- Email: [email protected]
Bioaugmentation as a method of biodegradation enhancement in oil hydrocarbons contaminated soil
Vol. 6 No 1- 4, 163-169 2006
Bioaugmentation as a method of biodegradation enhancement in oil hydrocarbons contaminated soil
Ecohydrology for Implementation of the European Water Framework Directive
Iwona Zawierucha, Grzegorz Malina Institute of Environmental Engineering, Czêstochowa University of Technology, BrzeŸnicka 60A, 42-200 Czêstochowa, Poland, e-mail: [email protected]
Abstract Respirometry studies using the 10-chamber Micro-Oxymax respirometer (Columbus, Ohio) were conducted to determine the effect of adding bacterial consortium for enhancing biodegradation in soils contaminated with oil hydrocarbons from the former military airport. Bioaugmentation was carried out using both indigenous and extraneous bacteria able to degrade hydrocarbons. The potentials of intrinsic and enhanced biodegradation were evaluated by the mean O2 uptake and CO2 production rates. The results demonstrated that the highest biodegradation rates were achieved using bacterial consortium containing 4.8×1015 Colony Forming Units (CFU) cm-3. Generally, in all cases, enhanced biodegradation rates were four times higher than intrinsic biodegradation rates. Moreover, application of indigenous bacterial consortia was more efficient in comparison to the employment of extraneous bacterial consortia. This study showed that bioaugmentation could be an effective method for enhancing intrinsic biodegradation of oil hydrocarbons in soil. Key words: intrinsic biodegradation, indigenous microorganisms, extraneous bacteria.
1. Introduction Dynamic development of transportation causes that more of petroleum substances may penetrate natural environment, resulting in degradation of soils, surface water and groundwater (Deska et al. 2003). Nearly 2.5 million tons of oil products is estimated to enter the environment yearly (Gaca 2000). Since late 70-ties intensive studies have been carried out in Poland to estimate the degree of soil and groundwater contamination. A number of locations have been identified and documented with very high contamination of oil hydrocarbons (Koœcielniak, Steininger 2001). Oil-hydrocarbon contaminants may appear locally and periodically as a consequence of leaks of crude oil from mining shafts,
damaged pipelines, cisterns, tankers, and storage and distribution stations of petroleum products. They can also appear permanently on definite areas and derive from refineries or engine industry (Malina et al. 1999). Moreover, floods are a serious factor of contamination spreading in the environment (Ró¿añski 1999). Oil products in soils affect vegetation, threaten quality of surface water and groundwater, and are also potentially hazardous for humans and the environment (Gaca 2000). According to the ecohydrology concept, elimination of threats without consideration of biological processes, cannot lead to any success. Thus, ecohydrology incorporates the use of ecosystem properties as a management tool in implementing a program of water resource man-
164
I. Zawierucha, G. Malina
agement (Zalewski 2007 in press.). In the face of that, soil remediation technologies based on natural attenuation (NA) are closely connected with ecohydrology concept, because they use biodegradation with the participation of indigenous microorganisms to prevent migration of contaminants, and consequently to protect groundwater from pollution. According to the US Environmental Protection Agency (EPA 1999), 'natural attenuation' is the use of natural processes to contain the spread of the contamination from chemical spills and reduce the concentration and amount of pollutants at contaminated sites. It is also known as intrinsic (bio)remediation, and (bio)attenuation. In this approach, the contaminants are left on site, and the naturally occurring processes are responsible for clean-up of the site. The NA includes physical, chemical and biological processes. Biochemical processes, especially biodegradation with participation of microorganisms, play the most important role in these natural self-purification processes. Biodegradation is the breakdown of organic contaminants by microbial organisms into smaller compounds. The microbial organisms transform the contaminants through metabolic or enzymatic processes. Biodegradation processes vary greatly, but frequently under aerobic conditions the final products are: carbon dioxide, water and biomass (Malina 1999). The activity of native microflora decides about efficiency of biodegradation, and it can be enhanced by providing optimal conditions for the microbial growth. Thus, Enhanced Natural Attenuation (ENA) is commonly achieved by supplying nutrients, oxygen (or other electron acceptors) and/or water to contaminated soils (biostimulation), and/or inoculating soil with oil-degrading bacteria to supplement the existing microbial population (bioaugmentation or seeding) (Coulon, Delille 2003; Ivshina et al. 2001; Odokuma, Dickson 2003; Ko³wzan 2000; Siuta 2000; www.epa.gov). The cheapest method of intrinsic biodegradation enhancement is inoculating soils with microorganisms. They can be either isolated, adapted and multiplied autochthonous microflora, or genetically modified microorganisms (Ko³wzan 2000; Marquez-Rocha et al. 2001). The potential success of bioaugmentation depends on bioavailability of contaminants, the activity of added microorganisms and soil physical-chemical characteristics (Vogel 1996). Multiplied autochthonous microflora is generally applied in remediation technologies but inoculating of soil with laboratory modified bacterial cultures still arouses many reservations. Genetically modified microorganisms should not only degrade hydrocarbons with high efficiency, but they should also be capable of quick adaptation
and development in new environments (Fantroussi, Agathos 2005). They ought to be neutral in the presence of autochthonous microflora and should not produce any toxic substances (metabolites) during biodegradation. There are a number of arguable problems related to inoculating of soils with microorganisms modified genetically, including: - not well-known, defined and predictable side effects of inoculation that may affect the environment and humans, - effective monitoring of microorganisms distribution in soil, - survival of laboratory bacteria under field conditions, - regulation of bacterial activities in uncontaminated zones, - setting up the risk level that is acceptable by the public. Besides, it should be pointed out that effects of augmenting of soil with laboratory-modified microorganisms could be visible in the environment after a long time. For that reason the use of microorganisms modified genetically should be limited rather to technologies based on ex situ methods, where it is possible to fully control the entire process. The ENA is an attractive approach of cleaning up oil hydrocarbons contaminated soils, because it is environmentally friendly, cost-effective, and may lead to the complete destruction of contaminants. The real advantage of ENA is the fact that it can be used both as a post-treatment, and as an independent remediation method. The goal of this work was to analyze the enhancement of biodegradation in soil contaminated with oil hydrocarbons at the former military airport in Kluczewo. The effect of supplying additional bacterial consortia (bioaugmentation) on the rates of oil hydrocarbons biodegradation in soil was studied.
