1. Appl Microbiol Biotechnol
DOI 10.1007/s00253-009-1868-0
ENVIRONMENTAL BIOTECHNOLOGY
Enhancement of the microbial community biomass
and diversity during air sparging bioremediation of a soil
highly contaminated with kerosene and BTEX
Nadja Kabelitz & Jirina Machackova & Gwenaël Imfeld &
Maria Brennerova & Dietmar H. Pieper &
Hermann J. Heipieper & Howard Junca
Received: 6 October 2008 / Revised: 9 January 2009 / Accepted: 10 January 2009
# Springer-Verlag 2009
Abstract In order to obtain insights in complexity shifts quantities and composition of the microbial communities
taking place in natural microbial communities under strong developed at different stages of the bioventing treatment
selective pressure, soils from a former air force base in the progress was performed. Depending on the length of the air
Czech Republic, highly contaminated with jet fuel and at sparging treatment that led to a significant reduction in the
different stages of a bioremediation air sparging treatment, contamination level, we observed a clear shift in the soil
were analyzed. By tracking phospholipid fatty acids and microbial community being dominated by Pseudomonads
16S rRNA genes, a detailed monitoring of the changes in under the harsh conditions of high aromatic contamination
to a status of low aromatic concentrations, increased
biomass content, and a complex composition with diverse
Electronic supplementary material The online version of this article
bacterial taxonomical branches.
(doi:10.1007/s00253-009-1868-0) contains supplementary material,
which is available to authorized users.
Keywords BTEX . Air sparging . Bioremediation .
N. Kabelitz : H. J. Heipieper (*)
Department of Bioremediation, Biodiversity . Microbiota
Helmholtz Centre for Environmental Research (UFZ),
Permoserstr. 15,
04318 Leipzig, Germany Introduction
e-mail: hermann.heipieper@ufz.de
J. Machackova The spillage of organic compounds represents one of the
Earth Tech CZ s.r.o., biggest problems of contamination in soils and ground-
Trojská 92, water, especially in eastern European countries. Military
171 00 Prague 7, Czech Republic
areas particularly represent a major problem due to their
G. Imfeld high pollutant concentration. Therefore, massive attempts
Department of Isotope Biogeochemistry, are being carried out to remediate such sites, commonly
Helmholtz Centre for Environmental Research (UFZ), highly polluted with alkanes and benzene, toluene, ethyl-
Permoserstr. 15,
04318 Leipzig, Germany benzene, and xylene (BTEX) compounds. One of the in situ
bioremediation technologies directed toward volatile hydro-
M. Brennerova carbons, mainly BTEX and gasoline relying on the aerobic
Institute of Microbiology (IMIC), Czech Academy of Sciences, stimulation of the catabolic capabilities of the autochtho-
Videnska 1083,
142 20 Prague 4-Krc, Czech Republic nous bacteria, is air sparging (Marley et al. 1992; Reddy et
al. 1995; Bass et al. 2000; Hall et al. 2000; Heron et al.
D. H. Pieper : H. Junca 2002; Yang et al. 2005). However, despite the wide
Biodegradation Research Group, application of this technique to enhance the bioremediation
Helmholtz Centre for Infection Research (HZI),
Inhoffenstrasse 7, of nonchlorinated aromatic contamination in situ, there is
38124 Braunschweig, Germany still a scarcity of knowledge on the biocatalysts being

2. Appl Microbiol Biotechnol
stimulated and the overall microbiological characteristics of site. Several principal source zones of petroleum pollution
the process. In contrast, bioremediation studies often used were identified at the site which has a size of 28.3 ha—
to be restricted to follow the disappearance of hazardous three storage areas and the jet-fuelling depot. The pollutants
pollutants (Frostegard et al. 1993a; Frostegard et al. 1996) migrated significant down-gradient distances due to more
and still regard this system as a black box. As it is known than 20 years of massive fuel leakages in source areas and
that traditional culture-dependent methods are highly biased high permeability of sandstones. The amount of total petrol
when analyzing environmental samples (Amann et al. hydrocarbon (TPH) released into soil and groundwater until
1995), culture-independent methods have been applied 1997 is estimated as 7,150 t. At the start of the treatment,
since two decades in order to characterize microbial light nonaqueous-phase liquid (LNAPL) phase was fre-
community structures and their shifts under changing quently present in the wells with a thickness >0.5 m. The
environmental conditions. Lipid biomarker-based techni- pollution consisted mainly of jet fuel (70%) with admixture
ques (Guckert et al. 1991; White 1993; Frostegard et al. of gasoline and diesel.
