Soil Biology & Biochemistry 40 (2008) 30403048

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Soil Biology & Biochemistry

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Regulation of extracellular protease activity in soil in response to different sources and concentrations of nitrogen and carbon
Daniel Geisseler*, William R. Horwath
Department 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 2008 Received in revised form 19 August 2008 Accepted 1 September 2008 Available online 9 October 2008 Keywords: Extracellular enzymes Protease activity Microorganisms Carbon Nitrogen

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 breakdown requires the activity of extracellular proteases and other decomposing enzymes. The goal of our study was to better understand how carbon (C) and N availability affect soil protease activity. Several aerobic incubations were carried out with ammonium (NH) and proteins as N sources and cellulose as the main 4 C source. A strong increase in protease activity was observed when proteins were added, the increase depending on the amount of protein added and its solubility. Protease synthesis was clearly substrate induced, as NH had no effect. During this substrate induced phase, the addition of glucose but not NH 4 4 resulted in protease repression, indicating that the level of protease synthesis was determined by the need for C rather than N. After 1 month of incubation, protease activity remained relatively constant over time and was closely related to microbial biomass N. Different concentrations of mineral N in soil solution had no direct effect on protease activity. However, during this stationary phase, protease activity could be repressed by glucose and NH in a treatment with low mineral N content while in treatments 4 with a higher N availability no repression was observed. We hypothesize that the need for N determined protease activity in the treatment with limited N availability. The addition of NH allowed for reallo4 cation of C and N away from protease synthesis, leading to the observed decrease in protease activity. The repression by glucose may be attributed to shifts in the pathway of microbial NH assimilation. The 4 results 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-derived elements essential for plant growth because of its complex biogeochemistry and the large amounts required by plants in comparison to other elements. In most terrestrial ecosystems N is the limiting nutrient for plant growth (Robertson and Groffman, 2007). More than 90% of the total N in soils occurs in the form of organic N compounds (Foth and Ellis, 1997), most of which are not directly available to plants. In living organisms most of the organic N is in the form of proteins (Kogel-Knaber, 2006). Despite being labile compounds with high turnover rates, proteins from plant and microbial tissues comprise a large part of soil N (Ladd and Jackson, 1982). It has been estimated that about 40% of the total soil N is proteinaceous material, including proteins, glycoproteins, peptides and amino acids (Sowden et al., 1977; Schulten and Schnitzer, 1998). Soil proteins are hydrolyzed to peptides and amino acids by extracellular proteases. Proteases are produced by a wide range of bacteria,
* Corresponding author. Tel.: 1 530 754 8226; fax: 1 530 752 1552. E-mail address: djgeisseler@ucdavis.edu (D. Geisseler). 0038-0717/$ see front matter 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.soilbio.2008.09.001

actinomycetes and fungi (Kumar and Takagi, 1999; Glenn, 1976) and usually have a wide substrate-specicity, degrading most nonstructural proteins (Kalisz, 1988). Due to the abundance of proteins and peptides in soil, proteases likely supply a large part of the bioavailable N. Enzyme production is N and energy intensive. Microbes that regulate enzyme production according to their needs and the availability of substrates should have a competitive advantage (Koch, 1985). Four different mechanisms have been described to regulate the synthesis and secretion of extracellular protease (Kalisz, 1988). (1) The presence of a substrate can induce protease secretion. (2) High levels of end products, such as amino acids, NH 4 and easily metabolizable sources of C may repress production. (3) On the other hand, protease production may be increased (derepressed) when insufcient levels of C, N or sulfur (S) are available. (4) Finally, extracellular enzymes may be secreted constitutively at low levels regardless of the availability of a substrate. The factors regulating protease activity have been studied extensively with single bacterial and fungal species (Glenn, 1976; Kumar and Takagi, 1999). Much less information is available about how differences in C and N availability affect protease activity in soil, with its complex microbial community. In inoculated sand

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cultures amended with dened nutrient media, Sims and Wander (2002) found evidence for the de-repression mechanism. The microbial community responded to limitations of N and S by increasing proteolytic activity and reducing biomass compared to the control. C limitation had no effect on proteolytic activity. Allison and Vitousek (2005) found that glycine aminopeptidase was induced by collagen addition to soil, but signicantly repressed by NH. Asmar et al. (1992) found that protease activity was increased 4 in soil samples amended with nutrients and glucose. In their study, protease activity was correlated with ATP content, respiration and bacterial biomass. Extracellular enzymes often catalyze the rate-limiting step of decomposition and nutrient cycling (Sinsabaugh, 1994), making their expression and kinetics potential useful parameters for nutrient turnover models. Several authors have presented conceptual models using enzyme activity to describe the acquisition of C and N by microbes (Sinsabaugh and Moorhead, 1994; Schimel and Weintraub, 2003; Allison, 2005). However, there is still little information available about the different feedback mechanisms and interaction of the various C and N pools that govern extracellular enzyme production in complex soil microbial communities. This study provides information on the inuence of C and N availability on protease expression. First, we determined the expression of protease based on amount and availability of proteins. Second, we studied the effect of C and N availability on soil protease activity over time. Third, we investigated the effects of non-polymeric C and N sources on soil protease activity. 2. Material and methods 2.1. Soil sampling Soil samples (015 cm) were collected in late fall 2006 and spring 2007 at the Long-Term Research on Agricultural Systems (LTRAS) site west of Davis, CA. The soil is a loam of the Rincon series classied as ne, smectitic, thermic Mollic Haploxeralf (Soil Survey Staff, 1997). The soil is under permanent turf grass cover and was preferred to a soil under row crops to eliminate possible effects of tillage 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 incubations within 24 h of collection. Selected soil properties are summarized in Table 1. 2.2. Soil incubations Field moist soil (68 g) was weighed into 50 ml centrifuge tubes. Amendments were either applied dissolved in DI water (ammonium chloride [NH4Cl], ammonium sulfate [(NH4)2SO4], Nacaseinate, glucose), or as a powder well mixed with the soil