2. Materials and methods Soil samples Soils samples contaminated with oil hydrocarbons originated from Baracska, Hungary (soil I) and the former military airport in Kluczewo, Poland (soil II). Soil I was collected at the depths of 2.0-2.5 m and 3.0-3.5 m, and soil II at 1.5 m (G1) and 2.0 m (G2). Intrinsic and enhanced biodegradation rates were evaluated for soil II. Soil I was used only to isolate non-indigenous (extraneous) microorganisms capable for hydrocarbons biodegradation. As a control 1, uncontaminated soil samples were collected at the depths of 1.5 and 2.0 m (G1R and G2R). As a control 2, contaminated soil samples were used (G1 and G2).
Bioaugmentation for biodegradation of oil hydrocarbons in soil
Soil characteristics Soil pH and Eh were determined according to PN-ISO 10390:1997, soil moisture - PN-ISO 11465:1999, organic matter (OM) content - PN78/C-04541. Contents of aromatic hydrocarbons in soil were determined using a headspace method and capillary gas chromatography with the mass detector (GC-MS). The Shimadzu GC-17 gas chromatograph was used coupled to the Shimadzu QP 5000 mass spectrometer. Samples (wet) were analysed using an internal standard for marked compounds. Quantitative analyses were made for ionic flows that are specific for aromatic hydrocarbons.
Microbial tests Number of bacteria A general number of bacteria in 1 g of soil were determined by the Koch's plague method that relies on executing of dilution series of soil suspension, and inoculating stable volumes of the dilutions on the solid medium (agar). The dilution series of soil suspension were made with sterile water. Bacteria were incubated at 28oC for 48 hours. Isolation of bacteria capable for hydrocarbons degradation Bacterial strains were isolated from soil I and II by the Koch's plague method that were capable to grow on the solid medium composed of: yeast extract - 2.5 g, agar - 15.0 g, trypthon - 5.0 g, K2HPO4 - 1.0 g, MgSO4 - 0.5 g, aromatic hydrocarbons - 5.0 cm3, demi water - 750 cm3, tap water - 250 cm3. The liquid media to isolate bacteria from soil I and II differed in contents of aromatic hydrocarbons. The composition was established with regard to three predominant compounds found in soil I and II. The liquid medium for isolating of bacteria from soil I was composed of (weight basis): benzene - 50%, toluene - 35%, xylene 15%, and from soil II: benzene - 10%, toluene 10%, xylene - 80%. Soil samples from all the depths tested were used, and tests were made on 1g soil in duplicate. Isolated bacterial strains were transferred and stored on solid medium (agar) plates Cultivation of bacteria Isolated bacterial strains were transferred from agar plates to a liquid medium composed of: yeast extract - 4.0 g, K2HPO4 - 1.0 g, MgSO4 - 0.5 g, aromatic hydrocarbons - 5.0 cm3, demi water 750 cm3, tap water - 250 cm3. The composition of aromatic hydrocarbons was established in a similar way as it was in the
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medium used for isolation of bacteria. Bacteria were grown as a consortium (several strains in one liquid medium) without identifying the strains. The aerobic bacteria were grown in flasks of 500 cm3 with aeration by mechanical mixing. The separation of bacterial suspension from the liquid medium was achieved by centrifuging. The concentrations of bacterial consortium (numbers of cells in 1 cm3 of a suspension) were checked using the Thom's chamber (Wójcik-Szwedziñska et al. 2000).
Respirometry tests Biodegradation of oil hydrocarbons under aerobic conditions was analysed using the 10chamber Micro-Oxymax respirometer (Columbus Instruments, Ohio, USA) in conjunction with an Ultra IBM-compatible computer to collect and record all the data obtained. This is a closed-circuit system capable of measuring on-line the oxygen consumed and carbon dioxide produced during hydrocarbons biodegradation. Two experiments were carried out, in which intrinsic biodegradation of oil hydrocarbons in soil II was enhanced. In the first experiment, biodegradation was enhanced using bacterial strains isolated from soil I (extraneous bacteria), and the liquid medium used to cultivate them. In the second experiment, biodegradation was enhanced using bacterial strains isolated from soil II (indigenous bacteria) and the liquid medium used to cultivate them. The experiments were carried out for soil samples G1 and G2. As a control, contaminated soil samples G1 and G2 without enhancement (control 2), as well as uncontaminated soil samples G1R and G2R (control 1) were used. Analyses of intrinsic and enhanced biodegradation were carried out in batch tests at a temperature range of 27-34oC (optimum for investigated microorganisms). Soil samples of 30 g were placed into 500 cm3 chambers, to which 8 cm3 of bacterial consortium containing 2.4×1015 Colony Forming Units (CFU) cm-3, or 4.8×1015 CFU cm-3, or 8 cm3 of the liquid medium only were added. All tests were done in triplicate. Biodegradation of oil hydrocarbons was evaluated based on the cumulative curves of O2 consumption and CO2 production. The mean rates of O2 uptake and CO2 production were calculated from the linear regressions of the cumulative curves.
3. Results and discussion The physico-chemical characteristics of soil and a general number of psychrophilic bacteria are presented in Table I. The determined soil physicalchemicals parameters are considered optimal for biodegradation of oil hydrocarbons under aerobic
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Table I. Soil samples characteristics and numbers of bacteria Sample symbol G1R G1 G2R G2
Sample description not contaminated (depth 1.5m) contaminated (depth 1.5m) not contaminated (depth 2.0m) contaminated (depth 2.0m)
pH
Eh [mV]
Moisture [%]
OM content [%]
Number of bacteria (CFU /1 g soil)
6.07
446.78
26.71
3.92
1×105
7.38
450.78
18.18
2.76
20×105
8.13
449.22
13.07
1.99
3×105
7.59
456.78
19.23
3.20
10×105
conditions (Karkulski, Zieñko 1997; Malina 1999; Ko³wzan 2000). The sufficient number of microorganisms for effective biodegradation was also present. Soil I and II were mainly contaminated with BTX hydrocarbons (benzene, toluene and xylene). Soil I contained these compounds in the following proportion: benzene - 50%, toluene - 35%, xylene 15%, soil II: benzene - 10%, toluene - 10%, xylene - 80%.