1996; White et al. 1996; Zelles 1997; MacNaughton et al. Figure 1 shows a scheme of the Hradčany site and the
1999) provide culture-independent insights into several clean-up procedure carried out since 1997, when in situ
important characteristics of microbial communities such as technologies have been gradually applied. LNAPL soil
viable biomass, community structure, nutritional status, or vapour extraction (SVE) and air sparging (AS) with
physiological stress responses of the bacteria (Guckert et al. application of nutrient solutions (N, P, and K) have been
1991; Heipieper et al. 1996; Pennanen et al. 1996; applied to the site (Masak et al. 2003). The first clean-up
MacNaughton et al. 1999). However, the insight gained phase focused on maximum removal of LNAPL by vacuum
from lipid biomarker analysis primarily concerns nutritional extraction, whereas the second phase aimed at creating
or physiological status with little differentiation among favorable conditions enabling aerobic degradation in the
bacterial species. Complementary genetic methods targeting entire contaminated profile by AS and SVE. In the time
and discerning the sequence complexity of 16S rRNA frame of 1997–2006, 3,667 t of TPH were removed from
genes as a bacterial taxonomical biomarker allow the the site and it was estimated that biodegradation accounts
monitoring of taxonomical shifts in microbial community for 93%, vacuum extraction of LNAPL for 5%, and SVE/
structure at greater details (Janssen 2006). AS for 2% of the TPH amounts eliminated (Machackova et
The present study shows the monitoring of a former air al. 2005). In this study, the development of microbial
force base in the Czech Republic highly contaminated with communities in samples taken from three locations of that
jet fuel that is currently under bioremediation by the air site representing different stages of the treatment progress
sparging technique (Bass et al. 2000; Hall et al. 2000). The was studied using microbial community analyses by
site is a part of the Bohemian Cretaceous Basin, the most phospholipid fatty acid (PLFA) profiling and 16S rRNA
important resource of high-quality groundwater in the gene library analysis.
Czech Republic (Masak et al. 2003; Machackova et al. The results presented in this polyphasic approach show
2005). The endangered aquifer is the only source of links between the depletion of contaminants (in this case, a
drinking water in the region and the presence of extensive strong selector) in natural setups due to oxygen amendment
contamination limits future use and revitalization of the and an increase of the abundance and complexity of the
Fig. 1 Schematic representation
of the clean-up procedure
carried out since 1997 at
Hradčany site (AS air sparging,
VE venting, GWT ground
water table)

3. Appl Microbiol Biotechnol
autochthonous microbial soil community. Most probably, the previously lyophilized for 24 h and was carried out using
observed changes in the microbial community are related accelerated solvent extraction in an ASE 200 apparatus
and associated with the successful remediation of the soil. (Dionex), allowing an efficient extraction of lipids from
soils under high temperatures and pressures. Methanol,
chloroform, and buffer were applied in ratios described by
Materials and methods Bligh and Dyer (1959).
For the extraction, from each of the samples, 30 g of soil
Sampling were lyophilized and filled in an extraction cell (volume=
22 mL) together with the mix of solvents, heated for 5 min,
All samples were taken from a site of high kerosene and pressurized to 120 bar. The temperature and pressure
contamination, located in the Czech Republic, referred here were kept constant for 10 min (static extraction, two
as the Hradčany site. Since the Second World War until cycles). The total amount of solvent used for each cell
1990, the site was used as a military airport, and the was about 25 mL. The extracts were collected and
military activities resulted in an extensive contamination of separated by addition of appropriate volumes of distilled
the soil and groundwater by petroleum products (mainly by water and chloroform. The chloroform phase, which
jet fuel). The upper layers of the site (0.5–3 m) are formed contained the total fatty acids, was isolated and dried over
by quaternary river sediments (sands, gravels); the aquifer anhydrous sodium sulfate. The PLFA fraction was separat-
is composed of middle- to fine-grained Middle Turonian ed by liquid chromatography using silica gel columns
sandstone with a thickness of 67 to 75 m. The base of the (Bakerbond spe, Baker). By subsequent elution with
aquifer consists of Lower Turonian siltstones and marlites chloroform, acetone, and methanol, neutral glycolipids
with a thickness of about 75 m. The groundwater table and phospholipids were collected separately according to
depth varies from 3 to 8 m below the surface. In 1997, a Zelles (1997). The methanol fraction containing the PLFA
full-scale clean-up was initiated (Masak et al. 2003; was transesterified to the respective fatty acid methyl esters
Machackova et al. 2005). Soil samples were taken using (FAMEs) with trimethylchlorosilane in methanol (1:9, v/v)
spiral auger drilling technique. The actual level of the at 60°C for 2 h. The solvent was evaporated under a gentle
groundwater table (GWT) was measured prior to drilling in stream of nitrogen, and residues were resuspended in
the adjacent monitoring point for preliminary setting of hexane.
sampling depth. The three sampling sites are located within
the Hradcãny area (approximately 30 ha) with a reasonable Analysis of fatty acid composition by GC-MS and GC-FID
distance of several hundred meters between each other
(HRB-3: highest contamination, beginning of clean-up; Analysis of FAMEs in hexane was performed using a
HRB-2: 2.5 years of treatment; HRB-1: 5 years of quadruple GC system (HP8690, Hewlett & Packard, Palo
treatment). Samples were taken in the depth of 0.5 m Alto, USA) equipped with a split/splitless injector. A BPX-
above–1 m under the actual GWT level from the 0.2-m 5 capillary column (SGE, Darmstadt Germany; length,
layer of maximum contamination. All samples represent 30 m; inner diameter, 0.32 mm; 0.25 μm film) was used
very similar soils, both from the geological (soil scientific) for separation where the injector temperature was held at
and hydrogeological aspects. From each of the three sites, 240°C. The injection was splitless and He was used as
approximately 2 kg of soil were taken. The soil of each site carrier gas at a flow of 2 mL/min. The temperature program
was then homogenized in a sterile bucket and then packed was: 40°C, 2 min isothermal; 4°C/min to 230°C; 5 min
into glass jars and stored at 4°C under aerobic conditions. isothermal at 230°C. The pressure was held constant at
Sampling for petroleum hydrocarbon quantification was 7,57 psi. Additionally, a GC system with flame ionization
performed prior to soil mixing as petroleum contamination detector was used (Agilent 6890N) with a special FAME
quickly volatilizes during homogenization. Two split column (CP-Sil88 Varian Chromopack; length, 50 m; inner
samples for contamination content analyses were taken diameter, 0.25 mm; 0.2 μL film) to reach better separation.
from the sampled interval. Content of TPH was measured The pressure program was as follows: start, 27,64 psi for
by standard gas chromatography and infrared detection 2 min; increase, 0.82 psi/min up to 45.7 psi; isobaric for
(ISO TR 11046 and ISO TR11046[2]); BTEX was analyzed 5.5 min. The temperature program started at 40°C (2 min),
by standard gas chromatographic methods (EPA 601). increased 8°C/min up to 220°C, and was held there for
5 min. Injector temperature was 240°C, detector temperature
Fatty acid extraction and separation 270°C.
The peak areas of the carboxylic acids in total ion
The extraction of total fatty acids was performed with the chromatograms (TIC) were used to determine their relative
soil samples (five split samples of each site) that were amounts. The fatty acids were identified by their mass

4. Appl Microbiol Biotechnol
spectra and retention time compared to coinjections of (synthesized by Invitrogen, Karlsruhe, Germany). For
authentic reference compounds obtained from Supelco thermal cycling, a Hybaid PCR Express Thermocycler
(Bellefonte, USA). (Thermo Electron, Waltham, MA, USA) was used as
follows: initial denaturation at 94°C for 4 min, 35 cycles
Statistical analysis of 95°C for 45 s, 55°C for 45 s, and 72°C for 1.5 min.
These cycles were followed by one elongation step at 72°C
Principal component analyses (PCA) were applied on the for 7 min. PCR products were purified by using the
basis of numerical data matrices converted using the QIAquick PCR purification kit (Qiagen, Hilden, Germany)
program R (R: Copyright 2005, The R Foundation for and cloned in pGEM-T easy vector system (Promega,
Statistical Computing Version 2.1.1). The relative amounts Madison, WI, USA). Plasmid inserts were amplified by
of PLFA data were subjected to PCA to investigate the PCR with vector-specific M13 forward and reverse primers
interrelationships between the soil samples and to deter- (Sambrook et al. 1989) on transformant colonies dissolved
mine the predominant PLFA species in the samples. In the in water and previously incubated at 95°C for 10 min.
first attempt, the investigated soil samples corresponded to Amplified ribosomal DNA restriction analysis (ARDRA)
the object represented in the multidimensional space and was performed as previously described (Junca and Pieper
the PLFA values to the descriptors of the multivariate 2004). The purified PCR products were used as DNA
analysis. In a second PCA, reciprocal analysis was carried templates in independent sequencing reactions of both
out with the soil samples corresponding to the descriptors strands using the BigDye terminator v1.1 cycle sequencing
of the analysis. kit (Applied Biosystems, Foster City, CA, USA) using M13
primers and primers annealing at four different conserved
DNA extraction regions in two directions inside the 16S rRNA gene
sequences as described previously (Lane 1991). Sequencing
For DNA extraction, fractions of the soil samples were reactions were analyzed in an Applied Biosystems 3130xl
frozen with solid carbon dioxide at the time of sampling Genetic Analyzer (Applied Biosystems, Foster City, CA,
and maintained in this condition during transportation. USA) and sequence contigs were assembled using
Later on, samples were stored at −70°C until further Sequencher version 4.0.5 (Genes Codes, Ann Arbor, MI,
processing. DNA was extracted with the FastDNA Spin USA). The sequences were cleaned of vector sequences
kit for soil (QBiogene, Carlsbad, CA, USA) from 800 mg using VecScreen Blast program (NCBI, USA) and oriented
of soil per reaction tube, according to the instructions of the in 5′–3′ of the 16S rRNA genes using OrientationChecker
manufacturer with the only exception that the final elution (Bioinformatics Toolkit, Cardiff School of Biosciences,
of DNA from the filter was with 75 μL of Tris–HCl buffer UK). Sequences were analyzed for potential chimeric
3.33 mM pH 8.0. Five DNA extractions, equivalent to 4 g sequences with the service available at the Ribosomal
of soil, were performed for each soil sample, and extracted Database Project II (Cole et al. 2003). Additional potential
DNA were pooled together in a single reaction tube. The chimeras were assessed with the program MALLARD
DNA was dried and the final volume adjusted to 40 μL (Ashelford et al. 2006). The final datasets were aligned
with MilliQ water. DNA concentrations were quantified with the multiple sequence alignment method MUSCLE
using the Quant-iT PicoGreen dsDNA quantitation kit (Edgar 2004). A block of sequence alignments was selected
(Invitrogen—Molecular Probes Europe BV, Leiden, The with GeneDoc multiple sequence alignment editor software
Netherlands). (Nicolas 1997). A collection of the nearest neighbors to the
sequences obtained against the 16S rRNA gene sequences
PCR amplification, cloning, sequencing, and analyses reported and classified in the Ribosomal Database Project II
were found using Seqmatch (Cole et al. 2003). Neighbor-
The pooled DNA extracts were used as template in joining trees were calculated from the composite align-
polymerase chain reaction (PCR) amplifications with ments together with calculated bootstrapped values of 1,000
primers targeting two highly conserved regions identified trials using the functions implemented inside Clustal W
on bacterial 16S rRNA genes (Marchesi et al. 1998) [63F: (Thompson et al. 1994). Tree files were graphically
5′-CAG GCC TAA CAC ATG CAA GTC-3′ and 1387R: displayed with MEGA 3.1 software (Kumar et al. 2004).
5′-GGG CGG WGT GTA CAA GGC-3′]. The final For calculation of rarefaction curves and Shannon diversity
amounts or concentrations of the reagents for PCR in a indexes, the program DOTUR was used (Schloss and
volume of 50 μL were: 1X colorless GoTaq reaction buffer Handelsman 2005) using the distance matrices computed
(Promega, Madison, WI, USA), 5 U of GoTaq polymerase with Dnadist program (Felsenstein 1989) from the nucleo-
(Promega, Madison, WI, USA), 200 μM of dNTPs (MBI tide sequence alignments of the sequence libraries obtained
Fermentas, Germany), and 10 pmol of each primer in this study.

5. Appl Microbiol Biotechnol
Microbiological culture techniques from HRB-3 were even lower and only detectable after
PCR amplification. Thus, compatible results were obtained
Fractions of the soil samples were kept at 4°C after when comparing, as biomarkers, total DNA extract con-
collection. Colony forming units were determined in R2A centrations, CFUs, or PLFA concentration, which all point
agar (Difco, Livonia, MI, USA) in triplicates after plating to an increase in living microbial biomass.
of the appropriate dilution that were carried out on
phosphate buffer (50 mM, pH 7.0). Phospholipid fatty acid composition of Hradčany soils
The PLFA composition of Hradcãny samples (Fig. 2b) showed
Results significant differences depending on the time of air sparging
treatment. In the untreated samples, saturated fatty acids
Biomass development in the Hradčany soil (16:0, 18:0) are predominant next to 18:1Δ9cis fatty acid.
The major difference in the PLFA profiles between the three
Soil samples from the Hradčany site representative for investigated soil sampling sites was the significantly higher
different steps of the air sparging bioremediation process relative abundance of 16:1Δ9cis and 18:1Δ11cis monoun-
were investigated for pollutant content. In general, the three saturated fatty acids as well as cyclo19:0 cyclopropane fatty
sites can be characterized as follows: HRB-3, soil from an acid in the treated compared to the untreated samples.
untreated site contains high organic contamination (concen- A PCA of the PLFA profiles underlined the results given
trations of TPH=6,400 mg/kg and BTEX=4,400 mg/kg dry above. The first PCA (Fig. 3a) allowed to clearly
weight); HRB-2, soil from a 3-year clean-up site exhibits a distinguish PLFA patterns associated with the soil from
moderate organic contamination (TPH=3,900 mg/kg and three sites differing in the level of BTEX and kerosene
BTEX=190 mg/kg dry weight); and HRB-1, soil from a contamination and treatment duration. The data of all the
5.5-year clean-up site contains low organic contamination three different sampling sites formed distinct clusters. This
(TPH=1,500 mg/kg and BTEX=9 mg/kg dry weight). From PCA showed a clear separation of the three conditions on
each site, five samples were investigated which were taken in the biplot of the first two principal components, emphasiz-
the same drilling campaign. As the geological and hydro- ing changes in the PLFA composition of the soils according
geological specificities of all three sites were similar, the to the length of treatment. A separation of the soil samples
differences in microbiota are most likely due to the from the lowest level of contamination to the highest one is
difference in pollution level and cannot be explained by operating along the first principal component.
geological or other aspects. The second PCA stresses the dominant PLFAs that are
In order to compare the abundance and complexity of associated with the difference in the analyzed soil samples
microbial biomass of the samples subject to different times (Fig. 3b). The amount of variation explained by the first
of air sparging treatment, the overall abundance of PLFA and second principal components reached 86.3% of the
was analyzed (Fig. 2a). On the other hand, in the nontreated total variation. This PCA relates the abundance of specific
soil, which contains very high toxic concentrations of PLFAs (16:0; 18:0; 18:1cisΔ9) with the level of contam-
BTEX compounds, PLFAs were only present in very low ination (prior clean-up, HRB-3). On the other hand, other
amounts; this content increased by more than two orders of PLFAs (18:1cisΔ11, cyclo19:0, 16:1Δ9cis) increase in
magnitude in the air sparging treated soils. As PLFA are response to the treatment time and were particularly
only present in living (micro)organisms (MacNaughton et associated with the 3 years treated (HRB-2) samples. The
al. 1999; Kindler et al. 2006), this is a clear indication that other PLFAs were found in a close vicinity of the origin of
this bioremediation treatment leads to a significant increase the PCA plot, indicating that the relative amounts of these
in overall microbial biomass. The increase in biomass was PLFAs were not substantially affected by the level of
also reflected in strong differences between the soil samples contamination and the length of treatment.
regarding quantities of heterotrophic bacteria as quantified
by the number of colony forming units per gram of soil Molecular biological analysis of the microbiota
(CFU/g) (Fig. 2a) with HRB-3 exhibiting CFUs/g two composition of Hradčany soil
orders of magnitude lower than HRB-1. Analysis of DNA
concentrations by fluorescence quantification (see the To correlate shifts in lipid composition occurring at the
“Materials and methods” section) revealed a concentration investigated site with changes in bacterial taxonomical
of 40 ng dsDNA per gram of HRB-1 soil, whereas DNA complexity, the microbial community structures of the three
from HRB-2 was observable after gel electrophoresis but sampling points were assessed by 16S rRNA gene libraries.
below the concentration of 0.5 ng/μL dsDNA which could PCR clone libraries of 16S rRNA gene were generated from
be accurately quantified. Amounts of DNA extractable total pooled DNA extracts of the soils and initial screenings

6. Appl Microbiol Biotechnol
Fig. 2 Effect of air sparging
treatment on PLFA abundance
a 1.E+07 1.E+08
and composition in Hradčany
soils. a Biomass development,
Overall abundance of PLFAs (mA)
given as the overall abundance 1.E+07
of PLFA (filled diamonds, rep-
resented by area counts) and 1.E+06
CFUs (cell g-1)
colony forming units (open 1.E+06
squares) in the Hradčany site
caused by the air sparging treat-
ment. b PLFA patterns of soils
from the Hradčany site. No 1.E+05
treatment (HRB-3), 1.E+05
total BTEX concentration=
17,000 mg/kg; 3 years treatment 1.E+04
(HRB-2), total BTEX concen-
tration=960 mg/kg; 5.5 years
treatment (HRB-1), total BTEX
concentration=70 mg/kg. From 1.E+04 1.E+03
0 1 2 3 4 5 6
each sampling point, five
independent soil samples were Years of air sparging treatment
extracted and analyzed for their b 50
PLFA content. Standard devia-
tion of these five independent 45
measurements are given as
error bars 40
35
PLFA (%)
30
25
20
15
10
5
0
:0
:0
:1 :0
:0
ns 0
19 :0
:0
o
o
17 is
0
0
1
:0
16 s
:1 d 9
:1 1
n
cl
cl
:
5:
6:
d1
d1
c
14
15
16
17
18
19
20
15
tra
cy
cy
i1
i1
18 i s
:1
s
c
a
ci
tr a
16
18
:1
18
5.5 Years Treatment 3 Years Treatment No Treatment
performed by ARDRA. For HRB-3, ARDRA screening supplementary material). A global alignment of the
with AluI on 96 clones showed identical patterns in 82 of sequences obtained together with the most closely related
the clones, suggesting the predominance of a single 16S rRNA gene sequences from type strains and selected
taxonomic group in the library. A similar ARDRA sequences retrieved from public databases indicated the
screening on HRB-1 and HRB-2 clone libraries did not presence of sequences related to diverse evolutionary
give evidence for any predominant pattern. Further screen- branches (Janssen 2006). The clones in the clone libraries
ing by random sequencing was performed on 79 clones were assigned to operational taxonomic units (OTUs) using
from HRB-1, 80 clones from HRB-2, and 28 clones from >99% (OTU0.01), >95% (OTU0.05), and >90% (OTU0.1)
HRB-3. The relationships of 187 assembled sequences, sequence identity as criteria, as sequences with greater than
comprising a common 1-kb length block covering variable those identities are typically excluding differences based on
regions V2 to V6 of the 16S rRNA genes (Neefs et al. operon heterogeneity or are typically assigned to the same
1993), corresponding to positions 103 to 1130 of Escher- genus or order, respectively (Acinas et al. 2004b; Schloss
ichia coli 16S rRNA gene (GenBank accession number and Handelsman 2004).
J01695), are shown in Fig. 4a (expanded view and detailed Rarefaction analysis on each sequence library showed
labeling of these results are available as Electronic that the higher number of clones sequenced from the HRB-

7. Appl Microbiol Biotechnol
Fig. 3 PCA analyses of PLFA
a cyclo 19:0
0.4
patterns obtained from 18:1cis∆11
Hradčany soils. a Ordination
plot representing the relationship
between the contamination sites
and the PLFA patterns. The
19:0
cross indicates the origin of HRB-2
0.0
coordinates and values on the
5
cyclo17:016:0
i16:0
axes indicate the percentage of
16:1trans 14:0
the total explained variation. b GGG a15:0
G 16:1cis 17:0 18:0
PCA showing loading values for 20:0 18:1cis∆9
18:1trans 18:1cis
individual PLFA. PLFAs found
i15:0 15:0
on the right in the plot had
-0.4
PC 1 (26.6%) 16:0
increased in the no treatment
-0.4 0.0 0.4
(HRB-3) soils, whereas the ones
found in the lower part of the L
0
L L
plot had increased the 5.5-year L
II
Iu
u
treatment (HRB-1) and 3-year u II L
treatment (HRB-2) soils
HRB-1 HRB-3
-5
-
-4 -2 0 2
PC 1 (41.6%)
-4 -2 0 2 4
b
4
0.4
2
18:1cis∆9 18:0
20:015:0
PC 2 (34.3%)
cyclo19:0
itrans
16:0
14:0
18:1trans
i 15:0
cyclo17:0 HRB-3
0.0
a15:0
0
17:0
16:0
16:1cis
-2
-0.4
cyclo19:0
HRB-1
HRB-2
-4
18:1cis∆11
- 0 .4 0.0 0 .4
PC 1 (52.0%)

8. Appl Microbiol Biotechnol

9. Appl Microbiol Biotechnol
ƒFig. 4 Taxonomical distribution of the 16S rRNA gene sequences Discussion
retrieved from the contaminated soils DNA under different bioreme-
diation treatments. a Neighbor-joining tree based on 16S rRNA gene
sequences obtained from HRB soil DNA amplifications. Circles The air sparging treatment of the Hradcãny site caused a
indicate sequences obtained by random screening of PCR clone significant increase in the amount of biomass and, at least
libraries of amplifications from DNA extracts of HRB-1 (blue), HRB- partially as a consequence, a decrease in organic contam-
2 (green), and HRB-3 (orange) soils. Light purple trapezoids indicate
ination of the soil. The increase in living microbial biomass
sequences of closely related bacterial type strains or cultured strains.
In cases where sequences with a similarity higher than 60% to an could be shown by us; compatible results were obtained
observed HRB-derived sequence were not available from bacterial when comparing, as biomarkers, total DNA extract con-
type strains, sequences from uncultured bacteria were included for centrations, CFUs, or PLFA concentration, which all point
orientation (violet triangles). Rarefaction curves for different distance
levels (OTU0.01, OTU0.05, and OTU0.10) for each of the analyzed
to an increase in living microbial biomass.
libraries were calculated by DOTUR (Schloss and Handelsman 2005) The major difference in the PLFA profiles between the
and are given below the dendrogram. Coverages (C) at 95% distances three investigated soil sampling sites was the significantly
were calculated according to Turing’s formula (Good 1953) where C= higher relative abundance of 16:1Δ9cis and specifically
100 represents complete coverage. b Relative clone frequencies in
18:1Δ11cis monounsaturated fatty acids in the treated
major phylogenetic groups (Order–Class) of the clone libraries
analyzed. HRB-derived 16S rRNA gene sequences were assigned to compared to the untreated samples. As cis-vaccenic acid
bacterial classes using the RDP-naïve Bayesian classifier according to (18:1Δ11cis) is synthesized via the so-called anaerobic
the taxonomical hierarchy of Garrity and Lilburn (release 6.0) with the pathway of fatty acid synthesis that is exclusively present in
default confidence threshold of 80%. The colors used in the stack
several Gram-negative bacteria (Keweloh and Heipieper
column diagram correspond to bacterial Orders as defined to the right
of the columns. Orders were grouped as Classes as shown to the very 1996), this indicates the high abundance of Gram-negative
right of the figure, except for Bacillales and Clostridiales that, for bacteria in the treated samples. The predominance of Gram-
simplicity, were grouped in the higher rank (phylum) of Firmicutes. negative bacteria in the treated samples is further supported
Sequences that could not be classified and that were retrieved only in
very low amounts were collectively indicated as “Unclassified
by the high abundance of Gram-negative-specific cyclo-
ribosomal genes.” For additional details of the sequences obtained propane fatty acids (cy17:0 and cy19:0) and the low
and control sequences used in this figure, see the accompanying abundance of Gram-positive-specific iso- and anteiso-
Electronic supplementary material branched fatty acids (i15:0, a15:0, i16:0, and i17:0).
However, inspection of discriminatory fatty acids shows
1 and HRB-2 libraries were indeed necessary to obtain the presence of Gram-negative-specific cyclopropane fatty
coverage comparable to the one for the HRB-3 library (see acids accounting for approximately 5% up to 40% of the
Fig. 4). total fatty acids, whereas abundance of Gram-positive-
Almost all the sequences obtained from HRB-3 were specific acids was negligible. This indicates also the
tightly clustering (>95% overall sequence similarity) inside untreated site to be dominated by Gram-negative organ-
the genus Pseudomonas with the majority of these sequences isms, and treatment to exert a significant effect on the
closely related, but not identical (identities >1,022/1,030, composition of the Gram-negative microbial community
99%), to those found in Pseudomonas cedrina or Pseudo- fraction. In fact, taking the combined abundance of the
monas azotoformans type strains inside the Pseudomonas Gram-positive-specific fatty acids and the Gram-negative-
fluorescens group (Anzai et al. 2000). Such clusters of specific fatty acids as a measure of the ratio between Gram-
sequence microdiversity in ribosomal genes are commonly positive and Gram-negative bacteria (Margesin et al. 2007),
observed in amplifications of environmental samples (Acinas the relative abundance of Gram-positive organisms was
et al. 2004a); however, its interpretation and significance is highest in the 5.5 years treated soil. The absence of
still under discussion. Nevertheless, it is very likely that, due polyunsaturated fatty acids shows that eukaryotes are
to a strong selection caused by the hazardous environmental practically absent in these soils, which indicates that a
conditions, only members of a bacterial genus tolerant to normal soil microflora has not been completely established
high solvent concentrations and possibly with the potential to by the so far carried out bioremediation process.
