Protective Effects of Cryoprotectants and Lyoprotectants on the Survival of Persipeptide Producing Streptomyces zagrosensis UTMC 1154

Abstract

Streptomyces sp. are bacteria recognized as the producers of more than half of the known bioactive compounds. Developing appropriate preservation methods for industrial strains of Streptomyces is necessary, as continuous subculture could have significant negative effects on their characteristics, including their potential to produce secondary metabolites. The effects of two common preservation methods on a bioactive metabolite producer, Streptomyces zagrosensis UTMC 1154, were studied. In the cryopreservation method, glycerol and dimethyl sulfoxide (DMSO) were evaluated as cryoprotectants. Three different suspending fluids including skimmed milk, sucrose+gelatin and Mist. dessicans were compared as the freeze-drying methods. Freeze-dried samples were stored at 4°C for 6 months and at 37°C for 1 and 2 weeks in an accelerated storage stability study, which approximately correspond to storage at 4°C for 10 and 20 years, respectively. Frozen samples were stored at −20°C, −70°C and in the vapor phase of liquid nitrogen for 6 months. Skimmed milk and DMSO were the most efficient protectants for survival and functional maintenance of the strain during the lyophilization and cryopreservation processes (p < 0.05), respectively. The survival rate of S. zagrosensis was 95.0% and 99.3% after 6 months of preservation by using skimmed milk as lyoprotectant and DMSO as the cryoprotectant, respectively. The obtained results showed that cryopreservation is the method of choice for long-term preservation of S. zagrosensis. Cryopreservation also led to only 1%–3% reduction in the biological activity of the strain after 6 months preservation in vapor phase of the liquid nitrogen.

Introduction

Long-term preservation of bacteria without phenotypic and genotypic changes is essential for all biotechnological applications such as the development of industrial processes, taxonomical studies, biological assays, agriculture or biodiversity conservation.¹ It is also necessary to sustain the productivity of bioactive compounds resources at the highest level in the biotech and pharmaceutical industries. Therefore, in parallel to the procedures for isolation, identification, collection and genetic engineering, a requirement arises for maintaining the vitality, genetic activity, immunogenicity and other properties of microorganisms in laboratory conditions.²

Members of the genus Streptomyces are well recognized as the producers of more than half of the known bioactive compounds such as antibiotics, anticancer agents, antifungal or antiparasitic drugs, herbicides, immune suppressants, etc.³ Metabolically active preservation methods such as sub-culture and maintenance under oil can lead to a decrease in the production of these valuable secondary metabolites.² On the other hand, preservation of strains by metabolically inactive techniques is based on the suppression of metabolism in the cell, which is achieved by reducing the water available to cells through dehydration or freezing. These methods may also effectively prevent deterioration and loss of properties like the production of bioactive compounds.⁴ Preservation leads the bacteria to an anabiotic state, the condition of reversible cessation in vital activities, while the cells keep their vitality to a minimum in continuous growth methods.² Since secondary metabolite synthesis only decreases during active preservation methods, utilizing inactive methods are necessary. However, cell damage occurs during the preservation process and even during the storage of inactive cultures. Therefore, preservation of highly productive variants even at efficient conditions can impede the production of biologically active substances.⁴

For the protection of the cells against oxidative stresses, osmotic stress and other harmful damage during freezing or drying as well as during storage, different suspending fluids are used. Selection of suitable methods and appropriate protectants allows not only the achievement of higher cell viability but also the long-term conservation of stability in the production of bioactive metabolites.⁵ In fact, for the secure maintenance of the physiological and genetic characteristics involved in secondary metabolites production, industrial strains should be subjected to an optimal preservation protocol.

Biological resource centers mainly use methods that suspend metabolism and keep the cells in the anabiosis phase, including cryopreservation and freeze-drying. These techniques ensure optimal long-term genetic stability of the strains in addition to the maintenance of their viability⁶ and lead the microorganisms to an anabiotic state, whereas methods such as storage under mineral oils, in aqueous solution or inactivation in dryers lead microorganisms to a hypobiotic state.² Freeze-dried microorganisms can be effectively preserved and maintained through the removal of water under reduced pressure in the presence of lyoprotectants.⁷ Cryopreservation is the storage at low temperatures below −70°C to inhibit chemical reactions in cells. The addition of cryoprotective agents is essential to protect the cells during the freezing process.⁷ However, handling, storage and distribution of freeze-dried biomaterials are more convenient and less costly in comparison to cryopreserved cells.^8,9

Parallel with storage methods, a multitude of factors influence the cell resistance and survival of preserved microorganisms. These factors include cultivation method, nutrient composition, pH, incubation temperature, aeration, cell water content, physiological culture condition, cell concentration, protective agents, suspension equilibration and recovery media.²

The suitability and efficiency of protectants largely depend on the type of microorganism; however, there are a few protectants that work relatively well with almost all species in most taxa. They consist of skimmed milk, horse serum, trehalose, glycerol, and dimethyl sulfoxide (DMSO), betaine, adonitol, sorbitol, sucrose, glucose, lactose and polymers such as dextran and polyethylene glycol.¹⁰

The aim of the present investigation was to study the impact of the principal storage methods (freeze-drying and cryopreservation) including the subsequent selection of the usual protectants (skimmed milk, serum, glucose, sucrose, gelatin, DMSO and glycerol) in the functional preservation of Streptomyces zagrosensis UTMC 1154. This model strain was considered to be representative of a potent actinobacterium with 36 secondary metabolite pathways (unpublished data) and a number of bioactive secondary metabolites including persipeptides.¹¹

Materials and Methods

Bacterial strains

An active culture of S. zagrosensis UTMC 1154 was obtained from the University of Tehran Microorganisms Collection (UTMC) and was used throughout the study as the producer of persipeptides. Bacillus subtilis UTMC 1416 (ATCC 6633TM) and methicillin resistant Staphylococcus aureus (MRSA) UTMC 1401 (DSM 23622) were used as test microorganisms in the determination of antibacterial activity.

Culture media

The spore production was performed on ISP2 agar (Merck) (0.4% glucose, 0.4% yeast extract, 1% malt extract and 1.5% agar, pH 7.2 ± 0.2). Seeding and fermentation broths were prepared using ISP2 broth medium.

Preparation of primary cultures

S. zagrosensis UTMC 1154 was cultured on ISP2 medium and incubated at 28°C for 10–14 days.¹¹ The spore suspension of S. zagrosensis UTMC 1154 was prepared by adding 10 mL sterile distilled water to each pure culture bottle. The consistent cellular physiological state can enhance the accuracy of conclusions in comparative studies of preservation processes. In the case of spore-forming bacteria, the equilibrated metabolic state can be obtained by excluding the vegetative cells from the spore mass. To prepare the spore suspension as the initial cells for each preservation method, spores were harvested by softly scraping the surface of the culture with a sterile loop followed by aseptic filtration using non-absorbent cotton wool (1 g/cm³).¹²

Lyophilization

Three different suspending fluids were used in freeze-drying of the spores of S. zagrosensis consisting of skimmed milk 20% (w/v) (Merck), Mist. dessicans (horse serum 75% (v/v)+nutrient broth 25% (v/v)+glucose 7.5% (w/v) and 12% sucrose (w/v) +12% gelatin (w/v).

The spore suspension (1.5 × 10⁷ spores/mL) was mixed with an equal volume of each medium and 200 μL of the mixture were transferred to small sterile glass vials (11 × 44 mm). The glass vials were kept at −20°C for 1 hour and subsequently at −70°C for 2 hours. Then, vials were subjected to a primary drying (20 hours, 0.02–0.03 mbar, −50 ± 2°C) in the freeze dryer (Christ, Alpha 1–2 LD plus). The small glass vials were placed into 16 × 140 mm glass tubes containing 0.05 g crystals of silica gel with moisture indicator (Merck, 101925). After the constriction of the outer tubes, they were attached to the ampoule manifold of the freeze dryer for performing the secondary drying (4 hours under 0.05–0.07 mbar pressure, −50 ± 2°C). At the end, the ampoules were sealed under vacuum (<0.07 mbar) and were stored in the dark at 4°C for 6 months. Furthermore, they were maintained at 37°C for 7 and 14 days for accelerated storage tests, which are equal to 10 and 20 years of maintenance, respectively.¹³

