Antonie van Leeuwenhoek

, Volume 101, Issue 4, pp 819–835 | Cite as

Spatial and temporal analysis of estuarine bacterioneuston and bacterioplankton using culture-dependent and culture-independent methodologies

  • Juliana S. N. Azevedo
  • Isabel Ramos
  • Susana Araújo
  • Cláudia S. Oliveira
  • António Correia
  • Isabel S. Henriques
Original Paper


Bacterioneuston may play a key role in water–air exchange of gases and in processing organic matter and pollutants that accumulate at the sea-surface microlayer (SML). However, the phylogenetic diversity of bacterioneuston has been poorly characterized. We analyzed 24 samples each from the SML and underlying water (UW) at three sites in the Ria de Aveiro estuary, Portugal. Cultivation and culture-independent techniques were used to compare bacterioneuston and bacterioplankton. Culturable heterotrophic bacteria were enriched in the SML. The culturable community was dominated by Psychrobacter and Acinetobacter. The presence of high numbers of Psychrobacter was a notable result. Differences were confined to a few genera overrepresented in UW samples (Kocuria, Agrococcus and Vibrio). 16S rDNA DGGE profiles were highly stable in terms of number and position of bands between sampling sites and dates but cluster analysis revealed a slight tendency for grouping according to sampled layer. SML-specific DGGE bands affiliated with Actinobacteria, Cyanobacteria, Gammaproteobacteria and Bacteroidetes. Low similarity between nucleotide sequences of DGGE-bands and previously reported sequences suggest the occurrence of SML-specific populations. Enrichment of SML for Pseudomonas and Aeromonas was questioned and the diversity of both communities was analyzed. Consistent differences between SML and UW aeromonads communities were not identified. In terms of Pseudomonas, a culturable operational taxonomic unit was consistently overrepresented within SML samples. Taken together, our results indicate that the similarity between SML and UW communities depends on spatial and temporal factors.


Phylogenetic diversity biofilm Sea-surface microlayer Estuary DGGE 



This work was financed by the Fundacão para a Ciência e a Tecnologia (FCT) through grants SFRH/BPD/65820/2009 (CSO), SFRH/BD/64057/2009 (JSNA) and SFRH/BPD/21384/2005 (IH) and by the European Union Program of High Level Scholarships for Latin America––Program Alβan––through grant E07D403901BR (JSNA). The authors wish to thank Ângela Cunha and Adelaide Almeida for technical support (sampling device, conductivity meter, etc.). We are also grateful to A. Moura (Department of Biology, University of Aveiro) for reviewing English style and grammar.

Supplementary material

10482_2012_9697_MOESM1_ESM.doc (320 kb)
Supplementary material 1 (DOC 320 kb)


  1. Agogué H, Casamayor EO, Joux F, Obernosterer I, Dupuy C, Lantoine F, Catala P, Weinbauer MG, Reinthaler T, Herndl GJ, Lebaron P (2004) Comparison of samplers for the biological characterisation of the sea-surface microlayer. Limnol Oceanogr Methods 2:213–225CrossRefGoogle Scholar
  2. Agogué H, Casamayor EO, Bourrain M, Obernosterer I, Joux F, Herndl GJ, Lebaron P (2005) A survey on bacteria inhabiting the sea-surface microlayer of coastal ecosystems. FEMS Microbiol Ecol 54:269–280PubMedCrossRefGoogle Scholar
  3. Altschul S, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402PubMedCrossRefGoogle Scholar
  4. Calhau V, Tacão M, Morgado F, Correia A, Henriques I (2010) PCR-DGGE methodologies to study diversity and dynamics of Aeromonas communities. J Appl Microbiol 108:611–623PubMedCrossRefGoogle Scholar
  5. Cincinelli A, Stortini AM, Checchini L, Martellini T, Del Bubba M, Lepri L (2005) Enrichment of organic pollutants in the sea-surface microlayer (SML) at Terra Nova Bay, Antarctica: influence of SML on superficial snow composition. J Environ Monit 7:1305–1312PubMedCrossRefGoogle Scholar
  6. Clarke KR, Gorley RN (2001) PRIMER v5: User manual/tutorial. PRIMER-E, Plymouth, p 91Google Scholar
  7. Coelho FJRC, Sousa S, Santos L, Santos AL, Almeida A, Gomes NCM, Cunha A (2010) PAH Degrading bacteria in an estuarine system. Studies on environmental chemistry-biological responses to contaminants. In: Hamamura N, Suzuki S, Mendo S, Barroso CM, Iwata H, Tanabe S (eds) Interdisciplinary studies on environmental chemistry-biological responses to contaminants. Center for Marine Environmental Studies, Ehime University, Japan, Department of Biology, University of Aveiro, Portugal, pp 77–87Google Scholar
  8. Cole JR, Wang Q, Cardenas E, Fish J, Chai B, Farris RJ, Kulam-Syed-Mohideen AS, McGarrell DM, Marsh T, Garrity GM, Tiedje JM (2009) The ribosomal database project: improved alignments and new tools for rRNA analysis. Nucleic Acids Res 37:141–145CrossRefGoogle Scholar
  9. Conrad R, Seiler W (1988) Influence of the surface microlayer on the flux of nonconservative trace gases (CO, H2, CH4, N2O) across the ocean-atmosphere interface. J Atmos Chem 6:83–94CrossRefGoogle Scholar
  10. Cunliffe M, Murrell JC (2009) The sea-surface microlayer is a gelatinous biofilm. ISME J 3:1001–1003PubMedCrossRefGoogle Scholar
  11. Cunliffe M, Schafer H, Harrison E, Cleave S, Upstill-Goddard RC, Murrell JC (2008) Phylogenetic and functional gene analysis of the bacterial and archaeal communities associated with the surface microlayer of an estuary. ISME J 2:776–789PubMedCrossRefGoogle Scholar
  12. Cunliffe M, Harrison E, Salter M, Schäfer H, Upstill-Goddard RC, Murrell JC (2009) Comparison and validation of sampling strategies for the molecular microbial analysis of surface microlayers. Aquat Microb Ecol 57:69–77CrossRefGoogle Scholar
  13. Cunliffe M, Upstill-Goddard RC, Murrell JC (2011) Microbiology of aquatic surface microlayers. FEMS Microbiol Rev 35:233–246PubMedCrossRefGoogle Scholar
  14. Cuong DT, Karuppiah R, Obbard JP (2008) Distribution of heavy metals in the dissolved and suspended phase of the sea-surface microlayer, seawater column and in sediments of Singapore’s coastal environment. Environ Monit Assess 138:255–272PubMedCrossRefGoogle Scholar
  15. Doğruöz N, Göksay D, Ilhan-Sungur E, Cotuk A (2009) Pioneer colonizer microorganisms in biofilm formation on galvanized steel in a simulated recirculating cooling-water system. J Basic Microb 49:5–12CrossRefGoogle Scholar
  16. Franklin MP, McDonald IR, Bourne DG, Owens NJP, Upstill-Goddard RC, Murrell JC (2005) Bacterial diversity in the bacterioneuston (sea-surface microlayer): the bacterioneuston through the looking glass. Environ Microbiol 7:723–736PubMedCrossRefGoogle Scholar
