Removal and biodegradation of nonylphenol by different Chlorella species

Marine Pollution Bulletin 63 (2011) 445–451

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Marine Pollution Bulletin journal homepage: www.elsevier.com/locate/marpolbul

Removal and biodegradation of nonylphenol by different Chlorella species Q.T. Gao a, Y.S. Wong b, N.F.Y. Tam a,c,⇑ a

Department of Biology and Chemistry, City University of Hong Kong, Hong Kong SAR, China Department of Biology, The Hong Kong University of Science and Technology, Hong Kong SAR, China c State Key Laboratory in Marine Pollution, City University of Hong Kong, Hong Kong SAR, China b

a r t i c l e Keywords: Biosorption Contamination Growth Microalgae Removal mechanism

i n f o

a b s t r a c t All four Chlorella species, including one commercially available species, Chlorella vulgaris and three local isolates, Chlorella sp. (1uoai), Chlorella sp. (2f5aia) and Chlorella miniata (WW1), had a rapid and high ability to remove nonylphenol (NP). Among these species, C. vulgaris had the highest NP removal (nearly all NP was removed from the medium) and degradation abilities (more than 80% of NP was degraded) after 168 h, followed by WW1 and 1uoai; 2f5aia had the lowest NP degradation ability. The NP removal by C. vulgaris was less affected by growth conditions, but its biodegradation efficiency was significantly increased by temperature and light intensity, suggesting that the biodegradation ability was positively related to photosynthetic and metabolic activities. These results indicated that C. vulgaris was the most suitable species for effective removal and biodegradation of NP, especially under 25 °C with light illumination and initial biomass between 0.5 and 1.0 mg chlorophyll l1. Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction Nonylphenol (NP) is a metabolic product from the microbial transformation of a group of nonionic surfactants, nonylphenol ethoxylates (NPEOs), which are one of the most widely used and cost effective classes of nonionic surfactants in industrial, institutional, commercial and household applications. NPEOs are also important to a number of industrial applications, such as pulp and paper, textiles, coatings, agricultural pesticides, metals and plastics (Vazquez-Duhalt et al., 2005). Due to the wide and long term application of NPEOs, contamination of NP is nearly ubiquitous in the environment and significant concentrations of NP have frequently been detected in all environmental compartments that directly or indirectly receive NPEOs, such as wastewater effluents, surface water, estuaries and coastal water, groundwater, atmosphere, sludge-amended soils, organisms and food. Concentrations up to 110 ng m3 in atmosphere, up to 644 lg l1 in superficial waters, up to 13,700 lg kg1 in soils, up to 1350 lg l1 in untreated wastewater, up to 5.2 lg l1 in estuaries and up to 1840 mg dw kg1 in digested biosolids from wastewater treatment plants have been reported (Blackburn and Waldock, 1995; Rudel et al., 1998, 2003; Sole et al., 2000; Pryor and Walker, 2002; Falkenberg et al., 2003).

⇑ Corresponding author. Address: Department of Biology and Chemistry, City University of Hong Kong, Tat Chee Avenue, Kowloon, Hong Kong, China. Tel.: +852 34427793; fax: +852 34420522. E-mail address: [email protected] (N.F.Y. Tam). 0025-326X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.marpolbul.2011.03.030

The presence of NP in the environment has become an increasing concern because it has been shown to be persistent, toxic and is an endocrine disrupting compound (White et al., 1994). The toxicity data reviewed by Servos (1999) showed that algae were sensitive to NP with median lethal concentrations (LC50) varied between 27 and 2500 lg l1, followed by fish (17–3000 lg l1) and invertebrates (21–3000 lg l1). NPs are readily accumulated in a wide range of marine and aquatic life as they are lipophilic (Ahel et al., 1993; Snyder et al., 2001; Correa-Reyes et al., 2007; Lietti et al., 2007). Algae, particularly green microalgae, being the important primary producers in aquatic ecosystems could accumulate NP and pose significant impact to higher tropic levels via biomagnification along food chains (Correa-Reyes et al., 2007). As conventional and advanced wastewater treatment is either not suitable or inefficient for the removal of NP, new methods are needed for effectively removing NP from wastewater before it is discharged into the environment. The handling and running costs of physical and chemical removal methods are often high due to the relatively low contaminant concentration of NP in the environment (Jones et al., 2007; Liu et al., 2009). In recent decades, new technologies employing microorganisms, particularly bacteria, to remove NP from wastewater or environment via biosorption and biotransformation/biodegradation process has been intensively studied (Reviewed by Corvini et al., 2006). However, until now, there have only been a few reports on the role of microalgae in removing NP, despite the fact that microalgae have been employed to remove various organic matters and inorganic nutrients from wastewater and have been applied as tertiary or quaternary treatment units for several decades due to its low capital