Table 1 Selected properties of the soil samples used for the incubations Soil property pH EC Texture Total C Value 7.4 88 mS cm1 37% sand, 39% silt, 24% clay 13.0 g total C kg1 dry soil 1.18 g total N kg1 dry soil Method 1:1 soil:water (Thomas, 1996) 1:1 soil:water (Rhoades, 1996) Pipet method (Gee and Bauder, 1986) Dry combustion using a Carlo Erba CNS analyzer NA 1500 series 2 (Nelson and Sommers, 1996) Dry combustion using a Carlo Erba CNS analyzer NA 1500 series 2 (Bremner, 1996)

Total N

(cellulose, gluten, zein). The samples were incubated at a moisture content of 28% (g g1), which corresponds to 50% of the water holding capacity determined by the funnel method. During the incubations the centrifuge tubes containing the samples were kept in glass jars sealed with lids equipped with rubber septa for gas sampling. Incubation 1 was carried out to determine the expression of protease based on the amount and availability of proteins (objective 1). NH4Cl, Na-caseinate (from bovine milk), gluten (from wheat) or zein (from corn) were used as N sources (Table 2). Soil samples were amended with 0.05, 0.1, 0.15, or 0.2 mg N g1 dry soil. After 2 days, total carbon dioxide (CO2) production and protease activity were measured (see details in Section 2.3). To investigate effects of varying C and N availability on protease activity over time (objective 2), soil samples were amended with cellulose and different N sources. In incubation 2a, the amount of N was the same for all treatments and the cellulose addition was adjusted to obtain different C to N ratios of the amendments. For incubation 2b, the cellulose addition was kept constant, while the amount of N added was adjusted (Table 2). In incubation 2a, 0.1 mg N g1 dry soil of (NH4)2SO4, Nacaseinate, gluten and zein were added to separate samples. Different amounts of cellulose were added to the samples to attain C to N ratios of the amendment of 10, 25 and 40 (Table 2). These three C to N ratios were chosen because residues that have C to N ratio greater than about 30 result in net N immobilization, while residues with a C to N ratio below 20 normally lead to net N mineralization (Stevenson, 1986). Unamended samples served as control. After 3, 7, 14, 28, 49, and 70 days, four replicates per treatment were destructively sampled to determine protease activity, NH, nitrate (NO), and microbial biomass N. In addition, 4 3 CO2 evolution was measured frequently so that the CO2 concentration did not exceed 1.5% in the headspace of the glass jars in which the samples were kept. Incubation 2b contained the same C and N sources as incubation 2a but the amount of cellulose added was kept constant at 5 mg g1 dry soil while the addition of N was adjusted to obtain C to N ratios of 10 and 40. Two controls were included in the experiment, one without any amendments and a second with cellulose as the sole amendment. After 59 days, three replicates per treatment were analyzed for protease activity (two subsamples per replicate), mineral N (NH and NO), and microbial 4 3 biomass N. CO2 evolution was measured periodically (see below). Incubations 2a and 2b were set up as a two-way factorial with N source as one factor and the C to N ratio of the amendments as the second factor. Finally, two incubations (3a and 3b) were carried out to investigate the effects of non-polymeric C and N sources, namely glucose and NH, on soil protease activity (objective 3). In incubation 3a the 4 effect of glucose and NH on protease activity was studied 4 following the addition of protein, when the level of protease activity was determined by the protein added. In incubation 3b glucose and NH were added several weeks after the addition of 4 protein, when the initial protein addition was no longer the dominant factor determining protease activity. For incubation 3a, soil samples were amended with Nasoil), NH4Cl (0, 0.02, or caseinate (0.1 mg N g1 dry 0.04 mg N g1 dry soil) and glucose (0, 0.2, or 0.4 mg C g1 dry soil). After 1, 2, 3, 5 and 8 days the samples were analyzed for protease activity, mineral N, reducing sugars, and CO2 evolution (see below). An unamended sample served as control (Table 2). For incubation 3b, soil samples were amended with Na-caseinate as N source (0.1 mg N g1 dry soil) and cellulose as C source (varying amounts to reach C to N ratios of 10, 25 and 40). These treatments were identical to the Na-caseinate amended treatments in incubation 2a. After 48 days of incubation, however, NH4Cl (0 or 0.04 mg N g1 dry soil) and glucose (0 or 0.4 mg N g1 dry soil) were added. The

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Table 2 Overview of the substrates used for the different incubations Incubation N sources Main C source None Cellulose Cellulose None Cellulose N added (mg g1 dry soil) 0.05, 0.1, 0.15, 0.2 0.1 0.21, 0.05 0.1 0.1 C added with cellulose (mg g1 dry soil) 0 1, 2.5, 4 2.1 0 1, 2.5, 4 C to N ratiosb (mg g1 dry soil) 04.1 10, 25, 40 10, 40 3.6 10, 25, 40 NH4Cl and glucose added at day 0 NH4Cl and glucose added at day 48 Other additions

1 2aa 2b 3a 3b
a b

Na-caseinate, zein, gluten, NH4Cl, Na-caseinate, zein, gluten, (NH4)2SO4 Na-caseinate, zein, gluten, (NH4)2SO4 Na-caseinate Na-caseinate

The number refers to the objective addressed, the letter separates incubations carried out for the same objective. 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.

