Chemosphere 58 (2005) 515522 www.elsevier.

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Bioremediation of a weathered and a recently oil-contaminated soils from Brazil: a comparison study
P.V.O. Trindade a, L.G. Sobral b, A.C.L. Rizzo
a b

b,*

, S.G.F. Leite c, A.U. Soriano

Petrobras Research Center (CENPES), Quadra 7, Ilha do Fundao, Rio de Janeiro, RJ, CEP 21949-900, Brazil Center for Mineral Technology, Ilha da Cidade Universitaria, Ministry of Science & Technology (CETEM/MCT), Av. Ipe, 900, Ilha do Fundao, Rio de Janeiro, RJ, CEP 21941-590, Brazil c School of Chemistry, Technology Center, Federal University of Rio de Janeiro, Ilha do Fundao, Rio de Janeiro, RJ, CEP 21949-900, Brazil Received 16 June 2003; received in revised form 31 August 2004; accepted 9 September 2004

Abstract The facility with which hydrocarbons can be removed from soils varies inversely with aging of soil samples as a result of weathering. Weathering refers to the result of biological, chemical and physical processes that can aect the type of hydrocarbons that remain in a soil. These processes enhance the sorption of hydrophobic organic contaminants (HOCs) to the soil matrix, decreasing the rate and extent of biodegradation. Additionally, pollutant compounds in high concentrations can more easily aect the microbial population of a recently contaminated soil than in a weathered one, leading to inhibition of the biodegradation process. The present work aimed at comparing the biodegradation eciencies obtained in a recently oil-contaminated soil (spiked one) from Brazil and an weathered one, contaminated for four years, after the application of bioaugmentation and biostimulation techniques. Both soils were contaminated with 5.4% of total petroleum hydrocarbons (TPHs) and the highest biodegradation eciency (7.4%) was reached for the weathered contaminated soil. It could be concluded that the low biodegradation eciencies reached for all conditions tested reect the treatment diculty of a weathered soil contaminated with a high crude oil concentration. Moreover, both soils (weathered and recently contaminated) submitted to bioaugmentation and biostimulation techniques presented biodegradation eciencies approximately twice as higher as the ones without the aforementioned treatment (natural attenuation). 2004 Elsevier Ltd. All rights reserved.
Keywords: Bioremediation; Biostimulation; Bioaugmentation; Soil; Oil; Weathering

1. Introduction The risk of oil spillage, involved in many activities of the petroleum industry, poses a serious environmental

* Corresponding author. Tel.: +55 21 3865 7345; fax: +55 21 3865 7232. E-mail address: arizzo@cetem.gov.br (A.C.L. Rizzo).

problem, due to the possibility of air, water and soil contamination. Recent oil spill occurrences in clay-bearing Brazilian soils are motivating studies in the soil bioremediation eld. Bioremediation is an attractive process due to its cost eectiveness and the benet of pollutant mineralization to CO2 and H2O (da Cunha, 1996). This technology accelerates the naturally occurring biodegradation under optimized conditions such as oxygen supply, temperature, pH, the presence or addition of

0045-6535/$ - see front matter 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.chemosphere.2004.09.021

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P.V.O. Trindade et al. / Chemosphere 58 (2005) 515522 Table 1 Background characteristics of used soils Characteristics Water holding capacity (WHC) (%) pH Total petroleum hydrocarbons (TPH) (%) Polycyclic aromatic hydrocarbons (PAH) (%) Corganic (%) N (g kg1 soil) P (mg kg1 soil) Contaminated soil 26.9 4.8 5.4 1.7 4.8 0.6 2.0 Non-contaminated soil 32.8 5.4 1.0 0.6 2.0

