Available online at www.sciencedirect.com

Bioresource Technology 99 (2008) 2872–2880

Application of different organic amendments in a gasolinecontaminated soil: Effect on soil microbial properties

M. Tejada a,*, J.L. Gonzalez b, M.T. Hernandez c, C. Garcia c

a Departamento de Cristalografıa, Mineralogıa y Quımica Agrıcola, E.U.I.T.A. Universidad de Sevilla, Crta de Utrera km. 1, 41013 Sevilla, Spainb Departamento de Quımica Agrıcola y Edafologıa, Universidad de Cordoba, Campus de Rabanales, Edificio C-3,

Crta N-IV-a, km. 396, 14014 Cordoba, Spainc Departamento de Conservacion de Suelos y Agua y Manejo de Residuos Organicos, Centro de Edafologıa y Biologıa Aplicada del Segura,

CEBAS-CSIC, P.O. Box 4195, 30080 Murcia, Spain

Received 3 May 2007; received in revised form 11 June 2007; accepted 11 June 2007Available online 26 July 2007

Abstract

The effects of four organic wastes, including cotton gin crushed compost (CC), poultry manure (PM), sewage sludge (SS) and organicmunicipal solid waste (MSW) on some biological properties of a Xerollic Calciorthid soil polluted with gasoline at two loading rates (5%and 10%) were studied in an incubation experiment. Three hundred grams of sieved soil (<2 mm) were polluted with gasoline and mixedwith PM at a rate of 10%, CC at a rate of 17.2%, SS at a rate of 23.1%, or MSW at a rate of 13.1%, applying to the soil the same amountof organic matter with each organic amendment. An unamended soil, non polluted (C) and polluted with gasoline at 5% (G1) and 10%(G2) rate were used as reference. Soil samples were collected after 1, 30, 60, 90, 120, 180 and 270 d of incubation and analyzed for micro-bial biomass carbon, respiration and dehydrogenase, urease, b-glucosidase, phosphatase and arylsulfatase activities. At the end of theincubation period, soil biological properties were higher in organic amended soils than in C, G1 and G2 treatments. In particular, soilmicrobial biomass carbon and dehydrogenase, urease, b-glucosidase, phosphatase and arylsulfatase activities increased 87.1%, 92.9%,88.7%, 93.2%, 78.2% and 85.3%, respectively for CC-amended soils respect to G2, 85.7%, 82.3%, 87.3%, 92.2%, 76.7% and 83.6%, respec-tively for PM-amended soils; 82%, 90%, 84.8%, 89.9%, 74.1% and 80%, respectively for SS-amended soils; and 71.3%, 78.3% 26.2%,38.2%, 79.7% and 88.6%, respectively for MSW-amended soils. Since the adsorption capacity of gasoline was higher in CC than thePM, SS and MSW-amended soils, it can be concluded that the addition of organic wastes with higher humic acid concentration is morebeneficial for remediation of soils polluted with gasoline.� 2007 Published by Elsevier Ltd.

Keywords: Gasoline; Organic wastes; Soil biochemical properties

1. Introduction

Polycyclic aromatic hydrocarbons (PAHs) have beenrecognised as a potential health risk due to their intrinsicchemical stability, high recalcitrant to different types ofdegradation and high toxicity to living microorganisms(Alexander, 1999; Andreoni et al., 2004; Eibes et al.,2006). Soil microorganisms, being in intimate contact withsoil environment, are very sensitive to any ecosystem per-

0960-8524/$ - see front matter � 2007 Published by Elsevier Ltd.

doi:10.1016/j.biortech.2007.06.002

* Corresponding author.E-mail address: mtmoral@us.es (M. Tejada).

turbation, and therefore are considered to be the best indi-cators of soil pollution (Andreoni et al., 2004). Soilpollution with PAHs influences microbiota, producingchanges in enzyme activity, soil respiration, biomass andmicrobial counts (Margesin et al., 2000; Baran et al.,2004; Labud et al., 2007). It should be noted that PAHs(64 rings) are moderately toxic and degradable by micro-organisms, whereas the remaining group (>4 rings) has astrongly toxic, mutagenic and carcinogemic character,and their decomposition is only possible via cometabolism(Cernigilia, 1984; Kanaly and Harayama, 2000). The mea-surement of microbial parameters, such as soil respiration,

Table 1Initial soil characteristics and standard error in parenthesis (data are themeans of five samples)

pH (H2O) 7.7 (0.1)Electrical conductivity (dS m�1) 0.24 (0.05)Clay (g kg�1) 312 (17)Silt (g kg�1) 258 (24)Sand (g kg�1) 430 (32)Texture Clay loamDominant clay types Illite, illite-montmorillonite

(interstratified)CaCO3 (g kg�1) 349 (28)Bulk density (Mg m�3) 1.49 (0.03)Total N (g kg�1) 0.9 (0.03)Organic C (g kg�1) 6.3 (0.09)Humic acid – C (g kg�1) 1.9 (0.2)Fulvic acid – C (g kg�1) 0.5 (0.1)C/N ratio 7 (3)P (mg kg�1) 8.9 (0.3)K (g kg�1) 542 (37)

M. Tejada et al. / Bioresource Technology 99 (2008) 2872–2880 2873

microbial biomass carbon or enzyme activities, providesinformation on the presence and activity of viable microor-ganisms as well as on the intensity, kind and duration ofthe effects of hydrocarbon pollution on soil metabolicactivity; such measurements may serve as a good index ofthe impact of pollution on soil health (Eibes et al., 2006;Labud et al., 2007): Microbiological and biochemical prop-erties are very responsive and provide precise informationon small changes occurring in soil (Tejada et al., 2007).

In recent years, it has become common practice to addorganic matter to soils contaminated by PAHs for theirbioremediation. Addition of horticultural compost (Malis-zewska-Kordybach et al., 2000), straw (Kucharski et al.,2000) and manure (Coover and Sims, 1987) to soils havebeen found to immobilize PAHs and reduce the negativeeffects on soil microbial populations and enzyme activitiesprobably due to the role of organic matter for sorptionprocesses of organic pollutants (Kleineidam et al., 1999;Guanasekara and Xing, 2003). However, the influence oforganic matter on a soil’s biological and biochemical prop-erties depends on the amount, type, and size of the addedorganic materials (Tejada et al., 2007). In turn, the effectof each organic material on soil biological propertiesdepends on its dominant component.