The cumulative curves of O2 consumption and CO2 production during biodegradation of oil hydrocarbons in soil are presented in Fig. 1. The coefficients of the linear regression equations (Fig. 2) are the mean rates of O2 uptake and CO2 production during natural and enhanced biodegradation. The biodegradation rates estimated as the O2 uptake and CO2 production rates were the lowest in uncontaminated soil samples. Higher values of mean rates of O2 uptake and CO2 production in
25000
G1 (control 2) 15 -3 2B 2.4 10 CFU cm 15 -3 CFU cm 2C 4.8 10
cumulative O 2 (mm 3 )
20000
y = 331.73x R2 = 0.9707
15000 y = 296.35x R2 = 0.9625
10000 5000
y = 113.05x R2 = 0.873
0
cumulative CO 2 (mm 3 )
20000 y = 285.48x R2 = 0.9789
15000
y = 255,19x R2 = 0,9714
10000
y = 62.723x R2 = 0.8998
5000
0 0
9
18
27
36
45
54
63
72
81
90
time (h)
Fig. 1. Cumulative curves of O2 uptake and CO2 production during biodegradation tests. Notation: G1 - control 2 (contaminated soil without enhancement - depth of 1.5 m), 2B - indigenous bacterial consortium (2.4 1015 CFU cm-3), 2C - indigenous bacterial consortium (4.8 1015 CFU cm-3).
167
Bioaugmentation for biodegradation of oil hydrocarbons in soil
mean rates of O2 uptake and CO2 production, mm 3 min -1
6 oxygen uptake
5
carbon dioxide production
4 3 2 1 0
indigenous
extraneous
indigenous
indigenous
extraneous
extraneous
no enhancement
liquid bacteria bacteria medium 2.4 105 4.8 105 CFU CFU cm-3 cm-3 depth 1.5 m
G2R G2 1A 2A 1B 2B 1C 2C
uncontaminated
indigenous
extraneous
indigenous
extraneous
indigenous
extraneous
no enhancement
uncontaminated
G1R G1 1A 2A 1B 2B 1C 2C
liquid bacteria bacteria medium 2.4 105 4.8 105 CFU CFU cm-3 cm-3 depth 2.0 m Fig. 2. Mean rates of O2 uptake and CO2 production during intrinsic and enhanced biodegradation. Notation: G1R, G2R - control 1 (uncontaminated soil - depths of 1.5 and 2.0 m), G1, G2 - control 2 (contaminated soil without enhancement - depths of 1.5 and 2.0 m), 1A/2A - liquid medium used to cultivate extraneous/indigenous bacteria, 1B/2B - extraneous/indigenous bacterial consortium (2.4 1015CFU cm-3), 1C/2C - extraneous/indigenous bacterial consortium (4.8 1015CFU cm-3).
contaminated soil samples compared to uncontaminated soil samples indicated aerobic biodegradation of oil hydrocarbons. However, the intrinsic biodegradation rates in contaminated soil samples were considerably lower than in samples, where the bacterial consortia or liquid medium were added. The enhancement of biodegradation using the liquid medium resulted in increased rates of O2 uptake and CO2 production. Providing microorganism with micronutrients and additional source of carbon caused augmenting of microbial population and improved biodegradation. The higher efficiency of biodegradation as a result of bioaugmentation indicated that number and activity of oil-degrading bacteria, but not bioavailability of contaminant, were limiting factors for biodegradation. The highest biodegradation rates were observed in samples with bacterial consortium containing 4.8 1015 CFU cm-3. On an average, in all performed tests, enhanced biodegradation rates were four times higher than natural biodegradation rates. New bioaugmentation approaches described in the literature, as well as the selection and growing of microorganisms directly from the local site have shown to give the best results (Devinny, Chang 2000). Capelli et al. (2001) reported a decrease in Total Petroleum Hydrocarbons (TPH) of almost 70% in laboratory studies with soils inoculated with pre-selected bacteria. Bento et al.
(2005) noted that the addition of a bacterial consortium previously isolated from the Long Beach soil degraded 73-75% of the light (C12-C23) and heavy (C23-C40) fractions of the TPH present in the soil as a result of diesel oil contamination, but had no effect in the soil from Hong Kong. In our study, application of indigenous bacterial consortium was also more efficient in comparison to extraneous bacterial consortium (Fig. 3). When the indigenous bacterial consortium was applied, the increase of O2 uptake and CO2 production rates were about 2246% higher compared to extraneous bacterial consortium. This could be explained by the autochthonous adaptation that allows microorgani-sms to be physiologically compatible with their habitat, as compared to transient allochthonous organisms that do not occupy a functional niche (Atlas, Bartha 1998). Thus, the best bioaugmentation performance can be approached by the use of multiplied autochthonous microorganisms to increase their abundance in the soil. Taking into account that further doubling of microorganisms resulted in a subsequent increase of biodegradation rates; it may suggest that bioavailable contaminants still remained in studied soil samples. Therefore, for designing optimum bioaugmentation it is necessary to evaluate the fractions of bioavailable contaminants to determine the required concentration of degrading inoculum to be added.
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mean rates of O2 uptake and CO2 production, mm 3 min -1
6
oxygen uptake
5
carbon dioxide production
4 3 2 1 0 1B
2B
extraneous
indigenous 15
bacteria 2.4 10
CFU cm- 3
1C extraneous
2C indigenous
bacteria 4.8 1015 CFU cm- 3
Fig. 3. The effect of extraneous vs. indigenous bacteria application on biodegradation of oil hydrocarbons in soil sample G1. Notation: 1B/2B - extraneous/indigenous bacterial consortium (2.4 1015 CFU cm-3), 1C/2C - extraneous/indigenous bacterial consortium (4.8 1015 CFU cm-3).
Conclusion Bioaugmentation can be considered as an independent and effective method for enhancing biodegradation of oil hydrocarbons in soil. Higher biodegradation rates were achieved when inoculum contained bacterial consortium of 4.8 1015 CFU cm-3 than the concentration was 2.4 1015 CFU cm-3. Generally, in all cases, enhanced biodegradation rates were four times higher than intrinsic biodegradation rates. Application of indigenous bacterial consortia was more efficient in comparison to the extraneous bacterial consortia. Native microorganisms are well adapted to their environment, and a rapid growth of population guarantees better biodegradation.
