Journal of Environmental Management 168 (2016) 175e184

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Journal of Environmental Management

journal homepage: www.elsevier.com/locate/jenvman

Research article

Isolation of hydrocarbon-degrading and biosurfactant-producing

bacteria and assessment their plant growth-promoting traits
b
_
Magdalena Pacwa-Pociniczak a, *, Tomasz Pociniczak a, Joanna Iwan a, Monika Zarska
,
b
_
, Marzena Dzida b, Zoa Piotrowska-Seget a
Mirosaw Chora zewski
a
b

 ska 28, 40-032 Katowice, Poland

Department of Microbiology, University of Silesia, Jagiellon
Institute of Chemistry, University of Silesia, Szkolna 9, 40-006 Katowice, Poland

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 11 February 2015
Received in revised form
4 August 2015
Accepted 29 November 2015
Available online xxx

Forty-two hydrocarbon-degrading bacterial strains were isolated from the soil heavily contaminated
with petroleum hydrocarbons. Forty-one strains were identied based on their whole-cell fatty acid
proles using the MIDI-MIS method. Thirty-three of them belong to species Rhodococcus erythropolis,
while the others to the genera Rahnella (4), Serratia (3) and Proteus (1). Isolates were screened for their
ability to produce biosurfactants/bioemulsiers. For all of them the activity of several mechanisms
characteristic for plant growth-promoting bacteria was also determined. In order to investigate surface
active and emulsifying abilities of isolates following methods: oil-spreading, blood agar, methylene blue
agar and determination of emulsication index, were used. Among studied bacteria 12 strains (CD 112,
CD 126, CD 131, CD 132, CD 135, CD 147, CD 154, CD 155, CD 158, CD 161, CD 166 and CD 167) have been
chosen as promising candidates for the production of biosurfactants and/or bioemulsiers. Among them
2 strains (R. erythropolis CD 126 and Rahnella aquatilis CD 132) had the highest potential to be used in the
bioaugmentation of PH-contaminated soil. Moreover, 15 of tested strains (CD 105, CD 106, CD 108, CD 111,
CD 116, CD 120, CD 124, CD 125, CD 130, CD 132, CD 134, CD 154, CD 156, CD 161 and CD 170) showed the
activity of four mechanisms (ACC deaminase activity, IAA and siderophore production, phosphate solubilization) considered to be characteristic for plant growth-promoting bacteria. Two of them (R.
erythropolis CD 106 and R. erythropolis CD 111) showed the highest activity of above-mentioned mechanisms and thus are considered as promising agents in microbe assisted phytoremediation.
2015 Elsevier Ltd. All rights reserved.

Keywords:
Petroleum hydrocarbons
Biosurfactant production
Bioemulsiers
Plant growth-promoting bacteria

1. Introduction
Total petroleum hydrocarbons (TPH) are a broad family of
several hundred chemical compounds that originally come from
crude oil. Contamination of soil by petroleum hydrocarbons (PH) is
a serious environmental problem because they are harmful to living
organisms being toxic, mutagenic and/or carcinogenic. This is the
reason that PH are classied as priority environmental pollutants
by the US Environmental Protection Agency (USEPA, 1986). Therefore, the development of efcient remediation technologies for the

* Corresponding author.
E-mail addresses: magdalena.pacwa-plociniczak@us.edu.pl (M. PacwaPociniczak), tomasz.plociniczak@us.edu.pl (T. Pociniczak), joanna.iwan@hotmail.
_
com (J. Iwan), monika.zarska@us.edu.pl (M. Zarska),
miroslaw.chorazewski@us.
_
edu.pl (M. Chora zewski),
marzena.dzida@us.edu.pl (M. Dzida), zoa.piotrowskaseget@us.edu.pl (Z. Piotrowska-Seget).
http://dx.doi.org/10.1016/j.jenvman.2015.11.058
0301-4797/ 2015 Elsevier Ltd. All rights reserved.

removal of these contaminants from soil remains one of the major

goals of engineers, environmental biotechnologists and ecologists.
One of the most promising, inexpensive and environmentally
friendly methods that have been used for cleaning up environments polluted with organic compounds are bioaugmentation and
phytoremediation.
The rst approach relies on the improvement of the hydrocarbon degradative capacity of contaminated areas through the
introduction of specic strains or microbial consortia (Liu et al.,
2011). This technique has proven to be successful in cleaning up
hydrocarbon-polluted sites however, there are still many problems.
The most important and difcult issue is selecting the proper microorganisms, which should be able to use the hydrocarbons as a
carbon and energy source. The degradation of PH in soil is primarily
limited by the low solubility and high hydrophobicity of oil pollutants. These compounds strongly bind to soil particles and thus
are poorly accessible to bacterial cells (Johnsen et al., 2005). In

