CN110272850B - 具有溶藻能力的新属菌株及其对球形棕囊藻的应用 - Google Patents
具有溶藻能力的新属菌株及其对球形棕囊藻的应用 Download PDFInfo
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Abstract
本发明公开了一种具有溶藻能力的新属菌株,保藏编号为CCTCC NO:M 2019404,分类命名为Mesobaculum littorinae M0103。该菌株筛选简单、容易培养,发明人还建立了相应培养方法。研究表明,M0103菌株溶藻效果好,其发酵液对北部湾海域优势藻种球形棕囊藻具有很好的抑制及杀灭效果,24h处理条件下溶藻率达90%以上。由于M0103菌株来自北部湾海域螺类,因而还具有生态安全性好等特点,使用过程中不会造成二次污染。该菌株的发现丰富了我国的可利用微生物资源,其除藻效果稳定、高效、环境友好的优势,在控制球形棕囊藻赤潮方面具有很好的应用前景。
Description
技术领域
本发明属于环境微生物技术领域,尤其涉及一种具有溶藻能力的新属菌株及其对球形棕囊藻的应用。
背景技术
赤潮,是指海洋水体中某些浮游植物、原生动物或细菌在一定的环境条件下突发性增殖和聚集,引发一定范围和一段时间内水体变色的现象。赤潮的危害很大,主要体现在以下三个方面:第一,赤潮对海洋生态平衡的破坏。赤潮发生初期,水体出现高叶绿素、高溶解氧、高化学耗氧量,引起周围海洋环境剧烈改变,破坏了水体的生态平衡。第二,赤潮对海洋渔业和水产资源的破坏。赤潮发生初期,藻类生物大量繁殖,引起经济藻类变色或腐烂,分泌出的粘液,黏附于鱼类鳃部妨碍其呼吸,引起鱼类缺氧死亡。有些赤潮生物体内生物毒素能够引起鱼虾贝类中毒死亡;赤潮发生后期,藻类大量死亡后,释放有害气体和毒素,导致鱼类和其他海洋生物因缺氧或中毒死亡。第三,赤潮对人类健康的危害。有些赤潮生物毒素通过食物链在鱼虾贝类体内富集,如果人类不慎摄食,轻则中毒,严重可导致死亡。例如:2011年4月钦州市犀牛脚、三娘湾海域发生的一次球形棕囊藻赤潮,导致大量鱼类死亡。广西沿海目前所发生的赤潮相对我国东部沿海和相邻的广东珠江口海域来说其规模性虽尚小,但危害性已经开始显露,发生次数的速度也在加快。如不及早做好应对未来更大风险、规模、危害的赤潮发生的各项技术研发准备,一旦严重暴发,将对广西海洋生态环境及社会经济发展造成严重的影响和损失。
对于赤潮的控制目前主要有物理法、化学法和生物法。物理方法虽然见效很快并且不会对生态环境造成危害,但是极有可能对底栖生物造成负面影响,并且无法从根本上治理赤潮。化学方法是目前相较之下最有效的方法,但是它容易对环境造成二次污染,无法针对性的只对赤潮生物产生影响,会在杀死赤潮生物的同时对非赤潮生物也造成一定程度上的伤害。生物方法强调的是整个生态***的管理,其中溶藻细菌作为水生生态***生物种群结构和功能的重要组成部分,对防治水华和赤潮及对维持藻的生物量平衡起着至关重要的作用。
发明内容
本发明要解决的技术问题是提供一种具有溶藻能力的新属菌株及其对球形棕囊藻的应用。
为解决上述技术问题,本发明采用以下技术方案:
一种新属菌株,保藏编号为CCTCC NO:M 2019404,分类命名为Mesobaculumlittorinae M0103。
上述新属菌株,其16S rRNA基因具有序列表SEQ.ID.NO.1的碱基序列。
上述新属菌株在治理赤潮或水华中的应用。
上述新属菌株在溶藻方面的应用。
上述新属菌株的发酵液用于制备溶藻药剂。
上述新属菌株用于抑制或灭杀球形棕囊藻。
上述新属菌株的发酵液用于制备球形棕囊藻抑制剂或灭杀剂。
上述新属菌株的培养方法,将该菌接种于培养基中,在28-30℃,pH4-8.5的条件下进行培养。
上述培养方法,包括以下步骤:
(1)斜面培养:将M0103菌种接种于固体斜面培养基上,在28℃条件下培养24h;
(2)初级培养:无菌条件下取培养好的斜面,用接种环取一环于100mL液体培养基中,28℃,摇床180rpm培养24h,获得初级培养菌液;
(3)扩大培养:将初级培养菌液接种于500mL液体培养基中,接种量为5%,28℃,摇床180rpm培养72h,获得扩大培养菌液后。
液体培养基的配方为:酵母提取粉2.0g/L,麦芽提取粉2.0g/L,葡萄糖2.0g/L,海盐26.0g/L,121℃高温蒸汽灭菌20min;固体斜面培养基为液体培养基中添加终浓度1.3%的琼脂粉。
发明人在北海金海湾红树林的一只粗糙滨螺中分离得到本发明的溶藻微生物,它属于醋杆菌科(Acetobacteraceae),是新属菌株,保藏编号为CCTCC NO:M 2019404,分类命名为Mesobaculum littorinae M0103。该菌株筛选简单、容易培养,发明人还建立了相应培养方法。研究表明,M0103菌株溶藻效果好,其发酵液对北部湾海域优势藻种球形棕囊藻具有很好的抑制及杀灭效果,24h处理条件下溶藻率达90%以上。由于M0103菌株来自北部湾海域螺类,因而还具有生态安全性好等特点,使用过程中不会造成二次污染。该菌株的发现丰富了我国的可利用微生物资源,其除藻效果稳定、高效、环境友好的优势,在控制球形棕囊藻赤潮方面具有很好的应用前景,也为水华防治生物制剂的开发与研究领域奠定了坚实的基础,今后将在大面积水体爆发的有害藻华事件中大有所为。
附图说明
图1是基于16S rRNA基因序列分析构建菌株M0103与红杆菌科中邻近有效发表菌株的N-J***发育树。
保藏信息说明
Mesobaculum littorinae M0103,保藏编号为CCTCC NO:M 2019404,保藏日期:2019年05月29日,保藏地址为:中国.武汉.武汉大学,邮编430072,保藏单位:中国典型培养物保藏中心。
具体实施方式
下列实施例中未注明具体条件的实验方法,按照常规方法和条件,或按照商品说明书选择。
Tranquillimonas alkanivorans A34T购买至德国微生物菌种保藏中心(DSMZ)。
实施例1、本发明新菌株M0103的分离制备
取粗糙滨螺组织浸出液,将其稀释涂布于ISP4固体培养基中,30℃培养21天,挑取单菌落,然后划线纯化得到。
送至中国典型培养物保藏中心保藏,其保藏编号为CCTCC NO:M 2019404。
实施例2、本发明新菌株M0103的表观特征
取菌株M0103的单菌落,转接到ISP2+2.6%海盐固体培养基(琼脂)上,于30℃恒温培养箱中培养48h,分别观察其菌落的大小、颜色、边缘、凸起、光滑度、粘性、透明度等形态特点。
结果显示,菌株M0103在ISP2+2.6%海盐固体培养基上形成边缘整齐,微凸起,光滑,不透明,奶白色的圆形菌落,直径约2-3mm。
实施例3、本发明新菌株M0103的生长特性
挑取在ISP2+2.6%海盐固体培养基上培养24h的新鲜培养物,接种到R2A液体培养基,37℃摇床培养36h,作为种子。
ISP2+2.6%海盐固体培养基的组成:酵母提取粉2.0g/L,麦芽提取粉2.0g/L,葡萄糖2.0g/L,海盐26.0g/L,1.4%琼脂粉,去离子水1000mL。
R2A固体培养基:酵母粉0.5g/L,胰蛋白胨0.5g/L,酪蛋白氨基酸0.5g/L,葡萄糖0.5g/L,可溶性淀粉0.5g/L,磷酸氢二钾0.3g/L,七水硫酸镁0.05g/L,丙酮酸钠0.3g/L,琼脂13.0g/L,去离子水1000mL。
1.生长温度
将培养物接种于ISP2+2.6%海盐固体培养基,分别置于10℃、15℃、20℃、25℃、30℃、37℃、40℃和45℃培养,每个温度梯度做三个平行,分别在7和14天后观察生长情况。结果表明,菌株M0103生长温度范围为15-35℃,最适温度28-30℃。
2.生长NaCl耐受性
将培养物分别接种于含0-15%氯化钠的R2A固体培养基,浓度梯度为1%,28℃培养,每个NaCl浓度梯度做三个平行,分别在7和14天观察生长情况。结果显示,菌株M0103的NaCl耐受性为1-14%,最适盐度为2-3%。
3.生长pH范围
将R2A液体培养基调pH为4.0-12.0,梯度为0.5。将所培养的M0103种子按2%接种量接入,30℃培养,分别在7和14天观察生长情况。结果显示,菌株M0103生长的pH范围为4.0-8.5,最适pH为7.0。
