Skip to main content
SpringerLink
Log in

Quantitative Physiology Approaches to Understand and Optimize Reducing Power Availability in Environmental Bacteria

  • Protocol
  • First Online:
Hydrocarbon and Lipid Microbiology Protocols

Part of the book series: Springer Protocols Handbooks ((SPH))

Abstract

The understanding of how carbon fluxes are distributed through a metabolic network offers an overview of the pathways that a given microorganism uses to produce energy, reducing power, and biomass. These invaluable data are related to the physiological state of the cell and provide information about the metabolic potential of microorganisms for specific environmental and biotechnological applications such as the degradation of toxic compounds (e.g., hydrocarbons) or the targeted production of high value-added products (e.g., lipids). Here, we propose a general approach to explore the pathways involved in NADPH balance in bacteria, which are in turn responsible for maintaining redox homeostasis and endowing the microorganism with the ability to counteract oxidative stress. We focus on the fluxes catalyzed by NADP+-dependent enzymes in the metabolic network of the model soil bacterium Pseudomonas putida KT2440. This environmental microorganism is a promising cell factory for a number of NADPH-dependent biotransformations, including industrial and bioremediation processes. The relevant enzymes involved in redox balance in strain KT2440 are (1) glucose-6-phosphate dehydrogenase, (2) 6-phosphogluconate dehydrogenase, (3) isocitrate dehydrogenase, (4) malic enzyme, and (5) 2-keto-6-phosphogluconate reductase. NADPH can be generated or consumed by other enzymatic reactions depending on the microorganism; however, the first four enzymes listed above are recognized as a major source of reducing power in a wide variety of microorganisms. The present protocol includes a first stage in which the NADPH balance is derived from fluxomic data and in vitro enzymatic assays. A second step is then proposed, where the redox ratios of pyridine dinucleotides and the cell capacity to counter oxidative stress are qualitatively correlated.

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

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 109.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Nicholson JK, Lindon JC (2008) Metabonomics. Nature 455:1054–1056

    Article  CAS  PubMed  Google Scholar 

  2. Joyce AR, Palsson BØ (2006) The model organism as a system: integrating “omics” data sets. Nat Rev Mol Cell Biol 7:198–210

    Article  CAS  PubMed  Google Scholar 

  3. Zhang W, Li F, Nie L (2010) Integrating multiple “omics” analysis for microbial biology: application and methodologies. Microbiology 156:287–301

    Article  CAS  PubMed  Google Scholar 

  4. Blankenburg M, Haberland L, Elvers H-D, Tannert C, Jandrig B (2009) High-throughput omics technologies: potential tools for the investigation of influences of EMF on biological systems. Curr Genomics 10:86–92

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Gatherer D (2010) So what do we really mean when we say that systems biology is holistic? BMC Syst Biol 4:22

    Article  PubMed  PubMed Central  Google Scholar 

  6. Kell DB (2004) Metabolomics and systems biology: making sense of the soup. Curr Opin Microbiol 7:296–307

    Article  CAS  PubMed  Google Scholar 

  7. Winter G, Krömer JO (2013) Fluxomics - connecting omics analysis and phenotypes. Environ Microbiol 15:1901–1916

    Article  CAS  PubMed  Google Scholar 

  8. Liu L, Agren R, Bordel S, Nielsen J (2010) Use of genome-scale metabolic models for understanding microbial physiology. FEBS Lett 584:2556–2564

    Article  CAS  PubMed  Google Scholar 

  9. Kitano H (2002) Systems biology: a brief overview. Science 295:1662–1664

    Article  CAS  PubMed  Google Scholar 

  10. Chavarría M, Kleijn RJ, Sauer U, Pflüger-Grau K, de Lorenzo V (2012) Regulatory tasks of the phosphoenolpyruvate-phosphotransferase system of Pseudomonas putida in central carbon metabolism. mBio 3, e00028-12

