Laboratory 11



Laboratory 13 Anaerobic vs. Aerobic Treatment

Introduction

Bacterial metabolism is driven by redox reactions. Bacteria obtain energy by oxidizing reduced chemicals (sugars, hydrogen etc.) and transferring the resulting electrons to an acceptor molecule (oxygen, nitrate, organic molecules, etc.). When the electron acceptor is the same organic molecule that is being oxidized, the process is called fermentation. The amount of energy that the cell gains from these reactions is directly related to the electron potential of the electron acceptor. Acceptors with higher potentials will yield more energy than those with lower potentials, and the difference results in higher cell yields. Because of this, bacteria will tend to use the most favorable electron acceptor available to them.

In this lab you will be demonstrating differences in cell yield among cultures using different electron acceptors to metabolize sugar. Respiratory bacteria using oxygen will be compared to fermentative bacteria.

Materials

Equipment

- drying oven (100(C)

- incubator

- glass funnels and flasks

- shaking incubator (25(C)

Cultures

- sewage samples

Supplies

- minimal salts media

- glucose

- sterile graduated cylinders

- pipettes and bulbs

- paper filters

- foam plugs

- 150 ml serum bottles

Procedures

Period 1

1. Label two 150 ml serum bottles as “aerobic-A and B” and two bottles as “anaerobic-A and B”.

2. Using a graduated cylinder, carefully dispense 100 ml of medium into each of the 150 ml serum bottles.

3. Measure out 5 ml of sewage for each culture and dispense it into the bottles.

4. Weigh out 0.5 grams of glucose and pour it into each of the “A” bottles.

5. The “B” bottles will serve as un-fed controls.

6. Plug the aerobic bottles with foam plugs and the anaerobic bottles with rubber stoppers.

7. Incubate all of the bottles at 25(C while shaking at 150 rpm for 2 days.

Period 2

1. Set up a filtration apparatus for each of the samples.

2. Filter each of the cultures onto a separate filter.

3. Transfer each filter to a pre-weighed aluminum dish and put them in a 100(C oven to dry overnight.

4. After cooling, weigh each sample and subtract the weight of the dish.

5. Calculate the cell yield for each condition.

Lab #13 10 points

Name: Date:

Cell Yield = (mg of cell mass / mg of substrate) X 100

DATA

| |dish weight |dish+filter+cells |filter+cells |weight of fed – unfed |cell yield |

| | |(dry weight) |(dry weight) |cells | |

|Aerobic A | | | | | |

|B | | | |xxx | |

|Anaerobic A | | | | | |

|B | | | |xxx | |

Questions:

1.) Do you expect that the same bacteria are involved in breaking down the sugar in both the aerobic and anaerobic cultures? Why?

______________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________

2.) What factors besides the terminal electron acceptor could have caused differences in cell yield?

______________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________________

Laboratory 14 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 ammonium, nitrite and nitrate test kits

- Incubator

- Balance

Cultures

- soil samples

Supplies

- 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 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 one 0.1ml sample 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 60 ml of succinate - salts broth.

2. Shake vigorously and let the soil particles settle.

3. Carefully pour the supernatant fluid into serum bottles.

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 1.5 ml of each diluted sample into a new screw-cap tube and add 13.5 ml 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.

Lab #14 15 points

Name: Date:

Ammonification

|Sample |pH |Ammonia |

| | | |

|Soil | | |

| | | |

|Control | | |

Denitrification

|Sample |pH |Nitrite |Nitrate |

| | | | |

|Soil | | | |

| | | | |

|Control | | | |

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