CN113278558A - 一种高产脲酶的枯草芽孢杆菌及其应用方法 - Google Patents
一种高产脲酶的枯草芽孢杆菌及其应用方法 Download PDFInfo
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Abstract
本发明涉及微生物技术领域,且公开了一种高产脲酶的枯草芽孢杆菌及其应用方法,所述菌株为枯草芽孢杆菌,该菌能够生产脲酶,其主要应用于治理被重金属污染的土壤,所述菌株是从如下方法得到:从采集的重金属污染的土壤样品中分离出六株枯草芽孢杆菌在37℃培养36h‑48h后,对六株枯草芽孢杆菌进行生物量和脲酶活性的测定,得到一株脲酶减量较高、生长最佳的菌株。该高产脲酶的枯草芽孢杆菌及其应用方法,与化学修复重金属污染的土壤的方法具有成本低,本身对环境影响小,对技术及设备要求低等优势,与其他植物类生物修复法相比也具有周期短、见效快和耗力小的优势,同时降低环境污染。
Description
技术领域
本发明涉及微生物技术领域,具体为一种高产脲酶的枯草芽孢杆菌及其应用方法。
背景技术
近些年来,国内外众多学者利用微生物对已经被重金属污染得土壤进行矿化修复实验开展了很多研究。硫还原细菌可以通过呼吸作用使硫酸盐作为电子受体,进而在呼吸过程中将其还原成硫化物;它还可以在同化过程中利用硫酸盐合成氨基酸,如胱氨酸和蛋氨酸,再通过脱硫作用使S2-分泌于体外,S2-可以和重金属形成硫化物沉淀。某些研究发现细菌对尿素具有分解作用,从而能够使得重金属离子在方解石矿物中共沉淀出来,进而能够修复那些被重金属污染的地下水。还有学者基于生物矿化原理,利用细菌分离底物产生碳酸根离子,固结重金属离子,达到修复污染土壤的目的。
进一步研究发现,为了更有效固结重金属离子,最理想的状态就是在污染体系内部,重金属离子附近的微区域形成大量离子CO32-。碳酸盐矿化菌能够在其生长过程中产生脲酶,并通过脲酶分解底物尿素产生CO32-,矿化固结环境当中的重金属离子,使其由可提取态转变为较稳定的碳酸盐矿物态,降低其被植物吸收进入人类食物链的风险,达到修复土壤的目的。因此,实现碳酸盐矿化菌在上述领域的规模应用。
这种微生物修复重金属污染土壤不仅能够将滞留的重金属污染物快速降解,或降低重金属毒性,也能够减少污染现场污染物的浓度,为此我们提出了一种高产脲酶的枯草芽孢杆菌及其应用方法。
发明内容
针对现有技术的不足,本发明提供了一种高产脲酶的枯草芽孢杆菌及其应用方法,该菌株能够生产脲酶,并且在尿素的底物作用下,能够高效的降解土壤环境中的重金属,还能够相对于物理化学法修复重金属污染土壤具有对环境影响小,费用低且对技术设备要求都低的优点,解决了上述背景技术中所提出的问题。
为实现上述目的,本发明提供如下技术方案:一种高产脲酶的枯草芽孢杆菌及其应用方法,所述菌株为枯草芽孢杆菌,该菌能够生产脲酶,其主要应用于治理被重金属污染的土壤,所述菌株是从如下方法得到:从广西南宁周边采集的重金属污染的土壤样品中分离出六株枯草芽孢杆菌在37℃培养36 h-48h后,对六株枯草芽孢杆菌进行生物量和脲酶活性的测定,得到一株脲酶减量较高、生长最佳的菌株,命名为B-0,作为出发菌株。
将B-0进行培养,并且按照稀释涂布法稀释至105cfu/mL,取菌液0.1mL 均匀涂于无菌的空平板中,待风干后进行N+离子束注入,N+离子束注入剂量为(80、125、170、215、260)×2.6×1013N+/cm2,N+离子束注入能量为15 keV。辐照结束后用1mL无菌水洗涤细胞,按5倍稀释法稀释后涂入平板培养基,37℃倒置培养24h,待挑取单菌落,摇瓶检测,筛选出其中脲酶活性最强且生长最佳的那一株菌株,命名为枯草芽孢杆菌S-1。
优选的,所述的枯草芽孢杆菌S-1培养条件为碳源为葡萄糖;氮源为酵母粉、牛肉膏、蛋白胨、尿素、硫酸铵等材料;生长的最适温度范围为30~42℃, pH为6.5~8.0,培养过程中可添加磷酸二氢铵、氯化钙、硫酸镁等。
