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


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


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).



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 2011 The Authors


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, 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).



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



Time FC soil


Saturated


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


Day 36 FCP 34Æ11 6Æ24 2Æ51 12Æ57 7Æ66 4Æ91 44Æ57





ª 2011 The Authors


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



Time FC soil


Saturated


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


Day 68 FCP 36Æ01 8Æ45 2Æ53 13Æ01 0Æ97 12Æ04 40Æ00






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


Time FS soil


Saturated


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


Day 48 FSP 41Æ62 7Æ33 0Æ00 12Æ00 10Æ10 1Æ90 39Æ05


Day 56 FSP 40Æ65 7Æ05 0Æ00 10Æ89 10Æ15 0Æ74 41Æ41



FSP, contaminated and nonbioaugmented soil; FSP+B, contaminated and bioaugmented soil; ND, not determined.


ª 2011 The Authors


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



Time FS soil


Saturated


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


Day 64 FSP 44Æ11 9Æ44 0Æ00 10Æ40 4Æ16 6Æ24 36Æ05






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.



Time FS soil


Saturated


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


Day 88 FSP 44Æ14 12Æ00 0Æ00 11Æ01 2Æ88 8Æ13 32Æ85


Day 96 FSP 42Æ76 10Æ11 0Æ00 12Æ05 4Æ57 7Æ48 35Æ08





ª 2011 The Authors


Acknowledgements

This work was supported by grant no. N N305 049536

from the Polish Ministry of Science and Higher Educa-

tion.

References

Afzal, M., Yousaf, S., Reichenauer, T.G., Kuffner, M. and Sess-

itsch, A. (2011) Soil type affects plant colonization, activity

and catabolic gene expression of inoculated bacterial

strains during phytoremediation of diesel. J Hazard Mater

186, 1568–1575.

Agency for Toxic Substances and Disease Registry (ATSDR)

(1998) Toxicological Profile for Phenol (Update). Atlanta,

GA: Public Health Service, U.S. Department of Health and

Human Services.

Budavari, S. (ed.) (1996) The Merck Index: An Encyclopedia of

Chemicals, Drugs, and Biologicals. Whitehouse Station, NJ:

Merck.

Chen, W.M., Chang, J.S., Wu, C.H. and Chang, S.C. (2004)

Characterization of phenol and trichloroethene degrada-

tion by the rhizobium Ralstonia taiwanensis. Res Microbiol

155, 672–680.

Das, K. and Mukherjee, A.K. (2007) Crude petroleum-oil bio-

degradation efficiency of Bacillus subtilis and Pseudomonas

aeruginosa strain isolated from a petroleum-oil contami-

nated soil from North-East India. Bioresour Technol 98,

1339–1345.

Davis, J.W. and Madsen, S. (1996) Factors affecting the bio-

degradation of toluene in soil. Chemosphere 33, 107–130.

Dec, J., Kleemann, W., Miga, S., Shvartsman, V.V., Łukas-

iewicz, T. and Swirkowicz, M. (2007) Aging, rejuvenation,

and memory effects in the domain state of

Sr0.75Ba0.25Nb2O6. Phase Trans 80, 131–140.

Diefenbach, R., Heipieper, H.J. and Keweloh, H. (1992) The

conversion of cis into trans unsaturated fatty acids in

Pseudomonas putida P8: evidence for a role in the regula-

tion of membrane fluidity. Appl Microbiol Biotechnol 38,

382–387.

Dott, W., Feidieker, D., Kampfer, P., Schleibinger, H. and Stre-

chel, S. (1989) Comparison of autochthonous and com-

mercially available cultures with respect to their

effectiveness in fuel-oil degradation. J Ind Microbiol 4,

365–373.

Dunn, R.J.K., Welsh, D.T., Teasdale, P.R., Lee, S.Y., Lemckert,

C.J. and Meziane, T. (2008) Investigating the distribution

and sources of organic matter in surface sediment of Coo-

mbabah Lake (Australia) using elemental, isotopic and

fatty acid biomarkers. Cont Shelf Res 28, 2535–2549.

