CN106591198A - 陶厄氏细菌Thauera sp. K11及其在降解含酚类化合物污泥废水中的应用 - Google Patents
陶厄氏细菌Thauera sp. K11及其在降解含酚类化合物污泥废水中的应用 Download PDFInfo
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Abstract
本发明提供了一株陶厄氏细菌Thauera sp.K11及其在降解含酚类化合物污泥废水中的应用,属于环境保护技术领域,能够在具备降解多种芳香族化合物能力的同时,还具备反硝化的能力,从而有效应用在污水尤其是石化废水的处理中。该菌株于2016年7月19日保藏在中国典型培养物保藏中心(中国武汉,武汉大学),保藏编号为:CCTCC M 2016399。本发明能够应用于含酚类化合物污水、尤其是石化废水的处理中。
Description
技术领域
本发明属于环境保护技术领域,尤其涉及一株陶厄氏细菌Thauera sp.K11及其降解含酚类化合物污泥废水中的应用。
背景技术
在含酚类物质的污水处理尤其是石化废水的处理过程中,多需要微生物的参与。目前,所报道的酚类降解细菌绝大多数营异养生长,且为好氧或兼性好氧菌。研究表明,酚类的降解主要发生在有氧存在的环境下,影响其降解的限速步骤的最重要的一步是苯环的打开。在大多芳香族化合物的生物降解过程中,均生成中心代谢中间产物邻苯二酚,微生物在好氧条件下,降解途径的第一步是将苯胺氧化成邻苯二酚。而邻苯二酚降解可以通过两种代谢途径,即邻位和间位代谢途径,分别由邻苯二酚1、2-双加氧酶或邻苯二酚2、3-双加氧酶催化。在污水生物处理过程中,常常要改变环境条件(溶氧,pH等)来满足不同污染物的降解要求。如常见的A2/O(厌氧-缺氧-好氧,Anaerobic-Anoxic-Oxic)工艺,就是通过改变污水的溶氧,即厌氧-缺氧-好氧达到污水中氮和磷等污染物的溶氧进行脱除的。类似这样的工艺过程虽然比传统的活性污泥法的效果好,但却增加了污水处理的成本。因此,开发一种可具有广泛环境适应性的高效污染物降解菌对于本领域将产生重要意义。
陶厄氏细菌属(Thauera)是贝塔变形菌纲下的一类革兰氏阴性菌,于1993年由Macy等人第一次定义了该菌株。目前分离到的陶厄氏细菌属细菌60余种,大部分具有反硝化、降解芳香族化合物的能力,可作为活性污泥中优势菌群广泛应用在污水处理中。另外有报道该菌属的部分细菌具有脱氮和分泌胞外多聚物的能力,在工厂或者生活污水生物处理系统的脱氮、反硝化、降低COD以及重金属离子的吸附作用中具有重要意义。然而,目前为止,尚没有发现一株陶厄氏细菌在具备降解多种酚类化合物能力的同时,还具备反硝化的能力,从而可有效应用在污水尤其是石化废水的处理中。
发明内容
本发明提供了一株陶厄氏细菌Thauera sp.K11及其在降解含酚类化合物污泥废水中的应用,能够在具备降解多种芳香族化合物能力的同时,还具备反硝化的能力,从而有效应用在污水尤其是石化废水的处理中。
本发明的一方面提供了一株陶厄氏细菌Thauera sp.K11,所述细菌菌株于2016年7月19日保藏在中国典型培养物保藏中心(中国武汉,武汉大学),保藏编号为:CCTCC M2016399。
作为优选,所述陶厄氏细菌Thauera sp.K11分离自石化污水中,菌落呈圆形,乳白色,边缘整齐,革兰氏染色呈阴性,通过16S rRNA测序鉴定为陶厄氏细菌,16S rRNA序列分析结果为序列表中<400>1所示序列。
本发明的另一方面提供了一种如上述技术方案所述的陶厄氏细菌Thauerasp.K11在降解含多种酚类化合物污泥废水中的应用。
作为优选,所述酚类化合物选自苯酚、邻甲苯酚、间甲苯酚、对甲苯酚、2,3-二甲基苯酚、2,4-二甲基苯酚、2,5-二甲基苯酚、2,6-二甲基苯酚、3,4-二甲基苯酚、3,5-二甲基苯酚和3-乙基苯酚中的一种或多种。
作为优选,将所述陶厄氏细菌Thauera sp.K11投放至含有酚类化合物的污泥废水中,实现所述细菌降解酚类化合物的作用。
本发明的再一方面提供了一种以上述技术方案所述的陶厄氏细菌Thauerasp.K11为活性成分的降解剂。
与现有技术相比,本发明的优点和积极效果在于:
本发明通过对陶厄氏细菌Thauera sp.K11(保藏编号为:CCTCC M2016399)以及其对含酚类化合物的污泥废水、尤其是污泥石化废水中的降解研究,发现了陶厄氏细菌Thauera sp.K11的用途,该菌株在高浓度含酚类化合物污水、尤其是石化废水中可以有效地降解多种酚类化合物,有效地去除了污水中的石油类和挥发性酚类污染物,在COD为500mg/L的污泥石化废水中,对苯酚、邻甲苯酚、间甲苯酚、对甲苯酚、2,4-二甲基苯酚、2,5-二甲基苯酚、2,6-二甲基苯酚、3,4-二甲基苯酚、3-乙基苯酚的去除率在76%以上,尤其是对苯酚、间甲苯酚、2,4-二甲基苯酚、3,4-二甲基苯酚的降解能力可达到100%以上。本发明将推动降解酚类污染物的细菌在污水、尤其是石化废水处理及其相关领域中的应用。
附图说明
图1为本发明实施例所提供的陶厄氏细菌Thauera sp.K11的透射电镜照片;
图2为本发明实施例所提供的陶厄氏细菌Thauera sp.K11的16S rRNA序列系统进化树;
图3为本发明实施例所提供的陶厄氏细菌Thauera sp.K11在42小时内的增殖曲线;
图4为本发明实施例所提供的陶厄氏细菌Thauera sp.K11在气相色谱法分析中对甲基苯酚降解示意图。
具体实施方式
下面将对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
下述实施例中所用的各培养基配方如下:
(1)种子培养基:
种子培养基为LB培养基,其组成为:
5g酵母粉,10g胰蛋白胨,10g氯化钠(固体添加15g/L琼脂);
1L去离子水,pH 7.0,121℃条件下灭菌20分钟。
(2)基础培养基:
基础培养基为无机盐培养基MS,其组成为:
K2HPO4·3H2O 2.28g/L,NaH2PO4·2H2O 0.47g/L,(NH4)2SO41.32g/L,MgSO4.7H2O0.20g/L(最后加),1ml/L微量元素母液,调至pH值为7.0,微量元素母液:CaCl2 2.63g/L,FeSO4·7H2O 0.72g/L,ZnSO4·7H2O 0.46g/L,MnSO4·H2O 0.22g/L;
1L去离子水,调pH至6.8,121℃条件下灭菌20分钟。
(3)苯酚降解途径检测培养基:
苯酚降解途径的检测培养基为:无机盐培养基MS+苯酚
实施例1
菌株Thauera sp.K11的培养与鉴定
菌种培养和纯化:配制固体种子培养基;将菌体用无菌生理盐水进行稀释后涂布到上述固体种子LB培养基上,置于30℃的恒温箱中培养2-5天,用接种环挑取单菌落,得到纯菌。
(1)形态特征
菌株Thauera sp.K11的菌落在LB固体培养基上呈圆形,乳白色,边缘整齐,革兰氏染色呈阴性,菌体短杆状,大小约为0.4-0.5μm宽,1.0-1.5μm长,如图1所示。
(2)菌株16S rRNA鉴定
使用细菌16S rRNA通用引物27F和1492R作为扩增引物,采取PCR方法扩增菌株Thauera sp.K11的16S rRNA片段,电泳检测后,送上海生工生物工程技术服务有限公司进行测序,获得测序结果,并用美国国立生物技术信息中心(NCBI)的BLAST程序对该菌的16SrRNA序列和GenBank已收录的序列进行核苷酸同源性比对。如图2所示,发现与之序列相似性最大的是已知标准菌株Thauera aromatica DSM6984T(X77118),相似度为97.49%。K11为本发明实施例所采用的Thauera sp.K11菌株。该菌株的16S rRNA序列GenBank数据库登录号为KX953213。
(3)菌株生理生化特征
对菌株Thauera sp.K11进行了生理生化特征鉴定,结果如表1所示。
表1Thauera sp.K11的生理生化特征
注:+,阳性;–,阴性;W,弱阳性
实施例2
苯酚含量测定方法
利用酚类物质在pH=10.0±0.2的介质中,在铁氰化钾存在下,与4–氨基安替比林反应生成橙红色的吲哚酚安替比林染料,其水溶液在510nm波长处有最大吸收峰的特性,使用分光光度计测定其不同浓度下的吸光值,绘制出苯酚含量与吸光度的标准曲线,并得到拟合的计算公式。
①苯酚标准液:称取0.5g无色苯酚溶于蒸馏水,定容至500ml,配成浓度为1g/L苯酚溶液;
②氨缓冲溶液:称取20g NH4Cl白色固体溶于100ml的氨水中配成pH≈10的氨缓冲溶液;
③4–氨基安替比林溶液:称取2g 4–氨基安替比林固体粉末溶于蒸馏水,定容至100ml,配成质量浓度为20%的浅黄色溶液;
④铁氰化钾溶液:称取8g铁氰化钾红色颗粒溶于92ml的蒸馏水中,配成质量浓度为8%的铁氰化钾溶液。
用去离子水和①-④所属溶液配制体积为1ml的苯酚浓度梯度检测液。其中苯酚的含量分别为:0μg、0.1μg、0.2μg、0.3μg、0.5μg、1.0μg和2.0μg;氨缓冲溶液:均为10μl;4–氨基安替比林溶液:均为20μl;铁氰化钾溶液:均为20μl;不足1ml的部分用去离子水补足。各溶液混匀,静置10分钟后,使用紫外分光光度计在波长510nm处测定吸光值。经绘制苯酚含量与吸光度的标准曲线后,得到拟合度高(R2=0.9987)的计算公式:苯酚含量=5×(吸光度值-0.309)/3.8269,如图3所示。
细菌增殖测定方法:
根据生物类实验惯例,细菌的增殖状态通过细菌培养液在波长为600nm时的吸光度(OD600)进行表征。
①调整仪器吸光度:在分光光度计的光度模式下,将波长调整为600nm;
②仪器调零:将空白培养基装入1cm比色皿,装入光路,调整吸光值为零;
③OD值测定:将待测样品装入干净的培养基,读取吸光度数值。
实施例3
酚类化合物含量测定方法-气相色谱定量分析
色谱定量分析的依据是根据被测物质的量与它在色谱图上的峰面积(或峰高)成正比。常用色谱分析方法有百分比法、归一化方法、内标法、外标法和标准加入法。我们测定酚类化合物含量采用内标法,内标法的优点是定量精度高。
利用酚类化合物在气相色谱柱中不同的分离时间,可以把酚类化合物分离。具体操作方法是用待测组分的纯物质配成标准溶液,然后在等体积的这些标准溶液中加人浓度相同的内标物,混合后进行色谱分析。
色谱条件:选用HP-5柱子。分流进样,分流进样比设定为20:1;进样口温度320℃;FID检测器温度320℃;程序升温,初始温度为60℃,恒温1min,20℃/min升温到320℃,恒温3min;空气流量为400ml/min,H2流量为40ml/min,进样量1ul。
经过气相色谱检测,菌株Thauera sp.K11对酚类化合物降解效果见下表。
表2Thauera sp.K11对酚类化合物降解性能
由表2降解试验可知,以500mg/L上述物质计,Thauera sp.K11对苯酚、邻甲苯酚、间甲苯酚、对甲苯酚、2,4-二甲基苯酚、2,5-二甲基苯酚、2,6-二甲基苯酚、3,4-二甲基苯酚、3-乙基苯酚的降解能力最好,可达到76%以上,尤其是对苯酚、间甲苯酚、2,4-二甲基苯酚、3,4-二甲基苯酚的降解能力可达到100%以上;其次,对邻甲苯酚、2,5-二甲基苯酚、3-乙基苯酚的降解能力可达到80%以上;对对甲苯酚、2,6-二甲基苯酚的降解能力可达到70%以上;再次是对2,3-二甲基苯酚、3,5-二甲基苯酚的降解能力可达到57%以上。以1000mg/L上述物质计,Thauera sp.K11对大部分物质的降解率可达到50%及以上程度。
实施例4
Thauera sp.K11酚降解途径检测
在以500mg/L苯酚为唯一碳源的无机盐培养基中,30℃转速150rpm下,培养24小时后将菌液取出;培养液在10000r/min,4℃离心10分钟,沉淀物以冰冷的0.1mol几磷酸缓冲液(pH7.5)清洗两次,然后重悬。将悬浮液在冰水浴条件下超声波破碎:循环99次,每次35,振荡35,功率120W。悬浮液以2000r/min,4℃离心30分钟,离心后用50mM磷酸缓冲溶液(pH7.0)清洗菌种2次;将菌种在磷酸缓冲溶液中超声破碎15分钟;之后再高速离心,得到粗酶提取液;分别移取2.9ml缓冲溶液、0.1ml 0.01mol/L的邻苯二酚溶液及0.1ml的粗酶提取液于试管中摇匀,放入37℃的恒温水浴中。
在零时刻加入酶,并同时将试管放入沸水中煮沸3min,之后分别将试管在1-18min内每隔一分钟取出,并煮沸3min,直至酶失活,反应终止;然后,分别将其放入紫外分光光度计中连续(200nm-500nm)扫描其光谱吸收特征。
邻苯二酚存在两种分解方式,分别是:
1)1,2-双加氧酶起作用底物后,产物为内酯,在波长为在260nm处有吸收峰。
2)2,3-双加氧酶起作用底物后,产物为2-HMSA(2-羟基粘康酸半醛),在波长为在375nm处有吸收峰。
