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

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. 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. The metabolome of Chlamydomonas reinhardtii following induction of anaerobic H2 production by sulfur depletion.Journal of Biological ChemistryVol. 284Issue 51PreviewVOLUME 284 (2009) PAGES 23415–23425 Full-Text PDF Open Access A variety of unicellular eukaryotic green algae have the ability to produce H2 under anaerobic conditions (1.Boichenko V.A. Hoffmann P. Photosynthetica. 1994; 30: 527-552Google Scholar, 2.Timmins M. Thomas-Hall S.R. Darling A. Zhang E. Hankamer B. Marx U.C. Schenk P.M. J. Exp. Bot. 2009; 60: 1691-1702Crossref PubMed Scopus (57) Google Scholar). This ability is greatly enhanced in the light (3.Gaffron H. Rubin J. J. Gen. Physiol. 1942; : 219-240Crossref PubMed Scopus (457) Google Scholar). 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 (4.Happe T. Naber J.D. Eur. J. Biochem. 1993; 214: 475-481Crossref PubMed Scopus (246) Google Scholar, 5.Forestier M. King P. Zhang L. Posewitz M. Schwarzer S. Happe T. Ghirardi M.L. Seibert M. Eur. J. Biochem. 2003; 270: 2750-2758Crossref PubMed Scopus (210) Google Scholar). The electrons for the reduction of ferredoxin that is used in H2 production can come from endogenous substrates or from water oxidation (6.Stuart T.S. Gaffron H. Planta. 1972; 106: 101-112Crossref PubMed Scopus (43) Google Scholar, 7.Melis A. Planta. 2007; 226: 1075-1086Crossref PubMed Scopus (207) Google Scholar). 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 (3.Gaffron H. Rubin J. J. Gen. Physiol. 1942; : 219-240Crossref PubMed Scopus (457) Google Scholar, 8.Gfeller R.P. Gibbs M. Plant Physiol. 1984; 75: 212-218Crossref PubMed Google Scholar). 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 (9.Melis A. Zhang L. Forestier M. Ghirardi M.L. Seibert M. Plant Physiol. 2000; 122: 127-136Crossref PubMed Scopus (890) Google Scholar). 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 (9.Melis A. Zhang L. Forestier M. Ghirardi M.L. Seibert M. Plant Physiol. 2000; 122: 127-136Crossref PubMed Scopus (890) Google Scholar). 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 (10.Pollock S.V. Pootakham W. Shibagaki N. Moseley J.L. Grossman A.R. Photosynth. Res. 2005; 86: 475-489Crossref PubMed Scopus (50) Google Scholar). Microarray studies have been performed on C. reinhardtii following transfer to sulfur-free medium (11.Zhang Z. Shrager J. Jain M. Chang C.W. Vallon O. Grossman A.R. Eukaryot. Cell. 2004; 3: 1331-1348Crossref PubMed Scopus (154) Google Scholar) and in a sulfur-deplete anaerobic environment in which H2 production was observed (12.Nguyen A.V. Thomas-Hall S.R. Malnoë A. Timmins M. Mussgnug J.H. Rupprecht J. Kruse O. Hankamer B. Schenk P.M. Eukaryot. Cell. 2008; 7: 1965-1979Crossref PubMed Scopus (117) Google Scholar). 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. (12.Nguyen A.V. Thomas-Hall S.R. Malnoë A. Timmins M. Mussgnug J.H. Rupprecht J. Kruse O. Hankamer B. Schenk P.M. Eukaryot. Cell. 2008; 7: 1965-1979Crossref PubMed Scopus (117) Google Scholar) 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 (13.Grossman A.R. Croft M. Gladyshev V.N. Merchant S.S. Posewitz M.C. Prochnik S. Spalding M.H. Curr. Opin. Plant Biol. 2007; 10: 190-198Crossref PubMed Scopus (131) Google Scholar), 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 (14.Mus F. Dubini A. Seibert M. Posewitz M.C. Grossman A.R. J. Biol. Chem. 2007; 282: 25475-25486Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar). 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 and hundreds of the of used chromatography coupled to mass and were used in to as as key The of work was to first into the pathways to anoxia during and to H2 production. gas chromatography coupled to mass mass triacylglycerides H2 production conditions were by the method of sulfur depletion as reported by et al. (9.Melis A. Zhang L. Forestier M. Ghirardi M.L. Seibert M. Plant Physiol. 2000; 122: 127-136Crossref PubMed Scopus (890) Google Scholar). In cultures of C. reinhardtii C. L. A. Kruse O. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar) were phase by and in sulfur-depleted medium E. The Chlamydomonas Scholar). were resuspended to a of in sulfur-depleted medium and sealed in with gas were to 500 light and with O2 H2 gas and of the cultures were for 120 h sulfur depletion. were for metabolomic analysis and 120 h sulfur depletion. were time NMR were time In analysis by NMR required the of from the medium, medium was that was with of phosphate and and acetate was conditions were O2 and of algal cultures were using a with and was by with a and into an gas with a and was used as the culture was from the with a and in a of water for to culture was to a and was for NMR 500 of the was to an NMR and of was culture was to a and for The was and the was with 500 of with of for was added to the was performed by of in a was for and of the was in a for were by the addition of of a of in for of was then added and for a of an of and in was added for To the of amino for were with the which than for the amino use of the from analysis and for of of metabolites the amino acid were with of a of for of was then and were for were the amino that be that both and and the of the as well as the of the the of used for of and amino Open in a were and of was into an with an was used as the gas a of was was for then with a final of A was The transfer was was and was from to mass were with for were by and to or by the with metabolites were into and a was using and to were was performed to the and to the NMR was performed on a with time and water during the were with was and NMR were and using was performed on the formate analysis was using the The from to of NMR was into of were to the of the for The resulting were used for analysis with the analysis were on to in were from cultures were for the was and the were with of and of acetate The were in 500 of acetate and 120 for starch was by incubation with of was by and 500 of the was for analysis was on to and to starch culture was for were from the by the addition of of of and of were in a water for and and was performed by for and of the was and in a were resuspended to a volume of in and was a phase the was in an acid The was a and with with the was and were under cultures were for The was and the were and by with of a for h of acid was added as of and of was then and the was for and was performed by for of the was into an with a A was used with conditions as for acid were using the of the with that of the of acid was on mass to and time from method and by to those previously in C. reinhardtii by et al. J. E. 2004; PubMed Scopus Google Scholar). H2 production was in C. reinhardtii by sulfur depletion. H2 production, pH, and O2 levels were through 120 h of sulfur depletion for profiling were and 120 h of C. reinhardtii culture in sulfur-depleted medium was used for culture but for NMR analysis it was to with phosphate resulting in a reduction of H2 H2 production, and to of those in medium, as and O2 levels not C. reinhardtii cells were to sulfur depletion there was an increase in the O2 and of the h, the O2 to and the culture anoxic The to anoxic conditions were h, H2 be in the gas the H2 production of of culture h sulfur depletion and then 120 h, which H2 production were H2 production with a of from to In studies have shown to be in the of but to S. Seibert M. Ghirardi M.L. Plant Physiol. 2003; PubMed Scopus Google Scholar). The in increase but of metabolism that of H2 production. NMR analysis a culture medium was This procedure metabolites from the cells into the medium, both the and were analyzed as a This procedure in analysis of the However, experiments for the by the and were but the cells only to the not it to cells in under anaerobic we analyzed the were for NMR analysis over 120 h of sulfur-depleted H2 production. were integrated over and analysis was performed to the number of in the and to those that most to of that to the of changes in abundance are shown in was following to in abundance of metabolites during 120 h of sulfur depletion. from are were from in Analysis of the from the first two components revealed that the changes during the first 24 This is to the of acetate during which is the key to the time The of acetate was during the first 24 h then slowed 72 that The of in the and it is to the of 72 and 120 h, a of an production of acetate in H2 is The products and formate were the to products anoxic conditions were and in a to and the time to abundance in the first 24 h and to h, which it a and starch were to increase in the first 24 h and then the of the time the metabolome of C. reinhardtii was analyzed over 120 h of sulfur depletion. In were to and cells in sulfur-depleted a putative metabolites be of which were of these metabolites are shown in of metabolites from C. reinhardtii following 120 h of depletion Open in a of cells in sulfur-depleted medium and a number of metabolites in The of h and has two there were that of metabolites during is is for the decrease in abundance for many to the cells from the and is to in changes in are key to the observed increase in abundance for metabolites such as acid and analysis performed during work was and as such amounts have been However, from analysis of it be that amino were in abundance with most the amino and the by and and and and were only The results that the amino and in following transfer to sulfur-depleted the of amino generally in following the onset of anaerobic conditions. The general was for abundance to increase and 72 h and then in the h of sulfur depletion. was the to which 24 This is to that NMR in which the of the time NMR analysis was performed on of analysis was performed on cells