Acidified-flocculation process for harvesting of microalgae: Coagulant reutilization and metal-free-microalgae recovery

Accepted Manuscript Acidified-flocculation process for harvesting of microalgae: coagulant reutilization and metal-free-microalgae recovery Dong-Yeon Kim, Kyubock Lee, Jiye Lee, Young-Hee Lee, Jong-In Han, JiYeon Park, You-Kwan Oh PII: DOI: Reference:

S0960-8524(17)30660-0 http://dx.doi.org/10.1016/j.biortech.2017.05.021 BITE 18047

To appear in:

Bioresource Technology

Received Date: Revised Date: Accepted Date:

8 February 2017 2 May 2017 3 May 2017

Please cite this article as: Kim, D-Y., Lee, K., Lee, J., Lee, Y-H., Han, J-I., Park, J-Y., Oh, Y-K., Acidifiedflocculation process for harvesting of microalgae: coagulant reutilization and metal-free-microalgae recovery, Bioresource Technology (2017), doi: http://dx.doi.org/10.1016/j.biortech.2017.05.021

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Acidified-flocculation process for harvesting of microalgae: coagulant reutilization and metal-free-microalgae recovery Dong-Yeon Kim a,b,†, Kyubock Lee c,†, Jiye Lee a, Young-Hee Lee c, Jong-In Han b , Ji-Yeon Park a, You-Kwan Oh b* a

Biomass and Waste Energy Laboratory, Korea Institute of Energy Research, 152 Gajeong-ro, Yuseong-gu, Daejeon 34129, Republic of Korea

b

Department of Civil and Environmental Engineering, KAIST, 291 Daehak-ro, Yuseong-gu, Daejeon 34141, Republic of Korea

c

Graduate School of Energy Science and Technology, Chungnam National University, 99 Daehak-ro, Yuseong-gu, Daejeon 34134, Republic of Korea †

These authors equally contributed to this work.

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ABSTRACT Chemical flocculation is considered to be an overall low-cost and up-scalable process for harvesting of microalgae. In this study a new flocculation approach utilizing metal coagulant (Fe2(SO4)3) and sulfuric acid (H2SO4) was introduced for harvesting of Chlorella sp. KR-1, which overcome two main issues of contamination and reuse of coagulant. Reduction of pH successfully released precipitates attached to the microalgae, and the remaining acidic solution containing recovered ferric ions could be reused for harvesting up to three times with high, better-than 98% efficiencies. Moreover, the acid-treated microalgal biomass could be directly used for lipid extraction without additional catalyst. High extraction yield of around 32% were achieved with FAME conversion efficiencies of around 90%. The integrated approach devised in the present study is expected to make the best use of the age-old yet effective harvesting means of flocculation, which can be a practical and economical option in microalgal biorefinery.

Keywords: Microalgae · Harvesting · Extraction · Coagulation/flocculation · Acidification · Coagulant reutilization

