CHANGES IN NUMBER OF CHOSEN METABOLIC GROUPS IN MICROFLORA OF SOIL POLLUTED WITH BENZENE

Małgorzata Hawrot-Paw, Magdalena Czapla
Department of Microbiology and Environmental Biotechnology, West Pomeranian University of Technology, Szczecin, Poland

ABSTRACT

The following study evaluates the influence of benzene pollution on the number of specific metabolic groups of soil microflora. In our research we used the following pollution doses: 100, 1.000 and 10.000 mg · kg^-1 s.m. of soil. The results were analyzed. The proteolytic organisms showed up to be the most susceptible for benzene, the least – the organisms decomposing fats. The analysis revealed that the dose as well as the kind of a hydrocarbon and the time of exposure did not have any significant influence on the amount of amylolytic organisms, however those factors influenced significantly the lypolytic and proteolytic organisms.

Key words: benzene, pollution, soil microflora.

INTRODUCTION

The disturbance of balance between creation and decomposition of polycyclic aromatic hydrocarbons in the natural environments, including the soil, causes the increase of their concentration in ecosystems, and as a result of that, the disturbance of biologic balance [10]. The crude oil derivative compounds are substances that are hard to be decomposed, some of them may exist in the environment for a lot of years.

In the accessible literature there is a lot of data concerning the influence of the aromatic hydrocarbons on the amount and activity of certain groups of soil microorganisms which influence the stabilization – homeostasis of ecosystem, through creation or decomposition of organic compounds in the soil, as well as release and storage of foods [4]. The presence of toxins is the reason why most of the population is not able to develop. The depletion of soil microflora does not have reflection in quantitative changes of microorganisms. As a result of selection, there might be some development of microorganisms able to live in new conditions [9].

In case of certain metabolic groups in soil, for example proteolytic bacteria, the hydrocarbons may stimulate their development, however, usually in a larger amount of time this activity will be decreases. The initial stimulation may be a result of activation of proteolytic microorganism because of additional source of carbon in the form of hydrocarbons. We can observe some very similar dependences in case of activity of an enzyme decomposing carbohydrates – α-amylase [6].

The aim of this research, considering the very important role of microorganism in proper functioning of the soil environment, was the study of benzene's influence on quantity and enzymatic activity of chosen groups of soil microorganism.

MATERIAL AND METHODS

In order to perform the study we used the soil collected in Ostoja situated by Szczecin. It was a clay soil, the black soil type The material was collected from the depth of 0-15 cm of the humus level. After transportation to the laboratory, we dried and grounded the soil using the riddle with 2 mm orifices. The material was brought to 60% water holding capacity. This humidity was constant for the entire time of experiment, in case it was necessary the losses were replenished with distilled water.

The material was divided into small samples, each one of 300 g, and placed in polyethylene bags. Later on, the samples were contaminated with the following doses of benzene: 100, 1.000 i 10.000 mg · kg^-1 s.m. of soil. One of the samples, not contaminated with the hydrocarbons was left as a control sample. The soil samples were incubated in the room temperature (± 20ºC). On the following measurement days (0, 7, 14, 28, 56 and 112 day of incubation) we performed the analysis specifying: the amount of amylolytic microorganisms on the culture medium with addition of starch [2], amount of proteolytic microorganisms on the culture with addition of milk [3], amount of lipolytic microorganisms on the culture with tributirine [1]. We used the common method of inoculation of soil dilutions according to Koch. The samples were incubated in the room temperature for 5 days. All of the measurements had 3 repetitions.

The study results were subject of statistic analysis.

RESULTS

At the beginning of the study the quantity of amylolytic microorganisms in the control sample was a little bit over 700 thousands of cells in 1 g d.m. of soil. For the first measurement, despite of the dose of benzene, the amount of microorganisms increased in all three objects. However, the most extensive stimulation was observed in the sample with benzene concentration of 1.000 mg · kg^-1 s.m. of soil – 170% compared to the control group (Fig. 1). Starting from the 7th day of experiment, we could observe the decrease of amount of amylolytic organisms in all of the objects compared to the control sample. Another increase in the amount of amylolytic microorganism was noted on the 56^th day of incubation. The highest increase of cell number was observed after the use of the highest dose of benzene (10.000 mg · kg^-1 s.m. of soil) – 1500% compared to the control. After use of 1.000 mg · kg^-1 d.m. of soil, the amount of cells was 300% bigger than in control object and was 3 mln of cells in 1 g d. m. of soil.

After implementation of benzene we observed a mild increase of lipolytic microorganisms in all of the objects studied. The biggest quantity in the object infected with 100 mg of benzene was observed after 56 days of incubation (over 1800% compared to the control), and for the dose of 1.000 mg after 28 days. The biggest dosage of contamination had the smallest influence on the quantity of microorganism, that decompose fats (Fig. 2).

The soil contamination with benzene had a negative influence on the amount of proteolytic microorganism (Fig. 3). Only during 2 measurements carried out in the object with the smallest dosage of hydrocarbon the number of proteolytic microorganisms was higher than the values observed in control sample. Based on the results of analysis carried out in the next terms we assessed that inhibition noted on the day of benzene implementation remained constant for the entire time of incubation.

The statistic analysis revealed that the dose of benzene as well as the time of measurement did not have any significant influence only on the quantity of amylolytic microorganisms, however, for this group of microorganisms, just like for the other ones, the interaction of dose and date of measurement was significant (Table 1).

DISCUSSION

The results of this study point out the significant influence of benzene on the quantity of particular groups of soil microorganisms. Zabłocka-Godlewska and Buczkowska-Wesołowska [9] found out that the hydrocarbons implemented in the natural environment like soil can have a very variable influence on its biological balance, including the quality and quantity changes of microorganisms.

