Journal of Environmental Management 215 (2018) 216e229

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Journal of Environmental Management

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Research article

Degradation of 4-chlorophenol and microbial diversity in soil

inoculated with single Pseudomonas sp. CF600 and Stenotrophomonas
maltophilia KB2
Agnieszka Nowak*, Agnieszka Mrozik

ska 28, 40-032 Katowice, Poland
Department of Biochemistry, Faculty of Biology and Environmental Protection, University of Silesia, Jagiellon

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

Article history: Soil contamination with chlorophenols is a serious problem all over the world due to their common use
Received 21 July 2017 in different branches of industry and agriculture. The objective of this study was to determine whether
Received in revised form bioaugmenting soil with single Pseudomonas sp. CF600 and Stenotrophomonas maltophilia KB2 and
6 March 2018
additional carbon sources such as phenol (P) and sodium benzoate (SB) could enhance the degradation of
Accepted 12 March 2018
4-chlorophenol (4-CP). During the degradation experiment, the number of bacteria as well as the
structural and functional diversity of the soil microbial communities were determined. It was found that
the most effective degradation of 4-CP in the soil was observed after it was inoculated with CF600 and
Keywords:
Biodegradation
the addition of SB. The biodegradation of five doses of 4-CP in this soil proceeded within 100 days. At the
Bioaugmentation same time, the rate of the disappearance of 4-CP in the soil that had been bioaugmented with CF600 and
Survival of bacteria contaminated with 4-CP and P was 5e6.5 times lower compared to its rate of disappearance in the soil
Structural and functional diversity that had been contaminated with 4-CP. The biodegradation of 4-CP in all of the treated and untreated
soils was accompanied by a systematic decrease in the number of heterotrophic bacteria (THB) ranging
between 13 and 40%. It was also proven that the tested aromatic compounds affected the soil microbial
community structure through an increase in the marker fatty acids for Gram-negative bacteria (BG-) and
fungi (F). The essential changes in the patterns of the fatty acid methyl esters (FAMEs) for the polluted
soil included an increase in the fatty acid saturation and hydroxy fatty acid abundance. The obtained
results also indicated that the introduction of CF600 into the soil contaminated with 4-CP and SB or P
caused an increase in the functional diversity of the soil microorganisms. In contrast, in the soil that had
been inoculated with KB2 and in the non-inoculated soil, the addition of 4-CP and P decreased the
microbial activity. In conclusion, the inoculation of both strains into contaminated soil with aromatic
compounds caused irreversible changes in the functional and structural diversity of the soil microbial
communities.
© 2018 Elsevier Ltd. All rights reserved.

1. Introduction
The development of many branches of industry such as chem-
ical, pharmaceutical, textile, refinery and paper as well as agricul-
ture would not be possible without using phenolic compounds
such as chlorophenols (Igbinosa et al., 2013; Olaniran and Igbinosa,
2011). The global demand for chlorophenols is estimated to be
more than 100,000 tons per year (Veenagayathri and Vasudevan,
2013). Due to their acute toxicity, suspected carcinogenic and
endocrine disruptive properties, unpleasant taste and odor, the
* Corresponding author.
E-mail address: agnieszka.a.nowak@us.edu.pl (A. Nowak).
widespread use and release into the environment mainly through
improper disposal, wastes or accidental effluents cause many
ecological problems (Igbinosa et al., 2013; Sinkkonen et al., 2013).
Furthermore, the structure of chlorophenols, their different physi-
cochemical properties, which are dependent on environmental
parameters such as pH or temperature, result in their diverse
behavior, bioavailability and unpredictable effects in the contami-
nated ecosystems (Caliz et al., 2011; Spagnuolo et al., 2010). This is
the reason that chlorophenols are considered to be priority pol-
lutants by the World Health Organization (WHO), the United States
Environmental Protection Agency (USEPA) and the European Union
(EU). In order to prevent the negative impact of chlorophenols on
soil functioning, the creation and implementation of legal

https://doi.org/10.1016/j.jenvman.2018.03.052
0301-4797/© 2018 Elsevier Ltd. All rights reserved.
 A. Nowak, A. Mrozik / Journal of Environmental Management 215 (2018) 216e229
regulations such as the Soil Thematic Strategy by the European
Union requires its member states to prepare regulatory and eco-
nomic instruments to monitor, control and prevent soil pollution
(COM (2012) 46).
There are several physical and chemical methods for recovering
chlorophenols from the environment including adsorption onto
activated carbon and organoclays, ion exchange, oxidation and
thermal decomposition (Busca et al., 2008; Hamad, 2014; Lakshmi
et al., 2013; Nafees and Waseem, 2014). Despite the high efficiency
of the physicochemical processes, they can lead to the formation of
intermediates that are more harmful than the primary pollution.
Moreover, the extracted contaminants and incinerated soil need to
be treated further or disposed of. Compared to these methods,
bioremediation, which involves bioaugmentation, is considered by
its supporters to be a more attractive, eco-friendly, cost effective,
energy saving, less invasive process that does not introduce any
additional hazardous chemicals into the environment (Ghaly et al.,
2013). Bioaugmentation is defined as the improvement of the
degradative capacity of contaminated areas through the introduc-
tion of highly concentrated and specialised microorganisms. It is
generally practiced in soil in which the number of microorganisms
is insufficient or in which native populations do not have the
metabolic pathways that are necessary to metabolise the contam-
inants. Although bioaugmentation has been successfully applied in
practice, its standardisation and effectiveness is still problematic
and it is to the subject of a great many studies (Bahafid et al., 2017;
Benyahia and Embaby, 2016; Ghaly et al., 2013; Ma et al., 2015; Roy
et al., 2014). These difficulties result from the complexity of the
local edaphic and climate conditions, which strongly influence the
results of bioaugmentation and the low survival rate of the in-
oculants after their inoculation into the contaminated soil.
Furthermore, soil is often polluted with a mixture of substances
that have different physicochemical properties and biodegrad-
ability, which require the accurate selection of microorganisms that
have the desirable degradation capacity (Lagana et al., 2002;
Michałowicz, 2005).
Regardless of the method that is used for the decontamination
of chlorophenols, when accessing the quality and functioning of the
treated soil, it is important to monitor the activity and microbial
diversity of the soil. Due to methodological problems, a direct
process to identify and count the number of species in the envi-
ronment is impossible. Therefore, the European Commission
Directorate General for the Environment in the Technical Report
entitled “Soil biodiversity: functions, threats and tools for policy
makers” recommends using indicators as a way to present and
manage complex information in a simple and clear manner. It has
also been stated that these indicators should be meaningful,
standardised, measurable and cost-effective. For example, direct
counting, community-level physiological profiles (CLPPs) and fatty
acid analysis are listed as simple indicators of the microbial di-
versity in soil (Turbe
et al., 2010). CLPPs indicate the activity of
individual enzymes or substrate-induced respiration in a soil
sample and allow the functional diversity of microorganisms that
are adapting to stress conditions to be assessed (Swallow and
Quideau, 2015). In turn, analyses of the methyl ester fatty acids
(FAMEs) and the methyl esters of phospholipid fatty acids (PLFAs)
are useful for evaluating any changes in the structural diversity of
soil microorganisms based on the abundance of the marker fatty
acids for Gram-positive and Gram-negative bacteria, Actino-
mycetales and fungi (Piotrowska-Seget and Mrozik, 2003). As many
studies have indicated, microorganisms can alter their membrane
lipid composition and integrity in response to various environ-
mental disturbances such as temperature, pressure, pH, the avail-
ability of nutrients and harmful chemicals (Bla zina et al., 2010;
217
Murínov
a and Dercov
a, 2013; Segura et al., 2010).
Our previous studies demonstrated the degradative potential of
Pseudomonas sp. CF600 and Stenotrophomonas maltophilia KB2 on
the co-metabolic degradation of 4-chlorophenol (4-CP) in batch
liquid cultures (Nowak and Mrozik, 2016; Nowak et al., 2016). Based
on these results, in this study, an attempt was made to determine
whether bioaugmenting soil with these bacteria could enhance the
degradation of 4-CP alone as well as in the presence of additional
carbon sources such as phenol (P) and sodium benzoate (SB).
During the biodegradation studies, the number of total heterotro-
phic bacteria as well as the structural and functional diversity of the
soil microbiome were analysed.
2. Materials and methods
2.1. Bacterial strains
The bacterial strains used in this study were Pseudomonas sp.
CF600 (CCUG #32333) and Stenotrophomonas maltophilia KB2 (VTT
E-113197). Both strains have previously been described as able to
degrade 4-chlorophenol (4-CP) under co-metabolic conditions in
liquid cultures (Nowak and Mrozik, 2016; Nowak et al., 2016).
2.2. Soil
The soil was collected from the top layer (5e20 cm) of a
waterlogged meadow located in Da˛ browa Go
rnicza, Upper Silesia,
Poland (N 50,38, E 19,25). The selected physicochemical properties
of the soil, which were determined according to the Polish norms,
are presented in Table 1. The tested soil was free from phenol
contamination.
2.3. Experimental design
The biodegradation experiments were designed for use in soil
air-dried at room temperature and sieved (1.5 mm). Triplicate
portions of the soil were placed in pots (200 g) and amended with
4-chlorophenol (Sþ4-CP), as well as with two-component mixtures
consisting of 4-CP and P (Sþ4-CP þ P) and 4-CP and SB (Sþ4-
CP þ SB) at the concentrations of 260 mg g1, 260 and 560 mg g1
and 260 and 860 mg g1 soil, respectively. The concentrations of the
4-CP, P and SB stock solutions (in water) were 0.6 g l1, 38 g l1 and
29 g l1, respectively. The concentrations of aromatic compounds
added to the soil were doubled compared to the concentrations of
these compounds that were introduced to the liquid cultures of
Pseudomonas sp. CF600 and S. maltophilia KB2 in our previous
studies (Nowak and Mrozik, 2016; Nowak et al., 2016) and were
chosen based on the results of the preliminary research (data not
shown). The soil contamination with mixture of substances with
different physicochemical properties and susceptibility to biodeg-
radation was intended to reflect the conditions in many contami-
nated areas in which different aromatic compounds frequently
occur as a mixture. Another reason for using mixed aromatic
compounds was to induce the enzymes that are involved in the
decomposition of 4-CP in the presence of additional aromatic car-
bon source.
Next, some of the contaminated and non-contaminated soil
samples were bioaugmented with single strains of the bacteria
Pseudomonas sp. CF600 and S. maltophilia KB2. Before inoculation
into the soil, both strains were cultured in a mineral salts medium
(Mrozik et al., 2007) containing the correspondent aromatic sub-
strate in 500 ml flasks on a rotary shaker (130 rpm) at 30  C.
Pseudomonas sp. CF600 was cultured on P (280 mg l1) or SB
(430 mg l1), whereas S. maltophilia KB2 was only cultured in the
218 A. Nowak, A. Mrozik / Journal of Environmental Management 215 (2018) 216e229
Table 1
Selected physicochemical properties of the soil.
Parameter
Sand fraction: 2e0.05 mm, %
Dust fraction: 0.05e0.002 mm, %
Clay fraction: < 0.002 mm, %
Water content, %
pH
Electrical conductivity, mS cm1
Organic C, %
Total Kjeldahl N, %
Ammonium nitrogen N-NH4, mg kg1
Nitrate nitrogen N-NO-3, mg kg1
Available P, mg P2O5 g1
Available K, mg K2O g1
Cation exchange capacity (CEC), cmolþkg1
presence of P (280 mg l1). It was due to the preliminary research in
the batch liquid cultures which indicated that the co-metabolic
degradation of 4-CP in the presence of SB by S. maltophilia KB2
was inefficient (data not shown). The cells were adapted to each
substrate by transferring them on to the same substrate three
successive times, as was previously described (Nowak and Mrozik,
2016). Then, the cultures were centrifuged (4612 g, 20 min, 4  C),
the pellets were washed twice (0.9% NaCl) and resuspended in
20 ml of 0.9% NaCl. The obtained bacteria suspensions were added
to the soil resulting in a cell number of 108 g1 soil. All of the soil
samples were incubated at room temperature (22.0 ± 1.0  C) within
100 days. The final water content of the soils was adjusted to about
25% of the maximum water-holding capacity. The scheme of
experimental design is shown in Fig. 1.
2.4. Extraction and determination of aromatic compounds
concentration
To estimate the concentration of 4-CP, P and SB, the soil samples
(5 g) were collected on the 1, 4, 7 days and then at seven-day in-
tervals. Next, a mixture of 0.01% HCl, methanol and acetonitrile
(50:25:25, v/v/v) was added to the soil and shaken (130 rpm) for
1.5 h. Then, the samples were centrifuged (4612 g, 10 min, 4  C) and
the supernatant was analysed chromatographically using a Merck
Hitachi HPLC equipped with an Ascentis® Express C18 HPLC Col-
umn (100  4.6 mm), an Opti-Solw® EXP precolumn and a DAD
detector (Merck Hitachi). The mobile phase was a mixture of
acetonitrile, methanol and 1% acetic acid (20:20:60, v/v/v). The flow
rate was 1 ml min1. The chemical compounds in the supernatant
were identified and quantified by comparing their HPLC retention
times and UVevisible spectra with those of the external standards.
The detection wavelength for the detection of 4-CP, P and SB was
set at 272 nm.
2.5. Determining the bacteria cell number
On all of the sampling days, the number of total heterotrophic
bacteria (THB) in the bioaugmented and non-bioaugmented soils
were calculated. For this purpose, 2.5 g of soil were placed into an
Erlenmeyer flask containing 22.5 ml of 0.9% NaCl for shaking
(30 min, 130 rpm) and for preparing serial ten-fold dilutions for the
plate counts. The inoculated plates were incubated at 30  C for 48 h.
The number of bacteria was expressed as the log CFU g1 soil.
2.6. MIDI-FAME analysis
Fatty acid analyses were performed on days 1, 60 and 100 of the
experiment. Triplicate samples of each soil (5 g) were extracted
Value Method/source
88.0 ± 8.8 PN-R04032:1998
6.0 ± 0.6 PN-R04032:1998
6.0 ± 0.6 PN-R04032:1998
19.0 ± 1.5 PN-ISO 11465:1999
7.1 ± 0.2 PN-ISO 10390:1997
112 ± 3.0 PN-ISO 11265:1997
1.96 ± 0.2 PN-Z-15011-3
0.008 ± 0.0008 PN-ISO 11261:2000
21.8 ± 2.0 PN-ISO 14256e2:2010
0.6 ± 0.06 PN-ISO 14256e2:2010
0.104 ± 0.01 PN-R-04023:1996
0.08 ± 0.008 PN-R-04022:1996
2.71 ± 0.25 ISO 23470:2007
according to the procedure described by Kozdro
j (2000) and were
identified using the Microbial Identification System (MIS; Microbial
ID Inc., Newark). Fatty acid methyl esters (FAMEs) were separated
using a gas chromatograph (Hewlett-Packard 6890) equipped with
an HP-Ultra 2 capillary column (25 m, 0.22 mm ID) with hydrogen
as the carrier gas. The FAMEs were detected using a flame ionisa-
tion detector (FID) and identified using the MIDI Microbial Identi-
fication System software (Sherlock TSBA 6.1 method and TSBA6
library; MIDI Inc., Newark, DE, USA). To prevent the effect of fatty
acids that were only detected occasionally, the analysis of the
FAMEs included only fatty acids with a content of at least 1% in the
FAME profile.
Among the identified fatty acids, 17:0 cy, 19:0 cy, 16:1 u5c, 16:1
u7c, 17:1 u7c, 18:1 u7c were grouped as markers of Gram-negative
bacteria (BG-) (Moore-Kucera and Dick, 2008), whereas 11:0 iso,
11:0 anteiso, 13:0 iso, 13:0 anteiso, 14:0 iso, 15:0 iso, 15:0 anteiso,
16:0 iso, 17:0 iso, 17:0 anteiso were grouped as markers of Gram-
positive bacteria (BGþ) (Moore-Kucera and Dick, 2008). In turn,
16:0 10Me and 17:0 10Me were considered to be of an actinomycete
origin (Ac) (Moore-Kucera and Dick, 2008), while 18:2 u6,9c, 18:1
u9c were used as the biomarkers of fungi (F) (Kaiser et al., 2010).
2.7. Community level physiological profiling
The community level physiological profiles (CLPPs) in the soil
samples were obtained using Biolog® EcoPlates™ (BIOLOG Inc.,
Hayward, USA). Each plate contains 96 wells with 31 different
carbon sources plus a blank well in three replications. The rate of
the utilisation of the carbon sources was determined by the
reduction of tetrazolium violet redox dye, which changed from
colourless to purple when the added microorganisms utilised the
appropriate substrate (Stefanowicz, 2006). To prepare the Eco-
Plates™, 10 g of each tested soil were added to 90 ml of 0.9% NaCl
and shaken for 1 h (130 rpm) on days 1, 60 and 100 of the experi-
ment. Next, the soil suspension was diluted (1:9) with 0.9% NaCl
and 120 ml of the prepared suspension were used to inoculate each
well. The plates were incubated at 22  C for five days. The colour
development was followed by reading the absorbance at 590 nm at
12-h intervals using a MicroStation™ reader (Fra˛c et al., 2012). The
microbial activity in each microplate was expressed as the average
well colour development (AWCD) and the area under the curve
(AUC). Additionally, species richness (R) was determined as the
number of utilised substrates (Stefanowicz, 2006). To interpret the
obtained results, 31 carbon sources were divided into seven
groups: carbohydrates (Carb) (glucose-1-phosphate, D,L-a-glycerol
phosphate, b-methyl-D-glucoside, N-acetyl-D-glucosamine, a-D-
lactose, D-cellobiose, D-mannitol, D-xylose, i-erythritol), amino
acids (AA), (glycyl-L-glutamic acid, L-arginine, L-asparagine, L-
 A. Nowak, A. Mrozik / Journal of Environmental Management 215 (2018) 216e229
Fig. 1. The scheme of experimental design: treatment of the soil (S) bioaugmented
with Pseudomonas sp. CF600 (A), S. maltophilia KB2 (B) and non-bioaugmented soil (C).
Abbreviations: 4-CP e 4-chlorophenol, P e phenol, SB e sodium benzoate; S þ CF600
e S with Pseudomonas sp. CF600, Sþ4-CP þ CF600 e S with 4-CP and Pseudomonas sp.
CF600, Sþ4-CP þ P þ CF600 e S with 4-CP, P and Pseudomonas sp. CF600, Sþ4-
CP þ SB þ CF600 e S with 4-CP, SB and Pseudomonas sp. CF600, S þ KB2 - S with
S. maltophilia KB2, Sþ4-CP þ KB2 - S with 4-CP and S. maltophilia KB2, Sþ4-
CP þ P þ KB2 - S with 4-CP, P and S. maltophilia KB2, Sþ4-CP - S with 4-CP, Sþ4-
CP þ P - S with 4-CP and P, Sþ4-CP þ SB - S with 4-CP and SB.
219
phenyloalanine, L-threonine, L-serine), amines (A) (phenylethyl-
amine, putrescine), carboxylic acids (CA) (a-ketobutyric acid, D-
glucosaminic acid, D-galactonic acid g-lactone, D-galacturonic acid,
D-malic acid, pyruvic acid methyl ester, itaconic acid, g-hydroxy-
butyric acid), phenolic acids (PA) (2-hydroxybenzoic acid, 4-
hydroxybenzoic acid), surfactants (Surf) (Tween 40, Tween 80),
polymers (Poly) (glycogen, a-cyclodextrin). Furthermore, the ni-
trogen use index (N-index) and phosphorus use index (P-index)
were calculated as the percentage of total carbon substrate uti-
lisation, which was related to the substrates that contained nitro-
gen (amino acids, amines, N-acetyl-D-glucosamine, D-glucosaminic
acid) and phosphorus (glucose-1-phosphate, D,L-a-glycerol phos-
phate), respectively.
2.8. Data analyses
The degradation rate constant (k) of the 4-CP in the soil was
determined using the algorithm Ct/C0 ¼ e-kt, where C0 was the
substrate concentration at time 0 and Ct was the substrate con-
centration in the soil at time t. The average degradation rates (V) of
the aromatic substrates were calculated by dividing the net amount
of the degraded compound between t and 0. The theoretical
disappearance time 50 (DT50) was calculated from the linear
equation obtained from the regression between the 4-CP concen-
tration and time.
All of the identified FAMEs as well as the absorbance values on
the EcoPlates™ were used to calculate the Shannon's diversity in-
dex according to equation (1):
X
n
H0 ¼  pi log pi (1)
i¼1
where pi was a single FAME or the value of the absorbance of each
substrate divided by the sum of the absorbance that was recorded
for all of the substrates on the plate.
Additionally, the Shannon evenness (E) index was calculated
from H’ and R as follows (equation (2)):
H0
E¼ (2)
lnR
Data for the cluster analysis were normalised previously ac-
cording to equation (3):
ai  amin
N¼ (3)
amax  amin
where N (normalised data) scales all of the numeric variables in the
range between 0 and 1, ai e the initial raw data, amin e the mini-
mum value of each parameter and amax e the maximum value of
each parameter.
To determine whether the additional carbon source or bio-
augmentation influenced the k, V, and DT50 the independent
samples t-test (p < 0.05) was used to compare the values of
adequate pairs of obtained parameters. The obtained results were
also evaluated using the analysis of variance (ANOVA). The statis-
tical significance (p < 0.05) of any differences were treated using a
one-way ANOVA considering the effect of the 4-CP or additional
carbon source in the soil on the percentages of distinct FAME
markers as well as the functional diversity indices and assessed by a
post-hoc comparison of the means using the lowest significant
differences (LSD) test. In order to find any differences in the FAME
profiles between the tested soils, principal component analysis
(PCA) was used. All of the analyses were performed using the Sta-
tistica® 12.5 PL (StatSoft® Inc., USA) software package. For all of the
220 A. Nowak, A. Mrozik / Journal of Environmental Management 215 (2018) 216e229
analyses, data were represented as the mean ± the standard devi-
ation (SD) of three independent replicates.
3. Results
3.1. Degradation of 4-CP and number of bacteria in the soil
In the experiment, it was assessed whether soil bio-
augmentation with Pseudomonas sp. CF600 or S. maltophilia KB2
and the presence of P and SB could enhance the rate of the removal
of 4-CP in the contaminated soil. Biodegradation studies were run
for 100 days and were based on the premise that if any substrate
depletion would be confirmed in the soil, its subsequent identical
dose would be added. It was found that in the Sþ4-CP þ CF600, 4
doses of this compound were degraded completely and 75% of 5
dose was utilised (Fig. 2A). At the same time point (100 day), 3
doses and 80% of 4 dose of 4-CP were utilised in the Sþ4-CP þ KB2
(Fig. 3A). In comparison, in the Sþ4-CP the removal of 3 doses and
the 58% of 4 dose of 4-CP was established during this time (Fig. 3A).
The rate of the first dose of 4-CP disappearance (V), the rate con-
stant (k) and DT50 in all of the treated soil samples reached similar
values and V and k were significantly higher, whereas DT50 were
lower compared to the correspondent values for subsequent doses
(Table 2). It was also confirmed that the disappearance of 4-CP in
the Sþ4-CP þ P took longer compared to the Sþ4-CP. The utilisation
of the first dose of 4-CP in the presence of P in the bioaugmented
and control soils took 28 days (Figs. 2C and 3C), whereas the first
dose of 4-CP was degraded within 7 days (Figs. 2A and 3A). The
rates of the disappearance of 4-CP in the soil that had been
contaminated with 4-CP and P were 5e6.5 times lower compared
to its disappearance in the soil that had been contaminated with 4-
CP and DT50 values were 3 times higher. The 70%, 35% and 30% of
the second dose of 4-CP in the presence of P bacteria degraded in
the Sþ4-CP þ P þ CF600 (Fig. 2C), Sþ4-CP þ P þ KB2 and Sþ4-
CP þ P, respectively (Fig. 3C). There were also differences in the
parameters of the degradation kinetics (Table 2). In another
experiment, it was proven that SB clearly accelerated the removal
of 4-CP in the Sþ4-CP þ SB þ CF600. The complete biodegradation
of five doses of 4-CP in this soil was confirmed during 100 days
(Fig. 2E). The calculated parameters of the degradation kinetics (V
and DT50) of the 5 dose of 4-CP in Sþ4-CP þ SB þ CF600 and Sþ4-
CP þ CF600 also were significantly different (Table 2).
Analysis of the total number of heterotrophic bacteria (THB) in
all of the treated and untreated soils indicated a systematic decline
in the number of cells. In the Sþ4-CP þ CF600, Sþ4-CP þ KB2 and
Sþ4-CP, the initial numbers of THB were 1.0  108, 2.5  108 and
2.8  107 g1 soil, respectively. The decline in the bacterial count in
all of the tested soils was similar and ranged from 20 to 26%. In
comparison, the decrease in the THB numbers in the control soil (S)
was near to 50% (Figs. 2B and 3B). Data analyses of the THB counts
in the soil that had been contaminated with the mixtures of 4-CP
and P or 4-CP and SB showed that although the numbers of cul-
turable bacteria decreased over time, the bacteria survived better in
the soil that had been contaminated with the mixtures of these
compounds than in the non-contaminated soil. The decline in the
THB counts in the Sþ4-CP þ P þ CF600, Sþ4-CP þ P and Sþ4-
CP þ P þ KB2 constituted 13%, 18% and 21% of the initial number
of cells, respectively, at the end of the experiment (Fig. 2D, F and 3B,
D). In turn, a decrease in the THB counts in the S þ CF600, S þ KB2
and S was established at the level of about 40% (Figs. 2B and 3B).
3.2. FAME profiling
In parallel with the biodegradation studies, the impact of 4-CP
and the bioaugmentation of the soil with Pseudomonas sp. CF600
and S. maltophilia KB2 on the FAME composition was assessed. The
PCA analysis involved 15e19 fatty acids and explained
79.18e85.59% of the variance (Fig. 4). Although the most important
factor that had an influence on the variations in the FAME patterns
was the time, bioaugmentation did not distinguish the fatty acids
that were isolated from the soils (Fig. 4B, D, F). The PCA analysis
indicated that the FAMEs that were isolated from the bio-
augmented and non-bioaugmented soils (S þ CF600, S þ KB2, S) on
day 1 were distinct compared to those that were isolated from Sþ4-
CP þ CF600, Sþ4-CP þ KB2, Sþ4-CP, S þ CF600, S þ KB2 and S on
day 100 along the first axis and those extracted from the same soils
on day 60 along the second axis (Fig. 4B). The fatty acids 10:0 3OH,
12:0 3OH, 16:0 3OH and 19:0 cy u10c correlated positively with
PC1, whereas 15:0 iso, 16:0 iso, 16:1 u5c, 16:1 u7c, 18:1 u7c, 18:1
u9c correlated negatively (Fig. 4A). The result of observed changes
in the content of FAMEs was an increase in the sat/unsat ratio over
time (Table 3). A similar analysis of the FAMEs was conducted for all
of the soils that had been contaminated with 4-CP and P (Fig. 4C
and D). It was found that the S and S þ CF600 on day 1 were
grouped together and were distinguished from Sþ4-
CP þ P þ CF600, Sþ4-CP þ P þ KB2, Sþ4-CP þ P, S þ CF600,
S þ KB2 and S on day 100 along the first axis (Fig. 4D). The pro-
jection of the FAME profiles that were obtained for the soil that had
been contaminated with 4-CP and P indicated different local-
isations of the treated and control soils along the PC1 and PC2 axes.
The S and S þ CF600 on day 1 were grouped together and were
distinguished from Sþ4-CP þ P þ CF600, Sþ4-CP þ P þ KB2, Sþ4-
CP þ P, S þ CF600, S þ KB2 and S on day 100 along the first axis
(Fig. 4D). The FAME profiles on day 100 were characterised by a
higher content of 10:0 3OH, 12:0 3OH, 16:0 3OH, 17:0 iso 3OH and
19:0 cy u10c. The observed changes were reflected in the increase
of the sat/unsat ratio over time (Table 3). The variations in the FAME
profiles of the Sþ4-CP þ SB was not as strongly connected with the
time compared to the Sþ4-CP and Sþ4-CP þ P. The FAMEs that were
extracted from the S and S þ CF600 on day 1 were grouped together
and were differentiated from the FAMEs that were extracted from
the S, S þ CF600 on day 60 and the S þ CF600 on day 100 along the
second axis and from the Sþ4-CP þ SB þ CF600 on day 60, and Sþ4-
CP þ SB þ CF600 and Sþ4-CP þ SB on day 100 along the first axis
(Fig. 4F). As Fig. 4E indicates, 16:0, 18:0, 20:0, 15:0 iso, 16:0 iso, 16:1
u5c, 18:1 u9c, 18:2 u6,9c were positively correlated with PC1,
whereas 10:0 3OH, 12:0 2OH, 12:0 3OH had a negative correlation.
Only 16:1 u7c was positively correlated with PC2. In the 4-CP and
SB contaminated soil, the sat/unsat ratio increased over time,
although the values of this ratio for the Sþ4-CP, Sþ4-CP þ P and
Sþ4-CP þ SB on day 100 in the bioaugmented and non-
bioaugmented soils did not differ significantly (Table 3).
The influence of aromatic compounds and soil bioaugmentation
on microbial diversity in the tested soils was evaluated by analysing
the content of the markers of fatty acids for Gram-negative bacteria
(BG-), Gram-positive bacteria (BGþ), actinomycetes (Ac) and fungi
(F) (Table 3). It was observed that the participation of the FAME
markers for BG-increased over time and was the highest (28.42%)
for the Sþ4-CP þ CF600 on day 100 (Table 3). In contrast, the
content of the FAME markers for BGþ and F decreased over time. In
the Sþ4-CP þ SB þ CF600, Sþ4-CP þ CF600 and Sþ4-
CP þ P þ CF600, the content of the BG þ markers declined by
29%, 22% and 16%, respectively, on day 100, while in the all of the
bioaugmented soils with S. maltophilia KB2 and in all of the non-
bioaugmented soils, the observed changes were not significant.
The lowest content of fungal markers (2.82 and 3.17%) was recorded
for the Sþ4-CP þ SB þ CF600 and Sþ4-CP þ SB on day 100. As H’
indicated, the S þ KB2 were the most diverse among all of the
tested soils on day 100, while none of the bioaugmented soils with
Pseudomonas sp. CF600 had any significant changes over time
 A. Nowak, A. Mrozik / Journal of Environmental Management 215 (2018) 216e229 221

