Laboratory 15
Biodegradation of Xenobiotics
Introduction
Xenobiotics are chemical compounds that are foreign to the biosphere. Because they are
new to the environment, these chemicals often persist without being degraded. The native
bacterial populations have not evolved mechanisms to metabolize them. Because they persist,
xenobiotics can become important environmental pollutants.
Often these xenobiotics are slightly modified versions of naturally occurring compounds.
A small change in the structure can dramatically affect the biodegradability of a chemical. An
example of this is type of modification is chlorination. Very few naturally occurring organic
chemicals contain chlorine atoms so most bacteria are not able to metabolize chlorinated
compounds.
In this lab we will use an HPLC to measure the amounts of benzoate and 2chlorobenzoate remaining in soil slurries over time. We should be able to see an effect of the
chlorine substitution on degradation.
Materials
Equipment
- High pressure liquid chromatograph
(HPLC)
- shaking incubator
- centrifuge
Cultures
- soil samples
Supplies
- pipettes and tips
- microcentrifuge tubes
- 0.45 m filters
- 1 ml syringes
- HPLC vials and caps
- benzoate (100 mM)
- 2-chlorobenzoate (10 mM)
- 150 ml serum bottles and foam plugs
- minimal salt media
Procedures
Enrichment Culture
1. Label two 150 ml serum bottles as “benzoate-A and B”, two bottles as
“chlorobenzoate-A and B”, and two bottles as “benzoate + chlorobenzoate sterile-A
and B”.
2. Using a graduated cylinder, carefully dispense 50 ml of minimal salts medium
(MSM) into each of the 150 ml serum bottles.
3. Weigh out 5 grams of soil for each culture and pour it into the bottles.
4. Plug each bottle with a foam plug and autoclave all of the sterile controls.
5. After the bottles have cooled, add 0.5ml of benzoate or 5.0ml of chlorobenzoate to
the appropriate cultures.
6. Sample each bottle on Days 0, 2 and 7.
7. Analyze all samples on the HPLC.
Sampling
1. Remove 1ml of each culture using a pipette.
2. Transfer the sample to a microcentrifuge tube and spin for 3 min.
3. Remove the supernatant with a 1 ml syringe and filter it into an HPLC vial.
Minimal Salts Medium (MSM)
NH4Cl
1.0 g
K2HPO4
1.0 g
MgSO47H2O
0.2 g
CaCl22H2O
0.1 g
FeSO47H2O
0.1 g
NaSO4
0.5 g
Dissolve in 1 liter of distilled water and sterilize at 121C.
DATA Lab 15
15 points
Name:
Date:
Standard Curve
Concentration (mM)
Benzoate
Chlorobenzoate
Absorbance
Absorbance
2.0
1.0
0.5
0.25
0.125
Concentration of Substrate (mM)
Day 0
Day 2
Day 7
Sterile Cl-Benz. - A
/
/
/
Sterile Cl-Benz. - B
/
/
/
Benzoate - A
Benzoate - B
Cl-Benzoate - A
Cl-Benzoate - B
Attach Graphs
Laboratory 16
Nitrogen Cycle
Introduction
Nitrogen is an important nutrient in the biosphere and it is constantly being cycled from
one form to another by bacteria. These transformations include: fixation of gaseous N2 into
organic compounds, release of ammonia from amino acids, oxidation of ammonia to nitrite and
nitrate, and reduction of nitrate back to gaseous form. Different groups of bacteria are involved
in each of these processes.
In this lab we will examine two transformations that can take place under anaerobic
conditions. Ammonification is the release of ammonia from amino acids. Many microbes do this
as a fermentative process when they are grown with an excess of organic nitrogen.
Denitrification is a respiratory process whereby nitrate and nitrite are used as terminal electron
acceptors in the absence of oxygen. The result is gaseous N2.
Materials
Equipment
- Hach nitrate and nitrate test kits
- Incubator
- Balance
Cultures
- soil samples
Supplies
- nitrate-succinate salts medium
- Peptone broth
- 150 ml serum bottles
- beakers and flasks
- pipettes and bulbs
Procedures
Ammonification
Period 1
1. Add 1 gram of soil to a 150 ml serum bottle.
2. Fill the bottle 2/3 rds full with 4% Peptone broth.
3. Stopper the bottle and shake well.
4. Fill a separate bottle with Peptone broth but no soil to act as a control.
5. Check the pH of each culture.
6. Completely fill both of the bottles with medium and stopper tightly.
7. Incubate the bottles at 25C for one week.
Period 2
1. Use the HACH kit to measure the concentration of ammonia in each bottle.
a. Remove four 0.1ml samples from each of the cultures that you want to analyze
(Soil and Control) and transfer them into labeled screw-cap tubes.
b. Add 9.9ml of distilled water to each tube to dilute the samples 1:100.
c. Transfer 1ml of each diluted sample into a new screw-cap tube and add 9ml of
distilled water to a final dilution of 1:1000.
d. Follow the procedures for the “Ammonia Test” below to test each culture.
(NOTE: in order to save time, we will add the reagents to the screw-cap tubes and
run all of the reaction periods for each test at the same time and then transfer the
samples to the sample cells for reading.)
e. Convert the values by multiplying them by 1000 to get the original concentration.
2. Check the pH of each culture.
Denitrification
Period 1
1. Add 1 gram of soil to an ehrlenmyer flask along with 150 ml of nitrate-succinate salts
broth.
