Liquid Mineral Medium Research Articles

Monoaromatic Compounds Biodegradation by Hydrocarbon-Oxidizing Actinobacteria

Monoaromatic substances belong to the most widespread and dangerous environmental pollutants. They are components of wastewater from oil refineries and the coal and chemical industries and are often found in soil, groundwater, and surface water. The use of actinobacteria for bioremediation of natural and industrial objects contaminated by these substances is an environmentally and economically advantageous alternative to physicochemical cleaning methods. Therefore, it is actual to search for new actinobacteria strains capable to assimilate of monoaromatic compounds for their further use in biotechnologies of purification of the environment polluted by these substances. Aim. To determine the ability of hydrocarbon-oxidizing strains of actinobacteria of the Ukrainian Collection of Microorganisms to assimilate monoaromatic compounds − derivatives of benzene and phenol. Methods. The strain cultivation was carried out in a liquid mineral medium at an initial concentration of monoaromatic compounds of 500 mg/L as the only source of carbon and energy. The expression activity of the catA gene encoding catechol 1,2-dioxygenase, the key enzyme at the initial stage of the monoaromatic compounds biodegradation, was assessed under culture growth conditions with phenol and glucose. Relative expression of the catA gene was evaluated using RT-qPCR. Fatty acid methyl esters were obtained by hydrolysis of cells in a 5 % solution of acetyl chloride in methanol, followed by extraction with an ether-hexane mixture. Methyl esters were identified using an Agilent 6800N/5973 inert GC/MS system (Agilent Technologies, US). Fatty acid content was determined by AgilentChemStation software. Results. It was established that the studied strains of actinobacteria belonging to the species Dietzia maris, Gordonia rubripertincta, Rhodococcus aetherivorans, and Rhodococcus erythropolis differ in their ability to assimilate monocyclic aromatic compounds − benzene and phenol derivatives. Most strains grew better in media with benzene and its derivatives. All strains well grew on a mixture of ethylbenzene, ortho-, meta-, and para-xylene (EX), most of them grew with different intensities on a mixture of benzene, toluene, and ortho-xylene (BTX), and on monosubstrates – benzene, benzotriazole, and benzoate. Toluene was used by 50 % of the studied strains, and only one of them (R. aetherivorans UCM Ac-602) grew on ortho-xylene. A smaller amount of strains grew in media with phenol derivatives. They did not assimilate ortho-cresol and hydroquinone but grew with different intensities on phenol, paranitrophenol, resorcinol, and catechol. Only a few strains grew in the presence of meta- and para-cresols. The widest range of monoaromatic substances was used by R. aetherivorans UCM Ac-602. It was shown that in the process of phenol assimilation by strain R. aetherivorans UCM Ac-602, the level of transcriptional activity of the catA gene encoding the key enzyme of an ortho-pathway of aromatic ring cleavage, catechol 1,2-dioxygenase, increased almost 2-fold after 48 h compared to 24 h of strain growth on this substrate and 3-fold compared to glucose. The obtained data indicate the ability of R. aetherivorans UCM Ac-602 strains to assimilate monoaromatic substances through theortho-cleavage way. During growth on benzene, toluene, BTX and EX substances, the fatty acid pool of cells of this strain was dominated by hexadecanoic C16:0 (32.73-39.81 %), hexadecenoic C16:1 cis-9 (5.66-15.11 %), and octadecenoic C18:1 cis-9 (7.09-11.83 %) acids, as well as 10-methyloctadecanoic 10-Me-C18:0 (29.88-34.76 %) acid. The composition of fatty acids of cells grown on aromatic compounds, unlike those grown on glucose, contained 2.4-4.0 times less unsaturated C18:1 cis-9 acid and 2.8-3.2 times more methyl-branched acid 10Me-C18:0. Conclusions. Assimilation of monoaromatic compounds − derivatives of benzene and phenol - is a strain-specific characteristic of the actinobacteria species. Using strain R. aetherivorans UCM Ac-602 as an example, it was shown that the assimilation of monoaromatic substances in studied actinobacteria occurs through the ortho-cleavage pathway. One of the adaptation mechanisms of strain R. aetherivorans UCM Ac-602 to the assimilation of these substances is a significant decrease in unsaturated fatty acids and an increase in methyl branched acids, which contributes to an increase in membrane rigidity and cell resistance to the toxic effect of these substrates. The data obtained indicate a high level of adaptation of actinobacteria to the assimilation of monoaromatic compounds and the prospects for their further study to be used in biotechnologies for environmental purification from these pollutants.

