Bacterial Degradation of Trifluarlin
and 2,4-Dichlorophenoxyacetic acid
in York County
Gavin Durkin
Mentor: Dr. Carolyn Mathur
Chemical structure for Trifluralin
from: http://www.sigmaaldrich.com/catalog/product/riedel/45700?lang=en&region=US
Introduction
Bioremediation is the biological
degradation of toxic wastes into less
hazardous compounds. Naturally
occurring organisms are usually the
best candidates for bioremediation
(Antonious 2012). Furthermore,
bacteria are the most common organisms
which perform these processes. Some
bacterial species, such as those in
the Pseudomonas family, have been
found to persist in contaminated soil,
and several are known to degrade these
toxic residues into some less toxic
components (Ferreira-Guedes 2011).
Herbicides usage is increasing all
over the world, both in the commercial
and private realms (Ferreira-Guedes et
al. 2011). Two broadleaf herbicides,
Trifluralin and 2,4Dichlorophenoxyacetic acid, or 2,4-D,
are known to produce toxic effects on
the environment (Ferreira-Guedes et
al. 2011, Antonious 2012). These
herbicides, as well as their many
residues, may remain in the soil for
many years due to their affinity for
soil and low solubility (FerreiraGuedes et al. 2011, Antonious 2012).
While not so common, bacteria from
around the world, have been found to
degrade herbicides, such as several
species which can degrade Trifluralin
in Brazil to a strain of Comamonas
koreensis which can degrade 2,4-D (De
Lourdes Bellinaso et al. 2006, Wang et
al. 2009).
Almost 33% of the land in York county,
Pennsylvania is uses as farmland,
making it the county with the second
highest number of farms in
Pennsylvania (Agricultural Facts
2012). However, there are only about
50 organic farms (Pennsylvania
Certified Organic 2012). Therefore it
can be speculated that much of the
farmland utilizes herbicides.
To
Objective
find naturally occurring
bacteria which can degrade
Trifluralin.
To
find naturally-occurring
bacteria which can degrade 2,4-D.
Chemical structure of 2,4-D
from:http://www.sigmaaldrich.com/catalog/product/aldrich/d70724?lan
g=en&region=US#
Methods
Soil samples were collected from a
farm and garden in York County, PA.
Results
The
16s rDNA of T4 is 97% similar to
Comamonas koreensis strain KCTC 12005
1.Growth
a.Turbidity
b.Gram stains
1.Enrichment Culture Technique
(Stukus 1997)
Discussion
A similar strain, Comamonas koreensis
strain CY01, is capable of dechlorinating
2,4-D (Wang et al. 2009).
Two
Figure 3. Gram
stain of the
bacteria T4. The
cells are G- at
0.55 x 1.04um.
Figure 2. Gram
stain of the
bacteria T3. The
cells are G- rods
at 0.49 x 0.85um.
Figure 4. Gram
stain of the
bacteria 2,4Ds.
The cells are Gat 0.53 x 1.02um.
Figure 5. Gram
stain of the
bacteria 2,4D2.
The cells are Gat 0.58 x 1.02um.
2. Colony Morphology
T3 has a mucoid colony morphology while T4
has a non-mucoid colony morphology.
It is possible these might be two
isolates from the same species as
Comamoas koreensis has two colony
morphologies, mucoid and non-mucoid
(Willems and Gillis 2005).
Figure 1. A diagram displaying the Enrichment Culture Technique in
which each flask contains 100mL of Minimal Salts Medium, 0.1% of the
alternative carbon source, and a decreasing amount of nutrient broth,
until no nutrient broth is present. The first flask contains 1.0g of
soil. In this series of dilutions 1.0mL is transferred from the
previous flask to the next one.
A similar strain, Comamonas koreensis
strain CY01, is capable of dechlorinating
2,4-D (Wang et al. 2009).
2. Identification
a.Macroscopic
i. Colony morphology
b.Microscopic:
i. Cell shape
ii.Cell size
iii.Gram stain
Technique
c.16s rDNA analysis using the
Qiagen “Puregene” Kit
i. DNA was sequenced
with BLAST.
3. Confirmatory Tests:
bacterial isolates, T3 and T4, were
found to degrade Trifluralin.
Figure 6. Bacteria colonies of the bacteria
sample T3.
