significance of microbial urea turnover in n cycling of three danish agricultural soils

Signi¢cance of microbial urea turnover in N cycling of threeDanish agricultural soils

Tommy Harder Nielsen *, Torben Andreas Bonde, Jan SÖrensenSection of Genetics and Microbiology, Department of Ecology and Molecular Biology, The Royal Veterinary and Agricultural University,

Thorvaldsensvej 40, DK-1871 Frederiksberg C (Copenhagen), Denmark

Received 29 July 1997; revised 17 October 1997; accepted 27 October 1997

Abstract

The importance of microbial urea turnover in N cycling was investigated in three agricultural soils by comparison of gross Nmineralization determined by the 15N-NH�4 dilution technique and urea turnover determined by a new 14C-urea tracertechnique. Average urea turnover rates were 1.5 to 4.2 Wg N g31 d31 indicating that the soil urea pool was turned over every9 to 30 min. Urea turnover rates were generally lowest in set-aside soil with increasing activities in bulk and rhizosphere soilfrom a barley field. Gross N mineralization and urea turnover rates were correlated (r = 0.79, P6 0.005) and of similar size inthe three soils. The high urea turnover rates indicated that urea-N was immobilized directly in soil microorganisms, rather thanmineralized to the free NH�4 pool. Our study suggests that microbial urea turnover, by-passing the conventional mineralization-immobilization pathway involving a free NH�4 pool, has a significant role in N cycling of agricultural soils. z 1998 Feder-ation of European Microbiological Societies. Published by Elsevier Science B.V.

Keywords: Urea turnover; Gross N mineralization; N cycling; Urease activity

1. Introduction

In soil the production of urea, CO(NH2)2, mayarise from bacterial degradation of purines [1] andof the amino acid arginine; the latter reaction mayinvolve arginase, arginine decarboxylase and argi-nine oxidase [2]. A functional ornithine cycle hasalso been reported in fungi and a few bacteria [3].Altogether, little is known about the natural ureaproduction rates in soil, and virtually nothing isknown about the role of the microorganisms.

Hydrolysis of urea with subsequent release of the

free NH�4 (urea ammoni¢cation) has been widelystudied in soils [4^6] and its important role in Ncycling after urea fertilization has been documented[7]. In contrast, unfertilized agricultural ¢elds typi-cally harbor a very low urea pool [8] ; yet, urea am-moni¢cation may still be signi¢cant in the soil Ncycling if urea hydrolysis rates, by the matrix-boundor microbial enzymes, are high. It is well known thatseveral bacteria possess a potential for urea hydrol-ysis [9], including a high-a¤nity urea uptake system,and subsequent degradation by an `intracellular' ure-ase enzyme [10]. When urea is hydrolyzed intracellu-larly, the NH�4 product may become directly immo-bilized (assimilated) into new organic material. Inthis case, the microbial urea turnover in the soil rep-

0168-6496 / 98 / $19.00 ß 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V.PII S 0 1 6 8 - 6 4 9 6 ( 9 7 ) 0 0 0 9 1 - 3

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* Corresponding author. Tel. : +45 (35) 28 26 27;Fax: +45 (35) 28 26 06.

FEMS Microbiology Ecology 25 (1998) 147^157

resents a bypass from the conventional mineraliza-tion-immobilization pathway involving a turnover ofthe free NH�4 pool. Alternatively, if the NH�4 productdi¡uses out of the cell [11], the urea turnover may beconsidered as part of the gross N mineralization, i.e.the total NH�4 production from degradation of or-ganic N in the soil.

Determinations of urea ammoni¢cation in soilhave typically been based on urea additions in largeexcess of the natural pool, thus enabling a directquanti¢cation of the process rates by urea disappear-ance or NH�4 production [12,13]. It is clear that thisapproach merely provides potential rates of urea am-moni¢cation, rather than in situ rates at the naturalurea concentrations. Only recently has a 14C tracermethod been developed to determine in situ turnoverrates of urea in soil without enrichment of the natu-ral urea pool [8,14]. Using this method in oak forestsoil, the authors showed activities supporting turn-over times of 9^15 min for the indigenous soil ureapool (29 to 56 ng urea-N g31). The data showed thatN cycling through the urea pool was 3 times higherthan the net N mineralization measured by accumu-lation of NH�4 and NO3

3 in the soil [8]. Gross Nmineralization rates are typically higher than thoseof net mineralization [15] and investigations in ma-rine sediments have shown that urea turnover mayconstitute up to 80% of the gross N mineralization[16]. The work by Pedersen et al. [8,14] suggests thatthe urea turnover rates in soil may have been com-parable to those of gross N mineralization.

