微生物學

Nematophagous fungi can form different kind of trapping device to trap the nematodes when they show off. They may play a role for control of the plant and animal parasitic nematodes as an alterative choice beside regular practice. We attempt to investigate the adhesive’s attributes and the genes that encode trapping structures. Now we have already constructed the Arthrobotrys musiformis Fosmid library which will play a vital resource for specific genes analysis, cloning and characterization in the future. We have chosen two genes encoding protease and superoxide dismutase from Arthrobotrys musiformis, respectively, and will be used as probes to screen the Fosmid library. The relevent clone(s) will be subject to restrictive enzyme disection, Southern blotting or even whole Fosmid 40kb DNA fragment sequencing to discover the interesting and paramount genes. 線蟲捕捉菌在線蟲出現時可以產生型態各異的捕捉構造，捕捉或黏著線蟲。它是防治線蟲的另類選擇。本實驗是由生物的分生觀點切入，希望能夠了解線蟲捕捉菌Arthroborys musiformis於捕捉網表面之黏液生化特性以及控制產生捕捉構造的基因。目前已完成建 Arthrobotrys musiformis之Fosmid library，並且選擇兩組探針：蛋白質?(protease)以及超歧氧化?(superoxide dismutase)，將以PCR進行基因探針之DIG標定，之後篩檢Fosmid library，選殖出相關clone，進行限制?切割，南方氏雜合特性分析或40kbDNA全序列分析，尋找相關基因以利下游實驗工作之進行。

20 世紀初，石油的量產造就了人類文明前所未有的繁榮，然而由於運送、廢棄處理等因素，使得油類污染成為環境保護的重大議題。本實驗中，我們的研究主題為在受油污染的土壤中純化並鑑定出可分解油類之土壤菌和綠膿桿菌對可分解油類之土壤菌與這群土壤菌彼此之間的交互關係，藉此了解它們間的互動對環境生物復育的影響。我們由受油類污染的行道樹土壤中分離了約12 種的土壤菌，其中我們得到3 種對油類分解效果效果極佳的非綠膿桿菌(暫時命名為P7A、P7C、P7D)。經過菌種鑑定發現P7A、P7C、P7D 均為格蘭氏陽性菌。為了解這群可分解油類之土壤菌間的互動關係，我們針對分解效果最佳的P7A、P7C、P7D 作為研究對象，將菌落接種至含有鹽類與機油的液體培養基中震盪培養，並每隔一定時間測量其O.D 值。結果發現P7A、P7C、P7D 間的互動會導致其在以機油為單一碳源的培養液中之生長速度的改變，因此在行環境生物復育時須注意土壤菌間交互關係對其分解污染物速率的影響。此外我們由受油類污染的行道樹土壤中亦分離出了一些綠膿桿菌，因文獻指出，綠膿桿菌所分泌的綠膿素降低受油污染土壤中土壤微生物相的多樣性；因此，我們將由行道樹土壤中純化出的綠膿桿菌T3 與可分解油類P7A、P7C、P7D 進行交互作用觀察，發現T3 會侵占P7A、P7C、P7D 的既有菌落區，而平板培養基亦可清楚看出和T3 交接的P7A、P7C、P7D 菌落區寬度有明顯降低，因此我們認為T3 可抑制或殺死P7A、P7C、P7D，可得知綠膿桿菌會對可分解油類之土壤菌產生抑制或競爭關係。In early 20th century, the exploitation of petroleum transformed human civilization into a tremendously prosper stage. Because of the transportation and disposition of petroleum, the oil pollution has become a important issue in environmental protection. Besides, Chloropseudomonas spp. which can survive in many different environments and decompose lots of organic compounds. In this study, we want to find the bacteria which can utilize oil from machine oil-contaminated soil, investigating the interaction relations between Chloropseudomonas spp. and these oil-degrading soil bacteria. First, we classified these oil-degrading bacteria by the book called“Bergey’s Manual of Systematic Bacteriology.”We find three species of oil-degrading bacteria (P7A、 P7C、P7D) which are all grams-positive bacillus, possibly belonged to Aureobactreium、Curtobacterium、Cellulomonas、Oerskovia、Brochothrix、 Caryophanon. Second, in the study of the relationship between Chloropseudomonas spp. and the oil degrading soil bacteria, we found that Chloropseudomonas spp can considerably inhabit the growth of oil-degrading bacteria. Besides, there are also a great variety of interaction between three species of the oil-degrading bacteria. According to the result , the interaction might considerably affect the efficiency of oil bioremediation. Due to our analysis, we suggest that it is necessary to pay more attention to the interaction between bacteria when undertaking oil bioremediation.

Azotobacteraceae 為一單棲固氮菌科，包含Azotobacter 與Azomonas 兩菌屬，在農業上可用來改善缺氮的貧瘠土壤。在分離土壤中的Azotobacteraceae 時，發現非單棲固氮菌與單棲固氮菌間可能具有共生的情形。我們利用優勢培養(缺氮)的方法篩選土壤中的Azotobacteraceae，將優勢培養後所生成的菌落稀釋104~106倍後，能有效分離Azotobacter 與Azomonas，然而低於此稀釋倍率則會形成混合菌落，其中可同時發現單棲固氮菌與非單棲固氮菌存在，推測某些非固氮菌在優勢培養過程中可能可從單棲固氮菌獲得氮源，與之共生。此外亦從菌種形態的差異並配合顯微螢光雜合技術(fluorescence in situ hybridization, FISH)、分子遺傳標記(16S-rDNA)等方式，分析土壤中的Azotobacteraceae，探討單棲固氮菌及其他非單棲固氮菌在培養基上的生長情形、比例及親緣關係。The family Azotobacteraceace is group of free-living nitrogen-fixing bacteria that is found in soil. Two genera are within this family: Azotobacter and Azomonas. Agriculturally, it is often used to improve fertility for nitrogen deficient barren lands. We analyze the Azotobacteraceace according to molecular biology and traditional taxonomy. We used an enrichment procedure to culture the bacteria, and diluted it repeatedly. We found it most suitable to dilute it 104~106 times to best separate Azotobacter from Azomonas. If the concentration were to be higher than this, mixed flora containing many different bacteria species would be found. Moreover, we noticed that non nitrogen-fixing bacteria, symbiotic nitrogen-fixing bacteria, and free-living nitrogen-fixing bacteria would form a single colony on a nitrogen-deprived medium. This implies that a symbiotic relationship may exist between nitrogen-fixing bacteria and non nitrogen-fixing bacteria. We also discuss the growing situation, the group proportion, and the relationships between free-living nixtron fixing bacteria and other bacteria by morphology, fluorescence in situ hybridization (FISH), and molecular biology.

在先備知識中，我們知道在缺少營養鹽及紫外線傷害下，浮游藻類的葉綠體會因為過氧化物（R.O.S.）的增加而受到破壞，進而影響光合作用的進行，甚至導致死亡。所以確實了解常見浮游藻類生理狀態和環境的影響，以期待未來可利用大幅度提高浮游藻類生產力的方式有效降低溫室效應的影響為本實驗的主要目的。故實驗設計針對兩種常見的海洋種浮游藻類（Tetra、Ske），在不同紫外線光譜（UVAB、UVC）的照射下，觀察R.O.S.的產生量和T-T dimer的表現狀況，並對照兩者之間的關係。結果我們發現：綠藻（Tetra）和矽藻（Ske）在UVAB、UVC 的照射下皆會產生R.O.S.，且綠藻產生的量較少；但在UVC 照射下皆有DNA 損傷（產生T-T dimer）。故推估並不是綠藻（Tetra）擁有紫外線的特殊防禦機制，而是能較有效地代謝R.O.S.。As we know, under the condition of unorganized salt’s shortage and the harm of the ultraviolet ray, the phytoplankton’s chloroplast will be destroyed because of the increasing peroxide (R.O.S.). Furthermore, the ultraviolet ray will have an effect on the process of photosynthesis, and even result in the death of phytoplankton. So, we intend to promote the production of phytoplankton in order to lower the influence of greenhouse effect by probing into the environmental influence on the physiology of phytoplankton. The experimental is designed to observe two common marine phytoplankton: Tetra and Ske. By close observing Tetra and Ske exposed to different wavelength of ultraviolet way (UVAB and UVC ), we contrast the production of R.O.S. with the appearing of T-T dimer. We observe that both Tetra and Ske will produce R.O.S. after being exposed to UVAB and UVC , but Tetra produce less than Ske, and that UVC will do harm to both the DNA of Tetra and Ske (producing T-T dimer). Based on the result of the experiment we estimate that Tetra can catobolize R.O.S. efficiently instead of having a unique defensive mechanism against ultraviolet ray (UVAB and UVC under discussion in this experiment.)

