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David Hanson - Analytical Problem - Butanol Production from Clostridia fermentation

Recently, there has been a major interest to investigate different types of biologically produced solvents (biofuels) as substitutes for petroleum based fuels. One such biofuel that was of major importance as a transportation fuel during the First World War was butanol. Butanol has many properties that make it an ideal candidate for a petroleum substitute, most importantly being that butanol has a combustion temperature equal to that of gasoline. Meaning that butanol can fuel most of today's automobiles without a conversion to the modern engine. The biggest drawback to the use of butanol as a transportation fuel is its high cost of production.

Butanol is a fermentation byproduct of a family of Clostridia bacteria known as ABE fermenters. When provided a glucose rich feed stock this family of bacterium undergoes two lifecycles. The first lifecycle is known as acidogenesis. During acidogenesis, the Clostridia utilize glucose to produce acetic acid and butyric acid as waste products. As the two acids build up the surrounding pH in the environment lowers, when the environmental pH reaches a specific point the Clostridia switches to its second lifecycle called solventogensis. During solventogenesis, in the presence of glucose the Clostridia will then utilize the acetic and butyric acid to produce acetonitrile, butanol, and ethanol (ABE) as fermentation products. Solventogenesis will continue until all of the acetic and butyric acid has been exhausted at which point the Clostridia switches back to acidogenesis. Due to the constant switching between lifecycles, butanol is not produced continuously thus lowering the amount that can be produced leading to the high production cost.

Current research has shown that if acetic and butyric acid are initially added to the glucose feed stock the Clostridia will bypass acidogenesis and go directly into solventogenesis. Additionally, it has been shown that by changing the ratio of acetic to butyric acid will cause a change in the ratio of acetonitrile, butanol, and ethanol produced. As an analytical problem, this leads to three concerns: two are experimental and the third is engineering. The first is to determine which ratio of acetic/butyric acid will yield the highest the amount of butanol. The second: determine the rate at which the acids are utilized to determine how the acids should be continuously added to prevent exhaustion. The third is to determine or engineer a way in which the amount and ratios of two acids can be continuously monitored in the fermentation vessel in which the matrix contains glucose, acetonitrile, butanol, ethanol, acetic acid, butyric acid, and the Clostridia itself.

Reference:
Lee, S., Cho, M., Park, C., Chung, Y., Kim, J., Sang, B., et al. (2008). Continuous butanol production using suspended and immobilized Clostridium beijerinckii NCIMB 8052 with supplementary butyrate. Energy & Fuels, 22(5), 3459-3464.

UV-Vis absorption spectrometry
5a. As acetic acid and butyric acid are both organic molecules it follows that all must have a UV-Visble absorbance.

Since acetic acid and butyric acid have very similar structures it would be expected that they have similar absorption properties. Using a table of common chemical constants, carboxyl groups are listed to have a wavelength maximum that should be ~205nm with a molar absortivity of ~60. Unfortunately, the table did not list solvents used to determine these values. These estimates were confirmed for acetic acid, which the NIST lists as a wavelength maximum of 207nm and a molar absortivity of 76. The NIST's website did not list the solvent used to determine this number. The NIST did list the original source for this information; however, the source was from a German journal.

Due to the relatively low wavelength maximum of these acids, it could probably be assumed that water was the most likely solvent used to determine these values. Water has a absorbance cut-off of 180nm, most alcohols have cut-offs above 260nm; additionally, both acetic acid and butyric acid are known to be soluble in water.

4. Referances:
Skoog , D, et al. Principles of Instrumental Analysis, 6th ed.; Brooks/Cole: Belmont, CA, 2007.
National Institute of Standards and Technology. Chemistry WebBook: Acetic Acid. http://webbook.nist.gov/cgi/cbook.cgi?ID=64-19-7&Units=SI&cUV=on#Refs (accessed Sept. 28, 2011)
National Physical Laboratory. Kaye and Labby: Tables of Physical and Chemical Constants. http://www.kayelaby.npl.co.uk/chemistry/3_8/3_8_7.html (accessed Sept. 28, 2011)

Initial Search
I know that UV spectra data must be available for both acetic and butyric acid as these are very commonly used chemicals. I first tried to search using the KnowItAll database through the UofMN library site. When I tried to open this database, I continuously received error messages on both my home PC and a University PC. I sent Meghan an email about this and received no response; additionally, I asked her for help tracking other places to find spectral data and no response. Next, I tried searching for property data on Google. I was able to find UV spectra databases on both the University of Wisconsin and University of Texas library sites; however, I would need to be a student of those schools to access the information. After these roadblocks, I looked up my analytes on ChemSpider. ChemSpider had UV-Vis data available; except, the data is in '.jdx' format and I was unsuccessful in finding a free application I could download for this file type. My next course of action would have been to go to Walter library to find chemical encyclopedias; however, I work full-time and have two young children which makes it very difficult for me to be available during library hours. My last course of action was to find a table of common chromophores online.