By convention, gross N mineralization refers tothe total release of free NH�4 from organic N degra-dation. In environmental samples, the process maytherefore be determined by the 15N dilution tech-nique using a labelling of the NH�4 pool. Hence, todetermine if urea turnover was quantitatively signi¢-cant relative to gross N mineralization we comparedthe in situ urea turnover rate determined by 14Ctracer technique [8], and the gross N mineralizationdetermined by 15N dilution technique [17] in threeDanish agricultural soils. The comparison shouldalso indicate if in situ urea turnover is merely apart of gross N mineralization or if urea turnovershould be considered as an internal loop of organicN transformations in the soils. We ¢nally included acomparison of the in situ urea turnover rates withdeterminations of potential urea ammoni¢cation ac-

tivities, as the latter represents a much adoptedstandard assay of urease activity in soils.

2. Materials and methods

2.1. Soil sampling and preparation

All soil samples were collected in the autumns of1995 or 1996 from a ¢eld site comprising 3.0% clay,10% silt, 39.5% ¢ne sand and 47.5% course sand (wt/wt) at the experimental station HÖjbakkegaard(Taastrup, Denmark). The soil water content wasapproximately 12% (wt/dry wt) at all sampling occa-sions, except one (August 28, 1995) with only ap-proximately 1% water content. Some samples werecollected from the upper 5 cm of soil in a set-asideplot which had been uncropped for 3 years, withperiodical milling of the soil ; these samples are re-ferred to as set-aside bulk soil. Other samples werecollected from the upper 5 cm of bulk soil betweenplants in a barley (Hordeum vulgare L.) ¢eld; thesesamples are referred to as barley bulk soil. Finally,soil was collected from the roots of barley stubblesby gently digging up the roots with the adhering soil.The stubble was shaken by hand and the soil closelyassociated to the barley roots was released and col-lected; these samples are referred to as barley rhizo-sphere soil. In all experiments, the soil samples weresieved through a 2 mm sieve before preincubation inplastic containers at 20³C for 5 days. All studies ofmicrobial activities were conducted during the fol-lowing 1^3 day period. The gravimetric soil watercontent was determined after 24 h of incubation at105³C.

2.2. Gross N mineralization rate

Gross N mineralization was determined by a mod-i¢cation of the 15N dilution method described forsediments by Blackburn [17] and outlined below.Soils were 15N-labelled by spraying with a 24 mM(15NH4)2SO4 solution (98 atom % 15N; CambridgeIsotope Laboratories, MA, USA), while the soil wasgently mixed in a polyethylene bag. The water con-tent increased by less than 2% by the 15NH�4 additionusing this procedure. Subsamples of 5 g soil wereweighed into 50 ml polyethylene centrifuge tubes

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T. Harder Nielsen et al. / FEMS Microbiology Ecology 25 (1998) 147^157148

with loosely attached screw caps and incubated in awater bath at 20³C. Three replicate samples weresubsampled 4 or 5 times during the incubation peri-od, adding 24 ml 2 M KCl to each sample and shak-ing it for 1 h on an orbital shaker (1000 rpm). Aftercentrifugation at 4500Ug, the supernatant was ¢l-tered through KCl-washed GF/C ¢lters and frozenfor later analysis.

The NH�4 concentration in the KCl extracts wasanalyzed by the method of Verdouw et al. [18] asmodi¢ed by HÖjberg et al. [19]. The 15N analysisof the NH�4 pool was conducted by the microdi¡u-sion assay of Risgaard-Petersen et al. [20] and a tri-ple collector mass spectrometer (Tracermass model;Europa Scienti¢c LTD., Crewe, United Kingdom)using manual injection of the samples. The 15Natom fraction of the NH�4 pool was calculated bythe method of Nielsen [21] and the results wereused in the isotope dilution model for NH�4 turnover[17]. The method provides a determination of grossN mineralization, i.e. total NH�4 release (d) and totalconsumption of NH�4 (i) according to two equations:

