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UDC 579.663 https://doi.org/10.15407/biotech11.06.082
THE PROPERTIES OF SURFACTANTS SYNTHESIZED BY Acinetobacter calcoaceticus IMV B-7241 ON REFINED AND SUNFLOWER OIL WASTE
T. P. Pirog
D. A. Lutsai National University of Food Technologies, Kyiv, Ukraine
S. I. Antonuk I. V. Elperin
E-mail: [email protected]
Received 14.08.2018 Revised 06.11.2018 Accepted 28.12.2018
The aim of the work was to compare the antimicrobial and anti-adhesive activity (including the ability to destroy biofilms), as well as the effect on oil degradation of the surfactants synthesized by the culture of Acinetobacter calcoaceticus IMV B-7241 on refined and sunflower waste.
The microbial surfactants were extracted from supernatant of cultural liquid by mixture of chloroform and methanol (2:1). The number of attached cells and the degree of biofilm destruction were analyzed spectrophotometrically. Antimicrobial properties of the surfactants were determined by index of the minimal inhibiting concentration. The concentration of oil in water was analyzed by the gravimetric method after extraction with hexane.
It was shown that microbial surfactants synthesized in medium with 2% of both refined and fried oil were characterized by high antimicrobial (MIC with respect to bacterial test cultures 0.8-29 pg/ml, Candida albicans D-6 26 — 58 pg/ml) and anti-adhesive (decreasing number of bacterial and fungal cells of test cultures attached to abiotic surfaces by 35-70%, destruction of biofilms by an average of 40-44%) activity. Increasing concentration of waste oil in the medium to 4% was accompanied by the formation of surfactants with low antimicrobial activity, in the presence of which the degree of oil destruction in water (3-6 g/l) was 80-88% in 20 days, which is 10-16% higher than when using surfactants synthesized in a medium with 2% oil.
Obtained data indicate on the need for studies on the effect of cultivation conditions of producer on the properties of synthesized surfactants for the production of final product with stable predetermined properties, depending on the field of practical application.
Key words: microbial surfactants, sunflower oil waste, antimicrobial and anti-adhesive activity.
Biodegrading and nontoxic microbial surfactants (MS) are multifunctional due to their surfactant emulsifying properties, antimicrobial and anti-adhesive activities. MS are a worthwhile alternative to the chemical surfactants for various industrial and medical purposes, and in nature conservation technologies [1-5].
Nowadays, however, MS are industrially produced by only a few companies [6], although worldwide production of synthetic surfactants is 15 billion tons per year, expected to increase to 24 billion tons annually [7]. This situation is caused by insufficiently efficient microbial
technologies of producing MS, partly because of highly expensive biosynthesis process and low MS concentrations. One of approaches of decreasing the MS cost is using industrial waste such as waste oil [8-10].
Another disadvantage of MS is the possible changes of their properties depending on the producers cultivation conditions, because the MS are secondary metabolites synthesized as a complex of such substances, see our previous studies [11, 12]. In those studies we emphasized the fact that at present the influence of cultivation conditions on the properties of microbial surfactants escapes the
attention of researchers. In recent years the number of publications on the dependence of biological properties of MS on their chemical composition has increased [13-16]. However, the authors of these papers did not investigate the dependence between the biological properties of surfactant and the conditions of cultivating producers.
Earlier [17], we showed that replacing purified glycerol in the cultivation medium of Acinetobacter calcoaceticus producer IMV B-7241 with the biodiesel production waste (technical glycerol) was accompanied by a decrease in the antimicrobial and anti-adhesive activity of synthesized surfactants. In [17], it was suggested that this may be caused by the inhibiting effect of monovalent cations (components of biodiesel production) on the activity of NADP+-dependent glutamate dehydrogenase, the key enzyme in the biosynthesis of aminolipids responsible for these properties of the surfactants complex.
In previous studies [18], we have established the conditions for the cultivation of A. calcoaceticus IMV B-7241 on processed (fried) sunflower oil of different quality, which provide maximum rates of synthesis of surfactants. The maximum concentration of surfactant (7.9-8.5 g/l) was achieved during the cultivation of the IMV strain B-7241 on the waste after frying meat (4%) and sunflower potato (6%) oil, respectively. However, it is known [19, 20] that waste oils from frying potatoes and meat differ in composition, particularly in toxic substances. Thus, the iron cations from meat increase the degree of oxidation and thermal degradation in the waste oil [19]. In addition, when frying meat, toxic heterocyclic amines are formed [19, 20], which can act as inhibitors of NADP+-dependent glutamate hydrogen dehydrogenase. For example, in the presence of 10 mM 2,4-pyridinedicarboxylate (derivative of heterocyclic aromatic amine pyridine), the activity of that enzyme in Aspergillus niger NCIM 565 is reduced by 2-4 times [21, 22].
Hence, the aim of this work was to compare the antimicrobial and anti-adhesive activity (including the ability to destroy biofilms), as well as the impact on the destruction of oil pollution by surfactants synthesized during the cultivation of A. calcoaceticus IMV B-7241 on refined and fried sunflower oil.
Materials and Methods
Study objects. The main study object was strain of petrooxidizing bacteria isolated
from oil-contaminated soil and identified as Acinetobacter calcoaceticus K-4 [23]. The strain K-4 is registered in Depository of microorganisms of D. K. Zabolotny Institute of Microbiology and Virology of NAS of Ukraine under the number IMV B-7241.
