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Analysis of Soils to Investigate Microbial Biomass and Enzymatic Activities of Punjab Soils

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Soil sampling will be done from different districts of Punjab and from sites which will be following one of the following cropping rotations namely Wheat-Groundnut, Wheat-Rice, Wheat-cotton, Wheat-Gram and mix cropping system. Analysis will be done at PMAS Arid Agriculture University, Rawalpindi.


To 40 g air-dried soil (<2mm), added 40 ml 5 % sodium hexametaphosphate (NaHMP) solution and 150 ml deionized (DI) water in a beaker. Covered the suspension with watch glass and kept it overnight. After stirring for five minutes with high speed electrical stirrer, shifted the contents quantitatively into the 1000 ml capacity calibrated cylinder and raised the volume up to the mark by adding water. Mixed the suspension well in the cylinder with plunger and inserted the hydrometer (ASTM 152 H) immediately after its withdrawal. Froth at the surface of suspension in hydrometer jar, produced due to vigorous use of plunger, was dispersed by adding one drop of amyl alcohol. First reading for silt plus clay was taken after 40 seconds of plunger withdrawal from suspension. Once again mixed the suspension with plunger as before and left it undisturbed after careful withdrawal of plunger. After 4 hours hydrometer was inserted and reading was recorded for clay. Finally, the percent values of sand, silt and clay fractions were incorporated into the ISSS triangle and soil textural class was established.

Soil pH

Soil pH will be determined using soil to water ratio of 1:2.5 suspensions using calibrated pH meter. 20g of soil will be weighed in 100ml beaker, 50ml distilled water will be added and solution will be mixed until we get a homogenous mixture. Final readings will be noted by inserting pH meter.

Soil EC

Electrical conductivity of soil samples will be determined with 1:2.5 soil water suspensions. In 20g air dried soil 50ml distilled water will be added, solution will be mixed and final readings will be measured on EC meter.

Total organic carbon

In a wide-mouth Erlenmeyer flask of 500 ml capacity 1 g soil and 10 ml of 1N potassium dichromate solution will be added. The flask will then be softly swirled so as to mix the contents of the flask. Then, rapidly we will add 20 ml conc. H2SO4 into the flask and swirled it again for one minute to mix the soil and reagents. Put the flask aside for 30 minutes. After adding 200 ml of DI water and 10 ml of conc. phosphoric acid (H3PO4), the mixture in the flask was left to cool. Then, the contents of the flask were titrated against 0.5N ferrous sulphate (FeSO4) solution in the presence of o-phenanthroline indicator until the development of red/ maroon color.

Nitrate Nitrogen

Salicylic method will be used to determine nitrate nitrogen. 10 gram of soil and 20ml of distilled water will be taken into a 50ml Erlenmeyer flask. Then contents will be shaken for 1 hour and will be filtered through Whatman No.42 filter paper. Aliquot 0.5ml will be taken and 1ml of 5% salicylic acid reagent solution will be added into test tube and will be gently mixed and tubes will be left for 30 minutes. Afterwards, 10ml of 4 M NaOH reagent will be added to each test tube. After mixing the tubes will be left for 1 hour for full color development. Readings will be recorded using spectrophotometer at wavelength of 410nm.

Extractable Phosphorus

5g soil will be taken; 100ml of 0.5 M NaHCO3 will be added and will be shaken for about 30 minutes into 250ml Erlenmeyer flask. 10 mL of filtrate, 1mL of 5 N NH2SO4 will be added into 50 mL volumetric flask. Total volume will be made up to 40 mL by addition of DI water. Ammonium heptamolybdate, antimony potassium tartrate and ascorbic acid will be used to develop color. Absorbance will be recorded after 10 minutes using Spectrophotometer at 882 nm wavelength. Standard curve will be developed using range of standards for computing P value from reading output.

Extractable Potassium

5 g of soil and 33 mL 1 N ammonium acetate solution will be taken into 50 mL centrifuge tube and will be shaken for 5 minutes. The solution will be centrifuged until the supernatant liquid will be obtained. Then, the supernatant will be diluted to 100mL with 1 N ammonium acetate. The K concentration in the samples will be determined using a standard curve, which will be prepared from absorbance values of a series of K solution using flame photometer.

Microbial biomass carbon (MBC)

For the measurement of microbial biomass carbon and microbial biomass nitrogen contents in samples collected for this purpose fumigation-extraction technique was will be used (Vance et al., 1987). Two portions of 10 g moist soil (on dry weight basis) each will be taken in air tight glass bottles simultaneously. One portion will be placed in vacuum desiccator with opened lids and will be transferred to a fume hood. At the bottom the desiccator two wet tissues will be placed to recover the moisture reduced in the samples during the fumigation process. 30 mL ethanol-free chloroform (CHCl3) with some boiling chips will be placed in a glass beaker in the center of the desiccator. After the application of silicone grease at the edges as well as at the inner part of the knobs of the desiccator (to make it air tight), the lid of the desiccator will closed and will be attached to the vacuum pump. The vacuum pump will be turned on and beaker inside the desiccator will observed carefully. When chloroform in the beaker will start boiling, stop watch will be turned on and process of vacuum application will be continued for next 2 minutes. Then the knob of desiccator will be closed, vacuum pump will be turned off and the samples will be placed in an incubator for 24 hours at 25 °C.

After fumigation, CHCl3 will be removed from the samples through repeated (six times) evacuations. The samples will be extracted in 40 ml of 0.5 M potassium sulphate (K2SO4) by 30 minutes back-to-back shaking at 200 rpm and filtrate will be collected with Whatman no. 42 paper filter. The extraction of the un-fumigated 10 g portion of soil will be carried out in the same way at the time, the fumigation initiated. All extracts will be stored at -15o C in freezer prior to analysis. The measurement of organic carbon in the extracts will be made in the form of CO2 by infrared absorption after combustion at 720° C by means of a Total Organic Carbon Analyzer (Shimadzu Corp. Japan). The computation of MBC will be as below:

MBC = Ec / kEC

Where, Ec is the difference in the organic carbon extracted from fumigated soils and the un-fumigated soils and kEC = 0.45, the fraction of the killed biomass mineralized to CO2 over the incubation period.

Microbial biomass nitrogen (MBN)

The measurement of total N in the extracts was made as NO2 by chemiluminescence detection with Shimadzu’s Total Nitrogen Module (TNM-1). The computation of biomass N was as below:

MBN = EN / kEN

Where, EN is the difference between total N in fumigated soils and the un-fumigated soils, kEN = 0.54, the fraction of the killed biomass mineralized to nitrogen over the incubation period.

Microbial biomass phosphorus (MBP)

The fumigation-extraction technique was used for the measurement of soil MBP. For this purpose, pre-incubated moist soil was split into 3 portions of 2.5 g oven-dry weight and each of them was extracted with 50 ml 0.5M NaHCO3 at pH 8.5 following different pre-treatment. One portion/ sample was treated as fumigation treatment (as described above) while the 2nd one was used as non-fumigation treatment. Third portion was spiked with phosphorus @ 25 µg g-1 soil by adding KH2PO4 solution to the extractant so as to estimate the P sorbed by the soil during the process of fumigation-extraction. Phosphorus contents were analyzed by Murphy and Riley (1962) method. The computation of MBP was as below:

MBP = EP / kEP

EP = (A – B) / (C – B) × 25

Where, A = phosphorus in fumigated soil, B = phosphorus in un-fumigated soil, C = phosphorus in un-fumigated spiked soil.

Urease activity

For the analysis of soil urease activity, 5 g soil will be taken in an Erlenmeyer flask (100 ml) and 2.5 ml urea solution will be added. Then we will stopper the flask and incubate at 37o C for 2 hours. After incubation 50 ml of KCl solution will be added and will shake the flask for 30 minutes. After filtration, the filtrate will be analyzed for ammonium content. The blank will be performed as described above but with 2.5 ml distilled water and will add the urea solution at the end of the incubation and immediately before KCl addition. For ammonium estimation 1 ml of the clear filtrate will be taken into an Erlenmeyer flask (50 ml), then 9 ml of distilled water, 5 ml of Na salicylate/ NaOH solution and 2 ml of dichloroisocyanide solution will be added and will be allowed to stand at room temperature for 30 minutes and optical density will be measured at 690 nm.

Dehydrogenase activity

Dehydrogenase activity in soil will be determined by estimating the rate of production of tri- phenyl formazan (TPF) from tri-phenyl tetrazolium chloride (TTC). Briefly 5 g soil will be mixed with 5 ml triphenyl tetrazolium chloride (TTC) in test tube. Contents will be mixed and shall be incubated for 24 hours at 30 ºC. The control shall contain only 5 ml Tris buffer (without TTC). After incubation, 40 ml acetone will be added to each test tube and the tubes will be shaken thoroughly and will be further incubated at room temperature for 2 hours in the dark. The soil suspension will then be filtered and optical density of the clear supernatant will be measured at 546 nm. Due to the light sensitivity of triphenyltetrazolium chloride (TTC) and triphenyl formazan (TPF), all procedures were performed under diffused light.

Alkaline phosphatase activity

The soil alkaline phosphatase activity will be measured by taking 1 g soil in Erlenmeyer flask (50 ml) and will be treated with 0.25 ml of toluene, 4 ml of MUB (Modified Universal Buffer, pH of 11 for alkaline phosphatase) and 1 ml of p-nitrophenyl phosphate (PNP) solution made in the same buffer. After stopping the flask, contents will be mixed and will be incubated for 1 hour at 37o C. After incubation, 1 ml of CaCl2 (0.5 M) and 4 ml of NaOH (0.5 M) will be added. We will mix the contents and filter the soil suspension through Whatman no. 2v folded filter paper. To perform control, 1 ml of PNP solution will be added after the addition of 1 ml CaCl2 (0.5 M) and 4 ml of NaOH (0.5 M) and immediately before filtration of soil suspension and optical density will measured at 400 nm.

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Analysis of soils to investigate microbial biomass and enzymatic activities of Punjab soils. (2019, April 10). GradesFixer. Retrieved September 22, 2021, from
“Analysis of soils to investigate microbial biomass and enzymatic activities of Punjab soils.” GradesFixer, 10 Apr. 2019,
Analysis of soils to investigate microbial biomass and enzymatic activities of Punjab soils. [online]. Available at: <> [Accessed 22 Sept. 2021].
Analysis of soils to investigate microbial biomass and enzymatic activities of Punjab soils [Internet]. GradesFixer. 2019 Apr 10 [cited 2021 Sept 22]. Available from:
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