Impact of submerged culture different parameters on growth and naringinase activity of Bacillus amyloliquefaciens D1 isolated from citrus gardens of North-East India

The study aims at screening, isolation, and characterization of naringinase enzyme producing bacterial strains from soil samples. In this study, 45 bacterial isolates were screened for naringinase activity, and among them, 20 bacterial isolates are found to be capable of naringinase production in liquid mineral medium. Bacterial isolate D1 exhibited the highest naringinase activity in liquid mineral medium. Based on the morphological, biochemical, fatty acid profile, and molecular characterization, bacterial isolate D1 was identified as Bacillus amyloliquefaciens. Impact of different submerged culture parameters were evaluated in different experimental conditions. The study revealed that 48 hrs of incubation periods, pH 6.0 and temperature 350 C is optimal for naringinase activity when the culture medium is supplemented with starch and yeast extract as carbon and nitrogen sources. Thus, it can be concluded that due to the potential naringinase activity of the strain Bacillus amyloliquefaciens D1, further the identifying strain may be used in citrus processing and bioprocess industries upon large scale field applications.

Технология культивирования Limnospira fusiformis из озера Соленого (г. Омск)

Global food shortages make it necessary to look for alternative renewable bioresources. In the south of Western Siberia, the filamentous cyanoprokaryote Limnospira fusiformis triggers seasonal algae bloom in hypergaline alkaline Lake Solenoye. The species has valuable nutritional properties, and its phytomass is a potential source of proteins and biologically active substances. The O9.13F strain of L. fusiformis has a good potential as a bioadditive in animal feeding. The article offers a technology for cultivating O9.13F of L. fusiformis, isolated from Lake Solenoye. The research objectives were to establish the optimal cultivation conditions, medium, and periodicity.
 The study featured strain O9.13F of filamentous cyanoprokaryote L. fusiformis. The micropipette method made it possible to isolate pure culture from water samples taken from Lake Solenoye, Omsk, Russia, at the end of algae bloom. The cultivation involved a UT-6070 climatic chamber under uniform illumination with light intensity 10–30 μmol photons/m2s and 12-h light-dark circle at 20 ± 2°C on various media: natural habitat – water from the Solenoye Lake; mineral medium – liquid Zarrouk’s medium; agarized Zarrouk’s medium; composite variants, where the ratio of mineral medium vs. water varied from 1:9 to 9:1.
 Lake water inhibited the culture growth: the trichomes died and sank on day 10–15. Zarrouk’s agarized medium stopped the culture growth as early as on day 2. The most intensive growth and development of the culture was observed in the samples with Zarrouk’s mineral liquid medium and a composite mix of Zarrouk’s medium and sterilized water at a ratio of 5:5. Without stirring, full-fledged trichomes had no time to develop, and the increase in phytomass volume slowed down. O9.13F showed the highest rate of phytomass growth at a cultivation temperature of 20 ± 2°C and a light intensity of 10–30 μmol photons/m2s. The recommended light-dark circle was12:12 h. Zarrouk’s mineral liquid medium and a composite medium of Zarrouk’s medium and sterilized water proved to be optimal in a UT-6070 environmental chamber. Europolitext KV-06 or mechanical mixing could prevent sedimentation of phosphates after 20 days of cultivation. The optimal recultivation frequency was once every 5–7 days. 
 The new cultivation technology made it possible to obtain a significant volume of L. fusiformis phytomass in a short time and with low financial expenses.

Prospects for application of microorganisms in bioremediation of soils contaminated with pesticides

The contamination of soil with residual amounts of pesticides remains an urgent challenge for human community. The most efficient approach to address this challenge is the direct microbial degradation of a pesticide in agricultural lands. To this end, the selected microorganisms, which quickly and completely utilize pesticides, are employed. In the present work, two herbicides belonging to different classes of chemical compounds, that is, imazamox and chlorsulfuron were used. The screening of promising microorganisms was carried out among different strains of bacteria and fungi in a liquid mineral medium containing a pesticide as the only source of carbon. It was found that the most active microorganisms were capable of utilizing up to 90% of the active substance for a short time. The dynamics of pesticides degradation indicated that the maximum destruction of the studied substances occurred during the first two weeks of cultivation. Further, the rate of degradation dramatically dropped or stopped at all. An increase in the concentration of pesticides in the cultivation medium almost completely suppressed their degradation. It is interesting that the bacteria were more suitable for the degradation of imazamox, while the fungi rendered the destruction of chlorsulfuron.

