SOIL NITROGEN DYNAMICS IN RESPONSE TO CARBON

INCREASE IN A MEDITERRANEAN SHRUBLAND OF SW

SPAIN

ANTONIO GALLARDO1* and JOSEÂ MERINO2

1Area de EcologõÂ a, Facultad de Ciencias, Universidad de Vigo, Apdo. 874, 36200 Vigo, Spain and2Departamento de EcologõÂ a, Universidad de Sevilla, Apdo. 1095, 41080 Sevilla, Spain

(Accepted 3 November 1997)

SummaryÐMost models predict that high atmospheric CO2 concentrations will lead to an increase inthe C-to-N ratio of litter production in terrestrial ecosystems. The e�ect of an increase in the soil C-to-N ratio on the nitrogen dynamics in a Mediterranean shrubland was simulated by mixing with the litterlayer wood shavings with a high C-to-N ratio. Samples of mineral soil, taken subsequently eight timesduring 404 d, were analyzed for total C, total N, total soil carbohydrates, potential net N mineraliz-ation, potential net nitri®cation and microbial biomass-N. We found signi®cant increases in the concen-tration of total carbohydrates, C-to-N ratio and microbial biomass N in amended soils during theexperiment, while potential net N mineralization rate and net nitri®cation rate signi®cantly decreased;amounts of available nitrogen (NH4

+±N+NO3±N) were una�ected by the amendment treatment.However, by the end of the experiment, no signi®cant di�erences between amended and control soilsamples were found. The total carbohydrates-to-K2SO4-extractable total-N ratio was the best predictorof both net mineralization rate and microbial biomass N, showing that the available C-to-available-Nratio is a better indicator of N dynamics than the total C to total N ratio. Our results support the hy-pothesis that increasing C availability in soils leads to a decrease in N availability for plants throughthe immobilization of N in microbial biomass and to an increase in the temporal heterogeneity of soilproperties in a Mediterranean shrubland. # 1998 Elsevier Science Ltd. All rights reserved

INTRODUCTION

Nitrogen is the most limiting nutrient in many

plant communities (Vitousek et al., 1982; Vitousek

and Howarth, 1991). Several studies suggest that

the increase of atmospheric CO2 will a�ect below-

ground processes through its e�ects on plant carbon

allocation and tissue chemistry. Both leaf litter and

®ne root decomposition may decrease as a result of

the changes in the chemical composition of the lit-

ter. Chemical changes observed in leaves produced

under CO2 enrichment include increases in non-

structural C and secondary plant metabolites and

decreases in leaf N concentrations (Curtis et al.,

1989; KoÈ rner and Arnone III, 1992; Cipollini et al.,

1993; Owensby et al., 1993; KoÈ rner and Miglietta,

1994). Also N concentrations in roots are reduced,

and C-to-N ratios may increase, suggesting that ®ne

root decomposition rates may decline under CO2

enrichment, constraining the turnover of nutrients

for plant uptake (Curtis et al., 1990; Norby, 1994;

O'Neill, 1994).

These changes may alter N cycling and ecosystem

productivity. Higher C-to-N ratios in plant litter,

and higher C availability through root exudates,

will modify the sources of energy and nutrients for

the soil microbial community. Whether the response

of soil microbes to these indirect e�ects of rising at-

mospheric CO2 will lead to positive or negative

feedback in N availability for plants is still unclear.Although some models predict that the increase in

soil microbial biomass and activity with higher C

availability may result in higher N availability for

plants (Zak et al., 1993), other models hypothesize

that an increase in microbial biomass could lead to

decreased N availability, constraining the plant re-

sponse to elevated CO2 (DõÂ az et al., 1993). The re-

sponse of soil microbes to the increase in C-to-N

ratio in each plant community may depend on

whether they are limited by C availability, N avail-

ability or others factors (Schimel, 1986; Gallardoand Schlesinger, 1992, 1994).

In short-term experiments, extra C added to the

soil (usually as sugars), causes immobilization of

nutrients in microbial biomass (e.g. Gallardo and

Schlesinger, 1995). However, the response of soil

microbes to the addition of a labile source of C is

not conclusive, because labile C is not representa-tive of the complex C sources that reach the soil

and it may allow speci®c sugar-sensitive microor-

ganisms to outcompete the native microbial bio-

mass (Wardle and Parkinson, 1990).

