When cells are in suspension they can be easily perceived as particles randomly distributed in three dimensions and if the aim is to measure physical and chemical characteristics of these single cells, a flow cytometer is probably the best choice for the job. Concisely, this machine will order the sample’s single cells into a stream of single particles and carry them in a flow through one of more beams of light (usually a laser). The scattered light and the fluorescence emission (if the cells are labeled) are collected, filtered and converted into digitized values that can be further analyzed elsewhere. Although this machine and its associated methods have been around for a while it is only relatively recently that it has been applied in environmental and industrial microbiology. This dissertation is about flow cytometry in general and its applications in environmental microbiology in the particular case of studying bacterial stress.
Consequently, the introduction will start by shortly describing some crucial perceptions about flow cytometry, and some key terminologies and parameters used in connection with the technique that are fundamental in understanding this tool and the potential that it has for microbiological studies in general. An overview about stress in bacteria and its connection to bacterial heterogeneity is then outlined, followed by practical applications of flow cytometry based methods in the study of stress and heterogeneity in microbial populations. As a final point, the introduction will be followed by conclusions and future perspectives.
This PhD-thesis has resulted in 5 manuscripts of which 2 are published in peer reviewed journals and 3 are drafted manuscripts. These manuscripts are presented in a consecutive order rather than a chronological one. Manuscript 1 describes an innovative holistic approach to describe and study cell population heterogeneity in batch cultures based on experimental and theoretical methods, where the flow cytometer plays an important role in the gathering of experimental data for the construction of theoretical models that model heterogeneous microbial populations. The partially application of the experimental part of this described approach resulted in manuscript 2, where the combination of the use of fluorescent dyes and a whole cell biosensor were enlisted to monitor single-cell heterogeneity of Escherichia coli in batch cultures by flow cytometry. Aiming to develop a simple toolbox of combined single cell techniques for the assessment of cellular heterogeneity in industrial scale fermentations, a biosensor strain of E. coli MG1655 was grown with two different carbon sources (glucose and acetate) and metabolic, growth and physiology data was collected. Results show clearly that the biosensor’s growth dependent GFP expression is consistent with the observation of metabolic activity and that the strain is clearly more stressed in acetate than in glucose. The data analysis approach used highlighted and exposed heterogeneity in a more complete way than more traditional data analysis techniques. Analogously, in manuscript 3, a reporter system was developed to investigate physiological heterogeneity by mapping growth and cell membrane robustness towards freeze-thaw stress at different phases of the cell cultivation. A strong inverse correlation between growth and cell membrane robustness was detected, showing clearly that cellular resources are limited and need to be rationalized constantly and balanced between two functions: growth and robustness. Manuscripts 4 and 5 are examples of the application of flow cytometry and its associated techniques in environmental microbiology. In manuscript 4 these techniques are employed in the evaluation of the application of gamma radiation in wastewater treatment, as the technology inactivates microorganisms even when particle associated, promotes pollutants oxidation and odor nuisance. Among others, samples of active sludge were spiked with a biosensor that expresses GFP when in presence of IPTG, and irradiated at different radiation rates and doses. Amount of metabolic active bacteria after irradiation was measured by flow cytometry. This procedure showed the tremendous potential use of biosensors as biological dosimeters. Manuscript 5 describes the combination of cutting edge culture independent methods, among which flow cytometry, to study the impact in the bacterial composition and diversity of long term exposure to heavy metals, specifically, chromium, arsenic and copper. And amid the other results, one can state that despite the utilization of flow cytometry in this complex samples proved to be tricky and laborious; it showed clearly that it provides accurate population numbers when performed optimally and that surprisingly one of the most ready-used stains in flow cytometry shouldn’t be employed in soil studies.
Overall, the flow cytometer proved to be a resourceful and versatile technique in environmental and industrial microbiology, not only for the particular case of understanding bacterial stress but as well in all other fields of study.