Application and reactivation of magnetic nanoparticles in Microcystis aeruginosa harvesting

Bioresource Technology 190 (2015) 82–88

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Application and reactivation of magnetic nanoparticles in Microcystis aeruginosa harvesting Zhong Lin a,b,c, Yunfeng Xu d, Zhen Zhen a, Yu Fu d, Yueqiao Liu e, Wenyan Li f, Chunling Luo b, Aizhong Ding e, Dayi Zhang c,⇑ a

College of Agriculture, Guangdong Ocean University, Zhanjiang 524088, PR China Guangzhou Institute of Geochemistry, Chinese Academy of Sciences, Guangzhou 510640, PR China Lancaster Environment Centre, Lancaster University, Lancaster LA1 2YQ, UK d School of Environmental and Chemical Engineering, Shanghai University, Shanghai 200444, PR China e College of Water Sciences, Beijing Normal University, Beijing 100875, PR China f College of Natural Resources and Environment, South China Agricultural University, Guangzhou 510642, PR China b c

h i g h l i g h t s

g r a p h i c a l a b s t r a c t

 Effective cyanobacteria harvesting by

coprecipitation magnetic nanoparticles.  Ultrasonic solvent treatment reactivates MNPs and maintains harvesting performance.  Low dosage (0.2 g MNPs/g biomass) with high harvesting efficiency (>93%).

a r t i c l e

i n f o

Article history: Received 26 January 2015 Received in revised form 12 April 2015 Accepted 13 April 2015 Available online 23 April 2015 Keywords: Magnetic nanoparticles (MNPs) Microcystis aeruginosa Cyanobacteria harvesting Electrostatic attraction

a b s t r a c t This study developed a magnetic nanoparticles (MNPs) harvesting and reactivation technique for rapid cyanobacteria Microcystis aeruginosa separation. The harvesting of raw MNPs achieved high efficiency of 99.6% with the MNPs dosage of 0.58 g MNPs/g dry-biomass, but gradually decreased to 59.1% when directly reused 5 times. With extra ultrasonic chloroform:methanol solvent treatment, the MNPs can be effectively reactivated for M. aeruginosa harvesting with 60% efficiency after 5 times reactivation and the separation efficiency kept above 93% with 0.20 g MNPs/g dry-biomass dosage. The cyanobacteria–MNPs complex can be effectively disrupted by ultrasonic chloroform:methanol solvent treatment and the zeta potential was recovered for MNPs electrostatic attraction. The MNPs adsorption followed the Langmuir isotherm, and the maximum adsorption capacity and Langmuir constant was 3.74 g dry-biomass/g and 311.64 L/g respectively. This MNPs reactivation technique can achieve low energy separation and reduce MNPs consumption by 67%, providing potential engineering implementation for cyanobacterial biomass harvesting. Ó 2015 Elsevier Ltd. All rights reserved.

1. Introduction ⇑ Corresponding author at: B27, LEC3, Lancaster Environment Centre, Lancaster University, Lancaster LA1 2YQ, UK. E-mail address: [email protected] (D. Zhang). http://dx.doi.org/10.1016/j.biortech.2015.04.068 0960-8524/Ó 2015 Elsevier Ltd. All rights reserved.

Bioenergy is viewed as a sustainable energy source to solve the energy crisis due to its advantages of renewability and environmental friendliness. Of all the biological energy resources, algal

