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VIABILITY AND PHENOTYPIC HETEROGENEITY OF RHODOCOCCUS RHODO-CHROUS CNMN-Ac-05 IN THE PRESENCE OF FULLERENE C60
Olga POSTOLACHI, Inna RASTIMESINA, Valentina JOSAN Institute of Microbiology and Biotechnology, Republic of Moldova
Corresponding author: Olga Postolachi, e-mail: [email protected]
DOI: 10/38045/ohrm.2021.3.01 CZU: 579.87.083.13:615.281.9
Keywords: fullerene C60, rhodococci, viability, phenotypic heterogeneity.
Cuvinte cheie: fule-rena C60, rodococi, viabilitate, heteroge-nitate fenotipicâ.
Introduction. In recentyears, due to wide applications ofnanotechnologies in various fields, the safety of nanomaterials has become a pressing issue. Fullerene C60 is not an exception. Research on the activity of microorganisms and their interaction with nanoparticles is of major importance, both for microorganisms and for the ecosystem as a whole. Material and methods. Fullerene C60 powder was purchased from Sigma-Aldrich. The object of study was R. rhodochrous CNMN-Ac-05 strain. The number of viable bacterial cells was estimated by colony-forming units (CFU). The morphological features of the rhodococci colonies have been described according to the usual microbiological method. Results. It was established that fullerene C60 in concentrations of 1-25 mg/L fullerene C60 stimulated the growth of R. rhodochrous by 2.4-2.8 times. As the concentration of fullerene C60 increased up to 50-100 mg/L, the multiplication and growth of rhodococci decreased by 29.5% and 38% respectively. In the presence of 1-10 mg/L fullerene C60 the rhodococci population remained homogeneous, being composed of 100% S type colonies. The increase of fullerene C60 concentration led both to the decrease in the CFU number and to the appearance of R type colonies, up to 1.3% of population.
Conclusions. Fullerene C60 in concentrations 1-100 mg/L had no obvious toxic effect on the rhodococci strain. The optimum concentration is 10 mg/L. The concentrations higher than 25 mg/L led to the dissociation of rhodococcalpopulation and diminution in the CFU counts, but not to the total inhibition.
VIABILITATEA SI HETEROGENITATEA FENOTIPICÂ A TULPINII RHODOCOCCUS RHODOCHROUS CNMN-Ac-05 ÎN PREZENTA FULERENEI C 60
Introducere. Siguranta nanomaterialelor devine din ce în ce mai actualâ, având în vedere utilizarea tot mai intensâ a nanotehnologiilor în diferite domenii. Fullerena C60 nu este o exceptie. Cercetârile privind activitatea vitalâ a microorganismelor si interactiunea lor cu nanoparticulele are importantâ majorâ atât pentru fiecare microorganism în parte, cât si pentru ecosistem în totalitate.
Material si metode. Fulerena C60 a fost achizitionatâ de la Sigma-Aldrich. Obiectul cercetârii a servit tulpina R. rhodochrous CNMN-Ac-05. Numârul de celule bacterien e viabile a fost estimat prin unitâti formatoare de colonii (UFC). Caracterele morfologice ale coloniilor de rodococi au fost descrise conform metodei microbiologice uzuale. Rezultate. S-a stabilit câ fulerena C60, în concentratii de 1-25 mg/L, stimuleazâ cresterea tulpinii R. rhodochrous de 2,4-2,8 ori. Odatâ cu cresterea concentratieipânâ la 50-100 mg/L, multiplicarea si cresterea rodococilor a scâzut cu 29,5% si, respectiv, 38%. Tulpina de rodococi crescutâ în prezenta a 1 -10 mg/L fulerena C60 a râmas omogenâ, populatia fiind alcâtuitâ din 100% colonii de tip S. Mârirea concentratiei de fulerenâ C60 a dus nu doar la scâderea numârului de celule, dar si la aparitia coloniilor de tip R, pânâ la 1,3% din populatie.
Concluzii. Fulerena C60 în concentratiile 1-100 mg/L nu are efect toxic evident asupra tulpinii de rodococi. Concentratia optimalâ este de 10 mg/L. Concentratiile mai mari de 25 mg/L duc la disocierea populatiei si la scâderea semnificativâ a numarului CFU, dar nu si la inhibare totalâ.
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INTRODUCTION
Fullerenes are an allotropic form of carbon with many synthesized modifications. Due to its structure, composed of 60 carbon atoms, fullerene C60 are good heat and electricity conductor that possess an excellent tensile strength. These properties make it to be a unique functional material for electronics and optics, energy, biochemistry, and molecular medicine. More recently, fullerenes have been used for bioremediation of environment contaminated with polyethylene (1), pesticides (2, 3), or other xenobiotics of different nature (4).