Bacterial strains of Escherichia coli IEM1-, Bacillus subtilis BT2- (grown from spore cells in 24 hr), Staphylococcus aureus BMC-1, Pseudomonas sp. MI-2, Enterobacter cloacae C-8; yeasts Candida albicans D-6 and fungus Aspergillus niger P-3 were used to assess the antimicrobial and anti-adhesive activity of MS. The cultures were taken from the collection of microorganisms of the Department of biotechnology and microbiology of National university of food technologies.
Medium composition and cultivation conditions. The strain A. calcoaceticus IMV B-7241 was grown in liquid mineral medium as follows (g/l): 0.35 (NH2)2CO, 1.0 NaCl, 0.6 Na2HPO412H2O, 0.14 KH2PO4, 0.1 MgSO47H2O, distilled water up to 1 l, pH 6.8-7.0. Also, 0.5% yeast lysate (v/v) and 0.1% microelement solution (v/v), were added. The microelement solution contained (g/100 ml): 1.1 ZnSO4 7H2O; 0.6 MnSO4H2O; 0.1 FeSO4 7H2O; 0.004 CuSO4 5H2O; 0.03 CoSO4 7H2O; 0.006 H3BO3; 0,0001 KI; 0.5 EDTA (Trilon b). In one version, the concentration of urea in the medium was increased to 0.7-1.0 g/l.
Refined sunflower oil "Oleina" (Dnipropetrovsk oil and extraction plant) and waste oil from frying potatoes and meat (McDonald's, fast food restaurants network, Kyiv) (2-5%, volumetric) were used as a source of carbon and energy.
As inoculum we used the cultures in the exponential growth phase, grown on the medium of the described composition containing 0.5% (v/v) of the respective oil. The inoculation material (104-105 cells/ml) amounted to 10% of the volume of the culture medium.
The bacteria were cultivated in flasks of 750 ml with 100 ml of medium on the shaker (320 rpm) at 28-30 °C for 120 hr.
Determination of the antimicrobial and anti-adhesive activity of MS. The MS under study were extracted from supernatant of the culture liquid with Folch's method (chloroform and methanol mixture, 2:1).
The antimicrobial properties of MS were analyzed by the index of minimum inhibitory concentration (MIC). The MIC was determined using the method of double serial dilutions in
times (ratio 1: 1). The organic extract was evaporated to a constant mass on a rotary evaporator IR-1M2 (Russia) at a temperature of 55 °C and an absolute pressure of 0.4 atm.
All experiments were thrice replicated; the number of parallel determinations in the experiments was 3 to 5. The statistical treatment of the experimental data was carried out as described previously [24]. The differences between the means were considered significant at P < 0.05.
Results and Discussion
Antimicrobial activity of surfactants of A. calcoaceticus IMV B-7241. Table 1 includes data on the dependence of MIC index of synthesized surfactants on the conditions of cultivation of IMV strain B-7241 on oily substrates. It was showed that A. calcoaceticus IMV B-7241, cultured on the medium with concentration of fried sunflower oil of different quality increased by 4-5%, produced surfactants with extremely low antimicrobial activity: the MIC for the bacterial test cultures was > 600-900 pg/ml, and in the case of yeast > 1200-1700 pg/ml (see Table 1). In these studies, the concentration of the carbon source in the medium increased simultaneously with the content of the nitrogen source to maintain
Table 1. The influence of sunflower oil concentration and nitrogen source in the culture medium of A. calcoaceticus IMV B-7241 on the antimicrobial activity of surfactants
Oil as substrate Concentration in medium Minimum inhibiting concentration (pg/ml) against
oil, % Nitrogen source, g/l Escherichia coli IEM-1 Bacillus subtilis BT-2 (spores) Staphylococcus aureus BMC-1 Pseudomonas sp. MI-2 Candida albicans D-6
Refined 2 0.35 0.8 51.2 1.6 0.8 25.6
4 0.7 15 64 32 16 130
Waste of frying meat 4 0.7 > 670** > 670** > 670** > 670** >1340**
4 1.0 > 850** > 850** > 850** > 850** >1700**
Waste of frying potatoes 2 0.35 0.9* 28,8* 0.45* 0,45* 57.6*
4 0.7 > 600** > 600** > 600** > 600** >1200**
4 1.0 > 750** > 750** > 750** > 750** N.d.
5 0.7 > 850** > 850** > 850** > 850** N.d.
5 1.0 > 900** > 900** > 900** > 900** N.d.
Note. Here and after: the statistical error of the analysis of minimum inhibitory concentration did not exceed 5%. N.d. — not determined.
* — P < 0.05 compared to control (minimum inhibitory concentration of MS, synthesized on medium with 2% (v/v) refined oil).
** — P < 0.05 compared to control (minimum inhibitory concentration of MS, synthesized on medium with 4% (v/v) refined oil).
meat-peptone broth (MPB) for bacteria and liquid yeast as described earlier [11].
The anti-adhesive properties of surfactant were determined as described in [11]. The number of adherent cells (degree of adhesion) was found spectrophotometrically as the ratio of the optical density of the suspension obtained from substrates (tile, steel, linoleum) treated with the MS preparations to the optical density of the control samples (without surfactant processing), expressed as a percentage.
The MS effect on the biofilm destruction was studied according to the method shown in our previous publication [11].
