Environ Sci Pollut Res (2014) 21:25922602

DOI 10.1007/s11356-013-2139-2

RESEARCH ARTICLE

Bioremediation assessment of dieselbiodiesel-contaminated

soil using an alternative bioaugmentation strategy
Tatiana Simonetto Colla & Robson Andreazza & Francielle Bcker &
Marcela Moreira de Souza & Letcia Tramontini & Gernimo Rodrigues Prado &
Ana Paula Guedes Frazzon & Flvio Anastcio de Oliveira Camargo &
Ftima Menezes Bento

Received: 16 April 2013 / Accepted: 6 September 2013 / Published online: 5 October 2013
# Springer-Verlag Berlin Heidelberg 2013

Abstract This study investigated the effectiveness of succes-
sive bioaugmentation, conventional bioaugmentation, and
biostimulation of biodegradation of B10 in soil. In addition, the
structure of the soil microbial community was assessed by
polymerase chain reaction-denaturing gradient gel electrophore-
sis. The consortium was inoculated on the initial and the 11th
day of incubation for successive bioaugmentation and only on
the initial day for bioaugmentation and conventional
bioaugmentation. The experiment was conducted for 32 days.
The microbial consortium was identified based on sequencing of
16S rRNA gene and consisted as Pseudomonas aeruginosa,
Achromobacter xylosoxidans, and Ochrobactrum intermedium.
Nutrient introduction (biostimulation) promoted a positive effect
on microbial populations. The results indicate that the edaphic
community structure and dynamics were different according to
the treatments employed. CO2 evolution demonstrated no signif-
icant difference in soil microbial activity between biostimulation
and bioaugmentation treatments. The total petroleum hydrocar-
bon (TPH) analysis indicated a biodegradation level of 35.7 and
32.2 % for the biostimulation and successive bioaugmentation
treatments, respectively. Successive bioaugmentation displayed
Responsible editor: Zhihong Xu
T. S. Colla : F. Bcker : M. M. de Souza : L. Tramontini :
G. R. Prado : A. P. G. Frazzon : F. M. Bento
Department of Microbiology, Federal University of Rio Grande do
Sul, Porto Alegre, Rio Grande do Sul, Brazil
R. Andreazza (*)
Center of Engineer, Federal University of Pelotas, 1734 Almirante
Barroso, Pelotas 96010-280, Rio Grande do Sul, Brazil
e-mail: robsonandreazza@yahoo.com.br
F. A. d. O. Camargo
Department of Soil, Laboratory of Soil Microbiology, Federal
University of Rio Grande do Sul, Porto Alegre, Rio Grande do Sul,
Brazil
positive effects on biodegradation, with a substantial reduction in
TPH levels.
Keywords Biodegradation . Successive bioaugmentation .
Biostimulation . Microbial consortium
Introduction
An indication of petroleum reserve depletion and an environ-
mental preoccupation have promoted interest in renewable and
less polluting energy sources. Biodiesel is considered an alter-
native fuel to diesel because of its renewable nature, biodegrad-
ability, and lower emission of atmospheric pollutants (Knothe
2010). Biodiesel is chemically defined as a mixture of mono-
alkyl esters of fatty acids, derived from transesterification of
vegetable oils and animal fats (Knothe 2010). In Brazil, biodie-
sel is commercialized as a minor component in diesel blends.
Overall, it represents 5 % of the diesel formulation, projected to
increase concentration in the near future.
Due to the current insertion of dieselbiodiesel blends in
the world energy matrix, studies on their biodegradability in
natural compartments are needed. Bioremediation is attractive
compared to conventional methods for decontamination be-
cause this technology is compatible with natural biochemical
recycling in nutrient routes (El Fantroussi and Agathos 2005).
Biochemical recycling is recognized as a friendly environ-
mental approach to remediate impacted sites by promoting
conversion of persistent molecules into less toxic and less
complex compounds (Xu and Lu 2010). Furthermore, biore-
mediation presents efficient results, simplified maintenance,
wide applicability, and low operating costs (Bento et al. 2005;
Megharaj et al. 2011).
The presence of microorganisms potentially degrading pe-
troleum hydrocarbons in contaminated and uncontaminated
Environ Sci Pollut Res (2014) 21:25922602
soils is well-documented (Kaczorek and Olszanowski 2011).
However, with bioremediation involving the incorporation of
autochthonous or allochthonous specialized microorganisms
(bioaugmentation), the reduction rates of contaminants may
be unsatisfactory. The biodegradation kinetics show an initial
period of intense activity, followed by a reduction phase in
degradation rates (Lin et al. 2011; Tahhan et al. 2011). The
imbalance of abiotic factors can make the process unfeasible
under natural conditions (Tyagi et al. 2011), and reducing the
exposure of the inoculated specialized population to the nat-
ural conditions of predation and competition may also de-
crease the biodegradation efficiency (Lin et al. 2011; Tahhan
et al. 2011). Bioaugmentation has been used increasing effi-
cient microorganisms for bioremediation with high perfor-
mance, and also, biostimulation has good results for biodeg-
radation of many compounds and may be used as an efficient
treatment. However, successive bioaugmentation is a new
treatment that presents potential use for biodegradation.
Hence, in this study, an allochthonous consortium was added
in two stages of the biodegradation process to investigate the
effectiveness of successive bioaugmentation in the bioreme-
diation soil contaminated with B10 blend (90 % diesel:10 %
biodiesel). The bacterial population response after the biore-
mediation treatments was assessed by culture-dependent and
culture-independent methods.
Material and methods
Soil characteristics
The soil was sampled from a superficial layer (015 cm) in a
rural area without agricultural use and without petroleum hy-
drocarbon contamination, localized at Mariana Pimentel City,
Rio Grande do Sul State, Brazil. The physicochemical analysis
was performed by the Soil Analysis Laboratory of the Federal
University of Rio Grande do Sul. The most probable number
(MPN) estimative of heterotrophic microorganisms was carried
out in nutrient broth. The degrading microorganisms MPN
estimative was carried out in the minimum mineral medium
