SPE 107384

Biostimulation Treatments of Hydrocarbon-Contaminated Soil in Semiarid Patagonia,

Argentina
P.A. Haro, J.D. Perez, and A.F. Alvarez, Laboratorio-Investigación OIL M&S S.A., and R.A. Silva and H.M. Alvarez,
CRIDECIT, FCN-U. Nacional de la Patagonia San Juan Bosco

Copyright 2007, Society of Petroleum Engineers

Patagonia have the potential to degrade hydrocarbons after
This paper was prepared for presentation at the 2007 SPE Latin American and Caribbean amelioration of unfavorable environmental conditions.
Petroleum Engineering Conference held in Buenos Aires, Argentina, 15–18 April 2007.

This paper was selected for presentation by an SPE Program Committee following review of
information contained in an abstract submitted by the author(s). Contents of the paper, as
Introduction
presented, have not been reviewed by the Society of Petroleum Engineers and are subject to Las Heras is a place located in northwest Santa Cruz province
correction by the author(s). The material, as presented, does not necessarily reflect any
position of the Society of Petroleum Engineers, its officers, or members. Papers presented at (Patagonia, Argentina) with a very intensive activity of the oil
SPE meetings are subject to publication review by Editorial Committees of the Society of
Petroleum Engineers. Electronic reproduction, distribution, or storage of any part of this paper
industry. Crude oil spills have occurred at many sites in the
for commercial purposes without the written consent of the Society of Petroleum Engineers is region, and there is a need to remediate contaminated soil.
prohibited. Permission to reproduce in print is restricted to an abstract of not more than
300 words; illustrations may not be copied. The abstract must contain conspicuous Biodegradation by natural populations of microorganisms
acknowledgment of where and by whom the paper was presented. Write Librarian, SPE, P.O.
Box 833836, Richardson, Texas 75083-3836 U.S.A., fax 01-972-952-9435.
represents one of the primary mechanisms for eliminating
hydrocarbon pollutants from the environment [1, 2]. However,
Abstract the successful application of bioremediation depends on
Bioremediation is considered an appropriate technology for appropriate hydrocarbon-degrading microorganisms and
decontamination of polluted natural environments. The environmental conditions in situ [3]. The persistence of old
successful application of bioremediation depends on spilled hydrocarbons in this region suggests that the rate of
appropriate hydrocarbon-degrading microorganisms and natural degradation is very slow under the environmental
environmental conditions in situ. Las Heras is a place located conditions of semiarid soil. Several conditions may limit
in northwest Santa Cruz (Patagonia, Argentina) with a very hydrocarbon degradation by natural populations of
intensive activity of oil industry. In this region the conditions microorganisms in this area, including cold and fluctuating
most likely to limit hydrocarbon degradation include cold and temperatures (between -20° C in winter to 37° C in summer),
fluctuating temperatures, low moisture contents, low nutrient strong winds (up to 120 km/h), low moisture and low nutrient
levels and alkaline pH. In this work we describe three contents in soil (principally the nitrogen and phosphorous
representative cases of biostimulation treatments (nutrients sources) and alkaline pH. Low temperatures and desiccation
and water addition) applied to soils with different pollution normally reduce microbial metabolism and the nutrient
states: Case A (1680 m3) exhibited high values of total availability can influence the rate and extent of degradation in
petroleum hydrocarbons (TPH 33510 mg/kg); Case B (480 contaminated soils. These conditions represent a challenge to
m3) showed high values of total petroleum hydrocarbons (TPH the classic in situ bioremediation procedures, which were
27754 mg/kg) and polyaromatic hydrocarbons (PAH 1.64 μg/l developed for more temperate climates. One of the primary
in lixiviate); and Case C (1680 m3) exhibited high PAH values benefits of in situ bioremediation is that the procedure does
(PAH 1.94 μg/l in lixiviate). The controlled addition of not require excavation, although it involves minimal site
nitrogen and phosphorous sources and water increased disruption, so site disturbance is much less. This should make
bacterial counts of soil in all cases and resulted in a significant in situ bioremediation cost-competitive since the bulk of the
elimination of hydrocarbons: 32 % TPH in Case A after 7 transportation and excavation cost has been significantly
months of treatment (from May to December 2005); 39 % reduced or nullified [4]. In addition, this procedure is friendly
TPH and 88 % PAH in Case B after 5 months (from October to the environment.
2005 to February 2006) and 90 % of PAH after 3 months of Some studies have examined biodegradation of hydrocarbon-
treatment (from May 2005 to July 2005) were removed. In polluted soil in cold environments, such as Artic and Antartic
addition, we performed a comparative evaluation of natural soils. Mohn et al. [5] demonstrated that on-site bioremediation
attenuation, biostimulation and bioaugmentation (inoculation of fuel-contaminated soil at Artic tundra sites is feasible. On
of bacterial strains) for bioremediation of PAH-contaminated the other hand, other authors demonstrated that indigenous
soil in situ. Both, biostimulation and bioaugmentation microbial communities of Antartic soils are able to degrade
promoted significant degradation (> 90 %) of PAH after 6 petroleum hydrocarbons after the adequate management of the
weeks of treatment, whereas attenuation resulted in a reduced bioremediation procedure [6, 7]. All these studies indicate the
degradation rate (67 %). In conclusion, this study suggests that potential for in situ bioremediation of hydrocarbon-
autochthonous bacterial communities from semiarid soils in contaminated soils at sites with extreme environmental
conditions.
2 SPE 107384

