石油污染土壤文献

Iwona Zawierucha, Grzegorz Malina

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 O 2uptake and CO 2production rates. The results demonstrated that the highest biodegradation rates were achieved using bacterial consortium containing 4.8×1015Colony 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 consor-tia was more efficient in comparison to the employment of extraneous bacterial con-sortia. 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 degra-dation 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 esti-mate the degree of soil and groundwater contami-nation. A number of locations have been identified and documented with very high con-tamination of oil hydrocarbons (Ko?cielniak,Steininger 2001). Oil-hydrocarbon contaminants may appear locally and periodically as a conse-quence 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 indus-try (Malina et al.1999). Moreover, floods are a serious factor of contamination spreading in the environment (Ró?a?ski 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-

Vol. 6

No 1- 4, 163-1692006

Bioaugmentation as a method of biodegradation enhancement in oil hydrocarbons contaminated soil

Ecohydrology for Implementation of the European Water Framework Directive

Institute of Environmental Engineering, Czêstochowa University of Technology,

Brze?nicka 60A, 42-200 Czêstochowa, Poland,

e-mail: iwona_zawierucha@o2.pl

I. Zawierucha, G. Malina

agement (Zalewski 2007 in press.). In the face of that, soil remediation technologies based on natu-ral attenuation (NA) are closely connected with ecohydrology concept, because they use biodegra-dation with the participation of indigenous microorganisms to prevent migration of contami-nants, and consequently to protect groundwater from pollution.

According to the US Environmental Protec-tion Agency (EPA1999), '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 partici-pation of microorganisms, play the most impor-tant 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 Atten-uation (ENA) is commonly achieved by supplying nutrients, oxygen (or other electron acceptors) and/or water to contaminated soils (biostimula-tion), and/or inoculating soil with oil-degrading bacteria to supplement the existing microbial pop-ulation (bioaugmentation or seeding) (Coulon, Delille 2003; Ivshina et al.2001; Odokuma, Dick-son 2003; Ko3wzan 2000; Siuta 2000; https://www.360docs.net/doc/c35456142.html,). 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 (V ogel 1996).

Multiplied autochthonous microflora is gene-rally applied in remediation technologies but inoc-ulating of soil with laboratory modified bacterial cultures still arouses many reservations. Geneti-cally 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 neu-tral 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 microorgani-sms modified genetically, including:

- not well-known, defined and predictable side effects of inoculation that may affect the envi-ronment and humans,

- effective monitoring of microorganisms distribu-tion in soil,

- survival of laboratory bacteria under field condi-tions,

- regulation of bacterial activities in uncontami-nated 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 environ-ment 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 clean-ing up oil hydrocarbons contaminated soils, because it is environmentally friendly, cost-effec-tive, 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 contami-nated with oil hydrocarbons at the former military airport in Kluczewo. The effect of supplying addi-tional 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 hydro-carbons 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 (extrane-ous) 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, contami-nated soil samples were used (G1 and G2).

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Soil characteristics

Soil pH and Eh were determined according to PN-ISO 10390:1997, soil moisture - PN-ISO 11465:1999, organic matter (OM) content - PN-78/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 QP5000 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 hydro-carbons.

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 sus-pension, 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 28o C 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 hydro-carbons - 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 hydrocar-bons. 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 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 cm3with aeration by mechanical mixing. The sep-aration of bacterial suspension from the liquid medium was achieved by centrifuging. The con-centrations of bacterial consortium (numbers of cells in 1 cm3of a suspension) were checked using the Thom's chamber (Wójcik-Szwedzi?ska et al. 2000).

Respirometry tests

Biodegradation of oil hydrocarbons under aerobic conditions was analysed using the 10-chamber 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-cir-cuit 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, biodegra-dation was enhanced using bacterial strains iso-lated 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 (indige-nous bacteria) and the liquid medium used to cul-tivate them. The experiments were carried out for soil samples G1and G2. As a control, contami-nated soil samples G1and G2without enhance-ment (control 2), as well as uncontaminated soil samples G1R and G2R(control 1) were used.

Analyses of intrinsic and enhanced biodegra-dation were carried out in batch tests at a tempera-ture range of 27-34o C (optimum for investigated microorganisms). Soil samples of 30 g were placed into 500 cm3chambers, to which 8 cm3of bacter-ial consortium containing 2.4×1015Colony Form-ing Units (CFU) cm-3, or 4.8×1015CFU cm-3, or 8 cm3of the liquid medium only were added. All tests were done in triplicate. Biodegradation of oil hydrocarbons was evaluated based on the cumula-tive curves of O2consumption and CO2produc-tion. The mean rates of O2uptake and CO2 production were calculated from the linear regres-sions 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 physical-chemicals parameters are considered optimal for biodegradation of oil hydrocarbons under aerobic

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