Applied Biological Chemistry

, Volume 61, Issue 2, pp 153–161 | Cite as

Application of metagenome analysis to characterize the molecular diversity and saxitoxin-producing potentials of a cyanobacterial community: a case study in the North Han River, Korea

  • Keon Hee Kim
  • Youngdae Yoon
  • Woon-Young Hong
  • JaeBum Kim
  • Yung-Chul Cho
  • Soon-Jin Hwang


A wide variety of cyanobacterial species that inhabit freshwater systems are known to produce diverse toxins and off-flavor compounds during the development of environmentally harmful blooms. However, cyanobacterial community development and toxin production potential have not been well studied. In this study, we examined the taxonomic diversity and saxitoxin production potential of cyanobacteria in the water and sediments of a large river, the North Han River in South Korea, by metagenome analysis using next-generation sequencing (NGS) and molecular biological approaches, respectively. NGS revealed that the entire cyanobacterial community in the study area consisted of 39 genera and 47 species. The most abundant genera were Microcystis, Anabaena, Cyanobium, and Synechococcus, which accounted for more than 90% of the entire community. The saxitoxin production potential of the cyanobacterial community was assessed by detecting the sxtA and sxtG genes related to saxitoxin production. Eleven sxtA and 24 sxtG genes were identified through molecular cloning and sequencing. Phylogenic analysis revealed that three sxtA genes that grouped in one phylogenic branch with Scytonema sp. were distinctly separated from the sxtA genes of Anabaena, Aphanizomenon, Lyngbya, and Cylindrospermopsis. Sixteen of the detected sxtG genes were phylogenically similar to those of Anabaena circinalis (Dolichospermum circinale), Aphanizomenon gracile, and Aphanizomenon flos-aquae. Our study demonstrates the utility of the metagenomics approach for characterizing the natural community structure of cyanobacteria containing diverse and even rare species, and the evaluation of saxitoxin-producing potential in the cyanobacterial community.


Cyanobacteria Diversity Genes Metagenomics Next-generation sequencing Saxitoxin 



This study was financially supported by the Basic Environmental Research Program of the Han River System (2013–2015) of the Han River Watershed Environmental Office (the Ministry of Environment, Republic of Korea). The authors are grateful to the members of Limnology Laboratory in Konkuk University for their field assistance.


  1. 1.
    Paerl HW, Hall NS, Calandrino ES (2011) Controlling harmful cyanobacterial blooms in a world experiencing anthropogenic and climatic-induced change. Sci Total Environ 409(10):1739–1745CrossRefGoogle Scholar
  2. 2.
    Paerl HW, Fulton RS, Moisander PH, Dyble J (2001) Harmful freshwater algal blooms, with an emphasis on cyanobacteria. Sci World J 1:76–113CrossRefGoogle Scholar
  3. 3.
    WHO (2003) Cyanobacterial toxins: microcystin-LR in drinking-water. Background document for development of WHO Guidelines for drinking-water quality, 2nd edn. World Health Organization, GenevaGoogle Scholar
  4. 4.
    EPA (2015) Drinking water health advisory for the cyanobacterial microcystin toxins. United States Environmental Protection Agency, Washington, USAGoogle Scholar
  5. 5.
    NSW (2015) Blue-green algae blooms: risks to fishers. D.o.P. industries, EditorGoogle Scholar
  6. 6.
    Humpage A, Rositano J, Bretag AH, Brown R, Baker PD (1994) Paralytic shellfish poisons from Australian cyanobacterial blooms. Mar Freshw Res 45(5):761–771CrossRefGoogle Scholar
  7. 7.
    Pearson L, Mihali T, Moffitt M, Kellmann R, Neilan B (2010) On the chemistry, toxicology and genetics of the cyanobacterial toxins, microcystin, nodularin, saxitoxin and cylindrospermopsin. Mar Drugs 8(5):1650–1680CrossRefGoogle Scholar
  8. 8.
    Batoréu M, Dias E, Pereira P, Franca S (2005) Risk of human exposure to paralytic toxins of algal origin. Environ Toxicol Pharmacol 19(3):401–406CrossRefGoogle Scholar
  9. 9.
    Metcalf JS, Codd GA (2014) Cyanobacterial toxina (Cyanotoxin) in water, 2014. Foundation for Water Research, AmsterdamGoogle Scholar
  10. 10.
    Dos Anjos FM, Do Carmo Bittencourt-Oliveira M, Zajac MP, Hiller S, Christian B, Erler K, Luckas B, Pinto E (2006) Detection of harmful cyanobacteria and their toxins by both PCR amplification and LC-MS during a bloom event. Toxicon 48(3):239–245CrossRefGoogle Scholar
  11. 11.
    Foss AJ, Phlips EJ, Yilmaz M, Chapman A (2012) Characterization of paralytic shellfish toxins from Lyngbya wollei dominated mats collected from two Florida springs. Harmful Algae 16:98–107CrossRefGoogle Scholar
  12. 12.
    Ballot A, Fastner J, Wiedner C (2010) Paralytic shellfish poisoning toxin-producing cyanobacterium Aphanizomenon gracile in northeast Germany. Appl Environ Microbiol 76(4):1173–1180CrossRefGoogle Scholar
  13. 13.
    Hoff-Risseti C, Dorr FA, Schaker FD, Pinto E, Werner VR, Fiore MF (2013) Cylindrospermopsin and saxitoxin synthetase genes in Cylindrospermopsis raciborskii strains from Brazilian freshwater. PLoS ONE 8(8):e74238CrossRefGoogle Scholar
  14. 14.
    Moustafa A, Loram JE, Hackett JD, Anderson DM, Plumley FG, Bhattacharya D (2009) Origin of saxitoxin biosynthetic genes in cyanobacteria. PLoS ONE 4(6):e5758CrossRefGoogle Scholar
  15. 15.
    Srivastava A, Ko SR, Ahn CY, Oh HM, Ravi AK, Asthana RK (2016) Microcystin biosynthesis and mcyA expression in geographically distinct microcystis strains under different nitrogen, phosphorus, and boron regimes. Biomed Res Int 2016:13CrossRefGoogle Scholar
  16. 16.
    Boopathi T, Ki J-S (2014) Impact of environmental factors on the regulation of cyanotoxin production. Toxins 6(7):1951–1978CrossRefGoogle Scholar
  17. 17.
    Chorus I, Bartram J (1999) Toxic cyanobacteria in water: a guide to their public health consequences, monitoring and management. WHO, LondonCrossRefGoogle Scholar
  18. 18.
    New-Zealand Ministry of Health (2016) Guidelines for drinking-water quality management for New Zealand. Ministry of Health, WellingtonGoogle Scholar
  19. 19.
    Whitton BA (2012) Ecology of cyanobacteria II: their diversity in space and time. Springer, BerlinCrossRefGoogle Scholar
  20. 20.
    Park HJ (2013) Study on harmful cyanobacteria and off-flavor production in Lake Paldang. Konkuk University, SeoulGoogle Scholar
  21. 21.
    Baek JS (2015) The phytoplankton community and the waterquality assessment in Paldang-Lake. Kyonggi University, SeoulGoogle Scholar
  22. 22.
    Kim YJ, Baek JS, Youn SJ, Kim HN, Lee BC, Park S, You KA, Lee JK (2016) Cyanobacteria community and growth potential test in sediment of lake Paldang. Journal of Korean Society on Water Environment 32:9Google Scholar
  23. 23.
    Lee HS, Oh KH, Cho YC (2008) Isolation of cyanobacteria producing microcystin from lakes. Korean J Microbiol 44(3):6Google Scholar
  24. 24.
    Kim YG, Shin YK, Choi JK (2000) Studies on the phytoplankton community in the Paldang Lake. Environ Sci Eng Center Sang-ji Univ 6:11Google Scholar
  25. 25.
    Park H-K, Jheong WH, Kwon OS, Ryu J-K (2000) Seasonal succession of toxic cyanobacteria and microcystins concentration in Paldang reservoir. Algae 15(1):29–35Google Scholar
  26. 26.
    Jheong WH (2001) Characteristics of occurrence and control of cyanobacteria and phytoplankton in Lake Paldang. Dankook University, YonginGoogle Scholar
  27. 27.
    D’Agostino PM, Song X, Neilan BA, Moffitt MC (2016) Proteogenomics of a saxitoxin producing and non toxic strain of Anabaena circinalis (cyanobacteria) in response to extracellular NaCl and phosphate depletion. Environ Microbiol 18(2):461–476CrossRefGoogle Scholar
  28. 28.
    Lin X, Ding H, Zeng Q (2016) Transcriptomic response during phage infection of a marine cyanobacterium under phosphorus-limited conditions. Environ Microbiol 18(2):450–460CrossRefGoogle Scholar
  29. 29.
    Hii KS, Lim PT, Kon NF, Takata Y, Usup G, Leaw CP (2016) Physiological and transcriptional responses to inorganic nutrition in a tropical Pacific strain of Alexandrium minutum: implications for the saxitoxin genes and toxin production. Harmful Algae 56:9–21CrossRefGoogle Scholar
  30. 30.
    D’Agostino PM, Woodhouse JN, Makower AK, Ac Yeung, Ongley SE, Micallef ML, Moffitt MC, Neilan BA (2016) Advances in genomics, transcriptomics and proteomics of toxin producing cyanobacteria. Environ Microbiol Rep 8(1):3–13CrossRefGoogle Scholar
  31. 31.
    Lazarevic V, Gaïa N, Girard M, François P, Schrenzel J (2013) Comparison of DNA extraction methods in analysis of salivary bacterial communities. PLoS ONE 8(7):e67699CrossRefGoogle Scholar
  32. 32.
    Bolger AM, Lohse M, Usadel B (2014) Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30(15):170CrossRefGoogle Scholar
  33. 33.
    Mende DR, Waller AS, Sunagawa S, Järvelin AI, Chan MM, Arumugam M, Raes J, Bork P (2012) Assessment of metagenomic assembly using simulated next generation sequencing data. PLoS ONE 7(2):e31386CrossRefGoogle Scholar
  34. 34.
    Zerbino DR, Birney E (2008) Velvet: algorithms for de novo short read assembly using de Bruijn graphs. Genome Res 18(5):821–829CrossRefGoogle Scholar
  35. 35.
    Namiki T, Hachiya T, Tanaka H, Sakakibara Y (2012) MetaVelvet: an extension of Velvet assembler to de novo metagenome assembly from short sequence reads. Nucleic Acids Res 40(20):e155–e155CrossRefGoogle Scholar
  36. 36.
    Rho M, Tang H, Ye Y (2010) FragGeneScan: predicting genes in short and error-prone reads. Nucleic Acids Res 38(20):e191–e191CrossRefGoogle Scholar
  37. 37.
    Buchfink B, Xie C, Huson DH (2015) Fast and sensitive protein alignment using DIAMOND. Nat Methods 12(1):59–60CrossRefGoogle Scholar
  38. 38.
    Huson DH, Auch AF, Qi J, Schuster SC (2007) MEGAN analysis of metagenomic data. Genome Res 17(3):377–386CrossRefGoogle Scholar
  39. 39.
    Huson DH, Mitra S, Ruscheweyh HJ, Weber N, Schuster SC (2011) Integrative analysis of environmental sequences using MEGAN4. Genome Res 21(9):1552–1560CrossRefGoogle Scholar
  40. 40.
    Komárek J, Komárková J (2004) Taxonomic review of the cyanoprokaryotic genera Planktothrix and Planktothricoides. Czech Phycol 4:1–18Google Scholar
  41. 41.
    Komárek J, Zapomělová E (2007) Planktic morphospecies of the cyanobacterial genus Anabaena = subg. Dolichospermum–1. Part: coiled types. Fottea 7(1):1–31CrossRefGoogle Scholar
  42. 42.
    Komárek J, Zapomělová E (2007) Planktic morphospecies of the cyanobacterial genus Anabaena = subg. Dolichospermum–2. part: straight types. Fottea 8(1):1–14CrossRefGoogle Scholar
  43. 43.
    Felisberto SA, e Souza DBS (2014) Characteristics and diversity of cyanobacteria in periphyton from lentic tropical ecosystem, Brazil. Adv Microbiol 4(15):1076CrossRefGoogle Scholar
  44. 44.
    HRWEMD (2012) Distribution and eco-physiological characteristics of harmful algae in North Han River. Final Report, H.R.W.a.E.M. District)Google Scholar
  45. 45.
    HRWEMD (2013) Investigation of causes of off-flavor material production by harmful algae and management strategy. Final Report, H.R.W.a.E.M. DistrictGoogle Scholar
  46. 46.
