藉由拉曼顯微光譜技術研究培養基對紅球菌SD-74聚集體型態以及聚集體中類胡蘿蔔素增長的變化

Abstract

細菌的特性及功能常因為形成聚集體而與原先浮游狀態有所不同，在我們實驗室過去的研究中，利用在活體觀察的拉曼顯微光譜技術及成像法顯示出，在紅球菌SD-74體內的類胡蘿蔔素濃度會隨著聚集體的成長而增加，雖然這可以是了解細菌存活策略的一個研究發現，但目前這個結果的生物學意義、以及細菌體內類胡蘿蔔素的增長機制仍是未知，為了更了解這個現象，我們藉由不同的營養環境來了解對紅球菌SD-74體內類胡蘿蔔素的影響。 首先，我們決定利用相較之前研究的雷射波長(632.8 奈米)短的光源來當激發光源，藉由共振拉曼效應來提升類胡蘿蔔素的訊號，我們使用的是氬/氪混合氣體雷射中的531、488 奈米，兩者皆是落在共振拉曼範圍的雷射波長，而結果顯示488奈米的激發波長由於有光漂白的作用並不適合我們的實驗，最後我們還是使用以往的632.8波長來當激發光源。我們將紅球菌SD-74培養在不同營養水平的兩種培養基中: LB培養基以及TSB培養基。在九天的培養過程中，我們發現在兩種不同培養基中類胡蘿蔔素的拉曼光譜圖案皆沒有隨著時間有明顯變化，以及都隨著培養時間的上升，類胡蘿蔔素的濃度也隨著上升。然而，在培養的第一天我們發現，在兩種培養基中的類胡蘿蔔素增長速率不相同，而且在多天的觀察中，發現同一天、同一培養基中在不同聚集體的類胡蘿蔔素濃度有極大的差別。

Characteristic properties and functions of bacteria are often altered when bacterial cells form aggregates rather than exist in the free-floating, planktonic state. A previous study from our laboratory using in vivo Raman microspectroscopy and imaging revealed that the concentration of intracellular carotenoids in the bacterium Rhodococcus sp. SD-74 increases with the development of aggregates. Although it is likely that this interesting finding is a survival strategy of the bacterium, the biological significance and mechanisms of the carotenoid accumulation are still unclear. To better understand this phenomenon, in this study, we examined the effects of nutrition on the carotenoid accumulation in Rhodococcus sp. SD-74 aggregates. We first tried to use shorter wavelength light than that in the previous study (632.8 nm) in order to enhance carotenoid Raman signals through the resonance Raman effect. We used 531 nm and 488 nm light produced from an Ar/Kr-mixed gas laser, both of which fall into the category of rigorous resonance. It turned out that the 488 nm excitation is not the best choice of excitation wavelength among the three wavelengths tested because it causes photobleaching. Using the same 632.8 nm excitation as before, we next investigated how culture media affect aggregate formation and carotenoid accumulation in Rhodococcus sp. SD-74. We cultured Rhodococcus sp. SD-74 in two standard media with different nutritional levels: Lysogeny broth (LB) and Tryptic soy broth (TSB). During 9-day culture, there were no appreciable changes in the Raman spectral pattern, and the carotenoid Raman intensities increase with time in both media. However, the rates of carotenoid accumulation in the two media were found to be different at early stages (e.g. day 1), and aggregate-to-aggregate variation in carotenoid Raman intensity was also different in the two media.