Ecological Engineering 37 (2011) 1895–1900

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Ecological Engineering
journal homepage: www.elsevier.com/locate/ecoleng

Short communication

Bioremediation of soil polluted with fuels by sequential multiple injection of

native microorganisms: Field-scale processes in Poland
Maria Łebkowska a , Ewa Zborowska a , Ewa Karwowska a , Ewa Miaśkiewicz-Peska
˛ a
, Adam Muszyński a ,
a,∗ b c
˛
Agnieszka Tabernacka , Jeremi Naumczyk , Maciej Jeczalik
a
Warsaw University of Technology, Faculty of Environmental Engineering, Biology Division, Nowowiejska 20, 00-653 Warsaw, Poland
b
Warsaw University of Technology, Faculty of Environmental Engineering, Nowowiejska 20, 00-653 Warsaw, Poland
c
Hydrogeotechnika Sp. z o.o., ul. Ściegiennego 262A, 25-116 Kielce, Poland

a r t i c l e i n f o a b s t r a c t

Article history: Research was conducted to estimate impact of the multiple bioaugmentation on the treatment of soil
Received 14 March 2011 contaminated by fuels – diesel oil and aircraft fuel. The bacteria used to inoculate the remediation plots
Received in revised form 15 June 2011 were isolated from the polluted soil and proliferated in field conditions. The amount of biomass applied
Accepted 29 June 2011
to the polluted soil was set to ensure the total number of bacteria in soil 107 –108 cfu/g d.w. The multiple
Available online 30 July 2011
inoculation of soil with indigenous bacteria active in diesel oil and engine oil (plot A) degradation increased
bioremediation effectiveness by 50% in comparison to the non-inoculated control soil and by 30% in
Keywords:
comparison to the soil that was inoculated only once. The multiple inoculation of soil with indigenous
Soil pollution
Soil bioremediation
microorganisms was then applied in bioremediation of the soil polluted with double high concentration
Diesel oil of diesel oil (soil B) and in bioremediation of the soil polluted with aircraft fuel (soil C). The process
Bioaugmentation efficiency was 80% and 98% removal of TPH for soil B and C, respectively.
Biopiles © 2011 Elsevier B.V. All rights reserved.
Aircraft fuel

1. Introduction ganisms (Rojas-Avelizapa et al., 2007; Calvo et al., 2009; Haritash

The efficiency of the bioremediation of soil contaminated by
petroleum products (PP) and other pollutants depends on the
biotic and abiotic conditions of the process. Temperature, humid-
ity, addition of nutrients, concentration of oxygen, appropriate
size of soil particles, and the presence of (mainly biological) sur-
face active substances play important roles, shaping the abiotic
conditions that can enhance the hydrocarbon biodegradation pro-
cesses (Fountoulakis et al., 2009). Addition of various forms of
degradation rate limiting substances to the indigenous micro-
bial population in soil is called biostimulation. Bioaugmentation
involves introducing bacterial cultures to a contaminated soil, usu-
ally in combination with biostimulation (Boopathy, 2000). Some
authors believe that bioaugmentation does not significantly affect
the biodegradation efficiency for polycyclic aromatic hydrocarbons
(PAHs) with low molecular weight (Serrano Silva et al., 2010).
Others like Bento et al. (2005) have proved that biostimulation
(addition of nutrients) is less effective than natural attenuation and
bioaugmentation. Many authors have shown that bioaugmentation
and biostimulation enhance the biochemical activity of microor-
∗ Corresponding author. Tel.: +48 22 234 5943; fax: +48 22 621 2979.
E-mail address: agnieszka.tabernacka@is.pw.edu.pl (A. Tabernacka).
and Kanshik, 2009).
Application of biosurfactants (BS) may be realized by flushing
the soil with BS solutions (Urum et al., 2005). Bioemulsifiers can
emulsify hydrocarbons, thus enhancing their water solubility and
increasing the displacement of oily substances from soil particles,
such as in the treatment of oil in soils with a high percentage
of clay. Biosurfactants can be more ecologically acceptable than
synthetic surfactants in bioremediation due to their biodegradabil-
ity and lower toxicity. Biosurfactant producing microorganisms –
such as bacteria and fungi, which are also hydrocarbon degraders
– can be used to speed up the remediation of contaminated sites.
Many microorganisms are capable of producing surfactants: Pseu-
domonas, Nocardia, Rhodococcus, Bacillus, Arthrobacter, Torulopsis
and Candida (Christofi and Ivshina, 2002).
The process of hydrocarbon biodegradation is carried out by
various microorganisms present in soil, but there are relatively
few microorganisms that use hydrocarbons as the sole source of
carbon and energy compared to the general number of bacteria
(Łebkowska et al., 1995). It is a general belief that the bacteria
species active in hydrocarbon biodegradation should be isolated
and selected, grown and cultured into consortia, and used as an
inoculum in the polluted soil. It is necessary to eliminate the poten-
tial pathogens from the consortia, such as Pseudomonas aeruginosa
(Singer et al., 2005). The sources of extraneous and indigenous