Z. Lin et al. / Bioresource Technology 190 (2015) 82–88

biofuels are the most attractive products (Christenson and Sims, 2011). Besides the biomass yield and engineering operation, dewatering and harvesting are the main challenges for microalgal biomass production (Abdelaziz et al., 2013; Uduman et al., 2010). Various approaches have been investigated for algae harvesting, such as filtration (Zhang et al., 2010), flocculation (Gerde et al., 2014) and centrifugation (Chen et al., 2011). Though good performance and high harvesting efficiency were achieved from these studies, the energy consumption in filtration and centrifugation methods was the key technical problem in bioenergy production. As for flocculation, the high cost of flocculants and large area restricted its engineering application. Magnetic nanoparticles (MNPs) have been proved as an effective and widely applied approach to harvest microalgae (Prochazkova et al., 2013; Wang et al., 2014; Xu et al., 2011) and algae (Cerff et al., 2012). With the unique features of superparamagnetism and biological affinity, magnetic iron oxide (like Fe3O4 or b-FeOOH) can achieve rapid cells adsorption and isolation with high efficiency and minimal energy consumption via external magnet (Fakhrullin et al., 2010; Xu et al., 2014; Zhang et al., 2015). Nevertheless, seldom published work has been attempted for the magnetic harvesting of cyanobacteria, like Microcystis aeruginosa, which are also important biofuel hosts in bioenergy engineering (Jansson, 2012). Furthermore, there are some attempts to reactive MNPs for harvesting reuse and disposal reduction, by disrupting the electrostatic attraction between MNPs and algal cells in water phase under acidic (Prochazkova et al., 2013; Xu et al., 2011) or alkaline (Lee et al., 2014; Seo et al., 2014) conditions. Considering the synthesis cost, disposal contamination and further biofuel refinery processes, it is important and urgent to devise practical technologies on MNPs reactivation in organic solvent to benefit downstream industry. This research aimed to develop the new magnetic nanoparticles (MNPs) harvesting and reactivation technology for rapid and lowcost cyanobacterial M. aeruginosa separation. By effective ultrasonic chloroform:methanol solvent treatment, the MNPs can be reactivated and the harvesting efficiency remained 60% after 5 times reactivation. In the semi-continuous bioreactor, the new MNPs reactivation technology achieved stable M. aeruginosa harvesting efficiency over 93% with 67% MNPs dosage reduction, showing engineering feasibility in sustainable algal biomass harvesting and bioenergy production.

2. Methods 2.1. Strain and cultivation Cyanobacterial M. aeruginosa was used in this study and the M9 cultivation medium contained 1.0 g NH4Cl, 11.0 g Na2HPO47H2O, 3.0 g KH2PO4, 5.0 g NaCl, 4.0 g glucose, 120 mg MgSO4, 10 mg CaCl2, and water to 1.0 L (Howard-Flanders and Theriot, 1966). To study the impacts of pH value on MNPs harvesting efficiency on M. aeruginosa, the M9 cultivation medium was also adjusted with 1.0 M HCl or 1.0 M NaOH to the final pH value of 3.0, 5.0, 7.0, 9.0 and 11.0. A single colony of M. aeruginosa strain (around 105 cells) was transferred into 100 mL M9 medium and homologized by vortex. The cyanobacteria and algae strains were cultivated under 20 °C and 500 cd sr/m2 light condition. The 1.0 mL of M. aeruginosa suspension was collected at Day 0, 2, 4, 6, 8, 10 and 12 to monitor the growth curve and evaluate the MNPs harvesting efficiency at different stage. Three biological replicates were carried out for each treatment. Without specifically statement, all the investigated adsorption isotherms and MNPs reactivation efficiency used the M. aeruginosa samples on Day 10 in the cultivation medium of pH 7.0.

