Understanding the compositional changes in LB Lennox medium during growth of Escherichia coli DH5α in a 1L bioreactor

Abstract

Compositional changes in growth medium represents dynamic interplay between cell growth, biomass formation, and energy maintenance, with concomitant decrease in nutrients and increase in secreted metabolites and metabolic byproducts. Such information is important for quantifying microbial physiological response both at the population and cellular level, with respect to understanding subtle differences in microbial growth response, as well as supporting model building efforts in metabolic engineering. With the desire to understand molecular weight changes in components of LB Lennox medium as well as broth fractions present during cultivation of Escherichia coli DH5 (ATCC 53868) at 37 {degrees}C, 400 rpm stirring and 1 VVM aeration in a 1 L bioreactor, this study used a combination of gel filtration chromatography (GFC) and reversed phase high performance liquid chromatography (RP-HPLC) for determining changes to molecular weight of different fractions of the growth medium. Experiment results revealed the difficulty of fractionating the culture broth with RP-HPLC, where no distinct peaks of narrow retention time width were obtained. More importantly, the column used for GFC was unable to differentiate small molecular weight changes on the order of a few tens to few hundred Da through a refractive index detector. Together, GFC and RP-HPLC highlighted the difficulty of fractionating LB Lennox culture broth into different distinct fractions. Finally, the study validated the use of 194 nm as detection wavelength for visualizing the chromatogram of LB Lennox medium eluted from a C-18 reversed phase column during liquid chromatography. Collectively, GFC and RP-HPLC could not fractionate LB Lennox broth of E. coli DH5 into distinct fractions for further analysis by identification techniques such as mass spectrometry. Given the inherent complexity of complex medium such as LB Lennox, clean separation of the medium into every component with high purity may be impossible. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=47 SRC="FIGDIR/small/311373v1_ufig1.gif" ALT="Figure 1"> View larger version (7K): org.highwire.dtl.DTLVardef@2d0861org.highwire.dtl.DTLVardef@814ebcorg.highwire.dtl.DTLVardef@158328borg.highwire.dtl.DTLVardef@15e6844_HPS_FORMAT_FIGEXP M_FIG C_FIG Short descriptionBroad elution profile and lack of distinct peaks in chromatogram of LB Lennox broth at 6 hours post inoculation with Escherichia coli DH5 (ATCC 53868), after attempted fractionation by C-18 reversed phase high performance liquid chromatography (RP-HPLC), revealed that separation could not be achieved with a complex mixture such as a microbiology broth. Specifically, while the objective of understanding compositional changes to culture medium during growth presents tremendous opportunities for determining the dynamic conversion of nutrients into metabolites and byproducts, difficulty of profiling all chemical constituents in a complex broth mixture meant that current model building efforts for metabolic engineering remains primitive with respect to the consortia of metabolic reactions occurring in situ at the cellular level. Subject areasbiotechnology, biochemistry, cell biology, microbiology, analytical chemistry