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Study of ciprofloxacin biodegradation by a Thermus sp. isolated from pharmaceutical sludge
Accepted Manuscript Title: Study of ciprofloxacin biodegradation by a Thermus sp. isolated from pharmaceutical sludge Authors: Lan-jia Pan, Jie Li, Chun-xing Li, Xiao-da Tang, Guang-wei Yu, Yin Wang PII: DOI: Reference:
S0304-3894(17)30690-8 http://dx.doi.org/10.1016/j.jhazmat.2017.09.009 HAZMAT 18855
To appear in:
Journal of Hazardous Materials
Received date: Revised date: Accepted date:
15-6-2017 4-9-2017 6-9-2017
Please cite this article as: Lan-jia Pan, Jie Li, Chun-xing Li, Xiao-da Tang, Guang-wei Yu, Yin Wang, Study of ciprofloxacin biodegradation by a Thermus sp.isolated from pharmaceutical sludge, Journal of Hazardous Materialshttp://dx.doi.org/10.1016/j.jhazmat.2017.09.009 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.
Study of ciprofloxacin biodegradation by a Thermus sp. isolated from pharmaceutical sludge Lan-jia Pana,b, Jie Lia,b, Chun-xing Lia, Xiao-da Tanga,b, Guang-wei Yua, Yin Wanga,* aCAS
Key Laboratory of Urban Pollutant Conversion, Institute of Urban Environment, Chinese Academy of Sciences, Xiamen 361021, China
bUniversity
*
of Chinese Academy of Sciences, Beijing 100049, China
Corresponding author. E-mail address: [email protected] (Y. Wang) Abbreviations: CIP, ciprofloxacin; OFL, ofloxacin; NOR, norfloxacin; ENR, enrofloxacin; SA, sodium acetate; MMSM,
minimal mineral salt medium; ESI, electrospray interface; UPLC-MS/MS, ultra-performance liquid chromatography tandem mass spectrometry with triple quadrupole mass spectrometer. LB, Luria-Bertani medium. Graphical abstract
Highlights
1
A thermophilic fluoroquinolone-degrading bacterium was isolated from sludge.
The optimal conditions for ciprofloxacin biodegradation are 70 °C and pH 6.5.
Suitable concentrations of sodium acetate can promote the removal of ciprofloxacin.
Several biodegradation metabolites by strain C419 were proposed.
The antibacterial activities of fluoroquinolone metabolites were attenuated.
Abstract：Ciprofloxacin (CIP) is an antibiotic drug frequently detected in manure compost and is difficult to decompose at high temperatures, resulting in a potential threat to the environment. Microbial degradation is an effective and environmentally friendly method to degrade CIP. In this study, a thermophilic bacterium that can degrade CIP was isolated from sludge sampled from an antibiotics pharmaceutical factory. This strain is closely related to Thermus thermophilus based on 16S rRNA gene sequence analysis and is designated C419. The optimal temperature and pH values for CIP degradation are 70 °C and 6.5, respectively, and an appropriate sodium acetate concentration promotes CIP degradation. Seven major biodegradation metabolites were identified by an ultra-performance liquid chromatography tandem mass spectrometry analysis. In addition, strain C419 degraded other fluoroquinolones, including ofloxacin, norfloxacin and enrofloxacin. The supernatant from the C419 culture grown in fluoroquinolone-containing media showed attenuated antibacterial activity. These results indicate that strain C419 might be a new auxiliary bacterial resource for the biodegradation of fluoroquinolone residue in thermal environments.
Keywords：Antibiotic, Ciprofloxacin biodegradation, Thermophilic bacterium, Thermus sp., Fluoroquinolones
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1. Introduction The fluoroquinolones are a series of broad-spectrum synthetic antibiotic agents that are widely used as human and veterinary medicines [1]. Their major mode of action is inhibiting the activity of key enzymes related to DNA replication. As fluoroquinolones are only partially metabolized by animals, they are mainly eliminated as parent compounds and subsequently discharged into manure. However, the residual fluoroquinolones in animal manure can easily enter the terrestrial environment and are readily transported into aquatic environments via leaching and direct runoff, which further spreads pharmaceutical pollution. Several fluoroquinolones, such as ciprofloxacin (CIP), ofloxacin (OFL), norfloxacin (NOR) and enrofloxacin (ENR), are currently available; the most commonly used fluoroquinolone antibiotic is CIP [25]. CIP (Table 1), is a third-generation fluoroquinolone, and its high stability and resistance to degradation results in its frequent detection in the environment [5,7,8]. CIP concentrations in stream water have been determined to range from 0.01 to 0.03 mg L-1 [7], increasing to 0.75 mg kg-1 in agricultural soils [9] and 0.64 to 45.59 mg kg-1 in manure [10]. The effects of CIP on environmental processes and ecosystem services have resulted in increasing concern, as it has been found harmful to soil processes, the adaptation of the microbial communities and microbial catabolic diversities [2,11]. In addition, residual CIP in the environment can serve as a selecting pressure for the development of antibiotic-resistant bacteria. CIP-resistant bacteria and genes are prevalent in various environments [1214]. Many physical-chemical methods, such as advanced oxidation processes [15,16], sorption by special materials [17,18], and photodegradation [19], have been adopted to remove CIP. The biodegradation of CIP has also been reported in the literature, but to our knowledge, only a few 3
bacterium, fungus and microalga species can degrade CIP. The capability of Labrys portucalensis F11 to biodegrade CIP as a single substrate and mixed with other fluoroquinolones was investigated [20]. Several kinds of fungi were studied for their CIP biotransformation potential, and many metabolites were proposed [2123]. Xiong et al. (2016) explored CIP toxicity and its co-metabolic removal by a freshwater microalga, Chlamydomonas mexicana [24]. Moreover, information about CIP degradation by mixed bacterial cultures is limited because its high toxicity and inhibition to microbial activity makes its biodegradation difficult [25]. Liao et al. (2016) investigated the pathways and factors that influence CIP biodegradation by microbial populations enriched from the activated carbon particles (polluted with antibiotics) [26]. A microbial consortium with the ability to degrade fluorinated aromatic compounds was used to biodegrade CIP and other fluoroquinolones [27]. Liu et al. (2013) studied the inhibition and biotransformation of CIP under aerobic conditions by two kinds of poultry litter extracts [28]. Most previous studies mainly focused on CIP biodegradation or transformation at room temperature, while little work has been done at higher temperatures. However, CIP is highly resistant to thermal decomposition since it is difficult to remove CIP by composting [29]. It is therefore urgent to develop microorganisms that can remove CIP at higher temperatures and be further applied to CIP bioremediation. In this study, we reported the isolation of a thermophilic bacterial strain, C419, that can degrade CIP at temperatures from 6580 °C. The optimal conditions for the degradation of CIP and the effects of different concentrations of sodium acetate on bacterial growth and biodegradation were investigated. Major CIP biodegradation metabolites were proposed based on an ultra-performance liquid chromatography tandem mass spectrometry analysis. Moreover, strain C419 was used to degrade other types of fluoroquinolones, and the residual antibacterial activity of the biodegradation metabolite 4
mixture was also explored. To the best of our knowledge, this manuscript is the first report of a thermophilic bacterial strain that degrades fluoroquinolones. 2. Materials and methods 2.1. Chemicals and media Reagent grade CIP hydrochloride and analytical standards (CIP, ofloxacin-OFL, norfloxacin-NOR, enrofloxacin-ENR) were bought from the Aladdin Industrial Corporation (Shanghai, China). Acetonitrile and methanol (HPLC grade) were purchased from Fisher (USA), and yeast extract was obtained from the Sangon Biotech Co. Ltd. (Shanghai, China). All other chemicals, obtained from Sinopharm Chemical Reagent Co. Ltd. (China), were of analytical grade. Minimal mineral salt medium (MMSM) [30] containing 0.013 g of FeSO4·7H2O, 0.013 g of CaCl2·2H2O, 0.018 g of Na2EDTA·2H2O, 0.25 g of MgSO4·7H2O, 7.5 g of Na2HPO4, 5 g of KH2PO4, 5 g of NH4NO3 and 0.6 g of yeast extract were dissolved in 1000 mL of deionized water containing sodium acetate (03 g L-1). The Luria-Bertani medium (LB) consisted of 10 g of tryptone, 5 g of yeast extract and 10 g of NaCl, dissolved in 1000 mL of deionized water. In total, 1.52.0% (w/v) agar was added to prepare solid medium. All media were sterilized at 121 °C for 20 minutes prior to use. 2.2 Isolation and identification of a ciprofloxacin-degrading thermophilic strain The method of microorganism acclimation and enrichment was identical to that described in our previous study [31]. In brief, 5 g of sludge was added in 100 mL of MMSM medium with 1 mg L-1 CIP hydrochloride. The culture was placed on a rotary shaker (100 rpm) at 70 °C for 10 days in the dark, and the supernatant from the enrichment culture was then centrifuged and transferred into fresh sterile MMSM medium containing 5 mg L-1 CIP hydrochloride. The inoculation steps were repeated until the CIP concentration in the medium was 20 mg L-1. The final enriched culture was diluted and spread onto 5
MMSM agar plates containing 20 mg L-1 CIP. Bacterial colonies were streaked for purification and tested for their CIP-degrading capability. One strain, designated C419, that possessed CIP-degrading ability was selected for further investigation. Genomic DNA was extracted using the Bacterial Genome DNA Extraction Kit (Tiangen Biotech Co., Ltd.) according to the manufacturer’s instructions. The following primers were used for PCR amplification of the gene encoding 16S rRNA: 27f (5’-AGA GTT TGA TTC TGG CTC AG-3’) was the forward primer, and 1492r (5’-GGT TAC CTT GTT ACG ACT T-3’) was the reverse primer [32]. The PCR mixtures (20 µL) contained 2 µL of 10×Ex Taq buffer, 1.6 µL of 2.5 mM dNTP Mix, 0.8 µL of each primer, 0.5 µL of DNA template, 0.2 µL of Ex Taq and 14.1 µL of ddH2O. The thermocycling conditions consisted of a denaturation step at 95 °C for 5 min, 24 amplification cycles of 95 °C for 30 s, 55 °C for 30 s and 72 °C for 90 s, and a final polymerization for 10 min. The PCR products were sequenced at MajorBio (Shanghai, China). The sequence was compared against the available DNA sequences in EzTaxon (http://www.ezbiocloud.net/eztaxon) using the IDENTIFY tool. Phylogenesis was analyzed using MEGA version 4.0 software, and a phylogenetic tree was drawn using the neighbor-joining method. 2.3 Preparation of the microbial culture Strain C419 was grown to the exponential growth phase (approximately 15 h) in 50 mL of LB medium at 70 °C and 150 rpm. Bacterial cells were collected after centrifugation at 6000 rpm and room temperature for 5 min. The cell pellets were washed twice with sterilized 0.1 M phosphate buffer (pH 7.0) and resuspended in sterilized 0.9% NaCl solution to a concentration of OD600= 6.0. 2.4 Optimal conditions for ciprofloxacin degradation Temperature and pH values were selected to investigate the effects of environmental factors on the 6
