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May 26, 2018 - ... Wasif Farooq 3, Sang Goo Jeon 4, You-Kwan Oh 5,* and Young-Chul Lee 2,* ID. 1. Advanced Biomass R&D Center, KAIST, 291 Daehak-ro, ...

energies Article

Magnesium Aminoclay-Fe3O4 (MgAC-Fe3O4) Hybrid Composites for Harvesting of Mixed Microalgae Bohwa Kim 1,† , Vu Khac Hoang Bui 2,† , Wasif Farooq 3 , Sang Goo Jeon 4 , You-Kwan Oh 5, * and Young-Chul Lee 2, * ID 1 2 3 4 5

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Advanced Biomass R&D Center, KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon 34141, Korea; [email protected] Department of BioNano Technology, Gachon University, 1342 Seongnamdaero, Sujeong-gu, Seongnam-si, Gyeonggi-do 13120, Korea; [email protected] Department of Chemical Engineering, King Fahd University of Petroleum and Minerals (KFUPM), Dhahran 31261, Saudi Arabia; [email protected] Biomass and Waste Energy Laboratory, Korea Institute of Energy Research (KIER), Daejeon 34129, Korea; [email protected] School of Chemical and Biomolecular Engineering, Pusan National University, Busan 46241, Korea; [email protected] Correspondence: [email protected] (Y.-K.O.); [email protected] (Y.-C.L.); Tel.: +82-51-510-2395 (Y.-K.O.); +82-31-750-8751 (Y.-C.L.); Fax: +82-51-512-8563 (Y.-K.O.); +82-31-750-4748 (Y.-C.L.) Equally Contributing Authors.

Received: 5 April 2018; Accepted: 25 May 2018; Published: 26 May 2018

 

Abstract: In this paper, we describe the synthesis of magnesium aminoclay-iron oxide (MgAC-Fe3 O4 ) hybrid composites for microalgae-harvesting application. MgAC-templated Fe3 O4 nanoparticles (NPs) were synthesized in different ratios of MgAC and Fe3 O4 NPs. The uniform distribution of Fe3 O4 NPs in the MgAC matrix was confirmed by transmission electron microscopy (TEM). According to obtained X-ray diffraction (XRD) patterns, increased MgAC loading leads to decreased intensity of the composites’ (311) plane of Fe3 O4 NPs. For harvesting of Chlorella sp. KR-1, Scenedesmus obliquus and mixed microalgae (Chlorella sp. KR-1/ Scenedesmus obliquus), the optimal pH was 4.0. At higher pHs, the microalgae-harvesting efficiencies fell. Sample #1, which had the highest MgAC concentration, showed the most stability: the harvesting efficiencies for Chlorella sp. KR-1, Scenedesmus obliquus, and mixed microalgae were reduced only to ~50% at pH = 10.0. The electrostatic interaction between MgAC and the Fe3 O4 NPs in the hybrid samples by microalgae, as confirmed by zeta potential measurements, were attributed to the harvesting mechanisms. Moreover, the zeta potentials of the MgAC-Fe3 O4 hybrid composites were reduced as pH was increased, thus diminishing the microalgae-harvesting efficiencies. Keywords: magnesium aminoclay (MgAC); magnetite; Chlorella sp. KR-1; Scenedesmus obliquus; microalgae harvesting; biorefinement

1. Introduction Microalgae convert sunlight and carbon oxide to essential materials such as biofuels, pharmaceutical bioactives, and fertilizers [1–5]. Indeed, lipids obtained from microalgae promise to replace fossil fuels [6]. However, such potential remains limited by the costs of microalgae cultivation, harvesting, dewatering, lipid extraction, and conversion to biodiesel [6]. Among the various nanomaterials that could be applied for microalgae cultivation and harvesting, magnesium aminoclay (MgAC) is a potential candidate [7]. MgAC, which was first introduced Energies 2018, 11, 1359; doi:10.3390/en11061359

