BIODEGRADATION AND ANTIMICROBIAL ACTIVITY OF GUANIDINE-CONTAINING POLYETHYLENE OXIDE HYDROGEL

The aim of the study was to determine the biodegradation of guanidine-containing polyethylene oxide hydrogel under the action of bacteria and to

The aim of the study was to determine the biodegradation of guanidine-containing polyethylene oxide hydrogel under the action of bacteria and to study its antimicrobial properties. The antimicrobial activity of newly synthesized polyethylene oxide hydrogel was studied by a disco-diffusion method. The enzymatic activities of bacteria were determined by spectrophotometry. To study the resistance of guanidine-containing polyethylene oxide hydrogel to microbial destruction, bacteria were grown in Tauson's liquid medium with the addition of meat peptone broth at a temperature of 28±2 C. Changes in the chemical composition of the studied materials were analyzed by infrared Fourier spectroscopy and 1H NMR spectroscopy.
Guanidine-containing polyethylene oxide hydrogels were obtained by reacting oligooxyethylene glycol MW 6000 with toluene diisocyanate and guanidine-containing oligomer, which acted as a crosslinking, ion-containing and antibacterial-agent. The synthesized hydrogel showed antimicrobial activity against both gram-positive and gram-negative bacteria. The biodegradation of hydrogels under the action of various bacterial strains and enzymes that they synthesize was studied. The presence of the tested materials lead to a decrease in the enzymatic activity of bacteria in 2.5 times compared with the control.
Synthesized guanidine-containing polyethylene oxide hydrogel showed antimicrobial activity against the studied test cultures. The introduction of the studied materials into Tauson's environment as additional sources of carbon and energy helped to reduce the catalase and lipase activities of hydrocarbon-oxidizing bacteria. Under the influence of the studied bacteria, the hydrogels were destroyed up to 88.4% from the initial value.
influenced by environmental conditions, as well as the composition and chemical structure of the material. The bacteria of Cytophaga, Bacillus, Rhodococcus, Streptomyces, Mycobacterium, Pseudomonas genera and filamentous fungi of Aspergillus, Alternaria, Penicillium, Trichoderma genera, and others play the greatest role in the microbial destruction of materials [3].
In the current literature, there are a lack of data about enzymatic activity of polymerdestroying bacteria and mainly about on the activity of redox and hydrolytic enzymes in micromycetes that damage building materials, foams, rubbers, etc. [7,8]. However, the lipolytic and catalase activities of bacteria, which result in microbial destruction of materials, reduce their strength and elasticity, remain poorly studied [7,8].
In modern polymer chemistry, one of the promising areas is the obtaining and studying the properties of hydrogel systems, among which the polyethylene oxide hydrogels take a significant place. They are widely used in various fields of science and technology due to non-toxicity, high degree of swelling [9][10][11], as well as the ability to biodegradation [12][13][14][15][16]. One method of polyethylene oxide hydrogels producing is based on the reaction of urethane formation in the presence of a crosslinking agent. Particular importance is attached to the construction of pHsensitive hydrogels, which are carried out by introducing a controlled number of ionic groups by copolymerization of acrylic derivatives of oligooxyethylene glycols with acrylic monomers containing such groups, or the introduction of ionic additives in the urethane formation reaction. The obtaining of hydrogels using derivatives of such a highly basic compound as guanidine, which has antimicrobial activity is of particular interest. Among guanidinium-containing polymers, polyhexamethylene guanidinium chloride attracts attention due to its biocide properties, which are important for practical use [17,18]. It belongs to the fourth security class. The high reactivity of the guanidine fragment stimulated research in the direction of chemical modification of polyhexamethylene guanidine chloride in order to provide it with additional functionality. Іts derivatives with methylene, acrylate groups are used for obtaining pH-sensitive hydrogels [19]. Guanidinecontaining polyethylene oxide hydrogels have not yet been described in the literature. It can be assumed that such hydrogels should have antibacterial and biodegradable properties as well as polyhexamethylene guanidine chloride will be low-toxic.
The aim of this work was to determine the antimicrobial properties of materials against bacteria isolated from pathogenic material of sick people and biodegradation of guanidinecontaining polyethylene oxide hydrogel under the action of bacteria previously isolated and identified from damaged protective coatings of gas pipelines.
Guanidine-containing oligomer (GO) was obtained at a temperature of 50 C for two hours by reaction of 0.1 M of epoxy oligomer in 70% ethanol solution while stirring with an alcoholic solution of 0.2 M guanidine obtained immediately before the reaction. The yield of the final product was 95%.