Biological Activity of Acinetobacter calcoaceticus IMV B-7241 Surfactants Synthesized in the Presence of Competitive Bacteria Bacillus subtilis BT-2

Currently, the effectiveness of technologies for microbial surfactants, which are characterized by a complex of practically valuable physicochemical and biological properties is lower than that of synthetic analogues. To reduce the cost of these products of microbial synthesis, industrial waste is used as substrates for their biosynthesis. In previous studies, it has been established that surfactants synthesized by Acinetobacter calcoaceticus IMV B-7241 on crude glycerol have lower antimicrobial activity compared to that obtained on purified glycerol. The main approaches to the regulation of the biological activity of microbial surfactants are their post-fermentation chemical modification, as well as the improvement of producer strains by methods of metabolic and genetic engineering. In recent years, numerous studies have appeared on the co-cultivation of producers of antimicrobial compounds with competitive microorganisms (biological inductors), in response to the presence of which the antimicrobial activity of the final product increases. Aim. To study the effect of live and inactivated cells of Bacillus subtilis BT-2, as well as the corresponding supernatant, on the antimicrobial and anti-adhesive activity and the ability to destroy biofilms of A. calcoaceticus IМV B-7241 surfactants synthesized in a medium with glycerol of different degrees of purification. Methods. The A. calcoaceticus IMV B-7241 strain was grown in a liquid mineral medium with purified and crude glycerol, into which live and inactivated B. subtilis BT-2 cells as well as the supernatant after growing the B. subtilis BT-2 strain (2.5—10%, v/v) were added. Surfactants were extracted from the supernatant of the culture liquid with Folch’s mixture. Anti-adhesive activity and the degree of destruction of biofilms were determined by the spectrophotometric method, and antimicrobial activity — by the indicator of the minimum inhibitory concentration. The activity of enzymes of surface-active aminolipids biosynthesis (NADP+-dependent glutamate dehydrogenase) and glycolipids (phosphoenolpyruvate (PEP)-carboxylase, PEP-synthetase, PEP-carboxykinas, trehalose-phosphate synthase) was analyzed in cell-free extracts obtained after сells sonication. Results. It was established that the introduction of inactivated B. subtilis BT-2 cells and supernatant into the medium with both substrates did not affect the indicators of the surfactant synthesis, while in the presence of live cells of the B. subtilis BT-2 strain in the medium with purified glycerol, a decrease in the concentration of the final product by 1.5 times, and in the culture medium with crude glycerol — an increase by1.4 times were observed compared to the indicators with no inductor. The study of the antimicrobial activity of surfactants showed that the most effective of the used inductors (live, inactivated cells, supernatant) were live cells of B. subtilis BT-2. The introduction of B. subtilis BT-2 strain live cells into the culture medium with both substrates was accompanied by the formation of surfactants, the minimum inhibitory concentrations of which in relation to bacterial (Bacillus subtilis BT-2, Staphylococcus aureus BMS-1, Proteus vulgaris PA-12, Enterobacter cloacae С-8) and yeast (Candida albicans D-6, Candida tropicalis PE-2) test-cultures were 3—23 times lower than established for those synthesized on the medium with no inductor. Anti-adhesive activity of surfactants obtained on purified and crude glycerol in the presence of all types of inductors was higher compared to those synthesized in the culture medium without inductors (cells adhesion of bacterial and yeast test-cultures on polyvinyl chloride was 13—70 and 33—96%, respectively). Introduction of live and inactivated B. subtilis BT-2 cells or the supernatant into A. calcoaceticus IMV B-7241 cultivation medium was accompanied by the synthesis of surfactants, in the presence of which the disruption of bacterial biofilms was on average 10-20% higher compared to using surfactants synthesized without an inductor. In the presence of B. subtilis BT-2 in the medium, in the cells of the A. calcoaceticus IMV B-7241 strain, the activity of NADP+-dependent glutamate dehydrogenase (a key enzyme of aminolipids biosynthesis) increased by 1.5—2 times, while the activity of biosynthesis of glycolipids enzymes remained practically at the same level as without an inductor. Such data indicate that the higher biological activity of surfactants obtained by A. calcoaceticus IMV B-7241 in the presence of biological inductors might be due to an increase in the content of aminolipids in their composition. Conclusions. This research has established the possibility of regulating the antimicrobial and anti-adhesive activity as well as the ability to disrupt biofilms of A. calcoaceticus IМV B-7241 surfactants by introducing competitive bacteria B. subtilis BT-2 into the culture medium. It is important that under such cultivation conditions, the antimicrobial activity of surfactants synthesized on toxic crude glycerol significantly increases.

