Bacteria associated with Oak and Ash on a TCE-contaminated site: characterization of isolates with potential to avoid evapotranspiration of TCE

Article (PDF Available)inEnvironmental Science and Pollution Research 16(7):830-43 · May 2009with116 Reads
DOI: 10.1007/s11356-009-0154-0 · Source: PubMed
Abstract
Along transects under a mixed woodland of English Oak (Quercus robur) and Common Ash (Fraxinus excelsior) growing on a trichloroethylene (TCE)-contaminated groundwater plume, sharp decreases in TCE concentrations were observed, while transects outside the planted area did not show this remarkable decrease. This suggested a possibly active role of the trees and their associated bacteria in the remediation process. Therefore, the cultivable bacterial communities associated with both tree species growing on this TCE-contaminated groundwater plume were investigated in order to assess the possibilities and practical aspects of using these common native tree species and their associated bacteria for phytoremediation. In this study, only the cultivable bacteria were characterized because the final aim was to isolate TCE-degrading, heavy metal resistant bacteria that might be used as traceable inocula to enhance bioremediation. Cultivable bacteria isolated from bulk soil, rhizosphere, root, stem, and leaf were genotypically characterized by amplified rDNA restriction analysis (ARDRA) of their 16S rRNA gene and identified by 16S rRNA gene sequencing. Bacteria that displayed distinct ARDRA patterns were screened for heavy metal resistance, as well as TCE tolerance and degradation, as preparation for possible future in situ inoculation experiments. Furthermore, in situ evapotranspiration measurements were performed to investigate if the degradation capacity of the associated bacteria is enough to prevent TCE evapotranspiration to the air. Between both tree species, the associated populations of cultivable bacteria clearly differed in composition. In English Oak, more species-specific, most likely obligate endophytes were found. The majority of the isolated bacteria showed increased tolerance to TCE, and TCE degradation capacity was observed in some of the strains. However, in situ evapotranspiration measurements revealed that a significant amount of TCE and its metabolites was evaporating through the leaves to the atmosphere. The characterization of the isolates obtained in this study shows that the bacterial community associated with Oak and Ash on a TCE-contaminated site, was strongly enriched with TCE-tolerant strains. However, this was not sufficient to degrade all TCE before it reaches the leaves. A possible strategy to overcome this evapotranspiration to the atmosphere is to enrich the plant-associated TCE-degrading bacteria by in situ inoculation with endophytic strains capable of degrading TCE.
COST ACTION 859 PHYTOREMEDIATION RESEARCH ARTICLE
Bacteria associated with oak and ash on a TCE-contaminated
site: characterization of isolates with potential to avoid
evapotranspiration of TCE
Nele Weyens & Safiyh Taghavi & Tanja Barac &
Daniel van der Lelie & Jana Boulet & Tom Artois &
Robert Carleer & Jaco Vangronsveld
Received: 18 November 2008 /Accepted: 26 April 2009 /Published online: 29 April 2009
#
Springer-Verlag 2009
Abstract
Background, aim, and scope Along transects under a mixed
woodland of English Oak (Quercus robur) and Common
Ash (Fraxinus excelsior) growing on a trichloroethylene
(TCE)-contaminated groundwater plume, sharp decreases
in TCE concentrations were observed, while transects
outside the planted area did not show this remarkable
decrease. This suggested a possibly active role of the trees
and their associated bacteria in the remediation process.
Therefore, the cultivable bacterial communities associated
with both tree species growing on this TCE-contaminated
groundwater plume were investigated in order to assess the
possibilities and practical aspects of using these common
native tree species and their associated bacteria for
phytoremediation. In this study, only the cultivable bacteria
were characterized because the final aim was to isolate
TCE-degrading, heavy metal resistant bacteria that might be
used as traceable inocula to enhance bioremediation.
Materials and methods Cultivable bacteria isolated from bulk
soil, rhizosphere, root, stem, and leaf were genotypically
characterized by amplified rDNA restriction analysis
(ARDRA) of their 16S rRNA gene and identified by 16S
rRNA gene sequencing. Bacteria that displayed distinct
ARDRA patterns were screened for heavy metal resistance,
as well as TCE tolerance and degradation, as preparation for
possible future in situ inoculation experiments. Furthermore, in
situ evapotranspiration measurements were performed to
investigate if the degradation capacity of the associated
bacteria is enough to prevent TCE evapotranspiration to the air.
Results and discussion Between bo th tree species, the
associated populations of cultivable bacteria clearly differed
in composition. In English Oak, more species-specific,
most likely obligate endophytes were found. The majority
of the isolated bacteria showed increased tolerance to TCE,
and TCE degradation capacity was observed in some of the
strains. However, in situ evapotranspiration measurements
revealed that a significant amount of TCE and its
metabolites was evaporating through the leaves to the
atmosphere.
Conclusions and perspectives The characterization of the
isolates obtained in this study shows that the bacterial
community associated with Oak and Ash on a TCE-
contaminated site, was strongly enriched with TCE-
tolerant strains. However, this was not sufficient to degrade
all TCE before it reaches the leaves. A possible strategy to
overcome this evapotranspiration to the atmosphere is to
enrich the plant-associated TCE-degrading bacteria by in
situ inoculation with endophytic strains capable of degrad-
ing TCE.
Environ Sci Pollut Res (2009) 16:830843
DOI 10.1007/s11356-009-0154-0
Responsible editors: Peter Schröder, Jean-Paul Schwitzguébel
N. Weyens
:
T. Barac
:
J. Boulet
:
J. Vangronsveld (*)
Environmental Biology, Hasselt University,
Agoralaan Building D,
3590 Diepenbeek, Belgium
e-mail: jaco.vangronsveld@uhasselt.be
T. Artois
Biodiversity, Phylogeny and Population Studies,
Hasselt University,
Agoralaan Building D,
3590 Diepenbeek, Belgium
R. Carleer
Applied Chemistry, Hasselt University,
Agoralaan Building D,
3590 Diepenbeek, Belgium
S. Taghavi
:
D. van der Lelie
Biology Department, Brookhaven National Laboratory (BNL),
Building 463,
Upton, NY 11973, USA
Keywords Chlorinated solvents
.
Fraxinus excelsior
.
Phytoremediation
.
Plant-associated bacteria
.
Quercus robur
.
TCE
.
Evapotranspiration
.
