Bioresource Technology 100 (2009) 3221–3227

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Bioresource Technology

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Application of aerobic microorganisms in bioremediation in situ of soilcontaminated by petroleum products

Dorota Wolicka a,*, Agnieszka Suszek b, Andrzej Borkowski c, Aleksandra Bielecka d

a Institute of Geochemistry, Mineralogy and Petrology, Faculty of Geology, University of Warsaw, _Zwirki i Wigury 93, 02-089 Warsaw, Polandb Department of General Microbiology, Institute of Microbiology, University of Warsaw, ul. Miecznikowa 1, 02-096 Warsaw, Polandc Department of Soil Sciences, Faculty of Agriculture and Biology, University of Life Sciences (SGGW), Warsaw, Polandd GEO-KAT Sp. z o. o., Al. Prymasa Tysiaclecia 145/149, 01-424 Warsaw, Poland

a r t i c l e i n f o

Article history:Received 24 November 2008Received in revised form 9 February 2009Accepted 12 February 2009Available online 16 March 2009

Keywords:In situ bioremediationAerobic bacterial communitiesBTEX

0960-8524/$ - see front matter � 2009 Published bydoi:10.1016/j.biortech.2009.02.020

* Corresponding author. Fax: +48 225540000.E-mail address: d.wolicka@uw.edu.pl (D. Wolicka)

a b s t r a c t

Aerobic microorganisms able to biodegrade benzene, toluene, ethylbenzene, xylene (BTEX) have beenisolated from an area contaminated by petroleum products. The activity of the isolated communitieswas tested under both laboratory and field conditions. Benzene, toluene, ethylbenzene and xylene wereadded to the cultures as the sole carbon source, at a concentration of 500 mg/L. In batch cultures underlaboratory conditions, an 84% reduction of benzene, 86% of toluene and 82% of xylene were achieved. Incultures with ethylbenzene as the sole carbon source, the reduction was around 80%. Slightly lower val-ues were observed under field conditions: 95% reduction of benzene and toluene, 81% of ethylbenzeneand 80% of xylene. A high biodegradation activity of benzene (914 lM/L/24 h), toluene (771 lM/L/24 h), xylene (673 lM/L/24 h) and ethylbenzene (644 lM/L/24 h) was observed in the isolatedcommunities.

� 2009 Published by Elsevier Ltd.

1. Introduction

In industrialized countries, contamination of soil by crude oiland petroleum products has become a serious problem. The mainsources of this contamination are: oil field installations, petroleumplants, liquid fuel distribution and storage devices, transportationequipment for petroleum products, airports and illegal drillingsin pipe lines. The scale of the hazards imposed on the natural envi-ronment depends on the surface of the area contaminated bypetroleum products, their chemical composition, and the depthat which pollutants occur.

Crude oil and petroleum products contain many kinds of organ-ic compounds, dominated by aliphatic and aromatic hydrocarbons(Fukui et al., 1999). The most toxic components, with mutagenicand carcinogenic potential activity, include the aromatic com-pounds benzene, toluene, ethylbenzene and xylene (BTEX), whicheasily pass into the groundwater and may pose a hazard to organ-isms using it (Bogan and Sullivan, 2003; Kasai et al., 2006; Wolickaand Suszek, 2008). It is commonly known that BTEX compoundsare biodegradable under aerobic conditions (Nielsen et al., 2006;Sublette et al., 2006). Thus, aerobic conditions have been shownto be highly effective in the remediation of many oil spills.

Many soil microorganisms transform oil hydrocarbons into non-toxic compounds or mineralize them to inorganic compounds (Lea-

Elsevier Ltd.

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hy and Colwell, 1990). Hydrocarbons are degraded in soil mainly bybacteria (0.13–50% of the total of heterotrophic soil microorgan-isms) and fungi (6–82%) (Leahy and Colwell, 1990; Wolicka,2008). This natural microbiological activity is applied in bioremedi-ation to reduce the concentration and/or toxicity of various pollu-tants, including petroleum products (Dua et al., 2002). Theseprocesses take place in the natural environment, and their end-products are carbon dioxide and water (Olliver and Magot, 2005).

