焦化废水中苯酚降解菌的筛选与降解性能的研究
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摘要
中国是焦化产品生产、消费、出口大国,由焦化废水的排放所造成的环境污染问题和焦化废水的治理技术已成为整个焦化行业发展的瓶颈。2010年我国积极推动焦化产业的优化升级,由扩大产能规模为主向高附加值焦化产品深加工为主、向“低碳”、“绿色”方向转变,焦化废水的深度处理受到高度重视。
焦化废水含挥发酚、多环芳烃、杂环化合物、焦油等有机成分,还含有氰、硫、氨氮等无机化合物,成分复杂、色度高、毒性大,是典型的难生物降解处理的有机废水。2010年7月1日起执行的《炼焦工业污染物排放标准》(GB 16171-2011)规定COD直接排放限值,现有企业由原来的150 mg/L改为100 mg/L,新建企业由原来的100 mg/L改为50 mg/L,对COD的排放要求越来越严格。酚类物质作为COD的主要贡献物,其降解效率对于焦化废水的达标排放尤为重要。本文就此开展以下内容研究：
1.从理论方面探讨了构建用于处理焦化废水的基因工程菌的必要性和可行性。仅从活性污泥中分离筛选得到的菌株,对焦化废水的降解能力往往有限,利用基因操作技术对筛选得到的菌株进行遗传学改造,能大幅提高降解酶的活性并扩大降解底物范围。在焦化废水中由于酚类物质相对于难降解的含氮杂环化合物物质有较高的降解率,所以拟通过构建基因工程菌,降低微生物之间的拮抗作用,提高降解效率,实现对酚类物质和含氮杂环物质的双重降解。
2.以太原煤气化公司第二焦化厂废水处理车间的活性污泥作为菌源,选用两种不同的培养方案,利用不同的液体培养基,采用选择性富集培养技术、逐级提高苯酚浓度的菌株驯化方法,筛选方案(一)当苯酚浓度1800mg/L时筛选得到苯酚降解菌A1,筛选方案(二)当苯酚浓度2000mg/L时筛选得到苯酚降解菌C1、D2、D3。筛选得到的四株菌株均以苯酚为唯一碳源且对苯酚有较高的耐受力。
3.对筛选得到的四株降酚菌,通过菌体的染色观察、菌落形态特征观察、常规的生理生化特征实验,初步鉴定菌株A1为球杆菌属、菌株C1为不动杆菌属、D2丛毛单胞菌属、D3新鞘脂菌属。同时由生物公司完成细菌16srDNA的分子鉴定,进一步鉴定A1为Acinetobacter baumannii(鲍曼不动杆菌),D2为Comamonas testosteroni(睾丸酮丛毛单胞菌),D3为Novosphingobium naphthalenivorans。
4.通过对四株菌株在500mg/L、1000mg/L、1500mg/L、2000mg/L不同苯酚浓度的降解率的测定,发现菌株D2不同浓度下的降酚率均优于其他菌株。对菌株D2做环境因子对降酚率的影响实验,改变温度、酸碱度、摇床转速等环境条件探讨环境因子的改变对D2降酚率的影响,从而实现对D2降酚环境条件的优化。
综上所述,本研究共筛选得到四株降酚菌,均对苯酚具有较高耐受力和降解力,可作为下一步构建焦化废水基因工程菌的受体菌的备选菌株,其中菌株A1并不是常见的降酚菌,丰富了降酚菌的微生物资源。通过菌株D2环境影响实验,优化了其降酚条件,对焦化废水活性污泥处理实践提供了数据参考。
China is the main country of the coking production, coking consumption and coking export. However, the outdated production techniques and equipments seriously polluted the water environment. In 2010, something has been done toactively promote the optimization and upgrading of coking industry in China. It is planned and ruled that the development mode would change from the mainly expanding capacity to the higher-valued coking products in the way of "low carbon" and "green" direction. The coking wastewater treatment are paid more attentions.
Coking wastewater mainly contains phenol, polycyclic aromatic hydrocarbons, heterocyclic compounds, tar and other organic compounds except for cyanide, sulphide, high concentrations of NH3-N and other inorganic compounds. Due to the complicated compositions, heavy chromaticity and strong toxicity, the coking wastewater is a typical organic wastewater difficult to treat. Since July 1,2010, the implementation of coking industry emission standards (GB 16171-2011) provides the limits of direct discharge of COD, for the existing factory is from 150 mg/L to 100 mg/L, the new enterprise from 100 mg/L to 50 mg/L. Clearly, phenolic substances as the main contribution of COD, their degradation efficiency has the direct relation with meeting the requirements of the coking wastewater discharge standards. For this purpose, it is investigated the treatment process and selected the bacteria to degradate the phenolic substances in this thesis. The details is as follows:
1. It theoretically discusses the necessity and feasibility of constructing gene engineering bacteria to degrade the coking wastewater effectivly. The capacity of activated sludge strains isolated to degrade the coking wastewater is often limited. The use of genetic technique for genetic transformation strains obtained can significantly increase the degradation enzyme activity and expand the scope of the degradation substrate. The phenolic compounds has higher degradation rate than nitrogen heterocyclic compounds in the coking wastewater during the microbial degradation process, while gene engineering bacteria reduce the antagonism among microorganisms, increase the degradation efficiency and get the double degradation of phenolic compounds and heterocyclic substances.
2. Activity sludge from Taiyuan Coal Gasification Company's second coke plant is served as the bacterial source. Two training programs are developed bydifferent liquid medium, selective enrichment culture technology and the increase of phenol concentration. In the screening program (1), when phenol concentration is 1800 mg/L we get phenol degradation bacterium A1; in the screening program (2) when phenol concentration is 2000 mg/L we get phenol degradation bacterium C1, D2 and D3. These four strains screened have higher tolerance to phenol and take phenol as sole carbon source.
3. By the means of biomass staining, colony morphology observation, investigation of physiological and biochemical characteristics, these four phenol degrading bacteria were identified. A1 is as Sphaerobacter, C1 as Acinetobacter, D2 as Comamonas and D3 as Novosphingobium. Biotechnology company (Genolab Biotech Co.,Ltd.) was commissioned to do 16s rDNA molecular identification, C1 is Acinetobacter baumannii, D2 is Comamonas testosteroni and D3 is Novosphingobium naphthalenivorans.
4. Four strains in different phenol concentration 500mg/L, 1000mg/L, 1500mg/L and 2000mg/L were determined the efficiency of phenol degradation. It was found that strain D2 had better results than the other three. D2 strain was further selected and studied the environmental factors on the rate of phenol degradation experiments, such as temperature, pH and rotation speed. The optimization of environmental conditions was.
In summary, four strains of phenol degrading bacteria has been screened out during this study. All of them have higher tolerance and better degradation ability for phenol. All of these can be as the candidate recipient strain in constructing genetic engineering strain using in the coking wastewater degration. In addition to, Strain A1 is not a common strain as usually we used, that means it can enrich microbial resources for phenol cracking. Through the environmental impact experiments, the environmental conditions of strain D2 have been optimized in the process of degradation of phenol. It provides data as reference for coking wastewater treatment by activated sludge.
焦化废水含挥发酚、多环芳烃、杂环化合物、焦油等有机成分,还含有氰、硫、氨氮等无机化合物,成分复杂、色度高、毒性大,是典型的难生物降解处理的有机废水。2010年7月1日起执行的《炼焦工业污染物排放标准》(GB 16171-2011)规定COD直接排放限值,现有企业由原来的150 mg/L改为100 mg/L,新建企业由原来的100 mg/L改为50 mg/L,对COD的排放要求越来越严格。酚类物质作为COD的主要贡献物,其降解效率对于焦化废水的达标排放尤为重要。本文就此开展以下内容研究：
1.从理论方面探讨了构建用于处理焦化废水的基因工程菌的必要性和可行性。仅从活性污泥中分离筛选得到的菌株,对焦化废水的降解能力往往有限,利用基因操作技术对筛选得到的菌株进行遗传学改造,能大幅提高降解酶的活性并扩大降解底物范围。在焦化废水中由于酚类物质相对于难降解的含氮杂环化合物物质有较高的降解率,所以拟通过构建基因工程菌,降低微生物之间的拮抗作用,提高降解效率,实现对酚类物质和含氮杂环物质的双重降解。
2.以太原煤气化公司第二焦化厂废水处理车间的活性污泥作为菌源,选用两种不同的培养方案,利用不同的液体培养基,采用选择性富集培养技术、逐级提高苯酚浓度的菌株驯化方法,筛选方案(一)当苯酚浓度1800mg/L时筛选得到苯酚降解菌A1,筛选方案(二)当苯酚浓度2000mg/L时筛选得到苯酚降解菌C1、D2、D3。筛选得到的四株菌株均以苯酚为唯一碳源且对苯酚有较高的耐受力。
3.对筛选得到的四株降酚菌,通过菌体的染色观察、菌落形态特征观察、常规的生理生化特征实验,初步鉴定菌株A1为球杆菌属、菌株C1为不动杆菌属、D2丛毛单胞菌属、D3新鞘脂菌属。同时由生物公司完成细菌16srDNA的分子鉴定,进一步鉴定A1为Acinetobacter baumannii(鲍曼不动杆菌),D2为Comamonas testosteroni(睾丸酮丛毛单胞菌),D3为Novosphingobium naphthalenivorans。
4.通过对四株菌株在500mg/L、1000mg/L、1500mg/L、2000mg/L不同苯酚浓度的降解率的测定,发现菌株D2不同浓度下的降酚率均优于其他菌株。对菌株D2做环境因子对降酚率的影响实验,改变温度、酸碱度、摇床转速等环境条件探讨环境因子的改变对D2降酚率的影响,从而实现对D2降酚环境条件的优化。
综上所述,本研究共筛选得到四株降酚菌,均对苯酚具有较高耐受力和降解力,可作为下一步构建焦化废水基因工程菌的受体菌的备选菌株,其中菌株A1并不是常见的降酚菌,丰富了降酚菌的微生物资源。通过菌株D2环境影响实验,优化了其降酚条件,对焦化废水活性污泥处理实践提供了数据参考。
China is the main country of the coking production, coking consumption and coking export. However, the outdated production techniques and equipments seriously polluted the water environment. In 2010, something has been done toactively promote the optimization and upgrading of coking industry in China. It is planned and ruled that the development mode would change from the mainly expanding capacity to the higher-valued coking products in the way of "low carbon" and "green" direction. The coking wastewater treatment are paid more attentions.
