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Thermoactinomycetes isolated from geothermal springs in Armenia capable of producing extracellular hydrolases

  • Hovik PanosyanEmail author
Original Article
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Abstract

Microbes colonizing high elevation hot springs have been extensively studied in last decades. In this sense, Armenian highland geothermal springs are in the center of attention for exploring new thermophilic microbes with biotechnological prospects. In present paper, the identification and characterization of two thermoactinomycetes isolated from Armenian geothermal springs, namely Akhurik and Tatev, were reported. The isolates, designated as AkhA-12 and Tatev 35a were studied for phenotypic and phylogenetic profiling. The aerial and substrate mycelia formed by strain AkhA-12 were yellow-white and greyish-yellow respectively. The strain Tatev 35a developed white aerial and substrate mycelia. The endospores produced by thermoactinomycetes were round in shape (0.7–0.9 µm in diameter) and located on short unbranched sporophores. The strains grew aerobically at 35–60 °C (Topt 50–55 °C). The pH range for growth was observed between 5.0 and 8.0 with optimum pH 7.0–7.4. Both strains were able to grow at 0–8% NaCl (optimum 2–3%). Based on phenotypic characteristics and phylogenetic analyses (16S rRNA gene sequence analysis) both strains were identified as representatives of the genus Thermoactinomyces. The strain AkhA-12 demonstrated close relationship to Thermoactinomyces sp. JIR-004 (99.7% similarity) and T. daqus DSM 45914T (99.4% similarity), while the strain Tatev 35a was closely related to T. vulgaris DSM 43016T (99.01%). The ability to synthesize extracellular hydrolases (amylase, lipase and protease) was evaluated by growth of strains on solid media supplemented with appropriate substrate at different temperatures. The active production of thermostable hydrolytic enzymes by strains indicates their potential in biotechnology.

Keywords

Geothermal springs Thermoactinomyces 16S rRNA gene sequences Thermostable extracellular hydrolases 

Introduction

Thermal springs are selective habitats of thermophilic microbes serving valuable sources of unique biomolecules and thermostable enzymes (Raddadi et al. 2015; DeCastro et al. 2016; Sahay et al. 2017). Thermophilic microorganisms taxonomically are very diverse and spread widely among prokaryotes (Archaea and Bacteria) (Charliera and Droogmansb 2005; Mehta and Satyanarayana 2013). A separate group of thermophilic bacteria is represented by thermoactinomycetes. Thermoactinomycetes have been isolated from different environments including natural terrestrial hot springs (Carrillo and Benitez-Ahrendts 2014; Sahay et al. 2017). The genus Thermoactinomyces, as one of genera within order Bacillales, includes aerobic, endospore-forming, Gram-positive bacteria (Carrillo and Benitez-Ahrendts 2014). Recently based on phenotypic, phylogenetic and chemotaxonomic analyses species of Thermoactinomyces have been reclassified (Yoon et al. 2005). Currently genus Thermoactinomyces includes five valid species: T. vulgaris (Goodfellow and Jones 2009) T. intermedius (Goodfellow and Jones 2009), T. daqus (Yao et al. 2014), T. guangxiensis (Wu et al. 2015) and T. khenchelensis (Mokrane et al. 2016). T. khenchelensis sp. nov. has been recently isolated from an Algerian hot spring (Mokrane et al. 2016).

In the last decade, several strains of thermoactinomycetes have been isolated from geothermal areas in Bulgaria (Derekova et al. 2008), India (Sahay et al. 2017), Russia (Rozanov et al. 2017), Turkay (Uzel et al. 2011; Aksoy et al. 2012), Pakistan (Jadoon et al. 2014), Kenya (Waithaka et al. 2017) and Japan (Nishiyama et al. 2013).

Attention of biotechnologists have been focused on thermoactinomcetes taking in account their ability to produce thermozymes, like thermostable amylases (Jadoon et al. 2014), proteases (Aksoy et al. 2012; Verma et al. 2016), cellulases (Chaudhary and Prabhu 2016) and bioactive molecules.

Despite comprehensive studies on microbial composition of terrestrial geothermal springs, limited attention has been paid toward diversity of thermoactinomycetes harboring high elevation hot springs. From this point of view, geothermal springs discovered in Armenia represent a source to isolate new and undescribed microbes with biotechnological prospects. Recently microbiological investigations to evaluate bacterial and archaeal diversity in some Armenian geothermal springs were carried out (Panosyan and Birkeland 2014; Shahinyan et al. 2017; Panosyan 2017; Panosyan et al. 2018). Thus, many strains identified as representatives of genus Bacillus and related genera have been isolated from Armenian hot springs and studied to discover their biotechnological potency (Shahinyan et al. 2017; Panosyan 2017). The diversity of thermoactinomycetes in Armenian hot springs remains uninvestigated. The present study reports the isolation of thermoactinomycetes from Akhurik and Tatev geothermal springs and their identification based on phenotypic peculiarities and 16S rRNA gene sequence analysis. The potential of isolates to produce thermostable hydrolases (lipase, protease and amylase) was explored as well.

Materials and methods

Description of study sites and sampling

Sediment/soil samples were aseptically collected from terrestrial geothermal springs located in Akhurik and Tatev region of Armenia. In situ water temperature, pH and conductivity were measured using portable combined pH/EC/TDS/Temperature tester (HANNA HI98129/HI98130). Geographical locations and elevations of these springs were determined by portable GPS (GERMIN 64 s). The Akhurik spring is situated in Shirak region of Armenia at 40°44′34.04″N, 43°46′53.95″E, 1490 m above sea level (Fig. 1a, b). Outlet water temperature was 30 °C and pH was 6.5. Dissolved mineral content was relatively high (2490 μS/cm). The Tatev spring is situated in Syunik region of Armenia at 39°23′76.00″N 46°15′48.00″E, 960 m above sea level (Fig. 1a, c). Outlet water temperature, pH and dissolved mineral content were 27.5 °C, 7.50 and 1920 μS/cm, respectively. Spring in Akhurik was classified as a HCO3/SO42−/Na+/Mg2+-type, while spring in Tatev was classified as a CO32−/HCO3/Ca2+-type (Mkrtchyan 1969).
Fig. 1

Location of study sites, a Map of Armenia showing locations of Akhurik (1) and Tatev (2). Close up photographs of the source pools, b Akhurik hot spring (vigorous degassing and cyanobacterial mats are visible), c Tatev hot spring

Enrichment and isolation

In order to obtain enrichment for only endospore-forming bacteria 10 ml suspension of sediment/soil samples (1 g) were pasteurized at 80 °C for 10 min. Erlenmeyer flasks (100 ml) containing 20 ml of nutrient broth (NB, Difco) was inoculated by 1 ml aliquots of pasteurized suspension and incubated overnight at 55 °C on shaker (250 rpm). Then 100 μL of turbid enrichment was streaked on plates of NB solid medium containing 2% (w/v) agar by standard serial dilution plating technique and incubated at 55 °C for 48 h. The pure cultures were obtained by sub culturing of picked single colonies showing typical morphology. NB containing 20% glycerol was used for preserving/maintaining bacterial cultures at − 80 °C.

Phenotypic characteristics

Properties of microbial colonies, like colours of substrate and aerial mycelia, size, shape, surface, margins, ability to produce any soluble pigments, were described. The cell morphology, endospore’s form and location was determined by phase-contrast microscopy (Nikon, Eclipse E400 light microscope). Biochemical tests including utilization of d-glucose, d-fructose, d-lactose, l-arabinose, d-xylose, d-inositol, d-mannitol, coagulation and peptonization of milk, tests for catalase, oxidase and tyrosinase, liquefaction of gelatin, production of H2S, nitrate reduction to nitrite were performed by earlier described methods (Williams et al. 1983; Tindall et al. 2007).

The growth temperature range was determined by incubation of thermoactinomycetes at 10–75 °C by 5 °C intervals. The growth of bacterial isolates was evaluated at various values of pH (at 5.0–10.0, with an interval of 1.0). Salt tolerance of microbes was determined by inoculation of cells in NB supplemented by 0–10% (w/v) of NaCl and by incubation at optimal growth teperature. Each experiment was carried out in triplicate.

The ability to produce extracellular hydrolytic enzymes (amylase, lipase and protease) were checked qualitatively by growth of isolates on corresponding solid media. Thermostability of selected hydrolases was monitored by incubation of cultures at 50, 55 and 60 °C growth temperatures for 24–48 h. The amylase production was screened on medium containing (w/v) soluble starch (2%), peptone (1%), KH2PO4 (0.5%), agar (1.5%), pH 7.0–7.2 (Panosyan 2017). The clear halo zone around the colony formed after staining with Lugol’s solution (0.5% I2 and 1.0% KI (w/v) in distilled water) confirmed the existence of amylase activity (Jadoon et al. 2014). Extracellular protease production was determined by streaking thermoactinomycetes on medium containing 0.5% of skim milk powder, 0.5% of glucose, 2% of agar, pH 7.0–7.2. The appearance of a clear zone around bacterial streak resulting from decomposition of milk casein was indicative for protease activity (Aksoy et al. 2012). The production of lipolytic enzymes was evaluated by streaking strains on nutrient agar medium supplemented with 1% (v/v) of Tween 20 or Tween 80 and CaCl2·2H2O (0.01% w/v). The presence of lipase activity was indicated by visible precipitates of fatty acid calcium salts (Panosyan 2017).

Extraction of DNA and PCR amplification

Genomic DNA extracted by GenElute™ Bacterial Genomic DNA Kit (Sigma) from bacterial strains was used as template for PCR amplification. Universal primer pairs 27f (5′-GAGTTTGATCCTGGCTCA-3′) and 1525r (5′-GAAAGGAGGAGATCCAGCC-3′) (Escherichia coli numbering) were used to amplify 16S rRNA genes. Composition of mixtures used for PCR amplification was following: 10 ng DNA, 5 µl 10× PCR buffer, 5 µl 10 mM dNTP (dATP, dGTP, dCTP and dTTP), 1 µl each primer (25 pmol/µl), 1.5 mM MgCl2, 0.2 µl Taq DNA polymerase, and sterile water up to the final volume of 50 µl. DNA Engine thermal cycler (BIO RAD) was applied for amplification of target gene. Following regime of PCR amplification was used: initial denaturation of templates for 3 min at 96 °C, then 30 cycles of steps including denaturation for 30 s at 96 °C, annealing for 30 s at 55 °C, and extension for 2.5 min at 72 °C (final extension for 10 min at 72 °C). PCR amplicons were visualized by gel electrophoresis under UV light. GenElute™ PCR Cleanup Kit (Sigma) was applied to purify PCR products.

Sequencing and phylogenetic analysis

ABI PRISM capillary sequencer was used to sequence 16S rDNA amplicons following the recommendations of the ABI Prism Big-Dye Terminator kit (Perkin Elmer). Raw data of DNA sequences was interpreted by Chromas and BioEdit software. BLASTn search was performed to find phylogenetically closest relatives (National Center for Biotechnology Information, http://www.ncbi.nlm.nih.gov/Blast) (Altschul et al. 1997). MEGA X software was applied to construct phylogenetic tree (Tamura et al. 2004; Kumar et al. 2018). The bootstrap test (1000 replicates) was used to determine confidence in branching points (Felsenstein 1985).

Nucleotide sequence accession numbers

The 16S rRNA gene sequences of strains were deposited in NCBI GenBank database. The accession numbers obtained are MK418253 and MK418408.

Results and discussion

Due to active volcanic and tectonic processes numerous geothermal mineral springs are found in Lesser Caucasus (Mkrtchyan 1969; Henneberger et al. 2000). In this paper, we focused on two Armenian high-elevated geothermal springs, namely Akhurik and Tatev, aiming to isolate and describe thermoactinomycetes from sampled sediments. Both studied springs are mesothermal, circumneutral and highly mineralized (Mkrtchyan 1969; Henneberger et al. 2000).

Two thermophilic isolates showing typical colony morphology for thermoactinomycetes were isolated from above mentioned mesothermal springs. Isolated thermophilic bacterial strains were studied based on phenotypic and phylogenetic characteristics. Isolates obtained from Akhurik and Tatev springs were designated as AkhA-12 and Tatev 35a, respectively. Isolate AkhA-12 formed yellow–white aerial and greyish-yellow substrate mycelia, while aerial and substrate mycelia developed by isolate Tatev 35a were white. The endospores produced by both isolates were round in shape, located on short unbranched sporophores and had 0.7–0.9 µm sizes in diameter. Both isolates were Gram-positive and produced catalase and oxidase. No diffusible or soluble pigments were observed on used media.

Physiological and biochemical features of studied strains are reported in Table 1. Phenotypic characteristics of reference strains T. daqus DSM 45914T, 2, T. vulgaris DSM 43016T, 3, T. guangxiensis ATCC BAA-2630T, 4, T. intermedius DSM 43846T, 5, T. khenchelensis DSM 45951T were provided for comparison.
Table 1

Comparison of phenotypic properties of thermoactynomycetes isolated from Armenian geothermal springs with Thermoactinomyces species (1, T. daqus DSM 45914T, 2, T. vulgaris DSM 43016T, 3, T. guangxiensis ATCC BAA-2630T, 4, T. intermedius DSM 43846T, 5, T. khenchelensis DSM 45951T)

Characteristics

Strains

AkhA-12

Tatev 35a

1a

2a,b

3c

4a,b,c,d

5b

Aerial mycelium

Yellow–white

White

Yellow–white

White

White

White

White

Spore form

S, R

S, R

S, R

Nd

S, Sp

S,

S, R

 Diameter µm

0.7–0.9

0.7–0.9

0.8–0.9

 

0.8–1.0

0.6–1.0

0.8–1.0

Sporophores

Short

Short

Short

Short

Long

Short

Short

Soluble pigment

+

Temperature range (Topt) (°C)

30–60 (50)

35–60 (50–55)

45–60 (55)

35–60 (55)

37–55 (45–50)

37–65 (50–55)

37–55 (50–55)

NaCl (w/v) range (optimum) (%)

0–8.0 (0–3.0)

0–5.0 (0–2.0)

Nd

0–7.0 (Nd)

0–2.0 (0–1.0)

Nd

0–7.0 (0–2)

pH range (optimum)

5.0–8.5 (7.0–7.2)

5.0–8.0 (7.2–7.4)

5.0–9.0 (7.0)

5.0–8.0 (7.2–7.4)

6.0–11.0 (7.0–9.0)

5.0–8.0 (7.2–7.4)

7.0–9.0 (8.0)

Catalase

+

+

Nd

Nd

+

Nd

Nd

Oxidase

+

+

Nd

Nd

Nd

Nd

Tyrosinase

+

Utilization of

       

 d-glucose

+

+

+

 l-arabinose

 d-fructose

+

+

W

+

+

+

 d-xylose

Nd

 d-lactose

W

 d-inositol

W

Nd

Nd

Nd

 d-mannitol

+

+

+

+

 Citrate

W

W

Nd

Nd

+

Nd

Nd

 Propionate

W

W

Nd

Nd

+

Nd

Nd

Gelatin liquefaction

+

+

+

+

+

+

Milk coagulation

+

+

+

+

+

+

+

Milk peptonization

+

+

+

+

+

+

Degradation of hypoxanthine

+

+

Nitrate reduction

+

+

+

H2S Production

Nd

aData from Yao et al. (2014)

bData from Mokrane et al. (2016)

cData from Wu et al. (2015)

dData from Goodfellow and Jones (2009)

+ positive, − negative, w weakly positive result, Nd not determined, S single, R round, Sp spherical

Isolate AkhA-12 was capable to grow at wide range of temperatures from 37 to 60 °C with optima at 50 °C. Growth occurred at pH range from 5.0 to 8.0 (pHopt 7.0–7.2). The isolate was tolerant up to 8% of NaCl. Growth of isolate Tatev 35a wasn’t observed at temperature under 35 °C and above 60 °C. Optimal growth temperature of isolate Tatev 35a was 50–55 °C. The pH range of isolate Tatev 35a occurred between pH 5.0 and 8.0 (pHopt 7.2–7.4). This isolate was able to stand up to 5% (w/v) NaCl concentration (optimum 0–2.0%).

Temperature of outlet water of studied mesothermal springs was around 28–30 °C, while isolates were able to grow optimally at temperature of 50–55 °C. Resent geophysical investigations confirmed that water temperature of the source increases with the depth and reaches up to 99 °C at deeper levels (Henneberger et al. 2000). This evidence confirms that detected thermoactinomycetes are autochthonous inhabitants of thermal springs able to grow at reservoir temperatures.

Both isolates demonstrated positive results for catalase, oxidase, gelatin liquefaction, milk coagulation and peptonization. Tests for tyrosinase and H2S production were negative for both isolates. Isolates were not able to hydrolyze hypoxanthine. In contrast to the isolate AkhA-12, the isolate Tatev 35a was able to reduce nitrate. As sole carbon sources d-fructose and d-mannitol were utilized by both isolates, but l-arabinose and d-xylose were not. The isolate AkhA-12 was able to utilize d-glucose and d-lactose (weakly positive), but not d-inositol. In contrast to AkhA-12, the isolate Tatev 35a utilized d-inositol (weakly positive), but not d-glucose and d-lactose. Test describing utilization of propionate and citrate for both isolates showed weak positive result.

For further identification of the isolates, their 16S rRNA genes were amplified and sequenced. BLAST results of 16S rRNA gene sequences of thermoactimycetes are reported in Table 2. The isolate AkhA-12 exhibited 99.7% similarity to Thermoactinomyces sp. JIR-004 (AB899820), a strain isolated from deep subseafloor sediment samples (www.ncbi.nlm.nih.gov) and 99.4% similarity to T. daqus strain H-18 (KF590624), a thermophilic bacterium isolated from a high-temperature Daqu (Yao et al. 2014). 16S rRNA gene sequence similarities of AkhA-12 to type strains of Thermoactinomyces species were following: T. vulgaris (96.0%), T. intermedius (95.88%), T. daqus (99.38%) and T. guangxiensis (94.42%). T. khenchelensis (94.01%).
Table 2

BLAST results of 16S rRNA gene sequences of thermoactimycetes isolated from Armenian hot springs and accession numbers

Isolates

Sequence length (bp)

Closest match taxonomic affiliation, phylotype accession no.

% Similarity to closest match

Accession no.

AkhA-12

804

Thermoactinomyces sp. JIR-004, AB899820

99.7

MK418253

T. daqus strain H-18, KF590624

99.4

Tatev 35a

921

T. vulgaris strain LJTC-1 KT454966

98.9

MK418408

The isolate Tatev 35a demonstrated 98.9% similarity to T. vulgaris strain LJTC-1 KT454966, a thermophile bacterium isolated from Tangchi Spring in Lujiag (www.ncbi.nlm.nih.gov). 16S rRNA gene sequence similarities of Tatev 35a to type strains of Thermoactinomyces species were following: T. vulgaris (99.01%), T. intermedius (98.36%), T. daqus (95.19%) and T. guangxiensis (94.72%). T. khenchelensis (96.16%).

The phylogenetic tree using neighbor-joining method was constructed (Fig. 2). The phylogenetic tree confirmed that isolate AkhA-12 constitutes a part of the cluster with T. daqus strain H-18 (KF590624), while the isolate Tatev 35a were branched with T. vulgaris (AF138739) indicating their close relationship. Nevertheless, the differences on physiological and biochemical properties between new isolates and close relatives were observed (Table 1). The evidence indicates that additional studies like whole genome analysis should be applied to confirm taxonomic affiliation.
Fig. 2

Phylogenetic tree based on 16S rRNA gene sequences showing relationships between Thermoactinomyces sp. AkhA-12, T. vulgaris Tatev 35a and other Thermoactinomyces species. The optimal tree with the sum of branch length = 3.11 is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches. Scale bar represents 0.2 substitutions per site

Representatives of the genus Thermoactinomyces are commonly considered the species most frequently isolated from similar habitats (Carrillo and Benitez-Ahrendts 2014; Mokrane et al. 2016; Sahay et al. 2017). As chemoorganoheterotrophic aerobic thermophiles they are actively involved in degradation processes. In addition, thermoactinomycetes have huge input in biogeochemical cycles of carbon, sulfur and nitrogen under extreme conditions. Set of physical–chemical and edaphic conditions is decisive for natural selection of bacteria able to inhabit extreme habitats. Being metabolically very flexible and active reducers in food chain, thermoactinomycetes take part in bacterial saprotrophic complexes and promote decomposition of both autochthonous and allochthonous biopolymers of ecosystem. The results obtained indicate the importance of further investigation of geothermal springs to discover ecological role of thermoactinomycetes in extreme ecosystems and their diversity. The description of major microbial components of thermal ecosystems can be helpful to understand their role for maintaining environmental sutainability.