MM1 (0.7 g L1 of KCl, 2.0 g L1 of KH2PO4, 3.0 g L1 of
Na2HPO4, and 1.0 g L1 of NH4NO3 and micronutrient solution
with 4.0 mL L1 of MgSO4, 0.2 mL L1 of FeSO4, 0.2 mL L1
of MnCl2, and 0.2 mL L1 of CaCl2) amended with 2,3,5-
triphenyltetrazolium chloride, as described by Braddock and
Catterall (1999). The remaining soil sample was dried at room
temperature (20 C5) for 2 days, sieved with 3-mm mesh and
stored at 4 C until the start of the bioremediation experiment.
The soil had a sandy loam texture (61 % sand, 23 % silt, and
16 % clay) and an acidic pH (4.7). Soil nutrients were quantified
as follows: 3.5 mg dm3 P, 34 mg dm3 K, 4.3 mg dm3 S,
0.07 % N, 0.87 % Corganic, and 1.5 % organic matter. The total
heterotrophic microorganism estimate was 4.6104 MPN mL1,
2593
with 5.2 % representing degrading microorganisms (2.4103
MPN mL1). Thus, degrader consortium inoculation was re-
quired in order for bioremediation treatments to occur at signif-
icant rates (Mishra et al. 2001).
Fuels
The fuels used were metropolitan diesel oil with 50 ppm sulfur
(B0) and soybean methyl biodiesel (B100). The fuels were
obtained from Ipiranga Produtos de Petrleo S.A. Diesel and
biodiesel mixture 10 % (B10) was prepared in the laboratory
from B0 and B100 fuels. The oil sterilization was carried out by
vacuum filtration, in sterilized kitassato, with a filter adapted to
place membrane filters and a pore size of 0.22 m. After this,
the fuels were stored at room temperature in glass vials and
wrapped in aluminum wrapped to prevent photo-oxidation.
Degrader bacterial consortium composition
The bacterial strains used in this study were isolated from diesel
biodiesel-contaminated soil collected from a gas station during
the replacement of underground storage tanks. Total petroleum
hydrocarbon (TPH) concentration in this sample was
1,980 mg kg1 of soil. The microbial isolation method used
was the enrichment method according to the procedure outlined
by Bento et al. (2005). Erlenmeyer flasks were inoculated with
10 g of contaminated soil and 1 % B0, B10, or B100 in a final
volume of 100 mL of MM1. The flasks were maintained with
shaking at 190 rpm and 30 C. Every 7 days, a grown culture
aliquot (10 mL) was transferred to a new flask containing the
same MM1 sterile medium and 1 % fuel and incubated under the
same conditions. After three transfers, the grown culture was
serially diluted and subjected to surface spreading and streak
plating on nutrient agar. Purified isolates were pre-selected
according to their ability to metabolize different fuels (B0,
B10, and B100) as carbon sources. Combinations among the
strains were established to select the consortium most efficient at
B10 biodegradation in liquid medium tests, when considering
their growth kinetics. The consortium selected for soil
bioaugmentation assays consisted of three microorganisms.
The bacterial cultures were identified based on the sequencing
of the 16S rRNA gene, using the primers F-C27 (5-AGA GTT
TGA TCC TGG CTC AG-3) and R-530 (5-CCG CGG CTG
CTG GCA CGT A-3) (Gontang et al. 2007). The polymerase
chain reactions (PCR) were optimized in 25 L of deionized
sterile water, containing 2 mM MgCl2, 2.5 pmol of each oligo-
nucleotide, 1 U of Taq DNA polymerase (Invitrogen), 1 reac-
tion buffer, 300 M deoxyribonucleotides, and 1 L of genomic
DNA. Amplification was performed using an automated thermal
cycler (Mastercycler Personal, Eppendorf) as follows: one cycle
at 94 C for 5 min, followed by 35 cycles of 45 s at 94 C, 45 s at
58 C, and 45 s at 72 C and a final extension step at 72 C for
5 min.
Environ Sci Pollut Res (2014) 21:25922602
soils is well-documented (Kaczorek and Olszanowski 2011).
However, with bioremediation involving the incorporation of
autochthonous or allochthonous specialized microorganisms
(bioaugmentation), the reduction rates of contaminants may
be unsatisfactory. The biodegradation kinetics show an initial
period of intense activity, followed by a reduction phase in
degradation rates (Lin et al. 2011; Tahhan et al. 2011). The
imbalance of abiotic factors can make the process unfeasible
under natural conditions (Tyagi et al. 2011), and reducing the
exposure of the inoculated specialized population to the nat-
ural conditions of predation and competition may also de-
crease the biodegradation efficiency (Lin et al. 2011; Tahhan
et al. 2011). Bioaugmentation has been used increasing effi-
cient microorganisms for bioremediation with high perfor-
mance, and also, biostimulation has good results for biodeg-
radation of many compounds and may be used as an efficient
treatment. However, successive bioaugmentation is a new
treatment that presents potential use for biodegradation.
Hence, in this study, an allochthonous consortium was added
in two stages of the biodegradation process to investigate the
effectiveness of successive bioaugmentation in the bioreme-
diation soil contaminated with B10 blend (90 % diesel:10 %
biodiesel). The bacterial population response after the biore-
mediation treatments was assessed by culture-dependent and
culture-independent methods.
Material and methods
Soil characteristics
The soil was sampled from a superficial layer (015 cm) in a
rural area without agricultural use and without petroleum hy-
drocarbon contamination, localized at Mariana Pimentel City,
Rio Grande do Sul State, Brazil. The physicochemical analysis
was performed by the Soil Analysis Laboratory of the Federal
University of Rio Grande do Sul. The most probable number
(MPN) estimative of heterotrophic microorganisms was carried
out in nutrient broth. The degrading microorganisms MPN
estimative was carried out in the minimum mineral medium
MM1 (0.7 g L1 of KCl, 2.0 g L1 of KH2PO4, 3.0 g L1 of
Na2HPO4, and 1.0 g L1 of NH4NO3 and micronutrient solution
with 4.0 mL L1 of MgSO4, 0.2 mL L1 of FeSO4, 0.2 mL L1
of MnCl2, and 0.2 mL L1 of CaCl2) amended with 2,3,5-
triphenyltetrazolium chloride, as described by Braddock and
Catterall (1999). The remaining soil sample was dried at room
temperature (20 C5) for 2 days, sieved with 3-mm mesh and
stored at 4 C until the start of the bioremediation experiment.
The soil had a sandy loam texture (61 % sand, 23 % silt, and
