Skip to main content

Advertisement

SpringerLink
Log in

Enhancement of photoheterotrophic biohydrogen production at elevated temperatures by the expression of a thermophilic clostridial hydrogenase

  • Applied genetics and molecular biotechnology
  • Published:
Applied Microbiology and Biotechnology Aims and scope Submit manuscript

Abstract

The working temperature of a photobioreactor under sunlight can be elevated above the optimal growth temperature of a microorganism. To improve the biohydrogen productivity of photosynthetic bacteria at higher temperatures, a [FeFe]-hydrogenase gene from the thermophile Clostridium thermocellum was expressed in the mesophile Rhodopseudomonas palustris CGA009 (strain CGA-CThydA) using a log-phase expression promoter P pckA to drive the expression of heterogeneous hydrogenase gene. In contrast, a mesophilic Clostridium acetobutylicum [FeFe]-hydrogenase gene was also constructed and expressed in R. palustris (strain CGA-CAhydA). Both transgenic strains were tested for cell growth, in vivo hydrogen production rate, and in vitro hydrogenase activity at elevated temperatures. Although both CGA-CThydA and CGA-CAhydA strains demonstrated enhanced growth over the vector control at temperatures above 38 °C, CGA-CThydA produced more hydrogen than the other strains. The in vitro hydrogenase activity assay, measured at 40 °C, confirmed that the activity of the CGA-CThydA hydrogenase was higher than the CGA-CAhydA hydrogenase. These results showed that the expression of a thermophilic [FeFe]-hydrogenase in R. palustris increased the growth rate and biohydrogen production at elevated temperatures. This transgenic strategy can be applied to a broad range of purple photosynthetic bacteria used to produce biohydrogen under sunlight.

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

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

References

  • Assa P, Özkan M, Özcengiz G (2005) Thermostability and regulation of Clostridium thermocellum l-lactate dehydrogenase expressed in Escherichia coli. Ann Microbiol 55:193–197

    CAS  Google Scholar 

  • Barbosa MJ, Rocha JMS, Tramper J, Wijffels RH (2001) Acetate as a carbon source for hydrogen production by photosynthetic bacteria. J Biotechnol 85:25–33

    Article  CAS  Google Scholar 

  • Boran E, Özgür E, van der Burg J, Yücel M, Gündüz U, Eroglu I (2010) Biological hydrogen production by Rhodobacter capsulatus in solar tubular photo bioreactor. J Clean Prod 18:S29–S35

    Article  CAS  Google Scholar 

  • Cammack R (1999) Bioinorganic chemistry: hydrogenase sophistication. Nature 397:214–215

    Article  CAS  Google Scholar 

  • Carlozzi P, Pushparaj B, Degl'Innocenti A, Capperucci A (2006) Growth characteristics of Rhodopseudomonas palustris cultured outdoors, in an underwater tubular photobioreactor, and investigation on photosynthetic efficiency. Appl Microbiol Biotechnol 73:789–795

    Article  CAS  Google Scholar 

  • Chen C-Y, Lee C-M, Chang J-S (2006) Feasibility study on bioreactor strategies for enhanced photohydrogen production from Rhodopseudomonas palustris WP3-5 using optical-fiber-assisted illumination systems. Int J Hydrogen Energy 31:2345–2355

    Article  CAS  Google Scholar 

  • Daday A, Lambert GR, Smith GD (1979) Measurement in vivo of hydrogenase-catalysed hydrogen evolution in the presence of nitrogenase enzyme in cyanobacteria. Biochem J 177:139–144

    CAS  Google Scholar 

  • Donohue TJ, Kaplan S (1991) Genetic techniques in Rhodospirillaceae. Methods Enzymol 204:459–485

    Article  CAS  Google Scholar 

  • Ducat DC, Sachdeva G, Silver PA (2011) Rewiring hydrogenase-dependent redox circuits in cyanobacteria. Proc Natl Acad Sci U S A 108:3941–3946

    Article  CAS  Google Scholar 

  • Eberly JO, Ely RL (2008) Thermotolerant hydrogenases: biological diversity, properties, and biotechnological applications. Crit Rev Microbiol 34:117–130

    Article  CAS  Google Scholar 

  • Eroglu I (2008) Hydrogen production by Rhodobacter sphaeroides O.U.001 in a flat plate solar bioreactor. Int J Hydrogen Energ 33:531–541

    Article  CAS  Google Scholar 

  • Fisher K, Newton WE (2002) Nitrogen fixation—a general overview. Elsevier, Leigh, pp 1–34

    Google Scholar 

  • Girbal L, von Abendroth G, Winkler M, Benton PM, Meynial-Salles I, Croux C, Peters JW, Happe T, Soucaille P (2005) Homologous and heterologous overexpression in Clostridium acetobutylicum and characterization of purified clostridial and algal Fe-only hydrogenases with high specific activities. Appl Environ Microbiol 71:2777–2781