amendments were dissolved in 0.25 ml DI water and injected with a syringe. DI water alone was added to a control. Protease activity, mineral N, reducing sugars, and CO2 evolution were measured before 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 method described by Ladd and Butler (1972). Briey, 1 g of soil was weighed into a glass vial and 2.5 ml of Tris buffer (0.2 M, pH 8.0) and 2.5 ml of a 2% Na-caseinate solution (20 g l1 DI water) were added. The capped vials were incubated in a water bath at 50  C for 2 h. After the incubation, the remaining casein was precipitated with 5 ml 10% trichloroacetic acid. 1.5 ml of the solution was pipetted into a microcentrifuge tube and centrifuged at 13 000 rev min1 for 1 min. 0.5 ml of the clear supernatant was mixed with 0.75 ml Na2CO3 (1.4 M) and 0.25 ml threefold diluted FolinCiocalteu reagent in a microcuvette. The tyrosine concentration was measured colorimetrically at 680 nm after exactly 5 min. For the controls, the same procedure was followed, with the exception that the Na-caseinate was added after the incubation and the addition of the trichloroacetic acid. To measure CO2 evolution, a maximum of four centrifuge tubes, each containing 68 g of soil were placed into 0.95 L glass jars sealed with lids equipped with rubber septa for gas sampling. To each jar 30 ml of DI water was added to minimize evaporation from the soil samples during the incubation. The jars were kept in the dark for the duration of the incubation. A randomly selected subset of four jars per treatment was used for CO2 measurements. Headspace CO2 was analyzed with a Qubit CO2 analyzer (model S-151, Qubit Systems Inc., Kingston, Canada). After each analysis, the jars were opened and ushed with ambient air. A blank was used to correct for background CO2. The ideal gas law was applied to calculate the amount of organic C released based on the CO2 concentration (Zibilske, 1994). CO2 measurements were always taken before the CO2 in the headspace reached a concentration of 1.5% by volume. Soils were extracted with 0.5 M K2SO4 (5 ml g1 soil; Mulvaney, 1996) and the suspension ltered (Fisherbrand, Q5) for the analysis of NH, NO, total N in solution and reducing sugars. 4 3 NO was analyzed using a single reagent method (Doane and 3 Horwath, 2003). The NH concentration was determined using the 4 salicylate method (Verdouw et al., 1978; Foster, 1995). Reducing sugars were measured colorimetrically with an anthron reagent (Fales, 1951). A standard curve was prepared with glucose. Microbial biomass N was determined using the chloroform fumigation extraction method (Horwath and Paul, 1994) followed by the determination of total N in the extracts with the alkaline persulfate oxidation method (Cabrera and Beare, 1993). Dissolved organic 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 N content between the fumigated and unfumigated sample by 0.68 to account 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 at 105  C for 24 h. 2.4. Data analysis Statistical analyses were conducted with the SAS program (SAS Institute, 1990), using the general linear model (GLM) procedure for analysis of variance and the REG procedure for regression analyses. The assumptions of the statistical models were tested for every data set. Normality of the residuals was evaluated graphically and with the ShapiroWilk test. Homogeneity of variances was tested by plotting the residuals vs. the predicted values and with Levenes test. If necessary, the data were transformed. Mean comparisons were performed using the Tukey test, which controls the experimentwise type I error rate a (SAS Institute, 1990). Effects were considered signicant for P < 0.05. 3. Results 3.1. Relationship between protein degradation and protease activity Increasing additions of Na-caseinate, gluten and zein in incubation 1 resulted in linear increases in protease activity. In contrast, the addition of NH4Cl had no effect on protease activity. The evolution of CO2 was positively and linearly correlated to protease activity for all three proteins (P < 0.05; R2 > 0.78; Fig. 1). CO2 evolution was not correlated with NH4Cl additions (P 0.84; R2 0.02). CO2 evolution was much higher when Na-caseinate was added compared to gluten and zein, however, less CO2 was evolved per unit protease activity for Na-caseinate than the other proteins (Fig. 1). 3.2. Inuence of C and N availability on protease activity over time For incubation 2a, cellulose was added in increasing amounts together with (NH4)2SO4, Na-caseinate, gluten, or zein to formulate C to N ratios of 10, 25 and 40. The addition of proteins increased protease activity by at least 80% within 3 days (Fig. 2). In contrast, there was no increase in protease activity in the NH treatments 4 within the rst 2 weeks. As in incubation 1, Na-caseinate had the most pronounced effect on protease activity; however, protease activity decreased quickly after 3 days. In the treatments with gluten and zein, protease activity increased slowly, but the activity was maintained at an elevated level for a longer time.

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Fig. 1. Relationship between protease activity and CO2 evolution with different N sources after 2 days of incubation. A: Na-caseinate (R2 0.99; P < 0.001). B: gluten (R2 0.78; P 0.046), zein (R2 0.86; P 0.023) and NH4Cl (R2 0.02; P 0.84). Data shown are means (n 4) standard error of the means (SE). Some error bars for CO2 evolution are smaller than the icon.

Throughout the incubation, the N source continued to be a signicant factor determining protease activity (P for factor N source <0.001 throughout the incubation). In general, protease activity was highest in the samples with Na-caseinate and lowest in the samples amended with (NH4)2SO4. After the second week of incubation, higher C to N ratios resulted in higher protease activities (P for factor C to N ratio <0.001 for sampling dates 28, 49, and 70). This was not only the case in the protein amended treatments but also in the NH amended treatments where protease activity 4 increased signicantly in the treatments with a C to N ratio of 25 and 40. After the rst 4 weeks of incubation, protease activity remained relatively stable in all treatments and there was no signicant interaction between C to N ratio and protein source (P for day 49: 0.93; for day 70: 0.06). Differences in the C to N ratio not only affected protease activity but higher C to N ratios also increased microbial biomass N and respiration rate during the second half of the experiment (data not shown), due to the fact that higher C to N ratios were obtained by increasing the amount of C added. On the other hand, the mineral N content in soil solution decreased with increasing C to N ratio. In order to determine which of these factors were related to protease activity, incubation 2b was carried out with the same substrates. In contrast to incubation 2a, however, the cellulose addition was the same for all treatments but the amount of N added was adjusted to reach C to N ratios of 10 and 40. After 59 days of incubation, protease activity was higher in the protein amended treatments compared to the NH amended 4 treatments. There were no signicant differences between treatments amended with different proteins (Table 3). Over all treatments, the C to N ratio had no effect on protease activity (P 0.78).

