Kazimierz Grabas Wroclaw University of Technology, Wroclaw, Poland

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1 Review paper APPLICATION OF MICROBIOLOGY IN REMEDIATION OF ENVIRONMENT POLLUTED WITH HYDROCARBONS Adam Pawełczyk, Barbara Kołwzan, Kazimierz Grabas Wroclaw University of Technology, Wroclaw, Poland Two bioremediation methods of grounds polluted with hydrocarbons at the former military air bases have been presented. The ex situ method consisted in cleaning the ground excavated first and stored in areated windrows. The biodegradation of xenobiotics proceeded under aerobic condition when selected and cultured strains of microorganisms were applied to the ground. In situ method eliminated the pollution directly at the site where it occured, without excavating the ground. Microbiological preparation along with biogens, and air was applied through a system of piezometers inserted into ground. Both methods appeared to be effective and enabled to restore biological equilibrium at the previously contaminated area. The concentrations of pollutants met the obligatory standards set by the Ministry of Environment. The applied technique made it possible to reduce the concentration of hydrocarbons from 5,500 mg/kg to the level ranging from 18 to 163 mg/kg. Keywords: bioremediation, hydrocarbons, xhenobiotics, in-situ method, ex-situ method Introduction During last years, examples of serious devastation of environment have been revealed on the areas occupied by the Russian Military Troops in Poland. Also, a number of Polish air bases and many civil enterprises have polluted ground and water environment at areas where fuel stations were located. The pollution was caused by careless handling the petroleum by staff responsible for its storage, transportation and distribution. Ground contamination with petroleum products generates land degradation and deprives soils of any agricultural value. Moreover, it creates health hazards for both people and animals since, in most part, it toxically affects living organisms. Contamination of soils with kerosene products changes their physical properties, destroys the structure, causes particle dispersion as well as it limits filtration inducing water retention [1]. Components of kerosene are also harmful to plants and this is not only because of its direct toxicity but also due to microorganisms' activities. Hydrocarbon biodegradation in soil often causes oxygen-free conditions in the root zone. The lack of oxygen and resulting from it rise of the aggressive sulphate destroys roots of plants, including large trees which have a well developed root system. The most dangerous components of kerosene products are aromatic compounds, such as the multi-ring aromatic hydrocarbons as well as some metabolites created during the hydrocarbon decomposition. It is well known that some 26

2 hydrocarbons, such as benzene and benzo[a]pyrene show cancerous effect even in low concentrations. The above has been confirmed during the epidemiological research, which also shows that many of the petroleum's compounds play an important role in etiology of tumour disease in humans [1]. Considering the toxic effect of petroleum components, it is necessary that areas polluted are quickly subjected to rehabilitation processes. In recent years the remediation methods based on the biological methods have found a wider application. Bioremediation is a soil and water purification process, during which microorganisms (fungi and bacteria) are utilized. They carry out the degradation of harmful substances down to less or non-toxic. The microorganisms utilize the environment-polluting organic compounds as food substrate. The main role in the biological process of purification is played by the microorganisms capable of utilizing hydrocarbons as the source of carbon and energy. It has been estimated that about 100 different species of microorganisms representing 30 genera are capable of hydrocarbon decomposition [2]. The number of heterotrophic microorganisms capable of using hydrocarbons as a source for carbon and energy varies for individual soil and water ecosystems. The frequency of occurrences of such types of organisms within the soil-fungi fluctuates between 0.13 and 50% and in the case of the soil-bacteria between 6-82% in relation to the total number of microorganisms [3]. Microorganisms are capable of metabolising only the selected types of hydrocarbons, out of which a predominant number is capable of aliphatic and aromatic hydrocarbon biodegradation [4]. Moreover, only some of the mixed populations of microorganisms with a diverse enzymatic outfit have the ability to break up complex mixtures such as crude oil. Bacteria from the following genera: Achromobacter, Acinetobacter, Alcaligenes, Arthrobacter, Bacillus, Flavobacterium, Nocardia, Pseudomonas and Corynebacterium play the most important role in the process of hydrocarbon decomposition within the soil and water environments [2, 4]. Of fungi deteriorating hydrocarbons within the water environment Aureobasidium, Rhodotorula and Sporobolomyces play the crucial role, whereas within the soil environments the most common ones are Trichoderma and Mortierella. Aspergillus and Penicillium fungus which decompose isolated hydrocarbons came from both water and soil environments [3]. The speed and efficiency of the hydrocarbon break down within the environment depends on many physical-chemical and biological factors [5]. The crucial factor that affects the susceptibility to biodegradation is the chemical structure of a particular xenobiotic reflected by: presence of oxygen within the particle of hydrocarbon length of the chain degree of polymerization number of rings and structure number and length of branchings presence and the place of substitution (halogens, nitro, amino, sulfonates) 27