16 % clay) and an acidic pH (4.7). Soil nutrients were quantified
as follows: 3.5 mg dm3 P, 34 mg dm3 K, 4.3 mg dm3 S,
0.07 % N, 0.87 % Corganic, and 1.5 % organic matter. The total
heterotrophic microorganism estimate was 4.6104 MPN mL1,
2593
with 5.2 % representing degrading microorganisms (2.4103
MPN mL1). Thus, degrader consortium inoculation was re-
quired in order for bioremediation treatments to occur at signif-
icant rates (Mishra et al. 2001).
Fuels
The fuels used were metropolitan diesel oil with 50 ppm sulfur
(B0) and soybean methyl biodiesel (B100). The fuels were
obtained from Ipiranga Produtos de Petrleo S.A. Diesel and
biodiesel mixture 10 % (B10) was prepared in the laboratory
from B0 and B100 fuels. The oil sterilization was carried out by
vacuum filtration, in sterilized kitassato, with a filter adapted to
place membrane filters and a pore size of 0.22 m. After this,
the fuels were stored at room temperature in glass vials and
wrapped in aluminum wrapped to prevent photo-oxidation.
Degrader bacterial consortium composition
The bacterial strains used in this study were isolated from diesel
biodiesel-contaminated soil collected from a gas station during
the replacement of underground storage tanks. Total petroleum
hydrocarbon (TPH) concentration in this sample was
1,980 mg kg1 of soil. The microbial isolation method used
was the enrichment method according to the procedure outlined
by Bento et al. (2005). Erlenmeyer flasks were inoculated with
10 g of contaminated soil and 1 % B0, B10, or B100 in a final
volume of 100 mL of MM1. The flasks were maintained with
shaking at 190 rpm and 30 C. Every 7 days, a grown culture
aliquot (10 mL) was transferred to a new flask containing the
same MM1 sterile medium and 1 % fuel and incubated under the
same conditions. After three transfers, the grown culture was
serially diluted and subjected to surface spreading and streak
plating on nutrient agar. Purified isolates were pre-selected
according to their ability to metabolize different fuels (B0,
B10, and B100) as carbon sources. Combinations among the
strains were established to select the consortium most efficient at
B10 biodegradation in liquid medium tests, when considering
their growth kinetics. The consortium selected for soil
bioaugmentation assays consisted of three microorganisms.
The bacterial cultures were identified based on the sequencing
of the 16S rRNA gene, using the primers F-C27 (5-AGA GTT
TGA TCC TGG CTC AG-3) and R-530 (5-CCG CGG CTG
CTG GCA CGT A-3) (Gontang et al. 2007). The polymerase
chain reactions (PCR) were optimized in 25 L of deionized
sterile water, containing 2 mM MgCl2, 2.5 pmol of each oligo-
nucleotide, 1 U of Taq DNA polymerase (Invitrogen), 1 reac-
tion buffer, 300 M deoxyribonucleotides, and 1 L of genomic
DNA. Amplification was performed using an automated thermal
cycler (Mastercycler Personal, Eppendorf) as follows: one cycle
at 94 C for 5 min, followed by 35 cycles of 45 s at 94 C, 45 s at
58 C, and 45 s at 72 C and a final extension step at 72 C for
5 min.
2594
The PCR products were analyzed with 1 % agarose gel
electrophoresis. The amplicons obtained were purified and
sequenced. Nucleotide sequence similarity searches were
conducted using GenBank nucleotide collection BLAST
searches. Phylogenetic analysis was conducted using RDP
software, release 10 (Cole et al. 2009).
Bacterial inoculum
The bacterial inoculum was prepared in 20 mL of sterile
nutrient broth for 24 h at 30 C in shaker (190 rpm). The
cells were centrifuged at 9,000 rpm for 10 min at 4 C
and twice washed in sterile saline solution (0.85 %).
Subsequently, the cell extract was resuspended in saline
solution (0.85 %) and maintained in shaker incubation for
24 h at 190 rpm and 30 C to deplete energetic reserves
(starvation). The standardization of the cellular concentra-
tion of the isolates was determined with a spectrophotom-
eter at =600 nm. The microbial consortium (three strains)
was formulated by mixing equal proportions of each bac-
terial strain (1.0107 cells mL1).
Experimental design
All treatments for the evaluation of B10 soil bioreme-
diation are summarized in Table 1. The microcosms
were set up in quadruplicate using 1.5-L hermetically
sealed glass flasks that contained 150 g of soil. During
32 days of experimental analysis, the systems were
maintained at controlled temperature (28 C 1) and
protected from light. The inoculum and nutrient solution
(NH4NO3 and KH2PO4) volumes added to the treat-
ments were adjusted to maintain the soil field capacity
at 80 %. In treatments with no addition of nutrients and
consortium, sterile distilled water was used to maintain
the soil moisture. The purpose of the addition of nutritional
Table 1 Experimental design with amendment of B10 blend, moisture
adjustment, pH adjustment at pH 6.5, nutrients (N and P), and inoculation
with the bacterium consortium in the soil
Bioremediation B10 Moisture pH N, P Bacterial consortium
strategy (1107 UFC g1 solo)
Time 0 Time 11
Control + obtained through the equation: CO2 generated (mg kg1
Natural attenuation + +
Biostimulation + + + +
Conventional + + + + + treatment in milliliters, respectively; eq is the gram-
bioaugmentation
Successive + + + + + +
bioaugmentation
+ presence, absence
Environ Sci Pollut Res (2014) 21:25922602
solution to the bioaugmentation and biostimulation treatments
was to correct the C/N/P ratio to 100:10:1. The soil samples
that were to be contaminated with the dieselbiodiesel blend
B10 were treated with 5 % (w/w ) of fuel, corresponding to
5,000 g kg1 of soil. During the inoculation and contamination
processes, microcosms were individually homogenized, using
a sterilized spatula.
The soil used for the biostimulation (BS), conventional
bioaugmentation (BA CO), and successive bioaugmentation
(BA SC) treatments received additional CaCO3, according to
the amount recommended by Bissani et al. (2008) to adjust the
pH value to 6.5. Subsequently, the remaining soil was incu-
bated for 2 days at room temperature (20 C5).
Microbial activity was evaluated during the degradation
process. The most probable number estimates of the total
heterotrophic and degrading population were carried out.
