CN116218684A - 蚜虫莫氏黑粉菌z7菌株及其产品、应用 - Google Patents
蚜虫莫氏黑粉菌z7菌株及其产品、应用 Download PDFInfo
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Abstract
本发明公开蚜虫莫氏黑粉菌Z7菌株及其产品、应用。蚜虫莫氏黑粉菌(Moesziomyces aphidis)Z7菌株CGMCC No.40434自采集自成都市某河边花岗岩矿石堆附近土壤样本中筛选得到,具有分解硅酸盐/浸出硅的工业用途。在实验室条件下经7d培养,菌液硅离子浓度较对照组增加117.90%。Z7菌株适用的脱硅条件是pH 6。Z7菌株的脱硅反应没有副产品,对环境高度友好安全;仅须单一菌株常规条件培养即可应用于硅酸盐分解,工业适用性强。本发明是首次公开蚜虫莫氏黑粉菌的硅酸盐分解用途。本发明同时提供蚜虫莫氏黑粉菌Z7菌株的菌剂,以及菌株或菌剂的多种产品及用途。
Description
技术领域
本发明涉及一种硅酸盐微生物,特别是涉及一种蚜虫莫氏黑粉菌Z7菌株,属于工业微生物技术、生物浸出领域。
背景技术
硅酸盐细菌是指能够分解硅酸盐类矿物的微生物。作为广受重视的工业微生物,硅酸盐细菌的工业用途价值主要包括几方面:(1)硅元素(Si)是地壳中含量最多的固体元素,是“取之不尽”的资源。在现代工业体系中,硅的应用覆盖从传统领域到芯片领域,十分广泛。硅酸盐细菌提供凭借清洁技术从自然资源中获取硅原料的途径。(2)硅在各种工业固体废弃物中(如粉煤灰、高炉渣、赤泥等)含量丰富。硅酸盐细菌提供了这类固废的生物处置方法,是获取硅原料实现固废资源化的洁净生产手段。例如,粉煤灰中含有莫来石、石英、无定型玻璃体等大量硅酸盐化合物,现有技术将芽孢杆菌、黑曲霉、解淀粉芽孢杆菌等硅酸盐细菌用于粉煤灰生物浸矿脱硅。将粘液芽孢杆菌与圆形芽孢杆菌对电解锰残留物协同浸出,使矿样中Si含量大幅度降低。(3)自然界中大量的硅是存在于矿石与岩石中的硅氧化合物,统称硅酸盐。其中较重要的有长石KAlSi3O8、高岭土Al2Si2O5(OH)4、滑石Mg3(Si4O10)(OH)2、云母KAl2(AlSi3O10)(OH)2、石棉H4Mg3Si2O9、钠沸石Na2(Al2Si3O10)·2H2O、石榴石Ca3Al2(SiO4)3、锆石英ZrSiO4、绿柱石Be3Al2Si6O18等。各类硅酸盐中结合固定有丰富多样的其它元素(如K、P、Al等),但因结合状态稳定,不能被直接利用。硅酸盐细菌通过分解硅酸盐能够使化合态的元素溶解释放到土壤中,提升其利用效率。现有研究已表明,硅酸盐细菌是重要的土壤功能菌。硅酸盐细菌通过分解土壤中硅酸盐,可以促进K释放,提高土壤含K水平。此外,硅酸盐细菌还可以分解长石与云母等硅酸盐类的原生态矿物,分解磷灰石中的磷素,使土壤中难溶性的K、P、Si等元素转变为可溶性,提升土壤营养水平;同时,硅酸盐细菌的代谢产物如生长刺激素、多糖、有机酸和蛋白质等能抑制病原菌生长、改善土壤肥力,破坏土壤结晶构造,有利于农作物的营养吸收,从而综合性地达到促进作物生长、增产等效应。(4)硅酸盐细菌可应用于微生物矿物工程(浸矿、选矿、矿物加工),典型的是铝土矿的生物脱硅工艺,即利用硅酸盐细菌及其代谢产物与铝土矿发生吸附、溶蚀、氧化还原等反应,脱出铝土矿中无用成分硅,从而提升矿料的铝硅比,达到氧化铝生产工艺要求。
现有技术所称硅酸盐细菌实际不单指细菌,而是包含了范围更广的微生物门类。目前发现鉴定的硅酸盐细菌多属细菌门物种,即硅酸盐细菌。最主要的是:环状芽孢杆菌(Bacillus circulans)、胶质芽孢杆菌(Bacillus mucilaginosus)(有另鉴定其为胶冻样类芽孢杆菌(Paenibacillus mucilaginosus))、土壤芽胞杆菌(Bacillus edaphicus)(有另鉴定其为土壤类芽孢杆菌(Paenibacillus edaphicus))3种。