Coking wastewater mainly contains phenol, polycyclic aromatic hydrocarbons, heterocyclic compounds, tar and other organic compounds except for cyanide, sulphide, high concentrations of NH3-N and other inorganic compounds. Due to the complicated compositions, heavy chromaticity and strong toxicity, the coking wastewater is a typical organic wastewater difficult to treat. Since July 1,2010, the implementation of coking industry emission standards (GB 16171-2011) provides the limits of direct discharge of COD, for the existing factory is from 150 mg/L to 100 mg/L, the new enterprise from 100 mg/L to 50 mg/L. Clearly, phenolic substances as the main contribution of COD, their degradation efficiency has the direct relation with meeting the requirements of the coking wastewater discharge standards. For this purpose, it is investigated the treatment process and selected the bacteria to degradate the phenolic substances in this thesis. The details is as follows:
1. It theoretically discusses the necessity and feasibility of constructing gene engineering bacteria to degrade the coking wastewater effectivly. The capacity of activated sludge strains isolated to degrade the coking wastewater is often limited. The use of genetic technique for genetic transformation strains obtained can significantly increase the degradation enzyme activity and expand the scope of the degradation substrate. The phenolic compounds has higher degradation rate than nitrogen heterocyclic compounds in the coking wastewater during the microbial degradation process, while gene engineering bacteria reduce the antagonism among microorganisms, increase the degradation efficiency and get the double degradation of phenolic compounds and heterocyclic substances.
2. Activity sludge from Taiyuan Coal Gasification Company's second coke plant is served as the bacterial source. Two training programs are developed bydifferent liquid medium, selective enrichment culture technology and the increase of phenol concentration. In the screening program (1), when phenol concentration is 1800 mg/L we get phenol degradation bacterium A1; in the screening program (2) when phenol concentration is 2000 mg/L we get phenol degradation bacterium C1, D2 and D3. These four strains screened have higher tolerance to phenol and take phenol as sole carbon source.
3. By the means of biomass staining, colony morphology observation, investigation of physiological and biochemical characteristics, these four phenol degrading bacteria were identified. A1 is as Sphaerobacter, C1 as Acinetobacter, D2 as Comamonas and D3 as Novosphingobium. Biotechnology company (Genolab Biotech Co.,Ltd.) was commissioned to do 16s rDNA molecular identification, C1 is Acinetobacter baumannii, D2 is Comamonas testosteroni and D3 is Novosphingobium naphthalenivorans.
4. Four strains in different phenol concentration 500mg/L, 1000mg/L, 1500mg/L and 2000mg/L were determined the efficiency of phenol degradation. It was found that strain D2 had better results than the other three. D2 strain was further selected and studied the environmental factors on the rate of phenol degradation experiments, such as temperature, pH and rotation speed. The optimization of environmental conditions was.
In summary, four strains of phenol degrading bacteria has been screened out during this study. All of them have higher tolerance and better degradation ability for phenol. All of these can be as the candidate recipient strain in constructing genetic engineering strain using in the coking wastewater degration. In addition to, Strain A1 is not a common strain as usually we used, that means it can enrich microbial resources for phenol cracking. Through the environmental impact experiments, the environmental conditions of strain D2 have been optimized in the process of degradation of phenol. It provides data as reference for coking wastewater treatment by activated sludge.
引文
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