Wide applications use of nanotechnologies in various fields, including the field of remediation and reduction of environmental contamination, the safety of nanomaterials has become a current issue. Regarding the action of fullerene C60 on living cells, scientists observed some effects: on the one hand, the antioxidant action of fullerene was shown (5, 6, 7), on the other hand the antimicrobial activity was demonstrated (6, 8-11). For example, a nano-composite containing fullerene was effective in degrading pesticides Imidaclo-prid, Isoproturon and Malathion, but at the same time, it was used for inactivating Pseudomonas aureus, Staphylococcus aureus, Pseudomonas fluo-rescens, Bacillus subtilis and Escherichia coli bacteria (2).
Numerous studies of fullerene C60 have revealed that its biological activity is rather complex and multilateral, due to several factors, such as tro-pism for cell membranes, due to its lipophilicity, interaction with free radicals, and fullerene's ability to transfer energy to the oxygen molecule and convert it to single oxygen (7, 12, 13). It has been shown that the toxicity of fullerene C60 is determined by a number of factors, such as particle size, organic matter and ionic strength of the medium, fullerene C60 concentration and time of exposure, bacterial growth conditions, the age of C60 used, and the bacterial species tested (5, 8, 14).
Over time, it has been shown that the advantages of immobilized microorganisms, compared to free cells, are the enhanced stability of the bioca-talyst and posibility of recovering and reusing microorganisms. Moreover, immobilization could also protect the cells and thus increase the tolerance to high concentration of pollutants (15, 16). Voznyakovskii, et al. (2020) (17) demonstrated
the possibility of using microorganisms immobilized on carbon structures for eliminating the consequences of contamination with petroleum products.
The Rhodococcus rhodochrous CNMN-Ac-05 strain was retrieved from the stock of our laboratory, which is a destructor of benzothiazole and its metabolites (18). The purpose of our research was to evaluate the effects of fullerene on the viability and phenotypic heterogeneity of R. rhodochrous CNMN-Ac-05 in order to estimate the possibility of using fullerene C60 along with the rho-dococcal strain in bioremediation procedures.
MATERIAL AND METHODS
Chemicals. Fullerene C60 powder (purity 98%) was purchased from Sigma-Aldrich. The particle size is approximately 0.7 nm in diameter.
Bacterial strain and culture conditions.
Rhodococcus rhodochrous CNMN-Ac-05 was deposited within the National Collection of Non-Pathogenic Microorganisms of the Republic of Moldova, being able to degrade benzothiazoles and persistent organic pollutants (18). R. rhodochrous was grown in 100 mL portions of Tryptic soy (TS) broth (Sigma-Aldrich) in 300 mL Erlenmeyer flasks incubated at 28°C and 200 rpm. The cells were harvested over 36 h of culture and centrifuged at 6.000 rpm for 20 min. The bacterial pellet was washed first with a NaCl solution (0.8%) and then with distillated water.
Determination of the effects of fullerene C60.
Bacterial biomass was resuspended in distillated water (pH 7.2) to prepare cell suspension 6 mg/mL (1.4 mg cell dry weight/mL). The concentration of the cell biomass was determined spectrophotometrically by measuring the optical density of culture at A=540 nm, with subsequent recalculation to cell dry weight according to the calibration curve. Colloidal aqueous suspension of fullerene C60, 2 mg/mL was prepared on ultrasonic cleaner at 50 kHz for 5 min. For experiments, 5 mL of bacterial cells suspension was added in 250 mL Erlenmeyer flasks containing 95 mL of medium PAS and fullerene C60 in following concentrations (mg/L): 1, 10, 25, 50, and 100. The PAS medium contained (g/L): 4.35 K2HPO4, 1.7 KH2PO4, 2.1 NH4CL 0.2 MgSO4, 0.05 MnSO4, 0.01 FeSO4 7H2O, and 0.03 CaCl2 2H2O. pH adjusted at 7.2. Inoculated flasks were
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incubated in a rotary shaker (180 rpm) at 28°C for 24 h. After a serial dilution, the 50 [iL of suspension was spread on agar plates with TS medium; afterwards the plates were incubated at 28°C for 96 h until the bacterial colonies appeared. The number of viable bacterial cells was estimated by colony-forming units (CFU) inoculated on agar plates.
The morphological features of the rhodococci colonies were described according to Egorov method (19), by using a magnifying glass (8-fold magnification).
Statistical analysis was performed via MS Excel. All results were expressed as means of three individual replicates ±CI (confidence intervals). All the differences were considered statistically significant at P<0.05.
RESULTS
The results of the action of fullerene Ceo on the viability of R. rhodochrous CNMN-Ac-05 are shown in Figure 1. These results indicated that the concentration of the test substance was of major importance. The optimal concentrations for rhodo-coccal cell multiplication were 1-25 mg/L. At these concentrations, the growth of the strain was stimulated by 2.4-2.8 times compared to the control samples. The highest CFU count value was obtained at a concentration of 10 mg/L. However, an increase in the fullerene Ceo concentration up to 50 and 100 mg/L, obviously decreased the rhodo-cocci capacity of multiplication and growth. The values of the CFU number were lower by 29.5% and 38.0% compared to the control samples.
Figure 1. Influence of fullerene Ceo on the viability R. rhodochrous CNMN-Ac-05.
