Photosynthesis Research

, Volume 41, Issue 1, pp 157–164 | Cite as

Separation of bacteriochlorophyll homologues from green photosynthetic sulfur bacteria by reversed-phase HPLC

  • C. M. Borrego
  • L. J. Garcia-Gil
Group 5: Chlorosomes and Pigments Regular Papers


A reversed-phase High Performance Liquid Cromatography (HPLC) method has been developed to accurately separate bacteriochlorophyllsc, d ande homologues in a reasonably short run time of 60 minutes. By using this method, two well-defined groups of bacteriochlorophyll homologue peaks can be discriminated. The first one consists of 4 peaks (min 24 to 30), which corresponds to the four main farnesyl homologues. The second peak subset is formed by a cluster of up to 10 minor peaks (min 33 to 40). These peaks can be related with series of several alcohol esters of the different chlorosome chlorophylls. The number of homologues was, however, quite variable depending on both, the bacteriochlorophyll and the bacterial species. The method hereby described, also provides a good separation of other photosynthetic pigments, either bacterial (Bacteriochlorophylla, chlorobactene, isorenieratene and okenone) or algal ones (Chlorophylla, Pheophytina and β-carotene). A preliminary screening of the homologue composition of several green photosynthetic bacterial species and isolates, has revealed different relative quantitative patterns. These differences seem to be related to physiological aspects rather than to taxonomic ones. The application of the method to the study of natural populations avoids the typical drawbacks on the pigment identification of overlapping eukaryotic and prokaryotic phototrophic microorganisms, giving further information about their physiological status.

Key words

bacterial pigments bacteriochlorophyll homologues chlorobiaceae HPLC pigment analyses 









