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Combining the Power of Living Cells and Non-living Catalysts for the Production of Valuable Chemicals Ava Brook Ananya Gandhi [email protected] [email protected] Sophia Ginet Justin Ma Kirmina Monir [email protected] [email protected] [email protected] Dr. Haoran Zhang* [email protected] New Jersey’s Governor’s School of Engineering and Technology July 20, 2019 *Corresponding Author Abstract—Phenol is widely used in the chemical industry, and serves as a precursor for alkylates important to the production of plastics, explosives, pharmaceuticals, and wood preservatives. This study focused on establishing a pathway for the biosynthesis of phenol within Escherichia coli (E. coli) cells. During this study, five strains of E. coli were genetically engineered to produce phenol from tyrosine, which serves as a more reliable and environmentally friendly alternative to current production methods that require petroleum. The five different strains of E. coli were compared for their ability to produce phenol. BL21(DE3) was determined to be the most efficient strain for producing phenol, as it produced the largest amount of phenol per cell density unit. The high specific production value of the strain indicated that BL21(DE3) is a robust candidate for producing phenol in a more environmentally conscious manner. Considering the toxicity of phenol to E. coli cells, this project also evaluated the effectiveness of tributyrin as an extractive solvent. This study also compared the performance of cerium(IV) oxide and P25 as catalysts for the reaction between phenol and tert- butyl alcohol, which produces valuable tert-butyl phenols and ethers. I. I NTRODUCTION Phenol is an organic compound that serves as a precursor for various valuable chemicals, such as 2-tert-butylphenol (2- TBP), 4-tert-butylphenol (4-TBP), 2,4-tert-butylphenol (2,4- TBP), 2,6-tert-butylphenol (2,6-TBP), 2,4,6-tert-butylphenol (2,4,6-TBP), and tert-butylphenyl ether (TBPE). These alky- lated phenols are produced from the reaction between phenol and tert-butyl alcohol (TBA), which requires the presence of a catalyst. Alkylated phenols are extremely valuable, as they have unique properties and are used industrially as ultraviolet stabilizers, antioxidants, food additives, fragrances, etc. [1]. As phenol itself has a wide variety of applications and can be synthesized into even more valuable chemicals, the adoption of a more environmentally friendly method of generating phenol would be immensely impactful. This project aimed to investigate an inexpensive and en- vironmentally conscious way to produce phenol by harness- ing the biosynthetic power ofE. colicells. As the metabolic pathway that produces tyrosine, an amino acid, from sugars such as glucose and xylose exists naturally withinE. colicells, this project demonstrated thatE. coli could be genetically engineered to take this pathway a step further to produce phenol directly from simple carbon substrates. This study then compared the performance of cerium(IV) oxide and P25 as catalysts in the reaction between phenol and tert-butyl alcohol. II. BACKGROUND A. Metabolic Engineering Metabolic engineering is the practice of enhancing existing genetic and regulatory processes within cells to optimize their production of a specific substance [2]. Metabolic engi- neers develop high-performance microbial strains in order to synthesize valuable compounds from renewable biomass [3]. Within these strains, metabolic pathways are manipulated to overexpress enzymes that produce specific valuable chemicals [2]. During this process, metabolic engineers determine which pathways could be used to produce a certain compound, analyze the rates at which the pathways operate and how they work, and propose methods for modification of the pathways [4]. The products of these experiments often serve as precur- sors for valuable chemicals, fuels, and pharmaceuticals. While 1
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Page 1: Combining the Power of Living Cells and Non-living ... · produced from the oxidation of cumene with molecular oxy-gen, producing cumene hydroperoxide, which is decomposed into phenol

Combining the Power of Living Cells andNon-living Catalysts for the Production of

Valuable Chemicals

Ava Brook Ananya [email protected] [email protected]

Sophia Ginet Justin Ma Kirmina [email protected] [email protected] [email protected]

Dr. Haoran Zhang*[email protected]

New Jersey’s Governor’s School of Engineering and TechnologyJuly 20, 2019

*Corresponding Author

Abstract—Phenol is widely used in the chemical industry, andserves as a precursor for alkylates important to the productionof plastics, explosives, pharmaceuticals, and wood preservatives.This study focused on establishing a pathway for the biosynthesisof phenol within Escherichia coli (E. coli) cells. During thisstudy, five strains of E. coli were genetically engineered toproduce phenol from tyrosine, which serves as a more reliableand environmentally friendly alternative to current productionmethods that require petroleum. The five different strains ofE. coli were compared for their ability to produce phenol.BL21(DE3) was determined to be the most efficient strain forproducing phenol, as it produced the largest amount of phenolper cell density unit. The high specific production value ofthe strain indicated that BL21(DE3) is a robust candidate forproducing phenol in a more environmentally conscious manner.Considering the toxicity of phenol to E. coli cells, this project alsoevaluated the effectiveness of tributyrin as an extractive solvent.This study also compared the performance of cerium(IV) oxideand P25 as catalysts for the reaction between phenol and tert-butyl alcohol, which produces valuable tert-butyl phenols andethers.

I. INTRODUCTION

Phenol is an organic compound that serves as a precursorfor various valuable chemicals, such as 2-tert-butylphenol (2-TBP), 4-tert-butylphenol (4-TBP), 2,4-tert-butylphenol (2,4-TBP), 2,6-tert-butylphenol (2,6-TBP), 2,4,6-tert-butylphenol(2,4,6-TBP), and tert-butylphenyl ether (TBPE). These alky-lated phenols are produced from the reaction between phenoland tert-butyl alcohol (TBA), which requires the presence ofa catalyst. Alkylated phenols are extremely valuable, as theyhave unique properties and are used industrially as ultravioletstabilizers, antioxidants, food additives, fragrances, etc. [1].

