Elucidating the mechanism of bacteria-mediated oil degradation by imaging biofilm formation on oil drops in microfluidic traps

2021 Fiscal Year Annual Research Report

• Project/Area Number: 21H01720
• Research Institution: University of Tsukuba
• Principal Investigator: UTADA ANDREW 筑波大学, 生命環境系, 准教授 (90776626)
• Co-Investigator(Kenkyū-buntansha): 尾花 望 筑波大学, 医学医療系, 助教 (00722688)
• Project Period (FY): 2021-04-01 – 2024-03-31
• Keywords: microfluidics / biofilm / interface / active matter

Outline of Annual Research Achievements

During this first year, we first endeavored to achieved AIM 1, which was the establishment of a microfluidic platform to generate, trap, and store oil drops over a period of days. We achieved this by implementing a combination of surface-tension based microfluidic traps that can trap in-place oil drops. We combined this surface-tension device with a surface coating within our channels to prevent the polydimethylsiloxane (PDMS) channels from absorbing the oil directly. After confirming that we could store drops for at least one week, we then were able to commence our tests using bacteria on oil drops.

In parallel, we began to profile the wild-type Alcanivorax borkemensis bacterium to understand the characteristics of its growth and and that enables days-long observation of biofilm-mediated oil degradation. We have collected growth curves and have noted differences in growth depending on time of culture using oil.

After having collected this initial data on Alcanivorax borkemensis, we have begun to tackle AIM 2, which was to analyze high spatio-temporal image data by adapting cell-tracking algorithms to determine cell-surface and cell-cell interaction energies. By storing the drops for long periods, we have begun to infuse cells and observe their attachment and biofilm development on the drop surface.

Current Status of Research Progress

2: Research has progressed on the whole more than it was originally planned.

Reason

We are currently on target because of our successful development of a microfluidic platform that traps and stores oil drops, enabling days-long observation of biofilm-mediated oil degradation. We have aalso profiled our bacteria to understand how they grow using oil. We are labeling the cells with fluorescent dyes, which is allowing us to image the cells.

Strategy for Future Research Activity

To investigate cell attachment and early stages of biofilm formation on drops, we will adapt cell tracking algorithms to follow cells in the biofilms. We will correlate motion between surface-attached cells to reveal interaction energies.

We will start construction of fluorescent, reporter, and deletion mutant strains. The A. borkumensis genome has been sequenced and many genes necessary for oil degradation are known, which we will target for deletion. We plan to run RNA-sequencing tests on WT cultured under different conditions to identify genes important for oil adhesion that also may be targeted for deletion.

We aim to determine the drop shape and oil degradation rate using fluorescent strains with high spatial-resolution confocal imaging to generate a detailed map of the drop surface; and (2) the differences in physico-chemical properties such as the cell hydrophobicity and zeta-potential of WT and mutant strains to determine the role of genes in surface attachment. We will also correlate the interfacial tension of bacterial suspensions and oil to surface accumulation.