線蟲為植物發生病蟲害感染的病源之一，而台灣的松樹，目前正面臨著松材線蟲入侵的危機。從文獻的探討中，發現線蟲有其自然界的天敵 － 線蟲捕捉菌。本實驗著重在探討線蟲捕捉菌特殊的捕捉機制。當線蟲捕捉菌附近出現線蟲時，會展生誘引線蟲的物質，並設計了一步步的實驗，去探討此誘引物質的捕蟲效能及其成分。現在，已經發現此誘引物質為一揮發性氣體。往後將會設法增加其誘引氣體的產量，並使用氣相層析儀分析之。最後希望可以將此物質應用到微生物防治上，期望能解決台灣松樹被線蟲感染的問題。 In Taiwan, all of the pine trees have one common problem - nematodes, which causes diseases in plants. And this experiment focuses on the nematodes’ natural enemy - nematophagous fungi and its “peculiarly caused mechanism.” When nematodes appear near nematophagous fungi, the latter will produce some substance to tempt the former. To investigate this alluring substance, a series of experiments are done and systematic steps are taken. The first finding is that this substance is a volatility gas. Later in this research, measures will be taken to make “rematophagous fungi” produce more of this gas. And “gas chromatograph” will be used to analyze this gas in the future. Finally, the possibility of applying this substance to the defensive measure of microbiology will be discussed.

RNA 病毒在複製過程中容易產生錯誤，導致其族群具中有很大的遺傳歧異度，累積的錯誤再加上選汰的壓力造成往後之變異。由於RNA 基因體之病毒變異較大，使得RNA 病毒在單一寄主上具有quasispecies 的特性，提供病毒產生新基因體的機會以適應環境或演化成新病毒。例如流行性感冒病毒與之前造成恐慌的嚴重急性呼吸道症候群病毒(severe acute respiratorysyndrome,SARS)以及禽流感病毒 (avain influenza virus) 皆為RNA 病毒，意味著RNA 病毒知不穩定性，並容易造成一些目前我們無法及時反應的危害。大部分的植物病毒又為RNA 病毒，本研究將以竹嵌紋病毒 ( Bamboo mosaic virus , BaMV )及其衛星核酸 (satellite RNA, satBaMV)為材料，進一步探討核?酸序列之變異對其族群在複製競爭上的影響。

Purpose of research. Microbial fuel cells (MFCs) are bioelectrochemical systems that generate electrical energy by exploiting the extracellular electron transport (EET) capabilities of electrochemically active bacteria (EAB) (Logan 2009). This investigation aims to identify genes involved in driving bacterial EET with a new procedure that enables rapid screening of a side array of genes. These insights may lead to improved MFC performance through enhancing reactor design or genetic engineering EABs (Alfonta 2009). Procedures. MFC metagenomic analysis. Twelve MFCs incubated with four different bacterial samples were operated for approximately one year. The bacterial DNA from before and after incubation was extracted and the 16S rRNA regions were PCR amplified and sequenced. The bacterial community changes were analyzed using the QIIME program to identify bacteria that were being selected. Fosmid Clone Isolation. An E. coli fosmid library (Mewis et al. 2013) that contained genes from EAB inferred in the previous step was incubated in three MFCs. After a 48 hour enrichment period, biofilm samples from the MFCs were extracted and individual clones were isolated and screened in the MFCs individually. An E. coli DH5α strain with no insert DNA was incubated separately as the control. DNA sequencing. Fosmid insert DNA from high-performing clones were extracted, purified using gel electrophoresis, constructed into sequencing libraries and sequenced. Bioinformatics Analysis. The sequences were constructed into larger contigs using the Velvet algorithm package. The open reading frames (ORFs) were inferred and translated into amino acid sequences and annotated with proteins identified from the KEGG, and SEEDs databases using Metapathways 2.5. Results. The changes in bacterial communities from the metagenomic analysis revealed increases in relative abundance in numerous genera from Firmicutes and Bacteroidetes. The MFCs incubated with the fosmid clones generated about 4 times more peak power than the MFCs incubated with the E. coli DH5α. Polarization curves generated for the MFCs demonstrated that the fosmid clones were able to sustain a higher current. Incubation of pure cultures of individual clones yielded four clones with significant performance improvements over the control strain. Protein data from Metapathways outputs reveled both novel and previously reported EET genes encoding for Type IV pilus structures, c-type cytochromes, soluble cytochromes, flavoproteins, and porins. Taxonomy inferences of the gene inserts by the Green Genes database reveal the genes most likely came from the same EABs that were inferred from the metagenomic analysis. Conclusions. The increased performance of the fosmid clone-powered MFCs suggest that the clones carried genes that enhanced their performance in the MFCs. This is further confirmed by polarization curves generated for the MFCs. The results of the taxonomy inferences suggest that the bacteria being selected for in the environmental samples carried genes that enhanced their performance in the MFCs, and that these genes were successfully identified in the subsequent steps. The results of this study demonstrate that using a gain of function approach to rapidly screen a wide array of genes in a gene library may be an efficient method to identify genes that enhance power generation of EABs in MFCs.

線蟲捕捉菌Arthrobotrys musiformis 是一種可經線蟲誘導產生捕捉網來捕捉線蟲的真菌，本實驗即針對A. musiformis 的捕捉網黏液相關基因：Manosyltransferase（AH73）, β-1,3-glucan transferase（AH102）, fimbrin（AH121）及mannose-specific lectin precursor（AH338）進行選殖與功能界定，希望建立這方面的研究基礎，將來能應用在松材線蟲的生物防治上。首先我們大量培養A. musiformis，萃取菌絲體的DNA；接著進行聚合?連鎖反應 (Polymerase Chain Reaction，PCR) ，利用專一性引子對 (primer) 大量增幅AH73、AH102、AH121 及AH338之基因片段；增幅後的產物經過純化、選殖，定序並進行分析比對，確認增幅之序列無誤後，以 Digoxigenin (DIG) 標示當為探針，篩檢A. musiformis 的Fosmid Library﹔目前已成功選殖出AH73 之可能基因，完成AH73 之探針製備，並以其篩檢A. musiformis 的Fosmid Library﹔呈雜合正反應之選殖株 (clones) 將以散彈槍方法（shotgun）定序，作序列組合，探索相關的基因；接下來用 Rapid Amplification of cDNA Ends（RACE) 做出互補DNA (complementary DNA , cDNA) 全長度後；最後建構基因缺失株，驗證此基因所調控的生理以及生化機能。 Nematode trapping fungus Arthrobotrys musiformis can capture nematodes by producing adhesive nets when nematodes go through. Many kinds of nematodes, including pine wood nematode (Bursaphelencus xylophilus), can be captured. Pine wood nematode causes serious pine wood disease. Therefore, A. musiformis has the potential of biocontrol in pine wood nematode. Our research focused on adhesion and adhesive relevant genes of A. musiformis ：Manosyltransferase (AH73), β-1,3-glucan transferase (AH102), fimbrin (AH121), and mannose-specific lectin precursor (AH338). We try to clone these genes and carry out functional analysis. In order to achieve this goal, we used specific primers derived from previously obtained complementary DNA (cDNA), by Polymerase Chain Reaction (PCR) to amplify these genes and gained adequate quantity of genomic DNA products. After sequencing and verifying of the identity of the genomic DNA, we use Digoxigenin （DIG） to label them and use them as probes to screen the constructed A. musiformis Fosmid Library. Currently, the Southern colony hybridization is undergoing. The positive Fosmid clones against the specific probes will be sequenced completely by shotgun library to monitor the existence of adhesion related gene cluster. After working out the full length cDNA of these genes, we will use them to construct replacement vectors to knockout the adhesion related genes, creating mutants and further verify their functions through genotype or phenotype bioassay.

