Scientific innovation
First time in the multi-level system of oil-contaminated soil bioassays were included
indicators of cyanobacteria-algal cenoses (CAC).
Developed by coefficient of biological soil activity (BSA).
BSA includes the activity level of enzyme, the number of heterotrophic and
hydrocarbon-oxidizing
microorganisms
(HOM),
microscopic
fungi,
algae
and
cyanobacteria.
Practical significance of the work
The developed multi-component test system allows you to get reliable results when
assessing the nature and extent of oil pollution of the soil. It can be enabled as part of the
system of ecological monitoring. The results of researches can be used in courses of
lectures «Protection of nature», «Industrial ecology», «Ecology and rational nature
management». The developed rate BSA allows you to compare the functional state of soils
at different stages of oil biodegradation, and may serve as an additional argument for
choosing a particular method of remediation.
Acknowledgements
The author expresses his deep appreciation and gratitude to the supervisor: Professor
Yang Haizhen and PhD, Associate Professor Lu Zhibo, for their invaluable help in
carrying out this research, as well as all colleagues and members of the laboratory.
RESEARCH
Research on oil-contaminated and reclaimed soils were conducted from August 2012
to November 2012 in an industrial landfill for disposal and recycling of waste oil and
Laboratory of Eco-Technics Company in Kazakhstan, Atyrau.
In laboratory and field conditions was investigated of changes in soil biological
activity (gray forest, black soil) contaminated with oil, and soil at various stages of
reclamation microbial agents. For bioremediation were used:
1) Biological product «Lenoyl» on the based on the natural microbial consortium of
Bacillus and Arthrobacter.
2) Biological product «Azolen» on the based on the natural microbial consortium of
Arthrobacter.
3) Biological product «Belvitamil» is aerobic-anaerobic microorganisms containing
hydrocarbon-oxidizing microorganisms of the genera Arthrobacter, Bacillus, Candida,
Pseudomonas (only 10 species).
RESULTS AND DISCUSSION
Changing the biological activity of the soil under the influence of oil pollution
The biological activity of oil-contaminated soil was in a depressed state due to
inhibitory action of oil into its constituent components. Oil pollution has a significant
effect on the enzymatic activity of soils. Catalase activity fell sharply in soil contamination
by oil, and the degree of inhibition of enzyme activity was directly dependent on the
concentration of the pollutant. In contrast of catalase, oil pollution in small and medium
concentrations after an initial period of decline, has a stimulating effect on the activity of
soil lipase. The activity of chitinase and cellulase initially declined under the influence of
oil and gradually recovered with increasing periods of incubation.
Under the influence of oil pollution of soil microbial parameters were changed.
Adding oil contributed to the rapid growth of the number of HOM (Table 1). Heterotrophic
microbial complex part of the oil-contaminated soil, which was originally located in a
depressed state, gradually recovered over time.
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In soil contaminated with oil at a concentration of 8%, an increase in species
diversity of microscopic fungi isolated from the expanded list of contaminated soil types,
apparently involved in the disposal of oil or simply resistant to this type of pollution. In
contrast to the control options, where representatives of the genera Aspergillus and
Penicillium were typical sparse along with representatives of other genera (Trichoderma,
Mucor, Scopulariopsis), in heavily contaminated soil increased the proportion of species
belonging to these two genera, and they moved into the category of typical frequent and
dominant.
Oil pollution at a concentration of 1% already impacted heavily on quantitative
indicators of germination, speed of germination and seed vigor. In soils with contamination
8% mortality of seeds reached almost 100%.
Numerous experiments carried out using two types of Collembola (Folsomia
candida, Onychiurus stachianus) over all possible scenarios of stressful situations, have
shown that they respond to both direct and indirect effects on oil pollution and died in
«fresh» pollution.
Evaluating the effectiveness of remediation of contaminated soils using
microbial preparations
We studied three methods of bio augmentation with microbial preparations Lenoyl,
Azolen and Belvitamil. The efficiency of soil treated with these drugs was estimated
separately for each of the parameters studied and compared with similar values of
background soil and soil contaminated with oil without biologic treatment.
