Enhanced of Phenol Degradation by Soil Bioaugmentacion With Pseudomonas Sp JS150
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Transcript of Enhanced of Phenol Degradation by Soil Bioaugmentacion With Pseudomonas Sp JS150
ORIGINAL ARTICLE
Enhancement of phenol degradation by soilbioaugmentation with Pseudomonas sp. JS150A. Mrozik1, S. Miga2 and Z. Piotrowska-Seget3
1 Department of Biochemistry, University of Silesia, Jagiellonska 28, Katowice, Poland
2 Institute of Materials Science, University of Silesia, Bankowa 12, Katowice, Poland
3 Department of Microbiology, University of Silesia, Jagiellonska 28, Katowice, Poland
Introduction
Industrial activities such as oil refineries, gas stations, and
production of pesticides, explosives, paints, textiles, wood
preservatives and agrochemicals release phenol and its
derivatives into the environment. These compounds are
also the products of auto exhaust, and therefore, areas of
high traffic likely contain increased level of phenol (Bud-
avari 1996). Although there is no consistent evidence that
phenol causes cancer in humans, it is stated that long-
term or repeated exposure may cause harmful effects on
the central nervous system, heart, liver, kidney and skin
(Agency for Toxic Substances and Disease Registry 1998).
The additional effect for the toxicity of phenol may be
the formation of phenoxyl radicals (Hanscha et al. 2000).
This is a reason why cleaning up of phenol-contaminated
sites is of a great ecological concern.
There are many methods for the detoxification of
phenol from contaminated soils. As an alternative to
physico-chemical treatments, the use of micro-organism
Keywords
Bioaugmentation, biodegradation, FAMEs,
phenol, Pseudomonas sp. JS150, survival.
Correspondence
Agnieszka Mrozik, Department of
Biochemistry, Faculty of Biology and
Environmental Protection, University of Silesia,
Jagiellonska 28, 40-032 Katowice, Poland.
E-mail: [email protected]
2011 ⁄ 1025: received 21 June 2011, revised
12 August 2011 and accepted 17 August
2011
doi:10.1111/j.1365-2672.2011.05140.x
Abstract
Aims: To test whether bioaugmentation with genetically modified Pseudomonas
sp. JS150 strain could be used to enhance phenol degradation in contaminated
soils.
Methods and Results: The efficiency of phenol removal, content of humic car-
bon, survival of inoculant, number of total culturable autochthonous bacteria
and changes in fatty acid methyl esters (FAME) profiling obtained directly
from soils were examined. Bioaugmentation significantly accelerated phenol
biodegradation rate in tested soils. Phenol applied at the highest concentration
(5Æ0 mg g)1 soil) was completely degraded in clay soil (FC) within 65 days,
whereas in sand soil (FS) within 72 days. In comparison, phenol biodegrada-
tion proceeded for 68 and 96 days in nonbioaugmented FC and FS soils,
respectively. The content of humic carbon remained at the same level at the
beginning and the end of incubation time in all soil treatments. The number
of introduced bacteria (2Æ50 · 109 g)1 soil) markedly decreased during the first
4 or 8 days depending on contamination level and type of soil; however, inocu-
lant survived over the experimental period of time. Analysis of FAME patterns
indicated that changes in the percentages of cyclopropane fatty acids 17:0 cy
and 19:0 cy x10c and branched fatty acids might be useful markers for moni-
toring the progress of phenol removal from soil.
Conclusions: It was confirmed that soil bioaugmentation with Pseudomonas sp.
JS150 significantly enhanced soil activity towards phenol degradation. Cyclo-
propane and branched fatty acids were sensitive probes for degree of phenol
utilization.
Significance and Impact of the Study: In future, genetically modified Pseudo-
monas sp. JS150 strain could be of use in the bioaugmentation of phenol-con-
taminated areas.
Journal of Applied Microbiology ISSN 1364-5072
ª 2011 The Authors
Journal of Applied Microbiology 111, 1357–1370 ª 2011 The Society for Applied Microbiology 1357
processes has become the most promising approach in
remediation technology. Bioremediation is really suited
to vast and moderately contaminated soils and decreases
usually high energy demand and consumption of chemi-
cal reagents (Khan et al. 2004). In recent years, there has
been an increasing interest in developing new techniques
for bioremediation of soils contaminated with toxic
organic pollutants. One of the ways to enhance the effi-
cacy of contaminant removal is bioaugmentation. This
strategy is based on the inoculation of given soils with
micro-organisms either being pure or mixed cultures
characterized with desired catalytic capabilities (Heinaru
et al. 2005; Silva et al. 2009; Guimaraes et al. 2010).
Moreover, genetically modified micro-organisms exhibit-
ing enhanced degradative potential are considered to be
attractive for soil bioaugmentation. It is thought that
bioaugmentation should be applied when the biostimula-
tion and bioattenuation did not bring expected results
(Vogel 1996). Soils that need to be clean may be inocu-
lated with both wild indigenous or allochthonous strains
or laboratory-constructed strains carrying necessary deg-
radative pathways. Several bacterial strains have been
reported to posses the metabolic pathways for the degra-
dation of phenol. The most effective bacteria studied are
represented by strains from genera Burkholderia (Schro-
der et al. 1997), Pseudomonas (Kargi and Serkan 2004;
Yang and Lee 2007; Mrozik et al. 2010), Acinetobacter
(Paller et al. 1995; Mazzoli et al. 2007), Serratia (Prad-
ham and Ingle 2007), Rhodococcus (Goswami et al. 2005;
Nagamani and Lowry 2009) and Ralstonia (Chen et al.
2004).
Effective hydrocarbon-degrading strains are often used
as commercial inocula to enhance the bioremediation of
hydrocarbon-contaminated sites. For example, significant
increase in aromatic compounds biodegradation rate was
achieved by using commercial products such as Sybron
1000, Biozyn 301 and DBC-plus� (Dott et al. 1989;
Sobiecka et al. 2009). Several studies have successfully
applied this strategy for cleaning up polluted soils;
however, results of other experiment indicated its major
limitations (Simon et al. 2004).
A success of bioaugmentation depends on both biotic
and abiotic factors. The most important is a strain selec-
tion (Thompson et al. 2005). Bacteria for bioaugmenta-
tion should survive and multiply in soil as well as to
compete with autochthonous micro-organisms for nutri-
ents and oxygen. Moreover, after soil inoculation, they
should not lose their degradative capacity. Mineralization
rate of organic contaminants is also strongly influenced
by many physico-chemical environmental parameters.
They include chemical structure, bioavailability and con-
centration of pollutants accompanied with soil type, pH,
temperature, salinity, water and oxygen content (Leahy
and Colwell 1990; Davis and Madsen 1996; Stalwood
et al. 2005).
