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Microscale investigation of key bacterial phenotypes enhancing collection by rising bubbles and aerial dispersal

Periodic Reporting for period 1 - BactoBubble (Microscale investigation of key bacterial phenotypes enhancing collection by rising bubbles and aerial dispersal)

Reporting period: 2018-06-01 to 2020-05-31

Air currents provide a rapid means of dispersal for live microbes on very large scales, with the potential to spread diseases, influence microbial population composition over hundreds of kilometers, and impact atmospheric phenomena. While the field of aerobiology has explored the distribution and diversity of microbes found in the air, our understanding of microbial interactions between the atmosphere and aquatic systems is severely lacking. Specifically, the dynamics of microbial fluxes into the atmosphere are poorly understood. The mechanisms by which marine microbes leave the ocean give us our first avenue for investigating this microbial flux: above the ocean, microbial aerosolisation arises largely from bursting bubbles. Bubbles are produced in the ocean by crashing waves and these bubbles capture microbial cells on their surfaces as they rise through the water. These cells are subsequently trapped in micro-droplets ejected into the air when the bubbles burst on the ocean surface. This ‘scavenging’ of microbes by a rising bubble is key, as it can lead to a thousand-fold increase in the microbial content of aerosols. Understanding the microbial characteristics which lead to capture by bubbles is therefore fundamental to characterizing the ocean-atmosphere microbial flux. This would further support aerobiology models and our understanding of disease outbreaks, as well as contribute to elements of atmospheric research like cloud formation physics, given the very effective ice nucleation activity of some microbes. Finally, the separation of microbes from their suspending medium by rising bubbles, or ‘flotation’, could offer a powerful technique for the growing industry of microbial biosynthesis. These broad impacts demonstrate that the value of this work extends beyond understanding the nature and abundance of microbial aerosols. This project aims to combine microfluidic techniques with microscopic imaging to address the present knowledge gap, by performing the first direct observations of microbial collection by bubbles at the microscale, thus providing a major step forward in our understanding of the interaction between bubbles and microbes. Specifically, the objectives of this project are to investigate two hypotheses.

First, we investigate whether microbial motility (or ability to swim) increases microbial collection by rising bubbles. Indeed motility sets microbes starkly apart from inert particles, likely promoting collection by increasing the likelihood of encountering bubbles, and also influencing attachment as swimming appendages modify the cell surface properties. Second, we investigate whether starvation of microbes increases microbial collection by rising bubbles. Starving bacteria modify their surface properties and size, which may make them ‘stickier’ and hence enhance collection by bubbles –this might promote dispersal and escape from nutrient poor areas. Aerosolisation could then represent an active stage in the life cycle and ecological dynamics of bacteria in oceans.

To investigate these hypotheses, this project’s goal is to develop a novel microfluidic flow channel containing a pinned bubble, and use advanced optical microscopy to quantify collection rates for a range of microbes on a single bubble in flow. By bringing together pioneering quantitative microscopy and characterisation of flow regimes and microbial surface properties, the proposed research will determine the role of key factors influencing microbial collection by bubbles and develop new mathematical models for microbial aerosolisation.
We developed a novel millifluidic flow chamber to study the interaction between microbes and a rising bubble. This device creates a single, sub-millimeter scale, air bubble inside a small flow chamber, and keeps this bubble pinned at the tip of a small glass tube while exposing it to a flowing microbial suspension, mimicking the rising bubble process. The entire chamber can be mounted on top of a microscope and the encounters and attachments of individual microbes identified by imaging the bubble with fluorescence microscopy. In developing this technique, we had to address multiple challenges.

First, our investigation of the flow patterns of the interface revealed that the attached microbes do not accumulate at the downflow pole of the bubble, as fluid mechanical theory would predict. Instead, the attached microbes are constantly recirculated and distributed over the entire bubble surface. The absence of accumulation points makes the identification of individual cells more delicate: instead of imaging a restricted area at high magnification, knowing that cells located there are effectively attached, we had to image a complete hemisphere to obtain suitable statistics on cell attachment.

Moreover, we found that to distinguish between attached cells and cells flowing just above the bubble surface required the development of advanced image analysis techniques. We developed a novel focus-stacking pipeline for analyzing our fluorescence data, which reconstructs a flattened view of the bubble interface, thus enabling the accurate estimation of the number of attached cells.

Delays due to the COVID-19 pandemic prevented us from completing laboratory experiments investigating the role of microbial motility and starvation on microbial collection by rising bubbles. We plan to use our established techniques to finish testing these hypotheses and ultimately publish our results in peer-reviewed journals. A presentation of this technique at both the Gordon Research Conference and Seminar on Marine Microbes in 2020 was postponed due to the pandemic, and rescheduled to 2022.

A secondary goal of this project was to promote the fruitful interaction of modelling with experimental and field ocean microbial research. I therefore contributed to several projects, providing a theoretical support to experimental work. In particular, I contributed to a mathematical model of the carbon pump, that is the sinking of organic carbon particles in the ocean, which included the novel coupling of sinking speed with microbial degradation observed in the lab. I also provided the theoretical basis to a new quantification of bacterial motility in situ. Both contributions are part of upcoming publications.
The novel techniques we developed for imaging and quantifying the attachment of micron-sized fluorescent microbes to a rising bubble can readily be adapted to the study of model microorganisms other than those chosen for this project. It could potentially be improved in resolution to even investigate the collection of viruses by rising bubbles, to substantially improve our understanding of viral transport and marine disease spread. Similarly, this visualization technique could be adapted to investigate at the microscale how bubbles could capture mineral particles or other artificial material, such as microplastics. This could inform research in the industrial technique of flotation for separating particles from a bath, but also help understand the fate of microplastics in the ocean and their potential aerosolization.

This project also provided the opportunity for the principal investigator to train and supervise several BSc and MSc students, and develop and organize outreach events to introduce a young audience to the complexity of microbial life in lakes and oceans, thus more broadly contributing in the effort of scientific education and public outreach.
2D-view of individual bacterial cells (yellow) attached at the air-water interface of a bubble