CN112852675B - 枯草芽孢杆菌sk01及其在降解塑料中的应用 - Google Patents
枯草芽孢杆菌sk01及其在降解塑料中的应用 Download PDFInfo
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Abstract
本发明公开了枯草芽孢杆菌SK01及其在降解塑料中的应用,该枯草芽孢杆菌SK01保藏于中国典型培养物保藏中心,保藏编号CCTCC NO:M2020812,具有较强的降解塑料的能力,可用于制备可降解塑料,能够隔离高温对枯草芽孢杆菌SK01的破坏,缩小与聚烯类塑料的比重差异,保存期可达1年以上,确保枯草芽孢杆菌SK01产生的微生物酶在堆肥、土壤掩埋的环境中153天有机固体物总降解量49.11%、阳光照射(光降解)或浸泡水中可缓速降解,排除以上的环境,添加枯草芽孢杆菌SK01的聚烯类塑料成品的保存期与无降分解塑料是一致的,可应付各种聚烯类塑料的需求。
Description
技术领域
本发明涉及微生物技术领域,具体涉及一种新型的枯草芽孢杆菌SK01,及其在降解塑料中的应用。
背景技术
塑料制品背负着白色污染的恶名,但也是人类文明生活不可或缺的用品,更是工业之母,因此在全球大声疾呼解决白色污染的同时,生物降解材料如雨后春笋般百家争鸣。
生物降解塑料是指一类由自然界存在的微生物如细菌、霉菌(真菌)和藻类的作用而引起降解的塑料。理想的生物降解塑料是一种具有优良的使用性能、废弃后可被环境微生物完全分解、最终被无机化而成为自然界中碳素循环的一个组成部分的高分子材料。“纸”是一种典型的生物降解材料,而“合成塑料”则是典型的高分子材料。因此,生物降解塑料是兼有“纸”和“合成塑料”这两种材料性质的高分子材料。
要大规模使用,最终需要以成本为第一考虑重点,目前生物降解塑料的研发成果还无法做到低成本、可工业化生产的程度。
发明内容
本发明的目的在于提供一种特性优异的枯草芽孢杆菌SK01,以及该枯草芽孢杆菌SK01在降解塑料中的应用。
本发明技术方案详述如下:
枯草芽孢杆菌(Bacillus subtilis)SK01,保藏于中国典型培养物保藏中心,保藏编号CCTCC NO:M2020812。
该菌株的生物学特性如下:
(1)该株枯草芽孢杆菌菌体增殖快速,与其他菌种并存时具有繁殖优势。
(2)其耐温度特性如下:
1)活跃温湿度:温度15~85℃/湿度30~100%;
2)休眠湿温度:温度零下60℃~14℃、86℃~119℃/湿度30~100%;
3)死亡温湿度:温度零下60℃以下、120℃以上/湿度30~100%;
4)死亡温湿度:温度340℃/湿度0.01~0.2%/20秒。
(3)孢子抗逆境能力强,在活跃温湿度孢子出芽快速。
(4)有机物质分解力特强:具有高活性的淀粉分解酶、蛋白质分解酶、脂肪分解酶、纤维素分解酶、及其它种类的分解酶。
(5)针对腐败菌、恶臭菌及病源菌的抑制力特高:对金黄色葡萄球菌(含MRSA)、沙门氏菌、大肠菌(含O157:H7)、赤痢菌、肠炎弧菌、绿脓菌、退伍军人杆菌等重要病原细菌;镰刀菌(Fusarium)、格孢菌Alternarium、Botrytis、Pestalotia等植物病原性真菌具有拮抗能力。
本发明还提供了该枯草芽孢杆菌在降解塑料方面的应用。塑料主要指聚烯类塑料。
聚烯类塑料作为枯草芽孢杆菌SK01产生的各类微生物酶的载体,当塑料制品被使用后,物性渐近老化,待埋入土壤或处于堆肥中,通过带有酸性水分的浸湿以及表面氧化,枯草芽孢杆菌SK01产生的微生物酶便从聚烯类塑料中释出,从而诱发环境土著菌在塑料制品本体上生长扩繁,造成塑料制品的加速老化,再经过环境中的后生动物吞食代谢,最终达到降分解的目的,此作用简称为生物能反蚀载体“生物便当”。
优选的,做上述应用时,塑料中枯草芽孢杆菌SK01的添加量为塑料总重的0.05~0.2%(0.5~2kg/1000kg)。
