DEVELOPMENT OF A SENSITIVE POLYMERASE CHAIN REACTION (PCR) APPROACH TO DETECT ENTAMOEBA DISPAR IN INDUSTRIAL WASTEWATER

Deep injection wells are considered among the most efficient, environmentally-friendly and cost-effective techniques to dispose of wastewater. However, formation of biofilms in the casing pipe can reduce the effective diameter, which in turn, can lower the injectivity of wastewater and ultimately results in injection failure. A class 1 deep injection well located at the Solid Waste Authority of Palm Beach County was revealed to be getting clogged due to the development of a microbial community where Entamoeba dispar, a protozoan species was found to be the abundant microorganism in the biofilm. The injection well is used to discharge industrial wastewater coming from several sources at the facility which are discharged to a collection chamber, known as the wet well, before being disposed down the deep injection well pipe. Prior to design and implementation of a suitable treatment technique to inactivate the protozoan species, it is imperative to reveal the origins of the microorganism coming to the deep injection well. Therefore, the objective of the current research was to develop a technique to identify potential sources of Entamoeba dispar. In this study, samples were collected from the seven sources as well as from the wet well. Initially, a number of onsite and laboratory experiments were conducted to monitor the water quality parameters of the collected samples. In case of microbiological investigations, microscopic analysis was carried out to detect the microorganism in the wastewater specimens followed by polymerase chain reaction (PCR) and gel-electrophoresis assays. In addition, the number of DNA copies in each of the tested samples was determined using the ImageJ app. From the microscopic analysis, no samples were found to be Entamoeba dispar positive. However, PCR and gel electrophoresis tests results indicated that wet well, NEFCO effluent, class 1, REF 1 and groundwater dilution samples were positive and the calculated number of DNA copies were 6545, 6849, 16763, 6351 and 5635 in 100 mL of the wastewater specimens respectively. The PCR technique used in this study is sensitive enough to detect even 4 DNA copies of the target microorganism. All the positive samples have one thing in common, which is they all contain local groundwater from site, indicating a potential source for further investigation.