degradation of CIP by strain C419. The microbial suspensions were inoculated (3.0%, v/v) into MMSM liquid medium supplemented with 5 mg L-1 CIP and then incubated at various temperatures (65, 70, 75 and 80 °C) with an initial pH value of 7.0 or various initial pH values (6.0, 6.5, 7.0, 7.5 and 8.0) at the determined optimal temperature. Sterilized water was added instead of strain C419 in control experiments. The concentrations of CIP were detected after culturing for 72 h. All of the experiments were performed in triplicate on a rotary shaker and incubated in the dark to avoid photodegradation. Because of evaporation, sterilized water was added by weighing prior to sampling. 2.5 Effect of sodium acetate (SA) on the growth and biodegradation ability of strain C419 The removal of CIP (5 mg L-1) exposed to sodium acetate, mannitol, lactose, sucrose and glucose was investigated in 100 mL Erlenmeyer flasks containing 50 mL MMSM and inoculated with 3.0% of bacterial suspension to determine the best organic substrate for the co-metabolism of CIP by strain C419. The effect of sodium acetate (SA) on the removal of CIP was selected for further investigation as it caused the highest removal rate (Fig. S1). Different SA concentrations (0, 0.5, 1, 2, and 3 g L-1) were prepared in MMSM media with 5 mg L-1 CIP, and these solutions were inoculated with the bacterial suspension (3.0%). Samples were taken at specific times (0, 12, 24, 36, 48, 72, 96 and 120 h) to assess bacterial growth and the residual concentration of CIP. All the experiments were performed according to the method mentioned in Section 2.4. The kinetics of CIP removal by strain C419 at various SA concentrations were fit to the first-order kinetic model, whose concentration-time relationship is described by the following equation:
lnCt -kt lnC0 where C0 represents the concentration of CIP at time zero, Ct is the concentration of CIP at time t and k 7
is the rate constant (h-1). The half-live (h) can then be expressed as:
t1/ 2 ln 2 / k 2.6 Extraction of biodegradation metabolites The supernatants from C419 culture medium with CIP and the control were collected, subsequently filtered with a 0.22-µm membrane three times to remove impurities and then extracted with three equal volumes of ethyl acetate [33]. The extracts were evaporated to dryness under a gentle stream of N2 in the dark. The concentrated extracts were dissolved in methanol for analysis. 2.7 Degradation of other fluoroquinolones The ability of strain C419 to degrade OFL, NOR and ENR was examined in MMSM containing 5 mg L-1 each compound, with the control lacking inoculation of strain C419. The samples, cultured for 72 h, were collected to examine the residual concentration of the three compounds and the bacterial growth. 2.8 Residual antibacterial activity assay The antibacterial activities of fluoroquinolones and the metabolites were evaluated on Escherichia coli K12 (Gram-negative) using the modified disk diffusion susceptibility test [21]. Briefly, sterile LB medium containing nutrient agar was inoculated with 1 mL of a bacterium suspension (the optical density of the suspension at 600 nm was 1.5) and poured into a plate. When the medium solidified, four Oxford cups (6 mm diameter) were placed on the medium. In total, 200 µL of sample was added to each cup, and the plates were incubated at 37 °C for 20 h. In parallel, 5 mg L-1 medicine solutions were added, and the initial inhibition (without biodegradation) of the four fluoroquinolones was observed. The residual antibacterial activity was evaluated by comparing the inhibition zone sizes of samples and medicine solutions. 8
2.9 Analytical methods Bacterial growth was monitored via measuring the optical density at 600 nm using a UV-1100 ultraviolet spectrophotometer (Mapada, China). Aliquots (5001000 µL) of sample cultures were filtered through a 0.45-µm membrane filter to remove impurities and biomass and were then stored at 4 °C for further analysis. The fluoroquinolone concentration was monitored using a high-performance liquid chromatography instrument (HPLC, Hitachi L-2000, Japan) equipped with an auto sampler. An Extend-C18 column (250 mm × 4.6 mm, 5-Micron 80A) from Agilent (USA) was used to achieve chromatographic separation. The fluoroquinolones (CIP, OFL, NOR and ENR) were detected using 0.02 M trichloroacetic acid, acetonitrile and methanol (74:22:4, v/v/v) as the mobile phase. The detection wavelength was set to 276 nm, and the column temperature was 30 °C. The column was equilibrated for 1020 min prior to injection. Ultra-performance liquid chromatography tandem mass spectrometry with a triple quadrupole mass spectrometer (UPLC-MS/MS, AB Sciex 6500, USA) was used to analyze the biodegradation metabolites of CIP. The separation was performed on a Shimadzu (Japan) C18 column (20 mm i.d. × 75 mm, 1.6 µm) at a temperature of 30 °C. The mobile phase consisted of (A) water with 0.1% formic acid and (B) acetonitrile with 0.1% formic acid and flowed at 0.3 mL min-1; the injection volume was 10 µL. The elution program comprised gradient elution from 10 to 100% (B) within 20 min and subsequent isocratic elution with 100% (B) for 5 min, followed by 10% (B) for 5 min. The mass spectrometer was equipped with an electrospray interface (ESI); the ESI source operated in positive ionization mode at 350 °C, and the ionspray voltage was set as 4.5 kV. Full scans from m/z 50700 were acquired for identification of the metabolites. Data were analyzed with software Analyst 1.6.3. 9
The identification of the metabolites was confirmed by consulting previous literature and the EAWAG-BBD Pathway Prediction System (http://eawag-bbd.ethz.ch/predict/) as well as product-ion analysis under low-mass conditions. 2.10 Statistical analysis Results corresponding to the removal rate and bacterial growth were analyzed using one-way analysis of variance (ANOVA) applying the LSD multiple comparison test using IBM SPSS Statistics software (version 20.0). Values were considered significant when p < 0.05. 3. Results and discussion 3.1 Isolation and identification of the ciprofloxacin-degrading strain After acclimation and enrichment with CIP, a bacterial strain, C419, was obtained with the ability to grow on solid MMSM medium supplemented with 20 mg L-1 of CIP. Analysis of the partial 16S rRNA gene sequence of strain C419 (1265 bp, GenBank accession number: KY784655) indicated an affiliation with the genus Thermus and showed 99.84% similarity to Thermus thermophilus HB8 (GenBank accession number AP008226, Bacteria; Deinococcus-Thermus; Deinococci; Thermales; Thermaceae; Thermus). A phylogenetic tree was constructed using the neighbor-joining method based on the 16S rRNA gene sequences of strain C419 and related strains (all the related strains are members of Thermaceae) (Fig. 1). Thermus thermophilus, as an extremely thermophilic bacterium, was originally isolated from a naturally high-temperature environment. Several of its enzymes were crystallized, and their structures were analyzed by X-ray crystallography [34]. Thermophilic bacteria are often used to produce thermostable enzymes for processing starch, organic synthesis, diagnostics, waste treatment, pulp and paper manufacturing and animal feed utilization [35]. This manuscript is the first report of a 10
thermophilic Thermus strain biodegrading fluoroquinolone. Several microorganisms possess the ability to biodegrade CIP, and some of them work by laccase [21,22,36]. Laccases participate in the degradation of polymers and the ring cleavage of aromatic compounds [37] and uses molecular oxygen to oxidize substrate during the degradation process. The genome databases of aerobic thermophilic bacteria were searched for laccases, and an open reading frame in Thermus thermophilus HB27, designated TTC1370, was identified [35]. Miyazaki (2005) also detected copper-inducible laccase activity in Thermus thermophilus HB27; this enzyme was the most thermophilic laccase reported so far [37]. In this study, strain C419 is closely related to Thermus thermophilus HB8 based on its 16S rRNA gene sequence. Accordingly, the CIP degradation by strain C419 might be attributed to a laccase. Further studies are needed to shed additional light on the molecular biology and enzymology of this bacterium. 3.2 Optimization of ciprofloxacin-degrading conditions Temperature and the initial pH value of the medium were selected as the key environmental factors for the degradation of CIP by strain C419. Temperature has a significant impact on bacterial growth and some important enzymes during biodegradation processes [38]. Thermus thermophilus is an extremely thermophilic bacterium, with an optimal growth temperature between 65 and 72 °C and a maximum temperature of 85 °C [39]. In this study, bacterial growth decreased with increasing temperature and significantly (p<0.05) declined at 80 °C (Fig. 2a). CIP degradation occurred from 65 to 80 °C, but the optimal degradation temperature was 70 °C (47%). The initial pH value of the medium was also a key factor governing compound transport to microbes and changing the solubility of pollutants [40]. As shown in Fig. 2b, there was no significant difference in bacterial growth at pH 6.5, 7.0 and 7.5 (p>0.05), and the maximum growth was observed at a pH value of 8.0. Marteinsson (1999) 11
