WO2022087870A1 - 一种根瘤菌及其在降解水环境中壬基酚的应用 - Google Patents

一种根瘤菌及其在降解水环境中壬基酚的应用 Download PDF

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WO2022087870A1
WO2022087870A1 PCT/CN2020/124251 CN2020124251W WO2022087870A1 WO 2022087870 A1 WO2022087870 A1 WO 2022087870A1 CN 2020124251 W CN2020124251 W CN 2020124251W WO 2022087870 A1 WO2022087870 A1 WO 2022087870A1
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nonylphenol
rhizobia
rhizobium
water environment
sewage
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PCT/CN2020/124251
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French (fr)
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黄开龙
张徐祥
杨庆
任洪强
叶林
阮在高
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南京江岛环境科技研究院有限公司
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    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F3/00—Biological treatment of water, waste water, or sewage
    • C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
    • C—CHEMISTRY; METALLURGY
    • C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
    • C02F2101/00—Nature of the contaminant
    • C02F2101/30—Organic compounds
    • C02F2101/34—Organic compounds containing oxygen
    • C02F2101/345—Phenols

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  • the invention relates to a rhizobia, and also relates to the application of the above-mentioned rhizobia in degrading nonylphenol in water.
  • the printing and dyeing industry is a major user of industrial water and wastewater in China. According to incomplete statistics, the discharge of printing and dyeing wastewater in China is about 3 to 4 million m 3 /d, accounting for about 35% of the total industrial wastewater discharge. For a long time, printing and dyeing wastewater has become a difficulty in the field of industrial wastewater treatment due to its high concentration of organic matter, high salt content, and difficult biochemical degradation of organic matter.
  • Nonylphenol ethoxylates are widely used as textile treatment auxiliaries and are important industrial nonionic surfactants.
  • concentration range of NPEO in textile wastewater is as high as 450-5680 ⁇ g/L.
  • NPEO is formed by the polymerization of NP and ethylene oxide.
  • the instability of NPEOs is related to its polyethoxylate chain composed of 1-20 ethoxy units. After entering the environment, it can be converted into NP and NP through various pathways.
  • (1-3) EO and NP (1-3) EC these substances have a certain estrogenic effect and are toxic to organisms in water, so they have received extensive attention.
  • Nonylphenol is one of the main products of NPEOs, which has estrogenic effects, neurotoxicity, immunotoxicity and reproductive toxicity. Its half-life in the environment varies from several days to one hundred days. NPs in the environment are widely distributed and have bioconcentration, and the cost of processing them by physical adsorption and chemical photolysis is high, so they are not suitable for large-scale applications. Biodegradation is a fast and efficient method for removing NPs in environmental media because of its low cost and environmental protection. Most of the reported microorganisms (Pseudomonas stutzeri, Leclercia adecarboxylata, etc.) only have a certain removal effect on low concentrations of NPs.
  • the degradation rate is 60%-75%, when the NP concentration is 50mg/L, the degradation rate is 20%-40%, and when the NP concentration is 100mg/L, the degradation rate is only 10%-20%. %, and residual NPs can still accumulate into the organism in the environment.
  • Purpose of the invention Aiming at the problems of low treatment concentration, low removal rate and slow degradation rate of nonylphenol in the microbial treatment of water bodies in the prior art, the present invention provides a rhizobia, which can quickly and efficiently degrade high-concentration in sewage. Nonylphenol.
  • Rhizobia described in the present invention is named as Rhizobium sp. NPLJ-2, classified as Rhizobium sp., and the strain number is NPLJ-2, which has been preserved in China in Wuhan, China Type Culture Collection, the deposit number is CCTCC M 2020603, and the deposit date is October 19, 2020.
  • strain-Rhizobium sp. NPLJ-2 grows well in LB medium under aerobic conditions at 30°C, the colony is round, the diameter is 0.2-1mm, the colony color is white, opaque, and the surface is dry , Gram stain was negative.
  • the technical problem to be solved by the present invention is to provide the application of the above-mentioned rhizobia in degrading nonylphenol in water environment.
  • the application is as follows: inoculating rhizobia in the expansion medium, aerobic cultivation at 28-30° C. for 24-48 hours, and obtaining the NPLJ-2 expansion product after culturing. Calculated by volume fraction, the inoculation amount of rhizobia is 1 % ⁇ 3%; the expanded culture product is inoculated into the sewage containing nonylphenol for treatment.
  • the inoculation amount of Rhizobium sp. NPLJ-2 in the sewage was 3 ⁇ to 5 ⁇ (mass fraction), and the mass concentration of nonylphenol in the sewage was not less than 100mg/L.
  • the temperature of the sewage containing nonylphenol is 25 ⁇ 30°C, and the pH value is 7.0 ⁇ 7.5. Under these conditions, it is beneficial to accelerate the degradation rate of nonylphenol by Rhizobium sp. NPLJ-2.
  • the rhizobia of the present invention can achieve a biodegradation rate of more than 80% to nonylphenol with an initial concentration of 100 mg/L in the water body under aerobic conditions, and also effectively shorten the biodegradation time.
  • the nonylphenol is directly converted and removed from the water body, thereby effectively reducing the concentration of COD in the water; the present invention utilizes biological methods to rapidly and efficiently degrade high-concentration nonylphenol in sewage, so the treatment of high-concentration environmental hormone-nonylphenol in sewage significant.
  • Fig. 1 is the phylogenetic tree of Rhizobium sp. NPLJ-2 of the present invention
  • Fig. 2 is the degradation rate diagram of rhizobia (Rhizobium sp.) NPLJ-2 to nonylphenol and COD in sewage of the present invention.
  • Rhizobium sp. NPLJ-2 is obtained by screening from the activated sludge in the aerobic section of a landfill leachate biological treatment in Nanjing.
  • step (3) Take out the sludge obtained in step (2), dilute the sludge in a gradient manner, apply it to an inorganic salt medium containing nonylphenol, and statically culture it at 30°C for 3-5 days to obtain a single colony ;
  • the mass concentration of nonylphenol in the inorganic salt medium is not less than 100 mg/L;
  • step (3) After picking out the single colony in step (3) for streaking and purification, streak it on the slant for preservation to obtain a pure strain.
  • the medium used for the expanded culture of pure strain is one of LB medium, inorganic salt medium or beef extract peptone medium.
  • the LB medium includes components of the following quality: each liter of water contains 5.0 g of yeast powder, 10 g of sodium chloride and 10 g of peptone, and the pH of the medium is 7.0-7.5.
  • the inorganic salt culture medium includes components of the following quality: 100 mg of nonylphenol, 0.262 g of ammonium sulfate and 0.025 g of disodium hydrogen phosphate per liter of water, and the pH of the culture medium is 7.0 to 7.5.
  • the beef extract peptone medium includes the following components: each liter of water contains 3 g of beef, 10 g of peptone and 5 g of sodium chloride, and the pH of the medium is 7.2-7.6.
  • NPLJ-2 obtained in step (4) (the inoculation amount is 1% to 3% (volume fraction)) in the expansion medium for expansion culture, and aerobic culture at 28 to 30 ° C 24-48h, the expanded product of NPLJ-2 was obtained after culturing.
  • the present invention selects NPLJ-2 with the best degradation effect and the fastest growth rate.
  • NPLJ-2 was identified as Rhizobium sp.
  • Rhizobium sp. NPLJ-2 grows well in LB medium under aerobic conditions at 30°C, the colonies are round, 0.2-1mm in diameter, white in color, opaque, dry on the surface, gram Staining was negative.
  • NPLJ-2 sequencing results were submitted to GenBank for BLAST comparison. From the BLAST results, the sequences of strains with more than 99% similarity to the NPLJ-2 base sequence were selected, and multiple serial alignments were performed by Clustal W, and MAGE was used to construct a phylogenetic tree to obtain NPLJ -2 and Rhizobium sp.strain k13 (accession number MN662624.1) have the closest relationship, so strain NPLJ-2 was identified as Rhizobium.
  • the rhizobium of the present invention (Rhizobium sp.) NPLJ-2 measures the degradation rate of pollutant nonylphenol, and the concrete steps are as follows:
  • step (2) take the bacterial cells obtained in step (1) according to the inoculum amount of 3 ⁇ (mass fraction), disperse, add to the simulated nonylphenol wastewater, the mass concentration of nonylphenol in the water is 100mg/L;
  • the treatment group of NPLJ-2 was used as a control;
  • step (3) put the control group and the treatment group in the step (2) into a shaker, and under aerobic conditions, shake the reaction at 180rpm and 28°C;
  • NPLJ-2 can efficiently degrade nonylphenol in water. 100mg/L nonylphenol degrades rapidly within 24h. With the decrease of nonylphenol concentration, the degradation rate slows down. After 48h, nonylphenol degrades rapidly. The removal rate reaches 80%. While the concentration of nonylphenol in the water body decreased, the concentration of COD in the sewage also decreased significantly, indicating that Rhizobium sp. NPLJ-2 can directly discharge nonylphenol from the water body, so that the nonylphenol can be completely removed from the water.
  • the present invention obtains Rhizobium sp. NPLJ-2 which has high removal efficiency for nonylphenol after step-by-step domestication of the activated sludge in the aerobic section of the landfill leachate.
  • the biodegradation rate of 100 mg/L nonylphenol by the rhizobia of the present invention can reach more than 80% under aerobic conditions, and the biodegradation time is significantly shortened; at the same time, the nonylphenol can be directly removed from the water body, thereby effectively reducing the The concentration of COD in water, the rhizobia of the present invention has important application value in the bioremediation of nonylphenol-contaminated water bodies.

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Abstract

一种根瘤菌，命名为根瘤菌(Rhizobium sp.)NPLJ-2，于2020年10月19日保藏于中国典型培养物保藏中心，保藏编号为CCTCC M 2020603。该根瘤菌在降解水环境中壬基酚的应用。该根瘤菌在好氧条件下对水体中初始浓度为100mg/L的壬基酚的生物降解率可达到80％以上，并且还有效缩短了生物降解时间，同时还有效降低了污水中由壬基酚贡献的COD；利用生物法快速高效降解污水中的壬基酚，对污水中高浓度环境激素-壬基酚的处理具有重要意义。

Description

一种根瘤菌及其在降解水环境中壬基酚的应用 技术领域
本发明涉及一种根瘤菌，还涉及上述根瘤菌在降解水中壬基酚的应用。
背景技术
印染行业是我国工业用水大户和废水排放大户，据不完全统计，我国印染废水的排放量约为300～400万m 3/d，约占整个工业废水排放量的35％。长期以来，印染废水由于有机物浓度高、含盐量高、有机物难生化降解，成为工业废水治理领域的难点。
壬基酚乙氧基化物(NPEOs)广泛用于纺织品处理助剂，是重要的工业非离子表面活性剂。NPEO在纺织废水中的浓度范围高达450～5680μg/L。NPEO由NP和环氧乙烷聚合生成，NPEOs的不稳定性与其由1-20个乙氧基单元组成的聚乙氧基化物链有关，其进入环境后通过多种途径可转化成NP、NP(1～3)EO和NP(1～3)EC，这些物质由于具有一定的雌激素效应，会对水体中生物产生毒性，因此受到广泛关注。
壬基酚(NP)是NPEOs的主要产物之一，其具有雌激素效应、神经毒性、免疫毒性及生殖毒性等，在环境中的半衰期在几天到一百天不等。环境中的NP分布范围广，具有生物富集性，用物理吸附和化学光解法对其进行处理时成本较高，因此不适合大规模应用。生物降解法因其成本低、绿色环保，是一种快速高效去除环境介质中NP的方法，目前已报道的微生物(Pseudomonas stutzeri、Leclercia adecarboxylata等)大多只对低浓度的NP有一定的去除效果，NP浓度为10mg/L以下时，降解率为60％～75％，NP浓度为50mg/L时，降解率为20％～40％，NP浓度为100mg/L时，降解率只有10％～20％，并且残余的NP仍可在环境中不断累积进入生物体。
发明内容
发明目的：本发明针对现有技术中微生物处理水体中壬基酚存在处理浓度低、去除率低以及降解速率慢等问题，提供了一种根瘤菌，该根瘤菌能够快速高效降解污水中高浓度的壬基酚。
技术方案：本发明所述的根瘤菌命名为根瘤菌(Rhizobium sp.)NPLJ-2，分类命名为根瘤菌(Rhizobium sp.)，菌株号为NPLJ-2，现已保藏于位于中国武汉的中国典型培养物保藏中心，保藏编号为CCTCC M 2020603，保藏日期为2020年10月19日。
上述菌株-根瘤菌(Rhizobium sp.)NPLJ-2，在LB培养基中，30℃好氧条件下生长良好，菌落呈圆形，直径大小为0.2～1mm，菌落颜色为白色，不透明，表面干燥，革兰氏染色呈阴性。
本发明还要解决的技术问题是提供上述根瘤菌在降解水环境中壬基酚的应用。
所述的应用为：将根瘤菌接种于扩大培养基中，于28～30℃下好氧培养24～48h，培养后得到NPLJ-2扩培产物，按体积分数算，根瘤菌接种量为1％～3％；将扩培产物接种于含壬基酚的污水中进行处理。
其中，污水中根瘤菌(Rhizobium sp.)NPLJ-2的接种量为3‰～5‰(质量分数)，污水中壬基酚的质量浓度为不低于100mg/L。
菌体应用时，含壬基酚的污水的温度为25～30℃，pH值为7.0～7.5，此条件下有利于加快根瘤菌(Rhizobium sp.)NPLJ-2对壬基酚的降解速率。
有益效果：本发明的根瘤菌在好氧条件下对水体中初始浓度为100mg/L的壬基酚的生物降解率可达到80％以上，并且还有效缩短了生物降解时间，同时通过将水体中的壬基酚直接转化并从水体中去除，从而有效降低水中COD的浓度；本发明利用生物法能够快速高效降解污水中高浓度的壬基酚，因此对污水中高浓度环境激素-壬基酚的处理具有重要意义。
附图说明
图1为本发明根瘤菌(Rhizobium sp.)NPLJ-2的系统发育树；
图2为本发明根瘤菌(Rhizobium sp.)NPLJ-2对壬基酚及污水中COD的降解速率图。
具体实施方式
以下结合附图和具体实施例对本发明的技术方案做进一步说明。
本发明中，是从南京某垃圾渗滤液生物处理好氧段的活性污泥中筛选获得根瘤菌(Rhizobium sp.)NPLJ-2。
具体过程如下：
(1)从南京某垃圾焚烧厂渗滤液生物处理段采集好氧段活性污泥；
(2)将得到的泥水混合物取250mL盛装于锥形瓶中，在磁力搅拌器上常温搅拌，每天向其中加入100mg壬基酚；每次加药前倒掉上部分上清液(检测上清液中是否含有目标污染物-壬基酚)，然后用自来水补足，如此驯化直至溶液中检测不到目标污染物，则完成驯化；
(3)取出步骤(2)中得到的污泥，将污泥经过梯度稀释后，分别涂布于含壬基酚的无机盐培养基中，30℃条件下静态培养3～5d，获得单菌落；无机盐培养基中壬基酚的质量浓度为不低于100mg/L；
(4)挑取步骤(3)中的单菌落划线纯化后，于斜面上划线保存，得到纯菌株。
纯菌株的扩大培养采用的培养基为LB培养基、无机盐培养基或牛肉膏蛋白胨培养 基中的一种。
LB培养基包括如下质量的组分：每升水中含酵母粉5.0g、氯化钠10g和蛋白胨10g，培养基的pH＝7.0～7.5。
无机盐培养基包括如下质量的组分：每升水中含壬基酚100mg、硫酸铵0.262g和磷酸氢二钠0.025g，培养基的pH＝7.0～7.5。
牛肉膏蛋白胨培养基包括如下质量的组分：每升水中含牛肉3g、蛋白胨10g和氯化钠5g，培养基的pH＝7.2～7.6。
将步骤(4)得到的根瘤菌(Rhizobium sp.)NPLJ-2接种(接种量为1％～3％(体积分数))于扩大培养基中进行扩培，于28～30℃下好氧培养24～48h，培养后得到NPLJ-2的扩培产物。
对上述筛选的菌种(步骤(4)得到的纯菌株)进行鉴定。
本发明筛选得到降解效果最佳、生长速度最快的NPLJ-2。其中，经鉴定，NPLJ-2为根瘤菌(Rhizobium sp.)。
根瘤菌(Rhizobium sp.)NPLJ-2，在LB培养基中，30℃好氧条件下生长良好，菌落呈圆形，直径大小0.2～1mm，菌落颜色为白色，不透明，表面干燥，革兰氏染色呈阴性。
通过PCR扩增和Sanger法测序，得到NPLJ-2菌株16S rRNA全序列如下(SEQ ID NO.1)：
Figure PCTCN2020124251-appb-000001
NPLJ-2测序结果提交GenBank进行BLAST比较，从BLAST结果中挑选与NPLJ-2 碱基序列相似度99％以上的菌株序列，通过Clustal W进行多重系列比对，并利用MAGE构建系统发育树得到NPLJ-2与根瘤菌Rhizobium sp.strain k13(登录号为MN662624.1)，亲缘关系最近，因此鉴定菌株NPLJ-2为根瘤菌。
本发明根瘤菌(Rhizobium sp.)NPLJ-2对污染物壬基酚降解速率进行测定，具体步骤如下：
(1)将NPLJ-2经过扩大培养后菌液于6000rpm下离心10min，倒掉上清液，得到菌体；
(2)取步骤(1)中得到的菌体按3‰(质量分数)接种量，打散，加入模拟壬基酚废水中，水中壬基酚的质量浓度为100mg/L；并以不加NPLJ-2的处理组做对照；
(3)将步骤(2)中的对照组与处理组放入摇床中，好氧条件下，在180rpm，28℃下振荡反应；
(4)定时取样检测步骤(3)中处理组与对照组中壬基酚的去除率及水体中的COD浓度，结果见图2。
如图2所示，NPLJ-2能够高效降解水中的壬基酚，100mg/L的壬基酚在24h内快速降解，随着壬基酚浓度的降低，降解速率变缓，48h后壬基酚去除率达到80％。水体中壬基酚浓度降低的同时，污水中的COD浓度也显著降低，表明根瘤菌(Rhizobium sp.)NPLJ-2可将壬基酚直接从水体排出，从而使壬基酚从水中完全去除。
本发明通过对垃圾渗滤液好氧段的活性污泥逐级驯化后，获得对壬基酚有高效去除效率的根瘤菌(Rhizobium sp.)NPLJ-2。本发明的根瘤菌在好氧条件下对100mg/L壬基酚的生物降解率可达到80％以上，且显著缩短了生物降解时间；同时还能将壬基酚直接从水体去除，从而有效降低水中COD的浓度，本发明根瘤菌在壬基酚污染水体的生物修复方面具有重要的应用价值。

Claims (8)

  1. 一种根瘤菌，其特征在于：命名为根瘤菌(Rhizobium sp.)NPLJ-2，于2020年10月19日保藏于中国典型培养物保藏中心，保藏编号为CCTCC M 2020603。
  2. 权利要求1所述的根瘤菌在降解水环境中壬基酚的应用。
  3. 根据权利要求2所述的根瘤菌在降解水环境中壬基酚的应用，其特征在于：根瘤菌降解水体中的壬基酚在好氧条件下进行。
  4. 根据权利要求2所述的根瘤菌在降解水环境中壬基酚的应用，其特征在于：将根瘤菌接种于扩大培养基中，于28～30℃下好氧培养24～48h，培养后得到NPLJ-2扩培产物，按体积分数算，根瘤菌接种量为1％～3％；将扩培产物接种于含壬基酚的污水中进行处理。
  5. 根据权利要求4所述的根瘤菌在降解水环境中壬基酚的应用，其特征在于：按质量分数算，污水中根瘤菌的接种量为3‰～5‰。
  6. 根据权利要求4所述的根瘤菌在降解水环境中壬基酚的应用，其特征在于：污水中壬基酚的质量浓度不低于100mg/L。
  7. 根据权利要求4所述的根瘤菌在降解水环境中壬基酚的应用，其特征在于：菌体应用时，污水的温度为25℃～30℃，pH值为7.0～7.5。
  8. 根据权利要求4所述的根瘤菌在降解水环境中壬基酚的应用，其特征在于：扩大培养基为LB培养基、无机盐培养基或牛肉膏蛋白胨培养基中的一种。
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