Bacterial luminescence of manganese- and cobalt-containing ultrafine particles (Mn2O3 and Co3O4) in the rumen fluid

Along with the main nutrients, proteins, fats and carbohydrates, mineral elements are important in feeding farm animals, including cattle and poultry (D.V. Mashnin et al., 2022; T.M. Okolelova et al., 2018). Their inorganic or organic forms are components of premixes (M.Y. Mishanin et al., 2021; O.S. Koschaeva 2018). However, nanocompositions are more promising, the properties of which can be modeled by changing the shape, synthesis paths and size of ultrafine particles (UFP) (S. Miroshnikov et al., 2019). However, the use of UFPs has a number of limitations related to their potential toxicity (E. Rusakova et al., 2015). It is also known that the symbiotic microflora forms a multicomponent suspension of organic substances, intermediate and final metabolites of the microbiome, capable of interacting with UFP (B.S. Nurzhanov et al., 2019). In this work, the dynamics of luminescence of a bacterial test object was established for the first time when a complex of UFPs and rumen fluid was introduced into the nutrient medium. This combination has been shown to neutralize the toxicity of nano-structures. The purpose of our work was to evaluate the properties of ultrafine particles using the example of various concentrations of manganese and cobalt oxide in the biochemical environment of the ruminal community based on the method of inhibition of bacterial luminescence. The study was conducted on the basis of the center Nanotechnology in Agriculture of the FRC BST RAS (Orenburg) in 2022. Chemically pure manganese oxides Mn2O3 and cobalt Co3O4 (99 %) for analysis in the amount of 157.8 and 240.7 mg were dispersed by ultrasound at a frequency of 35 kHz in 1 ml of distilled water for 30 minutes at 25 ºC. The rumen fluid (RF) was collected through a chronic rumen fistula (d = 80 mm, ANKOM Technology Corporation, USA) 3 hours after feeding in a Kazakh white-headed bull, whose main diet was 30 % concentrates and 70 % coarse feed without the addition of UFP. The luminescent bacterial test Ecolum (a lyophilized culture of Escherichia coli microorganisms carrying a hybrid plasmid pUC19 with lux CDABE cloned P. leiognathi 54D10 genes, SIS IMMUNOTECH, Russia) was used. In a bioluminescent plan, a series of double dilutions of the UFP and RF suspension was prepared starting from 50 ml Mn2O3 (1 mol/l) + 50 ml RF; 50 ml Co3O4 (1 mol/l) + 50 ml RF; 50 ml Mn2O3 (1 mol/l) + 50 ml distilled water; 50 ml Co3O4 (1 mol/l) + 50 ml distilled water; 100 ml RF; 100 ml distilled water (control). Then 100 ml of the Ecolume test system were added to each cell to a total concentrations of UFPs from 0.25 to 0.00025 mol/l and dilution of RF from 1_2 to 1:2048 in a pure test and from 1:4 to 1:4096 in the test with UFPs. The toxicity of the studied samples was determined on a multifunctional micro-lancet reader TECAN Infinite F200 (Tecan Austria GmbH, Austria), fixing the luminescence value of the bacterial strain E. coli K12 TG1 at different concentrations of ultrafine particles and rumen fluid for 3 hours with a period of 5 minutes. Based on the obtained data, graphs reflecting the dynamics of bioluminescence inhibition were constructed and the toxicity index (T) and the relative value of bioluminescence (A) was calculated. It was found that UFPs in their pure form cause dose-dependent inhibition of bacterial luminescence, suppressing over 50 % of the luminescence (EC50) even when diluted by 2048 times (0.00025 mol/l). The values of the toxicity index, when calculating which the control is taken as 100 %, clearly indicate a decrease in the toxic properties of suspensions with a decrease in the proportion of UFPs in them. For Mn2O3, this value ranged from 89.76 % at a concentration of 0.25 mol/l to 38.57 % at 0.00025 mol/l at the 1st minute of the exposure and from 95.16 to 52.85 % at the end of the 3rd hour; for Co3O4 -99.44 and 32.80 %, respectively, at the 1st minute, and 99.43 and 54.72 % at the end of the 3rd hour. Similar indicators in the experiment with rumen fluid appeared only in the first minutes of exposure, after which the luminosity increased significantly, reaching 769.10 % to the control at 64-fold dilution. When combining rumen fluid with UFPs, a regression of the toxic properties of the latter was observed, although the maximum luminosity in combination with Mn2O3 was only 43.28 % of those for native RF, in combination with Co3O4 36.44 %. The observed changes in luminescence were divided into three types. The first type is control (luminescence changes in proportion to the growth phases of the bacterial culture; without additives).