From commercial fisheries to international trade, millions of people worldwide rely on marine ecosystems for their livelihood, food security, and way of life. Yet, the thousands of oil spills occurring annually in the U.S. pose a serious threat, impeding growth, destroying habitats, and poisoning our food. Oil spills have also been documented to have serious impacts on human health. Exposure to oil spills has led to reduced lung function, heart attacks, memory loss, and confusion (Sandifer et al., 2021).
In relation to our team, last year, at the Port of Wilmington, just a 15-minute drive from our school, over 3500 liters of oil spilled into the local Christina River Basin, which provides over 300 million liters of drinking water to half a million people daily (Baker & Stonesifer, 2024; CBCWP – Facts, 2015). Current existing solutions on degrading oil have limitations. Oil-absorbing materials pose difficulties with disposal, can harm marine ecosystems, and may have trouble dealing with larger oil spills (Sharma et al., 2024). Concentrating oil with booms and picking it up with skimmers is a time-consuming process and is greatly dependent on ocean currents and spill size (Etkin & Nedwed, 2021). Burning oil spills, while it can be a relatively fast way to clean up oil spills, can increase harmful air pollution in the surrounding areas, and oily residues remaining in the water can cause damage to the environment (United States Environmental Protection Agency, 2018).
Alcanivorax borkumensis, a halophic and hydrocarbonoclastic bacterium that is adapted to oil-polluted environments, is a promising candidate for bioremediation.
Marine oil spills and plastic pollution pose recurring threats to ocean ecosystems. Alcanivorax borkumensis – a halophic and hydrocarbonoclastic bacterium that has evolved to thrive around oil-contaminated environments – acts as a promising candidate for bioremediation. According to past studies, the concentration of A. borkumensis increases naturally and becomes abundant after oil spills (Karmainski et al., 2023). A. borkumensis efficiently degrades alkanes, utilizing a variety of specialized enzymes which include the following: alkane monooxygenases, cytochrome P450s, and biosurfactant-producing pathways. Additionally, its salt tolerance, metabolic abilities for long-chain hydrocarbons, and ease of culturing, along with its minimal reliance on complex nutrients, make it a perfect model organism. Within this study, we explored the biofilm formation and alkane oxygenation of A. borkumensis, identifying that it had overall better performance compared to champion isolates. Furthermore, by analyzing a proteomic study, we identified secreted proteins that are involved in the first step of alkane degradation and tested their degradation abilities through a biochemical NPD-hydrazine assay. Through these experiments, we deem the flavin-binding monooxygenase AlmA a promising protein in alkane degradation. The study also focuses on creating a genetic toolbox for A. borkumensis by studying conjugation and broad-host range plasmid transformation. The findings from this study give insight into the enzymes responsible for the first steps of degradation and set the foundation to create more complex genetic toolboxes for A. borkumensis. Future implementation, more efficient oil spill cleanup, has the potential to save marine life, protect coastal industries, and mitigate economic harm from oil spills.
Karmainski,. T., Dielentheis-Frenken, M. R. E., Lipa, M. K., Phan, A. N. T., Blank, L. M., & Tiso, T. (2023). High-quality physiology of Alcanivorax borkumensis SK2 producing glycolipids enables efficient stirred-tank bioreactor cultivation. Frontiers in Bioengineering and Biotechnology, 11, 1325019. https://doi.org/10.3389/fbioe.2023.1325019
Marine spills and plastic pollution represent two of the most common threats to marine ecosystems. Polyethylene (PE), a major component of plastic waste, is extremely difficult to naturally degrade, while hydrocarbons from oil spills tend to accumulate and disrupt the balance of marine ecosystems. Current physical remediation efforts and methods are costly, inefficient, or environmentally damaging. Our team decided to focus on utilizing environmentally friendly bioremediation strategies that could reduce contamination in marine environments and its effects. However, nature already has a partial solution. Certain bacteria have evolved to contain biodegrading proteins and have the ability to metabolize oil. The problem, however, lies within our ability to efficiently implement them in large-scale clean-ups. In addition, degrading plastics remains even more stubborn, with hardly any microbes that have the ability to do that. However, one of these microbes, A. borkumensis, thrives exclusively on hydrocarbons by naturally secreting enzymes capable of oxidizing long-chain alkanes, which is the first step in breaking down crude oil. On the other hand, research in the mealworm microbiome revealed additional information on bacteria capable of degrading plastics. The ability to identify A. borkumensis’s native degradation mechanisms and compare it to plastic-degrading microbes found in mealworms allows for endless possibilities of engineering a bioremediation chassis capable of both targeting oil and plastic pollution.
This project advances in three main steps. First, we aimed to determine whether A. borkumensis secreted extracellular enzymes responsible for the initial oxidation and degradation of hydrocarbons. We also identified and selected specific proteins we wanted to target and potentially engineer to increase efficacy. To test this, we selected candidate enzymes based on proteomic data and literature on alkane degradation pathways (Zadjelovic et al., 2022). These proteins were individually expressed in E. coli, verified in SDS-PAGE and western blotting, and then structurally analyzed through modeling to confirm functional relevance. Second, we confirmed A. borkumensis' innate bioremediation abilities by experimentally measuring its ability to degrade plastic-like substances and employed assays such as biofilm formation on hydrophobic surfaces, growth on oil or plastic as the pure carbon source, and FTIR spectroscopy. Finally, we explored methods to enhance biodegradation performance through synthetic biology. Using conjugation and broad-host-range plasmids, we introduced additional genes that assist in plastic degradation. Transformation success was indicated through antibiotic resistance markers and followed by repeated biodegradation tests to quantify improvements. These experiments collectively test our hypothesis that A. borkumensis can serve as a versatile bioremediation pathway for oil & plastic degradation.