Accepted Manuscript Coupling process study of lipid production and mercury bioremediation by biomimetic mineralized microalgae Yang Peng, Aosong Deng, Xun Gong, Xiaomin Li, Yang Zhang PII: DOI: Reference:
S0960-8524(17)31069-6 http://dx.doi.org/10.1016/j.biortech.2017.06.165 BITE 18402
To appear in:
Bioresource Technology
Received Date: Revised Date: Accepted Date:
12 June 2017 28 June 2017 29 June 2017
Please cite this article as: Peng, Y., Deng, A., Gong, X., Li, X., Zhang, Y., Coupling process study of lipid production and mercury bioremediation by biomimetic mineralized microalgae, Bioresource Technology (2017), doi: http:// dx.doi.org/10.1016/j.biortech.2017.06.165
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Title Coupling process study of lipid production and mercury bioremediation by biomimetic mineralized microalgae
Author names and affiliations Yang Peng, Aosong Deng, Xun Gong*, Xiaomin Li, Yang Zhang State Key Laboratory of Coal Combustion, School of Energy and Power Engineering, Huazhong University of Science and Technology, 1037 Luoyu Road, Wuhan 430074, PR China
*Corresponding author Tel: +86-27-87542417-8301, Fax: +86-27-87545526 (X. Gong) E-mail address:
[email protected] (X. Gong)
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Abstract Considering the high concentration of mercury in industrial wastewater, such as coal-fired power plants and gold mining wastewater, this research study investigated the coupling process of lipid production and mercury bioremediation using microalgae cells. Chlorella vulgaris modified by biomimetic mineralization. The cultivation was divided in two stages: a natural cultivation for 7 days and 5 days of Hg2+ addition (10~100 μg/L) for cultivation at different pH values (4~7) after inoculation. Next, the harvested cells were eluted, and lipid was extracted. The fluorescein diacetate (FDA) dye tests demonstrated that the mineralized layer enhanced the biological activity of microalgae cells in Hg2+ contaminated media. Hg distribution tests showed that the Hg removal capacity of modified cells was increased from 62.85% to 94.74%, and 88.72% of eluted Hg2+ concentration was observed in modified cells compared to 48.42% of raw cells, implying that more mercury was transferred from lipid and residuals into elutable forms. Keywords: lipid production, mercury bioremediation, biomimetic mineralization, microalgae
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1. Introduction Mercury and its compounds are regarded as the most toxic substances in the ecological environment. A series of physiological damage caused by mercurialism, including mental retardation, reproductive disturbance, antibiotic resistance and other symptoms have been confirmed over several decades (Yarlagadda V, 2007). With the development of human industrialization, large amounts of mercury emissions have been detected in the form of gaseous and aqueous compounds, which are primarily discharged in coal combustion and gold mining (Bakatula et al., 2014; Luo et al., 2011; Xu et al., 2003). In the past few years, large quantities of advanced technologies have been applied for mercury removal in power plants. Catalytic oxidation of elemental mercury with a selective catalytic reduction (SCR) denitrification system followed by oxidized mercury removal in wet flue gas desulfurization (WFGD) system has been identified as one of the most promising methods for mercury removal in flue gas from power plants (Li et al., 2009; Sun et al., 2014a; Sun et al., 2014b; Zhao et al., 2016). Nevertheless, ionic species of mercury (Hg2+) leached from flue gas can be concentrated and maintained in industrial wastewater, such as ash-flushing and slag wastewater (Pavlish et al., 2003). It is vital to remove the Hg compounds in wastewater before being discharged. With the study of marine microbiological chemistry, which includes microalgal bioactive product chemistry and renewable energy from microalgae, microalgae have persistently attracted considerable attention for their capacity to eliminate heavy metals.
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These microscopic photosynthetic organisms are primarily observed in aquatic environments, such as marine and freshwater. The organisms make up the largest group of primary producers all over the world, accounting for more than 32% of global photosynthesis (Priyadarshani I, 2011). Microalgae cultivation requires large amounts of water, high concentrations of nutrients, and suitable light and pH. As a novel biomass resource, microalgae can provide quantities of precious metabolites, including lipids, vitamin, proteins and others (Bingöl et al., 2012; Tedesco et al., 2013; Zeng et al., 2015). Moreover, the remarkable specialty, including high efficiency, faster growth rate compared to higher plants, high selectivity, no additional toxic pollution and environmental friendliness, contributes to microalgae being one of the most ideal biosorbents for fuel gas purification and wastewater treatment (Ajjabi & Chouba, 2009; Menger-Krug et al., 2012; Wilczak & Keinath, 1993). Strong interest has been inspired in coupling process studies on biodiesel microalgae cultivation in wastewater. Many aspects, including application of microalgae in industrial, domestic wastewater and nutrient removal, have been reviewed (Abinandan & Shanthakumar, 2015; Brennan & Owende, 2010). Furthermore, the co-process of microalgae biofuel production and contaminant removal in wastewater is attracting increasing attention all over the world (Orfield et al., 2014; Pittman et al., 2011; Richards & Mullins, 2013; Wu et al., 2014; Yang et al., 2014; Zhou et al., 2012). As a type of photoautrophic microorganisms, living microalgae could remove nitrate, phosphate, pathogens and even heavy metals by physiological synthesis and passive transformation (Brennan & Owende, 2010). Most
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wastewater, including agricultural wastewater, municipal wastewater containing high chemicals and organic substances, have been shown to be suitable growth media for certain species of microalgae, but numerous studies have shown that most heavy metals in those effluents have a significant effect on the growth of microalgae (Wu et al., 2014). However, few studies have focused on microalgae lipid production using mercury-containing wastewater. In general, the toxicity of heavy metals varies considerably for most microalgae species. The order of toxicity is reported to be Hg＞ Cd≈Cu＞Zn＞Pb＞Co＞Cr with several highly special cases (Mallick & Mohn, 2003; Shanmugaprakash & Sivakumar, 2013). Overall, how to obtain high content of biofuels from contained microalgae in heavy metal-contaminated wastewater along with removing the toxic metals remains a great global challenge. Several advanced gene technologies, including chemical and radiation mutagenesis and gene modification, have been employed for biodiesel production from microalgal cultivation, but the low survival rate is still the primary obstacle to broad application (Beacham et al., 2015; Mehtani et al.; Rashid et al., 2014). A novel pretreatment, biomimetic mineralization, which is a technique that synthesizes materials with morphologies and structures similar to those of natural creatures through utilizing bio-structures as templates for mineralization (Bhatnagar & Sillanpää, 2010b; Choi et al., 2016b; Fu et al., 2016; Lloyd-Jones et al., 2004), was first employed to remove the mercury in wastewater and to protect the microalgae from being poisoned by metals during wastewater cultivation. S-doped calcium thiophosphate crystals, in which one
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oxygen atom is replaced by a sulfur atom (Choi et al., 2016a), were first introduced for synthesis as the biomimetic mineralization shell wrapping around the algae cells. Bivalent mercury (Hg2+) was selected as the target metal in simulated wastewater because of its strong toxicity among the heavy metals for microalgae cultivation. The residual quantity of mercury in the extracted lipid and desorption characteristics were investigated, as well. 2. Methods 2.1. Microalgae cultivation and mineralization Individuals of Chlorella vulgaris, a kind of chlorophyta, were isolated from fresh water samples obtained from the Institute of Hydrobiology, Chinese Academy of Sciences. Algae cells were first grown in BG11 medium, adjusted to pH 7.0 without any additions, and maintained at 25±1゜C with 12:12 h of light – dark cycle using 2000 lux light intensity of fluorescent lamps. After the algae density reached approximately 107 cells/mL, which occurred around the 7th day, the cells were harvested by centrifugation at 1200 g for 7 min. Each algae suspension contains 100 mL. After being washed 3 times with 0.05 mol/L NaCl, the algae cells were ordinally impregnated for 15 min at 30 °C in two polyelectrolytes with opposite charges, 1 mg/mL Poly dimethyl diallyl ammonium chloride (PDADMAC, Mw = 10,000-20,000 Dalton, Aldrich) and 1 mg/mL sodium polyacrylate (PAAS, Mw = 5,100 Dalton, Aldrich) in 0.05 mol/L NaCl (Aladdin Ltd., AR, China), to form the structure of cross-linked macromolecules (Shi et al., 2015). This process was performed at crystallization nucleation sites. The alternate immersion
