1. Acknowledgements
Results
Conclusions and Future Research
Analysis
Monod Equation:
• dS/d t = rate of substrate consumption
• 𝒌 𝒎𝒂𝒙 = maximum specific growth rate
• 𝑺 = concentration of substrate
• KS = half rate constant
• Chlorinated ethenes such as trichloroethene (TCE), cis-dichloroethene
(cis-DCE), and vinyl chloride (VC) are common groundwater
contaminants.
• The Mycobacterium ELW-1 is a newly isolated organism from North
Carolina State University that was found to have important cometabolic
capabilities to transform the above contaminants into less toxic
substances.
o Grows on Isobutene:
Introduction
C4H8+6O2→4CO2+4H2O
Materials and Methods
The following materials were used in all experiments:
• 26mL batch reactors with Teflon-lined rubber septa and
aluminum crimp tops.
• Concentrated culture samples grown in either batch or
steady state chemostat environments.
• Growth media containing trace minerals such as cobalt.
10mL of growth media was prepared in each reactor, and
specific concentrations of biomass were added to the media
such that the metabolic rates were not mass transfer limited.
Isobutene
Chlorinated Ethenes
Final Monod Curves:
Final Transformation Rates:Isobutene:
Chlorinated Ethene Transformation:
0
5
10
15
20
25
30
35
40
45
0 25 50 75 100 125 150
Rate
(μmol/mgprotein/day)
CL0 (μM)
Batch
𝒌 𝒎𝒂𝒙 = 38.17 µmol/day/mg protein
KS = 5.55µM
TCE cis-DCE VC
• Low concentrations of isobutene gas (5-
15µL) were mixed into batch reactors with
around 1.0-1.5mg of biomass present.
• Higher concentrations of isobutene gas (25-
500µL) were mixed into batch reactors with
around 4.3-4.8mg of biomass present.
• Various initial concentrations of
TCE, cis-DCE, and VC were
added to batch reactors containing
about 4.0-10.9mg according to the
table to the right.
Substance Initial Mass (umol)
VC 9.1-10.7
cis-DCE 14.7-17.1
TCE 2.7-3.0
𝒅𝑺
𝒅𝒕
= 𝑿𝑲 𝒎𝒂𝒙
𝑺
𝑲 𝒔 + 𝑺
0.0
1.0
2.0
3.0
4.0
5.0
6.0
7.0
8.0
9.0
10.0
11.0
12.0
13.0
0.00 0.03 0.05 0.08 0.10 0.13 0.15 0.18
Time (days)
Batch High Concentrations
100 uL
200uL
300 uL
400 uL
• Rates of isobutene consumption with a
constant amount of ELW1 biomass were
calculated using best linear fit estimates.
• Analysis performed using a wide range of
initial isobutene concentrations for further
analysis using the Monod Equation.
Cell Growth Compound Initial Liquid
Concentration
(umol/L)
Rate of
Transformation
(umol/day/mg TSS)
Batch VC 421 5.1
Chemostat VC 382 3.2
Batch c-DCE 1330 4.1
Chemostat c-DCE 1400 3.5
Batch TCE 198 0.2
Batch Isobutene 118 15.9
Chemostat Isobutene 129 11.2
• Based on our results, it is uncertain whether the ELW-1 culture would be
effective for TCE bioremediation.
• However, cis-DCE and VC would be effectively treated using ELW-1.
• It is also apparent from our results that cultures grown in a batch
environment would be better to use for bioremediation than cultures
grown in a chemostat environment.
0
5
10
15
20
VC cisDCE TCE Isobutene
Rate(umol/day/mgTSS)
Future Research:
• The ELW-1 culture also has the
ability to transform 1,4-dioxane
into less toxic substances as well.
• Future kinetic analyses with
ELW-1 and 1,4-dioxane will be
conducted in a similar fashion.
0.0
0.2
0.4
0.6
0.8
1.0
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0
Mass(µmol)
Time (days)
Batch TCE
B1
B2
Control
Investigation Objectives:
1.To determine the rates of transformation
when the ELW-1 organism is exposed to
high contaminant concentrations.
2.To compare the rates of transformation of
chemostat and batch grown cultures to
determine if different growth conditions
affect the rate of transformation.
Batch Rate Comparison
0
5
10
15
20
Batch
Chemostat
Chemostat v.s. Batch
Rate(umol/day/mgTSS)
Isobutenecis-DCEVC
1,4-Dioxane
Photo of ELW-1 Culture
Chemostat with ELW-1
0
5
10
15
20
25
30
0 5 10 15 20 25 30 35 40
Rate
(μmol/mgprotein/day)
CL0 (μM)
Chemostat
𝒌 𝒎𝒂𝒙 = 26.52 µmol/day/mg protein
KS = 1.40 µM
0.0
2.0
4.0
6.0
8.0
10.0
12.0
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0
Mass(µmol)
Time (days)
Batch VC
B1
B2
Control
1
0.0
5.0
10.0
15.0
20.0
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0
Mass(µmol)
Time (days)
Batch cis-DCE
B1
B2
Control
TCE:
• There was approximately 10.6mg of
total biomass in the reactors for the
shown TCE test.
• High amounts of sorption in control
TCE reactors created difficulties with
rate calculation.
cis-DCE:
• 4.9mg of total biomass in
transformation data shown.
• Lower amounts of sorption in controls
than TCE.
• High initial concentrations of cis-DCE
because higher rates were expected.
VC:
• Highest transformation rates per
milligram biomass calculated.
• Least amount of sorption in controls.
• 5.9mg of biomass in transformation
data shown.
Batch Monod Curve with Isobutene Substrate Chemostat Monod Curve with Isobutene Substrate
Isobutene Consumption Rate Comparisons
Batch Grown ELW-1
MassofIsobutene(mg)
I would like to thank Dr. Lewis Semprini and Dr. Mohammad Azizian for their
dedication towards mentoring my development in research as well as Jenny Green for
always helping me run experiments. I would also like to thank Pete and Rosalie
Johnson and Dr. Skip for making this internship possible, as well as Dr. Mike Hyman
and SERDP for facilitating resources for the investigation of this phenomenon.
The Aerobic Cometabolic Transformation of Chlorinated Ethenes by
the Mycobacterium ELW1 Grown on Isobutene
Stephanie Rich1, Dr. Mohammad Azizian1, Dr. Lewis Semprini1, Dr. Michael Hyman2
1School of Chemical, Biological, and Environmental Engineering, Oregon State University
2Department of Microbiology, North Carolina State University