Geoderma 221–222 (2014) 40–49

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Changes in the functional properties of a sandy loam soil amendedwith biosolids at different application rates

Luigi Sciubba a,⁎, Luciano Cavani a, Andrea Negroni b, Giulio Zanaroli b, Fabio Fava b,Claudio Ciavatta a, Claudio Marzadori a

a Department of Agricultural Sciences, Alma Mater Studiorum-University of Bologna, viale Fanin, 40, I-40127 Bologna, Italyb Department of Civil, Chemical, Environmental and Materials Engineering, Alma Mater Studiorum-University of Bologna, via Terracini, 28, I-40131, Bologna, Italy

⁎ Corresponding author. Tel.: +39 051 2906214.E-mail address: luigi.sciubba@unibo.it (L. Sciubba).

0016-7061/$ – see front matter © 2014 Elsevier B.V. All rihttp://dx.doi.org/10.1016/j.geoderma.2014.01.018

a b s t r a c t

a r t i c l e i n f o

Article history:Received 8 May 2013Received in revised form 9 January 2014Accepted 21 January 2014Available online xxxx

Keywords:BiosolidsRice huskSewage sludgeSoil functionalityDose effectDGGE

The goal of this research was to study the impact of the application rate of biosolids from municipal sewagesludge on soil functionality. The biosolids originated from the composting of aerobic or anaerobicmunicipal sew-age sludge with rice husk in the ratio 1/1 v/v. The products were applied at increasing doses, 50 (1×), 150 (3×),and 300 (6×)mgN kg−1

ds, on a sandy loam soil. In order to highlight their impact on soil properties and evaluatetheir possible deleterious effects, soil functional parameters (soil microbial biomass, soil enzyme activities, andsoil bacterial population) were used. Outcomes showed that the increase of the application rate had significantimpact on microbial biomass carbon, which increased by 5%, 9% and 21% in 1×, 3× and 6× with respect to theuntreated soil. Biosolid application rate influenced soil enzyme activities, such as β-glucosidase, dehydrogenase,protease and alkaline phosphomonoesterase which sharply increased at 3× and 6×, especially in the soilsamended with the aerobic biosolid. Soil total bacterial population proved to be stable and not affected, at anydose, by biosolid addition.Concerning total trace metals, no dose effect was registered, as their concentrationswere the same for each doseand treatment; on the contrary, available copper diminishedwith application rate. On thewhole, soil functional-ity was not negatively affected by biosolid application.

© 2014 Elsevier B.V. All rights reserved.

1. Introduction

The use of organic amendments, such as municipal solid waste(MSW) and sewage sludge (SS), is a common practice to improve phys-ical, chemical and biological properties of depleted soils by supplyingorganic matter (Carbonell et al., 2011). In particular, sandy soils, thatare poor in clayminerals and organic colloids, are affected by low fertil-ity caused by low water holding capacity and shortage of nutrients.Good agricultural practices involve frequent applications of organic fer-tilizers, both conventional, such as manure or plant residues, as well asnon-conventional, such as peat, brown coal and SS. Their addition notonly improves soil properties but also helps to solve serious environ-mental problems concerning disposal of large quantities of differentwastes (Weber et al., 2007). Depending on the nature and treatment,organic amendments may affect size and activity of soil microflora; in-deed soil microorganisms are essential to agricultural systems, playingan important role in cycling carbon and nutrients and therefore affect-ing soil fertility (Nannipieri et al., 2002).

On the other hand, current guidelines and regulations require MSWandSS to beproperly treated before land application to reduce pathogens,

ghts reserved.

minimize environmental risks and enhance agronomic performances(CEC, 1986; Franco-Otero et al., 2012; US EPA, 1993; US EPA, 1994).Composting represents an established treatment option, by which MSWand SS are subjected to controlled aerobic conditions designed to pro-mote biological degradation and transformations of organic matter intoa humus-like product (Epstein, 1996). Moreover, community legislationin the European Union (EU) considers that biosolids, MSW and SS com-posts may substantially benefit the climate change given their action oncarbon sequestration (European Community, 2001).

However, according to several works, the main sources of metalinput in agricultural soils include the use of SS compost (Carbonellet al., 2009) and MSW compost (Smith, 2009), which are of particularconcern because of their potential risk for the environment (Carbonellet al., 2011). Thus, the beneficial aspect of compost amendment shouldbe assessed together with the potentially detrimental ones (Weberet al., 2007).

MSW and SS composts in recent decades have received great atten-tion (Senesi et al., 2007) and the literature on the effects of organicamendments on soil microbial biomass activity is extensive. Most stud-ies have consistently shown enhancement of soil microbial biomasscarbon, basal respiration and enzyme activities, with variations in soilmicrobial community structure (Bastida et al., 2008; Garcia-Gil et al.,2004, 2000), but, in some cases, also an increase of heavymetal concen-tration has been found (Baldantoni et al., 2010; Garcia-Gil et al., 2000).

Table 1Main physical and chemical characteristics of the employed soil.