Fischer, J., Schauer, F. and Heipieper, H.J. (2010) The trans ⁄ cis

ratio of unsaturated fatty acids is not applicable as biomar-

ker for environmental stress in long-term contaminated

habitats. Appl Microbiol Biotechnol 87, 365–371.

Goldstein, R.M., Mallory, L.M. and Alexander, M. (1985) Rea-

sons for possible failure of inoculation to enhance biodeg-

radation. Appl Environ Microbiol 50, 977–983.

Goswami, M., Shivaraman, N. and Singh, R.P. (2005) Micro-

bial metabolism of 2-chlorophenol, phenol and p-cresol by

Rhodococcus erythropolis M1 in co-culture with Pseudomo-

nas fluorescens P1. Microbiol Res 160, 101–109.

Guimaraes, B.C.M., Arends, J.B.A., van der Ha, D., Van de

Wiele, T., Boony, N. and Verstraete, W. (2010) Microbial

services and their management: recent progresses in soil

bioremediation technology. Appl Soil Ecol 46, 157–167.

Haigler, B.E., Pettigrew, C.H.A. and Spain, J.C. (1992) Biodeg-

radation of mixtures of substituted benzenes by Pseudomo-

nas sp. JS150. Appl Environ Microbiol 58, 2237–2244.

Hanscha, C., McKarnsb, S.C., Smith, C.J. and Doolittle, D.J.

(2000) Comparative QSAR evidence for a free-radical

mechanism of phenol-induced toxicity. Chem Biol Interact

127, 61–72.

Heinaru, E., Merimaa, M., Viggor, S., Lehiste, M., Leito, I.,

Truu, J. and Heinaru, A. (2005) Biodegradation efficiency

of functionally important populations selected for bioaug-

mentation in phenol- and oil-polluted area. FEMS Micro-

biol Ecol 51, 363–373.

Heipieper, H.J. and de Bont, J.A.M. (1994) Adaptation of Pse-

domonas putida S12 to ethanol and toluene at the level of

fatty acid composition of membranes. Appl Environ Micro-

biol 60, 4440–4444.

Islam, Md.R., Trivedim, P., Palaniappan, P., Reddy, M.S. and

Sa, T. (2009) Evaluating the effect of fertilizer application

on soil microbial community structure in rice based crop-

ping system using fatty acid methyl esters (FAME) analy-

sis. World J Microbiol Biotechnol 25, 1115–1117.

Juhanson, J., Truu, J., Heinaru, E. and Heinaru, A. (2009) Sur-

vival and catabolic performance of introduced Pseudomo-

nas strains during phytoremediation and bioaugmentation

field experiment. FEMS Microbiol Ecol 70, 446–455.

Karamalidis, A.K., Evangelou, A.C., Karabika, E., Koukku, A.I.,

Drainas, C. and Voudrias, E.A. (2010) Laboratory scale

bioremediation of petroleum-contaminated soil by indige-

nous microorganisms and added Pseudomonas aeruginosa

strain Spet. Bioresour Technol 101, 6545–6552.

Kargi, F. and Serkan, E. (2004) Toxicity and batch biodegrada-

tion kinetics of 2,4 dichlorophenol by pure Pseudomonas

putida culture. Enz Microb Technol 35, 424–428.

Kaur, A., Chaudhary, A., Kaur, A., Choudhary, R. and Kaus-

hik, R. (2005) Phospholipid fatty acids – a bioindicator of

environment monitoring and assessment in soil ecosystem.

Curr Sci 89, 1103–1112.

Khan, F.I., Husain, T. and Hejazi, R. (2004) An overview and

analysis of site remediation technologies. J Environ Manage

71, 95–122.

Kojima, Y., Itada, N. and Hayaishi, O. (1961) Metapyrocate-

chase a new catechol cleaving enzyme. J Biol Chem 236,

2223–2231.



ª 2011 The Authors

Kozdroj, J. (2000) Microflora of technogenous wastes charac-

terized by fatty acid profiling. Microbiol Res 155, 149–156.

