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Biodegradation of chlortetracycline by acclimated microbiota
Accepted Manuscript Title: Biodegradation of chlortetracycline by acclimated microbiota Authors: Xiaobin Liao, Rusen Zou, Bingxin Li, Tianli Tong, Shuguang Xie, Baoling Yuan PII: DOI: Reference:
S0957-5820(17)30081-2 http://dx.doi.org/doi:10.1016/j.psep.2017.03.015 PSEP 1006
To appear in:
Process Safety and Environment Protection
Received date: Revised date: Accepted date:
15-11-2016 2-3-2017 7-3-2017
Please cite this article as: Liao, Xiaobin, Zou, Rusen, Li, Bingxin, Tong, Tianli, Xie, Shuguang, Yuan, Baoling, Biodegradation of chlortetracycline by acclimated microbiota.Process Safety and Environment Protection http://dx.doi.org/10.1016/j.psep.2017.03.015 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 chlortetracycline by acclimated microbiota
Xiaobin Liao1, Rusen Zou1, Bingxin Li2, Tianli Tong2, Shuguang Xie2,*, Baoling Yuan1,*
1. Institute of Municipal and Environmental Engineering, College of Civil Engineering, Huaqiao University, Xiamen, Fujian 361021, P. R. China
2. State Key Joint Laboratory of Environmental Simulation and Pollution Control, College of Environmental Sciences and Engineering, Peking University, Beijing 100871, China
* Corresponding author. Tel: 86-10-62758599. Fax: 86-10-62758599. Email: [email protected] (Shuguang Xie); [email protected] (Baoling Yuan)
Abstract The wide presence of antibiotic chlortetracycline in the environment has aroused increasing ecological and human health concerns. Biodegradation can be a promising strategy to dissipate chlortetracycline. However, there is a paucity of knowledge on the biodegradation of chlortetracycline. The present study investigated the biodegradation of chlortetracycline by a microbial community in liquid culture, the potential 1
intermediates, the influences of temperature, external carbon and nitrogen sources, and the composition of chlortetracycline-degrading microbial community. At the initial chlortetracycline level of 100 μg l-1, the average removal rates of 48.7% and 84.9% were achieved by acclimated microbial populations in one and four weeks, respectively. Four potential intermediates were identified using LC/MS/MS analysis. Moreover, microbial
growth
was
observed
with
chlortetracycline
biodegradation.
Chlortetracycline could be used as sole carbon and nitrogen sources by the microbial community, while temperature rise and addition of external nitrogen source favored chlortetracycline biodegradation. Illumina MiSeq high-throughput sequencing analysis indicated that bacterial community structure considerably changed with the degradation of chlortetracycline. Firmicutes, Proteobacteria and Bacteroidetes were the dominant phylum groups in chlortetracycline-degrading bacterial community.
Keywords: Antibiotic; Biodegradation; Biological activated carbon; Tetracyclines
1. Introduction Chlortetracycline is a broad-spectrum antibiotic of the tetracycline family that is extensively used for animal husbandry, aquaculture and human disease control. The widespread use of chlortetracycline has led to the presence of this antibiotic compound in various aquatic and soil environments (Huang et al. 2013; Patyra et al. 2015; Tong et al. 2014; Wang et al. 2014; Xiong et al. 2015; Zhang et al. 2011). High levels of chlortetracycline can be found in surface water (122.3 ng l−1) (Tong et al. 2014) and 2
wastewater (1.8±0.5 mg l−1) (Hou et al. 2016).The antibiotic residue might have several adverse effects, such as inhibition of microbial activity and growth (Liu et al. 2015; Zielezny et al. 2006), phytoplankton toxicity (Guo and Chen 2012), and change of microbial community structure (Stone et al. 2011). Biodegradation has been proven to be a feasible strategy to remove various organic contaminants (Bach et al. 2015; Hou et al. 2015; Kuppusamy et al. 2016; Maddela et al. 2016; Varjani et al. 2015). Several previous studies have also documented the biological removal of antibiotics (Amorim et al. 2014; Cetecioglu et al. 2014; Jiang et al. 2014; Lin et al. 2015; Reis et al. 2014). These studies indicated the feasibility of biodegradation of antibiotics using microorganisms. Moreover, microbial consortia with a history of acclimation can enhance the antibiotic biodegradation (Liao et al. 2016a,b). However, direct information on the biological degradation of chlortetracycline is still very limited. Chang and Ren (2015) reported the effective biodegradation of chlortetracycline in river sediment with a previous exposure to chlortetracycline contamination, and they also found that the level of chlortetracycline could affect the bacterial community structure. However, there is still a paucity of knowledge on the biodegradation intermediates of chlortetracycline and the influential factors. Phylogenetic information on the composition of chlortetracycline-degrading bacterial community is still lacking. Therefore, the major aim of the present study was to investigate chlortetracycline biodegradation by acclimated microbial populations. The influential factors and the potential biodegradation intermediates were also explored. Moreover, the structure of chlortetracycline-degrading microbial community was characterized. 3
2. Materials and methods 2.1. Reagents and Instruments Chlortetracycline hydrochloride was purchased from Aladdin (>99.99%). R2A culture medium and inorganic salts (analytical reagent grade) were purchased from Anpu Biological Technology Co. Ltd. The pH of R2A culture medium was 7.2 and was not regulated in this study. Methanol and other solvents (analytical reagent grade) were purchased from Merck (Germany). Microbial growth was monitored by measuring the absorbance at 600 nm using V-1100 spectrometer (Mapada, China). Chlortetracycline concentration was quantified using high performance liquid chromatography (HPLC) (Shimadzu, LC-20A, Japan) coupled with a triple quadrupole mass spectrometer (AB SCIEX 3200 Q-Trap, USA).
2.2. Biodegradation test In the present study, the acclimated microbial cultures and the microcosms used for biodegradation experiments were prepared according to our previous studies (Liao et al. 2016a,b) with some minor modifications. Briefly, granular activated carbon (GAC) particles were collected from one biological activated carbon (BAC) filter system treating antibiotic-contaminated lake water (containing about 1 μg l-1 tetracyclines) (Liao et al. 2013). Biomass was detached from GAC particles (referred to original microbiota) and then incubated (100 rpm, 25oC) in basic salt medium (BSM) (pH 7.0) for a week. The composition of BSM was as follows (mg l-1): FeSO4 0.01, CaCl2 0.1, NaCl 0.2, KCl 4, MgSO4·7H2O4, K2HPO4 0.5, and KH2PO4 0.5 (Liao et al., 2015). 4
Aliquots (10% volume) was subcultured into chlortetracycline-supplemented BSM solution every a week. The enrichment of microbial consortia continued for four passages. The level of chlortetracycline in BSM solution gradually increased in these four passages, from 1μg l-1 to 100 μg l-1, 1mg l-1 and then 10 mg l-1. In this study, the final culture was referred to acclimated microbiota.
To compare the chlortetracycline biodegradation with different inocula, three different treatments was used, including treatment I: chlortetracycline (100 μg l-1), treatment II chlortetracycline (100 μg l-1) + original microbiota (1.03*108 CFU bacteria l-1), and treatment III: chlortetracycline (100 μg l-1) + acclimated microbiota (1.03*108 CFU bacteria l-1). To assess the influence of temperature on chlorotetracycline biodegradation, the temperature of the microcosm containing chlortetracycline (100 μg l-1) and acclimated microbiota (1.03*108 CFU bacteria l-1) was adjusted to 5, 25, and 45oC, respectively. Moreover, to further assess the influences of carbon or nitrogen sources on chlortetracycline biodegradation, glucose (1000 μg l-1) or ammonia nitrogen (100 μg l-1) was added into the microcosm containing chlortetracycline (100 μg l-1) and acclimated microbiota (1.03*108 CFU bacteria l-1) and incubated at 25oC for four weeks. All these biodegradation experiments were performed in batch mode. The batch tests were conducted in 1000 ml brown glass bottles. All microcosms were incubated at 120 r/min under aerobic condition. During the incubation, biomass was quantified using optical density (OD) at 600 nm (OD600 n m).
5
2.3. Chemical analysis Before chemical analysis, liquid samples were filtered through 0.22-μm nylon membrane and concentrated. The chlorine concentration was detected by Ion chromatography (930 Compact IC Flex, Metrohm) with a conductivity detector. Gradient separation was achieved with an anion exchange column (Metrosep A Supp 4-250/4.05.2) at the flow rate of 1.0 ml min-1. MSM Rotor Suppressor was used to reduce the conductivity of the background. 3.6 mmol Na2CO3 was used as the leaching solution, and the 50 mmol l-1 H2SO4 was the regeneration fluid. The injection volume of each sample was set at 20 μl.
The methods of concentrating and detecting chlortetracycline were in accordance with the previous study (Shelver et al. 2012). The chlortetracycline concentration was quantified using HPLC coupled with a triple quadrupole mass spectrometer (AB SCIEX 3200 Q-Trap). Gradient separation was achieved with a Thermo Scientific Hypersil GOLD C18 column (150 mm × 2.1 mm, 3 μm) at the flow rate of 0.35 ml min1
. The mobile phase consisted of solvent A (EDTA-McIlvaine buffer: 0.01 mol l-1
NaH2PO4 + 0.001 mol l-1 EDTA) and solvent B (methanol) with the volume ratio of 6:4. The injection volume of each sample was set at 20 μl. The mass spectrometry was obtained using a triple-quadrupole mass spectrometer equipped with an electrospray ionization source (ESI). The mass analyzer was operated in positive ionization mode, and the detailed parameters was described in our previous study (Liao et al. 2016a). The standard curve, with a correlation coefficient (R2) of 0.99, was linear within a 6
concentration range of 0.1–100 μg l-1 for chlortetracycline. The recovery rates were 82– 94% and the instrument detection limits (IDLs) was 0.08 μg l-1. The MS analysis of intermediates was performed using the same condition as previously reported in the literature (Liao et al. 2016b). The metabolite products of chlortetracycline biodegradation were confirmed by the previous literatures (Chen et al. 2011; McCormick et al. 1957) as well as by the Chem-Bio Draw Ultra software.
2.4. Molecular analysis At days 0 and 28, the samples from the microcosm with treatment III were collected for molecular analysis. Microbial cells were retained using a 0.22-μm pore-size membrane (diameter 50 mm; Millipore). The genomic DNA was extracted using the E.Z.N.A. Water DNA kit (Omega, USA) and amplified using the bacterial primer set 515F (5’GTGCCAGCMGCCGCGG-3’)/
R907
(5’-CCGTCAATTCMTTTRAGTTT-3’)
(Xiong et al. 2012; Guan et al. 2015; Wang et al. 2015). The amplicons of triplicate samples were mixed in equal amounts for Illumina MiSeq high-throughput sequencing. The obtained reads were deposited in the NCBI short-read archive under accession numbers SRR2140660 and SRR2140661. The quality filtering of reads was conducted following the protocol as previously described by Caporaso et al. (2010). The taxonomic identities of bacterial sequences were assigned using the Silva 16S rRNA database (Quast et al. 2013).
7
2.5. Statistical analysis Shapiro-Wilk test was applied to determine the data normality of chlortetracycline level or biomass, and Levene was used to determine the homogeneity of variance. The results supported the usage of one-way analysis of variance (ANOVA), followed by Student– Newman–Keuls test to check the significant difference (P<0.05) in the chlortetracycline level or biomass.
