應用靜電微粒懸浮系統評估生物氣膠-酵母菌活性

Abstract

過去在微生物活性探討上，多將其置於液體基質內或物體表面，然而，此仍與懸浮在空氣中之微生物狀態有所不同，因兩者所受環境因子影響程度不一。透過電力與微粒重力達平衡，靜電微粒懸浮腔可有效懸浮單一微粒，故從過去至現今，多使用在微粒物化特性探討上。近年來由於自動控制系統、影像擷取裝置的結合與進步，使靜電微粒懸浮腔穩定性更為增加，並有效節省在長時間研究人力的花費。本研究即將電微粒懸浮腔與自動控制系統做結合，應用於生物氣膠-酵母菌活性探討，而此也為首次靜電微粒懸浮腔研究。 實驗系統主要分為下列幾部分︰1.雙環型靜電微粒懸浮腔(利用直流與交流電電長懸浮單一微粒) 2.雷射光光源(0~100 mW, λ= 532nm)(使微粒產生散射光，而被影像擷取系統捕捉影像) 3.自動控制系統(Labview 8.2人機介面程式對懸浮單一微粒做位置上控制)。在生物氣膠活性測試前，本研究先對5, 10, 15 μm壓克力微粒做系統穩定性測試，其操作條件為直流與交流電電壓分別是200~1000 V和1.3 kV、頻率350~1000 Hz。由於酵母菌(Saccharomyces cerevisiae YPH499)在粒徑上可達5~10 μm，且適合本系統可捕捉之粒徑範圍(3~15 μm)，故被作為實驗使用之生物氣膠菌種選擇。生物氣膠產生方式採用改良式霧化產生器，使其可有效產生酵母菌微粒。單一懸浮酵母菌以帶電金屬棒吸引並收集至固態培養基中培養，最終對其活性做判斷。系統中影響生物氣膠活性因子之變項有雷射光強度、直流與交流電電場、相對濕度與懸浮時間。 結果顯示，不同粒徑壓克力微粒與酵母菌皆可長時間被懸浮，即本系統表現穩定。靜電微粒懸浮腔內相對濕度、懸浮時間對酵母菌存活率表現，當相對濕度由20至75%時，其存活率會下降約21% (4%至27%)；而懸浮時間由2分鐘上升至60分鐘時，存活率下降約17% (31%至14%)。上述酵母菌存活率結果可發現，其明顯與經氣膠產生器生成要低，推測應與系統雷射光、電場、靜電微粒懸浮腔內相對濕度、懸浮時間、金屬棒吸引電壓與培養處理方式有關。綜合實驗結果可知，改善培養處理方式後可有效增加酵母菌細胞存活率，即在相同相對濕度(55%)與懸浮時間(2分鐘)下，由原本31%增加至56%。由於此分析方法為使用傳統培養法所得到之結果，故與亞甲基藍染劑法判斷細胞活性之存活率有所差異。

Previous studies on survivability of microorganisms were mainly carried out by placing microbial-laden liquid, such as serum, on the surface. However, these viability test data could not reflect the survivability of microorganisms suspended in the air because the environmental stress levels would be different. Electrodynamic balance (EDB) has long been used to characterize the physical and chemical properties of single particle fixed and airborne in the chamber by electrostatic forces. With the assistance of video capture and automatic feedback control system, the EDB became more advanced and versatile. In the present study, an EDB system was used, probably for the first time, to evaluate the survivability of yeast cell. The experimental system consisted of (1) a double-ring EDB chamber which was designed to levitate one particle with DC and AC field, (2) a laser beam (I= 0~100 mW, λ= 532nm) to illuminate particle, and a digital camera (400X) to capture image of the target particle, and (3) an automatic control system written in Labview 8.2. Before introducing bioaerosol to EDB, the system was tested and optimized by using monodisperse (5, 10, 15 μm) acrylic powders. The operating DC and AC voltages were 200~1000 V and 1.3 kV/frequency= 350~1000 Hz, respectively. Yeast cell (Saccharomyces cerevisiae YPH499) was chosen for its size, falling right into the working range of EDB (3 to tens μm). The yeast aerosols were generated by using a Wright nozzle modified for maximum aerosol output. A retrieving probe was made to capture the yeast cell levitated in the EDB. The retrieved cell was then cultivated to examine the viability. Laser light intensity, chamber temperature, humidity, and retention time in the chamber are among the principal operating parameters. The results showed that the EDB system could retain supermicrometer-sized (5, 10, 15 μm) acrylic powders and yeast aerosols in the chamber for hours or even days. The survival rate of yeast cells was around 5%, after retrieved from the EDB chamber with RH= 20% and then cultivated on YEPD agar. The survival rate increased (from 4 to 27%) with increasing relative humidity (from 20 to 75%) in the EDB chamber. The survival rate decreased with increasing retention in the chamber, for example, from 31% of 2 min down to 14% of 60 min. These survival rates are much lower than yeast cells in distilled water before aerosolization (90%) and yeast cells after aerosolization (80%). However, it should be noticed that both high survival rates were based on viability test following methylene blue staining procedure, which might not be exactly the same with YEPD agar cultivation method.