from culture medium, it that is into the medium during the time This effect was for and it that the of these metabolites into the is a C. reinhardtii employs to with anaerobic to the response observed for most amino and in abundance 24 or h of and then the of the acid from 24 to 120 h of sulfur but with an for which is to an decrease and an observed from the most in C. reinhardtii were and and showed change during the first 72 h of but the of the time two are to be relatively but the of during of the that have been by The major in C. in abundance the with a can be to a that is a amino and abundance was reduced during sulfur depletion. The of sulfur the increase in due to an for it to be of and has been to be a key for cells anoxia H. Gibbs J. Plant Biol. 2003; 30: PubMed Scopus Google Scholar). of these functions can in of and and of such as and through and The of of these under anaerobic conditions are not but are to be in of Analysis of the metabolome of C. reinhardtii during sulfur-depleted H2 production revealed that amino a in The of amino are not and of change be a of a protein degradation and or a in energy and detailed analysis of amino acid metabolism was performed by cells in medium with by in medium sulfur depletion. the changes in abundance of and amino through h of sulfur depletion. sulfur the medium was with amino were an increase in amino and a decrease in amino be This was for and amino and showed an increase in both and The increase in amino come from protein or synthesis from amino the increase in amino that were and showed change in levels but levels of the these amino in through synthesis. The increase in abundance of these amino during anaerobic H2 production with levels is of a under anoxic conditions or H2 production. The of C. reinhardtii was analyzed following sulfur depletion by abundance and 24 h of sulfur depletion and the of the time showed an increase during the H2 production The from was and to the for analysis by of is shown in to analysis by an increase in acid was abundance of 24 h in sulfur-free medium and the of the time changes be observed in of and most of the are from the of energy in was greater than the in starch during the first 24 h of sulfur it be that starch were made from cells in a medium for to NMR analysis and not H2 acid of C. reinhardtii cells during 120 h of sulfur of sulfur depletion acid Open in a To a of the changes in the metabolome during to anaerobic H2 production, of of metabolites were a from in the of and and and in the from both NMR and analysis were and of metabolites 72 h sulfur which was through the H2 production were with are on the in green during anaerobic H2 production and in are of a to the of the metabolome during H2 production as with to anaerobic indicate a general of the acid and an increase in abundance of amino and is down to but there is that is used for a small increase in was observed, that the increase not from of but from synthesis pathways. The of an increase of in the but not in the that cells during anaerobic This is in by a of in in the acid and a of in green in a production The metabolome of C. reinhardtii during sulfur depletion be as changes in The first is an energy during the first 24 the an anaerobic H2 production phase from 24 to 72 and the phase a stabilization of and in H2 production to 120 During the first 24 h of sulfur cells accumulate energy in the of starch and This is due to of sulfur for synthesis of amino and components required for in which carbon is in a that is sulfur becomes This is a well response and is depletion L. A. Plant Scopus Google Scholar). The phase is the anaerobic H2 production during time cells to respond to the of O2 by down endogenous substrates and some amino and fermentative The is by a decrease in of the medium and in amounts of products and The amounts of metabolites of and some amino were acid were a in oxidative The and final results in a of starch stabilization of pH, and a reduction in of H2 production. This phase be as a response to energy the medium acetate and the cells starch and It of some products of the phase the of sulfur which and of key or By O2 the ability to in the is and the of to by is becomes the major of and for to there is a to during to and formate can and is a under such anaerobic conditions is in to pathways of carbon operative to In addition to fermentative carbon to be amino and synthesis. for the oxidation of a in under anaerobic conditions. The of to can an the of to be to during of In the of to for the oxidation of of In addition to the amino and were during sulfur-depleted H2 production and to levels than were during as by the following transfer from to reserves of these amino not during sulfur that were not down or into abundance during there was of from the medium into amino of by the E. A. Eukaryot. Cell. 2008; 7: PubMed Scopus Google Scholar), and it be that synthesis is to a to that of amino acid synthesis. The decrease in abundance of as a of a decrease in the of We not but that to acid are in a reduced than acid and production under anaerobic conditions have of the following to as a carbon and conditions are to or or to a of increase of under dark anaerobic conditions was observed by Dubini et al. A. F. Seibert M. Grossman A.R. Posewitz M.C. J. Biol. Chem. 2009; Full Text Full Text PDF PubMed Scopus Google Scholar). fermentative production to be an of allowing of and a of This is in and the of an increase of in the by which is not a relatively sensitive that an production is In the work of Dubini et al. showed that the of was The ability to fermentative pathways C. reinhardtii a clear in under anaerobic conditions. during sulfur which have been in responses to and and as J.D. Plant Full Text PDF Google Scholar). amounts observed during anoxia of a general the of to reduced to and as a energy It be in work to results from metabolomic studies as to such changes can a to these The reduction of the through from oxidation of endogenous substrates has been R.P. Gibbs M. Plant Physiol. 1984; 75: 212-218Crossref PubMed Google Scholar, L. C. F. S. L. J. PubMed Scopus Google Scholar, A. S. L. Happe T. Planta. 2008; PubMed Scopus Google Scholar). can the then that these the of H2 that can be that oxidation have the to in and amino acid synthesis to be of the of that be to the However, as is a key during anoxic cells from an energy the ability to by is not to the with fermentative or In the work we starch to decrease with H2 production. This as well as studies M.C. S. A. Seibert M. Ghirardi M.L. Plant Cell. 2004; PubMed Scopus (131) Google Scholar, O. Rupprecht J. Thomas-Hall S. Schenk P.M. Hankamer B. J. Biol. Chem. 2005; Full Text Full Text PDF PubMed Scopus Google Scholar), a clear of starch in H2 production. starch degradation to reduction of the or it is for anoxic conditions through reduction of O2 during is to be In H2 production is This work has been with the that H2 production is a of and for H2 production is that it be a of The of anaerobic conditions a number of of which is the of the H. Gibbs J. Plant Biol. 2003; 30: PubMed Scopus Google Scholar). has been with an ability to in and F. L. M. N. E. Plant Physiol. PubMed Scopus Google Scholar). C. reinhardtii use the to The reduction of for a to a and metabolites were to be reduced in under anoxic conditions. O2 and is under of during H2 production is to come from pathways. of O2 a of and limits the oxidation of acid This the of to acid A reduction of through the acid results in production of and can have for H2 production. levels of through the and in lead to a of and reduction of the photosynthetic the photosynthetic reduced and of through can as an of in oxidative to the photosynthetic and for generation through of a that can be by The of acetate to H2 production to be H2 production can only be observed in amounts acetate or light not it is that both in a that acetate does H2 production, it does only during the period h of sulfur depletion. The increase in acetate 72 h decreases the of it used for H2 production increase is due to on to the fermentative products formate and ethanol. of acetate during the first 24 h through oxidative the glyoxylate be H2 production by O2 It is that cells acetate production, a large increase in was h and 24 h of sulfur depletion. The abundance of is of due to the for energy reserves to be H2 production and the of the algal culture to be used for production following H2 production. as an cells an ability to production during anaerobic is the most that can both light and II be for O2 generation and to that the and but O2 be to of of O2 endogenous the of H2 that can be produced that by the endogenous the and additional can the for amounts of H2 it be to the of that can be electrons from and of down metabolites to the of 120 h of sulfur depletion there energy reserves in the of starch, and amino To increase H2 production it is to these components down and the during these processes through the photosynthetic and to of H2 production a of the of from oxidation to of the is key to In to H2 produced in the to be to in the these are large to the of for into the pathways that with for has the to increase H2 production. The fermentative pathways that Chlamydomonas of Key fermentative pathways that C. reinhardtii employs to be production of and or for key in the production of these fermentative products in H2 levels the cells not from an energy a is of genes not in Chlamydomonas be to down some metabolites to formate can be down to H2 and in some species using formate and H2 production be formate is in C. reinhardtii. This work used metabolomic to the operative pathways during anaerobic H2 production. The model to a that can in the of key components for to H2 production

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