Applied Microbiology and Biotechnology

, Volume 103, Issue 5, pp 2113–2120 | Cite as

Perspectives for the biotechnological production of biofuels from CO2 and H2 using Ralstonia eutropha and other ‘Knallgas’ bacteria

  • Christopher BrighamEmail author


With global CO2 emissions at their highest in several years, mitigation and possibly reduction of greenhouse gas buildup and concomitant production of renewable fuel molecules for growing transportation fuel needs are urgent challenges for renewable energy scientists and engineers. Knallgas bacteria provide a biocatalyst platform for utilization of CO2 and production of diverse and some high-energy density biofuel molecules, requisite for drop-in transportation fuels. The most well-studied Knallgas bacterium, Ralstonia eutropha, has been engineered to produce n-butanol, isobutanol, and terpene molecules under chemolithoautotrophic conditions. There are other representatives of this group of bacteria that potentially have the capabilities for CO2-based fuel molecule synthesis. In principle, fermentative production of biofuel from CO2 could rival the “power-to-gas” (non-biological production of fuels using CO2 and H2) production methods. However, challenges remain for both methods in order to compete with currently priced petroleum-based fuels. With continued streamlining of processes and attention to Industrial Ecology principles, biofuel synthesis by Knallgas bacteria could represent a viable part of a nation’s energy portfolio.


Carbon dioxide Dihydrogen Knallgas bacteria Ralstonia eutropha Biofuel 



CJB thanks Prof. Alexander Steinbüchel and the editorial team of Applied Microbiology and Biotechnology for the opportunity to write and publish this work. CJB also thanks Mr. John Quimby and Ms. Jayashree Chakravarty for critical review of the manuscript. Continued thanks to Prof. Anthony Sinskey of Massachusetts Institute of Technology for the opportunity to work on a biofuel production project, which serves as the inspiration for continued interest in this topic.

Compliance with ethical standards

This article does not contain any studies with human participants or animals performed by any of the authors.

Conflict of interest

The author declares that he has no conflict of interest.


