屬於紅細菌科(Rhodobacteraceae)且具去硝化(denitrification)能力的假弧菌屬(Pseudovibrio)細菌被認為廣泛分佈在各種海洋環境中，例如海水、海洋沉積物和海洋無脊椎動物。此屬菌重複地由海洋無脊椎生物中分離出，被發現許多能生產具有生物活性的二次代謝物。然而，其多樣性與抗菌活性仍大多未被研究。本研究中，吾人採集了台灣附近沿海及國立海洋生物博物館水族實驗中心戶外池的沉積物樣本，透過培養方法分離假弧菌屬細菌。吾人總共由海洋沉積物樣本中分離出1200株細菌，其中226株(18.83%)可能具有去硝化能力。根據16S rDNA基因序列，其中1株(分離自龜山島)與169株分離株分別被確定為假弧菌屬細菌與紅細菌科細菌，但是這170株細菌均未顯示抗微生物活性。使用假弧菌屬專一性引子對所有海洋沉積物樣本抽取的細菌DNA進行PCR擴增，吾人重複確認假弧菌屬細菌僅存在龜山島樣本中。16S rDNA基因之親緣關係分析顯示紅細菌科分離株包含Rugeria屬(142株)、Labrenzia屬(10)、Thalassobius屬(3)與Shimia屬(3)。
為了解與珊瑚伴生的假弧菌屬細菌多樣性，本研究還分析了國立海洋生物博物館水族實驗中心七個軟珊瑚樣本，使用不培養方法與假弧菌屬專一引子，發現四種珊瑚Sinularia sandensis、Briareum excavatum、Sarcophytum sp.與Sinularia brassica含有假弧菌屬細菌。使用基因選殖方法總共獲得74條假弧菌屬序列，親緣關係分析顯示其為Pseudovibrio denitrificans與Pseudovibrio ascidiaceicola。
研究結果顯示，假弧菌屬細菌在海洋沉積物中並不廣泛存在，這與以前的報告不同。但海洋無脊椎動物如珊瑚卻被發現含有此屬菌，它們或許能成為具生物活性二次代謝物的假弧菌屬菌株獨特來源。

Denitrifying bacteria of the genus Pseudovibrio, a member of the family Rhodobacteraceae, are considered to be widely distributed in various marine habitats such as seawater, marine sediments, and marine invertebrates. In addition, they are found to be repeatedly isolated from marine invertebrates and many can produce secondary metabolites with antimicrobial activity against a broad-spectrum of pathogens. However, their diversity and antimicrobial activity are largely unexplored. In the present study, nine marine sediment samples collected from the coastal area near Taiwan and the outdoor pool of National Museum of Marine Biology and Aquarium (NMMBA) were used to screen for Pseudovibrio strains by culture-based method. A total of 1200 bacterial strains were isolated from marine sediments, among which 226 (18.83%) strains displayed possibly denitrifying activity and within which one (isolated from Kueishan Island) and 169 isolates were confirmed to be Pseudovibrio and Rhodobacteraceae, respectively, based on 16S rRNA gene sequences. However, none of the 170 strains showed antimicrobial activity. Using PCR amplification of bacterial DNA extracted from all marine sediment samples with Pseudovibrio-specific primers, we reconfirmed that Pseudovibrio only existed in Kueishan Island. Phylogenetic analysis based on 16S rRNA genes suggested Rhodobacteraceae isolates belong to genera Rugeria (142 isolates), Labrenzia (10), Thalassobius (3), and Shimia (3).
To understand the diversity of Pseudovibrio associated with coral, we investigate the Pseudovibrio communities of seven soft coral samples collected from the husbandry center, NMMBA. Using culture-independent method and Pseudovibrio-specific primers, Pseudovibrio were detected in four coral species, namely Sinularia sandensis, Briareum excavatum, Sarcophytum sp., and Sinularia brassica. A total of 74 Pseudovibrio clones identified from coral cloning library were sequenced. Phylogenetic analysis showed they belong to Pseudovibrio denitrificans and Pseudovibrio ascidiaceicola.
Our results demonstrate that Pseudovibrio strains do not widely exist in marine sediments, different from previous reports. However, marine invertebrates such as corals are a unique source of diverse Pseudovibrio which may be use as a source of secondary metabolites with biological activities.

第一章 緒論 1
第一節 前言 1
第二節 海洋微生物與其二次代謝物 1
第三節 假弧菌屬簡介 2
第四節 假弧菌屬菌分離與其二次代謝物 3
第五節 紅細菌科Rhodobacteraceae與其分類簡介 5
第六節 細菌之16S rRNA基因鑑定(identification)與親緣關係(phylogenetic)分析 7
第七節 不培養(culture- independent)方法分析環境中微生物 8
第八節 研究動機與目的 9
第二章 實驗材料與方法 11
第一節 藥品、器材及培養基 11
第二節 樣本採集、菌株分離與培養 18
第三節 16S rDNA 序列鑑定 20
第四節 菌株抗菌活性測試實驗 23
第五節 不可培養方法分析海洋沉積物與珊瑚所含假弧菌屬菌 24
第六節 序列生物資訊與親緣關係分析 29
第三章 結果 31
第一節 樣本採集與鑑種 31
第二節 海洋沉積物樣本選擇性培養基培養與篩選 33
第三節 分離株16S rDNA序列鑑定 38
第四節 分離株抗菌活性測試 46
第五節 以不培養方法分析海洋沉積物樣本中所含假弧菌屬細菌 46
第六節 以不培養方法分析珊瑚樣本 49
第四章 討論 57
第一節 不同環境、類型的樣本與假弧菌屬細菌 57
第二節 以相同培養基比較沉積物與海綿菌數 57
第三節 具去硝化能力的細菌 59
第四節 假弧菌屬細菌在海洋沉積物中的分布 59
第五節 紅細菌科細菌在海洋沉積物中的分布 60
第六節 抗微生物活性結果 60
第七節 珊瑚與假弧菌屬細菌 61
第八節 16S rDNA親緣關係分析 62
第五章 結論與建議 65
參考文獻 67
附錄 77

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