Ann Microbiol

DOI 10.1007/s13213-014-0856-5

ORIGINAL ARTICLE

Rapid remediation of soil heavily contaminated

with hydrocarbons: a comparison of different approaches
Angelos Dados & Michalis Omirou &
Kyproula Demetriou & Chara Papastephanou &
Ioannis M. Ioannides

Received: 22 September 2013 / Accepted: 28 February 2014

# Springer-Verlag Berlin Heidelberg and the University of Milan 2014

Abstract To improve our understanding of the dissipation patterns were noted among the different n-alkane fractions,
kinetics of total petroleum hydrocarbons (TPH), we tested with short chain molecules (up to 14 carbon atoms) being
two bioremediation strategies in soil heavily contaminated rapidly reduced within the first 21 days, whereas long chain
with hydrocarbons. In one strategy, different fertilizers and molecules were more recalcitrant. In summary, we demon-
composted winery products were used to stimulate a local strated that Pseudomonas-like strains and in situ soil incorpo-
microbial community capable of dissipating petroleum hydro- ration of compost derived from winery byproducts can be
carbons. In the second approach, polluted soil was used for the effectively used for the rapid bioremediation of soil heavily
enrichment and isolation of potential hydrocarbon-degrading polluted with hydrocarbons.
bacteria that were subsequently used for implementing a bio-
augmentation strategy. The efficacy of both strategies was Keywords TPH and n-alkane dissipation kinetics .
evaluated using the Hockey-Stick and first-order kinetics Bioremediation . Biostimulation . Bioaugmentation .
models. Two Pseudomonas isolates designated as el20 and Pseudomonas sp.
el15 were able to readily degrade TPH and n-alkanes both
in vitro and in a microcosm study. Phylogenetic analysis based
on 16sRNA gene sequencing revealed that both strains Introduction
showed a high similarity with Pseudomonas otitidis and
P. stutzeri. Enrichment with both compost and nitrogen fertil- Diesel is one of the most important sources of energy world-
izer enhanced the dissipation of TPH. Overall, compost- wide. Its extensive use, however, has inevitably led to various
treated soils exhibited the highest degradation rates, with environmental problems mainly due to improper waste dis-
half-life (T½) values ranging from 13 to 37 days. Urea- posal practices, accidental spills, and leakage from storage
treated soils exhibited the lowest TPH T½ values among the tanks as well as leaching landfills. Diesel pollutants can cause
soils treated with inorganic fertilizers. The addition of glucose serious disturbance to the ecological balance which usually
to soils treated with inorganic nitrogen fertilizers retarded the takes years to recover.
degradation rate of TPH, with estimated T½values measure- Physical, chemical and biological methods have been de-
ments ranging from 70 to 140 days. Different dissipation veloped over the years to remediate petroleum spills.
Bioremediation has proven to be an effective and cheap ap-
proach for the remediation diesel pollutants in a variety of
Electronic supplementary material The online version of this article environments. The bioremediation of hydrocarbons in pollut-
(doi:10.1007/s13213-014-0856-5) contains supplementary material, ed soils can be promoted by stimulating the native microbial
which is available to authorized users. community through the addition of nutrients or/and oxygen
M. Omirou : I. M. Ioannides (*) (biostimulation) or through inoculation with an appropriate
Department of Agrobiotechnology, Agricultural Research Institute, microbial consortium (bioaugmentation) (Seklemova et al.
1516 Nicosia, Cyprus 2001; El Fantroussi and Agathos 2005; Delille and Coulon
e-mail: [email protected]
2008; Liu et al. 2010; Nikolopoulou and Kalogerakis 2009).
A. Dados : K. Demetriou : C. Papastephanou The availability of nutrients, nitrogen in particular, and
cp FOODLAB Ltd, Polyfonti 25, Strovolos, Nicosia, Cyprus various other environmental factors control the degradation
 Ann Microbiol