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1. Introduction Microalgae, offering several advantages over conventional oil crops such as high productivity, non-edibility and high lipid content, have been considered to be an attractive feedstock for sustainable production of biofuel (Milano et al. 2016). The process of biodiesel production from microalgae biomass mainly comprises cultivation, harvesting (and drying), lipid extraction, and biodiesel conversion. In order to make the commercialization of microalgal biodiesel feasible, every step of the process needs to be improved, though far more work is required for the cultivation and harvesting steps (Barros et al. 2015). Especially, harvesting is energy-intensive, mainly because of the small size of microalgal cells and their negatively charged surfaces, both of which facts lead to a stable, sedimentation-resisting colloidal suspension. Since the typical final cell concentration of cultivated microalgae (mass concentration less than 2 g/L in photobioreactors; far less in open ponds) is too low to be directly used in the subsequent oil-extraction step, an efficient biomass-concentration technique is essential. Depending on the biomass/water ratio, concentrating methods can be classified as harvesting (1~5%), dewatering (5~25%) or drying (> 25%) (Barros et al. 2015). There are various methods of microalgal harvesting; centrifugation, filtration, flotation, flocculation, magnetic separation and electrical processes or their combinations (Kim et al. 2015a). Centrifugation is the most conventional means, providing biomass concentrations up to a dewatering level of 25% with high recovery more than 95% when a centrifugal force equivalent to 5,000-10,000 g-force is applied. Its main drawbacks are the high financial and energy costs for applying high g-force and low scalability (Christenson and Sims 2011). Filtration is another well-known physical method and the efficiency is highly dependent on algal species. Moreover, clogging or fouling is another significant issue causing low final concentration (<1%) of harvested microalgae (Bilad et al. 2013). Electricity-driven physico-chemical methods such as dissolved-air flotation and electro-coagulation-flotation are highly energy consuming for supplying or producing microbubble (air or H2) with significant equipment costs (Kim et al. 2015a). It has been reported that a 10.2 L dispersed air flotation-foam fractionation could achieve maximum biomass concentration round 2.5 % with an energy consumption of 0.015 kWh/m3 (Coward et al. 2013). For more practical applications, particularly on a large scale and at low cost, coagulation-flocculation using 3

metal coagulant such as aluminum and ferric salts, commonly known as inorganic chemical flocculation, is considered to be the most promising, owing specifically to its simplicity and low energy requirement (Vandamme et al. 2013). It has been reported that high harvesting efficiencies of more than 98% and around 90% extraction yields were achieved for Chlorella sp. KR-1 by using ferric chloride of 0.56 g/L and ferric sulfate of 1.06 g/L with addition of 1% H2O2, respectively (Kim et al., 2015b). Choi et al (2016) also reported similar result for Chlorella sorokiniana; harvesting efficiency of around 97% with ferric nitrate dosage of 0.9 g/L. The remained nitrate ion after flocculation was recycled as nitrogen source in second cultivation to save cost in cultivation. The mechanism in metal coagulant-based flocculation process includes charge neutralization and sweep flocculation. In charge neutralization, the positively charged metal coagulants are attracted to the negatively charged colloids via electrostatic interaction, which is followed by the formation of large microfloc agglomerations (Ebeling et al. 2013). Flocs start to form during the neutralization step as particle collisions occur. Adding excess coagulants beyond charge-neutralization initiates the formation of metal precipitates. These metal hydroxide compounds, such as Al(OH)3 and Fe(OH)3, are heavy, sticky and large in particle size. Sweep flocculation takes place when colloidal materials are entrapped and swept down by precipitates settling in the suspension (Ghernaout et al. 2012). Conventionally, this process is widely used in the wastewater industry, specifically for removal of suspended solids by means of chemical agents such as alum or ferric chloride/sulfate. Direct application to microalgae harvesting of this otherwise promising option, however, entails two critical issues. First, the metal compound attached to biomass has an adverse effect on products such as oil and high-value chemicals (Gerardo et al. 2015). Second, the coagulants, though cheap, normally are not reusable. Until now, it has been rarely tried to remove the contaminants of metal coagulants from biomass to reuse them in microalgae harvesting process. Iron-based coagulants such as ferric chloride and ferric sulfate, once dissolved in water, are transformed into precipitates of metal hydroxide (Me(OH)x) and, if phosphate ions are available, also into those of metal phosphate (Mex(PO4)y) (Keeley et al. 2016). These ferric-based precipitates are solubilized, releasing ferric ions (Fe3+) as long as pH is sufficiently low (Edzwald and Haarhoff 2011). This simple chemistry can indeed offer a 4

means of iron-precipitate removal from microalgae-precipitate flocs and, thereby, ferric-ion recovery. The purpose of this study therefore was to develop a low-cost harvesting method that offers the added benefits of metal-free-biomass production and repetitive use of coagulants. To this end, ferric sulfate and sulfuric acid were chosen for the flocculation of microalgae and the release of the attached and/or precipitates, respectively. The integration of the two processes was expected to provide the following two advantages. First, the separated ferric ions can be directly recycled for the next round of harvesting; the recycled ferric-ion solution moreover, with its high acidity, maintains the culture pH sufficiently low for generation of efficient microalgal flocculation. Second, the residual acid in microalgal biomass after separation of iron precipitates works as a catalyst in the subsequent oilextraction process via hot-water extraction, which is known to be effective for all types of microalgae and, in particular, cells with cellulosic cell wall (Lee et al. 2014, Park et al. 2014).

