摘要
为了深入认识污泥中蛋白质提取过程机理,了解不同的蛋白质提取与分离方法特点,以及考察分离得到的蛋白质性质,探索污泥中蛋白质资源化方法,本研究以纪庄子污水厂剩余污泥为研究对象,主要进行了以下工作。
污泥中蛋白质溶出机理的研究。依据污泥中优势细菌结构特点、污泥颗粒性质以及蛋白质结构特点,采用微生物细胞壁水解原理、污泥吸附/脱附机理以及蛋白质水解理论,建立了关于污泥浓度、温度、酸或碱浓度以及操作时间对提取蛋白质浓度影响的13参数动力学模型。通过实验,采集了大量的不同条件下的蛋白质浓度数据。利用非线性优化技术获得了动力学模型参数。最后,利用动力学模型及其参数,分析了各影响因素对蛋白质浓度的影响规律。结果发现:本研究建立的蛋白质溶出动力学模型效果良好;适当增加污泥浓度、温度以及酸或碱浓度有利于污泥中蛋白质的提取,但必须选择最佳操作时间,否则,蛋白质提取效果可能适得其反。
污泥中蛋白质提取方法、蛋白质结晶工艺探索及蛋白质产品性质分析。主要考察了盐酸处理方法对污泥中无机物的溶解效果,盐酸或氢氧化钠提取污泥中蛋白质过程特点和现象,探索了提取液中蛋白质的结晶方法,并对蛋白质产品进行了紫外(UV)光谱分析、蛋白质含量分析、傅立叶红外(FTIR)分析以及X-射线荧光分析。结果表明:盐酸预处理可以降低污泥中无机物的含量,有利于提高污泥中有机物含量;碱法存在美德拉反应,而且固液分离困难,提取液质量较差等问题;高温下盐酸提取法不仅蛋白质提取效果较好,而且有利于固液分离,提取液黏度小,颜色较好;高温酸法提取液中的金属离子沉淀后,在等电点下加入2倍体积的无水乙醇,蛋白质收率可达78.5%,蛋白质含量达103.8%,而且产品中未检出《饲料卫生标准》(GB13078-2001)所规定的5种重金属;蛋白质干燥需在40℃以下进行,否则,容易变性,溶解性能较差;傅立叶红外(FTIR)数据证明,无论碱法还是酸法提取,最终得到的蛋白质主要为-螺旋结构。
通过对污泥中蛋白质提取机理、蛋白质结晶方法以及蛋白质产品性质的研究,认为污泥中蛋白质具有作为饲料用途的潜在可能性。本研究可为加热条件下盐酸或氢氧化钠提取污泥中蛋白质提供动力学模型指导,同时对蛋白质分离提纯具有良好的参考价值,有利于促进我国剩余污泥中蛋白质的资源化研究。
To study the process of protein extraction from excess sludge, the characteristics among different protein extraction and separation methods, the properties of isolated proteins, and protein resources methods, the following work was done based on the excess sludge from Jizhuangzi wastewater plant.
The protein dissolution mechanism from sludge was researched. The dynamic model, with thirteen parameters and four factors including sludge concentration, temperature, acid or alkali concentration and operating time, was built by using the principle of microbial cell wall hydrolysis, sludge adsorption / desorption mechanism and the theory of protein hydrolysis, which took the characteristics of the main bacteria structural, sludge particle and protein structure into consideration. A certain amount of the protein concentration data was collected by experiments. The parameters of kinetic model were obtained through nonlinear optimization techniques and could be used to investigate the effects of various factors on the protein concentration. The results confirmed the dynamic model and concluded that appropriate increase of sludge concentration, temperature and concentration of acid or alkali could facilitate the process of protein extraction.
The protein extraction method in sludge, protein crystallization process and protein products nature analysis were carried out. Inorganic sludge dissolution by hydrochloric acid, protein extraction process characteristics and phenomena using hydrochloric acid or sodium hydroxide, and protein products analysis including ultraviolet (UV) spectroscopy, protein content analysis, fourier transform infrared (FTIR) analysis, and X-ray fluorescence analysis were investigated. The results showed that: hydrochloric acid pretreatment could reduce the inorganic content of the sludge, and it could help improve the organic content. Alkali method possessed many problems such as Maillard reaction, the difficulty of solid-liquid separation and poor quality of extract. Hydrochloric acid extraction method under high temperature could not only promote the protein extraction, but also benefit the solid-liquid separation, when the extract viscosity was small, and the color could be better. After precipitation of metal ions from the extracted hydrochloric acid at high-temperature, two times volume of ethanol was added into solution at isoelectric point. The protein yield and protein content were 78.5% and 103.8% respectively, while the five heavy metals limited in "feed hygiene standards" (GB 13078-2001) was not detected in the products. Proteins products need to be dried under 40℃, otherwise protein would be easily denatured and its solubility would be poor. Fourier transform infrared (FTIR) data proved that the obtained protein was mainly ?- helices whether using alkaline or acid extraction method.
The sludge protein shows the potential possibility to be used as feed by studying the mechanism of sludge protein extraction, protein crystallization methods and protein products properties. The work could provide kinetic model guidance for the protein extraction from sludge by hydrochloric acid or sodium hydroxide under heating conditions and could have certain reference value in proteins separation and purification, which is also conducive to promoting studies on the protein resource from the excess sludge in China.
引文
[1]崔小浩,李五勤,谢文征,等.北京市污水处理厂污泥处理与处置探讨[J].给水排水, 2010, 36(6): 17-20.
[2]陈蓉蓉,李庆新.剩余污泥处理存在问题及发展趋势[J].科技创业月刊, 2010(6): 142-143.
[3]朱书景,薛改凤,张垒.污泥处理技术与发展趋势[J].武钢技术, 2010, 48(3): 1-3.
[4]李家麟.利用活性污泥提取蛋白质技术的可行性分析[J].资源节约, 2007, 23(101): 23-24.
[5] Monique R, Elisabeth G N, Etienne P. A high yield multi-method extraction protocol for protein quantification in activated sludge[J]. Bioresource Technology, 2008, 99(16): 7464-7471.
[6] Park C, Novak J T, Helm R F, et al. Evaluation of the extracellular proteins in full-scale activated sludges[J]. Water Research, 2008, 42(148): 3879-3889.
[7] Neyens E, Baeyens J, Creemers C. Alkaline thermal sludge hydrolysis[J]. Hazardous Material, 2003, 97(1-3): 295-314.
[8]周健,柴宏祥.活性污泥胞外聚合物EPS的影响因素研究[J].中国给水排水, 2005, 31(8): 19-21.
[9]肖本益,刘俊新.不同预处理方法对剩余污泥性质的影响研究[J].环境科学, 2009, 29(2): 327-331.
[10] Choi H B, Hwang K Y, Shin B B. Effects on anaerobic digestion of sewage sludge pretreatment[J]. Water Science and Technology, 1997, 35(10): 200-207.
[11] Kepp U, Machenbach I, Weisz N, et al. Enhanced stabilisation of sewage sludge through thermal hydrolysis-three years of experience with full scale plant[J]. Water Science and Technology, 2000, 42(9): 89-96.
[12] Chu C P, Chang B V, Liao G S, et al. Observations on changes in ultrasonically treated waste activated sludge[J]. Water research, 2001, 35(4): 1038-1046.
[13] Sohn K D, Neppolian B, Choi H. Effect of soil organic matter (SOM) and soil texture on the fatality of indigenous microorganisms in intergrated ozonation and biodegradation[J]. Journal of Hazardous Materials, 2008, 150(3): 809-817.
[14] Azize A. Enzymatic treatment effects on dewaterability of anaerobically digested biosolids-I: Performance evaluations[J]. Process Biochemistry, 2005, 40(7): 2427-2434.
[15] Nah I W, Kang Y W, Hwang K Y, et al. Mechanical pretreatment of waste activated sludge for anaerobic digestion process[J]. Water Research, 2000, 34(8): 2362-2368.
[16]戴前进,方先金,邵辉煌.城市污水处理厂污泥厌氧消化的预处理技术[J].中国沼气, 2006, 25(2): 11-14.
[17]冯若,李化茂.声化学及其应用[M].合肥:安徽科学技术出版社, 1992.