In order to estimate the potential of in situ bioremediation The objective of our study was to analyze the response of the
procedure in soil chronically contaminated with hydrocarbons indigenous bacterial communities of contaminated soil in Las
in Las Heras area some field experiences in addition to an Heras area to the addition of nutrients and water for
experimental assay in a real polluted site were achieved. The supporting a bioremediation process. For this purpose, we
existence of well-documented reports demonstrating on-site evaluated the efficiency of biostimulation treatment in three
bioremediation of soil in semiarid Patagonia is of importance. field experiences in the region. In addition, we isolated and
characterized two indigenous Rhodococcus strains from
Statement of Theory and Definitions: contaminated soil and evaluated their capacity for degrading
The degradation of certain contaminants may take place if a hydrocarbons in a real polluted site. Then, we compared in a
specific population of microorganisms is present in the small scale experiment the efficiency of different
contaminated site. Microbial degradation of organic bioremediation alternatives, attenuation, biostimulation and
contaminants normally provides the microorganisms with a bioaugmentation, for the treatment of a soil contaminated with
source of carbon and energy for their growth and polyaromatic hydrocarbons.
reproduction. However, hydrocarbon degradation by
microorganisms is influenced by environmental conditions [3]. Description and Application of Equipment and
As indicated above, microbial biodegradation in soil may be Processes:
significantly limited by unfavourable environmental Study site: The study was conducted in Las Heras area,
conditions, although the microbial communities have the located in northwest Santa Cruz province (S 46º 5’ and WO
potential for supporting a bioremediation process. We 68º 95’), Patagonia, Argentina. This semiarid region possesses
hypothesize that the indigenous bacterial species may be well an intensive activity of the oil industry. The region possesses a
adapted to the soil and climate of this semiarid region, so semiarid climate which is characterized by great thermal
amelioration of unfavourable environmental conditions by an amplitude along the year with temperatures ranging between -
adequate management of the site will be a good strategy for 20° to 37° C. There are also strong winds mainly during
bioremediation of hydrocarbon-contaminated soil in semiarid autumn months (March-June) (up to 120 km/h) and low level
Patagonia. Biostimulation is a bioremediation alternative, of precipitation, under 200 mm/year. Soil is from clay to
which aims at enhancing the activities of indigenous sandy loam structure with low content of organic matter (< 1
microorganisms that are capable of degrading the contaminant %). The vegetation consists of grass and xerophile plants.
by the addition of nutrients and water [4]. This procedure
leads to an increase in degradation potential without Biostimulation treatments: A field study was conducted in
increasing genetic diversity of soil. In addition, some members three selected soils of the Las Heras area. The selected sites
of the natural microbial population occurring in a were old pits (formerly called mud pits), usually located near
contaminated site may represent good candidates for oil wells, replenished with contaminated soil and wastes. In
bioaugmentation procedures. Bioaugmentation can be these sites the contamination had accumulated in the past as a
considered the inoculation of contaminated soil or water with result of crude oil extraction operations. The aim of this study
specific strains or consortia of microorganisms to improve the was to improve of the biodegradation rate obtained by intrinsic
biodegradation capacity of the system for a specific pollutant bioremediation using the biostimulation treatment in three
organic compound [4]. Among several well known different cases of soil contamination: Case A (1680 m3)
hydrocarbon-degrading microorganisms, we are especially exhibited high values of total petroleum hydrocarbons (TPH
interested in bacteria belonging to Rhodococcus genus. The 33510 mg/kg); Case B (480 m3) showed high values of total
genus Rhodococcus is a very diverse group of bacteria that petroleum hydrocarbons (TPH 27754 mg/kg) and
possesses the ability to degrade a large number of organic polyaromatic hydrocarbons (PAH 1.64 μg/l in lixiviate); and
compounds, including some of the most recalcitrant Case C (1680 m3) exhibited high PAH values (PAH 1.94 μg/l
compounds [8]. The biodegradation of diverse hydrocarbons in lixiviate). In all cases, mechanical homogenization of soil
by Rhodococcus and other related actinomycetes has been plots was performed using an excavator unit before
demonstrated even under unbalanced growth conditions such application of bioremediation technology. In order to evaluate
as nitrogen-starved conditions which are usually found in soil the contamination level of each field plot and to predict the
environments [9]. Cultivation of these bacteria under natural capabilities of soils to biodegrade hydrocarbon
unfavourable conditions frequently promotes the incomplete contaminants, chemical, physical and microbiological
degradation of hydrocarbons and the incorporation of parameters were analyzed before treatments. Quantification of
oxidation intermediates into neutral lipids [9, 10, 11]. In total petroleum hydrocarbons (TPH), total polycyclic
addition, Rhodococcus bacteria combine their genetic and hydrocarbons (PAH) and their components, nutrient contents
metabolic versatility with the ability to occupy many niches (N and P), the presence of heavy metals, humidity and pH
and environments [8]. In a previous study, we reported the were analyzed in addition to microbiological studies. Soils
high tolerance to desiccation, which is one of the most critical were supplemented by adding a dry commercial nitrogen-rich
environmental factors in semiarid Patagonia, in a member of fertilizer (8 g per m2 of soil), which consisted of a mixture of
this genus (Rhodococcus opacus strain PD630) and described urea and phosphorous pentoxide, P2O5 (70:30, w/w). Nutrients
some morphological and physiological mechanisms to were added manually on the top of the soil plots before adding
withstand desiccation [12]. water and mixing soil as described by Jorgensen et al. [13].
Addition of water and oxygenation of field plots were
SPE 107384 3