2. Materials and methods 2.1. Strain and culture conditions Chlorella sp. KR-1 (Sung et al. 1999), an indigenous, freshwater, high-CO2-tolerant oleaginous microalga, was cultured using modified N8 medium (constituents: KNO3, 3 mM; KH2PO4, 5.44 mM; Na2HPO4, 1.83 mM; MgSO4·7H2O, 0.20 mM; CaCl2, 0.12 mM; FeNaEDTA, 0.03 mM; ZnSO4·7H2O, 0.01 mM; MnCl2·4H2O, 0.07 mM; CuSO4, 0.07 mM; Al2(SO4)3·18H2O, 0.01 mM) adjusted to a pH of 6.5. The strain was cultivated for seven days at 30°C in a Pyrex bubble-column reactor (working volume: 6 L) equipped with 12 fluorescent lamps at the front and back (light intensity: 80 µmol photons/m2/s). The reactor was supplied with 10% (v/v) CO2 in air at a rate of 0.6 L/min. The detailed conditions are available elsewhere (Praveenkumar et al. 2014).

2.2. Microalgal harvesting, precipitate removal, and coagulant recycling The concentration of microalgae culture used for harvesting experiments was determined to be 1.52±0.07 g/L based on optical density (OD) measurements and the OD/dry 5

cell weight (DCW) relationship previously established for Chlorella sp. KR-1 (Praveenkumar et al. 2014). The range of Fe2(SO4)3 concentrations selected for the harvesting test was 0.5 – 1.2 g/L. Harvesting experiments were carried out in Falcon 50mL conical centrifuge tubes containing 50 mL of microalgae culture. The coagulants in the aforementioned concentrations were added, with the 2 M stock (Fe2(SO4)3, Sigma-Aldrich, USA), into each tube. After stirring for 5 mins using a vortex machine (Vortex Genius 3, IKA, Italy), the tubes containing microalgae and coagulants were left undisturbed for 30 mins so as to allow coagulation/flocculation to take place. In terms of harvesting efficiency, the optimal coagulant concentration was found to be 1.0 g/L. The harvesting efficiency was calculated based on the equation (1) Harvesting ef iciency % =

  

·····················································Eq. 1

× 100

where ODi and ODf are the OD of the initial microalgal culture and the supernatant liquid after microalgal harvesting, respectively. The tests of coagulant recycling were conducted, as shown in Fig. 1, with the optimal concentration of ferric sulfate (1.0 g/L). First, 50 mL of algal culture (a) was 10-times concentrated (approximately: 15 g/L) using 1.0 g/L Fe2(SO4)3 (b-c), and the supernatant was removed so that the remaining volume was 5 mL. Then, 1 M sulfuric acid (d) (Junsei, Japan) was added to 1% (wt./wt.), which solution was well mixed for 1 min using the vortex machine and centrifuged at 400×g for 5 mins (e) to separate the water-soluble precipitates (g) from the microalgae (f). The separated, ferric-ion-containing liquid (g) was then reused as a coagulant without pH control (h). This experiment was repeated three times. Thereafter, the iron-less microalgae-containing sulfuric acid residue (f) was directly used for oil extraction without further treatment. In order to obtain microalgae-free precipitates for analytical characterization, 1.0 g/L Fe2(SO4)3 was added to the supernatant after centrifugation-removal of the culture solution’s microalgae at 400×g for 5 mins.