Endophytes
1 Background, aim, and scope
Beneficial associations between plants and microorganisms
are important in the development of the host plant (Mastretta
et al. 2006) and its adaptation to environmental conditions. In
addition to the root zone (rhizosphere), where the microbial
biomass can be one order of magnitude or more higher than
that in bulk soil, bacteria can colonize the interior of their
host plant without causing symptoms of disease. These
endophytic bacteria, which have been found in numerous
plant species, often belong to genera commonly found in
soil, including Pseudomonas, Burkholderia, Bacillus,and
Azospirillum (Lodewyckx et al. 2002; Mastretta et al. 2006).
Like rhizosphere bacteria, endophytes can affect plant
growth and development by fixing atmospheric nitrogen
(diazotrophy; Döbereiner et al. 1995; Triplett 1996) and/or
synthesizing phytohormones and enzymes involved in plant
growth hormone metabolism, such as ethylene (e.g., 1-
aminocyclopropane-1-carboxylic acid (ACC) deaminase),
auxins, acetoin, 2,3-butanediol and cytokinins (Arshad and
Frankenberger 1991; Glick et al. 1994 ; Glick et al. 1998;
Ryu et al. 2003; Glick 2004; Kuklinsky-Sobral et al. 2004).
They can also exhibit strong antifungal activities (Hebbar et
al. 1992a, b) and antagonisze bacterial pathog ens (Coombs
and Franco 2003).
In addition to their beneficial effects on plant growth,
endophytic bacteria can be exploited for improving phytor-
emediation of organic contamina nts. The fate of organic
contaminants in the rhizosphereroot system largely
depends on their physicalchemical properties. Plants
readily take up organics with a log K
ow
between 0.5 and
3.5. These compounds seem to enter the xylem faster than
the soil and rhizosphere microflora can degrade them, even
if the latter is enriched with degradative bacteria (Trapp et
al. 2000). Once these contaminants are taken up, plants may
metabolize them, although some of them or their metabo-
lites can be toxic (Doucette et al. 1998). For example, TCE
can be transformed into TCA. Alternatively, some plants
preferentially release volatil e pollutants (such as TCE and
BTEX) and/or their metabolites into the environment by
evapotranspiration via the leaves. This raises questions
about the merits of phytoremediation (Burken and Schnoor
1999; van der Lelie et al. 2001; Schwitzguébel et al. 2002;
Ma and Burken 2003). The use of engineered endophytic
bacteria, which complement the metabolic properties of their
host, has the potential to overcome these limitations: while
contaminants move through the plants vascular system,
endophytic bacteria, colonizing the xylem (Germaine et al.
2004), can promote their degradation. This may result in
both decreased phytotoxicity and evapotranspiration, pro-
vided the bacteria have the genetic information required for
efficient degradation of the contaminants. These bacteria
can be isolated, subsequently equipped with desirable
characteristics and re-inoculated in the host plant to
enhance their beneficial effects. Proof of concept was
provided by inoculating Lupine plants (Barac et al. 2004)
and poplar cuttings (Taghavi et al. 2005) with endophytic
bacteria able to degrade toluene, which resulted in
decreased toluene phytotoxicity and a significant decrease
in toluene evapotranspiration.
Before endophyte-assisted phytoremediation of volatile
organic contaminants can be successfully applied under field
conditions, several obstacles need to be overcome (Newman
and Reynolds 2005). One major point of concern is the
persistence and stability of the engineered organisms and
their degradation capabilities in field-grown plants, as
phytoremediation projects often last for decades. Porteous
Moore et al. (2006) have investigated the diversity of
endophytic bacteria associated with hybrid poplar trees
growing on a BTEX-contaminated site. They have demon-
strated that within the diverse bacterial communities found in
poplar, several endophytic strains are capable of degrading
BTEX compounds. However, Barac et al. (2009)have
demonstrated that there only will be an advantage for those
endophytic community members possessing the appropriate
degradation characteristics as long as a selection pressure is
present. Furthermore, this selection pressure is not a
guarantee that inoculated strains equipped with the appro-
priate degradation pathway will become an integrated part of
the endogenous endophytic community. Horizontal gene
transfer has been shown to play an important role in rapidly
adapting a microbial community to a new environmental
stress factor (Dong et al. 1998; van Elsas et al. 1998;
Ronchel et al. 2000; Top et al. 2002;Deversetal.2005).
Taghavi et al. (2005) have reported the first in planta
horizontal gene transfer among poplar-associated endophytic
bacteria and demonstrated that such transfer can be used to
change natural endophytic microbial communities in order to
improve the remediation of organic contaminants.
For this work, a site was chosen where TCE was present in
the groundwater at concentrations up to 100 mg l
1
due to
former large-scale use of TCE as a degreaser during the
production of metal barrels. TCE concentrations were
determined in two transects through a small woodland of
English Oak (Quercus robur)andCommonAsh(Fraxinus
excelsior) planted about 25 years ago (Fig. 1). The presence
of this woodland seems to be responsible for a sharp decrease
in TCE concentrations (Fig. 2) along these transects, since
transects outside the planted area did not show this
remarkable decrease; concentrations there were stable around
9.5 mg l
1
. The main objectives of this work were to
Environ Sci Pollut Res (2009) 16:830843 831
investigate (1) the role bacteria that are associated with both
tree species can play in the TCE-degradation and (2) if the
natural bacterial community is sufficient to prevent evapo-
transpiration from the leaves to the atmosphere. Given that
TCE is one of the most widespread groundwater contami-
nants, and that English Oak and Common Ash are both
native tree species, it is important to explore the cultivable-
associated bacterial diversity, and its TCE degradation
capacity as part of a larger study on the potential of in situ
inoculation with (cultivable) plant-associated bacteria to
enhance phytoremediation. Additionally, in 2006, rows of
hybrid poplar trees were planted perpendicularly to the
contamination plume (see Fig. 1)inordertoaugmentthe
already existing bioscreen of English Oak and Common Ash.
2 Materials and methods
2.1 Sampling
In June 2007, the following samples were taken in order to
isolate cultivable bacteria associated with the different
compartments of English Oak and Common Ash growing
in the high TCE concent ration zone, nearby monitoring
well 1: bulk soil, rhizosphere, and roots were sampled at a
depth of 1.5 m and stored in sterile Falcon tubes (50 ml)
filled with 20 ml sterile 10 mM MgSO
4
; leaf and stem
samples were transferred in separate plastic bags. For every
compartment, samples were taken from three different trees,
and they were pooled for further analysis.
