Methods for Generating Microbial Cocultures that Grow in the Absence of Fixed Carbon or Nitrogen

  • Matthew J. Smith
  • Matthew B. FrancisEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1772)


This work is motivated by both (1) the desire to make interesting products without the reliance on fixed carbon or fixed nitrogen using microbial cocultures, and (2) the desire to develop new methods for growing microbial communities. The bioplastic polyhydroxybutyrate (PHB), for example, is considered prohibitively expensive to make from sugar (compared to polypropylene). Utilizing and building on engineered strains of Synechococcus elongatus and Azotobacter vinelandii, we have combined a nitrogen-fixing organism with a carbon-fixing organism to make PHB from air, water, sunlight, and trace minerals. Our observations of coculture growth in batch culture led us to develop an improved system based on manipulating the osmotic pressure within a hydrogel. The methods we used to develop this coculture are described in detail in the following chapter, including notes detailing some of our additional observations or thoughts on this system.


Azotobacter Synechococcus Coculture Metabolic crossfeeding Hydrogel 


  1. 1.
    Traxler MF, Watrous JD, Alexandrov T, Dorrestein PC, Kolter R (2013) Interspecies interactions stimulate diversification of the Streptomyces coelicolor secreted metabolome. MBio 4(4). CrossRefGoogle Scholar
  2. 2.
    Bader J, Mast-Gerlach E, Popovic M, Baipai R, Stahl U (2010) Relevance of microbial coculture fermentations in biotechnology. J Appl Microbiol 109:371–387CrossRefGoogle Scholar
  3. 3.
    Goers L, Freemont P, Polizzi KM (2014) Co-culture systems and technologies: taking synthetic biology to the next level. J R Soc Interface 11:20140065CrossRefGoogle Scholar
  4. 4.
    Smith MJ, Francis MB (2016) A designed A. vinelandii-S. elongatus coculture for chemical photoproduction from air, water, phosphate and trace metals. ACS Synth Biol 5(9):955–961CrossRefGoogle Scholar
  5. 5.
    Setubal JC et al (2009) Genome sequence of Azotobacter vinelandii, an obligate aerobe specialized to support diverse anaerobic metabolic processes. J Bacteriol 191:4534–4545CrossRefGoogle Scholar
  6. 6.
    El-Shanshoury AE, Kenawy E, Amara AA, Salama AF, Kishk SS (2013) Optimization of Polyhydroxybutyrate (PHB) production by Azotobacter vinelandii using experimental design. Int J Curr Microbiol Appl Sci 2:227–241Google Scholar
  7. 7.
    Brewin B, Woodley P, Drummond M (1999) The basis of ammonium release in nifL mutants of Azotobacter vinelandii. J Bacteriol 181(23):7356–7362PubMedPubMedCentralGoogle Scholar
  8. 8.
    Ortiz-Marquez JC, Nascimento MD, Dublan M, Curatti L (2012) Association with an ammonium-excreting bacterium allows diazotrophic culture of oil-rich eukaryotic microalgae. Appl Environ Microbiol 78:2345–2352CrossRefGoogle Scholar
  9. 9.
    Ducat DC, Avelar-Rivas JA, Way JC, Silver PA (2012) Rerouting carbon flux to enhance photosynthetic productivity. Appl Environ Microbiol 78:2660–2668CrossRefGoogle Scholar
  10. 10.
    Smith MJ, Francis MB (2017) Improving metabolite production in microbial cocultures using a spatially-constrained hydrogel. Biotechnol Bioeng 114(6):1195–1200CrossRefGoogle Scholar
  11. 11.
    Stanier R, Kunisawa R, Mandel M, Cohen-Bazire G (1971) Purification and properties of unicellular blue-green algae (order Cchroococcales). Bacteriol Rev 35:171–205PubMedPubMedCentralGoogle Scholar
  12. 12.
    D’Mello R, Hill S, Poole RK (1994) Determination of the oxygen affinities of terminal oxidases in Azotobacter vinelandii using the deoxygenation of oxyleghaemoglobin and oxymyoglobin: cytochrome bd is a low-affinity oxidase. Microbiology 140:1395–1402CrossRefGoogle Scholar
  13. 13.
    Engler C, Kandzia R, Marillonnet S (2008) A one pot, one step, precision cloning method with high throughput capability. PLoS One 3:e3647CrossRefGoogle Scholar
  14. 14.
    Porra RJ (2002) The chequered history of the development and use of simultaneous equations for the accurate determination of chlorophylls a and b. Photosynth Res 73:149–156CrossRefGoogle Scholar
  15. 15.
    Law JH, Slepecky RA (1961) Assay of poly-β-hydroxybutyric acid. J Bacteriol 82:33–36PubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  1. 1.Department of ChemistryUniversity of CaliforniaBerkeleyUSA
  2. 2.Materials Sciences DivisionLawrence Berkeley National LaboratoryBerkeleyUSA

Personalised recommendations