2.3. Oil extraction 6

After microalgal harvesting by flocculation and acidification, hot-water treatments for oil extraction were carried out in a 100 mL autoclave reactor at 120°C for 30 mins. Crude oil was extracted from the treated cells by means of liquid–liquid extraction using hexane for 2 h at 750 rpm. Hexane was evaporated using a vacuum evaporator (EZ2 PLUS, Genevac, UK) to recover oil, and then the lipid-extraction yield was estimated by weighing the remaining oil. The lipid content extracted from 10 mg of freeze-dried cells using chloroform/methanol (2/1, v/v) was measured and assumed to be the total fatty acid content accumulated in the microalgae, which was used as a reference throughout (Cho et al., 2011). The freeze-dried cells were prepared by centrifugation (800×g, 10 mins) and three-times washing with deionized water followed by freeze-drying (FD5512, IlShin BioBase Co., Korea) for four days or longer. All of the experiments were performed in triplicate.

2.4 Fatty acids methyl ester (FAME) analysis FAME profiles for each samples were analyzed using a modified transesterification method (Cho et al. 2011). In detail, a 10 mL Pyrex glass tube was filled with around 10 mg of lyophilized cells or extracted crude oil and sealed with a Teflon-covered screw cap, after which 2 mL of chloroform/methanol (2/1, v/v) was added. The tube was then vortex-mixed for 10 mins at room temperature. Subsequently, 1 mL of chloroform (including heptadecanoic acid as an internal standard), 1 mL of methanol and 300 µL of H2SO4 were sequentially added to the tube, which was vortex-mixed for 5 mins. Following this, the tube was reacted in a 100°C water bath for 10 min, after which it was cooled to room temperature, supplemented with 1 mL of deionized water, and intensely mixed for 5 mins. Preparatory to phase separation, the mixture was centrifuged at ×800 g for 10 mins. The lower layer (organic phase) was extracted using a disposable plastic syringe (NORM-JECT, Henke Sass, Wolf GmBH, Germany) and filtered with a disposable 0.22 mm PVDF syringe (Millex-GV; Millipore, USA). Methyl esters of fatty acids were analyzed using a gas chromatograph equipped with a flame ionization detector and a 30 m × 0.32 mm (ID) HP-INNOWax capillary column (Agilent Technologies, USA). Mix RM3, Mix RM5, GLC50, GLC70, heptadecanoic acid, and c-linolenic acid were used as internal standards. Regarding the unit, FAMEs per oil (mg 7

FAME/g oil) was converted into FAMEs per cell (mg FAME/g cell) by multiply extraction yield. All of the other reagents used were of analytical grade. The experiments were performed in triplicate and then averaged.

2.5. Analytical methods The concentrations of total iron in the solution were determined by the 1,10 phenanthroline method (Total Iron Kit, Humas, Korea). The zeta potentials of the microalgae and of the precipitates with and without microalgae were measured using a Zetasizer (ZS90, Malvern, UK). The data were recorded as the average values of three measurements. The morphologies and the elemental compositions of the microalgae before and after removal of precipitates were characterized by field emission scanning electron microscopy (FE-SEM, S4800, HITACHI, Japan) equipped with energy-dispersive spectroscopy (EDS, X-MAX50, HORIBA, Japan). Differential interface contrast (DIC) microscopy (Microscope Axio Imager A2, Carl Zeiss Microscopy GmbH, Jena, Germany) images of the precipitates, microalgaeprecipitate flocs, and precipitate-removed microalgae were obtained at magnifications of ×20 and ×100.

3. Results and discussion 3.1 Harvesting of Chlorella sp. KR-1 using ferric sulfate Fig. 2 shows the harvesting efficiency, pH and Fe concentration in the supernatant with respect to the concentration of ferric sulfate. The harvesting efficiency increased with dosage, reaching over 98% with more than 0.9 g/L ferric sulfate. Considering the initial concentration of culture (1.52 g/L), around 1.7 g of biomass was harvested with 1 g of ferric sulfate. It is remarkable that the pH of the culture was reduced to 3 by the addition of ferric sulfate, a strong Lewis acid (Kim et al. 2015b). This addition of ferric sulfate, which elevated the harvesting efficiency, resulted in a reduced concentration of ferric ions in the bulk phase, indicating, significantly, that ferric ions had been consumed for floc formation. The lowest 8

level of ferric ions in the spent medium was obtained at 1.0 g/L ferric sulfate; therefore, this dose was used for the subsequent experiments. The final biomass concentration, after gravitational sedimentation followed by supernatant removal, reached approximately 15 g/L (a roughly 10-fold concentration).