Structural changes in the bacterial community were assessed
through denaturing gradient gel electrophoresis (DGGE) of
16S rRNA gene fragments. Fuel degradation was character-
ized by CO2 evolution and by gas chromatography (GC)
analysis of TPH.
Microbial growth
Microbial population growth monitoring was carried out at
0, 6, 12, 18, 24, and 32 experimental days. The total
numbers of heterotrophic and degrading microorganisms
were estimated using the MPN method in microtiter plates
according to the method described by Bento et al. (2005).
The microbial population was determined using MPN
tables (APHA 1995).
CO2 evolution
To determine the metabolic activity in each microcosm,
respirometric analyses were periodically performed
using CO 2 evolution. The carbon dioxide produced
during microbial activity was captured with a 0.5-M
NaOH solution (20 mL) in 50 mL flasks in the respi-
rometric flasks. Periodically, the NaOH solution was
replaced, and 1 mL of 30 % BaCl2 solution and four
drops of 1 % phenolphthalein indicator were added to
the solution that had been removed. The residual
NaOH was titrated with 1 M HCl standardized solu-
tion. The amount of carbon dioxide produced was
solo) = [(B T ) eq M HCl CF]/M c, where B and T are
the volume of 1 M HCl used to titrate the blank and the
equivalent, equal to 6; M HCl is the molar concentration
of HCl standard solution; CF is the correction factor for
acid/base molarity (M HCl/M KOH); and M c is the dry soil
mass in kilograms.
Environ Sci Pollut Res (2014) 21:25922602
Gas chromatography
Microcosm samples, totaling 30 g of soil, were sent to Innolab
do Brasil S.A. (Rio de Janeiro, Brazil) for TPH chromato-
graphic analysis. These samples were taken at 0, 11, and
32 days. Analysis procedures followed the terms established
by the International Standard Organization, ISSO/DIS
16703:2004 method.
Briefly, samples (20 g) were added to 20 mL acetone
and 10 mL extraction solvent. Then, the suspension was
incubated in the ultrasonic bath for 30 min at 45 C. After
cooling, 30 mL of distilled water was added, and the
suspension was homogenized for 1 min. The organic phase
was washed with distilled water to eliminate interfering
compounds. Extract aliquots (5 mL) were concentrated to
0.8 mL. The sample was analyzed in a gas chromatograph
equipped with a flame ionization detector, CP-3800
(Varian) model. Extract aliquots were injected into a CP-
SIL8CB chromatographic column with temperature pro-
gramming. TPH quantification was carried out by the ex-
ternal standard technique, using diesel and lube oils as the
reference standard. The degradation percentage was calcu-
lated as follows: %TPH degradation =[(DTi DTf)/DTi]100,
where DTi and DTf are the TPH values at the initial and
final time points, respectively.
Microbial diversity
Microbial diversity analysis was carried out to monitor
the bacterial community and structural dynamics dur-
ing the bioremediation process. Samples were taken at
0, 11, and 32 days. The sample from day 11 was taken
before inoculation of the second consortium into the BA
SC treatment.
Soil DNA extraction and PCR amplification
Total DNA was extracted from 300 mg of soil samples using a
PowerSoil DNA Isolation Kit (MoBio Inc.), following the
manufacturers instructions. A fragment of the V3 region of
the bacterial 16S rRNA gene was amplified from
metagenomic DNA using the primers BA 338 F-GC (5-
CGC CCG CCG CGC GCG GCG GGC GGG GCG GGG
GCA CGG GGG GAC TCC TAC GGG AGG CAG CAG-3)
and UN518R (5-ATTACCGCGGCTGCTGG-3) (Ovreas
et al. 1997). Amplification was performed in Taq DNA
Polymerase buffer, containing 20 ng L1 of DNA template,
1 U Taq DNA Polymerase Platinum (Invitrogen), 3 mM
MgCl2, 0.2 mM dNTP, and 5 pmol of each primer at a final
volume of 25 L. The amplification conditions were as fol-
lows: 5 min at 95 C, 30 cycles for 1 min at 95 C, 1 min at
55 C, and 1 min at 72 C and a final extension at 72 C for
10 min.
2595
Denaturing gradient gel electrophoresis
Amplicons obtained from the PCR amplification were
analyzed by DGGE according to the method outlined by
Ovreas et al. (1997). An 8 % polyacrylamide (acrylamide/
bis-acrylamide mix 37.5:1w /v) gel containing a 1555 %
ureaformamide gradient was used (Ovreas et al. 1997).
The electrophoresis was performed in 1 TAE buffer at a
constant voltage of 200 V for 3 h and 30 min at 60 C in
the DCode TM System (Bio-Rad Inc., Hercules, CA, USA).
After electrophoresis, the gel was stained with 3 L Syber
Green I (Invitrogen) in 300 mL of Milli-Q water for 30 min
and photographed using a Photodocumentor GL2200
(Kodak).
The DGGE profiles were compared using Gel Compar
software, followed by visual analysis. The resulting banding
patterns were used to estimate the diversity via the
ShannonWeaver (H) index. Each band was considered an
operational taxonomic unit. Data analysis was performed
using the DivEsSpecies Diversity program, version 2.0.
Dendrograms were created by the evaluating a binary array
generated from the band profiles and subjected to statistical
analysis using the Dice coefficient.
pH measurement and moisture monitoring
The determination of pH was carried out at 0, 10, and 32 days,
using a digital pH meter (Digimed, MD-22). Gravimetric
moisture content was monitored throughout the incubation
period. Water content adjustment was performed when neces-
sary to maintain soil field capacity at 80 %, along with the
addition of sterile distilled water.
Statistical analysis
Descriptive statistics (means and standard deviations (N 1))
were utilized. ANOVA was used to test differences among the
treatments. The means of the treatments were evaluated by
Tukeys test with a 5 % error probability.
Results and discussion
Bacterial identification
Results of the partial sequencing of the 16S rRNA gene of the
three bacteria used in this study are listed in Table 2 and Fig. 1.
The isolate UFRGS4 exhibited 99 % similarity to
Achromobacter xylosoxidans and to other Achromobacter
species. The isolate UFRGS9 exhibited 89 % similarity to
Pseudomonas aeruginosa in the GenBank match; however,
the evolutionary distance presented almost 100 % of similarity
to P. aeruginosa NBAII CK-4 D-2 within my rdp database. The
2596 Environ Sci Pollut Res (2014) 21:25922602

