Elsevier

Chemosphere

Volume 211, November 2018, Pages 600-607
Chemosphere

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

https://doi.org/10.1016/j.chemosphere.2018.08.004Get rights and content

Highlights

  • Bradyrhizobium sp. GLC_01 was isolated from activated sludge.

  • GLC_01 removed CIP via cometabolism with primary carbon source.

  • High concentration of the primary carbon source induced better removal of CIP.

  • The primary carbon source degradability affected CIP removal.

  • GLC_01 could remove 70% of CIP at environmentally relevant concentration.

Abstract

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.

Introduction

The occurrence of trace organic contaminants (TrOCs) including pharmaceuticals, personal care products, steroid hormones, and industrial chemicals in sewage and sewage-impacted water bodies is of considerable human health and ecological concern. Some of these compounds, such as pharmaceuticals and personal care products, are indispensable in our modern society. Others, such as steroid hormones, are naturally and excreted continuously by mammals including human beings, and thus their release is unavoidable. There is a growing concern that the occurrence of TrOCs in the environment can affect aquatic ecology due to their biologically active properties (Clara et al., 2012; Dong et al., 2015; Luo et al., 2014; Tran et al., 2018). Another notable effect is the spread and proliferation of microbes that are persistent to antibiotics in the environment (Halling-Sørensen et al., 2000; Martínez, 2008).

Antibiotics are widely used in medicine and agriculture. However, only a small portion can be metabolised by humans and animals, and the rest is released into the environment (Nguyen et al., 2017). As an example, ciprofloxacin (CIP) is commonly used to treat bacterial infection and is frequently detected at elevated concentration in secondary effluent and hospital wastewater (ca. 10–200 μg L−1) and pharmaceutical manufacturing wastewater (ca. 6.5–31 mg L−1) (Larsson et al., 2007; Nguyen et al., 2017; Tran et al., 2018). Indeed, CIP concentrations in some of these wastewaters exceed the predicted no-effect concentrations for several aquatic organisms (Robinson et al., 2005). CIP has also been suspected to cause the development and transmission of antibiotic resistance genes in environmental microbiota (Martínez, 2008; Turolla et al., 2018; Zhang et al., 2013).

Biological treatment plays a crucial role in the removal of TrOCs prior to effluent discharge into the environment (Luo et al., 2014). Concerted research efforts in recent years have significantly improved our understanding of the biodegradation of TrOCs by biological (including both aerobic and anaerobic) treatment. For example, it has been established that biodegradation of TrOCs is governed by their physicochemical properties, especially the presence of either electron-withdrawing or donating functional groups in their molecular structure (Tadkaew et al., 2011; Wijekoon et al., 2015). TrOCs with electron-withdrawing functional groups are expected to be poorly removed (i.e. < 20%) while those with electron-donating functional groups are expected to be well removed (i.e. > 70%) by activated sludge treatment (Tadkaew et al., 2011). Based on this theory, Tadkaew et al. (2011) has developed a qualitative framework for the prediction of TrOC removal by activated sludge treatment.

Although the qualitative prediction framework proposed by Tadkaew et al. (2011) has been successfully validated by other authors (Li et al., 2015; Naghdi et al., 2018; Tran et al., 2018), it has not yet been able to account for occasionally peculiar and unusually high removal values of persistent TrOCs reported in the literature. Indeed, negligible removal efficiency (<15%) of CIP by activated sludge treatment has been widely reported (Jia et al., 2012; Li and Zhang, 2010; Lindberg et al., 2006) possibly due to the presence of fluoro which is a strong electron-withdrawing functional group in its molecular structure. On the other hand, CIP removal as high as 52.8% by a laboratory-scale membrane bioreactor has been reported by (Dorival-García et al., 2013). Recent research suggests that these occasionally and unusually high removal values of persistent TrOCs by biological treatment might be attributed to the microbial composition of the biomass (Vuono et al., 2016). In other words, there are rare microbial strains that can effectively metabolise otherwise poorly biodegradable TrOCs. The identification of these microbial strains and elucidation of their metabolic pathways can provide new insights into a bioaugmentation approach for the treatment of persistent TrOCs.

Although CIP is poorly biodegradable, a few CIP-degrading strains have been reported. A fluorobenzene-degrading bacterium Labrys portucalensis F11 could substitute the fluoride group in CIP with a hydroxyl group. This strain was isolated from an industrially contaminated site, however, the site characteristics were not provided (Amorim et al., 2014). Another CIP-degrading strain Thermus sp. was isolated via a serial enrichment of pharmaceutical sludge with CIP concentration of 1, 5 and 20 mg L−1 (Pan et al., 2018). This strain was a thermophilic microbe (70 °C), making it difficult to apply in wastewater treatment which commonly operate at 20–30 °C (Pan et al., 2018). Freshwater microalgae Chlamydomonas mexicana showed 13% removal of CIP after 11 days of cultivation (Xiong et al., 2017). A mixture of anaerobic sulfate-reducing bacteria showed moderate degree of CIP biodegradation (Jia et al., 2018). Liao et al. (2016) reported that activated sludge could harbour CIP-degrading strains in the classes of Gammaproteobacteria, Bacteroidia and Betaproteobacteria. Identifying CIP-degrading strains from activated sludge is an important step towards the improvement of CIP removal.

Previous studies have demonstrated that long-term exposure of activated sludge microbiome to TrOCs can alter the microbial community and in some cases selectively enrich specific microbes with enhanced affinity for TrOCs biodegradation (Moreira et al., 2014; Navaratna et al., 2012; Qu and Spain, 2010; Terzic et al., 2018; Zhou et al., 2013). An early example was observed for acesulfame (ACE), a synthetic sweetener. ACE was reportedly persistent to biological degradation in German WWTPs with less than 5% removal in 2010 (Kahl et al., 2018) when it was first introduced in the market. Over time, the activated sludge microbial community seemingly evolved to biotransform ACE. Recently, more than 85% ACE removal by conventional wastewater treatment has been reported (Kahl et al., 2018). Exposure of activated sludge microbiome to TrOCs (at a level that is higher than the environmentally relevant concentration) could increase selective pressure and shorten the evolution time. The initial activated sludge was unable to degrade macrolide antibiotics at concentration of 1–10 mg L−1. After two months of exposure at 10 mg L−1, the removal efficiency was increased to 99% (Terzic et al., 2018). Accordingly, TrOC-degrading strains have been identified for the removal of previously reported persistent compounds (Moreira et al., 2014; Mulla et al., 2016; Pan et al., 2018; Yu et al., 2007).

This study aims to retrieve CIP-degrading strains from activated sludge and subsequently characterise the degradation of CIP by the strains. The strains are obtained using enrichment and isolation methods, and then further characterised in terms of their genotypes using 16S rRNA gene-based sequencing. Phenotypes of the strains are characterised against a number of abiotic factors (i.e. CIP concentrations, concentration and types of primary carbon sources). CIP removal mechanisms (i.e. abiotic, adsorption and biodegradation) are evaluated. By identifying and comprehensively examining CIP-degrading strains from activated sludge, this study provides new insights that can be used to enhance the removal persistent TrOCs by biological treatment.

Section snippets

Chemicals

Analytical grade (>98% purity) of ciprofloxacin hydrochloride monohydrate was purchased from Sigma-Aldrich (Singapore). A stock solution containing 1 g L−1 was prepared in Milli-Q water for all subsequent experiments. A growth medium containing glucose (1.8 g L−1), urea (35 mg L−1), KH2PO4 (17.5 mg L−1), MgSO4 (17.5 mg L−1), and FeSO4 (10 mg L−1) was prepared following a procedure previously described by Oh et al. (2013). R2A agar was purchased from (DB Diagnostics, Singapore).

Enrichment protocol

Three identical

Identification of a CIP-degrading bacteria

Two bacterial strains were retrieved by enrichment and isolation (Section 2.2) from activated sludge continuously exposing to 5 mg L−1 of CIP in the feed. However, only strain GLC_01 showed the ability to degrade CIP in a growth medium. The DNA sequencing of the 16S rRNA gene (1326 bp) showed that strain GLC_01 shares over 99% nucleotide identity with the genus Bradyrhizobium (Fig. 1). This strain was classified as Bradyrhizobium sp. strain GLC_01. Species of the genus Bradyrhizobium have been

Conclusion

CIP-degrading Bradyrhizobium sp. GLC_01 was isolated from activated sludge. Our quantitative analyses revealed that biotransformation was the major removal pathway of CIP by Bradyrhizobium sp. GLC_01, and biotransformation occurred via cometabolism with the presence of another primary carbon source, rather than direct metabolism. Concentration and biodegradability of the primary carbon substrate affected the extent and rate of CIP biotransformation. Higher concentration of the primary carbon

Conflicts of interest

The authors declare that they have no competing interests.

Acknowledgements

This research work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (No. NRF-2017R1C1B5076367).

References (44)

  • S.I. Mulla et al.

    Degradation of triclocarban by a triclosan-degrading Sphingomonas sp. strain YL-JM2C

    Chemosphere

    (2016)
  • M. Naghdi et al.

    Removal of pharmaceutical compounds in water and wastewater using fungal oxidoreductase enzymes

    Environ. Pollut.

    (2018)
  • D. Navaratna et al.

    Impact of herbicide Ametryn on microbial communities in mixed liquor of a membrane bioreactor (MBR)

    Bioresour. Technol.

    (2012)
  • T.-T. Nguyen et al.

    Removal of antibiotics in sponge membrane bioreactors treating hospital wastewater: comparison between hollow fiber and flat sheet membrane systems

    Bioresour. Technol.

    (2017)
  • S. Oh et al.

    Metagenomic characterization of biofilter microbial communities in a full-scale drinking water treatment plant

    Water Res.

    (2018)
  • L.-j. Pan et al.

    Study of ciprofloxacin biodegradation by a Thermus sp. isolated from pharmaceutical sludge

    J. Hazard Mater.

    (2018)
  • T. Paul et al.

    Photolytic and photocatalytic decomposition of aqueous ciprofloxacin: transformation products and residual antibacterial activity

    Water Res.

    (2010)
  • G.U. Semblante et al.

    Trace organic contaminants in biosolids: impact of conventional wastewater and sludge processing technologies and emerging alternatives

    J. Hazard Mater.

    (2015)
  • N. Tadkaew et al.

    Removal of trace organics by MBR treatment: the role of molecular properties

    Water Res.

    (2011)
  • S. Terzic et al.

    Biotransformation of macrolide antibiotics using enriched activated sludge culture: kinetics, transformation routes and ecotoxicological evaluation

    J. Hazard Mater.

    (2018)
  • N.H. Tran et al.

    Occurrence and fate of emerging contaminants in municipal wastewater treatment plants from different geographical regions-a review

    Water Res.

    (2018)
  • A. Turolla et al.

    Antibiotic resistant bacteria in urban sewage: role of full-scale wastewater treatment plants on environmental spreading

    Chemosphere

    (2018)
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