176

M. Pacwa-Pociniczak et al. / Journal of Environmental Management 168 (2016) 175e184

order to overcome this obstacle and increase the bioavailability of

PH in soil, the application of hydrocarbon-degrading and
biosurfactant-producing bacteria for bioaugmentation is proposed.
The biosurfactants enhance the desorption and solubilisation of
petroleum hydrocarbons and thus facilitate their assimilation by
microbial cells (Pacwa-Pociniczak et al., 2011).
The second approach includes phytoremediation, which consists of a set of innovative technologies for environmental cleanup
that takes advantage of the unique extractive and metabolic capabilities of plants (Campos et al., 2008). Organic pollutants can be
degraded into less toxic forms or even mineralized completely by
plants. The mechanisms that are active in hydrocarbon decontamination include plant uptake and phytodegradation, which can
lead to volatilisation (Walton et al., 1994). Moreover, plant roots
exude into soil organic compounds which increase the density,
diversity and activity of microorganisms that are mainly responsible for hydrocarbon degradation. It is believed that the combined
activity of roots and soil microorganisms is the most effective
method for the removal of PH from soil (Liu et al., 2013). Some of
the these bacteria exhibit features of plant growth-promoting
bacteria (PGPB) and can increase the biomass of plants via several
mechanisms that include: the production of phytohormones,
siderophores, 1-aminocyclopropane-1-carboxylic acid deaminase
(ACCD), nitrogen xation as well as the solubilisation of phosphorus (Glick, 2003; Gerhardt et al., 2009; Kukla et al., 2014).
Furthermore, the production of biosurfactants by PGPB increases
the bioavailability of hydrocarbons to plants, which will further
facilitate their removal. The ability of bacteria to promote plant
growth and enhance the bioavailability of hydrocarbons through
the production of biosurfactants is crucial in supporting the phytoremediation of soil contaminated with PH (Zhang et al., 2010).
There are several approaches that allow for the selection of
microorganisms that are useful for both bioaugmentation and
phytoremediation. In the rst method, bacteria that exhibit the
desired properties are isolated from contaminated soils and after
culturing under laboratory conditions are returned to the same soil.
The second method is the selection of the appropriate microorganisms from soil polluted with contaminants that are similar to
those that are present in the soil that is to be cleaned up by the
selected microorganisms (Thompson et al., 2005). The aim of our
study was to isolate microorganisms from polluted soil and determine their potential to degrade hydrocarbons, produce biosurfactants/bioemulsiers and promote the growth of the plants
that could be promising agents for cleaning up environments
contaminated with petroleum compounds. Blood agar, methylene
blue agar, oil-spreading methods and determination of emulsication index were used for the selection of the biosurfactantproducing bacteria. In order to assess the plant growthpromoting activities of the isolates, their ability to synthesise
siderophores, ACCD, ammonia, HCN and IAA as well as to solubilise
phosphate were determined.
2. Materials and methods
2.1. Isolation and identication of hydrocarbon-degrading bacteria
Hydrocarbon-degrading bacteria were isolated from petroleumpolluted soil taken from the vicinity of a 100-year-old oil renery in
Czechowice-Dziedzice, Upper Silesia, Poland using an enrichment
technique. Ten g of soil was initially suspended in 100 ml of an M9
mineral salt medium (Na2HPO4 6 g, KH2PO4 3 g, NaCl 0.5 g, NH4Cl
1 g, MgSO4
7H2O 0.24 g and CaCl2 0.01 g per litre of deionised
water) (Viramontes-Ramos et al., 2010) supplemented with 1% (v/
v) of crude oil as a carbon and energy source. The incubation was
performed at 28  C on a rotary shaker at 120 rpm for seven days.

Continuous sub-culturing and shaking was carried out four times

for enrichment. Isolation was performed by inoculation of soil
suspension on M9 agar plates supplemented with 1% (v/v) of crude
oil as a carbon and energy source. Morphologically distinct colonies
were isolated and identied using the MIDI microbial identication
system (MIDI Inc., Newark, DE, USA) as described in PiotrowskaSeget et al. (2005).
2.2. Screening for biosurfactant/bioemulsier-producing bacteria
The isolated strains were studied for the production of biosurfactants and emulsication activity using the various methods.
Test of haemolytic activity was carried out as described by Carrillo
et al. (1996). Strains were streaked onto blood agar plates that
contained 40 g of a blood agar base (Becton Dickinson, Sparks, MD)
and 50 ml of sheep blood (BIOMED-LUBLIN) per litre. Plates were
incubated for 48 h at 28  C. The plates were inspected for zones of
clearing around the colonies, which are indicative of the production
of biosurfactants. The synthesis of any extracellular glycolipid
biosurfactant was detected using the Siegmund and Wagner (1991)
technique. A spot of cultures grown on LB medium were placed on
methylene blue agar plates (cetyltrimethylammonium bromide
(CTAB) 0.2 g, methylene blue 0.005 g, glucose 20 g, KH2PO4 0.7 g,
Na2HPO4 0.9, NaNO3 2 g, MgSO4
7H2O 0.4 g, CaCl2
2H2O 0.1 g,
agar 20 g and trace elements solution containing 2 g of
FeSO4
7H2O, 1.5 g of MnSO4
H2O and 0.6 g of
(NH4)6Mo7O24
4H2O 2 ml per litre of deionised water). After 48 h
of incubation at 28  C, the plates were observed for the formation of
a dark blue halo around the culture spot, which indicated the formation of an insoluble ion pair between the anionic glycolipid
biosurfactant and the cationic CTAB-methylene blue agar complex.
The oil spreading technique was carried out according to Morikawa
et al. (2000) and Youssef et al. (2004). Fifty ml of distilled water was
added to Petri dishes, followed by the addition of 100 ml of crude oil
onto the surface of the water. Then, 10 ml of each culture supernatant was put onto the surface of crude oil after which the diameter
of the clear zone on the oil surface was measured.
The emulsifying capacity was determined using a modication
of the method described by Cooper and Goldenberg (1987). Three
ml of hydrocarbons (n-hexadecane, Diesel oil and p-xylene) was
added to 2 ml of the bacterial cultures in a screw-cap tube and
vortexed at high speed for 2 min. The stability of the emulsion was
determined after 24 h. The emulsication index (E24) was calculated as the percentage height of the emulsion layer to the total
height of the liquid column. The experiments were carried out in
triplicate.
2.3. Determination of surface tension
Bacterial strains with the best results of drop-collapse and
emulsifying tests were also evaluated for the reduction of surface
tension. Strains were grown in an M9 broth with crude oil (1% v/v)
added at 28  C on a rotary shaker at 120 rpm for ve days. Then, the
cultures were centrifuged at 5000 rpm for 20 min. The surface oil
layer was discarded and the culture supernatant was used for the
surface tension measurements using a drop shape analyser (Kruss
DSA100) at 25  C. This equipment consists of a camera with a light
source and the a measuring cell that is located between them. A
pendant drop of the investigated liquid is formed at the end of a
needle placed in thermostated cell. The shape of the pendant drop
is analysed and the surface tension is determined using the LaplaceeYoung formula. The diameter of the needle and the density of
investigated liquid are required for the calculations. Therefore, the
density of liquids have been measured using an oscillating
densimeter (Anton Paar DMA5000) at the same temperature with

