Bioresource Technology 100 (2009) 32213227

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Bioresource Technology
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Application of aerobic microorganisms in bioremediation in situ of soil contaminated by petroleum products

Dorota Wolicka a,*, Agnieszka Suszek b, Andrzej Borkowski c, Aleksandra Bielecka d
_ Institute of Geochemistry, Mineralogy and Petrology, Faculty of Geology, University of Warsaw, Zwirki i Wigury 93, 02-089 Warsaw, Poland Department of General Microbiology, Institute of Microbiology, University of Warsaw, ul. Miecznikowa 1, 02-096 Warsaw, Poland c Department of Soil Sciences, Faculty of Agriculture and Biology, University of Life Sciences (SGGW), Warsaw, Poland d GEO-KAT Sp. z o. o., Al. Prymasa Tysiaclecia 145/149, 01-424 Warsaw, Poland
a b

a r t i c l e

i n f o

a b s t r a c t
Aerobic microorganisms able to biodegrade benzene, toluene, ethylbenzene, xylene (BTEX) have been isolated from an area contaminated by petroleum products. The activity of the isolated communities was tested under both laboratory and eld conditions. Benzene, toluene, ethylbenzene and xylene were added to the cultures as the sole carbon source, at a concentration of 500 mg/L. In batch cultures under laboratory conditions, an 84% reduction of benzene, 86% of toluene and 82% of xylene were achieved. In cultures with ethylbenzene as the sole carbon source, the reduction was around 80%. Slightly lower values were observed under eld conditions: 95% reduction of benzene and toluene, 81% of ethylbenzene and 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 isolated communities. 2009 Published by Elsevier Ltd.

Article history: Received 24 November 2008 Received in revised form 9 February 2009 Accepted 12 February 2009 Available online 16 March 2009 Keywords: In situ bioremediation Aerobic bacterial communities BTEX

1. Introduction In industrialized countries, contamination of soil by crude oil and petroleum products has become a serious problem. The main sources of this contamination are: oil eld installations, petroleum plants, liquid fuel distribution and storage devices, transportation equipment for petroleum products, airports and illegal drillings in pipe lines. The scale of the hazards imposed on the natural environment depends on the surface of the area contaminated by petroleum products, their chemical composition, and the depth at which pollutants occur. Crude oil and petroleum products contain many kinds of organic compounds, dominated by aliphatic and aromatic hydrocarbons (Fukui et al., 1999). The most toxic components, with mutagenic and carcinogenic potential activity, include the aromatic compounds benzene, toluene, ethylbenzene and xylene (BTEX), which easily pass into the groundwater and may pose a hazard to organisms using it (Bogan and Sullivan, 2003; Kasai et al., 2006; Wolicka and Suszek, 2008). It is commonly known that BTEX compounds are biodegradable under aerobic conditions (Nielsen et al., 2006; Sublette et al., 2006). Thus, aerobic conditions have been shown to be highly effective in the remediation of many oil spills. Many soil microorganisms transform oil hydrocarbons into nontoxic compounds or mineralize them to inorganic compounds (Lea* Corresponding author. Fax: +48 225540000. E-mail address: d.wolicka@uw.edu.pl (D. Wolicka). 0960-8524/$ - see front matter 2009 Published by Elsevier Ltd. doi:10.1016/j.biortech.2009.02.020

hy and Colwell, 1990). Hydrocarbons are degraded in soil mainly by bacteria (0.1350% of the total of heterotrophic soil microorganisms) and fungi (682%) (Leahy and Colwell, 1990; Wolicka, 2008). This natural microbiological activity is applied in bioremediation to reduce the concentration and/or toxicity of various pollutants, including petroleum products (Dua et al., 2002). These processes take place in the natural environment, and their endproducts are carbon dioxide and water (Olliver and Magot, 2005). Numerous microbes present in soil contaminated by oil hydrocarbons are able to grow, despite the high toxicity of these compounds. The ability to degrade and/or utilize oil hydrocarbons has been observed in numerous types of bacteria and fungi, and in yeast e.g. Candida, Saccharomyces (Bento and Gaylarde, 2001; Prenafeta-Boldu et al., 2002), some Cyanobacteria e.g. Oscillatoria, Anabaena, Nostoc, Microcoleus, Chlamydomonas, Scenedesmus, Phormidium and green algae e.g. Chlorella, Microcoleus, Chlamydomonas, Ulva, Scenedesmus, Phormidium ( Antizar-Ladislao et al., 2004). However, in soil bioremediation mainly bacteria are applied, because they are distinguished by high frequency, fast growth 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 an industrial infrastructure, create good conditions for microorganisms which can biodegrade aromatic hydrocarbons (Wolicka and Borkowski, 2007). Hence, these environments seem to be a properly for isolation of microorganisms which can be potentially applied in bioremediation in situ.