4.光照
将培养物接种于ISP2+2.6%海盐固体培养基,分别放置在光照和黑暗培养箱中,28℃培养,每个因子做三个平行,分别在3和7天观察生长情况。结果显示,菌株M0103的生长不受光照的影响,对光不敏感。
实施例4、本发明新菌株M0103的生理生化特性
利用API ZYM和API 50CH鉴定***(bioMérieux)对菌株M0103及相关的菌株进行生理生化特征鉴定。其中,
API ZYM是一个半定量的微量方法***,专为研究酶活所设计的。它可***和快速地研究19种酶的活性。实验步骤如下:
1)样品准备:在最小体积稀释标本:可用2mL无菌蒸馏水或其它如普通的生理盐水不需要缓冲液。制备一个菌悬液,其混浊度在McFarland NO.5-6-。从斜面或离心肉汤培养物的纯菌种都能用来制备菌悬液。
2)试验条的准备:准备一个培养盘和盖子。记标本号于盘的侧面。可使用一个塑料洗瓶,分装约5ml蒸馏水于培养盘内,为了培养时能保持一定的湿度。从密封的包装中取出API ZYM试验条,置培养盘内。
3)试验条的接种:用吸管接种,于试验条的每个杯中接入2滴标本(65μL)。
4)试验条培养:接种后,托盘上放塑料盖子,37℃培养4h。所有的测定条件(时间、温度、培养基、悬液浓度)要保持一致。接种的试验条避光保存。
5)试验条的结果观察:培养后,加1滴ZYM A试剂和1滴ZYM B试剂。5分钟后生色,如果是阳性,将试验条置于一个强光源(1000瓦灯泡)下10s(秒,下同),把灯泡放在杯上4s。这是为消除杯中多余坚牢兰的黄色。暴光后阴性反应变为无色。
6)记录反应:0-5标记相应的颜色深度。0相当于阴性反应,5为最强反应。2-4是二者之间的强度。从颜色深度可知近似的微毫克分子浓度(nM):1相当释放5微毫克分子浓度(5nM),2相当于10nM,3为20nM,4为30nM,5为40nM或更高。
结果见表1。
表1 M0103的API ZYM试纸条的酶活反应
注:“+”为阳性,“w”为弱阳性,“-”为阴性。
API 50CH是一个标准化的鉴定***,它采用50个生化试验来对微生物的碳水化合物的代谢进行研究。实验步骤如下:
1)样品准备:挑取少量生长对数后期的菌落置于10mL API 50CHL液体培养基中,制备一个菌悬液,其混浊度在McFarland NO.5-6。
2)试验条的准备:准备一个培养盘和盖子。记标本号于盘的侧面。可使用一个塑料洗瓶,分装少量蒸馏水于培养盘内,保持培养过程中湿度。从密封的包装中取出API 50CH试验条,置培养盘内。
3)试验条的接种:用吸管接种,于试验条的每个杯中接入适量样本,加满管的部分,管上部分不加菌悬液,这样管内可保持厌氧环境,加入两滴石蜡油进行密封。
4)试验条培养:接种后,托盘上放塑料盖子,37℃培养12和24h。所有的测定条件(时间、温度、培养基、悬液浓度)要保持一致。接种的试验条避光保存。
5)试验条的结果观察:试条孵育过程中,试条上的小管内在厌氧环境下产酸,PH改变而使培养基中指示剂颜色变化,由此可观察到发酵作用。试条上第一孔中不含任何活性成分,以此作为阴性控制。
6)记录反应:以阳性(+),弱阳性(w),阴性(-)来解释每一个试验,将结果记录在结果记录单上(表2)。
表2 M0103的API 50CH试纸条的利用唯一碳源产酸反应结果
底物成分 | 结果 | 底物成分 | 结果 | 底物成分 | 结果 |
控制 | - | 肌醇 | - | D-松三糖 | + |
甘露醇 | - | 甘露醇 | - | D-棉子糖 | + |
赤藻糖醇 | - | 山梨醇 | - | 淀粉 | - |
D-***糖 | w | 甲基-D-吡喃甘露糖苷 | + | 糖原 | - |
L-***糖 | + | 甲基-D-吡喃葡萄糖苷 | + | 木糖醇 | - |
D-核糖 | + | N-乙酰葡萄糖胺 | w | D-龙胆二糖 | + |
D-木糖 | + | 苦杏仁苷 | w | D-土伦糖 | + |
L-木糖 | w | 熊果苷 | + | D-来苏糖 | + |
