Footprint area analysis of binary imaged Cupriavidus necator cells to study PHB production at balanced, transient, and limited growth conditions in a cascade process.
Statistical distribution of cell and poly[3-(R)-hydroxybutyrate] (PHB) granule size and number of granules per cell are investigated for PHB production in a five-stage cascade (5CSTR). Electron microscopic pictures of cells from individual cascade stages (R1-R5) were converted to binary pictures to visualize footprint areas for polyhydroxyalkanoate (PHA) and non-PHA biomass. Results for each stage were correlated to the corresponding experimentally determined kinetics (specific growth rate μ and specific productivity π). Log-normal distribution describes PHA granule size dissimilarity, whereas for R1 and R4, gamma distribution best reflects the situation. R1, devoted to balanced biomass synthesis, predominately contains cells with rather small granules, whereas with increasing residence time τ, maximum and average granule sizes by trend increase, approaching an upper limit determined by the cell's geometry. Generally, an increase of intracellular PHA content and ratio of granule to cell area slow down along the cascade. Further, the number of granules per cell decreases with increasing τ. Data for μ and π obtained by binary picture analysis correlate well with the experimental results. The work describes long-term continuous PHA production under balanced, transient, and nutrient-deficient conditions, as well as their reflection on the granules size, granule number, and cell structure on the microscopic level.
Department of Biochemical Engineering, Faculty of Food Technology and Biotechnology, University of Zagreb, Pierottijeva 6, 10000, Zagreb, Croatia.
Denis
D
Vadlja
Office of Research Management and Service, c/o Institute of Chemistry, NAWI Graz, University of Graz, Heinrichstrasse 28/III, 8010, Graz, Austria. martin.koller@uni-graz.at.|ARENA Arbeitsgemeinschaft für Ressourcenschonende & Nachhaltige Technologien, Inffeldgasse 21b, 8010, Graz, Austria. martin.koller@uni-graz.at.
Martin
http://orcid.org/0000-0002-9251-1822
M
Koller
Department of Biochemical Engineering, Faculty of Food Technology and Biotechnology, University of Zagreb, Pierottijeva 6, 10000, Zagreb, Croatia.
Mario
M
Novak
ARENA Arbeitsgemeinschaft für Ressourcenschonende & Nachhaltige Technologien, Inffeldgasse 21b, 8010, Graz, Austria.
Gerhart
G
Braunegg
Department of Biochemical Engineering, Faculty of Food Technology and Biotechnology, University of Zagreb, Pierottijeva 6, 10000, Zagreb, Croatia.
Predrag
P
Horvat
D019149:Bioreactors / Q000382:microbiology*; D020118:Cupriavidus necator / Q000378:metabolism*; D006885:Hydroxybutyrates / Q000378:metabolism*; D008854:Microscopy, Electron; D061848:Optical Imaging / Q000379:methods*; D011091:Polyesters / Q000378:metabolism*; D000091402:Prohibitins; D000094122:Polyhydroxybutyrates
D006885:Hydroxybutyrates; D011091:Polyesters; D000091402:Prohibitins; C000717328:PHB protein, human; C003182:poly-beta-hydroxybutyrate; D000094122:Polyhydroxybutyrates
Binary imaging; Bioreactor cascade; Cupriavidus necator; Footprint area analysis; PHA granules; Poly[3-(R)-hydroxybutyrate] (PHB)
J Biotechnol. 2015 Apr 10;199:17-20
25661838
Nat Biotechnol. 1997 Jan;15(1):63-7
9035108
Biomacromolecules. 2009 Apr 13;10(4):660-9
19275166
Chem Soc Rev. 2009 Aug;38(8):2434-46
19623359
Biosci Biotechnol Biochem. 2015;79(8):1369-77
25971301
Appl Microbiol Biotechnol. 2011 Jul;91(2):295-304
21503760
Microbiology. 2005 Mar;151(Pt 3):825-33
15758228
Appl Environ Microbiol. 1997 Sep;63(9):3703-6
16535699
Microbiology. 2002 Aug;148(Pt 8):2413-26
12177335
J Bacteriol. 2002 Jan;184(1):59-66
11741844
J Mol Biol. 1968 Aug 14;35(3):489-502
4970600
J Gen Microbiol. 1964 Mar;34:441-6
14135548
Environ Microbiol. 2015 May;17(5):1765-76
25297625
Appl Microbiol Biotechnol. 2004 Jun;64(5):611-7
14758517
Environ Microbiol. 2014 Aug;16(8):2357-73
24329995
Microbiol Rev. 1990 Dec;54(4):450-72
2087222
J Bacteriol. 2012 Nov;194(21):5909-21
22923598
Appl Environ Microbiol. 2007 Jan;73(2):586-93
17085698
FEMS Microbiol Lett. 2016 May;363(10):null
27190240
Biotechnol Bioeng. 2015 Dec;112(12):2475-84
26059321
Int J Biol Macromol. 1999 Jun-Jul;25(1-3):3-19
10416645
Appl Environ Microbiol. 2013 May;79(9):2989-99
23435892
Nat Prod Rep. 2003 Oct;20(5):445-57
14620841
J Gen Appl Microbiol. 2015;61(2):63-6
26018503
Curr Opin Biotechnol. 2014 Dec;30:59-65
24976377
Annu Rev Microbiol. 2002;56:403-32
12213937
J Bacteriol. 2005 Jun;187(11):3814-24
15901706
Appl Environ Microbiol. 2015 Jul;81(13):4411-22
25911471
Proc Natl Acad Sci U S A. 1995 Jul 3;92(14):6279-83
7603982
Int J Biol Macromol. 2002 Apr 8;30(2):97-104
11911900
Adv Drug Deliv Rev. 2001 Dec 3;53(1):5-21
11733115
BMC Microbiol. 2012 Nov 16;12:262
23157596
Biomacromolecules. 2005 Mar-Apr;6(2):552-60
15762612
Bioprocess Biosyst Eng. 2013 Sep;36(9):1235-50
23135491
Mol Microbiol. 2011 Nov;82(4):936-51
22023320