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investment and operation costs and high efficiency (de la Noüe et al., 1992). A number of recent studies have demonstrated that microalgae could remove a variety of toxic organic pollutants (Lei et al., 2002; Pinto et al., 2002; Tam et al., 2002; Hirooka et al., 2005), but the ability of microalgae to remove and degrade NP has never been reported. Among different microalgae, Chlorella genus has been widely reported for its ability to remove various types of pollutants from wastewater, and different Chlorella species have different removal abilities (Munoz and Guieysse, 2006). Additionally, it is well accepted that microalgae grown in polluted environments might be more resistant to the pollutant, and such tolerant species may have a higher accumulation and biotransformation capacity (Wong and Pak, 1992). The present study aims to compare the NP removal and degradation ability among four different Chlorella species, including one commercially available species, Chlorella vulgaris, and three local isolates, Chlorella minata (WW1), Chlorella sp. (1uoai) and Chlorella sp. (2f5aia) and identify the most effective species. The study also attempts to understand the mechanisms involved in NP removal and biodegradation and how growth conditions affecting the NP removal and biodegradation efficiency by the most effective Chlorella species. 2. Materials and methods

were incubated on a rotary shaker at a speed of 160 rpm at 25 °C (unless otherwise stated), a light intensity of 40 lmol s1m2, and a 16/8 h light/dark cycle. At time intervals of 12, 24, 96 and 168 h, triplicate flasks from each algal culture were retrieved for the measurement of residual NP in the medium, adsorbed onto the cell surface and absorbed inside the cell, as well as the determination of chlorophyll a concentration and dry weight. At each sampling time, 5 ml samples were also collected from each of the triplicate control flasks for the measurement of residual NP in the BM medium. 2.2.2. Removal of NP by dead cells of C. vulgaris The same experimental set-up as above was used, except the cells were killed by autoclaving. In brief, algal cells, after harvesting and washing, were re-suspended in sterilized BM at a chlorophyll a concentration of 1 mg l1 as the stock culture, and an appropriate amount of the stock was then autoclaved at 121 °C for 15 min. The cell death was confirmed by the loss of auto-fluorescence of chlorophyll while the cell still maintained its shape and the cell wall was intact under the fluorescence microscope (Axiostop Zeiss, Germany). After cooling down to room temperature, the culture was distributed into a 250 ml conical flask containing 100 ml sterilized BM. At time intervals of 24 and 96 h, triplicate flasks from each treatment were retrieved for the determination of residual NP.

2.1. Chlorella species and culture conditions Four freshwater Chlorella species, namely C. vulgaris, C. miniata (WW1), Chlorella sp. (1uoai) and Chlorella sp. (2f5aia), were used in the present study. The first species was commercially available and purchased from Carolina Biological Supply Company, US, with a spherical shape and a diameter ranging from 3.0 to 6.0 lm, while the other three species were isolated from polluted water in Hong Kong. Chlorella sp. (1uoai) and Chlorella sp. (2f5aia) were spherical shapes with diameters ranging from 2.0 to 5.0 lm and 2.5 to 5.0 lm, respectively. WW1 was spherical to ellipsoidal in shape with a dimension of 3.5–7.5  7.5–12 lm. The axenic microalgal culture of each species was cultivated in 2 l conical flasks containing 1000 ml Bristol medium (BM) (James, 1978), in an environmental chamber illuminated with cool white fluorescent tubes at a light intensity of 175 lmol s1 m2, a diurnal cycle of 16 h light and 8 h dark and at a temperature of 25 ± 2 °C. The culture was aerated with 0.2 lm filtered air at a rate of 35 ml min1 through a mechanical air pump. The cells were harvested at the middle of the log phase of growth, at around 7 days of cultivation, by centrifuging at 5,000g for 10 min at 20 °C. The supernatant was discarded and the pellets were washed by sterilized deionized water, twice, and then re-suspended in deionized water.

