Uncovering High-Yield Ectoine Producers from Extreme Environments: Insights from Tuz Lake

Mustafa Çiçek; Aytac Kocabas

doi:10.18016/ksutarimdoga.vi.1653239

EN TR

Uncovering High-Yield Ectoine Producers from Extreme Environments: Insights from Tuz Lake

Abstract

Ectoine, a compatible solute produced by halophile microorganisms, is widely used in various industries due to its protective properties against environmental stress. This study aimed to isolate and characterize bacterial strains capable of producing high levels of ectoine without medium optimization from Tuz Lake, an extremely saline environment in Turkey. Bacterial isolates were obtained by selective culturing on meat peptone agar containing NaCl concentrations ranging from 3% to 21%, using water and soil samples collected from randomly selected sites in the Şereflikoçhisar and Cihanbeyli regions. A total of 22 isolates were screened for salt tolerance and their potential for ectoine production. Among them, 10 isolates with the highest salt tolerance and distinctive colony morphology were selected for further analysis. Molecular characterization via 16S rRNA sequencing identified these isolates as belonging to the genera Halomonas, Chromohalobacter, and Salinivibrio. High-performance liquid chromatography (HPLC) analysis revealed that Salinivibrio sp. (Isolate 12) and Halomonas sp. (Isolate 21) exhibited the highest ectoine production, yielding 296.88 µg mL-1 and 202.49 µg mL-1, respectively. To optimize ectoine yield, a Plackett-Burman experimental design was applied, evaluating the effects of different nitrogen sources. Peptone was identified as a statistically significant factor (p < 0.05) for enhancing ectoine production in Salinivibrio sp. This study highlights the biotechnological potential of halophilic bacteria from Tuz Lake for industrial ectoine production and emphasizes the importance of medium optimization in improving ectoine yields. Further optimization may enable the development of scalable processes for commercial ectoine production.

Keywords

Aşırı Çevrelerden Yüksek Verimli Ektoin Üreten Mikroorganizmaların Keşfi: Tuz Gölü Örneği

Abstract

Ektoin, halofil mikroorganizmalar tarafından üretilen ve çevresel streslere karşı koruyucu özellikleri nedeniyle çeşitli endüstrilerde yaygın olarak kullanılan uyumlu bir çözünendir. Bu çalışmada, Türkiye’deki aşırı tuzlu bir ortam olan Tuz Gölü’nden, besiyeri optimizasyonu yapılmaksızın yüksek düzeyde ektoin üretebilen bakterilerin izolasyonu ve karakterizasyonu amaçlanmıştır. Şereflikoçhisar ve Cihanbeyli bölgelerinde rastgele seçilen alanlardan toplanan su ve toprak örneklerinden, %3 ila %21 arasında değişen NaCl konsantrasyonlarına sahip et-pepton agar kullanılarak yapılan seçici kültürleme ile bakteri izolatları elde edilmiştir. Toplamda 22 izolat tuz toleransı ve ektoin üretim potansiyeli açısından taranmış, en yüksek tuz toleransına ve belirgin koloni morfolojisine sahip 10 izolat detaylı analize alınmıştır. 16S rRNA dizilemesiyle yapılan moleküler karakterizasyon, bu izolatların Halomonas, Chromohalobacter ve Salinivibrio cinslerine ait olduğunu göstermiştir. Yüksek performanslı sıvı kromatografisi (HPLC) analizleri, Salinivibrio sp. (İzolat 12) ve Halomonas sp. (İzolat 21) türlerinin sırasıyla 296.88 µg mL-1 ve 202.49 µg mL-1 ektoin üretimiyle en yüksek verimi gösterdiğini ortaya koymuştur. Ektoin verimini artırmak için farklı azot kaynaklarının etkisini değerlendiren Plackett-Burman deney tasarımı uygulanmış ve Salinivibrio sp. için peptonun ektoin üretimini anlamlı şekilde artıran bir faktör olduğu (p <0.05) belirlenmiştir. Bu çalışma, Tuz Gölü'nden izole edilen halofilik bakterilerin endüstriyel ektoin üretiminde biyoteknolojik potansiyelini vurgulamakta ve ektoin verimini artırmada besiyeri optimizasyonunun önemini göstermektedir. İleri optimizasyon çalışmaları, ticari ektoin üretimi için ölçeklenebilir süreçlerin geliştirilmesine olanak sağlayabilir.

