Journal of Culture Collections National Bank for Industrial Microorganisms and Cell Cultures ISSN: 1310-8360 Vol. 4, Num. 1, 2005, pp. 43-47

Journal of Culture Collections, Volume 4, No. 1, 2004-2005, pp. 43-47 

Characterization of three bacterial strains– biodegradants of aromatic substrates

Lilia Tserovska*, Tanya Yordanova and Lilia Mehandjiyska

National Bank for Industrial Microorganisms and Cell Cultures, 1113 Sofia, P.O.Box 239, Bulgaria;

Code Number: cc05005

Summary

Three bacterial strains, isolated from contaminated soil, were adapted to degrade increasing concentrations of xenobioticaromatic substrates. Morphological, physiological and biochemical characteristics define them as belonging to the genera Pseudomonas, Alcaligenes and Citrobacter. The strain with highest biodegradative activity is taxonomically related as the closest to Pseudomonas pseudoalcaligenes.

Introduction

Ecological problems of worldwide importance are extended every year with the accumulation of greater amounts and variety of anthropogenic contaminants. In most of the cases they resist physical and chemical influences and the biological factor is the one that could accomplish their degradation. Bacteria are the group of microorganisms with the greatest role in the biopurifying processes [4]. Their function is determined by the wide catabolic potential and adapting abilities to assimilate different xenobiotic substrates [3, 9]. Often the conditions of contaminated regions lead to phenotype and genotype changes that trouble the biodegrading strain identification. Despite that, the taxonomical determination is an important stage of the biotechnological approach in the environmental purifying.

The aim of the present work was the investigation and the taxonomical determination of three strains biodegrading aromatic substrates.

Materials and Methods

Microorganisms and cultivation. Six bacterial strains were investigated for biodegradative abilities. They were isolated and adapted for assimilation of xenobiotic substrates with aromatic structure. The adaptation was made in periodical cultivationin mineral medium with different substrates as a sole carbon and energy source – methylbenzoate  and dimethyltherephthalate  from 100 to 1000 mg/l [7]. The study of the substrate assimilation was carried out in liquid medium with minimal mineral composition (g/l): K₂HPO₄ – 8.25; KH₂PO₄ - 1.82; NH₄NO₃ – 1.0; MgSO₄ x 7H₂O – 0.2; CaCl₂ x 2H₂O – 0.02; FeSO₄ x 7H₂O – 0.0006; NaMoO₄ x 2H₂O – 0.06 and MnSO₄– 0.06. The following substrates were added: B-ester; pT– ester; Zumpf- ester; therephthal acid (TA); dimethyltherephthalate (DMT) and dimethylisophthalate (DMI) in working concentration 250 mg/l.

Identification of the bacterial strains.The strain determination was made by means of morphological, physiological and biochemical characteristics, according to the procedures in Bergey’s manual [1]. API 20 (bioMerieux) tests were used for identification of nonfermenting Gram - negative bacteria. Two strains from CNCTC – Pseudomonas alcaligenes 152 and P. pseudoalcaligenes 160, were used as control for species defining.

Results and Discussion

Forty five microbial cultures with biodegradative activity to the limiting aromatic substrate were isolated from the contaminated biotops of textile factory “D. Dimov” purifying station - Yambol. They were adapted by increasing the substrate concentration [7]. Cultures with the following designations – 109, 112, 169, 170, 185 and 189, were selected for further studies.

The substrate specificity of the mentioned above strains was of interest, because of the arising polluting. Microorganisms were cultivated in mineral medium with addition of different aromatic compounds as a sole carbon and energy source. Substrate type and assimilation of adapted bacterial strains are presented in Table 1.

Table 1. Aromatic compounds assimilation as a sole carbon source by the bacterial strains 109, 112, 169, 170, 185 and 189.

Legend: presence of growth (+); good growth (++); very good growth (+++).

Envisage the wide substrate specificity and biodegradation efficiency, the most active three strains - 109, 170 and 189 were investigated. The morphological and some physiological characteristics are shown in Table 2.

Table 2. Morphological and physiological properties of bacterial strains 109, 170, 189, 152 and 160.

Legend: positive reaction (+); negative reaction (-); no data (ND).

The biochemical properties of the tested strains were studied by means of specific media. The results are presented in Table 3.

The obtained data referred these strains according to Bergey’s manual as follows: strain 109 – to genus Alcaligenes, strain 170 – to genus Pseudomonas and strain 189 – to genus Citrobacter. The presence of these bacteria [2, 5, 6] is connected to the well known adaptive and biodegradative abilities of the genera Pseudomonas and Alcaligenes. The Citrobacter strain is explained with the technological mixing of industrial and dailyfaecal wasted waters, during the purifying process. For the last strain the results from IMFIC - test were: formation of indol (-); methylrot test (+); VP - test (-); Simons’ citrate (+).

Strain 170 was enlisted in group A (RNA group I, section I, 2b) of Pseudomonas [1]. Two control type strains 152 and 160 were investigated for a comparison and more accurate taxonomical determination. The results from the morphologicalstudy of the three cultures are included in Table 2 and the biochemical characteristics in Table 4.

Table 3. Biochemical properties of bacterial strains 109, 170 and 189.

Table 3. Biochemical properties of bacterial strains 109, 170 and 189 (continued).

Table 4. Biochemical properties of bacterial strains 152, 160 and 170.

Table 4. Biochemical properties of bacterial strains 152, 160 and 170 (continued).

The present results revealed, that strain 170 could not be defined correctly by the methods of classical taxonomy. Obviously it endured variety of adaptive changes (probably on a genetic base) as a result of the toxic aromatic pollution in the region. The strain could be related as the closest to P. pseudoalcaligenes, but distinguished by the assimilation of arabinose, manose, maltose, citrate, malonate and betaine. The investigation of Whiteley and Bailey on strains actively degrading phenol announced, that a large amount of them also belonged to P. pseudoalcaligenes [8].

The taxonomical determination of “wild” strains, exposed to continuous “toxic pressure” as a result of polluted environment, faces difficulties using the classical methods. The adaptive changes of these microorganisms give an advantage in evolutional aspect, but differ them from the collections strains, cultivated at optimal conditions. It is necessary to apply a number of modern molecular genetic methodsfor determination that will be a future task.

Regardless of not entirely correct identification, the “wild” strains are the best naturalpurifiers of the environment, therefore future investigations have to be continued and extended.

References

1. Bergey’s Manual of Systematic Bacteriology, 1984. N. Krieg (Eds.), vol. 1, 2, Baltimore: Williams and Wilkins.
2. Hill, G., B. Milne, P. Nawrocki, 1996. Appl. Microbiol. Biotechnol., 46, 163-168
3. Johnson, R., R. Olsen, 1997. Appl. Envir. Microbiol., 63 (10), 4047-4052
4. Powlowski, J., V. Shinger, 1994. Biodegradation,5, 219-235.
5. O’Reilly, K., R. Crawford, 1989. Appl. Envir. Microbiol., 55 (4), 866-870.
6. Richter, M., R. M. Wittich, 1994. Biodegradation,5, 63-69.
7. Tserovska, L., R. Dimkov, Y. Topalova, 1995. J. Cult. Coll., 1,23-27.
8. Whiteley, A., M. Bailey, 2000. Appl. Envir. Microbiol., 66 6, 2400-2407.
9. Widada, J., H. Nojiri, T. Omori, 2002. Appl. Microbiol. Biotechnol., 60(1-2), 45-59.

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