suitable microbial population (bioaugmentation) and nutrients (biostimulation), water content and mixing. The biodegradation of an oil-contaminated soil can also be seriously aected by the hydrocarbons structure and by the contamination time, due to weathering processes, which decrease the bioavailability of pollutants to microorganisms. Weathering refers to the result of biological, chemical and physical processes that can aect the type of hydrocarbons that remain in a soil (Loehr et al., 2001). Those processes enhance the sorption of hydrophobic organic contaminants (HOCs) to the soil matrix, decreasing the rate and extent of biodegradation (Bosma et al., 1997). Generally, only the fraction of HOCs dissolved in the aqueous phase is available for microbiological degradation while the sorbed fraction has a low bioavailability (Allan et al., 1997). Moreover, a weathered oil-contaminated soil normally contains a recalcitrant fraction of compounds composed, basically, of high molecular weight hydrocarbons (higher than C25 compounds), which could not be degraded by indigenous microorganisms. In contrast, a recently oil-contaminated soil contains a higher amount of saturated and aliphatic compounds, which are the most susceptible to the microbial degradation. However, pollutant compounds in a recently contaminated soil are potentially more toxic to the native microorganisms, leading to longer adaptation time (lag phase) before degrading the pollutant and even to an inhibition of the biodegradation process. The present work aimed at comparing the biodegradation eciencies obtained applying bioaugmentation and biostimulation techniques in a weathered crude oil-contaminated Brazilian soil (real situation; four years of contamination) and in a recently contaminated one (a spiked soil), both at tropical conditions (high temperature and clay content). In previous investigations (Rizzo et al., 2002; Trindade, 2002; Trindade et al., 2002), the authors determined the best strategies of biostimulation, bioaugmentation and moisture adjustment to be applied in the biological treatment of the Brazilian contaminated soil used in this study. A dened microbial consortium, in an inoculum size of 108 counting forming unit (CFU) g1 of soil, was used and the C:N:P ratio was adjusted to 100:1.25:1 (optimized conditions). Both soils were contaminated with 5.4% of total petroleum hydrocarbons. The main challenge of this work was to evidence the eect of the weathering process in the biodegradation of a heavy crude oil present in a typical tropical soil, containing high content of clay material.

years ago, and a non-contaminated one collected in the same site (Sao Paulo, Brazil). After sampling, both soils were stored at 5 C. Some physicochemical characteristics of contaminated and non-contaminated soils were obtained previously by the authors (Rizzo et al., 2002) and the background information for them is summarized in Table 1. The soil pH and water holding capacity were measured according to Alef and Nannipieri (1995) for oil-contaminated and non-contaminated samples. Samples of old contaminated soil were extracted with n-hexane (EPA 3550B, 1996) and the organic extracts were analyzed in terms of total petroleum hydrocarbons TPH (EPA 8015B, 1996) and polycyclic aromatic hydrocarbonsPAHs (Fitzgerald et al., 1970). Additionally, fertility analyses were done (organic C, P, and N) to determinate if there were any limiting nutrients to biodegradation experiments (EMBRAPA, 1997). In conditions where the recently contaminated soil was evaluated, the crude oil was added to the noncontaminated soil in order to obtain a concentration around of 5.4% of TPH, as observed in the weathered contaminated soil. The crude oil used was similar to the heavy crude oil that contaminated the older weathered soil. 2.2. Bioaugmentation A microbial consortium constituted by two crude oildegrading microorganisms (Nocardia nova and Rhodotorula glutinis var. dairenesis), previously isolated from the crude oil-contaminated soil, was added to the soil samples in a inoculum size of 108 CFU g1 soil (Sa, 2002; Trindade, 2002). The inoculum was obtained in the exponential growth phase of the strains, determined by previous growth kinetics studies (Sa, 2002). 2.3. Biostimulation In conditions where the biostimulation technique was employed the carbon: nitrogen:phosphorous (C:N:P)

2. Materials and methods 2.1. Soils The soils investigated were a Brazilian clay bearing soil contaminated with crude oil, approximately four