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by Mann et al. (1997) [8], is attractive due to its propylamine functionalities, structures, as well as high dispersity in water [9,10]. We have already reported on several studies using MgAC for microalgae-cultivation improvement [11], harvesting [12,13], recovery [14], and also lipids extraction from microalgae biomass [15]. MgAC significantly enhances microalgae-harvesting efficiencies [6] while is not effected by inhibition factors such as humic acid [16]. For example, in one study conducted several years ago, 100% of Chlorella sp. KR-1 (1.7 g/L microalgae feedstocks) was harvested 30 min after injection of MgAC at a concentration above 0.6 g/L while maintaining the neutral pH [12]. Farooq et al. (2013) achieved flocculation of microalgae by electrostatic interaction with high-positive-surface-charged MgAC, as confirmed by the obtained zeta potentials [12]. Ji et al. (2016) more recently utilized CeAC with MgAC in a two-ACs-mixture system for cyanobacteria harvesting. The harvesting efficiency of the mixed aminoclays reached 100% within 1 h. Moreover, the mixture loading was 10 times lower than for single-aminoclay treatment [13]. MgAC has the additional potential to be incorporated with other nanomaterials for microalgae-harvesting improvement. To overcome the limitation of MgAC in reusability, we have performed MgAC-templated zero-valent iron (MgAC-nZVI) synthesis for harvesting of Chlorella sp. KR-1. This composite showed a high positive-surface charge (+40 mV) and ferromagnetic properties (30 emu/g). On the laboratory-scale, MgAC-nZVI enabled harvesting of Chlorella sp. KR-1 (concentration: 20 g/L) within 3 min under a magnetic field, the microalgae-harvesting efficiencies having reached ~100% [14]. We also have already conjugated MgAC with TiO2 for simultaneous harvesting and wet-disruption of Chlorella sp. KR-1 [17]. MgAC played a key role in microalgae flocculation, while TiO2 contributed to direct cell disruption under UV-light irradiation (365 nm). Within 10 min, the injection of MgAC into the microalgae feedstocks at a concentration of 1.5 g/L effected an ~85% harvesting efficiency; subsequently, the harvested wet-microalgae biomass was irradiated under UV light for 4 h, thus enabling ~95% cell-disruption efficiencies [17]. A previous study indicated the potential of combination of MgAC with metal oxide nanoparticles for microalgae harvesting purpose [17]. In the present study, we utilized magnetic Fe3 O4 NPs as another candidate for templating with MgAC and, thereby, harvesting. Fe3 O4 NPs have been used previously for harvesting of Botryococcus braunii and Chlorella ellipsoidea [18]. The microalgae-harvesting mechanism has been attributed to the electrostatic interaction between Fe3 O4 NPs and microalgae. Meanwhile, the development of in-situ magnetic separation technology provide the potential for further microalgae-harvesting improvement [18]. 2. Results 2.1. Characterization of MgAC-Fe3 O4 Hybrid Composites On transmission electron microscopy (TEM) images (Figure 1), the Fe3 O4 NPs had approximately crystalline sizes of ~3.50 nm (sample #1), ~3.57 nm (sample #2), ~4.28 nm (sample #3), ~6.42 nm (sample #4), and ~7.14 nm (sample #5, only Fe3 O4 NPs). The Fe3 O4 NPs were distributed uniformly in the MgAC matrix [17,19,20]. According to the X-ray diffraction (XRD) patterns (Figure 2), all of the as-prepared samples had seven main peaks, at 18◦ , 29.98◦ , 35.3◦ , 42.9◦ , 54◦ , 56.37◦ , and 62◦ , belonging to (002), (200), (311), (400), (422), (511), and (440) of Fe3 O4 magnetite (JCPDS-00-019-0629; JCPDS: Joint Committee on Power Diffraction Standard) [21], respectively. Meanwhile, from sample #1 to sample #3, there were additional peaks at 10.81◦ and 22.33◦ belonging to (002) and (020,110) of MgAC [22], respectively. In the sample #4, the peaks of MgAC were disappeared due to its low ratios in the hybrid composites (Tables 1 and 2). It was apparent that in the hybrid composite, from sample #1 to sample #3, the increase of the MgAC loading concentration resulted in the decrease of the intensity of the (311) plane of Fe3 O4 . The appearance of NaCl halite (JCPDS-00-005-0628) indicated remaining NaCl, even after washing.

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Figure 1. Transmission electron microscopy (TEM) images of (a) sample #1; (b) sample #2; (c) sample #3; Figure 1. Transmission electron (TEM)sizes images of (a) #1; (b) sample #2;are (c) shown sample (d) sample #4; and (e) sample #5.microscopy The crystalline of the Fe3sample O4 nanoparticles (NPs) #3; (d) the sample #4;red andcircles. (e) sample #5. The crystalline sizes of the Fe3O4 nanoparticles (NPs) are shown within dotted within the dotted red circles.