Cryopreservation

Glycerol (Merck) at the concentration of 15% or 30% (v/v) and DMSO (Merck) at the concentration of 5% (v/v) were used as cryoprotective agents in this study. The double strength of cryoprotectants was prepared in one-fifth strength diluted ISP2 medium and 250 μL of this sterile cryoprotectant solution was dispensed into sterile 2.5 mL cryotubes (JetBiofil). The same volume (250 μL) of spore suspension (containing 1.5 × 10⁷ spores/mL) was added to each cryotube in a dropwise manner to attain the final intended concentration. Augmentations of the spore suspension with cryoprotectant were carried out at room temperature in the case of glycerol as the protectant, while they were mixed on the ice when DMSO was used as the protectant.

When glycerol was used as a protectant, the equilibration between spores and glycerol was conducted at room temperature for 60 minutes to allow glycerol to be taken up before preservation. The equilibration treatment with DMSO was executed at 4°C for 15 minutes. The cryotubes containing DMSO were immediately transferred to ordinary/ultra low freezers or nitrogen tanks (MVE, Bio-Medical System, USA), to avoid its toxic effects on cells at ambient temperature.¹³ Filled cryotubes of glycerol and spore suspensions were kept at −20°C for 30 minutes. Then, one-third of the tubes were transferred to −70°C for an additional 30 minutes. After 30 minutes half of these cryotubes were transferred from −70°C to the vapor phase of liquid nitrogen.

Assessment of cell resuscitation

The resuscitation of the spore suspension of S. zagrosensis was determined by the conventional plate count method before and after preservation. The serial dilution of spore suspension was prepared using sterilized saline (0.9% NaCl) containing 0.1% Tween 80. Each spore dilution in a volume of 100 μL was spread on plates of ISP2 agar media and incubated at 28°C for 10 days.

Lyophilized spores were rehydrated at room temperature and revived by the addition of 400 μL of ISP2 medium after 2 weeks or 6 months and also after storage at 37°C for 1 and 2 weeks.^14,15

Cryopreserved suspensions were also resuscitated after 2 weeks and 6 months. Cryovials were thawed at 37°C. The viable spores in the rehydrated suspensions were determined as already described. All the experiments were conducted in triplicate and the results were expressed as CFU/mL (colony-forming units per milliliter) of the survived spores.

Antibacterial activity assay

The antibacterial activity of S. zagrosensis UTMC 1154 was determined by the agar diffusion assay method before and following the preservation shock.

The spore suspension (100 μL) at the concentration of 10⁷ spore/mL was inoculated into 100 mL Erlenmeyer flasks containing 10 mL seeding medium and incubated at 28°C for 26 hours on a rotary shaking at 200 rpm. The fermentation broth was inoculated with 1% (v/v) of seeding material and incubated at 28°C for 7 days, with shaking at 200 rpm. Antimicrobial activities of 7-day old cultures of S. zagrosensis against B. subtilis UTMC 1416 and MRSA UTMC 1401 were determined by the diffusion method on Muller-Hinton agar and results were measured as diameter of the inhibition zone in mm.¹⁶

Statistical analysis

The data obtained from three replications were analyzed using the statistical program SPSS16.0 and the one-way analysis of variance test was used to determine if significant differences (p ≤ 0.05) existed between mean values.

Results

The resuscitation value of S. zagrosensis UTMC 1154 before preservation was 1.5 × 10⁷ CFU/mL. The lyophilization process reduced the viable spore counts by less than one log₁₀ unit. After 6 months of storage at 4°C, all lyophilized cells using investigated lyoprotectants showed a similar survival behavior. The resuscitation rate of the cells lyophilized in various protectants was steadily maintained (p > 0.05) for up to 6 months of preservation.

The storage of ampoules at 37°C for 2 weeks, which corresponds approximately with normal storage at 4°C for 20 years, caused more than two log₁₀ unit reductions in numbers of viable spores. However, the survival rate of spores lyophilized in skimmed milk was significantly better (p < 0.05) than the other two lyoprotectants, as is presented in Figure 1.

FIG. 1.

Comparative viability study of S. zagrosensis UTMC 1154 after freeze drying in various protective media: skimmed milk (■), horse serum+nutrient broth+glucose (Mist. dessicans) (), and gelatin+sucrose (□). Spore counts I: Before lyophilization, II: After lyophilization, III: 6 months after lyophilization, IV: After 1 week storage at 37°C, V: After 2 weeks storage at 37°C. CFU, colony-forming unit; UTMC, University of Tehran Microorganisms Collection.

The resuscitation of S. zagrosensis UTMC 1154 in different concentrations of glycerol and DMSO did not show a significant reduction (p > 0.05), after 6 months storage at −20°C, −70°C and in the vapor phase of liquid nitrogen. The number of viable spores were slightly higher when DMSO had been applied as a cryoprotectant. However, the rate of recovery was not statistically significant (p > 0.05) at different low temperatures during the time frame for up to 6 months of storage (Fig. 2). Accordingly, all type of these tested protectants and variable low temperatures can efficiently protect the cells from inactivation up to 6 months unless other detrimental factors such as freeze-thaw stress are involved.

FIG. 2.

Effects of various protective media and different freezing temperatures on viability of the S. zagrosensis UTMC 1154 after freezing in various protective media: glycerol 15% (■), glycerol 30% (□), and dimethyl sulfoxide (Me₂SO) 5% (). The viability of the strain was measured before (I), immediately after freezing (II), 6 months after freezing (III).

The assessment of the antibacterial activity of S. zagrosensis UTMC 1154 following the preservation stress showed that with skimmed milk the biological activity was better (p < 0.05) preserved than with the other protectants after 14 days of storage at 4°C. In contrast, the antibiotic activity of the strain declined significantly (p < 0.05) when the gelatin-sucrose mixture was used as the protective agent. However, long-term preservation in the lyophilized state did not significantly influence the average antibiotic biosynthesis of S. zagrosensis UTMC 1154 in tested protective media (Table 1).

Table 1.

Impact of Lyophilization Protective Media on Conservation of Antibacterial Activity in Streptomyces zagrosensis UTMC 1154 Against Methicillin Resistant Staphylococcus aureus and Bacillus subtilis UTMC 1416

Protective medium	Average antibacterial activity (mm of inhibition zone)
Protective medium	Test strain	Before preservation	After 2 weeks preservation	After 6 months	After 1 week treatment at 37°C (10 years)	After 2 week treatment at 37°C (20 years)
Skim milk	MRSA	29.1 ± 0.05 (100%)	28.8 ± 0.04 (99%)	27.7 ± 0.06 (95%)	25.9 ± 0.1 (89%)	25.3 ± 0.08 (87%)
Skim milk	B. subtilis	29.8 ± 0.04 (100%)	29.6 ± 0.09 (99%)	28.4 ± 0.05 (95%)	27.9 ± 0.03 (94%)	27.9 ± 0.11 (94%)
Horse serum+glucose+nutrient broth	MRSA	29.1 ± 0.06 (100%)	28.3 ± 0.09 (97%)	27.4 ± 0.05 (94%)	25.6 ± 0.1 (88%)	25.1 ± 0.04 (86%)
Horse serum+glucose+nutrient broth	B. subtilis	29.8 ± 0.07 (100%)	29.1 ± 0.04 (98%)	27.8 ± 0.05 (93%)	27.3 ± 0.08 (92%)	27.1 ± 0.09 (91%)
Gelatin+sucrose	MRSA	29.1 ± 0.12 (100%)	27.3 ± 0.09 (94%)	27.3 ± 0.15 (94%)	26.1 ± 0.08 (90%)	25.6 ± 0.03 (88%)
Gelatin+sucrose	B. subtilis	29.8 ± 0.08 (100%)	28.9 ± 0.05 (97%)	27.9 ± 0.05 (94%)	27.3 ± 0.1 (92%)	27 ± 0.09 (91%)

MRSA, methicillin resistant Staphylococcus aureus.

