TY - BOOK

T1 - Physiological Studies of Lactococcus lactis

T2 - Exploring the Potential of Flow Cytometry

AU - Hansen, Gunda

PY - 2016

Y1 - 2016

N2 - Aiming at a superior performance, survival and stability of dairy starter cultures requires deeperinsights into physiological dynamics and relationships. This PhD thesis contributes to a morecomprehensive physiological understanding of Lactococcus lactis under conditions encounteredduring industrial production by employing flow cytometry for viability assessment, cell sizecomparison, intracellular pH (pHi) determination and cell sorting. The physiological studies ofL. lactis were complemented by examining the growth behavior, glucose consumption, lactateproduction, culturability on solid medium and (specific) acidification activity in milk in response tothe extracellular pH (pHex) during batch fermentations and in response to stress during downstreamprocessing and storage as frozen, freeze- and spray-dried cells.In this PhD thesis, in situ flow cytometric viability assessment was found to facilitate thedifferentiation and accurate quantification of L. lactis cells in different physiological states, whichagreed with the reproductive viability of reference samples and of exponential cells. The highviability of one particular L. lactis strain demonstrated its robustness during fermentation,downstream processing and storage in the absence of a protectant. However, storing freeze-driedL. lactis cells at 30 °C negatively affected the culturability and acidification activity.The reactivation of freeze-dried cells in fermentation medium prior to flow cytometric viabilityassessment, cell size comparison and pHi determination reflected the increasing physiologicalimpairment during this accelerated stability test, while a preincubation in buffer led to inconsistentflow cytometric results. The comparison of reproductive and growth-independent viabilitysuggested the presence of cells showing metabolic activity and membrane integrity but beingnon-culturable during storage.Comparing the size of L. lactis cells during pH-controlled fermentations facilitated theidentification of relationships to growth behavior, glucose metabolism and specific acidificationactivity. For instance, a higher specific acidification activity was maintained during fermentationat pHex 5.5 which was linked to higher cell sizes and ongoing glucose metabolism.In this PhD thesis, flow cytometric pHi determination was employed to examine the responseof L. lactis cells towards different pHex under non-growing conditions in buffer and showed therelevance of the glucose availability and of the growth phase. Kinetics of pHi and pH gradients(pHi – pHex) were recorded during L. lactis fermentations and provided valuable insights into therelationships between pHi perturbations and recovery with regard to growth, growth inhibition andsurvival. On the one hand, cells were able to tolerate and grow despite small negative or evenabsent pH gradients at a high pHex of 7.5 although lag phase was prolonged and growth wasdelayed. On the other hand, challenging cells by shifting the pHex upwards or downwards duringfermentations caused an immediate adaptation of the pHi and the growth behavior. The impactof pHex on the physiology of L. lactis during fermentations was further shown by the earlierinhibition of growth at low or decreasing pHex and by the continuation of growth until glucoselimitation at higher pHex. In this context, the pHi heterogeneity was found to reflect thepH-dependent stress level causing these differences in growth which were presumably related tothe differences in non-dissociated lactic acid concentrations.Because a negative impact of fluorescent labeling and cell sorting on L. lactis physiology wasdetected in this PhD thesis, it was not possible to take advantage of applying flow cytometriccell sorting for the subsequent characterization and recultivation of sorted cells.In conclusion, this PhD thesis demonstrates both the complexity and the potential of flow cytometryin physiological studies of L. lactis. An improved understanding of L. lactis physiology at thesingle-cell level as well as of cell population heterogeneity will provide the basis for optimizingindustrial production processes in terms of biomass and activity.

AB - Aiming at a superior performance, survival and stability of dairy starter cultures requires deeperinsights into physiological dynamics and relationships. This PhD thesis contributes to a morecomprehensive physiological understanding of Lactococcus lactis under conditions encounteredduring industrial production by employing flow cytometry for viability assessment, cell sizecomparison, intracellular pH (pHi) determination and cell sorting. The physiological studies ofL. lactis were complemented by examining the growth behavior, glucose consumption, lactateproduction, culturability on solid medium and (specific) acidification activity in milk in response tothe extracellular pH (pHex) during batch fermentations and in response to stress during downstreamprocessing and storage as frozen, freeze- and spray-dried cells.In this PhD thesis, in situ flow cytometric viability assessment was found to facilitate thedifferentiation and accurate quantification of L. lactis cells in different physiological states, whichagreed with the reproductive viability of reference samples and of exponential cells. The highviability of one particular L. lactis strain demonstrated its robustness during fermentation,downstream processing and storage in the absence of a protectant. However, storing freeze-driedL. lactis cells at 30 °C negatively affected the culturability and acidification activity.The reactivation of freeze-dried cells in fermentation medium prior to flow cytometric viabilityassessment, cell size comparison and pHi determination reflected the increasing physiologicalimpairment during this accelerated stability test, while a preincubation in buffer led to inconsistentflow cytometric results. The comparison of reproductive and growth-independent viabilitysuggested the presence of cells showing metabolic activity and membrane integrity but beingnon-culturable during storage.Comparing the size of L. lactis cells during pH-controlled fermentations facilitated theidentification of relationships to growth behavior, glucose metabolism and specific acidificationactivity. For instance, a higher specific acidification activity was maintained during fermentationat pHex 5.5 which was linked to higher cell sizes and ongoing glucose metabolism.In this PhD thesis, flow cytometric pHi determination was employed to examine the responseof L. lactis cells towards different pHex under non-growing conditions in buffer and showed therelevance of the glucose availability and of the growth phase. Kinetics of pHi and pH gradients(pHi – pHex) were recorded during L. lactis fermentations and provided valuable insights into therelationships between pHi perturbations and recovery with regard to growth, growth inhibition andsurvival. On the one hand, cells were able to tolerate and grow despite small negative or evenabsent pH gradients at a high pHex of 7.5 although lag phase was prolonged and growth wasdelayed. On the other hand, challenging cells by shifting the pHex upwards or downwards duringfermentations caused an immediate adaptation of the pHi and the growth behavior. The impactof pHex on the physiology of L. lactis during fermentations was further shown by the earlierinhibition of growth at low or decreasing pHex and by the continuation of growth until glucoselimitation at higher pHex. In this context, the pHi heterogeneity was found to reflect thepH-dependent stress level causing these differences in growth which were presumably related tothe differences in non-dissociated lactic acid concentrations.Because a negative impact of fluorescent labeling and cell sorting on L. lactis physiology wasdetected in this PhD thesis, it was not possible to take advantage of applying flow cytometriccell sorting for the subsequent characterization and recultivation of sorted cells.In conclusion, this PhD thesis demonstrates both the complexity and the potential of flow cytometryin physiological studies of L. lactis. An improved understanding of L. lactis physiology at thesingle-cell level as well as of cell population heterogeneity will provide the basis for optimizingindustrial production processes in terms of biomass and activity.

UR - https://soeg.kb.dk/permalink/45KBDK_KGL/fbp0ps/alma99122542178005763

M3 - Ph.D. thesis

BT - Physiological Studies of Lactococcus lactis

PB - Department of Food Science, Faculty of Science, University of Copenhagen

ER -