Keywords

Supporting Institution

Karamanoglu Mehmetbey University

Project Number

10-M-21

Ethical Statement

This study does not require approval from an ethics committee.

Thanks

This research was supported by the project number 10-M-21 accepted by Karamanoglu Mehmetbey University Scientific Research Projects Coordination Office.

References

Altschul, S. F., Gish, W., Miller, W., Myers, E. W., & Lipman, D. J. (1990). Basic local alignment search tool. Journal of Molecular Biology, 215(3), 403–410. https://doi.org/10.1016/S0022-2836(05)80360-2
Apprill, A., Mcnally, S., Parsons, R., & Weber, L. (2015). Minor revision to V4 region SSU rRNA 806R gene primer greatly increases detection of SAR11 bacterioplankton. Aquatic Microbial Ecology, 75(2), 129–137. https://doi.org/10.3354/ame01753
Ayadi, H., Frikha-Dammak, D., Fakhfakh, J., Chamkha, M., Hassairi, I., Allouche, N., Sayadi, S., & Maalej, S. (2020). The saltern-derived Paludifilum halophilum DSM 102817T is a new high-yield ectoines producer in minimal medium and under salt stress conditions. 3 Biotech, 10(12), 533. https://doi.org/10.1007/s13205-020-02512-x
Balderrama-Subieta, A., & Quillaguamán, J. (2013). Genomic studies on nitrogen metabolism in Halomonas boliviensis: Metabolic pathway, biochemistry and evolution. Computational Biology and Chemistry, 47, 96–104. https://doi.org/10.1016/j.compbiolchem.2013.08.002
Bourot, S., Sire, O., Trautwetter, A., Touzé, T., Wu, L. F., Blanco, C., & Bernard, T. (2000). Glycine betaine-assisted protein folding in a lysA mutant of Escherichia coli. Journal of Biological Chemistry, 275(2), 1050–1056. https://doi.org/10.1074/jbc.275.2.1050
Brown, A. D. (1976). Microbial water stress. Bacteriological Reviews, 40(4), 803. https://doi.org/10.1128/br.40.4.803-846.1976
Burg, M. B., & Ferraris, J. D. (2008). Intracellular organic osmolytes: Function and regulation. In Journal of Biological Chemistry (Vol. 283, Issue 12, pp. 7309–7313). American Society for Biochemistry and Molecular Biology. https://doi.org/10.1074/jbc.R700042200
Canli Taşar, Ö., Erhan TAŞAR, G., Teknoloji Uygulama ve Araştırma Merkezi YÜTAM, Y., Teknik Üniversitesi, E., Meslek Yüksek Okulu, K., & Üniversitesi, A. (2023). Optimization of Keratinase Enzyme synthesized by Micrococcus luteus using Taguchi DOE Method. Kahramanmaraş Sütçü İmam Üniversitesi Tarım ve Doğa Dergisi, 26(5), 1027–1033. https://doi.org/10.18016/ksutarimdoga.vi.1128064