    Kim KH, Lim BJ, You KA, Park MH, Park JH, Kim KH, Hwang SJ (2014) Identification and analysis of geosmin production potential of Anabaena strain isolated from North Han River. Korean J Ecol Environ 47(4):7Google Scholar
  47. 47.
    Tamura K, Stecher G, Peterson D, Filipski A, Kumar S (2013) MEGA6: molecular evolutionary genetics analysis version 6.0. Mol Biol Evol 30(12):2725–2729CrossRefGoogle Scholar
  48. 48.
    Han MS, Auh YY, Ryu JK, Yoo KI, Choi YK (1995) Ecological studies on Pal'tang River-reservoir system in Korea 2. Changes in phytoplankton community structure. Korean J Limnol 28:335–344Google Scholar
  49. 49.
    Hong SS, Auh YY (2002) Ecological studies on Pal'tang river—reservoir system in Korea 4. Dynamics on inorganic nutrients, POM and phytoplankton succession in the lower stream Kyungan. Korean J Ecol Environ 35(1):9Google Scholar
  50. 50.
    Han MS, Jheong WH, Park JD, Kim JM (2005) Correlation between phytoplankton dynamics and water quality in Paldang Reservoir. Korean J Limnolo 38(2):7Google Scholar
  51. 51.
    Park H, Lee HJ, Kim EK, Jung DI (2005) Characteristics of algal abundance and statistical analysis of environmental factors in Lake Paldang. J Korean Soc Water Qual 21:584–594Google Scholar
  52. 52.
    HRWEMD (2015) Investigation of causes of off-flavor material production by harmful algae and management strategy. Final Report, H.R.W.a.E.M. District, EditorGoogle Scholar
  53. 53.
    Casero MC, Ballot A, Agha R, Quesada A, Cirés S (2014) Characterization of saxitoxin production and release and phylogeny of sxt genes in paralytic shellfish poisoning toxin-producing Aphanizomenon gracile. Harmful algae 37:28–37CrossRefGoogle Scholar
  54. 54.
    Kellmann R, Mihali TK, Jeon YJ, Pickford R, Pomati F, Neilan BA (2008) Biosynthetic intermediate analysis and functional homology reveal a saxitoxin gene cluster in cyanobacteria. Appl Environ Microbiol 74(13):4044–4053CrossRefGoogle Scholar
  55. 55.
    Al-Tebrineh J, Mihali TK, Pomati F, Neilan BA (2010) Detection of saxitoxin-producing cyanobacteria and Anabaena circinalis in environmental water blooms by quantitative PCR. Appl Environ Microbiol 76(23):7836–7842CrossRefGoogle Scholar
  56. 56.
    Beltran EC, Neilan BA (2000) Geographical segregation of the neurotoxin-producing cyanobacterium Anabaena circinalis. Appl Environ Microbiol 66(10):4468–4474CrossRefGoogle Scholar
  57. 57.
    Mahmood NA, Carmichael WW (1986) Paralytic shellfish poisons produced by the freshwater cyanobacterium Aphanizomenon flos-aquae NH-5. Toxicon 24(2):175–186CrossRefGoogle Scholar
  58. 58.
    Cirés S, Wörmer L, Ballot A, Agha R, Wiedner C, Velázquez D, Casero MC, Quesada A (2014) Phylogeography of cylindrospermopsin and paralytic shellfish toxin-producing Nostocales cyanobacteria from Mediterranean Europe (Spain). Appl Environ Microbiol 80(4):1359–1370CrossRefGoogle Scholar
  59. 59.
    Smith FM, Wood SA, van Ginkel R, Broady PA, Gaw S (2011) First report of saxitoxin production by a species of the freshwater benthic cyanobacterium, Scytonema Agardh. Toxicon 57(4):566–573CrossRefGoogle Scholar
  60. 60.
    Ongley SE, Pengelly JJ, Neilan BA (2016) Elevated Na+ and pH influence the production and transport of saxitoxin in the cyanobacteria Anabaena circinalis AWQC131C and Cylindrospermopsis raciborskii T3. Environ Microbiol 18(2):427–438CrossRefGoogle Scholar
  61. 61.
    Wang D-Z, Zhang SF, Zhang Y, Lin L (2016) Paralytic shellfish toxin biosynthesis in cyanobacteria and dinoflagellates: a molecular overview. J Proteom 135:132–140CrossRefGoogle Scholar
  62. 62.
    McGregor GB (2013) Freshwater cyanobacteria of north-eastern Australia: 2. Chroococcaes (Phytotaxa 133). Magnolia Press, Auckland, p 130Google Scholar

Copyright information

© The Korean Society for Applied Biological Chemistry 2018

Authors and Affiliations

  • Keon Hee Kim
    • 1
  • Youngdae Yoon
    • 1
  • Woon-Young Hong
    • 3
  • JaeBum Kim
    • 2
  • Yung-Chul Cho
    • 4
  • Soon-Jin Hwang
    • 1
  1. 1.Department of Environmental Health ScienceKonkuk UniversitySeoulRepublic of Korea
  2. 2.Department of Biomedical Science and EngineeringKonkuk UniversitySeoulRepublic of Korea
  3. 3.Department of Animal BiotechnologyKonkuk UniversitySeoulRepublic of Korea
  4. 4.Department of Environmental EngineeringChungbuk National UniversityCheongjuRepublic of Korea

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