Numerous microbes present in soil contaminated by oil hydro-carbons are able to grow, despite the high toxicity of these com-pounds. The ability to degrade and/or utilize oil hydrocarbonshas been observed in numerous types of bacteria and fungi, andin yeast e.g. Candida, Saccharomyces (Bento and Gaylarde, 2001;Prenafeta-Boldu et al., 2002), some Cyanobacteria e.g. Oscillatoria,Anabaena, Nostoc, Microcoleus, Chlamydomonas, Scenedesmus, Pho-rmidium and green algae e.g. Chlorella, Microcoleus, Chlamydo-monas, Ulva, Scenedesmus, Phormidium ( Antizar-Ladislao et al.,2004). However, in soil bioremediation mainly bacteria are ap-plied, because they are distinguished by high frequency, fastgrowth and a wide spectrum of the utilized petroleum products.The natural environments contaminated by aromatic compounds,such as areas where oil is mined and exploited, or areas with anindustrial infrastructure, create good conditions for microorgan-isms which can biodegrade aromatic hydrocarbons (Wolicka andBorkowski, 2007). Hence, these environments seem to be a prop-erly for isolation of microorganisms which can be potentially ap-plied in bioremediation in situ.

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This paper is focused on the isolation and selection of aerobic con-sortia able to biodegrade BTEX, from an area contaminated by petro-leum products, and on testing their activity under field conditions.

2. Methods

2.1. Enrichment of BTEX degrading consortia

The microorganisms were isolated from the area of a petrol sta-tion contaminated by petroleum products, located in north-easternPoland. Contamination by BTEX in the study area has occurred forat least 30 years.

Samples of contaminated soil (ca. 5 g) were inserted in 300 mlflasks, to which a substrate with a suitable carbon source (benzene,toluene, ethylbenzene or xylene) was added as the only electron do-nor to the culture. The flasks were sealed with cotton wool, and thenshaken in a shaker for 72 h in order to assure aerobic conditions.Next, from the culture, of 0.1 ml aliquots were spread on the surfaceof M9 agar medium with a suitable carbon sources. The cultures ob-tained were transformed on a liquid medium, shaken for 24 h andinoculated again on Petri dishes. This process was repeated severaltimes, in order to obtain a sufficient content of specific consortia toestablish fluid cultures and apply it under field conditions. Abioticcontrols were established to test anaerobic chemical biodegrada-tion of BTEX. Isolated specific consortia were put on into contami-nated soils (depth: 1.5 m). The volume of added liquid mediumwith bacteria was strictly 30 L for each of three piezometers.

2.2. Culture conditions

The cultures were grown at room temperature (ca. 20 �C). Theinoculum to medium ratio was 1:10, in 300 ml glass bottles. Afterthis stage, the inoculum was increased to have 30 L of the solutionswith specific consortia.

2.3. Media

M9 medium: (Na2HPO4 � H2O – 0.134 g/L, KH2PO4 – 0.03 g/L,NaCl – 0.5 g/L, NH4Cl – 3.982 g/L, salts: MgSO4 � 7H2O – 2.47 g/100 ml – 1 ml salt/100 ml medium, CaCl2 – 111 mg/100 ml –1 ml salt/100 ml medium), minimal medium: (NH4Cl – 1.0 g/L),Davis medium: (K2HPO4 – 35 g/L, KH2PO4 – 10 g/L, MgSO4 � 7H2O– 0.5 g/L, (NH4)SO4 – 2 g/L).

Benzene (0.5 g/L), toluene (0.5 g/L), ethylbenzene (0.5 g/L) orxylene (0.5 g/L) were added to the medium as the sole carbonsource.

2.4. Analyses

Biological determinations: Viable bacterial counts were esti-mated by bacterial colony counts on agar M9 media. Fluorescenceand scanning electron microscopy (SEM) were used in order tocharacterize the bacterial consortia.

Chemical determinations: Hydrocarbons were tested using gaschromatography with a flame ionization detector GC-FID. The sam-ples were extracted by a petroleter (according to DIN EN ISO 9377-2 H53). BTEX analyses were made using the gas chromatographymethod, with mass spectrometer GS–MS (according to DIN38407 F9, F5).

2.5. Characteristics of the study area

2.5.1. Geological settingThe geological setting of the study area is linked with the re-

treat of the ice-sheet of the Vistulian Glaciation. This recession

caused the formation of dead-ice moraines, which were next cov-ered by sandur sands. Therefore, the area is covered by tills, onwhich lie fluvioglacial sands, the top of which is located at differentlevels. Field studies carried out in March 2007 included drilling ofeight wells and detailed recognition of the geological setting in theremediated area (Fig. 1). Anthropogenic embankments are presentnear wells P1, Pz1 and Pz3, which are underlain by fine- and med-ium-grained sands. Below occur gravels, in some cases with boul-ders and pebbles. The gravels are underlain by sandy tills, observedbetween 0.2 (P3) to 18 m (Pz2) below surface level; the tills werenot drilled through. In wells P1 and Pz2, 0.5 m thick alternationsof sandy tills (3–3.5 m below surface level in P1, 13.5–14 m belowsurface level in Pz2) have been observed (Fig. 2).