2.2.3. Effect of temperature, light and initial biomass amounts on NP removal efficiency of C. vulgaris The same experimental set-up, as described in the above section, was used, except the flasks were incubated at two temperatures, 15 and 25 °C, for determining the temperature effect. For the light effect, half of the flasks were wrapped with aluminium foil to prevent any light penetration, i.e., the flasks were kept in dark. For the effects of biomass, different initial chlorophyll a concentrations, 0.5, 1.0, 2.0, 4.0 and 6.0 mg l1, were added to each flask at the beginning of the experiment. Samples were collected at time intervals of 24 and 96 h, as described above. 2.3. Measurement of cell growth Growth was monitored by the changes of chlorophyll a concentration. A 5 ml culture was harvested by centrifugation at 4,500g for 10 min. The supernatant was discarded and the pellet was resuspended in 5 ml of 95% methanol, incubated at 60 °C for 5 min. and centrifuged again for 10 min. The absorbance of the supernatant at 665 and 652 nm wavelengths was determined with an Agilent 8453 UV–visible spectrophotometer, and the chlorophyll a concentration of the extract was calculated following the formula described by Porra et al. (1989):

2.2. Experimental set-up

Chlorophyll a ðmg l Þ ¼ 16:29  A665  8:54  A652

2.2.1. NP removal by live cells NP, purchased from Sigma–Aldrich (St. Louis, MO, USA) was dissolved in methanol as the stock solution at a concentration of 1 mg ml1. The stock solution was added into 250 ml conical flasks, each containing 100 ml sterilized BM, to achieve an initial NP concentration of 1 mg l1. An appropriate amount of algal cells of each species was then inoculated into the flask to obtain an initial biomass concentration equivalent to 1.0 mg chlorophyll l1. The control flasks, each containing 1 mg l1 NP in 100 ml sterilized BM, but without algae, were also prepared for monitoring the abiotic loses of NP. Solvent control flasks, each containing 100 ml BM with methanol at its final concentration of 0.1% (v/v) and microalgal cells, but without NP, were also setup to ensure that methanol used to dissolve NP had no significant effects on Chlorella cells. All flasks

For dry weight (DW) measurement, aliquots of 20 ml were filtered through a pre-dried (24 h at 105 °C) and pre-weighed Whatman GF-52 filter (47 mm diameter). After filtration, the cells were washed with deionized water and dried in an oven at 105 °C for 24 h. After cooling to room temperature in desiccators, the filters with algal cells were weighed again and the dry weight was calculated and expressed as mg l1.

1

2.4. Determination of residual NP 2.4.1. NP in the medium At each sampling time, 5 ml cultures were withdrawn from the flasks, cells were separated from the culture through centrifugation at 4,500g for 15 min at 4 °C. The supernatant was extracted with

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liquid–liquid microextraction (DLLME), as described by Rezaee et al. (2006), with some modifications. In brief, a 5 ml of sample was injected with 0.2 ml mixture of chlorobenzene and acetone (1:2) in a 10 ml screw cap glass test tube with a conical bottom. After gently shaking, a milky cloudy solution (water/chlorobenzene) was formed in the test tube. The sample was then centrifuged for 5 min at 4,500g. The dispersed fine particles of extraction phase which settled in the bottom of the conical test tube were withdrawn using a 50 ll microsyringe (zero dead volume, cone tip needle). This extraction process was repeated three times and the sediment fractions were combined for further analysis with GC/MS. All the extraction was kept at room temperature (23 ± 2 °C). 2.4.2. NP adsorbed onto cell surface The cell pellets from the above section were washed with 5 ml deionized water, the NP contained in the water was considered as the surface adsorbed NP (Chan et al., 2006) and then extracted with DLLME, as described above and analyzed with GC/MS. 2.4.3. NP absorbed into cells After an appropriate amount of anhydrous Na2SO4 was added, the cell pellets obtained from the above section were mixed with 3 ml dichloromethane–methanol (1:2 v/v); after sonication for 20 min., the sample was centrifuged for 5 min at 3,500g. The cell pellets were extracted two more times and the solvent fractions were combined for further analysis with GC/MS (Correa-Reyes et al., 2007). 2.4.4. Determination of NP with GC/MS Before the Gas Chromatography/Mass Spectrometry (GC/MS) analysis, all the extracted samples were dried with N2 stream, and 50 ll N-methyl-N-(tert-butyldimethylsilyl) trifluoroacetamide (MTBSTFA) (Sigma–Aldrich, St. Louis, MO, USA) was added into the GC vial to dissolve all the analyst; the vial was then incubated at 65 °C for 90 min. After cooling to room temperature, an appropriate volume of acetone was added into the GC vial to make up a final volume to 0.1 ml and the sample was analyzed with GC/MS. The GC–MS analysis was performed using a GC (Agilent technologies, 7890A GC) coupled with a MS (Agilent technologies, 5975C MSD). The capillary column of 30 m  0.25 mm  0.25 lm HP-5 MS was used. The GC injector port was equipped with a 4-mm i.d. glass liner and used in the ‘splitless’ mode, held isothermally at 280 °C. The mass selective detector (MSD) was operated in selected ion monitoring (SIM) mode. The carrier gas was helium and set at a constant flow rate of 1.0 ml min1. The GC column temperature program was as follows: the initial oven temperature was set at 120 °C, held for 5 min, increased from 120 to 300 °C at a rate of 15 °C min1 and maintained at 300 °C for 1 min. The quantitative ion m/z at 277 was selected for the detection of [–C (CH3)3 + ions]. Quantification of NP was based on the internal standard and the calibration curve (0.01–20 mg l1). 2.5. Statistical analyzes The mean and standard deviation of three replicates from each treatment and control, at each sampling time, was calculated. A parametric three-way analysis of variance (ANOVA) was used to test any difference among different Chlorella species, sampling times and treatments. The effect of growth conditions on C. vulgaris was determined by a two-way ANOVA with time and condition as the two main factors. If the interaction factor was significant, a one-way ANOVA was used to test the treatment effect. If the statistical test was significant at p 6 0.05, a Tukey test was employed to find out where the difference occurred (Zar, 1999). All tests were