M. Pacwa-Pociniczak et al. / Journal of Environmental Management 168 (2016) 175e184

an uncertainty of 0.05 kg m3. The uncertainty of the surface tension measurement is 0.1 mN m1, while the uncertainty of the
temperature measurement is 0.1 K. All samples were degassed for
10 min in an ultrasonic cleaner just before immediately prior to
each measurement. Uninoculated M9 medium supplemented with
crude oil (1% v/v) was used as a control. For each measurement,
three replicates were used.
2.4. Evaluation of plant growth-promoting activities
ACCD activity was assayed according to a modied Honma and
Shimomura (1978) method as described by Saleh and Glick (2001).
In this method the amount of a-ketobutyrate released by ACCD
from 1-aminocyclopropane-1-carboxylic acid was estimated. ACCD
activity was expressed in nmol of a-ketobutyrate mg1 h1. The
protein concentration of microbial cell suspensions was determined using the Bradford (1976) method. Siderophore secretion by
the tested strain was detected by the Schwyn and Neilands method
(1987) using blue agar plates containing the Chrome azurol S (CAS)
dye. Orange zones around the colonies on blue agar were considered to be a positive reaction for siderophore production. IAA
production was determined according to the modied method of
Brick et al. (1991) using Salkowski's reagent. The IAA concentration
in cultures was calculated using a calibration curve of pure IAA
(1e100 mg ml1 of medium) as the standard. The phosphate solubilising ability of the bacterial strains was determined on an NBRIP
agar medium. Three replicates of the bacterial strains per plate
were stabbed using a sterile needle. The halo size and colony diameters were measured after 14 days of incubation at 28  C. Halo
size was calculated by subtracting the colony diameter from the
total diameter. The production of hydrogen cyanide by bacteria was
tested by the Lorck method (1948). Bacteria were cultured on a
broth agar medium amended with 4.4 g l1 of glycine. A Whatman
lter paper soaked in 2% sodium carbonate in 0.5% picric acid solution was placed on top of the agar. Plates were sealed with paralm and incubated at 28  C for four days. Development of an
orange to red colour on the lter paper indicated HCN production.
For each measurement, three replicates were used.
2.5. Statistical analyses
Statistical analysis was done using STATISTICA 10.0 PL software
(StatSoft, Tulsa, USA). The relationships between tested properties
of the studied strains were analysed with the use of the principal
components analysis (PCA) multivariate statistical technique. For
all analyses, data were represented as mean standard deviation
(SD) of 3 replicates.
3. Results
3.1. Screening for biosurfactant/bioemulsier-producing bacteria
A total of 42 hydrocarbon-degrading bacterial strains were
isolated from the soil collected in the vicinity of a 100-year-old oil
renery in Czechowice-Dziedzice in Poland. Among those isolates,
41 were identied using the MIDI-MIS method. Thirty-three of
them belong to species Rhodococcus erythropolis, while the others
belong to the genera Rahnella (4), Serratia (3) and Proteus (1).
Cluster analysis (CA, unweighted pair matching) of the FAME proles revealed two main clusters. The rst involved unidentied
isolate CD167, while in the second two sub clusters were found. The
rst sub cluster included members of the Rhodococcus genus from
the Nocardiaceae family, whereas the second bacteria from the
Enterobacteriaceae family (Fig. 1).
The results concerning the ability of the strains for the