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This paper is focused on the isolation and selection of aerobic consortia able to biodegrade BTEX, from an area contaminated by petroleum products, and on testing their activity under eld conditions.

2. Methods 2.1. Enrichment of BTEX degrading consortia The microorganisms were isolated from the area of a petrol station contaminated by petroleum products, located in north-eastern Poland. Contamination by BTEX in the study area has occurred for at least 30 years. Samples of contaminated soil (ca. 5 g) were inserted in 300 ml asks, to which a substrate with a suitable carbon source (benzene, toluene, ethylbenzene or xylene) was added as the only electron donor to the culture. The asks were sealed with cotton wool, and then shaken 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 surface of M9 agar medium with a suitable carbon sources. The cultures obtained were transformed on a liquid medium, shaken for 24 h and inoculated again on Petri dishes. This process was repeated several times, in order to obtain a sufcient content of specic consortia to establish uid cultures and apply it under eld conditions. Abiotic controls were established to test anaerobic chemical biodegradation of BTEX. Isolated specic consortia were put on into contaminated soils (depth: 1.5 m). The volume of added liquid medium with bacteria was strictly 30 L for each of three piezometers. 2.2. Culture conditions The cultures were grown at room temperature (ca. 20 C). The inoculum to medium ratio was 1:10, in 300 ml glass bottles. After this stage, the inoculum was increased to have 30 L of the solutions with specic 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) or xylene (0.5 g/L) were added to the medium as the sole carbon source. 2.4. Analyses Biological determinations: Viable bacterial counts were estimated by bacterial colony counts on agar M9 media. Fluorescence and scanning electron microscopy (SEM) were used in order to characterize the bacterial consortia. Chemical determinations: Hydrocarbons were tested using gas chromatography with a ame ionization detector GC-FID. The samples were extracted by a petroleter (according to DIN EN ISO 93772 H53). BTEX analyses were made using the gas chromatography method, with mass spectrometer GSMS (according to DIN 38407 F9, F5). 2.5. Characteristics of the study area 2.5.1. Geological setting The geological setting of the study area is linked with the retreat of the ice-sheet of the Vistulian Glaciation. This recession

caused the formation of dead-ice moraines, which were next covered by sandur sands. Therefore, the area is covered by tills, on which lie uvioglacial sands, the top of which is located at different levels. Field studies carried out in March 2007 included drilling of eight wells and detailed recognition of the geological setting in the remediated area (Fig. 1). Anthropogenic embankments are present near wells P1, Pz1 and Pz3, which are underlain by ne- and medium-grained sands. Below occur gravels, in some cases with boulders and pebbles. The gravels are underlain by sandy tills, observed between 0.2 (P3) to 18 m (Pz2) below surface level; the tills were not drilled through. In wells P1 and Pz2, 0.5 m thick alternations of sandy tills (33.5 m below surface level in P1, 13.514 m below surface level in Pz2) have been observed (Fig. 2). 2.5.2. Hydrogeological conditions The main useful aquifer occurs at depths between 15 and 45 m below the till. This is a mosaic type aquifer, linked with the variable depth at which occur non-permeable deposits. Thus, in the southern part of the contaminated area the groundwater ow is towards the south-east, whereas in the northern towards the northeast. Therefore, two independent contaminated areas occur.

3. Results and discussion 3.1. Selection of optimal medium for microorganisms able to biodegrade BTEX Based on the analyses carried out at the water samples collected from the piezometers, the study area is contaminated mainly by BTEX, particularly by xylene, which occurred in the highest abundances: 5500 lg/L in P1, 5330 lg/L in P2, and 780 lg/L in P3. High concentrations 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) (Table 1). The total content of bacteria in soil able to biodegrade of BTEX was 106 cfu/g d.w. of soil. Taking into account the high concentration of BTEX in the study area, the selected medium had to contain the smallest concentration of chemical compounds able to disturb the already unbalanced biological equilibrium in the soil. On the other hand, we looked for a medium that did not contain compounds easily accessible to the microorganisms that could be used as the carbon source in the rst stage of bioremediation. Only after these compounds were used would, adaptation to BTEX take place, an unfavourable condition. In most papers referring to bioremediation, the medium contained e.g. yeast extract (Carla et al., 2004) or citrate (Harayama et al., 1999), which might be the cause of the longer adaptation of microorganisms to utilizing BTEX as the sole carbon source. In order to eliminate additional factors, such as an easily accessible carbon source, and at the same time shorten the time of adaptation to high concentrations of BTEX in the initial part of the study, three media (Davis, minimal and M9) have been applied. Based on growth results on a liquid medium and in Petri dishes, obtained during the rst passage, the M9 medium was considered optimal for the selected microorganisms able to biodegrade BTEX in this study. Therefore this medium was applied in further analyses. Benzene, toluene, ethylbenzene or xylene was applied as the sole carbon source. In effect, four microorganism communities able to utilize these compounds were obtained. Four cultures were established that were passed several times through a medium containing 0.5 g/L of benzene, toluene, ethylbenzene or xylene. A fth culture was also established with all the compounds (BTEX) at a concentration of 0.5 g/L, giving a primary concentration of 2 g/L. The culture was inoculated with four earlier selected communities able to biodegrade BTEX. The inoculum/medium ratio was 1:10. Seven days after inoculation, a chro-

