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Available online at www.sciencedirect.com
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ENVIRONMENT INTERNATIONAL

ELSEVIER

Environment Intemational31 (2005) 163-166
www.el sevier. corn/locate! en vi n t

Application of biopreparation "Rhoder" for remediation of oil polluted polar marshy wetlands in Komi Republic
ValentinaP. Muryginaa,*,Maria Y. Markarovab, SergeyV. Kalyuzhnyia
"Department o/Chemical Enzymology, ChemicalFaculty,MoscowState University,119992Moscow,Russia blnstitute0/ Biology Russian Academy Science,Ural Division, Siktivkal; Komi Republic,Russia Availableonline II November2004

Abstract This paper describes the testing and corresponding results of the preparation "Rhoder" in comparison with several other bioremediation variants during the field trials in Komi Republic throughout 2002-2003. All bioremediation trials were performed on one vast polar marshy wetland polluted by accidental crude oil spill and uncovered by grass. After application of the "Rhoder" at the site, with an area of -2000 m2, during the cold and rainy summer of 2002 (1.5 months), the level of oil contamination decreased by 20-51%, depending on initial oil pollution (458-738 g/kg dry weight of soil). In the middle of September 2002, the treated site was covered by 70-85% with green grass. Though, during 2003, the "Rhoder" treatment was not practiced, at the end of August 2003, the site was already covered by 85-95% with green grass and the level of oil contamination further decreasedby 54-79% from the initial level of oil pollution at the beginning of 2002. These results were much better compared to those from other bioremediation variants applied at this spill. @ 2004 Elsevier Ltd. All rights reserved.
Keywords: Bioremediation; Oil polluted polar marshy wetland; Komi Republic; Preparation "Rhoder"
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1. Introduction Most of functioning oil fields in Russiaare situatedin the northernregionsof the European part of Russia(Komi Republic) and in Western Siberia. Severe climatic and geological conditions make not only oil production and transportationdifficult, but also the application of remediation methodsfor accidental spills. A specific climate oil with shortand cold summers lastingonly 1-1.5 monthsand long and severewinters lasting 6-7 months with temperaturefluctuationsfrom plus 30-35 DCin July to minus 4555 DCin Januarycauses pipeline ageingand damageeven for the modernpipelineswith specialanticorrosioncovering. As a result, accidental oil spills unfortunately are frequent in theseregions of Russia.There are more than 200,000 ha of contaminated soils with a thicknessof oil

layer greaterthan 5 cm in WesternSiberia (GreenPeace, 1998). Thus, extensiveareasof tundra, forest-tundraand taiga are heavily contaminated and need remediation works. However, vast and impenetrable wetlands and marshy soils made impossible an application of the majority of conventional methods for land reclamation suchas landfarming(ex-situ),bioventing,composting, etc., there, Harsh geological and climatic conditions in such region in Komi Republicimpelledthe company"Lukoil" to seek for the most effective and economically sound technologies remediationof vast oil polluted territories. for This paperdescribes resultsof the preparation the "Rhodef" applied to field tests on the polar marshy wetland during 2002-2003 in comparison with other bioremediation variants.

2. Materials and methods ~sponding author. +7095 Tel.: 0305083; +70959395417. fax: E-mail address: vpm@enzyme.chem.msu.ru (V.P. Murygina).
0160-4120/$ - see front matter ~ 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.envint.2004.09.010

The preparation "Rhodef" (Certificate No. 77.99.04. 515D.004855.08.0 issued by the Russian Ministry of 1


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~p. Murygina et at. / Environment International31 (2005) 163-166

Health)developed us during the 1990swas successfully by tested in Western Siberia on different types of oil contaminatedwetlands, marshy soils and water surfaces between1995and 2000 (Murygina et al., 2000), as well as in laboratoryand pilot testsin Komi Republicduring 2001 (Mouryguinaand Kalyuzhnyi, 2001). The preparation "Rhoder" consistsof two Rhodococcus oil degradingstrainsand represents concentrated a sion of living bacterialcells with concentration 109_1010 of suspencells/mlthat arereadyfor applicationwithout a preliminary
activation, Murygina et al. (2000), Mouryguina and Kalyuzhnyi (2001). The working suspension of "Rhoder" for treatment contained 106-107 oil degrading cells/ml in addition to fertilizer N IS/P1S/K1S (0.2% solution).
The field tests were performed in 2002 on the oil

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polluted wetland about 2 ha (accidentalspills occurred810 yearsbefore) in the polar region, in a cold and humid zone of the high northerntaiga of the Komi Republic.All plants died at this wetland. The depth of oil contamination (458-738 gikg dry soil) fluctuated from 15 to 25 cm. The area was divided into nine sites (-2000 m2 each,size-25x 80 m), two sites from those were used as negative controls and seven ones were chosen to test several bioremediationvariants. One of these sites was provided for testing the preparation"Rhoder". About 57 cm of the upper layer of oil-contaminated peat was cut off previously. Each site was tilled twice by a pontonstepped-machine and lime was added (400 kg/site) to increase the pH from 6.0 to 7.0. The Moscow State University (MSU) experimental site was treated three times (once every 2 weeks)with the preparation"Rhoder" and the fertilizer by sprinkling, using vacuum pump machinery.Before the secondtreatmentwith "Rhoder", a biosorbent(-4 kg on 0.02 ha of soil) was put on some piecesof crude oil, left after site tilling. Before the third treatmentwith "Rhoder", the Gramineaeseedsof Avena sativa and timothy-grass (16 and 5 kg per 0.2 ha, respectively)were sowed by hand, 1 month later than at the other sites. The seedswere preliminarily activated 800
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Fig. 2. Microorganismcountsduring bioremediation with the preparation . "Rhoder"