177

production of biosurfactants/bioemulsiers are presented in

Table 1. All of the isolates generated clear zones in the oil-spreading
test; however, the size of area of displacement differed considerably between strains. Thirteen of these strains gave clear zones of
less than 1.5 cm, 13 gave diameters that ranged from 1.5 to 2.0 cm
and 16 generated an area of displacement larger than 2.0 cm.
Among the 42 strains, ten showed hemolytic activity, whereof 2
strains (CD 107 and CD 131) exhibited alpha haemolysis and eight
(CD 130, CD 148, CD 154, CD 155, CD 157, CD 158, CD 161 and CD
167) beta haemolysis. In addition, four strains (CD 107, CD 109, CD
111 and CD 130) formed dark blue halo zone in the methylene blue
agar plate supplemented with CTAB. The values of the emulsication index ranged from 0.00 to 20.00% for n-hexadecane,
0.00e37.14% for diesel oil and 2.86e68.57% for p-xylene. The values
of the emulsication index were higher than 20.00% for more than
one tested hydrocarbon for only six strains (CD 105, CD 126, CD 131,
CD 132, CD 161 and CD 166) and among those only one (CD 126)
had the ability to form emulsions (E24  20.00%) with all three
hydrocarbons. Thirteen of the 42 isolates showed the highest
values of halos in the oil-spreading test. Among all of the isolates, 13
generated clear zones in the oil-spreading test that were greater
than 2.0 cm, gave positive results in the haemolytic test and also
showed emulsication activity of higher than 20.00% for at least
one of hydrocarbons that were tested. In order to conrm their
good surface active properties, they were further tested for the
reduction of the surface tension of the cultivation medium. It was
observed that seven of the selected strains (CD 106, CD 154, CD 155,
CD 157, CD 158, CD 166 and CD 167) reduced the surface tension of
the M9 medium with crude oil to values below 60 mN m1, while
the remaining strains reduced the surface tension to values in the
range from 60.35 to 64.85 mN m1 (Fig. 2).
PCA (Fig. 3) with multivariate ordination show the relationship
between surface active and bioemulsifying properties of analysed
strains. This analysis enabled to distinguish bacterial strains positively associated with (i) generating high oil-displaced area and
forming efcient emulsion with diesel oil (CD 161, CD 166 and CD
167) and (ii) forming efcient emulsion with n-hexadecan and pxylene (CD 112, CD 126, CD 131, CD 135, CD 147, CD 154, CD 155 and
CD 158). It has been also noticed that strain CD 132, situated between these two groups of bacteria, was positively associated with
both generating high oil-displaced area and forming efcient
emulsion with n-hexadecan and p-xylene.
3.2. Evaluation of plant growth-promoting activities
The isolates were also screened for their plant growthpromoting properties. Among the 42 isolates, 23 strains showed
an activity of ACCD that was more than 20 nmol a-ketobutyrate
mg1 protein h1 and the highest value (99.60 nmol a-ketobutyrate
mg1 protein h1) was observed for CD 163. Twenty-two strains
developed large and clear orange haloes around colonies when
grown on a CAS agar medium, thus indicating siderophore production. Thirty-three strains were able to produce IAA, but 32 of
them were considered to be low IAA producers (IAA content was
lower than 2.5 mg IAA ml1 of the medium). The highest amount of
IAA (13.54 nmol a-ketobutyrate mg1 protein h1) was observed
for CD 106. Thirty-one strains were capable of solubilising
Ca3(PO4)2 in an NBRIP medium. The diameter of the halo size
ranged from 3 to 16 mm. None of the bacterial isolates showed an
ability to release hydrocyanic acid (Table 2).
PCA (Fig. 4) with multivariate ordination was also performed to
analyse relationship between strains due to their plant growth
promoting features. It has been observed that among tested bacteria strains positively associated with high solubilization of
phosphate (CD 101, CD 110, CD 123, CD 126, CD 147 and CD 163)

178

M. Pacwa-Pociniczak et al. / Journal of Environmental Management 168 (2016) 175e184

Fig. 1. Cluster analysis using unweighted pair matching of FAME proles of isolates.

were negatively associated with secretion of siderophores. On the

other hand, strain CD 111, as an exception, was positively associated
with both, high solubilization of phosphate and production of
siderophores and additionally was positively related to high activity of ACC deaminase. In turn, strain CD 106, located in the far
distance from the other strains, was positively associated with both,
high production of IAA and ACC deaminase activity.
4. Discussion
4.1. Surface-active abilities
In this study 42 isolates from the genera Rhodococcus, Rahnella,
Serratia and Proteus, which are able to degrade hydrocarbons and
produce biosurfactants, were isolated. Members of these genera

have also been identied as crude oil degraders and biosurfactant

producers in a wide range of studies (Ma et al., 2006; Zhukov et al.,
2007; Anyanwu et al., 2011; Ibrahim et al., 2013). Since biological
surface active compounds are considered to be very useful in
improving the bioavailability of hydrocarbon pollutants in soil,
bacterial strains that have the ability to produce biosurfactants in
combination with the capacity to degrade hydrocarbons have
become one of the most useful and promising tools in the bioremediation of petroleum-contaminated environments (Barin et al.,
2014). Nevertheless, the evaluation of the ability of bacteria to
produce biosurfactants faces some difculties. The methods that
are used for this purpose involve the culturing of bacteria on specic media, as well as an analysis of surface activity, bacterial
emulsifying ability or a measurement of the surface/interfacial
tension of a medium after bacterial cultivation (Satpute et al., 2010).

