Article pubs.acs.org/est
Pyrene Degradation Accelerated by Constructed Consortium of Bacterium and Microalga: Effects of Degradation Products on the Microalgal Growth Shusheng Luo,†,‡,∥ Baowei Chen,†,∥ Li Lin,† Xiaowei Wang,† Nora Fung-Yee Tam,§ and Tiangang Luan*,† †
MOE Key Laboratory of Aquatic Product Safety, School of Marine Sciences, School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, People’s Republic of China ‡ Chemistry Department, South University of Science and Technology of China, Shenzhen 518055, People’s Republic of China § Department of Biology and Chemistry, City University of Hong Kong, Tat Chee Avenue, Kowloon, Hong Kong SAR, China S Supporting Information *
ABSTRACT: Abundant microbes including bacteria, fungi, or algae are capable of biodegrading polycyclic hydrocarbons (PAHs). However, pure cultures never occur in the contaminated environments. This study aimed to understand the general potential mechanisms of interactions between microbes under pollution stress by constructing a consortium of PAHdegrading microalga (Selenastrum capricornutum) and bacterium (Mycobacterium sp. strain A1-PYR). Bacteria alone could grow on the pyrene, whereas the growth of algae alone was substantially inhibited by the pyrene of 10 mg L−1. In the mixing culture of algae and bacteria, the growth rate of algae was significantly increased from day 4 onward. Rapid bacterial degradation of pyrene might mitigate the toxicity of pyrene to algae. Phenolic acids, the bacterial degradation products of pyrene, could serve as the phytohormone for promoting algal growth in the coculture of algae and bacteria. In turn, bacterial growth was also enhanced by the algae presented in the mixing culture. Consequently, the fastest degradation of pyrene among all biodegradation systems was achieved by the consortium of algae and bacteria probably due to such interactions between the two species by virtue of degradation products. This study reveals that the consortium containing multiple microbial species is high potential for microbial remediation of pyrene-contaminated environments, and provides a new strategy to degrade the recalcitrant PAHs.
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algae.20,25 In addition, cell surface of microalgae can provide a stable habitat for the bacteria.26 Pollutants absorbed on the microalgal cell surface could also create a pollutant-enriching zone to facilitate bacterial degradation.27,28 In view of the high degradation rate of organic pollutants achieved collaboratively by phototrophic microalgae and heterotrophic bacteria, algal− bacterial consortium has been successfully applied in the biodegradation of organic solvents,24,29,30 crude oil,23,31 phenol,29 salicylate,32,33 etc. In spite of great advantages, limited studies on PAH biodegradation by the algal−bacterial consortium are available. Munoz constructed an algal−bacterial consortium of Chlorella sorokiniana and phenanthrene-degrading Pseudomonas migulae for phenanthrene biodegradation in two-phase partitioning bioreactors.34 Although the authors optimized the operating conditions of phenanthrene biodegradation, little attention has been paid on the underlying mechanism for the cooperation of
INTRODUCTION Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous in various environmental niches and have been of great concern due to their high risks to animals and humans. 1−3 Biodegradation is considered as one of the most important routes for removing PAHs from the contaminated sites.4−7 A broad spectrum of microorganisms including bacteria,8,9 fungus,10,11 and microalgae12−14 have exhibited strong ability to degrade PAHs. However, biodegradation efficiency of PAH by a single pure culture was found to be low.15,16 Aiming to achieve high biodegradation rate of PAHs, an alternative strategy is to construct a consortium that is comprised of several PAH-degraders.15,17−19 In recent years, the consortium consisting of microalgae and bacteria has been considered for biological degradation of organic contaminants.20,21 Some algal−bacterial consortiums were constructed to investigate their abilities of degrading toxic organic contaminants.20,22−24 The mutualistic symbiosis of microalgae and bacteria offers many advantages over single species. For instance, microalgae can provide oxygen and carbon source, which are required for aerobic bacteria to metabolize organic pollutants, and in turn CO2 released from bacteria respiration is utilized by micro© XXXX American Chemical Society
Received: August 1, 2014 Revised: November 4, 2014 Accepted: November 9, 2014
