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Applied Soil Ecology 61 (2012) 92– 99

Contents lists available at SciVerse ScienceDirect

Applied Soil Ecology

journa l h o me page: www.elsev ier .com/ locate /apsoi l

ffects of tannery sludge application on physiological and fatty acid profiles ofhe soil microbial community

ndré S. Nakatania, Marco A. Nogueirab, Alexandre M. Martinesa, Cristiane A. Dos Santosa,uis F. Baldesina, Petra Marschnerc, Elke J.B.N. Cardosoa,∗

Universidade de São Paulo, ESALQ, Avenida Pádua Dias, 11, CEP 13418-900, Piracicaba, SP, BrazilEMBRAPA Soja, Caixa Postal 231, CEP 86001-970, Londrina, PR, BrazilThe University of Adelaide, School of Agriculture, Food and Wine, Adelaide, SA 5005, Australia

r t i c l e i n f o

rticle history:eceived 24 April 2011eceived in revised form 27 April 2012ccepted 8 May 2012

eywords:iologarbon source utilizationitrogenHLFA

a b s t r a c t

The impact of tannery sludge application on soil microbial community and diversity is poorly understood.We studied the microbial community in an agricultural soil following two applications (2006 and 2007) oftannery sludge with annual application rates of 0.0, 2.3 and 22.6 Mg ha−1. The soil was sampled 12 and 271days after the second (2007) application. Community structure was assessed via a phospholipid fatty acidanalysis, and the physiological profile of the soil microbial community via the Biolog method. Tannerysludge application changed soil chemical properties, increasing the soil pH and electrical conductivityas well as available P and mineral N concentrations. The higher sludge application rate changed thecommunity structure and the physiological profile of the microbial community at both sampling dates.However, there is no clear link between community structure and carbon substrate utilization. Accordingto the Distance Based Linear Models Analysis, the fatty acids 16:0 and i17:0 together contributed 84%

ludge land utilization to the observed PLFA patterns, whereas the chemical properties available P, mineral N, and Ca, andpH together contributed 54%. At 12 days, tannery sludge application increased the average well colordevelopment from 0.46 to 0.87 after 48 h, and reduced the time elapsed before reaching the midpointcarbon substrate utilization (s) from 71 to 44 h, an effect still apparent nine months after application ofthe higher sludge application rate. The dominant signature fatty acids and kinetic parameters (r and s)were correlated to the concentrations of available P, Ca, mineral N, pH and EC.

. Introduction

The tanning industry generates great quantities of waste duringeather processing (Pacheco, 2005). Producing “wet blue” leather inarticular requires a broad array of chemical compounds (Martinest al., 2010) and generates a nutrient-rich and high pH waste sludge.hus, this sludge, if carefully managed, could be recycled as fertil-zer and acidity-neutralizer, making them a potentially useful tooln agriculture and for the amelioration of degraded soils (Kray et al.,008). In recent years, tanneries have improved the tanning processo reduce the amount of chromium in the waste; hence chromiumollution is no longer the leading concern regarding the disposalf tannery waste. However, there is still concern about the highitrogen and sodium concentrations in the sludge (Nakatani et al.,

011a).

The soil microbial community plays a pivotal role in nutri-nt cycling by mineralizing organic matter and transforming

∗ Corresponding author. Tel.: +55 19 3417 2118; fax: +55 19 3417 2110.E-mail address: ejbncard@esalq.usp.br (E.J.B.N. Cardoso).

929-1393/$ – see front matter © 2012 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.apsoil.2012.05.003

© 2012 Elsevier B.V. All rights reserved.

nutrients. Because it can respond quickly to environmental change,it is considered a sensitive indicator of soil health and of anthro-pogenic disturbance (Bending et al., 2004). However, little isknown about the effect of the application of tannery sludge onsoil biological properties. Trasar-Cepeda et al. (2000) reportedlower microbial biomass and enzyme activities in areas pol-luted with tannery effluent, compared to unpolluted control areasin Spain. On the other hand, the microbial biomass increasedin soils treated with tannery waste in Mexico (Alvarez-Bernalet al., 2006). Barajas-Aceves et al. (2007) also observed tannerysludge stimulated soil microbial activity in Mexico. The micro-bial community structure based on PLFA profiling changed fromgram-positive bacteria to gram-negative bacteria in heavily tan-nery waste-polluted soils in Australia (Kamaludeen et al., 2003).These contrasting results suggest that more studies need to beconducted to assess the effects of tannery sludge of different ori-gins on microbial communities under different soil and climatic

conditions.

Biotic and abiotic factors modulate metabolic diversity andbiological activity (Marschner et al., 2003), and consequently themicrobial community structure (Zak et al., 1994). Therefore, the

ied Soil Ecology 61 (2012) 92– 99 93

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Table 1Physical and chemical attributes of the tannery sludge used in the experiment.

Variable 1stapplication

2ndapplication

Rangec

pHa 12.7 9.7 7.7–11.8EC (dS m−1)a 29.5 16.6 –Total solids, at 65 ◦Ce 53.3 55.4 120.0–390.0Volatile solidse 442.0 554.0 –Neutralization power(CaCO3 eq.)e

262.0 361.0 160.0–315.0

Organic Ce 308.0 321.0 65.0–257.8Total Ne 35.7 53.2 9.8–53.4NH4

+-Ne 20.4 21.9 –NO3

−-Ne 0.2 0.2 –C/N ratio 8.7 6.0 4.1–13.8Cae 78.9 88.0 20.0–210.0Mge 0.7 1.0 0.2–7.5Ke 0.1 3.3 0.1–1.7Pe 3.9 3.8 1.1–7.5Se 36.1 43.0 13.0–15.0Nae 10.0 66.9 8.1–59.9Mnf 2858.0 3340.0 128.0–6350.0Fef 408.0 1249.0 –Bf 4.5 5.6 –Znf 43.3 73.0 48.0–176.0Cuf 4.5 16.0 14.0–81.0Mof 3.3 <0.5b –Alf 2257.0 13,440.0 –Asf <1.0b <0.5b –Cdf <1.0b <0.5b 0.1–4.0Pbf <1.0b 9.3 15.0–35.0Hgf <1.0b <0.5b –Nif 3.0 7.8 1.3–20.0Sef <1.0b <0.5b –Total Crf,d 1613.0 580.0 798.0–22,200.0

a Measured in in natura samples.b Concentrations below the detection limit.c Values obtained from Kray et al. (2008), Alcântara et al. (2007), Barajas-Aceves

et al. (2007), Martines et al. (2006) and Barajas-Aceves and Dendooven (2001).d EU upper limit value of chromium allowed for a sludge to be incorporated

in agricultural soil according to Directive 86/278/EEC = 1000 mg kg−1 (European

A.S. Nakatani et al. / Appl

ssessment of the functional diversity is as important as assessmentf the microbial species diversity (Tótola and Chaer, 2002).

The aim of this study was to assess the metabolic profile andicrobial community structure of soils treated with tannery sludge.ur hypothesis was that the PLFA analysis and carbon substrateonsumption profile of the soil microbial community are sensitivendicators of the effects of tannery sludge application.

. Materials and methods

.1. Experimental design

The experiment was carried out in an agricultural area in theunicipality of Rolândia in Paraná state (23◦17′S, 51◦29′W, 650 m),

razil. The climate at the site is classified as Cfa under the Köp-en system, with hot summers, undefined dry season, and averageemperature of 21 ◦C (means ranging from 16 to 27 ◦C), with meannnual rainfall of 1600 mm falling mostly between September andarch. The study area has been managed for more than 10 years

nder a no-till system with rotating crops (soybean/corn in summernd wheat/oats in winter). The soil has a high clay content (74%)nd is classified as Rhodic Kandiudult (US Soil Taxonomy). Sludgeas first applied at the site in 2006 and reapplied in 2007. The

xperiment had a completely randomized block design with foureplications (15 m × 6 m plots) that had the following treatments:ero, 2.3 and 22.6 Mg ha−1 of tannery sludge (dry weight basis)nnually, corresponding to zero (no sludge), 120 and 1200 kg ha−1

f total N. These relatively high rates are commonly used in Brazilue to lack of federal laws that regulate the limits to be appliedo the soil. The tannery sludge was incorporated into the soil bylowing to 0–20 cm depth 89 days after the first application. Cornas then planted in summer receiving 48 kg ha−1 of P (triple super-hosphate) and 42 kg ha−1 of K (KCl), and black oats in winter, bothithout irrigation. After oat cultivation, sludge was applied again

93 days after the first application and the sludge was kept on theoil surface for 87 days, after which it was plowed and planted withorn.

For this study, soil samples were collected 12 and 271 days afterhe second application of tannery sludge. Nine subsamples wereollected at 0–10 cm of soil depth in each experimental plot andooled. Soil samples were stored at 4 ◦C and sent to the laboratoryor analysis.

.2. Tannery sludge and soil chemical analyses

The tannery sludge used in the experiment was obtained fromhe Curtume Vanzella (Rolândia, Paraná, Brazil) and was composedf equal parts of liming sludge and primary waste treatment (WTP)ludge. Table 1 provides details of the sludge used in both applica-ions and results were expressed in dry basis, after drying at 65 ◦Cor 48 h. The concentrations of N-NH4

+ and N-NO3− + N-NO2 were

etermined by steam distillation (Mulvaney, 1996); pH and elec-rical conductivity (EC) were read directly in samples, and totalnd volatile solids were obtained by drying at 65 ◦C and 500 ◦C,espectively (APHA, 2005). Total organic carbon was analyzed byxidation with dichromate under external heating (Nelson andommers, 1996). Total N was determined by the Kjeldahl methodfter sulfuric digestion (Bremner, 1996); neutralization power vialkalimetry (Brazil, 2007); total Ca, Mg, K, P, S, Na, Mn, Fe, B, Zn,u, Mo, Al, As, Cd, Pb, Hg, Ni, Se, Cr concentrations were deter-ined by ICP-AES (model Vista MPX, Varian, Mulgrave, Australia)

fter nitric digestion in a microwave oven (USEPA, 1986); K anda in the digest were determined by flame photometer. The tan-ery sludge used in this study had a relatively low Cr concentrationompared to sludges used in other studies (Table 1).

Community, 1986).e g kg−1.f mg kg−1.

Soil chemical properties were determined as described inNakatani et al. (2011a). Briefly, the pH and electrical conductivity(EC) were determined in CaCl2 (0.01 mol l−1) and water, respec-tively. Total organic carbon (Corg) was determined by oxidationwith dichromate. Ca, Mg, K, Na, and P were extracted with ionexchange resins. Ca and Mg were determined by atomic absorp-tion spectrometry with flame atomization, Na and K by atomicflame photometry, and P by atomic absorption spectrophotome-try. Ammonium and nitrate were extracted with KCl (2 mol l−1)extracts at a 1:10 ratio (m:v). N-NH4

+ was determined in a con-tinuous flow injection analysis system with spectrophotometricreading at 605 nm (Kamogawa and Teixeira, 2009), while N-NO3

−

was determined at 220 nm and 275 nm (APHA, 2005).In a previous study we showed that tannery sludge increased

soil pH and electrical conductivity and concentrations of available Pand mineral N with the strongest increase shortly after application(12 days) (Nakatani et al., 2011b).

2.3. Phospholipid fatty acid (PLFA) analysis

The PLFA were extracted from 4 g of soil (dry weight) using a pro-cedure based on Frostegard et al. (1993) and Bardgett et al. (1996).Lipids were extracted with a monophasic chloroform–methanol-

buffer citrate solvent, and the PLFA fraction separated usingsilicic acid columns before transesterification with mild alkaliand final capture in dichloromethane. Methyl-nonadecanoate(C19:0) was added to each sample as an internal standard. Fatty

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4 A.S. Nakatani et al. / Appl

cid methyl esters (FAME) were separated by gas chromatogra-hy with flame ionization detector (GC-FID) (HP6890, Hewlettackard, Palo Alto, USA) using a fused silica capillary column60 m, 0.25 mm × 0.25 mm film thickness; Supelco, Sigma–Aldrich,ustralia) and helium as a transport gas. Temperature specifica-

ions were set at 250 ◦C for the injector, 260 ◦C for the detector,ith a temperature program of 140 ◦C for 5 min and a gradient of

40–240 ◦C at 4 ◦C min−1, 240 ◦C for 15 min. Individual PLFA peaksere identified by comparing retention time with the peaks of the

tandard mixture Supelco 37 (Supelco, Bellefonte, PA), and thosedentified by GC–MS (gas chromatography combined with an HP973 mass spectrometer, Hewlett Packard, Palo Alto, USA) using theame column, temperature program and gas transport conditionsescribed above. Identification of the mass spectrometer peaks wasased on comparison with the database in NIST02.L software.

Fatty acid nomenclature is based on Frostegard et al. (1993). TheLFA markers were used as indicators of specific microbial groupsbacteria: 14:0, 15:0, 17:0, i15:0, i16:0; fungi: 18:3�3c, 18:2�6c or, 18:3�6c) (Zelles, 1999; Zak et al., 2000; Graham et al., 1995).

.4. Community level physiological profile (CLPP) analysis

The CLPP analysis was carried out using the Biolog Ecoplate®ystem (Biolog Inc., EUA). To standardize inoculum density, we esti-ated bacterial density by the most probable number method in a

utrient agar medium kept at 28 ◦C for 48 h, using the drop plateethod (Jahnel et al., 1999). Each well was inoculated with 150 �L

oil suspension prepared with 10 g of soil diluted in sterile salineolution (NaCl; 0.85%), to obtain approximately 1000 cells mL−1.he microplates were incubated at 28 ◦C and analyzed after 12 hnd subsequently every 24 h for the next 7 days, using an auto-atic microplate reader (Model 550, Biorad Laboratories, Hercules,

A, USA), adjusted to 590 nm. The optical density data (OD590 nm) ofach well were corrected by the readings taken immediately afternoculation and also by the control well of each microplate.

The corrected data were transformed by dividing each value byhe average well color development (AWCD) of the plate (Garlandnd Mills, 1991). Those corrected values for the entire plate and forhe six groups of substrates (carbohydrates, carboxylic acid, aminocids, miscellaneous, polymers, and amines) were used to assessean heterotrophic metabolism and to estimate the kinetic param-

ters, via the logistic growth equation proposed by Lindström et al.1998) to adjust the absorbance curve to 590 nm in relation to thencubation time of the plates:

K

D590 nm =

1 + e−R(T−S)(1)

here K (asymptote) is the maximum degree of color developmentOD590 nm), R (degradation rate) the exponential rate of change of

able 2acterial and fungal fatty acids and bacterial-to-fungal ratio in soil treated with tan-ery sludge (0.0, 2.3 and 22.6 Mg ha−1), 12 and 271 days after the second applicationf sludge.

Treatment Bacterialfatty acids(�g kg−1 soil)

Fungalfatty acids(�g kg−1 soil)

B/F ratio(�g kg−1

soil)

Day 120.0 9.9 b 1.3 ab 7.5 abc2.3 10.9 b 1.5 b 7.1 abc22.6 9.9 b 1.8 b 5.5 a

Day 2710.0 11.7 b 1.4 ab 8.4 c2.3 10.7 b 1.4 ab 8.1 bc22.6 5.9 a 1.0 a 6.1 ab

eans followed by different letters, for each column, differ significantly accordingo the LSD test (P < 0.05).

il Ecology 61 (2012) 92– 99

OD590 nm (h−1), T the inoculation time of the plates (h), and S thetime to reach the midpoint of the exponential portion of the curve,at K/2 (h).

Shannon’s diversity index (H’) and substrate consumption rich-ness (Ss) were calculated based on Zak et al. (1994), considering theAWCD for each reading, during the incubation. Greater richness anddiversity of substrate consumption indicate the metabolic capacityof the microbial community to deal with xenobiotic carbon sourcesadded to the soil (e.g. tannery sludge).

2.5. Data analyses

The PLFA data were log transformed(x + 1) and analyzed viaANOVA (followed by an LSD test) with SAS, version 9.2 (SASInstitute Inc., 2008) and plotted using non-metric multidimen-sional scaling (NMDS) with Primer-E (PRIMER-E, 2001). NMDS plotswith a 2D stress < 0.2 show that the graphic representation has agood quality for the correct interpretation of the data and providea good reflection of the structure of the microbial community. Sig-nificant differences in community structure between treatmentswere assessed using an analysis of similarity – ANOSIM (PRIMER-E,2001). The DistLM procedure in Primer was used to determine thesoil properties most strongly related with the observed patterns.This procedure was also used to assess the contribution of differentfatty acids to the observed patterns. For all tests the significancelevel was P ≤ 0.05.

Differences between kinetic parameters of the CLPP data treat-ments were assessed using ANOSIM in the Primer software. TheCLPP data (kinetic parameters, the Shannon’s diversity index (H’)and the substrate consumption richness (Ss)) were examined viaANOVA and means compared using the LSD test (P < 0.05). Statisti-cal analyses were carried out with SAS, version 9.2.

A redundancy analysis (RDA) with Monte Carlo’s permutationtests was used to check for the existence of significant relation-ship between PLFA data or kinetic parameters of the CLPP data andsoil chemical attributes using CANOCO 4.5 (Ter Braak and Smilauer,1988). The dataset was also subjected to Pearson’s correlation anal-ysis with the soil chemical attributes.

3. Results

3.1. Phospholipid fatty acid (PLFA) analysis

Twelve days after sludge application there were no significantdifferences in bacterial and fungal fatty acid concentration and theB/F ratio among the treatments (Table 2). From day 12 to day 271,the concentrations of bacterial and fungal fatty acids decreased sig-nificantly only in the soil with the high sludge application rate.On day 271, the concentrations of bacterial fatty acids and the B/Fratio were lower in the soil with the high sludge application ratecompared to the unamended soil.

The dominant signature fatty acids responded differently tosludge application. The concentrations of the fatty acids iC15:0,iC16:0, and iC17:0 decreased with increasing rates, whereas theconcentrations of aC15:0, C16:0, and C18:1�9 increased (Table 3).In addition the concentrations of the fatty acids iC15:0, iC16:0, andiC17:0 correlated negatively with soil pH, available P and Ca. Con-versely aC15:0 and C18:1�9 correlated positively with soil mineralN and EC (Supplementary Table S1).

The NMDS plot shows a clear separation of the 22.6 Mg ha−1

sludge treatment at both sampling times compared to the control

and the 2.3 Mg ha−1 treatment (Fig. 1). ANOSIM confirmed that thecommunity structure at the highest sludge rate was significantlydifferent from the other two treatments at both sampling times(Table 4).

A.S. Nakatani et al. / Applied Soil Ecology 61 (2012) 92– 99 95

Fig. 1. Non-metric multidimensional scaling (NMDS) analysis of phospholipid fatty acid (PLFA) profiles of the microbial community in agricultural soils treated with tannerysludge, at two sampling times after the second application of waste (12 and 271 days). 0,

(B) chemical attributes of soil which explained the PLFA patterns. The concentration or

vector length indicates relative weight of the attribute to the explanation of the phenome

Table 3Percentage of dominant signature fatty acids in soil treated with tannery sludge (0.0,2.3 and 22.6 Mg ha−1), 12 and 271 days after the second application of sludge.

Treatment iC15:0(%)

aC15:0(%)

iC16:0(%)

C16:0(%)

iC17:0(%)

C18:1�9(%)

Day 120.0 27.5 b 10.7 ab 15.6 bc 21.5 abc 9.5 bc 7.9 a2.3 26.9 b 12.0 c 15.9 bc 20.9 a 8.8 b 8.4 a22.6 24.1 a 14.8 d 13.2 a 22.1 c 7.8 a 11.1 b

Day 2710.0 27.5 b 10.0 a 16.9 c 21.2 ab 9.9 c 7.3 a2.3 27.9 b 9.9 a 16.8 c 20.8 a 9.8 bc 7.5 a22.6 26.8 b 11.1 b 15.1 b 21.8 c 9.3 bc 9.0 a

Means followed by different letters, for each column, differ significantly accordingto the LSD test (P < 0.05).

Table 4Pairwise test (R statistics) of PLFA patterns and kinetic parameters from AWCD curvefitting of soil that received tannery sludge (2.3 and 22.6 Mg ha−1) and control soil(0.0 – no sludge) in two sampling dates (12 and 271 days) after the second sludgeapplication.

Treatment R value

PLFA patterns AWCD kineticparameters

Dose effects0.0-12 d, 2.3-12 d −0.09 0.51*

0.0-12 d, 22.6-12 d 0.73* 0.98*

2.3-12 d, 22.6-12 d 0.72* −0.020.0-271 d, 2.3-271 d 0.02 −0.070.0-271 d, 22.6-271 d 0.96* 0.57*

2.3-271 d, 22.6-271 d 0.70 0.18Time effects

0.0-12 d, 0.0-271 d 0.02 0.422.3-12 d, 2.3-271 d 0.06 0.3122.6-12 d, 22.6-271 d 0.97* 0.41

* P < 0.05.

2.3 and 22.6 are rates of tannery sludge application, in Mg ha−1. (A) Fatty acids andvalue of the attribute increases according to the direction of the vector, while thenon.

The RDA showed that the PLFA patterns were related to soilchemical properties (F value = 8.93, P value = 0.002). According tothe DistLM analysis, the fatty acids 16:0 and i17:0, together con-tributed 84% to the observed PLFA patterns (Fig. 1A). Among thechemical properties, the concentrations of available P, mineral N,and Ca, and pH together contributed 54% to the observed patterns(Fig. 1B).

3.2. Carbon substrate utilization patterns

Carbon substrate utilization, assessed via Biolog microplates,showed that tannery sludge modified the metabolic potential ofthe soil microbial community, and that this effect was strongest 12days after sludge application (Table 4), when the AWCD between 24and 96 h was higher in soils with sludge than in the control (Fig. 2A).On the other hand, at 271 days, only the higher sludge rate differedfrom the control (Table 4), showing a smoother pattern of the fittedcurves (Fig. 2B).

The maximum substrate consumption (K) did not differ amongtreatments at both sampling times (Table 5). Twelve days afterapplication, the treatments that received sludge had a greatersubstrate consumption rate (r) and reached the midpoint (s) ofexponential growth earlier than the control. After 271 days, r ands were significantly different than the control only at the highersludge rate (Table 5).

The Monte Carlo’s permutation test showed that soil chemi-cal properties significantly influenced the kinetic parameters forcarbon substrate utilization (F value = 19.88, P value = 0.002). In

general, relationship between soil properties and parameter r waspositive whereas that with parameter s was negative (Table 6).

Among the six different groups of carbon substrates, the clear-est effects on the kinetic parameters r and s were observed for

96 A.S. Nakatani et al. / Applied Soil Ecology 61 (2012) 92– 99

Fig. 2. Kinetics of average well colour development (AWCD) curve fitting in soil treatedapplication of sludge. Different symbols indicate different treatments, as follows: Fillesludge. Lines represent the fitted equations; symbols represent the means of each treatme

Table 5Kinetic parameters of the AWCD curve fitting of the heterotrophic microbial com-munity from soil treated with tannery sludge (0.0, 2.3, and 22.6 Mg ha−1), 12 and271 days after the second application of sludge, at Rolândia, Brazil.

Treatment K r s

12 days0.0 1.35 a 0.05 a 70.83 c2.3 1.37 ab 0.08 cd 46.39 a22.6 1.38 ab 0.09 c 44.14 a

271 days0.0 1.50 b 0.04 a 77.47 c2.3 1.50 b 0.05 ab 65.72 bc22.6 1.39 ab 0.07 bc 54.55 ab

Means followed by different letters, for each column, differ significantly accordingto the LSD test (P < 0.05). K, maximum degree of substrate consumption; r, rate ofsubstrate consumption; s, time elapsed before reaching the midpoint of substrateconsumption.

Table 6Pearson’s correlations between metabolic kinetic parameters and chemical soilattributes.

pH P Ca Na N min EC

K −0.38 −0.14 −0.28 −0.21 −0.17 −0.16r 0.75** 0.30 0.54** 0.76** 0.72** 0.70**

s −0.81** −0.40* −0.55** −0.62** −0.57** −0.57**

K, maximum degree of substrate consumption; r, rate of substrate consumption; s,time to reach the midpoint of substrate consumption; pH, active acidity; P, availablep

catcwp(atT

nwpiwTw

hosphorus; N min, mineral nitrogen; EC, electrical conductivity.* P < 0.1.

** P < 0.05.

arboxylic acids (COOH), amino acids (AAS), polymers (POL), andmines (NH2), especially at the highest dose for both samplingimes. For these groups, r values generally were higher than in theontrol; whereas s values were lower (Table 7). The RDA analysesith Monte Carlo’s permutation test showed that the soil chemicalroperties significantly influenced the kinetic parameters of COOHP = 0.002), AAS (P = 0.002), POL (P = 0.018), and NH2 (P = 0.002). Inddition, there was positive (parameter r) and negative (parame-er s) correlations with soil pH, mineral N and EC (Supplementaryable S2).

The effect of sludge application on diversity index (H’) and rich-ess (Ss) of the carbon substrate utilization of microbial communityas greater at the first sampling date (Fig. 3) than at the later sam-ling. Twelve days after sludge application, the diversity index (H’)

ncreased rapidly in the first 72 h, with diversity being greater in soilith the high sludge application rate than in the control (Fig. 3A).

he substrate richness also increased strongly in the first 72 h andas higher in the sludge treatments than in the control during this

with tannery sludge (0–22.6 Mg ha−1), (A) 12 and (B) 271 days after the secondd circles = control; empty circles = 2.3 Mg ha−1 sludge; filled triangles = 22.6 Mg ha−1

nt; and vertical bars represent standard deviations (n = 4).

time (Fig. 3B). After 271 days, sludge treatments had no effect onrichness or diversity (data not shown).

4. Discussion

4.1. Microbial community structure based on PLFA analysis

The total PLFA concentration of soils can be considered anindicator for microbial biomass (White, 1993). In this and in ourprevious study based on the same field experiment (Nakatani et al.,2011a), sludge application had no significant effect on the soilmicrobial biomass. Despite addition of C and nutrients, sludgeapplication did not increase soil organic C (Nakatani et al., 2011a),which may be due to the high variability of soil organic C contentand/or rapid decomposition of the added C. Martines et al. (2006)found that more than 50% of the carbon in tannery sludge addedto soil was mineralized in the first 10 days. The lack of response tothe sludge application may be due to the high electrical conductiv-ity. Previous work has shown that the addition of organic residuesto the soil stimulates the microbial activity but not the micro-bial biomass in a short-term (Tejada and Gonzalez, 2007). Understress, the efficiency by which C is utilized for growth decreases,i.e. relative more C is respired to counteract the stress (Yuan et al.,2007). This latter explanation may also explain that the capacityto utilize the C compounds in the Biolog test was increased bysludge application. Although microbial biomass was not affected,sludge application changed microbial community structure at thehigher sludge application rate and its substrate utilization pattern(Table 4). Changes in microbial community structure induced bythe high sludge application rate were apparent even 9 monthsafter sludge application indicating a long-lasting effect if high ratesof sludge are applied. The changes in soil microbial communitystructure based on PLFA profile in the present study are in agree-ment with previous studies with tannery sludge (Kamaludeen et al.,2003) and sewage sludge (Abaye et al., 2005; Baath et al., 1998).

As indicated by the Monte Carlo’s permutation test (RDA) andthe DistLM analysis, these changes in microbial community struc-ture based on PLFA patterns are likely to be related to the increaseof pH and available P, mineral N, Ca, concentrations by the sludge.The soil pH in the control was about 0.7 unit lower than in the high-est sludge dose for both sampling dates, while the soil mineral Nconcentration was 12.0 and 1.8 times higher in the highest sludgedose compared with the control treatment, at 12 and 271 days after

the second sludge application, respectively (Nakatani et al., 2011b).Zhong et al. (2010) also noted that changes in soil chemical prop-erties were important determinants for changes in the PLFA profilein soils treated with mineral (urea, P2O5 and K2O) and organic

A.S. Nakatani et al. / Applied Soil Ecology 61 (2012) 92– 99 97

Table 7Kinetic parameters of the AWCD curve fitting of substrate groups (CHO: carbohydrates; COOH: carboxylic acids; AAS: amino acids; MIS: miscellaneous; POL: polymers; NH2:amines) from soil treated with tannery sludge (0.0, 2.3, and 22.6 Mg ha−1), 12 and 271 days after the second application of sludge, at Rolândia, Brazil.

Dose (Mg ha−1) CHO COOH AAS MIS POL NH2

K

Day12

0.0 1.55 ab 1.12 ab 1.67 0.91 1.56 1.322.3 1.60 ab 1.09 b 1.76 0.99 1.56 1.44

22.6 1.46 b 1.17 ab 1.60 1.01 1.66 1.54

Day271

0.0 1.44 b 1.18 ab 1.68 0.86 1.72 1.312.3 1.59 ab 1.36 a 1.78 1.05 1.75 1.43

22.6 1.71 a 1.10 b 1.58 0.92 1.79 1.22r

Day12

0.0 0.06 0.05 d 0.05 b 0.11 0.05 bc 0.05 c2.3 0.09 0.14 a 0.07 b 0.16 0.05 ab 0.13 ab

22.6 0.09 0.11 b 0.11 a 0.13 0.06 a 0.15 a

Day271

0.0 0.08 0.04 d 0.05 b 0.09 0.04 c 0.05 c2.3 0.09 0.05 d 0.05 b 0.11 0.04 bc 0.05 c

22.6 0.09 0.08 c 0.06 b 0.14 0.05 ab 0.10 bs

Day12

0.0 60.87 ab 68.89 b 80.58 a 44.78 ab 82.65 ab 95.11 a2.3 40.20 b 40.63 c 57.94 cd 33.85 b 70.97 bc 46.02 c

22.6 40.05 b 43.23 c 44.45 d 34.24 b 64.13 c 43.67 c

Day271

0.0 69.40 a 90.99 a 88.80 a 52.48 a 98.05 a 87.09 a2.3 46.01 ab 75.09 b 75.10 ab 40.50 ab 86.49 ab 74.37 ab

M differ

c midpo

(eois

4

setbNisohbahe

Fs

22.6 50.20 ab 50.89 c

eans followed by different letters, for each column and each kinetic parameter,

onsumption; r, rate of substrate consumption; s, time elapsed before reaching the

composted pig manure) fertilizer. In agreement with Baumannt al. (2009), who reported that N supply and availability is onef the main drivers of soil microbial community composition,ncreased N availability in soils after the application of tanneryludge was one of the primary factors affecting the PLFA patterns.

.2. Community level physiological profile (CLPP)

The metabolic diversity of the microbial community is a con-equence of genetic diversity, environmental effects on genexpression, and ecological interactions between different popula-ions (Zak et al., 1994). Therefore, the physiological potential maye a promising indicator of changes in the biological quality of soils.evertheless, more detailed studies need to be conducted to ver-

fy its feasibility. Due to the high substrate concentrations and thehort incubation times, the physiological profile is mainly basedn copiotrophic bacteria (Hu et al., 1999). The Biolog Ecoplate®as been shown to be useful in quantifying the dynamics of car-

on substrate utilization by microbial communities following thepplication of wastes including biosolids (Sullivan et al., 2006),ousehold solid waste compost (Gomez et al., 2006), and sewageffluent (Paula et al., 2010). Quantity and quality of the organic

ig. 3. Shannon’s diversity index (H’) and richness (Ss) of carbon substrate consumptionecond sludge application. Means with the same letter, for each incubation time, do not d

62.66 bc 39.36 ab 70.70 bc 51.47 bc

significantly according to the LSD test (P < 0.05). K, maximum degree of substrateint of substrate consumption.

material are primary drivers of the structure, function and activityof the soil microbiota (Marschner et al., 2003; Ndaw et al., 2009).In our study, application of tannery sludge increased the activityof copiotrophic bacteria native to the soil or introduced with thesludge, increasing the functional potential of the community asshown in the accelerated (higher r value) and intensified C substrateutilization (lower s value) at the first sampling time (12 days afterapplication).

The increased activity after tannery sludge application is inagreement with other studies (Martines et al., 2006; Barajas-Aceveset al., 2007; Nakatani et al., 2011a). However, at the low sludgeapplication rate, this effect was transient. It can be assumed thatby the second sampling time (271 days after application), mostof the immediately available substrate introduced with the sludgehad been decomposed, particularly at the lower sludge applica-tion rate, leaving only the more recalcitrant forms. This change insubstrate availability may have decreased the abundance of copi-otrophic bacteria which could explain the decrease in r and the

increase in s at the low sludge application rate to values similar as inthe control (Table 5). On the other hand, at the high sludge applica-tion rate this effect is still evident 271 days after sludge application(Table 5).

of CLPP from soil treated with tannery sludge (0–22.6 Mg ha−1), 12 days after theiffer from each other by the LSD test (P < 0.05) (n = 4).

9 ied So

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8 A.S. Nakatani et al. / Appl

Application of tannery sludge increased substrate consumptioniversity (H’) and richness (Ss). Gomez et al. (2006) reported simi-

ar results in soils treated with solid domestic waste and Paula et al.2010) in soils irrigated with treated sewage effluent, which couldndicate greater functional diversity in the soil microbial com-

unity. However, these indices do not necessarily reflect higherenetic diversity.

The kinetic parameters r and s, which describe the metabolicrofile of the microbial community, correlated with the same soilhemical properties (pH and mineral N concentration) that con-ributed to the change in microbial community structure assessedy PLFA, which emphasizes the importance of soil chemical prop-rties for soil microorganisms (Aon and Colaneri, 2001).

The strong effect of pH on microbial community structure andunction is in agreement with previous studies (White et al., 2005;nderson and Joergensen, 1997; Frostegard et al., 1993; Marschnert al., 2005). According to Paula et al. (2010), adding nitrogen viarganic waste can be an important stimulus to microbial activitynd can increase the potential heterotrophic metabolism of theoil microbial community. The significant correlations between Nand EC with the parameters r and s are probably effects of theigh concentrations of Na in the sludge, which in addition to otherolutes, lead to an increase in the soil EC. Such correlations werenexpected, since these chemical properties, above certain limits,egatively affect biological activity (Rietz and Haynes, 2003). Theignificant relationship to r and s is therefore surprising but may beelated to the fact that high Na concentrations and EC were foundith the high sludge application rate which also induced high N

oncentrations which may override the negative effect of EC anda (Nakatani et al., 2011a).

Studies of the physiology and structure of microbial communi-ies are important for understanding the sustainability of naturalnd agricultural ecosystems, since they determine the rates ofrganic material decomposition and nutrient cycling (Frey et al.,999), as well as having direct influence on the functional redun-ancy and thus resilience of soils.

Monitoring the soil microbial community in response to tan-ery sludge application is pivotal to reduce the environmentalisks of such practice because key microbial functional groupsay be affected (Alvarez-Bernal et al., 2006; Barajas-Aceves et al.,

007). Furthermore, high doses of tannery sludge might causeitrate leaching, emissions of greenhouse gases (N2O), N losses bymmonia volatilization, degradation of soil structure by high saltoncentrations (Alvarez-Bernal et al., 2006; Trujillo-Tapia et al.,008; Martines et al., 2010), and may decrease plant growthCardoso et al., 2011). In the present study, the highest sludgepplication rate did not affect negatively the corn grain yieldcontrol = 9590 kg ha−1; lowest rate = 9830 kg ha−1; and highestate = 9290 kg ha−1; F value = 0.79; P > 0.05). However, long-termtudies should be done to determine the doses of tannery sludgehat do not impair the biological health and crop yield.

Our results suggest that there is no clear link between microbialommunity structure assessed by PLFA and substrate utilizationy Biolog method. In the present study both methods detectedhanges, however they did not match. There is evidence that theLFA analysis is more sensitive than the CLPP in detecting changesYao et al., 2000; Elfstrand et al., 2007). Such inconsistency maye due to the fact that the Biolog method is based on microbialrowth. Many soil microorganisms are not able to grow in the arti-cial medium. In addition, the number of substrates on the plates isery small compared to that present in soils (Ramsey et al., 2006).oreover, care must be taken regarding misinterpretations of PLFA

rofiles, because the same fatty acids may occur in the differenticrobial groups and the same microbial group may change the

omposition of fatty acids under a particular environmental condi-ion (Frostegard et al., 2011). Therefore, more studies are needed

il Ecology 61 (2012) 92– 99

to evaluate the meaning of changes in the microbial communitystructure and their effects on the physiological profile (Chaer et al.,2009).

5. Conclusions

This study showed that high doses of tannery sludge changethe soil microbial community structure and physiological profile,which persists even nine months after application. The effects of thesludge on PLFA and CLPP are mainly related to the increase in soil pHand available N concentration. Both methods detected changes dueto the sludge application. However, there was no clear link betweenmicrobial community structure and substrate utilization. Despitethe sensitivity of both methods in detecting effects of sludge appli-cation on soil, long-term studies are needed to ascertain the safetyof this practice.

Acknowledgements

We thank Fundac ão de Amparo à Pesquisa do Estado de SãoPaulo (FAPESP) for providing funding for this project and forsupporting A.S. Nakatani with a Ph.D. grant. We thank CurtumeVanzella Ltda. for providing the sludge and the experimental area.Thanks are due to the staff of the Microbial Ecology Lab at theUniversidade Estadual de Londrina (UEL), the Soil MicrobiologyLab at the Escola Superior de Agricultura Luiz de Queiroz (ESALQ),and Soil Biology Lab at the University of Adelaide (Australia) fortheir assistance in conducting the experiment and laboratory anal-yses. We thank Conselho Nacional de Desenvolvimento Científicoe Tecnológico (CNPq) for research grants to M.A. Nogueira, E.J.B.N.Cardoso.

Appendix A. Supplementary data

Supplementary data associated with this article can befound, in the online version, at http://dx.doi.org/10.1016/j.apsoil.2012.05.003.

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