Measuring photosynthesis of both oxygenic and anoxygenic photosynthetic organisms using pulse amplitude modulation (PAM) fluorometry in wastewater ponds

Abstract

Oxygenic photosynthesis can be measured easily using O2 or CO2 gas exchange, oxygen electrodes, Winkler titration, 14CO2-fixation and by PAM (pulse amplitude modulation) fluorometry. PAM estimates the photosynthetic electron transport rate (ETR) by measuring the variable fluorescence of chlorophyll (Chl) a (> 695 nm) induced by absorption of blue or red light. Anoxygenic photosynthetic bacteria (APB) do not use water as an electron source and are typically photoheterotrophic rather than photoautotrophic and so 14CO2 fixation is a misleading estimate of photosynthetic electron transport in APB photosynthesis. In vivo bacteriochlorophyll a (BChl a) absorbs blue light similar to Chl a but its characteristic longer-wavelength absorption is in the infrared and fluorescence is at > 800 nm. Blue light-induced PAM fluorescence can be used to measure the ETR in purple non-sulphur anoxygenic photobacteria and purple sulphur photobacteria because their RC-2 type BChl a complexes fluoresce similarly to PSII but at longer wavelengths than Chl a. Conventional PAM fluorometers using blue light cannot readily distinguish between oxygenic and RC-2 type anoxygenic photosynthesis because they use a simple > 700 nm highpass filter in front of the detector diode. We modified one fluorometer to use a 695–750-nm bandpass filter to measure Chl a fluorescence from PS-II, representing oxygenic photosynthesis. Similarly, we modified another fluorometer to use a highpass filter (> 830 nm) to measure BChl a fluorescence, representing anoxygenic photosynthesis. However, the fluorescence bands of Chl a and BChl a were found to be too wide to unambiguously distinguish between oxygenic and anoxygenic photosynthesis purely by fluorometry. Treatment with the specific PS-II inhibitor DCMU (Diuron) did enable discrimination of the two types of photosynthesis in a mixture of oxygenic and anoxygenic organisms. Ecological niches made up of both oxygenic and anoxygenic organisms such as microbial mats and hypereutrophic environments such as sewage ponds, wastewater ponds and prawn farm ponds are much more common than often realized. Anoxygenic photosynthesis in such systems is significant yet largely unquantified.

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

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8
Fig. 9
Fig. 10
Fig. 11

References

  1. Belila B, Abbas B, Fazaa I, Saidi N, Snoussi M, Hassen A, Muyzer G (2013) Sulphur bacteria in wastewater stabilization ponds periodically affected by the ‘red-water’ phenomenon. Appl Microbiol Biotechnol 97:379–394

    CAS  Article  Google Scholar 

  2. Blankenship RE, Madigan MT, Bauer CE (1995) Anoxygenic photosynthetic bacteria. Kluwer Academic Publishers, Dordrecht

    Google Scholar 

  3. Brestic M, Zivcak M (2013) PSII fluorescence techniques for measurement of drought and high temperature stress signal in plants: protocols and applications. In: Rout GR, Das AB (eds) Molecular stress physiology in plants. Springer, Dordrecht pp, pp 87–131

    Google Scholar 

  4. Chandaravithoon P, Nakphet S, Ritchie RJ (2018) Oxygenic and anoxygenic photosynthesis in a sewage pond. J Appl Phycol 30:3089–3102

    CAS  Article  Google Scholar 

  5. Chandaravithoon P, Ritchie RJ, Runcie JW (2019) Measuring both oxygenic and anoxygenic photosynthetic organisms using pulse amplitude modulation (PAM) florometry in wastewater ponds. The 23rd International Seaweed Symposium (ISS2019), Jeju, Korea 28 April to 3 May 2019, Abstract ST 04-6, p 162

  6. Cherif J, Derbel N, Nakkach M, von Bergmann H, Jemal F, Ben Lakhdar Z (2010) Analysis of in vivo chlorophyll fluorescence spectra to monitor physiological state of tomato plants growing under zinc stress. J Photochem Photobiol B 101:332–339

    CAS  Article  Google Scholar 

  7. Clayton RK (1963) Toward the isolation of a photochemical reaction center in Rhodopseuodomonas spheroides. Biochim Biophys Acta 75:312–323

    CAS  Article  Google Scholar 

  8. Clayton RK (1966) Fluorescence from major and minor bacteriochlorophyll components in vivo. Photochem Photobiol 5:679–688

    CAS  Article  Google Scholar 

  9. Cochran WG, Snedecor GW (1989) Statistical methods, 8th edn. Iowa State University Press, Ames

    Google Scholar 

  10. Dewez D, Didur O, Vincent-Héroux J, Popovic (2008) Validation of photosynthetic-fluorescence parameters as biomarkers for isoproturon toxic effect on alga Scenedesmus obliquus. Environ Pollut 151:93–100

    CAS  Article  Google Scholar 

  11. Falkowski PG, Raven JA (2007) Aquatic photosynthesis, 2nd edn. Princeton University Press, Princeton

    Google Scholar 

  12. Genty B, Briantais JM, Baker NR (1989) The relationship between the quantum yield of photosynthetic electron transport and quenching of chlorophyll fluorescence. Biochim Biophys Acta 990:87–92

    CAS  Article  Google Scholar 

  13. Gitelson A, Stark R, Oron G, Dor I. (1997) Monitoring of polluted water bodies by remote sensing. In: Remote Sensing and Geographic Information Systems for Design and Operation of Water Resources Systems (Proceedings of Rabat Symposium S3, April 1997). IAHS Publ. No 242, pp 181–188

  14. Gitelson A, Stark R, Dor I, Michielson O, Yacobi YZ (1999) Optical characteristics of the phototroph Thiocapsa roseopersicina and implication for real time monitoring of the bacteriochlorophyll concentration. Appl Environ Microbiol 65:3392–3397

    CAS  Article  Google Scholar 

  15. Gloag RS, Ritchie RJ, Chen M, Larkum AWD, Quinnell RG (2007) Chromatic photoacclimation, photosynthetic electron transport and oxygen evolution in the chlorophyll d-containing oxyphotobacterium Acaryochloris marina Miyashita. Biochim Biophys Acta-Bioenergetics 1767:127–135

    CAS  Article  Google Scholar 

  16. Goericke R (2002) Bacteriochlorophyll a in the ocean: is anoxygenic bacterial photosynthesis important? Limnol Oceanogr 47:290–295

    CAS  Article  Google Scholar 

  17. Hayashi H, Miyao M, Morita S (1982) Absorption and fluorescence spectra of light-harvesting bacteriochlorophyll-protein complexes from Rhodopseudomonas palustris in the near-infrared region. J Biochem 91:1017–1027

    CAS  Article  Google Scholar 

  18. Hellingwerf KJ, de Vrij W, Konings WN (1982) Wavelength dependence of energy transduction in Rhodopseudomonas sphaeroides: action spectrum of growth. J Bacteriol 151:534–541

  19. Hohlmann-Marriott MF, Blankenship RE (2011) Evolution of photosynthesis. Annu Rev Plant Biol 62:515–548

    Article  Google Scholar 

  20. Hubas C, Jesus B, Passarelli C, Jeanthon C (2011) Tools providing new insight into coastal anoxygenic purple bacterial mats. Res Microbiol 162:858–868

    Article  Google Scholar 

  21. Irving DE, Dromgoole FI (1986) Algal populations and characteristics of oxygen exchange of effluent samples from a facultative oxidation pond. N Z J Mar Freshwat Res 20:9–16

    CAS  Article  Google Scholar 

  22. Kim M-K, Harwood CS (1991) Regulation of benzoate-CoA ligase in Rhodopseudomonas palustris. FEMS Microbiol Lett 83:199–203

    CAS  Google Scholar 

  23. Kim MK, Choi K-M, Yin C-R, Lee K-Y, Im W-T, Lim JH, Lee S-T (2004) Odorous swine wastewater treatment by purple non-sulfur bacteria, Rhodopseudomonas palustris, isolated from eutrophicated ponds. Biotechnol Lett 26:819–822

