CN113025536B - A strain of Pseudomonas PR1 and its preparation method and application - Google Patents
A strain of Pseudomonas PR1 and its preparation method and application Download PDFInfo
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- C02F1/00—Treatment of water, waste water, or sewage
- C02F1/52—Treatment of water, waste water, or sewage by flocculation or precipitation of suspended impurities
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Abstract
Description
技术领域technical field
本发明属于废水处理技术领域,特别是涉及一株假单胞菌PR1及其制备方法和应用。The invention belongs to the technical field of wastewater treatment, and in particular relates to a strain of pseudomonas PR1 and its preparation method and application.
背景技术Background technique
有色金属资源是重要的战略资源,但是开采过程极易污染环境,甚至影响人们的身体健康。如铅在人和动物体内蓄积,不仅影响身体健康甚至会致命;锌过量摄入会造成肠胃不适,严重的对肝功能有一定程度的损害。Non-ferrous metal resources are important strategic resources, but the mining process can easily pollute the environment and even affect people's health. For example, the accumulation of lead in the body of humans and animals will not only affect the health of the body and even cause death; excessive intake of zinc will cause gastrointestinal discomfort, and seriously damage the liver function to a certain extent.
絮凝法在铅锌金属浮选选矿污水处理上被广泛应用。常见的絮凝剂包括无机絮凝剂和有机高分子絮凝剂,常见的无机絮凝剂虽然能够去除含有有色金属的废水中的有色金属离子,但是极易造成二次污染。利用微生物制成的絮凝剂虽然能够避免二次污染,但是难以平衡絮凝的效率。The flocculation method is widely used in the wastewater treatment of lead-zinc metal flotation beneficiation. Common flocculants include inorganic flocculants and organic polymer flocculants. Although common inorganic flocculants can remove non-ferrous metal ions in wastewater containing non-ferrous metals, they can easily cause secondary pollution. Although flocculants made of microorganisms can avoid secondary pollution, it is difficult to balance the efficiency of flocculation.
发明内容Contents of the invention
为了解决上述问题,本发明提供了一株假单胞菌PR1及其制备方法和应用。本发明提供的假单胞菌PR1能够高效地絮凝有色金属离子,絮凝得到的产物具有环保、能够生物降解的特性,能够有效避免有色金属离子的二次污染。In order to solve the above problems, the present invention provides a strain of Pseudomonas PR1 and its preparation method and application. The Pseudomonas PR1 provided by the invention can efficiently flocculate the colored metal ions, and the product obtained by the flocculation has the characteristics of environmental protection and biodegradability, and can effectively avoid the secondary pollution of the colored metal ions.
为了实现上述目的,本发明提供了如下技术方案:In order to achieve the above object, the present invention provides the following technical solutions:
本发明提供了一株假单胞菌(Pseudomonas sp.)PR1,所述假单胞菌PR1的保藏号为GDMCC NO:61255。The present invention provides a strain of Pseudomonas sp. PR1, and the preservation number of the Pseudomonas PR1 is GDMCC NO: 61255.
本发明还提供了上述假单胞菌PR1的培养方法,包括以下步骤:The present invention also provides a method for cultivating the above-mentioned Pseudomonas PR1, comprising the following steps:
将上述假单胞菌PR1置于种子培养基中进行种子培养得到种子培养液,将种子培养液接种于发酵培养基中进行发酵培养得到含有假单胞菌PR1的发酵培养液。The above-mentioned Pseudomonas PR1 is placed in the seed medium for seed culture to obtain a seed culture liquid, and the seed culture liquid is inoculated into a fermentation medium for fermentation culture to obtain a fermentation culture liquid containing Pseudomonas PR1.
本发明还提供了上述假单胞菌PR1或上述培养方法培养得到的假单胞菌PR1在絮凝有色金属离子中的应用。The present invention also provides the application of the above-mentioned Pseudomonas PR1 or the Pseudomonas PR1 cultured by the above-mentioned culture method in flocculating non-ferrous metal ions.
本发明提供了一种有色金属离子絮凝剂,所述絮凝剂包括:上述假单胞菌PR1或上述培养得到含有假单胞菌PR1的发酵培养液或上述培养得到的发酵培养液分离得到的上清液或对所述上清液进行提取得到的上清液提取物。The present invention provides a non-ferrous metal ion flocculant. The flocculant includes: the above-mentioned Pseudomonas PR1 or the above-mentioned fermentation culture liquid containing Pseudomonas PR1 obtained from the above-mentioned cultivation or the above-mentioned separation obtained from the fermentation culture liquid obtained from the above-mentioned cultivation. The supernatant or the supernatant extract obtained by extracting the supernatant.
优选的,所述分离的方法包括离心;所述离心的温度为0℃~4℃,转速为8000~12000r/min,离心的时间为15~20min。Preferably, the separation method includes centrifugation; the temperature of the centrifugation is 0°C-4°C, the rotation speed is 8000-12000r/min, and the centrifugation time is 15-20min.