Most of C entering the soil (specially in nutrient-

poor ecosystems), is part of the cell wall or second-

Soil Biol. Biochem. Vol. 30, No. 10/11, pp. 1349±1358, 1998# 1998 Elsevier Science Ltd. All rights reserved

Printed in Great Britain0038-0717/98 $19.00+0.00PII: S0038-0717(97)00265-4

*Author for correspondence.

1349

ary compounds, such as tannins. These moleculesare slow to decompose, and can immobilize N in

recalcitrant compounds (Gallardo and Merino,1992). Some secondary compounds, such as tannins,have antimicrobial properties and high concen-

trations in soils may inhibit the activity of soilmicroorganisms (Scalbert, 1991). Secondary com-pounds are expected to increase at high concen-

trations of CO2. in nutrient-poor ecosystems(Bryant et al., 1983; Lambers, 1993).In the Mediterranean shrublands of SW Spain,

the C input to the soil system is markedly seasonal,but the leaf litter decomposition rate appears to befairly constant through the year (Merino et al.,1990, Gallardo and Merino, 1993). Extra C incor-

porated in the soil may increase temporal hetero-geneity of N dynamics, depending on how fast theC is respired by microorganisms and whether the

C-to-N ratio of the soil returns to the original levelor reaches a new, higher equilibrium value.We have studied the e�ect of an increase in the

litter soil C-to-N ratio on N dynamics and temporalheterogeneity in the mineral soil of a Mediterraneanshrubland of SW Spain. The litter was amended

with shavings of wood which has a high C-to-Nratio and is composed mainly of holocellulose, lig-nin and polyphenols.

METHODS

Area of study

Our study was conducted in DonÄ ana Biological

Reserve, southwest Spain (3787' N, 6812' W). Thearea has the typical winter-wet, summer-dry patternof a Mediterranean-type climate. Mean annual rain-

fall is 1500 mm with maxima in both winter andearly spring. Summer drought is severe, with noprecipitation during July and August, and little if

any in June and September. The period of the studywas drier than the mean year, with 395 mm of rain-fall during 1994 and 416 mm for the whole period(December 1993 to January 1995). Mean annual

temperature is 16.78C. Winter temperatures aremild with a daily mean of 9.38C during the coldestmonths (December and January). Summer tempera-

ture are high, with a daily mean of 23.98C for July,the warmest month. Monthly precipitation andmean temperature during the period of study are

shown in Fig. 1.Matorral (mediterranean shrubland) is the predo-

minant vegetation type in the DonÄ ana Reserve.Species composition appears to be controlled by

soil topography, which determines the depth to thewater Table (Gonzalez-Bernaldez et al., 1975).However, productivity of the matorral community

is limited by nutrient availability more than bywater availability (Merino et al., 1990). Our studywas undertaken at a site 120 m elevation above

mean sea level in an open shrubland dominated

mainly by Halimium halimifolium, H. commutatum,Cistus libanotis, and Rosmarinus o�cinalis. Biomass,

productivity, and successional changes in the areaare described by Merino and MartõÂ n-Vicente (1981)and Merino et al. (1990). The soils are sandy (95%

sand) and formed from weathering of holocenequartz parent rock of stabilized sand dunes.

Experimental design

We established three blocks at 30-m intervalsalong a ¯at area of matorral. In December 1993, weamended 15-m2 randomly selected subplots (®ve

replicates per block) with 220 g mÿ2 of C as woodshavings (Pinus pinaster wood) by gently mixingwith the litter layer. The size of the wood shavingsranged between 0.05 mm and 0.5 mm, and they con-

tained 71% holocellulose, 13.7% lignin, 11.9% totalpolyphenols, 0.2% of N and a C-to-N ratio 1250.Other randomly selected 15-m2 subplots served as

controls. This treatment was chosen to duplicatethe annual above- and below-ground inputs of C tothe soil, based on estimates obtained from Merino

et al. (1990) and MartõÂ nez-GarcõÂ a and RodrõÂ guez(1988). We removed the litter layer (0.2±0.4 cmdepth) before sampling with a spade the mineral

soil to a depth of 10 cm every 1±2 months, at 8sampling dates (Dec. 93, Feb. 94, Apr. 94, May 94,June 94, Sep. 94, Nov. 94 and Jan. 95). We selected®ve 200 g soil samples from each subplot and

block.

Laboratory procedures

Oven-dry (808C, 24 h) wood shavings were ana-

lyzed for ash, holocellulose, lignin, total polyphe-nols, and total N. Ash content was calculated afterheating the subsample in a mu�e furnace at 4508Cfor 4 h. Cellulose and lignin were analyzed bysequential analysis of ®ber (Robertson and VanSoest, 1981). Total phenolics were sequentially

Fig. 1. Monthly precipitation and mean temperatureduring the period of study in a matorral community at

DonÄ ana National Park (SW Spain).

A. Gallardo and J. Merino1350

extracted with 100% methanol (low molecular

mass) and 50% methanol (high molecular mass;

Glyphis and Puttick, 1988), and were colorimetri-

cally measured using the Folin±Ciocalteu reagent

with tannic acid as the standard (Singlenton and

Rosi, 1965). C content was taken as 50% of ash-

free dry mass (Schlesinger, 1977; McClaugherty et

al., 1985). Total N was determined on 0.2-g sub-

samples using standard Kjeldahl procedures.

Soil samples were sieved (<2 mm) in ®eld-moist

condition, air-dried (358C) and analyzed individu-

ally for total C, total N, total soil carbohydrates,

net potential nitri®cation and mineralization, and

soil microbial biomass N. Total soil C was deter-

mined by a modi®ed Mobius procedure following

Yeomans and Bremer (1988). Total soil N was ana-

lyzed by a dichromate procedure as outlined by

Flowers and Bremmer (1991). Soil carbohydrates

were analyzed by the phenol-sulfuric acid procedure

following Safarik and Santruckova (1992). This

method quanti®es soluble mono-, oligo- and poly-

saccharides as well as insoluble polysaccharides,

such as cellulose. Soil carbohydrates were expressed

in units of L-dextrose. Estimates of potential net N

mineralization and nitri®cation were obtained by

incubating in the dark 40-g soil subsamples at 80%

of water-holding capacity for 10 d at 208C. Samples

were extracted with 80 ml of 1 M KCl, ®ltered

through 0.45-mm Millipore ®lters, and analyzed for

inorganic N using a BuÈ chi 315 steam distillation

unit. Liberation of ammonium was carried out by

adding magnesium oxide to the extract. Nitrate in

the residue was reduced to ammonium by using

Devarda's alloy. Net N mineralization was de®ned

as the net increase in NH4+ÿN +NO3

ÿ±N over the

incubation interval; the net increase in NO3ÿ±N was

used to indicate net nitri®cation potentials. Initial

NH4+±N +NO3

ÿ±N in the samples was de®ned as

available N.

Soil microbial biomass N was analyzed by using

the fumigation-extraction method as outlined by

Brookes et al. (1985). Remoistened soil subsamples

(80% of water-holding capacity) were incubated for

5 d at 258C in the dark, and then exposed to

chloroform for 5 d. They were then extracted with

100 ml of 0.5 M K2SO4, and ®ltered through 0.45-

mm Millipore ®lters. Subsamples, extracted with

0.5 M K2SO4 immediately before fumigation served

as controls. Total N in the extracts was analyzed by

a dichromate procedure (Flowers and Bremner,

1991). Extracts (30 ml) were acidi®ed with 0.5 ml of

concentrated H2SO4 and heated in an aluminum

block (1158C) to reduce the volume to 4 to 5 ml.

After cooling, the tubes were amended with 5 ml of

1 N K2Cr2O7 and 8 ml of concentrated H2SO4 and

placed in a block digester heated to 1708C. After

45 min the cooled digest was amended with 30%

NaOH and connected to the steam distillation ap-

paratus for ammonia-N determination. Extractable-

N was de®ned as the initial total-N in the K2SO4-extracts in the unfumigated sample. N in microbial

biomass was calculated using a Kn of 0.69 (Brookeset al., 1985). All results are expressed on the basisof oven-dried soil.

Statistical analysis

Repeated measures ANOVA (type III sum of

squares) was used to determine signi®cant di�er-ences by block, treatment and sampling days. Thedata were ®rst log-transformed when Cochran's C

and Barlett's test indicated a lack of variance hom-ogeneity (P < 0.05). When unequal variances werefound after log-transformation, or log-transform-ation was not possible (negative values), we used a

non-parametric ANOVA (Kruskall±Wallis) to testfor di�erences between means. When interactionbetween sampling time and treatment was signi®-

cant, we examined di�erences between amendedand control samples for each sampling time.Tukey's honest signi®cant di�erences test was used

to determine individual signi®cant di�erences (atthe 0.05 level) between means.We performed repeated measures ANCOVAs

with microbial biomass-N, net N mineralizationand net nitri®cation as dependent variables, and or-ganic C, soil carbohydrates, total N, Extractable-N,as independent variables. Microbial biomass-N also

served as independent variable for net N mineraliz-ation and nitri®cation. Block and treatment vari-ables were included in the models. By examining

the residuals we extracted the % variationaccounted for each independent variable. Whenblock or treatment were signi®cant e�ects, we

yielded di�erent equations for each block and eachtreatment. We performed single linear correlationsbetween variables. Because the high partial corre-lations between variables, we only discuss the

models with independent variables explaining atleast 50% of the variation of the dependent variable(i.e. R2>0.5). All analyses were performed using

the statistical package SYSTAT 5 for Windows andSTATGRAPHICS.

RESULTS

Amendment e�ect on soil C and N

The mean organic C concentration of unamended

soil samples was 0.97% (20.02 S.E.). We found anincrease (but only signi®cant at P < 0.1), by up to16%, in the amounts of organic C in amended plots(Fig. 2, Table 1). The mean total carbohydrates

content of unamended soil samples was 3.1 mg gÿ1

(20.1 S.E.), with a maximum signi®cant increase(P < 0.01) of 43% in the amended subplots after

285 d. The mean total N concentration was 0.047%(20.001). The organic-amendment did not signi®-cantly increase the amounts of total N. The C-to-N

ratio increased from a mean value of 20.7 (20.3) in

Soil nitrogen dynamics in Mediterranean shrubland 1351

unamended plots to a maximum of 26.3 in amended

samples after 350 d. By the end of the experiment

(404 d), none of the di�erences between amended

and control soil samples was signi®cant for any of

these properties. (Fig. 2). Di�erences in total carbo-

hydrates and the C-to-N ratio between amended

and unamended soils were signi®cantly a�ected by

the interaction treatment� time (Table 1); these

treatment di�erences began to appear between 123

and 156 d after the addition of the organic amend-

ment (Fig. 2).

The ANOVA showed signi®cant di�erences in

the amounts of organic C and total N among

sampling dates, with the lowest values in the period

from February to April and in November (Fig. 2).

Di�erences between sampling dates for total carbo-

hydrates and the C-to-N ratio were signi®cant in

the amended soil samples (P< 0.05).

Amendment e�ect on N dynamics.

Average potential net N mineralization was

422 ng gÿ1 dÿ1 in unamended subplots, with a netnitri®cation rate of 310 ng gÿ1 dÿ1. Net N mineraliz-

ation was signi®cantly lowest during the monthsfrom May to September, with higher values during

both winters. However, net nitri®cation in una-mended soil samples was the lowest in both winters

(Fig. 3). The mean soil microbial biomass-N in una-mended subplots was 12.1 mg gÿ1, with minimum

values in winter and higher values from April toSeptember.

Net N mineralization and net nitri®cation rates,

and amounts of microbial biomass-N di�ered sig-ni®cantly between amended and control plots, and

the interactions between treatment and time werealso signi®cant. Both net N mineralization and net

nitri®cation rates decreased signi®cantly in amended

Fig. 2. Changes in organic C, total N, total soil carbohydrates and the C-to-N ratio with time inamended and control soil samples of a matorral community at DonÄ ana National Park (SW Spain).Asterisks show signi®cant di�erences between amended and control soil samples. Error bars are the

standard error of the mean (S.E.).

A. Gallardo and J. Merino1352

plots 123 d after the experiment started (Fig. 3).

When the e�ect of the amendment treatment was

most pronounced, net mineralization shifted to netimmobilization. However, net nitri®cation remained

positive in both amended and control subplots.

