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0ASSESSMENT OF ESCHERICHIA COLI SORPTION ON AMr6, ZnAl, AND L16 ALLOYS USING CERAMIC COATINGS: A DENSITOMETRIC APPROACH
1Sattarov M.E., 2Lee L.T., 3Akhmedova Z.R.
1Ph.D., Associate Professor, Head of the National Collection of Industrial Microorganisms, Tashkent Research Institute of Vaccines and Serums, Tashkent 2Head of the Laboratory of Microbiology, Tashkent Research Institute of Vaccines and Serums 3Doctor of Biology, Professor, Head of the Laboratory of Environmental Biotechnologies, Institute of Microbiology, Academy of Sciences of the Republic of Uzbekistan https://doi.org/10.5281/zenodo.14062401
Abstract. This study investigates the sorption efficiency of E.coli ATCC 25922 strain on ceramic-coated samples of AMr6, ZnAl, andL16 alloys. The results demonstrated variable sorption capacities across the alloys, with the ZnAl sample exhibiting the highest sorption rate. Specifically, the reduction percentages of bacterial presence in the microbial suspension were 5.7% for AMr6, 36.9% for ZnAl, and 12.7% for L16. The findings indicate that the ZnAl alloy, in particular, possesses superior sorptive properties compared to the other alloys, suggesting its potential application in bacterial containment and environmental safety measures. This research contributes valuable insights into the development of ceramic coatings for bacterial sorption applications.
Keywords: sorption, E.coli, aluminosilicate, sorbent, densitometer, microbial suspension, AMr6, ZnAl, L16.
INTRODUCTION
Globally, there is ongoing research focused on the targeted synthesis of new, highly efficient, functional nanostructured materials for various applications, such as catalysts, sorbents, drug delivery systems, membranes, composite fillers, and ceramics. Catalytic and adsorption processes frequently rely on materials that possess a well-developed nanoporous structure to be effective.
Oxide materials, containing elements like silicon and aluminum-both in natural and synthetic forms are extensively utilized and hold considerable scientific interest. The ability to control their porous structure and composition through synthesis and subsequent modifications is crucial for enhancing their desired functional properties [1].
In modern biotechnological processes, the technique of immobilizing microorganisms on solid surfaces has been a significant advancement. This method has been extensively applied in practical biotechnology, including food production. Notably, the immobilization of microorganisms has profoundly impacted fermentation technologies, particularly in the continuous flow production of sparkling wines [2].
However, natural layered aluminosilicates often contain foreign impurities that can adversely affect the functional activity of derived systems. Moreover, the composition of these aluminosilicates can vary even within the same deposit. Synthetic aluminosilicates can be produced under hydrothermal conditions at temperatures ranging from 100-400°C or by melting
initial oxide mixtures at temperatures between 800-1500°C. Synthesized under the former conditions, these materials generally exhibit superior textural characteristics.
Layered aluminosilicates feature active centers such as exchange cations, hydroxyl groups, and sorbet water molecules, which greatly broaden their practical applications when modified to enhance specific adsorptive and catalytic properties. Common modification techniques involve treating clays with acidic or alkaline solutions at elevated temperatures to remove undesirable inclusions. Such modifications typically increase the specific surface area and, consequently, the sorption capacity of the materials [3].
The primary chemical components of natural layered aluminosilicates, including clay minerals, are SiO2 (30-70%), AhO3 (10-40%), and H2O (5-10%). Their structure comprises a tetrahedral silicon-oxygen network (T-network) and an octahedral Al or Mg oxygen-hydroxyl network (O-network), which are interconnected into layers that form specific mineral combinations. These silicates are categorized into two types: 1:1 and 2:1 [4].
The adsorption of microbiological entities, which typically carry a negative charge in aqueous environments, is most effective on surfaces with a positive charge. However, options for such adsorbents are limited, especially following the cessation of chrysotile asbestos use. Thus, the development of new, positively charged sorbents capable of effectively adsorbing microorganisms remains a priority.
Recent advancements in nanotechnology have enabled the production of a new positively charged adsorbent-aluminum oxyhydroxide with a pseudoboehmite structure, available as porous, nearly spherical agglomerates. When these agglomerates are affixed to polymer microfibers, they form a fibrous material with a positively charged, accessible surface, enhancing the efficiency of bacterial and viral adsorption from aqueous and biological fluids.
