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Sci Rep
Sci Rep
Scientific Reports
2045-2322
Nature Publishing Group UK London

71529
10.1038/s41598-024-71529-6
Article
Efficacy assessment of different cryoprotectants for preserving the viability of Enterobacterales strains at − 20 °C
http://orcid.org/0000-0003-1636-2148
Tutrina Anastasia atutrina0@gmail.com

http://orcid.org/0000-0003-0678-1170
Zhurilov Pavel
grid.465383.f Federal Research Center for Virology and Microbiology, Branch in Nizhny Novgorod, 603950 Nizhny Novgorod, Russia
6 9 2024
6 9 2024
2024
14 208438 5 2024
28 8 2024
© The Author(s) 2024
2024
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The preservation of microorganisms is pivotal in microbiological practice. Currently, cryopreservation is assumed to be an effective and inexpensive approach for the storage of microorganisms, including bacteria. The key point of cryopreservation is optimal cryoprotectant selection. In the present study, different cryoprotectant compositions were tested for long-term storage of 15 Enterobacterales bacterial strains at − 20 °C. The survival rates of the bacterial strains were evaluated in four different cryoprotectant solutions containing 70% glycerin only (cryoprotectants 1 and 4), 10% dimethyl sulfoxide (DMSO) with 70% glycerin (cryoprotectant 2), and 10% DMSO (cryoprotectant 3). In addition, cryoprotectants 1 and 2 contained peptone and yeast extract as nutritional supplements. The general survival rates of the bacterial strains were evaluated after 12 months of storage. After 12 months, the survival rates of the different cryoprotectants were as follows: cryoprotectant 1—88.87%; cryoprotectant 2—84.85%; cryoprotectant 3—83.50%; and cryoprotectant 4—44.81%. Thus, the composition of cryoprotectant 1 (70% glycerin with nutrient supplements) was optimal for preserving 15 tested strains of the order Enterobacterales. Despite these findings, the biochemical properties of the tested strains changed after cryopreservation for 12 months in the presence of 1 or 3 cryoprotectants. Alterations in the biochemical profile could be related to changes in environmental conditions and cold adaptation. We assume that the composition of cryoprotectant 1 can be optimal for storing the order Enterobacterales at − 20 °C. However, further investigations are needed to elucidate the problem of cryopreservation and to support our assumption.

Keywords

Cryopreservation
Cryoprotectants
Enterobacteria
Long-term storage
Subject terms

Biotechnology
Microbiology
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pmcIntroduction

The storage of microorganisms is a crucial issue in microbiological practice. It is used both in industry and in scientific areas. Different types of biological entities, such as bacteria, fungi, viruses, eukaryotic cells and tissues, are usually stored. The storage of microorganisms allows the preservation of cultures in a viable state and maintains their native characteristics1. Different types of microorganisms, including bacteria, require special storage approaches and conditions. Currently, various approaches are used to preserve bacterial cultures: periodic subcultures, drying, freeze-drying, and cryopreservation2. This particular preservation method is chosen according to the individual characteristics of bacteria: morphological and physiological properties, biochemical activity, and genetic stability1,3.

The periodic subculture method is used for the storage of susceptible freezing and drying microorganisms4,5. The drying method is widely applied to bacterial storage, but it is primarily used to store spore-forming bacterial cultures6–8. The freeze-drying method is used to preserve only some species of non-spore-forming bacteria. To date, the latter approach is the most popular approach for long-term storage. Additionally, this method is applied to the preservation of most bacterial species and has a high survival rate for bacterial cells9,10. However, the freeze-drying method is commonly applied for large-scale processes because freeze-drying requires specialized and expensive equipment. This greatly reduces the availability of methods for low-scale use. On the other hand, the cryopreservation method is optimal for low-scale usage and does not require specialized and expensive equipment. Additionally, this method has comparable efficacy with freeze-drying for most bacterial species11,12. A wide temperature range (from − 20 to − 196 °C) is used for bacterial cryopreservation2,13.