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2.2. Magnetic nanoparticles synthesis and harvesting The synthesis of magnetic nanoparticles followed the modified chemical deposition methods (Zhang et al., 2011). The 2.0 mL of 1 M FeCl3 was mixed with 0.5 mL of 2 M FeCl2 and homologized by ultrasound bath (40 kHz, LNGF175, Langford Electronics Ltd., UK). The 25 mL of 2.0 M NaOH solution was then added by dropwise till the appearance of a dark iron oxide precipitate, with ultrasonic homologation throughout the process. The oxide suspension was kept homologation for 30 min. The precipitate was separated with a permanent magnet and washed with the same volume of deionized water until the supernatant reached pH 7.0 as the MNPs stock solution. MNPs concentration was measured by gravimetric method as 17.4 g/L. For cyanobacterial harvesting and isolation, 28 lL of MNPs stock solution was mixed with 972 lL M. aeruginosa suspension and cultivated for 5 min with 150 rpm shaking. The cyanobacteria–MNPs mixture was subsequently harvested by a permanent magnet for 5 min. The supernatant and MNPs pellets were then separated for further analysis. For the harvesting isotherm test, the original MNPs stock solution followed serial dilutions as the final concentration of 15.0, 12.0, 9.0, 4.5, 2.25, 1.25 and 0.5 g/L. The same addition volume (28 lL) of MNPs with different concentration was applied for M. aeruginosa harvesting. 2.3. Magnetic nanoparticles reactivation and harvesting batch reactor The batch experiment system was operated to test the harvesting efficiency of reactivated MNPs to minimise the MNPs consumption during harvest process, as illustrated in graphic abstract and Fig. 1. The bioreactor was a 500 mL flask containing M. aeruginosa strains in 100 mL M9 medium. The fresh cultivation M9 medium was pumped by peristaltic pump (205S/CA, Watson, UK) with the flow rate at 14 lL/min and the hydraulic retention time (HRT) was 10 days for the optimal cyanobacterial cultivation. The 20 mL cyanobacterial effluent was collected every day in harvesting chamber, subsequently mixed with 576 lL MNPs stock solution (or reactivated MNPs suspension) and cultivated for 5 min with 150 rpm shaking. The 20 mL cyanobacteria–MNPs suspension was then transferred in the separation system with magnetic isolation for 5 min. The supernatant was discarded as the waste and the cyanobacteria–MNPs pellets were put in the extraction chamber to separate M. aeruginosa cells and reactivate MNPs. Roughly 0.1 g of cyanobacteria–MNPs pellets (from 10 mL cyanobacteria–MNPs suspension) was suspended in 1.0 mL chloroform:methanol (2:1, v/v) solvent and the 40 kHz ultrasonic treatment (LNGF175, Langford Electronics Ltd., UK) was applied for 60 s separation (Wahlen et al., 2011). The reactivated MNPs were harvested by a permanent magnet for 5 min and resuspended in 500 lL deionized water. The reactivated MNPs suspension was applied directly in the harvesting chamber for reactivated MNPs harvesting or mixed with fresh MNPs stock solution (2:1, v/v) for optimised MNPs reuse. 2.4. Chemical and biological analysis The MNPs concentration was determined gravimetrically. The 1.0 mL MNPs stock solution was dried at 120 °C for 12 h, and the MNPs concentration was then calculated by the ratio of dry MNPs weight (mg) to volume (mL). Zeta potential of MNPs materials was determined by ZetaSizer Nano ZS90 (Malvern, UK) equipped with a DTS1060c zeta cell (Malvern, UK) at 25 °C with previous protocols (Jain et al., 2008). The amount of biomass was determined by gravimetric method. The 10.0 mL M. aeruginosa suspension was centrifuged at 6000 rpm for 10 min and washed by deionized water three times. The cell pellet was then dried at

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Fig. 1. Schematic figure for magnetic nanoparticles reactivation and harvesting batch reactor. Fresh cultivation medium (M9) flow rate was 14 lL/min and the HRT in bioreactor was 10 days. The light intensity for M. aeruginosa cultivation was 500 cd sr/m2. The applied MNPs for M. aeruginosa harvesting include raw MNPs, reactivated MNPs and the raw/reactivated MNPs mixture in different treatments, and the dosage was 0.58 g MNPs/g dry-biomass (0.76 g/L). The reaction time was 5 min, 5 min and 60 s in the harvesting, separation and extraction chamber respectively.

105 °C for 4 h. The biomass concentration was then calculated by the ratio of dry M. aeruginosa weight (mg) to the total volume (mL). A Philips EM400 electron microscope operating at 100 kV was used to obtain the transmission electron microscopy (TEM) images of MNPs after harvesting M. aeruginosa and reactivation. The Raman spectrum was obtained by LavRAM Aramis (Horiba, UK). A 632.8 nm He–Ne laser was used as the light source for Raman measurement, and all the samples were washed by deionized water three times before detection. 2.5. Data analysis The MNPs harvesting efficiency was calculated by the ratio of M. aeruginosa dry biomass after separation to that before separation. The attached residual biomass on MNPs was measured after reactivation procedure via the same method as the whole biomass. The detachment efficiency was calculated as the ratio of attached residual biomass to the harvested biomass. All the M. aeruginosa cultivation and MNPs harvesting were conducted in biological triplicates. All the efficiencies were analysed by one-way ANOVA and the significance level was p = 0.05. 3. Results and discussion