优选的,所述提取的方法包括醇沉。Preferably, the extraction method includes alcohol precipitation.
优选的,所述醇沉包括以下步骤:将上清液与预冷的无水乙醇混合后冷藏静置,冷藏静置结束后进行离心,离心后舍弃上清液,将沉淀冷冻干燥,得到上清液提取物。Preferably, the alcohol precipitation includes the following steps: mixing the supernatant with pre-cooled absolute ethanol and then refrigerating and standing still, centrifuging after the refrigerating and standing, discarding the supernatant after centrifuging, and freeze-drying the precipitate to obtain the above Serum extract.
优选的,所述预冷的无水乙醇的温度为0℃~4℃,所述上清液与无水乙醇的体积比为1:(2~4);所述冷藏静置的温度为4℃,冷藏静置的时间为12~24h;沉淀分离时,离心的转速为6000~8000r/min,温度为0℃~4℃,离心的时间为10~15min。Preferably, the temperature of the pre-cooled absolute ethanol is 0° C. to 4° C., and the volume ratio of the supernatant to absolute ethanol is 1:(2 to 4); ℃, the time for refrigerating and standing is 12-24h; when the sedimentation is separated, the centrifugation speed is 6000-8000r/min, the temperature is 0℃-4℃, and the centrifugation time is 10-15min.
本发明提供了一种有色金属溶液的处理方法,所述处理方法包括以下步骤:将权利要求4~8任一项所述的絮凝剂与有色金属溶液混合。The invention provides a treatment method for non-ferrous metal solution, the treatment method comprising the following steps: mixing the flocculant according to any one of claims 4 to 8 with the non-ferrous metal solution.
优选的,所述絮凝剂与有色金属溶液混合的体积比为1:(40~50);Preferably, the volume ratio of the flocculant mixed with the non-ferrous metal solution is 1:(40-50);
所述混合后,包括搅拌和静置,所述搅拌包括第一搅拌和第二搅拌;After the mixing, including stirring and standing, the stirring includes the first stirring and the second stirring;
所述第一搅拌的转速为190~210r/min,时间为25~35s;The rotational speed of the first stirring is 190-210r/min, and the time is 25-35s;
所述第二搅拌的转速为50~70r/min,时间为4~5min;The rotational speed of the second stirring is 50-70r/min, and the time is 4-5min;
所述静置的时间为5~10min。The standing time is 5-10 minutes.
本发明提供了一株假单胞菌(Pseudomonas sp.)PR1,所述假单胞菌PR1的保藏号为GDMCC NO:61255。本发明提供的假单胞菌PR1具有高效絮凝有色金属离子的作用。由实施例数据可知,向含有Pb2+和/或Zn2+的有色金属废水中,添加本发明提供的含有假单胞菌PR1的絮凝剂能够起到高效絮凝的作用,对Pb2+的絮凝率为99.35%;对Zn2+的絮凝率为55.90%;对Pb2+和Zn2+的混合废水中,Pb2+的絮凝率为92.94%,对Zn2+的絮凝率为80.67%;均高于聚氯化铝絮凝剂(PAC)的絮凝率。The present invention provides a strain of Pseudomonas sp. PR1, and the preservation number of the Pseudomonas PR1 is GDMCC NO: 61255. The Pseudomonas PR1 provided by the invention has the function of efficiently flocculating the colored metal ions. As can be seen from the data of the examples, in the non-ferrous metal wastewater containing Pb 2+ and/or Zn 2+ , adding the flocculant containing Pseudomonas PR1 provided by the present invention can play the role of high - efficiency flocculation. The flocculation rate is 99.35%; the flocculation rate for Zn 2+ is 55.90%; for the mixed wastewater of Pb 2+ and Zn 2+ , the flocculation rate for Pb 2+ is 92.94%, and the flocculation rate for Zn 2+ is 80.67% ; All higher than polyaluminum chloride flocculant (PAC) flocculation rate.
生物保藏说明Biological Deposit Instructions
假单胞菌(Pseudomonas sp.)PR1,于2020年10月29日保藏在广东省微生物物种保藏中心(GDMCC),保藏地为广州市先烈中路100号大院59号楼5楼,保藏编号为GDMCC NO:61255。Pseudomonas sp. PR1 was deposited in the Guangdong Provincial Microbiological Species Collection Center (GDMCC) on October 29, 2020, at the 5th Floor, Building 59, Compound, No. 100 Xianlie Middle Road, Guangzhou, and the preservation number is GDMCC NO: 61255.