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test was conducted 3 times to ensure that the polyelectrolyte layers could be formed completely. After the LBL self-assembly of polyelectrolytes, the cells were centrifuged and washed with 0.05 mol/L NaCl followed by mineralization. Next, 100 mL 0.111 g/L prepared CaCl2 (Aladdin Ltd., AR, China) ionized water was mixed with the washed algae cells for 6 h to ensure that Ca2+ ions were completely trapped by polyelectrolyte layers. Next, the Ca2+ trapped cell suspension was centrifuged and washed with deionized water until the dissociative Ca2+ ions were totally removed. The subsequent formation of a biomimetic mineral compound (i.e., LBL3-M in further discussion) was initialed by rapid addition of 100 mL 0.108 g/L Na3PO3S (Aladdin Ltd., AR, China) at pH = 9, adjusted with 25% (w/v) ammonium hydroxide (Aladdin Ltd., AR, China) for 4 h. To certify that the S atoms doped in coated shells contributed to mercury biosorption, a blank group with mercury biosorption by Ca-PO4 biomimetic mineralized microalgae synthesized under the same conditions was conducted. A scanning electron microscope (SEM) system, JSM-7100F (JOLE Co. Ltd, Japan), was employed for mineralized shell characterization of freeze-dried cells. A transmission electron microscopy (TEM) system TECNAI G2 20 TWIN (FEI Co. Ltd, Japan) was applied for ultrathin section image observation. X-Ray Diffraction (XRD) measurement (Empyrean, PANalytical B.V., Netherlands) was used for crystal structure analysis. 2.2. Cultivation under simulated mercury contamination The completely mineralized algae cells were separately inoculated in 100 mL undefiled and mercury-contaminated BG11 medium with varied initial Hg2+
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concentrations (10 ug/L, 50 ug/L, 100 ug/L) and were cultivated for 5 days, and three parallel sets were performed for each sample. The pH value and light intensity were the same as described in section 2.1. To investigate the effect of pH, another group was independently run, with pH varying from 4 to 7, and sampling occurred on the 5th day. 100 mL algae cell suspensions were cultivated under regular and mercury stress and were subsequently separated at 1200 g for 7 min, followed by fluorescein diacetate (FDA) (molecular Probes, Sigma) dye tests for metabolic activity assessment. The concentrated algae cells was stained with 100 ug/mL dissolved FDA solution in a dark environment for 15 min. Prior to scanning, the sample containing chlorella cells was placed on a glass slide and covered with a glass slip. A confocal laser scanning microscopy system (CLSM), FV1000 (Olympus Co. Ltd, Japan), equipped with an inverted microscope IX81, was used to obtain all images. A 488 nm Ar laser was responsible for excitation. Emission was obtained by setting the detection band between 500 and 520 nm for FDA fluorescence. Images were scanned at 2 μm z-intervals with two-frame averaging. The optimized parameters were not changed during the subsequent images acquisition. 2.3. Lipid extraction and mercury distribution 100 mL algae cells suspension after regular and mercury stress of varied concentration cultivation separately were cultivated by centrifugation at 1200 g for 10 min. The separated algae cells were then dried in a freeze dryer (80 Pa, -50゜C) and desorbed by agitating with 100 mL of 10% (w/v) EDTA at 150 rpm for 2 hours to
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investigate the desorption capacity in a horizontal shaker. The desorbed cells were subsequently washed with deionized water three times and extracted by n-hexane at 85 ゜C for 36 hours using the Bligh-Dyer(Lu et al., 2008) method to determine the lipid content. The liquid phase of medium, crude lipid extracts, algae residuals and eluents were digested by mixing acids (HNO3:H2O2= 8:2) at 180゜C for 2 h in a muffle furnace. The Hg2+ concentration in each product was determined using an inductively coupled plasma mass spectrometer (ICP-MS), an ELAN DRC-e produced by PerkinElmer Co. Ltd, USA. A subtraction formula was applied to investigate the removal efficiency of mercury by pretreating cells:
Co Ct 100% Co
(1)
