Photodegradation of aniline in aqueous suspensions ...

Journal of Photochemistry and Photobiology B: Biology 87 (2007) 49–57 www.elsevier.com/locate/jphotobiol

Photodegradation of aniline in aqueous suspensions of microalgae Lei Wang, Changbo Zhang, Feng Wu, Nansheng Deng

*

School of Resources and Environmental Science, Wuhan University, Wuhan 430079, PR China Received 3 August 2006; received in revised form 6 December 2006; accepted 12 December 2006 Available online 10 January 2007

Abstract The photodegradation of aniline was investigated using freshwater algae suspended in aqueous media under metal halide light (250 W). Four algal species were used: Nitzschia hantzschiana, Chlorella vulgaris, Chlamydomonas sajao and Anabaena cylindrica. Reactions were carried out under aerobic conditions. The photodegradation rate of aniline was accelerated by the algae. In the A. cylindrica suspensions, with cell density ranging from 2.5 · 105 cells mL1 to 6.5 · 106 cells mL1, the photodegradation rate of aniline was increased from 10% to 80% and rate constant k increased from 1.86 · 103 min1 to 9.66 · 103 min1. Reactive oxygen species were thought to be the main reason for the degradation of aniline. Hydroxyl radicals and singlet oxygen photogenerated in the algal suspensions were detected. The maximum singlet oxygen yield was 75 lM in the presence of 1.0 · 106 cells mL1 C. sajao. About 5 lM hydroxyl radicals were generated in the 4-h reaction. Oxygen played an important role in the formation of reactive oxygen species in the algal suspensions. The nature of the algae facilitating the photodegradation of aniline was also investigated. 2007 Elsevier B.V. All rights reserved. Keywords: Aniline; Nitzschia hantzschiana; Chlorella vulgaris; Chlamydomonas sajao; Anabaena cylindrica; Photodegradation

1. Introduction Aniline is an important industrial chemical used as an intermediate in the production of a very wide range of synthetic organic chemicals and polymers including polyurethanes, rubber additives, dyes, pharmaceuticals, pesticides and herbicides. Its occurrence in the environment is associated in part with its industrial manufacture and use, but it can also be released as a result of the partial biodegradation of xenobiotic compounds including certain azo dyes and herbicides (e.g. acylanilides, phenylureas and phenylcarbamates) [1,2]. About 300 chemical products and intermediates are currently manufactured from aniline [3,4]. Estimated annual production of aniline in the USA alone was 1.04 · 105 tons in 2003 [5]. The annual discharge to the environment worldwide was nearly 3.0 · 104 tons. Wide-scale production and use of aniline ensures that it is present in many effluents from the chemical industry.

*

Corresponding author. Tel./fax: +86 27 68778511. E-mail address: [email protected] (N. Deng).

1011-1344/$ - see front matter 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.jphotobiol.2006.12.006

Hence, aniline is a major environmental pollutant. Many methods, including biological [6–10], chemical [11,12], photochemical ones [13–17], have been used to remove this pollutant, and they have received increasing attention. Photolysis in the presence of algae is a possible alternative technology for the destruction of aquatic environmental pollutants. Algae, given their substantial biomass, extensive range of habitat and diversity, play an important role in the fate of organic compounds in aquatic ecosystems [18]. Rontani et al. [19] reported the rate and mechanism of light-dependent degradation of sterols in senescent cells of Skeletonema costatum, a diatom widespread in coastal waters. Zepp and Schlotzhauer [20] studied the influence of algae on photolysis rates of chemicals in water. Results showed that algae could accelerate the sunlightinduced transformation of nonionic organic chemicals. But there have been no reports on the mechanism of photodegradation in the presence of algae. In our previous work, we demonstrated that algae (Nitzschia hantzschiana, Anabaena cylindrica) could accelerate the photodegradation of 17a-ethynylestradiol (EE2) and 17b-estradiol (E2) in aqueous solution exposed to high-pressure mercury

