EXPERIMENT ONE: DEHYDROGENASE ACTIVITY IN SOILS

1. Prepare a standard calibration line (concentration of TPF (ug) against absorbance).

Determine linear equation and THE regression coefficient.

Prepared data for standard calibration line:

No. TPF Concentration (g/mL) UV Absorbance (Abs)

1 1 0.0256
2 5 0.238
3 10 0.391
4 25 0.7731
5 50 1.4028
Table 1. UV absorbance values for known conc. of TPF solution

Plot for the calibration line on excel

UV Absorbance (Abs) vs TPF Conc (g/mL)

1.6

1.2
y = 0.027x + 0.0745
UV Abs (Abs)

R = 0.9917
0.8

0.4

0
0 15 30 45 60
TPF Concentration (g/mL)

Figure 1. Standard calibration line

The linear trend line graph plotted is = 0.027 + 0.0745. With the high R2 value of
0.99168, the equation = 0.027 + 0.0745 is a good representation of the points plotted.

2. Calculate the concentration of TPF in the samples (accounting for blanks) and
determine the concentration of TPF produced per hour per Gram of DRY soil.

For the experiment, each sample of soil has 2 replications; 1 and 2. Using the equation
obtained in Figure. 1, the TPF concentration in the samples can be obtained.

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These are the information given for the experiment:
Soil moisture content = 25%
Volume of 3% TTC = 1 mL
Volume of water/nutrient = 1 mL
Volume of Analar methanol = 25 mL
Weight of fresh wet soil = 5.5 g
Incubation time = 48 hr

Soil Content of the Soil Sample Glucose (g) 4% NH4NO3 (g) 4% K2HPO4 (g)
5g Soil + 1ml 3% TTC +25 mL 0.025
1
methanol + 0.5% Glucose
5g Soil + 1ml 3% TTC +25 mL 0.125
2
methanol +2.5% Glucose
5g Soil + 1ml 3% TTC +25 mL 0.125 0.2
3 methanol +2.5% Glucose + 4%
NH4NO3
5g Soil + 1ml 3% TTC +25 mL 0.125 0.2
4
methanol +2.5% Glucose + 4%
Table 2. Soil Sample Content

Sample Calculation for sample 1A

TPF Concentration = 17.628 x 1.08

= 19.038 mg/L

(/) (20+1+1)
TPF produced = (0.8 5 ) (48 )

19.038 22
= (0.8 5) 48
= 1.983 g/g dry soil/hr

Concentration of TPF
Soil TPF produced / gram
UV Absorbance (Abs) in Samples (mg/L or
Sample of soil/hr
ug/mL)
Soil 1 A 1.08 19.038 1.983

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 B 1.58 27.852 2.901
Mean 1.330 23.445 2.442
A 1.47 25.913 2.699
Soil 2 B 2.7 47.596 4.958
Mean 2.085 36.754 3.829
A 0 0.000 0.000
Soil 3 B 0 0.000 0.000
Mean 0.000 0.000 0.000
A 1.56 27.500 2.865
Soil 4 B 2.68 47.243 4.921
Mean 2.120 37.371 3.893
Table 3. Results for the concentration of TPF

For this experiment four soil sample with different nutrients amount and types were tested
the breakdown are listed below along with the properties and the environment condition of
the lab that was given.

3. Present the TPF results for each of the soils (Soil 1 - 8) in tabular and graphical form,
showing the duplicate results, mean and standard deviation.

For the calculation of mean, the formula x = x / n

For the calculation of standard deviation, the following formula is used:

Sample Calculation for sample A 1

19.038 +27.852
Mean, x = 2
= 23.445 mg/L

(19.03823.445)2 +(27.85223.445)2
Standard deviation = 21
=6.232

{DHA rate Mean based Std Dev

Concentration Mean of
Soil {TPF (ug)} / on TPF based on
of TPF in DHA rate
Sample Samples
{dry soil wt formed TPF formed
(ug/mL)
(g) * hr} (ug/mL) (ug/mL)