Genome Analysis and Physiology of Pseudomonas sp. Strain OVF7 Degrading Naphthalene and n-Dodecane.

The complete genome of the naphthalene- and n-alkane-degrading strain Pseudomonas sp. strain OVF7 was collected and analyzed. Clusters of genes encoding enzymes for the degradation of naphthalene and n-alkanes are localized on the chromosome. Based on the Average Nucleotide Identity and digital DNA-DNA Hybridization compared with type strains of the group of fluorescent pseudomonads, the bacterium studied probably belongs to a new species. Using light, fluorescent, and scanning electron microscopy, the ability of the studied bacterium to form biofilms of different architectures when cultured in liquid mineral medium with different carbon sources, including naphthalene and n-dodecane, was demonstrated. When grown on a mixture of naphthalene and n-dodecane, the strain first consumed naphthalene and then n-dodecane. Cultivation of the strain on n-dodecane was characterized by a long adaptation phase, in contrast to cultivation on naphthalene and a mixture of naphthalene and n-dodecane.

Nitrogen sources alter ligninase and cellulase activities of thermophilic fungi isolated from compost and vermicompost.

Fungi harboring lignocellulolytic activity accelerate the composting process of agricultural wastes; however, using thermophilic fungal isolates for this process has been paid little attention. Moreover, exogenous nitrogen sources may differently affect fungal lignocellulolytic activity. A total of 250 thermophilic fungi were isolated from local compost and vermicompost samples. First, the isolates were qualitative assayed for ligninase and cellulase activities using Congo red (CR) and carboxymethyl cellulose (CMC) as substrates, respectively. Then, twenty superior isolates harboring higher ligninase and cellulase activities were selected and quantitatively assayed for both enzymes in basic mineral (BM) liquid medium supplemented with the relevant substrates and nitrogen sources including (NH4)2SO4 (AS), NH4NO3 (AN), urea (U), AS + U (1:1), or AN + U (1:1) with final nitrogen concentration of 0.3g/L. The highest ligninase activities of 99.94, 89.82, 95.42, 96.25, and 98.34% of CR decolorization were recorded in isolates VC85, VC94, VC85, C145, and VC85 in the presence of AS, U, AS + U, AN, and AN + U, respectively. Mean ligninase activity of 63.75% in superior isolates was achieved in the presence of AS and ranked the highest among other N compounds. The isolates C200 and C184 exhibited the highest cellulolytic activity in the presence of AS and AN + U by 8.8 and 6.5 U/ml, respectively. Mean cellulase activity of 3.90 U/mL was achieved in AN + U and ranked the highest among other N compounds. Molecular identification of twenty superior isolates confirmed that all of them are belonging to Aspergillus fumigatus group. Focusing on the highest ligninase activity of the isolate VC85 in the presence of AS, the combination can be recommended as a potential bio-accelerator for compost production.

Изучение возможности использования водных композиций полиакриламида с катионами цинка и меди для снижения рисков микробной контаминации объектов внутрибольничной среды

Microbial contamination means that infectious agents are identified on objects in the hospital environment. This serious issue is the most significant for healthcare organizations. Covering abiotic surfaces with a thin polymer film can be a promising way to fight against microbial adhesion and colonization. This film acts as a depot of an antibacterial substance. In this study, our aim was to investigate antimicrobial effects of new water compositions of polyacrylamides (PAM) with CuSO4 and ZnSO4. We examined antibacterial activity of 5%-solutions of CuSO4 and ZnSO4 and their compositions with various PAM types in a concentration equal to 0.075 % against such reference cultures as Escherichia coli, Klebsiella pneumoniaе, Pseudomonas aeruginosa, and Staphylococcus aureus. We estimated use of PAM as a growth substrate as well as antimicrobial activity of the analyzed solutions and compositions in agar and liquid nutrient media. As a result, we established that bacterial cultures did not use PAM as sole nutrition source when growing in a liquid mineral medium and on PAM-films covering glass and plastic surfaces. More apparent inhibitory effects were produced on microorganisms cultivated on solid and liquid nutrient media by 5%-solution of ZnSO4. When PAM Praestol 857 and PAM Praestol were added to solutions of Cu2+ and Zn2+ cations, it resulted in an authentic increase in a diameter of a zone with inhibited bacterial growth in the agar medium. In the liquid medium, salts of both metals inhibited the growth and viability of all the analyzed microorganisms already in a concentration equal to 0.16 % or lower. Adding PAM Praestol 2530 led to a slight decrease in antibacterial efficiency of the examined metal salts whereas PAM Praestol 857 had practically no influence on bacteriostatic and bactericidal effects produced by them. Therefore, use of the obtained composite solutions where CuSO4 or ZnSO4, immobilized on a PAM matrix act as an antibacterial component seems a promising way to disinfect objects in the hospital environment. This can significantly reduce risks of hospital-acquired infections.

