微生物學

粒線體，『細胞內的發電廠』，它生產了我們生存所需的幾乎所有能量。除了產生能量，它也參與了細胞的凋亡的過程，或反應出自由基所引起細胞的老化。我們在此實驗中，利用酵母菌模式，跟兩個特殊的螢光染劑（JC-1與Rhodamine 123），來活體測量酵母菌裡的粒線體膜電位的變化，以了解細胞的早期凋亡的過程。並且，透過加入抗氧化劑與否，測量氧化游離基的產生，探討跟粒線體膜電位之間的關聯性。實驗結果顯示，的確酵母菌經過長期培養，且無提供適當的營養補充條件下，雖然外表還活著，但JC-1的螢光染色顯示，粒線體膜電位是有逐步遞減的現象，代表酵母菌活力慢慢衰弱。但是，另外氧化游離基的實驗結果卻沒有如預期般增加，反倒是跟JC-1的表現呈正相關現象。抗氧化劑的加入，非但沒有增加酵母菌的活力，反而是減弱了粒線體膜電位的表現，而氧化游離基也同時降低。這樣的實驗結果，雖然跟事前的預期結果不同，但也可能暗示1. 胡亂加入抗氧化劑，對生物不盡然是有幫忙的。2. 酵母菌在這一種生長條件中，可能需要一些氧化物的刺激或幫忙。3. 粒線體是細胞內氧化作用最強的單位，如果他生命力變弱了，自然產生氧化游離基的能力變弱，而非氧化游離基一開始去減弱粒線體的活性。此實驗的重大意義，是告訴我們，氧化游離基或抗氧化物的腳色並非一成不變的，甚至你原來認為的粒線體膜電位改變是細胞凋亡的起始原因，也可能相反，粒線體膜電位改變只是細胞凋亡後來發生的結果。科學的有趣也就是在這裡!我們藉助於【實驗】，不斷地實驗，去挑戰既有的知識跟原理，會讓我們發現更多的真理，一個單細胞的酵母菌就有這麼多問題需要去回答，那麼放眼將來不孕症醫師想嘗試『卵質轉移』，主要就是要取得健康的粒線體，增加懷孕的機會，那就可能需要更多的實驗跟努力，才能給出一個真正的正確答案。

雌激素為一種固醇類的環境賀爾蒙，若水生動物長時間暴露於低濃度的雌激素中，即會導致其生理及行為異常。我們企圖從迪化汙水處理廠中分離純化出能有效降解雌激素之菌株，希冀了解這些菌株的生理特性與降解雌激素的能力。首先在活性污泥中加入高濃度雌二醇(1mM)，以增進雌激素降解菌在菌群中的比例，接著利用十倍序列稀釋進行菌株純化。培養期間以薄層層析檢測雌激素降解活性。隨後以固態培養基培養法取得單一菌落，並進行聚合酶連鎖反應及核酸定序。之後我們亦將純化出之菌株進行其他固醇降解測定。目前純化出一株Novosphingobium屬的變形菌，其為一新的菌種，確實能降解雌二醇及其他固醇，包含雌酮、雄烯二酮、膽酸、孕酮和睪酮。然而，其並不能降解乙炔雌二醇。該菌株具有修復受汙染環境及製備雌激素相關藥品之應用潛力。

基因型的改變會導致表現型的改變，然而，有一種稱為遺傳緩衝的效應，能隱蔽基因型變異，維持表現型穩定。由於某些遺傳變異不適合生物的原始環境，卻能被保護，使在各種環境中持續演化。從前人的果蠅研究發現，若剔除熱休克蛋白90能夠使其不同遺傳品系間的基因型變異被顯露出來；而在酵母菌的研究中，剔除熱休克蛋白90會增加在環境擾動時的生長差異，因此熱休克蛋白90被認為有參與遺傳緩衝效應。更重要的是，熱休克蛋白家族普遍存在於所有真核生物中。為一窺遺傳緩衝效應機制的原貌，本研究誘發釀酒酵母菌菌種196的突變、增加其基因型的多樣化，並以doxycycline環境及高溫環境營造環境壓力，以降低酵母菌196及其突變株的基因Hsc82之表現量，找出含有因為突變而被熱休克蛋白90所保護的基因的突變株。希望透過找到這些突變株以了解，有哪些基因突變會被熱休克蛋白90所保護，並進而推論人類基因的突變是否也能依循此機制被隱蔽。

Saccharomyces cerevisiae 藉由出芽生殖的方式產生子代，而執行的次數並非無所限制。酵母菌一生的分裂次數(壽命)稱為 Replicative Life Span ( RLS )，指的是母細胞在停止分裂前所產生的子代數目。本實驗將老單倍體細胞( RLS 中位數: 6代 )和年輕單倍體細胞( RLS 中位數: 20代 )透過有性生殖的方式形成合子，發現合子的 RLS ( 中位數: 10.5代 )會受到老細胞的影響而縮短，並且接近兩單倍體親代 RLS 中位數的算術平均數( 13代 )，而其 F1 子代的 RLS 卻能回復與雙倍體相似的壽命。發現單倍體親代老化導致合子短命，可進一步推測人類生殖細胞老化對受精卵的影響模式。 

The redox reactions of bacteria metabolism have been extrinsically studied. These mechanisms allow certain types of bacteria to be able to synthesize extremely valuable extracellular byproducts. Other types of bacteria are able to extract toxic metals from water by donating electrons directly to those aqueous metal ions, thus turning them into solid precipitates. However, the problem of these microorganisms is that their efficiency rates and production speeds are exceptionally low. This study focuses on the properties of electrotrophs, which are bacteria that can feed on pure electrons directly from an electrode (Rabaey et al 2010). Compared to normal organic-feeding bacteria, electrotrophs direct the majority of the electrons obtained to the production of metabolic byproducts (Nevin et al 2010). Therefore, when electrotrophs are employed in bioelectrochemical systems (BESs) their metabolic redox reaction efficiency rates are dramatically increased. This makes it possible to produce large quantities of valuable compounds such as hydrocarbons, plastics and medicine or efficiently remediating the environment (He et al 2016). Moreover, the usage of electricity as an energy source compared to conventional organic substrates is immensely cheaper (Rabaey et al 2010). However, not all bacteria are electrotrophs nor do all electrotrophs have favourable metabolic traits. Thus, there is a need for a novel procedure to turn conventional bacteria into electrotrophs which is a crucial step to making the BES an aggressive competitor in the sustainable energy industry.

Saccharomyces cerevisiae 是常用來研究老化的模式生物。S. cerevisiae的壽命稱為replicative lifespan (RLS)，被定義為母細胞一生中可產生的子代總數。S. cerevisiae有一種缺少整套粒線體DNA的突變種，稱為petite。在這個研究中，我們利用附解剖針之複式顯微鏡測量年輕單倍體細胞和老單倍體細胞形成的合子、年輕單倍體petite細胞和老單倍體細胞形成的合子之RLS。此外，我們也使用ROS螢光探針DCFH-DA及DHE測量不同單倍體細胞的ROS含量。實驗結果顯示ROS本身並不足以降低合子的壽命。本研究可為人類生殖細胞影響受精卵的機制提供有用的資訊。

The rate and production of conventional petroleum based plastics is unsustainable and not eco-friendly. Plastics often end up in marine environments and can take hundreds of years to decompose in landfills. According to Statistica, in 2015 alone, global plastic production was approximately 322 million metric tonnes and is projected to increase in the future. PHB bioplastic or Polyhydroxybutyrate is both biologically produced and biodegradable and can serve as a viable alternative to conventional plastics. But can it be broken down by soil microbes within a reasonable time frame? I have set out to answer this question. My aim was to isolate and analyse microorganisms from the Rotorua area that are capable of degrading Polyhydroxybutyrate (PHB) bioplastic . I isolated PHB degrading microorganisms from Rotorua soils by culturing on an agar based mineral salt media supplemented with PHB powder (MSM PHB agar). Samples were taken from Mount Ngongotaha and Te Puia geothermal soils as well as Okareka, termite frass and termite guts. One isolate from the Te Puia sample (labelled G2) was found to successfully degrade PHB powder. After isolation and purification of the G2 isolate, it was cultured on a range of media types to examine properties exhibited under differing nutrient conditions. Multiple organisms were found to be involved in the degradation of PHB bioplastic and work together symbiotically, this included bacteria and fungi which was identified as penicillium. The sample isolated from Te Puia soils (site 2 – G2Clear) in the Rotorua environment was found capable of competently degrading PHB, clearing 8% of PHB after 26 days. The G2Clear isolate is a mixture of bacteria and fungi working in an endosymbiotic relationship to degrade PHB and are unable to successfully degrade PHB individually. It is through the secretion of an extracellular PHB depolymerase enzyme that PHB is degraded, conforming with my hypothesis. This proves that PHB bioplastic is a viable alternative to conventional petroleum based plastics as PHB can be relatively quickly broken down by soil microorganisms.

雌激素為一種環境賀爾蒙，若水生動物長期暴露於其中，即會導致其生理及行為異常。我們企圖從迪化汙水處理廠中純化出能降解雌激素之菌株，希冀了解其雌激素降解機制與生理特性。首先於菌液添加高濃度雌激素（1～2毫莫耳／升）培養以純化菌株，而後測定其16S核糖體核糖核酸序列確認純化出之菌株身分並設計引子，觀察此細菌之生長情形，並進行該菌株之生理測試，包含功能基因定序、代謝產物分析、脂肪酸組成分析、利用其它固醇為碳源之降解測定等。我們純化出一株新鞘氨醇桿菌屬（Novosphingobium）的變形菌，於雌激素濃度1毫莫耳／升的環境下能大量生長，確實能降解雌激素並生成代謝產物，具有與Sphingomonas sp. strain KC8相似之降解雌激素功能基因（oecC），且能降解其他固醇，包含雄烯二酮、膽酸、孕酮和睪酮，然而並不能降解乙炔雌二醇。該菌株具修復受汙染環境及製備雌激素相關藥品之應用潛力。

Microalgae-fungi biofuel has significantly less CO2 emissions than fossil fuels, making it much more environmentally friendly. As well, unlike traditional biofuel, microalgae-fungi does not require large masses of agricultural land for production. Thus, microalgae-fungi is an optimal option for biofuel production. This is a cost-effective renewable energy source that can be used in place of regular gas in cars and other means of transportation. By determining the most effective fungi for biofuel production, the threat of the impending environmental damage from pollution can be diminished. This novel experiment determines which fungi: Aspergillus niger, Rhizopus stolonifer or Saccharomyces cerevisiae, is the most effective bioflocculant in the microalgae-fungi co-pelletization process for biofuel production. We hypothesize that when paired with the microalgae Chlorella vulgaris, Rhizopus stolonifer will be the most effective. It has a high lipid content which could enhance the overall production of biofuel. Furthermore, its negative charge will aid with attracting and neutralizing the C. vulgaris colloidal particles resulting in an easier and more efficient removal of microalgae particles. Through the process of bioflocculation, pelletization, esterification and transesterification, the most effective fungi paired with C. vulgaris was determined. This experiment was carried out thoroughly and precisely resulting in a cost-effective solution for the world's current pollution crisis.

The synthesis of RNA using DNA (transcription) can be regulated by special proteins - transcription factors (TF) by binding to specific DNA regions - binding sites (BSTF). The purpose of the project is building a phylogenetic tree of orthologous groups of the each studying TF subfamilies, compare it with the tree of the corresponding BSTF motifs belonging to one orthologous group, find some common parts.