Assessment of biological destruction of oil. The oil pollution of water was modeled according to [9, 24]. The experiment was conducted for 20 days. The amount of oil was determined by weight method after triple extraction with hexane (ratio 1:1). To study the influence of surfactants on the decomposition of oil we used preparations in the form of a culture liquid (10%, volumetric).
Total number of living cells in water during the experiment (20 days) was determined by Koch's method on MPA.
The quantity of oil was determined by the weight method. For this, extraction of petroleum with hexane was carried out three
the carbon/nitrogen ratio at the optimal level for synthesis of surfactants.
At the same time, increase concentration of refined oil to 4 % in the culture medium of IMV B-7214 strain was accompanied by MS synthesis, antimicrobial activity of those MS (MIC 15-130 pg/ml) was higher in comparison with that established for surfactants obtained on the fried oils of similar concentration (MIC > 600-1700 pg/ml) (Table 1). These data confirm our assumption that the composition of fried oils includes the synthesis inhibitors in the MS complex, responsible for the antimicrobial properties of A. calcoaceticus IMV B-7241.
Each year, the number of publications on the antimicrobial properties of MS and the potential use of these products of microbial synthesis in medicine is constantly increasing [25, 26]. However, there are only a few reports of the antimicrobial properties of surfactants synthesized on the oil-containing industrial waste [27-29].
Thus, the rhamnolipid of Thermus thermophilus HB8 [27], synthesized on such substrates, inhibited the growth of Micrococcus lysodeikticus. The growth of bacteria of the genus Streptococcus decreased by 35-65% in the presence of Rufisan (6-12 mg/l), synthesized by Candida lipolytica UCP0988 on soybean oil wastes (6%), while that of the representatives of the genus Staphylococcus was inhibited by only 15-18% [28]. It should be noted that Rufisan did not exhibit antimicrobial activity on Candida albicans.
In [29] it was showed that rhamnolipid synthesized by Pseudomonas aeruginosa LBI on soybean oil wastes inhibited the growth of Bacillus cereus and Mucor miehei at concentration of 64 pg/ml, and the growth of Neurospora crassa, Staphylococcus aureus and Micrococcus luteus was inhibited at 256 pg/ml.
In previous studies [30, 31] we showed the dependence of the antimicrobial activity of Nocardia vaccinii IMV B-7405 on the quality of the frying oil and the duration of the process. The strongest antimicrobial effect on the phytopathogenic bacteria of the genera Pseudomonas, Xanthomonas and Pectobacterium was caused by the MS solutions synthesized by N. vaccinii IMV B-7405 on the fried potato waste oil (reducing the survival of phytopathogenic bacteria by 50-95%), as well as by IMV B-7405 strains cultivated for 7 days on all oil-containing substrates (minimum
inhibitory concentrations of 7-40 pg/ml, which is several times lower than that of MS synthesized in 5 days) [30].
The antibacterial (against B. subtilis BT2-, E. coli IEM1-, Proteus vulgaris PA12-, S. aureus BMC1-, Pseudomonas sp. MI-2, E. cloacae C-8, Erwinia aroideae H-3) and antifungal (against Candida tropicalis PE-2 and C. albicans D-6) MIC of MS synthesized by N. vaccinii IMV B-7405 on fried potato waste oil were lower in 2.5 to 8 times than those of MS produced on media with fried meat waste oil [31]. Longer cultivation (for 7 days instead of 5 days) of N. vaccinii IMV B-7405 on waste oil after frying potatoes was accompanied by the synthesis of surfactants, MIC of which against test cultures was lower by 1.4-4 times. The MIC indexes of N. vaccinii IMV-7405 surfactants correlated with the presence of aminolipids in the synthesized MS, and the activity of NADF+-dependent glutamate dehydrogenase enzyme, which is the key enzyme for their biosynthesis [31].
Note that in [30, 31], N. vaccinii IMV B-7405 was cultured on a medium containing 2% (volumetric) of fried oil.
Comparing the MIC of MS synthesized for 5 days on a medium with 2% fried potato oil revealed higher antimicrobial activity of the MS of A. calcoaceticus IMV B-7241 relative to the tested test cultures (0.4-58 pg/ml, Table 1) than that of MS of N. vaccinii IMV B-7405 (22-176 pg/ml) [31].
Anti-adhesive properties of MS. Since a 4-5% increase of the concentration of fried oil in A. calcoaceticus IMV B-7241 cultivation medium was accompanied by the formation of MS with low antimicrobial activity (Table 1), the anti-adhesive properties were determined for MS synthesized on a medium with 2% fried potatoes waste oil (Table 2).
According to the data in the Table 2, the adhesion of the tested test cultures to abiotic materials treatment with MS solutions was practically the same for MS synthesized on both refined and treated oils. Thus, the quantity of B. subtilis BT-2 cells attached to different surfaces was 27-48%, that of S. aureus BMS-1 cells was 25-58%, and that of C. albicans D-6 cells was 40-65%. Importantly, that significantly reduced adhesion of test cultures to materials treatmented with MS was achieved at extremely low concentrations of surfactants of A. calcoaceticus IMV-7241 (1.25-2.5 pg/ml).
Earlier [32] were found that the adhesion of B. subtilis BT-2, E. coli IEM-1 and C. albicans D-6 decreased by 30-60% on abiotic surfaces treated with solutions of MS synthesized by
Notes. The medium contained 2% (v/v) oil after frying potatoes. Statistical error of determination of adhesion did not exceed 5 %. N.d. — not determined. * — P < 0.05 compared to control (adhesion after treatment with 2.5 pg/ml surfactant, synthesized on the medium with 2 % of refined oil). ** — P < 0.05 compared to control (adhesion after treatment with 1.25 pg/ml surfactant, synthesized on medium with 2 % of refined oil).
Table 2. The influence of surfactants synthesized by A. calcoaceticus IMV B-7241 on refined or fried sunflower oil on the adhesion of microorganisms to abiotic surfaces
Test culture Oil substrate Surfactant concentration, pg/ml Adhesion,%
polystirene tile steel linoleum
Bacillus subtilis BT-2 Refined 2.5 34 36 41 33
1.25 27 47 48 41
Fried 2.5 32* 34* 48* 34*
1.25 29** 49** 44** 44**
Staphylococcus aureus BMC-1 Refined 2.5 57 58 47 25
1.25 46 81 56 36
Fried 2.5 56* 54* 45* 25*
1.25 53** 79** 51** 37**
Candida albicans D-6 Refined 2.5 46 52 34 47
1.25 42 49 47 65
Fried 2.5 51* 51* 40* 44*
1.25 n.d. 48** 43** 51**
Aspergillus niger P-3 Refined 2.5 60 N.d. N.d. n.d.
1.25 77 N.d. N.d. N.d.
Fried 2.5 54* N.d. N.d. N.d.
1.25 51** N.d. N.d. N.d.
N. vaccinii IMV B-7405 on refined and fried sunflower oil, at an order of magnitude higher concentrations of surfactants (10-40 pg/ml).
It should be noted that the publications of the anti-adhesive properties of surfactants, synthesized on oil-containing substrates, are not numerous. In [28] it was reported that Rufisan synthesized by C. lipolytica UCP0988 on waste products of soybean oil reduced the adhesion of bacteria of the genus Streptococcus and Lactobacillus on polystyrene plates.
For example, at the minimum investigated concentration of MS (0.75 mg/l), the adhesion of test cultures was 61-91%. With increased MS concentration in a solution up to 12 mg/l, Rufisan reduced the number of attached E. coli and C. albicans cells by 21-51%.
The probiotic strain Propionibacterium freudenreichii subsp. freudenreichii PTCC 1674 in cultures on various substrates including waste sunflower oil synthesized MS, which at a concentration of 10 mg/ml reduced the amount of E. coli cells attached
to plastic by 13% and Staphylococcus aureus by 37% [33].
The data in Table 2 show that A. calcoaceticus IMV В-7241 on refined and processed oils synthesized MS, which were more effective as anti-adhesive agents than Rufisan and MS produced by P. freudenreichii subsp. freudenreichii PTCC 1674, because they caused the 35-75% decrease of the adhesion of test cultures at a concentration of only 1.25-2.5 pg/ml.
The role of MS of A. calcoaceticus IMV В-7241 in biofilm destruction. The data presented in Table 3 indicate that the degree of destruction of bacterial biofilms in the presence of surfactant depended on the quality of the oil used as substrate (refined, fried), the concentration of MS and the type of test culture. For example, the degradation of B. subtilis BT-2 biofilm for all studied concentrations of surfactant (29-233 pg/ml) obtained on fried sunflower oil was 6-15% higher compared to the values established
Table 3. The influence of surfactants synthesized by A. calcoaceticus IMV В-7241 on refined and fried sunflower oil on the destruction of bacterial biofilms
Test culture Oil substrate Destruction (%) of biofilms treated with MS in concentrations (pg/ml)
29 58 116 233
Bacillus subtilis BT-2 Refined 5 7 37 48
Fried 11* 19* 47* 63*
Staphylococcus aureus BMC-1 Refined 52 53 53 55
Fried 36* 38* 57* 66*
Escherichia coli IEM-1 Refined 50 54 67 75
Fried 83* 42* 33* 25*
Enterobacter cloacae C-8 Refined 24 29 52 54
Fried 32* 47* 53* 55
Notes. The producer was grown on medium with 2% (v/v) oil after frying potatoes. The statistical error of determination of the biofilm destruction did not exceed 5 %.
* — P < 0.05 compared to control (biofilm destruction after treatment with solutions of MS synthesized on medium with 2% refined sunflower oil).
for MS synthesized on the refined substrate. The degree of destruction of the S. aureus BMS-1 biofilm in the presence of MS formed on fried oils was 4-11% higher compared with the effect of MS obtained on refined oil, only at higher concentrations of such surfactants (116-233 pg/ml). At the same time, the maximum destruction of E. coli IEM-1 biofilm (83%) was achieved at the minimum the concentration (29 pg/ml) of surfactants synthesized on the fried oil. The degree of destruction of E. cloacae C-8 biofilm in the presence of 116-233 pg/ml of surfactant, was similar for MS synthesized on refined and fried oil (52-55%). However, at lower concentrations (29-58 pg/ml), MS obtained on fried oils destroyed the biofilm of this test culture better.
Hence, based on the data presented in Table 3, it can be concluded that replacing the refined oil with the fried oil in the cultivated medium of A. calcoaceticus IMV B-7241 is accompanied by the synthesis of surfactant which at low concentrations effectively destroys bacterial biofilms.
Notably, there are practically no reports about destruction of biofilms in the presence of surfactants synthesized in oil-containing substrates, although there are many publications on the effect of microbial surfactants on the degree of their destruction. Such data were summed up in the review [34]. The same publication presents data on the destruction of biofilms
by surfactants synthesized by A. calcoaceticus IMV B-7241 on traditional substrates (ethanol, purified glycerin, hexadecane). Unlike MS derived from waste oils, surfactants produced by the IMV strain V-7241 on traditional substrates efficiently destroyed biofilms at higher concentrations (up to 1.28 mg/ml) [34].