P�t� � P0 � �d3i�t; �1�

ln�R315n� � ln�R0315n�3 d

d3i

� �Uln

�d3i�t� P0

P0

� ��2�

where P0 is the initial 14�15N pool and P(t) is the14�15N pool at time t, 15n is the natural abundanceof 15N (0.37 atom %) and R0 and R are the relativeabundances of 15N expressed as 15N/15N+14N at time0 and time t, respectively. When i and d are constant,P(t) in Eq. (1) is linear with time. Eq. (2) describesthat ln(R315n) is linear with ln[((d3i)t+P0)/P0], rep-resented by a slope of 3d/(d3i) and an intercept ofln(R0315n). The gross N mineralization (d) can becalculated by combination of Eqs. (1) and (2).

2.3. Urea pools

The urea concentration in the soils was determinedby a modi¢cation of the method developed by Pe-dersen et al. [8]. Soil samples (16 g) were weighedinto 50 ml centrifuge tubes and incubated at 20³C,in parallel to the incubations for 14C-urea turnover.

Three replicate soil samples were extracted 3 timesduring the incubation period by adding 5 ml 1 MKCl containing 0.5% ZnCl2 to inhibit microbial ac-tivity as described by Pedersen et al. [8]. After vor-texing the soil, each sample was transferred into adouble-chamber centrifugation unit [22] and centri-fuged 10 min at 3³C (6800Ug). The ¢ltrate was im-mediately frozen for subsequent urea analysis by themethod of Pedersen et al. [8] as modi¢ed from Priceand Harrison [23]. Absorbances were measured at540 nm in a Microplate reader (Bio-Tek Instru-ments) and blanks were prepared for each sampleby replacing the diacetylmonooxime reagent withdistilled water.

A recovery experiment to test the urea extractionprocedure was conducted. To avoid hydrolysis ofadded urea during this experiment, air-dried bulksoil from the barley ¢eld was autoclaved three timesfor 1.5 h each during a 3 day period [28]. Threesamples of the air-dried soil (each 100 g) were rewet-ted with 10 ml sterile distilled water or urea solutioncontaining 4.4 or 8.8 Wg urea-N. The samples weresubsequently incubated for 0.5 h at 20³C before ex-tracting the urea as described above.

2.4. Urea turnover rate

Turnover rates of 14C-urea were determined in9 ml polypropylene centrifuge tubes with a butylstopper in the lid. Samples of 1.5 g of soil wereweighed into each tube, covered with a perforatedsafran ¢lm, and incubated at 20³C for approximately15 h before use. A sample of 5 Wl 14C-urea tracersolution (56 mCi/mmol urea) was then added tothe soil and the tube was sealed with a lid. The tubeswere again incubated at 20³C and subsequentlystopped after 2, 4, 8, 10 and 15 min, respectively.The urea concentration of the added tracer solutionwas adjusted to the indigenous urea concentration inthe soil, since enrichment of the natural urea poolwas shown to in£uence the turnover of 14C-urea [8].In the standard protocol, the tracer addition in-creased the soil water content of the soil sample byless than 0.5%. One exception to this protocol wasmade on August 28, 1995, where the low soil watercontent required that the 14C-urea tracer was addedin a dry form as described by Pedersen et al. [14].This was accomplished by evaporating an aliquot of

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T. Harder Nielsen et al. / FEMS Microbiology Ecology 25 (1998) 147^157 149

tracer solution to dryness in a 9 ml polypropylenecentrifuge tube before the soil sample was added.The dry 14C-urea was transferred to the soil bygently shaking the tube, before incubation was con-tinued in a new vial. To determine the transfer of14C-urea tracer to the soil, 0.3 ml of the NaOH sol-ution was added directly into the scintillation liquid(see below). All incubations were stopped by inject-ing 3 ml NaOH with a hypodermic needle throughthe butyl rubber stopper and violently shaking thetube. The NaOH raised the pH to approximately 14in the soil slurry, thus terminating all microbial ac-tivity and converting the produced 14CO2 gas to 14C-labelled carbonate ions in solution. The tubes werethen centrifuged at 15 000Ug and the supernatantfrozen for analysis of 14C radioactivity in the carbo-nate pool.

