CN114806589B - 一种活体藻组合物及其制备方法和应用 - Google Patents
一种活体藻组合物及其制备方法和应用 Download PDFInfo
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- CN114806589B CN114806589B CN202210497235.0A CN202210497235A CN114806589B CN 114806589 B CN114806589 B CN 114806589B CN 202210497235 A CN202210497235 A CN 202210497235A CN 114806589 B CN114806589 B CN 114806589B
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Abstract
本发明涉及一种活体藻组合物及其制备方法和应用,所述的活体藻组合物中含有的活性成分包括以下体积份原料:小球藻1~15份、多肽1~10份和甘油酯成分1~10份,所述多肽的氨基酸序列为RSS,该活体藻组合物可以应用在改良土壤或刺激植物生根发芽方面,用于制备土壤改良剂或生粉营养液,能改善土壤和种植环境,增强土壤透气性和疏松度,多肽可以抑制其它植物有害病菌滋生繁殖产生定向抑菌作用,提高作物免疫力,还可以加速种籽发芽,提高出苗率,刺激根端分生组织细胞的分裂与增长;在甘油酯成分培养体系下,小球藻的功效和活力持久性大幅提高,达到“减量增效”的效果,对于促进种子萌发率可以得到有效提高,能有效的缩短育种时间。
Description
技术领域
本发明属于微生物学工程与技术领域,具体涉及一种活体藻组合物及其制备方法和应用。
背景技术
影响植物生长的因素主要有水分、空气、光照、温度和养分。其中,人为刺激植物生长的重要手段是为植物提供容易被植物根系或种子吸收的养分,而提供养分通常是通过改良土壤肥力实现。传统的操作方式主要向土壤中施加肥料或者土壤改良剂,肥料包含有机肥和无机肥,有机肥一般分为植物性氮肥与动物性氮肥,比如秸秆、豆渣、谷糠、麦麸等植物性氮肥,人畜粪便、动物残体、屠宰场废弃物等动物性氮肥;常见的无机肥铵态氮肥、硝态氮肥、酰胺态氮肥、水溶性磷肥、拘溶性磷肥、难溶性磷肥、氮磷钾肥等等。无论是有机肥还是无机肥,都不宜长期使用,容易使土壤中产生并累积大量亚硝态微,从而导致土壤板结,团粒减少,容重增加,通气透水性能差,使土壤中微团聚体结构破坏,质地变粗,易蚀性增加。
而土壤改良剂中通常含小球藻、枯草芽孢杆菌、地衣芽孢杆菌、巨大芽孢杆菌、胶冻样芽孢杆菌等,虽可以使土壤中的难溶性磷素如磷酸钙、磷酸镁、磷酸铁分解为作物能够吸收利用的可溶性磷PO4-,促进团粒结构形成,提高土壤保肥保水能力,增加土壤疏松度,促进根系生长;但由于土壤改良剂中含有大量的菌种和藻,生产成本高,而且要促进植物发芽以及根系生长,则要菌种产生大量类似细胞分裂素、植物生长激素的物质,因此,刺激植物生长发芽的时间比较长。
发明内容
本发明目的在于提供一种活体藻组合物及其制备方法和应用。
为实现上述目的,本发明提供如下技术方案:
本发明提供了一种活体藻组合物含有活性成分,所述活性成分包括以下体积份原料:小球藻1~15份、多肽1~10份和DHA型甘油酯成分1~10份,所述多肽的氨基酸序列为RSS。
本发明通过实验验证,氨基酸序列为RSS的多肽对加速番茄种籽发芽,提高出苗率,刺激根端分生组织细胞的分裂与增长,使幼苗发根快,次生根增多,具有良好的促进效果。
