Skip to main content
SpringerLink
Log in

Production of High-Value Polyunsaturated Fatty Acids Using Microbial Cultures

  • Protocol
  • First Online:
Book cover Microbial Lipid Production

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1995))

Abstract

Microbes can produce not only commodity fatty acids, such as palmitic acid (16:0) and stearic acid (18:0), but also high-value fatty acids (essential fatty acids). Most high value fatty acids belong to long chain polyunsaturated fatty acids (PUFA), such as omega-3 fatty acids (e.g., eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)) and omega-6 fatty acids (e.g., arachidonic acid (ARA) and γ-linolenic acid (GLA)). EPA (20:5n-3) is a 20-carbon fatty acid with five double bonds, and the first double bond is in the n-3 position. DHA (22:6n-3) is a 22-carbon fatty acid with 6 double bonds and the first double bond is also in the n-3 position. Both EPA and DHA play an essential role in cardiovascular health including prevention of atherosclerotic disease development (Zehr and Walker, Prostaglandins Other Lipid Mediat 134:131–140, 2018). ARA (20:4n-6) is a 20-carbon fatty acid with four double bonds, and the first double bond is in the n-6 position. GLA (18:3n-6) is an 18-carbon fatty acid with three double bonds, and the first double bond is in the n-6 position. ARA and GLA have multiple biological effects, such as lowering blood cholesterol, and lowering cardiovascular mortality (Poli and Visioli, Eur J Lipid Sci Technol 117(11):1847–1852, 2015). This chapter provides details on microbial production of EAP, DHA, ARA, and GLA.

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 109.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 139.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 199.99
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. Damude HG, Gillies PJ, Macool DJ, Picataggio SK, Pollak DMW, Ragghianti JJ, Xue Z, Yadav NS, Zhang H, Zhu QQ (2011) High eicosapentaenoic acid producing strains of Yarrowia lipolytica. Google Patents

    Google Scholar 

  2. Xue Z, Sharpe PL, Hong S-P, Yadav NS, Xie D, Short DR, Damude HG, Rupert RA, Seip JE, Wang J (2013) Production of omega-3 eicosapentaenoic acid by metabolic engineering of Yarrowia lipolytica. Nat Biotechnol 31(8):734–740

    Article  CAS  PubMed  Google Scholar 

  3. Hoshida H, Ohira T, Minematsu A, Akada R, Nishizawa Y (2005) Accumulation of eicosapentaenoic acid in Nannochloropsis sp. in response to elevated CO2 concentrations. J Appl Phycol 17(1):29–34

    Article  Google Scholar 

  4. Wen Z-Y, Jiang Y, Chen F (2002) High cell density culture of the diatom Nitzschialaevis for eicosapentaenoic acid production: fed-batch development. Process Biochem 37(12):1447–1453

    Article  CAS  Google Scholar 

  5. Chen C-Y, Chen Y-C, Huang H-C, Huang C-C, Lee W-L, Chang J-S (2013) Engineering strategies for enhancing the production of eicosapentaenoic acid (EPA) from an isolated microalga Nannochloropsis oceanica CY2. Bioresour Technol 147:160–167

    Article  CAS  PubMed  Google Scholar 

  6. Chen C-Y, Chen Y-C, Huang H-C, Ho S-H, Chang J-S (2015) Enhancing the production of eicosapentaenoic acid (EPA) from Nannochloropsis oceanica CY2 using innovative photobioreactors with optimal light source arrangements. Bioresour Technol 191:407–413

    Article  CAS  PubMed  Google Scholar 

  7. Wen Z-Y, Chen F (2002) Perfusion culture of the diatom Nitzschia laevis for ultra-high yield of eicosapentaenoic acid. Process Biochem 38(4):523–529

    Article  CAS  Google Scholar 

  8. Camacho-Rodríguez J, González-Céspedes A, Cerón-García M, Fernández-Sevilla J, Acién-Fernández F, Molina-Grima E (2014) A quantitative study of eicosapentaenoic acid (EPA) production by Nannochloropsis gaditana for aquaculture as a function of dilution rate, temperature and average irradiance. Appl Microbiol Biotechnol 98(6):2429–2440

