Utilization of industrial wastes with production of microbial surfactants of Rhodococcus erythropolis IMV Ac-5017, Acinetobacter calcoaceticus IMV В-7241 and Nocardia vaccinii K-8 with multifunctional application

ЭКОЛОГИЯ/ 2. Экологические и метеорологические

проблемы больших городов и промышленных зон

Konon A.D., Sofilkanych A.P., Andruschenko Ya.V., Labovka I.L., Pirog T.P.

National University of Food Technologies, Kyiv, Ukraine

Utilization of industrial wastes with production of microbial surfactants of Rhodococcus erythropolis IMV Ac-5017, Acinetobacter calcoaceticus IMV В-7241 and Nocardia vaccinii K-8 with multifunctional application

Microbial surface active substances (surfactants) are able to reduce surface and interfacial tension, absorb heavy metals, and increase the efficiency of remediation of oil polluted ecosystems, exhibit antimicrobial and antiadhesive effects against pathogenic microorganisms. Due to the unique properties of microbial surfactants they can be used in various industries [1]. The feasibility of their practical application depends on the economic efficiency of production. One of the ways to reduce the cost of microbial surfactant technology is to use the cheap growth substrates, such as wastes from other industries [2–4].

The oil-oxidizing bacteria were isolated from the oil-polluted samples of soil and identified as Rhodococcus erythropolis IMV Ac-5017, Acinetobacter calcoaceticus IMV В-7241 and Nocardia vaccinii K-8. The ability of these strains to synthesize the metabolites with surface-active and emulsifying properties during the cultivation on traditional hydrophobic and hydrophilic substrates was determined [5–7].

The aim of present work was to study the possibility of using various waste products as a cheap growth substrates for the surfactant synthesis by R. erythropolis IMV Ac-5017, A. calcoaceticus IMV B-7241 and N. vaccinii K-8, and also to determine the antimicrobial and antiadhesive properties of these preparations and their possible use for the remediation of ecosystems from oil and heavy metals.

Liquid paraffin, whey, fried and refined sunflower oil, and molasses were used as carbon and energy sources. In control variants strain IMV B-7241 were cultivated in medium with ethanol, strain IMV Ac- 5017 in medium with n-hexadecane, and strain K-8 – with glycerol. The indexes of surfactant synthesis and bacteria growth – concentration of biomass, the surface tension (σ_s) of cell-free cultural liquid, the conditional surfactant concentration (CSC*, dimensionless), the emulsification index of cultural liquid (Е₂₄, %) – were determined as it was described in our previous works.

The sterile supernatant of cultural liquid was used as a surfactant preparation. Bacillus subtilis BТ-2, Escherichia coli IEM-1, Candida tropicalis PBТ-5, Candida albicans D-6, Staphylococcus aureus BМС-1, Saccharomyces cerevisiae ОB-3 and Aspergillus. niger Р-3 were used as a test cultures.

Studding of antimicrobial properties of surfactants was carried out in culture suspension. Suspensions of test cultures were prepared in test tubes (1.5 mL), then 1.5 mL of preparation was added and kept within 1 and 2 h at the temperature optimal for growth of test culture. The quantity of living cells was determined by the Koch method [8]. The antiadhesive properties were determined as correlation between quantity of attached cells on surfaces treated with surfactant and the quantity of the cells on the control surfaces.

It was shown that R. erythropolis IMV Ac-5017, A. calcoaceticus IMV B-7241 and N. vaccinii K-8 were able to synthesize surfactants on all substrates tested, except the whey (Figure). The maximal rates of surfactants biosynthesis have been registered while the cultivation of strains IMV Ac-5017, IMV B-7241 and K-8 in oil containing medium: increasing of the conditional surfactant concentration by 1.5–2.5 folds compared to the medium with n-hexadecane, ethanol or glycerol. The increase of the quantity of biosurfactant of strains IMV B-7241 and K-8 by 80–196% and 40% was observed when molasses or liquid paraffin were used as carbon sources compared to the cultivation on ethanol- and glycerol containing medium.

Synthesis of surfactants of R. erythropolis IMV Ac-5017, A. calcoaceticus IMV B-7241 and N. vaccinii K-8 cultivated on industrial wastes

It was established that surfactant preparations of R. erythropolis IMV Ac-5017, A. calcoaceticus IMV B-7241 and N. vaccinii K-8 composed of the cultural liquid intensified the processes of oil degradation in contaminated water and soil. The degree of the crude oil degradation in presence of surfactant preparations (5 %) was 83–92 % and 51–86 % in water (2.6 g of oil/L) and soil (21.4 g of oil/kg) respectively, after 30 days. However, the pollution in ecosystems are often complex (crude oil and heavy metal cations), so at the next step we investigated the influence of surfactants of A. calcoaceticus IMV B-7241 and R. erythropolis IMV Ac-5017 on the degradation of petroleum in soil and water in the presence of Cu²⁺. It was determined that the oil destruction degree in the presence of Cu²⁺ (0.01–0.5 mM) and surfactants of strains IMV Ac-5017 and IMV B-7241 was 95–98 % and 91–92 % in contaminated water and soil, respectively, after 30 days.

It was shown that surfactants synthesized by strains IMV Ac-5017 and IMV B-7241 have antimicrobial effect against the number of bacterial and yeast test cultures. The highest antimicrobial activity of investigated surfactants was observed against test-culture of B. subtilis BТ-2. The death of over 90 % of test-culture cells was shown in presence of surfactant of R. erythropolis IMV Ac-5017 (in concentration of 0.98 mg/mL) after 1 hour of exposition; the addition of A. calcoaceticus IMV B-7241 surfactant (0.22 mg/mL) led to the 100 % death of B. subtilis BТ-2 cells independent on time of treatment (table).

Antibacterial influence of surfactant of R. erythropolis IMV Ac-5017 and A. calcoaceticus ІМV В-7241 on B. subtilis БТ-2* survival

Preparation producer	Surfactant concentration in preparation, mg/mL	Survival (%) after
Preparation producer	Surfactant concentration in preparation, mg/mL	1 h	2 h
R. erythropolis ІМV Ac-5017	0.61	55.0±2.70	53.0±2.60
R. erythropolis ІМV Ac-5017	0.98	1.95±0.10	1.8±0.09
A. calcoaceticus ІМV В-7241	0.15	1.4±0.07	0.52±0.03
A. calcoaceticus ІМV В-7241	0.22	0	0

* The initial quantity of B. subtilis БТ-2 cells was 4·10⁶ CFU/mL.

It was shown that antimicrobial activity of A. calcoaceticus IMV B-7241 surfactant against B. subtilis BТ-2 depended on physiological state of test-culture. Thus, surface-active preparation was more effective against spore culture (decreasing of quantity of living cells by 75 % after 2 hours of exposition) then against vegetative cells (decreasing by 47 % after 2 h).

It was determined that the quantity of C. tropicalis PBТ-5 cells in the presence of R. erythropolis IMV Ac-5017 surfactant decreased by 80 % due to the increasing of surfactant concentration up to 1.44 mg/mL and time of treatment up to 2 h. After treatment with the surfactant of R. erythropolis IMV Ac-5017 (0.92 and 1.44 mg/mL) the quantity of C. albicans D-6 cells that stayed alive was 26–33 and 44–77 %, respectively. Similar results were got after treatment with the surfactant of A. calcoaceticus K-4. In this case quantity of living cells of C. albicans D-6 also increased from 71 % to 89 % due to the increasing of surfactant concentration from 0.15 mg/mL to 0.22 mg/mL. So for investigated test-culture the higher concentration of surfactant showed to be less effective.

On the next stage of investigations we determined that surfactant of R. erythropolis IMV B-7241 amplified the antimicrobial activity of manuka oil against some microorganisms (C. albicans D-6, A. niger Р-3, S. aureus BМС-1) due to own antimicrobial and emulsifying properties. When emulsion of manuka oil (12.5 μL/mL) and surfactants (0.43 mg/mL) was added into test-cell suspension (10⁴–10⁵ cells/mL) it was shown that after 15 min of exposition quantity of living cells was 0.7–66.0 % lesser then after addition of pure manuka oil.

It was also shown that A. calcoaceticus IMV B-7241 surfactant preparations in concentration of 0.28 mg/mL led to the decreasing of quantity of attached B. subtilis BТ-2 (24 h culture) cells on glazed tile in 41.3 % and linoleum in 82.4 %. The decreasing of attached E. coli ІЕМ-1 cells was: on metal plates in 41 %, on plastic in 15 % and on glazed tile in 14 %. It was shown that antiadhesive activity of surfactant preparation was depended on physiological state of B. subtilis BТ-2 cells, and the attachment properties decreased with the increasing of culture age.

Thus, the results of this work show the possibility of utilization of industrial wastes and production of practically valuable surfactants with multifunctional application, which can be used as preparations for the oil polluted water and soil remediation, as well as an effective antimicrobial and antiadhesive agents.

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