Science of the Total Environment 508 (2015) 251–257

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Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv

Enhanced biodegradation of phthalate acid esters in marine sediments by benthic diatom Cylindrotheca closterium Ying Li a,b, Jing Gao a, Fanbo Meng a, Jie Chi a,⁎ a b

School of Environmental Science and Engineering, Tianjin University, Tianjin 300072, PR China College of Marine Science and Engineering, Tianjin Key Laboratory of Marine Resources and Chemistry, Tianjin University of Science and Technology, Tianjin 300457, PR China

H I G H L I G H T S • • • • •

Benthic diatom C. closterium increased PAE removal rates from marine sediments. The increment was more obvious in surface sediments than in bottom sediments. The increment was more obvious for DEP than for DBP. C. closterium stimulated the growth of aerobic bacteria indicated by PLFAs. Aerobic bacteria played a key role in C. closterium-promoted PAE degradation.

a r t i c l e

i n f o

Article history: Received 15 September 2014 Received in revised form 30 November 2014 Accepted 1 December 2014 Available online xxxx Editor: Thomas Kevin V Keywords: Marine sediments Marine benthic diatom Cylindrotheca closterium Phthalate acid esters Degradation

a b s t r a c t Cylindrotheca closterium, a marine benthic diatom, was inoculated on the surface of marine sediments spiked with diethyl phthalate (DEP) and dibutyl phthalate (DBP) to investigate the effects of benthic microalgae on the degradation of the contaminants. The elimination of DEP and DBP from unsterilized sediments with C. closterium (treatment BA) was compared with that from unsterilized sediments without C. closterium (treatment B), sterilized sediments with C. closterium (treatment A) and sterilized sediments without C. closterium (treatment N). The results showed that during the 8-day experiment, inoculation with C. closterium increased the removal rates of the contaminants from the sediments, and more significantly from the surface layer (top 0.5 cm) of sediments than from the bottom layer of sediments. In the surface sediments, the first-order elimination rate constants (k) of DEP and DBP were in the order of treatment BA (2.098 and 0.309 d−1) N treatment B (0.460 and 0.256 d−1) N treatment A (0.216 and 0.039 d−1) N treatment N (nil (no data)), indicating that microbial degradation played a major role in the removal of the contaminants from the sediments. A similar trend was also observed in bottom sediments (0.444 and 0.165 d−1 in treatment BA, 0.329 and 0.194 d−1 in treatment B, 0.129 d−1 and nil in treatment A), but the difference of k values between treatments BA and B was relatively small. The positive effect of C. closterium on total phospholipid fatty acid (PLFA) content in sediments was observed, which was mainly related to the increase of biomass of aerobic bacteria as a result of improved sediment oxygenation and release of exudates (e.g. exopolysaccharides) by C. closterium. Moreover, Pearson correlation analysis showed a significant positive correlation between the elimination ratios of the contaminants and abundance of total aerobic bacterial PLFAs, suggesting that aerobic bacteria played a key role in C. closterium-promoted degradation of the contaminants in sediments. © 2014 Elsevier B.V. All rights reserved.

1. Introduction Phthalic acid esters (PAEs) are widely used as plasticizers in polyvinyl chloride plastics. They can enter the environment through losses during manufacturing processes and by leaching from final products, because they are not chemically bonded to the polymeric matrix. The United States Environmental Protection Agency (USEPA) and its ⁎ Corresponding author. E-mail address: [email protected] (J. Chi).

http://dx.doi.org/10.1016/j.scitotenv.2014.12.002 0048-9697/© 2014 Elsevier B.V. All rights reserved.

counterparts in several other countries have classified the most commonly occurring PAEs as priority pollutants (Keith and Telliard, 1979) and endocrine disrupting compounds (Matsumoto et al., 2008). Once entering the aquatic environment, because of their strong hydrophobicity, PAEs are mainly found to be associated with organic matter in sediments and lipid-rich tissues of organisms (Staples et al., 1997). It has been reported that PAEs are transformed by a variety of different processes in the environment. Microorganism degradation has been expected to be the dominant process affecting the environmental fate of PAEs (Staples et al., 1997; Chang et al., 2007). In addition to bacteria and

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fungi, microalgae have been shown to play an important role in the degradation of organic contaminants. Microalgae are a class of autotrophic microorganisms capable of photosynthesizing. They produce oxygen through photosynthesis, which can meet the needs of heterotrophic bacteria and consequently stimulate activities of the bacteria degrading organic contaminants (Godos et al., 2010). Moreover, microalgae also have the ability to degrade organic contaminants directly (Semple and Cain, 1996; Semple et al., 1999; Yan and Pan, 2004). However, to date, most studies have been limited to planktonic microalgae. The benthic microalgae consist of photosynthetic algae and cyanobacteria that live on the sediment–water interface in salt marshes, estuaries and photic coastal zones. In many shallow ecosystems, the biomass of benthic microalgae often exceeds that of the planktonic microalgae in the overlying waters (MacIntyre and Cullen, 1996). As the major primary producer, benthic microalgae contribute significantly to the matter and energy transmission of intertidal ecosystem, in oxygen production, and in stabilizing the sediment (MacIntyre and Cullen, 1996; de Brouwer et al., 2003). Recently, Yamamoto et al. (2008) reported that replantation of marine benthic microalga Nitzschia sp. could enhance aerobic bacterial activity, accelerating the decomposition of sediment organic matter, indicated by chemical oxygen demand, ignition loss and organic nitrogen. The replantation of benthic microalgae might be a potential technology to remediate marine sediments contaminated by organic contaminants. However, to date, little research has been conducted in this area. Diatoms are the major components of benthic microalgae (MacIntyre and Cullen, 1996; de Brouwer et al., 2003; Araújo et al., 2010a). The marine benthic diatom Cylindrotheca closterium (Bacillariophyta, order Pennales) is a widely distributed benthic species and can be easily found on the surface of sediments (Moreno-Garrido et al., 2003, 2007). In this study, the marine benthic diatom C. closterium was selected as the test species and cultivated on sediments spiked with diethyl phthalate (DEP) and dibutyl phthalate (DBP). The purpose of this work is to elucidate the effects of C. closterium on the degradation of the two PAEs in marine sediments. Moreover, phospholipid fatty acids (PLFAs) were analyzed to explore the change of the microbial community structure in the sediment. Then the degradation mechanism of the contaminants in the presence of C. closterium was discussed.

2.2. Experiment design Fifteen glass beakers (diameter 4.5 cm, height 9 cm) were prepared with each treatment and sterilized before use. A portion of the sediment samples (about 1/10) was spiked with the stock solution of DEP and DBP in acetone at a ratio of 4:1 (w/v). When the acetone evaporated off, the spiked sediments were diluted with non-spiked sediments at a ratio of 1:9 (w/w). The non-spiked sediments had been replenished with 40% of artificial seawater which was filtered through a 0.45 μm cellulose acetate membrane, and kept in the dark for 7 days to restore sediment microbial diversity. Concentrations of DEP and DBP in the diluted sediments were determined to be 2.20 ± 0.17 and 6.25 ± 0.38 μg g−1 dry dry wt. About 27 g of the diluted sediments (on dry weight basis) and 30 mL of sterilized artificial seawater were filled in each beaker to form a 2-cm-thick sediment layer. The following treatments were made: (1) inoculating C. closterium on the sediment surface in the beakers at an initial density of 2 × 105 cells cm−2 (treatment BA); and (2) without inoculating on the sediment surface (treatment B). Similarly, a portion of the sediment samples (about 1/10) was spiked with the stock solution of DEP and DBP in acetone at a ratio of 4:1 (w/v). When the acetone evaporated off, the spiked sediments were diluted with non-spiked sediments at a ratio of 1:9 (w/w). About 27 g of the diluted sediments (on dry weight basis) were filled in each beaker to form a 2-cm-thick sediment layer. Filtered artificial seawater was added into the beakers to make the sediments saturated with water. Then the beakers were sealed with sealing membrane and sterilized three times (i.e. once a day for 3 days) by autoclaving at 121 °C for 20 min. The concentrations of DEP and DBP in the sterilized sediments were determined to be 3.40 ± 0.72 and 8.86 ± 1.89 μg g− 1 dry wt. Thereafter, 30 mL of sterilized artificial seawater was added in each of the beakers. The following treatments were made: (1) inoculating C. closterium on the sterilized sediment surface in the beakers at an initial density of 2 × 105 cells cm−2 (treatment A); and (2) without inoculating on the sterilized sediment surface (treatment N). Finally, all of the beakers prepared above were placed in the intelligent illumination incubator under the same conditions as those of algal cultivation. The experiments were conducted for 8 days. Triplicate beakers of each treatment (except treatment N) were removed after 1-, 2-, 4-, 6-, and 8-day incubation. For treatment N, triplicate beakers were removed after 4- and 8-day inoculation.

2. Materials and methods 2.3. Sampling and analysis 2.1. Materials The marine benthic diatom C. closterium was purchased from the Institute of Oceanology of the Chinese Academy of Sciences. The diatom was cultivated in sterile f/2 silicon-contained medium (Guillard and Ryther, 1962) made by artificial seawater (Araújo et al., 2010b) at 22 ± 1 °C, under a 16:8 light:dark cycle provided by an intelligent illumination incubator at an intensity of 60 μE s−1 m−2. The algal culture was maintained in mid-log exponential growth by serial transfers of subcultures to fresh medium every 7 days. Algal cells in the exponential phase of growth were used in the experiment. DEP and DBP with 99% purity were purchased from Sigma-Aldrich. All organic solvents used for the analysis were of analytical grade and obtained from Tianjin Chemical Reagent Factory. In addition, n-hexane and dichloromethane were rectified before use. A mixture stock solution of DEP and DBP (each of 400 mg L− 1) was prepared in acetone and stored at 4 °C prior to use. Surface sediments (top 0–10 cm) were collected from the intertidal flats of Bohai Bay, Tianjin (39° 8′ 34″ N, 117° 50′ 00″ E). The sediments were then air-dried and ground to pass through a 2-mm sieve to remove debris and small rocks. The characteristics of the sediment were as the following: pH 7.9, total organic content 2.29%, total nitrogen amount 20 mg kg−1 dry wt, and total phosphate amount 7 mg kg−1 dry wt. The sediment was composed of 18.4% sand, 43.6% silt and 38.1% clay.

After the beakers were collected, the overlying water was removed carefully with a pipette. The sediment in the beakers was then divided into two slices. One was the top 0.5 cm of the sediment, and the other was the 1.5 cm of the sediment left at the beaker bottom. A subsample of each slice was immediately freeze-dried, ground and passed through an 80-mesh sieve for the analysis of PAEs and PLFAs (only samples collected on days 0, 2 and 8 were used for PLFA analysis), and the rest was used for the analysis of chlorophyll content. Three replicates were conducted for each sample. Dried sediment samples (2 g) were sonically extracted with 5 mL dichloromethane. Each sample was extracted three times and each for 10 min. The extraction solution was centrifuged at 4000 rpm for 5 min. The combined dichloromethane solutions were concentrated to a volume of 0.5 mL under a gentle stream of nitrogen gas. The concentrated extracts were then analyzed for PAEs by an Agilent 6890N gas chromatograph fitted with a splitless injector, a fused-silica capillary column (HP-5, 0.32 μm × 30 m) and a flame ionization detector. The temperatures of injector and detector were both set at 250 °C. Nitrogen was used as a carrier gas at a flow rate of 50 mL min−1, while the flow rates of hydrogen and air were 37 and 550 mL min−1. Injection volume was 1 μL. DEP and DBP were eluted with the following temperature program: 100 °C (1 min) → 30 °C min−1 (6 min) → 280 °C (5 min). The retention times of DEP and DBP were 5.62 and 6.88 min.

Y. Li et al. / Science of the Total Environment 508 (2015) 251–257

PLFAs were extracted in three steps using a modified procedure (He et al., 2009). In brief, lipids were extracted from sediments by chloroform, methanol and citrate buffer, and then fractionated into neutral lipids, glycolipids and phospholipids on a silica gel column. The phospholipids were then subjected to alkaline methanolysis and analysis on GC–MS. The experimental details are described in the Supplementary material associated with this article. Chlorophyll was analyzed according to the method of Hagy et al. (2005). 2.4. Statistical analysis All the results were expressed as mean ± SE of three replicates. The treatment effects were compared using analysis of variance (ANOVA), and comparisons of means were carried out using Duncan's test. The significance level was P ≤ 0.05. Microbial community structure represented by PLFAs was analyzed by principal component analysis (PCA). All statistical analyses were performed using the software Statistical Package for Social Sciences (SPSS 19.0 for Windows). 3. Results 3.1. Algae growth The growth of C. closterium indicated by chlorophyll content in sediments was monitored regularly. During the 8-day experiment, there were no significant changes (P N 0.05) of chlorophyll contents in bottom sediments (Fig. 1). In contrast, the chlorophyll contents in surface sediments increased over time, and more significantly in unsterilized sediments with C. closterium (i.e. treatment BA) than in sterilized sediments with C. closterium (i.e. treatment A). At the end of the experiment, the chlorophyll contents in surface sediments of treatment BA and treatment A increased by 2.7 and 2.3 times. 3.2. The profiles of PLFAs in marine sediments Fourteen PLFAs ranging from C14 to C20 were identified in the sediment samples, including saturated (SAT), methyl-branched (BR), monounsaturated (MON), polyunsaturated (POL) and cyclopropyl (CYC) fatty acids (Table 1 and Table S1). In order to assess sediment microbial community, all of the individual PLFAs were classified into four groups: bacteria (the sum of SAT, BR, MON and CYC fatty acids in the range of C14 to C19; Zelles, 1999), aerobic bacteria (MON fatty acids; Rajendran et al., 1992, 1997), anaerobic bacteria (BR fatty acids;

5.0

BA-s BA-b A-s A-b

4.5

-1

Chlo contents ( µg g )

4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0

2

4

6

8

Time (d) Fig. 1. Chlorophyll contents in sediments inoculated with C. closterium. Letters BA and A represent unsterilized treatment with C. closterium and sterilized treatment with C. closterium. Letters s and b represent surface and bottom sediments.

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Rajendran et al., 1992, 1997), and fungi (POL fatty acid; Frostegård et al., 1993). The ubiquitous fatty acid 16:0 showed the highest abundance (31.1– 43.1%) in all sediment samples (Table 1), consistent with those reported by Su and Yang (2009) and Zhang et al (2012). The relative abundance of 18:2ω6,9, an indicator of fungi, was relatively low and in the range of ND–4.5%, suggesting that microeukaryotes were sparsely distributed in the sediments. This is similar to the result by Su and Yang (2009). The relative abundances of individual PLFAs in bottom sediments generally showed no significant change over time except for monounsaturated fatty acid 16:1ω7 and 18:1ω7 which increased obviously on day 8 in the bottom sediments of treatment BA. In surface sediments, the relative abundances of some PLFAs (such as monounsaturated fatty acid 16:1ω7 and 18:1ω7, and cyclopropyl fatty acid cy19:0) increased over time, while others (such as saturated fatty acid 15:0 and 17:0, branched fatty acid i15:0 and a15:0, and polyunsaturated fatty acid 18:2ω6,9) showed decreasing trends, and the rest of the PLFAs generally showed no significant changes over time. Cyclopropyl fatty acid cy19:0 was found only in the surface sediments of treatment BA. Contents of different PLFA groups, including total PLFAs, bacterial PLFAs, aerobic and anaerobic bacterial PLFAs, initially increased and then declined in surface sediments, but show relatively small variation in the bottom sediments (Table 2). In contrast, the content of fungal PLFA generally decreased over time. Compared with unsterilized sediments without C. closterium (i.e. treatment B), the contents of total PLFAs, bacterial PLFAs and aerobic bacterial PLFAs in the sediments of treatment BA increased by 11.5–40%, 10.6–45.5% and 36.4–271%, respectively, on day 2, and 47.1–54.3%, 46.8–70.3% and 89%, respectively, on day 8. However, the content of fungal PLFA was generally lower in the sediments of treatment BA than in the sediments of treatment B. For anaerobic bacteria, compared with treatment B, the PLFA content in treatment BA was lower in surface sediments, but higher in the bottom sediments. It can also be seen that the sum of the bacterial PLFAs was more than 96.4% of the total PLFAs in all the sediment samples. Aerobic bacteria were more dominant than anaerobic bacteria, especially in the surface sediments of treatment BA. The ratio of aerobic to anaerobic bacteria increased from the initial 1.21 to 7.98 and 3.65 in surface sediments of treatment BA and treatment B on day 8, but there was no significant change (P N 0.05) in the bottom sediments. The ratio of fungi to bacteria in all sediment samples ranged from 0.000 to 0.048 which was similar to the result of previous studies (Li et al, 2010; Zhang et al., 2012). In treatment BA, the ratio of fungi to bacteria decreased over time and more quickly in the surface sediments, while there was only small change in treatment B. The PCA for the profiles of PLFAs was conducted to analyze the changes of the microbial communities in different treatments. The first principal component (PC1) and the second principal component (PC2) explained 40.38% and 33.80% of the data variability (Fig. 2). It can be seen from Fig. 2a that all points were separated along PC2, but little apart along PC1. The scattered points of the bottom sediments mostly gathered near the initial sediment point and were separated along PC2 from most points of the surface sediments. This result indicated that during the experiment, the microbial community structure was relatively stable in the bottom layer of the sediments, and changed significantly in the surface layer of sediments, especially in the presence of C. closterium. In addition, the point of the bottom sediments of treatment BA on day 8 (i.e. BA-b (8 day)) was closer with the points of the surface sediments than with the other points of the bottom sediments. This suggested that cultivating C. closterium on the sediment surface could affect the microbial community structure in a deeper layer of sediments at the end of the experiment. The aerobic bacterial biomarkers (16:1ω7 and 18:1ω7), which lay on the upper side of the plot, accounted for much of the scores along PC2 (Fig. 2b). Besides, their abundances were also higher in all of the sediment samples (Table 1). The results in both Fig. 2a and b indicated sediment points scattered on the right-up side of the plot obviously contained relatively

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Table 1 Proportions of individual PLFA (mol%) to total PLFAs in unsterilized sediments. Treatment

Sediment layer

Time (d)

i14:0

14:0

i15:0

a15:0

15:0

i16:0

16:1ω7

With C. closterium

Surface

0 2 8 0 2 8 0 2 8 0 2 8

1.24 0.72 0.36 1.24 1.47 1.36 1.24 1.24 0.62 1.24 1.05 1.21

5.91 8.22 7.63 5.91 8.08 5.31 5.91 7.26 ND 5.91 7.02 5.52

5.50 3.12 2.05 5.50 6.07 6.47 5.50 4.40 2.86 5.50 4.61 4.59

7.47 3.91 2.31 7.47 7.33 8.13 7.47 5.60 3.85 7.47 5.25 6.53

4.26 2.24 1.82 4.26 4.11 2.62 4.26 5.59 1.82 4.26 4.56 4.23

2.34 1.12 0.55 2.34 2.09 2.29 2.34 2.00 2.22 2.34 2.76 2.16

8.10 14.57 18.71 8.10 8.44 11.27 8.10 5.43 10.65 8.10 6.11 8.30

Bottom

Without C. closterium

Surface

Bottom

Treatment

Sediment layer

Time (d)

16:0

17:0

18:2ω6,9

18:1ω7

18:0

cy19:0

20:0

With C. closterium

Surface

0 2 8 0 2 8 0 2 8 0 2 8

37.91 31.09 35.11 37.91 37.18 35.17 37.91 40.66 36.44 37.91 43.10 38.33

1.54 0.99 ND 1.54 1.54 ND 1.54 1.60 1.48 1.54 1.69 1.72

3.09 0.88d ND 3.09 2.79 1.18 3.09 3.24 3.87 3.09 2.03 2.62

11.85 23.77 23.12 11.85 10.48 15.00 11.85 8.95 22.54 11.85 9.34 12.31

10.80 7.37 8.34 10.80 10.42 9.54 10.80 12.67 11.14 10.80 12.49 12.48

ND 2.04 4.90 ND ND ND ND ND ND ND ND ND

ND ND ND ND ND 1.60 ND 1.39 1.64 ND ND ND

Bottom

Without C. closterium

Surface

Bottom

ND means not detected.

high biomass of aerobic bacteria than those located on the right-down side. 3.3. PAEs in marine sediments The concentrations of DEP and DBP in all the sediment samples are shown in Table S2. There were no significant changes of DEP and DBP concentrations in unsterilized sediments without C. closterium (i.e. treatment N) over time (P N 0.05), suggesting that the effect of abiotic processes (such as photolysis, hydrolysis, volatilization) on the PAE removal from the sediments was negligible. It can be seen from Fig. 3 that the elimination ratios of the PAEs increased rapidly in unsterilized sediments (i.e. treatment BA and treatment B) during the first 2 days and then slowly during the remaining 6 days. However, there were no significant changes (P N 0.05) of PAE elimination ratios in sterilized sediments with C. closterium (i.e. treatment A) during the first 2 days, but thereafter, the elimination ratios increased over time except that of DEP in the bottom sediments which showed no significant change (P N 0.05) during the remaining 6 days. Moreover, the elimination ratios were higher in the surface sediments than in the bottom sediments and higher for DEP than for DBP. After one day of experiment, the elimination ratios were in the order of the

surface sediments in treatment BA (BA-s; 89.0%) N bottom sediments in treatment BA (BA-b; 75.7%) N surface sediments in treatment B (B-s; 51.5%) N bottom sediments in treatment B (B-b; 23.0%) N surface sediments in treatment A (A-s; 10.6%) N bottom sediments in treatment A (A-b; 2.3%) for DEP, and BA-s (66.2%) N BA-b (61.7%) N B-s (11.8%) and B-b (10.2%) N A-s (− 3.2%) and A-b (− 3.2%) for DBP. On the 2nd day, there were no significant differences (P N 0.05) of the PAE elimination ratios among samples of BA-b, B-a and B-b. In addition, the concentration of DEP in the surface sediments of treatment BA was not detectable. The elimination ratios were in the order of BA-s (near 100.0%), BA-b (near 100.0%) and B-s (near 100.0%) N B-b (92.0%) N A-s (79.5%) N A-b (58.6%) for DEP, and BA-s (94.1%) N B-s (86.5%) N BA-b (81.4%) and B-b (77.0%) N A-s (12.8%) N A-b (3.8%) for DBP at the end. The plots of the decline of DEP and DBP in the sediments followed the first-order kinetic model. The kinetic data obtained are presented in Table 3. The elimination rate constants (k) of the PAEs were mostly in the order of treatment BA N treatment B N treatment A for both surface and bottom sediments, and surface sediments N bottom sediments in the same treatment. Inoculating C. closterium on the surface of unsterilized sediments obviously increased the k values of DEP and DBP in surface sediments, but changed the k values slightly in the bottom sediments.

Table 2 Contents of different microbial groups indicated by PLFAs (nmol·g−1 dry weight) and ratios of aerobic to anaerobic bacteria and fungal to bacteria in unsterilized sediments. Treatments

Sediment layer

Time (d)

Total PLFAs

Bacteria

Aerobic bacteria

Anaerobic bacteria

Fungi

Aerobic to anaerobic

Fungal to bacteria

With C. closterium

Surface

0 2 8 0 2 8 0 2 8 0 2 8

33.18 cd 73.21a 53.93b 33.18 cd 34.90 cd 42.40c 33.18 cd 52.32b 34.72 cd 33.18 cd 31.31d 28.82d

32.17d 72.58a 55.93b 32.17d 33.93 cd 41.89c 32.17d 49.89bc 32.85 cd 32.17d 30.67d 28.06d

6.55e 28.02a 22.35b 6.55e 6.60e 11.17d 6.55e 7.55d 11.83d 6.55e 4.84e 5.91e

5.49bc 6.43a 2.81d 5.49bc 5.89b 7.77a 5.49bc 6.94a 3.28c 5.49bc 4.28c 4.13c

1.00b 0.63bc NDd 1.00b 0.97 0.50c 1.00b 1.70a 1.78a 1.00b 0.64bc 0.76bc

1.21 4.35 7.98 1.21 1.12 1.44 1.21 1.09 3.65 1.21 1.13 1.43

0.032 0.009 0.000 0.032 0.029 0.012 0.032 0.033 0.041 0.032 0.021 0.027

Bottom

Without C. closterium

Surface

Bottom

Values in each column followed with different letters (a–e) indicate a significant difference (P b 0.05) among different sediment samples.

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Fig. 2. Principal component analysis (PCA) for PLFAs in unsterilized sediments with and without C. closterium. (a) variation in PLFA patterns; (b) loading values of individual PLFAs. Letters BA and B represent unsterilized treatment with and without C. closterium. Letters s and b represent surface sediments and bottom sediments. 2 day and 8 day in brackets represent the sampling days of 2 and 8.

