CN109161513B - 一株鞘氨醇杆菌及其应用 - Google Patents
一株鞘氨醇杆菌及其应用 Download PDFInfo
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- WWUZIQQURGPMPG-KRWOKUGFSA-N sphingosine Chemical compound CCCCCCCCCCCCC\C=C\[C@@H](O)[C@@H](N)CO WWUZIQQURGPMPG-KRWOKUGFSA-N 0.000 claims abstract description 34
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Abstract
本发明公开了一株鞘氨醇杆菌及其应用,属于生物工程、环境工程技术领域。本发明的一株鞘氨醇杆菌已于2018年10月17日保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏编号为CGMCC No.16596,该菌株具有异养硝化、好氧反硝化特性,能够应用于含氮污水处理、河道治理领域,在对氨氮的去除率大于90%并对环境无污染,达到快速生物脱氮、恢复河道自净能力的效果,在环境水处理领域具有重大意义。
Description
技术领域
本发明涉及一株鞘氨醇杆菌及其应用,属于生物工程、环境工程技术领域。
背景技术
河流、湖泊中氨氮含量超标常常导致水体富营养化,水中溶氧减少,这已成为我国水体污染的主要原因之一。水体中氨氮主要来源于农田化肥的使用,养殖业产生的动物粪便污水等等,这些污染物质通过地表径流进入湖泊河流带入大量的硝氮和氨氮,造成水体质量恶化和水生态环境结构的破坏。
传统的生物脱氮理论中,氨氮经自养硝化、亚硝化细菌逐步转化成亚硝态氮、硝态氮,再经反硝化菌作用转化成氮气从环境中去除。由于现实环境复杂,这两个部分在真实的生态***中,是比较难实现的。
兼性异养硝化和好氧反硝化菌不仅仅能使硝化过程和反硝化成过程同步进行成为可能,而且生长速率远大于自养菌,大大缩短了生长周期,。硝化过程的产物可直接成为反硝化过程的底物,避免了硝化产物积累对硝化反应的抑制作用,大大提高生物脱氮效率。整个过程能保持酸碱相对平衡,反硝化作用的产物能够补充环境碱度,从而是使pH保持在一定范围之内。大部分兼性异养硝化和好氧反硝化菌环境适应性强,适合治理大面积污染水域。
目前,研究者对于兼性异养硝化和好氧反硝化菌的研究还很少,且仅有的一些研究也多是聚焦在其降解机理。因此,对兼性异养硝化和好氧反硝化菌的生理生化特性及脱氮效能进行深入研究是必要的,具有重要的理论和实际应用价值。
发明内容
本发明的第一个目的是提供一株鞘氨醇杆菌(Sphingobacterium sp.),于2018年10月17日保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏编号为CGMCCNo.16596,保藏地址为北京市朝阳区北辰西路1号院3号,中国科学院微生物研究所。
本发明的第二个目的是提供含有鞘氨醇杆菌的微生物制剂。
在本发明的一种实施方式中,所述微生物制剂中鞘氨醇杆菌的活菌含量≥1×109cfu/g。
在本发明的一种实施方式中,所述微生物制剂为生物微胶囊。
在本发明的一种实施方式中,所述生物微胶囊的制备方式为:(1)将活性炭10-50g/L、壳聚糖5-50g/L、海藻酸钠10-50g/L称量溶解并混合均匀,得到凝胶液;(2)将50-100g固态菌体接入凝胶液中,得到菌体浓度为1×108-1×1011cfu/ml的微生物凝胶液;(3)将微生物凝胶液按照15-25滴/分钟的速度滴入质量浓度为1-50g/L的CaCl2溶液中,得到微生物活性颗粒;(4)将微生物活性颗粒于5%戊二醛溶液中在室温下交联24小时;(5)将(4)中交联后的微生物活性颗粒用无菌水浸泡24-48小时后,增殖得到鞘氨醇杆菌BT1生物微胶囊。
本发明的第二个目的是提供所述鞘氨醇杆菌的应用。
在本发明的一种实施方式中,所述应用包括降解河道、湖泊污染物或污水处理等。
在本发明的一种实施方式中,所述应用是对含氮污水进行处理。
在本发明的一种实施方式中,所述对含氮污水进行处理是将所述鞘氨醇杆菌BT1按终浓度10~30mg菌体/L污水投放至含氮污水中。
在本发明的一种实施方式中,所述鞘氨醇杆菌BT1以生物微胶囊的形式投放至水体中。
有益效果:本发明提供了一株鞘氨醇杆菌,该菌株具有异养硝化、好氧反硝化特性,能够应用于含氮污水处理、河道治理领域,在对氨氮的去除率大于90%并对环境无污染,达到快速生物脱氮、恢复河道自净能力的效果,在环境水处理领域具有重大意义。
生物材料保藏
一株鞘氨醇杆菌(Sphingobacterium sp.),于2018年10月17日保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏编号为CGMCC No.16596,保藏地址为北京市朝阳区北辰西路1号院3号,中国科学院微生物研究所。
附图说明
图1为鞘氨醇杆菌BT1生长及氨氮降解曲线;
图2为鞘氨醇杆菌BT1对硝氮降解变化曲线图;
图3为鞘氨醇杆菌BT1对亚硝氮降解变化曲线图;
图4为amoA基因验证的凝胶电泳图;其中,Marker:DL2000(100bp~2000bp);1:基因片段:amoA;
图5为nirK基因验证的凝胶电泳图;其中,Marker:DL2000(100bp~2000bp);1:基因片段为nirS;2:基因片段为nirK。
具体实施方式
异养硝化培养基(g/L):(NH4)2SO4 0.47,C4H4Na2O4 5.62,维氏盐溶液50ml,C/N=10,pH=7.0,琼脂2%。
维氏盐溶液(g/L):K2HPO4 5.0,MgSO4·7H2O 2.5,NaCl 2.5,MnSO4·4H2O 0.05,FeSO4·7H2O 0.05。
好氧反硝化培养基:硝酸盐培养基:KNO3 2g,柠檬酸钠5g,K2HPO4 1g,KH2PO4 1g,MgSO4·7H2O 0.2g,微量元素溶液2mL,补充蒸馏水至1L,pH7.2-7.5
亚硝酸盐培养基:NaNO2 2g,柠檬酸钠5g,K2HPO4 1g,KH2PO4 1g,MgSO4·7H2O0.2g,微量元素溶液2mL,补充蒸馏水至1L,pH7.2-7.5
微量元素溶液:EDTA 50.0g,ZnSO4 2.2g,CaCl2·2H2O 5.5g,MnCl2·4H2O 5.06g,FeSO4·7H2O 5.0g,(NH4)6Mo7O2·4H2O 1.1g,CuSO4·5H2O 1.57g,CoCl2·6H2O 1.61g,补蒸馏水至1L,pH 6.0
实施例1
垃圾渗滤液好氧发酵罐运行稳定的某周期的活性污泥作为分离用泥。在异养硝化培养基上,采用稀释混合平板法进行分离获得纯种鞘氨醇杆菌BT1菌株。提取菌株鞘氨醇杆菌BT1的基因组,进行16S rDNA测序。将测序结果与Genbank数据库中的已知序列进行相似度对比,结果显示,菌株鞘氨醇杆菌BT1与鞘氨醇杆菌属(Sphingobacterium)序列同源性最高,鉴定为鞘氨醇杆菌。
实施例2鞘氨醇杆菌(Sphingobacterium)鞘氨醇杆菌BT1氨氮降解能力测定
将鞘氨醇杆菌(Sphingobacterium)鞘氨醇杆菌BT1活化富集培养后,移取1ml悬浊液至含有100ml不含琼脂的异养硝化培养基中,在30℃,160rpm/min条件下培养48小时。每隔8小时取培养液。取部分培养液在600nm处测定鞘氨醇杆菌BT1的生长曲线,然后剩余培养液经0.22μm微孔滤膜过滤,检测滤液氨氮、亚硝氮、硝氮和COD等指标。结果如表1及图1所示。
表1鞘氨醇杆菌BT1对氨氮降解速率的变化
由表1OD600指标可以得到菌株的生长曲线。菌株只需要经过8h的迟缓期就可进入指数期,在24h内,OD值从0.01达到了1.349,24h后进入稳定期,与常见的硝化细菌生长周期相比,缩短了24-48小时。
在40h内,氨氮从90.21mg/L降至4.49mg/L,氨氮降解率达到95.01%,总氮从93.77mg/L降至5.11mg/L,总氮的降解率为94.65%。
需要注意的是,48小时内,亚硝氮含量从0mg/L积累至0.3mg/L,硝氮从0.6mg/L降低至0.05mg/L,表明鞘氨醇杆菌BT1具有积累亚硝氮的和降解硝氮的能力,由此我们推测鞘氨醇杆菌BT1可能具有好氧反硝化的能力。
与常见的硝化细菌或者厌氧反硝化细菌相比,鞘氨醇杆菌BT1兼具异养硝化和好氧反硝化功能,硝化过程和反硝化过程可同时进行,硝化的产物可直接作为反硝化的底物。从表1可以看出,鞘氨醇杆菌BT1生长周期短,24小时内即可达到平台期,生长速率远大于自养硝化菌;环境适应力强,对高浓度氨氮的耐受力强。
实施例3鞘氨醇杆菌(Sphingobacterium)BT1好氧反硝化能力测定
(1)采用硝酸盐培养基对鞘氨醇杆菌BT1的反硝化性能进行筛选和表征。
将鞘氨醇杆菌(Sphingobacterium)BT1活化富集培养后,移取1ml悬浊液至含有100ml不含琼脂的硝酸盐培养基或亚硝酸盐培养基中,在30℃,160rpm/min条件下培养,每隔8小时取培养液分别测定细菌浓度(OD600)、硝氮、亚硝氮。
表2鞘氨醇杆菌BT1对硝氮降解变化
从表2及图2可知,鞘氨醇杆菌BT1在第46小时开始生长,在80小时达到峰值。在100小时内,硝态氮从240.42mg/L下降至133.32mg/L,硝态氮降解率为44.5%,亚硝酸盐从0累积至40.59mg/L,表明鞘氨醇杆菌BT1对硝态氮的去除率较好,具有较强的反硝化功能。
(2)采用亚硝酸盐培养基对鞘氨醇杆菌BT1的反硝化性能进行筛选和表征。在硝酸盐培养基的基础上,将其中的硝酸钾替换为亚硝酸钠。
表3鞘氨醇杆菌BT1对亚硝氮降解变化
从表3和图3中可以看出,菌种在接种后第27小时开始生长,在68小时达到顶峰。在100小时内,亚硝氮含量从439.86mg/L降至375.97mg/L,亚硝酸盐含量下降了14.5%,硝态氮从80.27mg/L降至53.13mg/L,含量下降了33.8%。
综合鞘氨醇杆菌BT1降解硝酸盐和亚硝酸盐的效果,可以得出,鞘氨醇杆菌BT1以硝酸盐或亚硝酸盐为氮源时,会有一个25-40小时的潜伏期,菌株的对数生长期为12-22小时,30小时后开始进入稳定期和衰亡期。鞘氨醇杆菌BT1可以单纯利用硝酸盐或者亚硝酸盐作为氮源,达到脱氮的效果,从而证明鞘氨醇杆菌BT1具有较强的反硝化功能。
实施例4鞘氨醇杆菌(Sphingobacterium)鞘氨醇杆菌BT1好氧反硝化能力测定
(1)采用PCR验证鞘氨醇杆菌BT1中是否存在硝化功能基因amoA。引物序列如下:
amoA1F(5’-GGGGTTTCTACTGGTGGT-3’);
amoA2R(5’-CCCCTCKGSAAAGCCTTCTTC-3’)
反应条件:95℃3min;94℃1min,54.5℃45s,72℃1min,35个循环;72℃,10min;PCR产物使用0.8%琼脂糖凝胶电泳检测,结果如图4所示:
PCR产物为491bp大小的条带,经测序,与amoA基因序列一致,由此可以确认鞘氨醇杆菌BT1中存在amoA基因,具有硝化功能。
(2)反硝化功能基因Nir:Nir基因分为两种,分别是nirK和nirS。nirK基因编码Cu型亚硝酸盐还原酶,另一种nirS基因编码细胞色素还原酶。需要注意的是,这两种编码基因不会同时存在于同一株菌中。采用PCR验证鞘氨醇杆菌BT1中是否存在nirK和nirS基因。引物序列如下:
nirS-cd3aF(5’-GTSAACGTSAAGGARACSGG)
nirS-R3cd(5’-GASTTCGGRTGSGTCTTGA)
nirK-F1aCu(5’-ATCATGGTSCTGCCGCG)
nirK-R3Cu(5’-GCCTCG ATCAGRTTGTGGTT)
反应条件:95℃10min;95℃30s,56℃30s,72℃30s,35个循环;72℃,10min;PCR产物使用0.8%琼脂糖凝胶电泳检测,结果如图5所示。
从图中可以看出,菌株鞘氨醇杆菌BT1中不存在nirS基因,存在nirK基因。即表明鞘氨醇杆菌BT1中存在Cu型亚硝酸盐还原酶,基因型与其表现出来的好氧反硝化功能吻合。
实施例5
鞘氨醇杆菌BT1在异养硝化培养基上培养24小时后,按照1%的体积比例投入DN反应器中,DN反应器为高效脱氮反应器,内有布水***和三相分离器等核心构建,并设定高径比>2,可控参数为HRT,当HRT设定为48h时,反应器运行参数如下:
以上参数表明,鞘氨醇杆菌BT1在实际应用中能够高效的去除氨氮,,在48小时内,氨氮含量从2000mg/L降低至35mg/L,去除速率达到了40.9mg/L*h,同时,总氮(TN)含量在48h内从2500mg/L降低至200mg/L,COD从8000mg/L降低至700mg/L,总氮和COD的降解率分别达到了92%和91.25%。DO始终保持在小于1mg/L,这表明鞘氨醇杆菌BT1对溶氧的浓度要求低,适应性强,特别适用于治理富营养化、高氨氮水体。
实施例6菌株鞘氨醇杆菌BT1在含氮污水处理方面的应用
在使用鞘氨醇杆菌BT1对南京某污水处理厂含氮污水处理实验中,按照20ppm的浓度在好氧池前端流加鞘氨醇杆菌BT1菌液。48小时内,氨氮含量从20mg/L降低至5mg/L,氨氮去除率达到了75%,日处理污水量可达到55000-60000m3,表明鞘氨醇杆菌BT1用于处理大规模污水时,可以有效的减少含氮污水中的氨氮浓度。
实施例7菌株鞘氨醇杆菌BT1在微生物固定化方面的应用
将鞘氨醇杆菌BT1菌株与生物载体结合,制备成一种适用于河道水体并能缓慢释放的固定化微生物。
制备固定化菌株的步骤为:
菌株的发酵培养;采用液体发酵的方式在机械搅拌式发酵罐中进行鞘氨醇杆菌BT1的纯培养,培养基为异养硝化液体培养基。在30℃下进行纯培养达到12-48小时后,得到高浓度的鞘氨醇杆菌BT1纯培养菌液,菌液活菌计数在1×108-1×1011cfu/ml。
固态菌体的制备:菌株发酵结束后,离心去除发酵液,得到固态菌体。
生物载体的制备及固定化:(1)将活性炭10-50g/L、壳聚糖5-50g/L、海藻酸钠10-50g/L称量溶解并混合均匀,得到凝胶液。(2)将50-100g固态菌体接入凝胶液中,得到菌体浓度为1×108-1×1011cfu/ml的微生物凝胶液。(3)将微生物凝胶液按照15-25滴/分钟的速度滴入质量浓度为1-50g/L的CaCl2溶液中,得到微生物活性颗粒。(4)将微生物活性颗粒于5%戊二醛溶液中以1:50在室温下交联24小时。(5)将(4)中交联后的微生物活性颗粒用无菌水浸泡24-48小时后,增殖得到鞘氨醇杆菌BT1生物微胶囊。
获得的鞘氨醇杆菌BT1生物微胶囊在30天内的释放量达85%以上,菌存活率大于5%,有效活菌数经过验证后达到109cfu/g。
虽然本发明已以较佳实施例公开如上,但其并非用以限定本发明,任何熟悉此技术的人,在不脱离本发明的精神和范围内,都可做各种的改动与修饰,因此本发明的保护范围应该以权利要求书所界定的为准。
SEQUENCE LISTING
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Claims (8)
1.一株鞘氨醇杆菌(Sphingobacterium sp.),于2018年10月17日保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏编号为CGMCC No.16596,保藏地址为北京市朝阳区北辰西路1号院3号,中国科学院微生物研究所。
2.含有权利要求1所述鞘氨醇杆菌的微生物制剂。
3.根据权利要求2所述的微生物制剂,其特征在于,鞘氨醇杆菌的活菌含量≥1×109CFU/g。
4.根据权利要求2所述的微生物制剂,其特征在于,所述微生物制剂为生物微胶囊。
5.根据权利要求4所述的微生物制剂,其特征在于,所述生物微胶囊包含有壳体以及壳体内部芯材;壳体材料包括但不限于海藻酸钠凝胶;壳体内部芯材包括但不限于氯化钙溶液,所述壳体内部芯材中分散有1×108~1×1011CFU/mL所述鞘氨醇杆菌的细胞。
6.权利要求1所述鞘氨醇杆菌在化工、环境领域降解河道、湖泊含氮污染物或含氮污水处理方面的应用。
7.一种含氮污水处理方法,其特征在于,将权利要求1所述的鞘氨醇杆菌按终浓度10~30mg菌体/L污水投放至含氮污水中。
8.根据权利要求7所述的方法,其特征在于,所述鞘氨醇杆菌以生物微胶囊的形式投放至水体中。
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