0925-8574/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.ecoleng.2011.06.047
1896 M. Łebkowska et al. / Ecological Engineering 37 (2011) 1895–1900
microorganisms used in bioremediation are mainly soils polluted
with hydrocarbon derivatives and, sometimes, biocatalysts con-
taining genetically engineered microorganisms (GEMs).
The use of consortia containing microorganisms with unknown
substrate requirement may result in the failure of bioaugmenta-
tion process (Hesselsoe et al., 2008; Simon et al., 2004), as well as
competition between extraneous and indigenous microorganisms
and the impact of protozoan as predators (Bouchez et al., 2000).
Low bioremediation efficiency may also be caused by the lim-
ited survivability of the inoculum and the substrate bioavailability,
which depends on the type and the composition of soil (Balba et al.,
1998; Mrozik and Piotrowska-Seget, 2010; Scherr et al., 2007). The
application of GEMs in the environment has been limited, mostly
because of the risks associated with the uncontrolled growth and
proliferation of introduced biocatalysts and horizontal gene trans-
fer (Paul et al., 2005).
Currently, it is a general belief that bioaugmentation is a very
good option for further developing soil remediation methods. Li
et al. (2009) proved that bioaugmentation highly increases the
biodegradation rate of PAHs present in low concentrations in aged
contaminated soil. Introduction of Paracoccus sp. HPD-2 to the con-
sortium of soil microorganisms led to the removal of 23% PAHs
from an aged contaminated soil, polluted with 9942 ␮g PAH/kg of
dry weight of soil (Teng et al., 2010). Microorganisms from munic-
ipal wastewater introduced to a bioslurry phase reactor operated
in periodic discontinuous batch mode increased the degradation
rate of pyrene in soil. In the case of augmented reactors, the sys-
tem showed a pyrene degradation efficiency of almost 99% when
operated with low substrate loading (Mohan et al., 2008). The appli-
cation of isolated bacterial strains – two Pseudomonas strains and
Gordonia sp. – resulted in 65–85% degradation of kerosene from
an 8% solution in biostimulating conditions (Gouda et al., 2007).
Research by Ruberto et al. (2003) has proved that the indige-
nous microorganisms from Antarctic soils are able to degrade
an important fraction of gas oil, and that bioaugmentation with
the psychrotolerant strain B-2-2 previously isolated from this soil
improved the bioremediation efficiency.
The bioaugmentation of soil polluted with diesel oil and heavy
metals by isolated bacterial strains resistant to heavy metals
resulted in an overall reduction of about 75% of the total diesel
hydrocarbons in 42 days (Alisi et al., 2009). The application of
soil inoculation with bacterial strains active in pollutant degra-
dation is common not only when the pollutants are petroleum
hydrocarbons but also when other organic substances are present.
Rousseaux et al. (2003) researched the effect of inoculation with
the atrazine-mineralizing strain Chelatobacter heintzii Cit 1 on
soil polluted with atrazine (a widely used herbicide). It was
determined that in the case of soils adapted to atrazine degra-
dation, inoculation of C. heintzii did not accelerate the rate of
Fig. 1. Biopile on plot A: 1 – insulating foil; 2 – polluted soil; 3 – geogrid; 4 – geotextile; 5 – compressed airpipe PE 50 mm; 6 – gravel 8/32 layer depth 0.3–0.4 m; 7 – drainage
pipe PVC 80 mm; 8 – subsoil; 9 – geomembrane.
atrazine biodegradation which was performed by the indigenous
microflora. However, for soils that did not mineralize atrazine, the
introduction of 104 cfu/g resulted in a threefold increase in the
atrazine degradation capacity.
Enhancement of oil biodegradation involves a number of mech-
anisms including plant uptake and promotion of microbial growth
by plants’ presence. Plants with high root biomass might produce
considerable root exudates that may increase microbial number
and activity in the rhizosphere, which in turn increase the soil
bioremediation efficiency (Lin and Mendelssohn, 2009).
A literature review provides a few data concerning the efficiency
of bioremediation of soil polluted with petroleum derivatives in
relation to inoculation frequency. Usually, there is a single applica-
tion of bioaugmentation and biostimulation. The main goal of this
study is an assessment of the efficiency of treating soils polluted
