Development of an active bacterial formulation for degradation of complex crude oil wastes

Abstract

Oil refineries generate huge amounts of crude oil sludge, a complex waste product containing many carcinogens. The rapid increase in crude oil sludge pollution on land and water has been an enormous environmental challenge and a serious health concern over the years. Bioremediation of crude oil sludge has faced challenges, such as the availability of the contaminant molecules to the degrading microbial populations, survival of degrading microorganisms and the deficiency of required nutrients to sustain microorganisms' activities during the biodegradation process. To overcome these challenges and enhance the biodegradation of crude oil sludge, this study aimed to use adapted bacteria generated from composting to develop an active bacterial formulation (ABF) to degrade complex crude oil refinery wastes sludge. The active degrading bacteria generated from co-composting crude oil sludge with different animal manures were isolated and identified using molecular techniques, a culture-dependent and culture-independent metagenomic methods (Illumina MiSeq analysis), a high throughput deep sequencing of 16S rRNA genes approach. The selection of isolates for the development of the active bacteria formulation was based on their capabilities to produce enzymes and biosurfactant, possession of catechol-2, 3-dioxygenase genes, and 2,6 DCPIP assay/screening test. The active bacteria formulations were developed in 3 different sets, Wet/ABF-1, Air-dry/ABF-2, and Freeze-dry/ABF-3 consisting of a carefully selected bacteria consortium with specialized capabilities, modified and immobilized on corncob powder. The formulations were evaluated for their shelf life and viability before and during storage, which helped to observe changes in colony-forming units (CFUs) and determine their shelf life and viability during storage at room temperature. The formulations were applied to degrade complex crude oil waste sludge, and the efficacy was measured by the PAHs residual concentrations after treatment using automated Soxhlet extractor and Dichloromethane as the extraction solvent, quantified with GC/MSD. The composition of the active degrading bacteria community was analysed using culture-dependent and culture-independent approaches to elucidate the autochthonous microbial community present. The diversity composition was determined by high throughput deep sequencing of 16S rRNA genes (Illumina MiSeq next generation sequencer). After the treatment, the treated soil oil sludge mixture was tested for toxicity on sweet bell pepper plants to determine their safe disposal in the environment. Also, the effect of the untreated complex crude oil waste sludge was compared with that of the treated soil-oil sludge/ABF by application on sweet bell pepper plants in a greenhouse set-up. Co composting of crude oil waste sludge with animal manures efficiently generated bacteria capable of degrading PAHs. Parameters such as temperature, moisture content, pH level, ash content, carbon dioxide evolution, and oxygen consumption measured during the incubation period showed that microbial activities were enhanced. The culture-dependent method and Illumina high-throughput culture-independent sequencing platform completely elucidated the bacteria community composition in this study. The sequences classified under the bacterial domain yielded the top 4 dominant phyla Proteobacteria, Actinobacteria, Firmicutes, Bacteroidetes, and minor phyla (unclassified) in different proportions affiliated to over 90 genera. The dominant genera among the immobilized cells used for the active bacteria formulation were Rhodococcus sp., Bacillus sp., Gordonia sp., Dietzia sp., Ochrobactrum sp., Clostridium sp., Pseudomonas sp., and Microbacterium sp., which were detected in high relative abundance ranging from 1.0>10>. They have been consistently detected in petroleum contaminated environments because of their distinctive capabilities to degrade polycyclic aromatic hydrocarbons (PAHs), this suggests their highest abundance in these present experiments. The degradability-screening test, the 2,6-DCPIP assay, showed that 54% of the bacteria strains were positive to redox indicator reaction within the first 3 days and 77% of the degrading bacteria harboured the catechol 2,3-dioxygenase (cbzE gene). Corncob powder was an ideal carrier material for this study, as confirmed by the scanning electron micrograph (SEM) images that bacteria cells were firmly immobilized on the corncob surface. The application of the formulations to degrade complex crude oil sludge revealed a shift or differentiation in genera composition, attributed to materials, nutrient or carbon sources used for the experiment, as well as simultaneous factors such as symbiosis and competition to utilize different carbon sources. The treatment effect was evaluated via bacteria community composition and relative abundance, and PAH-residual level. The sweet bell pepper plant proved to take up PAHs revealing its phytoextraction ability in the process and exhibited rapid structural changes due to the PAH toxic effects. The results suggest that challenges such as the survival of microbial communities with degrading capabilities and nutrient deficiency in the treatment system were addressed by the application of the active bacteria formulation, and crude oil sludge degradation with some PAHs being thoroughly degraded, near-complete degradation (95-100%) but over 80% degradation in all the treatments was achieved within 90 days as compared to 10 months in the previous study. Thus, the aim of this study to improve bioremediation using the developed active bacteria formulation and effectively shorten the crude oil sludge biodegradation period was achieved.