Journal of Applied Phycology

, Volume 27, Issue 3, pp 1127–1136 | Cite as

Environmental manipulation of growth and energy carrier release from freshwater and marine Chlamydomonas species

  • Tyson A. Burch
  • William W. AdamsIIIEmail author
  • Benoît L. S. Degrenne
  • Calvin H. Englert
  • Brita R. Mines
  • Parker C. Nash
  • Emma C. Boone
  • Barbara Demmig-Adams


While many microbial species produce glycerol as an internal osmoregulant, only some species release glycerol into the external medium. The present study compared the synergistic effects of light intensity and salinity on rates of glycerol release and growth in two marine species of Chlamydomonas (C. euryale and C. hedleyi) versus a freshwater species (C. reinhardtii). High light intensity stimulated both glycerol release and algal growth in all species, presumably by stimulating photosynthesis and thereby the production of the energy carrier glycerol. The freshwater species exhibited a lower salinity threshold than the marine species for both glycerol release and growth retardation. These findings suggest (i) that there is competition between the production and release of glycerol into the medium versus the internal use of the products of photosynthesis for algal growth and (ii) that the freshwater species has a greater propensity for glycerol leakage into the external medium under saline conditions than the marine species. Furthermore, conditions that stimulated glycerol release increased the maximal rate of photosynthesis, suggesting that synthesis (from the direct products of photosynthesis) and removal of glycerol from the cell may alleviate feedback inhibition on photosynthetic capacity. The stimulation of glycerol synthesis and release by Chlamydomonas species via environmental manipulation offers an attractive option for continuous algal energy carrier production as a feedstock for biofuel generation under conditions eliminating energy carrier consumption by algal growth, while circumventing feedback inhibition of photosynthesis as well as the need for algal harvest and regrowth employed for extraction of lipid or carbohydrate feedstocks.


Biofuels Glycerol Green algae Light intensity Photosynthesis Salinity 



We thank Dr. Amy Palmer for generously providing access to the plate reader in her lab. We also gratefully acknowledge the guidance of Prof. William Henley during the initial phases of establishing cultures and protocols in our laboratory. This work was supported by an EAGER grant from the National Science Foundation (award number IOS-1044552), grants from ConocoPhillips (award numbers OCG5163B and OCG5388B), and the Undergraduate Research Opportunity Program of the University of Colorado.


