Team:CCA San Diego/Description

project description

Why are PAHs important?

Contamination of aquatic and terrestrial environments with crude oils (or petroleum) represents a fundamental problem worldwide that results in both immediate and long-term environmental damages, including toxicity to plants and soil invertebrates and with significant risks to humans and wildlife. Crude oils are predominantly composed of hydrogen and carbon. The massive ecological damage caused by oil spills in the ocean can largely be attributed to a subset of crude oils called polycyclic aromatic hydrocarbons. Polycyclic aromatic hydrocarbons account for approximately 80% of the total petroleum hydrocarbons in crude oils. Aromatic carbons such as polycyclic aromatic hydrocarbons (PAHs) tend to be the most toxic and persistent molecular compounds in oil.

More on PAHs

Polycyclic aromatic hydrocarbons (PAHs) are organic compounds found in crude oils. Crude oils vary in chemical composition, color, viscosity, specific gravity, and other physical properties with color ranges from light yellow-brown to black. Viscosity varies from free flowing to a thick substance. Specific gravity varies between 0.73 and 0.95. Refined oil products most often spilled in large quantities are aviation fuel, gasoline, No.2 fuel oil (diesel fuel), and No.6 fuel oil (bunker C). PAHs are comprised of two or more benzene rings bonded in linear, cluster, or angular arrangements. The major source of PAHs is the incomplete combustion of organic material such as coal, oil and wood. PAHs are not synthesized chemically for industrial purposes. The general uses of some PAHs are pigments, dyes, wood preservatives, asphalt, and also in the field of electronics and functional plastics.

OUR SOLUTION

We proposed a novel method of degradation of multiple PAHs through combinational implementation of these bacteria-derived pathways into E. coli. Thus, this treatment allows for a broad spectrum transformation of multiple PAHs within the same oil environment into safer residues. Isolation of the bacteria allows for its implementation into a spill site or in bioreactors, achieving efficient detoxification through combination genetic bioremediation.

The degradation pathway of each of these PAHs begins with oxygenation, followed by degradation of the aromatic ring. Each of these pathways includes a complex set of 10-15 genes typically organized in operons, encoding different classes of enzymes, mainly oxygenase, hydrogenase, and carboxylase. Additional genes are involved in the regulation of expression of the pathway (activator), secreting surfactants to allow the bacteria to mix well with crude oil, and the transport of PAHs inside the microorganism where the degradation takes place.

EXPERIMENTAL DESIGN

In order to assess whether the newly engineered E. coli strains containing the fluorene catabolic pathway was able to degrade their respective PAH, they were grown in minimal medium supplemented with fluorene as a sole source of carbon. For controls, the strains were grown in presence of glucose. In addition, E. coli strains containing the corresponding vector without insert was also grown in parallel. Fluorene was prepared as stock solution of 10 mg/mL and was initially dissolved in methanol. Because the stock solution showed a slight precipitate, stock solutions of 10 mg/mL was also prepared in the organic solvent dimethyl sulfoxide (DMSO). Growth comparisons using these 2 solvents were performed in parallel.

CULTURE SETUP

Cultures were started from glycerol stock in 4 mL of medium and incubated at 37°C. The OD readout of the overnight cultures was determined using a spectrophotometer according to the protocol shown above. All cultures were then diluted to 0.02 using the volume below and OD measurements were determined at the indicated time points.

CONCLUSION

The data show all absorbance measurements obtained during the biotransformation of fluorene by our recombinant E. coli in minimal medium supplemented with PAHs. To evaluate whether the recombinant cells had the ability to transform PAHs, growth experiments were set up with various clones expressing the fluorene catabolic pathway. The clones described above with the catabolic pathway under the control of 3 different constitutive promoters were set in cultures using minimal medium supplemented with fluorene (0.1 mg/mL) as sole source of carbon (figures below). Antibiotics were added as appropriately. Clones containing the catabolic pathway exhibited higher cell density at 48 hours compared to their respective controls (vector alone) with the strongest promoter given a greater advantage (clone 48 or K2491013 for fluorene). PAHs dissolved in DMSO appeared slightly more available than PAHs dissolved in methanol. This may be explained by the fact that PAHs stock solutions prepared in methanol exhibited some precipitates not observed with DMSO-based stocks.

[[Image:MMFluGrowth2017.png|600px]]

[[Image:MMFluGrowthMeth2017.png|600px]]

Figure 1. Growth of recombinant E. coli BL21DE3 cultures harboring the control plasmid pSB3T5 or the fluorene pathway under the control of 3 different constitutive promoters: BBa_J23100 (clone 48), BBa_J23101 (clone 51), and BBa_J23110 (clone 54) cloned into pSB3T5. Data points represent value averages of the duplicate of OD at 600 nm taken over time for 2 independent colonies per clone. Recombinant clones were grown in minimal medium supplement with tetracycline (15 µg/mL) and fluorene (0.1 mg/mL). Fluorene was dissolved in 100% DMSO (Top panel) or 100% methanol (Bottom panel).

Growth comparison of recombinant E. coli in minimal medium supplemented with PAHs and glucose. In order to evaluate the role of toxicity and/or the metabolic burden caused by the PAH catabolic genes and the PAHs, namely fluorene, cells were grown in minimal medium together with fluorene and glucose as carbohydrate sources (figures below). It appears that all 3 clones carried by a low copy plasmid number with the fluorene catabolic pathway under the control of 3 promoters of various strengths behaved similarly to the control strain harboring the corresponding vector pSB3T5 with no insert. The fluorene genes independently of their expression level do not appear to impact cell growth when carried by a low copy vector. In addition, fluorene (0.1 mg/mL) in presence of glucose is not toxic to the cells.

[[Image:MMGrowthOverTime.png|600px]]

Figure 3. Fluorene biotransformation experiment using recombinant E. coli BL21DE3 harboring the control plasmid pSB3T5 or the fluorene pathway under the control of 3 different constitutive promoters: BBa_J23100 (clone 48), BBa_J23101 (clone 51), and BBa_J23110 (clone 54) cloned into pSB3T5. Data points represent value averages of duplicate of OD at 600 nm taken over time for 2 independent colonies per clone. Recombinant clones were grown in minimal medium supplement with tetracycline (15 µg/mL), fluorene (0.1 mg/mL), and glucose (0.4%).

EXPERIMENTAL DESIGN

This study was aimed at determining the growth rate of the recombinant strains containing the fluorene catabolic pathway alone or together when using fluorene as sole source of carbon. Recombinant cells were grown in minimal medium supplemented with fluorene as a sole source of carbon. For controls, the strains were grown in presence of glucose. In addition, E. coli strains containing the corresponding vector without insert was also grown in parallel. PAHs were dissolved in DMSO.

CULTURE SETUP

Cultures were started from glycerol stock in 4 mL of medium and placed at 37°C. The OD readout of the overnight cultures was determined using a spectrophotometer according to the protocol shown above. All cultures were then diluted to 0.02 using the volume below and OD measurements were determined at the indicated time points.

[[Image:MoreGrowthTestFlur.png|600px]]

Graph 3. Average absorbance values measured in quadruplet at 600 nm of cultures of 2 independent colonies of control (vector) and catabolic plasmid (clone CCA-48 or K2491013 for fluorene) at 2, 4, 24, and 48 hours after inoculation of minimal media supplemented with fluorene at 0.1 mg/mL with or without glucose (0.4%) at 25. All media contained the surfactant Tween-20 (0.1%). Cultures were grown at 25°C or 37°C. MM= Minimal Medium, Glu= Glucose; Ave.= Average; OD=Optical Density; SD= standard deviation.

Results

Here are our results for our project...