Accepted Manuscript Title: Biodegradation of bisphenol A with diverse microorganisms from river sediment Author: Yu-Huei Peng Ya-Jou Chen Ying-Jie Chang Yang-hsin Shih PII: DOI: Reference:
S0304-3894(14)01035-8 http://dx.doi.org/doi:10.1016/j.jhazmat.2014.12.051 HAZMAT 16486
To appear in:
Journal of Hazardous Materials
Received date: Revised date: Accepted date:
1-10-2014 17-12-2014 28-12-2014
Please cite this article as: Yu-Huei Peng, Ya-Jou Chen, Ying-Jie Chang, Yang-hsin Shih, Biodegradation of bisphenol A with diverse microorganisms from river sediment, Journal of Hazardous Materials http://dx.doi.org/10.1016/j.jhazmat.2014.12.051 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Biodegradation of bisphenol A with diverse microorganisms from river sediment
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Yu-Huei Peng1, *, Ya-Jou Chen1, Ying-Jie Chang, and Yang-hsin Shih*
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Taiwan R.O.C.
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Department of Agricultural Chemistry, National Taiwan University, Taipei City 10617,
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* Corresponding author. Tel/ fax: +886 2 33669443
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E-mail address: [email protected] (Y.-H. Peng)
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[email protected] (Y.-h. Shih)
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These authors equally contributed to the work.
Highlights
► Six intermediates depict the metabolic pathways and the detoxification of BPA.
The novel BPA-degrading activity of Pseudomonas knackmussii is demonstrated.
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The mixture of isolated strains enhances the BPA-degradation kinetics.
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Microbial diversity is important for fast decomposition of the pollutant.
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ABSTRACT
of
its
endocrine-disrupting
characteristics
and
toxicity.
Developing
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because
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The wide distribution of bisphenol A (BPA) in the environment is problematic
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cost-effective remediation methods for wide implementation is crucial. Therefore, this
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study investigated the BPA biodegradation ability of various microorganisms from river
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sediment. An acclimated microcosm completely degraded 10 mg L-1 BPA within 28 h and transformed the contaminant into several metabolic intermediates. During the
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degradation process, the microbial compositions fluctuated and the final, predominant
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microorganisms were Pseudomonas knackmussii and Methylomonas clara. From the original river sediment, we isolated four distinct strains, which deplete the BPA over 7 to 9 days. They were all genetically similar to P. knackmussii. The degradation ability
of mixed strains was higher than that of single strain but was far less than that of the microbial consortium. The novel BPA degradation ability of P. knackmussii and its role in the decomposing microcosm were first demonstrated. Our results revealed that microbial diversity plays a crucial role in pollutant decomposition.
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Keywords: bisphenol A, biodegradation, intermediates, Pseudomonas knackmussii,
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biodiversity
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1. Introduction
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Bisphenol A (4,4'-Isopropylidenediphenol, BPA) is widely used in the
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manufacturing of polycarbonate plastics and epoxy resins, the end-products of which
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include food containers, thermal papers, lacquers, and dental fillings. Globally, more
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than 2.2 million tons of BPA is consumed annually in which approximately 25% is
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estimated to be released into the environment [1, 2]. BPA is also the degradation intermediate of tetrabromo-BPA, an emerging contaminant with large consumption,
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persistency, and bioaccumulation characteristics [3]. Exposure to BPA through diet or
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skin contact results in various biological side effects. BPA influences the growth, reproduction, and development of biota through interfering with the activities of several endocrine hormones, specific intracellular signaling pathways, and epigenetic regulators
[4]. BPA also causes acute toxicity to aquatic organisms (> 1 mg L-1) and the LD50 for mice (oral dose) is 2400 mg kg-1 [5-7]. The wide distribution and increasing concentration of BPA in the environment threaten the health of human and other biota [8, 9]. Research on BPA degradation in the environment is crucial for pollutant
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remediation. Generally, BPA in household wastewater can be removed by treatment
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plants. However, diverse releasing routes and the increasingly higher volumes of BPA
on
environmental
biodegradation,
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relies
particularly
microbial
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remediation
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released into the environment exceed the capacity of water treatment plants [10]. BPA
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decomposition [7, 11-13]. Biodegradation of BPA favors oxygenic environments, and
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the half-life may be up to one month [8]. The following three BPA-degrading bacteria
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and their degradation efficiencies have been reported by previous studies:
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Sphingomonas sp., Pseudomonas sp., and Bacillus sp. [11, 14]. However, bacteria with strong degradation ability are limited and typically depend on biostimulation [15, 16].
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For example, the removal of 115 mg L−1 of BPA from pre-cultivated Sphingomonas sp.
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strain AO1 took 5 days, and the efficiency elevated 20-fold with a supplement of a rich medium. The metabolic pathways of BPA degradation in specific bacterial strains were proposed according to the intermediates detected during the degradation process [11, 14,
16]. To date, the functions of bacterial enzymes in performing the specific step of transformation of BPA were based only on the findings of studies on cytochrome P450 monooxygenase and laccase [17, 18]. Although BPA can be degraded, some of its intermediates are even more toxic than the original component [5]. Despite the isolated pure strains and decomposing consortiums, the role of the specific degrader in the entire
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microcosm and the interaction between microorganisms remain unclear. Thus, how the
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composition of the microbial community and the interactions between microorganisms
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determine the degradation ability require further investigations.
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The aim of this study is to gain a greater insight into biodegradation of BPA.
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Through initially collecting a bacterial consortium or pure degraders from river
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sediment in Taiwan, the biodegradation ability toward BPA, the metabolic
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intermediates after BPA degradation, and the transformation efficiency were
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investigated. In addition, the roles of specific degraders in the microbial consortium were studied according to their enrichment during the degradation process and the effect
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of the mixed culture on BPA biodegradation. These results are critical for understanding
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the biodegradation of endocrine disruptors in nature and for facilitating the optimization of present remediation methods.
2.
Materials and Methods
2.1. Microcosm set-up Sediment (0-10 cm depth) was collected from the river bank of Hsin-Dian Creek, a tributary of the Dan-Shui River, Taipei, Taiwan. The sampling site was at the lower reaches of the river (25°0'37"N, 121°31'36"E). The pH value and texture of the
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sediment were measured using standard soil testing methods [19]. The element
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composition of carbon (C), hydrogen (H), nitrogen (N), sulfur (S), and oxygen (O) was
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analyzed using an elemental analyzer (Flash 2000, Thermo). Table 1 shows the basic
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characteristics of the sediment. Basal salt medium (BSM) supplemented with 10 mg L−1
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of BPA (97%, Acros Organics) was used as a working medium. The BSM contained 2.1
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g L−1 of K2HPO4, 0.4 g L−1 of KH2PO4, 0.5 g L−1 of NH4NO3, 0.2 g L−1 of
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MgSO4·7H2O, 0.023 g L−1 of CaCl2·2H2O, 0.002 g L−1 of FeCl3·2H2O, and was
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neutralized using NaOH to approximately pH 7.3. Eight grams of sediment samples were acclimated in 50 mL of the working medium for 5 days. The culture and all of the
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following experimental sets were incubated in an orbital shaker (50 rpm) at 30 °C.
