Removal of antibiotic resistance genes in an algal-based wastewater treatment system employing Galdieria sulphuraria: A comparative study
Graphical abstract
Introduction
Due to the massive use of antibiotics in human and veterinary health applications, domestic and agricultural wastewaters are now recognized as major environmental sources of antibiotic-resistant bacteria and genes (Andersson and Hughes, 2010, Bengtsson-Palme et al., 2017). Since all the domestic wastewaters eventually flow through wastewater treatment plants (WWTPs), they have been identified as large reservoirs for antibiotic-resistant bacteria (ARB) and antibiotic-resistant genes (ARGs) (Rizzo et al., 2013). In the US and in most countries worldwide, WWTPs depend on conventional treatment processes, (e.g. preliminary treatment, primary treatment, and secondary treatment) to remove traditional pollutants (e.g. suspended solids, dissolved organics, and nutrients). The activated sludge (AS) process is the most common secondary treatment processes where, high concentrations of bacteria are utilized to biologically oxidize dissolved organic carbon in wastewaters.
Previous studies have reported that biological processes, such as the AS process, could promote the spread of ARB and ARGs (Rizzo et al., 2013) resulting from sub-inhibitory concentrations of antibiotics in wastewater and the abundance of microorganisms in the AS process. First, the sub-inhibitory concentration of antibiotics in wastewater provides potentially suitable environment for developing antibiotic resistance and spreading in the AS process (Auerbach et al., 2007, Da Silva et al., 2006, Davies et al., 2006). Antibiotics with sub-inhibitory concentrations are considered as a trigger of the SOS response to DNA damage in which DNA repair and mutagenesis are induced. These sub-inhibitory effects may play a crucial role in the spread of antibiotic resistance accelerating the rates of mutation and horizontal gene transfer (HGT) (Baharoglu and Mazel, 2014). According to Andersson and Hughes, sub-inhibitory concentrations of antibiotics may be more relevant to the issue of antibiotic resistance than lethal concentrations of antibiotics (Andersson and Hughes, 2012).
Furthermore, the high abundance of microorganisms in the AS process (~4000 mg/L) would exacerbate the spread of antibiotic resistance. High nutrient load, normal pH and temperature in the AS process provide favorable environment for cell proliferation which would increase the chance of HGT (Di Cesare et al., 2016, Kim et al., 2014, Marano and Cytryn, 2017). A previous report has noted that secondary sludge contained higher number of resistant bacteria of 107 CFU/g secondary sludge (Gao et al., 2012b). Additionally, abundance of bacteriophage in WWTPs is also extraordinarily high, with over 1000 unique viral genomes identified in the activated sludge (Parsley et al., 2010), which increase the chance of gene transfer via transduction. Most wastewaters carry a bacteriophage content of 105–108 plaque forming units (PFU)/mL (McMinn et al., 2017, Yahya et al., 2015). Although conventional treatment could remove significant number of bacteria, it is not designed to remove bacteriophages in wastewater.
The mechanism of antibiotic resistance development could be explained by intrinsic resistance, acquisition through mutations, or HGT which could be from donor bacteria or bacteriophage. HGT represents gene transfer mediated by mobile genetic elements (MGEs) including plasmid, integrons and bacteriophage (Nwosu, 2001). Genetic exchange through generalized or specialized transduction is achieved by bacteriophage, whereby a fragment of DNA is carried by bacteriophage from a donor bacterial to a recipient cell (Balcazar, 2014). Although it was thought transduction occurs at low frequency, recent single cell studies (Touchon et al., 2017) observed transduction rates close to 1% per plaque forming units when natural communities served as recipients. Metagenomic data also show generalized transduction may contribute significantly to HGT in prokaryotes and these analyses indicated that bacteriophage could be a vital reservoir for ARGs (Schmieder and Edwards, 2012). It was demonstrated that bacteriophages play a vital role in the dissemination of ARGs in aquatic environment (Yang et al., 2018). Some recent studies that used both sequence and function-based metagenomic analyses revealed the occurrence of ARG-like genes in the virome of activated sludge, which could transfer resistance to some host bacteria (Balcazar, 2014).
