Abstract
Several communities in South Africa (SA) still depend on untreated surface water for different domestic purposes. Compliance of the effluents from wastewater treatment plants (WWTPs) to the regulatory standards, which mostly entails the removal/reduction of organic waste and deactivation of the potential microbial pathogens is of great importance. The detection of indicator parameters can be used to determine the effectiveness of a Wastewater Treatment Plant (WWTP) and the level of compliance with the South African regulatory standards. The aim of this research project was to study the impact of chlorine resistant Escherichia coli (E. coli) on the compliance of treated effluents to JB Marks Local Municipality’s WWTP regulatory standards. In achieving this aim, this research project was divided into four part namely: (i) Evaluation of fecal coliform prevalence and physicochemical indicators in the effluent; (ii) The effects of dry and wet season on the performance of the studied WWTP; (iii) Characterization of bacterial communities in wastewater through 16S ribosomal ribonucleic acid (rRNA) Next Generation Sequencing (NGS); and (iv) Molecular categorization and antimicrobial profiles of E. coli that survived chlorination at the studied plant. The first chapter intended in assessing the prevalence and compliance of indicator parameters to South African regulatory standards with regard to E. coli and physicochemical parameters as well as the efficiency of the studied WWTP in removing the studied indicator parameters. The E. coli ranged from 0 to 2420 count/100 mL in the final effluent. The recorded values for the physicochemical parameters were within the following ranges: pH (7.03–8.49), electrical conductivity (81.63–126.5 mS/m), suspended solids (0.40–20.4 mg/L), ammonia (0–22.15 mg/L), Chemical Oxygen Demand (COD) (1–73 mg/L), nitrate (0–16.1 mg/L), ortho-phosphate (0–8.58 mg/L) and free chlorine (0–3.21 mg/L). Furthermore, the concentration of toxic heavy metals was recorded to be between 1–10 ug/L for arsenic, cadmium, lead and mercury. In conclusion, all the parameters that were evaluated in this study indicate that the studied WWTP is performing in accordance with the prescribed general limits from GA, with limited compliance to the special limits of the same GA. The effect of seasonal variations of rainfall and temperature on the fate of E. coli and chemical parameters was investigated in the second chapter. Both seasons showed variations in terms of rainfall levels and temperature. The average temperatures measured at the final effluent were 14 and 22°C for the dry and wet season, while the rainfall averages ranged between 0.0 and 69.0 mm and 16.0 and 258.9 mm for the dry and wet season, respectively. The impact of rainfall within the two seasons presented a variation with an average inflow rate of 34 000 and 48 000 m3 during the dry and wet seasons, respectively. Higher E. coli concentrations were detected before and after chlorination in the wet season (1.86 x 105 and 8.40 x 101 MPN/100 mL) than in the dry season (2.26 x 104 and 5.10 x 101 MPN/100 mL). The recorded values for the chemical parameters in the dry and wet seasons were within the following ranges: ammonia (0.27 and 3.68 mg/L), COD (29.53 and
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22.10 mg/L), nitrate (9.21 and 2.40 mg/L) and ortho-phosphate (0.46 and 0.39 mg/L). Though the detections of these indicator parameters were affected differently by the seasonal variations, it is important to note that the efficiency of the WWTP in reducing these indicator parameters proved to be consistent across all seasons, except in the case of ammonia and nitrate. Most of the studied parameters showed effective compliance when measured against South African regulatory standards (GA, general limits) in both the dry and wet seasons, with the exception of ammonia during the wet season. Most of the water borne pathogens are spread through the fecal oral means. They are then found in wastewater via excreted feces from infected beings. The NGS technique was employed in this chapter to provide insights into the diversity of these bacterial constituencies using bacterial 16S rRNA. This chapter identified the presence of bacterial RNA from various bacterial families that include putative pathogens and fecal indicator bacteria at samples taken before disinfection and after disinfection. Proteobacteria, Firmicutes and Actinobacteria were the dominant phyla, while Betaproteobacteria and Clostridia were dominating the bacterial class level across both influent and effluent samples. At the final effluent, the study identified organisms used by the plant for organics removal thus confirming their survival to chlorine disinfection at the plant. Furthermore, the presence of potential pathogens was also confirmed by detection of E. coli commonly used as indicator bacteria for possible potential pathogens presence. These pathogenic organisms in the treated effluent pose a potential health risk to aquatic life and downstream users. Lastly, the resistance of different pathogenic variants of E. coli to antibiotics, is a health concern not only in SA, but globally. Therefore, the intention of this chapter was to determine the resistance of different E. coli pathotypes to 13 different antibiotics namely: cephazolin, gentamicin, ciprofloxacin, streptomycin, trimethoprim, amoxycillin, neomycin, kanamycin, chloramphenicol, erythromycin, sulphamethoxazol, nalidixic acid and tetracycline. A total of 90 E. coli isolates that survived chlorination, were analysed for their antibiotics resistant profiles using the Kirby-Bauer disk diffusion method. A high degree of resistance was observed against erythromycin (96.67%) and sulphamethoxazol (92.22%), while lower resistance degree was observed against Kanamycin (3.33%), chloramphenicol (5.56%) and ciprofloxacin (6.67%). Most of the isolates were resistant to at least three and more antibiotics, and multiple drug resistance was observed in 91% of the E. coli isolates that survived chlorination. The studied E. coli isolates were also assessed for their pathogenicity origin using polymerase chain reaction technique (PCR). The study confirmed the identification of ETEC, EPEC, EIEC, and from the six studied diarrheagenic pathotypes. The identified pathotypes also showed multiple resistance to 3 and more antibiotics. Even though the plant showed high reduction of E. coli under different seasonal conditions, the survival of the E. coli isolates to disinfection resulted to non-compliance as the plant did not meet its WUL conditions in some instances, thus disadvantaging the plant in obtaining its Green Drop (GD) certification. The presence of E. coli confirmed possible presence of pathogenic bacteria inclusive of diarrheagenic E. coli pathotypes, thus
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enhancing the risk to public health impairment, especially with the isolates inclusive of their pathotypes which showed resistance to multiple conventionally used antibiotics.