Exploring the changes of soil biological activity in the remediation of oil pollution,
we compared them with quantitative data of residual oil. Adding drugs contributed to a
significant increase in the rate of degradation of hydrocarbons. In soils Biotherapy amount
of residual oil was reduced to 90 days to 45-60% of the original, depending on the initial
concentration of oil.
All of the studied biological preparations have had a positive impact on soil catalase
activity, the growth of heterotrophic and hydrocarbon-oxidizing microorganisms. The
stimulating effect of drugs on the development of heterotrophic microorganisms due, most
likely, with a reduction in the overall toxicity of the soil with the simultaneous appearance
of additional resources. The LCS is a priori an incentive to the development due to the
appearance of hydrocarbons in the conditions with the application of biological products
exhibit more rapid growth, although in some cases there was antagonism of native and
inoculated hydrocarbon oxidizing micro biota.
Using Lenoyl for bioremediation led to some reduction in the number of
micromycetes through the introduction of hydrocarbon-oxidizing bacteria of the
consortium, which forms the basis of a biological product that contributed to the recovery
ratio of bacteria – fungi. The use of a biological product Lenoyl for cleaning oil-
contaminated black soil also has an impact on the species composition of the complex
micromycetes. In the version with low concentrations of oil were identified fungi are not
found in the other samples. In samples of contaminated soils with Belvitamil, compared
with those without a biological product, there was a significant stimulation of certain
micromycetes.
The most effective biological preparations for reducing phytotoxicity in this
experiment were the Azolen have a positive effect on the recorded performance
(germination, vigor and speed) throughout the experiment in all concentrations. These
results seem to be quite natural if we take into account that the Azolen was initially
developed as a bio-fertilizer. It is known that the microorganisms are included in it have
the ability to fix atmospheric nitrogen synthesis plant growth stimulants and antagonize
against phytopathogenic fungi.
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Table 1 – Effect of different concentrations of oil on phytotoxicity of soil
Note: the «+» denotes the appearance of offspring
Version
Collembola Survival in soils with different concentrations of oil (%)
Folsomia Candida
Onychiurus stachianus
3 Days
30 Days
90 Days
3 Days
30 Days
90 Days
C
100+
100+
100+
100+
100+
100+
C+1%
50
70
90
40
70
100+
C+4%
0
30
65
30
60
85
C+8%
0
0
0
0
0
5
1%+L
70
85
95+
90
95
100+
4%+L
10
40
75
60
80
95
8%+L
0
0
15
0
5
25
1%+A
45
55
65
40
70
90
4%+A
5
15
30
25
50
85
8%+A
0
0
10
0
0
15
1%+B
65
85
90
65
85
90+
4%+B
0
40
80
50
75
90
8%+B
0
5
20
0
10
25
The use of different biological preparations for remediation of contaminated soils has
provided an ambiguous effect on the species diversity of CAC in the background, and in
contaminated soil. Being in the surface layers of soil, cyanobacteria and algae quickly
reacted to the introduction of the test substances, while showing the various reactions of
different groups on different biological preparations.
The greatest variety of species of cyanobacteria and algae was observed in the
contaminated soil treated Lenoyl (Table 1). Under its action was a decrease toxicity of oil
introduced. Application Lenoyl contributed to the recovery of species diversity of algae
and cyanobacteria at 1% contamination.
Table 2 – Change qualitative and quantitative characteristics of the CAC
Terms of sampling, days
C
C+L
1%
1%+L
4%
4%+L
Number of species
30
13
9
8
7
0
0
90
15
15
10
15
0
0
180
19
19
16
19
3
9
All period of observation
26
24
22
23
3
9
Level of development CAC (points)
30
69
58
13
24
0
0
90
88
87
35
66
0
0
180
97
77
56
62
3
16
Reclamation of soil contaminated with oil at a concentration of 8%, did not lead to
the restoration of species diversity of algae. Only representatives of cyanobacteria of the
genus Nostoc detected these soil samples. Obviously, for the recovery of the species
composition of algae needs to re-process of biological preparations or longer to detoxify
pollutants.
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Table 3 – BSA values in various embodiments experiments (3 days)
Note: The test organism died = «-»
Variants
of the
experim
ent
Test – objects
BSA
Catal
ase
Heterotrop
hic
microorga
nisms
HO
M
Microsc
opic
fungi
Soil
alga
e
Plant
s
Invertebr
ates
Control
1,00
1,00
1,00
1,00
1,0
0
1,00
1,00
1,00
C+(1%)
0,62
0,87
1,06
0,96
0,8
2
0,88
0,45
0,83
C+(4%)
0,24
0,83
1,04
0,95
0,7
4
0,53
0,30
0,67
C+(8%)
0,16
0,75
1,08
1,21
-
0,05
-
0,57
1% + L
0,32
0,93
1,08
1,34
1,1
4
0,94
0,80
0,94
4% + L
0,22
0,88
1,11
1,25
1,0
5
0,54
0,35
0,77
8% + L
0,05
0,75
1,22
1,21
-
0,10
-
0,48
1% + A
0,76
1,04
1,05
1,58
1,0
0
1,15
0,43
1,00
4% + A
0,49
0,97
1,12
1,55
0,9
7
0,68
0,15
0,85
8% + A
0,18
0,81
1,25
1,53
-
0,13
-
0,56
1% + B
0,68
0,90
1,15
1,33
1,1
5
0,78
0,65
0,95
4% + B
0,46
0,87
1,24
1,37
0,9
6
0,54
0,25
0,81
8% + B
0,11
0,77
1,26
1,41
1,0
0
0,12
-
0,67
Using such an integrated indicator as BSA gives an indication of the generalized
reaction of the soil ecosystem on oil pollution and more reliable results about the
effectiveness of biological preparations used in bioremediation.
In Tables 3 and 4 shows the coefficient of BSA calculated for all the variants of the
experiment with incubation periods of 90 days and 3 days. As seen from the results (Table
3, 4), the total oil pollution of soil biological activity noticeably decreased. The degree of
suppression of the biological activity was dependent on the concentration of oil and the
time elapsed since entering it into the soil.
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Table 4 – BSA values in various embodiments experiments (90 days)
Note: The test organism died = «-»
Variants of
the
experiment
Test – objects
BSA
Catala
se
Heterotr
ophic
microorg
anisms
HOM
Micros
copic
fungi
Soil
alga
e
Plants
Inverteb
rates
Control
1,00
1,00
1,00
1,00
1,00
1,00
1,00
1,00
C+ (1%)
0,76
1,03
1,25
1,08
0,83
1,05
0,95
0,99
C+ (4%)
0,39
1,02
1,32
0,90
0,79
0,84
0,75
0,86
C+ (8%)
0,22
0,92
1,32
0,95
-
0,33
0,03
0,54
1% + L
1,13
1,07
1,28
1,36
1,19
1,31
1,00
1,19
4% + L
0.63
1,10
1,35
1,11
1,03
0,9
0,85
1,00
8% + L
0,33
0,96
1,38
1,00
-
0,23
0,2
0,59
1% + A
1,04
1,04
1,33
1,02
1,20
1,40
0,77
1,11
4% + A
0,57
1,04
1,35
1,05
1,04
0,85
0,57
0,92
8% + A
0,26
0,98
1,34
0,96
-
0,39
0,12
0,58
1% + B
1,22
0,94
1,09
1,01
1,18
1,19
0,9
1,08
4% + B
0,70
0,97
1,25
1,07
1,06
0,72
0,85
0,95
8% + B
0,43
0,94
1,28
1,10
0,83
0,37
0,22
0,74
In contrast to the soil exposed to the weak contamination (1%), biological activity in
90 days almost completely restored (BSA for 1% contamination after 90 days = 0.99), with
an average degree of soil contamination (4%) are not able to return to its original state at
the expense of internal resources for such a length of time (BSA for 4% of pollution =
0.67, 0.79 and 0.86 after 3, 30 and 90 days of incubation of the soil, respectively). At
higher concentrations of oil (8%) the value of the BSA during the observation period (3,
30, 90 days) remained virtually unchanged and ranged from 0,54-0,57 units.
Against this background, good monitor progress on the introduction of biologics.
Already 3 days meaning BSA soils biological preparations were significantly higher than
in untreated. Thus, in the soil with the introduction Lenoyl it equaled 0.77 Azolen – 0.85
Belvitamil – 0.81. The trend of recovery of biological activity in soils with 4% of oil and
biological preparations continued throughout the experiment and after 90 days BAP
indicators were as follows: 4% of the oil without processing – 0.86, with Lenoyl – 1.00,
with Azolen – 0.92 c Belvitamil – 0.95.
Based on these results, we can say the undeniable effectiveness of such specimens as
tools to restore the biological activity in soils affected by oil pollution medium (equal in
our case, 4%). Use of biological preparations in high concentrations of oil (8%) failed to
significantly alter the rate of recovery of biological activity. Perhaps in the long term, the
introduction of biological preparations into the soil with heavy pollution will play a
positive role, but as part of our experiment, significant increases in BSA values were
observed. The only drug that has shown good results in the restoration of BSA 8%
contamination was Belvitamil (BSA for 8% of pollution in 90 days = 0.54 with Belvitamil
– 0, 74). Most likely, this is due to its multi-component composition, which includes
proteins, vitamins and minerals that serve as energetic materials for soil micro biota, as
well as low molecular weight cellulose fiber and lignin having some oil capacity.
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CONCLUSION
1. A new test system, including indicators of the enzymatic activity (catalase),
number of heterotrophic and hydrocarbon-oxidizing microorganisms, quantitative and
qualitative composition of microscopic fungi, algae and cyanobacteria, as well as levels of
phyto and zoo toxicity. This system can be used to assess the state of soil and the
effectiveness of different remediation of contaminated soils and disturbed.
2. To determine the biological activity of the developed integrated indicator of the
biological activity of the soil (BAP). It allows you to more reliably assess the condition of
the soil, than the use of any of its individual characteristics.
3. The studied biologics Azolen, Lenoyl and Belvitamil proved effective in the
reduction of the biological activity of oil-contaminated soils. Bioremediation techniques
using these preparations had the highest efficiency in the initially low (1%) or average
(4%) contamination, with moderately heavy contamination (8%), the most active
preparation Belvitamil.
4. Biological preparations Azolen, Lenoyl and Belvitamil did not have their own
properties. When making biologics for remediation of contaminated soils toxic effects of
oil on soil algae and cyanobacteria decreased. The most effective recovery of
cyanobacteria-algal cenoses was Lenoyl with its introduction of the contaminated soil (1%
and 4% oil concentration) showed complete recovery of the species composition,
abundance and biomass of algae cells and cyanobacteria and with 8% of pollution all three
biological preparations were ineffective.
5. Azolen, Belvitamil and Lenoyl had similar stimulatory effects on the activity of
soil catalase. Dynamics of catalase activity is maintained throughout the experiment.
Samples in the original pollution 1% after 90 days, these values become higher than the
control. The use of drugs contributed to the recovery number of heterotrophic
microorganisms. After 90 days in a slightly (1%), and soiled (4%) soils, with the
introduction of Azolen and Lenoyl their number was greater than in controls. Belvitamil
positively influenced the growth of heterotrophic organisms in soils with an initially strong
(8%) contamination. Hydrocarbon-oxidizing microorganisms are encouraged to develop at
the expense of petroleum hydrocarbons, showed even more rapid growth in the use of
biologics, especially Belvitamil and Lenoyl. In soils treated with these drugs, after 30 days
the number of hydrocarbon-oxidizing microorganisms in the order of 107.
6. Application of biological preparations reduced the toxicity of zooplankton. Lenoyl
had a stimulating effect on the growth of radish seedlings at 1% contamination during the
experiment. Adding soil biological preparations with higher concentrations of oil (4% and
8%) affected only increase germination percentage, increased seedling lengths were
observed. The most effective biological preparation for reducing phytotoxicity in this
experiment was Azolen have a positive effect on the recorded performance (length of
seedlings emergence, energy and germination rate of the seeds) throughout the experiment
in all concentrations. Survival of Collembola depended on the time elapsed from the
moment of getting oil into the soil and the concentration of oil pollution, as well as the
specific features of their resistance to oil. In the soil with 8% concentration of oil
specimens of springtails.
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