Biomass nitrogen

Estimation of biomass nitrogen

Principle

Microbial biomass is determined by the fumigation technique in which a fresh soil sample is subjected to chloroform, which cause  cell wall to lyse and denature, the cellulose contents are extractable in 0.5 M K2SO4.

Apparatus required

1.      100 ml beaker

2.      Desiccator

  3.  250 ml conical flak

  4.  Digestion tube

  5.  250 ml volumetric flask

Reagents required

1.Chloroform solution

2.0.5 M K2SO4

3.0.2 M CuSO4

4.0.4 N K2Cr2O7

5.0.2 N Ferrous ammonium sulphate

6.Phenolphthalein indicator

7.Sulphuric - orthophosphoric acid mixture (2: 1)

8.0.01 N sulphuric acid

9.Methyl red indicator

10. Zinc and porcelain bits

11.40 % NaOH

12.10 % sodium sulphide

13. Red litmus paper

14.0.01 N KOH

Procedure

·         Take 25 g of air dried soil in a 100 ml beaker

·         Determine moisture content

·         Fumigate soil sample with chloroform for 72 hrs by using desiccator

·         Transfer the soil into a 250 ml conical flask

·         Add 100 ml of 0.5 M K2SO4 and shake well in a mechanical shaker for 1 hour.

Determination of nitrogen

a.      Digestion

·         Pipette out 50 ml of the filtrate in a digestion tube.

·         Add 5 ml 0.2 M CuSO4 and 10 ml of conc. H2SO4

·         Digest at 380°C for 3 hrs

·         Cool it and make up the volume up to 250 ml

·         Similarly run one control and prepare one EDTA standard

b.      Distillation

·         Pipette out 25 ml of the above digested sample in a digestion flask.

·         Pipette out 25 ml of 0.01 N sulphuric acid with 2-3 drops of methyl red indicator and keep it at the delivery end of the distillation unit

·         Add few zinc and porcelain bits to the distillation flask followed by 120 ml of 40 per cent NaOH till the contents become distinctly alkaline (Test with litmus paper) and add 10 ml of 10% sodium sulphide. Close the mouth of the distillation flask immediately and start the distillation.

·         Collect the ammonia evolved in a beaker containing 0.01 N H2SO4. If the indicator turns yellowish, pipette out another 25 ml of 0.01 N sulphuric acid into the beaker.

·         Test the completion of the distillation with the moist red litmus paper. Absence of blue colour indicates that all the ammonia has been distilled.

·         Then detach the delivery end and wash it with distilled water and collect the washings in an ice tumbler or beaker

·         Titrate the contents with 0.01 N KOH and the end point is the change of colour from pinkish red to straw yellow

·         From the volume of 0.01 N sulphuric acid actually consumed for absorbing the ammonia, calculate the percentage of nitrogen present in the soil.

Calculation

Weight of soil taken

=

25 g

Volume of 0.5M K2SO4 added

=

100 ml

Volume of filtrate pipetted out for digestion

=

50 ml

Volume made up

=

250 ml

Volume of digested sample pipetted out

=

25 ml

Volume of 0.01 N H2SO4 used

=

X ml

Volume of 0.01 N KOH consumed for titration

=

Y ml

Actual volume of 0.01 N H2SO4 used

=

(X-Y) ml

1 ml of 0.01 N H2SO4

=

 0.0014 g N

Biomass N Present in the sample (%)

=