4. References Atlas, R.M., Bartha, R., 1998. Microbial Ecology: Fundamentals and Applications. Benjamin/Cummings Publishing, Menlo Park, CA. Bento, F.M., Camargo, F.A.O., Okeke B.C., Frankenberger W.T., 2005. Comparative bioremediation of soils contaminated with diesel oil by natural attenuation, biostimulation and bioaugmentation. Bioresource Technology 96, 1049-1055. Capelli, S.M., Busalmen, J.P., Sanchez, S.R., 2001. Hydrocarbon bioremediation of a mineral-base contaminated waste from crude oil extraction by indigenous bacteria. Int. Biodet. Biod. 47, 233-238
Coulon, F., Delille, D. 2003. Effects of Biostimulation on Growth of Indigenous Bacteria in Sub-Antarctic Soil Contaminated with Oil Hydrocarbons. Oil and Gas Science and Technology - Rev. IFP. 58 (4), 469-479. Deska, I., Malina, G., Rad³o, A. 2003. Wp³yw uziarnienia gruntu oraz paliwa na ró¿nicê miêdzy pozorn¹ i rzeczywist¹ LNAPL na zwierciadle wody podziemnej. [The influence of Soil and Fuel Properties on the Difference between Apparent and Actual LNAPL Thickness on the Groundwater Table]. In¿ynieria i Ochrona Œrodowiska 6(2), 203-220 [Eng summ.]. Devinny, J., Chang, S.H., 2000. Bioaugmentation for soil bioremediation. In: Wise, D.L., Trantolo, D.J. [Eds], Bioremediation of Contaminated Soils. Marcel Dekker, New York, str. 465-488. Fantroussi, S.E., Agathos, S.N. 2005. Is bioaugmentation a feasible strategy for pollutant removal and site remediation? Current Opinion in Microbiology 8, 268-275. Gaca, J. 2000. Biodegradacja olejów i produktów naftowych i ich wp³yw na œrodowisko, Wp³yw zanieczyszczeñ naftowych i chemicznych na œrodowisko przyrodnicze. [Biodegradation of Oil and Petroleum products and their Influence on environment]. In: Wp³yw zanieczyszczeñ naftowych i chemicznych na œrodowisko przyrodnicze VII Miêdzynarodowe Sympozjum Szkoleniowe, PZITS, Pi³a, pp. 1-8 [Eng summ.]. Ivshina, B.I., Kuyukina, M.S., Ritchkova, M.I., Philp, J.C., Cunningham, C.J., Christofi, N. 2001. Oleophilic Biofertilizer Based on a Rhodococcus Surfactant Complex for the Bioremediation of Crude Oil-Contaminated Soil. Contaminated Soil, Sediment and Water 2001, 20-24.
Bioaugmentation for biodegradation of oil hydrocarbons in soil Karkulski, K., Zieñko, J. 1997. Substancje ropopochodne w œrodowisku przyrodniczym. [Petroleum substances in natural environment] Wydawnictwo Uczelniane Politechniki Szczeciñskiej, Szczecin, 1997. Ko³wzan, B. 2000. Biodegradacja produktów naftowych. [Biodegradation of petroleum products] In: Zanieczyszczenia naftowe w gruncie. Oficyna Wydawnicza Politechniki Wroc³awskiej, Wroc³aw, 207-235. Koœcielniak, S., Steininger, M. 2001. Dopuszczalne stê¿enia zanieczyszczeñ gruntów substancjami chemicznymi (propozycje zmian). [Permissible concentrations of chemical pollutants in soils (proposal of changes)] In: Wp³yw zanieczyszczeñ naftowych i chemicznych na œrodowisko przyrodnicze. Materia³y szkoleniowe cz. 1.,PZITS, Poznañ, str. 11-17. Malina, G. 1999. Biowentylacja strefy aeracji zanieczyszczonej substancjami ropopochodnymi. [The bioventing of unsaturated zone contaminated with oil compounds] Monografia 69. Wydawnictwo Poliechniki Czêstochowskiej, Czêstochowa. Malina, G., Derda, A., Derda, M. 1999. Ocena zagro¿enia u¿ytkowych zbiorników wód podziemnych substancjami ropopochodnymi w rejonie Czêstochowy. [Risk Assessment from Oil Compounds to Grounwater Reservoirs in Czêstochowa Region] In¿ynieria i Ochrona Œrodowiska 2 (3-4), 279-295 [Eng summ.]. Marquez-Rocha, J.F., Hernandez-Rodriguez, V., Lamela, T.M. 2001. Biodegradation of Diesel Oil in Soil by a Microbial Consortium. Water, Air and Soil Pollution, 128, 313-320. Odokuma, L.O., Dickson, A.A. 2003. Bioremediation of a crude oil polluted tropical rain forest soil. Global Journal of Environmental Sciences 2 (1), 29-40. PN-ISO 10390:1997 Jakoœæ gleby. Oznaczanie pH. [Soil quality. Determination of pH]. PN-ISO 11465:1999 Jakoœæ gleby. Oznaczanie zawartoœci suchej masy gleby i wody w glebie w przeliczeniu na such¹ masê gleby. Metoda wagowa. [Soil quality. Determination of soil dry matter and water content on a dry matter basis. The gravimetric method]. PN-78/C-04541 Woda i œcieki. Oznaczanie suchej pozosta³oœci, pozosta³oœci po pra¿eniu, straty przy
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pra¿eniu oraz substancji rozpuszczonych i lotnych. [Water and waste water. Determination of solid residua, residua after ignition, ignition loss, dissolved and volatile substances]. Ró¿añski, H. 1999. Zaburzenia sk³adu chemicznego roœlin leczniczych, pastwiskowych i warzywnych pochodz¹cych z gleb i wód zanieczyszczonych rop¹ naftow¹ oraz produktami ropopochodnymi. [The disorders of chemical composition of remedial, pasture and vegetable plants originating from soils contaminated with petroleum compounds] In: Techniczne i spo³eczne aspekty gospodarki odpadami. III Miêdzynarodowe Forum Gospodarki Odpadami, PZITS Poznañ, str. 365-373 [Eng summ.]. Siuta, J. 2000. Podstawy biodegradacji ropopochodnych sk³adników w glebach i w odpadach.[Fundamentals biodegradation of petroleum compounds in soils and waste]. In¿ynieria Ekologiczna 2, 23-30. USEPA. 1999. Use of Monitored Natural Attenuation at Superfund, RCRA Corrective Action, and Underground Storage Tank Sites. OSWER Directive 9200.4-17P,1999. US Environmental Protection Agency, Washington DC: Office of Solid Waste and Emergency Response. USEPA Science Advisory Board. 2001. Monitored natural attenuation; US Environmental Protection Agency Research Programme-an EPA Science Advisory Board Review. Washington DC: Science Advisory Board. Vogel, T.M. 1996. Bioaugmentation as a soil bioremediation approach. Current Opinion in Biotechnology, 7(3), 311-316. Wójcik - Szwedziñska M., Nowak D., Stañczyk Mazanek E., 2000. Elementy biologii sanitarnej. [The elements of sanitary biology]. Wydawnictwo Poliechniki Czêstochowskiej, Czêstochowa. Zalewski, M. 2007 in press. Ecohydrology as a concept and management tool. In: King, C., Ramkissoon, J., Clüsener-Godt, M., Zafar Adeel, Z. [Eds] Managing Water in Diverse Ecosystems to Ensure Human Wellbeing. United Nations University - International Network on Water, Environment and Health (UNUINWEH), Hamilton. pp. 39-50.
