Filtering Out Chlorobenzene

Tineer Ahmed
Purpose
To investigate the efficacy of in situ bioremediation plans for the
degradation of chlorobenzene in water by analyzing the
chlorobenzene concentration, chloride concentration, and pH of
five treatment types in a two-week span.
Research Question
Which bioremediation approach yields the most desirable results
for the degradation of chlorobenzene?
Hypothesis
If chlorobenzene undergoes Treatment #4, then it will deteriorate
at a greater concentration than in the other treatment conditions
because the stirring will stimulate the oxidant, hydrogen peroxide,
to chemically react with the contaminant at a quicker rate.
 Background
● Before the 21st century, chemical manufacturers suggested to industries the procedure of
disposing excess chlorinated solvents onto dry ground, allowing the materials to evaporate.
● Scientists did not notice the harmful effects of this practice until 1980s because of the lack of
technology directed toward detecting low concentration organic compounds.
● Consequently, there is debate surrounding which removal approach best treats hazardous
material, comparatively chlorobenzene.

● Chlorobenzene ( C6H5Cl ) has six chlorine atoms and is characterized by its almond-like smell
and colorless appearance. It is primarily used for adhesives and insecticides, but it can
function as a solvent for paints and dry-cleaning.
Background Continued
● In the environment, the water-insoluble liquid migrates in tiny quantities and
accumulates to form a long-term contamination in groundwater, which is water found
in soil pores and rock crevices.
● This issue is significant because chlorobenzene brings health effects, like damage to
central nervous system and irritation to respiratory tract.

● Bioremediation depends on the placement of microbes to degrade the pollutants. This

technique considers the chemical makeup and concentration of pollutants, accessibility
of microorganisms, and the physicochemical characteristics of the environment.
● Bioremediation is classified under ex situ and in situ treatments: Ex situ physically
removes contaminated material, while in situ involves treating the material at its
natural place.
● In situ is also favorable because it limits human exposure risk by reducing contaminant
concentration and remediation time.
Experimental Setup
The following amounts of chlorobenzenes Treatment bottles were prepared using a
were added to each bottle: simulated groundwater formulation
with the following composition:
• 50 mg/L monochlorobenzene
• 25 mg/L 1,2-dichlorobenzene Concentration
Compound
• 10 mg/L 1,2,4-trichlorobenzene (mg/L)
Chloride 0.81
Sulfate 10.17
Iron 0.26
Nitrogen 21.22
Phosphate 39.25
Potassium 12.30
Sodium 17.19
Calcium 1.71
Magnesium 2.46
Treatment # Condition Bacterial Inoculum Oxygen Source

1 Abiotic Control No bacteria. 200 mg/L 50 mL air in headspace (274 mg

NaN3 biocide O2 available)

2 Ambient Air 10 mL of enriched 50 mL air in headspace (274 mg

aerobic degrader culture O2 available)

3 Hydrogen 10 mL of enriched 316 g/L H2O2 + 50 mL air in

Peroxide aerobic degrader culture headspace (47 mg O2 available)

4 Oxygen releasing 10 mL of enriched 74 mg/L CaO(OH)2 + 50 mL air in

solid (mixed) aerobic degrader culture headspace (216 mg O2 available)

5 Oxygen releasing 10 mL of enriched 74 mg/L CaO(OH)2 + 50 mL air in

solid (unmixed) aerobic degrader culture headspace (216 mg O2 available)
 Materials

Vial bottles & Gas

Ion chromatography chromotography
tubes & machine

Five treatment types & Small & large

Contaminated groundwater centrifuge tube
pH probe
Methods
Before taking measurements of the treatment bottles, an interaction
between the bacteria and chlorobenzene needed to be verified:

● Treatment #1 contained 9.1uL chlorobenzene, #2 was 1mL bacteria, and #3 & #4 had
both amounts of chlorobenzene and bacteria
● The results exhibit that the concentration of chlorobenzene decreased in the
experimental bottles

For the preparation of the lab, bacteria needed to be produced:

● Five large centrifuge tubes contained .2 mL of its corresponding treatment and was put
in the centrifuge for 4300rpm, 18 Celsius, 10 minutes
● Liquid is drained out, but the bacteria pellet formed at the bottom of the tube is added
with 10mL of mineral media and vortexed for five seconds
● Add .2mL of the six minerals is added to the tubes, which go to their amber bottles
To test the hypothesis, the treatment types were contained in five separate 250mL amber
bottle:

● For each of the sampling dates, 1mL of the aqueous solution was transferred with the
micropipette to the ten vial bottles (labeled 1a, 1b, 2a, 2b, 3a, etc.)
● With a syringe, 10uL of each of the treatment bottles was inserted into its vial bottle (ex:
Treatment #1 went to its duplicate bottles- 1a and 1b)
● The vial bottles are vortexed for five minutes and are put in the fridge until they are put
in the gas chromatography to calculate chlorobenzene concentration
● With the micropipette, .5mL of the treatment bottles was sampled and placed in five
small centrifuge tubes to measure the pH with the pH probe
● Also, .5mL of the treatment bottles was sampled and place them in five IC tubes that are
put in the ion chromatography to calculate chloride concentration
● Throughout the two weeks, only Treatment #4 was put on the stirrer while the other
bottles were set side
Results
Treatments 1 & 2 and Treatments 4 & 5 were alike in
graph characteristics