dependent on the growing phase of M. aeruginosa that high efficiency (>98%) was only observed in exponential and stationary phases. Similar relationship between the harvesting efficiency and growth phase was also observed in microalgae Nannochloropsis maritima (Hu et al., 2013) and Chlorella zofingiensis (Zhang et al., 2012). In the lag (adaption) and early exponential phase, M. aeruginosa adapted to the new cultivation condition and limited activities were observed in the first several days (Dechatiwongse et al., 2014). The less activities and functional groups on the algal surface consequently resulted in lower electrostatic attraction capacity of M. aeruginosa cells, significantly reducing the MNPs harvesting efficiency as 34.2–86.3% in different treatments. Additionally, the high MNPs separation performance in the late exponential phase might also be attributed to the increasing collision capacity of higher cyanobacterial cell concentration (Ferreira et al., 2011). Different from the green algae, no significant harvesting efficiency decrease was found for cyanobacterial M. aeruginosa that the recovery behaviour maintained over 99% after 10 days cultivation. Considering the algae autolysis as the main reason causing decreasing biomass and the activities of membrane functional groups (Hu et al., 2013), cyanobacterial M. aeruginosa was more robust than green algae and there was no significant loss in electrostatic attraction to affect the harvesting effectiveness.

3.1. Cyanobacterial growth and MNPs harvesting efficiency

3.2. MNPs reactivation

The synthesised magnetic nanoparticles (MNPs) have strong capacity to adsorb M. aeruginosa cells and then separated by the permanent magnet. Before magnetic harvesting, the cyanobacterial suspension has a dark green colour uniformly distributed in the cultivation medium (as shown in graphic abstract). After MNPs harvesting, the cyanobacteria–MNPs complex can be effectively captured by the permanent magnet and further separated from the supernatant. The growth curve and MNPs harvesting efficiency of M. aeruginosa was illustrated in Fig. 2A. After 2 days lag phase, M. aeruginosa achieved the stationary phase after 8 days cultivation and its maximal biomass yield was 869 mg/L. From the ratio of M. aeruginosa dry-biomass to MNPs addition, the MNPs dosage was 0.58 g MNPs/g dry-biomass. The MNPs harvesting efficiency was highly

The stable M. aeruginosa biomass concentration in the effluent of the bioreactor was 865 mg/L and the MNPs harvesting performance was shown in Fig. 3(A). M. aeruginosa had high stability and there was no significant cell aggregates without MNPs addition (as illustrated in graphic abstract). Raw MNPs materials had strong electrostatic attraction capacity to adsorb M. aeruginosa cells and the harvesting efficiency was 99.6%. It was also found that the harvesting efficiency was positively related to the harvesting time (data not shown), and 5 min of the harvesting time was the optimal reaction time to obtain the short operation and high harvesting efficiency. The time dependant harvesting efficiency was explained previously that the cell aggregates had viscous drag and random Brownian force and required longer time to be captured by MNPs (Hu et al., 2014).

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Fig. 2. Magnetic nanoparticles harvesting efficiency at different M. aeruginosa cultivation stage (A) and under different pH conditions (B). The Microcystis aeruginosa biomass was taken and monitored at Day 0, 2, 4, 6, 8, 10 and 12.

