CN113151049B - 一种赖氨酸芽孢杆菌Owin-1 - Google Patents
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Abstract
本发明涉及一种赖氨酸芽孢杆菌Owin‑1,属于餐厨垃圾的生物处理技术领域。所述赖氨酸芽孢杆菌的分类命名为赖氨酸芽孢杆菌(Lysinibacillus sp.)Owin‑1,保藏于中国典型培养物保藏中心,保藏编号CCTCC NO:M 2021020;保藏日期:2021年1月6日;保藏地址:中国,武汉,武汉大学。本发明公开的赖氨酸芽孢杆菌Owin‑1应用于餐厨垃圾处理菌剂,处理效率相对于现有菌剂处理效率提高20%以上。
Description
技术领域
本发明涉及餐厨垃圾的生物处理技术领域,具体涉及一种赖氨酸芽孢杆菌Owin-1。
背景技术
餐饮垃圾是居民在生活消费过程中形成的,其来源主要是饭店、食堂、街头小吃等场所。所含的各种有机物质在夏天极易腐蚀,餐饮垃圾中剩菜汤、馊水等含量很大,容易在垃圾的收集、运输过程中造成污染。同时餐饮垃圾又是垃圾填埋场所渗滤液的主要来源,也是大气污染和苍蝇滋生重要原因。由餐饮垃圾喂养的猪和提炼的泔水油流向市场已经严重危害了市民的健康。大量的餐饮垃圾已经成为城市污染以及危害人类健康的主要来源,生活垃圾的无害化、减量化和资源化处理越来越受到社会重视。另外,区别于其他的有机垃圾,餐厨垃圾中盐分较高,酸性也较高,使用传统微生物菌剂进行降解菌种抗逆性差,效率较低。
发明内容
为解决现有技术中的问题,本发明专利提供了一种赖氨酸芽孢杆菌,所述赖氨酸芽孢杆菌的分类命名为赖氨酸芽孢杆菌(Lysinibacillus sp.)Owin-1,保藏于中国典型培养物保藏中心,保藏编号CCTCC NO:M 2021020;保藏日期:2021年1月6日;保藏地址:中国,武汉,武汉大学。
同时本发明公开了上述赖氨酸芽孢杆菌Owin-1在餐厨垃圾处理中的应用。
本发明还提供了含有上述赖氨酸芽孢杆菌Owin-1餐厨垃圾处理菌剂,所述菌剂包含:50g的Na2HPO4·7H2O,30g的KH2PO4,1g的CaCl2·6H2O,5g的NaCl,60g的葡萄糖,3g的乙酸钠,1g的ZnSO4·7H2O,10g的NH4CI,60g的Casamino Aci,10L蒸馏水H2O,以及1x1010cfu/ml的赖氨酸芽孢杆菌Owin-1。
上述餐厨垃圾处理菌剂平时以冻干粉的形式保存运输,使用时用无菌水溶解即可,并以液体菌剂的方式喷洒在破碎处理的餐厨垃圾上。
进一步的,所述赖氨酸芽孢杆菌Owin-1的菌剂用量为5%-8%。
相对于现有技术,本发明专利提供的一种赖氨酸芽孢杆菌Owin-1的进步之处在于:利用天然能够利用含氮化合物的细菌来转化有毒有害物质,以实现餐厨垃圾的“处理无害化”;同时,由于转化过程中伴有菌体内自身结构及功能物质的合成,有利于菌群的增殖,菌群可持续,无需频繁补充新菌;菌剂以冻干粉形式进行保存运输,解决了传统工作菌(液体培养基)长期储存和长途运输困难的技术难题,从而突破产品大范围、大规模应用的限制;本申请提供的菌株及菌剂应用效果佳,相比市售的餐厨垃圾处理菌剂效果提高近10%。
附图说明
图1是3种菌消除氨的时量曲线
图2是3种菌对氨水的消除率变化示意图
图3是3种菌转化吲哚的时量曲线
图4是3种菌对吲哚的消除率变化示意图
具体实施方式
下面结合附图和具体实施例对本发明做进一步的说明。对本发明实施例中的技术方案进行清楚、完整的描述,所描述的实施例仅仅是本发明创造一部分的实施例,而不是全部。基于本发明创造中的实施例,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,都属于本发明创造保护的范围。
实施例1
将餐厨垃圾处理厂污泥加入玉米粉、豆粉搅拌均匀,加无菌水后于无菌环境进行细菌增值。
