Pseudomonas species were grown in microcosms containing carbon tetrachloride and one of three biostimulants: baby formula, soil extract, or a mixture of both. Baby formula enhanced the growth of Pseudomonas in the presence of carbon tetrachloride. No growth was observed in the soil-only microcosms. While carbon tetrachloride was detected in all microcosms using GC/MS, its breakdown products could not be identified, possibly due to frequent sampling making conditions aerobic or interference from chemicals in the baby formula and soil. Further study is needed to optimize conditions for nitrate reduction and detection of breakdown products.

1. INTRODUCTION
The presence of xenobiotic chemicals in ground water systems pose a serious threat to
human health, and an even greater difficulty in their remediation. Utilizing bioaugmentation
techniques as a way to stimulate microbial growth and break down these dangerous
chemicals is a preferable method when compared to other expensive and ineffective
techniques, which typically involve the mechanical pumping and treating of water (Best et
al. 1998; Bouwer et al. 1983). Carbon tetrachloride (CT), a regulated industrial solvent
today, is a known groundwater contaminate. It is a carcinogen capable of causing damage
to the liver, kidneys, and nervous system at high enough concentrations (Criddle et al.
2013). Carbon tetrachloride is resistant to both oxidative processes and hydrolysis because
of its chemical nature and structure, making reductive dehalogenation the most effective
way to degrade the compound. Cometabolic nitrate reduction is one such process that
breaks down CT, reducing it into products such as chloroform, dichloromethane, and
methane (Boopathy 2002; Bouwer et al. 1983). Stimulating the growth of naturally occurring
nitrate reducers in groundwater systems would be a simple, effective, and non-invasive
procedure for CT degradation. Previous studies have shown Pseudomonads as capable of
breaking down CT through the process of nitrate reduction under anaerobic conditions.
Stimulating the growth and metabolic activity of Pseudomonads in a CT rich environment
under anaerobic conditions was the prime concern of this study. The intention of the study
was to identify a biostimulant that not only provided the necessary materials and energy
sources, but could also be store easily and not significantly shock contaminated ground
water sources.
Preliminary Bioremediation of Carbon
Tetrachloride Under Denitrifying
Conditions
!
!
Justin Amburgey, Nathan Putz, and Daniel French
University of Evansville
CONCLUSIONS
Pseudomonas species were subjected to high concentrations of CT, 10.8 mol/L environments.
The microcosms augmented with baby formula were capable of stimulating growth of the
Pseudomonads, but soil microcosms were unable to support life.The capabilities of baby formula
to stimulate microbial growth and improve survivorship of Pseuomonads in CT rich environments
were both shown in this study. The organisms utilized in this study’s ability to breakdown CT
using nitrate reduction remained undetected. The GC/MS was able to detect the high
concentrations of carbon tetrachloride, but the breakdown products were indiscernible. This may
have been due to frequent sample withdraws from the microcosms, making them aerobic. Further
steps to keep microcosms anaerobic must be taken in the future to stimulate nitrate reduction.
The complexity of chemicals in both baby formula and soil may have also interfered, and even
recombined with CT breakdown products, which would have made their discernment in GC/MS
difficult. Running microcosm samples through the GC/MS revealed a plethora of peaks, with only
the presence of CT being frequently represented. Breakdown products may have combined with
other organic chemicals present in the microcosms; this may have been observed as several
unidentifiable chlorinated compounds. A better understanding of the contents of baby formula and
how these interact with CT reduction products is necessary for measuring CT degradation. Given
this, it is possible the reduction of CT still may have occurred. Measuring the concentration of CT
present at the end was not an option because the baby formula samples would not give a pure
spectra reading. Extraction of CT using diethyl ether, and even the extraction of DNA using
InstageneTM Matrix, both produced a semi-solid in baby formula based microcosms. This caused
a problem during extractions, and required a second extraction in both instances. Should this be
the result of the proteins and/or lipids present in baby formula in large amounts, the semi-solid
could be degraded using proteinase and lipase before extractions. Pseudomonas was shown to
be able to survive high levels of CT in conjunction with baby formula as a stimulant, and CT laden
samples were able to be preserved in teflon sealed test tubes. Retaining CT in samples during
storage until extraction was a concern. Useful information was acquired for microcosm CT
application, CT extraction, bacterial DNA extraction from baby formula microcosms, and GC/MS
procedures.
ACKNOWLEDGMENTS
Special thanks to Dr. Kaufman for allowing the use of and assisting with the GC/MS
RESULTS
Pseudomonas growth in a CT rich environment was enhanced by the presence of baby formula
as a biostimulant in all microcosms augmented with baby formula. There were no observable
changes in growth in the soil only microcosms. The presence of Pseudomonas in CT
microcosms was identified using PCR (Fig.1c). Heating baby formula at 100°C for ten minutes
was determined to be ineffective at sterilizing baby formula for the microcosms from observing
the presence of both Pseudomonas and an unidentified gram positive bacteria in baby formula
and half controls (Fig.1b). It is possible that contamination may have occurred. Double liquid-
liquid extractions were required for microcosms which contained baby formula; this was
attributed to the formation of a semisolid with the addition diethyl ether. Carbon tetrachloride
was successfully identified in all microcosms using GC/MS, with main peaks at 117 and 82 (Fig.