As phenol itself has a wide variety of applications and can besynthesized into even more valuable chemicals, the adoption ofa more environmentally friendly method of generating phenolwould be immensely impactful.

This project aimed to investigate an inexpensive and en-vironmentally conscious way to produce phenol by harness-ing the biosynthetic power ofE. colicells. As the metabolicpathway that produces tyrosine, an amino acid, from sugarssuch as glucose and xylose exists naturally withinE. colicells,this project demonstrated thatE. coli could be geneticallyengineered to take this pathway a step further to producephenol directly from simple carbon substrates. This study thencompared the performance of cerium(IV) oxide and P25 ascatalysts in the reaction between phenol and tert-butyl alcohol.

II. BACKGROUND

A. Metabolic Engineering

Metabolic engineering is the practice of enhancing existinggenetic and regulatory processes within cells to optimizetheir production of a specific substance [2]. Metabolic engi-neers develop high-performance microbial strains in order tosynthesize valuable compounds from renewable biomass [3].Within these strains, metabolic pathways are manipulated tooverexpress enzymes that produce specific valuable chemicals[2]. During this process, metabolic engineers determine whichpathways could be used to produce a certain compound,analyze the rates at which the pathways operate and how theywork, and propose methods for modification of the pathways[4]. The products of these experiments often serve as precur-sors for valuable chemicals, fuels, and pharmaceuticals. While

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some chemicals have been synthesized and commercializedsuccessfully through metabolic engineering, there are stillnumerous other chemicals and materials that can be developedmetabolically as well.

B. Biosynthesis of Phenol

Most aromatic chemicals, or compounds with a benzenering, are currently produced from fossil fuels. Phenol is oftenproduced from the oxidation of cumene with molecular oxy-gen, producing cumene hydroperoxide, which is decomposedinto phenol and acetone [5]. Phenol production is integralfor the manufacturing of important chemicals and polymers,notably bisphenol A and phenolic resins. Although the processof creating phenol from cumene is relatively inexpensive,its reliance on petroleum is extremely detrimental to theenvironment. The environmental concerns regarding the pro-duction of phenol from cumene have drawn attention to thepotential of utilizing microbial strains to manufacture aromaticcompounds.

Metabolic engineers have spent decades attempting to ge-netically modify microorganisms to produce aromatic com-pounds on an industrial scale. While the biological productionof aromatic compounds from renewable resources has beendifficult to achieve due to their toxicity to microorganisms,recent advancements in metabolic engineering have createda possibility of engineering microorganisms to efficientlyproduce aromatic chemicals on an industrial scale [6].

C. Two-Phase Extractive Fermentation

New methods such as two-phase extractive fermentationhave successfully proven to remove products from cell culturesin order to avoid the harmful effects of toxic compoundson cell growth. Two-phase extractive fermentation producesgreater concentrations of phenol from bacteria such as E. coli.According to a study conducted by Liangtian Miao et al., cel-lular growth of E. coli was affected by phenol concentrationsof over 0.5 g/L, and completely inhibited at 1.6 g/L. The useof tributyrin and dibutyl phthalate for the extraction of phenolfrom E. coli cells proved to be the most successful in the study,as the compounds had higher extractive capacities than othersolvents such as dodecane, octanol, and isopropyl myristate.Additionally, tributyrin and dibutyl phthalate were ideal forimproving phenol production as the solvents were not toxic tothe E. coli cells, unlike other extractive solvents [7].

D. Escherichia coli

The bacterium Escherichia coli is a rod-shaped organismthat resides within the digestion system of warm-bloodedanimals. Discovered in 1885, E. coli is the most commoninfecting organism of the enterobacteriaceae family and isknown as the most-studied free-living organism. In 1973, Her-bert Boyer and Stanley Cohen discovered how two sections ofE. coli bacterial DNA could be cut, connected, and reinsertedback into the original cell. Since this discovery, E. coli hasbeen majorly significant in metabolic engineering, as it has

been used to store foreign DNA sequences and manufactureproteins [8].

The majority of microbially produced aromatic chemicalsare derived from shikimate (shk) and aromatic amino acidslike L-phenylalanine, L-tyrosine, and L-tryptophan. The shkpathway is the link between the central carbon metabolism tothe biosynthesis of aromatic amino acids. Aromatic chemicalsare characterized into intermediates, or derivatives of theshk pathway and aromatic amino acids [3]. The essentialaromatic compound phenol, a bulk chemical with a multitudeof applications in the chemical industry, can be produced fromE. coli cells that are genetically engineered to express tyrosinephenol-lyase (tpl). This enzyme converts tyrosine, an essentialamino acid, into phenol. To produce a high concentrationof phenol from this process, the products must be extractedthroughout the process, as phenol is toxic to E. coli cells [7].

E. Chemical Catalysis

A catalyst is a substance that increases the rate of reac-tion without being consumed by the reaction itself. Catalystschange the reaction rate by promoting a different reactionmechanism, which lowers the activation energy and allowsreactions that would normally never occur to take place.During a reaction, catalysts reacts with reactants, formingchemical intermediates that are able to react more readily witheach other and with other reactants. This process increasesthe rate of the reaction, enhancing the production of thedesired chemical. In this study, cerium(IV) oxide and P25were evaluated as catalysts for the reaction between phenoland TBA [9].