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L. Wang et al. / Journal of Photochemistry and Photobiology B: Biology 87 (2007) 49–57

lamp (HPML) or UV light [21,22]. Zepp et al. [23] studied the rate of decomposition and photoproduction of H2O2 by algae in water. Their results suggested that algae had an important influence on the environmental concentration of H2O2. We [24] have reported the photoproduction of OH in aqueous solution with freshwater algae under metal halide light. These results indicate that algae have the potential to destroy pollutants in natural waters. In this work, we investigated the photodegradation of aniline in the presence of the freshwater algae N. hantzschiana, C. vulgaris, C. sajao and A. cylindrica. These species are easily and cheaply available locally. The influence of initial aniline concentration, algal density, exposure time and oxygen were all investigated. The nature of the algae that facilitate the photodegradation of aniline will also be discussed. 2. Materials and methods 2.1. Chemicals and reagents HCl, FeCl3 Æ 6H2O, CaCl2 Æ 2H2O, H3BO3, K2HPO4 Æ 3H2O, KH2PO4, MgSO4 Æ 7H2O, MnCl2 Æ 4H2O, MnSO4 Æ 4H2O, NaOH, CuSO4 Æ 5H2O, NaCl, Na2CO3, NaHCO3, Na2EDTA, Na2SiO3 Æ 9H2O, ZnSO4 Æ 7H2O, (NH4)6Mo7O24 Æ 4H2O, NaNO3, ascorbic acid, ferric citrate, acetone and aniline were purchased from Tianjin Reagent Co., Ltd. Methanol was HPLC grade (Fisher Chemicals, Fair Lawn, New Jersey). Acetonitrile was analytical grade (Shanghai Lingfeng Chemical Reagent Co., Ltd). Doubledistilled water was used in the experiments. 2.2. Preparation of algae The algae N. hantzschiana, C. vulgaris, C. sajao and A. cylindrica were obtained from the Wuhan Hydrobiology Institute of the Chinese Academy of Sciences (Wuhan, PR China). The algae were raised in culture media at 25 C using a 24-h light cycle in a greenhouse equipped with daylight lamps, light intensity, 2000 Lux. N. hantzschiana was cultured in D1 medium that consisted of 120 mg L1 NaNO3, 70 mg L1 MgSO4 Æ 7H2O, 40 mg L1 K2HPO4 Æ 3H2O, 80 mg L1 KH2PO4, 20 mg L1 CaCl2 Æ 2H2O, 10 mg L1 NaCl, 100 mg L1 Na2SiO3 Æ 9H2O, 2 mg L1 MnSO4 Æ 4H2O, 5 mg L1 ferric citrate, 1 mL L1 A5 solution and 20 mL L1 soil extract. C. vulgaris, C. sajao and A. cylindrica were cultured in SE medium that consisted of 250 mg L1 NaNO3, 75 mg L1 K2HPO4 Æ 3H2O, 75 mg L1 MgSO4 Æ 7H2O, 25 mg L1 CaCl2 Æ 2H2O, 175 mg L1 KH2PO4, 25 mg L1 NaCl, 5 mg L1 FeCl3 Æ 6H2O, 100 mg L1 Fe-EDTA, 1 mL L1 A5 solution and 4 mL L1 soil extract. The medium was adjusted to pH 7.0–7.2 by using 0.1 M Na2CO3. The composition of the A5 solution was 2.86 mg L1 H3BO3, 1.81 mg L1 MnCl2 Æ 4H2O, 0.22 mg L1 ZnSO4 Æ 7H2O, 0.079 mg L1 CuSO4 Æ 5H2O and 0.039 mg L1 (NH4)6Mo7O24 Æ 4H2O. Fe-EDTA was the mixture of 1 g

Na2EDTA, 50 mL distilled water, 81 mg FeCl3 Æ 6H2O and 50 mL HCl (0.1 N). Soil of loess type was used to extract the soil extract. The culture media were free of bacteria. When taken for use in the experiments, the algae were in the logarithmic growth phase and at high density (normally 12–14 days of culture). Prior to the illumination experiments, a modified version of the procedure used in [20] was applied to remove colloidal ferric hydroxide particles that might have adsorbed on the algal cells. This procedure involved washing the cells by gentle agitation for 30 min with 0.01 M aqueous ascorbic acid adjusted to pH 3.0. Then the algae were washed three times with double-distilled water to remove all the ascorbic acid. The resulting algal suspension was diluted with double-distilled water to obtain different densities of algae. Cell counting was carried out, and the density of algae (cells mL1) was calculated. Absorbance at 680 nm was determined with a spectrophotometer, UV-120-02 (Shimadzu), to get the relationship between algal cell density and absorbance of the algal suspension. 2.3. Photochemical equipment Experiments were carried out in a homemade photochemical reactor (250 mL) which has an air inlet for a continuous stream of air, and a sample port. As shown in Fig. 1, a metal halide light (k P 350 nm, 250 W, Yaming, Shanghai, China) placed in the centre of the reactor was used as the light source. The distance between the lamp and the reactor was about 5 cm. During the photoreaction, a water jacket was used to maintain a constant temperature of about 25 C. The light intensity was 34 lW cm2, which was detected by an irradiance meter (FS type, Peixian Photoelectric Instruments Co., Jiangsu, PR China). The spec-

Fig. 1. Photochemical reactor.


trum of the metal halide light used in this work is shown in Fig. 2. The major emission wavelengths of the lamp are 365, 380, 406, 412, 438, 452, 532 and 587 nm. 2.4. Photoreaction procedure and analysis Aqueous solutions of aniline were made with known concentrations (0.02, 0.04, 0.05, 0.08 and 0.10 mmol L1) and all had a pH of 6.7 ± 0.1. Each aqueous solution of aniline was mixed thoroughly with an algal suspension. The resulting mixture was placed into the reactor and air was bubbled through it during the light irradiation process to maintain air-saturation. The lamp was always preheated for a 3-min period. At different time intervals (20 min), samples of about 10 mL were taken out of the reactor and were centrifuged at 3500 rpm for 20 min using centrifugation procedures that were discussed elsewhere [25] to remove the algae. The aniline solutions were analysed by a high pressure liquid chromatograph equipped with a column packed with VP-ODS (Shimadzu Co. Ltd.). An SPD10A UV–vis detector (Shimadzu Co. Ltd.) was used, and the wavelength was 270 nm. The mobile phase was a mixture of acetonitrile and water (40/60, v/v) with a flow rate of 1.0 mL min1. Spectroscopic analysis was done using a UV–vis spectrophotometer (UV-1601, Shimadzu). 2.5. Determination of aniline in the algal cells Algal cells were separated from the aqueous solution by centrifugation at 3500 rpm for 20 min after 4 h of irradiation. The cells were collected and crushed mechanically. Aniline in the algal cells was extracted for 1 h with methanol and analysed by HPLC. The recovery efficiency of methanol extraction was about 90%. 2.6. Detection of singlet oxygen and hydroxyl radicals Furfuryl alcohol (FFA) was used as the probe to detect singlet oxygen generated in the algal suspensions. It was recommended as an efficient trapping agent for singlet oxy-

R elative intensity

100 80 60 40 20 0 350

400

450

500

550

600

650

wavelength / nm Fig. 2. The spectrum of the metal halide light used in this work.

