ISOLATION AND IDENTIFICATION OF SOIL BACTERIA

ABLE TO EFFICIENTLY REMOVE COPPER

FROM CULTURE MEDIUMS

M. CONSTANTIN1,2, C.D. NEGUT1, C. BARNA1, C. CÎMPEANU1, I.I. ARDELEAN2

1
“Horia Hulubei” National Institute for Physics and Nuclear Engineering, Bucharest, Romania
2
Department of Microbiology, Institute of Biology Bucharest, Romanian Academy

Received May 28, 2015

Copsa Mica is one of the sites with the highest degree of heavy metal pollution in
Romania, the whole area being well known on national and international level for the
ecological lack of balance due to the non-ferrous smelter plant. Three bacterial strains
were isolated from the soil samples derived from the extraction sit. The growth and
development of these bacteria in high concentration of toxic metal environments (Pb,
Cd, Zn) represent an important research direction, the understanding of those
mechanisms involved in bacterial resistance against the toxic effects of heavy metals
and radionuclides contributes to the development of new technologies for treating
contaminated water. Concurrently, their capacity of absorbing and accumulating the
metals present in a liquid medium was analyzed.
In this paper we report the isolation, purification and identification of three bacterial
strains from metal contaminated soil (Copsa Mica), strains able to grow in high
copper concentration and efficiently remove copper from culture medium. The best
removal results were obtained with Bacillus megaterium which removes 99.8 % of the
copper present in growth media supplemented with 192 mM Cu2+.

Key words: bioremediation, biosorbtion, copper removal bacteria.

1. INTRODUCTION

Heavy metal contamination is one of the most important problems nowadays,

worldwide this type of pollution being a major threat for the ecosystem as well as
for the health of the population. The metallurgical, mining, fertilizer, pesticide and
electroplating industries generate and eliminate in the environment residues that
contain different toxic metals [1], including copper.
The decontamination of polluted sites is realized using two different
processes: physicochemical processes that include evaporation, filtration, ion-
exchange resins, reverse osmosis, chemical precipitation [2] and biotic methods
that imply the use of bacteria in decontamination processes.
Rom. Journ. Phys., Vol. 61, Nos. 3–4, P. 707–717, Bucharest, 2016
708 M. Constantin et al. 2

The use of physicochemical methods is widely spread but their efficiency is

low due to high costs, low recuperation and the secondary toxic compounds that
need to be neutralized [3].
A new approach on this problem is the use of viable/non-viable biomass with
special emphasis on bacteria as well as fungi and yeasts [4].
The general interest in biological methods is related to certain main
advantages: they can take place at the exact location of the contamination; they
have a low negative effect on the environment since they do not generate
secondary toxic compounds and are much cheaper than the chemical processes [5].
The bacterial surface is very large and has many ligands for specific heavy-
metals ions, the biomass can be regenerated by the desorption of metal ions using
mild acids, hence the bacterial biomass can be reutilized [6].
There are two major biological mechanisms in which the heavy metal ions
are introduced in the cytoplasm: the passive uptake and the active uptake.
The passive uptake (biosorbtion) is an independent method of the biological
metabolic cycle by which the heavy-metal ions are entrapped in the cellular structures
and slowly biosorbed onto the cell; this process includes physicochemical interactions
between metal ions and different ligands present on the surface of bacteria [4]. The
active uptake (bioaccumulation) is the process by which the metal ions are introduced
in the cytoplasm through cell metabolic cycle [3, 5, 7].
The bacterial growth in culture medium supplemented with toxic metals
occurs on a wide range of concentrations depending on the type of metal studied
[8, 9]. The presence of toxic metals can have considerable impact on microbial
population from an aquatic habitat, causing reduction of bacterial biomass in
chronical pollution cases or a decrease in the metabolic activity and even the death
of the entire bacterial population [8].
A better approach for this problem is to use a bacterial consortium capable to
grow in heavy metal contamination medium, and have a well-defined metabolism
for bioprecipitation processes [10]. Also the search for these types of bacteria
should start in heavy polluted environments where a possible strain can have all the
necessary capacities for bioprecipitation [11].
The aim of this study is to isolate bacteria or a bacterial consortium, from a
chronically metal polluted soil (Copsa Mica) [10], capable to grow in the presence
of high copper concentrations and to remove copper from the growing medium in
order to develop a practical method for heavy metal decontamination.