2.2. Pure strain isolation and identification The bacterial consortium collected from river sediment was serially diluted using
the BSM solution. A 0.2-mL aliquot of the appropriate dilution was spread onto selective agar plates containing 10 mg L−1 of BPA. Under 2 days of incubation at 30 °C, axenic colonies that were viable on the plates were selected to identify their BPA degradation abilities, as follows. A 1.5-mL overnight Luria-Bertani (LB) culture of isolated BPA-degrading strain
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was used to extract the genomic DNA by using a Tissue and Cell Genomic DNA
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Purification Kit (GeneMark Corp.). The 16S ribosomal RNA (rRNA) gene was
fragments
were
identified
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the sequences
the
BLAST
of the algorithm
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amplified
Subsequently,
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(5’-TACGGTTACCTTGTTACGACTT-3’).
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amplified by PCR with primer 27F (5’-AGAGTTTGATCCTGGCTCAG-3’) and 1492R
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(http://blast.ncbi.nlm.nih.gov/Blast.cgi) to compare them with known nucleotides
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deposited in the GeneBank. A phylogenetic tree was constructed using the maximum
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likelihood method and Mega software (Version 4). Bootstrap analysis (1000 replicates)
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was used to obtain confidence estimates for the phylogenetic tree topologies.
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2.3. Degradation kinetics analysis The acclimated bacterial consortium collected from river sediment was washed once in the BSM medium and then cultured in 50 mL of the working medium. The
non-inoculated BSM and autoclaved (121 °C, 30 min) microcosm were used as controls. Similarly, the overnight culture of isolated pure strain grown in the LB medium at 37 °C was washed once with an equal volume of BSM and then resuspended to an OD600 of 0.5 by using 50 mL of the working medium. For the mixed culture of specific strains,
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the concentrations of each strain were equal and to the sum of an OD600 of 0.5.
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2.4. Chemical Analysis
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At selected time intervals, the culture medium was removed and passed through a
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syringe filter with a 0.2-µm polypropylene membrane (PALL Corp.). The concentration
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of BPA was determined using a high performance liquid chromatography apparatus
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equipped with an Agilent TC-C18 column (4.6 × 25 mm, 5 µm), using a mixture of
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methanol and water (7:3) as the mobile phase at a flow rate of 1 mL min-1. BPA was detected by absorbance at 280 nm by using an ultraviolet–visible detector. The
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identification of intermediates during BPA biodegradation was performed using an
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ultra-performance liquid chromatography system (Ultimate 3000 RSLC, Dionex) and an electrospray ionization source of quadrupole/time-of-flight (TOF) mass spectrometer (maXis HUR-QTOF system, Bruker Daltonics). Separation was performed with a BEH
C18 column (2.1 × 100 mm, Walters). The elution started from the 99% mobile phase A (0.1% formic acid in ultrapure water) and 1% mobile phase B (0.1% formic acid in ACN), held at 1% B for 30 s, raised to 60% B in 6 min, further raised to 90% B in 30 s, held at 90% B for 90 s, and then lowered to 1% B in 30 s. The column was equilibrated
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by pumping 1% B for 4 min. The flow rate was 0.4 mL min-1 with injection volume of 2
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µL.
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2.5. Bacterial community analysis
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Genomic DNA of the microcosm was extracted using the UltraClean Microbial
16S
rRNA
genes
were
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The
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DNA Isolation Kit (MoBio Laboratories) according to the manufacturer’s instructions. amplified
with
primer
Univ1392R
to
a
GC-clamp
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attached
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(5’-ACGGGCGGTG-TGTAC-3’) and Eub968F (5’-AACGCGAAGAACCTTAC-3’) (5’-CGCCCGGGGCGCGCCCCGGGCGGGGCGGGG
GCACGGGGGGAACGCGAAGAACCTAC-3’).
Denaturing
gradient
gel
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electrophoresis (DGGE) analysis was conducted using the DCode Universal Mutation
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Detection System (Bio-Rad), with 6% acrylamide gel and 40%–60% denaturant gradient. Approximately 700 ng of PCR products were loaded per lane. The amplified DNA fragment was loaded and separated under 100 V for 16 h at 60 ° C. The DGGE
bands were excised from the gel, and each band was eluted in 50 µL of ddH2O overnight at 4 ° C. The extracted DNA was re-amplified using primers without a GC clamp, and the sequences were identified as described.
3. Results and Discussion
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3.1. BPA biodegradation by the aerobic microcosm
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The acclimated bacterial consortium collected from river sediment exhibited
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considerable BPA degradation ability. Half of the compound was eliminated by the
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microcosms within 6 h and the entire compound was depleted after 28 h of incubation
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(Fig. 1). The BPA concentration in the non-inoculated blank was constant throughout
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the experimental duration, indicating that this compound was stable in the BSM
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medium. The BPA concentration in sterilized control was reduced by 16% after 2 days
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of incubation. Previous studies have reported that BPA can be adsorbed by organic matter in soils or sediments [20, 21]. The reduction of BPA in the sterilized control may
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have resulted from the adsorption with the cell surface or sediment matter, which
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co-precipitated with the microorganisms during the experimental preparation. BPA decomposition rate varied among the different environmental microcosms according to the sampling sites and experimental settings. Spiked BPA (1 mg L-1) was
degraded within 10 days in water samples collected from Japan [22]. The bacterial consortium collected from river sediment in Southern Taiwan degraded 50 mg kg-1 of BPA within 3 days [13]. Another bacterial consortium collected from river sediment in China took 3 days to degrade 180 mg kg-1 of BPA [12]. The BPA degradation rate of the microcosm in this study was similar to previously reported samples. Such
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transformation ability may be the result of long-term acclimatization in the natural
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environment. The Hsin-Dian Creek flows through New Taipei City and the Taipei City,
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and has a drainage area of approximately 900 km2. A previous study reported that the
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illegal dumping of raw sewage and industrial waste polluted the middle and lower
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reaches of the river [23]. The concentration of BPA in Hsin-Dian Creek was under 0.1
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µg L−115 years ago; however, it increased to 0.508 µg L−1 over the span of a decade [9,
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24]. The long-term exposure of the contaminant could select and enrich the
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BPA-degrading microorganisms.
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3.2. Analysis of the biodegraded intermediates To elucidate the metabolic mechanisms of BPA within the aerobic microcosm, the
2-day degradation products were analyzed by liquid chromatography–mass spectrometry. All obtained mass spectra of these possible intermediates were compared
to the standard spectra of NIST Mass Spectral Library. Six compounds were tentatively identified as the intermediates of BPA biodegradation (Table 2 and Fig. 2) as follows: 2,2-bis(4-hydroxyphenyl)-l- propanol (A) and 1,2-bis(4-hydroxyphenyl)-2-propanol (B) were eluted at a retention time of 6.7 min; carbocationic isopropylphenol (C) and 4-isopropenylphenol (D) were eluted at retention times of 6.6 and 6.5 min, respectively;
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were eluted at retention times of 2.8 and 1.7 min, respectively.
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4,4-dihydroxy-α-methylstilbene (E) and 2,2-bis(4-hydroxyphenyl) propanoic acid (F)
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These six intermediates were previously identified as BPA degradation products
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from Sphingomonas sp. strains, Achromobacter xylosoxidans, and Cupriavidus
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basilensis but not from Pseudomonas sp. and Bacillus sp. [11, 14, 16, 18]. We examined
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whether these degraders were present in our microcosms. To further investigate the
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mechanism, the BPA degradation products of P450
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(1,2-bis(4-hydroxyphenyl)-2-propanol and 2,2-bis(4-hydroxyphenyl)-1-propanol) were detected in this study [17], whereas the degradation products of laccase
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(2,3-bis(4-hydroxyphenyl)-2- hydroxypropionaldehyde, 2,3-bis(4-hydroxyphenyl)-2,3-
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dihydroxypropionaldehyde, and p-1,2-dihydroxyisopropyl phenol) were absent [18], indicating that the BPA transformation in our microcosm might have occurred through the reaction of monooxygenase but not laccase. Known intermediates that are more
toxic than BPA but were not detected in this study are: p-hydroxyacetophenone, p-hydroxybenzaldehyde, p-hydroxybenzoic acid, hydroquinone, and phenol [5, 6]. The only known toxicity of our detected intermediates was 4-isopropenylphenol, which has an LD50 for rats (oral dose) of 585 mg kg-1 [25], which is considerably less than that of BPA [6]. Therefore, the bacterial consortium collected from river sediment in this study
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transformed the BPA and detoxified the endocrine disruptor after the degradation
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process.
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3.3. Microbial community fluctuation during the degradation process
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The microbial compositions in the sediment during the BPA degradation were
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analyzed using DGGE. In the original sediment, several predominant bacteria were
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initially detected (Fig. 3, Lane 1). After 5 days of acclimation, several major
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predominant bacteria were selected (Fig. 3, Lane 2). At the end of degradation, the
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number of major predominant bacteria was further reduced (Fig. 3, Bands 1 and 2 in Lane 3). Comparing the DGGE patterns during the BPA degradation (T0 and T2)
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revealed that the proportion of Band 1 remained constant while the proportion of Band
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2 increased. Such fluctuation in the microbial community during degradation typically results from the enrichment of specific degraders and the disappearance of microorganisms with low potential to survival [12, 26, 27].
We identified the exact identities of the predominant microorganisms. Band 1 was 100% identical to Pseudomonas knackmussii (Strain B13, Accession Number NR_041702). Band 2 was 99% identical to Methylomonas clara (Strain DSM 6330, Accession Number HF564897). During the acclimation and degradation process, BPA was the only carbon source for the bacterial consortium collected from river sediment.
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The predominance of these microorganisms suggests their utilization of BPA during
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these processes. P. knackmussii B13 is known to degrade chlorobenzoate and
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fluorobenzoate [28, 29]. Genomic analysis of this degrader revealed the presence of
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genes for chlorocatechol and 2-aminophenol degradation, aromatic ring dioxygenase,
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and aromatic compound transport protein [30]. These genes may function in the BPA
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degradation pathways. Methylomonas clara is an obligate methylotroph that uses
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methanol [31]. Its relative, Methylomonas methanica 68-1 oxidized naphthalene through
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soluble methane monooxygenase [32]. The BPA degradation abilities of these microorganisms have not yet been reported previously, although they have high
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potential.
3.4. Pure strain isolation and degradation ability assessment A collection of 10 colonies with distinct morphologies were selected from the
BPA-containing selection agar plates. Among them, only four (RB-F, G, H, and I) exhibited BPA degradation abilities. Based on the 16S rRNA gene sequences, these strains were the closest relative to P. knackmussii (Strain B13, Accession Number NR_041702), with identities exhibiting more than 99.7% similarity (Table 3 and Fig. 4). The 16S rRNA gene sequences of these strains were similar to each other with a
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difference of only two to five nucleotides (Table 3). RB-F, G, and H were relatively
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closer to each other, and RB-I was considerably further from the other three strains.
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presence of genetic polymorphism in the sediment.
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These genetically close bacterial strains with distinct metabolic activities indicated the
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This study compared the BPA degradation ability of these four strains. The RB-G
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strain was the most efficient degrader, requiring 7 days to remove all of the BPA (Fig.
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5). The other three strains took 9 days to deplete an identical amount of BPA (Fig. 5 for
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RB-I and data not shown). This result demonstrated the novel BPA degradation ability of P. knackmussii strains. In the DGGE analysis, P. knackmussii was among the major
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predominant microorganisms in the BPA degradation consortium (Fig. 3, Band 1 in
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Lane 2 and 3). The consistency in degrader isolations and predominant species characterization in the microbial consortium supported the critical role of P. knackmussii in our BPA degrading microcosm.
Compared with the degradation ability of known degraders, our isolated strains were prominent: Pseudomonas putida required 7 days to degrade 1 mg L−1 of BPA [22]; Cupriavidus basilensis JF1 exhibited a relatively slow degradation rate where the half-life of 38 mg L−1 of BPA was approximately 144 days [16]; Achromobacter xylosoxidans B-16 degraded half of the 10-mg L−1 BPA within 5 days, but failed to
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remove the residual compounds [33]. Only Sphingomonas sp. strain AO1 exhibited
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bacteria removed 115 mg L−1 of BPA within 5 days [15].
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greater degradation ability than our isolated strains, where that the pre-cultivated
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Bio-augmentation or co-cultivation with specific compounds can enhance the
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biodegradation ability of BPA degraders. The addition of glucose enhanced the BPA
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degradation rate of Sphingomonas sp. strain AO1 three-fold. This bacteria also
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decomposed an identical amount of BPA within 6 h when a rich medium was supplied
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[15]. Co-incubation with phenol accelerated the BPA degradation rate of C. basilensis JF1 approximately four-fold [16]. Further adjustment of co-incubation conditions would
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assist with defining the optimal degradation conditions for our isolated strains.
3.5. BPA degradation in mixed culture The effect of a mixed culture on BPA degradation and their interactions were
investigated according to degradation kinetics. The most efficient BPA degrading strain, RB-G, was selected and mixed with RB-I, the most divergent strain based on the16S rRNA gene sequence. The degradation rate of the mixed culture was faster than that of the single strain culture (Fig. 5, G + I): the mixed culture required 6 days to degrade all of the BPA. When all four of the isolated strains were mixed together, the BPA
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degradation rate was faster than that of the single strain culture, although it exhibited
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similar degradation efficiency with the G + I mixed culture (Fig. 5, mixed 4). A mixture
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of the various BPA-degrading strains examined in this study increased the
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decomposition rate of the single strain.
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Previous studies that investigated the same scenario reported diverse results. Kang
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and Kondo [22] reported that a mixture of two BPA degrading microorganisms, P.
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putida and Pseudomonas sp. strain, did not alter the BPA degradation efficiency. By
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contrast, with the assistance of the non-BPA degrading Pseudomonas sp. strain BP-14, the degradation period of Sphingomonas sp. strain BP-7 was reduced six-fold [34].
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Enhancement of the BPA degradation rate in the mixed culture may have been involved
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in the cooperation to decompose BPA and other downstream toxic intermediates, as well as the support in the metabolism of the degraders. The degradation rate of the mixed P. knackmussii strains in this study was considerably less than that of the original
microbial consortium (i.e. 6 days versus to 28 h). A DGGE analysis revealed that there were other predominant microorganisms in the fast BPA-degrading microcosm. A lack of other BPA (or intermediates)-degrading or -supporting microorganisms may limit the enhancement of the BPA degradation efficiency. Therefore, rich microbial diversity in
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the consortium is critical and should be considered as an option in bioremediation.
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4. Conclusions
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The BPA biodegradation ability of various microorganisms extracted from
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sediment were analyzed in this study. The microcosm required 28 h to degrade and
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transform the compound into six intermediates. The microbial community fluctuated
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and the final, predominant species were Pseudomonas knackmussii and Methylomonas
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clara. Distinct isolated strains exhibiting various BPA degradation abilities were genetically close and identical to P. knackmussii. On average, mixture of pure strains
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shortened the degradation period from 8 days to 6 days. However, microbial diversity
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was crucial in supporting rapid decomposition. This is the first report demonstrating the novel BPA-degrading activity of P. knackmussii and its role in the decomposing microcosm.
Acknowledgments The authors thank Ministry of Science and Technology, Taiwan for financial support under Contract No. NSC 102-2221-E-002 -015 -MY3. The authors also thank
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the Instrumentation Center, National Taiwan University for elemental analysis.
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252-260.
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[19] Methods of soil analysis, part II, 2 ed., A.L. Page, American Society of Agronomy, Inc., Soil Society of America, Inc., 1982. [20] K. Sun, B. Gao, Z. Zhang, G. Zhang, X. Liu, Y. Zhao, B. Xing, Sorption of endocrine disrupting chemicals by condensed organic matter in soils and sediments, Chemosphere, 80 (2010) 709-715. [21] Y.-h. Fei, X.-d. Li, X.-y. Li, Organic diagenesis in sediment and its impact on the adsorption of bisphenol A and nonylphenol onto marine sediment, Mar. Pollut. Bull., 63 (2011) 578-582.
[22] J.H. Kang, F. Kondo, Bisphenol A Degradation by Bacteria Isolated from River Water, Arch. Environ. Contam. Toxicol., 43 (2002) 0265-0269. [23] Y.-C. Chen, H.-C. Yeh, C. Wei, Estimation of River Pollution Index in a Tidal Stream Using Kriging Analysis, International Journal of Environmental Research and Public Health, 9 (2012) 3085-3100. [24] W.-H. Ding, C.-Y. Wu, Determination of Estrogenic Nonylphenol and Bisphenol a in River Water by Solid-Phase Extraction and Gas Chromatography-Mass Spectrometry,
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J. Chin. Chem. Soc., 47 (2000) 1155-1160. [25] M, Material Safety Data Sheet - 4-(1-methylethenyl) phenol, in: L.a.W. Ministry of Health, Japan (Ed.), Ministry of Health, Japan, 2012. [26] Y.H. Shih, H.L. Chou, Y.H. Peng, Microbial degradation of 4-monobrominated
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diphenyl ether with anaerobic sludge, J. Hazard. Mater., 213 (2012) 341-346. [27] Y.H. Shih, H.L. Chou, Y.H. Peng, C.Y. Chang, Synergistic effect of microscale zerovalent iron particles combined with anaerobic sludges on the degradation of
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decabromodiphenyl ether, Bioresour. Technol., 108 (2012) 14-20. [28] K. Misiak, E. Casey, C.D. Murphy, Factors influencing 4-fluorobenzoate degradation in biofilm cultures of Pseudomonas knackmussii B13, Water Res., 45 (2011) 3512-3520.
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[29] E. Dorn, M. Hellwig, W. Reineke, Knackmus.Hj, Isolation and characterization of a 3-chlorobenzoate degrading Pseudomonad, Arch. Microbiol., 99 (1974) 61-70. [30] M. Gaillard, T. Vallaeys, F.J. Vorholter, M. Minoia, C. Werlen, V. Sentchilo, A. Puhler, J.R. van der Meer, The clc element of Pseudomonas sp. strain B13, a
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genomic island with various catabolic properties, J. Bacteriol., 188 (2006) 1999-2013. [31] W. Hohnloser, F. Lingens, P. Präve, Characterization of a new methylotrophic strain, Methylomonas clara, Eur. J. Appl. Microbiol. Biotechnol., 6 (1978) 167-179. [32] S.C. Koh, J.P. Bowman, G.S. Sayler, Soluble Methane Monooxygenase Production and Trichloroethylene Degradation by a Type I Methanotroph, Methylomonas
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methanica 68-1, Appl. Environ. Microbiol., 59 (1993) 960-967.
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[33] C. Zhang, G. Zeng, L. Yuan, J. Yu, J. Li, G. Huang, B. Xi, H. Liu, Aerobic degradation of bisphenol A by Achromobacter xylosoxidans strain B-16 isolated from compost leachate of municipal solid waste, Chemosphere, 68 (2007) 181-190. [34] K. Sakai, H. Yamanaka, K. Moriyoshi, T. Ohmoto, T. Ohe, Biodegradation of bisphenol A and related compounds by Sphingomonas sp. strain BP-7 isolated from seawater, Biosci., Biotechnol., Biochem., 71 (2007) 51-57.
Figure captions Figure 1. Biodegradation kinetics of BPA for the bacterial consortium collected from river sediment.
Figure 2. Proposed degradation pathway of BPA for the bacterial consortium collected
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from river sediment.
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Figure 3. DGGE banding profile of 16S rDNA genes from the original bacterial
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consortium collected from river sediment (Ori.), the acclimated microcosm (T0) and the
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microcosm that performed 2 days of BPA degradation (T2).
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Figure 4. Phylogenetic relationships of isolated organisms with Pseudomonas
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knackmussii B13 (NR_041702) based upon the 1342-bp fragment of the 16S rRNA gene. Numbers on the branches indicate the bootstrap confidence estimates obtained
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with 1000 replicates. The scale bar represents 0.005% sequence divergence.
Figure 5. Biodegradation kinetics of BPA for isolated bacterium and mixed cultures.
Table 1. The characteristics and element composition of the river sediment Parameters
Value 6.1
pH
65.42 ± 1.78 26.26
Silt
8.32 ± 0.13
C
1.05 ± 0.07
H
0.51 ± 0.001
N
0.10 ± 0.001
S
0.08 ±0.006
O
2.42 ± 0.074
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Clay
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A
N
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Element composition (%)
±1.91
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Sand
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Texture (%)
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(n=2)
t R (min)
A
2,2-bis(4-hydroxyphenyl)-l-propanol
6.7
B
1,2-bis(4-hydroxyphenyl)-2-propanol
6.7
C
carbocationic isopropylphenol
6.6
D
4-isopropenylphenol
6.5
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Compound in Fig. 3
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Table 2. Intermediates of BPA biodegradation
4,4-dihydroxy-α-methylstilbene
2.8
F
2,2-bis(4-hydroxyphenyl) propanoic acid
1.7
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E
P. knackmussii B13 (NR_041702)
1408/1410 (99.8582%)
A
RB-I
(99.8596%) 1412/1414
D
RB-G
M
RB-H
(99.7171%) 1409/1413 (99.7169%) 1423/1425
N
1410/1414
RB-F
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(99.8586%)
A
RB-F
RB-H
RB-I
1410/1412
1409/1413
(99.8584%) 1409/1411 (99.8583%)
(99.7169%) 1409/1413 (99.7169%) 1411/1413
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ID
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Table 3. Identities between four BPA degradation bacteria and their closest match
(99.8585%)
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N
A
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Fig. 1
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A D
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A
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Fig. 2
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Fig. 3
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Fig. 5
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Fig. 4
S0304-3894(14)01035-8 http://dx.doi.org/doi:10.1016/j.jhazmat.2014.12.051 HAZMAT 16486
To appear in:
Journal of Hazardous Materials
Received date: Revised date: Accepted date:
1-10-2014 17-12-2014 28-12-2014
Please cite this article as: Yu-Huei Peng, Ya-Jou Chen, Ying-Jie Chang, Yang-hsin Shih, Biodegradation of bisphenol A with diverse microorganisms from river sediment, Journal of Hazardous Materials http://dx.doi.org/10.1016/j.jhazmat.2014.12.051 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Biodegradation of bisphenol A with diverse microorganisms from river sediment
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Yu-Huei Peng1, *, Ya-Jou Chen1, Ying-Jie Chang, and Yang-hsin Shih*
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Taiwan R.O.C.
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Department of Agricultural Chemistry, National Taiwan University, Taipei City 10617,
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* Corresponding author. Tel/ fax: +886 2 33669443
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E-mail address: [email protected] (Y.-H. Peng)
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[email protected] (Y.-h. Shih)
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1
These authors equally contributed to the work.
Highlights
► Six intermediates depict the metabolic pathways and the detoxification of BPA.
The novel BPA-degrading activity of Pseudomonas knackmussii is demonstrated.
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The mixture of isolated strains enhances the BPA-degradation kinetics.
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Microbial diversity is important for fast decomposition of the pollutant.
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ABSTRACT
of
its
endocrine-disrupting
characteristics
and
toxicity.
Developing
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because
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The wide distribution of bisphenol A (BPA) in the environment is problematic
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cost-effective remediation methods for wide implementation is crucial. Therefore, this
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study investigated the BPA biodegradation ability of various microorganisms from river
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sediment. An acclimated microcosm completely degraded 10 mg L-1 BPA within 28 h and transformed the contaminant into several metabolic intermediates. During the
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degradation process, the microbial compositions fluctuated and the final, predominant
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microorganisms were Pseudomonas knackmussii and Methylomonas clara. From the original river sediment, we isolated four distinct strains, which deplete the BPA over 7 to 9 days. They were all genetically similar to P. knackmussii. The degradation ability
of mixed strains was higher than that of single strain but was far less than that of the microbial consortium. The novel BPA degradation ability of P. knackmussii and its role in the decomposing microcosm were first demonstrated. Our results revealed that microbial diversity plays a crucial role in pollutant decomposition.
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Keywords: bisphenol A, biodegradation, intermediates, Pseudomonas knackmussii,
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biodiversity
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1. Introduction
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Bisphenol A (4,4'-Isopropylidenediphenol, BPA) is widely used in the
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manufacturing of polycarbonate plastics and epoxy resins, the end-products of which
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include food containers, thermal papers, lacquers, and dental fillings. Globally, more
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than 2.2 million tons of BPA is consumed annually in which approximately 25% is
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estimated to be released into the environment [1, 2]. BPA is also the degradation intermediate of tetrabromo-BPA, an emerging contaminant with large consumption,
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persistency, and bioaccumulation characteristics [3]. Exposure to BPA through diet or
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skin contact results in various biological side effects. BPA influences the growth, reproduction, and development of biota through interfering with the activities of several endocrine hormones, specific intracellular signaling pathways, and epigenetic regulators
[4]. BPA also causes acute toxicity to aquatic organisms (> 1 mg L-1) and the LD50 for mice (oral dose) is 2400 mg kg-1 [5-7]. The wide distribution and increasing concentration of BPA in the environment threaten the health of human and other biota [8, 9]. Research on BPA degradation in the environment is crucial for pollutant
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remediation. Generally, BPA in household wastewater can be removed by treatment
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plants. However, diverse releasing routes and the increasingly higher volumes of BPA
on
environmental
biodegradation,
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relies
particularly
microbial
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remediation
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released into the environment exceed the capacity of water treatment plants [10]. BPA
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decomposition [7, 11-13]. Biodegradation of BPA favors oxygenic environments, and
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the half-life may be up to one month [8]. The following three BPA-degrading bacteria
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and their degradation efficiencies have been reported by previous studies:
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Sphingomonas sp., Pseudomonas sp., and Bacillus sp. [11, 14]. However, bacteria with strong degradation ability are limited and typically depend on biostimulation [15, 16].
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For example, the removal of 115 mg L−1 of BPA from pre-cultivated Sphingomonas sp.