2.5.2. Hydrogeological conditionsThe main useful aquifer occurs at depths between 15 and 45 m

below the till. This is a mosaic type aquifer, linked with the vari-able depth at which occur non-permeable deposits. Thus, in thesouthern part of the contaminated area the groundwater flow is to-wards the south-east, whereas in the northern towards the north-east. Therefore, two independent contaminated areas occur.

3. Results and discussion

3.1. Selection of optimal medium for microorganismsable to biodegrade BTEX

Based on the analyses carried out at the water samples collectedfrom the piezometers, the study area is contaminated mainly byBTEX, particularly by xylene, which occurred in the highest abun-dances: 5500 lg/L in P1, 5330 lg/L in P2, and 780 lg/L in P3. Highconcentrations of ethylbenzene were also observed in P1 (1100 lg/L) and P2 (1200 lg/L), as well as of benzene in P2 (8000 lg/L) (Ta-ble 1). The total content of bacteria in soil able to biodegrade ofBTEX was 106 cfu/g d.w. of soil.

Taking into account the high concentration of BTEX in the studyarea, the selected medium had to contain the smallest concentra-tion of chemical compounds able to disturb the already unbalancedbiological equilibrium in the soil. On the other hand, we looked fora medium that did not contain compounds easily accessible to themicroorganisms that could be used as the carbon source in the firststage of bioremediation. Only after these compounds were usedwould, adaptation to BTEX take place, an unfavourable condition.In most papers referring to bioremediation, the medium containede.g. yeast extract (Carla et al., 2004) or citrate (Harayama et al.,1999), which might be the cause of the longer adaptation of micro-organisms to utilizing BTEX as the sole carbon source.

In order to eliminate additional factors, such as an easily acces-sible carbon source, and at the same time shorten the time of adap-tation to high concentrations of BTEX in the initial part of thestudy, three media (Davis, minimal and M9) have been applied.Based on growth results on a liquid medium and in Petri dishes,obtained during the first passage, the M9 medium was consideredoptimal for the selected microorganisms able to biodegrade BTEXin this study. Therefore this medium was applied in further analy-ses. Benzene, toluene, ethylbenzene or xylene was applied as thesole carbon source. In effect, four microorganism communities ableto utilize these compounds were obtained.

Four cultures were established that were passed several timesthrough a medium containing 0.5 g/L of benzene, toluene, ethyl-benzene or xylene. A fifth culture was also established with allthe compounds (BTEX) at a concentration of 0.5 g/L, giving a pri-mary concentration of 2 g/L. The culture was inoculated with fourearlier selected communities able to biodegrade BTEX. The inocu-lum/medium ratio was 1:10. Seven days after inoculation, a chro-

Fig. 1. Distribution of piezometer wells.

D. Wolicka et al. / Bioresource Technology 100 (2009) 3221–3227 3223

matographic analysis was made to check the degree of biodegrada-tion of the aromatic hydrocarbons applied as the sole carbonsource (Table 2).

In cultures where benzene, toluene or xylene were applied asthe sole carbon source, 84%, 86% and 82%, respectively, biodegrada-tion of the applied compounds was observed (in correlation withabiotic controls), whereas in the case of ethylbenzene biodegrada-tion reached 80%. In a culture containing all aromatic compounds(BTEX), the effectiveness of biodegradation for benzene 79.2%, fortoluene 78.6%, for xylene 80.4% and for ethylbenzene 73% were ob-served. The fact that the aerobic microorganisms are able to biode-grade BTEX is not surprising. However, it should be underlined thatthe initial concentration of BTEX was extremely high (2 g/L in thecultures), which shows that isolated bacterial communities canbe active even at such high concentration.