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Figure 2. Powder X-ray diffraction (PXRD) of#1–5 samples #1–5 l halite, Figure 2. Figure Powder X-ray diffraction (PXRD) patterns ofofsamples #1–5. (MgAC), (NaCl halite, 2. Powder X-ray diffraction (PXRD) patterns samples #1–5 l halite, Figure 2. Powder X-ray diffraction (PXRD) patterns ofpatterns samples l halite, Figure 2. Powder X-ray diffraction (PXRD) patterns of samples #1–5. (MgAC), (NaCl halite, JCPDSJCPDS-00-005-0628; JCPDS: Joint Committee on Power Diffraction Standard), and JCPDS-00-005-0628; JCPDS: Joint Committee on Power Diffraction Standard), and (Fe 3(Fe O 4 3O4 JCPDS-00-005-0628; JCPDS:Joint Joint Committee onPower Power Diffraction Standard), and (Fe 3 O 4 JCPDS-00-005-0628; JCPDS: Committee on Diffraction Standard), and (Fe O magnetite, 3 4 00-005-0628; JCPDS: Joint Committee on Power Diffraction Standard), and (Fe3O4 magnetite, magnetite, JCPDS-00-019-0629). magnetite, JCPDS-00-019-0629). magnetite, JCPDS-00-019-0629). JCPDS-00-019-0629). JCPDS-00-019-0629). Table 1. Elemental compositions of as-prepared samples by X-ray fluorescence (XRF) Table 1. Elemental compositions %) %) of as-prepared by fluorescence X-ray fluorescence Table 1. Elemental compositions (wt. %)(wt. of(wt. as-prepared samplessamples by X-ray (XRF) (XRF)

Table 1. Elemental compositions (wt. %) of as-prepared samples by X-ray fluorescence (XRF) spectrometry. spectrometry. spectrometry. Table 1. spectrometry. Elemental compositions (wt. %) of as-prepared samples by X-ray fluorescence (XRF) Sample Sample #1 #1 #3 #2 #2 #4 #3 #3 #5 #4 #4 #5 #5 spectrometry. Sample #1#1 #2 Sample #2 #3 #4 #5 Fe (%)77.7 72.672.682.8 77.777.786.0 82.882.894.7 86.086.094.794.7 Fe (%) Fe (%) 72.6

Sample #1 4.6 #23.8 #3 #4 2.0 0.2 #5 Fe (%) 72.6 82.8 86.0 94.7 (%) 5.3 0.2 (%) 5.3 77.7 4.6 4.6 3.8 3.8 0.2 2.0 Mg (%)MgMg 5.3 2.0 MgFe(%) 5.3 4.6 3.8 2.0 Si 0 (%) 72.61.5 77.7 82.8 Si (%) 2.2 2.2 1.5 1.5 1.3 1.386.0 1.7 0 0.2 Si (%) 2.2(%) 1.3 1.7 0 1.7 94.7 Si (%) Cl (%) 1.516.216.21.3 1.79.9 5.1 5.1 0 Cl 2.2 (%) 19.919.9 12.0 9.9 Cl (%) 12.0 3.8 9.912.02.0 5.1 Mg (%) 19.9 5.316.2 4.6 0.2 Cl (%) 19.9 16.2 12.0 9.9 5.1 Si (%)

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Table 2. Formulations of magnesium aminoclay (MgAC) and precursors in mL 90 mL volumes 2. Formulations of magnesium aminoclay and ironiron precursors in volumes 90 volumes for for Table 2.Table Formulations of magnesium aminoclay (MgAC)(MgAC) and iron precursors in 90 mL for Cl3O(%) 19.9 16.2 12.0 9.9 5.1 preparation of3O MgAC-Fe O4 hybrid composites. preparation of MgAC-Fe 4 3hybrid composites. preparation of MgAC-Fe 4 hybrid composites. Table 2. Formulations of magnesium aminoclay (MgAC) and iron precursors in 90 mL volumes for

preparation of MgAC-Fe composites. Reactants Sample 3 O4 hybrid Reactants #1 #1 Sample #2 #2 Sample #3 #3 Sample #4 #4 Sample #5 #5 SampleSample #1aminoclay Sample #2Sample Sample #3Sample Sample #4Sample Sample #5Sample Table 2. Reactants Formulations of magnesium (MgAC) and iron precursors in 90 mL volumes for MgAC (g/40 (g/40 mL)mL) 0.80 0.800.80 0.40 0.400.40 0.08 0.080.08 0.04 0.040.04 0 0 0 MgAC MgAC (g/40 mL) preparation ofNanosized MgAC-Fe 3O4 hybrid composites. Fe43by O4 by Sample #1 Nanosized Fe3O Reactants Sample #2 Sample #3 Sample #4 Sample #5 Nanosized Fe3O4 by g 2FeCl •6H 2FeCl O0.7  20.7 g 2FeCl 2•4H [1.9[1.9 FeCl 2Og g•4H FeCl 2•4H 2O]2O] [1.9 g FeCl 3g •6H O3•6H  30.7 O] precipitation method mL) precipitation method mL) precipitation method (40 mL)(40 (40 Reactants Sample Sample Sample Sample MgAC (g/40 mL) 0.80 #1 0.40 #2 0.08 #3 0.04 #4 10 mL 10 mL NaOH M) M) 10 mL NaOH (10NaOH M) (10 (10