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7−10 days of incubation, microalgal cells at the exponential growth phase were harvested by centrifugation at 2200 g (5000 rpm) for 10 min at 4 °C. The cell pellet was washed and resuspended in autoclaved deionized water. The cell number was counted using a hemocytometer and adjusted to 1 × 107 cells mL−1 for subsequent inoculation. M. A1-PYR was isolated from the mangrove surface sediment. This strain is able to utilize phenanthrene, fluoranthene and pyrene as the sole carbon source as described by Zhong et al.40 M. A1-PYR was incubated in nutrient broth (10 g of peptone, 3 g of beef extract, 5.0 g of NaCl, and 1.0 g of glucose in 1 L of distilled water) on a rotary shaker (150 rpm) at 30 ± 1 °C. The cells were harvested at the late exponential growth phase (about 48 h) by centrifugation at 3960 g (9000 rpm) for 10 min at 4 °C and washed twice with 8.5 g L−1 NaCl. The cell pellet was resuspended in NaCl solution (8.5 g L−1) to obtain a cell suspension with an optical density of 1.0 at 600 nm wavelength for inoculation (1.22 × 109 CFU mL−1, determining by counting CFU on nutrient agar plates). Experimental Setup. A stock solution of pyrene (5000 mg L−1) was prepared by dissolving appropriate amounts of pyrene in acetone. A 100 μL aliquot of pyrene stock solution was mixed with 50 mL of SE medium in a sterilized 150 mL Erlenmeyer flask with a final concentration 10 mg L−1 (49.5 μmol L−1). The level of pyrene is generally lower in an aquatic environment (below several hundred ng L−1) than that used in our experiments,41,42 but it is unexpectedly higher in some cases, e.g., oil spill accidents or coking wastewater (up to several hundred μg L−1).43,44 The high concentration of pyrene selected in this study could better estimate the pyrene degradation capacity of the mixing cultures containing microalgae and bacteria. The flasks were capped using plastic filter membranes (pore size, 0.2 μm) that facilitated gas exchange with ambient environment. Controls containing acetone and medium were also set up to understand its influence of acetone on the growth of microalga and bacterium. All flasks were kept in the dark for 4 days to allow the evaporation of added acetone prior to inoculation. To differentiate the contributions of microalgae and bacteria to the degradation of pyrene, three different biodegradation systems (algal−bacterial consortium, algal-only, and bacterialonly) were constructed by inoculating the mixing culture of S. capricornutum (0.5 mL, 1 × 105 cell mL−1) and M. A1-PYR (0.5 mL, 1 × 107 CFU mL−1) or the single culture containing each of the two species (0.5 mL) in the 50 mL pyrene-added SE medium (10 mg L−1). Flasks without inoculation with microbes were used as the controls to determine any abiotic loss of pyrene during incubation. In addition, another set of flasks devoid of pyrene were inoculated with S. capricornutum, M. A1PYR, and the mixture of them for investigating the interaction between algae and bacteria under natural conditions. All flasks were incubated in a light incubator under the same conditions as described above. To avoid the sedimentation of microorganisms, the flasks were shaken for 5 min on a rotary shaker every 12 h. Pyrene Degradation and Microbial Growth. Triplicate flasks (50 mL) from each of treatments were sacrificed at the day 1, 4, 7, 10, and 14 to determine pyrene residue, and 100 μL of medium was used to count the cell number of S. capricornutum and M. A1-PYR. The cell density of S. capricornutum was directly determined using ahemocytometer and M. A1-PYR by counting CFU on the agar plate. For measuring pyrene concentration, each of the flasks was spiked
microalgae and bacteria during phenanthrene degradation. Microalgae are not simply considered as a “helper” for bacteria in the algal−bacterial consortium during pollutant degradation because they are also capable of degrading various organic pollutants that range from monocyclic to polycyclic aromatic compounds.13,35−38 Since microalgae and bacteria are both able to degrade PAHs, the mixing culture of them would be more effective in processing recalcitrant PAHs. Warshawsky et al. exhibited that the initial oxidation of benzo[a]pyrene (Bap) performed by microalgae could in succession ease its further degradation by bacteria.39 This result revealed that the interactive compensation of microalgae and bacteria in PAH metabolic pathways could overcome some aspects of the rate-limiting step(s) observed for single species. However, microalgae and bacteria were separately incubated in different medium in the above study. Therefore, it was impossible to investigate the cometabolic effect on organic pollutant degradation between microalgae and bacteria and to explore the exact mechanism responsible for their synergistic cooperation. Constructing an effective algal−bacterial consortium for the PAH biodegradation is often challenged with several issues: microalgae are always more susceptible to hazard PAHs than bacteria; PAHdegrading bacteria are not always compatible with microalgae; the accumulation of PAHs intermediates could probably exert a great influence on the microalgal growth. In the present study, an algal−bacterial consortium consisting of microalgae (Selenastrum capricornutum) and bacteria (Mycobacterium sp. strain A1-PYR) that were both able to degrade PAH was constructed to understand their respective roles in pyrene degradation, as well as in turn the potential effects of pyrene metabolites on the microbial growth.