III. METHODS

A. Overview

In this experiment, plasmid DNA was extracted fromthe K12(DE3) strain of Escherichia coli, which had beenpreviously engineered to contain the tyrosine phenol-lyase(tpl) gene. The genetically engineered plasmid, pET28a-proC-tpl, was analyzed using gel electrophoresis to ensure thatthe plasmid had been correctly constructed. The plasmidwas then transformed into five different strains of E. coli:K12(DE3), BL21(DE3), BL21 Gold(DE3), BL21 Star(DE3),and JM109(DE3). These strains were cultivated overnight, andthe cell density of the cultures was standardized to comparethe respective phenol production by different strains. Aftertyrosine was added to the cultures and the cells grew fora given time, the production of phenol for each strain wascompared by running samples of the cultures through High-Performance Liquid Chromatography. The different strainswere analyzed for phenol production by calculating theirspecific phenol concentrations, tyrosine concentrations, andstandard error. As phenol is a precursor for several valuablealkylates, cerium(IV) oxide and P25 were compared for theireffectiveness in catalyzing the reaction between phenol andTBA.

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B. Extracting DNA from Escherichia coli

To begin the experiment, minipreparation of the plasmidDNA was conducted. Minipreparation, commonly referred toas miniprep, is a lab procedure that allowed for the isolation ofplasmid from a culture of E. coli cells. Two centrifuge tubeswere filled with 1.5 mL of K12(DE3, pET28a-proC-tpl) andplaced into a Biofuge centrifuge (Figure 1), a machine thatrapidly spins small tubes to separate different components ofa solution. The solution was spun for 1 minute at a speed of10,000 rotations per minute (rpm). The supernatant fluid wasdecanted from the vials into a waste beaker, leaving only theE. coli cells. Following this step, 480 µL of deionized waterwere added to each of the vials and mixed thoroughly in aVortex Genie 2, a machine that stirs substances by vibratingthe containers on a rotating disk.

Fig. 1. Biofuge Centrifuge

To rupture the cells and release the plasmid, 80 µL of alysis solution were added to each mixture and the vials wereinverted gently five times. 280 µL of a neutralization bufferwere added and the vials were inverted about ten times, whenthe solution turned yellow. The vials were centrifuged for4.5 minutes at a speed of 10,000 rpm. After the vials werecentrifuged, the supernatant fluid from each vial was pouredinto a special vial that contained a small tube that includedresin that binds with DNA. This was placed into the centrifugefor 1 minute at 10,000 rpm. The remaining fluid from eachvial was poured into a waste bucket. The tubes containing theresin were cleaned with 200 µL of 1 endo-wash buffer, and thevials were placed again in the centrifuge at 10,000 rpm for 1minute. The excess fluid was removed from the vials and 400µL of 2 wash buffer were added. The vials were centrifugedfor 2 minutes at 10,000 rpm, and the small tubes containingthe resin were removed. Using a pipette, 20 µL of deionizedwater were added to the tubes, and the tubes were allowed tosit for 1 minute before they were centrifuged at 10,000 rpmfor 1 minute. The step was repeated with 15 µL of water,resulting in a small, concentrated solution of plasmid DNA.

C. Running a DNA Gel

To create a 1% agarose solution, 500 mL of TAE buffer(Tris, Acetate, EDTA) and 5 g of agarose powder were mixed.This solution was put in a microwave for 5 minutes and thenadded to the DNA gel mold. 2 µL of Sybr Safe DNA stainwere mixed into the solution while cooling. After hardening,the gel was loaded into an electrophoresis unit filled with theTAE buffer. Purple loading dye was mixed into the solutionof the plasmid in order to increase the density of the plasmidand allow it to mix evenly within the gel. 5 µL of the plasmidand 1 µL of purple loading dye were added to four smalltubes and mixed with a pipette tip. The 6 µL of each solutionwere transferred to the wells of the DNA gel; this was donecarefully to ensure that the gel was not broken. 4 µL of DNAmarker 1-kb ladder were added into the fifth well. The DNAgel was placed into the gel electrophoresis unit, which utilizesan electric field to separate DNA molecules based on size. Thismachine ran for 30 minutes at 140 Volts [10]. After runningthe gel electrophoresis, the gel was placed onto a blue tray andinserted into a Gel Doc EZ Imager. The Gel Doc EZ Imagerutilized Image Lab software to analyze and retrieve an imageof the DNA gel (Figure 2). The DNA gel was analyzed toensure that it contained two bands, which indicated that theplasmids had been correctly engineered.

Fig. 2. Gel electrophoresis of the pET28a-proC-tpl plasmid.

D. Digestion of the Plasmid

The pET28a-proC-tpl plasmid was genetically engineeredbefore the extraction to incorporate the tyrosine phenol-lyase(tpl) gene. Using a pipette, each of the four centrifuge tubeswere filled with 17.6 µL of the plasmid DNA, 0.2 µL ofSpe1 enzyme, 0.2 µL of Xho1, and 2 µL of Cutsmart buffer.The vials were centrifuged for 10 seconds and placed intoa 37°Celsius incubator overnight, which was the optimumconditions for using the restriction enzymes.