Sample #5 0 MgAC (g/40 mL) 0.80 0.40 0.08 0.04 0 Nanosized Fe3 O4 by [1.9 g FeCl3 •6H2 O ⊕ 0.7 g FeCl2 •4H2 O] Nanosized Fe3O4(40 by mL) precipitation method Microalgae-Harvesting Efficiencies of 3MgAC-Fe 4 Hybrid Composites 2.2.2.2. Microalgae-Harvesting Efficiencies of MgAC-Fe 3O43O Hybrid Composites 2.2. Microalgae-Harvesting Efficiencies of MgAC-Fe O4[1.9 Hybrid Composites g FeCl 3•6H2O  0.7 g FeCl2•4H2O] precipitation method (40 mL) 10 NaOH (10samples M) optimal microalgae-harvesting of mL the MgAC-Fe 4 (#1~5) samples (#1~5) was TheThe optimal microalgae-harvesting for all all of the MgAC-Fe 3O43O (#1~5) was 4.0,4.0, at at The optimal microalgae-harvesting pH forpH allpH of for the MgAC-Fe 3O4 samples was 4.0, at 10 mL NaOH (10 M) which the harvesting efficiencies were better than 80% for Chlorella sp. KR-1, Scenedesmus obliquus,

the harvesting efficiencies were than better80% thanfor 80% for Chlorella sp. Scenedesmus KR-1, Scenedesmus obliquus, which which the harvesting efficiencies were better Chlorella sp. KR-1, obliquus, and mixed microalgae (Chlorella sp. KR-1 and Scenedesmus obliquus were mixed in a 1:1 ratio and mixed microalgae (Chlorella sp. KR-1 and Scenedesmus obliquus were mixed in a 1:1 ratio by by and mixed microalgae (Chlorella sp. KR-1 and Scenedesmus obliquus were mixed in a 1:1 ratio by 2.2. Microalgae-Harvesting Efficiencies of MgAC-Fe3 O4 Hybrid Composites 2.2. volume) Microalgae-Harvesting ofresulted MgAC-Fe 4microalgae-harvesting Hybrid Composites volume) (Figure 3a). Higher pHs in3O diminished microalgae-harvesting results 3). For volume) (Figure 3a).Efficiencies Higher pHs in diminished microalgae-harvesting (Figure (Figure 3a). Higher pHs resulted inresulted diminished resultsresults (Figure 3).(Figure For 3). For samples #3, #4, and #5, harvesting efficiencies Chlorella sp. KR-1 and mixed microalgae The optimal pH for all offor theChlorella MgAC-Fe Osp. (#1~5) was 4.0, at which samples #2, #3, #4, and #5, thethe harvesting efficiencies for for Chlorella KR-1 and mixed microalgae samples #2, #3,microalgae-harvesting #4,#2, and #5, the harvesting efficiencies sp. and mixed microalgae 3KR-1 4 samples The optimal microalgae-harvesting pHwhile for all of theScenedesmus MgAC-Fe 3O4 samples (#1~5) was 4.0, at were below at and pH =10.0, 7.0 10.0, those obliquus were at and pH =mixed 7.0 were below pH = 7.0 andand 10.0, while those for for Scenedesmus obliquus were 60–80% pH = 7.0 were below 20% at20% pH20% =at7.0 while those for Scenedesmus were 60–80% at60–80% pH =at7.0 the harvesting efficiencies were better than 80% for Chlorella sp.obliquus KR-1, Scenedesmus obliquus,

which the harvesting efficiencies were better than 80% for Chlorella sp. KR-1, Scenedesmus obliquus, microalgae (Chlorella sp. KR-1 and Scenedesmus obliquus were mixed in a 1:1 ratio by volume) (Figure 3a). and mixed microalgae (Chlorella sp. KR-1 and Scenedesmus obliquus were mixed in a 1:1 ratio by Higher pHs resulted in diminished microalgae-harvesting results (Figure 3). For samples #2, #3, #4, volume) (Figure 3a). Higher pHs resulted in diminished microalgae-harvesting results (Figure 3). For and #5, the harvesting efficiencies for Chlorella sp. KR-1 and mixed microalgae were below 20% at samples #2, #3, #4, and #5, the harvesting efficiencies for Chlorella sp. KR-1 and mixed microalgae pH = 7.0 and 10.0, while those for Scenedesmus obliquus were 60–80% at pH = 7.0 and 55–75% at were below 20% at pH = 7.0 and 10.0, while those for Scenedesmus obliquus were 60–80% at pH = 7.0

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and=55–75% at pH = 10.0, respectively. In the mixed-microalgae harvesting, sp.affected KR-1 was pH 10.0, respectively. In the mixed-microalgae harvesting, Chlorella sp. KR-1Chlorella was more by more by the pHenvironment condition of (Figure the environment 3d,e,f) than was Scenedesmus obliquus. the pHaffected condition of the 3d–f) than(Figure was Scenedesmus obliquus.

