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Title: Isotope labelling of bacteria for functional analysis in mixed microbial communities
Author: Chisanga, Malama
ISNI:       0000 0004 8505 105X
Awarding Body: University of Manchester
Current Institution: University of Manchester
Date of Award: 2020
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There is no doubt that microbes are important drivers of bioprocesses that have huge impact on ecosystems that support life on Earth. The prime goal of microbial ecology is to unravel microbial functional diversity and metabolic networks in natural habitats, aimed to improve the quality of life for plants and animals. The steady increase in technological advancements and their application in microbial ecology have greatly enhanced our understanding of functions and metabolic interactions of microbes. Stable isotope probing of bacteria has also emerged as a novel approach to unravel metabolic functions and interactions of microbes directly within environmental samples. The main goal of this thesis is to apply advanced techniques and isotope labelling to gain deeper insights into community functions of microbes, and how cells interact within mixed populations. Firstly, Raman spectroscopy and surface-enhanced Raman scattering (SERS) were optimised and applied in parallel with chemometrics to differentiate near isogenic mutants of Campylobacter jejuni. The optimised conditions for SERS were subsequently applied in combination with isotope labelling for quantitative metabolic fingerprinting of Escherichia coli at bulk and single-cell levels. The results of this study demonstrated the potential application of SERS imaging to characterise E. coli enriched with 13C, 15N and dual 13C and 15N isotopes in a bacterial mixture. Single-cell analysis revealed valuable biochemical information which, if coupled with DNA/RNA-based tools, may permit taxonomic resolution of novel functionally active microbes in future investigations. Furthermore, Raman, Fourier-transform infrared spectroscopies and isotope probing were employed to investigate the rate of incorporation of 13C-labelled substrate by E. coli. This kinetics study generated quantitative data which revealed additive isotope uptake by cells and different rates at which isotopes flow into cellular biomolecules at various time points. This study highlights the potential applicability of vibrational spectroscopy for accurate detection of primary substrate consumers, and to resolve cross-feeding of metabolic products in a microbial community. Finally, Raman spectroscopy, culture volatile metabolites profiling and reverse stable isotope labelling employing kinetics principles of substrate uptake, were used to identify Pseudomonas putida capable of degrading phenol in a two-species community with E. coli. Heavy water was used to investigate cellular metabolic activities and interactions. The results clearly indicate that P. putida was metabolically active in axenic and co-cultures. Whilst E. coli did not show any detectable growth or activity in axenic cultures, it became metabolically active in co-cultures with P. putida. This study also demonstrated the uncoupling of metabolic activity from substrate metabolism by E. coli in co-cultures. Together, these findings indicate the potential application of vibrational spectroscopy and isotope labelling to link bacterial identity to microbially-mediated bioprocesses and to elucidate metabolic interactions in microbial communities in situ.
Supervisor: Lockyer, Nicholas Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Raman spectroscopy ; SERS ; mass spectrometry ; chemometrics ; isotope labelling