The amount of 14C trapped in the carbonate poolwas determined by a di¡usion technique. A ¢lterpaper (10 cm2) was folded into a 1.5 ml centrifugetube and 0.4 ml Carbo-sorb E (Packard Instruments,Groningen, The Netherlands) was added to the ¢lter.The centrifuge tube was then installed in a stringhanging from the butyl rubber lid of a 100 ml infu-sion bottle. A 0.5 ml sample was placed in the in-fusion bottle and acidi¢ed by injecting 1.5 ml 5 NH2SO4 with a hypodermic needle through the lid torelease CO2 from the NaOH solution. The releasedCO2 was allowed to absorb to the Carbo-sorb over-night before the CO2 trap was transferred to a 20 mlscintillation vial and ¢lled with scintillation liquid(Ecoscient A, National Diagnostics, Atlanta, GA).The 14C activity was measured in a Beckman LS1801 scintillation counter with an automatic quenchcorrection software program. Radioactivity (dpmg31 soil) was calculated using the soil water contentand the amount of NaOH added. The 14CO2 back-ground in the 14C-urea tracer solution was sub-tracted from all results. The radioactivity of addedtracer was determined by adding 5 Wl 14C-urea tracersolution directly into the scintillation liquid.

The 14C-urea turnover rate was calculated usingthe steady-state model described by Lund and Black-burn [24]. This model is valid if the urea pool sizeremains constant during the incubation period andthe 14C-urea pool decreases exponentially with time.The model determines the turnover rate constant (k)as the slope of the natural logarithm (ln) of 14C-urea

content against time, and the turnover rate is deter-mined as k times the urea pool. The 14C-urea content(%) left in the soil during the incubation was calcu-lated knowing the amount of added 14C-urea tracerand the amount of produced 14CO2. As suggested byPedersen et al. [8] the turnover rate constant wascalculated from data points where 6 90% of theadded 14C-urea was hydrolyzed. The standard devia-tion on the turnover rate was determined using thestandard deviation on the urea pool, whereas nostatistical analysis was performed on the urea turn-over rate constant.

2.5. Potential urea ammoni¢cation

Potential urea ammoni¢cation activity was quan-ti¢ed as NH�4 production from added urea. Fromeach soil 4 replicates of 0.1 g soil samples wereweighed into 1.5 ml Eppendorf tubes and amendedwith 0.2 ml non-bu¡ered substrate solution of 5 mMurea. The soil slurries were shaken and incubated at20³C for 0.5 h. The incubations were stopped by theaddition of 0.8 ml ice-cold 2 M KCl solution andshaken for 1 min before centrifugating at 15 000Ugfor 5 min. The supernatant was collected and frozenfor later NH�4 analysis. Control samples (3 replicates)were prepared as described above, but incubationwas stopped immediately after substrate addition.

2.6. Statistics

The statistical analysis of correlations between theurea turnover rate, gross N mineralization and ureaammoni¢cation rates was conducted by using a sim-ple correlation analysis [25].

3. Results

3.1. Assay of gross N mineralization

Gross N mineralization rates were determined by15N isotope dilution technique during a 2^4 day soilincubation, in which the NH�4 concentration and 15Natom % in the NH�4 pool were followed. This isexempli¢ed in Fig. 1, showing progress curves (Aand B) for the analyzed parameters and a plot (C)of the slope of 3ln[((d3i)t+P0)/P0] versus ln(%

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15N30.37). The latter was used for calculation ofgross N mineralization rates (d) [17]. This regressionwas chosen since the multiple determination of NH�4concentration and 15N-atom % during the same in-cubation period gave us several data points based onsteady mineralization rates rather than a two-pointdetermination of gross mineralization rates [26,27].

Gross N mineralization rates were not expected tobe a¡ected by the 15NH�4 isotope addition, sinceNH�4 is itself a product rather than substrate of the

process [27]. However, the consumption activity (i)was clearly stimulated by the initial 15NH�4 additionleading to a steady decrease of the soil NH�4 poolduring the incubation period. Short incubation peri-ods (24 h) were therefore necessary when analysinggross N mineralization rates in the barley rhizo-sphere soil samples, in which NH�4 consumptionwas rapid (data not shown).

3.2. Assay of urea pools and urea turnover rates

The urea extraction procedure modi¢ed from Pe-dersen et al. [8] was tested by adding a knownamount of urea to autoclaved soil samples and meas-uring the recovery of extractable urea. As seen inTable 1 there was a slight increase in KCl-extractableurea when the air-dried soil was rewetted, but other-wise the urea content in the extracts was most similarto the expected values after addition of 44 ng N g31

(21% increase) or 88 ng N g31 (43% increase) urea tothe soil. The 100% extraction e¤ciency indicatedthat no binding of the added urea to soil matrixtook place, and an e¤cient extraction of the nativeurea pool was therefore likely.

