Exploration of Metagenomic Sequences Derived from Bacterial Assemblages in the Hypersaline Lonar Lake of India: Revelation of Distinctive Genomic Signatures Encoding Pharmaceutical Significance

Pradip P. Bawane^1,2, Santosh Yele²*

¹Department of Pharmacognosy, SVKM's NMIMS,

Shobhaben Pratapbhai Patel School of Pharmacy and Technology Management, Mumbai, 400056, India.

²Department of Pharmacognosy,

Shri Vile Parle Kelavani Mandal's Institute of Pharmacy, Dhule, India – 424001.

³Department of Pharmacognosy, SVKM's NMIMS,

School of Pharmacy & Technology Management, Telangana, Hyderabad, 509301, India.

*Corresponding Author E-mail: santoshyele@gmail.com

ABSTRACT:

Background: Lonar Soda Lake, formed in basaltic rock by a meteorite impact, possesses unique geochemical conditions that support microorganisms with distinct metabolic capabilities, including antibiotic, biosurfactant, and biofuel production. Exploring the genetic basis of these capabilities can uncover novel compounds and biotechnological innovations. Aim: To analyze metagenomic sequences from bacterial communities in Lonar Lake to identify genetic signatures associated with antibiotic production, biosurfactant synthesis, and biofuel pathways. Methods: Metagenomic DNA was extracted from Lonar Lake samples and sequenced using high-throughput techniques. Bioinformatic analyses identified genes related to antibiotic production, biosurfactant synthesis, and biofuel pathways. DNA extraction was performed using the Qiagen gDNA kit and quantified with the Qubit dsDNA HS Assay kit. 16S rRNA gene sequencing libraries were prepared with the Ion 16S Metagenomics Kit and sequenced on the Ion Torrent S5 system. Results: Metagenomic analysis revealed a highly diverse bacterial community, comprising 19 phyla. Dominant phyla included Proteobacteria (48.12%), Firmicutes (26.44%), and Actinobacteria (0.98%). We identified 31 Actinomycetes families and 161 species, including industrially significant genera such as Pseudonocardia, Saccharomonospora, Actinomadura, Amycolatopsis, Nocardia, and Saccharopolyspora. Notable species included Mycobacterium salmoniphilum, Microbispora rosea, and Nocardiopsis alba. Genomic signatures for antibiotic production, biosurfactant synthesis, and biofuel pathways were identified. Conclusion: The metagenomic analysis of Lonar Lake bacterial communities highlights their potential for novel antimicrobial, antiviral, biosurfactant, and biofuel production, offering promising avenues for biotechnological applications and industrial wastewater treatment.

Graphical Abstract:

Graphical Abstract. Exploration of Metagenomic Sequences Derived and their Applications from Bacterial Assemblages in the Hypersaline Lonar Lake of India

KEYWORDS: Bacterial Metagenomics, Lonar Soda Lake, Metagenomics Rapid Annotations using Subsystems Technology, Actinobacteria, Next Generation Sequencing Platform.

1. INTRODUCTION: 

These days, metagenomics methods such as functional gene sequencing and sequencing analysis make it feasible to research the microbial population from many sources¹. Highlights regarding the genetic and evolutionary relationships, functional activities, community profile, microbial diversity, and relationship with the environment are provided by metagenomics investigations^1-3. Since most studies on microbes have concentrated on cultivable microbes, it is extremely difficult to understand the structure, dynamics, and function of microbial consortia in natural environments^4,5. To address this, alternative methods, or culture-independent methods, are needed to investigate the realm of microbes that cannot be cultured. Recent advances in NGS have opened up new possibilities for research on the variety of bacteria in different environments through the discovery of the functional roles of microbes^5,6. This research can be conducted by parallel sequencing of distinct samples. High-throughput sequencing technologies like Next Generation Sequencing (NGS) are used to assemble microbial genome sequences quickly^6,7.