Fig. 2. Dynamics of 4-CP (A), 4-CP and P (C), 4-CP and SB (E) degradation and number of THB (B, D, F) in the soil bioaugmented with Pseudomonas sp. CF600 and the control soil.
222 A. Nowak, A. Mrozik / Journal of Environmental Management 215 (2018) 216e229
Fig. 3. Dynamics of 4-CP (A), 4-CP and P (C) degradation and number of THB (B, D) in the soil bioaugmented with S. maltophilia KB2 and the control soil.
(Table 3).
3.3. Metabolic activity of soil microorganisms
The functional diversity of microorganisms in the treated and
untreated soils is presented in Table 4. The calculated values of the
AWCD and AUC indicated that the bacteria in the S þ CF600,
S þ KB2 and S were most active on day 1 as well as those in the
Sþ4-CP þ SB þ CF600, Sþ4-CP þ SB, Sþ4-CP þ CF600, Sþ4-
CP þ CF600, Sþ4-CP on days 60 and 100. The highest value of H0
was noted for the Sþ4-CP þ P þ CF600 and S on days 60 and 100,
while the lowest H’ value was recorded for the S þ CF600 on day 1.
The R values were the highest for the Sþ4-CP þ SB and Sþ4-CP on
day 60 and the lowest for the Sþ4-CP þ SB on day 60. The highest E
value was recorded for the S on days 60 and 100, while the lowest E
value was calculated for the S þ CF600 and S þ KB2 on day 1.
To determine whether the type of available substrate affected
the utilisation patterns depending on the soil treatment, all of the
analysed substrates were grouped by their biochemical guild and
their N-index and P-index were calculated (Fig. 5). It was found that
the increases of the intensity of Carb usage at the end of the
experiment were 20, 30, 50 and 50% for the S þ KB2, Sþ4-
CP þ CF600, Sþ4-CP þ KB2 and S þ CF600, respectively,
compared to the initial values. In turn, 20, 30 and 40% decreases in
the intensity of AA utilisation were recorded for the S þ CF600, S
and S þ KB2, respectively. Furthermore, a high CA utilisation level
in the range of 40e60% was observed for the Sþ4-CP þ P þ CF600
on day 60, the Sþ4-CP þ P þ KB2 on day 100 and for the Sþ4-CP þ P
on days 60 and 100. Interestingly, a low level of Poly, Surf and PA
utilisation was recorded for all of the treated soils. High N-index
values (above 50%) were calculated for the S þ CF600, S þ KB2 and S
on day 1, for the Sþ4-CP þ SB þ CF600, Sþ4-CP þ SB, Sþ4-
CP þ CF600 on days 60 and 100 and for the Sþ4-CP þ P þ CF600
and Sþ4-CP on day 100.
A cluster analysis of all of the treated soils on different sampling
days, which was based on the pattern utilisation of the seven guilds
and FAME microbial markers, is presented in Fig. 6. Such an analysis
allowed two main separate clusters to be distinguished. One cluster
included the microbial markers A and AA, whereas Surf, PA, Poly, CA
 A. Nowak, A. Mrozik / Journal of Environmental Management 215 (2018) 216e229 223