In this study, the feasibility of controlling ARGs and ARB in a pilot-scale, algal-based wastewater treatment system utilizing Galdieria sulphuraria was investigated. Galdieria sulphuraria is a thermo-acidophilic, unicellular red alga, that can grow mixotrophically on dissolved organics in wastewaters (Gross, 2000, Gross and Schnarrenberger, 1995). Long-term operation of this pilot scale algal wastewater treatment system has been demonstrated previously as an energy-efficient and sustainable technology for removing biochemical oxygen demand (BOD), ammoniacal nitrogen, and phosphates in primary-settled domestic wastewater in a single step. In addition to treating the wastewater to the discharge standards, this algal system can produce energy-rich biomass which could be used as a feedstock for recovering biocrude oil (Henkanatte-Gedera et al., 2015). Superior performance of this algal system in concurrent inactivation of pathogenic bacteria in the primary-settled wastewater has also been demonstrated (Delanka-Pedige et al., 2019). Given the high pathogenic bacterial inactivation and the inclement culture conditions (low pH and high temperature) in the algal system, the current study was designed to test the follow-on idea that the system also reduces the occurrence of antibiotic resistance.
The specific goal of this study was to compare the reductions of ARGs and ARB in an algal system against that in a secondary treatment system at a conventional wastewater treatment plant. Five commonly used groups/classes of antibiotics were chosen to assess the fate of ARGs and ARB in the two systems. Given that Class 1 integrons can acquire and express multiple resistance genes by using site-specific recombination mediated by an integron-integrase (intⅠ) (Hall and Collis, 1995), the abundance of intⅠ1 gene in WWTPs was investigated as well. Additionally, the role of bacteriophages in ARG transfer was also assessed in the two systems.
Section snippets
Sample collection
Samples were obtained from the algal system deployed outdoors at the local wastewater treatment plant (Las Cruces, New Mexico), running in parallel with the existing plant. The extremophilic algal culture, G. sulphuraria (CCMEE 5587.1) was cultivated in a horizontal, enclosed photobioreactor with a working volume of 700 L, operated in batch mode. This pilot system has been operated at a pH of 4.0 within a temperature range of 27–46 °C (Delanka-Pedige et al., 2019, Henkanatte-Gedera et al., 2015
Occurrence of total bacteria in wastewater treatment processes
The qPCR results for batch 1 showed total bacterial numbers (copies/mL) of (4.33 ± 0.06) × 108 in the primary effluent; (1.18 ± 0.05) × 107 in the activated sludge effluent; and (5.00 ± 0.10) × 107 in the algal effluent. Corresponding results in batch 2 were (7.04 ± 1.05) × 107, (9.09 ± 1.09) × 105, and (2.53 ± 0.65) × 106. In both batches, bacterial concentration in the primary effluent was the highest and that in activated sludge effluent was the lowest. In the conventional system, bacterial
Conclusion
This study demonstrated that the algal-based wastewater treatment system utilizing Galdieria sulphuraria could reduce not only the abundance of ARB but also the relative abundance of ARGs (qnrA, qnrS, tetW) in surviving bacteria. These reductions in the algal system were superior to those in a conventional secondary treatment system. The role of bacteriophages in ARG transfer was also investigated in this study through detection of ARGs in bacteriophages and its correlation with relative ARGs
Acknowledgements
This work was supported in part by the National Institutes of Health Support of Competitive Research (SCORE) Pilot Project Award program (grant number 1SC2GM130432), the National Science Foundation Engineering Research Center for Reinventing the Nation's Urban Water Infrastructure (ReNUWIt) (award EEC 1028968), College of Engineering at New Mexico State University, Interdisciplinary IMPACT Mini-Grants Program at New Mexico State University, and New Mexico Water Resources Research Institute
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