CN104560728B - 一株具有乙酸乙酯降解能力的绿色木霉菌及其应用 - Google Patents
一株具有乙酸乙酯降解能力的绿色木霉菌及其应用 Download PDFInfo
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Abstract
本发明涉及一种具有乙酸乙酯降解能力的绿色木霉菌(Trichoderma viride)LW‑1，其保藏编号为CCTCC NO:M2014176。以及该绿色木霉菌(Trichoderma viride)LW‑1在降解乙酸乙酯、苯、甲苯、氯苯、α‑蒎烯或二氯甲烷中的应用。本发明对目标污染物高效降解、耐受能力强。
Description
技术领域
本发明属于环境污染物生物处理技术领域，具体涉及一株具有乙酸乙酯降解能力的绿色木霉菌(Trichoderma viride)LW-1，及其在降解乙酸乙酯、苯、甲苯、氯苯、α-蒎烯或二氯甲烷中的应用。
背景技术
乙酸乙酯(分子式：CH3CO2CH2CH3)是一种常见的有机溶剂，在印刷、胶粘剂等生产企业和工业过程中广泛使用。乙酸乙酯挥发性强，对眼睛、皮肤、粘膜和上呼吸道有刺激性，长时间接触对人有麻醉作用，可致头晕和头痛。因此寻找有效防治乙酸乙酯污染的对策刻不容缓，对人类健康和环境保护具有重大意义。
生物法处理乙酸乙酯污染以其简单高效、能耗低、费用低、无二次污染等特点越来越受到重视。与以细菌为核心的生物工艺相比，丝状真菌由于所产生的菌丝体容易捕捉气相中的疏水性污染物，同时又具备耐低pH值和适应干燥环境等特性，日益成为VOCs生物过滤技术中新的研究热点。大量研究表明从环境中分离筛选高效降解乙酸乙酯等VOCs的真菌，仍然是消除环境中该类污染物的重要方法之一。
木霉菌属于半知菌亚门、丝孢纲、丝孢目，粘孢菌类，是一类普遍存在的真菌。绿色木霉在自然界分布广泛，常腐生于木材、种子及植物残体上。绿色木霉能产生多种具有生物活性的酶系，如：纤维素酶、几丁质酶、木聚糖酶等，目前主要用于植物病理生物防治中。
专利号为200810027399.7，专利名称为“绿色木霉H1在降解甲醛中的应用”的中国专利中公开了一种绿色木霉H1在降解甲醛中的应用同时提供了绿色木霉H1在降解甲醛的应用中的相关数据。但在该专利中报道了该绿色木霉只能降解甲醛或者在其他常见碳源(葡萄糖、蔗糖等)存在的条件下降解甲醛，对于其他工业常见的污染物是否具有降解能力并未提及。本发明提及的乙酸乙酯和甲醛属于两类物质，结构迥异，降解甲醛的绿色木霉无法降解乙酸乙酯。
发明内容
本发明所要解决的技术问题是克服现有技术中的不足而提供一种高效、耐受能力强的具有乙酸乙酯降解能力的绿色木霉菌及其应用。
本发明解决上述技术问题采用的技术方案是：该具有乙酸乙酯降解能力的绿色木霉菌(Trichoderma viride)LW-1，其保藏编号为CCTCC NO:M2014176。保藏于中国典型培养物保藏中心，地址：中国，武汉，武汉大学，430072，保藏日期：2014年5月3日。
本发明所述的具有乙酸乙酯降解能力的绿色木霉菌(Trichoderma
viride)LW-1，其菌落特征为子座为半球形、扁碟状或盾状，直径1.5-7mm，表面绿色，内部近白色。
本发明所述的具有乙酸乙酯降解能力的绿色木霉菌(Trichodermaviride)LW-1在降解乙酸乙酯、苯、甲苯、氯苯、α-蒎烯或二氯甲烷中的应用。该菌株LW-1能降解初始浓度0～600mg/L的乙酸乙酯，最终降解产物为CO2、H2O和细胞生物量，其去除率大约为100％，即可以完全降解乙酸乙酯。该降解菌的发现对工业废气中乙酸乙酯的高效净化具有重要意义。该菌株LW-1还可降解其他工业常见有机污染物，尤其是苯、甲苯、1-氯苯、二氯甲烷等。
本发明所述的对工业常见有机污染物的降解优选在30℃、pH＝4.87条件下进行。上述条件下降解的好处是绿色木霉活性较大，能在较短时间内完全利用乙酸乙酯等工业常见的污染物。
本发明与现有技术相比具有以下优点：本发明中的绿色木霉(Trichodermaviride)LW-1取自废水处理单元，对于VOCs类有机污染物，尤其是乙酸乙酯具有较好地降解效果，可以较为完全地把乙酸乙酯转化为CO2、H2O、细胞生物量等无害物质；同时，该菌株也能不同程度地降解苯、甲苯、氯苯等工业常见的污染物，因而在工业废气废水的生物净化中具有广阔的应用前景。
本发明所述的绿色木霉能将乙酸乙酯完全降解为无机物(H2O和CO2)和细胞生物质，实现完全矿化，且对于乙酸乙酯的耐受浓度高达600mg/L。因此，该绿色木霉对于工业常见污染物(如乙酸乙酯等)具有高效的降解能力，且能承受较高浓度的污染物。
附图说明
图1为绿色木霉(Trichoderma viride)LW-1的乳酸酚棉兰染色照片。
图2为绿色木霉(Trichoderma viride)LW-1在PDA培养基上菌落形态照片。
图3为绿色木霉(Trichoderma viride)LW-1的系统发育树图。
图4为绿色木霉(Trichoderma viride)LW-1在不同初始浓度的乙酸乙酯条件下的降解效果图。
图5为绿色木霉(Trichoderma viride)LW-1在不同pH条件下对乙酸乙酯的降解效果图。
图6为绿色木霉(Trichoderma viride)LW-1在不同pH条件下的生物量变化。
具体实施方式
下面结合具体实施例对本发明进行进一步描述，但本发明的保护范围并不仅限于此，下述实施例中所使用的实验方法如无特殊说明，均为常规方法。
实施例1：绿色木霉(Trichoderma viride)LW-1的分离、纯化及其鉴定。
1.绿色木霉(Trichoderma viride)LW-1的分离及纯化。
绿色木霉(Trichoderma viride)LW-1是从浙江杭州市某污水处理厂曝气池中的活性污泥中驯化、分离得到的一株真菌。具体步骤如下：取浙江杭州市某污水处理厂曝气池中的活性污泥。自来水淘洗五次后，空曝48h，尽量去除残留的有机物。配制无机盐培养液，以乙酸乙酯为唯一碳源对活性污泥进行定向驯化，每3d更换新鲜无机盐培养液，40d左右即可分离。
配制乙酸乙酯初始浓度为50mg/L无机盐培养基，配制方法如下：KH2PO40.45g，Na2HPO40.47g，(NH4)2SO42.0g，MgSO4·7H2O 0.5g，无水CaCl20.01g，微量元素(MnSO4·H2O0.001g，ZnSO4·7H2O 0.001g，FeSO40.001g，CuSO4·5H2O0.001g)，蒸馏水，1000mL；pH＝3～6，分装于250mL的密封盐水瓶(50mL/个)，110℃灭菌40min。
从驯化瓶中取50ml上清液进行离心，将沉淀下来的污泥加入到装有50ml的无机盐培养基(已灭菌)的盐水瓶中，并且添加链霉素0.003g，庆大霉素0.001g。盖上瓶塞后加入乙酸乙酯(浓度为50mg/L)。放置在30℃，160rpm的摇床中培养。隔一段时间观察盐水瓶中菌体生长情况，之后在已降解完全的盐水瓶中取5mL菌悬液到装有新鲜的50mL的无机盐培养基的瓶中，仍添加链霉素0.003g，庆大霉素0.001g。盖上瓶塞后加入乙酸乙酯(浓度为100mg/L)。将培养瓶置于30℃，160rpm的摇床中培养。培养一定时间后，再取5ml菌悬液到装有新鲜的50ml的无机盐培养基的瓶中，添加链霉素0.003g，庆大霉素0.001g。盖上瓶塞后加入乙酸乙酯(浓度为200mg/L)。置于摇床中培养。
取2ml混合菌液,涂布于含有乙酸乙酯的固体无机盐培养基上,连续划线分离，最终获得纯化菌株，接种到PDA斜面培养基中，在4℃的冰箱中保存。
2.绿色木霉(Trichoderma viride)LW-1的鉴定。
通过真菌ITS序列分析，确定菌株为绿色木霉(Trichoderma viride)。具体步骤如下：PCR扩增反应体系见表1，PCR扩增的反应条件为95℃预变性5min，95℃变性30s，55℃退火30s，72℃延伸40s，循环32次；72℃延伸10min，10℃保存。
表1：ITS序列PCR扩增反应体系
| 反应体系成分 | 体积(μl) |
| 10×Ex Taq buffer | 2.0 |
| 2.5mM dNTP Mix | 1.6 |
| 5p Primer 1 | 0.8 |
| 5p Primer 2 | 0.8 |
| Template | 0.5 |
| 5u Ex Taq | 0.2 |
| 超纯水 | 14.1 |
| 总体积 | 20 |
将菌株LW-1的ITS序列上传到Genbank，获得Genbank登录号为KJ716342。同时该序列与NCBI数据库中的基因序列进行Blast对比。结果表明LW-1菌株的ITS序列与菌株Trichoderma viride strain Tri178(HQ229943)和Trichoderma viride strain AIBF002-1[GQ229073]具有67％的同源性。从结果中选取10株具有代表性的菌株,以ITS基因序列同源性为基础，结合采用Clustal X2.0和MEGA4.0(1000次抽样分析)软件构建系统发育树。通过遗传距离及ITS序列对比，鉴定为绿色木霉(Trichoderma viride)。
实施例2：菌株LW-1对Biolog FF板上95种碳源的利用能力。
利用Biolog自动微生物鉴定系统考察菌株对95种不同碳源的代谢情况：将菌株接种于真菌平板培养基，26℃恒温培养5天，用无菌棉签将平板上的菌体洗下，与接种液(FF-IF)混合，制成菌悬液，用浊度计调整至75％T/FF。用8孔电动加液器将菌悬液分别加在Biolog FF微孔鉴定板的各孔中，每孔100μl。将微孔鉴定板放在26℃培养箱中，分别在培养24h、48h、72h、96h、168h和240h后将其置于Biolog读数仪上读取结果。经Biolog读数仪分析代谢指纹，菌株LW-1可较强利用43种碳源，对其他52种碳源不能利用或利用能力较弱。Biolog系统给出48h鉴定结果，如表2所示。鉴定结果表明，该菌株与系统内Trichodermaviride SIM index符合度较好，表明分离得到的菌株LW-1属于Trichoderma viride。
表2：菌株LW-1对Biolog FF板上95种碳源的利用能力
表中:+,阳性反应；-,阴性反应；B,边界反应。
实施例3：优化菌株绿色木霉(Trichoderma viride)LW-1降解乙酸乙酯的环境因子。
1、不同pH值对绿色木霉(Trichoderma viride)LW-1降解乙酸乙酯的影响。
环境中的pH值对微生物生长的影响较大，pH能直接影响细胞膜电荷的变化，间接影响营养物质的离子化程度。事实表明，多数非离子状态下的化合物比离子状态的化合物更容易通过细胞壁进入细胞。另外，微生物中的酶在适宜的pH值条件下才能发挥活性。与细菌、放线菌相比，真菌喜欢在偏酸性的环境中生长。
从图5可以得出：当乙酸乙酯的初始浓度都是35.88mg/L时，pH为4.87的降解速率最快，其余降解速率是pH5.57>pH3.25>pH3.88。当摇瓶被培养3天时，pH为4.87的摇瓶基本将乙酸乙酯降解完全，特别是反应的第一天，pH为4.87的摇瓶对乙酸乙酯降解的效果相对其他是最好的。还有pH为4.87时，其去除率为96.88％。
2、不同pH值对绿色木霉(Trichoderma viride)降解乙酸乙酯时生物量的变化情况。
从图6可以看出：pH为4.87时，其瓶中生物量增长为最大值是316mg/L，其余生物量顺序是pH5.57(284mg/L)>pH3.25(82mg/L)>pH3.88(30mg/L)。这个顺序刚好和图5的降解曲线相一致，也进一步说明pH为4.87时真菌LW-1对瓶内的乙酸乙酯的降解效果是最佳的。
实施例4：绿色木霉(Trichoderma viride)LW-1对不同初始浓度乙酸乙酯的降解性能检测。
有机污染物对微生物降解的影响主要是指污染物本身的生物可利用性。污染物的化学结构、浓度、溶解性以及吸附性能都影响它的生物可利用性。这里主要考察不同乙酸乙酯初始浓度对真菌LW-1降解效率的影响。
如图4所示，乙酸乙酯作为一种有机污染物，具有一定的生物毒性。在最佳生长条件下，考察菌株LW-1对不同初始浓度乙酸乙酯的降解特性，结果表明真菌LW-1能完全降解浓度低于600mg/L的乙酸乙酯，且在这范围内随着浓度的增加，真菌降解的适应期开始趋长。
实施例5：绿色木霉(Trichoderma viride)LW-1降解底物的广谱性。
在实际应用中，不仅仅存在乙酸乙酯这一种有机污染物，工业废气中往往是多组分的有机污染物共存，因此，研究真菌LW-1对其他底物的降解效果很有必要。在pH值为5.50，温度是30℃的条件下，真菌LW-1对初始浓度为44mg/L的1-氯苯，53.2mg/L的二氯甲烷，34.4mg/L的α-蒎烯，79mg/L的异丙醇，90mg/L的乙酸乙酯的降解效果如表3所示。结果表明，真菌LW-1能不同程度的降解氯苯、二氯甲烷、α-蒎烯，以及异丙醇等类的碳氢化合物。
表3：真菌LW-1对不同碳源的降解效果。
| 物质名称 | 初始浓度(mg/L) | 去除率(％) |
| 1-氯苯 | 44 | 25.43 |
| 二氯甲烷 | 53.2 | 39.1 |
| α-蒎烯 | 34.4 | 51.52 |
| 异丙醇 | 79 | 48.63 |
| 乙酸乙酯 | 90 | 74.41 |
如表3所示：菌株只能少部分降解1-氯苯，二氯甲烷；乙酸乙酯和异丙醇溶于水，溶解度相对较大，所以其去除率要高许多。进一步表明，真菌LW-1能不同程度的降解氯苯、二氯甲烷、α-蒎烯，以及异丙醇等类的碳氢化合物。
虽然本发明已以实施例公开如上，但其并非用以限定本发明的保护范围，任何熟悉该项技术的技术人员，在不脱离本发明的构思和范围内所作的更动与润饰，均应属于本发明的保护范围。
Claims (4)
1.一株具有乙酸乙酯降解能力的绿色木霉菌（Trichoderma viride）LW-1，其保藏编号为CCTCC NO: M2014176。
2.根据权利要求1所述的具有乙酸乙酯降解能力的绿色木霉菌（Trichoderma viride）LW-1，其特征在于：其菌落特征为子座为半球形、扁碟状或盾状，直径1.5-7mm，表面绿色，内部近白色。
3.权利要求1所述的具有乙酸乙酯降解能力的绿色木霉菌（Trichoderma viride）LW-1在降解乙酸乙酯、氯苯、α-蒎烯或二氯甲烷中的应用。
4. 根据权利要求3所述的应用，其特征在于：所述的降解在30℃、pH= 4.87条件下进行。
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