Removal of fluoranthene and pyrene by different microalgal species

Bioresource Technology 98 (2007) 273–280

Removal of Xuoranthene and pyrene by diVerent microalgal species An-Ping Lei a, Zhang-Li Hu a, Yuk-Shan Wong b, Nora Fung-Yee Tam

b,¤

a

b

College of Life Sciences, Shenzhen University, Shenzhen 518060, China Department of Biology and Chemistry, City University of Hong Kong, Hong Kong SAR, China

Received 26 September 2005; received in revised form 10 January 2006; accepted 17 January 2006 Available online 6 March 2006

Abstract In this work, the eYciency of four microalgal species, namely, Chlorella vulgaris, Scenedesmus platydiscus, Scenedesmus quadricauda, and Selenastrum capricornutum to remove Xuoranthene (1.0 mg l¡1), pyrene (1.0 mg l¡1), and a mixture of Xuoranthene and pyrene (each at a concentration of 0.5 mg l¡1) was evaluated. Results showed that removal was algal species speciWc and was also toxicant-dependent. Se. capricornutum was the most eVective species while C. vulgaris was the least eYcient species in removing and transforming polycyclic aromatic hydrocarbons (PAHs). PAHs removal in 7-days of treatment was 78% and 48%, respectively by these two. All species, except S. platydiscus exhibited higher Xuoranthene removal eYciency than pyrene, indicating the latter PAH was generally more stable and recalcitrant. The removal eYciency of Xuoranthene and pyrene in a mixture was comparable, or higher than the respective single compound, suggesting that the presence of one PAH stimulated the removal of the other PAH. © 2006 Elsevier Ltd. All rights reserved. Keywords: Bioaccumulation; Biotransformation; Fluoranthene; Pyrene; Microalgae

1. Introduction Contamination of polycyclic aromatic hydrocarbons (PAHs) in the environment has attracted increasing public and academic concern because some of them are known or suspected mutagens or carcinogens. The major sources of PAHs contamination in the environment are from the coking of coal, distillation of wood, operation of gas works and oil reWneries, runoV from asphalt pavements, vehicular emission, petroleum spills, and the incomplete combustion of fossil fuels and organic matter (Canet et al., 2001; Tang et al., 2005). In recent years, new technology employing microorganisms to remove PAHs from the contaminated environment via biosorption and biotransformation has been proposed (Juhasz and Naidu, 2000; Chávez-Gómez et al., 2003). However, previous studies have been focused mainly on bacteria and fungi, relatively little attention has

*

Corresponding author. Tel.: +852 2788 7793; fax: +852 2788 7406. E-mail address: [email protected] (N.F.-Y. Tam).

0960-8524/$ - see front matter © 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2006.01.012

been paid on the role of microalgae despite algae have been applied in various wastewater treatment processes and have shown to be able to remove toxic organic pollutants by autotrophic growth (Semple et al., 1999; Lei et al., 2002; Tam et al., 2002). Most research work was on the uptake and/or metabolism of a single PAH contaminant, and very few reported the situation with two or more PAHs. In nature, it is common to have a mixture of PAH contamination, and PAH compounds would interact with each other (Canet et al., 2001; Tang et al., 2005). However, the possible stimulatory, antagonistic, and competitive uptake and metabolism of the combined PAHs received little exploration (Dean-Ross et al., 2002; Herwijnen et al., 2003). The present experiment therefore aims to evaluate the performance of diVerent microalgal species in the removal of PAHs, either as a single compound or in a mixture of two PAHs. Fluoranthene and pyrene were selected because they were the main representative PAHs, predominant in air, sediment and water, and were often existed together in contaminated environments (Tang et al., 2005). Fluoranthene is most ubiquitous and abundant pyrogenic PAH while pyrene is one of the most