[18]曹秀芹,陈珺.超声波技术在污泥处理中的研究及发展[J].环境工程, 2002, 20(4): 23-25.
[19] Petrier C, Francony A. Incidence of wave frequency on the reaction rates during ultrasonic wastewater treatment[J]. Water Science and Technology, 1997, 35(4): 175-180.
[20]吴臣军,黄少武,张建设.超声波在低声能密度下处理污泥的实验研究[J].环境科学与技术, 2009, 32(9): 170-172.
[21]王晓霞,吕树光,邱兆富.超声波处理、热处理及酸碱调节对剩余污泥溶解效果的对比研究[J].环境工程, 2010, 32(8): 56-61.
[22]汪群慧,刘建丽,艾恒雨,等.提高污泥厌氧消化效率的溶胞预处理技术[J].黑龙江大学自然科学学报, 2005, 22(5): 614-618.
[23]王伟.热水解预处理改善污泥的厌氧消化性能[J].环境科学, 2005, 26(1): 69-71.
[24]王治军,王伟.剩余污泥的热水解试验[J].中国环境科学, 2005, 25(z1): 56-60.
[25]乔玮,王伟,黎攀,等.城市污水污泥微波热水解特性研究[J].环境科学, 2008, 29(1): 152-157.
[26] Liao P H, Wong W T, Lo K V. Advanced oxidation process using hydrogen peroxide/microwave system for solubilization of phosphate[J]. Journal of Environmental Science and Health (PartA), 40(9): 1753-1761.
[27] Lerch R N, Barbarick K A, Azari P. Sewage sludge proteins: I. Extraction methodology[J]. Journal of Environmental Quality, 1993, 22(3): 620-624.
[28] Chishti S S, Hasnain S N, Khan M A. Studies on the recovery of sludge protein[J]. Water Research, 1992, 26(2): 241-248.
[29] Lau D C W. Utilization of sewage sludge as a resource for protein[J]. Conservation and Recycling, 1980, 4(3): 193-200.
[30]陈玉辉,华佳,霍苗,等.酸循环水解制备剩余污泥水解蛋白质的试验研究[J].湖北大学学报, 2006, 28(4): 417-419.
[31]赵顺顺,孟范平.酸水解法提取剩余污泥蛋白质的条件优化[J].环境科学研究, 2008, 21(3): 180-184.
[32] Ahn K H, Park K Y, Maeng S K, et al. Ozonation of wastewater sludge for reduction and recycling[J]. Water Science and Technology, 2002, 46(10): 71-77.
[33] Lee J W, Cha H Y, Park K Y, et al. Operational strategies for an activated sludge process in conjunction with ozone oxidation for zero excess sludge production during winter season[J]. Water Research, 2005, 39(7): l199-l204.
[34]王学创,泡沫分离法分离蛋白质的研究[D].天津:天津大学, 2004.
[35]孙墨珑,宋湛谦,方桂珍,等.减压蒸馏法提取核桃楸树皮中的胡桃醌[J].林业化学与工业, 2007, 27(6): 113-115.
[36]曹庆荣,王桂英,王广铨.乙烯基降冰片烯的分离[J].吉林化工学院学报, 1997, 14(3): 27-29.
[37]谢继宏.大豆蛋白质的泡沫分离研究[J].华东理工大学学报, 1997, 23(3): 270-280.
[38]王立新,石红,欧阳藩.连续泡沫分离蛋白质[J].化工冶金, 1996, 17(4): 343-347.
[39]周柏青.全膜水处理技术[M].北京:中国电力出版社, 2005.
[40]徐林.膜技术处理活性污泥水解蛋白液实验研究[D].天津:天津大学, 2008.
[41]王银珍.膜分离技术在蛋白质类生物产品分离领域的应用展望[J].山东化工, 2006, 35(2): 15-17.
[42]傅翠娥,杨旭明,林爱芬,等.弓形体抗原的纯化与分析[J].中国禽畜传染病, 1998, 20(5): 294-296.
[43]石彦国,郑佳,马永强,等.等电点冷沉法分离大豆11S的研究[J].中国食品学报, 2010, 10(1): 146-150.
[44]赵顺顺,孟范平.剩余污泥蛋白质作为动物饲料添加剂的营养性和安全性分析[J].中国饲料, 2008(15): 35-38.
[45] Chishtia S S, Hasnaina S N, Khanb M A. Studies on the recovery of sludge protein[J]. Water Research, 1992, 26(2): 241-248.
[46] Clevenger T E. Safety and efficacy of food processing sludges as animal feed: chemical characterization[J]. Research Journal of the Water Pollution Control Federation, 1990, 62(6): 820-827.
[47]李亚东,李海波.利用剩余活性污泥水解蛋白质制备蛋白质泡沫灭火剂的研究[J].湖北大学学报(自然科学版), 2005, 27(1): 91-93.
[48]王雅丽.剩余污泥溶胞过程数学模型[D].天津:天津大学, 2010.
[49]郭幸丽.污泥溶胞-水解酸化-CAS减量工艺中式设计及研究[D].天津:天津大学, 2010.
[50]尹军,谭学军.污水污泥处理处置与资源化利用[M].北京:化学工业出版社, 2005.
[51]贺延龄.废水的厌氧生物处理[M].北京:中国轻工业出版社, 1998.
[52]王淡兮,孙秀兰.蛋白质定量检测方法的探讨[J].粮食与食品工业, 2009, 16(4): 49-51.
[53]鲁健章,孙丽华,周彦钢.凯氏定氮法测定鱼蛋白质含量的干扰因素分析[J].食品科学, 2010, 31(19): 453-456.
[54]王爱军,王凤山,王友联,等.低浓度蛋白质含量测定方法的研究[J].中国生化药物杂志, 2003, 24(2): 78-80.
[55]周群英,高廷耀.环境工程微生物学[M].北京:高等教育出版社, 1999.
[56]车振明.微生物学[M].武汉:华中科技大学出版社, 2010.
[57]张自杰.活性污泥生物学和反应动力学[M].北京:中国环境科学出版社, 1989.
[58]朱宪,万雪亮,马艳华,等.花生壳在近临界水中催化水解反应及其动力学[J].化学反应工程与工艺, 2010, 26(6): 565-569.
[59]安红钢,吴冬青.催化剂与反应速率及化学平衡关系的论证[J].河西学院学报, 2005, 21(5): 43-45.
[60]武增华,许玲,王立新.催化剂对CaO固硫反应活性的影响研究[J].化学学报, 2001, 59(11): 1914-1918.
[61]崔静,董岸杰,张卫江,等.热碱水解提取污泥蛋白质的实验研究[J].环境工程学报, 2009, 3(10): 1889-1892.
[62]乔显亮,骆永明.我国部分城市污泥化学组成及其农用标准初探[J].土壤, 2001, (4): 205-209.
[63]李仙粉,周玉松,任福民,等.上海城市污泥成分特性及分析方法研究[J].中国环境监测, 2006, 22(6): 48-50.
[64]尹兵.剩余污泥中蛋白质分离方法的研究[D].天津:天津大学, 2011.
[65]黄梅丽,江小梅.食品化学[M].北京:中国人民大学出版社, 1986.
[66]紫外吸收法测定蛋白质含量, http://www.docin.com/p-41024040.html, 2010-01-06.
[67]谢孟峡,刘媛.红外光谱酰胺Ⅲ带用于蛋白质二级结构的测定研究[J].高等学校化学学报, 2003, 24(2): 226-231.
[68]王崇明,何欣翔,孙殿都.实用红外光谱[M].北京:石油工业出版社, 1982.
[69]杨旭红,钟林钧,陈博.废弃羊毛中蛋白质提取初探[J].毛纺科技, 2004, (4): 14-16.
[70]沈子威,孙克利,杨钧,等.应用傅里叶红外光谱研究强声波作用下植物壁蛋白质二级结构变化[J].光学学报, 1999, 28(7): 600-602.
[71] Klockenkamper R.全反射X-射线应该分析(王晓红,王毅民,王永奉译)[M].北京:原子能出版社, 2002.
[72]化合物的溶度积常数表, http://www.chinabaike.com/article/16/184/2007/2007021242596.html, 2007-02-12.