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Fig. 1. Distribution of piezometer wells.

matographic analysis was made to check the degree of biodegradation of the aromatic hydrocarbons applied as the sole carbon source (Table 2). In cultures where benzene, toluene or xylene were applied as the sole carbon source, 84%, 86% and 82%, respectively, biodegradation of the applied compounds was observed (in correlation with abiotic controls), whereas in the case of ethylbenzene biodegradation reached 80%. In a culture containing all aromatic compounds (BTEX), the effectiveness of biodegradation for benzene 79.2%, for toluene 78.6%, for xylene 80.4% and for ethylbenzene 73% were observed. The fact that the aerobic microorganisms are able to biodegrade BTEX is not surprising. However, it should be underlined that the initial concentration of BTEX was extremely high (2 g/L in the cultures), which shows that isolated bacterial communities can be active even at such high concentration. Based on literature data, it can be assumed that generally BTEX biodegradation is faster under aerobic than anaerobic conditions; therefore the optimalisation of the process of aerobic BTEX biodegradation in laboratory conditions is crucial, particularly due to the rather 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 of the biodegradation depends on many factors, of which the most important is the environment in which the microorganisms are isolated. 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 aerobic consortia may come from the fact that the environment in which microorganisms were isolated was soil contaminated by BTEX, and the contamination in the study area has occurred for at least 30 years. This fact can also be conrmed by the results obtained by Taki et al. (2007) for Rhodococcus sp., indicating a much higher activity of microorganisms isolated from soil contaminated by petroleum products, compared to the activity of microorganisms isolated from non-contaminated soil from Seminale County in Oklahoma, obtained by Nicholson and Fathepure (2004) (Table 3). It is rather surprising that all BTEX in the mixed culture were biodegraded at a rate of 86%. Results of BTEX biodegradation obtained by Attway and Schmidt (2002) for Pseudomonas putida TX1 and P. putida BTE1 univocally indicated that toluene was the rst to be utilized in microorganism cultures, where it occurred with benzene, ethylbenzene or xylene. Similar results for P. putida F1 were obtained by Reardon et al. (2000) in microorganism cultures on a medium with toluene, benzene and phenol. In each of these cultures, toluene was the rst to be utilized at a rate of 100%. It should be pointed out that only a few soil microorganisms can simultaneously decompose one or several hydrocarbons. Therefore, effective bioremediation of soil from petroleum products requires the application of mixed communities of microorganisms

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Fig. 2. Well logs.

Table 1 Concentrations of BTEX in piezometers before bioremediation. No. P P1 P2 P3 P BTEX (lg/L) 6400 14700 953.6 Benzene (lg/L) 241 8000 7.6 Toluene (lg/L) 5 190 26 Ethylbenzene (lg/L) 1100 1200 140 m,p-Xylene (lg/L) 4500 4400 570 o-Xylene (lg/L) 550 930 210

Table 2 BTEX reduction in batch cultures of the isolated microorganisms. Source of carbon Benzene (B) Toluene (T) Ethylbenzene (E) Xylene (X) BTEX Benzene Toluene Ethylbenzene Xylene Abiotic control Benzene Toluene Ethylbenzene Xylene BTEX Concentration to(lg/L) Concentration t7 (lg/L) 500,000 500,000 500,000 500,000 2,000,000 500,000 500,000 500,000 500,000 500,000 500,000 500,000 500,000 2,000,000 Reduction % <0.1 <0.1 22 000 190 264,000 59,000 62,000 90,000 53,000 420,000 430,000 420,000 440,000 1,820,000 real reduction % 100 100 95.6 100 86.8 88.2 87.6 82.0 89.4 16.0 14.0 16.0 12.0 9.0 84.0 86.0 79.6 82.0 77.8 79.2 78.6 73.0 80.4

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

ylbenzene). The application of four communities to the simultaneous biodegradation of benzene, toluene, ethylbenzene and xylene was less effective. However, in an environment contaminated by petroleum products, one pollutant almost never occurs

