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

我們使用由本實驗是所架設的高靈敏性共聚焦拉曼顯微鏡來研究由紅球菌SD-74在靜止的空氣與培養液的表面所生長的生物薄膜。，菌株紅球菌SD-74的研究獲得了許多的關注因為它能生產生物性界面活性劑。我們發現紅球菌SD-74傾向於形成聚集的生長形態，由浮游態的紅球菌SD-74所得到的拉曼光譜具有蛋白質及脂質的拉曼特徵峰。而與此有著顯著的對比，在聚集的紅球菌SD-74生物薄膜中所得到的拉曼光譜多了三個主要的拉曼特徵峰，分別位於1514、1154及1002波數。這三個拉曼特徵峰是來自於類胡蘿蔔素這類典型的天然色素，事實上紅球菌SD-74的菌落顏色為橘色。更令人感到有趣的是，我們發現這些來自類胡蘿蔔素的拉曼峰強度會隨著紅球菌SD-74的生長年齡增加而顯著的增加（一天、三天及七天的前置培養）。為了研究類胡蘿蔔素的濃度及分布的改變與時間的關係，我們做了拉曼顯微成像實驗於同一紅球菌SD-74生物薄膜，而其結果顯示類胡蘿蔔素不只是隨著生物薄膜的生長而累積，在後期它們也存在於生物薄膜的各個角落。因為胡蘿蔔素是一種眾所皆知的抗氧化劑，所以我們提出胡蘿蔔素可能是用於降低與生物薄膜生長年齡及由雷射引起的氧化壓力來解釋此研究發現，這提供了一個在有顏色的生物薄膜內的氧化壓力的定量研究方法。

We studied biofilms of Rhodococcus sp. SD-74 grown at air–medium interfaces under static conditions, using a laboratory-built high-sensitivity confocal Raman microscope. Rhodococcus sp SD-74 has attracted interest because of its ability to produce biosurfactants. We found that R. sp. SD-74 tends to form aggregates. The Raman spectrum of planktonic R. sp. SD-74 was well characterized by Raman bands of proteins and lipids. In sharp contrast, additional Raman bands at 1514, 1154, and 1002 cm? dominated the Raman spectrum of aggregates in R. sp. SD-74 biofilm. These Raman bands were attributed to carotenoids, which are typical natural pigments. In fact, colonies of R. sp. SD-74 are orange-colored. Interestingly, we observed that the intensities of the carotenoid bands markedly increased with the age of R. sp. SD-74 biofilm (one, three, and seven days postinoculation). To study time-dependent variation in carotenoid concentrations and distributions, we performed Raman imaging experiments on the same R. sp. SD-74 biofilm. The results reveal that carotenoids not only accumulated as the biofilm grew but they were also localized at the later stage to very small regions in the biofilm. Because carotenoids are well known as an antioxidant, our finding here suggests that carotenoids might be used to reduce aging-associated and/or laser-induced oxidative stress in the biofilm. This offers a quantitative tool for visualizing the level of oxidative stress in colored biofilms in vivo.