CN111676151A - 一株耐低温聚磷菌及其应用 - Google Patents
一株耐低温聚磷菌及其应用 Download PDFInfo
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Abstract
本发明公开了一株耐低温聚磷菌及其应用,属于生物工程和环境工程技术领域。本发明中耐低温聚磷菌株聚磷菌命名为HCSW‑3,经鉴定属于不动杆菌属(Acinetobacter sp.),保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏日期为2019年8月14日,保藏号为CGMCC No.18385。将菌株Acinetobacter sp.HCSW‑3进行活化培养后,投入试验中,在低温条件下(10℃),对初始磷浓度为11mg/L的污水中磷的去除率高达100%。因此,Acinetobacter sp.HCSW‑3在低温条件下仍然具有良好的除磷效能,可应用于污水生物除磷及黑臭河道治理等。
Description
技术领域
本发明属于生物工程和环境工程技术领域,涉及一株耐低温聚磷菌及其应用。
背景技术
水体中较高浓度的磷是导致“水体富营养化”的主要原因之一。水体中的磷主要来源于人类生产活动所产生的生活污水、工业废水、农田径流及农业排水。随着环境问题的日渐突出,污水排放标准的日趋严格,如何经济、高效的去除污水及黑臭河道中的磷污染已成为水体污染防治领域的研究热点。生物除磷技术因其经济、环保等优点而得到广泛应用。到目前为止,聚磷菌株要集中在Pseudomonas菌属、Klebsiella菌属、RahnellaIzard菌属、Acinetobacter菌属、 Aeromonas菌属,但已报道的聚磷菌的除磷效能参差不齐,且研究多集中在降解基因和降解机理方面,实际应用研究有限。
温度是污水处理过程中一个重要的生态因子,其对活性污泥中微生物群落结构及功能有较大影响。寒冷恶劣的气候条件会严重抑制污水处理厂活性污泥的中微生物的活性,尤其对聚磷菌的活性有较大影响,可直接导致污水处理厂在低温期除磷效能不合格。因此,筛选出一株耐低温的高效聚磷菌,对于保证低温条件下生物除磷的效果具有重要的实际意义。
发明内容
针对现有技术存在的上述问题,本发明所要解决的技术问题在于提供一株耐低温聚磷菌。本发明所要解决的另一技术问题在于提供所述耐低温聚磷菌在污水除磷中的应用。
为了解决上述技术问题,本发明所采用的技术方案如下:
一株耐低温聚磷菌Acinetobacter sp.HCSW-3,所述的耐低温聚磷菌Acinetobacter sp.HCSW-3即不动杆菌HCSW-3,保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏号为CGMCC No.18385,保藏日期为2019年8 月14日,保藏地址为北京市朝阳区北辰西路1号院3号。
耐低温聚磷菌Acinetobacter sp.HCSW-3为革兰氏阴性杆菌,兼性好氧,接触酶阳性,氧化酶阴性,在pH 5.0-11.0、10-30℃条件下生长,无鞭毛;在固体培养基上生长为圆形乳白色半透明菌落,表面光滑,边缘整齐有隆起,能以多种有机物为碳源,具有良好的聚磷效能,可用于污水生物除磷。
耐低温聚磷菌Acinetobacter sp.HCSW-3的16S rDNA序列如SEQ ID NO.1 所示。
耐低温聚磷菌Acinetobacter sp.HCSW-3的专性培养基为配方如下: CH3COONa,3.23g/L;KH2PO4,35mg/L;CaCl2,19.39mg/L;NH4Cl,305.52 mg/L;MgSO4·7H2O,91.26mg/L;PIPES buffer,8.5g/L;微量元素,2mL。 pH调至7.0。其中,微量元素配方为:FeSO4·7H2O5g/L、CuSO4·5H2O 1.6g/L、 MnCl2·4H2O 5g/L、(NH4)6Mo7O24·4H2O 1.1g/L、H3BO3 0.05g/L、CoCl2·6H2O 0.05g/L、KI 0.01g/L。121℃条件下灭菌20min,冷却至室温后使用。
耐低温聚磷菌Acinetobacter sp.HCSW-3在污水除磷中的应用。
所述的应用为:在污水处理的厌氧池中投加耐低温聚磷菌Acinetobacter sp.HCSW-3菌种,使耐低温聚磷菌Acinetobacter sp.HCSW-3菌在厌氧池中挂膜并吸磷。
所述的应用的具体步骤包括:
1)制备所述耐低温聚磷菌Acinetobacter sp.HCSW-3的冻干菌粉;
2)停止厌氧池进出水,并将步骤1)所得低温聚磷菌Acinetobacter sp. HCSW-3的冻干菌粉投加到污水处理的厌氧池中,开启厌氧池的内循环;
3)耐低温聚磷菌Acinetobacter sp.HCSW-3菌在厌氧池中挂膜并吸磷。