  1. Adams WW III, Cohu CM, Muller O, Demmig-Adams B (2013a) Foliar phloem infrastructure in support of photosynthesis. Front Plant Sci 4:194. doi: 10.3389/fpls.2013.00194 PubMedCentralPubMedGoogle Scholar
  2. Adams WW III, Muller O, Cohu CM, Demmig-Adams B (2013b) May photoinhibition be a consequence, rather than a cause, of limited plant productivity? Photosynth Res 117:31–44CrossRefPubMedGoogle Scholar
  3. Adams WW III, Cohu CM, Amiard V, Demmig-Adams B (2014) Associations between phloem-cell wall ingrowths in minor veins and maximal photosynthesis rate. Front Plant Sci 5:24. doi: 10.3389/fpls.2014.00024
  4. Agre P, Bonhivers M, Borgnia MJ (1998) The aquaporins, blueprints for cellular plumbing systems. J Biol Chem 273:14659–14662CrossRefPubMedGoogle Scholar
  5. Ahmed A, Zidan M (1987) Glycerol production by Dunaliella bioculata. J Basic Microbiol 27:419–425CrossRefGoogle Scholar
  6. Anderberg HI, Danielson JAH, Johanson U (2011) Algal MIPs, high diversity and conserved motifs. BMC Evol Biol 11:110CrossRefPubMedCentralPubMedGoogle Scholar
  7. Anderca MI, Suga S, Furuichi T, Shimogawara K, Maeshima M, Muto S (2004) Functional identification of the glycerol transport activity of Chlamydomonas reinhardtii CrMIP1. Plant Cell Physiol 45:1313–1319CrossRefPubMedGoogle Scholar
  8. Baldisserotto C, Giovanardi M, Ferroni L, Pancaldi S (2014) Growth, morphology and photosynthetic responses of Neochloris oleoabundans during cultivation in a mixotrophic brackish medium and subsequent starvation. Acta Physiol Plant 36:461–472CrossRefGoogle Scholar
  9. Bandyopadhyay A, Stöckel J, Min H, Sherman LA, Pakrasi HB (2010) High rates of photobiological H2 production by a cyanobacterium under aerobic conditions. Nat Commun 1:139. doi: 10.1038/ncomms1139 CrossRefPubMedGoogle Scholar
  10. Barbirato F, Bories A (1997) Relationship between the physiology of Enterobacter agglomerans CNCM 1210 grown anaerobically on glycerol and the culture conditions. Res Microbiol 148:475–484CrossRefPubMedGoogle Scholar
  11. Ben-Amotz A, Avron M (1981) Glycerol and β-carotene metabolism in the halotolerant alga Dunaliella: a model system for biosolar energy conversion. Trends Biochem Sci 6:297–299CrossRefGoogle Scholar
  12. Borowitzka MA, Borowitzka LJ (1988) Dunaliella. In: Borowitzka MA, Borowitzka LJ (eds) Micro-algal biotechnology. Cambridge University Press, Cambridge, pp 27–58Google Scholar
  13. Borowitzka LJ, Brown A (1974) Salt relations of marine and halophilic species of unicellular green alga, Dunaliella—role of glycerol as a compatible solute. Arch Microbiol 96:37–52CrossRefGoogle Scholar
  14. Brown A, Simpson J (1972) Water relations of sugar-tolerant yeasts: role of intracellular polyols. J Gen Microbiol 72:589–591CrossRefPubMedGoogle Scholar
  15. Choi WJ, Hartono MR, Chan WH, Yeo SS (2011) Ethanol production from biodiesel-derived crude glycerol by newly isolated Kluyvera cryocrescens. Appl Microbiol Biotechnol 89:1255–1264CrossRefPubMedGoogle Scholar
  16. Chow YYS, Goh SJM, Su Z, Ng DHP, Lim CY, Lim NYN, Lin H, Fang L, Lee YK (2013) Continual production of glycerol from carbon dioxide by Dunaliella tertiolecta. Bioresour Technol 136:550–555CrossRefPubMedGoogle Scholar
  17. Cohu CM, Muller O, Demmig-Adams B, Adams WW III (2013a) Minor loading vein acclimation for three Arabidopsis thaliana ecotypes in response to growth under different temperature and light regimes. Front Plant Sci 4:240. doi: 10.3389/fpls.2013.00240 PubMedCentralPubMedGoogle Scholar
  18. Cohu CM, Muller O, Stewart JJ, Demmig-Adams B, Adams WW III (2013b) Association between minor loading vein architecture and light- and CO2-saturated rates of photosynthetic oxygen evolution among Arabidopsis thaliana ecotypes from different latitudes. Front Plant Sci 4:264. doi: 10.3389/fpls.2013.00264 PubMedCentralPubMedGoogle Scholar
  19. Cohu CM, Muller O, Adams WW III, Demmig-Adams B (2014) Leaf anatomical and photosynthetic acclimation to cool temperature and high light in two winter versus two summer annuals. Physiol Plant 152:164–173. doi: 10.1111/ppl.121154
  20. Craigie JS, McLachlan J (1964) Glycerol as a photo-synthetic product in Dunaliella tertiolecta Butcher. Can J Bot 42:777–778CrossRefGoogle Scholar
  21. da Silva GP, Mack M, Contiero J (2009) Glycerol: a promising and abundant carbon source for industrial microbiology. Biotechnol Adv 27:30–39CrossRefPubMedGoogle Scholar