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MATERIALS AND METHODS Chemicals. Pyrene, 1-hydroxypyrene, 4-phenanthrencarboxyic acid, 4-phenanthrol, biphenyl,1-naphthylacetic acid (NAA), phenylacetic acid (PAA), salicylic acid (SA), hydrophenlyacetic acid (HPA), and benzoic acid (BA) were purchased from Sigma−Aldrich (St. Louis, MO). The silylation reagent, N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA), and ethyl chloroformate were purchased from Sigma. The other chemicals and reagents used in this study were analytical grade. Stock solutions of biphenyl (5000 mg L−1) and NAA (10 mg L−1) were prepared by dissolving appropriate amounts of each chemical in acetone. Microorganisms and Culture Conditions. The S. capricornutum was purchased from Carolina Biological Supply Co., Burlington, NC. The microalgae were cultured in 100 mL of soil extract (SE) medium in a 250 mL Erlenmeyer flask under axenic conditions in a light incubator at 25 ± 2 °C with a 16-h/8-h light/dark photoperiod at a light intensity of 65 μE s−1 m−2 as PAR (photosynthetically active radiation) using cool-white fluorescent lamps. The SE medium contained NaNO3 (25 g L−1), K2HPO4 (7.5 g L−1), KH2PO4 (17.5 g L−1), MgSO4·7H2O (7.5 g L−1), NaCl (2.5 g L−1), CaCl2·2H2O (2.5 g L−1), FeCl3·6H2O (0.5 g L−1), MnCl2·4H2O (0.181 g L−1), CuSO4·5H2O (0.0074 g L−1), ZnSO4·7H2O (0.0222 g L−1), Na2MoO4·2H2O (0.0015 g L−1), H3BO3 (0.282 g L−1), and soil extract (40 mL L−1). Soil extract was obtained by adding 0.5 kg garden soil in 1000 mL of H2O, boiling for 2 h, and filtering after cooling to room temperature. The SE medium was prepared in the deionized water, and the pH was adjusted to 7.0 using sodium hydroxide (NaOH, 1 M). After B
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with 100 μL of stock solution of biphenyl as the internal standard, and then the cells were digested with 20 mL of NaOH (6 M) for 30 min. The medium was extracted twice with equal volumes of 25 mL of ethyl acetate according to the literature.45 Finally, the pyrene was analyzed using an Agilent 6890A-5973N gas chromatography mass spectrometry (GC− MS, Agilent Technologies, Wilmington, DE) equipped with an HP-5MS fused silica capillary column (30 m × 0.25 mm i.d. × 0.25 μm). Helium was used as carrier gas, and the flow rate was at 1.0 mL min−1. The inlet temperature was maintained at 280 °C with the splitless mode. The oven temperature was programmed as follows: initially at 60 °C, increasing to 300 °C (hold for 10 min) at the rate of 15 °C min−1. Mass spectrometry was operated at the electron impact ionization at 70 eV with selected ion monitoring mode. Pyrene and biphenyl were identified by the characteristic ions of 202 and 156, respectively. Analysis of Pyrene Metabolites. Another set of triplicate flasks containing 50 mL of culture were used for the analysis of pyrene metabolites. A 10 mL aliquot of medium was collected for the extraction of pyrene metabolites at each sampling time and centrifuged at 3960 g at 4 °C for 10 min. The flasks were discarded when the residual medium was less than 30 mL. The supernatant was adjusted to pH 3.0 using 1 M HCl. Pyrene metabolites in the supernatant were extracted by automated onfiber silylation solid-phase microextraction (SPME) using 85 μm polyacrylate fiber and 100 μL BSTFA derivatization agent (performed by Gerstel MPS2 multipurpose autosampler). In brief, a 1.5 mL aliquot of supernatant was added into the 2 mL vial on the agitator for heating and agitation (45 °C, 300 rpm), and then a polyacrylate fiber was immersed directly into the sample solution for 40 min. Immediately following extraction, the fiber was inserted into a vial containing 100 μL BSTFA for 5 min headspace derivatization. The extract was analyzed using GC−MS following the reported method.19,40 The disperse liquid−liquid microextraction (DLLME) was used for quantifying phenolic acids metabolites.46 The supernatant (5 mL) was placed in a 10 mL conical bottom centrifuge tube and mixed with 25 μL of NAA (10 mg L−1) as internal standard. A 1 mL mixture of