Isolation and preliminary identification of actinomycetes isolated from a wastewater treatment plant and capable of growing on methyl ethyl ketone as a sole source of carbon and energy

Abstract

Volatile organic compounds are considered as major sources of air pollution. They cause toxicity problems, bad odors, global warming, etc. Methyl ethyl ketone (MEK) is used in the formulation of lacquer type paints, varnishes, cleaners, thinners, etc. and in many other industries such as the manufacture of synthetic leather and in the decaffeination of coffee. Released into the environment, it causes respiratory, eye, and skin health problems. At high concentrations, it poses a potential threat to public health. In recent years, effective, very environmentally sound, and economical organic biological waste gas treatment processes have emerged. The sources of degrading micro-organisms are diverse and activated sludge suspensions are widely used. Actinomycetes are known for their ability to degrade various polymers. In this study, we are interested in isolating actinobacteria from activated sludge from the wastewater treatment plant of El Athmania, Mila. Thus, five actinomycetes were isolated on ISP4 medium supplemented with nystatin at 100 μg/ml and nalidixic acid at 10 μg/ml. These isolates proved to degrade efficiently MEK in batch reactors. Growth kinetics were determined for each isolate. The time course of MEK consumption was also measured by gas chromatography. A strain named A5.7 stood out as the best degrading bacterium. Indeed, complete degradation of the substrate was achieved after only 72 h of incubation. The A5.7 isolate was assigned by morphological and cultural methods to the genus Streptomyces.

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Desalination and Water Treatment
ISSN: 1944-3994 (Print) 1944-3986 (Online) Journal homepage: http://www.tandfonline.com/loi/tdwt20
Isolation and preliminary identification of
actinomycetes isolated from a wastewater
treatment plant and capable of growing on methyl
ethyl ketone as a sole source of carbon and energy
S. Silini, H. Ali-Khodja, A. Boudemagh, A. Terrouche & M. Bouziane
To cite this article: S. Silini, H. Ali-Khodja, A. Boudemagh, A. Terrouche & M. Bouziane (2016)
Isolation and preliminary identification of actinomycetes isolated from a wastewater treatment
plant and capable of growing on methyl ethyl ketone as a sole source of carbon and energy,
Desalination and Water Treatment, 57:26, 12108-12117, DOI: 10.1080/19443994.2015.1046943
To link to this article: http://dx.doi.org/10.1080/19443994.2015.1046943
Published online: 19 May 2015.
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Isolation and preliminary identification of actinomycetes isolated from a
wastewater treatment plant and capable of growing on methyl ethyl ketone as
a sole source of carbon and energy
S. Silini
a
, H. Ali-Khodja
b,
*, A. Boudemagh
a
, A. Terrouche
b
, M. Bouziane
b
a
Laboratoire de Biologie Applique
´e et Sante
´, Faculte
´des Sciences de la Nature et de la Vie, De
´partement des Sciences de la Nature et
de la Vie, Universite
´Constantine 1, Constantine, Algeria, Tel. +213 0 551183203; email: soumi.21@hotmail.fr (S. Silini),
Tel. +213 0 771206765; email: boudemaghallaoueddine@yahoo.fr (A. Boudemagh)
b
Laboratoire de Pollution et de Traitement des Eaux, Faculte
´des Sciences Exactes, De
´partement de Chimie, Universite
´Constantine
1, Constantine, Algeria, Tel. +213 0 552682141; email: hocine_ak@yahoo (H. Ali-Khodja), Tel. +213 0 794554548;
email: terroucheahmed@gmail.com (A. Terrouche), Tel. +213 0 777980260; email: Kbouz@ymail.com (M. Bouziane)
Received 3 June 2014; Accepted 26 April 2015
ABSTRACT
Volatile organic compounds are considered as major sources of air pollution. They cause
toxicity problems, bad odors, global warming, etc. Methyl ethyl ketone (MEK) is used in the
formulation of lacquer type paints, varnishes, cleaners, thinners, etc. and in many other
industries such as the manufacture of synthetic leather and in the decaffeination of coffee.