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 g1 dry soil) and adjusted cellulose additions to result in C to N ratios of 10 (d:d), 25 (dAd), and 40 (d-d). Data shown are means SE (n 4).

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Table 3 Protease activity, microbial biomass N, CO2 evolution and mineral N in soil samples incubated for 59 days N source C to N ratio Protease activity (mg Tyrosine kg1 dry soil h1) 88.5 106.5 92.9 130.4 144.0 145.0 155.2 120.3 142.4 143.8 (12.93) a (1.47) ab (4.47) a (8.03) bc (7.30) c (5.29) c (1.10) c (2.99) abc (0.52) c (11.88) c Microbial biomass N (mg N kg1 dry soil) Cumulative CO2 evolution (mg C g1 dry soil) Days 032 55.6 66.8 61.6 65.5 83.7 81.4 79.4 81.6 79.8 83.3 (3.06) a (4.71) abc (2.30) ab (0.91) abc (7.15) c (2.05) c (4.04) bc (2.60) c (4.26) bc (1.14) c 585 925 1602 1339 2171 1516 2271 1607 2186 1623 (14) a (57) b (54) d (25) c (24) e (63) cd (73) e (15) d (45) e (46) d Days 3259 394 824 504 895 721 957 756 948 873 897 (29) a (95) c (116) ab (62) c (116) bc (75) c (76) c (17) c (48) c (106) c 20.5 0.7 73.3 4.2 62.4 5.1 61.5 3.2 74.6 4.3 (2.57) b (0.07) a (2.08) d (1.01) a (2.52) c (0.48) a (3.31) c (0.25) a (3.70) d (1.69) a NH and NO 4 3 (mg N kg1 dry soil)

Control Cellulose only (NH4)2SO4 (NH4)2SO4 Na-caseinate Na-caseinate Gluten Gluten Zein Zein

10 40 10 40 10 40 10 40

Note: all values are means (n 4) with SE given in parenthesis. Means followed by a different letter within each column are signicantly different at P < 0.05 according to Tukeys 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.

However, the higher C to N ratio signicantly increased protease activity in the NH amended treatment, but had the opposite effect 4 in the gluten amended treatment. The unamended control and the control with cellulose had low protease activities. In contrast to the protease activity, higher N additions resulted in signicantly increased mineral N contents. Compared to the differences between the two C to N ratios, the differences caused by the N sources were small. Protease activity and mineral N content were not correlated in this experiment (R2 0.01; P 0.74). A low C to N ratio signicantly increased CO2 evolution during the rst 30 days of the incubation (Table 3). However, after day 30 the CO2 evolution in these treatments dropped below the level of the treatments with higher C to N ratios. A regression analysis indicated a signicant but weak correlation between protease activity after 59 days of incubation and CO2 respired between days 30 and 59 (R2 0.46; P 0.03). In contrast to mineral N and CO2 evolution, microbial biomass N did not differ signicantly between the two C to N ratios, nor among the protein amended treatments. However, microbial biomass N was higher in the treatments where protein served as the N source compared to the NH amended treatments. A linear 4 regression analysis revealed a positive correlation between microbial biomass N and protease activity (R2 0.73; Fig. 3). Analysis of the data from incubation 2a showed a poor correlation between protease activity and microbial biomass N across all treatments and sampling dates (Table 4). However, when each sampling date was analyzed separately, a clear pattern emerged.

While the correlations between protease activity and microbial biomass N were weak for the rst three sampling dates, they improved considerable for sampling days 28, 49, and 70 (Table 4). While the slopes of the regression lines for these days were very similar, there were large differences between the intercepts due to differences in microbial biomass N. This was due to the fact that slight changes in microbial biomass N over time were not reected in 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 on protease activity, incubations 3a and 3b were conducted. In incubation 3a, glucose, NH4Cl or both together were added to soil samples with Na-caseinate. The addition of glucose lowered mineral N levels and increased CO2 evolution signicantly (Table 5). The positive effect on respiration was most pronounced during the rst day but persisted until day 8. In contrast, the addition of NH4Cl increased mineral N levels and lowered CO2 evolution. In all treatments, except in the unamended control, protease activity increased sharply for 2 days and decreased until day 8 to about 50% of its peak (Fig. 4). The addition of glucose signicantly reduced protease activity by about 13% during the rst 2 days. The addition of NH4Cl did not repress protease activity, but resulted in a slightly less steep decrease after the maximum activity was reached. These differences were small compared to the vefold increase during the rst 2 days. In incubation 3b, glucose and NH4Cl were added to soil samples that 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 mineral N concentration was already very low before the glucose addition (Table 6). After 4 days, less than 1% of the glucose added was left in solution.

Table 4 Correlation between protease activity and microbial biomass N during an incubation with four different N sources and three different C to N ratios Sampling day 3 7 14 28 49 70 3-70 28-70 n 14 14 14 14 14 14 84 42 Slope 3.70 0.86 2.35 2.24 2.56 2.51 1.51 1.49 Intercept 194 114 47 32 40 10 55 71 R2 0.48 0.04 0.51 0.72 0.78 0.84 0.28 0.42 P 0.006 0.498 0.004 <0.001 <0.001 <0.001 <0.001 <0.001

Fig. 3. Relationship between protease activity and microbial biomass N after 59 days of incubation (R2 0.73; P 0.002). Each treatment (except the control) received the same amount of cellulose, but different amounts of N. Data shown are means SE (n 3).

Two controls were included per sampling date.

D. Geisseler, W.R. Horwath / Soil Biology & Biochemistry 40 (2008) 30403048 Table 5 Effect of directly available C and N on cumulative CO2 evolution, and N mineralized during an 8-day incubation with Na-caseinate, glucose and NH4Cl added at day 0 C added (mg/kg dry soil) 0 200 400 0 200 400 0 200 400 ANOVA F-tests C N CN P 0.022 0.005 0.652 <0.001 <0.001 0.227 N added (mg/kg dry soil) 0 0 0 20 20 20 40 40 40 Cumulative CO2 evolution (mg C/kg dry soil) 531 (18.2) 590 (0.7) 706 (22.2) 517 (10.2) 552 (16.7) 653 (2.9) 496 (38.8) 576 (41.5) 667 (23.4) N mineralized (mg N/kg dry soil) 16.3 (0.11) 12.8 (0.49) 9.7 (0.31) 20.8 (0.36) 18.4 (0.93) 14.4 (0.47) 23.7 (0.27) 19.8 (0.98) 17.1 (1.47)

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4. Discussion The production of extracellular protease by microorganisms is regulated by different mechanisms. Most of the research investigating the regulation of protease activity has been carried out with pure cultures. The species studied and the conditions of the experiment may or may not be representative for complex microbial communities found in soil. We investigated the response of soil microbial communities to different availabilities of C and N with several aerobic soil incubations. 4.1. Substrate induced protease synthesis The temporal pattern of protease activity observed in incubation 2a can be divided into two phases: during the rst phase, protease activity was highly dependent on the type of the added N source. In line with results obtained by Allison and Vitousek (2005) only proteins, not NH, induced protease synthesis. This substrate4 induced phase lasted about 1 week for the readily available Nacaseinate, which is highly water soluble (Kinsella, 1984), and 2 weeks for proteins with limited availability due to their water insolubility, such as gluten and zein (ONeil et al., 2006). Substrate induction of protease has been reported for several fungal (Hanson and Marzluf, 1975; Ogrydziak et al., 1977; Leake and Read, 1990) and bacterial species (Glenn, 1976; Beg et al., 2002). In contrast to proteins, NH is a directly available N source, which doesnt have to 4 be broken down by extracellular enzymes. Protease activity not only depended on the type, but also on the amount of protein added. In incubation 1, protease activity was positively correlated with the amount of protein added. In addition, there was a positive relationship between protease activity and CO2 evolution. As the addition of NH did not result in an increase on 4 CO2 evolution, the C mineralized most likely originated from the proteins added. Therefore, the protease synthesized degraded the added proteins and provided the microorganisms with C and N. Interestingly, CO2 evolution per unit protease activity was much higher for gluten and zein than for Na-caseinate. This may be due to the fact that the rate of hydrolysis is substrate specic. Studies comparing properties of proteases synthesized by the same microbial species found that the proteases were able to hydrolyse a range of different proteins, but that the rate of hydrolysis was protease and substrate specic (Allison and Macfarlane, 1992; Aoki et al., 1995; El-Shanshoury et al., 1995). After 4 weeks of incubation, the levels of protease activity remained relatively constant. During this second phase, protease activity was related to the C to N ratio of the amendments and therefore, as the same amount of N was added at the beginning of the incubation, to the amount of C added. Protease activity was also signicantly correlated to microbial biomass N. This is in line with Asmar et al. (1992) who reported a correlation between protease activity and bacterial biomass. It has been suggested that microorganisms may constitutively produce and excrete low levels of extracellular enzymes, even in the absence of substrate, in order to increase the likelihood of some enzyme molecules being present in the surrounding medium when a suitable substrate appears, such as those released from lysed microbial cells (Burns, 1982). This mechanism may have determined protease activity during the stationary phase in our incubation. Another possibility is that protease synthesis was induced by proteins released from dead microorganisms. 4.2. Effect of NH and glucose on protease activity 4

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

The addition of NH4Cl had no signicant effect on CO2 evolution in 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 N ratio of 40. When NH4Cl was added without glucose, more than 75% of the added NH remained in solution after 4 days in the control 4 and the treatments with C to N ratios of 10 and 25, but less than half was left in solution in the treatment with a C to N ratio of 40. In general, the combined addition of glucose and NH4Cl increased the proportion of N taken up. The additions of glucose and NH4Cl had no signicant effect on protease activity after 2 days (data not shown). After 4 days, however, protease activity had decreased by 20, 13 and 16% in the treatment with a C to N ratio of 40 and addition of glucose, NH4Cl or their combination, respectively. The decrease caused by glucose was signicant at the 5% level (Fig. 5). In the other treatments, glucose and NH4Cl had no signicant effect on protease activity.

Fig. 4. Effect of glucose and NH4Cl on protease activity when added together with Nacaseinate. Data shown are means SE (n 4). The lower line is the unamended control.

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

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Table 6 CO2 evolution, mineral N content and reducing sugar content four days after the application of DI water, glucose (C) or NH4Cl (N) Treatment Control Injection Water C N C and N Water C N C and N Water C N C and N Water C N C and N CO2 evolution (mg C kg1 dry soil) 50.8 (1.30) 234.4 (3.91) 39.1 (1.30) 242.2 (0.99) 84.6 (2.60) 239.6 (0.00) 67.7 (2.60) 238.3 (2.60) 139.3 (2.60) 289.0 (0.00) 125.0 (5.21) 308.6 (3.91) 197.9 (0.00) 317.7 (22.13) 209.6 (3.91) 334.6 (9.11) Mineral N (mg N kg1 dry soil) 20.2 (0.52) 9.4 (1.38) 51.0 (0.64) 27.7 (1.30) 43.4 (4.52) 27.2 (0.78) 78.4 (3.44) 56.2 (4.10) 9.9 (1.57) 7.4 (0.41) 41.7 (1.98) 25.1 (1.27) 3.7 (0.25) 3.7 (0.33) 21.4 (1.40) 17.2 (1.38) P <0.001 <0.001 0.153 <0.001 <0.001 0.414 <0.001 <0.001 0.136 0.125 <0.001 0.158 Reducing sugars (mg glucose kg1 dry soil) 20.0 (4.25) 17.7 (2.12) 16.7 (2.18) 19.0 (2.81) 14.3 (3.90) 19.8 (0.77) 20.0 (3.48) 22.2 (1.92) 17.9 (2.41) 18.8 (2.99) 25.9 (2.03) 22.2 (2.19) 28.5 (4.42) 29.4 (2.87) 27.9 (4.41) 30.2 (3.02)

C to N 10a

C to N 25

C to N 40

Anova F-tests Control C N CN C N CN C N CN C N CN <0.001 0.495 0.031 <0.001 0.463 0.525 <0.001 0.101 0.091 <0.001 0.049 0.638 1.000 0.738 0.464 0.517 0.098 0.918 0.569 0.037 0.375 0.691 0.979 0.852

C to N 10

C to N 25

C to N 40

Note: all values are means (for CO2 evolution n 2; for mineral N and reducing sugars n 4) with SE given in parenthesis. Data were analyzed as a two-way factorial for each treatment 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.

levels of NH (Beg et al., 2002; Bascaran et al., 1990). In our study, 4 however, the addition of NH4Cl in combination with Na-caseinate did not repress protease activity during the substrate-induced phase (incubation 3a). In contrast, the addition of glucose as a directly available C source in combination with Na-caseinate repressed protease activity slightly compared to the samples with Na-caseinate addition only. Glucose repression of protease activity has been reported before (Ogrydziak et al., 1977; Beg et al., 2002). We hypothesize that the breakdown of Na-caseinate by protease present in the soil at the beginning of the experiment provided the microorganisms with a constant excess of N compared to C due to

Na-caseinates low C to N ratio of 3.6. Therefore, the increase in protease synthesis was driven by the need for C rather than N, hence the repression by glucose. These results emphasize the close links between the microbially mediated cycles of organic C and N. The glucose related reduction in protease activity of 718% was relatively small when compared to the vefold increase that resulted in response to the Na-caseinate addition. Substrate induction of protease seems to have been the stronger signal than repression by glucose. In the presence of NH and glucose the 4 allocation of C and N to protease synthesis seems to be uneconomical. However, in soils, where excess nutrient availability is

Fig. 5. Effect of glucose (C) and NH4Cl (N) on protease activity when added 48 days after the addition of Na-caseinate and cellulose. Data shown are means SE (n 4).

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a rather rare event, being able to exploit a nutrient pool, such as the added proteins, may be a more favorable strategy than saving C and N in situations when they are abundant. 4.3. Microbial adaptations to N limitations During the second phase of incubation 2a, the large differences in the mineral N content in the soil solution (predominantly NH) 4 had no effect on protease activity. In addition, during this phase of the incubation the addition of NH as well as glucose had no effect 4 on protease synthesis in the unamended control and the treatments with C to N ratios of 10 and 25. However, in the treatment with a C to N ratio of 40, the addition of glucose, NH4Cl and their combination all decreased protease activity. The negative effect of NH may be explained as follows: the high proportion of the 4 additional N taken up by microorganisms in this treatment suggests that N availability was sub-optimal. Therefore, the level of protease activity before the addition of NH4Cl may have been determined by the need for N. The addition of NH4Cl reduced the need for protease synthesis, hence the decrease in its activity. However, this doesnt explain the negative effect of the glucose addition on protease activity. We hypothesize that this was caused by shifts in the NH assimilation pathway of the microorganisms. 4 Nutrient limitations lead to metabolic adaptations in microorganisms. A well-documented adaptation to N limitation is a shift in the NH assimilation pathway from the low-afnity glutamate 4 dehydrogenase (GDH) reaction to the high-afnity glutamine synthetase/glutamate synthase (GS/GOGAT) pathway (Merrick and Edwards, 1995; Silberbach et al., 2005). While NH assimilation via 4 the GS/GOGAT pathway is more efcient at low NH concentra4 tions, it requires more energy than the GDH pathway (Schmid et al., 2000; Silberbach et al., 2005). Another adaptation to N limitation is an increased synthesis of proteins involved in scavenging of alternative N sources and protein degradation (Boer et al., 2003; Kolkman 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 shift in the pathway of NH assimilation and the production of extra4 cellular enzymes, are complementary, they compete for the same resources, namely C, energy and N. Which strategy is more favorable may therefore depend on the availability of C and N. Assuming that the shift to the GS/GOGAT pathway is the more energy intensive strategy, extracellular enzyme synthesis may be the preferred adaptation when C and energy are scarce. This would explain the decrease in protease activity in response to glucose addition. Glucose improved the energy status of the microbes, making the GS/GOGAT pathway more attractive, and leading to a decrease in protease activity. 4.4. Conclusions There is increasing evidence that plants are able to take up N not only in mineral form, but also in the form of simple organic molecules (Schimel and Bennett, 2004). When organic N can be taken up by plants without being mineralized rst, the depolymerization of N containing organic compounds becomes the central link between decomposition and N uptake by plants (Schimel and Bennett, 2004). Due to the abundance of protein in organic residues, protease plays a central role in providing bioavailable N. Even though our study, carried out with one soil and covering a relatively small part of the C to N ratios found in organic residues, cannot be seen as representative for soils in general, it revealed interesting patterns between soil protease activity and C and N availability: (1) the level of protease activity was regulated by both the availability of C and N. (2) Substrate induction was dominant in

