摘要
偶氮类活性染料是纺织印染工业中应用最为广泛的一类染料。偶氮染料和其代谢物具有的致癌、致畸、致突变的作用,对人类及其所生存的环境造成了极大的危害。细菌极强的繁殖力和环境适应性,为其在印染废水处理的应用中奠定了基础。
本研究采用梯度平板分离法,从活性污泥中筛选分离得到了一株高效脱色偶氮染料活性黑5的细菌RB5-M1。经细菌形态学观察、生理生化试验及16S rRNA基因序列分析,鉴定菌株RB5-M1为嗜水气单胞菌,命名为Aeromonas hydrophila strain RB5-M1。取10 mL菌液加入90 mL的染料培养基中,温度为35℃,pH 8.0,厌氧条件(氮气85%、二氧化碳6%)培养。24 h测定的平均脱色率可达94.1%,最高脱色率为99.8%。菌株RB5-M1在温度20-40℃、pH 5.0-9.0时,均可对染料脱色,其中脱色最适温度为35℃、最佳pH为8.0。且该菌可适当调节环境酸碱度,有降低染料废水碱污染的能力。
菌株RB5-M1在厌氧条件(氮气85%、二氧化碳6%)下培养时,在培养初期0-24 h内,脱色速率上升很快,脱色效果明显高于静置培养和好氧培养方式;而进入中后期24-72 h,静置培养方式的脱色速率显著上升;在培养72 h后静置培养与厌氧培养方式最终的脱色效率持平。从经济成本考虑,若实际生产中设计适当的反应器,采用静置培养方式来对染料废水进行脱色处理,相比于厌氧或好氧方式或可在达到预期效果的同时,大大节约处理成本。
当活性黑5初始浓度C0为50 mg/L时,菌体延滞期低于5 h,6h测得OD600为0.313;当C0为200 mg/L时,菌体的生长延滞期为12 h,当c0为300 mg/L时,菌体的生长延滞期接近60 h。初始浓度(C0)为50 mg/L~250 mg/L时,用Monod零级反应方程拟合具有较高的相关性,说明零级反应方程能够较好地描述脱色过程,即菌株RB5-M1能以较为恒定的速率脱色活性黑5。而初始浓度(C0)为300 mg/L时,零级反应方程、一级反应方程、二级反应方程的相关系数R2较为接近,考虑到数据点选取的不同可能造成的误差,因此初始浓度大于300 mg/L的染料溶液脱色规律或更符合一级以上动力学方程模型。
当冷却温度为30℃、PVA与海藻酸钠浓度比为10:1、氯化钙浓度为2%、明胶浓度为0.2%时,获得的固定化颗粒I1机械强度较好、包埋量较多,在SBR反应器中初期对模拟染料废水的脱色率较高,后期脱色率明显下降,运行10个周期依然具有脱色效果,颗粒物理形态良好。当氯化钙浓度2%、明胶浓度0.3%、PVA与海藻酸钠浓度9:2、冷却温度20℃,获得的固定化颗粒I2的渗漏量较低,在SBR反应器中运行7个周期内脱色率均可保持在66%以上。
固定化颗粒连续脱色10次以内,脱色率高于61%,渗漏量低于24.53×107个/mL。固定化颗粒在储存前30天中活性良好。脱色率稳定在80%以上。低温条件下,储存时间超过30天的固定化颗粒,直接进行脱色试验,脱色率约为65%。单独含有固定化颗粒或驯化污泥的反应器C1、C2,其在COD去除率、TOC去除率、脱色率方面的表现均不如同时含有两者的反应器C3。由此可见固定化优势脱色菌强化SBR法处理模拟染料废水试验有良好的应用前景。
Azo dye is the most widely used dye in textile and dyeing industry. Azo dyes and its metabolins usually are carcinogenic, teratogenic and mutagenic, which has made a great danger to nature and human survival environment. Bacterial, which was considered to have an exuberant fertility and strong adaptability, shows bright prospect in application of textile and dyeing wastewater treatment.
Through isolating by the gradient plate, we got a strain named RB5-M1 which has good decolorization ability. According to its morphological, physiological characteristics and the analysis of 16S rRNA gene analyses for strain identification,>99.86% of gene sequences in isolated strain RB5-M1 were similar to Aeromonas hydrophila compared to available gene sequences in the NCBI BLAST gene bank. Added 10 mL bacterial culture solution into 90 mL Dye-BPA medium, and cultivated in anaerobic cultural incubator (nitrogen 85%, carbon dioxide 6%),35℃, pH 8.0. The measurement of decolorization rate showed that decolorization rate performance is 94.1% for average value,99.8% for maximum value. The strain RB5-M1 can decolor reactive black 5 at 20-40℃, pH 5.0-9.0. The optimum conditions are 35℃, pH 8.0. What's more, the strain can also adjust pH value of environment and reduce pollution.
When we cultivated RB5-M1 in anaerobic cultural incubator (nitrogen 85%, carbon dioxide 6%), for the first 24 hours, decolorization rate increased fast, and it was much better than in static culture and aerobic culture. For the second and third 24 hours, decolorization rate in static culture increased rapidly; after 72 hours, decolorization rate in static culture approximated the decolorization rate in anaerobic culture. From an economic cost consideration, if we can design the appropriate reactor, using this strain by static fermentation, it probably achieves the desired results as anaerobic fermentation, with the significant cost savings.
When initial concentration of reactive black 5 is 50 mg/L, the growth of RB5-M1 hardly delay, the OD600 at 6h is 0.313; if initial concentration of reactive black is 200 mg/L, the lag phase will be 12h; if initial concentration of reactive black is 300 mg/L, the lag phase will need nearly 60 h. When initial concentration of reactive black 5 is 50,100,150,200,250 mg/L, kinetic processing of decoloring highly corresponded with the Monod zero level reaction model, the relative coefficient R2 is 0.9878,0.9725,0.9465,0.964 and 0.902, respectively. When initial concentration of reactive black 5 is 300 mg/L, the relative coefficient R2 of Monod zero level reaction model is close to R2 of Monod 1st level and 2nd reaction model, kinetic processing of decoloring may highly corresponded with the Monod 1st level reaction model.
When stain RB5-M1 was immobilized at the condition of 30℃cooling temperature, PVA: Sodium alginate equals to 10:1,2% concentration of CaCl2,0.2% concentration of gelatin, it could form immobilized globules with higher mechanical strength and embedding biomass. When stain RB5-M1 was immobilized at the condition of 2% concentration of CaCl2,0.3% concentration of gelatin, PVA:Sodium alginate equals to 9:2,20℃cooling temperature, it could form immobilized globules with lower leakage biomass. Meanwhile, result of experiment in SBR (Sequencing Batch Reactor) confirmed the conclusion about immobilization conditions.
The immobilized globules can reapply 10 times while the decolorization rate is higher than 61%, leakage biomass is less than 24.53×107 cells/mL. Immobilized globules are active in the first 30 days; decolorization rate is more than 80%. When the immobilized globules were stored more than 30 days at a low temperature (4℃), do the decoloring test immediately, the decolorization rate will be about 65%. The removal of COD, TOC and decolorization rate of reactor C1 and C2 are worse than C3. Thus it can be seen immobilized advantage decoloring bacteria to enhance the SBR process has a bright prospect in application of textile and dyeing wastewater treatment.
引文
[1]柳广飞.偶氮还原酶AZR的结构模型与功能研究[D].大连:大连理工大学,2009.
[2]Zollinger H. Color chemistry.New York:VCH Weinheim,1991.
[3]钟金汤.偶氮染料及其代谢产物的化学结构与毒性关系的回顾与前瞻[J].环境与职业医学,2004,21(1):58-61.
[4]刘华丽.印染废水的生物处理技术[J].环境科学与技术,1996,(1):42-45.
[5]Choudhary G. Human health perspectives on environmental exposures to Benzedrine: a review [J]. Chemosphere,1996,12:267-291.
[6]马春燕.印染废水深度处理及回用技术研究[D].上海:东华大学,2007.
[7]何强,龙腾锐.印染废水治理技术评价[J].给水排水,1995,5:47-51.
[8]梁彦秋,孙小寒,马俊.氧化钙改性粉煤灰对酸性橙的吸附脱色性能试验研究[J].安全与环境工程,2011,18(4):70-73.
[9]冯丽,葛小鹏,晏晓敏,等.载钯树脂对偶氮类染料的吸附降解及其产物对发光菌的毒性暴露试验[J].生态毒理学报,2011,6(3):265-270.
[10]史玲,董社英,柏世厚,等.常温常压催化湿式氧化降解偶氮染料废水的研究[J].环境工程学报,,2011,5(6):1325-1329.
[11]魏红,李克斌,赵锋,等.磷钨酸光催化降解直接大红4BE溶液的研究[J].中国环境科学,2011,31(6):921-926.
[12]付忠田,郑琳子,胡筱敏.活性艳蓝X-BR染料废水电化学脱色及其机理研究[J].环境工程,2011,29(3):44-52.
[13]宿程远,韦金尚,陈孟林,等.增强型内电解-H202催化氧化处理染料废水研究[J].水处理技术,2011,37(7):61-64.
[14]张丽.微电解-混凝和高效菌处理还原染料废水研究[J].环境科学与技术:2004,27(9):67-68.
[15]尤克非,高晓红,赵芳.镁盐对活性染料废水的混凝脱色研究[J].针织工业,2011,7:47-49.
[16]叶国祥,周烁灵,唐佳玙,等.Fe-C微电解法预处理高浓度印染废水的研究[J].高校化学工程学报,2011,25(3):489-494.
[17]罗方.复合纳滤膜的应用研究[D]浙江:浙江大学,2006.
[18]马江权,郭楠,许守勇,等.微滤/纳滤联用技术深度处理印染废水[J].水处理技术,2010,36(9):65-68.
[19]邱丽娟.厌氧颗粒污泥对活性黑KN-B染料的生物降解脱色研究[D]上海:东华 大学,2010.
[20]李博,熊小京,赵志新,等.缺氧-好氧生物滤池中高效菌对活性红KN-3B的降解特性[J].环境工程学报,2009,3(12):2170-2174.
[21]Horitsu H. Degradation of P-aminoazobenzene by Bacillus subtilis [J]. European Journal of Applied Microbiology,1977,4 (3):217-224.
[22]P.K.Wong. Decolorization and Biodegradation of Methyl red by Klebsiella pneumoniae RS-13[J]. Wat.Res.1996,30(7):1736-1744.
[23]Roxon J J, Ryan A J, Wright S E. Reduction of Water-soluble Azo Dyes by Zntestinal Bacteria [J]. Food Cosmet Toxicol,1967, (5):367-369.
[24]Mechsner K, Wuhrmann K. Cell permeability as a Rate Limiting Factors in the Microbial Reduition of sulfonated Azo Dyes [J]. Eur J Appl Microbiol Biotechnol, 1982,15:123-126.
[25]Heiss GS, B Gowan, ER Dabbs. Cloning of DNA from a Rhodococcus strain conferring the ability to decolorize sulfonated azo dyes. FEMS Microbiology Letters, 1999(2-3):221-226.
[26]Anjali Pandey, Poonam Singh, Leela Iyengar. Bacterial decolorization and degradation of azo dyes[J]. International Biodeterioration & Biodegradation,2007,59: 73-84.
[27]钱陈.偶氮染料脱色菌的筛选以及脱色条件优化研究[D].浙江大学,2009.
[28]柳广飞,周集体.细菌对偶氮染料的降解及偶氮还原酶的研究进展[J].环境科学与技术,2006,29(4):112-114.
[29]魏剑斌,付永胜,朱杰,等.印染废水生物脱色研究现状及展望[J].污染防治技术,2003.87-91.
[30]John A B. Oxidation of Persistent environmental Pollutants by a white rot fungus [J]. Seienee,1985,228:1434-1436.
[31]Cripps C, Bumpus JA, Aust SD. Biodegradation of azo and heterocyclic dyes by Phanerochaete chrysosporium [J]. Applied and Environmental Microbiology,1990,56 (4):1114-1118.
[32]肖继波,胡勇有.菌株HX5对多种染料的吸附作用[J].环境科学学报.2005.25(4):525-529.
[33]尹亮,陈章和,赵树进.微生物对偶氮染料的脱色及其基因工程研究进展[J].生物技术,2007,06:86-89
[34]荚荣,汤必奎,张晓宾.藜芦醇和吐温80对白腐菌产木质素降解酶的影响及在偶氮染料脱色中的作用[J].生物工程学报,2004,20(4):302-305.
[35]Young L, Yu J. Ligninase-catalyzed decolorization of synthetic dyes [J]. Water Research,1997,31(5):1187-1193.
[36]赵春芳,胡道伟.偶氮金属络合染料的微生物脱色研究[J].武汉化工学院学报, 2001,23(3):18-25.
[37]郑金来,李君文,晃福寰.生物降解常见染料的研究进展[J].环境污染治理技术与设备,2000,1(3):39-43.
[38]Smet B, Rittmamn E, Sthal DA. The role of genes in biological processes [J]. Environment Science Technology,1990,24(2):162-168.
[39]尹亮.混合菌群共培养对偶氮染料的协同脱色及降解的研究[D].华南理工大学,2009.
[40]宋智勇.偶氮染料高效降解基因工程菌构建与特性研究[D].大连:大连理工大学,2006.
[41]常忠义,周波,高红亮,等.基因工程菌在生物降解中的应用及其发展[J].生物技术,2003,13(5):46-48.
[42]Hattori T., Furusaka C.Chemical activities of Escherichia coli adsorbed on a resin[J]. Biochimica et BiophysicaActa,1959,31 (2):581-582.
[43]Umezawa H., Matsuhashi Y., Yagisawa M., et al Immobilization of phosphotransferases obtained from resistant bacteria [J]. Journal of Antibiotics,1974, 28(4):358-360.
[44]Keisuke Hanaki. Protect ion of m ethanogenic bacteria from low pH and toxic mat erials by immobilization using polyviny lalcohol [J]. Water Research,1994,28(4): 877-885.
[45]Stephen rothenburger. Hydroxylation and biodegradation of 6-methylquinoline by Pseudomonas in aqueous and nonaqueous im mobilized-cell bioreactors [J]. Applied and Enviromental Microbiology,1993,59(7):2139-2144.
[46]周定,侯文华.固定化微生物法处理含酚废水的研究[J].环境科学,1990,11(1):2-5.
[47]牛志卿,吴国庆,张琳,等.固定化紫色非硫光合细菌降解活性艳红X-3B的研究[J].环境科学,1994,15(5):49-52.
[48]张秀霞,房苗苗,赵朝成,等.固定化降解菌Q5去除喹啉的性能研究[J].环境工程学报,2008,2(9):1265-1268.
[49]张艳,郑媛,王海英,等.不同载体固定化海洋微生物酯酶ETM-b的性能比较[J].渔业科学进展,2010,31(5):110-116.
[50]巴淑丽.光合细菌固定化包埋颗粒产氢特性实验研究[D].重庆:重庆大学,2007.
[51]东秀珠,蔡妙英.常见细菌系统鉴定手册[M].北京:科学出版社.2001:162-167.
[52]George MGarrity, Julia ABell, Timothy GLilburn. Bergey's Manual of Systematic Bacteriology.2nd Edition. USA:Department of Microbiology and Molecular Genetics Michigan State University East Lansing,2004:112.
[53]Liu JS, Xie XH, Xiao SM. Isolation of Leptospirillum ferriphilum by single-layered solid medium [J]. Journal of Central South University of Technology,2007,4: 467-473.
[54]胡俊,万欢,吴根福.环境中未培养微生物的研究进展[J].农机化研究,2007,8:1-3.
[55]Rodrigo Otavio Alves de Lima, Ana Paula Bazo, Daisy Maria Favero Salvadori, et al. Mutagenic and carcinogenic potential of a textile azo dye processing plant effluent that impacts a drinking water source[J]. Mutation Research/Genetic Toxicology and Environmental Mutagenesis,2007,626(1-2):53-60.
[56]钟金汤.偶氮染料及其代谢产物的化学结构与毒性关系的回顾与前瞻[J].环境与职业医学,2004,21(1):58-61.
[57]Rau J, Knackmuss H J, Stolz A. Effects of different quinoide redox mediators on the anaerobic reduction of azo dye by bacteria[J]. Environmental Science and Technology, 2002,36(7):1497-1504.
[58]Keck A, J Klein, M Kudlich, et al. Reduction of azo dyes by redox mediators originating in the naphthalenesulfonic acid degradation pathway of Sphingomonas sp. Strain BN6[J]. Applied and Environmental Microbiology,1997,63(9):3684-3690.
[59]Tomei, M.C., Annesini, M.C. Biodegradation of phenolic mixtures in a sequencing batch reactor:A kinetic study[J]. Environmental Science and Pollution Research,2008, 15(3):188-195.
[60]聂麦茜,吴蔓莉,王晓昌,等.一株黄杆菌及其粗酶液对芘降解的动力学研究[J].环境科学学报,2006,26(2):181-185.
[61]邱丽娟,黄满红,杨煜东.活性黑KN-B染料的生物降解研究[J].环境科学与技术,2010,33(6):63-66.
[62]Reis M. A. Evidence for the Intrinsic Toxic of H2S to Sulphate-reducing Bacteria [J], Application Microbial,1991,36:145-147.
[63]李博.固定化Citrobacter sp. CK3对活性红偶氮染料的脱色研究[D].厦门:厦门大学,2009.
[64]贺江,樊明涛.固定化酵母粒子的制备及特性[J].酿酒科技,2007,11:29-33.
[65]刘树文,李华.改善固定化微生物细胞粒子机械强度的研究[J].微生物学杂志,2005,25(4):32-34.