isolated Thermus thermophilus Gy1211 from a deep-sea hydrothermal vent. It grew optimally at a pH value of 8.0, as did the strain in this study [41]. Maximum CIP degradation (55%) was achieved at a pH value of 6.5, but strain C419 also exhibited high degradation efficiency in neutral and alkaline conditions. The degradation of CIP by C419 occurred at a wide range of pH values, suggesting that this strain could be a good candidate for applications. 3.3 Effect of sodium acetate (SA) on the growth and biodegradation ability of strain C419 Co-substrate plays a dynamic role in the degradation of priority pollutants. Previous studies showed that antibiotics can be degraded faster in the presence of substrates that are easier to utilize, such as glucose and sodium acetate [24,31]. In this study, we examined the effects of different SA concentrations on bacterial growth and CIP biodegradation activity. As shown in Fig. 3, the removal of CIP and growth of C419 were significantly (p<0.05) inhibited by SA at a concentration of 3 g L-1. In the SA concentration range from 1 to 2 g L-1, the growth of C419 was inhibited during the initial 48 h, and CIP was slowly removed (Fig. 3b, c). After 48 h, the bacterium grew rapidly and demonstrated a high ability (more than 55%) to degrade CIP. A small amount of SA (0.5 g L-1) significantly (p<0.05) promoted CIP removal and bacterial growth compared to other treatments at earlier stages (036 h), but then its effect weakened. It is well known that suitable carbon and energy provision stimulates bacterial growth and co-metabolism. Carbon source concentrations that are too high or too low affect the ratio of carbon to nitrogen (C/N) and induce weak biodegradation [38]. In this study, 0.5 g L-1 SA was considered the best concentration for CIP degradation, although 12 g L-1 of SA resulted in relatively high bacterial growth and CIP removal rates after a 120-h incubation. The microorganism might take a long time to adapt to high concentrations (>1 g L-1) of SA. The removal of CIP (5 mg L-1) at different SA concentrations can be fitted using the first-order 12
kinetic model. As shown in Table 2, the removal rate constants (k) and half-lives (T1/2) of CIP (5 mg L-1) ranged from 0.0032 to 0.0157 h-1 and from 44.3 to 215.2 h, respectively. The R2 values ranged from 0.902 to 0.975, demonstrating that first-order kinetic model fit the data well. SA alone (without microorganisms) decreased the CIP concentration only slightly, suggesting that most of the CIP degradation was caused by the microorganism. SA serves as an additional carbon source to promote bacterial biomass production, and on the other hand, it can also act as an electron donor for the co-metabolism of the non-growth substrate [42]. In the present study, use of appropriate SA concentrations boosted bacterial growth and CIP degradation. 3.4 Identification of biodegradation metabolites Concentrated extracts were taken from the cultures to identify the potential biodegradation metabolites of CIP. The structures of CIP metabolites were proposed on the basis of previous papers and information from EAWAG-BBD Pathway Prediction System. Seven major metabolites of CIP were found in UPLC-MS spectrograms. All these metabolites were derived from the C419 culture that had grown with CIP for 120 h, and the blank control (without microorganism) did not show any peaks corresponding to these metabolites. The protonated molecules [M+H]+ of the metabolites are shown in Table 3. For further structure confirmation, the protonated molecules of the major metabolites were selected for product-ion analysis. The calculated formulas, proposed chemical structures and the product ions are also reported in Table 3. Metabolite M1, with a protonated molecule signal at m/z 360 and major product ions at m/z 342 (M+HH2O), 243 and 230, was identified as N-formylciprofloxacin. Metabolite M2, with a protonated molecule signal at m/z 348 and major product ions at m/z 330 (M+HH2O) and 217, was identified as desethylene-N-acetylciprofloxacin. The two metabolites were reported for the biotransformation of CIP 13
by the fungus Pestalotiopsis guepini [23]. Metabolite M3, with a protonated molecule signal at m/z 334 and major product ions at m/z 316 (M+HH2O) and 217 (M+HH2OC3H5NOCO), was identified as desethylene-N-formylciprofloxacin, which was first proposed by Čvančarová et al. (2015) to be one of the metabolite products by fungus tests [21]. Metabolite M4 ([M+H]+ at m/z 306) produced MS/MS product-ion signals at m/z 288 (M+HC2H4), 268, and 218, which agreed well with expectations for the metabolite desethylene-N-ciprofloxacin. Metabolite M5, with a protonated molecule signal at m/z 263 and major product ions at m/z 245 (M+HH2O), 216 and 204, was proposed to be 7-amino-1-cyclopropyl-6fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid. M4 and M5 are two kinds of common metabolites, and both of them have already been identified in the literature [21,22,27,43]. Metabolite M4 was also found when Labrys portucalensis F11 was cultured with CIP [20]. Metabolites M6 and M7, which were detected in this work, have not been previously described in the literature. Previous studies of the biotransformation of fluoroquinolones usually focused on the piperazine ring, which is preferably attacked by enzymes [44,45], resulting in metabolites M1 to M5. Metabolite M6, with a protonated molecule signal at m/z 223 and major product ions at m/z 207 (M+HNH2), 190 (M+HCHO2) and 178 (M+HNH2OH), may be 7-amino-6-fluoro-4-oxo-1,4dihydroquinoline-3-carboxylic acid. Biodegradation pathway analysis of CIP using the EAWAG-BBD Pathway Prediction System displays several routes for CIP transformation; it shows that metabolite M6 can derive from M5 by oxidative removal of the cyclopropyl group from the piperazine ring. M7, with a protonated molecule signal at m/z 190 and major product ions at m/z 172 (M+HH2O), 146 (M+HCHO2) and 130 (M+HCHO2NH2), matches the description of 4-oxo-1,4-dihydroquinoline-3 -carboxylic acid and might be obtained from the loss of a –F atom and a –NH2 group from the benzene 14
ring of M6. The information obtained from the UPLC-MS/MS was not sufficient to completely specify the chemical structures of these metabolites. However, nuclear magnetic resonance and/or the use of authentic standards will be adopted in future work to confirm the structures of CIP transformation metabolites. 3.5 Degradation of other fluoroquinolones Strain C419 possesses the ability to degrade OFL, NOR and ENR. Strain C419 removed ENR the most efficiently of these three fluoroquinolones. Nearly 74% of the ENR in the medium was removed after a 72-h incubation, while the efficiency of removing OFL and NOR were 70 and 63%, respectively. The ability of C419 to degrade different types of fluoroquinolones might be explained by the similar chemical structures of fluoroquinolones, but the various degradation abilities also reflect their different toxicity to microorganisms. In our previous work [31], a thermophilic sulfamethazine-degrading isolate was also able to degrade three other types of sulfonamides. Amorim et al. (2014) used an Alphaproteobacteria strain to degrade CIP, OFL, and NOR as single and mixed substrates, and all the tested drugs were degraded [20]. Müller et al. (2013) reported that the enzymes that require primary biodegradation are class-specific rather than compound-specific [46]. The adapted microbial culture was therefore able to biodegrade a set of structurally similar drugs. 3.6 Residual antibacterial activity assay The Gram-negative bacterium Escherichia coli K12 was selected as the test organism to determine the antibacterial activity of fluoroquinolones and biodegraded fluoroquinolones. The relative inhibition rate was evaluated by comparing the inhibition zone sizes resulting from the biodegraded fluoroquinolone and initial fluoroquinolone solution. As shown in Fig. 5, the inhibition by the four 15
biodegraded fluoroquinolones decreased by 2040% compared with that of the parent compounds (5 mg L-1). However, the antibacterial activities were still high, which may be due to incomplete transformation of the four fluoroquinolones by strain C419. Parent compounds that were not biodegraded may also contribute to the high antibacterial activities. Given the high activity of transformed fluoroquinolones, it is necessary to evaluate the toxicity of the metabolites. Čvančarová et al. (2015) measured the activity of biodegraded fluoroquinolones using various Gram-positive and Gram-negative bacteria isolated from the environment, and all the tested organisms were highly inhibited by the biodegraded fluoroquinolones, which indicated that the metabolites produced by the fungi still possessed high antibacterial activity [21]. Wetzstein et al. (1999) investigated the residual activity of transformed CIP on Escherichia coli ATCC 8739 and proved that the antibacterial activity of transformed CIP decreased [22]. 4. Conclusions A fluoroquinolone-degrading Thermus sp. strain, C419, was isolated from the sludge of an antibiotics pharmaceutical factory and used to biodegrade CIP at high temperature. The results showed that the best degradation conditions of CIP were 70 °C and pH 6.5. Addition of sodium acetate can obviously promote bacterial growth and the degradation of CIP, with more than 57% of the initial CIP in the media being removed after 5 days of incubation. Major biodegradation metabolites were identified by UPLC-MS/MS, and two new metabolites of CIP were proposed. The strain C419 possesses the ability to degrade three other types of fluoroquinolones and reduce antibacterial activity. Its outstanding thermotolerance and high CIP-degrading ability make the strain C419 a good candidate for remediation of fluoroquinolone-contaminated manure compost and materials found in other thermal environments. Acknowledgements 16
This work was supported by the China-Japan Research Cooperative Program [Grant No. 2016YFE0118000]; the National Natural Science Foundation of China [Grant No. 41373092]; the Key Project of Young Talent of IUE, CAS [Grant No. IUEZD201402]; the Scientific and Technological Major Special Project of Tianjin City [Grant No. 16YFXTSF00420]; the Industry Leading Key Projects of Fujian Province [Grant No. 2015H0044] and the Key Project of Young Talents Frontier of IUE, CAS [Grant No. IUEQN201501].
