Manual for Soil Analysis-Monitoring and Assessing Soil Bioremediation Phần 9 pps

290 R.G. Joergensen, P.C. Brookes

• Ninhydrin reagent: 2 g ninhydrin and 0.3 g hydrindantin dihydrate dis￾solved (for amino acid analysis) in 75 mL dimethylsulfoxide (DMSO),

25 mL of 4 M lithium acetate buffer then added (Moore 1968)

• Ethanol/water mixture (1 + 1, v/v)

• Standard solutions: 10 mM L-leucine prepared in 0.5 M K2SO4 and di￾luted within the range 0−1,000 µM

■ Sample Preparation

Use soil extract prepared as described in Sect. 14.2.

■ Procedure

1. Add 0.6 mL of standard solutions, K2SO4 soil extracts or blank, and

1.4 mL of citric acid buffer to 20 mL test tubes (Joergensen and Brookes

1990).

2. Add 1 mL of ninhydrin reagent slowly, mix thoroughly, and close with

loose aluminum lids.

3. Heat the test tubes for 25 min in a vigorously boiling water bath; any

precipitate formed during the addition of the reagents then dissolves.

4. After heating, add 4 mL of the ethanol-to-water mixture, mix the solu￾tions thoroughly, and read the absorbance at 570 nm.

■ Calculation

1. Calculation of extracted ninhydrin-reactive N (Nnin)

Nnin (µg/g soil) = (S − B) × N × (VK + SW)

L × DM (14.5)

S absorbance of the sample

B absorbance of the blank

N atomic mass of nitrogen (14)

VK volume of K2SO4 extractant (mL)

SW total amount of water in the soil sample (mL)

L millimolar absorbance coefficient of leucine

DM total mass of dry soil sample (g)

14 Quantification of Soil Microbial Biomass by Fumigation-Extraction 291

2. Calculation of microbial ninhydrin-reactive N

Bnin =

Nnin extracted from the fumigated soil

−

Nnin extracted from the non-fumigated soil (14.6)

3. Calculation of microbial biomass C

Biomass C = Bnin × 22

(for soils with a pH(H2O) above 5.0; Joergensen 1996b)

Biomass C = Bnin × 35

(for soils with a pH(H2O) of or below 5.0; Joergensen 1996b)

■ Notes and Points to Watch

• A reflux digestion is not required for ninhydrin N. This makes it very

suitable for situations with minimal laboratory facilities.

• In both biomass C and N measurements the fraction coming from the

biomassis determined following subtraction of an appropriate “control.”

With biomass C this value is often half of the total, while with biomass

ninhydrin N it is commonly about 10% or less. This causes considerably

less error in its determination.

• At 100 ◦C the reaction with free amino groups of proteins and amino

acids is essentially complete within 15 min (e.g., leucine reaches the max￾imum optical density after approximately 5 min). However the reaction

of hydrindantin with NH+

4 requires 25 min.

• The ratio between the volume of the sample and that of citric acid should

not be closer than 0.75:1.75 to avoid the formation of a precipitate after

the addition of the ninhydrin reagent.

• The most common solvent in the ninhydrin method is 2-methoxyethanol

(Amato and Ladd 1998). However, because it is an ether it tends to

form peroxides that destroy ninhydrin and hydrindantin. Dimethylsul￾foxide (DMSO) is peroxide free, has lower toxicity and a higher boil￾ing point (189 ◦C), and gives a more stable color development than

2-methoxyethanol.

• The ninhydrin method proposed by Amato and Ladd (1988) for 2 M

KCl extracts does not require the use of citric acid buffer. The optimum

reagent-to-sample ratio is 1:2.

292 R.G. Joergensen, P.C. Brookes

14.4.2

Total Nitrogen

Principle. Total nitrogen is measured under strong acidic conditions by

Kjeldahl digestion. Ammonium can be measured by distillation (see

Chapt. 16).

Theory. Ammonium is released from amines, peptides and amino acids in

0.5 M K2SO4 soil extracts of fumigated and non-fumigated soil samples. Ni￾trate is additionally reduced to ammonium under strong acidic conditions

in the presence of KCr(SO4)2, Zn powder, and CuSO4 as reducing agents.

■ Equipment

• Digestion block

• Steam distillation apparatus

• Burette or autotitrator

■ Reagents

• Reducing agent: 50 g of chromium(III) potassium sulfate dodecahydrate

(KCr(SO4)2 × 12H2O) dissolved in approx. 700 mL deionized water, and

after adding 200 mL conc. H2SO4, cooled and diluted to 1,000 mL

• Zn powder

• CuSO4 solution (0.19 M)

• Conc. H2SO4

• 10 M NaOH

• 2% H3BO3

• 10 µM HCl

■ Sample Preparation

Use soil extract prepared as described in Sect. 14.2.

■ Procedure

1. Add 10 mL of the reducing agent and approx. 300 mg Zn powder to 30 mL

of the K2SO4 soil extract and leave for at least 2 h at room temperature.

2. Add 0.6 mL of CuSO4 solution, 8 mL of conc. H2SO4, heat gently for 2 h

until all the water has disappeared, and then heat for 3 h at the maximum

temperature.