Journal: bioRxiv

Article Title: Rapid evolution in necromass use under resource limitation reduces persistence in producer-decomposer microbial biospheres

doi: 10.64898/2026.01.14.699458

Figure Lengend Snippet: Wild-type Escherichia coli (WT E. coli ) was first subjected to 60-day monoculture in two enclosed environments without added nutrients (HTG: spatially heterogeneous; HMG: spatially homogeneous). The evolved populations, along with the WT progenitor, were then assayed for motility, biofilm formation, pellicle formation, curli fimbriae expression, and growth curves on eight distinct carbon sources. Subsequently, each evolved population and the WT progenitor was individually paired with Chlamydomonas reinhardtii for co-culture in a spatially structured microbial biosphere established in 48-well plates. System persistence was tracked by continuous measurement of autofluorescence, and population dynamics were investigated through four destructive samplings.

Article Snippet: E. coli MC4100 was obtained from the Leibniz-Institut DSMZ (DSM 6574).

Techniques: Expressing, Co-Culture Assay

Journal: bioRxiv

Article Title: Rapid evolution in necromass use under resource limitation reduces persistence in producer-decomposer microbial biospheres

doi: 10.64898/2026.01.14.699458

Figure Lengend Snippet: (A) Log 10 -transformed viable cell counts of Escherichia coli ( E. coli ) at the initiation and upon destructive harvest after 60 days of monoculture in two distinct systems (HTG: spatially heterogeneous; HMG: spatially homogeneous). Thin semi-transparent lines and small points represent the three biological replicates per treatment ( n = 3). Thick lines and large points indicate the group means. While the mean cell concentration in the HMG at harvest was numerically higher than in the HTG, the difference was not statistically significant (Mann-Whitney U test, W = 9, p = 0.077). (B) Sankey diagram illustrating the nomenclature of E. coli MC4100 samples under three treatments (WT: untreated wild-type progenitor; HTG/HMG: subjected to 60-day monoculture in the respective systems) and the phenotypic divergence of evolved populations relative to the WT progenitor. The x-axis “biofilm” represents the qualitative assessment of biofilm formation using Congo Red agar (CRA), with results on a binary scale .

Article Snippet: E. coli MC4100 was obtained from the Leibniz-Institut DSMZ (DSM 6574).

Techniques: Transformation Assay, Concentration Assay, MANN-WHITNEY

Journal: bioRxiv

Article Title: Rapid evolution in necromass use under resource limitation reduces persistence in producer-decomposer microbial biospheres

doi: 10.64898/2026.01.14.699458

Figure Lengend Snippet:

Article Snippet: E. coli MC4100 was obtained from the Leibniz-Institut DSMZ (DSM 6574).

Techniques:

Journal: bioRxiv

Article Title: Rapid evolution in necromass use under resource limitation reduces persistence in producer-decomposer microbial biospheres

doi: 10.64898/2026.01.14.699458

Figure Lengend Snippet: (A) Comparison of the area under the curve (AUC) for growth curves on eight carbon sources between the wild-type progenitor (WT) and three evolved populations from each monoculture system (HTG: spatially heterogeneous; HMG: spatially homogeneous). Data were normalized to the AUC of the WT and log 10 -transformed. Consequently, the AUC for the WT is 0 for each carbon source; values > 0 indicate an increase relative to WT, while values < 0 indicate a decrease. CreiNecro and EcoNecro denote the necromass of Chlamydomonas reinhardtii and Escherichia coli , respectively. The original AUC data are presented as beeswarm plots in Supplementary Figures 2-4. (B) Heatmap comparing the AUC for growth curves on eight carbon sources among the three treatment groups (WT, HTG, HMG). Data were similarly normalized to the WT AUC but without log 10 transformation. Values are displayed as mean ± standard error (SE). (C) Correlations among three growth parameters, maximum growth rate (μ), carrying capacity (K), and AUC, for the three treatments across different carbon sources. The original data are presented as scatter plots in Supplementary Figure 5. Correlations were assessed using ordinary least squares (OLS) regression; only statistically significant results are shown. Blue and orange indicate positive and negative correlations, respectively. Point size represents the absolute value of the p -value, with larger points corresponding to smaller p -values.

Article Snippet: E. coli MC4100 was obtained from the Leibniz-Institut DSMZ (DSM 6574).

Techniques: Comparison, Transformation Assay

Journal: bioRxiv

Article Title: Rapid evolution in necromass use under resource limitation reduces persistence in producer-decomposer microbial biospheres

doi: 10.64898/2026.01.14.699458

Figure Lengend Snippet: (A) Time to chlorophyll fluorescence loss (TCFL) for microbial biospheres co-cultured with the wild-type progenitor (WT) and the six evolved Escherichia coli ( E. coli ) populations. TCFL represents the persistence of the co-culture system. Orange hues denote populations pre-cultured in the HTG (spatially heterogeneous) system; blue hues denote those from the HMG (spatially homogeneous) system. Means without a common letter differ significantly (Dunn’s test, χ ²(6) = 66.994, η ² = 0.436, p < 0.01). (B) Log 10 -transformed E. coli population densities (cells per biosphere) for the three treatments (WT, HTG, and HMG) at the four destructive harvest time points. At each time point, means without a common letter differ significantly among treatments (Dunn’s test: Day 10, χ ²(6) = 14.286, η ² = 0.315, p < 0.05; Day 16, χ ²(2) = 13.104, η ² = 0.285, p = 0.001; Day 23, χ ²(2) = 26.243, η ² = 0.622, p < 0.05; Day 28, χ ²(6) = 15.022, η ² = 0.723, p < 0.05). The dashed line at the top indicates the initial number of E. coli cells inoculated per biosphere. (C) The percentage of biospheres in each state relative to the total number harvested per time point for the three treatments across the four harvests. “both” indicates biospheres where both E. coli and Chlamydomonas reinhardtii ( C. reinhardtii ) survived; “neither” indicates the opposite. “C. reinhardtii only” and “E. coli only” indicate biospheres where only the alga or the bacterium survived, respectively. (D) Comparison of motility and biofilm formation capacity before and after co-culture for the five E. coli populations successfully recovered at the fourth harvest.

Article Snippet: E. coli MC4100 was obtained from the Leibniz-Institut DSMZ (DSM 6574).

Techniques: Fluorescence, Cell Culture, Co-Culture Assay, Transformation Assay, Comparison

Journal: bioRxiv

Article Title: Rapid evolution in necromass use under resource limitation reduces persistence in producer-decomposer microbial biospheres

doi: 10.64898/2026.01.14.699458

Figure Lengend Snippet: (A) Kaplan-Meier survival probability curves for microbial biospheres, grouped by four Escherichia coli ( E. coli ) phenotypic traits. Motility was assessed qualitatively using motility test media with results on a binary scale . Biofilm formation capacity was quantified using the tissue culture plate (TCP) method (72-hour data) . Populations were categorized based on comparing the raw OD570 values with those of the negative control. Pellicle formation was similarly assessed qualitatively, following the TCP protocol but with cultures grown in test tubes . (B) Forest plot presenting the results of the Cox proportional hazards model. HR = hazard ratio; CI = confidence interval; N = sample size. (C) Spearman’s rank correlation coefficients and statistical analysis results between the persistence of co-cultured microbial biospheres (characterized by time to chlorophyll fluorescence loss) and the following traits of the E. coli populations: the AUC of growth curves on eight carbon sources, motility, and biofilm formation capacity. Biofilm data for correlation analysis were raw OD570 values from the quantitative TCP assay (72-hour), not classified categories. The complete correlation heatmap is shown in Supplementary Figure 10. Orange and blue represent negative and positive correlations, respectively. Point size corresponds to p -values; all p -values were < 0.001.

Article Snippet: E. coli MC4100 was obtained from the Leibniz-Institut DSMZ (DSM 6574).

Techniques: Negative Control, Cell Culture, Fluorescence

Journal: bioRxiv

Article Title: Rapid evolution in necromass use under resource limitation reduces persistence in producer-decomposer microbial biospheres

doi: 10.64898/2026.01.14.699458

Figure Lengend Snippet: (A) Proposed changes in motility, biofilm formation capacity, and carbon source utilization of E. coli under nutrient-limited conditions in closed monoculture systems and in microbial biospheres. Shaded areas indicate inferred trends. (B) Hypothesized and experimentally inferred relationship between E. coli carbon source utilization capacity and the persistence of co-cultured microbial biospheres.

Article Snippet: E. coli MC4100 was obtained from the Leibniz-Institut DSMZ (DSM 6574).

Techniques: Cell Culture

Journal: Materials Today Bio

Article Title: Living probiotics-loaded wound matrices prepared by microchip electrospinning

doi: 10.1016/j.mtbio.2025.102403

Figure Lengend Snippet: A. Schematic illustration of microchip electrospinning set-up together with the scanning electron microscopy (SEM) micrograph of obtained electrospun (ES) living probiotics-loaded fiber matrix and confocal fluorescence microscopy (CFM) micrograph where the bacteria are shown in red within fibers and fibers in green, samples stained using FM 4–64 and SYTO 9, respectively; B. Polydimethylsiloxane (PDMS) chip design, the microfluidic chip (PDMS) included two inlet channels (inlet #1 and inlet #2) — inlet #1 connected to a syringe with the PLC/PEO polymer solution and inlet #2 to a syringe containing the agarose-bacterial dispersion. These channels converged into a common outlet channel, which was connected to a metal needle (21G). The electrospinning voltage was applied to the needle tip, and fibers were collected on a grounded collector plate at a distance of 13 cm. Flow direction is pointed out with an arrow. C - microcapsule with labelled E. coli BW25113 micrograph. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Gram-positive and gram-negative pathogenic bacteria isolated from wounds, namely E. coli DSM 1103, S. aureus DSM 2569, P. aeruginosa DSM 1117, S. epidermidis DSM 28319 (DSMZ, Germany), were used to assess the antimicrobial activity of probiotic bacteria (e.g.

Techniques: MicroChIP Assay, Electron Microscopy, Probiotics, Fluorescence, Microscopy, Bacteria, Staining, Polymer, Dispersion

Journal: Materials Today Bio

Article Title: Living probiotics-loaded wound matrices prepared by microchip electrospinning

doi: 10.1016/j.mtbio.2025.102403

Figure Lengend Snippet: Viability and functionality (including antimicrobial activity) of probiotic bacteria within electrospun fiber matrices. A. Bromocresol purple indicator incorporated into M17 agarose plates was used to visualize the decrease of pH in the surrounding medium caused by probiotic ( L. rhamnosus Fibro 2)-loaded fiber matrices electrospun directly onto glass discs. The color change of the media due to acidification is shown at different time points: B. 24 h, C . 48 h, D . 72 h. E. Schematics of agar overlay assay setup. F. Agar overlay assay using 6 mm diameter fiber matrix discs with and without different probiotic bacteria namely ( L. plantarum Fibro 1 and L. rhamnosus Fibro 2). Initial concentration of pathogenic bacteria in the soft agar was 10 6 CFU/mL. G. Schematics illustrating the L. rhamnosus Fibro 2-loaded fiber matrix disc, cut from the entire sample and placed on MRS base agar. The antimicrobial activity against E. coli DSM 1103 was assessed using the agar overlay assay immediately after electrospinning, as well as after 24 h and 4 months. Key : red dashed circles indicate zones of inhibition, representing the activity of probiotic bacteria against relevant pathogenic bacteria. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Gram-positive and gram-negative pathogenic bacteria isolated from wounds, namely E. coli DSM 1103, S. aureus DSM 2569, P. aeruginosa DSM 1117, S. epidermidis DSM 28319 (DSMZ, Germany), were used to assess the antimicrobial activity of probiotic bacteria (e.g.

Techniques: Activity Assay, Bacteria, Overlay Assay, Concentration Assay, Inhibition