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Feb 20, 2013 (4 years and 7 months ago)

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Bioremediation study with soils contaminated
by explosives at Adazhi military polygon


Olga Muter

(
corresponding author, e
-
mail: olga.muter@inbox.lv
)

Institute of Microbiology & Biotechnology, University of Latvia,

4
Kronvalda blvd., R
ī
ga, LV
-
1586, Latvia

The FT
-
IR absorption
spectra of liquid M8*
medium with amendments
after cultivation of the
bacteria consortia A43 (lines
1
-
3), as well as CLE (line 4)
and M8* medium without
amendments before
cultivation (line 5).

(+28

C, 6
days).

CLE
sample
*

Carbon,

vol. %

Total
nitroge
n, vol.
%

N/C

Sucrose,
g/l

Glucose,
g/l

Fructose,
g/l

Total
reducing
sugars, g/l

1

0.555

0.38

0.68

0.57

7.08

6.03

13.68

2

1.186

0.29

0.24

1.33

13.41

10.24

24.98

3

1.221

0.57

0.47

1.04

11.01

8.74

20.79

4

1.186

1.00

0.84

1.38

6.06

4.97

12.41

5

0.823

0.38

0.46

0.64

6.38

5.51

12.53

6

1.251

0.22

0.18

2.09

10.79

8.30

21.18

4.
Toxicity study

1.
Analytical chemistry

2.
Isolation of microorganisms

3.
Bioremediation

1.
Limane B., Juhanson J., Truu J., Truu M., Muter O., Dubova L., Zarina D. Changes in microbial population affected by
physico
-
chemical conditions of soils contaminated by explosives. . In: “Current Research Topics in Applied Microbiology
and Microbial Biotechnology", World Scientific Publishing Co. 200
9, 637
-
640
.

2.
Dubova L., Limane B., Muter O., Versilovskis A., Zarina D., Alsina I. Effect of nitroaromatic compounds to the growth of
potted plants. In: “Current Research Topics in Applied Microbiology and Microbial Biotechnology", World Scientific
Publishing Co. 200
9, 24
-
28
.

3.
Muter O., Versilovskis A., Scherbaka R., Grube M.; Zarina Dz.
Effect of plant extract on the degradation of nitroaromatic
compounds by soil microorganisms. J. Ind. Microbiol. Biotechol. 2008
, 35: 1539
-
1543.


4.
Grube M., Muter O., Strikauska S., Gavare M., Limane B. Application of FT
-
IR for control of the medium composition
during biodegradation of nitro aromatic compounds. J. Ind. Microbiol. Biotechol. 2008
, 35:

1545
-
1549
.

-50
-40
-30
-20
-10
0
10
20
30
40
RedOx, mV
1
2
3
4
5
6
6
6,2
6,4
6,6
6,8
7
7,2
7,4
7,6
7,8
8
8,2
Thick fraction
Liquid fraction
pH
1
2
3
4
5
6
1,0E+03
1,0E+05
1,0E+07
1,0E+09
1
2
3
4
5
6
1'
2'
3'
4'
5'
6'
Samples
cfu/ml
Work was supported by contract AĪVA 2004/288 from the Ministry of Defense, the Republic of
Latvia. We thank
National Armed Forces of the Republic of Latvia for providing chemicals, as well
as consulting and assistance in soil sampling at the polygon.
Work was partially financed by The
Latvian C
o
uncil of Sciences, Project
s

No. 05.1484
, 04.1100 and 04.1076.

Collaboration was
financially supported by the Estonian Academy of Sciences and University of Tartu.

We are grateful to Anna Zheiviniece for consulting in plant identification. We also acknowledge the
helpful discussions of Dr.Chem.Vadim Bartkevich from the National Diagnostics Centre.
Authors are
gratefull to Dr.Phys. Aloizijs Patmalnieks and Lidija Saulite for SEM micrographies.



