Segregostat: A novel concept to control phenotypic diversification dynamics on the example of Gram-negative bacteria

Abstract

Controlling and managing the degree of phenotypic diversification of microbial populations is a challenging task. This task not only requires detailed knowledge regarding diversification mechanisms but also advances technical setups for the real-time analyses and control of population behavior on single-cell level. In this work, setup, design and operation of the so called segregostat is described which, in contrast to a traditional chemostats, allows the control of phenotypic diversification of microbial populations over time. Two exemplary case studies will be discussed, emphasizing the applicability and versatility of the proposed approach. In detail the phenotypic diversification of Eschericia coli or Pseudomonas putida based on monitoring membrane permeability will be controlled. We show that upon nutrient limitation, cell population tends to diversify into several subpopulations exhibiting distinct phenotypic (non-permeablized and permeablized cells). On-line analysis leads to the determination of the ratio between cells in these two states, which in turn trigger the addition 2 of glucose pulses in order to maintain a pre-defined diversification ratio. These results prove that phenotypic diversification can be controlled by means of defined pulse-frequency modulation within continuously running bioreactor setups. This lays the foundation for systematic studies, not only of phenotypic diversification but also for all processes where dynamics single cell approaches are required, such as synthetic co-culture processes.

Measurement Techniques to Resolve and Control Population Dynamics of Mixed-Culture Processes

“…Optical measurement techniques are currently the most powerful tools that enable the monitoring of mixed cultures and will also have an integral role for active control of these systems ( Table 2). Available technologies are based on fluorescence, absorbance, and scattered light at a single wavelength or with the advanced ability to determine the spectra (Box 2 and Figure 3) [33,52,55,[73][74][75]. All technologies provide insight into the system without any relevant delay [76][77][78].…”

“…A benefit of this method is that the system automatically learns to fulfil the target objective, which could be either to establish a specified population ratio or to adapt the population ratio for maximization of product output [124]. One of the rare published examples of a full implementation of an external feedback control loop for population control was implemented by Sassi and colleagues, proposing the so-called 'Segregostat' concept, which comprises a FC that is coupled to a bioreactor to perform online FC analysis [73]. They used the setup for real-time monitoring and control of heterogeneity profiles of a continuous bioreactor process.…”

Reducing phenotypic instabilities of microbial population during continuous cultivation based on cell switching dynamics

“…According to the sequences controlled by the online FC software, pulse of arabinose was injected in order to quickly increase (approx. 30 seconds) the global arabinose concentration to 0.5 g/L [18]. On-line sampling was performed every 12 minutes and processed according to the dilution sequence set through the following series of steps.…”

“…In the present work, this specific behavioral feature was exploited for optimizing the GFP expression profiles for the whole population. The cell-machine interface set-up is inspired on the segregostat concept that has been previously used for minimizing phenotypic heterogeneity related to the membrane permeabilization process during E. coli continuous cultivation [18] [29]. In its current version, the segregostat comprises a continuous cultivation device connected to an on-line flow cytometry device allowing sampling the state of the population at regular time intervals.…”

“…The strategy relying on the use of environmental perturbations at given frequencies and amplitudes for stabilizing and directing microbial diversification processes was explored through an experimental device previously set-up and published, i.e. the segregostat [18]. The segregostat is a continuous cultivation device connected to on-line flow cytometry for tracking phenotypic diversification processes.…”