where Co and Ct are the initial and final Hg2+ concentration in cell suspensions, respectively. is the removal efficiency of mercury by algae cells. 3. Results and Discussion 3.1. Sample characterization The raw and mineralized cells observed in the optical microscope showed a sharp difference in the extracellular domain. The modified cells were wrapped in a transparent layer, and the raw cells were exposed in circumstances without any capsules. It can be seen that the outer layer had an amorphous mineral structure. The thickness of the outer layer seemed to be uneven in different locations on the cell surface, and the possible reason was that heterogeneous nucleation sites formed by the inhomogeneity of the self-assembled polyelectrolyte layers (Alslaibi et al., 2013). Furthermore, the ultrathin 9
section images showed delicate differences before and after mineralization. The modified cell wall became significantly thicker than that of the raw cell, which was mostly due to the combination of polyelectrolyte layers with the oxygen-containing functional groups on the cell wall (Bhatnagar & Sillanpää, 2010a). The mineral structure was observed on TEM, which was amorphous, rather than neatly arranged. With the coating of polyelectrolytes and mineral layers, the extracellular walls of the cells became more compact and restricted from the external environment. It was reported that two stages were involved in the combination of Ca2+ and PO3-4 in the presence of polyelectrolytes (Choi et al., 2016a): 1) the formation of spherical amorphous calcium phosphate, precipitation followed by a rapid mixing of Ca2+ and PO3-4 aqueous solutions, and 2) the metastable highly hydrated calcium phosphate particles join together rapidly into a chainlike structure and later gradually convert to more stable, needle-like hydroxyapatite (HA) crystals. SPO3-3 is considered a derivative anion group of PO3-4 in which an O atom is replaced by an S atom at the same position, which implies that the crystallization of Ca2+ and PO3-4 is performed in the same action(Hameed, 2006). As observed on TEM section images, the chainlike crystals suggest that the mineralization was terminated in the middle transition stage in the presence of PDADMAC and PAAS. This finding is primarily due to the rich distribution of acidic functional groups on the polyelectrolyte layers, which significantly inhibited mass transfer through surface binding. Nonetheless, this metastable phase exposed more activated S atoms than the stable phase, which allowed more Hg atoms to be trapped
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through interstitial voids and lattice imperfections. 3.2. Effect of mineralization on C. vulgaris growth under Hg(II) stress As various hazardous metals are being discharged with industrial sewerage, the viability of microalgae cells cultured in wastewater is an important concern. As a demonstration, the fluorescein diacetate (FDA) dye tests using a confocal laser scanning microscopy system (CLSM) was conducted to evaluate the disturbance of mineralized shells for living cell growth. The greater incorporation between fluorescein and enzymes that occurred in the cell body, the greener the points indicating living individuals. Both raw and LBL3-M exhibited exuberant vitality, which implies that the mineralized shells have no significant inhibition of the metabolic system. With the addition of 10 μg/L Hg(II) ions in medium, a rapid decline in cells was observed in the raw sample, but no particular variation was observed in the LBL3-M. With the increased Hg(II) concentration in the medium (50 μg/L , 100 μg/L), this comparison of living cell proportion between raw and LBL3-M samples turned out to be more remarkable, though the viability of LBL3-M was subdued slightly. In 50 and 100 μg/L Hg contaminated medium, almost no living cells in raw samples were observed, whereas a large number of LBL3-M cells survived in the extreme environment. It has been demonstrated that the biosorption of Hg2+ by living microalgae can be divided into three stages when metal ions are trapped by cells: the binding behaviors of exopolysaccharides, mass transfer by biotic exocellular ligands and detoxification by various metallothioneins (Qiu et al., 2009). One essential factor that dominates the
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toxicity of Hg for living cells is the incorporation of exopolysaccharides and metal ions. With a rich distribution of exopolysaccharides outside the cell wall, the biosorption capacity of heavy metals by microalgae has been reported to be relatively high (Singh et al., 2012), whereas the high concentration of heavy metals leads to aberrations, even death, for living microorganisms. During the PDADMAC-PAAS self-assembled modification mentioned above, most oxygen containing functional groups, which are responsible for metal binding, are occupied by polyelectrolyte layers (Shi et al., 2015; Wang et al., 2015), as observed in Fig. 1f. 3.3. Effect of mineralization on lipid extraction of microalgae C. vulgaris under Hg(II) stress The lipid yields of raw and LBL3-M samples under Hg-contaminated cultivation were investigated, as presented in Fig. 1. For the raw cells, the lipid yield cultivated under low Hg2+ concentration stress (i.e., 10 ug/L) was 9.17%, almost level with the blank group under no Hg addition (8.71%). This finding was probably due to the so-called Hormesis Effect (Kaiser, 2003; Mehta & Gaur, 2001), a dose-response action characterized by low-dose stimulation and high-dose inhibition. With increasing Hg2+ concentration in the medium, a sharp decline was observed, 6.01% and 5.12% under 50 ug/L and 100 ug/L, respectively. The cell wall was observed to have irreversible deformation, and the biological activity was significantly suppressed. For mineralized, modified cells, no obvious decrease in lipid yield was detected with increasing Hg2+ concentration. As presented in Fig. 1, the morphology of mineralized, modified cells
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after Hg2+ addition remained unchanged, which implies that the mineralized, modified surface showed strongly protection for intracellular organization to ensure normal physiological metabolism and fat accumulation. 3.4. Effect of mineralization on Hg2+ removal Not only is biological protection required, but extraordinary adsorption capacity for Hg removal is indispensable. The sulfo-calcium phosphate was shown to be a kind of extraordinary adsorbent for heavy metals. A series of Hg 2+ removal tests were conducted in detail to assess the promotion of sulfur with a mineralized surface for the biosorption ability of microalgae. The effect of pH and time on the removal of Hg2+ by raw and LBL3-M cells was investigated as shown in Fig. 2 (a). The range of pH was set from 4 to 7, close to the actual industrial wastewater conditions. With increasing pH, the adsorption of raw cells improved at lower pH values (from 4 to 5) followed by a slight decline with relatively higher pH values (from 5.5 to 7), and this trend agrees with several earlier studies(Ponce et al., 2015; Vilar et al., 2005). Briefly, the extent of competitive adsorption between Hg2+ and H+ dominated the adsorption capacity under lower pH, whereas complex formation of Hg(OH)2 in the liquid phase significantly affected the combination of Hg2+ and organic functional groups on the cell surface. No peak value was observed for LBL3-M, and the adsorption efficiency improved with the increase in pH. That result is attributed to a higher degree of crystallinity at relatively higher pH values, resulting in more sulfur atoms loaded on the outer mineralized surface (Tam et al., 2009). Three groups in 10, 50, and 100 ug/L were conducted. The
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optimal capacity for raw cells was observed at a pH of 5.0±0.2 at 62.85%, whereas the best capacity for LBL3-M occurred at a pH of 7.0, 94.74%. Both optimal values were obtained in the group that received 10 ug/L, showing that the increase in initial Hg2+ concentration had a negative impact on adsorption efficiency. The effect of contact time on Hg2+ removal efficiency was also investigated, as shown in Fig. 2 (b). For the raw cells, an increasing trend in adsorption efficiency was observed with the increase in cultivation time, approximately 60% on the 5th day. No significant change in removal efficiency for LBL3-M was found over 5 days, reaching 95%. The optimal efficiency for raw and LBL3-M were both obtained on the last day, 61.47% and 94.70%, respectively, in 10 ug/L Hg2+ contaminated medium. It was found that the biosorption of microalgae consists of two stages: a rapid extracellular passive adsorption and a slow physiological intracellular positive diffusion (Qiu et al., 2009). The adsorption equilibrium for raw cells was not easily observed, implying that the absorbed Hg2+ ions were constantly transferred into algae cells in certain ways (Hsu-Kim et al., 2013). From the kinetic data, the modified LBL3-M adsorption reached equilibrium rapidly in the earlier cultivating period (day 1), demonstrating that the adsorption rate of Hg2+ was dominated first by extracellular passive adsorption, and the intracellular transfer was completely inhibited. Moreover, aiming to investigate the positive effect of doped S atoms in mercury biosorption, a control biosorption group was included with Ca-PO4 biomimetic mineralized microalgae (as the same assembly process), as shown in Table 1. With the