51

gen determinations in natural waters [26]. Most (90%) of the singlet oxygen could be trapped by FFA. The initial concentration of FFA in the algal suspensions after dilution was 0.1 mM. Singlet oxygen concentrations can be quantified by determining the loss of the FFA. FFA was analysed by HPLC. The detection wavelength was 230 nm and the mobile phase was a mixture of methanol and water (50/50, v/v) with a flow rate of 1.0 mL min1. Benzene was used as the probe to detect hydroxyl radicals generated in the algal suspensions. Benzene is very unreactive toward singlet oxygen [24,27]. The hydroxylation of benzene by hydroxyl radicals to produce phenol is a fairly selective process. It is thought that the OH-mediated oxidation of benzene forms phenol with 100% yield and the phenol concentration represents the concentration of hydroxyl radicals [28]. 7 mM benzene was added to the algal suspension. Phenol concentration was detected by HPLC. The detection wavelength was 270 nm and the mobile phase was a mixture of acetonitrile and water (40/ 60, v/v) with a flow rate of 0.8 mL min1. 2.7. FT-IR measurements The KBr pressed disk technique was used [29]. Three hundred millilitre algal suspensions were filtered through a 0.45 lM filter membrane after 8 h of irradiation. The filtrate was concentrated by C18 column and washed with 5 mL methanol and 5 mL acetonitrile. The resulting solutions were concentrated by K–D concentrator (Kuderna– Danish concentrator kit) and evaporated to dryness by purging with N2. The dry samples were ground with KBr to a fine powder and then pressed to a disk under high pressure. Finally the samples were used for IR detection. Fourier-transform infrared spectroscopy (FT-IR 5700, Nicolet) was used to detect the algal exudates in the algal supernatants. 2.8. Pigment measurements After 4 h reaction, samples of about 5 mL were taken out of the reactor. Then algal cells were separated from the solutions by centrifugation at 3500 rpm for 20 min. Cells without irradiation were used as 0 h samples. After 24 h extraction with acetone, samples were detected by spectrophotometer UV-120-02 (Shimadzu). Lichtenthaler and Wellburn [30] reported the following equations to determine chlorophyll-a, chlorophyll-b and carotenoid content in 80% acetone extracts: Ca ¼ 12:21A663 –2:81A646 Cb ¼ 20:13A646 –5:03A663

ð1Þ ð2Þ

Ccarotenoid ¼ ð1000A470 3:27Ca 104Cb Þ=229

ð3Þ

Ca, Cb and Ccarotenoid represent the concentration of the chlorophyll-a, chlorophyll-b and carotenoid. A663, A646 and A470 are the absorbance of the pigments extraction at 663, 646 and 470 nm, respectively.

52


2.9. Data analysis All of the experiments were undertaken in triplicate. Data in the text, figures and tables are expressed as means of triplicate experiments, with coefficients of variation less than 10%. Statistical tests were carried out using OriginLab, version 7.5 (OriginLab Corporation, One Roundhouse Plaza, Northampton, MA 01060, USA). 3. Results and discussion 3.1. Photodegradation of aniline in aqueous solution with algae Fig. 3 shows the relationship between algal density and absorbance at 680 nm. It is obvious that at the same absorbance wavelength, the cell density of C. vulgaris, N. hantzschiana, C. sajao and A. cylindrica correlated with absorbance. Different algal species contain different amounts of the pigments chlorophyll-a, chlorophyll-b and carotenoid, and so can be compared by their absorbance.

Y=26.46846*X

6

-1

Algal density (10 ×cells mL )

14

3.2. Effect of initial concentration of aniline

12 10 8

Y=13.53454*X

6

Y=10.08889*X

4

Y=4.36911*X

2 0 0.00

The photodegradation of aniline in the algal solution fitted well the exponential decay curve, following a pseudo-first order reaction. As shown in Table 1, the photodegradation rate of aniline was quite different in different algal suspensions. The results suggest that environmental oxidative stress effects are different among algal species: the species have different cell wall structure, which can be related to their resistance to irradiation. The blue–green algae A. cylindrica is a prokaryote; its cell walls are made of murein and peptidoglycan [31] and are thin and easily soluble. So A. cylindrica is easily destroyed. But the green algae C. vulgaris and C. sajao are eucaryotes; the cell walls consist of fibrin and chitin and are stronger than that of A. cylindrica, and so they are difficult to destroy. The cell walls of the diatom N. hantzschiana, are made of pectic substances (inside layer) and chitin (outside layer), and contain inorganic substances such as SiO2 and CaCO3 [32]. This enhances the rigidity of the cell wall and makes it hard to destroy. As a result, the amounts of photosensitising substances (such as pigments, carboxylic acids) released into the reaction solution by the algal cells when they were irradiated were quite different. Herth [33,34] reported that several green algae can release intracellular components into the surrounding media. These substances were thought to be the main sources of the compounds responsible for photodegradation.