    Article  PubMed  CAS  Google Scholar 

  9. Xu F, Hu H-h, Cong W, Z-l C, Ouyang F (2004) Growth characteristics and eicosapentaenoic acid production by Nannochloropsis sp. in mixotrophic conditions. Biotechnol Lett 26(1):51–53

    Article  CAS  PubMed  Google Scholar 

  10. Athalye SK, Garcia RA, Wen Z (2009) Use of biodiesel-derived crude glycerol for producing eicosapentaenoic acid (EPA) by the fungus Pythium irregulare. J Agric Food Chem 57(7):2739–2744

    Article  CAS  PubMed  Google Scholar 

  11. Okuda T, Ando A, Negoro H, Muratsubaki T, Kikukawa H, Sakamoto T, Sakuradani E, Shimizu S, Ogawa J (2015) Eicosapentaenoic acid (EPA) production by an oleaginous fungus Mortierella alpina expressing heterologous the Δ17-desaturase gene under ordinary temperature. Eur J Lipid Sci Technol 117(12):1919–1927

    Article  CAS  Google Scholar 

  12. Gandhi S, Weete J (1991) Production of the polyunsaturated fatty acids arachidonic acid and eicosapentaenoic acid by the fungus Pythium ultimum. Microbiology 137(8):1825–1830

    CAS  Google Scholar 

  13. Shimiziu S, Kawashima H, Shinmen Y, Akimoto K, Yamada H (1988) Production of eicosapentaenoic acid by Mortierella fungi. J Am Oil Chem Soc 65(9):1455–1459

    Article  Google Scholar 

  14. Liang Y, Zhao X, Strait M, Wen Z (2012) Use of dry-milling derived thin stillage for producing eicosapentaenoic acid (EPA) by the fungus Pythium irregulare. Bioresour Technol 111:404–409

    Article  CAS  PubMed  Google Scholar 

  15. Yazawa K, Araki K, Okazaki N, Watanabe K, Ishikawa C, Inoue A, Numao N, Kondo K (1988) Production of eicosapentaenoic acid by marine bacteria. J Biochem 103(1):5–7

    Article  CAS  PubMed  Google Scholar 

  16. Ward AC, Glassey J (2014) Process development of eicosapentaenoic acid production. Biochem Eng J 82:53–62

    Article  CAS  Google Scholar 

  17. Yazawa K (1996) Production of eicosapentaenoic acid from marine bacteria. Lipids 31(1):S297–S300

    Article  CAS  PubMed  Google Scholar 

  18. Yu R, Yamada A, Watanabe K, Yazawa K, Takeyama H, Matsunaga T, Kurane R (2000) Production of eicosapentaenoic acid by a recombinant marine cyanobacterium, Synechococcus sp. Lipids 35(10):1061–1064

    Article  CAS  PubMed  Google Scholar 

  19. Damude HG, Gillies PJ, Macool DJ, Picataggio SK, Pollak DMW, Ragghianti JJ, Xue Z, Yadav NS, Zhang H, Zhu QQ (2014) High eicosapentaenoic acid producing strains of Yarrowia lipolytica. Google Patents

    Google Scholar 

  20. Pal D, Khozin-Goldberg I, Cohen Z, Boussiba S (2011) The effect of light, salinity, and nitrogen availability on lipid production by Nannochloropsis sp. Appl Microbiol Biotechnol 90(4):1429–1441

    Article  CAS  PubMed  Google Scholar 

  21. Pérez-López P, González-García S, Allewaert C, Verween A, Murray P, Feijoo G, Moreira MT (2014) Environmental evaluation of eicosapentaenoic acid production by Phaeodactylum tricornutum. Sci Total Environ 466:991–100

    Article  PubMed  CAS  Google Scholar 

  22. Shimizu S, Kawashima H, Akimoto K, Shinmen Y, Yamada H (1989) Conversion of linseed oil to an eicosapentaenoic acid-containing oil by Mortierella alpina 1S-4 at low temperature. Appl Microbiol Biotechnol 32(1):1–4

    Article  CAS  Google Scholar 

  23. Sukenik A (1991) Ecophysiological considerations in the optimization of eicosapentaenoic acid production by Nannochloropsis sp. (Eustigmatophyceae). Bioresour Technol 35(3):263–269

    Article  CAS  Google Scholar 

  24. Mitra M, Patidar SK, Mishra S (2015) Integrated process of two stage cultivation of Nannochloropsis sp. for nutraceutically valuable eicosapentaenoic acid along with biodiesel. Bioresour Technol 193:363–369

    Article  CAS  PubMed  Google Scholar 

  25. Wen Z, Chen S (2005) Prospects for eicosapentaenoic acid production using microorganisms. In: Cohen Z, Ratledge C (eds) Single cell oils, pp. 138–160. Champaign: AOCS Press, 2005

    Google Scholar 

  26. Sang M, Wang M, Liu J, Zhang C, Li A (2012) Effects of temperature, salinity, light intensity, and pH on the eicosapentaenoic acid production of Pinguiococcus pyrenoidosus. J Ocean Univ China 11(2):181–186. (English Edition)

    Article  CAS  Google Scholar 

  27. Chen F, Johns MR (1991) Effect of C/N ratio and aeration on the fatty acid composition of heterotrophicChlorella sorokiniana. J Appl Phycol 3(3):203–209

    Article  CAS  Google Scholar 

  28. Bajpai P, Bajpai PK (1993) Eicosapentaenoic acid (EPA) production from microorganisms: a review. J Biotechnol 30(2):161–183

    Article  CAS  PubMed  Google Scholar 

  29. Sevilla JF, Grima EM, Camacho FG, Fernandez FA, Perez JS (1998) Photolimitation and photoinhibition as factors determining optimal dilution rate to produce eicosapentaenoic acid from cultures of the microalga Isochrysis galbana. Appl Microbiol Biotechnol 50(2):199–205

    Article  CAS  Google Scholar 

  30. Chen CY, Yeh KL, Su HM, Lo YC, Chen WM, Chang JS (2010) Strategies to enhance cell growth and achieve high-level oil production of a Chlorella vulgaris isolate. Biotechnol Prog 26(3):679–686

    Article  CAS  PubMed  Google Scholar 

  31. Armenta RE, Valentine MC (2013) Single-cell oils as a source of Omega-3 fatty acids: an overview of recent advances. J Am Oil Chem Society 90(2):167–182

    Article  CAS  Google Scholar 

  32. Ren L-J, Ji X-J, Huang H, Qu L, Feng Y, Tong Q-Q, Ouyang P-K (2010) Development of a stepwise aeration control strategy for efficient docosahexaenoic acid production by Schizochytrium sp. Appl Microbiol Biotechnol 87(5):1649–1656

    Article  CAS  PubMed  Google Scholar 

  33. Qu L, Ji XJ, Ren LJ, Nie ZK, Feng Y, Wu WJ, Ouyang PK, Huang H (2011) Enhancement of docosahexaenoic acid production by Schizochytrium sp. using a two-stage oxygen supply control strategy based on oxygen transfer coefficient. Lett Appl Microbiol 52(1):22–27

    Article  CAS  PubMed  Google Scholar 

  34. Chang G, Gao N, Tian G, Wu Q, Chang M, Wang X (2013) Improvement of docosahexaenoic acid production on glycerol by Schizochytrium sp S31 with constantly high oxygen transfer coefficient. Bioresour Technol 142:400–406

    Article  CAS  PubMed  Google Scholar 

  35. Qu L, Ren L-J, Huang H (2013) Scale-up of docosahexaenoic acid production in fed-batch fermentation by Schizochytrium sp based on volumetric oxygen-transfer coefficient. Biochem Eng J 77:82–87

    Article  CAS  Google Scholar 

  36. Qu L, Ren L-J, Sun G-N, Ji X-J, Nie Z-K, Huang H (2013) Batch, fed-batch and repeated fed-batch fermentation processes of the marine thraustochytrid Schizochytrium sp for producing docosahexaenoic acid. Bioprocess Biosyst Eng 36(12):1905–1912

