凤眼莲秸秆生物脱胶的研究
摘要
凤眼莲(Eichhornia crassipes)在水体中的泛滥严重影响到了水生生态系统的生物多样性,并对人们的生产、生活、健康造成威胁,已被列为危害最严重的十大害草之一。如何对凤眼莲进行防治和资源化利用,已经越来越引起人们的关注。与其它植物秸秆类似,凤眼莲化学成分主要由纤维素、半纤维素和木质素组成。纤维素是自然界最重要的生物质资源之一。利用微生物手段分解掉凤眼莲成分中的非纤维素物质,即生物脱胶,得到较高纯度的纤维素,为纤维素的后续利用奠定基础,这将成为凤眼莲资源化利用新途径的重要内容。本课题从以下几个方面对凤眼莲秸秆生物脱胶进行了研究。
1.凤眼莲化学成分含量的测定
采用《苎麻化学成分含量测定方法GB5889-86》测定凤眼莲的化学成分表明,凤眼莲不同部位的各种化学成分含量不同。其中,凤眼莲叶中的腊脂质含量最高,果胶含量最低;茎的腊脂质含量最低,水溶物含量也最低;半纤维素在凤眼莲根中的含量最低,在叶中的含量最高,为52.94%;叶中的木质素含量最低,仅为12.65%;凤眼莲茎中的纤维素含量最高,为17.23%。
2.凤眼莲脱胶菌的筛选和脱胶实验
利用刚果红平板染色法从凤眼莲自身茎叶及其生长的池塘、南湖水中筛选出5株透明圈较大的菌株(A1、B1、B11、B3、B13),通过比较菌株分泌的果胶酶、木聚糖酶活性和脱胶率,确定A1菌为理想的凤眼莲脱胶菌。按接种量4%,在35℃,pH7.0,145 r/min振荡速度下发酵72h后,木聚糖酶和果胶酶活分别为72.69μg/mL·min,45.72μg/mL·min,脱胶率为31.89%。
3.不同处理方法得到的凤眼莲纤维的红外光谱分析
通过比较凤眼莲原料纤维、A1菌处理得到的纤维、碱化纤维这三种凤眼莲纤维的红外光谱后发现,经过A1菌发酵处理或碱化处理后,样品中的半纤维素和木质素成分与原料样品相比,大部分被降解掉或去除掉。碱化处理对于去除凤眼莲胶质(非纤维素物质)效果最为明显,但同时对凤眼莲纤维素的破坏也最大;A1菌处理凤眼莲茎纤维的结构变化介于原料纤维和碱化纤维之间。
4.A1菌最佳脱胶条件的选择
以凤眼莲作为脱胶培养基的碳源,根据不同的理化因子(包括氮源种类以及浓度、无机盐、温度、pH、接种量等),选择最佳发酵工艺。结果表明,凤眼莲脱胶菌A1的最佳脱胶培养基为:NH_4Cl 0.1%,KH_2PO_4 0.1%,MgSO_4·7H_2O 0.05%,pH7.5,接种量0.8%,35℃培养72h后,凤眼莲脱胶菌分泌的木聚糖酶活性达到75.05μg/mL·min,果胶酶活性达到63.51μg/mL·min,一次脱胶率达到33.37%。
The flooding of Eichhornia crassipes (E. crassipes) in water body had broken the biodiversity of aquatic ecosystem and threaten the human production, living and health. E. crassipes had been list as one of the ten kinds of harmful grass. How to control and utilize it as a kind of resource is attracting more and more attention. Similar to other plants, cellulose, hemicellulose and lignin are the main chemical constituents of this plant. Cellulose is the one of the main biomass resources. Microbial method can be used to remove noncellulose (called degumming) of this plant, to obtain high content cellulose, for further utilization of cellulose, it is important components for the new utilization approaches of E. crassipes. This paper on the Eichhornia crassipes's biological degumming included aspects as following.
1. Determination of chemical composition contents of E. crassipes
The determination of chemical composition contents of E. crassipes was conducted according to The Determination Method of Chemical Composition of Ramie GB5889-86. The results showed that the contents of different components are different in different parts of this plant. In the leaves, the content of lipid is the highest, pectin and lignin are all the lowest. In the stems, the contents of lipid and water soluble substances are all the lowest; the content of hemicellulose is the lowest in the roots, but highest in the leaves, being 52.94%. The lignin content in leaves, 12.65%, is the lowest. The cellulose content in stems is highest, being 17.23%.
2. Screening of degumming strains of E. crassipes and degumming test
Using congo red-broth agar plate to select five strains from the leaves and stems of plant, the ponds that E. crassipes grow near Lake Nanhu of Wuhan City. Through comparison of the activity of pectin and xylan, the degumming rate among these five strains, Al strain was determined as the ideal degumming strain of the E. crassipes. Under the condition of inoculation amount 4%, 35℃, pH 7.0, agitation speed 145 r/min, cultivating for 72 hours, the activity of pectin and xylan were 72.69μg/mL·min and 45.72μg/mL·min respectively, the degumming rate was 31.89%.
3. FTIR analysis of the celluloses obtained by different methods
The FTIR spectrum of raw fiber of E. crassipes, fiber treated by Al strain and alkali treated fiber were compared, the results indicated, the hemicellulose and lignin were mostly decomposed by two ways - treatment by A1 strain and alkalization by NaOH. The degumming efficiency of alkalization was most obvious, but the damage to cellulose was also largest. The structure change of plant treated by Al strain lied between that treated by alkali and raw material.