In 2017, a lipopeptide synthesized by the actinobacteria strain Nesterenkonia sp. MSA31, isolated from the marine sponge Fasciospongia cavernosa (Lendenfeld, 1889), was reported to affect the destruction of S. aureus biofilm [35]. The MSA31 strain was grown in medium with 10% olive oil, the lipopeptide was extracted with organic solvents (ethyl acetate, methanol, petroleum ether, dichloromethane) from a supernatant pre-acidified to pH 2.0. Surfactant solutions in the range of concentrations of 25 to150 pg/ml were used to determine the effect of the lipopeptide in destroying the biofilms. It was established that the maximum destruction of S. aureus biofilms (90%) was achieved in the presence of lipopeptide at a concentration of 125 pg/ml [35].
Effect of MS on the destruction of oil pollution in water. Previously [36], we have shown that MS synthesized by A. calcoace-ticus IMV B-7241 on traditional substrates intensified the decomposition of oil in water and soil, even in the presence of heavy metals. One of the determined mechanisms of intensification of oil decomposition
Table 4. Oil destruction in water and quantitative changes in microbiota in the presence of culture liquid after the cultivation of the A. calcoaceticus IMV B-7241 strain on refined and fried oil
Oil as substrate Oil concentration in medium,% Degree of oil destruction (%) at initial concentration (g/l) Microorganisms in water (CFU/ml) at oil concentration (g/l)
3.0 6.0 3.0 6.0
Refined 2 71* 70* (1.9 ± 0.09) 106** (2.2 ± 0.11)106**
Fried 2 72* 70* (2.0 ± 0.10)106** (2.2 ± 0.11)106**
4 88* 80* (3.1 ± 0.15)106** (2.9 ± 0.14)-106**
Notes. The producer was grown on medium with 2% (v/v) oil after frying potatoes. The statistical error of determination of the oil destruction did not exceed 5 %. * — P < 0.05 compared to control (8% — degree of oil destruction in water without the culture liquid). ** — P < 0.05 compared to control (2.6104 CFU/ml — initial number of cells in water before the addition of oil and culture liquid).
is the activation of natural oil-oxidizing microbiota under the influence of microbial surfactants.
However, high antimicrobial activity of microbial surfactants can be a significant barrier to their application in environmental technologies. For example, in some cases, highly concentrated MS can reduce the rate of purification process, adversely affecting destructor microorganisms. Whang et al. [37] note that surfactin at a concentration of 40 mg/l negatively affected the remediation of soils from diesel fuel, and at a concentration of 400 mg/l completely inhibited it. It should be noted that there are no data on the correlation of surfactant's antimicrobial activity and its role in the degradation of oil pollution.
Hence, the effect of surfactant A. calcoaceticus IMV B-7241 with different antimicrobial activity on decomposition of oil in water was investigated at the next stage of our study (Table 4).
It was established that the degree of oil destruction in water (both in concentrations of 3.0 and 6.0 g/l) and the total number of microorganisms which survived to the end of the experiment in the presence of MS, synthesized in medium with 4% of fried oil, were 10-16% and in 1.3-1.5 times higher than the values established for the MS, obtained in a medium with a
lower (2%) concentration of the substrate. This can be explained by the fact that increasing the concentration of waste oil up to 4% in the A. calcoaceticus IVB-7241 culture medium induces formation of the surfactants with low antimicrobial activity (Table 1). As can be seen from the data in Table 4, such surfactants are promising for the purification of the environment from xenobiotics, since they do not have a negative effect on natural destructor microorganisms.
Hence, it was found that the replacement of refined oil (2%) with a similar concentration of fried waste oil in the culture medium of A. calcoaceticus IMV B-7241 was accompanied by the synthesis of surfactants with high antimicrobial and anti-adhesive activity (including the ability to destroy biofilms).
Increasing the concentration of waste oil to 4-5% allows the production of surfactants with low antimicrobial activity, which can be used in environmental technologies for the destruction of xenobiotics.
The presented data confirm the necessity of research on the influence of culture conditions on the properties of synthesized surfactants in order to obtain a target product with stable predetermined properties depending on the field of practical application.