Chen, W. C., Hsu, C. C., Lan, J. C. W., Chang, Y. K., Wang, L. F., & Wei, Y. H. (2018). Production and characterization of ectoine using a moderately halophilic strain Halomonas salina BCRC17875. Journal of Bioscience and Bioengineering, 125(5), 578–584. https://doi.org/10.1016/j.jbiosc.2017.12.011
Chen, Y., Liu, Y., Meng, Y., Jiang, Y., Xiong, W., Wang, S., Yang, C., & Liu, R. (2024). Elucidating the salt-tolerant mechanism of Halomonas cupida J9 and unsterile ectoine production from lignocellulosic biomass. Microbial Cell Factories, 23(1), 1–12. https://doi.org/10.1186/s12934-024-02515-w
Czech, L., Hermann, L., Stöveken, N., Richter, A. A., Höppner, A., Smits, S. H. J., Heider, J., & Bremer, E. (2018). Role of the Extremolytes Ectoine and Hydroxyectoine as Stress Protectants and Nutrients: Genetics, Phylogenomics, Biochemistry, and Structural Analysis. Genes, 9(4). https://doi.org/10.3390/genes9040177
Çiçek, M., Çiçek, E., & Kocabaş, A. (2025). Ektoin: Mikrobiyal Hayatta Kalma Sırrından Biyoteknolojik Uygulamalara. Iğdır Üniversitesi Fen Bilimleri Enstitüsü Dergisi, 15(1), 92–109. https://doi.org/10.21597/jist.1522694
Deantas-Jahn, C., Mendoza, S. N., Licona-Cassani, C., Orellana, C., & Saa, P. A. (2024). Metabolic modeling of Halomonas campaniensis improves polyhydroxybutyrate production under nitrogen limitation. Applied Microbiology and Biotechnology, 108(1), 310. https://doi.org/10.1007/s00253-024-13111-8
Doğan, S. Ş., & Kocabaş, A. (2021). Metagenomic Assessment of Prokaryotic Diversity within Hypersaline Tuz Lake, Turkey. Microbiology (Russian Federation), 90(5), 647–655. https://doi.org/10.1134/s0026261721050118
Elbialy, H. A., Omara, A., Sharaf, A. M., el-hela, A., Shahin, A., & El-Fouly, M. (2019). Isolation and characterization of new ectoine-producers from various hypersaline ecosystems in Egypt. Journal of Nuclear Technology in Applied Science, 7(1), 221–236. https://doi.org/10.21608/jntas.2019.16407.1005
Galinski, E. A. (1995). Osmoadaptation in Bacteria. Advances in Microbial Physiology, 37(C), 273–328. https://doi.org/10.1016/s0065-2911(08)60148-4
García-López, M. L., Santos, J. A., Otero, A., & Rodríguez-Calleja, J. M. (2014). Psychrobacter. Encyclopedia of Food Microbiology: Second Edition, 261–268. https://doi.org/10.1016/b978-0-12-384730-0.00285-8
Graf, R., Anzali, S., Buenger, J., Pfluecker, F., & Driller, H. (2008). The multifunctional role of ectoine as a natural cell protectant. Clinics in Dermatology, 26(4), 326–333. https://doi.org/10.1016/j.clindermatol.2008.01.002
Gunde-Cimerman, N., Plemenitaš, A., & Oren, A. (2018). Strategies of adaptation of microorganisms of the three domains of life to high salt concentrations. In FEMS Microbiology Reviews (Vol. 42, Issue 3, pp. 353–375). Oxford University Press. https://doi.org/10.1093/femsre/fuy009
Kalia, V. C., Kumar, R., Kumar, P., & Koul, S. (2016). A Genome-Wide Profiling Strategy as an Aid for Searching Unique Identification Biomarkers for Streptococcus. Indian Journal of Microbiology, 56(1), 46–58. https://doi.org/10.1007/s12088-015-0561-5
Kanapathipillai, M., Lentzen, G., Sierks, M., & Park, C. B. (2005). Ectoine and hydroxyectoine inhibit aggregation and neurotoxicity of Alzheimer’s β-amyloid. FEBS Letters, 579(21), 4775–4780. https://doi.org/10.1016/j.febslet.2005.07.057
Kang, J. Y., Lee, B., Kim, J. A., Kim, M. S., & Kim, C. H. (2022). Identification and characterization of an ectoine biosynthesis gene cluster from Aestuariispira ectoiniformans sp. nov., isolated from seawater. Microbiological Research, 254, 126898. https://doi.org/10.1016/j.micres.2021.126898
Katoh, K. (2002). MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier transform. Nucleic Acids Research, 30(14), 3059–3066. https://doi.org/10.1093/nar/gkf436
Katoh, K., & Standley, D. M. (2013). MAFFT Multiple Sequence Alignment Software Version 7: Improvements in Performance and Usability. Molecular Biology and Evolution, 30(4), 772–780. https://doi.org/10.1093/molbev/mst010
Kempf, B., & Bremer, E. (1998). Uptake and synthesis of compatible solutes as microbial stress responses to high-osmolality environments. Archives of Microbiology, 170(5), 319–330. https://doi.org/10.1007/s002030050649
Le Rudulier, D., Strom, A. R., Dandekar, A. M., Smith, L. T., & Valentine, R. C. (1984). Molecular biology of osmoregulation. Science, 224(4653), 1064–1068. https://doi.org/10.1126/science.224.4653.1064