Kozdroj, J. and van Elsas, J.D. (2001) Structural diversity of

microbial communities in arable soils of a heavily indus-

trialised area determined by PCR-DGGE fingerprinting

and FAME profiling. Appl Soil Ecol 17, 31–42.

Leahy, J.G. and Colwell, R.R. (1990) Microbial degradation of

hydrocarbons in the environment. Microbiol Mol Biol Rev

54, 305–315.

Litynski, T., Jurkowska, H. and Gorlah, E. (1972) Chemical

and Agricultural Analysis. Warsaw: Polish Scientific Press.

Liu, Y.J., Zhang, A.N. and Wang, X.C. (2009) Biodegradation

of phenol by using free and immobilized cells of Acineto-

bacter sp. XA05 and Sphingomonas sp. FG03. Biochem Eng

J 44, 187–192.

Lurie, J. and Rybnikova, I. (1968) Chemical Analysis of Indus-

trial Sewages. Moscow: Gaschmizdat.

Mariano, A.P., Angelis, D.F., Pirollo, M.P.S., Contiero, J. and

Bonotto, D.M. (2009) Investigation about the efficiency of

the bioaugmentation technique when applied to diesel oil

contaminated soils. Braz Arch Biol Technol 5, 1297–1312.

Mazzoli, R., Pessione, E., Giuffrida, M.G., Fattori, P., Barello,

C., Giunta, C. and Lindley, N.D. (2007) Degradation of

aromatic compounds by Acinetobacter radioresistens S13:

growth characteristics on single substrates and mixtures.

Arch Microbiol 188, 55–68.

Mrozik, A. and Piotrowska-Seget, Z. (2010) Bioaugmentation

as a strategy for cleaning up of soils contaminated with

aromatic compounds. Microbiol Res 165, 363–375.

Mrozik, A., Piotrowska-Seget, Z. and Łabu_zek, S. (2008)

FAMEs profiles of phenol-degrading Pseudomonas stutzeri

introduced into soil. Int Biodeterior Biodegradation 62,

319–324.

Mrozik, A., Cycon, M. and Piotrowska-Seget, Z. (2010)

Changes of FAME profiles as a marker of phenol degrada-

tion in different soils inoculated with Pseudomonas sp.

CF600. Int Biodeterior Biodegradation 64, 86–96.

Nagamani, A. and Lowry, M. (2009) Phenol biodegradation by

Rhodococcus coprophilus isolated from semi arid soil sam-

ples of Pali, Rajasthan. Int J Appl Environ Sci 3, 294–302.

Nam, K. and Kim, J.Y. (2002) Role of loosely bound humic

substances and humin in the bioavailability of phenan-

threne aged soil. Environ Pollut 118, 427–433.

Nasseri, S., Kalantary, R.R., Nourich, N., Naddafi, K., Mahvi,

A.H. and Baradaran, N. (2010) Influence of bioaugmenta-

tion in biodegradation of PAHs-contaminated soil in

bio-slurry phase reactor. Iran J Environ Health Sci Eng 7,

199–208.

Paller, G., Hommel, R.K. and Kleber, H.P. (1995) Phenol deg-

radation by Acinetobacter calcoaceticus NCIB 8250. J Basic

Microbiol 35, 325–335.

Pradham, A. and Ingle, A.O. (2007) Mineralization of phenol

by a Serratia plymuthica strain GC isolated from sludge

samples. Int Biodeterior Biodegradation 60, 103–108.

Quezada, M., Buitron, G., Moreno-Andrade, I., Moreno, G.

and Lopez-Martin, L.M. (2007) The use of fatty acid

methyl esters as biomarkers to determine aerobic, faculta-

tively and anaerobic communities in wastewater treatment

systems. FEMS Microbiol Lett 266, 75–82.

Ruberto, L., Vazquez, S.C., Dias, R.L., Hernandez, E.A., Coria,

S.H., Levin, G., Balbo, A.L. and Mac Cormack, W.P.

(2010) Small-case studies towards a rational use of bioaug-

mentation in an Antarctic hydrocarbon-contaminated soil.

Antarct Sci 22, 463–469.

Sasser, M. (1990) Identification of Bacteria by Gas Chromatog-

raphy of Cellular Fatty Acids. MIDI Technical Note 101.

Newark, USA: Microbial ID, Inc.

Schroder, M., Muller, C., Posten, C., Deckwer, W.D. and

Hecht, V. (1997) Inhibition kinetics of phenol degradation

from unstable steady-state data. Biotechnol Bioeng 54, 567–

576.

Sejakova, Z., Dercova, K. and Tothova, L. (2009) Biodeg-

radation and ecotoxicity of soil contaminated by

pentachlorophenol applying bioaugmentation and

addition of sorbents. World J Microbiol Biotechnol 25,

243–252.

Silva, I.S., Santos, E.C., Menezes, C.R., Faria, A.F., Francis-

con, E., Grossman, M. and Durrant, L.R. (2009)

Bioremediation of a polyaromatic hydrocarbon contami-

nated soil by native soil microbiota and bioaugmentation

with isolated microbial consortia. Bioresour Technol 100,

4669–4675.

Simon, M.A., Bonnier, J.S., Page, C.A., Townsend, R.T., Muel-

ler, D.C., Fuller, C.B. and Autenrieth, R.L. (2004) Evalua-

tion of two commercial bioaugmentation products for

enhanced removal of petroleum from a wetland. Ecol Eng

22, 263–277.

Sobiecka, E., Cedzynska, K., Bielski, C. and Antizar-Ladislao,

B. (2009) Biological treatment of transformer oil using

commercial mixtures of microorganisms. Int Biodeterior

Biodegradation 63, 328–333.

Stalwood, B., Shears, J., Williams, P.A. and Hughes, K.A.

(2005) Low temperature bioremediation of oil-contami-

nated soil using biostimulation and bioaugmentation with

a Pseudomonas sp. from maritime Antarctica. J Appl Micro-

biol 99, 794–802.

Teng, Y., Luo, Y., Liu, Z. and Christie, P. (2010) Effect of bio-

augmentation by Pyrococcus sp. strain HPD-2 on the soil

microbial community and removal of polycyclic aromatic

hydrocarbons from an aged contaminated soil. Bioresour

Technol 101, 3437–3443.

Thompson, I.P., Van der Gast, C.J., Ciric, L. and Singer, A.C.

(2005) Bioaugmentation for bioremediation: the challenge

of strain selection. Environ Microbiol 7, 909–915.

Tyagi, M., da Fonseca, M.M.R. and de Carvalho, C.C.C.R.

(2011) Bioaugmentation and biostimulation strategies to

improve the effectiveness of bioremediation processes.

Biodegradation 22, 232–241.


ª 2011 The Authors


van Veen, J.A., van Overbeek, L.S. and van Elsas, J.D. (1997)

Fate and activity of microorganisms introduced into soil.

Microbiol Mol Biol Rev 61, 121–135.

Vinken, R., Schaffer, A. and Ji, R. (2005) Abiotic association

of soil-borne monomeric phenols with humic acids. Org

Geochem 36, 583–593.

Vogel, T.M. (1996) Bioaugmentation as a soil bioremediation

approach. Curr Opin Biotechnol 7, 311–316.

Wang, J.L., Mao, Z.Y., Han, L.P. and Qjan, Y. (2004) Bioreme-

diation of quinoline-contaminated soil using bioaugmenta-

tion in slurry-phase reactor. Biomed Environ Sci 17,

187–195.

Weber, F.J. and de Bont, J.A.M. (1996) Adaptation mecha-

nisms of microorganisms to the toxic effects of organic

solvents on membranes. Biochim Biophys Acta 1286,

225–245.

Yang, C.F. and Lee, C.M. (2007) Enrichment, isolation, and

characterization of phenol-degrading Pseudomonas resi-

novorans strain P-1 and Brevibacillus sp. strain P-6. Int

Biodeterior Biodegradation 59, 206–210.



ª 2011 The Authors

enhanced of phenol degradation by soil bioaugmentacion with pseudomonas sp js150

Documents