Systems-level characterization of probiotic bifidobacteria - Towards rational optimization of industrial production
Doctoral thesis, 2022
Probiotic Bifidobacterium strains contribute to a healthy gut microbiota of their hosts. Increasing public awareness of this positive effect has resulted in a growing demand for these microorganisms. During industrial production, probiotic microorganisms encounter environmental stressors, which can negatively impact their viability and health-promoting benefits. In this thesis, the current state of knowledge on robustness, stability, and stress physiology in bifidobacteria is reviewed, and the robust and stable Bifidobacterium animalis subsp. lactis BB-12® and the more sensitive Bifidobacterium longum subsp. longum BB-46 are investigated in detail.
The aim of this thesis was to compare the metabolism and physiology of BB-12® and BB-46, and to identify key determinants of growth and viability. The applied approach relied on the integration of constraint-based modeling, classical physiological analyses, and omics analyses. Strain-specific, thoroughly curated, genome-scale metabolic models were built for BB-12® and BB-46, and were applied to identity their nutritional requirements. This allowed for the formulation of a chemically defined medium that supported growth of both strains. The models and medium are valuable tools for optimizing industrial production of these two strains. BB-12® and BB-46 were studied in lab-scale cultivations in the newly formulated medium to identify correlations between cellular characteristics, robustness, and stability of bifidobacteria. Transcriptomic analysis revealed consistently higher expression of several stress-associated genes (e.g., chaperones) in BB-12® as compared to BB-46, which may explain the higher stress tolerance of BB-12®. Upregulation of genes related to DNA repair in BB-46 coincided with increased robustness and stability in stationary compared to exponential phase. The composition of the cultivation medium had a considerable impact on growth and stability of BB-12® and BB-46. The cell membrane fatty acid profile was identified as a key determinant of robustness and stability, by omitting Tween® 80 from the medium. An unsaturated to saturated fatty acid ratio below or around one was found to be beneficial. Moreover, a complex nitrogen source was found to reduce the survival of BB-46, and an increased cell size of BB-12® in complex MRS medium was proposed to contribute to its poor survival under this condition. To assess for possible correlations between gene content and the strain physiology under stress conditions, the genomes of 171 Bifidobacterium strains, including BB-12® and BB-46, were screened for the presence of known stress-associated genes, resulting in the postulation of putative genotype-phenotype correlations. The long-term objective is to use the knowledge gained in this work to guide rational optimization of industrial production processes involving probiotic bifidobacteria.
The aim of this thesis was to compare the metabolism and physiology of BB-12® and BB-46, and to identify key determinants of growth and viability. The applied approach relied on the integration of constraint-based modeling, classical physiological analyses, and omics analyses. Strain-specific, thoroughly curated, genome-scale metabolic models were built for BB-12® and BB-46, and were applied to identity their nutritional requirements. This allowed for the formulation of a chemically defined medium that supported growth of both strains. The models and medium are valuable tools for optimizing industrial production of these two strains. BB-12® and BB-46 were studied in lab-scale cultivations in the newly formulated medium to identify correlations between cellular characteristics, robustness, and stability of bifidobacteria. Transcriptomic analysis revealed consistently higher expression of several stress-associated genes (e.g., chaperones) in BB-12® as compared to BB-46, which may explain the higher stress tolerance of BB-12®. Upregulation of genes related to DNA repair in BB-46 coincided with increased robustness and stability in stationary compared to exponential phase. The composition of the cultivation medium had a considerable impact on growth and stability of BB-12® and BB-46. The cell membrane fatty acid profile was identified as a key determinant of robustness and stability, by omitting Tween® 80 from the medium. An unsaturated to saturated fatty acid ratio below or around one was found to be beneficial. Moreover, a complex nitrogen source was found to reduce the survival of BB-46, and an increased cell size of BB-12® in complex MRS medium was proposed to contribute to its poor survival under this condition. To assess for possible correlations between gene content and the strain physiology under stress conditions, the genomes of 171 Bifidobacterium strains, including BB-12® and BB-46, were screened for the presence of known stress-associated genes, resulting in the postulation of putative genotype-phenotype correlations. The long-term objective is to use the knowledge gained in this work to guide rational optimization of industrial production processes involving probiotic bifidobacteria.
genome-scale metabolic modeling
stability
robustness
industrial manufacturing
bifidobacteria
stress-associated genes
probiotics
nutritional requirement
interspecies variations
Lecture hall KB, Kemigården 4, Gothenburg
Opponent: Dr. Abelardo Margolles, Dairy Research Institute of Asturias (IPLA), Spanish National Research Council (CSIC), Department Microbiology and Biochemistry of Dairy Products, Spain
Author
Marie Schöpping
Chalmers, Biology and Biological Engineering, Industrial Biotechnology
Many people may associate bacteria with diseases; however, bacteria are extremely important for our health, with on average 38 trillion bacterial cells naturally inhabiting the human body, mainly the gut. Among other beneficial functions, bacteria that live in and on us can provide protection against harmful bacteria and promote our immune system. The consumption of sufficient amounts of probiotic bacteria may promote our well-being in situations such as after the intake of antibiotics. Importantly, these probiotics must be alive when they are consumed.
In this thesis, the focus lies on the production of probiotic bifidobacteria. The industrial production of probiotic bifidobacteria faces many challenges. Bifidobacteria are demanding microorganisms regarding nutrient requirements. Moreover, probiotic bifidobacteria are exposed to various stressors during the production process and subsequent storage, which can impact their survival and the delivery of alive bacteria in the probiotic product. The ability to cope with and overcome these perturbations (= robustness), as well as the ability to survive storage conditions (= stability) varies among Bifidobacterium strains, although, the reason for this is poorly understood.
To improve our understanding of the properties that determine robustness and stability in bifidobacteria, I studied two commercial Bifidobacterium strains that differed with respect to these traits. For this purpose, I combined computational and experimental analyses. This allowed me to identify the nutritional requirements of the strains and to formulate a medium with defined composition, which can be used for their industrial cultivation. Moreover, I applied the newly-formulated medium for a comprehensive comparison of the two strains which revealed considerable differences in their metabolism and physiology. I also investigated the effect of changing the medium’s composition on the cellular characteristics of the two strains, including their robustness and stability. Finally, I extended my scope to more than 150 Bifidobacterium strains and looked for correlations between their genetic information and their capability to cope with stressors.
Overall, the results presented in this thesis contribute to a better understanding of the variety in robustness and stability characterizing bifidobacteria, and are expected to provide guidance for the optimization of industrial production processes relating to probiotic bifidobacteria.
In this thesis, the focus lies on the production of probiotic bifidobacteria. The industrial production of probiotic bifidobacteria faces many challenges. Bifidobacteria are demanding microorganisms regarding nutrient requirements. Moreover, probiotic bifidobacteria are exposed to various stressors during the production process and subsequent storage, which can impact their survival and the delivery of alive bacteria in the probiotic product. The ability to cope with and overcome these perturbations (= robustness), as well as the ability to survive storage conditions (= stability) varies among Bifidobacterium strains, although, the reason for this is poorly understood.
To improve our understanding of the properties that determine robustness and stability in bifidobacteria, I studied two commercial Bifidobacterium strains that differed with respect to these traits. For this purpose, I combined computational and experimental analyses. This allowed me to identify the nutritional requirements of the strains and to formulate a medium with defined composition, which can be used for their industrial cultivation. Moreover, I applied the newly-formulated medium for a comprehensive comparison of the two strains which revealed considerable differences in their metabolism and physiology. I also investigated the effect of changing the medium’s composition on the cellular characteristics of the two strains, including their robustness and stability. Finally, I extended my scope to more than 150 Bifidobacterium strains and looked for correlations between their genetic information and their capability to cope with stressors.
Overall, the results presented in this thesis contribute to a better understanding of the variety in robustness and stability characterizing bifidobacteria, and are expected to provide guidance for the optimization of industrial production processes relating to probiotic bifidobacteria.
Subject Categories
Biological Sciences
ISBN
978-91-7905-697-1
Doktorsavhandlingar vid Chalmers tekniska högskola. Ny serie: 5163
Publisher
Chalmers
Lecture hall KB, Kemigården 4, Gothenburg
Opponent: Dr. Abelardo Margolles, Dairy Research Institute of Asturias (IPLA), Spanish National Research Council (CSIC), Department Microbiology and Biochemistry of Dairy Products, Spain