Microbial biomass-N, in contrast, showed a signi®-

cant increase by d 285 in the amended subplots.

For all these properties, di�erences between the

amended and control plots had disappeared by theend of the experiment (Fig. 3).

Available-N (NH4+±N + NO3

ÿ±N) and extracta-

ble-N did not di�er signi®cantly between theamended and control soils (Table 2). Mean avail-

able-N was 3.98 mg gÿ1, with low amounts of NO3ÿ±

N (0.59 mg gÿ1). Mean extractable-N was 11.2 mg gÿ1

soil, indicating that a large part of the total N in

the K2SO4 extract was in the organic form.

Amounts of available and extractable-N were high-est in the winter samplings and lower from April to

November (Fig. 3).

N dynamics in relation to soil properties

Soil carbohydrates, and the carbohydrate-to-

extractable-N ratio explained 63% and 68% of thevariation in microbial biomass N. (Fig. 4).ANCOVA showed no signi®cant e�ect of block andtreatment, and most of the variation in microbial

biomass N was explained by the total carbohydrate(34%), extractable-N (23%) and total C concen-trations (5%).

Net N mineralization was signi®cantly correlatedwith the carbohydrate-to-extractable N ratio(R2=0.71), and microbial biomass-N (R2=0.65;

Fig. 4). ANCOVA showed a signi®cant e�ect of

Table 1. Repeated measures analysis of variance for the dependent variables of soil organic C, total soil N, total soil carbohydrates andsoil C-to-N ratio, with block, organic amended and time since the beginning of the experiment in a matorral community at SW Spain

DF F-ratio Probability >F

Organic-CBetween subjectsBlock 2 0.882 0.427Treatment 1 3.433 0.076Block� treatment 2 0.026 0.974Error 24Within subjectsTime 7 5.14 0.000Time�block 14 0.48 0.943Time� treatment 7 0.80 0.587Time�block� treatment 14 1.36 0.176Error 168

Organic-NBetween subjectsBlock 2 0.052 0.950Treatment 1 0.001 0.972Block� treatment 2 0.447 0.645Error 24Within subjectsTime 7 5.52 0.000Time�block 14 0.48 0.942Time� treatment 7 0.28 0.962Time�block� treatment 14 0.89 0.569Error 168

Total CarbohydratesBetween subjectsBlock 2 1.10 0.349Treatment 1 8.57 0.007Block� treatment 2 0.62 0.544Error 24Within subjectsTime 7 1.74 0.104Time�block 14 0.76 0.705Time� treatment 7 2.08 0.048Time�block� treatment 14 1.04 0.420Error 168

C-to-N ratioBetween subjectsBlock 2 3.21 0.058Treatment 1 13.04 0.001Block� treatment 2 0.71 0.503Error 24Within subjectsTime 7 3.51 0.002Time�block 14 0.99 0.461Time� treatment 7 3.59 0.001Time�block� treatment 14 0.67 0.802Error 168

Soil nitrogen dynamics in Mediterranean shrubland 1353

treatment, and carbohydrates, extractable-N and

microbial biomass explained 5, 23 and 18%, re-

spectively, of the variation in net N mineralization

in the amended and 2, 42 and 6%, respectively, in

the control samples.

No single predictor explained signi®cantly the

variations of net nitri®cation rate. ANCOVA

showed a signi®cant e�ect of treatment on nitri®ca-

tion concentrations, and some of the variation of

net nitri®cation rate in amended soils was explained

by the concentrations of organic C (5%), and

extractable-N (5%). In the unamended soils, only

12% of the variation in net nitri®cation could be

explained by the organic C and extractable N con-

centrations.

DISCUSSION

The soil of the matorral community studied is

extremely nitrogen-poor. For example, the total N

concentration is about half of the N content found

in the soils of mesquite and creosotebush shrubland

communities of the Chihuahuan desert, which have

lower C-to-N ratios, and similar amounts of soil

microbial biomass N and net N mineralization

(Fisher et al., 1987; Gallardo and Schlesinger,

1992). Values of N availability are also below the

values found by Carreira et al. (1994) for di�erent

mediterranean shrublands in Spain. Since the shrub-

land at DonÄ ana is in a late succesional stage, we

tested the equation used to predict soil microbial

biomass N as a function of aboveground net pri-

Fig. 3. Changes in potential net N mineralization, potential net nitri®cation, soil microbial biomass-N,inorganic and K2SO4-extractable-N with time in amended and control soil samples of a matorral com-munity at DonÄ ana National Park (SW Spain). Asterisks show signi®cant di�erences between amended

and control soil samples. Error bars are 1 S.E.