此外还有邻单胞菌属(P.esianonas)、多粘芽孢杆菌(Paenibacillus polymyxa)、粘液芽孢杆菌(Paenibacillusmucilaginosus)、圆形芽孢杆菌(Bacillus circulans)、根瘤农杆菌(Agrobacteriumtumefaciens)、粘着剑菌M78(Ensifer adhaerens M78)、节杆菌H19(Arthrobacterpascens H19)、伯克氏菌G27(Burkholderia anthina G27)。被鉴定属于真菌物种的硅酸盐细菌数量较少,包括烟曲霉(Aspergillus fumigatus)、黑曲霉(Aspergillus niger)、小刺青霉(Penicillium spinulosum Thom)。
现已鉴定的硅酸盐细菌中,细菌门类远多于真菌门类。然而现有技术研究发现,由于生化代谢差异,真菌会分泌较多有机酸,实现质子交换,溶解矿物,因而真菌脱硅效果优于细菌。筛选培育真菌门类的硅酸盐细菌具有较高的工业用途价值。
现有技术尚未涉及莫氏黑粉菌属(Moesziomyces)微生物的脱硅作用,也没有公开过任何一株具有硅浸出作用的莫氏黑粉菌属菌株。
发明内容
本发明的目的就是提供一种蚜虫莫氏黑粉菌(Moesziomyces aphidis)菌株。该菌株具有硅浸出的工业用途。
为实现上述目的,本发明首先提供一种蚜虫莫氏黑粉菌菌株,其技术方案如下:
一种蚜虫莫氏黑粉菌(Moesziomyces aphidis)Z7菌株,保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏日期2022年12月08日,保藏编号CGMCC No.40434。
蚜虫莫氏黑粉菌Z7菌株CGMCC No.40434是自花岗岩风化形成的土壤样本中筛选得到。筛选过程为:土壤样品加入无菌水,28℃,130r/min振荡培养15min,取上清液梯度稀释10-2~10-5;分别取梯度稀释后的10-4、10-5两浓度培养液样品40μl,涂布到马铃薯固体培养基(PDA),28℃培养5d,挑取单菌落在PDA固体平板上分离纯化,直到平板上出现单一菌落;最终分离获得纯菌株。编号Z7菌株。
Z7菌株在显微镜下观察可见,菌落呈米白色,表面呈褶皱状,菌体呈杆状1.368μm×606.7nm。Z7菌株生理生化特点见表1。将Z7菌株的ITS基因序列(如SEQ ID NO.1)在Genebank进行序列同源性比较,结果显示Z7菌株属于莫氏黑粉菌属下蚜虫莫氏黑粉菌。因此初步鉴定Z7菌株为一株蚜虫莫氏黑粉菌,命名为蚜虫莫氏黑粉菌Z7菌株(Moesziomycesaphidis Z7)。
本发明实验数据证明,蚜虫莫氏黑粉菌Z7菌株CGMCC No.40434能够分解硅酸盐化合物。具体是浸出释放花岗岩粉中硅离子。在实验室条件下经7d培养,花岗岩粉中硅离子浸出释放效率提升超过117%。据此,本发明首先提供以蚜虫莫氏黑粉菌Z7菌株的菌剂产品,其技术方案如下:
菌剂,其特征在于:包含上述蚜虫莫氏黑粉Z7菌株CGMCC No.40434。
本发明同时提供如下应用类技术方案:
蚜虫莫氏黑粉Z7菌株CGMCC No.40434或上述菌剂在分解硅酸盐中的应用。
分解硅酸盐可以是生物脱硅、矿物硅释放、矿物硅资源化、硅酸盐矿物改性、花岗岩环境改造、土壤硅释放、土壤改良/调理、生物肥料、含硅垃圾处置、废弃物资源化、污水污泥处理。
蚜虫莫氏黑粉Z7菌株CGMCC No.40434或上述菌剂在环境硅酸盐资源化中的应用。
蚜虫莫氏黑粉Z7菌株CGMCC No.40434或上述菌剂在微生物矿物工程中的应用。
蚜虫莫氏黑粉Z7菌株CGMCC No.40434或上述菌剂在制备硅酸盐分解产品中的应用。
本发明提供的蚜虫莫氏黑粉Z7菌株或菌剂可以进一步依照常规加工工艺加相关产品,其技术方案如下:
蚜虫莫氏黑粉Z7菌株CGMCC No.40434或上述菌剂为效用成分的硅酸盐分解产品。
以现有技术对硅酸盐细菌工业用途的研究,硅酸盐分解产品可以是生物肥料、土壤调理/改良产品、矿物资源化产品、垃圾处置产品、土壤肥料产品、硅酸盐分解产品、土壤调理/改良产品、矿物资源化产品、垃圾处置产品、花岗岩环境改造产品、选矿脱硅提纯产品等多种。