Table 2
Degradation rate constant (k), disappearance rate (V) and disappearance time 50 (DT50) of 4-CP in the soil non-bioaugmented and bioaugmented with Pseudomonas sp. CF600
or S. maltophilia KB2.

Treatments Dosage number k, day1 V, mg g1 day1 DT50 (days) Regression equation R2

Soil bioaugmented with Pseudomonas sp. CF600

Sþ4-CP þ CF600 1 0.258 ± 0.038I 37.09 ± 0.29I 3.33 ± 0.14I ln(Ct/C0) ¼ /0.255tþ5.56 0.824
2 0.106 ± 0.003 II 4.87 ± 0.49 II,a 10.84 ± 1.09 II,a ln(Ct/C0) ¼ /0.106tþ5.64 0.987
3 0.190 ± 0.009a 4.66 ± 0.86b 4.89 ± 0.72 III,b ln(Ct/C0) ¼ /0.268tþ5.86 0.950
4 0.016 ± 0.007b 11.01 ± 1.81 9.02 ± 2.27c ln(Ct/C0) ¼ /0.051tþ5.36 0.829
5 0.076 ± 0.010 9.58 ± 0.69 III 15.10 ± 0.38 IV ln(Ct/C0) ¼ /0.068tþ5.63 0.955
Sþ4-CP þ P þ CF600 1 0.036 ± 0.014I,c 5.67 ± 0.71I,c 10.42 ± 0.37I ln(Ct/C0) ¼ /0.043tþ5.27 0.769
2 0.020 ± 0.001 II,d 2.88 ± 0.59 II,d 46.01 ± 6.51II,d ln(Ct/C0) ¼ /0.019tþ5.70 0.863
Sþ4-CP þ SB þ CF600 1 0.248 ± 0.015 36.61 ± 0.83 3.31 ± 0.07 ln(Ct/C0) ¼ /0.248tþ5.55 0.885
2 0.084 ± 0.013 II 7.49 ± 1.15 II,e 11.21 ± 0.98 ln(Ct/C0) ¼ /0.083tþ5.54 0.996
3 0.122 ± 0.064 7.49 ± 1.66 9.06 ± 0.63 III,e ln(Ct/C0) ¼ /0.096tþ5.50 0.955
4 0.027 ± 0.007 18.17 ± 0.28 8.34 ± 0.23 ln(Ct/C0) ¼ /0.027tþ5.53 0.990
5 0.071 ± 0.005 11.09 ± 0.13 III 11.96 ± 0.22 IV ln(Ct/C0) ¼ /0.071tþ5.64 0.862
Soil bioaugmented with S. maltophilia KB2
III
Sþ4-CP þ KB2 1 0.233 ± 0.025 36.71 ± 0.25 IV 3.40 ± 0.13V ln(Ct/C0) ¼ /0.231tþ5.55 0.996
IV
2 0.098 ± 0.005 2.46 ± 0.29 9.53 ± 1.17 VI,a ln(Ct/C0) ¼ /0.097tþ5.48 0.988
3 0.069 ± 0.039 7.98 ± 0.96 15.41 ± 1.83 ln(Ct/C0) ¼ /0.059tþ5.58 0.969
4 0.085 ± 0.004 3.10 ± 0.28 10.07 ± 1.60c ln(Ct/C0) ¼ /0.084tþ5.37 0.968
III,c
Sþ4-CP þ P þ KB2 1 0.040 ± 0.003 7.63 ± 0.52 IV 10.40 ± 1.10V ln(Ct/C0) ¼ /0.089tþ5.53 0.745
IV,d
2 0.006 ± 0.001 1.48 ± 0.32d 78.70 ± 2.30 VI ln(Ct/C0) ¼ /0.006tþ5.52 0.723
Non-bioaugmented soil
Sþ4-CP 1 0.380 ± 0.124V 36.49 ± 0.26V 3.22 ± 0.24 VII ln(Ct/C0) ¼ /0.293tþ5.55 0.876
2 0.097 ± 0.009 VI 2.47 ± 0.59 VI,a 19.60 ± 1.97 VIII,a ln(Ct/C0) ¼ /0.128tþ5.64 0.852
3 0.090 ± 0.014 VII,a 9.44 ± 0.18 VII,b 12.54 ± 0.25 IX,b ln(Ct/C0) ¼ /0.100tþ5.77 0.939
4 0.063 ± 0.016b 3.09 ± 0.49 30.50 ± 1.47c ln(Ct/C0) ¼ /0.077tþ5.79 0.883
Sþ4-CP þ P 1 0.088 ± 0.023V,c 6.60 ± 0.37V,c 9.86 ± 1.02 VII ln(Ct/C0) ¼ /0.047tþ5.66 0,940
2 0.013 ± 0.002 VI,d 0.50 ± 0.09 VI,d 83.88 ± 13.40 VIII,d ln(Ct/C0) ¼ /0.025tþ6.22 0.979
Sþ4-CP þ SB 1 0.225 ± 0.014 36.61 ± 0.83 3.40 ± 0.09 ln(Ct/C0) ¼ /0.220tþ5.55 0.982
2 0.084 ± 0.019 4.13 ± 0.33 VI,e 10.48 ± 0.44 VIII ln(Ct/C0) ¼ /0.077tþ5.41 0.989
3 0.060 ± 0.004 VII 6.39 ± 0.15 VII 18.52 ± 0.43 IX,e ln(Ct/C0) ¼ /0.077tþ5.59 0.898

The plus/minus values represent SD. In column with k, V and DT50 the pairs of means with the same letters are significantly different (p < 0.05, the independent samples t-test)
considering the effect of P and SB compared to the soil polluted with adequate dose of 4-CP (Roman numerals) and the effect of bioaugmentation compared to the non-
bioaugmented soil (lower case letters).