A rise in chloride concentration indicates that the

chlorobenzene is degrading

An acidic pH level (less than 7) means the water

contains more free hydrogen ions [H+] from the
breakdown of chlorobenzene

The addition of the organic compounds for Treatments

4 & 5 resulted in a greater number of hydroxide
ions (OH-) and a pH over 7

The spike in chloride concentration for treatment 5 on

March 30th could be the fault of human error
Treatment #3 consistently has the lowest
MCB concentration

The treatment types follow a similar pattern

for the DCB concentration

Overall, Treatments #1 & #4 are not ideal

Conclusion
The results of the experiment negate the hypothesis. Treatment #3 ended with the least amount of
chlorobenzene concentration and the most amount of chloride concentration, so it is most effective
of the treatment types. The stirring option did have an effect on the results because Treatment #5
had a lower monochlorobenzene, dichlorobenzene, and trichlorobenzene than Treatment #4,
meaning the the fifth treatment was more effective. It is inconclusive if Treatment #4 is the next
best plan because its pH and chloride concentration was indistinguishable among Treatments 1 & 2
and each of the treatments’ chlorobenzene graphs are sporadic. When sampling throughout the two
week period, duplicates were taken of each treatment type so that the average is later graphed. A
greater sampling size and thorough sterilization of equipment would minimize the amount of
human error and would result in a smaller standard deviation. Overcoming these limitations would
present a more reliable data with a better perspective of the ranking of the treatment types. This
experiment presents an in situ bioremediation approach that could be applied at a larger scale. In a
further investigation, questions to be addressed are: Will sunlight presence affect the degradation
process? Is there an optimal temperature setting? How much of the liquid evaporated during the
sampling days?
Internship
My hands-on experience at the Department of Environmental Engineering has exposed me to the
innovative process of a research student. Although I cannot travel to specific sites during my
internship, I employ aspects of my research when running and analyzing labs that replicate data of
contaminated water samples from SuperFund sites. Overall, I desired to intern at the Department of
Environmental Engineering because of my interest in creating new technology to enhance traditional
practices, and I am eager to continue fortifying my expertise as an undergraduate at JHU!
 Bibliography
Pictures were originally taken at the internship site.

Chlorobenzene molecule image: http://www.sigmaaldrich.com/catalog/product/sial/284513?lang=en&region=US

The pictures of the centrifuge tubes: https://www.membrane-solutions.com/micro_centrifuge_tubes.htm http://www.laboratorysales.net/centrifuge-tubes-15-and-50-ml.aspx

Background research:

APEC Water. (1999). Chlorobenzene- Drinking Water Contaminants, Facts/Removal Methods.

Retrieved from http://www.freedrinkingwater.com/water-contamination/chlorobenzene-contaminants-removal-water.html
Balcke, G. U., Turunen, L. P., Geyer, R., Wenderoth, Dirk. F. and Schlosser, D. (2004),
Chlorobenzene biodegradation under consecutive aerobic–anaerobic conditions. FEMS Microbiology Ecology, 49: 109–120. doi:10.1016/j.femsec.2003.08.014
Boopath, R. (2000). Factors limiting bioremediation technologies. Bioresource Technology, 74,
63-67. doi: 10.1016/S0960-8524(99)00144-3
El Fantroussi, S. & Agathos, S.N. (2005). Is bioaugmentation a feasible strategy for pollutant
removal and site remediation? Current Opinion in Microbial Biology, 8, 268-275. doi: 10.1016/j.mib.2005.04.011
John J. McKetta Jr. (1993). Chemical Processing Handbook. Austin, Texas: CRC Press.
Kueper, B.H., Leharne, S.A., Lerner, D.N., Smith, J.W.N., & Wealthall, G.P. (2003). An
illustrated handbook of DNAPL transport and fate in the subsurface. Environmental Agency, 133, 1-10. Retrieved from https://clu-in.org/conf/itrc/dnaplpa/dnapl_handbook_final.pdf
McGuire, T. M., McDade, J. M. and Newell, C. J. (2006). Performance of DNAPL Source
Depletion Technologies at 59 Chlorinated Solvent-Impacted Sites. Ground Water Monitoring & Remediation, 26: 73–84. doi:10.1111/j.1745-6592.2006.00054.
Montgomery, J. H. (2007). Groundwater Chemicals Desk Reference, Fourth Edition. Retrieved
from https://books.google.com/books?id=AbLpHq-V9j4C&pg=PA236&dq=Chloroben
zene&hl=en&sa=X&ved=0ahUKEwiUvsa73ZfQAhUj5YMKHRnOBjAQ6AEINTAF#v=onepage&q=Chlorobenzene&f=false
U.S. Environmental Protection Agency. (2000). Chlorobenzene. Retrieved from
https://www3.epa.gov/airtoxics/hlthef/chlorobe.html
U.S. Environmental Protection Agency. (2006). In Situ and Ex Situ Biodegradation Technologies
for Remediation of Contaminated Sites. Retrieved from https://clu-in.org/download/contaminantfocus/dnapl/Treatment_Technologies/epa_2006_engin_issue_bio.pdf