Team:Calgary/Project

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TITLE=A Biosensor for Naphthenic Acids|
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<h2>Naphthenic Acids in the Oil Sands</h2>
<h2>Naphthenic Acids in the Oil Sands</h2>
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<p>Naphthenic Acids (NAs) are a grouping of different carboxylic acids which may contain multiple hydrocarbon rings. Containing both a large hydrocarbon segment and a carboxylic acid group makes them a slightly amphipathic compound. NAs are natively found in oil sands deposits and their surfactant quality contributes to a higher efficiency of oil sands recovery in the hot water extraction process. The majority of the NAs end up in large tailings ponds with the water used in the bitumen extraction process. The NAs continuously accumulate in these large tailings ponds along with other wastes generated in the bitumen extraction process which are left to settle to the bottom of the pond.</p>
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</html>[[Image:UofC2011_Whitbey.png|thumb|600px|center|<b>Figure 1.</b> Structure of Naphthenic Acids as discussed in Whitby, C. <i>et al.</i> 2010.]]<html>
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Pic of tailings pond
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<p>Naphthenic Acids (NAs) are a group of recalcitrant and hydrophobic compounds comprising a variety of structures. All NAs contain a conserved carboxylic acid group followed by a hydrocarbon chain. Attached to this hydrophobic chain there may be between one and four hydrogenated ring systems. NA classification criteria are continually debated in the scientific community. With such diversity within this chemical class, the biodegradation of NAs is difficult to define due to the likelihood of biodegradation pathways interacting with very specific topological structures. Thus one pathway likely does not degrade all NAs, and NA research has yet to define any NA degrading pathways (Whitby, 2010).</p>
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</html>[[Image:UofC_TailingPond.png|thumb|600px|center|<b>Figure 2.</b> Courtesy of Pembina, a picture of a tailings pond]]<html>
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<h2>Bitumen Extraction Process</h2>
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<p>NAs naturally occur in all petroleum deposits and their surfactant quality is actually useful for higher efficiency of oil sands recovery in the hot water extraction process. Post processing, the majority of NAs from the tar sands separate into the waste water aqueous fraction and are then collected in large tailings ponds along with a variety of other toxic by-products of bitumen extraction. The NAs continuously accumulate in these large tailings ponds and are allowed to settle to the bottom of these pools.</p>
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</html>[[Image:UofC_Extraction.png|thumb|600px|center|<b>Figure 3.</b>The extraction process from bitumen to oil, showing where waste like naphthenic acids is extracted]]<html>
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<h2>The Toxicity of Naphthenic Acids</h2>
<h2>The Toxicity of Naphthenic Acids</h2>
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<p>Unfortunately NAs are a contributor to corrosion in equipment and pipelines. In addition, their surfactant nature also makes them toxic as it allows them to pass through cell membranes and adversely affect a plethora of local organisms. The higher the concentration of the NAs in the environment the greater the potential harm to the local ecosystem.</p>  
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<p>One of the major industry concerns with NAs is their contribution to corrosion of equipment and pipelines. In addition, their surfactant nature also makes them toxic in mammalian systems allowing them to pass through and potentially disrupt cell membranes. NAs with a higher molecular weight are typically less toxic than those with a lower molecular weight, however, the lower molecular weight compounds still have significant toxicity with LC<sub>50</sub> values of less than 50 mg/L in freshwater systems. The higher the concentration of NAs in the environment the greater the potential harm to the local ecosystem (Dokhoyan and Magomedov, 1984).</p>  
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</html>[[Image:UCalgary2011_LC50_values_for_fish_and_stuff.png|thumb|600px|center|<b>Figure 1.</b> LC50 values for Adult kutum, sturgeon, and roach when exposed to Napthenic Acids. Adapted from Dokholyan VK., Magomedov AK. (1983). ]]<html>
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</html>[[Image:UCalgary2011_LC50_values_for_fish_and_stuff.png|thumb|600px|center|<b>Figure 4.</b> LC<sub>50</sub> values for Adult kutum, sturgeon, and roach when exposed to Napthenic Acids. Adapted from Dokholyan VK., Magomedov AK. (1983). ]]<html>
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<h2>Monitoring Naphthenic Acids</h2>
<h2>Monitoring Naphthenic Acids</h2>