regulating protease activity. Changes in protease activity due to substrate induction dwarfed those caused by repression by directly available C and N sources. (3) In contrast to pure culture studies, the availability of NH in solution had little effect on the level of 4 protease activity. (4) Microorganisms have developed multiple strategies to respond to C and N limitations, one of which is the increased synthesis of extracellular protease. Which strategy is more favorable in a certain situation may depend on the availability of C and N. These results suggest that microorganisms regulate protease synthesis depending on their needs for C and N. The level of protease activity in turn directly affects the degradation rate of proteins. This relationship seems to apply to other polymers and their corresponding enzymes as well (Carreiro et al., 2000). Therefore, the degradation rate of organic material is not only a function of environmental conditions and substrate quality, as assumed in most decomposition models, but also of extracellular enzyme activity (Schimel and Weintraub, 2003). More research is needed to fully understand the mechanisms regulating soil protease activity at the community level and the role of protease in providing bioavailable N in soil ecosystems. Acknowledgements We would like to thank Patricia Lazicki and Tad Doane for their help with laboratory analyses as well as Kate Scow, Randy Dahlgren and two anonymous reviewers for their valuable comments on the manuscript. Funding and support were provided by the Fertilizer Research and Education Program of the California Department of Food and Agriculture, the California State Water Resources Control Board and the Agricultural Experimental Station at UC Davis. References
Allison, C., Macfarlane, G.T., 1992. Physiological and nutritional determinants of protease secretion by Clostridium sporogenes: characterization of six extracellular proteases. Applied Microbiology & Biotechnology 37, 152156. Allison, S.D., 2005. Cheaters, diffusion and nutrients constrain decomposition by microbial enzymes in spatially structured environments. Ecology Letters 8, 626635. Allison, S.D., Vitousek, P.M., 2005. Responses of extracellular enzymes to simple and complex nutrient inputs. Soil Biology & Biochemistry 37, 937944. Aoki, K., Miyamoto, K., Murakami, S., Shinke, R., 1995. Anaerobic synthesis of extracellular proteases by the soil bacterium Bacillus sp. AM-23: purication and characterization of the enzymes. Soil Biology & Biochemistry 27, 13771382. Asmar, F., Eiland, F., Nielsen, N.E., 1992. Interrelationship between extracellular enzyme activity, ATP content, total counts of bacteria and CO2 evolution. Biology & Fertility of Soils 14, 288292. Bascaran, V., Hardisson, C., Brana, A.F., 1990. Regulation of extracellular protease production in Streptomyces clavuligerus. Applied Microbiology & Biotechnology 34, 208213. Beg, Q.K., Saxena, R.K., Gupta, R., 2002. De-repression and subsequent induction of protease synthesis by Bacillus mojavensis under fed-batch operations. Process Biochemistry 27, 11031109. Boer, V.M., de Winde, J.H., Pronk, J.T., Piper, M.D.W., 2003. The genome-wide transcriptional response of Saccharomyces cerevisiae grown on glucose in aerobic chemostat cultures limited for carbon, nitrogen, phosphorus, or sulfur. Journal of Biological Chemistry 278, 32653274. Bremner, J.M., 1996. Nitrogen-total. In: Sparks, D.L. (Ed.), Methods of Soil Analysis. Part 3. Chemical Methods. Soil Science Society of America, American Society of Agronomy, Madison, WI, pp. 10851121. Burns, R.G., 1982. Enzyme activity in soil: location and a possible role in microbial ecology. Soil Biology & Biochemistry 14, 423427. Cabrera, M.L., Beare, M.H., 1993. Alkaline persulfate oxidation for determining total nitrogen in microbial biomass extracts. Soil Science Society of America Journal 57, 10071012. Carreiro, M.M., Sinsabaugh, R.L., Repert, D.A., Parkhurst, D.F., 2000. Microbial enzyme shifts explain litter decay responses to simulated nitrogen deposition. Ecology 81, 23592365. Doane, T.A., Horwath, W.R., 2003. Spectrophotometric determination of nitrate with a single reagent. Analytical Letters 36, 27132722. El-Shanshoury, A.R., El-Sayed, M.A., Sammour, R.H., El-Shouny, W.A., 1995. Purication and partial characterization of two extracellular alkaline proteases from Streptomyces corchorusii ST36. Canadian Journal of Microbiology 41, 99104. Fales, F.W., 1951. The assimilation and degradation of carbohydrates by yeast cells. Journal of Biological Chemistry 193, 113124.

3048

D. Geisseler, W.R. Horwath / Soil Biology & Biochemistry 40 (2008) 30403048 ONeil, M.J., Heckelman, P.E., Koch, C.B., Roman, K.J. (Eds.), 2006. The Merck Index: An Encyclopedia of Chemicals, Drugs, and Biologicals, Fourteenth Edition. Merck & Co., Inc., Whitehouse Station, NJ, 2564 pp. Rhoades, J.D., 1996. Salinity: electrical conductivity and total dissolved solids. In: Sparks, D.L. (Ed.), Methods of Soil Analysis. Part 3. Chemical Methods. Soil Science Society of America, American Society of Agronomy, Madison, WI, pp. 417435. Robertson, G.P., Groffman, P.M., 2007. Nitrogen transformations. In: Paul, E.A. (Ed.), Soil Microbiology, Ecology, and Biochemistry. Academic Press, Bulington, MA, pp. 341364. SAS Institute, 1990. SAS/STAT Users Guide, Version 6. SAS Institute, Inc., Cary, NC, 1848 pp. Schimel, J.P., Bennett, J., 2004. Nitrogen mineralization: challenges of a changing paradigm. Ecology 85, 591602. Schimel, J.P., Weintraub, M.N., 2003. The implications of exoenzyme activity on microbial carbon and nitrogen limitation in soil: a theoretical model. Soil Biology & Biochemistry 35, 549563. Schmid, R., Uhlemann, E.-M., Nolden, L., Wersch, G., Hecker, R., Hermann, T., Marx, A., Burkovski, A., 2000. Response to nitrogen starvation in Corynebacterium glutamicum. FEMS Microbiology Letters 187, 8388. Schulten, H.-R., Schnitzer, M., 1998. The chemistry of soil organic nitrogen: a review. Biology & Fertility of Soils 26, 115. Silberbach, M., Schafer, M., Huser, A.T., Kalinowski, J., Puhler, A., Kramer, R., Burkovski, A., 2005. Adaptation of Corynebacterium glutamicum to ammonium limitation: a global analysis using transcritome and proteome techniques. Applied & Environmental Microbiology 71, 23912402. Sims, G.K., Wander, M.M., 2002. Proteolytic activity under nitrogen or sulfur limitation. Applied Soil Ecology 19, 217221. Sinsabaugh, R.L., 1994. Enzymatic analysis of microbial pattern and processes. Biology & Fertility of Soils 17, 6974. Sinsabaugh, R.L., Antibus, R.K., Linkins, A.E., McClaugherty, C.A., Rayburn, L., Repert, D., Weiland, T., 1993. Wood decomposition: nitrogen and phosphorus dynamics in relation to extracellular enzyme activity. Ecology 74, 15861593. Sinsabaugh, R.L., Moorhead, D.L., 1994. Resource allocation to extracellular enzyme production: a model for nitrogen and phosphorus control of litter decomposition. Soil Biology & Biochemistry 26, 13051311. Soil Survey Staff, 1997. Ofcial Soil Series Descriptions Available from: http://soils. usda.gov/technical/classication/osd/index.html (accessed 23.10.07.). Sowden, F.J., Chen, Y., Schnitzer, M., 1977. The nitrogen distribution in soils formed under widely differing climatic conditions. Geochimica et Cosmochimica Acta 41, 15241526. Stevenson, F.J., 1986. Cycles of Soil. John Wiley & Sons, New York, NY, 380 pp. Thomas, G.W., 1996. Soil pH and soil acidity. In: Sparks, D.L. (Ed.), Methods of Soil Analysis. Part 3. Chemical Methods. Soil Science Society of America, American Society of Agronomy, Madison, WI, pp. 475490. Verdouw, H., van Echteld, C.J.A., Dekkers, E.M.J., 1978. Ammonium determination based on indophenol formation with sodium salicilate. Water Research 12, 399402. Zibilske, L.M., 1994. Carbon mineralization. In: Weaver, R.W., Angle, S., Bottomley, P., Bezdiecek, D. (Eds.), Methods of Soil Analysis. Part 2. Microbiological and Biochemical Properties. Soil Science Society of America, Madison, WI, pp. 851863.