In view of the above, the objective of this study was toinvestigate under laboratory conditions the effect of theadsorption capacity of four different organic wastes(a crushed cotton gin compost, a poultry manure, a sewagesludge and an organic municipal solid waste) on a soil con-taminated by gasoline at two loading rates (5% and 10%),studying their effect on soil microbial biomass carbon, res-piration, dehydrogenase activity and the activities of somehydrolases involved in the cycles of N (urease), C (b-gluco-sidase), P (phosphatase), and S (arylsulphatase).

2. Methods

2.1. Incubation experiment

The soil used in this experiment was a Xerollic Calciort-hid soil (Soil Survey Staff, 1987). The general properties ofthe soil (0–25 cm) are shown in Table 1. Three hundredgrams of sieved soil (<2 mm) were contaminated with gas-oline (originating of a petrol station) at two different load-ing rates (5% and 10% w/w). In order to get a good andhomogeneous distribution of gasoline in the soil, soils werepolluted by adding a suspension of the correspondingamount of gasoline in the amount of water necessary tobring the soil to 60% of its water holding capacity. Soilsamples were mixed with PM at a rate of 10%, CC at a rateof 17.2%, SS at a rate of 23.1%, or MSW at a rate of 13.1%,applying to the soil the same amount of organic matterwith each organic amendment. An unamended soil bothnon polluted (C) and polluted with gasoline at 5% (G1)and 10% (G2) rate were used as reference. Distilled waterwas added to each soil to bring it to 60% of its water-hold-

ing capacity. In summary, the treatments were thefollowing:

(1) C: no organic amendment and no contaminated withgasoline

(2) G1: soil contaminated with gasoline at 5%(3) G2: soil contaminated with gasoline at 10%(4) PM1: soil contaminated with gasoline at 5% and

amended with PM(5) PM2: soil contaminated with gasoline at 10% and

amended with PM(6) CC1: soil contaminated with gasoline at 5% and

amended with CC(7) CC2: soil contaminated with gasoline at 10% and

amended with CC(8) SS1: soil contaminated with gasoline at 5% and

amended with SS(9) SS2: soil contaminated with gasoline at 10% and

amended with SS(10) MSW1: soil contaminated with gasoline at 5% and

amended with MSW; and(11) MSW2: soil contaminated with gasoline at 10% and

amended with MSW.

The general properties of organic wastes are shown inTable 2.

The treatments were replicated three times. Soil sampleswere collected after 1, 30, 60, 90, 120, 180 and 270 daysafter the gasoline contamination. Soils from the pots werethoroughly homogenised and stored (fresh samples) at 4 �Cfor analysis.

2.2. Analytical determinations

2.2.1. Physical and chemical parametersSoil texture was determined by the Robinson’s pipette

method (SSEW, 1982) and dominant clay types were

Table 2Characteristics of the organic wastes used and standard error in parenthesis (data are the means of six samples)

CC PM SS MSW

pH (H2O) 7.0 (0.1) 7.1 (0.1) 6.6 (0.2) 6.2 (0.3)Total organic matter (g kg�1) 356 (24) 614 (26) 266 (14) 469 (15)Humic acid – C (g kg�1) 89.8 (1.5) 21.4 (2.5) 16.6 (0.9) 6.3 (0.7)Fulvic acid – C (g kg�1) 0.9 (0.1) 57.8 (0.2) 39.2 (1.4) 35.7 (1.7)Total N (g kg�1) 13.2 (1.2) 40.8 (0.9) 50.8 (2.8) 17.3 (1.3)P (g kg�1) 6.3 (1.1) 17.4 (1.9) 21.3 (2.1) 10.5 (1.3)K (g kg�1) 54.6 (9.2) 21.2 (3.1) 29.1 (3.2) 9.5 (1.8)Ca (mg kg�1) 275 (32) 549 (28) 86 (11) 124 (16)Mg (mg kg�1) 30.5 (8.2) 53.6 (4.7) 5.9 (1.2) 79.6 (7.2)

2874 M. Tejada et al. / Bioresource Technology 99 (2008) 2872–2880

determined by X-ray diffraction. Total CaCO3 was mea-sured by estimating the quantity of the CO2 produced byHCl addition to the soil (MAPA, 1986). Soil bulk densitywas determined using the core method. The soil wasweighed and dried at 105 �C for 48 h before determiningbulk density as the ratio between soil dry weight and thering volume, according to the official methods of the Span-ish Ministry of Agriculture (MAPA, 1986). In soil andorganic amendments pH was determined in distilled waterwith a glass electrode (soil:H2O 1:2.5 ratio), electrical con-ductivity was determined in distilled water with a glass elec-trode (soil:H2O ratio 1:5). Total N was determined by theKjeldahl method (MAPA, 1986). Organic carbon wasdetermined by oxidizing organic matter in soil samples withK2Cr2O7 in sulphuric acid (96%) for 30 min, and measur-ing the concentration of Cr3+ formed (Sims and Haby,1971). Humic and fulvic acids-C were extracted with a mix-ture of 0.1 M sodium pyrophosphate and 0.1 M sodiumhydroxide at pH 13 (Kononova, 1966). The supernatantwas acidified to pH 2 with HCl and allowed to stand for24 h at room temperature. To separate humic acids fromfulvic acids, the solution was centrifuged and the precipi-tate containing humic acids was dissolved with sodiumhydroxide (Yeomans and Bremner, 1988). The humic andfulvic acid C content was measured by spectrophotometricdetermination of Cr3+, after oxidation with K2Cr2O7 (Simsand Haby, 1971). Inorganic soluble P was determined bythe Willians and Stewart method, described by Guitianand Carballas. Available K was determined by MAPAmethods (1986) using ammonium acetate 1 N as extractant.

The general properties of organic wastes are shown inTable 2. Organic matter content was determined by thedry combustion method (MAPA, 1986). Humic and fulvicacids C were determined by the method previouslydescribed. Total P was determined by the Willians andStewart method, described by Guitian and Carballas(1976) after nitric-perchloric acid digestion. Calcium, Mg,and K were determined in the nitric-perchloric 1:1 diges-tion extract, Ca and Mg by atomic absorption spectrome-try and K by atomic emission spectrometry. Available Kwas determined by MAPA methods (1986) using ammo-nium acetate 1 N as extractant, and organic matter contentin the amendments was determined by the dry combustionmethod (MAPA, 1986).