    Article  PubMed  PubMed Central  Google Scholar 

  11. Fuhrer T, Fischer E, Sauer U (2005) Experimental identification and quantification of glucose metabolism in seven bacterial species. J Bacteriol 187:1581–1590

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Sauer U, Eikmanns BJ (2005) The PEP-pyruvate-oxaloacetate node as the switch point for carbon flux distribution in bacteria. FEMS Microbiol Rev 29:765–794

    Article  CAS  PubMed  Google Scholar 

  13. Dauner M, Bailey JE, Sauer U (2001) Metabolic flux analysis with a comprehensive isotopomer model in Bacillus subtilis. Biotechnol Bioeng 76:144–156

    Article  CAS  PubMed  Google Scholar 

  14. del Castillo T, Ramos JL, Rodríguez-Herva JJ, Fuhrer T, Sauer U, Duque E (2007) Convergent peripheral pathways catalyze initial glucose catabolism in Pseudomonas putida: genomic and flux analysis. J Bacteriol 189:5142–5152

    Article  PubMed  PubMed Central  Google Scholar 

  15. Berger A, Dohnt K, Tielen P, Jahn D, Becker J, Wittmann C (2014) Robustness and plasticity of metabolic pathway flux among uropathogenic isolates of Pseudomonas aeruginosa. PLoS One 9, e88368

    Article  PubMed  PubMed Central  Google Scholar 

  16. Sauer U, Lasko DR, Fiaux J et al (1999) Metabolic flux ratio analysis of genetic and environmental modulations of Escherichia coli central carbon metabolism. J Bacteriol 181:6679–6688

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Perrenoud A, Sauer U (2005) Impact of global transcriptional regulation by ArcA, ArcB, Cra, Crp, Cya, Fnr, and Mlc on glucose catabolism in Escherichia coli. J Bacteriol 187:3171–3179

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Stephanopoulos G (1999) Metabolic fluxes and metabolic engineering. Metab Eng 1:1–11

    Article  CAS  PubMed  Google Scholar 

  19. Blank LM, Ionidis G, Ebert BE, Bühler B, Schmid A (2008) Metabolic response of Pseudomonas putida during redox biocatalysis in the presence of a second octanol phase. FEBS J 275:5173–5190

    Article  CAS  PubMed  Google Scholar 

  20. Xiong W, Liu L, Wu C, Yang C, Wu Q (2010) 13C-Tracer and gas chromatography–mass spectrometry analyses reveal metabolic flux distribution in the oleaginous microalga Chlorella protothecoides. Plant Physiol 154:1001–1011

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Shi H, Shiraishi M, Shimizu K (1997) Metabolic flux analysis for biosynthesis of poly(β-hydroxybutyric acid) in Alcaligenes eutrophus from various carbon sources. J Ferment Bioeng 84:579–587

    Article  CAS  Google Scholar 

  22. Tyo KEJ, Fischer CR, Simeon F, Stephanopoulos G (2010) Analysis of polyhydroxybutyrate flux limitations by systematic genetic and metabolic perturbations. Metab Eng 12:187–195

    Article  CAS  PubMed  Google Scholar 

  23. Nanchen A, Fuhrer T, Sauer U (2007) Determination of metabolic flux ratios from 13C-experiments and gas chromatography–mass spectrometry data: protocol and principles. Methods Mol Biol 358:177–197

    Article  CAS  PubMed  Google Scholar 

  24. Fischer E, Sauer U (2005) Large-scale in vivo flux analysis shows rigidity and suboptimal performance of Bacillus subtilis metabolism. Nat Genet 37:636–640

    Article  CAS  PubMed  Google Scholar 

  25. Dauner M, Sauer U (2000) GC-MS analysis of amino acids rapidly provides rich information for isotopomer balancing. Biotechnol Prog 16:642–649

    Article  CAS  PubMed  Google Scholar 

  26. Sauer U (2006) Metabolic networks in motion: 13C-based flux analysis. Mol Syst Biol 2:62

    Article  PubMed  PubMed Central  Google Scholar 

  27. Fischer E, Zamboni N, Sauer U (2004) High-throughput metabolic flux analysis based on gas chromatography–mass spectrometry derived 13C constraints. Anal Biochem 325:308–316

    Article  CAS  PubMed  Google Scholar 

  28. Fischer E, Sauer U (2003) Metabolic flux profiling of Escherichia coli mutants in central carbon metabolism using GC-MS. Eur J Biochem 270:880–891

    Article  CAS  PubMed  Google Scholar 

  29. Kleijn RJ, Buescher JM, Le Chat L, Jules M, Aymerich S, Sauer U (2010) Metabolic fluxes during strong carbon catabolite repression by malate in Bacillus subtilis. J Biol Chem 285:1587–1596

    Article  CAS  PubMed  Google Scholar 

  30. Meijnen JP, de Winde JH, Ruijssenaars HJ (2012) Metabolic and regulatory rearrangements underlying efficient D-xylose utilization in engineered Pseudomonas putida S12. J Biol Chem 287:14606–14614

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Yang C, Hua Q, Shimizu K (2002) Metabolic flux analysis in Synechocystis using isotope distribution from 13C-labeled glucose. Metab Eng 4:202–216

    Article  CAS  PubMed  Google Scholar 

  32. Nissen TL, Schulze U, Nielsen J, Villadsen J (1997) Flux distributions in anaerobic, glucose-limited continuous cultures of Saccharomyces cerevisiae. Microbiology 143:203–218

    Article  CAS  PubMed  Google Scholar 

  33. Christensen B, Nielsen J (1999) Isotopomer analysis using GC-MS. Metab Eng 1:282–290

    Article  CAS  PubMed  Google Scholar 

  34. Nikel PI, Zhu J, San KY, Méndez BS, Bennett GN (2009) Metabolic flux analysis of Escherichia coli creB and arcA mutants reveals shared control of carbon catabolism under microaerobic growth conditions. J Bacteriol 191:5538–5548

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Emmerling M, Dauner M, Ponti A et al (2002) Metabolic flux responses to pyruvate kinase knockout in Escherichia coli. J Bacteriol 184:152–164

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Massou S, Nicolas C, Letisse F, Portais JC (2007) NMR-based fluxomics: quantitative 2D NMR methods for isotopomers analysis. Phytochemistry 68:2330–2340

    Article  CAS  PubMed  Google Scholar 

  37. Sekiyama Y, Kikuchi J (2007) Towards dynamic metabolic network measurements by multi-dimensional NMR-based fluxomics. Phytochemistry 68:2320–2329

    Article  CAS  PubMed  Google Scholar 

  38. Sauer U (2004) High-throughput phenomics: experimental methods for mapping fluxomes. Curr Opin Biotechnol 15:58–63

    Article  CAS  PubMed  Google Scholar 

  39. Nargund S, Joffe ME, Tran D, Tugarinov V, Sriram G (2013) Nuclear magnetic resonance methods for metabolic fluxomics. Methods Mol Biol 985:335–351

    Article  CAS  PubMed  Google Scholar 

  40. Chavarría M, Nikel PI, Pérez-Pantoja D, de Lorenzo V (2013) The Entner-Doudoroff pathway empowers Pseudomonas putida KT2440 with a high tolerance to oxidative stress. Environ Microbiol 15:1772–1785

    Article  PubMed  Google Scholar 

  41. Banga JR (2008) Optimization in computational systems biology. BMC Syst Biol 2:47

    Article  PubMed  PubMed Central  Google Scholar 

  42. Fuhrer T, Sauer U (2009) Different biochemical mechanisms ensure network-wide balancing of reducing equivalents in microbial metabolism. J Bacteriol 191:2112–2121

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Chen X, Li S, Liu L (2014) Engineering redox balance through cofactor systems. Trends Biotechnol 32:337–343

    Article  CAS  PubMed  Google Scholar 

  44. Singh R, Mailloux RJ, Puiseux-Dao S, Appanna VD (2007) Oxidative stress evokes a metabolic adaptation that favors increased NADPH synthesis and decreased NADH production in Pseudomonas fluorescens. J Bacteriol 189:6665–6675

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Berríos-Rivera SJ, Bennett GN, San KY (2002) Metabolic engineering of Escherichia coli: increase of NADH availability by overexpressing an NAD+-dependent formate dehydrogenase. Metab Eng 4:217–229

    Article  PubMed  Google Scholar 

  46. Berríos-Rivera SJ, Bennett GN, San KY (2002) The effect of increasing NADH availability on the redistribution of metabolic fluxes in Escherichia coli chemostat cultures. Metab Eng 4:230–237

    Article  PubMed  Google Scholar 

  47. Ruiz JA, de Almeida A, Godoy MS et al (2013) Escherichia coli redox mutants as microbial cell factories for the synthesis of reduced biochemicals. Comput Struct Biotechnol J 3, e201210019

    PubMed  PubMed Central  Google Scholar 

  48. Storz G, Imlay JA (1999) Oxidative stress. Curr Opin Microbiol 2:188–194

    Article  CAS  PubMed  Google Scholar 

  49. Cabiscol E, Tamarit J, Ros J (2000) Oxidative stress in bacteria and protein damage by reactive oxygen species. Int Microbiol 3:3–8

    CAS  PubMed  Google Scholar 

  50. Carmel-Harel O, Storz G (2000) Roles of the glutathione- and thioredoxin-dependent reduction systems in the Escherichia coli and Saccharomyces cerevisiae responses to oxidative stress. Annu Rev Microbiol 54:439–461

    Article  CAS  PubMed  Google Scholar 

  51. Masip L, Veeravalli K, Georgiou G (2006) The many faces of glutathione in bacteria. Antioxid Redox Signal 8:753–762

    Article  CAS  PubMed  Google Scholar 

  52. Romano AH, Conway T (1996) Evolution of carbohydrate metabolic pathways. Res Microbiol 147:448–455

    Article  CAS  PubMed  Google Scholar 

  53. Downs DM (2006) Understanding microbial metabolism. Annu Rev Microbiol 60:533–559

    Article  CAS  PubMed  Google Scholar 

  54. Sudarsan S, Dethlefsen S, Blank LM, Siemann-Herzberg M, Schmid A (2014) The functional structure of central carbon metabolism in Pseudomonas putida KT2440. Appl Environ Microbiol 80:5292–5303

    Article  PubMed  PubMed Central  Google Scholar 

  55. Conway T (1992) The Entner-Doudoroff pathway: history, physiology and molecular biology. FEMS Microbiol Rev 9:1–27

    Article  CAS  PubMed  Google Scholar 

  56. Poulsen BR, Nøhr J, Douthwaite S et al (2005) Increased NADPH concentration obtained by metabolic engineering of the pentose phosphate pathway in Aspergillus niger. FEBS J 272:1313–1325

    Article  CAS  Google Scholar 

  57. Lee WH, Park JB, Park K, Kim MD, Seo JH (2007) Enhanced production of ε-caprolactone by overexpression of NADPH-regenerating glucose 6-phosphate dehydrogenase in recombinant Escherichia coli harboring cyclohexanone monooxygenase gene. Appl Microbiol Biotechnol 76:329–338

    Article  CAS  PubMed  Google Scholar 

  58. Marino D, González EM, Frendo P, Puppo A, Arrese-Igor C (2007) NADPH recycling systems in oxidative stressed pea nodules: a key role for the NADP+-dependent isocitrate dehydrogenase. Planta 225:413–421

    Article  CAS  PubMed  Google Scholar 

  59. Rippa M, Giovannini PP, Barrett MP, Dallocchio F, Hanau S (1998) 6-Phosphogluconate dehydrogenase: the mechanism of action investigated by a comparison of the enzyme from different species. Biochim Biophys Acta 1429:83–92