Released into the environment, it causes respiratory, eye, and skin health problems. At high
concentrations, it poses a potential threat to public health. In recent years, effective, very envi-
ronmentally sound, and economical organic biological waste gas treatment processes have
emerged. The sources of degrading micro-organisms are diverse and activated sludge sus-
pensions are widely used. Actinomycetes are known for their ability to degrade various poly-
mers. In this study, we are interested in isolating actinobacteria from activated sludge from the
wastewater treatment plant of El Athmania, Mila. Thus, five actinomycetes were isolated on
ISP4 medium supplemented with nystatin at 100 μg/ml and nalidixic acid at 10 μg/ml. These
isolates proved to degrade efficiently MEK in batch reactors. Growth kinetics were
determined for each isolate. The time course of MEK consumption was also measured by gas
chromatography. A strain named A5.7 stood out as the best degrading bacterium. Indeed,
complete degradation of the substrate was achieved after only 72 h of incubation. The A5.7
isolate was assigned by morphological and cultural methods to the genus Streptomyces.
Keywords: Methy ethyl ketone; Biodegradation; Activated sludge; Actinomycetes; Streptomyces
1. Introduction
Methyl ethyl ketone (MEK) is a largely used sol-
vent that serves as a constituent of paints, varnishes,
inks, glues, and adhesives. It is released into the
environment from industrial and domestic uses
threatening, therefore, air quality and public health.
MEK appears to be mobile, bioaccumulative and it
can easily leach in to soil and cause alteration of
ground water quality. Its impact on human health
*Corresponding author.
1944-3994/1944-3986 Ó2015 Balaban Desalination Publications. All rights reserved.
Desalination and Water Treatment 57 (2016) 12108–12117
June
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doi: 10.1080/19443994.2015.1046943
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may range from irritation of mucous membranes
(ocular, nasal or pharyngeal) to digestive disorders or
headaches, even central nervous system dysfunction at
high concentrations [1]. MEK toxicity is relatively high
in the presence of other organic solvents [2]. It is thus
necessary to remove MEK from the environment in
order to avoid such effects.
The treatment of VOCs contaminated air has been
the subject of much research and experimentation over
the last decades in the hope of finding the most effec-
tive and less expensive solutions. In addition to the
diverse physical and chemical treatment technologies
in use, the last years have seen the emergence of very
efficient biological processes that are environmentally
and economically sound.
Activated sludge comprises both organic and
inorganic components. It is considered as an impor-
tant reservoir of micro-organisms. Actinomycetes are
present in this ecosystem [3–8]. Such bacteria are
widespread in nature and known to play a significant
role in the biodegradation of organic and inorganic
matter. Research works highlighting the responsibility
of micro-organisms in the biodegradation of MEK are
relatively scarce and fewer studies have concerned
actinomycetes.
The aim of this study is to isolate and identify
actinomycetes from activated sludge and determine
their ability to grow on MEK as the sole source of car-
bon and energy. It falls within a research program
which aims to highlight the role played by actino-
mycetes isolated from wastewater treatment plants in
the biodegradation of certain volatile organic com-
pounds. We present in this work preliminary results
of MEK biodegradation [8].
2. Materials and methods
2.1. Isolation and enumeration of actinomycetes
Bottles with a capacity of 250 ml were used to
store samples of activated sludge collected from the
wastewater treatment plant of El-Athmania in the
vicinity of the town of Mila in the east of Algeria.
Samples were stored in the refrigerator at 4˚C.
The ISP4 isolation medium was used. It consisted of
soluble starch, 10 g; K
2
HPO
4
, 1 g; MgSO
4
7H
2
O, 1 g;
NaCl, 1 g; (NH
4
)
2
SO
4,
2g; CaCO
3,
2 g; the trace ele-
ment solution, 1 ml; Agar, 20 g; pH, 7 to 7.4. Steriliza-
tion of this medium was carried out at 120˚C during
20 min. The saline solution consisted of: FeSO
4
7H
2
O,
0.1 g; MnCl
2
4H
2
O, 0.1 g; ZnSO
4
7H
2
O, 0.1 g; distilled
water, 100 ml. Nystatin at 100 μg/ml and nalidixic acid
at 10 μg/ml were added to the medium. The antibiotics
used were sterilized by filtration through a millipore
filter of 0.22 microns porosity. The inoculation was
carried out using a sterile Pasteur pipette; 0.1 ml of
each decimal dilution (until 10
−6
) was collected and
inoculated on the surface of a Petri dish containing an
ISP4 medium. The dishes were incubated at a tempera-
ture of 28˚C for three weeks. After 21 d of incubation,
bacterial colonies were counted using a colony counter.
Actinomycetes were identified by their characteristic
macroscopic appearance (hard colonies embedded in
agar) and by direct observation of colonies under an
optical microscope (Leica DMLS) at magnification ×10
(presence of thin filaments) [9]. These isolates were
then observed using the same microscope at a magni-
fication of ×100 after Gram staining. The actinomycetes
were Gram+ filamentous forms sometimes fragmented
into rods or cocci. Purified isolates of actinomycetes
were preserved by freezing at −20˚C in the presence of
20% glycerol.
2.2. Screening isolates of actinomycetes growing on MEK
A first test was performed on all isolates of actino-
mycetes to test their capacity to degrade the MEK as
the sole carbon source. The bioreactor used in this
study was in the form of a glass bottle with a total
volume of 500 ml, closed with a perforated rubber
stopper which allowed insertion of a syringe needle to
collect samples. The minimal medium used was that
of Vandermesse [10]. This medium was completely
devoid of carbon source. Its chemical composition was
as follows: KNO
3
, 13.76 g/l; KH
2
PO
4
, 1.78 g/l;
Na
2
HPO
4
2H
2
O, 4.66 g/l; Na
2
SO
4
, 9.68 g/l; MgSO
4
7H
2
O, 0.8 g/l; EDTA, 10 mg/l; FeSO
4
7H
2
O, 5 mg/l;
MnCl
2
4H
2
O, 1.22 mg/l; ZnSO
4
7H
2
O, 0.25 mg/l;
CuSO
4
5H
2
O, 0.2 mg/l; CaCl
2
2H
2
O, 1 mg/l;
Na
2
MoO
4
H
2
O, 0.2 mg/l.
The inoculum was prepared in the same manner
for all bacteria. From bacterial cultures, two platinum
loops were removed aseptically and transferred into a
test tube containing 15 ml of sterile distilled water.
The tubes were then agitated with a vortex for
homogenization and stored at 4˚C for a period not
exceeding 3 h. Strains in this state served as an inocu-
lum for the next subculture. The bacterial inoculum
(15 ml), 75 ml of culture medium and 100 μl of MEK
were introduced into the 500 ml sterile reactors
[7,11,12]. The latter was placed in an agitated water
bath and set at 30˚C under stirring at 150 rev/min.
Samples of 2 ml were collected every 6 h using a ster-
ile syringe. These samples were used to measure opti-
cal density (OD). The kinetics of growth (OD = f [t]) of
each isolate was then drawn using Microsoft Windows
OriginLab software.
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2.3. Batch culture
The preparation of the starter culture was carried
out as previously described. The incubation within the
reactor was carried out in an agitated water bath that
was adjusted to 30˚C. Samples were collected every
6 h to monitor the kinetics of growth of each isolate. A
volume of 15 ml of subculture giving the first cells in
their exponential phase was removed and centrifuged
at 400 gfor 20 min. The supernatant was removed and
the cell biomass was added to sterile distilled water to
15 ml. This amount was used to inoculate the aerobic
batch-operated reactors containing the above-men-
tioned volumes of medium and MEK. After incubation
in the same growth conditions, samples of 2 ml were
taken every 6 h and allowed two types of measure-
ments:
(1) −1.5 ml was used to monitor the bacterial
growth by reading the OD at 546 nm. The con-
version of results in g/l was achieved by com-
paring the optical densities with a previously
drawn calibration curve.
