Dataset Information

Morphological and physiological characterization of filamentous Lentzea aerocolonigenes: Comparison of biopellets by microscopy and flow cytometry.

ABSTRACT: Cell morphology of filamentous microorganisms is highly interesting during cultivations as it is often linked to productivity and can be influenced by process conditions. Hence, the characterization of cell morphology is of major importance to improve the understanding of industrial processes with filamentous microorganisms. For this purpose, reliable and robust methods are necessary. In this study, pellet morphology and physiology of the rebeccamycin producing filamentous actinomycete Lentzea aerocolonigenes were investigated by microscopy and flow cytometry. Both methods were compared regarding their applicability. To achieve different morphologies, a cultivation with glass bead addition (Ø = 969 ?m, 100 g L-1) was compared to an unsupplemented cultivation. This led to two different macro-morphologies. Furthermore, glass bead addition increased rebeccamycin titers after 10 days of cultivation (95 mg L-1 with glass beads, 38 mg L-1 without glass beads). Macro-morphology and viability were investigated through microscopy and flow cytometry. For viability assessment fluorescent staining was used additionally. Smaller, more regular pellets were found for glass bead addition. Pellet diameters resulting from microscopy followed by image analysis were 172 ?m without and 106 ?m with glass beads, diameters from flow cytometry were 170 and 100 ?m, respectively. These results show excellent agreement of both methods, each considering several thousand pellets. Furthermore, the pellet viability obtained from both methods suggested an enhanced metabolic activity in glass bead treated pellets during the exponential production phase. However, total viability values differ for flow cytometry (0.32 without and 0.41 with glass beads) and confocal laser scanning microscopy of single stained pellet slices (life ratio in production phase of 0.10 without and 0.22 with glass beads), which is probably caused by the different numbers of investigated pellets. In confocal laser scanning microscopy only one pellet per sample could be investigated while flow cytometry considered at least 50 pellets per sample, resulting in an increased statistical reliability.

SUBMITTER: Schrinner K

PROVIDER: S-EPMC7269266 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Morphological and physiological characterization of filamentous Lentzea aerocolonigenes: Comparison of biopellets by microscopy and flow cytometry.

Schrinner Kathrin K Veiter Lukas L Schmideder Stefan S Doppler Philipp P Schrader Marcel M Münch Nadine N Althof Kristin K Kwade Arno A Briesen Heiko H Herwig Christoph C Krull Rainer R

PloS one 20200603 6

Cell morphology of filamentous microorganisms is highly interesting during cultivations as it is often linked to productivity and can be influenced by process conditions. Hence, the characterization of cell morphology is of major importance to improve the understanding of industrial processes with filamentous microorganisms. For this purpose, reliable and robust methods are necessary. In this study, pellet morphology and physiology of the rebeccamycin producing filamentous actinomycete Lentzea a ...[more]

PMID: 32492063

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Project description:This study compared the relative cellular uptake of 80 nm silver nanoparticles (AgNP) with four different coatings including: branched polyethyleneimine (bPEI), citrate (CIT), polyvinylpyrrolidone (PVP), and polyethylene glycol (PEG). A gold nanoparticle PVP was also compared to the silver nanoparticles. Biophysical parameters of cellular uptake and effects included flow cytometry side scatter (SSC) intensity, nuclear light scatter, cell cycle distributions, surface plasmonic resonance (SPR), fluorescence microscopy of mitochondrial gross structure, and darkfield hyperspectral imaging. The AgNP-bPEI were positively charged and entered cells at a higher rate than the negatively or neutrally charged particles. The AgNP-bPEI were toxic to the cells at lower doses than the other coatings which resulted in mitochondria being transformed from a normal string-like appearance to small round beaded structures. Hyperspectral imaging showed that AgNP-bPEI and AgNP-CIT agglomerated in the cells and on the slides, which was evident by longer spectral wavelengths of scattered light compared to AgNP-PEG and AgNP-PVP particles. In unfixed cells, AgNP-CIT and AgNP-bPEI had higher SPR than either AgNP-PEG or AgNP-PVP particles, presumably due to greater intracellular agglomeration. After 24 hr. incubation with AgNP-bPEI, there was a dose-dependent decrease in the G1 phase and an increase in the G2/M and S phases of the cell cycle suggestive of cell cycle inhibition. The nuclei of all the AgNP treated cells showed a dose-dependent increase in nanoparticles following non-ionic detergent treatment in which the nuclei retained extra-nuclear AgNP, suggesting that nanoparticles were attached to the nuclei or cytoplasm and not removed by detergent lysis. In summary, positively charged AgNP-bPEI increased particle cellular uptake. Particles agglomerated in the peri-nuclear region, increased mitochondrial toxicity, disturbed the cell cycle, and caused abnormal adherence of extranuclear material to the nucleus after detergent lysis of cells. These results illustrate the importance of nanoparticle surface coatings and charge in determining potentially toxic cellular interactions.

Dataset Information

Morphological and physiological characterization of filamentous Lentzea aerocolonigenes: Comparison of biopellets by microscopy and flow cytometry.

Publications

Morphological and physiological characterization of filamentous Lentzea aerocolonigenes: Comparison of biopellets by microscopy and flow cytometry.

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