Role of amendments on N cycling in Mediterranean abandoned semiarid soils

applied soil ecology 41 (2009) 195–205

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Role of amendments on N cycling in Mediterranean abandoned semiarid soils F. Bastida a,*, A. Pe´rez-de-Mora b, K. Babic b, B. Hai b, T. Herna´ndez a, C. Garcı´a a, M. Schloter b a

Centro de Edafologı´a y Biologı´a Aplicada del Segura, CEBAS-CSIC. Department of Soil and Water Conservation, Campus Universitario de Espinardo, Aptdo. de Correos 164, Espinardo 30100 Murcia. Spain b Helmholtz Zentrum Mu¨nchen. German Research Centre for Environmental Health (GmbH); Department of Terrestrial Ecogenetics, Institute for Soil Ecology, Ingolsta¨dterlandstrasse 1, 85764 Neuherberg, Germany

article info

abstract

Article history:

Soils found in semiarid areas of the Mediterranean Basin are particularly prone to degrada-

Received 21 May 2008

tion due to adverse climatic conditions with annual rainfall <300 mm and high tempera-

Received in revised form

tures being responsible for the scant vegetal growth and the consequent lack of organic

22 October 2008

matter. A three-year field experiment was conducted to test the potential of two organic

Accepted 24 October 2008

amendments (sludge and compost) to improve soil quality and plant growth in a semiarid degraded Mediterranean ecosystem. Since little is known about N dynamics in such assisted ecosystems, we investigated the effects of this practice on key processes of the global N

Keywords:

cycle. Besides soil chemical and biological parameters and vegetation cover, we measured

AmoA

absolute and specific potential nitrification and denitrification rates and quantified the size

Denitrification

of the ammonia oxidising and denitrifying bacterial populations via quantitative PCR (amoA

Nitrification

and nirS genes). At the end of the experiment soil fertility, microbial activity and plant

NirS

growth had improved in treated plots. Amendments increased the amount of ammonia

Organic amendment

oxidisers and denitrifiers in soil, but the relative proportion of these groups varied in relation

QPCR

to the total microbial community, being higher in the case of ammonia oxidisers but not in the case of denitrifiers. As a consequence, significantly higher potential nitrification and denitrification rates were measured on a global basis in amended soils. Yet specific activities (potential rate/gene copy numbers) were lower for ammonia oxidisers in amended soils and for denitrifiers in sludge treated soils than those observed in control plots. Organic amendments influenced resource availability, the size and the activity patterns of microbial populations involved in long-term N dynamics. Therefore N cycling processes may play a key role to assist sustainable restoration practices in semiarid degraded areas. # 2008 Elsevier B.V. All rights reserved.

1.

Introduction

Soils found in semiarid areas of the Mediterranean Basin are particularly prone to erosion due to high climatic variability (long dry periods with significant fire risk and very low precipitation arriving as irregular torrential rainfall events) (Van Lynden, 1995; De Paz et al., 2006).

Additionally inappropriate agricultural practices, land use and construction activities can result in loss of soil organic matter and essential nutrients (Yassoglou et al., 1998). As a consequence of soil quality declines, vegetation development is constrained and soil erosion risk increases (Garcı´a et al., 1998; Xie and Steinberger, 2005; Bastida et al., 2007a).

* Corresponding author. E-mail address: [email protected] (F. Bastida). 0929-1393/$ – see front matter # 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.apsoil.2008.10.009

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Several studies have shown that the incorporation of organic amendments into semiarid soils can reverse this trend by improving aggregate stability, nutrient cycling and plant colonization (Ros et al., 2003; Pe´rez de Mora et al., 2006). Recycling of organic residues as amendments is a costeffective and sustainable alternative to improve the organic matter content of soils. However, their application must be adequately controlled since some of these amendments contain heavy metals, pathogens and unwanted odours. To minimize risks it has been suggested that organic wastes should be previously composted or stabilized (Pascual et al., 1997; Burton and Turner, 2003). Since organic C content is a critical criterion to evaluate the degree of desertification, most studies in semiarid areas have dealt with C-related processes. In contrast, N dynamics have received less attention, although nitrogen (N) is the only essential nutrient that is not released by the weathering of minerals and is a primary limiting nutrient for vegetation development in Mediterranean type ecosystems (Arianoutsou-Faraggitaki and Margaris, 1982; Schulten and Schnitzer, 1998). External N sources such as organic amendments can therefore ameliorate the N pool of such soils and consequently improve plant growth. In addition to ammonium (NH4+) and nitrate (NO3), organic amendments incorporate degradable organic compounds into the soil, which can in turn affect key steps of the N cycling such as nitrification and denitrification (De Wever et al., 2002). The main factors affecting these processes have largely been studied in agricultural systems (Vallejo et al., 2006), but little is known about the effects of incorporating amendments into highly degraded soils from semiarid areas. Biochemical and microbiological properties have traditionally been used to monitor changes in soil quality and quantify restoration activities in degraded semiarid soils (Garcı´a et al., 1998; Pe´rez de Mora et al., 2005). Recently, the development of culture independent techniques such as quantitative PCR has significantly improved the ability to study of microbial populations and microbial processes in soils. Nitrogen-related transformations are particularly good examples because there are many genes involved in the N cycle and the activity of the microorganisms responsible for such processes can be monitored (Ferguson, 1998; Bothe et al., 2000; Francis et al., 2007). The combination of measuring activity and using molecular tools can therefore provide quantitative and qualitative information essential for understanding ecosystem dynamics and for improving land management. In the present study, an experiment was set up to test the potential of organic amendments under field conditions to improve the soil status and vegetation cover in a semiarid degraded area. As little is known about N dynamics in ‘‘seminatural’’ managed ecosystems in arid or semiarid environments, our aim was to evaluate the impact of incorporating stabilized sludge and biosolid compost on key processes of the N cycle (nitrification and denitrification) three years after addition. Furthermore, we were interested to know whether amendments have a durable effect on the genetic potential of such soils and hence affect the population size and the activity patterns of the main players involved in these two processes (ammonia oxidising and denitrifying bacteria) to improve our current understanding of N dynamics in managed semiarid

