Metabolic and Morphological Aspects of Adaptation of Alkaliphilic Bacillus aequororis 5-DB and Alkali-Tolerant Bacillus subtilis ATCC 6633 to Changes in pH and Mineralization

The goal of the study is to evaluate metabolic and morphological changes of the facultative alkaliphile Bacillus aequororis 5-DB and the weakly alkali-resistant B. subtilis ATCC 6633 in a wide pH range and at different NaCl concentrations. The alkaliphile B. aequororis 5-DB is shown to have a broader general resistance to adverse factors (wide pH range, 50 g/L NaCl) than a weakly alkali-tolerant strain of the same genus. This alkaliphile is also shown to have a significantly greater resistance not only to high pH but also to low pH in comparison with B. subtilis ATCC 6633. The resistance of B. aequororis 5-DB to low pH was expressed in higher metabolic activity, maintenance of ΔpH, and no significant cell damage. The selected set of methods (reduction of resazurin to resorufin by cell dehydrogenases, bioluminescent method for determining ATP, AFM, and measurement of intracellular pH) allows us to adequately assess the ability of microbial cells to withstand harsh environmental factors. Nonspecific resistance of B. aequororis 5-DB was proven using a complex of selected methods. Tolerance to a wide range of pH and high salt concentrations may be useful for biotechnological applications of the strain.


Introduction
Soda lakes and artifcial alkaline biotopes, such as soda sludge reservoirs, harbor microorganisms that are resistant to high pH and salinity.Te microbial community of these environments includes extremophiles and tolerant to extreme factors organisms.Alkaliphiles require an alkaline environment to live, and their optimum pH for growth is about 10.0.Based on their preference for diferent environmental pH values, these microorganisms are grouped into two broad categories: alkali-tolerant organisms, which exhibit optimal growth at pH 7.0-9.0but cannot grow at pH above 9.5, and alkaliphilic organisms, which grow optimally at pH from 10.0 to 12.0.Tey are divided into facultative alkaliphiles, which grow well in the neutral pH range, but with an optimum at pH 10.0 or higher, and obligate alkaliphiles, which grow optimally at pH above 10.0, but do not grow at pH below 9.0 [1].
In addition to high pH, microorganisms in alkaline ecosystems are afected by high mineralization of the environment.Te consequence of high salt concentration in the medium is the loss of water from the cell and plasmolysis of the cell.Turgor pressure is the force within the cell that pushes the cytoplasmic membrane against the cell wall.To support tension, a cell maintains an intracellular osmotic pressure above that of the environment [2].To do this, halophiles carry out two strategies: (1) "salt in," which consists in the accumulation of molar concentrations of potassium chloride in the cell and (2) the synthesis of osmoprotectants by the cell.Weak halophiles grow optimally on the media containing 0.5% to 3% salt; moderate halophiles, 3% to 15% salt; and extreme halophiles, 15% to 30% salt.In addition, many halotolerant microorganisms grow both without the addition of salt and at a salt concentration of more than 12% [3].Survival and growth of Bacillus subtilis in an osmotically changing environment depends on adaptive reactions that are either part of the general stress response or specifc to osmotic stress.Specifc stress responses of many Bacillus spp.include the synthesis and absorption of certain organic osmolytes, in particular, proline, glycine betaine, and ectoine, under hyperosmotic conditions [4].