From the previous study (Zhang et al., 2011), the morphological structure of MNPs is the nano-scale round Fe3O4 nanoparticles with negative charge and b-FeOOH nanorods with positive charge. The average size of Fe3O4 (brown arrow) is approximately 20 nm and the b-FeOOH nanorods (blue arrow) are around 100–200 nm in length and 30–50 nm in diameter (Fig. S1B and S1C in Supplementary material). They are both much smaller than the average diameter (1 lm) of the cyanobacterial M. aeruginosa (Fig. S1A). The spheroid microstructures on the surface of M. aeruginosa can also effectively aggregate nanoparticles (Fig. S1D) and favour the adsorption process (Higgins et al., 2003). With the increasing reactivation times, MNPs gradually lost the adsorption capacities due to the residual biomass on the surface, as illustrated in Fig. 3(A). The MNPs harvesting efficiency dropped to 94.1% after one reactivation, and further declined to 61.7% and 59.1% after 4 and 5 times reactivation. The harvested biomass concentration therefore decreased from 865 mg/L (raw MNPs) to 532 mg/L (5 times reactivation). From the TEM images of MNPs

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Fig. 3. M. aeruginosa harvesting efficiency with MNPs reactivation. (A) For MNPs with solvent-extraction reactivation and (B) for raw/reactivation MNPs mixture (1:2, m/m). Magnetic nanoparticles dosage was 0.5 g/L and M. aeruginosa biomass was 865 mg/L. Reaction time for M. aeruginosa harvesting was 5 min and the MNPs reactivation solvent was chloroform:methanol (2:1, v/v).

after ultrasonic chloroform:methanol solvent reactivation, the whole M. aeruginosa cell was effectively removed but numerous cyanobacterial debris was captured and identified on the surface of MNPs (Fig. S1E and S1F), explaining the declining harvesting capacities. The detachment efficiency after the first reactivation process was calculated as 98.5% (12.6 mg/L residual biomass on MNPs), declining to 91.6% after 5 times reactivation (44.6 mg/L residual biomass on MNPs). Comparing to other reactivation protocols of acidic treatment for 180 min (Prochazkova et al., 2013), this chloroform:methanol solvent reactivation took only 60 s and the reactivated MNPs maintained the high harvesting performance. The detachment efficiency was relatively lower than previous detachment under acidic or alkaline conditions (Lee et al., 2014; Xu et al., 2011), because the disruption was carried out by ultrasonic treatment under pH 7.0 when the strong electrostatic attraction between M. aeruginosa and MNPs still existed. Some cyanobacterial debris might be strongly captured by MNPs and the cyanobacteria–MNPs complex was hard to be broken. More

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importantly, the advantage of this ultrasonic solvent treatment is that M. aeruginosa solvent extraction is the routine biomass pretreatment method and the suspension can be directly used for further biofuel refinement and production. Fig. S2 illustrated the Raman spectrum of M. aeruginosa and MNPs, before and after harvesting, further showing the interaction between cyanobacterial cells and magnetic nanoparticles. The synthesised MNPs have both characteristic bands for Fe3O4 (670 and 1320 cm1) and b-FeOOH nanorods (542 cm1) (Wang et al., 2009). Significant bands of carotenoids (1070 cm1) and chlorophyll (747 and 1376 cm1) are identified on M. aeruginosa (blue), similarly to previous research (Lahr and Vikesland, 2014; Vitek et al., 2014). However, the 1306 cm1 carboxylate band is also dominant on M. aeruginosa membrane (Choi et al., 2007), but showing weak Raman signal in our study. After MNPs harvesting, only carotenoids and chlorophyll bands are observed (green curve in Fig. S2), whereas strong carboxylate bands and lipids fingerprints (Lahr and Vikesland, 2014) exist on MNPs after reactivation (purple curve). It is reported that M. aeruginosa cells are rough with some negatively charged surface functional groups, including galactolipids, phospholipids and carboxylate. The positively charged b-FeOOH nanorods therefore can specifically recognise and bind these groups by electrostatic attraction (Fig. S1D). To mitigate the MNPs dosage for biomass harvesting and manage practical harvesting operation for engineering application, the mixture of raw and reactivated MNPs was applied in the harvesting chamber (Fig. 3(B)). With the mixing ratio of 2:1 (reactivatedMNPs/raw-MNPs, mass/mass), the M. aeruginosa harvesting efficiency remained at high level, ranging from 93.8% to 96.4% after 5 times cycle. The harvested biomass yield was from 811 to 833 mg/L. The usage amount was reduced by 67% from the harvesting with raw MNPs with this practical reactivation technique and MNPs mixture protocol. Compared to previous magnetic harvesting methods with various MNPs types (Hu et al., 2014; Lee et al., 2013; Prochazkova et al., 2013; Wang et al., 2014; Xu et al., 2011), the reactivated/raw-MNPs for M. aeruginosa harvesting had similar performance but low dosage of 0.20 g MNPs/g dry-biomass (Table 1). The high harvesting performance and low MNPs usage indicated that this MNPs reactivation technology can effectively mitigate the harvesting cost and further waste disposal. 3.3. pH dependent harvesting Fig. 2B showed the significant impacts of pH value on MNPs harvesting efficiency of M. aeruginosa. The MNPs harvesting performance decreased with the increasing pH value in the cultivation