进一步地,所述甘油酯成分包含甘油磷脂型二十二碳六烯酸、二十二碳六烯酸单甘油酯、二十二碳六烯酸甘油二酯、二十二碳六烯酸甘油三酯、破囊壶菌中的至少一种。当甘油酯成分中含有破囊壶菌,破囊壶菌细胞破裂分解时,破囊壶菌的细胞质中会释放出甘油酯型二十二碳六烯酸,促进小球藻微藻生物质数量和油脂含量增多,从而延长微藻的存活、作用时间,减少活体藻的施用频率。
进一步地,所述破囊壶菌含量为103~104cfu/ml。
进一步地,所述活体藻组合物由活性成分和辅剂组成,按重量份计活性成分为5份~35份,辅剂为65份~95份,其中小球藻的浓度为小球藻细胞3×106~4×106cfu/ml。其中,对于活性成分在活体藻组合物中的总含量以及辅剂在活体藻组合物中的含量无特殊要求,可以根据土壤改良或刺激植物生根发芽作用配制的不同剂型选择常规的用量。
进一步地,本发明中,对于活体藻组合物中的辅剂无特殊要求,可以根据需要选配本领域常用的成分,例如,分散剂、润湿剂、防冻剂、稳定剂、增稠剂、消泡剂、溶剂、培养基和固体惰性载体中的一种或几种。这些辅剂成分可以根据活体藻组合物配制的剂型选择常规的用量。
本发明中,对于上述活体藻组合物的成分均无特殊要求,可以采用本领域常用的物质,例如:分散剂可以为三聚磷酸钠、亚甲基双萘磺酸钠、六偏磷酸钠、三异丙醇胺环硼酸盐的至少一种;所述润湿剂可以为十二烷基苯磺酸钠、甘油、大豆卵磷脂、十二烷基磺酸钠中的至少一种;所述防冻剂可以为甘油、聚乙二醇、乙二醇、尿素、萘乙酸钠、硝酸铈中的至少一种;所述稳定剂可以为双氰胺、羧甲基纤维素钠、聚乙烯二醇、聚乙烯醇、聚丙烯酰胺中的至少一种;所述增稠剂选自阿拉伯胶、羧甲基纤维素钠、黄原胶、槐豆胶、瓜尔胶卡拉胶、魔芋胶中的一种或多种;所述消泡剂可以选择聚二甲基硅氧烷、苯乙醇油酸酯、甘油、磷酸三丁酯、二甲基硅油的一种或多种;所述溶剂为水、金属盐溶液、泥水混合物的至少一种;所述培养基可为TAP培养基、SPA培养基、GY液体培养基、水生4号和克诺普(Knop)培养液、BG-11培养基的至少一种;所述固体惰性载体可以为高岭土、沙土、二氧化硅、硅藻土、凹凸棒土、腐殖土、膨润土、泥碳、白碳黑和轻质碳酸钙中的至少一种。
进一步地,本发明对所述活体藻组合物的剂型无特殊要求,例如可以为悬浮剂、油剂、微囊悬浮剂、水分散粒剂或可湿性粉剂。
进一步地,一种的活体藻组合物制备方法,通过如下步骤制备:
步骤S1:培养小球藻:挑取小球藻均匀涂布在TAP平板培养基上,在光照培养箱中20~26℃培养3~5天后,挑取单藻落,转接到TAP斜面培养基中,在光照培养箱中20~26℃培养3~5天,放在冰箱中保种,温度3~7℃,每2个月进行传代一次;
步骤S2:活化:在锥形瓶中加入100mL~150mL的TAP培养基,灭菌,用接种环挑取小球藻接种到锥形瓶中,置于光照摇床,20~26℃振荡培养3天;
步骤S3:多肽的合成:往装有树脂的反应器中依次加入原料Fmoc-Ser(otbu)-OH、Fmoc-Ser(otbu)-OH和Fmoc-Arg(otbu)-OH,利用Fmoc法进行合成氨基酸序列为RSS的多肽;
步骤S4:甘油酯成分制备:在锥形瓶中加入GY液体培养基,灭菌,用接种环挑取破囊壶菌接种到锥形瓶中,置于摇床,20~26℃振荡培养3~7天;
步骤S5:混合:在锥形瓶中加入200mL的TAP培养基,取1~15mL上述经过活化的小球藻、以及1~10mL上述多肽接种TAP培养基中,然后置于摇床20~26℃振荡培养3~7天,再添加1~10mL甘油酯成分。