(2) −0.5 ml was collected in a sealed tube and then
centrifuged at 400 g for 20 min to obtain a
supernatant free from microbial cells. The sam-
ple was then stored at 4˚C in eppendorfs and
used to measure the concentration of the sub-
strate (MEK) by gas chromatography. Control
reactors were tested for each isolate. They
received the nutritive medium described
above, but no MEK.
The time course of the MEK concentration in the
liquid phase was determined by injecting 1 μlina
Schimadzu gas chromatograph, Model GC-17A,
equipped with a DB-5 capillary column (30 m long,
0.25 mm internal diameter). Nitrogen gas was used as
carrier gas at a flow rate of 45 ml/min and a split
ratio of five. The temperatures of the injector and the
flame ionization detector were 250 and 200˚C, respec-
tively. The analysis was carried out isothermally at
130˚C. A standard curve was established with an
external standard of 1,000 ppm of MEK. Hourly injec-
tions of 1 μl allowed establishing biodegradation
parameters of MEK consumption.
2.4. Specific growth rate (μ)
The specific growth rate is defined by Monod
equation (1) [13]:
l¼dx=dtðÞð1=xÞ(1)
where μ= specific growth rate (h
−1
), X= biomass
concentration (g/l), t= time (h). From hourly experi-
mental measurements, we can estimate the real value
of specific growth rate through Eq. (2):
ln¼1=Xn
ðÞXnþ1Xn
ðÞ=tnþ1tn
ðÞ (2)
2.5. Maximum growth rate μ
max
In order to determine the maximum growth rate
μ
max
, Eq. (3) was applied:
X¼P1=1þexp P2 þP3 tðÞ

þP4 (3)
where X= biomass concentration (g/l), t= time (h), P1,
P2, P3, P4: parameters
The parameter sets obtained for each batch reactor,
using Microsoft Windows OriginLab software, are
shown in Table 1.
Each of these parameters is defined as follows:
P1: absolute difference between the concentrations
(g
DW
/l); P1 is equal to the difference between the
initial and final biomass concentrations (A1 −A2).
P2: spreading of the curve along the x-axis
(unitless). It is equal to X
0
/dx.
P3: slope of the sigmoid (h
−1
); P3 is equal to 1/dx.
P4: final concentration of biomass (g
DW
/l); P4 is
represented by A2.
2.6. Specific rate of MEK consumption (r
x.MEK
)
The specific rate of consumption of the MEK
(r
x.MEK
) is defined as the mass of MEK degraded per
unit dry weight of biomass per unit of time (h). This
degradation rate was calculated by Eq. (4):
ðrx:MEKnÞ¼ð1=XnÞ:½Snþ1Sn=tnþ1tnÞ (4)
Table 1
Parameter values for each isolate obtained after parametric
adjustment of the experimental curves of growth
Strains P1 P2 P3 P4
A2.3 3.003 2.956 0.037 2.957
A3.2 3.589 4.269 0.042 3.738
A3.3 2.598 3.219 0.038 2.669
A3.9 3.534 5.382 0.090 3.755
A5.7 1.501 5.577 0.132 1.666
Note: P1: Absolute difference between the concentrations (g
DW
/l),
P2: spreading of the curve along the x-axis (unitless), P3: slope of
the sigmoid (h
−1
), P4: final concentration of biomass (g
DW
/l).
12110 S. Silini et al. / Desalination and Water Treatment 57 (2016) 12108–12117
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where n= order number of the MEK concentration
measurement, (r
x.MEK
)
n
= specific rate of MEK con-
sumption at time t
n
(g
MEK
/g
DW
h), X
n
= biomass con-
centration at time t
n
(g
DW
/l), S
n
= residual MEK
concentration in the liquid phase at time t
n
(ppm), S
n+1
= residual MEK concentration in liquid phase at time
t
n+1
(ppm), t
n
= time at the nth measurement of resid-
ual MEK concentration (h), and t
n+1
= time at the (n+1)
th measurement of residual MEK concentration (h).
2.7. Identification of the actinomycetes at the genus level
Several actinomycetes are identifiable only at the
genus level through the study of the macroscopic
appearance of colonies and morphological characters
of the aerial mycelium and the substrate [14–17].
The macroscopic appearance (color, shape, etc.) of
the colonies was observed after 21 d of incubation at
30˚C on the same medium. The color of the substrate
mycelium was determined as follows: a piece of agar
was cut from mature crops and then deposited on a
disinfected substrate. Excess agar was removed by a
razor blade and the color was noted [18].
The observation of the morphology of chains of
spores, substrate mycelium, and aerial mycelium was
performed according to the technique of culture on
lamella. This technique consisted in carefully inserting
sterile strips in an agar medium ISP2, ISP3, ISP4, or
ISP5, such that they formed an angle of 45˚C with the
surface of the latter. The bacterium was then inocu-
lated against the blade in contact with the medium.
After 14 d of incubation at 30˚C, the plate was
removed carefully from the agar, carrying with it frag-
ments of substrate and aerial mycelium; it was then
deposited on a slide and examined under an optical
microscope (G ×100) [18].
3. Results and discussion
3.1. Isolation
From the selective medium used (ISP4 containing
antibiotics), colonies of powdery, dry, and hard
appearance which are embedded in agar were col-
lected. Observed under an optical microscope, these
isolates showed a filamentous aspect with a Gram
positive staining characteristic of actinomycetes. After
subculturing, seven strains of actinomycetes were
selected. These isolates were referred to by a code
name as follows: A2.3, A3.2, A3.3, A3.9, A5.2, A5.3,
and A5.7. The ISP4 medium used in this study was
effective for the isolation of actinomycetes in this
ecosystem. The starch and ammonium sulfate present
in this medium, known as being favorable for the
isolation of actinomycetes from soils [9,19–21], are also
favorable for the isolation of these bacteria in this type
of aquatic ecosystem.
The addition of the mixture of nystatin (antifungal)
at 100 g/ml and of nalidixic acid (anti-Gram-) at
10 mg/ml to the selective medium has eliminated
almost all of the undesirable fungal and bacterial con-
taminants. Nystatin is a very effective antibiotic for
inhibiting the growth of fungi. Williams and Davies
[15] tested this antifungal substance on fungi
previously isolated from soil. They found that it inhib-
ited the growth of most fungi at a concentration of
50 μg/ml. However, when tested with actinomycetes,
this antibiotic had no inhibitory action toward their
growth although its concentration was increased to
100 μg/ml.
Nalidixic acid was used in the work of Takizawa
et al. [22] for the isolation of actinomycetes in marine
environments. Suzuki et al. [23] found that actino-
mycetes can withstand nalidixic acid up to a concen-
tration of 10 μg/ml; beyond the latter, growth
inhibition can occur. According to our results, these
two antibiotics are also indicated in the isolation of
actinomycetes from activated sludge.
3.2. Growth kinetics of actinomycetes using MEK as the
sole source of carbon and energy
Control reactors showed, for all the tested isolates
of actinomycetes, that bacterial growth was insignifi-
cant. Fig. 1shows the time course of the OD for the
seven strains of actinomycetes isolated from activated
sludge.
From these graphs (Fig. 1), isolates A2.3, A3.2,
A3.3, A3.9, and A5.7 were the most efficient in terms
of substrate consumption when MEK was the sole
source of carbon and energy. A5.2 and A5.3 bacteria
were not capable to degrade the same substrate in
these culture conditions. The profile of the growth
kinetics of the selected strains showed a generally
slow incubation rate. Indeed, the process of adapting
all isolates to growth conditions lasted from 12 to 36 h
of incubation. The log phase occurred immediately
after a lag phase which lasted between 100 and 150 h.
