CN108004170B - 一种气单胞菌属菌株r1及制备方法及其在溶藻降解微囊藻毒素上的应用 - Google Patents
一种气单胞菌属菌株r1及制备方法及其在溶藻降解微囊藻毒素上的应用 Download PDFInfo
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Abstract
本发明公开了一种气单胞菌属菌株R1及制备方法及其在溶藻降解微囊藻毒素上的应用。通过从镜湖中腐烂的水华中筛选得到了一株菌株R1,该菌株具有较强的耐镉能力、溶藻以及可降解微囊藻毒素,经16S rDNA序列对比分析鉴定,R1属于气单胞菌属(Aeromonas)。该菌株对Cd2+有很好的耐受性,在含有50mg/LCd2+的培养基中仍能正常生长;细菌通过分泌胞外物质溶解铜绿微囊藻,且该物质耐高温,非核酸和多糖类等物质;在0.1mg/L Cd2+存在的情况下,细菌溶藻能力增强;在微囊藻毒素的初始浓度为1.84mg/L时,7天时间降解率为43.5%。
Description
技术领域
本发明涉及一种气单胞菌属菌株R1及制备方法及其在溶藻降解微囊藻毒素上的应用。
背景技术
近年来,许多城市的河道、湖泊等水体都受到了污染。不仅氮磷超标,造成水体富营养化程度越来越严重,蓝藻水华频发,产生了许多种毒素,包括最危险的微囊藻毒素,而且重金属物质大量留存,影响人体健康。
对于这类复合污染水体,单一的物理、化学方法都难以修复或控制,还可能对水体造成二次污染,而微生物去除蓝藻和微囊藻毒素具有效率高、安全性强和环境友好等优势。已有研究发现的溶藻微生物包括:真菌类的赤霉菌属、青霉菌属,细菌类的假单胞菌、交替假单胞菌、寡养单胞菌属和降解微囊藻毒素的不动杆菌、芽孢杆菌、铜绿假单胞菌、假单胞菌、伯克氏菌等。但大多数研究只针对微生物的溶藻或降解藻毒素单一功能,对于复合污染水体,特别是重金属污染情况下的实际溶藻或降解藻毒素能力的研究较少。
发明内容
针对上述不足,本发明提供了一种气单胞菌属菌株R1及制备方法及其在溶藻降解微囊藻毒素上的应用。所述菌株R1具有较强的耐镉能力、溶藻以及可降解微囊藻毒素的能力。
本发明采取的技术方案为:
一种气单胞菌属(Aeromonas)菌株R1CGMCC No.14864,所述菌株R1已经于2017年11月6保藏于中国微生物菌种保藏委员会普通微生物中心 (CGMCC),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,保藏编号为CGMCC No.14864,建议的分类命名为气单胞菌Aeromonas sp.。
菌株R1的基因序列表如SEQUENCE LISTING<400>1所示。
菌株R1的菌落形态为圆形,乳白色,光滑半透明,边缘整齐,较粘稠,易挑起;革兰氏染色阴性,菌体呈短杆状,无芽孢;能利用葡萄糖产生有机酸但不产气;不能分解含硫有机物和不能分解色氨酸产生吲哚;与大肠杆菌,金黄色葡萄球菌和枯草芽孢杆菌共培养做抑菌实验,它们相互之间无影响。
本发明还提供了所述的气单胞菌属(Aeromonas)R1的应用,菌株R1可降解含镉废水中的微囊藻毒素并且具有溶藻的能力。其溶藻的机理是间接溶藻,通过分泌的丙氨酸和苯丙氨酸协同作用进行溶藻。
本发明还提供了所述气单胞菌属(Aeromonas)菌株R1的制备方法,其特征在于,所述制备方法包括以下步骤:
S1:将从安徽省芜湖市镜湖区镜湖中采集的腐烂的水华离心后,收集上清液;
S2:在无菌条件下将上清液接种到牛肉膏蛋白胨液体培养基中35~40℃震荡培养20~28小时得到菌液;
S3:将菌液接种到含镉的牛肉膏蛋白胨平板培养基上,35~40℃倒置培养 68~82小时;
S4:将长势良好的菌落纯化后接种到斜面培养基,2~6℃保存待用。
所述步骤S2中,上清液与牛肉膏蛋白胨液体培养基的体积之比为1:5~15。
所述步骤S3中,含镉的牛肉膏蛋白胨平板培养基中镉离子的浓度为 10~50mg/L。
所述牛肉膏蛋白胨液体培养基的成分为:每1L培养基中含有牛肉膏3g,蛋白胨10g,NaCl 5g,全部溶于蒸馏水,调节PH为7.4。
所述步骤S3中,含镉的牛肉膏蛋白胨平板培养基成分为:每1L培养基中含有990mL牛肉膏蛋白胨液体培养基和10mL的镉离子母液,配置成含镉浓度为10mg/L的固体培养基。
所述镉离子母液的制备方法为:称取1.000g光谱纯金属镉,用优级纯硝酸溶解,然后用水稀释定容至1000mL,其浓度为1g/L。
所述步骤S4中,纯化的方法为挑取牛肉膏蛋白胨平板培养基上的菌落,划线法接到含镉的牛肉膏蛋白胨平板培养基,平板倒置培养,再挑取菌落继续重复之前的步骤,直至菌落为单菌。
所述斜面培养基的成分为:每1L培养基中含有牛肉膏3g,蛋白胨10g,NaCl 5g,琼脂20g,全部溶于蒸馏水,PH7.4。
本发明通过从镜湖中腐烂的水华中筛选得到了一株菌株R1,该菌株具有较强的耐镉能力、溶藻以及可降解微囊藻毒素,经16S rDNA序列对比分析鉴定, R1属于气单胞菌属(Aeromonas)。该菌株对Cd2+有很好的耐受性,在含有50mg/L Cd2+的培养基中仍能正常生长;细菌通过分泌胞外物质溶解铜绿微囊藻,且该物质耐高温,非核酸和多糖类等物质,可能是丙氨酸和苯丙氨酸协同作用而溶藻;在0.1mg/L Cd2+存在的情况下,细菌溶藻能力增强;在微囊藻毒素的初始浓度为1.84mg/L时,7天时间降解率为43.5%。
附图说明
图1为菌株R1的菌落形态;
图2为R1菌体显微形态(×1000);
图3为R1与气单胞菌属的基因比对分析;
图4为R1菌株和镉离子对铜绿微囊藻的溶藻效果;
图5为菌株R1对微囊藻毒素降解的曲线;
图6为菌液不同处理方式的溶藻效果比较;
图7为乙醇沉淀各组分对铜绿微囊藻的溶藻效果;
图8为液体培养基中游离氨基酸图谱;
图9为R1发酵液游离氨基酸图谱;
图10丙氨酸和苯丙氨酸溶藻效果比较。
具体实施方式
下面结合实施例及说明书附图对本发明进行详细说明。
铜绿微囊藻(Microcystisaeruginosa):来源于武汉水生所藻种保藏中心,培养条件:25℃,光照强度2500lx、光暗比12h:12h。
铜绿微囊藻培养基采用BG11改良版,其成分为:NaNO3 15g,K2HPO4 0.4g, MgSO4·7H2O 0.75g,CaCl2·2H2O 0.36g,柠檬酸0.06g,EDTANa2 0.01g,Na2CO3 0.2g,A5液10mL,溶解到10L蒸馏水中;
A5液:H3BO3 0.286g,MnCl2·4H2O 0.186g,ZnSO4·7H2O,Na2MoO4·2H2O 0.039g,CuSO4·5H2O 0.008g,Co(NO3)2·6H2O 0.005g,溶解于100mL蒸馏水中,调节pH7.1。
牛肉膏蛋白胨培养基:牛肉膏3g,蛋白胨10g,NaCl 5g,全部溶于1L蒸馏水,调PH7.4。
M9培养基:取M9母液200mL加水700mL,加2mL1mol/L已灭菌的硫酸镁,加100μL已灭菌的1mol/L氯化钙;M9母液的成分为:64g磷酸氢二钠,15g磷酸二氢钾,2.5g氯化钠,5.0g氯化铵,加1L水灭菌。
斜面培养基:牛肉膏3g,蛋白胨10g,NaCl 5g,琼脂20g,全部溶于1L蒸馏水,调PH7.4,。
含镉离子的母液1g/L:称取1.000g光谱纯金属镉,用优级纯硝酸溶解,然后用水稀释定容至1000mL。
含镉的牛肉膏蛋白胨平板培养基:990mL牛肉膏蛋白胨培养基中加入10mL 的含镉离子的母液,配置成含镉浓度为10mg/L的固体培养基。
实施例1
菌株R1的筛选与鉴定
S1:将从安徽省芜湖市镜湖区镜湖中采集的腐烂的水华离心后,收集上清液;
S2:在无菌条件下将上清液10mL接种到90mL牛肉膏蛋白胨液体培养基中, 180r/min、37℃振荡培养24h得到菌液;