2.2.2. Biological and biochemical parameters

Soil microbial biomass was determined using the CHCl3fumigation-extraction method (Vance et al., 1987). Theactivity levels of five soil enzymes were measured.Dehydrogenase activity was measured by reduction of 2-p-iodo-3-nitrophenyl-5-phenyl tetrazolium chloride toiodonitrophenylformazan (Garcia et al., 1993). Ureaseactivity was determined using urea as substrate (Kandelerand Gerber, 1988). b-glucosidase activity was measuredusing p-nitrophenyl-b-d-glucopyranoside as substrate(Masciandaro et al., 1994). Alkaline phosphatase activityby was measured using p-nitrophenyl phosphate disodiumas substrate (Tabatabai and Bremner, 1969). Arylsulfataseactivity was measured using p-nitrophenylsulfate (pNPS)as substrate (Tabatabai and Bremner, 1970).

Soil respiration was measured in the laboratory and insamples at 1 and 270 days after gasoline contamination.For all treatments, soil respiration was measured byincubation for 3, 15, 45, 90 and 120 days. Total C–CO2 col-lected in the NaOH flasks was determined by the additionof an excess of 1.5 M BaCl2 followed by tritation with stan-dardized HCl using a phenolphthalein indicator (Zibilske,1994).

2.3. Statistical analysis

Analysis of variance (ANOVA) was performed for allvariables and parameters, considering all the data collected(columns corresponding to incubation days and rows cor-responding to soil treatments) using the Statgraphics v.5.0 software package (Statistical Graphics, 1991). Themeans were separated by the Tukey’s test, considering asignificance level of p < 0.05 throughout the study. Forthe ANOVA analysis, triplicate data were used of eachtreatment and every incubation time.

3. Results

Table 3 shows the evolution of soil microbial biomasscarbon during the experimental period. In unamendedsoils, soil microbial biomass carbon (MBC) decreased withthe contamination level. This inhibitory effect of gasolinewas noticeable immediately after contamination (1 day)and persisted 270 days after pollution. At the end of the

Table 3Soil microbial biomass (lg C g�1 dry soil) in soils polluted with gasoline

Incubation days

1 30 60 90 120 180 270

C 98abA (9) 134ab (10) 167ab (15) 195ab (13) 237ab (21) 255ab (19) 301b (17)G1 73ab (8) 59a (7) 40a (7) 79ab (9) 95ab (9) 83a (11) 74a (8)G2 58a (6) 38a (4) 24a (5) 65a (9) 80 (10) 72a (6) 63a (10)PM1 135ab (11) 206b (18) 293b (17) 337b (17) 416bc (19) 499bc (17) 589bc (20)PM2 129ab (18) 172ab (12) 240ab (19) 287b (21) 359b (23) 401b (19) 440bc (22)CC1 157ab (14) 231ab (16) 318b (16) 368b (15) 439bc (20) 540bc (24) 648c (20)CC2 140ab (17) 205ab (17) 269b (19) 310b (14) 368b (21) 425bc (18) 290bc (25)SS1 120ab (8) 181ab (13) 256b (12) 297b (14) 352b (18) 409b (10) 493bc (11)SS2 112ab (12) 145ab (14) 179ab (11) 210ab (14) 248ab (11) 293b (16) 350b (17)MSW1 105ab (11) 130ab (11) 211ab (14) 249ab (11) 298b (8) 337b (10) 401b (11)MSW2 105ab (10) 147ab (15) 159ab (10) 193ab (9) 230ab (9) 269b (8) 310b (10)

Standard error in parenthesis.A Different letters following the figures indicate a significant difference at P < 0.05. The significant differences apply to the rows and columns.

M. Tejada et al. / Bioresource Technology 99 (2008) 2872–2880 2875

incubation period, soil microbial biomass carbon decreased75.4% and 79.1% in G1 and G2, respectively, with respectto the control soil (no polluted and unamended soil). In theamended soils, MBC increased progressively during theincubation period. However, this increase differed depend-ing on the type of organic amendment. At the end of theincubation period, MBC increased 88.6%, 87.4%, 84.9%and 81.5% in soils amended with CC, PM, SS and MSW,respectively with respect to the gasoline polluted soil at5% (G1). This increase of MBC in amended soils withrespect to the unamended soil was lower for the 10% con-

Table 4Cumulative C–CO2 (mg kg soil�1) in soils polluted with gasoline

Incubation days

1 3 7

1 day after gasoline contamination

C 129 (7) 208 (9) 336 (11)G1 109 (9) 181 (15) 257 (12)G2 92 (7) 143 (14) 210 (14)PM1 241 (11) 342 (16) 615 (20)PM2 228 (12) 262 (12) 499 (16)CC1 276 (15) 393 (11) 699 (22)CC2 250 (10) 285 (17) 574 (19)SS1 206 (8) 295 (17) 540 (18)SS2 198 (11) 283 (14) 519 (14)MSW1 199 (13) 273 (15) 498 (13)MSW2 182 (15) 244 (18) 397 (12)

270 days after gasoline contamination

C 93 (9) 176 (11) 298 (12)G1 87 (10) 155 (10) 274 (15)G2 73 (8) 130 (11) 242 (14)PM1 278 (15) 384 (18) 609 (13)PM2 253 (14) 316 (19) 563 (19)CC1 319 (13) 472 (14) 778 (17)CC2 297 (16) 345 (15) 608 (16)SS1 241 (14) 328 (11) 560 (15)SS2 225 (11) 309 (14) 501 (16)MSW1 224 (10) 298 (15) 498 (20)MSW2 203 (11) 277 (17) 430 (19)

Standard error in parenthesis.A Different letters following the figures indicate a significant difference at P <

tamination level (87.1%, 85.7%, 82% and 79.7% for CC,PM, SS and MSW, respectively). The statistical analysesshowed important significant differences for all treatmentsduring the incubation period.

The soil respiration values (CO2 evolved during 60 daysincubation experiment) in soil samples taken immediatelyafter gasoline contamination (1 day) and at the end ofthe incubation period (270 days) are shown in Table 4.CO2 emissions were higher in the amended soils followedby C, G1 and G2 treatments. Similar to MBC, the effectof organic wastes in soil respiration depends of the type

15 45 60

459 (15) 693 (19) 765aA (18)362 (13) 605 (14) 680a (15)308 (14) 552 (11) 593a (11)860 (19) 1171 (33) 1422b (17)698 (18) 983 (28) 1096ab (29)976 (30) 1217 (35) 1652b (30)796 (24) 1098 (31) 1298b (28)712 (28) 996 (30) 1199ab (34)653 (21) 805 (24) 959ab (17)670 (20) 847 (18) 990ab (21)562 (15) 699 (14) 804ab (20)

416 (19) 634 (20) 740aA (15)393 (17) 608 (18) 675a (14)312 (14) 547 (17) 620a (18)976 (28) 1211 (22) 1509b (27)810 (24) 1107 (23) 1300ab (29)

1093 (31) 1386 (28) 1766b (33)897 (23) 1203 (24) 1494b (31)832 (22) 1099 (19) 1328ab (24)766 (26) 957 (17) 1125ab (28)772 (24) 879 (11) 1182ab (30)701 (21) 816 (15) 1003ab (23)

0.05. The significant differences apply only at 60 days of incubation.

2876 M. Tejada et al. / Bioresource Technology 99 (2008) 2872–2880

of organic amendment. Thus, at both 1 day and 270 daysafter contamination, soil respiration values were highestin CC, followed by PM, SS and MSW-amended soils.Again, the statistical analyses showed important significantdifferences for all treatments during the incubation period.

Dehydrogenase activity was significantly inhibited bygasoline contamination, the activity of this enzyme being67.8% and 76.9% in the uncontaminated soil (C) withrespect to G1 and G2, respectively, at the end of the incu-bation period. However, dehydrogenase activity was signif-icantly stimulated by the organic amendments, thisstimulation depending on the type of organic matter andrate of gasoline applied to the soil. Thus, soil dehydroge-nase activity increased 94.1%, 93.4%, 92.1% and 90.4% insoils amended with CC, PM, SS and MSW, respectivelywith respect to the G1 treatment, whereas increased92.9%, 92.3%, 90% and 88.6%, respectively respect to theG2 treatment.

As in the case of dehydrogenase activity, urease activitywas strongly inhibited by gasoline contamination in una-mended soils, mainly at the higher rate (Table 5), whilethe organic amendments stimulated the activity of thisenzyme, this stimulation being the highest in CC-amendedsoils, followed by PM, SS and MSW-amended soils. Again,the statistical analyses showed important significant differ-ences for all treatments during the incubation period.

After gasoline contamination, the response of b-glucosi-dase activity was irregular being either stimulated or inhib-

Table 5Dehydrogenase and urease activities in soils polluted with gasoline

Incubation days

1 30 60

Dehydrogenase activity (ng INTF g�1 h�1)

C 2343abA (18) 2235ab (15) 2128ab (11)G1 2145ab (20) 1903ab (10) 1614ab (13)G2 1747ab (19) 1322ab (12) 1077a (17)PM1 4152b (22) 4586b (17) 5039b (20)PM2 3133ab (24) 3385b (20) 3671b (22)CC1 4867b (30) 5432b (22) 6091bc (28)CC2 3602b (17) 3851b (24) 4077b (29)SS1 3776b (15) 4116b (28) 4603b (21)SS2 2803ab (12) 2919ab (17) 3045ab (19)MSW1 3608b (19) 3787b (16) 3992b (15)MSW2 2517ab (20) 2603ab (21) 2695ab (23)

Urease activity (nmol NHþ4 g�1 h�1)

C 1409abA (23) 1357a (17) 1421ab (15)G1 1083a (21) 902a (17) 838a (21)G2 845a (19) 765a (10) 701a (11)PM1 1961ab (18) 2314ab (23) 2894ab (25)PM2 1880ab (11) 2225ab (27) 2693ab (22)CC1 2256ab (19) 2608ab (21) 3186b (24)CC2 2093ab (18) 2495ab (27) 3035b (13)SS1 1752ab (15) 2022ab (20) 2603ab (19)SS2 1618ab (21) 1876ab (30) 2101ab (34)MSW1 1669ab (12) 1790ab (19) 2162ab (26)MSW2 1514ab (27) 1698ab (18) 2035ab (14)

INTF: 2-p-iodo-3-nitrophenyl formazan.Standard error in parenthesis.

A Different letters following the figures indicate a significant difference at P <

ited in G1 and G2 treatments (Table 6). So, in unamendedpolluted soils, b-glucosidase activity increased after 60–90days, decreasing by the end of the incubation experiment.At the end of the incubation period, the activity of thisenzyme decreased 40.6% and 45.4%, respectively in G1and G2 treatments respect to the control soil. In theamended soils, the evolution of this enzyme was very sim-ilar to that of the others enzymes studied. In this respectand at the end of the incubation period, b-glucosidaseactivity increased 94.5%, 93.4%, 92.1% and 88.7% in CC,PN, SS and MSW-amended soils with respect to G1, andincreased 93.2%, 82.2%, 89.9% and 87.8% respect to theG2.

Finally, the evolution of phosphatase and arylsulfataseactivities during the incubation period was similar to thatof the other enzymatic activities studied, emphasizing thehighest values in CC-amended soils, followed by PM, SSand MSW-amended soils. The values of these activitieswere also lower in the gasoline polluted unamended soilsthan in the control soil.

4. Discussion

The decrease observed in soil microbial population sizeand activity with increasing gasoline concentrations indi-cates that gasoline has a toxic effect on soil microorgan-isms. These results are in agreement with those of

90 120 180 270

1932ab (17) 1724ab (20) 1610ab (15) 1508ab (11)1277ab (11) 1005a (13) 782a (11) 485a (10)

852a (10) 558a (7) 415a (9) 348a (8)5641b (18) 6129bc (22) 6637bc (21) 7358bc (35)3897b (33) 4126b (31) 4377b (24) 4517b (22)

6553bc (30) 7094bc (39) 7487bc (37) 8279c (35)4295b (25) 4586b (27) 4712b (29) 4903b (31)5031b (23) 5447b (19) 5809bc (33) 6158bc (27)

3158ab (21) 3322b (24) 3419b (30) 3510b (24)4234b (17) 4557b (20) 4871b (27) 5032b (30)

2753ab (19) 2817ab (11) 2909ab (18) 3056ab (16)

1486ab (22) 1418ab (20) 1493ab (25) 1056ab (16)773a (19) 674a (13) 617a (11) 588a (15)658a (17) 604a (18) 566a (12) 529a (14)

3376b (21) 3982b (17) 4639bc (31) 5263c (35)3056b (24) 3424b (29) 3796bc (30) 4153bc (20)3714b (27) 4236bc (30) 4955bc (33) 5784c (27)3316b (17) 3744b (21) 4196bc (25) 4700bc (33)3017b (24) 3487b (25) 3781b (24) 4507bc (33)

2348ab (18) 2656ab (16) 2996b (30) 3479b (17)2329ab (29) 2976b (26) 3179b (29) 3639b (31)2279ab (17) 2450ab (18) 2687ab (21) 2937b (27)

0.05. The significant differences apply to the rows and columns.

Table 6b-Glucosidase, phosphatase and arylsulfatase activities in soils polluted with gasoline

Incubation days

1 30 60 90 120 180 270

b-Glucosidase activity (nmol PNP g�1 h�1)

C 1179abA (19) 1035a (22) 989a (17) 943a (14) 897a (15) 863a (17) 845a (19)G1 846a (11) 898a (13) 916a (17) 969a (10) 803a (14) 649a (11) 502a (15)G2 715a (10) 758a (21) 803a (14) 732a (15) 614a (18) 515a (16) 461a (20)PM1 2070ab (22) 2991ab (25) 4052b (21) 5012bc (27) 5934bc (25) 6821c (29) 7634c (25)PM2 1936ab (19) 2473ab (23) 3491b (25) 3987b (30) 4652b (19) 5299bc (30) 5947bc (22)CC1 2304ab (31) 3394b (27) 4995bc (29) 6122bc (28) 7354c (26) 8266c (22) 9115c (29)CC2 2171ab (25) 2946ab (30) 3692b (27) 4361b (22) 4982bc (10) 5839bc (17) 6740c (27)SS1 1785ab (27) 2551ab (24) 3447b (30) 4225b (17) 4930bc (34) 5774bc (31) 6387bc (22)SS2 1662ab (20) 2197ab (23) 2786ab (19) 3136b (21) 3672b (30) 4098b (25) 4586b (16)MSW1 1641ab (17) 2145ab (32) 2658ab (22) 3110b (29) 3593b (36) 4020b (32) 4436b (10)MSW2 1488ab (15) 1814ab (19) 2257ab (25) 2673ab (24) 2996ab (22) 3417b (27) 3780b (18)

Phosphatase activity (lmol PNP g�1 h�1)

C 10.5abA (1.2) 10.2ab (1.0) 9.9ab (0.8) 9.6ab (0.8) 9.3ab (0.7) 9.2ab (0.7) 9.0ab (0.5)G1 8.4a (0.8) 7.8a (0.6) 7.1a (0.5) 6.6a (0.4) 6.0a (0.4) 5.2a (0.5) 4.4a (0.4)G2 8.1a (0.7) 7.4a (0.5) 6.6a (0.7) 6.1a (0.5) 5.7a (0.3) 5.0a (0.4) 4.1a (0.3)PM1 15.8b (1.1) 16.9b (0.9) 18.0b (1.1) 19.2b (0.9) 20.0b (1.4) 21.0bc (1.3) 21.9bc (1.5)PM2 14.6b (1.2) 15.0b (0.9) 15.5b (1.0) 16.1b (1.1) 16.5b (0.8) 16.9b (1.0) 17.6b (1.1)CC1 17.9b (1.4) 19.0b (1.2) 19.6b (1.1) 20.2b (1.2) 20.9bc (1.2) 22.1bc (1.3) 23.0bc (1.6)CC2 16.6b (1.3) 16.9b (1.0) 17.3b (1.1) 17.6b (1.2) 18.0b (1.1) 18.4b (0.9) 18.8b (1.2)SS1 14.2b (0.9) 14.7b (1.0) 15.3b (0.9) 15.9b (1.1) 17.0b (1.2) 17.8b (0.9) 18.7b (1.0)SS2 13.3ab (0.8) 13.7ab (0.9) 14.2b (0.9) 14.8b (1.0) 15.2b (0.8) 15.5b (0.7) 15.8b (1.1)MSW1 13.8ab (1.0) 14.1b (0.8) 14.7b (0.7) 15.3b (0.8) 15.9b (0.9) 16.4b (0.5) 17.0b (1.0)MSW2 13.1ab (0.9) 13.3ab (0.8) 13.4ab (0.8) 13.6ab (0.7) 13.8ab (0.8) 14.1b (0.6) 14.3b (0.9)

Arylsulfatase activity (lmol PNF g�1 h�1)

C 3.9abA (0.7) 3.7ab (0.4) 3.4a (0.5) 3.1a (0.5) 2.9a (0.4) 2.7a (0.3) 2.6a (0.8)G1 3.1a (0.6) 2.8a (0.4) 2.3a (0.5) 2.0a (0.4) 1.8a (0.2) 1.5a (0.3) 1.2a (0.2)G2 2.9a (0.5) 2.6a (0.5) 2.2a (0.4) 1.8a (0.3) 1.6a (0.2) 1.2a (0.2) 1.0a (0.1)PM1 5.9b (1.1) 6.4b (0.7) 6.7b (0.8) 7.0b (0.5) 7.4b (0.8) 7.8b (0.6) 8.2b (0.7)PM2 5.3ab (1.0) 5.5b (0.6) 5.6b (0.5) 5.7b (0.6) 5.9b (0.7) 6.0b (0.5) 6.1b (0.4)CC1 6.5b (1.1) 6.9b (0.5) 7.5b (0.6) 8.0b (0.5) 8.6bc (0.9) 9.2bc (0.8) 9.7bc (0.6)CC2 5.8b (0.9) 6.0b (0.4) 6.1b (0.5) 6.2b (0.4) 6.4b (0.6) 6.6b (0.7) 6.8b (0.6)SS1 5.3ab (0.6) 5.5b (0.5) 5.8b (0.6) 6.0b (0.3) 6.2b (0.5) 6.5b (0.8) 6.8b (0.4)SS2 4.6ab (0.5) 4.7ab (0.6) 4.8ab (0.4) 4.8ab (0.4) 4.9ab (0.5) 4.9ab (0.6) 5.0ab (0.3)MSW1 5.0ab (0.6) 5.2ab (0.5) 5.4b (0.4) 5.7b (0.5) 5.9b (0.6) 6.1b (0.5) 6.4b (0.6)MSW2 4.0ab (0.4) 4.1ab (0.5) 4.2ab (0.6) 4.3ab (0.8) 4.4ab (0.4) 4.5ab (0.4) 4.6ab (0.5)

PNP: p-nitrophenol; PNF: p-nitrophenyl.Standard error in parenthesis.

A Different letters following the figures indicate a significant difference at P < 0.05. The significant differences apply to the rows and columns.

M.

Teja

da

eta

l./

Bio

resou

rceT

echn

olo

gy

99

(2

00

8)

28

72

–2

88

02877

2878 M. Tejada et al. / Bioresource Technology 99 (2008) 2872–2880

Andreoni et al. (2004), Baran et al. (2004) and Eibes et al.(2006).

The dynamic of some enzyme activities during the incu-bation period after gasoline addition may be related to thedynamics of the soil microbial populations. They decreasedon depletion of readily available substrate resulting fromgasoline toxicity and starved as the reserves wereexhausted, and decreased significantly in size up to 270days. The reduction in soil microbial biomass under thegasoline stress conditions was also due to the additionalenergy cost to soil microorganisms, which may result in adecrease in the amount of substrate that is available forgrowth.

Soil respiration is a good index of soil microbial activityand gives an idea of the quantity of easily mineralizablesubstrates (Anderson, 1982). Many authors have reportedan increase in soil respiration after application of hydrocar-bons probably due to the fact that soil microorganismsresisting hydrocarbon toxicity can degrade these newsources of carbon (Bauer et al., 1991; Boopathy, 2000;Labud et al., 2007). However, our results indicate that soilrespiration decreased in gasoline polluted soils. Accordingto Labud et al. (2007) differences in microbial communitystructure and colloids composition between soils may bean important factor in explaining these discrepancies.Our experimental soil has a low content in organic matter,although the clay content is high. The capacity of organicmatter to adsorb organic and inorganic compounds isgreater than that of clays. This can be due to the humicsubstances with higher molecular weight should be morestrongly sorbed because their carboxyl group content isgreater on a molar basis (Murphy et al., 1994). For this rea-son in our experiment, the adsorption of gasoline by theorganic matter is loss and therefore, this polluting agentbecomes soluble with time in the soil solution, which facil-ities its contact with microorganisms. Possibly this is thecause by which the soil respiration decreased during theincubation period.

The inhibitory effect of the gasoline on soil enzymaticactivities is probably due to the suppression of the micro-bial populations involved in the C, N, P and S cycles;and/or to the fact that they may cover both organic-min-eral and cell surfaces, thus hindering the interactionbetween enzyme active sites and soluble substrates withadverse effect on enzyme activity expression (Kiss et al.,1998).

The results obtained in this study indicate that the addi-tion of organic matter to the soil decreased the extent towhich soil microbial biomass, respiration and enzymaticactivities were inhibited by gasoline. These results are inagreement with those of Coover and Sims (1987), Ruther-ford et al. (1992), Kucharski et al. (2000) and Maliszewska-Kordybach et al. (2000), who observed a decrease in theinhibitory effects on biological parameters after the addi-tion of diverse sources of organic matter such as muck,peat, cellulose, manure, straw and horticultural compostto soils polluted with hydrocarbons.

The increase in microbial biomass C observed in theorganic wastes amended soils can be attributable to theincorporation of easily biodegradable compounds withthe organic material added, which stimulate the activityof the authoctonous soil microorganisms, and to the incor-poration of exogenous microorganisms (Tejada et al., 2006,2007). Soil microbial respiration, measured by carbondioxide emission is a direct indicator of microbial activity,and indirectly reflects the availability of organic material(Tejada et al., 2006, 2007). According to Marinari et al.(2000) the increase in soil respiration observed whenorganic matter is applied to soil can be due to a synergiceffect of the microorganisms of both soil and amendmentor to a stimulation of microbial growth by the organic sub-strates added with the amendments.

Dehydrogenase activity typically occurs in all intact,viable microbial cells. Thus, its measurement is usuallyrelated to the presence of viable microorganisms and theiroxidative capability (Trevors, 1984). According to Raoand Pathak (1996), the incorporation of organic amend-ments to soil stimulates dehydrogenase activity becausethe added material may contain intra- and extracellularenzymes and may also stimulate microbial activity in thesoil (Tejada et al., 2006, 2007). The high level of dehydro-genase activity in the soils treated with organic wastes sug-gests the existence of a high quantity of biodegradablesubstrates in these soils, which is in agreement with theirhigh content of labile C capable of stimulating soil micro-bial activity.

Urease catalyses the hydrolysis of urea to carbon diox-ide and ammonium, and it is widely distributed in microor-ganisms, plants and animals (Nannipieri et al., 2002). Thestimulation of this enzymatic activity with respect to thenon organic amended soil suggests that the amendmentused did not contain compounds toxic for these activities,it increased soil microbial growth due to the substratesadded, and added microbial cells and/or enzymes, whichcounteract any inhibitory effect of the toxic compounds.

The higher b-glucosidase activity in organic amendedsoils can be explained by the positive effect of the organicamendment on the activity of this enzyme, probably dueto the higher microbial biomass produced in response (Tej-ada et al., 2006, 2007).

Phosphatase and arylsulphatase release phosphate andsulfate, the main plant and microbial available P and Sforms, from various organic phosphate and sulfate esters(Nannipieri et al., 2002). The existence of phosphomonoes-terase activity in organic amended soils is interesting sincethis hydrolase manages to hydrolize phosphorus organiccompounds to render them inorganic and therefore moreuseable by microorganisms and plants (Speir and Ross,2000). The high activity detected in amended soils suggestseither the existence in organic wastes of phosphorus andsulphate compounds that can act as substrate for theenzyme, or the existence of microbial populations whichneed inorganic phosphorus for their own development,stimulating the enzyme synthesis.

M. Tejada et al. / Bioresource Technology 99 (2008) 2872–2880 2879

Besides the stimulating influence of organic matter onthe development of the metabolism of microorganismsand hence on the level of enzymatic activity, organic mattercan also have other effects. Numerous studies pointed tothe role of organic matter for sorption processes of organicpollutants (Murphy et al., 1990; Pignatello and Xing,1996), probably due to the functional groups of humic sub-stances, such as carboxyl, phenolic, alcohol, and carbonyl(Datta et al., 2001). In some specific cases, very stable com-binations of organic pollutants with organic matter can beformed. Xenobiotics are then unavailable to microorgan-isms, lowering their toxic effect on these microorganisms.In the literature these combinations are called ‘bound-resi-due’ (Alexander, 1995; Kastner and Richnow, 2001). Aside-effect of the above phenomena is the limitation ofthe remediation processes.

