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Soil Biology & Biochemistry 40 (2008) 3040–3048

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Soil Biology & Biochemistry

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Regulation of extracellular protease activity in soil in response to differentsources and concentrations of nitrogen and carbon

Daniel Geisseler*, William R. HorwathDepartment of Land, Air and Water Resources, University of California, Davis, 1 Shields Avenue, Davis, CA 95616, USA

a r t i c l e i n f o

Article history:Received 14 February 2008Received in revised form 19 August 2008Accepted 1 September 2008Available online 9 October 2008

Keywords:Extracellular enzymesProtease activityMicroorganismsCarbonNitrogen

* Corresponding author. Tel.: þ1 530 754 8226; faxE-mail address: djgeisseler@ucdavis.edu (D. Geisse

0038-0717/$ – see front matter � 2008 Elsevier Ltd.doi:10.1016/j.soilbio.2008.09.001

a b s t r a c t

A large proportion of the nitrogen (N) in soil is in the form of proteinaceous material. Its breakdownrequires the activity of extracellular proteases and other decomposing enzymes. The goal of our studywas to better understand how carbon (C) and N availability affect soil protease activity. Several aerobicincubations were carried out with ammonium (NH4

þ) and proteins as N sources and cellulose as the mainC source. A strong increase in protease activity was observed when proteins were added, the increasedepending on the amount of protein added and its solubility. Protease synthesis was clearly substrateinduced, as NH4

þ had no effect. During this substrate induced phase, the addition of glucose but not NH4þ

resulted in protease repression, indicating that the level of protease synthesis was determined by theneed for C rather than N. After 1 month of incubation, protease activity remained relatively constant overtime and was closely related to microbial biomass N. Different concentrations of mineral N in soilsolution had no direct effect on protease activity. However, during this stationary phase, protease activitycould be repressed by glucose and NH4

þ in a treatment with low mineral N content while in treatmentswith a higher N availability no repression was observed. We hypothesize that the need for N determinedprotease activity in the treatment with limited N availability. The addition of NH4

þ allowed for reallo-cation of C and N away from protease synthesis, leading to the observed decrease in protease activity. Therepression by glucose may be attributed to shifts in the pathway of microbial NH4

þ assimilation. Theresults emphasize the close links between the microbially mediated cycles of organic C and N.

� 2008 Elsevier Ltd. All rights reserved.

1. Introduction

Nitrogen occupies a unique position among the soil-derivedelements essential for plant growth because of its complexbiogeochemistry and the large amounts required by plants incomparison to other elements. In most terrestrial ecosystems N isthe limiting nutrient for plant growth (Robertson and Groffman,2007). More than 90% of the total N in soils occurs in the form oforganic N compounds (Foth and Ellis, 1997), most of which are notdirectly available to plants.

In living organisms most of the organic N is in the form ofproteins (Kogel-Knaber, 2006). Despite being labile compoundswith high turnover rates, proteins from plant and microbial tissuescomprise a large part of soil N (Ladd and Jackson, 1982). It has beenestimated that about 40% of the total soil N is proteinaceousmaterial, including proteins, glycoproteins, peptides and aminoacids (Sowden et al., 1977; Schulten and Schnitzer, 1998). Soilproteins are hydrolyzed to peptides and amino acids by extracel-lular proteases. Proteases are produced by a wide range of bacteria,

: þ1 530 752 1552.ler).

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actinomycetes and fungi (Kumar and Takagi, 1999; Glenn, 1976) andusually have a wide substrate-specificity, degrading most non-structural proteins (Kalisz, 1988). Due to the abundance of proteinsand peptides in soil, proteases likely supply a large part of thebioavailable N.

Enzyme production is N and energy intensive. Microbes thatregulate enzyme production according to their needs and theavailability of substrates should have a competitive advantage(Koch, 1985). Four different mechanisms have been described toregulate the synthesis and secretion of extracellular protease(Kalisz, 1988). (1) The presence of a substrate can induce proteasesecretion. (2) High levels of end products, such as amino acids, NH4

þ

and easily metabolizable sources of C may repress production.(3) On the other hand, protease production may be increased (de-repressed) when insufficient levels of C, N or sulfur (S) are available.(4) Finally, extracellular enzymes may be secreted constitutively atlow levels regardless of the availability of a substrate.

The factors regulating protease activity have been studiedextensively with single bacterial and fungal species (Glenn, 1976;Kumar and Takagi, 1999). Much less information is available abouthow differences in C and N availability affect protease activity insoil, with its complex microbial community. In inoculated sand

D. Geisseler, W.R. Horwath / Soil Biology & Biochemistry 40 (2008) 3040–3048 3041

cultures amended with defined nutrient media, Sims and Wander(2002) found evidence for the de-repression mechanism. Themicrobial community responded to limitations of N and S byincreasing proteolytic activity and reducing biomass compared tothe control. C limitation had no effect on proteolytic activity. Allisonand Vitousek (2005) found that glycine aminopeptidase wasinduced by collagen addition to soil, but significantly repressed byNH4þ. Asmar et al. (1992) found that protease activity was increased

in soil samples amended with nutrients and glucose. In their study,protease activity was correlated with ATP content, respiration andbacterial biomass.

