Molecular Characterisation of Bacteria and their Degradation Role on Polychlorinated Biphenyls (PCBs) and Polycyclic Aromatic Hydrocarbons (PAHs) in Wastewater from Gaborone (Botswana) and Mafikeng (South Africa)
This thesis is a study on biodegradation of polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs) in wastewater by bacteria isolated from wastewater as an alternative means of treating wastewater for safe recycling as a possible option to reduce water shortages. The research focused on selected wastewater treatment plants and raw water dams in Gaborone, Botswana and Mafikeng, South Africa. The thesis argues, amongst other things, that water is a scarce resource and this indicates the need to have measures in place to effectively and efficiently treat wastewater for safe recycling to curb the problem of water shortage. Wastewater contains recalcitrant compounds which are not easily removed from the environment through simple conventional processes. Some of these compounds like polycyclic aromatic compounds (PAHs) and polychlorinated biphenyls (PCBs) have carcinogenic and teratogenic effects. These compounds are also considered persistent organic pollutants (POPs) leading to a global agreements such as the Stockholm Convention of 2001 that called for nations to stop the production of these compounds as well as a decree for all nations to come up with a national plan on how these compounds will be eliminated. The objectives of this study was to isolate and characterise aerobic bacteria that possess biodegradation characteristics for inorganic compounds from wastewater samples obtained from Notwane Sewage Treatment Plant and Setumo/Modimola Dam and surface water samples from Gaborone dam and Disaneng Dam,. A further objective was to analyse the water for the presence of PAHs and PCBs as well as perform degradation tests on PAHs and PCBs using selected isolates In the present study, a total of 60 raw water samples and 50 wastewater samples were collected and analysed for bacterial diversity and presence of PCBs and PAHs. The samples and controls were taken from Gaborone and Mafikeng. The identities of 29 bacterial isolates were identified using preliminary (Gram staining, Analytical Profle Index 20E test) and confirmatory (16S rRNA and 16S rRNA gene sequence analysis using BLAST search). Bacteria species belonging to the genus Aeromonas, Bacillus, Pseudomonas, Exiguobacterium, Kurthia and Vibrio were detected. From these it is evident that wastewater has a highly diverse group of bacteria, some of which might be having potential biodegradation properties for recalcitrant compounds. Isolate MD2 which was identified as Pseudomonas aeruginosa and this was detected in wastewater samples obtained from both Gaborone and Mafikeng. The Pseudomonas aeruginosa (MD2 isolates) together with Serratia liquefacians and Aeromonas hydrophila were used to assess their potentials to degrade PAHs and PCBs at concentrations of 1.0 μg/mL. An aliquot of 1.0 μL of the bacterial suspension with an optical density of 1.0 at 600 nm was used as an inoculum of the assay. Isolates were analysed for their ability to degrade PCB (Aroclor 1260) by measuring a shift in the wavemax using Cary 300 UV-visible spectrophotometer for a period of 96 hours. The presence /absence of the compounds was checked using High Performance Liquid Chromatography (HPLC) UFLC Shimadzu using florescence detector pump RF-20A and system gold column C18 (CTO-20A) after 96 hours. PCBs were extracted from wastewater samples from both Gaborone and Mafikeng using the Quick, Easy, Cheap, Effective, Rugged and Safe (QuEChERS) extraction kit and analysis was performed using the Gas Chromatography Mass Spectrometer (GC-MS). The bacteria were able to degrade these compounds under different pH values of 5.0, 7.0, 8.0 and 9.0 and temperatures of 20°C, 27°C, 30°C and 35°C. These results showed that degradation occurred at the most at 35°C and the least at 20°C for all the PAH and PCB samples that were used in the study. At 27 and 30°C the activity for the bacteria similar. The bacteria strain MD2 was able to completely degrade Aroclor 1260 that was incoperated into the wastewater samples within 96 hours. This was supported from the fact that there was a shift in the wavelength from 224 nm to 270 nm which indicated that Aroclor 1260 was degraded and thefore forming a chlorobenzoate derivative. From this finding it can be concluded that the wastewater samples did not possess PCB (Aroclor 1260) after treatment with bacteria and can be safely recycled. The physico-chemical properties indicated that the pH (7.78 − 7.88), temperature (24.4 °C − 26.1 °C) and turbidity (0.01 NTU) values for control samples were within acceptable limits as per Botswana, South Africa and WHO drinking water standards. On the contrary, turbidity values were rather higher than the set standards for raw water and wastewater, with water samples from Modimola/Setumo dam having recorded the highest range of 25.0 − 200 NTU. The pH values of samples obtained from Modimola/Setumo dam were higher at 9.01 to 9.78. Wastewater effluent in Notwane Sewage Treatment Plant, Gaborone Dam (both in Gaborone) and Disaneng Dam (South Africa) have polychlorinated biphenyls (PCBs) below detectable levels by the Agilent Gas Chromatography Mass Spectrometer (GC-MS). Only traces of PCBs were detected from wastewater from Modimola dam in Mafikeng. This may be due to the different industries in Mafikeng producing various chemicals compared to Gaborone. The water in Modimola dam therefore requires thorough treatment before it can be returned for domestic consumption as PCBs are toxic compounds that have been found to trigger cancer in humans and also affect the reproduction system resulting in babies that have low inteligence quotient. These results indicate that the isolates obtained and screened in the study may be very useful in the biodegradation of recalcitrant PAHs and PCBs that are usually present in wastewater. In conclusion, the objectives of the study were fully achieved. Bacterial strains Serratia liquefacians and Aeromonas hydrophila, together with Pseudomonas aeruginosa and isolate ID MD2 were capable of degrading recalcitrant compounds under different environmental conditions. The result from this study also showed that wastewater from Setumo/Modimola dam (Mafikeng) is more polluted than the wastewater from Notwane Sewage Treatment Plant (Gaborone) with regard to the target compounds as well as taking into consideration the turbidity and pH values obtained from the samples. The wastewater from Setumo/Modimola was also found to contain traces of polychlorinated biphenyls. The results from this study therefore suggest that the wastewater from Setumo/Modimola need thorough treatment to render it safe for recycling for purposes of introducing it into treatment plant for potable water production. The target recalcitrant compounds were not detected in wastewater samples Notwane sewage treatment Plant and raw water from Disaneng and Gaborone dams. Although it may be concluded that the wastewater from Notwane Sewage Treatment Plant can be diverted to Gaborone dam to enhance the water level and thus be treated for portable water use, more studies still need to be carried out to check for the compounds through studying the vegetation, fish, and beef from cattle using those places as water holes, before a final recommendation can be forwarded to the Water Utilities Cooperation (WUC). This was the first study on use of bacterial isolates to breakdown PAHs and PCBs to be carried-out on wastewater in these two areas (Gaborone and Mafikeng). Moreover, the study was designed to target recalcitrant compounds, specifically the polycyclic aromatic hydrocarbons (PAHs) and polychlorinated biphenyls (PCBs).There is need for extensive research to screen for and identify bacteria strains among the isolates from wastewater treatment plants that can efficiently degrade recalcitrant compounds.. In addition, it is also important to thoroughly screen the wastewater to determine the type of compounds or metabolites that are produced during biodegradation of PAHs and PCBs as well as their effect on other microorganisms that are found in the water bodies. It is therefore suggested that there is the need to conduct further studies designed to detect specific genes that are actively involved in the production of enzymes that catalyse the biodegradation of PAHs and PCBs respectively.