Use of aqueous compositions of polyacrylamide with zinc and copper cations as a possible way to reduce the risks of microbial contamination in objects in the hospital environment

Microbial contamination means that infectious agents are identified on objects in the hospital environment. This serious issue is the most significant for healthcare organizations. Covering abiotic surfaces with a thin polymer film can be a promising way to fight against microbial adhesion and colonization. This film acts as a depot of an antibacterial substance. In this study, our aim was to investigate antimicrobial effects of new water compositions of polyacrylamides (PAM) with CuSO4 and ZnSO4. We examined antibacterial activity of 5%-solutions of CuSO4 and ZnSO4 and their compositions with various PAM types in a concentration equal to 0.075 % against such reference cultures as Escherichia coli, Klebsiella pneumoniaе, Pseudomonas aeruginosa, and Staphylococcus aureus. We estimated use of PAM as a growth substrate as well as antimicrobial activity of the analyzed solutions and compositions in agar and liquid nutrient media. As a result, we established that bacterial cultures did not use PAM as sole nutrition source when growing in a liquid mineral medium and on PAM-films covering glass and plastic surfaces. More apparent inhibitory effects were produced on microorganisms cultivated on solid and liquid nutrient media by 5%-solution of ZnSO4. When PAM Praestol 857 and PAM Praestol were added to solutions of Cu2+ and Zn2+ cations, it resulted in an authentic increase in a diameter of a zone with inhibited bacterial growth in the agar medium. In the liquid medium, salts of both metals inhibited the growth and viability of all the analyzed microorganisms already in a concentration equal to 0.16 % or lower. Adding PAM Praestol 2530 led to a slight decrease in antibacterial efficiency of the examined metal salts whereas PAM Praestol 857 had practically no influence on bacteriostatic and bactericidal effects produced by them. Therefore, use of the obtained composite solutions where CuSO4 or ZnSO4, immobilized on a PAM matrix act as an antibacterial component seems a promising way to disinfect objects in the hospital environment. This can significantly reduce risks of hospital-acquired infections.

Bioremediation of Oil-Contaminated Soil of the Republic of Kazakhstan Using a New Biopreparation.

A new biopreparation is developed to clean soils from oil pollution in the arid climate of the Republic of Kazakhstan. The biopreparation includes bacterial strains R. qingshengii F2-1, R. qingshengii F2-2, and P. alloputida BS3701. When using the biopreparation in a liquid mineral medium with 15% crude oil, laboratory studies have revealed degradation of 48% n-alkanes and 39% of PAHs after 50 days. The effectiveness of the biopreparation has been demonstrated in field experiments in the soil contaminated with 10% crude oil at the K-Kurylys landfill, Republic of Kazakhstan. During the six-month field experiment, the number of oil degraders reached 107 CFU/g soil, which degraded 70% of crude oil by the end of the experiment.

Structural characterization and physicochemical properties of the exopolysaccharide produced by the moderately halophilic bacterium Chromohalobacter salexigens, strain 3EQS1.

A strain, 3EQS1, was isolated from a salt sample taken from Lake Qarun (Fayoum Province, Egypt). On the basis of physiological, biochemical, and phylogenetic analyses, the strain was classified as Chromohalobacter salexigens. By 72h of growth at 25°C, strain 3EQS1 produced large amounts (15.1gL-1) of exopolysaccharide (EPS) in a liquid mineral medium (initial pH 8.0) containing 10% sucrose and 10% NaCl. The EPS was precipitated from the cell-free culture medium with chilled ethanol and was purified by gel-permeation and anion-exchange chromatography. The molecular mass of the EPS was 0.9 × 106Da. Chemical analyses, Fourier transform infrared spectroscopy, and nuclear magnetic resonance spectroscopy showed that the EPS was a linear β-D-(2 → 6)-linked fructan (levan). In aqueous solution, the EPS tended to form supramolecular aggregates with a critical aggregation concentration of 240µgmL-1. The EPS had high emulsifying activity (E24, %) against kerosene (31.2 ± 0.4%), sunflower oil (76.9 ± 1.3%), and crude oil (98.9 ± 0.8%), and it also had surfactant properties. A 0.1% (w/v) aqueous EPS solution reduced the surface tension of water by 11.9%. The levan of C. salexigens 3EQS1 may be useful in various biotechnological processes.

STUDY OF OIL DEGRADATION IN OIL-CONTAMINATED SOIL DURING PURIFICATION USING A BIOLOGICAL PRODUCT

During a scientific study, a laboratory experiment was conducted to investigate the degradation of oil by various microorganisms in a liquid mineral medium containing 2% oil. This experiment was conducted at temperatures of 24ºC and 5ºC for a duration of 10 days. Using infrared spectroscopy (IR spectroscopy), the degree of oil decomposition by each microorganism was determined. Based on these data, four associations of microorganisms were created, which proved to be effective in breaking down oil hydrocarbons. Using gravimetry and IR spectrometry, the degree of oil biodegradation by these microorganism associations was assessed, ranging from 30% to 70%. Based on the data obtained regarding microbial associations, a new bio-preparation was developed, containing the Pseudomonas sp. K3-H strain and two Rhodococcus strains (F2/2-Н4, F2/1-Н4). Subsequently, fieldwork was conducted on the premises of oil waste of the company "TOO Company – Daulet Asia." Over a period of 12 weeks, the population of oil-degrading microorganisms at the oil-contaminated site reached the required level, resulting in an 89.67% reduction in the amount of oil. It is worth noting that the ambient temperature varied from 05 to 39°C during the day and from 0 to 24°C at night during this period. The results of the field studies demonstrated the effectiveness of the developed bio-preparation based on the Rhodococcus erythropolis KZ1, Rhodococcus erythropolis KZ2, and Pseudomonas putida KZ3 strains. Thus, the proposed bioremediation strategy utilizing the developed bio-preparation successfully passed the testing phase at the landfill of "TOO Company - Daulet Asia" in the Kyzylorda region of the Republic of Kazakhstan.

Biodefluorination of Perfluorooctanesulphonate by Ensifer adhaerens M1

A strain of bacteria M1, defluorinating perfluorooctanesulfonic acid (PFOS), was isolated from soil taken from the territory for testing fire extinguishing agents. The analysis of cultural, morphological, physiological and biochemical characters, the 16S rRNA gene sequencing allowed us to identify strain M1 as Ensifer adhaerens. The uniquely skill of the isolated strain to utilize PFOS in a mineral liquid medium for 5 days of cultivation was shown. It was found that E. adhaerens M1 transforms perfluorooctanesulfonic acid into perfluoroheptanoic acid, releasing 152 mg/L of free fluoride ions from 1,000 mg/L of PFOS. The data obtained allow us to recommend E. adhaerens M1 as potential treatment agent of the environment from organofluorine compounds.

Pentachlorophenol Effect on Auxin Production by <i>Pseudomonas fluorescens</i> GU059580 and Its Application in Wastewater Bioremediation

Bioaugmentation by Pseudomonas strains is widely used for the removal of pollutants in wastewater. In this study, we aimed to determine the removal of pentachlorophenol (PCP, 800 mg·L<sup>–1</sup>) in secondary wastewater by the bioaugmentation process. We determined the effects of using three surfactants, namely sodium dodecyl sulfate (SDS), cetyl-tri-methyl-ammonium bromide (CTAB), and Tween 80 for PCP removal. We determined the effect of the role of PCP surfactant for the biofilm and auxin production of the selected bacterial strain of <em>P. fluorescens</em> GU059580. High-performance liquid chromatography and spectroscopic analysis were used to determine PCP removal and bacterial growth, respectively. Biofilm production was determined using 96-well polystyrene plates, and auxin production was determined using spectrophotometric measurement at 535 nm. The results showed the removal of PCP from wastewater by <em>P. fluorescens</em> GU059580 is about 90.12%. The PCP removal from wastewater showed an improvement of about 96.5% after the addition of Tween 80, whereas significant biofilm formation was found in the mineral liquid medium supplemented with PCP and Tween 80, with a value of 3.78. The highest concentration of auxin was found in the presence of PCP without surfactants, showing a value of 1.7 mg·L<sup>–1</sup>. To conclude, <em>P. fluorescens</em> GU059580 can be used in bioreactors or some specific wastewater treatment processes for bioremediation.

Effect of plant root exudate constituents on the degradation of phenanthrene by rhizobacterium Mycolicibacterium gilvum (Mycobacteriaceae, Actinobacteria)

The influence of the major components of plant root exudates, namely, carboxylic acids (succinic acid as an example) and secondary plant phenolic metabolites – flavonoids (rutin as an example), on the microbial degradation of the three-ring polycyclic aromatic hydrocarbon (PAH) phenanthrene by rhizobacterium Mycolicibacterium gilvum was studied. The destructive activity of this microorganism relative to PAH was studied by cultivating it in a liquid mineral medium containing phenanthrene (0.2 g/L), succinic acid (5 g/L), and rutin (0, 0.05, 0.1 or 0.2 mmol/L) at 30°C under aeration on a shaker (130 rpm) for 14 days. The stimulating effect of succinic acid and rutin on the microbial degradation of phenanthrene was revealed. It was found that carboxylate was utilized as the main growth substrate for the microorganism, while flavonol and PAH had little effect on bacterial growth. Rutin had no antimicrobial effect on the microorganisms studied; on the contrary, in combination with succinic acid, it significantly increased the biomass growth. At high concentrations (0.1 and 0.2 mmol/L), rutin inhibited the degradation of phenanthrene by 22 and 56%, respectively. However, at a concentration of 0.05 mmol/L rutin increased phenanthrene degradation by 10%. Thus, the results obtained showed the dependence of the PAH microbial degradation efficiency on the presence, combination, and concentration of the plant root exudate constituents.

Physio-chemical properties of methyl red-degrading strains isolated from textile wastewater

Methyl red (MR)-reducing strains isolated from textile wastewater were identified and tested for physio-chemical properties. The bacterial colonies picked from the mineral medium supplemented with textile wastewater were transferred onto the mineral medium containing MR for the test. Two strains, namely SYK and STXL2, demonstrating the ability of MR decolorization in the agar medium were selected and purified. The strains SYK and STXL2 incubated in the liquid mineral medium with MR were able to reduce 100 % and 95.5 % of MR after 4 days incubation, respectively. Gram staining of both strains confirmed these two isolates were gram positive. The SYK strain has white, irregular and dry colonies while the STXL2 strain has orange, opaque, circular and glossy colonies. Both strains have short rod shape of cell under SEM images. The two strains were able to grow in the media such as Luria-Bertani medium, Meat-peptone broth, and Nutrient broth. The strain SYK were able to grow quickly in the medium with maltose while the strain STXL2 used glucose as the best carbon source. Biochemical tests showed that both strains had positive reaction in Voges-Proskauer test and negative reactions in lipase, cellulose, amylase, citrate, and gelatin hydrolysis tests. The SYK strain had positive reactions in protease, urease, and ammonia tests while the STXL2 strain had positive reaction in nitrate reduction test. The sequences of 16S rNA genes identified the SYK and STXL2 strains as Streptomyces cellulosae (100 % of similarity) and Rhodococcus ruber (99 % of similarity), and registered in the GenBank via the accessing number MZ414193 and MZ414194, respectively.

СКРИНИНГ УГЛЕВОДОРОДОКИСЛЯЮЩИХ МИКРООРГАНИЗМОВ, ОБЛАДАЮЩИХ УСТОЙЧИВОСТЬЮ К ТЯЖЕЛЫМ МЕТАЛЛАМ И ХЛОРИДУ НАТРИЯ

In the process of extraction of hydrocarbon raw materials, in addition to mechanical disturbance of the soil cover, it is chemically contaminated with hydrocarbons and heavy metals contained in oil and oilfield wastewater, mainly of sodium chloride composition. Combined pollution has a more pronounced adverse effect on plants and soil biocenosis than each pollutant alone and leads to a very rapid loss of soil fertility. The aim of this work was to screen for new strains of hydrocarbon-oxidizing microorganisms resistant to elevated concentrations of sodium chloride and heavy metals. Isolation of strains of microorganisms from samples of technogenically contaminated soils from the territory of the Republic of Bashkortostan was carried out by the method of enrichment cultures on a liquid mineral medium of Raymond with oil. The hydrocarbon-oxidizing activity of the isolates was determined by the degree of destruction of the aliphatic oil fraction by the gas chromatographic method after extraction with hexane. The resistance of strains to the presence of sodium chloride and heavy metals (Zn, Co, Cd, Cu, Pb, Ni) was established by their growth on meat-peptone agar with various concentrations of NaCl or salts of these metals. Of the 14 isolated strains, 6 were selected, most actively growing on a solid medium with oil. The highest abundance and the most significant hydrocarbon-oxidizing activity during cultivation in a liquid medium with oil was found in strain 22 (the degree of oil biodegradation was 93.0%). All microorganisms were halotolerant and grew at 5 and 7% sodium chloride in the medium, but cultures 11 and 41, which withstood a NaCl concentration of 10%, turned out to be the most resistant to salinity. Strains 33.1 and 33.2 had the highest resistance to the action of metals. Lead and zinc were the least toxic to microorganisms. The results obtained should be considered preliminary, and the isolates themselves need further research with a view to their possible application in ecobiotechnology.

Biodegradation of polycyclic aromatic hydrocarbons by Acremonium sp. activity

Chihuahua State is the largest apple producer in Mexico. To prevent apples from freezing during the winter, petroleum-based fuels are used to heat the orchards. In this research work, a microorganism isolated from inside of a fuel plastic container was identified through morphological analysis as the fungus Acremonium sp. By cultivating the fungus in potato dextrose agar medium (PDA), we were able to study the biodegradation rates of three polycyclic aromatic hydrocarbons (PAHs). The isolated microorganism was incubated at 28 ºC in M9 mineral liquid medium using phenanthrene, anthracene, and pyrene as the only carbon source at a concentration of 50 µg/mL. Degradation of PAHs was monitored using an environmentally friendly reverse phase ultra-high-performance liquid chromatography (UHPLC) system, with a C18 column, 30% water acidified at 0.1% with trifluoroacetic acid, and 70% acetonitrile as the mobile phase, 40 ºC column temperature, and UV detection at 254 nm. After 30 days of biodegradation (optimal time), the final mean concentration for individual degradation experiments were 2.51 µg/mL phenanthrene, 3.02 µg/mL anthracene and 5.53 µg/mL pyrene. Those final concentrations indicate degradation percentile of 95%, 94%, and 89%, respectively. Biodegradation experiments combining all three PAHs showed similarly high degradation percentiles of 93%, 94%, and 85% for phenanthrene, anthracene, and pyrene, respectively. The dry weight average at day 30 in the individual and combined degradation experiments was 2.34 mg/mg PAHs degraded.

Composition of bacterial community and isolation of bacteria responsible for diuron degradation in sediment and soil under anaerobic condition.

The herbicide diuron is extensively used in the agriculture sector and is detected widely in the environment. Although several studies on the degradation of diuron by aerobic microorganisms have been reported, the degradation of diuron by anaerobic microorganisms has not been received much attention. Also, no pure culture that can degrade diuron under anaerobic conditions has yet been reported. The evaluation of diuron degradation in the soil and sediment slurries showed that diuron led to a decrease in the biodiversity of the bacterial communities. Two mixed bacterial cultures, one from the soil and the other from sediment slurries, were isolated from the enrichment media under anaerobic conditions. After 30days of incubation at 30°C, the mixed bacterial culture from the soil degraded 84.5 ± 5.5%, and that from the sediment slurry degraded 94.5 ± 3.0% of diuron in liquid mineral medium at an initial concentration of 20mg/L. 1-(3,4-dichlorophenylurea (DCPU), 3-(3-chlorophenyl)-1,1-dimethylurea (CPDMU), and 3,4-dichloroaniline (3,4-DCA) were the major diuron metabolites produced by both the indigenous microorganisms and the isolated bacteria.

PSEUDOMONAS VERONII STRAIN 7P-81 - A PROMISING DESTRUCTOR OF ALIPHATIC AND AROMATIC HYDROCARBONS

Pseudomonas veronii strain 7p-81 is a destructor of both aliphatic and aromatic hydrocarbons. The genes encoding the enzymes of degradation of PAHs and n-alkanes are localized on the conjugative plasmid. The degree of oil degradation by the studied strain in a liquid mineral medium was 20.8% for 7 days.