2. MATERIALS AND METHODS

2.1. SOIL SAMPLING, PROCESSING AND STRAIN ISOLATION AND PURIFICATIONS

Ten grams of soil collected from Copsa Mica (a well-known heavy metal
polluted site in Romania) [12] were aseptically sampled into a sterile Falcon tube
and transported to the laboratory for analysis.
3 Isolation and identification of soil bacteria able to remove copper 709

Using a sterile mortar the sample was homogenized, weighted and distributed
in ten Eppendorf sterile microtubes (one gram in each microtube). In order to
resuscitate the bacterial species present in soil, all ten aliquots were hydrated with
100 ml sterile peptone water, magnetically homogenized and incubated at 32 oC for
2 hours. After the incubation period from every aliquot tenfold dilutions were done
(1 ml initial suspension + 9 ml sterile peptone water). Because a high bacterial
contamination was expected, for every aliquot of soil sample six tenfold dilutions
were performed. The sixth and the fifth dilution from every aliquot was then
entirely filtrated through 0.45 µm (Merck-Millipore®) hydrophilic filter, and the
filters were inoculated at the surface of the Tryptic Soy Agar (Merck®) solid
culture medium and incubated at 32 oC.

2.2. SELECTION OF BACTERIAL STRAINS ABLE TO GROW

IN THE PRESENCE OF COPPER IONS

Initially, there were selected 11 bacterial colonies which were able to grow
on Casein Soy Broth (Merck®). The isolated colonies grown on filters were further
tested for their copper resistance. Thereby, from every type of colony a bacterial
suspension with optical density (λ = 600 nm) = 1 was performed.
One milliliter from every suspension was inoculated in 100 ml sterile liquid
culture medium, Casein Soy Broth (Merck®), supplemented with Cu2+ in final
concentration of 0.236 mM. Copper was added to culture mediums before
sterilization as copper acetate monohydrate (Merck®).
The inoculated bottles were incubated for 24–48 h at 32 oC to identify the
copper tolerant bacteria. After 48 h only three isolates were able to grow at this
relatively low copper concentration (0.236 mM); these three isolated and purified
strains were identified (see below) and used in further experiments.

2.3. QUANTIFICATION OF DOUBLING TIME FOR SELECTED BACTERIA

GROWN IN THE PRESENCE OF DIFFERENT COPPER CONCENTRATIONS

Every bacterial isolate was inoculated (in replicates) in liquid nutritive

medium at a ratio of 1:4 (50 µL inoculums with optical density, OD = 1, in 200 µL
nutritive medium). There were performed two types of controls (each was
replicated): the negative control, represented by uninoculated copper supplemented
culture medium, and the positive control, namely the inoculated culture medium
without copper supplement.
The growth of the isolated bacteria was measured as increase in OD (λ = 600)
in the growing medium supplemented with five incremental concentrations of
710 M. Constantin et al. 4

copper: C1 = 0.236, C2 = 0.472, C3 = 0.944, C4 =1.888, C5 = 3.776 mM Cu2+.

The inoculation was performed in 96 well microplates and the readings were done
hourly for 8 h, the last one being achieved after 28 h from the initial inoculation.
The measurements were performed in replicates and using these results the
doubling time was also calculated for each bacterial isolate.

2.4. COPPER ELIMINATION FROM CULTURE MEDIA

BY PURIFIED STRAINS

It was also determined the efficiency of copper removal for the three strains
by growing them in liquid culture mediums supplemented with copper to reach the
following concentrations: 0.100, 0.200, 0.300, and 0.400 mM Cu2+.
The experiment of measuring copper concentrations from culture mediums
lasted 21 days. The readings were performed weekly using the following protocol:
15 ml of inoculated culture medium were filtrated through 0.45 µm hydrophilic
filters into sterile glass tube and processed for atomic absorption analysis.
The atomic absorption analysis was performed with an AA Avanta ∑ (GBC
Scientific) spectrometer [13, 14].

3. RESULTS

3.1. IDENTIFICATION OF THE PURIFIED BACTERIAL STRAINS

Preliminary investigations on the morphology and Gram character of the

three soil isolates showed that all of them are Gram-positive rods. The biochemical
characteristics (BioMérieux API 50 CHB/E) identify them as belonging to genus
Bacillus.
An alternative method, based on bacterial fatty acids profile – MIDI
technique, identifies them as Bacillus megaterium, Bacillus subtilis and Bacillus
cereus (Table 1).
The predominance of iso and anteiso fatty acids having 12 to 17 carbons as
well as the high amount of C15:0 iso and anteiso and C17:0 iso and anteiso fatty
acids strengthen the identification of this isolate as Bacillus [15, 16]. The high
amount of fatty acids is explained by high membrane fluidity of Bacillus
genus [15].
The identifications were performed using a gas chromatograph (GC 6890N)
coupled with a mass spectrometer (Agilent Technologies). Fatty acids
interpretation was performed using an internal software (MIDI Sherlock v6.1)
against MIDI fatty acids library RTSBA 6.
5 Isolation and identification of soil bacteria able to remove copper 711

