CN112522143A - 一种复合酶制剂和苍白杆菌辅助的厨余垃圾高温堆肥新工艺 - Google Patents
一种复合酶制剂和苍白杆菌辅助的厨余垃圾高温堆肥新工艺 Download PDFInfo
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Abstract
本发明涉及微生物技术和环境工程领域,公开了一种复合酶制剂和苍白杆菌辅助的厨余垃圾高温堆肥新工艺。本发明的苍白杆菌保藏编号为CGMCC No.19230,微生物分类命名为Ochrobactrum sp.。本发明的复合酶制剂和菌株配合高温堆肥工艺可用于对厨余垃圾、农产品尾料、屠宰场废料等多种有机质进行高温好氧堆肥处理,使得有机质物料完全腐殖质化、脱水干化并减重70‑92%,最终形成品质优良的有机肥。本发明的高温堆肥新工艺还涉及到特殊的物理切割、酶法水解、菌株代谢快速产热等预处理手段,使肥堆能够在8小时内快速发热升温至70℃以上,从而快速进入堆肥的高温阶段,使得各类高温菌种快速成为肥堆体系内的绝对优势菌种,并以此为基础,实现可每日补料出料的半连续堆肥发酵工艺。
Description
技术领域
本发明涉及微生物技术和环境工程领域,尤其涉及一种复合酶制剂和苍白杆菌辅助的厨余垃圾高温堆肥新工艺。
背景技术
随着我国经济蓬勃发展,人民生活水平不断提高,国内厨余垃圾产量逐年上升,目前国内厨余垃圾年产量已经超过1亿吨。厨余垃圾含有大量有机质,包括油脂、纤维素、糖类、蛋白质等等。如不科学合理的处置,厨余垃圾不但会产生固体污染,腐烂后还会产生严重的废水污染和恶臭污染,严重破坏生活环境,影响居民生活。近年来利用耐高温微生物发展起来的高温堆肥技术可以有效地处置厨余垃圾,使其高度腐殖化产生可资源化利用的有机肥料。高温堆肥是一种在高温条件下利用耐热微生物新陈代谢分解有机物使其转变为腐殖质的过程。根据所使用微生物呼吸作用的类型的不同,高温堆肥可分为好氧高温堆肥和厌氧高温堆肥。目前国内外应用较为广泛的是好氧高温堆肥技术。相较于厌氧高温堆肥技术,好氧高温堆肥存在以下几个优点:一、好氧高温堆肥产生的恶臭物质少,对于空气基本没有影响。二、好氧高温堆肥对于有机物的降解速率远大于厌氧高温堆肥,即好氧高温堆肥降解速率更快。三、好氧高温堆肥适用于小型化肥堆,可应用于分散式垃圾处理,无需大规模肥堆来保证厌氧条件。
传统堆肥发酵温度一般不超过60℃,反应时间可持续15天左右,参与堆肥过程的微生物种类繁多,也包括不少会产生尸胺、腐胺等恶臭物质的菌种,因此传统堆肥发酵会产生令人不悦的异味。而高温堆肥发酵温度高于70℃,常规微生物已经无法生存,需使用Pseudomonas taiwanensis、 Chelativorans compostii等耐高温的特殊微生物。由于生长繁殖的微生物种类在高温条件下仅限于耐高温菌种,产恶臭的菌种死亡,发酵过程也就不会产生恶臭物质。且高温条件下发酵速度加快,水分蒸发更快,因此高温堆肥产生有机肥的速度和质量更高。
尽管高温堆肥有诸多优点,但是在实际工程应用过程中,由于耐高温微生物在常温下繁殖速率很慢,甚至无法繁殖,常常需要肥堆通过电辅助加热升温来确保肥堆温度处于耐高温菌种的生态优势地位,额外增加了能耗。此外,目前常用的耐高温菌种的底物谱较窄,多数仅能直接利用简单的有机酸和氨基酸等小分子物质,同时高温条件又杀灭了其他大部分微生物,使得复杂大分子底物的水解过程减缓。因此垃圾中复杂有机质尤其是脂类和蛋白质的水解成为了影响堆肥效率的限速过程。
