Smart and Reusable Biopolymer Nanocomposite ... - ACS Publications

Research Article pubs.acs.org/journal/ascecg

Smart and Reusable Biopolymer Nanocomposite for Simultaneous Microalgal Biomass Harvesting and Disruption: Integrated Downstream Processing for a Sustainable Biorefinery Ramalingam Dineshkumar,† Amrita Paul,‡ Moumita Gangopadhyay,‡ N. D. Pradeep Singh,‡ and Ramkrishna Sen*,† †

Department of Biotechnology and ‡Department of Chemistry, Indian Institute of Technology Kharagpur, Kharagpur−721302, West Bengal, India S Supporting Information *

ABSTRACT: One of the major technological challenges in developing a microalgal biorefinery is to minimize the fossil energy inputs, particularly in two important downstream unit operations, cell harvesting and disruption. Hence, this study involved synthesizing and applying biopolymer nanocomposite to achieve concomitant biomass harvesting, cell disruption, and nanocomposite recovery by exploiting its cationic, photocatalytic, and magnetic properties respectively, in an integrated and optimized process chain. Accordingly, dual-functionalized chitosan-TiO2 conjugated particles (CTC) and trifunctionalized magnetic nanocomposites (MNCs) namely, chitosan coated core−shell structures of Fe3O4−TiO2, were prepared and characterized. The harvesting efficiency of >98% was achieved at the optimal dosages of chitosan, CTC and MNCs of 0.11, 0.09, and 0.07 g g−1 Chlorella minutissima biomass, respectively. TiO2 driven photocatalysis could effectively disrupt harvested wet-biomass, when exposed to UV irradiation in the presence of either CTC or MNCs for 2 h, and when subjected to visible light with only MNCs for 3 h. Photocatalytic cell disruption helped recover 96−97% of the intracellular lutein and lipid, when compared to ultrasonication as control. Subsequently, the MNCs were separated from residual biomass by physicochemical treatment, resulting in over 98% detachment efficiency for reuse in the downstream process chain. To the best of our knowledge, this integrated green process is novel, in terms of meeting a contemporary technological challenge in downstream processing of microalgal biomass, and this research outcome may inspire the development of sustainable microalgal biorefinery for the production of lutein and biodiesel. KEYWORDS: Microalgal biorefinery, Magnetic nanocomposites, Biomass separation, Photocatalysis, Cell disruption, Biodiesel, Carotenoid lutein

■

INTRODUCTION With the increasing global energy demands and its associated impact on climate have motivated the scientific communities to provide innovative solutions for the development of green, renewable, and economically feasible biofuels. In recent years, microalgae have emerged as a sustainable feedstock for the production of biofuels and high-value carotenoids, due to their inherent abilities including high growth rate, CO2 sequestration, and effective land utilization, as compared to terrestrial energy crops.1−4 However, the production of biofuels and carotenoids from microalgal biomass is still not economically viable, owing to the significant fossil energy inputs required for downstream processes.5,6 For instance, the cost of harvesting microalgal biomass is estimated to be 20−30% of the total production cost.1,7 Moreover, intense energy inputs are required for further © 2016 American Chemical Society

downstream processing of biomass involving cell disruption, product extraction and purification.8,9 Hence, the development of an integrated process which concomitantly addresses subsequent downstream step is expected to improve the costeffectiveness of microalgal products. Among the various harvesting methods, flocculation using organic biopolymers has been considered as sustainable alternative to chemical flocculants and energy intensive processes like centrifugation and filtration.9,10 The cationic charged chitosan biopolymer that is commercially produced from chitin has emerged as a promising feedstock option for Received: September 12, 2016 Revised: November 22, 2016 Published: November 23, 2016 852

DOI: 10.1021/acssuschemeng.6b02189 ACS Sustainable Chem. Eng. 2017, 5, 852−861

Research Article

ACS Sustainable Chemistry & Engineering

washed with excess of water and dried under vacuum for overnight. The trifunctionalized MNCs was synthesized in two steps (Scheme 1):

harvesting microalgal biomass, due to the fact that it is nontoxic, biodegradable, and biocompatible.11,12 Recently, cationic polymers including chitosan, polyethylenimine, and polyacrylamide have been used in conjugation with magnetite particles for achieving dual purpose like microalgal biomass separation and subsequent reuse of flocculant.13−15 This could effectively reduce the cost of biopolymer involved in harvesting step, as the polymer conjugated with magnetite can be recovered and reused for many cycles.15,16 TiO2 nanophotocatalyst has been studied for various environmental applications, because of its inherent properties such as chemical inertness, cost-effectiveness, high photocatalytic efficiency, and high stability.17,18 Recently, Lee et al.19 observed that aminoclay-conjugated TiO2 nanoparticles could disrupt the microalgal cells, after UV-irradiation at 365 nm for 3 h. However, the use of chemical flocculant and UV-irradiation for harvesting and cell disruption may not be suitable at a large scale. Moreover, it is essential to recycle the TiO2 particles so as to ensure the viability and sustainability of the downstream process. In this context, we propose to design and synthesize multifunctionalized biopolymer nanocomposites that possess cationic, photocatalytic, and magnetic properties to achieve simultaneous biomass harvesting and disruption with nanocomposite recovery for reuse, respectively. In addition, the photocatalytic biomass disruption may be carried out at visible light regime, as the absorption band of TiO2 will also exist in visible light region, upon doping TiO2 with Fe3O4.17 Hence, this approach is expected to significantly improve the costeffectiveness and sustainability of microalgal products like biodiesel and high-value carotenoid lutein. Thus, the present study focuses on integrating downstream processing of microalgal biomass by implementing the following strategies: (1) synthesis and characterization of dual-functionalized chitosan−TiO2-conjugated (CTC) nanoparticles, and trifunctionalized magnetic nanocomposites (MNCs) namely, chitosan coated core−shell structures of Fe3O4−TiO2 nanoparticles, (2) comparison of microalgal biomass harvesting efficiency of CTC and MNCs, (3) evaluation of photocatalytic efficiency and its influence on extraction of commercially important microalgal products, lipid and lutein, and (4) assessment of multireusability of MNCs. The novelty of this study lies in the use of green and reusable nanocomposites for concomitant achievement of three goals like biomass separation, cell disruption by visible-light-driven photocatalysis, and nanocomposite recovery for recycling, in an integrated and optimized downstream process chain.