Peak name

Amount,
μg/ml

1,3
-
Dinitrobenzene

17.384

1,4
-
Dinitrobenzene

3.974

2
-
A
mino
-
6
-
N
itrotoluene

0.129

4
-
A
mino
-
2
-
N
itrotoluene

2.719

4
-
A
mino
-
2,6
-
D
initrotoluene

8 672.557

2,4
-
Dinitrotoluene

29.965

2,6
-
Dinitrotoluene

9.614

3,4
-
Dinitrotoluene

22.636

2
-
Nitrotoluene

1.237

A

brown

powder

(remaining

from

the

partial

detonation

of

munition
)
,

which

was

sampled

at

the

military

polygon,

was

further

identified

by

HPLC

as

a

mixture

of

nitroaromatic

compounds

and

used

for

plant

toxicity

and

bioremediation

studies

(Table

1
.
1
)
.

Composition of brown powder identified
by HP
LC


Visual inspection of flora distribution near detonation crater at the
military polygon provided an additional information on plants
resistance to toxic nitroaromatic compounds. For example,
Koeleria
glauca

was the sole plant species, which grew close to detonation
crater in the medium coarse sandy soils contaminated by explosives
(Fig.4.1)
[2]
. This fact could indicate to the resistance of this plant to
nitroaromatic compounds and further use in phytoremediation
process. This finding requires a further investigation.

Acknowledgements

References

Fig.4.1.

Fig.1.1.

Table 3.1.

Table 1.1.

Fig.4.2.

A

B

Koeleria glauca

under field conditions

Stimulati
on

and inhibition
effect of

brown powder to the
growth of barley

(A)

and
tomato

(B), respectively
.

Effect of nitroaromatic compounds to higher plants was studied using wheat
,
barley, tomato,
radish, cress salad as test
-
organisms. Soil and the mixture of nitroaromatics

(
brown powder,
BP)

were sampled at the military polygon (Table 1.1). A regular addition of an equivalent
dosage of nitroaromatics (i.e. 0.33 mg/kg for 26 times, total amount 8.54 mg
nitroaromatics/kg soil) to potted plants during two
-
month vegetation experiment was
provided.
After
58
-
day
vegetation experiment the changes in the plants growth where
estimated, i.e.: shoot height, plant wet and dry weight, root growth
.
A
treatment of wheat,
barley and radish

with BP resulted in enhanced growth, i.e. their shoot height was 62 %; 67
%; and 36 % higher, correspondingly, as compared to control samples. In turn, tomato and
cress salad seedlings were inhibited by BP up to 62 % and 80 %, respectively

[2]
.

4. Main conclusions:



Among tested plants, cress salad remains to be one of the most sensitive plant to
nitroaromatics (NA) and, therefore, appropriate test
-
organism for assessment of soil
phytotoxicity. Toxicity of tested compound can differ in dependence on the plant
development stage (i.e. long
-
term vegetation experiment, germination and root elongation
tests).



Plant response to NA was found to be species
-
specific. Stimulating effect of NA for wheat,
barley and radish needs to be studied in future experiments to reveal the processes occurred
during longterm interrelation between NA and plant. New series of experiments with higher
NA concentrations could reveal the level of contamination, which would be toxic also for
those plants, which were resistant to NA in experiments described in this work.



Further experiments with
Koeleria glauca

could provide additional data on resistance
mechanism of this plant, which plays a “pioneer” role in the soils have been freshly
contaminated by explosives.



Toxicity study was performed using different approaches: phytotoxicity testing (germination and
root elongation test, long
-
term vegetation experiment, field inspection), c
hronic and acute
Toxkits (MicroBioTests Inc.
,

Belgium): microalgae, protozoans, crustaceans as test
-
o
rganisms.


*CLE


cabbage leaf extract. 6 different cabbage cultivars

(
white
cabbage
Brassica oleracea

(1
-
3),

Savoy cabbage
Brassica oleracea

(4),
Chinese cabbage
Brassica rapa

(5),
red cabbage
Brassica oleracea

(6).


The content of carbon,
nitrogen and reducing sugars in different cabbage leaf extracts

Mobile laboratory at the polygon

Samples
s
orting at the polygon

Sample
heterogenity

Plant
Koeleria glauca

near
detonation crater

Representatives of Latvian state
armed forces and researchers at
Adazhi polygon

Detonation crater

Sampling procedure at
different sites of the
polygon

Effect of TNT and RDX to the growth of isolates on the
Saccharose agar acc.

to Kirsop. A
-

without explosives; B
-

with RDX (100mg/l); C
-

with TNT (100mg/l).

Fig.
2
.1.

2
. Main conclusions:



Soil samples from the polygon sites contaminated with explosives contain
microorganisms with a potential to degrade explosives, however, this
potential can remain unrevealed, if only the standard methods of cultivation
are used. It is necessary to vary conditions of cultivation for detection of
explosive
-
degrading activity.



Cross
-
resistance to different explosives is detected for isolates obtained
during isolation procedure. The same isolates were resistant and act
ive

in the
presence of toxic TNT and “brown powder”
. I
ts content is determined by
HPLC and it consists of 10 different explosives

(Table 1.1)
.



In comparison of
a
toxic effect of TNT and RDX
(
represented
nitroaromatic compounds and nitramines
)

to biota,
-

TNT was defined as the
most strong toxicant as
to

microorganisms, as
to

plants.


Soil samples collected at Adazhi polygon were tested for the presence of explosives
.

Colorimetric methods, GC
and HPLC were used (Fig.1.1).

Methods for the measurement of explosives used in
our study. A


HPLC; B


colorimetry for
nitramines; C


colorimetry for nitroaromatics.

A

C

B

Fig.
2
.
2
.


Bacteria isolated from soil
(purified or in consortia) were tested using
different approaches.

A


scanning micrograph

B


API identification
system

C


interrelation
between isolates

0
5
10
15
20
25
30
35
40
45
1
2
3
4
5
Samples
Nitroaromatic compounds, mg/l
2,3-DNT
2-Am-4,6-DNT
1,3-DNB
2,4,6-TNT
Fig.
3
.
1
.

Fig.
3
.
2
.

Remediation of soils contaminated by
nitroaromatic compounds and nitramines, i.e.
explosives, is known as very important,
complicated, and rapidly developing area of
biotechnology.


Application of different organic amendments, e.g. compost, manure, pulp sludge, molasses etc. for soil bioremediation
has become a common practice worldwide. All of them are highly variable by bio
-
chemical composition. Moreover,
development of microbial diversity in contaminated soil in the presence of organic amendment under real conditions can
be unpredictable. Experiments on real scale should be supported by data obtained in model experiments under laboratory
conditions.

Our results showed that cabbage leaf extract (CLE) added to the growth medium can noticeably promote the degradation
of nitro aromatic compounds by specific association of bacteria upon their growth
(Fig.3.1) [3]
. Complex, partly not
-
reproducible (among different cultivars and harvests) composition of this amendment makes this study rather difficult.

Q
uantitative differences in the composition of the studied CLE and the response of bacterial cells to the composition of
the growth media

was investigated using
FT
-
IR spectroscopy
and
conventional chemical methods

(Fig.3.2, Table 3.1)[4]
.

The

effect

of

amendments

on

the

change

of

micr
o
bial

community

in

soil

during

remediation

process

is

known

to

be

one

of

the

most

important

factors

finally

influenc
ing

the

outcome

of

remediation
.

T
he

impact

of

microbial

biomass

addition

and

various

amendments

on

changes

in

microbial

community

of

contaminated

soils

was

studied

in

the

slurry
-
type

experiment

(Fig
.
3
.
3
,

3
.
4
)

[
1
]
.

Contaminated

soil

was

sampled

at

the

military

polygon,

prepared

as

average

sample,

analyzed

for

identification

of

explosives

and

further

used

in

the

experiment
.

Results

of

16
S

rRNA

gene

based

DGGE

fingerprints

of

soil

samples

showed

the

impact

of

amendments

and

bacterial

biomass

addition

on

the

contaminated

soil

microbial

community

structure

(Fig
.
3
.
3
)
.

In future it is supposed to investigate the promoting effect of cabbage leaf extract to the soil bacteria with explosives
-
degrading activity more detailed to use it in soil remediation technologies.

3. Main conclusions:



C
abbage leaf extract (CLE) added to the growth medium can noticeably
promote the degradation of nitro aromatic compounds by specific
association of bacteria upon their growth

(Fig.3.1).


Nitroaromatic compounds can be identified in FT
-
IR spectra by a
characteristic peak at 1527 cm
-
1

(Fig.3.2)
.



Band at 1602 cm
-
1 was characteristic for CLE in FT
-
IR spectra and
correlated with the nitrogen content

(Fig.3.2)
.



The content of C, N and carbohydrates varied in different cabbage
cultivars

(Table 3.1)
.


For discrimination of CLE, conventional chemical analyses and FT
-
IR
spectroscopy

can be used
(Fig.3.2 , Table 3.1)
.



Variations of the C/N ratio in medium affected the content of
carbohydrates and lipids of bacterial cells.

Concentration of TNT degradation
products in M8* liquid medium with
different amendments after incubation of
the A43 (+28

C, 7 days).

The samples 1
-
5 contained 40mgTNT/l
and an inoculum in M8* liquid medium;

2


amended with 2 % sucrose; 3


amended with 2 % CLE; 4


amended
with 2 % sucrose and 2 % CLE; 5
-

amended with 1 % sucrose and 1 % CLE.

1413

1527

1641

1527

1084

993

861

1602

1078

1059

800

1000

1200

1400

1600

Wavenumber cm
-
1

0.0

0.2

0.4

0.6

0.8

1.0

A

B

C

1

2

3

4


5



Addition of buffered salt composition to the soils contaminated with
nitroaromatic compounds, resulted in decrease of redox potential,
which is known to

play an important role in the degradation of
explosives

(Fig.3.4
-
A)
.


The t
otal microbial count was considerably increased in the samples
amended with buffered salt composition, as compared to water. Other
tested amendments, i.e. carbohydrates and cabbage leaf extract, as
well as a mixture of bacteria with explosives
-
degrading ability, also
resulted in an increased total microbial count

(Fog.3.4
-
C)
.


Inoculation of soil samples with mixture of bacterial isolates had a
strong effect on microbial community composition revealed by 16s
rDNA
-
DGGE analysis. Several bacterial strains presented in inoculum
became dominant in TNT and RDX amended samples

(Fig.3.3)
.

.

1


soil + water;

2


soil + M8*;

3


soil + water + A
-
43;

4


soil + M8* + 1.25% CLE + 0.25% sucrose + A
-
43;

5


soil + M8* + A
-
Mix;

6
-

soil + M8* + 1.25% CLE + 0.25% sucrose + A
-
Mix.

Fig.
3
.
3
.

Fig.
3
.
4
.

Dendrogram

of

soil

samples

based

on

cluster

analysis

of

the

DGGE

profiles

of

microbial

communities
.

Abbreviations
:

S



soil,

M
8


M
8
*

salt

composition,

Glc



glucose,

CLE



cabbage

leaf

extract,

M



mixture

of

strains

A
-
Mix
.

Effect of various amendments to redox potential
(
A
)
,

pH (
B
)

and microbail count (C)

in

soil samples
incubated during 14 days at +28

C.


B

C

A

B

C

A

Multidisciplinary approach in this study was achieved
with participation of:



Ministry of Defense of the Republic of Latvia


National Armed Forces of the Republic of Latvia


Institute of Microbiology & Biotechnology,
University of Latvia


Institute of Solid State Physics


Latvian University of Agriculture


National Diagnostics Centre


University of Tartu