    Article  CAS  PubMed  Google Scholar 

  37. Ren L-J, Feng Y, Li J, Qu L, Huang H (2013) Impact of phosphate concentration on docosahexaenoic acid production and related enzyme activities in fermentation of Schizochytrium sp. Bioprocess Biosyst Eng 36(9):1177–1183

    Article  CAS  PubMed  Google Scholar 

  38. Chang G, Wu J, Jiang C, Tian G, Wu Q, Chang M, Wang X (2014) The relationship of oxygen uptake rate and k(L)a with rheological properties in high cell density cultivation of docosahexaenoic acid by Schizochytrium sp S31. Bioresour Technol 152:234–240

    Article  CAS  PubMed  Google Scholar 

  39. Ren L-J, Sun L-N, Zhuang X-Y, Qu L, Ji X-J, Huang H (2014) Regulation of docosahexaenoic acid production by Schizochytrium sp.: effect of nitrogen addition. Bioprocess Biosyst Eng 37(5):865–872

    Article  CAS  PubMed  Google Scholar 

  40. Ling X, Guo J, Liu X, Zhang X, Wang N, Lu Y, Ng IS (2015) Impact of carbon and nitrogen feeding strategy on high production of biomass and docosahexaenoic acid (DHA) by Schizochytrium sp LU310. Bioresour Technol 184:139–147

    Article  CAS  PubMed  Google Scholar 

  41. Sun X-M, Ren L-J, Ji X-J, Chen S-L, Guo D-S, Huang H (2016) Adaptive evolution of Schizochytrium sp by continuous high oxygen stimulations to enhance docosahexaenoic acid synthesis. Bioresour Technol 211:374–381

    Article  CAS  PubMed  Google Scholar 

  42. Zhang Y, Min Q, Xu J, Zhang K, Chen S, Wang H, Li D (2016) Effect of malate on docosahexaenoic acid production from Schizochytrium sp B4D1. Electron J Biotechnol 19:56–60

    Article  CAS  Google Scholar 

  43. Zhao X, Ren L, Guo D, Wu W, Ji X, Huang H (2016) CFD investigation of Schizochytrium sp impeller configurations on cell growth and docosahexaenoic acid synthesis. Bioprocess Biosyst Eng 39(8):1297–1304

    Article  CAS  PubMed  Google Scholar 

  44. Jia J, Zhang Q, Lu X, Huang C, Ji J (2015) Impact of pH on docosahexaenoic acid (DHA) production in fermentation of Schizochytrium limacinum. J Biol 32(4):16–19

    CAS  Google Scholar 

  45. Zeng Y, Ji X-J, Lian M, Ren L-J, Jin L-J, Ouyang P-K, Huang H (2011) Development of a temperature shift strategy for efficient docosahexaenoic acid production by a marine fungoid protist, Schizochytrium sp HX-308. Appl Biochem Biotechnol 164(3):249–255

    Article  CAS  PubMed  Google Scholar 

  46. Zhu L, Zhang X, Ji L, Song X, Kuang C (2007) Changes of lipid content and fatty acid composition of Schizochytrium limacinum in response to different temperatures and salinities. Process Biochem 42(2):210–214

    Article  CAS  Google Scholar 

  47. Wu S-T, Yu S-T, Lin L-P (2005) Effect of culture conditions on docosahexaenoic acid production by Schizochytrium sp. S31. Process Biochem 40(9):3103–3108

    Article  CAS  Google Scholar 

  48. Ren L-J, Huang H, Xiao A-H, Lian M, Jin L-J, Ji X-J (2009) Enhanced docosahexaenoic acid production by reinforcing acetyl-CoA and NADPH supply in Schizochytrium sp HX-308. Bioprocess Biosyst Eng 32(6):837–843

    Article  CAS  PubMed  Google Scholar 

  49. Ping WEI, Lujing REN, Xiaojun JI, Qianqian T, Yun F, He H (2011) Effect of reinforcing acetyl-CoA supply in docosahexaenoic acid production by Schizochytrium sp. J Chinese Biotechnol 31(4):87–91