■

Scheme 1. Preparation of Chitosan Coated Core−Shell Structures of Fe3O4−TiO2

first, Fe3O4−TiO2 core−shell particles were prepared by coating TiO2 layer onto the surface of Fe3O4 nanoparticles (1 g) by hydrolyzing 3 mL of tetrabutyl orthotitanate (TBOT) in ethanol/acetonitrile solvent mixture in the presence of ammonia.18 Subsequently, the prepared Fe3O4−TiO2 was conjugated with chitosan through Ti−O−CH2 linkage (Scheme 1) by adding 3 g of chitosan which was dispersed in 1% (v/v) acetic acid and sonicated for 30 min. The precipitate was then obtained by adjusting the pH to 10 and heated at 80 °C for 5 h, and the nanocomposites were collected using magnet and dried under vacuum. To confirm the successful synthesis of CTC and MNCs, their crystal structure and surface functionalization were determined by Xray diffraction (XRD) analysis (PW-3040/60 PANalytical high resolution XRD) and Fourier transform infrared (FTIR) spectra (PerkinElmer RXI spectrometer), respectively. Microalgae Harvesting. The microalgal biomass (3.02 ± 0.05 g L−1) that was grown under the optimal conditions (section Microalgae and Culture Conditions) was used in this harvesting experiment. The stock solutions of chitosan, CTC and MNCs were prepared at a concentration of 1% (w/v) using 1% acetic acid solution. Chitosan and synthesized nanocomposites (CTC and MNCs) at different concentrations were added to a 250 mL microalgal suspension (Jar test method). After stirring the cells for 1 min at 120 rpm, the beaker was undisturbed for settling the biomass. In case of magnetic separation, the beaker was placed near NdFeB permanent magnet (50 mm L × 25 mm W × 100 mm H) with the magnetic induction of 4000 G. Subsequently, the supernatant was collected from each experiment and OD750 nm was determined23 to calculate harvesting efficiency (eq 1). Further, the zeta potential values were measured using a Zetasizer (Malvern, UK). All experiments were carried out in triplicates under the following conditions: microalgal concentration 3.02 ± 0.05 g L−1; pH 7 ± 0.2; temperature 28 ± 2 °C.

MATERIALS AND METHODS

Microalgae and Culture Conditions. The oleaginous and luteinrich microalga Chlorella minutissima (MCC-27) was grown using optimized Bold’s Basal Medium20 in customized 2 L airlift photobioreactors under the following conditions: pH 7−8; temperature 30 ± 2 °C; light intensity 250 μmol m−2 s−1; CO2 3.5% (v/v), and air flow rate 0.425 vvm. Under optimal conditions, C. minutissima was found to accumulate significant amount of lutein and lipid for healthcare and biodiesel applications, respectively.21 Preparation of CTC and MNCs. The synthesis of TiO2 and Fe3O4 nanoparticles are given in the Supporting Information. For CTC preparation, TiO2 nanoparticles (1 g) were initially dispersed in 100 mL of 1% (v/v) acetic acid, where TiO2 changed into Ti4+ ions. To this, 1 g of chitosan was added and sonicated for 30 min and was stirred continuously until the solution became clear. The pH was maintained at 10 by adding 1 M NaOH dropwise and the precipitate thus formed was heated at 80 °C for 5 h.22 Finally, it was filtered,

harvesting efficiency = 1 −

ODf × 100 ODi

(1)

where ODi and ODf represents the optical densities of culture before and after cell separation, respectively. Photocatalytic Cell Disruption. The CTC and MNCs harvested biomass slurry were then irradiated at ≥320 nm using 125 W medium 853


Research Article


Scheme 2. Development of Integrated Downstream Processing for a Sustainable Microalgal Biorefinery Model

pressure Hg vapor lamp (light intensity, 20 mW cm−2) for different time intervals. Furthermore, the MNC harvested algal slurry was also exposed at visible light (400−700 nm) using cool-white fluorescent lamps (light intensity, 250 μmol m−2 s−1). In this study, a fixed amount of UV/visible irradiation was supplied as per the accessibility of the light setup used, but the time required for complete disruption of wet biomass was optimized for both CTC and MNCs. The efficiency of photocatalytic cell disruption was analyzed in terms of intracellular product recovery. After photocatalysis, the morphology of microalgal cells was examined using scanning electron microscope (Carl ZEISS SMT, Germany). Moreover, Nile Red staining of cells was carried out to examine lipid droplets, and the morphology of intact and disrupted cells was also studied using fluorescence microscopy (Olympus IX51, Japan). The photocatalytically disrupted biomass was then subjected for extracting lipid and lutein using hexane and ethanol solvent system.21 A cell disruption method involving sonication (Q125, Q SONICA, USA) was carried out to calculate the percentage product recovery achieved by photocatalytic cell disruption. In our previous study, we optimized the sonication protocol (10 min in pulse mode of 45 s on and 15 s off at 50% amplitude) for cell disruption, and it resulted in 6.44 ± 0.07 mg g−1 of lutein and 212.23 ± 2.5 mg g−1 of lipid. In addition, the total intracellular lipid was quantified following the spectrofluorometric methods of Chen et al.24 and De Bhowmick et al.,25 and it was calculated to be 239.6 ± 4.8 mg g−1. Although sonication based disruption method did not give 100% lipid extraction efficiency, it performed comparatively better as compared to other cell disruption techniques such as bead beater and homogenizer (data not shown). Hence, in this study, sonication was treated as the control method for calculating product recovery. For all experiments, the carotenoid lutein was quantified using reverse phase HPLC (Agilent, USA)26 and the total lipid content was measured gravimetrically. The percent product (lutein or lipid) recovery was calculated using eq 2

percent product recovery product obtained upon photocatalysis = × 100 product obtained upon control cell disruption