3 Bio-accessibility of many xenobiotics is limited by their solubility in water. Hydrocarbons are among those substrates that are not easily available due to their lipophilic character. The more water soluble hydrocarbon, the more biodegradable it is. Other physical-chemical factors affecting the biodegradation process are: the xenobiotics concentration in the contaminated environment, the presence of electron acceptor, temperature, ph, biogenic substances, water accessability etc. Biological factors affecting the course of biodegradation process include: - qualitative and quantitative composition of microorganisms that colonize the contaminated ecosystem, - their adaptive abilities in changed environmental conditions, - degradation activity in relation to particular types of xenobiotics, - biological nature of metabolites formed during the biodegradation process, - spread of bacteria predators and parasites in water and ground habitats. Experimental part Two practical examples of bioremediation methods developed by the authors and applied at former Russian air bases polluted with the petroleum products are presented in the experimental part. The main task of the remediation works was to reduce the petroleum products concentration in the contaminated ground to the level acceptable by obligatory standards. The scope of the work was: - analysing and investigating petroleum pollutants on the selected area - developing the biological process of polluted water and ground remediation - application of the selected bacteria strains and biogenic substances which stimulate petroleum products biodegradation - monitoring the fuel pollutants, biogens and bacteria bioactivity in the polluted grounds. Two remediation methods were developed. Less expensive in situ method was applied in the area where the risk of migration of remediation agents to not polluted locations was excluded. Ex situ method was used in the area where a full control of ground parameters was needed and where the risk of any secondary pollution of ground with biogens and microorganisms used could not be tolerated. In the first method, in situ, the point is that the pollution is eliminated directly at the site where it occurs, without excavating the ground. However, in the second methods the contaminated ground or waters are excavated before the actual regeneration procedures and placed on specially prepared and isolated beds. In situ techniques do not require excavation of the contaminated soils so they may be less expensive, create less dust, it is possible to treat a large volume of soil and cause less release of contaminants than by ex situ methods. But the in situ techniques are slower than ex situ, may be difficult to manage, and are most effective at sites with permeable soil. Ex situ methods are utilized where there is danger of toxic pollutants migration into the ground waters and when the process of detoxification must be conducted in a short period of time [6, 7]. As substances making up the environment's contamination at the areas subjected to the remediation procedure were composed out of many compounds a 28

4 complete soil cleanup required a special, carefully selected mixture of microorganisms. The multiplied natural autochthonous microorganisms selected from the contaminated ground were cultivated first in a microbiological laboratory. Then, inoculation of the contaminated soil was carried out after multiplication of microorganisms in a bioreactor installed next to the remediation site. The bio-preparation contained a mixture of bacteria suspended in water solution of nutrients. The following inoculation methods were applied [5]: - injections with the use of high-pressure equipment, - immobilization of microorganisms on solid supports and applied to the ground, - contaminated ground surface spraying. The ex situ method was implemented at the former Russian air base Bagicz in the North Poland. In this area a source of pollution were fuel leakages from damaged tanks and pipes (toluene, xylene, 1,3-dimethyl-p-xylene). Also, heavy ends of diesel oil were observed in the ground. As a result, 800 m 3 of the ground was contaminated by the pollutantsm which had to be neutralized. The average content of the petroleum products in the ground was about 5500 mg/kg of dry matter. Because of the recreation area located in the nearest neighbourhood at the sea side, there was necessary to develop such a remediation method that would eliminate danger of secondary pollution of the waters in this area. Fig. 1. Diagram of the windrow with the aeration lances and drainage system The assumptions accepted during elaborating the biological method were as follows: biological degradation of pollutants under the oxygen conditions using the autochthonous microflora ex situ method of remediation closed water circulation system. 29