6. Similar Problem: DEHP Leaching from PVC into Contents of Medical Devices

Similar Analytical Problem
a. The following problem is the most similar to mine:
DEHP Leaching from PVS into Contents of Medical Devices (Rajvi Mehta): Hypothesis- By understanding how and detecting DEHP leaching from medical devices into the body can help to prevent health risks; such as birth defects. Analytes: DEHP, Matrix: Blood/Plasma, Relevance: Health
b. Both my problem dealing with butanol production and the above problem will have a similar study in that they will need to measure changes over time. DEHP would leach slowly from the medical device. By measuring the rate that it takes for DEHP build up to harmful levels from the device will allow for a shelf-life for the device to be determined.
c. The DEHP problem differs from mine in that the location of a single analyte will change. DEHP will be moving from the medical device into the bloodstream; therefore, levels of DEHP will need to be detected and compared in both the medical device and the patient's blood stream. In my problem, the location the analytes will be found stays the same (fermentation vessel), but the analytes measured will change. While acetic and butyric acid will be detected to determine rate of change, butanol will measured as the analyte that will be built-up over time.

Blog #6 Chemical Structure and Standards
All of the standards/reagents that will be required for construction of calibration curves can be purchased through Sigma-Aldrich.
View image
Acetic Acid
Purity: 99.99%
Catalog #: 338826-25ML
Quantity: 25ml
Price: $32.50

View image
Butyric Acid
Purity: >99%
Catalog #: B103500-5ML
Quantity: 5mL
Price: $19.20

View image
1-Butanol
Purity: 99.8%
Catalog#: 281549-100ML
Quantity: 100mL
Price: $31.50

Reference:
Sigma-Aldrich. http://www.sigmaaldrich.com/united-states.html (accessed Oct 24, 2011)

Blog 7: Mass Spectrometry

View image
Mass Spectrum of Butyric (Butanoic) Acid

Blog 9: Chromatographic Techniques
1) Gas chromatography, Reverse phase LC, and HILIC would all be the most useful for separating my analytes: acetic acid, butyric acid, and butanol. Ion-exchange would not be useful as butanol typically carries a neutral charge and acetic and butyric acid would have too similar of charge to be separated. Size-exclusion is usually preformed on large molecular weight molecules(>100,000Da); all three of my analytes have molecular weights <90Da making them too small for SEC. Affinity chromatography could be used to separate butanol from the two acids as ligand beads could be produced that have affinity to carboxylic acids, but I doubt the acetic and butyric acids could be separately isolated. My analytes are not chiral or do not have r/s stereoisomers and therefore would not be applicable to chiral chromatography.
2) My first choice for separating my analytes would gas chromatography. While structurally, my analytes are similar they have different boiling points. Using the physical property of boiling point makes GC the perfect separation technique. Since, I will have large amounts of sample to work there is no need to worry about the destructiveness of the method.
3) Previous experiments utilizing GC to study butanol production have used the following column: HP-INNOwax column (Agilent Technologies Part#29091N-133LTM) Stationary Phase: bonded polyethylene glycol (high polarity); Particle Size: 0.25µm, Column Length: 30m; Column Diameter: 0.25mm; Stability Conditions: >1800°C.
4) Helium is listed as a carrier gas used for this type of experiment.
5) Previously described experiments have used a flame ionization detector. Flame ionization is suitable for hydrocarbons; which is the largest component of the three analytes in question.
6) NA
7) References:

Lee, S., Cho, M., Park, C., Chung, Y., Kim, J., Sang, B., et al. (2008). Continuous butanol production using suspended and immobilized Clostridium beijerinckii NCIMB 8052 with supplementary butyrate. Energy & Fuels, 22(5), 3459-3464.

Agilent Technology. HP-INNOwax Columns. http://www.chem.agilent.com/en-US/products/columns-supplies/gc-gc-mscolumns/jwhp-innowax/Pages/default.aspx (accessed Nov 10, 2011)

Power Point Presentation
Butanol Production from Clostridia Fermentation.ppt

Blog #13) Analytical Electrochemistry
1) Both acetic acid and butyric acid are electroactive. They can both be reduced to carry a single negative charge.
2) I would identify them in by using control standards to determine the E1/2 for each of the analytes.
3) I would quantify them by using control standards to create a standard curve. I would plot the limiting current versus the known concentrations of the standards. The slope of the curve would be the diffusion coefficient for the specific analyte when y-intercept is zero. Measuring the limiting current of the sample and dividing by the determined diffusion coefficient would calculate the concentration.
Reference:
Skoog , D, et al. Principles of Instrumental Analysis, 6th ed.; Brooks/Cole: Belmont, CA, 2007.

Comments

Blog 13. Good answers.

Blog #13) Analytical Electrochemistry
1) Both acetic acid and butyric acid are electroactive. They can both be reduced to carry a single negative charge.
2) I would identify them in by using control standards to determine the E1/2 for each of the analytes.
3) I would quantify them by using control standards to create a standard curve. I would plot the limiting current versus the known concentrations of the standards. The slope of the curve would be the diffusion coefficient for the specific analyte when y-intercept is zero. Measuring the limiting current of the sample and dividing by the determined diffusion coefficient would calculate the concentration.
Reference:
Skoog , D, et al. Principles of Instrumental Analysis, 6th ed.; Brooks/Cole: Belmont, CA, 2007.

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Blogs 9, 10, and 11. Good answers.

Blog 11: Capillary Electrophoresis Techniques
1) The CE techniques that will not be useful for my analytical problem would be capillary gel electrophoresis (CGE) and isoelectric focusing (cIEF). CGE is not useful as all of my analytes are small molecules and would not be impeded by the gel. cIEF is not applicable as none of my analytes are zwitterions. Capillary Zone Electrophoresis (CZE) and Micellular Electro-kinetic Chromatography (MEKC) could both be potentially used.
2) My first choice to use would be CZE. At specific pH’s certain analytes and matrix materials will have different charges. Those that have similar charges will have slightly different sizes. This technique will allow to separate by both charge and size.
3) I would use an uncoated capillary with a negative electric field. Using a basic buffer will cause the acetic and butyric acids to give up a hydrogen giving each a single negative charge. The butanol should maintain a neutral charge. The matrix components, like glucose and zylose would attract protons resulting in positive charges.
4) I would use a UV detector reading at 2 and possibly 3 different wavelengths. The UV detector will be the easiest to use as it can be placed on the capillary. Fluorescence is not an option as my analytes do not fluoresce. MS could be used; however, do to the small molecular weights of the analytes and the low concentrations that could be expected it is doubtful that the MS signal would be high enough for good detection.
References:
Sigma-Aldrich. http://www.sigmaaldrich.com/united-states.html (accessed Nov 19, 2011)
Skoog , D, et al. Principles of Instrumental Analysis, 6th ed.; Brooks/Cole: Belmont, CA, 2007.

Blog 10: Problems using Similar Techniques
1) The preferred technique for my analytical problem is to first utilize vacuum filtration to remove any solids from the sample. Next, the analytes of interest will then be separated by using Gas Chromotography with a thermal gradient to take advantage of the different boiling points of my analytes. Since, all of the analytes contain hydrocarbons a flame ionization detector will be used to collect the data.
2) While no other projects presented a flame ionization detector, three did choose to use GC as the method for separation. These included: ‘Silicones in Cosmetics’, ‘Liquefied Petroluem Gas in insecticide aerosol’, and ‘Vapor Intrusion in Buildings’. All three of these projects chose to use a form of Mass Spectrometry for detection. I question whether FID would not be suitable for these as well.
- ‘Silicones in Cosmetics’ - Dimethicone contains hydrocarbons and should be detectable by FID; since this project is to identify and track dimethicone in nature a total mass analysis seems sort of overkill.
- ‘Liquefied Petroluem Gas in insecticide aerosol’ - Liquified petroleum is an assortment of many different hydrocarbon containing components, using MS might result in so many mass fragments that analysis would be difficult. An FID should yield a fairly simple fingerprint or set of peaks for each componenet.
- ‘Vapor Intrusion in Buildings’ – Trichloro(ethylene) and Tetrachloro(ethylene) also contain hydrocarbons and should detectable by FID. However, these two chemicals are so similar GC might not be enough to separate them from each other and MS could be necessary as a semi-separation technique to identify them as two different mass objects.