0.15

0.30

0.45

0.60

0.75

Absorbance (at 680nm) Fig. 3. The relationship between algal density and absorbance at 680 nm. (n) Chlorella vulgaris; (.) Nitzschia hantzschiana; (m) Chlamydomonas sajao; (d) Anabaena cylindrica.

The algae A. cylindrica and C. sajao were used to investigate the effect of the initial concentration of aniline (0.02, 0.04, 0.05, 0.08 and 0.10 mmol L1 aniline) on the photodegradation rate. Analysis of reaction kinetics is shown in Table 1. Over the aniline concentration range studied, the photodegradation rate decreased with increasing initial concentration of aniline. In the presence of A. cylindrica, when the aniline concentration was 0.02 mmol L1, the rate constant k was 7.33 · 103 min1, but k decreased to 4.36 · 103 min1 as the aniline concentration increased to 0.10 mmol L1. As a result, t1/2 increased with increasing aniline concentration. As shown in Table 1, the same pattern was observed with C. sajao.

Table 1 Analysis of the kinetics of the photodegradation of aniline (N. Deng et al.) Abs680

Caniline (mmol L1)

kinetics equation

r1

SD

t1/2 (h)

0.569

0.02 0.05 0.10

ln(c/c0) = 0.00733 t ln(c/c0) = 0.00558 t ln(c/c0) = 0.00436 t

0.97847 0.99825 0.99649

0.01419 0.02653 0.02931

1.58 2.07 2.65

3.6 · 106 cells mL1

0.255

0.04 0.08 0.10

ln(c/c0) = 0.01126 t ln(c/c0) = 0.01013 t ln(c/c0) = 0.00806 t

0.98976 0.99839 0.99543

0.05686 0.06033 0.08506

1.03 1.14 1.92

Nitzschia hantzschiana: 5.0 · 105 cells mL1

0.037

0.10

ln(c/c0) = 0.0028 t

0.99228

0.03829

4.13

Chlorella vulgaris: 1.7 · 107 cells mL1

0.647

0.10

ln(c/c0) = 0.0032 t

0.97934

0.06985

3.61

Reaction systems Anabaena cylindrica: 2.5 · 106 cells mL1 Chlamydomonas sajao:


3.3. Biodegradation and bioaccumulation of aniline by algae

3.4. Effect of algal density on the photodegradation rate of aniline Experiments were conducted to investigate the effect of algal density on the photodegradation rate of aniline. A. cylindrica was used at densities from 2.5 · 105 cells mL1 to 6.5 · 106 cells mL1. The photodegradation rate of aniline increased with increasing density of A. cylindrica. The rate constant k increased from 1.86 · 103 min1 to 9.66 · 103 min1. As cell density is increased, it will affect the light dispersion. However, the reactive sites on and/or in the algae increase with density and so can enhance the photodegradation of aniline. The degradation rate becomes dependent on the cell density.

1.0

N2-saturated 0.8

C/C0

Dark reactions were carried out in the presence of C. sajao (3.55 · 106 cells mL1). Results show no evident biodegradation of aniline in the 4-h dark reaction. Dark reactions were also carried out under various conditions with the other algal species, but with the same conclusions. Less than 3% aniline was adsorbed by algae. It is impossible for the biodegradation of aniline to occur in such a short time. In the light the photodegradation rate of aniline increased with time. After 4 h of irradiation, nearly 70% of the aniline was degraded. Lai et al. [18] similarly found that under light conditions, 50% of estradiol was metabolised into an unknown product by C. vulgaris. Experiments were carried out to examine whether algae could bioaccumulate aniline in the 4-h light reaction. After 4 h of irradiation, algae were collected by centrifugation and then crushed. The cells were extracted for 1 h with methanol and analysed by HPLC. No aniline was detected in them. It can be concluded that biodegradation and bioaccumulation of aniline by the algae themselves could be neglected in such a short reaction time, and the main reason for the aniline degradation was the photodegradation induced by the algae.

53

0.6 0.4

air-saturated

0.2 0

50

100

150

200

250

Time/min Fig. 4. Photodegradation of aniline in aqueous solution with algae under different conditions, N2-saturated or air-saturated. In an aqueous solution of Anabaena cylindrica density was 4.0 · 106 cells mL1 and Caniline was 0.08 mmol L1.

3.6. Photoproduction of singlet oxygen and hydroxyl radicals in the algal solutions Since biodegradation and bioaccumulation were not responsible for the degradation of aniline, experiments were carried out to investigate how algae bring about photodegradation. As suggested above, some reactive species may be produced in the algal supernatants. Furfuryl alcohol (FFA) and benzene were separately used as trapping agents to determine singlet oxygen and hydroxyl radicals. Results are shown in Figs. 5 and 6, respectively. In the aqueous solution without algae there was no loss of FFA on direct irradiation. Singlet oxygen was photogenerated in different algal solutions. The yield of singlet oxygen was increased with increasing cell density. The maximum singlet oxygen yield was 75 lM in the presence of 1.5 · 106 cells mL1 C. sajao. Hydroxyl radicals have also been detected in the algal solutions. About 5 lM hydroxyl radicals were generated in the 4-h reaction.

75

Experiments were also carried out to investigate the effect of gas medium, N2 or air, on the photodegradation rate of aniline. The photodegradation experiments were repeated in the presence of A. cylindrica and the initial concentration of aniline was 0.08 mmol L1. Results are shown in Fig. 4. Photodegradation efficiency in the air-saturated solution was much higher than that in the N2-saturated solution, confirming that molecular oxygen plays an important role in the photodegradation reaction. When the algae were irradiated, they released many substances (such as pigments, carboxylic acids) into the aqueous solution, and molecular oxygen in the aqueous solution can help to form some active species, such as hydroxyl radicals, singlet oxygen and H2O2, which have the potential to degrade most of the organic pollutants [35,36].

60

a b c d e f g

45

1

2

C O (µM)

3.5. Control experiments in different gas media

30 15 0

h 0

50

100

150

200

250

Time / min Fig. 5. Determination of 1O2 in different algal solutions. Chlamydomonas sajao: (a) 1.5 · 106 cells mL1, (b) 1.0 · 106 cells mL1, (c) 7.5 · 105 cells mL1; Anabaena cylindrica: (d) 1.25 · 106 cells mL1, (f) 2.5 · 105 cells mL1; Chlorella vulgaris: (e) 1.5 · 107 cells mL1, (g) 5.0 · 106 cells mL1; (h) without algae.

54


6

COH (µM)

5 4 3 2 1 0 0

50

100

150

200

250

300

Time / min Fig. 6. Determination of hydroxyl radicals in different algae solutions. (n) Chlamydomonas sajao: 1.0 · 105 cells mL1; (m) Anabaena cylindrica: 6.6 · 105 cells mL1; (d) Chlorella vulgaris: 2.5 · 105 cells mL1.

3.7. Photodegradation of aniline related to the nature of the algae Photodegradation of aniline was tested with dead algae and with living algae of all four species. The results are shown in Fig. 7. The photodegradation rates of aniline in the presence of the dead algae were higher than with living