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carried out using the Statistical Package for Social Science (SPSS 16.0 for Windows, SPSS Inc., USA). 3. Results 3.1. Growth of different Chlorella species exposed to 1 mg l1 NP A significant decrease in growth, in terms of chlorophyll a concentration, and an apparent longer lag phase was observed in all four Chlorella species when exposed to 1 mg l1 NP, as compared with their respective control cultures (Fig. 1). Among four species, the growth of C. vulgaris was completely recovered to the control level at 96 h, whereas the growth of WW1, 2f5aia and 1uoai was still significantly inhibited at the end of the experiment (168 h), when compared to that of their corresponding control, suggesting that C. vulgaris was the most adaptive species to NP. 3.2. Removal of NP by different Chlorella species and its mechanisms The residual concentrations of NP in the medium in the control flasks (without microalgal inoculation) did not show any significant changes during the 168-h experiment (data not shown), indicating that abiotic loss was negligible. The amount of NP remained in the medium inoculated with Chlorella species all decreased substantially within the first 24 h (Table 1). More than 70% and 80% of NP were removed from the medium after 12 and 24 h, respectively (Fig. 2). The decrease of NP was then in a slower process, especially after a 96-h exposure. Further, more than 90% of NP was removed by all species at the end of the experiment (168 h). Among all species, C. vulgaris was the most effective species and removed more than 73%, even after a 12-h exposure; it also displayed the highest NP removal efficiency (99%) after 168 h, followed by WW1 (Fig. 2A and B). Compared to NP removal, the NP biodegradation was in a much slower process. After a 12-h exposure, only 16%, 21%, 35% and 36% of the spiked NP were degraded by C. vulgaris, 1uoai, WW1 and 2f5aia, respectively (Fig. 2), suggesting that most of the NP removed from the medium was due to adsorption/absorption rather than the biodegradation processes. For C. vulgaris, WW1 and 1uoai, the NP degradation efficiency increased progressively with exposure time, and these three Chlorella species had similar NP degradation ability (about 80% of the spiked NP was degraded) after 168 h (Fig. 2A–C). On the contrary, another local isolate of Chlorella, 2f5aia, not only displayed the lowest NP degradation ability but also a more or less constant NP degradation efficiency during the experiment, with only 38% NP degraded at the end of the experiment (Fig. 2D). All four Chlorella species also showed a very high NP bioconcentration capability in a short time. For instance, after 12 h, the cellular NP content varied from 7077 to 18283 lg g1 DW, with the highest biomass uptake found in C. vulgaris (18283 lg g1 DW), probably due to its low DW in the culture, as well as it high NP absorption ability. After the first 12 h of exposure, the content of NP in the biomass decreased gradually with time, due to the biodegradation of NP and the increase of the biomass (Table 1). The NP adsorbed, when compared to the high percentage of NP absorbed into the cells, was much lower in all four species, with less than 10% of the spiked NP being adsorbed onto cell surface and adsorption decreased with exposure time. 3.3. Effect of initial biomass on NP removal and biodegradation by C. vulgaris The amount of biomass, indicated by absolute chlorophyll content per unit of culture, increased with increases of initial biomass concentrations, but the growth of C. vulgaris (in terms of relative chlorophyll content) was significantly reduced with increasing

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NS

6 NS

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Fig. 1. Growth of (A) C. vulgaris (CV) and three local isolates of Chlorella, (B) WW1, (C) 1uoai and (D) 2f5aia, exposed to 1 mg l1 NP at the end of 0, 12, 24, 96 and 168 h (Mean and standard deviation of three replicates were shown. The mean with different letters at each exposure time for each algal species indicated that they were significantly different at p 6 0.05 according to one-way ANOVA test. NS: not significant).

Table 1 The changes of biomass, distribution of NP in medium, adsorbed on cell surface, absorbed into cells and NP uptake per biomass at the end of 12, 24, 96 and 168 h. Mean and standard deviation of three replicates are shown. Microalgal species

DW (mg l1)

Amount of NP (mg l1) NP remained in medium

NP adsorbed onto cell surface

NP absorbed into cells

NP uptake per g biomass (lg g1 DW)

Percentage of NPa Remained in medium

Uptake by cells

After12 h

CV WW1 1uoai 2f5aia

24.1 ± 1.6 42.5 ± 5.3 33.3 ± 2.3 47.1 ± 2.6

0.27 ± 0.05 0.16 ± 0.01 0.27 ± 0.00 0.31 ± 0.03

0.09 ± 0.01 0.04 ± 0.01 0.07 ± 0.01 0.08 ± 0.01

0.48 ± 0.12 0.54 ± 0.12 0.45 ± 0.03 0.26 ± 0.03

18283 ± 3607 13428 ± 1205 9008 ± 1139 7077 ± 336

27 16 27 31

57 58 52 33

After 24 h

CV WW1 1uoai 2f5aia

31.5 ± 2.7 50.0 ± 3.5 47.0 ± 4.2 56.7 ± 13.1

0.18 ± 0.01 0.08 ± 0.03 0.18 ± 0.03 0.19 ± 0.00

0.07 ± 0.00 0.02 ± 0.00 0.07 ± 0.01 0.06 ± 0.01

0.44 ± 0.02 0.45 ± 0.03 0.29 ± 0.02 0.42 ± 0.02

15481 ± 615 9449 ± 241 9739 ± 2781 8405 ± 1077

18 11 18 19

51 47 37 48

After 96 h

CV WW1 1uoai 2f5aia

77.9 ± 3.6 106.7 ± 4.6 232.0 ± 39.4 174.0 ± 13.1

0.08 ± 0.01 0.06 ± 0.01 0.10 ± 0.00 0.07 ± 0.01

0.05 ± 0.01 0.01 ± 0.00 0.01 ± 0.00 0.03 ± 0.01

0.28 ± 0.06 0.19 ± 0.05 0.09 ± 0.01 0.51 ± 0.02

4135 ± 815 1855 ± 350 943 ± 16 3107 ± 268

8 10 9 7

33 20 11 54

After 168 h

CV WW1 1uoai 2f5aia

140.0 ± 6.2 242.0 ± 28.2 397.3 ± 12.9 232.3 ± 17.8

0.01 ± 0.00 0.07 ± 0.02 0.11 ± 0.01 0.06 ± 0.00

0.01 ± 0.00 0.01 ± 0.00 0.01 ± 0.00 0.02 ± 0.00

0.17 ± 0.01 0.09 ± 0.01 0.08 ± 0.02 0.54 ± 0.07

1217 ± 86 380 ± 31 533 ± 16 2392 ± 138

1 7 8 6

18 10 12 56

DW, dry weight. a Percentage of NP in the medium = NP in the medium/Initial NP added  100%; and Percentage of NP uptake = NP adsorbed onto cell surface + NP absorbed into cells/Initial NP added  100%.

initial biomass concentrations after both 24- and 96-h exposures; the higher the initial biomass concentration, the lower the growth rate (Fig. 3A and B). A slight increase in the NP removal efficiency of C. vulgaris with increases in initial biomass concentrations was also found after these two exposure times (Fig. 3C). However, a decrease in the NP biodegradation efficiency was recorded with increases of initial biomass, especially in the treatment with initial biomass equal to or more than 2.0 mg l1 chlorophyll a after 96h of exposure (Fig. 3D).

3.4. Effects of biotic and abiotic factors The NP removal efficiency by dead cells of C. vulgaris was comparable to that of live cells after both 24- and 96-h exposures, with around 90% removal (Table 2). However, the NP biodegradation by dead cells was minimal, while the NP biodegradation percentage of live cells increased progressively with exposure times. These findings suggested that metabolic activity was needed for the degradation of NP by microalgal cells.

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Fig. 2. Removal and biodegradation of NP by (A) C. vulgaris(CV) and three local isolates of Chlorella, (B) WW1, (C) 1uoai and (D) 2f5aia, at the end of 12, 24, 96 and 168 h (Mean and standard deviation of three replicates are shown. The mean with different letters at each incubation time for each algal species indicated that they were significantly different at p 6 0.05 according to one-way ANOVA test. NP removal (%) = (NP conc. in medium of the control  NP conc. in medium with algae)/Initial NP added  100%; NP biodegradation (%) = (NP conc. in medium of control  NP conc. in medium with algae  NP in cells  NP onto cell surface)/Initial NP added  100%).

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Fig. 3. Effect of initial biomass on (A) growth, (B) relative growth, (C) removal and (D) biodegradation of NP by C. vulgaris (Mean and standard deviation of three replicates are shown. The mean with different letters at each incubation time for each algal species indicated that they were significantly different at p 6 0.05 according to one-way ANOVA test. NP removal (%) = (NP conc. in medium of the control  NP conc. in medium with algae)/Initial NP added  100%; NP biodegradation (%) = (NP conc. in medium of control  NP conc. in medium with algae  NP adsorbed/absorbed by cells)/Initial NP added  100%).

The growth of C. vulgaris was closely dependent on temperature, and the chlorophyll concentration at a lower temperature (15 °C) decreased to 60% and 54% of that at 25 °C after 24- and 96-h exposures, respectively (Table 2). Similar to growth, the NP biodegradation ability of C. vulgaris at a lower temperature also decreased to 76% of that at 25 °C, after 96 h. On the other hand, the

cellular NP content was higher at 15 °C than at 25 °C, while the NP removal efficiency was less affected by temperature. Chlorella cells did not grow in the dark, but the NP removal and biodegradation were comparable to that under light condition after 24 h of exposure (Table 2). Under light condition, the cellular NP content decreased with exposure time as more biomass was

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Table 2 Effect of cell viability, light and temperature on the growth, NP removal and biodegradation and NP content per biomass. Mean and standard deviation of three replicates are shown. Biotic/abiotic parameters

Growth (mg chlorophyll l1)

% Removala 96 h

% Biodegradationb

NP uptake (lg g1 DW)

24 h

96 h

24 h

24 h

96 h

24 h

96 h

Cell viability

Live cell Dead cell

1.98 ± 0.04 NA

5.12 ± 0.23 NA

88.2 ± 0.5 90.4 ± 1.2

87.1 ± 0.2 87.9 ± 0.3

23.4 ± 5.0 1.5 ± 0.7

44.6 ± 1.0 6.3 ± 0.5

9286 ± 854 NA

2321 ± 33 NA

Temperature

25 °C 15 °C

1.98 ± 0.04 1.19 ± 0.00

5.12 ± 0.23 2.74 ± 0.19

88.2 ± 0.5 82.1 ± 2.8

87.1 ± 0.2 86.2 ± 0.6

23.4 ± 5.0 16.1 ± 4.5

44.6 ± 1.0 38.9 ± 3.9

9286 ± 854 12171 ± 956

2321 ± 33 3104 ± 188

Light

Light Dark

2.09 ± 0.18 1.57 ± 0.02

4.17 ± 0.55 1.58 ± 0.01

72.5 ± 2.5 72.8 ± 1.2

88.6 ± 1.4 77.37 ± 2.1

22.5 ± 3.3 25.9 ± 7.9

51.6 ± 0.6 31.5 ± 2.0

7917 ± 371 6848 ± 559

2710 ± 164 6351 ± 954

Chl, chlorophyll; DW, dry weight; NA, not applicable. a NP removal (%) = (NP conc. in medium of control  NP conc. in medium with algae)/Initial NP added  100%. b NP biodegradation (%) = (NP conc. in medium of control  NP conc. in medium with algae  NP adsorbed/absorbed by cells)/Initial NP added  100%.

produced, while the content of NP in the biomass of cells grown in the dark kept constant with exposure time. After 96 h, NP biodegradation by cells grown under dark conditions decreased to 61% of that under light conditions, indicating that illumination was an important parameter affecting NP removal and degradation.

