CN116376707A - 一种草酸青霉菌0710-26及其应用 - Google Patents
一种草酸青霉菌0710-26及其应用 Download PDFInfo
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Abstract
本发明属于微生物领域,公开了一种高效抑藻活性的草酸青霉菌0710‑26及其应用。所述的草酸青霉菌0710‑26由大亚湾海水中分离纯化获得原始菌株。经中国普通微生物菌种保藏管理中心鉴定,分类命名为草酸青霉菌,保藏编号为CGMCC No.40217。本发明通过草酸青霉菌0710‑26液体发酵后获得大量菌体,本发明的草酸青霉菌0710‑26是高效抑藻微生物,具有较大的应用价值。
Description
技术领域
本发明涉及环境治理微生物领域,具体涉及一种草酸青霉菌0710-26及其应用。
背景技术
人类的生产及生活产生污水,营养了海洋的水体,从而使得水体富营养化,在温度达到藻类生长需求时,藻类及其他浮游生物将迅速繁殖生长,海洋即出现赤潮。赤潮爆发时,主要的藻类多种多样。其中,球形棕囊藻也是赤潮的一种主要藻类。球形棕囊藻爆发时将产生丙烯酸类物质,该类物质对于鱼类及水生甲壳均有一定的毒性,给当地的水产养殖业造成极大的危害。同时,球形棕囊藻爆发时会形成囊体,大的囊体会堵塞沿岸核电站冷源用水拦网,从而影响核电站的安全运行。
目前针对藻类引起的赤潮常采用化学及物理方法处理。化学方法处理藻类使用的试剂:金属试剂、光敏剂、除草剂和其他化学物质等。物理方法一般指超声技术、紫外线照射及吸附方法对藻类的控制方法。物理和化学方法可以有效去除藻类。然而,它们有明显的缺点,如成本高、二次污染和对水生生物和人类的有毒性。以微生物为基础的抑藻处理方法,安全、环保。
草酸青霉属于真菌,广泛存在于自然界中,具有可有诱导抗性、分泌酶解类物质等特性。通过代谢能产生抗真菌物质,能抑制多种病原真菌的生长。例如:能拮抗菌核病菌、抑制菌核病菌菌丝的生长。最新研究报道草酸青霉还具有溶磷、降解农药的功能,同时具有促生和改善农产品品质的作用。目前,在富营养化水体中有害藻类治理方面应用较少。
发明内容
本发明的目的在于提供一种草酸青霉菌0710-26及其应用。
本发明第一目的在于提供一种自主筛选的具有高抑藻活性的草酸青霉菌0710-26,分类命名为草酸青霉菌Penicilliutn oxalicum,该菌株已于2022年6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.40217。
本发明所述的草酸青霉菌0710-26是从大亚湾海水中分离纯化获得的原始菌株,所述的草酸青霉菌0710-26的筛选方法包括以下步骤:
(1)取大亚湾区域海水,采用无菌水对海水进行梯度稀释:101、102、103、104、105、106,每个梯度稀释液取0.1ml涂布在改良马丁培养基上,30℃培养5-7天;
(2)挑取不同菌落,划线于改良马丁培养基上,置于30℃培养3-5天;观察分离菌落是否纯化,若有杂菌,重复该步骤直至纯培养;
(3)将上述纯化后的菌株接种于液体培养基中,50-100rpm,30℃振荡培养3-5天;
(4)取1ml上述菌液接种于20ml指数生长的球形棕囊藻培养液中,于20±1℃,12h光照,12h黑暗,2400lux光照强度条件下,培养2天,改良马丁培养基为对照加入藻液中,分别设置3个平行,检测叶绿素a含量,判断藻细胞的数量,从而筛选出有抑藻活性最强菌株为草酸青霉菌0710-26。
其中,所述改良马丁培养基组分包括:酵母蛋白胨5.0g,酵母浸出粉 2.0g,葡萄糖20.0g,磷酸氢二钾 1.0g,硫酸镁 0.5g,琼脂14.0g,纯化水1000ml。
所述液体培养基组分包括:酵母蛋白胨5.0g,酵母浸出粉 2.0g,葡萄糖 20.0g,磷酸氢二钾 1.0g,硫酸镁 0.5g,纯化水1000ml。
本发明的第二目的在于提供一种草酸青霉菌0710-26在球形棕囊藻治理中的应用。
具体是将草酸青霉菌0710-26进行发酵培养得到草酸青霉菌菌液,并将所述菌液应用于球形棕囊藻治理。
