Atwood, J. | bioRxiv Science

bioRxiv2025

Cold-water gut isolate from threespine stickleback (Gasterosteus aculeatus) reveals polypropylene surface oxidation and co-culture inhibition

Polyethylene terephthalate (PET) and polypropylene (PP), two of the most widely produced plastics in the United States, persist in cold-water environments where plastic-degrading microbes have been poorly characterized. Understanding how gut microbes interact and contribute to plastic degradation is essential for developing potential microbiome-based bioremediation strategies. We isolated 184 microbes from wild Alaskan threespine stickleback (Gasterosteus aculeatus) guts across six lakes and screened for plastic degrading potential using lipase/esterase assays and biofilm formation on PET and PP. During the screen for microbes with plastic degrading potential, we discovered that stickleback gut microbiota members enhance and suppressed the lipase, esterase, and biofilm activity of other microbes. Isolates with the highest plastic degrading potential were incubated in minimal media with PET or PP as the sole carbon source to determine whether plastic degradation potential is enhanced. Surface analysis identified a Pseudomonas trivialis strain that exhibited degradation of PP in monoculture; however, this activity was suppressed in the presence of another gut isolate, Pseudomonas germanica. These results demonstrate that microbes associated with the wild threespine stickleback gut microbiome possess plastic degradation potential and provide insights into how microbial interactions can either promote or inhibit bioremediation of plastic pollution in cold-water environments.

microbiology↗

bioRxiv2024

Comparing Raman and NanoSIMS for heavy water labeling of single cells

Stable isotope probing (SIP) experiments in conjunction with Raman microspectroscopy (Raman) or nano-scale secondary ion mass spectrometry (NanoSIMS) are frequently used to explore single cell metabolic activity in pure cultures as well as complex microbiomes. Despite the increasing popularity of these techniques, the comparability of isotope incorporation measurements using both Raman and NanoSIMS directly on the same cell remains largely unexplored. This knowledge gap creates uncertainty about the consistency of single-cell SIP data obtained independently from each method. Here, we conducted a comparative analysis of 543 Escherichia coli cells grown in M9 minimal medium in the absence or presence of heavy water (2H2O) using correlative Raman and NanoSIMS measurements to quantify the results between the two approaches. We demonstrate that Raman and NanoSIMS yield highly comparable measurements of 2H incorporation, with varying degrees of similarity based on the mass ratios analyzed using NanoSIMS. The 12C2H/12C1H and 12C22H/12C21H mass ratios provide targeted measurements of C-H bonds but may suffer from biases and background interference, while the 2H/1H ratio captures all hydrogen with lower detection limits, making it suitable for applications requiring comprehensive 2H quantification. Importantly, despite its higher mass resolution requirements, the use of C22H/C21H may be a viable alternative to using C2H/C1H due to lower background and higher overall count rates. Furthermore, using an empirical approach to determining Raman wavenumber ranges via the 2nd derivative improved the data equivalency of 2H quantification between Raman and NanoSIMS, highlighting its potential for enhancing cross-technique comparability. These findings provide a robust framework for leveraging both techniques, enabling informed experimental design and data interpretation. By enhancing cross-technique comparability, this work advances SIP methodologies for investigating microbial metabolism and interactions in diverse systems. ImportanceAccurate and reliable measurements of cellular properties are fundamental to understanding the function and activity of microbes. This study addresses to what extent Raman microspectroscopy and nano-scale secondary ion mass spectrometry (NanoSIMS) measurements of single cell anabolic activity can be compared. Here, we study the relationship of the incorporation of a stable isotope (2H through incorporation of 2H2O) as determined by the two techniques and calculate a correlation coefficient to support the use of either technique when analyzing cells incubated with 2H2O. The ability to discern between the comparative strengths and limitations of these techniques is invaluable in refining experimental protocols, enhancing data comparability between studies, data interpretation, and ultimately advancing the quality and reliability of outcomes in microbiome research.

microbiology↗