**4. Discussion**

Perhaps, the most important results of the study are that in systems with minimum and maximum concentrations of inorganic ions, only analogs of the phosphate renal stone mineral phases were observed, while calcium oxalates were obtained under given conditions only with an increase in the concentration of oxalate ions up to the oxalatouria values, both in experiments with bacteria and without them. This result is in general agreement with the literature data on model crystallization experiments in the human urinary system [21,27,31]. Thus, according to the thermodynamic calculations and experiments in systems that simulate composition of the physiological liquid, calcium oxalates are formed in much smaller quantities than what is actually observed during pathogenic processes in the human body. Moreover, the weddellite phase (calcium oxalate dihydrate) does not form at all [31]. Introduction of bacteria and protein (ovalbumin) to the system leads to a similar result in all the experiments, increasing the portion of weddellite and increasing the amount of calcium oxalates in general. It should be also noted that, according to the unit cell parameters of weddellite crystals which are formed in the presence of bacteria, the amount of "zeolite" H2O molecules (x) falls into a rather narrow range of values, whereas those in the structures of weddellite crystals from human renal stones vary much more (from 0.13 to 0.37 p.f.u.).

According to our data, all bacteria initiate the nucleation of calcium oxalates and promote the crystallization of metastable calcium oxalate dihydrate (weddellite) in the oxalate system (containing only Ca2<sup>+</sup> and [C2O4] 2– ions). The initiation of calcium oxalate nucleation in the presence of bacteria is in agreement with the results of some recent studies, which describe an increase in the number of calcium oxalate crystals and their size in the presence of bacteria [32].

As it was shown by the results of phosphate crystallization experiments, the addition of bacteria and nutrient media leads to a change in the phase composition of the precipitate and to the shift of the phosphate phase's formation boundaries (Figures 3 and 4). The addition of the MHB medium to the model solution with the minimum concentration of inorganic impurities led to the disappearance of octacalcium phosphate and whitlockite, followed by the formation of brushite and rare struvite occurrences. The same addition to the model solution with the maximum concentration of inorganic impurities led to the crystallization of apatite, along with brushite and struvite, and to the significant shift of brushite and apatite formation areas toward higher pH values of the solution (~7.0).

The addition of the MPB medium to the model solution with a minimum concentration of inorganic impurities led to the formation of brushite and whitlockite and, in addition, crystallization of struvite was detected at a pH of 7.26, so the shift in the struvite phase formation boundary in this system moved toward being significantly more acidic. The brushite phase was observed in this system in a narrower pH range around 7.06. Since brushite did not form in experiments without organic additives at such a high pH, this suggests that the boundary of its formation expanded to the more alkaline side. The same addition to the model solution with the maximum concentration of inorganic impurities did not lead to any change in the phase composition of the synthesized products. At the same time, it can be stated that the boundary of the brushite formation area has shifted to the more alkaline region of solutions and the boundary of the struvite formation area shifted to the more acidic region (pH of 6.96).

The addition of bacteria to the appropriate media led to additional changes in the composition of the precipitates (Figures 3 and 4). Thus, in the syntheses with minimal concentrations of inorganic impurities, the appearance of *Escherichia coli* and *Pseudomonas aeruginosa* in an MHB medium led to the formation of struvite and shifted its starting crystallization boundary to the more acidic region (pH of 7.05). Although struvite was initially present in the synthetic products, the appearance of bacteria in the MHB medium contributed to the displacement of its crystallization area to the more acidic region. The effect of *Escherichia coli* and *Staphylococcus aureus* bacteria on the crystallization of brushite was also well demonstrated in systems containing MHB; the shift of the brushite initial crystallization area occurred toward the more alkaline region. The effect of the bacteria addition on the crystallization of apatite was clearly visible in the MPB medium; the appearance of bacteria promoted crystallization of apatite and shifted its formation boundary to the more acidic region (pH of 6.72).

The change in the pH values of the solution during the biomimetic syntheses process occurred in different directions, due to both the crystallization process of various phases and the effect of a certain protein medium type and all types of bacteria addition. The decrease in pH in systems that modeled urine using inorganic components can be explained by the result of phosphate phase crystallization, while an increase in systems with bacteria can be explained by the influence of metabolic products. The presence of urease-producing bacteria such as *Pseudomonas aeruginosa*, *Klebsiella pneumoniae* and *Staphylococcus aureus* in urea led to an increase in pH [11,12].

The displacement of struvite crystallization boundaries obtained in the experiments, which led to its intensive formation, once again underlines the involvement of bacteria in the formation of "infectious" renal stones, described in a number of works [3,12]. At the same time, the expansion of the brushite crystallization area boundaries and the crystallization of apatite, as well as the formation of weddellite in the oxalate system, shows that the influence of the presence and function of bacteria in the crystallization medium was not only limited to the alkalization of the urine and the formation of ammonium ions, but significantly affected the types of growing mineral phases and the size of their crystallization areas with natural variations in urine pH.