进一步地,在步骤S2中,可以挑取经过传代两次以上的小球藻接种到锥形瓶中,光照摇床的光照强度为8000LX~12000LX,振荡频率为150~300rpm/min。
进一步地,所述步骤S1中的TAP平板培养基、TAP斜面培养基,所述步骤S2、步骤S5中的TAP培养基均由TAP液体培养液制成,每1000mL的所述TAP液体培养液中包含三羟甲基氨基甲烷2.40~2.50g、盐溶液25~30mL、磷酸溶液0.360~0.380mL、微量元素溶液0.5~2mL、98%冰醋酸0.8~1.5mL。
进一步地,每1000mL所述盐溶液中包含NH4Cl 15.0g、MgSO4·7H2O 4.0g、CaCl2·2H2O 2.0g;每1000mL所述磷酸溶液中包含K2HPO4 28.8g、KH2PO4 14.4g;每1000mL所述微量元素溶液中包含Na2-EDTA 12.5g、ZnSO4·7H2O 2.2g、H3BO3 2.28g、MnCl2·4H2O 0.253g、CoCl2·6H2O 0.08g、CuSO4·5H2O0.078g、(NH4)2Mo2O7 0.05g、FeSO4·7H2O 0.249g。
进一步地,所述步骤S3包括以下步骤:
步骤3.1:根据目标多肽的重量计算每种原料的重量后,将5g树脂放入150mL反应器中,加入50mLDCM浸泡2h,浸泡后用DMF洗涤树脂,然后抽干,如此重复四次,将树脂抽干;
步骤3.2:称取0.02molFmoc-Lys(t Bu)-OH(C端第一个氨基酸)、10mLDCM和10mLDIEA置于30℃的摇床中反应2h,用50mL甲醇溶液封闭(甲醇:DIEA:DCM=1:1:2)30min,然后用DMF洗涤四次,抽干;
步骤3.3:加入20%哌啶溶液(哌啶/DMF=1:4),脱去Fmoc保护基团,再用DMF洗涤四次抽干、取少量树脂,用三酮法检测,若树脂有颜色,说明脱保护成功;
步骤3.4:称00.6molFmoc-Ala(otbu)-0H(C端第一个氨基酸)20mlHOBT和10mLDIC,置于30℃的摇床中反应1h,取少量树脂检测用茚三酮法检测,若树脂有颜色,说明缩合不完全,继续反应;若树脂为无色,说明反应完全;待反应完全后,用DMF洗涤树脂四次,抽干;
步骤3.5:加入20%哌啶(哌啶/DMF=1:4),放在脱色摇床上摇晃20min,脱去树脂上的Fmoc保护基团;脱完保护后用DMF洗涤四次,然后抽干检测保护是否脱去;取少量树脂用茚三酮法检测树脂有颜色,说明脱保护成功;
步骤3.6:按照前述步骤使用Fmoc-Met(otbu)-OH+HOBT+DIC原料,连接氨基酸,用切割试剂将多肽保护基团全部切除,并从树脂上切割下来,然后纯化。
进一步地,在步骤S4中,每1000mL的所述GY平板培养基中含甘蔗糖蜜5~20g、蛋白胨1~2g、酵母提取物0.1~0.5g、海盐2~7g。
进一步地,在步骤S4中,光照摇床的光照强度为8000LX~12000LX,振荡频率为150~300rpm/min。
本发明中,对于各种剂型的制备方法无特殊要求,可以采用本领域公知的各种方法,在此不再赘述。本领域技术人员应该理解的是,本发明中的各种剂型在实际使用时可以根据实际需要稀释使用。
进一步地,本发明还公开了所述的活体藻组合物在改良土壤或刺激植物生根发芽方面的用途。
本发明中,适用于所述活体藻组合物没有特别的限制,对于各类作物均可使用,例如,所述作物可以为果树、蔬菜、瓜类、藤类、草本类和花卉中的至少一种,可以应用在化肥、土壤改良剂、生根营养液。