3). The identification breakdown products using GC/MS did not occur, but several unknown
chlorinated compounds were observed.
METHODS
Species: Pseudomonas aeruginosa and P. stutzeri were grown in half strength LB
media for two days at room temperature.
!
Experimental Design: Cultures of Pseudomonas aeruginosa and P. stutzeri were grown
separately in prepared sterile microcosms. Microcosms contained 300mL of Parent’s
Choice Premium baby formula, soil extract, or a solution of half baby formula-half soil
extract. Baby formula was prepared at full strength according to container instructions,
43.62 g were mixed in ultra pure water and heated at 100°C for ten minutes (Best et al.
1998; Bouwer et al. 1983). A greater temperature would curdle the baby formula,
resulting in an unusable product. Soil extract was made by mixing 300g of potting soil
(manufacturer unknown) with 2L deionized water. Soil solution was left to settle
overnight. Supernatant was then autoclaved. Half baby formula and half soil extract
microcosms were made using a 1:1 ratio. Each microcosm contained 5mL of carbon
tetrachloride and were kept at room temperature. Microcosms were made in triplicate,
and controls of the three microcosm types were made without bacteria. Samples were
taken from each microcosm twice a week to test pH, presence of metabolically active
bacteria, and for GC/MS.
!
PCR: DNA was extracted from bacteria in microcosms using InstaGeneTM Matrix. PCR
was used to test for the presence of Pseudomonas aeruginosa, P. stutzeri, and general
bacteria (using universal primers). Primers for the Pseudomonads are shown in
Fig.1a (Bennasar 1998).
!
Chemistry: Liquid-liquid extractions were done on each sample using diethyl ether as
the solvent. Samples were injected into a Hewlett Packard GC/MS, Model 5890, using a
splitless injection. A 5 minute solvent delay was used. Initial oven temperature was set
to 35°C (below the boiling point of dichloromethane 39.6°C).
LITERATURE CITED
Bennasar G., and Lalucat J. 1998. Molecular Methods for the Detection and Identification of !! !
! Pseudomonas stutzeri in Pure Culture and Environmental Samples. Microbial Ecology. 35: ! !
! 22-33. !
Best, J. Salminen, E., Doddema, H., Janssen, D. and Harder W. 1998. Transformation of Carbon !
! Tetrachloride under Sulfate Reducing Conditions. Biodegradation. 8: 429-36.!
Boopathy, R. 2002. Anaerobic Biotransformation of Carbon under various Electron Acceptor !! !
Conditions. Bioresource Technology. 84: 69-73.!
Bouwer, E. and McCarty P. 1983. Transformation of Halogenated Organic Compounds under ! !
! Denitrification Conditions. Environmental Microbiology. 45: 1295-99.!
Criddle, C., DeWitt, J., Grbic-Galic, D., and McCarty, P. 1990. Transformation of Carbon ! ! ! !
! Tetrachloride by Pseudomonas sp. Strain KC under Denitrification Conditions. Applied and ! !
! Environmental Microbiology. 56: 3240-46.!
Figure 2. GC/MS results revealing the presence of carbon tetrachloride. Two
identifying CT peaks are found at 117m/z and 82m/z, with isotopes to either
side.
Figure 1. a) A table depicting the Pseudomonas primers used. Primers were ordered from
Integrated DNA Technologies. b) A gel depicting soil, half, and baby formula controls. Single
band at top shows presence of P. aeruginosa in the half control. Two bands at the bottom
show bacterial (16s universal primer) presence in the half and baby formula controls. c) Box 1
shows bacterial (16s universal primer) presence in half and baby formula. Box 2 shows no
bands for soil microcosms with P. stutzeri. Box 3 shows presence of P. stutzeri in half
microcosms. Box 4 shows that half sample 2 show a bacterial band that was not P. stutzeri
(16s universal primer).
OBJECTIVES
To determine the effectiveness of Baby Formula as a biostimulant and generate a method for
Baby Formula sterilization, extraction, and detection of CT breakdown products by GC/MS.
Justin Amburgey injecting a sample into the GC/MS for analysis.
SPECIES PRIMER
Pseudomonas
aeruginosa
5’ - GGG GGA TCT TCG GAC CTC - 3’!
5’ - TCC TTA GAG TGC CCA CCC - 3’
Pseudomonas stutzeri
5' - TTG CTG GGT GGA TTA GTG - 3’!
5’ - CAT CTC ACG ACA CGA GCT - 3’
a) b)
1.
2.
3.
4.c)