CN104630098B - 一种蒙氏假单胞菌ky‑05与应用 - Google Patents
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Abstract
本发明提供了一种蒙氏假单胞菌KY‑05，保藏号为CGMCC No.10172，具有纤维素降解能力；所述蒙氏假单胞菌KY‑05对于现有的污泥厌氧消化工艺的沼气产气量和厌氧反应启动速度具有提升作用，可用于开发增强厌氧消化产气效能的微生物制剂，并逐步在污泥厂进行实际应用和推广，具有广泛的应用前景。
Description
技术领域
本发明涉及生物技术领域，尤其是一种蒙氏假单胞菌KY-05与应用。
背景技术
随着城市生活污水排放量的逐年增长，我国城市污水厂污泥急剧增加，到2010年底，我国年污泥总产生量(以含水率80％计)已达到3000万吨左右，并以每年约10％的速度增长。然而大约80％的脱水污泥都是直接填埋或焚烧，造成环境的二次污染，大打折扣了污水处理厂的环境效益。因此，实现污泥的稳定化、资源化、无害化的处理与处置问题日益受到关注和重视。污泥厌氧消化处理因其处理费用较低、致病菌破坏率高、可产甲烷、减少污泥产量和改善污泥脱水性能等优点得到较广泛地应用，是污水厂污泥快速稳定化和资源化的一个重要方法。
污泥中的有机物质主要可溶性糖类、纤维素、木质素、脂肪、蛋白质等这些有机物是由长链分子构成，有机物的降解是污泥厌氧消化的限速步骤，其中可溶性糖类、蛋白质和脂肪可以直接被微生物水解利用，但是占干物质量10％～30％的纤维素很难被降解利用，而在厌氧消化过程中存在碳源不足的现象，因此将纤维素水解为可利用的可溶性糖类可以弥补污泥厌氧消化碳源不足的现象，对厌氧消化起到增效的作用。目前污泥中纤维素物质的水解主要是靠化学和物理的方法进行污泥预处理，但是该方法运行费用高，还存在二次污染，而利用纤维素分解菌对纤维素进行水解可以大大降到运行成本，避免了二次污染，同时不需要特定的设备。
蒙氏假单胞菌已报道在烟草普通花叶病毒防治方面具有很高的应用价值，可以抑制烟草普通花叶病毒(Tobacco mosaic virus，TMV)的侵染，但是该菌在纤维素降解方面的降解功能还未被报道。
发明内容
本发明所要解决的技术问题在于提供一种蒙氏假单胞菌KY-05，具有纤维素降解能力。
本发明所要解决的另一技术问题在于提供上述蒙氏假单胞菌KY-05的应用。
为解决上述技术问题，本发明的技术方案是：
一种蒙氏假单胞菌(Pseudomonas monteilii)KY-05，其保藏号为CGMCC No.10172(保藏单位:中国微生物菌种保藏管理委员会普通微生物中心，地址：北京市朝阳区北辰西路1号院3号、保藏日期：2014年12月15日、保藏编号：CGMCC No.10172)。
优选的，上述蒙氏假单胞菌KY-05，菌株呈革兰氏阴性，电子显微镜观察呈杆状，无芽胞；通过16SrDNA测序鉴定为蒙氏假单胞菌(Pseudomonas monteili)，其系统进化树如图1所示，16SrDNA序列分析结果为序列表中<400>1所示序列，即：
5’-TCTTCGGATTGTAAAGCACTTTAAGTTGGGAGGAAGGGCAGTAAGTTAATACCTTGCTGTTTTGACGTTACCGACAGAATAAGCACCGGCTAACTCTGTGCCAGCAGCCGCGGTAATACAGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCGCGTAGGTGGTTTGTTAAGTTGGATGTGAAAGCCCCGGGCTCAACCTGGGAACTGCATCCAAAACTGGCAAGCTAGAGTACGG-3’。
优选的，上述蒙氏假单胞菌KY-05，所述核苷酸序列为细菌16S rDNA序列的V3可变区。
优选的，上述蒙氏假单胞菌KY-05，分离自市政污泥，在分离平板上培养2d后，菌落直径在2～5mm，菌落表面和边缘光滑，隆起，呈浅黄色。
优选的，上述蒙氏假单胞菌KY-05，具有较强的纤维素降解能力。
上述蒙氏假单胞菌KY-05对厌氧消化产沼反应的增效用途。
优选的，上述蒙氏假单胞菌KY-05的应用，将所述蒙氏假单胞菌KY-05接种至增菌培养基中，30℃振荡培养至对数增长期，菌浓为106～1010个/ml，离心取菌液上清，按照DNS还原糖比色国标法GB/T 23881-2009测定上清液对纤维素等有机质的分解代谢能力，并以0.1～0.5％的接种量接种于厌氧消化反应体系中，实现该菌株对厌氧消化反应的增效作用。
本发明的有益效果是：
本发明提供了蒙氏假单胞菌KY-05(保藏号为CGMCC No.10172)，可产生降解纤维素类的物质，对于现有的污泥厌氧消化工艺的沼气产气量和厌氧反应启动速度具有提升作用，可用于开发增强厌氧消化产气效能的微生物制剂，并逐步在污泥厂进行实际应用和推广，具有广泛的应用前景。
附图说明
图1为本发明所述蒙氏假单胞菌KY-05的系统进化树，图中，Pseudomonasmonteilii为蒙氏假单胞菌。
图2为本发明所述蒙氏假单胞菌KY-05对市政污泥产气增效结果图，其中，上面曲线为市政污泥+菌，下面曲线为市政污泥。
保藏信息