Bioaugmentation as a method of biodegradation enhancement in oil hydrocarbons contaminated soil
Ecohydrology for Implementation of the European Water Framework Directive
Iwona Zawierucha, Grzegorz Malina Institute of Environmental Engineering, Czêstochowa University of Technology, BrzeŸnicka 60A, 42-200 Czêstochowa, Poland, e-mail: [email protected]
Abstract Respirometry studies using the 10-chamber Micro-Oxymax respirometer (Columbus, Ohio) were conducted to determine the effect of adding bacterial consortium for enhancing biodegradation in soils contaminated with oil hydrocarbons from the former military airport. Bioaugmentation was carried out using both indigenous and extraneous bacteria able to degrade hydrocarbons. The potentials of intrinsic and enhanced biodegradation were evaluated by the mean O2 uptake and CO2 production rates. The results demonstrated that the highest biodegradation rates were achieved using bacterial consortium containing 4.8×1015 Colony Forming Units (CFU) cm-3. Generally, in all cases, enhanced biodegradation rates were four times higher than intrinsic biodegradation rates. Moreover, application of indigenous bacterial consortia was more efficient in comparison to the employment of extraneous bacterial consortia. This study showed that bioaugmentation could be an effective method for enhancing intrinsic biodegradation of oil hydrocarbons in soil. Key words: intrinsic biodegradation, indigenous microorganisms, extraneous bacteria.
1. Introduction Dynamic development of transportation causes that more of petroleum substances may penetrate natural environment, resulting in degradation of soils, surface water and groundwater (Deska et al. 2003). Nearly 2.5 million tons of oil products is estimated to enter the environment yearly (Gaca 2000). Since late 70-ties intensive studies have been carried out in Poland to estimate the degree of soil and groundwater contamination. A number of locations have been identified and documented with very high contamination of oil hydrocarbons (Koœcielniak, Steininger 2001). Oil-hydrocarbon contaminants may appear locally and periodically as a consequence of leaks of crude oil from mining shafts,
damaged pipelines, cisterns, tankers, and storage and distribution stations of petroleum products. They can also appear permanently on definite areas and derive from refineries or engine industry (Malina et al. 1999). Moreover, floods are a serious factor of contamination spreading in the environment (Ró¿añski 1999). Oil products in soils affect vegetation, threaten quality of surface water and groundwater, and are also potentially hazardous for humans and the environment (Gaca 2000). According to the ecohydrology concept, elimination of threats without consideration of biological processes, cannot lead to any success. Thus, ecohydrology incorporates the use of ecosystem properties as a management tool in implementing a program of water resource man-
164
I. Zawierucha, G. Malina
agement (Zalewski 2007 in press.). In the face of that, soil remediation technologies based on natural attenuation (NA) are closely connected with ecohydrology concept, because they use biodegradation with the participation of indigenous microorganisms to prevent migration of contaminants, and consequently to protect groundwater from pollution. According to the US Environmental Protection Agency (EPA 1999), 'natural attenuation' is the use of natural processes to contain the spread of the contamination from chemical spills and reduce the concentration and amount of pollutants at contaminated sites. It is also known as intrinsic (bio)remediation, and (bio)attenuation. In this approach, the contaminants are left on site, and the naturally occurring processes are responsible for clean-up of the site. The NA includes physical, chemical and biological processes. Biochemical processes, especially biodegradation with participation of microorganisms, play the most important role in these natural self-purification processes. Biodegradation is the breakdown of organic contaminants by microbial organisms into smaller compounds. The microbial organisms transform the contaminants through metabolic or enzymatic processes. Biodegradation processes vary greatly, but frequently under aerobic conditions the final products are: carbon dioxide, water and biomass (Malina 1999). The activity of native microflora decides about efficiency of biodegradation, and it can be enhanced by providing optimal conditions for the microbial growth. Thus, Enhanced Natural Attenuation (ENA) is commonly achieved by supplying nutrients, oxygen (or other electron acceptors) and/or water to contaminated soils (biostimulation), and/or inoculating soil with oil-degrading bacteria to supplement the existing microbial population (bioaugmentation or seeding) (Coulon, Delille 2003; Ivshina et al. 2001; Odokuma, Dickson 2003; Ko³wzan 2000; Siuta 2000; www.epa.gov). The cheapest method of intrinsic biodegradation enhancement is inoculating soils with microorganisms. They can be either isolated, adapted and multiplied autochthonous microflora, or genetically modified microorganisms (Ko³wzan 2000; Marquez-Rocha et al. 2001). The potential success of bioaugmentation depends on bioavailability of contaminants, the activity of added microorganisms and soil physical-chemical characteristics (Vogel 1996). Multiplied autochthonous microflora is generally applied in remediation technologies but inoculating of soil with laboratory modified bacterial cultures still arouses many reservations. Genetically modified microorganisms should not only degrade hydrocarbons with high efficiency, but they should also be capable of quick adaptation
and development in new environments (Fantroussi, Agathos 2005). They ought to be neutral in the presence of autochthonous microflora and should not produce any toxic substances (metabolites) during biodegradation. There are a number of arguable problems related to inoculating of soils with microorganisms modified genetically, including: - not well-known, defined and predictable side effects of inoculation that may affect the environment and humans, - effective monitoring of microorganisms distribution in soil, - survival of laboratory bacteria under field conditions, - regulation of bacterial activities in uncontaminated zones, - setting up the risk level that is acceptable by the public. Besides, it should be pointed out that effects of augmenting of soil with laboratory-modified microorganisms could be visible in the environment after a long time. For that reason the use of microorganisms modified genetically should be limited rather to technologies based on ex situ methods, where it is possible to fully control the entire process. The ENA is an attractive approach of cleaning up oil hydrocarbons contaminated soils, because it is environmentally friendly, cost-effective, and may lead to the complete destruction of contaminants. The real advantage of ENA is the fact that it can be used both as a post-treatment, and as an independent remediation method. The goal of this work was to analyze the enhancement of biodegradation in soil contaminated with oil hydrocarbons at the former military airport in Kluczewo. The effect of supplying additional bacterial consortia (bioaugmentation) on the rates of oil hydrocarbons biodegradation in soil was studied.
2. Materials and methods Soil samples Soils samples contaminated with oil hydrocarbons originated from Baracska, Hungary (soil I) and the former military airport in Kluczewo, Poland (soil II). Soil I was collected at the depths of 2.0-2.5 m and 3.0-3.5 m, and soil II at 1.5 m (G1) and 2.0 m (G2). Intrinsic and enhanced biodegradation rates were evaluated for soil II. Soil I was used only to isolate non-indigenous (extraneous) microorganisms capable for hydrocarbons biodegradation. As a control 1, uncontaminated soil samples were collected at the depths of 1.5 and 2.0 m (G1R and G2R). As a control 2, contaminated soil samples were used (G1 and G2).