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ratio was adjusted to 100:1.25:1 by adding a K2HPO4 solution, as suggested by Trindade (2002). No nitrogen addition was necessary, as the nitrogen concentration in soil was sucient to maintain the aforementioned ratio. 2.4. Biodegradation experiments The biodegradation experiments were carried out in 250 ml conical asks (Pyrex lter ask for use in vacuum ltration applications), containing 50 g of soil and others additives such as nutrients and inoculum, hermetically closed with rubber stoppers. The soil moisture and pH were adjusted to 50% of water holding capacity and 7.0, respectively. The specic conditions used for each sample are described as follow: NCS: non-contaminated soil. AC (abiotic control): recently contaminated soil sample and sodium azide addition (0.3% w w1) to inhibit soil microorganisms and to monitor abiotic hydrocarbon losses. RCS: recently contaminated soil sample. RCSopt: recently contaminated soil sample, nutrient supplementation (biostimulation) and inoculum addition (bioaugmentation). WCS: weathered contaminated soil WCSopt: weathered contaminated soil, nutrient supplementation (biostimulation) and inoculum addition (bioaugmentation). The asks were incubated at 30 C for 41 days and, during this time, periodical renewals of asks headspaces were made by opening and aerating them for 2 min to keep sucient oxygen concentration. This aerations process occurred in 48 h intervals and after the sampling for chromatographic analyse of CO2. All conditions were performed in triplicates and, for each one, one additional sacrice ask was used to initial quantitative determination of microorganisms. In those experiments, the microbial activity was monitored by carbon dioxide production and by TPH removal. 2.5. Quantitative determination The carbon dioxide determination was performed in order to verify the mineralization occurrence, being also an indicative of microbial activity. For this purpose, a HewlettPackard 5890A II gas chromatograph was used for analyzing gas of the headspaces from hermetically stopper Pyrex lter ask containing soil samples. The carrier gas was ultra-high purity helium and a thermal conductivity detector set at 220 C and CHROMOSORB 102 column was used. The gas conned in Pyrex ltering asks was sampled, using a gas-tight syringe, in

volume of 500 ll through the suction of the internal atmosphere of the asks headspace before deaerating process. It could be assumed that, as a consequence of the heterotrophic aerobic metabolisms of the soil microorganisms, if two moles of organic carbon are consumed (from the weathered oil or from the soil natural organic matter), one carbon mol is employed in CO2 generation, and the second carbon mol is incorporated into the biomass. So, the CO2 quantied by gas chromatography represents only 50% of the total organic matter biodegraded. Based on this concept and using the accumulated value of CO2 generated during 41 days of the experiments, the biodegradation eciency (BE) could be calculated as: BE% = (Totally biodegraded carbon) 100/total organic carbon in the soil; Totally biodegraded carbon = 2 Carbon from CO2 generated during 41 days. It is also frequently assumed that the total organic carbon corresponds to 8090% of TPH content in the contaminated soil with crude oil. It is possible to conrm in Table 1 that the value obtained for organic carbon (Corganic, %) corresponds to 89% of the TPH (%) present in the weathered contaminated soil. So, in this work, it was considered that: Total organic carbon in the soil = 85% of TPH concentration at the beginning of the experiment. It was applied for both cases: weathered and recently contaminated soils. Samples of soil in the beginning and at the end of the experiments were extracted with n-hexane (EPA 3550B, 1996) and the organic extracts were analyzed in terms of total petroleum hydrocarbonsTPH (EPA 8015B, 1996). The results were used to calculate the percentage of TPH removal for each condition. The quantication of total heterotrophic microorganisms was accomplished, at the beginning and in the end of the experiments, by the pour plate technique (triplicates) in a solid organic medium (g l1): glucose, 10.0; peptone, 5.0; yeast extract, 2.0; NaCl, 5.0, AgarAgar, 20.0. The Petri dishes were incubated at 30 C for 48 h and the results were expressed in CFU g1 of dry soil. The quantication of oil-degrading microorganisms was made, at the beginning and in the end of the experiments, by the most probable number (MPN) technique (Oblinger and Koburger, 1975). The liquid mineral medium (Vecchioli et al., 1990), the crude oil (0.5% v v1) and the microorganism inoculum were added to the NMP tubes, which were incubated at 30 C for 7 days. The results were expressed in cells g1 soil.

3. Results and discussion The CO2 quantication accomplished during the biodegradation experiments allowed an evaluation of the CO2 evolution prole of each condition (Fig. 1).

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8.00

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NCS

A.C.

RCS

RCSopt

WCS

WCSopt

CO2 Evolution (mmol)

6.00

4.00

2.00

0.00 0 5 10 15 20 25 Time (days) 30 35 40 45

Fig. 1. Proles of CO2 evolution.

It was observed that the indigenous microorganisms responsible for the CO2 generation in the RCS condition presented an adaptation period (015 days) in which no destruction of the pollutant is evident. This behavior was not observed in conditions WCS, and WCSopt and it can be explained by the previous exposure of the native microorganisms to the contaminant for four years, leading to a selection of the oil-degrading and/or tolerant ones. As reported by Alexander (1999), once the indigenous community of microorganisms has become acclimated to the degradation of a chemical and the activity becomes marked, the potential for this activity may continue to remain higher than in comparable soils that have not acquired this capacity. The adaptation phase was also not present in the recently contaminated soil sample with phosphorus adjustment and inoculum addition (RCSopt), which presented relevant CO2 generation since the beginning of the experiment (Fig. 1). This behavior could be mainly associated to the inoculum addition (N. nova and R. glutinis var. dairenensis in an inoculum size of 108 CFU g1 soil) since it is well known that the addition of the exogenous microorganisms in the contaminated soils was extremely important in eliminating the adaptation phase (Davis and Madsen, 1996; Alexander, 1999). It is possible to note in Fig. 1 that the RCS and RCSopt conditions in day 35 reached the deceleration phase of the CO2 generation, probably caused by the beginning of the recalcitrant compounds degradation or by the inability of the microorganisms in degrading those compounds. Crude oil components are dierentially degraded: branched alkanes are more resistant to microbial degradation than n-alkanes due to their molecular structure. Moreover, the biologically inert fraction, as asphaltenes, also aects the degradation of crude oil in soils because they inhibit the transport of the biodegradable fraction, decreasing the rate and extent of biodegradation (Uraizee et al., 1997). The more recalcitrant fraction of contamination seems to be de-

graded with a rate that is about an order of magnitude lower than the one observed for other components. As a consequence, after an initial period of biological treatment, the hydrocarbon concentration tends to stabilize at a value that can be called residual concentration. One of the most accepted hypotheses proposes that pollutant transfer phenomena are probably involved in the rate-limiting step (Margesin et al., 2000; Nocentini et al., 2000). Under these circumstances, the residual concentration depends mainly on soil characteristics and contamination aging. Table 2 presents the TPH values obtained for the soil samples at the beginning and in the end of the experiments. It is possible to note that the amount of crude oil added to the non-contaminated soil to reproduce the contamination level present in the weathered contaminated soil was excessive, leading to a higher TPH value (67.8 mg g1 soil). The BE based on the total amount of CO2 generated during the biodegradation experiments and percentage of TPH removal for the tested conditions were calculated and are presented in Table 3. It could be observed that even presenting dierent values, the two parameters show the same behavior of biodegradation susceptibility for each condition, indicating that both CO2 and TPH

Table 2 Total petroleum hydrocarbon (TPH) content Conditions TPH (mg g1 soil)a Initial (t = 0 days) NCS AC RCS RCSoptimal WCS WCSoptimal
a

Final (t = 41 days) 0.00 0.02 66.89 0.04 66.19 0.03 63.93 0.02 47.62 0.03 46.13 0.01

0.00 0.01 67.77 0.03 67.77 0.03 67.77 0.03 53.80 0.02 53.80 0.02

Mean values of three experiments.

P.V.O. Trindade et al. / Chemosphere 58 (2005) 515522 Table 3 Biodegradation eciencies (BE, %) and TPH removal (%) for the tested conditions Conditions NCS AC RCS RCSopt WCS WCSopt
a

519

BE (%)a 0.5 0.1 0.0 0.0 1.0 0.2 2.4 0.1 3.7 0.6 7.4 0.1

TPH removal (%)a 0.0 0.0 1.3 0.0 2.3 0.0 5.7 0.0 11.5 0.0 14.3 0.0

Mean values of three experiments.

quantication are adequate to be employed in biodegradation process monitoring. However, is important to emphasize that BE and TPH removal give dierent interpretations, since comparatively, the mineralization phenomenon is slower than incomplete transformation of organic compounds, meaning that the biodegradation eciency based on CO2 generation is frequently lower than one based on chromatographic determination. This dierence can be explained analytically, since chromatographic determination takes into consideration both of transformation: formation of intermediary polar compounds (which can correspond or not to the appearance of an unresolved fraction) and mineralization (denitive peak disappearance). The WCSopt condition presented the highest biodegradation eciency (7.4%) which was approximately twice as high as the BE obtained in the control of natural attenuation of the WCS condition (Table 3). The WCSopt condition also presented the highest TPH removal (14.4%), which was 1.2 times higher than the value obtained for the WCS condition (11.5%). In addition, the optimized condition presented biodegradation eciency and percentage of TPH removal approximately three-times higher than the RCSopt. Although the same optimized conditions (inoculum addition and C:P adjustment) had been applied in those soils, the adapted native microorganisms in WCSopt were responsible for this dierential in the biodegradation eciency and TPH removal. Possibly, as mentioned before, previous adaptation of that native microbial population to the high pollutant concentration and its cometabolic interactions with the exogenous microbial population favoured the biodegradation of the crude oil, including more recalcitrant compounds. Both recently contaminated soil conditions (RCS and RCSopt) presented low biodegradation eciencies and TPH removals suggesting, as aforementioned, that the previous adaptation of the microbial population is extremely important and that the elevated crude oil concentration present in the samples (6.8% of TPHs) could have inhibited the microorganisms growth and, consequently, reduced their degradation potential. The available substrate concentration is one of the most

important parameters that inuences the biodegradation of xenobiotic pollutants. Although the biodegradation rate is directly related with the substrate concentration, high levels of chemical compounds can cause inhibitory or toxic eects to the microorganisms. As mentioned by DelArco and de Franca (2001), 4% is a threshold con centration of crude oil in soils above which biodegradation occurs in very low rate. However, even with the high oil concentration of 6.8%, RCSopt condition presented a biodegradation eciency and a percentage of TPH removal 141.3% and 144.6%, respectively, higher than the natural attenuation control of the RCS condition. As observed in Table 3, low biodegradation eciencies and TPH removal were reached for all conditions tested (BE ranging from 0.5% to 7.4% and TPH removal ranging from 0.00% to 14.3%) reecting the treatment diculty of a weathered soil contaminated with a high crude oil concentration. One point to be mentioned is the sorption of hydrophobic organic contaminants (HOCs) to soil matrix that is thought to lower the rate and extent of biodegradation by decreasing contaminant bioavailability (Allan et al., 1997; Bosma et al., 1997; Berg et al., 1998; Jorgensen et al., 2000; Conte et al., 2001; Kottler and Alexander, 2001). It is generally assumed that only the fraction of HOCs dissolved in the aqueous phase is available for microbial use while the sorbed fraction is inaccessible to microorganisms, although there are exceptions. The dierent soil compounds also inuences biodegradation of the organic pollutants. The characteristics, which may inuence bioavailability, are: organic matter content and mineralogy. The biodegradation rates are usually higher in sandy soils than aluminosilicate soils and, the organic matter content is inversely proportional to biodegradation rates. Moreover, another extremely important factor, which inuences the contaminant sorption to soil, is the weathering process. It can be noted that a pollutant fraction seems to be inaccessible to biodegradation in contaminated soils for a long time due to the weathering. According to Bosma et al. (1997), decreased bioavailability may result from: incorporation of the pollutant to the soil natural organic material due to the chemical oxidation reactions, slow diusion into very small pores, sorption into organic matter and formation of semi-rigid lms around non-aqueous-phase liquids, causing a high resistance to the mass transfer. However, the weathered fraction can represent an environmentally acceptable endpoint (EAE). Such an endpoint occurs when (Berg et al., 1998): the chemicals that remain in the soil do not have an adverse impact on human health and the environment, and any chemicals that are slowly released from the soil are mainly weathered by natural assimilative capacity of the soil. The EAE concept recognizes that: (1) high concentrations of chemicals in a soil may not be associated with