S3:吸取0.1mL菌液接种到含镉浓度为10mg/L的牛肉膏蛋白胨固体培养基上,37℃倒置培养72d;
S4:将长势良好的菌落纯化后接种到斜面培养基,2~6℃保存待用,并将其命名为R1。
所述步骤S4中,纯化的方法为挑取牛肉膏蛋白胨固体培养基上的菌落,划线法接到含镉的牛肉膏蛋白胨培养基,平板倒置培养,再挑取菌落继续重复之前的步骤,直至菌落为单菌
对R1菌株进行形态特征、生理生化特征鉴定,R1的菌落形态如图1所示,显微形态如图2所示。从图1、2中可见,R1的菌落形态为圆形,乳白色,光滑半透明,边缘整齐,较粘稠,易挑起;革兰氏染色阴性;菌体呈短杆状;无芽孢。对其进行生理生化鉴定的结果如下:能利用葡萄糖产生有机酸但不产气;不能分解含硫有机物和分解色氨酸产生吲哚;与大肠杆菌,金黄色葡萄球菌和枯草芽孢杆菌共培养做抑菌实验,它们相互之间无影响。
并对菌株进行16S rDNA序列分析,PCR引物为细菌通用引物:27F: 5’-AGAGTTTGATCCTGGCTCAG-3’,1492R: 5’-GGTTACCTTGTTACGACTT-3’,双向测通。其基因序列表如SEQUENCE LISTING<400>1所示,序列长度为1396bp。将序列在NCBI数据库比对分析,16S rDNA同源性比较见图3,结合形态学的观察,发现其与气单胞菌属 (Aeromonas)细菌的相似性高达99%。
实施例2
溶藻试验
将一环R1菌株接种到200mL牛肉膏蛋白胨液体培养基中,180r/min、37℃培养30h得到R1菌液,其在620nm下,吸光度值为1.0。以下实施例中涉及到的R1菌液的制备方法均同此。
所述铜绿微囊藻藻液的制备方法为:购入的藻种接入BG11培养基,每天摇藻,光照培养箱,25℃,光照强度2500lx、光暗比12h:12h培养。
溶藻试验按表1设计进行。
表1溶藻试验
每组实验做3个重复,于25℃、光照强度2500lx、光暗比12h:12h条件下培养,每天观察铜绿微囊藻细胞形态变化和测定藻细胞叶绿素a的含量,其结果如图4所示,所有实验组铜绿微囊藻初始叶绿素a含量为1.98mg/L。实验组 A藻液中含有0.1mg/LCd2+,在接入R1菌液的第2天开始黄化,并出现絮状沉淀,在第4天就彻底黄化了,叶绿素a降到0.38mg/L。实验组B中藻液中不含镉,接入R1菌液后,第4天叶绿素a含量为0.79mg/L;由此可知,铜绿微囊藻液中是否有重金属镉离子,影响R1的溶藻效果,在镉离子存在的情况下,R1 溶藻效果更好。实验组C中并没有接入菌液的含镉藻液在第3天也有黄化的趋势,是因为重金属镉离子破坏了藻细胞,影响铜绿微囊藻正常生长。实验组D 中只接入培养基,藻细胞正常生长,叶绿素a含量一直增加。
实施例3
R1菌株对藻毒素的降解试验
将培养24h的R1菌液按照5%的体积比投放到以微囊藻毒素为底物的M9 培养基中,藻毒素初始浓度1.84mg/L,镉浓度0.1mg/L;另一实验组,M9培养基只含有藻毒素,浓度1.84mg/L,设阴性对照组。37℃,180r/min于摇床中培养7d,每24h取一次样,通过Beacon公司生产的微囊藻毒素检测试剂盒检测藻毒素含量变化。检测方法:吸取50μl酶标记物到微孔板的小孔中;吸取50μl标样、样品、阴性对照到对应微孔中;在各空加入50μl抗体溶液,薄膜快速振荡孵育30min;清洗液清洗微孔5次,将孔里水拍干,加100μl底物,孵育30min;加100μl停止液,通过酶标仪(SP—Max2300A)在450nm下读板,并绘制标准曲线计算微囊藻毒素浓度。
菌株藻毒素降解结果如图5所示,菌株R1从第1天就开始降解藻毒素,到第7天,降解率为43.5%。在含有0.1mg/LCd2+存在的情况下,对降解效率有影响,但影响不大,7天仍能降解40.2%。镉存在并不影响R1降解藻毒素,可能是因为降解藻毒素是组织内某些酶起作用,镉离子并不影响这些酶活性。
实施例4
溶藻机理探索
在无菌操作下,按照5%接入量,分别将牛肉膏蛋白胨液体培养基(对照组)、 R1菌液、重悬的R1菌体(将R1菌液经10000r/min离心15min沉降后清洗三次菌体,使用0.9%的生理盐水制成菌悬液、过0.22μm滤膜的菌液、121℃高温加热的菌液和超声破碎的菌液,分别接入铜绿微囊藻藻液中,每天观察藻细胞形态变化和测定藻细胞叶绿素a的含量。结果如图6所示,从图6可以看出只有重悬的R1菌体失去了溶藻效果,其他处理方式仍具有很强的溶藻效果。
Molish反应:分别在试管中加入1%葡萄糖溶液、1%蔗糖溶液、1%淀粉溶液1mL,然后加两滴Molish试剂,摇匀;沿管壁缓缓加入1mL浓硫酸,切勿摇动,观察两层液面交界处的颜色变化。然后分别用R1发酵液(含菌体)以及上清液代替糖溶液,做三组同样的实验,观察结果。Molish反应显示,R1发酵液上清中无糖类物质。所述R1发酵液的制备方法为将一环R1菌株接种到200mL 牛肉膏蛋白胨液体培养基中,180r/min、37℃培养30h得到R1菌液,其在620nm 下,吸光度值为1.0;上清液的制备方法为:5000rmp离心发酵液,去除沉淀,发酵液过0.22um的微孔滤膜,得R1上清液。
乙醇沉淀:取R1发酵上清液(上清液)10mL,用30mL无水乙醇沉淀,静置10min,3500r/min离心10min,以上步骤重复三次,合并三次的上清和沉淀。合并后的上清于65℃经旋转蒸发仪处理后,用灭菌的BG11定容至10mL 并称之为溶解相;合并后的沉淀用10mL的BG11溶解并称之为沉淀相。对铜绿微囊藻的溶藻效果如图7所示,R1上清液经无水乙醇沉淀后,有极少量的沉淀,将沉淀溶解后并不具备溶藻能力,而溶解相和R1发酵上清液都显示出了一定的溶藻活性,说明R1的溶藻物质并不是易于在乙醇中沉淀的蛋白质、核酸、多糖类等物质。
氨基酸对比:利用氨基酸分析仪(依利特AAK)比较牛肉膏蛋白胨培养基和R1上清液的游离氨基酸含量对比。如图8和9所示,由图8、9可知,对比 R1发酵液上清和液体培养基的游离氨基酸种类差异,发现R1发酵液中多了丙氨酸和苯丙氨酸两种氨基酸。
苯丙氨酸(Phe)、丙氨酸(Ala)、R1发酵液上清溶藻活性比较:分别配置 0.1g/L的Phe、Ala溶液,实验组1为R1发酵液的上清按5%的接种量接入铜绿微囊藻液中光照培养;实验组2为0.1g/L Ala按5%的接种量接入铜绿微囊藻液中光照培养;实验组3为0.1g/L Phe按5%的接种量接入铜绿微囊藻液中光照培养;实验组4为Phe和Ala按1:1的比例混合,按5%的接种量接入铜绿微囊藻液中光照培养。对比五组实验数据,如图10可知,单独添加苯丙氨酸有溶藻效果,但同时添加丙氨酸和苯丙氨酸溶藻效果更显著,推测溶藻物质可能是丙氨酸和苯丙氨酸协同作用的结果。
上述参照实施例对一种气单胞菌属菌株R1及制备方法及其在溶藻降解微囊藻毒素上的应用进行的详细描述,是说明性的而不是限定性的,可按照所限定范围列举出若干个实施例,因此在不脱离本发明总体构思下的变化和修改,应属本发明的保护范围之内。
SEQUENCE LISTING
<110> 安徽师范大学
<120> 一种气单胞菌属菌株R1及制备方法及其在溶藻降解微囊藻毒素上的应用
<130> 1
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 1396
<212> DNA
<213> R1
<400> 1
cgagcggcag cgggaaagta gcttgctact tttgccggcg agcggcggac gggtgagtaa 60
tgcctgggga tctgcccagt cgagggggat aactactgga aacggtagct aataccgcat 120
acgccctacg ggggaaagca ggggaccttc gggccttgcg cgattggatg aacccaggtg 180
ggattagcta gttggtgagg taatggctca ccaaggcgac gatccctagc tggtctgaga 240
ggatgatcag ccacactgga actgagacac ggtccagact cctacgggag gcagcagtgg 300
ggaatattgc acaatggggg aaaccctgat gcagccatgc cgcgtgtgtg aagaaggcct 360
tcgggttgta aagcactttc agcgaggagg aaaggttggt agctaataac tgccagctgt 420
gacgttactc gcagaagaag caccggctaa ctccgtgcca gcagccgcgg taatacggag 480
ggtgcaagcg ttaatcggaa ttactgggcg taaagcgcac gcaggcggtt gggataagtt 540
agatgtgaaa gccccgggct caacctggga attgcattta aaactgtccg gctagagtct 600
tgtagagggg ggtagaattc caggtgtagc ggtgaaatgc gtagagatct ggaggaatac 660
cggtggcgaa ggcggccccc tggacaaaga ctgacgctca ggtgcgaaag cgtggggagc 720
aaacaggatt agataccctg gtagtccacg ccgtaaacga tgtcgatttg gaggctgtgt 780
ccttgagacg tggcttccgg agctaacgcg ttaaatcgac cgcctgggga gtacggccgc 840
aaggttaaaa ctcaaatgaa ttgacggggg cccgcacaag cggtggagca tgtggtttaa 900
ttcgatgcaa cgcgaagaac cttacctggc cttgacatgt ctggaatcct gcagagatgc 960
gggagtgcct tcgggaatca gaacacaggt gctgcatggc tgtcgtcagc tcgtgtcgtg 1020
agatgttggg ttaagtcccg caacgagcgc aacccctgtc ctttgttgcc agcacgtaat 1080
ggtgggaact caagggagac tgccggtgat aaaccggagg aaggtgggga tgacgtcaag 1140
tcatcatggc ccttacggcc agggctacac acgtgctaca atggcgcgta cagagggctg 1200
caagctagcg atagtgagcg aatcccaaaa agcgcgtcgt agtccggatc ggagtctgca 1260
actcgactcc gtgaagtcgg aatcgctagt aatcgcaaat cagaatgttg cggtgaatac 1320
gttcccgggc cttgtacaca ccgcccgtca caccatggga gtgggttgca ccagaagtag 1380
atagcttaac cttcgg 1396
Claims (2)
1.一种气单胞菌属(Aeromonas sp. )菌株R1,其保藏编号为CGMCC No.14864。
2.权利要求1所述的气单胞菌属菌株R1的应用,其特征在于,用于降解含镉废水中的微囊藻毒素,或用于溶解铜绿微囊藻。
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