分类名词：蒙氏假单胞菌(Pseudomonas monteili)
保藏单位名称：中国微生物菌种保藏管理委员会普通微生物中心
保藏单位地址：北京市朝阳区北辰西路1号院3号
保藏日期：2014年12月15日
保藏号：CGMCC No.10172
具体实施方式
下面结合具体实施例对本发明所述技术方案作进一步的说明。
下述实施例中不同的培养基及溶液配方如下：
1.筛选培养基：纤维素15～30g，磷酸二氢钾2g，硫酸铵1～2g，硫酸镁0.2～1g，氯化钙0.2～1g，硫酸亚铁3～6mg，硫酸锰1～2mg，氯化锌1.0～1.7mg，氯化钴1～2mg，蒸馏水1000mL，pH 7.0。
2.增菌培养基：蒸馏水1000mL，蛋白胨5.0～10g，牛肉膏2～5g，NaCl 5～15g，pH7.2。
实施例1
蒙氏假单胞菌(Pseudomonas monteilii)KY-05的筛选和鉴定。
实验材料来自天津纪庄子污水处理厂的厌氧活性污泥样品。
取样品稀释液(稀释倍数10-6)0.1ml，涂布于筛选培养基平板上，在30℃恒温箱中培养，挑取单菌落接种至筛选培养基，强化培养48h以上，重复划线纯化3次，得到纯化菌株；经过16S rDNA和Biolog微生物分类鉴定系统鉴定，确定为蒙氏假单胞菌KY-05，系统进化树如图1所示，通过PCR扩增其16SrDNA基因，测序结果如<400>1所示序列，即：
5’-TCTTCGGATTGTAAAGCACTTTAAGTTGGGAGGAAGGGCAGTAAGTTAATACCTTGCTGTTTTGACGTTACCGACAGAATAAGCACCGGCTAACTCTGTGCCAGCAGCCGCGGTAATACAGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCGCGTAGGTGGTTTGTTAAGTTGGATGTGAAAGCCCCGGGCTCAACCTGGGAACTGCATCCAAAACTGGCAAGCTAGAGTACGG-3’。
实施例2
蒙氏假单胞菌(Pseudomonas monteilii)KY-05对纤维素等大分子有机质的降解试验。
处理对象为筛选培养基，运行方式为摇瓶振荡培养。具体实施步骤如下：实施例1所述蒙氏假单胞菌KY-05接种至增菌培养基，30℃振荡培养至对数增长期(菌浓约为108个/ml)，以3％接种量接种于液体筛选培养基中，30℃振荡培养1天，维持溶氧为1.0～3.0ppm；离心取上清作为粗酶液，按照DNS比色法(GB/T 23881-2009)测定该菌株降解纤维素等大分子复杂有机质产生还原糖的量。结果显示，每mL该菌株粗酶液平均可产生还原糖557.92μg/min。
实施例3
蒙氏假单胞菌(Pseudomonas monteilii)KY-05对市政污泥厌氧消化的增效实验。
处理对象为厌氧市政污泥，采用400mL模拟厌氧反应体系运行。具体实施步骤如下：实施例1所述蒙氏假单胞菌KY-05接种至增菌培养基，30℃振荡培养至对数增长期(菌浓约为108个/ml)以0.2％接种量接种于模拟厌氧反应系统中，中温(35±2℃)厌氧条件下运行。如图2所示，对反应体系的产气效率进行监测，并与未接种组进行对比分析，结果显示，在300小时的反应时间内，巴氏葡萄球菌KY-06可使混合污泥产气量较对照组平均提升20.99％，生物产沼势能MP增加27.81％，证实该菌剂对厌氧消化均具有显著增效作用，并能显著提高污泥中可直接参与厌氧消化的有效底物转化率，使反应系统的整体产沼势能得到提升，从而达到产气量和污泥减量的双重强化效果。
上述参照实施例对该一种蒙氏假单胞菌KY-05与应用进行的详细描述，是说明性的而不是限定性的，可按照所限定范围列举出若干个实施例，因此在不脱离本发明总体构思下的变化和修改，应属本发明的保护范围之内。
Claims (5)
1.一种蒙氏假单胞菌(Pseudomonas monteilii)KY-05，其特征在于：其保藏号为CGMCCNo.10172。
2.根据权利要求1所述的蒙氏假单胞菌(Pseudomonas monteilii)KY-05，其特征在于：分离自市政污泥，在分离平板上培养2d后，菌落直径在2～5mm，菌落表面和边缘光滑，隆起，呈浅黄色。
3.权利要求1-2任一所述的蒙氏假单胞菌(Pseudomonas monteilii)KY-05在降解纤维素中的用途。
4.权利要求1-2任一所述的蒙氏假单胞菌(Pseudomonas monteilii)KY-05对厌氧消化产沼反应的增效用途。
5.根据权利要求3或4所述的用途，其特征在于：将所述蒙氏假单胞菌KY-05接种至增菌培养基中，30℃振荡培养至对数增长期，菌浓度为106～1010个/mL ，离心取菌液上清，按照DNS还原糖比色国标法GB/T 23881-2009测定上清液对纤维素的分解代谢能力，并以0.1～0.5％的接种量接种于厌氧消化反应体系中，实现该菌株对厌氧消化反应的增效作用；所述增菌培养基配方为：蒸馏水1000mL，蛋白胨5.0～10g，牛肉膏2～5g，NaCl 5～15g，pH 7.2。
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