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標題: 本土木質纖維分解菌之篩選與其在經超音波前處理稻殼之發酵研究
Isolation of Lignocellulolytic Microorganisms from Soil and its Fermentation with Rice Hull by Ultrasonic Pretreatment
作者: 楊珺堯
Yang, Chun-Yao
關鍵字: Rice hull;稻殼;Ultrasonic pretreatment;Lignocellulose;Xylooligosaccharide;超音波前處理;木質纖維素;木寡醣
出版社: 食品暨應用生物科技學系所
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本研究自土樣中篩選出32株可利用稻殼木質纖維素之菌株,並利用不同酵素活性的測試挑選出活性較高之菌株CY6-1進行後續實驗,此分離菌株CY6-1的四種酵素活性(CMCase, β-glucosidase, Avicelase, Xylanase) 活性皆優於對照組Phanerochaete chrysosporium BCRC36201。此分離菌株CY6-1經鑑定結果歸屬於Aspergillus japonicus var. japonicus。

本研究藉由超音波放射的方式來作為稻殼前處理之步驟,不僅可萃取稻殼中可用之纖維素與半纖維素,也可利用超音波所產生的噴流與局部熱點來震碎及破壞附著於稻殼表面的二氧化矽,此前處理可將稻殼前處理的時間由24小時縮短至1.5小時外,並提升1.4倍之半纖維素產率。所使用的超音波前處理條件為300W、28 kHz於80℃作用1.5小時,並以場發射掃描式電子顯微鏡觀察未處理與經超音波處理之稻殼的微結構變化,發現到經過超音波處理之稻殼表面較未處理之表面較光滑,但若超音波加上12% NaOH處理之稻殼表面則會有皺縮的現象產生。

在酵素活性測試方面,發現在不同稻殼克數、不同超音波前處理方式與不同時間的培養下,酵素活性皆以4 g /50 mL比例之超音波處理的稻殼(USRHM)培養時最佳,不僅有助於延長酵素活性的穩定性,且更可有效地提升β-glucosidase之活性,於培養第28天後可達5.62 U/mL,而xylanase的活性則於培養第10天達到7.37 U/mL。

以稻殼為基質之發酵液在經過28天的培養,其發酵液之組成主要以木四糖、木六糖與一些分子量較大之低聚木糖,其木寡醣產率可以達到1.17 mg/ mL(木四糖與木六糖)。

Rice hull is a common and enormous amount of agricultural waste in Taiwan. It contains an abundant lignocellulosic biomass and can be used as a substrate for microorganism. Developing the methods to utilize rice hull is essential in the field of bio-resources. The aim of this thesis is to explore the methodology of utilizing rice hull by the isolation of cellulolytic and xylanolytic microorganism, pretreatment of rice hull with ultrasonic irradiation, testing the enzyme activities, and analyzing the composition of hydrolysis products of the fermentation broth with the isolate on different culture times for various pretreatment methods.

Microorganism being able to hydrolyze the lignocellulose of rice hull was isolated from soil, and the isolate CY6-1 was selected from 32 strains by enzyme activity tests. Those enzyme activities of the isolate CY6-1 were better than Phanerochaete chrysosporium BCRC36201, including CMCase, β-glucosidase, avicelase, and xylanase. The isolate CY6-1 is identified as Aspergillus japonicus var. japonicus.

Using ultrasonic irradiation as pretreatment, it has been employed to not only extract cellulose and hemicellulose from rice hull, but also remove silica on the surface via liquid jet and hot spots. The pretreatment can shorten the treated time from 24 hours to 1.5 hours, and increase 1.4 fold in yield of hemicellulose. In this work, rice hull was treated at 80℃ for 1.5 hours in 300W and 28 kHz ultrasonic system. And field emission scanning electron microscope (FESEM) was used to observe the structural change of the rice hull before and after the pretreatments. The structure of rice hull treated with ultrasound was smoother than untreated rice hull, and the structure was corroded when using ultrasonic pretreatment in combination with 12% NaOH.

For enzyme activity test, the highest enzyme activity of isolate CY6-1 was observed at using 4 g USRHM/50 mL in different grams of rice hull and culture times by various pretreatments. In this culture condition, it could not only extend the stability of enzyme activity, but also increase β-glucosidase activity better than in other culture conditions. After 28- day culture by isolate CY6-1, β-glucosidase activity could achieve 5.62 U/mL, and the highest xylanase activity was 7.37 U/mL after 10 days of culture.

The fermentation broth of rice hull was cultured with isolate CY6-1in 28 days, and compositions of the broth were xylotetraose, xylohexaose, and higher molecular weight xylooligosaccharide, and the yield of xylotetraose and xylohexaose were 1.17 mg/mL.
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