Texture Sandy loam soil

Soil classification (soil taxonomy) Aquic XeropsammentSand (g kg−1) 790Silt (g kg−1) 100Clay (g kg−1) 110pH (pH unit) 7.3 ± 0.1CEC (cmol(+) kg−1) 15.3 ± 0.3Total carbonates (g CaCO3 kg−1) 440 ± 8Active carbonates (g CaCO3 kg−1) 75 ± 8Total organic C (g kg−1) 14.0 ± 0.5Total N (g kg−1) 1.38 ± 0.1C:N ratio 10.1

Data expressed on dry matter, mean ± standard error (n = 3).

41L. Sciubba et al. / Geoderma 221–222 (2014) 40–49

In the latest years Fernandez et al. (2007) focussed their attention onthe comparison of thermally dried and composted SS in a short-termlab-scale experiment and in a mid-term field study (Fernandez et al.,2009). Recently Franco-Otero et al. (2012) investigated the short-termeffect of such amendments on microbial biomass, activity and soilchemical properties.

In a previous work (Sciubba et al., 2013) we evaluated the effects onsoil fertility of biosolids obtained by composting urban SS and rice husk,which had, at the employed compost dose (50mgNkg−1

ds correspond-ing to 8 Mg ha−1

dm approximately), a weak effect on enzyme activitiesand heavy metal concentration.

Brown and Cotton (2011) carried out a field survey in California toquantify the benefits of applying compost to agricultural soils, in farmsites, at different application rates. Their results showed an improve-ment in some soil quality indicators (soil organic carbon, bulk density,nutrients availability, and soil respiration) as a result of compost appli-cation and underlined that the largest response to compost amendmentwas found in the sites that received the highest cumulative loadingrates. Moreover, in this study the effects of compost application rateswere less clear, despite the trends towards more pronounced differ-ences in soil properties with higher application rates, as other factorssuch as soil texture influenced the measured variables (Brady andWeil, 2002). It is likely that a higher control of other factors includingsoil typewould lead to amore linear response to increased compost ap-plication rates (Brown and Cotton, 2011) and this could be achieved in alab-scale experiment.

The effects of biosolids on soil quality can be expressed through theuse of indicators such asmicrobial biomass content, metabolic quotient,microbial C-to-organic C ratio, soil enzymatic activities (Anderson andDomsch, 1990; Baath, 1989; Brookes, 1995; Dick, 1994; Giller et al.,1998; Pavan Fernandes et al., 2005; Wardle and Ghani, 1995) andstudying the biodiversity of the soil indigenous microbial community(Sampedro et al., 2009), as the application of biosolids can stimulatesoil microbial activity, due to an increase in available carbon and nutri-ents, or inhibit activity, due to the presence of heavy metals and otherpollutants (Pavan Fernandes et al., 2005).

Particularly, microbial activity and soil fertility are closely related be-cause it is through the biomass thatmineralization of important organicelements (C, N, P, and S) occurs (Frankenberger and Dick, 1983; Garcia-Gil et al., 2000). Studies onmicrobial biomass carbon and enzyme activ-ities provide information on the biochemical processes occurring in soiland there is evidence that soil biological parameters are early and sen-sitive indicators of soil ecological stress and restoration (Dick andTabatabai, 1992), especially in soil treated with organic amendments.

Moreover, fingerprinting DNA-targeted molecular analyses areemployed to get more insights into the structure and dynamics of mi-crobial based processes and ecosystems, where just 1% of the total bio-mass is cultivable (Amann et al., 1995). In particular, PCR–DGGE(Denaturing Gradient Gel Electrophoresis) has been used to study bac-terial diversity in soils (Ding et al., 2013; Torsvik et al., 1998; Valánkováand Baldrian, 2009; Van Elsas et al., 2002) and during composting(Sampedro et al., 2009).

On the other hand, the measurement of biological activities of soilhas become an interesting subject of investigation, not only because oftheir importance to soil function and structure, but also as changes in bi-ological activity may be used as indicators of soil pollution (Lakhdaret al., 2011). The decrease of some enzyme activities, such as phospha-tase and urease, may be related to the presence of heavymetals inMSWand SS composts. The increase of total heavymetal concentration, espe-cially in sandy soils, should be considered as a matter of enhanced risk,as they usually form relatively soluble species in these soils (Kabata-Pendias and Pendias, 2001).

Considering all these issues, in the present work we investigated, ona laboratory scale, the impact on the functionality of a sandy loam soil ofbiosolids from municipal SS composted with rice husk (Sciubba et al.,2013) at three different and increasing application rates, in order to

better understand the response of soil properties to the amendmentand underline such effects, with special focus on microbial biomass,basal respiration rate, enzyme activities, total and available heavymetals, and bacterial community structure.

2. Materials and methods

2.1. Soil, biosolids, and chemicals

The biosolids A, B and Cwere used to amend a cultivated sandy loamsoil (Aquic Xeropsamment) whose superficial horizon (0–25 cm) wascollected and its features are reported in Table 1. The soil sampleswere taken in a farm located at 44° 06′ 29″ latitude Nord and 12° 31′05″ longitude Est (Torre Pedrera, Rimini, Emilia-Romagna region,Northern Italy).

The two biosolids frommunicipal SS compostedwith rice husk camefrom anaerobic (product A) and aerobic (product B) SS (both describedin a previous work, Sciubba et al., 2013), whilst the third product (C)was obtained through composting of solid waste, municipal sludge(5% w/w on dry matter) and green manure. A subsample for each bio-solid was analysed according to the European methods (EC Regulation2003/2003), described in Sciubba et al. (2013), and are listed in Table 2.