    Google Scholar 

  50. Eroshin V, Dedyukhina E, Chistyakova T, Zhelifonova V, Kurtzman C, Bothast R (1996) Arachidonic-acid production by species of Mortierella. World J Microbiol Biotechnol 12(1):91–96

    Article  CAS  PubMed  Google Scholar 

  51. Eroshin V, Satroutdinov A, Dedyukhina E, Chistyakova T (2000) Arachidonic acid production by Mortierella alpina with growth-coupled lipid synthesis. Process Biochem 35(10):1171–1175

    Article  CAS  Google Scholar 

  52. Hwang B-H, Kim J-W, Park C-Y, Park C-S, Kim Y-S, Ryu Y-W (2005) High-level production of arachidonic acid by fed-batch culture of Mortierella alpina using NH 4 OH as a nitrogen source and pH control. Biotechnol Lett 27(10):731–735

    Article  CAS  PubMed  Google Scholar 

  53. Jin M-J, Huang H, Xiao A-H, Zhang K, Liu X, Li S, Peng C (2008) A novel two-step fermentation process for improved arachidonic acid production by Mortierella alpina. Biotechnol Lett 30(6):1087–1091

    Article  CAS  PubMed  Google Scholar 

  54. Higashiyama K, Yaguchi T, Akimoto K, Fujikawaa S, Shimizu S (1998) Effects of mineral addition on the growth morphology of and arachidonic acid production by Mortierella alpina 1S-4. J Am Oil Chem Soc 75(12):1815–1819

    Article  CAS  Google Scholar 

  55. Yamada H, Shimizu S, Shinmen Y (1987) Production of arachidonic acid by Mortierella elongata 1S-5. Agric Biol Chem 51(3):785–790

    CAS  Google Scholar 

  56. Yuan C, Wang J, Shang Y, Gong G, Yao J, Yu Z (2002) Production of arachidonic acid by Mortierella alpina I~ 4~ 9-N~ 1~ 8. Food Technol Biotechnol 40(4):311–316

    CAS  Google Scholar 

  57. Koike Y, Cai HJ, Higashiyama K, Fujikawa S, Park EY (2001) Effect of consumed carbon to nitrogen ratio of mycelial morphology and arachidonic acid production in cultures of Mortierella alpina. J Biosci Bioeng 91(4):382–389

    Article  CAS  PubMed  Google Scholar 

  58. Shinmen Y, Shimizu S, Akimoto K, Kawashima H, Yamada H (1989) Production of arachidonic acid by Mortierella fungi. Appl Microbiol Biotechnol 31(1):11–16

    Article  CAS  Google Scholar 

  59. Park EY, Koike Y, Higashiyama K, Fujikawa S, Okabe M (1999) Effect of nitrogen source on mycelial morphology and arachidonic acid production in cultures of Mortierella alpina. J Biosci Bioeng 88(1):61–67

    Article  CAS  PubMed  Google Scholar 

  60. Gema H, Kavadia A, Dimou D, Tsagou V, Komaitis M, Aggelis G (2002) Production of [gamma]-linolenic acid by Cunninghamella echinulata cultivated on glucose and orange peel. Appl Microbiol Biotechnol 58(3):303

    Article  CAS  PubMed  Google Scholar 

  61. Conti E, Stredansky M, Stredanska S, Zanetti F (2001) γ-Linolenic acid production by solid-state fermentation of Mucorales strains on cereals. Bioresour Technol 76(3):283–286

    Article  CAS  PubMed  Google Scholar 

  62. Kavadia A, Komaitis M, Chevalot I, Blanchard F, Marc I, Aggelis G (2001) Lipid and γ-linolenic acid accumulation in strains of Zygomycetes growing on glucose. J Am Oil Chem Soc 78(4):341–346

    Article  CAS  Google Scholar 

  63. Ochsenreither K, Glück C, Stressler T, Fischer L, Syldatk C (2016) Production strategies and applications of microbial single cell oils. Front Microbiol 7