M. Pacwa-Pociniczak et al. / Journal of Environmental Management 168 (2016) 175e184

179

Table 1
Surface active ability and emulsication activity of the tested strains.
Isolate

CD
CD
CD
CD
CD
CD
CD
CD
CD
CD
CD
CD
CD
CD
CD
CD
CD
CD
CD
CD
CD
CD
CD
CD
CD
CD
CD
CD
CD
CD
CD
CD
CD
CD
CD
CD
CD
CD
CD
CD
CD
CD

101
105
106
107
108
109
110
111
112
114
116
120
123
124
125
126
128
130
131
132
133
134
135
138
139
146
147
148
149
151
154
155
156
157
158
159
161
163
164
166
167
170

Rhodococcus erythropolis
Serratia odorifera
Rhodococcus erythropolis
Rhodococcus erythropolis
Rhodococcus erythropolis
Rhodococcus erythropolis
Rahnella aquatilis
Rhodococcus erythropolis
Rahnella aquatilis
Rhodococcus erythropolis
Rhodococcus erythropolis
Proteus hauseri
Rhodococcus erythropolis
Rhodococcus erythropolis
Serratia odorifera
Rhodococcus erythropolis
Rhodococcus erythropolis
Rhodococcus erythropolis
Rhodococcus erythropolis
Rahnella aquatilis
Rhodococcus erythropolis
Rhodococcus erythropolis
Rhodococcus erythropolis
Serratia odorifera
Rhodococcus erythropolis
Rhodococcus erythropolis
Rhodococcus erythropolis
Rhodococcus erythropolis
Rhodococcus erythropolis
Rhodococcus erythropolis
Rhodococcus erythropolis
Rhodococcus erythropolis
Rahnella aquatilis
Rhodococcus erythropolis
Rhodococcus erythropolis
Rhodococcus erythropolis
Rhodococcus erythropolis
Rhodococcus erythropolis
Rhodococcus erythropolis
Rhodococcus erythropolis
Isolate
Rhodococcus erythropolis

Methylene blue agar plate

e
e
e

e
e
e
e
e
e
e
e
e

e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e
e

Type of hemolysis

g
g
g
a
g
g
g
g
g
g
g
g
g
g
g
g
g
b
a
g
g
g
g
g
g
g
g
b
g
g
b
b
g
b
b
g
b
g
g
g
b
g

Emulsication index [%]

Oil-spreading test [cm]

5.71 0.57
8.57 1.43
5.71 0.29
11.43 0.57
5.71 0.57
2.86 0.57
5.71 0.29
0.00
14.29 0.29
2.86 0.57
2.86 0.29
5.71 0.29
5.71 0.57
2.86 0.29
2.86 0.57
20.00 0.57
0.00
2.86 0.53
20.00 1.43
20.00 0.57
2.86 0.29
8.57 1.43
17.14 0.29
8.57 0.57
0.00
2.86 0.29
14.29 0.58
5.71 1.15
5.71 0.29
8.57 0.29
17.14 0.57
11.43 0.57
11.43 0.29
17.14 0.29
14.29 0.58
0.00
5.71 0.57
8.57 0.57
2.86 0.57
5.71 1.15
17.14 0.57
5.71 1.15

0.00
28.57 1.71
0.00
2.86 0.29
2.86 0.57
2.86 0.29
2.86 0.86
2.86 0.57
11.43 0.57
0.00
2.86 0.86
2.86 0.29
2.86 0.57
2.86 0.86
2.86 0.57
28.57 1.14
0.00
5.71 0.29
0.00
2.86 0.29
0.00
2.86 0.86
2.86 0.57
5.71 0.57
0.00
14.29 0.58
2.86 0.86
0.00
2.86 0.29
2.86 0.57
5.71 1.15
2.86 0.86
2.86 0.57
2.86 0.86
0.00
2.86 0.29
37.14 1.43
2.86 0.86
2.86 0.57
28.57 1.71
34.29 0.57
28.57 1.14

22.86
28.57
54.29
28.57
54.29
8.57
28.57
11.43
42.86
8.57
40.00
28.57
51.43
5.71
51.43
68.57
8.57
28.57
51.43
51.43
8.57
60.00
40.00
14.29
10.00
11.43
68.57
2.86
20.00
25.71
54.29
57.14
34.29
48.57
51.43
5.71
25.71
8.57
8.57
20.00
14.29
11.43

0.57
1.14
1.43
1.14
2.86
0.29
1.71
0.57
2.86
1.43
1.43
1.71
5.71
0.29
0.29
7.14
1.43
1.71
5.72
0.29
0.29
2.86
5.71
0.58
0.57
0.29
1.71
0.57
0.57
4.29
1.43
4.00
3.43
1.43
0.29
0.57
5.14
0.57
0.29
0.57
0.29
0.29

1.40
1.82
2.03
1.53
1.33
1.40
1.93
1.80
1.65
1.33
1.33
0.93
2.07
1.33
1.47
1.63
1.90
2.50
2.53
2.93
1.47
1.50
1.03
1.85
2.37
1.60
1.20
1.87
1.77
1.40
2.07
2.03
1.27
2.03
2.27
2.07
2.03
1.87
2.17
2.60
2.15
1.84

0.10
0.03
0.06
0.25
0.15
0.10
0.13
0.10
0.15
0.15
0.15
0.08
0.08
0.15
0.15
0.15
0.05
0.30
0.25
0.12
0.31
0.10
0.06
0.08
0.15
0.36
0 .010
0.60
0.15
0.10
0.14
0.15
0.06
0.15
0.21
0.21
0.10
0.12
0.15
0.23
0.10
0.06

Stand. Dev. of three independent experiments; H - n-hexadecane; O - diesel oil; X - p-xylene.

Fig. 2. Reduction of surface tension of bacterial cultivation medium; control e uninoculated M9 medium supplemented with crude oil (1% v/v).

However, results of this study and those that have been published
by other authors (Satpute et al., 2008; Thavasi et al., 2011), have
shown that these techniques are not equally reliable and sensitive.
Thus, only the use of a combination of various techniques enables
the efcient detection of biosurfactant-producing microorganisms.
The application of the blood agar lysis method enabled the

selection of 10 potential biosurfactant-producing strains although

not all of them gave positive results in the other tests that were
used. Furthermore, there were also strains that did not lyse blood
agar but formed clear zones in the oil spreading technique. Similarly, strains that gave positive results in only one of the methods
used (blood agar or the oil-spreading test) were reported by
Youssef et al. (2004) and Satpute et al. (2008). Due to the fact that
other microbial compounds, such as some of virulence factors, have
haemolytic activity and because some biosurfactants, which are
poorly diffusible, may not be able to lyse blood cells, this technique
can only be regarded as a preliminary screening method and has to
be supported by other techniques (Paza et al., 2006). The application of the blue agar plate method, developed by Siegmund and
Wagner (1991) for the detection of glycolipid biosurfactants, gave
positive results for only four of the 42 strains that were analysed
(CD 107, CD 109, CD 111 and CD 130). All of these strains belong to
the genus Rhodococcus, which is a known glycolipid producer. No
dark blue halos around the other Rhodococcus strains may be the
result of the fact that bacteria from this genus, with the exception of
glycolipids, also produce lipopeptides (Peng et al., 2008) and free
fatty acids (Peng et al., 2007, 2008), which exhibit the activity of
surface compounds. Moreover, the glycolipids that are produced by

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M. Pacwa-Pociniczak et al. / Journal of Environmental Management 168 (2016) 175e184

Fig. 3. PCA ordination of the studied strains based on their surface active and bioemulsifying abilities.

rhodococci can be bound to a cell during growth (Lang and Philip,

1998), and therefore, cannot form an insoluble complex with the
bromide salt present in the medium. The positive results of 42
strains in the oil-spreading test, which was indicated by Youssef
et al. (2004) as a reliable technique to detect the production of
biosurfactants, showed surface-active properties of these strains. It
was observed by Morikawa et al. (2000) that the oil-displaced area
that is formed by a surfactant-containing solution is directly proportional to the concentration of surfactant that is being tested.
Therefore, a different area of clear zones that were obtained for
strains that were analysed may provide information about the
different concentrations of the biosurfactants. The size of the
displacement areas that were formed by the 42 strains ranged from
0.93 to 2.93. Similar values of the diameters of the clear zone of
effective biosurfactant producers were reported by Thavasi et al.
(2011) and Almansoory et al. (2014). Another criterion that is
used to determine potential surface-active compound producers is
their ability to emulsify of hydrocarbons. The ability of a
biosurfactant-producing strain to emulsify non-aqueous liquids in
addition to reducing surface tension is a very practical measure of
their utility, especially when they are used for the bioremediation
of petroleum-polluted environments (Pacwa-Pociniczak et al.,
2014). Nevertheless, emulsication activity is specic for bioemulsiers and is not always exhibited by biosurfactant-producing
strains (Batista et al., 2006). Willumsen and Karlson (1997)
observed that emulsion formation did not correlate with a reduction of surface tension. In this study, strains with good

emulsication activity (>30%) constituted 45% of the isolates.

However, the results obtained by other authors showed that the
ability of bacteria to form a stable emulsion is not very common
(Satpute et al., 2008; Kumar et al., 2013; Tambekar and Gadakh,
2013). They detected this activity for a smaller number of bacteria
that were tested (they constituted 7, 14 and 29% of the isolates,
respectively). It has been observed that the most efcient emulsiers (with E24  60.00%) did not have high values of halos in the
oil-spreading test. The same observation was made by Ruggeri et al.
(2009), who reported that three of the tested strains that released
the most efcient emulsiers did not reduce the surface tension of
the medium. In order to conrm the ability of bacterial strains to
produce biosurfactant, a determination of the reduction of the
surface tension of the bacterial cultivation medium has been recommended (Paza et al., 2011; Dhail, 2012). According to Willumsen
and Karlson (1997), a microorganism that is considered to be a
promising biosurfactant producer should be able to reduce the
surface tension of the growth medium by  20 mN m1. None of the
strains that were analysed was able to decrease the surface tension
of the broth by that value. The best among them reduced the surface tension of the broth in the range of 10.38e14.9 mN m1. In
contrast to other studies (Afshar et al., 2008; Thavasi et al., 2011), a
direct correlation between the results obtained with the oilspreading and surface tension assays was not observed in our
studies. The differences may have resulted from the fact that the
determination of the surface tension of the supernatant posed
some difculties. The accuracy of the measurement in the drop

M. Pacwa-Pociniczak et al. / Journal of Environmental Management 168 (2016) 175e184

181

Table 2
Plant growth promoting properties of isolates.