  22. Davies P (1984) The role of zooxanthellae in the nutritional energy requirements of Pocillopora eydouxi. Coral Reefs 2:181–186Google Scholar
  23. Demmig-Adams B, Stewart JJ, Adams WW III (2014) Multiple feedbacks between chloroplast and whole plant in the context of plant adaptation and acclimation to the environment. Philos Trans R Soc B 369:UNSP 20130244. doi: 10.1098/rstb.2013.0244
  24. Fatichi S, Leuzinger S, Körner C (2014) Moving beyond photosynthesis: from carbon source to sink-driven vegetation modeling. New Phytol 201:1086–1095CrossRefPubMedGoogle Scholar
  25. Ganesh I, Ravikumar S, Hong SH (2012) Metabolically engineered Escherichia coli as a tool for the production of bioenergy and biochemicals from glycerol. Biotechnol Bioprocess Eng 17:671–678CrossRefGoogle Scholar
  26. Grant AJ, Rémond M, Starke-Peterkovic T, Hinde R (2006) A cell signal from the coral Plesiastrea versipora reduces starch synthesis in its symbiotic alga Symbiodinium sp. Comp Biochem Physiol A 144:458–463CrossRefGoogle Scholar
  27. Grizeau D, Navarro JM (1986) Glycerol production by Dunaliella tertiolecta immobilized within Ca-alginate beads. Biotechnol Lett 8:261–264CrossRefGoogle Scholar
  28. Hedfalk K, Bill RM, Mullins JGL, Karlgren S, Filipsson C, Bergstrom J, Tamas MJ, Rydstrom J, Hohmann S (2004) A regulatory domain in the C-terminal extension of the yeast glycerol channel Fps1p. J Biol Chem 279:14954–14960CrossRefPubMedGoogle Scholar
  29. Hutner SH, Provasoli L, Schatz A, Haskins CP (1950) Some approaches to the study of the role of metals in the metabolism of microorganisms. Proc Am Philos Soc 94:152–170Google Scholar
  30. Ianora A, Boersma M, Casotti R, Fontana A, Harder J, Hoffman F, Pavia H, Potin P, Poulet SA, Toth G (2006) New trends in marine chemical ecology. Estuar Coasts 29:531–551CrossRefGoogle Scholar
  31. Imai I (2012) Biology and ecology of harmful algal blooms (24): cell contacts and cyst formation. Aquabiology 34:577–582Google Scholar
  32. Körner C (2013) Growth controls photosynthesis—mostly. Nova Acta Leopold 114:273–283Google Scholar
  33. Krapp A, Stitt M (1995) An evaluation of direct and indirect mechanisms for the “sink-regulation” of photosynthesis in spinach: changes in gas exchange, carbohydrates, metabolites, enzyme activities and steady-state transcript levels after cold-girdling source leaves. Planta 195:313–323CrossRefGoogle Scholar
  34. Lee JJ (2006) Algal symbiosis in larger foraminifera. Symbiosis 42:63–75Google Scholar
  35. Lee JJ, Zucker W (1969) Algal flagellate symbiosis in foraminifer Archaivas. J Protozool 16:71–81CrossRefGoogle Scholar
  36. Lee JJ, Crockett LJ, Hagen J, Stone RJ (1974) The taxonomic identity and physiological ecology of Chlamydomonas hedleyi sp. nov. algal flagellate symbiont from the foraminifer Archaias angulatus. Br Phycol J 9:407–422CrossRefGoogle Scholar
  37. Lee JJ, Cervasco MH, Morales J, Billik M, Levy M, Fine O (2010) Symbiosis drove cellular evolution. Symbiosis 51:13–25CrossRefGoogle Scholar
  38. León R, Galván F (1994) Halotolerance studies on Chlamydomonas reinhardtii: glycerol excretion by free and immobilized cells. J Appl Phycol 6:13–20CrossRefGoogle Scholar
  39. León R, Galván F (1995) Glycerol photoproduction by free and Ca-alginate entrapped cells of Chlamydomonas reinhardtii. J Biotechnol 42:61–67CrossRefGoogle Scholar
  40. Luo Y-H, Mitsui A (1994) Hydrogen production from organic substrates in an aerobic nitrogen-fixing marine unicellular cyanobacterium Synechococcus sp. strain Miami BG 043511. Biotechnol Bioeng 44:1255–1260CrossRefPubMedGoogle Scholar
  41. Luyten K, Albertyn J, Skibbe W, Prior B, Ramos J, Thevelein J, Hohmann S (1995) Fps1, a yeast member of the MIP family of channel proteins, is a facilitator for glycerol uptake and efflux and is inactive under osmotic stress. EMBO J 14:1360–1371PubMedCentralPubMedGoogle Scholar
  42. Menzel K, Zeng A-P, Deckwer W-D (1997) High concentration and productivity of 1,3-propanediol from continuous fermentation of glycerol by Klebsiella pneumoniae. Enzym Microb Technol 20:82–86CrossRefGoogle Scholar