ethanol and pyridine (4:1, v/v; as dispersant and catalyzer, respectively) and 100 μL of chloroform (as extraction solvent) were added into the tube, and then pyrene metabolites in the aqueous sample were derivatized by 100 μL of ethyl chloroformate under ultrasonic conditions. The derivatives of phenolic acids in the chloroform phase were separated by centrifugation at 2200 g and subjected to GC−MS analysis Growth-Promoting Effects of Phenolic Acids on the Algae. The phenolic acids were investigated for their growthpromoting effects on S. capricornutum. Stock solutions of all metabolites (1.0 g L−1) were prepared by dissolving appropriate amounts of standards in acetone. Triplicate assays of each of phenolic acids were conducted at two levels (10 and 100 μg L−1). An equal amount of acetone in the medium was used as the control. The final concentration of acetone in the medium was below 0.1% (v/v) that has no significant effect on the S. capricornutum growth. All experiments were carried out in 150 mL Erlenmeyer flasks with 50 mL of SE medium under the same conditions as mentioned above. After the exponential growth stage (7−10 days), S. capricornutum was inoculated with an initial cell density of 1 × 105 cells mL−1. The growth of algal cells was microscopically monitored by directly counting cell numbers using a hemocytometer.
Statistical Analysis. The significance of differences in the pyrene degradation between different treatments was evaluated by one-way analysis of variance (ANOVA). All statistical analyses were performed using SPSS 13.0 for Windows (SPSS Inc.).
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RESULTS AND DISCUSSION Pyrene Degradation by Different Microbial Degradation Systems. The concentration of pyrene residue in different microbial degradation systems (algal-only, bacterialonly, and algal−bacterial consortium) was monitored over 14 days (Figure 1). The result demonstrated that the algal−
Figure 1. Degradation curves of pyrene by algal-only, bacterial-only and algal−bacterial consortium over 14 days. Error bar represents one standard deviation of triplicate assays. Letters indicate the significance of differences in the pyrene degradation between degradation systems on the level of p < 0.05.
bacterial consortium had the highest degradation rate of pyrene among all cultures, followed by bacterial-only and algal-only cultures. The fast pyrene degradation by the consortium was likely achieved by the cooperation or interaction between microalgae and bacteria. Pyrene was completely degraded by the algal−bacterial consortium in 10 days with an estimated rate of 5.05 ± 0.20 μmol L−1 d−1. In comparison to the algal− bacterial consortium (p < 0.05), bacterial-only culture took a longer time (14 days) to degrade all pyrene (4.36 ± 0.15 μmol L−1 d−1). This microalgal species could degrade up to 98% of added pyrene at a relative low concentration (4.95 μmol L−1) over 7 days.36 However, negligible amount of pyrene was removed by the algal-only culture in the present study. It was probably attributed to that the high pyrene concentration (49.5 μmol L−1) in this study posed a potential adverse effect on the algal growth. Algal and Bacterial Growth. The growth of S. capricornutum alone was severely inhibited by the pyrene of 10 mg L−1 (Figure 2a). This result was consistent with that pyrene was hardly degraded by the algal-only culture at such a high concentration. On the contrary, the growth of bacteria alone was substantially promoted with the addition of pyrene (Figure 2c), indicating that bacteria could growth on the pyrene.19,40 In the algal−bacterial consortium, the growth rate of S. capricornutum was significantly increased from day 4 with the addition of pyrene (Figure 2b), which suggested that the presence of M. A1-PYR could mitigate the inhibitory effect of C
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Figure 2. Growth curves of S. capricornutum and M. A1-PYR in the cultures of algae alone, bacteria alone, and algal−bacterial consortium with or without the addition of pyrene.