with fuels by using biostimulation and bioaugmentation where
the indigenous bacterial strains isolated from the polluted soils
were cultured and used in high concentration as an inoculum. The
inoculation process was repeated every three days. The developed
technology has been patented (Łebkowska and Kańska, 2000) and
applied in Poland on a field scale to treat over 150,000 tons of
soil.
2. Materials and methods
2.1. Soil
Soil samples were collected from aged soil polluted with diesel
oil and engine oil (A), diesel oil (B) and aircraft fuel (C). Soils A
and C were composed of sand and loam (85% of sand, 5% of silt,
10% of clay), and soil B of 65% of sand, 12% of loam and 23% of
clay. The porosity of soil A was 35%, soil B was 41% and soil C was
32% and the saturated hydraulic conductivity of soils A, B and C
were 5 × 10−4 , 7 × 10−5 and 6 × 10−4 m/s, respectively. The stones
and bigger solid particles were removed from soils and then the
samples were crumbled and ground to the particle size of about
5 cm. The bioremediation process was conducted in all soils ex situ
in biopiles.
2.2. Soil biopiles
Soil A was remediated on a plot with these overall dimensions:
12 m (width) × 33 m (length) × 0.5 m (height). The remediation plot
was provided with geomembrane (polyethylene foil), an aerating
grate composed of air pumps, each with a flow of 270 m3 /h, and
drainage to direct the leachates to the collecting well (Fig. 1). The
plot was divided into three areas each 4 m wide: a control area
(A1), an area with one inoculation of microorganisms (A2), and an
 M. Łebkowska et al. / Ecological Engineering 37 (2011) 1895–1900
area where the inoculation was repeated every three days (A3). To
protect the biopile from rain, a foil was used. Soils B and C were
remediated on plots with the same dimensions as that used for
remediating soil A. A land farming process was applied – digging
and ploughing the soil twice a week using plough machines. Soil
was put onto the geomembrane and the leachates were collected
to the canal surrounding the biopile. The bioremediation was car-
ried out from June to October at temperature ranging from 15 ◦ C to
30 ◦ C.
2.3. Microbial assay
2.3.1. Isolation and identification of microorganisms
One kilogram samples of soil were taken from five different
places from well-blended soils. They were prepared for deposition
and storage, and mixed together. The average sample was used
for plating on a mineral Grundmann medium solidified with agar
with the addition of 1% of diesel oil as the sole source of carbon
and energy (MD) (Sztompka, 1999; Tabernacka et al., 2010). Prior
to agar addition, the liquid medium with diesel oil was sonicated
by exposure to 35 kHz for 15 min. The plates were incubated at
temperature 26 ◦ C for 72 h and the number of bacteria (cfu/g of
d.w.) was determined. The pure bacterial strains were obtained by
passaging the strains several times on the solid MD medium. API
20 NE and API Staph tests (bioMérieux) were used for the iden-
tification. Four to five predominant bacterial strains were chosen
from each site. The opportunistic pathogens such as P. aeruginosa
were rejected. At site A two species Bacillus sp. including one pro-
ducing BS, Pseudomonas mendocina and Pseudomonas putida were
used. At site B two species of Bacillus sp., P. mendocina and Acine-
tobacter lwoffii were applied, while at site C two species of Bacillus
sp., Pseudomonas alcaligenes, Sphingomonas paucimobilis and Alcali-
genes xylooxidans were applied.
2.3.2. Preliminary cultures in laboratory conditions
Selected bacterial strains were proliferated in liquid MD
medium with the addition of 0.1% of yeast extract. Incubation was
held in a bath shaker in 300 mL Erlenmeyer flasks at 26 ◦ C for 24 h.
Bacteria were then proliferated for 48 h in 10 L glass bottles con-
taining MD medium at room temperature 20 ± 2 ◦ C.
2.3.3. Cultures in field conditions
The bacterial suspensions from the glass bottles were used to
inoculate a liquid medium in a 2 m3 bioreactor (operational vol-
ume). The bioreactor was equipped with air pumps to ensure a
concentration of dissolved oxygen of 2–6 mg O2 /L. The actual con-
centration of dissolved oxygen in the bioreactor depended on the
air temperature at the test site. The liquid medium was composed
of broth 0.011 g/L, Florovit (a mineral fertilizer) 5.3 mL/L, sugar
0.27 g/L and diesel oil (PKN Orlen) 1.6 mL/L. The mineral fertil-