优选的,所述的枯草芽孢杆菌优化发酵,固化重金属的应用。包括如下步骤:
1)种子培养:培养温度30~42℃,培养时间24~60h。
2)取步骤1)中种子液培养基为碳源12~60g/L,氮源12~48g/L,无机盐为1~9g/L,pH6.5~8.0。所述的碳源为葡萄糖,所述的氮源为酵母粉、牛肉膏、蛋白胨、尿素、硫酸铵中的至少一种。所述的无机盐为磷酸盐、钾盐、钙盐、镁盐中的任意一种或者几种的组合。
优选的,所述最佳生产方式为:37℃下发酵24h,在发酵过程中,通气比为2.5v/vm、搅拌300rpm,其中,所述的发酵培养基为:葡萄糖32g/L,蛋白胨18.8g/L,磷酸二氢铵5.3g/L,氯化钙2.5g/L,尿素15.9g/L,无水硫酸镁5g/L,其余为水,pH=7.8。
有益效果如下:
1、该高产脲酶的枯草芽孢杆菌及其应用方法,与化学修复重金属污染的土壤的方法具有成本低,本身对环境影响小,对技术及设备要求低等优势。
2、该高产脲酶的枯草芽孢杆菌及其应用方法,与其他植物类生物修复法相比也具有周期短、见效快和耗力小的优势,同时降低环境污染。
3、该高产脲酶的枯草芽孢杆菌及其应用方法,与其他菌种相比,本发明的枯草芽孢杆菌具备高产脲酶的能力,能够再固化重金属。
具体实施方式
基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
实施例1枯草芽孢杆菌的发酵生产脲酶及其对重金属固化效果的影响
发酵培养基:葡萄糖32g/L,蛋白胨18.8g/L,磷酸二氢铵5.3g/L,氯化钙2.5g/L,尿素15.9g/L,无水硫酸镁5g/L,其余为水,pH=7.8。500mL 摇瓶装液量为100mL,110℃灭菌温度20min。
将初始菌株进行平板活化,生长24h后,将种子液接入液态发酵优化培养基,接种量4%,37℃、转速200r/min下培养24h-36h。测定脲酶的活性及生长情况。尿素浓度120g/L;固化反应时间24h-36h;尿素添加时间2-3h,采用枯草芽孢杆菌进行重金属离子固化实验,得出重金属离子(铅、锌、镉、砷)去除率分别为80.4%、76.3%、62.8%、55.1%。枯草芽孢杆菌对重金属离子有一定的固化效果。
表1枯草芽孢杆菌生长情况及脲酶活性
表2重金属的降解效率
将培养好的枯草芽孢杆菌的发酵菌液取1mL放入摇管中,再其中添加1mL 的尿素溶液,加入尿素后充分振荡混匀,在加入尿素2h后分别加入不同浓度的四种Pb2+、Cd2+、As3+、Zn2+金属离子溶液,充分混匀,放置24h后,离心测定上清中金属离子的含量,每批8支,共四批,分别添加尿素1ml使其终浓度为120g/L,加入尿素后充分振荡混匀,放置20min,然后分别加入不同浓度的金属离子溶液,充分混匀,放入37℃170r/min的摇床中,每隔 12小时每种菌分别取2支,共取4次,离心测上清中金属离子的含量。发现最佳固化时间为48h。
表3固化时间对枯草芽孢杆菌菌液固化重金属去除情况
实施例3粪产碱杆菌固化重金属的土壤实验
分别称取 分别称取五种土壤各3g于小离心管中,向其中加入尿素溶液,使其中的终浓度为120g/L,然后向其中加入浓缩4倍的四种菌液,3g 的土壤总共加入1.2ml、3ml的菌-尿素混合溶液,放置于通风良好的地方48h (模拟实际环境),检测土壤中金属离子的浓度,确定固化效果。实验表明,有效态Pb重金属去除率达到86.5%,有效态Cd重金属去除率达到70.37%,有效态As重金属去除率达到63.20%,有效态Zn重金属去除率达到92.10%。
表6土壤实验重金属去除率
脲酶活性的测定:
(1)样本的前处理:按提取比例加入提取液,超声破碎细胞(冰浴,功率20%,超声3秒,间隔10秒,重复30次)4℃离心弃上清,置冰上待测。