soils and the sustainability of assisted restoration practices in such areas. We hypothesised that a single amendment application of sludge or biosolid compost would improve soil fertility and plant growth. The synergistic interaction of amendments and plants would in turn stimulate microbial biomass and activity in soil, resulting in enhanced nitrification and denitrification potentials. We also predicted that there would be differences in the magnitude and pattern of the above responses in the long-term, restoration practices changing the physical environment, the availability of nutrients and the genetic pool of the soil.

2.

Materials and methods

2.1.

Study site

The study was carried out in an experimental area agriculturally abandoned 10 years ago and largely eroded, situated in the province of Murcia (Southeastern Spain, 38810 N 18120 W). The climate is semiarid Mediterranean (mean annual rainfall of 333.2 mm and a mean annual temperature of 17.2 8C) with mild rainy winters and very hot, dry summers. The soil was classified as loam, Aridic calcisol (FAO-ISRIC-ISSS, 1998). The vegetation is characterised by open matorral species such as Asphodelus fistulosus L., Salsola genistoides Juss. ex Poir. and Rosmarinum officinalis L.

2.2.

Materials and experimental design

Experimental plots (4 m  5 m) were arranged in a complete randomised block design with two amended treatments and one control (unamended). Each treatment was replicated three times. The amendments tested were: sewage sludge (S) from a wastewater treatment plant (Murcia, SE, Spain), digested anaerobically and stabilized through the removal of ammonia and methane, and a biosolid compost (CM) made from the same material with straw as bulking agent. Amendments were incorporated into the topsoil (15 cm) at a

Table 1 – Characteristics of organic materials (values on dry weight basis). Sludge pH ECa Total organic Cb Total Nc Total Pc Total Kc Total Cdc Total Crc Total Cuc Total Nic Total Pbc Total Znc C/N ratio a b c

dS m1. g kg1. mg kg1.

6.6 2.85 378 43000 1600 3900 <2.5 14.0 247 14.8 80.8 718 8.73

Compost 6.8 2.46 406 30300 1700 4000 <2.5 11.0 187 11.9 54.9 510 13.5

applied soil ecology 41 (2009) 195–205

rate of 12 kg m2 using a rotovator in March 2004. The analytical characteristics of the organic materials are shown in Table 1. Control plots were also tilled using a rotovator. During the experimental period, plots were left under natural conditions without irrigation. Plots were sampled in June 2007. Four surface soil samples (0–15 cm), each consisting of 8 soil cores, were randomly collected within each plot. Subsamples for molecular determinations (sieved by a particle size of less than 2 mm) were frozen in situ on dry ice and then stored at 80 8C until analysis (one month after the sampling). Bulk soil samples were returned to the laboratory and subsamples for microbial biomass and biochemical determinations were sieved to a particle size of less than 2 mm and stored at 4 8C until analysis (within two weeks after the sampling). The rest was air dried for two days and sieved to a particle size of less than 4 mm diameter before the chemical properties and heavy metal concentrations were determined. The analysis of amended samples was performed in the same way.

2.3.

