catalysts

Article
The Potential Applications of Bacillus sp. and
Pseudomonas sp. Strains with Antimicrobial Activity
against Phytopathogens, in Waste Oils and the
Bioremediation of Hydrocarbons
Mariana-Gratiela Soare (Vladu) 1,2 , Elena Simina Lakatos 3, * , Nicoleta Ene 2,4 ,
Nereida Malo (Dalanaj) 5 , Ovidiu Popa 1 and Narcisa Babeanu 1
1 Faculty of Biotechnologies, University of Agronomic Sciences and Veterinary Medicine, Mărăs, ti Blv. 59,
011464 Bucharest, Romania; [email protected] (M.G.S.); [email protected] (O.P.);
[email protected] (N.B.)
2 National Institute for Chemical Pharmaceutical Research and Development-ICCF, Vitan Avenue 112,
031299 Bucharest, Romania; [email protected]
3 Institute for Research in Circular Economy and Environment “Ernest Lupan”, Dorobantilor Avenue 71–73,
400808 Cluj-Napoca, Romania
4 Biology Department, University of Bucharest, Regina Elisabeta Blv. 4–13, 050663 Bucharest, Romania
5 Research Institute of the University of Bucharest-ICUB, Regina Elisabeta Blv. 4–12,
030018 Bucharest, Romania; [email protected]
* Correspondence: [email protected]; Tel.: +40-742516554




Received: 27 September 2019; Accepted: 11 November 2019; Published: 15 November 2019


Abstract: Biodegradation is one of the primary mechanisms for the elimination of petroleum and
other hydrocarbon pollutants from the environment. This study presents the results obtained with
two newly isolated microorganisms and their potential applications in bioremediation, agriculture,
and industrial fields. Twenty-five strains of microorganisms were isolated from plant materials
and were subject to a selection process on the basis of antimicrobial activity. Two bacterial strains,
respectively Bacillus mycoides (Bm) and Pseudomonas putida (B1), were selected for further experiments,
based on the largest inhibition zones against the phytopathogens Erwinia carotovora and Xanthomonas
campestris. The production of biosurfactants and enzymes was evaluated in specific media. In
order to assess the production of biosurfactants, submerged bioprocesses were carried out on Yeast
Malt Peptone Glucose (YMPG), M44, Luria-Bertani (LB), and King B media (KB); the supernatants
were used to form emulsions with heptane, octane, and sunflower oil, and the emulsifying indices
were determined.

Keywords: antimicrobial activity; biosurfactants; emulsion index; sunflower oil; hydrocarbons

1. Introduction
The resistance of phytopathogens to a wide range of chemical synthesis compounds is currently
a widespread phenomenon, and finding solutions to replace these substances with new, natural
compounds is an ongoing concern in the field of research [1]. Many studies and experiments have
been carried out over time, both in the laboratory and in the field, to combat the phytopathogens with
the help of microbial antagonists. These antagonists, also known as biocontrol agents, are commonly
occurring nonpathogenic microbes such as fungi, bacteria, or yeast. Antagonists act directly on
phytopathogens through different mechanisms such as hyperparasitism, competition for a substrate,
and the secretion of metabolites with an antibiotic effect. Many of the bacteria belonging to the

Catalysts 2019, 9, 959; doi:10.3390/catal9110959 www.mdpi.com/journal/catalysts

Catalysts 2019, 9, 959 2 of 16

Bacillus and Pseudomonas genera have been studied for their use in phytopathogens’ biocontrol [2].
These types of bacteria are known for their versatility and their ability to populate various niches
and to grow on numerous substrates [3]. This is due to the fact that they are rich in enzymes
and also to the metabolites with antimicrobial properties they excrete as biosurfactants [4,5]. It is
known that, under normal conditions, biosurfactants are bioactive compounds with the property
to decrease the superficial tension at the interface between immiscible liquids. The biosurfactant
producing microorganisms have the advantage of being able to survive and grow on water immiscible
substrates [6]. The Pseudomonas, Bacillus, Rhodococcus, and Candida genera are the most widely studied
microorganisms as producers of different types of biosurfactants [7]. Having different molecular
structures and surface activities, the biosurfactants are divided into different classes: glycolipids
(Candida sp., Pseudomonas sp.), polymeric surfactants (Candida sp., Bacillus sp.), lipopeptides (Bacillus sp.,
Candida lipolytica, Pseudomonas fluorescens), fatty acids, particulate surfactants (Pseudomonas marginalis),
and phospholipids [6]. For example, Bacillus sp. are known to produce surfactins, iturins, fengycins,
and lichenysins; Pseudomonas sp. produce mostly rhamnolipids; Rhodococcus sp., trehalolipids; and
Candida sp. liposans and sophorolipids.
Many studies aim to demonstrate the connections between compounds with an antimicrobial effect
and their biosurfactant properties [8,9]. According to some researchers, the production of peptides with
antimicrobial activity is characteristic for Bacillus sp. [10]. Grossman suggests that peptide antibiotics
play an essential role in sporulation [11]. These peptides, commonly known as lipopeptides, are
capable of damaging the plasma membrane of the target microorganisms (e.g., R. solanacearum, X.
oryzae), forming pores and leading to their death [12]. Other studies indicate that biosurfactants have
an antagonistic effect against other microorganisms [13,14], are involved in adherence to different
surfaces [15], accelerate a composting process, hence providing favorable conditions for beneficial
microbes [16], and are involved in bioremediation of contaminated environments.
Bioremediation is related to the acceleration of natural biodegradation processes in contaminated
environments. It is often a cost effective method to treat oily soils and petroleum wastes containing
biodegradable hydrocarbons and involves mostly indigenous microbes. The high diversity of
biosurfactants produced by numerous microorganisms is noteworthy [17]. In this sense, the chemical
nature of a biosurfactant/bioemulsifier plays an important role in its function. Many researchers have
established direct connections between the emulsification capacity of a biosurfactant and bioremediation.
The biosurfactants that registered good emulsification indexes proved to be most capable of making
the insoluble substrate accessible for biodegradation. Such biosurfactants can be successfully used for
Microbial Enhanced Oil Recovery (MEOR). For example, Silva et al. found that Pseudomonas aeruginosa
UCP 0992 and the biosurfactant produced by this strain achieved degradation rates higher than 90%
for oil present in sand and seawater [18]. Other researchers have shown that, due to their remarkable
characteristics (such as dispersion, emulsification, and de-emulsification), the addition of biosurfactants
has improved the bioremediation of oil contaminated sites, by improving the biodegradation activity
of indigenous microorganisms [19,20].
The fact that biosurfactants have a biological origin implies both better biocompatibility and good
microbial biodegradability; consequently, there is a large number of potential applications for this type
of surfactants, especially when there is extensive interference with the environment, for example for
tertiary petroleum recovery, decontamination of oil polluted areas, crop protection, and for the cosmetic
and pharmaceutical industries [21]. Therefore, it is not surprising that a number of investigations have
been carried out, both in the laboratory [22,23] and in the field [24–26], aiming to reveal the production
of such compounds with potential applications in bioremediation, agriculture, and industry.
The present paper is structured as follows: Section 2 presents the results and discussions related
to the production of biosurfactants and enzymes, in specific media. Section 3 provides the concept of
the study, and methodological remarks are presented in Section 3. Section 4 gives the conclusions of
the study.
Catalysts 2019, 11, x FOR PEER REVIEW 3 of 16

Catalysts 2019, 11, x FOR PEER REVIEW 3 of 16

concept of the study, and methodological remarks are presented in Section 3. Section 4 gives the
conclusions
concept of the
of the study.
study, and methodological remarks are presented in Section 3. Section 4 gives the
Catalysts 2019, 9, 959 3 of 16
conclusions of the study.
2. Results and Discussion
2. Results
Results and
and Discussion
Discussion
2.1. Cultivation of B. mycoides and P. putida in Several Media
Cultivation
2.1. Cultivation
Newly isolated mycoides
of B. mycoides and
andP.P.putida
microorganisms putida
werein in
Several Media
Several
grown Media
submerged in the above mentioned media, in
orderNewly
to be isolated
tested for antimicrobial
isolatedmicroorganisms
microorganismswere activity
were grownand
grown for enzyme
submerged
submerged and
in the biosurfactant
in above
the production.
mentioned
above media,
mentioned inIn the
order
media, in
figures below,
to be tested
order the growth
for antimicrobial
to be tested results for B. mycoides
activityactivity
for antimicrobial and forand (Bm)
enzyme and P. putida (B1)
and biosurfactant
for enzyme are presented.
production.
and biosurfactant In the figures
production. In the
below,Inthe
figures thegrowth
NBthe
below, medium
results(Figure
growth for
results1),
forthe
B. strain
B. mycoides (Bm)P.
mycoides putida
and (B1)P.
P. putida
(Bm) and reached
(B1) the maximum
are presented.
putida (B1) growth after 16 h;
are presented.
then,Ina slow decrease was registered, measured
the NB medium (Figure 1), the strain in optical density.
P. putida (B1) reached the maximum
strain P. maximum growth
growth after
after 16
16 h;
h;
then, a slow decrease was registered, measured
measured in
in optical
optical density.
density.

Figure 1. Growth of P. putida (B1) and B. mycoides (Bm) strains in NB medium.

Figure
Figure 1. Growth
Growth of
of P.
P. putida (B1) and B. mycoides (Bm) strains in NB medium.
The overall trend for pH value was a slight increase from 7.25 to 8.57. For B. mycoides (Bm), the
The
maximum overall
growth
The overall trend
wasfor
trend pH
pH value
achieved
for after
value was
8 haaof
was slight increase
increase from
fermentation,
slight when
from 7.25
theto
7.25 8.57.
topH For
value
8.57. B.
B. mycoides
Foralso reached(Bm),
mycoides the
a higher
(Bm), the
maximum
value. Then, growth
both was
the ODachieved
and the after
pH 8 h of
started fermentation,
to decrease when
slowly. the pH
maximum growth was achieved after 8 h of fermentation, when the pH value also reached a higher value also reached a higher
value. Then,
value.The
Then, both
slight
both the OD
the OD and
disruptions andofthe
the pH
thepH startedtrend
general
started to decrease
to decrease
in ODslowly.
were probably due to the shift from one
slowly.
source
The
Theofslight
growth
slight to another.
disruptions
disruptions ofofthethegeneral
generaltrend
trendin OD werewere
in OD probably due to
probably thetoshift
due thefrom
shiftone
from source
one
of In
growthKing
to B (KB)
another. medium
source of growth to another. (Figure 2), the strain P. putida (B1) achieved its maximum biomass
growth
In at 32BB
In King
King h(KB)
after inoculation,
(KB)medium
medium (Figure and
(Figure2),then,
the the
2), a slow
strain
strain decrease
P. putida (B1)inachieved
P. putida OD achieved
(B1) value wasitsobserved.
its maximum During
biomass
maximum the
growth
biomass
growth
at
growth period,
32 h after
at the pH
32inoculation,
h after value wasand
and then,
inoculation, in the
a slow
then,range of 7.2–7.5
decrease
a slow in ODand,
decrease value
in ODafter
was that, rose observed.
observed.
value was slightly
Duringtotheeight. The
growth
During the
strain
period,B.
themycoides
pH value(Bm)wasshowed
in the significant
range of growth
7.2–7.5 until
and, 24
after h,
that, coinciding
rose with
slightly
growth period, the pH value was in the range of 7.2–7.5 and, after that, rose slightly to eight. The to a slight
eight. decrease
The strain in
B.
pH value.
mycoides (Bm) showed significant growth until 24 h, coinciding with a slight
strain B. mycoides (Bm) showed significant growth until 24 h, coinciding with a slight decrease in decrease in pH value.
pH value.

Growth of P. putida (B1) and B. mycoides

Figure 2. Growth mycoides (Bm)
(Bm) strains
strains in
in King
King BB (KB)
(KB) medium.
medium.
Figure 2. Growth
The significant of in
decrease P. putida (B1) and32
OD between B. mycoides
and 48 h(Bm)
wasstrains in King
probably dueBto
(KB)
themedium.
fact that the strain
The significant decrease in OD between 32 and 48 h was probably due to the fact that the strain
depleted the peptone from the medium and switched to glycerol metabolism. In the next 16 h, when
depleted the peptone
The significant from the
decrease medium
in OD between and
32 switched to glycerol
and 48 h was probably metabolism. In the
due to the fact thatnext 16 h,
the strain
glycerol was almost exhausted, the OD value began to decrease as a result of cell death because of the
when glycerol was almost exhausted, the OD value began to decrease as a result
depleted the peptone from the medium and switched to glycerol metabolism. In the next 16 h, of cell death
small amount
because of theof nutrients,
small amountremaining relatively
of nutrients, constant
remaining between
relatively 56 andbetween
constant 72 h. 56 and 72 h.
when glycerol was almost exhausted, the OD value began to decrease as a result of cell death
During fermentation in the LB medium (Figure 3), the strains’ growth was similar: Maximum
because of the small amount of nutrients, remaining relatively constant between 56 and 72 h.
growth was achieved at 16 h for both strains. However, B. mycoides (Bm) registered higher values for
OD. Almost similar values were registered for the pH, with small differences in the first 12 h, as the
general tendency for the Bm strain was the pH decrease in the logarithmic phase of growth.
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11, xx FOR
FOR PEER
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During
During fermentation
fermentation in in the
the LB
LB medium
medium (Figure
(Figure 3),
3), the
the strains’
strains’ growth
growth was
was similar:
similar: Maximum
Maximum
growth
growth was achieved at 16 h for both strains. However, B. mycoides (Bm) registered higher
was achieved at 16 h for both strains. However, B. mycoides (Bm) registered higher values
values
for
for OD.
OD. Almost
Almost similar
similar values
values were
were registered
registered for
for the
the pH,
pH, with
with small
small differences
differences in
in the
the first
first 12
12 h, as
Catalysts 2019, 9, 959 4h,
of as
16
the general tendency for the Bm strain was the pH decrease in the logarithmic phase
the general tendency for the Bm strain was the pH decrease in the logarithmic phase of growth. of growth.

Figure
Figure 3.
3. Growth
Growth of
Growth of P.
of P. putida (B1)
P. putida (B1) and
and B.
B. mycoides
mycoides (Bm)
(Bm) strains
strains in
strains in LB
in LB medium.
LB medium.
medium.

In
In the
the M44
M44 medium
medium (Figure
(Figure 4),
4), both
both strains
strains registered
registered the
the best
best values
values for
for OD,
OD, when
when compared
compared
with
with the
the results
results obtained
obtained for
for the
the other media. Maximum
other media. Maximum growth
Maximum growth for
growth for Bm
Bm was
was achieved
achieved at
at 44
44 h,
h, then
then
OD started to slowly decrease, probably because of the lack of a
OD started to slowly decrease, probably because of the lack of a nitrogen source and an excess of
nitrogen source and an excess of
glycerol.
glycerol. B1
B1 exhibited
B1 exhibited aa different
exhibited different behavior:
behavior: The
The growth
The growth curve
curve forfor this
this strain
strain remained
remained ascendant
ascendant atat
72
72 h, probably because
because the glycerol
the glycerol amount
glycerol amount did
amount did not
did not act
not act as
act as an
as an inhibitor.
an inhibitor.
inhibitor.

Figure
Figure 4.
4. Growth
Growth of
Growth of P.
of putida (B1)
P. putida
P. (B1) and
and B.
B. mycoides
mycoides (Bm)
(Bm) strains
strains in
strains in M44
in M44 medium.
M44 medium.
medium.

