Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/5235
標題: 可同時脫硝除磷菌種之篩選及其除磷特性之研究
ISOLATION AND BIOCHEMICAL CHARACTERIZATION OF DENITRIFYING PHOSPHORUS REMOVAL BACTERIA
作者: 史慧萍
Shi, Hoi-Ping
關鍵字: 脫硝除磷菌
denitrifying phosphorus removal bacteria
聚合磷酸鹽
生物除磷
polyphosphate
biological phosphorus removal
polyhydroxybutyrate (PHB)
Brachymonas sp. P12
Paracoccus denitrificans PP15
出版社: 環境工程學系所
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摘要: 厭氧好氧(或厭氧無氧)EBPR已廣為應用於生物處理中有效地去除磷酸鹽,但所篩選之除磷菌大多未具有完整的EBPR生物除磷特性,因此,本研究擬篩選脫硝除磷菌株並探討其脫硝除磷之生理特性,以釐清各種不同生長條件下的生物除磷機制,並希望藉由脫硝除磷菌之特性同時去除氮磷等污染源。本實驗篩選之污泥來源為養豬場廢水處理槽污泥,酒廠污泥,以及養豬場廢水處理槽污泥經厭氧無氧SBR馴化後之污泥。首先於篩選脫硝除磷菌之流程建議先以脫硝能力測試篩選出脫硝菌,進一步利用兩種電子接受者-氧氣與硝酸鹽同時存在的批次實驗測試對數生長期間之除磷能力,菌株若能利用氧氣以及硝酸鹽作為電子接受者,則之後的生物除磷機制便能於好氧或無氧的環境中進行以提高其應用性。篩選出於對數生長期中具高除磷效率之脫硝菌株,淘汰生長良好卻幾乎沒有除磷效率的菌株。大多數的脫硝除磷菌株並無法於厭氧環境下釋磷或是生長,此一現象與厭氧無氧SBR之混菌處理結果相似。而SBR之除氮與除磷效率分別為96%與86%。而脫硝除磷效率較佳的菌株經16S rRNA鑑定為Brachymonas sp.、Pseudomonas stutzeri及Paracoccus denitrificans。 利用脫硝除磷菌株Paracoccus denitrificans PP15探討在不同Tris緩衝濃度下之微生物誘導化學沈澱除磷機制,在添加12 g Tris/L的條件下pH僅由7.0上升至7.4,磷去除量亦只有10 mg P/L之生物除磷量,反之,在不添加Tris緩衝的條件下,pH上升至8.5且高達72 mg P/L去除量,藉由結合生物與化學除磷機制而提升磷去除率。在pH連續監控設備之輔助下,pH值於8.47時,磷濃度由108 mg P/L急劇下降至55 mg P/L,明顯地是化學除磷沈澱機制所導致,因為磷與陽離子產生沈澱反應,因此利用IC陽離子管柱分析陽離子濃度變化以及利用powder X-ray diffractometer分析沈澱物的主要組成發現沈澱物主要是由鎂磷錯合物所組成,此有利於磷回收之經濟效益。該菌株藉由脫硝作用以及醋酸鈉代謝作用導致pH值自發性地升高,使磷去除率由8 mg P/L(pH控制為7的批次試驗,僅生物除磷作用)增加至82 mg P/L(pH未控制,pH由7上升至8.8),除磷效率提高9倍以上,因此生物代謝反應導致pH值上升以誘導化學除磷,其可同時去除COD及氮磷等污染物,反之,若是只靠化學藥劑增加pH達到化學除磷時,只能除磷而不能同時去除COD與氮污染物。 利用生理特性分析得菌株Brachymonas sp. P12之較佳脫硝除磷條件是在無氧培養下加入2 g/L CH3COONa•3H2O (942 mg COD/L)、105 mg NH4+-N/L以及15 mg P/L (COD:N:P=100:11.2:1.6)。利用modified STS method進行體內磷物種之萃取與分類,發現金屬磷的含量最高,可高達10-13 mg P/g biomass。將體內累積之低分子量polyP與高分子量polyP濃度相加可達10 mg P/g biomass。相較於菌株由溶液中攝取25 mg P/g biomass,大約33%磷酸鹽用於polyP之合成。利用重覆無氧批次實驗探討菌株經由無氧預培養後之polyP合成能力是否提升,並利用31P核磁共振儀量測polyP與體內正磷酸鹽之濃度。結果顯示菌株可於對數生長期中累積polyP,但是並無法經由預培養增加polyP合成效率,因為第二次無氧培養之起始OD較高(OD of 1st: 0.1至0.4; OD of 2nd: 0.2至0.4),有一半的微生物族群來自第一次的預培養,因此群族呈現較為老化的現象,菌株能夠累積之polyP有限,所以第二次無氧培養時,磷攝取後進行polyP合成的比例減少,因此需要新生族群來提高除磷效率。此外,菌株進入靜止期時,polyP分解後體內正磷酸鹽僅佔polyP分解量的5%,因此推測polyP分解主要經由酵素PPK作用產生ATP用以維持細胞生長所用,而較少部分經由酵素PPX作用產生正磷酸鹽而釋磷。而由此實驗可發現無氧醋酸鹽代謝可提供足夠的ATP供PHB合成、polyP累積以及細胞生長,而不會有釋磷現象產生。 利用菌株Brachymonas sp. P12探討在無氧及好氧醋酸代謝條件下,PHB合成對磷攝取效率之影響。可發現在無氧或好氧條件下,對數生長期可分為兩個階段,第一個階段包括醋酸鈉與肝醣之消耗和PHB合成,磷攝取量約維持20-30 mg P uptake/g biomass左右;第二個階段則是醋酸鈉與PHB同時降解,磷攝取量明顯上升,其中部分批次可高達71 mg P uptake/g biomass。其攝磷模式與傳統Mino之厭氧好氧或是厭氧無氧EBPR模式相似,只是該脫硝除磷菌株可在單一無氧或是單一好氧情況下完成PHB合成以及攝磷之作用。由結果可發現PHB合成與polyP累積是以能量狀況進行調整,醋酸鈉消耗轉變成PHB合成,同時需消耗肝醣以提供還原能,此時polyP合成並不明顯,攝磷率有時有稍微下降的狀態,可能是polyP分解提供ATP供PHB合成之用。好氧或是高碳源環境下之PHB合成能力較高,佔對數生長期較高的比例,相對於後半部對數生長期的polyP合成時間則較少,因此攝磷量低於40 mg P uptake/g biomass。反之,在無氧且碳源添加適當濃度之條件下,前半部PHB累積時間很短,大部分之對數生長期間則同時利用醋酸鈉與PHB作為碳源,幾近好氧攝磷兩倍之磷攝取效率。而菌株Brachymonas sp. P12之PHB合成效率與生長速率有高度正相關,生長良好時,PHB合成能力愈佳。因此,脫硝除磷菌株Brachymonas sp. P12可於好氧環境下累積高達60% PHB供PHB生產之用,亦或是在無氧EBPR環境下,同時去除COD以及氮磷等污染物。此外,此一研究亦顯示單一好氧或是無氧除磷EBPR具有進一步發展之研究潛力。