Total N content, which was employed to establish the applicationrate, was 1.6%, 2.0% and 2.5% on dry matter, respectively for productsA, B and C. The C/N ratio was 13.0 for biosolids A and B, whilst it was10.3 for C (Table 2).

The stability of biosolids was evaluated by a modified Oxitop®method according to Grigatti et al. (2007). Briefly, an amount of freshproduct corresponding to 2.00 g of volatile solids was weighted in a 1L glass-bottle, with 10 mL of a complete nutrient solution, 10 mL ofphosphate buffer pH at 7.0, 180 mL of deionized water and 2.5 mL ofATU (allil-thio-urea) as nitrification inhibitor. The bottle capwas consti-tuted by a soda lime-trap for the adsorption of CO2 and by the Oxitop®head, able to continuously register the pressure drops due to the respi-ration of organic material. The system was incubated for 14 days at25 °C under continuous orbital shaking at 100 rpm. At the end of thisperiod, COU (cumulative oxygen uptake) and OUR (oxygen uptakerate)were calculated. The comparison of the obtained OURwith the ref-erence values of the method (Veeken et al., 2007) allowed us to classifythe biosolids according to their stability.

All chemical reagents were purchased from Sigma-Aldrich (Milan,Italy), Carlo Erba (Milan, Italy) and Merck (Darmstadt, Germany).

2.2. Experimental design

The soil, milled and sieved at 2 mm, was pre-incubated at 25 °C and66% of its water holding capacity (WHC) for 2 weeks. The biosolids,dried, milled and sieved at 0.5 mm were added to the soil at differentamounts according to the established dose: zero (no biosolid), 50,150, and 300 mg kg−1 of total N, corresponding approximately to zero(0×), 7.5 (1×), 23 (3×), and 46 (6×) Mg ha−1 of biosolid (dry matter

Table 2Main physical and chemical characteristics of the employed biosolids.

Characteristics A B C

Raw materials and mixture (%w)

Anaerobic municipal sewagesludges (72) + rice husk (28)

Aerobic municipal sewagesludges (72) + rice husk (28)

Green manure (50) + municipal solidwaste (45) + municipal sewage sludge (5)

Moisture (g kg−1) 615 617 474Volatile Solids (g kg−1) 468 588 463Total Organic C (g kg−1) 208 260 257Dissolved organic C (g kg−1) 1.0 6.5 5.0Total N (g kg−1) 16 20 25Ammoniacal N (g kg−1) 0.35 1.4 0.07Nitric N (g kg−1) 0.94 3.2 0.02NH4

+–N/NO3−–N 0.37 0.44 3.5

DOC/Nin 0.775 1.41 55.5Organic N (g kg−1) 14.7 15.4 24.9C:N ratio 13.0 13.0 10.3Electrical conductivity (dS m−1) 3.70 5.46 4.08pH in water (−log[H+]) 7.5 6.4 8.2Total Cd (mg kg−1) 0.19 ± 0.05 b0.1 b0.1Total Cr (mg kg−1) 56 ± 2 5.1 ± 1.1 6.6 ± 1.3Total CrVI (mg kg−1) b0.5 b0.5 b0.5Total Cu (mg kg−1) 212 ± 6 95 ± 3 134 ± 11Total Hg (mg kg−1) b0.5 b0.5 b0.5Total Mn (mg kg−1) 487 ± 19 318 ± 5 361 ± 30Total Ni (mg kg−1) 35 ± 1 32 ± 2 28 ± 1Total Pb (mg kg−1) 40 ± 3 16 ± 1 79 ± 5Total Zn (mg kg−1) 391 ± 17 118 ± 4 225 ± 20OUR (mmol O2 kg−1

VS h−1) 4.36 ± 0.60 6.49 ± 0.17 12.14 ± 0.91

Data expressed on dry matter content, excluding moisture.

42 L. Sciubba et al. / Geoderma 221–222 (2014) 40–49

basis). The supplies of N, DOC and trace metals, expressed as mass perarea unit, for each product at different doses, are reported in Table 3.

All pots were put in static incubation at 25 °C for about 14 weeks(98 days) and pots with unamended soils were incubated in the sameconditions of the amended ones. Moisture was kept constant duringthe tests by adding distilledwater, if necessary. At the endof the incuba-tion period, soil pH, electrical conductivity (EC), soil inorganic N (Ncum),soil microbial biomass C (Cmic), basal respiration rate (BAS), metabolicquotient (qCO2) and microbial biomass-C-to-total organic C ratio(Cmic:Corg ratio), fluorescein diacetate (FDA) hydrolysis and other soilenzyme activities, total and diethylen-triamine-penta-acetic acid(DTPA) extractable trace metals were determined. Moreover,metagenomic DNA was extracted from soil samples, bacterial 16S DNAgene was amplified by PCR and total bacterial populations werescreened in DGGE.

2.3. Methods

Soil pH and ECwere determined through International StandardizedMethods (ISO, 10390, 2005; ISO 11265, 1994).

Soil microbial biomass C (Cmic) was determined through thefumigation–extraction method (Vance et al., 1987), as describedin Sciubba et al. (2013).