步骤1)制备所述耐低温聚磷菌Acinetobacter sp.HCSW-3的冻干菌粉的具体方法为:使用专性培养基培养所述的耐低温聚磷菌Acinetobacter sp.HCSW-3,培养完成后,通过离心机离心去除70%的上清液,将剩下的高浓度菌液加入菌种保护剂玉米芯粉与海藻糖进行冷冻干燥,制成耐低温聚磷菌Acinetobacter sp. HCSW-3的冻干菌粉。
相比于现有技术,本发明的有益效果为:
本发明提供的耐低温聚磷菌Acinetobacter sp.HCSW-3在低温条件下仍然具有极强的除磷效能,将耐低温聚磷菌Acinetobacter sp.HCSW-3投入试验中,在低温条件下(10℃),对初始磷浓度高达11mg/L的污水中磷的去除率仍可达到100%。
附图说明
图1为菌株Acinetobacter sp.HCSW-3在低温条件下(10℃)的生长曲线图;
图2为菌株Acinetobacter sp.HCSW-3在低温条件下(10℃)对可溶性磷的降解净化结果图。
具体实施方式
下面结合具体实施例对本发明进一步进行描述。
实施例1:菌株HCSW-3的筛选及生理生化鉴定
(1)菌株来源
本发明所用菌源来源于低温条件下稳定运行的SBR中的活性污泥。
(2)培养基
为避免选用常规筛菌培养基进行菌种分离时得到菌落数目过多,并且得到菌落无法保证均有聚磷效果,徒增菌种分离的工作量。本发明选用专性培养基进行菌株的分离筛选。专性培养基配方如下:CH3COONa,3.23g/L;KH2PO4,35mg/L; CaCl2,19.39mg/L;NH4Cl,305.52mg/L;MgSO4·7H2O,91.26mg/L;PIPES buffer, 8.5g/L;微量元素,2mL。pH调至7.0。固体培养基加入1.8-2.0%的琼脂粉。其中,微量元素配方为:FeSO4·7H2O 5g/L、CuSO4·5H2O 1.6g/L、MnCl2·4H2O 5g/L、(NH4)6Mo7O24·4H2O 1.1g/L、H3BO3 0.05g/L、CoCl2·6H2O0.05g/L、KI 0.01g/L。所有培养基均于121℃条件下灭菌20min,冷却至室温后使用。
(3)筛选
通过涂布、划线分离等常规操作得到若干株初筛菌株。然后,通过好氧吸磷实验对菌株进行复筛。初步确定菌株是否有聚磷效能,并比较其聚磷能力的强弱。选择其中聚磷效能最好的菌株命名为HCSW-3。
(4)生理生化鉴定
通过对菌株HCSW-3进行一系列的生理生化实验,结果表明该菌株HCSW-3 为革兰氏阴性杆菌,兼性好氧,接触酶阳性,氧化酶阴性,在pH 5.0-11.0、10-30 ℃条件下生长,无鞭毛;HCSW-3杆菌在固体培养基上生长为圆形乳白色半透明菌落,表面光滑,边缘整齐有隆起,能以多种有机物为碳源,具有良好的聚磷效能,可用于污水生物除磷。
实施例2:菌株HCSW-3的种属鉴定
通过扩增该菌株的16S rDNA,得到了长度为1395bp的16S rDNA序列,如SEQ IDNO.1所示。PCR引物采用细菌通用引物7F 5′-CAGAGTTTGATCCTGGCT-3′和1540R 5′-AGGAGGTGATCCAGCCGCA-3′。用PCR仪进行扩增反应。
PCR反应体系(25μL):2.5μL10×PCR Buffer,1μL dNTPs(浓度为2.5 mmol/L),2种引物各0.5μL,0.5μL DNA模板,0.5μL rTaq DNA聚合酶(5U/μL),加双蒸水至25μL。
PCR扩增程序为:94℃预变性4min,94℃变性45s,55℃复性45s,72℃延伸1min,共30个循环,最后72℃延伸10min,1%琼脂糖凝胶电泳,EB染色后紫外检测。通过程序进行同源性比较,结果表明该菌株与Acinetobacter sp.的相似性为99%,说明HCSW-3属于Acinetobacter sp.。
实施例3:Acinetobacter sp.HCSW-3休眠细胞的制备
制备菌株Acinetobacter sp.HCSW-3的休眠细胞:将在专性培养基中培养好的菌株Acinetobacter sp.HCSW-3于8000r/min,4℃下离心10min,用磷酸缓冲液洗涤3次,用同样的缓冲液重悬该沉淀,使得休眠细胞浓度为10mg/L,于4 ℃冻存,即制得菌株Acinetobacter sp.HCSW-3的休眠细胞,备用,12个月内,再次活化的方法为使用专性培养基在36摄氏度下培养20小时。
实施例4:Acinetobacter sp.HCSW-3生长曲线测定
按照测定细菌生长曲线的标准方法对以实施例1中专性培养基培养的菌株Acinetobacter sp.HCSW-3的生长曲线进行测定,每隔8h取培养液,采用光电比浊法,在600nm波长处测定菌液OD600(optical Density),然后经0.22μm微孔滤膜过滤,检测滤液PO4 3-P,pH值等指标。获得菌株Acinetobacter sp.HCSW-3 生长曲线如图1所示。从图1中可以看出,低温条件下(10℃)培养,在0-32h 时,菌株HCSW-3处于生长阶段的延迟期,其生长速度缓慢,OD600无明显变化。当32-64h时,菌株HCSW-3进入对数生长期,其生长速度达到最大,OD600显著上升;在64h以后开始进入菌株生长的稳定期及衰亡期,稳定期后期OD600达到最大值(OD600=1.519),随后以微小差距逐渐下降,但其生长状态基本维持动态平衡。
实施例5:菌株Acinetobacter sp.HCSW-3除磷效能测定
将菌株Acinetobacter sp.HCSW-3按照实施例4中方法进行培养,在培养的过程中,每8h检测PO4 3--P浓度。结果如图2所示。
从图2中可以看出,在0-32h时,培养液中PO4 3--P浓度无变化,说明此阶段菌株未进行吸磷作用。当32-64h时,菌株HCSW-3进入对数生长期,培养液中PO4 3--P浓度在此阶段伴随菌株的生长逐渐下降,且吸磷速率逐渐升高,菌株培养到约56h可几乎完全将初始培养液中12mg/L的PO4 3--P浓度过量吸收至菌体内。在64h以后开始进入菌株生长的稳定期及衰亡期,但其生长状态基本维持动态平衡。
实施例6:Acinetobacter sp.HCSW-3应用于污水处理除磷
某企业污水主要为化工制药污水,污水排放要求总磷在5mg/L以内,COD 在400mg/L,氨氮在8mg/L以内,该企业原水COD在5000mg/L左右,总磷 135mg/L,氨氮95mg/L左右,采用的污水处理工艺为改良AO工艺,包括:预处理,厌氧处理,好氧处理,MBBR,沉淀池。在投加HCSW-3菌种前该企业污水的总磷与氨氮超标,出水总磷35mg/L,氨氮19mg/L。
将菌种的专性培养基(实施例1中专性培养基)在高压灭菌锅里121度灭菌 20分钟冷却至室温,菌种在实验室复壮,挑取好的单菌落接种到灭好菌的液体培养基里,在摇床上37度恒温培养36小时制取种子液,将做好的种子液接种到 5立方发酵罐,控制温度37度,转速150R/小时,pH在6.8-7.5之间,发酵48 小时,通过离心机离心去除70%的上清液,将剩下的高浓度菌液加入菌种保护剂玉米芯粉与海藻糖进行冷冻干燥最终制成菌株HCSW-3的粉剂产品。由于该企业污水不停,所以为了减少菌种的流失,2019年10月25日开始,停止厌氧池进出水3-5天,将HCSW-3菌种分10天投加投加厌氧池,按照池子溶积每立方投加200克菌株HCSW-3的粉剂产品,并打开厌氧池的内循环。菌种的投加在厌氧池挂膜成功,提高了厌氧的处理效率,在投加第五天出现明显的效果,投加十天后,出水总磷在2.5-3mg/L之间,氨氮在3.5-4.2mg/L之间,总磷与氨氮达到该企业污水进入管网的要求。
序列表
<110> 扬州市海诚生物技术有限公司
<120> 一株耐低温聚磷菌及其应用
<130> 100
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1395
<212> DNA
<213> 不动杆菌HCSW-3(Acinetobacter sp.HCSW-3)
<400> 1
tcgagcggat gaagtagctt gctactgaat ttagcggcgg acgggtgagt aatacttagg 60
aatctgccta ttagtggggg acaacatttc gaaaggaatg ctaataccgc atacgcccta 120
cgggggaaag caggggctct tcggaccttg cgctaataga tgagcctaag tcagattagc 180
tagttggtgg ggtaaaggcc taccaaggcg acgatctgta acgggtctga gaggatgatc 240
cgtcacactg ggactgagac acggcccaga ctcctacggg aggcagcagt ggggaatatt 300
ggacaatggg gggaaccctg atccagccat gccgcgtgtg tgaagaaggc cttttggttg 360
taaagcactt taagcgagga ggaggctcct atagataata cctatagaga gtggacgtta 420
ctcgcagaat aagcaccggc taactctgtg ccagcagccg cggtaataca gagggtgcaa 480
gcgttaatcg gatttactgg gcgtaaagcg tacgtaggtg gtcttttaag tcggatgtga 540
aatccctgag cttaacttag gaattgcatt cgatactggg agactagagt atgggagagg 600
atggtagaat tccaggtgta gcggtgaaat gcgtagagat ctggaggaat accgatggcg 660
aaggcagcca tctggcctaa tactgacact gaggtacgaa agcatgggga gcaaacagga 720
ttagataccc tggtagtcca tgccgtaaac gatgtctact agccgttggg gtctttgaga 780
ctttagtggc gcagctaacg cgataagtag accgcctggg gagtacggtc gcaagactaa 840
aactcaaatg aattgacggg ggcccgcaca agcggtggag catgtggttt aattcgatgc 900
aacgcgaaga accttacctg gtcttgacat agtaagaact ttccagagat ggattggtgc 960
cttcgggagc ttacatacag gtgctgcatg gctgtcgtca gctcgtgtcg tgagatgttg 1020
ggttaagtcc cgcaacgagc gcaacccttt tccttatttg ccagcgagta atgtcgggaa 1080
ctttaaggat actgccagtg acaaactgga ggaaggcggg gacgacgtca agtcatcatg 1140
gcccttacga ccagggctac acacgtgcta caatggtcgg tacaaagggt tgctacctag 1200
cgataggatg ctaatctcaa aaagccgatc gtagtccgga ttggagtctg caactcgact 1260
ccatgaagtc ggaatcgcta gtaatcgcgg atcagaatgc cgcggtgaat acgttcccgg 1320
gccttgtaca caccgcccgt cacaccatgg gaatttgttg caccagaagt aggtagtcta 1380
accgtaagga ggacg 1395
Claims (7)
1.一株耐低温聚磷菌Acinetobacter sp.HCSW-3,其特征在于,所述的耐低温聚磷菌Acinetobacter sp.HCSW-3,保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏号为CGMCC No.18385,保藏日期为2019年8月14日,保藏地址为北京市朝阳区北辰西路1号院3号。
2.权利要求1所述的耐低温聚磷菌Acinetobacter sp.HCSW-3的专性培养基,其特征在于,所述的专性培养基配方为:CH3COONa,3.23g/L;KH2PO4,35mg/L;CaCl2,19.39mg/L;NH4Cl,305.52mg/L;MgSO4·7H2O,91.26mg/L;PIPES buffer,8.5g/L;微量元素,2mL;pH调至7.0。
3.根据权利要求2所述的耐低温聚磷菌Acinetobacter sp.HCSW-3的专性培养基,其特征在于,所述的微量元素的配方为:FeSO4·7H2O 5g/L、CuSO4·5H2O 1.6g/L、MnCl2·4H2O5g/L、(NH4)6Mo7O24·4H2O 1.1g/L、H3BO3 0.05g/L、CoCl2·6H2O 0.05g/L、KI 0.01g/L。
4.权利要求1所述的耐低温聚磷菌Acinetobacter sp.HCSW-3在污水处理中除磷的应用。
5.根据权利要求4所述的耐低温聚磷菌Acinetobacter sp.HCSW-3在污水处理中除磷的应用,其特征在于,在污水处理的厌氧池中投加耐低温聚磷菌Acinetobacter sp.HCSW-3菌种,使耐低温聚磷菌Acinetobacter sp.HCSW-3菌在厌氧池中挂膜并吸磷。
6.根据权利要求5所述的耐低温聚磷菌Acinetobacter sp.HCSW-3在污水处理中除磷的应用,其特征在于,所述的应用的具体步骤包括:
1)制备耐低温聚磷菌Acinetobacter sp.HCSW-3的冻干菌粉;
2)停止厌氧池进出水,并将步骤1)所得低温聚磷菌Acinetobacter sp.HCSW-3的冻干菌粉投加到污水处理的厌氧池中,开启厌氧池的内循环;
3)耐低温聚磷菌Acinetobacter sp.HCSW-3菌在厌氧池中挂膜并吸磷。
7.根据权利要求6所述的耐低温聚磷菌Acinetobacter sp.HCSW-3在污水处理中除磷的应用,其特征在于,步骤1)制备所述耐低温聚磷菌Acinetobacter sp.HCSW-3的冻干菌粉的具体方法为:使用权利要求2所述的专性培养基培养所述的耐低温聚磷菌Acinetobactersp.HCSW-3,培养完成后,通过离心机离心去除70%的上清液,将剩下的高浓度菌液加入菌种保护剂玉米芯粉与海藻糖进行冷冻干燥,制成耐低温聚磷菌Acinetobacter sp.HCSW-3的冻干菌粉。
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