Acknowledgements

Results

Conclusions and Future Research

Analysis

Monod Equation:• dS/d t = rate of substrate consumption

• 𝒌𝒎𝒂𝒙 = 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 MethodsThe 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

(μm

ol/

mg p

rote

in/d

ay)

CL0 (μM)

Batch

𝒌𝒎𝒂𝒙 = 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= 𝑿𝑲𝒎𝒂𝒙

𝑺

𝑲𝒔 + 𝑺

0.01.02.03.04.05.06.07.08.09.010.011.012.013.0

0.00 0.03 0.05 0.08 0.10 0.13 0.15 0.18Time (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

(um

ol/

da

y/m

gT

SS

)

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

(µ

mol)

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

(um

ol/

da

y/m

gT

SS

)

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

(μm

ol/

mg p

rote

in/d

ay)

CL0 (μM)

Chemostat

𝒌𝒎𝒂𝒙 = 26.52 µmol/day/mg protein

KS = 1.40 µM

0.02.04.06.08.0

10.012.0

0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0

Mass

(µ

mo

l)

Time (days)

Batch VCB1

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

(µ

mo

l)

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

Mass

of

Iso

bu

ten

e (m

g)

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 University2Department of Microbiology, North Carolina State University