Table 3Amounts of organic carbon (C), nitrogen (N) and trace metals applied with biosolids at the dif

Product Dose BiosolidMg/ha

VSMg/ha

TKNkg/ha

DOCkg/ha

Cdkg/ha

A 1× 9.4 4.4 150 9.4 0.0023× 28 13 450 28 0.0056× 56 26 900 56 0.011

B 1× 7.5 4.4 150 49 0.0013× 23 13 450 146 0.0026× 45 26 900 293 0.005

C 1× 6.0 2.8 150 30 0.0013× 18 8.0 450 90 0.0026× 36 17 900 180 0.004

Basal respiration rate (BAS) and inorganic nitrogen (Ncum) were de-termined at the end of the incubation period as in Sciubba et al. (2013).

Metabolic quotient (qCO2) and biomass-C-to-total organic C ratio(Cmic:Corg ratio) were calculated respectively as the ratio betweenbasal respiration rate (BAS) and microbial carbon (Cmic) and as theratio between Cmic and Corg (Anderson and Domsch, 1990).

Fluorescein diacetate [3′-6′-diacetylfluorescein (FDA)] hydrolysiswas determined according to Adam and Duncan (2001). Dehydroge-nase activity was determined as described by Von Mersi and Schinner(1991). β-Glucosidase activity was determined with the method sug-gested by Eivazi and Tabatabai (1988). Alkaline phosophomonoesteraseactivity was estimated as described in Eivazi and Tabatabai (1977) andNannipieri et al. (2011). Protease activity was determined with themethod by Ladd and Butler (1972).

Metagenomic DNA was extracted and purified from 500 mgsoil withdrawn from each replicate pot with the UltraClean SoilDNA kit (MoBio Laboratories, Carlsbad, CA, USA) according to themanufacturer's instructions. Bacterial 16S rRNA genes were PCR-amplified with primers GC-357f and 907r (Sass et al., 2001) in 50 μLreaction mixtures containing 1× PCR buffer (Invitrogen, Paisley, UK),1.5 mM MgCl2, 0.12 mM each dNTP, 0.3 mM each primer, 0.5 mg/mLBovine Serum Albumin, 1% Formamide, 1.0 U of Taq polymerase(Invitrogen, Paisley, UK) and 10 ng of template DNA. The thermal

ferent rates (dry matter basis).

Cukg/ha

Mnkg/ha

Nikg/ha

Pbkg/ha

Znkg/ha

Hgkg/ha

Crkg/ha

2.0 4.6 0.33 0.38 3.7 0.005 0.536.0 13.7 0.98 1.13 11 0.014 1.5812 27.4 1.97 2.25 22 0.028 3.150.7 2.4 0.24 0.12 0.9 0.004 0.042.1 7.2 0.72 0.36 2.7 0.011 0.114.3 14.3 1.44 0.72 5.3 0.023 0.230.8 2.2 0.17 0.47 1.4 0.003 0.042.4 6.5 0.50 1.42 4.1 0.009 0.124.8 13.0 1.01 2.84 8.1 0.018 0.24

Table 4Summary of ANOVA for effect of dose and treatment on pH and electrical conductivity(EC).

Factor pH EC (dS m−1)

Dose *** ***0× 7.83 ab 0.328 c1× 7.88 a 0.318 c3× 7.81 bc 0.378 b6× 7.76 c 0.436 aLSD 0.066 0.175Biosolid * ***A 7.79 a 0.358 bB 7.83 a 0.380 aC 7.84 a 0.357 bLSD 0.051 0.136Interaction * **

† Signif. code of ANOVA: “***”, p ≤ 0.001; “**”, p ≤ 0.01; “*”, p ≤ 0.05; “NS”, p N 0.05;marginal mean, in bracket standard error of the mean; LSD:least significant difference.‡ Means with the same letter are not significantly different for p ≤ 0.05.

43L. Sciubba et al. / Geoderma 221–222 (2014) 40–49

cyclingwas as described in Zanaroli et al. (2010). PCR products were re-solved with a D-Code apparatus (Bio-Rad, Milan, Italy) on a 7% (w/v)polyacrylamide gel with a denaturing gradient from 40% to 60% dena-turant as described previously (Zanaroli et al., 2012).

Total trace metal concentrations in soil were determined after aciddigestion. Briefly, an amount of 0.250 g of crushed soil was weightedinto PTFE recipients, added with 6 mL of HCl 37% and 2 mL of HNO3

65% and digested in a microwave oven (Milestone, Shelton, CT, USA).The digested suspensionwasfiltered throughWhatman no.42 paperfil-ters and brought to 20 mL with deionized water. The resulting solutionwas analysed through OES-ICP spectrophotometer for the determina-tion of trace metals.

Bioavailable tracemetals were determined according to Linsday andNorwell (1969).

2.4. Data analysis

Statistical analysis followed a 2-way factorial design, were “dose”(4 levels) and “biosolid” (3 levels) were the main factors. ParametricANOVA assumptionswere verified through Levene's test for homogene-ity of variances and Shapiro–Wilks test for normality of distributions.The significance of all statistical tests was assessed at α = 0.05. Posthoc Holm adjusted Fisher's LSD test was performed to investigate spe-cific interactions between treatment differences when ANOVA returneda significant global test. All statistics were performed using R version2.15.2 (R Development Core Team, 2010).

