可降解聚丁烯琥珀酸己二酸酯的菌株篩選及特性探討

Screening of Poly butylene succinate-co-adipate (PBSA) degrading microorganisms and exploring their characteristics

蔡依庭(Yi-Ting Tsai)

指導教授 ： 劉啟德

國立臺灣大學/生物資源暨農學院/生物科技研究所/碩士(2021年)

https://doi.org/10.6342/NTU202101594

摘要

塑膠農膜廣泛應用在農業上用來改善作物生長條件，例如可降低水分、除草劑及肥料的施用量，同時也能控制雜草生長及減少土壤流失。然而，廢棄農膜的回收率低，在收穫後大多就地掩埋在農地中，衍生環境污染問題。近年來基於環保考量，對於生分解農膜的需求持續增加。有別於傳統之聚乙烯（PE）農膜，生分解農膜在收成後可直接翻攪進土壤中被微生物分解。在生分解原料當中，聚丁烯琥珀酸己二酸（PBSA）因具有較佳的機械性及延展性，可生產成為包裝材料及農膜。加上其結晶度及溶融溫度較低，使得PBSA在中溫環境也可被微生物分解。
為了篩選出適合台灣土壤環境之PBSA降解微生物，我們利用透化圈(Clear zone method)快速篩選平台從不同土壤中篩選出可降解PBSA的潛力菌株，並評估其分解效力。有兩株潛力菌株在PBSA固態平板培養基上呈現較大的分解圈，經由分子鑑定結果得知與麴菌屬的Aspergillus terreus及 Aspergillus fumigatus分別有高達99.78%及99.74%之序列相似性。
為了評估其降解能力，將兩菌株分別與PBSA塑膠片培養在無碳源液態培養基中，經過30天後發現A. terreus HC及A. fumigatus L30處理組的塑膠片重量分別下降了42% 與26%。也由此推斷兩株潛力菌株可將PBSA作為其生長所需之碳源。此外也發現於分解30天後添加新鮮培養基能提升兩株菌降解PBSA的效率。為了闡明生物降解過程中聚合物的結構與理化性質變化，我利用SEM進行觀察，並分析其FTIR及NMR圖譜。
為了探討潛力菌株在土壤環境中降解PBSA的特性與機制，我進行了實驗室規模之土壤掩埋分解試驗。 實驗結果發現，A. terreus HC菌株在夏季及冬季土壤樣本中分別呈現90%及75%之PBSA降解效率。接著使用降解後之土壤進行植物毒性試驗及酯酶 (Esterase)活性試驗，發現無論是PBSA 降解過程或是添加潛力菌株至土壤中皆對植物幼苗生長沒有不利的影響。兩株潛力菌株在培養基上均呈現較高的脂肪酶活性，其中A. terreus HC對中長碳鏈基質呈現較高之活性。此外也發現若在分解力較低的土壤中添加A. terreus HC培養液可以提升土壤的酯酶活性，其降解PBSA的效率也獲得顯著提升。本研究不僅增進了我們對於真菌降解PBSA塑膠農膜的了解，也對於提昇中溫土壤環境之生物降解提供了有用的線索。

關鍵字

塑膠農膜 ； 聚丁烯琥珀酸己二酸酯 ； 生物可分解 ； 麴黴屬 ； 脂解酶

並列摘要

Plastic mulch film are widely used for lowering consumption of water, herbicides, and fertilizer and also controlling weeds and minimizing soil erosion, which can improve the conditions for plant growth. However, disposed plastic mulch film with a low recovery rate is mostly buried in land after harvest and causes several adverse environmental impacts. In order to solve this difficult environmental problem, the demand for biodegradable plastic mulch film has been increasing in recent years. Different from the traditional polyethylene (PE) agricultural film, it can be directly tilled into the soil at the end of the season. Poly butylene succinate-co-adipate (PBSA) is a biodegradable polymer with good ductility, which can be applied to packaging and mulch film making. Because of the low crystallinity, PBSA shows lower melting temperature, which can be decomposed by microorganisms without high temperature environment.
In order to screen microbes for degrading PBSA, I used the clear zone method to select elite microbes from cultivated soils in Taiwan and evaluated their degradation abilities. Among the isolated strains, two of them formed relative large clear zones on the PBSA agar plate, indicating they possess better decomposition abilities. According to molecular species identification, these two strains have 99.78% and 99.74% sequence similarity with Aspergillus terreus and Aspergillus fumigatus, respectively. To evaluate the decomposition ability, I inoculated the individual elite strains into a carbon free basal medium with PBSA plastic film. A. terreus HC showed better plastic decomposition ability than that of A. fumigatus L30. The decreased weight of the plastic film degraded by A. terreus HC was ~42% within 30 days, while that of A. fumigatus L30 was 26%. It suggests that the two elite strains can use PBSA as their sole carbon resource. The degradation rates of the two strains were further increased while fresh medium was added into the culture fluid after 30 days of incubation. To clarify the changes in polymer structures and physicochemical properties during biodegradation, I conducted SEM, FTIR and NMR analyses.
For further elucidation of environmental degradation and the underlying mechanism, a soil burial test was carried out on a laboratory scale to examine the biodegradability and the enzymatic activities. A. terreus HC showed over 90% and 75% degrading rates for summer and winter soil environments, respectively. The phytotoxicity assessment and soil esterase activity analysis were conducted for in-depth evaluation of PBSA degradation in soil environment. There was no adverse influence of PBSA degradation or addition of fungal culture to the growth of plant seedlings.
According to the result of in vitro enzyme activity, both elite fungal strains showed high potential for lipase production in the presence of PBSA. Moreover, I noticed that the crude enzymes extracted from A. terreus HC showed high substrate specificity of lipolytic enzyme on medium (C8, C10) and long (≥C12)-chain fatty acids in the presence of PBSA. While high dose of A. terreus HC was added into the soil with PBSA film, the soil esterase activity was further increased.
This study not only advances our understanding of biodegradation of PBSA mulch film by fungi, but also provides hints to improve the efficiency of biodegradation in soil environments with moderate temperature.

並列關鍵字

plastic mulch film ； poly butylene succinate-co-adipate (PBSA) ； biodegradation ； Aspergillus ； lipolytic enzyme

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