Microbial Ecology

, Volume 54, Issue 3, pp 523–531 | Cite as

Monitoring Diel Variations of Physiological Status and Bacterial Diversity in an Estuarine Microbial Mat: An Integrated Biomarker Analysis

  • Laura VillanuevaEmail author
  • Antoni Navarrete
  • Jordi Urmeneta
  • Roland Geyer
  • David C. White
  • Ricardo Guerrero


Microbial mats are highly productive microbial systems and a source of not-yet characterized microorganisms and metabolic strategies. In this article, we introduced a lipid biomarker/microbial isolation approach to detect short-term variations of microbial diversity, physiological and redox status, and also characterize lipid biomarkers from specific microbial groups that can be further monitored. Phospholipid fractions (PLFA) were examined for plasmalogens, indicative of certain anaerobes. The glycolipid fraction was processed for polyhydroxyalkanoates (PHA) and the neutral lipid fraction was used to evaluate respiratory quinone content. Data demonstrate an increase in the metabolic stress, unbalanced growth, proportion of anaerobic bacteria and respiratory rate after the maximal photosynthetic activity. Higher accumulation of polyhydroxyalkanoates at the same sampling point also suggested a situation of carbon storage by heterotrophs closely related to photosynthetic microorganisms. Besides, the characterization of lipid biomarkers (plasmalogens, sphingolipids) from specific microbial groups provided clues about the dynamics and diversity of less-characterized mat members. In this case, lipid analyses were complemented by the isolation and characterization of anaerobic spore formers and sulfate reducers to obtain insight into their affiliation and lipid composition. The results revealed that temporal shifts in lipid biomarkers are indicative of an intense change in the physiology, redox condition, and community composition along the diel cycle, and support the hypothesis that interactions between heterotrophs and primary producers play an important role in the carbon flow in microbial mats.


Sphingoid Base Respiratory Quinone Ebro Delta Neutral Lipid Fraction Total PLFA 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.



This paper is dedicated to the memory of David C. White: “Thank you for being a friend and mentor, we will miss you.” We thank Mercè Piqueras and Wendy Ran for useful suggestions. We are grateful to the Center for Biomarker Analysis (TN, USA) staff for advice and technical assistance. This research was supported by Spanish MCyT grant BOS2002-02944 and MEC CGL2005-04990, and by grant DE-FC02-96ER62278, from the Office of Biological and Environmental Research (OBER) and the Natural and Accelerated Bioremediation Research (NABIR) Program. LV was recipient of a scholarship from the Spanish MECD (AP2001-0953).


  1. 1.
    Anderson, KL, Tayne, TA, Ward, DM (1987) Formation and fate of fermentation products in hot spring cyanobacterial mats. Appl Environ Microbiol 53: 2343–2352PubMedGoogle Scholar
  2. 2.
    Baker, GC, Smith, JJ, Cowan, DA (2003) Review and re-analysis of domain-specific 16S primers. J Microbiol Methods 55: 541–555PubMedCrossRefGoogle Scholar
  3. 3.
    Berlanga, M, Motero, MT, Fernández-Borrell, J, Guerrero, R (2006) Rapid spectrofluorometric screening of poly-ydroxyalkanoate-producing bacteria from microbial mats. Int Microbiol 9: 95–102PubMedGoogle Scholar
  4. 4.
    Bligh, EG, Dyer, WJ (1959) A rapid method of total lipid extraction and purification. Can J Biochem Physiol 37: 911–917PubMedGoogle Scholar
  5. 5.
    Brisbarre, N, Fardeau, ML, Cueff, V, Cayol, JL, Barbier, G, Cilia, V, Ravot, G, Thomas, P, Garcia, JL, Ollivier, B (2003) Clostridium caminithermale sp. nov., a slightly halophilic and moderately thermophilic bacterium isolated from an Atlantic deep-sea hydrothermal chimney. Int J Syst Evol Microbiol 53: 1043–1049PubMedCrossRefGoogle Scholar
  6. 6.
    Canfield, DE, Des Marais, DJ (1993) Biogeochemical cycles of carbon, sulfur, and free oxygen in a microbial mat. Geochim Cosmochim Acta 57: 3971–3984PubMedCrossRefGoogle Scholar
  7. 7.
    Collins, MD, Jones, D (1981) Distribution of isoprenoid quinone structural types in bacteria and their taxonomic implications. Microbiol Rev 45: 316–354PubMedGoogle Scholar
  8. 8.
    Doi, Y, Tamaki, A, Kunioka, M, Soga, K (1987) Biosynthesis of terpolyesters of 3-hydroxybutyrate, 3-hydroxyvalerate, and 5-hydroxyvalerate in Alcaligenes eutrophus from 5-chloropentanoic and pentanoic acids. Macromol Rapid Commun 8: 631–635CrossRefGoogle Scholar
  9. 9.
    Elhottová, D, Tríska, J, Petersen, SO, Santruckvá, H (2000) Analysis of poly-β-hydroxybutyrate in environmental samples by GC-MS/MS. Fresenius J Anal Chem 367: 157–164PubMedCrossRefGoogle Scholar
  10. 10.
    van Germerden, H (1993) Microbial mats: a joint venture. Mar Geol 113: 3–25CrossRefGoogle Scholar
  11. 11.
    Geyer, R, Peacock, AD, White, DC, Lytle, C, Van Berkel, GJ (2004) Atmospheric pressure chemical ionization and atmospheric pressure photoionization for simultaneous mass spectrometric analysis of microbial respiratory ubiquinones and menaquinones. J Mass Spectrom 39: 922–929PubMedCrossRefGoogle Scholar
  12. 12.
    Gottwald, M, Andersen, JR, Le Gall, J, Ljungdabl, LG (1975) Presence of cytochrome and menaquinone in Clostridium formicoaceticum and Clostridium thermoaceticum. J Bacteriol 122: 325–328PubMedGoogle Scholar
  13. 13.
    Grötzschel, S, de Beer, D (2002) Effect of oxygen concentration on photosynthesis and respiration in two hypersaline microbial mats. Microb Ecol 44: 208–216PubMedCrossRefGoogle Scholar
  14. 14.
    Guckert, JB, Antworth, CP, Nichols, PD, White, DC (1985) Phospholipid, ester-linked fatty acid profiles as reproducible assays for changes in prokaryotic community structure of estuarine sediments. FEMS Microbiol Ecol 31: 147–158Google Scholar
  15. 15.
    Guckert, JB, Hood, MA, White, DC (1986) Phospholipid, ester-linked fatty acids profile changes during nutrient deprivation of Vibrio cholerae: increases in the trans/cis ratio and proportions of cyclopropyl fatty acids. Appl Environ Microbiol 52: 794–801PubMedGoogle Scholar
  16. 16.
    Guerrero, R, Urmeneta, J, Rampone, G (1993) Distribution of types of microbial mats at the Ebro delta, Spain. BioSystems 31: 135–144PubMedCrossRefGoogle Scholar
  17. 17.
    Hedrick, DB, White, DC (1986) Microbial respiratory quinones in the environment. J Microbiol Methods 5: 243–254CrossRefGoogle Scholar
  18. 18.