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 (mg/L or
ug/mL)
A 19.038 1.983
Soil 1 23.445 2.442 6.232
B 27.852 2.901
A 25.913 2.699
Soil 2 36.754 3.829 15.332
B 47.596 4.958
A 0.000 0.000
Soil 3 0.000 0.000 0.000
B 0.000 0.000
A 27.500 2.865 37.371 3.893 13.961
Soil 4
B 47.243 4.921
Table 4. Tabulation of mean and standard deviation

The following is the graphical representation of table 4 :

50.000

45.000

40.000

35.000

30.000 Soil #1
Soil #2
25.000
Soil #3
20.000
Soil #4
15.000

10.000

5.000

0.000
A B Mean
Figure 2. Graphical form of the TPF results

4. What are some of the errors that could have occurred in this analysis?

Firstly, there may be human errors during the weighing of the soil for the samples. As the
weighing machine used in the lab is very sensitive, it will take into account the small amount
of soil that have fallen to the side and not into the test-tube. The imprecise amount of soil
content used in the experiment will inevitably affect the results.

This experiment is designed to be strictly anaerobic to enable accurate results. However,

during the transfer of soil and the adding of chemicals, the soil is already exposed to the
atmosphere. In addition, oxygen is present when the test tubes are capped, and some of the
caps are not tight fitting, hence oxygen may still seep into the test tubes. These will affect the
result of the experiment as the experiment is not based on aerobic conditions.

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 Micropipette is used to measure and add chemicals into the soil samples. Human error may
have caused the amount of chemicals added to be inaccurate. Air bubbles may be present
during pipetting causing the amount added to be lower than desired. The difference in
amount added may result in huge experimental error as the amount added is not a lot.

In the experiment, the soil used is assumed to be homogenous but that is not the case in
reality. The deeper part of the soil will have more organic matters as compared to the layer
on top and the microbial communities present will be different as well. Hence, the part of the
soil used for the sample will greatly affect the experimental result. In addition, the soil
contains dead leafs, roots and other organic matter which may affect the result as well.

The spectrophotometer is a very sensitive equipment and the cuvette used to contain the
sample may have dust or water droplets which may affect the reading. It is very difficult to
ensure that the surface of cuvette is completely clean as it is small and some particles present
on the surface may be no visible to the naked eye.

5. What is the most important nutrient in terms of stimulating microbial activity in soils?

From the experimental result, it is conclusive that the most important nutrient is inorganic
phosphorous. From the experiment, the addition of phosphorus is able to increase microbial
activity as seen from the difference in dehydrogenase activity between sample 2 and sample
4. Glucose is a crucial source of energy as seen from the experiment. From soil sample 1 to 2,
the increment of 0.025g of glucose to 0.125g have showed the most significant increase in
dehydrogenase activity. Even though on average, the addition of inorganic phosphorous
assisted in stimulating soil microbial activity, the increment is not as significant as the once as
glucose. Therefore in order to stimulate microbial activities effectively through the addition
of phosphorus, there must be enough food present in the environment.

6. Comment on the DHA activity results in terms of the effects of adding different nutrient
and glucose concentrations relative to the control. What does this tell you about the
microenvironment of soils and the microorganisms which live in soil? (500 words).

In all of the samples, glucose nutrients are added to assist the growth of microbes as it serves
to provide simple substrate which the microbes can use to metabolize as food.

Comparing the TPF concentration of sample 1 and 2, the values of sample 2 is significantly
higher than that of sample 1. Since the only difference between sample 1 and 2 is the amount
of glucose added, it is conclusive that the addition of glucose serve to act as substrate for the
microbes present to metabolize easier and hence increasing their activity. From this result, it
can be inferred that the soil sample used does not contain high amount of organic carbon
which means that the availability of carbon source is the limiting factor for the growth in that
soil.

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From sample 3, it is the only sample with no microbial activity at all despite having glucose
nutrients present as well. This could be due to the fact that the addition of ammonium nitrate
introduced nitrate ions into the sample. Nitrate ions are inhibitors to DHA activity.

Sample 4 has slightly higher TPF value as compared to soil sample 2. This is because the
introduction of inorganic phosphorus is effectively able to stimulate microbial activity. In soil,
the amount of inorganic phosphorus is limited, hence the addition of phosphorus is able to
encourage microbial growth. In the samples, the availability of carbon nutrients is limiting,
hence addition of phosphorous is does not cause a significant increase compared to adding
glucose.

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