Based on literature data, it can be assumed that generally BTEXbiodegradation is faster under aerobic than anaerobic conditions;therefore the optimalisation of the process of aerobic BTEX biodeg-radation in laboratory conditions is crucial, particularly due to therather common application of the method in practice. Bacteria(Alfreider and Vogt, 2007) and fungi (Prenafeta-Boldu et al.,2004) are applied in BTEX biodegradation. The effectiveness ofthe biodegradation depends on many factors, of which the mostimportant is the environment in which the microorganisms areisolated. In cultures of the isolated communities, a high biodegra-

dation activity of benzene (914 lM/L/24 h), toluene (771 lM/L/24 h), xylene (673 lM/L/24 h) and ethylbenzene (644 lM/L/24 h)was observed. Such a high activity of biodegradation by aerobicconsortia may come from the fact that the environment in whichmicroorganisms were isolated was soil contaminated by BTEX,and the contamination in the study area has occurred for at least30 years. This fact can also be confirmed by the results obtainedby Taki et al. (2007) for Rhodococcus sp., indicating a much higheractivity of microorganisms isolated from soil contaminated bypetroleum products, compared to the activity of microorganismsisolated from non-contaminated soil from Seminale County inOklahoma, obtained by Nicholson and Fathepure (2004) (Table 3).

It is rather surprising that all BTEX in the mixed culture werebiodegraded at a rate of 86%. Results of BTEX biodegradation ob-tained by Attway and Schmidt (2002) for Pseudomonas putidaTX1 and P. putida BTE1 univocally indicated that toluene was thefirst to be utilized in microorganism cultures, where it occurredwith benzene, ethylbenzene or xylene. Similar results for P. putidaF1 were obtained by Reardon et al. (2000) in microorganism cul-tures on a medium with toluene, benzene and phenol. In each ofthese cultures, toluene was the first to be utilized at a rate of 100%.

It should be pointed out that only a few soil microorganisms cansimultaneously decompose one or several hydrocarbons. There-fore, effective bioremediation of soil from petroleum products re-quires the application of mixed communities of microorganisms

Fig. 2. Well logs.

Table 1Concentrations of BTEX in piezometers before bioremediation.

No. PP

BTEX (lg/L) Benzene (lg/L) Toluene (lg/L) Ethylbenzene (lg/L) m,p-Xylene (lg/L) o-Xylene (lg/L)

P1 6400 241 5 1100 4500 550P2 14700 8000 190 1200 4400 930P3 953.6 7.6 26 140 570 210

Table 2BTEX reduction in batch cultures of the isolated microorganisms.

Source of carbon Concentration to(lg/L) Concentration t7 (lg/L) Reduction % real reduction %

Benzene (B) 500,000 <0.1 100 84.0Toluene (T) 500,000 <0.1 100 86.0Ethylbenzene (E) 500,000 22 000 95.6 79.6Xylene (X) 500,000 190 100 82.0BTEX 2,000,000 264,000 86.8 77.8

Benzene 500,000 59,000 88.2 79.2Toluene 500,000 62,000 87.6 78.6Ethylbenzene 500,000 90,000 82.0 73.0Xylene 500,000 53,000 89.4 80.4

Abiotic controlBenzene 500,000 420,000 16.0 –Toluene 500,000 430,000 14.0 –Ethylbenzene 500,000 420,000 16.0 –Xylene 500,000 440,000 12.0 –BTEX 2,000,000 1,820,000 9.0 –

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able to metabolize particular compounds (Korda et al., 1997). Theresults obtained indicate, however, that much more effective aremicroorganism communities adapted to one particular pollutant,where the effectiveness of bioturbation reached 100% (except eth-

ylbenzene). The application of four communities to the simulta-neous biodegradation of benzene, toluene, ethylbenzene andxylene was less effective. However, in an environment contami-nated by petroleum products, one pollutant almost never occurs

Table 3Activity of the isolated aerobic microorganisms in batch cultures.

Source of carbon Nicholson and Fathepure (2004) Taki et al. (2007) Own studies

to (lM/L) lM/L/24 h to (lM/L) lM/L/24 h to (lM/L) lM/L/24 h

Benzene 125 20.8 200 40 6400 914Toluene 125 8.9 200 50 5400 771Ethylbenzene 125 5.9 200 66 4700 644Xylene 125 5.9 200 50 4700 673

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by itself, therefore it is indispensable to apply mixed selectedmicroorganism communities able to biodegrade the particularcompounds constituting the main pollutant in the study area.

At this stage of it laboratory studies, four active communities ofmicroorganisms able to biodegrade BTEX under aerobic conditionswere isolated. The Gram-method colouring and SEM show that iso-lated bacterial consortia were rod- and coccus-shaped bacteria1.5 lm length. (Fig. 3).