REFERENCES
1. Fracchia L., Banat J. J., Cavallo M., Ceresa C., Banat I. M. Potential therapeutic applications of microbial surface-active compounds. AIMS Bioengineering. 2015, 2 (3), 144-162. https:// doi.org/10.3934/bioeng.2015.3.144
2. Paulino B. N, Pessôa M. G., Mano M. C., Molina G., Neri-Numa I. A., Pastore G. M. Current status in biotechnological production and applications of glycolipid biosurfactants. Appl. Microbiol. Biotechnol. 2016, 100 (24), 10265-10293. https://doi.org/10.1007/ s00253-016-7980-z
3. Irorere V. U., Tripathi L., Marchant R., McClean S., Banat I. M. Microbial rhamnolipid production: a critical reevaluation of published data and suggested future publication criteria. Appl. Microbiol. Biotechnol. 2017, 101 (10), 3941-3951. https://doi.org/10.1007/s00253-017-8262-0
4. Franco Marcelino P. R., da Silva V. L., Rodrigues Philippini R., Von Zuben C. J., Contiero J., Dos Santos J. C., da Silva S. S. Biosurfactants produced by Scheffersomyces stipitis cultured in sugarcane bagasse hydrolysate as new green larvicides for the control of Aedes aegypti, a vector of neglected tropical diseases. PLoS One. 2017, 12 (11), e0187125. https://doi.org/10.1371/journal. pone.0187125
5. Parthipan P., Preetham E., Machuca L. L., Rahman P. K., Murugan K., Rajasekar A. Biosurfactant and degradative enzymes mediated crude oil degradation by bacterium Bacillus subtilis A1. Front. Microbiol. 2017, V. 8, P. 193. https://doi.org/10.3389/ fmicb.2017.00193. 2017
6. Sekhon Randhawa K. K., Rahman P. K. Rhamnolipid biosurfactants — past, present, and future scenario of global market. Front. Microbiol. 2014, V. 5. P. 454. https://doi. org/10.3389/fmicb.2014.00454
7. Chong H., Li Q. Microbial production of rhamnolipids: opportunities, challenges and strategies. Microb. Cell Fact. 2017, 16 (1), 137. https://doi.org/10.1186/s12934-017-0753-2
8. Ebadipour N., Lotfabad T. B., Yaghmaei S., RoostaAzad R. Optimization of low-cost biosurfactant production from agricultural residues through response surface methodology. Prep. Biochem. Biotechnol. 2016, 46 (1), 30-38. https://doi.org/10.1080/10826 068.2014.979204
9. Pirog T. P., Shulyakova M. O., Nikituk L. V., Antonuk S. I., Elperin I. V. Industrial waste bioconversion into surfactants by Rhodococcus erythropolis IMV Ас-5017, Acinetobacter calcoaceticus IMV В-7241 and Nocardia vaccinii IMV В-7405. Biotechnol. acta. 2017, 10 (2), 22-33. https://doi.org/10.15407/ biotech10.02.022
10. Almeida D. G., Soares da Silva R. C., Luna J. M., Rufino R. D, Santos V. A., Sarubbo L. A. Response surface methodology for optimizing the production of biosurfactant by Candida tropicalis on industrial waste substrates. Front. Microbiol. 2017, V. 8, P. 157. https://doi.org/10.3389/ fmicb.2017.00157
11. Pirog T. P., Sidor I. V., Lutsai D. A. Calcium and magnesium cations influence on antimicrobial and antiadhesive activity of Acinetobacter calcoaceticus IMV B-7241 surfactants. Biotechnol. acta. 2016, 9 (6), 50-57. https://doi.org/10.15407/ biotech9.06.050
12. Pirog T. P., Nikituk L. V., Shevchuk T. A. Influence of divalent cations on synthesis of Nocardia vaccinii IMV B-7405 surfactants with high antimicrobial and anti-adhesion activity. Mikrobiol. Zh. 2017, 79 (5), 13-22. (In Ukrainian).
13. De Rienzo M. A., Martin P. J. Effect of mono and di-rhamnolipids on biofilms preformed by Bacillus subtilis BBK006. Curr. Microbiol. 2016, 73 (2), 183-189. https:// doi.org/10.1007/s00284-016-1046-4
14. Kim K., Lee Y., Ha A., Kim J. I., Park A. R., Yu N. H., Son H,, Choi G. J., Park H. W., Lee C. W., Lee T., Lee Y. W., Kim J. C. Chemosensitiza-tion of Fusarium graminearum to chemical fungicides using cyclic lipopeptides produced by Bacillus amyloliquefaciens strain JCK-12. Front. Plant Sci. 2017, V. 8, P. 2010. https://doi.org/10.3389/fpls.2017.02010
15. Tiso T., Zauter R., Tulke H., Leuchtle B., Li W. J., Behrens B., Wittgens A, Rosenau F., Hayen H., Blank L. M. Designer rhamnolipids by reduction of congener diversity: production and characterization. Microb. Cell Fact.
2017, 16 (1), 225. https://doi.org/10.1186/ s12934-017-0838-y
16. Ramachandran R., Shrivastava M., Narayanan N. N., Thakur R. L., Chakrabarti A, Roy U. Evaluation of antifungal efficacy of three new cyclic lipopeptides of the class bacillomycin from Bacillus subtilis RLID 12.1. Antimicrob. Agents Chemother.
2018, V. 62, P. e01457-17. https://doi. org/10.1128/AAC.01457-17
17. Pirog T. P., Lutsai D. A., Shevchuk T. A., Iutynska G. O., Elperin I. V. Antimicrobial and anti-adhesive activity of surfactants synthesized by Acinetobacter calcoaceticus IMV V-7241 on technical glycerol. Mikrobiol. Zh. 2018, 80 (2), 14-27. https://doi.org/10.15407/ microbiolj80.02.014 (In Ukrainian).
18. Pirog T. P., Nikituk L. V., Antonuk S. I., Shevchuk T. A., Iutynskaya G. A. Intensification of Acinetobacter calcoaceticus IMV B-7241 surfactants synthesis on waste sunflower oil. Mikrobiol. Zh. 2018,
80 (1), 15-26. https://doi.org/10.15407/ microbiolj80.01.015 (In Russian).
19. Choe E., Min D. B. Chemistry of deep-fat frying oils. J. Food Sci. 2007, 72 (5), R77-86.
20. Totani N., Ono M., Burenjargal M., Ojiri Y. Carbonyl compounds vaporize from oil with steam during deep-frying. J. Oleo Sci. 2007, 56 (9), 449-456.
21. Noor S., Punekar N. S. Allosteric NADP-glutamate dehydrogenase from aspergilli: purification, characterization and implications for metabolic regulation at the carbon-nitrogen interface. Microbiology. 2005, V. 151, P. 1409-1419. https://doi. org/10.1099/mic.0.27751-0
22. Choudhury R., Noor S., Varadarajalu L. P., Punekar N. S. Delineation of an in vivo inhibitor for Aspergillus glutamate dehydrogenase. Enzyme Microb. Technol. 2008, 42 (2), 151-159. https://doi. org/10.1016/j.enzmictec.2007.08.011
23. Pirog T. P., Shevchuk T. A., Voloshina I. N, Gregirchak N. N. Use of claydite-immobilized oil-oxidizing microbial cells for purification of water from oil. Appl. Biochem. Microbiol. 2005, 41 (1), 51-55. https://doi. org/10.1007/s10438-005-0010-z
24. Pirog T., SofilkanychA., KononA., Shevchuk T., Ivanov S. Intensification of surfactants' synthesis by Rhodococcus erythropolis IMV Ac-5017, Acinetobacter calcoaceticus IMV B-7241 and Nocardia vaccinii K-8 on fried oil and glycerol containing medium. Food Bioprod. Process. 2013, 91 (2), 149-157. http://dx.doi.org/10.1016/j. fbp.2013.01.001
25. Santos D. K., Rufino R. D., Luna J. M., Santos V. A., Sarubbo L. A. Biosurfactants: multifunctional biomolecules of the 21st century. Int. J. Mol. Sci. 2016, V. 17 (3). https://doi.org/10.3390/ijms17030401
26. Gudina E. J., Teixeira J. A., Rodrigues L. R. Biosurfactants produced by marine microorganisms with therapeutic applications. Mar. Drugs. 2016, V. 14 (2). https://doi.org/10.3390/md14020038
27. PantazakiA.A., Dimopoulou M. I., Simou O. M., Pritsa A. A. Sunflower seed oil and oleic acid utilization for the production of rhamnolipids by Thermus thermophilus HB8. Appl. Microbiol. Biotechnol. 2010, 88 (4), 939-951. https://doi.org/10.1007/s00253-010-2802-1
28. Rufino R. D., Luna J. M., Sarubbo L. A. Antimicrobial and anti-adhesive potential of a biosurfactant Rufisan produced by Candida lipolytica UCP 0988. Coll. Surf. B. Biointerfaces. 2011, 84 (1), 1-5. https://doi. org/10.1016/j.colsurfb.2010.10.045
29. Nitschke M., Costa S. G., Contiero J. Structure and applications of a rhamnolipid surfactant produced in soybean oil waste. Appl. Biochem. Biotechnol. 2010, 160 (7), 2066-2074. https://doi.org/10.1007/s12010-009-8707-8
30. Pirog T. P., Panasyuk E. V., Nikityuk L. V., Iutinska G. O. Influence of cultivation conditions on antimicrobial properties of Nocardia vaccinii IMV B-7405 surfactants. Biotechnol. acta. 2016, 9 (1), 38-47. https:// doi.org/10.15407/biotech9.01.038
31. Pirog T. P., Nikituk L. V., Antonuk S. I., Shevchuk T. A., Iutynska G. O. Peculiarities of Nocardia vaccinii IMV B-7405 surfactants synthesis on waste oil of different quality and their antimicrobial properties. Mikrobiol. Zh. 2017, 79 (2), 13-22. https:// doi.org/10.15407/microbiolj79.02.013 (In Ukrainian).
32. Pirog T. P., Nikituk L. V., Tymoshuk K. V., Shevchuk T. A., Iutynska G. O. Biological properties of Nocardia vaccinii IMV B-7405 surfactants synthesized on fried sunflower oil. Mikrobiol. Zh. 2016, 78 (2), 2-12. (In Ukrainian). http://microbiolj.org.ua/images/files/ magazine/2016/2/2016_78_2_01_Pirog.pdf
33. Hajfarajollah H., Mokhtarani B., Noghabi K. A. Newly antibacterial and antiadhesive lipopeptide biosurfactant secreted by a probiotic strain, Propionibacterium freudenreichii. Appl. Biochem. Biotechnol. 2014, 74 (8), 2725-2740. https://doi. org/10.1007/s12010-014-1221-7
34. Pirog T. P., Savenko I. V., Lutsay D. A. Microbial surface-active substances as antiadhesive agents. Biotechnol. acta. 2016, 9(3), 7-22, https://doi.org/10.15407/ biotech9.03.007
35. Kiran G. S., Priyadharsini S., Sajayan A., Priyadharsini G. B., Poulose N., Selvin J. Production of lipopeptide biosurfactant by a marine Nesterenkonia sp. and its application in food industry. Front. Microbiol. 2017, V. 8, P. 1138. https://doi.org/10.3389/ fmicb.2017.01138
36. Pirog T. P., Konon A. D., Savenko I. V. Microbial surfactants in environmental technologies. Biotechnol. acta. 2015, 8 (4), 21-39. https://doi.org/10.15407/ biotech8.04.021
37. Whang L. M., Liu P. W., Ma C. C., Cheng S. S. Application of biosurfactants, rhamnolipid, and surfactin, for enhanced biodegradation of diesel-contaminated water and soil. J. Hazard. Mater. 2008, 151 (1), 155-163.
