台灣本土根瘤菌Wautersia taiwanensis 184生產聚羥基丁酸醱酵策略之探討

A feasible fermentation strategy to enhance poly (3-ydroxybutyrates) production from an indigenous isolate Wautersia taiwanensis 184 strain

黃晉貴(Chin-Kuei Huang)

指導教授 ： 魏毓宏

元智大學/工程學院/生物科技暨生物資訊研究所/碩士(2005年)

摘要

本研究主要目的係探討並比較一系列Wautersia taiwanensis 184、185、186、187、204、208、209及Pseudomonas oleovoransa ATCC 10951等菌株經醱酵生產之生物可分解塑膠－聚羥基烷酸鹽(polyhydroxylalkanoates, PHA)之產能，藉以評估並決定最佳之PHA生產菌。並且針對其培養基的最適化，醱酵參數的最適化和兩階段醱酵策略應用對PHA量產做一系列之探討。首先本研究以Sudan Black B染色法，初步判斷細菌體內的脂類含量，藉以判斷PHA醱酵生產之潛力，研究顯示，眾多測試菌株中以Wautersia　taiwanensis系列菌株其菌體內含較多的脂類，尤其以Wautersia taiwanensis184脂質含量最高，顯示W. taiwanensis 184可能為較佳之PHA生產菌。以W. Taiwanensis 184醱酵生產PHs製程中，以葡萄糖酸為碳源和氯化銨為氮源，並且碳氮莫耳比為8時，在溫度30oC，200 rpm和pH 7條件下進行polyhydroxybutyrate（P(3HB)）之醱酵，可得到最佳菌體乾重及P(3HB)濃度，分別達4.15 g/L與2.44 g/L。搖瓶醱酵生產以兩階段醱酵策略，配合在第二階段限制培養基中Mg營養源將其濃度減半，可得到菌體乾重為10 g/L，P(3HB)濃度為5.22 g/L。以醱酵槽試產方面，當C/N比為8/1(mol/mol)時，在30oC、200 rpm和pH 7條件下進行P(3HB)之醱酵，可得到菌體乾重為7 g/L，P(3HB)濃度為3.6 g/L。於五公升醱酵槽生產製程，以兩階段醱酵策略，配合在第二階段限制培養基中Mg和氮營養源將其濃度減半，可得到菌體乾重為10 g/L，P(3HB)濃度為7 g/L。

關鍵字

Wautersia taiwanensis ； PHA ； P(3HB) ； Sudan Black B染色法 ； 兩階段醱酵策略

並列摘要

This study aimed to screen better production strains in order to obtain more efficient production of poly (3-hydroxybutyrate)(P(3HB)) from Wautersia taiwanensis 184, 185, 186, 187, 204, 208 and 209. This work shows that the best strain for P(3HB) production was Wautersia taiwanensis 184, based on the Sudan Black B staining and GC analysis. While the W. taiwanensis 184 strain grew on medium with a carbon to nitrogen molar ratio of 8 at 30oC and 200 rpm allowed the highest P(3HB) production. The carbon source and nitrogen source were gluconic acid and ammonium chlorine, respectively. Meanwhile, the best pH for P(3HB) production was also determined by observing the P(3HB) production under a culture pH of 6.0, 7.0 and 8.0. It turns out that pH 7.0 gave the highest P(3HB) production of 4.15 mg/L. The optimal culture condition (30oC, pH 7.0 and 200 rpm agitation) was used for the rest of experiments. Furthermore, two-stage fermentation strategies were carry out to investigate the effect of carbon source concentration on specific growth rate and the characteristics of P(3HB) produced by Wautersia taiwanensis 184. This study shows that the limiting condition was created by using 50% of Mg concentration at stage 2, at which a cell dry weight (CDW) and P(3HB) concentration of 10 g/L and 5.22 g/L were obtained, respectively. Moreover, in a 5L fermentor while the concentration of nitrogen source and Mg was half of stage 2, the cell dry weight (CDW) and P(3HB) concentration of 10 g/L and 7 g/L were obtained, respectively.

並列關鍵字

Wautersia taiwanensis ； PHA ； P(3HB) ； Sudan Black B staining ； Two stages fermentation strategy