While these results are important to figure out microbial community structure of geothermal springs and further taxonomic work, positive results on hydrolytic activities of isolates indicate their biotechnological potency. Because of their activity at high temperatures, enzymes of Thermoactinomyces species have attracted much interest. In this sense, capacity of production of extracellular hydrolases by newly isolated strains were determined. It was shown that the isolates produced extracellular proteases, amylases and lipases (Table 3). Moreover, bacterial isolates were able to actively produce not only one, but two or even all three enzymes. Both isolates showed the stability in enzymes activities at 50–60 °C. Highest production of enzymes was observed at 55 °C. Thermoactinomyces sp. AkhA-12 demonstrated high amylase and protease activities, while T. vulgaris Tatev 35a was good producer of amylase.
Table 3

Hydrolytic enzyme production by thermoactinomycetes obtained from Akhurik and Tatev hot spring at different temperatures

Isolates

Enzyme activitya

Protease

Amilase

Lipase

50 °C

55 °C

60 °C

50 °C

55 °C

60 °C

50 °C

55 °C

60 °C

Thermoactinomyces sp. AkhA-12

+

+++

++

+

+++

+++

+

++

+

T. vulgaris Tatev 35a

++

++

++

++

+++

++

+

++

+

aEnzyme activity was expressed by diameter of clear zones (in case of protease and amylase) and precipitation (in case of lipase) around colonies: (< 5 mm; +) (5–10 mm; ++) (> 10 mm; +++)

Obtained results are in good agreement with results achieved by other scientists demonstrating thermostable production of hydrolases by thermoactinomycetes (Aksoy et al. 2012; Jadoon et al. 2014; Verma et al. 2016; Sahay et al. 2017). The thermostable hydrolases have found applications in various industries due to their feature to act in extreme conditions (Kambourova 2018; Han et al. 2019).

The hydrolase producers belonging to representatives of genus Bacillus and related genera (Anoxybacillus, Brevibacillus, Geobacillus, Paenibacillus, Sporosarcina and Ureibacillus) isolated from Armenian geothermal springs have been well characterized recently (Panosyan 2017; Panosyan et al. 2018). Shahinyan and coauthors (Shahinyan et al. 2017) have reported primary structures of lipase protein of lipase-producing strains B. licheniformis Akhurik 107 and Geobacillus sp. Tatev 4 isolated from Akhurik and Tatev mesothermal springs, respectively. Despite this progress, similar attention has not been paid to thermoactinomycetes and their enzymes.

Although the main producers of hydrolases are still representatives of the genus Bacillus and related genera (Yadav et al. 2018), Thermoactinomyces strains could be realistic alternatives for production of resilient enzymes.

This work fills the gap regarding distribution and diversity of hydrolase producing thermoactinomycetes inhabiting geothermal springs in Armenia.

Conclusions

Two hydrolases producing microbes designated as Thermoactinomyces sp. AkhA-12 and T. vulgaris Tatev 35a were isolated from sediment samples of Armenian mesothermal springs. Positive results on amylase, protease and lipase activities are indicative of potential application of isolates in various biotechnologies. Obtained results show the importance of further exploration of thermoactinomycetes’ diversity in Armenian hot springs in order to isolate new strains with biotechnological potency. Extracellular thermozymes acting at harsh temperature conditions serve molecular models to reveal adaptation mechanisms of thermophiles. Studied hot springs are used by local people for many purposes like bathing or householding. Such anthropogenic pressure negatively effects on diversity of ecosystems. Thus, study and conservation of microbial diversity of thermal ecosystems is an urgent action to maintain the valuable resources and for the environmental sustainability.

Notes

Acknowledgements

This work was supported by the RA MES State Committee of Science, in the frames of the research projects no 15T-1F399 and no 18T-1F261, Armenian National Science and Education Fund based in New York, USA, to HP ANSEF-NS-microbio 4676 Grants.

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Copyright information

© Society for Environmental Sustainability 2019

Authors and Affiliations

  1. 1.Department of Biochemistry, Microbiology and BiotechnologyYerevan State UniversityYerevanArmenia

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