Regardless of the type of the applied protectants, the bioassay results showed that the antibacterial activity of S. zagrosensis against B. subtilis UTMC 1416 was better maintained compared to MRSA after preservation in an accelerated condition. This impact can be possibly attributed to the fact that effective compounds against MRSA are produced by biosynthetic pathways, and that their expression is more amenable to the influence of the long-term preservation of the producing strain. However, Table 1 shows that lyophilization in all tested protectants did not cause more than a 5%–8% reduction in antibacterial activity of the strain in comparison with the initial activity, while following 20 years of simulated preservation, the antibacterial activity of the initial culture decreased 14% and 9% against MRSA and B. subtilis, respectively. Consequently, skim milk could provide higher tolerance both for the cell to survive and maintain their secondary metabolite expression after the freeze-drying mode of preservation.

The average antibiotic activity of S. zagrosensis UTMC 1154 against B. subtilis UTMC 1416 and MRSA UTMC 1401 was significantly reduced (p < 0.05) during the cryopreservation process in the presence of 30% glycerol compared to 15% glycerol or 5% DMSO.

The antimicrobial activity of S. zagrosensis decreased from 100% before preservation to not <91% using the tested cryoprotectants after 6 months preservation at −20°C, −70°C or the vapor phase of liquid nitrogen. In general, the antibacterial potential of the strain was better conserved following the cryopreservation process using 5% DMSO and storage (Table 2) in the vapor phase of liquid nitrogen. This optimum condition to protect the functionality of the strain (DMSO/vapor phase of liquid nitrogen) was revealed to be different from what is sufficient for its sustainable viability (DMSO or glycerol/at −20°C).

Table 2.

Influence of Protective Media and Cryopreservation Temperature on Antibacterial Activity of S. zagrosensis UTMC 1154 Against Methicillin Resistant S. aureus and B. subtilis UTMC 1416

Protective medium	Average antibacterial activity (mm of inhibition zone)
Protective medium	Temperature condition (°C)	Test strains	Before preservation	After 2 weeks preservation	After 6 months
Glycerol 15%	−20	MRSA	29.1 ± 0.09 (100%)	28.6 ± 0.04 (98%)	28.1 ± 0.07 (96%)
	−20	B. subtilis	29.8 ± 0.1 (100%)	28.9 ± 0.05 (100%)	28.3 ± 0.11 (95%)
	−70	MRSA	29.1 ± 0.06 (100%)	28.9 ± 0.08 (99%)	26.9 ± 0.14 (92%)
	−70	B. subtilis	29.8 ± 0.01 (100%)	28.8 ± 0.07 (97%)	27.7 ± 0.09 (93%)
	Vapor phase of liquid nitrogen	MRSA	29.1 ± 0.8 (100%)	28.6 ± 0.4 (98%)	27.9 ± 0.8 (96%)
	Vapor phase of liquid nitrogen	B. subtilis	29.8 ± 0.05 (100%)	29.2 ± 0.13 (98%)	28.6 ± 0.15 (96%)
Glycerol 30%	−20	MRSA	29.1 ± 0.7 (100%)	28.2 ± 0.05 (97%)	27.4 ± 0.03 (94%)
	−20	B. subtilis	29.8 ± 0.11 (100%)	28.6 ± 0.07 (96%)	28.1 ± 0.08 (94%)
	−70	MRSA	29.1 ± 0.1 (100%)	28.2 ± 0.05 (97%)	26.4 ± 0.12 (91%)
	−70	B. subtilis	29.8 ± 0.06 (100%)	27.5 ± 0.08 (92%)	27.8 ± 0.05 (93%)
	Vapor phase of liquid nitrogen	MRSA	29.1 ± 0.05 (100%)	28.3 ± 0.1 (97%)	27.5 ± 0.06 (94%)
	Vapor phase of liquid nitrogen	B. subtilis	29.8 ± 0.03 (100%)	28.6 ± 0.9 (96%)	28.4 ± 0.07 (95%)
Me₂SO 5%	−20	MRSA	29.1 ± 0.04 (100%)	29 ± 0.05 (100%)	28.2 ± 0.05 (97%)
	−20	B. subtilis	29.8 ± 0.08 (100%)	29.1 ± 0.1 (98%)	28.6 ± 0.07 (96%)
	−70	MRSA	29.1 ± 0.05 (100%)	28.3 ± 0.05 (97%)	26.8 ± 0.05 (92%)
	−70	B. subtilis	29.8 ± 0.05 (100%)	28.4 ± 0.05 (95%)	28.2 ± 0.05 (95%)
	Vapor phase of liquid nitrogen	MRSA	29.1 ± 0.06 (100%)	28.8 ± 0.03 (99%)	28.4 ± 0.1 (98%)
	Vapor phase of liquid nitrogen	B. subtilis	29.8 ± 0.05 (100%)	29.3 ± 0.07 (98%)	28.7 ± 0.08 (97%)

Discussion

From among 16,000 discovered bioactive compounds, 12,400 are produced by Streptomyces sp.¹⁷ Therefore, maintaining the physiological state and secondary metabolites production of Streptomyces members following their long-term preservation process is of critical value.

It is reported that almost all tested strains, except five strains out of 2334 strains of yeasts, bacteria and actinomycetes were preserved well in freeze-dried form, indicating the effectiveness of freeze-drying as a suitable preservation method.⁷ However, optimal freeze-drying conditions are strain specific and a single optimal protocol suitable for all strains in a culture collection seems, therefore, unobtainable.¹⁸

Throughout the cryoprotection process, cryoprotectants act as prominent agents in keeping cells alive and productive. Even if many classical cryoprotectants, including glycerol, gelatin, skimmed milk, and horse serum, have been well known for the preservation of cells, for special species, particularly industrial strains, the cryoprotectants need to be re-optimized.¹⁹

The validation of conventional long-term preservation protectants in the conservation of the viability and secondary metabolite biosynthetic yield of a bioactive new Streptomyces sp. were examined in this report. The most efficient protectant for longer survival and maintenance of genetic stability required in the production of secondary metabolites was evaluated following preservation stress.

It was reported that three antibiotic producing Streptomyces strains were optimally preserved in skim milk, sucrose and gelatin after long-term storage. Revived Streptomyces strains from the lyophilized state demonstrated 80%–90% viability after 8–28 years.²⁰ Data from the current study indicated that skim milk had the most lyoprotective activity among the tested protectants for S. zagrosensis UTMC 1154 in the freeze-dried state. Compatibility and availability of skim milk, in addition to its low cost, justify its selection as a suitable protectant for large scale preservation of industrial strains. Since skim milk contains 32.0%–35.7% protein and 48.4%–54.1% lactose, it is a preferred protectant in the preservation of bacteria. The reason for this lies in the fact that proteins play a more important role in glass formation in comparison to the sugars. It can explain why skim milk powder is also found to be an efficient desiccation protectant.¹⁰

In the freezing state, the highest protection level for S. zagrosensis was obtained by 5% DMSO compared to glycerol 15% and 30%. Similarly, previous data have shown that DMSO can better retain the resuscitation state compared to glycerol or other protectants in some bacteria such as Spirillum volutans, Escherichia coli, Lactobacillus delbrueckii, methanotrophic bacteria, Saccharomyces exiguus, and some filamentous fungi.¹⁰

In cryopreservation processes up to 6 months, it was observed that the strain survival following preservation at −20°C was slightly higher than the strains which were preserved at −70°C. This deviation from the principle may be due to the additional cold shock (at −20°C for 30 minutes) before the maintenance at −70°C, which was not conducted for spores maintained at −20°C. This may indicate that gradual adaptation steps in lowering the temperature positively affect the preservation/resuscitation. However, survival rates are higher at lower temperatures including the vapor phase of liquid nitrogen in long-term studies.

In general, storage using freezing was a more reliable method compared to freeze-drying for preservation programs up to 6 months. It is inferred from the above comparison that for short-term preservation purposes (<6 months) of Streptomyces cultures, cryopreservation at −20°C is as effective as lower temperatures and this can be the selective temperature. In fact, intensive metabolism ceasing at lower temperatures can compensate for the initial cold shock harm of the preservation process only if the maintenance exceeds 6 months. However, when the duration of the preservation is <6 months, better cell preservation at −70°C may not dominate the effect of initial imposed cold stress. In conclusion, for preservation purposes aimed for <6 months (such as preparing the working stock in the industry of antibiotic production), it is recommended to preserve the culture at −20°C based on the results obtained in this study. It seems that approximately all preservation procedures are efficient during 6 months, but to reach long-term preservation tolerance, different methods and protectants need to be evaluated and optimized for any of target strain.

Footnotes

Acknowledgments

The authors thank Hamed Kazemi Shariat Panahi, Fahimeh Mohammadnia and Abbas Abbasi for their technical assistance. This research work has been carried out with the financial support from the Ministry of Science, Research and Technology of Iran) and International Foundation of Science.

Author Disclosure Statement

No conflicting financial interests exist.

References

Day

, Stacey

. Cryopreservation and Freeze-Drying Protocols. New York: Humana Press; 2007:198.

Uzunova-Doneva

, Donev

Anabiosis and conservation of microorganisms. J Cult Collect, 2004–2005;;4:17–28.

Bhasin

, Modi

. Streptomycetes: A storehouse of bioactive compounds and enzymes A: Production of glucose isomerase. Res J Recent Sci, 2013; 2:330–339.

Rifaat

HM.

Viability study of some locally isolated streptomycetes. J Cult Collect, 2008–2009; 6:38–41.

Morgan

, Herman

, White

, Vesey

. Preservation of microorganisms by drying: A review. J Microbiological Methods, 2006; 66:183–193.

Gherna

RL.

Culture preservation. In: Gerhardt

, Murray

RGE

, Wood

, Krieg

(eds). Methods for General and Molecular Bacteriology. Washington, DC: ASM Press; 1994: 278–292.

Nagai

, Tomioka

, Takeuchi

, Iida

, Kawada

, Sato

. Evaluation of preservation techniques of microorganism resources in the MAFF genebank. JARQ, 2005; 39:19–27.

Bozoglu

, Ozilgen

, Bakir

. Survival kinetics of lactic acid starter cultures during and after freeze drying. J Microbiol Tech, 1987; 9:531–537.

Miyamoto-Shinohara

, Imaizumi

, Sukenobe

, Murakami

, Kawamura

, Yasuhiko

Komatsu

. Survival rate of microbes after freeze-drying and long-term storage. J Cryobiology, 2000; 41:251–255.

10.

Hubalek

Protectants used in the cryopreservation of microorganisms. J Cryobiology, 2003; 46:205–229.

11.

Mohammadipanah

, Matasyoh

, Hamedi

, Klenk

, Laatsch

. Persipeptides A and B, two cyclic peptides from Streptomyces sp. UTMC 1154. J Bioorg Med Chem, 2011; 20:335–339.

12.

Kieser

, Bibb

, Buttner

, Chater

, Hopwood

. Practical Streptomyces Genetics. Norwich, England: The John Innes Foundation; 2000.

13.

Iino

, Suzuki

. Improvement of the L-drying procedure to keep anaerobic conditions for long-term preservation of methanogens in a culture collection. Microbio Cult Coll, 2006; 22:99–104.

14.

Yocheva

, Najdenova

, Doncheva

, Antonova-Nikolova

Influence of the long term preservation on some biological features of three streptomycetes strains, producers of antibiotic substances. J Cult Collect, 2000–2002;3:25–32.

15.

Sakane

, Kuroshima

. Viabilities of dried cultures of various bacteria after preservation for over 20 years and their prediction by the accelerated storage test. Microbiol Cult Coll, 1997; 13:1–7.

16.

Schwalbe

, Steele-Moore

, Goodwin

. Antimicrobial Susceptibility Testing Protocols. Florida: CRC Press; 2007.

17.

Bérdy

Current innovations and future trends. In: Sánchez

, Demain

(eds). Antibiotics. Poole, UK: Caister Academic Press; 2015: 49–64.

18.

Peiren

, Buyse

, De Vos

, et al. Improving survival and storage stability of bacteria recalcitrant to freeze-drying: A coordinated study by European culture collections. Appl Microbiol Biotechnol, 2015; 99:3559–3571.

19.

Liao

, Liu

Prevent the degradation of algicidal ability in Scenedesmus-lysing bacteria using optimized cryopreservation. Environ Sci Pollut Res Int, 2015:23:5925–5930.

20.

Hoefman

, Van Hoorde

, Boon

, Vandamme

, De Vos

, Heylen

. Survival or revival: Long-term preservation induces a reversible viable but non-culturable state in methane-oxidizing bacteria. PLoS One, 2012; 7:e34196.