Table 2 Molecular identification

of bacteria isolated and selected Bacterial ID 16S rDNA nucleotides GenBank match Identity Homology (%)
from contaminated soil for
bioremediation UFRGS4 494 GU014534.1 Achromobacter xylosoxidans 99
UFRGS9 484 HQ162486.1 Pseudomonas aeruginosa 89
UFRGS14 440 DQ833764.1 Ochrobactrum intermedium 99

isolate UFRGS14 exhibited 98 % of similarity to Ochrobactrum
intermedium in the GenBank match and also high similarity to
O. intermedium and Ochrobactrum sp.
The identified species have been isolated and used in
bioremediation trials of various xenobiotic compounds, such
as petroleum hydrocarbon derivatives (Ermakova et al. 2010;
Ghazali et al. 2004; Tiwari et al. 2010). In diesel degradation
studies, the potential of the Pseudomonas genus has been
demonstrated for both mono- and multi-specific consortia
(Lawniczak et al. 2011; Taccari et al. 2012). The combined
performance of Pseudomonas and Achromobacter can en-
hance bioremediation treatments of dieselbiodiesel-contam-
inated soils, as they are able to metabolize both hydrocarbon
and fatty acid fractions (Cyplik et al. 2011). The beneficial
association between O. intermedium and Achromobacter sp.
has also been demonstrated (Gargouri et al. 2011), indicating
that the use of a consortium formed by these three species (P.
aeruginosa, O . intermedium , and A. xylosoxidans) has great
promise for bioremediation assays.
B10-contaminated soil bioremediation
Microbial growth evaluation
The growth of the degrading and heterotrophic microorganism
was assessed by the MPN technique in the soil bioremediation
systems. The growth rates are in Fig. 2. Increases in both
degrading and heterotrophic microbial population were ob-
served over time for all treatments. The control population also
increased, mainly in the first 12 days. The increase in microbial
populations in the microcosms with B10 added demonstrated
that the initial TPH concentration (30,000 mg kg1 of soil

Fig. 1 Phylogenetic tree showing distance of the three isolates: UFRGS4 scale is the evolutionary distance value. The number at each node is the
(04FF in the Tree), UFRGS9 (09FF in the Tree), and UFRGS14 (14FR in bootstrap from 100 analyses
the Tree) from representative species using RDP release 10 software. The
Environ Sci Pollut Res (2014) 21:25922602 2597

11
a combined nutrients and inoculum addition. Bioaugmentation
soil)

10 and biostimulation association is a promising strategy to ac-

-1

celerate the bioremediation process because both indigenous

Degrading microorganisms (log MPN g

9
and exogenous microorganisms benefit from the regulation of
8
some environmental factors (El Fantroussi and Agathos
7 2005).
6 Degrading bacterial counts in inoculated soils were higher
compared to the microcosms without bioaugmentation treat-
5
ment over time, demonstrating the efficiency of the bacterial
4 consortium in establishing and remaining in the systems
3 (Fig. 2 a). Despite receiving an additional inoculation on the
2 11th day, BA SC degrading microorganisms exhibited growth
rates similar to that in BA CO microorganism. This result may
11 be due the limiting factors such as mineral and organic nutri-
Heterotrophic microorganisms (log MPN g-1 soil)

b ent bioavailability.
10

9
Figure 3 presents the proportion of degrading and hetero-
trophic populations (DG/HT). From the 18th day, the
8 degrading microbes begin to represent a larger proportion
7 within the heterotrophic population in the BS and
6 bioaugmentation (BA CO and BA SC) treatments. By the
end of the experiment, the DG/HT proportion rate in both
5
the BA CO and BA SC treatments was 1, indicating that both
4 populations exhibited the same microorganism concentrations
3 (MPN g1 soil) by the 30th day.
Considering that hydrocarbon degraders are heterotrophs,
2
0 6 12 18 24 30 they should represent a part of this population. However,
Time Course (days) surprisingly, the ratio of DG/HT exceeded 100 % in some
Fig. 2 Degrading (a) and heterotrophic (b) microorganisms most prob- treatments (Fig. 3). The reason for this apparent discrepancy is
able number estimative in bioremediation of B10-contaminated soil most likely related to the culturing conditions, in which the
during 32 days of incubation (control: ; natural attenuation: ; hydrocarbon degraders were incubated for 12 days at 30 C
biostimulation: ; conventional bioaugmentation: ; successive
bioaugmentation: )
and the heterotrophic populations growth was evaluated after
48 h of incubation. Similar observations were noted in a
bioremediation study with oily sludge-contaminated soil
2,000 mg kg1 of soil) did not inhibit the population growth for (Tahhan and Abu-Ateih 2009).
both native and exogenous microorganisms.
Natural attenuation results indicated that the presence
of fuel was able to stimulate the growth of the indige- 1.2
nous degrading population, most likely because of the
fuel represented an additional carbon source. The MPN 1.0