medium, 99.65% at pH = 3.0 and 82.70% at pH = 11.0 for raw MNPs materials. MNPs with different reactivations had similar trends to the raw MNPs and the reactivated/raw MNPs mixtures. The Fe3O4 nanoparticles behaved positively charged when the pH value below 6.0 (Prochazkova et al., 2013) and it can consequently increase the zeta potential of MNPs for higher electrostatic attraction with the negatively charged extracellular organic matters on the cell surface of M. aeruginosa (Qu et al., 2012). Similar to previous research on Botryococcus braunii (Xu et al., 2011), the results suggested that the optimal cultivation and harvesting condition for M. aeruginosa was the weak acidic medium from pH 5.0 to 7.0. After reactivation by ultrasonic chloroform:methanol solvent treatment, MNPs harvesting efficiency ranged from 85.46% (pH = 11.0) to 96.77% (pH = 3.0) (Fig. 2B). Additionally, the harvesting performance of reactivated/raw MNPs mixture was less affected by the pH value in the cultivation medium. The recovery rate was similar when the pH value was <9.0. 3.4. MNPs harvesting isotherm The adsorption isotherm of MNPs for M. aeruginosa harvesting reveals the equilibrium between the adsorbed cells and their equilibrium concentration in aqueous phase. The Langmuir isotherm model represents the monolayer adsorption mechanism, as described in Eq. (1). Freundlich isotherm model demonstrates both monolayer and multilayer adsorptions by considering the heterogeneous surfaces possessing different sorption energy sites, as shown in Eq. (2).

Q e ¼ Q max

K LCe 1 þ K LCe

ð1Þ

Q e ¼ K F C 1=n e

ð2Þ

Here, Qe (g/g MNPs) refers to the absorbed M. aeruginosa on the surface of MNPs, and Ce (g/L) represents the equilibrium M. aeruginosa biomass concentration in the suspension. Qmax (g/g MNPs) is the maximum adsorption capacity for monolayer adsorption in Langmuir isotherm model, and KL (L/g) is the Langmuir constant associated with adsorption energy. KF (g/g MNPs) represents the adsorption capacity in both monolayer and multilayer mechanism, and 1/n is the heterogeneous sorption sites. Either Langmuir or Freundlich isotherm model can be expressed in the linear form as shown in Eqs. (3) and (4), respectively. The adsorption isotherm of M. aeruginosa was illustrated in Fig. 4 and the key parameters were listed in Table 2.

Table 1 Comparison of different MNPs on microalgae and cyanobacteria harvesting. Microalgae or cyanobacteria

Biomass (g/L)

MNPs type

Reaction time (min)

Recovery efficiency (pH = 7)

MNPs dosage (g/L)

References

Botryococcus braunii Botryococcus braunii

1.80 1.80

2–3 10

>98% 95%

0.075 0.12

Xu et al. (2011) Wang et al. (2014)

Chlorella ellipsoidea Chlorella ellipsoidea Chlorella ellipsoidea

0.80 0.75 0.70

2–3 2 10

>98% >97% 96%

0.30 0.50 0.12

Xu et al. (2011) Hu et al. (2014) Wang et al. (2014)

Chlorella vulgaris

0.30

Raw MNPs Poly(diallyldimethylammonium chloride) functionalized MNPs Raw MNPs Polyethylenimine functionalized MNPs Poly(diallyldimethylammonium chloride) functionalized MNPs Raw MNPs