izer Florovit was obtained from INCO-VERITAS S.A. It was used
as the source of nitrogen and phosphorus. The chemical content
of Florovit was as follows: total N 7.0%; P2 O5 5.0%; K2 O 6.0%; B
0.02%; Cu 0.008%; Fe 0.03%; Mn 0.015%; Mo 0.002%; Zn 0.015%.
The microorganisms were proliferated for 24–48 h at field temper-
ature and then 7/8 of the suspension was used to inoculate the
remediation plots and 1/8 of the suspension was used to prepare
another batch of bacterial suspension from the MD medium. All
strains selected at each site remained in the bioreactor after the
cultivation. The exact ratio of strains in the consortia was not mea-
sured. The number of bacteria (cfu/g d.w.) in the treated soil was
determined using an average sample and the solid MD medium in
accordance with Section 2.3.1.
1897
2.3.4. Assessment of BS production by the isolated
microorganisms
Bacteria were proliferated using a medium composed of (g/L):
Na2 HPO4 6.0; KH2 PO4 3.0; NaCl 0.5; (NH4 )2 SO4 3.0. Additionally,
250 ␮g of yeast extract, 250 ␮g of peptone, 250 ␮g of starch and
2 mL of diesel oil were added to 1 L of medium. Incubation was
held for 24–48 h at a temperature of 26 ◦ C in a water bath shaker at
120 rpm. After the first 24 h, another dose of diesel oil was added.
The ability to produce biosurfactants was determined in test tubes
by adding 3 mL of diesel oil to 7 mL of bacterial suspension. The
content of the test tubes was mixed thoroughly for a minute and
then left at room temperature. After an hour, and again after 24 h,
the height of the emulsified phase was measured. The stability of
emulsion was measured by calculating the proportional ratio of the
emulsified phase to the hydrocarbon (oil) phase. Emulsion was con-
sidered stable if its percentage after 24 h (EI 24 h) was 50 percent
or more (Płaza et al., 2006).
2.4. Determination of hydrocarbon content
The amount of hydrocarbons and their degradation products in
the soil was determined by using four different procedures.
Volatile aromatic hydrocarbons (BTEX) were determined using
the head-space gas chromatographic method (Margesin et al.,
2003). A gas chromatograph Carlo Erba HRGC 5300 equipped with
FID detector and FFAP10% column was used. The column tempera-
ture was set at 70 ◦ C, injector temperature was 180 ◦ C and detector
temperature was 190 ◦ C. Nitrogen was used as the carrier gas. Chro-
matographic determination was conducted according to EN ISO
11423-1.
The content of hydrocarbons (C10 –C22 ) with retention times
between those of n-decane (C10 ) and docosane (C22 ) was deter-
mined by gas chromatography (GC). Hydrocarbons were extracted
(5 h) from soil (previously dried with anhydrous Na2 SO4 ) by
n-hexane in Soxhlet apparatus. The clean-up procedure and chro-
matographic determination were accomplished according to EN
ISO 9377-2. Aliphatic hydrocarbons were determined using the
capillary column DB-5 (diameter 0.53 mm) on the aforementioned
gas chromatograph. The following parameters were used: detector
temperature 310 ◦ C, injector cool on column, the column start tem-
perature 70 ◦ C for 4 min, then raise 4 ◦ C/min to 270 ◦ C. The carrier
gas was helium.
The total content of petroleum hydrocarbons (TPH) was deter-
mined using IR spectroscopy. A sample of soil (15–20 g) was dried
with anhydrous Na2 SO4 and extracted (6 h) with CCl4 in Soxhlet
apparatus. After drying with Na2 SO4 , the extract was concen-
trated to 2–3 mL by evaporation in Kuderna–Danish apparatus,
then introduced into a column filled with Florisil (previously con-
ditioned 2 h in 250 ◦ C) of 8 mm diameter and 100 mm bed height.
Hydrocarbons were eluted with 20 mL of CCl4 . Hydrocarbons con-
tent was measured with a UR 20 spectrometer at 2928 cm−1
(cuvette 10 mm). The calibration solutions were prepared by dilu-
tion of the standard solution of n-hexadecane + isooctane + benzene
(37.5% + 37.5% + 25%) in CCl4 .
The total content of substances extracted by CCl4 (TES−CCl4
extract) was measured using the same procedure (IR spectrome-
try) except for the cleaning-up procedure. The result was assumed
as the total content of hydrocarbons and degradation products of
low polarity.
All analyses were performed in triplicate.
2.5. Bioremediation in biopiles
The amount of hydrocarbons and the number of bacteria using
fuels as the sole sources of carbon and energy were determined in
1898 M. Łebkowska et al. / Ecological Engineering 37 (2011) 1895–1900