The presence of phenols shows harmful effect on the
biological properties of bacterial cell membrane. Particles
of phenolic substrates partition into phospholipid bilay-
ers resulting in the changes in cytoplasmic membrane
fluidity, stability and permeability (Weber and de Bont
1996). As a response to phenols, many bacteria can adapt
to unfavourable conditions by the modification of fatty
acid composition. The adaptive mechanisms include de
novo synthesis of fatty acids, cis to trans isomerization,
the increase in branched and cyclopropane fatty acid
content and alteration in lipid-to-protein ratio (Diefen-
bach et al. 1992; Heipieper and de Bont 1994; Kaur et al.
2005; Fischer et al. 2010). Based on these considerations,
changes in bacterial fatty acid composition may be used
as a marker for monitoring the process of bioremedia-
tion.
Materials and methods
Bacterial strain and culture conditions
Bacterial strain Pseudomonas sp. JS150 was kindly pro-
vided by Dr J. Spain from Air Force Civil and Engineer-
ing Support Agency, Tyndall Air Force Base, Florida,
USA. Pseudomonas sp. JS150 is a nonencapsulated mutant
of strain JS1 obtained after ethyl methanesulfonate muta-
genesis. It is known as an efficient degrader of phenol
and other aromatic compounds such as toluene, benzene,
benzoate, salicylate and naphthalene (Haigler et al. 1992).
This strain was routinely grown at 30�C on nutrient agar
medium and in Kojima mineral liquid medium (Kojima
et al. 1961) supplemented with phenol at the concentra-
tion of 752 mg l)1.
Soils
Soil samples were collected from the top layer of 5–20 cm
at two distinct sites localized close to Sosnowiec (Upper
Silesia, Poland). Soils came from mixed and pine forests
and were signed as FC and FS, respectively. No phenol
contamination was determined in these soils. Prior to
experiment, the air-dried soil samples at room tempera-
ture were sieved (2 mm) and transferred to plastic pots
(150 g). Physical and chemical properties of each soil are
presented in Table 1.
For experiment purpose, triplicate portions of FC and
FS soils were amended with phenol at three concentra-
tions: 1Æ7, 3Æ3 and 5Æ0 mg g)1 and pre-incubated for
1 day. Such phenol concentrations were significantly
(1000 times) higher than in similar biodegradation stud-
ies. Part of soil samples was additionally inoculated with
Soil bioaugmentation A. Mrozik et al.
1358 Journal of Applied Microbiology 111, 1357–1370 ª 2011 The Society for Applied Microbiology
ª 2011 The Authors
phenol-degrading Pseudomonas sp. JS150. For soil bioaug-
mentation, bacteria were cultured in 250 ml of nutrient
broth medium (Becton Dickinson, Franklin Lakes, NJ,
USA) at 28�C on rotary shaker at 125 rev min)1 to reach
the mid-logarithmic growth phase. Then, cultures were
centrifuged (8000 g), and pellets were washed twice
(0Æ85% NaCl). After this, the pellets were resuspended in
sterile NaCl. Next 15 ml of this suspension was poured
into pot resulting in 2Æ50 · 109 bacteria per gram of soil.
The final water content of the soils was adjusted to about
50% of the maximum water-holding capacity. All pots
were kept in a chamber cabinet at room temperature.
Biodegradation and survival experiments
To estimate phenol removal in bioaugmented and non-
bioaugmented FC and FS soils, samples were taken on 1,
4 and 8 days and next at 8-day intervals. Phenol was
extracted from soil with methanol, and its concentration
was determined by colorimetric method with diazoate
p-nitroaniline at the wavelength 550 nm (Lurie and
Rybnikova 1968).
For monitoring the survival of inoculant, the spontane-
ous rifampicin-resistant mutant of Pseudomonas sp. JS150
was used. On the sampling days, the numbers of inocu-
lant and total heterotrophic bacteria were calculated in
bioaugmented soils, whereas in nonbioaugmented, only
total heterotrophic bacteria were determined. For this
purpose, 5 g of soil was placed into Erlenmeyer flasks
containing 45 ml of 0Æ85% NaCl for shaking (30 min,
125 rev min)1) and preparing serial 10-fold dilutions for
plate counts. Nutrient agar supplemented with rifampicin
at the concentration of 100 lg ml)1 and nutrient agar
were used for counting the number of inoculant and total
number of bacteria, respectively. Inoculated plates were
incubated at 28�C for 48 h. Data are representative of
three individual experiments. At the beginning and the
end of experiments, organic matter, organic carbon and
humic carbon contents were determined in all soil treat-
ment. The procedure of humic substance extraction from
soil was described in detail in previous article (Mrozik
et al. 2008).
midi-FAME analysis
Fatty acid analyses were performed on the same days
when phenol concentration and survival of inoculants
were determined. Duplicate samples of 5 g of each soil
were extracted according the procedure by Kozdroj
(2000) and identified using the Microbial Identification
System (Microbial ID Inc., Newark, Delaware, USA) stan-
dard protocol (Sasser 1990). The procedure of fatty acid
extraction and methylation was carried out as described
previously (Mrozik et al. 2010). Fatty acids were analysed
by gas chromatograph (Hewlett-Packard 6890, Santa
Clara, CA, USA) equipped with capillary column Ultra
2-HP (5% phenylmethyl silicone; 25 m, 0Æ22 mm ID, film
thickness 0Æ33 mm) and flame ionisation detector. Peaks
from chromatograms were identified using midi software
(Sherlock aerobe method and TSBA library ver. 5.0).
Data analysis
Decay process can be described by several types of func-
tions, e.g. exponential, bi-exponential, stretched exponen-
tial, inverse logarithmic and power law (Dec et al. 2007).
The simplest function is the exponential one. This func-
tion g(t) = g0e)t ⁄ s (where g0 and g(t) are the initial and
after time t concentrations of phenol, respectively, and sis relaxation time of described process) depends on two
parameters g0 and s only. It is important for the analysis
of experimental data containing a few points only (see
Fig. 1a1). Relaxation time has very clear interpretation –
during this time, phenol concentration decreases by about
63Æ2%. The exponential function describes very well all
our temporary data (uncertainty of estimated parameters
is relatively low, and R2 coefficient is close to the unity).
Additionally, similar function was successfully used for
the analysis of degradation rate constant, rate of disap-
pearance and disappearance time for phenol in different
soils inoculated with Pseudomonas sp. CF600 (Mrozik
et al. 2010). Therefore, for quantitative analysis of phenol
degradation process, the exponential function has been
chosen. The least square method was used for fitting the
exponential function to an experimental data. This way
values of g0 and s and their uncertainty were estimated.