a

algae, even when the density of the living algae was higher than that of the dead algae (results for C. sajao). Matsumura and Esaac [37] have reported similar results. Blue– green algae could photosensitise several pesticides more quickly when the algae were denatured by heat. In dead phytoplankton cells [38], the photochemical process was also found to act intensively on the phytyl side chain of chlorophyll-a and chlorophyll-b. In order to investigate the nature of the algal cells in the photoreaction procedure, experiments were carried out to determine the substances released by the algal cells. The IR spectrum of the 8-h irradiated algae shows bands at 3300– 2900 cm1, 2850–2600 cm1 and 1700 cm1 attributed to vibrations of the carboxyl groups. It can be concluded that carboxylic acids were produced during the reaction. The bands at 2800–2500 cm1 and 1740–1630 cm1 correspond to the vibration of –NH3. At 3000–2700 cm1, 1500–1300 cm1, 1100–1000 cm1 and 700–550 cm1 bands of alkyl are observed. As shown in Fig. 8, all these bands appear in the different algal solutions. A number of studies have shown that visible light absorption by the photosynthetic apparatus of algae commonly results in the photometabolism of polar, ionic organic substances such as carboxylic acids, carbohydrates, and amino acids [39,40].

b

1.0

1.0

0.8

C/C0

C/C0

0.8 0.6 0.6

0.4 0.2

0.4

Chlamydomonas sajao

Nitzschia hantzschiana

0.0 0

50

100

150

200

250

0

50

Time/min

d

1.0

0.8

0.6

0.6

0.4

0.0 100

150

Time/min

200

250

Anabaena cylindrica

Chlorella vulgaris 50

250

0.4 0.2

0.2

0

200

1.0

0.8

0.0

150

Time/min

C/C0

C/C0

c

100

200

250

0

50

100

150

Time/min

Fig. 7. Photodegradation of aniline (0.08 mmol L1) by dead and living algae in an aqueous solution. (a) Chlamydomonas sajao: 2.7 · 106 cells mL1 (dead algae), 3.6 · 106 cells mL1 (living algae); (b) Nitzschia hantzschiana: 2.3 · 106 cells mL1; (c) Chlorella vulgaris: 2.0 · 107 cells mL1; (d) Anabaena cylindrica: 3.0 · 106 cells mL1. (n) dead algae; (m) living algae.

L. Wang et al. / Journal of Photochemistry and Photobiology B: Biology 87 (2007) 49–57 A.cylindrica

Algae hν

120

%Transmittance

1073 603 1382

2853

100 2925

80

3433

1648

2847

3433

596 1249 1073 1652 1389

C.vulgaris

40

Pigments (chlorophylls) hν

N.hantzschiana 1550

2927

60

1249 1652 1381 1073

2853

Algae exudates (carboxylic acids)

1

Chl Intersystem living crossing cells senescent 3Chl cells 3 O2 Photosynthesis

596

2927

20

1

3410

Chl

3750

3000

55

2250

1500

Cells fragments (cells wall, membranes)

hν O2 ROO.

O2, O2.-, HO., H2O2

750

-1

Wavenumbers (cm )

Fig. 10. The mechanism of photodegradation by algae in aqueous solutions.

Fig. 8. IR spectra of the algal species after 8 h irradiation.

a 1.6

1.6

b

Chlamydomonas sajao

1.4

1.2

1.2

1.0

Abs.

Abs.

1.0 0.8

0.8

0.6

0.6

0.4

0.4

0.2

0.2

0.0 200

300

400

0.0 200

500

wavelength / nm

c 1.4 1.2

Anabaena cylindrica

1.4

300

400

500

wavelength / nm

d

Nitzschia hantzschiana

2.5

Chlorella vulgaris 2.0

1.0

1.5

Abs.

Abs.

0.8 0.6

1.0

0.4

0.5

0.2 200

300

400

0.0 200

500

wavelength / nm

1.0

Abs.

0.8 0.6 0.4

f

Chlamydomonas sajao

0.4

Irradiation time 4h 3h 2h 1h 0h

500

Irradiation time

0.2 0.0 200

400

Chlorella vulgaris

0.3

Abs.

e

300

wavelength / nm

4h 3h 2h 1h 0h

0.2 0.1

250

300

wavelength / nm

350

400

200

250

300 350 wavelength / nm

400

Fig. 9. UV–vis absorption spectrum of aqueous solutions with different algal species. (—) after 40 min heat killing; (– – – –) before heat killing.