  1. Amils R (2014) Chemolithoautotroph. In: Gargaud M, Amils R, Cernicharo Quintillana J, Cleaves HJ, Irvine WM, Pinti D, Visco M (eds) Encyclopedia of astrobiology, 2nd edn. Springer, Berlin, HeidelbergGoogle Scholar
  2. Bailera M, Lisbona P, Romero LM, Espatolero S (2017) Power to gas projects review: lab, pilot and demo plants for storing renewable energy and CO2. Renew Sustain Energy Rev 69:292–312CrossRefGoogle Scholar
  3. Bengelsdorf FR, Straub M, Dürre P (2013) Bacterial synthesis gas (syngas) fermentation. Environ Technol 34:1639–1651CrossRefGoogle Scholar
  4. Bhujade R, Chidambaram M, Kumar A, Sapre A (2017) Algae to economically viable low-carbon-footprint oil. Annu Rev Chem Biomol Eng 8:335–357CrossRefGoogle Scholar
  5. Brigham CJ, Gai C, Lu J, Speth DR, Worden RM, Sinskey AJ (2012a) Engineering Ralstonia eutropha for production of isobutanol from CO2, H2, and O2. In: Li JW (ed) Advanced biofuels and bioproducts. Springer, Germany, pp 1065–1090Google Scholar
  6. Brigham CJ, Kehail AA, Palmer JD (2016) Ralstonia eutropha and the production of value added products: metabolic background of the wild-type strain and its role as a diverse, genetically-engineered biocatalyst organism. Recent Adv Biotechnol:265–347Google Scholar
  7. Brigham CJ, Speth DR, Rha C, Sinskey AJ (2012b) Whole genome microarray and gene deletion studies reveal regulation of the polyhydroxyalkanoate production cycle by the stringent response in Ralstonia eutropha H16. Appl Environ Microbiol 78(22):8033–8044CrossRefGoogle Scholar
  8. Burgdorf T, Lenz O, Buhrke T, van der Linden E, Jones AK, Albracht SP, Friedrich B (2005) [NiFe]-hydrogenases of Ralstonia eutropha H16: modular enzymes for oxygen-tolerant biological hydrogen oxidation. J Mol Microbiol Biotechnol 10:181–196CrossRefGoogle Scholar
  9. Croce S, Wei Q, D’Imporzano G, Dong R, Adani F (2016) Anaerobic digestion of straw and corn stover: the effect of biological process optimization and pre-treatment on total bio-methane yield and energy performance. Biotechnol Adv 34:1289–1304CrossRefGoogle Scholar
  10. Doud DFR, Holmes EC, Richter H, Molitor B, Jander G, Angenent LT (2017) Metabolic engineering of Rhodopseudomonas palustris for the obligate reduction of n-butyrate to n-butanol. Biotechnol Biofuels 10: 178–017–0864-3. eCollection 2017Google Scholar
  11. Dürre P, Richard T (2011) Microbial energy conversion revisited. Curr Opin Biotechnol 22:309–311CrossRefGoogle Scholar
  12. Fei Q, Fu R, Shang L, Brigham CJ, Chang HN (2014) Lipid production by microalgae Chlorella protothecoides with volatile fatty acids (VFAs) as carbon sources in heterotrophic cultivation and its economic assessment. Bioprocess Biosyst Eng 38:691–700CrossRefGoogle Scholar
  13. Fukui T, Ohsawa K, Mifune J, Orita I, Nakamura S (2011) Evaluation of promoters for gene expression in polyhydroxyalkanoate-producing Cupriavidus necator H16. Appl Microbiol Biotechnol 89:1527–1536CrossRefGoogle Scholar
  14. Ghimire A, Valentino S, Frunzo L, Pirozzi F, Lens PN, Esposito G (2016) Concomitant biohydrogen and poly-beta-hydroxybutyrate production from dark fermentation effluents by adapted Rhodobacter sphaeroides and mixed photofermentative cultures. Bioresour Technol 217:157–164CrossRefGoogle Scholar
  15. Goetz M, Lefebvre J, Moers F, Koch AM, Graf F, Bajohr S, Reimert R, Kolb T (2016) Renewable power-to-gas: a technological and economic review. Renew Energy 85:1371–1390CrossRefGoogle Scholar
  16. Haas T, Poetter M, Schaffer S (2016) Fatty acid derivatives and function. United States Patent Application US2016/0138061 A1: 19 May, 2016Google Scholar
  17. Hashimoto K (1994) Metastable metals for “green” materials for global atmosphere conservation and abundant energy supply. Mater Sci Eng A 267:200–206CrossRefGoogle Scholar
  18. Holder JW, Ulrich JC, DeBono AC, Godfrey PA, Desjardins CA, Zucker J, Zeng Q, Leach AL, Ghiviriga I, Dancel C, Abeel T, Gevers D, Kodira CD, Desany B, Affourtit JP, Birren BW, Sinskey AJ (2011) Comparative and functional genomics of Rhodococcus opacus PD630 for biofuels development. PLoS Genet 7:e1002219CrossRefGoogle Scholar
