JOURNAL OF CULTURE COLLECTIONS

Journal of culture collection= s

Volume 4, 2004-2005= , pp. 43-47

Characterization of three bacterial strains –

biodegradants of aromatic substrates <= /span>

&= nbsp;

Lilia Tserovska*, Tanya Yordanova and Lilia Mehandjiyska

National Bank for Industrial Microorganisms and C= ell Cultures, 1113 Sofia, P.O.Box 239, Bulgaria

Summar= y

Three bacterial = strains, isolated from contaminated soil, were adapted to degradе increasing concentrations of xenobiotic aromatic substra= tes. Morphological, physio-logical 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 closes= t to Pseudomonas pseudoalcaligenes.

Introd= uction

Ecological probl= ems 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 assimi= late 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 purif= ying.

The aim of the p= resent work was the investigation and the taxonomical determination of three strai= ns biodegrading aromatic substrates.

Materi= als and Methods

Microorganisms and cultivation. Six bacterial s= trains were investigated for biodegradative abilities. They were isolated and adapted f= or assimilation of xenobiotic substrates with aromatic structure. The adaptati= on was made in periodical cultivation in mineral medium= with different substrates as a sole carbon and energy source – methylbenzoate and dimethyltherephthalate from 1= 00 to 1000 mg/l [7]. The study of the substrate assimilation was carried out in liquid medium with minimal mineral composition (g/l): K₂HPO_=
4 – 8.25; KH₂PO₄ - 1.82; NH₄NO₃= – 1.0; MgSO₄ x 7H₂O – 0.2; CaCl₂&= nbsp;x 2H₂O – 0.02; FeSO₄ x 7H₂O –&nbs= p;0.0006; NaMoO₄ x 2H₂O –= 0.06 and MnSO₄ &= #8211; 0.06. The follo= wing 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<= span lang=3DEN-GB style=3D'font-size:12.0pt;mso-bidi-font-weight:normal'>. The strain deter= mination 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.<= /o:p>

Result= s and Discussion

Fort= y 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 adap= ted 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, becau= se 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 so= le carbon source by the bacterial strains 109, 112, 169, 170, 185 and 189.

Substrates	Strains
109	112	169	170	185	189
B-ester	-	-	-	+	-	-
pT-ester	+	-	-	+	-	-
Zumpf-ester	-	-	+	-	-	-
TA	+++	+	+++	++	+	++
DMT	+++	++	++	+++	++	++
DMI	+++	+	+	++	++	++

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

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

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

Strains

Characteristics

Cell morphology (mm)

Gram stain

Spore formation

Motility

Colony morphology

Growth in liquid medium

Catalase reaction

Oxidase reaction

Acid from glucose

Nitrate reduction

Urease reaction

Growth at t°C=

Growth at pH

pH opt

Temp opt (°= C)

5.5

7.0

8.0

109

Rod

0.7 x 1.6

G-

Round, regular, flat, smooth edge, opaque, muc= ous, to 2 mm

Mud slurry, not forming veil and sediment=

To nitrite

7.0

170

Rod

0.6 x 2.2

G-

Round, regular, flat, smooth edge, hemi-transl= ucent, mucous, to 4 mm

Mud slurry, forming ring and sediment

6.5

189

Rod

0.8 x 2.0

G-

Round, regular, low convex, smooth edge, opaqu= e, mucous, to 3 mm

Mud slurry, not forming veil and sediment=

To nitrite

7.2

152

Rod

0.5 x 2.3

G-

Round, regular, flat, wrinkled, hemi-transluce= nt, mucous, to 2 mm

Mud slurry, without veil or ring, forming sedi= ment

7.0

160

Rod

0.7 x 2.0

G-

Round, regular, flat, smooth edge, hemi-transl= ucent, mucous,

to 1 - 2 mm

Mud slurry, without veil or ring, forming sedi= ment

7.0

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

The biochemical properties of the tested strains were studied by means of speci= fic 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 Alcalig= enes, strain 170 – to genus Pseudom= onas and strain 189 – to genus Cit= robacter. The presence of these bacteria [2, 5, 6] is connected to the well known adaptive and biodegradative abilities of the genera Pseudomonas and Alcalig= enes. The Citrobacter strain is expla= ined with the technological mixing of industrial and daily = faecal wasted wa= ters, during the purifying process. For the last strain the results from IMFIC - test were: formation of indol (-); methylrot test (+); VP= - test (-); Simons’ citrate (+).

Stra= in 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 morphological study 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 a= nd 189.

Strains	Characteristics
Adonitol	Arabinose	Cellobiose	Citrate	Esculin	Fructose	Galactose	Glucose	Lactate	Lactose	Maltose	Mannitol	Mannose	Rhamnose	Ribose	Sorbitol	Sucrose	Trehalose	Xylose	Decarboxylation of L-lisine<= /p>	Decarbox= ylation of L-ornitine	Dehydrolysation of L-arginine	Desamination of phenylalanine	Hydrolysis of phenylalanine<= /p>	Hydrolysis of esculin	Hydrolysis of gelatin
109	-	-	+	-	-	+	+	+	+	-	-	-	+	-	-	-	-	-	-	-	-	-	-	-	-	+
170	-	+	-	+	ND	+	-	-	-	-	+	-	+	-	-	-	-	-	-	ND	-	-	-	-	-	-
189	-	+	+	+	-	+	+	+	ND	+	+	+	-	+	-	-	±	-	+	-	+	-	-	-	ND	-

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

Strains	Characteristics
L-valine	L-serine	Alanine	Phenylalanine	L-arginine	L-glutamate	Betaine	Acetate	Citrate	Succinate	Pyruvate	Malonate	Tartaric acid	Gluconate	Ethanol	Mannitol	Ethylamin	Fructose	Sorbitol	Glycerol
152	-	-	+	-	+	+	-	-	+	+	+	-	-	-	-	-	-	-	-	-
160	-	-	+	-	-	+	+	+	-	+	+	-	-	-	+	-	+	+	-	-
170	-	-	+	-	-	+	-	+	+	+	+	+	-	-	+	-	+	+	-	-

The = present results revealed, that strain 170 could not be defined correctly by the met= hods 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 assimilatio= n of arabinose, manose, maltose, citrate, malonate and betaine. The investigatio= n of Whiteley and Bailey on strains actively degrading phenol announced, that a large amo= unt of them also belonged to P. pseudoalcaligenes [8].

The taxonomical determination of “wild” strains, exposed to continu= ous “toxic pressure” as a result of polluted environment, faces difficulties using the classical methods. The a= daptive 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 methods = for determinatio= n that will be a future task.

Rega= rdless of not entirely correct identification, the “wild” strains are = the best natural purifiers of the environment, therefore future investigations have to be continued and extended.

Refere= nces

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.<= /o:p>

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.