Team:Lethbridge/Acetic Acid Production

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<li><a href="https://2012.igem.org/Team:Lethbridge/notebook">Notebook</a></li>
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<a href="https://2012.igem.org/Team:Lethbridge/Acetic Acid Production">Acetic Acid Production</a>
<a href="https://2012.igem.org/Team:Lethbridge/Acetic Acid Production">Acetic Acid Production</a>
<a href="https://2012.igem.org/Team:Lethbridge/ACK Production">ACK Production</a>
<a href="https://2012.igem.org/Team:Lethbridge/ACK Production">ACK Production</a>
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<h2 class="pagetitle">Judging Criteria</h2>
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<h2 class="pagetitle">Acetic Acid Production</h2>
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<p>Increasing global oil demands require new, innovative technologies for the extraction of unconventional oil sources such as those found in Alberta’s Carbonate Triangle. Carbonate oil deposits account for almost 50% of the world’s oil reserves and approximately 26% of the bitumen found in Alberta 1. Due to unstable oil prices in Western Canada, these vast reserves have historically been set aside in favour of less time consuming, more economical sites. Microbial enhanced oil recovery (MEOR) has been utilized across the world to increase the productivity of difficult resources including carbonate oil deposits. Using a synthetic biology approach, we have designed the CAB (CO2, acetic acid, and biosurfactant) extraction method that demonstrates a modified MEOR method for extracting carbonate oil deposits. CAB extraction will utilize the natural carbon fixation machinery in the cyanobacteria Synechococcus elongatus to convert CO2 into sugars to fuel acetic acid and biosurfactant production in Escherichia coli. Acetic acid applied to carbonate rock increases the pore sizes and allows for enhanced oil recovery. The reaction produces gases that will help pressurize the well site to facilitate extraction. The natural biosurfactant rhamnolipid will also be applied to the carbonate rock to further enhance extraction yields.</p>  
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<p>Acetic Acid Production and Export by E. coli</p><br>
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<p>Overview</p>
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<p>To test the effectiveness of overexpressing and co-localizing PTA and ACK with the transporter Aata, we first needed to determine intrinsic acid production by Escherichia coli BL21(DE3). Glucose was used as an energy source for E. coli, since this will be produced by Synechococcus elongatus in our optimized system.</p><br>
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<p>Experimental Setup</p>
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<p>Overnight cultures of E. coli were used to inoculate fresh LB media that was supplemented with various concentrations of glucose (0-16 g/L). Growth of the cultures was monitored by measuring the optical density at 600 nm every hour for 6 hours. In addition, 4 mL of each culture were collected and centrifuged to obtain a clear supernatant. Titrations were performed with standardized 0.05 M NaOH in order to determine the concentration of acid in the media.</p><br>
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<p><img src="https://static.igem.org/mediawiki/2012/7/79/Acetic_acid.JPG"></p>
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<p> Figure 1. Growth curves of E. coli BL21(DE3) in different starting concentrations of glucose.</p>
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<p>All cultures grew at a similar rate and to a similar final density (Fig. 1). This indicates that glucose concentration does not significantly affect the growth of E. coli. The amount of acid produced by the E. coli was also not significantly influenced by glucose concentration (Fig. 2). In the culture grown without the addition of glucose, an average amount of 13 mM acid was released into the media. This concentration remained stable over the six hour time window. However, a slight upward trend can be seen for most of the cultures supplemented with glucose, with the media for those cultures reaching a final acid concentration close to 30 mM, or approximately 2-fold when compared to the control culture. Further replication of this data will be necessary to determine acid production rates by E. coli.</p><br>
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<p><img src="https://static.igem.org/mediawiki/2012/4/4a/Acetic_acid2.JPG"></p>
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<p>Figure 2. Concentration of acid in the culture media of growing E. coli. Data points represent an average concentration from three separate trials ± standard deviation.</p>
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<p>The acid produced by these cultures was due entirely to intrinsic acid production by E. coli, and we will be able to use these values to compare acid production by E. coli after overexpression of the acetogenesis enzymes PTA and ACK, as well as after using a scaffold to co-localize those enzymes with the acetic acid transporter Aata. This will indicate the level of expression needed for optimal levels of acetic acid production for CAB extraction. With the overexpression of Aata, we will also expect increased resistance to low pH, which should be reflected in the growth curve of E. coli growing in higher concentrations of acetic acid.</p>
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<p>By coupling carbon capture with acetic acid and biosurfactant production, carbonate oil deposits can be mined with reduced greenhouse gas emissions. The use of carbon fixation to feed downstream systems can be tailored for use as a module in many applications requiring inexpensive methods for fueling biological systems. CAB extraction will be suitable for large-scale bioreactors, providing an alternative, inexpensive, and environmentally sustainable method for MEOR from Alberta’s oil deposits. Furthermore, developing the carbon capture module will be of interest in oil extraction strategies using steam, as it will help with the mitigation of CO2 release caused by steam production using for example natural gas. </p>
 
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Latest revision as of 03:58, 4 October 2012

2012 iGEM - University of Lethbridge

Acetic Acid Production

Acetic Acid Production and Export by E. coli


Overview

To test the effectiveness of overexpressing and co-localizing PTA and ACK with the transporter Aata, we first needed to determine intrinsic acid production by Escherichia coli BL21(DE3). Glucose was used as an energy source for E. coli, since this will be produced by Synechococcus elongatus in our optimized system.


Experimental Setup

Overnight cultures of E. coli were used to inoculate fresh LB media that was supplemented with various concentrations of glucose (0-16 g/L). Growth of the cultures was monitored by measuring the optical density at 600 nm every hour for 6 hours. In addition, 4 mL of each culture were collected and centrifuged to obtain a clear supernatant. Titrations were performed with standardized 0.05 M NaOH in order to determine the concentration of acid in the media.


Figure 1. Growth curves of E. coli BL21(DE3) in different starting concentrations of glucose.

All cultures grew at a similar rate and to a similar final density (Fig. 1). This indicates that glucose concentration does not significantly affect the growth of E. coli. The amount of acid produced by the E. coli was also not significantly influenced by glucose concentration (Fig. 2). In the culture grown without the addition of glucose, an average amount of 13 mM acid was released into the media. This concentration remained stable over the six hour time window. However, a slight upward trend can be seen for most of the cultures supplemented with glucose, with the media for those cultures reaching a final acid concentration close to 30 mM, or approximately 2-fold when compared to the control culture. Further replication of this data will be necessary to determine acid production rates by E. coli.


Figure 2. Concentration of acid in the culture media of growing E. coli. Data points represent an average concentration from three separate trials ± standard deviation.

The acid produced by these cultures was due entirely to intrinsic acid production by E. coli, and we will be able to use these values to compare acid production by E. coli after overexpression of the acetogenesis enzymes PTA and ACK, as well as after using a scaffold to co-localize those enzymes with the acetic acid transporter Aata. This will indicate the level of expression needed for optimal levels of acetic acid production for CAB extraction. With the overexpression of Aata, we will also expect increased resistance to low pH, which should be reflected in the growth curve of E. coli growing in higher concentrations of acetic acid.