[M+], 290 [M − TMS-CH3], 260 [M − TMS-3CH3], and 73 [TMS] in Figure S1 (Supporting Information), which were labeled as dihydroxypyrene I, II, and III, respectively.19 The occurrences of pyrene metabolites produced by the algal-only and bacterial-only cultures, as well as the algal−bacterial consortium, are tabulated in Table S1 (Supporting Information). Dihydroxypyrene (I, II, and III) and 1-hydroxypyrene were considered as the initial intermediates in the degradation pathways of pyrene.19,40 The 4-phenanthrenecarboxylic acid and 4-phenanthrol were identified as subsequent metabolites following the ring cleavage of dihydroxypyrene, which were only detectable in the presence of M. A1-PYR (Table S1). Although some initial pyrene degradation products including 1hydroxypyrene and dihydroxypyrenes II and III were detected in the algal-only culture probably due to cometabolism, these two ring-cleavage pyrene metabolites were not detected in the algal-only culture. This result corroborated that S. capricornutum was unable to cleave the rings of pyrene and even to utilize the ring-cleavage metabolites. The four phenolic acids (benzoic acid, phenylacetic acid, salicylic acid, and hydroxyphenlyacetic acid) with small molecular weight were considered as the end degradation products of pyrene. Temporal Profiles of Pyrene Degradation Metabolites by Microbes. Figure 3 demonstrates that significant differences in the temporal profiles of initial metabolites (dihydroxypyrenes and 1-hydroxypyrene) were observed between algae and bacteria over 14 days. There were negligible differences in the concentration of 1-hydroxypyrene among different degradation systems before day 4. The concentration of 1hydroxypyrene in the algal-only medium increased after day 4 and finally reached 0.19 μmol L−1 at day 14, which was up to 45 times higher than those in the absence of alga (Figure 3a). It has not been reported that 1-hydroxypyrene can be degraded or utilized by any microorganisms. Bacteria could produce more
pyrene on the algal growth via pyrene degradation. The nutrients in SE medium without additional pyrene could be supplied for bacterial growth, as shown in Figure 2c. The introduction of algae into the culture could considerably enhance bacterial growth (Figure 2d). This result implied that the algae probably provided a heterotrophic carbon source required for the growth of M. A1-PYR. The microbial growth curves were completely consistent with the corresponding degradation curves of pyrene. Microbial growths were accelerated at day 4 when the fast microbial degradation of pyrene was achieved. The major challenge to construct an algal−bacterial consortium for contaminant degradation is that the algae are generally susceptible to toxic organic pollutants and grow much more slowly than bacteria. Therefore, detailed information on microbial growth is essential to evaluating the effectiveness of constructed microbial consortium in pollutant degradation and in turn the toxicities of pollutants to microbes. The significant difference in the microbial growth between different degradation systems also provided a clue to understanding why the mixing culture of algae and bacteria exhibited a stronger ability in pyrene degradation than single species. Pyrene Metabolites by the Algae and Bacteria. A compound that was only detected in the medium inoculated with microbes instead of the controls was determined as a degradation intermediate or product of pyrene. Six peaks were found to be related to pyrene metabolism using the SPME method, and four peaks were found using the DLLME method. The identities of these peaks were determined by comparing the retention time and mass spectra with the authentic standards. Although dihydroxypyrene standard was not commercially available, three peaks at the retention time of 24.26, 27.39, and 27.75 min were determined as dihydroxypyrene according to ion fragment pattern in mass spectra (378 D
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Figure 3. Temporal profiles of concentrations of initial pyrene degradation products by algal-only, bacterial-only, and algal−bacterial cultures over 14 days. Dihydroxypyrenes I−III represent three compounds that had identical ion fragments with dihydroxypyrene ((378 [M+], 290 [M − TMS-CH3], 260 [M − TMS-3CH3], and 73 [TMS]) but different chromatographic behaviors with retention times at 24.26, 27.39, and 27.75 min, respectively. The concentrations of dihydroxypyrenes could not be quantified due the lack of authentic standards. The levels of dihydroxypyrenes in the medium were expressed as peak area. Error bar represents one standard deviation of triplicate assays.
dihydroxypyrenes than algae (Figure 3b−d). The levels of all dihydroxypyrenes were much higher in the bacterial-only culture than in the algal-only culture. Notably, the levels of both 1-hydroxypyrene and dihydroxypyrenes in the consortium were lower than those in the single microbe. It probably indicates that initial pyrene metabolites in the consortium were rapidly transformed into downstream products with the participation of both bacterium and alga. For instance, it was observed in Figure 3b that the reduction of dihydroxypyrene I in the mixing culture synchronized with the fast microbial growth and pyrene removal at day 4. The changes in the concentrations of 4-phenanthrenecarboxylic acid and 4-phenanthrolover 14 days are shown in Figure S2 (Supporting Information). The results demonstrated that the concentrations of these two metabolites were consistently higher in the consortium than in the bacterial-only culture over the whole incubation period. It is unknown from which upstream metabolites (dihydroxypyrenes or 1-hydroxypyrene) 4-phenanthrenecarboxylic acid and 4-phenanthrolwere exactly generated in the consortium. Our findings at least explained that the concurrence of M. A1-PYR and S. capricornutum in the consortium led to a higher efficiency in the ring cleavage of dihydroxypyrenes or 1-hydroxypyrene than those in single cultures. As a result, the initial pyrene metabolites were maintained at a low level in the consortium. Figure 4 demonstrated that the levels of all phenolic acids (benzoic acid (BA), phenylacetic acid (PAA), and hydrox-
yphenlyacetic acid (HPA)) in the algal−bacterial consortium were significantly higher than those in the single cultures. Trace amounts of phenolic acids were detectable in the algal-only culture, which were probably produced from the common processes of biosynthesis as the ring-cleavage pathway of pyrene were not found in this algal species. Indeed, phenolic acids are the essential precursors of secondary metabolites for biological system and their biosynthetic pathway (i.e., via Lphenylalanine) is commonly found in both eukaryotes and prokaryotes.47 A key issue was to determine whether such high levels of phenolic acids (0.44, 1.12, and 1.20 μmol L−1 for HPA, PAA, and BA at day 14, respectively) in the consortium were produced from common biological processes or from pyrene degradation. A set of assays were therefore conducted to compare the production of phenolic acids with and without the addition of pyrene in the consortium (Figure S3, Supporting Information). The result demonstrated that the concentrations of phenolic acids with pyrene addition were far higher than those without pyrene, suggesting that phenolic acids were mainly generated from the pyrene biodegradation instead of the common biological processes in S. capricornutum and/or M. A1PYR. Promoting Effects of Phenolic Acids on Algal Growth. It was noted that high levels of phenolic acids (BA, PAA, and HPA) were produced and accumulated in the algal−bacterial mixed culture containing pyrene. Phenolic acids, such as PAA and SA, have phytohormone-like activity toward microalgal E
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Figure 4. Production of phenolic acids by algal-only, bacterial-only, and algal−bacterial cultures over 14 days. Error bar represents one standard deviation of triplicate assays.