and Carb were grouped in the second cluster. Moreover, a cluster
analysis of the studied soils in terms of soil treatment over time
showed three separate clusters. The first one included the
S þ CF600, S þ KB2 and S on day 1, the Sþ4-CP and Sþ4-CP þ SB on
day 60 and the Sþ4-CP þ CF600, Sþ4-CP þ KB2 and Sþ4-
CP þ SB þ CF600 on days 60 and 100. In the second cluster, two
subgroups were distinguished and grouped together. The first
subgroup included the Sþ4-CP þ SB, Sþ4-CP þ P þ CF600, Sþ4-CP
on day 100 and the S þ CF600 and S on day 60, while the second
subgroup included the S þ CF600 on day 100 and the Sþ4-CP þ P
and S þ KB2 on days 60 and 100. In turn, the Sþ4-CP þ P þ CF600
on day 60, Sþ4-CP þ P þ KB2 on days 60 and 100 and the S on day
100 were grouped independently.
4. Discussion
Soil contamination with chlorophenols is an persistent problem
in many places in the world and therefore has been the subject of
numerous studies (Sinkkonen et al., 2013; Ivanciuc et al., 2006).
Although the attention of scientists is mainly focused on hardy,
degradable polychlorinated phenols (Chen et al., 2012; Diez et al.,
2012; Li et al., 2013), monochlorophenol contamination should
not be neglected. Such compounds are widespread not only in soil
but are also dangerous pollutants of sewage and rivers
(Michałowicz et al., 2005, 2008). In spite of many studies, our
knowledge on how to increase the efficiency of monochlorophenol
degradation in soil using biological methods is still limited.
Furthermore, little is known about how microorganisms that are
exposed to monochlorophenols survive in soil and how these
compounds affect their structural and functional diversity.
In our previous studies, it was indicated that Pseudomonas sp.
CF600 and S. maltophilia KB2 were able to co-metabolise 4-CP in the
presence of phenol or sodium benzoate in batch liquid cultures
(Nowak et al., 2016; Nowak and Mrozik, 2016). This prompted us to
introduce these bacteria into the soil. The 4-CP degradation ex-
periments in the S showed that the first dose of this compound was
metabolised within the same period of time in the bioaugmented
and non-bioaugmented soil. This indicated that a short-term
exposure of soil microorganisms to 4-CP did not affect their
degradative activity and that autochthonous microorganisms were
able to metabolise this compound. Similarly, Lallai and Mura (2004)
reported that in forest soil that had not previously been exposed to
any synthetic chemicals, the degradation of 2-chlorophenol (2-CP)
proceeded slower than in soil with a longer history of contamina-
tion. In this study, marked differences in the elimination of 4-CP in
the treated soils were recorded during the degradation of succes-
sive doses of 4-CP within 100 days. In this case, the most effective
degradation of 4-CP was observed in the soil that had been bio-
augmented with Pseudomonas sp. CF600, while no significant dif-
ferences were observed between the S inoculated with
S. maltophilia KB2 and the non-bioaugmented soil. The positive
effect of soil bioaugmentation with Arthrobacter chlorophenolicus
A6 on the acceleration of the removal of 4-CP (175 mg g1 soil) was
also observed by Elva €ng et al. (2001). Similarly, Teng et al. (2017)
indicated the positive effect of soil inoculation with Cupriavidus
sp. YNS-85 on the removal of pentachloronitrobenzene (2.5 mg g1
soil) from soil. In turn, the results of Kauppi et al. (2011) did not
confirm an enhanced degradation of diesel-fuel hydrocarbons
(2.7 g kg1 soil) in boreal soil that had been bioaugmented with a
pure Acinetobacter culture and an enriched microbial consortium.
As many studies have indicated, the effectiveness of bio-
augmentation depends on many factors such as optimal
224 A. Nowak, A. Mrozik / Journal of Environmental Management 215 (2018) 216e229

Fig. 4. Projection of individual fatty acids along PC1 and PC2 (A, C, E) and FAME profiles of the control soil, Sþ4-CP, Sþ4-CP þ CF600, Sþ4-CP þ KB2 (B), Sþ4-CP þ P, Sþ4-
CP þ P þ CF600, Sþ4-CP þ P þ KB2 (D) and Sþ4-CP þ SB, Sþ4-CP þ SB þ CF600 (F). The numbers in captions after underlying means the sampling day (B, D, F).
 A. Nowak, A. Mrozik / Journal of Environmental Management 215 (2018) 216e229 225

Table 3
The percentages of distinct FAME markers, sat/unsat ratio and Shannon index (H’) for the treated and untreated soils.

Treatments BG- BGþ Ac F Sat/unsat H0

Soil bioaugmented with Pseudomonas sp. CF600

S þ CF600_1 15.58 ± 0.20 16.66 ± 0.56 3.17 ± 0.68 8.08 ± 0.23 3.19 ± 0.09 2.69 ± 0.16
S þ CF600_60 25.10 ± 0.66 15.27 ± 1.99 2.69 ± 0.12ab 7.02 ± 0.01a 3.81 ± 0.02 2.89 ± 0.10
Sþ4-CP þ CF600_60 25.02 ± 0.84 13.13 ± 0.19 3.33 ± 0.21a 4.65 ± 0.60b 4.13 ± 0.20 2.64 ± 0.14
Sþ4-CP þ P þ CF600_60 26.17 ± 0.28 14.22 ± 1.71 2.18 ± 0.26b 6.20 ± 1.23a 3.67 ± 0.18 2.82 ± 0.12
Sþ4-CP þ SB þ CF600_60 25.16 ± 0.58 12.84 ± 1.88 2.74 ± 0.60ab 4.49 ± 0.49b 3.98 ± 0.18 2.80 ± 0.16
S þ CF600_100 27.37 ± 2.16 14.96 ± 0.34 1.39 ± 0.58ce 6.62 ± 1.75c 4.54 ± 0.17 2.87 ± 0.01
Sþ4-CP þ CF600_100 28.42 ± 0.48 12.95 ± 1.15 3.58 ± 0.12d 4.13 ± 0.36cd 5.56 ± 0.95 2.88 ± 0.10
Sþ4-CP þ P þ CF600_100 26.75 ± 1.31 13.92 ± 1.41 0.80 ± 0.04c 4.40 ± 0.18cd 5.24 ± 0.67 2.83 ± 0.14
Sþ4-CP þ SB þ CF600_100 25.15 ± 1.25 11.78 ± 1.60 2.25 ± 0.22e 2.82 ± 0.16d 5.76 ± 1.34 2.66 ± 0.10
Soil bioaugmented with S. maltophilia KB2
S þ KB2_1 22.02 ± 0.48 16.08 ± 1.26 3.43 ± 1.19 7.69 ± 1.60 3.88 ± 0.47 2.60 ± 0.12
S þ KB2_60 23.94 ± 0.50 13.40 ± 1.17 2.44 ± 0.90 5.38 ± 0.18 3.88 ± 0.20a 3.02 ± 0.25
Sþ4-CP þ KB2_60 22.19 ± 2.27 11.52 ± 2.54 3.02 ± 0.78 5.09 ± 1.56 3.77 ± 0.18a 2.69 ± 0.05
Sþ4-CP þ P þ KB2_60 22.77 ± 0.75 15.26 ± 2.14 2.67 ± 0.45 5.32 ± 0.92 4.51 ± 0.12b 2.87 ± 0.25
S þ KB2_100 23.94 ± 1.95 13.47 ± 1.76 2.10 ± 0.39 5.00 ± 1.37 4.75 ± 0.55 3.07 ± 0.03a
Sþ4-CP þ KB2_100 25.92 ± 3.00 12.69 ± 0.07 2.68 ± 0.54 3.94 ± 0.71 5.55 ± 0.39 2.82 ± 0.07b
Sþ4-CP þ P þ KB2_100 27.66 ± 1.08 14.15 ± 2.14 1.43 ± 0.17 5.21 ± 1.36 4.68 ± 0.39 2.75 ± 0.06b
Non-bioaugmented soil
S_1 20.00 ± 6.47 16.49 ± 1.61 2.45 ± 0.13 7.49 ± 0.44 3.29 ± 0.36 2.46 ± 0.06
S_60 22.40 ± 2.73 16.98 ± 0.94 2.84 ± 0.18fg 6.01 ± 0.50e 4.42 ± 0.21 2.98 ± 0.09
Sþ4-CP_60 23.21 ± 0.47 13.83 ± 1.05 3.80 ± 1.41f 4.93 ± 0.82ef 4.83 ± 0.27 2.91 ± 0.04
Sþ4-CP þ P_60 23.18 ± 1.03 15.34 ± 0.16 1.94 ± 0.57g 4.85 ± 0.77ef 4.74 ± 0.66 3.00 ± 0.30
Sþ4-CP þ SB_60 19.86 ± 3.66 14.02 ± 1.70 3.49 ± 0.78fg 4.39 ± 0.51f 4.96 ± 0.06 2.87 ± 0.29
S_100 24.97 ± 2.52 15.53 ± 1.05ab 0.98 ± 1.39 4.33 ± 0.11gh 5.30 ± 0.16 2.97 ± 0.06
Sþ4-CP_100 28.30 ± 1.56 17.31 ± 0.79a 2.89 ± 0.51 3.88 ± 0.40g 4.11 ± 0.16 2.87 ± 0.05
Sþ4-CP þ P_100 25.90 ± 0.44 15.48 ± 1.41ab 1.82 ± 0.13 5.23 ± 0.54h 4.66 ± 0.87 2.82 ± 0.19
Sþ4-CP þ SB_100 26.37 ± 1.97 13.22 ± 1.43b 2.89 ± 0.96 3.17 ± 0.51g 5.13 ± 0.58 2.81 ± 0.14

In each column the means with different letters are significantly different (p < 0.05, LSD test) considering the effect of 4-CP, P and SB in the soil bioaugmented with Pseu-
domonas sp. CF600, S. maltophilia KB2 and non-bioaugmented soil on days 60 and 100, analysed separately. The means without any letters did not differ among others in the
adequate soil and sampling day.

Table 4
The functional diversity indices for the treated and untreated soils.

Treatments AWCD H0 R E AUC

Soil bioaugmented with Pseudomonas sp. CF600

S þ CF600_1 0.719 ± 0.067 1.047 ± 0.031 23.22 ± 2.68 0.768 ± 0.019 663.81 ± 20.01
S þ CF600_60 0.076 ± 0.017a 1.291 ± 0.010a 23.43 ± 2.50a 0.945 ± 0.030a 70.73 ± 12.94a
Sþ4-CP þ CF600_60 1.013 ± 0.010b 1.237 ± 0.012a 29.11 ± 1.45b 0.845 ± 0.008b 512.88 ± 7.26b
Sþ4-CP þ P þ CF600_60 0.008 ± 0.001c 1.588 ± 0.220b 19.00 ± 1.00c 1.243 ± 0.180c 6.63 ± 0.27c
Sþ4-CP þ SB þ CF600_60 0.795 ± 0.049d 1.159 ± 0.040c 29.00 ± 1.93b 0.793 ± 0.020b 498.03 ± 6.78d
S þ CF600_100 0.026 ± 0.004e 1.450 ± 0.076d 19.00 ± 3.93d 1.147 ± 0.090d 19.24 ± 9.34e
Sþ4-CP þ CF600_100 1.010 ± 0.009f 1.178 ± 0.009e 24.56 ± 2.29e 0.849 ± 0.020e 511.51 ± 10.30f
Sþ4-CP þ P þ CF600_100 0.737 ± 0.022g 1.140 ± 0.015e 27.33 ± 2.18f 0.795 ± 0.012f 582.74 ± 27.63g
Sþ4-CP þ SB þ CF600_100 0.998 ± 0.090f 1.304 ± 0.040f 28.40 ± 2.19f 0.898 ± 0.028e 728.31 ± 61.56h
Soil bioaugmented with S. maltophilia KB2
S þ KB2_1 0.710 ± 0.044 1.132 ± 0.040 27.11 ± 1.61 0.790 ± 0.019 439.55 ± 71.34
S þ KB2_60 0.015 ± 0.001h 1.280 ± 0.020 24.20 ± 0.15g 0.930 ± 0.020g 137.31 ± 30.20i
Sþ4-CP þ KB2_60 0.727 ± 0.020i 1.271 ± 0.010 28.78 ± 0.44h 0.871 ± 0.440h 341.79 ± 9.46j
Sþ4-CP þ P þ KB2_60 0.021 ± 0.003h 1.307 ± 0.040 24.50 ± 4.80g 0,948 ± 0.045g 31.22 ± 4.14k
S þ KB2_100 0.024 ± 0.006j 1.364 ± 0.034g 22.33 ± 2.25ij 1.013 ± 0.013i 32.13 ± 2.96l
Sþ4-CP þ KB2_100 0.608 ± 0.030k 1.155 ± 0.020h 26.22 ± 2.22i 0.815 ± 0.012j 361.68 ± 17.83m
Sþ4-CP þ P þ KB2_100 0.003 ± 0.001l 1.185 ± 0.014i 17.33 ± 6.10j 0.981 ± 0.131i 1.43 ± 0.09n
Non-bioaugmented soil
S_1 0.660 ± 0.050 1.200 ± 0.013 24.44 ± 2.12 0.866 ± 0.020 367.52 ± 31.28
S_60 0.025 ± 0.003m 1.615 ± 0.100j 17.75 ± 3.50km 1.300 ± 0.040k 47.64 ± 6.50
Sþ4-CP_60 1.036 ± 0.010n 1.292 ± 0.006k 29.00 ± 0.87l 0.883 ± 0.004l 550.28 ± 6.03p
Sþ4-CP þ P_60 0.005 ± 0.000m 1.350 ± 0.038k 19.50 ± 2.12k 1.048 ± 0.068m 13.89 ± 0.19
Sþ4-CP þ SB_60 0.489 ± 0.030 1.136 ± 0.036l 15.89 ± 1.60m 0.949 ± 0.060n 384.32 ± 35.12r
S_100 0.035 ± 0.009p 2.100 ± 0.160m 20.33 ± 2.08n 1.605 ± 0.060 29.97 ± 5.92s
Sþ4-CP_100 0.591 ± 0.009r 1.124 ± 0.011n 21.22 ± 2.10n 0.849 ± 0.020p 412.20 ± 10.89t
Sþ4-CP þ P_100 0.016 ± 0.002p 1.260 ± 0.047 25.11 ± 1.90 0.900 ± 0.017r 4.80 ± 0.62s
Sþ4-CP þ SB_100 0.535 ± 0.093s 1.314 ± 0.041 24.44 ± 2.74 0.937 ± 0.019s 318.61 ± 44.11u

In each column the means with different letters are significantly different (p < 0.05, LSD test) considering the effect of 4-CP, P and SB in the soil bioaugmented with Pseu-
domonas sp. CF600, S. maltophilia KB2 and non-bioaugmented soil on days 60 and 100, analysed separately. The means without any letters did not differ among others in the
adequate soil and sampling day.

physicochemical conditions, indigenous microbial ecology, the which often undergo stress when they come in contact with the
procedure used to supply the remediation agents into the envi- complexity of the natural habitats (Mrozik et al., 2011; Tyagi et al.,
ronment as well as the individual properties of the inoculants, 2011; Nzila et al., 2016).
226 A. Nowak, A. Mrozik / Journal of Environmental Management 215 (2018) 216e229
Fig. 5. The pattern of carbon sources utilisation, N and P - use indexes for the treated
and untreated soils. The percentage of the absorbance of each group of substrates and
index with the reference to the total absorbance of the plate was presented as a colour
scale ranging from dark green (0e2%) to burgundy (62%). (For interpretation of the
references to colour in this figure legend, the reader is referred to the Web version of
this article.)
Our study also indicated that the presence of P inhibited the rate
of the removal of 4-CP from the treated soil. This was contrary to
the results that were obtained for the bacteria Pseudomonas sp.
CF600 and S. maltophilia KB2 that were cultivated in liquid batch
cultures in which the addition of P accelerated 4-CP degradation
(Nowak and Mrozik, 2016; Nowak et al., 2016). This could be a
result of the competitive inhibition of these two compounds. Such a
phenomenon was described by Hao et al. (2002), who studied the
degradation kinetics of a self-inhibitory growth substrate (P) and a
non-growth (4-CP) substrate in Acinetobacter sp. batch liquid cul-
tures. On the other hand, an intensification of the removal of 4-CP
in the presence of P by activated sludge that had been bio-
augmented with Pseudomonas putida CECT 4064 was reported by
Monsalvo et al. (2012). Contrary to P, the addition of SB to the soil
that had been bioaugmented with Pseudomonas sp. CF600 signifi-
cantly accelerated the removal of 4-CP compared to the control soil.
This stimulation may have resulted from the less toxic effect of SB
on microorganisms compared to P or its easier biodegradability
with the simultaneous induction of the enzymes that are involved
in the catabolic pathways (WHO, 2000; Guzik et al., 2009). The
decline of the total number of THB in all of the treated soils in the
range of 13e21% that was observed in this work was smaller to that
in phenol-contaminated soil (5 mg g1) that had been inoculated
with Pseudomonas sp. JS150 and PCP-contaminated soil
(0.75 mg g1) that had been bioaugmented with immobilised
R. chlorophenolicus PCP-1, which was about 30 and 10%, respectively
(Briglia et al., 1990; Mrozik et al., 2011). It is difficult to make a
comparative analysis of the THB count in bioaugmented soils that
have been exposed to monochlorophenol because there is not any
new information available in this field.
Soil contamination with 4-CP had a significant impact on the
structural and functional diversity of the soil microbial commu-
nities. A fatty acid analysis indicated that essential changes in the
FAME patterns for the polluted S included an increase in the satu-
ration of fatty acids, which was reflected in the membrane hydro-
philicity, mainly through an increase of the content of 3-hydroxy
fatty acids. An increase of the saturated to unsaturated fatty acid
ratio can be considered to be a good indicator of stress conditions.
Furthermore, the shifts in the proportion of the hydroxy, branched,
cyclopropane and straight-chain fatty acids allowed the optimum
hydrophilic-lipophilic balance of the membrane to be achieved,
which is considered to be an important parameter in the adaptation
of cells to aromatic hydrocarbon stress (Segura et al., 2010;
Kaczorek et al., 2013). Additionally, during the degradation of 4-
CP as well as 4-CP and P by soil microbial communities, a signifi-
cant increase in the 19:0 cy u10c percentages in the FAME profiles
was recorded. The appearance of 19:0 cy u10c in the FAME patterns
as an indicator of the removal of phenol from the soil was described
previously in several articles (Mrozik et al., 2008, 2010, 2011).
Interestingly, in the soil that had been contaminated with 4-CP and
SB, no cyclopropane fatty acids were detected. This could be
explained by the lower toxicity of SB compared to P and the
different mechanisms for regulating the membrane fluidity in the
presence of these compounds (Grogan and Cronan, 1997). The in-
crease in the content of 3-hydroxy fatty acids, which are a typical
structural component of the lipopolysaccharides in the outer
membrane of BG-, was in accordance with the increase in the
percentages of marker fatty acids for BG-in all of the studied soils.
At the same time, the increase of the BG-marker distribution was
followed by a decrease in the abundance of the F markers.
Regardless of the soil treatment, the contribution of the microbial
group markers over the course of the experiment can be ordered as
follows: BG- > BGþ>F > Ac. The domination of the BG-in envi-
ronments that have been contaminated with toxic compounds was
revealed in many studies (M€ € et al., 2001; Podio et al., 2008;
annisto
Wasilkowski et al., 2017). As the reported data indicates, the types
of environment and contamination usually determine the devel-
opment of the specified microbial groups and their dominance in
the ecosystem. However, an interpretation of the shifts in the
distinguished microbial groups that depend on contamination with
aromatic compounds has not yet been well documented. Moreover,
the observed changes in the FAME profiles of individual strains
growing on pure hydrocarbons in liquid cultures are often contra-
dictory with regard to those obtained for soil. For example, in our
previous studies, an important change in the cellular fatty acid
composition of S. maltophilia KB2 during the co-metabolic degra-
dation of monochlorophenols was an increase in the contribution
of branched fatty acids, which are considered to be markers of BGþ
(Nowak et al., 2016).
The analysis of the functional diversity of microorganisms in the
treated soil showed distinct differences in their activity, which was
manifested in the various utilisation patterns of the seven
biochemical guilds. The most active microorganisms were found in
the soil that had been contaminated with 4-CP and SB and bio-
augmented with Pseudomonas sp. CF600 as was evidenced by the
high values of the AUC, AWCD and R. The introduction of Pseudo-
monas sp. CF600 into the soil that had been contaminated with 4-
CP and P also caused an increase in the microbial activity but to a
lesser extent than in the corresponding soil that had been polluted
with 4-CP and SB. Meanwhile, in the soil that had been inoculated
with S. maltophilia KB2 and in the non-inoculated soil, the mixture
of 4-CP and P had a toxic effect on the microbial community
 A. Nowak, A. Mrozik / Journal of Environmental Management 215 (2018) 216e229 227

Fig. 6. Cluster display of the seven guilds of carbon sources and FAME microbial markers for the treated and untreated soils over time.

functions. The functional diversity of soil microorganisms that have differences in the utilisation patterns of carbon substrates between
been exposed to aromatic compounds is not well documented or the polluted and non-polluted soils confirmed that the introduction
discussed in the literature. The results of Qasemian et al. (2012), of aromatic compounds into the soil caused irreversible changes in
who found that the microbial activity during three months of the functional diversity of the soil microbial communities.
anthracene exposure remained constant, while H0 decreased, thus
indicating that the microbial communities were more specialised
5. Conclusions
and utilised carbon sources more intensively than those in the
control soil. In our study, the addition of 4-CP or 4-CP and P to the
Our work underscores the importance of soil bioaugmentation
bioaugmented soil did not result in changes in the H0 values;
in the removal of 4-CP, which poses a threat for the environment
however, the H’ index increased in the non-polluted soils on day
and human health. This study demonstrated that soil inoculation
100 of the experiment. These results might indicate that the
with Pseudomonas sp. CF600 or S. maltophilia KB2 as well as the
functional diversity of the microorganisms was primarily affected
addition of an additional aromatic carbon source (P or SB)
by the enrichment of the soil with the external carbon sources. A
enhanced the removal of 4-CP from the soil. Naturally occurring
more detailed analysis of the carbon source utilisation patterns
microflora also participated in the 4-CP degradation in the presence
indicated that the addition of 4-CP, P and SB to the soil caused a
of additional carbon source but to a much lesser extent up to 40%
more intensive utilisation of PA compared to the non-polluted soil.
compared to bioaugmented soils. Soil autochthonous microorgan-
This may indicate the induction of the enzymes that are involved in
isms enriched with Pseudomonas sp. CF600 exhibited better
the degradation of aromatic compounds in microorganisms that
degradative abilities towards aromatic compounds than microbial
exist in contaminated soils. Furthermore, the heterotrophic fraction
populations enriched with S. maltophilia KB2, what indicated a
of bacteria in the contaminated soils and the soil that had been
greater usefulness of Pseudomonas sp. CF600 in the bioremediation
bioaugmented with Pseudomonas sp. CF600 as well as in the non-
of 4-CP contaminated soil. This can also mean that indigenous
bioaugmented soil metabolised AA and A more intensively than
microorganisms in the soil bioaugmented with Pseudomonas sp.
in the non-contaminated soils, which was reflected in the higher N-
CF600 created more synergies between themselves than in the soil
use index. This could suggest that these microorganisms were
inoculated with S. maltophilia KB2. The important finding of this
versatile and were able to use unexpected inputs of various dis-
study was also that all of the tested aromatic compounds affected
solved organic carbon sources in the environment. All of these
the soil microbial community structure as well as they caused
228 A. Nowak, A. Mrozik / Journal of Environmental Management 215 (2018) 216e229
irreversible changes in the functional diversity of the soil microbial
populations. Future studies will be focused on the increasing in the
efficiency of 4-CP degradation in soil as well as the survival of in-
oculants through their immobilisation in different carriers.
Acknowledgment
This work was partly supported by National Science Centre N
N305 061640. We thank Michele Simmons for improving the En-
glish of the manuscript as well as Daniel Wasilkowski and Justyna
Michalska from the University of Silesia in Katowice for valuable
consultation and collaboration.
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