Table 1
The results of chemical and microbiological analyses of soil A during the bioremediation process.

Type of experiment Compound Results after day

0 2 5 10 17 25

A1 (control) BTEX nd nd nd nd nd nd
C10 –C22 1079 1009 1011 920 875 704
TPH 2509 2517 2382 2227 2004 1742
TES 2609 2598 2598 2503 2269 1810

A2 BTEX nd nd nd nd nd nd
inoculación simple C10 –C22 1079 1006 984 824 611 387
TPH 2509 2415 2286 1918 1530.5 1210
TES 2609 2473 2266 1823 1347 1005

A3 BTEX nd nd nd nd nd nd
inoculación múltiple C10 –C22 1079 1140 882 596 268 48.6
TPH 2509 2405 2118 1380 961 452
TES 2609 2663 2341 1395 682 398

TPH biodegradation A1 0 45 31 32 33
rate mg/(kg × d) A2 47 43 74 55 40
A3 52 96 148 60 64

Type of experiment Number of microorganisms growing on the medium with diesel oil as the sole source of carbon in bioremediation of soil A

After 2 days After 10 days After 17 days After 25 days

A1 (control) 2.6 × 104 2.9 × 104 3.8 × 104 3.8 × 104

A2 1.4 × 106 5.8 × 106 5.1 × 106 2.3 × 106
A3 1.6 × 107 9.9 × 108 6.9 × 108 2.0 × 108
Species of microorganisms Bacillus sp. – two species Bacillus sp. – two species Bacillus sp. – two species Bacillus sp. – two species
including one producing BS, including one producing BS, P. including one producing BS, P. including one producing BS, P.
Pseudomonas mendocina, mendocina, P. putida mendocina, P. putida mendocina, P. putida
Pseudomonas putida

Note: Concentration is given in mg/kg of soil. The number of microorganisms is given in cfu/g d.w. (dry weight). TPH biodegradation rate in mg/(kg × d). The number of
microorganisms in soil at the beginning of the process – 2 × 104 cfu/g d.w. B, benzene; T, toluene; E, ethylbenzene; X, xylene; C10 –C22 , aliphatic hydrocarbons C10 –C22 ; TPH,
total petroleum hydrocarbons; TES, total content of substances extracted by CCl4 ; nd, not detected.

biopiles at the beginning of the process. The decrease of TPH con-
centration and the number of microorganisms were determined
after the several days of process, and then at intervals of once a
week or less. Biopiles were inoculated with the bacteria culture
from the bioreactors every three days (excluding the A1 and A2
soils) to ensure the number of microorganisms in soil was in the
range of 107 –108 cfu/g d.w. Soil humidity was kept at 20–30% using
water from the liquid bacteria culture and from additional humid-
ification.
3. Results and discussion
The bioremediation of sandy–loam soils and sandy–clay soils
using multiple/repeated inoculation with indigenous bacteria
was effective considering both the decrease in the pollutant
concentration and the duration of process. The content of TES in
remediated soils was slightly higher than TPH; during the biore-
mediation the decrease of TPH was followed by the decrease of
TES. Research conducted in soil A showed that the inoculation of
soil with indigenous bacteria active in diesel oil and engine oil
degradation (A3) increased bioremediation effectiveness by 50%
in comparison to the non-inoculated control soil (A1) and by 30%
in comparison to the soil that was inoculated only once (A2). The
results are presented in Table 1. The maximum biodegradation rate,
achieved on the tenth day of the process, was two times higher in
soil A3 than in soil A2 and five times higher than in soil A1.
Lin et al. (2010) obtained similar results using bioaugmentation
with two bacterial strains and biostimulation with biosurfactant

Table 2
The results of the chemical and microbiological analyses of soil B during the bioremediation process.