The objective of our study is to investigate the sorption of the Escherichia coli strain ATCC 25922 on ceramic-coated samples made from AMr6, ZnAl, and L16 alloys using the densitometric method [5].
MATERIALS AND METHODS
Equipment and Consumables
The experiments were conducted using the following equipment: VK-75 autoclave, BINDER air sterilizer, Biosan densitometer, electronic scales, and a binocular microscope. Consumables used included physiological NaCl solution, Endo medium, trypticase soy broth, glycerol, E.coli culture ATCC 25922, Petri dishes, PH-16 tubes, measuring pipettes (1 ml, 5 ml, 10 ml), cryotubes (2.5 ml), tripods, an alcohol lamp, and bacteriological loops.
Ceramic Coating Samples. The materials tested were ceramic coatings on the following
alloys:
AMr6 alloy
ZnAl alloy
L16 alloy
Preparation for the Test Petri dishes, test tubes, pipettes, and ceramic coating samples in AMr6, ZnAl, and L16 alloys were prepared, wrapped, and sterilized using a BINDER air sterilizer. The Endo medium was prepared as per the manufacturer's instructions and a 0.9% saline solution of NaCl was also prepared. It was sterilized in an autoclave for 15 minutes at a temperature of 121°C.
The E.coli ATCC 25922 culture was preserved in trypticase soy broth with glycerol at -20°C for up to a year.
Experimental part
Testing was initiated by thawing the E.coli culture at room temperature. The culture was then inoculated onto two Petri dishes containing Endo medium from HiMedia Laboratories, India. A small quantity of the culture from the cryotube was carefully spread on the surface of the medium at the edge of the dish using a bacteriological loop. After application, the loop was incinerated to remove residual material.
The inoculation was performed by lightly touching the loop to the nutrient medium, and carefully drawing strokes along sectors that were approximately divided into four equal parts. Care was taken to ensure the strokes were as close to each other as possible to extend the overall inoculation line, which facilitates the growth of isolated microbial colonies. Following inoculation, the dishes were placed in a thermostat set at 37°C for 24 hours to promote colony development.
RESULTS AND DISCUSSION
Initial Densitometric Analysis. We began our experimental analysis by preparing a microbial suspension of E. coli with a 0.5 McFarland standard, indicating a specific turbidity correlating to bacterial concentration [6]. This suspension was created by adding 10 ml of saline solution into nine sterilized tubes, three for each sample alloy (AMr6, ZnAl, L16), to measure the initial optical density.
Table 1: Initial Densitometer Readings before E.coli addition
Sample Densitometer Readings before adding E.coli
AMr6 0.39
0.33
0.38
ZnAl 0.37
0.41
0.5
Li6 0.26
0.29
0.34
Following this, E. coli was added to achieve a final McFarland reading of 0.5 for each tube. The post-inoculation readings were taken to assess the increase in optical density due to bacterial addition.
Table 2: Densitometer Readings Before and After adding E.coli
Sample Before Adding E. coli After Adding E. coli (initial optical density of saline solution before adding bacteria)
AMr6 0.39 0.9
0.33 0.83
0.38 0.91
ZnAl 0.37 0.92
0.41 0.95
0.5 1.22
Li6 0.26 0.76
0.29 0.81
0.34
0.81
Differential Analysis of Optical Density. To analyze the differential impact of the ceramic coatings on E.coli sorption, we calculated the difference in densitometer readings before and after the addition of the bacterial culture.
Table 3: Difference in Densitometer Readings Before and After adding E.coli
Sample Before After Differences
AMr6 0.39 0.9 0.51
0.33 0.83 0.50
0.38 0.91 0.53
ZnAl 0.37 0.92 0.55
0.41 0.95 0.54
0.5 1.02 0.52
L16 0.26 0.76 0.50
0.29 0.81 0.52
0.34 0.81 0.47
Sorption Analysis. For sorption analysis, each ceramic sample was immersed in 30 ml of the prepared microbial suspension (divided among three test tubes) at room temperature for 30 minutes to allow E.coli sorption.
Table 4: Optical Density of Microbial Suspension in a Volume of 30 ml
Sample Optical Density (Average of 3 tubes)
AMr6 (0.9+0.83+0.91):3=0.88
ZnAl (0.92+0.95+1.02):3=0.96
L16 (0.76+0.81+0.81):3=0.79
Post-exposure, the microbial suspensions were transferred to clean sterile test tubes to measure the final optical density after sorption.