结果显示:在375nm处,1分钟时就有波峰出现,此后峰值越来越高,如图4所示。说明此酶分解邻苯二酚是从苯环两羟基的间位断开的,发现菌株Thauera sp.K11是通过邻苯二酚2、3-双加氧酶催化酚进行降解。
实施例5
现场微生物多样性测定和水质检测
对石化污水处理厂曝气池中的活性污泥进行三点采样。污泥样品经3000r/min离心15min后倒掉上清液、沉淀,立即保存于-80℃冰箱中。在48h内提取基因组DNA(采用美国OMEGA公司的E.Z.N.A.TM Soil DNA Kit试剂盒)。送上海美吉生物进行高通量测序(Miseq,引物对515F/907R)。在相似度为97%的水平上,对微生物的OTU(operational taxonomicunit)进行多样性分析。发现三点采样的活性污泥中Thauera sp.K11数目和总的OTU数目分别为:5865/41848(~14%);4896/36516(~13%)和5120/32298(~16%)。表明,Thauerasp.K11为此石化污水处理厂活性污泥中的优势菌群。采用中国环境科学出版社《水和废水监测分析方法》第四版进行污水处理厂进出水水质分析,结果如下:
进水:
COD:1224.6mg/L,氨氮:67.3mg/L,石油类:301.2mg/L,悬浮物:778.6mg/L,pH:7.68,挥发酚:31.3mg/L,硫化物:47.7mg/L。
出水:
COD:117.3mg/L,氨氮:15.2mg/L,石油类:4.6mg/L,悬浮物:57.1mg/L,pH:7.48,挥发酚:未检出,硫化物:0.029mg/L。
表明,在此石化污水处理厂中,Thauera sp.K11菌株作为优势菌群对于活性污泥中的石油类和挥发酚有很好的降解效果,从而可证明菌株K11在处理石化废水中有较好的开发利用前景。
序列表
<110> 中国石油大学(华东)
<120> 陶厄氏细菌Thauera sp.K11
<130> 0
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 1552
<212> DNA
<213> 陶厄氏细菌(Thauera sp.)
<400> 1
1 KACTCGGTAC GCGCGGATCT TCCAGAGATT AGAGTTTGAT CCTGGCTCAG ATTGAACGCT
61 GGCGGCATGC TTTACACATG CAAGTCGAAC GGCAGCGGGG GCTTCGGCCC GCCGGCGAGT
121 GGCGAACGGG TGAGTAATGC ATCGGAACGT GCCCATTCGT GGGGGATAAC GTATCGAAAG
181 GTACGCTAAT ACCGCATACG CCCTGAGGGG GAAAGCGGGG GATCTTCGGA CCTCGCGCGA
241 TTGGAGCGGC CGATGTCGGA TTAGCTTGTT GGTGAGGTAA AGGCTCACCA AGGCGACGAT
301 CCGTAGCGGG TCTGAGAGGA TGATCCGCCA CACTGGGACT GAGACACGGC CCAGACTCCT
361 ACGGGAGGCA GCAGTGGGGA ATTTTGGACA ATGGGGGCAA CCCTGATCCA GCCATGCCGC
421 GTGAGTGAAG AAGGCCTTCG GGTTGTAAAG CTCTTTCGGC CGGGAAGAAA TCGGACGCTC
481 TAACATAGCG TTCGGATGAC GGTACCGGAC TAAGAAGCAC CGGCTAACTA CGTGCCAGCA
541 GCCGCGGTAA TACGTAGGGT GCGAGCGTTA ATCGGAATTA CTGGGCGTAA AGCGTGCGCA
601 GGCGGTTTGG TAAGACAGGT GTGAAATCCC CGGGCTTAAC CTGGGAACTG CGCTTGTGAC
661 TGCCAAGCTG GAGTACGGCA GAGGGGGGTG GAATTCCACG TGTAGCAGTG AAATGCGTAG
721 AGATGTGGAG GAACACCGAT GGCGAAGGCA GCCCCCTGGG CCTGTACTGA CGCTCATGCA
781 CGAAAGCGTG GGGAGCAAAC AGGATTAGAT ACCCTGGTAG TCCACGCCCT AAACGATGTC
841 GACTAGTTGT TCGGTGCAGC AATGCGCTGA GTAACGCAGC TAACGCGTGA AGTCGACCGC
901 CTGGGGAGTA CGGCCGCAAG GTTAAAACTC AAAGGAATTG ACGGGGACCC GCACAAGCGG
961 TGGATGATGT GGATTAATTC GATGCAACGC GAAAAACCTT ACCTACCCTT GACATGCCAG
1021GAACCTTGGT GAGAGCCGAG GGTGCCTTCG GGAGCCTGGA CACAGGTGCT GCATGGCTGT
1081CGTCAGCTCG TGTCGTGAGA TGTTGGGTTA AGTCCCGCAA CGAGCGCAAC CCTTGTCACT
1141AGTTGCCATC ATTTGGTTGG GCACTCTAGT GAGACTGCCG GTGACAAACC GGAGGAAGGT
1201GGGGATGACG TCAAGTCCTC ATGGCCCTTA TGGGTAGGGC TTCACACGTC ATACAATGGT
1261CGGTACAGAG GGTTGCCAAG CCGCGAGGTG GAGCCAATCC CTTAAAGCCG ATCGTAGTCC
1321GGATCGTAGT CTGCAACTCG ACTGCGTGAA GTCGGAATCG CTAGTAATCG CAGATCAGCA
1381TGCTGCGGTG AATACGTTCC CGGGTCTTGT ACACACCGCC CGTCACACCA TGGGAGTGGG
1441TTTCACCAGA AGTAGGTAGC TTAACCTTCG GGAGGGCGCT TACCACGGTG AGATTCATGA
1501CTGGGGTGAA GTCGTAACAA GGTAACCAAT CGTCGAACGG CAGGCGTGCA AA
Claims (6)
1.一株陶厄氏细菌Thauera sp.K11,其特征在于,所述细菌菌株于2016年7月19日保藏在中国典型培养物保藏中心(中国武汉,武汉大学),保藏编号为:CCTCC M 2016399。
2.根据权利要求1所述的陶厄氏细菌Thauera sp.K11,其特征在于,所述陶厄氏细菌Thauera sp.K11分离自石化污水中,菌落呈圆形,乳白色,边缘整齐,革兰氏染色呈阴性,通过16S rRNA测序鉴定为陶厄氏细菌,16SrRNA序列分析结果为序列表中<400>1所示序列。
3.一种如权利要求1或2所述的陶厄氏细菌Thauera sp.K11在降解含多种酚类化合物污泥废水中的应用。
4.根据权利要求3所述的应用,其特征在于,所述酚类化合物选自苯酚、邻甲苯酚、间甲苯酚、对甲苯酚、2,3-二甲基苯酚、2,4-二甲基苯酚、2,5-二甲基苯酚、2,6-二甲基苯酚、3,4-二甲基苯酚、3,5-二甲基苯酚和3-乙基苯酚中的一种或多种。
5.根据权利要求3所述的应用,其特征在于,将所述陶厄氏细菌Thauerasp.K11投放至含有酚类化合物的污泥废水中,实现所述细菌降解酚类化合物的作用。
6.一种以权利要求1所述的陶厄氏细菌Thauera sp.K11为活性成分的降解剂。
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CN106047768A (zh) * | 2016-08-02 | 2016-10-26 | 浙江至美环境科技有限公司 | 一种陶厄氏菌菌株及其应用 |
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CN107828692A (zh) * | 2017-11-28 | 2018-03-23 | 广东省生态环境技术研究所 | 一株陶厄氏菌及其微生物菌剂制备与应用 |
CN107828692B (zh) * | 2017-11-28 | 2020-01-21 | 广东省生态环境技术研究所 | 一株陶厄氏菌及其微生物菌剂制备与应用 |
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