aerobically degrade such compounds were observed. HRB-2 The PCA carried out with data that stresses the dominant
exhibited a wider sequence diversification compared to the PLFAs associated with the difference in the analyzed soil
contaminated nontreated state (HRB-3). A distinct Pseudo- samples (Fig. 3b) clearly approves the tendencies visible
monas intragenus microdiversity was evidenced in this from the fatty acid profiles. The relation of specific PLFAs
sampling area with sequences highly similar to above- (16:0; 18:0; 18:1cisΔ9) with the highest level of contam-
mentioned P. cedrina/P. azotoformans cluster still being ination (prior clean-up, HRB-3) suggests that a highly
predominant (22% of clones) but 5% of the clone sequences specific microbiota is associated with these hazardous
being closely related (identities >1,016/1,030, 98%) to the environmental conditions. On the other hand, other PLFAs
recently described Pseudomonas rhizosphaerae type strain (18:1cisΔ11, cyclo19:0, 16:1Δ9cis) increase in response to
(Peix et al. 2003). the treatment time and were particularly associated with the

10. Appl Microbiol Biotechnol
3 years treatment (HRB-2) indicates a specific Gram- However, whereas there is only a small increase in the
negative bacterial community accumulating during the air Shannon diversity index from HRB-2 to HRB-1, both sites
sparging treatment. comprise very different microbial community compositions.
However, next to community shifts, changes in the Most importantly, Pseudomonas spp. was barely detectable
membrane fatty acid patterns of bacteria can also occur as (one out of 79 sequences) in HRB-1. In contrast, sequences
adaptive response to pollutant toxicity and environmental affiliated with Sphingomonadales, members of which had
stress conditions (Frostegard et al. 1993b; Heipieper and de been observed in various aromatic contaminated sites and
Bont 1994; Heipieper et al. 1996). Therefore, it is necessary which had been related to primary stages on polycyclic
to support the insights based on PLFA profiling also by other aromatic biodegradation (Leys et al. 2004, 2005), were
methods such as, e.g., molecular biological techniques. abundant only in HRB-1, comprising roughly 10% of the
Surprisingly, the expected trans–cis ratio of unsaturated fatty respective clone library, contrasting a single sequence in the
acids, a very useful parameter for stress monitoring in HRB-2 clone library.
bacterial cultures (Guckert et al. 1986; Guckert et al. 1991; In addition, Betaproteobacteria-affiliated sequences, a
Heipieper et al. 1992; Heipieper et al. 1996), did not show group only marginally detected (one sequence only) in
significant changes in the samples analyzed (data not HRB-2, are a significant fraction of the HRB-1 library,
shown), probably due to its transient identity. accounting for 20% of the total amount of sequences.
Although the PLFA analysis already demonstrated a shift Additional sequences exclusively observed in HRB-1
in the microbial community as well as an increase in living constitute a new branch inside the family Xanthomonada-
biomass, a detailed molecular biological analysis of the ceae (Gammaproteobacteria) with equal divergences (ap-
microbiota was necessary. Here, a clear increase in the proximately 15% of difference) against sequences from
microbial biodiversity of the site caused by the air sparging strains of the genera Frateuria and Rhodanobacter.
treatment was visible. Whereas almost all the sequences Bacterial assemblages similar to that of HRB-1 and
obtained from HRB-3 were clustering inside the genus consisting of Pseudomonas, Sphingomonas, Xanthomonas,
Pseudomonas, a tremendous increase in the identified Acidovorax, and Burkholderia sequences have been previ-
bacterial diversity occurred in the samples taken from 3 ously observed, for instance, at anthropogenic hydrocarbon-
and 5.5 years of treatment. contaminated coastal soils in Antarctica (Saul et al. 2005),
While Pseudomonas is a genus defined as ubiquitous while bacterial communities predominantly composed of
and of high environmental importance, these conclusions Pseudomonas, Sphingomonas, and Acidobacteria had been
are predominantly coming from observations using tradi- reported for instance in soil–groundwater ecosystems with
tional culture-dependent techniques (Moore et al. 2006) petroleum contamination (Popp et al. 2006).
which are generally accepted to include a severe bias As shown in Fig. 4b, a comparison of community
toward easy to culture microorganisms (Amann et al. composition at the level of bacterial classes, which is used
1995). However, our study and some other recent reports in many reports tracking shifts in microbial communities by
(Duineveld et al. 2001; Kaplan and Kitts 2004; Gerdes et al. FISH probes, T-RF sizes, or OTU definition (Pett-Ridge
2005; Popp et al. 2006; Ferguson et al. 2007) are showing and Firestone 2005; Yu et al. 2005; Watt et al. 2006; Allen
that Pseudomonas may be defined as a predominant et al. 2007; McGarvey et al. 2007) among others, would
member in communities of aerobic or microaerophilic direct to misleading conclusions as it would suggest the
ecosystems where high concentrations of crude oil are predominance and resilience of Gammaproteobacteria in
acting as a strong selector. the sites independent of the treatment. However, when
However, whereas HRB-3 sequences affiliated with comparisons are performed at the taxonomical scale Order,
Pseudomonas spp. comprise roughly 80% of all clones, only this shows to be an oversimplified assumption as strong
25% of HRB-2 clones were affiliated with that genus. Other shifts in composition inside the Order were observed. As an
predominant sequence types in HRB-2 were affiliated with example, among the Gammaproteobacterial sequences,
the classes of Actinobacteria, Acidobacteria, and Alphapro- only those affiliated with Pseudomonadales were predom-
teobacteria (predominantly members of the orders Rhizo- inant in the HRB-2 site but practically absent from HRB-1
biales and Rhodospirillales). The higher diversity observed where Xanthomonadales and Unclassified Gammaproteo-
in HRB-2 (Fig. 4) was also reflected by a higher Shannon bacteria are accounting for a relatively high amount of the
diversity index (H′, calculated for OTU0.05) of 2.67±0.25 total bacterial composition detected (>35%). This example
(95% confidence interval), compared to only 0.73±0.44 for shows how comparisons of taxonomical composition in
HRB-3. An even slightly higher value compared to HRB-2 bacterial communities should be at least at Order ranks.
was observed for HRB-1 (2.86±0.21), indicating diversity Lower resolution comparisons of Classes or even Phyla can
and balance of community composition to increase with be misleading and fail to detect significant community
bioremediation treatment time. changes.

11. Appl Microbiol Biotechnol
Integrating the information of overall microbial abun- necessarily able to degrade aromatics, but capable of
dance (Fig. 2a) with the sequences obtained (Fig. 4), it is surviving and growing in the cross-feeding mesh of
evident that even though the relative abundance of metabolites excreted from the initial biomass of degraders.
Pseudomonadales decreased in HRB-2 compared to HRB- In accordance with decreased concentrations of aromatics
3, the total number of Pseudomonadales cells per gram (and thus lower solvent stress), a higher variety of bacterial
of soil in HRB-2 is very likely by at least one order of taxonomical types and higher biomass content was ob-
magnitude higher. Thus, the initial predominance of served. This biodiversity restoration, which can be seen as
Pseudomonadales in HRB-3 may indicate a physiological an ecological succession, probably would not lead to the
advantage. These cells not only survive under these harsh same microbial composition of the soil as it was before the
conditions of low oxygen, high loads of aromatic carbon aromatic contamination occurred (Curtis et al. 2002).
pollutants, and high solvent concentrations, but obviously Here, it is shown that the bacterial community under
have been replicating under these conditions. This is in adaptation in these soils, concomitantly with the observed
accordance with culture-dependent studies on Pseudomo- degradation of the contaminants in situ, showed a dynamic
nadales (Heipieper and de Bont 1994; Sikkema et al. 1995; succession of Gram-negative bacteria with the community
Heipieper et al. 1996), which have shown members of this being initially restricted to Pseudomonadales at very low
group to be capable to replicate under harsh laboratory densities, developing an increased diversity comprising new
conditions and high solvent stress. It can thus be proposed proteobacterial types and Gram-positive bacteria. Compatible
that, at least at the site under study, Pseudomonads paved trends were observed using ordination methods, which
the road for other bacteria to be capable to replicate as showed the clear separation of the different fatty acid clusters
shown by the increased diversity observed in HRB-2. Thus, and indicated the predominance of Gram-negative bacteria
for a certain time, Pseudomonas is sharing its habitat in a able to resist the solvent concentrations at untreated
bacterial community of increasing complexity, as less contaminated soils and the diversification in samples where
restricting conditions for other phylotypes are being the treatment is applied. Future studies will focus on
generated during the clean-up process, leading to the catabolic activities in these sites and the relationship with
decrease in community predominance of Pseudomonas the phylogenetic changes observed by means of culture-
and the increase of other, previously not detectable dependent and culture-independent studies.
phylotypes. Particularly interesting is the increase in Acid-
obacterial sequence types, which are supposed to be Acknowledgments This work was supported by contract no.
003998 (GOCE) of the European Commission within its Sixth
selected in low-nutrient soil or in soil with a high amount Framework Program project BIOTOOL. We would like to thank the
of recalcitrant substrates (Torsvik and Ovreas 2002). As, excellent technical assistance of Silke Kahl.
moreover, soils with a high content of nutrients showed
positive selection for Alphaproteobacteria and specifically
Gammaproteobacteria (Amann et al. 1995), the ratio
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