进一步地,20公斤/亩,2~3次/月,优选稀释20~50倍后再使用,使用前先浇湿土壤。
与现有技术相比,本申请的活体藻组合物主剂成分简单,有利于降低生产成本,抗菌促生,效力持久;小球藻可以作用于土壤中的难溶性养分,能改善土壤和种植环境,增强土壤透气性和疏松度、保水能力,促进固氮菌、放线菌等有益菌的生长,多肽可以抑制其它植物有害病菌滋生繁殖产生定向抑菌作用,提高作物免疫力,还可以加速种籽发芽,提高出苗率,刺激根端分生组织细胞的分裂与增长;在甘油酯成分培养体系下,小球藻的功效和活力持久性大幅提高,达到“减量增效”的效果,因此,本申请的活体藻组合物对于促进种子萌发率可以得到有效提高,能有效的缩短育种时间。
附图说明:
图1为本发明30天培育后对比例与实施例的根系比对图;
图2为本发明30天培育后对比例与实施例的根系活力比对示意图;
图3为本发明a1、a2两组的发芽率统计示意图;
图4为本发明A盆、B盆的发芽势统计示意图;
图5为本发明土壤测试的结果统计示意图。
具体实施方式
以下通过具体实施方式的描述对本发明作进一步说明,但这并非是对本发明的限制,本领域技术人员根据本发明的基本思想,可以做出各种修改或改进,但是只要不脱离本发明的基本思想,均在本发明的范围之内。本发明中所涉及的材料、器皿、试剂以及设备,均可通过市售或本领域常规技术手段获得。
本发明中,实施例中公开的制备方法仅例示性说明本发明的原理及其功效,而非用于限制本发明,其中公开的参数数值仅为本实施例的其中一种方式,在本发明公开的参数范围内均落入本申请的保护范围。任何熟悉此技术的人士皆可在不违背本发明的精神及范畴下,对上述实施例进行修饰或改变,在此不再赘述。本领域技术人员应该理解的是,本发明中的制备方法中所使用的各种成分或设备运行参数在实际使用时可以根据实际需要调整使用。
一种活体藻组合物,通过如下步骤制备:
步骤S1:培养小球藻:挑取小球藻均匀涂布在TAP平板培养基上,在光照培养箱中25℃培养3天后,挑取单藻落,转接到TAP斜面培养基中,在光照培养箱中25℃培养3天,放在冰箱中保种,温度4℃,每2个月进行传代一次;
步骤S2:活化:在锥形瓶中加入100mLmL的TAP培养基,灭菌,用接种环挑取经过传代两次以上的小球藻接种到锥形瓶中,置于光照摇床,25℃振荡培养3天;光照摇床的光照强度为10000LX,振荡频率为150rpm/min;
步骤S3:多肽的合成:往装有树脂的反应器中依次加入原料Fmoc-Ser(otbu)-OH、Fmoc-Ser(otbu)-OH和Fmoc-Arg(otbu)-OH,利用Fmoc法进行合成氨基酸序列为RSS的多肽;
步骤S4:甘油酯成分制备:在锥形瓶中加入GY液体培养基,灭菌,用接种环挑取破囊壶菌接种到锥形瓶中,置于摇床,25℃振荡培养3天,光照摇床的光照强度为10000LX,振荡频率为150rpm/min;每1000mL的所述GY平板培养基中含甘蔗糖蜜10g、蛋白胨1.5g、酵母提取物0.1g、海盐5g;
步骤S5:混合:在锥形瓶中加入200mL的TAP培养基,取1~15mL上述经过活化的小球藻、以及1~10mL上述多肽接种TAP培养基中,然后置于摇床20~26℃振荡培养3~7天,再添加1~10mL甘油酯成分。