4. Discussion In the present study, most of the spiked NP was removed from contaminated BM by all four microalgal species within 24 h. There was no significant difference between dead and live C. vulgaris cells, and growth conditions did not change the NP removal. These results clearly indicated that the initial NP removal was just a passive physicochemical adsorption process. Lei et al. (2002) also reported that the removal of pyrene was comparable between live and dead microalgal cells. Similarly, the sorption efficiency of pesticides was also similar between live and heat-killed bacteria (MacRae, 1985). The capability of freshwater micro- and macro-algae to adsorb pollutants was highly dependent on the cell biovolume and surface area, in particular, the ratio of surface area to volume (Tang et al., 1998). However, the relationship between the amounts of NP uptake and the surface area/volume ratio in the present study was insignificant, as the NP removal efficiency among Chlorella species of different sizes and shapes were comparable. Tsezos and Bell (1989) found that the capacity of cell walls to remove toxic organic pollutants was less than the cell contents. These results suggested that in addition to cell volume and shape, other properties, such as composition and structure of the cell, might also be important in determining NP biosorption. The absorption of NP was also a significant process in the present study, with more than 7000 lg g1 DW taken up in cells after a 12-h exposure, and the highest accumulation was found in the biomass of C. vulgaris (18283 lg g1 DW) (Table 1). These high values were comparable to that reported for macroalgae, such as Cladophora (Ahel et al., 1993), and were much higher than that for a marine microalga, Isochrysis galbana (Correa-Reyes et al., 2007). After the rapid adsorption and absorption processes, NP was gradually degraded with more than 70% of the spiked NP degraded by three Chlorella species, C. vulgaris, WW1 and 1uoai at the end of the 168-h exposure. Such biodegradation was much faster than the previous results from two Microcystis aeruginosa strains, which exhibited more than 60% NP degradation after 12 days of incubation, with different concentrations of NP (Wang and Xie, 2007) and microalgal species from other taxa (Correa-Reyes et al., 2007). These findings suggested that Chlorella species, especially the commercial species C. vulgaris, were more capable of degrading NP than other algal genera, and the mechanisms involved two

processes, a rapid initial passive physiochemical adsorption followed by active absorption, accumulation and degradation process. The removal of organic contaminants was affected by various biotic and abiotic factors (Newsted, 2004; Chan et al., 2006). In the present study, the NP biodegradation was substantially influenced by various growth conditions, including growth temperature, initial biomass concentration and light, although the NP removal efficiency was less affected by these growth conditions. The NP biodegradation was decreased at lower light intensity and lower temperature, probably due to their negative effects on algal cell metabolic activity. On the other hand, high initial biomass significantly reduced NP biodegradation, which may also be related to light intensity, as high cell density could cause self-shading, thus reducing the light intensity per cell. The dependence on light on the removal of a variety of organic compounds was observed by previous researchers. Tsuji et al. (2003) demonstrated that the removal of 2,4-dichlorophenol (DCP) by C. fusca was dependent on photosynthesis. Hirooka et al. (2005) showed that a high removal of bisphenol A (BPA) by C. fusca occurred in light, while dark conditions reduced the removal. In addition to light intensity, the light regime was also important in the removal of q-chlorophenol by marine microalga, Tetraselmis marina (Petroutsos et al., 2007). It has been suggested that the removal of organic pollutants is an enzymatic process dependent on NADPH produced by photosynthesis, thus light becomes an important factor (Thies et al., 1996). A more in-depth study on the effect of various light intensities and inorganic carbons on the NP degradation efficiency by microalgal cells, as well as the relationship between the photosynthetic activity and NP degradation efficiency, should be conducted. The NP biodegradation pathway has been widely investigated in bacteria, while less has been conducted in plants, particularly in microalgae. In bacteria, two possible NP biodegradation pathways were suggested (Gabriel et al., 2007; De Weert et al., 2010). First, NP was transformed to a metabolite with a nitro-group at the ortho-position of the phenol ring. Second, the C-4 atom of the aromatic ring to which the nonyl chain attached was hydroxylated (ipso-hydroxylation). The formed products were then further degraded by bacteria. In higher plants, the NP biodegradation appeared to follow the metabolism of other xenobiotics suggested by Sandermann (1994) and Coleman et al. (1997), that is, hydroxylation followed by subsequent conjugations. The hydroxylation led to the formation of 4-(hydroxy)- and 4-(dihydroxy)- nonylphenols, which were further glucosylated at the phenolic OH-group, and/or further glucosylated, glucuronidated and acylated with molecules such as glucuronic acid or malonic acid, forming several products (Bokern and Harms, 1997; Schmidt et al., 2004). Until now, information related to the metabolism of NP by microalgae has never been published. Nevertheless, the metabolism of other