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The procedure previously mentioned in Part C was repeatedwith the newly digested plasmid strain, to ensure that thetpl gene had been successfully incorporated into the plasmid.After the DNA gel was analyzed and proved that the genehad been successfully incorporated, the remaining sample ofplasmid was introduced into five different strains of E. coli,specifically K12(DE3), BL21(DE3), BL21 Gold(DE3), BL21Star(DE3), and JM109(DE3). A culture tube containing 2 mLof each strain was prepared. The culture tubes were put on icefor 30 minutes, weakening the cell membranes to transformthe plasmid into the cells.

E. Transformation and Recovery

While the cell cultures were on ice, LB agar gel, consistingof 25 g/L of LB powder and 10 g/L of agar powder, washeated in a microwave for 2 minutes. 8 mL of the LB agargel liquid and 8 µL of 50 g/L kanamycin was transferred tofive 50 mL centrifuge tubes. The mixtures were gently shakenand transferred to five Petri dishes. After the gel solutionsolidified, 1 mL of each cell culture was transferred fromthe culture tube to five different microcentrifuge tubes, whichwere centrifuged for 1 minute at 10,000 rpm. The supernatantfluid was removed from the tubes, leaving only the E. colicells. To purify the cell samples, 1 mL of sterile deionizedwater was added to each tube and the solutions were mixedusing a Vortex machine. The tubes were centrifuged for 1minute at 10,000 rpm, and the supernatant fluid was removed.This process of purifying the sample was repeated three moretimes for each of the five tubes. Following the purificationstep, 400 µL of sterile deionized water were added to thetubes and mixed using the Vortex machine. Cell cultures fromeach of the tubes were transferred to cuvettes, and 1 µLof the genetically engineered plasmid was added to each ofthe cuvettes. The solutions within the cuvettes were mixedusing a pipette and placed in an Eporator machine whereelectroporation was performed. Electroporation is where anelectric shock is applied to cells in order to weaken theirmembranes, making them more permeable to the plasmid.

After electroporation was performed, 1 mL of fresh LBmedium was added immediately to each of the cuvettes.Afterward, the mixture was transferred from the cuvettes toculture tubes, which were labeled with the names of the E.coli strains. These tubes were put into a 37 °C shaker for 45minutes. After being put in the shaker, 400 µL of the cellcultures were transferred from the culture tubes to the Petridishes. The Petri dishes were gently swirled to make sure thegel was fully coated with the cell culture solutions. The disheswere closed and left in a 37 °C incubator to cultivate overnight.

F. Preparation for Production

Two colonies from each of the agar plates were isolatedand transferred to two culture tubes. The culture tubes werelabeled with the name of the E. coli strain and either 1 or2 to distinguish the culture tubes from one another. The cellswere suspended in a 30% glycerol stock solution to protect thecells while being stored in a freezer at -80 °C overnight. After

being removed from the freezer, 400 µL of the five cell culturesolutions were placed into pre-sterilized centrifuge tubes alongwith 400 µL of the 30% glycerol stock solution. The tubeswere mixed gently with a pipette, labeled with the plasmidstrain and date, and put in the freezer.

To prepare for the production step, a medium known as M9medium was created. To generate 500 mL of a modified M9medium, 430.25 mL of deionized water, 50 mL of M9 salts,500 µL of MgSO4 and 12.5 mL of 10 g/L yeast extract wereautoclaved individually at 121C and 100 kPa and mixed. Tothis solution, 6.25 mL of 200 g/L glucose and 250 L of traceelements were added and the solution was mixed. 250 L of 50g/L kanamycin stock solution were also added. To thoroughlydissolve the M9 salts within the solution, the mixture wasadded to a sonicator for 20 minutes. A sonicator uses soundwaves to disrupt the solution and properly dissolve compoundswithin a solution.

After the cells were cultivated overnight in the LB medium,the tubes were centrifuged at 25°C for 5 minutes, at aspeed of 3900 rotations per minute. The undesired toxins andnutrients were removed from the culture tubes by decantingthe supernatant fluid into a waste beaker. 2 mL of the modifiedM9 medium were added to the culture tube using a pipette.To properly mix the solution, the culture tubes were placed ina vortex for 1 minute.

To compare the production of the five strains, the celldensity of each of the cultures was standardized. A V-1200Spectrophotometer, an instrument that emits light in order tomeasure absorbance, was used to measure the optical densityof a 2 mL sample of each culture in a cuvette. The opticaldensity of the ten cultures was measured and recorded, andcalculations were performed to determine the ratio at which thecultures should be diluted with varying amounts of deionizedwater, which would standardize the cell density. The opticaldensity was standardized at 0.300 for all five of the strains toensure that the spectrophotometer readings were accurate, asthe measurements of cell density are most accurate within therange of 0.300-0.700. This process was repeated until therewere two new solutions for each strain of E. coli. The tenculture tubes were added to a 37°C shaker to grow for 4 hours.

G. Adding Tyrosine

After the standardization of the optical density of the cellcultures, 5.0 g/L tyrosine stock solution was prepared to add tothe cultures. The stock solution was prepared by mixing 0.05g tyrosine and 10 mL deionized water and was placed in VWRSonicator for 20 minutes to dissolve the tyrosine. 200 µL of5.0 g/L tyrosine stock solution were added to each culture tubeand mixed with a pipette tip. After adding tyrosine, the cellcultures were placed in a 37°C shaker for 65 hours. 1 mL ofthe culture sample was added to two microcentrifuge tubes,which were labeled with the strain, number, and either A or Bto distinguish the two tubes from one another. The tubes wereplaced into the centrifuge for five minutes at 10,000 rpm.