Table 1 Characteristics of soils
Soil property
Clay
(FC)
Sand
(FS) Method ⁄ source
Sand (%) 59 96 PN-R-04032:1998
Silt (%) 31 4 PN-R-04032:1998
Clay (%) 10 0 PN-R-04032:1998
Density (g cm)3) 0Æ57 1Æ17 PN-88 ⁄ B-04481
pH (H2O) 6Æ02 6Æ89 PN-ISO 10390:1997
Organic matter
(% d.w)
29Æ5 1Æ9 Combustion
Total organic
carbon (% d.w)
7Æ9 0Æ61 PN-Z-15011-3
C hum (% d.w)* 1Æ38 0Æ24 Litynski et al. (1972)
CEC (cmol + kg)1) 13Æ7 1Æ9 ISO 23470:2007
P2O5 (mg 100 g)1) 0Æ05 0Æ06 PN-R-04023:1996
K2O (mg 100 g)1) 41Æ5 5Æ0 PN-R-04022:1996
Conductivity (lS cm)1) 99Æ3 30Æ3 PN-ISO 11265 + AC1:1997
*Total humic and fulvic acids.
A. Mrozik et al. Soil bioaugmentation
ª 2011 The Authors
Journal of Applied Microbiology 111, 1357–1370 ª 2011 The Society for Applied Microbiology 1359
Results
Biodegradation studies
Phenol degradation experiments were carried out in clay
(FC) and sand (FS) soils varied in their physico-chemical
parameters (Table 1). Such soils were chosen to compare
the degradation rate in soils distinctly differed in organic
matter and carbon content. Owing to phenolic com-
pounds participate in forming of humic and fulvic acid
structure, additionally the content of total humic carbon
was determined at the beginning and the end of the
experiments (Tables 2 and 3). To assess the impact of
phenol-degrading bacteria on phenol biodegradation rate,
Table 2 Selected FC soil parameters at the beginning and the end of experiment
Soil parameter
FC soil
FCP1Æ7 FCP1Æ7+B FCP3Æ3 FCP3Æ3+B FCP5Æ0 FCP5Æ0+B
Day 1 Day 24 Day 1 Day 8 Day 1 Day 36 Day 1 Day 24 Day 1 Day 68 Day 1 Day 56
Organic matter (% d.w.) 29Æ50 28Æ78 29Æ84 29Æ23 29Æ61 28Æ71 29Æ91 28Æ76 30Æ21 28Æ12 30Æ31 27Æ91
Organic carbon (% d.w.) 7Æ90 7Æ66 7Æ99 7Æ80 8Æ04 7Æ44 8Æ14 7Æ68 8Æ32 7Æ20 8Æ37 7Æ51
C hum (% d.w.) 1Æ39 1Æ37 1Æ38 1Æ39 1Æ40 1Æ42 1Æ41 1Æ43 1Æ42 1Æ45 1Æ41 1Æ45
pH 5Æ82 6Æ12 5Æ91 6Æ55 5Æ54 6Æ33 5Æ66 6Æ18 5Æ42 6Æ27 5Æ51 6Æ23
Values are the means of three replicates (standard errors <5%).
Table 3 Selected FS soil parameters at the beginning and the end of experiment
Soil parameter
FS soil
FSP1Æ7 FSP1Æ7+B FSP3Æ3 FSP3Æ3+B FSP5Æ0 FSP5Æ0+B
Day 1 Day 56 Day 1 Day 32 Day 1 Day 64 Day 1 Day 48 Day 1 Day 96 Day 1 Day 72
Organic matter (% d.w.) 1Æ99 1Æ89 2Æ06 1Æ97 2Æ16 2Æ07 2Æ22 2Æ00 2Æ21 1Æ97 2Æ28 1Æ84
Organic carbon (% d.w.) 0Æ66 0Æ62 0Æ68 0Æ62 0Æ68 0Æ59 0Æ71 0Æ67 0Æ79 0Æ69 0Æ70 0Æ62
C hum (% d.w.) 0Æ24 0Æ23 0Æ22 0Æ21 0Æ22 0Æ22 0Æ22 0Æ23 0Æ22 0Æ25 0Æ23 0Æ25
pH 6Æ59 6Æ72 6Æ51 6Æ82 6Æ44 6Æ64 6Æ40 6Æ69 6Æ19 6Æ61 6Æ21 6Æ71
Values are the means of three replicates (standard errors <5%).
0
1
2
3
4
5 (a1)P
heno
l (m
g g–
1 )
0 20 400
1
2
3
4
5
(a2)
= 12 ± 1 days= 10·8 ± 1·8 days
= 3·6 ± 1·1 days
= 26·2 ± 2·7 days
= 6·1 ± 0·7 days = 16·6 ± 1·6 days = 25·3 ± 3·0 days
= 33·4 ± 3·2 days= 24·4 ± 1·7 days
= 7·3 ± 0·8 days
40Time (Days)
(a3)
= 33·4 ± 3·2 days
= 25·3 ± 3·0 days
4060 80 0 20 60 80 0 20 60 80
(b3)(b2)(b1)
τFC5·0
τFC5·0+B
τFC3·3+Bτ
FC1·7+B
τFS1·7+B
τFS3·3+B
τFS5·0+B
τFS5·0
τFS3·3
τFS1·7
τFC1·7
τFC3·3
Figure 1 Dynamics of phenol degradation in
bioaugmented (j) and nonbioaugmented (h)
FC and FS soils contaminated with phenol at
the concentrations of 1Æ7 mg g)1 (a1, b1),
3Æ3 mg g)1 (a2, b2) and 5Æ0 mg g)1 (a3, b3).
Soil bioaugmentation A. Mrozik et al.
1360 Journal of Applied Microbiology 111, 1357–1370 ª 2011 The Society for Applied Microbiology
ª 2011 The Authors
a part of soil samples were inoculated with Pseudomonas
sp. JS150. This allowed us to compare the rate of phenol
removal by indigenous micro-organisms exhibiting tested
soils and enriched with inoculated strain with high cata-
bolic potential.
The results clearly showed that bioaugmentation signif-
icantly accelerated phenol degradation in tested soils and
was correlated with the type of soil. In FC soil contami-
nated with the concentrations of 1Æ7, 3Æ3 and 5Æ0 mg g)1
soil (FC1Æ7, FC3Æ3 and FC5Æ0) and inoculated with Pseudo-
monas sp. JS150 (FC1Æ7+B, FC3Æ3+B and FC5Æ0+B), phenol
was completely degraded within 8, 24 and 56 days,
respectively (Fig. 1a1,a2,a3). The same doses of phenol in
contaminated and inoculated FS soils (FS1Æ7+B, FS3Æ3+B
and FS5Æ0+B) were degraded slower, within 32, 48 and
72 days, respectively (Fig. 1b1,b2,b3). In contrast, in both
nonbioaugmented FC and FS soils, phenol removal lasted
2–3 weeks longer (Fig. 1). Moreover, in soils inoculated
with Pseudomonas sp. JS150, the removal of 50% of phe-
nol (DT50 = )sÆln 0Æ5) proceeded significantly faster as
compared to nonbioaugmented soils. For example, DT50
for FC1Æ7+B and FC5Æ0+B were 2Æ5 and 7Æ1 days, whereas in
nonbioaugmented FC soils, FC1Æ7 and FC5Æ0 were 7Æ5 and
14 days, respectively. In comparison, in FS1Æ7+B and
FS5Æ0+B treated with the same phenol concentrations,
DT50 reached the value of 4Æ2 and 17Æ5 days for bioaug-
mented soils and 18Æ2 and 23Æ2 days for FS1Æ7 and FS5Æ0,
respectively.