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mercury(II) concentration increased, the biosorption, desorption and residual content had the same trend, that is, a significant increase was observed. More mercury ions were absorbed on S-doped shells and easily desorbed as well with the EDTA elution. 3.5. Effect of mineralization on Hg distribution in microalgae cells To investigate the proportion of mercury contained in products after lipid extraction, mass balances of Hg2+ for raw and LBL3-M cells were conducted as illustrated in Fig. 3. In raw cells cultivated in 10 ug/L Hg2+ contaminated medium, the ratio of Hg in the liquid phase, lipid extracts, cell residuals and eluent were 38.53%, 8.74%, 7.42% and 48.42%, respectively. With the increase in Hg2+ added to the medium, the proportion in the liquid phase increased to 52.2%, owing to the decline of adsorption capacity. Hg2+ in lipid extracts and residuals increased to 11.58% and 8.42% at 100 ug/L, respectively. For LBL3-M, there were sharp decreases in Hg2+ concentration in the liquid phase, 11.28%, 18.97% and 29.42%, cultivated in 10, 50, and 100 ug/L Hg2+ medium, respectively. The Hg2+ that remained in lipid extracts and residuals in LBL3-M cells also greatly decreased, which was in contrast to the raw cells. This observation implies that the mineralized surface efficiently adsorbed more Hg2+ in recyclable forms, preventing ionic mercury transfer into the lipid and solid phase during lipid extraction, which can inhibit secondary emissions of mercury in the following resource utilization, such as bio-oil production and biomass pyrolysis. 4. Conclusions In this study, a novel pretreatment, polyelectrolyte self-assembly followed by
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biomimetic mineralization, was first introduced to modify Chlorella vulgaris with the aim of decreasing the toxicity of mercury for microalgae cells in simulated wastewater and enhancing heavy metal biosorption. Tests showed that mineralized shell succeeded in keeping living cells alive and significantly increased the lipid yield in Hg 2+ contaminated medium. The Hg removal efficiency of modified cells was enhanced to 94.74% compared to the raw cells at 62.85%. The study of Hg distribution in products after lipid extraction showed that less Hg2+ content remained in lipids and residuals after modification. Supplementary data Supplementary data associated with this article can be found, in the online version. Acknowledgments This research was supported by the National Natural Science Foundation of China (51661125011) and the Foundation of State Key Laboratory of Coal Combustion (FSKLCCB1406). The authors are grateful to the Graduates' Innovation Fund and the Analytical and Testing Center at Huazhong University of Science and Technology. References 1.
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Figure captions Fig. 1. Lipid yield of raw and mineralization-modified C. vulgaris LBL3-M in different Hg(II) contaminated BG11 medium on the 12th day (pH = 7, light intensity = 2000 lux). Fig. 2. Effect of pH (a) and time (b) on the removal of Hg2+ by raw and LBL3-M cells (a: cultivated at 12th day, b: pH = 7). Both test groups were cultivated under a light intensity of 2000 lux. Fig. 3. Hg2+ distribution in RAW and LBL3-M cells.
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Fig. 1
Lipid yield of raw and mineralization-modified C. vulgaris LBL3-M in different Hg(II) contaminated BG11 medium on the 12th day (pH = 7, light intensity = 2000 lux).
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Fig. 2
Effect of pH (a) and time (b) on the removal of Hg 2+ by raw and LBL3-M cells (a: cultivated at 12th day, b: pH = 7). Both test groups were cultivated under a light intensity of 2000 lux.
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Fig. 3
Hg2+ distribution in RAW and LBL3-M cells.
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List of tables Table 1. Comparison of mercury biosorption from Ca-PO3S and Ca-PO4 mineral shell-coated microalgae (cultivated at 12th day, pH = 7, light intensity = 2000 lux). 10 µg/L
50 µg/L
100 µg/L
remova
desorpt
residual
removal
desorpt
residual
removal
desorpt
residu
l (%)
ion (%)
(%)
(%)
ion (%)
(%)
(%)
ion (%)
al (%)
Ca-PO3S shell Ca-PO4 shell
94.7
88.72
3.69
90.1
81.03
4.81
81.3
70.58
4.68
62.48
58.34
4.14
50.22
42.76
7.46
48.72
40.71
8.01
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Highlights 1. Novelty biomimetic mineralized microalgae cultivated in Hg(II) wastewater. 2. Mineralized layer enhanced activity of cells in Hg(II) wastewater. 3. Higher lipid yield was observed in mineralized modified cells. 4. Removal of Hg(II) by modified microalgae was proved to be 94.74%.
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Graphical abstract
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