Soil management of copper mine tailing soils — Sludge amendment and tree vegetation could improve biological soil quality

Science of the Total Environment 456–457 (2013) 82–90

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Soil management of copper mine tailing soils — Sludge amendment and treevegetation could improve biological soil quality

Verónica Asensio a,b,⁎, Emma F. Covelo a, Ellen Kandeler b

a Department of Plant Biology and Soil Science, Faculty of Biology, University of Vigo, As Lagoas-Marcosende, 36310 Vigo, Pontevedra, Spainb Institute of Soil Science and Land Evaluation, Soil Biology Section, University of Hohenheim, D-70593 Stuttgart, Germany

H I G H L I G H T S

• Soils at a copper mine were reclaimed with tree vegetation and sludge amendment.• Sludge treatment recovered both the functions and abundance of soil microorganisms.• Heavy metal pollution in soils still negatively influenced bacteria and fungi.• The microbial community clearly shifted towards a fungally dominated system.• We recommend the periodic addition of sludges and plantation of legume species.

⁎ Corresponding author at: Department of Plant BioloBiology, University of Vigo, As Lagoas-Marcosende, 36Tel.: +34 986 812630; fax: +34 986 812556.

E-mail address: [email protected] (V. Asensio).

0048-9697/$ – see front matter © 2013 Elsevier B.V. Allhttp://dx.doi.org/10.1016/j.scitotenv.2013.03.061

a b s t r a c t

Article history:Received 9 January 2013Received in revised form 4 March 2013Accepted 16 March 2013Available online xxxx

Keywords:Enzyme activityErgosterolMine tailingTree vegetationSludge amendment

Mine soils at the depleted copper mine in Touro (Northwest Spain) are physico-chemically degraded and pol-luted by chromium and copper. To increase the quality of these soils, some areas at this mine have been veg-etated with eucalyptus or pines, amended with sludges, or received both treatments. Four sites were selectedat the Touro mine tailing in order to evaluate the effect of these different reclamation treatments on thebiological soil quality: (1) Control (untreated), (2) Forest (vegetated), (3) Sludge (amended with sludges)and (4) Forest + Sludge (vegetated and amended). The new approach of the present work is that we evalu-ated the effect of planting trees or/and amending with sludges on the biological soil quality of mine sites pol-luted by metals under field conditions. The addition of sludges to mine sites recovered the biological qualityof the soil, while vegetating with trees did not increase microbial biomass and function to the level ofunpolluted sites. Moreover, amending with sludges increased the efficiency of the soil's microbial communityto metabolize C and N, which was indicated by the decrease of the specific enzyme activities and the increasein the ratio Cmic:Nmic (shift towards predominance of fungi instead of bacteria). However, the high Cu and Crconcentrations still have negative influence on the microorganisms in all the treated soils. For the futureremediation of mine soils, we recommend periodically adding sludge and planting native legume species.

© 2013 Elsevier B.V. All rights reserved.

1. Introduction

Mine soils have numerous restrictions affecting their future devel-opment into natural soils, such as extreme pH, high concentrations ofmetals and low organic matter (Akala and Lal, 2001; Krzaklewski andPietrzykowski, 2002; Barrutia et al., 2011; Asensio et al., 2013). Theimportance of soil microbial communities for successful plant estab-lishment and growth has been demonstrated by numerous studies(Ehrenfeld et al., 2005; Kulmatiski et al., 2008). However, the ex-treme soil conditions caused by soil pollution usually have a negative

gy and Soil Science, Faculty of310 Vigo, Pontevedra, Spain.

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influence on soil biological activity (Frouz et al., 2001; Liao and Xie,2007). Therefore, several strategies have been developed to improvethe soil quality of former mine tailing areas in recent years. Thefirst strategy involves using different waste amendments in orderto complex considerable amounts of metals in the polluted sites(Tandy et al., 2009; Baker et al., 2011; Karami et al., 2011). The sec-ond strategy is to establish plants that are adapted to these quiteharsh soil conditions (Dary et al., 2010; Barrutia et al., 2011). However,little information is available on the combined effect of waste amend-ment and vegetation on soil properties during the reclamation ofthese areas (Pérez-de-Mora et al., 2006; de Varennes et al., 2010).

Microbial and enzymatic soil properties respond relatively quicklyto small changes in soil conditions (Zhang et al., 2010). Consequently,soil enzyme activities reflect changes in soil quality before they can bedetected by other soil analyses (Izquierdo et al., 2005; Gómez-Sagasti

83V. Asensio et al. / Science of the Total Environment 456–457 (2013) 82–90

et al., 2012). The activity of soil enzymes is an important index of soilfertility, and is often used to monitor the remediation of polluted sites(Zhang et al., 2010; Baker et al., 2011). From a range of studies it isobvious that arylsulfatase as well as phosphatase belong to a set ofenzymes that are highly sensitive to metal pollution (Kandeler et al.,1996; Hinojosa et al., 2010). On the other hand, other authors havefound phosphatase insensitive to soil degradation (Moreno-Jiménezet al., 2012). Apart from the characterisation of different functionsof the soil microbial community, ecotoxicological studies make useof methods to estimate the abundance of the total soil microbial com-munity or each different microbial population (i.e., bacteria, fungi).Since bacteria as well as fungi have clear pH preferences (Rousk et al.,2009; Aciego Pietri and Brookes, 2009), any treatment that couldchange the acidity of the soil environmentmay alter the fungal/bacterialratio. Fungi are more efficient than bacteria at assimilating and storingnutrients and, moreover, the lower C:N of fungi means they take lessN from the soil than bacteria, which means more N available for plants.