Lonar Lake is situated at a latitude and longitude of 19°59′N and 76°31′E, respectively, in the Buldhana district of Maharashtra, India. The Lonar Lake is the only lake created by a hypervelocity meteoritic impact in basaltic rock^8,9. The present study is designed to explore the microbial diversity of the lake in composite samples¹⁰. The outcome of this metagenomics study will be insightful for the researcher to explore the microbial diversity to obtain the scientifically important microbial population associated with pharmaceutical and industrial applications. Our understanding of the distinctive community makeup of this complex and exceptional lake ecosystem will be improved by using metagenomics studies using next-generation sequencing to provide a complete picture of the bacterial diversity and community structure in the Lonar Lake ecosystem.

The metagenomic data of the composite sample is uploaded on NCBI Bioproject with Biosample accession: SAMN35564812. The findings of this research have the potential to contribute significantly to the field and advance knowledge in microbial assemblages of extreme ecology.

The present study is designed to explore metagenomics sequences derived from bacterial assemblages in the Hypersaline Lonar Lake of India. To discover distinctive genomic signatures encoding antibiotics, biosurfactants, and biofuel synthesis. The outcome of this metagenomics study will be insightful for the researcher to explore the microbial diversity to obtain the scientifically important microbial population associated with the production of antibiotics. Our understanding of the distinctive community makeup of this complex and exceptional lake ecosystem will be improved by using metagenomics studies using next-generation sequencing to provide a complete picture of the bacterial diversity and community structure in the Lonar Lake ecosystem.

2. MATERIALS AND METHODS:

2.1 Location Description, Sampling Point, and Sample Optimization:

The sample was collected from six different sampling points in sterile glass bottles and transported to the laboratory in ambient conditions (kept in a thermostatic box with ice packs below 25°C) from Lonar Lake, India. The sampling was done in May, September, and December 2021 as part of a seasonal sample collection. A total of six samples, each of water and sediment, were collected from different sampling points (Figure 1). The samples were mixed to obtain a composite sample. The samples were stored in the refrigerator at 4°C - 8°C. The water pH and temperature at the time of collection were recorded.

Figure 1. Google image of Sampling site Lonar Lake, India, Buldhana District of Maharashtra, latitude and longitude of 19°59′N and 76°31′E with sampling points

2.2 DNA Extraction and Ion Torrent Sequencing:

Qiagen gDNA kit was used to isolate DNA, and the quality of gDNA was checked on a 0.8% agarose gel (loaded 5μl) for a single intact band. The gel was run at 110 V for 30 minutes. The sample of 2μl was loaded in the BioTek Epoch to determine the A260/280 ratio. The DNA was quantified using a Qubit dsDNA HS Assay kit (Life Tech). The concentration was determined by Qubit® 2.0 Fluorometer. The DNA was amplified using the Ion 16S Metagenomics Kit (Thermo Fisher Scientific, MA, USA). The kit includes two primer sets that selectively amplify the corresponding hypervariable regions of the 16S region in bacteria (primer set V2-4-8 and primer set V3-6,7-9). After amplification, the reactions obtained from each sample with the two primer sets were pooled and mixed with Agentcourt AMPure XP reagent (Beckman Coulter, CA, USA) for purification, using a magnetic rack as described by the manufacturer's workflow. Targeted 16S sequencing libraries were prepared using the Ion 16S Metagenomics Kit (Thermo Fisher Scientific, MA, USA) in combination with the Ion Plus Fragment Library kit (Thermo Fisher Scientific, MA, USA) and loaded on a 530 chip and sequenced using the Ion Torrent S5 system (Thermo Fisher Scientific, MA, USA). The protocol was designed for microbiome analysis using Ion Torrent 510/520/530/540 Kit-chef template preparation system (Thermo Fisher Scientific, MA, USA) and included two primer sets that selectively amplified seven hypervariable regions (V2, V3, V4, V6, V7, V8, V9) of the 16S gene. The equimolar pool of libraries was estimated using an Agilent Bioanalyzer 2100 using a DNA1000 chip (Agilent Technologies, CA, USA)^11-13. The sequence statistic for the composite sample of Lonar Lake is summarized in Table 1.