3. Results 3.1. Chlortetracycline biodegradation In the present study, two mixed cultures were used as inocula for chlortetracycline biodegradation tests, namely original microbiota (without acclimation using chlortetracycline-supplemented BSM solution) and acclimated microbiota (with four passages of acclimation using chlortetracycline-supplemented BSM solution). Only a slight attenuation of chlortetracycline (with the removal efficiency of 6.3±0.01%) occurred in the non-inoculated microcosm after four weeks’ incubation at 25oC, while a significant decrease of chlortetracycline was found in the microcosm added with original microbiota (P< 0.05) (Figure 1), suggesting the occurrence of chlortetracycline biodegradation. The chlortetracycline removal efficiency of 15.9±3.45% and 32.5±2.57% was observed in the microcosm added with original microbiota at days 7 and 28, respectively. Compared to the other two microcosms, the microcosm inoculated with acclimated microbiota displayed higher chlortetracycline removal (P<0.05) at each sampling date, and it showed the removal efficiency of 48.7±3.29% and 84.9±0.72% at 8
days 7 and 28, respectively. These results indicated that acclimated microbiota could more effectively biodegrade antibiotic chlortetracycline. In addition, the increase of the concentration of acclimated microbiota was found to be able to promote the chlortetracycline biodegradation (Figure S1). During the incubation, the chlorine level considerably increased in the microcosm inoculated with acclimated microbiota (Figure 2), which was consistent with the biodegradation of chlortetracycline. During the incubation, no significant change of biomass was found in the microcosm added with original microbiota (P>0.05), while the microcosm inoculated with acclimated microbiota illustrated a significant increase of biomass (P< 0.05) (Figure 3).
3.2. Influences of temperature, carbon and nitrogen sources The chlortetracycline removal rate at 45oC (89.8%) was significantly higher those at 25oC (84.9%) and at 5oC (18.8%) (P< 0.05) (Figure 4). The addition of glucose (1000 μg l-1) did not significantly enhance the chlortetracycline removal by acclimated microbiota (P> 0.05), while the addition of ammonia nitrogen (100 μg l-1) could significantly promote the chlortetracycline degradation (P< 0.05) (Figure 5). On day 28, the removal efficiency of 87.8±0.72% was observed in the microcosm with the addition of glucose, while a nearly complete removal occurred in the microcosm with the addition of ammonia nitrogen.
9
3.3. Biodegradation intermediates In the present study, the triplicate samples from the microcosm with treatment III on day 28 were further used to identify the potential intermediates of chlortetracycline biodegradation. Based on LC/MS/MS analysis, a total of four putative by-products were identified by comparing their molar masses with those of the compounds reported in the previous studies (Chen et al. 2011; McCormick et al. 1957). The strongest peak (compound A) appeared at m/z 332.1, and its elemental composition was elucidated as C18H20O6 (Figure 6). Compound B, detected at m/z 445.1, was tetracycline (C22H23 N2O8), as the result of the loss of Cl atom from chlortetracycline. Moreover, the demethyl product (compound C) was formed due to the loss of a methyl group from the –N(CH3)2 of chlortetracycline and could be named as 6-demethyl- chlortetracycline (McCormick et al. 1957). Compound D represented by the MS/MS fragment at m/z 511 might be derived from the addition of two OH groups to chlortetracycline.
3.4. Bacterial community composition In the present study, the liquid samples from the microcosm with treatment III at days 0 and 28 were referred to Samples D0 and D28, respectively. Illumina high-throughput sequencing was applied to characterize the bacterial community structure of acclimated microbiota and its change with chlortetracycline biodegradation. A total of 26,437 or 24,139 high-quality bacterial sequences retrieved from these two samples. A total of 5 abundant bacterial phyla (with relative abundance > 3% in at least one sample) were identified, including Proteobacteria, Bacteroidetes, Firmicutes, Actinobacteria and 10
Armatimonadetes (Figure 7a). Sample D0 was dominated by Bacteroidetes (41%), Proteobacteria (40%) and Actinobacteria (13%), while Sample D28 mainly included Firmicutes (45%), Proteobacteria (40%) and Bacteroidetes (15%). The proportions of Bacteroidetes and Actinobacteria considerably decreased with chlortetracycline biodegradation, while the fraction of Firmicutes showed a remarkable increase. Moreover,
classes
Sphingobacteriia
(39%),
Gammaproteobacteria
(21%),
Actinobacteria (13%) and Alphaproteobacteria (11%) dominated in Sample D0, while Bacilli (45%) and Gammaproteobacteria (30%) were the dominant bacterial classes in Sample D28 (Figure 7b). The dominant genera were different in Samples D0 and D28 (Figure 7c). Hydrotalea (31%), Pseudoxanthomonas (16%) and Mycobacterium (13%) were the most abundant genera in Sample D0, while genera Bacillus (44%) and Stenotrophomonas (28%) dominated in Sample D28. These results displayed an evident shift in bacterial community structure with chlortetracycline biodegradation.
4. Discussion To date, very limited information exists on the biodegradation of chlortetracycline. Chang and Ren (2015) showed the effective biodegradation of chlortetracycline in river sediment that was previously exposed to chlortetracycline contamination. In this study, the original microbiota used for biodegradation experiments was obtained from one drinking water BAC filter system that could effectively remove low level of tetracyclines (about 1 μg l-1), although it was unclear whether the removal of tetracyclines in BAC filter was achieved by microbial degradation or by GAC 11
adsorption (Liao et al. 2013). The original microbiota was found to be able to effectively dissipate high level of chlortetracycline (100 μg l-1), which was consistent with the microbial growth. This suggested that microbial community previously exposed to low level of tetracyclines had the potential of biodegrading much higher level of chlortetracycline. Several previous studies also suggested that the history of microbial community’s previous exposure to sulfonamides antibiotics could improve its biodegradation efficiency (Islas-Espinoza et al. 2012; Richter et al. 2008). In this study, the acclimated microbiota showed much higher chlortetracycline removal efficiency than the original microbiota. This further confirmed that acclimated microbial consortia could accelerate the antibiotic biodegradation. Our recent study also revealed that the microbial consortia previously acclimated to high level of antibiotics (sulfanilamide and ciprofloxacin) could have a much more rapid biodegradation rate (Liao et al. 2016a,b). Moreover, in the present study, the temperature rise was found to favor the chlortetracycline biodegradation. This was consistent the results of sulfanilamide and ciprofloxacin biodegradation using acclimated microbial consortia in our recent study (Liao et al. 2016a,b). In addition, both carbon and nitrogen sources were absent in the BSM used for biodegradation experiments. To the authors’ knowledge, this current study provided the direct evidence for the first time that chlortetracycline could be used by microorganisms as sole carbon and nitrogen sources. In this study, addition of nitrogen source was found to be able to stimulate chlortetracycline biodegradation. Our previous studies also showed the positive effect of external nitrogen source on the biodegradation of antibiotic sulfanilamide and ciprofloxacin by microbial community 12
(Liao et al. 2016a,b). Further efforts will be necessary in order to elucidate the effects of other factors (e.g., pH) on chlortetracycline biodegradation.