附图说明Description of drawings
图1为上清液提取物状态的絮凝剂;Fig. 1 is the flocculant of supernatant liquid extract state;
图2为假单胞菌(Pseudomonas sp.)PR1的平板菌落形态图;Fig. 2 is the plate colony morphology figure of Pseudomonas (Pseudomonas sp.) PR1;
图3为假单胞菌(Pseudomonas sp.)PR1的系统发育树;Fig. 3 is the phylogenetic tree of Pseudomonas sp. PR1;
图4为含有假单胞菌PR1的微生物絮凝剂及普通PAC絮凝剂处理含有铅、锌离子的模拟废水的絮凝率;Fig. 4 is the flocculation rate of the microbial flocculant containing Pseudomonas PR1 and the common PAC flocculant treating the simulated wastewater containing lead and zinc ions;
图5为含有假单胞菌PR1的微生物絮凝剂处理模拟铅锌混合废水的絮凝率。Figure 5 shows the flocculation rate of simulated lead-zinc mixed wastewater treated with microbial flocculants containing Pseudomonas PR1.
具体实施方式Detailed ways
本发明提供了一株假单胞菌(Pseudomonas sp.)PR1,所述假单胞菌PR1的保藏号为GDMCC NO:61255。本发明提供的假单胞菌PR1的菌落形态如图2所示,菌落颜色为微黄色,菌落四周边缘呈凹凸不平状,表面有些微光泽,较湿润;系统发育树如图3所示;假单胞菌PR1的测序序列为SEQ ID NO:1。本发明提供的假单胞菌PR1具有高效絮凝有色金属离子(铅离子、锌离子)的技术效果。The present invention provides a strain of Pseudomonas sp. PR1, and the preservation number of the Pseudomonas PR1 is GDMCC NO: 61255. The colony form of Pseudomonas PR1 provided by the present invention is as shown in Figure 2, and the color of the colony is yellowish, and the edges around the colony are uneven, and the surface is slightly glossy and moist; the phylogenetic tree is as shown in Figure 3; The sequencing sequence of Monas PR1 is SEQ ID NO:1. The Pseudomonas PR1 provided by the invention has the technical effect of efficiently flocculating non-ferrous metal ions (lead ions, zinc ions).
本发明还提供了上述假单胞菌PR1的培养方法,包括以下步骤:The present invention also provides a method for cultivating the above-mentioned Pseudomonas PR1, comprising the following steps:
将上述假单胞菌PR1置于种子培养基中进行种子培养得到种子培养液,将种子培养液接种于发酵培养基中进行发酵培养得到含有假单胞菌PR1的发酵培养液。本发明提供的培养方法有培养基配方简单、原料来源广、经济廉价的优势。The above-mentioned Pseudomonas PR1 is placed in the seed medium for seed culture to obtain a seed culture liquid, and the seed culture liquid is inoculated into a fermentation medium for fermentation culture to obtain a fermentation culture liquid containing Pseudomonas PR1. The cultivation method provided by the invention has the advantages of simple medium formula, wide sources of raw materials, and low cost.
在本发明中,所述种子培养基优选包括以下成分:葡萄糖10.0g/L、磷酸二氢钾0.5g/L、磷酸氢二钾5.0g/L、硫酸铵0.2g/L、氯化钠0.1g/L、尿素0.5g/L、酵母膏0.5g/L和硫酸镁0.2g/L;所述种子培养基的pH值优选为7。本发明提供的种子培养基方便易得,还能够有效促进菌株的生长繁殖。In the present invention, the seed medium preferably includes the following components: glucose 10.0 g/L, potassium dihydrogen phosphate 0.5 g/L, dipotassium hydrogen phosphate 5.0 g/L, ammonium sulfate 0.2 g/L, sodium chloride 0.1 g/L, urea 0.5g/L, yeast extract 0.5g/L and magnesium sulfate 0.2g/L; the pH value of the seed medium is preferably 7. The seed culture medium provided by the invention is convenient and easy to obtain, and can effectively promote the growth and reproduction of bacterial strains.
在本发明中,所述种子培养过程中,温度优选为30℃,培养的时间优选为22~24h。在上述此培养温度和时间下,培养得到的菌株生物酶活性较高。In the present invention, during the seed cultivation process, the temperature is preferably 30° C., and the cultivation time is preferably 22-24 hours. Under the above-mentioned cultivation temperature and time, the biological enzyme activity of the cultured bacterial strain is relatively high.
在本发明中,所述发酵培养基优选包括以下组分:葡萄糖10.0份、磷酸二氢钾0.5份、磷酸氢二钾5.0份、硫酸铵0.2份、氯化钠0.1份、尿素0.5份、酵母膏0.5份和硫酸镁0.2份,进一步优选为葡萄糖10.0g、磷酸二氢钾0.5g、磷酸氢二钾5.0g、硫酸铵0.2g、氯化钠0.1g、尿素0.5g、酵母膏0.5g和硫酸镁0.2g;所述发酵培养基的pH值优选为7。本发明提供的发酵培养基组成简单,成本低廉,能够有效促进菌的增殖。In the present invention, the fermentation medium preferably includes the following components: 10.0 parts of glucose, 0.5 parts of potassium dihydrogen phosphate, 5.0 parts of dipotassium hydrogen phosphate, 0.2 parts of ammonium sulfate, 0.1 parts of sodium chloride, 0.5 parts of urea, yeast 0.5 parts of cream and 0.2 parts of magnesium sulfate, more preferably glucose 10.0g, potassium dihydrogen phosphate 0.5g, dipotassium hydrogen phosphate 5.0g, ammonium sulfate 0.2g, sodium chloride 0.1g, urea 0.5g, yeast extract 0.5g and Magnesium sulfate 0.2g; The pH value of the fermentation medium is preferably 7. The fermentation medium provided by the invention has simple composition, low cost and can effectively promote the proliferation of bacteria.
