Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/5809
標題: 以功能性菌株探討薄膜生物反應槽中SMP與生物膜積垢之形成機制並評估二級訊號物質c-di-GMP與污泥生物膜積垢潛力之相關性
Exploring the membrane biofouling mechanisms of soluble microbial product (SMP) and biofilm by functional bacteria investigation and the relevance between c-di-GMP concentration and biofilm-fouling propensity in membrane bioreactor (MBR).
作者: 吳向宸
Wu, Siang-Chen
關鍵字: 薄膜生物反應器;Membrane Bioreactor;薄膜生物積垢;溶解性微生物產物;生物膜;二級訊號物質;食微比;Biofouling;Soluble Microbial Products;Biofilm;c-di-GMP;F/M ratio
出版社: 環境工程學系所
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摘要: 
本研究共分為兩個階段,第一階段為探討細菌分泌SMP所造成的薄膜積垢情形,先分離僅具有SMP分泌但並非具有生物膜生成能力之菌株以簡化其探討的複雜性,待暸解菌株SMP的分泌現象後將不同的SMP溶液以批次式的薄膜反應槽進行積垢測試,最終以SBR活性污泥系統進行不同食微比的操作,以暸解基質對活性污泥SMP分泌的影響。第二階段之實驗目的與第一階段雷同,探討對象改為生物膜所引起的薄膜積垢。針對三株生物膜標準菌株進行生理特性之探討,並以實驗室規模MBR搭配具時序採樣特性之薄膜組件,探討不同食微比下各類薄膜積垢物所貢獻的含量與變化情形。另外,也試圖探討c-di-GMP二級訊號傳遞物質與活性污泥生物膜積垢含量間的相關性,以期建立可適用於MBR生物膜積垢潛力的新式評估方法。
  於第一階段SMP積垢探討方面,以RCV與CR雙重選擇性培養基篩選分離出僅具有SMP分泌能力之菌株Microbacterium trichotecenolyticum B4-1,根據試驗結果發現,菌株B4-1之SMP分泌種類包含UAP與BAP,於UAP部分其分泌含量與基質含量成正比,於10X之合成廢水中其蛋白質UAP分泌含量可達到846.1 mg/L。另外,菌株B4-1於細胞衰敗過程會釋放BAP物質,其每毫克的細胞瓦解量會釋出約0.4毫克之蛋白質BAP物質。比較兩種SMP物質之積垢潛力發現,由BAP物質具有最高的薄膜積垢潛力,批次式薄膜積垢試驗中經20小時過濾後其薄膜總阻抗上升至89.3 x 1011 m-1,其餘UAP組別之薄膜總阻抗最高僅上升至33.7 x 1011 m-1。而EEM分析結果指出,菌株B4-1分泌之UAP與BAP物質含有酪胺酸或色胺酸之蛋白質物質,另外還包含部份棕黃酸、疏水性酸性物質與腐植酸等物質。在SBR活性污泥SMP積垢試驗方面,不同食微比之操作條件下可發現,食微比0.2 mg-COD/mg-MLSS-day (後續以day-1為單位)組別溶液中,聚醣類SMP含量約累積至23.2 ± 2.0 mg/L,而蛋白質SMP含量約累積至13.7 ± 0.4 mg/L,將SBR換至食微比0.05 day-1組別時,聚醣類與蛋白質含量分別下降至11.3 ± 1.5與3.0 ± 0.7 mg/L,將兩組SMP溶液與廢棄污泥組別進行積垢試驗發現,仍是以廢棄污泥所收集之BAP溶液具有最高的薄膜積垢潛力,於過濾試程31小時後達到40.7 ± 2.2 x 1011 m-1。由此可知細胞衰敗所釋出的BAP物質其溶液對薄膜會造成嚴重的積垢情形。
  於第二階段方面,本階段亦分離具有生物膜生成能力之菌株B2-1與B2-10。經生理特性探討發現,其生物膜生成受到基質濃度突然轉變而誘發,且其他三株生物膜標準菌株中也觀察到相同現象,由此可推論生物膜的形成與基質含量也具有直接的相關性。利用實驗室規模MBR進行不同食微比之操作結果可知,於食微比0.5 day-1組別時,其薄膜組件之TMP由0.03 bar (3 kPa)經20天後上升至0.28 bar (28 kPa),其中污泥顆粒為薄膜生物積垢主要的貢獻來源,其最終造成的薄膜阻抗為67.0 x 1011 m-1且貢獻百分比為85.6%,其他生物膜與孔洞阻塞的貢獻度則不顯著。於食微比0.05 day-1組別方面,於操作前9天中薄膜組件的TMP變化穩定,TMP由0.03 bar上升至0.06 bar,當試程繼續操作時,薄膜阻件的TMP急速上升並於試驗20天後達到0.52 bar,其薄膜積垢情形較為嚴重,於試驗前期即觀察到生物膜所造成的積垢,且其上升的趨勢與薄膜組件的TMP上升趨勢相符,於最終的阻抗貢獻百分比中,污泥顆粒、生物膜、與孔洞阻塞分別佔了59.6%、30.7%與7.7%,此結果顯示低食微比操作條件下容易促使生物膜生長,附著於薄膜表面造成嚴重的生物膜積垢。綜合本研究兩階段試驗之成果可知,污泥代謝活性與其基質利用情形對各類薄膜生物積垢的變動上具有重要的影響性,因此MBR操作過程應額外增加污泥諸類特性之監控與最佳化,以達到薄膜最少量之生物積垢情形。
  於c-di-GMP與生物膜積垢相關性方面,於生物膜標準菌株中可發現,其生物膜生成含量與細胞體內c-di-GMP含量成正相關之趨勢,菌株P. putida於生物膜累積的過程中,其體內的c-di-GMP含量由1.82 pmole/mg-cell上升至3.45 pmole/mg-cell,同樣地,菌株S. enterica體內之c-di-GMP含量也從0.01 pmole/mg-cell上升至8.21 pmole/mg-cell,由此可知,c-di-GMP於不同的生物膜生成菌株中,其與生物膜之生成含量確實具有其相關性。然而,於MBR活性污泥之評估結果卻發現,污泥細胞之c-di-GMP含量與薄膜生物膜積垢之情形相悖,於污泥細胞中,當MBR之操作食微比為0.5 day-1時,此時污泥細胞c-di-GMP之平均含量高達22.62 pmole/mg-sludge,但此時薄膜之生物積垢中生物膜所佔比例為11.0%,相對地,當MBR之操作食微比為0.05 day-1時,其污泥細胞c-di-GMP之平均含量降至2.79 pmole/mg-sludge,但此時薄膜生物積垢中生物膜所佔比例卻提升至30.7%,由此可推論,於混合族群中c-di-GMP訊號物質所調控之生理特性仍需要進一步地研究。

Membrane bioreactor (MBR) is a key technology for wastewater reuse because of the high-quality effluent, low sludge yield, and small reactor footprint. However, membrane biofouling in MBRs is a major obstacle that reduces the filtration efficiency, increases the cost-effectiveness, as well as discourages for wide applications. The aims of this study were focused on the mechanism of membrane biofouling attributed to soluble microbial product (SMP) or biofilm attachment by means of investigation of functional bacterial strains. In addition, the relevance between cyclic-di-GMP (c-di-GMP) amount, an second messenger compound, and biofilm fouling of sludge was also evaluated, tried to develop an novel method to predict the propensity of membrane biofilm fouling in an real MBR process.