72小时后,称取混合物料,使用无菌水进行稀释,划线涂布于选择培养基上,培养基的组成包括酵母膏10g、蛋白胨10g、琼脂10g、葡萄糖10g、硫酸镁1g、氯化钠1g、氯化钙1g、磷酸钾30g。
于37℃,培养24小时;升温至45℃再培养24℃;完成菌落的初步筛选。
选取长势良好的菌落于液体培养基进行摇床培养,液体培养基包含牛肉膏3g、蛋白胨10g、NaCl 5g和水1000ml,于37℃培养24小时;
向液体培养基中加入5g氯化钠,继续于37℃,200r/h摇床培养36小时;
接着再将菌种接种于含有15gNaCl的液体培养基中,37℃,200r/h摇床培养36小时;
重复以上操作,每次增加10g氯化钠至100g,每次培养条件37℃,200r/h摇床培养36小时;
待菌种进入对数生长期后,将菌种接种到新的含有3g牛肉膏、10g蛋白胨、5g氯化钠的液体培养基上,37℃摇床培养,24小时后加入1ml的醋酸,并每隔12小时加入1ml的醋酸,连续添加两次;
一共培养72小时后收集菌种稀释,接种于新的液体培养基上继续进行摇床培养,12小时后加入2ml醋酸,24小时后再加入2ml醋酸;
36小时后收集菌种,稀释后使用接种环接种于固体培养基上进行多次划线培养,直至长出单株菌落,固体培养基含有酵母膏10g、蛋白胨10g、琼脂10g、氯化钠5g。
得到的菌株革兰氏阳性,杆状,无荚膜,产芽孢,芽孢呈椭圆形至柱状。送青岛西弥斯检测技术服务有限公司进行16srDNA检测,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 TGA TCA TGG CTC AG-3′和F27:5′-AGA GTT TGATCA TGG CTC AG-3′的引导下进行16S rDNA基因的PCR扩增。扩增条件为:95℃预变性3min,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,所得序列如序列表SEQ No.1所示。Blast结果同源性最高的为赖氨酸芽孢杆菌,命名为赖氨酸芽孢杆菌Owin-1。
SEQ.No.1:
GCCGTCCCGAAGGTTACCCCACCTACTTCTTTTGTTACACACTCCCATGGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGTGGCATTCTGATCCACGATTACTAGCGATTCCGACTTCATGGAGTCGAGTTGCAGACTCCAATCCGAACTACGAACCACTTTATGAGATCAGCTTGCTCTCGCGAGGTCGCTTCCCTTTGTATGCTCCATTGTAGCACGTGTGTAGCCCTGGTCATAAGGGGCATGATGACTTGACGTCATCCCCACCTTCCTCCGGTTTATCACCGGCAGTCTCCTTTGAGTGCCCGGCCGAACGATGGCAACTAAAAGAAAAGGGTGCGCTCGTTGCGGGACTTTAACCCACATCTTCACACACGAACTGAACAACACCATGCACCACCTGTCATCGTAGCCCCCGAAGGGGAAACTATATCTCTACAGTGGTCAACGGAATGTCAAGACATGGTAAGGTTCTTCGCGTTGCTTCAAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCACTCTTGCGACCGTACTCCCCAAGCCGTCGTGCTTAATGCGTTAGCTCCAAAGCTAACGC
实施例2
1、取扩增的实施例1分离筛选得到的赖氨酸芽孢杆菌Owin-1和市购的两种餐厨垃圾处理用菌的菌悬液,离心,菌体分别用5%葡萄糖溶液重悬,以保证体系中无其它氮元素来源。加入一定浓度的氨水、吲哚溶液后,置于37℃,220rpm摇床振摇,以相同体积的无菌体系为阴性对照。按一定时间间隔取样,用比色法测定离心所得上清中的含氮化合物含量。以5%葡萄糖溶液为空白对照。绘制含氮化合物浓度-时间曲线(时效曲线),分析比较结果。
氨水
反应机理:铵态氮在碱性介质中与次氯酸钠作用生成靛酚蓝;
主要试剂:水杨酸、次氯酸钠溶液、氢氧化钠;
显色时间:20min
测定波长:580nm
吲哚
反应机理:吲哚与PDAB反应生成玫瑰红色的希夫氏碱;
主要试剂:PDAB的乙醇溶液、盐酸;
显色时间:90℃10min;22℃10min
测定波长:555nm
2、菌种消除氨水的定量测定
将菌密度OD600=0.75的3种菌悬液与1:3200稀释的氨水溶液(氨浓度74.8mg/L)混合,分别编号实验组、市购1组和市购2组,留取0h样品作氨水初始浓度,后分别放入恒温摇床37℃,220rpm培养,以无菌的5%的葡萄糖溶液混合氨水为阴性对照;其余间隔取样3ml,12000rpm离心2min后取上清2.5ml,待显色完全后测定580nm处的OD值,并同时定量检测氨水的转化率。测定结果如图1、2所示。
由图1可以看出,分离得到的赖氨酸芽孢杆菌Owin-1的实验组的OD值在开始出现下降后迅速回升,并一直保持在相对恒定的数值,说明赖氨酸芽孢杆菌Owin-1具有很好的持续性,菌体在处理的过程中能够进行有效的自我增值,处理能力更加的持久,而市购的两组芽孢杆菌随着时间的推移,菌密度都存在明显的下降,自我增值更新能力不足,持久性差。
从图2可以看出分离得到的赖氨酸芽孢杆菌Owin-1对氨水的消除率一直保持稳定的增长状态。而市购的两种菌株,在初始阶段保持了一定的消除率,到后期基本不变,说明大部分菌体已不再增值。
3、菌种消除吲哚水溶液的定量测定
将菌密度OD600=0.75的3中菌悬液与浓度为100mg/L的吲哚溶液混合,留取0h样品作吲哚初始浓度,后分别放入恒温摇床37℃,220rpm培养,以无菌的5%的葡萄糖溶液为阴性对照;其余间隔取样2ml,12000rpm离心2min后取上清1.5ml,待显色完全后测定555nm处的吸光度值。测定结果如图3、4所示。
由图3可以看出,分离得到的赖氨酸芽孢杆菌Owin-1实验组的OD值在起初出现明显的下降后进入平稳期,30min后,才开始出现又一次的明显下降,下将趋势缓慢,而市购的2两菌,在起初明显下降后也进入短暂的平稳期,但在第15-20min时又出现明显的下降,并且下将快速。说明赖氨酸芽孢杆菌Owin-1的持久力较好。
由图4也可以看出,赖氨酸芽孢杆菌Owin-1对吲哚的消除率更佳。
实验结果表明,赖氨酸芽孢杆菌Owin-1体外条件下对氨水和吲哚的消除能力强,且与现有市售菌相比更具优势,能够最大限度地消除含氮化合物,同时菌体继续保持扩增状态。
实施例3
使用实施例1分离筛选得到的赖氨酸芽孢杆菌Owin-1及两种市购餐厨垃圾处理用菌制备液体菌剂,菌剂包含50g的Na2HPO4·7H2O,30g的KH2PO4,1g的CaCl2·6H2O,5g的NaCl,60g的葡萄糖,3g的乙酸钠,1g的ZnSO4·7H2O,10g的NH4CI,60g的Casamino Aci,10L蒸馏水H2O,以及1x1010cfu/ml的活菌。
称取餐厨垃圾30kg充分破碎后分成3份,充分翻搅混合,置于处理槽中,每份添加菌剂150g。并每1小时翻动一次,每6小时检测一次餐厨垃圾的有机质降解率,检测结果如表1所示,。
表1:两组菌剂不同时间的有机质降解率(%)
组别 | 12 | 18 | 24 | 30 | 36 | 40 | 48 |
Owin-1 | 13.2 | 28.6 | 50.8 | 61.4 | 77.3 | 80.2 | 83.2 |
市购1组 | 10.3 | 25.1 | 42.4 | 52.2 | 60.6 | 67.4 | 73.3 |
市购2组 | 9.8 | 23.5 | 38.6 | 50.1 | 58.2 | 63.5 | 71.1 |