Most Bacillus spp.are moderately halotolerant and can grow under salinity over the 60 g/L.Tus, B. spizizenii was able to grow at mineralization above 57 g/L but with a long growth retardation time [5].However, the adaptive responses of bacilli during growth at high levels of mineralization have not been studied.Zhang et al. studied morphological changes during adaptation of Bacillus sp.YM1 to increased salt concentrations.It was shown that in a medium with 30-50 g/L salt, the cell surface was strongly wrinkled, sticky secretions appeared on the cell surface, and sporulation increased, but the shape and size of the cells did not change signifcantly [6].Te adaptation of alkaliphilic bacilli has been studied in detail, mainly from the point of view of energetics.Alkaliphiles demonstrate multiple adaptive responses that allow them to live at high pH [7].When growing in an alkaline environment with a low proton content and a high sodium ion content, alkaliphilic microorganisms, in addition to osmoadaptation, need to maintain intracellular pH homeostasis.In an alkaline environment, the formation of a chemiosmotic reverse pH gradient (∆pH) is provided by an electrochemical Na + gradient using Na + /H + antiporters, which are involved in the replacement of sodium ions with protons [8].It was reported that in the facultative alkaliphile Bacillus pseudofrmus OF4, ATP synthesis occurs faster at high alkaline medium than at a pH close to neutral [9].Te efect of pH has been studied mostly in terms of its impact on growth rate [10], while the morphological responses of bacilli, ATP content, and dehydrogenases activity are poorly understood.Zhang et al. studied the expression of bacilli stress genes under salt stress [6]; however, there are insufcient data on the combined efects of high salt concentrations and high pH on bacilli metabolism and morphology.In this study, we studied the efect of pH and high NaCl concentration on the metabolic intensity and ATP content of bacilli cells from both the log phase of growth and the stationary phase under starvation conditions and with the addition of glucose.In addition, changes in intracellular pH and cell morphology were studied.We hypothesized that alkaliphilic bacilli have a more pronounced tolerance to high pH and salt concentration than alkali-tolerant representatives of the Bacillus sp.However, our experiments showed that alkaliphilic bacilli had a greater resistance not only to high but also to low pH, which could be related to a more pronounced nonspecifc resistance.Te role of spore formation in the adaptation of bacilli to extreme pH values and high salt concentration was also studied, and it was shown that adaptation is provided mainly in vegetative cells.
From the soil of the soda sludge storage with a high salt content, we previously isolated and identifed Bacillus aequororis 5-DB by 16S rRNA sequencing [11], which has similarity of 99.87% with the new species Bacillus aequororis M-8T isolated by Singh et al. from a marine sediment [12].Bacillus aequororis 5-DB has lipase and amylase activity, and grows at pH 11 and 50 g/L NaCl [13].Bacilli have great biotechnological potential and are used as producers of hydrogen and polyhydroxyalkanoates [14], as probiotics [15], enzyme producers [16][17][18][19][20][21], and in food fermentation [22].For biotechnological applications, it is important that bacteria withstand extreme conditions (high or low pH, low water activity, the presence of solvents, and suboptimal temperatures), so it is important to evaluate the metabolic activity of strains under harsh conditions.We have selected a set of methods that allow us to comprehensively assess the metabolic and morphological changes in the cells of an alkaliphilic strain of bacilli, which has the prospect of biotechnological use, in comparison with a weakly alkalitolerant collection strain of B. subtilis ATCC 6633.We adapted a method for determining dehydrogenase activity using the PrestoBlue HS Cell Viability Reagent dye, based on the reduction of resazurin to fuorescent resorufn, a bioluminescent method for determining the ATP content in the cell, and an AFM method for assessing the morphological changes that occur in response to changes in pH and mineralization of the medium.We also assessed intracellular pH (pH in ) using a 5-(and-6)-carboxyfuorescein diacetate succinimidyl ether fuorescent probe and visualized sporulation by phase-contrast microscopy.
Te aim of the investigation is to evaluate the metabolic intensity and morphological changes of the facultative alkaliphile B. aequororis 5-DB in a wide range of pH and at diferent concentrations of NaCl in the medium in comparison with the weakly alkali-tolerant B. subtilis ATCC 6633.

Assessment of ATP Content.
To determine the ATP concentration in cells, 1 ml of the sample was centrifuged for 5 min at 15400 g, the supernatant was removed, and the cells were destroyed with 1 mL of DMSO for 15 min.Te lyophilized reagent containing frefy luciferin and luciferase (ATP reagent, ZAO BKhM ST, Moscow, Russia) was diluted with Tris-acetate bufer (pH 7.8) according to the manufacturer's protocol, and after that, additionally, it was diluted with 25-fold dilution with Tris-acetate bufer (pH 7.8) and mixed in a 1 : 1 ratio with 10-fold dilution with deionized water samples, and 100 μl of the mixture was placed in the wells of a white opaque fat-bottom plate (Nunc, Denmark).
The luminescence intensity was measured on an Infnite M1000 Pro plate reader (Tecan, Switzerland).Te ATP concentration in the samples was estimated using a calibration graph of the dependence of luminescence intensity on the known ATP concentration.

