Scale up performance of polyhydroxybutyrate production from Caldimonas manganoxidans in 3 L fermentor

塑膠帶給我們生活很大的便利性，但塑膠完成它的任務之後該何去何從卻是個需要解決的問題。生物可降解塑膠因其能夠被生物降解以及具有可再生特性，讓它再取代現有塑膠上有很大的發展潛力。屬於生物可降解塑膠之一的聚羥基丁酸酯因其性質與聚丙烯相似而受到高度的關注。在先前的研究中，Caldimonas manganoxidans在PHB生產上的表現被認為具有工業化的潛力，包含高PHB含量以及高的甘油容忍度。因此，本研究使用3公升發酵槽研究規模放大之後的操作參數(溶氧及酸鹼值)對C. manganoxidans PHB生產表現的影響，並利用饋料批次發酵來進一步提升PHB的產量。因為生質柴油工業的蓬勃發展，副產物的甘油豐富且價格低廉，故用來做為本實驗中的碳源以達到降低PHB成本的目的。53%的PHB含量能夠在固定轉速200 RPM下獲得，但因為菌體密度低而只能得到低的PHB濃度(1.9 g/L)。高菌體密度能夠在10%溶氧條件下獲得，但因酸鹼值的快速降低導致PHB的累積受到嚴重的限制，僅有0.48克/升。再進行酸鹼值的控制之後，能夠在酸鹼值7的條件下得到4.3 克/升的PHB，而酸鹼值在提升到8之後因為PhaZ(負責PHB降解的基因)的活躍而造成PHB濃度不如酸鹼值7來的高。相較於批次發酵，使用饋料批次發酵後PHB的濃度提升了140%，生產力達到0.388克/升/小時。PHB無法進一步累積可能是由於培養液中的甘油濃度累積太高而造成抑制現象的發生，並可以透過饋料策略的改良來達到改善的目的。與先前的搖瓶實驗相比，本菌株的產率在放大之後仍然維持在0.17克PHB/克甘油，這在工業應用上是一大優點。從饋料批次發酵中得到的PHB分子量僅160±23 kDa，可能是長時間接觸甘油所導致的結果。把碳源從精製甘油換成粗製甘油後，菌體的生長延遲了12小時但最後的菌體密度能達到與精緻甘油相近的結果(約6.5 克/升)，PHB的表現則有小幅度的下降(約33%)，能夠使用粗製甘油來進行PHB的生產讓本菌株在工業上的應用潛力大幅提升。Petroleum-derived plastics improved people’s lives a lot, but the disposal of petroleum-derived plastics was a difficult issue that was needed to be solved. Biodegradable plastics are excellent material to replace petroleum-derived plastics because they are not only biodegradable but also renewable. In previous study, performance of Caldimonas manganoxidans was proof to be potential for industrial application, including high PHB content and high glycerol tolerance. Therefore, the effect of operation parameters after scaling up was investigated in this study, including dissolved oxygen and pH value. Glycerol was used as carbon source because it was cheap and abundant as a by-product of bio-diesel industry. High PHB content could be obtained under constant 200 rpm agitation speed, but low cell density brought out low PHB concentration which was 1.9 g/L. high biomass could be obtained with 10% dissolved oxygen, but the rapid decrease of pH seriously restricted the accumulation of PHB which was 0.43 g/L only. With the control of pH, the PHB concertation could increase from 0.48 g/L under no pH control to 4.3 g/L under pH 7. Lower PHB concentration under pH 8 was caused by the increased activity of PhaZ. Further improvement in PHB concentration was obtain with pH-stat fed-batch fermentation. The PHB concentration increased by 140% with the productivity of 0.388 g/L/h. Stopping of PHB accumulation may be inhibited by the increased glycerol concentration in medium and this could be improved by the adjustment of feeding strategy. Compared to the previous shake flask experiment, the yield of PHB remained unchanged after scaling up, which was an advantage for industrial application. The MW of PHB obtained by fed-batch fermentation was only 160±23, which was possibly caused by the long exposure to glycerol. The PHB production from bio-diesel derived glycerol (raw glycerol) was examined. 12 hours delayed growth was observed and possibly caused by the impurity of raw glycerol. The ability of producing PHB from raw glycerol is another huge advantage for industrial application.摘要 i Abstract ii Contents iv Table contents v Figure contents vi Chapter 1 Motivation 1 Chapter 2 Introduction 2 2.1 Background 2 2.2 Biodegradable plastic 2 2.3 Thermophilic PHA producer 5 2.4 Fed-batch fermentation 7 Chapter 3 Experimental 10 3.1 Chemicals 10 3.2 Equipment 10 3.3 Strain, cultivation medium and condition 11 3.4 Preparation of bio-diesel-derived glycerol 12 3.5 pH-stat fed-batch fermentation 12 3.6 Analytical method 12 Chapter 4 Results 14 4.1 Identification the performance of PHB production after scaling up 14 4.2 Effect of pH on the performance of PHB production 14 4.3 Effect of DO on the performance of PHB production 15 4.4 pH-stat fed-batch fermentation 16 4.5 Molecular weight of PHB at different fermentation condition 17 4.6 Batch PHB production using bio-diesel-derived glycerol 17 Chapter 5 Discussion 19 Chapter 6 Future perspective 24 Reference 37 Appendix 4