    Heipieper, H-J, Diefenbach, R, Keweloh, H (1992) Conversion of cis unsaturated fatty acids to trans, a possible mechanism for the protection of phenol-degrading Pseudomonas putida P8 from substrate toxicity. Appl Environ Microbiol 58: 1847–1852PubMedGoogle Scholar
  19. 19.
    Hiraishi, A (1999) Isoprenoid quinones as biomarkers of microbial populations in the environment. J Biosci Bioeng 88: 449–460PubMedCrossRefGoogle Scholar
  20. 20.
    Hiraishi, A, Iwasaki, M, Kawagishi, T, Yoshida, N, Narihiro, T, Kata, K (2003) Significance of Lipoquinone as quantitative biomarkers of bacterial populations in the environment. Microb Environ 18: 89–93CrossRefGoogle Scholar
  21. 21.
    Iwasaki, M, Hiraishi, A (1998) A new approach to numerical analyses of microbial quinone profiles in the environment. Microb Environ 13: 67–76Google Scholar
  22. 22.
    Jonkers, HM, Abed, RMM (2003) Identification of aerobic heterotrophic bacteria from the photic zone of a hypersaline microbial mat. Aquat Microb Ecol 30: 127–133CrossRefGoogle Scholar
  23. 23.
    Jonkers, HM, Koh IO, Behrend, P, Muyzer, G, De Beer, D (2005) Aerobic organic carbon mineralization by sulfate-reducing bacteria in the oxygen-saturated photic zone of a hypersaline microbial mat. Microb Ecol 49: 1–10CrossRefGoogle Scholar
  24. 24.
    Kato, M, Muto, Y, Tanaka-Bandoh, K, Watanabe, K, Ueno, K (1995) Sphingolipid composition in Bacteroides species. Anaerobe 1: 135–139PubMedCrossRefGoogle Scholar
  25. 25.
    Krumbein, WE, Gorbushina, AA, Holtkamp-Tacken, E (2004) Hypersaline microbial systems of sabkhas: examples of life’s survival in “extreme” conditions. Astrobiology 4: 450–459PubMedCrossRefGoogle Scholar
  26. 26.
    Lauro, FM, Bertoloni, G, Obraztsova, A, Kato, C, Tebo, BM, Bartlett, DH (2004) Pressure effects on Clostridium strains isolated from a cold deep-sea environment. Extremophiles 8: 169–173PubMedCrossRefGoogle Scholar
  27. 27.
    Leung, KT, Chang, YJ, Gan, YD, Peacock, A, Macnaughton, SJ, Stephen, JR, Burkhalter, RS, Flemming, CA, White, DC (1999) Detection of Sphingomonas spp. in soil by PCR and sphingolipid biomarker detection. J Ind Microbiol Biotech 23: 252–260CrossRefGoogle Scholar
  28. 28.
    Mayberry, WR, Lane, JR (1993) Sequential alkaline saponification/acid hydrolysis/esterification: a one-tube method with enhanced recovery of both cyclopropane and hydroxylated fatty acids. J Microbiol Methods 18: 21–32CrossRefGoogle Scholar
  29. 29.
    Mayberry, WR, Smith, PF, Langworthy, TA, Plackett, P (1973) Identification of the amide-linked fatty acids of Acholeplasma axanthum S743 as d(-)3-hydroxydecanoate and its homologues. J Bacteriol 116: 1091–1095PubMedGoogle Scholar
  30. 30.
    Mir, J, Martínez-Alonso, M, Esteve, I, Guerrero, R (1991) Vertical stratification and microbial assemblage of a microbial mat in the Ebro Delta (Spain). FEMS Microbiol Ecol 86: 59–68CrossRefGoogle Scholar
  31. 31.
    Moore, LVH, Borne, DM, Moore, WEC (1994) Comparative distribution and taxonomic value of cellular fatty acids in thirty-three genera of anaerobic Gram-negative bacteria. Int J Syst Bacteriol 44: 338–347PubMedCrossRefGoogle Scholar
  32. 32.
    Mountfort, DO, Rainey, FA, Burghardt, J, Kaspar, HF, Stackebrandt, E (1997) Clostridium vincentii sp. nov., a new obligately anaerobic, saccharolytic, psychrophilic bacterium isolated from low-salinity pond sediment of the McMurdo Ice Shelf, Antarctica. Arch Microbiol 167: 54–60PubMedCrossRefGoogle Scholar
  33. 33.
    Navarrete, A, Peacock, A, Macnaughton, SJ, Urmeneta, J, Mas-Castellà, J, White, DC, Guerrero, R (2000) Physiological status and community composition of microbial mats of the Ebro delta, Spain, by Signature Lipid Biomarkers. Microb Ecol 39: 92–99PubMedCrossRefGoogle Scholar
  34. 34.
    Navarrete, A, Urmeneta, J, Cantu, JM, Vegas, E, White, DC, Guerrero, R (2004) Signature lipid biomarkers of microbial mats of the Ebro delta (Spain), Camargue and Étang de Berre (France): an assessment of biomass and activity. Ophelia 58: 175–188Google Scholar