3. Results and discussion

3.1. Biosolids

Biosolid B obtained from aerobic municipal SS had the highest con-tent in volatile solids and TOC, whilst biosolid A, from anaerobic munic-ipal SS, showed the lowest concentrations of TOC and TN (Table 2). TheC:N ratio was similar in all biosolids and ranged from 10 to 13. EC wasgenerally lower than 5.5 dS m−1 and was higher in B than in the otherproducts. The pH was sub-alkaline for A and C and sub-acid for B. TheDOC content, an indirect measure of biosolid maturity (Zmora-Nahumet al., 2005), was low for biosolid A and moderate for biosolids B andC, respectively (Table 2), therefore sample A should have a higher de-gree of maturity than the other biosolids. Inorganic N (ammonia and ni-trate) was 1.3 and 4.6 g kg−1

dm for samples A and B, respectively, whilstit was very low for sample C. All the biosolids showed values of totalheavymetals within the limits fixed by the current European legislationfor the use of SS in agriculture (ECDirective 86/278/EEC) and by the Ital-ian legislations (D.Lgs. 99/1992 and 75/2010). The other propertieswere discussed in a previous work by Sciubba et al. (2013).

The calculated values of OUR were quite low, being 4.36, 6.49 and12.14mmol O2 kgvs−1 h−1 for biosolids A, B and C, respectively. In agree-mentwith Veeken at al. (2007), theOUR of the anaerobic biosolid Awasless than 5 mmol O2 kgvs−1 h−1 and therefore it can be considered “verystable”, whilst the aerobic biosolid B and the conventional compost Ccan be considered “stable”, because their OUR values were in therange 5–15 mmol O2 kgvs−1 h−1. The higher OUR values calculated forbiosolids B and C are probably related to their higher content in dis-solved organic carbon than biosolid A.

3.2. Soil pH and EC

At the end of the incubation period there were slight differences inpH values (Table 4). On the other hand, EC (Table 4) was found to havea significant variation with doses, ranging from 0.318 dS m−1 of the1× application rate to 0.378 dS m−1 of the 3× one and 0.436 dS m−1

of the 6× one; the aerobic biosolid was the one significantly increasingthe EC value, whilst the A amended soils showed EC values similar to

the one of the conventionally amended soil. These results are in agree-ment with the EC of biosolids.

3.3. Microbial biomass carbon

As shown in Table 5, significant differences in Cmic were found in 6×dose, which increased Cmic up to 232 mg kg−1

ds (+30% with respect tothe untreated soil). Regarding treatments, only aerobic biosolid B wasshowing a significant increase in Cmic with respect to the others; suchincrease was mainly evident at the 6× dose, which had 273 μg C g−1,with respect to 193 and 211 μg C g−1 of 1× and 3× doses (Fig. 1a).These values were of the same order as of the ones reported byGarcia-Gil et al. (2000) with MSW compost at different doses (20 and80 Mg ha−1), even if lower with respect to the ones described byFranco-Otero et al. (2012) with SS compost and thermally dried com-post. However, the addition of such biosolids did not have inhibitory ef-fects onmicrobial biomass up to the studied doses, as instead registeredin other works, probably due to heavy metal content (Brookes andMcGrath, 1984; Pedra et al., 2007).

3.4. Basal respiration rate and eco-physiological indicators

Corgwas (Table 5) in the range 13–15 g C kg−1ds; also in this case, the

clearest differenceswere found at the highest application rate, whilst nodifferences were found amongst the treatments.

BAS (Table 5)was significantly higher in the 6× dosewith respect tothe other application rates; moreover it was higher for the B amendedsoil (0.188, 0.202, 0.334 μg CO2-C g−1 h−1 at 1×, 3× and 6× doses re-spectively) than for theA andC ones, at each dose. Therefore the highestdose better underlined the different behaviour of different products, asit stressed their effects on basal respiration.

As Cmic had the values discussed in the previous paragraph, qCO2

(Table 5) was almost the same for all the treatments and doses; indeedthe highest respiration rate, registered at 6× dose and with the aerobicbiosolid B, corresponded to the highest microbial carbon concentration.Therefore no significant differences were found for qCO2. For Cmic-to-Corg ratio, there were no significant differences as the increase of micro-bial biomass carbon at the higher application rates was balanced by theincrease of total organic carbon; only B amended soils had Cmic-to-Corgratios significantly higher with respect to other treated soils.

The effects of biosolid addition on basal respiration, due to micro-bial biomass and nutrient supply, were described in many works,which usually reported higher increases in respiration rate, especial-ly at elevated doses, with respect to those measured in the presentwork (Brown and Cotton, 2011; Huang and Chen, 2009; PavanFernandes et al., 2005). Indeed, the impact of these biosolids onBAS, qCO2 and Cmic:Corg ratio was observed especially at the highest

Table 5Summary of ANOVA for effect of dose and biosolid treatment on Corg, Cmic, basal respiration rate (BAS), qCO2, Cmic-to-Corg ratio, and Ncum.