在本发明中,所述发酵培养过程中,种子培养液的接种量优选为2.5%~5%,温度优选为30℃,培养时间优选为24~48h,转速优选为180r·min-1。在上述培养温度和时间下,培养得到的菌株生物酶活性较高,对铅离子和锌离子的絮凝率相对较高。In the present invention, during the fermentation culture process, the inoculation amount of the seed culture solution is preferably 2.5%-5%, the temperature is preferably 30°C, the culture time is preferably 24-48h, and the rotation speed is preferably 180r·min -1 . Under the above-mentioned culture temperature and time, the cultured strains have higher bioenzyme activity and relatively higher flocculation rates for lead ions and zinc ions.
本发明还提供了上述假单胞菌PR1或上述培养方法培养得到的假单胞菌PR1在絮凝有色金属离子中的应用。在本发明中,所述有色金属离子优选包括铅离子和/或锌离子。本发明提供的应用能够有效避免有色金属离子造成二次污染,还有絮凝率高和成本低廉的优势。The present invention also provides the application of the above-mentioned Pseudomonas PR1 or the Pseudomonas PR1 cultured by the above-mentioned culture method in flocculating non-ferrous metal ions. In the present invention, the colored metal ions preferably include lead ions and/or zinc ions. The application provided by the invention can effectively avoid secondary pollution caused by non-ferrous metal ions, and has the advantages of high flocculation rate and low cost.
本发明提供了一种有色金属离子絮凝剂,所述絮凝剂包括:上述假单胞菌PR1或上述培养得到含有假单胞菌PR1的发酵培养液或上述培养得到的发酵培养液分离得到的上清液或对所述上清液进行提取得到的上清液提取物。本发明提供絮凝剂不仅具有极高的絮凝铅离子和锌离子的效率和低廉的成本,在使用后不会对人体健康和生态环境造成危害,产生二次污染,残余物不具备“三致”效应,而且产生的絮体能够进行生物降解。The present invention provides a non-ferrous metal ion flocculant. The flocculant includes: the above-mentioned Pseudomonas PR1 or the above-mentioned fermentation culture liquid containing Pseudomonas PR1 obtained from the above-mentioned cultivation or the above-mentioned separation obtained from the fermentation culture liquid obtained from the above-mentioned cultivation. The supernatant or the supernatant extract obtained by extracting the supernatant. The flocculant provided by the present invention not only has extremely high efficiency of flocculating lead ions and zinc ions and low cost, but also does not cause harm to human health and ecological environment after use, and produces secondary pollution, and the residue does not have "three causes" effect, and the resulting flocs are biodegradable.
在本发明中,当絮凝剂为上清液时,所述上清液的分离方法优选包括离心;所述离心的温度优选为0℃~4℃,转速优选为8000~12000r/min,离心的时间优选为15~20min。In the present invention, when the flocculant is the supernatant, the separation method of the supernatant preferably includes centrifugation; the temperature of the centrifugation is preferably 0° C. to 4° C., and the rotation speed is preferably 8000 to 12000 r/min. The time is preferably 15 to 20 minutes.
在本发明中,当絮凝剂为上清液提取物时,所述上清液提取物的提取的方法优选包括醇沉。在本发明中,所述醇沉优选包括以下步骤:将上清液与预冷的无水乙醇混合后冷藏静置,冷藏静置结束后进行离心,离心后舍弃上清液,将沉淀冷冻干燥,得到上清液提取物。在本发明中,所述上清液提取物在实际应用中优选进行冷冻干燥处理。所述预冷的无水乙醇的温度优选为0℃~4℃,所述上清液与无水乙醇的体积比优选为1:(2~4);所述冷藏静置的温度优选为4℃,冷藏静置的时间优选为12~24h;沉淀分离时,离心的转速优选为6000~8000r/min,温度优选为0℃~4℃,离心的时间优选为10~15min。本发明提供的絮凝剂的制备方法简单易操作,能够有效提高絮凝剂中的有效成分,提高絮凝剂的使用效果。In the present invention, when the flocculant is a supernatant extract, the extraction method of the supernatant extract preferably includes alcohol precipitation. In the present invention, the alcohol precipitation preferably includes the following steps: mixing the supernatant with pre-cooled absolute ethanol and then refrigerating and standing still, centrifuging after the refrigerating and standing, discarding the supernatant after centrifuging, and freeze-drying the precipitate , to obtain the supernatant extract. In the present invention, the supernatant extract is preferably freeze-dried in practical applications. The temperature of the precooled absolute ethanol is preferably 0°C to 4°C, and the volume ratio of the supernatant to absolute ethanol is preferably 1:(2 to 4); the temperature of the cold storage is preferably 4 °C, the time for refrigerated standing is preferably 12-24h; during precipitation separation, the centrifugation speed is preferably 6000-8000r/min, the temperature is preferably 0°C-4°C, and the centrifugation time is preferably 10-15min. The preparation method of the flocculant provided by the invention is simple and easy to operate, can effectively increase the active ingredients in the flocculant, and improve the use effect of the flocculant.
本发明还提供了一种有色金属溶液的处理方法,所述处理方法包括以下步骤:将上述絮凝剂与有色金属溶液。在本发明中;所述絮凝剂与有色金属溶液混合的体积比优选为1:(40~50);所述混合后,优选包括搅拌和静置,所述搅拌优选包括第一搅拌和第二搅拌。The present invention also provides a treatment method for non-ferrous metal solution, which includes the following steps: mixing the above-mentioned flocculant with the non-ferrous metal solution. In the present invention; the volume ratio of the flocculant mixed with the non-ferrous metal solution is preferably 1:(40-50); after the mixing, it preferably includes stirring and standing, and the stirring preferably includes the first stirring and the second Stir.
所述第一搅拌的转速优选为190~210r/min,进一步优选为200r/min,时间优选为25~35s,进一步优选为30s;The rotation speed of the first stirring is preferably 190-210r/min, more preferably 200r/min, and the time is preferably 25-35s, more preferably 30s;
所述第二搅拌的转速优选为50~70r/min,进一步优选为60r/min,时间优选为4~5min,进一步优选为4min;The rotation speed of the second stirring is preferably 50-70r/min, more preferably 60r/min, and the time is preferably 4-5min, more preferably 4min;
所述静置的时间优选为5~10min,进一步优选为5min。本发明提供的使用方法简单易操作,能够有效提高絮凝率。The standing time is preferably 5-10 minutes, more preferably 5 minutes. The usage method provided by the invention is simple and easy to operate, and can effectively improve the flocculation rate.
为了进一步说明本发明,下面结合附图和实施例对本发明提供的一株假单胞菌PR1及其制备方法和应用进行详细地描述,但不能将它们理解为对本发明保护范围的限定。In order to further illustrate the present invention, a strain of Pseudomonas PR1 provided by the present invention and its preparation method and application are described in detail below in conjunction with the accompanying drawings and examples, but they should not be construed as limiting the protection scope of the present invention.
实施例1Example 1
细菌的筛选Screening of bacteria
(1)培养基的制备:(1) Preparation of medium:
(A)分离培养基(A) Separation medium
牛肉膏蛋白胨(g/L):牛肉膏3.0、蛋白胨10.0、氯化钠5.0和琼脂20.0,pH=7.0;Beef extract peptone (g/L): beef extract 3.0, peptone 10.0, sodium chloride 5.0 and agar 20.0, pH=7.0;
LB培养基(g/L):蛋白胨10.0、酵母膏5.0、氯化钠6.0和琼脂20.0,pH=7.0。LB medium (g/L): peptone 10.0, yeast extract 5.0, sodium chloride 6.0 and agar 20.0, pH=7.0.
(B)发酵培养基(g/L):葡萄糖10.0、磷酸二氢钾0.5、磷酸氢二钾5.0、硫酸铵0.2、氯化钠0.1、尿素0.5、酵母膏0.5和硫酸镁0.2,pH=7。(B) Fermentation medium (g/L): glucose 10.0, potassium dihydrogen phosphate 0.5, dipotassium hydrogen phosphate 5.0, ammonium sulfate 0.2, sodium chloride 0.1, urea 0.5, yeast extract 0.5 and magnesium sulfate 0.2, pH=7 .
(C)种子培养基(g/L):葡萄糖10.0、磷酸二氢钾0.5、磷酸氢二钾5.0、硫酸铵0.2、氯化钠0.1、尿素0.5、酵母膏0.5和硫酸镁0.2,pH=7。(C) Seed medium (g/L): glucose 10.0, potassium dihydrogen phosphate 0.5, dipotassium hydrogen phosphate 5.0, ammonium sulfate 0.2, sodium chloride 0.1, urea 0.5, yeast extract 0.5 and magnesium sulfate 0.2, pH=7 .
上述培养基经120℃灭菌30min,放置24h后,若无污染方可使用。The above-mentioned culture medium was sterilized at 120°C for 30 minutes and left for 24 hours before use if there is no contamination.
(2)菌源准备:土壤采于桂林阳朔县思的村受铅锌污染土壤,在受污染的重、中、轻三个区域随机采集5~10cm土壤。(2) Bacterial source preparation: The soil was collected from lead-zinc-contaminated soil in Sidi Village, Yangshuo County, Guilin, and 5-10 cm of soil was randomly collected in heavily polluted, moderately and lightly polluted areas.
(3)将90mL无菌水装入带有少量玻璃珠的无菌锥形瓶中,再称取10g受铅锌污染土样加入其中,放入200r/min的摇床中水平震荡20min,使水与土壤充分混匀,将细菌分散,即成10-1土壤悬液。将10-1土壤悬液取出1mL放入装有9ml无菌水的离心管中,充分摇匀,即成10-2土壤悬液,并以同样的方法逐级稀释成(10-3)~(10-6)浓度梯度的土壤稀释液。(3) Put 90 mL of sterile water into a sterile conical flask with a small amount of glass beads, then weigh 10 g of lead-zinc-contaminated soil sample into it, put it into a shaker at 200 r/min and shake it horizontally for 20 min, so that Mix the water and soil thoroughly to disperse the bacteria to form a 10 -1 soil suspension. Take out 1mL of the 10 -1 soil suspension and put it into a centrifuge tube filled with 9ml of sterile water, shake it well to form a 10 -2 soil suspension, and dilute it step by step in the same way to (10 -3 )~ (10 -6 ) Soil dilution with concentration gradient.
(4)取10-3、10-4、10-5、10-6的土壤稀释液,用经灭菌移液枪取0.1mL稀释液放在平板分离培养基(牛肉膏蛋白胨培养基)的中心,后用涂布器涂布均匀,每个浓度梯度做三个平行样。涂布结束后将培养皿倒置,置于30℃恒温培养箱中培养72h。(4) Take 10 -3 , 10 -4 , 10 -5 , 10 -6 soil dilutions, use a sterilized pipette gun to take 0.1mL of the dilutions and put them on the plate separation medium (beef extract peptone medium) center, and spread evenly with a spreader, and make three parallel samples for each concentration gradient. After coating, the petri dish was inverted and placed in a constant temperature incubator at 30°C for 72 hours.
(5)将培养后生长出的单株菌落用接种环分别挑取少量细胞划线接种到平板培养基上,并于30℃恒温培养,培养72h,再次挑取单菌落划线并进行培养,观察菌落的形态特征是否相同,若发现有杂菌存在,则需进一步进行分离纯化,直至获得纯的菌落。将分离得到的纯菌株接种到试管斜面上,于30℃恒温培养,待长出菌落后放置于4℃冰箱下保存备用。筛选得到的菌为假单胞菌(Pseudomonas sp.)PR1,保藏编号为GDMCC NO:61255。(5) Use an inoculation loop to pick a small amount of cells from the individual colonies grown after cultivation and inoculate them on the plate medium, and culture them at a constant temperature of 30°C for 72 hours. Pick a single colony again and culture them. Observe whether the morphological characteristics of the colonies are the same. If there are miscellaneous bacteria, further separation and purification are required until pure colonies are obtained. The isolated pure strains were inoculated on the slant of the test tube, cultured at a constant temperature of 30°C, and stored in a refrigerator at 4°C after the bacteria grew out. The bacterium obtained by screening is Pseudomonas sp. PR1, and the preservation number is GDMCC NO: 61255.
将筛选得到的假单胞菌PR1接种到种子培养基,培养22h后,以5%(V/V)的接种量将种子液接种到发酵培养基中,置于温度30℃,转速为180r·min-1的振荡培养箱内振荡培育48h后得到发酵液。即为本发明提供的含有假单胞菌PR1的絮凝剂。The Pseudomonas PR1 obtained by screening was inoculated into the seed medium, and after cultivating for 22 hours, the seed solution was inoculated into the fermentation medium with an inoculation amount of 5% (V/V), placed at a temperature of 30° C., and a rotating speed of 180 r. The fermentation broth was obtained after 48 hours of shaking cultivation in a shaking incubator at min -1 . That is, the flocculant containing Pseudomonas PR1 provided by the present invention.
应用例1Application example 1
使用所制得的发酵培养液在0~4℃条件下以8000~12000r/min离心15~20min得到上清液。随后向上清液加入2~4倍体积的预冷的无水乙醇(0℃~4℃),在4℃冰箱静置12~24h,然后在0~4℃条件下以6000~8000r/min离心10~15min,舍弃上清液,将沉淀冷冻干燥,即得含有上清液提取物的絮凝剂,如图1所示。The prepared fermentation culture liquid is centrifuged at 8000-12000 r/min for 15-20 min under the condition of 0-4° C. to obtain a supernatant. Then add 2 to 4 times the volume of pre-cooled absolute ethanol (0°C to 4°C) to the supernatant, let it stand in a refrigerator at 4°C for 12 to 24 hours, and then centrifuge at 6000 to 8000r/min at 0 to 4°C After 10-15 minutes, the supernatant was discarded, and the precipitate was freeze-dried to obtain a flocculant containing the supernatant extract, as shown in Figure 1.