  35. 35.
    Ollivier, B, Caumette, P, García, J-L, Mah, RA (1994) Anaerobic bacteria from hypersaline environments. Microbiol Rev 58: 27–38PubMedGoogle Scholar
  36. 36.
    Peacock, AD, Chang, Y-J, Istok, JD, Krumholz, L, Geyer, R, Kinsall, B, Watson, D, Sublette, KL, White, DC (2004) Utilization of microbial biofilms as monitors of bioremediation. Microb Ecol 47: 284–292PubMedCrossRefGoogle Scholar
  37. 37.
    Rothermich, MM, Guerrero, R, Lenz, RW, Goodwin, S (2000) Characterization, seasonal occurrence, and diel fluctuations of poly(hydroxyalkanoate) in photosynthetic microbial mats. Appl Environ Microbiol 66: 4279–4291PubMedCrossRefGoogle Scholar
  38. 38.
    Rütters, H, Sass, H, Cypionka, H, Rullkötter, J (2001) Monoalkylether phospholipids in the sulfate-reducing bacteria Desulfosarcina variabilis and Desulforhabdus amnigenus. Arch Microbiol 176: 435–442PubMedCrossRefGoogle Scholar
  39. 39.
    Smith, LD (1970) Clostridium oceanicum, sp. nov., a sporeforming anaerobe isolated from marine sediments. J Bacteriol 103: 811–813PubMedGoogle Scholar
  40. 40.
    Spring, S, Merkhoffer, B, Weiss, N, Kroppenstedt, RM, Hippeand, H, Stackebrandt, E (2003) Characterization of novel psychrophilic clostridia from an Antarctic microbial mat: description of Clostridium frigoris sp. nov., Clostridium lacusfryxellense sp. nov., Clostridium bowmanii sp. nov. and Clostridium psychrophilum sp. nov. and reclassification of Clostridium laramiense as Clostridium estertheticum subsp. laramiense subsp. nov. Int J Syst Evol Microbiol 53: 1019–1029PubMedCrossRefGoogle Scholar
  41. 41.
    Vestal, JR, White, DC (1989) Lipid analysis in microbial ecology: quantitative approaches to the study of microbial communities. BioScience 39: 535–541PubMedCrossRefGoogle Scholar
  42. 42.
    Villanueva, L, Navarrete, A, Urmeneta, J, White, DC, Guerrero, R (2004) Combined phospholipid biomarker-16S rRNA gene denaturing gradient gel electrophoresis analysis of bacterial diversity and physiological status in an intertidal microbial mat. Appl Environ Microbiol 70: 6920–6926PubMedCrossRefGoogle Scholar
  43. 43.
    White, DC, Bobbie, RJ, Heron, JS, King, JD, Morrison, SJ (1979) Biochemical measurements of microbial mass and activity from environmental samples. In: Costerton, JW, Colwell, RR (Eds.) Native Aquatic Bacteria: Enumeration, Activity and Ecology. ASTM STP 695, American Society for Testing and Materials, Philadelphia, PA, pp 69–81Google Scholar
  44. 44.
    Wieland, A, Kühl, M (2006) Regulation of photosynthesis and oxygen consumption in a hypersaline cyanobacterial mat (Camargue, France) by irradiance, temperature and salinity. FEMS Microbiol Ecol 55: 195–210PubMedCrossRefGoogle Scholar
  45. 45.
    Wilkinson, SG (1988) Gram-negative bacteria. In: Ratledge, C, Wilkinson, SG (Eds.) Microbial Lipids, Academic Press, London, UK, pp 299–488Google Scholar
  46. 46.
    Yamamoto, K, Murakami, R, Takamura, Y (1998) Isoprenoid quinone, cellular fatty acid composition and diamopimelic isomers of newly classified thermophilic anaerobic gram-positive bacteria. FEMS Microbiol Lett 161: 351–358CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2007

Authors and Affiliations

  • Laura Villanueva
    • 1
    Email author
  • Antoni Navarrete
    • 1
  • Jordi Urmeneta
    • 1
  • Roland Geyer
    • 2
    • 3
  • David C. White
    • 2
  • Ricardo Guerrero
    • 1
  1. 1.Department of MicrobiologyUniversity of BarcelonaBarcelonaSpain
  2. 2.Center for Biomarker AnalysisUniversity of TennesseeKnoxvilleUSA
  3. 3.Department of Environmental MicrobiologyUFZ Centre for Environmental ResearchLeipzig–HalleGermany

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