We further tested the extraction e¤ciency for14CO2 produced in the soil after a complete turnoverof the added 14C-urea tracer. Fig. 2A shows the rap-id accumulation of 14CO2 during incubation of a set-aside bulk soil sample with 14C-urea tracer; 90% ofthe added 14C-urea was hydrolyzed to 14CO2 withinthe ¢rst 30 min, indicating a high turnover rate con-stant. A 85^95% recovery was found in all incuba-tions suggesting that most of the added tracer wasaccessible for hydrolysis. Subsequent incubationswere made with incubation periods of only 15 min.Fig. 2B shows the % of added 14C-urea, which wasleft in the three di¡erent soils during 15 min of in-cubation. The results gave a linear progress curve on

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Table 1Recovery of urea added to sterilized set-aside bulk soila

Urea pool size Dry soil Dry soil+water Dry soil+1Uureac Dry soil+2Uuread

Measured (ng N g31) 134 þ 3 206 þ 3 255 þ 6 306 þ 6Expected rangeb (ng N g31) 244^256 288^300

aValues are means (n = 3) þ standard deviation.bExpected concentration range is calculated as: Urea pool in dry soil+water þ 2Ustd+urea pool added.c1Uurea is 44 ng N g31.d2Uurea is 88 ng N g31.

Fig. 1. Measurements of NH�4 concentration (A) and 15N atom% (B) during the gross N mineralization assay in set-aside bulksoil sampled on October 23, 1995. Part C shows parameters usedfor the calculation of gross N mineralization rate (see text).


the log-scale, which allowed the turnover rate con-stant to be determined from the slope of a linearregression. Finally, to determine if the short-termincubations (15 min) for determining turnover rates

were reporting a steady-state activity, a series of ureahydrolysis assays were conducted for di¡erent peri-ods (17^21, 22^26 and 41^45 h) during the long-termassay for gross N mineralization. The results showedthat there were no signi¢cant changes in averageurea concentrations or turnover rate constants dur-ing the 2 day incubation period (Table 2).

3.3. Comparisons of process rates in three agriculturalsoils

As seen in Table 3, the average water content wasbetween 9 and 14% in the three soils during thesampling period in September^December of 1995^1996. This gave a comparable water content betweenthese samples, which in turn allowed a direct com-parison of their process rates at an incubation tem-perature of 20³C. The data from the dry soil col-lected on August 28, 1995 are not included inTable 3, but reported separately below. During thesampling period, urea concentrations were constantat approximately 20 ng g31 in the set-aside bulk soil.Similar concentrations were found in the barley bulksoil, except at one occasion when a higher level of 34ng g31 was reached. Finally, the barley rhizospheresoil had a concentration level between 25 and 36 ngg31. The urea turnover rate constant was highly var-iable within each soil, although the rate constantswere generally similar in the three soils.

Table 3 also shows the calculated urea turnoverrates and the gross N mineralization rates deter-mined by 15N isotope dilution technique. Duringthe autumn, the urea turnover rate in the set-asidebulk soil varied from 1.5 to 3.3 (mean 2.1) Wg N g31

d31 while rates in the barley bulk and rhizospheresoils were higher, ranging from 2.1 to 2.2 (mean 2.2)and 1.6 to 4.2 (mean 2.9) Wg N g31 d31, respectively.

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Table 2Urea concentrations, urea turnover rate constants and urea turnover rates in the set-aside bulk soil sampled on December 6, 1995 andincubated over a 46 h period (see text)a

Period ofdetermination (h)

Water contentb Urea pool size(ng N g31)

Turnover rateconstant (k)

Urea turnover(Wg N g31 d31)

17^21 12.1 þ 0.2 19.4 þ 8.5 0.050 1.4 þ 0.622^26 12.1 þ 0.2 22.9 þ 8.1 0.048 1.6 þ 0.641^45 12.1 þ 0.2 19.6 þ 8.3 0.054 1.5 þ 0.6

aValues are means (n = 3) þ standard deviation.bWater content is indicated as % (wt/dry wt).

Fig. 2. Part A shows measurements of % 14CO2 production origi-nating from 14C-urea hydrolysis in a set-aside bulk soil sampledon October 23, 1995. Part B shows a logarithmic plot of % re-maining 14C-urea versus incubation time, used for determiningthe turnover rate constant (see text) in the set-aside bulk, barleybulk and barley rhizosphere soil sampled on October 23, 1995.


By comparison, the set-aside bulk soil showed grossN mineralization rates varying from 0.7 to 1.5 (mean1.0), whereas the rates in the barley bulk and rhizo-sphere soils were again higher, from 1.5 to 2.0 (mean1.9) Wg N g31 d31 and from 2.6 to 4.8 (mean 3.8) WgN g31 d31, respectively.

Finally, Table 3 shows that the potential rates ofurea ammoni¢cation also measured when comparing

set-aside bulk, barley bulk and rhizosphere soils. Thepotential urea ammoni¢cation activities were from8.5 to 52.6 (mean 30.6), 47.7 to 56.4 (mean 52.4)and 117.6 to 169.2 (mean 138.5) Wg N g31 d31, re-spectively.

4. Discussion

4.1. Urea turnover in soil

The urea concentrations of 20^35 ng N g31 in thethree agricultural soils were comparable, yet slightlylower than those of 30^90 ng N g31 reported in agrassland soil by Pedersen et al. [14]. Quantitativeextraction of the small urea pools from the soilwas important for determination of in situ urea turn-over rates. Test experiments with added urea poolsshowed extraction e¤ciencies close to 100% (Table1), which suggested an e¤cient extraction of thesmall, native urea pool. The linearity of 14C-ureahydrolysis, as expressed on a log-scale (Fig. 2B) dur-ing the initial 15 min of incubation, indicated thatthe urea existed as a single pool in the soil. Similarresults have been found in 14C-urea tracer experi-ments in forest soil [8], whereas two urea pools

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Table 3Soil water contents, urea concentrations, urea turnover rate constants, urea turnover rates, gross N mineralization rates, and potentialurea ammoni¢cation rates in the set-aside bulk, barley bulk and barley rhizosphere soilsa

Soil Watercontentb

Urea pool size(ng N g31)

Turnover rateconstant (k)

Urea turnover rate(Wg N g31 d31)

Gross N mineral. rate(Wg N g31 d31)

Potential urea ammon. rate(Wg N g31 d31)

Set-aside bulk

B 9.4 þ 0.1 21.0 þ 5.4 0.110 3.3 þ 0.9 1.5 þ 0.2 52.6 þ 5.5C 12.6 þ 0.0 17.1 þ 7.8 0.061 1.5 þ 0.7 0.7 8.5D 12.1 þ 0.2 20.7 þ 8.1 0.051 1.5 þ 0.6 0.8 NDBarley bulk

A 12.0 þ 0.0 20.1 þ 3.2 0.078 2.2 þ 0.4 2.0 53.0 þ 6.0B 10.4 þ 0.1 16.0 þ 2.1 0.090 2.1 þ 0.3 1.5 þ 0.2 56.4 þ 5.3C 13.0 þ 0.1 33.6 þ 7.5 0.044 2.2 þ 0.5 2.1 47.7Barley rhizosphere

A 13.1 þ 0.1 35.7 þ 3.5 0.082 4.2 þ 0.8 4.0 169.2 þ 24.5B 13.0 þ 0.1 25.0 þ 8.0 0.082 2.9 þ 0.9 4.8 þ 0.6 117.6 þ 14.4C 13.7 þ 0.1 31.5 þ 7.6 0.035 1.6 þ 0.4 2.6 128.6

aValues are means (n = 3) þ standard deviation.bWater content is indicated as % (wt/dry weight).ND, not determined.Sampling times were: A: September 13, 1995; B: October 23, 1995; C: November 6, 1996; D: December 6, 1995. Measurements onDecember 6, 1995 are average values of data from Table 2.

Fig. 3. Comparison of urea turnover rates and gross N minerali-zation rates in the set-aside bulk, barley bulk and barley rhizo-sphere soil sampled during autumn 1995 and 1996, including `no-activity' data points observed in dry soils sampled on August 28,1995.


with distinctively di¡erent turnover rates seemed tooccur in grassland soil [14].