of microorganisms able to metabolize the fuel increased

from 1.0103 (initial levels) to 1.0106 MPN g1 of soil over
0.8
DG/HT ratio

time (Fig. 2 a). In a current oil spill contamination scenario, 0.6

this microbial profile is interesting, as it represents the survival C
AN
ability and adaptive capacity of endogenous microorganisms 0.4 BS
BA CL
to toxic conditions. However, this microbial profile does not BA SC

0.2
exclude the degrading consortium inoculation, as the initial
degrading microorganisms concentration (1.0103 MPN g1
0.0
soil) would not support a bioremediation treatment at signif- 0 6 12 18 24 30
icant rates (Mishra et al. 2001). Time Course (days)

Nutritional supplementation demonstrated beneficial ef-
fects on both degrading and heterotrophic population growth
(Fig. 2 a, b). A considerable population increase can be
showed for both the BA CO and BA SC treatments, which
Fig. 3 Degrader (DG) and heterotrophic (HT) populations ratio estimat-
ed by most probable number technique in bioremediation of B10-con-
taminated soil during 32 days. C control, AN natural attenuation, BS
biostimulation, BA CO conventional bioaugmentation, BA SC successive
bioaugmentation
2598 Environ Sci Pollut Res (2014) 21:25922602

Microbial activity Table 3 Total petroleum hydrocarbon biodegradation by different bio-

remediation strategies

All B10-contaminated microcosms presented higher levels of Strategies Biodegradation (%)

respiration activity compared to the negative control (p <0.05)
over the time course (Fig. 4). This result indicates a high micro- T0T11 T11T32 T0T32
bial ability to metabolize the fuel contaminants. C-CO2 accumu- Natural attenuation 0.89 a 0.16
lated quantification started to differ significantly between the
Biostimulation a
40.17 35.7
control and the BS, BA CO, and BA SC treatments by the fifth
Conventional bioaugmentation a 21.75 17.1
day and by the 11th day compared to the AN treatment.
Successive bioaugmentation a 45.77 32.2
The soil community treated with BS, BA CO, and BA SC
exhibited no difference in C-CO 2 evolution (p > 0.05). a
There was no decrease in TPH levels at this sample period
However, these treatments demonstrated more metabolic ac-
tivity than the soil treated with AN (p <0.05). These results
suggest that under appropriate abiotic conditions, microbial be underestimated if a portion of the degraded organic matter
metabolism was stimulated to achieve similar levels of C-CO2 is used in biomass production. It can also be overestimated if
evolution from the soils with inoculum addition (BA CO and the CO2 produced is from degradation of a soil-available
BA SC). As already demonstrated, in these soils, microbial carbon source rather than the contaminant (Horel and
growth was higher than the growth in the natural attenuation Schiewer 2009). With this, hydrocarbon degradation was
and control soils, indicating a direct relationship between analyzed by gas chromatography.
population density and C-CO2 evolution.
C-CO2 evolution and population growth rates were not sig- Petroleum hydrocarbon degradation
nificantly different between the conventional bioaugmentation
(one inoculation) and successive bioaugmentation (inoculations The TPH degradation was obtained by gas chromatography, and
at 0 and 11 days) strategies. In our study, a second exponential it is summarized in Table 3, which lists the TPH biodegradation
phase of C-CO2 evolution after the additional consortium inoc- percentage for each treatment, and in Fig. 5, which presents the
ulation was not observed, in contrast with some other reports quantitative variations of TPH levels at 0, 11, and 32 days. The
(Tahhan et al. 2011). However, both analyses demonstrated that soils treated by biostimulation and bioaugmentation strategies
the soil microbial community was active throughout the exper- exhibited higher biodegradation rates than natural attenuation
imental period. (Table 3). In some periods, especially during the first 11 days
Mineralization studies involving CO2 quantification as an of incubation, there was a TPH increase, possibly due to the
indicator of microbial basal respiration can provide informa- release of intermediate organic compounds derived from the
tion about hydrocarbon biodegradability in contaminated soils degradation process (Fig. 5).
(Silva et al. 2009). However, degraded fuel quantification can

50000
8000
AN
BS
Accumulated C-CO2 (mg Kg soil-1)

BA CO
C 40000 BA SC
AN
TPH (mg Kg-1 soil)