by the anti-stress agent "Symbiont" (4 mL for all seeds). The other six bioremediation variants were performed according to the authors instructions: (1) agricultural engineeringmethod without addition of any preparation ("Komimeliovodhozproekt"); oil-degradingpreparation (2) "Universal" (Institution of Biology of RAS); (3) lignosorbent with immobilized oil-degrading preparation "Unirem" (company "Recultivation"); (4) oil-degrading preparation "Petrolan" (Company "Priborservice"); (5) peat-compost preparation"Bamil" (company "Nika"); (6) oil-degradingpreparation"Deconta" (company"Deconta", Czech Republic). By the end of June 2002, when comparisontests started,the seedsmentionedabovewere sowed in all experimentalsites, exceptingthe MSU site. The weather in summer2002 was cold (+6 to +10 °C), rainy and windy, it was permafrostunder a depth of 2025 cm of oil-contaminated peat. The warm weatherlasted only for 10-12 days in the middle of July. Then, it was rainy and cold again. In 2003, dry fertilizers (~20 kg! site) were spreadout without any addition of preparations at the experimentalsites. In 2002, samplesfrom the MSU site were taken for analyses beforeeachtreatmentas well as 1.5 monthsafter the lastapplication,according a 5 point scheme a rule, to (as one sampleis taken from the centre of the site and four others are taken along the edges of the site). The total
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hydrocarbon content(THC) wasdeterrnined a gravimetby ric method.Concentrations microorganisms the treated of in soilswereestimated MPN methodusingthe meat-pepton by agar for heterotrophic bacteria (HT) and Raymond's medium with oil for hydrocarbon-oxidizing bacteria (HCO) (Nazinaet al., 1988). The efficiency of each bioremediation variants was evaluatedwith indexesof bacterial trophic groups, enzymatic soil activity, length of grassand percentage grass of coveringeachsite (Gerhardt,1984).

3. Results and discussion The results obtained(Fig. 1) showedthat the application of the preparation"Rhoder" decreased THC by the 20-51%, depending on the initial (458-738 g/kg) oil contaminationon the site during a short warm period in 2002. The supplementaryTHC decrease(residual THC content 147-336 g/kg) was observed in 2003 without addition of the "Rhoder" due to phytoremediationand fertilizers supply (Fig. 1). The MPNs of HCO and HT bacteria during the bioremediationin 2002 were rather high and only decreased slightly in 2003 (Fig. 2). By the middle of September2002, the treated MSU site was coveredwith 70-85% of green grass.This grasscovering increaseduntil 85-95% in August 2003. At the same period, at the siteswherethe other bioremediation variants were applied,grasscovering accounted only for 50-70%,

except at the site of the company"Priborservice",where such covering was 90-95%. However, the quantity of seedswas tenfold higher in comparisonwith the other bioremediation technologies. The results concerning specific characteristics soil of restoration(indexesof bacterialtrophic groups,enzymatic soil activity) showed that the application of the oildegradingpreparations ("Rhoder","Universal", "Petrolan", "Unirem") was more efficient than the use of preparation "Bamil" developedas an enhancer agriculturalsoils or for tilling with additionof fertilizersand seeds only (Figs.3-6), especiallywhen the initial THC was about 740 g/kg dry soil.

4. Conclusions The results obtainedduring the field tests in the polar region of Komi Republicthroughout2002 showedthat the triple applicationof the preparation"Rhoder" was able to degrade spilled crudeoil by 20-51% (meanvalue-40%) a underthe initial THC of 458-738 gikg dry soil evenwhen the weatherwas unfavourablefor bioremediation.In this case,it was not expected that the bioremediation would be completedduring one season. Phytoremediation introand duction of fertilizers during the summer of 2003 further decreased THC by 9-50% (147-336 gikg dry soil). It the meantthat the total decrease THC accounted 55-79%, of for meanvalue-67% for 2002-2003.Generally,the bioremediation technologywith using the preparation "Rhoder",the biosorbentand the anti-stressagent for the seedsshowed superiorresultscompared the other optionstestedat this to
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"Komimeliowodkhozproekf' for the assistance with field trials, Dr. Irina Perrninova for the biosorbent delivery and Salem Qayid for the technical assistance.


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I-:P.Murygina et al. / Environment International 3/ (2005) /63-/66

References Gerhardt F, editor. Methods of common bacteriology; 1984. (in Russian). Green Peace, Newspaper No. 27, 1998 (in Russian). Mouryguina V, Kalyuzhnyi S. Comparison of two methods ofbioremediation of oil polluted wetland and marshy soils in Western Siberia: bioaugmentation with preparation "Rhoder" versus stimulation of indigenous microorganisms. In: Kalogerakis N, editor. The First European Bioremediation Conference, Crete, Greece; 200 I,

Murygina V, Arinbasarov M, Kalyuzhnyi S. Bioremediation of oil polluted aquatic systems and soils with novel preparation "Rhoder". Biodegra~ation 2000;11(6):385-9. Nazlna T, Rozanova Ye, Belyayev S, Ivanov M. Chemical and microbiological methods of investigation of liquid and solid samples from oil fields. Pushchino, Russia: Biological Centre Press; 1988. (in Russian).

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