3.3. Determination of some parameters of the growth of
selected isolates of actinomycetes
3.3.1. Evolution of the concentration of the biomass as a
function of incubation time
Fig. 2shows a remarkable reduction of the lag
phase in reactor five. These results differ significantly
from those found in the preliminary study. Indeed,
S. Silini et al. / Desalination and Water Treatment 57 (2016) 12108–12117 12111
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the duration of this phase was between 12 and 36 h
for preliminary tests and a few hours to 18 h for the
incubation period when the culture starter was used.
3.4. Determination of the specific growth rate
The time course of the specific growth rate for the
different strains is shown in Fig. 3.
3.5. Determination of maximum growth rate μ
max
The parameter sets obtained for each batch reactor
are shown in Table 1.
The quickest growing strain was A5.7. Growth lasted
72 h (3 d) (Fig. 2). The highest maximum growth rate
μ
max
was calculated for this strain (0.132 h
−1
)(Table1).
The A5.7 strain entered the exponential phase after an
acclimation period of 12 h. Once the division started, the
growth of this bacterium was rapid and steady up to
42 h and then it began to slow down until stabilization
occurred in the third day. The time course of the specific
growth rate μconfirms these results (Fig. 2). This rate
reached a maximum of 0.23 h
−1
. A progressive reduction
of growth rate down to a value of 0.16 h
−1
was observed
(Fig. 3). The maximum biomass concentration was 1.67
g
DW
/l; it was recorded after 66 h of incubation (Fig. 2).
0 20 40 60 80 100 120 140 160 180
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
OD
time (h)
(a)
0 20 40 60 80 100 120 140 160
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
OD
time (h)
(b)
0 20 40 60 80 100 120 140 160 180
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
OD
time (h)
(c)
0 102030405060
0.00
0.02
0.04
0.06
0.08
0.10
0.12
0.14
0.16
OD
time (h)
(f)
0 1020304050
0.00
0.02
0.04
0.06
0.08
0.10
0.12
0.14
0.16
OD
time(h)
(g)
0 20 40 60 80 100 120 140 160
0.0
0.2
0.4
0.6
0.8
1.0
OD
time (h)
(d)
0 20 40 60 80 100 120 140 160
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
OD
time (h)
(e)
Fig. 1. Growth kinetics of different strains of actinomycetes. (a) A2.3, (b) A3.2, (c) A3.3, (d) A3.9, (e) A5.7, (f) A5.2, and
(g) A5.3.
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3.6. Specific consumption rate of MEK (r
x,MEK
) for the
A5.7 strain
The results of the time course of the biomass con-
centration Xand the residual MEK concentration for
the A5.7 strain are presented graphically in Fig. 4. The
profiles of the time course of the experimental specific
consumption rate of MEK (r
x,MEK
) are shown in Fig. 5.
3.7. The specific growth rate vs. the MEK concentration
The profiles of the specific growth rates vs. the
residual MEK concentration are shown in Fig. 6. The
growth of A5.7 strain varied regularly. Its specific
growth rate quickly reached a maximum and then
diminished gradually with the concomitant substrate
concentration decrease (Fig. 4). The results show that
0 20 40 60 80 100 120 140 160
0.0
0.5
1.0
1.5
2.0
2.5
3.0
[X] (gDW /l)
time (h)
(a)
0 20 40 60 80 100 120 140
0.0
0.5
1.0
1.5
2.0
2.5
3.0
time (h)
(b)
0 20406080100120
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0
time (h)
(c)
020406080100
0.0
0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
time (h)
(d)
0 1020304050607080
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
time (h)
(e)
[X] (gDW /l)
[X] (gDW /l)
[X] (gDW /l)
[X] (gDW /l)
Fig. 2. Time course of the biomass concentration X(g
DW
/l) for strains. (a) A2.3, (b) A3.2, (c) A3.3 (d) A3.9, and (e) A5.7.
S. Silini et al. / Desalination and Water Treatment 57 (2016) 12108–12117 12113
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there is a decrease in the MEK consumption rate over
time (Fig. 5).
Strain A5.7 seemed to be efficient in terms of MEK
utilization. Indeed, the complete substrate degradation
occurred after only 72 h of incubation (Fig. 4). Growth
evolved smoothly with MEK consumption and was
characterized by the highest maximum growth rate of
0.132 h
−1
in comparison with the rest of the isolates
(Table 1). The strain A5.7 was therefore selected
among the set of the isolated actinobacteria.
Studies dealing with the biodegradation of MEK
by a pure culture in batch reactors are very scarce
[24]. Mixed cultures and consortia are commonly used
[7,12,25,26]. Moreover, optimal biodegradation of vola-
tile organic compounds is not necessarily better with a
consortium. Indeed, micro-organisms isolated from
activated sludge achieved faster complete methanol
biodegradation than the entire sludge as an inoculum
[7]. In addition, in the presence of activated sludge,
complete degradation of the MEK occurred after 8 d
at a concentration of 200 mg/l (200 ppm) and after 9 d
at a concentration of 400 ppm [23]. At an MEK
concentration of 20 mg/l in river water containing
micro-organisms acclimatized to MEK, complete con-
sumption was achieved after 2.5 d [27]. Delfino and
Miles [26] reported a slower rate of aerobic
0 20 40 60 80 100 120 140
0.00
0.02
0.04
0.06
0.08
0.10
0.12
0.14
0.16
0.18
0.20
0.22
0.24
0.26
time (h)
(a)
020406080100120
0.00
0.02
0.04
0.06
0.08
0.10
0.12
0.14
0.16
0.18
0.20
0.22
0.24
0.26
time(h)
(b)
020406080100120
0.00
0.05
0.10
0.15
0.20
0.25
time (h)
(c)
0 20 40 60 80 100
0.00
0.02
0.04
0.06
0.08
0.10
0.12
0.14
0.16
0.18
0.20
0.22
0.24
0.26
time (h)
(d)
010203040506070
0.00
0.05
0.10
0.15
0.20
time (h)
(e)
µ (h
-1
)
µ (h
-1
)
µ (h
-1
)
µ (h
-1
)
µ (h
-1
)
Fig. 3. Time course of the experimental specific growth rate μ(h
−1
) for different strains. (a) A2.3, (b) A3.2, (c) A3.3, (d)
A3.9, and (e) A5.7.
0 1020304050607080
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
time (h)
[X] (gDW/l)
0
200
400
600
800
1000
residual [MEK] (ppm)
Fig. 4. The time course of the biomass concentration X
(g
DW
/l) and the MEK residual concentration (ppm) for the
A5.7 strain.
12114 S. Silini et al. / Desalination and Water Treatment 57 (2016) 12108–12117
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decomposition in groundwater; 1 mg/l was com-
pletely degraded after 14 d. Our results were quite
satisfactory. Indeed, actinomycetes A5.7, which were
isolated from activated sludge at El-Athmania station
degraded alone the entire concentration of 900 ppm in
just 72 h of incubation.
3.8. Morphological identification of strain A5.7
3.8.1. Macroscopic appearance
Owing to its speed of growth, A5.7 strain proved
to be the fastest degrading bacterium compared to the
other four selected isolates. The first signs of growth
consisted in the appearance of round, pasty colonies
which were slightly sunken in the agar after 2–3 d of
incubation. After 7 d of incubation, this strain had a
powdery white appearance which constituted the aer-
ial mycelium that is characteristic of actinomycetes.
From the 14th day, the aerial mycelium was gradually
taking a gray color. This corresponded to the forma-
tion of mature spores at the end of aerial hyphae.
3.8.2. Microscopic appearance
The results of the microscopic observation of the
selected strain by the inclined blades method are
shown in Fig. 7. The strain A5.7 was composed of a
well-developed substrate mycelium and a less dense
and thicker aerial mycelium bearing long chains of
spores (generally 20 spores per chain). The spore
chains of cylindrical shape were either straight or
spiral.
The taxonomy of actinomycetes is based on mor-
phological, chemical, physiological, and molecular cri-
teria. Identifying genres is facilitated by morphological
studies while chemical, physiological, and molecular
criteria separate species [17]. According to Williams
et al. [28], certain types of actinomycetes (Streptomyces
Streptoverticillium Micromonospora, Microbispora … ) can
be identified with a greater degree of accuracy com-
pared to other genera (Nocardia, Actinomadura … ) sim-
ply by microscopic observation. Several authors have
determined the types of actinomycetes from morpho-
logical characteristics [14] simply by microscopic
observation of actinomycetales strains grown on the
Czapek medium. Others have identified Actinomyc-
etales genera by observation of spore chains under an
optical microscope [29].
The A5.7 strain develops colonies after 2–3 d of
incubation, which is a characteristic of fast-growing
actinomycetes. Nodwel and Losick [30] found that
colonies of Streptomyces coelicolor established aerial
hyphae in 24 h. The genus Streptomyces is part of fast-
growing actinomycetes.
The A5.7 isolate constituted round-shaped colonies
with a powdery appearance, which were slightly sun-
ken in the agar. The shape of the spore chains was
spiral or straight. These characteristics are typical of
Streptomyces according to Myadoh et al. [31].
Schematized morphological aspects that character-
ize the genera belonging to the actinomycetes as pro-
posed by Lechevalier [16] and which appeared in the
ninth edition of Bergey’s Manual, identify most of
these bacteria. The appearance of aerial mycelia and
A5.7 isolate (Fig. 7) is exactly the same as the genus
0 10203040506070
0
20
40
60
80
100
120
rx,MEK (gMEK/ gDW.h)
time (h)
Fig. 5. The time course of the experimental specific MEK
consumption rate (r
x,MEK
)(g
MEK
/g
DW
h) for the A5.7
strain.
800 600 400 200 0
0.00
0.05
0.10
0.15
0.20
µ (h-1)
residual [MEK] (ppm)
Fig. 6. The experimental specific growth rate μ(h
−1
) vs. the
residual MEK concentration for the A5.7 strain.
S. Silini et al. / Desalination and Water Treatment 57 (2016) 12108–12117 12115
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Streptomyces. Therefore, on the basis of all these
similarities, the isolate in question is assigned to the
genus Streptomyces.
The role of actinomycetes in the biological treat-
ment of volatile organic compounds is particularly
known in biofilters [32–34].
4. Conclusion
Waste management is one of the most complex
problems for both developed and developing coun-
tries. In addition, globalization puts new challenges
for waste management within societies. Waste man-
agement is a key element for sustainable development,
economic issues, health concerns, and environmental
problems.
Biological treatment of VOCs containing gases is at
present the most widely used technique [24,35]. Its
use and development grow exponentially on an indus-
trial scale.
In this study, several actinomycetes were isolated
from activated sludge collected from the wastewater
treatment plant of El Athmania, Mila. These bacteria
were capable of degrading MEK. A strain assigned to
the genus Streptomyces was the most efficient degrad-
ing bacterium of all identified isolated actinomycetes.
The entire substrate degradation was achieved after
72 h of batch incubation. The calculated growth rate
μ
max
and some culture parameters reached maximum
values for this strain.
Acknowledgment
We would like to express our deepest gratitude to
Professor Abderrahmane Boulahrouf of the Laboratoire
de Ge
´nie Microbiologie et Applications, Universite
´
Constantine 1, for allowing us to use his laboratory
facilities in order to perform some of this work.
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