  19. House KZ, Baclig AC, Ranjan M, van Nierop EA, Wilcox J, Herzog HJ (2011) Economic and energetic analysis of capturing CO2 from ambient air. Proc Natl Acad Sci U S A 108:20428–20433CrossRefGoogle Scholar
  20. International Energy Agency (2017) Biofuels for transport: Tracking Clean Energy Progress.Google Scholar
  21. IRENA (2018) Renewable power generation costs in 2017. International Renewable Energy Agency. Abu DhabiGoogle Scholar
  22. Jannson C, Carr CAM, Reed JS (2016) Microorganism for biosynthesis of limonene on gaseous substrates. United States Patent 9,506,086: 29 Nov, 2016Google Scholar
  23. Jungert JR, Borisova M, Mayer C, Wolz C, Brigham CJ, Sinskey AJ, Jendrossek D (2017) Absence of ppGpp leads to increased mobilization of intermediately accumulated poly(3-hydroxybutyrate) (PHB) in Ralstonia eutropha H16. Appl Environ Microbiol 83(13):e00755–e00717Google Scholar
  24. Kalkus J, Reh M, Schlegel HG (1990) Hydrogen autotrophy of Nocardia opaca strains is encoded by linear megaplasmids. J Gen Microbiol 136:1145–1151CrossRefGoogle Scholar
  25. Khan NE, Myers JA, Tuerk AL, Curtis WR (2014) A process economic assessment of hydrocarbon biofuels production using chemoautotrophic organisms. Bioresour Technol 172:201–211CrossRefGoogle Scholar
  26. Khan NE, Nybo SE, Chappell J, Curtis WR (2015) Triterpene hydrocarbon production engineered into a metabolically versatile host—Rhodobacter capsulatus. Biotechnol Bioeng 112:1523–1532CrossRefGoogle Scholar
  27. Kiely PD, Call DF, Yates MD, Regan JM, Logan BE (2010) Anodic biofilms in microbial fuel cells harbor low numbers of higher-power-producing bacteria than abundant genera. Appl Microbiol Biotechnol 88:371–380CrossRefGoogle Scholar
  28. Kim K, Chiba Y, Kobayashi A, Arai H, Ishii M (2017) Phosphoserine phosphatase is required for serine and one-carbon unit synthesis in Hydrogenobacter thermophilus. J Bacteriol 199:e00409–e00417CrossRefGoogle Scholar
  29. Kokkonen P, Bednar D, Dockalova V, Prokop Z, Damborsky J (2018) Conformational changes allow processing of bulky substrates by a haloalkane dehalogenase with a small and buried active site. J Biol Chem 293:11505–11512CrossRefGoogle Scholar
  30. Kopke M, Mihalcea C, Bromley JC, Simpson SD (2011) Fermentative production of ethanol from carbon monoxide. Curr Opin Biotechnol 22:320–325CrossRefGoogle Scholar
  31. Krieg T, Sydow A, Faust S, Huth I, Holtmann D (2018) CO2 to terpenes: autotrophic and electroautotrophic alpha-humulene production with Cupriavidus necator. Angew Chem Int Ed Engl 57:1879–1882CrossRefGoogle Scholar
  32. Kumar S, Spiro S (2017) Environmental and genetic determinants of biofilm formation in Paracoccus denitrificans. mSphere 2: eCollection 2017 Sep-Oct
  33. Kurosawa K, Boccazzi P, de Almeida NM, Sinskey AJ (2010) High-cell-density batch fermentation of Rhodococcus opacus PD630 using a high glucose concentration for triacylglycerol production. J Biotechnol 147:212–218CrossRefGoogle Scholar
  34. Kurosawa K, Laser J, Sinskey AJ (2015) Tolerance and adaptive evolution of triacylglycerol-producing Rhodococcus opacus to lignocellulose-derived inhibitors. Biotechnol Biofuels 8: 76–015–0258-3. eCollection 2015Google Scholar
  35. Kurosawa K, Wewetzer SJ, Sinskey AJ (2013) Engineering xylose metabolism in triacylglycerol-producing Rhodococcus opacus for lignocellulosic fuel production. Biotechnol Biofuels 6: 134–6834–6-134Google Scholar
  36. Lenz O, Ludwig M, Schubert T, Burstel I, Ganskow S, Goris T, Schwarze A, Friedrich B (2010) H2 conversion in the presence of O2 as performed by the membrane-bound [NiFe]-hydrogenase of Ralstonia eutropha. ChemPhysChem 11:1107–1119CrossRefGoogle Scholar
  37. Li H, Opgenorth PH, Wernick DG, Rogers S, Wu TY, Higashide W, Malati P, Huo YX, Cho KM, Liao JC (2012) Integrated electromicrobial conversion of CO2 to higher alcohols. Science 335:1596CrossRefGoogle Scholar