Table 1

Three unknown bacterial soil isolates, identified by fatty acids profile as resulted from GC-MS
readings, using RTSBA6 library
Fatty acid concentration
Fatty acid name
Unknown 1 Unknown 2 Unknown 3
11:0 iso 0.00 0.15 0.00
12:0 iso 0.00 0.83 0.00
12:0 (acid lauric) 0.00 0.52 0.00
13:0 iso 0.14 8.94 0.43
13:0 anteiso 0.00 1.23 0.31
13:0 0.00 0.15 0.00
14:0 iso (12-methyltridecanoic) 0.94 4.16 5.36
14:0 (acid miristic) 0.24 3.35 2.17
15:0 iso (13-methyltetradecanoic) 21.27 28.20 31.88
15:0 anteiso (12-methyltetradecanoic) 39.09 4.86 41.71
15:1 w5c 0.15 0.67 0.00
16:1 w7c alcohol 0.27 0.68 0.70
14:0 3OH/16:1 iso I 0.00 2.87 0.00
16:0 iso (14-methylpentadecanoic) 3.80 7.36 1.80
16:1 w11c 0.43 0.41 2.87
16:1 w6c/16:1 w7c 0.00 9.39 0.00
16:1 w5c 0.00 0.11 0.00
16:0 (acid palmitic) 2.83 5.43 5.32
15:0 2OH 0.00 0.31 0.00
17:1 iso w10c 0.91 1.93 0.36
17:1 iso w5c 0.00 3.87 0.00
17:0 iso 3OH 0.08 0.00 0.00
17:0 2OH 0.15 0.00 0.00
17:1 anteiso A 0.00 0.91 0.00
17:1 anteiso B/iso I 0.57 0.00 0.00
17:0 iso (15-methyltetradecanoic) 13.07 10.61 2.30
17:0 anteiso (14-methylhexadecanoic) 15.36 2.01 4.16
17:0 (acid margaric) 0.00 0.14 0.00
18:0 iso 0.11 0.19 0.00
18:1 w9c 0.00 0.22 0.00
18:1 2OH 0.11 0.00 0.00
18:0 (acid stearic) 0.24 0.36 0.24
19:0 iso 0.11 0.20 0.00
19:0 anteiso 0.14 0.00 0.00
Fatty acid identification (base on GS-MS Bacillus Bacillus Bacillus
analysis) subtilis cereus megaterium
Index similarities compared with RTSBA 6
0.903 0.770 0.814
database
712 M. Constantin et al. 6

3.2. THE GROWTH OF ISOLATED BACTERIAL STRAINS IN ENVIRONMENTS WITH HIGH

CONCENTRATIONS OF COPPER

2,0 2,0

1,8 1,8
1,6
1,6
Optical density (600nm)

1,4
1,4

Optical density (600nm)

1,2
1,2
1,0
1,0
0,8
0,8
0,6
0,6
0,4

Growth evolution in medium without Cu

0,4
0,2 Bacillus subtilis ( Boltzmann)
Growth evolution in medium with 0,236 mM Cu
Bacillus cereus ( Boltzmann)
Bacillus megaterium ( Boltzmann) 0,2 Bacillus subtilis ( Boltzmann)
0,0 Bacillus cereus ( Boltzmann)
Bacillus megaterium ( Boltzmann)
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 0,0
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30
Incubation time (h)
Incubation time (h)

1 2

2,0 2,4

1,8 2,2

2,0
1,6
1,8
1,4
Optical density (600nm)
Optical density (600nm)

1,6
1,2
1,4
1,0 1,2

0,8 1,0

0,8
0,6
0,6
0,4
Growth evolution in medium with 0,472 mM Cu 0,4 Growth evolution in medium with 0,944 mM Cu
Bacillus subtilis ( Boltzmann fit)
0,2 Bacillus cereus ( Boltzmann fit) Bacillus subtilis ( Boltzmann)
Bacillus megaterium ( Boltzmann fit) 0,2 Bacillus cereus ( Boltzmann)
Bacillus megaterium ( Boltzmann)
0,0 0,0
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30
Incubation time (h)
Incubation time (h)

3 4

2,0 1,4

1,8 1,2

1,6 1,0
Optical density (600 mM)
Optical density (600nm)