A. Gallardo and J. Merino1354

mary productivity (Zak et al., 1994). The value of

soil microbial biomass N estimated by this equation

was 5.4 g N mÿ2, versus 2 g N mÿ2 measured in the

®eld. The di�erence may be explained by the low

clay content of the DonÄ ana soil (<2%), which in-

¯uences the size of the soil microbial biomass (Van

Veen et al., 1985), and by the Kn (0.69) chosen in

this study.

Di�erences in the amounts of C and N among

sampling times may be driven by the litter inputs

and by the typical seasonality of a mediterranean

climate (Hart and Firestone, 1991). Aboveground

litter inputs are maximum in spring (Merino et al.,

1990), while belowground litter inputs are likely to

be greatest in late winter, after the maximum ®ne

root biomass is reached (MartõÂ nez-GarcõÂ a and

Table 2. Repeated measures analysis of variance for the dependent variables of net N mineralization rate, net nitri®cation rate, soil mi-crobial biomass-N, available-N (NÐNO3+NÐNH4), and K2SO4-extractable total N, with block, organic amended and time since the

beginning of the experiment in a matorral community at SW Spain

DF F-ratio Probability >F

Potential mineralizationBetween subjectsBlock 2 1.91 0.171Treatment 1 23.2 0.000Block� treatment 2 1.73 0.199Error 24Within subjectsTime 7 44.2 0.000Time�block 14 0.84 0.628Time� treatment 7 3.66 0.001Time�block� treatment 14 0.76 0.712Error 168

Potential nitri®cationBetween subjectsBlock 2 2.62 0.094Treatment 1 20.5 0.000Block� treatment 2 0.07 0.930Error 24Within subjectsTime 7 19.8 0.000Time�block 14 0.41 0.969Time� treatment 7 9.46 0.000Time�block� treatment 14 1.20 0.283Error 168

Microbial biomass-NBetween subjectsBlock 2 1.88 0.174Treatment 1 6.20 0.020Block� treatment 2 1.17 0.329Error 24Within subjectsTime 7 17.6 0.000Time�block 14 0.63 0.836Time� treatment 7 3.65 0.001Time�block� treatment 14 0.82 0.644Error 168

Available-NBetween subjectsBlock 2 0.27 0.763Treatment 1 0.00 0.977Block� treatment 2 0.36 0.699Error 24Within subjectsTime 7 19.6 0.000Time�block 14 0.62 0.844Time� treatment 7 3.83 0.001Time�block� treatment 14 1.07 0.380Error 168

Extractable-NBetween subjectsBlock 2 0.21 0.810Treatment 1 0.33 0.568Block� treatment 2 0.64 0.538Error 24Within subjectsTime 7 64.3 0.000Time�block 14 0.49 0.938Time� treatment 7 2.88 0.007Time�block� treatment 14 1.11 0.355Error 168

Soil nitrogen dynamics in Mediterranean shrubland 1355

RodrõÂ guez, 1988). The intense autumn and winter

precipitation during the time of the study is coinci-dent with the decrease of C and N in the soils in

November. Sampling-time di�erences in soil carbo-

hydrates, the C-to-N ratio, net N mineralization

and microbial biomass N were higher in amendedsoil than in the controls (Figs 2 and 3). These di�er-

ences were caused by to a rapid increase followed

by a rapid decrease, resulting from the amendment,

even though the substrate of wood shavings is fairlyrecalcitrant. The magnitude of the responses in

total C and total carbohydrates in the mineral soil,

given that the wood was added only to the litter

layer, suggests gradual physical movement of thesmall wood particles to the top of the soil. Soil ani-

mals may be involved in this movement of the

wood particles.

The di�erences between the amended and control

soils in organic C concentration, total carbo-

hydrates, and the C-to-N ratio disappeared after

404 d. The extra C added to the soil may have been

incorporated into microbial biomass, respired, orleached during the rainfall events (see Fig. 1). The

increase in the size of the soil microbial biomass N

in the amended soil supports the hypothesis that

the extra C was respired through microbial metab-olism. The addition of C decreased potential net N

mineralization and nitri®cation, but left una�ected

the amounts of available NH4+±N and NO3

ÿ±N,

probably because leaching, and plant and microbialuptake, kept the available N to a minimum.

Microbial biomass N values and net N mineraliz-ation were highly correlated with the total soil

carbohydrate and with the extractable-N content of

soils. Organic C contributed to explain variation in

microbial biomass, indicating that microorganismswere dependent on sources of C other than carbo-

hydrates. Extractable-N was a good predictor of

both microbial biomass N and net N-mineraliz-

ation, suggesting that only a small part of the total

Fig. 4. Relationships between soil microbial biomass-N and net N mineralization as dependent variablesand total carbohydrate content, the ratio carbohydrates-to-extractable-N, and soil microbial biomass N

as independent variables. All regressions are highly signi®cant (P < 0.001).

A. Gallardo and J. Merino1356

N is used by microorganisms and that there is a

substantial exchange of N between extractable-N

and microbial biomass N (Hart and Firestone,

1991). Gallardo and Schlesinger (1992) also found

in various plant communities of the Chihuahuan

desert that the ratio of total C to extractable-N

determined whether C or N was limiting to the soil

microorganisms. Others authors have shown that N

mineralization was less sensitive to the ratio of total

C-to-N than to the ratio of available-C to avail-

able-N (e.g. Baath et al., 1978; Foster et al., 1980).

Unlike net N mineralization, net nitri®cation

showed a poor correlation with soil microbial bio-

mass N, as well as with net N mineralization (data

not shown). This is not surprising, because mi-

crobial biomass has a low potential to immobilize

nitrate (e.g. Schimel and Firestone, 1989; but see

Hart et al., 1994). Although net nitri®cation was

signi®cantly lower in the amended soil samples, the

variation in nitri®cation values was not well

explained by the total organic C, soil carbohydrate,

and extractable N values of the samples. Other soil

variables, such as PO4±P supply (Pastor et al.,

1984), polyphenol concentrations (Horner et al.,

1988; Barford and Lajtha, 1992), or increased CO2

production (Keeney et al., 1985) may also in¯uence

nitri®cation but were not examined in this study.

If potential net N mineralization in DonÄ ana soils

showed the same pattern under ®eld conditions, we

may conclude that a seasonal input of plant litter

with higher C-to-N ratio into the soil may cosider-

ably reduce the availability of N for plants by im-

mobilization in microbial biomass. Experiments

conducted in other ecosystems lead to similar con-

clusions. For example, Jonasson et al. (1996) found

that sugar amendment to a grassland±shrub ecosys-

tem in Denmark reduced the pools of soil inorganic

N and P, whereas the pools of N and P in the mi-

crobial biomass increased, and N and P uptake by

herbs declined.

However, this conclusion has several constraints.

First, we only looked at total soil microbial biomass-

N. The long-term response of free-living N2-®xers and

mycorrhizal fungi to increasing C availability can also

in¯uence the response of soil microbial biomass.

Second, we assumed that microorganisms outcompete

plants for inorganic-N (the microorganism maximiz-

ing hypothesis, Harte and Kinzig, 1993), but there is

much evidence that plants roots may be signi®cant

competitors for both NH4+ and NO3

ÿ (Jackson et al.,

1989, Schimel et al., 1989). Third, the quantitative and

qualitative response of microorganisms to increased

C in the soil may depend upon soil water conditions,

as suggested by Rice et al. (1994). In the mediterra-

nean area changes in precipitation may cause longer

e�ects on N dynamics than changes in C availability.

Our results support the hypothesis that increasing

C availability in soils leads to a decrease in N avail-

ability for plants and an increase in temporal het-

erogeneity of soil properties in a mediterraneanshrubland.

AcknowledgementsÐWe thank William H. Schlesinger andLawrence E. Woods for earlier review of the manuscript,Ana Lomba for help in the laboratory and SalustianoMato for providing the steam distillation unit. Thisresearch was ®nanced by the Spanish Ministry ofEducacion and Science and Xunta of Galicia.

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