species.48,49 It could be considered that phenolic acid produced from the bacterial degradation of pyrene had a significant effect on the growth of S. capricornutum. Hence, each of the phenolic acids (HPA, PAA, and BA) was separately incubated with S. capricornutum to investigate their individual effects on the algal growth (Figure 5). HPA and PAA at the low concentration (10 μg L−1) promoted the S. capricornutum growth by 44.5% and 36.6%, respectively, while BA accelerated the algal growth by 39.6% over 14 days as its concentration reached up to 100 μg L−1. It has been reported that the compounds secreted by algal growth-promoting bacteria (AGPB) have phytohormone-like activity to promote the algal growth; e.g., indole-3-acetic acid (auxin) produced by the bacterium Azospirillum spp. exhibited the growth-promoting effect on coimmobilized Chlorella vulgaris.50 Similarly, phenolic acids that were generated from the bacterial degradation of pyrene exerted a promoting-effect on the algal growth in the algal−bacterial consortium. Cooperation of Algae and Bacteria in Pyrene Degradation. In fact, the real ecosystem is far more complex than our constructed pyrene biodegradation system only containing two microbial species. However, it is a good starting point to understand the interaction of different species under the stress of pollution using simple artificially constructed consortium. The consortium of alga and bacterium could achieve a higher pyrene degradation rate than either of single species. It remains to be elucidated how these two different microbes cooperate in pyrene degradation. First, the bacteria could substantially promote the algal growth. It was probably attributed to that the fast elimination of pyrene by bacteria reduced its toxicity to the algae and that phenolic acids produced from the bacterial degradation of pyrene functioned as phytohormones for the algal growth. However, the high algal population in the consortium could not lead to the
Figure 5. Promoting effects of phenolic acids on the algal growth at the levels of 10 and 100 μg L−1 over 14 days. BA, PAA, and HPA represent benzoic acid, phenylacetic acid, and hydroxyphenlyacetic acid, respectively. Letters indicate the significance of differences in the algal growth between degradation systems on the level of p < 0.05.
accumulation of 1-hydroxypyrene. This result could be explained by two reasons: one was that mono-oxygenation was not the predominant pathway of pyrene degradation in the mixing cultures; another was that 1-hydroxypyrene was utilized and further degraded by the bacteria. Our unpublished data verified that M. A1-PYR could not utilize 1-hydroxypyrene as the sole carbon and energy source. Therefore, pyrene degradation may be initiated mainly via dioxygenation in the mixed cultures, which was evidenced by the fact that higher levels of pyrene degradation products associated with dioxygenation pathway (such as HPA, PAA, and BA) were observed in the consortium than in the single cultures. Dihydroxypyrene, the dioxygenation product of pyrene, was detectable in both algal-only and bacterial-only cultures. It could be expected that dihydroxypyrene was accumulated in the consortium due to the high population of alga and the additive effect of alga and bacterium on dihydroxypyrene production. As a matter of fact, dihydroxypyrene was rapidly depleted in the consortium of algae and bacteria, and converted into the subsequent metabolites. It seemed that alga and bacterium collaboratively accelerated the biotransformation of dihydroxypyrene into downstream products. In the coculture of algae and bacteria, dihydroxypyrene I underwent a transitory accumulation and was further degraded from day 4 onward F
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as both algae and bacteria rapidly grew. Although alga itself was unable to degrade dihydroxyprene, it can provide oxygen and carbon source, which are essential for aerobic M. A1-PYR to metabolize organic pollutants.20 Consequently, such the intimate interactions between algae and bacteria in terms of growth promotion eased the biodegradation of pyrene in the mixing cultures. The algal−bacterial consortium constructed by S. capricornutum and M. A1-PYR exhibited a higher degradation rate of pyrene than either of single species. The fast algal growth in the consortium favorably accelerated the production of dihydroxypyrene, and also potentially played as a “helper” in the further degradation of dihydroxypyrene by bacterium. This study suggested that the algal−bacterial consortium had a high potential for application in the remediation of PAHcontaminated environments. It also implied that the diverse and complex ecosystem in the real aquatic environment with mixed species might exhibit a higher tolerance to pollution stress than the simple ones with single species.
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ASSOCIATED CONTENT
S Supporting Information *
Table S1 and Figures S1−S3. This material is available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*Tel: +86-20-84112958. Fax: +86-20-84037549. E-mail:
[email protected]. Author Contributions ∥
These authors contributed equally to this work.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This research was financially supported by the National Natural Science Foundation of China (NSFC, No. 21277177, 21307167), Foundation for High-level Talents in Higher Education of Guangdong Province, and Administration of Ocean and Fisheries of Guangdong Province, China.
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REFERENCES
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