The
The maximum
maximumgrowth
maximum growthfor
growth forB1
for B1was
B1 wasreached
was reached
reached at at
32 32
at h in
32 h the
h in YMPG
in the
the YMPG
YMPG medium
medium
medium(Figure 5). In5).
(Figure
(Figure 5).the
Infirst
In the 24
the h,
first
first
the
24 pHthe
24 h,
h, value
the pH slowly
pH value decreased
value slowly
slowly to 6.88 and
decreased
decreased to thenand
to 6.88
6.88 slowly
and thenincreased
then slowly to 7.51 at to
slowly increased
increased the7.51
to end at
7.51 of the
at the bioprocess.
end
end ofof the
the
The cellular
bioprocess. growth
The for
cellular the Bm
growth strain
for was
the greatest
Bm strainat 32
was h and significantly
greatest at 32 h decreased
and between
significantly
bioprocess. The cellular growth for the Bm strain was greatest at 32 h and significantly decreased 40 h and
decreased
48 h, as a40
between
between result
40 h of 48
h and
and cellh,
48 h,death,
as remaining
as aa result
result of cellrelatively
of cell death, constantrelatively
death, remaining
remaining between 48
relatively and 72between
constant
constant h.
between 48
48 and
and 7272 h.
h.

Figure
Figure 5.
5. Growth
Growth of
Growth of P.
of P. putida
P. putida (B1) and B.
(B1) and B. mycoides
mycoides (Bm)
(Bm) strains
strains in
in YMPG
YMPG medium.
medium.
2.2. Antimicrobial Activity

2.2.1. Antimicrobial Activity of P. putida and B. mycoides against E. carotovora and X. campestris
The results obtained during the experiments confirmed that, in both cases, the antagonists exerted
antimicrobial action against the phytopathogens E. carotovora ICCF 138 and X. campestris ICCF 274. A
Catalysts 2019, 9, 959 5 of 16

better antimicrobial activity (Table 1) was noticed in Experiment I, where the diameters of the inhibition
zones were larger than those registered in Experiment II.

Table 1. Antimicrobial activity of antagonists after 48 h of growth together with phytopathogens E.

carotovora ICCF 138 and X. campestris ICCF 274.

Inhibition Zones (mm) against Inhibition Zones (mm) against

Bacterial Strain E. carotovora ICCF 138 X. campestris ICCF 274
Experiment I Experiment II Experiment I Experiment II
P. putida (B1) 35 ± 0.58 24 ± 0.29 36 ± 0.58 22 ± 0.29
B. mycoides (Bm) 60 ± 0.58 27 ± 0.58 43 ± 0.58 30 ± 0.29
Control - - - -
Note: Experiment I, antagonists added in the same day with phytopathogen; Experiment II, antagonists added 24 h
after phytopathogen inoculation. Data are the means of three replicates (n = 3) ± standard error.

2.2.2. Antimicrobial Activity of the Biosurfactants from P. putida and B. mycoides Supernatants against
E. carotovora and X. campestris
In Table 2, the results are presented as the average of the values together with the standard errors,
from three independent replications.

Table 2. Antimicrobial activity (as zones of inhibition) of the biosurfactants after 48 h of growth together
with the phytopathogens.

Inhibition Zone Inhibition Zone

Supernatants of the Strains Diameters (in mm) Diameters (in mm)
Against E. c. ICCF138 Against X. c. ICCF274
1* 7 ± 0.87 8 ± 0.29
2* 10 ± 0.58 10 ± 0.29
P. putida (B1)/Medium * 3* 10 ± 0.58 9 ± 0.58
4* 11 ± 0.58 12 ± 0.29
5* 8 ± 0.58 9 ± 0.58
1* 8 ± 0.58 7 ± 0.58
2* 10 ± 0.29 9 ± 0.58
B. mycoides
3* 9 ± 0.58 10 ± 0.58
(Bm)/Medium *
4* 10 ± 0.58 11 ± 0.87
5* 8 ± 0.58 7 ± 0.58
Medium *: 1 *, NB; 2 *, KB; 3 *, LB; 4 *, M44; 5 *, YMPG. Data are the means of three replicates (n = 3) ± standard error.

According to some researchers [27], the production of biosurfactants is influenced by the type
of carbon source present in the medium. The results obtained during this study confirmed their
observations, especially in the case of the B. mycoides (Bm) strain, which grew better in the M44 medium
containing glycerol.

2.3. Enzyme Production

Extracellular enzyme and metabolite production is often associated with antagonism
properties [28]. Most of the species of the Bacillus and Pseudomonas genera have the ability to
produce extracellular enzymes and secondary metabolites [29]. For this reason, the selected strains
were tested in several media in order to study the production of such compounds. Enzyme production
was revealed on agarized media, containing the appropriate carbon sources. Tests results revealed
that B. mycoides (Bm) produced amylases, P. putida (B1) produced catalase (Figures 6 and 7), and both
strains produced lipases (Figure 8).
results
productionrevealedwas that B. mycoides (Bm) produced amylases, P.the
putida (B1) produced catalase (Figures
6 and 7), and bothrevealed on agarized
strains produced media,
lipases containing
(Figure 8). appropriate carbon sources. Tests
6results
and 7),revealed
and both strains produced
that obtained
B. mycoides lipases (Figure 8).
The inoculum in (Bm) produced
all five media amylases,
produced P. putida
halos (B1) produced
around catalase
the colonies, (Figures
which is an
andThe
6indicator inoculum
7), and both obtained
strains in all lipases
produced five media produced
(Figure 8). halos around the colonies, which is an
of lipase production and potential application of the strains in the bioremediation of
indicator
The of lipase production
inoculum andallpotential
obtained in five mediaapplication
producedof the strains
halos in thethe
around bioremediation of is an
colonies, which
waste oils.
waste oils.
indicator of lipase
Catalysts 2019, 9, 959 production and potential application of the strains in the bioremediation of 6 of 16
waste oils.

(A) (B)
(A) (B)
(A) (A) B. mycoides (Bm) positive;
Figure 6. Amylase production: (B) (B) P. putida (B1) negative.
Figure 6. Amylase production: (A) B. mycoides (Bm) positive; (B) P. putida (B1) negative.
Figure 6. Amylase production: (A) B. mycoides (Bm) positive; (B) P. putida (B1) negative.
Figure 6. Amylase production: (A) B. mycoides (Bm) positive; (B) P. putida (B1) negative.

(A) (B)
(A) (B)
Figure 7. Catalase
Figure 7. (A) (A)
Catalase production:
production: (A) B.
B. mycoides
mycoides (Bm) (B)
(Bm) insignificant;
insignificant; (B)
(B) P. putida (B1)
P. putida (B1) positive.
positive.
Figure 7. Catalase production: (A) B. mycoides (Bm) insignificant; (B) P. putida (B1) positive.
Figure 7. Catalase production: (A) B. mycoides (Bm) insignificant; (B) P. putida (B1) positive.

