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ApproachtoaProblemofBioremediationOil
pollutedRaisedBogsintheWesternSiberia
(Russia)
SergeyGaydamaka1,ValentinaMurygina2
LomonosovMoscowStateUniversity,ChemistryFaculty,DepartmentofChemicalEnzymology.119991,Moscow,
Leninskygory1/11,fax:+74959395417
s.gaidamaka@gmail.com;2vp_murygina@mail.ru
Abstract
impassablebogspollutedwithoilisalmostimpossible
technically and economically unfavorable. Besides a
severe climate with cold and long winters and short
cool summers,it is caused also by theabsence of any
road in tundra and foresttundra as well as by
emergency oil spills on raised bogs impassable for
special machinery devices. A depth of oil penetration
on bogs doesnt exceed 0.20.5 m (Kurchenko 1999)
andisoftenproppedupwithwaterorpermafrost.The
oil pollution can extend on width and the irreparable
damage will be caused to the Nature of this Region.
Processes of selfrestoration of such bogs can be
prolongedforseveralhundredyears.Therefore,huge
raisedoligotroficbogsintheWesternSiberiaownvery
important ecological function, supporting a
biodiversityofallinhabitantsofsuchbogsandhaving
influenceonaclimateandqualityofsurfacewater.So
an elimination of such oil spills and their
consequencesonthebogsisaveryactualanddifficult
problem.
Keywords
Bog, Moss, Oil, Augmentation, Microorganisms, Preparation
Rhoder,OilDegradation
Introduction
ThemainoilproductionareasinRussiaaresituatedin
the Westtern Siberia, and in the same places where
there are huge bogs polluted with oil. Application of
remediation technologies, developed in Russia, on
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www.ijer.orgInternationalJournalofEnvironmentandResource(IJER)Volume2Issue3,August2013
theaimofthisinvestigationisrepresentationresultsof
thebioremediationbogpollutedwithoilbyusingthe
Rhoder and also the reasons which propel us to be
engaged in development of a new bioremediation
technologywhichwouldbecarefulandsolicitousone
forraisedbogspollutedwithoil.
Material and Methods
TheOilOxidizingPreparationRhoder
TheRhoderconsistsoftwobacterialstrainsbelonging
tothegenusRhodococcus,(R.ruberAc1513DandR.
erythropolis Ac1514 D), isolated from soils polluted
with crude oil. The strains are nonpathogenic and
nonmutagenic to humans, animals, plants and
bacteria. The Rhoder is approved for wide use in the
Nature and it has been successfully used for
bioremediation of oil refinery sludge, soils, wetlands
and water surfaces polluted with oil (Murygina,
Korotaeva, Stolyarova, Peterson, Arinbasarov 1996;
Murygina, Arinbasarov, Kalyuzhnyi 2000; Murygina,
Markarova, Kalyuzhnyi 2005; Gally, Murygina,
Kalyuzhnyi2005;Ouyang,YongYong,Liu,Murygina,
Kalyuzhnyi, ZengDe 2005; Ouyang, Liu, YongYong,
Murygina, Kalyuzhnyi, ZengDe 2006; DeQing, Jian,
ZhaoLong,Jian,TianLi,Murygina,Kalyuzhnyi2007)
and the Rhoder is used in these described fieldscale
test.
TheWesternSiberia,MuravlenkoTown
In 2011, an impassable bog located near the
Muravlenko town with a size about 0.8 hectares
(Figure 1) polluted with spring accidental oil spill
because of corrosion of pipe line and halved by high
knolls was offered for the bioremediation with using
the Rhoder only Typical marsh plants (moss,
cloudberry, wild rosemary) existed on the knolls,
which were practically not affected by the oil spill.
Large spots of the oil were situated on swampy
impassable depressions. Vegetation (moss, sedge) on
thesedepressionsperishedalmostcompletely.Alayer
of the oil with a thickness about 1 cm and more was
presentedonthewatersurfacesonthesedepressions.
Whilesampling,wediscoveredthatanpenetrationof
the oil into the moss was modified from 25 to 40 cm.