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adverse eects, (2) at a site with contaminated soil, risk to human health and the environment is related directly to the release and availability of a chemical, and (3) what is released and the rate of release is as important as the type and concentration of pollutant that is in the soil. Loehr et al. (2001) reported that one soil containing aged crude oil was not able to be remediated using solid-phase bioremediation (land treatment) and, although substantial concentrations of hydrocarbons remained in the soil, it was determined that those hydrocarbons were highly weathered and unable to be released from the soil. Based in the aforementioned concept and analyzing Fig. 2, which presents the representative chromatograms of the crude oil that caused the contamination of the soil and the extract obtained after four years of contamination, the remaining fraction of crude oil in the contaminated soil can represent an EAE. Possibly, the best biodegradation eciency and TPH removal gotten in the experiment (7.4% and 14.3%, respectivelyWCSopt) had been enough to eliminate the rest of potential toxic

compounds that still remained in the weathered contaminated soil, facts that should be exploited in a future work, by performing toxicity and leaching tests before and after soil remediation. Another way to verify the eect of the conditions tested on the biodegradation process is evaluating the percentage of oil-degrading microorganisms in comparison to the total amount of heterotrophic microorganisms, in soils samples, at the beginning and at the end of the experiments (Table 4). The NCS and RCS conditions presented the lowest oil-degrading microorganisms percentages during all the experiment. Despite the exogenous microorganism inoculation, the degrading percentage of the RCSopt decreased in the end of the test. Possibly, the low native microbial population present in the sample (103 CFU g1 soil) was not enough to support the metabolic interactions necessary to the growth and maintenance of exogenous oil-degrading microorganisms. In the WCS and WCSopt conditions it was observed a low reduction in the degrading percentage at the end

FID1 A, (GUAMAREM\SIG10008.D) counts 60000 50000 40000 30000 20000 10000

(a)

10

20

30

40

50

min

FID1 A, (AJFINAL\SIG10018.D) counts 18000 16000 14000 12000 10000 8000 6000 0 10 20 30 40 50

(b)

min

Fig. 2. Representative chromatograms of the crude oil that caused the contamination of the soil (a) and the oil extract obtained after four years of contamination (b).

P.V.O. Trindade et al. / Chemosphere 58 (2005) 515522 Table 4 Degrading microorganisms percentage (logarithmic base) Conditions Initiala total heterotrophic microorganisms (CFU g1 soil) 2.70 105 3.30 104 1.13 105 3.40 108 2.38 106 2.45 108 Initiala degrading microorganisms (cells g1 soil) 1.43 103 0.00 2.38 103 2.38 108 4.28 105 2.28 108 Initiala Degrading microorganisms (%) 64.61 0.00 72.30 98.37 89.90 99.67 Finalb total heterotrophic microorganisms (CFU g1 soil) 6.08 107 5.60 103 1.35 108 6.45 108 3.10 108 1.90 108 Finalb degrading microorganisms (cells g1 soil) 7.13 105 0.00 9.03 105 9.03 106 1.90 107 4.28 107

521

Finalb degrading microorganisms (%) 78.02 0.00 76.18 81.10 87.22 93.02

NCS AC RCS RCSopt WCS WCSopt

a b

Initial: time = 0 days. Final: time = 41 days.

of the experiment. The highest concentration of degrading microorganisms obtained in the end of experiment in the WCSopt condition can explain the best eciency and TPH removal reached also in this condition, 7.4% and 14.3%, respectively.

4. Conclusions The application of the optimized conditions stimulated the biodegradation of both weathered and recently oil-contaminated soils from Sao Paulo, Brazil, leading to the increase of oil mineralization and TPH removal in those soils. Although the weathering process was responsible for the low levels of biodegradation eciencies and TPH removal obtained after 41 days of experiments, the well adapted native microbial consortium was responsible for better decontamination results than those obtained for a recently contaminated soil. This work gave a contribution in terms of understanding the occurrence of oil biodegradation in tropical soils, in which the weathering phenomenon is especially relevant.

Acknowledgements This study was supported by Conselho Nacional de Desenvolvimento Cientco e Tecnologico (CNPq) and Petroleo Brasileiro S.A. (PETROBRAS).

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