(A) (B)
(A) (B)
8. Lipase
Figure 8. Lipaseproduction
productionwith withstrains P. putida
strains (B1)(B1)
P. putida (A) B.
(A) and mycoides
and (Bm) (B)
B. mycoides (inocula
(Bm) from media:
(B) (inocula from
Figure 8. Lipase (A)
production with strains P. M44
putida (B)(Bm) (B) (inocula from
NA (1 and
media: NA11), KB
(1 and (2 and
11), KB12), LB (3
(2 and and
12), LB13),
(3 and (4 (B1)
13), and (A) YMPG
M4414),
and B. mycoides
(4 and 14), (5 and 15)).
YMPG (5 and 15)).
media:
Figure NA (1 andproduction
8. Lipase 11), KB (2 andwith12), LB (3 P.
strains and 13), M44
putida (B1) (4
(A)and
and14),
B.YMPG
mycoides(5 and
(Bm)15)).
(B) (inocula from
The
media:inoculum
NA (1 obtained
and 11), KB in
(2 all
and five
12), media
LB (3 produced
and 13), M44 halos
(4 andaround
14),
The enzymatic activities measured for amylase (enzymatic assay of α-Amylase the
YMPG colonies,
(5 and which
15)). EC is 3.2.1.1,
an indicator
with
of The enzymatic
lipase production activities measured
and potential for amylase
application the(enzymatic
of EC strains assay of α-Amylase of EC 3.2.1.1, with
DNS, [30]), lipase (enzymatic assay of lipase 3.1.1.3, intitrimetric
the bioremediation
method, [31]), wasteandoils.
catalase
DNS,The [30]), lipase
The enzymatic (enzymatic assay of lipase EC 3.1.1.3, titrimetric method, [31]), and catalase
(enzymatic assay ofactivities
enzymatic activities
catalase ECmeasured
1.11.1.6,for
measured for amylase
amylase
[32]), (enzymatic
following(enzymatic assay
assay of
the Sigma-Aldrich of α-Amylase
α-Amylase
protocols, EC
EC 3.2.1.1,
3.2.1.1,
revealed with
with
that:
(enzymatic
DNS, [30]),assay
DNS,After
[30]), lipase
lipase of catalase EC
(enzymatic
(enzymatic 1.11.1.6,
assay
assay of of
lipase[32]),
lipase
EC following
EC
3.1.1.3, the Sigma-Aldrich
3.1.1.3, titrimetric
titrimetric method, protocols,
method,
[31]), and revealed
[31]), and
catalase that:
catalase
(enzymatic
72 h of development in SNA medium, B. mycoides (Bm) registered 0.75 U/mL, and the
assayAfter
(enzymatic 72 h ofEC
assay
of catalase ofdevelopment
catalase
1.11.1.6, EC in following
SNA[32]),
1.11.1.6,
[32]), medium, B. mycoides
following
the (Bm)
the Sigma-Aldrich
Sigma-Aldrich registered
protocols, 0.75that:
protocols,
revealed U/mL,
revealedandthat:
the
amylase activity of the P. putida (B1) strain was insignificant.
amylase
After activity of the P. putida (B1) strain was insignificant.
After 72
After 48 hhh of
72 ofdevelopment
of developmentin
development inSNA
in SNA medium,
medium, B.
LA medium, B. mycoides
B. mycoides
mycoides (Bm)(Bm)
(Bm) registered
registered 0.75
registered 0.75 U/mL,
0.39 U/mL, and
U/mL, and the
and the
the
Afteractivity
amylase
amylase 48 h of
activity of
of development
the
the P.
P. putida in LA
(B1) strainmedium,
was B. mycoides (Bm) registered 0.39 U/mL, and the
insignificant.
lipase activity of the P. putida (B1) strain registered 0.36 U/mL.
lipaseAfter
activity hh of of
thedevelopment
P. putida (B1) in strain registered 0.36 U/mL.
After 48
After 72 hof ofdevelopment
developmentinon LA LA medium,
medium,
M44 solid medium,B. mycoides
B. mycoides P. (Bm) (Bm)
putida registered
registered
(B1) 0.39
2.3U/mL,
0.39 U/mL,
registered and the
U/mL, and the
lipase
and the
lipaseAfter
activity of 72
activity
the h of
of
P. development
the
putida P. putida
(B1) (B1)
strainon M44
strain
registered solid medium,
registered
0.36 0.36
U/mL. P. putida (B1) registered 2.3 U/mL, and the
U/mL.
catalase activity of the B. mycoides (Bm) strain was insignificant.
catalase
Afteractivity
After 72 of
72 hh of the B. mycoideson
of development
development (Bm)
on M44
M44 strain
solidwas
solid insignificant.
medium,
medium, P.
P. putida
putida (B1)
(B1) registered
registered 2.3
2.3 U/mL,
U/mL, and and the
the
catalase
catalase activity
activity of
of the
the B.
B. mycoides
mycoides (Bm)
(Bm) strain
strain was
was insignificant.
insignificant.

2.4. Emulsification Index

The cultivation dynamics of the selected strains, tested on KB, LB, M44, and YMPG media
for biosurfactant production, was monitored by regular pH and ODλ = 550nm measurements. After
72 h of fermentation, the media were centrifuged, and the supernatants were used for the following
experiments. In the Figure 9 are presented the emulsions obtained by these supernatants with sunflower
oil, heptane and octane.
 2.4. Emulsification
following experiments. Index
In the Figure 9 are presented the emulsions obtained by these supernatants
with sunflower oil, heptane and octane.
The cultivation dynamics of the selected strains, tested on KB, LB, M44, and YMPG media for
biosurfactant production, was monitored by regular pH and ODλ = 550nm measurements. After 72 h of
fermentation, the media were centrifuged, and the supernatants were used for the
following
Catalysts experiments.
2019, 9, 959 In the Figure 9 are presented the emulsions obtained by these supernatants
7 of 16
with sunflower oil, heptane and octane.

Figure 9. Emulsions obtained with the supernatants of the two strains cultivated on LB and KB
media: supernatants of P. putida with: sunflower oil 1 and 2, heptane 3 and 4, octane 5 and 6;
supernatants of B. mycoides with sunflower oil 7 and 8, heptane 9 and 10, octane 11 and 12.

The figures below present the values of the emulsifying index obtained after 24 h and 30 days,
Figure 9. 9. Emulsions
Emulsionsobtained
obtainedwithwith thethe supernatants
supernatants of the
of the two two strains
strains cultivated
cultivated on LBon andLBKBand KB
media:
respectively,
media:
following
supernatants
the of
vigorous
P. putida
stirring
with:
of supernatants
sunflower oil 1
with
and 2,
sunflower
heptane 3
oil, heptane,
and
supernatants of P. putida with: sunflower oil 1 and 2, heptane 3 and 4, octane 5 6; supernatants of 6;
4, octane 5 and B.
and octane.
supernatants
mycoides withof B. mycoides
sunflower oil with
7 andsunflower
8, heptaneoil 7 and
9 and 10,8,octane
heptane119and
and12.
10, octane 11 and 12.
Supernatants of the P. putida (B1) strain grown in the LB medium produced stable emulsions
with sunflower
The figures oil (Figure 10); thethe
below present value of the
values of emulsifying
emulsifying
the emulsifying index was obtained
index
index 56.7 afterafter
obtained 24 h 24
after and
24 hh 54.76
and 30
and after
30 days,
days,
30 respectively,
days, respectively.
respectively, following
followingIn thetheKB
the medium,
vigorous
vigorous the strain
stirring
stirring of produced biosurfactants
of supernatants
supernatants with sunflowerthat
withsunflower oil,emulsified
oil, heptane,
heptane,and theoctane.
oil,
butandtheoctane.
emulsion formed
Supernatants of the P. was
putidaless
(B1) stable; the emulsifying
strain grown in the LB medium indexproduced
was 19.18 after
stable 30 days.
emulsions with
Supernatants
sunflower of the
oil (Figure
Supernatants B. mycoides
of 10);
the the (Bm)
value (B1)
P. putida strain grown
of thestrain
emulsifyingon LB
grown index and
in thewasKB media produced
56.7 after produced
LB medium emulsions
24 h and 54.76
stableafter with
30 days,
emulsions
sunflower
with oil (Figure
respectively.
sunflower In oil 10)
KBthat
the(Figure remained
medium,
10); thethe stable
strain
value for 24
produced
of the h. biosurfactants
emulsifying index wasthat 56.7emulsified
after 24 h theandoil,
54.76butafter
the
30 P. putida
emulsion (B1)
days, respectively.produced
formed was In less biosurfactants
stable;
the in the
the emulsifying
KB medium, YMPG
the strainindex and M44
was 19.18
produced media (Figure
after 30 days.
biosurfactants 11), which
thatSupernatants formed
emulsified the of oil,
the
emulsions
B. the with
butmycoides (Bm)
emulsion sunflower
strain grown
formed oil was
that
on remained
LBless
andstable; stable
KB media for 24 h,
theproduced
emulsifying while
indexthose
emulsions with of19.18
B. mycoides
wassunflower 30(Bm)
oil (Figure
after 10)
days.
remained stable of
that remained
Supernatants for 30 days,
stable
the 24when
formycoides
B. h. (Bm)the strain
strainwas grown
grown onon LBM44.
and KB media produced emulsions with
sunflower oil (Figure 10) that remained stable for 24 h.
P. putida (B1) produced biosurfactants in the YMPG and M44 media (Figure 11), which formed
emulsions with sunflower oil that remained stable for 24 h, while those of B. mycoides (Bm)
remained stable for 30 days, when the strain was grown on M44.

Figure
Figure 10.10. Emulsifying
Emulsifying index
index forfor sunflower
sunflower oil oil
(LB(LB
andand KB).
KB).

P. putida (B1) produced biosurfactants in the YMPG and M44 media (Figure 11), which formed
emulsions with sunflower oil that remained stable for 24 h, while those of B. mycoides (Bm) remained
stable for 30 days, when the strain was grown on M44.
Figure 10. Emulsifying index for sunflower oil (LB and KB).
 Catalysts 2019, 9, 959 8 of 16
Catalysts 2019, 11, x FOR PEER REVIEW
Figure 11. Emulsifying index for sunflower oil (YMPG and M44). 8 of 16

The emulsifying indices registered for the P. putida (B1) strain grown in the LB medium were
higher in the case of heptane (Figure 12) and were also stable: After 24 h, it was 68.49 and 63.23 after
30 days, respectively. The biosurfactants produced in the KB medium also formed relatively stable
emulsions with heptane; the emulsifying indices were 34.29 after 24 h and 20.9 after
30 days, respectively.
The emulsions of the supernatants from B. mycoides (Bm) grown in both the LB and KB media
(Figure 12) with heptane were not stable for even 24 h.
The emulsions of the biosurfactants from the P. putida (B1) strain (on YMPG and M44) with
heptane (Figure 13) remained stable for 24 h, while the Bm supernatants from the fermentations in
the M44 medium (Figures 13) formed emulsions that remained stable for 30 days.
Biosurfactants of the P. putida (B1) strain grown in both the KB and LB media (Figure 14)
registered higher values of emulsifying indices with octane at 24 h and 30 days, respectively.
Biosurfactants produced by B. mycoides (Bm) in the KB and LB media (Figure 14) did not seem to
have the ability to emulsifyFigure
Figure
the
11. 11.
octane,
Emulsifying
but
Emulsifying those
index
index
produced
for for sunflower
sunflower
onoilM44
(YMPG
oil (YMPG
(Figure
andand
15) registered, at 24 h
M44).
M44).
and 30 days, good values for emulsifying indices.
TheThe
The emulsifying
emulsions
emulsifying indices
ofindices
the registeredfor
biosurfactants
registered for the P.
from
the P. putida
the (B1)
(B1)strain
P. putida
putida (B1)
straingrown
strain
grown in in
(on the LB LB
YMPG
the medium
and werewere
M44)
medium higher
with
in the
heptane
higher case of heptane
in(Figure
the case 13) and(Figure
of heptane octane 12) and
12) were
(Figure
(Figure also stable:
15) were
and remained AfterAfter
stable
also stable: 24 h,
for ith,
2424 was it68.49
h,while
wastheandBm
68.4963.23 after 30
supernatants
and 63.23 days,
after
respectively.
from the The
fermentationsbiosurfactants
in the M44 produced
medium in the
(FiguresKB medium
13 and also
15) formed
formed
30 days, respectively. The biosurfactants produced in the KB medium also formed relatively stable relatively
emulsions stable
that emulsions
remained
stable
withfor
emulsions 30 days.
heptane;
with the emulsifying
heptane; indices were
the emulsifying indices 34.29
wereafter 24 hafter
34.29 and24 20.9 after20.9
h and 30 days,
after respectively.
30 days, respectively.
The emulsions of the supernatants from B. mycoides (Bm) grown in both the LB and KB media
(Figure 12) with heptane were not stable for even 24 h.
The emulsions of the biosurfactants from the P. putida (B1) strain (on YMPG and M44) with
heptane (Figure 13) remained stable for 24 h, while the Bm supernatants from the fermentations in
the M44 medium (Figures 13) formed emulsions that remained stable for 30 days.
Biosurfactants of the P. putida (B1) strain grown in both the KB and LB media (Figure 14)
registered higher values of emulsifying indices with octane at 24 h and 30 days, respectively.
Biosurfactants produced by B. mycoides (Bm) in the KB and LB media (Figure 14) did not seem to
have the ability to emulsify the octane, but those produced on M44 (Figure 15) registered, at 24 h
and 30 days, good values for emulsifying indices.
The emulsions of the biosurfactants from the P. putida (B1) strain (on YMPG and M44) with
heptane (Figure 13) and octane (Figure 15) remained stable for 24 h, while the Bm supernatants
from the fermentations in the M44 medium (Figures 13 and 15) formed emulsions that remained
stable for 30 days.
Figure 12. Emulsifying index for heptane (LB and KB).
Figure 12. Emulsifying index for heptane (LB and KB).
The emulsions of the supernatants from B. mycoides (Bm) grown in both the LB and KB media
(Figure 12) with heptane were not stable for even 24 h.
The emulsions of the biosurfactants from the P. putida (B1) strain (on YMPG and M44) with
heptane (Figure 13) remained stable for 24 h, while the Bm supernatants from the fermentations in the
M44 medium (Figure 13) formed emulsions that remained stable for 30 days.

Figure 12. Emulsifying index for heptane (LB and KB).

Catalysts 2019, 9, 959 9 of 16
Catalysts 2019, 11, x FOR PEER REVIEW 9 of 16

Catalysts 2019, 11, x FOR PEER REVIEW 9 of 16

Catalysts 2019, 11, x FOR PEER REVIEW 9 of 16

Figure Emulsifying
13.13.
Figure Emulsifyingindex
index for heptane(YMPG
for heptane (YMPG and
and M44).
M44).

Biosurfactants of the P. putida (B1) strain grown in both the KB and LB media (Figure 14) registered
higher values of emulsifying indices with octane at 24 h and 30 days, respectively. Biosurfactants
produced by B. mycoides (Bm) in the KB and LB media (Figure 14) did not seem to have the ability
to emulsify the octane, but those produced on M44 (Figure 15) registered, at 24 h and 30 days, good
values for emulsifying indices.
Figure 13. Emulsifying index for heptane (YMPG and M44).
Figure 13. Emulsifying index for heptane (YMPG and M44).

Figure 14. Emulsifying index for octane (LB and KB).

Figure 14.14.Emulsifying
Figure Emulsifying index foroctane
index for octane(LB
(LB and
and KB).
KB).
Figure 14. Emulsifying index for octane (LB and KB).

Figure 15. Emulsifying index for octane (YMPG and M44).

Figure Emulsifying
15.15.
Figure Emulsifyingindex for octane
index for octane(YMPG
(YMPG and
and M44).
M44).

Figure 15. Emulsifying index for octane (YMPG and M44).

Catalysts 2019, 9, 959 10 of 16

The emulsions of the biosurfactants from the P. putida (B1) strain (on YMPG and M44) with
heptane (Figure 13) and octane (Figure 15) remained stable for 24 h, while the Bm supernatants from
the fermentations in the M44 medium (Figures 13 and 15) formed emulsions that remained stable for
30 days.
The values of the emulsifying index obtained with sunflower oil indicated that the biosurfactants
produced by B. mycoides (Bm) could have applications in the preparation of oil based cosmetics and
foods, as well as in the bioremediation of oil waste.
Some authors have shown that Pseudomonas sp. strains are good producers of biosurfactants that
can be used in many industrial applications [33–35]. According to Chong and Lee, the key enzymes
for rhamnolipids biosynthesis are almost exclusively limited to Pseudomonas sp. and Burkholderia sp.
Although many authors have claimed that P. aeruginosa is the best producer of biosurfactants, there
remain some concerns about its pathogenicity in the case of large scale production and applications.
Our results showed that the P. putida (B1) strain was a good producer of biosurfactants, and
in addition, the strain did not present a danger for future applications. The emulsions formed by
supernatants of the newly isolated strains with sunflower oil were stable, and many authors considered
that the emulsion stability is one of the most important properties of a biosurfactant. In their opinion,
the emulsion remains stable, and the biosurfactant could have numerous potential applications, if its
E24 corresponds to 50% or more [36–39].
The values of the emulsifying indices obtained for octane were better than those obtained for
heptane. This observation is in accordance with the observations made by Pathak and Keharia
(2014) [40], who obtained an emulsifying index of 50.0 for octane and an emulsifying index of 40.0 for
heptane, with the emulsions remaining stable for two days.
Many researchers agree with the observation of Pathak and Keharia that the emulsification index
decreases with the reduction of hydrocarbon chain length [40,41].

3. Materials and Methods

3.1. Biologic Material

The microorganisms of interest were isolated from various plant materials (hay, beans, potatoes,
cabbage). The plant materials were collected from different areas of Romania (Valcea, Bucharest, Ilfov)
in sterile receptacles, kept in a refrigerator until processing, and prepared according to the protocol
described elsewhere: approximately 1 g of each material was sterile inoculated in 500 mL flakes
with 100 mL liquid medium, specific for bacteria, yeasts, and fungi [42]. After 24 h of incubation at
30 ± 1 ◦ C and 220 rpm, the microorganisms were isolated as a pure culture using the serial dilution
method and the streak plate method, on specific media: bacteria on Nutrient Agar (NA), yeasts on
Yeast Malt Peptone Glucose (YMPG), and fungi on Potato Dextrose Agar (PDA). Twenty-five strains
of microorganisms were isolated from the plant collected and the samples processed: 20 strains of
bacteria, 3 of yeasts, and 2 of fungi.
These microorganisms were tested for their antimicrobial activity against two phytopathogens,
from the collection of the National Institute for Chemical Pharmaceutical Research and
Development-ICCF, Erwinia carotovora ICCF 138 and Xanthomonas campestris ICCF 274.
Two bacterial strains were the most interesting, being identified as belonging to Pseudomonas
putida and Bacillus mycoides, by Maldi Biotyper MSP Identification Standard Method 1.1 and Rep-PCR.

3.2. Culture Media

The following media were used in the experiments [43]:
NA (Nutrient Agar) % (g/v): 0.50 peptone, 0.30 yeast extract, 1.50 agar, 0.50 NaCl;
Bacillus thuringiensis medium is denoted here as NB (Nutrient Broth) % (g/v): 0.10 beef extract,
0.20 yeast extract, 0.50 peptone, 0.50 NaCl;
Catalysts 2019, 9, 959 11 of 16

YMPG (Yeast malt peptone glucose) % (g/v): 0.30 yeast extract, 0.30 malt extract, 0.50 peptone,
1.00 glucose, 2.00 agar;
PDA % (g/v): 20.00 potato infusion, 2.00 dextrose, 2.00 agar;
LA (lipase Agar) % (g/v): 0.50 peptone, 0.30 beef extract, 1.00 tributyrin, 2.00 agar;
M44 (and M44 broth without agar) % (g/v): 1.00 yeast extract, 1.00 bacteriological peptone, 5.00
glycerol, 2.00 agar;
LB (Luria-Bertani) broth % (g/v): 1.00 tryptone, 0.50 yeast extract, 0.50 NaCl;
KB (King’s Medium B) broth % (g/v): 2.00 proteose-peptone, 1.00 glycerol, 0.15 K2 HPO4 , 0.15
MgSO4 * 7H2 O;
SNA (Starch Nutrient Agar) % (g/v): 0.30 beef extract, 0.10 peptone, 0.50 NaCl, 1.00 starch,
2.00 agar.
The chemicals were purchased from Sigma and Difco. All culture media were prepared with
distilled water, adjusted to a pH range of 6.5–7.2, and sterilized for 20 min at 121 ◦ C. For the submerged
bioprocesses, Erlenmeyer flasks of 500 mL capacity, with 100 mL medium, were used.

3.3. Preservation of Strains and Cultivation Conditions

The phytopathogen strains used in experiments, X. campestris ICCF 274 and E. carotovora ICCF
138, were from the collection of the National Institute for Chemical Pharmaceutical Research and
Development-ICCF. The microorganisms of interest, B. mycoides (Bm) and P. putida (B1), were isolated
from various plant materials collected from different areas of Romania (Valcea, Bucharest, Ilfov).
Except for the X. campestris ICCF 274 strain, which was maintained on YMPG medium, all the
other strains used in experiments, respectively E. carotovora ICCF 138, B. mycoides (Bm), and P. putida
(B1), were grown on M44 medium.
The pre-inoculum consisted of bacterial strains incubated at 28–30 ◦ C for 48–72 h on YMPG,
respectively M44 agarized medium. The inoculum medium (YMPG for X. campestris ICCF 274 and M44
for all the other strains) was seeded with 2.0 mL pre-inoculum containing 9 × 108 CFU/mL (McFarland
Standard No. 3), and the fermentation medium was inoculated with 10.0 mL of inoculum. The strains
were cultivated 24–48 h for inoculum and 48–72 h for bioproduction, at 28–30 ◦ C and 220 rpm.
Bacterial cell growth was expressed as Optical Density (OD) at 550 nm.

3.4. Antimicrobial Activity Assay

The isolated strains and their supernatants were tested for their antimicrobial activity in two
parallel experiments, conducted in triplicate. The antimicrobial activity assay was performed by
the double culture method, in Petri plates [42] (Soare et al., 2017). YMPG medium in the case of
Xanthomonas campestris ICCF 274 and M44 medium for Erwinia carotovora ICCF 138 were used, being
the best growth media for these strains. The first experiment (Experiment I) followed an approach
described by Soare et al.: One millimeter of inoculum from the broth culture of phytopathogens
(containing 3 × 108 UFC/mL, McFarland Standard No. 1) was added by pipette to the center of the
Petri dish, over the agar medium (cooled, but still molten) and rotated gently, till they mixed. After
solidification, 20 µL of inoculum containing 9 × 108 CFU/mL (McFarland Standard No. 3), from the
broth culture of B. mycoides (Bm), respectively P. putida (B1), were put in the middle of the plate, allowed
to be adsorbed in the medium, and the plates were incubated at 30 ± 1 ◦ C. The inhibition zones were
checked daily.
In the second experiment (Experiment II), the phytopathogens (1 mL of inoculum from the broth
culture of phytopathogens, containing 3 × 108 UFC/mL) were first added to the culture medium
according to the same method. The plates were maintained at room temperature (20 ± 1 ◦ C) for 24 h,
and then, the antagonists (B. mycoides (Bm), respectively P. putida (B1)) were inoculated. The antagonists
were added in the center of the Petri dishes containing the phytopathogen and were incubated for 48 h
at 30 ± 1 ◦ C. 1 m L of the phytopathogen suspension (containing 3 × 108 UFC/mL), and 20 µL of the
antagonists inoculum, containing 3 × 108 UFC/mL were added in every Petri plate. The antimicrobial
Catalysts 2019, 9, 959 12 of 16

activity was checked daily, and the inhibition zones were recorded. The Zones Of Inhibition (ZOI)
were calculated using the formula:
Catalysts 2019, 11, x FOR PEER REVIEW 12 of 16

where ZOI = Zones Of Inhibition and ZOId ==distance

colony diameter
between + 2xd (1)
the edge of the antagonist’s colony and
the edge of the zone of inhibition.
where ZOI = Zones Of Inhibition and d = distance between the edge of the antagonist’s colony and the
For the antimicrobial activity assay of the biosurfactants, the strains of B. mycoides (Bm) and P.
edge of the zone of inhibition.
putida (B1) were grown submerged, for 72 h, at 28–30 °C and 220 rpm, on five media: NB, KB, LB,
For the antimicrobial activity assay of the biosurfactants, the strains of B. mycoides (Bm) and
M44, and YMPG. At the end of the fermentations, the ◦broths were centrifuged for 40 min at 4 °C
P. putida (B1) were grown submerged, for 72 h, at 28–30 C and 220 rpm, on five media: NB, KB, LB,
and 9000 rpm.
M44, and YMPG. At the end of the fermentations, the broths were centrifuged for 40 min at 4 ◦ C and
The supernatants used in experiments were sterilized for 30 min at 115 °C, and after
9000 rpm.
sterilization, no significant changes were registered in the emulsification◦ activity. Many authors
The supernatants used in experiments were sterilized for 30 min at 115 C, and after sterilization,
mention that some biosurfactants are heat resistant [44], while some are not, and the main criterion
no significant changes were registered in the emulsification activity. Many authors mention that some
in their tests was verifying the emulsification activity after autoclaving the samples [45]. The
biosurfactants are heat resistant [44], while some are not, and the main criterion in their tests was
antimicrobial activity of the biosurfactants was evaluated using the agar diffusion method against
verifying the emulsification activity after autoclaving the samples [45]. The antimicrobial activity
the aforementioned phytopathogens. For each sample, 0.25 mL of supernatant were added in metal
of the biosurfactants was evaluated using the agar diffusion method against the aforementioned
cylinders. After 24 h of incubation at 30 ± 1 °C, the inhibition zones were measured from the edge of
phytopathogens. For each sample, 0.25 mL of supernatant were added in metal cylinders. After 24 h of
the cylinder to the edge of the inhibition zone.
incubation at 30 ± 1 ◦ C, the inhibition zones were measured from the edge of the cylinder to the edge
of the inhibition zone.
3.5. Enzyme Production
3.5. Enzyme Production
The ability of the two bacterial strains to produce certain enzymes was tested on differential
media, according
The ability of the to the
twoprotocols described
bacterial strains below;certain
to produce then, the enzymatic
enzymes activities
was tested were measured
on differential media,
for: amylase (enzymatic assay of α-amylase EC 3.2.1.1, with DNS, [30]), lipase
according to the protocols described below; then, the enzymatic activities were measured for: amylase (enzymatic assay of
lipase EC 3.1.1.3, titrimetric method, [31]), and catalase (enzymatic assay
(enzymatic assay of α-amylase EC 3.2.1.1, with DNS, [30]), lipase (enzymatic assay of lipase EC 3.1.1.3,of catalase EC 1.11.1.6,
[32]), following
titrimetric method, the Sigma-Aldrich
[31]), and catalase protocols.
(enzymatic assay of catalase EC 1.11.1.6, [32]), following the
In order to investigate
Sigma-Aldrich protocols. amylases’ production, B. mycoides (Bm) and P. putida (B1) were grown
on Petri plates with Starch Nutrient
In order to investigate amylases’ production, Agar (SNA) B. medium.
mycoidesThe(Bm)medium was spot
and P. putida (B1) inoculated
were grownand on
incubated for 48 h at 30 °C. Following incubation, the plates were flooded
Petri plates with Starch Nutrient Agar (SNA) medium. The medium was spot inoculated and incubated with 2 mL of Gram’s
iodine,
for 48 hwhich
at 30 ◦forms a dark blue-colored
C. Following incubation, the complex
plates in theflooded
were presencewith
of starch.
2 mL of Colorless zones around
Gram’s iodine, which
the colonies revealed starch hydrolysis.
forms a dark blue-colored complex in the presence of starch. Colorless zones around the colonies
For amylase
revealed activity determination, the strains were cultivated in SNA broth medium, for 72 h,
starch hydrolysis.
at 28–30 °C and 220
For amylase activity rpm,determination,
till B. mycoidesthe (Bm) reached
strains werean OD of 8.15
cultivated in SNAand broth
P. putida (B1) reached
medium, for 72 h,anat
OD
28–30 ◦ C and 220 rpm, till B. mycoides (Bm) reached an OD of 8.15 and P. putida (B1) reachedrpm),
of 4.25. After centrifugation of the culture broths (30 min at 4 °C and 8000 an ODtheof
supernatants were used for
4.25. After centrifugation theculture
of the determination
broths (30of min
the atenzymatic
4 ◦ C and activity,
8000 rpm), by thea spectrophotometric
supernatants were
method, based on starch hydrolysis into maltose [30].
used for the determination of the enzymatic activity, by a spectrophotometric method, based on starch
One unit
hydrolysis into of amylase
maltose [30].activity is defined as the amount of enzyme that liberates 1.0 mg of
maltose
Onefromunit starch in 3 min,
of amylase at pH
activity 6.9, at 20as°C.
is defined the amount of enzyme that liberates 1.0 mg of maltose
from starch in 3 min, at pH 6.9, at 20important
The production of catalase is ◦ C. in neutralizing the bactericidal effects of hydrogen
peroxide, which can harm
The production the microorganisms.
of catalase is important in Toneutralizing
check the ability of B. mycoideseffects
the bactericidal (Bm) and
of hydrogen
peroxide, which can harm the microorganisms. To check the ability of B. mycoides (Bm)3%
P. putida (B1) to produce catalase, a small amount of bacterial isolate was mixed with andhydrogen
P. putida
peroxide solution. The bacterial isolates were considered as catalase positive
(B1) to produce catalase, a small amount of bacterial isolate was mixed with 3% hydrogen peroxide if oxygen
bubbles
solution.occurred.
The bacterial isolates were considered as catalase positive if oxygen bubbles occurred.
To investigatecatalase
To investigate catalaseproduction
productionatat a quantitative
a quantitative level,
level, B. mycoides
B. mycoides (Bm)(Bm)
and and P. putida
P. putida (B1)
(B1) were
were grown on M44 solid medium, for 72 h, at 30 °C. After that, the
grown on M44 solid medium, for 72 h, at 30 ◦ C. After that, the surfaces of the media containing the surfaces of the media
containing
cultures of thethestrains
cultures(6 ×of10the strains (6were
8 UFC/mL) × 10washed
8 UFC/mL) were washed with 3 mL of distilled water.
with 3 mL of distilled water. The cell suspensions
The cell
were suspensions
subjected were subjected
to ultrasonication to ultrasonication
to extract the enzymeto(35 extract the enzyme
kHz frequency (35 kHz frequency
ultrasound, Bandelin
ultrasound, Bandelin Sonorex digital 10 p ultrasonic bath dk 255 P),
Sonorex digital 10 p ultrasonic bath dk 255 P), then centrifuged, and the supernatant was analyzed then centrifuged, and the
for
supernatant
spectrophotometric determination of the enzymatic activity [32], based on the following reaction:[32],
was analyzed for spectrophotometric determination of the enzymatic activity
based on the following reaction:

One unit of catalase will decompose 1.0 µm of H2O2 per minute at pH 7.0 at 25 °C, while the
H2O2 concentration falls from 10.3 mM to 9.2 mM. The rate of disappearance of H2O2 was followed
by observing the rate of decrease in the absorbance at 240 nm.
For testing the microorganisms’ ability to produce lipases, the strains were grown in
submerged culture for 72 h at 30 °C and 220 rpm, on several media (NB, KB, LB, M44, YMPG).
 Catalysts 2019, 9, 959 13 of 16

One unit of catalase will decompose 1.0 µm of H2 O2 per minute at pH 7.0 at 25 ◦ C, while the
H2 O2 concentration falls from 10.3 mM to 9.2 mM. The rate of disappearance of H2 O2 was followed by
observing the rate of decrease in the absorbance at 240 nm.
For testing the microorganisms’ ability to produce lipases, the strains were grown in submerged
Catalysts 2019, 11, x FOR PEER
culture for 72 h at 30 ◦ CREVIEW 13 of 16
and 220 rpm, on several media (NB, KB, LB, M44, YMPG). These constituted
the inoculum used in the lipase assay. To show lipase production, the strains were then cultivated
These constituted the inoculum used in the lipase assay. To show lipase production, the strains
(spot inoculated) on Petri plates with LA medium containing tributyrin. After 48 h of incubation, the
were then cultivated (spot inoculated) on Petri plates with LA medium containing tributyrin. After
plates were analyzed as regards the diameters of the clear zones around the colonies, which signifies
48 h of incubation, the plates were analyzed as regards the diameters of the clear zones around the
lipase production.
colonies, which signifies lipase production.
Triglyceride + H2O Lipase Diglyceride + Fatty Acid
T = 37 ° C, pH = 7.2

One unit of enzyme activity is defined as that amount of enzyme that liberates the equivalent
One unit of enzyme activity is defined as that amount of enzyme that liberates the equivalent of
of 1 µm of fatty acid per minute from the substrate emulsion (olive oil in the presence of
1 µm of fatty acid per minute from the substrate emulsion (olive oil in the presence of thymolphthalein
thymolphthalein indicator) under the described assay conditions.
indicator) under the described assay conditions.
3.6.3.6.
Emulsification Index
Emulsification Test
Index Test
In In
order
orderto toevaluate
evaluate thetheproduction
productionof ofbiosurfactants bybythetheselected
biosurfactants selectedstrains,
strains,submerged
submerged
bioprocesses in 500 mL flasks with 100 mL medium on a rotary shaker at 220
bioprocesses in 500 mL flasks with 100 mL medium on a rotary shaker at 220 rpm were performed.rpm were performed.
The strains
The were
strains weregrown
grownforfor7272hhatat30
30°C,
◦ C,in
inYMPG,
YMPG, KB,KB, LB, and M44
LB, and M44 media.
media. After
Aftercentrifugation
centrifugationofofthe
theculture
culturebroths
broths (30(30 min
min at at
4 ◦4C°C and
and 9000
9000 rpm),
rpm), thethe supernatants
supernatants were
were used
used for for
the the determination
determination of the
of emulsifying
the emulsifying indexes of heptane, octane, and
indexes of heptane, octane, and sunflower oil.sunflower oil.
Emulsions
Emulsions werewereprepared
prepared as as
described
described byby Pathak
Pathakand
andKeharia
Keharia(2014)
(2014)[40].[40].SixSix
milliliters of of
milliliters
sunflower
sunflower oil/heptane/octane
oil/heptane/octane and
and4 mL
4 mL of of
supernatant
supernatantwere added
were added inin
the
thetest tubes.
test tubes.
The tubes were vigorously stirred, and the height of the emulsions
The tubes were vigorously stirred, and the height of the emulsions formed were formed were measured
measured after
after 24 h and 30 days, respectively. The emulsifying index was calculated using
24 h and 30 days, respectively. The emulsifying index was calculated using the following formula:the
following formula:
E24
E24 = (Height
= (Height of of emulsion
emulsion layer/Height
layer/Height of of total
total liquid
liquid column)
column) × 100
× 100 (2) (2)
The experiments
The were
experiments performed
were in in
performed triplicate.
triplicate.

4. Conclusions
4. Conclusions
Regarding
Regarding thethe growthofofthe
growth B. mycoides
theB. mycoides (Bm) and the theP.P.putida
putida(B1)
(B1)strains,
strains,thethe
best results
best were
results
obtained
were in the
obtained M44M44
in the medium
medium as the
as ODs werewere
the ODs 40.3540.35
and 24.35, respectively,
and 24.35, after 72
respectively, h of72the
after bioprocess.
h of
Newly isolated strains and their supernatants exerted antimicrobial activity against the
the bioprocess.
phytopathogens
Newly isolated Erwinia
strainscarotovora
and their 138 and Xanthomonas
ICCFsupernatants exerted campestris
antimicrobialICCFactivity
274. against the
Initial tests
phytopathogens on solid
Erwinia media showed
carotovora ICCF 138that andB.Xanthomonas
mycoides (Bm) produced
campestris ICCF the274.
enzymes amylase and
lipase. After
Initial tests72on
h of submerged
solid development
media showed that B. inmycoides
SNA medium
(Bm) and 48 h in the
produced enzymes B.
LA medium, mycoides
amylase (Bm)
and
produced
lipase. After small
72 h ofamounts
submergedof amylases (as the in
development amylase activity was
SNA medium and 0.75
48 hU/mL)
in LA and lipasesB.(0.39
medium, U/mL),
mycoides
which
(Bm) meanssmall
produced that the fermentation
amounts media
of amylases (asneeded to be optimized.
the amylase activity wasThe 0.75amylase
U/mL) and activity of P.(0.39
lipases putida
(B1) was
U/mL), insignificant:
which means thatthe thelipase activity of
fermentation the strain
media neededregistered 0.36 U/mL,
to be optimized. Theand the catalase
amylase activity
activity of
P. registered
putida (B1)2.3was
U/mL. insignificant: the lipase activity of the strain registered 0.36 U/mL, and the
catalaseThe biosurfactants
activity registeredof 2.3both
U/mL.strains formed emulsions with sunflower oil. The emulsions formed
byThe
the supernatants
biosurfactantsofof P. both
putidastrains
(B1), grown
formed inemulsions
the LB medium, presented oil.
with sunflower the The
mostemulsions
significantformed
values in
byboth
the cases (after 24 of
supernatants andP.30 days,(B1),
putida respectively).
grown inThe the emulsifying
LB medium,indices (E24)the
presented for octane reached the
most significant
highest
values values:
in both 73.97%
cases in24
(after theand
LB medium
30 days, and 71.26% in the
respectively). TheKB medium. indices (E24) for octane
emulsifying
reachedAlthough
the highestsupernatants
values: 73.97% theB.LB
frominthe mycoides
medium (Bm)
andstrain
71.26%grown
in theinKBthemedium.
KB and LB media formed
emulsions
Althoughwith sunflowerfrom
supernatants oil (with
the B. an emulsifying
mycoides index
(Bm) strain of 60.56
grown and
in the KB56.76,
and LB respectively),
media formed they
remainedwith
emulsions stable for only 24
sunflower oilh,(with
whileanthose from M44index
emulsifying and YMPG remained
of 60.56 and 56.76,stablerespectively),
for 30 days. they
remained stable for only 24 h, while those from M44 and YMPG remained stable for 30 days.
These results suggest the possibility of using these newly isolated strains in agriculture (for
preventing the diseases caused by several phytopathogens) and in the bioremediation of waste oils
and hydrocarbons, as well as in the food, cosmetic, chemical, and pharmaceutical industries.
However, further investigations are required to improve the media, mainly with waste oils and
hydrocarbons. Being able to grow on these kinds of substrates, the strains could be used both to
Catalysts 2019, 9, 959 14 of 16

These results suggest the possibility of using these newly isolated strains in agriculture (for
preventing the diseases caused by several phytopathogens) and in the bioremediation of waste oils
and hydrocarbons, as well as in the food, cosmetic, chemical, and pharmaceutical industries. However,
further investigations are required to improve the media, mainly with waste oils and hydrocarbons.
Being able to grow on these kinds of substrates, the strains could be used both to reduce the pollutants
and to produce biosurfactants with many potential applications.

Author Contributions: M.-G.S. (Vladu) conceived of and designed the research and drafted and finalized the
paper; E.S.L. was responsible for the supervision and methodology and was the project coordinator; N.E. performed
the research and the analysis; N.M. (Dalanaj) performed the analysis; O.P. performed the writing, review, and
editing; N.B. performed the writing, review, and editing; all authors contributed to discussing the research, writing
parts of the paper, and commenting on draft versions.
Funding: This research was funded by Erasmus +, Grant Number 2019-1-RO01-KA105-062720. Research: “Act for
sustainable consumption and production in the context of circular economy in line with sustainable development
goals–ACT4SCP”.
Acknowledgments: We would like to express our gratitude to Diana Smarandache (Pelinescu) for her valuable
work to identify the surfactant producing strains.
Conflicts of Interest: The authors declare that there is no conflict of interest regarding the publication of this article.

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