4. Discussion 4.1. Effect of algae growth on the profiles of PLFAs in marine sediments In this work, obvious yellow-green algal mat was observed on the surface sediments of both treatments BA and A, indicating that the benthic diatom C. closterium could be inoculated easily at the

sediment–water interface and grew well, consistent with that reported by Miller et al. (1996). An exponential decrease in chlorophyll content with depth below the sediment–water interface has been found by previous studies (Sun et al., 1994; Hagy et al., 2005). Accordingly, most of C. closterium should be presented in the top 0.5 cm of the sediments. The chlorophyll content in the surface sediments (top 0.5 cm) significantly increased, but no obvious algae growth in the bottom sediments

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Fig. 3. Elimination ratios of (a) DEP and (b) DBP in sediments of different treatments. Letters BA and B represent unsterilized treatment with and without C. closterium. Letters A and N represent sterilized treatment with and without C. closterium. Letters s and b represent surface sediments and bottom sediments.

was observed (Fig. 1). This is consistent with the previous findings. Moreover, in the presence of bacteria (i.e. treatment BA) C. closterium grew a little better than in the absence of bacteria (i.e. treatment A) as

Table 3 Elimination rate constants (kb) of DEP and DBP in sediments of different treatments. PAEs

Treatments

Layer

kb (d−1)

R2

DEP

Sterilized, with C. closterium

Surface Bottom Surface Bottom Surface Bottom Surface Bottom Surface Bottom Surface Bottom

0.216 0.129 0.460 0.329 2.098a 0.444 0.039 –b 0.256 0.194 0.309 0.165

0.9565 0.9344 0.8393 0.8494 – 0.7349 0.8973 – 0.9309 0.7363 0.8703 0.7789

Unsterilized, without C. closterium Unsterilized, with C. closterium DBP

Sterilized, with C. closterium Unsterilized, without C. closterium Unsterilized, with C. closterium

a The value of kb was estimated by kb = lnC0 − lnC1, where C0 and C1 are DEP concentrations on days 0 and 1. b The value of kb was unavailable because there was no obvious change (P N 0.05) of DBP concentration during the experiment. – indicates no data available.

a result of the stimulating effect of heterotrophic bacteria on algae growth (Yamamoto et al., 2008). It can be seen from Table 1 and Table S1 that in the presence of C. closterium, both contents and relative abundances of PLFAs in the surface sediments of treatment BA varied more significantly, which was mainly related to the increase of the biomass of aerobic bacteria indicated by 16:1ω7 and 18:1ω7. The ratio of the aerobic to anaerobic bacteria also increased significantly (Table 2), indicating that aerobic bacteria were more dominant in the microbial community. Similar results were also found in the PCA plots. The reason is that C. closterium could produce oxygen through photosynthesis and release algal exudates (e.g. exopolysaccharides), meeting the needs of aerobic bacteria, and consequently stimulating the bacterial growth in the sediments. Moreover, the negative effect of C. closterium on the ratio of fungi to bacteria was found, showing growth inhibition of fungi by the alga. Zhang et al. (2012) found that compared with non-rhizospheric soils, the presence of Scirpus triqueter, a dominant species in wetland of Huangpu–Yangtze River estuary, increased the ratio of fungi/bacteria in rhizospheric soils. Su and Yang (2009) reported that no significant difference of the ratio of fungi/bacteria between rhizospheric and non-rhizospheric soils of rice (Oryza sativa) was observed. It seems that different plant species had different effects on fungal biomass. However, in the absence of C. closterium, there was less increase of the biomass of aerobic bacteria in the surface sediments (treatment B). This is because the oxygen in the surface sediments of treatment B was mainly from overlying water via diffusion. The positive effect of C. closterium on the growth of aerobic bacteria was also observed to some extent in the bottom sediments of treatment BA at the end of the experiment (i.e. the 8th day), indicating that improved sediment oxygenation by C. closterium could stimulate the growth of aerobic bacteria in deeper sediment layer. The possible reasons for the lag of aerobic bacterial growth in the bottom sediments are as follows: firstly, oxygen uptake by the microbes in the surface sediments left little oxygen to penetrate the sediment at the initial period; secondly, the diffusion rate of oxygen in the submerged sediments was low, which is up to four orders of magnitude less than that in soils (Good and Patrick, 1987; Huesemann and Truex, 1996); lastly, the biomass of C. closterium was initially smaller. With the growth of C. closterium, oxygen production by the alga increased, and consequently the oxygen flux into the sediment increased over time. 4.2. Effect of C. closterium on PAE degradation in marine sediments From the sterilization control test without C. closterium, abiotic losses of the two PAEs were negligible. Moreover, elimination rate constants of the PAEs in sterilization control test with C. closterium were much smaller than those in unsterilized tests (Table 3), indicating that C. closterium played a minor role in the PAE removal from sediments. Therefore, it can be concluded that the elimination of the PAEs in sediments was mainly by microbial degradation. From Fig. 3, it can be seen that inoculating C. closterium on the sediment surface could enhance the degradation of the PAEs in both surface and bottom layers of unsterilized sediments, and more significantly in the surface layer than in the bottom layer. This is similar to the change of trend of the biomass of aerobic bacteria. Pearson correlation analysis showed a positive correlation between elimination efficiency of DBP and abundance of total aerobic bacterial PLFAs (r = 0.727, n = 8, p b 0.01). However, the Pearson correlation analysis for DEP was not performed because DEP was mostly undetected at the end of the experiment due to rapid elimination of DEP from the sediments. Numerous studies indicate that PAEs are degraded by a wide range of bacteria under both aerobic and anaerobic conditions (Staples et al., 1997). However, the rate of degradation under anaerobic conditions has been found to be retarded relative to aerobic tests (Staples et al., 1997). The submerged sediments are often low in oxygen and thus do not provide favorable conditions for the biodegradation of PAEs by

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aerobic bacteria. In this study, increased sediment oxygenation by C. closterium stimulated the growth of aerobic bacteria as well as increased their relative abundance in total PLFAs, leading to an enhancement of PAE degradation. Therefore, it can be concluded that aerobic bacteria were mainly responsible for the C. closterium-promoted degradation of the PAEs in the sediments. The elimination efficiency of DEP was significantly higher than that of DBP, indicating that DBP was more persistent. This is mainly due to the stronger hydrophobic nature of DBP, which reduces its bioavailability as the result of being adsorbed on the sediments (Cousins and Mackay, 2000). 5. Conclusions Accelerated removal of DEP and DBP in sediments was observed in the presence of marine benthic diatom C. closterium. During the 8-day experiment, the elimination ratios of the PAEs increased more significantly in the surface layer (top 0.5 cm) of sediments than in the bottom layer of the sediments, and were higher for DEP than for DBP. Moreover, the microbial degradation was found to be the dominant process for PAE removal from the sediments because of lower PAE degradation rates by C. closterium (treatment A). In order to better understand the microbial mechanism involved, the profiles of PLFAs in the sediments were analyzed. The results revealed that both PLFA contents and microbial community structure changed significantly in the presence of C. closterium, which was mainly related to the increase of the biomass of aerobic bacteria as a result of improved sediment oxygenation by C. closterium. Furthermore, significant positive correlations between the elimination ratios of the PAEs and abundances of total aerobic bacterial PLAFs were obtained, suggesting that aerobic bacteria played a key role in C. closterium-promoted PAE degradation in the sediments. This study provides valuable information for the potential remediation of PAE-contaminated marine sediments by benthic microalgae as well as for the remediation mechanism. Further studies in the field need to be undertaken. Acknowledgments The authors are grateful for the financial support from the Tianjin Key Laboratory of Marine Resources and Chemistry, China (contract/ grant number: 201208). Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.scitotenv.2014.12.002. References Araújo, C.V.M., Blasco, J., Moreno-Garrido, I., 2010a. Microphytobenthos in ecotoxicology: a review of the use of marine benthic diatoms in bioassays. Environ. Int. 36, 637–646. Araújo, C.V.M., Tornero, V., Lubián, L.M., Blasco, J., van Bergeijk, S.A., Cañavate, P., Cid, Ángeles, Franco, D., Prado, R., Bartual, A., López, M.G., Ribeiro, R., Moreira-Santos, M., Torreblanca, A., Jurado, B., Moreno-Garrido, I., 2010b. Ring test for wholesediment toxicity assay with -a-benthic marine diatom. Sci. Total Environ. 408, 822–828. Chang, B.V., Wang, T.H., Yuan, S.Y., 2007. Biodegradation of four phthalate esters in sludge. Chemosphere 69, 1116–1123.

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