Elsevier

Bioresource Technology

Volume 267, November 2018, Pages 303-310
Bioresource Technology

Response of antibiotic and heavy metal resistance genes to two different temperature sequences in anaerobic digestion of waste activated sludge

https://doi.org/10.1016/j.biortech.2018.07.051Get rights and content

Highlights

  • Response of ARGs and HMRGs to TPAD of WAS were investigated.

  • Highest removal of total ARGs was achieved by mesophilic-thermophilic AD.

  • TPAD was effective removing human bacterial pathogens.

  • Some HBPs showed a positive correlation with ARGs, HMRGs and intI1.

Abstract

Response of antibiotic resistance genes (ARGs) and heavy metal resistance genes (HMRGs) to two different temperature sequences (i.e., mesophilic-thermophilic and thermophilic-mesophilic) were investigated. Higher removal of total ARGs (twenty-one targeted subtypes) and HMRGs (three targeted subtypes) was achieved by the mesophilic-thermophilic sequence than by the thermophilic-mesophilic sequence. The sequence of mesophilic-thermophilic showed the highest removal of total ARGs, but the sequence of thermophilic-mesophilic proved more suitable for removal of class 1 integrons (intI1). Correlation analysis indicated that intI1 correlated significantly with tetG, tetQ, tetX, sul2, aac(6′)-lb-cr, blaTEM, ermB and floR. High-throughput sequencing revealed that the mesophilic-thermophilic sequence TPAD removed more human bacterial pathogens (HBPs) than did the thermophilic-mesophilic sequence. Also, significantly positive correlation was observed between ARGs and HBPs. For instance, Mycoplasma pneumonia showed significantly positive correlation with several ARGs including tetE, tetQ, tetX, tetZ, sul1, sul2, aac(6′)-lb-cr and floR.

Introduction

One of the major drawbacks of any activated sludge process is that huge amounts of waste activated sludge (WAS) are produced as a by-product (Appels et al., 2011). According to Zhang et al. (2017b), approximately 45 million dry tons of global sewage sludge are produced annually. Prior to 2013, the cost for sludge disposal accounted for nearly 50% of total wastewater treatment plant (WWTP) operating costs (Appels et al., 2011), representing up to 10 billion USD annually (Zhang et al., 2017b). To reduce this cost, various physical, chemical and biological processes can be applied. Among these, anaerobic digestion (AD) is considered as one of the most cost-effective alternatives for WAS treatment because of its ability to convert organic matter to biogas, especially methane (Appels et al., 2011). About 90% and 66% of sewage sludge are treated via AD in Germany and the UK, respectively (Tao et al., 2017).

According to recent studies (Auerbach et al., 2007, LaPara et al., 2011, Munir et al., 2011), proliferation of antibiotic resistance genes (ARGs) has become a serious threat to both animals and humans and a growing body of evidence has suggested that WWTPs are considered to be potential reservoirs of diverse ARGs because most ARGs-contaminated wastewater originate from livestock, agriculture, industry and hospitals. Moreover, environmental factors (e.g., high microbial diversity and nutrition) in WWTPs may promote the proliferation of ARGs through vertical or horizontal gene transfer (HGT) with mobile genetic elements such as plasmids, transposable elements or integrin-integrase gene cassettes (Schlüter et al., 2007). Results of the high-throughput sequencing approach has recently suggested that a large proportion of ARGs in WWTPs are concentrated in WAS (Yang et al., 2014). For these reasons, considerable attention has been paid to the efficient treatment of WAS via AD, especially, because it is considered essential to reduce environmental burdens as well as operating costs.

In a positive sense, large amounts of ARGs can be reduced by AD. Recently, significant efforts have been made to evaluate the effect of operating parameters on the removal of ARGs in AD. Diehl and LaPara (2010) reported that digestate from thermophilic AD (TAD) showed a lower quantity of genes encoding tetracycline resistance (tetA, tetL, tetO, tetW, tetX) and class 1 integrons (intI1) than that in mesophilic AD (MAD). Also, Ma et al. (2011) achieved more effective reduction of two erythromycin (ermB, ermF) and tetracycline (tetO, tetW) resistance genes in TAD compared to MAD. However, MAD showed higher removal of other resistance genes (sul1, sul2, tetC, tetG, tetX) and intI1 than that obtained in TAD. These results suggest that temperature is critical to determining the distribution of ARGs in AD, but their response to temperature in AD is not universal (Zhang et al., 2015). By extension, various effects by pretreatment (Ma et al., 2011, Zhang et al., 2016b) of AD or two-stage (acidogenic-methanogenic phase or different temperature phased) AD (Ghosh et al., 2009, Wu et al., 2016) on ARGs have been reported. However, understanding of the fate of different types of ARGs in temperature-phased anaerobic digestion (TPAD) is still limited, although the presence of three tetracycline resistance genes (tetA, tetO, tetX) and intI1 in TPAD has been reported by Ghosh et al. (2009).

Interestingly, experimental evidence indicates that heavy metals can exert selection pressure not only on heavy metal resistance genes (HMRGs) but also ARGs (Berg et al., 2010, Di Cesare et al., 2016, Seiler and Berendonk, 2012). There are two potential mechanisms involved in the co-selection of antibiotic and heavy metals resistance (Baker-Austin et al., 2006): (1) cross-resistance (genes share a common route of access to their respective targets) and (2) co-resistance (genes specifying resistant phenotypes are located together on the same genetic element such as a plasmid, transposon or integron). Considering the characteristics of domestic wastewater (i.e., generally higher concentration and lower degradability of heavy metals than for antibiotics) (Di Cesare et al., 2016), co-selection of resistance genes might be enhanced.