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A.-P. Lei et al. / Bioresource Technology 98 (2007) 273–280

common pyrolytic PAHs and the ratio of Xuoranthene to pyrene has been used to indicate the origin of PAHs (Li et al., 1996; Rababah and Matsuzawa, 2002). These two 4ring compounds are therefore frequently chosen as the model compound for ex situ biodegradation studies of high molecular weight PAHs (Sepic et al., 2003). They have the same molecular weight and octanol–water partition coeYcients (Kow), but the solubility of Xuoranthene (0.26 mg l¡1) was nearly double of that of pyrene (0.14 mg l¡1) (Mackay et al., 1992). 2. Methods 2.1. Algal species and culture conditions Four diVerent freshwater microalgal species of the division Chlorophyta were examined (Table 1). The culture conditions including medium for each algal species were the same as described by Lei et al. (2003). All algae were grown under axenic conditions in an environmental chamber illuminated with cool white Xuorescent tubes at a light intensity of 175 mol s¡1 m¡2 in room temperature (23 § 1 °C) and a diurnal cycle of 16 h light and 8 h dark. Cultures were continuously aerated with 0.2 m Wltered air through a mechanical pump for 7–10 days (exponential growth phase) and harvested by centrifugation at 5000 rpm for 10 min at 4 °C. The concentrated cell mass was stored at 4 °C for a maximum of two weeks prior to the start of the experiment. 2.2. Experimental set-up For each microalgal species, 36 conical Xasks (250 ml), each containing 90 ml culture medium, were prepared and autoclaved. After sterilization, 81 l stock solution of Xuoranthene or pyrene, or a mixture of Xuoranthene and pyrene each at 40.5 l were spiked into conical Xasks to obtain the concentrations of Xuoranthene, pyrene, and a mixture of Xuoranthene and pyrene of 1.0, 1.0, and (0.5 + 0.5) mg l¡1, respectively. The Xasks without the addition of Xuoranthene or pyrene were used as the controls. A total of 144 Xasks were prepared for the four algal species. The Xasks were shaken on a rotary shaker at 160–170 rpm

at a light intensity of 50 mol s¡1 m¡2 under the same room temperature and diurnal cycle as in the environmental chamber. Appropriate amounts of algal mass from each species were then inoculated to each conical Xask to obtain an initial biomass of 34.5 mg dry wt l¡1. Triplicate Xasks of the control and the PAH treatments were retrieved after 1-, 4- and 7-days of incubation. Simultaneously, 27 conical Xasks, each containing 90 ml autoclaved SE culture medium, were prepared. These were divided into three groups, nine with Xuoranthene, nine with pyrene and the remaining nine with a mixture of Xuoranthene and pyrene, to monitor the photo-degradation and abiotic removal of PAHs. The procedure was the same as the algal experiments except no algae were added. Triplicate Xasks from each group were collected after 1-, 4-, and 7-days incubation. 2.3. Sampling and analysis The standard methods described by Lei et al. (2002) were employed for algal analysis. After the Xasks were collected, the algal pellet was separated from the medium by centrifugation at 5000 rpm for 10 min at 4 °C. The supernatant (i.e., the medium) was extracted twice with 40 ml ethyl acetate in a separating funnel on a shaker, and the extracted samples were then concentrated to 8 ml by a vacuum rotary evaporator (WMZK-18E). The algal pellets were extracted three times with 4 ml ethyl acetate in an ultrasonic bath for 30 min. The reference standard m-terphenyl (a total of 90 g) was added before extraction to estimate the recovery rate. The organic extract was dried over anhydrous Na2SO4, and the samples were stored at 4 °C prior to gas chromatography (GC) analysis. A Hewlett Packard 5890 GC with a Xame ionization detector (FID) was used. The fused capillary column was 30 m long, 0.25 mm internal diameter, coated with methyl silicone, 0.25 m thickness Wlm. The temperature programme was maintained at 120 °C for 1 min and increased to 280 °C at a rate of 4 °C min¡1 then at 280 °C for 1 min. The injector and detector temperatures were 280 °C and 300 °C, respectively. Helium was used as the carrier gas. QuantiWcation was based on the external standard, Xuoranthene and pyrene calibration curves.

Table 1 Characteristics of the four microalgal species used in this study Algal species

Culture mediuma

Cell size (m)

Surface area S (m2)

Volume V (m3)

S/V ratio

Cell shape

Lipid (g/mg dry wt)

Protein (g/mg dry wt)

C. vulgarisb S. platydiscusc

Bristol SE

3–6 30 £ 70

89.9 238.8

80.1 1490.0

1.10 0.16

208 279

256 302

S. quadricaudab

SE

30 £ 50

258.5

290.8

0.89

165

203

Se. capricornutumb

SE

2 £ 10

126.5

69.6

1.82

Spherical Colonial 4 or 8 cells Colonial 4 cells attached Spiral

232

221

a b c

Bristol medium (James, 1978), SE medium (Song and Liu, 1999). Commercially available species. Local isolate.