本发明第三目的在于提供一种草酸青霉菌菌液制备的方法,包括以下步骤:
将草酸青霉菌0710-26接入固体斜面培养基中进行活化,培养温度为30-37℃,静置培养5-7 d;活化后的固体平板菌株接入一级种子培养基中,进行一级种子扩大培养,培养温度为30-37℃,转速100-200rpm,培养2-4d;按1-5%的接种量将一级种子液接入发酵培养基进行扩大培养,培养温度为30-37℃,转速50-100rpm,培养2-4d,收集菌液备用。
上述培养基中包含碳源和氮源,所述碳源包括葡萄糖、玉米浆粉、糖蜜、甘油和葡萄糖浆中的至少一种;所述氮源包括硫酸铵、大豆粉、酵母提取物、蛋白胨、硝酸钠、谷氨酸钠、氨水中的至少一种。
上述培养基中还包括无机盐,所述无机盐包括硫酸镁、氯化钾、氯化钠、氯化钙、磷酸二氢钾、磷酸氢二钾中的至少一种。
优选的,所述固体平板培养基组分包括:葡萄糖 1-3%(w/v)、酵母粉 0 .15-0 .3%(w/v)、酵母蛋白胨0.25-0.75%(w/v)、磷酸氢二钾0.05-0.15%(w/v)、琼脂粉 1-2%(w/v)、硫酸镁0.025-0.075%(w/v)。
优选的,所述一级种子培养基组分包括:葡萄糖 1.5-3%(w/v)、酵母粉 0 .15-0.3%(w/v)、酵母蛋白胨0.25-0.75%(w/v)、磷酸氢二钾0.05-0.15%(w/v)、硫酸镁0.025-0.075%(w/v)。
优选的,所述发酵培养基组分包括:葡萄糖 1.5-3%(w/v)、酵母粉0 .15-0 .3%(w/v)、酵母蛋白胨0.25-0.75%(w/v)、磷酸氢二钾0.05-0.15%(w/v)、硫酸镁0.025-0.075%(w/v)。
更进一步地,根据本发明实施方案,所述固体平板培养基组分包括:葡萄糖 2%(w/v)、酵母粉 0.2%(w/v)、酵母蛋白胨0.5% (w/v)、磷酸氢二钾0.1%(w/v)、琼脂粉 1.5%(w/v)、硫酸镁0.05%(w/v)。
更进一步地,根据本发明实施方案,所述一级种子培养基组分包括:葡萄糖 1.5%(w/v)、酵母粉 0.2%(w/v)、酵母蛋白胨0.5% (w/v)、磷酸氢二钾0.1%(w/v)、硫酸镁0.05%(w/v)。
更进一步地,根据本发明实施方案,所述发酵培养基组分包括:葡萄糖 1.5%(w/v)、酵母粉 0.2%(w/v)、酵母蛋白胨0.5%(w/v)、磷酸氢二钾0.1%(w/v)、硫酸镁0.05%(w/v)。
本发明还提供上述草酸青霉菌0710-26在球形棕囊藻治理方法,包括以下步骤:取1ml菌液加入20ml球形棕囊藻的培养液中,培养2天,取样品检测其抑藻效果。
与现有技术相比,本发明具有以下有益效果:
本发明的草酸青霉菌0710-26作为一种基本菌株,原料易得,成本低廉;作为“抑藻剂”,采用天然生物材料,环境亲和性强,草酸青霉菌产生的物质能够被环境所自然降解,使用中无二次污染,操作简便,可实施性强,安全性能高,抑制效果良好,抑藻率可达到80%;能在较长时间内保持一定的抑藻效率,抑制藻类爆发,有利于实际应用。草酸青霉菌0710-26经二级放大发酵培养后,可获得大量发酵液,且大规模生产的菌液抑藻应用后,抑藻率仍能达到80%,未见明显下降。说明该菌株的抑藻活性高效且稳定,为利用生物方法处理水体藻华问题提供新的方法和方向。
附图说明
图1为本发明不同草酸青霉菌株抑藻效率对比图。
图2为本发明草酸青霉菌种在MEA培养基形态。
图3为本发明草酸青霉菌丝显微图片。
图4为本发明草酸青霉菌生长曲线。
图5为不同生长阶段草酸青霉菌株对球形棕囊藻抑制作用。
图6为草酸青霉菌0710-26发酵液对球形棕囊藻抑制作用。
具体实施方式
下面结合具体实施例,进一步阐述本发明。应理解这些实施例仅用于说明本发明而不用于限制本发明的保护范围,在阅读了本发明之后,本领域技术人员对本发明的各种等价形式的修改,或直接或间接运用在其他相关的技术领域,均落于本申请所附权利要求所限定的范围。
实施例1
1、菌株的筛选
(1)取大亚湾区域的海水样品10ml,溶解于湿热灭菌后的90ml无菌水中,混匀。