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phenolic compounds by microalgae displayed similar patterns as that in higher plants. Different freshwater microalgae were found to metabolize BPA to BPA glycosides, which were then released into the culture medium (Nakajima et al., 2007). The metabolic pathway of q-chlorophenol (q-CP) in a marine microalga, T. marina involved glucosyl transfer followed by malonyl transfer (Petroutsos et al., 2007). The diatom Skeletonema costatum was able to detoxify 2,4-dichlorophenol by conjugation to glutathione catalyzed by glutathione S-transferase (Yang et al., 2002). Further studies are needed to identify the major metabolic products and the biodegradation pathways of NP by microalgae. 5. Conclusions All the four Chlorella species investigated in the present study could efficiently remove NP at a concentration of 1 mg l1 from water (almost the highest NP concentration detected in the environment), within a short exposure time (within 24 h), under photoautotrophic conditions. The mechanisms included initial rapid adsorption and absorption, followed by accumulation and biodegradation. The NP biodegradation ability was species-specific and significantly dependent on growth conditions, particularly light, while the removal of NP from contaminated water was less affected by species and growth conditions. Among four Chlorella species, the commercially available species, C. vulgaris was the most suitable one for the NP removal and degradation. Acknowledgment The work described in this paper was supported by the Areas of Excellence Scheme established under the University Grants Committee of the Hong Kong SAR, China (Project No. AoE/P-04/2004). References Ahel, M., McEvoy, J., Giger, W., 1993. Bioaccumulation of the lipophilic metabolites of non-ionic surfactants in freshwater organisms. Environ. Pollut. 79, 243–248. Blackburn, M.A., Waldock, M.J., 1995. Concentrations of alkylphenols in rivers and estuarines in England and Wales. Water Res. 29, 1623–1629. Bokern, M., Harms, H.H., 1997. Toxicity and metabolism of 4-n-nonylphenol in cell suspension cultures of different plant species. Environ. Sci. Technol. 31, 1849– 1854. Chan, S.M.N., Luan, T.G., Wong, M.H., Tam, N.F.Y., 2006. Removal and biodegradation of polycyclic aromatic hydrocarbons by Selenastrum capricornutum. Environ. Toxicol. Chem. 25, 1772–1779. Coleman, J.O.D., BlakeKalff, M.M.A., Davies, T.G.E., 1997. Detoxification of xenobiotics by plants: chemical modification and vacuolar compartmentation. Trends Plant Sci. 2, 144–151. Correa-Reyes, G., Viana, M.T., Marquez-Rocha, F.J., Licea, A.F., Ponce, E., VazquezDuhalt, R., 2007. Nonylphenol algal bioaccumulation and its effect through the trophic chain. Chemosphere 68, 662–670. Corvini, P., Schäffer, A., Schlosser, D., 2006. Microbial degradation of nonylphenol and other alkylphenols – our evolving views. Appl. Microbiol. Biotechnol. 72, 223–243. de la Noüe, J., Laliberté, G., Proulx, D., 1992. Algae and wastewater. J. Appl. Phycol. 4, 247–254. De Weert, J., Viñas, M., Grotenhuis, T., Rijnaarts, H., Langenhoff, A., 2010. Aerobic nonylphenol degradation and nitro-nonylphenol formation by microbial cultures from sediments. Appl. Microbiol. Biotechnol. 86, 761–771. Falkenberg, J.A., Persson, B., Hojsholt, U., Rokkjaer, A., Wahid, M., 2003. Typical values for diffuse soil pollution in Danish urban soil. Report to the Danish Environmental Protection Agency, NIRAS, Allerod, Denmark. Gabriel, F.L.P., Cyris, M., Jonkers, N., Giger, W., Guenther, K., Kohler, H.P.E., 2007. Elucidation of the ipso-substitution mechanism for side-chain cleavage of alpha-quaternary 4-nonylphenols and 4-t-butoxyphenol in Sphingobium xenophagum Bayram. Appl. Environ. Microbiol. 73, 3320–3326. Hirooka, T., Nagase, H., Uchida, K., Hiroshige, Y., Ehara, Y., Nishikawa, J., Nishihara, T., Miyamoto, K., Hirata, Z., 2005. Biodegradation of bisphenol a and disappearance of its estrogenic activity by the green alga Chlorella fusca var. vacuolata. Environ. Toxicol. Chem. 24, 1896–1901. James, D.E., 1978. Culturing Algae. Carolina Biological Supply Company, USA. Jones, O.A.H., Green, P.G., Voulvoulis, N., Lester, J.N., 2007. Questioning the excessive use of advanced treatment to remove organic micropollutants from wastewater. Environ. Sci. Technol. 41, 5085–5089.

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