Assessment of the Viable Cells and Spores. Te number of viable cells of B. aequororis 5-DB and B. subtilis ATCC 6633
was assessed by inoculation in the rich agar medium with pH 11 and 8, respectively.Te viable cell concentration was calculated and expressed as colony-forming units per mL (CFU/mL).Te number of spores was determined in CFU/ ml after heating the suspension at 80 °C for 10 min to inactivate vegetative cells [23].

Assessment of Intracellular pH. Intracellular pH (pH in )
was assessed using a 5-(and-6)-carboxyfuorescein diacetate succinimidyl ether (cFDASE) (Sigma-Aldrich, Slovakia) fuorescent probe [24].Bacterial cultures were grown for 7 days at 25 °C in the medium of the composition given above at pH 8 and 11, and cells were washed with 50 mM HEPES bufer (pH 8.0).Te cell's pellets were resuspended in the same bufer.Ten, the cells were incubated for 10 min at 30 °C in the presence of 1.0 μM cFDASE.Next, the cells were washed and resuspended in bufers with extracellular pH (pH out ) values ranging from 3.0 to 13.0.10 mM of glucose was added to eliminate unconjugated cFDASE, and the cells were incubated for 30 min at 30 °C.Ten, the cells were washed twice and resuspended in bufers with pH 3.0-13, and fuorescence was measured on Infnite M1000 Pro plate reader (Tecan, Switzerland) at excitation wavelengths of 490/440 nm and emission wavelengths of 525 nm.Te extracellular fuorescence signal (background) was determined by fltering the cell suspension through a 0.22 μm membrane flter, followed by measurement.Calibration curves for bacterial cultures were determined in bufers with pH values ranging from 4 to 10 (Figure 1).External and internal pH values were balanced by adding valinomycin (1 μm) and nigericin (1 μm).
2.6.Microscopy.Te morphology of bacterial cells and surface profles was studied using an Asylum MFP-3D-BIO atomic force microscope (AFM) (Asylum Research, USA) in the laboratory of atomic force microscopy and confocal microscopy at the Rhodococcus-Center of the Perm State University.Scanning was performed in the air in semicontact mode using OMCL-AC240TS-R3 silicon cantilevers (Olympus, Taiwan) coated with aluminum, with a resonant frequency of 70 (50-90) kHz, a needle curvature radius of 7 nm, and a stifness constant of 2 (0.6-3.5) N/m.Two-and three-dimensional topographic images of bacteria were obtained to determine the linear dimensions of cells (length, width, and height) and characterize the structure of the cell surface (roughness).Preparations for AFM scanning were made immediately after placing the cells in the appropriate bufer (1-2 min) and after 24 hours.Te microphotographs were processed using the Igor Pro 6.22A program (Wave-Metrics, USA) [25].Sporulation was studied using phase-contrast microscopy (S1).Te native samples in the form of a crushed drop without additional staining were examined at a magnifcation of 1000 using a Leica DM LS microscope (Germany).Spores were distinguished from vegetative cells by the opalescence of the cell under phase contrast.

Statistical Analysis.
Te presented data are the results of three independent experiments.Te results obtained were processed statistically, and the means, standard deviations, and confdence intervals were determined.Te signifcance of diferences was assessed using Student's t-test, p < 0.05 (n � 9).

Assessment of the Metabolic Intensity of B. aequororis 5-DB and B. subtilis ATCC 6633 Cells Depending on the pH and
NaCl Concentrations.Te metabolic intensity of bacilli cells was assessed using PrestoBlue HS Cell Viability Reagent (Invitrogen, Termo Fisher Scientifc, USA).Te reagent contains resazurin, which is reduced by living cell dehydrogenases to fuorescent resorufn.Bacilli were grown to the logarithmic and stationary growth phases, and the metabolic intensity was assessed after 2, 24, and 48 h in a medium with diferent pH and 0.5 and 50 g/L NaCl concentrations under starvation and in the presence of glucose.
International Journal of Microbiology B. aequororis 5-DB and B. subtilis ATCC 6633 cells from the logarithmic growth phase were incubated in a medium with pH from 3 to 13 with two concentrations of NaCl (0.5 and 50 g/L) for 2, 24, and 48 h.B. aequororis 5-DB remains metabolically active at pH 7-11 with 0.5 g/L NaCl and pH 7-9 at 50 g/L NaCl (Figure 2).At both salt concentrations, the metabolic activity manifests itself in 2 h of incubation in a medium with pH 5 and 13, but sharply decreases with increasing incubation time.At pH 7-9, activity increases by 24 and 48 h of incubation at 50 and 0.5 g/L NaCl.Te metabolic activity of B. subtilis ATCC 6633 is also manifested at pH 3 with 0.5 g/L NaCl; however, the specifc fuorescence units are signifcantly lower than those of B. aequororis 5-DB (Figure 3).B. subtilis ATCC 6633 showed an increase in metabolic intensity with increasing incubation time.
B. aequororis 5-DB and B. subtilis ATCC 6633 cells from the stationary growth phase were incubated in a medium with pH 3-13 with two concentrations of NaCl (0.5 and 50 g/ L).Te metabolic intensity of B. aequororis 5-DB at 0.5 g/L NaCl increases noticeably by the end of the 24-hour incubation period compared to 2-hour, except for pH 11 and 13, but by the end of the 48-hour period the metabolic intensity decreases and becomes lower than the intensity at the 2-hour incubation (Figure 4).Such regularity is not observed at 50 g/L NaCl.Te metabolic activity of B. subtilis ATCC 6633 cells from the stationary phase of growth at pH 5 increases sharply after 2 h of incubation, but then, by 24 and 48 h, it decreases signifcantly (Figure 5).Te metabolic intensity is low at pH 11 and 13; unlike B. aequororis 5-DB, there is no increase of metabolic intensity at the frst hours of incubation.
B. aequororis 5-DB and B. subtilis ATCC 6633 cells were transferred from the stationary growth phase into a medium with pH 3-13 with 0.5 and 50 g/L NaCl and 1 g/L of glucose.Te metabolic intensity of B. aequororis 5-DB in most cases increases by the end of a 24-hour incubation period of incubation with a further decrease, and this pattern is also observed at 50 g/L NaCl (Figure 6).Tis trend is observed to a lesser extent in B. subtilis ATCC 6633 at 0.5 g/L NaCl, but not at 50 g/L NaCl (Figure 7).Te metabolic activity of B. subtilis ATCC 6633 is practically absent at pH 11 and 13.Compared to starvation, the metabolic activity of B. subtilis ATCC 6633 cells collected from the stationary growth phase in the presence of glucose in the medium signifcantly increases as shown from the units of fuor./OD600ratio.An increase in metabolic intensity is also observed at extremely low pH values; however, glucose in the medium does not afect metabolic intensity of B. subtilis ATCC 6633 at high pH.
Te number of viable cells of B. aequororis 5-DB and B. subtilis ATCC 6633 from the stationary phase was determined under various pH and mineralization values.CFU/ ml of B. aequororis 5-DB was shown to decrease by two orders of magnitude at pH 11 and 50 g/L NaCl and to decrease by one order of magnitude after 24 and 48 hours of incubation at pH 5 and 0.5 g/L (S2).B. subtilis ATCC 6633 did not form colonies after 48 hours of incubation at pH 5 and pH 11 (0.5 and 50 g/L NaCl), after 24 hours of incubation at pH 5 and pH 11 (50 g/L NaCl) and after 24 and 48 h of incubation at pH 8 and 50 g/L NaCl.After heating the suspension at 80 °C for 10 min, B. aequororis 5-DB and B. subtilis ATCC 6633 did not form colonies.Te ATP content in B. aequororis 5-DB and B. subtilis ATCC 6633 cells from the stationary growth phase at the initial time in a medium with pH 5 and 11 (0.5 g/L NaCl) was signifcantly higher than at pH 8, which may be associated with the response to stress.Te ATP content in B. aequororis 5-DB cells decreased after 24-48 hours of incubation (Figure 9).Adding 1 g/L glucose to the medium has virtually no efect on B. aequororis 5-DB cells (Figure 10(a)) harvested from the stationary growth phase but courses to a signifcant increase in the ATP content in B. subtilis ATCC 6633 cells in  Moreover, if glucose is added to a medium with 50 g/L NaCl, it does not course to an increase in the ATP content in cells.11).Morphology changes of cells were investigated immediately and after 24 h of adaptation.Cell lysis     International Journal of Microbiology and cell debris were observed after 24 h of incubation of B. subtilis ATCC 6633 cells in the medium with pH 5. On the contrary, B. aequororis 5-DB was more resistant to pH 5; the cells were even and had a smooth surface after a short exposure in that kind of medium.Te surface roughness increased after 24 h of exposure, but a cell lysis was insignifcant.Te morphometric parameters (length, width, height, and surface roughness) of bacilli cells were assessed after incubation in the medium with pH 5 and 11.Te control conditions were the same as those of cultivation (0.5 g/L NaCl and pH 11 for B. aequororis 5-DB; 0.5 g/L NaCl and pH 8 for B. subtilis ATCC 6633).Te surface roughness of B. aequororis 5-DB did not difer signifcantly from the control when cells were incubated in an acidic medium (pH 5).In this case, the cell volume increased by 40% (Table 3).During incubation in this medium for 24 hours, a slight increase in roughness and a signifcant elongation of cells were observed.Te roughness of B. subtilis ATCC 6633 cells increased by 31-35% under pH 5, regardless of the time of incubation in this media.Slight shortening of cells and an increase in transverse dimensions relative to the control were also observed.

Discussion
B. aequororis 5-DB was isolated from an alkaline highly mineralized medium from the soil surface of a soda sludge storage in the rich medium with pH 11 [11].Te strain grew very poorly in the rich medium with pH 8 and was classifed as a facultative alkaliphile.Its adaptive reactions were considered in comparison with the weakly alkali-tolerant B. subtilis ATCC 6633.Te following methods were chosen for comprehensive assessment of adaptive reactions under various pH values and 50 g/L NaCl.Te metabolic state of the cell was assessed by PrestoBlue HS Cell Viability Reagent dye.Te dye contains resazurin, which is reduced to fuorescent resorufn by dehydrogenases in living cells.ATP content was assessed using the bioluminescent method.Viability, number of spores, and vegetative cells were assessed by CFU/mL.Intracellular pH was assessed by fuorescent cFDASE probe.Bacterial cells were visualized by AFM and phase-contrast light microscopy.
Metabolic activity at diferent pH and high mineralization was assessed in three options.First and second options: cells were obtained from (1) logarithmic growth phase and (2) stationary growth phase and placed in a bufer with diferent pH values and 0.5 or 50 g/L NaCl.Tird option: International Journal of Microbiology cells were obtained from the stationary growth phase and placed in a bufer with diferent pH values, 0.5 or 50 g/L NaCl, and 1 g/L glucose.Alkaliphiles are known to exhibit multiple adaptive responses that allow them to live at high pH [7].When growing in an alkaline medium characterized by a low content of protons and a high content of sodium ions, in addition to osmoadaptation, microorganisms need to maintain intracellular pH homeostasis.Protons removed from the cell during respiration are not able to do useful work when they return to the cytoplasm, since they will move against the concentration gradient.Replacement of sodium ions by protons is carried out by Na + /H + antiporters.Formation of chemiosmotic reverse ΔpH is provided by a Na + electrochemical gradient.A major strategy for bacterial pH homeostasis is the use of transporters that catalyze active proton transport.Tese transporters include primary proton pumps, such as proton pumping respiratory chain complexes or proton coupled ATPases, and secondary active transporters, such as cation-proton antiporters, which use the proton motive force (PMF), generated by respiration or ATPases to energize active uptake of protons in exchange for cytoplasmic cations, such as Na + or K + [8,26].In the  8 International Journal of Microbiology stationary phase of growth, bacteria are already preadapted to adverse changes, since in this phase, RpoS becomes the main subunit of RNA polymerase [27].