In the present study, a total of four putative by-products were identified. Moreover, chloride liberation with the biodegradation of chlortetracycline also suggested the removal of chlortetracycline via dehalogenation. However, further efforts will be necessary in order to elucidate the intermediates of chlortetracycline biodegradation and the degradation pathways for chlortetracycline.
Although several previous studies have investigated the impacts of chlortetracycline on soil microbial communities (Liu et al. 2012; Stone et al. 2011; Zielezny et al. 2006), little is known about the chlortetracycline-degrading microbial community. Chang and Ren (2015) applied denaturing gradient gel electrophoresis (DGGE) analysis to characterize the chlortetracycline-degrading bacterial community in river sediment, and they found that the level of chlortetracycline could affect the bacterial community structure. In the present study, Illumina MiSeq high-throughput sequencing was used to characterize the bacterial communities before and after chlortetracycline biodegradation. A remarkable shift in bacterial community structure at phylum, class and genus levels occurred with chlortetracycline biodegradation. Phyla Firmicutes, Proteobacteria and Bacteroidetes dominated in chlortetracycline-degrading bacterial community. The dominance of these three bacterial groups was also found in aquaculture sediment that was contaminated by various antibiotics including 13
chlortetracycline and other tetracyclines (Xiong et al. 2015). In contrast, Song et al. (2015) revealed that Proteobacteria predominated in activated sludge bacterial community exposed to antibiotic tetracycline. In this study, Bacillus and Stenotrophomonas were the largest genera in chlortetracycline-degrading bacterial community, while Azoarcus was the most dominant genus in tetracycline-containing activated sludge bacterial community (Song et al. 2015). The isolation of chlortetracycline-degrading microorganisms could be used for design of more elaborate studies on biodegradation mechanisms and influential factors, and also used to test their bioaugmentation efficiency in bioreactors.
5. Conclusions Both original microbiota and acclimated microbiota could biodegrade antibiotic chlortetracycline that was used as sole carbon and nitrogen sources. However, acclimated microbiota showed a more rapid chlortetracycline removal as well as microbial growth. Temperature rise and addition of nitrogen sources could further promote chlortetracycline biodegradation by acclimated microbiota. A remarkable shift in bacterial community structure was observed after a considerable chlortetracycline biodegradation. Chlortetracycline-degrading bacterial community was mainly composed of phyla Firmicutes, Proteobacteria and Bacteroidetes.
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Conflict of interest statement The authors declare no financial or commercial conflicts of interest.
Acknowledgments This work was financially supported by National Natural Science Foundation of China (Nos. 51508209, 51178117), China Post-Doctoral Funding (No.145723), Natural Science Foundation of Fujian province (No. 2015J05102), Scientific Research project for Young teachers supported by Education Department of Fujian Province (No. JA15033), the Science Research Foundation of Huaqiao University (No.15BS105), and Key Project of International Cooperation of Science and Technology of Fujian (No. 2014I0013).
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Microbiol. Biotechnol. 99, 3259–3268. Xiong, J.B., Liu, Y.Q., Lin, X.G., Zhang, H.Y., Zeng, J., Hou, J.Z., Yang, Y.P., Yao, T.D., Knight, R., Chu, H.Y., 2012. Geographic distance and pH drive bacterial distribution in alkaline lake sediments across Tibetan Plateau. Environ. Microbiol. 14SI., 2457–2466. Zhang, D.D., Lin, L.F., Luo, Z.X., Yan, C.Z., Zhang, X., 2011. Occurrence of selected antibiotics in Jiulongjiang River in various seasons, South China. J. Environ. Monit. 13, 1953–1960. Zielezny, Y., Groeneweg, J., Vereecken, H., Tappe, W., 2006. Impact of sulfadiazine and chlortetracycline on soil bacterial community structure and respiratory activity. Soil Biol. Biochem. 38, 2372–2380.
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Figure captions Figure 1 Removal efficiency of chlortetracycline in the microcosms at 25°C with three different treatments. Treatment I: chlortetracycline (100 μg l-1); treatment II: chlortetracycline (100 μg l-1) + original microbiota (1.03*108 CFU bacteria l-1); and treatment III: chlortetracycline (100 μg l-1) + acclimated microbiota (1.03*108 CFU bacteria l-1). Values are the average of three independent experiments. Vertical bars indicate standard deviations.
Figure 2 The change of chlorine concentration in the microcosm with treatment III at 25°C. Values are the average of three independent experiments. Treatment III: chlortetracycline (100 μg l-1) + acclimated microbiota (1.03*108 CFU bacteria l-1). Vertical bars indicate standard deviations.
Figure 3 Microbial growth in the microcosms with three different treatments at 25°C. Treatment II: chlortetracycline (100 μg l-1) + original microbiota (1.03*108 CFU bacteria l-1); and treatment III: chlortetracycline (100 μg l-1) + acclimated microbiota (1.03*108 CFU bacteria l-1). Values are the average of three independent experiments. Vertical bars indicate standard deviations.
Figure 4 Effect of temperature on the biodegradation of chlorotetracycline. Values are the average of three independent experiments. Vertical bars indicate standard deviations.
22
Figure 5 Chlortetracycline removal efficiency with different carbon and nitrogen sources after 28-day incubation at 25°C. Values are the average of three independent experiments. Vertical bars indicate standard deviations.
Figure 6 The results of the LC/MS/MS analysis and the proposed intermediates of chlortetracycline biodegradation
Figure 7 The relative abundance of major phyla (a), classes (b), and genera (c).