56


Table 2 The changes in pigment contents after 4 h reaction (N. Deng et al.) Pigments contents (mg L1)

Chlorella vulgaris 0h

4h

0h

4h

0h

4h

Chlorophyll-a Chlorophyll-b Carotenoid

0.277 0.076 0.045

0.080 0.005 0.032

0.599 0.323 0.052

0.105 0.071 0.002

0.906 0.025 0.281

0.144 0 0.038

Chlamydomonas sajao

The variation of the UV–vis absorption spectra of algal solutions was also studied. Aqueous solutions containing living algae, heat-killed algae and irradiated algae were all scanned. Fig. 9 illustrates the UV–vis absorption spectra for the different algal species. At the same wavelength (k < 400 nm), the absorbance of solutions with the heatkilled algae or the irradiated algae was much higher than that of the living algae. But when k > 400 nm, there were no differences in absorbance. It can be concluded that algae exposed to high temperature or irradiation released many substances (such as pigments, carboxylic acids) into the aqueous solution. Further, the pigments (chlorophyll-a, chlorophyll-b, carotenoid) of the algal cells were not completely destroyed. This observation suggests that photochemical reactions can still occur in the aqueous solutions under irradiation. The photochemical processes involved are shown in Fig. 10. During the irradiation process, the algal cells were gradually destroyed and many substances could be released by them. Results showed that the contents of pigments (chlorophyll-a, chlorophyll-b, carotenoid) in the algal solutions decreased after 4 h reaction (Table 2). In the healthy cells, the primary route for energy from the 1Chl is the fast reactions of photosynthesis. Only a small proportion (less than 1%) of 1Chl may undergo intersystem crossing to form the longer-lived triplet state (3Chl). It may also generate 3 active oxygen species (1O2, O 2 , O2). However, many kinds of antioxidants (ascorbate, glutathione and a-tocopherol) can form a photoprotective system in the algal cells [41– 43]. In the senescent algal cells, as the fast photochemical reactions of photosynthesis are not functional, an accelerated rate of 3Chl (chlorophyll in the triplet state) formation would thus be expected. The rates of the formation of 3Chl and reactive oxygen species (1O2, O 2 , HO , HOO ) (formed 3 by reaction of Chl with ground state oxygen (3O2)) can then exceed the quenching capacity of the photoprotective system of the algal cells, and the cell membrane could be partially disrupted. Singlet oxygen and hydroxyl radicals have been detected in the algal solutions. Carboxylic acids also produced in the reaction, which associated with many cell fragments (algal cell walls, algal cell membranes), must play an important role in the formation of some peroxy radicals (ROO). These reactive oxygen species produced by the algal cells caused the rapid photolysis of aniline. 4. Conclusions The photodegradation rate of aniline could be accelerated by the microalgae N. hantzschiana,, C. vulgaris, C.

Anabaena cylindrica

sajao and A. cylindrica under irradiation. Algal cell density, initial aniline concentration, exposure time and oxygen all have effects on the photodegradation rate of aniline. The reason for aniline removal was not biosorption or bioconcentration by the algae. The reactive oxygen species produced by the algal cells were thought to be the main cause of the degradation of aniline. Singlet oxygen and hydroxyl radicals have been detected in the reaction. The maximum singlet oxygen yield was 75 lM in the presence of 1.5 · 106 cells mL1 C. sajao. About 5 lM hydroxyl radicals were generated in the 4-h reaction. This study can help to explain one of the degradation paths of organic materials in natural aquatic environments such as lakes and rivers. 5. Abbreviations HPML High-pressure mercury lamp HPLC High pressure liquid chromatograph FFA Furfuryl alcohol EE2 17a-ethynylestradiol E2 17b-estradiol N. hantzschiana Nitzschia hantzschiana C. vulgaris Chlorella vulgaris C. sajao Chlamydomonas sajao A. cylindrica Anabaena cylindrica

Acknowledgements This research was supported by the National Natural Science Foundation of PR China (No. 20477031). The authors gratefully acknowledge many helpful suggestions from the editor and the reviewers of this article, as well as substantial support in final language related preparation of the manuscript by several unnamed sources.

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