Effect of nutrients on the biodegradation of tributyltin (TBT) by alginate immobilized microalga, Chlorella vulgaris, in natural river water

Journal of Hazardous Materials 185 (2011) 1582–1586

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Effect of nutrients on the biodegradation of tributyltin (TBT) by alginate immobilized microalga, Chlorella vulgaris, in natural river water Jing Jin a,b,1 , Lihua Yang a,1 , Sidney M.N. Chan b , Tiangang Luan a,b,∗ , Yan Li a , Nora F.Y. Tam b,∗∗ a b

MOE Key Laboratory of Aquatic Product Safety, School of Marine Sciences, Sun Yat-sen University, Guangzhou 510275, PR China Department of Biology and Chemistry, City University of Hong Kong, Tat Chee Avenue, Kowloon Tong, Kowloon, Hong Kong, PR China

a r t i c l e

i n f o

Article history: Received 19 August 2010 Received in revised form 17 September 2010 Accepted 19 September 2010 Available online 29 September 2010 Keywords: Biodegradation Tributyltin Chlorella vulgaris Nutrients River water

a b s t r a c t The removal and degradation of tributyltin (TBT) by alginate immobilized Chlorella vulgaris has been evidenced in our previously published work. The present study was further to investigate the effect of spiked nutrient concentrations on the TBT removal capacity and degradation in the same alginate immobilized C. vulgaris. During the 14-d experiment, compared to the control (natural river water), the spiked nutrient groups (50% or 100% nutrients of the commercial Bristol medium as the reference, marked as 1/2N or 1N) showed more rapid cell proliferation of microalgae and higher TBT removal rate. Moreover, significantly more TBT was adsorbed onto the alginate matrix, but less TBT was taken up by the algal cells of the nutrient groups than that of the control. Mass balance data showed that TBT was lost as inorganic tin in the highest degree in 1N group, followed by 1/2N group and the least was in the control, but the relative abundance of the intermediate products of debutylation (dibutyltin and monobutyltin) were comparable among three groups. In conclusion, the addition of nutrients in contaminated water stimulated the growth and physiological activity of C. vulgaris immobilized in alginate beads and improved its TBT degradation efficiency. © 2010 Elsevier B.V. All rights reserved.

1. Introduction Organotin compounds (OTCs) are a large class of synthetic compounds with widely varying chemical properties. The trisubstituted OTCs, in particular tributyltin (TBT, tri-n-butyltin, (CH3 CH2 CH2 CH2 )3 Sn+ , CAS Reg. 688-73-3), is a toxic chemical used for various industrial purposes, such as slime control in paper mills, disinfection of circulating industrial cooling waters, antifouling agents, and the preservation of wood [1]. However, TBT is extremely toxic to a number of aquatic organisms, such as mollusc [2,3] and fish [4,5]. Moreover, TBT can bio-accumulate along food chains, and contaminate aquatic food, thereby pose high health risks for humans [6]. In the European Union, the use of TBT is currently banned [7], however, large back-log of TBT is still a concern [8–10]. Due to decades of leaching from antifouling paints applied on boat

∗ Corresponding author at: MOE Key Laboratory of Aquatic Product Safety, School of Marine Sciences, Sun Yat-sen University, Guangzhou 510275, PR China. Tel.: +86 20 84112958; fax: +86 20 84112199. ∗∗ Corresponding author at: Department of Biology and Chemistry, City University of Hong Kong, Tat Chee Avenue, Kowloon Tong, Kowloon, Hong Kong, PR China. Tel: +852 27887793; fax: +852 27887406. E-mail addresses: [email protected] (T. Luan), [email protected] (N.F.Y. Tam). 1 These authors contributed equally to this work. 0304-3894/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jhazmat.2010.09.075

hulls and other underwater structures, TBT has been a common contaminant of marine and freshwater ecosystems exceeding acute and chronic toxicity levels [1]. Therefore, effective remediation of the TBT contaminated water environment is urgently. In a recent decade, biological degradation has been suggested to be the major pathway for the removal of TBT from the natural environment. In addition to bacteria and fungi, microalgae can also biosorb and biodegrade TBT [11–14]. As compared with free algal suspension, cells immobilized in alginate matrix have a comparable or even higher efficiency for the removal of TBT and other environmental contaminants [14–22]. In previous experiments, the alginate immobilized Chlorella emersonni cells had higher respiratory and growth rates than free cells in contaminated water, probably because the immobilized matrix could adsorb the toxicants and protect the cells from direct toxic exposure [22]. Thus, the immobilization technology that entraps the small-sized cells into a matrix not only solves the harvesting problem, it also offers a greater degree of operational flexibility than the free cells [17,23,24]. In natural aquatic environment, nutrients as nitrogen (N), phosphorus (P) and sulfur (S) are major elements, and play important roles in the survival, growth and reproduction of microalgae [25]. The concentration and proportion of nutrients in natural water body, especially the N:P ratio, have direct impacts on algal growth and their ability to adsorb, accumulate and detoxify pollutants

J. Jin et al. / Journal of Hazardous Materials 185 (2011) 1582–1586

[26–30]. However, contaminated water is often limited in nutrients, and the addition of nutrients would enhance cell growth and stimulate cell activity, thereby improve their removal efficiency of pollutants. However, the effects of nutrients on algal growth and debutylation of TBT have been seldom reported. The present study aims to evaluate the effect of different amounts of nutrients on the removal and degradation of TBT in contaminated river water by alginate immobilized Chlorella vulgaris beads. The fate and distribution of TBT during algal treatments was also determined. 2. Materials and methods 2.1. Preparation of immobilized algal beads C. vulgaris purchased from Carolina Biological Supply was mass cultivated in 2 L conical flasks containing Bristol medium with pH maintained at 6.8–7.2. The medium consisted of the following chemicals (mg L−1 ): NaNO3 (250), K2 HPO4 (75), KH2 PO4 (175), MgSO4 ·7H2 O (118), NaCl (25), CaCl2 (25) and trace elements (a mixture of microelements). The cultures were aerated with 0.2 mm filtered air and incubated in an environmental chamber illuminated with cool white fluorescence lights by a light intensity of 175 ␮mol s−1 m−2 with a 16-h light:8-h dark cycle at 22 ± 2 ◦ C. After 1 week of culture, algal cells at their log phase were harvested by centrifugation at 5000 rpm for 10 min at 4 ◦ C. The cell residues were washed, re-suspended in sterilized deionized water. The concentrated algal suspension was then immobilized in alginate matrix followed the method described by Luan et al. [14]. In brief, cell suspension was mixed with 4% (w/v) sodium alginate solution in 1:3 (v/v) to yield a mixture of 3% algal–alginate suspension with a cell density of 3.7 × 108 cells mL−1 . The algal–alginate mixture was dropped into calcium chloride solution (2.5%) using a eight-channel peristaltic pump at a flow speed at 0.6 rpm and a tube internal diameter of 0.5 mm to form uniform algal beads, each of 3 mm in diameter and an initial cell number of 1 × 106 cells bead−1 . The algal beads were left in CaCl2 solution for 12 h for hardening. 2.2. Experimental set-up Natural river water was collected from Lam Tsuen River in Hong Kong SAR. The river was considered as polluted according to 2-year monitoring on the river water quality done by the Environmental Protection Department of the Hong Kong Government. The sample was filtered through Whatman no. 42 filter paper and the background nutrient concentrations were determined by standard Flow Injection analysis (FIA, Lachat QuikChem® 8000, Lachat Instruments, USA). The river water was divided into three groups, each in triplicate, they were: (i) 1N group with nutrients added by the same amount and composition as the Bristol medium mentioned above; (ii) 1/2N group which contained half amount of the nutrients in Bristol medium; and (iii) 0N group as the control in which no nutrient was added. The nutrients composition of three groups was listed in Table 1. TBT in the form of tributyltin chloride (CH3 CH2 CH2 CH2 )3 SnCl purchased from Aldrich Chemical Co., USA was then added to the river water at a concentration of Table 1 Concentrations of nutrients (mg L−1 ) in TBT-contaminated water in two nutrient groups (1/2N and 1N) and the control (0N, that is Lam Tsuen River water). Nutrients

Control (0N)

1/2N Nutrient group

1N Nutrient group

Nitrate N TKNa TPb

1.216 0.931 0.218

10.334 2.990 2.632

19.451 5.049 5.045

a b

Total Kjeldahl nitrogen: organic nitrogen and ammonia. Total phosphorus.

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100 ␮g Sn L−1 , which has been illustrated as an optimal concentration in the investigation of cellular degradation of TBT in a similar system [14]. Immobilized algal beads were mixed with 100 mL TBTcontaminated river water at a density of 10 beads per mL. pH of the medium was adjusted to 7 and the temperature was 22 ± 2 ◦ C. The flasks were shaken on a rotary shaker in an environmental chamber for 14 d. A blank flask without any bead but containing TBT-contaminated river water was prepared, also in triplicate, for the determination of any abiotic loss of TBT during the experiment. Ten milliliters medium together with 100 beads were collected from each flask at 1-, 4-, 8- and 14-d. The residual butyltin concentrations in the medium were analyzed using solid phase microextraction–gas chromatography with flame photometric detector (SPME–GC-FPD) as described below. Ten algal beads were dissolved in 0.1 M sodium citrate, and the cell viability was verified by red fluorescence under a fluorescent microscope at 568 nm, by 400× magnification. The number of viable cells was then counted using an improved Neubauer Haemocytometer (Boeco, Germany) under a light microscope (Zeiss, Axioskopp). Only when survival rate of cells was more than 90%, the following analyses would be performed. Fifty beads were separated into alginate, extracellular (cell wall) and intracellular fractions according to the method described by Tam et al. [31]. The beads were dissolved in 5 mL sodium citrate (0.2 M) and centrifuged at 5000 rpm for 10 min. The supernatant was collected as the alginate fraction. The cell pellets were re-suspended in 1 mL HCl (1 M) and shaken on a rotary shaker at 100 rpm for 1 h, then centrifuged at 4000 rpm for 10 min. The supernatant representing the extracellular fraction was collected and neutralized to pH 7. The cell residues were re-suspended in 1 mL NaOH (6 M) and shaken at 100 rpm overnight, then centrifuged at 4000 rpm for 10 min. The alkaline-washed supernatant representing the intracellular fraction was collected and neutralized to pH 7. The concentrations of TBT, DBT (dibutyltin) and MBT (monobutyltin) in each fraction were determined by the SPME–GCFPD. Because the concentration of organotin in intracellular fraction was very low, the value was combined with that in the cell wall fraction.

2.3. SPME–GC method for analysis of butyltin The sample was added into a 40 mL glass amber vial containing 9 mL nanopure water and 10 mL acetate buffer (0.1 M at pH 5.0). 50 ␮L sodium tetraethylborate (NaBEt4 , 5%) and 20 ␮L tetrabutyltin (TeBT at 1 mg L−1 , as an internal standard) were then added. The vial was immediately placed on a magnetic stirrer to maintain a constant agitation. A SPME fiber coated with 30 ␮m thickness polydimethylsiloxane (PDMS) purchased from Supelco (Bellefonte, PA, USA) was exposed to the headspace over the vigorously stirred sample for 20 min at room temperature. The fiber was then placed in the GC injector, desorbed at 250 ◦ C for 3 min. The quantities and speciation of butyltins were measured using a capillary gas chromatography equipped with a flame photometric detection (GC-FPD, Hewlett-Packard 5890). A 30 m × 0.25 mm i.d. × 0.25 ␮m HP-5MS fuse-silica capillary column coated with 5% biphenyl and 95% dimethylpolysiloxane (from HP Inc.) was employed with splitless injection mode. Both injector and detector temperatures were at 250 ◦ C. The column was held at 70 ◦ C for the first 1 min, increased to 190 ◦ C at 30 ◦ C min−1 , and to 270 ◦ C at 15 ◦ C min−1 , and the final temperature was held for 6 min. Helium was used as the carrier gas at a flow rate of 0.8 mL min−1 . The flame photometer was operated by a hydrogen–air–nitrogen flame and was equipped with a 610 nm optical filter that was selective for tin-containing compounds.

J. Jin et al. / Journal of Hazardous Materials 185 (2011) 1582–1586

250

100

200

80

TBT removal (%)

Cell number (104 cells bead-1)

1584

150

100 0N

50

0

0

2

4

6

8

1/2N

10

1N

12

60

40

20

14

Experimental period (d) Fig. 1. Growth of Chlorella vulgaris cells in alginate immobilized beads at d 0, 1, 4, 8 and 14 in TBT-contaminated water with different concentrations of nutrients. 1N means the 100% nutrients as the Bristol medium; 1/2N means the half nutrients as the Bristol medium; 0N is the control and without any nutrients supplemented. Data are mean ± S.D, n = 3.

2.4. Statistical analyses Mean and standard deviation of the triplicates in each treatment are calculated. The differences among three nutrient groups were evaluated firstly by a parametric one-way analysis of variance (ANOVA). If significant difference was found at p ≤ 0.05, multiple comparison test of Tukey–Kramer was employed. All statistical analyses were performed using SigmaStat Software (Version 2.0, Jandel Scientific, USA). 3. Results and discussion 3.1. Growth of immobilized C. vulgaris cells In all groups, C. vulgaris immobilized in alginate beads were highly viable and continuously multiplied, and the beads remained intact and cell leakage was slight during the 14 d experiment. The cell number of the nutrient groups (1N and 1/2N) increased faster than that of the control (Fig. 1), which is in accordance to the previous results using free microalgae [28,32,33]. However, no significant difference of cell number was observed between the two nutrient groups, showing the nutrients level of the 1/2N group was sufficient to satisfy the maximum nutrients requirement of cell growth of the immobilized algal beads. This also agrees with the previous finding that when sufficient nutrients were added in the medium or the contaminated water, the addition of extra N and P did not show any obvious effect on algal growth [34].

0

0N

0

4

6

8

10

1N

12

14

Experimental period (d) Fig. 2. TBT removal percentages during the 14 d experiment by alginate immobilized C. vulgaris beads at d 0, 1, 4, 8 and 14 in TBT-contaminated water with different concentrations of nutrients. 1N means the 100% nutrients as the Bristol medium; 1/2N means the half nutrients as the Bristol medium; 0N is the control and without any nutrients supplemented. Data are mean ± S.D, n = 3.

plementation has been demonstrated to improve the growth and also physiological activity of algae [28,32,33], thus we hypothesized that the microalgae cells of nutrient groups could have higher TBT degradation than the control, although the latter could uptake the same amount of TBT as the nutrient groups from media during the 14 d experiment. 3.3. Distribution and degradation of TBT To test our above hypothesis, the distribution and degradation of TBT in the system were measured at the end of the experiment. The amounts of residual TBT in the media were comparable among three groups, but the TBT adsorbed onto the alginate of the nutrient groups (1/2N and 1N) were significantly higher than that of the control (Table 2). On the contrary, the residual TBT in the cells significantly decreased with the addition of nutrients. The total TBT remained in the nutrient groups was also less than that in the control, and negatively related to the nutrients level. At the end of the 14 d experiment, the total DBT remained in the system was lower than TBT and MBT (Table 2). The DBT amounts in all fractions of the nutrient groups were significantly lower than that of the control. The amounts of MBT remained in the media of the 1N group were the lowest among three groups, but no significant differences Table 2 Distribution of TBT, DBT and MBT (expressed as ␮g Sn) in different fractions of the immobilized algal bead with different nutrient treatments after 14 d.

3.2. Percentages of TBT removal In all groups, the removal of TBT was very rapid in the first day, and then the residual TBT in media maintained at relatively stable levels during the experiment (Fig. 2), which agrees with the results of our previous study [14]. At d 1, 4 and 8, the removal percentages of TBT in the 1/2N and 1N groups were significantly higher than that of the control (0N). Of note, however, the cell number at d 1 and 4 was similar among three groups (Fig. 1), indicating that the higher TBT removal rates achieved by the nutrient groups at d 1 and 4 were potentially associated with cell physiological activity, but not cell number. At the end of the 14 d experiment, although the control had significantly lower cell number than the nutrient groups, the removal percentages of TBT were similar among three groups. However, considering the removed TBT from media probably was only adsorbed into alginate matrix or cells, the removal rate could not reflect the final bio-degradation of TBT in cells. Nutrient sup-

2

1/2N

TBT Medium Alginate Cell Mean total DBT Medium Alginate Cell

0N

1/2N

1N

2.76 ± 0.15 1.13 ± 0.03a 4.33 ± 0.87c

3.04 ± 0.28 2.53 ± 0.31c 1.33 ± 0.12b

2.42 ± 0.44 1.82 ± 0.14b 0.84 ± 0.08a

8.22

6.90

5.08

0.21 ± 0.02b 0.31 ± 0.01b 0.21 ± 0.06c

0.14 ± 0.03a 0.25 ± 0.02a 0.09 ± 0.01b

0.17 ± 0.02ab 0.25 ± 0.02a 0.06 ± 0.01a

Mean total MBT Medium Alginate Cell

0.73

0.48

0.48

0.11 ± 0.01ab 0.84 ± 0.11 0.04 ± 0.00

0.16 ± 0.07b 0.72 ± 0.18 0.04 ± 0.01

0.08 ± 0.02a 0.71 ± 0.04 0.03 ± 0.00

Mean total

0.99

0.92

0.82

Results are expressed as mean ± S.D, n = 3. Mean values in the same row followed by different letter at the superscript position indicate significant difference at p ≤ 0.05.

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immobilized beads were found to be improved by the addition of nutrients. 4. Conclusions In conclusion, the present study indicated that the addition of nutrients in contaminated water could stimulate the growth and physiological activity of C. vulgaris immobilized in alginate beads, thus improved its capability to degrade TBT to DBT, MBT and inorganic tin. The results give some guidelines for the employment of immobilized microalgae in the remediation of TBT-contaminated groundwater. However, for the real practical application of immobilized microalgae, the effects of other environmental factors such as light cycle, temperature and dissolved oxygen concentration are still waiting for further investigation. Acknowledgements Fig. 3. The mass balance calculation of the fate of TBT in contaminated water treated by alginate immobilized C. vulgaris beads at the end of 14 d experiment. 1N means the 100% nutrients as the Bristol medium; 1/2N means the half nutrients as the Bristol medium; 0N is the control and without any nutrients supplemented. Data are means of triplicate.

were found for the MBT adsorbed onto the alginate and in the cells (Table 2). In order to clearly illustrate the relative abundance of TBT and its debutylation products between three groups, the mass balance was calculated. It showed that TBT in the contaminated water was not only taken up by alginate and cells, but also debutylated to DBT, MBT and even to inorganic tin (Fig. 3). The percentages of DBT and MBT to the total amounts of TBT in the contaminated water were similar among three groups, but the percentages of TBT lost as inorganic tin increased significantly with the amounts of nutrients added, nearly 30% TBT lost in the 1N group, 20% lost in the 1/2N group and only less than 5% lost in the control. On the contrary, the percentage of TBT to total contamination was the highest in the control. These results certified our above hypothesis that the addition of nutrients stimulated cell activity and enhanced TBT debutylation. 3.4. Mechanisms related to the removal of TBT using immobilized algal beads In using immobilized algal beads for the removal of TBT from contaminated water, two main mechanisms were probably involved. The first process was physico-chemical adsorption of TBT onto alginate matrix and algal cell wall. The alginate adsorption was important in removing TBT by immobilized algal beads because the adsorption would enhance the cellular uptake as well as protecting the cells from direct exposure to TBT toxicity. Table 2 indicated that alginate adsorbed more TBT, DBT and MBT than cell fraction (including both adsorption and absorption). Our previous work also found that immobilized algal beads had higher efficiency in the removal of TBT than free cells [11,13]. After physico-chemical adsorption, TBT on the alginate and cell wall was gradually absorbed into cells by an active process. In short, the intracellular TBT was debutylated to DBT and MBT by viable cells, and these two metabolites were then released into the medium. The significance of these biological processes related to TBT degradation in free cells had been reported by Dowson et al. [35]. Other studies also showed that free cells of C. emersonni had the ability to debutylate TBT to DBT then to MBT even to inorganic tin through a cascade of enzymatic reactions [12,31,36]. The present study showed that the TBT removal mechanism in immobilized algal beads was similar to that in free cells. Both adsorption and biodegradation processes of the

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 (Project No. AoE/P-04/2004), National Natural Science Foundation of China (NSFC, no. 20977116 and no. 40903046). We also thank Dr. Zhen-Yu Du (Bergen, Norway) for proofreading of the manuscript. References [1] B. Antizar-Ladislao, Environmental levels, toxicity and human exposure to tributyltin (TBT)-contaminated marine environment. A review, Environ. Int. 34 (2008) 292–308. [2] J.A. Hagger, M.H. Depledge, T.S. Galloway, Toxicity of tributyltin in the marine mollusc Mytilus edulis, Mar. Pollut. Bull. 51 (2005) 811–816. [3] J. Zhou, X.S. Zhu, Z.H. Cai, Tributyltin toxicity in abalone (Haliotis diversicolor supertexta) assessed by antioxidant enzyme activity, metabolic response, and histopathology, J. Hazard. Mater. (2010) 428–433. [4] K. Mochida, K. Ito, K. Kono, T. Onduka, A. Kakuno, K. Fujii, Molecular and histological evaluation of tributyltin toxicity on spermatogenesis in a marine fish, the mummichog (Fundulus heteroclitus), Aquat. Toxicol. 83 (2007) 73–83. [5] C.A. Ribeiro, J. Padros, F.X. Domingos, F.M. Akaishi, E. Pelletier, Histopathological evidence of antagonistic effects of tributyltin on benzo[a]pyrene toxicity in the Arctic charr (Salvelinus alpinus), Sci. Total Environ. 372 (2007) 549–553. [6] P. Rantakokko, T. Kuningas, K. Saastamoinen, T. Vartiainen, Dietary intake of organotin compounds in Finland: a market-basket study, Food Addit. Contam. 23 (2006) 749–756. [7] European Commission, Regulation (EC) no 782/2003 of the European Parliament and of the Council of the European Union, of 14 April 2003, on the prohibition of organotin compounds on ships., Off. J. Eur. Union L 115/1. 2003. [8] C.M. Barroso, M.H. Moreira, Spatial and temporal changes of TBT pollution along the Portuguese coast: inefficacy of the EEC directive 89/677, Mar. Pollut. Bull. 44 (2002) 480–486. [9] A.C. Birchenough, N. Barnes, S.M. Evans, H. Hinz, I. Kronke, C. Moss, A review and assessment of tributyltin contamination in the North Sea, based on surveys of butyltin tissue burdens and imposex/intersex in four species of neogastropods, Mar. Pollut. Bull. 44 (2002) 534–543. [10] J. Novak, S. Trapp, Growth of plants on TBT-contaminated harbour sludge and effect on TBT removal, Environ. Sci. Pollut. Res. Int. 12 (2005) 332–341. [11] A.M.Y. Chong, Toxicity of organotin, and its biosorption and biodegradation by microalgae, MPhil. Thesis, City University of Hong Kong. 2001. [12] N.F.Y. Tam, A. Chong, Y.S. Wong, Removal of tributyltin (TBT) from wastewater by microalgae, in: C.A. Brebbia, D. Almorza, D. Sales (Eds.), Water Pollution Vii – Modelling, Measuring and Prediction, International Series on Progress in Water Resources, Vol. 9, 2003, pp. 261–271. [13] N.F.Y. Tam, A.M.Y. Chong, Y.S. Wong, Removal of tributyltin (TBT) by free and immobilized Chlorella sorokiniana, in: Proceedings of the 4th World Water Congress and Exhibition, Marrakech, Morocco, 2004, CD-Rom format. [14] T.G. Luan, J. Jin, S.M.N. Chan, Y.S. Wong, N.F.Y. Tam, Biosorption and biodegradation of tributyltin (TBT) by alginate immobilized Chlorella vulgaris beads in several treatment cycles, Process Biochem. 41 (2006) 1560–1565. [15] P.S. Lau, N.F.Y. Tam, Y.S. Wong, Effect of algal density on nutrient removal from primary settled waste-water, Environ. Pollut. 89 (1995) 59–66. [16] V. Shashirekha, M.R. Sridharan, M. Swamy, Biosorption of trivalent chromium by free and immobilized blue green algae: kinetics and equilibrium studies, J. Environ. Sci. Health A: Toxic Hazard. Subst. Environ. Eng. 43 (2008) 390–401. [17] N.F.Y. Tam, Y.S. Wong, C.G. Simpson, Repeated removal of copper by alginate beads and the enhancement by microalgae, Biotechnol. Tech. 12 (1998) 187–190. [18] S.K. Mehta, J.P. Gaur, Use of algae for removing heavy metal ions from wastewater: progress and prospects, Crit. Rev. Biotechnol. 25 (2005) 113–152.

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