Isolation of New Strains of Microorganisms for Bio-Purification of Polluted Reservoirs of Northern Kazakhstan

In the course of the study, the following theoretical methods were used: analysis of scientific and methodological literature of Kazakh and Western European researchers; synthesis and specification of theoretical material over the past 3-5 years; generalisation of research material on microbiology, water purification technologies, ecology, general biology; analogies and modelling. An empirical investigation of the experience of research organisations, normative and educational documentation was conducted during a laboratory experiment, which took place in three stages: ascertaining, forming, and controlling. The methods of mathematical statistics using the programmes Statistica 6.0 and Microsoft Excel 97, graphical images of the results, and drawings were used.

At the ascertaining stage, the study identified the objects of research, which were bacterial strains isolated from the water bodies of Astana – BolshoyTaldykol (BT) and Ulmes (U) lakes and collection test strains of bacteria taken from the Biobank of Industrial Microorganisms of the Republican Collection of Microorganisms: Escherichia coli ATCC 25922 B–RKM 0447, Staphylococcus aureus ATCC-6538 B-RKM 0470, Salmonella enteriditis B-RKM 0680, Klebsiella pneumoniae B-RKM 0444, Enterococcus faecium, Pseudomonass taiwanensis CB2R-1B-RKM 0726, Pseudomonas aeruginosa G13 B-RKM 0427, Aeromonas punctata 30 B-RKM 0287 [2, 3]. Isolation of bacterial isolates from the studied water samples was conducted by the method of marginal dilutions, followed by seeding on pre-prepared Petri dishes with nutrient agar, dry nutrient agar, Czapek, Sabouraud, and Endo media. The cups were incubated at 30℃ and 37℃ for 1-3 days. Pure cultures were obtained by re-streaking the plates with the appropriate one for each microorganism species. The morphological features and purity of the isolated isolates were checked by staining with a GramStrains-Kit, Himedia staining kit for microorganisms using a MicrosMC300X laboratory microscope (Austria) with immersion oil and ×100 magnification. Cultural signs were considered by standard methods.

At the formative stage, the study determined proteolytic, lipolytic, and amylolytic activities, and carbohydrate fermentation (CF) was conducted according to standard methods used in microbiology. Determination of the phosphate-mobilising activity (PMA) of bacteria was detected on Muromtsev's medium by the Gerretson precipitation method. Antibiotic sensitivity was determined by the standard disco-diffusion method using available antibiotics. Antagonistic activity (AnA) was determined by the method of diffusion in agar along the width of the zone of no growth of available opportunistic cultures. The isolated bacterial pure cultures were identified by mass spectrometry on a MALDI-TOF analyser (Bruker). At the control stage of the experiment, the study drew conclusions and proposed recommendations for the introduction of new virus strains for the bio-purification of polluted reservoirs of Northern Kazakhstan. Statistical processing of the results was conducted using the software package Statistica 6.0, Microsoft Excel 97. In total, 14 isolates of various bacteria were isolated: 8 isolates from Bolshoy Taldykol lake, 6 isolates of bacteria from Ulmes lake, represented mainly by gram-negative bacteria, except for isolate U3.

The main source of life on Earth is water, it is of particular importance both economically and socially. Water as a source of life forms the climate on the planet, thus affecting all living beings and ensuring a continuous cycle of nutrients. This interrelation of the components of the biosphere makes them dependent on the ecological state of each other and biogeocenoses, in particular [11]. The ecological state of the hydrosphere was understudied, its state is dynamically developing with the advent of technological progress. Unfortunately, modern requirements for water resources management at industrial enterprises are ignored, environmental protection recedes into the background but should be paramount: research in the field of global changes in the biosphere and the climate of the planet. The method of bio-purification of water resources is based on the ability of heterotrophic and autotrophic microorganisms to use a variety of organic compounds as food sources, subjecting the latter to biochemical transformations. Microorganisms are a good indicator for monitoring the ecological state of aquatic ecosystems [12]. Due to their large number, they are easy to obtain in the laboratory for a test sample. The presence of certain microorganisms positively correlates with certain types of pollution. Being a kind of pollution indicator, some microorganisms, when exposed to pollutants, release stress proteins, such as cadmium and benzene. Bacteria with bioluminescent properties are used to detect toxins in the water. The cellular metabolism of the bacterium is immobilised by identifying toxins, which affects the amount of light emitted by the bacteria [13, 14]. To isolate various groups of microorganisms, the study used water samples taken from the following surface sources of Astana:

1. Ulmes is a natural lake recreated as a result of the reclamation of the Taldykol sewage evaporator. The maximum depth is 3.0 m. The category of the lake being reconstructed is a water object of cultural and community water use.

2. Bolshoy Taldykol is a natural lake recreated as a result of the reclamation of the Taldykol wastewater storage evaporator, located in the southwestern part of the city of Astana. The water area of the lake is 292 hectares, the maximum depth is 2.6 m. The category of the lake being reconstructed is a water object of cultural and community water use (Figures 3 and 4).

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Figure 3. Bolshoy Taldykol lake

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Figure 4. Ulmes lake

Characterisation of bacteria by cultural, morphological, and biochemical characteristics is one of the key conventional taxonomic methods. These features are quite species-specific; their examination is used to determine the species affiliation of the culture under study. The cultural-morphological and physiological-biochemical properties of isolates and collection strains were studied (Table 4).