Bioaugmentation for biodegradation of oil hydrocarbons in soil
Soil characteristics Soil pH and Eh were determined according to PN-ISO 10390:1997, soil moisture - PN-ISO 11465:1999, organic matter (OM) content - PN78/C-04541. Contents of aromatic hydrocarbons in soil were determined using a headspace method and capillary gas chromatography with the mass detector (GC-MS). The Shimadzu GC-17 gas chromatograph was used coupled to the Shimadzu QP 5000 mass spectrometer. Samples (wet) were analysed using an internal standard for marked compounds. Quantitative analyses were made for ionic flows that are specific for aromatic hydrocarbons.
Microbial tests Number of bacteria A general number of bacteria in 1 g of soil were determined by the Koch's plague method that relies on executing of dilution series of soil suspension, and inoculating stable volumes of the dilutions on the solid medium (agar). The dilution series of soil suspension were made with sterile water. Bacteria were incubated at 28oC for 48 hours. Isolation of bacteria capable for hydrocarbons degradation Bacterial strains were isolated from soil I and II by the Koch's plague method that were capable to grow on the solid medium composed of: yeast extract - 2.5 g, agar - 15.0 g, trypthon - 5.0 g, K2HPO4 - 1.0 g, MgSO4 - 0.5 g, aromatic hydrocarbons - 5.0 cm3, demi water - 750 cm3, tap water - 250 cm3. The liquid media to isolate bacteria from soil I and II differed in contents of aromatic hydrocarbons. The composition was established with regard to three predominant compounds found in soil I and II. The liquid medium for isolating of bacteria from soil I was composed of (weight basis): benzene - 50%, toluene - 35%, xylene 15%, and from soil II: benzene - 10%, toluene 10%, xylene - 80%. Soil samples from all the depths tested were used, and tests were made on 1g soil in duplicate. Isolated bacterial strains were transferred and stored on solid medium (agar) plates Cultivation of bacteria Isolated bacterial strains were transferred from agar plates to a liquid medium composed of: yeast extract - 4.0 g, K2HPO4 - 1.0 g, MgSO4 - 0.5 g, aromatic hydrocarbons - 5.0 cm3, demi water 750 cm3, tap water - 250 cm3. The composition of aromatic hydrocarbons was established in a similar way as it was in the
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medium used for isolation of bacteria. Bacteria were grown as a consortium (several strains in one liquid medium) without identifying the strains. The aerobic bacteria were grown in flasks of 500 cm3 with aeration by mechanical mixing. The separation of bacterial suspension from the liquid medium was achieved by centrifuging. The concentrations of bacterial consortium (numbers of cells in 1 cm3 of a suspension) were checked using the Thom's chamber (Wójcik-Szwedziñska et al. 2000).
Respirometry tests Biodegradation of oil hydrocarbons under aerobic conditions was analysed using the 10chamber Micro-Oxymax respirometer (Columbus Instruments, Ohio, USA) in conjunction with an Ultra IBM-compatible computer to collect and record all the data obtained. This is a closed-circuit system capable of measuring on-line the oxygen consumed and carbon dioxide produced during hydrocarbons biodegradation. Two experiments were carried out, in which intrinsic biodegradation of oil hydrocarbons in soil II was enhanced. In the first experiment, biodegradation was enhanced using bacterial strains isolated from soil I (extraneous bacteria), and the liquid medium used to cultivate them. In the second experiment, biodegradation was enhanced using bacterial strains isolated from soil II (indigenous bacteria) and the liquid medium used to cultivate them. The experiments were carried out for soil samples G1 and G2. As a control, contaminated soil samples G1 and G2 without enhancement (control 2), as well as uncontaminated soil samples G1R and G2R (control 1) were used. Analyses of intrinsic and enhanced biodegradation were carried out in batch tests at a temperature range of 27-34oC (optimum for investigated microorganisms). Soil samples of 30 g were placed into 500 cm3 chambers, to which 8 cm3 of bacterial consortium containing 2.4×1015 Colony Forming Units (CFU) cm-3, or 4.8×1015 CFU cm-3, or 8 cm3 of the liquid medium only were added. All tests were done in triplicate. Biodegradation of oil hydrocarbons was evaluated based on the cumulative curves of O2 consumption and CO2 production. The mean rates of O2 uptake and CO2 production were calculated from the linear regressions of the cumulative curves.
3. Results and discussion The physico-chemical characteristics of soil and a general number of psychrophilic bacteria are presented in Table I. The determined soil physicalchemicals parameters are considered optimal for biodegradation of oil hydrocarbons under aerobic
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I. Zawierucha, G. Malina
Table I. Soil samples characteristics and numbers of bacteria Sample symbol G1R G1 G2R G2
Sample description not contaminated (depth 1.5m) contaminated (depth 1.5m) not contaminated (depth 2.0m) contaminated (depth 2.0m)
pH
Eh [mV]
Moisture [%]
OM content [%]
Number of bacteria (CFU /1 g soil)
6.07
446.78
26.71
3.92
1×105
7.38
450.78
18.18
2.76
20×105
8.13
449.22
13.07
1.99
3×105
7.59
456.78
19.23
3.20
10×105
conditions (Karkulski, Zieñko 1997; Malina 1999; Ko³wzan 2000). The sufficient number of microorganisms for effective biodegradation was also present. Soil I and II were mainly contaminated with BTX hydrocarbons (benzene, toluene and xylene). Soil I contained these compounds in the following proportion: benzene - 50%, toluene - 35%, xylene 15%, soil II: benzene - 10%, toluene - 10%, xylene - 80%.
The cumulative curves of O2 consumption and CO2 production during biodegradation of oil hydrocarbons in soil are presented in Fig. 1. The coefficients of the linear regression equations (Fig. 2) are the mean rates of O2 uptake and CO2 production during natural and enhanced biodegradation. The biodegradation rates estimated as the O2 uptake and CO2 production rates were the lowest in uncontaminated soil samples. Higher values of mean rates of O2 uptake and CO2 production in
25000
G1 (control 2) 15 -3 2B 2.4 10 CFU cm 15 -3 CFU cm 2C 4.8 10
cumulative O 2 (mm 3 )
20000
y = 331.73x R2 = 0.9707
15000 y = 296.35x R2 = 0.9625
10000 5000
y = 113.05x R2 = 0.873
0
cumulative CO 2 (mm 3 )
20000 y = 285.48x R2 = 0.9789
15000
y = 255,19x R2 = 0,9714
10000
y = 62.723x R2 = 0.8998
5000
0 0
9
18
27
36
45
54
63
72
81
90
time (h)
Fig. 1. Cumulative curves of O2 uptake and CO2 production during biodegradation tests. Notation: G1 - control 2 (contaminated soil without enhancement - depth of 1.5 m), 2B - indigenous bacterial consortium (2.4 1015 CFU cm-3), 2C - indigenous bacterial consortium (4.8 1015 CFU cm-3).