采用无菌水对海水进行梯度稀释:101、102、103、104、105、106,每个梯度稀释液取0.1ml涂布在改良马丁培养基上(培养基配方为:酵母蛋白胨5.0g,酵母浸出粉 2.0g,葡萄糖 20.0g,磷酸氢二钾 1.0g,硫酸镁 0.5g,琼脂14.0g,纯化水1000ml),30℃培养5-7天。
(2)挑取不同菌落,划线于改良马丁培养基上(培养基配方为:酵母蛋白胨5.0g,酵母浸出粉 2.0g,葡萄糖 20.0g,磷酸氢二钾 1.0g,硫酸镁 0.5g,琼脂14.0g,纯化水1000ml),置于30℃培养3-5天。观察分离菌落是否纯化。若有杂菌,重复该步骤直至纯培养。
(3)将上述纯化后的菌株接种于液体培养基中(培养基配方为:酵母蛋白胨5.0g,酵母浸出粉 2.0g,葡萄糖 20.0g,磷酸氢二钾 1.0g,硫酸镁 0.5g,纯化水1000ml),50-100rpm,30℃振荡培养3-5天。
(4)取1ml上述菌液接种于20ml指数生长的无菌球形棕囊藻培养液中,于20±1℃,12h光照,12h黑暗,2400lux光照强度条件下,培养2天,液体培养基为对照加入藻液中,分别设置3个平行,检测叶绿素a含量,判断藻细胞的数量,从而筛选出抑藻活性的最高的菌株,如图1。多株菌株通过抑制藻测试,结果显示,菌株0710-23、0710-24、0710-25、0710-26、0710-27中0710-26菌株抑藻效率最高,达到80%。此菌株为高效抑藻菌株,对该菌株进行鉴定。
2、菌种的鉴定
取上述抑藻效果最高的菌株草酸青霉菌0710-26,菌种在MEA培养基上,25℃黑暗条件下培养7天,菌落直径45mm,暗绿色,绒毛状,如附图2所示。显微镜下观察:菌丝无色,具隔膜,多分枝。分生砲子梗发生于气生菌丝,无色,平滑,分枝。分生孢子椭圆形,单孢,无色,光滑,链生,如附图3所示。
由中国科学院微生物研究所进行测序,菌种16S rRNA基因序列测定结果如SEQ IDNO:1所示。根据菌种的培养特征、显微特征、rRNA基因序列等实验数据综合分析,菌株的鉴定结果为:Penicilliutn oxalicum 草酸青霉。
所述草酸青霉菌0710-26的16S rDNA序列如下(SEQ ID NO:1):
caacctggtt aagattgatg gtgttcgccg gcgggcgccg gccgggccta cagagcgggtgacgaagccc catacgctcg aggaccggac gcggtgccgc cgctgccttt cgggcccgcc ccccggaagcggggggcgag agcccaacac acaagccgtg cttgagggca gcaatgacgc tcggacaggc atgccccccggaataccagg gggcgcaatg tgcgttcaaa gactcgatga ttcactgaat tctgcaattc acattacttatcgcatttcg ctgcgttctt catcgatgcc ggaaccaaga gatccgttgt tgaaagtttt aactgatttagtcaagtact cagactgcaa tcttcagaca agagttcgtt tgtgtgtctt cggcgggcgc gggcccgggggcggatgccc cccggcggcc gtgaggcggg cccgccgaag caacaaggta cgataaacac gggtgggaggttggacccag agggccctca ctcggtaatg atccttccgc aggttcacct acggaaacct tgttcatttttttttttttc caaagggg。
实施例2
草酸青霉菌0710-26生长曲线绘制及不同生长阶段抑藻测试
(1)将草酸青霉菌0710-26菌株接种于新鲜的改良马丁培养培养基上划线分离(培养基配方为:酵母蛋白胨5.0g,酵母浸出粉 2.0g,葡萄糖 20.0g,磷酸氢二钾 1.0g,硫酸镁0.5g,琼脂14.0g,纯化水1000ml),于30℃培养3-5天;