Factor Corgmg C g−1

Cmic

μg C g−1BASμg CO2-C g−1 h−1

qCO2

mg CO2-C g−1 Cmic h−1Cmic:Corgmg g−1

Ncum

mg N kg−1

† ‡

Dose *** ** *** ** NS ***0× 12.9 c 186 b 0.152 c 0.835 b 14.5 107 c1× 13.6 bc 170 b 0.174 b 1.040 a 12.6 103 c3× 14.0 b 188 b 0.184 b 0.990 ab 13.5 124 b6× 15.6 a 232 a 0.259 a 1.122 a 14.9 141 aLSD 0.8 39 0.013 0.186 3.1 10Biosolid NS ** *** NS * ***A 14.1 184 b 0.181 b 1.000 13.1 b 114 bB 14.0 217 a 0.220 a 1.002 15.6 a 128 aC 13.9 182 b 0.177 b 0.989 13.0 b 115 bLSD 0.6 30 0.010 0.143 2.4 8Interaction NS NS *** NS NS **

† Significance code of ANOVA: “***”, p ≤ 0.001; “**”, p ≤ 0.01; “*”, p ≤ 0.05; “NS”, p N 0.05; marginal mean, in bracket standard error of the mean; LSD: least significant difference.‡ Means with the same letter are not significantly different for p ≤ 0.05.

a

0

50

100

150

200

250

300

350

Dose

C m

ic (

µgC

g-1

)

A

B

C

b

0.0

20.0

40.0

60.0

80.0

mg

flu

ore

scei

n k

g-1

h-1

A

B

C

c

0.0

20.0

40.0

60.0

80.0

100.0

mg

INT

kg

-1h

-1

A

B

C

6x3x1x

Dose6x3x1x

Dose6x3x1x

Fig. 1. Soil quality indicators microbial biomass carbon (a), fluorescein diacetate hydrolysis (b), dehydrogenase (c), β-glucosidase (d), alkaline phosphomonoesterase (e) and protease(f) activities at the end of the incubation period for soils amended with biosolids A (white bars), B (black bars) and C (grey bars) at different doses (1×, 3×, and 6×).

44 L. Sciubba et al. / Geoderma 221–222 (2014) 40–49

d

0.0

10.0

20.0

30.0

40.0

50.0

Dose

mg

PN

P k

g-1

h-1

mg

PN

P k

g-1

h-1

A

B

C

e

0

50

100

150

200

250

300

A

B

C

f

0

25

50

75

100

125

150

175

200

mg

tyr

kg

-1h

-1

A

B

C

6x3x1x

Dose6x3x1x

Dose6x3x1x

Fig. 1 (continued).

45L. Sciubba et al. / Geoderma 221–222 (2014) 40–49

application rate, where BAS had an increase of 70% with respect tothe control.

3.5. Inorganic nitrogen release

The cumulative inorganic N release (Ncum) at the end of the incuba-tion period is reported in Table 5.

Ncum was found in the soils mostly as nitrate (data not shown);amongst doses, the highest valueswere registered in the 6× applicationrate, followed by 3× and 1×, which was not significantly different fromthe control. This behaviour is clear as the different doses are character-ized by different N supplies. Amongst treatments, only aerobic biosolidB was significantly affecting Ncum release, which was higher with re-spect to the other amended soils. As amatter of fact, the anaerobic prod-uct (A) and especially the aerobic one (B) had 1.3 and 4.5 g kg−1

dm ofinorganic N respectively, whilst inorganic N concentration in the tradi-tional compost was negligible; therefore the final Ncum concentrationshould be ascribed both to the mineralization of added organic N andto the initial inorganic N occurrence in the products.

Therefore these results confirmed the behaviour of the biosolids de-scribed in our previouswork (Sciubba et al., 2013), which showed a ten-dency to accumulate nitrate and a low mineralization percentage (lessthan 10%). Indeed, the employed soil is sandy loamy and its pH is sub-alkaline (7.3), therefore it mineralizes organic N as nitrate; howeverNcum final concentration is lower than the one reported by Huang andChen (2009) in awork on SS compost applied on different soils at differ-ent application rates (the highest of them comparable with our 6×dose), suggesting a low N mineralization capability of biosolids fromMSW composted with rice husk.

3.6. Fluorescein diacetate hydrolysis and other soil enzyme activities

The values and ANOVA for fluorescein diacetate (FDA) hydrolysisand other soil enzyme activities are reported in Table 6.

FDA hydrolysis was significantly higher at 6× dose than at 3× and1× ones. The soils amended with B had the highest activity (37.1,40.8, 56.7 mg fluorescein kg−1 h−1 at 1×, 3× and 6× respectively, asshown in Fig. 1b) with respect to those amended with C and A, withthe highest differences shown at the highest application rate. As FDA

Table 6Summary of ANOVA for effect of dose and treatment on diacetil fluoresceine hydrolase acitivity (FDA), β-glucosidase activity (β-GLU), dehydrogenase activity (DH), alkaline phosphataseactivity (ALK P), and protease activity (PROT).