Figure 3. 3. Microalgae-harvesting (%, top) top) of of respective respective Chlorella Chlorella sp. sp. KR-1, KR-1, Scenedesmus Scenedesmus Figure Microalgae-harvesting efficiencies efficiencies (%, obliquus and and mixed mixedmicroalgae microalgae(Chlorella (Chlorellasp. sp.KR-1 KR-1and andScenedesmus Scenedesmus obliquus were mixed a 1:1 ratio obliquus obliquus were mixed in ain1:1 ratio by by volume) by samples #1 #5 at pH 4.0 (a); pH 7.0 (b); and pH 10.0 (c) and corresponding proportions volume) by samples #1 - #5 at pH 4.0 (a); pH 7.0 (b); and pH 10.0 (c) and corresponding proportions (%, bottom) bottom) of of respective respective Chlorella Chlorella sp. sp. KR-1 KR-1 and and Scenedesmus Scenedesmus obliquus obliquus in in mixed mixed microalgae microalgae by by samples samples (%, #1 #5 at pH 4.0 (d); pH 7.0 (e); and pH 10.0 (f). Control is proportions of Chlorella sp. KR-1 and #1–#5 at pH 4.0 (d); pH 7.0 (e); and pH 10.0 (f). Control is proportions of Chlorella sp. KR-1 and Scenedesmus obliquus obliquus in in initial initial media. media. Scenedesmus

Sample #1 showed the most stability, which harvesting efficiencies for single Chlorella sp. KR-1, Sample #1 showed the most stability, which harvesting efficiencies for single Chlorella sp. KR-1, Scenedesmus obliquus, and mixed microalgae were reduced from >80% at pH = 4 to around 60% at pH Scenedesmus obliquus, and mixed microalgae were reduced from >80% at pH = 4 to around 60% at = 7.0 and 50% at pH = 10.0 (Figure 3). The greater stability of sample #1 under the high-pH condition pH = 7.0 and 50% at pH = 10.0 (Figure 3). The greater stability of sample #1 under the high-pH was owed to its highest concentration of MgAC (Table 1), which is less affected by high pH [12]. condition was owed to its highest concentration of MgAC (Table 1), which is less affected by high The mechanism of microalgae harvesting is the electrostatic interaction between MgAC and pH [12]. Fe3O4 NPs in the hybrid samples (which have a positively charged surface) with microalgae (which The mechanism of microalgae harvesting is the electrostatic interaction between MgAC and have a negatively charged surface) [13,18]. The decreased harvesting efficiency of the MgAC-Fe3O4 Fe3 O4 NPs in the hybrid samples (which have a positively charged surface) with microalgae (which hybrid composites under the high-pH condition could be confirmed by the decreased zeta potential. have a negatively charged surface) [13,18]. The decreased harvesting efficiency of the MgAC-Fe3 O4 For example, the zeta potential of sample #4 decreased from +30 (mV) at pH = 4.0 to 0 (mV) at pH = hybrid composites under the high-pH condition could be confirmed by the decreased zeta potential. 8.0 and −4 (mV) at pH = 10, while that of sample #1, which showed the best stability, decreased from For example, the zeta potential of sample #4 decreased from +30 (mV) at pH = 4.0 to 0 (mV) at pH = 8.0 +30 (mV) at pH = 4.0 to +20 at pH = 8.0 and +8.5 (mV) at pH = 10.0 (Figure 4). Farooq et al. (2013) and −4 (mV) at pH = 10, while that of sample #1, which showed the best stability, decreased from explained that the degree of harvesting efficiency of aminoclay is attributable to its high positive zeta +30 (mV) at pH = 4.0 to +20 at pH = 8.0 and +8.5 (mV) at pH = 10.0 (Figure 4). Farooq et al. (2013) potential [12]. explained that the degree of harvesting efficiency of aminoclay is attributable to its high positive zeta potential [12].