Our results pointed to differences in the percentage ofinhibition of soil enzymatic activities in organic wastesamended soils. The principal difference between theseorganic wastes was the humic and fulvic acid concentra-tion, CCGC having a higher humic acid concentration,followed by PM, SS and MSW. It is clear that materialsthat are formed through accelerated fermentation pro-cesses, such as composting, are able to immobilizeenzymes in their humic fraction in a higher proportionthan fresh materials (Pascual et al., 1998). It is possiblethat the organic material itself influences such complexformation. The PM, SS and MSW has little humifiedmaterial available (extractable carbon with sodium pyro-phosphate), and this carbon type may be the one that col-laborates in the formation of enzymatic complexes in ourstudy.

In addition, humic and fulvic acids differ in their struc-tures. Fulvic acids are macromolecules with a lower poly-merization index, are more highly charged, more polar,and have a lower molecular weight than humic acids.Hayes (1991) suggested that humic substances with highermolecular fractions contain more strong acid groups thanthe lower molecular weight materials. Humic acids havegreater aromaticity than fulvic acids, and this is also inkeeping with the concept of greater numbers of aromaticcarboxylic acids in the humic acids. For this reason, thebinding of humic acids with gasoline is higher than in fulvicacids. These results are in agreement with those of Murphyet al. (1990) and Kollist-Siigur et al. (2001) who suggestedthat humic acids had greater binding affinity for PAHsthan fulvic acids. These results also suggest that soil enzy-matic activities are higher in humic acid – than in fulvicacid-amended soil.

5. Conclusions

It can be concluded that the contamination of soil withgasoline has a negative effect on soil ecosystems and nutri-ent cycles. The application of CC, PM, SS and MSW in thedoses studied to a soil polluted with gasoline under labora-tory conditions improved the soil enzymatic activities com-

pared with no organically amended soils. The microbialbiomass carbon, respiration and enzyme activities studiedwere inhibited by gasoline and the four organic amend-ments decreased gasoline toxic effects significantly. How-ever, this increase was different depending of the organicmatter type applied to soil. The inhibition of the soil bio-chemical properties was lowest in CC-amended soils, fol-lowed by PM, SS and MSW-amended soils. Therefore,our results suggest that: (1) the addition of these organicmaterials may be considered a good strategy for remediat-ing gasoline contaminated soils, and (2) the addition oforganic materials with a high humic acid content is benefi-cial for such remediation.

References

Alexander, M., 1995. How toxic are chemicals in soil. Environ. Sci.Technol. 29, 2713–2717.

Alexander, M., 1999. Biodegradation and bioremediation. AcademicPress, Inc., San Diego.

Anderson, J.P.E., 1982. Soil respiration. In: Page, A.L., Miller, R.H.,Keenney, D.R. (Eds.), Methods of soil analysis, chemical andmicrobiological properties. Part 2, 2nd ed. Agron. Monogr. 9. ASAand SSSA, Madison, WI, pp. 831–871.

Andreoni, V., Cavalca, L., Rao, M.A., Nocerino, G., Bernasconi, S.,Dell’Amico, E., Colombo, M.m., Gianfreda, L., 2004. Bacterialcommunities and enzyme activities of PAHs polluted soils. Chemo-sphere 57, 401–412.

Baran, S., Bielinska, J.E., Oleszczuk, P., 2004. Enzymatic activity in anairfield soil polluted with polycyclic aromatic hydrocarbons. Geo-derma 1118, 221–232.

Bauer, E., Pennerstorfer, C., Holubar, P., Plas, C., Braun, R., 1991.Microbial activity measurements in soil: a comparison of methods. J.Microbiol. Methods 14, 109–117.

Boopathy, R., 2000. Factors limiting bioremediation technologies. Biores.Technol. 74, 63–67.

Cernigilia, C.E., 1984. Microbial metabolism of polycylic aromatichydrocarbons. Adv. Appl. Microbiol. 30, 31–71.

Coover, M.P., Sims, R., 1987. The rate of benzo(a)pyrene apparent loss innatural and manure amended clay loam soil. Hazard. Waste Hazard.Mater. 4, 69–82.

Datta, A., Sanyal, S.K., Saha, S., 2001. A study on natural and synthetichumic acids and their complexing ability towards cadmium. Plant Soil235, 115–125.

Eibes, G., Cajthaml, T., Moreira, M.T., Feijoo, G., Lema, J.M., 2006.Enzymatic degradation of anthracene, dibenzothiophene and pyreneby manganese peroxidase in media containing acetone. Chemosphere64, 408–414.

Garcia, C., Hernandez, T., Costa, F., Ceccanti, B., Masciandro, G. 1993.The dehydrogenase activity of soils and ecological marker inprocesses of perturbed system regeneration. In: XI InternationalSymposium Environmental Biogeochemistry Salamanca, Spain, pp.89–100.

Guanasekara, A.S., Xing, B., 2003. Sorption and desorption of naphtha-lene by soil organic matter: Importance of aromatic and aliphaticcomponents. J. Environ. Qual. 32, 240–246.

Guitian, F., Carballas, T., 1976. In: Sacro, Picro (Ed.), Tecnicas de analisisde suelos. Santiago de Compostela, Espana.

Hayes, H.B.H., 1991. Concepts of the origins, composition, and structuresof humic substances. In: Wilson, W.S. (Ed.), Advances in soil organicmatter research: The impact on agriculture and the environment. TheRoyal Society of Chemistry, Cambridge, UK, pp. 3–22.

Kanaly, R.A., Harayama, S., 2000. Biodegradation of high-molecular-weight polycyclic aromatic hydrocarbons by bacteria. J. Bacteriol. 182,2059–2067.

2880 M. Tejada et al. / Bioresource Technology 99 (2008) 2872–2880

Kandeler, E., Gerber, H., 1988. Short-term assay of soil urease activityusing colorimetric determination of ammonium. Biol. Fertil. Soils 6,68–72.

Kastner, M., Richnow, H.H., 2001. Formation of residues of organicpollutants within the soil matrix – mechanisms and stability. In:Stegmann, R., Brunner, G., Calmano, W., Matz, G. (Eds.), Treatmentof Contaminated Soil. Springer-Verlag, Berlin, pp. 221–250.

Kiss, S., Pasca, D., Dragan-Bularda, M., 1998. Development in SoilScience 26: Enzymology of disturbed soils. Elsevier, Amsterdam.

Kleineidam, S., Ruegner, H., Ligouis, B., Grathwohl, P., 1999. Organicmatter facies and equilibrium sorption of phenanthrene. Environ. Sci.Technol. 33, 1637–1644.

Kollist-Siigur, K., Nielsen, T., Grøn, C., Hansen, P.E., Helweg, C.,Jonassen, K.E.N., Jørgensen, O., Kirso, U., 2001. Sorption ofpolycyclic aromatic compounds to humic and fulvic acid HPLCcolumn materials. J. Environ. Qual. 30, 526–537.

Kononova, M.M., 1966. Soil organic matter, second ed. Pergamon Press,Oxford.

Kucharski, J., Jastrzebska, E., Wyszkowska, J., Hlasko, A., 2000. Effect ofpollution with diesel oil and leaded petrol on enzymatic activity of thesoil. Zesz. Probl. Postpep. Nauk Rol. 472, 457–464.

Labud, V., Garcia, C., Hernandez, T., 2007. Effect of hydrocarbonpollution on the microbial properties of a sandy and a clay soil.Chemosphere 66, 1863–1871.

Maliszewska-Kordybach, B., Smreczak, B., Martyniuk, S., 2000. Theeffect of polycyclic aromatic hydrocarbons (PAHs) on microbialproperties of soils of different acidity and organic matter content.Rocz. Glebozn. 3, 5–18.

MAPA, 1986. Metodos oficiales de analisis. Ministerio de Agricultura,Pesca y Alimentation. 1, 221–285.

Margesin, R., Walder, G., Schinner, F., 2000. The impact of hydrocarbonremediation (diesel oil and polycyclic aromatic hydrocarbons) onenzyme activities and microbial properties of soil. Acta Biotechnol. 20,313–333.

Marinari, S., Masciandaro, G., Ceccanti, B., Grego, S., 2000. Influence oforganic and mineral fertilisers on soil biological and physical proper-ties. Biores. Technol. 72, 9–17.

Masciandaro, G., Ceccanti, B., Garcia, C., 1994. Anaerobic digestion ofstraw and piggery waste waters. II. Optimization of the process.Agrochimica 38, 195–203.

Murphy, E.M., Zachara, J.M., Smith, S.C., 1990. Influence of mineral-bound humic substances on the sorption of hydrophobic organiccontaminants. Environ. Sci. Technol. 24, 1507–1516.

Murphy, E.M., Zachara, J.M., Smith, S.C., Phillips, J.L., Wietsma, T.,1994. Interaction of hydrophobic organic compounds with mineral-bound humic substances. Environ. Sci. Technol. 28, 1291–1999.

Nannipieri, P., Kandeler, E., Ruggiero, P., 2002. Enzyme activities andmicrobiological and biochemical processes in soil. In: Burns, R.G.,

Dick, R.P. (Eds.), Enzymes in the Environment. Activity, Ecology andApplications. Marcel Dekker, New York, pp. 1–33.

Pascual, J.A., Hernandez, T., Garcia, G., Ayuso, M., 1998. Enzymaticactivities in an arid soil amended with urban organic wastes:Laboratory experiment. Biores. Technol. 64, 131–138.

Pignatello, J.J., Xing, B., 1996. Mechanism of slow sorption of organicchemicals to natural particles. Environ. Sci. Technol. 30, 1–11.

Rao, D.L.N., Pathak, H., 1996. Ameliorative influence of organic matteron the biological activity of salt affected soils. Arid Soil Res. Rehab.10, 311–319.

Rutherford, D.W., Chiou, C.T., Kile, D.E., 1992. Influence of soil organicmatter composition on the partition of organic compounds. Environ.Sci. Technol. 26, 336–340.

Sims, J.R., Haby, V.A., 1971. Simplified colorimetric determination of soilorganic matter. Soil Sci. 112, 137–141.

Soil Survey Staff. 1987. Keys to Soil Taxonomy. SMSS TechnicalMonograph No. 6, 3rd ed. New York.

Speir, T.W., Ross, D.J., 2000. Hydrolytic enzyme activities to assess soildegradation and recovery. In: Burns, R.G. (Ed.), Enzymes in theEnvironment Activity, Ecology, and Applications. Marcel Dekker,New York, pp. 407–433.

SSEW, Soil Survey of England and Wales, 1982. Soil Survey Laboratorymethods. Tech. Monogr. 6. SSEW, Harpenden, UK.

Statiscal Graphics Corporation, 1991. Statgraphics 5.0. Statistical Graph-ics System. Educational Institution Edition, USA, p. 105.

Tabatabai, M.A., Bremner, J.M., 1969. Use of p-nitrophenol phosphate inassay of soil phosphatase activity. Soil Biol. Biochem. 1, 301–307.

Tabatabai, M.A., Bremner, J.M., 1970. Arylsulfatase activity of soils. SoilSci. Soc. Am. Proc. 34, 225–229.

Tejada, M., Garcia, C., Gonzalez, J.L., Hernandez, M.T., 2006. Organicamendment based on fresh and composted beet vinasse: influence onphysical, chemical and biological properties and wheat yield. Soil Sci.Soc. Am. J. 70, 900–908.

Tejada, M., Hernandez, M.T., Garcia, C., 2007. Application of twoorganic wastes in a soil polluted by lead: Effects on the soil enzymaticactivities. J. Environ. Qual. 36, 216–225.

Trevors, J.T., 1984. Dehydrogenase in soil: a comparison between the INTand TTC assay. Soil Biol. Biochem. 16, 673–674.

Vance, E.D., Brookes, P.C., Jenkinson, D.S., 1987. An extraction methodfor measuring soil microbial biomass C. Soil Biol. Biochem. 19, 703–707.

Yeomans, J.C., Bremner, J.M., 1988. A rapid and precise method forroutine determination of organic carbon in soil. Commun. Soil Sci.Plant Anal. 19, 1467–1476.

Zibilske, L.M., 1994. Carbon mineralization. In: Weaver, R.W. (Ed.),In: Methods of Soil Analysis. Part 2. Microbiological and BiochemicalProperties. SSSA Book Series 5, SSSA, Madison, WI, pp. 835–863.