Lippert, K., & Galinski, E. A. (1992). Enzyme stabilization be ectoine-type compatible solutes: protection against heating, freezing and drying. Applied Microbiology and Biotechnology, 37(1), 61–65. https://doi.org/10.1007/bf00174204
Ma, H., Zhao, Y., Huang, W., Zhang, L., Wu, F., Ye, J., & Chen, G.-Q. (2020). Rational flux-tuning of Halomonas bluephagenesis for co-production of bioplastic PHB and ectoine. Nature Communications, 11(1), 3313. https://doi.org/10.1038/s41467-020-17223-3
Mahansaria, R., Choudhury, J. D., & Mukherjee, J. (2015). Polymerase chain reaction-based screening method applicable universally to environmental haloarchaea and halobacteria for identifying polyhydroxyalkanoate producers among them. Extremophiles, 19(5), 1041–1054. https://doi.org/10.1007/s00792-015-0775-9
Orhan, F., Ceyran, E., & Akincioğlu, A. (2023). Optimization of ectoine production from Nesterenkonia xinjiangensis and one-step ectoine purification. Bioresource Technology, 371, 128646. https://doi.org/10.1016/j.biortech.2023.128646
Parada, A. E., Needham, D. M., & Fuhrman, J. A. (2016). Every base matters: Assessing small subunit rRNA primers for marine microbiomes with mock communities, time series and global field samples. Environmental Microbiology, 18(5), 1403–1414. https://doi.org/10.1111/1462-2920.13023
Plackett, R. L., & Burman, J. P. (1946). The Design of Optimum Multifactorial Experiments. Biometrika, 33(4), 305–325. https://doi.org/10.1093/biomet/33.4.305
Roessler, M., & Muller, V. (2001). Osmoadaptation in bacteria and archaea: common principles and differences. Environmental Microbiology, 3(12), 743–754. https://doi.org/10.1046/j.1462-2920.2001.00252.x
Sadeghi, A., Soltani, B. M., Nekouei, M. K., Jouzani, G. S., Mirzaei, H. H., & Sadeghizadeh, M. (2014). Diversity of the ectoines biosynthesis genes in the salt tolerant Streptomyces and evidence for inductive effect of ectoines on their accumulation. Microbiological Research, 169(9–10), 699–708. https://doi.org/10.1016/j.micres.2014.02.005
Sahin Dogan, S., & Kocabaş, A. (2023). Profiling the genes associated with osmoadaptation and their variation by seasonally in Tuz Lake. Communications Faculty of Science University of Ankara Series C Biology Geological Engineering and Geophysical Engineering, 32(2), 174–191. https://doi.org/10.53447/communc.1206230
Şahi̇n Doğan, S., & Kocabaş, A. (2024). Seasonal Gene Profiling in Tuz Lake with Regard to Biogeochemical Cycling. Kahramanmaraş Sütçü İmam Üniversitesi Tarım ve Doğa Dergisi, 27(2), 273–284. https://doi.org/10.18016/ksutarimdoga.vi.1212062
Sayers, E. W., Bolton, E. E., Brister, J. R., Canese, K., Chan, J., Comeau, D. C., Connor, R., Funk, K., Kelly, C., Kim, S., Madej, T., Marchler-Bauer, A., Lanczycki, C., Lathrop, S., Lu, Z., Thibaud-Nissen, F., Murphy, T., Phan, L., Skripchenko, Y., … Sherry, S. T. (2022). Database resources of the national center for biotechnology information. Nucleic Acids Research, 50(D1), D20–D26. https://doi.org/10.1093/nar/gkab1112
Stamatakis, A. (2014). RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics, 30(9), 1312–1313. https://doi.org/10.1093/bioinformatics/btu033
Van Thuoc, D., Hien, T. T., & Sudesh, K. (2019). Identification and characterization of ectoine-producing bacteria isolated from Can Gio mangrove soil in Vietnam. Annals of Microbiology, 69(8), 819–828. https://doi.org/10.1007/s13213-019-01474-7
Wei, Y. H., Yuan, F. W., Chen, W. C., & Chen, S. Y. (2011). Production and characterization of ectoine by Marinococcus sp. ECT1 isolated from a high-salinity environment. Journal of Bioscience and Bioengineering, 111(3), 336–342. https://doi.org/10.1016/j.jbiosc.2010.11.009
Xie, Y., Tian, X., He, Y., Dong, S., & zhao, K. (2023). Nitrogen removal capability and mechanism of a novel heterotrophic nitrification–aerobic denitrification bacterium Halomonas sp. DN3. Bioresource Technology, 387, 129569. https://doi.org/10.1016/j.biortech.2023.129569
Yancey, P. H., Clark, M. E., Hand, S. C., Bowlus, R. D., & Somero, G. N. (1982). Living with water stress: Evolution of osmolyte systems. Science, 217(4566), 1214–1222. https://doi.org/10.1126/science.7112124
Zhang, T., Cui, T., Cao, Y., Li, Y., Li, F., Zhu, D., & Xing, J. (2022). Whole genome sequencing of the halophilic Halomonas qaidamensis XH36, a novel species strain with high ectoine production. Antonie van Leeuwenhoek, International Journal of General and Molecular Microbiology, 115(4), 545–559. https://doi.org/10.1007/s10482-022-01709-9