On the first day of incubation benzene introduced into the soil caused increase of number of cells of amylolytic microorganisms in the amount of 70 up to almost 180% compared to the control sample. Małachowska-Jutsz et al. [6] and Surygała [8] revealed very similar dependences. Those authors, who were studying the influence of contamination with crude oil derivatives also observed the increase of quantity at the beginning of their work (from 50 to 250% during the first week of experiment). Such reaction of the microorganism could be a result of self-activation resulting from additional source of carbon in a form of hydrocarbons, however, the quantity of amylolytic microorganisms kept decreasing significantly in the next measurements. The inhibition remained until the 4^th measurement (28 day) – the quantity decreased from 15 to 90% below the values observed in the control object at that time. Maliszewska-Kordybach et al. [5] revealed that soil contamination with PAH’s on the level of 10 mg · kg^-1 d. m. of soil caused decrease of enzymatic activity. The decrease of number of amylolytic organisms below the control after 112 days of experiment could have been caused by depletion of food components for amylolytc microorganism and selection leading to displacement of such organisms. [7].

The presence of benzene in soil usually stimulated the quantity of lipolytic microorganisms. We noted a significantly high value after 90 days of experiment for the concentration of 1.000 mg · kg^-1 d. m. of soil (over 2000% of value observed in the control object) and after 120 days of incubation for the dose of 100 mg · kg^-1 d. m. of soil (over 1900% of control value). Later on the increase of quantity of microorganism inoculated on the culture with tributirine compared to the control was not very high. Just like for amylolytic organisms the increase of cell number could have been caused by decomposition of benzene to the components that are better assimilated by the microorganism, being an additional source of carbon and energy, or by selection leading to displacement of lypolytic microorganisms.

The implementation of benzene into the soil, except a small (6%) stimulation of growth in the object with the smallest dosage, caused reduction of proteolytic microorganisms up to 70% compared to the control. In case of dosage of 1.000 and 10.000 mg · kg^-1 d. m. of soil the inhibition remained for the entire period of incubation. Małachowska-Jutsz et al. [6] had different results. After implementation of crude oil to the soil, the authors observed a visible, clear stimulation of proteolytic microorganisms (from 20 to 60% compared to the control). The significant increase of quantity, just like in other groups of microorganisms, was observed after 56 days of incubation (only for the dose of 100 mg · kg^-1 d. m. of soil – 350% of control values).

CONCLUSIONS

1. During incubation of the soil with benzene increase and decrease in the amylolytic microorganisms count was observed.

2. The presence of benzene usually stimulated the quantity of lipolytic microorganisms.

3. Benzene, despite of dose, had a negative influence on the amount of proteolytic microorganisms.

REFERENCES

1. Burbianka M., Pliszka A., 1977. Mikrobiologia żywności: mikrobiologiczne metody badania produktów żywnościowych [Food microbiology: microbiologic methods of food examination]. Państ. Zakł. Wydaw. Lekarskich, Warszawa [in Polish].

2. Conney D.G., Emerson R., 1964. Termophilic fungi. Freeman and Co., London.

3. Kędzia W., Konar H., 1974. Diagnostyka mikrobiologiczna [Microbiologic diagnostics]. Państ. Zakł. Wydawnictw Lekarskich, Warszawa [in Polish].

4. Kowalik P., 2001. Ochrona środowiska glebowego [Protection of soil environment]. PWN, Warszawa [in Polish].

5. Maliszewska-Kordybach B., Smreczak B., Martyniuk S., 2000. Wpływ wielopierścieniowych węglowodorów aromatycznych na mikrobiologiczne właściwości gleb o zróżnicowanej kwasowości i zawartości substancji organicznych [The influence of policyclic aromatic hydrocarbons on the microbiological properties of soils of different acidity and different content of organic substances]. Rocz. Glebozn., LI (3/4), 5–18 [in Polish].

6. Małachowska-Jutsz A., Mrozowska J., Kozielska M., Miksch K., 1997. Aktywność enzymatyczna w glebie skażonej związkami ropopochodnymi w procesie jej detoksykacji [Enzymatic activity of soil contaminated with crude oil derivative substances in the process of its detoxication]. Biotechnologia, 1 (36), 79–91 [in Polish].

7. Przystaś W., Miksch K., Małachowska-Jutsz A., 2000. Zmiany aktywności enzymatycznej gleby w procesie biodegradacji zanieczyszczeń ropopochodnych przy zastosowaniu biopreparatów [Changes of enzymatic activity of soil in the process of biodegradation of crude oil derivatives contamination with the use of biopreparations]. Arch. Ochr. Środ., 26 (2), 59–70 [in Polish].

8. Surygała J., 2000. Zanieczyszczenia naftowe w gruncie [Paraffin oil contamination of soils]. Wyd. Politech., Wrocław [in Polish].

9. Zabłocka-Godlewska E., Buczkowska-Wesołowska K., 1998. Ocena wpływu WWA oraz modyfikacji układu na zmiany jakościowo-ilościowe głównych grup mikroorganizmów w glebie piaszczysto-bielicowej [The evaluation of influence of PAH's and modification of system due to quality and quantity changes of main groups of microorganisms in sandy soil]. Mat. Ogólnopol. Symp. Nauk. – Techn. "Bioremediacja gruntów"; Wisła-Bukowa; 59–73 [in Polish].

10. Zabłocka-Godlewska E., Mrozowska J., 1997. Wpływ WWA na aktywność mikrobiologiczną gleby w zmodyfikowanych układach modelowych [The influence of PAHs on the microbiological activity of soils in modified model systems]. Mat. V Ogólnopol. Symp. Nauk.-Techn. "Biotechnologia środowiskowa"; Ustroń-Jaszowiec, 61–72 [in Polish].

Accepted for print: 22.06.2010

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