Figure 2 shows the estimated values of the relaxation
time (s) for biodegradation processes. Bacterial inocula-
tion of soil with phenol at the concentration of
1Æ7 mg g)1 soil increased 3- and 4-fold the rate of phenol
removal for FC1Æ7+B and FS1Æ7+B, respectively. In turn, in
bioaugmented FC and FS soils with higher phenol dos-
ages, the biodegradation rate was lower; however, it was
still remarkably higher as compared to nonbioaugmented
FC and FS soils. In both soils with low phenol concentra-
tion, biodegradation rate was almost constant, but above
3Æ3 mg of phenol g)1 soil, its degradation rate was lower.
In bioaugmented FC and FS soils, time for phenol
removal was almost linear function of initial phenol
concentration.
Microbial numbers
During biodegradation studies, the survival of Pseudomo-
nas sp. JS150 and total heterotrophic bacteria was deter-
mined in both phenol-contaminated FC and FS soils. The
number of total culturable bacteria was also counted in
phenol-polluted and nonbioaugmented soils. Obtained
data indicated that Pseudomonas sp. JS150 introduced to
FC and FS soils survived during experimental period;
however, cell number decreased over time. The observed
decrease strongly depended on soil type and the level of
phenol contamination. In FC1Æ7+B soil, the number of
inoculant decreased from 2Æ5 · 109 g)1 soil on day 0 to
3Æ2 · 107 g)1 on day 8, when phenol was completely
degraded (Fig. 3a1). In comparison, in FS1Æ7+B soil on day
8, the number of Pseudomonas sp. JS150 cells reached the
value of 5Æ9 · 106 g)1 and on day 32 after phenol degra-
dation, it reached 5Æ0 · 105 g)1 soil (Fig. 3b1). In turn, in
FC5Æ0+B and FS5Æ0+B soils, number of inoculant on day 56
declined to 6Æ4 · 103 and 5Æ3 · 102 g)1, respectively, and
finally on day 72, it was equal to 2Æ5 · 102 g)1 in FS5Æ0+B
soil (Fig. 3a3,b3).
Similarly as for Pseudomonas sp. JS150, the number of
total autochthonous bacteria decreased in both contami-
nated and bioaugmented FC and FS soils. The more phe-
nol pollution, the stronger decline in bacterial counts was
observed. In FC1Æ7+B soil, the number of heterotrophic
bacteria was reduced from initial 2Æ5 · 108 to
3Æ3 · 106 g)1 on day 24, whereas in the same soil exposed
to the highest phenol concentration, it was reduced to
1Æ5 · 104 g)1 on day 56. However, in FS1Æ7+B bacteria,
number decreased from 8Æ0 · 105 to 1Æ0 · 104 CFU g)1
soil on day 32 and in FS5Æ0+B to 1Æ0 · 102 on day 72
(Fig. 3).
Data analysis of bacterial numbers in nonbioaugmented
and contaminated soils showed that phenol applied at
increasing concentrations in different degree decreased
the number of autochthonous bacteria. The highest
decline in bacterial counts was observed during the first
4 days of the experiment in both FC5Æ0 and FS5Æ0 soils.
From that sampling time till the end of phenol degrada-
tion, numbers of bacteria maintained at the similar level
(Fig. 3a3,b3). In turn, the smallest decrease in bacterial
number was determined in soils polluted with the lowest
phenol concentration.
2 3 4 50
10
20
30
τ (d
ays)
Phenol (mg g–1)
Figure 2 Phenol concentration dependences of relaxation time. ( )
FCP; ( ) FCP+B; ( ) FSP and ( ) FSP+B.
A. Mrozik et al. Soil bioaugmentation
ª 2011 The Authors
Journal of Applied Microbiology 111, 1357–1370 ª 2011 The Society for Applied Microbiology 1361
Fatty acids analysis
In the study, the impact of phenol contamination and
bioaugmentation with Pseudomonas sp. JS150 on soil fatty
acid profiles was analysed. To make the comparison of
fatty acid methyl esters (FAME) profiles, all extracted
fatty acids were grouped into five major classes: straight-
chain, branched, hydroxylated, cyclopropane and unsatu-
rated fatty acids. Both phenol and bioaugmentation
influenced the soil FAME profiles that changed over the
experimental period. The most visible changes under phe-
nol exposure between augmented and nonbioaugmented
soils involved the abundance of branched and cyclopro-
pane fatty acids. For example, at the beginning of the
experiment in FC1Æ7+B soil, the percentage of branched
fatty acids in FAME profiles composed 22Æ47%, while in
nonbioaugmented, it was significantly lower and consti-
tuted 3Æ48% of total fatty acids only (Table 4). In com-
parison, in control soil (nonbioaugmented and
nonpolluted), their content reached the value of 2Æ83%.
Over phenol degradation, the content of branched fatty
acids increased in FC1Æ7+B to 28Æ53% on day 8 and to
Table 4 The percentages of distinct groups of fatty acids isolated from nonbioaugmented and bioaugmented FC soil during phenol degradation
at the concentration 1Æ7 mg g)1
Time FC soil
Total fatty acids (weight %)
Saturated
UnsaturatedStraight-chain Branched Hydroxylated
Total
cyclopropane
Cyclopropane
17:0 cy 19:0 cy x10c
Day 1 FCP 33Æ05 3Æ48 2Æ29 12Æ38 12Æ38 0Æ00 48Æ80
FCP+B 37Æ96 22Æ47 4Æ39 15Æ38 15Æ38 0Æ00 19Æ80
Day 4 FCP 36Æ49 8Æ03 3Æ05 14Æ65 0Æ51 14Æ14 37Æ78
FCP+B 39Æ23 28Æ53 1Æ54 17Æ25 0Æ66 16Æ59 13Æ45
Day 8 FCP 38Æ07 8Æ94 3Æ33 19Æ21 0Æ17 19Æ04 30Æ45
FCP+B 38Æ78 25Æ51 1Æ41 14Æ98 0Æ99 13Æ99 19Æ32
Day 16 FCP 37Æ48 10Æ67 3Æ68 16Æ69 0Æ00 16Æ69 31Æ48
FCP+B ND ND ND ND ND ND ND
Day 24 FCP 34Æ66 6Æ15 2Æ39 16Æ48 3Æ57 12Æ91 40Æ32
FCP+B ND ND ND ND ND ND ND
Values are the means of three replicates (standard errors <5%).
FCP, contaminated and nonbioaugmented soil; FCP+B, contaminated and bioaugmented soil; ND, not determined.
FS soil
Time (days)
109
107
105
103
109
107
105
103
109
107
105
103
FC soil (a1) (b1)
(b2)
(b3)
(a2)
(a3)
CF
U g
–1 s
oil
0 10 20 30 40 50 60 70 80 900 10 20 30 40 50 60
Figure 3 The number of introduced and
total bacteria in bioaugmented and nonbio-
augmented FC and FS soils contaminated
with phenol at the concentration 1Æ7 mg g)1
(a1, b1), 3Æ3 mg g-1 (a2, b2) and 5Æ0 mg g)1
(a3, b3). (—); control soil; ( ) with phenol;
( ) with phenol and bacteria (total) and
( ) with phenol and bacteria (inoculant).
Soil bioaugmentation A. Mrozik et al.
1362 Journal of Applied Microbiology 111, 1357–1370 ª 2011 The Society for Applied Microbiology
ª 2011 The Authors
10Æ68% on day 16 in FC1Æ7. Similarly, in FC5Æ0+B, the
highest increase in percentages of branched fatty acids
(29Æ94%) was noticed during the first 4 days of the incu-
bation and then maintained at the similar level. In con-
trast, in FC5Æ0, content of branched fatty acids increased
about four times from day 1 to day 24 and then gradually
declined (Table 6). Phenol contamination caused also the
appearance of new fatty acids that were not present in
untreated soil. They were mainly represented by branched
fatty acids such as 13:0 iso, 13:0 anteiso, 14:0 iso,
16:0 anteiso, 17:0 anteiso and hydroxylated 16:0 2OH and
16:0 3OH; however, their contribution in FAME profiles
was low and did not exceed 1% of total fatty acids (data
not shown).
The another significant changes in FAME profiles of
tested soils were related to cyclopropane fatty acid abun-
dance. In FC soil, independently of phenol concentrations
used the content of 17:0 cy strongly declined during the
first 4 days of the experiment. In the following days, its
contents in FAME patterns still decreased, even to 0%.
Interestingly, the other cyclic fatty acid 19:0 cy x10c
appeared only from day 4. In general, its abundance
depended on the degree of phenol utilization. In bioaug-
mented FC soils, the highest contents of 19:0 cy x10c
were detected between 4 and 8 days when about 60% of
phenol added was degraded. However, in nonbioaug-
mented FC soil polluted with lower phenol doses (1Æ7and 3Æ3 mg g)1), the highest content of this fatty acid was
found when about 50% of phenol was removed (day 8),
whereas in soil with phenol at the concentration of
5Æ0 mg of g)1, it was found when above 70% of this pol-
lutant was degraded (day 32) (Tables 4–6).
Similar responses of bacterial communities to phenol
were found in both augmented and nonbioaugmented FS
soil. Observed changes included alterations in the amount
of branched and cyclopropane fatty acids. In augmented
FS soil, phenol treatment caused the increase in branched
fatty acid content from day 1 to day 8 (1Æ7 mg g)1) and
day 24 (3Æ3 and 5Æ0 mg g)1) (Tables 7–9). In nonbioaug-
mented soil, the abundance of branched fatty acids also
increased at the first days of incubation; however, it was
about four times lower as compared to bioaugmented FS
soil. During the following days till the end of the experi-
ment in both soils, the amount of branched fatty acids in
FAME profiles decreased (Tables 7–9).
Changes in cyclopropane fatty acids were related to a
decrease in 17:0 cy content and appearance of 19:0 cy
x10c over the experimental period. In phenol-polluted
soil with the dosages of 1Æ7 and 3Æ3 mg g)1 and bioaug-
mented FS soil, the highest abundance of 19:0 cy x10c in
FAME profiles was observed on day 16 when 60–80% of
contaminant was degraded, whereas in FS5Æ0+B, it was
observed on day 56 when phenol was almost completely
degraded (Tables 7–9). No such effect was observed in
phenol-contaminated but nonbioaugmented soils. In con-
trast to contaminated FC soils, in FS soils under phenol
Table 5 The percentages of distinct groups of fatty acids isolated from nonbioaugmented and bioaugmented FC soil during phenol degradation
at the concentration 3Æ3 mg g)1
Time FC soil
Total fatty acids (weight %)
Saturated
UnsaturatedStraight-chain Branched Hydroxylated
Total
cyclopropane
Cyclopropane
17:0 cy 19:0 cy x10c
Day 1 FCP 33Æ55 3Æ41 2Æ26 12Æ33 12Æ33 0Æ00 48Æ45
FCP+B 38Æ11 24Æ11 4Æ37 15Æ49 15Æ49 0Æ00 17Æ92
Day 4 FCP 36Æ86 8Æ28 3Æ19 17Æ02 1Æ27 15Æ75 34Æ65
FCP+B 39Æ94 30Æ16 1Æ67 16Æ63 0Æ41 16Æ22 11Æ60
Day 8 FCP 37Æ09 9Æ67 3Æ26 20Æ85 0Æ80 20Æ05 29Æ13
FCP+B 38Æ11 30Æ27 1Æ30 20Æ06 0Æ00 20Æ06 10Æ26
Day 16 FCP 37Æ64 11Æ07 3Æ17 19Æ62 1Æ01 18Æ61 28Æ50
FCP+B 36Æ50 28Æ80 1Æ84 14Æ92 1Æ58 13Æ34 17Æ94
Day 24 FCP 35Æ18 8Æ47 3Æ12 16Æ26 1Æ77 14Æ49 36Æ97
FCP+B 37Æ02 27Æ67 1Æ94 14Æ35 3Æ51 10Æ84 19Æ02
Day 32 FCP 34Æ88 7Æ06 2Æ84 13Æ33 3Æ12 10Æ21 41Æ89
FCP+B ND ND ND ND ND ND ND
Day 36 FCP 34Æ11 6Æ24 2Æ51 12Æ57 7Æ66 4Æ91 44Æ57
FCP+B ND ND ND ND ND ND ND
Values are the means of three replicates (standard errors <5%).
FCP, contaminated and nonbioaugmented soil; FCP+B, contaminated and bioaugmented soil; ND, not determined.
A. Mrozik et al. Soil bioaugmentation
ª 2011 The Authors
Journal of Applied Microbiology 111, 1357–1370 ª 2011 The Society for Applied Microbiology 1363
exposure, any new fatty acids as compared to untreated
FS soil were detected.
Discussion
In this study, we demonstrated that soil inoculation with
Pseudomonas sp. JS150 characterized by high catabolic
potential towards many aromatic compounds is an effec-
tive way to enhance the rate of phenol removal and soil
restoration. Its capability to degrade a wide range of con-
taminants was achieved by genetic modification through
mutagenesis (Haigler et al. 1992). Bacteria from genus
Pseudomonas are known to have versatile metabolic capa-
bilities, and therefore, they are often used to increase the
efficiency of aromatic compounds mineralization in con-
taminated sites (Stalwood et al. 2005; Das and Mukherjee
2007; Juhanson et al. 2009; Karamalidis et al. 2010; Afzal
et al. 2011).
Degradation studies revealed that soil bioaugmentation
significantly accelerated the rate of phenol degradation as
compared to nonbioaugmented soils. While in FC5Æ0 soil
this process lasted 68 days, in FC5Æ0+B, it proceeded
2 weeks shorter. In FS5Æ0+B, phenol was completely
degraded 24 days faster than in nonbioaugmented soil.
The enhanced catabolic potential by soil inoculation with
specialized bacterial single strain was well documented in
many studies. For example, Teng et al. (2010) reported
that soil bioaugmentation by Paracoccus sp. strain HPD-2
decreased total polycyclic aromatic hydrocarbons (PAHs)
concentrations from 9942 to 7638 lg kg)1 dry soil after
28 days, whereas it decreased only to 9601 lg kg)1 in
noninoculated control soil. In other study, Wang et al.
(2004) showed that after introduction of Burkholderia
picketti into soil, quinoline at the concentration of
1 mg g)1 soil was completely removed within 6 and 8 h
with and without combined effect of indigenous bacteria.
The final effect of soil bioaugmentation depends on the
ability of inoculant to colonize soil niches and compete
with autochthonous micro-organisms. Data on the sur-
vival of introduced cell are contradictory. In most studies,
a sharp decrease in inoculant number was observed
immediately after inoculation (between 4 and 7 days after
Table 6 The percentages of distinct groups of fatty acids isolated from nonbioaugmented and bioaugmented FC soil during phenol degradation
at the concentration 5Æ0 mg g)1
Time FC soil
Total fatty acids (weight %)
Saturated
UnsaturatedStraight-chain Branched Hydroxylated
Total
cyclopropane
Cyclopropane
17:0 cy 19:0 cy x10c
Day 1 FCP 33Æ56 3Æ39 2Æ38 12Æ27 12Æ27 0Æ00 48Æ40
FCP+B 38Æ76 22Æ96 4Æ11 15Æ36 15Æ36 0Æ00 18Æ81
Day 4 FCP 35Æ72 8Æ35 2Æ97 16Æ78 0Æ56 16Æ22 36Æ18
FCP+B 41Æ19 29Æ94 0Æ65 18Æ36 0Æ00 18Æ36 9Æ86
Day 8 FCP 37Æ13 10Æ93 2Æ66 21Æ33 0Æ00 21Æ33 27Æ95
FCP+B 40Æ10 28Æ09 0Æ52 23Æ44 0Æ00 23Æ44 7Æ85
Day 16 FCP 38Æ76 12Æ40 2Æ80 22Æ29 0Æ00 22Æ29 23Æ75
FCP+B 40Æ31 28Æ66 0Æ57 22Æ41 0Æ00 22Æ41 8Æ05
Day 24 FCP 38Æ88 12Æ73 2Æ77 22Æ99 0Æ00 22Æ99 22Æ63
FCP+B 40Æ05 28Æ89 0Æ83 21Æ66 0Æ00 21Æ66 8Æ57
Day 32 FCP 38Æ51 12Æ66 2Æ74 24Æ71 0Æ00 24Æ71 21Æ38
FCP+B 41Æ16 29Æ19 0Æ84 18Æ44 0Æ00 18Æ44 10Æ37
Day 40 FCP 38Æ01 12Æ57 2Æ63 22Æ61 0Æ00 22Æ61 24Æ18
FCP+B 39Æ55 28Æ02 0Æ93 16Æ22 0Æ00 16Æ22 15Æ28
Day 48 FCP 37Æ87 12Æ41 2Æ85 19Æ44 0Æ00 19Æ44 27Æ43
FCP+B 39Æ04 28Æ16 1Æ12 15Æ18 0Æ47 14Æ71 16Æ50
Day 56 FCP 37Æ74 11Æ10 2Æ66 17Æ21 0Æ00 17Æ21 31Æ29
FCP+B 38Æ99 27Æ01 1Æ54 12Æ56 2Æ26 10Æ40 19Æ90
Day 64 FCP 36Æ84 9Æ15 2Æ49 15Æ01 0Æ00 15Æ01 36Æ51
FCP+B ND ND ND ND ND ND ND
Day 68 FCP 36Æ01 8Æ45 2Æ53 13Æ01 0Æ97 12Æ04 40Æ00
FCP+B ND ND ND ND ND ND ND
Values are the means of three replicates (standard errors <5%).
FCP, contaminated and nonbioaugmented soil; FCP+B, contaminated and bioaugmented soil; ND, not determined.
Soil bioaugmentation A. Mrozik et al.
1364 Journal of Applied Microbiology 111, 1357–1370 ª 2011 The Society for Applied Microbiology
ª 2011 The Authors
inoculation) and then maintained at the similar level for
a long time, while others found that number of intro-
duced cell slightly decreased or even increased over time.
In our studies, the number of Pseudomonas sp. JS150
instantly decreased during the first 4 days in contami-
nated FC and FS soils. The observed decrease depended
on the concentrations of phenol added. The higher phe-
nol doses were applied, and the higher cell count decline
was observed. Similarly, initial decreasing of CFU of Pseu-
domonas aeruginosa was found by Nasseri et al. (2010),
who studied the effect of bioaugmentation on phenan-
threne degradation. However, in contrast to our study,
the decline in introduced bacteria was followed by the
4- to 6-fold increase in bacterial counts during 2 months.
As showed by Juhanson et al. (2009), introduced Pseudo-
monas strains could survive and demonstrate their cata-
bolic traits at phenol-contaminated soil even 40 months
after inoculation. The correlation between CFU numbers
of inoculants and contamination level was observed by
Sejakova et al. (2009) studying the effect of Comamonas
testosteroni CCM7530 inoculation on pentachlorophenol
(PCP) biotransformation. In bioaugmented Fluvisol soil
containing 10 mg PCP kg)1, number of CFUs decreased
over 7 days and then increased till day 17, whereas in soil
with 100 mg PCP kg)1, number of CFUs rapidly
increased from day 1 to 17. The observed decrease in
introduced cells may be explained by the fact that bacte-
rial inoculants cultured in laboratory optimum conditions
undergo stress when enter natural soil. The fate of intro-
duced strains depends on several abiotic and biotic factors
such as fluctuations in temperature, water content, pH,
lack of nutrients as well a level of contaminants and
interactions with indigenous organisms (Mrozik and
Piotrowska-Seget 2010; Tyagi et al. 2011). What is impor-
tant, stress because of drastic changes in environmental
conditions may lead to loss of microbial viability and
even death of inoculated cells (Goldstein et al. 1985; van
Veen et al. 1997; Liu et al. 2009). Sometimes unfavour-
able environmental circumstances, especially during the
first days after inoculation, may be a reason that bio-
augmentation does not enhance the degradative potential
of contaminated soil (Mariano et al. 2009; Silva et al.
2009; Ruberto et al. 2010).
A success of microbial survival, phenols removal and
subsequently the efficiency of their degradation were
strongly correlated with soil organic matter content. In
our study, we revealed that phenol biodegradation rate
was significantly faster in soil with higher organic matter
content what had especially seen in soils polluted with
the highest phenol dose. The organic matter, especially
Table 7 The percentages of distinct groups of fatty acids isolated from nonbioaugmented and bioaugmented FS soil during phenol degradation
at the concentration 1Æ7 mg g)1
Time FS soil
Total fatty acids (weight %)
Saturated
UnsaturatedStraight-chain Branched Hydroxylated
Total
cyclopropane
Cyclopropane
17:0 cy 19:0 cy x10c
Day 1 FSP 39Æ26 6Æ21 0Æ00 11Æ00 11Æ00 0Æ00 43Æ53
FSP+B 38Æ66 26Æ81 1Æ48 12Æ41 12Æ41 0Æ00 20Æ24
Day 4 FSP 38Æ70 6Æ54 0Æ00 11Æ95 11Æ95 0Æ00 42Æ81
FSP+B 40Æ06 27Æ21 1Æ01 11Æ21 11Æ21 0Æ00 20Æ51
Day 8 FSP 40Æ13 6Æ89 0Æ00 10Æ78 5Æ77 5Æ01 42Æ20
FSP+B 44Æ06 28Æ13 1Æ28 11Æ15 3Æ94 7Æ21 15Æ38
Day 16 FSP 42Æ64 7Æ77 0Æ00 11Æ16 6Æ17 4Æ99 38Æ43
FSP+B 47Æ38 26Æ72 1Æ93 12Æ25 1Æ66 10Æ59 11Æ72
Day 24 FSP 42Æ44 7Æ90 0Æ00 11Æ63 7Æ12 4Æ51 38Æ03
FSP+B 45Æ33 25Æ59 1Æ16 10Æ21 2Æ68 7Æ53 17Æ71
Day 32 FSP 41Æ76 7Æ59 0Æ00 11Æ90 9Æ16 2Æ74 38Æ75
FSP+B ND ND ND ND ND ND ND
Day 40 FSP 41Æ54 7Æ10 0Æ00 12Æ28 10Æ01 2Æ27 39Æ08
FSP+B ND ND ND ND ND ND ND
Day 48 FSP 41Æ62 7Æ33 0Æ00 12Æ00 10Æ10 1Æ90 39Æ05
FSP+B ND ND ND ND ND ND ND
Day 56 FSP 40Æ65 7Æ05 0Æ00 10Æ89 10Æ15 0Æ74 41Æ41
FSP+B ND ND ND ND ND ND ND
Values are the means of three replicates (standard errors <5%).
FSP, contaminated and nonbioaugmented soil; FSP+B, contaminated and bioaugmented soil; ND, not determined.
A. Mrozik et al. Soil bioaugmentation
ª 2011 The Authors
Journal of Applied Microbiology 111, 1357–1370 ª 2011 The Society for Applied Microbiology 1365
humic substances, are considered to be growth-promoting
and protective factors against harmful organic com-
pounds for soil micro-organisms. The protective character
of humic acids is connected with their ability to bind
recalcitrant contaminants, reduce their bioavailability and
limit the toxicity for soil microbiota (Nam and Kim
2002). It is known that phenolic carbon, which is enzy-
matically incorporated into humic acids, is much more
stable against biodegradation in soil than the carbon of
free phenols (Vinken et al. 2005). In this study, the con-
tent of humic carbon did not change during biodegrada-
tion experiments suggesting that aromatic carbon did not
strongly incorporate into humic substances. Phenol could
reversibly associate with humic substances and after
releasing phenolic particles were subjected to biodegrada-
tion. Similarly, we did not observe significant changes in
the humic material content during phenol dissipation in
contaminated sterile MF and WM soils inoculated with
Pseudomonas stutzeri (Mrozik et al. 2008).
In many environmental studies, analysis of FAME pro-
files has been used to determine changes in microbial
populations and their activity in soil (Kozdroj and van
Elsas 2001), wastewater treatment (Quezada et al. 2007)
and sediments (Dunn et al. 2008). Moreover, alterations
in FAME patterns obtained directly from soil may indi-
cate the response of microbial communities to natural
and antropogenic stress (Kozdroj 2000; Islam et al. 2009).
In this study, we successfully applied FAME analysis to
assess the progress in phenol degradation in both nonbio-
augmented FC and FS soils and bioaugmented with
Pseudomonas sp. JS150.
Phenol contamination as well as soil inoculation shifted
microbial communities’ FAME profiles, and the most sig-
nificant alterations were connected with the distribution
of branched and cyclopropane fatty acids. Studying phe-
nol biodegradation in sterile soils inoculated with P. stut-
zeri and Pseudomonas sp. CF600, we found that
cyclopropane fatty acid 19:0 cy x8c apparent at substan-
tial amount when more than 50% of phenol added was
degraded (Mrozik et al. 2008, 2010). It was interesting to
check whether the similar effect can occur in nonsterile
soil bioaugmented with Pseudomonas sp. JS150. Results of
our study confirmed that appearance of a new cyclopro-
pane fatty acid 19:0 cy x10c is connected with the degree
of phenol removal. Depending on soil type and phenol
contamination in both augmented and nonbioaugmented
Table 8 The percentages of distinct groups of fatty acids isolated from nonbioaugmented and bioaugmented FS soil during phenol degradation
at the concentration 3Æ3 mg g)1
Time FS soil
Total fatty acids (weight %)
Saturated
UnsaturatedStraight-chain Branched Hydroxylated
Total
cyclopropane
Cyclopropane
17:0 cy 19:0 cy x10c
Day 1 FSP 39Æ66 6Æ24 0Æ00 11Æ41 11Æ41 0Æ00 42Æ69
FSP+B 39Æ47 28Æ01 1Æ54 12Æ36 12Æ36 0Æ00 18Æ62
Day 4 FSP 40Æ74 7Æ12 0Æ00 12Æ01 12Æ01 0Æ00 40Æ13
FSP+B 45Æ84 29Æ11 1Æ02 12Æ41 12Æ41 0Æ00 11Æ62
Day 8 FSP 42Æ77 9Æ66 0Æ00 10Æ63 4Æ19 6Æ44 36Æ94
FSP+B 46Æ94 31Æ01 1Æ78 11Æ23 3Æ82 7Æ41 9Æ04
Day 16 FSP 44Æ55 11Æ99 0Æ00 12Æ16 1Æ72 10Æ44 31Æ30
FSP+B 46Æ99 31Æ04 1Æ66 12Æ55 0Æ98 11Æ57 7Æ76
Day 24 FSP 44Æ99 13Æ00 0Æ00 12Æ63 0Æ77 11Æ86 29Æ38
FSP+B 46Æ91 31Æ42 0Æ00 12Æ06 1Æ55 10Æ51 9Æ61
Day 32 FSP 46Æ71 13Æ04 0Æ00 12Æ70 0Æ00 12Æ70 27Æ55
FSP+B 44Æ11 29Æ16 1Æ56 12Æ27 4Æ11 8Æ16 12Æ90
Day 40 FSP 45Æ11 13Æ00 0Æ00 11Æ42 0Æ94 10Æ48 30Æ47
FSP+B 44Æ55 28Æ05 1Æ59 12Æ02 5Æ13 6Æ89 13Æ79
Day 48 FSP 46Æ70 11Æ91 0Æ00 10Æ09 1Æ94 8Æ15 31Æ30
FSP+B 42Æ16 28Æ44 1Æ61 11Æ98 5Æ77 6Æ21 15Æ81
Day 56 FSP 46Æ80 11Æ15 0Æ00 10Æ90 5Æ15 5Æ75 30Æ15
FSP+B ND ND ND ND ND ND ND
Day 64 FSP 44Æ11 9Æ44 0Æ00 10Æ40 4Æ16 6Æ24 36Æ05
FSP+B ND ND ND ND ND ND ND
Values are the means of three replicates (standard errors <5%).
FSP, contaminated and nonbioaugmented soil; FSP+B, contaminated and bioaugmented soil; ND, not determined.
Soil bioaugmentation A. Mrozik et al.
1366 Journal of Applied Microbiology 111, 1357–1370 ª 2011 The Society for Applied Microbiology
ª 2011 The Authors
soils, the highest content of this fatty acid in FAME pro-
files was detected when phenol was biodegraded in 60–
80%. Our earlier results (Mrozik et al. 2008, 2010)
showed that bacteria from genus Pseudomonas used cyclo-
propane ring formation in response to high phenol con-
centration. Therefore, we indicate that it is an important
adaptive mechanism of bacteria to chemical stress
although the protective role of cyclopropane fatty acids in
cytoplasmic membrane is not understood in details.
Moreover, cyclopropane fatty acids are very useful mark-
ers for monitoring phenol degradation in soil.
Essential changes in FAME patterns of sterile and non-
sterile soils inoculated with different strains from genus
Pseudomonas included also the alterations in branched
fatty acid contents (Mrozik et al. 2008, 2010). In this
study immediately after inoculation, the abundance of
this fatty acid group increased and then gradually
declined in parallel with the overall decrease in phenol
concentrations. The active remodelling of fatty acid com-
position including alterations in cyclopropane and
branched fatty acids in the membrane structure and regu-
lation of membrane functions allows bacteria to adapt
and survive in unfavourable conditions.
In conclusion, Pseudomonas sp. JS150 did not lose
capability of phenol degradation after soil inoculation,
well adapted to stress conditions and survived over the
experimental time. For these reasons, it seems to be an
attractive micro-organism for bioremediation technology
to enhance the capability of soil towards phenol degrada-
tion. Moreover, cyclopropane and branched fatty acid
contents are sensitive probes for monitoring the progress
of phenol removal from soil.
Table 9 The percentages of distinct groups of fatty acids isolated from nonbioaugmented and bioaugmented FS soil during phenol degradation
at the concentration 5Æ0 mg g)1
Time FS soil
Total fatty acids (weight %)
Saturated
UnsaturatedStraight-chain Branched Hydroxylated
Total
cyclopropane
Cyclopropane
17:0 cy 19:0 cy x10c
Day 1 FSP 38Æ78 6Æ13 0Æ00 11Æ14 11Æ14 0Æ00 43Æ95
FSP+B 38Æ77 27Æ64 1Æ54 12Æ73 12Æ73 0Æ00 19Æ32
Day 4 FSP 41Æ64 8Æ66 0Æ00 12Æ17 12Æ17 0Æ00 37Æ53
FSP+B 45Æ20 29Æ51 0Æ97 11Æ89 11Æ89 0Æ00 12Æ43
Day 8 FSP 44Æ97 11Æ63 0Æ00 12Æ27 4Æ11 8Æ16 31Æ13
FSP+B 46Æ40 31Æ24 0Æ90 11Æ89 0Æ00 11Æ89 9Æ57
Day 16 FSP 45Æ90 13Æ70 0Æ00 13Æ66 2Æ09 11Æ57 26Æ64
FSP+B 46Æ24 31Æ66 0Æ76 13Æ68 0Æ00 13Æ68 7Æ66
Day 24 FSP 45Æ25 14Æ21 0Æ00 13Æ99 0Æ00 13Æ99 26Æ55
FSP+B 45Æ02 31Æ21 0Æ92 15Æ11 0Æ00 15Æ11 7Æ74
Day 32 FSP 45Æ98 14Æ68 0Æ00 14Æ19 0Æ00 14Æ19 25Æ25
FSP+B 46Æ94 30Æ43 0Æ84 15Æ21 0Æ00 15Æ21 6Æ55
Day 40 FSP 45Æ63 14Æ84 0Æ00 14Æ72 0Æ00 14Æ72 24Æ81
FSP+B 46Æ56 29Æ95 0Æ91 15Æ33 0Æ00 15Æ33 7Æ25
Day 48 FSP 46Æ31 15Æ44 0Æ00 16Æ77 0Æ00 16Æ77 21Æ48
FSP+B 45Æ17 29Æ91 0Æ98 15Æ44 0Æ00 15Æ44 8Æ50
Day 56 FSP 45Æ59 15Æ77 0Æ00 17Æ17 0Æ00 17Æ17 21Æ47
FSP+B 45Æ12 29Æ51 0Æ82 15Æ62 0Æ00 15Æ62 8Æ93
Day 64 FSP 46Æ29 16Æ06 0Æ00 13Æ77 0Æ00 13Æ77 23Æ88
FSP+B 44Æ77 29Æ12 1Æ12 13Æ11 0Æ00 13Æ11 11Æ88
Day 72 FSP 44Æ01 14Æ15 0Æ00 11Æ85 1Æ57 10Æ28 29Æ99
FSP+B 44Æ05 28Æ88 1Æ21 11Æ91 0Æ00 11Æ91 13Æ95
Day 80 FSP 44Æ00 12Æ16 0Æ00 11Æ55 2Æ44 9Æ11 32Æ29
FSP+B ND ND ND ND ND ND ND
Day 88 FSP 44Æ14 12Æ00 0Æ00 11Æ01 2Æ88 8Æ13 32Æ85
FSP+B ND ND ND ND ND ND ND
Day 96 FSP 42Æ76 10Æ11 0Æ00 12Æ05 4Æ57 7Æ48 35Æ08
FSP+B ND ND ND ND ND ND ND
Values are the means of three replicates (standard errors <5%).
FSP, contaminated and nonbioaugmented soil; FSP+B, contaminated and bioaugmented soil; ND, not determined.
A. Mrozik et al. Soil bioaugmentation
ª 2011 The Authors
Journal of Applied Microbiology 111, 1357–1370 ª 2011 The Society for Applied Microbiology 1367
Acknowledgements
This work was supported by grant no. N N305 049536
from the Polish Ministry of Science and Higher Educa-
tion.
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