Obtaining polyethylene oxide hydrogel was performed by melting oligooxyethylene glycol MW 6 000 and mixing it with guanidinecontaining oligomer at 85 C to homogenize the mixture at a molar ratio of the original components of OEG:GO-1.5:(1-1.5), then mixing with TDI at a molar ratio of the original components of 1.5:(1-1.5):2. The formation of the hydrogel was performed at 25 C for one hour on a substrate, curing -at 80 C for 4 hours. Two types of hydrogel are obtained, which differ in the molar content of guanidine-containing oligomer: hydrogel 1ratio of the initial components of OEG:GO:TDI (1.5:1:2) and hydrogel 2 -ratio of the initial components OEG:GO:TDI (1.5:1.5:2). The degree of crosslinking of the obtained hydrogel was determined by the content of the gel fraction in the Soxhlet apparatus in 93-95% acetone solution.
Disco-diffusion method was used to determine the bactericidal activity of the original guanidine-containing oligomer and polyethylene oxide hydrogels against the bacteria Staphylococcus aureus 451, Escherichia coli 475, Pseudomonas mirabilis 494. These strains have been isolated from biomaterial of urogenital system of sick people and stored in the Laboratory of Bacteriological Research of the Institute of Urology of the National Academy of Medical Sciences of Ukraine. Also, hydrocarbon-oxidizing bacteria Pseudomonas pseudoalcaligenes 109, Rhodococcus erythropolis 102, Bacillus subtilis 138, which were previously isolated and identified by us from damaged protective coatings of gas pipelines [4,6] and stored in the Сollection of the Department of General and Soil Microbiology of the Zabolotny Institute of Microbiology and Virology of the National Academy of Sciences of Ukraine were used. Guanidine-containing oligomer in the form of a 0.1 ml aqueous solution was used with standard paper disks with a diameter of 6 mm on the surface of meat-peptone agar inoculated with the appropriate bacterial testculture. Polyethylene oxide hydrogels have been obtained based on guanidine-containing oligomer in the form of a film with a size of 10x10x0.5 mm. They were applied to the surface of meat-peptone agar inoculated with the appropriate test culture of bacteria. Incubation was performed for 18 hours at 28 ± 2 С. Antimicrobial activity was calculated in diameter (mm) of the zones of growth inhibition of microorganisms. Antimicrobial activity was estimated by the following scale: zone 0-10 mm -non sensitive, 15-25 mmsensitive, up to 25 mm -high sensitive.
Microbial degradation of guanidinecontaining polyethylene oxidehydrogel was studied with the use of hydrocarbonoxidizing bacteria P. pseudoalcaligenes 109, R. erythropolis 102 and B. subtilis 138 strains. Bacteria had grown in Tauson's liquid medium at 28 ± 2 C [5] with the addition of 20% meatpeptone broth (MPB) as a source of Nitrogen and Carbon Test materials' samples with the sizes of 10100.5 mm were weighed on electronic scales (ANG-200, AXIS) and sterilized with 72% solution of ethyl alcohol (30 min) and with UV rays with a wavelength of 256 nm (15 min) and were put in Tauson medium, inoculated with one of the abovementioned bacterial strains. The amount of cells were 10 6 cells/ml calculated with serial dilution method. Variants with Tauson's medium. with materials without the addition of bacteria were used as control. The term of the experiment was 60 days. The destruction of the samples was determined gravimetrically by loss of their mass. To perform this, after the exposure, the material samples were removed from the cultural liquid, air-dried, then weighed and the weight loss of control and test samples was determined. The amount of bacteria in the cultural liquid was determined with serial dilution method [20].
Enzymatic activity. To determine the effect of materials on the enzymatic activity of bacteria, cultural liquid was centrifuged for 20 min at 8 000 rp/min (2 000 g) with the use of Eppendorf 5810R centrifuge (Germany). Lipolytic activity was determined by reaction with p-nitrophenylpalmitate [21], catalase activity was determined using 0.03% hydrogen peroxide, which formed a stable colored complex with 4% molybdenum phosphate solution [22]. Enzymatic activity was determined in the supernatant spectrophotometrically on KFK-3 (Russian Federation). Total protein concentration in the cultural liquid was determined by the Lowry method. The specific activity of the studied enzymes was calculated according to the formulas specified in [18,19] and was expressed in u·mg -1 protein.
IR spectra of the investigated materials with Fourier transform were obtained on a spectrophotometer TENSOR 37 (BrukerOptik GmbH, Germany) in the spectral range of 6 000-400 cm -1 in KBr tablets. 1 H NMR spectra were recorded on a Varian VXR-400 MHz instrument in CDCI 3 solution.
The degree of swelling of the obtained polyethylene oxide hydrogels in water for injections at pH 4.0, 7.0 and 9.8 at room temperature to constant values was measured by weighing on the torsion scales. The degree of swelling of the samples was calculated with the formula:  = (m -m 0 )/m 0 , where m 0 is the mass of the sample before swelling, m is the mass of the swollen sample.