以加工成除硅菌剂为例,可以麸皮为填充剂,将蚜虫莫氏黑粉Z7菌株在扩大发酵液体培养基中适宜条件下培养制得液体除硅菌剂,再将液体除硅菌剂与灭菌麸皮按质量比1:1(W/W)混合后,在恒温箱中烘干至控制水分,即为固体除硅菌剂。除硅菌剂又可进一步加工包装成多类除硅产品。
上述产品以加工成土壤调理剂或土壤改良剂为例,可以将蚜虫莫氏黑粉Z7菌株在扩大发酵液体培养基中适宜条件下培养制得液体土壤调理剂或土壤改良剂,再将液体土壤调理剂或土壤改良剂加入到土壤中,即达到调理土壤或改良土壤的目的。
上述产品以加工成生物肥料为例,可以将蚜虫莫氏黑粉Z7菌株在发酵液体培养基中适宜条件下培养制得液体生物肥料菌剂,再将液体生物肥料菌剂加入到种有植物的土壤中,即可释放土壤中营养元素以供植物吸收利用,达到生物肥料作用。
上述产品以加工成选矿脱硅提纯产品为例,可以将蚜虫莫氏黑粉Z7菌株在发酵液体培养基中适宜条件下培养制得液体脱硅提纯菌剂,再将液体脱硅提纯菌剂加入到含有矿物的培养基中,即可对矿物进行脱硅提纯处理,提高矿物的品位。
与现有技术相比,本发明的有益效果是:(1)提供了新的真菌门类的硅酸盐细菌——菌株蚜虫莫氏黑粉Z7菌株CGMCC No.40434,该菌株具有分解硅酸盐/浸出硅的工业用途。(2)在现有技术中,蚜虫莫氏黑粉菌只被公开过经发酵生产甘露糖赤藓糖醇脂(MELs)、合成脂肪酶的工业用途,本发明提供了蚜虫莫氏黑粉菌新工业用途。(3)Z7菌株的脱硅效率高,培养条件温和,工业适用性强。(4)Z7菌株是自完全天然花岗岩风化土壤环境中筛选得到,浸出硅反应没有副产品,对周围环境不具有生态入侵威胁,环境高度友好安全,符合生物安全法规。(5)仅须单一菌株即可应用于硅酸盐分解,具有使用方便,包装、运输便利,易形成商品化的明显的技术优势。
附图说明
图1是Z7菌株菌落形态。
图2是Z7菌株显微镜观察形态。
图3是Z7菌株电镜扫描形态。
图4是Z7菌株ITS同源性对比结果。
图5是Z7菌株ITS系统进化树发育树。
图6是Z7菌株生长曲线。
图7是Z7菌株生长最适pH。
具体实施方式
下面结合附图,对本发明的技术方案作进一步的描述。
实施例一
菌株筛选与鉴定。
1、菌株筛选
1.1样品、培养基
花岗岩风化土壤样品:采集自成都市某河边花岗岩矿石堆附近土壤。
马铃薯固体培养基(PDA):马铃薯浸出粉8.0g/L,葡萄糖20.0g/L,琼脂20g/L。
Z7菌悬液:向100mL PD培养基中接入Z7菌株制成种子菌液,pH=7,28℃,条件,培养至对数期(OD600=1),制成Z7菌悬液。
稀盐酸:1mol/L。
1.2分离纯化菌株
1.2.1初筛
在超净无菌操作台中称量土壤样品5g,加入50ml无菌水,28℃,130r/min振荡培养15min,取上清液梯度稀释10-2~10-5。
吸取1ml梯度稀释后的10-4、10-5培养液40μl,涂布到PDA固体培养基,28℃培养5d。
1.2.2分离纯化
挑取PDA固体培养基上疑似菌落的单菌落接种至PDA固体培养基进行分离纯化。28℃静止培养,直到平板上出现单一的菌落。编号为Z7菌株。图1是Z7菌株菌落形态。
2、菌株鉴定
2.1菌株形态、生理生化特性
菌落呈米白色,表面呈褶皱状,菌体呈杆状1.368μm×606.7nm。图2是Z6菌株显微镜观察形态,图3是Z7菌株电镜扫描形态。
2.2菌株ITS基因序列鉴定
取Z7菌悬液2ml于无菌离心管中,12000r/min离心2min,弃上清后下部样品用于分析菌株基因组。
序列分析采用细菌全基因组抽提试剂盒(TSINGKE植物DNA提取试剂盒(通用型))提取上述纯菌株的全基因组。以试剂盒提供引物进行纯化菌株全基因组ITS序列扩增。扩增的PCR产物进行纯化处理,用琼脂糖凝胶电泳和紫外光分光光度计检测浓度与纯度合格后进行测序分析。将所得ITS基因序列(如SEQ ID NO.1)在Genebank进行序列同源性比较。Z7与Moesziomyces aphidis相似性大于99%,通过MEGA11.0软件,建立系统发育树。图4是Z7菌株ITS同源性对比结果,图5是Z7菌株ITS系统进化树发育树。
Z7菌株鉴别是一株蚜虫莫氏黑粉菌(Moesziomyces aphidis)菌株。
2.3确定纯化菌株的种名