    Google Scholar 

  64. Fakas S, Papanikolaou S, Batsos A, Galiotou-Panayotou M, Mallouchos A, Aggelis G (2009) Evaluating renewable carbon sources as substrates for single cell oil production by Cunninghamella echinulata and Mortierella isabellina. Biomass Bioenergy 33(4):573–580

    Article  CAS  Google Scholar 

  65. Sukrutha S, Adamechova Z, Rachana K, Savitha J, Certik M (2014) Optimization of physiological growth conditions for maximal gamma-linolenic acid production by cunninghamella blakesleeana-JSK2. J Am Oil Chem Soc 91(9):1507–1513

    Article  CAS  Google Scholar 

  66. Jangbua P, Laoteng K, Kitsubun P, Nopharatana M, Tongta A (2009) Gamma-linolenic acid production of Mucor rouxii by solid-state fermentation using agricultural by-products. Lett Appl Microbiol 49(1):91–97

    Article  CAS  PubMed  Google Scholar 

  67. Nisha A, Venkateswaran G (2011) Effect of culture variables on mycelial arachidonic acid production by Mortierella alpina. Food Bioprocess Technol 4(2):232–240

    Article  CAS  Google Scholar 

  68. Shu C-H, Lung M-Y (2004) Effect of pH on the production and molecular weight distribution of exopolysaccharide by Antrodia camphorata in batch cultures. Process Biochem 39(8):931–937

    Article  CAS  Google Scholar 

  69. Somashekar D, Venkateshwaran G, Sambaiah K, Lokesh B (2003) Effect of culture conditions on lipid and gamma-linolenic acid production by mucoraceous fungi. Process Biochem 38(12):1719–1724

    Article  CAS  Google Scholar 

  70. Dyal SD, Bouzidi L, Narine SS (2005) Maximizing the production of γ-linolenic acid in Mortierella ramanniana var. ramanniana as a function of pH, temperature and carbon source, nitrogen source, metal ions and oil supplementation. Food Res Int 38(7):815–829

    Article  CAS  Google Scholar 

  71. Chen HC, Chang CC (1996) Production of γ-linolenic acid by the fungus Cunninghamella echinulata CCRC 31840. Biotechnol Prog 12(3):338–341

    Article  CAS  Google Scholar 

  72. Wynn JP, Hamid AA, Li Y, Ratledge C (2001) Biochemical events leading to the diversion of carbon into storage lipids in the oleaginous fungi Mucor circinelloides and Mortierella alpina. Microbiology 147(10):2857–2864

    Article  CAS  PubMed  Google Scholar 

  73. Certik M, Megova J, Horenitzky R (1999) Effect of nitrogen sources on the activities of lipogenic enzymes in oleaginous fungus Cunninghamella echinulata. J Gen Appl Microbiol 45(6):289–293

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

This work was supported by “Natural Science Foundation of Jiangsu Province,” Grant No. BK20170037. “National Key R&D Program of China,” Grant No. 2016YFE0105400, and “The Fundamental Research Funds for the Central Universities,” Grant No. 30916011202.

Author information

Authors and Affiliations

  1. School of Environmental and Biological Engineering, Nanjing University of Science and Technology, Nanjing, China

    Mingjie Jin, Rui Zhai, Zhaoxian Xu & Zhiqiang Wen

Authors
  1. Mingjie Jin
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Rui Zhai
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Zhaoxian Xu
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Zhiqiang Wen
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Mingjie Jin .

Editor information

Editors and Affiliations

  1. Department of Engineering Technology, Biotechnology Program College of Technology, University of Houston, Houston, TX, USA

    Venkatesh Balan

Rights and permissions

Reprints and permissions

Copyright information

© 2019 Springer Science+Business Media, LLC, part of Springer Nature

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Jin, M., Zhai, R., Xu, Z., Wen, Z. (2019). Production of High-Value Polyunsaturated Fatty Acids Using Microbial Cultures. In: Balan, V. (eds) Microbial Lipid Production. Methods in Molecular Biology, vol 1995. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9484-7_15

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-9484-7_15

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-4939-9483-0

  • Online ISBN: 978-1-4939-9484-7

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation