2.1 Molecular Identification of Species

A total of 41 isolates were selected according to the medium used for their isolation including actinomycetes-specific media: actinomycetes isolation agar, starch casein agar, international Streptomyces project-2 medium and Reasoner’s 2A agar. DNA of these isolates was isolated using the Invitrogen^TM PureLink^TM Genomic DNA Mini Kit K182001 (Invitrogen^TM, USA) according to the manufacturer’s procedure. To amplify the 16s rRNA gene, the polymerase chain reaction (PCR) was run using universal primers 27F (5’-AGA GTT TGA TCC TGG CTC AG-3’) and 1492R (5’- ACG GCT ACC TTG TTA CGA CTT-3’) under these conditions: initial denaturation at 94°C for 30 s, followed by 30 cycles of 94°C for 30 s, 58°C for 30 s, and 72°C for 60 s, then a final extension step was performed at 72°C for 10 min. The resulting PCR products were run on 1% agarose gel by electrophoresis before sequencing using the Sanger DNA sequencing method.

2.2 Screening for Non-Ribosomal Peptide Synthases and Polyketide Synthase Genes in Actinobacteria from Parsık Cave

Primers coding the NRPS and PKS gene regions of bacteria were used to screen the enzyme-encoding gene clusters in the identified Actinobacteria. They consist of A7R (5′-SASGTCVCCSGTSCGGTAS-3′) / AF3 (5′-GCSTACSYSATSTACACSTCSGG-3′) for NRPS gene cluster and DegKS2R (5’-GTICCIGTICCRTGISCYTCIAC-3’) / DegKS2F (5’-GCIATGGAYCCICARCARMGIVT-3’) for PKS gene cluster (23). PCR was run following the methods proposed by Amos et al. (23) with some modification. For the A7R/AF3 primers, PCR was run under the following conditions: primary denaturation at 95°C for 5 min followed by 35 cycles (denaturation at 94°C for 35 s, annealing at 55°C for 40 s and extension at 72°C for 1 min) and final elongation at 72°C for 8 min. PCR for the PKS region was run under the following conditions: primary denaturation at 94°C for 4 min followed by 35 cycles of (denaturation at 94°C for 35 s, annealing at 55.6°C for 40 s and primary extension at 72°C for 75 s) and elongation at 72°C for 8 min. The PCR products were run in a 1.2% gel electrophoresis to visualise the bands.

2.3 Antimicrobial Production Potentiality of the Isolates

Identified Actinobacteria were tested against selected standard microorganisms constituted of pathogens or opportunistic pathogens of Gram-negative and Gram-positive bacteria and one fungi strain: Escherichia coli (ATCC^® 8739^TM), methicillin-resistant Staphylococcus aureus (MRSA) (ATCC^® 33591^TM), Pseudomonas aeruginosa (ATCC^® 9027^TM), Staphylococcus aureus (ATCC^® 6538^TM), Staphylococcus epidermidis (ATCC^® 12228^TM) and C. albicans (ATCC^® 10231^TM). Fresh culture of each isolate was used to prepare 0.5 McFarland and 100 μl of each suspension was spread onto 1/2 tryptic soy agar. The incubation was at 30°C for 48 h. 0.5 McFarland of each standard microorganism was also prepared and 100 μl of each suspension was spread to tryptic soy agar medium and incubated at room temperature for 3–5 min. After that, discs of the isolates (6 mm) were cut off from the 48 h cultures and inversely placed onto the plates containing standard microorganisms. After 24 h at 30°C, every standard Petri plate was observed for the inhibition zone around the isolate’s discs. Isolates with phenotypical antimicrobial activities were selected for characterisation of bioactive compounds.

2.4 Characterisation of Bioactive Compounds

Fresh cultures were inoculated on tryptic soy broth for 24 h. The broth was chromatographically separated using an Agilent 1260 Infinity II LC System (Agilent Technologies Inc, USA) instrument and Agilent Poroshell 120 EC-C18 Threaded Column (3.0 mm × 150 mm, 2.7 μm particle size). A gradient elution of 5 mM ammonium formate in water (A) and methanol (B) was used to construct the mobile phase system. This was as follows: 0–0.5 min, 10% B; 0.5–5 min, 70% B; 5–7 min, 95% B; 7–10 min, 95% B; 10–15 min, 10% B. Sample injection was 2 μl with a flow rate of 0.5 ml min^–1 and column oven maintained at 25°C.

Mass spectroscopy was performed using an Agilent 6550 iFunnel equipped with the high‐resolution accurate mass quadrupole time of flight/mass spectroscopy sheath gas flow (nitrogen 12 l min^–1) and the selected scanning range m/z was between 50 and 1800. Samples were screened using Agilent MassHunter METLIN Metabolite Personal Compound Database and Library (Metlin_AM_PCDL.cdb).

2.5 Screening of Enzymatic Activities of Parsık Cave Actinobacteria

The isolates were screened for seven enzymatic activities: amylase, lipolytic activity (Lip.A), cellulase, casein hydrolase (Cas.Hydrolase), DNase, urease, proteinase and gelatinase. Except for DNase and gelatinase activities, a fresh culture of each isolate was inoculated in media composed of minimal salt agar medium and enzyme specific substrates. 1000 ml of minimal salt agar medium was composed of 0.1 g NaCl, 1 g KH₂PO₄, 0.1 g MgSO₄, 5 g (NH₄)₂SO₄ and 15 g agar. For DNase and gelatinase activities, respectively, DNase test agar and gelatine were directly used. During the incubation period (3–10 days) at 30°C, the growth of isolates was checked daily and clear zones, growth or change in the colour were checked for positive or negative activities (9).

3.1 Identification of Isolates

The identified species in our study are presented in Table I. From the 41 selected isolates, 28 have been identified as Actinobacteria. The remaining isolates are constituted of Proteobacteria, Firmicutes and three others which were not identified in the National Center for Biotechnology Information (NCBI, Table I). As previously mentioned, the bacterial diversity of sediment and surface samples from different karstic caves has been reported with a high rate of Actinobacteria (10–12). Some Actinobacteria strains like Streptomyces species are known for their role in the formation of caves and cave speleothems (14, 17). This can be achieved through their metabolic reactions in the presence of some chemicals in the cave ecosystems (14). Genera like Nocardia and Rhodococcus identified in our study have been reported with potential for degradation of hazardous chemicals and organic matter (24, 25). They may play an important role in the bioremediation of natural ecosystems and polluted environments.

3.2 Screening of Antimicrobial Production Potential of the Isolates

Our study aimed to determine the bioactive potentials of Actinobacteria isolated from Parsık Cave. 53% of the isolates showed bands for NRPS gene after running their PCR products on an agarose gel electrophoresis. 32% of the identified Actinobacteria showed bands for PKS gene after running their PCR products on an agarose gel electrophoresis. Isolate P1 showed a band for both regions while neither region was amplified in P16. These genes encode enzyme groups that play an important role in the synthesis of different bioactive compounds including some with antimicrobial, antioxidant and anticancer activities (26–28). These observations bring us back to the potential uses of Actinobacteria isolated from the Parsık Cave in the biotechnological and pharmaceutical industries.

However, further tests are recommended to identify the different biological activities of these isolates. One of these tests is the antimicrobial production assay. This test was done by testing the phenotypical response of the isolates against some standard bacteria and one Candida species. Only two isolates showed antimicrobial activity. These were the P1 and P16 isolates. The isolate P1, identified as Streptomyces exfoliatus, showed antimicrobial activity against the fungal strain C. albicans. Antimicrobial potential in Streptomyces species has been observed previously in different studies with Streptomyces isolated from different environments (29–32). Sharma et al. showed through in vitro analyses the antifungal potential of Streptomyces exfoliates MT9 against wood rotting fungi (33). Inhibition zones were observed around isolate P16, identified as Micrococcus sp. TM4_2, when tested against S. aureus and S. epidermidis. The growth inhibition of the pathogen S. aureus and the opportunistic pathogen S. epidermidis by isolate P16 shows the antibacterial potential of this isolate and promises biomedical uses for this isolate and its extracts.

Results observed through the genotypic test, contrary to the antimicrobial tests, showed high potential bioactive compound production in our isolates. This great difference between the phenotypical and genotypical results may be explained through different hypotheses such as the laboratory conditions (culture medium or temperature) which may not be favourable to phenotypical expression in the other isolates. Besides that, these isolates may show antimicrobial activities against other standard microorganisms which were not used in our study. The presence of biological activities other than antimicrobial activity like anticancer or antioxidant activity may also explain this observation.

3.4 Parsık Cave as a House of Actinobacteria with High Diversity of Enzymes

The enzymatic activities of microorganisms indicate which organic compounds are present in the studied ecosystem. In addition, studying these activities allows us to understand the potential use of these microorganisms for human or other applications. Enzymes can be used in environmental bioremediation and in other industries such as detergents, cosmetics, leather and textiles production. Different enzymatic activities from cave bacteria have been reported in different studies. Using enzymes secreted by psychrotolerant bacteria is thought to be economically valuable to save energy in large scale applications (9). Our isolates showed different enzymatic activities (Table I).

The physiological role of amylase enzyme, which is mainly used in the textile, food, fermentation and paper industries, is to hydrolyse starch molecules into dextrins and glucose molecules (36). It is secreted by different biological sources of which the most important is the Streptomyces species from Actinobacteria (36). 29% of our isolates showed amylase activity. Different strains of these isolates showed clear zones around their colonies after incubation in starch agar plates and staining with iodide. They include S. exfoliates, Arthrobacter humicola, Kocuria rosea and Oerskovia turbata NBRC 15015 (Table I).

Protease activity was also tested in our study. Proteases are a group of enzymes directed to hydrolyse the peptide bonds of protein molecules into amino acid chains or simple peptide molecules (37). They are synthesised from different sources including plants, animals and microorganisms. They are used in different industrial and biotechnological areas including waste treatment, pharmaceuticals, detergent production, photographic and food industries (37, 38). Microbial proteases are obtained both from fungi and bacterial sources. Most of the fungi proteases are acidic while the bacterial ones are mainly active in alkaline environments (39). Proteases from Actinobacteria are reported from different strains including Streptomyces and Nocardia isolated from different ecosystems (39).

In our study, protease activity was tested through gelatin and casein hydrolysing profiles of the isolates. 5% of our isolates showed gelatinase activity while 48% showed casein hydrolase activity (Table I). The species identified as Nocardia and Nocardioides showed gelatin hydrolysing activity while those identified as Streptomyces, Nocardia, Nocardioides, Microbacterium, K. Rosea and Arthrobacter showed protease activity by hydrolysing casein molecules. These isolates might be considered candidates for the previously mentioned areas as they can produce cold-tolerant and alkaline protease enzymes in oligotrophic areas.

Among the investigated enzymatic activities in this study is the ureolytic activity. It consists of the degradation of urea molecules to generate ammonia which provides an alkaline environment at the end of the reaction (40). The urease activity of cave microorganisms has been previously demonstrated from karstic cave isolates and they are often studied for their possible significant role in cave and speleothem formation (9, 19, 40). Cave bacteria that show urease activity mostly include strains of Proteobacteria, Firmicutes and Actinobacteria (4, 9, 19). Some Streptomyces species isolated from moonmilk of the Springtails’ Cave were reported with ureolytic activity through phenotypical and genotypical analyses (19). Micrococcus luteus from Magura Cave, Bulgaria, also showed pink colour after incubation on urea-based agar plates, related to the urease secretion ability of the bacteria (9). The precipitation of calcium by bacteria is thought to take place through different mechanisms which include the urease reaction (9). The isolation of urease positive bacteria and their in vitro calcium precipitation ability were shown in previous cave microbiological studies (4).

In our study, 65% of the selected isolates showed positive results for urease activity. They include species of Streptomyces, Arthrobacter, Propionicimonas, Microbacterium and Nocardioides. These bacteria may play a significant role in construction activities like self-healing concrete production or concrete bioremediation. In addition, they may be used to remove calcium ions from wastewater. The calcium precipitation ability of these isolates should be the subject of future studies for further industrial and biotechnological applications.

Three of the identified Actinobacteria (S. exfoliatus, O. turbata NBRC 15015 and K. rosea), which represent 9.7% of our isolates, showed positive lipolytic activity in the basal agar medium supplemented with 1% Tween 80 and CaCl_2·2H₂O (Table I). They formed precipitation zones around their colonies that indicate the complex formed by liberated fatty acids and calcium present in the media. Different Actinobacteria isolated from the Hampoeil Cave, Iran, were also reported with esterase activity which is one of lipolytic enzymes (41). In our previous study in Parsık Cave, only strains of Proteobacteria were identified with lipolytic activity through the VITEK^® test (11). In the present study, species of the same genera show different results. This suggests the lipolytic activity of the bacteria isolated from the same cave (same and different points) may be related to the substrate. Furthermore, it may be necessary to study the activities from different isolation points in order to establish the different hydrolytic activities shown by the ecosystem isolates. Lipolytic enzymes are used in industrial fields such as detergent production (11, 41). Such lipolytic enzymes isolated from cold-tolerant bacteria might be preferred in detergent production to reduce energy use during washing.

Two of our identified Actinobacteria showed a clear zone around their colonies incubated on DNA agar plates for five days (Table I). This zone was formed after flooding 1 N hydrochloric acid on the agar plate. The clear zone is caused by the reaction of DNase secreted by the isolates on the DNA (depolymerisation) present in the medium. DNase enzymes are known for different functions depending on the microbial sources: they can cause disruption of biofilms or play a role in bacterial predation, inhibition of natural transformation or the recovery of nutrients such as carbon and phosphate (42, 43). DNase activity was previously reported from Actinobacteria species isolated from other cave ecosystems (9, 43).

Cellulases are hydrolytic enzymes that hydrolyse cellulose by hydrolysing the B-1,4 glycosidic bonds into sugars (44, 45). These enzymes are important in different applications such as the degradation of cellulosic waste, bioethanol production, textile polishing and the production of detergent, food and feed (44, 45). A diversity of bacteria including Actinobacteria isolated from different ecosystems are reported with cellulase secretion ability (44, 45). In our study, three of the identified Actinobacteria (P1, P21 and P26 isolates) showed positive results at the end of a cellulase enzyme test (Table I). Their culture on carboxymethylcellulose agar plates were stained with Congo red dye, showing a yellow hallo against a red background. Interestingly, most of our isolates did not grow on the carboxymethylcellulose agar plates. This may be explained by inappropriate incubation conditions for most of the isolates. Furthermore, the medium used was not favourable for most of these oligotrophic isolates which may cause eutrophication.

The isolate P1 which was identified as S. exfoliatus exhibited the greatest enzymatic potential (Table I). As previously shown, most actinobacterial species that exhibit high bioactive compound potential belong to the genus Streptomyces (46). A strain of S. exfoliates isolated from a soil environment in Saudi Arabia has been analysed with potential lipase and protease activities (47). In addition, in our study, both PKS and NRPS cluster genes were amplified in this isolate. This result suggests the high potential for bioactive compounds of this isolate. The isolate P1 should be further investigated as a potentially industrially useful Actinobacteria from Parsık Cave.