Most of the studies on the reclamation ofmetal polluted soils throughrevegetation orwaste amendments have been performed under lab con-ditions and focused on changes in chemical or biochemical soil proper-ties. The new aspect of the current study is that we evaluated the effectof planting trees and/or amending with sludges on the biological soilquality of mine sites polluted by metals and under field conditions.Evaluating the biological quality of the soil implies taking physical,chemical and biological soil properties into account. For this purpose,we selected sites in Touro (Galicia, Northwest Spain) that were usedbetween 1973 and 1988 for copper mining. Since 1988, the remainingmaterial has been used for road construction. In order to reclaim themine soils, three different treatments were established: planting trees,adding sludges and a combination of both treatments. The plantedtreeswere Pinus pinasterAiton and Eucalyptus globulus Labill. Thewastesthat were the most frequently used as organic amendments to improvesoil quality were sewage sludge and paper mill residues. Untreatedsoils at Touro were polluted by Cr and Cu, and the amended sites notonly contained Cr and Cu, but also comparably higher amounts of Zn(Asensio et al., 2011, 2013). Untreated soils were extremely acidic,lacked N, P and K and were very poor in organic C (Asensio et al.,2011). Our aimwas to discoverwhether differentmanagement practicescan reduce the toxic effects of copper on soil functioning. We carriedout measurements of both the microbial biomass and fungal biomass(ergosterol), and the enzymes involved in C-, N- and P-cycles usingfluorogenic substrates. The measurements of urease and arylsulfatasewere selected as indicators of possible metal pollution (Kandeler et al.,1996). We hypothesized that vegetating with E. globulus Labill andP. pinaster Aiton, and amending with sludges at the same time mayincrease the biological soil quality in mine soils polluted by metals.

2. Material and methods

2.1. Description of the study area

The sampling area is located at the mine in Touro (Galicia, north-west Spain) (Lat/Lon (Datum ETRS89, European Terrestrial ReferenceSystem, EPSG, 1989): 8° 20′ 12.06″ W 42° 52′ 46.18″ N) (Fig. 1). Theclimate of the experimental site is Atlantic (oceanic) with precipita-tion reaching 1886 mm per year (an average of 157 mm permonth) and a mean daily temperature of 12.6 °C. The average relativehumidity is 77% (AEMET, 2013).

Four areas were selected at the Touro mine tailing (Fig. 1): (1) Con-trol (untreated soil), (2) Forest (vegetated), (3) Sludge (amended) and(4) Forest + Sludge (vegetated + amended). The control soilwas clas-sified as Spolic Technosol (FAO, 2006) and did not have vegetation. TheControl area was 1.20 ha in size and 336 m above sea level. The Forestsoil is a Spolic Technosol (FAO, 2006) vegetated with Pinus pinasterAiton 22 years before the sampling date. This soil also had naturalvegetation consisting of Ulex sp., Erica sp., Agrostis sp. and bryophytes.

The Forest soil was 0.58 ha in size and 340 m above sea level. Thetrees that were planted at Forest site looked thinner and smaller thanother pines with that age, but they did not show foliar symptoms oftoxicity. The Sludge soil is an Urbic Technosol (FAO, 2006) made ofsludges of different origins (a wastewater treatment plant, a papermill and an aluminium plant). The different sludges were mixed witha compost turner at a 45:45:10 ratio (dry weight basis) and then spreadon the soil surface in the Sludge site (Fig. 1), without being mixed withthe mine soil. This mixture of sludges was added to the soil 3 monthsbefore the sampling date. The amount of sludge applied was 158 tonsper ha and the final depth of this new layer was around 3 m. Thechemical properties of this sludge amendment were similar to thosedescribed by Camps Arbestain et al. (2008). Briefly, these sludges gener-ally had a pH of 8–10, more than 150 g kg−1 of total organic C, morethan 100 mg kg−1 of total Cu and more than 300 mg kg−1 of totalZn. Due to the huge amount of sludge needed for the reclamation ofmine sites, heterogeneous sources had to be used (i.e. sewage sludges,paper mill residues) that largely differ in their pH, nutrient and metalconcentration values. The Sludge site covered an area of 0.8 ha at178 m above sea level and it had only natural herbaceous vegetation.Forest + Sludge soil is also an Urbic Technosol (FAO, 2006) vegetatedwith Eucalyptus globulus Labill and amended with sludges 11 years be-fore the sampling date. The sludges used in the Forest + Sludge sitewere from a wastewater treatment plant and a paper mill, whichwere mixed in a 50:50 ratio with a compost turner and then spreadon the soil surface. The physico-chemical composition of these sludgeswas very similar to the Sludge soil. The amount of sludge added in theForest + Sludgewas 297 tons per ha. This area also had natural vegeta-tion (Ulex sp., Rubus sp., seedlings of P. pinaster Aiton and bryophytes).The Forest + Sludge soil was 1.5 ha in size and 336 m above sealevel. The trees planted at the Forest + Sludge site had a size similarto other with that age and they did not show toxicity symptoms.

2.2. Soil sampling

Five subsamples for each soil were randomly collected at a depth of10 cm on 24th March 2011, removing the litter layer during samplingwhen necessary. Each subsample was collected in a polyethylene bagandwere all transported to the laboratory in a cooling box. The sampleswere stored at−20 °C until biological analysis, and onepart of the sam-pleswas then dried at room temperature for chemical analysis. Sampleswere sieved to b2 mm prior to being analysed.

2.3. Soil chemical analyses

Soil pHwas determinedwith a pH electrode in 1:2.5 0.01 M CaCl2 tosoil extracts. The available content of Al, Ca, Co, Cr, Cu, Fe, K,Mg,Mn, Na,Ni, P, Pb and Znwas extractedwith 0.01 MCaCl2 in soil solution (Houbaet al., 2000). Pseudototal metal contents were extractedwith aqua regiaby acid digestion in a microwave oven (Milestone ETHOS 1, Italy). Thecertified reference material CRM026-050 was also analysed in parallelwith samples to check the analysis. Total Kjeldahl-N (TN) was deter-mined according to Bremner (1996). Both soil organic and inorganiccarbon (SOC and IC) were determined in a solid module (ShimadzuSSM-5000, Japan) coupled with a TOC analyser (Shimadzu TNM-1,Japan).

2.4. Soil microbial biomass

Microbial biomass carbon (Cmic) and nitrogen (Nmic) were deter-mined by the fumigation–extraction method according to Vance et al.(1987a). A sample of 2 g was fumigated with 0.1 mL of chloroform,added directly to the soil sample at 25 °C for 24 h. The chloroformwas then removed from the samples. The samples and non-fumigatedcontrols were extracted with 8 mL of 0.5 M K2SO4 solution for 30 minon a rotatory shaker and then centrifuged for 30 min at 3900 rpm.

Fig. 1. Location of sampled areas in the Touro mine. Source: ©Instituto Geográfico Nacional de España.

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Carbon and nitrogen in the fumigated and non-fumigated extracts weredetermined with a TOC analyser (Shimadzu TNM-1, Japan). Microbialbiomass C was calculated as: Biomass C = EC/kEC; where EC is thedifference between organic C extracted from fumigated soils andorganic C extracted from non-fumigated soils, kEC = 0.45 for soilswith pH > 4.5 and kEC = 0.30 for soils pH b 4.5 (Vance et al., 1987b;Joergensen, 1996). Microbial biomass N was calculated as C but usingthe factor kEN = 0.54 (Brookes et al., 1985).

2.5. Soil fungal biomass

Soil fungal biomass was determined by the extraction and quanti-fication of the ergosterol content using the method of Djajakiranaet al. (1996) with slight modifications. One gram of soil (5 g forControl sample by its probable low content in fungi) was suspendedin 25 mL of ethanol (HPLC-grade) in a 100 mL wide-mouth brownbottle and extracted in a shaker for 30 min at 250 rpm · min−1

followed by centrifugation in a 50 mL tube at 4560 rpm min−1 for30 min. An aliquot of 10 mL (20 mL for Control sample) was trans-ferred into a test tube and evaporated in a rotary evaporator at50 °C under vacuum. The dry extract was then dissolved in 1 mLmethanol and percolated through a syringe filter (cellulose–acetate,0.45 mm pore size) into a brown glass HPLC-vial. The extracts were

measured by injecting 20 μL into a HPLC autosampler (BeckmannCoulter, System Gold 125 Solvent Module, USA), then passing themthrough a column (250 mm × 4.6 mm, 5 μm diameter = solid phase,Spherisorb ODS II). Pure methanol was used as the mobile phase at aflow rate of 1 mL·min−1. The column was conditioned with methanoleluent at a flow rate of 1 mL·min−1 for 30 min before measurementbegan. Detectionwas carried outwith a UV-detector (Beckmann CoulterSystem Gold 166, USA) at a wavelength of 282 nm. Identification ofergosterol was performed by retention time and quantification bypeak area.

2.6. Soil enzyme activities

The activity of the urease and arylsulfatase enzymes was determinedfollowing the methods described by Kandeler and Gerber (1988) andStrobl and Traunmüller (1996), respectively. The urease and arylsulfatasemeasurements were used as early indicators of metal pollution.

A range of hydrolytic enzyme soil processes were measured usinga fluorimetric microplate assay (Marx et al., 2001). The activitiesof β-D-glucosidase, β-N-acetyl-glucosaminidase, β-xylosidase, acidphosphomonoesterase and L-leucine-aminopeptidase were mea-sured using 4-methylumbelliferon (MUB-) and 7-amido-4-methyl-coumarin (AMC-) labelled substrates. Enzyme activitieswere determined

Table 1Mean and confidence interval (CI) of pH, carbon, nitrogen, Olsen P, different ratios, elements extracted with CaCl2 (mg kg−1) and pseudototal heavy metal contents (mg kg−1).