2.2 Isol ation of bacteria associated with English Oak
and Common Ash
Soil samples were diluted up to 10
7
in 10 mM MgSO
4
solution and plated on 1/10 strength 869 solid medium
(Mergeay et al. 1985) in order to isolate soil bacteria.
Rhizosphere samples were vortexed, roots were removed,
and serial dilutions up to 10
7
were prepared in 10 mM
MgSO
4
solution and plated on 1/10 strength 869 solid
medium. After 7 days incubation at 30°C, colony-forming
units (CFU) were counted and calculated per gram of bulk
or rhizosphere soil.
To isolate the endophytic bacteria, English Oak and
Common Ash samples were surface sterilized for 10 (roots
and leaves) or 5 (stems) min in a 2% (roots and leaves) or a
1% (stems) active chloride solution supplemented with one
droplet Tween 80 (Merck) per 100 ml solution, and were
subsequently rinsed three times for 1 min in sterile distilled
water. The third rinsing solution was plated on 869 medium
to check surface sterility (if no grow th was observed after
7 days, surface sterilization was considered to be success-
ful). Surface sterile English Oak and Common Ash samples
were macerated during 60 (roots and leaves) or 90 (stems)s
in 10 ml 10 mM MgSO
4
using a Polytron PR1200 mixer
(Kinematica A6). Serial dilutions were plated on 1/10
strength 869 solid media and incubated for 7 days at 30°C
before the CFU were counted and calculated per gram fresh
plant weight.
Fig. 2 TCE concentrations crossing the 2 transects through the
woodland (monitoring wells are indicated in Fig. 1) of English Oak
and Common Ash
Fig. 1 Map of the TCE-
contaminated site. The green
and the red lines indicate the
TCE European background val-
ue (0.5µg l
1
) and remediation
treshhold (70µg l
1
), respec-
tively. Sampling of English Oak
and Common Ash was done
near monitoring well 1
832 Environ Sci Pollut Res (2009) 16:830843
All morphologically different bacteria were purified three
times and plated on selective 284 medi um supplemented
with C-mix (per liter medium, 0.52 g glucose, 0.35 g lactate,
0.66 g gluconate, 0.54 g fructose, and 0.81 g succinate). The
284 medium contains per liter distilled water 6.06 g Tris
HCl, 4.68 g NaCl, 1.49 g KCl, 1.07 g NH
4
Cl, 0.43 g NaSO
4
,
0.20 g MgCl
2
×6H
2
O, 0.03 g CaCl
2
×2H
2
O, 40 mg
Na
2
HPO
4
×2H
2
O, 0.48 mg Fe(III)NH
4
citrate, 1 ml
microelements solution , final pH7. T he microe lement
solution contains per liter distilled water: 1.3 ml 25%
HCl, 144 mg ZnSO
4
×7H
2
O, 100 mg MnCl × 4 2H
2
O,
62 mg H
3
BO
3
, 190 mg CoCl
2
×6H
2
O, 17 mg CuCl
2
×
2H
2
O, 24 mg NiCl
2
×6H
2
O and 36 mg NaMoO
4
×2H
2
O.
2.3 Genotypic characterization
Total genomic DNA was extracted from the purified
bacteria (Bron and Venema, 1972). Polymerase chain
reaction (PCR) amplification of 16 S rRNA genes was
carried out in mixtures containing 100 ng μl
1
DNA,
High Fidelity PCR buffer (Invitrogen, Carlsbad, CA, USA),
0.2 mM of each of the four deoxynucleoside triphosphates,
2 mM MgCl
2
, 0.2μM each of the forward and reverse
primers, and 1 U of High Fidelity Platinum Taq DNA
polymerase (Invitrogen, Carlsbad, CA, USA) per 50μl. The
universal 1392R (5-ACGGGCGGTGTGTRC-3) and the
bacteria-specific 26F (5-AGAGTTTGATCCTGGCTCAG-
3) primers were used for prokaryotic 16 S rRNA gene
amplification. Cycling conditions consisted of: one dena-
turation cycle at 94°C for 5 min, followed by 30 cycles at
94°C for 1 min, 45°C for 45 s, and 72°C for 1.5 min, and
completed with an extension cycle of 10 min at 72°C. PCR
products were purified using the QIAquick PCR purifica-
tion columns (Qiagen, Valencia, CA, USA) and quantified
spectrophotomet rically, using the Nanodrop spectropho-
tometer (ND-1000, Isogen Life Science). The PCR prod-
ucts were directly used for ARDRA and sequencing.
For amplified 16 S rDNA restriction analysis (ARDR A),
aliquots of the PCR products were digested overnight at
37°C with 1 U of the four-base-specific restriction
endonuclease HpyCH4 IV in NEB buffer 1 (New
England Biolabs, Beverly, MA, USA). The digestion
products obtained were separated by electrophoresis in a
1.5% agarose gel, and visualized by ethidium bromide
staining and UV illumination. Bacterial strains with the
same ARDRA patt erns were grouped, and one representa-
tive strain of each group was selected for sequencing.
Purified PCR products (QIAquick column) of 16 S rRNA
genes were sequenced using the Prism Big Dye Terminator
sequencing kit (Applied Biosystems, Foster City, CA, USA)
with 100 ng of template DNA. The extended sequences were
obtained with universal primers 26F and 1392R. DNA
sequences were determined on a 16 Capillary DNA Sequencer
(Applied Biosystems, Foster City, CA, USA). Sequence
Match at the Ribosome Database Project II (http://rdp.cme.
msu.e du/index.jsp) was used for nearest neighbor and
species identification. In order to verify the identification, a
neighbor-joining analysis was performed. Prior to this
analysis, the sequences were aligned using Clustal X
(Thompson et al. 1997). A neighbor-joining tre e was
constructed with PAUP
*
4.0b10 (Swofford 2003), using
default settings. In order to assess branch supports, bootstrap
values were calculated with 2,000 pseudoreplicates.