Burova [15], upon investigating the effect of temperature and pH on the state of rhizosphere microorganisms, established that bacteria from the genus Pseudomonas are characterised by rapid growth. In particular, Pseudomonas chlororaphissub sp. aureofaciens are characterised by rapid growth and the ability to settle in the plant ecosystem and thereby negatively affect micro-organisms that hinder plant growth. Bacteria of this genus are widely used for bio-purification of reservoirs, as they are capable of producing growth hormone and compounds with fungicidal or fungistatic properties, assimilating nitrogen from the atmosphere and phosphorus compounds against phytopathogenic fungi. Important in the isolation of new strains of microorganisms of this kind is the presence of certain factors for their development: the degree of aeration, the acidity of the medium, temperature. However, current research focuses on the use of bioremediation to clean water bodies of pollutants, taking into account these beneficial results.

Snegirev and Karepina [16] examined the photo-disinfecting effect of sensitisers of Escherichia coli culture strains depending on organic water pollution. The museum stick strain (Escherichia coli) is a classic indicator among microorganisms in obtaining sanitary indicators of water. Microorganisms were sown on differential nutrient media by direct seeding and membrane filtration in accordance with the species. In this experiment, Snegirev and Karepina [16] found that the highest efficiency of inactivation of E. coli photodisinfection at a concentration of 0.5 mg/l operates with organic contamination of water with proflavine acetate and methylene blue sensitisers during transillumination for 30 minutes. The authors suggest that these findings demonstrate the potential of sensitisers for the bio-purification of water contaminated with organic pollutants. Annual water consumption on the planet is from 3300-3500 km³, the demand for this most valuable natural resource is increasing [17, 18]. The authors of the current study recognize the importance of addressing issues related to water pollution and highlight the potential of biotechnologies, such as the use of microorganisms, to help solve these issues. Therefore, biotechnologies use the properties of adaptation of microorganisms to successfully solve the issues of bio-treatment of industrial wastewater, which includes complex organic compounds [19, 20]. The authors of the current study note that the use of microorganisms in biotechnologies is a promising approach for the treatment of industrial wastewater, but caution that careful monitoring is needed to prevent the development of antibiotic resistance. The ability of microbes to quickly acquire resistance to antibiotics requires monitoring their use and finding new means to combat their reproduction [21]. Thus, substances of natural origin are able to regulate the ratios of groups of microorganisms in microbiocenosis. The detrimental effect of organic suspensions on the process of self-purification of waters lies in the sediments with which they cover the bottom and delay or completely stop the vital activity of microorganisms. As they are able to selectively act on various wastewater containing suspensions of organic origin and thus regulate the dissolved organic matter, they negatively affect the state of water bodies [22-24]. Such a mechanism makes it difficult for light to penetrate to depth and hinders the processes of photosynthesis. The authors of the current study acknowledge the importance of photosynthesis in the maintenance of healthy water bodies and suggest that their findings on the use of sensitisers for the bio-purification of water could contribute to the development of sustainable solutions to address water pollution.

Thu et al. [25] investigated the ability of microorganisms to immobilise for the bio-treatment of wastewater with a high concentration of waste from the petrochemical industry. Bio-purification using new strains of microorganisms is ineffective in oil refining production. It is necessary to pre-clean from the active effects of oil refining products, namely, the use of highly active specialised microbial complexes and devices before the wastewater is transported to the general plant treatment facilities. For this purpose, the technology of immobilisation of microorganisms on a polymer carrier was used, which maintains a high concentration of destructor microorganisms and increased the resistance of the biocenosis to adverse factors in the purification system [26, 27]. Thu et al. [25] used a cumulative culture of microorganisms adapted to high concentrations of wastewater components. In the first period (about 20 months), the effectiveness of wastewater pretreatment without using a carrier with fixed microflora was examined. In the second period, lasting from 37 to 46 months, various immobilising carriers in the form of fibreglass ruffs, polyurethane foam, and activated carbon were used. As a result, the breeding community of microorganisms, which are distinguished by a high degree of neutralisation and decomposition of organic sewage pollution, are attached to the fibreglass ruff in the bioreactor. The findings of Thu et al. [25] regarding the use of immobilising carriers to maintain a high concentration of destructor microorganisms and increase the efficiency of bio-purification are relevant to this study.

In turn, Rehman et al. [1] substantiated the method of modifying polyfunctional sorption materials based on the residual biomass of Chlorella sorokiniana microalgae in relation to oil products and heavy metal ions. It was established that the sorption material successfully binds water pollution with oil and petroleum products using composite magnetosorbents, which are easily removed from the water surface using a magnetic field. The development of new technologies to protect the environment from waste pollution from various industrial productions and the cleaning of already polluted areas is an important area of biotechnological research. The efforts of most researchers from different countries are aimed at solving this problem. Therefore, the creation of biotechnologies aimed at detoxification and disposal of pollution of water bodies involves the study of the microbiological status of this anthropogenic ecosystem. Among them, biological methods of pollution purification occupy an important place – the creation of consortia of strains of destructive microorganisms, including immobilised ones [28, 29]. Currently, bacterial cocktails are offered in a wide range by biotech companies in Europe, the USA, Japan, and are in great demand. However, the practice of using foreign bacterial preparations is ineffective due to different climatic and environmental conditions. The potential of biological species is to bring changes to the ecology of the environment [30]. Therefore, the settlement of microorganisms unusual for this water area is one of the biggest threats in the world.

Thus, to prove the effectiveness of microorganisms in wastewater treatment, the article presents the results of screening and characteristics of 14 isolated strains of bacteria, among which 8 strains have high reliability and showed the greatest activity in biological wastewater treatment. Such criteria as antagonistic, phosphate-mobilizing, proteolytic, lipolytic, amylolytic, nitrifying activity, carbohydrate fermentation and sensitivity to antibiotics were used for screening.