Control Forest Sludge Forest + Sludge

pH 3.30 ± 0.02c 3.42 ± 0.0.3c 8.17 ± 0.23a 4.20 ± 0.05 bTexture Sandy loam Sandy loam Sandy clay loam Sandy loamIC (g kg−1) n.d. n.d. 14.55 ± 2.4 n.d.Bulk density (g cm−3) 1.15b 1.88a 0.93c 0.84dTOC (g kg−1) 2.60 ± 0.59d 7.75 ± 0.59c 106 ± 4.05a 13.16 ± 1.33bTN (g kg−1) 0.24 ± 0.04c 0.52 ± 0.2b 1.69 ± 0.3a 0.36 ± 0.09bcC:N 10.75 ± 1.90c 16.62 ± 8.04c 72.76 ± 13.71a 38.39 ± 12.14bErgosterol: Cmic 0.05 ± 0.03c 9.97 ± 3.97a 3.26 ± 1.2b 10.39 ± 2.8aCmic:Nmic 1.42 ± 0.45c 3.69 ± 0.89b 5.03 ± 1.18a 3.91 ± 0.44bCmic:TOC (%) 0.22 ± b0.01a 0.24 ± b0.01a 0.29 ± b0.01a 0.26 ± b0.01aNmic:TN (%) 1.7 ± 0.01b 1.1 ± 0.01b 3.9 ± 0.01a 2.5 ± 0.01abOlsen P (mg kg−1) n.d. 3.84 ± 0.54 c 265 ± 7.29a 126 ± 5.1bCaCl2-extractable Al 0.61 ± 0.08b 0.21 ± 0.05c n.d. 1.18 ± 0.31a

Ca 167 ± 40.71d 763 ± 22.04c 1209 ± 37.52b 1942 ± 83.16aCo 1.01 ± 0.16b 0.61 ± 0.05c 0.09 ± 0.01d 1.58 ± 0.31aCr 0.38 ± 0.04a 0.40 ± 0.05a 0.17 ± 0.14c 0.26 ± 0.03bCu 20.48 ± 2.11b 28.64 ± 1.95a 0.52 ± 0.11d 5.72 ± 2.25cFe 483 ± 55.25a 504 ± 9.58a n.d. 146 ± 18.59bK 27.49 ± 1.02b 47.87 ± 8.77b 401 ± 69.10a 41.27 ± 10.20bMg 21.37 ± 3.62b 24.56 ± 2.87b 2155 ± 333a 5.16 ± 5.34bMn 15.88 ± 1.09c 9.05 ± 1.55c 256 ± 34.07a 76.00 ± 15.43bNa 36.03 ± 7.69b 33.68 ± 2.43b 727 ± 187a 7.34 ± 2.76bNi 0.20 ± 0.08c 0.53 ± 0.03b 4.98 ± 0.43a 0.70 ± 0.20bP n.d. n.d. 7.07 ± 3.16 n.d.Pb 0.71 ± 0.20c 0.89 ± 0.11b n.d. 2.81 ± 0.23aZn 0.64 ± 0.18a 0.64 ± 0.02a 0.19 ± 0.09b 0.81 ± 0.42a

Pseudototal Co 23.64 ± 1.61a 31.46 ± 16.94a 8.61 ± 0.72b 28.50 ± 0.99aCr 130 ± 9.99a 129 ± 6.50a 79.98 ± 15b 76.95 ± 4.98bCu 644 ± 90.35a 637 ± 71.16a 89.53 ± 3.53b 61.46 ± 5.41bNi 14.64 ± 1.67d 23.47 ± 5.64c 154 ± 4.86a 46.59 ± 1.94bPb 21.99 ± 2.53b 16.51 ± 2.26b 46.69 ± 2.79a 39.04 ± 21.60aZn 113 ± 13.36b 88.18 ± 4.55c 178 ± 7.82a 108 ± 4.01b

Values followed by different letters in each row differ significantly with P b 0.05. n.d.: not detected. IC: inorganic carbon, TOC: total organic carbon, Cmic: microbial biomass carbon,Nmic: microbial biomass nitrogen, Olsen P: phosphorus extracted by Olsen method.

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as the release rate of fluorescent MUB and AMC from the MUB- orAMC-labelled substrates, respectively. Aqueous soil suspensions (freshweight soil: water = 0.5:50) were prepared from each soil sample.A mixture of 50 μL of sample, 50 μL of buffer (carbohydrases, acid phos-phomonoesterase: 0.1 M MES buffer, pH 6.1, aminopeptidase: 0.05 MTRIZMA buffer, pH 7.8) and 100 μL of substrate were added in triplicateinto a 96-well microplate, yielding to a final substrate concentration of500 μM. Microplates were incubated at 30 °C and the release of MUFand AMC was measured after 0, 30, 60, 120 and 180 min using an auto-mated luminescence spectrophotometer (BioLumin TM, 960 MolecularDynamics, USA) at emission 446 nm and excitation 377 nm. The fluo-rescence produced was converted into an amount of MUB:AMCaccording to a soil-specific standard calibration. This consisted of in-creasing the amounts of substrates (0, 10, 20, 50, 80 and 120 μL) andconstant amounts of soil solution (50 μL), which corrects for the possi-ble quenching effects on the fluorescence intensity of MUB:AMC.Enzyme activities were reported as nmol MUB:AMC g−1 h−1 DW soil.The specific enzyme activity was calculated by dividing the measuredabsolute enzyme activity by the microbial biomass of the respectivesample (Table 2).

2.7. Statistical analyses

All of the analytical determinationswere performed in triplicate andthe data obtained were treated statistically using the programme SPSSversion 15.0 for Windows. Analysis of variance (ANOVA) and homoge-neity of variance test were carried out. In case of homogeneity, a post-hoc least significant difference (LSD) test was carried out. If there wasno homogeneity, Dunnett's T3 test was performed. TheMann–Whitneytestwas usedwhen the datawere not parametric. A correlated bivariateanalysis was also carried out with data from all of the soil samples. Themost commonly used bivariate correlation technique, the Pearson cor-relation (r) was used, which measures the association between two

quantitative variables without distinguishing between the independentand dependent variables. The obtained correlation (r) and the usedprobability (95% confidence = P b 0.05) are given in the results or dis-cussion section.

3. Results

3.1. Soil chemical properties

The amendment with sludges as well as tree planting had a signif-icant impact by increasing the soil pH and available plant nutrients,and reducing metal contents in the mine tailing soil. The Controlsite and the vegetated soils showed a pH value in the range of 3.3 to4.2, which is extremely acid according to the USDA (1998), but thesoil receiving recent sludge amendment (Sludge) showed an increaseof this value to 8.2 due to the addition of high amounts of inorganic C(carbonates) and basic cations (Ca2+, K+, Mg2+, Na+) (Table 1). Sig-nificant differences in nutrient contents were also observed betweenthe recently amended soil and the rest of the mine sites: sludgeamendment increased both the potassium and calcium content by afactor of around 100, and Olsen phosphorus by more than 200. Thevegetated soils (Forest and Forest + Sludge) also significantly in-creased Ca and P contents (Table 1).

Pseudototal concentrations of some of the analysed metals (Ni, Pband Zn) were still as high in some of the sites after the application oftreatments (Forest, Sludge and Forest + Sludge) as in the Control(Table 1). The generic reference level for Galicia (Macías and Calvode Anta, 2009) established the following threshold limit values formetals in soils: 40 mg kg−1 for cobalt, 80 mg kg−1 for chromium,50 mg kg−1 for copper, 75 mg kg−1 for nickel and 200 mg kg−1 forzinc. According to these threshold values, all of the sites in thisstudy can be categorised as polluted by Cu and Cr. The following ex-ceptions were found: the Forest + Sludge site was not polluted by

Table 2Specific enzyme activities in soil samples.

Control Forest Sludge Forest + Sludge

Arylsulfatasea 0.11 ±0.08b

0.05 ±0.02b

0.09 ± 0.04b 0.85 ± 0.15a

Ureaseb 0.82 ±0.34a

0.32 ±0.11d

0.57 ± 0.26b 0.39 ± 0.08c

β-glucosidasec 2.36 ±1.26c

5.51 ±2.38a

3.72 ± 1.57b 1.40 ± 0.46c

β-xylosidasec 1.22 ±0.27a

0.64 ±0.39bc

0.73 ± 0.20b 0.27 ± 0.15c

β-acetyl-glucosaminidasec 1.42 ±0.32c

5.53 ±2.85a

3.51 ± 0.60b 1.25 ± 0.45c

Phosphatasec 96.45 ±19.18a

28.46 ±13.52b

1.28 ± 1.09c 17.43 ± 5.20b

Leucine aminopeptidasec n.d. n.d. 8.86 ± 5.14 n.d.

n.d.; not detected.Mean ± confidence interval (CI) of the mean of fifteen replicates. Values followed bydifferent letters in each row of each mine area differ significantly with P b 0.05.

a g p-nitrophenol g−1 Cmic h−1.b g N g−1 Cmic 2 h−1.c nmol MUF g−1 Cmic h−1.

Fig. 2. Carbon and nitrogen contents in the microbial biomass and ergosterol in the soilsamples. Bars with the same letters are not significantly different at P b 0.05. Means offifteen replicates are reported with confidence interval (CI).

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Cr, and the Sludge sitewas also polluted by Ni. However, due to the highpH in Sludge, the content ofmetals in available formwasmuch lower inthe Sludge site than in the others. The CaCl2-extractable metal contentswere below the threshold limits in all soils.

3.2. Microbial and fungal biomass

Themicrobial biomass C (Cmic) of themine tailing soils did not signif-icantly increase after 22 years of planting Pinus pinaster Aiton, as thevalues in the Control and the Forest site show (Fig. 2a). In contrast, veg-etating with Eucalyptus globulus Labill together with sludge addition for11 years (Forest + Sludge) enlarged the pool of microbial biomass by afactor of six. The highest enrichment of Cmic was observed after the re-cent addition of sludge to the mine soil (Sludge). The ratio betweenCmic and total organic C (TOC) was in the range of 0.22 and 0.29% anddid not differ significantly between treatments (Table 1). Pearson corre-lation analyses revealed that Cmicwas positively correlatedwith TOC, TNand soil pH (r = 0.99, 0.95 and 0.98, respectively, P b 0.01). This analy-sis reveals a negative correlation between soil microbial biomass andavailable Cr and Cu contents (r b −0.7, P b 0.01).

The microbial biomass N (Nmic) only increased significantly in therecently amended soil (Fig. 2b). The ratio between Nmic and total Nwas less than 2% in both the Control and Forest sites, but higherthan 2.5% in the two amended sites. Microbial biomass N was alsopositively correlated with TOC, TN and soil pH (r = 0.98, 0.91 and0.97, respectively, P b 0.01), and negatively correlated with availableCr and Cu (r b −0.7, P b 0.01). The Cmic:Nmic ratio was significantlyhigher in all of the treated soils than in the Control soil, but Sludgehad the highest value (Table 1).

The ergosterol content showed a similar pattern as microbial bio-mass carbon (Fig. 2c). This fungal biomarker significantly increased inall treated sites. Ergosterol was positively correlated with TOC, TNand soil pH (r = 0.93, 0.89 and 0.92, respectively, P b 0.01), and neg-atively correlated with available Cr and Cu (r = −0.60 and −0.70,respectively, P b 0.01). Calculating the ergosterol to Cmic ratio re-vealed that all of the remediation treatments shifted the fungal tototal microbial biomass ratio towards a more fungally-dominatedcommunity (Table 1).

3.3. Enzymes involved in the C cycle

Enzymes involved in the carbon cycle (β-glucosidase, β-xylosidaseand β-N-acetyl-glucosaminidase) significantly increased their activityin the recently amended soil (Fig. 3a, b and c). The β-glucosidase activ-ity also significantly increased in the two vegetated soils, but only

slightly in comparison to the Control soil (Fig. 3a). The activity of thethree enzymes involved in C cycling was positively correlated withTOC content (r > 0.97 in all cases, P b 0.01), as well as with K, Mg,Mn, Na, Olsen P, TN and soil pH (r > 0.86 in all cases, P b 0.01). The ac-tivity of these enzymes was negatively correlated with available Cr andCu (r b −0.65 in all cases, P b 0.01).

3.4. Enzymes involved in N cycle

Urease activity only increased significantly in the recently amendedsoil (Fig. 3d). The activity of this enzymewas negatively correlatedwithavailable Cr and Cu (r = −0.51 and−0.66, respectively, P b 0.01) andpositively with K, Mg, Mn, Na, Olsen P, TN and soil pH (r > 0.87 in allcases, P > 0.01). Leucine aminopeptidase activity was only detected inSludge (2778 nmol MUF g−1 soil h−1).

3.5. Enzymes involved in P and S cycling

Phosphatase activity did not differ between the treatments (Fig. 3e),although the phosphorus contents (Olsen and CaCl2-extractable) weredifferent between the four sites. This phosphorus fraction could not bedetected in the Control site (Table 1).

Arylsulfatase activity increased significantly in the two amendedsoils by a factor of 47, whereas the Forest site did not differ from theControl site (Fig. 3f). The activity of this enzyme was negatively corre-lated with available Cr and Cu contents (r = −0.87 in both cases,P b 0.01) and positively correlated with all nutrients (Ca, K, Mg, Mn,

Fig. 3. Activities of β-D-glucosidase, β-D-xylosidase, β-N-acetyl-glucosaminidase, urease, phosphatase and arylsulfatase in the soil samples. Bars with the same letters are not sig-nificantly different at P b 0.05. Means of fifteen replicates are reported with confidence interval (CI).

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Na, Olsen P, TN) and soil pH (r = 0.81, 0.55, 0.53, 0.72, 0.51, 0.83, 0.87and 0.65, respectively, P b 0.05).

4. Discussion

4.1. Microbial and fungal responses to vegetation and sludge amendmentin the mine soil

The microbial biomass C (Cmic) in the mine soil without treatment(Control) and the site vegetated with pines for 22 years (Forest)werelow, but its value was significantly higher in the recently amended(Sludge) and the vegetated and amended (Forest + Sludge). This indi-cates that the use of sludges and combination of them with eucalyptusplantation increased the amount of soil microbial biomass (Cmic) in themine soil. However, the ratio between biomass C and soil total organic Cwas significantly the same between treatments and still much lowerthan expected in microbially active soils, which is 1–4% according toseveral studies (Sparling, 1992; Kandeler et al., 1996). This trendcould indicate that regardless of the high nutrients and organic C con-tents in the treated sites, microorganisms were not able to overcomethe high concentration of metals in soils. It appears that this pollutionbymetalsmasked the beneficial effect of the reclamation strategies pro-vided by organic amendments and vegetation (both root exudates anddebris), even when they are combined.

The microbial biomass N (Nmic) only increased in Sludge and it wasnegatively correlated with available Cr and Cu, which indicates that itwas also negatively affected by the high concentrations of availablemetals in soils. We calculated the Nmic:TN ratio, because it expressesthe amount of potentially available soil inorganic N in the short term(Recous et al., 1990; Yu et al., 2008). The unamended sites (Control

and Forest) had a much lower Nmic:TN ratio than other microbially ac-tive soils (Jenkinson, 1988). After sludge amendment, the Nmic:TNratio changed to characteristic levels for microbiologically active sites(2–6%). Therefore, the turnover of the microbial biomass in amendedsites may release between 60 and 120 kg N ha−1 y−1 for plant growthin mine soils (this calculation is based on Nmic content, bulk density,sampling depth and a turnover time of soil microbial biomass of twicea year).

Comparing the Cmic:Nmic ratio of the different sites (higher in alltreated soils than in Control) revealed that remediation increasedthe C:N ratio of the soil microbial community. A shift in the C:Nratio can be explained on the basis of differences in C and N contentsof bacteria and fungi, and while the C:N ratio of bacteria is expected tobe in the range of 3–6, fungi are characterised by a wider C:N ratio ofabout 5–15 (Paul and Clark, 1996; Harris et al., 1997). The increase ofthe C:N ratio in amended soils revealed that the soil microbial com-munity shifts towards a more fungally-dominated community. Thisresult was supported by our ergosterol data showing significantlyhigher contents in the remediation sites than in the Control. Themajor dominance of ergosterol in the Sludge site could be partlydue to other sterols that may be co-extracted with ethanol anddetected by HPLC with a similar retention time. Nevertheless, sincethe ratio of ergosterol to Cmic was still in the same range than theother treatments, we do not expect a high overestimation of ergoster-ol in the Sludge.

The higher dominance of fungi after sludge amendment could bedue to their feeding behaviour and their sensitivity towards pH aswell as metals. It has previously been reported that an increase insoil organic C increases the ergosterol content (Bastida et al., 2008;Enowashu et al., 2009). Fungi are themajor decomposers of recalcitrant

88 V. Asensio et al. / Science of the Total Environment 456–457 (2013) 82–90

substances in soils (De Boer et al., 2005). Although recalcitrant com-pounds have not been analysed in the present work, it has beenreported that both tree species used (pines and eucalyptus) and theadded sludges (sewage sludges and paper mill residues) contain highconcentrations of lignin, cellulose and hemicellulose (Hattori andMukai, 1986; Martins et al., 1999; Li et al., 2001; Corbeels et al., 2005),which were probably an important source of nutrients for fungi in themine soils.

Although many studies have shown a clear preference of fungiunder acidic soil conditions (Bárcenas-Moreno et al., 2011), the ergos-terol to Cmic ratio did not correlate with the pH values. Therefore, thenutrient limitation of the Control soil may have limited fungal growtheven under acidic soil conditions. In contrast, the relative contributionof fungi depends on pH under conditions of nutrient additions (Sludge,Forest and Forest + Sludge).

Since microbial biomass and ergosterol content were significant-ly negatively correlated with the available fractions of Cr and Cu(r b −0.62 in all cases, P b 0.01), bacteria as well as fungi seemedto be sensitive to these metals. Our results support previous resultsshowing that soil microorganisms are more sensitive to high metalconcentrations in soils than plants or animals (Kandeler et al.,1996; Belyaeva et al., 2005; Moreno et al., 2009).

4.2. Enzyme activity responses to vegetation and sludge amendment inthe mine soil

The highest activities of enzymes involved in the C cycle(β-glucosidase, β-xylosidase andβ-N-acetyl-glucosaminidase)wereob-served in the Sludge site. This suggests that the amendment with sludgesstimulated the activity of the enzymes involved in the carbon cycle muchmore than tree vegetation.We obtained positive significant correlation ofthese enzymes with TOC content. The enzyme β-glucosidase is mainly apart of the cellulose enzyme system and catalyses short chain oligosac-charides and cellobiose (Bhatia et al., 2002) and β-xylosidase catalysesthe hydrolysis of hemicellulose (Zhang et al., 2007). β-glucosaminidaseis a key enzyme involved in the hydrolysis of N-acetyl-β-D-glucosamineresidues from the terminal non-reducing ends of chitooligosaccharidesand it plays an important role in both C and N cycling in soil (Parhamand Deng, 2000; Seidl, 2008). The high concentrations of hemicellulose,cellulose, sugars and carbohydrates in the sludge added to mine soil(Hattori and Mukai, 1986; Li et al., 2001) probably stimulated microor-ganisms to increase β-glucosidase and β-xylosidase production. Sincefungi are important producers of chitin, we expected that the higherβ-N-acetyl-glucosaminidase activity under sludge amendment can be re-lated to thehigher substrate availability derived fromdecaying fungal bio-mass. Due to the lower metal concentrations in the amended soils incomparison with the untreated, microorganisms were able to grow andmake use of the substrates provided by the sludges.

The activity of enzymes involved in N cycling (urease and leucineaminopeptidase) only significantly increased in the recently amendedsoil (Sludge) due to the high concentration of nitrogen in the sludges.However, the activities of enzymes involved in the N cycle in vegetatedsites (eucalyptus and pine trees) did not differ from the Control, even inthe Forest + Sludge. This implies that the nitrogen supplied by thesludges used decreases its concentration in the soil over time and, con-sequently, the enzymes involved in the N cycle are lower in the soilamended for 10 years than in the soil amended for 6 months. Toavoid N limitation for tree growth, the regular addition of sludges mayoffer a possibility to provide organic nitrogen compounds for microbialN mobilisation. Alternatively, the plantation of legume species, i.e., na-tive plants such as Trifolium pratense L. (red clover) or Medicago sativaL. (alfalfa) (Pedrol et al., 2010), could support the nitrogen nutrition oftrees. It is well known that legume species helps to increase nitrogenconcentration in soils because they can fix atmospheric N significantlymore than other plant species and, therefore, helps to increase its con-centration in soils.

Similar acid phosphatase activities were detected in all sites. It ispossible that this enzyme was independent of substrate availabilityin the different sites, since the pH of the different soils may havebeen the most important factor regulating its production and activity.It is well known that the optimum pH for phosphatase is around 4 forthe acid form, 6–7 for the neutral form and 10–11 for the alkalineform (Arevalo et al., 1993; Garcia et al., 1993). Since soil pH increasedup to 8.2 in the Sludge site, we expect that microorganisms may haveproduced different isoenzymes characterised by an alkaline pH opti-mum (Garcia et al., 1993). In addition, the activity of the acid phos-phatase could have been negatively affected in the Sludge site dueto the feedback inhibition by inorganic phosphate usually found insewage sludges (Nannipieri et al., 1979; Smith et al., 2006). Thearylsulfatase activity of unpolluted forest soils has been reported asbeing in the range of 23–75 μg p-nitrophenol g−1 soil h−1 and ureasein the range of 20–30 μg N g−1 soil 2 h−1 (Dilly and Paolo Nannipieri,2001; Hinojosa et al., 2004; Gelsomino and Azzellino, 2011). Thearylsulfatase and urease activities of the Control and Forest sites wereclearly affected by the metal pollution in the soils, as their activitieswere much lower than previously described (Table 1, Fig. 3d and f).The arylsulfatase activities of the sites amendedwith sludgewerewithinthe range of unpolluted sites. Urease activity was much higher thanexpected in the forest sites, probably due to the high fraction of organicnitrogen in the sludge.

The specific enzyme activity is a valid indicator of microbial effi-ciency in the utilization of energy and the degree of substrate limita-tion for soil microorganisms (Gavrichkova et al., 2010). The sitestreated with sludges decreased the specific activity of all enzymes in-volved in the C and N cycle (Table 2), indicating that their microbialcommunity is characterised by higher C and N use efficiency thanunamended soils.

5. Conclusions

The application of sludges to mine soil contaminated by metalswas a suitable treatment to increase both bacterial and fungal abun-dance (Cmic and ergosterol content) as well as microbial functions(arylsulfatase, urease, glucosidase, xylosidase and glucosaminidaseactivities). We also observed that amending with sludges increasedthe efficiency of the soil's microbial community to metabolize C andN. This was indicated by the decrease of the specific enzyme activitiesand the increase in the ratio Cmic:Nmic (shift towards predominance offungi instead of bacteria). Planting eucalyptus or pine trees also stim-ulated soil microbial activity, but did not increase microbial biomassand function to the level of unpolluted sites. In the sludge amend-ment site, the possible negative effects of metals were overcome byhigh substrate availability for microorganisms. Regardless of thefavourable effect of treatments, the negative influence of pollutionby metals could still be noticed in all of the treated soils, since themicrobial biomass C to soil organic C ratio was lower than usual inunpolluted sites. Since tree vegetationwithout additional nutrient sup-ply did not fully recover soil microbial communities, it is recommendedfor the future remediation of mine soils to periodically add sludge or toimprove nutrient availability for trees by planting native legume speciesto support nitrogen nutrition. Studies like this should be carried out inother seasons to observe differences in soil biological characteristicsdue to different weather conditions.

Conflict of interest

The authors declare that there are no conflicts of interest.

Acknowledgements

This work was supported by the Spanish Ministry of Educationand Science through project CGL2009-07843 and by the University

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of Vigo through a pre-doctoral fellowship awarded to V. Asensio. Theauthors would like to thank Heike Halswimmer for helping with theergosterol and MUF analyses, to Sabine Rudolph for her assistancewith the arylsulfatase and urease determinations, and to the mem-bers of Professor Kandeler's team at the University of Hohenheimfor their help in the biological analyses. We thank F.A. Vega and M.L.Andrade for their valuable advices. We also thank the anonymous re-viewers for their comments which triggered some new thoughts andthus helped to improve the quality of this paper.

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