From the centrifuge tubes, 300 µL of the supernatant liquidwere added to a cuvette along with 600 µL of water. The

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cuvette was inserted into a spectrophotometer and the opticaldensity value was measured to determine which strain of E.coli grew the most. 1 mL was taken from each of the centrifugetubes and filtered into two small bottles, leaving only a concen-trated solution of phenol and other cellular metabolites. Thisstep ultimately resulted in four tubes of phenol solution foreach of the five strains of E. coli. The bottles were analyzed ina High Performance Liquid Chromatography machine, whichran the solution through a column at high pressure to separatethe components of the solution and measure the concentrationof both tyrosine and phenol. The integral of the graph ofIntensity vs. Time was analyzed for both tyrosine and phenol:the intensity of tyrosine was measured at a retention time ofabout 4 minutes, and the intensity of phenol was measured ata retention time of about 10 minutes [11].

Fig. 3. High Performance Liquid Chromatography (HPLC) Machine

H. Evaluating the Effectiveness of Tributyrin

Five different solutions of phenol and water were createdby diluting a solution of 10 g/L phenol stock solution. Thesolutions had concentrations of 2 g/L, 4 g/L, 6 g/L, 8 g/L,and 10 g/L. Two samples of each solution were generated byfilling ten centrifuge tubes with 2 mL of each phenol solutionand 2 mL of tributyrin stock solution, an organic phase used toextract phenol from water. These tubes were mixed in a Vortexmachine, then centrifuged for 5 minutes at 3,900 rotations perminute. 1 mL of the bottom layer of liquid was removed fromeach solution using a pipette and placed into a small tube. 100µL were taken from the solutions of 2 or 4 g/L phenol anddiluted with 900 µL of pure methanol. 50 µL of the solution

from the tubes with 6, 8, or 10 g/L phenol were taken anddiluted with 950 µL pure methanol. Each new diluted solutionwas put in an HPLC bottle and placed in the HPLC machine.The HPLC machine was placed on the 0.80 mL/min settingto ensure that the pressure on the solution was not too high.This data was analyzed to evaluate the effectiveness of usingtributyrin to extract phenol.

I. Producing Alkylated Phenols

To determine which catalyst is more effective in catalyzingthe reaction between phenol and tert-butyl alcohol (TBA),catalysts cerium (IV) oxide and P25 were tested. Pure phenoland TBA were mixed in a 10:1 ratio. Three 20 µL samplesof each solution were taken and diluted with toluene in 3separate 20 mL volumetric flasks. The diluted solutions werethen analyzed for their respective concentrations of phenol andTBA in an Agilent Technologies 7890B Gas ChromatographyMachine (GC). The Gas Chromatography Machine uses aneedle to inject samples into a 30 m column, where the liquidsamples are vaporized and separated in order to measure theirconcentrations. The GC was initially started at 50 °C, andwas set to heat up at 25 °C/minute until it reached 300 °C.Toluene was utilized both to dilute the liquid samples as wellas for solvent washes in the GC. Each of the 3 initial sampleswas injected 3 times into the GC to ensure an accurate andconsistent measurement.

The reaction was carried out in two 15 mL pressure vesselsequipped with a thermoplunger, allowing for the use of athermocouple to precisely control reaction temperature. 10 mLsolutions of the aforementioned mixture of phenol and TBAwere placed in these vessels along with a stir bar. Catalystloading was decided to be 3% by weight, which correspondedto 0.3126 g of cerium (IV) oxide and 0.3131 g of P25.Cerium(IV) oxide was added to one vessel and P25 was addedto the other, and the reaction vessels were then sealed tightlyand submerged in a 200 mL oil bath. The system was heated to120°C and stirred at 1050 rpm. The reaction was stopped aftertwo hours, and the final reaction mixtures were centrifuged for13 minutes at 3900 rpm to separate the liquid mixture fromthe solid catalyst. As done previously, three 20 µL sampleswere taken from each final reaction mixture and diluted withtoluene in 5 mL volumetric flasks. 3 samples of each of thediluted solutions were injected into the GC to determine theconcentrations of the alkylated products.

IV. RESULTS

A. Optical Densities

Optical density measures how easily light can pass through asolution, which correlates to the concentration of cells in a so-lution. After running samples through the Spectrophotometerand comparing them to the initial optical densities recorded,it was apparent that K12(DE3) had the highest cell growthrate while BL21(DE3) experienced the least growth. The tableillustrates that the initial optical densities of the samples werestandardized at 0.300, and then the final optical density valuewas measured in order to determine the cellular growth. With

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the results from Figure 1, the optical density values for eachsample were averaged to generate Figure 4. As indicated byFigure 4, K12(DE3) had an average optical density of 3.171and BL21(DE3) had an average optical density of 1.5175.These results were important in determining the cell culturewith the highest cell growth and helped in analyzing the phenolproduction rate.