Blog 10: Problems using Similar Techniques
1) The preferred technique for my analytical problem is to first utilize vacuum filtration to remove any solids from the sample. Next, the analytes of interest will then be separated by using Gas Chromotography with a thermal gradient to take advantage of the different boiling points of my analytes. Since, all of the analytes contain hydrocarbons a flame ionization detector will be used to collect the data.
2) While no other projects presented a flame ionization detector, three did choose to use GC as the method for separation. These included: ‘Silicones in Cosmetics’, ‘Liquefied Petroluem Gas in insecticide aerosol’, and ‘Vapor Intrusion in Buildings’. All three of these projects chose to use a form of Mass Spectrometry for detection. I question whether FID would not be suitable for these as well.
- ‘Silicones in Cosmetics’ - Dimethicone contains hydrocarbons and should be detectable by FID; since this project is to identify and track dimethicone in nature a total mass analysis seems sort of overkill.
- ‘Liquefied Petroluem Gas in insecticide aerosol’ - Liquified petroleum is an assortment of many different hydrocarbon containing components, using MS might result in so many mass fragments that analysis would be difficult. An FID should yield a fairly simple fingerprint or set of peaks for each componenet.
- ‘Vapor Intrusion in Buildings’ – Trichloro(ethylene) and Tetrachloro(ethylene) also contain hydrocarbons and should detectable by FID. However, these two chemicals are so similar GC might not be enough to separate them from each other and MS could be necessary as a semi-separation technique to identify them as two different mass objects.

Blog 10: Problems using Similar Techniques
1) The preferred technique for my analytical problem is to first utilize vacuum filtration to remove any solids from the sample. Next, the analytes of interest will then be separated by using Gas Chromotography with a thermal gradient to take advantage of the different boiling points of my analytes. Since, all of the analytes contain hydrocarbons a flame ionization detector will be used to collect the data.
2) While no other projects presented a flame ionization detector, three did choose to use GC as the method for separation. These included: ‘Silicones in Cosmetics’, ‘Liquefied Petroluem Gas in insecticide aerosol’, and ‘Vapor Intrusion in Buildings’. All three of these projects chose to use a form of Mass Spectrometry for detection. I question whether FID would not be suitable for these as well.
- ‘Silicones in Cosmetics’ - Dimethicone contains hydrocarbons and should be detectable by FID; since this project is to identify and track dimethicone in nature a total mass analysis seems sort of overkill.
- ‘Liquefied Petroluem Gas in insecticide aerosol’ - Liquified petroleum is an assortment of many different hydrocarbon containing components, using MS might result in so many mass fragments that analysis would be difficult. An FID should yield a fairly simple fingerprint or set of peaks for each componenet.
- ‘Vapor Intrusion in Buildings’ – Trichloro(ethylene) and Tetrachloro(ethylene) also contain hydrocarbons and should detectable by FID. However, these two chemicals are so similar GC might not be enough to separate them from each other and MS could be necessary as a semi-separation technique to identify them as two different mass objects.

Good answers.

Blog 8: Sample Preparation Procedures
1) Filtration: Solids and cellular debris will be removed by using a vacuum filter with a (0.2μm pore size) SFCA membrane (Cat No. 161-0020, NALGENE Lab ware).
2) Evaporation: Glucose and residual salts will be removed from filtered solution by evaporation/condensation using a rotary evaporator (Cat No. 8024701, IKA). Heating temperature ~165°C.
3) Storage: Solution collected in the condensation vessel will be transferred to 50mL centrifuge tubes (Cat No. 89039-656, VWR) and stored at room temperature until analysis.
References:
IKA Technology. http://www.ika.com/ (accessed Nov 3, 2011)
NALGENE Labware. http://www.nalgenelabware.com/ (accessed Nov 3, 2011)
Sigma-Aldrich. http://www.sigmaaldrich.com/united-states.html (accessed Nov 3, 2011)
VWR. https://www.vwrsp.com/index.cgi (accessed Nov 3, 2011)

Blogs 6 and 7. Good answers.