D. Wolicka et al. / Bioresource Technology 100 (2009) 32213227 Table 3 Activity of the isolated aerobic microorganisms in batch cultures. Source of carbon Nicholson and Fathepure (2004) to (lM/L) Benzene Toluene Ethylbenzene Xylene 125 125 125 125 Taki et al. (2007) to (lM/L) 200 200 200 200 Own studies

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lM/L/24 h
20.8 8.9 5.9 5.9

lM/L/24 h
40 50 66 50

to (lM/L) 6400 5400 4700 4700

lM/L/24 h
914 771 644 673

by itself, therefore it is indispensable to apply mixed selected microorganism communities able to biodegrade the particular compounds constituting the main pollutant in the study area. At this stage of it laboratory studies, four active communities of microorganisms able to biodegrade BTEX under aerobic conditions were isolated. The Gram-method colouring and SEM show that isolated bacterial consortia were rod- and coccus-shaped bacteria 1.5 lm length. (Fig. 3). 3.2. In situ bioremediation Heavy pollution of soils and surface waters results in changes to the physical and chemical properties of the contaminated soil

through changes in pH, oxygen content, or concentration of nutrients. Deciency of nutrients is the result of a high content of carbon in the contaminated area in relation to other elements indispensable 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 ebkowska, 2005). Therefore, before commencing in situ remediation, the C:N:P ratio was optimized, which was achieved by the addition of inorganic fertilizers such as: potassium nitrate (KNO3) and sodium orthophosphate (Na3PO4). Four isolated communities of microorganisms were applied in the in situ bioremediation of the area contaminated by petroleum products. Most of the described communities are mesophilous

Fig. 3. Isolated aerobic bacterial communities biodegrading xylene (AC), toluene (D), benzene (E) and ethylobenzene (F) as sole carbon source.

3226 Table 4 Pollution reduction (%). Piezometer P1 P2 P3 P BTEX 45.5 95 99.7

D. Wolicka et al. / Bioresource Technology 100 (2009) 32213227

Benzene 0 97 93.4

Toluene 0 92 99.2

Ethylbenzene 48 95 99.8

m,p-Xylene 49 91.6 99.7

o-Xylene 47 92.2 99.9

(Aeckersberg et al., 1998; Vieth et al., 2005; Fahy et al., 2006); however papers have recently appeared describing BTEX biodegradation by psychrophilous microorganisms (Aislabie et al., 2006). Their application in eld conditions seems groundless at the present level of study due to the long time of generation and remediation, exceeding the duration of the winter season in temperate climate areas. Taking this into account, the bioremediation was commenced in April and ended in November. The in situ bioremediation lasted for 7 months. Selected communities of microorganisms were introduced each month into piezometers P1, P2 and P3, according to the scheme presented in Fig. 1. The introduction method was linked to the existing pollutant; i.e. to P1 were introduced consortia able 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. The different method of introducing the particular consortia was focused on the removal of pollutants occurring at high concentrations in a given place. The effectiveness of bioremediation was controlled by analysis of BTEX concentrations 30 days after we added isolated consortia during total research period (during 7th months). The highest reduction was noted in the case of P3, and also P2, where the soil was almost completely puried. The lowest reduction was observed in P1 (45.5% for BTEX). With regard to benzene and toluene in P1, their concentrations did not change after the bioremediation processes nished, whereas the general concentration of BTEX underwent reduction (Table 4). Evaluation of the effectiveness of bioremediation in the eld is much more difcult than under laboratory conditions. One of the difculties, for example is the uneven distribution of pollutants, which requires the collection a larger number of samples in order to obtain statistically signicant results. In soil, microorganisms often occur in consortia encompassing several species that are in syntrophic and spatial relationships. This fact often obstructs obtaining in pure from the bacterial communities responsible for 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 Sikkema et al. (1995), only cultures of the Pseudomonas have shown resistance to high concentrations of cyclic aromatic hydrocarbons. This was conrmed by Attaway and Schmidt (2002) and Shim et al. (2002, 2005), who showed the ability of P. putida and Pseudomonas uorescens, respectively, to utilize high concentrations of BTEX. 4. Conclusions 1. To achieve a maximal rate of BTEX biodegradation, and to minimize the introduction with the medium of different chemical compounds in the initial phase of analysis, an optimal medium was selected for the autochthonous microorganisms isolated from soil contaminated by petroleum products. 2. Optimization of BTEX biodegradation in laboratory conditions provides an opportunity to obtain a high activity of the consortia able to biodegrade BTEX. The selected media should not contain 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 process through utilization of simple organic compounds as the source of carbon in the rst place. 3. A high effectiveness of in situ biodegradation results from the application of selected bacterial communities adapted to utilize one type of BTEX compound. Nevertheless, this does not exclude the ability to utilize other BTEX by selected microorganisms able to biodegrade e.g. benzene.

Acknowledgement We thank for Professor Raymond Macdonald for his help with English. References
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