CD
CD
CD
CD
CD
CD
CD
CD
CD
CD
CD
CD
CD
CD
CD
CD
CD
CD
CD
CD
CD
CD
CD
CD
CD
CD
CD
CD
CD
CD
CD
CD
CD
CD
CD
CD
CD
CD
CD
CD
CD
CD

101
105
106
107
108
109
110
111
112
114
116
120
123
124
125
126
128
130
131
132
133
134
135
138
139
146
147
148
149
151
154
155
156
157
158
159
161
163
164
166
167
170

Strain

Siderophore
secretion

IAA production [mg ml1]

Phosphate solubilizing ability [mm]

ACC deaminase
[a-ketobutyrate mg1 protein h1]

Rhodococcus erythropolis
Serratia odorifera
Rhodococcus erythropolis
Rhodococcus erythropolis
Rhodococcus erythropolis
Rhodococcus erythropolis
Rahnella aquatilis
Rhodococcus erythropolis
Rahnella aquatilis
Rhodococcus erythropolis
Rhodococcus erythropolis
Proteus hauseri
Rhodococcus erythropolis
Rhodococcus erythropolis
Serratia odorifera
Rhodococcus erythropolis
Rhodococcus erythropolis
Rhodococcus erythropolis
Rhodococcus erythropolis
Rahnella aquatilis
Rhodococcus erythropolis
Rhodococcus erythropolis
Rhodococcus erythropolis
Serratia odorifera
Rhodococcus erythropolis
Rhodococcus erythropolis
Rhodococcus erythropolis
Rhodococcus erythropolis
Rhodococcus erythropolis
Rhodococcus erythropolis
Rhodococcus erythropolis
Rhodococcus erythropolis
Rahnella aquatilis
Rhodococcus erythropolis
Rhodococcus erythropolis
Rhodococcus erythropolis
Rhodococcus erythropolis
Rhodococcus erythropolis
Rhodococcus erythropolis
Rhodococcus erythropolis
Isolate
Rhodococcus erythropolis















0.44 0.05
1.70 0.20
13.54 2.11
0.30 0.02
0.37 0.02
0.00
1.38 0.28
0.48 0.15
2.11 0.32
0.56 0.14
1.24 0.10
1.31 0.29
0.73 0.23
1.44 0.33
0.65 0.11
1.18 0.05
0.04 0.02
1.89 0.31
0.10 0.03
1.12 0.17
0.16 0.04
0.13 0.07
0.00
1.38 0.21
0.00
0.49 0.17
1.94 0.37
0.00
0.00
0.00
0.30 0.05
0.07 0.03
1.02 0.17
0.05 0.11
0.00
0.03 0.08
0.18 0.03
0.23 0.19
0.00
0.00
0.52 0.21
0.31 0.12

10.00 1.56
4.50 1.10
5.50 0.53
0.00
3.00 0.64
0.00
16.00 2.14
13.00 1.42
0.00
4.00 0.39
4.00 0.53
6.00 0.15
13.00 0.00
4.00 0.20
6.50 0.84
14.00 1.34
0.00
9.00 0.94
5.00 0.43
7.50 0.33
0.00
3.70 0.63
5.20 1.28
12.50 2.34
4.00 0.44
0.00
11.50 0.00
7.50 0.33
4.50 0.37
0.00
6.00 0.53
0.00
9.50 0.86
7.50 0.85
4.20 1.39
5.20 1.24
5.00 0.40
9.50 1.34
0.00
0.00
0.00
4.50 0.34

58.60 4.24
22.30 1.27
96.40 6.87
44.60 5.26
12.80 2.11
9.50 2.30
88.60 7.97
66.30 6.84
0.00
12.80 3.27
18.60 2.43
8.60 1.67
48.90 6.93
55.30 4.65
24.60 2.31
36.80 6.39
44.80 7.27
26.80 5.54
24.70 1.37
19.80 4.27
4.50 1.00
3.60 0.44
12.80 2.13
3.30 0.42
68.30 5.57
33.90 3.77
41.50 4.42
18.30 3.14
11.50 1.21
22.40 1.07
37.50 7.09
19.30 4.02
22.10 2.28
8.70 2.63
33.50 3.73
33.20 4.41
15.40 3.70
99.60 12.74
11.70 3.24
32.60 6.94
4.60 0.52
15.60 2.29

Stand. Dev. of three independent experiments.

shape method is dependent on the uid density. In our experiment,

the strains that were cultured in the M9 medium supplemented
with crude oil generated emulsions and the oil molecules remained
in the culture liquid even after centrifugation of broth and separation of obtained supernatant from the oil layer. These molecules
interfered with the measurement of the liquid density and thereby
inuenced the results of the surface tension. For this reason, the
values of the surface tension that were obtained with culture supernatants of the strain that was tested could not be interpreted
quantitatively. They can only be analysed qualitatively by
comparing the studied samples.
In our study, based on the PCA analysis, 12 strains of studied
bacteria (CD 112, CD 126, CD 131, CD 132, CD 135, CD 147, CD 154, CD
155, CD 158, CD 161, CD 166 and CD 167) have been chosen as
promising candidates for the production of biosurfactants and/or
bioemulsiers. Three of them (CD 161, CD 166 and CD 167) have
potential for the production of biosurfactants and bioemulsiers
which are active against organic compounds included in diesel oil.
Furthermore, 8 strains (CD 112, CD 126, CD 131, CD 135, CD 147, CD
154, CD 155 and CD 158) were efcient in forming emulsion with nhexadecan and p-xylene. Among them only 4 strains (CD 131, CD
154, CD 155 and CD 158) generated in the oil-spreading test an area

of displacement larger than 2.0 cm, indicating their high ability to

produce biosurfactants. On the other hand, strain CD 126 was
efcient in forming emulsion with both diesel oil, n-hexadecan and
p-xylene, but was less efcient in producing of biosurfactants.
Furthermore, it has been observed that strain CD 132 was efcient
bioemulsiers of n-hexadecan and p-xylene and generated very
high clear zones in the oil-spreading method. Because all the
screening assays used in our study were performed using a cell-free
culture supernatant, it can be concluded that studied strains secrete
biosurfactants/bioemulsiers into the growth medium. This
observation is very interesting from an industrial point of view
because product recovery can be simplied (Cameotra and Makkar,
1998; Kuyukina et al., 2001). Moreover, they showed also a good
surface and bioemulsier activity on the medium supplemented
with crude oil with unchanged parameters of pH and with no
additional carbon sources, which signicantly reduces the costs of
the production of biosurfactants/bioemulsiers.
4.2. Plant growth-promoting abilities
The combined use of plants and hydrocarbon-degrading and/
or biosurfactant-producing PGPB is a relatively new concept in