Table 1. Sequencing Statistics

bp Count	221,138,786 bp
Total number of reads	1,03,0418
Mean Sequence Length	215 ± 69 bp
Mean GC percent	56 ± 3 %
Number of valid reads	6,22,677
Number of reads ignored	2,51,151 (due to a low number of copies <10)
Mapped reads in a sample	3,71,192
Unmapped reads in the sample	334

3. RESULTS:

3.1 Bacterial Composition – Ion Reporter Analysis:

The bacterial composition of the sample was found to be highly diverse, comprising a total of 19 bacterial phyla. Proteobacteria (48.12%) were the most abundant phylum. Firmicutes (26.44%), unclassified bacteria (18.75%), indicating the existence of some novel and unidentified bacterial community, Arthropoda (2.09%), Actinobacteria (0.98%), Chlorobi (0.93%), Bacteroidetes (0.80%), followed and other phyla are below 0.1% abundance (Figure 2 A). At the family taxonomic level, 87.7% of the species were successfully classified. Halomonadaceae (42.92%), unclassified derived from bacteria (21.52%), Clostridiaceae (14.94%), Bacillacae (4.63%), Rhinotermitidae (2.39%) were among the most abundant families (Figure 2B).

Figure 2 A

Figure 2 B

Figure 2 A. Phylum Diversity Classification of Lonar Lake composite sample, and Figure 2 B. Family Level Classification of the Composite Sample of Lonar Lake

Among the classes, the report reveals the dominance of Gammaproteobacteria (47.18%), other major classes include unclassified sequences (19.62%), Clostridia (17.84%), Bacilli (6.46%), and Insecta (2.18%). The most abundant species (Extremophiles) found in the sample based on read counts are Halomonas mongoliensis (43670), Halomonas pantelleriensis (31175), Methylonatrum kenyense (23224), Halomonas shengliensis (10463) and Alkaliphilus metalliredigens (7483). The microbial diversity concerning bacteria and archaea is summarized by the Krona chart and OTU Sequence count in Figures 3A and 3B, respectively.

Figure 3A

Figure 3b

Figure 3A: Krona chart indicating relative abundance of the bacterial community in a composite sample of Lonar Lake, and Figure 3B. OTU Indicating distribution of major families Present in the composite sample of Lonar Lake

3.2 Alpha Diversity Classification:

Variations of microbes present in the composite sample, species richness, and diversity were studied by plotting the rarefaction curve in Figure 4. The rarefaction curve was plotted by the number of species against the number of samples. This curve was created by randomly resampling the pool of N samples several times and then plotting the average number of species found on each sample. It initially grows rapidly (as the most common species are found) and then slightly flattens (as the rarest species remain to be sampled). It indicates that many rare species may exist in the composite sample from Lonar Lake. The variation in the species may be due to some genetic modification occurring because of the unusual ecology of the lake.

Figure 4. Rarefaction curve variation at the Genus and Species level

4.3 Actinobacteria – MG-RAST Analysis

Looking into Actinomycetals commonly referred to as Actinomycetes, MG-RAST has predicted and classified 31 families of Actinomycetes and a total of 161 on the species level (Figure 5). The most abundant species are Mycobacterium salmoniphilum (517), Microbispora rosea (422), Nocardiopsis alba (394), Nocardiopsis metallicus (326), Kribbella jejuensis (259), Acidothermus cellulolyticus (158). Mycobacterium salmoniphilum is a fish pathogen that causes mycobacteriosis in cold-water living fish, especially in salmonids ¹⁴. Microbispora rosea is among the rare thermophilic actinomycetes that exhibit antibacterial activity and are producers of secondary metabolites. Genus Microbispora, a member of the family Streptosporangiaceae, was recognized for its role in providing humus and nutrients for plants through the biodegradation process.

Figure 5. Actinobacterial Diversity of Lonar Lake Ecosystem

It also contributed as a biocontrol agent by producing bioactive compounds against plant pathogens. Other potent secondary metabolites known to be produced by the members of the genus Microbispora are antibiotics, enzyme inhibitors, and antidiabetic compounds¹⁵. Several other known genera of (thermophilic) actinomycetes are found in the sample, i.e., Pseudonocardia, Saccharomonospora, Actinomadura, Amycolatopsis, Nocardia, Saccharopolyspora, etc. Antibiotic-producing rare genera were also found in the sample such as Streptomyces and Actinomadura¹⁶. Streptomyces carpaticus, a new species, produced exopolysaccharide which exhibits free radical scavenging activity and antitumor activity against both breast and colon cell lines¹⁷. It was also reported that one of the strains of Streptomyces carpaticus contains antioxidant and antimicrobial compounds, which could be potentially used for the aquaculture industries¹⁸. The identified reads for genus and species, probable applications for the production of antibiotics, biofuel, and biosurfactant are summarized in Table 2.