Factor FDAmg fluor. kg−1 h−1

DHmg INT kg−1 h−1

β-GLUmg PNP kg−1 h−1

ALK Pmg PNP kg−1 h−1

PROTmg Tyr kg−1 h−1

Dose † ‡

*** *** *** *** ***0× 30.9 c 46.3 c 27.3 c 160 c 105 c1× 35.0 bc 48.1 c 27.3 c 173 bc 102 c3× 37.7 b 53.3 b 30.1 b 182 b 128 b6× 49.5 a 67.4 a 34.2 a 232 a 151 aLSD 4.7 6.9 2.6 20 16Biosolid ** *** ** * *A 37.1 b 52.5 b 28.7 b 189 ab 123 abB 41.6 a 60.8 a 31.4 a 196 a 127 aC 36.1 b 48.0 b 28.9 b 176 b 114 bLSD 3.6 5.3 2.0 16 13Interaction NS ** * * *

† Significance code of ANOVA: “***”, p ≤ 0.001; “**”, p ≤ 0.01; “*”, p ≤ 0.05; “NS”, p N 0.05; marginal mean, in bracket standard error of the mean; LSD: least significant difference.‡ Means with the same letter are not significantly different for p ≤ 0.05.

46 L. Sciubba et al. / Geoderma 221–222 (2014) 40–49

activity is inversely correlated to biosolid stability (Adam and Duncan,2001; Sanchez-Monedero et al., 2008), A and C confirmed to be morestable than B. In a previouswork (Sciubba et al., 2013)we demonstratedthat the effect of biosolids frommunicipal SS on FDA hydrolysis is weakif compared to other organic amendments (Sanchez-Monedero et al.,2008). The analysis of FDA hydrolysis confirmed that the aerobic biosol-id B mostly affected this soil property and the highest application ratebetter underlined this behaviour.

Dehydrogenase activity was also affected by the application rate, asit varied from 48.1 mg INTF kg−1 h−1 of the lowest dose to67.4 mg INTF kg−1 h−1 of the highest dose. The aerobic biosolid B was,also in this case, the one showing significantly higher activity at each ap-plication rate, being 50, 61 and 84 mg INTF kg−1 h−1 at 1×, 3× and 6×doses respectively (Fig. 1c). Dehydrogenase was found to be a good indi-cator of soil microbial activity, as its increase is positively correlatedwithsoil biomass carbon (Garcia-Gil et al., 2000) whilst it is inhibited byheavy metals, particularly Pb (Marzadori et al., 1996).

β-glucosidase activity was clearly affected by the application rate; theaerobic biosolid B was the only one that was significantly different fromthe other treatments, particularly at the highest dose (Fig. 1d). Indeed,β-glucosidase activity is usually increased by organic amendments, dueto labile C occurring in organic matter (Garcia-Gil et al., 2000).

The same considerations can be made for alkaline phosphomonoes-terase activity. In this case, the increase of application rate from 50 to150 mg N kg−1

ds caused little effects on enzymatic activity, whilst thehighest dose (300mgN kg−1

ds) mostly affected soil response, stimulat-ing enzymatic activity, especially with product B, which reached morethan 250mg PNP kg−1 h−1 at 6× dose (Fig. 1e), but without any nega-tive effect; as a matter of fact, phosphatase can be inhibited not only byheavy metals such as Cu and Zn (Tyler, 1974) added with compost tosoil, but also by inorganic P (Nannipieri et al., 1979). However the im-pact of biosolid addition on phosphomonoesterase activity resulted ina little stimulation as in other short-term studies (Franco-Otero et al.,2012; Lakhdar et al., 2011); no negative effects were shown, as insteadobserved in some long-term experiments (Garcia-Gil et al., 2000).

Protease activity was included between 102 mg tyr kg−1 h−1 of the1× dose and the 151mg tyr kg−1 h−1 of the 6× one; the aerobic biosol-id B was, also in this case, the one significantly affecting this parameter(Fig. 1f). Indeed, this enzyme activity, involved in the hydrolysis of Ncompounds to ammonium (Garcia-Gil et al., 2000) was affected, differ-ently from other short-term work (Franco-Otero et al., 2012), by com-post addition and clearly responded to the increase of application rate.

The analysis of soil enzyme activities showed a general increasewithapplication rate, as expected. The aerobic biosolid B mainly increasedtheir values, confirming its higher content in bioavailable substrates.

For this reason, soil enzyme activities are confirmed to be interestingsoil quality indicators as they promptly responded to system variations,such as compost application, at different doses, also in a short-timeperiod.

3.7. Soil bacterial population

DGGE profiles ofmicrobial populations in soils at the endof the incu-bation are reported in Fig. 2. The same profilewas obtained by each rep-licate of all the treatments and of the unamended controls. None of thebiosolids significantly modified the biodiversity of the indigenous bac-terial population, regardless the dose applied. In addition, no relevantshift in relative band intensity was ever observed, thus suggesting thatthe treatments do not alter the relative abundance of the indigenousspecies. In otherwords, the employed biosolids did not impose selectivepressure on resident bacterial community, despite their positive effecton the metabolic activity and biomass growth observed.

However, it was already reported that DGGE analysis of the totalbacteria might fail in detecting little variations of community composi-tion (Neilson et al., 2013), especially in stable populations characterizedby high species diversity such as in soils; still, it is possible that somespecies actually benefitted or were inhibited by some treatments. Fur-thermore, whilst bacterial profile shifts are observed during composting(Sampedro et al., 2009), significant variations of total bacteria profile areusually found only between different soils (Cotta et al., 2013). In partic-ular, soil type and texture are the main factors which drive bacterial di-versity in agricultural systems by determining local conditions, nutrientavailability and consequently habitable niches (Garbeva et al., 2004).The biosolids employed in this studywere not able to bring a visible im-pact in terms of bacterial diversity even at the higher dose tested.