Extracellular enzymes often catalyze the rate-limiting step ofdecomposition and nutrient cycling (Sinsabaugh, 1994), makingtheir expression and kinetics potential useful parameters fornutrient turnover models. Several authors have presentedconceptual models using enzyme activity to describe the acquisi-tion of C and N by microbes (Sinsabaugh and Moorhead, 1994;Schimel and Weintraub, 2003; Allison, 2005). However, there is stilllittle information available about the different feedback mecha-nisms and interaction of the various C and N pools that governextracellular enzyme production in complex soil microbialcommunities.

This study provides information on the influence of C and Navailability on protease expression. First, we determined theexpression of protease based on amount and availability ofproteins. Second, we studied the effect of C and N availability on soilprotease activity over time. Third, we investigated the effects ofnon-polymeric C and N sources on soil protease activity.

2. Material and methods

2.1. Soil sampling

Soil samples (0–15 cm) were collected in late fall 2006 andspring 2007 at the Long-Term Research on Agricultural Systems(LTRAS) site west of Davis, CA. The soil is a loam of the Rincon seriesclassified as fine, smectitic, thermic Mollic Haploxeralf (Soil SurveyStaff, 1997). The soil is under permanent turf grass cover and waspreferred to a soil under row crops to eliminate possible effects oftillage and crop rotation on biochemical and biological processes.The samples were stored on ice for transport back to the laboratory.The soil was sieved through a 4-mm sieve prior to starting incu-bations within 24 h of collection. Selected soil properties aresummarized in Table 1.

2.2. Soil incubations

Field moist soil (6–8 g) was weighed into 50 ml centrifuge tubes.Amendments were either applied dissolved in DI water (ammo-nium chloride [NH4Cl], ammonium sulfate [(NH4)2SO4], Na-caseinate, glucose), or as a powder well mixed with the soil

Table 1Selected properties of the soil samples used for the incubations

Soilproperty

Value Method

pH 7.4 1:1 soil:water (Thomas, 1996)EC 88 mS cm�1 1:1 soil:water (Rhoades, 1996)Texture 37% sand,

39% silt, 24% clayPipet method (Gee and Bauder, 1986)

Total C 13.0 g totalC kg�1 dry soil

Dry combustion using a CarloErba CNS analyzer NA 1500 series 2(Nelson and Sommers, 1996)

Total N 1.18 g total N kg�1

dry soilDry combustion using a Carlo ErbaCNS analyzer NA 1500 series 2(Bremner, 1996)

(cellulose, gluten, zein). The samples were incubated at a moisturecontent of 28% (g g�1), which corresponds to 50% of the waterholding capacity determined by the funnel method. During theincubations the centrifuge tubes containing the samples were keptin glass jars sealed with lids equipped with rubber septa for gassampling.

Incubation 1 was carried out to determine the expression ofprotease based on the amount and availability of proteins(objective 1). NH4Cl, Na-caseinate (from bovine milk), gluten (fromwheat) or zein (from corn) were used as N sources (Table 2). Soilsamples were amended with 0.05, 0.1, 0.15, or 0.2 mg N g�1 dry soil.After 2 days, total carbon dioxide (CO2) production and proteaseactivity were measured (see details in Section 2.3).

To investigate effects of varying C and N availability on proteaseactivity over time (objective 2), soil samples were amended withcellulose and different N sources. In incubation 2a, the amount of Nwas the same for all treatments and the cellulose addition wasadjusted to obtain different C to N ratios of the amendments. Forincubation 2b, the cellulose addition was kept constant, while theamount of N added was adjusted (Table 2).

In incubation 2a, 0.1 mg N g�1 dry soil of (NH4)2SO4, Na-caseinate, gluten and zein were added to separate samples.Different amounts of cellulose were added to the samples to attainC to N ratios of the amendment of 10, 25 and 40 (Table 2). Thesethree C to N ratios were chosen because residues that have C to Nratio greater than about 30 result in net N immobilization, whileresidues with a C to N ratio below 20 normally lead to net Nmineralization (Stevenson, 1986). Unamended samples served ascontrol. After 3, 7, 14, 28, 49, and 70 days, four replicates pertreatment were destructively sampled to determine proteaseactivity, NH4

þ, nitrate (NO3�), and microbial biomass N. In addition,

CO2 evolution was measured frequently so that the CO2 concen-tration did not exceed 1.5% in the headspace of the glass jars inwhich the samples were kept. Incubation 2b contained the same Cand N sources as incubation 2a but the amount of cellulose addedwas kept constant at 5 mg g�1 dry soil while the addition of N wasadjusted to obtain C to N ratios of 10 and 40. Two controls wereincluded in the experiment, one without any amendments anda second with cellulose as the sole amendment. After 59 days, threereplicates per treatment were analyzed for protease activity (twosubsamples per replicate), mineral N (NH4

þ and NO3�), and microbial

biomass N. CO2 evolution was measured periodically (see below).Incubations 2a and 2b were set up as a two-way factorial with Nsource as one factor and the C to N ratio of the amendments as thesecond factor.

Finally, two incubations (3a and 3b) were carried out to inves-tigate the effects of non-polymeric C and N sources, namely glucoseand NH4

þ, on soil protease activity (objective 3). In incubation 3a theeffect of glucose and NH4

þ on protease activity was studiedfollowing the addition of protein, when the level of proteaseactivity was determined by the protein added. In incubation 3bglucose and NH4

þ were added several weeks after the addition ofprotein, when the initial protein addition was no longer thedominant factor determining protease activity.

For incubation 3a, soil samples were amended with Na-caseinate (0.1 mg N g�1 dry soil), NH4Cl (0, 0.02, or0.04 mg N g�1 dry soil) and glucose (0, 0.2, or 0.4 mg C g�1 dry soil).After 1, 2, 3, 5 and 8 days the samples were analyzed for proteaseactivity, mineral N, reducing sugars, and CO2 evolution (see below).An unamended sample served as control (Table 2). For incubation3b, soil samples were amended with Na-caseinate as N source(0.1 mg N g�1 dry soil) and cellulose as C source (varying amountsto reach C to N ratios of 10, 25 and 40). These treatments wereidentical to the Na-caseinate amended treatments in incubation 2a.After 48 days of incubation, however, NH4Cl (0 or 0.04 mg N g�1 drysoil) and glucose (0 or 0.4 mg N g�1 dry soil) were added. The

Table 2Overview of the substrates used for the different incubations

Incubation N sources Main Csource

N added(mg g�1 dry soil)

C addedwith cellulose(mg g�1 dry soil)

C to N ratiosb

(mg g�1 dry soil)Other additions

1 Na-caseinate, zein,gluten, NH4Cl,

None 0.05, 0.1, 0.15, 0.2 0 0–4.1

2aa Na-caseinate, zein,gluten, (NH4)2SO4

Cellulose 0.1 1, 2.5, 4 10, 25, 40

2b Na-caseinate, zein,gluten, (NH4)2SO4

Cellulose 0.21, 0.05 2.1 10, 40

3a Na-caseinate None 0.1 0 3.6 NH4Cl and glucose added at day 03b Na-caseinate Cellulose 0.1 1, 2.5, 4 10, 25, 40 NH4Cl and glucose added at day 48

a The number refers to the objective addressed, the letter separates incubations carried out for the same objective.b The calculation of the C to N ratio takes the C added with proteins into account. When no cellulose was added, the number represents the C to N ratio of the N source.

D. Geisseler, W.R. Horwath / Soil Biology & Biochemistry 40 (2008) 3040–30483042

amendments were dissolved in 0.25 ml DI water and injected witha syringe. DI water alone was added to a control. Protease activity,mineral N, reducing sugars, and CO2 evolution were measuredbefore as well as 2 and 4 days after the injections (Table 2).

2.3. Soil analyses

Protease activity (EC 3.4.2.21-24) was assayed using the methoddescribed by Ladd and Butler (1972). Briefly, 1 g of soil was weighedinto a glass vial and 2.5 ml of Tris buffer (0.2 M, pH 8.0) and 2.5 mlof a 2% Na-caseinate solution (20 g l�1 DI water) were added. Thecapped vials were incubated in a water bath at 50 �C for 2 h. Afterthe incubation, the remaining casein was precipitated with 5 ml10% trichloroacetic acid. 1.5 ml of the solution was pipetted intoa microcentrifuge tube and centrifuged at 13 000 rev min�1 for1 min. 0.5 ml of the clear supernatant was mixed with 0.75 mlNa2CO3 (1.4 M) and 0.25 ml threefold diluted Folin–Ciocalteureagent in a microcuvette. The tyrosine concentration wasmeasured colorimetrically at 680 nm after exactly 5 min. For thecontrols, the same procedure was followed, with the exception thatthe Na-caseinate was added after the incubation and the addition ofthe trichloroacetic acid.

To measure CO2 evolution, a maximum of four centrifuge tubes,each containing 6–8 g of soil were placed into 0.95 L glass jarssealed with lids equipped with rubber septa for gas sampling. Toeach jar 30 ml of DI water was added to minimize evaporation fromthe soil samples during the incubation. The jars were kept in thedark for the duration of the incubation. A randomly selected subsetof four jars per treatment was used for CO2 measurements. Head-space CO2 was analyzed with a Qubit CO2 analyzer (model S-151,Qubit Systems Inc., Kingston, Canada). After each analysis, the jarswere opened and flushed with ambient air. A blank was used tocorrect for background CO2. The ideal gas law was applied tocalculate the amount of organic C released based on the CO2

concentration (Zibilske, 1994). CO2 measurements were alwaystaken before the CO2 in the headspace reached a concentration of1.5% by volume.

Soils were extracted with 0.5 M K2SO4 (5 ml g�1 soil; Mulvaney,1996) and the suspension filtered (Fisherbrand, Q5) for the analysisof NH4

þ, NO3�, total N in solution and reducing sugars.

NO3� was analyzed using a single reagent method (Doane and

Horwath, 2003). The NH4þ concentration was determined using the

salicylate method (Verdouw et al., 1978; Foster, 1995). Reducingsugars were measured colorimetrically with an anthron reagent(Fales, 1951). A standard curve was prepared with glucose.

Microbial biomass N was determined using the chloroformfumigation extraction method (Horwath and Paul, 1994) followedby the determination of total N in the extracts with the alkalinepersulfate oxidation method (Cabrera and Beare, 1993). Dissolvedorganic N was extracted with K2SO4 as described above. Controls

were treated identically except that they were not fumigated.Microbial biomass N was calculated by dividing the difference in Ncontent between the fumigated and unfumigated sample by 0.68 toaccount for an incomplete N extraction (Horwath and Paul, 1994).

All results reported are expressed on a moisture-free basis.Moisture content was determined after drying the soil samples at105 �C for 24 h.

2.4. Data analysis

Statistical analyses were conducted with the SAS program (SASInstitute, 1990), using the general linear model (GLM) procedure foranalysis of variance and the REG procedure for regression analyses.The assumptions of the statistical models were tested for every dataset. Normality of the residuals was evaluated graphically and withthe Shapiro–Wilk test. Homogeneity of variances was tested byplotting the residuals vs. the predicted values and with Levene’stest. If necessary, the data were transformed.

Mean comparisons were performed using the Tukey test, whichcontrols the experimentwise type I error rate a (SAS Institute,1990). Effects were considered significant for P< 0.05.

3. Results

3.1. Relationship between protein degradation and protease activity

Increasing additions of Na-caseinate, gluten and zein in incu-bation 1 resulted in linear increases in protease activity. In contrast,the addition of NH4Cl had no effect on protease activity. Theevolution of CO2 was positively and linearly correlated to proteaseactivity for all three proteins (P< 0.05; R2> 0.78; Fig. 1). CO2

evolution was not correlated with NH4Cl additions (P¼ 0.84;R2¼ 0.02).

CO2 evolution was much higher when Na-caseinate was addedcompared to gluten and zein, however, less CO2 was evolved perunit protease activity for Na-caseinate than the other proteins(Fig. 1).

3.2. Influence of C and N availability on protease activity over time

For incubation 2a, cellulose was added in increasing amountstogether with (NH4)2SO4, Na-caseinate, gluten, or zein to formulateC to N ratios of 10, 25 and 40. The addition of proteins increasedprotease activity by at least 80% within 3 days (Fig. 2). In contrast,there was no increase in protease activity in the NH4

þ treatmentswithin the first 2 weeks. As in incubation 1, Na-caseinate had themost pronounced effect on protease activity; however, proteaseactivity decreased quickly after 3 days. In the treatments withgluten and zein, protease activity increased slowly, but the activitywas maintained at an elevated level for a longer time.

Fig. 1. Relationship between protease activity and CO2 evolution with different Nsources after 2 days of incubation. A: Na-caseinate (R2¼ 0.99; P< 0.001). B: gluten(R2¼ 0.78; P¼ 0.046), zein (R2¼ 0.86; P¼ 0.023) and NH4Cl (R2¼ 0.02; P¼ 0.84). Datashown are means (n¼ 4)� standard error of the means (SE). Some error bars for CO2

evolution are smaller than the icon.

Fig. 2. Protease activity in soil amended with (NH4)2SO4 (A), Na-caseinate (B), gluten(C), and zein (D) as N sources (0.1 mg N g�1 dry soil) and adjusted cellulose additions toresult in C to N ratios of 10 (d:d), 25 (dAd), and 40 (d-d). Data shown aremeans� SE (n¼ 4).

D. Geisseler, W.R. Horwath / Soil Biology & Biochemistry 40 (2008) 3040–3048 3043

Throughout the incubation, the N source continued to bea significant factor determining protease activity (P for factor Nsource <0.001 throughout the incubation). In general, proteaseactivity was highest in the samples with Na-caseinate and lowest inthe samples amended with (NH4)2SO4. After the second week ofincubation, higher C to N ratios resulted in higher protease activi-ties (P for factor C to N ratio <0.001 for sampling dates 28, 49, and70). This was not only the case in the protein amended treatmentsbut also in the NH4

þ amended treatments where protease activityincreased significantly in the treatments with a C to N ratio of 25and 40. After the first 4 weeks of incubation, protease activityremained relatively stable in all treatments and there was nosignificant interaction between C to N ratio and protein source (Pfor day 49: 0.93; for day 70: 0.06).

Differences in the C to N ratio not only affected protease activitybut higher C to N ratios also increased microbial biomass N andrespiration rate during the second half of the experiment (data notshown), due to the fact that higher C to N ratios were obtained byincreasing the amount of C added. On the other hand, the mineral Ncontent in soil solution decreased with increasing C to N ratio. Inorder to determine which of these factors were related to proteaseactivity, incubation 2b was carried out with the same substrates. Incontrast to incubation 2a, however, the cellulose addition was thesame for all treatments but the amount of N added was adjusted toreach C to N ratios of 10 and 40.

After 59 days of incubation, protease activity was higher in theprotein amended treatments compared to the NH4

þ amendedtreatments. There were no significant differences between treat-ments amended with different proteins (Table 3). Over all treat-ments, the C to N ratio had no effect on protease activity (P¼ 0.78).

Table 3Protease activity, microbial biomass N, CO2 evolution and mineral N in soil samples incubated for 59 days

N source C to N ratio Proteaseactivity(mg Tyrosine kg�1

dry soil h�1)

Microbial biomass N(mg N kg�1 dry soil)

Cumulative CO2 evolution(mg C g�1 dry soil)

NH4þ and NO3

�

(mg N kg�1 dry soil)

Days 0–32 Days 32–59

Control 88.5 (12.93) a 55.6 (3.06) a 585 (14) a 394 (29) a 20.5 (2.57) bCellulose only 106.5 (1.47) ab 66.8 (4.71) abc 925 (57) b 824 (95) c 0.7 (0.07) a(NH4)2SO4 10 92.9 (4.47) a 61.6 (2.30) ab 1602 (54) d 504 (116) ab 73.3 (2.08) d(NH4)2SO4 40 130.4 (8.03) bc 65.5 (0.91) abc 1339 (25) c 895 (62) c 4.2 (1.01) aNa-caseinate 10 144.0 (7.30) c 83.7 (7.15) c 2171 (24) e 721 (116) bc 62.4 (2.52) cNa-caseinate 40 145.0 (5.29) c 81.4 (2.05) c 1516 (63) cd 957 (75) c 5.1 (0.48) aGluten 10 155.2 (1.10) c 79.4 (4.04) bc 2271 (73) e 756 (76) c 61.5 (3.31) cGluten 40 120.3 (2.99) abc 81.6 (2.60) c 1607 (15) d 948 (17) c 3.2 (0.25) aZein 10 142.4 (0.52) c 79.8 (4.26) bc 2186 (45) e 873 (48) c 74.6 (3.70) dZein 40 143.8 (11.88) c 83.3 (1.14) c 1623 (46) d 897 (106) c 4.3 (1.69) a

Note: all values are means (n¼ 4) with SE given in parenthesis. Means followed by a different letter within each column are significantly different at P< 0.05 according toTukey’s test. The same amount of cellulose was added to all samples except the control. The N addition was adjusted to reach C to N ratios of 10 and 40.

D. Geisseler, W.R. Horwath / Soil Biology & Biochemistry 40 (2008) 3040–30483044

However, the higher C to N ratio significantly increased proteaseactivity in the NH4

þ amended treatment, but had the opposite effectin the gluten amended treatment. The unamended control and thecontrol with cellulose had low protease activities. In contrast to theprotease activity, higher N additions resulted in significantlyincreased mineral N contents. Compared to the differencesbetween the two C to N ratios, the differences caused by the Nsources were small. Protease activity and mineral N content werenot correlated in this experiment (R2¼ 0.01; P¼ 0.74). A low C to Nratio significantly increased CO2 evolution during the first 30 daysof the incubation (Table 3). However, after day 30 the CO2 evolutionin these treatments dropped below the level of the treatments withhigher C to N ratios. A regression analysis indicated a significant butweak correlation between protease activity after 59 days of incu-bation and CO2 respired between days 30 and 59 (R2¼ 0.46;P¼ 0.03).

In contrast to mineral N and CO2 evolution, microbial biomass Ndid not differ significantly between the two C to N ratios, noramong the protein amended treatments. However, microbialbiomass N was higher in the treatments where protein served asthe N source compared to the NH4

þ amended treatments. A linearregression analysis revealed a positive correlation betweenmicrobial biomass N and protease activity (R2¼ 0.73; Fig. 3).Analysis of the data from incubation 2a showed a poor correlationbetween protease activity and microbial biomass N across alltreatments and sampling dates (Table 4). However, when eachsampling date was analyzed separately, a clear pattern emerged.

Fig. 3. Relationship between protease activity and microbial biomass N after 59 days ofincubation (R2¼ 0.73; P¼ 0.002). Each treatment (except the control) received thesame amount of cellulose, but different amounts of N. Data shown are means� SE(n¼ 3).

While the correlations between protease activity and microbialbiomass N were weak for the first three sampling dates, theyimproved considerable for sampling days 28, 49, and 70 (Table 4).While the slopes of the regression lines for these days were verysimilar, there were large differences between the intercepts due todifferences in microbial biomass N. This was due to the fact thatslight changes in microbial biomass N over time were not reflectedin protease activity.

3.3. Effect of simple C and N compounds on protease activity

In order to study the effect of readily available C and N onprotease activity, incubations 3a and 3b were conducted. In incu-bation 3a, glucose, NH4Cl or both together were added to soilsamples with Na-caseinate. The addition of glucose loweredmineral N levels and increased CO2 evolution significantly (Table 5).The positive effect on respiration was most pronounced during thefirst day but persisted until day 8. In contrast, the addition of NH4Clincreased mineral N levels and lowered CO2 evolution.

In all treatments, except in the unamended control, proteaseactivity increased sharply for 2 days and decreased until day 8 toabout 50% of its peak (Fig. 4). The addition of glucose significantlyreduced protease activity by about 13% during the first 2 days. Theaddition of NH4Cl did not repress protease activity, but resulted ina slightly less steep decrease after the maximum activity wasreached. These differences were small compared to the fivefoldincrease during the first 2 days.