    CAS  Article  Google Scholar 

  24. Kolber ZS, Van Dover CL, Niederman RA, Falkowski PG (2000) Bacterial photosynthesis in surface waters of the open ocean. Nature 407:177–179

    CAS  Article  Google Scholar 

  25. Larimer FW, Chain P, Hauser L, Lamerdin J, Malfatti S, Do L, Land ML, Pelletier DA, Beatty JT, Lang AS, Tabita FR, Gibson JL, Hanson TE, Bobst C, Torres JLT, Peres C, Harrison FH, Gibson J, Harwood CS (2004) Complete genome sequence of the metabolically versatile photosynthetic bacterium Rhodopseudomonas palustris. Nat Biotechnol 22:55–61

    CAS  Article  Google Scholar 

  26. Larkum AWD, Ritchie RJ, Raven JA (2018) REVIEW: living off the sun: chlorophylls, bacteriochlorophylls and rhodopsins. Photosynthetica 56:11–43

    CAS  Article  Google Scholar 

  27. Papineau D, Walker JJ, Mojzsis SJ, Pace NR (2005) Composition and structure of microbial communities from stromatolites of Hamelin Pool in Shark Bay, Western Australia. Appl Environ Microbiol 71:4822–4832

  28. Pedrós R, Moya I, Goulas Y, Jacquemoud S (2008) Chlorophyll fluorescence emission spectrum inside a leaf. Photochem Photobiol Sci 7:498–502

    Article  Google Scholar 

  29. Porra RJ (2006) Spectrophotometric assays for plant, algal and bacterial chlorophylls. In: Grimm B, Porra RJ, Ruediger W, Scheer H (eds) Chlorophylls and bacteriochlorophylls: biochemistry, biophysics, functions and applications. Springer, Dordrecht, pp 95–106

    Google Scholar 

  30. Prášil O, Bína D, Medová H, Řehaková K, Zapomělová E, Veselá J, Oren A (2009) Emission spectroscopy and kinetic fluorometry studies of phototrophic microbial communities along a salinity gradient in solar saltern evaporation ponds of Eilat, Israel. Aquat Microb Ecol 56:285–296

    Article  Google Scholar 

  31. Ralph PJ, Gademann R (2005) Rapid light curves: a powerful tool to assess photosynthetic activity. Aquat Bot 82:222–237

    CAS  Article  Google Scholar 

  32. Rascher U, Liebig M, Lüttge U (2000) Evaluation of instant light-response curves of chlorophyll fluorescence parameters obtained with a portable chlorophyll fluorometer on site in the field. Plant Cell Environ 23:1397–1405

    CAS  Article  Google Scholar 

  33. Ritchie RJ (2008) Fitting light saturation curves measured using PAM fluorometry. Photosynth Res 96:201–215

    CAS  Article  Google Scholar 

  34. Ritchie RJ (2013) The use of solar radiation by a photosynthetic bacterium living as a mat or in a shallow pond or flatbed reactor. Photochem Photobiol 89:1143–1162

  35. Ritchie RJ (2018) Measurement of chlorophylls a and b and Bacteriochlorophyll a in organisms from hypereutrophic auxinic waters. J Appl Phycol 30:3075–3087

    CAS  Article  Google Scholar 

  36. Ritchie RJ, Larkum AWD (2012) Modelling photosynthesis in shallow algal production ponds. Photosynthetica 50:481–500

    CAS  Article  Google Scholar 

  37. Ritchie RJ, Mekjinda N (2015) Measurement of photosynthesis using PAM technology in a purple Sulphur bacterium Thermochromatium tepidum (Chromatiaceae). Photochem Photobiol 91:350–358

    CAS  Article  Google Scholar 

  38. Ritchie RJ, Runcie JW (2013) Measurement of the photosynthetic electron transport rate in an anoxygenic photosynthetic bacterium Afifella (Rhodopseudomonas)marina using PAM fluorometry. Photochem Photobiol 89:370–383

  39. Ritchie RJ, Runcie JW (2014) A portable reflectance-Absorptance-transmittance (RAT) meter for vascular plant leaves. Photosynthetica 52:614–626