2.4 Phenotypic characterization
Bacterial strains that displayed distinct ARDRA patterns
were screened for heavy metal resistance (these strains can
be used in future experiments to trace inoculated bacteria), as
well as TCE and toluene (as a model BTEX compound)
tolerance and degrada tion, as preparation for possible future
in situ inoculation experiments (see discussion). To test
metal resistance, all different bacteria were plated on
selective 284 medium with the addition of 1 mM nickel,
2 mM zinc, or 0.8 mM cadmium. A carbon mix (per liter
medium, 0.52 g glucose, 0.35 g lactate, 0.66 g gluconate,
0.54 g fructose, and 0.81 g succinate) was added, and
cultures were incubated at 30°C for 7 days. In order to screen
the bacteria for toluene and TCE tolerance and degradation,
strains were plated on selective medium and incubated for
7 days at 30°C in sealed 10-l vessels with addition of 600µl
toluene or TCE to have a TCE or toluene-saturated
atmosphere. To detect autotrophic strains, bacteria were also
plated on selective medium without any carbon source.
After this screening, head space gas chromatography
was used in order to confirm tolu ene and TCE degradation.
For this experiment, bacteria were grown in 40 ml Schatz
medium (Schatz and Bovell 1952) with the addition of
100 mg l
1
toluene, 100 mg l
1
TCE, or 100 mg l
1
toluene
and 100 mg l
1
TCE, and in Schatz medium supplemented
with C-mi x (per liter medium, 0.52 g glucose, 0.35 g
lactate, 0.66 g gluconate, 0.54 g fructose, and 0.81 g
succinate) and 100 mg l
1
TCE. Samples of 10 ml were taken
at the beginning of the experiment and after 3 days, and
placed in 20-ml head space vials to which 4 g Na
2
Cl was
added to stop all bacterial activity. Samples were analyzed by
head space (Teledyne Tekmar HT3) gas chromatography
(T race GC Ultra, Interscience). The volatilization of toluene
and TCE was taken into account by measuring control
samples (without addition of bacteria), and degradation was
calculated as a percentage of the nonvolatilized fraction.
2.5 In situ evapotranspiration
In June 2008, the in situ evapotranspiration was measured for
English Oak and Common Ash. The system designed for these
Environ Sci Pollut Res (2009) 16:830843 833
measurements is shown in Fig. 3. Gas sampling pumps (ADC
BioScientific) were connected to Teflon sampling bags
(Chemware Laboratory products) via Teflon tubes and
Chromosorb 106 traps. A column with CaCl
2
was placed
between the sampling bags and the Chromosorb traps to
prevent water condensation in the traps. In order to have an
inflow free of TCE, a column with CaCl
2
and Chromosorb
106 traps were also placed before the inflow of the sampling
bags. For each tree species, three measurements were
performed. Twigs with five to six leaves were placed into
the sampling bag which was made gas-tight around the twig,
and an airflow of 5 l h
1
was created for 3 h. After sampling,
the leaves were collected in plastic bags and stored at 4°C
until leaf surface area analysis. The Chromosorb traps were
analyzed by gas chromatography-mass spectrometry (GC-
MS) with an ATD400 automatic thermal desorption system,
an Auto System XLL gas chromatograph, and a Turbo mass
spectrometer (Perkin-Elmer). The amount of evapotranspired
TCE was calculated per hour and unit of leaf surface.
3 Results
3.1 Isol ation of bacteria associated with English Oak
and Common Ash
Bacteria were isolated from bulk soil, rhizosphere, root,
stem, and leaf from English Oak and Common Ash. For
both tree species, the number of cultivable bacteria
recovered was an order of magnitude higher for rhizosphere
than for soil samples (Table 1). The number of endophytic
bacteria recovered was the highest in roots and stems, and
was lower in the leaves. For both tree species, the number
of different bacterial morphotypes was the highest in the
rhizosphere, rather similar in roots and stems, and the
lowest in the leaves. For soil, the number of morpholog-
ically different species was clearly higher in association
with Common Ash than in association with English Oak.
3.2 Genotypic characterization
After purification, all morphologically different bacteria
were characterized by ARDRA using HpyCH4 IV. Closely
related strains were determined, and out of these strains,
16S rRNA genes of representative members were se-
quenced for species identification by means of Sequence
Match at the Ribosome Database Project II (Fig. 4). The
sequence match numbers marked in Fig. 4 were all (except
bacterial strain 2) higher than 0.900, which indicated that
the identification to the genus level was confident.
Moreover, in the neighbor-joining tree, strains belonging
to the same genus cluster together in distinct clades
(bootstrap values of 100%), which confirmed the results
of the 16S rRNA identification procedure. The 16S rRNA-
based identification resulted in 41 and 30 genotypically
different bacterial strains associated with English Oak and
Common Ash, respectively. We have numbered the
Table 1 The total numbers of CFU calculated per gram fresh weight
of soil, rhizosphere or plant material isolated from English Oak and
Common Ash, growing on the TCE-contaminated groundwater plume
Compartment cfu/g fresh weight
Oak Soil 14.72×10
4
(12)
Rhizosphere 37.09×10
5
(17)
Root 94.74×10
3
(16)
Shoot 28.19×10
4
(16)
Leaf 13.89×10
3
(10)
Ash Soil 19.11×10
4
(17)
Rhizosphere 14.77×10
5
(18)
Root 18.00×10
4
(14)
Shoot 28.19×10
4
(16)
Leaf 13.89×10
3
(10)
The number of phenotypically distinct colony morphologies observed
is marked in parentheses
Fig. 3 Schematic setup for
measuring in situ TCE evapo-
transpiration
834 Environ Sci Pollut Res (2009) 16:830843
different bacterial strains (numbers 156 in Fig. 4). These
numbers are further used in Figs. 5 and 6 and in Table 2.
To visualize the diversity of cultivable bacteria associ-
ated with Oak and Ash (Fig. 5), soil and rhizosphere
bacteria were distinguished from endophytic bacteria. The
relative abundance of each genotypically different strain
was expressed as a percentage of the total number of
cultivable isolates per gram fresh weight in soil and
rhizosphere (Oak, 100%=14.7×10
4
+37.1×10
5
=38.6×10
5
;
Ash, 100%=19.1×10
4
+14.8×10
5
=16.7×10
5
; see Table 1)
or inside the plant (Oak, 100%=94.7×10
3
+28.2×10
4
+
13.9×10
3
=39.1×10
4
; Ash, 100%=18.0×10
4
+28.2×10
4
+
13.9×10
3
=47.6×10
4
; see Table 1).