3.8. Total and bioavailable trace metals

Total metal content (data here not shown) showed the same valuesfor all doses and treatments:Mnwas found in the highest concentration(1300–1500 mg kg−1

ds), Zn was in the range 115–125 mg kg−1ds,

whilst Cu, Pb and Ni were under 100 mg kg−1ds at each dose. Total Cr

had a concentration in the range 30–40 mg kg−1ds, Cd in the range

0.29–0.37 mg kg−1ds, whilst Hg was under 0.1 mg kg−1

ds at eachdose. The amounts of trace metals added with biosolids (Table 3)were quite low with respect to the metal concentration of the controlsoil (0.1–3% according to the metal and product, at the 1× dose), mak-ing it difficult to observe differences amongst treatments. Only at the 6×dose the added metal amount (1–20% of the control) was enough highto underline differences in their concentration which, however, were

Fig. 2. Soil bacterial population analyses. Total bacteria DGGE analyses on treated and control soils at the end of incubation in dose 1× (a), 3× (b), and 6× (c). CONT=dose 0× (untreatedcontrol); A, B, C stand for the three different biosolids. Replicates of each soil were run one close to the other.

47L. Sciubba et al. / Geoderma 221–222 (2014) 40–49

not observed, ruling outmetal accumulation in soil. Moreover the addedmetal amount (expressed as kg ha−1 yr−1) was under the limits fixedby the current European legislation (EC Directive 86/278/EEC, 1986).

The concentration of bioavailable trace metals is shown in Table 7.These data suggested that there were significant dose effects for Cu, thatdecreased at higher doses, probably as itwas linked to the organicmatter,Mn, Ni and Pb, which, on the contrary, were significantly higher at 6×dose. As for the treatments, the behaviour is less clear, as the highest avail-ability value for Cuwas found in A amended soils, whilst forMn and Zn inB ones, for Ni in C ones and no effects of treatmentwere registered for Pb.Cr, Cd and Hg were not found in the DTPA extracts.

The response of soil in terms of tracemetal content to compost addi-tion is really important as, in this study, therewere no sharp increases intheir concentrations after compost application, even at the highest dose(300 mg N kg−1

ds), as it has been instead observed in other works onthe use of SS compost (Baldantoni et al., 2010; Pedra et al., 2007), nornegative effects of metal on enzyme activities were found (Garcia-Gilet al., 2000; Marzadori et al., 1996).

4. Conclusions

The impact on soil functionality of biosolids from municipal SScomposted with rice husk, at three different application rates, was

Table 7Summary of ANOVA for effect of dose and treatment on available (DTPA) trace metals.

Factor Cumg kg-1

Mnmg kg-1

Nm

Dose † ‡

*** *** **0× 20.8 ab 10.7 b 0.1× 21.4 a 10.8 b 0.3× 20.5 b 11.5 a 0.6× 19.4 c 11.4 a 0.LSD 0.6 0.4 0.Biosolid NS *** **A 20.7 10.3 c 0.B 20.6 12.0 a 0.C 20.3 11.1 b 0.LSD 0.5 0.3 0.Interaction * *** N

† Significance code of ANOVA: “***”, p ≤ 0.001; “**”, p ≤ 0.01; “*”, p ≤ 0.05; “NS”, p N 0.05; ma‡ Means with the same letter are not significantly different for p ≤ 0.05.

investigated in this work. Amongst the different treatments, the aerobicbiosolid was the one significantly affecting soil quality indicators as itmostly increased enzyme activities, microbial biomass carbon and res-piration rate; on the other hand, the anaerobic biosolid had a behavioursimilar to the one of the conventional compost.

As to application rate, the highest dose (6×) had a significant impacton soil quality indicators, beinguseful to stress the effects of biosolids onsoil properties and underline the differences amongst them; all enzymeactivities, microbial biomass carbon and respiration rate, at this applica-tion rate, were significantly higher with respect to the other doses. Theemployed parameters, especially soil enzyme activities, were confirmedto be useful soil quality indicators for their prompt response to systemvariation caused by compost application.

Further, bacterial populations of the soils were not impacted by theaddition of these biosolids, thus suggesting a resilience of the indige-nous communities which can withstand a foreign biomass loadwithoutbeing altered or shocked.

Finally, it is important to underline that trace metal concentrationswere not negatively affected by compost application, as it could occurwith the use of municipal SS compost.

Therefore, we can conclude that the biosolids from municipal sew-age sludge compostedwith rice husk, investigated in this work, seemedto be suitable for agricultural use as they did not show any negative

ig kg-1

Pbmg kg-1

Znmg kg-1

* ** NS628 c 7.77 ab 12.9651 b 7.18 b 12.9648 b 7.14 b 12.6695 a 8.40 a 13.0019 0.93 0.6* NS **631 c 7.57 12.4 b660 b 7.52 12.9 a767 a 7.78 13.2 a015 0.72 0.5S NS *

rginal mean, in bracket standard error of the mean; LSD: least significant difference.

48 L. Sciubba et al. / Geoderma 221–222 (2014) 40–49

effect on soil quality indicators in the low term, nor did they have anyevident drawback regarding the diversity of resident bacterial popula-tions, not even at the highest application rate.

Acknowledgements

The authors wish to thank Herambiente S.p.A. (Italy) for financiallysupporting the project.

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