Chlorophyll Extraction Poster

8/12/2019 Chlorophyll Extraction Poster

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Chlorophyll Extraction for

Bioremediation Applications

Larry Rountree,* Dean Archer*, TadWhitesides** and Delana A. Nivens***

*undergraduate students** collaborator, Savannah River National

Laboratory at the Savannah River Site*** faculty advisor



Abstract:Bacteria that are involved in the bioremediation ofchlorinated wastes at the Savannah River Site (SRS)need a source of nitrogen for metabolic processes. Acheap and abundant source of nitrogen is chlorophyll.In this experiment, multiple variables were tested to

obtain the optimal conditions for the extraction ofchlorophyll from local plant species. Magnolia, Live Oak,and Saw Palmetto leaves were extracted using ediblesoybean oil. Varying concentrations of triethylphosphate

(TEP) were incorporated into the solutions. Theextractions were run at room temperature and 37 0C.Optimal chlorophyll extraction was obtained by usingmagnolia leaves extracted at 37 0C with the highestconcentration of TEP.



Introduction:

• In industry, a common problem encountered is how toproperly dispose of waste, particularly chlorinated andorganic solvents (i.e. BTEX -benzene, toluene,ethylbenzene and xylene; trichloroethylene,perchloroethylene) without the cause of risk to theenvironment.

• To rectify the problem, researchers at the SRS and

Savannah River National Laboratory (SRNL) areinvestigating the use of bioremediation.



Background: SRS and SRNL

• The SRS and the SRNL are located in Aiken, SC.• The SRS constructed during the early 1950’s, in support of our nation's defense

programs, to produce materials used in the fabrication of nuclear weapons, mainlytritium and plutonium-239.

• Over the years, it has had many missions.• Currently its mission is: We serve the nation through safe, secure, cost-effective

management of our nuclear weapons stockpile, nuclear materials, and theenvironment. SRS will be a modernized DOE site, recognized for performance andexcellence in support of our national security and as a responsible steward of theenvironment.

• The Savannah River National Laboratory (SRNL) is the applied research and

development laboratory at the U.S. Department of Energy’s (DOE) Savannah RiverSite (SRS).• Numerous sites at SRS contain buried tanks of hazardous waste, some of which are

leaking into the ground around the tanks.• Researchers are interested in finding novel ways to clean up (remediate) the waste.



Background: Bioremediation

• A process whereby bacteria are able to ingest toxicchemicals and metabolize them into less harmfulbyproducts, such as carbon dioxide, salts and water.

• This is accomplished by injecting edible soybean oil intothe ground near a spill, allowing the solvent to dissolveinto the oil and allowing the bacteria to eat the oil, whichcontains the solvent to be remediated.

• In order to hasten the process, we want to introducenitrogen into the soil, since the bacteria utilized are not

nitrogen fixing. We are investigating if this is feasible bydissolving a non-toxic nitrogen containing compound intothe soy bean oil prior to injection. A cheap, abundant andnon-toxic source of nitrogen is chlorophyll.



Background: Chlorophyll a

• Found in all green plants• Chlorophyll a absorbance – 440 nm• Chlorophyll a fluorescence – 685 nm• Harvests light from the sun• Contains 4 nitrogens



Background: Fluorescence

• Fluorescence is the ability of aparticular molecule to emit aphoton at a longer wavelengthafter it has been excited by aphoton at a shorter wavelength.

• The wavelength at which amolecule emits a photon isdependent upon that molecule.

• The intensity measured from theemission of the photon is directlyproportional to the concentrationof that molecule. This allows forquantitative measurements.



Purpose:

• Our goal is to help the researchers at SRS todetermine the optimum plants and materials forthe extraction of the nitrogen into the edible oil.

• We analyzed the amount of chlorophyll, as anindicator of the amount of nitrogen, in eachsample via fluorescence



Procedure: Chlorophyll Extraction

• Leaves of three local plants (Live Oak, Magnolia andSaw Palmetto) were obtained

• Leaves were removed from the stems and cut into small

pieces• 1 gram samples of each plant were prepared in triplicateunder the following conditions:-Room temperature and 37 oC

-1, 2, and 5% triethylphosphate in soybean oil (totalvolume of 10 mL combined)-Incubated for 24 hours and 0.5 mL samples were takenat various times and frozen until analysis



Procedure: Chlorophyll analysis

• Prepared standards of chlorophyll in heptane solution. – Samples diluted in spectrophotometric grade n-

heptane – Excitation of samples at 615 nm – Linear calibration curve made by graphing

fluorescence emission intensity at 664 nm – Error bars represent one standard deviation

– Calibration curve used to obtain the concentration ofchlorophyll in soybean oil samples, diluted in n-heptane



Fluorescence Analysis ofChlorophyll Standards

Fluorescence Emission Spectra ofChlorophyll a Standards

0

75

150

225

300

625 640 655 670 685 700

Wavelength (nm)

F l u o r e s c e n c e

I n t e n s

i t y

( n m

)

1.1 uM

2.8 uM

5.6 uM

8.4 uM

11.2 uM

22.4 uM

Chlorophyll Calibration Curve y = 19.114xR2 = 0.9768

0

50

100

150

200

250

0 3 6 9 12

Concentration (µµµµM)

F l u o

r e s c e n c e

I n t e n s i t y

( a r b i t r a r y

u n i t )



Data Analysis:

• Using fluorescence intensity obtained from fluorometricanalysis of samples, we obtained the amount ofchlorophyll extracted by fitting the intensities to thecalibration plot to find the concentration and then back

calculated to micro-grams of chlorophyll per gram of leaf



Results: Magnolia

Room Temperature

43.2 ± 6.94.9 ± 1.05

18.5 ± 7.14.4 ± 1.92

15.0 ± 1.64.9 ± 0.81

24 hr2 hr%

TEP

37oC

74.4 ± 28.030.5 ± 8.75

74.5 ± 26.934.2 ± 8.32

43.8 ± 5.618.5 ± 1.31

24 hr2 hr%TEP

* the unit of the measurement is micrograms of nitrogen pergram of leaf



Results: Saw Palmetto

Room Temperature

12.5 ± 1.45

8.6 ± 1.825.4 ± 1.11

24 hr% TEP

37oC

46.3 ± 7.59.1 ± 0.65

28.4 ± 1.05.1 ± 0.6230.3 ± 12.85.0 ± 1.61

24 hr2 hr% TEP




Results: Live OakRoom Temperature

31.3 ± 3.05

10.6 ± 6.229.8 ± 3.51

24 hr% TEP

37oC

37.9 ±1.619.7 ± 1.25

25.9 ± 2.46.8 ± 2.32

29.0 ± 1.611.5 ± 1.81

24 hr2 hr% TEP




Conclusions:

• Only Magnolia showed appreciable amountsextracted at room temperature at times shorterthan 24 hours

• Optimal chlorophyll extraction can be obtainedat higher temperatures and longer extractiontimes

• Magnolia leaves displayed the greatest contentof chlorophyll amongst the leaves tested

• The incorporation of a higher concentration ofTEP allows for a relative increase in the amountof chlorophyll extracted in most cases



Future Work:

• Examine other (higher) concentrations ofTEP

• Examine other local plants• Have samples analyzed by a second

method to confirm amounts of nitrogen

found



Acknowledgements:

•Tad Whitesides (SRNL)•Chemistry and Physics Department of AASU

•Dr. Delana Nivens

Chlorophyll Extraction Poster

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