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

• Project/Area Number: 21H01720
• Research Category: Grant-in-Aid for Scientific Research (B)
• Allocation Type: Single-year Grants
• Section: 一般
• Review Section: Basic Section 27040:Biofunction and bioprocess engineering-related
• 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
• Project Status: Granted (Fiscal Year 2023)
• Budget Amount: ¥17,420,000 (Direct Cost: ¥13,400,000、Indirect Cost: ¥4,020,000); Fiscal Year 2023: ¥5,460,000 (Direct Cost: ¥4,200,000、Indirect Cost: ¥1,260,000); Fiscal Year 2022: ¥5,070,000 (Direct Cost: ¥3,900,000、Indirect Cost: ¥1,170,000); Fiscal Year 2021: ¥6,890,000 (Direct Cost: ¥5,300,000、Indirect Cost: ¥1,590,000)
• Keywords: biofilm / microfluidics / oil degradation / oil-water interface / bioremediation / active matter / living liquid crystal / interface / nematic ordering

Outline of Research at the Start

Environmental bacteria biodegrade significant quantities of global spilled oil. Many genetic and biochemical pathways responsible for oil-degradation are known but, the detailed mechanisms that bacteria use to degrade the oil are still unclear. Harnessing bacterial function for next-generation control will require a deeper understanding of both chemical pathways and biophysical mechanisms. I aim to elucidate the biophysical mechanisms by clarifying cell-attachment dynamics, biofilm formation, and oil-degradation rate using microfluidics, high-resolution imaging and cell-tracking.

Outline of Annual Research Achievements

We have investigated cell attachment and early stages of biofilm formation on drops. We have correlated motion between surface-attached cells on the interface. We have found clear liquid crystal ordering of cells on drop surfaces, which indicates that the biofilm is elastic. Further aiding in our interfacial imaging, we have constructed a set of fluorescent and deletion mutant strains. Our constitutive green fluorescent protein (GFP) and mCherry (red fluorescent protein) expressing cells have facilitated imaging of the oil-water interface throughout the process. Moreover, we have measured and analyzed the interfacial properties of the cells and their conditioned components, finding that the cells are more hydrophobic the longer they are cultured using oil and that they develop the ability to enact surface changes.

In addition, we have completed RNA-sequencing tests on WT cultured under different conditions and have identified a number of genes that are important for oil consumption. We are currently testing the deletion mutants to see if we can determine the genetic basis of the changes we see.

We have also completed a manuscript that describes our results and have uploaded it to the bioRxiv and have submitted it. We are currently undergoing the peer-review process.

Current Status of Research Progress

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

Reason

We have made significant progress in imaging of the interface due to improvements we have made in our imaging process as well as due to the fluorescent strains that we have constructed. We have analyzed 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 the differences in physico-chemical properties (e.g. hydrophobicity, zeta-potential) of WT and mutant strains. However, we have not yet determined the role of genes in surface attachment, which we are still investigating by analyzing deletion mutants.

Strategy for Future Research Activity

In our final year of the project, we aim to first complete the review process of our manuscript. Upon completion of this important step, we will further pursue optimization and redesign of the channels to increase throughput. We will also use this optimized device to search for the phenotypic changes to deletion mutants. We aim to clarify the genetic basis for the physical changes we observe during biofilm development.

Research Products

• [Journal Article] Alcanivorax borkumensis Biofilms Enhance Oil Degradation By Interfacial Tubulation (2022)
  • Author(s): M. Prasad, N. Obana, S.-Z. Lin, S. Zhao, K. Sakai, C. Blanch-Mercader, J. Prost, N. Nomura, J.-F. Rupprecht*, J. Fattaccioli*, A. S. Utada*
  • Journal Title: bioRxiv Volume: 0 Pages: 0-0
  • DOI: 10.1101/2022.08.06.503017
  • Open Access / Int'l Joint Research