CN101962619B - 一种毛栓菌及其固定化方法和应用 - Google Patents
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Abstract
本发明属于微生物工程及废水处理技术领域,具体涉及一种毛栓菌(Fungaliasp.DD616)及其固定化方法和在印染与造纸废水脱色中的应用。该菌株保藏号为CGMCC No.3680;该菌株用玉米芯、小麦秸秆、水稻秸秆、芦苇叶天然载体进行固定化处理;该菌固定化处理后可用于造纸和印染废水等的脱色。
Description
一、技术领域
本发明属于微生物工程及废水处理技术领域,具体涉及一株毛栓菌及其固定化方法和在印染废水与造纸废水脱色中的应用。
二、背景技术
气候变暖和环境污染是当今世界人类面临的最主要问题。据报道,全世界每年生产的染料有100,000种,700,000吨,中国每年生产的染料达150,000吨,而印染和造纸行业在染料的使用过程中有10%~15%的染料会随水排放,中国每年大约有15,000~22,500吨的印染和造纸等废水排出,印染和造纸废水已成为自然水体生态系统中最重要的污染源之一。
印染和造纸废水具有色度高、COD高、盐度高和毒性大等特点。目前多采用化学法(如:化学混凝法、湿式空气氧化法、光催化氧化法等)、物理法(如:吸附法、膜分离等)、生化法和电化学法等进行印染和造纸废水处理,但许多方法单独处理印染和造纸废水往往不能达到废水排放标准,且处理费用较高,有些方法甚至存在二次污染的风险。因此,利用生物技术来进行印染和造纸废水的处理逐渐受到人们的关注,然而,由于印染和造纸废水的特殊性,使得一般微生物无法或很难在这类废水中生长繁殖和发挥降解作用,这也是目前常规生物处理技术(如活性污泥法、生物膜法)处理印染和造纸废水效率低下的根本原因之一。有证明表明,好氧细菌在厌氧条件下脱色因偶氮还原酶对底物的专一性较低,Km值较高,无法彻底降解染料,且处理后的水中含有大量的毒性和致癌中间产物,如芳香胺化合物等,严重制约了其在印染和造纸废水处理中的应用。
70年代从腐烂的木材中得到的能降解木质素的担子菌——白腐真菌,因其细胞外降解能力较强,已成为印染废水脱色降解的理想菌种。本专利使用的毛栓菌属于白腐真菌的一种,对染料降解谱较宽,不需经过特定污染物的预处理,培养条件简单,抗逆性强,环境适应性好,是一株处理印染和造纸废水的理想菌种。
白腐真菌为好氧真菌,在脱色反应器中需要考虑氧气因素。目前解决白腐真菌好氧培养主要通过两条途径,一是通过搅拌或通入空气而实现,但因白腐真菌对剪切力十分敏感,搅拌处理对其脱色活性影响很大,严重限制了其大规模应用。二是多采用固定化技术,增强真菌的耐受能力,提高脱色效率。目前使用的固定化载体主要为琼脂,海藻酸钙,角叉菜胶,钢丝网,高分子材料等惰性材料,脱色效果较好,但都存在载体需要提前合成、成本较高和回收处理困难等问题。为此,本发明提供用天然材料为载体的白腐真菌固定化方法及其在印染废水脱色中的应用技术,为生产环保型生物脱色剂及其在印染废水、造纸废水等工业废水处理过程中的应用提供关键材料和技术。
三、发明内容
本发明需要解决的问题是提供一种具有高效、广谱脱色活性的毛栓菌(Fungalia sp.)DD616菌株及其固定化方法与在印染和造纸废水脱色中的应用。
本发明的具体技术方案:
1、一种毛栓菌(Fungalia sp.)DD616的分离及鉴定
将含有浓度为0.3mg/ml橙G的PDA平板暴露于空气中30min,然后于28℃恒温培养箱中培养,观察脱色圈的发生,并从脱色圈中挑出菌丝;该菌种在PDA平板上初生菌丝白色,随着时间的延长菌丝变黄。对该菌株染色体DNA中核糖体基因内部转录间隔区序列的PCR扩增和测序分析,将该菌株鉴定为毛栓菌(Fungalia sp.),菌株编号为DD616,该菌株已于2010年4月8日保藏在中国微生物菌种保藏管理委员会普通微生物中心,保藏号为CGMCC No.3680。
2、一种毛栓菌的固定化方法
将玉米芯、稻草秸秆、小麦秸秆、芦苇叶等粉碎,称量0.5~5.0g装入250ml锥形瓶中,再向瓶中加入5~15ml水和0.5~5.0%的葡萄糖,121℃高压灭菌30min后,接入在PDA培养基上培养5天的毛栓菌DD616菌株的菌饼3~8块,菌饼直径为0.7cm,28℃~30℃摇床培养培养3~7天,使真菌在载体上生长,去除培养液,即为毛栓菌DD606菌株的固定化。
3、一种固定化的毛栓菌在印染和造纸废水脱色中的应用
将固定化毛栓菌DD616菌株按干重的10~40g/L接种到三角瓶中,分别加入浓度为0.5~1.0g/L的活性蓝K-GC、活性翠蓝KN-G、活性红紫X-2R、分散橙S-4SL、分散蓝2BLN、橙K-GN、活性红K-2BP、活性X黄K-6G、粉碎大红S-R、橙G等,28C摇床培养2d,脱色率高达40~99%。用该固定化的毛栓菌DD616菌株对橙G进行连续脱色,每3d排出脱色液,继续加入含有相同浓度和含量的橙G,依次循环,共可脱色6批(18d),并保持每批脱色率为98%,之后脱色率有所降低。
本发明与现有技术相比其有益效果是:首先,毛栓菌DD616菌株具有较宽的脱色谱和较强的脱色能力,对橙G可连续脱色18d而保持98%的脱色率;其次,本发明采用的固定化载体为常见的农业废弃物,环境友好,无污染,而且可以对毛栓菌提供一定的营养物质;第三,本发明的固定化过程简单,毛栓菌可在这些天然废弃物上直接生长,不需要合成载体的复杂操作;因此,固定化毛栓菌应用于造纸废水处理、印染废水处理和服装行业等废水脱色处理等,具有广阔的产业化前景。
四、附图说明
图1.固定化毛栓菌的脱色谱
图2.固定化毛栓菌的脱色持续时间
图3.固定化毛栓菌对不同浓度染料的脱色效率
五、具体实施方案
实施例1、毛栓菌的分离与鉴定
将含有浓度为0.3mg/ml橙G的PDA平板暴露于空气中30min,然后于28℃恒温培养箱中培养,观察脱色圈的发生,并从脱色圈中挑出菌丝;该菌种在PDA平板上初生菌丝白色,随着时间的延长菌丝变黄。对该菌株染色体DNA中核糖体基因内部转录间隔区序列的PCR扩增和测序分析,将该菌株鉴定为毛栓菌(Fungalia sp.),菌株编号为DD616,该菌株已保藏在中国微生物菌种保藏管理委员会普通微生物中心,保藏号为CGMCC No.3680。
(1)DNA提取:将DD616菌株在新鲜的PDA培养基上活化后转接到摇瓶中,震荡培养3d后,过滤收集菌丝体,用无菌水冲洗3次,无菌滤纸吸干残余水分。将菌丝体置于研钵中,加入石英砂和1mL高盐缓冲液充分研磨。将研磨好的匀浆置于eppendorf管中,10000rpm离心10min,收集上清液,而后加入等体积的氯仿-异戊醇抽提,10000rpm离心10min移取上清,重复用氯仿-异戊醇抽提并离心,而后用70%乙醇沉淀,溶于适量的TE溶液中得到总提取溶液。
(2)PCR扩增:针对真核微生物核糖体基因内部转录间隔区序列设计特异性引物ITS1和ITS4(White等1990),ITS1为:5’-TCCGTAGGTGAACCTGCGG-3’,ITS4为5’-TCCTCCGCTTATTGATATGC-3’。PCR反应的混合液的总体积为50μL,包括:1×PCR buffer,MgCl2 3mM,4种dNTP各200mM,上下游引物各0.3μM,Taq酶2.0U,DNA模板2μL。将反应体系放于PCR仪上,反应程序如下:94℃预变性,然后进入循环,94℃变性1min,55℃复性45s,72℃延伸1min,共35个循环,最后72℃延伸7min。反应结束后,取5μL扩增产物经1%琼脂糖凝胶电泳,EB染色,紫外检测。同时设置不含模板DNA的阴性对照。
(3)PCR产物序列测定:PCR产物以ITS1为测序引物,送南京基天生物科技有限公司测序,经与Genbank数据库的序列进行同源性比对分析,鉴定该菌为毛栓菌(Fungalia sp.)。
实施例2、毛栓菌的固定化方法
将玉米芯、稻草秸秆、小麦秸秆、芦苇叶等粉碎,称量0.5~5.0g装入250ml锥形瓶中,再向瓶中加入5~15ml水和0.5~5.0%的葡萄糖,121℃高压灭菌30min后,接入在PDA培养基上培养5天的毛栓菌DD616菌株的菌饼3-8块,菌饼直径为0.7cm,28℃~30℃摇床培养培养3~7天,使真菌在载体上生长,去除培养液,即为毛栓菌的固定化;
(1)不同天然载体的脱色效率:用玉米芯、稻草秸秆、芦苇叶和小麦秸秆为载体,以橙G染料为脱色对象,固定化毛栓菌DD616菌株两天的脱色效率分别为99%、99%、90%和80%。
(2)固定时间与脱色的关系:用玉米芯为载体,以橙G染料为脱色对象,检测固定化时间与脱色的关系,发现固定化5d后脱色效果最好,对橙G的脱色率为99%。
(3)糖对固定化的作用:在毛栓菌DD616菌株固定化过程中加入不同浓度的葡萄糖(1~10g/ml),发现添加0.1~3.0g/L的葡萄糖,有利于固定化毛栓菌DD616菌株对橙G的脱色效率,处理2天的脱色率在90%以上。
实施例3、毛栓菌固定化后对印染和造纸废水脱色中的应用
(1)脱色谱:将固定化毛栓菌DD616菌株按干重的30g/L接种到三角瓶中,分别加入浓度为1.0g/L的活性蓝K-GC、活性翠蓝KN-G、活性红紫X-2R、分散橙S-4SL、分散蓝2BLN、橙K-GN、活性红K-2BP、活性X黄K-6G、粉碎大红S-R,28C摇床培养2d,脱色率高达40~99%(图1),显示了很好的广谱性。
(2)连续脱色效率:用固定化的毛栓菌DD616菌株对橙G进行连续脱色,每3d排出脱色液,继续加入含有相同浓度和含量的橙G,依次循环,橙G浓度为0.6g/L;连续脱色6批,共18d,脱色率>98%,第7批(21d)降至73.8%(图2)。
(3)对不同染料浓度的脱色效率:用固定化的毛栓菌DD616菌株处理不同浓度的橙G(1~10g/ml),28℃摇床培养培养2d,检测其脱色率。脱色结果见图3。以1g/ml浓度脱色率最高。
Claims (3)
1.一种毛栓菌DD616菌株,该菌株已于2010年4月8日保藏在中国微生物菌种保藏管理委员会普通微生物中心,保藏号为CGMCC No.3680。
2.根据权利要求1所述的毛栓菌DD616菌株的天然载体固定化方法,其特征是将玉米芯或稻草秸秆或小麦秸秆或芦苇叶粉碎,称量0.5~5.0g装入250ml锥形瓶中,再向瓶中加入5~15ml水和0.5~5.0%的葡萄糖,121℃高压灭菌30min后,接入在PDA培养基上培养5天的毛栓菌DD616菌株的菌饼3~8块,菌饼直径为0.7cm,28℃摇床培养3~7天,使真菌在载体上生长,去除培养液,即为毛栓菌DD606菌株的固定化。
3.根据权利要求1所述的毛栓菌DD616菌株在印染废水和造纸废水脱色中的应用。
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