title: Pitfalls in optical on-line monitoring for high-throughput screening of microbial systems.
abstract: BACKGROUND
  New high-throughput screening systems for microbial systems, e.g. the BioLector technology, are simple to handle and offer various options of optical online measurements. The parallelization and small scale in microtiter plates allow economical high throughput and, hence, to screen many parameters in reasonable time. Fluorescent proteins as fluorescent tags made the tracking of cellular proteins in-vivo a routine task. All these tools significantly contribute to the understanding of bioprocesses. But, there are some pitfalls which might mislead the user of such techniques.
  
  
  RESULTS
  In this work the bacterium E. coli and the yeast K. lactis expressing the recombinant fluorescent proteins GFP, YFP, FbFP and mCherry were investigated. Cultivations were performed applying special microtiter plates with optodes for dissolved oxygen tension (DOT) and pH measurement in the BioLector system. In this way, microbial growth, protein formation, DOT and pH were monitored on-line via optical signals. During these studies it became obvious that fluorescent proteins can interfere with the optical signals leading to incorrect results. In this work these effects are characterized in detail and possibilities are presented how such adverse effects can be corrected or minimized by mathematical procedures or modification of the measuring method. Additionally, it is shown that morphological changes of cells can affect the biomass on-line monitoring via scattered light.
  
  
  CONCLUSIONS
  The here reported phenomena refer to typical experiments in biotechnological labs. For this reason these aspects are highlighted in this work to make operators of such valuable techniques as the BioLector aware for potential pitfalls and resulting misinterpretations. With the right approach it is possible to minimize existing problems and deal with them.
authors.forename: Martin
authors.initials: M
authors.lastname: Kunze
authors.forename: Simon
authors.initials: S
authors.lastname: Roth
authors.forename: Esther
authors.initials: E
authors.lastname: Gartz
authors.affiliation: AVT-Chair for Biochemical Engineering, RWTH Aachen University, Worringerweg 1, 52074 Aachen, Germany. Jochen.Buechs@avt.rwth-aachen.de.
authors.forename: Jochen
authors.initials: J
authors.lastname: Büchs
mesh_terms: D061249:Batch Cell Culture Techniques; D018533:Biomass; D019149:Bioreactors / Q000382:microbiology; D004926:Escherichia coli / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007218:Industrial Microbiology / Q000295:instrumentation*; D007700:Kinetics; D007716:Kluyveromyces / Q000378:metabolism*; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification
chemical_list: D008164:Luminescent Proteins; D011994:Recombinant Proteins
references.citation: Biotechnol Prog. 2004 Jul-Aug;20(4):1062-8
references.pmid: 15296430
references.citation: Bioprocess Biosyst Eng. 2005 Nov;28(2):75-81
references.pmid: 16240130
references.citation: Anal Chem. 2008 Dec 15;80(24):9435-42
references.pmid: 19006407
references.citation: Biotechnol Bioeng. 2008 Aug 15;100(6):1144-55
references.pmid: 18404769
references.citation: BMC Biotechnol. 2012 Mar 28;12:11
references.pmid: 22455624
references.citation: Biotechnol Prog. 2011 Jan-Feb;27(1):2-14
references.pmid: 21312350
references.citation: Chem Soc Rev. 2009 Oct;38(10):2887-921
references.pmid: 19771335
references.citation: BMC Biotechnol. 2012 Sep 21;12:65
references.pmid: 22999206
references.citation: Biotechnol Bioeng. 2003 Mar 30;81(7):829-36
references.pmid: 12557316
references.citation: Biotechnol Bioeng. 2009 Oct 15;104(3):554-61
references.pmid: 19517521
references.citation: Biotechnol Bioeng. 2001 Apr 20;73(2):95-103
references.pmid: 11255157
references.citation: Biotechnol Bioeng. 2005 Oct 5;92(1):61-8
references.pmid: 15988771
references.citation: Trends Biotechnol. 2012 Jan;30(1):8-16
references.pmid: 21924508
references.citation: Biotechnol Bioeng. 2006 Oct 20;95(3):433-45
references.pmid: 16736531
references.citation: Biotechnol Prog. 2012 Jan-Feb;28(1):103-13
references.pmid: 22012896
references.citation: Microb Cell Fact. 2009 Aug 01;8:42
references.pmid: 19646274
references.citation: Biotechnol Bioeng. 2005 Mar 5;89(5):512-23
references.pmid: 15669089
references.citation: Biotechnol Bioeng. 2010 Oct 15;107(3):497-505
references.pmid: 20517981
references.citation: Trends Biotechnol. 2005 Dec;23(12):605-13
references.pmid: 16269193
references.citation: Trends Microbiol. 2007 Oct;15(10):469-75
references.pmid: 17920276
references.citation: Biotechnol Bioeng. 2003 Jan 20;81(2):178-86
references.pmid: 12451554
references.citation: Microb Cell Fact. 2009 Dec 22;8:68
references.pmid: 20028556
references.citation: Anal Chem. 2007 Nov 15;79(22):8486-93
references.pmid: 17948966
references.citation: Science. 2006 Apr 14;312(5771):217-24
references.pmid: 16614209
references.citation: Appl Environ Microbiol. 2010 Sep;76(17):5990-4
references.pmid: 20601504
references.citation: Cell. 2013 Jun 20;153(7):1602-11
references.pmid: 23768684
references.citation: Nat Methods. 2013 Aug;10(8):751-4
references.pmid: 23770755
references.citation: Biotechnol Prog. 2002 Jul-Aug;18(4):821-30
references.pmid: 12153317
references.citation: Biochem Eng J. 2001 Mar;7(2):113-115
references.pmid: 11173298
references.citation: Lab Chip. 2006 Sep;6(9):1229-35
references.pmid: 16929403
references.citation: Bioprocess Biosyst Eng. 2005 Nov;28(2):109-19
references.pmid: 16049713
references.citation: Biotechnol Bioeng. 1992 Mar 15;39(6):635-46
references.pmid: 18600993
references.citation: Sensors (Basel). 2007 Dec 20;7(12):3472-3480
references.pmid: 28903306
references.citation: Chem Soc Rev. 2009 Oct;38(10):2833-41
references.pmid: 19771330
references.citation: Microb Cell Fact. 2005 Nov 10;4:31
references.pmid: 16283927
references.citation: Biotechnol Bioeng. 2008 Jun 15;100(3):430-8
references.pmid: 18383124
references.citation: Curr Opin Biotechnol. 2006 Dec;17(6):611-8
references.pmid: 17084609
references.citation: Biotechnol Bioeng. 1990 Mar 25;35(7):668-81
references.pmid: 18592563
references.citation: Biotechnol Bioeng. 2004 Feb 20;85(4):376-81
references.pmid: 14755555
references.citation: Biotechnol Bioeng. 2009 Aug 15;103(6):1118-28
references.pmid: 19449392
references.citation: Appl Environ Microbiol. 2000 Jun;66(6):2641-6
references.pmid: 10831450
references.citation: Chem Commun (Camb). 2013 Mar 14;49(21):2139-41
references.pmid: 23389463
references.citation: Biotechnol Bioeng. 2005 Mar 20;89(6):698-708
references.pmid: 15696519
references.citation: Nat Biotechnol. 2007 Apr;25(4):443-5
references.pmid: 17351616