A.-P. Lei et al. / Bioresource Technology 98 (2007) 273–280

2.4. Statistical analysis A parametric analysis of variance (ANOVA) test followed by Tukey multiple comparisons were conducted. A three-way ANOVA was Wrst employed to test any signiWcant eVect of diVerent PAH treatments, incubation time and microalgal species on the amount of PAHs uptake by the microalgal cells, remained in medium, or loss from the system. If the interaction factor of microalgal species was signiWcant, a two-way ANOVA would be used to test any signiWcant diVerence among diVerent PAH treatments and incubation time for each microalgal species. If the interaction between PAH treatments and incubation time was statistically signiWcant, the eVect of diVerent PAH treatments on the data collected at each sampling time for each microalgal species would be evaluated by one-way ANOVA test. Similarly, the eVect of incubation time for each microalgal species and each individual PAH treatment would be explored by one-way ANOVA test. All statistical analyses were performed using the software called SPSS (SPSS 10.0 for Windows, SPSS Inc., USA) and Prism (Prism 3.03 for Windows, GraphPad Software, Inc., USA). 3. Results 3.1. Comparison of the removal of pyrene, Xuoranthene and a mixture of these two PAH compounds by microalgal species The percentages of abiotic loss of single Xuoranthene, single pyrene, Xuoranthene in mixture and pyrene in mixture at the end of 7-days incubation were 9.7 § 0.9%, 10.9 § 1.9%, 16.3 § 6.1% and 12.4 § 1.5% (mean and standard deviation of triplicates), respectively. These values were not high and abiotic losses due to photo-degradation

275

were, therefore, ignored in the present study. The three-way ANOVA analyses results suggested that the amount of PAHs uptake by microalgal cells or remained in medium, and the percentage of PAHs loss from the system were dependent on the microalgal species, the incubation time and PAH treatments (P 6 0.05). The amount of PAHs remained in medium decreased signiWcantly from 1- to 7days incubation, indicating that PAHs could be removed by all four microalgal species eVectively (Table 2). The residual PAHs amount in medium also varied among microalgal species. Chlorella vulgaris was least eVective to remove PAHs while Selenastrum capricornutum was the most eVective species. The total amount of residual PAHs in medium containing a mixture of Xuoranthene and pyrene was less than that of a single PAH, suggesting these two PAHs might have some stimulatory eVect. The amount of PAHs uptake by microalgal cells also varied among species, higher in the two Scenedesmus species than in C. vulgaris, and Se. capricornutum exhibited the least amount of PAH uptake (Table 2). Similar to temporal changes of the amount of PAHs remained in medium, the amount uptake by microalgae also decreased signiWcantly from 1- to 7-days incubation, indicating the added pyrene or Xuoranthene was biodegraded or biotransformed by the algal culture. The presence of pyrene had an enhancement eVect on degradation and transformation of Xuoranthene as the percentage loss of Xuoranthene in single PAH algal culture was statistically less than that in a mixture of Xuoranthene and pyrene. Similarly, pyrene alone had lower percentages of loss than that in a mixture with Xuoranthene (Table 2). Under both single and mixed conditions, Xuoranthene had higher percentages of loss than pyrene, suggesting that Xuoranthene was less recalcitrant and was easier to biotransform. The percentage of PAHs losses increased from 1- to 7-days incubation, and varied among microalgal

Table 2 The amount of PAHs remained in medium and uptake by cells, and the percentage of PAHs loss in four microalgal species cultures at diVerent incubation time Factors

Treatments

Amount of PAHs (g)

% PAHs loss

In medium

By microalgae

Species

C. vulgaris S. platydiscus S. quadricauda Se. capricornutum

26.3a 17.1b 18.3b 11.4c

21.8a 28.2b 27.8b 8.9c

47.8a 50.4a 50.5a 77.5b

Time

1-D 4-D 7-D

28.8a 15.9b 10.1c

25.7a 22.1b 17.3c

39.9a 58.5b 71.3c

PAHs

Flu-single Pyr-single Mixture

19.3a 21.2a (Flu + Pyr) 14.4b

18.8a 24.2b (Flu + Pyr) 22.1b

57.7b 49.6c (Flu-mixture) 63.3a (Pyr-mixture) 55.6b

According to three-way ANOVA, the values in “Species” factor were the averages for the three incubation time of a speciWc algal species, in “Time” factor were the averages for all four algal species at each incubation time, and in “PAHs” factor were the averages for all four algal species in each PAH treatment; the mean values of each column under each factor followed by the same letter in the superscript position indicate they were not signiWcantly diVerent at P 6 0.05.

276

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species with Se. capricornutum be the most eYcient species in PAHs metabolism (Table 2). 3.2. By Chlorella vulgaris When only a single PAH was present, the percentages of Xuoranthene and pyrene uptake by C. vulgaris or remained in medium did not change signiWcantly with incubation time (Fig. 1). However, the percentage of pyrene uptake by cells increased while the percentage of residual Xuoranthene in medium decreased signiWcantly during incubation. The

(a) In cells

% PAH in cells

40

NS ab b

NS

30

NS

3.3. By Scenedesmus platydiscus

a

20

10

0

Flu-S

Pyr-S

Flu-M

Pyr-M

PAH treatments (b) In medium

% PAH in medium

75

50

NS

1-D 4-D 7-D

NS

a

NS

b 25

0

c

Flu-S

Pyr-S

Flu-M

Pyr-M

PAH treatments (c) Loss 75

NS

50

a

a

NS

25

0

Under single PAH condition, the percentage of Xuoranthene and pyrene, either uptake by S. platydiscus, remained in medium, or loss from the algal culture did not vary signiWcantly during incubation (Fig. 2). DiVerent trends of temporal changes were found when Xuoranthene and pyrene were in a mixture, the percentage of each PAH uptake by microalgae or remained in medium reduced signiWcantly, while their loss from the system increased during incubation, in particular, from 4- to 7-days incubation (Fig. 2). These results suggested that S. platydiscus was more eYcient in removing Xuoranthene and pyrene from the medium when these two PAH compounds were coexisted in a mixture. The two-way ANOVA results revealed that both the percentages of PAH uptake by cells and loss from the algal culture were comparable among PAH treatments (Table 3). These results suggested that S. platydiscus exhibited comparable eYciency in the removal of Xuoranthene and pyrene, irrespective to whether it was added as a single PAH or mixed with another PAH.

b NS

% PAH loss

percentage of pyrene uptake by cells was higher than that of Xuoranthene, while the percentage of these two PAHs remained in medium was comparable (Table 3; Fig. 1). These results indicated that C. vulgaris was able to uptake more pyrene than Xuoranthene. Although the percentage loss was comparable between Xuoranthene and pyrene under single or mixed condition, the loss of Xuoranthene in PAHs mixture was signiWcantly higher than that of pyrene as a single PAH (Table 3). The percentage loss of PAHs did not change statistically during incubation except the loss of Xuoranthene under mixed condition increased signiWcantly from 4- to 7-days incubation (Fig. 1). These results suggested that C. vulgaris exhibited comparable eYciency in metabolizing Xuoranthene and pyrene under single condition, but it might have higher ability to metabolize Xuoranthene when pyrene was present.

Flu-S

Pyr-S

Flu-M

Pyr-M

PAH treatments Fig. 1. The % PAH uptake by cells, remained in medium and loss from C. vulgaris culture system at 1-, 4- and 7-days incubation (Flu, Xuoranthene; Pyr, pyrene; S, single PAH; M, mixture of Flu and Pyr; mean and standard deviation values of triplicates are shown; NS, not signiWcant; diVerent letters on the top of the bar indicate the means among three incubation time were signiWcantly diVerent at P 6 0.05 according to oneway ANOVA).

