CN116286523A - 一株枯草芽孢杆菌及其应用 - Google Patents
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Abstract
本发明公开了一株枯草芽孢杆菌,所述的枯草芽孢杆菌命名为枯草芽孢杆菌LY60(Bacillus subtilis LY60),保藏号为CGMCC NO.26033。本发明枯草芽孢杆菌首次从堆肥高温期样品中筛选分离得到的,该菌株耐高温,生长繁殖快,能促进堆肥腐熟;进一步的,本发明提供的含枯草芽孢杆菌LY60能够提高堆肥温度,加速有机质降解,提高凯氏氮含量,促进堆肥腐熟。
Description
技术领域
本发明涉及植物促生菌技术领域,具体涉及一株枯草芽孢杆菌及其应用。
背景技术
化肥在现代农业生产中起着增加产量,提升产品品质的作用。但是随着化肥的大量使用。农药和化肥发挥生产作用之后会释放大量的有害物质,对自然界生物的生存造成长远影响。因此,寻找一种能够代替化肥的肥料,适应现代农业发展;保障食品的安全供应已是当务之急。微生物肥料可以使土壤的营养成分增加;减少农作物病害;可以替代传统化肥发挥作用,是一种应用前景十分良好的新型肥料。
微生物肥料主要是依靠其中蕴含的微生物发挥作用,加入之后,能够获得特定的肥料效应。目前,一般将微生物肥料制品分为两大类:第一类是狭义的微生物肥料,指通过微生物的生命活动,增加土壤和生产环境中植物营养元素的供应总量,从而达到增产,提高品质的目的。这一类微生物肥料的代表是根瘤菌肥;第二类是广义的微生物肥料,广义的微生物肥料通过肥料组成中微生物的生命活动,不但能提高土壤中营养元素的含量;还能产生植物生长激素,促进植物对营养元素的吸收利用或抵抗某些病原微生物的致病作用,减轻农作物病虫害,提高作物产量。与传统化肥相比,微生物肥料具有安全,高效,成本低,环保等优点
微生物肥料是一种有机肥料,将其用于农业可以改善土壤的理化性质和生物学性状,长时间施用有机肥,可缓慢提高土壤有机质含量,使土壤生物活性变得旺盛,土壤含有更多的养分,抑制土壤病害的发生;再者可以提高作物产量,并保证稳产高产。
传统的堆肥存在发酵周期长、养分损失多、恶臭和温室气体排放量大、肥效较低等问题,制约了好氧堆肥的应用。而微生物是堆肥发酵的主体,其种类和数量变化对有机垃圾的发酵影响很大。因此在堆肥中添加微生物可以起到很好的效果,升高温度加快腐熟、将更多大分子分解成小分子增加肥效,减少废气排放等,具有良好的应用前景。
大量研究实验表明,芽孢杆菌添加剂不仅能够明显改善机体内菌群的组成,而且还具有促进机体生长的功效。此外,芽孢杆菌在颗粒料、粉料的加工过程中以及在酸性环境中均具备较高的稳定性,且能够分泌高活性的蛋白酶和淀粉酶,进而促进饲料营养物质的消化。芽孢杆菌还可以减少粪便和消化道中的大肠埃希氏菌的数量。
发明内容
为解决上述技术问题,本发明提供一株枯草芽孢杆菌及其应用,旨在得到一种加速有机质降解,提高凯氏氮含量,促进堆肥腐熟,快捷方便的腐熟、堆肥方法。
一株枯草芽孢杆菌,命名为枯草芽孢杆菌LY60(Bacillus subtilis LY60),于2022年11月02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,保藏号为CGMCCNO.26033。
如上所述枯草芽孢杆菌在促进堆肥腐熟中的应用。
如上所述枯草芽孢杆菌的菌剂在促进堆肥腐熟中的应用。
其中,所述枯草芽孢杆菌的菌剂的制备方法为将所述的枯草芽孢杆菌按1%(v/v)的接种量接种到500mL灭菌后的LB培养液中,在50℃、180rpm摇床中培养24h,收集培养液,即得菌剂。