由上可知,目前高温堆肥技术的效率主要限制于物料的水解环节,导致发酵效率不能大幅提高,堆肥温度难以达到更高和稳定。因此有必要优化发酵工艺,对物料进行预处理,使其更适合嗜热功能微生物利用。
发明内容
为了解决上述技术问题,本发明提供了一种复合酶制剂和苍白杆菌辅助的厨余垃圾高温堆肥新工艺。
本发明的复合酶制剂包括:漆酶,即多酚氧化酶(ρ-二元酚氧化酶, EC1.10.3.2),属于铜蓝氧化酶;碱性酯酶混合酶,包括脂酶(EC 3.1.1.3)、磷脂酶A(EC 3.1.1.4)、磷脂酶B(EC 3.1.1.5);蛋白酶K,是一种切割活性较广的丝氨酸蛋白酶,切割脂族氨基酸和芳香族氨基酸的羧基端肽键。本发明所述的苍白杆菌为一类新的苍白杆菌株系,保藏于中国微生物菌种保藏管理委员会普通微生物中心,其保藏编号为CGMCC No.19230,微生物分类命名为Ochrobactrumpseudintermedium,该菌株于2019年12月23 日保藏于北京市朝阳区北辰西路1号院3号的中国普通微生物菌种保藏管理中心,该CGMCC No.19230及其培养物(包括传代培养物)、突变体、子代在下文中均称为本发明的假中间苍白杆菌株。
本发明所述的高温堆肥工艺为一种复合酶和特殊菌株共同作用对厨余垃圾物料进行水解预处理的新型工艺,其使用漆酶、碱性酯酶、蛋白酶以及苍白杆菌对粉碎物料进行发酵前的预先氧化和水解处理,并快速产热,使得高温堆肥过程中可以实现迅速升温,在8小时内达到70℃以上的高温,帮助高温菌种跳过水解过程直接利用预处理产生的小分子底物从而实现肥堆的高效发酵。
本发明的具体技术方案为:本发明提供了一种复合酶制剂和本发明苍白杆菌辅助的厨余垃圾高温堆肥新工艺,其工艺流程及特征如下:
(1)厨余垃圾物料的含水率要求宽泛,60%-95%均可,有别于一般其他高温堆肥工艺,无需对物料进行除油和压榨脱水处理。
(2)物料通过分拣去除金属、塑料等不可降解物后,经过粉碎装置对大块物料进行粉碎,粉碎后的物料颗粒直径在10cm以下。
(3)粉碎后的物料加入物料总质量20–40%的稻壳、粉碎秸秆、粉碎玉米芯或堆肥熟料,以及占物料总质量1-2%的CGMCC No.19230,投入带搅拌功能的发酵设备中混合均匀。
(4)混合物料中加入占物料总质量1-2‰的漆酶粉剂,在室温条件下搅拌30–90分钟。
(5)漆酶处理后的物料中加入占物料总质量1-2%的生石灰搅拌充分,调节pH在9.0–11.0。
(6)调节pH后的物料加入物料总质量1-2‰的脂酶(EC 3.1.1.3)粉剂、0.2-0.8‰的磷脂酶A(EC 3.1.1.4)粉剂、0.2-0.8‰的磷脂酶B(EC 3.1.1.5) 粉剂,保持4–8小时。该过程中,每隔30分钟搅拌一次肥堆进行通气。肥堆会在CGMCC No.19230作用下快速升温至70℃。
(7)升温后的物料中投入市场上常用的高温堆肥菌种,包括但不限于芽孢杆菌属、栖热菌属、假单胞菌属等菌种,自然产热升温至80–105℃进行高温堆肥。
(8)堆肥2-3天后,肥堆完成腐殖化和减量化,排出20–40%熟料,留80-60%熟料在发酵装备内。
(9)重复步骤(1)到步骤(6),补充肥堆至初始体积,实现半连续发酵。
传统高温堆肥的发酵周期一般在7天以上,否则不能完全熟化。而本发明中所述复合酶制剂和苍白杆菌对物料进行预处理的工艺可以缩短2-5 天高温堆肥周期,提高处理效率,减小所需发酵装备的规模,有利于厨余垃圾处置装备的小型化和推广。
作为优选,(2)中所述切割的原因是物料粒径越小越容易分解,但是,考虑到在增加物料表面积的同时,还必须保持一定程度的孔隙率,以便于通风而使物料能够获得充足的氧供应,因此此处将有机物料切割粉碎成3-5cm的碎块为最优大小。
作为优选,(3)中所述添加稻壳、粉碎秸秆、粉碎玉米芯或堆肥熟料的目的是调节固液比例,避免肥堆中出现液体积聚,形成完全厌氧区域,可能导致硫化物等恶臭物质生成。因此稻壳、粉碎秸秆、粉碎玉米芯或堆肥熟料的添加量以混合后物料没有明显滴水现象为准,不宜添加过多,影响肥堆热值。通常的最佳添加量为25%左右。
作为优选,(4)中所述的漆酶添加量根据肥堆中纤维素含量多少适当增减。即当植物性废料占多数(即,半数以上)时,漆酶添加量增加至2‰;当动物性废料占多数时,漆酶添加量可减少至1‰。
作为优选,(5)中所述的pH值最佳为9.5,兼顾了酯酶活性和苍白杆菌的代谢。
作为优选,(6)中所述的酯酶添加量根据肥堆中油脂含量多少适当增减。物料在收集时若未进行隔油处理,则(6)中各类酯酶添加量可以取范围最大值;若物料已进行隔油处理,则(6)中各类酯酶添加量可以取范围最小值。
作为优选,(6)所述苍白杆菌还可以为前述CGMCC No.19230的培养物或者传代后的培养物。
本发明还提供了一种苍白杆菌的突变体,所述突变体为对前述 CGMCC No.19230进行诱变、驯化、基因重组或者经自然突变而获得的突变体。
本发明还提供了一种含有上述CGMCC No.19230或含有上述突变体的细菌培养物。
本发明还提供了一种细菌培养物,所述细菌培养物为菌液、菌剂或活性污泥等。
本发明还提供了一种含有上述CGMCC No.19230或上述突变体的酶制剂或蛋白质制剂。
本发明还公开了可将上述CGMCC No.19230或上述突变体应用于污水处理、垃圾处理、堆肥、酶制剂生产、蛋白质制剂生产中。
与现有技术对比,本发明的有益效果是:
1)本发明的苍白杆菌株能够在10-80℃、pH 3-11、盐度0-5%(w/v) 范围内正常代谢生长,可利用多种单糖、寡糖、多糖、有机酸、醇作为碳源,以及可利用胰蛋白胨、鱼蛋白胨、牛肉膏等多种复合氮源进行生长,底物谱十分宽广。
2)本发明的复合酶制剂和菌株配合高温堆肥工艺可用于对厨余垃圾、农产品尾料、屠宰场废料等多种有机质进行高温好氧堆肥处理,使得有机质物料完全腐殖质化、脱水干化并减重70-92%,最终形成品质优良的有机肥。
3)本发明的高温堆肥新工艺还涉及到特殊的物理切割、酶法水解、菌株代谢快速产热等预处理手段,使肥堆能够在8小时内快速发热升温至 70℃以上,从而快速进入堆肥的高温阶段,使得各类高温菌种快速成为肥堆体系内的绝对优势菌种,并以此为基础,实现可每日补料出料的连续堆肥发酵工艺。
在下面的附图和具体实施方案中进一步说明本发明。然而,这些附图和具体实施方案不应被认为限制本发明的范围,并且本领域技术人员容易想到的改变将包括在本发明的精神和所附权利要求的保护范围内。
附图说明
图1为本发明的苍白杆菌株的菌落形态图。
图2为复合酶剂对本发明的苍白杆菌的快速升温的影响曲线图。
具体实施方式
以下结合实施例对本发明进行详细描述。所用的所有实验试剂和仪器设备,如无特别说明均为普通市售试剂和设备。
除非另外限定,本文中所用的全部术语具有本发明所属技术领域的普通技术人员通常理解的含义,在使用部分以下术语的定义时,以单数形式使用的术语也可以包括复数,并且反之亦然。本文给出的部分术语的定义仅是为了描述具体的实施方案,并非旨在限制。
术语“约/大约”是指在给定值或范围的50%内,优选在25%内,更优选在10%-1%内;或指在平均数的可接受标准差内。
下面结合实施例对本发明作进一步的描述。
实施例1苍白杆菌CGMCC No.19230的筛选及鉴定
(1)样品采集
微生物样品采集自浙江省余杭区余杭高级中学食堂餐厨垃圾高温堆肥试验样品。
(2)菌株CGMCC No.19230的分离、纯化及保种
自主设计、配制的“酵母蔗糖YZ培养基”:酵母提取物5g/L、蔗糖10g/L、蛋白胨10g/L、氯化钠5g/L,pH=7.0。作固体培养基时加入琼脂20g/L。
将1g样品投加入装有100mL无菌蒸馏水及些许无菌玻璃珠的锥形瓶中,在120rpm(转/分钟)条件下震荡10分钟,得悬浊液。取悬浊液1mL 稀释涂布于无菌YZ固体培养基上,置于60℃恒温培养箱内培养2-3天,待平板上呈现出明显菌落后挑取单菌落,反复划线纯化后将单菌落接种于无菌YZ液体培养基中,并置于120rpm、60℃条件下培养至对数期。将处于对数期的菌液进行30%甘油保藏及冷冻干燥保藏。
(3)菌株CGMCC No.19230的多相分类学鉴定
1)表型特征
将菌株划线于无菌YZ固体培养基上,置于60℃条件下,培养3天后,菌落呈现豆浆色、直径约1-2mm、表面凸起且湿润、边缘光滑呈圆形、不透明(图1)。经革兰氏染色,菌株为革兰氏阴性菌。
2)生理生化特征
将对数期的菌液按照1%的接种量接种于无菌的YZ液体培养基中,将其置于不同温度(4℃、10℃、20℃、30℃、40℃、50℃、60℃、70℃、80℃)、不同pH(2.0、3)0、4.0、5.0、6.0、7.0、8.0、9.0、10.0、11.0、12.0)及不同NaCl盐度(0%、1%、2%、3%、4%、5%、6%、7%、8%)条件下,查看菌株生长范围。结果显示,菌株可在10-80℃、pH 3-11、NaCl盐度0-5%范围内生长。
将对数期的菌液按照1%的接种量接种于具有唯一碳源(葡萄糖、半乳糖、核糖、海藻糖、木糖、果糖、麦芽糖、纤维二糖、淀粉、甲醇、乙醇、乙酸钠、柠檬酸钠、草酸钠)培养基中,置于最佳培养条件中,查看菌株对碳源的利用情况。将对数期的菌液按照1%的接种量接种于具有唯一氮源 (胰蛋白胨、鱼蛋白胨、牛肉膏、硫酸铵、硝酸钠)培养基中,置于最佳培养条件中,查看菌株对氮源的利用情况。结果显示,菌株可利用多种单糖类、寡糖类、多糖类、有机酸、醇类作为碳源,可利用胰蛋白胨、鱼蛋白胨、牛肉膏等多种复合氮源进行生长。
将对数期的菌液按照1%的接种量接种于含有一定规格的滤纸片,置于最佳培养条件中,查看菌株对滤纸的降解情况。将对数期的菌液按照1%的接种量接种于含有1g/L的羧甲基纤维素的YZ固体培养基上,待出现明显菌落后,倾倒0.2%的刚果红,静置12小时,查看平板上是否有透明圈形成。结果显示,菌株能够降解滤纸及羧甲基纤维素。
其他生化实验结果显示,菌株具有过氧化氢酶活性、氧化酶活性、淀粉水解酶活性、酪蛋白水解酶活性及酯酶活性。
经以上鉴定,菌株为Ochrobactrumpseudintermedium。
实施例2复合酶对苍白杆菌的快速升温影响
漆酶(EC 1.10.3.2)粉剂:市售济南东轩生物工程有限公司食品级漆酶。
脂酶(EC 3.1.1.3)粉剂:市售隆大牌食品级脂肪酶(米曲霉,10万单位酶活)。
磷脂酶A(EC 3.1.1.4)粉剂:市售Sigma-Aldrich磷脂酶A2 P6534酶 9001-84-7。
磷脂酶B(EC 3.1.1.5)粉剂:SUNSON/夏盛牌溶血磷脂酶(食品级)。
取1吨餐厨垃圾,经粉碎至约5cm的碎块后,加入20%的干燥粉碎秸秆,充分搅拌后,加入2%的CGMCC No.19230菌液,加入2‰的漆酶粉剂充分搅拌后调节pH=9.0,再加入1‰的脂酶(EC 3.1.1.3)粉剂、0.5‰磷脂酶A(EC 3.1.1.4)粉剂、0.5‰磷脂酶B(EC3.1.1.5)粉剂,保持5小时后,每隔30分钟搅拌通气一次。设置不加漆酶及碱性酯酶粉剂的一组为对照组,每隔1小时检测温度,以查看复合酶剂对菌种升温的影响。结果显示(图2),复合酶组可促进苍白杆菌快速升温,堆肥8小时即可达 70℃,并能维持在70-80℃。
实施例3:复合酶制剂和苍白杆菌在高温堆肥中的应用
1:厨余垃圾堆肥处理
取2吨厨余垃圾,固液混合物经过切割搅拌使之固形物变成大约直径 5cm的碎块。将固液垃圾投入带有搅拌功能的发酵装置中,并加入600Kg 的干燥稻壳,充分混合搅拌均匀。投入20Kg CGMCC No.19230,加入3Kg 漆酶粉剂,以每分钟一次的频率搅拌60分钟。将40Kg生石灰分多次加入肥堆中,搅拌均匀使得pH达到9.5,并加入5Kg酯酶混合物,搅拌均匀。然后肥堆每隔30分钟搅拌一次通气,维持6小时后,温度上升至70℃。投入耐热芽孢杆菌进行高温堆肥,维持温度在70-100℃之间,2天后完成堆肥发酵,物料减重70%。排出一半熟料后,以剩下的熟料代替一半干燥稻壳重新补充1.8吨厨余垃圾,重复上述步骤,实现半连续发酵。
2:屠宰废料堆肥
取1吨屠宰场屠宰肥料,经过切割搅拌使之固形物变成大约直径3cm 的碎块。将固液垃圾投入带有搅拌功能的发酵装置中,并加入30%的干燥秸秆粉碎料,充分混合搅拌均匀。投入10Kg CGMCC No.19230,加入1Kg 漆酶粉剂,以每分钟一次的频率搅拌60分钟。将5Kg生石灰分多次加入肥堆中,搅拌均匀使得pH达到9.5,并加入2Kg酯酶混合物,搅拌均匀。然后肥堆每隔30分钟搅拌一次通气,维持8小时后,温度上升至70℃。投入市售EM菌剂进行高温堆肥,维持温度在70-80℃之间,3天后完成堆肥发酵。
3:农副产品肥料堆肥
取10吨秸秆,加入带切割搅拌的发酵装置进行连续切割搅拌为6cm 左右的碎片,投入100Kg CGMCC No.19230,加入20Kg漆酶粉剂,以每分钟一次的频率搅拌60分钟。将80Kg生石灰分多次加入肥堆中,搅拌均匀使得pH达到9.5,并加入1Kg酯酶混合物,搅拌均匀。然后肥堆每隔30分钟搅拌一次通气,维持4小时后,温度上升至70℃。投入市售秸秆堆肥菌剂进行高温堆肥,维持温度在80-95℃之间,4天后完成堆肥发酵。
本发明中所用原料、设备,若无特别说明,均为本领域的常用原料、设备;本发明中所用方法,若无特别说明,均为本领域的常规方法。
以上对本发明的一个实施例进行了详细说明,但所述内容仅为本发明的较佳实施例,不能被认为用于限定本发明的实施范围,本领域的专业技术人员在不脱离本发明原理的前提下对还可以对本发明进行修饰,这些修饰同样属于本发明的保护范围。
