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標題: Rhodopseudomonas palustris WP3-5細胞生長、產氫與PHB累積之關係及共培養試驗
Relationship between cell growth, hydrogen production and poly-β-hydroxybutyrate (PHB) accumulation by Rhodopseudomonas palustris WP3-5 and co-culture test
作者: 陳盈孜
Chen, Ying-Tzu
關鍵字: 生物產氫
biological hydrogen production
Rhodopseudomonas palustris WP3-5
purple non-sulfur photosynthetic bacteria
Rhodopseudomonas palustris WP3-5
soluble microbial product
continuous photosynthetic hydrogen production
出版社: 環境工程學系所
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摘要: 「氫氣」是一種乾淨的能源,其熱值含量高(122 kJ/g),且燃燒後無二氧化碳排放之問題,而以紫色不含硫光合菌於光異營條件下進行生物產氫為具潛力的方式之一。近年來,許多研究嘗試提升紫色不含硫光合菌之產氫效率,poly-β-hydroxybutyrate (PHB)為胞內聚合物,其合成可能會與產氫競爭能量及電子,且產氫多半是發生在微生物進入生長靜止期時,因此,紫色不含硫光合菌於產氫、細胞生長及PHB累積之能量分配是相當重要的。此外,暗醱酵出流水含有數種有機酸,適合做為紫色不含硫光合菌產氫之基質,如何有效地以生物方式進行處理及能源回收也為重要的課題。 本篇研究便希望藉由Rhodopseudomonas palustris WP3-5之PHB synthase-deficient mutant Rps. palustris M23以批次試驗去瞭解不同條件下能量分布的情形。另一方面,也嘗試分離、純化紫色不含硫光合菌,測試其利用各種有機酸產氫之情形,評估與Rps. palustris WP3-5共培養能否提升產氫及廢水處理之效率,並以光合反應槽培養達連續產氫。 實驗結果發現,Rps. palustris WP3-5於生長過程中伴隨著PHB之累積,PHB含量累積至最大值後,有被微生物再利用之情形,可供給Rps. palustris WP3-5額外的碳源及能源。當氮源耗盡時,細胞便會停止生長,此時細胞會將多餘之能量進行產氫。碳源濃度較低時,由於耗盡較快,Rps. palustris WP3-5無多餘之碳源合成PHB,因此,與突變株Rps. palustris M23產氫情形無明顯差異。當碳源濃度提高時,Rps. palustris WP3-5產氫量較多,基質轉換率也較高,突變株Rps. palustris M23產氫情形無明顯提升,其他不同條件之實驗也可觀察到相同結果,但以蘋果酸為碳源時,Rps. palustris WP3-5利用速率慢,產氫量較低,且不累積PHB。 不同紫色不含硫光合菌對同種類碳源之代謝途徑可能有差異,導致PHB累積量皆不相同。Rps. palustris WP3-5進入生長靜止期時會將大部份能量用於合成氫氣,而其PHB累積量不高,佔微生物獲得能量5%以下,可能為導致氫氣合成與PHB累積競爭還原能關係不明顯之原因。實驗也發現,SMP合成可能也會競爭還原能,PHB synthase-deficient mutant重新分配能量時不會完全分配至氫氣合成。 本實驗從環境中分離純化出數株紫色不含硫光合菌,經產氣測試及產氫試驗,各菌株對於各種有機酸利用情形無明顯差異,共培養未改善產氫量,可能有競爭能量之情形。而Rps. palustris WP3-5以新設計之光合反應槽進行連續流操作,以適合紫色不含硫光合菌產氫之鎢絲燈為光源,且水力停留時間由2天提高至3天,以初期產氣率較佳,達317.6 mL gas/L culture-day,試程中MLSS皆在220-360 mg/L,改變操作策略無法改善細胞生長量,因操作反應槽需考量光穿透率及因高細胞濃度造成之光遮蔽問題,此反應槽連續流產氫結果不如預期,可能不適合紫色不含硫光合菌進行連續流產氫。
Hydrogen is a clean energy that generates only water upon burning and it releases high amount of energy (122 kJ/g) by combustion. Photo-biological hydrogen production by purple non-sulfur photosynthetic bacteria is one of promising methods. Purple non-sulfur photosynthetic bacteria can produce hydrogen by nitrogenase under photo-heterotrophic condition. In recent years, many researchers make efforts to improve its efficiency of biological hydrogen production. Poly-β-hydroxybutyrate (PHB) is accumulated as carbon and energy storage material under unbalanced growth. The synthesis of PHB may compete with hydrogen production for energy and reducing power. In addition, maximum hydrogen production rate usually takes place during early stationary phase. Therefore, energy distribution between cell growth, hydrogen production and PHB accumulation is an important issue. Besides, dark fermentation effluent containing various volatile organic acids is suitable for purple non-sulfur photosynthetic bacteria to produce hydrogen. To treat wastewater and recycle clean energy efficiently by biological method are also essential. In this research, Rhodopseudomonas palustris WP3-5 and its PHB synthase-deficient mutant Rps. palustris M23 were used in batch experiments to explore energy distribution between cell growth, hydrogen production and PHB accumulation under different culture conditions. This study also tried to isolate purple non-sulfur photosynthetic bacteria from environment, and tested their ability to produce hydrogen using different volatile organic acids. Then, hydrogen production and treatment efficiency in co-culture system with Rps. palustris WP3-5 were evaluated and continuous photosynthetic hydrogen production was performed. From the results, Rps. palustris WP3-5 accumulated PHB in growth phase, and PHB could be utilized as another carbon and energy source when its content reached maximum value. Microorganism ceased growing when nitrogen source was exhausted, and used excess energy to produce more hydrogen in stationary phase. Rps. palustris WP3-5 could not synthesize PHB under low concentration of carbon source because substrate was degraded rapidly, and there were no difference of hydrogen production between wild-type strain and mutant Rps. palustris M23. When concentration of carbon source was three times higher, cumulated hydrogen volume and substrate conversion efficiency of Rps. palustris WP3-5 were better than Rps. palustris M23. This result coincided with other experiments in different culture conditions. But when using malate as carbon source, Rps. palustris WP3-5 had lower substrate degrading rate and cumulated hydrogen volume, and PHB was not accumulated. Different species of purple non-sulfur photosynthetic bacteria had different substrate assimilation pathway when using same carbon source and accumulated PHB content might be different. Rps. palustris WP3-5 used most energy to produce hydrogen in stationary phase and PHB content was below 10% cell dry weight, accounting for less than 5% of the substrate electrons utilized. This portion of energy might partially redistribute to synthesize soluble microbial product (SMP). Therefore, the competition relationship between hydrogen production and PHB accumulation was insignificant. In second part, several purple non-sulfur photosynthetic bacteria were isolated, and there were no obvious difference in utilizing volatile organic acids to produce hydrogen between these strains. Hydrogen production was not improved and competition for energy might exist in co-culture system. Additionally, new designed photo-bioreactor was used for Rps. palustris WP3-5 to produce hydrogen continuously when using tungsten filament lamp as light source. Operating hydraulic retention time from 2 day to 3 day, gas production rate was highest in initial period, up to 317.6 mL gas/L culture-day. MLSS was always between 220 and 360 mg/L, and MLSS wasn't enhanced by changing operational strategies. Thinking of light transmission efficiency and problem of light shielding effect caused by high biomass concentration, experimental results was not as expected, and this photo-bioreactor might not favorable for continuous hydrogen production by purple non-sulfur photosynthetic bacteria.
其他識別: U0005-2007201118365000
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