The constant urea concentrations observed duringeach of the standard 14C-urea incubation periodswere also maintained during longer incubations of2^4 days, corresponding to the long-term incubationassay for gross N mineralization (Table 2). Thesmall, but constant urea pools in the soil impliedthat the urea hydrolysis rate was balanced by a sim-ilar rate of urea production. Direct measurements ofturnover rate constants (Table 3) by the 14C assaysuggested a turnover time of 9 to 30 min for the ureapool. Purine catabolism [1] and amino acid hydrol-ysis via arginine degradation [2] are likely bacterialsources of urea in the soil. Indigenous urea produc-tion by bacteria containing the complete ornithinecycle has also been described [3], but so far only afew bacteria have been demonstrated to harbor thistrait. It may be added, however, that much attentionhas recently been paid to urea production by severalgroups of bacteria, including denitrifying, sulfatereducing or fermenting bacteria [29]. Signi¢cantbacterial production of urea has further been dem-onstrated in marine sediments [30]. In soil microfun-gi, the role of urea production is yet unknown buturea production and metabolism have been demon-strated in fungal species [31,32]. In contrast, proto-zoa and nematodes have not yet been shown to pro-duce urea in soil (Bryan Gri¤ths, personalcommunication).

In the present study, the soil water content wasapproximately 12%, which corresponded to a con-centration range for urea in the soil water of 10^20WM. The low urea concentration in the three soilssuggested that the urea hydrolysis was controlledby microbial activity, since microbial urease enzymesare reported to have low Km values [33]. Urea hy-drolysis may take place intracellularly in microor-ganisms [4,9], and a urea uptake system has beenobserved in both Klebsiella pneumoniae and Alcali-genes eutrophus, with low Km values of 13 and 38WM, respectively [10]. These values are one order ofmagnitude lower than the Km values of 280 and 650WM, respectively, for the urease enzyme per se [10].Hence, urea hydrolysis rather than urea uptake mayregulate the overall rate of urea turnover at the verylow concentrations in soils. By comparison, reportedKm values for total urease activity, including both

intracellular and matrix-bound enzymes, in varioussoils are as high as 1.3 to 62.5 mM [34].

In the assay of potential urea ammoni¢cation (ure-ase activity), the addition of 5 mM urea resulted inrates which were approx. 80% of Vmax values in thethree soils ; apparent Km values were approx. 2 mM(data not shown). Hence, the apparent Km value fortotal urease activity in the soils was higher than re-ported values for microbial uptake or hydrolysis ofurea, but at the low end of values reported for wholesoil samples. This suggests that both microbial (in-tracellular) and matrix-bound (extracellular) compo-nents are involved in urease activity of the soils. Thisis con¢rmed by the results of Pettit et al. [4], suggest-ing that the matrix-bound urease activity in soil maysometimes be up to 60% of the total activity. Thepotential urea ammoni¢cation rates (Table 3)showed an increasing level of activity from the set-aside bulk soil to the barley bulk and rhizospheresoils (in this order), which could re£ect a generalincrease of organic content [5] or microbial biomassand activity [35]. However, the absence of correla-tion between potential ammoni¢cation rates (ureaseactivity) and microbial urea turnover rates (P6 0.2,Table 3) con¢rmed that the former gave no speci¢cor detailed information on microbial urea transfor-mations in the soils, possibly because of a variablematrix-bound component.

4.2. Comparison of urea turnover and gross Nmineralization

Urea turnover rates of 1.5 to 4.2 Wg N g31 d31

observed in the three soils (Table 3) were comparableto turnover rates of 3.7 Wg N g31 d31 in an oakforest soil [8] and 0.3 Wg N g31 d31 to 13.0 Wg Ng31 d31 in a grassland soil [14]. Gross N mineraliza-tion rates of 0.7 to 4.8 Wg N g31 d31 in the presentstudy (Table 3) were also similar to results fromgrassland soils (1.4 Wg N g31 d31) reported by Da-vidson et al. [27] and from control soil (0.72 Wg Ng31 d31), oil-seed rape residues (0.87 to 2.2 Wg N g31

d31) and winter wheat residues (0.92 Wg N g31 d31)reported by Watkins and Barraclough [36].