We compared the adaptive response of the alkaliphilic B. aequororis 5-DB and the weakly alkali-tolerant B. subtilis ATCC 6633 in diferent growth phases.It was shown that the metabolic intensity of B. aequororis 5-DB cells harvested from the log phase of growth increases with increasing exposure time under a pH value diferent from the pH of the culture medium, while the metabolic intensity of cells harvested from the stationary phase of growth decreases after 48 hours of exposition.Te presence of glucose in the medium allowed B. aequororis 5-DB to signifcantly increase its metabolic intensity after 24 hours of incubation at 50 g/L NaCl, although the strain did not utilize glucose as a carbon source.In almost all the variants of the experiment, metabolic intensity increases after 24 hours of cell adaptation under these pH conditions.Despite the fact that B. aequororis 5-DB was grown at pH 11, only cells harvested from the log growth phase exhibit high metabolic intensity  International Journal of Microbiology under these conditions.In the active growth phase, the cell at pH 11 is able to receive energy and function actively due to the Na + /H + antiporter, while in the stationary phase, after the growth substrate is depleted, its metabolic activity decreases.B. subtilis ATCC 6633 cells harvested from the stationary growth phase showed metabolic activity at pH 3 and 5 in the presence of glucose.B. subtilis ATCC 6633, unlike B. aequororis 5-DB, utilizes glucose, which allows this bacterium to adapt to low pH.Te efect of glucose on the adaptation of B. subtilis ATCC 6633 is confrmed by data on ATP content.Te addition of 1 g/L glucose to the medium has virtually no efect on the ATP content in B. aequororis 5-DB cells taken from the stationary growth phase, but courses to a signifcant increase in the ATP content in B. subtilis ATCC 6633 cells even at pH 5 and 11.Tese data are consistent with the fact that B. subtilis ATCC 6633 grows on glucose, while B. aequororis 5-DB does not utilize glucose.B. aequororis 5-DB cells harvested from the stationary growth phase become more adapted to low pH, and the strain exhibits metabolic activity at pH 3 and 5, while the activity was absent in cells from the logarithmic growth phase.Te ATP content in B. subtilis ATCC 6633 cells from the stationary growth phase under low and high pH at 0.5 g/ L approaches that at optimal pH and, in some cases, even exceeds it.Te ATP content in the cells of both B. aequororis 5-DB and B. subtilis ATCC 6633 from the logarithmic growth phase at pH 5 and 0.5 g/L NaCl is signifcantly higher than at pH 11.Accumulation of ATP in the cell may result from the uncoupling of constructive and energy metabolism at low pH, which leads to the accumulation of ATP in the cell.In this case, cell death does not occur.
Statistical analysis showed signifcant diferences (Student's t-test, p < 0.05) of the metabolic intensity of B. aequororis 5-DB cells from the logarithmic growth phase under all pH and 0.5 g/L salt in the medium from that under pH 7. At 50 g/L salt in the medium, there is a signifcant diference only under extreme pH (3, 5, 11, and 13).For B. subtilis ATCC 6633, the diferences are signifcant in the frst 2 hours of incubation; the metabolic intensity is also signifcantly diferent at extreme pH from pH 7. In B. aequororis 5-DB cells from the stationary phase of growth, a signifcant increase in metabolic intensity is observed at 24 hours compared to 2 hours of incubation.At 48 hours, there is a signifcant decrease in metabolic intensity both with the addition of glucose and under starvation conditions.In B. subtilis ATCC 6633 cells from the stationary growth phase, a signifcant decrease in the metabolic rate was observed at pH >8 compared to pH 7. A signifcant decrease in the intracellular ATP content was shown in both B. aequororis 5-DB and B. subtilis ATCC 6633 at 50 g/L NaCl compared to 0.5 g/L.
Te diference between pH in and pH out in B. aequororis 5-DB is maximal at pH 11 with 0.5 and 50 g/L NaCl and is not equal to 0 at pH 3. It was noted that cells of B. subtilis ATCC 6633 at pH 3 are not viable, and pH in equals to pH out .Te pH in value of B. subtilis ATCC 6633 is higher than the pH in value of B. aequororis 5-DB at pH out 11.It is known that the Na + /H + antiporter plays the major role in the pH homeostasis of alkaliphiles [28]; therefore, bacteria need Na + ions for their vital activity.We have shown that the maximum ΔpH is observed at pH 11 and 50 g/L NaCl in both B. aequororis 5-DB and B. subtilis ATCC 6633.High concentration of Na + in the medium coupled with high pH out course to activating Na + /H + antiporter.Na + enters the cell and is exchanged for H+ during antiporter's work, which leads to an increase in the pH gradient.In addition, other Na + transporters are known.Comparison of the wholegenome sequence of B. halodurans C-125 with that of B. subtilis revealed that in addition to the F 1 F 0 -ATP synthase operon, an operon for a Na + -transporting ATP synthase is also revealed in both B. halodurans C-125 and B. subtilis [9].Strong alkalization of the cytoplasm (up to pH 9) is observed in B. aequororis 5-DB after 48 h.Tis correlates with a decrease in the metabolic activity of cells after 48 h of incubation in this medium.