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carbon nanotubes, J. Hazard. Mater. 310 (2016) 235245. [19] S. Babić, M. Periša, I. Škorić, Photolytic degradation of norfloxacin, enrofloxacin and ciprofloxacin in various aqueous media, Chemosphere, 91 (2013) 16351642. [20] C.L. Amorim, I.S. Moreira, A.S. Maia, M.E. Tiritan, P.M. Castro, Biodegradation of ofloxacin, norfloxacin, and ciprofloxacin as single and mixed substrates by Labrys portucalensis F11, Appl. Microbiol. Biot. 98 (2014) 31813190. [21] M. Čvančarová, M. Moeder, A. Filipová, T. Cajthaml, Biotransformation of fluoroquinolone antibiotics by ligninolytic fungi-Metabolites, enzymes and residual antibacterial activity, Chemosphere, 136 (2015) 311320. [22] H.G. Wetzstein, M. Stadler, H.V. Tichy, A. Dalhoff, W. Karl, Degradation of ciprofloxacin by basidiomycetes and identification of
metabolites generated by the brown rot fungus Gloeophyllum
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23 (2016) 79117918. [27] A.S. Maia, A.R. Ribeiro, C.L. Amorim, J.C. Barreiro, Q.B. Cass, P.M. Castro, M.E. Tiritan, Degradation of fluoroquinolone antibiotics and identification of metabolites/transformation products by liquid chromatography-tandem mass spectrometry, J. Chromatogr. A, 1333 (2014) 8798. [28] Z.G. Liu, P.Z. Sun, S.G. Pavlostathis, X.F. Zhou, Y.L. Zhang, Adsorption, inhibition, and biotransformation of ciprofloxacin under aerobic conditions, Bioresour. Technol.144 (2013) 644651. [29] H.B. Zhang, Y.M. Luo, L.H. Wu, Y.J. Huang, P. Christie, Residues and potential ecological risks of veterinary antibiotics in manures and composts associated with protected vegetable farming, Environ. Sci. Pollut. R. 22 (2015) 59085918. [30] S. Larcher, V. Yargeau, Biodegradation of sulfamethoxazole by individual and mixed bacteria, Appl. Microbiol. Biot. 91 (2011) 211218. [31] L.J. Pan, X.D. Tang, C.X. Li, G.W. Yu, Y. Wang, Biodegradation of sulfamethazine by an isolated thermophile-Geobacillus sp. S-07, World J. Microb. Biot. 33 (2017) 85. [32] C.L. Guo, H.W. Zhou, Y.S. Wong, N.F. Tam, Isolation of PAH-degrading bacteria from mangrove sediments and their biodegradation potential, Mar. Pollut. Bull. 51 (2005) 10541061. [33] A.J. Williams, J. Deck, J.P. Freeman, M.P. Chiarelli, M.D. Adjei, T.M. Heinze, J.B. Sutherland, Biotransformation of flumequine by the fungus Cunninghamella elegans, Chemosphere, 67 (2007) 240243. [34] C. Vieille, G.J. Zeikus, Hyperthermophilic enzymes: sources, uses, and molecular mechanisms for thermostability, Microbiol. Mol. Biol. R. 65 (2001) 143. [35] A. Henne, H. Brüggemann, C. Raasch, A. Wiezer, T. Hartsch, H. Liesegang, A. Johann, T. Lienard, O. Gohl, R. Martinez-Arias, C. Jacobi, V. Starkuviene, S. Schlenczeck, S. Dencker, R. Huber, 21
H.P. Klenk, W. Kramer, R. Merkl, G. Gottschalk, H.J. Fritz, The genome sequence of the extreme thermophile Thermus thermophilus, Nat. Biotechnol. 22 (2004) 547553. [36] A. Blánquez, F. Guillén, J. Rodríguez, M.E. Arias, M. Hernández, The degradation of two fluoroquinolone based antimicrobials by SilA, an alkaline laccase from Streptomyces ipomoeae, World J. Microb. Biot. 32 (2016) 52. [37] K. Miyazaki, A hyperthermophilic laccase from Thermus thermophilus HB27, Extremophiles 9 (2005) 415-425. [38] X.X. Peng, X.D. Qu, W.S. Luo, X.S. Jia, Co-metabolic degradation of tetrabromobisphenol A by novel strains of Pseudomonas sp. and Streptococcus sp, Bioresour. Technol. 169 (2014) 271276. [39] T. Oshima, K. Imahori, Description of Thermus thermophilus (Yoshida and Oshima) comb. nov., a nonsporulating thermophilic bacterium from a Japanese thermal spa, Int. J. Syst. Evol. Microbiol. 24 (1974) 102112. [40] V. Patel, J. Patel, D. Madamwar, Biodegradation of phenanthrene in bioaugmented microcosm by consortium ASP developed from coastal sediment of Alang-Sosiya ship breaking yard, Mar. Pollut. Bull. 74 (2013) 199207. [41] V.T. Marteinsson, Isolation and characterization of Thermus thermophilus Gy1211 from a deep-sea hydrothermal vent, Extremophiles 3 (1999) 247251. [42] W. Luo, Y.H. Zhao, H.T. Ding, X.Y. Lin, H.B. Zheng, Co-metabolic degradation of bensulfuron-methyl in laboratory conditions, J. Hazard. Mater. 158 (2008) 208214. [43] A. Prieto, M. Möder, R. Rodil, L. Adrian, E. Marco-Urrea, Degradation of the antibiotics norfloxacin and ciprofloxacin by a white-rot fungus and identification of degradation products, Bioresour. Technol. 102 (2011) 1098710995. 22
[44] M.D. Adjei, T.M. Heinze, J. Deck, J.P. Freeman, A.J. Williams, J.B. Sutherland, Transformation of the antibacterial agent norfloxacin by environmental mycobacteria, Appl. Environ. Microbiol. 72 (2006) 57905793. [45] H.G. Wetzstein, J. Schneider, W. Karl, Metabolite proving fungal cleavage of the aromatic core part of a fluoroquinolone antibiotic, AMB Express, 2 (2012) 3. [46] E. Müller, W. Schüssler, H. Horn, H. Lemmer, Aerobic biodegradation of the sulfonamide antibiotic sulfamethoxazole by activated sludge applied as co-substrate and sole carbon and nitrogen source, Chemosphere, 92 (2013) 969978.
23
Figure captions： Fig. 1 Phylogenetic tree constructed using the neighbor-joining method based on the 16S rRNA gene sequences of strain C419 and associated strains. Escherichia coli was used as an out-group. Bootstrap values (based on 1000 replications) are shown at branch points. Bar, 0.05 substitutions per nucleotide position. Fig. 2 Effect of temperature (a) and pH (b) on the growth of strain C419 and the degradation of CIP. Error bars represent standard deviations (n= 3). Data with different capital letters indicate significant differences (p < 0.05) of bacterial growth among treatments. Data with different lowercase letters indicate significant differences (p < 0.05) of removal rate among treatments. Fig. 3 Effect of sodium acetate (SA) on CIP removal kinetics (a), CIP removal (b) and growth of strain C419 (c) in a 120-h incubation. Error bars represent standard deviations (n= 3). Data with different letters indicate significant differences (p < 0.05) among treatments. Fig. 4 Degradation of three other fluoroquinolones by strain C419 (initial concentration of the fluoroquinolones: 5 mg L-1, 70 °C, pH= 6.5, 150 rpm). Error bars represent standard deviations (n= 3). Data with different letters indicate significant differences (p < 0.05) within a treatment. Fig. 5 The relative inhibition of four fluoroquinolones in MMSM liquid media after 72 h of incubation with strain C419 (the relative inhibition rate was evaluated by comparing the inhibition zone sizes resulting from the biodegraded fluoroquinolone and initial fluoroquinolone solution). Error bars represent standard deviations (n= 3).
24
Fig. 1
25
Fig. 2
26
Fig. 3
27
Fig. 4
28
Fig. 5
29
Table 1 Physico-chemical properties of ciprofloxacin. Properties of Ciprofloxacin Molecular Formula
C17H18FN3O3
CAS Number
85721-33-1
Molecular Weight
331.35 g mol-1
a
log Kow
0.4
a
pKa
3.01 ± 0.30
Chemical Structure
6.14 ± 0.13 8.70 ± 0.09 10.58 ± 0.30 Therapeutic Class a
Fluoroquinolone
The log Kow and pKa values are taken from reference [6].
30
Table 2 Kinetic parameters and removal of CIP (5 mg L-1) at different sodium acetate concentrations after a 120-h incubation. SA Concentration (g L-1)
k (h-1)
T1/2 (h)
R2
Removal (%)
0
0.0120
57.9
0.913
51.45±0.56
0.5
0.0157
44.3
0.910
57.12±0.94
1
0.0103
67.0
0.975
56.50±1.21
2
0.0072
95.7
0.902
59.23±0.46
3
0.0032
215.2
0.951
16.73±1.00
k - kinetic removal rate constant (h-1). T1/2 - degradation half-life (h). R2 - correlation coefficient.
31
Table 3 Structures and mass spectral data for ciprofloxacin and postulated biodegradation metabolites, as determined from UPLC-MS/MS. [M+H]+
Product ions
Calculated
(m/z)
(m/z)
Formula
CIP
332
314, 270, 245
C17H19FN3O3
M1
360
342, 243, 230
C18H19FN3O4
[23]
M2
348
330, 217
C17H19FN3O4
[23]
M3
334
316, 217
C19H21FN3O4
[21]
M4
306
288, 268, 218
C15H17FN3O3
[2022, 27, 43]
M5
263
245, 204, 216
C13H12FN2O3
[21, 22, 27, 43]
M6*
223
207, 190, 178
C10H8FN2O3
This study
M7*
190
172, 146, 130
C10H8NO3
This study
Compound
32
Proposed structure
References
S0304-3894(17)30690-8 http://dx.doi.org/10.1016/j.jhazmat.2017.09.009 HAZMAT 18855
To appear in:
Journal of Hazardous Materials
Received date: Revised date: Accepted date:
15-6-2017 4-9-2017 6-9-2017
Please cite this article as: Lan-jia Pan, Jie Li, Chun-xing Li, Xiao-da Tang, Guang-wei Yu, Yin Wang, Study of ciprofloxacin biodegradation by a Thermus sp.isolated from pharmaceutical sludge, Journal of Hazardous Materialshttp://dx.doi.org/10.1016/j.jhazmat.2017.09.009 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.
Study of ciprofloxacin biodegradation by a Thermus sp. isolated from pharmaceutical sludge Lan-jia Pana,b, Jie Lia,b, Chun-xing Lia, Xiao-da Tanga,b, Guang-wei Yua, Yin Wanga,* aCAS
Key Laboratory of Urban Pollutant Conversion, Institute of Urban Environment, Chinese Academy of Sciences, Xiamen 361021, China
bUniversity
*
of Chinese Academy of Sciences, Beijing 100049, China
Corresponding author. E-mail address: [email protected] (Y. Wang) Abbreviations: CIP, ciprofloxacin; OFL, ofloxacin; NOR, norfloxacin; ENR, enrofloxacin; SA, sodium acetate; MMSM,
minimal mineral salt medium; ESI, electrospray interface; UPLC-MS/MS, ultra-performance liquid chromatography tandem mass spectrometry with triple quadrupole mass spectrometer. LB, Luria-Bertani medium. Graphical abstract
Highlights
1
A thermophilic fluoroquinolone-degrading bacterium was isolated from sludge.
The optimal conditions for ciprofloxacin biodegradation are 70 °C and pH 6.5.
Suitable concentrations of sodium acetate can promote the removal of ciprofloxacin.
Several biodegradation metabolites by strain C419 were proposed.
The antibacterial activities of fluoroquinolone metabolites were attenuated.
Abstract：Ciprofloxacin (CIP) is an antibiotic drug frequently detected in manure compost and is difficult to decompose at high temperatures, resulting in a potential threat to the environment. Microbial degradation is an effective and environmentally friendly method to degrade CIP. In this study, a thermophilic bacterium that can degrade CIP was isolated from sludge sampled from an antibiotics pharmaceutical factory. This strain is closely related to Thermus thermophilus based on 16S rRNA gene sequence analysis and is designated C419. The optimal temperature and pH values for CIP degradation are 70 °C and 6.5, respectively, and an appropriate sodium acetate concentration promotes CIP degradation. Seven major biodegradation metabolites were identified by an ultra-performance liquid chromatography tandem mass spectrometry analysis. In addition, strain C419 degraded other fluoroquinolones, including ofloxacin, norfloxacin and enrofloxacin. The supernatant from the C419 culture grown in fluoroquinolone-containing media showed attenuated antibacterial activity. These results indicate that strain C419 might be a new auxiliary bacterial resource for the biodegradation of fluoroquinolone residue in thermal environments.
Keywords：Antibiotic, Ciprofloxacin biodegradation, Thermophilic bacterium, Thermus sp., Fluoroquinolones
2
1. Introduction The fluoroquinolones are a series of broad-spectrum synthetic antibiotic agents that are widely used as human and veterinary medicines [1]. Their major mode of action is inhibiting the activity of key enzymes related to DNA replication. As fluoroquinolones are only partially metabolized by animals, they are mainly eliminated as parent compounds and subsequently discharged into manure. However, the residual fluoroquinolones in animal manure can easily enter the terrestrial environment and are readily transported into aquatic environments via leaching and direct runoff, which further spreads pharmaceutical pollution. Several fluoroquinolones, such as ciprofloxacin (CIP), ofloxacin (OFL), norfloxacin (NOR) and enrofloxacin (ENR), are currently available; the most commonly used fluoroquinolone antibiotic is CIP [25]. CIP (Table 1), is a third-generation fluoroquinolone, and its high stability and resistance to degradation results in its frequent detection in the environment [5,7,8]. CIP concentrations in stream water have been determined to range from 0.01 to 0.03 mg L-1 [7], increasing to 0.75 mg kg-1 in agricultural soils [9] and 0.64 to 45.59 mg kg-1 in manure [10]. The effects of CIP on environmental processes and ecosystem services have resulted in increasing concern, as it has been found harmful to soil processes, the adaptation of the microbial communities and microbial catabolic diversities [2,11]. In addition, residual CIP in the environment can serve as a selecting pressure for the development of antibiotic-resistant bacteria. CIP-resistant bacteria and genes are prevalent in various environments [1214]. Many physical-chemical methods, such as advanced oxidation processes [15,16], sorption by special materials [17,18], and photodegradation [19], have been adopted to remove CIP. The biodegradation of CIP has also been reported in the literature, but to our knowledge, only a few 3
bacterium, fungus and microalga species can degrade CIP. The capability of Labrys portucalensis F11 to biodegrade CIP as a single substrate and mixed with other fluoroquinolones was investigated [20]. Several kinds of fungi were studied for their CIP biotransformation potential, and many metabolites were proposed [2123]. Xiong et al. (2016) explored CIP toxicity and its co-metabolic removal by a freshwater microalga, Chlamydomonas mexicana [24]. Moreover, information about CIP degradation by mixed bacterial cultures is limited because its high toxicity and inhibition to microbial activity makes its biodegradation difficult [25]. Liao et al. (2016) investigated the pathways and factors that influence CIP biodegradation by microbial populations enriched from the activated carbon particles (polluted with antibiotics) [26]. A microbial consortium with the ability to degrade fluorinated aromatic compounds was used to biodegrade CIP and other fluoroquinolones [27]. Liu et al. (2013) studied the inhibition and biotransformation of CIP under aerobic conditions by two kinds of poultry litter extracts [28]. Most previous studies mainly focused on CIP biodegradation or transformation at room temperature, while little work has been done at higher temperatures. However, CIP is highly resistant to thermal decomposition since it is difficult to remove CIP by composting [29]. It is therefore urgent to develop microorganisms that can remove CIP at higher temperatures and be further applied to CIP bioremediation. In this study, we reported the isolation of a thermophilic bacterial strain, C419, that can degrade CIP at temperatures from 6580 °C. The optimal conditions for the degradation of CIP and the effects of different concentrations of sodium acetate on bacterial growth and biodegradation were investigated. Major CIP biodegradation metabolites were proposed based on an ultra-performance liquid chromatography tandem mass spectrometry analysis. Moreover, strain C419 was used to degrade other types of fluoroquinolones, and the residual antibacterial activity of the biodegradation metabolite 4
mixture was also explored. To the best of our knowledge, this manuscript is the first report of a thermophilic bacterial strain that degrades fluoroquinolones. 2. Materials and methods 2.1. Chemicals and media Reagent grade CIP hydrochloride and analytical standards (CIP, ofloxacin-OFL, norfloxacin-NOR, enrofloxacin-ENR) were bought from the Aladdin Industrial Corporation (Shanghai, China). Acetonitrile and methanol (HPLC grade) were purchased from Fisher (USA), and yeast extract was obtained from the Sangon Biotech Co. Ltd. (Shanghai, China). All other chemicals, obtained from Sinopharm Chemical Reagent Co. Ltd. (China), were of analytical grade. Minimal mineral salt medium (MMSM) [30] containing 0.013 g of FeSO4·7H2O, 0.013 g of CaCl2·2H2O, 0.018 g of Na2EDTA·2H2O, 0.25 g of MgSO4·7H2O, 7.5 g of Na2HPO4, 5 g of KH2PO4, 5 g of NH4NO3 and 0.6 g of yeast extract were dissolved in 1000 mL of deionized water containing sodium acetate (03 g L-1). The Luria-Bertani medium (LB) consisted of 10 g of tryptone, 5 g of yeast extract and 10 g of NaCl, dissolved in 1000 mL of deionized water. In total, 1.52.0% (w/v) agar was added to prepare solid medium. All media were sterilized at 121 °C for 20 minutes prior to use. 2.2 Isolation and identification of a ciprofloxacin-degrading thermophilic strain The method of microorganism acclimation and enrichment was identical to that described in our previous study [31]. In brief, 5 g of sludge was added in 100 mL of MMSM medium with 1 mg L-1 CIP hydrochloride. The culture was placed on a rotary shaker (100 rpm) at 70 °C for 10 days in the dark, and the supernatant from the enrichment culture was then centrifuged and transferred into fresh sterile MMSM medium containing 5 mg L-1 CIP hydrochloride. The inoculation steps were repeated until the CIP concentration in the medium was 20 mg L-1. The final enriched culture was diluted and spread onto 5
MMSM agar plates containing 20 mg L-1 CIP. Bacterial colonies were streaked for purification and tested for their CIP-degrading capability. One strain, designated C419, that possessed CIP-degrading ability was selected for further investigation. Genomic DNA was extracted using the Bacterial Genome DNA Extraction Kit (Tiangen Biotech Co., Ltd.) according to the manufacturer’s instructions. The following primers were used for PCR amplification of the gene encoding 16S rRNA: 27f (5’-AGA GTT TGA TTC TGG CTC AG-3’) was the forward primer, and 1492r (5’-GGT TAC CTT GTT ACG ACT T-3’) was the reverse primer [32]. The PCR mixtures (20 µL) contained 2 µL of 10×Ex Taq buffer, 1.6 µL of 2.5 mM dNTP Mix, 0.8 µL of each primer, 0.5 µL of DNA template, 0.2 µL of Ex Taq and 14.1 µL of ddH2O. The thermocycling conditions consisted of a denaturation step at 95 °C for 5 min, 24 amplification cycles of 95 °C for 30 s, 55 °C for 30 s and 72 °C for 90 s, and a final polymerization for 10 min. The PCR products were sequenced at MajorBio (Shanghai, China). The sequence was compared against the available DNA sequences in EzTaxon (http://www.ezbiocloud.net/eztaxon) using the IDENTIFY tool. Phylogenesis was analyzed using MEGA version 4.0 software, and a phylogenetic tree was drawn using the neighbor-joining method. 2.3 Preparation of the microbial culture Strain C419 was grown to the exponential growth phase (approximately 15 h) in 50 mL of LB medium at 70 °C and 150 rpm. Bacterial cells were collected after centrifugation at 6000 rpm and room temperature for 5 min. The cell pellets were washed twice with sterilized 0.1 M phosphate buffer (pH 7.0) and resuspended in sterilized 0.9% NaCl solution to a concentration of OD600= 6.0. 2.4 Optimal conditions for ciprofloxacin degradation Temperature and pH values were selected to investigate the effects of environmental factors on the 6
degradation of CIP by strain C419. The microbial suspensions were inoculated (3.0%, v/v) into MMSM liquid medium supplemented with 5 mg L-1 CIP and then incubated at various temperatures (65, 70, 75 and 80 °C) with an initial pH value of 7.0 or various initial pH values (6.0, 6.5, 7.0, 7.5 and 8.0) at the determined optimal temperature. Sterilized water was added instead of strain C419 in control experiments. The concentrations of CIP were detected after culturing for 72 h. All of the experiments were performed in triplicate on a rotary shaker and incubated in the dark to avoid photodegradation. Because of evaporation, sterilized water was added by weighing prior to sampling. 2.5 Effect of sodium acetate (SA) on the growth and biodegradation ability of strain C419 The removal of CIP (5 mg L-1) exposed to sodium acetate, mannitol, lactose, sucrose and glucose was investigated in 100 mL Erlenmeyer flasks containing 50 mL MMSM and inoculated with 3.0% of bacterial suspension to determine the best organic substrate for the co-metabolism of CIP by strain C419. The effect of sodium acetate (SA) on the removal of CIP was selected for further investigation as it caused the highest removal rate (Fig. S1). Different SA concentrations (0, 0.5, 1, 2, and 3 g L-1) were prepared in MMSM media with 5 mg L-1 CIP, and these solutions were inoculated with the bacterial suspension (3.0%). Samples were taken at specific times (0, 12, 24, 36, 48, 72, 96 and 120 h) to assess bacterial growth and the residual concentration of CIP. All the experiments were performed according to the method mentioned in Section 2.4. The kinetics of CIP removal by strain C419 at various SA concentrations were fit to the first-order kinetic model, whose concentration-time relationship is described by the following equation:
lnCt -kt lnC0 where C0 represents the concentration of CIP at time zero, Ct is the concentration of CIP at time t and k 7
is the rate constant (h-1). The half-live (h) can then be expressed as:
t1/ 2 ln 2 / k 2.6 Extraction of biodegradation metabolites The supernatants from C419 culture medium with CIP and the control were collected, subsequently filtered with a 0.22-µm membrane three times to remove impurities and then extracted with three equal volumes of ethyl acetate [33]. The extracts were evaporated to dryness under a gentle stream of N2 in the dark. The concentrated extracts were dissolved in methanol for analysis. 2.7 Degradation of other fluoroquinolones The ability of strain C419 to degrade OFL, NOR and ENR was examined in MMSM containing 5 mg L-1 each compound, with the control lacking inoculation of strain C419. The samples, cultured for 72 h, were collected to examine the residual concentration of the three compounds and the bacterial growth. 2.8 Residual antibacterial activity assay The antibacterial activities of fluoroquinolones and the metabolites were evaluated on Escherichia coli K12 (Gram-negative) using the modified disk diffusion susceptibility test [21]. Briefly, sterile LB medium containing nutrient agar was inoculated with 1 mL of a bacterium suspension (the optical density of the suspension at 600 nm was 1.5) and poured into a plate. When the medium solidified, four Oxford cups (6 mm diameter) were placed on the medium. In total, 200 µL of sample was added to each cup, and the plates were incubated at 37 °C for 20 h. In parallel, 5 mg L-1 medicine solutions were added, and the initial inhibition (without biodegradation) of the four fluoroquinolones was observed. The residual antibacterial activity was evaluated by comparing the inhibition zone sizes of samples and medicine solutions. 8
2.9 Analytical methods Bacterial growth was monitored via measuring the optical density at 600 nm using a UV-1100 ultraviolet spectrophotometer (Mapada, China). Aliquots (5001000 µL) of sample cultures were filtered through a 0.45-µm membrane filter to remove impurities and biomass and were then stored at 4 °C for further analysis. The fluoroquinolone concentration was monitored using a high-performance liquid chromatography instrument (HPLC, Hitachi L-2000, Japan) equipped with an auto sampler. An Extend-C18 column (250 mm × 4.6 mm, 5-Micron 80A) from Agilent (USA) was used to achieve chromatographic separation. The fluoroquinolones (CIP, OFL, NOR and ENR) were detected using 0.02 M trichloroacetic acid, acetonitrile and methanol (74:22:4, v/v/v) as the mobile phase. The detection wavelength was set to 276 nm, and the column temperature was 30 °C. The column was equilibrated for 1020 min prior to injection. Ultra-performance liquid chromatography tandem mass spectrometry with a triple quadrupole mass spectrometer (UPLC-MS/MS, AB Sciex 6500, USA) was used to analyze the biodegradation metabolites of CIP. The separation was performed on a Shimadzu (Japan) C18 column (20 mm i.d. × 75 mm, 1.6 µm) at a temperature of 30 °C. The mobile phase consisted of (A) water with 0.1% formic acid and (B) acetonitrile with 0.1% formic acid and flowed at 0.3 mL min-1; the injection volume was 10 µL. The elution program comprised gradient elution from 10 to 100% (B) within 20 min and subsequent isocratic elution with 100% (B) for 5 min, followed by 10% (B) for 5 min. The mass spectrometer was equipped with an electrospray interface (ESI); the ESI source operated in positive ionization mode at 350 °C, and the ionspray voltage was set as 4.5 kV. Full scans from m/z 50700 were acquired for identification of the metabolites. Data were analyzed with software Analyst 1.6.3. 9
The identification of the metabolites was confirmed by consulting previous literature and the EAWAG-BBD Pathway Prediction System (http://eawag-bbd.ethz.ch/predict/) as well as product-ion analysis under low-mass conditions. 2.10 Statistical analysis Results corresponding to the removal rate and bacterial growth were analyzed using one-way analysis of variance (ANOVA) applying the LSD multiple comparison test using IBM SPSS Statistics software (version 20.0). Values were considered significant when p < 0.05. 3. Results and discussion 3.1 Isolation and identification of the ciprofloxacin-degrading strain After acclimation and enrichment with CIP, a bacterial strain, C419, was obtained with the ability to grow on solid MMSM medium supplemented with 20 mg L-1 of CIP. Analysis of the partial 16S rRNA gene sequence of strain C419 (1265 bp, GenBank accession number: KY784655) indicated an affiliation with the genus Thermus and showed 99.84% similarity to Thermus thermophilus HB8 (GenBank accession number AP008226, Bacteria; Deinococcus-Thermus; Deinococci; Thermales; Thermaceae; Thermus). A phylogenetic tree was constructed using the neighbor-joining method based on the 16S rRNA gene sequences of strain C419 and related strains (all the related strains are members of Thermaceae) (Fig. 1). Thermus thermophilus, as an extremely thermophilic bacterium, was originally isolated from a naturally high-temperature environment. Several of its enzymes were crystallized, and their structures were analyzed by X-ray crystallography [34]. Thermophilic bacteria are often used to produce thermostable enzymes for processing starch, organic synthesis, diagnostics, waste treatment, pulp and paper manufacturing and animal feed utilization [35]. This manuscript is the first report of a 10
thermophilic Thermus strain biodegrading fluoroquinolone. Several microorganisms possess the ability to biodegrade CIP, and some of them work by laccase [21,22,36]. Laccases participate in the degradation of polymers and the ring cleavage of aromatic compounds [37] and uses molecular oxygen to oxidize substrate during the degradation process. The genome databases of aerobic thermophilic bacteria were searched for laccases, and an open reading frame in Thermus thermophilus HB27, designated TTC1370, was identified [35]. Miyazaki (2005) also detected copper-inducible laccase activity in Thermus thermophilus HB27; this enzyme was the most thermophilic laccase reported so far [37]. In this study, strain C419 is closely related to Thermus thermophilus HB8 based on its 16S rRNA gene sequence. Accordingly, the CIP degradation by strain C419 might be attributed to a laccase. Further studies are needed to shed additional light on the molecular biology and enzymology of this bacterium. 3.2 Optimization of ciprofloxacin-degrading conditions Temperature and the initial pH value of the medium were selected as the key environmental factors for the degradation of CIP by strain C419. Temperature has a significant impact on bacterial growth and some important enzymes during biodegradation processes [38]. Thermus thermophilus is an extremely thermophilic bacterium, with an optimal growth temperature between 65 and 72 °C and a maximum temperature of 85 °C [39]. In this study, bacterial growth decreased with increasing temperature and significantly (p<0.05) declined at 80 °C (Fig. 2a). CIP degradation occurred from 65 to 80 °C, but the optimal degradation temperature was 70 °C (47%). The initial pH value of the medium was also a key factor governing compound transport to microbes and changing the solubility of pollutants [40]. As shown in Fig. 2b, there was no significant difference in bacterial growth at pH 6.5, 7.0 and 7.5 (p>0.05), and the maximum growth was observed at a pH value of 8.0. Marteinsson (1999) 11
isolated Thermus thermophilus Gy1211 from a deep-sea hydrothermal vent. It grew optimally at a pH value of 8.0, as did the strain in this study [41]. Maximum CIP degradation (55%) was achieved at a pH value of 6.5, but strain C419 also exhibited high degradation efficiency in neutral and alkaline conditions. The degradation of CIP by C419 occurred at a wide range of pH values, suggesting that this strain could be a good candidate for applications. 3.3 Effect of sodium acetate (SA) on the growth and biodegradation ability of strain C419 Co-substrate plays a dynamic role in the degradation of priority pollutants. Previous studies showed that antibiotics can be degraded faster in the presence of substrates that are easier to utilize, such as glucose and sodium acetate [24,31]. In this study, we examined the effects of different SA concentrations on bacterial growth and CIP biodegradation activity. As shown in Fig. 3, the removal of CIP and growth of C419 were significantly (p<0.05) inhibited by SA at a concentration of 3 g L-1. In the SA concentration range from 1 to 2 g L-1, the growth of C419 was inhibited during the initial 48 h, and CIP was slowly removed (Fig. 3b, c). After 48 h, the bacterium grew rapidly and demonstrated a high ability (more than 55%) to degrade CIP. A small amount of SA (0.5 g L-1) significantly (p<0.05) promoted CIP removal and bacterial growth compared to other treatments at earlier stages (036 h), but then its effect weakened. It is well known that suitable carbon and energy provision stimulates bacterial growth and co-metabolism. Carbon source concentrations that are too high or too low affect the ratio of carbon to nitrogen (C/N) and induce weak biodegradation [38]. In this study, 0.5 g L-1 SA was considered the best concentration for CIP degradation, although 12 g L-1 of SA resulted in relatively high bacterial growth and CIP removal rates after a 120-h incubation. The microorganism might take a long time to adapt to high concentrations (>1 g L-1) of SA. The removal of CIP (5 mg L-1) at different SA concentrations can be fitted using the first-order 12
kinetic model. As shown in Table 2, the removal rate constants (k) and half-lives (T1/2) of CIP (5 mg L-1) ranged from 0.0032 to 0.0157 h-1 and from 44.3 to 215.2 h, respectively. The R2 values ranged from 0.902 to 0.975, demonstrating that first-order kinetic model fit the data well. SA alone (without microorganisms) decreased the CIP concentration only slightly, suggesting that most of the CIP degradation was caused by the microorganism. SA serves as an additional carbon source to promote bacterial biomass production, and on the other hand, it can also act as an electron donor for the co-metabolism of the non-growth substrate [42]. In the present study, use of appropriate SA concentrations boosted bacterial growth and CIP degradation. 3.4 Identification of biodegradation metabolites Concentrated extracts were taken from the cultures to identify the potential biodegradation metabolites of CIP. The structures of CIP metabolites were proposed on the basis of previous papers and information from EAWAG-BBD Pathway Prediction System. Seven major metabolites of CIP were found in UPLC-MS spectrograms. All these metabolites were derived from the C419 culture that had grown with CIP for 120 h, and the blank control (without microorganism) did not show any peaks corresponding to these metabolites. The protonated molecules [M+H]+ of the metabolites are shown in Table 3. For further structure confirmation, the protonated molecules of the major metabolites were selected for product-ion analysis. The calculated formulas, proposed chemical structures and the product ions are also reported in Table 3. Metabolite M1, with a protonated molecule signal at m/z 360 and major product ions at m/z 342 (M+HH2O), 243 and 230, was identified as N-formylciprofloxacin. Metabolite M2, with a protonated molecule signal at m/z 348 and major product ions at m/z 330 (M+HH2O) and 217, was identified as desethylene-N-acetylciprofloxacin. The two metabolites were reported for the biotransformation of CIP 13
by the fungus Pestalotiopsis guepini [23]. Metabolite M3, with a protonated molecule signal at m/z 334 and major product ions at m/z 316 (M+HH2O) and 217 (M+HH2OC3H5NOCO), was identified as desethylene-N-formylciprofloxacin, which was first proposed by Čvančarová et al. (2015) to be one of the metabolite products by fungus tests [21]. Metabolite M4 ([M+H]+ at m/z 306) produced MS/MS product-ion signals at m/z 288 (M+HC2H4), 268, and 218, which agreed well with expectations for the metabolite desethylene-N-ciprofloxacin. Metabolite M5, with a protonated molecule signal at m/z 263 and major product ions at m/z 245 (M+HH2O), 216 and 204, was proposed to be 7-amino-1-cyclopropyl-6fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid. M4 and M5 are two kinds of common metabolites, and both of them have already been identified in the literature [21,22,27,43]. Metabolite M4 was also found when Labrys portucalensis F11 was cultured with CIP [20]. Metabolites M6 and M7, which were detected in this work, have not been previously described in the literature. Previous studies of the biotransformation of fluoroquinolones usually focused on the piperazine ring, which is preferably attacked by enzymes [44,45], resulting in metabolites M1 to M5. Metabolite M6, with a protonated molecule signal at m/z 223 and major product ions at m/z 207 (M+HNH2), 190 (M+HCHO2) and 178 (M+HNH2OH), may be 7-amino-6-fluoro-4-oxo-1,4dihydroquinoline-3-carboxylic acid. Biodegradation pathway analysis of CIP using the EAWAG-BBD Pathway Prediction System displays several routes for CIP transformation; it shows that metabolite M6 can derive from M5 by oxidative removal of the cyclopropyl group from the piperazine ring. M7, with a protonated molecule signal at m/z 190 and major product ions at m/z 172 (M+HH2O), 146 (M+HCHO2) and 130 (M+HCHO2NH2), matches the description of 4-oxo-1,4-dihydroquinoline-3 -carboxylic acid and might be obtained from the loss of a –F atom and a –NH2 group from the benzene 14
ring of M6. The information obtained from the UPLC-MS/MS was not sufficient to completely specify the chemical structures of these metabolites. However, nuclear magnetic resonance and/or the use of authentic standards will be adopted in future work to confirm the structures of CIP transformation metabolites. 3.5 Degradation of other fluoroquinolones Strain C419 possesses the ability to degrade OFL, NOR and ENR. Strain C419 removed ENR the most efficiently of these three fluoroquinolones. Nearly 74% of the ENR in the medium was removed after a 72-h incubation, while the efficiency of removing OFL and NOR were 70 and 63%, respectively. The ability of C419 to degrade different types of fluoroquinolones might be explained by the similar chemical structures of fluoroquinolones, but the various degradation abilities also reflect their different toxicity to microorganisms. In our previous work [31], a thermophilic sulfamethazine-degrading isolate was also able to degrade three other types of sulfonamides. Amorim et al. (2014) used an Alphaproteobacteria strain to degrade CIP, OFL, and NOR as single and mixed substrates, and all the tested drugs were degraded [20]. Müller et al. (2013) reported that the enzymes that require primary biodegradation are class-specific rather than compound-specific [46]. The adapted microbial culture was therefore able to biodegrade a set of structurally similar drugs. 3.6 Residual antibacterial activity assay The Gram-negative bacterium Escherichia coli K12 was selected as the test organism to determine the antibacterial activity of fluoroquinolones and biodegraded fluoroquinolones. The relative inhibition rate was evaluated by comparing the inhibition zone sizes resulting from the biodegraded fluoroquinolone and initial fluoroquinolone solution. As shown in Fig. 5, the inhibition by the four 15
biodegraded fluoroquinolones decreased by 2040% compared with that of the parent compounds (5 mg L-1). However, the antibacterial activities were still high, which may be due to incomplete transformation of the four fluoroquinolones by strain C419. Parent compounds that were not biodegraded may also contribute to the high antibacterial activities. Given the high activity of transformed fluoroquinolones, it is necessary to evaluate the toxicity of the metabolites. Čvančarová et al. (2015) measured the activity of biodegraded fluoroquinolones using various Gram-positive and Gram-negative bacteria isolated from the environment, and all the tested organisms were highly inhibited by the biodegraded fluoroquinolones, which indicated that the metabolites produced by the fungi still possessed high antibacterial activity [21]. Wetzstein et al. (1999) investigated the residual activity of transformed CIP on Escherichia coli ATCC 8739 and proved that the antibacterial activity of transformed CIP decreased [22]. 4. Conclusions A fluoroquinolone-degrading Thermus sp. strain, C419, was isolated from the sludge of an antibiotics pharmaceutical factory and used to biodegrade CIP at high temperature. The results showed that the best degradation conditions of CIP were 70 °C and pH 6.5. Addition of sodium acetate can obviously promote bacterial growth and the degradation of CIP, with more than 57% of the initial CIP in the media being removed after 5 days of incubation. Major biodegradation metabolites were identified by UPLC-MS/MS, and two new metabolites of CIP were proposed. The strain C419 possesses the ability to degrade three other types of fluoroquinolones and reduce antibacterial activity. Its outstanding thermotolerance and high CIP-degrading ability make the strain C419 a good candidate for remediation of fluoroquinolone-contaminated manure compost and materials found in other thermal environments. Acknowledgements 16
This work was supported by the China-Japan Research Cooperative Program [Grant No. 2016YFE0118000]; the National Natural Science Foundation of China [Grant No. 41373092]; the Key Project of Young Talent of IUE, CAS [Grant No. IUEZD201402]; the Scientific and Technological Major Special Project of Tianjin City [Grant No. 16YFXTSF00420]; the Industry Leading Key Projects of Fujian Province [Grant No. 2015H0044] and the Key Project of Young Talents Frontier of IUE, CAS [Grant No. IUEQN201501].
17
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Figure captions： Fig. 1 Phylogenetic tree constructed using the neighbor-joining method based on the 16S rRNA gene sequences of strain C419 and associated strains. Escherichia coli was used as an out-group. Bootstrap values (based on 1000 replications) are shown at branch points. Bar, 0.05 substitutions per nucleotide position. Fig. 2 Effect of temperature (a) and pH (b) on the growth of strain C419 and the degradation of CIP. Error bars represent standard deviations (n= 3). Data with different capital letters indicate significant differences (p < 0.05) of bacterial growth among treatments. Data with different lowercase letters indicate significant differences (p < 0.05) of removal rate among treatments. Fig. 3 Effect of sodium acetate (SA) on CIP removal kinetics (a), CIP removal (b) and growth of strain C419 (c) in a 120-h incubation. Error bars represent standard deviations (n= 3). Data with different letters indicate significant differences (p < 0.05) among treatments. Fig. 4 Degradation of three other fluoroquinolones by strain C419 (initial concentration of the fluoroquinolones: 5 mg L-1, 70 °C, pH= 6.5, 150 rpm). Error bars represent standard deviations (n= 3). Data with different letters indicate significant differences (p < 0.05) within a treatment. Fig. 5 The relative inhibition of four fluoroquinolones in MMSM liquid media after 72 h of incubation with strain C419 (the relative inhibition rate was evaluated by comparing the inhibition zone sizes resulting from the biodegraded fluoroquinolone and initial fluoroquinolone solution). Error bars represent standard deviations (n= 3).
24
Fig. 1
25
Fig. 2
26
Fig. 3
27
Fig. 4
28
Fig. 5
29
Table 1 Physico-chemical properties of ciprofloxacin. Properties of Ciprofloxacin Molecular Formula
C17H18FN3O3
CAS Number
85721-33-1
Molecular Weight
331.35 g mol-1
a
log Kow
0.4
a
pKa
3.01 ± 0.30
Chemical Structure
6.14 ± 0.13 8.70 ± 0.09 10.58 ± 0.30 Therapeutic Class a
Fluoroquinolone
The log Kow and pKa values are taken from reference [6].
30
Table 2 Kinetic parameters and removal of CIP (5 mg L-1) at different sodium acetate concentrations after a 120-h incubation. SA Concentration (g L-1)
k (h-1)
T1/2 (h)
R2
Removal (%)
0
0.0120
57.9
0.913
51.45±0.56
0.5
0.0157
44.3
0.910
57.12±0.94
1
0.0103
67.0
0.975
56.50±1.21
2
0.0072
95.7
0.902
59.23±0.46
3
0.0032
215.2
0.951
16.73±1.00
k - kinetic removal rate constant (h-1). T1/2 - degradation half-life (h). R2 - correlation coefficient.
31
Table 3 Structures and mass spectral data for ciprofloxacin and postulated biodegradation metabolites, as determined from UPLC-MS/MS. [M+H]+
Product ions
Calculated
(m/z)
(m/z)
Formula
CIP
332
314, 270, 245
C17H19FN3O3
M1
360
342, 243, 230
C18H19FN3O4
[23]
M2
348
330, 217
C17H19FN3O4
[23]
M3
334
316, 217
C19H21FN3O4
[21]
M4
306
288, 268, 218
C15H17FN3O3
[2022, 27, 43]
M5
263
245, 204, 216
C13H12FN2O3
[21, 22, 27, 43]
M6*
223
207, 190, 178
C10H8FN2O3
This study
M7*
190
172, 146, 130
C10H8NO3
This study
Compound
32
Proposed structure
References