CN1587379A - 非灭菌环境白腐真菌降解活性染料的抑制细菌生长培养基 - Google Patents
非灭菌环境白腐真菌降解活性染料的抑制细菌生长培养基 Download PDFInfo
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Abstract
本发明涉及非灭菌环境白腐真菌降解活性染料的抑制细菌生长培养基，属于应用微生物领域。该液体培养基是在现有培养白腐真菌液体培养基的基础上，通过调整碳氮比(C/N＝56/8.7mM)和微量元素(Fe)，并利用白腐真菌本身在酸性环境(pH＝4.4)生长最优的特点，开发出在白腐真菌降解活性染料时能有效抑制细菌生长的液体培养基。经过对比试验，本液体培养基培养出的白腐真菌在非灭菌环境下对活性艳红K－2BP的脱色率与灭菌环境得到的结果基本相当。并且镜检液体培养基未发现有大量细菌。本发明使用方便，为实际工程应用白腐真菌处理含染料废水提供了可能，并在实际中降低了因灭菌条件带来的运行成本。
Description
技术领域
本发明属于应用微生物领域，特别涉及在非灭菌环境应用白腐真菌降解活性染料时抑制细菌生长的液体培养基的研制。
背景技术
含染料废水是目前国内外公认的难处理的工业废水。近年来，许多研究表明在含染料废水处理中白腐真菌(White rot fungi)是很有发展前景的微生物，其中研究和讨论得最多的是黄孢原毛平革菌(Phanerochaete chrysosporium)。黄孢原毛平革菌因其具有非特异性、无需底物诱导的酶，对许多有机污染物和不同类型的人工合成染料(如偶氮，三苯甲烷，酞菁染料)具有广谱的降解能力(Heinfling A，et al.Biodegradation of azo andphthalocyanine dyes by Trametes versicolor and Bjerkandera adusta.Appl MicrobiolBiotechnol，1997，48：261-266.)。采用黄孢原毛平革菌对一些偶氮染料进行脱色已经发现了非常有意义的矿化(20％-48％)现象(Spadaro JT，et al.Degradation of azo dyes bythe lignin-degrading fungus Phanerochaete chrysosporium.Appl Environ Microbiol，1992，58：2397-2401.)。但是，虽然在灭菌环境中黄孢原毛平革菌对染料有较高的脱色效果，并且脱色效果不随时间而降低，但是如果在非灭菌环境应用黄孢原毛平革菌，细菌污染却很容易发生，一旦发生将引起脱色效率的急剧下降(Heinfling A，et al.Transformationof industrial dyes by manganese peroxidases from Bjerkandera adusta and Pleurotuseryngii in a manganese-independent reacxtion.Appl Environ Microbiol，1998，64：2788-2793.)。因此，如果在实际含染料废水处理中应用白腐真菌，必须要解决的问题就是反应体系染菌。如果对实际工程中的反应器、培养液、载体以及废水都进行灭菌处理并保证处理过程不染菌，显然将大大增加处理工艺的运行成本，并且在实际工程中也是行不通的。因此，如何解决白腐真菌降解含染料废水过程中的染菌问题是该工艺能否应用到实际工程中的瓶颈。不解决染菌问题，该工艺则很难在实际工程中使用，将会严重制约该项技术的发展。
通过检索中外文献，只发现两篇研究应用白腐真菌降解染料时的抑菌技术，一个为采用聚乙烯醇包埋法来保护白腐真菌Trametes versicolor和细胞外产生的过氧化物酶免受细菌攻击，从而达到对染料Poly R-478的连续生物氧化。但包埋法的问题是，由于白腐真菌不断生长逐渐会挣破聚乙烯小球，使菌丝体暴露出来，影响抑菌效果(Leidig E，
et al.Biotransformation of Poly R-478 by continuous cultures of PVAL-encapsulatedTrametes versicolor under non-sterile conditions.Bioprocess Eng，1999，21：5-12.)。另一篇为通过控制低pH值，氮限制培养基和天然载体研究应用白腐真菌Trametesversicolor在非灭菌环境降解活性染料的控制策略，但文中提到单单依靠控制低pH和限氮培养基很难获得长期的抑菌效果(Judy A，et al.Competition strategies for thedecolorization of a textile-reactive dye with the white-rot fungi Trametesversicolor under non-sterile conditions.Biotechnilogy and Bioengineering，2003，82(6)：736-744.)。以上两种抑菌技术均是采用Trametes versicolor菌种，目前国内外尚没有一种有效抑制细菌生长的黄孢原毛平革菌的生长液体培养基。
发明内容
本发明提供了在自然环境下活性染料降解过程中有效抑制细菌生长的液体培养基。
本发明提供的非灭菌环境白腐真菌降解活性染料的抑制细菌生长培养基，其特征在于：所述培养基利用黄孢原毛平革菌在氮限制和碳限制条件下能够分泌降解染料等难降解有机污染物过氧化物酶的特点，通过同时控制培养基中(1)碳源和氮源的浓度；(2)pH值；(3)微量元素，使在该液体培养基下生长的黄孢原毛平革菌降解活性染料时染菌几率大大降低，从而达到非灭菌环境对染料的脱色效果近似等于灭菌环境。
白腐真菌采用黄孢原毛平革菌，所述抑制细菌生长的液体培养基为氮限制液体培养基(C/N＝56/2.2～56/8.7mM)，其主要成分包括葡萄糖(碳源，占67.8％～76.1％)、酒石酸铵(氮源，占1.5％～5.4％)、磷酸二氢钾(占7.6％～13.6％)，其余为微量元素和藜芦醇(占4.9％～23.1％)等。具体配方如表1所示。
表1培养基配方
成分 浓度 成分 浓度
2.0mg/L～3.5
葡萄糖 10g/L FeSO4.7H2O
mg/L
酒石酸铵 0.2g/L～0.8g/L CoCl2 7mg/L
藜芦醇 1.5mM ZnSO4.7H2O 7mg/L
醋酸-醋酸钠缓冲
20mM CuSO4 7mg/L
液
KH2PO4 1.0g/L～2.0g/L A1K(SO4)2.12H2O 0.7mg/L
MgSO4 0.71g/L H3BO3 0.7mg/L
CaCl2 0.1g/L Na2MoO4.2H2O 0.7mg/L
MnSO4 35mg/L Nitrilotriacetate 0.105g/L
维生素B1(盐酸硫
NaCl 70mg/L
1.0g/L
胺)
加入醋酸-醋酸钠缓冲液后，此液体培养基的pH值在4.4～4.5左右，是白腐真菌的最佳生长和产酶pH环境，同时该pH值环境本身也不利于大部分细菌生长。但，该液体培养基并不适用在非灭菌环境培养白腐真菌孢子，它仅适于在灭菌环境培养白腐真菌后在非灭菌环境对活性染料进行降解。
采用本发明液体培养基在非灭菌环境对活性染料进行降解，获得了稳定、高效的脱色效果，其脱色率与灭菌环境得到的结果基本一致。并且在脱色结束时也未发现有大量细菌感染。因此，本发明使用方便，为实际工程应用白腐真菌处理含染料废水提供了可能，并在实际中降低了因灭菌条件带来的运行成本。
具体实施方式
下面结合实施例对本发明做进一步说明：
实施例一：
将配制好未加维生素Bl的液体培养基分装在6个250ml锥形瓶中，每瓶含有100ml液体培养基(此培养基C/N＝56/8.7mM，即酒石酸铵浓度为0.8g/L；另，磷酸二氢钾浓度为2.0g/L，FeSO4.7H2O为3.5mg/L)。将含有液体培养基的锥形瓶放入灭菌锅中在113℃下灭菌30min。灭菌结束后，用注射器和针头式过滤器(带膜灭菌)向100ml液体培养基过滤加入1ml 100mg/L维生素B1溶液，保持液体培养基中维生素B1的终浓度为1mg/L。然后，将生长在37℃PDA平板(200g/L土豆汁、20g/L葡萄糖和20g/L琼脂)上的黄孢原毛平革菌BKM-F-1767孢子等量无菌接入液体培养基中，接种量为1×105个孢子/ml。然后将锥形瓶放入温度为37℃的恒温摇床中，转速设为160rpm/min，在空气条件下培养。培养5d后，分别在灭菌环境和非灭菌环境下向含有白腐真菌和液体培养基的锥形瓶中加入未经灭菌处理的活性艳红K-2BP溶液(1000mg/L)2ml，保持锥形瓶内溶液中活性艳红K-2BP的浓度为20mg/L。24h后，非灭菌环境投加活性艳红K-2BP的3个锥形瓶内活性艳红K-2BP的脱色率均在80％以上，3d后脱色率升到88％；与灭菌环境投加活性艳红K-2BP相比，二者脱色率基本相同。并对3d后非灭菌环境投加活性艳红K-2BP的培养液镜检，只发现有少量酵母菌，并未发现有细菌。
实施例二：
改变液体培养基部分成分，进行非灭菌环境脱色活性艳红K-2BP实验。液体培养基中酒石酸铵浓度为0.2g/L，磷酸二氢钾浓度为1.0g/L，FeSO4.7H2O为2.0mg/L；其余成分含量同实施例一。具体培养黄孢原毛平革菌和脱色活性艳红K-2BP过程同实施例一。结果显示，在非灭菌环境仍然具有较好的脱色活性艳红K-2BP的效果，加入活性艳红K-2BP 2d后脱色率在82％以上；通过对液体培养基镜检，只发现少量酵母菌，并未发现有细菌。
实施例三：
仍然改变液体培养基部分成分，进行非灭菌环境脱色活性艳红K-2BP实验。液体培养基中酒石酸铵浓度为0.4g/L，磷酸二氢钾浓度为1.5g/L，FeSO4.7H2O为3.0mg/L；其余成分含量同实施例一。具体培养黄孢原毛平革菌和脱色活性艳红K-2BP过程同实施例一。结果显示，在非灭菌环境仍然具有较好的脱色活性艳红K-2BP的效果，加入活性艳红K-2BP 2d后脱色率在85％以上；液体培养基镜检结果，只发现少量酵母菌，并未发现有细菌。
Claims (2)
1、非灭菌环境白腐真菌降解活性染料的抑制细菌生长培养基，其特征在于：所述培养基利用黄孢原毛平革菌在氮限制和碳限制条件下能够分泌降解染料等难降解有机污染物过氧化物酶的特点，通过同时控制培养基中碳源和氮源的浓度，pH值及微量元素，使在该液体培养基下生长的黄孢原毛平革菌降解活性染料时染菌几率大大降低，从而达到非灭菌环境对染料的脱色效果近似等于灭菌环境，所述白腐真菌采用黄孢原毛平革菌，所述抑制细菌生长的液体培养基为氮限制液体培养基(C/N＝56/2.2～56/8.7mM)，其主要成分包括葡萄糖(碳源，占67.8％～76.1％)、酒石酸铵(氮源，占1.5％～5.4％)、磷酸二氢钾(占7.6％～13.6％)，其余为微量元素和藜芦醇(占4.9％～23.1％)。
2、按照权利要求1所述的非灭菌环境白腐真菌降解活性染料的抑制细菌生长培养基，其特征在于：所述培养基中pH值为4.4～4.5。
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100451112C (zh) * | 2005-12-05 | 2009-01-14 | 清华大学 | 在非灭菌环境抑制杂菌生长的白腐真菌固定化方法 |
| CN103018409A (zh) * | 2012-12-11 | 2013-04-03 | 湛江师范学院 | 一种检验野生仙人掌多糖抑菌效果的方法 |
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| CN100451112C (zh) * | 2005-12-05 | 2009-01-14 | 清华大学 | 在非灭菌环境抑制杂菌生长的白腐真菌固定化方法 |
| CN103018409A (zh) * | 2012-12-11 | 2013-04-03 | 湛江师范学院 | 一种检验野生仙人掌多糖抑菌效果的方法 |
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