This dissertation aimed to investigate the biochemical characteristics of the isolated denitrifying phosphorus removal bacteria (DPB) which were able to remove nitrogen and phosphorus simultaneously. For screening procedures, determination of denitrification ability was suggested as the first priority. Isolated denitrifiers were then cultured under an aerobic-anoxic single-stage process by using both oxygen and nitrate as electron acceptors to examine P removal capacity. The denitrifiers with higher phosphorus removal efficiency in the log phase were chosen. On the contrary, the well-grown denitrifiers without obvious P removal were excluded owing to P release by considerable PHB formation. Most of the isolated DPBs had no anaerobic P release, but did have significant anoxic P uptake. Similar to the results observed from inoculation of enriched sludge, pure isolated DPB cultures showed no anaerobic P release but achieved N and P removal efficiencies of 96% and 86%, respectively. The isolated DPB strains were indentified as Brachymonas sp., Pseudomonas stutzeri, and Paracoccus denitrificans. Paracoccus denitrificans PP15 was chosen as a model organism to investigate microorganism-mediated enhanced phosphorus chemical precipitation by varying Tris buffer concentrations. When Tris buffer was sufficiently provided up to 12 g/L, pH only increased from 7.0 to 7.4, and P. denitrificans PP15 removed only 10 mg P/L biologically. On the contrary, pH value increased to 8.5 without adding Tris buffer, and about 72 mg/L phosphorus was removed involved with biological and chemical mechanisms. When pH was higher than the breakpoint (pH 8.47) which was shown in the continuous pH curve via pH automatic online monitoring, phosphate concentration decreased suddenly from 108 to 55 mg P/L owing to chemical precipitation. Combined cation determination with powder X-ray diffractometer analyses, the major chemical precipitates in the environment were magnesium-phosphate compounds, used for P recovery. The strain could increase pH value spontaneously due to denitrification and acetate metabolism, and phosphorus removal was enhanced from 8 mg P/L (pH 7, pH controlled test) to 82 mg P/L (pH 8.8, pH-uncontrolled test), over 9-fold enhancement. The biological process treatment, accompanying with chemical precipitation, could remove COD, nitrogen, and phosphorus simultaneously, whereas chemical precipitation with pH increase might probably remove phosphorus only. An anoxic condition, which was conducted with 2 g/L CH3COONa•3H2O (942 mg COD/L), 105 mg NH4+-N/L, and 15 mg P/L, had a better efficiency of P removal in Brachymonas sp. P12. Based on the results from a modified STS method for intracellular phosphorus extraction and fractionation, metal phosphates had the highest content with 10-13 mg P/g biomass. The sum of low molecular weight (LMW) polyP and high molecular weight (HMW) polyP increased to 10 mg P/g biomass. Compared to P uptake of 25 mg P/g biomass from bulk solution, about 33% of P uptake was for polyP accumulation. Repeated anoxic batch cultivations were conducted to examine the enhancement of polyP accumulating capacity after an initial anoxic preculture, and polyP concentrations were quantified by using a 31P NMR spectroscopy. Data from the repeated anoxic batch examinations indicated that polyP accumulation occurred in the log phase under anoxic conditions and the efficiency of polyP accumulation was not enhanced by repeated batch cultivations. In the second anoxic batch, polyP-accumulating enhancement was limited in an aging population even though sufficient nutrients were provided the same as the first batch. More new-generated cells and higher cell growth rates promoted higher proportion of polyP to P uptake. An increased polyP concentration in the log phase indicated that anoxic acetate metabolism improved polyP accumulation. Anoxic acetate metabolism of Brachymonas sp. P12 provided sufficient ATP for polyP accumulation, PHB formation, and cell growth, simultaneously. The denitrifying phosphorus removal bacterium Brachymonas sp. P12 was used to investigate the enhanced biological phosphorus removal (EBPR) mechanism involved with polyhydroxybutyrate (PHB), glycogen, and phosphorus uptake in the presence of acetate under anoxic or aerobic conditions. The results showed that excess acetate concentration and aerobic cultivation could enhance PHB formation efficiency, and PHB formation might be stimulated by glycogenolysis of the cellular glycogen. The efficiency of the uptake of anoxic phosphorus was greater when PHB production was lower. The EBPR mechanism of Brachymonas sp. P12 for PHB, P and glycogen was similar to the conventional anaerobic-aerobic (or anaerobic-anoxic) EBPR models, but these models were developed under anoxic or aerobic conditions only, without an anaerobic stage. The anoxic or aerobic log phase of growth was divided into two main phases: (1) the early log phase, where acetate and glycogen were consumed to supply enough energy and reducing power for PHB formation and cell growth (P assimilation), and (2) the late log phase, which concluded the simultaneous degradation of PHB and remaining acetate for polyP accumulation. Glycogenolysis played a significant role in the alternate responses between PHB formation and P uptake under anoxic or aerobic conditions. For application of the denitrifying phosphorus removal bacterium Brachymonas sp. P12, aerobic cultivation would increase the level of PHB production, with anoxic cultivation further increasing P uptake.
URI: http://hdl.handle.net/11455/5235
其他識別: U0005-3001200718065300
文章連結: http://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-3001200718065300
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