环境友好型土壤保水剂聚γ-谷氨酸高吸水性树脂的制备
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摘要
聚γ-谷氨酸(γ-PGA)是一种由D-谷氨酸和L-谷氨酸通过酰胺键聚合而成的水溶性和生物可降解性高分子材料,具有吸附性强,对环境友好,可食无害等优点,因此近年来聚γ-谷氨酸及其衍生物的研究引起了国内外的关注。它在农业,食品,医药,化妆品,环保,合成纤维和涂膜等领域显示出广阔的应用前景,是一种极具开发价值的天然高分子材料。尤其是它可以作为不可降解的水凝胶强有力的替代品,为长期使用不可降解的原料来生产高吸水性树脂而带来的环境污染和资源短缺问题,提供了一个友好的解决思路。
本文对地衣芽胞杆菌Bacillus licheniformis WBL-3固体发酵生产聚γ-谷氨酸进行了研究,通过单因素试验方法得到了固体发酵聚γ-谷氨酸的优化培养基,培养基的配比组成为:豆渣:麸皮:营养液=1:1:1。优化培养条件为:初始pH为7.5,接种量为10%,装量为20g,发酵温度37℃,γ-PGA的产量最高可达到10.45g/kg。
建立了一种检测聚γ-谷氨酸的新方法,通过γ-PGA与氯化十六烷基吡啶(CPC)的乙酸钠水溶液反应形成混悬液,采用分光光度计于波长680nm处比浊,研究γ-PGA浓度与吸收度之间的线性关系,并研究了本方法测定γ-PGA的稳定性、重现性和回收率。在一定pH值和离子强度下,γ-PGA在12.5-50ug/ml范围内与CPC的乙酸钠水溶液生成的混悬液在波长680nm处的吸收度与其浓度呈线性关系,R~2=0.9939。本方法在2h内吸收度保持稳定(RSD=0.154%,n=10),CPC法测定浓度为5、10和40ug/mL时的平均回收率分别为86%,77%和99.75%,RSD分别为0.14%,0.23%和0.025%。应用比浊法测定γ-PGA的含量方便、简洁、重现性好,可用于γ-PGA浓度的检测。
使用化学和物理两种方法对B.licheniformis WBL-3发酵生产的γ-PGA进行交联,化学交联的的最适γ-PGA浓度为400mg/mL,最适交联剂的体积为100μL,反应的最适pH为5.0,反应时间在16h的时候交联效果最好,其特定水含量可以达到156g/g。
经过试验证明,物理交联与化学交联相比,具有形成的水凝胶溶胀特性高,无环境污染等优点,物理交联最适辐射总剂量为25KGy,最适γ-PGA的浓度为8%,在最适条件下形成的水凝胶中特定水含量为1396g/g。
交联形成的水凝胶具有一定耐温保水性能。γ-PGA在土壤中的实际吸水率最高为90g/g,并且经过试验证明。随着土壤中γ-PGA高吸水性树脂浓度的增高,土壤水分保持的效果越好,γ-PGA高吸水性树脂在脱水干燥过程中能有效的抑制土壤蒸发,从而在较长时间内使土壤保持较多的水分。
Poly-γ-glutamaic acid(γ-PGA) is a kind of biological macromolecules that made of L-and D-glutamic units linked through theα-amine and theγ-carboxylic acid groups,It is water-soluble,biodegradable,edible and non-toxic toward humans and the environment. Therefore,potential applications ofγ-PGA and its derivatives have been of interest in the past few years in the fields of medicine,food,cosmetic,plastics,and water treatment.Especially it can be the best substitute of undegradable hydrogel.In this article,the study began with theγ-PGA that B.licheniformisWBL-3 produced,used chemical and physical two methods to yield hydrogels,then studied the structure and its application in water retention properties of soil.
This article deals with biosynthesis ofγ-PGA by fermentation with B.licheniformis WBL-3 which was reserved in our laboratory.The solid state fermentation was carried out for the production ofγ-PGA.The maximumγ-PGA production was 10.45g/kg.it was achieved in the conditions that describes as follows:the ratio of oybean dregs and wheat bran and nutrition liquid is 1:1:1(w/w),the best fermentate condition is:the incolnm the amount of substrate was 10%,the incolum was 20g,the initial pH was 7,the temperature was 37℃.
A novel method was established to detectγ-PGA.A turbid solution was formed through the reaction betweenγ-PGA and cetylpyridinium chloride(CPC) and the turibidity was measured by using ultraviolet-visible spectroscopy at 680nm.The linearity between the concentration ofγ-PGA and its absorbance,the stability,repeatability and recovery of the method were studied. Through the reaction ofγ-PGA with CPC,a homogeneous turbid solution was formed.The turbid solution was stable in 3h under proper pH value and ion strength,the absorbance of the solution at 680nm had a good linear relationship with the concentration ofγ-PGA in the range 12.5-50ug/mL(R~2=0.9939).The recovery was within the range of 86%-99.75%.
Crosslinkedγ-PGA hydrogels were prepared by chemical method and by ~(60)Co-γ-irradiation.Compared the hydrogel made by chemical method and made by physical method,it is result that the hydrogel made by ~(60)Co-γ-irradiation has the higher water content.γ-PGA was crosslinked to yield high water sorption material with specific water content of about 1396 times their dry volume,it was achieved in the conditions that describes as follows: irradiation dose was 25KGy,γ-PGA concentration was 8%,pH9.0.
The application ofγ-PGA in agricultural filed was studied,γ-PGA could absorb 900 times water in solid,theγ-PGA solution had better water-holding and sustained release ability.
本文对地衣芽胞杆菌Bacillus licheniformis WBL-3固体发酵生产聚γ-谷氨酸进行了研究,通过单因素试验方法得到了固体发酵聚γ-谷氨酸的优化培养基,培养基的配比组成为:豆渣:麸皮:营养液=1:1:1。优化培养条件为:初始pH为7.5,接种量为10%,装量为20g,发酵温度37℃,γ-PGA的产量最高可达到10.45g/kg。
建立了一种检测聚γ-谷氨酸的新方法,通过γ-PGA与氯化十六烷基吡啶(CPC)的乙酸钠水溶液反应形成混悬液,采用分光光度计于波长680nm处比浊,研究γ-PGA浓度与吸收度之间的线性关系,并研究了本方法测定γ-PGA的稳定性、重现性和回收率。在一定pH值和离子强度下,γ-PGA在12.5-50ug/ml范围内与CPC的乙酸钠水溶液生成的混悬液在波长680nm处的吸收度与其浓度呈线性关系,R~2=0.9939。本方法在2h内吸收度保持稳定(RSD=0.154%,n=10),CPC法测定浓度为5、10和40ug/mL时的平均回收率分别为86%,77%和99.75%,RSD分别为0.14%,0.23%和0.025%。应用比浊法测定γ-PGA的含量方便、简洁、重现性好,可用于γ-PGA浓度的检测。
使用化学和物理两种方法对B.licheniformis WBL-3发酵生产的γ-PGA进行交联,化学交联的的最适γ-PGA浓度为400mg/mL,最适交联剂的体积为100μL,反应的最适pH为5.0,反应时间在16h的时候交联效果最好,其特定水含量可以达到156g/g。
经过试验证明,物理交联与化学交联相比,具有形成的水凝胶溶胀特性高,无环境污染等优点,物理交联最适辐射总剂量为25KGy,最适γ-PGA的浓度为8%,在最适条件下形成的水凝胶中特定水含量为1396g/g。
交联形成的水凝胶具有一定耐温保水性能。γ-PGA在土壤中的实际吸水率最高为90g/g,并且经过试验证明。随着土壤中γ-PGA高吸水性树脂浓度的增高,土壤水分保持的效果越好,γ-PGA高吸水性树脂在脱水干燥过程中能有效的抑制土壤蒸发,从而在较长时间内使土壤保持较多的水分。
Poly-γ-glutamaic acid(γ-PGA) is a kind of biological macromolecules that made of L-and D-glutamic units linked through theα-amine and theγ-carboxylic acid groups,It is water-soluble,biodegradable,edible and non-toxic toward humans and the environment. Therefore,potential applications ofγ-PGA and its derivatives have been of interest in the past few years in the fields of medicine,food,cosmetic,plastics,and water treatment.Especially it can be the best substitute of undegradable hydrogel.In this article,the study began with theγ-PGA that B.licheniformisWBL-3 produced,used chemical and physical two methods to yield hydrogels,then studied the structure and its application in water retention properties of soil.
This article deals with biosynthesis ofγ-PGA by fermentation with B.licheniformis WBL-3 which was reserved in our laboratory.The solid state fermentation was carried out for the production ofγ-PGA.The maximumγ-PGA production was 10.45g/kg.it was achieved in the conditions that describes as follows:the ratio of oybean dregs and wheat bran and nutrition liquid is 1:1:1(w/w),the best fermentate condition is:the incolnm the amount of substrate was 10%,the incolum was 20g,the initial pH was 7,the temperature was 37℃.
A novel method was established to detectγ-PGA.A turbid solution was formed through the reaction betweenγ-PGA and cetylpyridinium chloride(CPC) and the turibidity was measured by using ultraviolet-visible spectroscopy at 680nm.The linearity between the concentration ofγ-PGA and its absorbance,the stability,repeatability and recovery of the method were studied. Through the reaction ofγ-PGA with CPC,a homogeneous turbid solution was formed.The turbid solution was stable in 3h under proper pH value and ion strength,the absorbance of the solution at 680nm had a good linear relationship with the concentration ofγ-PGA in the range 12.5-50ug/mL(R~2=0.9939).The recovery was within the range of 86%-99.75%.
Crosslinkedγ-PGA hydrogels were prepared by chemical method and by ~(60)Co-γ-irradiation.Compared the hydrogel made by chemical method and made by physical method,it is result that the hydrogel made by ~(60)Co-γ-irradiation has the higher water content.γ-PGA was crosslinked to yield high water sorption material with specific water content of about 1396 times their dry volume,it was achieved in the conditions that describes as follows: irradiation dose was 25KGy,γ-PGA concentration was 8%,pH9.0.
The application ofγ-PGA in agricultural filed was studied,γ-PGA could absorb 900 times water in solid,theγ-PGA solution had better water-holding and sustained release ability.
引文
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[2]S.R法内斯托克,A.斯泰因比歇尔主编生物高分子[M].北京:化学工业出版社,2005,117-163.
[3]Ing-lung Shih,Yi-Tsong Van The production of poly(L-glutamic acid) from microorganisms and its various application[J]Bioresource T echnology,2001,79:207-225.
[4]董旭卿,张小红.生物降解高吸水性树脂的研究进展[J].广东化工,2005,(5):1-3.
[5]戈进杰,生物降解高分子材料及其应用[M].北京:化学工业出版社.2002,1.
[6]Ishihara K.,Kobayashi M,Polym.[J].1984,29:211-215.
[7]张新民,游庆红,徐虹,等.生物可降解型聚谷氨酸高吸水树脂的制备[J].高分子材料科学与工程,2003,19(2):203-205.
[8]陈坚,堵国成,陆卫功,华兆哲,编著.微生物重要代谢产物-发酵生产与过程解析[M1.北京:化学工业出版社,2005,234-280.
[9]张新民,γ-聚谷氨酸及高吸水树脂的制备研究(博士学位论文D).南京:南京农业大学,2003.
[10]Macaskie,L E,Basnakova,G.Microbially-enhanced chemisorption of heavy metal:a method for the bioremediation of solutions containing long-lived isotopesf neptunium and plutonium[J].Environ.Sci Technol,1998,32:184-187.
[11]GiannosS.A,Shah D,Gross R.A.[M]ACSPolymPrepr,1990,31-209.
[12]Gonzales D,Fan K,Sevoian M.Synthesis and swelling characterizations of a poly (gamma-glutamic acid) Hydrogen[J]Journal of polymer science;part A;polymer chemistry,1996,(34):2019-2027.
[13]Kanioka M,FurusawA k-Poly(γ-glutamic acid) Hydrogel prepared from microbial poly (γ-glutamic acid) and alkanediamine with water-solublec arbodiimide[J].Appl.Polym Sci,1997,65(10):1889-1896.
[14]Hara,T(200b) Greening by utilization of microbicalmacramolecules,Kobunshi[J].Desert greening,49,367-370.
[15]Kakinoki,K.,Kishida,A,Akashi,M.,Endo,T.(1993)synthesis and swelling characterization of a poly(γglutamic acid)hydrogel synthesis of poly(γ-glutamicacid)hydrogel[J].Chem.Soc.Jpa.Prep65 Ⅱ.258.
[16]Kunioka,M hydrogel,Chem.Soc.Jpn.Prep.65Biosynthesis and chemical reactions poly (aminio)s from microorganism[J].Appl.Microbiol Biotechnol.47,469-475.
[17]Atsuo G;Masaok Biosynthesis and hydrolysis of poly(γ-glutamic acid) from Bacillus Subtilis IFO3335.Biosci Biotech Biochem,1992,56(7):1031-1035.
[18]Do,J,H.,Chang,H.N.,Lee,S,Y.(2001b) Effect recovery of γ-poly(glutamic acid) from highly viscous culture broth[J].Biotechnol.Bioeng.76,219-223.
[19]林尚安,陆耕,梁兆熙,主编.高分子化学[M].北京:科学出版社,1982,675-690.
[20]Tseng,Y C,Hyson,S H,etal.In vivo evaluation of cyanoacrylates as surgical adhesives [J].Appl.Biomater,1990,1:111-122.
[21]Otani Yuto,Tabata Yasuhiko,Ikada Yoshito.Effects of additives on gelation and tissue adhesion of gelatin-poly(L-glutamic acid) mixture[J].Biomaterials,1998,19(23):2167-2173.
[22]Fanta G F,Barr Graft R C.Copolymers of starch Ⅱ copolymerization of gelatinized wheat starch reaction conditions on copolymer composition[J].J Polymer Sci,1996,B 4:765-767.
[23]Fanta G F,Barr Graft R C.Copolymers of starch.Ⅱ copolymerization of gelatinized wheat starch reaction conditions on copolymer composition[J].J Polymer Sci,1996,B4:765-767.
[24]Choi,H.J,Kunioka,M.Preparation conditions and swelling equilibria of hydrogel pre-pared by γ-irradition from microbial poly glutamic acid,Rradiat.Phys Chem 46,175-179.
[25]Toshio H.Water absorption polymer and manufacturing method[P].J P 200118137,3 July,2001.
[26]朱济群,肖祥熊,高吸水性高分子材料的研究进展[J].化工之友,2006,(02):37-38.
[27]刘静,金映红,杨超,等.利用~(60)Coγ-射线制备微生物聚谷氨酸类吸水剂及其吸水性能研究[J].离子交换与吸附,2007,23(2):129-136.
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