(2)挑取平板上的单菌落接种于液体培养基中(培养基配方为:酵母蛋白胨5.0g,酵母浸出粉 2.0g,葡萄糖 20.0g,磷酸氢二钾 1.0g,硫酸镁 0.5g,纯化水1000ml),50-100rpm,30℃振荡培养3-5天;
(3)菌株在摇瓶培养基中培养,不同时间段取样检测菌体湿重,并绘制生长曲线。如图4所示,接种后,进行菌株培养,培养时间50小时以内为延迟期;培养时间为50小时至96h为指数期;培养时间96小时至144h为平台期;
(4)不同生长阶段对球形棕囊藻抑制作用:依据菌株的生长曲线,选取延迟期,指数期及平台期的菌液作为测试组,培养基为对照组;
(5)取1ml菌液或培养基加入20ml球形棕囊藻的培养液中,培养48h,取样测试抑藻效率。
如图5所示,实验结果表明,菌株处于指数期,抑藻效果最明显,可能是菌株在此阶段活性较高。
实施例3
草酸青霉菌0710-26发酵液的制备,包括以下步骤:
(1)将草酸青霉菌0710-26菌株接入固体平板培养基中进行活化,培养温度为30℃,静置培养5d;
(2)活化后的固体平板菌株接入1L摇瓶中进行一级种子扩大培养,装液量为300mL,培养温度为35℃,转速100rpm,培养3d;
(3)按1%的接种量将一级种子液接入2L瓶中进行发酵培养,装液量为1.2L,培养温度为35℃,转速80rpm,培养3d,得到发酵液;
其中,培养基组成比例分别为:
固体平板培养基(w/v):葡萄糖2%、酵母粉0.2%、酵母蛋白胨0.5%、磷酸氢二钾0.1%、琼脂粉1.5%、硫酸镁0.05%。
一级种子培养基(w/v):葡萄糖1.5%、酵母粉0.2%、酵母蛋白胨0.5%、磷酸氢二钾0.1%、硫酸镁0.05%。
发酵培养基(w/v):葡萄糖1.5%、酵母粉0.2%、酵母蛋白胨0.5%、磷酸氢二钾0.1%、硫酸镁0.05%。
实施例4
草酸青霉菌0710-26发酵液的制备,包括以下步骤:
(1)将草酸青霉菌0710-26菌株接入固体平板培养基中进行活化,培养温度为35℃,静置培养6d;
(2)活化后的固体平板菌株接入1L摇瓶中进行一级种子扩大培养,装液量为300mL,培养温度为30℃,转速100rpm,培养4d;
(3)按1%的接种量将一级种子液接入2L瓶中进行发酵培养,装液量为1.2L,培养温度为37℃,转速50rpm,培养2d,得到发酵液;
其中,培养基组成比例分别为:
固体平板培养基(w/v):葡萄糖2%、酵母粉0.2%、酵母蛋白胨0.5%、磷酸氢二钾0.1%、琼脂粉1.5%、硫酸镁0.05%。
一级种子培养基(w/v):葡萄糖1.5%、酵母粉0.2%、酵母蛋白胨0.5%、磷酸氢二钾0.1%、硫酸镁0.05%。
发酵培养基(w/v):葡萄糖1.5%、酵母粉0.2%、酵母蛋白胨0.5%、磷酸氢二钾0.1%、硫酸镁0.05%。
实施例5
草酸青霉菌0710-26发酵液的制备,包括以下步骤:
(1)将草酸青霉菌0710-26菌株接入固体平板培养基中进行活化,培养温度为37℃,静置培养5d;
(2)活化后的固体平板菌株接入1L摇瓶中进行一级种子扩大培养,装液量为300mL,培养温度为30℃,转速100rpm,培养4d;
(3)按1%的接种量将一级种子液接入2L瓶中进行发酵培养,装液量为1.2L,培养温度为30℃,转速80rpm,培养4d,得到发酵液;
其中,培养基组成比例分别为:
固体平板培养基(w/v):葡萄糖2%、酵母粉0.2%、酵母蛋白胨0.5%、磷酸氢二钾0.1%、琼脂粉1.5%、硫酸镁0.05%。
一级种子培养基(w/v):葡萄糖1.5%、酵母粉0.2%、酵母蛋白胨0.5%、磷酸氢二钾0.1%、硫酸镁0.05%。
发酵培养基(w/v):葡萄糖1.5%、酵母粉0.2%、酵母蛋白胨0.5%、磷酸氢二钾0.1%、硫酸镁0.05%。
实施例6
草酸青霉菌0710-26在球形棕囊藻治理中的应用
将实施3~5制备的草酸青霉菌0710-26发酵液,取1ml菌液或培养基加入20ml球形棕囊藻的培养液中,于20±1℃,12h光照,12h黑暗,2400lux光照强度条件下,培养2天,取样测试抑藻效率。如图6所示,草酸青霉菌0710-26菌株先经一级放大培养,随后进行进一步的放大发酵培养,得到经过二次放大培养的菌液,随后采用菌液:藻液(1:20)比例投放测试,2天后草酸青霉菌0710-26的菌液抑藻效率可达到80%。经过多次的放大培养后的菌液,抑藻效率较初筛时未明显下降,有利于后期大规模培养及应用。
Claims (10)
1.一种草酸青霉菌0710-26,其特征在于,分类命名为草酸青霉菌Penicilliutn oxalicum,该菌株已于2022年6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.40217。
2.如权利要求1所述草酸青霉菌0710-26的筛选方法,其特征在于,包括以下步骤:
(1)取大亚湾区域海水,采用无菌水对海水进行梯度稀释:101、102、103、104、105、106,每个梯度稀释液取0.1ml涂布在改良马丁培养基上,30℃培养5-7天;
(2)挑取不同菌落,划线于改良马丁培养基上,置于30℃培养3-5天;观察分离菌落是否纯化,若有杂菌,重复该步骤直至纯培养;
(3)将上述纯化后的菌株接种于液体培养基中,50-100rpm,30℃振荡培养3-5天;
(4)取1ml上述菌液接种于20ml指数生长的球形棕囊藻培养液中,于20±1℃,12h光照,12h黑暗,2400lux光照强度条件下,培养2天,改良马丁培养基为对照加入藻液中,分别设置3个平行,检测叶绿素a含量,判断藻细胞的数量,从而筛选出有抑藻活性最强菌株为草酸青霉菌0710-26。
3. 如权利要求2所述的筛选方法,其特征在于,所述改良马丁培养基组分包括:酵母蛋白胨5.0g,酵母浸出粉 2.0g,葡萄糖 20.0g,磷酸氢二钾 1.0g,硫酸镁 0.5g,琼脂14.0g,纯化水1000ml。
4. 如权利要求2所述的筛选方法,其特征在于,所述液体培养基组分包括:酵母蛋白胨5.0g,酵母浸出粉 2.0g,葡萄糖 20.0g,磷酸氢二钾 1.0g,硫酸镁 0.5g,纯化水1000ml。
5.权利要求1所述的草酸青霉菌0710-26在球形棕囊藻治理中的应用。
6.如权利要求5所述的应用,其特征在于,将草酸青霉菌0710-26进行发酵培养得到草酸青霉菌菌液,并将所述菌液应用于球形棕囊藻治理。
7.一种制备权利要求6所述草酸青霉菌菌液的方法,其特征在于,包括如下步骤:
将草酸青霉菌0710-26接入固体平板培养基中进行活化,培养温度为30-37℃,静置培养5-7 d;活化后的菌株接入一级种子培养基中,进行一级种子扩大培养,培养温度为30-37℃,转速100-200 rpm,培养2-4 d;按1-5%的接种量将一级种子液接入发酵培养基进行扩大培养,培养温度为30-37℃,转速50-100rpm,培养2-4d,收集菌液备用。
8. 如权利要求7所述的方法,其特征在于:所述固体平板培养基组分包括:葡萄糖 1-3%、酵母粉 0.15-0.3%、酵母蛋白胨0.25-0.75%、磷酸氢二钾0.05-0.15%、琼脂粉 1-2%、硫酸镁0.025-0.075%;优选为葡萄糖 2%、酵母粉 0.2%、酵母蛋白胨0.5% 、磷酸氢二钾0.1%、琼脂粉 1.5%、硫酸镁0.05%。
9. 如权利要求7所述的方法,其特征在于:所述一级种子培养基组分包括:葡萄糖1.5-3%、酵母粉 0.15-0.3%、酵母蛋白胨0.25-0.75% 、磷酸氢二钾0.05-0.15%、硫酸镁0.025-0.075%;优选为葡萄糖 1.5%、酵母粉 0.2%、酵母蛋白胨0.5% 、磷酸氢二钾0.1%、硫酸镁0.05%。
10. 如权利要求7所述的方法,其特征在于:所述发酵培养基组分包括:葡萄糖 1.5-3%、酵母粉 0.15-0.3%、酵母蛋白胨0.25-0.75%、磷酸氢二钾0.05-0.15%、硫酸镁0.025-0.075%;优选为葡萄糖 1.5%、酵母粉 0.2%、酵母蛋白胨0.5%、磷酸氢二钾0.1%、硫酸镁0.05%。
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