微生物胺基酸碳同位素之組成型式與生理代謝關係研究

Abstract

胺基酸是生物體內重要的有機物質，在生物體和環境樣品中的組成比例與穩定碳氮同位素成分組合變化，被廣泛應用於生物地球化學和生態學的研究。前人研究顯示使用不同代謝途徑的微生物，其各種胺基酸的碳同位素成分組合，可大致依自營作用、異營作用和醋酸分解作用分群，而各種不同初級生產者的胺基酸之碳同位素成分組合，也可加以區別，並做為環境樣品中有機碳來源的指標。由於現有研究所涵蓋的微生物代謝途徑並不完整，對於使用各種代謝途徑微生物的生物體胺基酸的碳同位素組成型式變化，仍須進一步工作。本研究以環境中重要的厭氧微生物為目標，所選定進行生物體胺基酸碳同位素分析的菌株，皆分離自台灣本地自然環境中，包括使用乳酸或是醋酸作為碳源的硫酸鹽還原菌 (接近Desulfovibrio marrakechensis)、利用甲基類有機碳進行代謝作用的產甲烷菌 (接近Methanolobus profundi)、以及以酵母萃取物作為碳源的元素硫還原菌 (接近Thermococcus acidaminovorans)，這些菌株均與前人文獻曾分析的菌株不同，且使用不同代謝途徑，在生地化循環中均扮演重要角色。 研究結果顯示在大多數的培養組別中，各種胺基酸碳同位素的變化型式呈現相似的結果，表示不同種類微生物之體內進行胺基酸合成的路徑差異並不大，不過不同的物種在進行不同碳源代謝時，其體內酵素所造成的同位素分化程度仍有所差別，所以導致個別胺基酸間的同位素值差異不同。與前述結果不同的是利用醋酸作為碳源的硫酸還原菌培養組別，可以發現leucine、isoleucine以及lysine的碳同位素組成相較於其他培養組別變得較重，而此三種胺基酸的合成與醋酸上的羧基相關，可以推測此一碳同位素組成型式的變化趨勢與使用碳同位素值可能較大的醋酸有關。各種菌株培養若在不同生長階段取樣分析，其胺基酸碳同位素的組成型式與總有機碳同位素值皆沒有明顯改變，表示微生物處於不同生長階段對於胺基酸合成時的同位素分化並無影響。 本研究綜合前人文獻所分析之不同微生物的各種胺基酸碳同位素值與本研究所得的資料進行線性判別分析，結果顯示線性判別分析能夠依據胺基酸碳同位素組成型式，將使用不同代謝途徑的微生物加以分群。未來可將這項技術應用於環境樣品中，作為現地所進行的微生物代謝途徑指標。

Amino acids represent one of the most important categories of biomolecules. Their abundance and isotopic patterns have been broadly used to address issues related to biogeochemistry and ecology. Previous studies have shown that various carbon assimilative pathways of microorganisms (e.g. autotrophy, heterotrophy and acetotrophy) could be distinguished by their carbon isotopic patterns of amino acids. However, the taxonomic and metabolic coverage are limited in previous examination. This study aims to uncover the carbon isotopic patterns of amino acids for microorganisms remaining uncharacterized but bearing biogeochemical and ecological significance in anoxic environments. To fulfill the purpose, three anaerobic strains isolated from riverine wetland and hot spring system in Taiwan have been examined in this study. One strain is a sulfate reducing bacterium (related to Desulfovibrio marrakechensis), which is capable of utilizing either acetate or lactate, the other one is a methanogen (related to Methanolobus profundi), which grows solely with methyl-group compounds, and the third one is a sulfur oxidation bacterium (related to Thermococcus acidaminovorans). The compound specific isotopic analysis (CSIA) of amino acids for carbon isotope (δ13CAA) of these cultures was performed in this study. The pattern of δ13CAA was similar for most cultures and the result might imply the biosynthesis pathways of amino acids are also similar among these strains. However, a different pattern was observed in the culture of sulfate reducing bacterium utilizing acetate as carbon source. The δ13C values of leucine, isoleucine, and lysine were larger than that of other cultures. Since the synthesis pathway of these three amino acids were related to the carboxyl group from acetate, which was provided in substrate as demanded carbon source, the carbon isotopic composition of acetate may determine the δ 13C values of leucine, isoleucine and lysine. Besides, no significant difference of δ13CAA patterns and δ13C values of TOC ware observed for each culture grown in different stages. At last, the microbial δ13CAA values of published data and this study could be well classified into three groups (heterotrophs, autotrophs and acetotrophs) by LDA (linear discriminant analysis). The CSIA of amino acids and statistical techniques could be applied in environmental samples as the index of microbial metabolic pathways.