In addition to cell multiplication, the phenotypic modification of Rhodococcus colonies in the presence of fullerene Ceo was described. Macroscopi-cally observable features and colony types of R. rhodochrous CNMN-Ac-05 are presented in Table 1 and Figure 2.
Basic morphological features of R. rhodochrous CNMN-Ac-05 colonies were similar to the S type (smooth). Thus, cultivation on TS medium without fullerene (control), resulted in the formation of 100% S type colonies. The colonies of S type were dominant in all the experimental variants, regardless of fullerene Ce0 concentration, ranging between 98.7-100% (fig. 3).
The population of R. rhodochrous cultivated in the presence of 1 and 10 mg/L of fullerene Сбо remained homogeneous, being composed of 100% S type colonies, similar to the control sample. Moreover, cultivation in the presence of 25 mg/L fullerene Сбо, under conditions of active multiplication, resulted in phenotypic dissociation of strain into two types of colonies, S (99.6%) and R (0.4%) types.
The further increase of fullerene Сб0 concentration (50-100 mg/L) caused both a substantial decrease in the cell number and an increase in the phenotypic heterogeneity of the rhodococci. The highest percentage of R type cells - 1.3% was observed at the concentration of 100 mg/L.
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Table 1. Types of colonies formed by R. rhodochrous CNMN-Ac-05.
Morphological Colony type
features S R
Form Round Irregular
Size, mm 1.0-4.0 1.0-4.0
Margin Entire Undulate
Elevation Convex Convex
Surface Smooth and glistening Rough and dull
Color Pink Pink
Opacity Opaque Opaque
Type S
Type R
Figure 2. Types of colonies formed by R. rhodochrous CNMN-Ac-05 cultivated in the presence of fullerene Ceo.
Figure 3. Dissociation of R. rhodochrous CNMN-Ac-05 grown in the presence of fullerene Ce
DISCUSSIONS
The genus Rhodococcus belongs to the phylum and class Actinobacteria, the order Actinomy-cetales, and the family Nocardiaceae. Rhodococci possess a broad catabolic diversity and numerous enzymatic capabilities. They are able to use a wide range of organic compounds as sole sources of carbon and energy for growth, and that makes them well-equipped for biotransformation and biodegradation of xenobiotic compounds (15, 20, 21).
Since rhodococci are nocardioform bacteria, the phenotypic heterogeneity is quite common among them. According to Goodfellow et al. (22), rhodococci colonies may be mucoid, rough or
smooth, as well as a pigmented, yellow, orange, red, or colorless buff and cream. The splitting of a homogeneous rhodococci population into variants with different morphological, physiological, biochemical, and genotypic properties was observed by many researchers (23-26).
Phenotypic heterogeneity refers to the phenomenon when individual cells within an isogenic population, that have a uniform genetic background, can nevertheless display differences in phenotype (27, 28). Non-genetic variations, that exist within an isogenic population, benefit the population through division of labor and improving the ability to exhibit a high level of metabolic activity. Therefore, phenotypic heterogeneity allows bacterial populations to
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improve their ability to adapt to changing environments (29, 30).
In the present study, the concentration-toxicity ratio of fullerene C60 to rhodococcal cells was clearly established. In case of concentrations of 1 and 10 mg/L, the population of rhodococci was homogeneous and grew very actively, whereas starting with 25 mg/L, R type colonies appeared. Since typical S colonies are produced only under optimal cultural conditions, the appearance of R type colonies indicates a stressful condition,
caused by the presence of increased concentrations of fullerene C60. This phenomenon was confirmed by the experimental variants, when rhodococci were grown in the presence of 50 and 100 mg/L of fullerene C60. The number of R type colonies increased in these variants, additionally to the decrease in the CFU count. Similar results were obtained by Sah et al. (2010) (1) that reported a moderately decrease in the CFU counts of bacterial strains at the concentration of 100 mg/L fullerene C60.
CONCLUSIONS
1. The fullerene C60 in tested concentrations did not display any obvious toxic effects on R. rhodo-chrous CNMN-Ac-05 cells. The addition of fullerene C60 in concentrations up to 25 mg/L, stimulates the growth and multiplication of rhodococcal cells. The optimum concentration was 10 mg/L.
2. Concentration increases beyond 25 mg/L caused a dissociation of the rhodococcal population, expressed by the appearance of R type colonies, as well as a significant decrease of the CFU counts (29.5-38.0%), no total inhibition occurs. Higher fullerene C60 concentrations resulted in lower growth activity and higher phenotypic heterogeneity of the strain.
CONFLICT OF INTERESTS
The authors declare no conflict of interest.
ACKNOWLEDGEMENTS
The study was performed within the MoldovaBelarus Bilateral Project 19.80013.51.07.09A/BL.
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Date of receipt of the manuscript: 29/11/2020 Date of acceptance for publication: 06/04/2021
Olga POSTOLACHI, ORCID ID: 0000-0002-2240-7376 Inna RASTIMESINA, ORCID ID: 0000-0002-3303-4771 Valentina JOSAN, ORCID ID: 0000-0002-6971-7638