3.2 Precipitate formation and flocculation mechanism The addition of ferric sulfate resulted in destabilization of the microalgal suspension, ending in the formation of white precipitates and, concomitantly, the gravitational sedimentation of microalgal flocs, as shown in Fig. 1. The obtained optical microscopy and SEM images clearly show that nano-sized precipitates had attached to the microalgal cells, thus forming microalgae-precipitate flocs (Figs. S1a-b). Precipitates also formed in the absence of microalgal cells (Figs. S1c-d). It has been reported that ferric sulfate or chloride salts are rapidly disassociated in water, thereby forming ferric hydroxides (Fe(OH)3) and, if phosphorus is present, ferric phosphate (Fe2(PO4)3) as well (Keeley et al. 2016). With introduction of 5 mM of ferric ions into a BG-11 medium containing 8.44 mM phosphate ion, theoretically 2.5 mM ferric phosphate can be produced if formation of ferric hydroxide is prevented. Another potential ligand in this system could be microalgae- and bacteria-secreted extracellular polymeric substances (EPS), which are known to have negative charges and thus to be able to attract the positive charge of ferric ions (Tian et al. 2006). Therefore, these precipitates were speculated to be the complex of ferric hydroxide, ferric phosphate and EPS. The zeta potentials of the microalgae, precipitates and microalgae-precipitate flocs were measured to determine their surface charges (Fig. S2). Interestingly, all of them showed negative charges, in contrast to the results of previous research. Henderson et al. (2008) reported that adding metal coagulants to a microalgal culture resulted in a zeta potential increase as high as zero or the positive level. Aktas et al. (2012) reported a similar result: the best harvesting efficiency was achieved once the zeta potential of poly aluminum chloride (PAC) was markedly increased. On the other hand, in this experiment, the zeta potential of the microalgae precipitates was relatively low (-14 mV) compared with those of earlier studies; nevertheless, high harvesting efficiency could be achieved. It can be possibly 9

explained that ferric ions (Fe3+) added in the form of ferric sulfate to a culture play two different roles in microalgal harvesting (Fig. 1). First, Fe3+ ions work as linkers between the two negatively charged materials, partially initiating flocculation by electrostatic interaction. However, this is not the main factor impacting on microalgae harvesting. It can be easily confirmed that microalgal flocculation is inefficient when microalgae are washed by distilled water and then re-dispersed in distilled water. Second, and more importantly, Fe3+ ions not only form ferric hydroxide (Fe(OH)3) but also bind with phosphate ions (PO43-) and EPS to form precipitates that sweep microalgae in the form of microalgae-precipitate flocs. This physical entrapment of microalgae within precipitate networks followed by sweeping down effectively explains how negatively charged microalgae can be efficiently harvested by equivalently charged precipitates. Indeed, in the present experimentation, the microalgae washed in distilled water failed to form suitably rigid flocs even with ferric sulfate (data not shown), proving that the Fe3+ ions needed additional components such as phosphate ions and EPS to form visible flocs. It is also worth pointing out that sweep flocculation took place only when the precipitates were formed above a critical concentration, under which condition, microalgae could be entrapped within the precipitate network and settle (Fig. 1c). Interestingly, even with the microalgae concentration four-times increased and all of the medium conditions the same, a high harvesting efficiency (more than 90%) could still be obtained by adding 1.0 g/L ferric sulfate (Fig. 3). A plausible explanation is that the optimum coagulant concentration is dependent not on the concentration of microalgae but on the pH as well as the concentrations of the other ions with which the precipitates are formed.

3.3 Recycling of coagulants One of the main goals of this study was to obtain metal-free, clean biomass in such a way that coagulant can be reused. A simple approach entailing acid-assisted dissolution of ferric compounds was adopted. By lowering the pH (to around pH 1) using sulfuric acid, the ferric-containing precipitates could be completely solubilized and split into ferric ions and counter cations, resulting in a biomass free of metals (Figs. S1e, f). EDS analysis revealed that this was the case: the Fe had disappeared from the microalgal cells after the acid treatment (Table 1). 10

The liquid component (Fig. 1g) remaining after harvesting, containing still-high ferric-ion levels, was found to be reusable; indeed, over the course of three reuses, the harvesting efficiency remained high (more than 98%), dropping thereafter (Fig. 4). The total Fe concentration in the bulk solution was, after each flocculation, around 34 mg/L (~0.61mM) (Fig. 2), which meant that the concentration of elemental iron decreased from the initial value of 279 mg/L to, after the third reuse, 177 mg/L (Fig. 4). In fact, at a similar iron concentration level (i.e., 167 mg/L at 0.6 g/L of ferric sulfate, Fig. 2), the harvesting efficiency also was very low (~40%). In addition to ferric sulfate, various metal coagulants such as ferric and aluminum salts have been reported in flocculation-based harvesting process for several microalga species. Ferric salts (ferric chloride, ferric sulfate, ferric nitrate) were used for flocculation of Chlorella minutissma (Papazi et al. 2010), Chlorella sorokiniana (Choi et al. 2016), Chlorella zonfingiensis (Wyatt et al. 2012), Tetraselmis sp. (Kown et al. 2014). For aluminum salts, aluminum chloride and aluminum sulfate were used for flocculation of Chlorella sp. and Scenedesmus (Aragon et al. 1992), Tetraselmis sp. (Kown et al. 2014), Chaetoceros calcitrans, Tetraselmis chui, Skeletonema costarum and Isochrysis goibana species (Millamena et al. 1990). Likewise, microalgae flocculation using these metal salts works by charge neutralization between positively charged flocculent and negatively charged microalgae, implying the modified flocculation method would be applicable for various microalgal species that have negative charge as most of microorganisms do.

3.4 Lipid extraction and fatty acid profiles In addition to cleaning harvested biomass and enabling repeated coagulant use, acidification is also advantageous to the subsequent extraction process. Indeed, disruption of the Chlorella sp. cell walls, composed of cellulosic materials, is difficult (Domozych et al. 2012), typically requiring harsh conditions such as effected by high temperature/pressure along with strong acid/base catalysts (Lee et al. 2014). For example, a method known as acidcatalyzed hot-water extraction treats cells with 1~2% (wt./wt.) sulfuric acid at elevated temperature for sufficient lipid extractability (Park et al. 2014). It has been reported that 11

about 40 - 50% of extraction yield was increased by acid-treatment when the oil extraction yields with and without acid-treatment were compared for the same species, Chlorella sp. KR-1 (Kim et al. 2015b). Since acid-assisted cleaning approach in this study used approximately only 1% sulfuric acid, lipid extraction could directly proceed with no need of additional acids. The lipid contents extracted in this way were found to be 31.85 and 32.05% with FAME-conversion efficiencies of 89.82 and 90.31% for the 1st use and 2 nd use (reuse) of coagulants (Table S1), equivalent to 285.53 and 289.45 mg FAME/g cell of extracted FAME content, respectively (Table 2). These results are comparable to the intrinsic FAME content, 314.64 ± 5.89 mg FAME/g cell, obtained by modified transesterification from dry microalgal biomass (Table 2).