ВЛАСТИВОСТ1 ПОВЕРХНЕВО-АКТИВНИХ РЕЧОВИН, СИНТЕЗОВАНИХ
Acinetobacter calcoaceticus 1МВ В-7241 НА РАФ1НОВАН1Й I В1ДПРАЦЬОВАН1Й СОНЯШНИКОВ1Й ОЛП
Т. П. Пирог, Д. А. Луцай, С. I. Антонюк, I. В. ЕльперЬн
Нащональний ушверситет харчових технологш, Кшв, Укра1на
E-mail: [email protected]
Метою роботи було порiвняти антимшроб-ну та антиадгезивну актившсть (зокрема й здатшсть до руйнування бiоплiвок), а також дослщити вплив на деструкщю нафтових за-бруднень поверхнево-активних речовин, синте-зованих у процем культивування Acinetobacter calcoaceticus 1MB В-7241, на рафшованш та вiдпрацьованiй соняшниковш олiï.
Поверхнево-активнi речовини екстрагували i3 супернатанта культуральноï рiдини сумiшшю хлороформу i метанолу (2:1). Кiлькiсть адгезо-ваних клiтин i ступiнь руйнування бiоплiвки визначали спектрофотометричним методом, антимшробш властивостi — за показником мь нiмальноï iнгiбувальноï концентрацiï (MIK). Концентращю нафти у водi вимiрювали ваговим методом тсля екстрагування гексаном.
Встановлено, що мiкробнi поверхнево-ак-тивнi речовини, синтезоваш у середовищi з 2% як рафшовано^ так i вiдпрацьованоï олiï, характеризувалися високою антимiкробною (MIK щодо бактерiйних тест-культур 0,8-29 мкг/мл, щодо Candida albicans Д-6 — 26-58 мкг/мл) та антиадгезивною (зниження кшькосм при-крiплених до аб^тичних поверхонь клiтин тест-культур бактерiй i грибiв на 35-70%, руйнування бiоплiвок у середньому на 40-44%) актившстю. Пiдвищення концентрацiï вщпра-цьованоï олiï у середовищi до 4% супроводжу-валося утворенням мiкробних поверхнево-ак-тивних речовин з невисокою антимшробною активнiстю, за наявностi яких стутнь деструк-цiï нафти у водi (3-6 г/л) на 20-ту добу досягав 80-88%, що на 10-16% вище, шж у разi вико-ристання поверхнево-активних речовин, синте-зованих у середовишД з 2% оли.
Наведенi данi свiдчать про необхщшсть проведення дослiджень впливу умов культивування продуценив на властивостi синтезо-ваних мiкробних поверхнево-активних речовин з метою одержання щльового продукту з1 стабiльними наперед заданими властивостями залежно вщ галузi практичного застосування.
Ключовi слова: мшробш поверхнево-активш речовини, вщпрацьована соняшникова олiя, антимiкробна та антиадгезивна активнiсть.
СВОЙСТВА ПОВЕРХНОСТНО-АКТИВНЫХ ВЕЩЕСТВ, СИНТЕЗИРОВАННЫХ Acinetobacter calcoaceticus ИМВ В-7241
НА РАФИНИРОВАННОМ И ОТРАБОТАННОМ ПОДСОЛНЕЧНОМ МАСЛЕ
Т. П. Пирог, Д. А. Луцай, С. И. Антонюк, И. В. Эльперин
Национальный университет пищевых технологий, Киев, Украина
E-mail: [email protected]
Целью работы было сравнить антимикробную и антиадгезивную активность (в том числе и способность к разрушению биопленок), а также исследовать влияние на деструкцию нефтяных загрязнений поверхностно-активных веществ, синтезированных при культивировании Acinetobacter calcoaceticus ИМВ В-7241, на рафинированном и отработанном подсолнечном масле.
Поверхностно-активные вещества экстрагировали из супернатанта культуральной жидкости смесью хлороформа и метанола (2:1). Количество адгезированных клеток и степень разрушения биопленки определяли спектрофотометрическим методом, антимикробные свойства — по показателю минимальной ингибирующей концентрации (МИК). Концентрацию нефти в воде измеряли весовым методом после экстракции гексаном.
Установлено, что поверхностно-активные вещества, синтезированные в среде с 2% как рафинированного, так и отработанного масла, характеризовались высокой антимикробной (МИК по отношению к бактериальным тест-культурам 0,8-29 мкг/мл, Candida albicans Д-6 — 26-58 мкг/мл) и антиадгезивной (снижение количества прикрепленных к абиотическим поверхностям клеток тест-культур бактерий и грибов на 35-70%, разрушение биопленок в среднем на 40-44%) активностью. Повышение концентрации отработанного масла в среде до 4% сопровождалось образованием поверхностно-активных веществ с невысокой антимикробной активностью, в присутствии которых степень деструкции нефти в воде (3-6 г/л) на 20-е сутки достигала 80-88%, что на 10-16% выше, чем при использовании поверхностно-активных веществ, синтезированных в среде с 2% масла.
Приведенные данные свидетельствуют о необходимости проведения исследований влияния условий культивирования продуцентов на свойства синтезированных поверхностно-активных веществ для получения целевого продукта со стабильными заданными свойствами в зависимости от области практического применения.
Ключевые слова: микробные поверхностно-активные вещества, отработанное подсолнечное масло, антимикробная и антиадгезивная активность.