3.4. By Scenedesmus quadricauda The percentage of Xuoranthene uptake by cells of S. quadricauda dropped signiWcantly during incubation irrespective to whether it was a single PAH or mixed with pyrene. However, the percentage of pyrene uptake by cells remained at a similar level during incubation (Fig. 3). The percentage of each PAH remained in medium decreased signiWcantly, while their loss from the system increased during incubation (Fig. 3). Under both single and mixed conditions, higher percentage of Xuoranthene was lost from S. quadricauda cultures than that of pyrene although the percentage of pyrene remained in medium was comparable to that of Xuoranthene. More pyrene was taken up and accumulated in algal cells than that of Xuoranthene (Table 3; Fig. 3).

A.-P. Lei et al. / Bioresource Technology 98 (2007) 273–280

277

Table 3 EVects of diVerent PAH treatments and incubation time on % PAH accumulated in cells, remained in medium and loss from each of the microalgal system Factors

Treatments

Algal species C. vulgaris

S. platydiscus

S. quadricauda

Se. capricornutum

Cells

Medium

Loss

Cells

Medium

Loss

Cells

Medium

Loss

Cells

Medium

Loss

PAHs

Flu-single Pyr-single Flu-mixture Pyr-mixture

18.7a 30.2b 20.9a 26.7c

34.6b 28.5ab 23.9a 25.2a

46.7ab 41.3a 55.2b 48.1ab

32.6a 27.6a 33.8a 33.7a

19.8bc 23.9c 12.2a 14.7ab

47.6a 48.5a 54.0a 51.5a

24.4a 39.8b 22.8a 34.3b

20.1b 25.0b 12.8a 18.9ab

55.5c 35.2a 64.4d 46.7b

8.0a 9.7ab 11.2bc 13.1c

11.1a 16.8b 9.2a 10.9a

80.9c 73.5a 79.6c 76.1b

Time

1-D 4-D 7-D

20.0a 25.3b 27.2b

35.6b 24.7a 23.8a

44.4a 50.0a 49.1a

34.3b 38.5b 23.1a

22.6b 18.6b 11.8a

43.1a 42.9a 65.1b

36.5b 31.8b 22.8a

34.6c 19.6b 3.4a

29.0a 48.5b 73.8c

26.5c 4.0b 0.9a

30.4b 3.6a 2.0a

43.1a 92.4b 97.1b

According to two-way ANOVA, the values in “PAHs” factor were the averages for three incubation time of each algal species, in “Time” factor were the averages for PAHs treatments of each algal species; the mean values of each column under each factor followed by the same letter in the superscript position indicates they were not signiWcantly diVerent at P 6 0.05.

The percentage loss of pyrene under single or mixed condition remained at a similar level from 1- to 4-days incubation but increased signiWcantly at the end of 7-days, while the percentage loss of Xuoranthene increased rapidly from 1- to 4-days and more than 80% Xuoranthene were lost at 7-days incubation (Fig. 3). The percentage loss of Xuoranthene or pyrene under mixed condition was signiWcantly higher than that under single PAH condition (Table 3; Fig. 3). These results suggested that Xuoranthene present either as a single PAH or mixed with pyrene was easier to be metabolized by S. quadricauda than pyrene. S. quadricauda was also more eVective in metabolizing Xuoranthene and pyrene when they were added together. 3.5. By Selenastrum capricornutum Fig. 4 shows that the percentages of PAHs uptake by microalgal cells and remained in medium dropped sharply from 1- to 4-days incubation, with 88–98% loss or metabolized at the end of 7-days incubation. Similar patterns of temporal changes were found in all treatments, suggesting that Xuoranthene and pyrene could be rapidly metabolized by this species. In spite of the high eYciency in removing, both Xuoranthene and pyrene, the percentage of PAHs uptake by Se. capricornutum cells or remained in medium varied signiWcantly among PAH treatments (Table 3; Fig. 4). The percentage of pyrene uptake by cells was comparable to that of Xuoranthene. However, the percentage of single pyrene remained in medium was statistically higher than that of single Xuoranthene. The percentage loss of Xuoranthene under both single and mixed conditions was signiWcantly higher than that of pyrene (Table 3; Fig. 4), indicating that Se. capricornutum was more eVective in metabolism of Xuoranthene than pyrene. There was no signiWcant diVerence in the percentage loss of Xuoranthene between single and mixed conditions, but the percentage loss of pyrene in mixed PAHs was signiWcantly higher than that of single pyrene (Table 3; Fig. 4). These results suggest that the presence of Xuoranthene might enhance pyrene metabolism by Se. capricornutum.

4. Discussion 4.1. Removal eYciency among microalgal species Bioaccumulation of synthetic organic compounds by a variety of algal species has been widely demonstrated, and the rate or magnitude of bioaccumulation of organic toxicants in algae is species speciWc. The size and morphology of algal cells have been shown to play an important role in many physiological activities, such as nutrient uptake, photosynthesis, respiration, and regulation of waste products (Lee, 1999). Tang et al. (1998) suggested that high surface area to biovolume ratio of freshwater algae possessed high potential for sorption after examined eight species in accumulation of atrazine. Pirszel et al. (1995) explained that the reason why microalgae displayed considerably higher metal biosorption capacities than macroalgae might partly be attributed to their greater surface area to volume ratios. In the present study, the four microalgal species examined exhibited diVerent degrees of cellular uptake and metabolism of pyrene and Xuoranthene, either as a single or a mixture of two PAHs (Table 2), but the relationship between the percentage PAH loss and the surface area to volume ratio was insigniWcant (P > 0.05). Similarly, no signiWcant relationship was found between lipid content and removal of pyrene and Xuoranthene (P > 0.05). On the other hand, the removal and transformation of PAHs seemed to be growth-dependent. The biomass of the four species at the end of 7-days incubation under single pyrene treatment followed the order of Se. capricornutum > S. platydiscus > C. vulgaris > S. quadricauda, and the respective biomass was 219.2, 198.9, 147.4 and 49.9 mg dry wt l¡1, respectively. The order of the percentages of pyrene removal as well as its loss by the four algal cultures was the same (Table 3). Our previous work also reported that the removal of pyrene was dependent on the concentration of algal biomass used, the more the biomass the higher the removal percentages (Lei et al., 2002). Similarly, Chan et al. (in press) found that the removal of mixed phenanthrene, Xuoranthene and pyrene

278

A.-P. Lei et al. / Bioresource Technology 98 (2007) 273–280

(a) In cells

75

(a) In cells

50

NS

NS

NS a

a a a

ab

b

b

25

% PAH in cells

% PAH in cells

50

40

NS

a a

30

b b

20

c

10 0

Flu-S

Pyr-S

Flu-M

Pyr-M

0 Flu-S

PAH treatments

Pyr-S

Flu-M

Pyr-M

PAH treatments (b) In medium (b) In medium

30

NS NS

1-D 4-D 7-D

a a a

20

c

10

0

b

b Flu-S

Pyr-S

Flu-M

50

% PAH in medium

% PAH in medium

40

a

40

a

b

20

10

Flu-S

Pyr-S

a

a a

25

0

% PAH loss

% PAH loss

75

a

Flu-M

(c) Loss

Pyr-M

c

c

b

b NS

b

b PAH treatments

100

NS

b

b

c

(c) Loss

50

1-D 4-D 7-D

a

PAH treatments

100

a

a

30

0

Pyr-M

a

b

b

75

b

b a

50

a

a a

a

a

25

Flu-S

Pyr-S

Flu-M

Pyr-M

PAH treatments Fig. 2. The % PAH uptake by cells, remained in medium and loss from S. platydiscus culture system at 1-, 4- and 7-days incubation (Flu, Xuoranthene; Pyr, pyrene; S, single PAH; M, mixture of Flu and Pyr; mean and standard deviation values of triplicates are shown; NS, not signiWcant; diVerent letters on the top of the bar indicate the means among three incubation time were signiWcantly diVerent at P 6 0.05 according to oneway ANOVA).

by Se. capriconutum increased with initial cell densities ranging from 5 £ 104 to 1 £ 107 cells ml¡1. In addition to biomass, cell density and lipid content, other properties like cell wall composition and enzymes involved in PAH detoxiWcation and degradation might be important in determining the species-speciWc diVerence in the removal of Xuoranthene and pyrene. Various detoxiWcation enzymes were recorded in nine species of Antarctic and Arctic macroalgae (PXugmacher et al., 1999). Kirso and Irha (1998) suggested that the species-speciWc biotrans-

0

Flu-S

Pyr-S

Flu-M

Pyr-M

PAH treatments Fig. 3. The % PAH uptake by cells, remained in medium and loss from S. quadricauda culture system at 1-, 4- and 7-days incubation (Flu, Xuoranthene; Pyr, pyrene; S, single PAH; M, mixture of Flu and Pyr; mean and standard deviation values of triplicates are shown; NS, not signiWcant; diVerent letters on the top of the bar indicate the means among three incubation time were signiWcantly diVerent at P 6 0.05 according to one-way ANOVA).

formation of benzo(a)pyrene, a priority PAH, by six diVerent macroalgal species was dependent on the unique enzyme system of each species including o-diphenol oxidase, cytochrome P450, and peroxidase. Lei et al. (2003) reported that glutathione S-transferase (GST) played an important role in biotransformation of pyrene by microalgal species and their activities varied from species to species. Although it has been suggested that PAH-degrading bacterial strains utilized a common enzyme to degrade two

A.-P. Lei et al. / Bioresource Technology 98 (2007) 273–280

(a) In cells

% PAH in cells

40

a a

30

a

a 20

b

10

b

b

c

b

b b

b

0 Flu-S

Pyr-S

Flu-M

Pyr-M

PAH treatments (b) In medium

% PAH in medium

50

1-D 4-D 7-D

a

40 30

a

a a

20 10

b

b b

b

b b

b b

0 Flu-S

Pyr-S

Flu-M

Pyr-M

PAH treatments (c) Loss

b

% PAH loss

100

b b

b c

b c

c

75

a 50

a a

a

25

0

Flu-S

Pyr-S

Flu-M

Pyr-M

PAH treatments Fig. 4. The % PAH uptake by cells, remained in medium and loss from Se. capricornutum culture system at 1-, 4- and 7-days incubation (Flu, Xuoranthene; Pyr, pyrene; S, single PAH; M, mixture of Flu and Pyr; mean and standard deviation values of triplicates are shown; diVerent letters on the top of the bar indicate the means among three incubation time were signiWcantly diVerent at P 6 0.05 according to one-way ANOVA).

or more PAHs (Stringfellow and Aitken, 1995), it is still unclear whether microalgae also use a common enzyme system. More in-depth studies to understand the enzyme systems involved in the removal and degradation of PAHs by microalgae are needed. 4.2. Removal eYciency between Xuoranthene and pyrene Not only species-speciWc, the removal of organic compounds by algal uptake and metabolism is also toxicant-

279

dependent. The molecular weight, water solubility and lipophilicity of the compound would aVect the bioaccumulation and degradation by microorganisms. The 4-ring PAHs, Xuoranthene and pyrene, were easier to be removed and degraded by Se. capricornutum than phenanthrene, a 3-ring PAH (Chan et al., in press). Similar to microalgae, Potin et al. (2004) reported that two Wlamentous fungi isolated from PAH-contaminated soil, namely Coniothyrium sp. and Fusarium sp. preferentially degraded high molecular weight PAHs (5–6 ring) than low molecular weight PAHs. On the contrary, PAHs with low molecular weight such as 2-ring naphthalene and 3-ring phenanthrene were more susceptible to bacterial degradation than PAHs with more than 3rings (Juhasz and Naidu, 2000; Yu et al., 2005). Although the molecular weight and Kow value of Xuoranthene and pyrene are the same, the solubility of the former PAH was nearly double (Mackay et al., 1992). In the present study, except S. platydiscus that exhibited the same removal eYciency for both Xuoranthene and pyrene, all the other three microalgal species especially S. quadricauda showed higher eYciency in the removal of Xuoranthene than pyrene. These results indicate that pyrene was generally more stable, recalcitrant, and more diYcult to be removed by microalgae. It is rather unusual for natural environments to be polluted by a single PAH compound (Tang et al., 2005). When two or more PAHs are present together, one PAH compound has the capacity to inXuence the rate and extent of biodegradation of the other. Tiehm and Fritzsche (1995) showed that the degradation of soluble pyrene by a bacterial strain, Mycobacterium, was enhanced in the presence of another PAH, phenanthrene. Similarly, naphthalene could stimulate degradation of phenanthrene and pyrene by a bacterial isolate Pseudomonas putida KBM-1 (McNally et al., 1999). However, inhibitory interaction and competition between mixed PAHs were frequently reported (Stringfellow and Aitken, 1995; Bouchez et al., 1995). Dean-Ross et al. (2002) found that utilization of pyrene by a bacterial strain, Mycobacterium Xavescens, was slower in the presence of Xuoranthene than in its absence, although it could utilize the two PAHs simultaneously. There is little information available on the removal of mixed PAH by microalgae despite microalgae have shown to be able to remove individual PAH compounds (Semple et al., 1999; Lei et al., 2002). Chan et al. (in press) recently reported the positive interaction between pyrene and two PAHs (phenanthrene and Xuoranthene) when degraded by Se. capricornutum. In the present study, the presence of Xuoranthene was found to stimulate the removal of pyrene and vice versa, suggesting some positive interaction might occur when these two PAHs were mixed together. As only two PAHs at the same concentration were investigated in the present study, more detailed research must be conducted to understand the interactions. The interactions need to be taken into account when estimating the removal rates in contaminated environments.