3.5 Economic feasibility The currently available harvesting techniques and their costs are summarized in Table S2, which are roughly estimated based on only chemical or electricity consumed in the process. Among them, coagulation/flocculation appears to be the best option in terms of energy consumption, operational cost and scalability (Vandamme et al. 2013). Department of Energy (DOE) reported algae production logistic costs specifically divided in production, harvest, preprocessing and recycle (water and nutrients) costs (Davis. 2015). According to this report, total algal feedstock cost, in other word extracted oil cost, would be decreased from 18.22 $/GGE (GGE, Gasoline Gallon Equivalent) in 2010 to 3.27 $/GGE in 2022. In particularly, harvesting cost was targeted to be 0.67 $/GGE in 2022 from 2.99 $/GGE in 2010. According to a rough estimation based on our experimental results of chemical dosage and extraction yield, the harvesting cost was estimated to be 1.46 $/GGE by the developed approach as shown in supplementary data, which is not very unrealistic considering the targeted cost by 2022. Furthermore, if the metal-free clean biomass can be used for other applications such as food additives, aquaculture and husbandry utilizations, the cost will be reduced more.

4. Conclusion 12

In summary, a means of revitalizing the age-old harvesting method of flocculation by way of acid-assisted coagulant recycling was devised. This is achieved by lowering the solution pH with sulfuric acid. The pH drop, which enabled producing metal-free biomass and repeated use of coagulants, also was found to provide acidic condition allowing hot-water extraction without additional acid treatment. It is anticipated that the integrated approach can make flocculation, which is superbly cost-effective and up-scalable process, a realistic harvesting option for microalgae-derived biodiesel production.

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Acknowledgement This study was conducted within the framework of the Research and Development Program of the Korea Institute of Energy Research (KIER) (B7-2437-01). Further support was received from the Advanced Biomass R&D Center (ABC) of the Global Frontier Project funded by the Ministry of Science, ICT and Future Planning (ABC- 2012M3A6A205388).

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20. Lee, I., Park, J.-Y., Choi, S.-A., Oh, Y.-K., Han, J.-I., 2014. Hydrothermal nitric acid treatment for effectual lipid extraction from wet microalgae biomass. Bioresour. Technol. 172, 138-142. 21. Milano, J., Ong, H.C., Masjuki, H.H., Chong, W.T., Lam, M.K., Loh, P.K., Vellayan, V., 2016. Microalgae biofuels as an alternative to fossil fuel for power generation. Renew. Sustainable Energy Rev. 58, 180-197. 22. Millamena, O., Aujero, E., Borlongan, I., 1990. Techniques on algae harvesting and preservation for use in culture as larval food. Aquac Eng 9, 295-304. 23. Papazi, A., Makridis P., Divanach, P., 2010. Harvesting Chlorella minutissima using cell coagulants, J. Appl. Phycol. 22, 349-355. 24. Park, J.-Y., Oh, Y.-K., Lee, J.-S., Lee, K., Jeong, M.-J., Choi, S.-A., 2014. Acidcatalyzed hot-water extraction of lipids from Chlorella vulgaris. Bioresour. Technol. 153, 408-412. 25. Praveenkumar, R., Kim, B., Choi, E., Lee, K., Cho, S., Hyun, J.-S., Park, J.-Y., Lee, Y.-C., Lee, H.U., Lee, J.-S., Oh, Y.-K., 2014. Mixotrophic cultivation of oleaginous Chlorella sp KR-1 mediated by actual coal-fired flue gas for biodiesel production. Bioprocess and Biosyst. Eng. 37, 2083-2094. 26. Sung, K.D., Lee, J.S., Shin, C.S., Park, S.C., 1999. Isolation of a new highly CO2 tolerant fresh water microalga Chlorella sp. KR-1. Renew. Energy 16, 1019-1022. 27. Tian, Y., Zheng, L., Sun, D.-Z., 2006. Functions and behaviors of activated sludge extracellular polymeric substances (EPS): a promising environmental interest. J. Environ. Sci. 18, 420-427. 28. Vandamme, D., Foyubert, I., Muylaert K., 2013. Flocculation as a low-cost method for harvesting microalgae for bulk biomass production. Trends Biotechnol. 31, 233239.