167
Bioaugmentation for biodegradation of oil hydrocarbons in soil
mean rates of O2 uptake and CO2 production, mm 3 min -1
6 oxygen uptake
5
carbon dioxide production
4 3 2 1 0
indigenous
extraneous
indigenous
indigenous
extraneous
extraneous
no enhancement
liquid bacteria bacteria medium 2.4 105 4.8 105 CFU CFU cm-3 cm-3 depth 1.5 m
G2R G2 1A 2A 1B 2B 1C 2C
uncontaminated
indigenous
extraneous
indigenous
extraneous
indigenous
extraneous
no enhancement
uncontaminated
G1R G1 1A 2A 1B 2B 1C 2C
liquid bacteria bacteria medium 2.4 105 4.8 105 CFU CFU cm-3 cm-3 depth 2.0 m Fig. 2. Mean rates of O2 uptake and CO2 production during intrinsic and enhanced biodegradation. Notation: G1R, G2R - control 1 (uncontaminated soil - depths of 1.5 and 2.0 m), G1, G2 - control 2 (contaminated soil without enhancement - depths of 1.5 and 2.0 m), 1A/2A - liquid medium used to cultivate extraneous/indigenous bacteria, 1B/2B - extraneous/indigenous bacterial consortium (2.4 1015CFU cm-3), 1C/2C - extraneous/indigenous bacterial consortium (4.8 1015CFU cm-3).
contaminated soil samples compared to uncontaminated soil samples indicated aerobic biodegradation of oil hydrocarbons. However, the intrinsic biodegradation rates in contaminated soil samples were considerably lower than in samples, where the bacterial consortia or liquid medium were added. The enhancement of biodegradation using the liquid medium resulted in increased rates of O2 uptake and CO2 production. Providing microorganism with micronutrients and additional source of carbon caused augmenting of microbial population and improved biodegradation. The higher efficiency of biodegradation as a result of bioaugmentation indicated that number and activity of oil-degrading bacteria, but not bioavailability of contaminant, were limiting factors for biodegradation. The highest biodegradation rates were observed in samples with bacterial consortium containing 4.8 1015 CFU cm-3. On an average, in all performed tests, enhanced biodegradation rates were four times higher than natural biodegradation rates. New bioaugmentation approaches described in the literature, as well as the selection and growing of microorganisms directly from the local site have shown to give the best results (Devinny, Chang 2000). Capelli et al. (2001) reported a decrease in Total Petroleum Hydrocarbons (TPH) of almost 70% in laboratory studies with soils inoculated with pre-selected bacteria. Bento et al.
(2005) noted that the addition of a bacterial consortium previously isolated from the Long Beach soil degraded 73-75% of the light (C12-C23) and heavy (C23-C40) fractions of the TPH present in the soil as a result of diesel oil contamination, but had no effect in the soil from Hong Kong. In our study, application of indigenous bacterial consortium was also more efficient in comparison to extraneous bacterial consortium (Fig. 3). When the indigenous bacterial consortium was applied, the increase of O2 uptake and CO2 production rates were about 2246% higher compared to extraneous bacterial consortium. This could be explained by the autochthonous adaptation that allows microorgani-sms to be physiologically compatible with their habitat, as compared to transient allochthonous organisms that do not occupy a functional niche (Atlas, Bartha 1998). Thus, the best bioaugmentation performance can be approached by the use of multiplied autochthonous microorganisms to increase their abundance in the soil. Taking into account that further doubling of microorganisms resulted in a subsequent increase of biodegradation rates; it may suggest that bioavailable contaminants still remained in studied soil samples. Therefore, for designing optimum bioaugmentation it is necessary to evaluate the fractions of bioavailable contaminants to determine the required concentration of degrading inoculum to be added.
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mean rates of O2 uptake and CO2 production, mm 3 min -1
6
oxygen uptake
5
carbon dioxide production
4 3 2 1 0 1B
2B
extraneous
indigenous 15
bacteria 2.4 10
CFU cm- 3
1C extraneous
2C indigenous
bacteria 4.8 1015 CFU cm- 3
Fig. 3. The effect of extraneous vs. indigenous bacteria application on biodegradation of oil hydrocarbons in soil sample G1. Notation: 1B/2B - extraneous/indigenous bacterial consortium (2.4 1015 CFU cm-3), 1C/2C - extraneous/indigenous bacterial consortium (4.8 1015 CFU cm-3).
Conclusion Bioaugmentation can be considered as an independent and effective method for enhancing biodegradation of oil hydrocarbons in soil. Higher biodegradation rates were achieved when inoculum contained bacterial consortium of 4.8 1015 CFU cm-3 than the concentration was 2.4 1015 CFU cm-3. Generally, in all cases, enhanced biodegradation rates were four times higher than intrinsic biodegradation rates. Application of indigenous bacterial consortia was more efficient in comparison to the extraneous bacterial consortia. Native microorganisms are well adapted to their environment, and a rapid growth of population guarantees better biodegradation.
4. References Atlas, R.M., Bartha, R., 1998. Microbial Ecology: Fundamentals and Applications. Benjamin/Cummings Publishing, Menlo Park, CA. Bento, F.M., Camargo, F.A.O., Okeke B.C., Frankenberger W.T., 2005. Comparative bioremediation of soils contaminated with diesel oil by natural attenuation, biostimulation and bioaugmentation. Bioresource Technology 96, 1049-1055. Capelli, S.M., Busalmen, J.P., Sanchez, S.R., 2001. Hydrocarbon bioremediation of a mineral-base contaminated waste from crude oil extraction by indigenous bacteria. Int. Biodet. Biod. 47, 233-238
Coulon, F., Delille, D. 2003. Effects of Biostimulation on Growth of Indigenous Bacteria in Sub-Antarctic Soil Contaminated with Oil Hydrocarbons. Oil and Gas Science and Technology - Rev. IFP. 58 (4), 469-479. Deska, I., Malina, G., Rad³o, A. 2003. Wp³yw uziarnienia gruntu oraz paliwa na ró¿nicê miêdzy pozorn¹ i rzeczywist¹ LNAPL na zwierciadle wody podziemnej. [The influence of Soil and Fuel Properties on the Difference between Apparent and Actual LNAPL Thickness on the Groundwater Table]. In¿ynieria i Ochrona Œrodowiska 6(2), 203-220 [Eng summ.]. Devinny, J., Chang, S.H., 2000. Bioaugmentation for soil bioremediation. In: Wise, D.L., Trantolo, D.J. [Eds], Bioremediation of Contaminated Soils. Marcel Dekker, New York, str. 465-488. Fantroussi, S.E., Agathos, S.N. 2005. Is bioaugmentation a feasible strategy for pollutant removal and site remediation? Current Opinion in Microbiology 8, 268-275. Gaca, J. 2000. Biodegradacja olejów i produktów naftowych i ich wp³yw na œrodowisko, Wp³yw zanieczyszczeñ naftowych i chemicznych na œrodowisko przyrodnicze. [Biodegradation of Oil and Petroleum products and their Influence on environment]. In: Wp³yw zanieczyszczeñ naftowych i chemicznych na œrodowisko przyrodnicze VII Miêdzynarodowe Sympozjum Szkoleniowe, PZITS, Pi³a, pp. 1-8 [Eng summ.]. Ivshina, B.I., Kuyukina, M.S., Ritchkova, M.I., Philp, J.C., Cunningham, C.J., Christofi, N. 2001. Oleophilic Biofertilizer Based on a Rhodococcus Surfactant Complex for the Bioremediation of Crude Oil-Contaminated Soil. Contaminated Soil, Sediment and Water 2001, 20-24.
Bioaugmentation for biodegradation of oil hydrocarbons in soil Karkulski, K., Zieñko, J. 1997. Substancje ropopochodne w œrodowisku przyrodniczym. [Petroleum substances in natural environment] Wydawnictwo Uczelniane Politechniki Szczeciñskiej, Szczecin, 1997. Ko³wzan, B. 2000. Biodegradacja produktów naftowych. [Biodegradation of petroleum products] In: Zanieczyszczenia naftowe w gruncie. Oficyna Wydawnicza Politechniki Wroc³awskiej, Wroc³aw, 207-235. Koœcielniak, S., Steininger, M. 2001. Dopuszczalne stê¿enia zanieczyszczeñ gruntów substancjami chemicznymi (propozycje zmian). [Permissible concentrations of chemical pollutants in soils (proposal of changes)] In: Wp³yw zanieczyszczeñ naftowych i chemicznych na œrodowisko przyrodnicze. Materia³y szkoleniowe cz. 1.,PZITS, Poznañ, str. 11-17. Malina, G. 1999. Biowentylacja strefy aeracji zanieczyszczonej substancjami ropopochodnymi. [The bioventing of unsaturated zone contaminated with oil compounds] Monografia 69. Wydawnictwo Poliechniki Czêstochowskiej, Czêstochowa. Malina, G., Derda, A., Derda, M. 1999. Ocena zagro¿enia u¿ytkowych zbiorników wód podziemnych substancjami ropopochodnymi w rejonie Czêstochowy. [Risk Assessment from Oil Compounds to Grounwater Reservoirs in Czêstochowa Region] In¿ynieria i Ochrona Œrodowiska 2 (3-4), 279-295 [Eng summ.]. Marquez-Rocha, J.F., Hernandez-Rodriguez, V., Lamela, T.M. 2001. Biodegradation of Diesel Oil in Soil by a Microbial Consortium. Water, Air and Soil Pollution, 128, 313-320. Odokuma, L.O., Dickson, A.A. 2003. Bioremediation of a crude oil polluted tropical rain forest soil. Global Journal of Environmental Sciences 2 (1), 29-40. PN-ISO 10390:1997 Jakoœæ gleby. Oznaczanie pH. [Soil quality. Determination of pH]. PN-ISO 11465:1999 Jakoœæ gleby. Oznaczanie zawartoœci suchej masy gleby i wody w glebie w przeliczeniu na such¹ masê gleby. Metoda wagowa. [Soil quality. Determination of soil dry matter and water content on a dry matter basis. The gravimetric method]. PN-78/C-04541 Woda i œcieki. Oznaczanie suchej pozosta³oœci, pozosta³oœci po pra¿eniu, straty przy
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pra¿eniu oraz substancji rozpuszczonych i lotnych. [Water and waste water. Determination of solid residua, residua after ignition, ignition loss, dissolved and volatile substances]. Ró¿añski, H. 1999. Zaburzenia sk³adu chemicznego roœlin leczniczych, pastwiskowych i warzywnych pochodz¹cych z gleb i wód zanieczyszczonych rop¹ naftow¹ oraz produktami ropopochodnymi. [The disorders of chemical composition of remedial, pasture and vegetable plants originating from soils contaminated with petroleum compounds] In: Techniczne i spo³eczne aspekty gospodarki odpadami. III Miêdzynarodowe Forum Gospodarki Odpadami, PZITS Poznañ, str. 365-373 [Eng summ.]. Siuta, J. 2000. Podstawy biodegradacji ropopochodnych sk³adników w glebach i w odpadach.[Fundamentals biodegradation of petroleum compounds in soils and waste]. In¿ynieria Ekologiczna 2, 23-30. USEPA. 1999. Use of Monitored Natural Attenuation at Superfund, RCRA Corrective Action, and Underground Storage Tank Sites. OSWER Directive 9200.4-17P,1999. US Environmental Protection Agency, Washington DC: Office of Solid Waste and Emergency Response. USEPA Science Advisory Board. 2001. Monitored natural attenuation; US Environmental Protection Agency Research Programme-an EPA Science Advisory Board Review. Washington DC: Science Advisory Board. Vogel, T.M. 1996. Bioaugmentation as a soil bioremediation approach. Current Opinion in Biotechnology, 7(3), 311-316. Wójcik - Szwedziñska M., Nowak D., Stañczyk Mazanek E., 2000. Elementy biologii sanitarnej. [The elements of sanitary biology]. Wydawnictwo Poliechniki Czêstochowskiej, Czêstochowa. Zalewski, M. 2007 in press. Ecohydrology as a concept and management tool. In: King, C., Ramkissoon, J., Clüsener-Godt, M., Zafar Adeel, Z. [Eds] Managing Water in Diverse Ecosystems to Ensure Human Wellbeing. United Nations University - International Network on Water, Environment and Health (UNUINWEH), Hamilton. pp. 39-50.