  The SMP-secreted strain Microbacterium trichotecenolyticum B4-1 was isolated by selective RCV and CR mediums from acclimated wastewater slugde. From the results, the strain B4-1 contained two SMP secreting types. One is the type of substrate-utilization-associated product (UAP), which revealed a direct proportional to substrate concentrations (R2 = 0.988). The strain B4-1 could produce soluble proteins at the concentration of approximately 846.1 mg/L while cultivating with F/M ratio of 8.1. On the other hand, the intercellular polymer proteins of strain B4-1 was released as biomass-associated product (BAP) in the content of 0.4 mg-proteins/mg-cell during the status of cell decay. Comparing the membrane fouling propensity between these two types of SMP solutions, the results showed that the BAP solution had the highest membrane fouling propensity and raised the membrane resistance to 89.3 x 1011 m-1 after 20-hrs filtration. Moreover, the order of fouling propensity was identical in terms of activated sludge in a sequencing batch reactor (SBR). BAP solution from waste sludge storage tank also revealed the highest fouling propensity with the membrane resisitance of 40.7 ± 2.2 x 1011 m-1 for 31-hr filtration.
  With respect of biofilm fouling, the results of biofilm formation assay using 96-wells plate with two self-isolated and three widly studied biofilm-forming strains indicated that switching the substrate loadings in a short time could trigger their biofilm-forming state. In addition, it is observed that the intercellular c-di-GMP levels of P. putida and S. enterica were both direct proportional to their bioiflm biomass. In order to understand the correlation between membrane biofilm fouling and substrate loading, a lab-scale MBR under two food to microorganism (F/M) ratios was carried out. The observing results showed that cake layer was the dominant contributor (85.6%) to the membrane biofouling under F/M ratio of 0.5 day-1. The contributions from biofilm as well as pore blocking were only 11.0% and 7.1% respectively ,which were relatively lower than that of cake layer. Combined with microscopic observation, particle size of the sludge flocs was considerably below 20 μm and most of microorganisms grew in the planktonic state. Otherwise, the membrane biofouling distribution was totally distinct and the membrane resistance was almost twice as high as that under F/M ratio of 0.5 day-1 when the lab-scale MBR was operated under F/M ratio of 0.05 day-1. Although cake layer was still the dominant contributor causing 59.6% of total membrane resistance, the portion coming from biofilm was dramatically increased to 30.7% of total membrane resisitance and the particle size of sludge flocs also expanded over 150 μm. Those results elucidated that the biofilm-forming state of sludge was more active under the low F/M ratio condition and the biofilm attachment on the membrane surface caused serious membrane fouling. However, comparing with P. putida and S. enterica strains, the intercellular c-di-GMP level extracted from sludge cells exhibited an opposite tendency toward the propensity of membrane biofilm fouling. The average c-di-GMP level under F/M ratio of 0.5 day-1 achieved 22.62 pmole/mg-sludge, which was nearly 10-fold higher than that under F/M ratio of 0.05 day-1 (2.79 pmole/mg-sludge). Although it showed the opposite tendency, the intercellular c-diGMP level of sludge cell under each operating condtion was stable and the correlation efficiency between operating F/M ratio and intercellular c-di-GMP level was 0.942. These results indicated that using intercellular c-di-GMP level in activated sludge as an novel technique to predict the biofilm fouling propensity should need more investigation in terms of sludge characteristics such as microbial community or substrate affinity.
URI: http://hdl.handle.net/11455/5809
其他識別: U0005-0807201316392100
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