periodically performed. The response of the microbial from a nutrient-rich complex agar plate used for cell counts.
population to the nutrient addition and the treatment efficiency After growth, colonies were isolated and purified by re-
were monitored periodically to keep a check on the treatment streaking in MSM medium with phenanthrene as sole carbon
progress. source.
The strains were analyzed to obtain a preliminary taxonomic
Chemical analyses: The contaminated soils were sampled by characterization. The criteria and tests were as follows: cell
the Simple Random Sampling technique (EPA SW-846). shape, colony shape and colour, and Gram stain. For the
Composite samples were obtained by mixing 8-10 subsamples taxonomic identification of strains, bacterial 16S rRNA gene
(each 25 x 25 m2 area). A portion of the composite soil (500 g) fragments were amplified from genomic DNA by PCR using
was placed in sterile bottles for chemical and microbiological prokaryotic universal primers. Sequences were determined
analysis. Total petroleum hydrocarbons (TPH) were directly from these PCR products by Macrogen Inc., Korea.
determined using a modified EPA 418.1 technique with a FT- The 16S rRNA gene sequences were aligned with published
IR Spectrophotometer, Perkin-Elmer, Spectrum RX I. Carbon sequences from the GenBank data base using NCBI Blast
tetrachloride was used as solvent instead Freon 113. TPH were comparison software [14].
extracted from soil using EPA SW-846, method 3550. EPA Mineralization studies based on measuring the time-dependent
SW-846 method 8310 was applied to analyze PAH using high- release of CO2 by bacterial cells associated with the utilization
performance liquid chromatography (HPLC), Perkin-Elmer of phenanthrene as carbon source were performed according
Series 200 with fluorescence and UV/VIS detectors. to a modified method described by Fredrickson et al. [15].
Soil moisture was measured gravimetrically by drying at 105º Cells were grown aerobically at 25° C overnight in 10 ml
C to constant weight. Nutrients (N, P) were analyzed by the nutrient broth medium on a rotary shaker. After growth, cells
Kjeldahl method (total N) and Bray & Kurtz spectrometric were harvested, washed once with sterile NaCl solution (0.85
method (extractable P). Heavy metal contents in soil (lixiviate %, w/v), and resuspended to an A436 of 1 in sterile NaCl
fraction) was determined using an Atomic Absorption solution. Zero-point-five millilitre aliquots were used to
Spectrum, Perkin-Elmer, Analyst 700, according to the inoculate 500 ml flask containing 50 ml MSM with the
respective EPA methods: Arsenic (EPA 7061A), Silver (EPA hydrocarbon as sole carbon source. Each flask was designed to
7760), Barium (EPA 7080), Cadmium (EPA 7130), Total receive with a vial containing 2 ml of 1 M NaOH to absorb
Chromium (EPA 7190), Cupper (EPA 7210), Mercuric (EPA CO2 produced by cells. These vials were removed every 24 h
7471), Nickel (EPA 7520), Lead (EPA 7420), Selenium (EPA and replaced by new vials containing fresh NaOH solution.
7741A), Zinc (EPA 7950). pH of samples was measured by The flask were tightly sealed with wrapped rubber stoppers
the method SM 4500 H-B (Decreto Nacional 831/93). and incubated for 24 h at 25° C on a rotary shaker. CO2
production was monitored by titration with 0.1 M HCl.
Microbiological analysis: Bacterial cell number was
determined by plate-count on two different growth medium Bioremediation experiments: In order to compare the
after the adequate serial dilutions. The first was a rich complex efficiency of attenuation, biostimulation and bioaugmentation
agar medium designed for growing most chemoheterotroph treatments in semiarid chronically contaminated soils of
bacteria. The composition of this medium was 5 g peptone, 3 g Patagonia, we performed a field bioremediation test in a real
yeast extract, 1 g NaCl and 14 g agar-agar, per litre. The polluted site. The site selected for the experiment (S 46º 26’
second medium was a mineral salt medium (MSM) with 21.4” and WO 68º 52’ 22.5”) contained approximately 3.22
commercial diesel as sole carbon and energy source. The μg/l of PAH (in lixiviate), mainly at expenses of phenanthrene
composition of this medium was 1.5 g KH2PO4; 9 g (2.89 μg/l) and minor amounts of fluorene (0.33 μg/l). Five
Na2HPO4.12H2O; 0.2 g MgSO4.7H2O; 1 g NH4Cl, 1 ml CaCl2- soil plots of 1 m2 each one were used for the study as detailed
Fe solution; 0.1 ml oligoelement solution (SL6) y 14 g below:
ultrapure agar-agar, per litre. Briefly, samples of 1 g of soil a) Plots 1 and 2 were inoculated with cells of strains
were added to 10 ml of sterile NaCl solution (0.85 %, w/v) Rhodococcus sp. F7 and Rhodococcus sp. 006, respectively,
and vortexed vigorously. After appropriate dilutions, 0.1 ml of and supplemented by adding water and fertilizers as described
the suspension were spread over the surface of duplicate agar above.
plates with the media and incubated at 25° C. Total CFU/g b) Plot 3 was inoculated with a microbial consortium
(Colony Forming Units) was determined after 7 days by consisting of cells of strains F7 and 006 simultaneously. This
counting the bacterial colonies. soil was also fertilized by the addition of nutrients and water.
c) Plot 4 (biostimulation) was used for testing the ability of the
natural soil to degrade hydrocarbons after addition of
Isolation and characterization of indigenous nutrients. This soil was supplemented by addition of water and
microorganisms: Two hydrocarbon-degrading bacterial fertilizers.
strains were isolated from two different chronically d) Plot 5 (attenuation) was used for testing the intrinsic
contaminated soils in Las Heras area. Soil samples (1 g) were bioremediation of this soil after addition of water.
suspended in 10 ml of sterile NaCl solution (0.85 %, w/v) and Fig. 1 shows a view of experimental plots. Nutrients, which
vortexed vigorously. Aliquots of these suspensions (0.1 ml) consisted of a mixture of urea and phosphorous pentoxide,
were spread over the surface of MSM agar plates containing 5 P2O5 (70:30, w/w), were added manually on the top of the
ppm of phenanthrene as sole carbon and energy source. Plates plots before adding water and mixing soil as described by
were incubated at 25° C for 7 days. Strain 006 was isolated
4 SPE 107384

Jorgensen et al. [13]. Addition of water and homogenization bioremediation processes, did not occur in any case. Case A
(oxygenation) were performed weekly. The mechanic aeration exhibited high values of total petroleum hydrocarbons (TPH)
of soil was preformed using small agriculture devices (up to as shown in Table 1.
15 cm of profundity).
Table 1: Hydrocarbon concentration, percentage of
Figure 1: View of the soil plots used for the small-scale hydrocarbon degradation and changes in the
bioremediation treatments in Las Heras area number of total heterotrophic microorganisms in
soil samples of Case A after biostimulation
treatment.

Time elapsed TPH Hydrocarbon Total cell counts

after treatment (mg/kg) losses (%) (log CFU/g soil)
initiation (months)
0 33510 4.28
7 22813 32 6.00

Approximately, 32 % degradation of the TPH was achieved

after 7 months of biostimulation treatment (from May to
December 2005). During that period, total cell counts
increased 1.72 orders of magnitude (Table 1). In addition, the
results of biostimulation field tests demonstrated a high
efficiency for treatment of semiarid soil contaminated with
polyaromatic hydrocarbons (PAH). In case B, which showed
high values of both, TPH and PAH, the addition of nutrients
induced an increase of total bacterial numbers, and 39 % of
TPH and 88 % of PAH reduction after 5 months of treatment
(from October 2005 to February 2006) (Table 2).
Inocula were prepared from cultures grown on nutrient broth
containing phenanthrene (5 ppm) incubated at 25° C. Cells of
Table 2: Hydrocarbon concentration, percentage of
the isolated Rhodococcus strains grow well in complex media,
hydrocarbon degradation and changes in the
and the presence of phenanthrene in the culture may induce
the respective degrading-enzymes since these microorganisms number of total heterotrophic microorganisms in
soil samples of Case B after biostimulation
lack the catabolite-repression regulatory system. After 48 h
treatment.
cells were centrifuged (10.000 x g, 15 min) and the pellet was
resuspended in saline solution (NaCl 0.85 %, w/v) to a cell
density of approximately 109 CFU/ml before inoculating. Time elapsed TPH TPH PAH PAH Total cell
after treatment (mg/kg) losses (μg/l) losses counts
These suspensions of cells were added to soil in the respective initiation % % (log CFU/g soil)
plots 1, 2 and 3. For preparing the consortium, both (months)
Rhodococcus strains were separately cultivated under the same 0 27754 1.64 4.77
conditions described above, then harvested by centrifugation 5 16984 39 < 0.2 88 5.00
of the cultivation broth and mixed together (1:1, w/w). The
cell suspension of both bacteria contained a cell density of Case C exhibited high PAH values before treatment.
approximately 109 CFU/ml before inoculating. Two Stimulation of the indigenous microbial population present in
inoculation procedures of bacterial cells were performed in all this soil resulted in the disappearance of approximately 90 %
bioaugmentation tests (Plots 1, 2 and 3): the first at the of PAH after 3 months (from May to July 2005) (Table 3).
beginning of the experiment and the second after 15 days of The microbiological analysis of the soil samples showed an
treatment. increase of the total cell counts of 1.55 orders of magnitude
Samples for microbiological and chemical analysis were during that period (Table 3).
collected from soil plots. The same microbiological and
chemical techniques described above were applied in this field Table 3: Hydrocarbon concentration, percentage of
experience. hydrocarbon degradation and changes in the
number of total heterotrophic microorganisms in
soil samples of Case C after biostimulation
Presentation of Data and Results: treatment.
Biostimulation field treatments
The aim of these treatments was the improvement of the Time elapsed PAH Hydrocarbon Total cell counts
biodegradation rate of the indigenous microbial population after treatment (μg/l) losses (%) (log CFU/g soil)
present in contaminated semiarid soils. In situ biostimulation initiation (months)
treatments were applied to chronically contaminated soils of 0 1.94 4.56
3 < 0.2 90 6.11
Patagonia with three different representative pollution
situations. The presence of heavy metals, which could inhibit
SPE 107384 5

It should be noted that the relative proportion of the inoculated together as a microbial consortium in other
hydrocarbon-degrading bacterial population of these soils was separated plot. It should be noted that a second inoculation of
always near to 100 % relative to the total number of bacterial cells were performed after 15 days of treatment in all
heterotrophic bacteria. This suggested that the bacterial bioaugmentation tests. Biostimulation and bioaugmentation
population of these sites has been probably selected due to the promoted significant degradation (> 90 %) of contaminated
chronic presence of hydrocarbons. These bioremediation soil after 6 week of treatment, whereas attenuation had the
examples suggested that the indigenous microorganisms of least effect on the degradation of PAH (66.8 %). The high
semiarid Patagonia may be efficient enough to biodegrade the biodegradation rates obtained in this study may be partly due
contaminants when favourable conditions are provided during to the homogeneous work conditions regarding the
treatment. Biostimulation treatment seems to be a practical oxygenation procedure, the nutrient and water distribution in
approach to the restoration of desert soils contaminated with the small soil plots, which are more difficult to reach in a
hydrocarbons in Patagonia. large-scale field treatment. Among the bioremediation
treatments, the highest degradation rates of soil contaminated
Comparative bioremediation of soils by attenuation, with PHA (phenanthrene) was observed when the indigenous
biostimulation and bioaugmentation strains F7 (99.1 %) and 006 (96.8 %) were added.
In this study we evaluated the efficiency of all three Bioaugmentation with the microbial consortium and the
bioremediation technologies in the field in a small scale addition of nutrient (biostimulation) promoted similar
experiment. For the tests, we selected a site with high values degradation rates, (92.2 %) and (93.1 %), respectively. During
of PAH (3.22 μg/l in lixiviate), at expenses of phenanthrene this period, the area registered temperatures between – 3° and
(2.89 μg/l) as main compound, and fluorene (0.33 μg/l). We 13° C, moderated winds (30 km/h in average) and
isolated from a soil sample of this site a bacterial strain (strain approximately 29.7 mm of precipitation. These results
F7) with the ability for growing on phenanthrene as sole suggested that the isolated indigenous Rhodococcus bacteria
carbon and energy source. We also included in this study other may be able to maintain degradative activities under the local
autochthonous strain (strain 006) isolated from a soil sample environmental conditions. In addition, they probably possess
collected in other contaminated site in Las Heras area. Both specific physiological properties involved in the degradation
selected strains were Gram positive, aerobic, non-sporulating, of hydrophobic hydrocarbons with low bioavailability in the
non-motile filamentous cells. Comparison of the 16S rRNA environment, such as phenanthrene. Among these
gene sequences indicated that both strains belonged to the mechanisms, we can mention the production of biosurfactants
genus Rhodococcus. These microorganisms are grouped or the use of specific cell surface components with
within the nocardioform actinomycetes and are common in emulsifying properties; or the occurrence of specific cell
many environmental niches [16, 17]. They are known to surface structures which promote adhesion to hydrophobic
degrade several hydrocarbons and other organic compounds compounds sorbed on soil particles. In this context, the
[8]. occurrence of a very hydrophobic cell wall is a known feature
Both indigenous bacterial strains showed similar in bacteria belonging to the Nocardia-Rhodococcus-
mineralization rates during cultivation on phenanthrene as sole Mycobacterium group [18]. For these reasons, this bacterial
carbon source (Table 4). group is considered specialized microorganisms in degrading
such less-bioavailable compounds [18]. A better
Table 4: Mineralization of phenanthrene by the understanding of the physiology of the indigenous
indigenous Rhodococcus sp. strains F7 and 006. Rhodococcus bacteria used in this study and their interactions
1
mM total CO2 after 9 days of incubation;
2
mM CO2/day during the with contaminants and the semiarid environment will
first 3 days of cultivation. hopefully permit successful large-scale applications of these
inocula to effectively bioremediate soils in semiarid
Strain F7 Strain 006 Patagonia.
1
mM total CO2 0.5715 0.5398
Conclusions:
mM CO2/day
2
0.1196 0.1241 Environmental conditions in situ seem to be mostly
suboptimal for hydrocarbon biodegradation in semiarid soils
of Patagonia, Argentina. The rate of natural degradation is so
These results confirmed the ability of both bacteria for slow that spilled hydrocarbons persist for long time in soil
complete oxidizing phenanthrene to CO2 and H2O under environments in the region. Results of this study suggest that
laboratory conditions. In addition, strains F7 and 006 were in general indigenous bacterial communities from semiarid
also able to degrade complex hydrocarbon mixtures, as crude soils in Patagonia have the potential to degrade hydrocarbons
oil and gas-oil; and other pure hydrocarbons, such as after amelioration of unfavourable environmental conditions.
hexadecane and naphthalene. Nutrient addition, oxygenation and moisture were required for
Five soil plots of 1 m2 each one were used for a comparative effective bioremediation treatments in our study. On the other
bioremediation study by attenuation, biostimulation and hand, field tests showed a high efficiency of the indigenous
bioaugmentation. Three alternative bioaugmentation Rhodococcus strains isolated in this study for remediation of
treatments were achieved: strains F7 and 006 were inoculated soil chronically contaminated with polyaromatic hydrocarbons
individually in the respective plots and both strains were (phenanthrene) in this semiarid environment. However,
biostimulation treatment resulted also a highly efficient
6 SPE 107384