Sporulation plays an important role in the adaptation of bacilli [29].B. subtilis is able to grow at a pH of 4.8 to 9.2 and a water activity above 0.929.Gauvry et al. reported that the transition from favorable conditions to unfavorable conditions results in a delay and even a cessation of spore formation, while in favorable conditions, the sporulation process resumes.Tis can be explained by the fact that low pH courses to inhibition of the expression of sporulation genes and slows down enzymatic reactions.Vegetative cells sporulate more synchronously, and in higher proportions under suboptimal conditions for sporulation, the level of phosphorylated Spo0A increases to a high level in stress cells, which allows of more efcient initiation of the sporulation  [30].It was shown that lower temperatures and pH slowed down the sporulation process [31].Although B. aequororis [12] and B. subtilis [32] are the sporulating bacteria, we showed that exposure to the conditions studied did not result in active sporulation in the bacilli.In our opinion, extreme pH values and high salt concentrations did not cause active sporulation, frstly, because these conditions were not suboptimal but critical for the cells.Secondly, the cells were incubated in a medium with diferent pH values and mineralization in the absence of a nutrient substrate, which negatively afected all metabolic processes in the cell and gene expression, including sporulation genes.
According to the results of AFM, the cells of the alkaliphile B. aequororis 5-DB are less damaged in the medium with low pH than the weakly alkali-tolerant B. subtilis ATCC 6633.Under low pH, the roughness of B. subtilis ATCC 6633 cell surface increases relative to the control (pH 8) by 36%.Under low pH, the surface roughness of B. aequororis 5-DB cells increases relative to the control (pH 11) by 26%.An increase in surface roughness indicates a decrease in cell turgor.Low pH has little efect on cell size in both strains.
Te limitations of this study are the experimental conditions, namely, exposure to high pH and mineralization under starvation conditions, while conditions close to the natural habitat will expand our understanding of the adaptation of alkaliphilic bacilli.Furthermore, research directions may concern changes in the conditions of exposure to pH and mineralization and analysis of gene expression under such efects.
Tus, an interesting fact is the increased resistance of the facultative alkaliphile B. aequororis 5-DB to low pH, which confrms the general nonspecifc adaptive ability of this strain to adverse environmental conditions.We previously reported that B. aequororis 5-DB exhibits lipase and amylase activities under conditions of high alkalinity and mineralization [13].Tis strain can be used in feed production and in the production of agricultural probiotics and enzymes included in detergents, and processing of biogenic raw materials and waste from the food industry and agriculture.

Conclusion
Based on the proposed methods, we assessed the efect of changes in pH and mineralization of the medium on the metabolic intensity, morphology, and pH in of cells of two strains of alkaliphilic and weakly alkali-tolerant bacilli.We showed a signifcantly greater resistance of alkaliphilic B. aequororis 5-DB not only to high but also to low pH in comparison with B. subtilis ATCC 6633.Te resistance of B. aequororis 5-DB to low pH values was expressed in higher metabolic intensity, estimated by reduction of resazurin to resorufn by dehydrogenases, maintenance of ΔpH, no signifcant cell damage, and slight increase in surface roughness.Alkaliphilic B. aequororis 5-DB has a broader general resistance to adverse factors (wide pH range, 50 g/L NaCl) than a weakly alkali-tolerant strain of the same genus.Such nonspecifc resistance may be promising for the biotechnological application of B. aequororis 5-DB.
Content.Incubation in a medium with 50 g/L NaCl in all experimental variants for B. aequororis 5-DB and B. subtilis ATCC 6633 courses to a severalfold decreases in the intracellular ATP content.When B. aequororis 5-DB cells are harvested from the logarithmic growth phase and incubated in a medium with pH 5 and 0.5 g/L NaCl, the ATP content exceeds that at pH 8 and 11, but with increasing incubation time, it decreases signifcantly (Figure 8(a)).Te ATP content in B. subtilis ATCC 6633 cells is lower than in B. aequororis 5-DB cells (Figure 8(b)).

Table 1 :
PH in and ΔpH in B. aequororis 5-DB with diferent pH out and salt concentrations.

Table 2 :
PH in and ΔpH in B. subtilis ATCC 6633 with diferent pH out and salt concentrations.