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strain AO1 took 5 days, and the efficiency elevated 20-fold with a supplement of a rich medium. The metabolic pathways of BPA degradation in specific bacterial strains were proposed according to the intermediates detected during the degradation process [11, 14,
16]. To date, the functions of bacterial enzymes in performing the specific step of transformation of BPA were based only on the findings of studies on cytochrome P450 monooxygenase and laccase [17, 18]. Although BPA can be degraded, some of its intermediates are even more toxic than the original component [5]. Despite the isolated pure strains and decomposing consortiums, the role of the specific degrader in the entire
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microcosm and the interaction between microorganisms remain unclear. Thus, how the
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composition of the microbial community and the interactions between microorganisms
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determine the degradation ability require further investigations.
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The aim of this study is to gain a greater insight into biodegradation of BPA.
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Through initially collecting a bacterial consortium or pure degraders from river
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sediment in Taiwan, the biodegradation ability toward BPA, the metabolic
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intermediates after BPA degradation, and the transformation efficiency were
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investigated. In addition, the roles of specific degraders in the microbial consortium were studied according to their enrichment during the degradation process and the effect
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of the mixed culture on BPA biodegradation. These results are critical for understanding
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the biodegradation of endocrine disruptors in nature and for facilitating the optimization of present remediation methods.
2.
Materials and Methods
2.1. Microcosm set-up Sediment (0-10 cm depth) was collected from the river bank of Hsin-Dian Creek, a tributary of the Dan-Shui River, Taipei, Taiwan. The sampling site was at the lower reaches of the river (25°0'37"N, 121°31'36"E). The pH value and texture of the
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sediment were measured using standard soil testing methods [19]. The element
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composition of carbon (C), hydrogen (H), nitrogen (N), sulfur (S), and oxygen (O) was
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analyzed using an elemental analyzer (Flash 2000, Thermo). Table 1 shows the basic
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characteristics of the sediment. Basal salt medium (BSM) supplemented with 10 mg L−1
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of BPA (97%, Acros Organics) was used as a working medium. The BSM contained 2.1
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g L−1 of K2HPO4, 0.4 g L−1 of KH2PO4, 0.5 g L−1 of NH4NO3, 0.2 g L−1 of
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MgSO4·7H2O, 0.023 g L−1 of CaCl2·2H2O, 0.002 g L−1 of FeCl3·2H2O, and was
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neutralized using NaOH to approximately pH 7.3. Eight grams of sediment samples were acclimated in 50 mL of the working medium for 5 days. The culture and all of the
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following experimental sets were incubated in an orbital shaker (50 rpm) at 30 °C.
2.2. Pure strain isolation and identification The bacterial consortium collected from river sediment was serially diluted using
the BSM solution. A 0.2-mL aliquot of the appropriate dilution was spread onto selective agar plates containing 10 mg L−1 of BPA. Under 2 days of incubation at 30 °C, axenic colonies that were viable on the plates were selected to identify their BPA degradation abilities, as follows. A 1.5-mL overnight Luria-Bertani (LB) culture of isolated BPA-degrading strain
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was used to extract the genomic DNA by using a Tissue and Cell Genomic DNA
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Purification Kit (GeneMark Corp.). The 16S ribosomal RNA (rRNA) gene was
fragments
were
identified
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the sequences
the
BLAST
of the algorithm
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amplified
Subsequently,
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(5’-TACGGTTACCTTGTTACGACTT-3’).
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amplified by PCR with primer 27F (5’-AGAGTTTGATCCTGGCTCAG-3’) and 1492R
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(http://blast.ncbi.nlm.nih.gov/Blast.cgi) to compare them with known nucleotides
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deposited in the GeneBank. A phylogenetic tree was constructed using the maximum
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likelihood method and Mega software (Version 4). Bootstrap analysis (1000 replicates)
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was used to obtain confidence estimates for the phylogenetic tree topologies.
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2.3. Degradation kinetics analysis The acclimated bacterial consortium collected from river sediment was washed once in the BSM medium and then cultured in 50 mL of the working medium. The
non-inoculated BSM and autoclaved (121 °C, 30 min) microcosm were used as controls. Similarly, the overnight culture of isolated pure strain grown in the LB medium at 37 °C was washed once with an equal volume of BSM and then resuspended to an OD600 of 0.5 by using 50 mL of the working medium. For the mixed culture of specific strains,
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the concentrations of each strain were equal and to the sum of an OD600 of 0.5.
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2.4. Chemical Analysis
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At selected time intervals, the culture medium was removed and passed through a
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syringe filter with a 0.2-µm polypropylene membrane (PALL Corp.). The concentration
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of BPA was determined using a high performance liquid chromatography apparatus
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equipped with an Agilent TC-C18 column (4.6 × 25 mm, 5 µm), using a mixture of
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methanol and water (7:3) as the mobile phase at a flow rate of 1 mL min-1. BPA was detected by absorbance at 280 nm by using an ultraviolet–visible detector. The
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identification of intermediates during BPA biodegradation was performed using an
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ultra-performance liquid chromatography system (Ultimate 3000 RSLC, Dionex) and an electrospray ionization source of quadrupole/time-of-flight (TOF) mass spectrometer (maXis HUR-QTOF system, Bruker Daltonics). Separation was performed with a BEH
C18 column (2.1 × 100 mm, Walters). The elution started from the 99% mobile phase A (0.1% formic acid in ultrapure water) and 1% mobile phase B (0.1% formic acid in ACN), held at 1% B for 30 s, raised to 60% B in 6 min, further raised to 90% B in 30 s, held at 90% B for 90 s, and then lowered to 1% B in 30 s. The column was equilibrated
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by pumping 1% B for 4 min. The flow rate was 0.4 mL min-1 with injection volume of 2
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µL.
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2.5. Bacterial community analysis
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Genomic DNA of the microcosm was extracted using the UltraClean Microbial
16S
rRNA
genes
were
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The
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DNA Isolation Kit (MoBio Laboratories) according to the manufacturer’s instructions. amplified
with
primer
Univ1392R
to
a
GC-clamp
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attached
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(5’-ACGGGCGGTG-TGTAC-3’) and Eub968F (5’-AACGCGAAGAACCTTAC-3’) (5’-CGCCCGGGGCGCGCCCCGGGCGGGGCGGGG
GCACGGGGGGAACGCGAAGAACCTAC-3’).
Denaturing
gradient
gel
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electrophoresis (DGGE) analysis was conducted using the DCode Universal Mutation
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Detection System (Bio-Rad), with 6% acrylamide gel and 40%–60% denaturant gradient. Approximately 700 ng of PCR products were loaded per lane. The amplified DNA fragment was loaded and separated under 100 V for 16 h at 60 ° C. The DGGE
bands were excised from the gel, and each band was eluted in 50 µL of ddH2O overnight at 4 ° C. The extracted DNA was re-amplified using primers without a GC clamp, and the sequences were identified as described.