Cryoprotectants are solution systems used to protect cells from damage caused during cryopreservation. The main cryoprotectant component is the cryoprotective agent (CPA), which protects the integrity of the cell membrane and the intracellular environment. CPAs are divided into two types depending on cell membrane permeability: permeable and non-permeable14,15. One of the most popular CPAs are carbohydrates, which are non-permeable CPAs that do not penetrate cells. For example, frequently used glucose increases the viscosity of the extracellular environment and inhibits water flow between the inside and outside parts of the cell2,16,17. Alternatively, dimethyl sulfoxide (DMSO) and glycerin are frequently used as permeable CPAs that are capable of penetrating the cell and preventing the formation of ice crystals both outside and inside the cell, thus preventing cell destruction13,15,18–20. In addition to CPAs, cryoprotectants can contain nutrient supplements (for example, vitamins, peptides, amino acids, etc.) to improve the viability of bacteria during the cryopreservation process21. pH is a pivotal parameter of bacterial cell survival during cryopreservation and long-term storage. Harsh pH variations can disrupt cell membrane integrity, inhibit the activity of enzymes and membrane transport proteins, and cause bacterial cell death21,22. Phosphate buffer is widely used to stabilize the pH of cryoprotectant solutions23.

Currently, the optimization of the cryopreservation method is based on improvements in cryoprotectant solutions as well as freezing and thawing modes. An important cryopreservation stage is the selection of the optimal cryoprotectants for different bacterial cultures. Cryoprotectant selection takes into account the special features of different bacterial taxa24. For example, various CPAs contained in cryoprotectants may entirely suppress the growth of some bacterial species while being more or less tolerated by other bacterial species25. On the basis of actual literature analysis, the optimal cryoprotectant compositions for order Enterobacterales species are limited.

In the present study, we evaluated four different cryoprotectants for long-term storage of 15 Enterobacterales species at − 20 °C. The following cryoprotectants containing various CPAs were tested: glycerin only (cryoprotectants 1 and 4), glycerin and DMSO (cryoprotectant 2) and DMSO only (cryoprotectant 3). As nutrient supplements, peptone and yeast extract were added to cryoprotectants 1 and 2. Cryoprotectants 3 and 4 did not contain nutrient supplements. Additionally, 8% (m/v) glucose was added to all the samples. Storage efficacy was evaluated over 12 months.

The relevance of this study is determined by the importance of preserving microbial cultures in microbiological practice, particularly in the context of modern scientific research and biotechnology. Cryopreservation is an effective method for storing microorganisms; however, the selection of optimal cryoprotectants for various taxa, such as Enterobacterales, remains insufficiently explored. Some cryoprotectants may not provide adequate viability for bacteria after frozen, highlighting the need for this investigation. The results may contribute to the improvement of cryopreservation methods and enhance bacterial viability, which is of significant importance for microbiology and biotechnology.

Materials and methods

Bacterial strains

15 bacterial strains of the Enterobacterales order were used (Table 1). The bacterial strains had different origins: raw milk (ML-2, ML-9, ML-16, ML-17, ML-18, ML-31, ML-36, ML-49, ML-55, ML-56, and ML-60); the internal organs of birds (B-28 and B-75); and the internal organs of mice (M-4 and M-12). Strains were stored in the local collection of Nizhny Novgorod Research Veterinary Institute—Branch of Federal Research Center for Virology and Microbiology. Cultures were frozen in Trypticase Soy Broth (HiMedia, India) supplemented with 15% glycerin at − 80 °C. Analysis of the 16S rRNA gene sequence was used for strain identification. The tested strains belonged to the following families: Enterobacteriaceae (n = 10), Hafniaceae (n = 1), Morganellaceae (n = 2), and Yersiniaceae (n = 2).Table 1 Tested bacterial strains.

Family	
Enterobacteriaceae (n = 10)	Hafniaceae (n = 1)	Morganellaceae (n = 2)	Yersiniaceae (n = 2)	
Citrobacter freundii ML-18	Hafnia paralvei ML-17	Morganella morganii B-75	Serratia liquefaciens ML-2	
Citrobacter braakii ML-36		Proteus mirabilis M-4	Serratia marcescens B-28	
Citrobacter braakii ML-49				
Citrobacter freundii ML-60				
Enterobacter asburiae ML-55				
Enterobacter tabaci ML-56				
Escherichia marmotae M-12				
Klebsiella pneumoniae ML-9				
Klebsiella aerogenes ML-16				
Klebsiella variicola ML-31				

Cryoprotectant compositions

Despite the large number of CPA combinations used for the cryopreservation of bacterial cells, popular CPAs include DMSO and glycerin. Additionally, the use of nutrient supplements increases the survival rate of bacteria during long-term storage. However, the optimal and universal cryoprotective compositions for Enterobacteriales cryopreservation are still unknown. Therefore, in this study, four different cryoprotectants were tested (Table 2). The cryoprotectants included CPAs: glycerin only (cryoprotectants 1 and 4), glycerin with DMSO (cryoprotectant 2), and DMSO only (cryoprotectant 3). In cryoprotectants 1 and 2, nutrient supplements (peptone and yeast extract) were added, as well as phosphate-buffered saline solution (PBS) (HiMedia, India) to stabilize pH and osmolarity. Additionally, all cryoprotectants had 8% (m/v) glucose as a non-permeable CPA, as listed in Table 2. The cryoprotectant solutions were sterilized by autoclaving at 112 °C for 15 min.Table 2 Cryoprotectant compositions.

Cryoprotectant 1	Cryoprotectant 2	Cryoprotectant 3	Cryoprotectant 4	
Component	% (m/v)	Component	% (m/v)	Component	% (m/v)	Component	% (m/v)	
Glycerin	70	Glycerin	70	DMSO	10	Glycerin	70	
PBS*	14.5	PBS*	10.5	Glucose	8	Glucose	8	
Distilled water	6	DMSO	10	Distilled water	82	Distilled water	22	
Glucose	8	Glucose	8	pH 7.2**		pH 7.2**		
Peptone	1	Peptone	1					
Yeast Extract	0.5	Yeast Extract	0.5					
pH 7.2**		pH 7.2**						
*PBS-Phosphate buffered saline.

**Start pH level of cryoprotectant.

Inoculum preparation

Overnight plate cultures were used for inoculum preparation. Inocula (suspensions) were prepared in PBS at pH 7.2 and a density of 0.5 McFarland units. To concentrate the biomass, the bacterial cells were sedimented at 10,000 × g at 20 °C for 10 min via centrifugation. The sediments were resuspended in 5 ml of the different cryoprotectant compositions listed in Table 2. 500 µl cryoprotectant-bacteria suspensions were added to 1.5 ml test tubes (cryotubes) for further cryoprotective testing. The viable bacterial cell number was determined via the standard plate counting (SPC) method.

Freezing technique

Equilibration of the cryoprotectant-bacteria suspensions was carried out at 4–6 °C for 30 min26. Then, the cryotubes were frozen at − 20 °C. Cryoprotectant 3 had a solid state of solution (crystallizing) at − 20 °C, whereas cryoprotectants 1, 2, and 4 had liquid aggregate states of solution (non-crystallizing).

Survival rate and counting of live bacteria assay

The cryotubes with bacterial cells were rapidly defrosted at 37 °C for 3–5 min with mild shaking. The rapid defrosting approach protects membranes from disruption. The latter can be caused by ice crystal recrystallization or extended exposure to high extracellular concentrations of cryoprotectants in bacterial cells27,28.

After storage, the number of viable bacterial cells was determined via the standard plate counting (SPC) method. Briefly, 100 µl of serial dilutions (×10) of cryoprotectant-bacteria suspensions were streaked onto the Nutrient agar (NA) (HiMedia, India) plates. The plates were incubated for 18–22 h at 37 °C. The number of viable cells was expressed as colony-forming units per ml (CFU/ml). The experiment was repeated three times.

Viable cell number counting was performed every two months within one year (six measurement points). The 0 month time point is median values 1.8 × 108 CFU/ml of bacterial cultures counted before start storage.

The survival rate was calculated according to equation29: Survivalrate(%)=log(numberoflifebacteriaaftercryopreservation)log(numberoflivebacteriabeforecryopreservation)×100(%)

Statistical analysis

The significance of differences was determined via the Wilcoxon nonparametric signed rank test. The Wilcoxon test for comparison-related groups (before storage and after storage) was used. The significant values were if the p-value ≤ 0.05.

Kruskal–Wallis30 and Dunn’s tests31 were applied to assess the differences among groups (cryoprotectants 1–4). The statistical significance was calculated via the Kruskal–Wallis test, which is a non-parametric test for comparison of samples from two or more groups of independent observations. This test does not require the groups to be normally distributed and is more stable to outliers. The significant values were if the p-value < 0.05. Dunn’s test was used for multiple comparisons and determining which groups differed from each other.

The statistical significance of differences calculations was performed with Microsoft Office Excel 2012.

Biochemical tests

Biochemical tests were used for the characterization of bacterial strains before and after long-term storage in cryoprotectants 1 and 3. The biochemical reactions for Enterobacteriaceae described in Bergey’s Manual of Systematic Bacteriology were performed via classical approaches32–34. The following reactions were tested: indole, methyl red, Voges–Proskauer, sodium citrate, hydrogen sulfide, urea, phenylalanine deaminase, lysine decarboxylase, arginine decarboxylase, ornithine decarboxylase, malonate, esculin, tartrate, sodium acetate, nitrate, oxidase, ONPG (β-galactosidase), yellow pigment, d-glucose, d-adonitol, l-arabinose, dulcitol, glycerin, inositol, lactose, maltose, d-mannitol, d-mannose, melibiose, raffinose, l-rhamnose, salicin, d-sorbitol, sucrose, trehalose, d-xylose, and catalase.

The average numbers of changed biochemical characteristics were calculated among all tested cultures after storage in cryoprotectants 1 and 3 according to the following equation:x=Nn

x -The average number of changed biochemical characteristics among all cultures;

N -The number of changed biochemical characteristics among a n-number of cultures;

n -Number of cultures.

Results

Survival rates of bacterial cultures in different cryoprotectants

15 bacterial strains of the Enterobacterales order were used in the present study (Table 1). Suspensions of 15 bacterial cultures were prepared with 108–109 CFU/ml in four different cryoprotectants (Table 2). The median values of all bacterial cultures before storage (1.8 × 108 CFU/ml) were used as 100% survival rates, as shown in Figs. 1, 2, 3, 4, for all cryoprotectants at the 0 month time point (Table S1, Table S2, Table S3, Table S4). The survival rates of all bacterial cultures tested with different cryoprotectants are presented as median values (Table 3).Fig. 1 The survival rate of bacterial cultures in cryoprotectant 1 during 12 months of cryopreservation.

Fig. 2 The survival rate of bacterial cultures in cryoprotectant 2 during 12 months of cryopreservation.

Fig. 3 The survival rate of bacterial cultures in cryoprotectant 3 during 12 months of cryopreservation.

Fig. 4 The survival rate of bacterial cultures in cryoprotectant 4 during 12 months of cryopreservation.

Table 3 Survival rate of bacterial cultures in different cryoprotectants depending on storage times.

Cryoprotectants	The survival rate* (%)	
2 months	4 months	6 months	8 months	10 months	12 months	
Cryoprotectant 1	95.18	95.61	95.79	95.43	94.89	88.87	
Cryoprotectant 2	90.21	90.57	91.23	91.89	86.29	84.85	
Cryoprotectant 3	96.47	98.56	93.81	94.32	92.97	83.50	
Cryoprotectant 4	78.80	70.30	61.44	60.37	54.07	44.81	
*Median was showed.

Survival rate of bacterial cells in cryoprotectant 1

The survival rates of all the tested bacterial cultures in cryoprotectant 1 are shown in Fig. 1. The survival rate of the bacterial cultures decreased by 4.81% after 2 months of storage (to 95.18%). After 4 months of storage, the survival rate increased by 0.43% (to 95.61%). The rate increased gradually to 0.18% (to 95.79%) after 6 months of storage. At 10 months, the survival rate slightly decreased to 0.9% (94.89%). After 12 months of storage, the survival rate of the bacterial cultures was 88.87% in cryoprotectant 1.

Among the tested bacterial strains, the Hafnia paralvei ML-17 strain had the maximum survival rate with cryoprotectant 1. The rate was 100.11% after 12 months of storage. At the same time, the minimum survival rate of the Proteus mirabilis M-4 strain with cryoprotectant 1 was 69.93%.

The Wilcoxon test was used to verify the statistical significance. Data in all points of storage, except the point after 8 months, was statistically significant (p ≤ 0.05). In 8 months of storage, the results were not statistically significant. This means that the bacterial cultures did not have an overall trend of increasing or decreasing survival35. The decreases of the survival rate of bacterial cultures tested were observed after 10 and 12 months of storage (p ≤ 0.05).

Survival rate of bacterial cells in cryoprotectant 2

The survival rates of bacterial cultures in cryoprotectant 2 are shown in Fig. 2. The survival rate decreased by 9.79% (to 90.21%) over the 2 months of preservation. The survival rate was observed to increase by 1.68% (to 91.89%) between 2 and 8 months of bacterial culture storage. The rate decreased by 7.04% (to 84.85%) at 12 months.

Hafnia paralvei ML-17 strain had the greatest survival rate (96.86%) in cryoprotectant 2 among all bacterial strains tested. Proteus mirabilis M-4 and Morganella morganii B-75 strains had the lowest survival rates: 47.28% and 36.24%, respectively.

Received data in all points of storage, except the point after 8 months, was statistically significant (p ≤ 0.05) (Wilcoxon test). This means that the bacterial cultures did not have an overall trend of increasing or decreasing survival after 8 months of storage.

Survival rate of bacterial cells in cryoprotectant 3

The general survival rate of cryoprotectant 3 is shown in Fig. 3. The survival rate decreased by 3.53% (to 96.47%) after 2 months of storage. This rate increased by 2.09% (to 98.56%) in 4 months. The survival rate decreased by 4.75% (to 93.81%) at 6 months and increased by 0.51% (to 94.32%) after 8 months of storage. The survival rate decreased by 1.35% (to 92.97%) and 9.47% (to 83.50%) after 10 and 12 months of bacterial culture storage, respectively.

The greatest survival rate was exhibited by the Enterobacter asburiae ML-55 strain (96.12%), and the minimum survival rate was in Citrobacter braakii ML-49 strain (40.95%).

No statistical significance was observed after 6 and 8 months of storage (p ≥ 0.05) (Wilcoxon test). This indicates that the cultures tested had no general trend toward increased or decreased survival rates.

Survival rate of bacterial cells in cryoprotectant 4

The survival rate in cryoprotectant 4 is shown in Fig. 4. The survival rate decreased sharply by 21.20% (to 78.80%) after 2 months of storage, and by 8.5% (to 70.30%) after 4 months. The survival rate decreased by 25.49% (to 44.81%) between 4 and 12 months of bacterial culture storage.

The greatest survival rate was observed for Serratia liquefaciens ML-2 (88.16%) among the bacterial cultures tested. Proteus mirabilis M-4 strain had a minimal survival rate of 12.14%.

The death of bacterial cultures was detected for the following strains: Citrobacter braakii ML-49 and Citrobacter freundii ML-60 after 8 months of storage. Morganella morganii B-75 strain lost vitality after 12 months.

No statistical significance was observed after 8 months of storage (p ≥ 0.05) (Wilcoxon test). This indicates that the cultures tested had no general trend toward increased or decreased survival rates.

Analysis of different cryoprotectant efficiency after 12 months of storage

15 bacterial strains of the Enterobacterales order were used to study the cryoprotective features of different cryoprotectants (Table 3). The maximum survival rate of the cultures was detected in cryoprotectant 1. It was higher at 4.02%, 5.37%, and 44.06% than that in cryoprotectants 2, 3 and 4, respectively.

Statistical processing of the data was performed via the Kruskal‒Wallis and Dunn’s tests. The Kruskal–Wallis test revealed statistically significant differences in survival rates among all the tested cryoprotectants. Statistically significant differences among the cryoprotectant groups were determined via Dunn’s multiple comparison test. All the cryoprotectants were compared to each other.

The quality critical (Qc) value is the standard table value for Dunn’s test and depends on the number of compared groups and the significance level (p-value). In this study, we used 4 of the compared groups at p < 0.05. The value of Qc was 2.394 for the four compared groups. The practical quality (Qp) is the value calculated via the formula of Dunn’s test: Qp=R_A-R_BN(N+1)121nA+1nB

R_A-The average ranks of the two samples being compared;

nA, nB-The numbers of compared samples;

Where N-the total number of all compared samples36.

Differences between groups were considered statistically significant if Qp > Qc. The results of Dunn’s test were as follows: Qp was 1.176 for cryoprotectant groups 1 and 2; Qp was 1.615 for groups 1 and 3; and Qp was 0.44 for groups 2 and 3. These Qp values indicated no statistically significant differences among the studied groups.

Statistically significant differences were found among the groups: cryoprotectants 1 and 4 (Qp = 4.56), 2 and 4 (Qp = 3.38), and 3 and 4 (Qp = 2.94) (Fig. 5).Fig. 5 Survival rates of strains in the tested cryoprotectants after 12 months of storage. The median value of the general survival rate is depicted in boxes. Different letters indicate significant differences (Dunn, p < 0.05). Differences between groups were considered statistically significant if Qp > Qc. Cryoprotectants 1, 2, and 3 have no statistically significant differences with each other (Qp < Qc). Cryoprotectant 4 has statistically significant differences from cryoprotectants 1, 2, and 3 (Qp > Qc).

Biochemical characteristics of strains

The biochemical characteristics of the tested strains were evaluated before and after cryopreservation in cryoprotectants 1 and 3. These cryoprotectants have been tested according to their various aggregate states during long-term storage. The results of the biochemical tests are shown in Table S5.

Biochemical properties such as d-glucose, Voges–Proskauer, nitrate, oxidase, ONPG (β-galactosidase), yellow pigment, and catalase did not change among the tested strains in cryoprotectants 1 and 3 after 12 months of storage. However, other biochemical characteristics of the tested strains changed after storage (Fig. 6). The most frequently altered biochemical characteristic was carbohydrate utilization. Fewer alterations were detected for other fermentative activities.Fig. 6 The number of altered biochemical features.

The biochemical properties of all the tested strains were altered in cryoprotectants 1 and 3 (Fig. 7). Citrobacter braakii ML-49 had a minimum number of changed biochemical properties in cryoprotectant 1 (four reactions) and cryoprotectant 3 (five reactions). Escherichia marmotae M-12 had a maximum number of changed biochemical properties in cryoprotectant 1 (17 reactions) and cryoprotectant 3 (16 reactions).Fig. 7 The number of altered biochemical features among the 15 tested strains.

The average numbers of strains whose biochemical characteristics changed among the tested strains after storage in cryoprotectants 1 and 3 were x1 = 10.53 and x3 = 10.4, respectively. On average, 10 changes in biochemical characteristics per strain occurred after long-term storage in cryoprotectants 1 and 3.

Discussion

Cryopreservation is one of the most popular approaches for the storage of microorganisms in laboratory practice. It is used to preserve intact living cells and tissues at low temperatures13. For this process, special substances called cryoprotectants are used. They provide cytoprotective properties and protect cell membrane integrity and the intracellular environment. In the present study, we tested the viability of 15 enterobacteria strains after 12 months of storage at − 20 °C in four different cryoprotectants.

In this study, we used four cryoprotectants with different compositions that were manually assembled. Glycerine was used as a permeable CPA only in cryoprotectants 1 and 4. For cryoprotectant 2, both glycerin and dimethyl sulfoxide (DMSO) were used as permeable CPAs. Cryoprotectant 3 contained only DMSO as a CPA. As nutrient supplements, peptone and yeast extract were used in cryoprotectants 1 and 2. Cryoprotectants 3 and 4 did not contain nutrient supplements. Additionally, 8% (m/v) glucose was added to all cryoprotectants.

After 2 months of storage, a decrease in the survival rate was observed for all the tested cryoprotectants. The survival rate was 95.18–90.21% for cryoprotectants 1–3 and 78.88% for cryoprotectant 4. The reduced survival rate of all cryoprotectants could be caused by freezing damage to bacterial cells, stress factors such as changes in nutrient composition and environmental conditions, etc.37. In the case of cryoprotectant 3, an additional reason for the decrease in survival may be that ice crystals formed during the transition of the cryoprotectant to the solid stage (crystallization). Ice crystals can mechanically damage cell membranes and cause osmotic stress and dehydration, leading to cell death. Meanwhile, in cryoprotectant 4 the survival rate decrease was 21.12%. Notably, it consisted of 70% glycerin and did not contain nutrient supplements. However, cryoprotectant 1 consisted of 70% glycerin and nutrient components (peptone, yeast extract) and had greater cryoprotectant properties than cryoprotectant 4. This observation suggests that nutrient supplements are important components of cryoprotectants, which consist of only glycerin as a CPA38,39.

After 4 months of storage, the bacterial survival rate increased insignificantly (0.36–2.09%) in cryoprotectants 1, 2, and 3. We suppose that this difference could be associated with the bacterial cell cycle changing during storage. In our study, we did not confirm why there was a slight increase in the survival rate in bacterial cultures during this period of cryopreservation. However, it was reported previously that the bacterial cell cycle can be slowed but not halted under harsh conditions, including a decrease in temperature40,41. Some bacterial cells could be in the C and D stages of the cell cycle during the initial stages of long-term storage. We assume that after 2 months of storage, bacterial cells gradually complete their cell cycle (i.e., cell division), which ultimately increases their survival rate from 4 to 8 months of storage. However, there are no results confirming this hypothesis in this study.

From 10 to 12 months of storage, the survival rate of the bacterial cultures decreased for all the cryoprotectants. By 12 months, it was > 83.85% in cryoprotectants 1–3 and 44.81% in cryoprotectant 4. Moreover, in the latter, the extinction of 3 tested cultures was observed. Therefore, cryoprotectants 1, 2, and 3 were generally optimal for storing the tested bacterial cultures for 12 months. Notably, statistically significant differences were not found among cryoprotectants 1–3 (Dunn, p < 0.05). However, cryoprotectant 1 had the maximum median value of survival rate of the tested bacterial cultures (88.87%). In addition, the survival data range (between the minimum and maximum survival rates) was lower for cryoprotectant 1 (69.93–100.11%) than for cryoprotectants 2 (40.95–96.48%), and 3 (36.24–96.86%). Thus, it may be suggested that cryoprotectant 1 had an optimal composition for the survival of the tested bacterial cultures of the Enterobacterales order at − 20 °C for 12 months.

Interestingly, only cryoprotectant 3 crystallized and had the additional effect of damaging bacterial cells with ice crystals. However, despite this, it did not result in a sharp decrease in the survival rate of bacterial cells. Our findings are consistent with previous observations. In 2003, on the basis of pairwise comparisons of the cryoprotective activity of the more common CPAs, Hubálek noted that DMSO is one of the most successful CPAs26. It was also observed that DMSO more effectively stabilizes bacterial cell membranes than glycerin does through the formation of hydrogen bonds with the polar surface of the membrane17,20,42. It is thought that DMSO displaces water molecules from the membrane and therefore protects it from damage by extracellular ice crystals43.

The survival rates of the tested strains in cryoprotectants 1, 2 and 3 were different. H. paralvei ML-17, E. marmotae M-12, S. liquefaciens ML-2, and E. asburiae ML-55 had high survival rates (90–100%) after 12 months of storage. Cryoprotectants 1, 2, and 3 probably can also effectively maintain the viability of other strains of these species. The survival rates of M. morganii B-75 were significantly greater for cryoprotectant 1 (84%) than for cryoprotectants 2 (36%) and 3 (69%).

The differences in survival data between cryoprotectant 1 (69.93–100.11%) and cryoprotectant 2 (40.95–96.48%) may be related to the 10% DMSO supplement along with the 70% glycerin in the latter. This may be explained by the toxicity effect of high concentration of CPAs. Other researchers reported that DMSO may be an additional stress factor for bacterial cells: it is capable of causing suppression of the cell's immunity under stressful conditions, including freezing. Suppression can be caused by reduced gene expression, blocking of the enzyme’s active sites, and altering the synthesis levels of essential components of the cellular membrane in microorganisms43,44. Additionally, high concentrations of DMSO (> 5%) can cause toxic effects on some bacteria45. Moreover, 10% DMSO is recommended for use in cryopreservation17,26. This discrepancy indicates that more in-depth investigations of the effects of DMSO on cells are needed.

Additionally, we tested alterations in the biochemical profile of bacterial cultures during cryopreservation in cryoprotectants 1 and 3 (crystallizing and non-crystallizing). Notably, regardless of the cryoprotectant and aggregate state of the solution, the average number of altered biochemical characteristics per strain was ~ 10 (x1 = 10.53 and x3 = 10.4). However, it is impossible to detect a correlation of changes in the biochemical properties of strains from the composition of the cryoprotectants or aggregate states on the basis of our results. Probably alterations in the biochemical profile could be related to changes in environmental conditions and cold adaptation46,47. Moreover, we tested only the first passages of strains after storage, and during further passages, the initial biochemical profiles may be restored.

The problem of cryopreservation of microorganisms is complex, and we recognize the limitations of our study. We used only 15 strains of the Enterobacterales order and only four cryoprotectant compositions. The duration of long-term storage was also restricted to 12 months. Additionally, biochemical profile alterations during long-term storage should be investigated more precisely. In general, different parameters, such as equilibration regimens, the effectiveness of other CPAs and their concentrations, and the physiology and biochemistry of microorganisms during freezing, should be investigated further.

In conclusion, the present study investigated the viability of 15 enterobacteria strains during 12 months of storage at − 20 °C in four cryoprotectants with different cryoprotectant compositions. 15 tested bacterial cultures belonged to four families: Enterobacteriaceae, Hafniaceae, Morganellaceae, and Yersiniaceae. For the tested bacterial cultures, cryoprotectant 1 had the greatest effectiveness in terms of storage: the general survival rate was 88.87%. Despite the small number of different bacterial species tested in our study, we assume that the cryoprotectant 1 composition can be effective for storing the majority of members of the order Enterobacterales at − 20 °C. However, further experimental studies are necessary to elucidate the problem of cryopreservation and to support our assumption.

Supplementary Information

Supplementary Table S1.

Supplementary Table S2.

Supplementary Table S3.

Supplementary Table S4.

Supplementary Table S5.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-024-71529-6.

Author contributions

Conceptualization, methodology, validation, formal analysis, and investigation: A.T., P.Z.; writing-original draft preparation: A.T.; writing-review and editing, supervision: P.Z.; visualization: A.T.; project administration: P.Z.

Data availability

All data generated or analyzed during this study are included in this article.

Competing interests

The authors declare no competing interests.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Publisher's note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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