In the soil and rhizosphere associated with Eng lish Oak
(see Fig. 5A), 58.6% of the total number of isolates were
Actinobacteria mostly of the genera Streptomyces (48.9%)
and Arthrobacter (9.7%). Firmicutes made up 31.4% of the
total number of isolates represented by Paenibacillaceae
(18.6%) and Bacillaceae (12.8%). The remaining 10.0% of
the collection was represented by Proteobacteria with a
Fig. 4 Neighbor-joining tree of 16 S rDNA of cultivable bacteria
associated with English Oak and Common Ash growing on the TCE-
contaminated groundwater plume. On the right of the ARDRA
fingerprint, the sequence match number, the 16 S rDNA identification
(with unique number), the accession number of the closest reference
strain, and the associated tree are shown. QR Quercus robur (Oak), FE
Fraxinus excelsior (Ash). The TCE-degrading strains are marked with
a black box
Environ Sci Pollut Res (2009) 16:830843 835
Fig. 5 a Diversity of cultivable
bacteria in the soil and rhizo-
sphere associated with English
Oak; b Diversity of cultivable
endophytic strains associated
with English Oak. c Diversity of
cultivable bacteria in the soil
and rhizosphere associated with
Common Ash; d Diversity of
cultivable endophytic strains as-
sociated with Common Ash.
Central pie shows percentages
by phyla; each outer ring pro-
gressively breaks these down by
finer taxonomic levels with the
bacterial number (see Fig. 4)in
the outermost ring. Numbers in
parentheses indicate the relative
abundance, expressed as a per-
centage, of the total number of
cultivable isolates per gram
fresh weight that are present in
the soil and rhizosphere (a) and
inside (b) of English Oak, and in
the soil and rhizosphere (c) and
inside (d) of Common Ash. Pie
diagrams were generated using
sigmaplot
836 Environ Sci Pollut Res (2009) 16:830843
Fig. 5 (continued)
Environ Sci Pollut Res (2009) 16:830843 837
majority of gamma-Proteobacteria (8.3%) with Pseudomonas
as dominant genus, and 1.7% of beta-Proteobacteria. The
cultivable endophytic bacterial community associated with
English Oak (see Fig. 5B) was also dominated by Actino-
bacteria (65.1%), with Frigobacterium spp. (45.0%) and
Okibacterium spp. (13.0%) forming the majority of the
group. Arthrobacter (3.7%) and Streptomyces (2.0%) were
much less represented. Proteobacteria represented 23.1% of
the endophytic collection associated with English Oak and
were dominated by gamma-Proteobacteria (17.9%) including
Pseudomonas spp. (9%), Xanthomonas spp. (4.6%), Enter-
obacter spp. (3.4%), and Erwinia (0.8%). The remaining part
of the endophytic community associated with English Oak
were Firmicutes (11.8%) with 8.8% Bacillaceae and 3.0%
Paenibacillaceae. The compartmentalization of the dominant
endophytic taxa in the different plant parts associated with
English Oak is shown in Fig. 6A.
The community of cultivable soil and rhizosphere
bacteria associated with Common Ash (see Fig. 5C) was
dominated by Bacteroidetes, more specifically species of
Flavobacterium (36.1%), and by Firmicutes (35.0%),
including 29.4% Bacillaceae and 5.6% Paenibacillaceae.
Actinobacteria made up 18.9% of the community (9.5%
Arthrobacter spp. and 9.4% Streptomyces spp.) and
Proteobacteria represented 10.1% (5.1% Pseu domonas
spp. and 5.0% Collimonas). Proteobacteria accounted for
67.4% of the Common Ash endophyte isolates (see
Fig. 5D). Nearly all of these (67.3%) were gamma-
Proteobacteria of the genus Pseudomonas; the remaining
0.1% were identified as alpha-Proteobacterial Sinorhi-
zobium. Further, Actinobacteria made up 22.1% of the
endophytic community, including a majority of 19.9%
Streptomyces and a minority of 2.2% Arthrobacter. The
remaining part of the Common Ash associated endophytic
Fig. 6 Schematic representation
of endophytic strains as appear-
ing within the different com-
partments of English Oak (a)
and Common Ash (b). The
numbers in parentheses refer to
the bacterial strain numbers used
in Fig. 4
838 Environ Sci Pollut Res (2009) 16:830843
Table 2 Growth characteristics of cultivable bacterial strains isolated in association with English Oak and Common Ash
Strain 284 +
cmix
284 284 +
tol
284 +
TCE
284 +
cmix
+Ni
284 +
cmix
+Cd
284 +
cmix
+Zn
Strain 284 + cmix 284 284 +
tol
284 +
TCE
284 +
cmix
+Ni
284 +
cmix + Cd
284 +
cmix
+Zn
Strain 284 +
cmix
284 284 +
tol
284 +
TCE
284 +
cmix
+Ni
284 +
cmix
+Cd
284 +
cmix
+Zn
1 ++++++++––– 20 ++ ++ ++ + ––39 ++ ++ –––––
2+––––– 21 ++ ++ ++ ++ –– 40 ++ ++ + + + ––
3 ++++++++––– 22 + –––– 41 ++ ++ ++ ++ ––
4+
a
+
a
++
a
++
a
a
a
a
23 ++ + –––+ + 42 ++ ++ ++ + ++ ––
5 ++++++++––– 24 ++ ++ ++ ++ –– 43++++–––
6 ++++++++++–– 25 ++ ++ ++ ++ –– 44 ++ ++ ++ ++ –––
7 ++++++++––– 26 ++ + + + –– 45++++–––
8 ++++++++––– 27 +
a
+
a
++
a
+
a
a
a
a
46 ++ ++ ++ ++ –––
9 ++++++++––– 28 ++ ++ ++ ++ –– 47 ++ ++ ++ ++ –––
10 ++ + + + ––– 29 ++ ++ ++ ++ –– 48 ++ ++ ++ ++ –––
11 ++ ++ ++ ++ ––– 30 ++ ++ ++ + –– 49 ++ ++ ++ + ++ ––
12 ++ ++ ++ + ––– 31 ++ ++ ++ ––– 50 ++ ++ ++ + –––
13 ++ ++ ++ ++ ––– 32 + + + + –– 51 ++ ++ + ++ –––
14 ++ ++ ++ ++ ––– 33 ++ + + –– 52 ++ ++ ++ ++ –––
15 + + ––––– 34 +
a
+
a
a
++
a
a
a
a
53 ++ ++ ++ ++ –––
16++++––– 35 + –––– 54 ++ ++ ++ + –––
17 ++ + ––––– 36 ++ ++ ++ ++ + ––55 ++ ++ ++ ++ –––
18 + + ––––– 37 ++
a
+
a
++
a
++
a
a
a
a
56 + + + –––
19 +
a
+
a
++
a
++
a
a
a
a
38 ++ ++ ++ ++ ––
The numbers refer to the strain numbers used in Fig. 4 and the five strains that were selected for testing TCE and toluene degradation using head space gas chromatography are indicated with
a
.+:
growth (few colonies); ++: very good growth (many colonies)
Environ Sci Pollut Res (2009) 16:830843 839
community were Firmicutes (10.5%), comprising mainly
Bacillaceae (9.9%) , and a small fraction of Paenibacillaceae
(0.6%). The compartmentalization of the bacterial endo-
phytic community associated with Comm on Ash is pre-
sented in Fig. 6B. No typical leaf strains could be isolated
from Common Ash.
