可促進植物生長之光合菌Rhodopseudomonas palustris PS3的基因體分析與培養條件優化

Abstract

沼澤紅假單胞菌 (Rhodopseudomonas palustris) PS3菌株是一株由台灣水稻田土壤所篩選出的紫色非含硫光合菌。在先前研究中已證明該菌株具有促進各種作物生長的能力，並且可以提升植物氮肥使用效率。本論文的第一部份是從微生物全基因體的角度來探討光合菌與促進植物生長的關係。藉由與另一株不具促進植物生長功能之YSC3光合菌 (Rhodopseudomonas palustris)做比較分析，依基因構造、微生物生理反應以及基因表現的結果找出PS3菌株可能參與促進植物生長的相關基因。本研究結合了短片段與長片段定序技術，並完成序列組裝與註解後，分別得到PS3與YSC3菌株的基因體全長為527萬與537萬鹼基對，含有4,799以及4,907編碼序列。PS3與YSC3菌株有極高的序列相似度 （約95.11%），而且大部分的基因群的組成與排列方式類似，都有固氮、溶磷、吲哚乙酸合成、氨基環丙烷羧酸脫胺酶等代表性的植物生長促進相關基因群。雖然PS3與YSC3菌株的生長速率沒有差異，在微生物生理試驗結果發現，添加植物的根分泌液至兩株菌的培養液中會促使PS3菌株的生物膜生成量以及化學趨向性的相關基因表現量都較YSC3菌株高。這意味著PS3菌株對於植物的反應可能是其促進植物生長的重要關鍵。 PS3光合菌是具有產業化潛力的菌株，本論文的第二部分藉由數學統計模式探討最適化發酵條件，找出高產量與低成本的生產方式。在本研究中評估了各種較低成本的基質，包括了工業發酵常用的碳氮源以及農產加工副產物等作為培養基配方的可行性，並藉由回應曲面法優化培養條件。實驗結果顯示，當以39.41 mL/L的玉米浸漬液加上32.35 g/L的蔗糖糖蜜作為氮源和碳源，利用5公升桌上型發酵槽於38°C，pH 7以及溶氧濃度30%的條件下進行24小時培養，得到 PS3菌株的最大生物量約為2.18 ± 0.01 g/L。該新配方所生產的PS3發酵液產量約為使用傳統光合菌培養條件的8倍，成本卻只需原先的30%，而且在植物盆栽試驗上證實可以促進作物生長。 在本研究中所建立的基因體資訊，可作為探討光合菌與植物間的交互作用以及促進生長機制的研究平台。此外，新開發的培養基配方是以農產加工副產物作為光合菌的主要營養基質，不僅可有效降低生產成本，也促進了農業資源的加值與循環再利用。

Rhodopseudomonas palustris PS3 is one of purple non-sulfur phototrophic bacteria that was isolated from Taiwanese paddy soils. It has been demonstrated that PS3 can promote plant growth and increase the agronomic nitrogen use efficiency of the host plant. The first part of this dissertation focuses on the elucidation of the relationship between the phototrophic bacteria and plant growth-promotion from the view of whole genomes of microorganisms. I conducted comparative analyses of genomic structures, physiological responses of microbes, and gene expression profiles with those of an ineffective R. palustris YSC3 strain. Based on the differential data, many putative genes that were associated with known plant growth promotional traits were identified. In this study, Illumina short-reads and PacBio long-reads technologies were integrated and assembled to obtain the whole genome sequences. PS3 and YSC3 individually contains a one circular chromosome with 5.27 and 5.37 Mb bp in size, with 4,799 and 4,907 protein-coding genes, respectively. The PS3 and YSC3 strains are closely related to each other with high identity (95.11%), and have similar genomic structures and compositions. Both strains contain the genes associated with plant growth-promotion, such as nitrogen fixation, phosphate solubilization, indole acetic acid synthesis, 1-aminocyclopropane-1-carboxylate deaminase, etc. Although there was no difference in the growth rate of PS3 and YSC3 strains, both the production of biofilm and the gene expressions of chemotaxis of PS3 were higher than those of YSC3 by the addition of root exudate in culture broth. Since PS3 is an elite strain with commercialization potential, the second part of this dissertation focuses on the optimal fermentation conditions for PS3 through mathematical and statistical models to find out a high-yield and low-cost production strategy. In this study, I evaluated various substrates, including the carbon and nitrogen sources commonly used in industry as well as the agricultural processing by-products. The culture condition was optimized by response surface methodology. The optimum culture condition was found to be at corn steep liquor, 39.41 mL/L; molasses, 32.35 g/L; temperature, 38°C; pH, 7.0; and DO 30%. Under this condition, the maximum yield of PS3 strain was up to 2.18 ± 0.01 g/L, which was approximately 8-fold higher than that with original medium, and the medium cost was approximate 70% reduced. Moreover, the beneficial effect of the new PS3 broth on plant growth was verified by pot experiments. The genomic information established in this study can be used as a research platform to investigate the interaction between phototrophic bacteria and plants as well as the molecular mechanisms of plant growth promotion. In addition, the newly developed medium uses agricultural processing by-products as the main nutrient substrates for phototrophic bacterium growth, which not only effectively reduces production costs, but also promotes the value-added and recycling of agricultural resources.