The potential of plant growth-promoting bacteria to enhance rhizoremediation of diesel fuel-contaminated sites

Crude oil and fuel spillages are the most persistent environmental menace resulting from oil and gas exploration, production and utilisation. The United States Environmental Protection Agency (US EPA) estimated that rehabilitation can cost over $US1 million per hectare. Traditional solutions for remediation are expensive and environmentally unfriendly. Consequently, more cost-effective and eco-friendly remediation technologies are needed. The use of plants to clean up contaminated sites is a cost-effective and an environmentally-friendly approach. However, the toxicity of petroleum hydrocarbons to most plants, coupled with the slow rate of natural attenuation limits the effectiveness of this approach. Therefore, the identification of hydrocarbon tolerant plants and the isolation of microbial consortium and isolates capable of plant growth promotion and hydrocarbon degradation is crucial to the success of plant-based remediation techniques. This is the crux of this research. In the first part, I examine how ethanol addition to diesel fuel affects the leaching potentials of diesel fuel hydrocarbons. Since rhizoremediation of hydrocarbons depends largely on rhizodegradation of contaminants by the root-associated microbiome, the leaching of petroleum hydrocarbons beyond the rooting zones of plants may limit the effectiveness of this process as a reclamation strategy. The results revealed that while 5% (by volume) ethanol addition had a limited effect on aromatic hydrocarbons, 10% ethanol addition resulted in the elution of all classes of aromatic hydrocarbons studied beyond a 90 cm column. This revealed the need for choosing plants with adequate rooting system for an effective rhizoremediation of organic contaminants. Secondly, through phytotoxicity bioassays, I selected Medicago sativa as the most suitable species for rhizoremediation of diesel fuel. Dose-response analysis revealed that increasing diesel fuel concentrations in the soil generally led to a monotonically-decreasing biomass in all other studied plant species (P < 0.001), with EC10 values (±SE) ranging from 0.36 ± 0.18 g/kg to 12.67 ± 2.13 g/kg. On the other hand, hydrocarbons had a statistically significant hermetic influence on M. sativa (f = 3.90 ± 1.08; P < 0.01). Interestingly, exposure to diesel fuel contaminated soil up to 10 g/kg did not affect the viability of M. sativa seeds, although time to seed emergence was delayed. These factors position M. sativa as the most-promising plant species for microbially-enhanced rhizoremediation of diesel fuel. In the third part of the research, I successfully isolated a bacterial consortium and single isolates that can metabolize diesel fuel hydrocarbons as their sole carbon and energy source, while promoting the growth of host plants. In addition, I elucidated the genes and metabolic pathways involved in these reactions. I also reconstructed a number of metagenome-assembled genomes, many of which contained genes putatively involved in hydrocarbon degradation, with potentials for bioremediation application. Finally, I examined the rhizoremediation effectiveness of M. sativa inoculated with either the consortium or M. sativa inoculated with Paraburkholderia tropica single isolate. The results indicated that M. sativa–P. tropica symbionts successfully enhanced the rhizodegradation of diesel fuel hydrocarbons. The geochemical analysis of residual hydrocarbons revealed that the combined action of M. sativa and P. tropica resulted in 96% degradation of the total diesel fuel hydrocarbons within 60 days. Biodegradation was further confirmed using parameters such as nC17/pristane, nC18/phytane, nC16/nor-pristane and total petroleum hydrocarbons/unresolved complex mixture ratios. Molecular analysis of biodegradation revealed that the polycyclic aromatic hydrocarbon components of the diesel fuel were almost completely degraded by the plant-microbe symbionts. I am confident that the results of this research will revolutionize the way diesel spills and other organic contaminants are cleaned up, and facilitate the reclamation of petroleum contaminated sites.