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SodBid Biocht-m.Vol. 22,No.il.pp.1023-1027.1990 Printed in Great Britain. At1 rightsreserved

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NINHYDRIN-REACTIVE NITROGEN MEASUREMENTS OF MICROBIAL BIOMASS IN 0.5 M K,SO, SOIL EXTRACTS R. G. JOERGENSEN’ and P. C. BROOKES~ Soil Science Department, AFRC Institute of Arable Crops Research, Rothamsted

Experimental Station,

Harpenden, Herts AL5 UQ, England (Accepted 25 June 1990) Summary-Amato and Ladd showed that the amount of ninhydrin-reactive nitrogen (ninhydrin-N) extracted from soil by 2 M KCI following CHCI, fumigation is a reliable and sensitive indicator of the amount of soil microbial biomass. However, when we attempted the assay in 0.5 M K,SO, soil extracts, serious analytical problems (e.g. precipitation of CaSO, and KrSO,) occurred. which invalidated the ninhydrin-N determinations. As measurement of biomass C (and probably biomass N) in soils by fumigation-extraction is more reliable when KrSO,. rather than KCI, is used as the extractant, we modified the ninhydrin-N assay procedure to permit measurements in 0.5~ K,SO,. Our modified procedure is described. A strong relationship (r = 0.99) between ninhydrin-N measured in KCI and KrSO, soil extracts was obtained (KCI-ninhydrin-N = 0.90 K,SO,-ninhydrin-N). There were also strong linear relationships (r = 0.91-95) between biomass C, biomass N and biomass ninhydrin-N (all extracted with 0.5 M K,SO,). We conclude that reliable ninhydrin-N measurements are obtained in 0.5 M K,SO, soil extracts by our modified procedure.

the u.v.-pcrsulphate oxidation methods used to measure biomass C cxtractcd during FE (Powlson Chloroform fumigation of soil lyscs the cells of the and Jenkinson. 1976; Vance et al., 1987; Wu et al.. soil microbial biomass and makes them partially 1990). Also. at lcast theoretically. NH,CI could be extractable by various rcagcnts. Thus, soil microbial volatilized during Kjeldahl digestion of KCI soil biomass C (Vance ef ul.. 1987; Sparling and West, extracts for measurement of biomass N. 1988). biomass N (Brookcs et al., 1985). P (Brookcs However, KISOd dots have some disadvantages. It et al., 1982; Hcdley and Stewart, 1982) and S (Saggar is less soluble than KCI and a white precipitate of rf ui.. 1981) can all be measured in this way [usually CaSO, forms in K,SO, extracts of neutral soils, tcrmcd fumigation+zxtraction (FE)]. especially if they are frozen and then thawed before Hydrolytic enzymes remain active during CHCI, use, or if organic solvents are added. When we fumigation (Brookes ef al., 1982) and Amato and attempted to use the method of Moore and Stein Ladd (1988) reported that the amounts of ninhydrin(1948) to measure biomass ninhydrin-N in 0.5 M reactive compounds (i.e. ammonium, amino acids, KrSO,, as suggested by Amato and Ladd (1988). a peptides and proteins) released from the biomass heavy precipitate (presumably CaSO, and K:SO,) during the CHCI, fumigation and extracted by 2 M formed, causing erratic calorimetric analyses and KCI are strongly related to the initial soil biomass C invalidating the measurements. However, we finally content. Thus ninhydrin-reactive N (biomass ninhy developed a modified method to measure ninhydrindrin-N) released during fumigation (essentially CL- N in K,SO,, which quantitatively measured amamino N and ammonium-N) provides a rapid, monium-N also. So biomass C, biomass N and sensitive estimate of the amount of soil biomass. biomass a-amino N and ammonium-N (biomass They proposed the relationship: biomass C = 21 ninhydrin-N) may all be measured in a single K,SO, ninhydrin-N. extract during FE. In this paper our proposed We have often used Amato and Ladd’s ninhydrin method to measure biomass ninhydrin N is described method in our work. It gives a quick, simple and and some relationships between soil microbial reliable measure of soil biomass. In particular, FE biomass C, N and ninhydrin-reactive N (ammoniumcan reliably measure changes in soil biomass C and N and a-amino N) are discussed. N immediately following substrate addition (Ocio and Brookes, 1990). and the ninhydrin method has MATERIALS AND METHODS proved a valuable addition to these procedures. However, we invariably use 0.5 M K*SO, as the extractant Soils in FE measurements, rather than 2M KCI as Twelve soils, of different properties and under suggested by Amato and Ladd (1988) because different managements, were sampled to a depth of chloride interferes with the dichromate digestion and IO cm (Table 1). Before use they were sieved (C 2 mm), adjusted to 40% of full water holding capacity, incubated at 15°C for 21 days, then stored at 4°C until use. *Institute of Soil Science, von-Siebold-Str. 4, 3400 Total soil organic C and total N were measured after Goettingen. F.R.G. dry combustion by a Carlo Erba ANA 1400 analyser. tTo whom all correspondence should be addressed. lNTRODUCXlON

1023

1024

R.

G. JOERGENSEN and P. C. B~ootcss Table I. Dcscrimion of soils used

Soil No.

I 2 3 4 S 6 7 8 9 IO II I?

Site and cropping history Wobum fallow Wobum amble Wobum 8 yr Icy Broadbalk Continuous Wheat (FYM) Broadbalk Continuous Wheat (Nil) ~ Fosters arable Fostersgrass Finland grass Highfield grass Geescroft Wilderness (woodland) Northfield U.K. (arable) Northfield A&ha (arible)

Microbial biomass measurements Fumigation and soil extraction. Each moist soil (containing 20g soil on an oven-dry basis) was fumigated with ethanol-free CHCl, for 24 h. Following fumigant removal, the soil was extracted with 0.5 M KzSOI or 2 M KC1 (4: 1 solution:soil ratio) by shaking for 30 min on an oscillating shaker. It was then filtered through a Whatman No. 42 filter paper. Other non-fumigated portions were extracted similarly. at the time fumigation commenced. All results are expressed on an oven-dry soil basis (IOYC. 24 h) and are the means of triplicate determinations. Biomass C. Organic C in the K,SO, soil extracts was measured by an automated u.v.-persulphate oxidation procedure (Wu ef al., 1990). Briefly, 5 ml of soil extract was mixed with 5 ml of sodium hexametaphosphate (S%, acidified to pH 2 with concentrated H,P09) and the organic C in the extracts determined using a Dohrman DC80 automatic organic C analyser, with potassium persulphate (2%) as the oxidant. Biomass C (Bc) was calculated from Bc = 2.22 Ec, where E, = [(C extracted from fumigated soil) - (C extracted from non-fumigated soil)] (Vance et al., 1987; Wu et al., 1990). Biomass N. Total N in the K$O, soil extracts was measured by Kjeldahl digestion, as described by Brookes et al. (1985) but modified to include nitrateN (Pruden er al., 1985). Thus, 10ml of a reducing reagent [composed of 50 g KCr(SO,)r in 200 ml cont. H$O, I-‘] and zinc powder (300mg) was added to 15 ml of the K$O, soil extract and left for at least 2 h, before Kjeldahl digestion commenced (Pruden ef al., 1985). Biomass N (BN) was calculated from: BN = 2.22 E,, where E, = [(total N extracted from fumigated soil) - (total N extracted from nonfumigated soil)] (Jenkinson, 1988). Proposed method for measuring ninhydrin-reactice N in KJO, soi/ extracls Principles of the ninhydrin method. Ninhydrin decarboxyiates and deaminates n-amino groups and forms a purple complex with the a-amino N containing molecule. The colour is formed not only with amino acids, but also with peptides, proteins, ammonium and other compounds with free e-amino groups. The presence of reduced ninhydrin (hy drindantin) is essential to obtain quantitative colour development with ammonium (Moore and Stein,

Reference giving details of experiment (where available) Johnston Johnston Johnston Johnston Johnston Johnston

(1973) (1973) (1969) (1969) (1973j (1973)

Johnston (1973) Jenkinson (1971) Graham cf al. (1976) Amato and Laid (Ib88)

Organic C (%)

PH 5.5 6.4 6.8 7.6 7.9 7.7 6.0 5.6 5.8 3.9 6.1 7.9

Total N

1.16 1.2s I .46 3.20 1.09 1.63 2.44 2.44 4.55 3.72 3.21 I .92

0.109 0.130 0.136 0.311 0.117 0.161 0.233 0. I72 0.406 0.253 0.326 0.169

1948; Lamonthe and McCormick, 1973). At lOO”C, the reaction with free amino groups of proteins and amino acids is essentially complete within 15 min, e.g. leucine reaches the maximum colour yield after co 5 min. However, the reaction of hydrindantin with ammonium requires 25 min. To measure ninhydrin-N reproducibly, it is essential that the colour responses for ammonium and amino acids are as similar as possible. Special care also has to be taken to ensure that the reaction with ammonium and hydrindantin is complete. Ninhydrin-reactive N (or ninhydrin-N) is the sum of a-amino N, primary amines and ammonium-N. If required, the amount of ammonium-N is usually determined separately by autoanalysis and x-amino N can then be determined by difference. Choice of reagents Solvent. 2-methoxyethanol is the most common solvent used in the ninhydrin reaction. However, because it is an ether it tends to form peroxides which destroy ninhydrin and hydrindantin. Differences in the colour development, especially for ammonium between different batches of solvent, are a result of varying peroxide contents. Dimethylsulphoxide (DMSO) is peroxide-free. It is a better solvent for hydrindantin, has a high boiling point (189”C), and is of lower toxicity. Also, the ninhydrin reagent prepared in DMSO (see below) and the coloured complex produced are more stable than with 2-methoxyethanol. Therefore, we used DMSO as the solvent in the ninhydrin method. Buffer. In Moore and Stein’s (1948) original method [as recommended for KCI extracts by Amato and Ladd (1988)], the low solubility of CaSO, and of KrSO, in the presence of organic solvents caused serious analytical problems. The use of citric acid overcame this by complexing the Na+, K+ and Ca”. It was found that the optimum ratio [ninhydrin reagent-to-citric acid buffer + sample] was 1:2. If a wider ratio was used the colour intensity (especially for ammonium) decreased. The ratio between the volumes of sample and citric acid buffer should not be closer than 0.75: 1.75, or the solution becomes cloudy after the addition of the ninhydrin reagent. Complete details of our method for measuring ninhydrin-N are given below.

Ninhydrin biomass N

1025

2.5r

Reagents

All reagents are amino acid analytical grade. Ninhydrin reagent. Ninhydrin (20 g) and hy drindantin (3 g) were dissolved in DMSO (750 ml). Lithium acetate buffer (250 ml, 4 M. pH 5.2) was then added (Moore, 1968) and the mixture flushed for 30 min with O,-free N,. The reagent was prepared the day before use and kept airtight at room temperature. Citric acid buffer. Citric acid (42 g) and NaOH (16 g) were dissolved in water (900 ml). adjusted to pH 5 with 10 M NaOH, if required, then finally made up to 1 I. with water. Ethanol-water. Ethanol (95%) was diluted 1: 1 with water. Standark Standard solutions of leucine and (NH&SO, were separately prepared, in 0.5 M K,SO, or 2 M KCI, within the range O-1000 PM N. Operating procedure KJO, extructs. The standard N solutions, K,SO, soil extracts or blanks (0.75 ml) and the citric acid buffer (1.75 ml) were added to 20 ml test tubes. The ninhydrin reagent (1.25 ml) was then slowly added and thoroughly mixed, and the test tubes then heated for 25 min in a vigorously boiling water bath. Any precipitate which formed during addition of the reagents then dissolved. The temperature of the water bath should be sufficient for the water temperature to return to 100°C within 2 min after insertion of a full rack of 32 test tubes. The solutions were then cooled to room temperature. the ethanol-water (4.5 ml) added, the solutions thoroughly mixed and the absorbances then read at 570 cm (I cm path Icngth) against a water blank. Measurement of ammoniumN in the soil extracts was by automated calorimetric analysis. KC1 soil extracts

The procedure was similar, except that citric acid buffer was not used and the volumes changed to: blanks, standards or samples (I ml), ninhydrin reagent (0.5 ml). ethanol-water (9.5 ml). Biomass ninhydrin-N (BNIN)was calculated from: BN,N= [(ninhydrin-N in extracts of fumigated soil) mmus (ninhydrin-N in extracts of non-fumigated ammonium-N was calculated soil)]. Biomass similarly, and biomass a-amino N by difference. Thus, biomass a-amino N = [(ninhydrin-N minus ammonium-N in extracts of fumigated soil)(ninhydrin-N minus ammonium-N in extracts of non-fumigated soil)].

4

50100 250 N

500 concentration

750

1000

pM

Fig. I. Calibration curves of ammonium-N (0) and leucineN (0) in 0.5 M K,SO, measured with the modified ninhydrin reagent. Reagent blank not subtracted.

differences were