Statistical analysis of the obtained results was performed using the software package MS Excel 2010 and OriginPro 2016 (ver. B 9.3.226. www.originlab.com/). The obtained data were represented as M ± n (n = 3, at P  0.05).

Results and Discussion
To obtain a polyethylene oxide hydrogel, a guanidine-containing oligomer of linear structure with terminal guanidine fragments has been synthesized by the reaction between a bifunctional aromatic oligoepoxide (MW 365) and guanidine at a molar ratio of 1:2 (Fig. 1).
The synthesized guanidinium-containing oligoether with terminal guanidine moieties was used for the synthesis of polyethylene oxide hydrogels (1 and 2) as a crosslinking and antimicrobial agent. According to the results of microbiological studies, the obtained guanidine-containing oligoethers showed antimicrobial activity (Table 1; Fig. 2) against the studied bacterial test-cultures P. mirabilis 494, E. coli 475, P. pseudo alcaligenes 109 and S. aureus 451.
The diameters of inhibition zone of bacterial growth were in the range of 13-20 mm. The largest zones of growth inhibition were observed for strains of R. erythropolis 102 and B. subtilis 138 (24-26 mm) under the influence of hydrogels 1 and 2. The obtained two variants of polyethylene oxide hydrogels showed antimicrobial activity against the studied test-cultures. The diameters of the zone growth inhibition for strains E. coli 475, P. mirabilis 494, S. aureus 451, P. pseudoalcaligenes 109 were in the range 10-11 and 15-16 mm for hydrogel 1 and hydrogel 2, respectively. Hydrocarbon-oxidizing bacteria R. erythropolis 102 and B. subtilis138 were more sensitive to synthesized hydrogels because inhibition zones were 15-19 and 20-24 mm for hydrogel 1 and 2, respectively. Hydrogel 1 had low antibacterial activity against the studied test cultures due to lower content of guanidine-containing oligoether in hydrogel 1, which acts as a crosslinking and antibacterial agent ( Table 1). The scheme of synthesis of polyethylene oxide hydrogel is shown on Fig. 3.
The structure of the obtained hydrogels was confirmed by IR spectroscopy (Fig. 4).
The difference between the IR spectra of hydrogels 1 and 2 is the different ratio of the intensity of the absorption bands of the valence vibrations of the -CH bonds to the valence vibrations of the hydroxyl (-OH) and amino(-NH) groups. There was an increase in the absorption band of NH groups, because the molar content of GO in the hydrogel 2 was in 1.5 times higher than in hydrogel 1.
The degree of swelling of the polyethylene oxide hydrogels were obtained for various conditions, i.e. pH, temperature and swelling duration (Fig. 5).
The degree of swelling under different pH was in the range 8.05-11.5 (Fig. 5, a). The data show that both hydrogels are highly swellable and this parameter depends on the pH of the medium, the high degree of swelling 10.2-11.1 is observed under pH 4.0. The dependence of the degree of swelling of polyethylene oxide hydrogel 2 in water on temperature (Fig. 5, b) showed that high values 14 is observed at 56 C.The study of the duration of swelling of hydrogel 2 (Fig. 5, с) showed that under pH 4.0 and pH 7.0 during 180-210 min there was complete swelling and values were 11.1 and 9.0, respectively.
The hydrocarbon-oxidizing bacteria P. pseudoalcaligenes 109, R. erythropolis 102 and B. subtilis 138 produced extracellular polyand lipopolysaccharides, due to which these bacteria adhere to the surface of protective materials, ensuring their rapid reproduction and promoting survival of bacterial population in extreme conditions. The identified exopolysaccharides differed in monosaccharide composition and fatty acid content. Analysis of the fatty acid composition of the exopolymer complex of bacteria revealed the presence of saturated, unsaturated fatty acids and hydroxyl-acids with a carbon chain length of 11 to 18 carbon atoms [7].
In Tauson's liquid medium (with the addition of MPB, inoculated with bacterial strains and without hydrogels (control), increasing the number of hydrocarbonoxidizing bacteria to 10 9 cells/ml over 60 days of the experiment was observed. In the experimental variants in the presence of the samples of studied hydrogels it was observed  Thus, we studied the influence of obtained hydrogels on the enzymatic activity of bacteria. Bacterial cultures in the presence of the studied hydrogels had different enzymatic activity depending on the hydrogels (Fig. 6).
The specific catalase activity in the control (nutrient media with bacteria and without the hydrogels) was higher than in presence of bacteria and materials. The highest specific catalase activity was in B. subtilis 138 and R. erythropolis 102 (5.3 ± 0.5 and 5.2 ± 0.4 u·mg -1 protein, respectively), but in the presence of the studied hydrogels enzymatic activity was decreased in 1.1-1.9 times for different bacterial cultures. Catalase activity of P. pseudoalcaligenes 109 in the presence of hydrogels 1 and 2 was significantly lower from 2.4 to 5.0 times, respectively (Fig. 6, a). In Tauson's medium without the introduction of materials, higher lipase as well as catalase activities were found in B. subtilis138 (19.25 ± 2.1 u·mg -1 protein) and R. erythropolis 102 (18.6 ± 1.9 u·mg -1 protein), compared with other bacterial cultures. In the presence of the studied hydrogels 1 and 2 lipase activities decreased in 1.4-1.8 times, respectively. The specific lipase activity of P. pseudoalcaligenes 109 with guanidine-containing hydrogels in cultural liquid was decreased in 1.3-1.9 times compared to control. In the presence of hydrogel 1, compared to hydrogel 2 the lipase activity of bacterial test-cultures was lower (7.8 ± 1.4-12.7 ± 2.3 u·mg -1 protein).
It is known from literature data that changes in catalase activity are one of the indicators of processes intensity of organic matter oxidation [23,24]. There is a close correlation between the reduction degree of catalase activity of destructive strains and the efficiency of oil consumption. Active destructive bacteria have characterized in increasing their amount and decreasing catalase activity in contact with the petroleum products [25]. According to several reports, hydrocarbon-oxidizing bacteria activate lipase activity of soils, while in parallel with the activation of lipolysis, there is an increase in the number of hydrocarbon-oxidizing bacteria and a decrease of petroleum products [25].
The utilization of different materials after their use is an important aspect of the problem of materials biodamages. Significant indicator of material degradation is loss of the sample mass due to bacterial action ( Table 2).
After 60 days of the experiment, the largest weight loss 0.35 ± 0.1 g (85.4% from the initial mass) of hydrogel 1 samples was observed under the influence of B. subtilis 138. In the variant of the experiment with hydrogel 2, the weight loss of the samples under the influence of bacteria was from 0.12 ± 0.01 to 0.22 ± 0.05 g (59.5-88.4%). In the control, the weight loss of hydrogel 1 was 0.06 ± 0.01 g (17%).
The difference was that for hydrogels 1 and 2 under the influence of R. erythropolis 102 and B. subtilis 138 after 60 days, decreased the absorption band of 3500 cm -1 decreased, which was responsible for the valence fluctuations of hydroxyl (-OH) and amino (-NH) groups (Fig. 5). Because crosslinking of polyethylene oxide hydrogels occurs through the formation of amide bonds that are resistant to hydrolysis and biodegradation, it can be assumed that the presence of a significant number of secondary amino groups in the crosslinking agent contributes as a catalyst to biodegradation of hydrogels, especially under influence of the R. erythropolis 102.
The mechanism оf biocidal action of polyguanidines is similar to quaternary ammonium compounds and is of membranetoxic nature: guanidine polycations are adsorbed on the negatively charged surface of bacterial cells; diffuse through the cell wall; bind to acidic phospholipids, proteins of the cytoplasm membrane, which causes its breakage. As a result, the microbial cell dies. The increase in the biocidal activity of PHMGC as compared to the low-molecular biocides is due to the cooperative interaction of the adjacent guanidine polycation group links with the microbial cell. The increase in the activity of polyguanidines as compared to quaternary ammonium salts (QAS) is also due to the peculiarities of the guanidine group structure: unlike QAS cation, in which a large positive charge is localized on a single nitrogen atom, guanidine cation has a positive charge distributed between three nitrogen atoms, and additionally delocalized according to the -links system. It is well known that guanidinium polymers are less toxic than guanidine and belong to the third class of hazard [17,18].
The antimicrobial properties and biodegradation of newly synthesized guanidine-containing polyethylene oxide hydrogels, in which the linear guanidinecontaining oligomer simultaneously acts as a crosslinking, ion-containing and antimicrobial agent, were studied. The synthesized hydrogel showed antimicrobial activity against the studied test cultures of bacteria. The largest zones of growth inhibition were observed for S. aureus 451, R. erythropolis 102, B. subtilis 138. The introduction of the studied materials into the Tauson's medium as additional sources of Carbon and energy, contributed to the reduction of catalase and lipase activities of hydrocarbon-oxidizing bacteria. Under the influence of the studied bacteria, destruction of hydrogels, which is appeared in a decreasing of the studied samples mass, was observed. Infrared spectroscopy showed that the degradation of guanidine-containing hydrogels is affected by the presence of a significant number of secondary amino groups in the crosslinking agent, which can serve as catalysts for the biodegradation of hydrogels.
The research was carried out within the framework of the projects of the National Academy of Sciences of Ukraine "Functional activity and prospects of use in biotechnology soil microorganisms from natural, mancaused and agro systems" (2016-2019, State registration number GDR 0116U006319) and "Development of methods of synthesis of monomers containing perfluorated and highly basic nitrogenic and other functional groups and fragments" (2018-2022, GDR0118U002055).