2. Shake vigorously and let the soil particles settle.
3. Carefully pour the supernatant fluid into a 150 ml serum bottle.
4. Fill a separate bottle with medium but no soil to act as a control.
5. Incubate the bottles at 25C for one week.
Period 2
1. Examine the bottles for the presence of nitrogen gas.
2. Check the pH of each culture.
3. Use the HACH kit to measure the concentrations of nitrate and nitrite in each culture.
a. Remove four 0.1ml samples from each of the cultures that you want to analyze
(Denitrification and Control) and transfer them into labeled screw-cap tubes.
b. Add 9.9ml of distilled water to each tube to dilute the samples 1:100.
c. Transfer 1ml of each diluted sample into a new screw-cap tube and add 9ml of
distilled water to a final dilution of 1:1000.
d. Follow the procedures for the “Low-Range Nitrate Test” and “Low-Range Nitrite
Test” below to test each culture for both compounds. (NOTE: in order to save
time, we will add the reagents to the screw-cap tubes and run all of the reaction
periods for each test at the same time and then transfer the samples to the sample
cells for reading.)
e. Because the “Nitrate Test” measures both nitrate and nitrite nitrogen, subtract the
nitrite value from the nitrate value to get a corrected concentration.
f. Convert the values by multiplying them by 1000 to get the original concentration.
g. Compare the values for the experimental cultures to the control cultures.
Ammonia [Method 8155, Salicylate (0 – 0.50 mg/L NH3-N)]
1. Press [PRGM] key. The display will read “PRGM?”.
2. Press [6] [4] [ENTER]. The display will read “mg/L, NH3-N” and the ZERO
icon.
3. Fill a sample cell with 10 ml of deionized water (the blank).
4. Fill a second sample cell with 10 ml of the sample.
5. Add the contents of one “Ammonia Salicylate” packet to each sample cell. Cap
the cells and shake gently until dissolved.
6. Press [TIMER] [ENTER]. A 3-minute reaction period will begin.
7. When the timer beeps, add the contents of one “Ammonia Cyanurate” packet to
each sample cell. Cap the cells and shake gently to dissolve.
8. The display will read “15:00 TIMER 2”. Press [ENTER]. A 15-minute reaction
period will begin.
9. When the timer beeps, place the blank into the cell holder and tightly cover with
the instrument cap.
10. Press [ZERO]. The cursor will move to the right and the display will read “0.00
mg/L NH3-N”
11. Remove the blank and place the sample into the cell holder. Cover the sample
cell with the instrument cap.
12. Press [READ]. The cursor will move to the right and the result will be displayed.
Record this value.
Nitrate [Method 8192, Cadmium reduction, Low Range (0 - 0.50 mg/L NO3-N)]
1. Press [PRGM] key. The display will read “PRGM?”.
2. Press [5] [5] [ENTER]. The display will read “mg/L, NO3-N” and the ZERO
icon.
3. Fill a 25 ml graduated mixing cylinder to the 15 ml mark with sample.
4. Add the contents of one “NitraVer 6” packet to the cylinder and stopper.
5. Press [TIMER] [ENTER]. A 3-minute reaction period will begin. Shake the
cylinder vigorously throughout this time period.
6. When the timer beeps, the display will read “2:00 TIMER 2”. Press [ENTER].
A 2-minute reaction period will begin.
7. When the timer beeps, pour 10 ml of the sample into a sample cell (be careful not
to transfer any cadmium particles).
8. Add the contents of one “NitraVer 3” packet to the sample cell. Cap the cell and
shake gently for 30 seconds.
9. The display will read “15:00 TIMER 3”. Press [ENTER]. A 15-minute reaction
period will begin.
10. Fill another sample cell (the blank) with 10 ml of sample.
11. When the timer beeps, place the blank into the cell holder and tightly cover with
the instrument cap.
12. Press [ZERO]. The cursor will move to the right and the display will read “0.00
mg/L NO3-N”
13. Remove the blank and place the sample into the cell holder. Cover the sample
cell with the instrument cap.
14. Press [READ]. The cursor will move to the right and the result will be displayed.
Record this value.
Nitrite [Method 8507, Diazotization, Low Range (0 - 0.350 mg/L NO2-N)]
1. Press [PRGM] key. The display will read “PRGM?”.
2. Press [6] [0] [ENTER]. The display will read “mg/L, NO2-N” and the ZERO
icon.
3. Fill a sample cell with 10 ml of the sample.
4. Add the contents of one “NitraVer 3” packet to the sample cell. Cap the cell and
shake gently until dissolved.
5. Press [TIMER] [ENTER]. A 15-minute reaction period will begin.
6. Fill another sample cell (the blank) with 10 ml of sample.
7. When the timer beeps, place the blank into the cell holder and tightly cover with
the instrument cap.
8. Press [ZERO]. The cursor will move to the right and the display will read “0.000
mg/L NO2-N”
9. Remove the blank and place the sample into the cell holder. Cover the sample
cell with the instrument cap.
10. Press [READ]. The cursor will move to the right and the result will be displayed.
Record this value.
DATA Lab 16
10 points
Name:
Date:
Ammonification
Sample
pH
Ammonia
Soil
Control
Denitrification
Sample
Soil
Control
pH
Nitrite
Nitrate