Details

Primary Language

English

Subjects

Microbiology (Other)

Journal Section

Research Article

Authors

Mustafa Çiçek
0000-0002-7109-6500
Türkiye

Aytac Kocabas ^*
0000-0001-7622-1932
Türkiye

Early Pub Date

July 25, 2025

Publication Date

September 11, 2025

Submission Date

March 7, 2025

Acceptance Date

June 27, 2025

Published in Issue

Year 2025 Volume: 28 Number: 5

DOI

https://doi.org/10.18016/ksutarimdoga.vi.1653239

IZ

https://izlik.org/JA27AY68EG

Cite

RIS / Bibtex

APA

Çiçek, M., & Kocabas, A. (2025). Uncovering High-Yield Ectoine Producers from Extreme Environments: Insights from Tuz Lake. Kahramanmaraş Sütçü İmam Üniversitesi Tarım Ve Doğa Dergisi, 28(5), 1186-1196. https://doi.org/10.18016/ksutarimdoga.vi.1653239

AMA

1.Çiçek M, Kocabas A. Uncovering High-Yield Ectoine Producers from Extreme Environments: Insights from Tuz Lake. KSU J. Agric Nat. 2025;28(5):1186-1196. doi:10.18016/ksutarimdoga.vi.1653239

Chicago

Çiçek, Mustafa, and Aytac Kocabas. 2025. “Uncovering High-Yield Ectoine Producers from Extreme Environments: Insights from Tuz Lake”. Kahramanmaraş Sütçü İmam Üniversitesi Tarım Ve Doğa Dergisi 28 (5): 1186-96. https://doi.org/10.18016/ksutarimdoga.vi.1653239.

EndNote

Çiçek M, Kocabas A (September 1, 2025) Uncovering High-Yield Ectoine Producers from Extreme Environments: Insights from Tuz Lake. Kahramanmaraş Sütçü İmam Üniversitesi Tarım ve Doğa Dergisi 28 5 1186–1196.

IEEE

[1]M. Çiçek and A. Kocabas, “Uncovering High-Yield Ectoine Producers from Extreme Environments: Insights from Tuz Lake”, KSU J. Agric Nat., vol. 28, no. 5, pp. 1186–1196, Sept. 2025, doi: 10.18016/ksutarimdoga.vi.1653239.

ISNAD

Çiçek, Mustafa - Kocabas, Aytac. “Uncovering High-Yield Ectoine Producers from Extreme Environments: Insights from Tuz Lake”. Kahramanmaraş Sütçü İmam Üniversitesi Tarım ve Doğa Dergisi 28/5 (September 1, 2025): 1186-1196. https://doi.org/10.18016/ksutarimdoga.vi.1653239.

JAMA

1.Çiçek M, Kocabas A. Uncovering High-Yield Ectoine Producers from Extreme Environments: Insights from Tuz Lake. KSU J. Agric Nat. 2025;28:1186–1196.

MLA

Çiçek, Mustafa, and Aytac Kocabas. “Uncovering High-Yield Ectoine Producers from Extreme Environments: Insights from Tuz Lake”. Kahramanmaraş Sütçü İmam Üniversitesi Tarım Ve Doğa Dergisi, vol. 28, no. 5, Sept. 2025, pp. 1186-9, doi:10.18016/ksutarimdoga.vi.1653239.

Vancouver

1.Mustafa Çiçek, Aytac Kocabas. Uncovering High-Yield Ectoine Producers from Extreme Environments: Insights from Tuz Lake. KSU J. Agric Nat. 2025 Sep. 1;28(5):1186-9. doi:10.18016/ksutarimdoga.vi.1653239