  17. Frette L, Johnsen K, Jorgensen N, Nybroe O, Kroer N (2004) Functional characteristics of culturable bacterioplankton from marine and estuarine environments. Int Microbiol 7:219–227PubMedGoogle Scholar
  18. Hardy JT (1982) The sea-surface microlayer: biology, chemistry and anthropogenic enrichment. Prog Oceanogr 11:307–328CrossRefGoogle Scholar
  19. Harwati TU, Kasai Y, Kodama Y, Susilaningsih D, Watanabe K (2007) Characterization of diverse hydrocarbon-degrading bacteria isolated from Indonesian seawater. Microbes Environ 22:412–415CrossRefGoogle Scholar
  20. Henriques IS, Almeida A, Cunha A, Correia A (2004) Molecular sequence analysis of prokaryotic diversity in the middle and outer sections of the Portuguese estuary Ria de Aveiro. FEMS Microbiol Ecol 49:269–279PubMedCrossRefGoogle Scholar
  21. Henriques IS, Alves A, Tacão M, Almeida A, Cunha A, Correia A (2006) Seasonal and spatial variability of free-living bacterial community composition along an estuarine gradient (Ria de Aveiro, Portugal). Estuar Coast Shelf S 68:139–148CrossRefGoogle Scholar
  22. Hervàs A, Casamayor EO (2009) High similarity between bacterioneuston and airborne bacterial community compositions in a high mountain lake area. FEMS Microbiol Ecol 67:219–228PubMedCrossRefGoogle Scholar
  23. Hervàs A, Camarero L, Reche I, Casamayor EO (2009) Viability and potential for immigration of airborne bacteria from Africa that reach high mountain lakes in Europe. Environment Microbiol 11:1612–1623CrossRefGoogle Scholar
  24. Joux F, Agogue H, Obernosterer I, Dupuy C, Reinthaler T, Herndl GJ, Lebaron P (2006) Microbial community structure in the sea-surface microlayer at two contrasting coastal sites in the northwestern Mediterranean Sea. Aquat Microb Ecol 42:91–104CrossRefGoogle Scholar
  25. Keddy PA (2000) Wetland ecology: principles and conservation. Cambridge University Press, Cambridge, pp 3–77Google Scholar
  26. Laiz L, Pinãr G, Lubitz W, Saiz-Jimenez C (2003) Monitoring the colonization of monuments by bacteria: cultivation versus molecular methods. Environ Microbiol 5:72–74PubMedCrossRefGoogle Scholar
  27. Lane DJ (1991) 16S/23S rRNA Sequencing. In: Stackebrandt E, Goodfellow M (eds) Nucleic acid techniques in bacterial systematics. John Wiley and Sons, New York, pp 115–175Google Scholar
  28. Liss PS, Watson AJ, Bock EJ, Jahne B, Asher WE, Frew NM, Hasse L, Korenowski GM, Merlivat L, Philips LF, Schluessel P, Woolf DK (1997) Physical processes in the microlayer and the air–sea exchange of trace gases. In: Liss PS, Duce RA (eds) The sea surface and global change. Cambridge University Press, Cambridge, pp 1–34CrossRefGoogle Scholar
  29. Lo Giudice A, Casella P, Caruso C, Mangano S, Bruni V, De Domenico M, Michaud L (2010) Occurrence and characterization of psychrotolerant hydrocarbon-oxidizing bacteria from surface seawater along the Victoria Land coast (Antarctica). Polar Biol 33:929–943CrossRefGoogle Scholar
  30. Maki JS (2002) Neuston microbiology: life at the air–water interface. In: Bitton G (ed) Encyclopedia of environmental microbiology. John Wiley and Sons, New York, pp 2133–2144Google Scholar
  31. Monteiro M, Quintaneiro C, Nogueira AJA, Morgado F, Soares AMVM, Guilhermino L (2007) Impact of chemical exposure on the fish Pomatoschistus microps Krøyer (1838) in estuaries of the Portuguese Northwest coast. Chemosphere 66:514–522PubMedCrossRefGoogle Scholar
  32. Muyzer G, De Waal EC, Uitterlinden AG (1993) Profiling of complex microbial populations by denaturing gradient gel electrophoresis of polymerase chain reaction-amplified genes coding for 16S rRNA. Appl Environ Microbiol 59:695–700PubMedGoogle Scholar
  33. Naumann E (1917) Beiträge zur Kenntnis des Teichnannoplanktons. II. Über das Neustons des Süsswasser. Biologie Zentralbl 37:98–106Google Scholar
  34. Obernosterer I, Catala P, Lami R, Caparros J, Ras J, Bricaud A, Dupuy C, Van Wambeke F, Lebaron P (2008) Biochemical characteristics and bacterial community structure of the sea-surface microlayer in the South Pacific Ocean. Biogeosciences 5:693–705CrossRefGoogle Scholar
  35. Peltzer RD, Griffin OM, Barger WR, Kaiser JAC (1992) High-resolution measurement of surface-active film redistribution in ship wakes. J Geophys Res 97:5231–5252CrossRefGoogle Scholar
  36. Prabagaran SR, Manorama R, Delille D, Shivaji S (2007) Predominance of Roseobacter, Sulfitobacter, Glaciecola and Psychrobacter in sea water collected off Ushuaia, Argentina sub-Antartica. FEMS Microbiol Ecol 59:342–355PubMedCrossRefGoogle Scholar
  37. Reche I, Ortega-Retuerta E, Romera O, Pulido-Villena E, Morales-Baquero R, Casamayor EO (2009) Effects of Saharan dust inputs on bacterial activity and community composition in Mediterranean lakes and reservoirs. Limnol Oceanogr 54:869–879CrossRefGoogle Scholar
  38. Rodrigues DF, Jesus EC, Ayala-Del-Río HL, Pellizari VH, Gillichinsky D, Sepulveda-Torres L, Tiedje JM (2009) Biogeography of two cold-adapted genera: Psychrobacter and Exiguobacteria. ISME J 3:658–665PubMedCrossRefGoogle Scholar
  39. Santos AL, Mendes C, Gomes NCM, Henriques I, Correia A, Almeida A, Cunha A (2009) Short-term variability of abundance, diversity and activity of estuarine bacterioneuston and bacterioplankton. J Plankton Res 31:1545–1555CrossRefGoogle Scholar
  40. Santos AL, Henriques I, Gomes NCM, Almeida A, Correia A, Cunha A (2011a) Effects of ultraviolet radiation on the abundance, diversity and activity of bacterioneuston and bacterioplankton: insights from microcosm studies. Aquatic Sci 73:63–77CrossRefGoogle Scholar
  41. Santos AL, Lopes S, Baptista I, Henriques I, Gomes NCM, Almeida A, Correia A, Cunha A (2011b) Diversity in UV sensitivity and recovery potential among bacterioneuston and bacterioplankton isolates. Lett Appl Micróbio 52:360–366CrossRefGoogle Scholar
  42. Sieburth JM, Willis PJ, Johnson KM, Burney CM, Lavoie DM, Hinga KR, Caron DA, French FW III, Johnson PW, Davis PG (1976) Dissolved organic matter and heterotrophic microneuston in the surface microlayers of the North Atlantic. Science 194:1415–1418PubMedCrossRefGoogle Scholar
  43. Tamaki H, Sekiguchi Y, Hanada S, Nakamura K, Nomura N, Matsumura M, Kamagata Y (2005) Comparative analysis of bacterial diversity in freshwater sediment of a shallow eutrophic lake by molecular and improved cultivation-based techniques. Appl Environ Microbiol 71:2162–2169PubMedCrossRefGoogle Scholar
  44. Upstill-Goddard RC, Frost T, Henry GR, Franklin M, Murrell JC, Owens NJP (2003) Bacterioneuston control of air-water methane exchange determined with a laboratory gas exchange tank. Global Biogeochem Cy 17:1–15CrossRefGoogle Scholar
  45. Weiner RM, Hussong D, Colwell RR (1980) An estuarine agar medium for enumeration of aerobic heterotrophic bacteria associated with water, sediment and shellfish. Can J Microbiol 26:1355–1369CrossRefGoogle Scholar
  46. Zemmelink HJ, Houghton L, Sievert SM, Frew NM, Dacey JWH (2005) Gradients in dimethylsulfide, dimethylsulfoniopropionate, dimethylsulfoxide, and bacteria near the sea surface. Mar Ecol Prog Ser 295:33–42CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2012

Authors and Affiliations

  • Juliana S. N. Azevedo
    • 1
  • Isabel Ramos
    • 2
    • 3
  • Susana Araújo
    • 2
  • Cláudia S. Oliveira
    • 1
  • António Correia
    • 1
  • Isabel S. Henriques
    • 1
  1. 1.CESAM and Department of BiologyUniversity of AveiroAveiroPortugal
  2. 2.Department of BiologyUniversity of AveiroAveiroPortugal
  3. 3.Department of Microbiology, Faculty of MedicineUniversity of PortoPortoPortugal

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