Compound Concentration of hydrocarbons after the day

0 3 5 10 20 35 65

BTEX <2 nd nd nd nd nd nd
C10 –C22 4261 4004 3844 2892 2210 1955 nd
TPH 5336 5012 4799 3918 3100 2310 1084
TES 5568 5193 4700 3903 3251 2310 1193
TPH biodegradation rate 108 106 176 82 53 41
The numbers of 3.4 × 107 na 8.7 × 108 na 5.1 × 108 4.8 × 108
microorganisms active in soil
bioremediation
Species of microorganisms Bacillus sp. – two species, na na na na Bacillus sp. – two
Pseudomonas mendocina, species, P. mendocina,
Acinetobacter lwoffii P. alcaligenes,
Micrococcus sp.

Note: The concentration of hydrocarbons in mg/kg of soil, TPH biodegradation rate in mg/(kg × d), number of microorganisms in cfu/g d.w. The number of microorganisms
in soil at the beginning of process–0.16 × 105 cfu/g d.w. B, benzene; T, toluene; E, ethylbenzene; X, xylene; C10 –C22 , aliphatic hydrocarbons C10 –C22 ; TPH, total petroleum
hydrocarbons; TES, total content of substances extracted by CCl4 ; nd, not detected; na, not analysed.
 M. Łebkowska et al. / Ecological Engineering 37 (2011) 1895–1900 1899

Table 3
The results of chemical and microbiological analyses of soil C during the bioremediation process.

Compound Concentration of hydrocarbons after the day

0 5 10 22

T 5 <2 nd nd
X 12.1 <2 nd nd
EB 5.6 <2 nd nd
C10 –C22 632 253 nd nd
TPH 3336 1660 717 81
TES 3440 1812 881 166
TPH biodegradation rate 335 189 53
Number of microorganisms 3.6 × 104 2.1 × 108 8.6 × 107 3.4 × 107
Species of microorganisms Comamomonas testosteroni C. testosteroni Bacillus sp. – na Bacillus sp. – four species, S.
Bacillus sp. – two species, two species, P. alcaligenes, paucimobilis, A. xylooxidans
Pseudomonas alcaligenes, S. paucimobilis, A.
Sphingomonas paucimobilis, xylooxidans
Alcaligenes xylooxidans

Note: The concentration of hydrocarbons in mg/kg of soil, TPH biodegradation rate in mg/(kg of soil × d), the number of microorganisms in cfu/g d.w. The number of
microorganisms in soil at the beginning of process – 3.6 × 104 cfu/g d.w. T, toluene; X, xylene; C10 –C22 , aliphatic hydrocarbons C10 –C22 ; TPH, total petroleum hydrocarbons;
TES, total content of substances extracted by CCl4 ; nd, not detected; na, not analysed.