Fig. 4. Average Optical Density of Each Strain

B. Calculating Concentrations

The concentration of phenol within the unknown solutionswas derived from standardized samples. Five samples ofknown concentration of phenol were analyzed by the HPLCmachine, and the integral of the graph of Intensity vs. Timewas taken at 4 minutes for tyrosine and 10 minutes for phenol.The area beneath each of these peaks is directly proportionalto the concentration of the substance, and thus these peakareas were used to generate a calibration curve. The calibrationcurve allowed for the concentration of unknown samplesof phenol and tyrosine to be determined. To generate thecalibration curve, samples of different known concentrationsof phenol were used: 50 mg/L, 100 mg/L, 250 mg/L, 500mg/L, and 1000 mg/L. This process was repeated with tyrosinesamples with concentrations of 10 mg/L, 50 mg/L, 100 mg/L,200 mg/L, and 500 mg/L. After obtaining the areas of thepeaks from the HPLC for each solution, the results weregraphed (Figure 5). After the data was plotted, two calibrationcurves of best fit were generated with y = 3.9345x for tyrosineand y = 8.4218x for phenol, where y represents the area ofthe peak, x represents the concentration, and the slope is theconversion factor. These equations, along with the peak areaobtained for each unknown sample, were used to derive theconcentration of phenol and tyrosine in each sample. Theconcentrations were averaged for each strain and representedin Figures 6 and 7. As indicated in Figure 5, the R2 value fortyrosine and phenol was determined to be 0.9991 and 0.9982,respectively, which shows that the derived concentrations ofthe unknown solutions are accurate.

After the data was plotted, two calibration curves of bestfit were generated with y = 3.9345x for tyrosine and y =8.4218x for phenol, where y represents the area of the peak,

x represents the concentration, and the slope is the conversionfactor. These equations, along with the peak area obtained foreach unknown sample, were used to derive the concentrationof phenol and tyrosine in each sample. The concentrationswere averaged for each strain and represented in Figures 6and 7. As indicated in Figure 5, the R2 value for tyrosine andphenol was determined to be 0.9991 and 0.9982, respectively,which shows that the derived concentrations of the unknownsolutions are accurate.

Fig. 5. Area of Peak vs. Concentrations of Phenol (above) and Tyrosine(below)

C. Production of Phenol

High Performance Liquid Chromatography (HPLC) wasused to determine the concentration of phenol produced byeach strain. Samples of phenol generated by the E. coli cultureswere run through the HPLC machine, and the calibration curvegenerated earlier was used to determine the concentrationof phenol in these samples. K12(DE3) produced the highestconcentration of phenol with an average amount of 128.8mg/L. The high production value for K12(DE3) was largelydue to the high cellular density of the strain, as the presenceof more cells allowed the culture to produce more phenol. Onthe other hand, BL21(DE3) had the lowest raw production ofphenol at only 89.28 mg/L. The raw production of phenol,however, is not indicative of the efficiency of the strain, asit does not take the cell concentration of each sample intoaccount. This step was important, however, in determiningwhich strain was the most effective in producing phenol atthe lowest cell density unit.

D. Production of Phenol Normalized by Cell Density

The production of phenol normalized by cell density, orspecific production, was used to determine the amount ofphenol produced per cell density unit. This was derived bydividing the raw production of phenol by the optical density.

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Fig. 6. Raw Production of Phenol

Figure 6 illustrates that the average specific production ofphenol by E. coli strain JM109(DE3) was the highest, at61.25 mg/L/OD, with BL21(DE3) as a close second at 59.25mg/L/OD. K12(DE3) had the lowest specific production ofphenol, despite having the highest raw phenol production. Thisindicated that within the K12(DE3) strain, each cell densityunit did not produce much phenol.

Fig. 7. Production of Phenol Normalized by Cell Density

E. Tyrosine Concentration

The concentration of tyrosine remaining in the culturesamples was calculated using High Performance Liquid Chro-matography. The tyrosine concentration describes how effec-tive each E. coli sample was at converting tyrosine into phenol.A high remaining concentration of tyrosine indicated that theculture was not efficient in metabolizing tyrosine into phenol,as it did not effectively use the substrate added. As illustratedby Figure 8, the JM109(DE3) samples contained the highestremaining concentration of tyrosine at an average of 102 mg/L.BL21 Star(DE3) had the lowest average concentration of ty-rosine remaining at 22.3 mg/L. The other strains, BL21(DE3),K12(DE3), and BL21 Gold(DE3) had more moderate valuesof 47.89 mg/L, 58.55 mg/L, and 41.47 mg/L, respectively.

Fig. 8. Remaining Concentration of Tyrosine Per Strain

F. Effectiveness of Tributyrin

To avoid the toxicity that phenol poses to E. coli cells,tributyrin was evaluated as a method of extraction. As shownby Table 3, tributyrin was far more effective at extractingphenol from water at lower concentrations of phenol. At 6g/L of phenol, tributyrin became much less effective, as theextraction efficiency dropped to 21.47%. The results indicatethat tributyrin should be used during the process when phenolconcentration is low, rather than at the end of the productionprocess once phenol has accumulated.

G. Catalysis

P25 was determined to be the most effective at cat-alyzing the reaction between phenol and tert-butyl alcohol.During the experiment, conversion of TBA was observedusing both cerium(IV) oxide and P25 as catalysts (Fig-ure 9). However, cerium(IV) oxide was largely ineffectiveas a catalyst, as the reaction between phenol and TBAin the presence of cerium(IV) oxide did not yield any ofthe desired products, specifically 2-tert-butylphenol (2-TBP),4-tert-butylphenol (4-TBP), 2,4-tert-butylphenol (2,4-TBP),2,6-tert-butylphenol (2,6-TBP), 2,4,6-tert-butylphenol (2,4,6-TBP), or tert-butylphenyl ether (Figure 10 and 11). The useof cerium(IV) oxide likely produced some unknown minorproducts, such as isobutylene, that were not accounted forby the GC software due to lack of calibration data. Thus,P25 was determined to be more effective for the reactionbetween phenol and TBA, as it was able to convert 38% ofthe tert-butyl alcohol. Additionally, P25 was far more effectivein producing the desired products, as it produced 0.0654 Mof tert-butylphenyl ether. As a catalyst, P25 demonstrateda high selectivity towards producing tert-butylphenyl ether(TBPE), as 97.8% of the products consisted of TBPE. Minorselectivity toward the production of 2-TBP and 4-TBP wasalso observed. Thus, P25 would be ideal for the production oftert-butylphenyl ether on an industrial scale, as in mere 2 hoursit demonstrated an extremely high selectivity towards TBPE.Changing reaction conditions, such as increasing reaction timeor catalyst loading, may allow for increased conversion andgeneration of the desired alkylated products.

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H. Analysis

In order to analyze which strain was most effective inproducing phenol, the production of phenol normalized bycell density and the remaining concentration of tyrosine weretaken into consideration. The high specific production val-ues for JM109(DE3) and BL21(DE3) indicated that the twostrains were the most effective in producing phenol. Whilethe K12(DE3) strain experienced significant cell growth, theculture was not nearly as effective in producing phenol asBL21(DE3) or JM109(DE3). Although K12(DE3) had anextremely high average optical density value of 3.171, itwas ineffective in producing phenol and had a low spe-cific production value of only 40.630 mg/L/OD. Addition-ally, the remaining concentration of tyrosine indicates theoverall ineffectiveness of strains such as JM109(DE3). WhileJM109(DE3) had the highest specific production value, it alsocontained the highest remaining concentration of tyrosine,indicating an inefficiency in converting tyrosine into phenol(Figure 8). Additionally, JM109(DE3) had a non-negligiblemarginal error, demonstrating that the high specific productionvalue for the strain was not necessarily the most reliable. BL21Star(DE3) had a very low optical density and specific pro-duction amount compared to the other strains which demon-strates an overall ineffectiveness in producing phenol. BL21Gold(DE3) had a high optical density value, indicating fastgrowth, and moderate specific production and tyrosine con-centration values, showing neither efficiency nor inefficiencyin producing phenol. Overall, BL21(DE3) had a very highspecific production value (59.424 mg/L/OD) and a moderatetyrosine concentration (47.89 mg/L), which demonstrates ahigh efficiency in metabolizing tyrosine into phenol. The highspecific production value and moderate tyrosine concentration,combined with its low marginal error, led to the conclusion thatBL21(DE3) is the best strain for the biosynthesis of phenol.Additionally, P25 was determined to be an ideal catalyst forthe production of tert-butylphenyl ether, as in a mere 2 hoursit demonstrated an extremely high selectivity towards TBPEand was able to produce 0.0654 M of the ether.

V. CONCLUSION

This project explored an alternative to the industrial methodof producing phenol that relies on fossil fuels. Out of the fivestrains tested during this study, BL21(DE3) was determined tobe the most suitable for the production of phenol. BL21(DE3)was the most efficient in its use of tyrosine to produce phenol,and had one of the lowest marginal errors. P25 was alsodetermined to be an effective catalyst for the production ofalkylated phenols.

The biosynthesis of phenol is far more environmentallyfriendly than producing the desired aromatic compound fromnonrenewable resources such as fossil fuels. Additionally,because E. coli cells are readily available, can grow exponen-tially, and can be engineered and stored for long term use, thisprocess for production is extremely sustainable and econom-ically efficient. Furthermore, this method relies on tyrosine,which can be derived from carbon sources such as carbon

dioxide in the air, making this process chemically green. Whilethe technique that uses fossil fuels utilizes harsh conditions,most of the production of phenol in the E. coli strains occursin conditions that are much easier to maintain. Overall, thisstudy proved that the proposed method of phenol productionthrough metabolic engineering is a robust alternative to currentpetroleum-based production methods.

Ultimately, this study indicated that the production of alky-lated phenols can be optimized with the biosynthesis of phenolwithin the BL21(DE3) strain of E. coli and the tert-butylationof phenol with catalyst P25. In the future, this experimentcould be replicated on a larger scale with the use of tributyrinin the extraction process. The use of tributyrin would makethe phenol extraction process much more efficient in removinglarge quantities of produced phenol from the cultures. Consid-ering the toxicity of phenol to E. coli cells, using tributyrin forextraction would allow the cultures to continue growing andproducing phenol. Successful production of phenol throughmetabolic engineering would also encourage the production ofother useful chemicals through biological means. Additionally,with more time, this project could be extended to experimentwith other E. coli strains. For this experiment, five randomstrains were selected for testing out of eighteen availablestrains. Utilizing all eighteen of the strains would allow fora more conclusive result as to which of the strains most effi-ciently produces phenol. Lastly, the use of catalysts to producetert-butyl phenol would be another aspect of this project toexplore further. As alkylated phenols are extremely valuablecompounds with applications to a wide variety of fields, suchas petrochemicals, pharmaceuticals, and agricultural products,it would be extremely valuable to determine the ideal catalystfor the production of tert-butyl phenols. Further kinetic studiesare required to optimize and completely understand the tert-butylation of phenol using heterogeneous catalysts. Ultimately,the results of this project can provide insight on the beststrain of E. coli for the biosynthesis of phenol, as well as themost effective catalyst for the production of valuable alkylatedphenols.

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APPENDIXStrain of E. coli Initial Optical Density Final Optical DensityBL21 1-A (DE3) 0.300 1.854BL21 1-B (DE3) 0.300 1.485BL21 2-A (DE3) 0.300 1.410BL21 2-B (DE3) 0.300 1.320BL21 Star (DE3) 1-A 0.300 1.878BL21 Star (DE3) 1-B 0.300 2.004BL21 Star (DE3) 2-A 0.300 3.072BL21 Gold (DE3) 1-A 0.300 2.040BL21 Gold (DE3) 1-B 0.300 2.646BL21 Gold (DE3) 2-A 0.300 2.664BL21 Gold (DE3) 2-B 0.300 3.018K12 (DE3) 1-A 0.300 2.976K12 (DE3) 1-B 0.300 3.498K12 (DE3) 2-A 0.300 2.826K12 (DE3) 2-B 0.300 3.384JM109 Star (DE3) 1-A 0.300 1.587JM109 Star (DE3) 1-B 0.300 1.557JM109 Star (DE3) 2-A 0.300 1.515JM109 Star (DE3) 2-B 0.300 1.617

Table 1. Initial and Final Optical Densities Per Sample

Phenol (mg/L) Area Tyrosine (mg/L) Area50 523.5 10 39.3100 904.6 50 196.2250 2214.2 100 370.9500 4390 200 750.91000 8293.7 500 1986.2

Table 2. Peak Areas for Standard Samples of Phenol and Tyrosine

Phenol (g/L) Percent Yield2 51.43%4 51.37%6 21.47%8 21.60%10 20.58%

Table 3. Percent Phenol Extracted by Tributyrin

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Fig. 9. This graph shows how much TBA was used in each catalyst solution

Fig. 10. This graph shows the quantities of each product made in each of the catalyst solutions.

Fig. 11. This graph gives the ratios of products created in each catalyst solution

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ACKNOWLEDGMENTS

The authors of this paper would like to express their grat-itude towards Professor Haoran Zhang, who was an integralsource of guidance and expertise in the lab and provided thestudents with comprehensive lectures and continued support intheir research endeavors. They would also like to thank PhDstudent Xiaonan Wang for her help in the lab and for supple-menting Professor Zhang's role as a mentor. The students arealso thankful for Adam Zuber's help and lessons for the catal-ysis portion of this study. Additionally, they greatly appreciateResidential Teaching Assistant (RTA) Liaison, Kylie Lew, forher assistance and encouragement throughout the researchprocess, as well as for her countless hours put into the projectin order to ensure that it ran smoothly. Furthermore, theywish to thank Research Coordinator and RTA Helen Saggesand Head RTA Michael Higgins for their overall dedicationtowards the Governor's School of Engineering and Technology(GSET) program. The authors would also like to thank DeanJean Patrick Antoine, Director of the NJ GSET, for providingthe scholars the chance to be a part of the program and theopportunity to conduct this compelling research project. Ad-ditionally, the students would like to thank Rutgers Universityand Rutgers School of Engineering for providing them witha unique opportunity to use renowned laboratory facilities.Lastly, they appreciate and thank Lockheed Martin, the NewJersey Governor's School of Engineering and TechnologyAlumni, and the New Jersey Space Grant Consortium for

their generous contributions towards the Governors School ofEngineering and Technology.

REFERENCES

[1] ”2-Tert-butylphenol.” National Center for Biotechnology Information.PubChem Compound Database. Accessed July 24, 2019.

[2] Y.T. Yang, G. N. Bennet, and K.Y. San. Genetic and Metabolic Engineer-ing. Electronic Journal of Biotechnology. December 15, 1998. AccessedJuly 18, 2019.

[3] D. Huccetogullari, Z. W. Luo, and S. Y. Lee. ”Metabolic Engineeringof Microorganisms for Production of Aromatic Compounds.” MicrobialCell Factories. February 26, 2019. Accessed July 16, 2019.

[4] S. Edgar, G. Stephanopoulos, and B. M. Woolston. ”Metabolic Engi-neering: Past and Future.” Annual Reviews. March 27, 2013. AccessedJuly 16, 2019.

[5] Production of Phenol from Cumene. Chemical Engineering Science.September 28, 2001. Accessed July 23, 2019.

[6] B. Kim and S. Y. Lee. ”Metabolic Engineering of Escherichia coli forthe Production of Phenol from Glucose - Kim - 2014 - BiotechnologyJournal - Wiley Online Library.” Biotechnology Journal. October 11,2013. Accessed July 16, 2019.

[7] L. Miao, Q. Li, A. Diao, et al. ”Construction of a Novel Phenol SyntheticPathway in Escherichia coli through 4-hydroxybenzoate Decarboxyla-tion.” SpringerLink. March 12, 2015. Accessed July 16, 2019.

[8] ”E. coli the Biotech Bacterium.” Science Learning Hub. Accessed July24, 2019.

[9] Fogler, H. (2011). Elements of chemical reaction engineering. UpperSaddle River, NJ: Pearson Education Internat.

[10] ”What Is Gel Electrophoresis?” Facts. January 25, 2016. Accessed July20, 2019.

[11] ”High Performance Liquid Chromatography (HPLC).” HiQ. AccessedJuly 20, 2019.

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