应用例2Application example 2
使用含有假单胞菌PR1的絮凝剂分别处理铅锌废水:制备Pb2+的浓度为30mg/L,Zn2 +的浓度为30mg/L,pH值为7(使用浓度为3%CaC12溶液调节)的两种模拟废水各200mL,放入250mL的烧杯中。量取5mL的絮凝剂加入废水样品中先以200r/min搅拌30s,然后以转速为60r/min搅拌4min后,静置5min,取液面下2cm左右处的水样,用电感耦合等离子质谱仪测其处理后水中的浓度,并计算絮凝率。对照组为添加了相同剂量的PAC絮凝剂。Use the flocculant containing Pseudomonas PR1 to process lead-zinc wastewater respectively: the concentration of preparing Pb 2+ is 30mg/L, the concentration of Zn 2+ is 30mg/L, and the pH value is 7 (use concentration is 3%CaCl 2 solution 200mL each of the two simulated waste waters for adjustment) were put into a 250mL beaker. Measure 5mL of flocculant and add it to the wastewater sample, first stir at 200r/min for 30s, then stir at 60r/min for 4min, then let it stand for 5min, take a water sample about 2cm below the liquid surface, and use inductively coupled plasma mass spectrometry Measure the concentration in the treated water and calculate the flocculation rate. The control group was added with the same dose of PAC flocculant.
经由以下公式计算铅和锌的絮凝率。The flocculation rates of lead and zinc were calculated via the following formulas.
T=(D-E)/E×100%T=(D-E)/E×100%
式中:In the formula:
D——空白组在ICP中测量的铅或锌的浓度值;D - the concentration of lead or zinc measured in the ICP of the blank group;
E——待测样品中在ICP中测量的铅或锌的浓度值;E - the concentration value of lead or zinc measured in ICP in the sample to be tested;
T——为样品相对于空白组中铅或锌溶液的絮凝率;T——is the flocculation rate of the sample relative to the lead or zinc solution in the blank group;
重复测定三次絮凝率,取三次数据的平均值,最后由公式计算出样品絮凝率。实验结果如表1和图4所示。Repeat the determination of the flocculation rate three times, take the average value of the three data, and finally calculate the flocculation rate of the sample by the formula. The experimental results are shown in Table 1 and Figure 4.
表1本发明提供的絮凝剂和PAC絮凝剂处理Pb2+、Zn2+废水的絮凝率(%)Table 1 flocculant provided by the present invention and PAC flocculant process Pb 2+ , the flocculation rate (%) of Zn 2+ waste water
由表1和图4可知,本发明提供的絮凝剂的絮凝铅离子和锌离子的技术效果优越,而且比PAC絮凝剂的絮凝效果更好。It can be seen from Table 1 and Figure 4 that the flocculant provided by the present invention has superior technical effect of flocculating lead ions and zinc ions, and is better than the flocculation effect of PAC flocculant.
应用例3Application example 3
使用含有假单胞菌PR1的絮凝剂处理铅锌混合废水:制备Pb2+的浓度为30mg/L、Zn2 +的浓度为10mg/L,pH值为7的混合废水,放入250mL的烧杯中。量取5mL的絮凝剂加入废水样品中先以200r/min搅拌30s,然后以转速为60r/min搅拌4min后,静置5min,取液面下2cm左右处的水样,使用应用例1提供的检测方法进行检测,并计算絮凝率。检测结果如表2和图5所示。Use a flocculant containing Pseudomonas PR1 to treat lead-zinc mixed wastewater: prepare mixed wastewater with a Pb 2+ concentration of 30mg/L, a Zn 2+ concentration of 10mg/L, and a pH value of 7, and put it into a 250mL beaker middle. Measure 5mL of flocculant and add it to the wastewater sample, stir at 200r/min for 30s, then stir at 60r/min for 4min, then let it stand for 5min, take a water sample about 2cm below the liquid surface, use the sample provided in Application Example 1 The detection method is used to detect and calculate the flocculation rate. The test results are shown in Table 2 and Figure 5.
表2本发明提供的絮凝剂和铅锌混合废水的絮凝率(%)The flocculation rate (%) of flocculant provided by the present invention and lead-zinc mixed waste water of table 2
由表2和图5可知,本发明提供的絮凝剂能够有效处理混合废水中的铅离子和锌离子,起到絮凝有色金属离子的作用。It can be seen from Table 2 and Figure 5 that the flocculant provided by the present invention can effectively treat lead ions and zinc ions in mixed wastewater, and play the role of flocculating non-ferrous metal ions.
虽然本发明已以较佳的实施例公开如上,但其并非用以限定本发明,任何熟悉此技术的人,在不脱离本发明的精神和范围内,都可以做各种改动和修饰,因此本发明的保护范围应该以权利要求书所界定的为准。Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore The scope of protection of the present invention should be defined by the claims.
序列表sequence listing
<110> 桂林理工大学<110> Guilin University of Technology
<120> 一株假单胞菌PR1及其制备方法和应用<120> A strain of Pseudomonas PR1 and its preparation method and application
<160> 1<160> 1
<170> SIPOSequenceListing 1.0<170> SIPOSequenceListing 1.0
<210> 1<210> 1
<211> 1310<211> 1310
<212> DNA<212>DNA
<213> 假单胞菌PR1(Pseudomonas sp.)<213> Pseudomonas PR1 (Pseudomonas sp.)
<400> 1<400> 1
cgggagcttg ctccttgatt cagcggcgga cgggtgagta atgcctagga atctgcctgg 60cgggagcttg ctccttgatt cagcggcgga cgggtgagta atgcctagga atctgcctgg 60
tagtggggga caacgtttcg aaaggaacgc taataccgca tacgtcctac gggagaaagc 120tagtggggga caacgtttcg aaaggaacgc taataccgca tacgtcctac gggagaaagc 120
aggggacctt cgggccttgc gctatcagat gagcctaggt cggattagct agttggtggg 180aggggacctt cgggccttgc gctatcagat gagcctaggt cggattagct agttggtggg 180
gtaatggctc accaaggcga cgatccgtaa ctggtctgag aggatgatca gtcacactgg 240gtaatggctc accaaggcga cgatccgtaa ctggtctgag aggatgatca gtcacactgg 240
aactgagaca cggtccagac tcctacggga ggcagcagtg gggaatattg gacaatgggc 300aactgagaca cggtccagac tcctacggga ggcagcagtg gggaatattg gacaatgggc 300
gaaagcctga tccagccatg ccgcgtgtgt gaagaaggtc ttcggattgt aaagcacttt 360gaaagcctga tccagccatg ccgcgtgtgt gaagaaggtc ttcggattgt aaagcacttt 360
aagttgggag gaagggcagt aagttaatac cttgctgttt tgacgttacc gacagaataa 420aagttggggag gaagggcagt aagttaatac cttgctgttt tgacgttacc gacagaataa 420
gcaccggcta actctgtgcc agcagccgcg gtaatacaga gggtgcaagc gttaatcgga 480gcaccggcta actctgtgcc agcagccgcg gtaatacaga gggtgcaagc gttaatcgga 480
attactgggc gtaaagcgcg cgtaggtggt tcgttaagtt ggatgtgaaa gccccgggct 540attackgggc gtaaagcgcg cgtaggtggt tcgttaagtt ggatgtgaaa gccccgggct 540
caacctggga actgcatcca aaactggcga gctagagtac ggtagagggt ggtggaattt 600caacctggga actgcatcca aaactggcga gctagagtac ggtagagggt ggtggaattt 600
cctgtgtagc ggtgaaatgc gtagatatag gaaggaacac cagtggcgaa ggcgaccacc 660cctgtgtagc ggtgaaatgc gtagatatag gaaggaacac cagtggcgaa ggcgaccacc 660
tggactgata ctgacactga ggtgcgaaag cgtggggagc aaacaggatt agataccctg 720tggactgata ctgacactga ggtgcgaaag cgtggggagc aaacaggatt agataccctg 720
gtagtccacg ccgtaaacga tgtcaactag ccgttggaat ccttgagatt ttagtggcgc 780gtagtccacg ccgtaaacga tgtcaactag ccgttggaat ccttgagatt ttagtggcgc 780
agctaacgca ttaagttgac cgcctgggga gtacggccgc aaggttaaaa ctcaaatgaa 840agctaacgca ttaagttgac cgcctgggga gtacggccgc aaggttaaaa ctcaaatgaa 840
ttgacggggg cccgcacaag cggtggagca tgtggtttaa ttcgaagcaa cgcgaagaac 900ttgacggggg cccgcacaag cggtggagca tgtggtttaa ttcgaagcaa cgcgaagaac 900
cttaccaggc cttgacatgc agagaacttt ccagagatgg attggtgcct tcgggaactc 960cttaccaggc cttgacatgc agagaacttt ccagagatgg attggtgcct tcgggaactc 960
tgacacaggt gctgcatggc tgtcgtcagc tcgtgtcgtg agatgttggg ttaagtcccg 1020tgacacaggt gctgcatggc tgtcgtcagc tcgtgtcgtg agatgttggg ttaagtcccg 1020
taacgagcgc aacccttgtc cttagttacc agcacgttat ggtgggcact ctaaggagac 1080taacgagcgc aacccttgtc cttagttacc agcacgttat ggtgggcact ctaaggagac 1080
tgccggtgac aaaccggagg aaggtgggga tgacgtcaag tcatcatggc ccttacggcc 1140tgccggtgac aaaccggagg aaggtgggga tgacgtcaag tcatcatggc ccttacggcc 1140
tgggctacac acgtgctaca atggtcggta cagagggttg ccaagccgcg aggtggagct 1200tgggctacac acgtgctaca atggtcggta cagagggttg ccaagccgcg aggtggagct 1200
aatctcacaa aaccgatcgt agtccggatc gcagtctgca actcgactgc gtgaagtcgg 1260aatctcacaa aaccgatcgt agtccggatc gcagtctgca actcgactgc gtgaagtcgg 1260
aatcgctagt aatcgcgaat cagaatgtcg cggtgaatac gttcccgggc 1310aatcgctagt aatcgcgaat cagaatgtcg cggtgaatac gttcccgggc 1310
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