rates of hydrocarbons due to imbalance in the ratio between the soil from which they were isolated compared to alien
carbon and nitrogen as well as denitrification leading to a species. Thus, an evaluation of local isolates for their ability
suppression of soil microbial activity (Walworth and to enhance hydrocarbon degradation is a prerequisite for the
Reynolds 1995; Xu et al. 1995). Indeed, in their study, efficient bioremediation of heavily polluted soils.
Raffaldi et al. (2006) showed that the increased degradation The main objective of our study was to evaluate the effi-
rate of petroleum hydrocarbons following the addition of cacy of bioaugmentation using indigenous diesel-degrading
nitrogen was related to microbial activity. The effect of the bacteria for the remediation of long-term diesel-polluted soil.
nitrogen form on biodegradation of hydrocarbons is still con- At the same time we tested different inorganic nitrogen fertil-
troversial. It has been reported that ammonium is inhibitory to izers and composts derived from winery byproducts as mate-
hydrocarbon biodegradation, even at low levels of application, rial for biostimulating the dissipation of hydrocarbons in the
despite this form of nitrogen being the most efficacious in same soil.
terms of microbial growth and metabolism (Wrenn et al. 1994;
Foght et al. 1999). In a similar trend, Brook et al. (2001)
showed that nitrate-nitrogen was also less efficient, and in Materials and Methods
their study the hydrocarbon degradation rate was inhibited at
elevated concentrations of nitrate-nitrogen. In addition to the Soil and compost characteristics
influence of nitrogen form on microbial performance, the
extensive use of inorganic fertilizers that are able to provide The chemical characteristics of the soil and composts used
readily available nitrogen can cause serious environmental during this study are presented in Table 1. The soil was
problems, especially in coastal areas of semi-arid regions collected from a former refinery site in Larnaka, Cyprus, that
due to nitrate leaching. Thus, alternative strategies for the is currently used for the storage of refined petroleum products.
treatment of hydrocarbon-polluted soils by biostimulation The heavily contaminated soil (31,299 mg TPH/kg) was ho-
are needed. mogenized by hand, air dried and passed through a 2-mm
The addition of different organic amendments, including mesh sieve. The moisture content of the soil was adjusted to
composted material, has been shown to enhance hydrocarbon 30 % of the water-holding capacity and left standing for 4 days
degradation (Hupe et al. 1996; Sarkar et al. 2005; Hickman at room temperature to restore soil equilibrium. The compost
and Reid 2008; Kobayashi et al. 2009; Gandolfi et al. 2010). was prepared in the composting facilities of the Agricultural
Compost is characterized as a microbial active substrate which Research Institute of Cyprus. Winery by-products (seeds and
can be used for the improvement of soil fertility and also for skins) were collected from a local winery, air dried for 3 days
the remediation of agrochemicals, such as pesticides (Omirou and composted in 1-m3 bins for 4 months. Upon completion
et al. 2012). The low content of readily available nitrogen in of composting, the composts were air-dried, passed through a
compost but high total nitrogen content makes it an attractive 4-mm mesh sieve and stored in the dark at room temperature
alternative to inorganic nitrogen fertilizers which are used for until use.
hydrocarbon remediation.
Bacteria have been found to be the most active microbial
group, acting as primary degraders of spilled hydrocarbon Table 1 Physicochemic-
al characteristics of the Parameter Soil Compost
pollutants in the environment (Das and Chandran 2011). It
soil and compost used in
has been suggested that pre-selection and in situ application of the study pH 7.2 8.1
indigenous microbial populations can lead to increased hydro- Total N (%) 0.087 2.7
carbon removal rates (Bento et al. 2005; Thompson et al. Organic C (%) 3.45 29.2
2005). Indigenous bacteria of the genera Micrococcus, C/N 39.6 10.6
Bacillus, Anrthrobacter and Streptomycetes have been de- Total P (mg/kg) 184.2 3,495.1
scribed to enhance hydrocarbon degradation in polluted soils As (mg/kg) 4.0 1.01
(Das and Mukherjee 2007; Radwan 2008). Pucci et al. (2000) Cd (mg/kg) 4.5 0.07
studied soils heavily contaminated with crude oil and found Co (mg/kg) 5.4 0.1
that the most predominant phylotypes of bacteria present were Cr (mg/kg) 47.5 1.8
Gram-negative Pseudomonas strains, whereas much lower Cu (mg/kg) 44.9 43.1
proportions of Actinomycetales were isolated. The authors Hg (mg/kg) 0.11 <0.02
interpreted these results as indicating a preferential selection Ni (mg/kg) 19.5 0.9
and shift of the total petroleum hydrocarbon (TPH)-degrading Pd (mg/kg) 195.8 <0.17
microbial community. Hamdi et al. (2007) attributed this shift V (mg/kg) 35.9 0.3
to the inherent abilities of the isolates, such as selectivity and
Zn (mg/kg) 134.1 13.9
specialization, and to their ability to survive and propagate in
Ann Microbiol

Isolation and characterization of hydrocarbon-degrading Biology Laboratory database under accession number
Pseudomonas bacteria HG315717 up to HG315729. Phylogenetic analysis of the
deposited sequences was carried out using MEGA5 software
The microorganisms used in bioaugmentation tests were iso- (Tamura et al. 2011).
lated from the same diesel-contaminated soil as mentioned
above. The bacteria present in the samples were enriched by Evaluation of the degradation potential of the isolated
cultivation in a carbon-free mineral salt medium (MSM) con- Pseudomonas strains
taining 12.5 g/L K2PO4, 3.5 g/L KH2PO4, 1 g/L (NH4)2SO4,
0.1 g/L MgSO4⋅7H2O and 5 mL of trace element solution Thirteen selected Pseudomonas strains were grown aerobical-
(0.23 g/L H 3 BO 3, 0.174 g/L ZnSO 4 ⋅7H 2 O, 0.116 g/L ly in batch culture in 250-mL Erlenmeyer flasks containing
FeSO 4 ⋅6H 2 O, 0.096 g/L CoSO 4 ⋅7H 2 O, 0.022 g/L 100 mL of liquid culture. The strains were grown on MSM
NH4Mo7O24⋅4H2O, 8 mg/L CuSO4⋅5H2O and broth containing 100 mg/L TPH as the sole carbon source for
MnSO4⋅4H2O). Soil samples (2 g) were cultivated in 300-mL 24 h at room temperature. The cells were pelleted by centri-
Erlenmeyer flasks in MSM (200 mL), supplemented with fugation at 5,000 g for 10 min, washed twice with fresh MSM
120 mg crude diesel as the sole source of carbon and energy. and then used as inoculum. The dissipation studies were
Cultivation was carried out at 27 °C with shaking, and when carried out in MSM broth containing 200 mg/L TPH which
the concentration of the TPH reached half of the initial con- was inoculated with the isolated strains at a concentration of
centration 20 mL of the broth was poured into a fresh MSM 106 CFU/mL. Just after TPH addition and at 7, 14, 21, 28 and
(100 mL) supplemented with 120 mg crude oil. The enrich- 35 days after the application (DAA), subsamples of the MSM
ment cultures were carried out for 8 weeks and repeated four broth were removed for TPH analysis. The most efficient
times during the culture period for the selection of TPH strains were then selected for the microcosm bioaugmentation
degraders. The selective cetrimide/fucidin/cephalosporin me- experiments in diesel-contaminated soil.
dium (supplemented with 200 mg crude oil) was used to
isolate the Pseudomonas strains present in the enrichment Evaluation of the bioaugmentation potential
culture. Pure cultures were then selected based on colony of hydrocarbon-degrading Pseudomonas bacteria
morphology, and further purification was achieved with culti-
vation on bacteriological agar. The isolated strains were stored Unsterilized diesel-contaminated soil (250 g) was placed in 1-
at −20 °C in tryptone soy agar broth supplemented with 30 % L glass bottles (cleaned with dichloromethane prior to use) at
glycerol. room temperature and the soil water content adjusted to 60 %
DNA was extracted from overnight pure bacterial cultures of the water-holding capacity. Two of the isolated bacterial
using the NucleoSpin–Tissue kit (Macherey-Nagel GmbH, strains, el20 and el15, and a mixture of these two strains were
Düren, Germany) according to the manufacturer’s instruc- inoculated into the soil to a concentration of 106 microorgan-
tions. The isolated DNA was further purified using the isms per gram of soil. Each treatment was repeated three times
Nucleospin Extract II kit (Macherey-Nagel GmbH). Three in a completely randomized design, and samples were collect-
replicate DNA extractions were carried for each culture. The ed at 0, 7, 14, and 28 DAA.
respective DNA samples were used as template for PCR
reactions in a total volume of 50 μL containing 1× polymerase Microcosm biostimulation study
buffer, 1.5 mM MgCl2, 20 pmol of each primer, 200 μM of
each dNTP, 200 ng/ μL of acetylated bovine serum albumin Diesel-polluted soils (500 g/sample) were placed in 2-L glass
(GE Healthcare, Little Chalfont, UK) and 1.2 U of polymerase bottles (treated a priori as described above) at room tempera-
(Illustra; GE Healthcare). The primers PsF311-PsR1459 ture and the soil water content adjusted to 60 % of the water-
(approx. 1,150 bp) were used under the thermocycling condi- holding capacity. Water loss was replenished with deionized
tions described by Karpouzas et al. (2011). PCR amplicons sterilized water twice a week up to the end of the experiment.
were purified using the Nucleospin Extract II kit (Macherey- Three different types of biostimulation schemes were
Nagel GmbH) and sequenced. Sequencing reactions were set employed using the following inorganic and organic mate-
up using a PRISM BigDye Terminator v3.1 Cycle Sequence rials: (1) nitrogen chemical fertilizers [i.e NH 4 NO 3 ,
reaction kit (Applied Biosystems Inc., Foster City, CA). The (NH4)2SO4 and urea (NH2CONH2)]; (2) the specified fertil-
products were resolved on an ABI 3730XL genetic analyzer izers supplemented with glucose at the rate of 0.5 g/kg of soil;
(Applied Biosystems). Chromatograms were visually (3) compost derived from winery by-products in four different
inspected with Chromas ver. 2.33 software (Technelysium soil:compost ratios (1:1, 1:0.5, 1:0.3, 1:0.1). Ten treatments of
Pty Ltd, South Brisbane, Australia), and a standard BLAST each bioremediation strategy were employed in a completely
alignment against standard sequences was conducted. randomized design and repeated three times. Inorganic fertil-
Sequences were deposited in the European Molecular izers were added to obtain a soil C:N ratio of 100:1. The ratio
 Ann Microbiol

of polluted soil to compost was calculated on a wet weight °C, respectively. n-Alkanes were quantified by comparing the
basis in all samples. Samples were collected at 0, 7, 14, 21, 32, peaks with standards of the individual compounds.
40, 47, 59, 80 and 90 DAA. The number of colony-forming
heterotrophic bacteria in soil samples was determined by
plating a series dilutions on plate count agar medium (Oxoid Results and discussion
Ltd., Basingstoke, Hampshire, UK) and incubating the inoc-
ulated plates for 72 h at 30 °C. Each dilution was plated in Isolation, identification and characterization of the degrading
duplicate. capacity of hydrocarbon-degrading Pseudomonas strains

Dissipation kinetics and statistics In our study, the Pseudomonas isolates were extracted from
the heavily polluted soil and enriched using specific culture
The modified Hockey–Stick model (FOCUS 2006) was used medium in the presence of TPH by a continuous process.
for fitting the dissipation patterns of the TPH in soils. The Bacteria were identified by 16sRNA sequencing (approx.
following equations were used: 1,150 bp), with the phylogenetic analysis of the obtained
sequences showing that the majority of isolates had a high
C ¼ C0 ; for t≤ tb similarity with Pseudomonas aeruginosa. Bacteria belonging
to Pseudomonas genus are characterized by versatile meta-
C ¼ C0 e−K ðt−tbÞ for t > tb bolic capabilities, and Pseudomonas strains are often used in
the bioremediation of aromatic compounds from heavily pol-
The TPH half-life (T½) was calculated according the fol- luted sites. Zhang et al. (2011) recently isolated a
lowing equation: P. aeruginosa strain that was able to accelerate the degradation
of TPH, which the authors attributed to the production of
T1=2 ¼ tb þ ln2=k surfactants. In total, 13 isolates demonstrated a relatively high
degradation capacity with a TPH degradation T1/2 ranging
The n-alkane dissipation patterns and the dissipation po- from 7 to 67 days. Isolates el15 and el20 exhibited the highest
tential of the isolated strains were evaluated using the first- TPH degradation capacity with T1/2 values of 12 and 7 days,
order kinetic model: respectively (Fig. 1a, b). The high TPH dissipation capacity of
isolates el15 and el20 showed high degree of similarity with
C ¼ C0 e−kt
Pseudomonas stutzeri and P. otitidis respectively (Fig. 2).
Overall, both strains exhibited the same degradation capacity
The T1/2 of each n-alkane was calculated from:
of TPH, but they did differ in their ability to dissipate different
T1=2 ¼ ln2=k n-alkane fractions with isolate el15 showing a significantly
higher dissipation rate than el20 isolate for the ΣnC15–17,
where C is the concentration of the pollutant at a given time ΣnC18–21 and ΣnC21–25 n-alkane fractions (Fig. 3).
(t), C0 is the initial concentration of the pollutant in the soil, k
is the dissipation rate of the pollutant and tb is the breakpoint at Evaluation of the bioaugmentation potential
the time that dissipation starts. of the hydrocarbon-degrading Pseudomonas strains

Chemical analysis Pseudomonas isolates el15 and el20 showing the highest
degradation potential in liquid cultures were tested for their
A modified U.S. Environmental Protection Agency method bioaugmentation potential in a diesel-polluted soil. Both iso-
(EPA 8015b) was used for the hydrocarbon analysis. In brief, lates separately and a mixture of the two isolates effectively
5 g soil was mixed with 5 g Na2SO4 and extracted three times reduced the concentration of TPH in heavily polluted soils.
with 20 mL of dichloromethane in an ultrasonic bath. The gas The dissipation of TPH in these soils, with more than half the
chromatography-flame ionizer detector (GC-FID) analyses pollutants being efficiently removed in fewer than 28 days
were performed on a Thermo Focus GC system equipped with compared to the non-inoculated control where no dissipation
a FID detector (Thermo Fisher Scientific, Waltham, MA). of TPH was seen during the duration of the study (Fig. 4). The
Compounds were separated on a DB-5 capillary column (i.d. results of the bioaugmentation experiment revealed that com-
30 × 0.32 mm i.d., film thickness 0.25 μm), and helium was pared to isolate el15, isolate el20 exhibited higher dissipation
used as the carrier gas (1 mL/min). The column temperature rates of TPH but its ability to dissipate n-alkane fractions was
was held at 50 °C for 4 min and then programmed to 250 °C at lower. This observation is in line with the findings from other
steps of 4 °C /min; the final temperature was held for 10 min. studies where different bacterial species and isolates were
The injector and detector temperatures were 240 °C and 270 found to have different dissipation potential for TPH and n-
Ann Microbiol

a C=143.5e-0.057t
(p<0.05) throughout the experiment (Fig. 5) and, consequent-
200 Isolate el15
R2 = 0.78
ly, the dissipation pattern was dependent on the substrate
180
amended into the soil. By the end of the incubation period,
160
residual TPH concentrations ranged from 2 to 46 % of the
140
TPH (mg/L)

initial amount measured in the polluted soil. In contrast, TPH

120
dissipation in non-treated soils was very low (11 % of the
100
initial amount). In all cases, dissipation of TPH could be
80
effectively (R 2 = 0.74-0.99) described by the modified
60
Hockey–Stick model. This model is used to describe dissipa-
40
tion patterns with a lag-phase where the concentration of the
20
pollutant is not constant but declines very slowly up to a
0
0 7 14 21 28 35
breakpoint (tb) (FOCUS 2006).
Days Soils that were enriched with (NH4) 2SO4, urea and
b C=150.4e-0.098t
NH4NO3, either with or without glucose (GL) supplements,
200 Isolate el20 initially showed a lag phase characterized by a negligible
180 R2 = 0.90
degradation rate (Fig. 5a, b). Urea-treated soils exhibited the
160
lowest T1/2 values among the soils treated with inorganic
140
nitrogen fertilizers. Gallego et al. (2001) evaluated bioreme-
TPH (mg/L)

120 diation techniques in situ and demonstrated that under labora-

100 tory conditions it is possible to degrade up to 90 % of diesel oil
80 using inorganic nitrogen. Brook et al. (2001) studied different
60 sources of inorganic nitrogen and observed increased hydro-
40 carbon degradation in urea-treated soils. These authors attrib-
20 uted the increased degradation rate to both ammonium avail-
0 ability and the preference of the microbial community for
0 7 14 21 28 35
Days ammonium. Microbial degradation is the major and most
Fig. 1 Degradation of total petroleum hydrocarbons (TPH) by decisive natural mechanism for the removal of petroleum
Pseudomonas isolates el15 (a) and el20 (b). Error bars Standard error hydrocarbon pollutants from the environment, and bacteria
of the mean (SEM; n=3), lines and exponential equation estimates of the are the most active microbes in terms of petroleum degrada-
first-order kinetics model where R2 is the correlation coefficient tion, acting as primary degraders of spilled oil in the environ-
ment (Rahman et al. 2003). Our findings are in line with those
alkanes (Obuekwe and Al-Zarban 1998; Del’Arco and de of previous studies that also showed the beneficial effect of
Franca 2001; Al-Mailem et al. 2010). Several studies have nutrient addition on hydrocarbon degradation. Specifically, in
also shown that a microbial consortium was able to enhance our study the total viable bacterial count at the beginning of
the dissipation of petroleum hydrocarbon fractions (Bacossa the experiment was very low (average 4.7 × 104 CFU), indi-
et al. 2010; Tahhan et al. 2011). We observed that the combi- cating that the size of the microbial population in the soil
nation of both strains exhibited the highest dissipation, with a samples was very low. In comparison, the culture-dependent
calculated TPH T½ of 14.7 days. The TPH T1/2 in soils bacterial population was significantly higher in all treatments
bioaugmented using Pseudomonas isolates el20 and el15 than in the untreated soils at the end of the incubation period
separately was 16.9 and 21.0 days, respectively. The higher [Electronic Supplementary Material (ESM) Table 1], further
dissipation capacity that was noticed in soils co-inoculated supporting the notion that nutrient addition enhanced micro-
with el20 and el15 isolates could be related to different levels bial biomass in the treated soils. Delille and Coulon (2008)
of the metabolic activities involved during degradation. reported that the population of heterotrophic bacteria in-
However, more detailed experiments are needed to reveal creased in soils heavily polluted with TPH after nutrient
the biochemical and the molecular basis underlying these addition. Other studies have shown only a transient increase
differences. of the heterotrophic bacteria population during the initial
stages of biostimulation (Bento et al. 2005).The addition of
TPH dissipation pattern and kinetics in microcosm studies one or more rate-limiting nutrients to the system has been
found to increase microbial biomass and improve the biodeg-
At the beginning of the experiment the average measured TPH radation potential (Prince 1997; Hutchinson et al. 2001;
values of the soil samples was 31,299 mg/kg, which indicated Nikolopoulou and Kalogerakis 2009). Indeed, in our study
that the soil was extremely polluted. The concentration of the total viable bacterial population of the polluted soil signif-
TPH was significantly influenced by soil amendments icantly increased after nutrient addition.
 Ann Microbiol

Fig. 2 Evolutionary relationships el21

of isolates obtained and
Pseudomonas taxa available in el2
NCBI GenBank. The optimal tree el11
with a total branch length=
1.95467765 is shown. Number el13
next to branches Percentage of el18
replicate trees in which the
el10
associated taxa clustered together
in the bootstrap test (1,000 el12
replicates) (Felsenstein 1985). 99 el7
The tree is drawn to scale, with
el19
branch lengths in the same units
as those of the evolutionary 89 el5
distances used to infer the 69 el9
phylogenetic tree. The 82
evolutionary distances were Pseudomonas aeruginosa LMG 1242T
computed using the Jukes–Cantor 79 Pseudomonas otitidis MCC10330
method (Jukes and Cantor 1969) 99 el20
and are in the units of the number 82
of base substitutions per site. All Pseudomonas alcaligenes IAM12411
positions containing alignment Pseudomonas indica IMT37 DSM 14015
gaps and missing data were
Pseudomonas nitroreducens IAM1439
eliminated only in pairwise
sequence comparisons (Pairwise Pseudomonas citronellolis ATCC 13674T
96
deletion option). Phylogenetic Pseudomonas jinjuensis Pss 14
81
analyses were conducted in
100 Pseudomonas jinjuensis Pss 26
MEGA5 software (Tamura et al.
2011) 100 Pseudomonas cremoricolorata IAM 1541
Pseudomonas fulva IAM 1541
Pseudomonas putida IAM 1236
Pseudomonas monteilii CIP 104883T
Pseudomonas stutzeri CCUG 11256
99 el15
Pseudomonas parafulva TY32McD
Pseudomonas azotoformans Ka19
98
Pseudomonas synxantha IHB B 1322
95 Pseudomonas gessardii SR10
Pseudomonas gessardii CLRI1
95 Pseudomonas synxantha IAM12356
Pseudomonas marginalis pv. marginalis
84
Pseudomonas extremorientalis KMM 3447
Pseudomonas fluorescens IAM 12022
Pseudomonas mandelii CAT10

72 Pseudomonas mandelii NW28

Pseudomonas corrugata E60
Pseudomonas thermotolerans CM3 DSM 14292
Cupriavidus necator gi173698 ( outgroup )

0.02

The T1/2 of the TPH in the fertilizer-treated soils increased variance revealed that glucose addition had a significant neg-
after glucose addition (Table 2): 14.8-, 17.1- and 16.5-fold ative effect on TPH degradation (p<0.05) and that this effect
decrease of the TPH dissipation rate was observed in was independent of enrichment with fertilizer since no inter-
(NH4)2SO4-, urea- and NH4NO3- treated soils, respectively, action was noticed (p=0.52). In addition, the estimated lag
following the addition of glucose. Two-way analysis of phase (tb) of the glucose-treated soils was significantly longer
Ann Microbiol

ΣnC15-C17
Fig. 3 Degradation of n-alkane fractions by Pseudomonas isolates el15„
(filled diamond) and el20 (filled square) and by control samples (filled
triangle). Error bars SEM (n=3) 100

than that of the soils not treated with glucose, leading to an 80

extended TPH T½. Surprisingly, and in contrast previously
reported results (Sabate et al. 2004), the presence of glucose in

% reduction
soils treated with inorganic nitrogen caused reduced TPH 60 el15
dissipation (Fig. 5; Table 2). The retardation of TPH disap- el20
pearance from glucose-treated soils may be associated with Control
40
the preferential consumption of glucose by microbes rather
than TPH. As described in other studies, the addition of
20
glucose can lead to reduced degradation rates of xenobiotics
in soil environment (Entry et al. 1993; Carmichael and
Pfaender 1997). It has been suggested that the carbon source 0
in the amendment materials must not represent an easily 0 7 14 21 28 35
Time (days)
consumed source that would preempt the degradation of the
target pollutant (LaGrega et al. 1994; Cookson 1995). This is ΣnC18-C21
further supported by the significantly shorter lag phase that
was estimated in compost-treated soils (Table 2). The amount 100
of readily degradable organic carbon in mature composted
material is relatively low due to the stability of the organic
matter of this kind of amendment. However, more detailed 80
studies are needed to examine the impact of a readily degrad-
% reduction

able organic carbon on hydrocarbon degradation. 60

Although the availability of nitrogen in fertilizer-treated el15
soils is expected to be higher, we observed a greater percent- el20
age of hydrocarbon degradation in the compost-treated soils 40
Control
throughout the experiment (Fig. 5c). The T1/2 of TPH in
compost-treated soils ranged from 13 to 43 days, with an 20
increase in the quantity of compost incorporated in soils
resulting in a significant increase in the calculated dissipation
rates. The highest dissipation rate was noted in soil treated 0
0 7 14 21 28 35
with a 1:1 soil:compost ratio (Table 2). Further analysis of the
Time (days)
dissipation data showed a positive and significant correlation
between the estimated dissipation T1/2 and the amount of ΣnC21-C25
compost incorporated in the soil (r=0.87, p<0.01). This cor-
relation is in agreement with previous reports that the addition 100
of composted material enhanced the dissipation of petroleum
hydrocarbons (Namkoong et al. 2002; Sarkar et al. 2005) and
is likely associated with an increased microbial activity and 80
biomass caused by compost addition (Park et al. 2001).
% reduction

Indeed, in our study, compost-treated soils had a higher bac- 60 el15

terial population than nitrogen-treated and untreated soils el20
(ESM Table 1). Composted winery by-products have been Control
previously proposed as an efficient substrate for pesticide 40
bioremediation (Omirou et al. 2012). Thus, the positive effect
of compost on TPH dissipation compared to inorganic nitro- 20
gen fertilizers could be attributed to compost-derived meta-
bolically active microorganisms.
TPH are mainly mixtures of long and branched chain 0
0 7 14 21 28 35
hydrocarbons (alkanes), cycloalkanes and aromatic hydrocar- Time (days)
bons. We observed differences in the degradation rates of the
 Ann Microbiol

1.2 1.2

1.0
1.0

0.8
0.8
0.6
Ct/C0

0.6
0.4 (NH4)2SO4
NH2CONH2
NH4NO3
0.4 0.2
Control

el 20 0.0
0.2 el 15 + el 20 1.2
el 15
Control
1.0
0.0
0 7 14 28
Time (days) 0.8
Fig. 4 Normalized concentration of TPH in soil after inoculation with

Co/Ct
Pseudomonas isolates el15 (inverted filled triangle) and el20 (filled 0.6
circle) separately, a combination of both isolates (open circle) and control
samples (open triangle). Error bars SEM (n=3). Ct Concentration of the 0.4 (NH4)2SO4
pollutant in the soil at a given time (t), C0 initial concentration of the NH2CONH2
pollutant in the soil NH4NO3
0.2
Control

different TPH fractions, with low-molecular fractions dissi- 0.0

pating more rapidly than high-molecular TPH fractions. The 1.2

measurements revealed that the levels of n-alkanes fell signif- 1.0

icantly over time in all treatments. The dissipation of n-
alkanes was better described using a first-order model, with 0.8

the exception of pristane and phytane, in soils treated with

0.6
NH4NO3, NH4NO3 + GL and (NH4)2SO4 +GL (Table 3).
Short chain molecules (up to nC14) were rapidly reduced 0.4
within the first 21 days. The highest dissipation rate was
calculated in the nC10–C14 fraction of soils enriched with 0.2

(NH4)2SO4 (Table 3). Of the remaining n-alkane fractions, the

0.0
highest dissipation rate was calculated in urea- and compost-
treated soils (Table 3). The effect of compost addition on n-
0 20 40 60 80 100
alkane dissipation was related to the soil:compost ratio:n- time (days)
alkane dissipation rates increased significantly with increases Soil:Compost ratio (1:0.1)
Soil:Compost ratio (1:0.3)
in the amount of compost incorporated into the soil (Table 3). Soil:Compost ratio (1:0.5)
The most persistent compounds were pristane and phytane, Soil:Compost ratio (1:1)
Control
with T1/2ranging from 17 to 58 and 18 to 77.0 days, respec-
Fig. 5 Normalized concentration of TPH in soil after the addition of
tively. The dissipation of pristane and phytane was higher in
nitrogen chemical fertilizers (a), nitrogen chemical fertilizers with glucose
compost-treated soils than in nitrogen-treated and untreated (b) and compost at various ratios (c). Error bars SEM (n=3)
soils. During the course of the experiment there was a signif-
icant increase in the nC18/nC16 ratio and a significant decline
in the nC18/nC20 ratio in soils treated with nitrogen, indicat- significantly higher than that in non-supplemented soils
ing preferential dissipation of shorter chain length n-alkanes. (Table 3). Namkoong et al. (2002) also described n-alkane
On the contrary, no changes in the nC18/nC16 and nC18/ dissipation using a first-order kinetic model and reported
nC20 ratios were observed over time in compost-treated soils. much higher dissipation rates than those calculated in our
Glucose addition had a similar effect on n-alkane dissipation study, most likely related with the different organic material
rates as on TPH since glucose incorporation resulted in a that was used in their study, indicating the importance of this
significant reduction of the dissipation rate of the different n- factor on the dissipation potential of organic material that can
alkane fractions. The T1/2 of soils treated with inorganic be used. In nitrogen-treated soils, the nC18/C16 ratio in-
nitrogen fertilizers supplemented with glucose was creased over time whereas the nC20/C18 ratio decreased,
Ann Microbiol

Table 2 Modified Hockey–Stick dissipation kinetic parameters of total due to bioavailability (De Jonge et al. 1997). However, the
petroleum hydrocarbons in soil heavily polluted with diesel oil
disappearance of the low-molecular weight TPH fractions can
Total petroleum M0 k (days−1) tb (days)a T1/2 (days) R2b not only be attributed to their higher bioavailability
hydrocarbon (Seklemova et al. 2001). The high volatility of compounds
with a relatively small number of C atoms results in the
(NH4)2SO4 0.89 0.011 20.24 82.91 0.91
partitioning of a fraction of these compounds into the soil
NH2CONH2 0.93 0.024 13.37 42.12 0.94
gas phase. The type of n-alkane fraction and the inherent
NH4NO3 0.87 0.017 14.58 55.76 0.93
chemical properties of this fraction determine the persistence
(NH4)2SO4 +glucose 0.95 0.0074 40.75 140.11 0.74
of these compounds in the environment. We demonstrated that
NH2CONH2+glucose 0.92 0.014 22.27 70.01 0.84
the incorporation of nitrogen fertilizers and compost into soil
NH4NO3 +glucose 0.85 0.0103 30.72 98.01 0.87
resulted in a significant decrease of of the nC17:Pr and
Soil:compost ratio nC18:Ph ratios, indicating that the n-alkanes were preferen-
1:0.1 0.96 0.027 11.15 36.71 0.91 tially degraded compared with their respective isomers (ESM
1:0.3 0.89 0.037 7.73 26.37 0.99 Table 2). The preferential dissipation of n-alkanes has been
1:0.5 0.94 0.053 7.34 20.39 0.99 previously demonstrated (Seklemova et al. 2001; Coulon et al.
1:1 0.92 0.057 1.02 13.18 0.98 2005) and has been related with their susceptibility to micro-
M0, Normalized Initial amount of total petroluem hydrocarbon (TPH); tb,
bial degradation compared with pristane and phytane (Diaz
breakpoint at the time that dissipation starts; k, dissipation rate of the et al. 2002).
pollutant; T1/2, TPN half-life In summary, we have examined the efficiency of different
a
Constant rate from T=tb nutrient sources, both inorganic and organic, to stimulate the
b
Coefficient of determination of the modified Hockey–Stick model dissipation of hydrocarbons in a long-term heavily polluted
soil. Our results clearly show that the biostimulation potential
further supporting previously reported results (Coulon et al. was significantly increased in soil amended with composted
2005). In our study no significant differences were noticed material compared with soil amended with the inorganic fer-
between the treatments in terms of the dissipation rate of the tilizers tested. Alternatively, bioaugmentation of the polluted
ΣnC10–C14 fraction during the biostimulation studies. This soil with hydrocarbon-degrading Pseudomonas strains isolat-
implies that the dissipation of this fraction is also controlled by ed from the polluted soil showed high bioremediation poten-
other factors that are related with the chemical properties of tial and accelerated the dissipation of TPH and n-alkanes,
low chain hydrocarbons. It has been suggested that differences indicating the potential use of these microbial strains for in
in water solubility may result in different degradation rates situ bioremediation of hydrocarbon-polluted soils.

Table 3 First-order dissipation kinetic parameters of different n-alkane fractions in soil heavily polluted with diesel oil

n-Alkane fractions ΣnC10–C14 ΣnC15–C17 ΣnC18–C20 ΣnC21–C25 Pristane Phytane

ka R2b T1/2c k R2 T1/2 k R2 T1/2 k R2 T1/2 k R2 T1/2 k R2 T1/2

(NH4)2SO4 0.085 0.89 8.2 0.040 0.89 17.3 0.021 0.90 33.0 0.016 0.77 43.3 0.012 0.91 57.8 0.009 0.91 77,0
NH2CONH2 0.059 0.91 11.7 0.043 0.91 16.1 0.035 0.93 19.8 0.031 0.95 22.4 0.013 0.97 53.3 0.011 0.92 63.0
NH4NO3 0.056 0.84 12.4 0.027 0.87 25.7 0.018 0.77 38.5 0.017 0.84 40.8 -NA- -NA-
(NH4)2SO4 +glucose 0.049 0.86 14.1 0.026 0.92 26.7 0.017 0.75 40.8 0.016 0.71 43.3 -NA- -NA-
NH2CONH2 +glucose 0.052 0.96 13.3 0.032 0.94 21.7 0.026 0.97 26.7 0.023 0.93 30.1 0.0081 0.90 85.6 0,0074 0.91 93.7
NH4NO3 +glucose 0.053 0.93 13.1 0.023 0.91 30.1 0.017 0.89 40.8 0.014 0.85 49.5 -NA- -NA-
Soil:compost ratio
1:0.1 0.029 0.89 23.9 0.035 0.81 19.8 0.031 0.93 22.4 0.031 0.91 22.4 0.012 0.96 57.8 0.011 0.91 63.0
1:0.3 0.049 0.87 14.1 0.049 0.84 14.,1 0.045 0.87 15.4 0.030 0.90 23.1 0.019 0.94 36.5 0.021 0.92 33.0
1:0.5 0.051 0.90 13.6 0.047 0.91 14.,7 0.037 0.93 18.7 0.046 0.89 15.1 0.034 0.91 20.4 0.030 0.93 23.1
1:1 0.062 0.92 11.2 0.066 0.87 10.5 0.051 0.94 13.6 0.046 0.94 15.1 0.041 0.89 16.9 0.038 0.97 18.2

NA, Not available

a
Dissipation rate of n-alkanes fractions as estimated by first-order kinetics model
b
Coefficient of determination of the first – order kinetic model
c
Half-life of n-alkane fractions
 Ann Microbiol

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