    CAS  Article  Google Scholar 

  40. Ritchie RJ, Runcie JW (2018) Separately measuring photosynthesis of oxygenic and anoxygenic photosynthetic organisms using pulse amplitude modulation (PAM) fluorometry. Siam Physics Congress 2018, Topland Hotel, Phitsanulok, Thailand, 21st – 23rd May 2018, p 6

  41. Ritchie RJ, Chandaravithoon P, Runcie JW (2018) Separately measuring photosynthesis of oxygenic and anoxygenic photosynthetic organisms using pulse amplitude modulation (PAM) fluorometry. Australian Society for Biophysics, 2 to 6th December 2018, RMIT, Melbourne, Australia. Doi: https://doi.org/10.13140/RG.2.2.36471.27042

  42. Siefert E, Irgens RL, Pfennig N (1978) Phototrophic purple and green bacteria in a sewage treatment plant. Appl Environ Microbiol 35:38–41

    CAS  Article  Google Scholar 

  43. Sinning I (1992) Herbicide binding in the bacterial photosynthetic reaction center. TIBS 17:150–154

    CAS  PubMed  Google Scholar 

  44. Sinning I, Michel H, Mathis P, Rutherford AW (1989) Terbutryn resistance in a purple bacterium can induce sensitivity toward the plant herbicide DCMU. FEBS Lett 256:192–194

    CAS  Article  Google Scholar 

  45. Sinning I, Koepke J, Michel H (1990) Recent advances in the structure analysis of Rhodopseudomonas viridis reaction center mutants. In: Michel-Bayerle M-E (ed) Reaction centers of photosynthetic bacteria. Springer, Berlin, pp 199–208

  46. Yurkov VV, Beatty JT (1998) Aerobic anoxygenic phototrophic bacteria. Microbiol Molec Biol Res 62:695–724

    CAS  Article  Google Scholar 

  47. Zhang D, Yang H, Huang Z, Zhang W, Liu S-J (2002) Rhodopseudomonas faecalis sp. nov., a phototrophic bacterium isolated from an anaerobic reactor that digests chicken faeces. Int J Syst Evol Microbiol 52:2055–2060

    CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This study follows the study of Chandaravithoon et al. (2018) which showed that mixtures of oxygenic green algae and anoxygenic photosynthetic bacteria were present in sewage ponds but it was difficult to quantitatively distinguish the two types of photosynthesis. The O2 and non-O2 fluorometers developed in this study were designed to address this problem (Aquation Pty Ltd., Australia). Some elements of this study were previously reported at the SIAM Physics 2018 Conference, Topland Hotel, Phitsanulok, Thailand, May 21 to 23, 2018 (Ritchie and Runcie 2018), at the Australian Biophysics Conference in Melbourne: Ritchie, Chandaravithoon and Runcie, (2018), Australian Society for Biophysics, December 2 to 6, 2018, RMIT, Melbourne, Australia. https://doi.org/10.13140/RG.2.2.36471.27042 and Chandaravithoon et al. (2019). The 23rd International Seaweed Symposium (ISS2019), International Convention Centre, Jeju, Korea April 28 to May 3, 2019.

Funding

This project was partially funded by the Interdisciplinary Graduate School of Earth System Science and Andaman Natural Disaster Management (ESSAND), Andaman Environment and Natural Disaster Research Center (ANED) Prince of Songkla University, Phuket Campus.

Author information

Affiliations

Authors

Corresponding author

Correspondence to R. J. Ritchie.

Additional information

Publisher’s note

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

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Chandaravithoon, P., Ritchie, R.J. & Runcie, J.W. Measuring photosynthesis of both oxygenic and anoxygenic photosynthetic organisms using pulse amplitude modulation (PAM) fluorometry in wastewater ponds. J Appl Phycol (2020). https://doi.org/10.1007/s10811-020-02171-8

Download citation

Keywords

  • Anoxygenic photosynthetic bacteria
  • Oxygenic photosynthesis
  • Bacteriochlorophyll
  • Chlorophyll
  • PAM fluorometer
  • Research and development