Isolation and characterization of Bacillus spp. for use in the remediation of petroleum waste residues

Petroleum hydrocarbons are toxic to all forms of life; therefore, environmental pollution caused by petroleum is of great concern. Environmentally friendly strategies are required for the remediation of the contaminated sites. Microbial populations comprising of several different genera have been detected in soil and water environments that have been contaminated with petroleum. This suggests that these organisms are able to use hydrocarbon compounds as a substrate for survival and could be harnessed in bioremediation of contaminated sites. The first stage of this research was focussed on the isolation, purification, screening and selection of putative Bacillus spp from environmental samples. Samples were collected from different sites around the Gauteng province in South Africa. Samples from both soil and water were obtained from selected sites including environments that were contaminated by oil. Isolate selection was based on the growth rate of the isolates, the degree of sporulation and the rate of oil degradation. The identities of the potential isolates as well as their safety status were clarified in order to reduce possible risk to end users or the environment. Once suitable isolates were identified, those that possessed inherently strong biodegradation ability were assessed for their efficacy as well as compatibility to perform in a consortium. Various organism combinations were assessed and compared to the efficacy of individual isolates, in order to formulate a bioremediation consortium. Of the 115 isolates, the top performing isolates, identified as GPA 11.2, GPA 7.1, GPA 3.5, GPA 8.3 and GPB 4.4, were obtained from a car workshop in Midrand and a taxi rank in Silverton. GPA 8.3 and GPA 4.4 were, however, eliminated due to their low sporulation efficiency. The selected Bacillus isolates were identified using 16s rDNA sequencing and GPA 7.1 and GPA 11.2 were identified as B. subtilis, while GPA 3.5 was identified as B. methylotrophicus. These isolates 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 bioremediation potential of the consortium was evaluated using industrial effluents that contained hydrocarbons. Degradation of hydrocarbons using all three consortiums (Gen 3.1, Gen 3.2, Gen 3.3) in the respective industrial effluents were determined by measuring the rate of degradation for each hydrocarbon compound using Gas Chromatography (GC). Results indicated that the bulk of the contaminants were removed during the first 48 hours; and removal (%) did not increase significantly after 72 hours. The total petroleum hydrocarbons (TPH) (C8-C28) removal rates from synthetic effluent after 48 hours of treatment using the Gen 2, Gen 3.1, Gen 3.2 and Gen 3.3 consortia were 0.8, 0.26, 0.07 and 0.58 mg.L-1.h-1, respectively. The TPH (C8- C28) removal rate from true effluents after 48 hours of treatment using the Gen 2 (benchmark), Gen 3.1, Gen 3.2 and Gen 3.3 consortia were 0.23, 0.25, 0.12 and 0.17 mg.L-1.h-1, respectively. The results showed that the best performing consortium was Gen 3.1.This study has demonstrated the potential application of Bacillus as bioremediation agents for the treatment of hydrocarbon-contaminated sites. This technology could potentially also be utilised for addressing the challenges of a wider range of different hydrocarbon effluents.