In this study, TPAD of WAS using two different sequences (i.e., MAD-TAD, TAD-MAD) was operated to investigate variations in ARGs, HMRGs and intI1. Using quantitative PCR (qPCR), twenty-one ARGs encoding tetracycline resistance (tetA, tetB, tetD, tetE, tetG, tetH, tetM, tetQ, tetX, tetZ, tetBP), sulfonamide resistance (sul1, sul2), quinolone resistance (qnrD, aac(6′)-Ib-cr), β-lactam antibiotics resistance (blaSHV, blaTEM, blaCTX), macrolide resistance (ermB), florfenicol resistance (floR) and multidrug resistance (oqxA) and intI1 were quantified. In addition, three HMRGs encoding copper resistance (copA, pcoA, tcrB) were quantified by qPCR. To gain insight into the potential mechanisms impacting the fate of ARGs, HMRGs and intI1 in TPAD, the bacterial community structure was characterized using high-throughput sequencing of 16S rRNA genes. To our knowledge, limited studies to date have reported the fates and co-occurrence of ARGs, HMRGs and intI1 during TPAD. Thus, current research is essential to obtain insight into the potential mechanisms of these genes in TPADs.

Section snippets

Preparation of WAS and inoculum

The highly concentrated WAS and inoculum used in this study were collected from the secondary clarifier and mesophilic anaerobic digester in a municipal wastewater treatment plant in Daegu, Korea. The physico-chemical characteristics of WAS and inoculum were as follows: pH 5.59 ± 0.11 and 7.24 ± 0.11; total solids (TS) 45.60 ± 0.28 and 24.33 ± 0.89 g/L; volatile solids (VS) 33.67 ± 0.09 and 16.21 ± 0.57 g/L; total chemical oxygen demand (TCOD) 48.34 ± 0.44 and 26.65 ± 0.13 g/L; soluble COD

Responses of ARGs, HMRGs and intI1 to TPAD

The profile of the residual gene for tetracycline resistance is shown in Fig. 1a. The abundance of tetH and tetBP decreased below the limit of quantification (LOQ) (4.35 × 102 copies/µL for tetH and 5.96 × 104 copies/µL for tetBP) after all types of AD in this study. In addition, abundance of tetE decreased below LOQ (4.67 × 103 copies/µL) in T1st and T2nd, but the residual gene fractions of tetE in M1st and M2nd were 0.59 and 0.50, respectively. Interestingly, proliferation and rebound (i.e.

Conclusion

Considering the abundance of total ARGs as well as the rebound of some ARGs after a mesophilic stage, the application of TAD as the final digestion stage of TPAD could be reasonable. However, in terms of iniI1, MAD as the final digestion stage of TPAD might be appropriate. An internal or external recycle could solve this dilemma. Given that TAD is usually used in the first stage of TPAD to achieve higher methane production, recycling digested sludge from M2nd to T1st might result in the

Acknowledgements

This work was supported by GIST Research Institute (GRI) grant funded by the GIST in 2018.

References (46)

  • F. Raposo et al.

    Anaerobic digestion of solid organic substrates in batch mode: an overview relating to methane yields and experimental procedures

    Renew. Sustain. Energy Rev.

    (2012)
  • Z. Saribas et al.

    Prevalence of erm genes encoding macrolide-lincosamide-streptogramin (MLS) resistance among clinical isolates of Staphylococcus aureus in a Turkish university hospital

    Clin. Microbiol. Infect.

    (2006)
  • B. Tao et al.

    Enhancement of microbial density and methane production in advanced anaerobic digestion of secondary sewage sludge by continuous removal of ammonia

    Bioresour. Technol.

    (2017)
  • Y. Wu et al.

    Influence of two-phase anaerobic digestion on fate of selected antibiotic resistance genes and class I integrons in municipal wastewater sludge

    Bioresour. Technol.

    (2016)
  • R. Xu et al.

    Rapid startup of thermophilic anaerobic digester to remove tetracycline and sulfonamides resistance genes from sewage sludge

    Sci. Total Environ.

    (2018)
  • Y. Yang et al.

    Fate of antibiotic resistance genes in sewage treatment plant revealed by metagenomic approach

    Water Res.

    (2014)
  • J. Zhang et al.

    Impacts of addition of natural zeolite or a nitrification inhibitor on antibiotic resistance genes during sludge composting

    Water Res.

    (2016)
  • J. Zhang et al.

    Fate of antibiotic resistance genes and its drivers during anaerobic co-digestion of food waste and sewage sludge based on microwave pretreatment

    Bioresour. Technol.

    (2016)
  • J. Zhang et al.

    Enhanced anaerobic digestion of food waste by adding activated carbon: fate of bacterial pathogens and antibiotic resistance genes

    Biochem. Eng. J.

    (2017)
  • Q. Zhang et al.

    Sludge treatment: current research trends

    Bioresour. Technol.

    (2017)
  • R.G. Amachawadi et al.

    Occurrence of the transferable copper resistance gene tcrB among fecal enterococci of U.S. feedlot cattle fed copper-supplemented diets

    Appl. Environ. Microbiol.

    (2013)
  • APHA-AWWA-WEF

    Standard Methods for the Examination of water and Wastewater

    (1998)
  • J. Berg et al.

    Cu exposure under field conditions coselects for antibiotic resistance as determined by a novel cultivation-independent bacterial community tolerance assay

    Environ. Sci. Technol.

    (2010)
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    Current affiliation: Texas A&M AgriLife Research Center, 1229 North US Highway 281, Stephenville, TX 76401, USA.

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    Current affiliation: Department of Surface Waters-Research and Management, Eawag: Swiss Federal Institute of Aquatic Science and Technology, Kastanienbaum, Switzerland.

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