Table 2. Identified Reads for Genus and Species Probable Application for Production of Biomolecules of Pharmaceutical Significance

S. No	Genus	Species	Number of Reads	Compounds Produce	Applications
1	Microbispora	Microbispora rosea	422	β-Carbolins and Indoles	Antibacterial
2	Pseudonocardia	Pseudonocardia xinjiangensis	55	Macrolides	Antibacterial
3	Saccharomonospora	Saccharomonospora glauca	43	β-Lactamases	Cytotoxic
4	Actinomadura	Actinomadura formosensis	32	Genus reported to produce chandrananimycins A, B, and C,	by   Genus is known to produce cytotoxic compounds, Antibiotics, and antifungals.
5	Amycolatopsis	Amycolatopsis lactamdurans	31	Genus reported to produce Mutactimycin A, D, E, Amycolactam, Rifamorpholines A–E, Dipyrimicins A and B, Amycophthalazinone A	by   Genus is known to produce cytotoxic compounds, antibiotics, and antimicrobials.
6	Nocardia	Nocardia miyunensis	27	Species not explored for the production of metabolites. Genus reported to produce Nocardicins, nargenicin, Tubelactomycin A, brasilinolide A, brasilidine A	Antibacterial, antifungal, anti-mycobacterial, immunosuppressive, cytotoxic.
7	Saccharopolyspora	Saccharopolyspora pogona	8	Butenyl-spinosyn	Antibiotic, Insecticide,
8	Streptomyces	Streptomyces carpaticus	5	Unidentified compounds (polyphenolic bioactive metabolites)	Antimicrobial, antiviral, antioxidant etc.
9	Streptomyces	Streptomyces macrosporus	3	Unidentified compounds	Antibiotics, vitamins, enzymes etc.
10	Streptomyces	Streptomyces aureofaciens	2	Tetracycline, auricine	Antibiotic
11	Streptomyces	Streptomyces fulvorobeus	2	1-epimanzamine D, Relative species reported to produce Chromomycins	Antibiotic
12	Streptomyces	Streptomyces griseus	2	Streptomycin	Antibiotic
13	Streptomyces	Streptomyces lienomycini	2	Unidentified compounds	Antimicrobial
14	Kutzneria	Kutzneria viridogrisea	1	Unidentified compounds genus reported to produce epemicins A and B, phenol, 2,4-bis (1,1-dimethylethyl)	Antibiotic, antifungal
15	Streptomyces	Streptomyces caeruleus	1	Caerulomycin, Caerulomycin A	Antibiotic, antifungal
16	Streptomyces	Streptomyces fradiae	1	Neomycin, Tylosin A	Antibiotics
17	Streptomyces	Streptomyces nitrosporeus	1	Virantmycin, Nitrosporin, Benzastatins A, B, C, and D	Antiviral, antibiotic, antioxidant
18	Streptomyces	Streptomyces stelliscabiei	1	Rotihibin C and D	Antibacterial
19	Amycolatopsis	Amycolatopsis mediterranei	1	Rifamycin SV	Antibiotic

4. DISCUSSION:

Lonar Lake is a unique geological formation resulting from a meteorite impact in basaltic rock approximately 52,000 years ago. It is the only known hypersaline soda lake situated on basalt. Lonar Lake presents an exceptional and ideal environment for studying microbial diversity. In earlier research, scientists have mainly focused on exploring the microbial communities in Lonar Lake through culture-dependent methods. These studies have provided valuable insights, but they are limited by the inability to culture many microorganisms in laboratory conditions. Consequently, a significant portion of the lake's microbial diversity remains unexplored. Additionally, changes in microbial communities have been observed, which are thought to be affected by seasonal variations in environmental conditions, such as fluctuations in salinity, pH, and nutrient availability.