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Figure 4. 4. Zeta to pH. pH. Figure Zeta potentials potentials of of samples samples #1–5, #1–5, Chlorella Chlorella sp. sp. KR-1 KR-1 and and Scenedesmus Scenedesmus obliquus obliquus according according to

3. Discussion 3. Discussion Due to tothe thedifficulty difficulty single microalgae cultivation a large scale because goes against Due of of single microalgae cultivation on a on large scale because it goes it against natural natural ecological tendencies, mixed microalgae cultivation could be alternative approach due to its ecological tendencies, mixed microalgae cultivation could be alternative approach due to its potential in potential in both of increase biomass yields, and the crop protection requirements for commercial both of increase biomass yields, and the crop protection requirements for commercial applications [23]. applications [23]. Besides cultivation, the of harvesting process of mixed alsoIn needs to be Besides cultivation, the harvesting process mixed microalgae also needsmicroalgae to be studied. this paper, studied. In this paper, we chose mixed microalgae (Chlorella sp. KR-1 and Scenedesmus obliquus were we chose mixed microalgae (Chlorella sp. KR-1 and Scenedesmus obliquus were mixed in a 1:1 ratio by mixed in a 1:1 ratio by volume) for testing the harvesting efficiency of MgAC-Fe 3O4 hybrid volume) for testing the harvesting efficiency of MgAC-Fe3 O4 hybrid nanocomposites. nanocomposites. Fe3 O4 NPs, which is well-known by its microalgae harvesting ability due to electrostatic Fe3O4and NPs, which properties is well-known by its after microalgae harvesting ability due to the electrostatic attraction magnetic for recovery harvesting process [18]. However, common attraction and magnetic properties for recovery after harvesting process [18]. However, the common disadvantage of Fe3 O4 NPs is its decrease of surface charge at neutral and alkaline pH conditions [18]. disadvantage of Fe 3O4 NPs is its decrease of surface charge at neutral and alkaline pH conditions [18]. Gao et al. (2009) developed montmorillonite-Cu(II)/Fe(III) for the harvesting of Microcystis aeruginosa, Gao the et al.harvesting (2009) developed montmorillonite-Cu(II)/Fe(III) the harvesting but efficiencies still decreased at high pHforvalues [24]. Geof etMicrocystis al. (2015) aeruginosa, overcame but the harvesting efficiencies still decreased at high pH values [24]. Ge et al. (2015) overcame the the limitation of Fe 3 O4 NPs by coating polyethylenimine (PEI) on the surface of Fe3 O4 NPs. limitation of Fe3O4 still NPsremained by coating (PEI)toward on theScenedesmus surface of dimorphus Fe3O4 NPs. This This nanocomposite the polyethylenimine harvesting efficiencies around nanocomposite remained the harvesting Scenedesmus dimorphus around 80% 80% at pH of 7.0still [25]. But as indicated from efficiencies our results,toward the harvesting efficiencies of Scenedesmus at pH of 7.0 [25]. But as indicated from our results, the harvesting efficiencies of Scenedesmus species by Fe3 O4 NPs just slightly reduced (harvesting efficiency of Scenedesmus obliquus byspecies Fe3 O4 by Fe 3O4 NPs just slightly reduced (harvesting efficiency of Scenedesmus obliquus by Fe3O4 NPs and NPs and MgAC-Fe3 O4 hybrid nanocomposites was around ~60–80% at pH of 7.0 (Figure 3b)) by the MgAC-Fe 4 hybrid nanocomposites was around ~60–80% at pH of 7.0 (Figure 3b)) by the increase increase of3O pH value compared to Chlorella species (Chlorella sp. KR-1 in this paper). of pHMgAC value compared to Chlorella (Chlorella sp. KR-1 harvesting in this paper). has been showed its species potential for microalgae in previous reports [12,13]. MgAC has been showed its potential for microalgae harvesting in reports process. [12,13]. However, due to its high dispersion in water [6], it could not be recycled previous after harvesting However, due to high dispersion in water [6], it could not be recycled after harvesting process. In In this paper, weitssynthesized MgAC-Fe 3 O4 hybrid nanocomposites to take advantages of both this paper, we synthesized MgAC-Fe 3O4 hybrid nanocomposites to take advantages of both nanomaterials in microalgae harvesting while reducing their drawbacks. Synthesized MgAC-Fe3 O4 nanomaterials in microalgae reducing theirwere drawbacks. MgAC-Fe 3O4 hybrid nanocomposites easily harvesting prepared bywhile the sol-gel method applied Synthesized for microalgae harvesting. hybrid nanocomposites easily prepared by the sol-gel method were applied for microalgae Fe3 O4 NPs were found to be of uniform distribution in the MgAC matrix; their crystalline sizes, harvesting.were Fe3Oreduced 4 NPs were found to be of uniform distribution in the MgAC matrix; their crystalline moreover, by the presence of MgAC. The mechanism of this phenomenon should be sizes, moreover, wereworks. reducedThe by the presence The mechanism of this phenomenon should elucidated in future optimal pH of forMgAC. the harvesting of Chlorella sp. KR-1, Scenedesmus be elucidated in future works. The optimal pH for the harvesting of Chlorella sp. KR-1, Scenedesmus obliquus, and mixed microalgae was 4.0, beyond which level, the microalgae-harvesting efficiencies obliquus, and mixed microalgaemechanism, was 4.0, beyond which level, the microalgae-harvesting efficiencies were reduced. The harvesting as confirmed by zeta potential measurements, was the were reduced. The harvesting mechanism, confirmed byinzeta measurements, was the electrostatic interaction between MgAC andasthe Fe3 O4 NPs thepotential hybrid samples with microalgae. electrostatic interaction between MgAC and the Fe 3O4 NPs in the hybrid samples with microalgae. The zeta potentials of the MgAC-Fe3 O4 hybrid composites were reduced as pH was increased, The zeta potentials of the MgAC-Fe 3O4 hybrid composites were reduced as pH was increased, and and thereby, the harvesting efficiencies were diminished. The combination of MgAC with Fe3 O4 thereby, the harvesting efficiencies were diminished. The combination of MgAC with Fe3O 4 NPs NPs improved the harvesting efficiencies but still not completely overcome the limitation of Fe 3 O4 NPs improved the harvesting efficiencies but still not completely overcome the limitation of Fe 3O4 NPs at high pH condition. The MgAC-Fe3O4 hybrid composites require improvement in order to overcome

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at high pH condition. The MgAC-Fe3 O4 hybrid composites require improvement in order to overcome the problem of reduced harvesting efficiency at high pH before testing their magnetic properties for reusability in future studies. In conclusion, meanwhile the presence of MgAC in MgAC-Fe3 O4 hybrid nanocomposite could improve the microalgae harvesting of single and mixed microalgae, this strategy still not completely overcome the limitation of Fe3 O4 NPs at high pH values. We also indicated that between Chlorella sp. KR-1 and Scenedesmus obliquus, the harvesting of Fe3 O4 NPs and MgAC-Fe3 O4 hybrid nanocomposite towards Scenedesmus obliquus seems less affected by pH value of environment. 4. Materials and Methods 4.1. Materials (3-aminopropyl)triethoxysilane (APTES; ≥98%, 221.37 g/mol), iron(II) chloride tetrahydrate (FeCl2 •4H2 O, 98%), and iron(III) chloride hexahydrate (FeCl3 •6H2 O, 97%) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Magnesium chloride hexahydrate (MgCl2 •6H2 O; 98%) was obtained from Junsei Chemical (Tokyo, Japan). Ethanol (18 L, 95%) was purchased from Samchun Pure Chemicals (Pyungtack, Korea). NaOH (pellet, >97%) was obtained from Daejung Chemicals & Metals (Siheung, Korea). Distilled-deionized water (DI; resistance: >18 mΩ was employed in all of the experiments. 4.2. Preparation of Magnesium Aminoclay (MgAC) A total of 1.68 g of MgCl2 •6H2 O was diluted in 40 mL of ethanol (95%), mixed with 2.6 mL APTES, and continuously stirred for 8 h. The resulting white-solid was centrifuged and washed three times by ethanol and dried at 60 ◦ C, after which it was ground into powder [26]. 4.3. Preparation of Aminoclay-Fe3 O4 Hybrid Composites [1.9 g FeCl3 •6H2 O ⊕ 0.7 g FeCl2 •4H2 O] was dissolved in 40 mL of DI water and continuously stirred for 30 min before 10 mL of NaOH 10 M was added; stirring thereafter was continued overnight (12 h). MgAC in amounts of 0.80, 0.40, 0.08, 0.04, and 0 g was dissolved separately in 40 mL of DI water and then mixed with 40 mL of [1.9 g FeCl3 •6H2 O ⊕ 0.7 g FeCl2 •4H2 O] solution, again with stirring overnight (12 h). The mixture was then centrifuged, washed with water (3 × 50 mL), dried at 60 ◦ C, and ground to form MgAC-Fe3 O4 hybrid composites (Table 2). 4.4. Characterization of MgAC-Fe3 O4 Hybrid Composites The crystallography of the MgAC-Fe3 O4 hybrid composites was investigated in their X-ray diffraction (XRD) patterns using a Rigaku D/max-2500 (18 kW, Tokyo, Japan) incorporating a θ/θ goniometer equipped with a 40 kV and 30 mA CuKα radiation generator. The morphological of the MgAC-Fe3 O4 hybrid composites were examined by transmission electron microscopy (TEM; JEM-2100F, JEOL LTD, Tokyo, Japan). Elemental compositions of as-prepared samples were analyzed by X-ray fluorescence spectrometry (XRF; MiniPal 2, PANanalytical, Almelo, Netherlands). The zeta potentials of the MgAC-Fe3 O4 hybrid samples and microalgae were measured by dynamic laser-light scattering (DLS; Malvern Zetasizer NanoZS, Malvern Instruments, Malvern, UK). 4.5. Microalgae Cultivation Chlorella sp. KR-1 was cultured in nutrient media (constituents: KNO3 , 3 mM; KH2 PO4 , 5.44 mM; NaHPO4 , 1.83 mM; MgSO4 •7H2 O, 0.20 mM; CaCl2 , 0.12 mM; FeNaEDTA, 0.03 mM; ZnSO4 •7H2 O, 0.01 mM; MnCl2 •4H2 O, 0.07 mM; CuSO4 , 0.07 mM; Al2 (SO4 )3 •18H2 O, 0.01 mM). A Pyrex bubble column reactor (working volume: 6 L) equipped with 12 fluorescent lamps in the front and right/left sides (light intensity: 80 µmol/m2 /s) was used to cultivate the Chlorella sp. KR-1, which were

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maintained in a constant-temperature room (30 ◦ C). A 10% (v/v) volume of CO2 in air at a rate of 0.75 L/min was used to supply the reactor [15,27]. The oil content of Chlorella sp. KR-1 has been reported to be around 36.5–41% [28]. The average dry-biomass concentration of Chlorella sp. KR-1 in the present growth culture was 1.75 g/L. Scenedesmus obliquus was cultured in another nutrient medium (constituents: Ca(NO3 )2 •4H2 O, 4.23 mM; KH2 PO4 ; 1.91 mM; MgSO4 •7H2 O, 2.48 mM; KCl, 3.35 mM; Fe2 SO4 •7H2 O, 0.07 mM; EDTA•2Na, 0.53 mM; H3 BO3 , 0.04 mM; ZnCl2 , 0.0008 mM; MnCl2 •4H2 O, 0.009 mM; (NH4 )6 Mo7 O24 •4H2 O, 0.000015 mM; and CuSO4 •5H2 O, 0.0003 mM). The light intensity was about 60 µmol/m2 /s, and 20% (v/v) CO2 was fed at rate of 4 L/min [29]. The reactor was maintained in a constant-temperature room (30 ◦ C). The average dry-biomass concentration of Scenedesmus obliquus in the growth media was 2.0 g/L. In order to prepare mix microalgae, Chlorella sp. KR-1 and Scenedesmus obliquus were mixed in a 1:1 ratio by volume. 4.6. Microalgae-Harvesting Procedure The obtained MgAC-Fe3 O4 hybrid composites were dispersed in 1 mL of DI water (loading concentrations: #1: 4.72 g/L, #2: 4.43 g/L, #3: 4.32 g/L, #4: 4.19 g/L, #5: 4.42 g/L) and injected into 12 mL test tubes containing 9 mL of microalgae at a concentration of 1.7–2.0 g/L. The pH of the solutions was adjusted within the range of 2–12. The mixture was slowly mixed by hand (about 1 min) and stirred (800 rpm) for 10 min [15]. UV-Vis spectroscopy (Optizen 2120UV, Mecasys Co., Daejeon, Korea) was used to measure the optical density (OD) of the supernatant of each of the samples (the sampling height was 2/3 of the tube from the bottom). The harvesting efficiency (%) was evaluated by the equation [12]:  Efficiency (%) =

 ODf 1− × 100 ODi

where ODf and ODi were the final and initial ODs of each sample. The experiments were each repeated three times. The proportions of Chlorella sp. KR-1 and Scenedesmus obliquus in mixed microalgae were analyzed by a coulter counter (MultisizerTM 4, Beckman Coulter, Fullerton, CA, USA) due to the difference of cell sizes. Chlorella sp. KR-1 have cells size range of 2–4 µm while Scenedesmus obliquus have cells size range of 6–12 µm (Figure S1). Supplementary Materials: The following are available online at http://www.mdpi.com/1996-1073/11/6/1359/ s1, Figure S1. Number size distribution of mixed microalgae by Beckman Coulter. Author Contributions: Y.-C.L. and Y.-K.O. conceived and designed the experiments; B.K. and W.F. performed the experiments; V.K.H.B. and B.K. analyzed the data; V.K.H.B. and Y.-C.L. principally wrote the paper; S.G.J., Y.-C.L., and Y.-K.O. commented on and improved the manuscript. Acknowledgments: This work was supported by the Advanced Biomass R&D Center (ABC) of the Global Frontier Project funded by the Ministry of Science and ICT, Republic of Korea (ABC-2012M3A6A205388) and also by the Basic Science Research Program through the National Research Foundation of Korea funded by the Ministry of Education (NRF-2017R1D1A1A09000642). Conflicts of Interest: The authors declare no conflict of interest.

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