Table 5: Sorption of Microbial Suspension on Samples
Sample Exposure for sorption Room temperature
AMr6 30 minutes +24оС
ZnAl 30 minutes +24оС
L16 30 minutes +24оС
After 30 minutes, a microbial suspension was added from each dish into 3 clean sterile test tubes to measure the optical density after sorption.
Table 6: Microbial suspension densitometer readings before and after sorption
Sample Before After Differences
AMr6 0.88 0.83 0.05
ZnAl 0.96 0.65 0.31
L16 0.79 0.69 0.1
These results illustrate a reduction in optical density, indicating the adsorption of E.coli by the samples, with varying efficiencies across different alloys.
Table 7: Decrease in Optical Density of Microbial Suspension (%)
Sample Decrease in optical density of microbial suspension % of
(%) sorption
AMr6 5.7= (0.88-0.83)x100/0.88 5.7
ZnAl 36.9=(0.96-0.65)x100/0.96 32.3
L16 12.7=(0.79-0.69)x100/0.79 12.7
Moreover, the McFarland standard was recalculated to approximate the number of microbial cells based on the fact that 1 ml of a 0.5 McFarland microbial suspension contains approximately 1.5 x 108 E.coli bacteria. This quantification further supported the sorption dynamics observed across the different ceramic coatings.
Table 8: Optical density indicator before and after sorption/number of microbial cells in 1 ml
Sample Optical density before sorption/number of microbial cells in 1 ml Optical density after sorption/number of microbial cells in 1 ml Difference in indicators before and after sorption/number of adsorbed microbial cells in 1 ml Number of non- adsorbed microbial cells in 1 ml
AMr6 0.88/2.64x108 0.83/1.11 x108 0.05/1.5x107 2.49x108
ZnAl 0.96/2.88x108 0.65/1.95x108 0.31/9.3x107 1.95x108
L16 0.79/2.37x108 0.69/2.07x108 0.1/3x107 2.07x108
Additionally, for the experiment, ceramic coatings in the alloy AMr6, ZnAl and L16 were taken. Their sorption of a microbial suspension of E.coli was studied in dynamics. The characteristics of the microbial suspension of E.coli were determined before and after addition to ceramic coatings in the alloy AMr6, ZnAl and L16.
Furthermore, as can be seen from this table, after 30 minutes of adding a microbial suspension of E. coli to ceramic coatings in the alloy AMr6, ZnAl and L16, it was observed that the sorption of the bacterial suspension on them was different.
Table 9: Evaluation of sorption from microbial suspension of E.coli by ceramic coatings in
AMr6, ZnAl and L16 alloy
Sample Densitometer readings before adding E.coli Densitometer readings after adding E.coli Average value of 3 tubes Sorption exposure Densitometer readings of microbial suspension after sorption Decrease in optical density of microbial suspension % of sojgtion
AMl's 0.39 0.9 0.88 30 minutes 0.83 0.05 5,7
0.33 0.83
0.38 0.91
ZnAl 0.37 0.92 0.96 30 minutes 0.65 0.31 32.3
0.41 0.95
0.5 1.02
Li6 0.26 0.76 0.79 30 minutes. 0.69 0.1 12.7
0.29 0.81
0.34 0.81
Note: the bacterial content in 1 ml of microbial suspension is 0.5 according to McFarland (1.5x108).
CONCLUSION
In this study, the sorption efficiency of ceramic coatings on AMr6, ZnAl, and L16 alloys was evaluated using densitometric readings of microbial suspensions after exposure to E.coli. The post-sorption densitometer readings indicated optical densities of 0.83 for the AMr6 alloy, 0.65 for the ZnAl alloy, and 0.69 for the L16 alloy. Analysis of the percentage reduction in bacterial count
revealed that the ZnAl alloy achieved the highest reduction at 36.9%, followed by the L16 alloy at 12.7%, and the AMr6 alloy at 5.7%.
These results underscore the superior sorption capabilities of the ZnAl sample compared to the other tested alloys. Consequently, the ZnAl alloy demonstrates a significant potential for application in the sorption of E.coli bacteria from various environments. This capability positions the ZnAl alloy as a promising material for enhancing microbial safety in relevant industrial and environmental applications.
Acknowledgments
This research was supported by the fundamental project codes FL-7923051836 in Uzbekistan and T23UZB-015 in Belarus, highlighting the collaborative efforts in advancing material sciences in these regions.
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