CN116891809B - 一株亚洲假单胞菌及微生物菌剂和应用 - Google Patents
一株亚洲假单胞菌及微生物菌剂和应用 Download PDFInfo
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- CN116891809B CN116891809B CN202211556971.5A CN202211556971A CN116891809B CN 116891809 B CN116891809 B CN 116891809B CN 202211556971 A CN202211556971 A CN 202211556971A CN 116891809 B CN116891809 B CN 116891809B
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
- C12N9/1229—Phosphotransferases with a phosphate group as acceptor (2.7.4)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
- C12Y207/04—Phosphotransferases with a phosphate group as acceptor (2.7.4)
- C12Y207/04001—Polyphosphate kinase (2.7.4.1)
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2101/00—Nature of the contaminant
- C02F2101/10—Inorganic compounds
- C02F2101/105—Phosphorus compounds
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2101/00—Nature of the contaminant
- C02F2101/10—Inorganic compounds
- C02F2101/16—Nitrogen compounds, e.g. ammonia
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
- Y02W10/00—Technologies for wastewater treatment
- Y02W10/10—Biological treatment of water, waste water, or sewage
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- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
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- General Engineering & Computer Science (AREA)
- Medicinal Chemistry (AREA)
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- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Molecular Biology (AREA)
- Biodiversity & Conservation Biology (AREA)
- Hydrology & Water Resources (AREA)
- Environmental & Geological Engineering (AREA)
- Water Supply & Treatment (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Purification Treatments By Anaerobic Or Anaerobic And Aerobic Bacteria Or Animals (AREA)
Abstract
本发明涉及微生物技术领域,特别涉及一株亚洲假单胞菌及微生物菌剂和应用。该菌株的保藏编号为CCTCC NO:M2022729。该菌株对废水中的磷和氮均有显著的去除效果,除磷效果尤其显著,可达100%,可用于天然水体生态改造或原位修复。且具有广泛碳源浓度适应性,可用于低碳源废水的处理。
Description
技术领域
本发明涉及微生物技术领域,特别涉及一株亚洲假单胞菌及微生物菌剂和应用。
背景技术
随着人类的活动,水体富营养化已成为全球性问题,许多研究发现导致富营养化的主要原因之一是富含磷的生活废水、农业废水、工业废水排放至水体中。因此,从水体中除去积聚的磷是防治水体富营养化的关键,污水处理目前主要使用化学除磷和生物除磷,强化生物除磷法(EBPR)利用除磷菌(PAOs)在厌氧条件下水解胞内多聚磷酸盐(poly-P)释放磷,在好氧条件下从污水中将过量磷聚集到胞内积累poly-P的特性,最后将含有除磷菌的富磷污泥分排出而使磷从污水中去除。其中增强型生物除磷***由于其成本低、效益高而被广泛应用。除磷菌是一类对磷超量吸收的细菌,不是单一的微生物而是由不同的微生物群落组成,它们能将磷以多聚磷酸盐颗粒的形式存在于细胞内。Poly-P的形成在除磷微生物“超量吸磷”过程中起到至关重要的作用,科学家研究发现许多的生物体内都有poly-P存在,poly-P可作为生物的磷酸储备用于提供磷酸酐键。研究发现poly-P的形成与多聚磷酸盐激酶(PPK1)密切相关,在PPK1的作用下,ATP末端的磷酸残基转移到poly-P链上,并且这种催化是可逆的。2002年,Ishige K等从铜绿假单胞菌中发现另一种多聚磷酸盐激酶(ppk2),这种酶既能催化GTP上的磷酸基团转移到poly-P上,同时又可催化poly-P生成GTP的酶PPK2,然而其除氮除磷效果并不理想。
发明内容
有鉴于此,本发明提供了一株亚洲假单胞菌及微生物菌剂和应用,该菌株对废水中的磷和氮具有显著的去除效果,且具有广泛碳源浓度适应性,可用于低碳源废水的处理。
本发明随机采集了浙江、江苏和山东等地的污水处理厂污泥样品,总共获得85株初筛菌株,有41株菌株的除磷能力达到20%以上,其中Pseudomonas asiatica ZM16对人工废水最高除磷率和去氮率分别达到100%和75.9%,通过对该菌株全基因组测序后,并从中鉴定出了1个ppk1基因、1个ppk2基因,为富营养化水体的快速、高效处理提供了有效的菌种资源和基因资源。
本发明提供了保藏编号为CCTCC NO:M2022729的亚洲假单胞菌(Pseudomonasasiatica)ZM16。
ZM16菌株按照以下方法分离鉴定:
(1)分别配制培养基A-E:
培养基A为菌株分离、纯化、保藏培养基,其组成为:每升培养基A包含蛋白胨10g,酵母提取物5g,乙酸钠2g,琼脂19g,其余为水,调节pH至7.0~7.2。
培养基B为低P(1.8mg/L)培养基,其组成为:每升培养基B包含CH3COONa 3.42g,K2HPO410 mg,(NH4)2SO41.18 g,NH4Cl 0.955g,CaCl260mg,MgSO482 mg,HEPES 7g,微量元素2mL,其余为水,调节pH至7.0~7.2。
培养基C为富P(19.9mg/L)培养基,其组成为:每升培养基C包含CH3COONa 3.42g,MgSO482 mg,FeSO43.7 mg,CaCl260 mg,(NH4)2SO41.18g,NH4Cl 0.955g,酵母提取物0.1g,KNO31.81 g(N 250mg/L)、NaNO21.22g(N 250mg/L)、KH2PO456 mg,K2HPO440 mg,HEPES 7g,微量元素2mL,其余为水,调节pH至7.0~7.2。
培养基D为富磷硝酸盐培养基,其组成为:每升培养基D包含CH3COONa 3.42g,MgSO482 mg,FeSO43.7 mg,CaCl260 mg,NH4Cl 191.1mg,KNO31.81 g(N 250mg/L),(NH4)2SO4235 mg,酵母提取物0.1g,KH2PO456mg,K2HPO440 mg,HEPES 7g,微量元素2mL,其余为水,调节pH至7.0~7.2。
培养基E为富磷亚硝酸盐培养基,其组成为:每升培养基E包含CH3COONa 3.42g,MgSO482 mg,FeSO43.7 mg,CaCl260 mg,NH4Cl 191.1mg,NaNO21.22 g(N 250mg/L),(NH4)2SO4235 mg,酵母提取物0.1g,KH2PO456 mg,K2HPO440 mg,HEPES 7g,微量元素2mL,其余为水,调节pH至7.0~7.2。
所述微量元素的组成为:每升微量元素包含FeSO4·7H2O 100mg,H3BO320mg,CuSO4·5H2O20 mg,AlK(SO4)2·12H2O 15mg,KI 100mg,MnSO4·7H2O11mg,60mg,ZnSO4·7H2O100mg,CoCl2·6H2O 100mg,Na2MoO450 mg,乙二胺四乙酸10g,其余为水。
(2)富集培养
取不同处理厂污水处理池活性污泥20g至100mL无菌水中于30℃振荡培养12h,取10mL振荡培养后的混合液转接到100mL磷浓度为5mg/L的培养基B中,30℃富集培养3d,转接至培养基C中培养3天为一轮,共转接4轮,富集培养24d,得到富集培养液。
(3)分离纯化
将所述富集培养液稀释至10-6~10-3,吸取500μl稀释液于分离纯化平板中,在40℃~42℃下与培养基A混合,凝固后倒置,于30℃恒温培养出菌落;挑取所述平板上的单菌落在平板上进行多次划线纯化,观察显示无杂菌,即得到纯化菌株;挑取生长迅速的菌落接种于涂布100μL的BCIP显色剂的培养基培养,将生长迅速的蓝绿色菌落作为候选菌株。
(4)除磷菌筛选
将所述纯化菌株接种于装有100mL培养基B的锥形瓶中,在130r/min,30℃下振荡培养24h,得预培养液,将所述预培养液按10%转接到装有100mL培养基C的锥形瓶中,在130r/min,30℃下振荡培养2d;其中,先厌氧培养24h再好氧培养24h;培养完成后取培养液8000g离心10min取上清液,使用钼锑抗分光光度法测量液体中的磷含量,考察菌株对总磷的去除率,从而筛选出一株具有较高除磷效果的除磷菌ZM16,其16S rRNA基因的核苷酸序列如SEQ ID NO.1所示。
本发明中ZM16菌株分离自浙江杭州天子岭垃圾处理厂污水处理池污泥,在LB培养基和无机盐乙酸钠碳源培养基上呈淡黄色圆形菌落,光泽圆润,边缘整齐。经革兰氏染色、DAPI染色和尼罗红染色后,鉴定该菌为革兰氏阴性菌,菌体中有被染成绿色的异染粒(polyP)和被染成红色类脂性颗粒(PHB)。扫描电镜观察菌体呈杆状,无鞭毛、菌毛、微荚膜等特殊的细胞结构,生理生化特征如表1。
表1:ZM16菌株生理生化鉴定
根据ZM16的形态特征和生理生化特征,结合菌株ZM16的16S rRNA序列比较鉴定,表明其与Pseudomonas asiatica RYU5(T)(GeneBank:MH517510.1)的16S rRNA序列的相似性最高,达到99.44%,最终将其归类于假单胞菌属,命名为Pseudomonas asiatica ZM16,保藏在中国典型培养物保藏中心,保藏编号为CCTCC NO:M2022729,保藏日期为2022年5月26日。
本发明进一步解析了菌株Pseudomonas asiatica ZM16的除磷机制,采用二代+三代测序技术联用完成高效除磷菌株Pseudomonas asiatica ZM16的基因组扫描测序,利用Unicycler拼接软件对优化序列进行多个Kmer参数的拼接,得到最优的组装结果后,再运用Pilon软件对组装结果进行局部内洞填充和碱基校正。依据拼接序列的总长、scaffold的数量以及scaffoldN50等技术指标,对多个Kmer的组装结果进行综合评定,最终ZM16基因组测序分析获得了基因组大小为5543632bp,GC含量58.12%。
利用Glimmer 3.02软件进行细菌的基因预测,将预测基因的蛋白序列分别与NR、GENES、STRING和GO数据库进行blastp比对(BLAST 2.2.28+),鉴定菌株ZM16含1个ppk1基因和1个ppk2基因,命名为ppk1-zm16(如SEQ NO.2所示)和ppk2-zm16(如SEQ NO.3所示),开放阅读框(ORF)大小分别为2241bp和981bp,分别编码747个氨基酸(如SEQ NO.4所示)和327个氨基酸(如SEQ NO.5所示)。
多聚磷酸盐激酶基因ppk1-zm16和ppk2-zm16,其特征序列扩增方法如下:
以菌株Pseudomonas asiatica ZM16基因组为模板,利用PCR的方法获得完整的基因片段ppk1-zm16和ppk2-zm16。
其中,上述PCR使用的引物如表2:
表2:ppk1和ppk2基因引物序列信息表
实验表明,ZM16菌株对农污出水、农污原水和乳品厂原水的去氮除磷率均为100%。
本发明还提供了所述的亚洲假单胞菌在废水除磷、除氮中的应用。
其中,所述废水包括农业污水、农村生活污水、乳制品厂污水、化肥厂污水、厨余废水中的至少一种。所述废水中的含磷量≤40mg/L,含硝氮亚硝氮量≤100mg/L。
本发明还提供一种除磷除氮的微生物菌剂,包含亚洲假单胞菌ZM16菌株。
本发明还提供一种处理废水的方法,包括向含氮和/或磷的废水中施加所述的亚洲假单胞菌ZM16菌株或所述的微生物菌剂。
本发明提供的处理废水的方法中,向废水中施加所述亚洲假单胞菌至亚洲假单胞菌的OD值≥0.1。
本发明提供了多聚磷酸盐激酶基因,其核苷酸序列为1)~3)中任意一种:
1)、SEQ ID NO:2或SEQ ID NO:3所示的核苷酸序列;
2)、在1)所示的核苷酸序列中取代、缺失或添加一个或多个核苷酸,且与1)所示的核苷酸序列编码相同的蛋白或编码的蛋白功能相同或相似的核苷酸序列;
3)、与1)或2)所示的核苷酸序列至少有90%同源性且编码的蛋白功能相同或相似的核苷酸序列。
本发明还提供了所述的多聚磷酸盐激酶基因编码的蛋白,其氨基酸序列为:
I)、SEQ ID NO:4或SEQ ID NO:5所示的氨基酸序列;
II)、在I)所示的氨基酸序列中取代、缺失或添加一个或多个氨基酸且与I)所示氨基酸序列的蛋白功能相同或相似的氨基酸序列;
III)、与I)或II)所示的氨基酸序列至少有90%同源性且蛋白功能相同或相似的氨基酸序列。
本发明提供的亚洲假单胞菌Pseudomonas asiatica ZM16对废水中的磷和氮具有显著的去除效果,除磷率达100%,可用于天然水体生态改造或原位修复;且具有广泛碳源浓度适应性,可用于低碳源废水的处理。
附图说明
图1是Pseudomonas asiatica ZM16菌体染色图;1-a:PHB染色图;1-b:DAPI多聚磷酸盐颗粒染色图;
图2是Pseudomonas asiatica ZM16菌体的扫描电镜图,2-a为5,000倍扫描电镜下的菌株ZM16观察结果,2-b为30,000倍扫描电镜下的菌株ZM16观察结果;
图3是除磷基因琼脂糖凝胶电泳图;3-a为多聚磷酸盐激酶1基因琼脂糖凝胶电泳图,ppk1-zm16基因PCR产物;3-b为多聚磷酸盐激酶2基因琼脂糖凝胶电泳图,ppk2-zm16基因PCR产物;
图4是不同碳氮磷比的除磷性能图;
图5是不同碳源的除磷性能图;
图6是不同pH的除磷性能图;
图7是最优除磷反硝化性能图,7-a为除磷结果,7-b为反硝化结果;
图8是除磷范围性能图;
图9是人工合成废水除磷反硝化性能图,9-a为除磷结果,9-b为反硝化结果;
图10是污水处理厂废水除磷反硝化性能图;10-a为除磷结果,10-b为对硝氮的去除效果,10-c为对亚硝氮的去除效果。
生物保藏说明
Pseudomonas asiatica ZM16,于2022年5月26日保藏在中国典型培养物保藏中心,地址为:中国,武汉,武汉大学,保藏编号为CCTCCNO:M 2022729。
具体实施方式
为了便于对本发明的进一步理解,下面提供的实施例对其做了更详细的说明。但是这些实施例仅供更好的理解发明,而并非用来限定本发明的范围或实施原则,本发明的实施方式不限于以下内容。
以下实施例中所用的酶、试剂盒及其他试剂均购自南京诺唯赞生物科技有限公司。
以下实施例中所用引物由北京擎科生物科技有限公司合成。
实施例1:除磷菌Pseudomonas asiaticaZM16的分离与鉴定
1.样品处理
在无菌条件下,取浙江杭州天子岭垃圾处理厂污水处理池污泥活性污泥20g至已灭菌的含玻璃珠的100mL无菌水中于30℃振荡培养12h,取10mL振荡培养后的混合液转接到100mL磷浓度为5mg/L的培养基B中,30℃富集培养3d,转接至100mL磷浓度为49.5mg/L培养基C中培养3天为一轮,无菌条件下共转接4轮,富集培养24d,得到富集培养液。
2.分离纯化
将所述富集培养液在无菌条件下稀释至10-6~10-3,吸取500μl稀释液于分离纯化平板中,在40℃~42℃下与20mL培养基A混合,凝固后倒置,于30℃恒温培养出菌落;挑取所述平板上的单菌落在平板上进行多次“z字型”划线纯化,至观察显示无杂菌,即得到纯化菌株;挑取生长迅速的菌落接种于涂布100μL的BCIP显色剂的培养基B培养,将生长迅速的蓝绿色菌落作为候选菌株。结合细菌的形态特征和生理生化特征和16S rRNA序列对上述筛选的菌株进行菌种鉴定。
3.除磷菌筛选
将所述纯化菌株接种于装有100mL培养基B的锥形瓶中,在130r/min,30℃厌氧下振荡培24h,得预培养液,将所述预培养液按10%转接到装有100mL培养基C的锥形瓶中,在130r/min,30℃下振荡培养2d;其中,先厌氧培养24h再好氧培养24h;培养完成后取培养液8000g离心10min取上清液,使用钼锑抗分光光度法710nm测量液体中的磷含量,考察菌株对总磷的去除率,从而筛选出的具有较高除磷效果的除磷菌。
4.染色鉴定
1)菌体厌氧培养后PHB鉴定
取厌氧培养至对数生长期后期的菌液适量,8000g离心5min,去除上清,沉淀菌体用0.1M PBS缓冲溶液洗两次,重悬细胞至OD600=0.8,1mL菌液加15μL尼罗红染料(质量浓度为0.1mg/mL丙酮溶液),室温下混匀染色5min,激发波长480nm,吸收波长为575nm,荧光显微镜下观察,细胞内红色为PHB。结果如图1-B。
2)菌体好氧培养后poly-P鉴定
取好氧培养至对数生长期后期的菌液适量,8000g离心5min,去除上清,沉淀菌体用0.1M PBS缓冲溶液洗两次,重悬细胞至OD600=0.5,1mL菌液加100μL DAPI染液(质量浓度为1mg/mL溶液),室温下避光染色10min,激发波长480nm,吸收波长为575nm,荧光显微镜下观察,细胞内绿色荧光为poly-P颗粒。结果如图1-C。
5.形态特征的鉴定
1)扫描电镜
用接种环挑取单菌落于5mL LB培养基中,30℃,130rpm培养12h后,4℃,6000×g离心5min收集500μL菌液,用预冷的0.1MPBS洗涤菌体3次后,将菌体悬浮在500μL预冷的2.5%戊二醛固定液中,4℃避光固定24h;0.1M PBS清洗三次;加入400μL 1%锇酸固定2h;先后用30%、40%、50%、60%、70%、80%、90%、95%乙醇洗脱15min,再用无水乙醇脱水15min(重复二次);在通风厨中,将样品浸入六甲基二硅胺烷(HMDS)中15min,重复两次;室温下,将样品放入临界干燥仪中进行CO2置换干燥60min,用剪刀剪裁盖玻片,将适合的样品贴在铜板上;在溅射仪中进行金属铂镀膜,镀金厚度约20-30nm;在扫描电子显微镜下,选取适宜的放大倍数观察样品,并拍照,结果如图2。
2)基因组DNA提取
使用微生物基因组DNA快速抽提试剂盒进行提取,并通过1.0%琼脂糖电泳检测,结果如图3-A所示。
3)16S rRNA的PCR扩增与鉴定
选用细菌16S rRNA序列通用引物F27/R1492进行PCR扩增,F27引物序列为:5-AGAGTTTGATCCTGGCTCAG-3’,R1492引物序列为:5’-GGTTACCTTGTTACGACTT-3’;
PCR反应体系组成:PCR mix buffer 25μL,3’Primer 1μL,5’Primer 01μL,DNA模版2μL,ddH2O 21μL,总体积为50μL。
PCR扩增条件为:94℃预变性5min;30个循环:94℃变性1min,50℃退火1min,72℃延伸2min;最后72℃温育10min,4℃保存。
取6μL DNA产物用1.0%琼脂糖胶电泳检测。
经BLAST软件与GenBank中已登录的16S rRNA序列进行比对,菌株ZM16与Pseudomonas asiatica的同源性为99.6%,ANI值为98.72%,属于假单胞菌属。
实施例2本发明菌株ZM16的最佳去氮除磷条件
1.钼锑抗分光光度法测定总磷,具体方法如下:
配制磷标准使用溶液。分别取0、0.2、0.4、0.8、1.2、1.6、2.0、3.0、4.0、5.0mL磷标准溶液于带盖试管中,加入蒸馏水至总体积为10mL,加入1.6mL过硫酸钾,混匀,120℃消解30min。冷却后,加入0.4mL抗坏血酸溶液,混匀;30s后,加入0.8mL钼酸盐混合液,混匀,室温静置15min。700nm波长下,以1号为空白参照,测定其吸光度,并绘制标准曲线。
2.最佳碳氮磷比例测定
用本发明保藏的菌株ZM16富集培养后接种于不同碳氮磷比例的培养基C中,初始OD600=0.1,C:N:P分别为25:10:1、50:10:1、100:10:1、200:10:1和300:10:1,其中N源分别由NH4CL-N:KNO3-N:NaNO2-N=1:2:2组成;30℃下振荡培养48h,其中先厌氧培养24h,再好氧培养24h,48h后检测培养基的OD600值及除磷率,检测结果如图5所示。由图1结果可知,本发明菌株具有广泛碳源浓度适应性,在C:N:P 50:10:1至300:10:1内都有较好的除磷效果,可用于低碳源条件下的废水处理。
3.最佳碳源测定
用本发明保藏的菌株ZM16富集培养后接种于不同碳源的富磷培养基C中,初始OD600=0.1,在30℃振荡培养48h,其中先厌氧培养24h,再好氧培养24h,48h后检测培养基的OD600值及除磷率,检测结果如图6所示。上述不同碳源的富磷培养基是指培养基C,以等含碳量葡萄糖替代培养基C中乙酸钠所得到的培养基C1,以等含碳量果糖替代培养基C中乙酸钠所得到的培养基C2,以等含碳量蔗糖替代培养基C中乙酸钠所得到的培养基C3,以等含碳量乳糖替代培养基C中乙酸钠所得到的培养基C4;以等含碳量淀粉替代培养基C中乙酸钠所得到的培养基C5。由图6结果可知,本发明菌株能很好的利用各种碳源,最适碳源为乙酸钠。
4.最佳pH值测定
用本发明保藏的菌株ZM16富集培养后接种于不同pH的培养基C中,初始OD600=0.1,在20℃下振荡培养48h,其中先厌氧培养24h,再好氧培养24h,48h后检测培养基C的OD600值及除磷率,检测结果如图7所示。上述不同pH值的培养基分别指:培养基C;培养基C6:成分与培养基C相同,pH5.0;培养基C7:成分与培养基C相同,pH6.0;培养基C8:成分与培养基C相同,pH8.0;培养基C9:成分与培养基C相同,pH9.0;培养基C10:成分与培养基C相同,pH10.0;由图7结果可知,本发明菌株在pH7-9的范围内,具有很强的除磷活性。
5.最佳条件下除磷能力测定
用本发明保藏的菌株ZM16富集培养后接种于培养基C中,初始OD600=0.1,20℃下振荡培养48h,其中先厌氧培养24h,再好氧培养24h,整个培养过程中的实时OD值和除磷率、反硝化率和反亚硝化率如图7所示,48h后除磷率达到100%,反硝化率和反亚硝化率分别达到85.6%和92.3%。
实施例3:除磷菌除磷范围的测定
用本发明保藏的菌株富集培养后接种于最适条件培养基C中,初始OD600=0.1,pH=7.5,保持C含量不变,调整C:N为10:1,其中N源分别由NH4Cl-N:KNO3-N:NaNO2-N=1:2:2组成,并分别设置P含量为1mg/L、2mg/L、4mg/L、8mg/L、16mg/L、24mg/L、32mg/L和40mg/L;30℃下振荡培养48h,其中先厌氧培养24h,再好氧培养24h,48h后检测培养基的OD600值及除磷率,检测结果如图8所示。由图8可知,本发明菌株48h内能去除至多28.16mg/L的P,对P含量20mg/L以内的水体具有100%的除磷率。
实施例4:人工合成废水除磷率的测定
1.合成废水培养基配方如下:
三水合醋酸钠890mg、酵母粉10mg、蛋白胨100g、K2HPO456.25mg、KH2PO444 mg、KNO3288 mg、NaNO2197 mg、CaCl228 mg、NaCl 50mg、NaHCO375 mg、MgSO475 mg、ddH2O 1L。P含量为20mg/L。
2.菌株的除磷率:用本发明保藏的菌株接种于培养基C中,富集培养12h后厌氧培养12h,共培养24h后接种于200ml合成培养基中,初始OD600=1,好氧培养24h后检测培养基的OD600值及去氮除磷量,检测结果如图9所示。计算ZM16的除磷量,结果为20mg/L(100%),硝氮和亚硝氮去除量分别平均为33.3mg/L(83.5%)和27.3mg/L(68.3%)。
实施例5:污水处理厂污水去氮除磷
取浙江金华一处农业污水原水和出水以及一处乳品厂污水,分别测定pH、总磷、硝氮和亚硝氮含量如下:
表3
将上述ZM16菌株分别接种于装有100mL农污原水、农污出水和乳品污水的锥形瓶中,初始OD600=1,在130r/min,30℃下好氧振荡培养6h;培养完成后取培养液8000g离心10min取上清液,测定上清液的总磷、硝氮和亚硝氮浓度,考察菌株对总磷、硝氮和亚硝氮的去除率,检测结果如图10所示。结果ZM16的去氮除磷率均为100%。
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
Claims (8)
1.亚洲假单胞菌(Pseudomonas asiatica),其特征在于,其保藏编号为CCTCC NO:M2022729。
2.根据权利要求1所述的亚洲假单胞菌,其特征在于,其16S rRNA基因的核苷酸序列如SEQ ID NO. 1所示。
3.权利要求1或2所述的亚洲假单胞菌在废水除磷、除氮中的应用。
4.根据权利要求3所述的应用,其特征在于,所述废水包括农业污水、农村生活污水、乳制品厂污水、化肥厂污水、厨余废水中的至少一种。
5.根据权利要求3所述的应用,其特征在于,所述废水中的含磷量≤40 mg/L,含硝氮亚硝氮量≤ 100 mg/L。
6.一种除磷除氮的微生物菌剂,其特征在于,包含权利要求1或2所述的亚洲假单胞菌。
7.一种处理废水的方法,其特征在于,包括向含磷和/或氮的废水中施加权利要求1或2所述的亚洲假单胞菌或权利要求6所述的微生物菌剂。
8.根据权利要求7所述的方法,其特征在于,向废水中施加所述亚洲假单胞菌至亚洲假单细胞菌的OD值≥0.1。
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