    Article  CAS  Google Scholar 

  • Gorwa M, Croux C, Soucaille P (1996) Molecular characterization and transcriptional analysis of the putative hydrogenase gene of Clostridium acetobutylicum ATCC 824. J Bacteriol 178:2668–2675

    CAS  Google Scholar 

  • Gosse JL, Engel BJ, Rey FE, Harwood CS, Scriven LE, Flickinger MC (2007) Hydrogen production by photoreactive nanoporous latex coatings of nongrowing Rhodopseudomonas palustris CGA009. Biotechnol Prog 23:124–130

    Article  CAS  Google Scholar 

  • Gosse JL, Engel BJ, Hui JC-H, Harwood CS, Flickinger MC (2010) Progress toward a biomimetic leaf: 4,000 h of hydrogen production by coating-stabilized nongrowing photosynthetic Rhodopseudomonas palustris. Biotechnol Prog 25:907–918

    Google Scholar 

  • Hoekema S, Bijmans M, Janssen M, Tramper J (2002) A pneumatically agitated flat-panel photobioreactor with gas re-circulation: anaerobic photoheterotrophic cultivation of a purple non-sulfur bacterium. Int J Hydrogen Energ 27:1331–1338

    Article  CAS  Google Scholar 

  • Inui M, Nakata K, Roh JH, Zahn K, Yukawa H (1999) Molecular and functional characterization of the Rhodopseudomonas palustris no. 7 phosphoenolpyruvate carboxykinase gene. J Bacteriol 181:2689–2696

    CAS  Google Scholar 

  • Inui M, Roh JH, Zahn K, Yukawa H (2000) Sequence analysis of the cryptic plasmid pMG101 from Rhodopseudomonas palustris and construction of stable cloning vectors. Appl Environ Microbiol 66:54–63

    Article  CAS  Google Scholar 

  • Kim JS, Ito K, Takahashi H (1982a) Production of molecular hydrogen in outdoor batch cultures of Rhodopseudomonas sphaeroides. Agric Biol Chem 46:937–941

    Article  CAS  Google Scholar 

  • Kim JS, Yamauchi H, Ito K, Takahashi H (1982b) Selection of a photosynthetic bacterium suitable for hydrogen production in outdoor cultures among strains isolated in the Seoul, Taegu, Sendai and Bangkok areas. Agric Biol Chem 46:1469–1478

    Article  CAS  Google Scholar 

  • Kim E-J, Kim M-S, Lee JK (2008a) Hydrogen evolution under photoheterotrophic and dark fermentative conditions by recombinant Rhodobacter sphaeroides containing the genes for fermentative pyruvate metabolism of Rhodospirillum rubrum. Int J Hydrogen Energy 33:5131–5136

    Article  CAS  Google Scholar 

  • Kim E, Lee M, Kim M, Lee J (2008b) Molecular hydrogen production by nitrogenase of Rhodobacter sphaeroides and by Fe-only hydrogenase of Rhodospirillum rubrum. Int J Hydrogen Energ 33:1516–1521

    Article  CAS  Google Scholar 

  • Krahe M, Antranikian G, Märkl H (1996) Fermentation of extremophilic microorganisms. FEMS Microbiol Rev 18:271–285

    Article  CAS  Google Scholar 

  • Lamed R, Zeikus JG (1981) Thermostable, ammonium-activated malic enzyme of Clostridium thermocellum. BAA-Enzymology 660:251–255

    CAS  Google Scholar 

  • 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 JLTy, 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

    Article  CAS  Google Scholar 

  • Lee I-H, Park J, Kho D, Kim M-S, Lee J (2002) Reductive effect of H2 uptake and poly-β-hydroxybutyrate formation on nitrogenase-mediated H2 accumulation of Rhodobacter sphaeroides according to light intensity. Appl Microbiol Biotechnol 60:147–153

    Article  CAS  Google Scholar 

  • Maness PC, Weaver P (2001) Evidence for three distinct hydrogenase activities in Rhodospirillum rubrum. Appl Microbiol Biotechnol 57:751–756

    Article  CAS  Google Scholar 

  • McKinlay JB, Harwood CS (2010) Inaugural article: carbon dioxide fixation as a central redox cofactor recycling mechanism in bacteria. Proc Natl Acad Sci U S A 107:11669–11675

    Article  CAS  Google Scholar 

  • Nordlund S, Eriksson U (1975) Nitrogenase from Rhodospirillum rubrum. Relation between ‘switch-off’ effect and the membrane component. Hydrogen production and acetylene reduction with different nitrogenase component ratios. Biochim Biophys Acta 547:419–437

    Google Scholar 

  • Ozkan M, Erhan E, Terzi O, Tan I, Ozoner SK (2009) Thermostable amperometric lactate biosensor with Clostridium thermocellum L-LDH for the measurement of blood lactate. Talanta 79:1412–1417

