complete harvesting of microalgae. The kinetics of magnetophoretic harvesting is not comparable to that of gravitational harvesting. It has been reported that microalgal harvesting by settling only with chitosan as flocculants required one hour to harvest 97% of microalgae (Farid et al., 2013). Flocculation was observed when CS/MNP was mixed with microalgal solution. As soon as the magnet was externally applied to the vial containing the mixture, the magnet pulled flocs of CS/MNP and microalgae toward the magnet (Fig. S1a). Fig. S1b shows the differential interference contrast (DIC) microscopic image of CS/MNP composite synthesized with brownish aggregated Fe3O4 nanoparticles embedded in the chitosan matrix. The transparent chitosan matrix could be observed in DIC mode, which enhances the contrast of chitosan by the principle of interferometry. After mixing CS/MNP with microalgal solution, it was observed that microalgal cells were attached onto CS/MNP forming flocs (Fig. S1a and c). SEM image clearly shows microalgal cells attached onto CS/MNP as indicated by arrows in Fig. S1d. The protonated amino groups of chitosan in water are positively charged to present NH3+ ions and, therefore, induce a charge neutralization, which means attachment of negatively charged microalgal cells (Renault et al., 2009; Sirin et al., 2012). Nanosized Fe3O4 particles were also observed, and they were confirmed to be a Fe-rich region by energy-dispersive X-ray spectroscopy (EDAX) analysis and mapping (Figs. S1d and S2).
3.2. Harvesting efficiency and kinetics of magnetophoretic separation of microalgae This section investigates the relationships between the harvesting efficiency of microalgae and the concentration of the composites as well as the ratio of chitosan to magnetic nanoparticles. The harvesting efficiencies were improved to almost 100% by increasing the dosage of CS/MNP (Fig. 1a). Just as Rashid et al. (2013) reported, the zeta-potential of Chlorella vulgaris increased from 21 to +1.9 mV with the chitosan dosage. The charge neutralization seems to have occurred when the harvesting efficiency reached 100%. When the composite with a higher ratio of CS to MNP was used, the higher harvesting efficiency was obtained with same dosage. In the case of CS/MNP 0.54, a 99% harvesting efficiency was obtained by the 1.4 g/L dosage. On the other hand, only 95% of microalgae were harvested with 3.4 g/L of CS/MNP 0.13 because chitosan provides adsorption sites for microalgae and, therefore, the composite with a higher ratio of CS to MNP has more adsorption sites.
Fig. 1. (a) Plots of harvesting efficiency of CS/MNP with various ratio of CS to MNP as a function of the concentration of CS/MNP in 1 g/L of microalgal solution. (b) Microagal growth in the reused medium after harvesting microalgae by using CS/MNP 0.40 ( ), 0.9 mM ( ) and 5 mM of Alum ( ) as flocculants after adjusting the concentration of nitrate to the level of new N8 medium (d).
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that the composites do not affect the growth of microalgae, thus they could be considered as biocompatible materials. In addition, microalgae were successfully cultured by using microalgae-attached CS/MNP as an inoculum (black slurry, the 4th image of Graphical Abstract, Figs. S3a and S4b). The cell growth showed a typical pattern of microalgal growth in 1 L bubblecolumn with exponential growth followed by the stationary pattern whose OD reached 9.15 in 7 d (Supplementary materials, Fig. S4a). The optical microscope image of microalgae-attached CS/MNP after re-culture of 7 d shows that green microalgal cells are still alive and attached onto the composites (Supplementary materials, Fig. S4c). These results imply that CS/MNP composites are biocompatible with Chlorella sp. It is worth noting that a large portion of the cost in microagal culture is attributed to water, since the concentration of microalgae in culture medium is very low, less than 1 g/L (Mata et al., 2010). It is important, therefore, to reuse the water effectively to decrease the cost of microalgal biodiesel. It has been reported that the harvesting of microalgae by using chitosan as flocculants is most effective when pH is around 10 (Beach et al., 2012; Sirin et al., 2012). In our study, harvesting efficiency reached over 99% without changing the pH of the culture medium. The averaged pH of the culture medium was 6.90 ± 0.16 after harvesting with various ranges of dosage of CS/MNP. In addition, the effect of pH on the harvesting performance was not significant and the efficiency was kept over 95% in the ranges of pH 2–12 (data not shown). This is a favorable condition for medium recycling and cost saving cultivation. Farid et al. (2013) have estimated the cost for harvesting microalgae by using nano-chitosan to be about $0.0246/(kg dry microalgal biomass), which is promising based on the consideration of the economic viability of algal biodiesel and the minimum cost for harvesting microalgae ($0.08/kg dry mass). Even though we composite chitosan with additionally costing magnetic nanoparticles and need more dosage of chitosan than that of nano-chitosan for the flocculation, the economic viability of the present microalgal harvesting method is expected with taking account of rapid harvesting as well as recycling the composites and the culture medium. For future research, we are exploring the sustainability of a long-term medium recycling and the possibility of separating microalgae from CS/MNP composites. 4. Conclusions Chitosan–Fe3O4 nanoparticle composites were successfully applied to a biocompatible and rapid magnetophoretic harvesting of oleaginous microalgae. Over 99% harvesting efficiency of Chlorella sp. KR-1 was obtained without changing the pH of the culture medium by using the composites. Furthermore, the composites had no adverse effects on microalgal growth to be reused as culture medium after harvesting. This means that medium recycling is now made possible in microalgal culture. The present results show potential cost saving in microalgal diesel production by using the magnetophoretic harvesting process and biocompatible flocculants. Acknowledgements This work was supported by the Advanced Biomass R&D Center (ABC) of Global Frontier Project funded by the Ministry of Education, Science and Technology’’ (ABC-2012-053880), the Korea
Institute of Energy Technology Evaluation and Planning (KETEP) and Ministry of Knowledge Economy (MKE) as a parts of the Project of ‘‘Process demonstration for bioconversion of CO2 to highvalued biomaterials using microalgae’’ (2012-T-100201516) in ‘‘Energy Efficiency and Resources R&D project’’, and the Development of Biohydrogen Production Technology using the Hyperthermophilic Archaea program of the Ministry of Oceans and Fisheries.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.biortech.2013. 09.074.
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