10

95%

0.12

Chlorella sp. KR-1 Chlorella sp. Nannochloropsis maritima Microcystis aeruginosa Microcystis aeruginosa

1.0 0.19 1.02

Chitosan functionalized MNPs Chitosan functionalized MNPs Raw MNPs

3 6 4

99% >95% 97.5%

1.4 0.30 0.12

Prochazkova et al. (2013) Lee et al. (2013) Toh et al. (2014) Hu et al. (2013)

0.869

Raw MNPs

5

99.6%

0.50

This study

0.869

Reactivated/raw MNPs mixture

5

>93.8%

0.17

This study

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Table 3 Parameters of Qmax and KL in Langmuir isotherms model for magnetic nanoparticles after reactivation. Reactivation times

Qmax (g/g MNPs)

KL (L/g)

1 2 3 4

3.42 3.23 2.84 2.95

20.57 10.40 3.79 1.64

Fig. 5. Zeta potential of MNPs harvested M. aeruginosa and reactivated MNPs.

Fig. 4. Adsorption isotherms of M. aeruginosa on raw magnetic nanoparticles (A) and magnetic nanoparticles with different reactivation times (B). Magnetic nanoparticles dosage was 0.5 g/L. Reaction time for M. aeruginosa harvesting was 5 min.

Table 2 Parameters of Qmax and KL in Langmuir isotherms model and KF and n in Freundlich isotherms model. Isotherm

Model equation

Langmuir

Q¼

Freundlich

Q ¼ K F C 1=n

KL C Q max 1þK LC

Parameter

Value

Qmax (g/g MNPs) KL (L/g) R2

3.74 311.64 0.9964

KF (g/g MNPs) 1/n R2

30.34 0.582 0.9385

Ce 1 Ce ¼ þ Q e Q max  K L Q max

ð3Þ

1 lg Q e ¼ lg K F þ C e n

ð4Þ

The harvesting efficiency has negative relationship with M. aeruginosa biomass concentration. At low concentration of 50 mg/L, the MNPs harvesting efficiency achieves 99.8%, and gradually declines to 99.6% and 97.2% when biomass concentration is 900

and 1800 mg/L. Langmuir isotherm equation fits better with the adsorption isotherm of M. aeruginosa, indicating the monolayer adsorption of cyanobacterial cells on MNPs surface. Considering the smaller size of M. aeruginosa cells than microalgae, M. aeruginosa have significant higher specific surface area for more adsorption of MNPs, showing remarkable lower adsorption capacity (Qmax, 3.74 g/g MNPs) than B. braunii (55.9 g/g MNPs) and Chlorella ellipsoidea (5.83 g/g MNPs) (Xu et al., 2011). The high KL value (311.64 L/g) further shows the high binding affinity of M. aeruginosa to MNPs, suggesting strong binding strength between M. aeruginosa cells and b-FeOOH nanorods. As for Freundlich isotherm model, the 1/n value is 0.585, much higher than the normal value of microalgae from previous research (Xu et al., 2011). Though it is still between 0.1 and 1 to show easy adsorption, the high value suggests its less favourability for MNPs harvesting but feasibility for desorption and reactivation. Based on the concentration of M. aeruginosa biomass in the bioreactor, 0.5 g/L (0.58 g MNPs/g dry-biomass) raw MNPs is suggested as the optimal dosage for continuous cyanobacterial cultivation and harvesting. The Qmax and KL values of reactivated MNPs (Table 3) have strong negative relationship with the reactivation times. The Qmax decreases from 3.74 g/g raw-MNPs to 2.95 g/g reactivated-MNPs after 4 times reactivation, and the KL decreases to only 1.64 L/g. The results indicate that MNPs have lower adsorption capacity after reactivation treatment due to the insufficient disruption of cyanobacteria–MNPs complex in the ultrasonic solvent treatment. Further zeta potential analysis provided more evidence on the adsorption capacity change of MNPs after reactivation, as illustrated in Fig. 5. The zeta potential of M. aeruginosa and raw MNPs is 17.6 and 28.6 mV (pH 7.0) in this study. The zeta potential