其中,所述LB培养液组成为蛋白胨5g,酵母粉2.5g,氯化钠5g,去离子水500mL。
与现有技术相比,本发明的有益效果:
本发明枯草芽孢杆菌首次从堆肥高温期样品中筛选分离得到的,该菌株耐高温,生长繁殖快,能促进堆肥腐熟;进一步的,本发明提供的含枯草芽孢杆菌LY60能够提高堆肥温度,加速有机质降解,提高凯氏氮含量,促进堆肥腐熟。
保藏信息说明
枯草芽孢杆菌LY60(Bacillus subtilis LY60)于2022年11月02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(简称CGMCC),保藏号为CGMCC No.26033。
附图说明
图1为温度随堆肥时间变化曲线;RT:室温,空白组(CK),实验组(Kcy)。
图2为pH随堆肥时间变化曲线;空白组(CK),实验组(Kcy)。
图3为含水率随堆肥时间变化曲线;空白组(CK),实验组(Kcy)。
图4为有机质含量随堆肥时间变化曲线;空白组(CK),实验组(Kcy)。
图5为凯氏氮含量随堆肥时间变化曲线;空白组(CK),实验组(Kcy)。
图6为枯草芽孢杆菌(Bacillus subtilis)LY60菌落图。
图7为筛选出菌株的凝胶电泳图谱。
具体实施方式
下面结合附图具体实施方式进行详细描述,但应当理解本发明的保护范围并不受具体实施方式的限制。实施例中采用的原料、试剂若无特殊说明,皆为市售所得。
实施例1
枯草芽孢杆菌(Bacillus subtilis)LY60的筛选
1.实验材料与设备
1.1主要仪器与设备
表1主要仪器与设备
1.2培养基
细菌培养基:营养琼脂8g,去离子水500mL;
LB培养液:蛋白胨5g,酵母粉2.5g,氯化钠5g,去离子水500mL。
实施例2
枯草芽孢杆菌的分离筛选
2.1取样
筛菌的样品从堆肥高温期取得,在堆体翻堆时用五点取样法取样,样品混匀后装入样品袋中于-4℃的冰箱里保存。
2.2耐高温菌的筛选及纯化
称取在冰箱保存的样品10g,加入到装有90mL经高压灭菌处理的去离子水(0.9%w/v)的三角瓶中,置于摇床上振荡30min,目的是打散菌胶团,使细菌呈单细胞状态分散于溶液中;将灭菌处理后的去离子水分别加到7支试管中(同样进行灭菌处理),每支加入9mL,取震荡后的菌液1mL分别加入试管,即分别稀释到10-1、10-2、10-3、10-4、10-5、10-6、10-7,再分别将各稀释度下的菌液取0.1mL涂布在细菌培养基平板上,50℃恒温培养,培养36小时后菌落形成分布在划线附近,长势良好;每种稀释梯度的平板均作3个平行;可以观察到细菌群落在10-5稀释度下长势较好,在三个平行平板中选取菌落较大的进行分离培养并多次划线纯化。如图6所示,多次划线纯化后所得编号为LY60的菌株菌落形态为圆形,菌落较小,表面光滑,较湿润,稍透明,乳白色,菌落边缘为圆弧形。
实施例3
菌株的鉴定
鉴定对象:实施例2中多次划线纯化后所得编号为LY60的菌株;
3.1提取DNA
DNA使用杭博《Biospin细菌基因组DNA提取试剂盒》提取,具体操作步骤详见其说明书。
3.2PCR扩增
表2PCR扩增反应体系
表3PCR循环条件
3.3PCR产物电泳结果
使用1%浓度的琼脂糖凝胶电泳,电压120V,电泳40min,每孔上样1u L,电泳图谱见图7;如图7所示,PCR产物电泳结果表明细菌的16SrDNA片段扩增成功;
3.4测序及比对结果
测序使用引物27F/1492R双向测序,引物序列信息见表4(引物由北京擎科生物技术有限公司(南宁)进行合成):
表4引物序列信息
实施例2中多次划线纯化后所得编号为LY60的菌株序列如SEQ ID NO.1所示;在NCBI基因库进行BLASTN比对,得菌种鉴定结果,结果见表5;
表5枯草芽孢杆菌鉴定数据
菌株编号 | 相似度最高的菌株 | 一致性 |
LY60 | Bacillus subtilis | 100% |
由表5所示,菌株LY60与Bacillus subtilis相似度达到100%,因此,将该菌株命名为枯草芽孢杆菌(Bacillus subtilis)LY60。
实施例4
含枯草芽孢杆菌菌株的菌剂在促进堆肥发酵中的应用
4.1菌剂制备
把枯草芽孢杆菌LY60种子液按1%(v/v)的接种量,接种到500mL灭菌后的LB培养液中,在摇床(50℃,180rmp)中培养24h,收集培养液即为菌剂;添加菌剂组为实验组(Kcy),空白组(CK)添加500mL灭菌后的LB培养液;
4.2堆肥实验
堆肥原料选用猪粪和蔗髓、干草、木糠,质量比15:2.6:1.2:1,共20kg;堆肥进行36d,分别在第0,4,10,17,26,36d取样,测定了温度、pH、含水率、有机质、凯氏氮;整个堆肥过程在0d接种菌剂一次(空白组在0d接种等体积的LB培养液),堆体翻堆4次,分别是4d、10d、17d、26d;
4.3实验结果
枯草芽孢杆菌LY60耐高温,增长繁殖快;与不添加菌剂的堆肥相比(CK),添加枯草芽孢杆菌LY60的堆肥(Kcy)具有以下优势:
(1)从堆肥过程的温度看,添加枯草芽孢杆菌LY60菌剂的堆体温度比不添加菌剂的堆体温度高,有利于灭杀堆体中病原体及加速堆肥物料的高温降解,促进堆体发酵(图1);
(2)由图2、图3可知,添加枯草芽孢杆菌LY60菌剂的堆肥的pH降得更慢,水分降得更快,即添加菌剂的堆体反应更加剧烈从而导致NH3大量释放减缓了pH下降,产热更多带走大量的水分;
(3)从有机质(OM)的降解率看,不添加菌剂的OM从82.42%下降到70.22%,降解率为14.80%,添加枯草芽孢杆菌LY60菌剂的OM从84.24%下降到68.99%,降解率为18.10%(图4)。
综上所述,添加枯草芽孢杆菌LY60菌剂能够提高堆肥温度,加速有机质降解和水分蒸发,提高凯氏氮的含量,促进堆肥发酵。
前述对本发明的具体示例性实施方案的描述是为了说明和例证的目的。这些描述并非想将本发明限定为所公开的精确形式,并且很显然,根据上述教导,可以进行很多改变和变化。对示例性实施例进行选择和描述的目的在于解释本发明的特定原理及其实际应用,从而使得本领域的技术人员能够实现并利用本发明的各种不同的示例性实施方案以及各种不同的选择和改变。本发明的范围意在由权利要求书及其等同形式所限定。
Claims (5)
1.一株枯草芽孢杆菌,其特征在于,所述的枯草芽孢杆菌命名为枯草芽孢杆菌LY60(Bacillus subtilis LY60),保藏号为CGMCC NO.26033。
2.如权利要求1所述枯草芽孢杆菌在促进堆肥腐熟中的应用。
3.如权利要求1所述枯草芽孢杆菌的菌剂在促进堆肥腐熟中的应用。
4.根据权利要求3所述枯草芽孢杆菌的菌剂在促进堆肥腐熟中的应用,其特征在于:所述枯草芽孢杆菌的菌剂的制备方法为将所述的枯草芽孢杆菌按1%的接种量接种到灭菌后的LB培养液中,摇床中培养,收集培养液,即得菌剂。
5.根据权利要求3所述枯草芽孢杆菌的菌剂在促进堆肥腐熟中的应用,其特征在于:所述LB培养液组成为蛋白胨5g,酵母粉2.5g,氯化钠5g,去离子水500mL。
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