5 The bioremediation process was carried out in three stages. I stage Selection of bioactive microorganism strains in relation to the pollutants present in the ground. The microorganisms applied were selected from the natural environment polluted by the petroleum products. The taxonomy and pathogenic as well as relatively pathogenic properties of the strains were examined. II Stage - Real bioremediation process carried out in an areated windrow reactor. The diagram of the formed windrow (pile) is shown on Fig.1. The polluted ground was displaced in the windrow equipped with a system of aeration drainage. During the process the windrow was sprayed with water and aerated to maintain optimal living conditions for bacteria. Waste water collected in a well was introduced to a stripping tower and then recirculated to the windrow. The hydrocarbon vapours were adsorbed in a charcoal filter (fig. 2.). Growth of micro flora was stimulated by - inoculation of autochthonous bacteria reproduced in a bioreactor - intensive aeration of the windrow using system of drainage and vertical air lances - supplying the ground with biogenic substances - maintaining proper humidity of the ground. III stage Displacing the ground from the windrow to the original site after completing the bioremediation process. Fig. 2. Diagram of the remediation plant: 1 windrow with treated ground, 2 spraying system, 3 isolating foil, 4- charcoal filter, 5 nozzle, 6 stripping column, 7 well, 8 ph, temperature, oxygen and biogens concentration control, 9 heater, 10 air, 11 bioreactor. The bioremediation process lasted 7 months and resulted in very good effects in spite of unfavourable properties of the ground, which was clayey and not permeable. The final level of hydrocarbons in the ground before its displacement amounted from 30

6 18 to 163 mg/kg of dry matter. Constant recirculation of water in the system of windrow-bioreactor-stripping enabled a proper purification of waste waters and reproduction of active organisms applied for the ground inoculation. The in situ method was applied at the former Szprotawa air base. The contaminated area was located in the neighbourhood of a public road. The petroleum products originated from the fuel base, 200 m away, South-East. The source of the pollution were fuel tanks with plating damages which resulted in the migration of contaminated ground water and its enlargement to the area of 250 m 2. The contaminated area consisted of clayey ground and fine-grained sand. Ground water level was m. Petroleum products contents in the ground water amounted to mg/dm 3. In the first step of the remediation process the fuel was pumped out from several technological wells drilled in the ground. The second step consisted in biological remediation of the ground. 12 piezometers and technological wells were placed in the contaminated area. Additionally two depression wells were also installed. Southwards, close to the road, holes composing so called oxygen barrier were placed. Its aim was to protect the ground waters against the migration of the pollutants toward a water flow and a farm with water wells in the yard. The assumptions applied for the in situ bioremediation were: - Isolation of the bacteria capable to neutralize the petroleum products - Taxonometry of the bacteria and elimination of the pathogenic strains - Reproduction of the selected cultures in the field reactors - Inoculation the ground waters with the reproduced bacteria - Maintaining the optimal conditions for the growth of microorganisms in ground waters by aeration and supplying them with biogenic substances. The bacteria strains were applied to the ground with biogens and air pumped through a network of piezoneters inserted into holes drilled in the treated area. The undertaken measures created set of local bioreactors in the piezometers and in their surroundings, where the neutralization of petroleum products took place. The remediation process enabled to reduce the level of pollutants in the ground waters to mg/dm 3. The works carried out have protected the area against propagating the pollutants and reduced the danger of bacteria and the biogenic substances migration. Conclusions The rehabilitation methods presented in the paper refer to different objects and technologies developed on demand of the National Found of Environmental Protection and local governments. The aim of these works was to remediate grounds and restore the biological life on the areas formerly occupied by military troops in Poland. The task was extremely difficult as the polluted land were very widespread. Moreover, the occurring pollutants were of different qualitative and quantitative composition. This is why the usage of standard technological methods have been useless and individual technology adjusted to the local hydrological, geological, microbiological and physical-chemical conditions had to be developed for both specific 31

7 cases. The bioremediation processes carried out resulted in reduction of hydrocarbons concentration in the area treated to the obligatory standards set by the Ministry of Environment. Literature [1] B. Kolwzan Oficyna Wydawnicza Politechniki Wroclawskiej, seria: Monografie, no 44, Wroclaw 2005 [2] M. Alexander Academic Press. A Division of Harcout Brace & Company, 1994 [3] J.G Leahy, R.R Colwell Microbiological Reviews Vol. 54, No. 3, 1990, p [4] C.E. Cernigilia Biochemical Toxicology, vol.3 Eds. E. Hodgson, J.R. Bend., R.M. Philpot. Elsevier/ North Holand, N.Y [5] M. Lebkowska Gaz, Woda i Technika Sanitarna 3, 1996, p [6] G. Malina Biowentylacja Wyd. Politechniki Czestochowskiej, Czestochowa 1999 [7] G. Malina, A Szczepanski Biblioteka Monitoringu Srodowiska, Warszawa

ENVIRONMENTAL GEOLOGY - GEOL 406/506

ENVIRONMENTAL GEOLOGY - GEOL 406/506 Glossary of useful Terms: 1. Abiotic: not living. 2. A b s o r p t i o n: the penetration of atoms, ions, or molecules into the bulk mass of substrate. 3. Acclimation: