CN113913344A - 一种有机物料腐熟剂及其制备方法 - Google Patents
一种有机物料腐熟剂及其制备方法 Download PDFInfo
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Abstract
本发明公开了一种有机物料腐熟剂及其制备方法。本发明首先自辣椒田中分离到一株暹罗芽孢杆菌(Bacillus siamensis)PAPM‑L226,保藏编号为CGMCC No.23538。该菌株具有较强有机物料腐解能力、促进作物生长能力和拮抗作物病害能力。将采用上述菌株制备的菌粉与黑曲霉菌粉、枯草芽孢杆菌菌粉和黄粉虫粪沙复配而成有机物料腐熟剂,具有加速有机物料腐熟、提高腐熟质量、促进作物生长和预防作物病害的作用。
Description
技术领域
本发明涉及一种有机物料腐熟剂,特别涉及一种以暹罗芽孢杆菌(Bacillussiamensis)、黑曲霉(Aspergillus niger)和枯草芽孢杆菌(Bacillus subtilis)等有益菌作为有效成分的有机物料腐熟剂,属于农业生物工程与技术领域。
背景技术
我国是农业生产大国,种植业与养殖业产能巨大。据统计,我国种植业年产秸秆数量多达7亿吨,养殖业年产畜禽粪污约38亿吨。作物秸秆和畜禽粪便是农业生态系统中十分宝贵的生物质资源,其综合利用对农民增收、环境保护、资源节约以及农业经济可持续发展意义重大。近年来我国作物秸秆和畜禽粪污的综合利用率逐年提高,据报道全国秸秆综合利用率已达85%左右,畜禽粪污综合利用率接近60%。其中,最主要的利用方式是肥料化,包括直接还田和腐熟后制备有机肥料等多种方式。无论是作物秸秆还是畜禽粪污,不腐熟直接还田都会带来许多负面效应,例如影响作物根系生长、引发病虫害、污染土壤环境等。因此,将作物秸秆和畜禽粪污等有机物料先腐熟再还田或加工成有机肥料是最理想的利用方式。
有机物料腐熟剂是一种添加到作物秸秆和畜禽粪污等有机物料中,可达到促进有机物料腐熟进程的生物制剂,主要是微生物活菌制剂。使用有机物料腐熟剂可以加快作物秸秆和畜禽粪污等有机物料生物降解速度,缩短腐熟周期;减少腐熟过程中臭味和有害气体的产生,减少环境污染;促进有害物质的降解和腐植酸类物质的合成,提高腐熟质量。
目前市售有机物料腐熟剂应用广泛,种类繁多,但在生产应用中还普遍存在一些急需解决的问题。例如:对高纤维有机废弃物腐熟效果不够理想,特别是在温度较低的季节,腐熟速度较慢,腐熟周期长,纤维素及半纤维素类大分子物质降解率低。此外,目前市售腐熟剂中的菌种功能单一,往往只考虑其对有机物料的腐解能力,未关注其在农业生产中的其他作用,例如促进作物生长、拮抗作物病害的能力。事实上,如果采用具有较强腐解能力、同时具有较强促生抗病能力的菌种生产腐熟剂,会大幅度提升其在农业生产中的综合应用效果。这是因为,有机物料的腐熟过程,也是微生物生长繁殖和并分泌代谢产物的过程,腐熟产品中除含有有机质外,还含有大量的微生物及其代谢产物。
发明内容
针对现有腐熟剂产品与生产技术中存在的问题,本发明提供一种新型高效的有机物料腐熟剂,由具有较强有机物料腐解能力、促进作物生长能力和拮抗作物病害能力的暹罗芽孢杆菌PAPM-L226、黑曲霉、枯草芽孢杆菌复配而成,具有加速有机物料腐熟、提高腐熟质量、促进作物生长和预防作物病害的作用。
本发明首先提供了一株暹罗芽孢杆菌(Bacillus siamensis)PAPM-L226,该菌株分离自山东省德州市夏津县的辣椒田,该菌株已于2021年10月8日保藏在中国微生物菌种保藏管理委员会普通微生物中心,地址为: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,保藏编号为CGMCC No.23538。该菌株具有在解蛋白、解磷、解钾,拮抗尖孢镰刀菌、层出镰刀菌、串珠镰刀菌和白菜炭疽病菌等作物病原菌,产IAA、产纤维素酶、木聚糖酶和蛋白酶等有益特性,可以用于腐解有机物料、促进作物生长和提高植物抗逆性。
本发明还提供了一种有机物料腐熟剂,按照质量份数计,由暹罗芽孢杆菌PAPM-L226菌粉20~40份,黑曲霉菌粉5~15份,枯草芽孢杆菌菌粉5~15份,黄粉虫粪沙30-70份组成,要求各菌粉活菌含量均≥1.0×1010cfu/g。优选的,暹罗芽孢杆菌PAPM-L226菌粉30份、黑曲霉菌粉10份、枯草芽孢杆菌菌粉10份和黄粉虫粪沙50份。所述黄粉虫粪沙是黄粉虫养殖产业的副产物,是黄粉虫排泄的像细沙一样的粪便,又称沙粪。
其中,暹罗芽孢杆菌PAPM-L226菌粉为暹罗芽孢杆菌PAPM-L226菌株的固体培养物,黑曲霉菌粉为黑曲霉(Aspergillus niger)的固体培养物,枯草芽孢杆菌菌粉为枯草芽孢杆菌(Bacillus subtilis)的固体培养物。
其中暹罗芽孢杆菌PAPM-L226菌粉为:经菌种活化、三角瓶种子制备、发酵罐种子制备和经33℃~42℃固态发酵培养,培养物烘干、粉碎得到暹罗芽孢杆菌PAPM-L226菌粉。其中,所述固态发酵培养基的配方为:按质量比计,麸皮45~55%,小麦秸秆粉44~54%,Ca(OH)20.8~1.2%,加水调整初始含水量50~60%,优选为:麸皮50%,小麦秸秆粉49%,Ca(OH)2 1%,加水调整初始含水量50~60%。其中,菌种活化采用LB固体培养基试管斜面,培养温度33℃~37℃;三角瓶种子制备、发酵罐种子制备采用LB液体培养基,培养温度33℃~37℃。
腐熟剂制备方法:按照质量份数计,将暹罗芽孢杆菌PM-L226菌粉、黑曲霉菌粉、枯草芽孢杆菌菌粉和黄粉虫粪沙混合均匀。
上述有机物料腐熟剂在加速有机物料腐熟、提高腐熟质量、促进作物生长和预防作物病害方面的用途。
本发明还公开了上述有机物料腐熟剂的使用方法,其特征是,以重量份数计,将本发明所述有机物料腐熟剂1~2份与水20~100份混匀后,均匀洒入1000~2000份待腐熟的有机物料中,混合均匀。
上述待腐熟的有机物料包括小麦秸秆、玉米秸秆、水稻秸秆和蔬菜秸秆等各类作物秸秆,猪粪、牛粪、鸡粪和羊粪等各类畜禽粪便,酒糟、糠醛渣、蘑菇渣、餐厨垃圾等各类工农业下脚料。建议采用不同碳氮比和含水量的有机废弃物混合腐熟,以使初始碳氮比接近20~30,初始含水量接近50~60%。必要时可采用尿素调整碳氮比。
本发明的有益效果:
1、本发明有机物料腐熟剂含有多功能菌种暹罗芽孢杆菌PAPM-L226,该菌株不仅对有机物料具有较强的腐解能力,还具有促进作物生长和预防作物病害的作用。
2、本发明有机物料腐熟剂对各类有机物料的适用性强,腐熟速度快,腐熟周期短,腐熟产物安全性好,肥力强。因此,采用本发明有机物料腐熟剂腐熟有机物料,在农业生产中可获得较好的综合应用效果。
3、本发明采用黄粉虫粪沙作为载体。黄粉虫粪沙极为干燥,几乎不含水分,没有任何异味,具有自然气孔率很高的微小团粒结构,表面涂有黄粉虫消化道分泌液形成的微膜,且富含源自黄粉虫肠道的微生物。以黄粉虫粪沙作为载体,有利于改善产品外观,提高生物稳定性。此外,源自黄粉虫肠道中的微生物,也对有机物料的降解腐熟具有助力作用,这有利于提高腐熟剂产品的应用效果。
附图说明
图1为暹罗芽孢杆菌PAPM-L226的菌落形态;
图2为暹罗芽孢杆菌PAPM-L226的菌体形态;
图3为基于16SrDNA部分序列构建的系统发育树;
图4为暹罗芽孢杆菌PAPM-L226溶解无机磷形成的透明圈;其中内环为菌落,外环为因磷酸钙被溶解而形成的透明圈;
图5暹罗芽孢杆菌PAPM-L226分解脱脂奶粉形成的透明圈;其中内环为菌落,外环为脱脂奶粉被分解而形成的透明圈;
图6为暹罗芽孢杆菌PAPM-L226接种于硅酸盐细菌培养基,形成光滑透明油滴状菌落;
图7为暹罗芽孢杆菌PAPM-L226产IAA的能力;从左至右依次为:阳性对照、PAPM-L226;
图8为堆肥过程中的温度变化。
具体实施方式
下面结合实施例对本发明作进一步说明。
本实施例所用的黑曲霉(Aspergillus niger)ACCC 32589菌株,可从中国农业微生物菌种保藏管理中心获得,保藏编号为ACCC 32589,系面向社会公开提供的菌种资源。
上述枯草芽孢杆菌(Bacillus subtilis)ACCC 19373菌株,可从中国农业微生物菌种保藏管理中心获得,保藏编号为ACCC 19373,系面向社会公开提供的菌种资源。
实施例1:暹罗芽孢杆菌PAPM-L226的筛选
暹罗芽孢杆菌PAPM-L226分离自辣椒田土壤。土壤于2019年6月取样自山东省德州市夏津县辣椒田,当季施用了由秸秆和猪粪经堆肥腐熟后制备的有机肥料,有机质含量丰富。具体分离方法为:称取土样5g,放入盛有95mL无菌水和10粒玻璃珠的三角瓶中,于37℃、180rpm振荡30min。取悬液1mL进行10-1-10-8系列浓度梯度稀释,然后取10-5、10-6、10-7三个稀释度涂布至LB培养基平板上,于37℃恒温培养箱中培养48h。试验多次重复,从多个土样中进行分离。共初筛选到236株菌株。所述LB培养基的配方为:蛋白胨10g、酵母膏5g、NaCl10g、琼脂20g、蒸馏水1000mL、pH7.0,121℃灭菌20min。
解蛋白细菌筛选:将上述初筛菌株分别点种到脱脂奶粉培养基平板上,每个平板点4个菌株,37℃恒温培养72h,筛选可产生透明圈的菌株。测量不同菌株的菌落和透明圈直径,根据透明圈直径(D)与菌落直径(d)比值(HC)的大小,评价各菌株的解蛋白能力。共获得具有解蛋白能力的菌株142株,用于后续筛选。所述脱脂奶粉培养基配方为:脱脂奶粉3.0g、琼脂3.2g、去离子水200mL,pH=7.0-7.2,108℃灭菌15min。
解磷细菌筛选:将所筛具有解蛋白能力的菌株用灭菌的牙签接种到无机磷培养基平板上,28℃培养4d后,筛选产生透明圈的菌落。测量菌落直径d和透明圈直径D,并计算HC值(D/d),并据此评价各菌株的解磷能力。共获得具有解磷能力的菌株68株,用于后续筛选。所述无机磷细菌培养基(蒙金娜无机磷细菌培养基)的配方为:葡萄糖10.0g、(NH4)2SO40.5g、MgSO4·7H2O 0.3g、MnSO4·4H2O 0.03g、KCl 0.3g、FeSO4·7H2O 0.03g、NaCl 0.3g、Ca3(PO4)2 10.0g、琼脂1.6%、蒸馏水1000mL,pH=7.0~7.5。
解钾细菌筛选:选用硅酸盐细菌培养基作为筛选培养基,采用三区划线的方法,将上述步骤筛选到的具有解蛋白、解磷能力的菌株进行划线培养,37℃恒温培养72h,选取能在平板上生长并形成光滑透明油滴状菌落的菌株。共获得具有解钾能力的菌株22株。所述硅酸盐细菌培养基的配方为:蔗糖5g、MgSO4 0.5g、CaCO3 0.1g、Na2HPO4 2g、FeCl3 0.005g、玻璃粉1g、琼脂16g、蒸馏水1000mL、pH7.0。
拮抗菌株的筛选:采用平板对峙法,以可引起多种作物病害的尖孢镰刀菌为指示菌,从所筛22株具有解蛋白、解磷、解钾能力的菌株中筛选拮抗菌株。将尖孢镰刀菌接种于PDA平板,28℃培养7天。用直径5mm的打孔器在尖孢镰刀菌菌落边缘打取菌块,接种到另一PDA平板中央。培养3d后,在距离尖孢镰刀菌菌块2cm处接种待筛菌株,于28℃恒温培养7d,观察指示菌尖孢镰刀菌与待筛菌菌落间是否存在抑菌间隙。如存在抑菌间隙,则说明待筛菌对尖孢镰刀菌具有拮抗作用。测量拮抗菌菌落直径与抑菌间隙宽度,拮抗效果以抑菌圈与拮抗菌菌落直径的比值表示(抑菌圈直径=2×抑菌间隙宽度+拮抗菌菌落直径),比值越大,说明拮抗作用越大。共获得对尖孢镰刀菌具拮抗作用的菌株3株。
通过上述多重筛选,综合考虑各菌株的生长速度、产蛋白酶能力、解磷解钾能力和拮抗病原菌的能力,筛选出一株综合性能较好的菌株,标记为PAPM-L226。
实施例2:暹罗芽孢杆菌PAPM-L226的鉴定
(1)形态与生理生化特征
所述PAPM-L226菌株的形态特征为:在LB平板培养基上培养24h,菌落呈圆形,中心隆起形成褶皱,湿润,呈白色,边缘不规则(图1);菌体呈杆状,有芽孢(图2)。
所述PAPM-L226菌株的生理生化特征为:革兰氏阳性,产芽孢,丙二酸利用试验阳性,V-P试验阴性,葡萄糖发酵试验阳性,麦芽糖发酵试验阳性,乳糖发酵试验阳性,蔗糖发酵试验阳性,甲基红试验阴性,甘露醇试验阳性,水解淀粉试验阳性,柠檬酸盐利用试验阳性。
(2)16SrDNA序列分析
将PAPM-L226菌株接种到LB液体培养基中,于37℃、180r/min摇床培养24h。收集菌体,提取总DNA,然后以其为模板,在原核生物16SrRNA基因的通用引物F27:5′-AGA GTT TGATCA TGG CTC AG-3′和F27:5′-AGA GTT TGA TCA TGG CTC AG-3′的引导下进行16SrDNA基因的PCR扩增。扩增条件为:95℃预变性3min,94℃变性1min,55℃复性1min,72℃延伸1.5min,共30个循环,72℃延伸10min。扩增产物经1%琼脂糖凝胶电泳分离后,采用胶回收试剂盒回收,交由上海生工生物技术有限公司测序,所得序列如序列表SEQ No.1所示。将所测16SrDNA序列与GenBank数据库中的序列比对,并用MEGA7.0软件进行多序列同源性分析,并构建系统发育树,如图3所示。
通过形态、生理生化特征和16S rDNA序列分析可知,该菌株为暹罗芽孢杆菌,命名为暹罗芽孢杆菌(Bacillus siamensis)PAPM-L226。该菌株已于2021年10月8日保藏在中国微生物菌种保藏管理委员会普通微生物中心(CGMCC),地址为: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,保藏编号为CGMCC No.23538。
实施例3:暹罗芽孢杆菌PAPM-L226的有益特性测定
(1)暹罗芽孢杆菌PAPM-L226的解磷能力
将暹罗芽孢杆菌PAPM-L226用灭菌的牙签接种到无机磷培养基平板上,28℃培养3d,测量菌落直径d和透明圈直径D,并计算HC值(D/d)。试验结果如图4所示,暹罗芽孢杆菌PAPM-L226菌落直径为6.6mm,透明圈直径为16.5mm,HC值为2.5。
所述无机磷细菌培养基(蒙金娜培养基)配方为:葡萄糖10.0g、(NH4)2SO4 0.5g、MgSO4·7H2O 0.3g、MnSO4·4H2O 0.03g、KCl 0.3g、FeSO4·7H2O 0.03g、NaCl 0.3g、Ca3(PO4)2 10.0g、琼脂1.6%、蒸馏水1000ml、121℃灭菌20min,pH=7.0-7.5。
(2)暹罗芽孢杆菌PAPM-L226的解蛋白能力
将暹罗芽孢杆菌PAPM-L226用灭菌的牙签接种到脱脂奶粉培养基平板上,37℃培养24h,测定透明圈及菌落直径,计算透明圈与菌落直径比(HC)。培养结果如图5所示,透明圈直径为9.0mm,菌落直径为2.0mm,HC值为4.5,说明该菌株具有较强的产蛋白酶的能力。
所述脱脂奶粉培养基配方为:脱脂奶粉3.0g、琼脂3.2g、去离子水200mL,pH=7.0-7.2,108℃灭菌15min。
(3)暹罗芽孢杆菌PAPM-L226的解钾能力
将暹罗芽孢杆菌PAPM-L226在硅酸盐细菌培养基进行划线接种,37℃恒温培养72h。培养结果如图6所示,暹罗芽孢杆菌PAPM-L226能够在硅酸盐细菌培养基上生长并形成光滑透明油滴状菌落,这说明该菌株具有解钾能力。
所述硅酸盐细菌培养基的配方为:蔗糖5g、MgSO4 0.5g、CaCO3 0.1g、Na2HPO4 2g、FeCl3 0.005g、玻璃粉1g、琼脂1.6%、蒸馏水1000mL,121℃灭菌15min、pH=7.0。
(4)暹罗芽孢杆菌PAPM-L226产IAA的能力
向含L-色氨酸(200mg/L)的LB液体培养基中接入暹罗芽孢杆菌PAPM-L226,于28℃、180r/min震荡培养4d。取50μL菌悬液滴于白色陶瓷板上,同时加50μL Salkowski比色液。阳性对照CK中以50μL 50mg/L IAA代替50μL菌悬液。白色陶瓷板于室温避光放置30min,含暹罗芽孢杆菌L-226菌悬液的处理颜色呈深红色,与阳性对照颜色基本一致,这说明暹罗芽孢杆菌L-226具有较强的分泌IAA的能力(图7)。
所述的Salkowski比色液配方为:35%HClO4 50mL、0.5mol/L FeCl31mL。
(5)暹罗芽孢杆菌PAPM-L226的产酶的能力
取麸皮与玉米秸秆的混合物(1:1,w/w)10g,装入250mL三角瓶,加水12mL,混匀,121℃湿热灭菌30min,自然冷却,以5%(w/w)的接种量接入暹罗芽孢杆菌PAPM-L226菌悬液(活菌数1×109cfu/mL),35℃恒温培养48h,于12h、24h、36h各摇瓶一次,发酵完毕,置于通风干燥箱中60℃干燥24h,磨碎。称取1.0g,加50mL蒸馏水,100rpm震荡浸提10min,过滤,滤渣再重复浸提二次,合并3次滤液,即为粗酶液,用于测定纤维素酶和木聚糖酶。
纤维素酶与木聚糖酶酶活力测定采用DNS法:分别将1.5mL 1.00%CMC或1.5mL1.00%木聚糖溶液和经适当稀释的粗酶液置于50℃水浴锅中预热2min,吸取酶液0.5mL加入CMC溶液或木聚糖溶液中,精确计时,反应10min,迅速加入DNS试剂3mL终止反应。然后在沸水浴中显色15min,取出后用蒸馏水定容至25mL,摇匀,冷却,于540nm波长处测定吸光度。
空白对照:先向CMC溶液或木聚糖溶液中加入DNS试剂,置于沸水浴中,随后加入稀释酶液,其他条件和正常测定方法一致。
纤维素酶酶活力单位定义:在50℃、pH5.0的条件下,反应10min,每分钟水解羧甲基纤维素钠生成1μg还原糖所需的酶量为一个酶活力单位,以U表示。
木聚糖酶酶活力单位定义:在50℃、pH5.0的条件下,反应10min,每分钟水解木聚糖生成1μmol还原糖所需的酶量为一个酶活力单位,以U表示。
实验结果表明:暹罗芽孢杆菌L-226培养物中的纤维素酶活力为1265U/g,木聚糖酶活力为356U/g,这表明该菌株具有较高的产纤维素酶与木聚糖酶能力,这有利于其对有机物料的分解与腐熟。
(6)暹罗芽孢杆菌PAPM-L226拮抗作物病原菌的能力
采用平板对峙法研究暹罗芽孢杆菌PAPM-L226菌株对常见作物病原菌的拮抗能力。将尖孢镰刀菌(Fusarium oxysporum)、层出镰刀菌(F.proliferatum)、串珠镰刀菌(F.moniliforme)、白菜炭疽病菌(Colletotrichum higginsianum)等作物病原菌接种于PDA平板,28℃培养7天。用直径5mm的打孔器在病原菌菌落边缘打取菌块,接种到另一PDA平板中央。培养3d后,在距离病原菌菌块2cm处接种待筛菌株,于28℃恒温培养7d,观察病原菌与暹罗芽孢杆菌PAPM-L226菌落间是否存在抑菌间隙。测量暹罗芽孢杆菌PAPM-L226菌落直径与抑菌间隙宽度w,拮抗效果以抑菌圈与暹罗芽孢杆菌PAPM-L226菌落直径的比值D/d表示(抑菌圈直径D=2×抑菌间隙宽度+暹罗芽孢杆菌PAPM-L226菌落直径d),比值越大,说明拮抗作用越大。实验结果表明,暹罗芽孢杆菌PAPM-L226菌株与尖孢镰刀菌、层出镰刀菌、串珠镰刀菌和白菜炭疽病菌等作物病原菌间均可产生拮抗圈,其D/d值依次为2.67、2.12、1.85、2.03,这表明该菌株对多种病原菌均具有一定的拮抗作用。
实施例4:有机物料腐熟剂的制备
本发明有机物料腐熟剂的制备方法包括以下步骤:1、暹罗芽孢杆菌菌粉制备;2、黑曲霉菌粉制备;3、枯草芽孢杆菌菌粉制备;4、有机物料腐熟剂成品配制。
1、暹罗芽孢杆菌菌粉制备
1)将暹罗芽孢杆菌PAPM-L226菌株转接入LB固体培养基试管斜面,于37℃培养24h进行活化;
2)然后转接于三角瓶LB液体培养基,于180r/min、37℃震荡培养24h;
3)再将三角瓶种子以3%的接种量转接入含LB液体培养基的发酵罐中,37℃培养24h。所述发酵罐体积为10L,装液量为7L,搅拌转速250r/min,通风量为7L/min。
4)将发酵罐培养的种子液以5%的接种量转接入固体培养基,发酵培养36h。发酵过程中控制品温35~42℃,含水量45%~50%。发酵结束后,培养物于42℃烘干,粉碎至80~100目,即为暹罗芽孢杆菌PAPM-L226菌粉,活菌含量优选为(2.0~3.0)×1010cfu/g。
固态发酵培养采用55cm×100cm的不锈钢浅盘,发酵培养基的配方为(质量比):麸皮50%,小麦秸秆粉49%,Ca(OH)2 1%,加水调整初始含水量55%。将培养基于121℃灭菌30min,冷却后平铺在预灭菌的浅盘中,料层厚度4cm。
2、黑曲霉菌粉制备
1)将黑曲霉ACCC 32589转接入PDA培养基试管斜面,于27℃培养72h进行活化;
2)用接种环刮取菌苔,接种于PDB培养基中,于180r/min、27℃震荡培养48h;
3)将三角瓶种子以5%的接种量转接入含PDB培养基的发酵罐中,27℃培养36h。其中,所述发酵罐体积为10L,装液量为7L,搅拌转速220r/min,通风量为7L/min。
4)将发酵罐种子液以8%的接种量转接入固体培养基,发酵培养72h;发酵过程中控制品温28~32℃。发酵结束后,培养物于40℃烘干,粉碎至80~100目,即为黑曲霉菌粉,活菌含量优选为(1.0~2.0)×1010cfu/g。
黑曲霉固态发酵培养采用55cm×100cm的不锈钢浅盘。发酵培养基的配方为(质量比):麸皮40%,玉米粉10%,小麦秸秆粉49.8%,MnSO4 0.1%,ZnSO4 0.1%,加水调整初始含水量50%。将培养基于121℃灭菌30min,冷却后平铺在预灭菌的浅盘中,料层厚度4cm。
3、枯草芽孢杆菌菌粉制备
1)将枯草芽孢杆菌ACCC 19373转接入LB培养基试管斜面,于37℃培养24h进行活化;
2)用接种环刮取菌苔,接种于LB液体培养基中,于180r/min、37℃震荡培养24h;
3)将三角瓶种子以5%的接种量转接入含LB液体培养基的发酵罐中,37℃培养24h;其中,所述种子罐体积为10L,装液量为7L,搅拌转速250r/min,通风量为7L/min;
4)将发酵罐种子液以5%的接种量转接入发酵培养基,发酵培养36h。发酵过程中控制品温33~42℃,含水量45%~50%;发酵结束后,培养物于45℃烘干,粉碎至80~100目,即为枯草芽孢杆菌菌粉,活菌含量优选为(2.0~3.0)×1010cfu/g。
枯草芽孢杆菌固态发酵采用55cm×100cm的不锈钢浅盘。发酵培养基配方为:麸皮60%、小麦秸秆粉38.4%、尿素0.5%、Ca(OH)2 1%、MnSO4 0.1%、加水调整初始含水量55%。将培养基于121℃灭菌30min,冷却后平铺在预灭菌的浅盘中,料层厚度4cm。
4、有机物料腐熟剂成品制备
按照质量份数计,将暹罗芽孢杆菌PAPM-L226菌粉30份、黑曲霉菌粉10份、枯草芽孢杆菌菌粉10份和黄粉虫粪沙50份混合均匀,即为本发明所述有机物料腐熟剂。
应用例1:有机物料腐熟剂对作物秸秆的腐熟效果
为考察本发明有机物料腐熟剂对小麦等作物秸秆的腐解效果,2020年6~8月于山东省平阴县安城镇进行了腐熟试验。实验方法参照中华人民共和国农业行业标准NY/T2722-2015《秸秆腐熟菌剂腐解效果评价技术规程》进行。
随机选取粗细相近、完整无损的小麦秸秆,去掉叶片,裁成3cm~5cm的秸秆段,置于85℃鼓风干燥箱中烘干12h。取出称量(精确到0.01g),装入尼龙网袋,每袋秸秆质量50g。每处理按此要求称取15袋秸秆样品,各袋间的秸秆质量极差不超过0.5g。
试验共设3个处理。处理T1:称取本发明有机物料腐熟剂(实施例4制备)和尿素,用水稀释50倍后浸润秸秆;腐熟剂用量为秸秆重量的0.1%,尿素用量为秸秆重量的0.5%;充分浸润后,剩余的悬液均匀撒施于本处理小区地表。处理T2试验方法同T1,但使用的腐熟剂不含暹罗芽孢杆菌PAPM-L226,即本发明有机物料腐熟剂样品制备时,以等量枯草芽孢杆菌ACCC 19373菌粉替代暹罗芽孢杆菌PAPM-L226菌粉而特制的样品,用以对比暹罗芽孢杆菌PAPM-L226与枯草芽孢杆菌ACCC 19373对有机物料的腐熟效果。处理T3试验方法同T1,但不添加任何腐熟剂。
选择地势平坦、土壤肥力均匀、浇排水条件良好的试验地。将同一处理的15袋秸秆样品分成5组,每组3袋用细绳拴在一起。将5组样品按五点梅花形均匀埋置于小区,均匀散开袋中秸秆,埋入深度为5cm~15cm,保持土壤湿度维持在田间持水量的60%~80%。
在试验的第10d、第20d、第30d取样。每次取样时,从同一处理的各组中随机取出秸秆样品1袋,每处理共计5袋。将取得的样品用自来水冲洗干净,置鼓风干燥箱于85℃下烘干12h,称量,其质量记作Nx。
秸杆失重率计算:以质量分数ωx计,数值以%表示,按下式计算每袋样品的秸秆失重率:ωx=(N0-Nx)/N0×100。N0—腐解前秸秆干重,单位为克(g);Nx——某腐解时间秸秆干重,单位为克(g)。
不同处理的小麦秸秆的失重率见下表。
表1小麦秸秆的失重率
实验结果表明,与不施用腐熟剂(T3)相比,施用本发明有机物料腐熟剂(T1)可显著提高小麦秸秆的失重率,这说明本发明有机物料腐熟剂对小麦秸秆具有很好的腐熟效果。与不含暹罗芽孢杆菌L-226的腐熟剂(T2)相比,本发明有机物料腐熟剂(T1)具有更好的腐熟效果。
以同样的方法,还评价了本发明有机物料腐熟剂对水稻秸秆、玉米秸秆、番茄秸秆等作物秸秆的腐解能力,试验结果均能说明本发明有机物料腐熟剂具有较好的有机物料腐熟能力。
应用例2:有机物料腐熟剂对食用菌菌糠、畜禽粪污和番茄秸秆的腐熟效果
试验方法:为验证本发明有机物料腐熟剂在堆肥中的应用效果,以金针菇菌糠、畜禽粪污和番茄秸秆2:1:1混合为原料,于2020年4~6月在山东省平阴县山东佐田氏生物科技有限公司试验大棚内进行堆肥试验。堆肥反应器为内长60cm、内宽45cm、内高40cm、壁厚3cm的EPP泡沫箱,装料量40kg/箱,初始含水量55%。试验共设2个处理,对照处理CK接种普通市售有机物料腐熟剂(含芽孢杆菌、乳酸菌、酵母菌、光合细菌、放线菌,有效活菌数≥200亿个/克),处理T接种本发明实施例4有机物料腐熟剂,接种量均为0.1%(w/w)。每个处理设3个平行。每日14:00—16:00翻动1次,以供给氧气并混匀物料。堆肥过程中,每天9:00—10:00、15:00—16:00测量堆体温度,取平均值作为当日堆体温度。堆肥结束后,取样并在40℃的干燥箱中烘干,粉碎至60目,用于成品养分含量分析。
氮、磷、钾和有机质的含量根据中国农业部有机肥料产品标准进行测定(NY 525-2012)。总腐殖质碳(THC)、腐殖酸碳(HAC)和黄腐酸碳(FAC)的含量按照参考文献(Yu,K.,Sun,X.,Li,S.,Cai,L.,Zhang,P.,Kang,Y.,Yu,Z.,Tong,J.,Wang,L.,2018.Applicationof quadratic regression orthogonal design to develop a composite inoculum forpromoting lignocellulose degradation during green waste composting.WasteManag.79,443-453.https://doi.org/10.1016/j.wasman.2018.08.018.)中描述的方法进行测定。
试验结果:接种本发明有机物料腐熟剂对堆肥温度的影响如图8所示。与CK相比,接种本发明有机物料腐熟剂的处理T升温更快,高温期(50℃以上)更长,为7天,而CK仅3天,这有利于加速有机物料的腐熟进程,缩短腐熟周期。接种本发明有机物料腐熟剂对堆肥产品养分含量的影响下表2所示。与CK相比,处理T的堆肥产品总养分(N+P2O5+K2O)、总腐殖质碳(THC)和腐殖酸碳(HAC)含量更高,黄腐酸碳(FAC)含量更低,种子发芽指数更高,这说明其腐熟质量更好,具有更高的肥效和生物安全性。
表2接种本发明有机物料腐熟剂对堆肥产品养分含量的影响
项目 | CK | T |
有机质(%) | 64.35 | 63.15 |
N(%) | 2.25 | 2.48 |
P<sub>2</sub>O<sub>5</sub>(%) | 3.35 | 3.53 |
K<sub>2</sub>O(%) | 2.16 | 2.21 |
总腐殖质碳(THC,g/Kg) | 159.36 | 186.72 |
腐殖酸碳(HAC,g/Kg) | 140.62 | 165.87 |
黄腐酸碳(FAC,g/Kg) | 15.68 | 13.94 |
种子发芽指数(GI,%) | 112 | 137 |
应用例3:有机物料腐熟剂堆肥产品的促生与抗病抗逆作用
采用盆栽实验评价应用例2所制备堆肥的应用效果。试验所用花盆规格为上内径20cm、下内径14cm、高16cm,装土3.5Kg。试验设3个处理:对照CK不施用堆肥产品,处理T1施用应用例2中CK处理所制备的堆肥产品,处理T2施用应用例2中T处理所制备的堆肥产品。堆肥施用量均为1%(有机肥干重/土壤干重)。每盆定植2株辣椒幼苗。
盆栽试验进行到初果期时,将辣椒植株从盆中取出,尽量不要破坏根系,用清水冲洗根系后,测定下述指标:株高、茎粗、干重、鲜重、叶片叶绿素、根系抗氧化酶活性(超氧化物歧化酶、过氧化氢酶、过氧化物酶)和根系丙二醛(MDA)含量。
株高和茎粗分别使用米尺和游标卡尺测量;植株鲜重和干重采用称重法测量;叶片叶绿素采用紫外分光光度法测量;根系超氧化物歧化酶(SOD)采用氮蓝四唑(NBT)还原法测量;过氧化物酶(POD)采用愈创木酚法测量;过氧化氢酶(CAT)采用紫外分光光度法测量;根系丙二醛(MDA)含量采用硫代巴比妥酸(TBA)显色法测量。试验结果下表所示。
表3有机物料腐熟剂堆肥产品的促生与抗病抗逆作用实验结果
CK | T1 | T2 | |
株高(cm) | 40.58 | 45.2 | 48.89 |
茎粗(mm) | 7.91 | 8.48 | 9.26 |
鲜重(g) | 45.75 | 58.74 | 62.52 |
干重(g) | 13.49 | 16.49 | 18.08 |
叶绿素(mg/g) | 1.55 | 1.67 | 1.74 |
SOD(U/g) | 32.58 | 52.93 | 87.14 |
POD(U/FW·min·g) | 38.36 | 42.21 | 48.34 |
CAT(U/FW·min·g) | 0.58 | 0.66 | 0.79 |
MDA(mg/g·d) | 0.281 | 0.223 | 0.206 |
试验结果表明,与CK相比,施用应用例2所制备的堆肥产品,可以促进番茄的生长,株高、茎粗、鲜重和干重都明显增加,其中施用本发明有机物料腐熟剂堆肥产品的处理(T2)的促生效果最好。与CK相比,施用应用例2所制备的堆肥产品,可以提高番茄根系的抗氧化酶活力,这说明番茄的抗病与抗逆能力增强,其中施用本发明有机物料腐熟剂堆肥产品的处理(T2)的根系抗氧化活力最高。
SEQUENCE LISTING
<110> 山东佐田氏生物科技有限公司
<120> 一种有机物料腐熟剂及其制备方法
<130> 0
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 1427
<212> DNA
<213> 暹罗芽孢杆菌(Bacillus siamensis)PAPM-L226的16S rDNA序列
<400> 1
atgggagctt gctccctgat gttagcggcg gacgggtgag taacacgtgg gtaacctgcc 60
tgtaagactg ggataactcc gggaaaccgg ggctaatacc ggatggttgt ctgaaccgca 120
tggttcagac ataaaaggtg gcttcggcta ccacttacag atggacccgc ggcgcattag 180
ctagttggtg aggtaacggc tcaccaaggc gacgatgcgt agccgacctg agagggtgat 240
cggccacact gggactgaga cacggcccag actcctacgg gaggcagcag tagggaatct 300
tccgcaatgg acgaaagtct gacggagcaa cgccgcgtga gtgatgaagg ttttcggatc 360
gtaaagctct gttgttaggg aagaacaagt gccgttcaaa tagggcggca ccttgacggt 420
acctaaccag aaagccacgg ctaactacgt gccagcagcc gcggtaatac gtaggtggca 480
agcgttgtcc ggaattattg ggcgtaaagg gctcgcaggc ggtttcttaa gtctgatgtg 540
aaagcccccg gctcaaccgg ggagggtcat tggaaactgg ggaacttgag tgcagaagag 600
gagagtggaa ttccacgtgt agcggtgaaa tgcgtagaga tgtggaggaa caccagtggc 660
gaaggcgact ctctggtctg taactgacgc tgaggagcga aagcgtgggg agcgaacagg 720
attagatacc ctggtagtcc acgccgtaaa cgatgagtgc taagtgttag ggggtttccg 780
ccccttagtg ctgcagctaa cgcattaagc actccgcctg gggagtacgg tcgcaagact 840
gaaactcaaa ggaattgacg ggggcccgca caagcggtgg agcatgtggt ttaattcgaa 900
gcaacgcgaa gaaccttacc aggtcttgac atcctctgac aatcctagag ataggacgtc 960
cccttcgggg gcagagtgac aggtggtgca tggttgtcgt cagctcgtgt cgtgagatgt 1020
tgggttaagt cccgcaacga gcgcaaccct tgatcttagt tgccagcatt cagttgggca 1080
ctctaaggtg actgccggtg acaaaccgga ggaaggtggg gatgacgtca aatcatcatg 1140
ccccttatga cctgggctac acacgtgcta caatggacag aacaaagggc agcgaaaccg 1200
cgaggttaag ccaatcccac aaatctgttc tcagttcgga tcgcagtctg caactcgact 1260
gcgtgaagct ggaatcgcta gtaatcgcgg atcagcatgc cgcggtgaat acgttcccgg 1320
gccttgtaca caccgcccgt cacaccacga gagtttgtaa cacccgaagt cggtgaggta 1380
acctttatgg agccagccgc cgaaggtgga cagatgattg ggggaag 1427
Claims (10)
1.一株暹罗芽孢杆菌(Bacillus siamensis)PAPM-L226,所述菌株的保藏编号为CGMCCNo.23538。
2.权利要求1所述的暹罗芽孢杆菌PAPM-L226在解蛋白、解磷、解钾,拮抗尖孢镰刀菌、层出镰刀菌、串珠镰刀菌和白菜炭疽病菌,产IAA、产纤维素酶、木聚糖酶和蛋白酶方面的应用。
3.一种有机物料腐熟剂,其特征是,按照质量份数计,由权利要求1所述的暹罗芽孢杆菌PAPM-L226菌粉20~40份,黑曲霉菌粉5~15份,枯草芽孢杆菌菌粉5~15份,黄粉虫粪沙30-70份组成,要求各菌粉活菌含量均≥1.0×1010cfu/g。
4.如权利要求3所述的一种有机物料腐熟剂,其特征是,按照质量份数计,暹罗芽孢杆菌PAPM-L226菌粉30份、黑曲霉菌粉10份、枯草芽孢杆菌菌粉10份和黄粉虫粪沙50份。
