БИОЛОГИЧЕСКИЕ НАУКИ / 6. МИКРОБИОЛОГИЯ

Antoniuk S.О., Sofilkanych А.P., Pirog T.P.

National University of Food Technologies, Кyiv

DESTRUCTION OF AROMATIC COMPOUNDS BY THE OIL-OXIDIZING BACTERIA NOCARDIA VACCINII К-8 AND ACENITOBACTER CALCOACETICUS ІМВ В-7241

Up to 13 million lives could be saved each year by reducing environmental risks, according to the World Health Organization’s first country-by-country analysis of the impact of environmental factors on human health. Environmental pollution is a great concern to all countries around the world. China has made great efforts in this area and plans to invest $175 billion in environmental protection between 2006 and 2010, according to the National Development and Reform Commission of China [2].

Aromatic compounds are serious pollutants, being present mainly in industrial wastewater from chemical, petrochemical, pharmaceutical, textile and steel industries. Due to their ubiquitous occurrence, recalcitrance, bioaccumulation potential and carcinogenic activity, the aromatic compounds have gathered significant environmental concern [3]. They are widely distributed environmental contaminants that have detrimental biological effects, toxicity, mutagenecity and carcinogenicity [6].

Although aromatic hydrocarbons may undergo adsorption, volatilization, photolysis and chemical degradation, microbial degradation is the major degradation process. Bioremediation is the tool to transform the compounds to less hazardous or nonhazardous forms with less input of chemicals, energy and time [1]. Besides microbiological methods are economically advantageous, do not require large capital investments and operating costs, and local sewage treatment plants take low areas and very easy to maintain [3].

Numerous reports [1–6] have described the ability of bacteria species to degrade aromatic and hydrocarbons such as Nocardia vaccinii and Acenitobacter calcoaceticus.

Previously it was shown that the bacteria Nocardia vaccinii K-8 and Acenitobacter calcoaceticus IMV B-7241 intensified the processes of oil degradation in contaminated sites. Since crude oil always contains aromatic hydrocarbons (10–50%), we assumed that the studied strains may be potential destructors of aromatic compounds. In this regard, the aim of our work was to study the ability of strains IMV B-7241 and K-8 to grow on nutrient media containing substrates of aromatic nature as a carbon and energy source.

In the previous work the oil-oxidizing bacteria identified as Nocardia vaccinii К-8 and Acenitobacter calcoaceticus К-4 were isolated from the oil-polluted samples of soil. The ability of the strains to synthesize the metabolites with surface-active and emulsifying activity (biosurfactants) during their cultivation on different hydrophobic (n-hexadecane, liquid paraffin) and hydrophilic (glucose, ethanol) substrates was determined. The strain K-4 was deposited in the Depositary of microorganisms of the Institute of Microbiology and Virology of National Academy of Sciences of Ukraine at the number of IMV B-7241.

A. calcoaceticus IMV В-7241 was cultivated on the nutrient medium of the following composition (g/L): NaCl – 1.0; Na₂HPO₄ – 0.6; (NH₂)₂CO – 0.35; KH₂PO₄ – 0.14; MgSO₄´7H₂O – 0.1; рН 6.8–7.0; the yeast autolysate – 0.5 % (v/v) and trace elements solution – 0.1 % (v/v) were also added.

N. vacinii K-8 strain was grown on the synthetic nutrient medium containing (g/L): NaNO₃ – 0.5; MgSO₄´7H₂O – 0.1; СaCl×2H₂O – 0.1; KH₂PO₄ – 0.1; FeSO₄´7H₂O – 0.1, yeast autolysate – 0.5 % (v/v).

Phenol, 4-chlorphenol, hexachlorobenzene, naphthalene, benzoic, sulfanilic and N-phenilantranilic acids 0.3–0.5 % (v/v), benzene and toluene 0.3–0.5% (w/v) were used as soul carbon and energy sources.

The variants of the inoculum were the following. Variant 1 – 24-hour culture cultivated on the meat infusion agar (MIA). Variant 2 – culture cultivated on the liquid mineral medium containing above-mentioned nutrients and aromatic compounds (0.1–0.25%) as soul carbon and energy sources. The inoculum preparation included the adaptation of bacteria to aromatic compounds by gradual increase of their concentration from 0.1 to 0.25 % in nutrient medium followed by bacteria inoculating on mineral medium with 0.3–0.5% of the substrate. For acclimatization a loopful of organisms cultivated on the MIA (24 h) was directly inoculated into flasks containing aromatic compounds (0.1%) and all the required nutrients. The culture was kept in a rotary shaker for 72 h.

This formed the primary culture. The secondary acclimatized inoculum was prepared in the same way, wherein 10% (v/v) of primary culture was used instead of the subculture to inoculate the medium containing aromatic compounds (0.15%) and in this case the culture was incubated for 48 h.

This was continued for the third and fourth acclimatization by gradual increase of aromatic compound concentration in nutrient medium to 0.2 and 0.25%, respectively. Variant 3 – 48-hour culture incubated in the liquid mineral medium containing above-mentioned nutrients and aromatic compounds (0.3–0.5%) as soul carbon and energy sources. The inoculum was used in a concentration of 10% (v/v).

The cultivation of bacteria took place in the 750 ml Erlenmeyer flasks with 100 ml of medium on rotor shaker (320 rpm) at 28–30 ºС during 72–96 h.

The quantity of synthesized surfactant was evaluated by such indexes: conditional surfactant concentration (CSC*) and emulsification index (Е₂₄, %) of the cultural liquid. The number of viable cells was determined by the Koch method on MIA, biomass – by the optical density of cultural liquid, followed by recalculation to absolutely dry biomass by calibration graph.

It was determined that N. vaccinii K-8 and A. calcoaceticus IMV B-7241 intensively grew on phenol, hexachlorobenzene, naphthalene, N-phenilantranilic and benzoic acid, slightly worse on toluene, benzene and sulfanilic acid and died on 4-chlorphenol.

The utilization of aromatic compounds accompanied by the formation of extracellular metabolites with surface-active and emulsifying properties (Table).

Table. Surfactant synthesis during Acenitobacter calcoaceticus ІМВ В-7241 cultivating on aromatic compounds

Substrate	Concentration, %	CSC*	Е24, %
Phenol	0.3	3.2±0,16	65±3,2
	0.5	3.6±0,18	75±3,7
Benzene	0.3	1.6±0,08	50±2,5
	0.5	1.5±0,08	50±2,5
Toluene	0.3	1.7±0,09	55±2,7
	0.5	1.2±0,06	50±2,5
Benzoic acid	0.3	2.1±0,1	55±2,7
	0.5	2.8±0,14	52±2,6
N-phenilantranilic acid	0.3	1.9±0,09	45±2,2
	0.5	2.0±0,1	50±2,5
Hexachlorobenzene	0.3	1.5±0,08	45±2,2
	0.5	1.7±0,09	53±2,7
Ethanol (control)	0.3	0.8±0,04	40±2,0
	0.5	1.0±0,05	43±2,1

Note. The inoculum – 24-hour culture cultivated on the meat infusion agar, the cultivation took place during 96 h.

Thus, during A. calcoaceticus IMV B-7241 cultivation on phenol (0.5%) the highest conditional surfactant concentration (CSC*) and emulsification index (E₂₄, %) were 3.6 and 70%, respectively (while on ethanol CSC* – 1.0 and E₂₄ – 43%).

The maximum indexes of surfactant synthesis by N. vaccinii K-8 were observed as a result of strain growth on the media with naphthalene (0.5%): CSC* – 2.6 and E₂₄ – 70%, while on glycerol (0.5%) – 2.0 and 60%, respectively.

Similar results were described by Nitschke et al. [4] testing polycyclic aromatic hydrocarbon degradation by Pseudomonas aeruginosa strains. So, P. aeruginosa N43 synthesized extracellular metabolites with surface-active and emulsifying properties on phenantrene (0.5%): conditional surfactant concentration and emulsification index were 3,3 and 75%, respectively.

Moreover, it was shown that the increased concentrations of biomass (30–40 % of control) have been observed in the case of three consecutive inoculating of strains K-8 and IMV B-7241 on medium containing aromatic compounds (0.3–0.5%).

Based on this result, further the inoculum preparation included the adaptation of bacteria to aromatic compounds by gradual increase of their concentration from 0.1 to 0.25 % in nutrient medium followed by bacteria inoculating in mineral medium with 0.3–0.5% of the substrate. It was determined, that the process of cultivation was accompanied by 1.5–2.0-fold biomass increase compared to using the inoculum cultivated on the meat infusion agar (figure).

Effect of the inoculum quality on growth of N. vaccinii K-8 on aromatic compounds (0.3%)

It should be noticed, that in the case of the consecutive cultivation of the inoculum in liquid medium containing aromatic compounds (0.1–0.25%) biomass concentration of the strain K-8 was 0.4 g/L while Shetty et al. [5] reported that biomass of Nocardia hydrocarbonoxydans on phenol (0.5%) was 0.25 g/L subject to use above-mentioned method.

It was determined, that N. vaccinii K-8 and A. calcoaceticus IMV B-7241 showed a particular ability to assimilate aromatic compounds as soul carbon and energy sources and to synthesize practically valuable surfactants. The growth of strains K-8 and IMV B-7241 on aromatic compounds was intensified in 1.5–2.0 times in the case of the consecutive cultivation of the inoculum on liquid mineral medium with aromatic compounds (0.1–0.25%) compared to using the inoculum cultivated on the meat infusion agar. These strains are promising for use in the remediation of water and soil polluted with crude oil and aromatic xenobiotics.

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