1,4
0,8
1,2
0,6
1,0
0,4
0,8
0,2
0,6
0,0
0,4
Growth evolution in medium with 1,888 mM Cu
Bacillus subtilis ( Boltzmann)
-0,2 Growth evolution in medium with 3,776 mM Cu
0,2 Bacillus cereus ( Boltzmann) Bacillus subtilis ( Boltzmann)
Bacillus megaterium ( Boltzmann) Bacillus cereus ( Boltzmann)
-0,4 Bacillus megaterium ( Boltzmann)
0,0
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30
Incubation time (h) Incubation time (h)

5 6

Fig. 1 – Growth of B. subtillis (■), B. cereus (●), B. megaterium (▲) in 5 copper concentrations
(2–6) compared with the control (1).
7 Isolation and identification of soil bacteria able to remove copper 713

The growth of the three soil isolates in the presence of copper is different for
each, thus, B. cereus and B. megaterium grow faster in copper supplemented
culture mediums in concentrations ranging from 0.236 to 1.888 mM. Their growth
can be compared to that of the control (no copper addition) and after about 4 h their
initial concentration is doubled. These two species are better adapted for growing
in high metal concentrations and they develop a cellular mechanism in order to
neutralize cooper’s toxic effects. Among these mechanisms, metal complexation
and bacterial immobilization are the most common.
For B. cereus and B. megaterium the growth started after 3–4 hours from the
initial inoculation and after 23 hours the stationary phase occurred. The growth can
be described by a Boltzmann sigmoidal function [17].
B. subtilis showed an atypical growth evolution. The growth started with a
delay of 5 hours and after 23 h the stationary phase occurred. After 26 hours
another growth phase was noticed. This pattern was detected for all concentrations
of copper supplemented mediums.
The growth of all three Bacillus species in copper supplemented culture
medium started after a latency of about 2 hours. Based on these experimental
results the doubling time was calculated (Table 2) with the following formula
N(t)=N0* ,
where: N0 – initial concentration,
N(t) – concentration at the time t,
t2 – doubling time.

Table 2

Doubling time of Bacillus megaterium, Bacillus cereus and Bacillus subtilis grown in mediums
supplemented with different copper concentrations compared to the positive control (no copper
addition); data calculated according to Ref. [18]

Positive Copper concentration (mM)

control 0.236 0.472 0.944 1.888 3.776
doubling time, t2 (h)
Bacillus megaterium 1.47 1.28 1.22 1.29 1.47 1.9
Bacillus cereus 1.38 1.41 1.32 1.31 1.33 1.56
Bacillus subtilis 1.74 3.76 2.15 4.05 3.34 6.51

As shown in Table 2, in this range of concentrations the doubling time of

Bacillus megaterium and Bacillus cereus is practically not influenced by the
presence of copper in the culture mediums, a phenomenon which deserves further
attention. In both cases, at the end of the growth experiments a bacterial deposit
was noticed at the bottom of the inoculated bottles, representing bacteria that
absorbed copper.
714 M. Constantin et al. 8

3.3. COPPER REMOVAL BY COPPER RESISTANT BACTERIA

The interaction with copper of the three isolates is macroscopically visible

both in culture liquid and in solidified culture medium, suggesting that the dark
aspect of the colonies (results not shown) in the presence of copper is related to
copper removal from the growing medium.
For all three isolates the copper absorption capacity was further tested at four
experimental concentrations: 0.08, 0.192, 0.308, 0.414 mM Cu2+. These
concentrations and the incubation temperature were selected in agreement with
literature [9, 19].
Figure 3 shows the evolution of all four copper concentrations in the presence
of B. subtilis, B. cereus, and B. megaterium for 21 days.
After 48 hours from the inoculation, the culture medium became turbid thus
indicating bacterial growth; daily, the pH of the culture mediums was measured
because the solubility of toxic heavy metal increases with the decrease of pH
[13, 14]. After 21 days a decrease of the pH was registered, from an initial value of
7.2 to 5.3–5.6, with an intense decrease in the first 10 days (pH about 5.9).
The copper concentration, at all values, in the presence of all three Bacillus
strains displayed a rapid decrease in the first two weeks after the inoculation, when
the copper concentration acquired the lowest values. After 14 days equilibrium was
acquired and the copper concentrations remained constant until the end of the
experiment.
As shown above, at all concentrations, Bacillus megaterium has the highest
copper removal capacity from the growing medium.
The capacity of Bacillus megaterium to remove copper from the growing
medium, initially having 100% copper concentration, is also presented in Table 3.

Table 3

Copper removal by Bacillus megaterium from growing medium supplemented with different copper
concentrations

removed
Initial copper removed copper Final copper
copper after 14
Strain concentration after 7 days concentration
days
(mM Cu2+) (%) (%)
(%)
Bacillus 0.080 88.75 99.2 0.07
megaterium 0.192 79.16 99.8 0.01
0.308 48.05 75.32 29.87
0.414 37.19 51.69 47.58
9 Isolation and identification of soil bacteria able to remove copper 715

0,22
0,08 B.subtilis B.cereus B.megaterium B.subtilis B.cereus B.megaterium
0,20
0,07 0,18

Copper concentration (mM)

Copper concentration (mM)

0,06 0,16

0,14
0,05
0,12
0,04
0,10
0,03 0,08

0,02 0,06

0,04
0,01
0,02
0,00
0,00
0 2 4 6 8 10 12 14 16 18 20 22 0 2 4 6 8 10 12 14 16 18 20 22

Readings (days) Readings (days)

0,32
0,40
0,30 B.subtilis B.cereus B.megaterium
0,28
0,35
0,26
Copper concentration (mM)

0,24
Copper concentration (mM)

0,30
0,22
0,20 0,25
0,18
0,16 0,20
0,14
0,12 0,15

0,10
0,10
0,08
0,06
0,05
0,04 B.subtilis B.cereus B.megaterium
0,02 0,00
0,00 0 2 4 6 8 10 12 14 16 18 20 22
0 2 4 6 8 10 12 14 16 18 20 22
Readings (days)
Readings (days)

Fig. 2 – The evolution of copper absorption by B. subtillis, B. cereus and B. megaterium,

grown in medium supplemented with 0.080, 0.192, 0.308, and 0.414 mM Cu2+.

4. DISCUSSION

In this paper we report the isolation, purification and identification of three

bacterial isolates, namely B. subtillis, B. cereus and B. megaterium able to grow in
high copper concentration and able to remove copper from culture medium.
Each strain has its own profile for copper removal from growing mediums
supplemented with different copper concentrations.
The largest decrease in copper concentration in the growing medium is
always found in the case of B. megaterium, with a removal capacity of 99 % for
lower copper loadings (0.080 or 0.192 mM Cu2+) and of 50% for higher loadings
(0.414 mM Cu2+).
The results obtained with these three isolates, namely B. subtillis, B. cereus,
and B. megaterium, are in agreement with the performance of a strain of Bacillus
716 M. Constantin et al. 10

cereus, which removes copper with a maximum efficiency of 70 % for initial

concentrations between 25–75 mg/L, as well as with other results obtained with
bacteria belonging to genus Bacillus [19, 20].
The mechanisms involved in processes of copper removal by use of bacteria
are categorized as active and passive [8].
In the Gram-positive cell walls, anionic sites that are important in binding
metals are associated with the following: free carboxyl groups of the
peptidoglycan, hydroxyl groups of sugar in both peptidoglycan and teichoic acids,
phosphodiesters in teichoic acids and amide groups in peptides [8]. For example,
the relative affinities of the cell wall of Bacillus subtilis for metal ions can be
divided into three classes.
The greatest binding capacity occurs with Na+, K+, Mg2+, Fe3+, and Cu2+ with
lesser amounts of binding by Mn2+, Zn2+, Ca2+, and Ni2+. Cations that showed either
very little or no binding includes Hg2+, Ag+, Sr2+, Pb2+, Li+, Ba2+, Cd2+, and Al3+ .
With respect to the mechanism(s) involved in copper removal by the bacterial
strains used in this experiment, copper removal by growing cells suggests that
active mechanisms are involved [5]. However, further research is needed for a
deeper understanding of the processes, especially in the following directions. The
copper removal capacity also in the presence of dead cells and the possible
variations of the valence of copper ions added to the culture mediums with growing
cells.
Consequently, further improved research and study will lead to the
development of an experimental model for future applications, including
bioremediation of polluted environments with different toxic metals like
radionuclides.

5. CONCLUSIONS

This paper shows the results concerning the isolation, purification and
identification of three soil bacteria (Bacillus subtillis, Bacillus cereus, and
Bacillus megaterium) able to grow in high copper concentration, 0.236, 0.472,
0.944, 1.888, and 3.776 mM as well as copper removal capacity during bacterial
growth in mediums supplemented with copper in the range 0.080–0.414 mM Cu2+.
Out of the three bacterial isolates, B. megaterium showed the best capacity of
copper removal: 99 % for lower copper loadings (0.080 or 0.192 mM), 75% for
intermediate loadings (0.308 mM) and 50% for higher loadings (0.414 mM),
abilities which turn it into a very promising candidate for further research.
11 Isolation and identification of soil bacteria able to remove copper 717

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