Metagenomics offers a detailed understanding of microbial diversity and functional potential by sequencing and analyzing the genomes of all microorganisms present in a sample. This analysis is particularly effective for studying complex microbial ecosystems, such as those present in Lonar Lake. The unique environmental conditions of Lonar Lake are likely to harbor microorganisms with distinctive metabolic capabilities, like microbial arrays involved in the production of antibiotics, biosurfactants, and biofuels. These microbial products have significant industrial and pharmaceutical applications, ranging from the development of new antimicrobial agents to the production of environmentally friendly biofuels and biosurfactants. Understanding the genetic basis for these capabilities can lead to the discovery of novel compounds and biotechnological innovations. In this study, we employ metagenomic sequencing to explore the bacterial assemblages in water and sediment samples from Lonar Lake. By analyzing the genomic sequences, we aim to uncover the diversity of bacterial communities and identify genomic signatures associated with the synthesis of antibiotics, biosurfactants, and biofuels.

This research will enhance our understanding of the microbial ecology of hypersaline environments and help to explore the potential for discovering novel bioactive compounds and biotechnologically relevant genes. Furthermore, we intended to explore the microbial diversity of Lonar Lake by the Metagenomic Rapid Annotations using Subsystems Technology (MG-RAST) and the Next Generation Sequencing Platform (NGS).  Previous studies reported that the microbial diversity of the Lonar Lake water and sediment is different and dominated by certain phyla of bacteria. The composite sample obtained from the water and sediment was evaluated for the first time. The composite sample shows the presence of Halomonas mongoliensis and Halomonas kenyensis are denitrifying bacteria, true alkaliphiles, and grow in the pH ranges of 8.0–10.5 and 7.5–10.6, respectively. They are facultative anaerobes with an oxidative type of metabolism, able to utilize a wide range of organic substrates and reduce nitrate, nitrous oxide, and, to a lesser extent, nitrite to gaseous nitrogen ^[19]. Halomonas pantelleriensis is a Gram-negative haloalkaliphilic rod that shows optimal growth in high salt concentrations (3–15% w/v), displaying optimal growth between pH 9 and 10. To survive in these harsh conditions, extremophiles have developed several strategies that allow the microorganisms to thrive. Polyphasic approaches were used to determine the natural taxonomic position of species belonging to the genus Halomonas, including 16S rRNA and 23S rRNA genes ²⁰.

One of the important classes of bacteria, Actinobacteria, is found to be 10% in the sample, i.e., 8140 reads. Among these 0.22% (809 reads) are Actinomycetes. Composite samples collected were analyzed for actinomycete diversity using culture-independent techniques. Seven operational taxonomic units (OTUs) were identified on a family level, which are Cellulomonadaceae, Glycomycetaceae, Kineosporiaceae, Nocardioidaceae, Nocardiopsaceae, Pseudonocardiaceae, and Streptosporangiaceae. Two bacteria are identified on the species level, i.e., Nocardiopsis halophila and Nocardiopsis metallicus. Nocardiopsis metallicus is a metal-mobilizing, alkaliphilic bacterium reported to exhibit metal leaching activities²¹. Actinobacteria (Actinomycetes) are gram-positive, facultative anaerobic, fungus-like filamentous bacteria. They are extensively distributed in the natural habitat and are involved in different biological and metabolic processes, such as useful for producing extracellular enzymes. In addition, almost 90% of the Actinomycetes genera have been isolated from soil, which is innocuous for different fields: industrial and pharmaceutical sectors²².