6000 BS
BASC
BA CL 30000

4000
20000

2000 10000

0
0 0 5 10 15 30
0 5 10 15 20 25 30
Time Course (days) Time Course (days)
Fig. 4 C-CO2 accumulated release quantification by soil microbial pop- Fig. 5 Total petroleum hydrocarbon (in milligrams per kilogram soil)
ulation from B10-contaminated soil measured during 32 days of biore- quantification by gas chromatography of B10-contaminated soil in bio-
mediation assay. Control: ; natural attenuation: ; biostimulation: remediation assay. Measurements were performed at 0, 11, and 32 days to
; conventional bioaugmentation: ; successive bioaugmentation: treatments natural attenuation (AN), biostimulation (BS), conventional
. Error bars are the standard deviation of the means bioaugmentation (BA CO), and successive bioaugmentation (BA SC)
Environ Sci Pollut Res (2014) 21:25922602
Among the reductions of TPH percentage showed, the BS
and BA SC treatments are notable with their 35.7 and 32.2 %
reductions, respectively (Table 3). These results suggest that
the bacterial consortium promoted hydrocarbon degradation
only when it has been added two times. However, the degra-
dation activity of the introduced consortium was similar to the
native microbial community, which satisfactorily responded
to the generated environmental condition-related stimulation
from pH, C/N ratio, and moisture.
Generally, bioaugmentation is conducted through a
single consortium application at the initiation of the
treatment (Bento et al. 2005). There is a paucity of studies
concerning the bioremediation efficiency of petroleum
derivative-contaminated soil evaluating successive inocula-
tions. However, current studies with multiple inoculations
have demonstrated satisfactory results with contaminated
soil treatment, achieving 30 % higher efficiency than
conventional bioaugmentation in bioremediation of diesel-
(Lebkowska et al. 2011) and oil sludge-contaminated (Tahhan
et al. 2011) soils.
The results indicated that the inoculation performed on the
11th day produced a positive effect on degradation compared
to conventional bioaugmentation; however, BA SC degrada-
tion levels were similar to those at BS strategy (Table 3).
When examining the hydrocarbon reduction levels between
days 11 and 32, the BA SC treatment achieved 45.77 %
biodegradation efficiency, and the soil treated with one inoc-
ulation only achieved 21.75 % efficiency (Table 3). Thus, the
second inoculation can overcome the initial negative impact
on degradation rates promoted by the first inoculation. The
negative impact may be consequence of allochthonous con-
sortia use (El Fantroussi and Agathos 2005; Xu and Lu 2010).
Ruberto et al. (2009) tested the degradation metabolic activity
of three bacterial consortiums. They have verified that exog-
enous microorganisms promoted higher perturbations in the
system, leading to negative impacts on bioremediation.
In this study, the levels of hydrocarbon reduction were
relatively low compared to other soil bioremediation studies
(Silva et al. 2012). However, the degradation percentages
were consistent with studies that examined bioremediation
of highly hydrocarbon-contaminated soils (30,000 mg kg1
solo) analyzed for a long period (110 days) (Li et al. 2007).
According to Li et al. (2007), hydrocarbon degradation
proceeds are faster when the microbes are exposed to lower
contamination levels, such as 500, 1,000, and 5,000 mg kg1
of soil.
The hydrocarbon degradation rate is commonly limited by
a lack of nutrients (N, P). Highly contaminated soils often
possess a carbon excess, unbalancing the soil carbon rate
(Ruberto et al. 2009). It is possible to optimize the C/N/P ratio
by the addition of appropriate nutritional supplementation
(Peltola et al. 2006). The biostimulation strategy has demon-
strated substantial efficiency; however, it does not always
2599
exhibit significant differences in the TPH removal rates
when comparing biostimulated and bioaugmented soils
(Ruberto et al. 2009). Generally, this result is associat-
ed with the bioremediation of naturally contaminated
soils, which have an adequate microbial population to
address the respective pollutant. However, in the pres-
ent results, the microbial populations in the treated soil
have not previously been exposed to petroleum deriva-
tives. Nevertheless, they were able to more efficiently
degrade compounds at higher levels than the soil treat-
ed with adapted inoculum.
pH
The pH correction due CaCO3 application effectively pro-
vided adequate conditions to promote microbial population
growth by increasing the pH to 6.0 in the BS, BA CO,
and BA SC treatments (Table 4). Biostimulated and
bioaugmented soil pH maintained significantly different
soil pH than the control soil throughout the time course
(p <0.05). Within the BS, BA CO and BA SC treatments
pH measurements did not differ over time (p >0.05), most
likely due to the prolonged action of CaCO3 combined with
soil buffering properties. These pH changes of the soil did not
represent a negative effect on microbial development because
their values were close to neutral, a condition conducive to
bacterial growth.
The importance of appropriate environmental physical
conditions for microbial action and growth is well document-
ed (Horel and Schiewer 2009). The pH directly affects micro-
organism activity through the effect of H+ ions on cell perme-
ability and enzyme activity, as well as indirectly by influenc-
ing macro- and micronutrient availability and metal solubility
(Caldwell 2000).
Bacterial community dynamics and structure analysis
To understand how bioremediation influences microbial bio-
diversity changes occurring during the incubation period (0,
11, and 32 days), it was analyzed the microbial changes by
Table 4 pH values measured at 0 (T0), 10 (T10), and 32 (T32) days of
B10-contaminated soil in bioremediation assay
Treatments pH values
T0 T10 T32
Control 4.9 5.2 5.5
Natural attenuation 5.0 4.7 5.3
Biostimulation 6.1 6.4 6.9
Conventional bioaugmentation 6.1 6.5 6.7
Successive bioaugmentation 6.0 6.4 6.6
2600 Environ Sci Pollut Res (2014) 21:25922602

Fig. 6 Banding profile obtained a

by DGGE from V3 region
amplification of the a bacterial
16S rRNA gene and b bacterial
communities dendrogram based
on DGGE banding profile
generated from soil treated by
natural attenuation (AN),
biostimulation (BS), conventional
bioaugmentation (BA CO), and
successive bioaugmentation (BA
SC). Numbers under
abbreviations treatments are 0,
11, and 32 days of incubations.
Axis X represents community
similarities, based on Dice
coefficient