3.3 Phenotypic characterization
In a first test, all genotypically different bacteria associated
with English Oak and Common Ash were phenotypically
characterized for their heavy metal resistance and for their
tolerance to the target pollutants TCE and toluene (see
Table 2). Metal resistance was also tested because, in future
experiments, metal-resistant strains may allow an easier
tracebility. From all the isolated bacteria, six strains (6, 20,
36, 40, 41, and 49) could grow in the presence of Ni, and
one strain (23) was resistant to Cd and Zn. The screening
for TCE and tolu ene tolerance resulted in 82% of the
bacteria that could grow in the presence of TCE, and 77%
in the presence of toluene. In order to test TCE and toluene
degradation capacity, a selection was made of bacteria that
probably degrade TCE and/or toluene, based on the fact
that these bacter ia were growing better in a TCE and/or
toluene-saturated atmosphere compared to autotrophic
conditions, which suggests that they were able to use these
components as a carbon source. These bacteria (strains 4,
19, 27, 34 and 37) were screened for TCE and/or toluene
degradation. Strains 4 (Arthrobacter sp.) and 19 (Strepto-
myces turgidiscabies), both rhizosphere bacteria associated
with Common Ash, showed a 100% toluene degrada tion
capacity and a maximum TCE degradation capacity of 22%
and 14%, respectively, over a 3-day testing period in Schatz
medium wi th addition of 100 mg l
1
TCE (Fig. 7). The
other bacterial strains (27, 34, and 37) did not show any
TCE or toluene degradation capacity.
3.4 In situ evapotranspiration
In Common Ash 10.84×10
3
±1.17×10
3
ng TCE cm
²h
1
was evapotranspired to the atmosphere. The amount of
transpired TCE from the leaves of English Oak was 6.35×
10
3
±0.18×10
3
ng cm
²h
1
. Since (1) the most common
TCE degradation products in plant tissues are trichloroe-
thanol, trichloroacetic acid, dichloroacetic acid, and tri-
chloroethanol glycoside (Burken and Ma 2006) and (2)
aerobic degradation of TCE has been reported for bacterial
strains possessing the toluen e o rtho-monooxygena se
(TomA) genes with TCE -epoxide, dichloroacetate, glyox-
ylate, and formate as metabolites, these oxidative metabo-
lites were also analyzed. Although they have been
identified in controlled cell culture experiments, whole-
plant laboratory experiments, and full-scale field settings,
none of these degradation products could be detected in
these evapotranspiration measurements.
4 Discussion
Poplar trees are frequently used for phytoremediation of
groundwater contaminated with organic solvents (Schnoor
et al. 1995; Ferro et al. 1997; Barac et al. 2009), Their fast
growth and large transpiration potential make them trees
of choice for phytoremediation purposes (Schnoor et al.
1995; Shim et al. 2000). In this study, the potential of
English Oak and Common Ash and their associated
microorganisms for the phytoremediation of a TCE-
contaminated site was investigated. Since they are both
widely distributed native tree species in Europe possessing
a relatively high transpiration capacity, it seemed very
interesting to explore the diversity and reme diation poten-
tial of their associated bacterial community. Since this work
is part of a larger study on the potential role of traceable
Fig. 7 Toluene and TCE degradation tested by head space chromatog-
raphy. Bacteria were grown in Schatz medium with addition of 100 mg l
1
toluene, 100 mg l
1
TCE or 100 mg l
1
toluene and 100 mg l
1
TCE,
and in Schatz medium supplemented with C-mix (per liter medium,
0.52 g glucose, 0.35 g lactate, 0.66 g gluconate, 0.54 g fructose, and
0.81 g succinate) and 100 mg l
1
TCE. Samples were taken at the
beginning of the experiment and after 3 days. The volatilization of
toluene and TCE was taken into account by measuring control samples
(without addition of bacteria), and degradation was calculated as a
percentage of the nonvolatilized fraction
840 Environ Sci Pollut Res (2009) 16:830843
plant-associated bacteria to enhance in situ phytoremedia-
tion with an ultimate aim in situ (re-)inoculation of plant-
associated bacteria equipped with the required degradation
pathways, only the cultivable bacteria were characterized.
Cultivable bacteria were isolated from bulk soil, rhizo-
sphere, root, stem, twig, and leaf from English Oak and
Common Ash (see Table 1). For both tree species, the total
amount of bacteria in the rhizosphere was one order of
magnitude higher than in the bulk soil which can be
explained by the rhizosphere effect. The number of
cultivable endophytes was highest in roots and stems and
was lower in leaves, suggesting that colonization was
mainly taking place through the roots.
The 16S rDNA identification of all isolated bacteria that
were found to be different after ARDRA analysis resulted
in 56 different bacterial strains comprising 26 strains
exclusively associated with English Oak, 15 strains exclu-
sively associated with Common Ash and 15 strains
associated with both tree species (see Fig. 4). This indicates
that both tree species were associated with both a specific
bacterial population and a nonspecific bacterial population.
In case of English Oak, the bacterial diversity (number of
different genera) in soil and rhizosphere seemed similar to the
endophytic bacterial diversity, except that the fraction of
Proteobacteria was higher, and the fraction of Firmicutes was
lower in the endophytic community (see Fig. 5A,B). However,
a closer look at the results shows that some taxa indeed were
occurring in the bulk soil and the rhizosphere as well as
inside the plant (e.g., Arthrobacter , Str eptomyces, Bacillus,
Paenibacillus, Pseudomonas). Nevertheless, other taxa were
exclusively found inside the plant (Frigobacterium, Okibac-
terium, Curtobacterium, Aeromicrobium, Enterobacter,and
Erwinia). This suggests that the endophytic community of
English Oak might be composed of both facultative
endophytes colonizing the tree via the roots and obligate
endophytes transferred from one generation to the other
through the seeds. Furthermore, the endophytes exhibited a
marked spatial compartmentalization, suggesting that in
English Oak, some taxa of bacteria only occur in a specific
plant part (e.g., Paenibacillus), while others did not show any
compartment specificity (e.g., Pseudomonas;seeFig.6A).