5. CONCLUSION:

The present investigation deals with a composite sample of habitats from Lonar Lake from a microbiological perspective. Extensive 16S rRNA gene sequencing using the high-throughput Ion Torrent platform demonstrated a vast and varied outlook of microbial biota with the predominance of the phyla Proteobacteria, Firmicutes, Arthropoda, Actinobacteria, and unclassified sequences.  MG-RAST analysis provides breakthroughs concerning the microbial community of the Lonar lake, specifically concerning the pharmaceutically and industrially important species of Actinobacteria present in this extreme ecology. From the existing research data, many members of the bacterial community of Lonar Lake are well-identified and reported. The unclassified sequences which found to be about 18.75% of the phyla and 21.52% of the bacterial family, indicate that an infinite number is yet to be identified and explored further. The unclassified sequences designate uncommon, modified, and unexplored microbial species present in the Lonar Lake ecosystem. The Lonar Lake ecosystem indicates the seasonal variation and dominance of different bacterial diversity, which needs to be discovered with a systematic scientific approach to achieve the isolation of potential species from this unique ecology. The unexplored strains of Actinobacteria from this unique soda lake may come up as a leading solution for the treatment of infections caused due to Multidrug Resistant species of microorganisms. The actinomycetes from Lonar Lake need the attention of the researcher for their exploration in the production of antibiotics, bio-surfactants, biofuels, and many more including industrial waste treatment.

6. LIST OF ABBREVIATIONS:

DNA	Deoxyribonucleic acid
gDNA	Genomic Deoxyribonucleic acid
16S rDNA	Ribosomal Deoxyribonucleic acid
DNASIP	DNA stable-isotope probing
16S rRNA	16S ribosomal RNA
ITS	Internal Transcribed Spacer
NCBI	National Center for Biotechnology Information
bp Count	Base pair count
OTU	Operational taxonomic unit
MG-RAST	Metagenomic Rapid Annotations using Subsystems Technology
NGS	Next Generation Sequencing
EDTA	Ethylenediamine tetraacetic acid
TE buffer	Tris Ethylenediamine tetraacetic acid buffer

7. AUTHORS CONTRIBUTION:

Study conception and design: Pradip Bawane, Santosh Yele; Data collection: Pradip Bawane; Analysis and interpretation of results: Pradip Bawane; Draft manuscript: Santosh Yele.

8. CONFLICT OF INTEREST:

The author(s) declare no conflict of interest, financial or otherwise.

9. ACKNOWLEDGMENT:

The authors are grateful to the Chancellor, NMIMS (Deemed-to-be University), and management of SVKM's NMIMS, School of Pharmacy and Technology Management, for providing various facilities for implementing this work. As well as the authors are grateful to the Genexplore Diagnostic and Research Centre Pvt. Ltd, Ahmedabad, Gujarat, India, for performing Metagenomics analysis.

10. REFERENCES:

1. Fadiji AE, Babalola OO. Metagenomics methods for the study of plant-associated microbial communities: a review. J Microbiol Methods. 2020; 170: 105860.

2. Ghosh S, Das AP. Microbial metagenomics: current advances in investigating microbial ecology and population dynamics. Front soil Environ Microbiol. 2020; 247–54.

3. Xu J. Invited review: microbial ecology in the age of genomics and metagenomics: concepts, tools, and recent advances. Mol Ecol. 2006; 15(7): 1713–31.

4. Vishwakarma K, Kumar N, Shandilya C, Mohapatra S, Bhayana S, Varma A. Revisiting plant–microbe interactions and microbial consortia application for enhancing sustainable agriculture: a review. Front Microbiol. 2020; 11: 560406.

5. Ghosh S, Chowdhury R, Bhattacharya P. Mixed consortia in bioprocesses: role of microbial interactions. Appl Microbiol Biotechnol. 2016; 100: 4283–95.

6. Forbes JD, Knox NC, Ronholm J, Pagotto F, Reimer A. Metagenomics: the next culture-independent game changer. Front Microbiol. 2017; 8: 1069.

7. Kumar KR, Cowley MJ, Davis RL. Next-generation sequencing and emerging technologies. In: Seminars in thrombosis and hemostasis. Thieme Medical Publishers; 2024.

8. R. Chandran S, James S, Aswathi J, Padmakumar D, Kumar RBB, Chavan A, et al. Lonar Impact Crater, India: the Best-Preserved Terrestrial Hypervelocity Impact Crater in a Basaltic Terrain as a Potential Global Geopark. Geoheritage. 2022; 14(4): 130.

9. Chandran SR, James S, Aswathi J, Padmakumar D, Marjan TS, Kumar RBB, et al. A compendium of the best-preserved terrestrial hypervelocity impact crater in a basaltic terrain: The Lonar, India. Earth-Science Rev. 2023;104508.

10. Kanekar PP, Kanekar SP. Alkaliphilic, Alkalitolerant Microorganisms. In: Diversity and Biotechnology of Extremophilic Microorganisms from India. Springer; 2022; 71–116.

11. Guo F, Zhou Y, Song H, Zhao J, Shen H, Zhao B, et al. Next generation sequencing of SNPs using the HID-Ion AmpliSeqTM Identity Panel on the Ion Torrent PGMTM platform. Forensic Sci Int Genet. 2016; 25: 73–84.

12. Milani C, Hevia A, Foroni E, Duranti S, Turroni F, Lugli GA, et al. Assessing the fecal microbiota: an optimized ion torrent 16S rRNA gene-based analysis protocol. PLoS One. 2013; 8(7): e68739.

13. Malapelle U, Vigliar E, Sgariglia R, Bellevicine C, Colarossi L, Vitale D, et al. Ion Torrent next-generation sequencing for routine identification of clinically relevant mutations in colorectal cancer patients. J Clin Pathol. 2015; 68(1): 64–8.

14. Behra PRK, Das S, Pettersson BM, Shirreff L, DuCote T, Jacobsson KG, et al. Extended insight into the Mycobacterium chelonae-abscessus complex through whole genome sequencing of Mycobacterium salmoniphilum outbreak and Mycobacterium salmoniphilum-like strains. Sci Rep. 2019; 9(1): 1–15.

15. Ningsih F, Sari D, Yabe S, Yokota A, Oetari A, Sjamsuridzal W. Antibacterial activity of Microbispora rosea subsp. rosea SL3-2-R-1 grown on different media and solidifying agents. In: Journal of Physics: Conference Series. IOP Publishing; 2021. 52011.

16. Grasso LL, Martino DC, Alduina R. Production of antibacterial compounds from Actinomycetes. Actinobacter Basics Biotechnol Applic. 11: 177–198. 2016;

17. Selim MS, Amer SK, Mohamed SS, Mounier MM, Rifaat HM. Production and characterisation of exopolysaccharide from Streptomyces carpaticus isolated from marine sediments in Egypt and its effect on breast and colon cell lines. J Genet Eng Biotechnol. 2018; 16(1): 23–8.

18. Subramanian D, Kim MS, Kim DH, Heo MS. Isolation, characterization, antioxidant, antimicrobial and cytotoxic effect of marine Actinomycete, Streptomyces carpaticus MK-01, against fish pathogens. Brazilian Arch Biol Technol. 2017; 60.

19. Boltyanskaya Y V, Kevbrin V V, Lysenko AM, Kolganova T V, Tourova TP, Osipov GA, et al. Halomonas mongoliensis sp. nov. and Halomonas kenyensis sp. nov., new haloalkaliphilic denitrifiers capable of N2O reduction, isolated from soda lakes. Microbiology. 2007;76(6):739–47.

20. Corsaro MM, Gambacorta A, Iadonisi A, Lanzetta R, Naldi T, Nicolaus B, et al. Structural determination of the O‐chain polysaccharide from the lipopolysaccharide of the haloalkaliphilic Halomonas pantelleriensis bacterium. 2006;

21. Kushmaro A, Banin E, Loya Y, Stackebrandt E, Rosenberg E. Vibrio shiloi sp. nov., the causative agent of bleaching of the coral Oculina patagonica. Int J Syst Evol Microbiol. 2001; 51(4): 1383–8.

22. Savitha T, Khalifa A, Sankaranarayanan A. Isolation of Actinobacteria from Soil and Marine Sediment Samples. In: Methods in Actinobacteriology. Springer; 2022. 1–5.

Received on 28.05.2025      Revised on 12.09.2025

Accepted on 26.11.2025 Published on 01.07.2026

Available online from July 04, 2026

Research J. Pharmacy and Technology. 2026;19(7):2985-2991.

DOI: 10.52711/0974-360X.2026.00425

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