AN0
b
BS0

AN11

BA CO0

BA SC0

BA CO11

BA SC11

BS11

BS32

BA CO32

BA SC32

AN32

0.39 0.51 0.62 0.74 0.86

Dice Coefficient

DGGE (Fig. 6). The soil microbial community of the control
treatment was not subjected to analysis because the absence of
the fuel contaminant provided a different situation for DNA
extraction compared to the other samples.
The natural attenuation and biostimulation initial samples
(AN0, BS0, and AN11) were presented in the same cluster. This
clusters banding pattern exhibited a uniform profile with
68 % similarity. In this case, it can be inferred that the profiles
generated by DGGE (Fig. 6a) from the AN and BS samples
reflect the native soil population. The AN11 sample, clustered
with AN0 e BS0, indicated that native biota has a contaminant
tolerance, i.e., B10 contamination caused no toxic effects in
the indigenous population.
The different profile and structure of the AN community
were more evident throughout the time course, as the commu-
nity grouped separately from the day 32 samples (Fig. 6b). This
is not in accordance with the results of other studies. Taccari
et al. (2012) demonstrated that soil treated with natural attenu-
ation and soil treated with nutrients, a microbial consortium,
and biosurfactants were clustered with 75 % similarity at
40 days. However, the accumulation of metabolites derived
from contaminant oxidation can reduce the viability of some
groups of hydrocarbon degraders throughout the process
(Reddy et al. 2011). This fact, associated with the specific soil
microbial groups growth with ability to use the fuel efficiently
as a carbon source, may be the reason for the AN 32
communitys detachment from the other samples (Evans et al.
2004). As shown in Fig. 6a, two intense bands appeared by
day 32 that were not observed at 0 and 11 days of incubation.
The effects caused by nutrient addition reflect changes in
the community structure between 0 and 11 days during
biostimulation treatment (Fig. 6). However, no differences
Environ Sci Pollut Res (2014) 21:25922602
were detected by the end of the experimental period, as the
BS11 and BS32 communities were clustered, demonstrating
similarity. Thus, biostimulation treatment likely led to struc-
tural changes in the soil microbial populations mainly during
the first 11 days after nutrient supplementation. Nutrient in-
corporation did not lead to significant changes in population
by the final experimental phase because assessments on the
11th and 32th day indicated that the BS11 and BS32 samples
formed a cluster with 80 % similarity, i.e., the communities
remained stable during the latter half of the experimental
period. Similar results were noted by Evans et al. (2004).
They reported a population that stabilized after 15 days, after
comparing profiles of communities biostimulated by nutrient
supplementation in the presence and absence of oil. They
concluded that the changes found were predominantly due
to the introduction of nutrients.
The cluster formed by the BA CO0, BA SC0, BA CO11, and
BA SC11 samples indicated that the bacterial community
structure remained similar in the initial experimental phase,
remaining clustered with 70 % similarity at 0 and 11 days of
incubation. The communities responded similarly to alloch-
thonous consortium addition. Population composition
changes were observed only at 11 and 32 days in the conven-
tional bioaugmentation and successive bioaugmentation treat-
ments. Community dynamics and structures were different
from the initial conditions, but they remained similar between
these two treatments. These results indicate that the additional
inoculation in the BA SC treatment on the 11th day did not
cause a significant community change in relation to the
bioaugmented soil at day 0 (BA CO) because the BA SC32
and BA CO32 samples were clustered at 32 day with 65 %
similarity. Vias et al. (2005) developed a bioremediation
study of creosote-contaminated soil. Among the strategies
employed, biostimulation and bioaugmentation were used
with five microbial consortium inoculations over 200 days.
Unlike the results observed by authors who reported similar-
ities between the soil communities inoculated and stimulated
by nutrient addition over 45 days, in the present study, the
biostimulated (BS) and bioaugmented (BA CO and BA SC)
communities were distinct from each other during the initia-
tion of the study. This indicates that the presence of inoculum
can affect the structural dynamics of indigenous biota.
Otherwise, the bioaugmentation treatments were more closely
related to the BS treatment (Ciric et al. 2010). However, the
consortiums in our experiment demonstrated no difference in
representative bands during the incubation period (Fig. 6a).
The results of this study allow us to identify bacterial com-
munity changes in soil bioremediated with different strategies
over 32 days. According to Li et al. (2007), oil concentrations
over 10,000 mg kg1 soil can significantly affect both soil
microbial activity and community structure. As shown in
Table 5, except for the BA CO treatment, all other microcosms
showed a reduction in species diversity. This effect was also
2601
Table 5 ShannonWeaver (H) diversity index of bioremediated soils at
0 (T0), 11 (T11), and 32 (T32) days
Treatments ShannonWeaver (H) diversity index
T0 T11 T32
Natural attenuation 2.48 2.30 2.08
Biostimulation 2.30 1.39 1.79
Conventional bioaugmentation 1.94 1.79 2.19
Successive bioaugmentation 1.94 2.08 1.39
observed by Taccari et al. (2012). The authors verified biodiver-
sity losses for both biostimulated and bioaugmented soils
resulting from toxic fuel action. However, the inverse occurred
in soil bioremediation studies conducted by Aleer et al. (2011).
The authors revealed that bacterial population diversity increased
in the first month of incubation, noting a decline only in the final
process phase (between the fourth and 12th weeks). These data
variations can be related to several factors. Among them, the
differences may be the result of the soils used in each experiment.
Aleer et al. (2011) investigated bioremediation effects on a
microbial community from previously contaminated soil.
Taccari et al. (2012) and Li et al. (2007) performed their biodeg-
radation studies in soils without historic contamination, similar to
this study. The presence of adapted microorganisms in naturally
contaminated soil can result in the exclusion of a community
adaptation phase. Consequently, the biodiversity reduction in the
initial period of the process would not be observed.
Conclusions
This study demonstrated that TPH reduction is higher when
biostimulation and successive bioaugmentation strategies are
employed, but there were no significant differences between
the treatments. The results of this study support that soil
microbial communities, even without previous petroleum con-
taminants, are able to express their degrading potential with an
appropriate stimulus, and a strategy using an additional inoc-
ulation of degrading consortium microbes produces positive
effects on biodegradation that are more pronounced than what
is exhibited with conventional bioaugmentation. Hence, suc-
cessive bioaugmentation could be a powerful tool for use in
biodegradation and bioremediation strategies.
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