本发明还提供了一种枯草芽孢杆菌微粒胶囊,由上述枯草芽孢杆菌SK01包覆在衣膜中构成,所述衣膜为乳酸或壳聚糖,和甲基硅油。
优选的,上述枯草芽孢杆菌微粒胶囊,以干燥固体形态重量百分比计,各成分用量为:枯草芽孢杆菌SK0149~53%,乳酸或壳聚糖45~47%,甲基硅油1~4%。
本发明还提供了上述枯草芽孢杆菌微粒胶囊的制备方法,是将枯草芽孢杆菌SK01和衣膜经混合、干燥、研磨、过筛、精磨后添加甲基硅油混炼,形成衣膜包覆枯草芽孢杆菌SK01的微粒胶囊。
本发明还提供了一种生物降解塑料母粒,是由上述枯草芽孢杆菌微粒胶囊、聚烯烃、碳酸钙、分散剂和硬脂酸锌经密炼造粒制成。
优选的,上述生物降解塑料母粒,以干燥固体形态重量百分比计,各成分用量为:枯草芽孢杆菌微粒胶囊0.3~1%,聚烯烃12~16%、碳酸钙80~87%,分散剂2%,硬脂酸锌1%。
本发明还提供了上述生物降解塑料母粒的制备方法,包括以下步骤:
(1)将枯草芽孢杆菌微粒胶囊、聚烯烃、碳酸钙、分散剂和硬脂酸锌搅拌混匀,得混合物;
(2)混合物经密炼机进行密炼熔合后,螺杆挤出机造粒;
(3)送风冷却,过筛,得产品。
优选的,上述制备方法中,步骤(2)中密炼熔合温度为170~202℃,时间25~35分钟;螺杆挤出机造粒的温度190~202℃,时间2~5分钟。在此温状态下其活性并不被灭失或减少。
本发明还提供了一种生物降解塑料,由上述生物降解塑料母粒和聚烯烃材料制成,生物降解塑料母粒的添加量为生物降解塑料总重量的5~50%。
与现有技术相比,本发明具有以下有益效果:
本发明的枯草芽孢杆菌SK01能够产生多种酶类,对有机质的分解能力较强,适用于降解塑料等用途。
枯草芽孢杆菌微粒胶囊使用乳酸衣膜、或壳聚糖衣膜进行包覆,使其在极度厌氧和高温的环境下,如干热情况下能承受340℃/0.35hr高温,成分不被破坏。确保其后续制作生物可降解塑料时能够发挥降解作用。
生物降解塑料母粒用于制备成品可降解塑料,该母粒能够隔离高温对枯草芽孢杆菌SK01微生物酶的成分破坏,缩小与聚烯类塑料的比重差异,保存期可达1年以上,确保枯草芽孢杆菌SK01产生的微生物酶在堆肥、土壤掩埋的环境中(湿度、温度)153天有机固体物总降解量49.11%、阳光照射(光降解)或浸泡水中可缓速降解,排除以上的环境,添加枯草芽孢杆菌SK01微生物酶的聚烯类塑料成品的保存期与无降分解塑料是一致的,可应付各种聚烯类塑料的需求。
保藏信息:
培养物名称:枯草芽孢杆菌SK01,拉丁文名称:Bacillus subtilis SK01;
保藏机构:中国典型培养物保藏中心,地址:中国.武汉.武汉大学;
保藏日期:2020年12月2日,保藏编号CCTCC NO:M2020812。
附图说明
图1为生物降解塑料母粒结构示意图;
图2为枯草芽孢杆菌SK01在塑料制品中的3D穿隧式摄像图;
图3为枯草芽孢杆菌SK01产生的微生物酶与塑料作用后使塑料膨胀发泡的3D穿隧式摄像图;
图4为枯草芽孢杆菌SK01在塑料制品中的尺寸展示(3D穿隧式摄像图);
图5为2012E0568试样和参比材料纤维素降解过程中二氧化碳释放量曲线图。
图6为2012E0568试样和参比材料纤维素降解过程中生物分解率曲线图。
具体实施方式
实施例1枯草芽孢杆菌SK01的获取及特性检测
本发明所用的枯草芽孢杆菌SK01原生菌株取自枯干稻草,其筛选具体步骤如下:
1、枯草芽孢杆菌SK01培养基优化、培养条件的筛选
(1)C源基础培养基:酵母膏3g/L,蛋白胨10g/L,NaCl 5g/L,pH7.2-7.4。
(2)N源基础培养基:葡萄糖5g/L,NaCl 5g/L,pH7.2-7.4。
培养筛选出来的菌株在C源基础培养基中,以葡萄糖为C源,配制含不同葡萄糖浓度的培养基,250mL三角瓶内分装培养基25mL,考察葡萄糖浓度对原生菌株芽孢形成的影响。
以葡萄糖浓度为C源,在N源基础培养基中分别添加有机氮:蛋白胨、酵母膏、玉米浆粉、黄豆粉、黄豆饼粉、花生饼粉、鱼粉和酵母粉各10.0g/L;无机氮:尿素、NH4Cl和(NH4)2SO4各20.0g/L,250mL三角瓶内分装培养基25mL,考察不同氮源对原生菌株芽孢形成的影响。
在优化的碳源和氮源的培养基中,分别添加NaCl、CaCO3,MgSO4.7H2O,KH2PO4、K2HPO4、K2HPO4+KH2PO4,配制培养基,250mL三角瓶内分装培养基25mL,考察不同无机盐对原生菌株芽孢形成的影响。
在优化的培养基中,通过单因子试验考察接种量、通气量对发酵菌数及芽孢形成的影响。设培养基pH为7.0,250mL三角瓶内分装培养基25mL,接种前述步骤中筛选出来的芽孢率90%以上的菌株,使摇瓶中芽孢数量分别为104,105,106,107数量级,摇瓶培养24h,考察接种量对芽孢杆菌芽孢形成的影响。在250mL摇瓶中,分别分装培养基25mL和50mL,考察通气量对菌体生长和芽孢形成的影响。
经过多轮筛选,获得一株在有机物激活条件下能产生偏酸性生物酶的枯草芽孢杆菌SK01,其能够依环境营养源生成多种蛋白酶(特别是碱性蛋白酶)、糖化酶、脂肪酶、淀粉酶。
经研究发现,该枯草芽孢杆菌SK01被植入高分子塑料中,塑料受到环境中酸碱或温度变化或紫外线照射的影响,发生物性劣化,植入在塑料高分子的枯草芽孢杆菌SK01因载体劣化而释放(例如:破裂的食品包装破裂后,食品受到霉菌寄生而发霉),被释放的枯草芽孢杆菌SK01以环境中有机物为营养源迅速扩繁生长,环境中的有机物种类不一,因此被转换形成的生物酶属于多样性,原作为枯草芽孢杆菌SK01载体的塑料高分仔,其有机物同步进入营养食物链,最终被分降解为二氧化碳、无机盐、水。该枯草芽孢杆菌SK01在受到辅料保护的情况下能够取得抗高温以及促成塑料发泡的特性。
该枯草芽孢杆菌SK01的保藏分类命名为枯草芽孢杆菌(Bacillus subtilis)SK01。
实施例2生物降解塑料制备及降解检测
1、枯草芽孢杆菌微粒胶囊制备
枯草芽孢杆菌SK01微生物(固体物重量百分比49~53%)和壳聚糖(固体物重量百分比45~47%)混合后经过43~45℃低温烘干→研磨→1250~1500目过筛→精磨(达到次微米级,10-8m)→加入甲基硅油(固体物重量百分比1~4%)混炼→包覆完成→枯草芽孢杆菌微粒胶囊。
壳聚糖取自虾蟹壳经过酸溶后取得,也可以直接够买商品,具有耐高温、高温下形成聚合物的特点,与枯草芽孢杆菌SK01混合为一体后,可有效保护枯草芽孢杆菌SK01在340℃以下其活性不被灭失。
加入甲基硅油混炼的作用是防止枯草芽孢杆菌SK01和壳聚糖混合后经过低温烘干→研磨→过筛→精磨后其粉末达到次微米级(10-8)的状态不会被挥发,甲基硅油分解温度在316℃左右,随着甲基被丙基取代量增加,分解温度亦增加。丙基含量为30%时,分解温度达到400℃。
因塑料制品其制备过程中需要提供170℃以上的高温才能熔解聚合,因此为保护其活性不被灭失,利用壳聚糖高分子作为寄生的载体再通过甲基硅油的包覆达到高分散、高聚合力、抗高温的特性。
枯草芽孢杆菌微粒胶囊干热情况下能承受340℃/0.35hr高温,成分不被破坏。
2、生物降解塑料母粒制备
将枯草芽孢杆菌微粒胶囊0.3~1%、聚烯烃树脂塑料12~15%、细度达到1250网目以上的碳酸钙粉75~81%、分散剂2%和硬脂酸锌1%投入到料桶中,搅拌混合均匀。
混合物送入高温密炼机,在170~202℃温度条件下密炼熔合25~35分钟,然后再经螺杆挤出机190~202℃、2~5min条件下挤出造粒,得料粒。
料粒依次经送风冷却、过筛、集料、秤重、装袋,得生物降解塑料母粒。
如图1所示,生物降解塑料母粒的微观结构为气囊包裹形态,在制备过程中,枯草芽孢杆菌SK01自体吸收气体,气体因密炼熔融被高分子所封锁形成气囊发泡体,当温度升高气囊越发膨胀,气囊内的枯草芽孢杆菌SK01漂浮在气体中,当枯草芽孢杆菌SK01接近气囊壁的高温,本能地向温度低的空间移动。请参考图2-4,从3D穿隧式摄像图可以窥视,气囊的体积介于200~250nm,枯草芽孢杆菌SK01体积介于8~25nm,因此气囊中的气体间接或直接保护枯草芽孢杆菌SK01在高温时维持体温的恒定,并且可通过物理性调节来减少温度对枯草芽孢杆菌SK01的影响甚至灭失。
该生物降解塑料母粒能够缩小与聚烯类塑料的比重差异,由于枯草芽孢杆菌SK01与聚烯烃树脂塑料密炼熔融过程中,温度达到65℃~75℃时嗜氧扩繁生长,使聚烯类塑料产生微细气泡,此特性致使聚烯类塑料膨胀发泡,在同样的体积上,添加枯草芽孢杆菌SK01的聚烯类塑料的比重为0.919~0.926,比不添加枯草芽孢杆菌SK01的聚烯类塑料(比重为0.923~0.93)轻0.04。
经检测,由生物降解塑料母粒制成的塑料在堆肥、土壤掩埋的环境中(湿度、温度)153天有机固体物总降解量49.11%、阳光照射(光降解)或浸泡水中可缓速降解,排除以上的环境,由生物降解塑料母粒制成的塑料成品的保存期与无降分解塑料是一致的,保存期可达1年以上。
3、降解检测
生物降解塑料母粒可制造聚烯类成品,如膜、袋、吸塑品、硬质塑料,可依照物性需求,调整聚烯烃材料使用量范围介于50~95%,生物降解塑料母粒使用量范围介于5~50%。本实施例中数值及数值范围百分比均为重量百分比。
根据三次实验,每次两年的验证表明(北京国家塑料制品质量监督检验中心认证,国塑检〔2013〕C0292),聚烯烃材料添加生物降解塑料母粒,达到5%以上即具有诱蚀生物降解聚烯烃有机物的性能,添加比例与物性需求成反比。生物降解塑料母粒添加量越多,诱蚀生物降解性越强,物性的横向、纵向拉力与延伸力越差。根据实物测试,塑膜厚度0.008mm、母粒添加比例5%为基点,每增加0.001mm,添加比可增加1~1.2%,50%是最高的添加极限。
取成品聚烯烃材料含量54%,生物降解塑料母粒含量46%,制成0.035mm膜片试样,在153天对聚烯烃材料(碳酸钙为无机物)的聚烯烃降解率达到90.9%。参比材料为纤维素。
表1试样基本特性
表2二氧化碳释放量和生物分解百分率
表3二氧化碳产生量及生物分解百分率
图5为2012E0568试样和参比材料纤维素降解过程中二氧化碳释放量曲线图。
本发明中生物降解塑料母粒诱蚀生物降解聚烯烃高分子,其主要作用原理是在聚烯烃类材料中植入经过深加工的枯草芽孢杆菌SK01,此枯草芽孢杆菌SK01具有承受高温的性能,在造粒、压出、射出、成膜或制袋的高温下成分不被破坏,当袋、膜使用后被埋入土壤中或堆肥内,枯草芽孢杆菌SK01生长繁殖,产生的微生物酶诱引土著菌集结并进行分解,使袋、膜产生裂解成细片状(4~5cm),经过生态化学螯合袋、膜分解成粉粒状(0.01~0.1mm),受生态中的后生动物群吞食、消化,最终成为二氧化碳、水以及碳酸钙,还原为生态环境中的无害物。
添加生物降解塑料母粒的制品,通过FDA、RoHS、12类重金属、21类化学毒性检验、黄变4级、横纵向拉力的最高等级测试,取得了降解性能、安全与物性的优质评价。
添加生物降解塑料母粒的制品,铅、镉、汞、六价铬、多溴联苯(PBB)、多溴二苯醚(PBDE)的测试结果符合欧盟RoHS指令2002/96/EC的重订指令2011/65/EU附录Ⅱ的限值要求。
根据美国FDA法规要求,测定与食品接触而用于涂料的组分生物降解塑料母粒的聚乙烯的最大可萃取量,测试方法参考US FDA 21CFR 177.1520d(3)(ⅱ)&d(4)(ⅱ),测试结果如下表:
常规模拟液 | 时间 | 温度 | 最大允许限值 | 样本检测值 |
正己烷 | 2hr | 50℃ | 53%(w/w) | 3.7(w/w) |
二甲苯 | 2hr | 25℃ | 75%(w/w) | 5.7(w/w) |
加入生物降解塑料母粒的包装袋,长度700±5mm,宽度510±5mm,缝线处为双边,100mm内单丝数不少于47条,经机械性能检测结果,物理性能如下表所示:
牢固度:随机抽取5个包装嗲,2.5m±2mm高空自由落体后破包率≤4袋。
包装袋适用温度:≥100℃。
纸袋材料对水泥强度的影响:3天抗折强度比≥93%、3天抗压强度比≥95%.
包装袋防潮性能:3天抗压强度比≥90%。
单丝拉力检验结果:单丝拉力合格率≥80%。
生物降解塑料母粒与聚乙烯作为原材料制备的包装袋,采用GB13735-92标准进行产品检验的结果如下表所示:
生物降解塑料母粒有别于粮食性材料(以淀粉等天然物质为基础的生物降解塑料目前主要包括以下几种产品:聚乳酸(PLA)、聚羟基烷酸酯(PHA)、淀粉塑料、生物工程塑料、生物通用塑料:聚烯烃和聚氯乙烯等),以诱蚀生物降解聚烯高分子为机制,在生产工艺、设备不需要做任何变动、增加或修改即可生产生物降解塑料制品,平价的成本具有强大的市场竞争力。
本文中应用了具体个例对发明构思进行了详细阐述,以上实施例的说明只是用于帮助理解本发明的核心思想。应当指出,对于本技术领域的普通技术人员来说,在不脱离该发明构思的前提下,所做的任何显而易见的修改、等同替换或其他改进,均应包含在本发明的保护范围之内。
Claims (10)
1.枯草芽孢杆菌(Bacillus subtilis)SK01,保藏于中国典型培养物保藏中心,保藏编号CCTCC NO:M2020812。
2.权利要求1所述枯草芽孢杆菌SK01在降解塑料方面的应用,其特征在于,塑料中枯草芽孢杆菌SK01的添加量为塑料总重的0.05~0.2%。
3.一种枯草芽孢杆菌微粒胶囊,其特征在于,由权利要求1所述的枯草芽孢杆菌SK01包覆在衣膜中构成,所述衣膜为乳酸或壳聚糖,和甲基硅油。
4.根据权利要求3所述的枯草芽孢杆菌微粒胶囊,其特征在于,以干燥固体形态重量百分比计,各成分用量为:枯草芽孢杆菌SK01 49~53%,乳酸或壳聚糖45~47%,甲基硅油1~4%。
5.权利要求3或4所述枯草芽孢杆菌微粒胶囊的制备方法,其特征在于,是将枯草芽孢杆菌SK01和衣膜经混合、干燥、研磨、过筛、精磨后添加甲基硅油混炼,形成衣膜包覆枯草芽孢杆菌SK01的微粒胶囊。
6.一种生物降解塑料母粒,其特征在于,由权利要求3或4所述的枯草芽孢杆菌微粒胶囊、聚烯烃、碳酸钙、分散剂和硬脂酸锌经密炼造粒制成。
7.根据权利要求6所述的生物降解塑料母粒,其特征在于,以干燥固体形态重量百分比计,各成分用量为:枯草芽孢杆菌微粒胶囊0.3~1%,聚烯烃12~16%、碳酸钙80~87%,分散剂2%,硬脂酸锌1%。
8.权利要求6或7所述生物降解塑料母粒的制备方法,其特征在于,包括以下步骤:
(1)将枯草芽孢杆菌微粒胶囊、聚烯烃、碳酸钙、分散剂和硬脂酸锌搅拌混匀,得混合物;
(2)混合物经密炼机进行蜜炼熔合后,螺杆挤出机造粒;
(3)送风冷却,过筛,得产品。
9.根据权利要求8所述的制备方法,其特征在于,步骤(2)中熔合温度为170~202℃,时间25~35分钟;螺杆挤出机造粒的温度190~202℃,时间2~5分钟。
10.一种生物降解塑料,其特征在于,由权利要求6或7所述的生物降解塑料母粒和聚烯烃材料制成,生物降解塑料母粒的添加量为生物降解塑料总重量的5~50%。
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