D-侧金盏花醇 | - | 七叶灵柠檬酸铁 | + | D-塔格糖 | + |
甲基-β-D吡喃木糖苷 | + | 水杨苷 | + | D-岩藻糖 | + |
D-半乳糖 | + | D-纤维二糖 | + | L-岩藻糖 | + |
D-葡萄糖 | + | D-麦芽糖 | + | D-***醇 | + |
D-果糖 | + | D-乳糖 | + | L-***醇 | w |
D-甘露糖 | + | D-密二糖 | + | 葡萄糖酸钾 | w |
L-山梨糖 | + | D-蔗糖 | + | 2酮基葡萄糖酸钾 | - |
L-鼠李糖 | - | D-海藻糖 | + | 5酮基葡萄糖酸钾 | + |
卫茅醇 | - | 菊粉 | - |
从表1和表2中可见,菌株M0103的主要生理生化特征:好氧型细菌;能产脲酶、氧化酶和过氧化氢酶;β-半乳糖苷酶阳性,碱性磷酸酶、类脂酯酶(C8)、酸性磷酸酶、β-半乳糖苷酶、α-葡萄糖苷酶均呈阳性;酯酶(C4)、亮氨酸芳基氨肽酶、胱氨酸芳基氨肽酶、α-半乳糖苷酶、β-葡萄糖苷酶均呈弱阳性;类脂酶(C14)、缬氨酸芳基氨肽酶、α-岩藻糖苷酶、胰蛋白酶、胰凝乳蛋白酶、N-乙酰-葡萄糖胺酶、α-甘露糖苷酶、β-糖醛酸苷酶、萘酚-AS-BI-磷酸水解酶均呈阴性;不水解Tween20、Tween40和Tween80,水解七叶灵;不能水解纤维素;能利用D-***糖、L-***糖、D-核糖、D-木糖、L-木糖、甲基-β-D吡喃木糖苷、D-半乳糖、D-葡萄糖、D-果糖、D-甘露糖、L-山梨糖、甲基-D-吡喃甘露糖苷、甲基-D-吡喃葡萄糖苷、N-乙酰葡萄糖胺、苦杏仁苷、熊果苷、七叶灵柠檬酸铁、水杨苷、D-纤维二糖、D-麦芽糖、D-乳糖、D-密二糖、D-蔗糖、D-海藻糖、D-松三糖、D-棉子糖、D-龙胆二糖、D-土伦糖、D-来苏糖、D-塔格糖、D-岩藻糖、L-岩藻糖、D-***醇、L-***醇、葡萄糖酸钾、5酮基葡萄糖酸钾等作为碳源产酸;不利用甘露醇、D-侧金盏花醇、D-侧金盏花醇、L-鼠李糖、卫茅醇、赤藻糖醇、肌醇、菊粉、淀粉、2酮基葡萄糖酸钾、糖原、木糖醇、甘露醇、山梨醇等唯一碳源产酸。
且菌株M0103在碱性磷酸盐酶、缬氨酸芳胺酶、胱氨酸芳胺酶、酸性磷酸酶、萘酚-AS-BI-磷酸水解酶、β-半乳糖甙酶和α-葡萄糖甙酶等活性均与同源性接近的菌株Tranquillimonas alkanivorans A34T的相关酶活性具有显著差异。
实施例5、本发明新菌株M0103的16S rRNA基因的PCR扩增和序列测定及16S rRNA***发育特征。
1.提取基因组DNA
将M0103接种至ISP2+2.6%海盐固体培养基,生长至对数后期,挑取一个菌落,加入100ul 10%Chelex-100溶液中,100℃加热10min(分,下同),冷却后,5000rmp(转速)离心10min,取液体上层作为模板DNA。
2.16S rRNA基因的PCR扩增及测序
扩增和测序引物均为细菌通用引物27F(5'-AGAGTTTGATCCTGGCTCA-3')和1492R(5'-GGTTACCTTGTTACGACTT-3'),PCR反应条件:94℃预变性8min,94℃变性50s,55℃退火45s,72℃延伸90s,共30个循环,循环结束后,72℃延伸维持10min。扩增产物经1%琼脂糖凝胶电泳检测,Gel Logic 2200Pro凝胶成像仪检验条带合格后,委托上海美吉生物医药技术有限公司广州分公司进行测序。
测序结果经DNA Star软件整理,利用EzTaxon服务器(http://www.eztaxon.org/)及Blast网站进行在线比对;选取同源性最高菌株的序列作为参比对象,运用MEGA5.0软件,采用Neighbor-Joining法构建***树,Boostrap1000次检测各分支的置信值,对各菌株的***发育地位进行分析。
测序结果表明,菌株M0103的16S rRNA基因序列长度为1458bp。其16S rRNA基因的核苷酸序列如序列为SEQ.ID.NO.1所示的碱基序列。