The oil contamination of the bog was unequal. The
boghadaslightbiastowardsasandbankwhichhad
been made to prevent spreading of the oil pollution
and, in fact, turned into the road. Two previously
digging pits to collect oil with the pump were
presented on the bog. However, oil was gathered
poorly, and these pits still had much of oil. The
76
InternationalJournalofEnvironmentandResource(IJER)Volume2Issue3,August2013www.ijer.org
program,withthecolumnOV101lengthof50mand
internal diameter of 0.22 mm, with thickness of the
phase of 0.50 microns and FID detector, at the
temperature of the detector 300oC and the evaporator
temperature of 280. The gradient was from 80 to
270andthevelocityofraisingtemperaturewas12
C per minute. Mixture of Undecane, Dodecane,
Tetradecane, Hexadecane and Squalane were used as
external standards in concentrations of 5 g/L for
eachsubstance(Drugov,Zenkevich,Rodin2005).
Severalsamples(8samples)ofmoss(No.1,2,3,12,18,
22, 24, 26) from the bog in the Western Siberia,
Muravlenko town, were excessively polluted with
crude oil. The oil from these samples at first was
extracted by chloroform (150 mL) in chemical flasks
(each flask with a capacity of 400 mL) which were
shakenfor30minutesatroomtemperature.Received
solutionsoftheoilweretransferredtotheotherflasks
through waterless sodiumsulfate (Na2SO4) to remove
remains of water. The chloroform was evaporated at
75. Each sample of the moss excessively polluted
with oil was then extracted three times as described
above. The chloroform extracts in flasks were heated
at 105 till a constant weight. Samples of the moss
after oil extraction were dried at 75, weighed and
the total oil was calculated per 1 kg of a dry moss.
ChemicalanalysesofHCinallothersamplesandthe
driedsamplesofthemossafterpreviousextractionby
chloroformatroomtemperaturewerecarriedoutbya
gravimetricmethodwiththeuseoftheSocksletdevice
and column chromatography with Silica gel (Drugov,
Zenkevich,Rodin2005).
of each sample, humidity and the general total
contentoftheavailablenitrogenandphosphoruswere
determined with colorimetric methods (Mineev Ed.
2001).
FIG1.THEIMPASSABLEBOGPOLLUTEDWITHSPRING
ACCIDENTALOILSPILL,THEWESTRENSIBERIA,
MURAVLENKO,2011
MicrobiologicalAnalyses
MicrobiologicalMonitoring
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www.ijer.orgInternationalJournalofEnvironmentandResource(IJER)Volume2Issue3,August2013
No
Depth
of
samp
ling
p
H
HT
CFU/g
of
moss
HCO,
cellsg
of
moss
NNH4+
mg/kg
ofmoss
PO43
mg/kg
of
moss
(010)
(1025)
(010)
(1025)
(010)
(1025)
(010)
(025)
(010)
(1025)
(2540)
(010)
(1025)
(010)
(1025)
(010)
(1025)
(010)
(1025)
(010)
5.2
4.9
4.9
5.0
5.4
5.1
5.2
5.0
4.9
5.
5.3
4.9
4.9
5.0
5.0
4.9
5.1
2.8*107
2.5*107
6.1*108
3.8*108
2.8*108
6.1*107
1.1*107
5.1*107
1.8*107
2.5106
1.9*107
2.6*107
7.6*107
6.4*106
1.1*108
8.9*105
3.6*104
4.3*104
8.1*107
6.0*104
1.1*108
3.8*107
4.9*107
8.0*105
7.9*105
6.0*104
8.4*104
5.0*104
8.0*104
7.7*103
7.1*103
8.1*105
1.0*104
5.08
2.99
11.58
9.18
17.40
7.56
21.02
15.91
9.67
6.2
10.72
16.01
11.14
7.07
4.83
7.56
(1025)
5.2
2
3
4
5
6
7
8
9
10
11
12
13
(010)
(1025)
5.0
(015)
(1530)
4.9
(010)
7.3*107
2.8*10
6
5.1*106
1.0*107
6.35
6.54
7.7*105
3.30
33.11
31.80
22.28
33.53
20.81
16.64
19.10
5.9*107
1.2*106
18.51*
9.6*10
6.1*107
8.35
Note:notdetectedbecausesampleswereunabletodeterminedue
totheirhighoilcontentoraninsufficientamountofit
(1025)
5.0
AfterthreetimesintroductionoftheRhoder,thetotal
number of HT microorganisms as a whole didnt
decrease and even increased in some samples by 1
order.TheMPNofHCObacteriaincreasedbyabout2
orders and more in the majority of the samples
(TABLE2).Thenegativeinfluenceoftheoiloxidizing
preparation Rhoder on indigenous microorganisms
wasntobserved.
78
TABLE2.MICROBIOLOGICALANDAGROCHEMICALCHARACTERISTICSOF
MOSSSAMPLESSELECTEDAFTERAUGMENTATIONWITHTHERHODER
Depth
of
samp
ling
pH
MPNof
HT
CFU/g
ofmoss
MPNof
HCO
cells/g
ofmoss
NNH4
+
mg/kg
of
moss
PO43
mg/k
gof
moss
(010)
5.2
2.8*107
4.4*106
172.5
5.17
(1025)
6.3
2.5*10
4.5*106
99.6
8.84
(010)
6.4
3
(1025)
6.3
(010)
5.7
4
(025)
5.8
(010)
5
(1025)
6.0
(010)
7
(1025)
6.0
(010)
6.3
8
(1025)
4.9
Note:notanalyzed
2.2*107
3.8*108
1.7*107
7.1*106
2.4*106
3.2*107
2.4*107
1.8*107
4.3*106
5.1*106
5.9*107
8.9*106
3.7*106
1.1*106
6.6*106
1.0*106
284.9
66.0
275.8
163.3
88.8
139.9
240.7
71.0
9.98
9.46
12.46
2.60
3.55
3.58
2.15
1.83
No
HCDegradation
Somesamplesofthemossselectedforthepreliminary
examination of the bog were visually represented by
oil slightly contaminated with moss. Several samples
were examined relatively nonpolluted, while the
others were moderately polluted. 27 samples were
selected from the different depths of the bog and
analyzed before the bioremediation of this bog with
theRhoder.
On the right side of the bog in some places, the
preliminaryconcentrationofthecrudeoilinthemoss
layers of 010c m was from 35.13 to 14.35 kg/kg DM
andresidualconcentrationofHCinthesamesamples
after extraction of the crude oil at the room
temperature was from 290.6 to 66.9 g/kg DM. The
concentration of HC on the right side in two samples
(010cm)variedfrom543.1to522.99g/kgDM.Inthe
soillayersof1025cm,theconcentrationofHCvaried
from516.6 to 43.6 g/kg DM. In soillayer of1530 cm,
the concentration of HC was about 300.0 g/kg DM.
Thispartofthebogwasheavilypollutedwiththeoil
(TABLE3,sampleswithaletterR).Ontheleftsideof
the bog in the moss layer of 010 cm, the crude oil
concentration was 29.0 kg/kg DM in one place and
after extraction of this crude oil under room
temperature the residual HC concentration became
173.3g/kgDM.Intheothersamples,theconcentration
of HC varied from 508.1 to 567.2 g/kg DM. In the
depth of 1025 cm, the concentration of HC varied
from9.3to82.3g/kgDM.Inthesoillayerof2540cm,
the concentration of HC was about 27 g/kg DM. This
part of the bog visually seemed a little bit purer than
therightone(TABLE3,sampleswithletterL).
InternationalJournalofEnvironmentandResource(IJER)Volume2Issue3,August2013www.ijer.org
TABLE3.CONTENTOFCRUDEOILANDSATURATEDHYDROCARBONSINMOSSSAMPLESBEFOREANDAFTERAUGMENTATIONOFTHEBOGWITHTHE
RHODER
Beforeaugmentation
No
1_R
Samplingdepthcm
(010)
Afteraugmentation
Freecrudeoilkg/kg
HC,g/kg**
Freecrudeoilkg/kg
HC,g/kg**
Degradati
on%
15.2
66.9
5.39
105.5
(1025)
2.94
51.3
2.94
59.8
0
(010)
35.1
73.7
6.37
234.1
0
2_R
(1025)
*
516.6
*
470.9
8.8
(010)
*
543.1
*
312.4
40.8
3_R
(1025)
*
84.7
*
45.3
31.2
(010)
*
567.2
*
567.8
0
4_L
(1025)
*
38.1
*
24.4
35.9
(010)
*
546.7
15.6
230.7
57.8
5_L
(1025)
*
11.8
*
5.1
56.8
(2540)
*
27.1
*
207.6
0
(010)
29.0
173.3
*
47.8
72.4
6_L
(1025)
*
82.3
*
433.9
0
(010)
*
515.0
*
11.3
97.8
7_L
(1025)
*
38.8
*
339.7
0
(010)
*
522.9
*
27.6
94.7
8_R
(1025)
*
43.6
11.4
217.1
0
(010)
25.8
77.8
*
26.5
65.9
9_R
(1025)
*
76.9
*
260.9
0
(010)
*
508.1
*
8.02
98.4
10_L
(1025)
*
9.3
7.18
330.2
0
(010)
14.4
280.0
*
190.7
32.1
11_R
(1025)
*
196.7
4.96
314.1
0
(010)
25.0
187.6
*
123.5
34.2
12_R
(1025)
*
53.5
8.47
301.5
0
(015)
14.5
290.6
*
318.0
0
13_R
(1530)
*
308.0
*
384.1
0
Note:Rrightsideofthebog,Lleftsideofthebog,*freeoilisabsent;**residualsaturatedHCinthesamplesafterseparatedthecrudeoil
samplesofthemosswithconcentrationHCof850460
g/kgDMcontainedthesaturatedHCof61.81.3%,the
aromatic HC of 16.70.3%, resins and asphaltenes of
8.71.9%.Suchcontaminationalsorepresentsthehigh
quality oil and such oil should be gathered as well.
The oil in moss samples from the layers of 1025 cm,
1530 cm and 2540 cm contained saturated HC of
49.41.12%, aromatic HC of 19.62.3%, resins and
asphaltenesof13.45.2%and18%ofnonHC(oxidized
substances). Such HC composition of the pollution
indicated that the processes of the oil biodegradation
with indigenous anaerobic microorganisms had been
inside of these layers. Obtained results showed that
theinitialhugeamountofthecrudeoilinsomeplaces
wasdecreasedafterbioaugmentationwiththeRhoder
(TABLE3andFIGURE2),buttheoilhadappearedin
someotherplaces,wherepreviouslyitwasabsent.In
these places, content of the total saturated HC
increased.Probably,suchchangesintheamountofthe
crudeoilandmoreimpregnationofthetoplayers(0
10 cm) of the moss with the oil could be due to
movement and displacement of the oil because of the
smallbiastoabulkofthesandyroad.Nevertheless,
FIGURE2.BIOREMEDIATIONOFTHEBOGPOLLUTEDWITH
OILBYUSINGTHERHODER
Theoilinthesamplesofthemosswhichwasseverely
contaminated by the real crude oil (35.114.5kg/kg
DM), contained the saturated HC of 62.51.7%, the
aromatic HC of 19.31.4%, resins and asphaltenes of
11.80.8% and from 5 to 7% of non HC (oxidized
substances).Suchcompositionoftheoilisatypicalfor
any high quality oil and such oil should be gathered
and directed to a refinery plant. Oil contaminating
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www.ijer.orgInternationalJournalofEnvironmentandResource(IJER)Volume2Issue3,August2013
(A)THEDEPTHOF010CM
(A1)THEDEPTHOF010CM
(B)THEDEPTHOF1025CM
(C)THEDEPTHOF2540CM
(B1)THEDEPTHOF1025CM
(C1)THEDEPTHOF2540CM
FIG.3GCANALYSISOFTHEMOSSWITHEXTREMELYHIGHOILPOLLUTION,SELECTEDFROMTHEDIFFERENTDEPTHSBEFORE
(TOP)ANDAFTER(BOTTOM)AUGMENTATIONWITHTHERHODER
positiveinfluenceoftheRhoderonthebioremediation
of the bog was pronounced. This conclusion was
confirmed by results of the HC analysis on GC and
HPLC.Asforqualityoilonabogsurface,ofcourse,it
should be collected and transferred to processing,
however, it is impossible because of impassable bogs
fortheequipmentofgatheringofoilandaccordingto
the oil companies, it is economically unprofitable.
ChromatogramsofHCofthecontaminatedmosswith
extremelyhighandmediumlevelsoftheoilpollution
before and after bioaugmentation are presented on
FIGURE3ACandA1C1andconfirmresultsthatare
describedaboveandbelow.