As to Common Ash, a clear difference could be noticed
in the composition of the bacterial community in the soil
and rhizosphere and the endophytic bacter ial community,
since Bacteroidetes represented 36.1% of the soil and
rhizosphere bacteria, while they were completely lacking
in the endophytic bacterial community (see Fig. 5C,D).
Moreover, species of Proteobacteria dominate the endo-
phytic bacterial communi ty, while they only represented
10.1% of the soil and rhizosphere bacteria. In contrast with
the bacterial endophytes associated with English Oak, the
endophytic bacterial community associated with Common
Ash contained almost no species exclusively present inside
the plant, which might indicate that the endophytic
population consists mainly of facultative endophytes. This
hypothesis is supported by the compartmentalization of the
Common Ash associated endophytes (see Fig. 6B), since
most of the endophytes were localized in the roots and the
stems, which is typical for fa cultative endophytes
(Mastretta et al. 2006). Given that obligate endophytes
have to be transferred through the seeds, this difference in
endophyte-type between English Oak and Common Ash
can possibly be explained by both the size and the structure
of the seeds. Firstly, the seed is distinctly bigger and more
robust in case of English Oak. In addition, in seeds of
English Oak, the embryo possesses large cotyledons filling
up the entire space within the seed coat, while in case of
Common Ash, the cotyledons are tiny, and the embryos
food reserve is almost entirely locat ed in the extra-
embryonic endosperm. Due to these clear differences,
survival sites for endophytes might be different. Therefore,
in a future experiment, the seed endophytes of English Oak
and Common Ash growing on the TCE-contaminated field
site will be isolated and characterized. The importance of
seed endophytes as a vector for beneficial bacteria has
already been demonstrated by Cankar et al. (2005) and
Mastretta et al. (2006). Beside plant species-specific
bacteria (see Fig. 4), several taxa (e.g., Pseudomonas,
Bacillus, Paenibacillus,andArthrobacter) that were found
in association with both English Oak and Common Ash
were found in association with hybrid poplar (Populus
trichocarpa x deltoides; Porteous Moore et al. 2006).
Moreover, we will characterize the bacterial community
associated with the hybrid poplar trees newly planted on the
TCE-contaminated site and compare this with the popula-
tionsassociatedwithEnglishOakandCommonAsh
growing on the same site and with the bacterial community
associated with hybrid poplar growing on a BTEX-
contaminated site (Porteous Moore et al. 2006).
In order to determine whether there was selection for
specific bacterial phenotypes in the presence of TCE,
representatives of all ARDRA types were tested for TCE
and toluene tolerance. It is obvious that TCE concentrations
in the groundwater up to 100 mg l
1
resulted for both tree
species in a bacterial population that was dominated by
TCE tolerant strains (see Ta ble 2; Barac et al. 2009). This
enrichment of TCE-tolerant strains in response to the TCE
contamination is consistent with the observations of
Siciliano et al. (2001). Beside TCE tolerant bacteria, there
was also an enrichment of toluene-tolerant bacteria. The
combined occurrence of TCE and toluene tolerance might
be due to common mechanisms, which have been demon-
strated for aerobic degradation of these compounds.
Aerobic degradation of both toluene and TCE has been
reported for bacterial strains possessing the toluene ortho-
monooxygenase (TomA) genes of Burkholderia cepacia G4
Environ Sci Pollut Res (2009) 16:830843 841
(Mars et al. 1996; Yee et al. 1998), and for toluene-o-xylene
monoxygenase (TomO) of Pseudomonas stutzeri OX1
(Ryoo et al. 2000, 2001; Shim et al. 2000, 2001). The fact
that some strains (e.g., strains 4, 19, 34, 37) showed better
growth on the 284 medi um in the presence of TCE (and
toluene; see Table 2) suggests that TCE (and toluene) can
be used as a substrate. The results of the degradation
experiments indeed showed TCE (and toluene) degradation
capacity (see Fig. 7), which was already suggested by the
remarkable decrease in TCE concentration through the
transects at the field site (see Fig. 2). Although these results
support the hypothesis of a causal relationship between the
strong decrease in TCE concentration through the transects
(see Fig. 1) and the TCE (and/or toluene) degradation
capacity of the bacterial population associated with the
English Oak and Common Ash, still a signi ficant amount of
TCE (Engli sh Oak, 6.35×10
3
±0.18×10
3
ng cm
²h
1
;
Common Ash, 10.84×10
3
±1.17×10
3
ng TCE cm
²h
1
)
was evapotranspired from the leaves to the atmosphere.
This implies that the natural bacterial community has
insufficient capacity to degrade all the TCE taken up by
the roots before it reaches the leaves. Therefore, it might be
worth attempting to inoculate the TCE-degrading bacteria
that were isolated from Oak and Ash growing on this field,
in order to enrich the quantity of degrading strains resulting
in an improved remediation capacity. Furthermore, the
newly planted poplar trees could be inoculated with TCE-
degrading poplar endophytes, such as Pseudomonas putida
W619 (Taghavi et al. 2005), to improve the degradation
capacity of the endogenous endophytic populations through
horizontal gene transfer.
For these inoculation experiments, it is favorable to work
with bacteria that can easily be re-isolated, such as bacteria
possessing heavy metal resistance. Such strains may allow
an easie r traceability for bioaugmentation of polluted sites.
For this reason, all bacteria that wer e found to be different
after ARDRA analysis were also tested for heavy metal
resistance. Only a very limited number of strains were
found to be resistant to the heavy metals Ni, Cd, and/or Zn,
which can be explained by the absence of selection
pressure. Metal concentrations measured were indeed
within the range of background values.
5 Concl usions and perspectives
The results obtained in this study show that the bacterial
community associated with EnglishOakandCommon
Ash growing on a TCE contaminated grou ndwa ter plume,
was strongl y e nric hed with tol ue ne and/or TCE tolerant
strains, but that this was no t sufficie nt to degrade all TCE
before it reaches the leaves. Although both tree species
were exposed to the same type and level of contamination
and were growing side by side in the same woodland,
their associated bacterial populations c learly differed in
composition. The endophytic bacterial community associ-
ated with English Oak contained significantly more
species-specific, most likely obligate endophytes. This
might be related to the seed type. Furthermore, the
remediation capacity of E nglishOakandCommonAsh
possibly might be improved by in situ inoculation of the
TCE degra ding, plant-assoc iated bacteria that were isolat-
ed. Since English Oak and Common Ash are both
common native and widely spread species in Europe, this
in situ inoculation s trategy could have a large application
potential. In addition, the newly planted poplar trees can
be inoculated with Pseudo mona s putida W619 (Tag ha vi et
al. 2005), to improve t he degradation capacity of the
endogenous endophytic populations through horizontal
gene transfer.
Acknowledgements This research was funded by the Institute for the
Promotion of Innovation through Science and Technology in Flanders
(IWT-Vlaanderen) for N.W. and by the Fund for Scientific Research
Flanders (FWO-Vlaanderen), Ph.D. grant for J.B. and postdoc grant for
T.B. This work was also supported by the UHasselt Methusalem project
08M03VGRJ. D.v.d.L. and S.T. are supported by the US Department of
Energy, Office of Science, BER, project number KP1102010 under
contract DE-AC02-98CH10886, and by Laboratory Directed Research
and Development funds (LDRD05-063) at the Brookhaven National
Laboratory under contract with the U.S. Department of Energy. We
thank A. Wijgaerts and C. Put for their help with the isolation and J. Put,
J. Czech, and R. Carleer for GC analysis.
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constitutively. Appl Environ Microbiol 64:112118
Environ Sci Pollut Res (2009) 16:830843 843
    • "The ability of plants to remove low molecular mass compounds from soil or water and release them to the atmosphere through leaves via evapotranspiration and at low concentration ,volatilize into the atmosphere ((Mueller et al. 1999; Gerhardt et al. 2009; Weyens et al. 2009). It is process of adoption, transport and release of pollutants, via the mechanism of transpiration in higher plants with the release of pollutants in the same, or modified form, in the atmosphere (EPA 2000; Pilipović et al. 2002). "
    [Show abstract] [Hide abstract] ABSTRACT: Phytoremediation is promising economic effective and ecofriendly technology that uses plants for the remediating heavy metals contaminated of pollutants affects of the biosphere: soil, water and air. It also significantly influenced on properties, such as acidity, content of macro and micro nutrients as well as great efforts have been made to reduce, remove or stabilize contaminants in polluted sites. Few heavy metals (Cadmium (Cd), Chromium (Cr), Nickel (Ni), Mercury (Hg), Arsenic (As), Thallium (TI), Lead (Pb) and etc) are toxic and lethal in trace concentrations and can be teratogenic, mutagenic, endocrine disruptors while others can cause behavioral and neurological disorders among infants and children. The mechanisms of remediation, several methods of phytoremediation can be distinguished such as phytoextraction/ Phytoaccumulation, phytotransformation, phytostimulation, phytovolatilization, phytorhizodegration and phytostabilization. An attempt has been made in this article to review the methods, prospects and future of the phytoremediation for remediating heavy metals from polluted land surface and water.
    Chapter · Oct 2016 · Journal of hazardous materials
    • "However, the stem showed a higher number of genera specific for this compartment.. Root and stem bacterial communities showed a low correlation coefficient (CC = 0.06), indicating the presence of different bacterial genera. Several authors reported significant differences in bacterial communities between below-ground and above-ground plant parts, demonstrating that the organs of the plants have different bacterial communities associated with them (Lindow and Brandl, 2003; Izumi et al., 2008; Weyens et al., 2009a; Croes et al., 2013). "
    [Show abstract] [Hide abstract] ABSTRACT: The interaction between plant growth-promoting bacteria (PGPB) and plants can enhance biomass production and metal tolerance of the host plants. This work aimed at isolating and characterizing the cultivable bacterial community associated with Brassica napus growing on a Zn contaminated site, for selecting cultivable PGPB that might enhance biomass production and metal tolerance of energy crops. The effects of some of these bacterial strains on root growth of B. napus exposed to increasing Zn and Cd concentrations were assessed. A total of 426 morphologically different bacterial strains were isolated from the soil, the rhizosphere, the roots and stems of B. napus. The diversity of the isolated bacterial populations was similar in rhizosphere and roots, but lower in soil and stem compartments. Burkoholderia, Alcaligenes,Agrococcus, Polaromonas, Stenotrophomonas, Serratia, Microbacterium and Caulobacter were found as root endophytes exclusively. The inoculation of seeds with Pseudomonas sp. strains 228 and 256, and Serratia sp. strain 246 facilitated the root development of B. napus at 1000 µM Zn. Arthrobacter sp. strain 222, Serratia sp. strain 246, and Pseudomonas sp. 228 and 262 increased the root length at 300 µM Cd.
    Full-text · Article · May 2016
    • "Plants can selectively stimulate the growth of indigenous endophytic microbial strains equipped with specific catabolic genes in order to cope with pollutants [54]. In this context, many studies [27,40,55,56] have demonstrated that host plants growing on a contaminated soil harbor many tolerant endophytic strains. Besides the plant genotype, the concentration of the contaminant may influence the metabolic potential of the in planta community. "
    [Show abstract] [Hide abstract] ABSTRACT: A phytoremediation pilot emulating a shallow aquifer planted with Juncus acutus showed to be effective for remediating Bisphenol-A (BPA) contaminated groundwater. Biostimulation with root exudates, low molecular weight organic acids, of J. acutus did not improve BPA-degradation rates. Furthermore, the endophytic bacterial community of J. acutus was isolated and characterized. Many strains were found to possess increased tolerance to metals such as Zn, Ni, Pb and Cd. Moreover, several endophytic bacterial strains tolerated and even used BPA and/or two antibiotics (ciprofloxacin and sulfamethoxazole) as a sole carbon source. Our results demonstrate that the cultivable bacterial endophytic community of J. acutus is able to use organic contaminants as carbon sources, tolerates metals and is equipped with plant-growth promoting traits. Therefore, J. acutus has potential to be exploited in constructed wetlands when co-contamination is one of the restricting factors.
    Full-text · Article · May 2016
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