Our direct measurements of both urea turnoverrates and gross N mineralization rates by tracer tech-niques make it possible to compare the two processesin the soils. In Fig. 3, all measurements of urea turn-

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over and gross N mineralization rates of the presentstudy are shown, including those of the dry barleybulk and rhizosphere soils on August 28, 1995,where none of the processes seemed to be active. Amost important observation was that in situ ureaturnover and gross N mineralization rates were com-parable in absolute numbers. To our knowledge,however, the high signi¢cance of urea turnover inrelation to gross N mineralization has not previouslybeen demonstrated directly in soils.

The results in Fig. 3 show that the process rateswere well correlated (r = 0.79, P6 0.005) using datafrom all soils, but further analysis of the data indi-cates that the signi¢cance of urea turnover relative togross N mineralization is di¡erent in the three soils.The rhizosphere soil showed urea hydrolysis activ-ities which were similar to or only 60% of the grossN mineralization rates. In the latter case, at leastpart of the organic N was degraded to NH�4 withoutinvolvement of the urea pool. In the barley bulk soil,the urea turnover was approximately 100% of thegross N mineralization, which indicated that all Nmineralizations could, in theory, have passed via theurea pool. Finally, in the set-aside soil, the urea hy-drolysis was always 200% of the gross N mineraliza-tion, suggesting that urea hydrolysis did not result inconcomitant NH�4 release, but was assimilated di-rectly into the microbial biomass.

In the set-aside bulk soil, the recorded di¡erencebetween urea turnover and total NH�4 release bygross N mineralization suggested that 0.7^1.8 Wg Ng31 d31 could have been directly assimilated intomicrobial cells. Assuming a C/N ratio of 4.2 as de-rived from data of Bakken [37] in the microbial bio-mass the N immobilization would require a C assim-ilation of 2.5^6.4 Wg C g31 d31 corresponding to 0.1^0.3 Wg C g31 h31. To test whether this C assimilationis likely, data from measurements of bacterialgrowth in soils using the [3H]thymidine incorpora-tion technique may be useful. Reported C assimila-tion rates using this technique are 0.1^ 4 Wg C g31

h31 in bulk sand [38], 0.7^ 1.0 Wg C g31 h31 inunplanted soil, and 0.6^0.8 Wg C g31 h31 in bulksoil [39]. These numbers indicate that the above hy-pothesis of direct immobilization of N released byurea hydrolysis is indeed a plausible explanationfor the urea turnover exceeding the gross N miner-alization in the set-aside bulk soil.

4.3. Signi¢cance of urea turnover in soil N cycling

It has previously been stated that organic N com-pounds in soils may be mineralized via two pathwaysor a combination of both of them: (1) In the conven-tional mineralization-immobilization turnover (MIT)pathway, organic N compounds are mineralized byextracellular soil enzymes and released to the freeNH�4 pool before immobilization into microorgan-isms takes place [40]. (2) In the `direct' hypothesis,organic compounds are taken into the microorgan-isms, deaminated, and only the surplus N is released(i.e. mineralized) into the free NH�4 pool [40,41]. As-similation of organic N directly into microorganismshas been reported in soils incubated with glycine andleucine [41]. (3) Finally, in a `parallel' hypoth-esis suggested by Barraclough [43], both of thetwo above models are operative simultaneouslyin the soils. Findings by Hadas et al. [42] alsosuggested both pathways operated concurrently. Fi-nally, glutamic acid, leucine and NH�4 were alsoshown to be immobilized together in soil bacteria[44].

When urea turnover is sometimes higher than thegross N mineralization as seen in the set-aside soil,we propose that this could be due to an intracellularurea hydrolysis and immobilization of the NH�4 ,without a release (i.e. mineralization) into the freeNH�4 pool (`direct' hypothesis). Such a reactionthus by-passes the conventional mineralization-im-mobilization turnover (MIT model), proceeding viathe free extracellular NH�4 pool. Since the urea turn-over rates are high, and sometimes higher than themeasured gross N mineralization rates, we furthersuggest that the urea cycle in the soils has a pro-found signi¢cance in soil N cycling.

Acknowledgments

The authors thank Niels O.G. JÖrgensen for hisassistance in 14CO2 analysis, Henning Pedersen forcritically reading the manuscript and Bryan Gri¤thsfor commenting on urea production in soil protozoaand nematodes. This work was supported by theDanish Strategic Environmental Research Pro-gramme 1992^1996.

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significance of microbial urea turnover in n cycling of three danish agricultural soils

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