Hellerschmied, C. | bioRxiv Science

bioRxiv2025

Single-cell mass accumulation reveals bacterioplankton growth rate in native seawater

AO_SCPLOWBSTRACTC_SCPLOWThe growth of marine microbial communities drives biogeochemical cycling of carbon and other elements, yet the growth rates of individual species within complex ocean ecosystems remain poorly understood. In particular, the coexistence of a large diversity of copiotrophic bacteria, which are capable of fast growth but typically remain at low abundance, has been interpreted as a feast or famine existence. Here we show that contrary to the notion of infrequent growth, Vibrio bacteria exhibited consistent growth rates in coastal ocean samples, despite representing only a small fraction of the total community. These observations were enabled by a suspended microchannel resonator (SMR), which we adapted to function as a single-cell chemostat. By maintaining a continuous supply of native seawater around each trapped cell, we prevented nutrient depletion and used the SMRs high mass precision to resolve growth rates that are otherwise undetectable. Vibrio species displayed significantly larger cell mass and faster growth than other community members across samples collected at different temporal intervals from days to years. Surprisingly, their growth was consistently limited by carbon, contrary to the expectation that heterotrophic bacteria in the euphotic zone would be limited by nitrogen and phosphorus due to competition with algae. The correlation between cell mass and growth rate of Vibrionaceae in seawater followed established growth laws derived from laboratory conditions, suggesting that growth physiology observed in pure cultures is applicable to wild bacterial populations. Overall, our findings suggest that rare species may play a disproportionately large role in the marine carbon cycle, with rapid biomass turnover driven by a combination of high growth rates balanced by intense predation.

microbiology↗

bioRxiv2023

Single-cell mass distributions reveal simple rules for achieving steady-state growth

Optical density is a common method for measuring exponential growth in bacterial batch cultures. However, there is a misconception that such exponential growth is equivalent to steady-state growth, which is a distinct physiological state that improves experimental reproducibility. Determining precisely when steady-state growth occurs is technically challenging and is aided by paired single-cell and population-level measurements. Using microfluidic mass sensors and optical density, we explore when in typical laboratory batch cultures steady-state growth occurs. We show that cell mass increases by an order of magnitude within a few hours of dilution into fresh medium and that steady-state growth is only achieved when cultures are inoculated with high dilutions from overnight stationary phase cultures. At high dilutions, Escherichia coli and Vibrio cyclitrophicus grown in different rich media achieve steady-state growth approximately 4 total biomass doublings after inoculation. We can decompose these dynamics into 3 doublings of average cell mass and 1 doubling of cell number for both species. We also show that batch cultures in rich media depart steady-state growth early in their growth curves at low cell and biomass concentrations. Achieving and maintaining steady-state growth in batch culture is a delicate balancing act, and we provide general guidance for commonly used rich media. Quantifying single-cell mass outside of steady-state growth is an important first step towards understanding how microbes grow in their natural context, where fluctuations pervade at the scale of individual cells. ImportanceMicrobiologists have watched clear liquid turn cloudy for over 100 years. While the cloudiness of a culture is proportional to its total biomass, growth rates using such optical density measurements are challenging to interpret when cells change size. Many bacteria adjust their size at different steady-state growth rates, but also when shifting between starvation and growth. Optical density cannot disentangle how mass is distributed among cells of different sizes, and directly measuring how mass is distributed among cells has been a major challenge. Here we use single-cell mass measurements to demonstrate that a population of cells in batch culture achieves a stable mass distribution for only a short period of time. Achieving steady-state growth in rich medium requires low initial biomass concentrations and enough time for the coordination of individual cell and population growth. Steady-state growth is important for reliable cell mass distributions in a culture and we discuss how mass variation outside of steady-state can impact physiology, ecology, and evolution experiments.

microbiology↗