Aerobic biotransformation of the antibiotic ciprofloxacin by Bradyrhizobium sp. isolated from activated sludge.

Ciprofloxacin (CIP) is an antibiotic that is widely used to treat bacterial infections and is poorly biodegraded during wastewater treatment. In this study, a CIP-degrading bacterial strain (GLC_01) was successfully retrieved from activated sludge by enrichment and isolation. The obtained bacterial strain shares over 99% nucleotide identity of the 16S rRNA gene with Bradyrhizobium spp. Results show that Bradyrhizobium sp. GLC_01 degraded CIP via cometabolism with another carbon substrate following a first-order kinetics degradation reaction. CIP degradation by Bradyrhizobium sp. GLC_01 increased when the concentration of the primary carbon source increased. The biodegradability of the primary carbon source also affected CIP degradation. The use of glucose and sodium acetate (i.e. readily biodegradable), respectively, as a primary carbon source enhanced CIP biotransformation, compared to starch (i.e. relatively slowly biodegradable). CIP degradation decreased with the increase of the initial CIP concentration. Over 70% CIP biotransformation was achieved at 0.05 mg L-1 whereas CIP degradation decreased to 26% at 10 mg L-1. The phylogenetic identification and experimental verification of this CIP-degrading bacterium can lead to a bioengineering approach to manage antibiotics and possibly other persistent organic contaminants during wastewater treatment.


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The occurrence of trace organic contaminants (TrOCs) including pharmaceuticals, personal 41 care products, steroid hormones, and industrial chemicals in sewage and sewage-impacted 3.5 days and the system achieved a steady period with sCOD removal >90% and VSS 0.98 ± 138 0.2 g L -1 after 30 days. 139 The reactors were then fed with growth medium containing 5 mg L -1 CIP for 4 months to 140 encourage the proliferation of CIP degrading bacterial strains. The sludge was then obtained 141 from these reactors, mixed together into an inoculum source, and incubated on agar plate (R2A 142 agar) supplementing with 5 mg L -1 CIP. The derived colonies were subsequently transferred to 143 another agar plate four times by repeated streaking culture until a single colony was confirmed.  The potential removal routes of CIP including biotransformation (experiment I), adsorption 152 (experiment II), and utilization of CIP as sole carbon source (experiment III) by the strain 153 GLC_01 were elucidated by batch experiments. In experiment I, the growth medium was 154 inoculated with active GLC_01 cells at the initial OD620 nm of 0.1 that was equivalent to 10 5 155 colony forming unit (CFU/mL). In experiment II, inactive (heat-killed) GLC_01 cells were 156 added to get an OD620 nm of 1.6 ± 0.01. This value was pre-determined to maintain the same 157 level of cell biomass in experiment I and II, and thus adsorption of CIP would be comparable 158 in these two experiments. In experiment III, the growth medium was inoculated with same 159 amount of active GLC_01 cells as in experiment I but did not have any glucose -which was 160 the primary carbon source. All experiments were prepared with 50 mL growth medium into 161 250 mL-sterile flasks. Experiment IV was prepared without GLC_01 cells to determine the removal of CIP by abiotic factors (i.e. photolysis, hydrolysis and volatilization). CIP was added 163 in all experiments at concentration of 4.89 ± 0.01 mg L -1 (n= 12). All experimental flasks were 164 covered and incubated on a rotary shaker at 25 ˚C and 150 rpm. Samples were collected at 165 interval time of 1 day for 8 consecutive-days. All laboratory apparatuses were autoclaved at 166 121˚C and 15 min to avoid any contamination. Residues of CIP in each experiment were 167 measured using a HPLC method (see Section 2.5.2). COD removal was measured in 168 experiment I (active cells). Cell growth rate in experiment I, II and III was measured using 169 methods as described in Section 2.5.1.

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Influence of primary carbon sources on the performance of strain GLC_01 was elucidated in 178 this study. Glucose, sodium acetate and starch were selected to have diverse chemical structures 179 and biodegradable levels. Each carbon source was prepared to achieve 2000 mg L -1 initial 180 COD. Other experimental conditions were maintained as in experiment I (see Section 2.3).

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Impact of initial CIP concentrations on the performance of strain GLC_01 was investigated.

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The concentration of CIP varied from 0.05 to 10 mg L -1 . This range was selected to represent    To improve MS outcomes, several preliminary experiments were conducted to optimize the 211 LC-MS parameters. The target components were separated on a C18 column (particle size 1.5 212 µm, ID 2.1 µm, L 10 cm). Two eluents, A (acetonitrile + 0.1% (v/v) formic acid) and B (water 213 + 0.1% (v/v) formic acid) were delivered at 0.2 mL min -1 through the column for 11 min in the     (Table S1), 280 indicating that CIP has low volatility. The log octanol-water partition coefficient (log Kow) of 281 CIP is 0.28 suggesting that it is hydrophilic and adsorption to activated sludge is insignificant.

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The results further showed that the biotransformation of CIP occurred via cometabolism. than that of experiment I (3.0 ± 1.5% vs. 46.7 ± 1.9) (Fig. 2a). CIP removal in experiment I 291 increased sharply after 3 days incubation (from 8.3 ± 0.23%, day 1 to 37.4 ± 2.3, day 3), which 292 coincided with high COD removal and cell growth rate (Fig 2b). Then, CIP removal was stable 293 at 45 ± 2% until the end of incubation period (Fig 2a)  currently study provides ample evidence that an activated sludge strain can perform 306 biotransformation of CIP through cometabolism rather than direct metabolism.
wastewater. If a compound is not present for an extended period, the specific compound