Population heterogeneity in microbial bioprocesses: origin, analysis, mechanisms, and future perspectives

Heins, Anna‐Lena; Weuster‐Botz, Dirk

doi:10.1007/s00449-018-1922-3

Cited by 65 publications

(80 citation statements)

References 277 publications

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“…Due to technical and economic constraints for bioreactor operation, the demand for high oxygen levels is hardly satisfied. Imperfect mixing in large‐scale bioreactor regions with low oxygen concentrations can also appear (Heins & Weuster‐Botz, ). Oxygen limitation is undesirable for E. coli because fermentation pathways are activated with the concomitant accumulation of metabolic byproducts.…”

Section: Introductionmentioning

confidence: 99%

Design of a microaerobically inducible replicon for high‐yield plasmid DNA production

Jaén

Velázquez

Sigala

et al. 2019

Biotech & Bioengineering

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A pUC-derived replicon inducible by oxygen limitation was designed and tested in fed-batch cultures of Escherichia coli. It included the addition of a second inducible copy of rnaII, the positive replication control element. The rnaII gene was expressed from P trc and cloned into pUC18 to test the hypothesis that the ratio of the positive control molecule RNAII to the negative control element, RNAI, was the determinant of plasmid copy number per chromosome (PCN). The construct was evaluated in several E. coli strains. Evaluations of the RNAII/RNAI ratio, PCN and plasmid yield normalized to biomass (Y pDNA/X ) were performed and the initial hypothesis was probed. Furthermore, in high cell-density cultures in shake flasks, an outstanding amount of 126 mg/L of plasmid was produced. The microaerobically inducible plasmid was obtained by cloning the rnaII gene under the control of the oxygen-responsive Vitreoscilla stercoraria hemoglobin promoter. For this plasmid, but not for pUC18, the RNAII/RNAI ratio, PCN and Y pDNA/X efficiently increased after the shift to the microaerobic regime in fed-batch cultures in a 1 L bioreactor. The Y pDNA/X of the inducible plasmid reached 12 mg/g at the end of the fed-batch but the original pUC18 only reached ca. 6 mg/g. The proposed plasmid is a valuable alternative for the operation and scale-up of plasmid DNA production processes in which mass transfer limitations will not represent an issue. K E Y W O R D Smicroaerobic induction, oxygen limitation, plasmid DNA, pUC replicon, rnaII

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Section: Introductionmentioning

confidence: 99%

Design of a microaerobically inducible replicon for high‐yield plasmid DNA production

Jaén

Velázquez

Sigala

et al. 2019

Biotech & Bioengineering

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“…Nowadays, it is known that microbial cell populations in industrial‐scale, biotechnological production processes, though originating from pure, isogenic cultures, are heterogeneous. Due to nonideal mixing, gradients of process parameters like substrate, dissolved oxygen, and pH arise, creating different local microenvironments that are experienced by cells traveling throughout the reactor . This induces dynamic cell responses on genetic, metabolic, and physiological level, and consequently causes development of population heterogeneity .…”

Section: Introductionmentioning

confidence: 99%

“…Hence, potentially less productive or less robust subpopulations can occur that reduce overall process efficiency. Even though these consequences of population heterogeneity and acetate formation are well known, the underlying cellular phenomena are poorly understood . One reason might be that studies of the role of acetate and diauxic shift have mostly been performed in shake flasks instead of bioreactors, or have focused on population level not taking into account single cell physiology .…”

Section: Introductionmentioning

confidence: 99%

The effect of acetate on population heterogeneity in different cellular characteristics of Escherichia coli in aerobic batch cultures

Heins

Lundin

Nunes

et al. 2019

Biotechnology Progress

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Acetate as the major by-product in industrial-scale bioprocesses with Escherichia coli is found to decrease process efficiency as well as to be toxic to cells, which has several effects like a significant induction of cellular stress responses. However, the underlying phenomena are poorly explored. Therefore, we studied time-resolved population heterogeneity of the E. coli growth reporter strain MG1655/pGS20PrrnBGFPAAV expressing destabilized green fluorescent protein during batch growth on acetate and glucose as sole carbon sources. Additionally, we applied five fluorescent stains targeting different cellular properties (viability as well as metabolic and respiratory activity). Quantitative analysis of flow cytometry data verified that bacterial populations in the bioreactor are more heterogeneous in growth as well as stronger metabolically challenged during growth on acetate as sole carbon source, compared to growth on glucose or acetate after diauxic shift. Interestingly, with acetate as sole carbon source, significant subpopulations were found with some cells that seem to be more robust than the rest of the population. In conclusion, following batch cultures population heterogeneity was evident in all measured parameters.Our approach enabled a deeper study of heterogeneity during growth on the favored substrate glucose as well as on the toxic by-product acetate. Using a combination of activity fluorescent dyes proved to be an accurate and fast alternative as well as a supplement to the use of a reporter strain. However, the choice of combination of stains should be well considered depending on which population traits to aim for. K E Y W O R D Sflow cytometry, growth on acetate, metabolic activity, population heterogeneity, reporter strain, viability

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“…Metabolic gradients were found as key regulators in zonation of cellular metabolic regimes, that is excess, limitation, and starvation status of the limiting nutrient, most likely leading to ensemble reduction of productivity and relevant yields (Haringa et al, ), and phenotypic heterogeneity has been evolved as bet‐hedging adaptation strategies to actively increase fitness of a subpopulation in an unpredictable scenario (Grimbergen, Siebring, Solopova, & Kuipers, ). A recent review article by Heins and Weuster‐Botz () has summarized several mechanisms, such as (a) bet‐hedging; (b) noise in gene expression; (c) persistence; (d) bistability, behind population heterogeneity in microbial bioprocesses. Also, recent findings have concluded that stochastic fluctuations in the molecular level can propagate by both regulatory proteins and metabolic reactions and the inherently stochastic cellular metabolism could be a generic source of phenotypic heterogeneity (Kiviet et al, ; Takhaveev & Heinemann, ).…”

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Coupled metabolic‐hydrodynamic modeling enabling rational scale‐up of industrial bioprocesses

Wang

Haringa

Tang

et al. 2019

Biotech & Bioengineering

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Metabolomics aims to address what and how regulatory mechanisms are coordinated to achieve flux optimality, different metabolic objectives as well as appropriate adaptations to dynamic nutrient availability. Recent decades have witnessed that the integration of metabolomics and fluxomics within the goal of synthetic biology has arrived at generating the desired bioproducts with improved bioconversion efficiency. Absolute metabolite quantification by isotope dilution mass spectrometry represents a functional readout of cellular biochemistry and contributes to the establishment of metabolic (structured) models required in systems metabolic engineering. In industrial practices, population heterogeneity arising from fluctuating nutrient availability frequently leads to performance losses, that is reduced commercial metrics (titer, rate, and yield). Hence, the development of more stable producers and more predictable bioprocesses can benefit from a quantitative understanding of spatial and temporal cell-to-cell heterogeneity within industrial bioprocesses. Quantitative metabolomics analysis and metabolic modeling applied in computational fluid dynamics (CFD)-assisted scale-down simulators that mimic industrial heterogeneity such as fluctuations in nutrients, dissolved gases, and other stresses can procure informative clues for coping with issues during bioprocessing scale-up. In previous studies, only limited insights into the hydrodynamic conditions inside the industrial-scale bioreactor have been obtained, which makes case-by-case scale-up far from straightforward. Tracking the flow paths of cells circulating in large-scale bioreactors is a highly valuable tool for evaluating cellular performance in production tanks. The "lifelines" or "trajectories" of cells in industrial-scale bioreactors can be captured using Euler-Lagrange CFD simulation. This novel methodology can be further coupled with metabolic (structured) models to provide not only a statistical analysis of cell lifelines triggered by the environmental fluctuations but also a global assessment of the metabolic response to heterogeneity inside an industrial bioreactor. For the future, the industrial design should be dependent on the computational framework, and this integration work will allow bioprocess scale-up to the industrial scale with an end in mind. K E Y W O R D SCFD, Euler-Langrange, metabolic model, metabolomics, population heterogeneity, scale down

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Population heterogeneity in microbial bioprocesses: origin, analysis, mechanisms, and future perspectives

Cited by 65 publications

References 277 publications

Design of a microaerobically inducible replicon for high‐yield plasmid DNA production

Design of a microaerobically inducible replicon for high‐yield plasmid DNA production

The effect of acetate on population heterogeneity in different cellular characteristics of Escherichia coli in aerobic batch cultures

Coupled metabolic‐hydrodynamic modeling enabling rational scale‐up of industrial bioprocesses

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