Soil properties and heavy metals

Texture analysis was performed according to the method of Guitian and Carballas (1976). The pH and electrical conductivity (EC) were measured in 1/5 (w/v) aqueous suspensions after shaking for 1 h using a Crison mod.2001 conductivimeter and pH meter. Total organic carbon (TOC) was estimated by dichromate oxidation and titration with ferrous ammonium sulphate (Yeomans and Bremmer, 1989). Total N was assessed by the Kjeldahl method modified by Bremner and Mulvaney (1978). Total P and K were determined in nitric–perchloric digestion extracts, P by colorimetry and K by flame photometry using a Jenway PFP7 flame photometer (Olsen and Sommers, 1982; Page, 1982). The nitrate (NO3-N) concentration was determined in water extracts 1/10 (w/v) via high pressure liquid chromatography (UVIKON 720LC Shimadzu C-R3A). Ammonium (NH4+-N) was measured in 1/5 1 M KCl extracts following colorimetric reaction with sodium salicylate and dichlorisocyanuric acid 0.1% (Keeney and Nelson, 1982). Heavy metals were analysed by ICP-MS following nitric–perchloric acid digestion (ICP-MS Termo-iris Intrepid II XDL). The percentage of plant cover was estimated by a grid-line intersect method.

2.4.

197

shaking in Precellys-Keramik-Kit Tubes (PeqLab, Erlangen, Germany) with a Precellys 241 Lysis and homogenisation automated equipment (Bertin Technologies, France). Extracted nucleic acids were resuspended in 50 ml Milli-Q water (pH 6.8) and the concentration of total DNA was measured by means of a Nanodrop1 ND-1000 spectrometer (Nanodrop Technologies, Wilmington, DE, US) at 260 nm. Extractions were carried out in duplicate. The quality of the DNA extracted was checked by comparing OD 260/280 and OD 260/230 indices between samples. For the preparation of genomic DNA from pure cultures, cells were grown over night and harvested by centrifugation. DNA was extracted using the Wizard1 Genomic DNA Purification Kit from Promega (Madison, WI, USA) following the manufacturer’s instructions.

2.5.2. Quantification of amoA and nirS genes by quantitative PCR (qPCR) To quantify potential ammonia oxidisers and denitrifiers in soil, the amoA and nirS gene copy numbers were quantified by qPCR (Le Roux et al., 2008; Sharma et al., 2006, 2007). The primers used for amoA amplification were amoA-1R (50 -GGGGTTTCTACTGGTGGT-30 ) and amoA-2R (50 -CCCCTCKGSAAAGCCTTCTTC-30 ) (Le Roux et al., 2008). The qPCR protocol for amoA quantification was as follows: 15 min at 95 8C and then 39 cycles consisting of 1 min at 94 8C, 1 min at 60 8C and 1 min at 72 8C. The primers used for nirS amplification were nirScd2af (50 GTSAACGTSAAGGARACSGG-30 ) and nirSR3cd (50 -GASTT CGGRTGSGTCTTGA-30 ) (Sharma et al., 2006). The qPCR protocol for nirS quantification was as follows: 15 min at 95 8C and then 39 cycles consisting of 1 min at 94 8C, 1 min at 57 8C and 1 min at 72 8C. Reactions were carried out in an ABI Prism 7700 sequence detector (Applied Biosystems). The PCR reaction mixture (final volume of 25 ml) included: 5 ml of Power SYBR Green PCR Master Mix (Applied Biosystems), 0.06% BSA (bovine serum albumin), 7.5 pmol of each primer (amoA-1R and amoA-2R) for amoA, and 12 pmol of each primer (nirScd2af and nirSR3cd) and 2 ml of template. The reaction mix for nirS, also included 5% DMSO (dimethyl-sulphoxide). The efficiency of qPCR for both genes ranged between 92 and 94%. Standard curves were made from serial dilutions of amoA gene from Nitrosomonas sp. and nirS gene from Pseudomonas stutzeri 20 Bell 14405.

Microbiological properties 2.5.3.

Microbial biomass C (MBC) was determined by the fumigation– extraction method (Vance et al., 1987). Soil respiration was analysed as described in Herna´ndez and Garcı´a (2003). Soil dehydrogenase activity was determined as reported by Von Mersi and Schinner (1991) and soil urease activity was estimated by the method of Kandeler and Gerber (1988). To determine the potential nitrification rate the method of Beck (1976) was used. The potential denitrification in soil slurries rate was estimated under aerobic conditions, as described by Ryden et al. (1979).

2.5.

Molecular biological methods

2.5.1.

Preparation of environmental and genomic DNA

Total DNA from soil samples (0.5 g dw) was extracted using the method of Griffiths et al. (2000). Cells were lysed by mechanical

Preparation of standards for qPCR

Genomic DNA from pure cultures of Nitrosomonas sp. and of P. stutzeri 20 Bell 14405 were amplified using the primer system described above. PCR products were purified using the QIAquick1 PCR Purification Kit (Quiagen GmbH, Hilden, Germany). Amplicons were checked on agarose gel to ensure amplification of the right fragment. Purified products were cloned into the pCR12.1 vector and transformed into E. coli TOP10 competent cells using the TA Cloning1 Kit (Invitrogen, Karlsruhe, Germany). Plasmids from transformants were prepared as proposed by Birnboim and Doly (1979). Plasmids were checked by PCR for inserts of the expected size. In addition, inserts were sequenced on an ABI 3730 48-capillary sequencer (Applied Biosystems, Foster City, CA, USA). Fragments for sequencing were prepared with the BigDye Terminator Kit 3.1. Sequences were compared with NCBI BLAST.

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Table 2 – Characteristics of the amended soils (values on dry weight basis) at the end of the experiment.

pH ECd Bulk densitye Water holding capacityf Total Organic Cg Total Nh Total Ph Total Kg NO3-Nh NH4+-Nh C/N ratio Total Cdh Total Crh Total Cuh Total Nih Total Pbh Total Znh Percentage vegetal cover Microbial biomass Ch Basal respirationi Dehydrogenase activityj Urease activityk

Control

Sa

CMb

7.55a 0.34a 2.61b 40.2a 15.3a 1.80a 535a 5.68a 0.92a 3.89a 8.51b <2.5 37.5a 40.0a 6.20a <5.0 24.9a 68.0a 300a 41.7a 7.53a 0.90a

7.87b 0.43b 2.57b 47.6b 21.0b 3.50b 692b 6.24b 5.28b 25.3b 6.01a <2.5 38.9a 52.3b 7.89b <5.0 39.9b 93.7b 385b 71.1ab 10.4b 1.28a

7.52a 0.94c 2.40a 55.3b 27.6b 4.32b 741b 6.41b 4.40b 23.2b 6.39a <2.5 37.8a 55.9b 7.11b <5.0 32.1ab 92.5b 441c 85.8b 13.9c 1.80b

Limitsc

3.00 150 210 112 300 540

For each parameter, data followed by the same letter are not significantly different according to the LSD test (P  0.05). S (sewage sludge). b CM (compost). c Heavy metal limits for soil (European Directive 86/278/CEE). d EC, electrical conductivity (dS m1). e g cm3. f g 100 g1. g g kg1. h mg kg1. i mg CO2-C g1 d1. j mg INTF g1 h1. k mmol NH4+-N g1 h1. a

2.6.

Statistical analysis

A multiple range test (95% confidence interval) based on Fisher’s least significant difference (LSD) method was performed to test for differences in chemical, microbiological and genetic properties between treatment types.

3.

Results

3.1.

General soil properties

The incorporation of amendments into the soil had no relevant effect on soil pH, which remained near neutrality (approximately 7.5) in all treatments (Table 2). The electrical conductivity was significantly higher in amended soils than in control plots, but below 1 dS cm1. Hence no salinity problems were expected in treated soils. The addition of compost reduced the bulk density of the topsoil, while no clear effect was observed for the sludge treatment (Table 2). The amendments significantly increased the maximum water holding capacity of the soil by 1.2 (sludge) and 1.4 fold (compost) (Table 2). In general, the nutrient content and soil fertility were still better in treated soils than in control plots three years after the incorporation of the amendments. Total C and N contents

were 1.4–1.9 and 1.9–2.4 times higher in sludge and compost amended soils respectively than in controls (Table 2). The incorporation of the amendments resulted in total concentrations of P of 700 mg kg1 in contrast to the 535 mg kg1 of control plots (Table 2). Total K concentrations were also greater in amended plots (6.24 g kg1 in the sludge amended soil and 6.41 g kg1 in the compost amended soil) than in controls (5.68 g kg1) (Table 2). The effect of organic amendments on labile N forms was even greater than that reported for total N concentrations. Nitrate (N-NO3) and ammonia (NH4+) were significantly increased by approximately 5 fold in amended soils compared to controls (Table 2). Of the metals analyses, the concentrations of copper and zinc were highest in sludge and compost (Table 1). However, the concentrations of these elements in treated soils, although higher than in the controls, were far below levels considered dangerous for soils. The values of the above heavy metals were below the limits established by the EU (Directive 86/278(CEE) for soil.

3.2.

Biological properties

The colonization of soils by wild plants was higher in amended plots than in control plots, as reflected by the higher vegetal

applied soil ecology 41 (2009) 195–205

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Fig. 1 – Mean values and standard deviations of (A) amoA copy numbers (per gram of soil), (B) amoA copy numbers (per ng of DNA), (C) potential nitrification rate and (D) specific potential nitrification rate (potential nitrification rate: amoA copy number ratio) in control and amended plots. For each parameter, data followed by the same small letter are not significantly different according to the LSD test (P = 0.05).

cover densities recorded (Table 2). Sludge increased microbial biomass C in the soil by 30% and compost by almost 50% (Table 2). As a consequence higher CO2 production (basal respiration) was measured in the amended soils, being these values significantly different from those measured in controls (Table 2). Amended plots also showed higher potential dehydrogenase and urease activities (Table 2). The addition of sludge resulted in a 1.5 fold increase in these activities, whereas plots treated with compost showed a 2 fold increase (Table 2). The number of gene copies per gram of soil indicates the size of the ammonia oxidiser or denitrifier populations, while the number of gene copies per ng of DNA shows the relative abundance of these communities in relation to the whole microbial community. The number of amoA gene copies and hence of potential ammonia oxidisers, regardless of the units used (number of copies per gram of soil or number of copies per ng of DNA), was found to be significantly higher in amended soils than in control plots (Fig. 1a and b). The difference between organic treatments and controls was approximately two orders of magnitude when referring to grams of soil and one order of magnitude when in the case of total DNA (Fig. 1a and b). Potential nitrification rates in treated soils were significantly higher (25–30 mg NO3-N g1 d1) than those found in controls (5 mg NO3-N g1 d1) (Fig. 1c). In contrast, the specific potential rate (production of NO3-N per amoA copy number) was significantly higher in control than in amended plots by more than one order of magnitude (Fig. 1d). The incorporation of sludge and compost resulted in higher nirS gene copy numbers per gram of soil (Fig. 2a), but

no concomitant increase was found when nirS was referred to ng of DNA (Fig. 2b). In the latter case, nirS copy numbers were highest in the control followed by the compost treatment (Fig. 2b). Significant differences were reported for control and compost in relation to sludge treated plots (Fig. 2b). Interestingly, nirK genes were up to 3 orders of magnitude lower than nirS gene copy numbers, indicating that denitrification in these soils is dominated by nirS (data not shown). Significantly higher potential denitrification rates were measured in amended soils than in control plots (Fig. 2c). Sludge and compost treatments showed similar denitrification rates (2 mg N2O-N g1 d1), while N2O production in controls did not exceed 0.5 mg N2O-N g1 d1 (Fig. 2c). The specific potential denitrification rate was found to be similar in the control and the sludge treated soils, whereas lower values were recorded in soils amended with compost (2  106 mg N2O-N nirS copies1 d1) (Fig. 2d).

3.3.

Correlations

In general, soil fertility parameters (total N, ammonia and nitrate), biological indicators and vegetation cover were highly and positively correlated (Table 3). All of these variables were in turn positively correlated with the total amoA and nirS gene copy numbers and potential nitrification and denitrification rates, when the data was referred to grams of soil (Table 3). This trend was, however, not observed in the case of amoA and nirS expressed as gene copy numbers per ng DNA. The same situation was reported for the specific potential nitrification and denitrification rates (Table 3).

200

applied soil ecology 41 (2009) 195–205

Fig. 2 – Mean values and standard deviations of (A) nirS copy numbers (per gram of soil), (B) nirS copy numbers (per ng of DNA), (C) potential denitrification rate and (D) specific potential denitrification rate (potential denitrification rate: nirS copy number ratio) in control and amended plots. For each parameter, data followed by the same small letter are not significantly different according to the LSD test (P = 0.05). Table 3 – Correlation matrix between the different parameters analysed. Total N PNR PDR amoA-soil amoA-DNA nirS-soil nirS-DNA MBC RESP DH UA Vcov NO3-N NH4+-N TOC

0.93*** 0.68* 0.89** 0.87** 0.87** NS 0.93*** 0.71* 0.88** 0.87** 0.87** 0.83** 0.85** 0.71*

PNR

PDR

0.83** 0.95*** 0.94*** 0.94*** NS 0.99*** 0.70* 0.92*** 0.76** 0.98*** 0.84** 0.87** 0.86**

0.89** 0.83** 0.80** NS 0.80** NS NS NS 0.90*** 0.82** 0.80** 0.92***

amoA- amoAsoil DNA

0.98*** 0.90** NS 0.95*** 0.72* 0.81** 0.71* 0.97*** 0.89** 0.89** 0.92***

NS NS NS 0.94*** 0.70* 0.84** 0.74* 0.84** 0.84** 0.87**

nirSsoil

nirSDNA

MBC

RESP

DH

UA

Vcov

0.95*** 0.70* 0.87** 0.69* 0.87** 0.68* 0.74* 0.73*

NS NS NS NS NS NS NS

0.68* 0.93*** 0.75* 0.92*** 0.80* 0.83** 0.79*

0.74* 0.81** 0.75* NS 0.72* 0.68*

0.86** 0.78* 0.67* 0.72* NS

0.68* 0.67* 0.69* NS

0.91** 0.94*** 0.94***

NO3-N NH4+-N

0.96*** 0.93***

0.92***

Potential nitrification rate (PNR), potential denitrification rate (PDR), amoA copy numbers g1 soil (amoA-soil), nirS copy numbers g1 soil (nirSsoil), amoA copy numbers ng1 DNA (amoA-DNA), nirS copy numbers ng1 DNA (nirS-DNA), microbial biomass C (MBC), dehydrogenase activity (DH), urease activity (UA), vegetal cover (Vcov). NS: not significant. * Significant at P < 0.05. ** Significant at P < 0.01. *** Significant at P < 0.001.

4.

Discussion

4.1.

General soil properties, soil fertility and plant cover

One of the most important aspects of incorporating amendments into degraded soils is the durability of the treatment,

which will determine the need for future applications (doses and rate) and the success of restoration practices. In this study, the effects of organic amendments on soil fertility were still noticeable three years after their incorporation into the soil (Table 2). Total organic C and total nutrient (N, P and K) concentrations were significantly higher in amended soils

applied soil ecology 41 (2009) 195–205

than in the control plots (Table 2). These results have even greater value if we assume that soils with a total organic C of less than 2% are in a pre-desertification stage (COM, 2002), which is the case of the original soil. Given the low nutrient content of semiarid areas, improving soil fertility may be crucial to enhancing plant colonization in the first stages of the restoration process. The amendments used did not only improve the N, P and K contents but also those of readily available N forms (N-NO3 and NH4+), which can be directly used by plants and microbes. In addition, it is important to stress that these effects are noticeable even after one application. Madejo´n et al. (2006) observed better plant colonization and growth under field conditions in bare acid semiarid soils following the incorporation of organic amendments, and suggested that amended soils performed better due to soil alkalinization and improved nutrient content. Another interesting feature of organic treatments relevant to plant cover is that they can reduce the bulk density and increase the water holding capacity of the soil (Table 2). Given that low rainfall is the primary constraint on biological activity in arid and semiarid environments (Schaeffer and Evans, 2005), improving water retention in the soil may improve the conditions for plant survival and even for microbial development. As has been observed in other degraded areas, amendment resulted in higher microbial biomass and enhanced microbial activity (basal respiration, dehydrogenase and urease enzyme activities) (Liang et al., 2005; Ros et al., 2006; Bastida et al., 2007b) (Table 2). It has been suggested that the stimulatory effect of amendments on biochemical properties is mainly related to the greater availability of labile C sources (Deng and Tabatabai, 1997). In addition, Garcı´a et al. (1998) showed that a substantial amount of microbial biomass is already present in the amendments, although the actual fraction than remains active is unclear. It is interesting to note that organic amendment increases the microbial biomass but could affect microbial community structure and plant diversity, effects which should be taken into account in long term. Bastida et al. (2008) showed a change in the microbial community structure of amended soils compared to control soils by analysing phospholipid fatty acids (PLFAs). Pe´rez de Mora et al. (2006) observed differences in the microbial community of a contaminated degraded soil following the application of various amendments. Although the authors did not reveal whether such changes were due to a shift in the original microbial community or the introduction of new individuals through the amendments or a combination of both, other studies suggest that the incorporation of amendments does not leave a direct microbial imprint in the soil (Saison et al., 2006). Regardless of a direct or indirect influence of amendments on the soil microbial community structure, organic treatments may improve soil fertility and enhance microbial activity in semiarid soils by triggering nutrient cycling and accelerating vegetation growth (Bastida et al., 2007a). Once plants start colonizing the soil, they release labile C compounds through the root system, which serve as substrates for soil microorganisms. Although competition between microorganisms and plants for nitrate may also exist, the overall result is that both benefit and the recovery of such soils can be achieved at a low cost and in a sustainable way. The action

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range of amendments is therefore ample, covering physical, chemical and biological properties, all of which can positively influence vegetation growth. Indeed, we observed positive correlations between vegetal cover and several chemical and microbiological parameters (Table 3). It is, however, important to ensure that no collateral effects such as heavy metal contamination and salinization occur. In the present experiment, despite high experimental amendment doses, heavy metal concentrations and salt content were relatively similar in control and treated plots and therefore below the limits considered as problematic (Table 2).

4.2. amoA gene copy numbers and potential nitrification rates Since diminishing organic levels are of particular concern in Mediterranean areas, research efforts have been mainly focused on C dynamics. Conversely, N has received less attention despite being a limiting nutrient crucial to plant survival in semiarid regions. At the same time, most studies evaluating the effects of amendments on different processes of the N cycle, both chemical and genetic level, have been carried out in agricultural soils (Sˇimek and Hopkins, 1999; Wolsing and Prieme´, 2004; Chu et al., 2007). As a consequence, little is known about the implications of incorporating amendments into non-agricultural ecosystems. Nitrogen cycles in arid and semiarid regions are characterised by relatively low N availability, low rates of inputs via atmospheric deposition and N2-fixation, and relatively high rates of gaseous loss of NOx, N2O, N2 and NH3 (Peterjohn and Schlesinger, 1990). Therefore, the incorporation of organic amendments should alter the N pool and N processes in these soils since these materials contain high amounts of Ncompounds. The main aim of this work was to investigate the effect of amendments on key processes of the global N cycle such as nitrification and denitrification. Nitrification is a two-step process which involves the transformation of ammonia to nitrite and the oxidation of nitrite to nitrate (Bothe et al., 2000). The first step is considered the bottleneck of the nitrogen cycle, since only a limited number of bacteria and archaea are able to perform this reaction (Leininger et al., 2006). As the turnover rates of ammonia-oxidising archaea are much lower than those found in ammonium-oxidising bacteria (Okano et al., 2004; Ko¨nneke et al., 2005), attention was focused on the bacterial amoA gene. This gene encodes a subunit of ammonia oxygenase (the subunit carrying the active site), which catalyses the oxygenation of ammonia to hydroxylamine. Our results showed higher number of amoA copies per gram of soil and hence of potential ammonia oxidisers in amended soils than in the controls (Fig. 1a). Similar results were observed when amoA was expressed on the basis of total DNA in soil (Fig. 1b). Therefore, the incorporation of amendments resulted in a quantitative and significant change of ammonia oxidisers with regard to the original soil microbial community. From our results, it is evident that the greater availability of organic compounds and ammonium in amended soils, independently of their origin (plant or amendments), could have stimulated ammonia oxidisers. Hence, positive correlations coefficients were observed between the ammonia

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content and potential nitrification and amoA copy numbers (Table 3). It is also possible that ammonia-oxidising bacteria (AOB) had been incorporated with the amendments, contributing to larger amoA yields in treated soils. Kowalchuk et al. (1999) showed that several groups of AOB are present in composts and postulated that these materials are not biologically inert to nitrification and consequently the fate of nitrogen during composting and compost storage may be affected by the presence of these organisms. However, Innerebner et al. (2006) observed that the AOB community of various composts did not reflect that found in composttreated soils. These authors suggested that composts affected the original soil microbial community indirectly through changes in soil organic matter decomposition and/or different allocations of plant material to the soil. Such changes are not only detectable in the short-term, but can persist (Okano et al., 2004). Concomitantly with an increase in the number of ammonia oxidisers, significantly higher potential nitrification rates were observed in amended soils than in the controls (Fig. 1). This is not surprising as higher organic N and ammonia contents may be expected to stimulate the growth of ammonia oxidisers. Nevertheless, the specific potential nitrification rate, that is, the ratio of potential nitrification to amoA gene copy numbers was significantly higher in controls than in amended soils, by one order of magnitude (Fig. 1). This pattern indicates that the lower availability of suitable substrates results in more efficient nitrification rates. In semiarid regions, microorganisms are adapted to respond to resource pulses coinciding with infrequent rain events that provide access to energy and nutrients (carbon and nitrogen) (Gebauer and Ehleringer, 2000). Thus, when sufficient moisture is present the metabolic machinery of microbes must react quickly and efficiently to utilize these substrates before other competitors do so, or a new drought period begins. Since amendments improve water retention and nutrient content in the soil, pulse-response traits could be partly lost in treated soils. A community structure analysis would provide further information regarding the main groups responsible for this phenomenon and whether the same microorganisms become less effective when the substrate is highly available or compositional changes occur and more opportunistic species proliferate under organic amendment. The incorporation of amendments significantly increased the amount of nitrate in the soil (Table 2). During precipitation events, nitrate excess may leach to stream channels or down through the soil profile, resulting in groundwater contamination or eutrophication of water bodies (Bauhus and Melwes, 1991). Although the fate of N is highly influenced by plant and microbial N uptake, the positive effect of amendments on vegetation development may be reduced by increased nitrification. In this case, amended areas could act as sources of N during wet periods. Whatever the case, precipitation is scarce in semiarid areas and leaching should not be an important problem.

4.3.

nirS gene copy numbers and denitrification rates

Denitrification is a respiratory process in which oxidised nitrogen compounds are used as alternative electron accep-

tors for energy production when oxygen is limited. It is the major mechanism through which fixed nitrogen returns to the atmosphere from soil and water, thus completing the N cycle. It is a stepwise reduction of nitrate to dinitrogen gas (N2). The process includes several steps, each of them controlled by various environmental factors. The most important are oxygen limitation, and the availability of NO3 and organic C (Mahmood et al., 2005; Dambreville et al., 2006). Genes involved in denitrification (nar, nor, nirS, nirK and nos) contain highly conserved DNA regions which have been successfully exploited for developing gene probes. The conversion of nitrite to nitric oxide (or nitrous oxide) is the crucial step in the reaction sequence because it leads to gas formation (Bothe et al., 2000) and is closely related to denitrification rates (Sharma et al., 2005). Denitrifying bacteria possess either a cytochrome cd1 (cd1 NIR) encoded by nirS or a Cu-containing enzyme (Cu NIR) encoded by nirK to perform this reaction. In our soil samples, nirK only occurred in low numbers (<105 copies per gram of dry weight soil) and was not influenced by the different treatments (data not shown). In contrast, nirS showed a significant response to the incorporation of amendments and was detected in larger amounts than nirK. These results contrast with those of other authors, who successfully amplified nirK from soil samples but not nirS (Avrahami et al., 2002; Wolsing and Prieme´, 2004). Such trends are difficult to explain since current knowledge of the environmental preferences of nirS- and nirK-containing denitrifying bacteria is very limited, probably because very few of these organisms have been cultivated. When the number of nirS copies referred to grams of soil, a greater abundance was observed in amended soils compared with controls (Fig. 2a). Apart from the fact that organic amendment enrich the soil with potential substrates for denitrification, direct incorporation of potential denitrifiers with the amendments has been proposed (You, 2005). However, other studies suggest that the incorporation of amendments does not leave a direct microbial imprint in the soil (Saison et al., 2006). In this sense, the survival of exogenous microbial populations in these soils could be constrained by edaphoclimatic properties. In contrast, when nirS referred to total DNA instead of gram of soil no significant differences were found between control and compost samples, whereas lower copy numbers were recorded in sludge amended samples (Fig. 2b). These results suggest that organic amendments increase the size of the denitrifying population in soil, while the relative abundance of denitrifiers in relation to the whole microbial community does not necessarily change. The total number of nirS copies per gram of soil and hence of potential denitrifiers was positively correlated with the potential denitrification rate (Table 3). This was significantly higher in amended soils than in the control (Fig. 2c). As was indicated in the materials and methods section, the denitrification assay was carried out using soil slurries under aerobic conditions. In such conditions (He atmosphere) higher rates were measured but no significant differences between control and treated samples were observed (data not shown). Since oxygen limitation is crucial for denitrification it is clear that amendments favour such conditions and this may be as relevant, or even more, so than the size of the denitrifying community. Organic amendments may reduce O2 availability

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in two ways: (i) by creating additional microsites where O2 diffusion is restricted (He´nault et al., 2001) and (ii) by increasing the amount of readily available C, which in turn enhances O2 consumption in soil pores, thereby creating anaerobic microenvironments (Sikora and Yakovchenko, 1996). In general, organic amendments improve the soil structure (Bastida et al., 2007b), so probably the second mechanism (enhancement of O2 consumption) was the most important in these experiments. The specific potential denitrification rate, that is, the denitrification potential with respect to nirS copy numbers, however, was significantly higher in control and sludge plots than in those amended with compost (Fig. 2d). In a similar way as was reported for amoA and ammonia-oxidising bacteria, the incorporation of amendments may alter the efficiency of potential denitrifiers in soil. Yet this was not observed in the sludge amended samples. This may be partly explained by the nature of denitrifying bacteria which, in contrast to ammonia oxidisers, are facultative microorganisms with high metabolic versatility adapted to changing environments. In this manner, the specific activity of the denitrifying community would not be as affected as that of AOB following amendment. One of the most important aspects of denitrification is the emission of N2O, a greenhouse gas. In Mediterranean areas soil moisture is generally very low during most of the year and the water table is usually several metres below the soil surface (Butturini et al., 2003). Due to such unfavourable conditions denitrification plays a lesser role in semiarid soils in comparison with temperate regions. Therefore, the use of organic amendments may not be so relevant for N2O emissions on a global basis. Only during the rainy season, a significant effect may be expected, as demonstrated in the denitrification assay. Although the total production of N2O may be higher in treated soils, the incorporation of organic amendments reduces the N2O to N2 ratio compared to inorganic fertilizers or non-treated soils (Vallejo et al., 2006). This is due to higher organic C availability in amended soils, which increases biological O2 demand, stimulating the consumption of N2O, which yields N2 (Rochette et al., 2000). Hence, the effect of N2O emissions from amended areas is palliated in part by the organic nature of the amendments.

5.

Conclusions

The use of organic wastes as resources to restore degraded semiarid areas has important implications for predicting the response of ecosystem N dynamics. While the incorporation of organic amendments may enhance important processes such as nitrification and denitrification in an overall way, the specific activity of the microorganisms responsible for these reactions may be reduced. Furthermore, organic amendments can directly or indirectly increase the population of ammoniaoxidisers and denitrifiers in soil, but the relative proportion of these groups may vary in relation to the total microbial community, although the proportion of ammonia oxidisers is always greater than that of denitrifiers. The timing and intensity of precipitation events will determine the activity patterns and response of these communities to amendment doses and rates. Incorporating amendments into the soil

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seems an effective measure to trigger N cycling and enhance plant growth in semiarid areas. Given its low cost and the high erosion risk of these areas, the benefits of this technique seem to be greater than its drawbacks. Setting coherent management practices in motion and using stabilized or composted amendments with a low metal content will ensure the sustainability of this practice.

Acknowledgements Dr. Felipe Bastida and Dr. Pe´rez-de-Mora thank the MEC Spanish Ministry of Education and Science for the financial support. The project ‘‘Indicators and Threshold for desertification, soil quality, and remediation’’ is funded by the EU.

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