The natural fruits of soap berry are worth-while detergent in the aspect of environmental conservation. It has been shown that the soap berry fruits can reduce the itching of head and inhibit the growth of fungi. Here we would like to test the inhibitory activity of soap berry fruits on the bacteria. This might benefit to the further application of the soap berry fruits. First,the soap berry extract was prepared and mixed with E.coli or S. aureaus. The number of bacteria was counted to realize the inhibitory activity of the soap berries. We found that the soap berry extract can inhibit the growth of E.coli and S. aureaus. Higher concentration of the soap berry extract and longer duration for the mixture make better inhibitory activities. 天然的無患子果實是一種極具環保價值的清潔劑，已知它有抑制黴菌生長的能力，可以 去頭皮屑、止頭皮癢。因此，我們想進一步探討無患子果實對於細菌生長的抑制能力，以期 了解無患子清潔劑在廣泛推廣上的應用價值。 在實驗設計上，我們自製無患子萃取液，並選擇日常生活中常見的大腸桿菌及金黃色葡 萄球菌，並然後使菌液與適量的無患子萃取液混合，計算菌液在加入無患子之前、後的細菌 個數，藉以了解無患子的殺菌力。我們發現無患子萃取液對大腸桿菌及金黃色葡萄菌有明顯 的殺菌力，且無患子濃度愈高、作用時間愈長，殺菌力愈好。

The objectives of this investigation were to evaluate the growth conditions and the antioxidant activities of fermented black bean soy milk(BBSM) with Bifidobacterium longum B6 and 15708 cultured in four media, namely, ( BBSM ( 100%)+ 1% glucose ), ( BBSM (100%)), ( BBSM (50%) + milk (50%)), (milk (100%)) . These results indicated that; (1) both strains attained viable cell numbers about 7.19~9.53 log CFU/ml after 18 hrs of incubation and were in the order of ( milk (100%))＞( BBSM (50%) + milk (50%))＞ ( BBSM (100%) + 1% glucose)＞( BBSM (100%)), (2) both strains in ( BBSM (100%)) exhibited higher pH value ranging from 4.79 to 5.50 , but lower titratable acidity(%) ranging from 0.27% to 0.61% than the three other media after 48h of fermentation, (3) both strains displayed an even smaller tolerance to simulated gastric juice at pH = 2.0 for 3h, especially in ( BBSM(100%)), while in simulated gastric juice at pH =3.0 for 3h had higher tolerance , (4) both strains had high resistance ranging from 72.51% to 92.62% to 0.3% bile solution for 12h, (5) the reducing power of ( BBSM (100%)) was more excellent than that of ( milk (100%)), (6) the scavenging effect of yogurt (BBSM ( 100%) + 1% glucose) on DPPH radicals was significantly higher than that of ( milk (100%)), (7) In general, at ten- fold dilution the chelating effect on copper ions of these four un-fermented media except ( milk (100%)) was significantly higher than that of fermented media with B.longum B6 or 15708. 本研究是探討雙叉桿菌(Bifidobacterium longum B6及15708)在四種發酵基質(【黑豆奶(100%)+1%葡萄糖】、【黑豆奶(100%)】、【黑豆奶(50%)+牛奶(50%)】、【牛奶(100%)】)中的生長情形及抗氧化活性。結果顯示： (一) 兩株菌在四種培養基中的生長菌數大小順序如下：【牛奶(100%)】＞【黑豆奶(50%)+牛奶(50%)】＞【黑豆奶(100%)+1%葡萄糖】＞【黑豆奶(100%)】。 (二) 兩株菌在【黑豆奶(100%)】的pH值比較高於其他三種優酪乳，而最終發酵可滴定酸度比較低於其他三種優酪乳。(三) 兩株菌於pH2.0環境下，在【黑豆奶(100%)】優酪乳中耐酸性很低，而於pH3.0環境下卻有很高的耐酸性。(四) 兩株菌對0.3%膽鹽之耐受性均很高為72.52%~92.62%。(五) 在稀釋10倍的四種基質中，不論發酵前或發酵後的還原力皆以【黑豆奶(100%)】為最高，【牛奶(100%)】為最低。(六) 在濃度稀釋10倍時，【黑豆奶(100%)+1%葡萄糖】對DPPH‧自由基清除率明顯比【牛奶(100%)】高。(七) 在濃度稀釋10倍的四種優酪乳中，除【牛奶(100%)】外，發酵後比未發酵的銅離子螯合率明顯降低。