Owin-1菌组,在24h后有机质的降解率已超过50%,48小时降解率已超过80%,相较于两组市购菌株,其降解效率和最终降解率均有明显的提高,降解率提高近10%。
本实施例制作的赖氨酸芽孢杆菌Owin-1液体菌剂经浓缩、喷雾、真空冷冻干燥后可制成菌剂冻干粉,进行长期保存,方便运输。
上述内容仅为本发明创造的较佳实施例而已,不能以此限定本发明创造的实施范围,即凡是依本发明创造权利要求及发明创造说明内容所做出的简单的等效变化与修饰,皆仍属于本发明创造涵盖的范围。
序列表
<110> 江苏欧尔润生物科技有限公司
<120> 一种赖氨酸芽孢杆菌Owin-1
<141> 2021-03-08
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 617
<212> DNA
<213> Bacillus sp.
<400> 1
gccgtcccga aggttacccc acctacttct tttgttacac actcccatgg gtgtgacggg 60
cggtgtgtac aaggcccggg aacgtattca ccgtggcatt ctgatccacg attactagcg 120
attccgactt catggagtcg agttgcagac tccaatccga actacgaacc actttatgag 180
atcagcttgc tctcgcgagg tcgcttccct ttgtatgctc cattgtagca cgtgtgtagc 240
cctggtcata aggggcatga tgacttgacg tcatccccac cttcctccgg tttatcaccg 300
gcagtctcct ttgagtgccc ggccgaacga tggcaactaa aagaaaaggg tgcgctcgtt 360
gcgggacttt aacccacatc ttcacacacg aactgaacaa caccatgcac cacctgtcat 420
cgtagccccc gaaggggaaa ctatatctct acagtggtca acggaatgtc aagacatggt 480
aaggttcttc gcgttgcttc aaattaaacc acatgctcca ccgcttgtgc gggcccccgt 540
caattccttt gagtttcact cttgcgaccg tactccccaa gccgtcgtgc ttaatgcgtt 600
agctccaaag ctaacgc 617
Claims (6)
1.一种赖氨酸芽孢杆菌Owin-1,其特征在于,所述赖氨酸芽孢杆菌的分类命名为赖氨酸芽孢杆菌(Lysinibacillus sp.)Owin-1,保藏于中国典型培养物保藏中心,保藏编号CCTCC NO:M 2021020,保藏日期:2021年1月6日,保藏地址:中国,武汉,武汉大学。
2.一种如权利要求1所述的赖氨酸芽孢杆菌Owin-1在餐厨垃圾处理中的应用。
3.一种餐厨垃圾处理菌剂,其特征在于,所述菌剂包含:50g的Na2HPO4·7H2O ,30g的KH2PO4,1g的CaCl2·6H2O,5g的NaCl,60g的葡萄糖,3g的乙酸钠,1g的ZnSO4·7H2O,10g的NH4CI,60g的Casamino acids,10L蒸馏水H2O,以及1x1010cfu/ml的赖氨酸芽孢杆菌Owin-1。
4.根据权利要求3所述的餐厨垃圾处理菌剂,其特征在于,所述菌剂以冻干粉的形式保存运输,使用时用无菌水溶解即可。
5.根据权利要求3所述的餐厨垃圾处理菌剂在餐厨垃圾处理中的应用,其特征在于,所述处理菌剂以液体菌剂的方式喷洒在破碎的餐厨垃圾上。
6.根据权利要求5所述的餐厨垃圾处理菌剂在餐厨垃圾处理中的应用,其特征在于,所述处理菌剂的用量为5%-8%。
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