High Performance Liquid Chromatography


ion pairing agent




photodiode array detector


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  1. Abaychi JK and Riley JP (1979) The determination of phytoplankton pigments by high performance liquid chromatography. Anal Chem Acta 107: 1–11Google Scholar
  2. Bidigare RR, Kennicutt MC and Brooks JM (1985) Rapid determination of chlorophylls and their degradation products by high performance liquid chromatography. Limnol Oceanogr 30: 432–435Google Scholar
  3. Brockmann H (1976) Bacteriochlorophylle: Structure and stereochemistry of a new type of chlorophyll from Chlorobiaceae. Phil Trans R Soc London B 273: 277–285Google Scholar
  4. Caple MB, Chow H and Strouse CE (1978) Photosynthetic pigments of green sulfur bacteria. The esterifying alcohols of bacteriochlorophyllsc fromChlorobium limicola. J Biol Chem 253: 6730–6737Google Scholar
  5. Chow H, Caple MB and Strouse CE (1978) Polyethylene powder as stationary phase for preparative scale reversed-phase HPLC. J Chromatogr 151: 357–362Google Scholar
  6. Gloe A, Pfennig N, Brockman H and Trowitsch W (1975) A new bacteriochlorophyll from brown-colored Chlorobiaceae. Arch Microbiol 102: 103–109Google Scholar
  7. Hurley JP and Watras CJ (1991) Identification of bacteriochlorophylls in lakes via reverse-phase HPLC. Limnol Oceanogr 36: 307–315Google Scholar
  8. Huster M and Smith KM (1990) Biosynthetic studies of substituents homologation in Bacteriochlorophyllsc andd. Biochemistry 29: 4348–4355Google Scholar
  9. Jensen A, Aasmundrud O and Eimjhellen KE (1964) Chlorophylls from photosynthetic bacteria. Biochem Biophys Acta 88: 466–479Google Scholar
  10. Kondratieva EN, Pfennig N and Trüper HG (1992) The phototrophic prokaryots. In: Ballows A, Trüper HG, Dworkin M, Harden W and Scleifer KH (eds) The Prokaryots. A Handbook on the Biology of Bacteria: Ecophysiology, Isolation, Identification, Applications, pp 312–331, Second Edition. Springer-Verlag, New YorkGoogle Scholar
  11. Korthals HJ and Steenbergen CLM (1985) Separation and quantification of pigments fron natural phototrophic microbial populations. FEMS Microbial Ecology 31: 177–185Google Scholar
  12. Kraay GW, Zapata M and Veldhuis MJW (1992) Separation of chlorophyllsc 1,c 2 andc 3 of marine phytoplankton by reversedphase C18-High Performance Liquid Chromatography. J Phycol 28: 708–712Google Scholar
  13. Liaaen-Jensen S and Andrewes AG (1972) Microbial carotenoids. Annu Rev Microbiol 26: 227–247Google Scholar
  14. Liaaen-Jensen S, Heggem E and Jackman LM (1964) Bacterial Carotenoids: The carotenoids from photosynthetic green bacteria. Acta Chemica Scandinavica 18: 1703–1718Google Scholar
  15. Mantoura RFC and Llewellyn CA (1983) The rapid determination of algal chlorophyll and carotenoid pigments and their breakdown products in natural waters by reverse-phase high performance liquid chromatography. Anal Chem Acta 151: 297–314Google Scholar
  16. Montesinos E, Guerrero R, Abella CA and Esteve I (1983) Ecology and physiology of the competition for light betweenChlorobium limicola andChlorobium phaeobacteroides in natural habitats. Appl Environ Microbiol 46: 1007–1016Google Scholar
  17. Nelis HJ and DeLeenheer P (1989) Profiling and quantitation of bacterial carotenoids by liquid chromatography and photodiode array detection. Appl Environ Microbiol 55: 3065–3071Google Scholar
  18. Otte SCM, van deMeent EJ, vanVeelen PA, Pundsnes A and Amesz J (1993) Identification of the major chlorosomal bacteriochlorophylls of the green sulfur bacteria Chlorobium vibrioforme and Chlorobium phaeovibrioides; their functions in lateral energy transfer. Photosynth Res 35: 159–169Google Scholar
  19. Pfennig N (1989) Ecology of phototrophic purple and green sulfur bacteria. In: Schlegel HG and Bowien B (eds) Autotrophic Bacteria, pp 97–116. Springer-Verlag, BerlinGoogle Scholar
  20. Repeta DJ, Simpson DJ, Jørgensen BB and Jannasch HN (1989) Evidence for anoxygenic photosynthesis from the distribution of bacteriochlorophyll in the Black Sea. Nature (London) 342: 68–72Google Scholar
  21. Roy S (1987) High Performance Liquid Chromatographic analyses of chloropigments. J Chromatogr 391: 19–34Google Scholar
  22. Scholz B and Ballschmitter K (1981) Preparation and reversed-phase high performance liquid chromatography of chlorophylls. J Chromatogr 208: 148–155Google Scholar
  23. Smith KM (1994) Nomenclature of the bacteriochlorophyllsc, d ande. Photosynth Res 41: 23–26 (this issue)Google Scholar
  24. Stanier RY and Smith JHC (1960) The chlorophylls of green bacteria. Biochim Biophys Acta 41: 478–484Google Scholar
  25. Uehara K and Olson JM (1992) Aggregation of bacteriochlorophyll c homologs to dimers, tetramers, and polymers in water-saturated carbon tetrachloride. Photosynth Res 33: 251–257Google Scholar
  26. VanNiel CB (1971) Techniques for the enrichment, isolation and maintenance of the photosynthetic bacteria. In: SanPietro (ed) Methods in Enzymology, Vol 23, pp 3–28. Academic Press, New YorkGoogle Scholar
  27. Wright SW, Jeffrey SW, Mantoura RFC, Lewelyn CA, Bjørnland T, Repeta D and Welschmeyer N (1991) Improved HPLC method for the analysis of chlorophylls and carotenoids from marine phytoplankton. Mar Ecol Prog Ser 77: 183–196Google Scholar
  28. Yacobi Y Z, Eckert W, Trüper HG and Berman T (1990) High Performance Liquid Chromatography detection of phototrophic bacterial pigments in aquatic environments. Microbial Ecology 19: 127–136Google Scholar

Copyright information

© Kluwer Academic Publishers 1994

Authors and Affiliations

  • C. M. Borrego
    • 1
  • L. J. Garcia-Gil
    • 1
  1. 1.Laboratory of Microbiology, Deptartment of Biology and Institute of Aquatic EcologyUniversity of Girona, Hospital 6GironaSpain

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