将16S rRNA序列使用软件MEGA version5.0.software package绘制进化关系树。采用maximum-likelihood计算,并以neighbor-joining、maximum-parsimony和进行验证计算,bootstrap设置为1000个循环。结果如图1所示。同源性分析表明,菌株M0103属于Mesobaculum sp.,Tranquillimonas alkanivorans A34T聚在一个分支,16S rRNA的同源性最高,相似性为95.05%。通常两个菌株的16S rRNA同源性小于96%时可界定为不同的属,但多相分类学研究认为一个属或种的确定不应只依据一个单一的标准,有待其他生理生化指标、化学特征分析和全基因组鉴定等指标的分析,才能确定菌株M0103的分类地位。
实施例6本发明新微生物的脂肪酸含量特征
菌株M0103的总脂肪酸含量的测定。
配置如下溶液:Reagent 1,45g氢氧化钠溶于150mL甲醇及150mL双蒸水;Reagent2,190mL浓盐酸,275mL甲醇溶于135mL双蒸水;Reagent 3,200mL正己烷与200mL***混合均匀;Reagent 4,10.8g氢氧化钠溶于900mL双蒸水;Reagent 5,饱和氯化钠溶液,40g氯化钠+100mL双蒸水。
1)取适量细菌培养物,置于8mL螺口玻璃管中,加入1mL Reagent 1溶液,拧紧螺盖,沸水浴5min,取出振荡5-10s,再度拧紧螺盖,继续沸水浴25min;
2)待样品管冷却后,加入2mL Reagent 2溶液,盖严振荡5-10s,随后精确控制80±1℃水浴10min,冰浴冷却;此步骤需严格控制温度和时间,以免羟基酸和环式脂肪酸受到破坏;
3)在冷却的样品管中加入1.25mL Reagent 3溶液,快速振荡10min左右,弃去下层水相;
4)在剩余有机相中加入2mL Reagent 4溶液及4mL Reagent 5溶液,快速振荡5min左右,取上层有机相置气相色谱样品瓶中备用。
HP 6890气相色谱仪与质谱仪联用分析,委托中国科学院华南植物园进行测试。
结果显示,菌株M0103的主要细胞脂肪酸为C18:1ω7c和C16:0,百分含量分别为63.87%和20.12%。符合醋杆菌科主要的脂肪酸类型。而且同相似菌株脂肪酸种类与含量上,均有差异,以此判断同相似菌株属于不同种类。
实施例7本发明新微生物的G+C mol%含量特征
委托上海美吉生物医药科技有限公司进行全基因组测序。结果显示,待测菌株M0103的G+C mol%为68.2mol%。
实施例8不同浓度待测菌株M0103对球形棕囊藻的溶藻效果
球形棕囊藻培养条件:球形棕囊藻(Phaeocystis globosa)用f/2培养基进行培养,培养于20℃光照培养箱中,光照强度100μmol photons·m-2·s-1,光暗周期14h:10h(光:暗)。
溶藻实验的棕囊藻制备:取1000mL对数生长期的藻液,通过20μm孔径的膜过滤除去囊体,留下棕囊藻单细胞,以4×104cells/mL藻细胞浓度为初始浓度进行实验。
溶藻效率计算方法:溶藻效率(%)=(1-实验组藻细胞浓度/对照组藻细胞浓度)×100%。其中球形棕囊藻的藻细胞浓度用0.1mL水样计数板通过显微镜计数。
M0103的发酵方法:
(1)菌种:采用所述的待测菌株M0103;
(2)斜面培养:将步骤(1)中的菌种接种于固体斜面培养基上,在28℃培养48h;
(3)初级培养:无菌条件下取培养好的斜面,取一环菌体接种于100mL海盐液体培养基中,28℃,摇床180rpm培养24h,获得初级培养菌液;
(4)扩大培养:将初级培养菌液接种于500mL上述液体培养基中,接种量为5%,28℃,摇床180rpm培养72h,获得扩大培养菌液后,收集发酵液。
步骤(3)和(4)中所述的液体培养基配方:酵母提取粉2.0g/L,麦芽提取粉2.0g/L,葡萄糖2.0g/L,海盐26.0g/L,去离子水1000mL,121℃高温蒸汽灭菌20min;步骤(2)所述固体斜面培养基为上述液体培养基中添加终浓度1.3%的琼脂粉。
将溶藻细菌M0103发酵培养的产物以体积终浓度为0.1%、0.5%、1%、2%的细菌发酵培养物和藻液的比例进行共培养。设置空白对照,对照组为加入0.1%、0.5%、1%、2%体积无菌的ISP2+2.6%海盐液体培养基的同时期藻液。实验组与对照组均设置3个重复。在0h、6h、24h和48h取样,每次取2mL培养液,进行藻细胞浓度计数,并计算溶藻效率(结果列于表3)。
表3 M0103在不同浓度溶藻菌对球形棕囊藻的溶藻效率
处理时间/样本 | 0h(%) | 6h(%) | 24h(%) | 48h(%) |
0.1% | 0.27±0.12 | 16.25±7.13 | 27.40±10.10 | 50.78±9.72 |
0.5% | 3.51±1.52 | 23.67±5.22 | 54.33±6.92 | 79.33±8.83 |
1% | 2.89±1.08 | 40.54±11.35 | 73.98±4.50 | 87.22±1.23 |
2% | 5.77±1.93 | 38.77±8.05 | 91.41±4.14 | 98.04±1.25 |
从表3可知,以0.1%初始菌浓度将菌株M0103发酵液加入对数期生长期的球形棕囊藻中,48h后溶藻率为50.78%;以0.5%初始菌浓度添加时,24h溶藻效果为54.33%,48h为79.33%;以1.0%初始菌浓度添加时,24h溶藻效果为73.98%,48h为87.22%;以2.0%浓度添加时,24h溶藻活性可达91.41%,48h可达98.04%。可见,M0103对球形棕囊藻具有较高的溶藻效率。
序列表
<110> 广西科学院
<120> 具有溶藻能力的新属菌株及其对球形棕囊藻的应用
<160> 3
<170> SIPOSequenceListing 1.0
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<211> 1458
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<213> 人工序列(Artificial Sequence)
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tagagtttga tcctggctca gaacgaacgc tggcggcagg cttaacacat gcaagtcgag 60
cgaagacttc ggtcttagcg gcggacgggt gagtaacacg tgggaacatg cccttcactg 120
caggatagtc ccgggaaact gggtttaata ctgcatacgc ccttcggggg aaagatttat 180
cggtgaagga ttggcccgcg ctggattagg tagttggtgg ggtaatggcc taccaagccg 240
acgatccata gctggtttga gaggatgatc agccacactg ggactgagac acggcccaga 300
ctcctacggg aggcagcagt ggggaatctt agacaatggg cgcaagcctg atctagccat 360
gccgcgtgag tgacgaaggc cttagggtcg taaagctctt tcgctgggga agataatgac 420
tgtacccagt aaagaaaccc cggctaactc cgtgccagca gccgcggtaa tacggagggg 480
gttagcgttg ttcggaatta ctgggcgtaa agcgcgcgta ggcggattag tcagtcagag 540
gtgaaatccc agggctcaac cctggaactg cctttgatac tgctagtctt gagttcgaga 600
gaggtgagtg gaactccgag tgtagaggtg aaattcgtag atattcggaa gaacaccagt 660
ggcgaaggcg gctcactggc tcgatactga cgctgaggtg cgaaagcgtg gggagcaaac 720
aggattagat accctggtag tccacgccgt aaacgatgaa tgccagacgt cgggcagcat 780
gctgttcggt gtcacaccta acggattaag cattccgcct ggggagtacg gtcgcaagat 840
taaaactcaa aggaattgac gggggcccgc acaagcggtg gagcatgtgg tttaattcga 900
agcaacgcgc agaaccttac caacccttga catggatatc gtagttccga gagatcggtt 960
cgtcagttcg gctggatatc acacaggtgc tgcatggctg tcgtcagctc gtgtcgtgag 1020
atgttcggtt aagtccggca acgagcgcaa cccacatcct tagttgccag cagttcggct 1080
gggcactcta gggaaactgc ccgtgataag cgggaggaag gtgtggatga cgtcaagtcc 1140
tcatggccct tacgggttgg gctacacacg tgctacaatg gcatctacag tgggttaatc 1200
ccaaaaagat gtctcagttc ggattgaggt ctgcaactcg accccatgaa gtcggaatcg 1260
ctagtaatcg cggaacagca tgccgcggtg aatacgttcc cgggccttgt acacaccgcc 1320
cgtcacacca tgggagttgg gtttacccga aggcggtgcg ccaacctttt ggaggcagcc 1380
gaccacggta agctcagcga ctggggtgaa gtcgtaacaa ggtagccgta ggggaacctg 1440
cggctggatc acctcctt 1458
<210> 1
<211> 19
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agagtttgat cctggctca 19
<210> 2
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
ggttaccttg ttacgactt 19
Claims (9)
1.一种菌株M0103,保藏编号为CCTCC NO:M 2019404,分类命名为Mesobaculum littorinae。
2.权利要求1所述菌株在治理赤潮或水华中的应用。
3.权利要求1所述菌株在溶藻方面的应用。
4.权利要求1所述菌株的发酵液用于制备溶藻药剂。
5.权利要求1所述菌株用于抑制或灭杀球形棕囊藻(Phaeocystis globosa scherffel)。
6.权利要求1所述菌株的发酵液用于制备球形棕囊藻抑制剂或灭杀剂。
7.权利要求1所述菌株的培养方法,其特征在于:将该菌接种于培养基中,在28-30℃,pH4-8.5的条件下进行培养。
8.根据权利要求7所述的培养方法,其特征在于包括以下步骤:
(1)斜面培养:将M0103菌种接种于固体斜面培养基上,在28℃条件下培养24h;
(2)初级培养:无菌条件下取培养好的斜面,用接种环取一环于100mL液体培养基中,28℃,摇床180rpm培养24h,获得初级培养菌液;
(3)扩大培养:将初级培养菌液接种于500mL液体培养基中,接种量为5%,28℃,摇床180rpm培养72h,获得扩大培养菌液。
9.根据权利要求8所述的培养方法,其特征在于:所述液体培养基的配方为:酵母提取粉2.0g/L,麦芽提取粉2.0g/L,葡萄糖2.0g/L,海盐26.0g/L,121℃高温蒸汽灭菌20min;所述固体斜面培养基为液体培养基中添加终浓度1.3%的琼脂粉。
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