Metagenomic analyses uncover the differential effect of azide treatment on bacterial community structure by enriching a specific Cyanobacteria present in a saline-alkaline environmental sample


Treatment of environmental samples under field conditions may require the application of chemical preservatives, although their use sometimes produces changes in the microbial communities. Sodium azide, a commonly used preservative, is known to differentially affect the growth of bacteria. Application of azide and darkness incubation to Isabel soda lake water samples induced changes in the structure of the bacterial community, as assessed by partial 16S rRNA gene pyrosequencing. Untreated water samples (WU) were dominated by gammaproteobacterial sequences accounting for 86%, while in the azide-treated (WA) samples, this group was reduced to 33% abundance, and cyanobacteria-related sequences became dominant with 53%. Shotgun sequencing and genome recruitment analyses pointed to Halomonas campanensis strain LS21 (genome size 4.07 Mbp) and Synechococcus sp. RS9917 (2.58 Mbp) as the higher recruiting genomes from the sequence reads of WA and WU environmental libraries, respectively, covering nearly the complete genomes. Combined treatment of water samples with sodium azide and darkness has proven effective on the selective enrichment of a cyanobacterial group. This approach may allow the complete (or almost-complete) genome sequencing of Cyanobacteria from metagenomic DNA of different origins, and thus increasing the number of the underrepresented cyanobacterial genomes in the databases.

This is a preview of subscription content, log in to check access.

Access options

Buy single article

Instant unlimited access to the full article PDF.

US$ 39.95

Price includes VAT for USA

Fig. 1
Fig. 2


  1. Aguirre-Garrido JF, Ramírez-Saad HC, Toro N, Martínez-Abarca F (2016) Bacterial diversity in the soda saline crater lake from Isabel Island, Mexico. Microb Ecol 71(1):68–77.

  2. Alcocer J, Lugo A, Sánchez MR, Escobar E (1998) Isabela crater-lake: a Mexican insular saline lake. Hydrobiologia 381:1–7

  3. Alvarenga DO, Fiore MF, Varani AM (2017) A metagenomic approach to Cyanobacterial genomics. Front Microbiol 8:809.

  4. Aronesty E (2011) Ea-utils: command-line tools for processing biological sequencing data;

  5. Audicana A, Perales I, Borrego JJ (1995) Modification of kanamycin-esculin-azide agar to improve selectivity in the enumeration of fecal streptococci from water samples. Appl Environ Microbiol 61:4168–4183

  6. Bowyer JW, Skerman WBD (1968) Production of axenic cultures of soil-borne and endophytic blue-green algae. J Gen Microbiol 54:299–306

  7. Bundy DA, , Golden MH. (1985). Sodium azide preservation of faecal specimens for Kato analysis. Parasitology 90 3:463–469

  8. Burgsdorf I, Slaby BM, Handley KM, Haber M, Blom J, Marshall CW, Gilbert JA, Hentschel U, Steindler L (2015) Lifestyle evolution in cyanobacterial symbionts of sponges. MBio. 6(3):e00391–e00315.

  9. Case RJ, Boucher Y, Dahllöf I, Holmström C, Doolittle WF, Kjelleberg S (2007) Use of 16S rRNA and rpoB genes as molecular markers for microbial ecology studies. Appl Environ Microbiol 73(1):278–288.

  10. Dufresne A, Ostrowski M, Scanlan DJ, Garczarek L, Mazard S, Palenik BP, Paulsen IT, de Marsac NT, Wincker P, Dossat C, Ferriera S, Johnson J, Post AF, Hess WR, Partensky F (2008) Unraveling the genomic mosaic of a ubiquitous genus of marine cyanobacteria. Gen Biol 9:R90.

  11. Edgar RC, Haas BJ, Clemente JC, Quince C, Knight R (2011) UCHIME improves sensitivity and speed of chimera detection. Bioinformatics 27:2194–2200.

  12. Ferris M, Hirsch CF (1991) Method for isolation and purification of cyanobacteria. Appl Environ Microbiol 57:1448–1452

  13. Flynn JM, Brown EA, Chain FJJ, MacIsaac HJ, Cristescu ME (2015) Toward accurate molecular identification of species in complex environmental samples: testing the performance of sequence filtering and clustering methods. Ecol Evol 5:2252–2266.

  14. Gao ZM, Wang Y, Tian RM, Wong YH, Batang ZB, Al-Suwailem AM, Bajic VB, Qian PY (2014) Symbiotic adaptation drives genome streamlining of the cyanobacterial sponge symbiont “Candidatus Synechococcus spongiarum”. MBio 5(2):e00079-14.

  15. Garza DL, Dutilh BE (2015) From cultured to uncultured genome sequences: metagenomics and modeling microbial ecosystems. Cell Mol Life Sci 72:4287–4308.

  16. Gerencser VF, Weaver RH (1958) A new technique for the use of sodium azide (hydrazoic acid) as inhibitive agent. Appl Microbiol 7:113–115

  17. Ghai R, Martín-Cuadrado AB, Gonzaga-Molto A, García-Heredia I, Cabrera R, Martin J, Verdú M, Deschamps P, Moreira D, López-García P, Mira A, Rodríguez Valera F (2010) Metagenome of the Mediterranean deep chlorophyll maximum studied by direct and fosmid library 454 pyrosequencing. ISME J 4:1154–1166.

  18. Heaney SI, Jaworski GHM (1977) A simple separation technique for purifying micro-algae. Br Phycol J 12:171–174

  19. Kearse M, Moir R, Wilson A, Stones-Havas S, Cheung M, Sturrock S, Buxton S, Cooper A, Markowitz S, Duran C, Thierer T, Ashton B, Meintjes P, Drummond A (2012) Geneious basic: an integrated and extendable desktop software platform for the organization and analysis of sequence data. Bioinformatics. 28:1647–1649.

  20. Kurtz S, Phillippy A, Delcher AL, Smoot M, Shumway M, Antonescu C, Salzberg SL (2004) Versatile and open software for comparing large genomes. Genome Biol 5(2):R12.

  21. Lichstein HC, Soule MH (1943a) The action of sodium azide on microbic growth and respiration: I. The action of sodium azide on microbic growth. J Bacteriol 47(3):221–230

  22. Lichstein HC, Soule MH (1943b) The action of sodium azide on microbic growth and respiration: II. The action of sodium azide on bacterial catalase. J Bacteriol 47(3):231–238

  23. Llopis MB, Marugán MR, Althaus RL, Pons MP (2013) Effect of storage and preservation of milk samples on the response of microbial inhibitor tests. J Dairy Res 80(4):475–484.

  24. Parks DH, Tyson GW, Hugenholtz P, Beiko RG (2014) STAMP: statistical analysis of taxonomic and functional profiles. Bioinformatics 30:3123–3124.

  25. Ramírez-Saad H, Akkermans WL, Akkermans ADL (2004) DNA extraction from actinorhizal nodules. In: Kowalchuk G, de Bruijn F, Head IA, Akkermans ADL, van Elsas JD (eds) Molecular microbial ecology manual II. Kluwer Academic Publishers, Dordrecht

  26. Rippka R (1988) Isolation and purification of Cyanobacteria. Methods Enzymol 167:3–27

  27. Schloss PD, Westcott SL, Ryabin T, Hall JR, Hartmann M, Hollister EB, Lesniewski RA, Oakley BB, Parks DH, Robinson CJ, Sahl JW, Stres B, Thallinger GG, Van Horn DJ, Weber CF (2009) Introducing mothur: open-source, platform-independent, community-supported software for describing and comparing microbial communities. Appl Environ Microbiol 75:7537–7541.

  28. Schmieder R, Edwards R (2011) Quality control and preprocessing of metagenomic datasets. Bioinformatics 27:863–864.

  29. Sena L, Rojas D, Montiel E, González H, Morett J, Naranjo L (2011) A strategy to obtain axenic cultures of Arthrospira spp. Cyanobacteria. W J Microbiol Biotechnol 27:1045–1053.

  30. Sitz TO, Schmidt RR (1973) Purification of Synechococcus lividus by equilibrium centrifugation and its synchronization by differential centrifugation. J Bacteriol 115:43–46

  31. Vaara T, Vaara M, Niemela S (1979) Two improved methods for obtaining axenic cultures of cyanobacteria. Appl Environ Microbiol 38:1011–1014

  32. Vandeputte D, Tito RY, Vanleeuwen R, Falony G, Raes J. (2017). Practical considerations for large-scale gut microbiome studies. FEMS Microbiol. Rev. 41 (Suppl. 1):S154–S167. doi:

  33. Winter C, Kerros ME, Weinbauer M (2012) Effects of sodium azide on the abundance of prokaryotes and viruses in marine samples. PLoS One 7:e37597.

  34. Yilmaz P, Parfrey LW, Yarza P, Gerken J, Pruesse E, Quast C, Schweer T, Peplies J, Ludwig W, Glöckner FO (2014) The SILVA and ball-species Living Tree Project (LTP): taxonomic frameworks. Nucleic Acids Res 42:D643–D648.

  35. Yue H, Ling C, Yang T, Chen X, Chen Y, Deng H, Wu Q, Chen J, Chen G-Q (2014) A seawater-based open and continuous process for polyhydroxyalkanoates production by recombinant Halomonas campaniensis LS21 grown in mixed substrates. Biotechnol Biofuels 7:108–119.

Download references


DML and HRS acknowledge to the Mexican Consejo Nacional de Ciencia y Tecnología (CONACyT) for fellowships numbers 291062 Becas Mixtas de Movilidad en el Extranjero Programme and 710228 Estancias Sabática al Extranjero Programme, respectively. We specially thank Dr. Antonio J. Fernández-González and Mario R. Mestre for their valuable help with drawings of the recruitment plots.

Funding information

This work was supported by the Spanish Ministerio de Ciencia, Innovación y Universidades (research grant BIO2017-82244-P).

Author information

Correspondence to Hugo Ramírez-Saad.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Additional information

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Electronic supplementary material


(PDF 150 kb)


(PDF 84 kb)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Aguirre-Garrido, J.F., Martínez-Abarca, F., Montiel-Lugo, D. et al. Metagenomic analyses uncover the differential effect of azide treatment on bacterial community structure by enriching a specific Cyanobacteria present in a saline-alkaline environmental sample. Int Microbiol (2020) doi:10.1007/s10123-020-00119-z

Download citation


  • 16S rRNA analysis
  • Amplitag-pyrosequencing
  • Genome recruitment
  • Halo-alkalophile bacteria
  • Halomonas
  • Synechococcus