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South Africa’s freshwater resources, including rivers, man-made lakes and groundwater are under severe threat due to an ever-expanding population and economy, which is depleting these resources. The increase in population has a direct correlation with the increase in wastewater generated. The remaining fresh water resources need to be preserved therefore recycling of wastewater, to replenish our water supplies and preserve the environment, is a solution to the problem. For a developing country, it is important to use treatment methods that are cost effective and do not exert a negative impact on the environment, such as biological wastewater treatment options. One of the systems commonly used in biological wastewater treatment is the fluidized-bed bioreactor (FBBR) due to its advantages such as higher biomass concentration and a higher mass transfer thus resulting in a higher rate of biodegradation. This study focused on evaluating the efficacy of augmenting with Bacillus spp. to enhance the bioremediation of wastewater using a FBBR. Bacillus spp. used in this study were isolated from a municipal wastewater treatment plant (10 isolates) and the remaining three isolates were selected from the CSIR Bacillus database. The isolates (13 in total) were screened for 1) their ability grow in wastewater, 2) ability to reduce high concentrations of COD, ammonium, nitrates and phosphates in flask studies containing synthetic wastewater (SWW) and 3) ability to produce common enzymes such as amylase, cellulase, lipase and protease. Isolates showed varying bioremediation potential for different compounds analysed. Isolate B006 showed the highest phosphate removal rate (3.290 mg.L-1.h-1) where as D005 showed the highest growth rate (0.955 h-1), COD reduction rate (55 mg.L-1.h-1) and cellulase activity (5.485 mm) among all the isolates. Isolate D014 presented the highest ammonium removal rate (12.43 mg.L-1.h-1), amylase (5.00 mm) and protease (10.00 mm) activity whilst B001 displayed the highest nitrate removal rate (9.4 mg.L-1.h-1). The results for the individual assays were assessed and weighted in a matrix and the isolates that scored above 50% were selected for consortium studies. Four Bacillus spp. that scored above 50% in the scoring matrix were then evaluated for their ability to co-exist as a consortium. The consortium studies were then compared with results obtained for individual isolates. The selected Bacillus isolates were identified and assessed for their safety to the environment and to the end user. Identification was conducted using 16s rDNA sequencing and results showed that B006 identified as B. cereus, D005 as B. cereus and D014 as B. subtilis. Isolates, B006 and D005 were further assessed for enterotoxin production and the presence of anthrax virulent plasmids pX01 and pX02. After conducting the biosafety assays, the isolates were rendered safe for use. The isolates were then cryopreserved as spores in 25% glycerol and stored at -80 °C. The impact of the cryopreservation method and the storage conditions on the viability of the isolates was assessed after six months of storage and it was established that the isolates were still viable and that the method was adequate. The bioremediation potential of the consortium was further evaluated using a 17 L Pilot scale fluidised-bed bioreactor. The reactors were fed at three different flow rates of 1.5 L.h-1, 2 L.h-1 and 3 L.h-1 over steady state conditions (~3months). The results showed that the FBBR augmented with the selected Bacillus isolates, resulted in improved nutrient (COD, ammonium and phosphates) removal efficiencies compared to the non-bioaugmented control. The highest ammonium removal (62.8%) was observed at a flow rate of 1.5 L.h-1 (11.30 h retention time), whereby there was an overall 29.8% improvement in ammonia removal in comparison to the non-augmented control. Similarly, an overall improvement in phosphate (14.73%) was observed at a flow rate of 2 L.h-1 (8.48 h retention time) with 50% removal efficiency. The highest COD removal was observed at a flow rate of 1.5 L.h-1 (11.30 h retention time) whereby 74.5% COD was reduced with a 32.6% improvement when compared to the non-bioaugmented control. Our work has demonstrated the potential application of Bacillus as bioaugmentation agents to enhance wastewater treatment efficiency as a potential solution to water challenges in developing countries. This technology could also be utilized for addressing the challenges of a wider range of different effluents.

Bioaugmentation with Bacillus spp. for bioremediation of synthetic wastewater using a fluidized-bed reactor