Theme: 06. Petroleum and Heavy Hydrocarbon Site Strategies

Topic Area: 6e. Surfactant-Enhanced Remediation

Formatted Title:

Impact of Surfactant Use and Cell Immobilization on the Bioremediation of Polycyclic Aromatic Hydrocarbons by Rhodococcus rhodochrous ATCC 21198

Background/Objectives:

Polycyclic aromatic hydrocarbons (PAHs) are a class of ubiquitous environmental contaminants associated with adverse ecological and human health effects. Bioremediation of PAHs, although often the least expensive and invasive remediation strategy for PAHs, is not without its own unique challenges. Firstly, microorganisms that metabolize PAHs cannot degrade PAHs to treatment goal concentrations, as they are dependent on the PAHs for their activity and survival. Secondly, PAHs sorb strongly to soil and sediment in the environment due to their hydrophobicity, significantly limiting their bioavailability. The objectives of this research were to address these two challenges using a bacterial pure culture, Rhodococcus rhodochrous ATCC 21198 (21198) that has been recently discovered by our research group to transform PAHs via cometabolism. To increase bioavailability of PAHs, the surfactant Tween ® 80 was used to solubilize PAHs and aid in their transport to the cells. Cell immobilization techniques using hydrogels were also investigated to protect cells from the surfactant and retain biomass in the batch system.

Approach/Activities:

Laboratory-scale batch experiments were conducted in this research. A pure culture of 21198 grown on isobutane was used for all bioremediation experiments. Studies were conducted in batch bottles with a synthetic groundwater solution and a mixture of four PAHs: phenanthrene, fluorene, anthracene, and pyrene. 21198 was introduced to batch bottles either as suspended cells or immobilized cells in polyvinyl alcohol/sodium alginate hydrogel beads. A subset of batch bottles contained Tween ® 80. Samples were taken over the course of the experiments and analyzed for PAHs using liquid-liquid extraction and gas-chromatography-mass spectrometry (GC-MS). Additional samples were taken simultaneously to monitor PAH degradation and the formation of PAH metabolites with a non-targeted fluorescent spectroscopy technique. Endpoint samples were taken and extracted with solid phase extraction for investigation of PAH metabolites using both targeted and non-targeted approaches with ultra-high-performance liquid chromatography coupled with high resolution mass spectrometry (UPLC-HRMS).

Results/Lessons Learned:

Preliminary work with 21198 and a mixture of PAHs has demonstrated that 21198 can transform all PAHs studied, although the presence of Tween ® 80 was required for observable pyrene degradation. 21198 was found to have a moderate tolerance (~1 g/L) for Tween ® 80 both in suspended and immobilized forms, although Tween ® 80 was also found to be degraded by 21198 during remediation experiments. Fluorescent spectroscopy and parallel factor analysis results corroborate GC-MS findings for PAH transformation and indicate the formation and accumulation of PAH transformation products. UPLC-HRMS results revealed a complex mixture of hydroxylated products, quinones, and ring fission products, suggesting transformation pathways involve several enzymatic steps. At the time of presentation, results from non-targeted UPLC-HRMS analysis will be available to further elucidate potential PAH transformation used by 21198. Similarly, preliminary results from expanded studies with immobilized cells will be available to demonstrate the utility of this bioremediation system beyond batch-scale systems.

Jason Schindler

Affiliation

Refine Results

(Group 2, Poster Board #62) Impact of Surfactant Use and Cell Immobilization on the Bioremediation of Polycyclic Aromatic Hydrocarbons by (ital)Rhodococcus (ital)rhodochrous ATCC 21198