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Table 1 Energy-dispersive X-ray spectroscopy (EDS) analysis for precipitates, Chlorella sp. KR-1 with precipitates and precipitate-removed Chlorella sp. KR-1

Element

Weight%

Atomic%

Weight%

Atomic%

Precipitate-removed microalgae Weight% Atomic%

C O P S K Fe

10.35 43.75 16.59 0.92 1.48 26.91

18.42 58.42 11.45 0.61 0.81 10.30

42.74 36.24 7.71 0.80 1.35 11.16

56.20 35.78 3.93 0.39 0.55 3.16

59.76 36.22 0.89 2.38 0.76 0.00

Totals .

100.00

Precipitates

Microalgae-precipitate flocs

100.00

67.59 30.75 0.39 1.01 0.26 0.00

100.00

Table 2 FAME profile for Chlorella sp. KR-1 from modified transesterification using dry biomass and extracted oils by hot-water extraction from harvested wet biomass (1st use and reuse of coagulant)

Unit: (mg FAME/g cell)

(Myristate)

st

Standard

1 use

Reuse

(Dry biomass)

(Wet biomass)

(Wet biomass)

C14:0

0.40 ±

0.01

0.57 ± 0.01

0.61 ±

0.01

(Pentadecanoate) C15:0

0.00 ±

0.00

0.29 ± 0.00

0.28 ±

0.00

(Palmitate)

C16:0

81.77 ±

1.84

82.97 ± 0.29

83.68 ±

0.12

(Palmitoleate)

C16:1

0.91 ±

0.03

0.56 ± 0.01

0.77 ±

0.01

(Stearate)

C18:0

27.05 ±

0.65

30.47 ± 0.42

31.11 ±

0.16

(Oleate)

C18:1n9c

70.41 ±

1.20

63.36 ± 0.20

63.73 ±

0.20

(Linoleate)

C18:2n6c

74.10 ±

1.19

59.92 ± 0.15

61.01 ±

0.07

C18:3n6

0.48 ±

0.48

0.53 ± 0.16

0.69 ±

0.01

C18:3n3c

18.01 ±

0.22

12.27 ± 0.28

12.92 ±

0.05

Others

41.51 ±

1.22

34.59 ± 0.71

34.64 ±

0.24

Total

314.64 ±

5.89

285.53 ± 0.17

289.45 ±

0.87

(Gammalinoleate) (Linoleate)

19

Fig. 1. Overall experimental scheme of proposed system

6.0

5.5

100 Harvesting efficiency pH Fe conc. in supernatant

80

45

5.0 40 4.5

pH

Harvesting efficiency

50

60

35

4.0 40

30 3.5

20

3.0

0

2.5 0.4

0.6

0.8

1.0

1.2

1.4

Concentration of ferric sulfate (g/L)

Fig. 2. Effects of coagulation concentration on pH and harvesting efficiency

20

25

20

Fe concentration in supernatant (mg/L)

120

7 6

Harvesting efficiency (%)

100 5 90 4 80

3 Harvesting efficiency Algal concentration in supernatant

70

2 1

60 0

Algal concentration in supernatnat (g/L)

110

50 0

2

4

6

8

10

12

14

Algal concentration (g/L)

Fig. 3. Effect of algal concentration on harvesting efficiency at constant coagulant dosage (1.0 g/L)

4.0

100

40 3.5

80 30 60

3.0

pH

Harvesting efficiency (%)

50

Harvesting efficiency pH Fe conc. in supernatant

20 40 2.5 10

20

0

2.0 1st

2nd

3rd

4th

0

5th

Fig. 4. Effect of repetitive reutilizations of coagulant on harvesting precipitation efficiency

21

Fe concentration in supernatant (mg/L)

120

Research highlights

•

Metal-free biomass and repeated coagulant use was achieved by lowering pH.

•

High harvesting efficiency more than 98% was achieved using recycled coagulant.

•

Extraction yield and FAME contents reached to 32% and 289 mg/g cell, respectively.

22