  38. Liang X, Whitham JM, Holwerda EK, Shao X, Tian L, Wu YW, Lombard V, Henrissat B, Klingeman DM, Yang ZK, Podar M, Richard TL, Elkins JG, Brown SD, Lynd LR (2018) Development and characterization of stable anaerobic thermophilic methanogenic microbiomes fermenting switchgrass at decreasing residence times. Biotechnol Biofuels 11: 243–018–1238-1. eCollection 2018Google Scholar
  39. Long M, Ilhan ZE, Xia S, Zhou C, Rittmann BE (2018) Complete dechlorination and mineralization of pentachlorophenol (PCP) in a hydrogen-based membrane biofilm reactor (MBfR). Water Res 144:134–144CrossRefGoogle Scholar
  40. Luque R (2010) Algal biofuels: the eternal promise? Energy Environ Sci 3:254–257CrossRefGoogle Scholar
  41. Muller J, Maceachran D, Burd H, Sathitsuksanoh N, Bi C, Yeh YC, Lee TS, Hillson NJ, Chhabra SR, Singer SW, Beller HR (2013) Engineering of Ralstonia eutropha H16 for autotrophic and heterotrophic production of methyl ketones. Appl Environ Microbiol 79:4433–4439CrossRefGoogle Scholar
  42. Paoli GC, Tabita FR (1998) Aerobic chemolithoautotrophic growth and RubisCO function in Rhodobacter capsulatus and a spontaneous gain of function mutant of Rhodobacter sphaeroides. Arch Microbiol 170:8–17CrossRefGoogle Scholar
  43. Peplinski K, Ehrenreich A, Doring C, Bomeke M, Reinecke F, Hutmacher C, Steinbüchel A (2010) Genome-wide transcriptome analyses of the ‘Knallgas’ bacterium Ralstonia eutropha H16 with regard to polyhydroxyalkanoate metabolism. Microbiology 156:2136–2152CrossRefGoogle Scholar
  44. Pfeiffer D, Wahl A, Jendrossek D (2011) Identification of a multifunctional protein, PhaM, that determines number, surface to volume ratio, subcellular localization and distribution to daughter cells of poly(3-hydroxybutyrate), PHB, granules in Ralstonia eutropha H16. Mol Microbiol 82:936–951CrossRefGoogle Scholar
  45. Pisciotta JM, Zaybak Z, Call DF, Nam JY, Logan BE (2012) Enrichment of microbial electrolysis cell biocathodes from sediment microbial fuel cell bioanodes. Appl Environ Microbiol 78:5212–5219CrossRefGoogle Scholar
  46. Raberg M, Volodina E, Lin K, Steinbüchel A (2018) Ralstonia eutropha H16 in progress: applications beside PHAs and establishment as production platform by advanced genetic tools. Crit Rev Biotechnol 38:494–510CrossRefGoogle Scholar
  47. Reed J, Geller J, McDaniel R (2015) CO2 conversion by Knallgas microorganisms. In: CEC-500-2017-005Google Scholar
  48. Riedel SL, Bader J, Brigham CJ, Budde CF, Yusof ZA, Rha C, Sinskey AJ (2012) Production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) by Ralstonia eutropha in high cell density palm oil fermentations. Biotechnol Bioeng 109:74–83CrossRefGoogle Scholar
  49. Schatz A, Bovell C Jr (1952) Growth and hydrogenase activity of a new bacterium, Hydrogenomonas facilis. J Bacteriol 63:87–98Google Scholar
  50. Schiebahn S, Grube T, Robinius M, Tietze V, Kumar B, Stolten D (2015) Power to gas: technological overview, systems analysis and economic assessment for a case study in Germany. Int J Hydrog Energy 40:4285–4294CrossRefGoogle Scholar
  51. Schlegel H, Lafferty R (1971) Novel energy and carbon sources. Adv Biochem Eng 1:143–168CrossRefGoogle Scholar
  52. Sensfuss C, Reh M, Schlegel HG (1986) No correlation exists between the conjugative transfer of the autotrophic character and that of plasmids in Nocardia opaca strains. J Gen Microbiol 132:997–1007Google Scholar
  53. Silva FT, Moreira LR, de Souza Ferreira J, Batista FR, Cardoso VL (2016) Replacement of sugars to hydrogen production by Rhodobacter capsulatus using dark fermentation effluent as substrate. Bioresour Technol 200:72–80CrossRefGoogle Scholar
  54. Sterner M (2009) Bioenergy and renewable power methane in integrated 100% renewable energy systems. Kassel University Press, KasselGoogle Scholar
  55. Sznajder A, Pfeiffer D, Jendrossek D (2015) Comparative proteome analysis reveals four novel polyhydroxybutyrate (PHB) granule-associated proteins in Ralstonia eutropha H16. Appl Environ Microbiol 81:1847–1858CrossRefGoogle Scholar
