Antibiotic resistance genes, bacterial communities, and functions in constructed wetland-microbial fuel cells: Responses to the co-stresses of antibiotics and zinc

Highlights

• ARGs loss occurred during the continuous exposure to high mass accumulation of Zn.

• Eight model methods revealed factors related to the changes in ARGs and community.

• Factors determining community composition and their functions were revealed.

• Closed circuit mode contributed to antibiotic and Zn removal and accumulation.

• Co-occurrence of ARGs, their potential hosts and bacterial functions were revealed.

Abstract

The effects of the continuous accumulation of Zinc (Zn) on the fate of antibiotic resistance genes (ARGs) in constructed wetland-microbial fuel cells (CW-MFCs) remain unclear. In this study, the impacts of Zn addition and a circuit mode on antibiotic removal, occurrence of ARGs, the bacterial community, and bacterial functions were investigated in three groups of CW-MFCs. The results showed that continuous Zn exposure enriched the target ARGs during the initial stage, while excessive Zn accumulation decreased antibiotic removal and the abundance of ARGs. A principal component analysis demonstrated that ARGs and the bacterial community distribution characteristics were significantly impacted by the mass accumulation of antibiotics and Zn, as well as the circuit mode. A redundancy analysis, partial least squares path modeling, and Procrustes analysis revealed that the accumulation of antibiotics and Zn, the composition of the bacterial community, the circuit mode, and the abundance of intI associated with horizontal gene transfer jointly contributed to the distributions of ARGs in the electrodes and effluent. Moreover, continuous exposure to Zn decreased the bacterial diversity and changed the composition and function of the bacterial community predicted using PICRUSt tool. The co-occurrence of ARGs, their potential hosts and bacterial functions were further revealed using a network analysis. A variation partition analysis also showed that the accumulation of target pollutants and the circuit mode had a significant impact on the bacterial community composition and functions. Therefore, the interaction among ARGs, the bacterial community, bacterial functions, and pollutant accumulations in the CW-MFC was complex. This study provides useful implications for the application of CW-MFCs for the treatment of wastewater contaminated with antibiotics and heavy metals.

Introduction

Veterinary antibiotics such as sulfanilamide, tetracycline, and quinolone, as well as heavy metals, have been extensively adopted to prevent infections, and these substances are often used as food additives for animals. However, most antibiotics and heavy metals are barely metabolized, and high amounts of antibiotics and heavy metals are frequently detected together in the dung and urine of animals (Ji et al., 2012; Zhang et al., 2017). In addition, it is noteworthy that emerging antibiotic resistance genes (ARGs) can occur not only due to selective pressure caused by antibiotics but also because of heavy metals that promote the dissemination of genetic elements via cross-resistance and co-resistance (Dickinson et al., 2019). However, some studies have found that the horizontal transfer frequencies of plasmid carrying ARGs decreased under high levels of heavy metals (Klümper et al., 2017). This would limit the dissemination of ARGs among different bacteria. Thus, it is crucial to investigate the effects of continuous heavy metal exposure on the enrichment and dissemination of ARGs.

Most previous studies have focused only on the impacts of heavy metals on the horizontal transfer of ARGs in pure culture systems (Klümper et al., 2017; Zhang et al., 2018). However, ARG fate is also influenced by bacterial community evolution based on the hosting relationship during the continuous bacterial proliferation process (Yuan et al., 2018). In addition, it has been demonstrated that low bacterial community density is not conducive to ARG transfer (Qiu et al., 2012). Notably, one previous study reported that a high mass accumulation of Zn can cause negative impacts on bacterial growth and even lead to cell apoptosis in bacteria (Su et al., 2019). In these systems, the diversity of the bacterial community is significantly reduced (Di Cesare et al., 2016). Therefore, it is necessary to understand how continuous exposure to antibiotics and heavy metals in a wastewater treatment system may affect ARGs and the bacterial community composition. In addition, a technology should be developed to limit the dissemination and enrichment of antibiotics and heavy metals in these systems.

Constructed wetlands are a well-known technology for treating livestock wastewater that contains high concentrations of antibiotics and heavy metals. However, the removal performance of a traditional CW is relatively low (Li et al., 2019a). Constructed wetland-microbial fuel cells (CW-MFC) are an advanced bioremediation technology that has shown potential for sustainable bioremediation of antibiotic wastewater due to its low operational costs (Di et al., 2020; Li et al., 2020a; Song et al., 2018). The coupling effects of CWs and microbial fuel cells (MFCs) in CW-MFC systems result in the enrichment of electrochemically active bacteria (EAB), achieving enhanced wastewater treatment efficiency by accelerating electron transfer from organics to electrodes (Guan et al., 2019; Wang et al., 2019a). There is little information regarding the impacts of heavy metals on antibiotic removal in CW-MFCs, and it is critical to obtain a comprehensive understanding of antibiotic and heavy metal removal when they co-exist in CW-MFCs composed of water, substrates, and bacteria. It has been reported that the stable operation of CW-MFCs mainly depends on the bacterial community composition, and the functions of bacteria play an important role in pollutant removal (Li et al., 2019a; Srivastava et al., 2015; Rathour et al., 2019). To fully understand the impacts of typical heavy metal additions on the fate of ARGs, the bacterial community, and bacterial functions, comprehensive investigations of these factors and their interactions are required.

Copper and zinc are commonly used in pig and fish farms. Moreover, it has been reported that the detected concentration of zinc in animal manure is even higher than Cu (Hölzel et al., 2012). Therefore, typical heavy metal zinc was selected to be examined in this study. The fate of target antibiotics [i.e., sulfadiazine (SDZ) and ciprofloxacin (CIP)] and zinc (Zn), as well as the abundance of ARGs, the bacterial community dynamics, bacterial functions, and electricity generation performances were investigated in this study. Moreover, the key variables that determined the ARG contents and the correlation between ARGs, bacterial community, and bacterial functions were determined. Additionally, the contributions of the characteristics of the circuit mode, pollutant accumulation, and ARGs to the shifts in the bacterial communities and functions were illuminated. The correlations between ARGs, bacterial community, and the accumulation of target pollutants were studied. The results obtained from this study provide a reference for the application CW-MFCs in the treatment of wastewater contaminated with antibiotics and heavy metals.