Foster, J.C., 1995. Soil nitrogen. In: Alef, K., Nannipieri, P. (Eds.), Methods in Applied Soil Microbiology & Biochemistry. Academic Press, San Diego, CA, pp. 7987. Foth, H.D., Ellis, B.G., 1997. Soil Fertility. CRC Press, Boca Raton, FL, 290 pp. Gee, G.W., Bauder, J.W., 1986. Particle-size analysis. In: Klute, A. (Ed.), Methods of Soil Analysis. Part 1. Physical and Mineralogical Methods. American Society of Agronomy, Soil Science Society of America, Madison, WI, pp. 383411. Glenn, A.R., 1976. Production of extracellular proteins by bacteria. Annual Review of Microbiology 30, 4162. Hanson, M.A., Marzluf, G.A., 1975. Control of the synthesis of a single enzyme by multiple regulatory circuits in Neurospora crassa. Proceedings of the National Academy of Science of the United States of America 72, 12401244. Horwath, W.R., Paul, E.A., 1994. Microbial biomass. In: Weaver, R.W., Angle, S., Bottomley, P., Bezdiecek, D. (Eds.), Methods of Soil Analysis. Part 2. Microbiological and Biochemical Properties. Soil Science Society of America, Madison, WI, pp. 753773. Kalisz, H.M., 1988. Microbial proteinases. Advances in Biochemical Engineering/ Biotechnology 36, 165. Kinsella, J.E., 1984. Milk proteins: physicochemical and functional properties. Critical Reviews in Food Science & Nutrition 21, 197262. Koch, A.L., 1985. The macroeconomics of bacterial growth. In: Fletcher, M., Floodgate, G.D. (Eds.), Bacteria in Their Natural Environments. Academic Press, London, pp. 142. Kogel-Knaber, I., 2006. Chemical structure of organic N and organic P in soil. In: Nannipieri, P., Smalla, K. (Eds.), Nucleic Acids and Proteins in Soil. Springer Verlag, Berlin Heidelberg, pp. 2348. Kolkman, A., Daran-Lapujade, P., Fullaondo, A., Olsthoorn, M.M.A., Pronk, J.T., Slijper, M., Heck, A.J.R., 2006. Proteome analysis of yeast to various nutrient limitations. Molecular Systems Biology 2, 116. Kumar, C.G., Takagi, H., 1999. Microbial alkaline proteases: from a bioindustrial viewpoint. Biotechnology Advances 17, 561594. Ladd, J.N., Butler, J.H.A., 1972. Short-term assays of soil proteolytic enzyme activities using proteins and dipeptide derivatives as substrates. Soil Biology & Biochemistry 4, 1930. Ladd, J.N., Jackson, R.B., 1982. Biochemistry of ammonication. In: Stevenson, F.J. (Ed.), Nitrogen in Agricultural Soils. American Society of Agronomy, Crop Science Society of America, Soil Science Society of America, Madison, WI, pp. 173228. Leake, J.R., Read, D.J., 1990. Proteinase activity in mycorrhizal fungi. II. The effects of mineral and organic nitrogen sources on induction of extracellular proteinase in Hymenoscyphus ericae (Read) Korf & Kernan. New Phytologist 116, 123128. Merrick, M.J., Edwards, R.A., 1995. Nitrogen control in bacteria. Microbiological Reviews 59, 604622. Mulvaney, R.L., 1996. Nitrogen-inorganic forms. In: Sparks, D.L. (Ed.), Methods of Soil Analysis. Part 3. Chemical Methods. Soil Science Society of America, American Society of Agronomy, Madison, WI, pp. 11231184. Nelson, D.W., Sommers, L.E., 1996. Total carbon, organic carbon, and organic matter. In: Sparks, D.L. (Ed.), Methods of Soil Analysis. Part 3. Chemical Methods. Soil Science Society of America, American Society of Agronomy, Madison, WI, pp. 9611010. Ogrydziak, D.M., Demain, A.L., Tannenbaum, S.R., 1977. Regulation of extracellular protease production in Candida lipolytica. Biochimica et Biophysica Acta 497, 525538.