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The Metabolome of Chlamydomonas reinhardtii following Induction of Anaerobic H2 Production by Sulfur Depletion*

  • Timmins Matthew
    Affiliations
    School of Biological Sciences

    Institute for Molecular Bioscience, The University of Queensland, St. Lucia, Queensland 4072, Australia

    ARC Centre of Excellence in Plant Energy Biology
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  • Wenxu Zhou
    Affiliations
    ARC Centre of Excellence in Plant Energy Biology

    Centre of Excellence for Plant Metabolomics, The University of Western Australia, 35 Stirling Highway, Crawley, Western Australia 6009, Australia
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  • Jens Rupprecht
    Affiliations
    Institute for Molecular Bioscience, The University of Queensland, St. Lucia, Queensland 4072, Australia
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  • Lysha Lim
    Affiliations
    Institute for Molecular Bioscience, The University of Queensland, St. Lucia, Queensland 4072, Australia
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  • Skye R. Thomas-Hall
    Affiliations
    School of Biological Sciences
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  • Anja Doebbe
    Affiliations
    Department of Biology, AlgaeBioTech Group, University of Bielefeld, 33615 Bielefeld, Germany
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  • Olaf Kruse
    Footnotes
    Affiliations
    Department of Biology, AlgaeBioTech Group, University of Bielefeld, 33615 Bielefeld, Germany
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  • Ben Hankamer
    Correspondence
    To whom correspondence may be addressed: Institute for Molecular Bioscience, The University of Queensland, St. Lucia, Queensland 4072, Australia. Tel.: 61-7-33462012; Fax: 61-7-33462101;
    Footnotes
    Affiliations
    Institute for Molecular Bioscience, The University of Queensland, St. Lucia, Queensland 4072, Australia
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  • Ute C. Marx
    Correspondence
    To whom correspondence may be addressed: Bruker BioSpin, Silberstreifen, Rheinstetten 76287, Germany. Fax: 49-721-516-1297;
    Footnotes
    Affiliations
    Institute for Molecular Bioscience, The University of Queensland, St. Lucia, Queensland 4072, Australia
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  • Steven M. Smith
    Correspondence
    To whom correspondence may be addressed: The University of Western Australia, Stirling Highway, Crawley, Western Australia 6009, Australia. Fax: 61-8-6488-4401;
    Affiliations
    ARC Centre of Excellence in Plant Energy Biology

    Centre of Excellence for Plant Metabolomics, The University of Western Australia, 35 Stirling Highway, Crawley, Western Australia 6009, Australia
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  • Peer M. Schenk
    Affiliations
    School of Biological Sciences
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  • Author Footnotes
    * This work was supported in part by the Australian Research Council (ARC Grants FF0457721 and CE0561495). This work was also supported by the Solar Biofuels Consortium, the Centres of Excellence Program of the Government of Western Australia, the German Federal Ministry of Education and Science (BMBF-ForSys Partner 0315165A), and the Deutsche Bundesstiftung Umwelt (Grant DBU 20006/828).
    1 Supported by ARC Grant DP0877147.
Open AccessPublished:May 28, 2009DOI:https://doi.org/10.1074/jbc.M109.003541
      The metabolome of the model species Chlamydomonas reinhardtii has been analyzed during 120 h of sulfur depletion to induce anaerobic hydrogen (H2) production, using NMR spectroscopy, gas chromatography coupled to mass spectrometry, and TLC. The results indicate that these unicellular green algae consume freshly supplied acetate in the medium to accumulate energy reserves during the first 24 h of sulfur depletion. In addition to the previously reported accumulation of starch, large amounts of triacylglycerides were deposited in the cells. During the early 24- to 72-h time period fermentative energy metabolism lowered the pH, H2 was produced, and amino acid levels generally increased. In the final phase from 72 to 120 h, metabolism slowed down leading to a stabilization of pH, even though some starch and most triacylglycerides remained. We conclude that H2 production does not slow down due to depletion of energy reserves but rather due to loss of essential functions resulting from sulfur depletion or due to a build-up of the toxic fermentative products formate and ethanol.
      A variety of unicellular eukaryotic green algae have the ability to produce H2 under anaerobic conditions (
      • Boichenko V.A.
      • Hoffmann P.
      ,
      • Timmins M.
      • Thomas-Hall S.R.
      • Darling A.
      • Zhang E.
      • Hankamer B.
      • Marx U.C.
      • Schenk P.M.
      ). This ability is greatly enhanced in the light (
      • Gaffron H.
      • Rubin J.
      ). Studies using the model species Chlamydomonas reinhardtii have shown that H2 generation stems from the use of two oxygen (O2) sensitive Fe-hydrogenase enzymes, HydA1 and HydA2, that use reduced ferredoxin to catalyze the reduction of protons to yield H2 (
      • Happe T.
      • Naber J.D.
      ,
      • Forestier M.
      • King P.
      • Zhang L.
      • Posewitz M.
      • Schwarzer S.
      • Happe T.
      • Ghirardi M.L.
      • Seibert M.
      ). The electrons for the reduction of ferredoxin that is used in H2 production can come from endogenous substrates or from water oxidation (
      • Stuart T.S.
      • Gaffron H.
      ,
      • Melis A.
      ).
      H2 production was originally studied by making algal cultures anaerobic by purging with inert gases or by incubation in the dark and then exposure to light (
      • Gaffron H.
      • Rubin J.
      ,
      • Gfeller R.P.
      • Gibbs M.
      ). H2 was produced during these experiments in small amounts and for a relatively short period of time. More recently, a method of sulfur depletion was devised in which cells are resuspended in sulfur-depleted medium, allowing H2 production to be observed for numerous days and in larger quantities (
      • Melis A.
      • Zhang L.
      • Forestier M.
      • Ghirardi M.L.
      • Seibert M.
      ). By depleting a sealed culture of sulfur, photosynthetic O2 evolution decreases and it becomes microxic, leading to anaerobic pathways becoming operative and to the onset of H2 production. It is proposed that sulfur depletion preferentially limits synthesis of the D1 protein of photosystem II (
      • Melis A.
      • Zhang L.
      • Forestier M.
      • Ghirardi M.L.
      • Seibert M.
      ). Sulfur depletion has to date proven to be the best procedure for inducing H2 production in terms of volume and purity.
      Sulfur is transported into C. reinhardtii cells primarily as the sulfate anion, SO42−, and is required for a variety of lipids, proteins, and metabolites (
      • Pollock S.V.
      • Pootakham W.
      • Shibagaki N.
      • Moseley J.L.
      • Grossman A.R.
      ). Microarray studies have been performed on C. reinhardtii following transfer to sulfur-free medium (
      • Zhang Z.
      • Shrager J.
      • Jain M.
      • Chang C.W.
      • Vallon O.
      • Grossman A.R.
      ) and in a sulfur-deplete anaerobic environment in which H2 production was observed (
      • Nguyen A.V.
      • Thomas-Hall S.R.
      • Malnoë A.
      • Timmins M.
      • Mussgnug J.H.
      • Rupprecht J.
      • Kruse O.
      • Hankamer B.
      • Schenk P.M.
      ). In both studies, extensive changes in transcript abundance were observed, with over 20% of analyzed transcripts showing a change of greater than 2-fold, many of them with putative or unknown functions. Both studies showed sulfur depletion to lead to a general increase in transcripts involved in sulfur assimilation, protein degradation and stress, a decrease in most transcripts encoding components of the photosynthetic apparatus, a decrease in transcripts for carbon metabolism through the Calvin-Benson cycle, an increase in those of the oxidative pentose phosphate cycle, and an increase in those of starch synthesis. Key additional observations made in work by Ngyuen et al. (
      • Nguyen A.V.
      • Thomas-Hall S.R.
      • Malnoë A.
      • Timmins M.
      • Mussgnug J.H.
      • Rupprecht J.
      • Kruse O.
      • Hankamer B.
      • Schenk P.M.
      ) showed that the added effect of anaerobicity led to repression of genes of the glyoxylate cycle, an up-regulation of the major light harvesting complex Lhcbm9 protein and transcript, hydrogenase-encoding transcripts HydA1, HydA2, and HydEF, and an up-regulation of genes of fermentative pathways.
      The up-regulation of genes of fermentative pathways and hydrogenase-encoding transcripts are key processes that C. reinhardtii employs to respond to anaerobic conditions. However, these are not the sole processes, and it has been shown that there is a “whole cell” response such that hundreds to thousands of genes are regulated to deal with anoxic conditions. Analysis of the genome of C. reinhardtii has shown a large number of peptides to be involved in anaerobic metabolism (
      • Grossman A.R.
      • Croft M.
      • Gladyshev V.N.
      • Merchant S.S.
      • Posewitz M.C.
      • Prochnik S.
      • Spalding M.H.
      ), and microarray analysis has revealed over 500 transcripts involved in diverse processes such as transcription/translation regulators, prolylhydroxylases, hybrid cluster proteins, proteases, transhydrogenases, and catalases to be up-regulated by the onset of anoxia (
      • Mus F.
      • Dubini A.
      • Seibert M.
      • Posewitz M.C.
      • Grossman A.R.
      ). It is clear that photosynthetic organisms respond to anoxia by adopting an integrated response rather than activating or inactivating only a few select pathways.
      To better understand the response of C. reinhardtii to sulfur depletion and to anoxia as well as how such responses underpin H2 production, we undertook detailed metabolomic studies of C. reinhardtii. Metabolomics employs a non-targeted profiling approach and can potentially detect and quantify hundreds of metabolites, particularly the more abundant intermediates of primary metabolism. NMR, GC/MS,
      The abbreviations used are: GC/MS
      gas chromatography coupled to mass spectrometry
      CPMG
      Carr-Purcell-Meiboom-Gill
      MBTSTFA
      N-methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide
      MSD
      mass selective detector
      MSTFA
      N-methyl-N-(trimethylsilyl)trifluoroacetamide
      TAG
      triacylglycerides
      Tris
      tris(hydroxymethyl)aminomethane.
      5The abbreviations used are: GC/MS
      gas chromatography coupled to mass spectrometry
      CPMG
      Carr-Purcell-Meiboom-Gill
      MBTSTFA
      N-methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide
      MSD
      mass selective detector
      MSTFA
      N-methyl-N-(trimethylsilyl)trifluoroacetamide
      TAG
      triacylglycerides
      Tris
      tris(hydroxymethyl)aminomethane.
      and TLC were used in parallel to obtain as much information as possible about key metabolites. The aim of this work was to provide first insight into the metabolic pathways employed to survive anoxia during illumination and to support H2 production.

      Conclusion

      This work used metabolomic data to map the operative pathways during anaerobic H2 production. The developed model aims to provide a framework that can assist researchers in the process of identifying key components for metabolic engineering to improve H2 production rates.

      Acknowledgments

      We are grateful to work performed by personnel employed through Metabolomics Australia at the University of Western Australia.

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