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5. Conclusions All the four microalgal species investigated in the present study were capable of removing Xuoranthene and pyrene from medium via biosorption and biotransformation, and the eYciency was species-speciWc and toxicantdependent. Se. capricornutum was the most eVective species while C. vulgaris was the least eYcient one. All microalgal species except S. platydiscus, showed higher eYciency in the removal of Xuoranthene than pyrene, indicating that pyrene was a more recalcitrant PAH compound. The microalgal species also showed comparable or higher eYciency in the removal of Xuoranthene and pyrene in a mixture than the respective single PAH, suggesting a stimulatory eVect between Xuoranthene and pyrene. Acknowledgement We would like to thank all the group members of Prof. Nora Tam and Prof. Yuk-Shan Wong for their co-operation. This research was supported by the High-tech Research and Development Program of China (Grant No. 2001AA641030-06), grants from the Bureau of Science and Technology in Shenzhen, China (Grant No. 200433, 200439) and a grant from CityU, HKSAR (Project No. 7001542). References Bouchez, M., Blanchet, D., Vandecasteele, J.P., 1995. Degradation of polycyclic aromatic hydrocarbons by pure strains and by deWned strain associations: inhibition phenomena and cometabolism. Appl. Microbiol. Biotechnol. 43, 156–164. Canet, R., Birnstingl, J.G., Malcolm, D.G., Lopez-Real, J.M., Beck, A.J., 2001. Biodegradation of polycyclic aromatic hydrocarbons (PAHs) by native microXora and combinations of white-rot fungi in a coal-tar contaminated soil. Bioresour. Technol. 76, 113–117. Chan, S.M.N., Luan, T.G., Wong, M.H., Tam, N.F.Y., in press. Removal and biodegradation of polycyclic aromatic hydrocarbons by Selenastrum capricornutum. Environ. Toxicol. Chem. Chávez-Gómez, B., Quintero, R., Esparza-García, F., Mesta-Howard, A.M., Zavala Díaz de la Serna, F.J., Hernández-Rodríguez, C.H., Gillén, T., Poggi-Varaldo, H.M., Barrera-Cortés, J., Rodríguez-Vázquez, R., 2003. Removal of phenanthrene from soil by co-cultures of bacteria and fungi pregrown on sugarcane bagasse pith. Bioresour. Technol. 89, 177–183. Dean-Ross, D., Moody, J., Cerniglia, C.E., 2002. Utilization of mixtures of polycyclic aromatic hydrocarbons by bacteria isolated from contaminated sediment. FEMS Microbiol. Ecol. 41, 1–7. Herwijnen, R., van de Sande, B.F., van der Wielen, F.W.M., Govers, H.A.J., Parsons, J.R., 2003. InXuence of phenanthrene and Xuoranthene on the degradation of Xuorene and glucose by Sphingomonas sp. strain LB126 in chemostat cultures. FEMS Microbiol. Ecol. 46, 105–111. James, D.E., 1978. Culturing Algae. Carolina Biological Supply Company, USA.

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