5.如权利要求3或4所述的一种有机物料腐熟剂,其特征是,所述暹罗芽孢杆菌PAPM-L226菌粉为:经菌种活化、三角瓶种子制备、发酵罐种子制备和经33℃~42℃固态发酵培养,培养物烘干、粉碎得到暹罗芽孢杆菌PAPM-L226菌粉。
6.如权利要求5所述的一种有机物料腐熟剂,其特征是,所述固态发酵培养基为:按质量比计,麸皮45~55%,小麦秸秆粉44~54%,Ca(OH)2 0.8~1.2%,初始含水量50~60%。
7.如权利要求6所述的一种有机物料腐熟剂,其特征是,所述菌种活化采用LB固体培养基试管斜面,培养温度33℃~37℃;三角瓶种子制备、发酵罐种子制备采用LB液体培养基,培养温度33℃~37℃。
8.权利要求3或4所述的有机物料腐熟剂在加速有机物料腐熟、提高腐熟质量、促进作物生长和预防作物病害方面的用途。
9.权利要求3或4所述的有机物料腐熟剂的使用方法,其特征是,以重量份数计,将有机物料腐熟剂1~2份与水20~100份混匀后,均匀洒入1000~2000份待腐熟的有机物料中,混合均匀。
10.如权利要求9所述的有机物料腐熟剂的使用方法,其特征是,所述有机物料包括作物秸秆、畜禽粪便和工农业下脚料中的至少一种。
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Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN114591865A (zh) * | 2022-03-24 | 2022-06-07 | 江西益地生物科技有限公司 | 一株解钾抗病的暹罗芽孢杆菌ky758及其应用 |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN108018233A (zh) * | 2017-12-12 | 2018-05-11 | 广州融新科技发展有限公司 | 一株暹罗芽孢杆菌及其微生物菌剂的制备与应用 |
CN110964654A (zh) * | 2018-09-29 | 2020-04-07 | 福建省农业科学院农业生物资源研究所 | 一种拮抗枯萎病的芽孢杆菌及其应用 |
CN111073839A (zh) * | 2020-01-16 | 2020-04-28 | 中化农业(临沂)研发中心有限公司 | 暹罗芽孢杆菌、菌剂及其应用 |
CN111484949A (zh) * | 2019-01-28 | 2020-08-04 | 福建省农业科学院农业生物资源研究所 | 一种促生解磷固氮的耐热芽孢杆菌及其应用 |
-
2021
- 2021-11-16 CN CN202111355460.2A patent/CN113913344B/zh active Active
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN108018233A (zh) * | 2017-12-12 | 2018-05-11 | 广州融新科技发展有限公司 | 一株暹罗芽孢杆菌及其微生物菌剂的制备与应用 |
CN110964654A (zh) * | 2018-09-29 | 2020-04-07 | 福建省农业科学院农业生物资源研究所 | 一种拮抗枯萎病的芽孢杆菌及其应用 |
CN111484949A (zh) * | 2019-01-28 | 2020-08-04 | 福建省农业科学院农业生物资源研究所 | 一种促生解磷固氮的耐热芽孢杆菌及其应用 |
CN111073839A (zh) * | 2020-01-16 | 2020-04-28 | 中化农业(临沂)研发中心有限公司 | 暹罗芽孢杆菌、菌剂及其应用 |
Non-Patent Citations (3)
Title |
---|
IMEN ZOUARI,ET AL: "Biocontrol and plant growth-promoting potentiality of bacteria isolated from compost extract.", ANTONIE VAN LEEUWENHOEK ., pages 2107 - 2122 * |
林志楷等: "暹罗芽孢杆菌研究进展", 亚热带植物科学, no. 04, pages 391 - 396 * |
莫云等: "添加菌剂YX-3对猪粪堆肥氮素转化的影响", 湖南农业科学, no. 03, pages 57 - 60 * |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN114591865A (zh) * | 2022-03-24 | 2022-06-07 | 江西益地生物科技有限公司 | 一株解钾抗病的暹罗芽孢杆菌ky758及其应用 |
CN114591865B (zh) * | 2022-03-24 | 2023-08-22 | 江西益地生物科技有限公司 | 一株解钾抗病的暹罗芽孢杆菌ky758及其应用 |
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