综合Z7菌株形态特征与ITS序列分析结果,确定该Z7菌株为蚜虫莫氏黑粉菌新菌株,命名为蚜虫莫氏黑粉菌Z7菌株(Moesziomyces aphidis.Z7)。
上述菌株保藏于中国微生物菌种保藏管理委员会普通微生物中心(CGMCC),保藏地址: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菌保中心,邮编100101;保藏日期2022年12月08日,保藏编号CGMCC No.40434。
实施例二
本发明蚜虫莫氏黑粉Z7菌株CGMCC No.40434最适生长条件试验。
1、培养基
马铃薯液体培养基(PD):同实施例一马铃薯固体培养基,无琼脂。
所有培养基在使用前高温高压灭菌冷却备用。
2、实验方法
2.1制备种子液培养
将纯化好的Z7菌株接种至装有100ml PD液体培养基的250ml三角瓶中,28℃,130r/min振荡培养2d,得Z7菌种子液。
2.2最适培养pH值确定
取5ml种子液接种至装有100ml PD液体培养基的250ml三角瓶中,28℃,130r/min振荡培养。培养过程中取样测定菌液的分光光度值,确定菌株的生长情况。图6是Z7菌株生长曲线。
取5组装有100ml PD液体培养基的250ml三角瓶,分别接种5ml种子液接种,分别设置培养基pH=5、6、7、8、9,28℃,130r/min振荡培养,培养过程中取样测量菌液吸光度,以确定菌株最适生长pH。图7是Z7菌株生长最适pH。
综合确定,Z7菌最适生长pH=6.0。
实施例三
本发明蚜虫莫氏黑粉Z7菌株CGMCC No.40434应用于浸出硅。
1、培养基与材料
花岗岩粉:在花岗岩石堆中采集花岗岩,研磨过200目筛。
PD培养基同实施例二。
2、实验方法
取花岗岩粉样本1g加入装有100ml PD液体培养基的250ml三角瓶中,振荡均匀至菌悬液,按体积比5%接种实施例二制备Z7菌种子液,28℃,130r/min振荡培养,7d后,吸取6ml培养液于10ml离心管中,5000rpm、10min离心,吸取5ml上清液于10ml离心管中,通过ICP-OES测Si浓度。设置3组平行试验。
同时设置不接种Z7菌做对照。
表1菌株脱硅效果
表1结果表明Z7菌株在培养7d后,菌液硅离子浓度(3组平行试验平均值)较对照组增加117.90%。
Claims (10)
1.蚜虫莫氏黑粉菌(Moesziomyces aphidis)Z7菌株,保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏日期2022年12月08日,保藏编号CGMCC No.40434。
2.菌剂,其特征在于:包含权利要求1所述蚜虫莫氏黑粉Z7菌株CGMCC No.40434。
3.权利要求1所述蚜虫莫氏黑粉Z7菌株CGMCC No.40434或权利要求2所述菌剂在分解硅酸盐中的应用。
4.根据权利要求3所述应用,其特征在于:所述分解硅酸盐包括生物脱硅或矿物硅释放或矿物硅资源化或硅酸盐矿物改性或花岗岩环境改造或土壤硅释放或土壤改良/调理或生物肥料或含硅垃圾处置或废弃物资源化或污水污泥处理。
5.权利要求1所述蚜虫莫氏黑粉Z7菌株CGMCC No.40434或权利要求2所述菌剂在环境硅酸盐资源化中的应用。
6.权利要求1所述蚜虫莫氏黑粉Z7菌株CGMCC No.40434或权利要求2所述菌剂在微生物矿物工程中的应用。
7.根据权利要求6所述的应用,其特征在于:是矿物硅的浸出。
8.权利要求1所述蚜虫莫氏黑粉Z7菌株CGMCC No.40434或权利要求2所述菌剂在制备硅酸盐分解产品中的应用。
9.以权利要求1所述蚜虫莫氏黑粉Z7菌株CGMCC No.40434或权利要求2所述菌剂为效用成分的硅酸盐分解产品。
10.根据权利要求8所述应用或权利要求9所述产品,其特征在于:所述硅酸盐分解产品是生物肥料或土壤调理/改良产品或矿物资源化产品或垃圾处置产品或土壤肥料产品或土壤硅酸盐分解产品或土壤调理/改良产品或矿物资源化产品或垃圾处置产品或花岗岩环境改造产品或选矿脱硅提纯产品。
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