CN103361281B - 一种高温降解菌及其应用 - Google Patents
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Abstract
本发明提供了一种高温降解菌，其为地芽孢杆菌属(Geobacillus)菌株HN-5，保藏号为CGMCC NO.5852。该菌可在约70℃好氧条件下同时降解纤维素、蛋白质和淀粉三种大分子有机物，为高温好氧堆肥快速腐熟提供微生物资源和技术支撑。
Description
技术领域
本发明涉及微生物领域及生物降解领域，具体地说，涉及一种高温降解菌及其应用。
背景技术
高温菌是一类生活在高温环境中的微生物。在自然界中，高温菌通常分布在地热区和人工高温环境中。高温菌栖息的地热环境分三种类型：(1)酸性硫磺区。具有丰富的硫、酸性土壤、酸性温泉和沸泥塘等。(2)淡水温泉和中性、碱性的间歇温泉。(3)深海火山口。pH中性，温度在20℃～350℃。除此之外，被太阳能加热的各种池塘、土壤、干草堆、厩肥中生物本身也可达到相当高的温度，但维系时间较短，只适合生长较快的芽孢菌。人工的高温环境主要包括热水管道、燃烧的废煤堆、污水处理厂等。
高温菌具有代谢快、活性高、代时短、酶的热稳定性高、营造高温条件杀死病原菌等特点，对于堆肥高温期有机质的降解、病原微生物的杀灭具有不可估量的作用。研究表明，在堆肥中接种高温降解菌，可以有效地缩短堆肥周期、提高堆肥质量，为有机废弃物堆肥更好的实现减量化、无害化、稳定化，及资源化利用的工业化生产创造了有利条件，具有很高的应用前景。因此，高温菌的筛选及其酶学性质的研究具有很高的研究价值，意义重大。
目前已有研究者从堆肥中分离出高温降解菌。但能同时降解多种有机物的高温菌的相关报道较少。
发明内容
本发明的目的是针对堆肥高温期微生物种类及数量少从而导致有机大分子降解困难的问题，提供一种能在高温下生长并有效降解有机大分子的菌种。
为了实现本发明目的，本发明的一种高温降解菌，其为地芽孢杆菌属(Geobacillus)菌株HN-5，该菌分离自海南省澄迈县海南裕沃生物科技公司出垛堆置10d的滤泥堆肥中。现已保藏于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号中国科学院微生物研究所，保藏日期2012年3月7日，保藏号CGMCCNO.5852。
经人工富集培养、分离纯化得到的菌株HN-5在琼脂平板上的菌落呈白色、圆形，菌落表面干燥、较厚、不透明、边缘圆整；培养24小时后显微镜下菌体形态为长杆状，有芽孢产生，孢子内生，椭圆，位于菌体一端；革兰氏染色呈阳性。
本发明还提供由上述高温降解菌产生的纤维素酶、淀粉酶和蛋白酶。
本发明进一步提供上述高温降解菌在促进堆肥腐熟、提高堆肥品质中的应用，其是将地芽孢杆菌属HN-5接种到堆肥中，18～63.5℃下进行腐熟。
本发明的地芽孢杆菌属HN-5可在约70℃好氧条件下同时降解纤维素、蛋白质和淀粉三种大分子有机物，为高温好氧堆肥快速腐熟提供微生物资源和技术支撑。
附图说明
图1为本发明地芽孢杆菌属HN-5降解大分子有机物效果图及菌落形态；其中，a为纤维素刚果红培养基，b为蛋白水解培养基，c为淀粉水解培养基；d为牛肉膏蛋白胨培养基。
图2为本发明地芽孢杆菌属HN-5在不同时间的产酶能力。
图3本发明地芽孢杆菌属HN-5的生长曲线。
图4为接种本发明地芽孢杆菌属HN-5对堆肥过程温度的影响。
具体实施方式
以下实施例用于说明本发明，但不用来限制本发明的范围。若未特别指明，实施例中所用的技术手段为本领域技术人员所熟知的常规手段，所用原料均为市售商品。
实施例1 地芽孢杆菌属HN-5的分离
采集海南省澄迈县海南裕沃生物科技公司出垛堆置10d的滤泥堆肥样品，经过若干代高温驯化，在牛肉膏蛋白胨(NR)平板上用梯度稀释法涂布菌液后70℃培养，分离得到耐高温菌株，经过若干代的划线纯化，得到纯菌。NR配方：牛肉膏5g，蛋白胨10g，NaCl5g，琼脂15g～20g，水1000mL，pH 7.4～7.6。
将分离得到的多株纯菌接种至固体选择培养基中鉴定其降解能力，观察菌落周围是否有水解圈出现，如有水解圈则说明菌株对该种底物具有降解能力，反之则不具有降解能力，最后得到一株能够降解纤维素、蛋白质、淀粉的地芽孢杆菌属(Geobacillus)HN-5，保藏号为CGMCC NO.5852。
所述固体选择培养基包括：
(1)纤维素刚果红培养基：CMC-Na 1.88g，KH2PO4 0.5g，MgSO4·7H2O 0.25g，明胶2.0g，刚果红0.2g，植物凝胶10g，琼脂15g，H2O 1000mL，pH 7.0～7.2，121℃灭菌20min。
(2)蛋白水解培养基：蛋白胨10g，牛肉膏3g，NaCl 5g，酪蛋白5g，CaCl2 0.1g，L-酪氨酸0.1g，植物凝胶15g，H2O 1000mL，pH7.0-7.2，121℃灭菌冷却至60℃，加入1％115℃灭菌的吐温80。
(3)淀粉水解培养基：可溶性淀粉10g，蛋白胨10g，酵母粉5g，NaCl10g，pH7.0，植物凝胶15g-20g，曲利苯蓝0.05-0.1g，H2O 1000mL，121℃灭菌30min。
图1a为高效降解菌HN-5的纤维素降解效果图；图1b为高效降解菌HN-5的蛋白质降解效果示意图；图1c为高效降解菌HN-5的淀粉降解效果示意图；图1d为高效降解菌HN-5的菌落形态。从图1中可以看出，分离得到的地芽孢杆菌属HN-5可同时降解纤维素、淀粉、蛋白质三种有机大分子。
实施例2 地芽孢杆菌属HN-5产酶能力的研究
制备HN-5种子液(70℃，NR液体培养基中培养至OD值0.4左右)，取1ml种子液接种至纤维素、蛋白质、淀粉发酵培养基(配方见下)中，在70℃培养，24小时以后开始测定菌株的纤维素酶活力、淀粉酶活力和蛋白酶活力。
纤维素发酵培养基：CMC-Na 5g，NaNO3 2.5g，KH2PO4 1g，MgSO40.6g，NaCl 0.1g，CaCl2 0.1g，FeCl3 0.01g，明胶2g，酵母粉0.1g，H2O1000mL，pH 6.8-7.2。
淀粉发酵培养基：可溶性淀粉10g，蛋白胨10g，酵母粉5g，NaCl10g，pH 7.0，植物凝胶15g-20g，曲利苯蓝0.05-0.1g，H2O 1000mL，121℃灭菌30min。
蛋白发酵培养基：干酪素5％，Mandel A盐10％，Mandel B盐1％，pH6.0；Mandel A盐：KH2PO4 2g/L，(NH4)2SO4 1.4g/L，MgSO4 0.3g/L，CaCl2 0.3g/L；MandelB盐：FeSO4 5mg/L，MnSO4 1.6mg/L，ZnSO4·7H2O1.4mg/L，CoCl2 2mg/L。
纤维素酶活力采用DNS法测定，将1cm×6cm的滤纸条对称剪成32片分别放入4支25mL具塞刻度试管中，加1.0ml柠檬酸缓冲液浸润滤纸片，70℃水浴平衡10min，向1号试管中加入2.0mL 3，5-二硝基水杨酸试剂以钝化酶活性，作为空白对照。4支试管同时加入经过70℃预热的适当稀释的酶液，摇匀。70℃水浴中保温60min，取出后立即向2、3、4号试管中各加入2.0mL 3，5-二硝基水杨酸试剂(动作要迅速)以终止酶反应。将4支试管充分摇匀后在沸水浴中加热5min，取出冷却后用蒸馏水定容至25ml。以1号试管溶液为空白对照，在波长540nm处测定吸光度并记录结果。
淀粉酶活力采用改良的Yoo法测定。取5ml 0.5％的可溶性淀粉溶液，在70℃水浴中预热10min，加入适当稀释的酶液0.5ml(对照加0.5mlpH6.0的醋酸缓冲液)，准确反应5min后，用5ml 0.1mol/L的H2SO4终止反应。取0.5ml反应液与5ml稀碘液显色，以0.5ml水代替0.5ml反应液为空白在620nm处测吸光值。
蛋白酶活力采用福林试剂法测定。取四支试管分别加入1.0ml酶液，放置于70℃的水浴中预热2min，1号试管中加入10％三氯乙酸溶液2.0ml终止反应，然后向各试管中分别加入预热好的0.5％的酪素溶液1.0ml，摇匀，70℃下保温10min，1号试管加酪素溶液1.0ml，2、3、4号试管分别加入10％三氯乙酸溶液600μl终止反应，与室温下静置30min，然后5000rpm离心10min，取上清液1.0ml，加入碳酸钠溶液5.0mL，福林试剂1.0ml混匀后，70℃水浴显色20min，冷却后以1号试管为对照测定680nm处的吸光值并记录。
实验结果如图2所示。从图2中可以看出，HN-5在发酵前期由于受到菌体生物量的限制，酶活力较低；随着菌体生长，菌株产酶能力不断增大；随着发酵时间的延长，菌体开始死亡，酶活力也随之下降。发酵第三天纤维素酶活力达到最大值7.31U/ml；发酵第五天蛋白酶活力达到最大值53.71U/ml；发酵第四天淀粉酶活力最高，可达到70.02U/ml。
实施例3 地芽孢杆菌属HN-5的生长曲线
取待测细菌的新鲜细菌培养平板，以无菌操作挑取1环菌苔，接入装有100ml培养液的250ml的锥形瓶中，置于70℃静止培养24h作为种子培养液。用无菌移液管吸取1ml种子培养液至装有100ml灭菌后培养液的250ml的三角瓶中，70℃静止培养。每隔4小时取培养液测定660nm波长下吸光值，以时间为横坐标，OD值为纵坐标即可绘制菌株的生长曲线(图3)。
实验结果如图3所示，HN-5潜伏期较短，很快进入对数生长期，且对数期持续时间较长(≥24h)，36h后菌株停止生长。
实施例4 地芽孢杆菌属HN-5在促进堆肥腐熟、提高堆肥品质中的应用
物料及菌株：堆肥物料为鸡粪和锯末，两者的主要性质见表1；接种菌株为实施例1中筛得的菌株HN-5。
鸡粪来自中国农业大学动物科技学院养鸡场，锯末来自北京市海淀区永丰木器厂。
表1 堆肥原料的基本性质
根据鸡粪与锯末的性质，将水分调节至60％左右，C/N比调节至25-30之间。充分混合均匀后，装入容积为4L的泡沫盒中。堆肥接种时先将高温菌HN-5种子液(OD值0.4左右)以物料湿重的5‰与玉米面按1∶1(v/w)充分混匀，待堆体温度上升至40℃以上时接种至堆肥物料中，对照中以等量无菌水代替种子液与玉米面混合。将装有堆肥物料的泡沫盒置于18℃左右的室内进行腐熟。
分别在每天上午10:30和下午16:30进行温度测定，每种处理各取3个点测定，并记录当时的环境温度(即室温)，取1天中测定值的平均值作为当天温度值。
实验结果如图4所示，实验组在2天后进入高温期(＞50℃)，比对照组提前1天；由于实验装置小，堆体体积较小，因此堆体温度上升快，下降也快，处理组高温期维持时间为4d，明显比对照组高温期维持时间长；表明接种HN-5可加速堆肥进入高温期，延长堆肥高温期。
实施例5 地芽孢杆菌属HN-5所产酶的分离纯化
将1ml HN-5种子液(70℃，NR液体培养基中培养至OD值0.4左右)接种至纤维素、蛋白质、淀粉发酵培养基(配方同实施例2)中，在70℃发酵24小时得到发酵液。将发酵液10000rpm离心20min，取上清液用于酶的分离纯化。取发酵上清液加硫酸铵至饱和度为30％，4℃静置过夜，10000rpm离心20min；再取上清液加硫酸铵至饱和度为45％，用0.05mol/L，pH4.8的柠檬酸-柠檬酸钠缓冲液溶解沉淀，10000rpm离心5min，收集上清透析脱盐，在聚乙二醇-1000中浓缩。脱盐浓缩后的粗酶液经Sephadex G-100(规格：2.6cm×100cm)层析，用0.05mol/L，pH4.8的柠檬酸-柠檬酸钠缓冲液洗脱，流速1ml/min，收集洗脱液(每管5ml)。在280nm紫外检测仪下检测光密度值和酶活性。收集酶活较高的蛋白质峰并浓缩，经DEAESephadex A50(柱规格：14mm×200mm)层析，用0.5mol/L的NaCl溶液洗脱，流速为1ml/min，收集洗脱液(每管5ml)。分别收集各个蛋白质峰，在280nm紫外检测仪下检测光密度值和酶活性，合并酶活较高的蛋白质峰。
虽然，上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述，但在本发明基础上，可以对之作一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。
Claims (3)
1.一种高温降解菌，其为地芽孢杆菌属(Geobacillus)菌株HN-5，保藏号为CGMCC NO.5852。
2.权利要求1所述的高温降解菌在促进堆肥腐熟、提高堆肥品质中的应用。
3.根据权利要求2所述的应用，其是将地芽孢杆菌属HN-5接种到堆肥中，18～63.5℃下进行腐熟。
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