Claims (8)
1.一种苍白杆菌,其特征在于所述苍白杆菌已在2019年12月23日保藏于中国微生物菌种保藏管理委员会普通微生物中心,其保藏编号为CGMCC No.19230,微生物分类命名为Ochrobactrum sp.。
2.权利要求1所述苍白杆菌的培养物或者传代后的培养物。
3.权利要求1所述苍白杆菌的突变体,其特征在于所述突变体为对权利要求1所述苍白杆菌进行诱变、驯化、基因重组或者经自然突变而获得的突变体。
4.含有权利要求1所述苍白杆菌或含有如权利要求3所述突变体的细菌培养物。
5.一种包含权利要求4所述的细菌培养物的混合物,其特征在于所述混合物为菌液、菌剂或活性污泥。
6.一种含有如权利要求1所述苍白杆菌或含有如权利要求3所述突变体的酶制剂或蛋白质制剂。
7.权利要求1所述苍白杆菌或权利要求3所述突变体在污水处理、垃圾处理、堆肥、酶制剂生产、蛋白质制剂生产中的应用,其中所述堆肥优选为高温堆肥发酵。
8.一种使用权利要求1所述苍白杆菌或权利要求3所述突变体的高温堆肥发酵方法,其特征在于包括以下步骤:
(1)收集厨余垃圾物料,其中所述厨余垃圾物料的含水率为总物料的约60%-95%;
(2)对步骤(1)中的厨余垃圾物料进行分拣,去除不可降解物后,进行粉碎,至物料颗粒直径为10cm以下,优选为3-5cm,其中所述不可降解物优选为金属、塑料等;
(3)步骤(2)得到的粉碎后的物料中加入占所述粉碎后的物料总质量约20–40%,优选25%的稻壳、粉碎秸秆、粉碎玉米芯或堆肥熟料,以及占所述粉碎后的物料总质量约1-2%的权利要求1所述苍白杆菌或权利要求3所述突变体,然后混合均匀,其中所述权利要求1所述苍白杆菌或权利要求3所述突变体优选为液体状态,所述混合均匀步骤优选在带搅拌功能的发酵设备中进行;
(4)步骤(3)得到的混合物料中加入占所述混合物料总质量约1-2‰的漆酶粉剂,在室温条件下搅拌约为30–90分钟,优选当植物性废料占半数以上时,漆酶添加量为所述混合物料总质量的2‰;当动物性废料占半数以上时,漆酶添加量为所述混合物料总质量的1‰;
(5)步骤(4)得到的漆酶处理后的物料中加入占所述处理后的物料总质量约1-2%的生石灰,充分搅拌,调节pH至约9.0–11.0,优选为9.5;
(6)步骤(5)得到的调节pH后的物料中加入占所述该调节pH后的物料总质量约1-2‰的脂酶(EC 3.1.1.3)粉剂、约0.2-0.8‰的磷脂酶A(EC 3.1.1.4)粉剂、约0.2-0.8‰的磷脂酶B(EC 3.1.1.5)粉剂,保持4-8小时,其中每隔30分钟搅拌一次物料进行通气,至所述物料升温至约70℃,优选地,物料在收集时若未进行隔油处理,则上述酶添加量取范围最大值;若物料已进行隔油处理,则上述酶添加量可以取范围最小值;
(7)步骤(6)得到的升温后的物料中加入市场上常用的高温堆肥菌种,所述高温堆肥菌种优选包括但不限于芽孢杆菌属、栖热菌属、假单胞菌属等菌种,至所述物料升温至80–105℃;
(8)步骤(6)得到的物料堆肥2–3天后,完成腐殖化和减量化,排出20–40%熟料,留80-60%熟料在发酵装备内;
(9)重复步骤(1)到步骤(6),补充肥堆至初始体积,实现半连续发酵。
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