  56. Tabata T, Yoshiba Y, Takashina T, Hieda K, Shimizu N (2017) Bioethanol production from steam-exploded rice husk by recombinant Escherichia coli KO11. World J Microbiol Biotechnol 33: 47–017-2221-x. Epub 2017 Feb 7Google Scholar
  57. Tanaka K, Ishizaki A, Kanamaru T, Kawano T (1995) Production of poly(D-3-hydroxybutyrate) from CO(2), H(2), and O(2) by high cell density autotrophic cultivation of Alcaligenes eutrophus. Biotechnol Bioeng 45:268–275CrossRefGoogle Scholar
  58. Tibbs HBC (1992) Industrial Ecology: an environmental agenda for industry. Whole Earth Rev:4–19Google Scholar
  59. Torella JP, Gagliardi CJ, Chen JS, Bediako DK, Colon B, Way JC, Silver PA, Nocera DG (2015) Efficient solar-to-fuels production from a hybrid microbial-water-splitting catalyst system. Proc Natl Acad Sci U S A 112:2337–2342CrossRefGoogle Scholar
  60. Toyoda K, Yoshizawa Y, Arai H, Ishii M, Igarashi Y (2005) The role of two CbbRs in the transcriptional regulation of three ribulose-1,5-bisphosphate carboxylase/oxygenase genes in Hydrogenovibrio marinus strain MH-110. Microbiology 151:3615–3625CrossRefGoogle Scholar
  61. US EIA (2018) Levelized cost and levelized avoided cost of new generation resources in the annual energy outlook 2018. United States Energy Information Association. WashingtonGoogle Scholar
  62. Uyar B, Gurgan M, Ozgur E, Gunduz U, Yucel M, Eroglu I (2015) Hydrogen production by hup(−) mutant and wild-type strains of Rhodobacter capsulatus from dark fermentation effluent of sugar beet thick juice in batch and continuous photobioreactors. Bioprocess Biosyst Eng 38:1935–1942CrossRefGoogle Scholar
  63. Vo Hoang Nhat P, Ngo HH, Guo WS, Chang SW, Nguyen DD, Nguyen PD, Bui XT, Zhang XB, Guo JB (2018) Can algae-based technologies be an affordable green process for biofuel production and wastewater remediation? Bioresour Technol 256:491–501CrossRefGoogle Scholar
  64. Volova TG, Kalacheva GS, Altukhova OV (2002) Autotrophic synthesis of polyhydroxyalkanoates by the bacteria Ralstonia eutropha in the presence of carbon monoxide. Appl Microbiol Biotechnol 58:675–678CrossRefGoogle Scholar
  65. Weisz H, Suh S, Graedel TE (2015) Industrial Ecology: the role of manufactured capital in sustainability. Proc Natl Acad Sci U S A 112:6260–6264CrossRefGoogle Scholar
  66. Wilde E (1962) Untersuchungen uber Wachstum und Speikerstoffsynthese von Hydrogenomonas. Arch Mikrobiol 43:109–137CrossRefGoogle Scholar
  67. Wong YM, Brigham CJ, Rha C, Sinskey AJ, Sudesh K (2012) Biosynthesis and characterization of polyhydroxyalkanoate containing high 3-hydroxyhexanoate monomer fraction from crude palm kernel oil by recombinant Cupriavidus necator. Bioresour Technol 121:320–327CrossRefGoogle Scholar
  68. Xiong B, Li Z, Liu L, Zhao D, Zhang X, Bi C (2018) Genome editing of Ralstonia eutropha using an electroporation-based CRISPR-Cas9 technique. Biotechnol Biofuels 11: 172–018–1170-4. eCollection 2018Google Scholar
  69. Yan J, Liu Y, Wang K, Li D, Hu Q, Zhang W (2018) Overexpression of OsPIL1 enhanced biomass yield and saccharification efficiency in switchgrass. Plant Sci 276:143–151CrossRefGoogle Scholar
  70. Yeh YC, Singer SW, Chhabra SR, Beller HR, Mueller J (2017) Hybrid organic-inorganic system for producing biofuels. United States Patent 9,777,300: 3 October, 2017Google Scholar
  71. Yu J, Si Y (2004) Metabolic carbon fluxes and biosynthesis of polyhydroxyalkanoates in Ralstonia eutropha on short chain fatty acids. Biotechnol Prog 20:1015–1024CrossRefGoogle Scholar
  72. Yu J, Wang J (2001) Metabolic flux modeling of detoxification of acetic acid by Ralstonia eutropha at slightly alkaline pH levels. Biotechnol Bioeng 73:458–464CrossRefGoogle Scholar
  73. Zhao S, Li G, Zheng N, Wang J, Yu Z (2018) Steam explosion enhances digestibility and fermentation of corn stover by facilitating ruminal microbial colonization. Bioresour Technol 253:244–251CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Interdisciplinary EngineeringWentworth Institute of TechnologyBostonUSA

Personalised recommendations