微藻類於微型生物反應器之培養及分析

Cultivation and Analysis of Microalgae in Microscale Bioreactor

王群彥(Chun-Yen Wang)

指導教授 ： 王翔郁

國立清華大學/原子科學院/工程與系統科學系/碩士(2017年)

摘要

本研究將柵藻在不同培養條件下於微型生物反應器內進行連續式培養，並利用微量盤分光光譜儀對培養在微型生物反應器內的柵藻進行掃描，以光學密度（Optical Density, O.D.）在波長為440 nm及682 nm的吸收度結果以及色素萃取定量來探討微藻於微型生物反應器培養的反應變化。
一般傳統對微藻培養條件最適化往往耗時而步驟複雜，並且測試微藻所需的樣品量較大，再加上微藻測試樣品需經傳統實驗室培養方式數天後才可使用，因此本研究使用微型生物反應器進行柵藻的培養，並可同時進行色素以及細胞素（葉綠素）的O.D.掃描及分析。實驗首先證明微型生物反應器可適用於微藻掃描分析，我們利用不同濃度的柵藻做為測試樣品，比較UV分光光度計和微量盤分光光譜儀的O.D.吸收值。結果顯示兩台儀器測出來的吸收值呈現良好的回歸性（R2>0.98），代表微型生物反應器可適用於檢測微藻的分析，並可利用O.D.對色素及細胞數的檢量線進行推算，以比較微型生物反應器與傳統實驗生物培養方式內微藻的細胞數及色素含量。
本研究接著施加不同培養條件來觀察柵藻在微型生物反應器內色素及細胞數的變化情形，在不同碳源和不同氮源的培養實驗中分別加入10 g/L的葡萄糖及蔗糖作為不同碳源並分別加入1.25 g/L的硝酸鉀、0.21 g/L的氨及0.74 g/L的尿素作為不同氮源進行實驗，結果發現蔗糖及氨為最佳的碳氮源組合，經過96小時的培養後，其生物量可達到2.126 g/L，培養48小時後的類胡蘿蔔色素含量為1.014 mg/L。在不同酸鹼值的培養實驗中，設置pH=5.5、pH=7及pH=8.5三個組別進行實驗，結果顯示pH=7組別為最佳的培養組別，比較培養96小時後的生物量，pH=5.5以及pH=8.5均少了約7%的生物量；比較培養48小時後的類胡蘿蔔色素含量，pH=5.5和pH=8.5各自下降了22%和33%的類胡蘿蔔色素含量。在鹽度壓力的培養實驗中，0.2 M的氯化鈉伴隨在培養液中作為鹽度壓力，和相同培養條件無鹽度壓力的組別相比，鹽度壓力使培養96小時後的生物量下降了14.5%，培養48小時候的類胡蘿蔔色素下降29%。在不同光源的培養實驗中，選定了紅、藍、綠、黃、白色LED燈作為不同光源進行實驗，結果顯示綠光為生物量的最佳培養光源，其培養96小時後的生物量可達到2.412 g/L；而紅光則為累積類胡蘿蔔色素的最佳光源，其培養48小時後的類胡蘿蔔色素可達0.2185 mg/L。
以上實驗結果我們可以證實微藻可在微型生物反應器中培養，並透過微量盤分光光度儀利用O.D.值進行對微藻色素及細胞數的掃瞄。不僅可以提供快速及少量樣品的檢測方法，而且可同時間進行多種培養條件的篩選。此外以蔗糖作為碳源、氨作為氮源並以綠光LED作為光源為柵藻的最適化培養條件，相較於通常實驗室使用的葡萄糖作為碳源、硝酸鉀作為氮源並以白光作為光源的培養清況，其培養96小時後的生物量可達2.009倍，培養48小時後的類胡蘿蔔色素含量可達3.625倍。

關鍵字

微藻培養 ； 微流體裝置 ； 氮源 ； 酸鹼值 ； 鹽度壓力 ； 不同光源

並列摘要

This study presents the cultivation and monitoring of Scenedesmus abundans GH-D11 on a microbioreactor, which is compatible to commercially and readily available plate reader. The properties of cell, including microalgae cell density (O.D.682) and carotenoids (O.D.440), were determined by absorbance read by a plate reader during cultivation. Conventional screening methods for optimization of microalgae culture are time-consuming and complicated. Therefor, this research aims for developing prompt culture and rapid quantification of microalgae cellular contents using microbioreactor.
In this study, the effects of nitrogen resource, pH, salinity stress and spectral light on microalgae cultivation are investigated. The results show microalgae were cultivated better, indicated by more carotenoids and cell density, by applying suitable nitrogen source, adjusting a suitable pH value and using suitable light source. Scenedesmus abundans had high cell density (biomass = 2.126 g/L after 96 hour of cultivation) and carotenoids (total carotenoids = 1.014 mg/L) with sucrose as carbon source and ammonia as nitrogen source. The suitable pH value is 7 for Scenedesmus abundas cultivation and any shift of pH value may decrease the cell density and carotenoids. Salinity stress also decreased the cell density and carotenoids.The cultivation with salinity stress had the low cell density (biomass = 2.087 g/L after 96 hour of cultivation) and carotenoids (total carotenoids = 0.3580 mg/L) compared to the cultivation without salinity stress (biomass = 2.437 g/L after 96 hour of cultivation, total carotenoids = 0.504 mg/L). From the experimental results of effects of spectral light on microalgae cultivation, green LED exhibited the highest cell density (biomass = 2.412 g/L after 96 hour of cultivation) and red LED exhibited the highest carotenoids (total carotenoids = 0.2185 mg/L).In summary, our microbioreactor provides not only fast and convenient way to determining microalgae cellular contents but also considerably decreases the amount of microalgae cell culture size for screening cultivation parameters.

並列關鍵字

microalgae cultivation ； microfluidic bioreactor ； nitrogen source ； pH effect ； salinity stress ； spectral light

參考文獻

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