3.2. In situ bioremediation

Heavy pollution of soils and surface waters results in changes tothe physical and chemical properties of the contaminated soil

Fig. 3. Isolated aerobic bacterial communities biodegrading xylene (A–C),

through changes in pH, oxygen content, or concentration of nutri-ents. Deficiency of nutrients is the result of a high content of car-bon in the contaminated area in relation to other elementsindispensable to cell synthesis. The most crucial is the C:N:P ratio;its correct values are assumed to be at the level of 100:9:2,100:10:1 or 250:10:3, respectively (Klimiuk and Łebkowska,2005). Therefore, before commencing in situ remediation, theC:N:P ratio was optimized, which was achieved by the additionof inorganic fertilizers such as: potassium nitrate (KNO3) and so-dium orthophosphate (Na3PO4).

Four isolated communities of microorganisms were applied inthe in situ bioremediation of the area contaminated by petroleumproducts. Most of the described communities are mesophilous

toluene (D), benzene (E) and ethylobenzene (F) as sole carbon source.

Table 4Pollution reduction (%).

PiezometerP

BTEX Benzene Toluene Ethylbenzene m,p-Xylene o-Xylene

P1 45.5 0 0 48 49 47P2 95 97 92 95 91.6 92.2P3 99.7 93.4 99.2 99.8 99.7 99.9

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(Aeckersberg et al., 1998; Vieth et al., 2005; Fahy et al., 2006);however papers have recently appeared describing BTEX biodegra-dation by psychrophilous microorganisms (Aislabie et al., 2006).Their application in field conditions seems groundless at the pres-ent level of study due to the long time of generation and remedia-tion, exceeding the duration of the winter season in temperateclimate areas. Taking this into account, the bioremediation wascommenced in April and ended in November. The in situ bioreme-diation lasted for 7 months.

Selected communities of microorganisms were introducedeach month into piezometers P1, P2 and P3, according to thescheme presented in Fig. 1. The introduction method was linkedto the existing pollutant; i.e. to P1 were introduced consortiaable to biodegrade xylene, ethylbenzene and toluene; to P2 –those able to biodegrade benzene, ethylbenzene and toluene;and to P3 – those able to biodegrade xylene and benzene. Thedifferent method of introducing the particular consortia was fo-cused on the removal of pollutants occurring at high concentra-tions in a given place. The effectiveness of bioremediation wascontrolled by analysis of BTEX concentrations 30 days after weadded isolated consortia during total research period (during7th months).

The highest reduction was noted in the case of P3, and also P2,where the soil was almost completely purified. The lowest reduc-tion was observed in P1 (45.5% for BTEX). With regard to benzeneand toluene in P1, their concentrations did not change after thebioremediation processes finished, whereas the general concentra-tion of BTEX underwent reduction (Table 4).

Evaluation of the effectiveness of bioremediation in the field ismuch more difficult than under laboratory conditions. One of thedifficulties, for example is the uneven distribution of pollutants,which requires the collection a larger number of samples in orderto obtain statistically significant results. In soil, microorganismsoften occur in consortia encompassing several species that arein syntrophic and spatial relationships. This fact often obstructsobtaining in pure from the bacterial communities responsiblefor the biodegradation of petroleum products. Due to this fact,the communities obtained were recognized only at genus level.Most belong to the genus Pseudomonas. According to Sikkemaet al. (1995), only cultures of the Pseudomonas have shown resis-tance to high concentrations of cyclic aromatic hydrocarbons. Thiswas confirmed by Attaway and Schmidt (2002) and Shim et al.(2002, 2005), who showed the ability of P. putida and Pseudomo-nas fluorescens, respectively, to utilize high concentrations ofBTEX.

4. Conclusions

1. To achieve a maximal rate of BTEX biodegradation, and to min-imize the introduction with the medium of different chemicalcompounds in the initial phase of analysis, an optimal mediumwas selected for the autochthonous microorganisms isolatedfrom soil contaminated by petroleum products.

2. Optimization of BTEX biodegradation in laboratory conditionsprovides an opportunity to obtain a high activity of the consor-tia able to biodegrade BTEX. The selected media should not con-tain simple chemical compounds (such as: lactate, ethanol,acetate) that could act as a potential carbon source for the bac-

teria, because they could greatly inhibit the biodegradation pro-cess through utilization of simple organic compounds as thesource of carbon in the first place.

3. A high effectiveness of in situ biodegradation results from theapplication of selected bacterial communities adapted to utilizeone type of BTEX compound. Nevertheless, this does notexclude the ability to utilize other BTEX by selected microor-ganisms able to biodegrade e.g. benzene.

Acknowledgement

We thank for Professor Raymond Macdonald for his help withEnglish.

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