23
Figure 1
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Figure 3
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Figure 5
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Figure 6
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Figure 7
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Burkholderia Bacillus
S0957-5820(17)30081-2 http://dx.doi.org/doi:10.1016/j.psep.2017.03.015 PSEP 1006
To appear in:
Process Safety and Environment Protection
Received date: Revised date: Accepted date:
15-11-2016 2-3-2017 7-3-2017
Please cite this article as: Liao, Xiaobin, Zou, Rusen, Li, Bingxin, Tong, Tianli, Xie, Shuguang, Yuan, Baoling, Biodegradation of chlortetracycline by acclimated microbiota.Process Safety and Environment Protection http://dx.doi.org/10.1016/j.psep.2017.03.015 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 chlortetracycline by acclimated microbiota
Xiaobin Liao1, Rusen Zou1, Bingxin Li2, Tianli Tong2, Shuguang Xie2,*, Baoling Yuan1,*
1. Institute of Municipal and Environmental Engineering, College of Civil Engineering, Huaqiao University, Xiamen, Fujian 361021, P. R. China
2. State Key Joint Laboratory of Environmental Simulation and Pollution Control, College of Environmental Sciences and Engineering, Peking University, Beijing 100871, China
* Corresponding author. Tel: 86-10-62758599. Fax: 86-10-62758599. Email: [email protected] (Shuguang Xie); [email protected] (Baoling Yuan)
Abstract The wide presence of antibiotic chlortetracycline in the environment has aroused increasing ecological and human health concerns. Biodegradation can be a promising strategy to dissipate chlortetracycline. However, there is a paucity of knowledge on the biodegradation of chlortetracycline. The present study investigated the biodegradation of chlortetracycline by a microbial community in liquid culture, the potential 1
intermediates, the influences of temperature, external carbon and nitrogen sources, and the composition of chlortetracycline-degrading microbial community. At the initial chlortetracycline level of 100 μg l-1, the average removal rates of 48.7% and 84.9% were achieved by acclimated microbial populations in one and four weeks, respectively. Four potential intermediates were identified using LC/MS/MS analysis. Moreover, microbial
growth
was
observed
with
chlortetracycline
biodegradation.
Chlortetracycline could be used as sole carbon and nitrogen sources by the microbial community, while temperature rise and addition of external nitrogen source favored chlortetracycline biodegradation. Illumina MiSeq high-throughput sequencing analysis indicated that bacterial community structure considerably changed with the degradation of chlortetracycline. Firmicutes, Proteobacteria and Bacteroidetes were the dominant phylum groups in chlortetracycline-degrading bacterial community.
Keywords: Antibiotic; Biodegradation; Biological activated carbon; Tetracyclines
1. Introduction Chlortetracycline is a broad-spectrum antibiotic of the tetracycline family that is extensively used for animal husbandry, aquaculture and human disease control. The widespread use of chlortetracycline has led to the presence of this antibiotic compound in various aquatic and soil environments (Huang et al. 2013; Patyra et al. 2015; Tong et al. 2014; Wang et al. 2014; Xiong et al. 2015; Zhang et al. 2011). High levels of chlortetracycline can be found in surface water (122.3 ng l−1) (Tong et al. 2014) and 2
wastewater (1.8±0.5 mg l−1) (Hou et al. 2016).The antibiotic residue might have several adverse effects, such as inhibition of microbial activity and growth (Liu et al. 2015; Zielezny et al. 2006), phytoplankton toxicity (Guo and Chen 2012), and change of microbial community structure (Stone et al. 2011). Biodegradation has been proven to be a feasible strategy to remove various organic contaminants (Bach et al. 2015; Hou et al. 2015; Kuppusamy et al. 2016; Maddela et al. 2016; Varjani et al. 2015). Several previous studies have also documented the biological removal of antibiotics (Amorim et al. 2014; Cetecioglu et al. 2014; Jiang et al. 2014; Lin et al. 2015; Reis et al. 2014). These studies indicated the feasibility of biodegradation of antibiotics using microorganisms. Moreover, microbial consortia with a history of acclimation can enhance the antibiotic biodegradation (Liao et al. 2016a,b). However, direct information on the biological degradation of chlortetracycline is still very limited. Chang and Ren (2015) reported the effective biodegradation of chlortetracycline in river sediment with a previous exposure to chlortetracycline contamination, and they also found that the level of chlortetracycline could affect the bacterial community structure. However, there is still a paucity of knowledge on the biodegradation intermediates of chlortetracycline and the influential factors. Phylogenetic information on the composition of chlortetracycline-degrading bacterial community is still lacking. Therefore, the major aim of the present study was to investigate chlortetracycline biodegradation by acclimated microbial populations. The influential factors and the potential biodegradation intermediates were also explored. Moreover, the structure of chlortetracycline-degrading microbial community was characterized. 3
2. Materials and methods 2.1. Reagents and Instruments Chlortetracycline hydrochloride was purchased from Aladdin (>99.99%). R2A culture medium and inorganic salts (analytical reagent grade) were purchased from Anpu Biological Technology Co. Ltd. The pH of R2A culture medium was 7.2 and was not regulated in this study. Methanol and other solvents (analytical reagent grade) were purchased from Merck (Germany). Microbial growth was monitored by measuring the absorbance at 600 nm using V-1100 spectrometer (Mapada, China). Chlortetracycline concentration was quantified using high performance liquid chromatography (HPLC) (Shimadzu, LC-20A, Japan) coupled with a triple quadrupole mass spectrometer (AB SCIEX 3200 Q-Trap, USA).
2.2. Biodegradation test In the present study, the acclimated microbial cultures and the microcosms used for biodegradation experiments were prepared according to our previous studies (Liao et al. 2016a,b) with some minor modifications. Briefly, granular activated carbon (GAC) particles were collected from one biological activated carbon (BAC) filter system treating antibiotic-contaminated lake water (containing about 1 μg l-1 tetracyclines) (Liao et al. 2013). Biomass was detached from GAC particles (referred to original microbiota) and then incubated (100 rpm, 25oC) in basic salt medium (BSM) (pH 7.0) for a week. The composition of BSM was as follows (mg l-1): FeSO4 0.01, CaCl2 0.1, NaCl 0.2, KCl 4, MgSO4·7H2O4, K2HPO4 0.5, and KH2PO4 0.5 (Liao et al., 2015). 4
Aliquots (10% volume) was subcultured into chlortetracycline-supplemented BSM solution every a week. The enrichment of microbial consortia continued for four passages. The level of chlortetracycline in BSM solution gradually increased in these four passages, from 1μg l-1 to 100 μg l-1, 1mg l-1 and then 10 mg l-1. In this study, the final culture was referred to acclimated microbiota.
To compare the chlortetracycline biodegradation with different inocula, three different treatments was used, including treatment I: chlortetracycline (100 μg l-1), treatment II chlortetracycline (100 μg l-1) + original microbiota (1.03*108 CFU bacteria l-1), and treatment III: chlortetracycline (100 μg l-1) + acclimated microbiota (1.03*108 CFU bacteria l-1). To assess the influence of temperature on chlorotetracycline biodegradation, the temperature of the microcosm containing chlortetracycline (100 μg l-1) and acclimated microbiota (1.03*108 CFU bacteria l-1) was adjusted to 5, 25, and 45oC, respectively. Moreover, to further assess the influences of carbon or nitrogen sources on chlortetracycline biodegradation, glucose (1000 μg l-1) or ammonia nitrogen (100 μg l-1) was added into the microcosm containing chlortetracycline (100 μg l-1) and acclimated microbiota (1.03*108 CFU bacteria l-1) and incubated at 25oC for four weeks. All these biodegradation experiments were performed in batch mode. The batch tests were conducted in 1000 ml brown glass bottles. All microcosms were incubated at 120 r/min under aerobic condition. During the incubation, biomass was quantified using optical density (OD) at 600 nm (OD600 n m).
5
2.3. Chemical analysis Before chemical analysis, liquid samples were filtered through 0.22-μm nylon membrane and concentrated. The chlorine concentration was detected by Ion chromatography (930 Compact IC Flex, Metrohm) with a conductivity detector. Gradient separation was achieved with an anion exchange column (Metrosep A Supp 4-250/4.05.2) at the flow rate of 1.0 ml min-1. MSM Rotor Suppressor was used to reduce the conductivity of the background. 3.6 mmol Na2CO3 was used as the leaching solution, and the 50 mmol l-1 H2SO4 was the regeneration fluid. The injection volume of each sample was set at 20 μl.
The methods of concentrating and detecting chlortetracycline were in accordance with the previous study (Shelver et al. 2012). The chlortetracycline concentration was quantified using HPLC coupled with a triple quadrupole mass spectrometer (AB SCIEX 3200 Q-Trap). Gradient separation was achieved with a Thermo Scientific Hypersil GOLD C18 column (150 mm × 2.1 mm, 3 μm) at the flow rate of 0.35 ml min1
. The mobile phase consisted of solvent A (EDTA-McIlvaine buffer: 0.01 mol l-1
NaH2PO4 + 0.001 mol l-1 EDTA) and solvent B (methanol) with the volume ratio of 6:4. The injection volume of each sample was set at 20 μl. The mass spectrometry was obtained using a triple-quadrupole mass spectrometer equipped with an electrospray ionization source (ESI). The mass analyzer was operated in positive ionization mode, and the detailed parameters was described in our previous study (Liao et al. 2016a). The standard curve, with a correlation coefficient (R2) of 0.99, was linear within a 6
concentration range of 0.1–100 μg l-1 for chlortetracycline. The recovery rates were 82– 94% and the instrument detection limits (IDLs) was 0.08 μg l-1. The MS analysis of intermediates was performed using the same condition as previously reported in the literature (Liao et al. 2016b). The metabolite products of chlortetracycline biodegradation were confirmed by the previous literatures (Chen et al. 2011; McCormick et al. 1957) as well as by the Chem-Bio Draw Ultra software.
2.4. Molecular analysis At days 0 and 28, the samples from the microcosm with treatment III were collected for molecular analysis. Microbial cells were retained using a 0.22-μm pore-size membrane (diameter 50 mm; Millipore). The genomic DNA was extracted using the E.Z.N.A. Water DNA kit (Omega, USA) and amplified using the bacterial primer set 515F (5’GTGCCAGCMGCCGCGG-3’)/
R907
(5’-CCGTCAATTCMTTTRAGTTT-3’)
(Xiong et al. 2012; Guan et al. 2015; Wang et al. 2015). The amplicons of triplicate samples were mixed in equal amounts for Illumina MiSeq high-throughput sequencing. The obtained reads were deposited in the NCBI short-read archive under accession numbers SRR2140660 and SRR2140661. The quality filtering of reads was conducted following the protocol as previously described by Caporaso et al. (2010). The taxonomic identities of bacterial sequences were assigned using the Silva 16S rRNA database (Quast et al. 2013).
7
2.5. Statistical analysis Shapiro-Wilk test was applied to determine the data normality of chlortetracycline level or biomass, and Levene was used to determine the homogeneity of variance. The results supported the usage of one-way analysis of variance (ANOVA), followed by Student– Newman–Keuls test to check the significant difference (P<0.05) in the chlortetracycline level or biomass.
3. Results 3.1. Chlortetracycline biodegradation In the present study, two mixed cultures were used as inocula for chlortetracycline biodegradation tests, namely original microbiota (without acclimation using chlortetracycline-supplemented BSM solution) and acclimated microbiota (with four passages of acclimation using chlortetracycline-supplemented BSM solution). Only a slight attenuation of chlortetracycline (with the removal efficiency of 6.3±0.01%) occurred in the non-inoculated microcosm after four weeks’ incubation at 25oC, while a significant decrease of chlortetracycline was found in the microcosm added with original microbiota (P< 0.05) (Figure 1), suggesting the occurrence of chlortetracycline biodegradation. The chlortetracycline removal efficiency of 15.9±3.45% and 32.5±2.57% was observed in the microcosm added with original microbiota at days 7 and 28, respectively. Compared to the other two microcosms, the microcosm inoculated with acclimated microbiota displayed higher chlortetracycline removal (P<0.05) at each sampling date, and it showed the removal efficiency of 48.7±3.29% and 84.9±0.72% at 8
days 7 and 28, respectively. These results indicated that acclimated microbiota could more effectively biodegrade antibiotic chlortetracycline. In addition, the increase of the concentration of acclimated microbiota was found to be able to promote the chlortetracycline biodegradation (Figure S1). During the incubation, the chlorine level considerably increased in the microcosm inoculated with acclimated microbiota (Figure 2), which was consistent with the biodegradation of chlortetracycline. During the incubation, no significant change of biomass was found in the microcosm added with original microbiota (P>0.05), while the microcosm inoculated with acclimated microbiota illustrated a significant increase of biomass (P< 0.05) (Figure 3).
3.2. Influences of temperature, carbon and nitrogen sources The chlortetracycline removal rate at 45oC (89.8%) was significantly higher those at 25oC (84.9%) and at 5oC (18.8%) (P< 0.05) (Figure 4). The addition of glucose (1000 μg l-1) did not significantly enhance the chlortetracycline removal by acclimated microbiota (P> 0.05), while the addition of ammonia nitrogen (100 μg l-1) could significantly promote the chlortetracycline degradation (P< 0.05) (Figure 5). On day 28, the removal efficiency of 87.8±0.72% was observed in the microcosm with the addition of glucose, while a nearly complete removal occurred in the microcosm with the addition of ammonia nitrogen.
9
3.3. Biodegradation intermediates In the present study, the triplicate samples from the microcosm with treatment III on day 28 were further used to identify the potential intermediates of chlortetracycline biodegradation. Based on LC/MS/MS analysis, a total of four putative by-products were identified by comparing their molar masses with those of the compounds reported in the previous studies (Chen et al. 2011; McCormick et al. 1957). The strongest peak (compound A) appeared at m/z 332.1, and its elemental composition was elucidated as C18H20O6 (Figure 6). Compound B, detected at m/z 445.1, was tetracycline (C22H23 N2O8), as the result of the loss of Cl atom from chlortetracycline. Moreover, the demethyl product (compound C) was formed due to the loss of a methyl group from the –N(CH3)2 of chlortetracycline and could be named as 6-demethyl- chlortetracycline (McCormick et al. 1957). Compound D represented by the MS/MS fragment at m/z 511 might be derived from the addition of two OH groups to chlortetracycline.
3.4. Bacterial community composition In the present study, the liquid samples from the microcosm with treatment III at days 0 and 28 were referred to Samples D0 and D28, respectively. Illumina high-throughput sequencing was applied to characterize the bacterial community structure of acclimated microbiota and its change with chlortetracycline biodegradation. A total of 26,437 or 24,139 high-quality bacterial sequences retrieved from these two samples. A total of 5 abundant bacterial phyla (with relative abundance > 3% in at least one sample) were identified, including Proteobacteria, Bacteroidetes, Firmicutes, Actinobacteria and 10
Armatimonadetes (Figure 7a). Sample D0 was dominated by Bacteroidetes (41%), Proteobacteria (40%) and Actinobacteria (13%), while Sample D28 mainly included Firmicutes (45%), Proteobacteria (40%) and Bacteroidetes (15%). The proportions of Bacteroidetes and Actinobacteria considerably decreased with chlortetracycline biodegradation, while the fraction of Firmicutes showed a remarkable increase. Moreover,
classes
Sphingobacteriia
(39%),
Gammaproteobacteria
(21%),
Actinobacteria (13%) and Alphaproteobacteria (11%) dominated in Sample D0, while Bacilli (45%) and Gammaproteobacteria (30%) were the dominant bacterial classes in Sample D28 (Figure 7b). The dominant genera were different in Samples D0 and D28 (Figure 7c). Hydrotalea (31%), Pseudoxanthomonas (16%) and Mycobacterium (13%) were the most abundant genera in Sample D0, while genera Bacillus (44%) and Stenotrophomonas (28%) dominated in Sample D28. These results displayed an evident shift in bacterial community structure with chlortetracycline biodegradation.
4. Discussion To date, very limited information exists on the biodegradation of chlortetracycline. Chang and Ren (2015) showed the effective biodegradation of chlortetracycline in river sediment that was previously exposed to chlortetracycline contamination. In this study, the original microbiota used for biodegradation experiments was obtained from one drinking water BAC filter system that could effectively remove low level of tetracyclines (about 1 μg l-1), although it was unclear whether the removal of tetracyclines in BAC filter was achieved by microbial degradation or by GAC 11
adsorption (Liao et al. 2013). The original microbiota was found to be able to effectively dissipate high level of chlortetracycline (100 μg l-1), which was consistent with the microbial growth. This suggested that microbial community previously exposed to low level of tetracyclines had the potential of biodegrading much higher level of chlortetracycline. Several previous studies also suggested that the history of microbial community’s previous exposure to sulfonamides antibiotics could improve its biodegradation efficiency (Islas-Espinoza et al. 2012; Richter et al. 2008). In this study, the acclimated microbiota showed much higher chlortetracycline removal efficiency than the original microbiota. This further confirmed that acclimated microbial consortia could accelerate the antibiotic biodegradation. Our recent study also revealed that the microbial consortia previously acclimated to high level of antibiotics (sulfanilamide and ciprofloxacin) could have a much more rapid biodegradation rate (Liao et al. 2016a,b). Moreover, in the present study, the temperature rise was found to favor the chlortetracycline biodegradation. This was consistent the results of sulfanilamide and ciprofloxacin biodegradation using acclimated microbial consortia in our recent study (Liao et al. 2016a,b). In addition, both carbon and nitrogen sources were absent in the BSM used for biodegradation experiments. To the authors’ knowledge, this current study provided the direct evidence for the first time that chlortetracycline could be used by microorganisms as sole carbon and nitrogen sources. In this study, addition of nitrogen source was found to be able to stimulate chlortetracycline biodegradation. Our previous studies also showed the positive effect of external nitrogen source on the biodegradation of antibiotic sulfanilamide and ciprofloxacin by microbial community 12
(Liao et al. 2016a,b). Further efforts will be necessary in order to elucidate the effects of other factors (e.g., pH) on chlortetracycline biodegradation.
In the present study, a total of four putative by-products were identified. Moreover, chloride liberation with the biodegradation of chlortetracycline also suggested the removal of chlortetracycline via dehalogenation. However, further efforts will be necessary in order to elucidate the intermediates of chlortetracycline biodegradation and the degradation pathways for chlortetracycline.
Although several previous studies have investigated the impacts of chlortetracycline on soil microbial communities (Liu et al. 2012; Stone et al. 2011; Zielezny et al. 2006), little is known about the chlortetracycline-degrading microbial community. Chang and Ren (2015) applied denaturing gradient gel electrophoresis (DGGE) analysis to characterize the chlortetracycline-degrading bacterial community in river sediment, and they found that the level of chlortetracycline could affect the bacterial community structure. In the present study, Illumina MiSeq high-throughput sequencing was used to characterize the bacterial communities before and after chlortetracycline biodegradation. A remarkable shift in bacterial community structure at phylum, class and genus levels occurred with chlortetracycline biodegradation. Phyla Firmicutes, Proteobacteria and Bacteroidetes dominated in chlortetracycline-degrading bacterial community. The dominance of these three bacterial groups was also found in aquaculture sediment that was contaminated by various antibiotics including 13
chlortetracycline and other tetracyclines (Xiong et al. 2015). In contrast, Song et al. (2015) revealed that Proteobacteria predominated in activated sludge bacterial community exposed to antibiotic tetracycline. In this study, Bacillus and Stenotrophomonas were the largest genera in chlortetracycline-degrading bacterial community, while Azoarcus was the most dominant genus in tetracycline-containing activated sludge bacterial community (Song et al. 2015). The isolation of chlortetracycline-degrading microorganisms could be used for design of more elaborate studies on biodegradation mechanisms and influential factors, and also used to test their bioaugmentation efficiency in bioreactors.
5. Conclusions Both original microbiota and acclimated microbiota could biodegrade antibiotic chlortetracycline that was used as sole carbon and nitrogen sources. However, acclimated microbiota showed a more rapid chlortetracycline removal as well as microbial growth. Temperature rise and addition of nitrogen sources could further promote chlortetracycline biodegradation by acclimated microbiota. A remarkable shift in bacterial community structure was observed after a considerable chlortetracycline biodegradation. Chlortetracycline-degrading bacterial community was mainly composed of phyla Firmicutes, Proteobacteria and Bacteroidetes.
14
Conflict of interest statement The authors declare no financial or commercial conflicts of interest.
Acknowledgments This work was financially supported by National Natural Science Foundation of China (Nos. 51508209, 51178117), China Post-Doctoral Funding (No.145723), Natural Science Foundation of Fujian province (No. 2015J05102), Scientific Research project for Young teachers supported by Education Department of Fujian Province (No. JA15033), the Science Research Foundation of Huaqiao University (No.15BS105), and Key Project of International Cooperation of Science and Technology of Fujian (No. 2014I0013).
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Figure captions Figure 1 Removal efficiency of chlortetracycline in the microcosms at 25°C with three different treatments. Treatment I: chlortetracycline (100 μg l-1); treatment II: chlortetracycline (100 μg l-1) + original microbiota (1.03*108 CFU bacteria l-1); and treatment III: chlortetracycline (100 μg l-1) + acclimated microbiota (1.03*108 CFU bacteria l-1). Values are the average of three independent experiments. Vertical bars indicate standard deviations.
Figure 2 The change of chlorine concentration in the microcosm with treatment III at 25°C. Values are the average of three independent experiments. Treatment III: chlortetracycline (100 μg l-1) + acclimated microbiota (1.03*108 CFU bacteria l-1). Vertical bars indicate standard deviations.
Figure 3 Microbial growth in the microcosms with three different treatments at 25°C. Treatment II: chlortetracycline (100 μg l-1) + original microbiota (1.03*108 CFU bacteria l-1); and treatment III: chlortetracycline (100 μg l-1) + acclimated microbiota (1.03*108 CFU bacteria l-1). Values are the average of three independent experiments. Vertical bars indicate standard deviations.
Figure 4 Effect of temperature on the biodegradation of chlorotetracycline. Values are the average of three independent experiments. Vertical bars indicate standard deviations.
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Figure 5 Chlortetracycline removal efficiency with different carbon and nitrogen sources after 28-day incubation at 25°C. Values are the average of three independent experiments. Vertical bars indicate standard deviations.
Figure 6 The results of the LC/MS/MS analysis and the proposed intermediates of chlortetracycline biodegradation
Figure 7 The relative abundance of major phyla (a), classes (b), and genera (c).
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Figure 1
100
Treatment Ⅰ Treatment Ⅱ Treatment Ⅲ
90
Removal efficiency (%)
80 70 60 50 40 30 20 10 0
1
3
5 7 14 Incubation time (days)
24
21
28
Figure 2
Chlorine ion concentration (ug/L)
2.5
2.0
1.5
1.0
0.5
0.0
0
1
3
5
7
14
21
28
Incubation time (days)
25
Figure 3
Optical densiity at 600 nm (cm-1)
0.30
Treatment Ⅱ Treatment Ⅲ
0.25
0.20
0.15
0.10
0.05
0.00
0
1
3 5 7 14 Incubation time (days)
26
21
28
Figure 4
Removal efficiency (%)
100
80
60
40
20
0 5
25
45
Temperature (℃)
27
Figure 5
Removal efficiency (%)
100
80
60
40
20
0
Chlortetracycline
Chlortetracycline + glucose
28
Chlortetracycline + ammonia
Figure 6
29
Figure 7
Relative abundance
(a)
100% 80% 60% 40% 20% 0%
Others Armatimonadetes Actinobacteria Firmicutes D0
D28
Bacteroidetes Proteobacteria
Sample
Relative abundance
(b)
100%
Others
50%
Sphingobacteriia
0% D0
D28 Sample
Gammaproteobact eria
(c)
Others
Relative abundance
100%
Terrimonas
80%
Stenotrophomonas Pseudoxanthomonas
60%
Nubsella 40%
Mycobacterium
20%
Hydrotalea Dysgonomonas
0% D0
D28 Sample
30
Burkholderia Bacillus