  43. Metsoviti M, Paramithiotis S, Drosinos EH, Galiotou-Panayotou M, Nychas G-JE, Zeng A-P, Papanikolaou S (2012) Screening of bacterial strains capable of converting biodiesel-derived raw glycerol into 1,3-propanediol, 2,3-butanediol and ethanol. Eng Life Sci 12:57–68CrossRefGoogle Scholar
  44. Min H, Sherman LA (2010) Hydrogen production by the unicellular, diazotrophic cyanobacterium Cyanothece sp. strain ATCC 51142 under conditions of continuous light. Appl Environ Microbiol 76:4293–4301CrossRefPubMedCentralPubMedGoogle Scholar
  45. Miura Y, Ohta S, Mano M, Miyamoto K (1986) Isolation and characterization of a unicellular marine green alga exhibiting high activity in dark hydrogen production. Agric Biol Chem 50:2837–2844CrossRefGoogle Scholar
  46. Miyasaka H, Ohnishi Y, Akano T, Fukatsu K, Mizoguchi T, Yagi K, Maeda I, Ikuta Y, Matsumoto H, Shioji N, Miura Y (1998) Excretion of glycerol by the marine Chlamydomonas sp. strain W-80 in high CO2 cultures. J Ferment Bioeng 85:122–124CrossRefGoogle Scholar
  47. Muller O, Cohu CM, Stewart JJ, Protheroe JA, Demmig-Adams B, Adams WW III (2014a) Association between photosynthesis and contrasting features of minor veins in leaves of summer annuals loading phloem via symplastic versus apoplastic routes. Physiol Plant 152:174–183. doi: 10.1111/ppl.12155
  48. Muller O, Stewart JJ, Cohu CM, Polutchko SK, Demmig-Adams B, Adams WW III (2014b) Leaf architectural, vascular, and photosynthetic acclimation to temperature in two biennials. Physiol Plant. doi: 10.1111/ppl.12226 Google Scholar
  49. Muscatine L (1967) Glycerol excretion by symbiotic algae from corals and Tridacna and its control by host. Science 156:516–519CrossRefPubMedGoogle Scholar
  50. Natrah FMI, Kenmegne MM, Wiyoto W, Sorgeloos P, Bossier P, Defoirdt T (2011) Effects of micro-algae commonly used in aquaculture on acyl-homoserine quorum sensing. Aquaculture 317:53–57CrossRefGoogle Scholar
  51. Parekh SR, Pandey NK (1985) Production of glycerol by Hansenula anomala. Biotechnol Bioeng 27:1089–1091CrossRefPubMedGoogle Scholar
  52. Porra RJ, Thompson WA, Kriedemann PE (1989) Determination of accurate extinction coefficients and simultaneous equations for assaying chlorophylls a and b extracted with 4 different solvents: verification of the concentration of chlorophyll standards by atomic absorption spectroscopy. Biochim Biophys Acta 975:384–394CrossRefGoogle Scholar
  53. Solomon BO, Zeng A-P, Biebl H, Schlieker H, Posten C, Deckwer W-D (1995) Comparison of the energetic efficiencies of hydrogen and oxychemicals formation in Klebsiella pneumoniae and Clostridium butyricum during anaerobic growth on glycerol. J Biotechnol 39:107–117CrossRefPubMedGoogle Scholar
  54. Suescún-Bolívar LP, Iglesias-Prieto R, Thomé PE (2012) Induction of glycerol synthesis and release in cultured Symbiodinium. PLoS One 7:e47182CrossRefPubMedCentralPubMedGoogle Scholar
  55. Teplitski M, Chen H, Rajamani S, Gao M, Merighi M, Sayre RT, Robinson JB, Rolfe BG, Bauer WD (2004) Chlamydomonas reinhardtii secretes compounds that mimic bacterial signals and interfere with quorum sensing regulation in bacteria. Plant Physiol 134:137–146CrossRefPubMedCentralPubMedGoogle Scholar
  56. Venn AA, Loram JE, Douglas AE (2008) Photosynthetic symbioses in animals. J Exp Bot 59:1069–1080CrossRefPubMedGoogle Scholar
  57. Vijaikishore P, Karanth NG (1984) Glycerol production by fermentation. Appl Biochem Biotechnol 9:243–253CrossRefGoogle Scholar
  58. Yang Z-K, Ma Y-H, Zheng J-W, Yang W-D, Liu J-S, Li H-Y (2014) Proteomics to reveal metabolic network shifts towards lipid accumulation following nitrogen deprivation in the diatom Phaeodactylum tricornutum. J Appl Phycol 26:73–82CrossRefPubMedCentralPubMedGoogle Scholar
  59. Yu KO, Jung J, Ramzi AB, Kim SW, Park C, Han SO (2012) Improvement of ethanol yield from glycerol via conversion of pyruvate to ethanol in metabolically engineered Saccharomyces cerevisiae. Appl Biochem Biotechnol 166:856–865CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2014

Authors and Affiliations

  • Tyson A. Burch
    • 1
  • William W. AdamsIII
    • 1
    Email author
  • Benoît L. S. Degrenne
    • 1
  • Calvin H. Englert
    • 1
  • Brita R. Mines
    • 1
  • Parker C. Nash
    • 1
  • Emma C. Boone
    • 1
  • Barbara Demmig-Adams
    • 1
  1. 1.Department of Ecology and Evolutionary BiologyUniversity of ColoradoBoulderUSA

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