3. Results and Discussion
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3.1. BPA biodegradation by the aerobic microcosm
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The acclimated bacterial consortium collected from river sediment exhibited
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considerable BPA degradation ability. Half of the compound was eliminated by the
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microcosms within 6 h and the entire compound was depleted after 28 h of incubation
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(Fig. 1). The BPA concentration in the non-inoculated blank was constant throughout
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the experimental duration, indicating that this compound was stable in the BSM
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medium. The BPA concentration in sterilized control was reduced by 16% after 2 days
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of incubation. Previous studies have reported that BPA can be adsorbed by organic matter in soils or sediments [20, 21]. The reduction of BPA in the sterilized control may
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have resulted from the adsorption with the cell surface or sediment matter, which
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co-precipitated with the microorganisms during the experimental preparation. BPA decomposition rate varied among the different environmental microcosms according to the sampling sites and experimental settings. Spiked BPA (1 mg L-1) was
degraded within 10 days in water samples collected from Japan [22]. The bacterial consortium collected from river sediment in Southern Taiwan degraded 50 mg kg-1 of BPA within 3 days [13]. Another bacterial consortium collected from river sediment in China took 3 days to degrade 180 mg kg-1 of BPA [12]. The BPA degradation rate of the microcosm in this study was similar to previously reported samples. Such
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transformation ability may be the result of long-term acclimatization in the natural
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environment. The Hsin-Dian Creek flows through New Taipei City and the Taipei City,
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and has a drainage area of approximately 900 km2. A previous study reported that the
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illegal dumping of raw sewage and industrial waste polluted the middle and lower
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reaches of the river [23]. The concentration of BPA in Hsin-Dian Creek was under 0.1
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µg L−115 years ago; however, it increased to 0.508 µg L−1 over the span of a decade [9,
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24]. The long-term exposure of the contaminant could select and enrich the
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BPA-degrading microorganisms.
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3.2. Analysis of the biodegraded intermediates To elucidate the metabolic mechanisms of BPA within the aerobic microcosm, the
2-day degradation products were analyzed by liquid chromatography–mass spectrometry. All obtained mass spectra of these possible intermediates were compared
to the standard spectra of NIST Mass Spectral Library. Six compounds were tentatively identified as the intermediates of BPA biodegradation (Table 2 and Fig. 2) as follows: 2,2-bis(4-hydroxyphenyl)-l- propanol (A) and 1,2-bis(4-hydroxyphenyl)-2-propanol (B) were eluted at a retention time of 6.7 min; carbocationic isopropylphenol (C) and 4-isopropenylphenol (D) were eluted at retention times of 6.6 and 6.5 min, respectively;
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were eluted at retention times of 2.8 and 1.7 min, respectively.
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4,4-dihydroxy-α-methylstilbene (E) and 2,2-bis(4-hydroxyphenyl) propanoic acid (F)
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These six intermediates were previously identified as BPA degradation products
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from Sphingomonas sp. strains, Achromobacter xylosoxidans, and Cupriavidus
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basilensis but not from Pseudomonas sp. and Bacillus sp. [11, 14, 16, 18]. We examined
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whether these degraders were present in our microcosms. To further investigate the
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mechanism, the BPA degradation products of P450
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(1,2-bis(4-hydroxyphenyl)-2-propanol and 2,2-bis(4-hydroxyphenyl)-1-propanol) were detected in this study [17], whereas the degradation products of laccase
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(2,3-bis(4-hydroxyphenyl)-2- hydroxypropionaldehyde, 2,3-bis(4-hydroxyphenyl)-2,3-
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dihydroxypropionaldehyde, and p-1,2-dihydroxyisopropyl phenol) were absent [18], indicating that the BPA transformation in our microcosm might have occurred through the reaction of monooxygenase but not laccase. Known intermediates that are more
toxic than BPA but were not detected in this study are: p-hydroxyacetophenone, p-hydroxybenzaldehyde, p-hydroxybenzoic acid, hydroquinone, and phenol [5, 6]. The only known toxicity of our detected intermediates was 4-isopropenylphenol, which has an LD50 for rats (oral dose) of 585 mg kg-1 [25], which is considerably less than that of BPA [6]. Therefore, the bacterial consortium collected from river sediment in this study
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transformed the BPA and detoxified the endocrine disruptor after the degradation
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process.
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3.3. Microbial community fluctuation during the degradation process
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The microbial compositions in the sediment during the BPA degradation were
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analyzed using DGGE. In the original sediment, several predominant bacteria were
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initially detected (Fig. 3, Lane 1). After 5 days of acclimation, several major
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predominant bacteria were selected (Fig. 3, Lane 2). At the end of degradation, the
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number of major predominant bacteria was further reduced (Fig. 3, Bands 1 and 2 in Lane 3). Comparing the DGGE patterns during the BPA degradation (T0 and T2)
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revealed that the proportion of Band 1 remained constant while the proportion of Band
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2 increased. Such fluctuation in the microbial community during degradation typically results from the enrichment of specific degraders and the disappearance of microorganisms with low potential to survival [12, 26, 27].
We identified the exact identities of the predominant microorganisms. Band 1 was 100% identical to Pseudomonas knackmussii (Strain B13, Accession Number NR_041702). Band 2 was 99% identical to Methylomonas clara (Strain DSM 6330, Accession Number HF564897). During the acclimation and degradation process, BPA was the only carbon source for the bacterial consortium collected from river sediment.
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The predominance of these microorganisms suggests their utilization of BPA during
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these processes. P. knackmussii B13 is known to degrade chlorobenzoate and
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fluorobenzoate [28, 29]. Genomic analysis of this degrader revealed the presence of
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genes for chlorocatechol and 2-aminophenol degradation, aromatic ring dioxygenase,
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and aromatic compound transport protein [30]. These genes may function in the BPA
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degradation pathways. Methylomonas clara is an obligate methylotroph that uses
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methanol [31]. Its relative, Methylomonas methanica 68-1 oxidized naphthalene through
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soluble methane monooxygenase [32]. The BPA degradation abilities of these microorganisms have not yet been reported previously, although they have high
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potential.
3.4. Pure strain isolation and degradation ability assessment A collection of 10 colonies with distinct morphologies were selected from the
BPA-containing selection agar plates. Among them, only four (RB-F, G, H, and I) exhibited BPA degradation abilities. Based on the 16S rRNA gene sequences, these strains were the closest relative to P. knackmussii (Strain B13, Accession Number NR_041702), with identities exhibiting more than 99.7% similarity (Table 3 and Fig. 4). The 16S rRNA gene sequences of these strains were similar to each other with a
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difference of only two to five nucleotides (Table 3). RB-F, G, and H were relatively
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closer to each other, and RB-I was considerably further from the other three strains.
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presence of genetic polymorphism in the sediment.
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These genetically close bacterial strains with distinct metabolic activities indicated the
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This study compared the BPA degradation ability of these four strains. The RB-G
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strain was the most efficient degrader, requiring 7 days to remove all of the BPA (Fig.