AfterendingofthebioaugmentationwiththeRhoder
on the right side of the bog there were defined
60.50.7% of the saturated HC and 21.50.7% of the
aromatic HC, and 10.00.01% of resins and
asphaltenes and about 8% of non HC (oxidized
substances)insamplesofmossfromthedepthof010
cm,whichinitiallycontainedalotofcrudeoil.Theoil
contained saturated HC of 54.00.01% and aromatic
HC of 19.58.5% and resins and asphaltenes of
6.80.4%and about 20% of non HC insamples of the
soilfromlayersof1025cm.Itisinterestingthatinthe
depth of 1025 cm (anaerobic conditions) degradation
80
InternationalJournalofEnvironmentandResource(IJER)Volume2Issue3,August2013www.ijer.org
(B)
(A)
(C)
FIG4HPLCANALYSISOFTHEMOSSBEFOREANDAFTERAUGMENTATIONWITHTHERHODER(A)FROMTHEDEPTHOF010CM;
(B)FROMTHEDEPTHOF1025CM;(C)FROMTHEDEPTHOF1025CM
afterbioaugmentationwiththeRhodershowedtheoil
degradation (FIGURE 4 AC) in the layers of 010 cm
and1025cmofthemoss(exceptthelayerof2540cm)
and confirmed that the degradation of the aromatic
HC was observed in these layers of soil. Tentatively,
theaverageefficiencyoftheRhoderapplicationcanbe
estimated as 55.226.2% for not so favorable weather
conditionsifanaveragepercentageofoildegradation
be calculated (TABLE 3). It is significant that the oil
spill on the bog was the fresh (in spring), and the
Rhoder was prepared as a liquid concentrate of cells
with a high hydrocarbon oxidizing activity (1.0*1011
cellsper1mLoftheconcentratedproduct).
Conclusion
The described bioremediation technology (insitu)
cannot be considered as the effective one for
impassable raising bogs polluted with oil behind the
Polar Circle in the Western Siberia, because it really
requires 34 years or more to restore bogs in severe
climatic conditions. Nevertheless, the oiloxidizing
preparation Rhoder during insitu bioremediation is
capable to degrade oil (55.226.2%) in extreme
conditions: a super high level of oil pollution (from
14.435.1 kg of oil per kg of dry moss to 516.6
43.6g/kg of dry moss) for unfavorable weather
conditions without milling which is used for any
bioremediation.
Obtainedresultsalsoshowedthattheprocessesofoil
biodegradation had been inside of the bottommost
layers of the bog due to indigenous anaerobic
microorganisms. So it is necessary to develop a new
option of bioremediation technology with the
81
www.ijer.orgInternationalJournalofEnvironmentandResource(IJER)Volume2Issue3,August2013
Murygina
V.,
Arinbasarov
M.,
Kalyuzhnyi
S.
Bioremediationofoilpollutedaquaticsystemsandsoils
with novel preparation Rhoder. Biodegradation,
(2000),11,6,385389.
Murygina Valentina P., Markarova Maria Y., Kalyuzhnyi
KomiRepublic.EnvironmentalInternational,(2005),31,
2,163166.
NazinaT.,RozanovaYe.,BelyayevS.,IvanovM.Chemical
and microbiological research methods for reservoir
DeQing S., Jian Z., ZhaoLong G., Jian D., TianLi W.,
liquidsandcoresofoilfields.PreprintBiologicalCentre
Press,Pushchino.135(1988)(inRussian).
Netrusov A.I., Ed. Practical handbook on microbiology
Academia,Moscow,Russia(2005)(inRussian).
I.G.,
Rodin
A.A.
82
Intren.OilSpillConfer,Seattle,231238(1999).
REFERENCES
Yu.S.,
Russia,No.8,1619(1999)(inRussian).
Drugov
Biochemistry,(2005)40,No12,37633768.
Ouyang Wei, Liu Hong, YongYong Yu, Murygina V.,
Kalyuzhnyi S., ZengDe Xu. Fieldscale study on
performance comparison of bioaugmentation and
compost
treatment
of
oily
sludge.
Huanjing
Kexue/EnvironmentalScience.(2006)27(1),160164.