    Article  CAS  Google Scholar 

  • Peters JW (1999) Structure and mechanism of iron-only hydrogenases. Curr Opin Struct Biol 9:670–676

    Article  CAS  Google Scholar 

  • Rey FE, Oda Y, Harwood CS (2006) Regulation of uptake hydrogenase and effects of hydrogen utilization on gene expression in Rhodopseudomonas palustris. J Bacteriol 188:6143–6152

    Article  CAS  Google Scholar 

  • Rey FE, Heiniger EK, Harwood CS (2007) Redirection of metabolism for biological hydrogen production. Appl Environ Microbiol 73:1665–1671

    Article  CAS  Google Scholar 

  • Riederer A, Takasuka TE, Makino S, Stevenson DM, Bukhman YV, Elsen NL, Fox BG (2011) Global gene expression patterns in Clostridium thermocellum as determined by microarray analysis of chemostat cultures on cellulose or cellobiose. Appl Environ Microbiol 77:1243–1253

    Article  CAS  Google Scholar 

  • Sambrook J, Fritsch E, Maniatis T (1989) Molecular cloning: a laboratory manual, vol 18, 2nd edn. Cold Spring Harbor Laboratory, New York, p 58

    Google Scholar 

  • Sasikala C, Ramana CV (1997) Biodegradation and metabolism of unusual carbon compounds by anoxygenic phototrophic bacteria. Adv Microb Physiol 39:339–377

    Article  Google Scholar 

  • Sato Y, Nakaya A, Shiraishi K, Kawashima S, Goto S, Kanehisa M (2001) SSDB: Sequence Similarity Database in KEGG. Genome Inform 12:230–231

    CAS  Google Scholar 

  • Singh SP, Srivastava SC (1991) Isolation of non-sulphur photosynthetic bacterial strains efficient in hydrogen production at elevated temperatures. Int J Hydrogen Energ 16:403–405

    Article  CAS  Google Scholar 

  • Tian X, Liao Q, Liu W, Wang YZ, Zhu X, Li J, Wang H (2009) Photo-hydrogen production rate of a PVA-boric acid gel granule containing immobilized photosynthetic bacteria cells. Int J Hydrogen Energ 34:4708–4717

    Article  CAS  Google Scholar 

  • Tian X, Liao Q, Zhu X, Wang Y, Zhang P, Li J, Wang H (2010) Characteristics of a biofilm photobioreactor as applied to photo-hydrogen production. Bioresour Technol 101:977–983

    Article  CAS  Google Scholar 

  • Wang Y-Z, Liao Q, Zhu X, Tian X, Zhang C (2010) Characteristics of hydrogen production and substrate consumption of Rhodopseudomonas palustris CQK 01 in an immobilized-cell photobioreactor. Bioresour Technol 101:4034–4041

    Article  CAS  Google Scholar 

  • Wood BE, Ingram LO (1992) Ethanol production from cellobiose, amorphous cellulose, and crystalline cellulose by recombinant Klebsiella oxytoca containing chromosomally integrated Zymomonas mobilis genes for ethanol production and plasmids expressing thermostable cellulase genes from Clostridium thermocellum. Appl Environ Microbiol 58:2103–2110

    CAS  Google Scholar 

Download references

Acknowledgments

We thank Dr. Hideaki Yukawa from The Research Institute of Innovative Technology for the Earth in Japan for supplying the shuttle vector pMG105, Chia-Ching Lin for constructing plasmid pMG105P, Gincy Marina Mathew for proofreading the manuscript, and professor Chin-Ho Lin for helping us complete the in vivo hydrogenase activity assays. This work was funded by The National Science Council (95-2221-E-005-057-MY2) and ATU plan from the Ministry of Education, Republic of China (Taiwan).

Author information

Authors and Affiliations

  1. Department of Life Sciences, National Chung Hsing University, 250 Kuo Kuang Road, Taichung, 402, Taiwan, Republic of China

    Shou-Chen Lo, Shau-Hua Shih, Chun-Ying Wang & Chieh-Chen Huang

  2. Genomics Research Center, Academia Sinica, 128 Academia Road, Section 2, Nankang, Taipei, 115, Taiwan, Republic of China

    Jui-Jen Chang

  3. Agricultural Biotechnology Center, National Chung Hsing University, 250 Kuo Kuang Road, Taichung, 402, Taiwan, Republic of China

    Chieh-Chen Huang

Authors
  1. Shou-Chen Lo
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Shau-Hua Shih
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jui-Jen Chang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Chun-Ying Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Chieh-Chen Huang
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Chieh-Chen Huang.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Lo, SC., Shih, SH., Chang, JJ. et al. Enhancement of photoheterotrophic biohydrogen production at elevated temperatures by the expression of a thermophilic clostridial hydrogenase. Appl Microbiol Biotechnol 95, 969–977 (2012). https://doi.org/10.1007/s00253-012-4017-0

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00253-012-4017-0

Keywords

Search

Navigation