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5). The other three strains took 9 days to deplete an identical amount of BPA (Fig. 5 for
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RB-I and data not shown). This result demonstrated the novel BPA degradation ability of P. knackmussii strains. In the DGGE analysis, P. knackmussii was among the major
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predominant microorganisms in the BPA degradation consortium (Fig. 3, Band 1 in
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Lane 2 and 3). The consistency in degrader isolations and predominant species characterization in the microbial consortium supported the critical role of P. knackmussii in our BPA degrading microcosm.
Compared with the degradation ability of known degraders, our isolated strains were prominent: Pseudomonas putida required 7 days to degrade 1 mg L−1 of BPA [22]; Cupriavidus basilensis JF1 exhibited a relatively slow degradation rate where the half-life of 38 mg L−1 of BPA was approximately 144 days [16]; Achromobacter xylosoxidans B-16 degraded half of the 10-mg L−1 BPA within 5 days, but failed to
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remove the residual compounds [33]. Only Sphingomonas sp. strain AO1 exhibited
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bacteria removed 115 mg L−1 of BPA within 5 days [15].
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greater degradation ability than our isolated strains, where that the pre-cultivated
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Bio-augmentation or co-cultivation with specific compounds can enhance the
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biodegradation ability of BPA degraders. The addition of glucose enhanced the BPA
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degradation rate of Sphingomonas sp. strain AO1 three-fold. This bacteria also
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decomposed an identical amount of BPA within 6 h when a rich medium was supplied
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[15]. Co-incubation with phenol accelerated the BPA degradation rate of C. basilensis JF1 approximately four-fold [16]. Further adjustment of co-incubation conditions would
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assist with defining the optimal degradation conditions for our isolated strains.
3.5. BPA degradation in mixed culture The effect of a mixed culture on BPA degradation and their interactions were
investigated according to degradation kinetics. The most efficient BPA degrading strain, RB-G, was selected and mixed with RB-I, the most divergent strain based on the16S rRNA gene sequence. The degradation rate of the mixed culture was faster than that of the single strain culture (Fig. 5, G + I): the mixed culture required 6 days to degrade all of the BPA. When all four of the isolated strains were mixed together, the BPA
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degradation rate was faster than that of the single strain culture, although it exhibited
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similar degradation efficiency with the G + I mixed culture (Fig. 5, mixed 4). A mixture
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of the various BPA-degrading strains examined in this study increased the
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decomposition rate of the single strain.
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Previous studies that investigated the same scenario reported diverse results. Kang
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and Kondo [22] reported that a mixture of two BPA degrading microorganisms, P.
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putida and Pseudomonas sp. strain, did not alter the BPA degradation efficiency. By
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contrast, with the assistance of the non-BPA degrading Pseudomonas sp. strain BP-14, the degradation period of Sphingomonas sp. strain BP-7 was reduced six-fold [34].
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Enhancement of the BPA degradation rate in the mixed culture may have been involved
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in the cooperation to decompose BPA and other downstream toxic intermediates, as well as the support in the metabolism of the degraders. The degradation rate of the mixed P. knackmussii strains in this study was considerably less than that of the original
microbial consortium (i.e. 6 days versus to 28 h). A DGGE analysis revealed that there were other predominant microorganisms in the fast BPA-degrading microcosm. A lack of other BPA (or intermediates)-degrading or -supporting microorganisms may limit the enhancement of the BPA degradation efficiency. Therefore, rich microbial diversity in
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the consortium is critical and should be considered as an option in bioremediation.
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4. Conclusions
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The BPA biodegradation ability of various microorganisms extracted from
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sediment were analyzed in this study. The microcosm required 28 h to degrade and
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transform the compound into six intermediates. The microbial community fluctuated
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and the final, predominant species were Pseudomonas knackmussii and Methylomonas
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clara. Distinct isolated strains exhibiting various BPA degradation abilities were genetically close and identical to P. knackmussii. On average, mixture of pure strains
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shortened the degradation period from 8 days to 6 days. However, microbial diversity
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was crucial in supporting rapid decomposition. This is the first report demonstrating the novel BPA-degrading activity of P. knackmussii and its role in the decomposing microcosm.
Acknowledgments The authors thank Ministry of Science and Technology, Taiwan for financial support under Contract No. NSC 102-2221-E-002 -015 -MY3. The authors also thank
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the Instrumentation Center, National Taiwan University for elemental analysis.
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References
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Figure captions Figure 1. Biodegradation kinetics of BPA for the bacterial consortium collected from river sediment.
Figure 2. Proposed degradation pathway of BPA for the bacterial consortium collected
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from river sediment.
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Figure 3. DGGE banding profile of 16S rDNA genes from the original bacterial
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consortium collected from river sediment (Ori.), the acclimated microcosm (T0) and the
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microcosm that performed 2 days of BPA degradation (T2).
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Figure 4. Phylogenetic relationships of isolated organisms with Pseudomonas
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knackmussii B13 (NR_041702) based upon the 1342-bp fragment of the 16S rRNA gene. Numbers on the branches indicate the bootstrap confidence estimates obtained
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with 1000 replicates. The scale bar represents 0.005% sequence divergence.
Figure 5. Biodegradation kinetics of BPA for isolated bacterium and mixed cultures.
Table 1. The characteristics and element composition of the river sediment Parameters
Value 6.1
pH
65.42 ± 1.78 26.26
Silt
8.32 ± 0.13
C
1.05 ± 0.07
H
0.51 ± 0.001
N
0.10 ± 0.001
S
0.08 ±0.006
O
2.42 ± 0.074
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Clay
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Element composition (%)
±1.91
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Sand
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Texture (%)
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(n=2)
t R (min)
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2,2-bis(4-hydroxyphenyl)-l-propanol
6.7
B
1,2-bis(4-hydroxyphenyl)-2-propanol
6.7
C
carbocationic isopropylphenol
6.6
D
4-isopropenylphenol
6.5
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Compound in Fig. 3
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Table 2. Intermediates of BPA biodegradation
4,4-dihydroxy-α-methylstilbene
2.8
F
2,2-bis(4-hydroxyphenyl) propanoic acid
1.7
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E
P. knackmussii B13 (NR_041702)
1408/1410 (99.8582%)
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RB-I
(99.8596%) 1412/1414
D
RB-G
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RB-H
(99.7171%) 1409/1413 (99.7169%) 1423/1425
N
1410/1414
RB-F
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(99.8586%)
A
RB-F
RB-H
RB-I
1410/1412
1409/1413
(99.8584%) 1409/1411 (99.8583%)
(99.7169%) 1409/1413 (99.7169%) 1411/1413
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ID
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Table 3. Identities between four BPA degradation bacteria and their closest match
(99.8585%)
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Fig. 1
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Fig. 2
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Fig. 3
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Fig. 5
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Fig. 4