(2)

Assessment of Multireusability of MNCs and Culture Medium. The detachment of MNCs from microalgal cells was performed by adopting a combined physicochemical treatment.6,27 The residual biomass slurry containing 0.2 g of MNCs was treated with 100 mL of 0.1 M HCl and sonicated for short time (60 s). The slurry was then agitated at 120 rpm for different time intervals at ambient temperature (30 °C). Afterward, the MNCs were precipitated by adjusting the pH to 7−7.5 using ammonia. The detached nanocomposites were then collected using an external magnet and dried for repeated use. The detachment efficiency was calculated as the ratio of amount of MNCs recovered (desorbed) to amount of MNCs added (adsorbed) to the microalgal culture. The spent acid solution was then recycled for trans-esterification of microalgal lipid into fatty acid methyl esters (FAME, Biodiesel) in the presence of methanol at 70 °C for 5 h and extracted into hexane phase. The composition of FAME was analyzed using gas chromatography (Thermo Fisher Scientific, Chemito Ceres 800 plus) as described earlier.21,28 The reuse of culture medium after magnetophoretic separation was also investigated by culturing C. minutissima with similar process conditions. The recycled medium was replenished with fresh nutrients as per the medium composition (section Microalgae and Culture Conditions). Subsequently, the biomass concentration was measured gravimetrically at the time of harvest. All experiments were performed in triplicates and expressed as mean with standard deviation. Scheme 2 illustrates the integrated downstream process flow for concomitant biomass harvesting and disruption with consequent reuse of MNCs. 854


Research Article


■

RESULTS AND DISCUSSION Characterization of CTC and MNCs. Size and Morphology. The morphology and size of the biopolymer nanocomposites were examined using transmission electron microscopy (TEM) FEI Tecnai G220S-Twin (FEI, USA) at 200 kV. The average diameter of TiO2 and Fe3O4 were determined to be 130−140 and 8−10 nm, respectively (Figure S1 in the Supporting Information). All the synthesized particles were found to be relatively uniform in size and spherical in shape. The average particle size of CTC and MNCs were observed to be 90−100 nm and 10−15 nm, respectively (Figure 1). The structure of CTC shows that the TiO2

Figure 1. TEM images of (A) CTC and (B) MNCs.

nanoparticles are almost uniformly bound to the surface of chitosan. This is evident from the TEM image of CTC that the dark and bright areas indicate crystalline TiO2 particles and amorphous chitosan polymer, respectively (Figure 1A), as postulated by Haldorai and Shim.22 This may be attributed to high electron density of TiO2 nanoparticles. The TEM image of MNCs indicates that the inner dark region corresponds to magnetite and it is covered by TiO2 and chitosan with almost uniform size (Figure 1B). Crystal Phases. The crystal phases of chitosan, TiO2, Fe3O4, CTC, and MNC are shown in Figure 2. The XRD profile of chitosan showed a broad peak at 2θ = 20.1°, indicating that the polymer is amorphous. The XRD pattern of TiO2 is characterized by six primary peaks located at the 2θ values of 25.4°, 37.8°, 48.0°, 53.9°, and 55.1°, which correspond to the (101), (004), (200), (105), and (211) lattice planes of anatase phase of TiO2 (JCPDS 21-1272), respectively. The crystal phase of Fe3O4 exhibited peaks positioned at the 2θ values of 30.0°, 35.3°, 42.9°, 53.5°, 57.0°, and 62.4°, which were related to their corresponding indices (220), (311), (400), (422), (511), and (440), respectively, indicating the cubic phase of Fe3O4 (JCPDS 19-0629). The XRD of CTC confirms that the TiO2 nanoparticles were attached with chitosan. Similarly, the diffraction pattern of MNC exhibits the characteristic lattice planes of both Fe3O4 and TiO2 along with a characteristic broad peak of chitosan (2θ = 20.1°). The weaker diffraction profiles of CTC and MNCs show that the respective nanoparticles were suitably conjugated with the amorphous chitosan, without affecting the crystal structure of both Fe3O4 and TiO2. Characterization of Surface Functionalization. Surface functionalization of chitosan, TiO2, Fe3O4, CTC, and MNC were analyzed by FTIR spectra (Figure 3). The chitosan polymer was characterized by the following absorption bands: peaks at 3435 and 1653 cm−1 corresponds to the N−H stretching and bending vibrations respectively of the (N−H) backbone of the polymer, the peak at 1424 cm−1 is attributed to C−O stretching vibration of primary alcoholic group, and the peaks at 2874 and 2940 cm−1 are corresponding C−H

Figure 2. XRD pattern of chitosan, TiO2, Fe3O4, CTC, and MNCs.

stretching and bending vibrations. In TiO2 nanoparticle, a broad peak at 500−600 cm−1 corresponds to Ti−O stretching vibration. The peak at 570 cm−1 corresponds to Fe−O stretching vibration in the FTIR spectrum of Fe3O4 nanoparticles. In the spectrum of CTC, a broad band at 500−600 cm−1and the peak at 1650 cm−1 relate to respective Ti−O and N−H stretching vibrations, indicating that the TiO2 nanoparticles are conjugated with chitosan. The FTIR spectrum of MNC displays the characteristic peaks of Ti−O, Fe−O, and N−H stretching vibrations. As compared to the spectra of chitosan, TiO2 and Fe3O4, the peaks relating to OH, N−H, Fe−O, and Ti−O groups in the spectrum of MNC were shifted. This indicates the promising interaction between Fe3O4/TiO2 particles and chitosan. Magnetic Behavior. The magnetic property of MNCs and Fe3O4 nanoparticles was measured using a SQUID VSM DC magnetometer (Quantum Design, USA) with an applied field of −20 000 to 20 000 Oe. The saturation magnetization values of Fe3O4 and magnetic flocculant (MNC) were 50.88 and 28.5 emu g −1 , respectively (Figure S2 in the Supporting Information). The decrease in magnetization of MNC is primarily due to decreased proportion of Fe3O4 in the prepared magnetic flocculant after coated with TiO2 and chitosan. However, the synthesized MNCs still exhibit significant magnetization with superparamagnetic characteristics, which indicates the ease of MNCs recovery from the slurry or solution by using an external magnet. Microalgal Biomass Harvesting. As a preliminary study, we examined the self-flocculating ability of C. minutissima at the time of harvest. We found that the test microalga did not 855