(2)测定步骤:分光光度计或酶标仪预热30min以上,调节波长至578nm,蒸馏水调零;酶促反应:按照试剂盒加入并混匀,放入37℃水浴1h后, 10000r/min 25℃离心10min,取上清液;将上清液稀释10倍后测氨量:在微量石英比色皿96孔板中加入下列试剂,充分混匀,室温放置20min。混匀,于578nm处,蒸馏水调零,读吸光值A,计算A-A测定管-A对照管,每个测定管设一个对照管;UE活力计算:按细菌或细胞密度计算,标准条件下测定的回归方程为y=0.0915x+0.0373;x为标准品浓度(ug/ml),y为吸光值A。
单位的定义:每1万个细菌或细胞每分钟产生1ugNH3-N定义为一个酶活力单位。
这种微生物修复重金属污染土壤不仅能够将滞留的重金属污染物快速降解,或降低重金属毒性,减少污染现场污染物的浓度。
碳酸盐矿化菌是指其能在生长繁殖过程中产生某一酶化特性,将周围环境中Ca2+,Ba2+,Cu2+等离子以碳酸盐的形式沉积矿化出来的一类菌种。因此,碳酸盐矿化菌固结重金属离子过程可用如下反应式来表达:
H2CO3→H++HCO3 -
NH3+H2O→NH4 ++OH-
HCO3 -+H++2NH4 ++2OH-→2NH4 ++CO3 2-+2H2O
Cell-重金属离子+CO3 -2→Cell-重金属碳酸盐
需要说明的是,在本文中,诸如第一和第二等之类的关系术语仅仅用来将一个实体或者操作与另一个实体或操作区分开来,而不一定要求或者暗示这些实体或操作之间存在任何这种实际的关系或者顺序。而且,术语“包括”、“包含”或者其任何其他变体意在涵盖非排他性的包含,从而使得包括一系列要素的过程、方法、物品或者设备不仅包括那些要素,而且还包括没有明确列出的其他要素,或者是还包括为这种过程、方法、物品或者设备所固有的要素。
尽管已经示出和描述了本发明的实施例,对于本领域的普通技术人员而言,可以理解在不脱离本发明的原理和精神的情况下可以对这些实施例进行多种变化、修改、替换和变型,本发明的范围由所附权利要求及其等同物限定。
Claims (6)
1.一种高产脲酶的枯草芽孢杆菌及其应用方法,其特征在于:所述菌株为枯草芽孢杆菌,该菌能够生产脲酶,其主要应用于治理被重金属污染的土壤,所述菌株是从如下方法得到:从采集的重金属污染的土壤样品中分离出六株枯草芽孢杆菌在37℃培养36h-48h后,对六株枯草芽孢杆菌进行生物量和脲酶活性的测定,得到一株脲酶减量较高、生长最佳的菌株,命名为B-0,作为出发菌株;
将B-0进行培养,并且按照稀释涂布法稀释至105cfu/mL,取菌液0.1mL均匀涂于无菌的空平板中,待风干后进行N+离子束注入,N+离子束注入剂量为(80、125、170、215、260)×2.6×1013N+/cm2,N+离子束注入能量为15keV。辐照结束后用1mL无菌水洗涤细胞,按5倍稀释法稀释后涂入平板培养基,37℃倒置培养24h,待挑取单菌落,摇瓶检测,筛选出其中脲酶活性最强且生长最佳的那一株菌株,命名为枯草芽孢杆菌S-1。
2.根据权利要求1所述的一种高产脲酶的枯草芽孢杆菌及其应用方法,其特征在于:将枯草芽孢杆菌活化,在30~42℃下培养24~60h;
其中,发酵培养基为:碳源12~60g/L,氮源12~48g/L,无机盐为1~9g/L,pH6.5~8.0。
3.根据权利要求2所述的一种高产脲酶的枯草芽孢杆菌及其应用方法,其特征在于:所述的碳源为葡萄糖。
4.根据权利要求2所述的一种高产脲酶的枯草芽孢杆菌及其应用方法,其特征在于:所述的氮源为酵母粉、牛肉膏、蛋白胨、尿素、硫酸铵中的至少一种。
5.根据权利要求2所述的一种高产脲酶的枯草芽孢杆菌及其应用方法,其特征在于:所述的无机盐为磷酸盐、钾盐、钙盐、镁盐中的任意一种或者几种的组合。
6.根据权利要求1所述的一种高产脲酶的枯草芽孢杆菌及其应用方法,其特征在于:将所述的枯草芽孢杆菌进行活化,在37℃下发酵24h,在发酵过程中,通气比为2.5v/vm、搅拌300rpm,其中,所述的发酵培养基为:
葡萄糖32g/L,蛋白胨18.8g/L,磷酸二氢铵5.3g/L,氯化钙2.5g/L,尿素15.9g/L,无水硫酸镁5g/L,其余为水,pH=7.8。
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