procedure for eliminating this contaminant as revealed our [12] Alvarez, H.M. et al.: ″Physiological and Morphological
small scale study. These results suggest that in situ Responses of the Soil Bacterium Rhodococcus opacus Strain
bioremediation of hydrocarbon-contaminated soil at semiarid PD630 to Water Stress″, FEMS Microbiology Ecology (2004)
Patagonia using biostimulation procedure is feasible. Although 75
the bioaugmentation alternative using potent indigenous [13] Jorgensen, K.S. et al.: ″Bioremediation of Petroleum
hydrocarbon-degrading bacteria seemed to be highly efficient,
amelioration of unfavourable environmental conditions when Hydrocarbon-Contaminated Soil by Composting in Biopiles″,
there are sufficient hydrocarbon degraders in the soil seems to Environmental Pollution (2000) 245
be more beneficial from an economical and also ecological [14] Altschul, S.F. et al.: ″Basic Local Alignment Search
point of view. If the indigenous bacterial population of soil Tool″, Journal of Molecular Biology (1990) 403
possesses the potential to degrade the contaminant; the [15] Fredrickson, J.K. et al.: ″Isolation and Characterization of
addition of hydrocarbon-degrading specialist may only reduce Subsurface Bacterium Capable of Growth on Toluene
the time necessary for bioremediation. Naphthalene, and other Aromatic Compounds″, Applied and
Environmental Microbiology (1991) 796
[16] Finnerty, W.R.: ″The Biology and Genetics of the Genus
Acknowledgments: Rhodococcus″, Annual Review Microbiology (1992) 193
We are grateful to Repsol-YPF for providing a site for this [17] Warhurst, A.M. and Fewson, C.A.: ″Biotransformation
study. We wish to thank Dr. Nelda Olivera (CENPAT-
Catalyzed by the Genus Rhodococcus″, Critical Reviews of
CONICET) for collaboration in the molecular identification of
Biotechnology (1994) 29
the studied bacterial strains. We especially thank all
[18] Bastiaens, L. et al.: ″Isolation of Adherent Polycyclic
technicians of Oil M&S S.A. who provided technical
Aromatic Hydrocarbon (PAH)-Degrading Bacteria Using
assistance in this study.
PAH-sorbing Carriers″, Applied and Environmental
Microbiology (2000) 1834
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