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for Fe3O4 nanoparticles and b-FeOOH nanorods are previously reported from 10 to 20 mV and 10–20 mV at pH 7.0, respectively (Hadjoudja et al., 2010; Kumagai et al., 2007; Prochazkova et al., 2013; Xu et al., 2011). The apparent zeta potential of synthesised MNPs in this study is therefore the combined effects of Fe3O4 and b-FeOOH. Though behaving negatively charged, the MNPs still have high proportion of positively charged b-FeOOH nanorods to specifically bind negative M. aeruginosa cells. During the harvesting and reactivation process, the zeta potential shows a cycling behaviour. The values of harvested cyanobacteria–MNPs complex range from 20.5 to 28.4 mV, whereas they are 14.4 to 18.3 mV for the reactivated MNPs. Together with previous detachment analysis, the ultrasonic chloroform:methanol solvent treatment has acceptable detachment efficiency to disrupt the cyanobacteria–MNPs complex and recover the zeta potential of MNPs, particularly the b-FeOOH nanorods, to reactive its harvesting capacity. 4. Conclusions This research developed a new technique of magnetic nanoparticles reactivation to harvest cyanobacteria. The harvesting efficiency for M. aeruginosa was above 99% with 0.58 g MNPs/g drybiomass dosage, and the b-FeOOH nanorods held the positive charge to electrostatically capture negatively charged M. aeruginosa cells. The isotherm and morphological analysis revealed the monolayer adsorption equilibrium between cyanobacterial cells and MNPs. Ultrasonic solvent treatment could disrupt the cyanobacteria–MNPs complex and reactivate MNPs for reuse. In continuous batch reactor, M. aeruginosa harvesting efficiency successfully achieved >98%. Reducing 67% MNPs consumption, this method is suitable for sustainable bioenergy recovery with minimal disposal contamination. Acknowledgement The authors would like to thank National Natural Science Foundation of China (Nos. 41301331, 41301252) for financial support. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.biortech.2015.04. 068. References Abdelaziz, A.E.M., Leite, G.B., Hallenbeck, P.C., 2013. Addressing the challenges for sustainable production of algal biofuels: II. Harvesting and conversion to biofuels. Environ. Technol. 34, 1807–1836. Cerff, M., Morweiser, M., Dillschneider, R., Michel, A., Menzel, K., Posten, C., 2012. Harvesting fresh water and marine algae by magnetic separation: screening of separation parameters and high gradient magnetic filtration. Bioresour. Technol. 118, 289–295. Chen, C.-Y., Yeh, K.-L., Aisyah, R., Lee, D.-J., Chang, J.-S., 2011. Cultivation, photobioreactor design and harvesting of microalgae for biodiesel production: a critical review. Bioresour. Technol. 102, 71–81. Choi, J., Kim, J.C., Lee, Y.B., Kim, I.S., Park, Y.K., Hur, N.H., 2007. Fabrication of silicacoated magnetic nanoparticles with highly photoluminescent lanthanide probes. Chem. Commun., 1644–1646 Christenson, L., Sims, R., 2011. Production and harvesting of microalgae for wastewater treatment, biofuels, and bioproducts. Biotechnol. Adv. 29, 686–702. Dechatiwongse, P., Srisamai, S., Maitland, G., Hellgardt, K., 2014. Effects of light and temperature on the photoautotrophic growth and photoinhibition of nitrogenfixing cyanobacterium Cyanothece sp. ATCC 51142. Algal Res. 5, 103–111. Fakhrullin, R.F., Shlykova, L.V., Zamaleeva, A.I., Nurgaliev, D.K., Osin, Y.N., GarciaAlonso, J., Paunov, V.N., 2010. Interfacing living unicellular algae cells with biocompatible polyelectrolyte-stabilised magnetic nanoparticles. Macromol. Biosci. 10, 1257–1264. Ferreira, L.S., Rodrigues, M.S., Monteiro de Carvalho, J.C., Lodi, A., Finocchio, E., Perego, P., Converti, A., 2011. Adsorption of Ni2+, Zn2+ and Pb2+ onto dry biomass

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