培養條件對Haloferax mediterranei生產Poly-β-hydroxybutyrate的影響之探討

Abstract

本研究係利用Haloferax mediterranei菌株進行250 ml錐形瓶及5 L發酵槽發酵實驗，探討培養基組成以及發酵條件對菌體生長及PHA累積量的影響以尋找大量生產PHA之發酵控制策略。實驗結果發現，鹽類濃度對菌體生長及PHA累積有很大的影響，隨著NaCl、MgSO4、MgCl2濃度增高，菌量也隨之提高，但PHA的累積量卻越低。對於氮源的利用，Haloferax mediterranei對yeast extract利用效果會比利用無機氮有效，並限氮條件並不能促進PHA累積。 在pH影響方面，pH控制在6.5時，菌體生長狀況較佳。在溫度影響方面，當溫度由37 ℃提升到52 ℃時，PHA累積量及PHA含量從0.06 g/L 及0.88 %提升到0.64 g/L及16.6 %，因此提高溫度可加速H. mediterranei累積PHAs。然而，溫度的提升會加速菌體生長及PHA之累積，但對菌量有相當的抑制；在溫度37 ℃時，菌量可達8.8 g/L，溫度在52 ℃時，菌量達5.3 g/L。為使菌體生長不受限制以利於之後大量生產PHA，提出一兩階段溫度控制模式；第一階段控制在37 ℃使菌量大量生長，並在對數生長期中後期在提高溫度至52 ℃，使PHA迅速累積。結果顯示，在此溫度控制策略下，菌量、PHA累積量及PHA含量分別為12.4 g/L、2.44 g/L及20.4 %比溫度控制在52 ℃的結果8.3 g/L、1.14 g/L及15.5 %來的高，因此此溫度控制策略的確可在不抑制菌體生長下，增加PHA的累積量。另外，pH stat批次饋料實驗中，菌量及PHA累積量都有提升，最高分別可達23.23 g/L及4.66 g/L。

Effects of various substrates and fermentation conditions, cell growth and accumulation of polyhydroxyalkanoates (PHAs) were investigated using 250 ml shake flasks and a 5 L fermentor. The strain used was Haloferax mediterranei. It was found that the higher the concentrations of NaCl , MgSO4 and MgCl2 , the faster the cell growth and the lower the amount of PHA produced. The cells grew better by using yeast extract as nitrogen source compared to the inorganic nitrogen sources, and PHA accumulation didn’t enhance under nitrogen limitation. The cells grew fastest at pH 6.5. The effect of temperature on cell growth and PHA accumulation was remarkable. PHA production can be stimulated by raising the temperature. The amount of PHA accumulated and PHA content obtained were 0.64 g/L and 16.6 % dry weight, respectively at 52 ℃ which was higher than the 0.06 g/L and 0.88 % obtained at 37 ℃. However, cell mass concentration was lowered as temperature increased. Cell mass concentration at 37 ℃ and 52 ℃ were 8.8 g/L and 5.3 g/L, respectively. To alleviate suppression of cell growth and increase the amount of PHA produced, we proposed a two-step temperature control strategy. At first stage, temperature was controlled at 37 ℃ to enhance cell mass proliferation, and then adjusted to 52 ℃ in the second stage to stimulate the accumulation of PHA. It was found that this strategy was effective to improve the amount of PHA accumulation without suppression of cell growth. Cell mass concentration, amount of PHA produced and PHA content were 12.4 g/L, 2.44 g/L and 20.4 % dry weight, respectively by using this strategy. The results obtained were higher than those under temperature controlled at 52 ℃ throughout. Besides, cell concentration and amount of PHA accumulation can be improved by pH-stat fed-batch operation. The results were 23.23 g/L and 4.66 g/L, respectively.