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M. Pacwa-Pociniczak et al. / Journal of Environmental Management 168 (2016) 175e184

Fig. 4. PCA ordination of the studied strains based on their plant growth promoting traits.

the eld of the remediation of contaminated soil. For example,

Cowie et al. (2010) reported that PGPB enhanced hydrocarbon
removal from soil, primarily due to improving plant growth, the
density of the bacterial population and its activity. Most studies
attribute a stimulation in plant growth and biomass to the production of phytohormones (such as indoleacetic acid, IAA), the
suppression of stress ethylene production (due to ACC deaminase
activity) or an improvement in the nutritive status of plants due to
the presence of N2 xers, PO4-solubilisers or siderophore producers
(Sessitsch et al., 2013). The activity of bacterial ACC deaminase is
regarded to be one of the most important mechanisms that improve
the growth of plants in contaminated environments. It has been
observed that plants inoculated with PGPB with a high activity of
ACC deaminase are signicantly more resistant to the deleterious
effects of the stress caused by hydrocarbons, heavy metals and high
salt content in comparison to non-inoculated plants (Weyens et al.,
2009). PGPB that produce ACC deaminase signicantly lowered the
level of ACC in the stressed plants, thereby limiting the amount of
stress ethylene synthesis, which led to improved plant growth and
development (Hontzeas et al., 2006; Zahir et al., 2008). The tested
strains exhibited several mechanisms that are associated with the
promotion of plant growth and an increase in plant biomass. Among
the twelve strains that were found to be effective in the production
of biosurfactants and/or bioemulsiers, only three (CD 132, CD 154
and CD 161) showed the activity of the four mechanisms that are
considered to be characteristic for plant growth-promoting bacteria.

However, considering all studied strains activity of PGP mechanisms

estimated for CD 132, CD 154 and CD 161 reached the moderate
values. Among these 3 strains the highest ACCD activity (37.5 nmol
a-ketobutyrate mg1 protein h1) was observed for CD 154. In the
present study, the activity of ACCD measured for 42 strains varied
from 3.3 (Serratia odorifera CD 138) to 99.60 nmol a-ketobutyrate
mg1 h1 (R. erythropolis CD 163). It was observed that the level of
ACCD activity that was determined for 23 of the isolates, approximately 20 nmol a-ketobutyrate mg1 protein h1, is sufcient to
permit a bacterium to grow on ACC and to act as a PGPB. Furthermore, organisms with a higher ACCD activity (300e400 nmol
a-ketobutyrate mg1 protein h1) do not necessarily promote root
elongation more than the strains that are characterised by a smaller
enzyme activity (Penrose and Glick, 2003).
Khan et al. (2013) reported that hydrocarbon-degrading bacteria
that have the ability to produce biosurfactants as well as to promote
the growth of plants enhanced plant biomass production and hydrocarbon remediation for more than the bacteria that had only a
hydrocarbon degrading activity. Thus, the synergistic action of the
plants and inoculated bacteria, which is called rhizodegradation,
was more efcient in the removal of hydrocarbons from soil, as
compared to microbial remediation and non-supported phytoremediation. PCA analysis showed that strains CD 106 and CD 111,
regardless of moderate production of biosurfactants/bioemulsiers,
may be considered as promising agents for plant growth promotion, especially as consortium with strains that are effective in

M. Pacwa-Pociniczak et al. / Journal of Environmental Management 168 (2016) 175e184

secretion of surface active compounds (e.g. CD 126 and/or CD 132).

5. Conclusions
The results of this study indicate that from 42 hydrocarbondegrading isolates, two strains (R. erythropolis CD 126 and Rahnella aquatilis CD 132) have the highest potential to be used in the
bioaugmentation of PH-contaminated soil. Among them strain CD
126 was able to form efcient emulsion with all tested hydrocarbons and strain CD 132 generated high oil-displaced area and
formed efcient emulsion with diesel oil and n-hexadecane. The
above-mentioned strains do not show high activity of mechanisms
potentially considered as useful in plant growth promotion so they
are not suitable to be used in microbe assisted phytoremediation.
Thus, the application of consortium composed of strains CD 126
and CD 132 together with strains R. erythropolis CD 106 (siderophores secrection, moderate IAA production and high ACCD activity) and R. erythropolis CD 111 (siderophores secrection, high
phosphate solubilizing ability and high ACCD activity) seems to be
reasonable for enhanced phytoremediation of petroleumcontaminated soil.
Acknowledgements
The research was supported by grant No. 2011/03/N/NZ9/02089
nanced by National Science Centre (Poland). Author M.P.P. is a
scholarship holder within the DoktoRIS projectescholarship program for the innovation of the Silesia region, which is supported by
the European Community from the European Social Fund.
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