其中,所述步骤S1中的TAP平板培养基、TAP斜面培养基,所述步骤S2、步骤S5中的TAP培养基均由TAP液体培养液制成,每1000mL的所述TAP液体培养液中包含三羟甲基氨基甲烷2.42g、盐溶液25mL、磷酸溶液0.375mL、微量元素溶液1.0mL、98%冰醋酸1.0mL;每1000mL所述盐溶液中包含NH4Cl 15.0g、MgSO4·7H2O 4.0g、CaCl2·2H2O 2.0g;每1000mL所述磷酸溶液中包含K2HPO428.8g、KH2PO4 14.4g;每1000mL所述微量元素溶液中包含Na2-EDTA12.5g、ZnSO4·7H2O 2.2g、H3BO3 2.28g、MnCl2·4H2O 0.253g、CoCl2·6H2O 0.08g、CuSO4·5H2O 0.078g、(NH4)2Mo2O7 0.05g、FeSO4·7H2O 0.249g。
实施例1:
一种活体藻组合物,其含有活性成分,所述活性成分包括以下体积份原料:小球藻10份、多肽2份和甘油酯成分1份,所述多肽的氨基酸序列为RSS,其中,所述活体藻组合物中小球藻的浓度为小球藻细胞3×106cfu/ml,多肽浓度为50mg/mL,所述甘油酯成分为破囊壶菌,所述破囊壶菌含量为103cfu/ml。
实施例2:
一种活体藻组合物,其含有活性成分,所述活性成分包括以下体积份原料:小球藻10份、多肽4份和甘油酯成分1份,所述多肽的氨基酸序列为RSS,其中,所述活体藻组合物中小球藻的浓度为小球藻细胞3×106cfu/ml,多肽浓度为100mg/mL,所述甘油酯成分为破囊壶菌,所述破囊壶菌含量为103cfu/ml。
实施例3:
一种活体藻组合物,其含有活性成分,所述活性成分包括以下体积份原料:小球藻10份、多肽6份和甘油酯成分1份,所述多肽的氨基酸序列为RSS,其中,所述活体藻组合物中小球藻的浓度为小球藻细胞3×106cfu/ml,多肽浓度为150mg/mL,所述甘油酯成分为破囊壶菌,所述破囊壶菌含量为103cfu/ml。
实施例4:
一种活体藻组合物,其含有活性成分,所述活性成分包括以下体积份原料:小球藻10份、多肽8份和甘油酯成分1份,所述多肽的氨基酸序列为RSS,其中,所述活体藻组合物中小球藻的浓度为小球藻细胞3×106cfu/ml,多肽浓度为200mg/mL,所述甘油酯成分为破囊壶菌,所述破囊壶菌含量为103cfu/ml。
实施例5:
一种活体藻组合物,其含有活性成分,所述活性成分包括以下体积份原料:小球藻10份、多肽10份和甘油酯成分1份,所述多肽的氨基酸序列为RSS,其中,所述活体藻组合物中小球藻的浓度为小球藻细胞3×106cfu/ml,多肽浓度为250mg/mL,所述甘油酯成分为破囊壶菌,所述破囊壶菌含量为103cfu/ml。
实施例6
一种活体藻组合物,其含有活性成分,所述活性成分包括以下体积份原料:小球藻10份、多肽6份和甘油酯成分10份,所述多肽的氨基酸序列为RSS,其中,所述活体藻组合物中小球藻的浓度为小球藻细胞3×106cfu/ml,多肽浓度为150mg/mL,所述甘油酯成分为破囊壶菌,所述破囊壶菌含量为104cfu/ml。
实施例7
一种活体藻组合物,其含有活性成分,所述活性成分包括以下体积份原料:小球藻15份、多肽6份和甘油酯成分10份,所述多肽的氨基酸序列为RSS,其中,所述活体藻组合物中小球藻的浓度为小球藻细胞4×106cfu/ml,多肽浓度为150mg/mL,所述甘油酯成分为破囊壶菌,所述破囊壶菌含量为104cfu/ml。
对比例1
根据专利申请号为201611225878.0公开的一种小球藻土壤改良剂及其制备方法中实施例3制得土壤改良剂。
研究活体藻组合物催芽试验:
试验对象:
(1)以实施例1~实施例7制得的活体藻组合物分别稀释50倍,分别记为序号处理组1~7;
(2)以对比例1制得小球藻土壤改良剂,稀释50倍,记为对比组8;
(3)将蒸馏水作为空白对照例,记为空白组9。
试验方法:
随机向每组试验对象中投放100粒Micro Tom番茄种子,浸种8h后,分别置于铺有滤纸的培养皿中,保持湿润,在25℃恒温培养箱中催芽2天,每24h记录一次种子发芽数,计算2天后种子的发芽数、发芽势、发芽率、发芽指数。
发芽率Gr=(种子发芽粒数/种子总粒数)×100%
发芽势=日发芽数最高时的总发芽种子粒数/种子总粒数×100%
发芽指数(GI)=∑(Gt/Dt)式中,Gt为t日的发芽数,Dt为发芽天数
试验结果:
催芽试验结果如表1所示:
表1催芽试验结果
由表1可以得到,处理组1~处理组7对于促进种子萌发的效果由于对比组8和空白组9,能有效的缩短育种时间。
生长试验:
试验方法:
(1)番茄种子萌芽后分为两组分别移栽到土壤中,其中一组施加测试组溶液,另一组施加对照组溶液,培养30天后,观察其根部生长情况(为便于观察比较,将两棵番茄植株分别置于两个烧杯中,如图1,并用TTC法进行根系活力的测定。
(2)随机选取20粒Micro Tom番茄种子,分为a1、a2两组,每组10颗,分别置于铺有黑布的培养皿中,向a1组的Micro Tom番茄种子表面喷洒10ml对照组溶液,向a2组的MicroTom番茄种子表面喷洒10ml测试组溶液,再将两组培养皿置于25℃恒温培养箱中催芽2天。
(3)随机选取20粒Micro Tom番茄种子,分为两组,每组10颗,分别种于A、B两个盆中,每天分别向A、B两个盆中土壤表面喷洒20ml对照组溶液、测试组溶液,在光照、温度相同的培养环境下培养5天,观察植株的生长情况。
试验对象:
测试组溶液采用实施例7的活体藻组合物稀释50倍得到;对照组溶液采用对比例1的土壤改良剂稀释50倍得到。
试验结果:
(1)图1为两棵番茄植株培养30天后根系的比对图,可以看出,采用测试组溶液进行培植的番茄植株根系远发达于采用对照组溶液进行培植,番茄植株生长更快,侧根更丰富,有利于吸收营养和水分,叶片大小和茂密程度可以提高50%~60%,通过TTC法进行根系活力的测定,可以得到,采用测试组溶液进行培植的番茄植株根系活力高达0.935mg/g·h,详细可参考图2。
(2)通过图3可以看出,在相同的培育条件下,采用测试组溶液进行培植的番茄种子发芽率以及芽长均优于采用对照组溶液进行培植的番茄种子。
(3)通过图4可以看出,在相同的培育条件下,采用测试组溶液进行培植的番茄种子成活率均优于采用对照组溶液进行培植的番茄种子。
土壤测试:
测试方法:
随机选取种植土壤500g搅拌均匀,并将其平均分为测试组、对照组两组,每组250g,分别向测试组、对照组喷洒测试组溶液、对照组溶液50ml,搅拌均匀后,静置3天,分别测定经过喷洒测试组溶液、对照组溶液处理后的土壤中碱解氮、速效磷以及速效钾含量;其中,测试组溶液采用实施例7的活体藻组合物稀释50倍得到;对照组溶液采用对比例1的土壤改良剂稀释50倍得到;
碱解氮采用碱解扩散法测定;
速效磷采用碳酸氢钠浸提-钼锑抗分光光度法;
速效钾采用乙酸铵浸提-火焰光度法测定。
测试结果:
测试结果参考图5,采用测试组溶液处理的土壤中,碱解氮的含量比对照组高40%,且采用测试组溶液处理土壤中速效磷和速效钾的含量也对照组高2倍、3倍,因此,采用测试组溶液处理的土壤富集植物生长所需的微量元素,促进根系的生长。
上述实施例仅例示性说明本发明的原理及其功效,而非用于限制本发明。任何熟悉此技术的人士皆可在不违背本发明的精神及范畴下,对上述实施例进行修饰或改变。因此,举凡所属技术领域中具有通常知识者在未脱离本发明所揭示的精神与技术思想下所完成的一切等效修饰或改变,仍应由本发明的权利要求所涵盖。
Claims (10)
1.一种活体藻组合物,该活体藻组合物含有活性成分,其特征在于,所述活性成分包括以下体积份原料:小球藻1~15份、多肽1~10份和甘油酯成分1~10份,所述多肽的氨基酸序列为RSS,所述甘油酯成分包含破囊壶菌。
2.如权利要求1所述的活体藻组合物,其特征在于,所述甘油酯成分还包含甘油磷脂型二十二碳六烯酸、二十二碳六烯酸单甘油酯、二十二碳六烯酸甘油二酯、二十二碳六烯酸甘油三酯中的至少一种。
3.如权利要求2所述的活体藻组合物,其特征在于,所述破囊壶菌含量为103~104cfu/ml。
4.如权利要求1所述的活体藻组合物,其特征在于,由活性成分和辅剂组成,按重量份计活性成分为5份~35份,辅剂为65份~95份,其中小球藻的浓度为小球藻细胞3×106~4×106/ml。
5.如权利要求4所述的活体藻组合物,其特征在于,所述辅剂选自分散剂、润湿剂、防冻剂、稳定剂、增稠剂、消泡剂、溶剂、培养基和固体惰性载体中的至少一种。
6.如权利要求1所述的活体藻组合物,其特征在于,所述活体藻组合物为悬浮剂、油剂、水分散粒剂或可湿性粉剂。
7.一种如权利要求1~6任一项所述的活体藻组合物制备方法,其特征在于,通过如下步骤制备:
步骤S1:培养小球藻:挑取小球藻均匀涂布在TAP平板培养基上,在光照培养箱中20~26℃培养3~5天后,挑取单藻落,转接到TAP斜面培养基中,在光照培养箱中20~26℃培养3~5天,放在冰箱中保种,温度3~7℃,每2个月进行传代一次;
步骤S2:活化:在锥形瓶中加入100mL~150mL的TAP培养基,灭菌,用接种环挑取小球藻接种到锥形瓶中,置于光照摇床,20~26℃振荡培养3天;
步骤S3:多肽的合成:往装有树脂的反应器中依次加入原料Fmoc-Ser(otbu)-OH、Fmoc-Ser(otbu)-OH和Fmoc-Arg(otbu)-OH,利用Fmoc法进行合成氨基酸序列为RSS的多肽;
步骤S4:甘油酯成分制备:在锥形瓶中加入GY液体培养基,灭菌,用接种环挑取破囊壶菌接种到锥形瓶中,置于摇床,20~26℃振荡培养3~7天;
步骤S5:混合:在锥形瓶中加入200mL的TAP培养基,取1~15mL上述经过活化的小球藻、以及1~10mL上述多肽接种TAP培养基中,然后置于摇床20~26℃振荡培养3~7天,再添加1~10mL甘油酯成分。
8.如权利要求7所述的活体藻组合物制备方法,其特征在于,每1000mL的所述TAP平板培养基、TAP斜面培养基、TAP培养基中分别包含三羟甲基氨基甲烷2.40~2.50g、盐溶液25~30mL、磷酸溶液0.360~0.380mL、微量元素溶液0.5~2mL、98%冰醋酸0.8~1.5mL。
9.如权利要求8所述的活体藻组合物制备方法,其特征在于,在所述步骤S4中,每1000mL的所述GY液体培养基中含甘蔗糖蜜5~20g、蛋白胨1~2g、酵母提取物0.1~0.5g、海盐2~7g、琼脂8~15g。
10.一种根据权利要求1所述的活体藻组合物的应用,其特征在于,用于改良土壤或刺激植物生根发芽。
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