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<p>Having the ability to monitor the levels of NAs in an area would allow for the monitoring of levels of NAs in tailings ponds, and would be useful in assessing whether or not any future detoxification or remediation efforts were working. In addition, the surrounding areas could also be tested for seepage of NAs into ground water. Currently, the only ways to test for the presence of NAs are mass spectrometry and gas chromatography. These methods are both costly and inconvenient since samples must be taken off site for processing and interpreting results would take time. The University of Calgary’s iGEM team is working on developing a novel electrochemical biosensor which would allow for convenient on site monitoring of NAs. Certain components of the system would be re-useable while the actual bacteria involved in the sensing would be disposed after use.</p>
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<p>Having the ability to monitor the levels of NAs is mandated by Canadian law and would be useful in assessing whether or not any future detoxification or remediation efforts are effective. Other applications include examining the surrounding areas for seepage of NAs into ground water, and determining the extent of damage caused by oil based chemical spills. Currently, the main way to test for the presence of NAs is through gas chromatography-mass spectrometry. This method is costly, requires the experience of a trained technician, takes up to several hours per sample, and the samples must be shipped to a facility with the proper equipment for analysis. The University of Calgary’s iGEM team is working on developing a novel electrochemical biosensor which would allow for cheap, quick, and convenient on site monitoring of NAs. The bacteria used to sense the naphthenic acids would be contained inside a device, and respond specifically to these compounds.  Their response would signal a change in electrochemical potential in the solution that could be used as a read-out for our device in a quantitative manner.</p>
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Perhaps a pic of the system? Similar to the one on the ismos poster?
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</html>[[Image:TeamCalgary2011 The Vision Image 2.png|thumb|600px|center|<b>Figure 5.</b> The Vision of our project.  The University of Calgary team imagined a device which could allow for on-site testing of water samples for naphthenic acids.  Solutions would be placed into the device, where bacteria would recognize the concentration of naphthenic acids in solution and generate a response.  This can be detected by the device and a quantitative interpretation of the data would be viewable by the user.]]<html>
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<h2>Engineering the Biosensor</h2>
<h2>Engineering the Biosensor</h2>
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<p>The electrochemical system would consist of two parts: an NA inducible promoter and a suitable reporter. For the reporter we chose the LacZ gene which produces β -galactosidase which can cleave certain substrates. When β -galactosidase cleaves the substrate chlorophenol red β -D-galactopyranoside (CPRG) it produces chlorophenol red (CPR). CPR can be oxidized when exposed to a current and the voltage can be measured which give a value corresponding to the CPR molecules being oxidized. In this way the quantity of CPR being produced could be monitored, which in turn would be based on how much β-galactosidase was present. You can read more about this system under Project Electro.</p>
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<p>Engineering the biosensor involved three main components: finding an appropriate sensory element, characterizing a novel reporter and selecting and designing tools for an appropriate chassis for our system. Information on the sensory element can be found on our <a href="https://2011.igem.org/Team:Calgary/Project/Promoter">Promoter Project</a> page. More details on our reporter can be found on the <a href="https://2011.igem.org/Team:Calgary/Project/Reporter">Reporter Project</a> page. More details on our chassis can be found on our <a href="https://2011.igem.org/Team:Calgary/Project/Chassis">Chassis Project</a> page.</p>
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A diagram similar to the ismos poster one showing the two gene elements i.e. promoter and reporter might be nice
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<p>In order to find the promoter a couple of different approaches were employed. Firstly we attempted to create a genomic library of pseudomonas species known to degrade NAs and then screen it for NA degrading elements which would likely contain a promoter. We also created RT-qPCR experiments to search for genes of interest in those pseudomonas species which were upregulated in response to NAs. The genes selected for the RT-qPCR experiment (to observe upregulation) were selected through  an intense bioinformatics search. A CHIP-SEQ experiment was also conducted to search for any genes or proteins (possible gene regulatory elements) which would bind NAs in the pseudomonas species. Information on these approaches can be found under Project Pseudomonas.</p>
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<p>In addition, a microalgae species which was known to have some ability to degrade NAs was also examined for a possible promoter. However, this project only involved making a library (for screening purposes) of the chloroplast DNA only. Read more on this project by reading up Project Microalgae.</p>
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Latest revision as of 04:31, 29 September 2011


A Biosensor for Naphthenic Acids

Naphthenic Acids in the Oil Sands

Figure 1. Structure of Naphthenic Acids as discussed in Whitby, C. et al. 2010.

Naphthenic Acids (NAs) are a group of recalcitrant and hydrophobic compounds comprising a variety of structures. All NAs contain a conserved carboxylic acid group followed by a hydrocarbon chain. Attached to this hydrophobic chain there may be between one and four hydrogenated ring systems. NA classification criteria are continually debated in the scientific community. With such diversity within this chemical class, the biodegradation of NAs is difficult to define due to the likelihood of biodegradation pathways interacting with very specific topological structures. Thus one pathway likely does not degrade all NAs, and NA research has yet to define any NA degrading pathways (Whitby, 2010).

Figure 2. Courtesy of Pembina, a picture of a tailings pond

Bitumen Extraction Process

NAs naturally occur in all petroleum deposits and their surfactant quality is actually useful for higher efficiency of oil sands recovery in the hot water extraction process. Post processing, the majority of NAs from the tar sands separate into the waste water aqueous fraction and are then collected in large tailings ponds along with a variety of other toxic by-products of bitumen extraction. The NAs continuously accumulate in these large tailings ponds and are allowed to settle to the bottom of these pools.

Figure 3.The extraction process from bitumen to oil, showing where waste like naphthenic acids is extracted

The Toxicity of Naphthenic Acids

One of the major industry concerns with NAs is their contribution to corrosion of equipment and pipelines. In addition, their surfactant nature also makes them toxic in mammalian systems allowing them to pass through and potentially disrupt cell membranes. NAs with a higher molecular weight are typically less toxic than those with a lower molecular weight, however, the lower molecular weight compounds still have significant toxicity with LC50 values of less than 50 mg/L in freshwater systems. The higher the concentration of NAs in the environment the greater the potential harm to the local ecosystem (Dokhoyan and Magomedov, 1984).


Figure 4. LC50 values for Adult kutum, sturgeon, and roach when exposed to Napthenic Acids. Adapted from Dokholyan VK., Magomedov AK. (1983).


Monitoring Naphthenic Acids

Having the ability to monitor the levels of NAs is mandated by Canadian law and would be useful in assessing whether or not any future detoxification or remediation efforts are effective. Other applications include examining the surrounding areas for seepage of NAs into ground water, and determining the extent of damage caused by oil based chemical spills. Currently, the main way to test for the presence of NAs is through gas chromatography-mass spectrometry. This method is costly, requires the experience of a trained technician, takes up to several hours per sample, and the samples must be shipped to a facility with the proper equipment for analysis. The University of Calgary’s iGEM team is working on developing a novel electrochemical biosensor which would allow for cheap, quick, and convenient on site monitoring of NAs. The bacteria used to sense the naphthenic acids would be contained inside a device, and respond specifically to these compounds. Their response would signal a change in electrochemical potential in the solution that could be used as a read-out for our device in a quantitative manner.

Figure 5. The Vision of our project. The University of Calgary team imagined a device which could allow for on-site testing of water samples for naphthenic acids. Solutions would be placed into the device, where bacteria would recognize the concentration of naphthenic acids in solution and generate a response. This can be detected by the device and a quantitative interpretation of the data would be viewable by the user.

Engineering the Biosensor

Engineering the biosensor involved three main components: finding an appropriate sensory element, characterizing a novel reporter and selecting and designing tools for an appropriate chassis for our system. Information on the sensory element can be found on our Promoter Project page. More details on our reporter can be found on the Reporter Project page. More details on our chassis can be found on our Chassis Project page.