Blog 7: Atomic and Mass Spectrometries
2.) Acetic Acid, Butyric Acid, and Butanol are all small molecule arrangements of carbon, hydrogen, and oxygen. As such as, they are unlikely to be amenable to the various types of atomic spectrometry.
4.) Mass spectrometry can be used to analyze all three of my analytes. The molecular weights for each of the analytes is as follows: Acetic Acid (Nominal MW: 60; Exact MW:60.0522), Butyric Acid (88; 88.1056) Butanol (74; 74.1220) As all three analytes are found as liquid, they will need to be nebulized into a vapor before they can be ionized. The best type of ionization source for this would be to use an Eletrospray Ionizer or ESI. All of the analytes are relatively small molecules; therefore ,their mass ion signal should be fairly weak and will need to be concentrated to give a good signal. The best mass analyzer for concentrating weak signals is the ion trap-FT-ICR. Another method to concentrate the signal would be use mass analyzers in tandem; specifically a quadrapole followed by non-linear time of flight with a refractron. The refractron will allow the ions to be refocused concentrating the signal. Therefore the two types of mass spectrometry I would use are ESI-linear trap-FT-ICR and ESI-Quad-TOF.
The mass spectrum of butyric (butanoic) acid was available on the NIST Chemistry WebBook and referenced from a paper which utilized a MALDI-liner TOF to determine the mass spectrum. The matrix material consisted of 2,5-dihydroxybenzoic acid(DHB) dissolved in water to produce a 10mg/ml solution. Butyric acid was mixed with water to give the sample concentration and was mixed with the matrix material solution.

MALDI would seem to be poor a choice for the analysis of a low molecular weight molecule as the ion signal can be lost within the ion signal of the matrix itself. However, the authors demonstrate that MALDI can be used for the quantification of low molecular weight molecules by lowering the extraction and acceleration voltages of the ions entering the TOF. By lowering the voltage, the ions are extracted more slowly from the matrix allowing the ions to build up in the desorption plume. This build up causes the analyte ions to be more concentrated prior to entering the TOF and improving resolution.

References:
Goheen, S.C., K. L. Wahl, et al. (1997). Mass Spectrometry of Low Molecular Mass Solids by Matrix-assisted Laser Desorption/Ionization. Journal of Mass Spectrometry, 32(8), 820-828.
National Institute of Standards and Technology. Chemistry WebBook: Butanoic Acid. http://webbook.nist.gov/cgi/cbook.cgi?ID=107-92-6&Units=SI&cMS=on (accessed Oct. 27, 2011)
WebQC.Org Chemical Portal. Calculate Molecular Weight-Molar Mass Calculator. http://www.webqc.org/mmcalc.php (accessed Oct. 26, 2011)

Blog 7: Atomic and Mass Spectrometries
2.) Acetic Acid, Butyric Acid, and Butanol are all small molecule arrangements of carbon, hydrogen, and oxygen. As such as, they are unlikely to be amenable to the various types of atomic spectrometry.
4.) Mass spectrometry can be used to analyze all three of my analytes. The molecular weights for each of the analytes is as follows: Acetic Acid (Nominal MW: 60; Exact MW:60.0522), Butyric Acid (88; 88.1056) Butanol (74; 74.1220) As all three analytes are found as liquid, they will need to be nebulized into a vapor before they can be ionized. The best type of ionization source for this would be to use an Eletrospray Ionizer or ESI. All of the analytes are relatively small molecules; therefore ,their mass ion signal should be fairly weak and will need to be concentrated to give a good signal. The best mass analyzer for concentrating weak signals is the ion trap-FT-ICR. Another method to concentrate the signal would be use mass analyzers in tandem; specifically a quadrapole followed by non-linear time of flight with a refractron. The refractron will allow the ions to be refocused concentrating the signal. Therefore the two types of mass spectrometry I would use are ESI-linear trap-FT-ICR and ESI-Quad-TOF.
The mass spectrum of butyric (butanoic) acid was available on the NIST Chemistry WebBook and referenced from a paper which utilized a MALDI-liner TOF to determine the mass spectrum. The matrix material consisted of 2,5-dihydroxybenzoic acid(DHB) dissolved in water to produce a 10mg/ml solution. Butyric acid was mixed with water to give the sample concentration and was mixed with the matrix material solution.

MALDI would seem to be poor a choice for the analysis of a low molecular weight molecule as the ion signal can be lost within the ion signal of the matrix itself. However, the authors demonstrate that MALDI can be used for the quantification of low molecular weight molecules by lowering the extraction and acceleration voltages of the ions entering the TOF. By lowering the voltage, the ions are extracted more slowly from the matrix allowing the ions to build up in the desorption plume. This build up causes the analyte ions to be more concentrated prior to entering the TOF and improving resolution.

References:
Goheen, S.C., K. L. Wahl, et al. (1997). Mass Spectrometry of Low Molecular Mass Solids by Matrix-assisted Laser Desorption/Ionization. Journal of Mass Spectrometry, 32(8), 820-828.
National Institute of Standards and Technology. Chemistry WebBook: Butanoic Acid. http://webbook.nist.gov/cgi/cbook.cgi?ID=107-92-6&Units=SI&cMS=on (accessed Oct. 27, 2011)
WebQC.Org Chemical Portal. Calculate Molecular Weight-Molar Mass Calculator. http://www.webqc.org/mmcalc.php (accessed Oct. 26, 2011)

Both answers to Blogs 4 and 5 are ok. Nice and succinct hypothesis.

Blog 5: Fluorescence Techniques
Acetic Acid and Butyric acid are both carboxylic acids; as such, are linear acidic compounds and not likely to fluoresce. In order for the chemicals to be measured by fluorescence they will first need to be derivatized with a compound that will readily fluoresce. A compound of use that will chemically react with carboxylic acids is marketed by Invitrogen under the trade name Alexa Fluor/u00AE 350 C5-aminooxyacetimide, trifluoroacetate Salt. This compound will interact with the single bonded O- of the carboxy end of the acetic acid and butyric acid. This complex should give a maximum excitation of 350nm and maximum emission of 450nm when analyzed in deionized water.
Since, I will be taking many samples at different time points and the derivatization process will have some complication it would be best to test all the samples at once. To do this it would be best to use a microtiter plate reader for analysis.
References
Invitrogen. Life Technologies: Alexa Fluor® 350 C5-aminooxyacetamide, trifluoroacetate salt (Alexa Fluor® 350 hydroxylamine). http://products.invitrogen.com/ivgn/product/A30627 (accessed Oct 17,2011)

Blog 4: Studies needed to investigate the analytical problem
1. Hypothesis: Currently, butanol production by Clostridia fermentation is not a continuous process. Hypothesize that by determining the rate by which acetic acid and butyric acid are utilized by the Clostridia and adding the acids at this rate will aloe butanol to be produced continuously.
Studies:
A) Determine the ratio of acetic acid to butyric acid that will yield the optimal purity of butanol to be produced.
B) Determine the individual rates by which acetic acid and butyric acid are utilized when added in the optimal ratio.
C) Monitor butanol production over a period of time to ensure that production will be continuous as the acids are added continuously at the specified rates.

2. It is difficult to estimate the levels of analyte in the matrix as these levels will be changing over time. Acetic acid and butyric acid will decrease as butanol increases. That said in lab scale experiments (~2 liter batches) acetic acid and butyric acid are each typically added initially at concentrations between 9-36 mM. Butanol levels are then measured after all of the available glucose has been exhausted. Literature lists lab batches as having produced a total of 9.4-11.2 grams of butanol per liter or as it is typically measured 0.32-0.45 grams of butanol per 1 gram of glucose.

Industrial batches (>100m3) typically yield 0.38 grams of butanol per 1 gram of glucose. Since, this is a discontinuous process and the Clostridia produces its own acids, there is very little data available on acid levels produced industrially.

References:
Lee, S., Cho, M., Park, C., Chung, Y., Kim, J., Sang, B., et al. (2008). Continuous butanol production using suspended and immobilized Clostridium beijerinckii NCIMB 8052 with supplementary butyrate. Energy & Fuels, 22(5), 3459-3464.

Schmid, R.D., Pocket Guide to Biotechnology and Genetic Engineering; Wiley-VCH: Weinheim, Germany, 2003.

The description on the background problem is not succinct, but worth reading.

You mention three concerns but there is no definition of the hypothesis that you want to test.

Posted 24 hours late. Not graded.