Chlorophyll Extraction for

Bioremediation Applications
Larry Rountree,* Dean Archer*, Tad
Whitesides** 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 of
chlorinated wastes at the Savannah River Site (SRS)
need a source of nitrogen for metabolic processes. A
cheap and abundant source of nitrogen is chlorophyll.
In this experiment, multiple variables were tested to
obtain the optimal conditions for the extraction of
chlorophyll from local plant species. Magnolia, Live Oak,
and Saw Palmetto leaves were extracted using edible
soybean oil. Varying concentrations of triethylphosphate
(TEP) were incorporated into the solutions. The
extractions were run at room temperature and 37
0
C.
Optimal chlorophyll extraction was obtained by using
magnolia leaves extracted at 37
0
C with the highest
concentration of TEP.
Introduction:
In industry, a common problem encountered is how to
properly dispose of waste, particularly chlorinated and
organic solvents (i.e. BTEX -benzene, toluene,
ethylbenzene and xylene; trichloroethylene,
perchloroethylene) without the cause of risk to the
environment.
To rectify the problem, researchers at the SRS and
Savannah River National Laboratory (SRNL) are
investigating the use of bioremediation.
Background: SRS and SRNL
The SRS and the SRNL are located in Aiken, SC.
The SRS constructed during the early 1950s, in support of our nation's defense
programs, to produce materials used in the fabrication of nuclear weapons, mainly
tritium 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 the
environment. SRS will be a modernized DOE site, recognized for performance and
excellence in support of our national security and as a responsible steward of the
environment.
The Savannah River National Laboratory (SRNL) is the applied research and
development laboratory at the U.S. Department of Energys (DOE) Savannah River
Site (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 toxic
chemicals and metabolize them into less harmful
byproducts, such as carbon dioxide, salts and water.
This is accomplished by injecting edible soybean oil into
the ground near a spill, allowing the solvent to dissolve
into the oil and allowing the bacteria to eat the oil, which
contains the solvent to be remediated.
In order to hasten the process, we want to introduce
nitrogen into the soil, since the bacteria utilized are not
nitrogen fixing. We are investigating if this is feasible by
dissolving a non-toxic nitrogen containing compound into
the soy bean oil prior to injection. A cheap, abundant and
non-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 a
particular molecule to emit a
photon at a longer wavelength
after it has been excited by a
photon at a shorter wavelength.
The wavelength at which a
molecule emits a photon is
dependent upon that molecule.
The intensity measured from the
emission of the photon is directly
proportional to the concentration
of that molecule. This allows for
quantitative measurements.
Purpose:
Our goal is to help the researchers at SRS to
determine the optimum plants and materials for
the extraction of the nitrogen into the edible oil.
We analyzed the amount of chlorophyll, as an
indicator of the amount of nitrogen, in each
sample via fluorescence
Procedure: Chlorophyll Extraction
Leaves of three local plants (Live Oak, Magnolia and
Saw Palmetto) were obtained
Leaves were removed from the stems and cut into small
pieces
1 gram samples of each plant were prepared in triplicate
under the following conditions:
-Room temperature and 37
o
C
-1, 2, and 5% triethylphosphate in soybean oil (total
volume of 10 mL combined)
-Incubated for 24 hours and 0.5 mL samples were taken
at 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 of
chlorophyll in soybean oil samples, diluted in n-
heptane
Fluorescence Analysis of
Chlorophyll Standards
Fluorescence Emission Spectra of
Chlorophyll 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 = 19.114x
R
2
= 0.9768
0
50
100
150
200
250
0 3 6 9 12
Concentration ( M)
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 fluorometric
analysis of samples, we obtained the amount of
chlorophyll extracted by fitting the intensities to the
calibration plot to find the concentration and then back
calculated to micro-grams of chlorophyll per gram of leaf
Results: Magnolia
Room Temperature
43.2 6.9 4.9 1.0 5
18.5 7.1 4.4 1.9 2
15.0 1.6 4.9 0.8 1
24 hr
2 hr %
TEP
37
o
C
74.4 28.0 30.5 8.7 5
74.5 26.9 34.2 8.3 2
43.8 5.6 18.5 1.3 1
24 hr 2 hr %
TEP
* the unit of the measurement is micrograms of nitrogen per
gram of leaf
Results: Saw Palmetto
Room Temperature
12.5 1.4 5
8.6 1.8 2
5.4 1.1 1
24 hr
% TEP
37
o
C
46.3 7.5 9.1 0.6 5
28.4 1.0 5.1 0.6 2
30.3 12.8 5.0 1.6 1
24 hr 2 hr % TEP
* the unit of the measurement is micrograms of nitrogen per
gram of leaf
Results: Live Oak
Room Temperature
31.3 3.0 5
10.6 6.2 2
9.8 3.5 1
24 hr
% TEP
37
o
C
37.9 1.6 19.7 1.2 5
25.9 2.4 6.8 2.3 2
29.0 1.6 11.5 1.8 1
24 hr 2 hr % TEP
* the unit of the measurement is micrograms of nitrogen per
gram of leaf
Conclusions:
Only Magnolia showed appreciable amounts
extracted at room temperature at times shorter
than 24 hours
Optimal chlorophyll extraction can be obtained
at higher temperatures and longer extraction
times
Magnolia leaves displayed the greatest content
of chlorophyll amongst the leaves tested
The incorporation of a higher concentration of
TEP allows for a relative increase in the amount
of chlorophyll extracted in most cases
Future Work:
Examine other (higher) concentrations of
TEP
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