In incubation 3b, glucose and NH4Cl were added to soil samplesthat had been incubated for 48 days. When only glucose was added,CO2 evolution increased and mineral N concentration decreased,except in the treatment with a C to N ratio of 40 where the mineralN concentration was already very low before the glucose addition(Table 6). After 4 days, less than 1% of the glucose added was left insolution.

Table 4Correlation between protease activity and microbial biomass N during an incubationwith four different N sources and three different C to N ratios

Sampling day n Slope Intercept R2 P

3 14 3.70 �194 0.48 0.0067 14 0.86 114 0.04 0.49814 14 2.35 �47 0.51 0.00428 14 2.24 �32 0.72 <0.00149 14 2.56 40 0.78 <0.00170 14 2.51 �10 0.84 <0.0013-70 84 1.51 55 0.28 <0.00128-70 42 1.49 71 0.42 <0.001

Two controls were included per sampling date.

Table 5Effect of directly available C and N on cumulative CO2 evolution, and N mineralizedduring an 8-day incubation with Na-caseinate, glucose and NH4Cl added at day 0

C added(mg/kg dry soil)

N added(mg/kg dry soil)

Cumulative CO2

evolution(mg C/kg dry soil)

N mineralized(mg N/kg dry soil)

0 0 531 (18.2) 16.3 (0.11)200 0 590 (0.7) 12.8 (0.49)400 0 706 (22.2) 9.7 (0.31)0 20 517 (10.2) 20.8 (0.36)200 20 552 (16.7) 18.4 (0.93)400 20 653 (2.9) 14.4 (0.47)0 40 496 (38.8) 23.7 (0.27)200 40 576 (41.5) 19.8 (0.98)400 40 667 (23.4) 17.1 (1.47)

ANOVA F-testsP

C 0.022 <0.001N 0.005 <0.001C�N 0.652 0.227

Note: all values are means (n¼ 4) with SE given in parenthesis. Data were analyzedas a two-way factorial.

D. Geisseler, W.R. Horwath / Soil Biology & Biochemistry 40 (2008) 3040–3048 3045

The addition of NH4Cl had no significant effect on CO2 evolutionin the control and the treatments with C to N ratios of 10 and 25,but slightly increased CO2 evolution in the treatment with a C to Nratio of 40. When NH4Cl was added without glucose, more than 75%of the added NH4

þ remained in solution after 4 days in the controland the treatments with C to N ratios of 10 and 25, but less than halfwas left in solution in the treatment with a C to N ratio of 40. Ingeneral, the combined addition of glucose and NH4Cl increased theproportion of N taken up.

The additions of glucose and NH4Cl had no significant effecton protease activity after 2 days (data not shown). After 4 days,however, protease activity had decreased by 20, 13 and 16% inthe treatment with a C to N ratio of 40 and addition of glucose,NH4Cl or their combination, respectively. The decrease caused byglucose was significant at the 5% level (Fig. 5). In the othertreatments, glucose and NH4Cl had no significant effect onprotease activity.

Fig. 4. Effect of glucose and NH4Cl on protease activity when added together with Na-caseinate. Data shown are means� SE (n¼ 4). The lower line is the unamendedcontrol.

4. Discussion

The production of extracellular protease by microorganisms isregulated by different mechanisms. Most of the research investi-gating the regulation of protease activity has been carried out withpure cultures. The species studied and the conditions of theexperiment may or may not be representative for complex micro-bial communities found in soil. We investigated the response of soilmicrobial communities to different availabilities of C and N withseveral aerobic soil incubations.

4.1. Substrate induced protease synthesis

The temporal pattern of protease activity observed in incubation2a can be divided into two phases: during the first phase, proteaseactivity was highly dependent on the type of the added N source. Inline with results obtained by Allison and Vitousek (2005) onlyproteins, not NH4

þ, induced protease synthesis. This substrate-induced phase lasted about 1 week for the readily available Na-caseinate, which is highly water soluble (Kinsella, 1984), and 2weeks for proteins with limited availability due to their waterinsolubility, such as gluten and zein (O’Neil et al., 2006). Substrateinduction of protease has been reported for several fungal (Hansonand Marzluf, 1975; Ogrydziak et al., 1977; Leake and Read, 1990)and bacterial species (Glenn, 1976; Beg et al., 2002). In contrast toproteins, NH4

þ is a directly available N source, which doesn’t have tobe broken down by extracellular enzymes.

Protease activity not only depended on the type, but also on theamount of protein added. In incubation 1, protease activity waspositively correlated with the amount of protein added. In addition,there was a positive relationship between protease activity and CO2

evolution. As the addition of NH4þ did not result in an increase on

CO2 evolution, the C mineralized most likely originated from theproteins added. Therefore, the protease synthesized degraded theadded proteins and provided the microorganisms with C and N.Interestingly, CO2 evolution per unit protease activity was muchhigher for gluten and zein than for Na-caseinate. This may be due tothe fact that the rate of hydrolysis is substrate specific. Studiescomparing properties of proteases synthesized by the samemicrobial species found that the proteases were able to hydrolysea range of different proteins, but that the rate of hydrolysis wasprotease and substrate specific (Allison and Macfarlane, 1992; Aokiet al., 1995; El-Shanshoury et al., 1995).

After 4 weeks of incubation, the levels of protease activityremained relatively constant. During this second phase, proteaseactivity was related to the C to N ratio of the amendments andtherefore, as the same amount of N was added at the beginning ofthe incubation, to the amount of C added. Protease activity was alsosignificantly correlated to microbial biomass N. This is in line withAsmar et al. (1992) who reported a correlation between proteaseactivity and bacterial biomass. It has been suggested that micro-organisms may constitutively produce and excrete low levels ofextracellular enzymes, even in the absence of substrate, in order toincrease the likelihood of some enzyme molecules being present inthe surrounding medium when a suitable substrate appears, suchas those released from lysed microbial cells (Burns, 1982). Thismechanism may have determined protease activity during thestationary phase in our incubation. Another possibility is thatprotease synthesis was induced by proteins released from deadmicroorganisms.

4.2. Effect of NH4þ and glucose on protease activity

When directly available C and N sources are abundant, theallocation of resources to protease production may be repressed.Several studies reported repression of protease synthesis by high

Table 6CO2 evolution, mineral N content and reducing sugar content four days after the application of DI water, glucose (C) or NH4Cl (N)

Treatment Injection CO2 evolution(mg C kg�1 dry soil)

Mineral N(mg N kg�1 dry soil)

Reducing sugars(mg glucose kg�1 dry soil)

Control Water 50.8 (1.30) 20.2 (0.52) 20.0 (4.25)C 234.4 (3.91) 9.4 (1.38) 17.7 (2.12)N 39.1 (1.30) 51.0 (0.64) 16.7 (2.18)C and N 242.2 (0.99) 27.7 (1.30) 19.0 (2.81)

C to N 10a Water 84.6 (2.60) 43.4 (4.52) 14.3 (3.90)C 239.6 (0.00) 27.2 (0.78) 19.8 (0.77)N 67.7 (2.60) 78.4 (3.44) 20.0 (3.48)C and N 238.3 (2.60) 56.2 (4.10) 22.2 (1.92)

C to N 25 Water 139.3 (2.60) 9.9 (1.57) 17.9 (2.41)C 289.0 (0.00) 7.4 (0.41) 18.8 (2.99)N 125.0 (5.21) 41.7 (1.98) 25.9 (2.03)C and N 308.6 (3.91) 25.1 (1.27) 22.2 (2.19)

C to N 40 Water 197.9 (0.00) 3.7 (0.25) 28.5 (4.42)C 317.7 (22.13) 3.7 (0.33) 29.4 (2.87)N 209.6 (3.91) 21.4 (1.40) 27.9 (4.41)C and N 334.6 (9.11) 17.2 (1.38) 30.2 (3.02)

Anova F-testsP

Control C <0.001 <0.001 1.000N 0.495 <0.001 0.738C�N 0.031 0.153 0.464

C to N 10 C <0.001 <0.001 0.517N 0.463 <0.001 0.098C�N 0.525 0.414 0.918

C to N 25 C <0.001 <0.001 0.569N 0.101 <0.001 0.037C�N 0.091 0.136 0.375

C to N 40 C <0.001 0.125 0.691N 0.049 <0.001 0.979C�N 0.638 0.158 0.852

Note: all values are means (for CO2 evolution n¼ 2; for mineral N and reducing sugars n¼ 4) with SE given in parenthesis. Data were analyzed as a two-way factorial for eachtreatment separately. Prior to the application, the samples, amended with Na-caseinate and cellulose, had been incubated for 48 days.a Ratio of the total C and N added in the form of Na-caseinate and cellulose at day 0.

D. Geisseler, W.R. Horwath / Soil Biology & Biochemistry 40 (2008) 3040–30483046

levels of NH4þ (Beg et al., 2002; Bascaran et al., 1990). In our study,

however, the addition of NH4Cl in combination with Na-caseinatedid not repress protease activity during the substrate-inducedphase (incubation 3a). In contrast, the addition of glucose asa directly available C source in combination with Na-caseinaterepressed protease activity slightly compared to the samples withNa-caseinate addition only. Glucose repression of protease activityhas been reported before (Ogrydziak et al., 1977; Beg et al., 2002).We hypothesize that the breakdown of Na-caseinate by proteasepresent in the soil at the beginning of the experiment provided themicroorganisms with a constant excess of N compared to C due to

Fig. 5. Effect of glucose (C) and NH4Cl (N) on protease activity when added 48 days af

Na-caseinate’s low C to N ratio of 3.6. Therefore, the increase inprotease synthesis was driven by the need for C rather than N,hence the repression by glucose. These results emphasize the closelinks between the microbially mediated cycles of organic C and N.

The glucose related reduction in protease activity of 7–18% wasrelatively small when compared to the fivefold increase thatresulted in response to the Na-caseinate addition. Substrateinduction of protease seems to have been the stronger signal thanrepression by glucose. In the presence of NH4

þ and glucose theallocation of C and N to protease synthesis seems to be uneco-nomical. However, in soils, where excess nutrient availability is

ter the addition of Na-caseinate and cellulose. Data shown are means� SE (n¼ 4).

D. Geisseler, W.R. Horwath / Soil Biology & Biochemistry 40 (2008) 3040–3048 3047

a rather rare event, being able to exploit a nutrient pool, such as theadded proteins, may be a more favorable strategy than saving C andN in situations when they are abundant.

4.3. Microbial adaptations to N limitations

During the second phase of incubation 2a, the large differencesin the mineral N content in the soil solution (predominantly NH4

þ)had no effect on protease activity. In addition, during this phase ofthe incubation the addition of NH4

þ as well as glucose had no effecton protease synthesis in the unamended control and the treat-ments with C to N ratios of 10 and 25. However, in the treatmentwith a C to N ratio of 40, the addition of glucose, NH4Cl and theircombination all decreased protease activity. The negative effect ofNH4þ may be explained as follows: the high proportion of the

additional N taken up by microorganisms in this treatmentsuggests that N availability was sub-optimal. Therefore, the level ofprotease activity before the addition of NH4Cl may have beendetermined by the need for N. The addition of NH4Cl reduced theneed for protease synthesis, hence the decrease in its activity.However, this doesn’t explain the negative effect of the glucoseaddition on protease activity. We hypothesize that this was causedby shifts in the NH4

þ assimilation pathway of the microorganisms.Nutrient limitations lead to metabolic adaptations in microor-

ganisms. A well-documented adaptation to N limitation is a shift inthe NH4

þ assimilation pathway from the low-affinity glutamatedehydrogenase (GDH) reaction to the high-affinity glutaminesynthetase/glutamate synthase (GS/GOGAT) pathway (Merrick andEdwards, 1995; Silberbach et al., 2005). While NH4

þ assimilation viathe GS/GOGAT pathway is more efficient at low NH4

þ concentra-tions, it requires more energy than the GDH pathway (Schmid et al.,2000; Silberbach et al., 2005). Another adaptation to N limitation isan increased synthesis of proteins involved in scavenging of alter-native N sources and protein degradation (Boer et al., 2003; Kolk-man et al., 2006). When N sources consist mainly of polymers,extracellular protease is produced at higher rates (Sinsabaugh et al.,1993; Sims and Wander, 2002). While these two strategies, the shiftin the pathway of NH4

þ assimilation and the production of extra-cellular enzymes, are complementary, they compete for the sameresources, namely C, energy and N. Which strategy is more favor-able may therefore depend on the availability of C and N. Assumingthat the shift to the GS/GOGAT pathway is the more energyintensive strategy, extracellular enzyme synthesis may be thepreferred adaptation when C and energy are scarce. This wouldexplain the decrease in protease activity in response to glucoseaddition. Glucose improved the energy status of the microbes,making the GS/GOGAT pathway more attractive, and leading toa decrease in protease activity.

4.4. Conclusions

There is increasing evidence that plants are able to take up N notonly in mineral form, but also in the form of simple organicmolecules (Schimel and Bennett, 2004). When organic N can betaken up by plants without being mineralized first, the depoly-merization of N containing organic compounds becomes thecentral link between decomposition and N uptake by plants(Schimel and Bennett, 2004). Due to the abundance of protein inorganic residues, protease plays a central role in providingbioavailable N.

Even though our study, carried out with one soil and coveringa relatively small part of the C to N ratios found in organic residues,cannot be seen as representative for soils in general, it revealedinteresting patterns between soil protease activity and C and Navailability: (1) the level of protease activity was regulated by boththe availability of C and N. (2) Substrate induction was dominant in

regulating protease activity. Changes in protease activity due tosubstrate induction dwarfed those caused by repression by directlyavailable C and N sources. (3) In contrast to pure culture studies, theavailability of NH4

þ in solution had little effect on the level ofprotease activity. (4) Microorganisms have developed multiplestrategies to respond to C and N limitations, one of which is theincreased synthesis of extracellular protease. Which strategy ismore favorable in a certain situation may depend on the availabilityof C and N. These results suggest that microorganisms regulateprotease synthesis depending on their needs for C and N. The levelof protease activity in turn directly affects the degradation rate ofproteins. This relationship seems to apply to other polymers andtheir corresponding enzymes as well (Carreiro et al., 2000).Therefore, the degradation rate of organic material is not onlya function of environmental conditions and substrate quality, asassumed in most decomposition models, but also of extracellularenzyme activity (Schimel and Weintraub, 2003).

More research is needed to fully understand the mechanismsregulating soil protease activity at the community level and the roleof protease in providing bioavailable N in soil ecosystems.

Acknowledgements

We would like to thank Patricia Lazicki and Tad Doane for theirhelp with laboratory analyses as well as Kate Scow, Randy Dahlgrenand two anonymous reviewers for their valuable comments on themanuscript. Funding and support were provided by the FertilizerResearch and Education Program of the California Department ofFood and Agriculture, the California State Water Resources ControlBoard and the Agricultural Experimental Station at UC Davis.

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