利用拉曼顯微光譜技術原位探討在各種生長條件下生物膜之特徵及化學組成

Abstract

本計畫之目的期望藉由拉曼顯微光譜與影像技術發展一有效方法，使生物膜(biofilm) 得以進行在分子層級上的特性分析。 生物膜是細菌菌落生長附著於生物或非生物的表面上時，細菌群體所分泌具結構性 之包覆膜狀物質。因為生物膜在細菌感染，食品工業及環境科學等中扮演著重要的角 色，因此關於生物膜的研究已引起不同科學領域的高度關注。基於以上原因，目前極 需發展一套能在生物樣本原位進行分子層級分析的方法。在本計畫我們將應用拉曼成 像技術所能擷取之高度化學專一性的三維影像來分析不同條件下生物膜形成過程中的 生物分子，進而從分子層級的觀點來瞭解生物膜的組成對生物膜的形成及降解可能牽 涉的功能。 在過去的研究指出生物膜的生成速率與生長環境的養份和培養的流體條件有高度的 相關性，因此在操作拉曼顯微成像實驗時必須控制這些條件。為良好控制生物膜的生 長條件以易於拉曼影像量測、我們將發展一套相容於壓電奈米定位控制平台的流體計 量系統來擷取拉曼顯微成像。未來將應用此系統研究一些主要常見且具實際應用性的 細菌株所形成之生物膜，例如大腸桿菌，伯克氏菌及紅球菌。

The objective of this proposal is to develop an effective means based on Raman microspectroscopy and imaging for molecular-level characterization of biofilms. Biofilms, structured communities of bacteria attached to a biotic or abiotic surface, have been attracting much attention in diverse areas in science because they play central roles in bacterial infections, food industry, and environmental science. Thus the development of in situ molecular-level characterization methods has been highly demanded. The Raman imaging method used in this work will enable us to visualize with high chemical specificity the three-dimensional (3D) distribution of biomolecules in biofilms grown under various conditions and hence to understand the functions of these constituents at the molecular level in relation to biofilm formation and/or degradation. The rate of biofilm formation is known to depend largely on nutrition and hydrodynamic conditions, so that it is important to control these conditions during a Raman imaging experiment. To facilitate Raman measurements under well-controlled growth conditions of biofilms, we will develop a flow cell system that is compatible with a piezo nanopositioning stage used for Raman imaging. We will then apply the apparatus to the biofilms of several bacterial species of both fundamental and practical interest. They include Escherichia coli, Burkholderia multivorans, and Rhodococcus sp. In our previous Raman imaging study, we discovered that high levels of the amino acid leucine are localized within microcolonies in a nascent E. coli biofilm grown in LB medium. To understand the role of the leucine localization in biofilm formation, we will perform Raman imaging of the E. coli biofilm grown in distinct culture media (both nutrient and minimal media) flowing continuously, using the flow cell system developed. We will provide chemical insight into the interplay between the amino acid and biofilm formation. In the Raman imaging study of B. multivorans, a focus will be placed on chemical visualization of extracellular DNA (eDNA). eDNA has been shown to play positive roles in the formation and/or maintenance of biofilms in many species. Very recently, however, it has been shown biochemically that, as opposed to the prevailing notion, the biofilm formation of B. multivorans ATCC 17616 is stimulated by the degradation of eDNA. As a first step to understand the mechanistic details of this interesting phenomenon, we will do Raman imaging on the B. multivorans biofilm and reveal the 3D distribution of eDNA. Rhodococcus sp. SD-74 has been shown to produce crystalline cellulose in biofilms in the presence of n-hexadecane. However it remains to be seen why the bacterium produces cellulose, which is usually thought of as not advantageous for the bacterium survival. It will be interesting to investigate the effects of biofilm growth conditions on the production of cellulose.