Research Article


Figure 3. FTIR spectra of chitosan, TiO2, Fe3O4, CTC, and MNCs.

autoflocculate even after 12 h of settling time, as it is evident from zeta potential value of −14.1 mV. This implies that a suitable harvesting technique needs to be developed for effective biomass separation. Hence, we investigated the use of dual and trifunctionalized biopolymer nanocomposites for biomass harvesting and disruption. Figure 4A shows the harvesting efficiency of synthesized nanocomposites along with chitosan at different dosages. The biomass harvesting efficiency improved significantly with increase in concentration of the tested flocculants. The optimal dosages of chitosan, CTC and MNCs required to harvest per unit gram biomass were found to be 0.11, 0.09, and 0.07 g g−1, respectively (Figure 4A). The dry biomass concentration obtained before and after harvesting with optimal dosages was calculated to be 3.02 ± 0.05 and 2.97−2.99 ± 0.03 g L−1 respectively (Figure S4 in the Supporting Information), which corresponds to 98.5−99.1% of biomass harvesting. The time taken for almost complete flocculation using chitosan and CTC nanoparticle was 5 and 2 min, respectively. In case of MNCs, the time required for attachment of almost all microalgae cells to the external magnet (NdFeB) was within 2 min. A slight decline in harvesting efficiency was observed, when the concentration of chitosan and nanocomposites increased beyond the optimal range (Figure 4A). This is probably due to the electrostatic repulsion of amino groups present on chitosan that results in destabilization of microalgal flocs formation.12 The biomass separation by flocculation and magnetophoretic methods are shown in Figure 4B. The optimal time and dosage requirements of MNCs (0.07 g g−1 biomass) and CTC (0.09 g g−1 biomass) to achieve

Figure 4. (A) Plot of harvesting efficiency as a function of mass ratio of flocculant to biomass. (B) Flocculation and magnetophoretic biomass separation using CTC and MNCs, respectively. Operating conditions: volume 150 mL; microalgae 3.02 ± 0.05 g L−1; concentration and flocculation time of CTC 0.09 g g−1 and 2 min; concentration and separation time of MNCs 0.07 g g−1 and 1.5 min. (C) Change in zeta potential with increase in mass ratio of flocculant to biomass. Data represent average of three independent runs ± standard deviation.

maximum harvesting efficiency were found to be lower than chitosan (0.11 g g−1 biomass). This is evident from the higher zeta potential values of the synthesized CTC (43.6 ± 1.2 mV) and MNCs (54.7 ± 2.5 mv), as compared to chitosan (9.1 ± 0.4 mV). The obtained higher zeta potential illustrates the presence of increased surface charge on nanocomposites, which is adequate for complete flocculation of microalgal cells (zeta potential, −14.1 ± 0.7 mV), without adjusting the culture pH (7 ± 0.2).7 This indicates that the primary mechanism of cell 856


Research Article


Table 1. Comparison of different magnetic nanoparticles and its dosage requirements reported in the literature for achieving maximum microalgal biomass separation microalgal species

initial algal concentration (g L−1)

type of magnetic nanoparticles

optimal dose (g/g biomass)

harvesting efficiency (%)

ref

C. vulgaris C. ellipsoidea Chlorella sp. S. dimorphus Chlorella sp. C. minutissima

1.3 0.7 1.6 0.8 1.5 3.02

silica coated Fe3O4 CPAM−Fe3O4 APTES−Fe3O4@SiO2 PEI-conjugated Fe3O4 CTAB- decorated Fe3O4 chitosan−TiO2 coated Fe3O4

0.22 0.046 1.6 0.075 0.46 0.07

>90% 96% >98% 85% 96.6% >98%

29 14 7 6 9 this study

separation is due to electrostatic attraction, which is corroborated by correlating the zeta potential values with flocculant concentration (Figure 4C). The zeta potential was found to be increased from −14.1 mV (before harvesting) to −2.17 mV, −0.09 mV and −0.32 mV for the corresponding optimal dosages of chitosan, CTC and MNCs, respectively (Figure 4C). This elucidates the charge neutralization behavior or decreased electrostatic repulsion between cells that subsequently results in complete biomass separation. As compared to most of the relevant studies reported in literature, the dosage requirements of multifunctionalized nanoparticles used in this study to achieve similar harvesting efficiency was found to be either lower or almost similar (Table 1). Nevertheless, it has been reported that the optimal concentrations of magnetic particles depends on the microalgal species and type of magnetic flocculants used, and it was found to be varied from 0.015 to 12.8 g g−1.7,16 Photocatalytic cell disruption. The primary goal in the present study is to simultaneously accomplish biomass separation and subsequent downstream step like cell disruption by utilizing the photocatalytic activity of biocompatible TiO2 nanoparticles. Accordingly, the nanocomposites which comprise photocatalyst TiO2 was successfully prepared in this study and tested for its efficacy toward cell disruption. The solid-state UV−visible absorption spectra of CTC and MNCs were determined using Shimadzu UV-2450 spectrophotometer and it is shown in Figure S5 in the Supporting Information. The CTC nanoparticle showed a maximum absorption peak at 325 nm. On the other hand, MNC showed two absorption peaks at 313 and 490 nm, indicating that the polymer chitosan and TiO2 nanoparticles were successfully incorporated onto Fe3O4 nanoparticles. This suggests that the prepared CTC and MNCs could be effectively used for photocatalytic cell disruption using UV irradiation >320 nm. As the MNCs prepared in this study showed a broad absorption peak at 400−550 nm, photocatalysis was also carried out at visible region (400−700 nm) for only MNCs. Figure 5 shows the morphology of intact, mechanically disrupted (ultrasonication), and photocatalytically disrupted cells. Upon photocatalysis, the cell wall of microalgae was observed to be disrupted or destabilized, as compared to intact and mechanically disrupted cells. The cell disruption was found to be more pronounced when increasing the UV irradiation time from 1 to 2 h for both CTC and MNCs (Figure 5C−E) and on visible light exposure to 3 h for MNCs (Figure 5F). Upon irradiation, TiO2 photocatalyst generates free radicals,17 which subsequently act as potential reactive species responsible for cell wall disruption. This is corroborated with the study of Lee et al.,19 which reported that around 95% of Chlorella sp. KR-1 cells were found to be disrupted by TiO2 photocatalysis under UV irradiation for 3 h. Further, it was postulated that the hydroxyl radicals that were generated by TiO2 photoexcitation

Figure 5. Scanning electron microscopic images of C. minutissima cells under (A) intact, (B) ultrasonicated, (C) 1 h of UV irradiation (>320 nm) with CTC (0.09 g g−1), (D) 2 h of UV irradiation (>320 nm) with CTC (0.09 g g−1), (E) 2 h of UV irradiation (>320 nm) with MNCs (0.07 g g−1), and (F) 3 h of visible irradiation (400−700 nm) with MNCs (0.07 g g−1). Magnification 5.00 KX; scale 3 μm.

are responsible for nonselective and strong attackers of cell ́ surfaces. In another study, Rodriguez-Gonzá lez et al.30 reported the photocatalytic performance of silver TiO2 nanocomposites on inducing fatal damages to marine harmful algae. The photocatalysis driven cell disruption was also corroborated upon staining the intracellular lipid droplets using Nile Red dye. The optical and corresponding fluorescence images of Nile Red stained C. minutissima cells under intact, mechanical and photocatalytic disrupted conditions are illustrated in Figure 6. It should be noted that the dye Nile Red binds to internal oil droplets, which results in emission of yellow color. The fluorescence images of mechanically and photocatalytically disrupted cells confirms the presence of oil droplets either at the cell surface or outside the cell, as compared to the intact cells, wherein oil droplets are located inside. This shows the successful cell wall disruption by photocatalysis. The efficiency of photocatalytic cell disruption was analyzed in terms of intracellular product recovery (Figure 7). Upon increasing the UV irradiation time from 1 to 2 h for both CTC and MNCs, the intracellular product recovery was found to be significantly improved from 78−80% of lutein and 70−72% of lipid to 94−95% of lutein and 94−96% of lipid (Figure 7). 857


Research Article


extraction. The recovery of lutein and lipid obtained after solvent extraction (without cell disruption) was 8.1% and 9.5% respectively, which is comparatively very low with respect to sonication as control disruption technique (Figure 7). This confirms that cell disruption was predominantly accomplished by photocatalysis, which consequently results in maximum product recovery upon adopting relevant solvent extraction procedures. Recently, the use of chemical surfactant cetyltrimethylammonium bromide9,31 has been reported to achieve simultaneous biomass harvesting and disruption. However, our study stands out in its approach toward the use of a biopolymer, chitosan that was conjugated with biocompatible nanoparticles Fe3O4−TiO2 for concomitant biomass separation and disruption with consequent reuse of the nanocomposites. Evaluation of Reusability of MNCs and Culture Medium. The main aim of incorporating Fe3O4 magnetic nanoparticles in this study is to reuse the cationic and photocatalytic properties of nanocomposite. As magnetite (Fe3O4) nanoparticles are biocompatible, the reusability of MNCs was investigated after photocatalysis and extraction of intracellular products. Generally, the detachment of magnetic nanoparticles from microalgal slurry is carried out by establishing electrostatic repulsion between biomass slurry and nanoparticles with the use of acid or base, and adopting physical treatments including sonication or heating at 40 °C.32,33 Nevertheless, it has been reported that the use of strong acid or base solutions for detachment of magnetic nanoparticles may not be suitable in large scale.6 Hence, we attempted to separate MNCs from residual microalgal slurry by using dilute acid (0.1 M HCl) with short pulse of sonication (60 s), and the time required for attaining complete detachment efficiency was optimized. As shown in Figure 8A, the detachment efficiency of MNCs from algal biomass slurry significantly improved from 45.8% to 98.1%, when increasing the mixing time from 1 to 4 h. It was observed that the longer sonication exposure (>60 s) did not further decrease the mixing time required to achieve optimal detachment efficiency (data not shown). We also found that an experiment involving only dilute-acid treatment (0.1 M HCl) without sonication could reach maximum detachment efficiency (95.4%) at 7 h of mixing time (Figure 8A). This shows that a short pulse of sonication could adequately boost the disengagement of MNCs from cells during dilute-acid treatment, and consequently reduces mixing time so as to accomplish complete detachment. This is corroborated with the study of Prochazkova et al.,33 which reported that the additional physical treatment could considerably enhance detachment. Further, the recovered polymer nanocomposite was tested for its reusability toward biomass separation and photocatalytic disruption using visible light. On recycling MNCs for five consecutive batches, the harvesting efficiency of 90−97%, lutein recovery of 89−94%, and lipid recovery of 90−95% were achieved (Figure 8B). This implies that the MNCs could be recovered without significantly affecting the cationic, photocatalytic and magnetic properties for at least five cycles of unit operations. Meanwhile, the spent dilute-acid solution obtained after MNCs detachment was recycled as a catalyst for transesterification of microalgal lipid to FAME. It should be noted that the spent acid solution was adjusted to pH 2−3 before reusing it for lipid transesterification,34 as the acid dilution could hamper transesterification reaction. It was observed that there were no statistically significant differences in the FAME composition obtained between ultrasonicated and

Figure 6. Optical and respective fluorescence images of Nile Red stained cells of C. minutissima under (A) intact, (B) ultrasonicated, and (C) photocatalytically disrupted using MNCs (0.07 g g−1) under 3 h of visible irradiation (400−700 nm). Magnification 100X; scale 5 μm.

Figure 7. Percentage recovery of lutein and lipid, upon CTC (0.09 g g−1) and MNC (0.07 g g−1) driven photocatalysis using UV−visible irradiation at different time intervals vis-à-vis nondisrupted conditions. Data represent average of three independent runs ± standard deviation.

Almost complete recoveries of intracellular products were achieved at 2 h of UV exposure for both the nanocomposites. In case of visible light driven photocatalysis by MNCs, the maximum recovery of lutein (96.8%) and lipid (97.1%) was obtained at 3 h. One of the main advantages of using MNCs is the possible utilization of natural sunlight as a cost−effective and renewable source for photocatalytic wet biomass disruption. Moreover, the scalability of microalgal biomass harvesting and disruption under natural sunlight conditions seems to be viable and it will be investigated in our future study. As the extraction solvent ethanol may also slightly destabilize (permeate) cell walls, an experiment involving only solvent extraction (without cell disruption) was also performed. This was investigated to substantiate the effect of photocatalysis on cell disruption and consequent product recovery by solvent 858


Research Article


separation could be reused without causing adverse effects on algae. Techno-economic Implications. A preliminary cost analysis was performed based on operational energy requirements for biomass harvesting, drying and disruption at laboratory scale (Table S3 in the Supporting Information). As compared to conventional unit operations, the total energy required to harvest and disrupt 1 kg biomass by this integrated process was found to be significantly reduced from 184.6 to 2.37 kWh, indicating that this downstream process is energy efficient. Upon considering the cost of optimal MNCs dosage and electricity cost ($0.15 per kWh38,39), it was estimated that $7.53 was required to harvest and disrupt 1 kg biomass (Table S3 in the Supporting Information). Hence, the cost involved in this integrated process is 3.7 times lesser than that of conventional process of biomass harvesting, drying and disruption ($27.69 per kg biomass). It is evident that this cost could be further reduced while reusing the MNCs. The economic analysis of magnetic particles performed by Ge et al.6 suggested that $8.9 was required to only harvest 1 kg biomass at laboratory scale. Thus, the MNCs employed in this study could be more beneficial, as it integrates two critical downstream processes like biomass harvesting and disruption with consequent reuse of nanocomposites and spent medium. The life cycle assessment of this process will be investigated in our future work, along with scaleup studies of magnetophoretic biomass separation and disruption.

■

CONCLUSIONS In this study, we successfully implemented an innovative idea of developing an integrated and energy-efficient downstream process that utilizes reusable biopolymer nanocomposite for concurrent biomass separation and disruption. The optimal concentrations of nanocomposites resulted in over 98% biomass harvesting efficiency. Further, the harvested wet biomass was efficiently disrupted by both UV and visible light-driven photocatalysis, as it is corroborated from the corresponding lipid fluorescence and scanning electron images of microalgal cells. Subsequently, almost complete recoveries of intracellular lutein and lipid were achieved, indicating the photocatalytic efficiency of the nanocomposites. Moreover, this study showed the recyclability of nanocomposites for at least five consecutive batches. The preliminary techno-economic analysis indicated that this integrated downstream process could reduce the cost by 73%, as compared to conventional biomass harvesting, drying and disruption. This integrated process could effectively eliminate the use of chemicals and energy-intensive unit operations for two important downstream operations, cell harvesting, and disruption. One of the major highlights of this work is the possible utilization of sunlight as the renewable source for photocatalytic biomass disruption. This downstream processing strategy may also be judiciously extended to yeast biomass based biorefineries. To the best of our knowledge, this is the first time that a green and reusable nanocomposite was successfully employed for the development of an integrated downstream process for sustainable microalgal biorefinery.

Figure 8. (A) Time-course profile of detachment efficiency by physicochemical treatment. (B) Variation in harvesting efficiency and product recovery upon reusing detached MNCs. Loading of MNCs at each cycle: initial 0.07 g g−1; first 0.068 g g−1; second 0.067 g g−1; third, 0.065 g g−1; fourth, 0.064 g g−1; and fifth, 0.063 g g−1. Data represent average of three independent runs ± standard deviation.

photocatalytic disrupted biomass (Table S1 in the Supporting Information). The percentage of saturated fatty acid content (60.7%) was higher than the unsaturated content (39.3%), and the major fatty acids were found to be palmitic (C16:0), stearic (C18:0), oleic (C18:1), and linolenic (C18:3). The higher saturated fatty acid content obtained in this study is corroborated with the microalgal strains such as Chlamydomonas sp (78.61%) and Scenedesmus obliquus (70.83%) reported by Nascimento et al.35 and also with Chlorella sp MCCS 040 (>90%) reported by Rasoul-Amini et al.36 This high saturated FAME content would be beneficial in improving some specific fuel properties including oxidative stability, cetane number, and heating value.36,37 As the high saturated fatty acid content shows poor cold flow properties, the biodiesel quality can be further enhanced if it is suitably blended with unsaturated esters obtained from nonfood crops based feedstock.35 Furthermore, the reusability of spent medium was tested by cultivating C. minutissima under similar process conditions. It was observed that there was no drastic change in final biomass concentrations (3.1 ± 0.08 g L−1), while reusing the medium for five successive batches (Table S2 in the Supporting Information). Thus, these findings indicate that spent medium obtained after biomass

■

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acssuschemeng.6b02189. 859


Research Article


■

(10) Lee, Y.-C.; Lee, K.; Oh, Y.-K. Recent nanoparticle engineering advances in microalgal cultivation and harvesting processes of biodiesel production: A review. Bioresour. Technol. 2015, 184, 63−72. (11) Brennan, L.; Owende, P. Biofuels from microalgaeA review of technologies for production, processing, and extractions of biofuels and co-products. Renewable Sustainable Energy Rev. 2010, 14 (2), 557− 577. (12) Rashid, N.; Rehman, M. S. U.; Han, J. I. Use of chitosan acid solutions to improve separation efficiency for harvesting of the microalga Chlorella vulgaris. Chem. Eng. J. 2013, 226, 238−242. (13) Toh, P. Y.; Ng, B. W.; Chong, C. H.; Ahmad, A. L.; Yang, J.-W.; Chieh Derek, C. J.; Lim, J. Magnetophoretic separation of microalgae: the role of nanoparticles and polymer binder in harvesting biofuel. RSC Adv. 2014, 4 (8), 4114−4121. (14) Wang, S. K.; Wang, F.; Hu, Y. R.; Stiles, A. R.; Guo, C.; Liu, C. Z. Magnetic Flocculant for High Efficiency Harvesting of Microalgal Cells. ACS Appl. Mater. Interfaces 2014, 6 (1), 109−115. (15) Ge, S.; Agbakpe, M.; Wu, Z.; Kuang, L.; Zhang, W.; Wang, X. Influences of surface coating, UV irradiation and magnetic field on the algae removal using magnetite nanoparticles. Environ. Sci. Technol. 2015, 49 (2), 1190−6. (16) Wang, S. K.; Stiles, A. R.; Guo, C.; Liu, C. Z. Harvesting microalgae by magnetic separation: A review. Algal Res. 2015, 9, 178− 185. (17) Schneider, J.; Matsuoka, M.; Takeuchi, M.; Zhang, J.; Horiuchi, Y.; Anpo, M.; Bahnemann, D. W. Understanding TiO2 photocatalysis: mechanisms and materials. Chem. Rev. 2014, 114 (19), 9919−86. (18) Li, L.; Li, X.; Duan, H.; Wang, X.; Luo, C. Removal of Congo Red by magnetic mesoporous titanium dioxide-graphene oxide coreshell microspheres for water purification. Dalton Trans. 2014, 43 (22), 8431−8. (19) Lee, Y. C.; Lee, H. U.; Lee, K.; Kim, B.; Lee, S. Y.; Choi, M. H.; Farooq, W.; Choi, J. S.; Park, J. Y.; Lee, J.; Oh, Y. K.; Huh, Y. S. Aminoclay-conjugated TiO2 synthesis for simultaneous harvesting and wet-disruption of oleaginous Chlorella sp. Chem. Eng. J. 2014, 245, 143−149. (20) Dineshkumar, R.; Dhanarajan, G.; Dash, S. K.; Sen, R. An advanced hybrid medium optimization strategy for the enhanced productivity of lutein in Chlorella minutissima. Algal Res. 2015, 7, 24− 32. (21) Dineshkumar, R.; Dash, S. K.; Sen, R. Process integration for microalgal lutein and biodiesel production with concomitant flue gas CO2 sequestration: a biorefinery model for healthcare, energy and environment. RSC Adv. 2015, 5 (90), 73381−73394. (22) Haldorai, Y.; Shim, J. J. Novel Chitosan-TiO2 Nanohybrid: Preparation, Characterization, Antibacterial, and Photocatalytic Properties. Polym. Compos. 2014, 35 (2), 327−333. (23) Griffiths, M. J.; Garcin, C.; van Hille, R. P.; Harrison, S. T. L. Interference by pigment in the estimation of microalgal biomass concentration by optical density. J. Microbiol. Methods 2011, 85 (2), 119−123. (24) Chen, W.; Zhang, C.; Song, L.; Sommerfeld, M.; Hu, Q. A high throughput Nile red method for quantitative measurement of neutral lipids in microalgae. J. Microbiol. Methods 2009, 77 (1), 41−7. (25) De Bhowmick, G.; Vegesna, N.; Sen, R. Process design for augmentation and spectrofluorometric quantification of neutral lipid by judicious doping of pathway intermediate in the culture of marine Chlorella variabilis for biodiesel application. Bioresour. Technol. 2015, 198, 781−788. (26) Dineshkumar, R.; Subramanian, G.; Dash, S. K.; Sen, R. Development of an optimal light-feeding strategy coupled with semicontinuous reactor operation for simultaneous improvement of microalgal photosynthetic efficiency, lutein production and CO2 sequestration. Biochem. Eng. J. 2016, 113, 47−56. (27) Mohseni-Bandpi, A.; Kakavandi, B.; Kalantary, R. R.; Azari, A.; Keramati, A. Development of a novel magnetite−chitosan composite for the removal of fluoride from drinking water: adsorption modeling and optimization. RSC Adv. 2015, 5 (89), 73279−73289.

Synthesis and TEM images of TiO 2 and Fe 3 O 4 nanoparticles; magnetization curves of Fe3O4 and MNCs; observation of microalgae flocculation; solidstate UV−visible absorption spectra of CTC and MNCs; SEM images of microalgae before and after photocatalysis; comparison of FAME composition obtained under various cell disrupted conditions; biomass yield obtained upon reuse of culture medium; and technoeconomic analysis of the integrated downstream processing (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail address: [email protected]. Tel.: 91-3222-283752. ORCID

Ramkrishna Sen: 0000-0002-6207-3307 Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS R.D. gratefully acknowledges DST-INSPIRE, Government of India, for his fellowship. R.D. and R.S. acknowledge the Department of Science and Technology, Government of India, for the financial support (project grant no. DST/IS-STAC/ CO2-SR-160/13(G); date 08.07.2013). R.D. is also thankful to Dr. S. Karthik for his valuable technical inputs towards this research.

■

REFERENCES

(1) Razzak, S. A.; Hossain, M. M.; Lucky, R. A.; Bassi, A. S.; de Lasa, H. Integrated CO2 capture, wastewater treatment and biofuel production by microalgae culturing-A review. Renewable Sustainable Energy Rev. 2013, 27, 622−653. (2) Subramanian, G.; Dineshkumar, R.; Sen, R. Modelling of oxygenevolving-complex ionization dynamics for energy-efficient production of microalgal biomass, pigment and lipid with carbon capture: an engineering vision for a biorefinery. RSC Adv. 2016, 6 (57), 51941− 51956. (3) Gong, J.; You, F. Q. Value-Added Chemicals from Microalgae: Greener, More Economical, or Both? ACS Sustainable Chem. Eng. 2015, 3 (1), 82−96. (4) Orfield, N. D.; Levine, R. B.; Keoleian, G. A.; Miller, S. A.; Savage, P. E. Growing Algae for Biodiesel on Direct Sunlight or Sugars: A Comparative Life Cycle Assessment. ACS Sustainable Chem. Eng. 2015, 3 (3), 386−395. (5) Williams, P. J. L.; Laurens, L. M. L. Microalgae as biodiesel & biomass feedstocks: Review & analysis of the biochemistry, energetics & economics. Energy Environ. Sci. 2010, 3 (5), 554−590. (6) Ge, S.; Agbakpe, M.; Zhang, W.; Kuang, L. Heteroaggregation between PEI-coated magnetic nanoparticles and algae: effect of particle size on algal harvesting efficiency. ACS Appl. Mater. Interfaces 2015, 7 (11), 6102−8. (7) Lee, K.; Na, J.-G.; Seo, J. Y.; Shim, T. S.; Kim, B.; Praveenkumar, R.; Park, J.-Y.; Oh, Y.-K.; Jeon, S. G. Magnetic-Nanoflocculant-Assisted Water−Nonpolar Solvent Interface Sieve for Microalgae Harvesting. ACS Appl. Mater. Interfaces 2015, 7 (33), 18336−18343. (8) Orr, V. C. A.; Plechkova, N. V.; Seddon, K. R.; Rehmann, L. Disruption and Wet Extraction of the Microalgae Chlorella vulgaris Using Room -Temperature Ionic Liquids. ACS Sustain. ACS Sustainable Chem. Eng. 2016, 4 (2), 591−600. (9) Seo, J. Y.; Praveenkumar, R.; Kim, B.; Seo, J.-C.; Park, J.-Y.; Na, J.-G.; Jeon, S. G.; Park, S. B.; Lee, K.; Oh, Y.-K. Downstream integration of microalgae harvesting and cell disruption by means of cationic surfactant-decorated Fe3O4 nanoparticles. Green Chem. 2016, 18, 3981−3989. 860


Research Article

ACS Sustainable Chemistry & Engineering (28) Nayak, M.; Karemore, A.; Sen, R. Performance evaluation of microalgae for concomitant wastewater bioremediation, CO2 biofixation and lipid biosynthesis for biodiesel application. Algal Res. 2016, 16, 216−223. (29) Cerff, M.; Morweiser, M.; Dillschneider, R.; Michel, A.; Menzel, K.; Posten, C. Harvesting fresh water and marine algae by magnetic separation: Screening of separation parameters and high gradient magnetic filtration. Bioresour. Technol. 2012, 118, 289−295. (30) Rodríguez-González, V.; Alfaro, S. O.; Torres-Martínez, L. M.; Cho, S.-H.; Lee, S.-W. Silver−TiO2 nanocomposites: Synthesis and harmful algae bloom UV-photoelimination. Appl. Catal., B 2010, 98 (3−4), 229−234. (31) Coward, T.; Lee, J. G. M.; Caldwell, G. S. Harvesting microalgae by CTAB-aided foam flotation increases lipid recovery and improves fatty acid methyl ester characteristics. Biomass Bioenergy 2014, 67, 354−362. (32) Seo, J. Y.; Lee, K.; Lee, S. Y.; Jeon, S. G.; Na, J. G.; Oh, Y. K.; Park, S. B. Effect of barium ferrite particle size on detachment efficiency in magnetophoretic harvesting of oleaginous Chlorella sp. Bioresour. Technol. 2014, 152, 562−6. (33) Prochazkova, G.; Safarik, I.; Branyik, T. Harvesting microalgae with microwave synthesized magnetic microparticles. Bioresour. Technol. 2013, 130, 472−7. (34) De Bhowmick, G.; Subramanian, G.; Mishra, S.; Sen, R. Raceway pond cultivation of a marine microalga of Indian origin for biomass and lipid production: A case study. Algal Res. 2014, 6, 201− 209. (35) Nascimento, I. A.; Marques, S. S. I.; Cabanelas, I. T. D.; Pereira, S. A.; Druzian, J. I.; de Souza, C. O.; Vich, D. V.; de Carvalho, G. C.; Nascimento, M. A. Screening Microalgae Strains for Biodiesel Production: Lipid Productivity and Estimation of Fuel Quality Based on Fatty Acids Profiles as Selective Criteria. BioEnergy Res. 2013, 6 (1), 1−13. (36) Rasoul-Amini, S.; Montazeri-Najafabady, N.; Mobasher, M. A.; Hoseini-Alhashemi, S.; Ghasemi, Y. Chlorella sp.: A new strain with highly saturated fatty acids for biodiesel production in bubble-column photobioreactor. Appl. Energy 2011, 88 (10), 3354−3356. (37) Knothe, G. ″Designer″ biodiesel: Optimizing fatty ester (composition to improve fuel properties. Energy Fuels 2008, 22 (2), 1358−1364. (38) Gunaseelan, D.; Ramkrishna, S. Cost Analysis of Biosurfactant Production from a Scientist’s Perspective. Biosurfactants 2014, 153− 162. (39) Chopra, J.; Dineshkumar, R.; Bhaumik, M.; Dhanarajan, G.; Kumar, R.; Sen, R. Integrated in situ transesterification for improved biodiesel production from oleaginous yeast: a value proposition for possible industrial implication. RSC Adv. 2016, 6 (74), 70364−70373.

861


Smart and Reusable Biopolymer Nanocomposite ... - ACS Publications

Recommend Documents