•
•
•
•
Bacteria within the Comamonas genus are
known to contribute to many
bioremediation processes due to their
abilities to degrade complex organic
compounds. C. testosteroni is capable of
degrading 4-toluenesulfonic acid while
Comamonas strains JS46 and JS47 can
degrade m- and p-nitrobenzoate (Willems
and Gillis 2005).
T3: Unpigmented, convex, shiney, smooth, and wet
T4: Unpigmented, convex, dull, smooth, and dry
2,4D2: Unpigmented, umbonate, shiney, and smooth
2,4Ds: Unpigmented, umbonate, shiney, and smooth
3.PCR bands
T3
T4
24D2 24Ds

2,4D2 and 2,4Ds, are isolates of the
same bacterial colony and can degrade
2,4-Dichlorophenoxyacetic acid,
however, since their DNA was unable to
be successfully sequenced, they remain
unknown.
Growth of Isolates in Pure Chemicals,
from Sigma Aldrich.
Each sample was shaken for 24hours at
30oC, then left to grow for one week
without a photocycle.
a. 0.1% pure 2,4-D in MSM
broth
i. 2,4D2
ii.2,4Ds
iii.Control: No
bacteria
b. 0.1% ethanol/pure
Trifluralin solution
i. T3
ii.T4
iii.Control No
bacteria
c. Control: 0.1% ethanol in
MSM broth
i. T3
ii.T4
iii.No Bacteria
d.Gram stain Procedure
Future Studies
Figure 7. DNA bands from a PCR of
the four bacterial samples. The
bands at 1500bp.
4.BLAST search:
• The 16s rDNA sequence of T4 yielded a
97% match to Comamonas koreensis strain
KCTC 12005
5.Pictures of confirmatory gram stains
•
•
•
•
•
•
•
Figure 8. T3
grown in pure
Trifluralin.
Figure 9. T4
grown in pure
Trifluralin.
Figure 10. 2,4Ds
grown in pure
2,4-D.
What mechanisms do these bacteria utilize to degrade
these herbicides?
•
What is the efficiency at which these bacteria can
degrade their specific herbicide?
Literature Cited
•
Figure 11. 2,4Ds
grown in pure
2,4-D.
•
•
Agricultural Facts-York County, PA. Available from: http://yorkcountypa.gov/images/pdf/Agricultural-FactsYork-County-PA.pdf. Accessed 2012 November 19.
Antonious, G. F. (2012, May 4). On-farm bioremediation of dimethazone and trifluralin residues in runoff
water from an agricultural field [Electronic version]. Journal of Environmental Science and Health, Part B:
Pesticides, Food Contaminants, and Agricultural Wastes, 47(7), 608-621.
Bergey's manual of systematic bacteriology 9th ed. (2005). Baltimore, MD: The Williams and Wilkins Company.
De Lourdes Bellinaso, M., Greer, C. W., do Carmo Peralba, M., Henriques, J. A. P., & Gaylarde, C. C. (2006,
January). Biodegradation of the herbicide trifluralin by bacteria isolated from soil [Electronic version].
FEMS: Microbiology Ecology, 43(2), 191-194.
Ferreira-Guedes, S., Mendes, B., & Leitão, A. L. (2012, February). Degradation of 2,4-dichlorophenoxyacetic
acid by a halotolerant strain of Penicillium chrysogenum: antibiotic production [Electronic version].
Environmental Technology, 33(4-6), 677-686.
Pennsylvania Certified Organic. Available from: http://www.paorganic.org/farmfinder.
Accessed 2012
November 19.
Stukus, P. E. (1997). Investigating microbiology: A laboratory manual for general microbiology (pp. 305309). N.p.: Harcourt Brace & Company.
Wang, Y., Wu, C., Wang, X., & Zhou, S. (2009, May 30). The role of humic substances in the anaerobic
reductive dechlorination of 2,4-dichlorophenoxyacetic acid by Comamonas koreensis strain CY01 [Electronic
version]. Journal of Hazardous Materials, 164`(2-3), 941-947.
Willems, A., & Gillis, M. (2005). Genus I. Comamonas. In D. J. Brenner, N. R. Krieg, & J. T. Staley (Eds.),
Bergey's Manual of Systematic Bacteriology (2nd ed., Vol. 2, pp. 689-696). East Lansing, MI: Springer.
Acknowledgments
I would like to thank Dr. David Singleton for all of his help and guidance.