4. Single factor screening experiments for optimum fermentation conditions of Al strain
The screening experiments for optimum fermentation conditions of Al strain were conducted, using the plant as carbon source. The different factors for screening included form and concentration of nitrogen and, inorganic salt, temperature, and inoculation amount. The results indicated that the optimum degumming medium of Al strain was that NH_4Cl 0.1%, KH_2PO_4 0.1%, MgSO_4·7H_2O 0.05% and pH 7.5. After cultivating for 72 hours, the activity of pectin and xylan were75.05μg/mL·min and 63.51μg/mL·min, respectively, the degumming rate was 33.37%.
1.凤眼莲化学成分含量的测定
采用《苎麻化学成分含量测定方法GB5889-86》测定凤眼莲的化学成分表明,凤眼莲不同部位的各种化学成分含量不同。其中,凤眼莲叶中的腊脂质含量最高,果胶含量最低;茎的腊脂质含量最低,水溶物含量也最低;半纤维素在凤眼莲根中的含量最低,在叶中的含量最高,为52.94%;叶中的木质素含量最低,仅为12.65%;凤眼莲茎中的纤维素含量最高,为17.23%。
2.凤眼莲脱胶菌的筛选和脱胶实验
利用刚果红平板染色法从凤眼莲自身茎叶及其生长的池塘、南湖水中筛选出5株透明圈较大的菌株(A1、B1、B11、B3、B13),通过比较菌株分泌的果胶酶、木聚糖酶活性和脱胶率,确定A1菌为理想的凤眼莲脱胶菌。按接种量4%,在35℃,pH7.0,145 r/min振荡速度下发酵72h后,木聚糖酶和果胶酶活分别为72.69μg/mL·min,45.72μg/mL·min,脱胶率为31.89%。
3.不同处理方法得到的凤眼莲纤维的红外光谱分析
通过比较凤眼莲原料纤维、A1菌处理得到的纤维、碱化纤维这三种凤眼莲纤维的红外光谱后发现,经过A1菌发酵处理或碱化处理后,样品中的半纤维素和木质素成分与原料样品相比,大部分被降解掉或去除掉。碱化处理对于去除凤眼莲胶质(非纤维素物质)效果最为明显,但同时对凤眼莲纤维素的破坏也最大;A1菌处理凤眼莲茎纤维的结构变化介于原料纤维和碱化纤维之间。
4.A1菌最佳脱胶条件的选择
以凤眼莲作为脱胶培养基的碳源,根据不同的理化因子(包括氮源种类以及浓度、无机盐、温度、pH、接种量等),选择最佳发酵工艺。结果表明,凤眼莲脱胶菌A1的最佳脱胶培养基为:NH_4Cl 0.1%,KH_2PO_4 0.1%,MgSO_4·7H_2O 0.05%,pH7.5,接种量0.8%,35℃培养72h后,凤眼莲脱胶菌分泌的木聚糖酶活性达到75.05μg/mL·min,果胶酶活性达到63.51μg/mL·min,一次脱胶率达到33.37%。
The flooding of Eichhornia crassipes (E. crassipes) in water body had broken the biodiversity of aquatic ecosystem and threaten the human production, living and health. E. crassipes had been list as one of the ten kinds of harmful grass. How to control and utilize it as a kind of resource is attracting more and more attention. Similar to other plants, cellulose, hemicellulose and lignin are the main chemical constituents of this plant. Cellulose is the one of the main biomass resources. Microbial method can be used to remove noncellulose (called degumming) of this plant, to obtain high content cellulose, for further utilization of cellulose, it is important components for the new utilization approaches of E. crassipes. This paper on the Eichhornia crassipes's biological degumming included aspects as following.
1. Determination of chemical composition contents of E. crassipes
The determination of chemical composition contents of E. crassipes was conducted according to The Determination Method of Chemical Composition of Ramie GB5889-86. The results showed that the contents of different components are different in different parts of this plant. In the leaves, the content of lipid is the highest, pectin and lignin are all the lowest. In the stems, the contents of lipid and water soluble substances are all the lowest; the content of hemicellulose is the lowest in the roots, but highest in the leaves, being 52.94%. The lignin content in leaves, 12.65%, is the lowest. The cellulose content in stems is highest, being 17.23%.
2. Screening of degumming strains of E. crassipes and degumming test
Using congo red-broth agar plate to select five strains from the leaves and stems of plant, the ponds that E. crassipes grow near Lake Nanhu of Wuhan City. Through comparison of the activity of pectin and xylan, the degumming rate among these five strains, Al strain was determined as the ideal degumming strain of the E. crassipes. Under the condition of inoculation amount 4%, 35℃, pH 7.0, agitation speed 145 r/min, cultivating for 72 hours, the activity of pectin and xylan were 72.69μg/mL·min and 45.72μg/mL·min respectively, the degumming rate was 31.89%.
3. FTIR analysis of the celluloses obtained by different methods
The FTIR spectrum of raw fiber of E. crassipes, fiber treated by Al strain and alkali treated fiber were compared, the results indicated, the hemicellulose and lignin were mostly decomposed by two ways - treatment by A1 strain and alkalization by NaOH. The degumming efficiency of alkalization was most obvious, but the damage to cellulose was also largest. The structure change of plant treated by Al strain lied between that treated by alkali and raw material.
4. Single factor screening experiments for optimum fermentation conditions of Al strain
The screening experiments for optimum fermentation conditions of Al strain were conducted, using the plant as carbon source. The different factors for screening included form and concentration of nitrogen and, inorganic salt, temperature, and inoculation amount. The results indicated that the optimum degumming medium of Al strain was that NH_4Cl 0.1%, KH_2PO_4 0.1%, MgSO_4·7H_2O 0.05% and pH 7.5. After cultivating for 72 hours, the activity of pectin and xylan were75.05μg/mL·min and 63.51μg/mL·min, respectively, the degumming rate was 33.37%.
引文
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