rhamnolipid produced by P. aeruginosa, which is a dominant strain
in indigenous consortia. After 28 days of the process, diesel oil
and fuel oil were degraded by up to 70% and 63% respectively
in one of the small biopiles. Removal rate in these biopiles was
approximately 74–96 mg TPH/(kg × d). In this study, the maximum
removal rate by multiple inoculation was 148 mg TPH/(kg × d). The
number of bacteria in a biopile was about 108 cfu/g d.w. (Table 1);
in research conducted by Lin et al. (2010) the number was 106 cfu/g
d.w.
The results obtained for soil A, and from previous experiments
conducted by the authors, were used to plan the field application of
multiple bioaugmentation in soils B and C. It was the field applica-
tion; therefore, the control plot was not used. The soil was treated
until the legal requirements concerning soil parameters at the site
were fulfilled.
Soil B contained clay and the concentration of TPH was double
that in soil A. The bioremediation process was continued for more
than twice as long as that used for soil A, and its efficiency was very
high – 100% removal of C10 –C22 hydrocarbons and 80% removal of
TPH (Table 2). The bioremediation rate was greatest at the begin-
ning of the process – at the tenth day it was 176 mg/(kg × d). The
content of TES in remediated soils was slightly higher than TPH;
during the bioremediation the decrease of TPH was followed by
the decrease of TES. The dominant bacterial strains were the strains
introduced as an inoculum – Bacillus sp., which produced biosur-
factants, Acinetobacter lwoffi and P. mendocina. At the end of the
process, P. alcaligenes and Micrococcus sp. appeared in large num-
bers and the soil samples did not contain A. lwoffi. The number of
bacteria was 108 cfu/g d.w.
The positive impact on bioremediation efficiency of increasing
the biopile inoculation rate was observed by Ouyang et al. (2005).
The biocatalyst Rhoder was applied as a working solution with
MPN of oil oxidizing bacteria (1 × 106 –1 × 107 cells/ml) every two
weeks for bioremediation of soil polluted with oil and oily sludge.
After three applications of biocatalysts, the hydrocarbons concen-
tration decreased by 53% in soil and 46% in the oily sludge, while on
the positive controls (activation by indigenous microorganisms) it
decreased by only 13%. Ouyang’s team obtained very good biore-
mediation effects considering the high hydrocarbon concentration
at the beginning of the process – 94 g/kg in soil and 251.3 g/kg in
oily sludge.
A very high concentration of TPH – 61 g/kg at the beginning
of the process – was used by Salinas-Martinez et al. (2008). They
carried out research on soil bioremediation using a heap leach-
ing process. At a flow rate of 200 mL/h, the TPH concentration
decreased to 1800 mg/kg (98.5%) in 15 days. Heap leaching was
carried out in laboratory conditions, so the costs and the efficiency
of the process at field scale are still unknown. Kołwzan (2005) con-
ducted similar research to Salinas-Martinez et al. (2008) but at the
beginning of the process the TPH concentration was much lower –
2543 mg/kg – and the process was conducted in field. The bacte-
rial suspension, nutrients and water were applied continuously to
the biopile in closed circle. Within two months, TPH concentration
decreased to 21.3 mg/kg d.w. and the number of microorganisms
was at in the range of 107 –108 cfu/g d.w. Dominant were bacteria
strains introduced to soil as inoculum – Stenotrophomonas mal-
tophila and P. putida. The process efficiency obtained by Kołwzan is
similar to the efficiency of the remediation of soil A3.
Soil C contained the aviation fuel. The concentration of aromatic
and aliphatic hydrocarbons in this soil was low and the concentra-
tion of TPH was 3336 mg/kg. Multiple bioaugmentation resulted in
the fast removal of hydrocarbons; after 22 days, 98% of TPH was
removed (Table 3). The number of bacteria was greatest during the
first 10 days of the process – 108 –109 cfu/g d.w. Dominant were
bacteria strains introduced to soil as inoculum – Comamomonas
testosteroni, Bacillus sp., which produced BS, P. alcaligenes, S. pauci-
mobilis and A. xylooxidans. At the end of the process, P. alcaligenes
and C. testosteroni were not present in the consortium; however,
two strains of Bacillus sp. appeared.
The high efficiency of the bioremediation process in the soil con-
taining aviation fuel is connected to the fact that many species of
microorganisms are able to use this fuel as a source of carbon and
energy. Bacteria and fungi grow very easily in aviation fuel storage
tanks and aircraft wing tanks (Rauch et al., 2006; Itah et al., 2009).
The addition of biocide to the fuel does not protect from micro-
bial growth. Research conducted by Rabaev et al. (2009) has shown
that the degradability in soil of jet fuel with the anti-icing inhibitor
diethylene glycol monomethyl ether (DiEGME) is tenfold higher
than that of fuel without the inhibitor. The biocide promoted, rather
than retarded, microbial growth.
Moreover, during the soil bioremediation different interme-
diates may appear in the treated soil. This fact may explain the
changes in the qualitative content of the bacteria present in soil
and changes in the consortium.
Laboratory experiments have to be effectively extrapolated to
the field scale (Diplock et al., 2009). Our work was conducted with
a cooperative industrial partner, which carried out bioremediation
programme for each polluted soil.
1900 M. Łebkowska et al. / Ecological Engineering 37 (2011) 1895–1900
4. Conclusions
Bioremediation is a cost-effective and environmentally sound
soil remediation technology. Multiple inoculation of microbial con-
sortia from the polluted soil can increase the biomass of degrading
microorganisms present in the biopiles and shorten the bioreme-
diation process. Introducing microorganisms that are capable of
degrading pollutants and biosurfactant producing microorganisms
into the soil, as well as encouraging good biostimulation conditions,
increases the efficiency of bioremediation systems.
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