    Article  CAS  PubMed  Google Scholar 

  60. Moritz B, Striegel K, De Graaf AA, Sahm H (2000) Kinetic properties of the glucose-6-phosphate and 6-phosphogluconate dehydrogenases from Corynebacterium glutamicum and their application for predicting pentose phosphate pathway flux in vivo. Eur J Biochem 267:3442–3452

    Article  CAS  PubMed  Google Scholar 

  61. Minard KI, McAlister-Henn L (2005) Sources of NADPH in yeast vary with carbon source. J Biol Chem 280:39890–39896

    Article  CAS  PubMed  Google Scholar 

  62. Miyagi H, Kawai S, Murata K (2009) Two sources of mitochondrial NADPH in the yeast Saccharomyces cerevisiae. J Biol Chem 284:7553–7560

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. Wynn JP, Ratledge C (1997) Malic enzyme is a major source of NADPH for lipid accumulation by Aspergillus nidulans. Microbiology 143:253–257

    Article  CAS  Google Scholar 

  64. Ayala A, F-Lobato M, Machado A (1986) Malic enzyme levels are increased by the activation of NADPH-consuming pathways: detoxification processes. FEBS Lett 202:102–106

    Article  CAS  PubMed  Google Scholar 

  65. Blank LM, Lehmbeck F, Sauer U (2005) Metabolic-flux and network analysis in fourteen hemiascomycetous yeasts. FEMS Yeast Res 5:545–558

    Article  CAS  PubMed  Google Scholar 

  66. Remize F, Andrieu E, Dequin S (2000) Engineering of the pyruvate dehydrogenase bypass in Saccharomyces cerevisiae: role of the cytosolic Mg2+ and mitochondrial K+ acetaldehyde dehydrogenases Ald6p and Ald4p in acetate formation during alcoholic fermentation. Appl Environ Microbiol 66:3151–3159

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  67. Nikel PI, Kim J, de Lorenzo V (2014) Metabolic and regulatory rearrangements underlying glycerol metabolism in Pseudomonas putida KT2440. Environ Microbiol 16:239–254

    Article  CAS  PubMed  Google Scholar 

  68. Sauer U, Canonaco F, Heri S, Perrenoud A, Fischer E (2004) The soluble and membrane-bound transhydrogenases UdhA and PntAB have divergent functions in NADPH metabolism of Escherichia coli. J Biol Chem 279:6613–6619

    Article  CAS  PubMed  Google Scholar 

  69. Rühl M, Le Coq D, Aymerich S, Sauer U (2012) 13C-Flux analysis reveals NADPH-balancing transhydrogenation cycles in stationary phase of nitrogen-starving Bacillus subtilis. J Biol Chem 287:27959–27970

    Article  PubMed  PubMed Central  Google Scholar 

  70. Singh R, Lemire J, Mailloux RJ, Appanna VD (2008) A novel strategy involved anti-oxidative defense: the conversion of NADH into NADPH by a metabolic network. PLoS One 3, e2682

    Article  PubMed  PubMed Central  Google Scholar 

  71. Sprenger GA (1995) Genetics of pentose-phosphate pathway enzymes of Escherichia coli K-12. Arch Microbiol 164:324–330

    Article  CAS  PubMed  Google Scholar 

  72. Dean AM, Golding GB (1997) Protein engineering reveals ancient adaptive replacements in isocitrate dehydrogenase. Proc Natl Acad Sci U S A 94:3104–3109

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  73. Eyzaguirre J, Cornwell E, Borie G, Ramirez B (1973) Two malic enzymes in Pseudomonas aeruginosa. J Bacteriol 116:215–221

    CAS  PubMed  PubMed Central  Google Scholar 

  74. Nelson KE, Weinel C, Paulsen IT et al (2002) Complete genome sequence and comparative analysis of the metabolically versatile Pseudomonas putida KT2440. Environ Microbiol 4:799–808

    Article  CAS  PubMed  Google Scholar 

  75. Nikel PI, Martínez-García E, de Lorenzo V (2014) Biotechnological domestication of pseudomonads using synthetic biology. Nat Rev Microbiol 12:368–379

    Article  CAS  PubMed  Google Scholar 

  76. Zamboni N, Fendt S-M, Rühl M, Sauer U (2009) 13C-based metabolic flux analysis. Nat Protoc 4:878–892

    Article  CAS  PubMed  Google Scholar 

  77. Zamboni N, Fischer E, Sauer U (2005) FiatFlux - a software for metabolic flux analysis from 13C-glucose experiments. BMC Bioinformatics 6:209

    Article  PubMed  PubMed Central  Google Scholar 

  78. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    Article  CAS  PubMed  Google Scholar 

  79. Bernofsky C, Swan M (1973) An improved cycling assay for nicotinamide adenine dinucleotide. Anal Biochem 53:452–458

    Article  CAS  PubMed  Google Scholar 

  80. Nikel PI, Pettinari MJ, Ramirez MC, Galvagno MA, Méndez BS (2008) Escherichia coli arcA mutants: metabolic profile characterization of microaerobic cultures using glycerol as a carbon source. J Mol Microbiol Biotechnol 15:48–54

    Article  CAS  PubMed  Google Scholar 

  81. Nikel PI, Pettinari MJ, Galvagno MA, Méndez BS (2010) Metabolic selective pressure stabilizes plasmids carrying biosynthetic genes for reduced biochemicals in Escherichia coli redox mutants. Appl Microbiol Biotechnol 88:563–573

    Article  CAS  PubMed  Google Scholar 

  82. Leonardo MR, Dailly Y, Clark DP (1996) Role of NAD in regulating the adhE gene of Escherichia coli. J Bacteriol 178:6013–6018

    CAS  PubMed  PubMed Central  Google Scholar 

  83. Miller JH (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor

    Google Scholar 

  84. Nikel PI, de Lorenzo V (2013) Engineering an anaerobic metabolic regime in Pseudomonas putida KT2440 for the anoxic biodegradation of 1,3-dichloroprop-1-ene. Metab Eng 15:98–112

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Systems and Synthetic Biology Program, Centro Nacional de Biotecnología (CNB-CSIC), 28049, Cantoblanco, Madrid, Spain

    Pablo I. Nikel

  2. Escuela de Química & Centro de Investigaciones en Productos Naturales (CIPRONA), Universidad de Costa Rica, 11501-2060, San José, Costa Rica

    Max Chavarría

  3. Centro Nacional de Innovaciones Biotecnológicas (CENIBiot), CeNAT-CONARE, 1174-1200, San José, Costa Rica

    Max Chavarría

Authors
  1. Pablo I. Nikel
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Max Chavarría
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Max Chavarría .

Editor information

Editors and Affiliations

  1. University of Essex Dept. Biological Sciences, Colchester, Essex, United Kingdom

    Terry J. McGenity

  2. Institute of Microbiology, Technical University Braunschweig, Braunschweig, Germany

    Kenneth N. Timmis

  3. Department of Biology, University of the Balearic Islands and Mediterranean Institute for Advanced Studies (IMEDEA, UIB-CSIC), Palma de Mallorca, Spain

    Balbina Nogales

Rights and permissions

Reprints and permissions

Copyright information

© 2015 Springer-Verlag Berlin Heidelberg

About this protocol

Cite this protocol

Nikel, P.I., Chavarría, M. (2015). Quantitative Physiology Approaches to Understand and Optimize Reducing Power Availability in Environmental Bacteria. In: McGenity, T., Timmis, K., Nogales, B. (eds) Hydrocarbon and Lipid Microbiology Protocols . Springer Protocols Handbooks. Springer, Berlin, Heidelberg. https://doi.org/10.1007/8623_2015_84

Download citation

  • DOI: https://doi.org/10.1007/8623_2015_84

  • Published:

  • Publisher Name: Springer, Berlin, Heidelberg

  • Print ISBN: 978-3-662-50430-7

  • Online ISBN: 978-3-662-50432-1

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation