探討以紫外光發光二極體結合自由餘氯提升對於MS-2 coliphage與Bacillus subtilis孢子之抑制效果

Abstract

紫外光發光二極體(UV-C LED)是新型的紫外光光源具有能夠選擇特定波長的能力，本研究將利用紫外光發光二極體結合自由餘氯的高級氧化處理程序，評估其能否有效提升對於指標微生物MS-2 coliphage與Bacillus subtilis孢子的抑制能力，探討在不同紫外光波長、pH及水樣參與反應下對於殺菌能力的影響及自由基於反應中所扮演的角色，並以碘化丙啶即時定量聚合酶連鎖反應(PMA qPCR)檢視Bacillus subtilis孢子細胞膜在不同消毒方式下的損傷程度。由批次式殺菌實驗的結果可以發現將紫外光結合3 mg/L之自由餘氯對於MS-2 coliphage的抑制效果即有協同作用(synergistic)出現，而對於Bacillus subtilis孢子則需將初始自由餘氯提升到6 mg/L及9 mg/L才會有較明顯地協同作用(synergistic)出現，若以反應動力常數(k, obs)進行比較，以UV-C LED (275 nm)結合自由餘氯對於指標微生物的抑制效果皆會優於LPUV(254 nm)，顯示以波長275 nm的紫外光更加適合作為本研究中UV/chlorine反應的紫外光光源使用；當在UV/chlorine反應中加入t-BuOH做為radical scavenger後，LPUV/chlorine和UV-C/chlorine對於抑制MS-2 coliphage生長能力的增強效果會分別減少69.2%與84.4%，而Bacillus subtilis孢子再添加t-BuOH後原本的增強效果更是幾乎消失，顯示UV/chlorine反應中自由基的生成與否確實是提升微生物抑制能力的關鍵；在碘化丙啶即時定量聚合酶連鎖反應的實驗結果發現，以UV-C LED在紫外光劑量17.28 mJ/cm2及初始餘氯濃度6 mg/L中暴露12分鐘下皆不會對Bacillus subtilis 孢子細胞膜的通透性產生影響，但在相同反應時間下經UV-C LED /chlorine處理之spo0A功能性基因的濃度會由107.2 copy numbers/mL下降至104.8 copy numbers/mL，顯示經UV-C LED/chlorine處理下能大幅地提昇對於Bacillus subtilis 孢子細胞膜的破壞程度。

Ultraviolet light-emitting diode (UV-LED) is a novel UV light source which has the ability to select specific UV wavelength. Many studies have demonstrated the effectiveness of water disinfection by UV-LED. Recently, UV/chlorine also an emerging advanced oxidation process (AOPs), has been applied in waste water treatment procedures to promote the removal pollution. This study is undertaken to evaluate UV-C LED/chlorine process to check whether it could effectively enhance the inactivation of MS-2 coliphage and Bacillus subtilis spores. Moreover, we also discuss possible influence on the inactivation mechanism under different UV wavelengths, pH and water samples. At last, propidium monozide quantitative real-time PCR (PMA qPCR) was used to detect the degree of microbial cell membrane damage by UV-C LED/chlorine treatment and compare it with general disinfection methods. According to the results of batch inactivation experiments, it can be concluded that the combination of ultraviolet light (LPUV or UV-C LED) and 3 mg/L of free residual chlorine has a synergistic effect on MS-2 coliphage. However, for Bacillus subtilis spores, it is necessary to increase the initial free residual chlorine to 6 mg/L and 9 mg/L to have a significant synergistic effect. Comparing fluence-based inactivation rate constant (k, obs), UV-C LED (275 nm) /chlorine is better than LPUV (254 nm)/chlorine. The data shows that the UV wavelength at 275 nm may be more suitable for UV/chlorine inactivation process than 254 nm. When t-BuOH is added as a radical scavenger in the UV/chlorine reaction, the enhancement effect of LPUV/chlorine and UV-C LED/chlorine on the inactivation of MS-2 coliphage is reduced by 69.2% and 84.4%, respectively. As for the Bacillus subtilis spores after adding t-BuOH, the enhancement effect almost disappeared. This phenomenon indicates that the generation of radicals in the UV/chlorine reaction is indeed a key factor for enhancing the inactivation of microorganisms. Based on the results of propidium monozide real-time quantitative polymerase chain reaction(PMA-qPCR), we found that exposure of UV-LED to UV dose of 17.28 mJ/cm2 and initial residual chlorine concentration of 6 mg/L for 12 minutes does not affect the permeability of Bacillus subtilis spores cell membrane. However, under the same reaction time, the concentration of UV-C/chlorine-treated spo0A functional genes decrease from 107.2 copy numbers/mL to 104.8 copy numbers/mL. This data shows that UV-C LED/chlorine can greatly increase the damage to the cell membrane of Bacillus subtilis spores.