CN113976619B - Screening and application of a strain resistant to lead and cadmium pollution - Google Patents
Screening and application of a strain resistant to lead and cadmium pollution Download PDFInfo
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Abstract
Description
技术领域technical field
本发明属于菌株功能及应用技术领域,具体涉及耐辐射甲基杆菌JB18在修复重金属污染土壤中的应用。The invention belongs to the technical field of strain function and application, and in particular relates to the application of Methylobacterium radioduran JB18 in repairing heavy metal polluted soil.
背景技术Background technique
人类活动与工业化的发展不断地向土壤、水与大气中释放有毒有害物质,使生态破坏和环境质量恶化。重金属是危害较大的污染物之一,对整个人类生存环境的影响越来越突出。重金属污染的植物通过人类和动物的日常摄入进入到食物链中,导致动物和人类体中堆积有毒金属,许多致癌和其他有害影响都与此有关。The development of human activities and industrialization has continuously released toxic and harmful substances into the soil, water and atmosphere, causing ecological damage and environmental quality deterioration. Heavy metals are one of the most harmful pollutants, and their impact on the entire human living environment is becoming more and more prominent. Heavy metal-contaminated plants enter the food chain through daily ingestion by humans and animals, leading to the accumulation of toxic metals in animals and humans, which is associated with many carcinogenic and other harmful effects.
发明内容Contents of the invention
有鉴于此,本发明的目的在于提供耐辐射甲基杆菌JB18在修复重金属污染土壤中的应用,JB18具有较好的耐铅镉性,可为微生物修复重金属污染的土壤提供菌种资源。In view of this, the object of the present invention is to provide the application of Methylobacterium radiodurans JB18 in remediation of heavy metal-contaminated soil. JB18 has better resistance to lead and cadmium, and can provide strain resources for microbial remediation of heavy metal-contaminated soil.
为了实现上述发明目的,本发明提供以下技术方案:In order to achieve the above-mentioned purpose of the invention, the present invention provides the following technical solutions:
本发明提供了耐辐射甲基杆菌(Methylobacterium radiotolerans)JB18在修复重金属污染土壤中的应用,所述耐辐射甲基杆菌(Methylobacterium radiotolerans)JB18的保藏编号为 CGMCC NO.22961。The invention provides the application of Methylobacterium radiotolerans JB18 in repairing heavy metal-contaminated soil. The preservation number of Methylobacterium radiotolerans JB18 is CGMCC NO.22961.
优选的,所述JB18的ITS基因序列登录号为MZ723900。Preferably, the accession number of the ITS gene sequence of JB18 is MZ723900.
优选的,所述重金属包括铅或镉。Preferably, the heavy metal includes lead or cadmium.
优选的,利用所述JB18修复铅污染的土壤时,所述土壤的溶液中铅的浓度不高于200 mg/L;Preferably, when using the JB18 to restore lead-contaminated soil, the concentration of lead in the solution of the soil is not higher than 200 mg/L;
利用所述JB18修复镉污染的土壤时,所述土壤的溶液中镉的浓度不高于400mg/L;When using the JB18 to restore cadmium-contaminated soil, the concentration of cadmium in the soil solution is not higher than 400mg/L;
利用所述JB18修复铅和/或镉污染的土壤时,所述土壤的溶液中铅的浓度不高于200 mg/L,镉的浓度不高于20mg/L。When the JB18 is used to remediate lead and/or cadmium contaminated soil, the concentration of lead in the soil solution is not higher than 200 mg/L, and the concentration of cadmium is not higher than 20 mg/L.
本发明还提供了一种修复铅和/或镉污染的土壤的菌剂,所述菌剂的活性成分包括所述耐辐射甲基杆菌JB18。The present invention also provides a bacterial agent for remediating soil polluted by lead and/or cadmium, the active ingredient of the bacterial agent includes the Methylobacterium radioduran JB18.
本发明提供了耐辐射甲基杆菌JB18在修复重金属污染土壤中的应用,所述JB18分离筛选自黑龙江省齐齐哈尔市齐齐哈尔大学生命科学与农林学院环境修复实验室的铅镉污染土壤,经阶梯性筛选,JB18能在含有200mg/L Pb2+、400mg/L Cd2+培养基中存活,具有较好的耐铅镉性,能够利用淀粉、麦芽糖作为碳源生长,不能水解纤维素,在甲基红和明胶液化实验中皆显示为阴性,ITS序列鉴定为耐辐射甲基杆菌(Methylobacteriumradiotolerans)。本发明实施例中,将所述JB18在含铅的LB平板培养基上进行培养时,铅离子的最低抑制浓度为200mg/L;在含镉的LB平板培养基上进行培养时,镉离子的最低抑制浓度为400mg/L;在含铅和镉的LB培养基平板上进行培养时,铅镉复合浓度为200/20mg/L时,菌株JB18仍能存活。证明所述JB18具有较好的耐铅镉性,可为微生物修复重金属污染的土壤提供菌种资源。The invention provides the application of Methylobacterium radioduran JB18 in remediating heavy metal-contaminated soil. The JB18 is isolated and screened from the lead-cadmium-contaminated soil of the Environmental Remediation Laboratory of the College of Life Sciences and Agriculture and Forestry, Qiqihar University, Qiqihar City, Heilongjiang Province, and is screened stepwise. , JB18 can survive in the medium containing 200mg/L Pb 2+ and 400mg/L Cd 2+ , has good resistance to lead and cadmium, can use starch and maltose as carbon sources for growth, cannot hydrolyze cellulose, and can grow in methyl Both red and gelatin liquefaction tests were negative, and the ITS sequence was identified as Methylobacterium radiotolerans. In the embodiment of the present invention, when the JB18 is cultured on the LB plate medium containing lead, the minimum inhibitory concentration of lead ion is 200mg/L; The minimum inhibitory concentration was 400mg/L; when cultured on the LB medium plate containing lead and cadmium, the strain JB18 could still survive when the compound concentration of lead and cadmium was 200/20mg/L. It is proved that the JB18 has good resistance to lead and cadmium, and can provide strain resources for microbial remediation of heavy metal-contaminated soil.
生物保藏信息Biological deposit information
耐辐射甲基杆菌(Methylobacterium radiotolerans)JB18,保藏于中国微生物菌种保藏管理委员会普通微生物中心,具体地址为北京市朝阳区北辰西路1号院3号中国科学院微生物研究所,保藏编号为CGMCC NO.22961,保藏日期为2021年7月26日。Methylobacterium radiotolerans JB18, preserved in the General Microbiology Center of the China Committee for the Collection of Microbial Cultures, the specific address is the Institute of Microbiology, Chinese Academy of Sciences, No. 1, No. 1, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC NO .22961, with a deposit date of July 26, 2021.
附图说明Description of drawings
图1为利用ITS序列构建得到的进化树。Figure 1 is the phylogenetic tree constructed using ITS sequences.
具体实施方式Detailed ways
本发明提供了耐辐射甲基杆菌(Methylobacterium radiotolerans)JB18在修复重金属污染土壤中的应用,所述耐辐射甲基杆菌(Methylobacterium radiotolerans)JB18的保藏编号为 CGMCC NO.22961。The invention provides the application of Methylobacterium radiotolerans JB18 in repairing heavy metal-contaminated soil. The preservation number of Methylobacterium radiotolerans JB18 is CGMCC NO.22961.
本发明所述JB18的ITS基因序列登录号优选为MZ723900,其ITS基因序列优选如SEQ ID NO.1所示:The ITS gene sequence accession number of JB18 of the present invention is preferably MZ723900, and its ITS gene sequence is preferably as shown in SEQ ID NO.1:
GGCTCAGAGCGAACGCTGGCGGCAGGCTTAACACATGCAAGTCGAGCGGGCCCTTCGG GGTCAGCGGCGGACGGGTGAGTAACGCGTGGGAACGTGCCTTCTGGTTCGGAATAACC CTGGGAAACTAGGGCTAATACCGGATACGCCCTTTTGGGGAAAGGTTTACTGCCGGAAG ATCGGCCCGCGTCTGATTAGCTAGTTGGTGGGGTAACGGCCTACCAAGGCGACGATCAG TAGCTGGTCTGAGAGGATGATCAGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGCAAGCCTGATCCAGCCATGCCG CGTGAGTGATGAAGGCCTTAGGGTTGTAAAGCTCTTTTATCCGGGACGATAATGACGGTA CCGGAGGAATAAGCCCCGGCTAACTTCGTGCCAGCAGCCGCGGTAATACGAAGGGGGC TAGCGTTGCTCGGAATCACTGGGCGTAAAGGGCGCGTAGGCGGCGTTTTAAGTCGGGGG TGAAAGCCTGTGGCTCAACCACAGAATGGCCTTCGATACTGGGACGCTTGAGTATGGTA GAGGTTGGTGGAACTGCGAGTGTAGAGGTGAAATTCGTAGATATTCGCAAGAACACCGG TGGCGAAGGCGGCCAACTGGACCATTACTGACGCTGAGGCGCGAAAGCGTGGGGAGCA AACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAATGCCAGCTGTTGGGGT GCTTGCACCGCAGTAGCGCAGCTAACGCTTTGAGCATTCCGCCTGGGGAGTACGGTCGC AAGATTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTT AATTCGAAGCAACGCGCAGAACCTTACCATCCTTTGACATGGCGTGTTACCCAGAGAGA TCTGGGGTCCCCTTCGGGGGCGCGCACACAGGTGCTGCATGGCTGTCGTCAGCTCGTGT CGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCACGTCCTTAGTTGCCATCAT TCAGTTGGGCACTCTAGGGAGACTGCCGGTGATAAGCCGCGAGGAAGGTGTGGATGACGTCAAGTCCTCATGGCCCTTACGGGATGGGCTACACACGTGCTACAATGGCGGTGACAG TGGGAGGCGAAGGAGCGATCTGGAGCAAATCCCCAAAAGCCGTCTCAGTTCGGATTGC ACTCTGCAACTCGAGTGCATGAAGGCGGAATCGCTAGTAATCGTGGATCAGCATGCCAC GGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTTGGTCTTA CCCGACGGCGCTGCGCCAACCGCAAGGAGGCAGGCGACCACGGTAGGGTCAGCGACTGGGGTGAAGTCGTAA。本发明利用所述ITS序列与GenBank中已有的ITS序列进行BLAST 同源性分析,利用软件MEGA X构建进化树,如图1所示,该菌株与耐辐射甲基杆菌(Methylobacterium radiotolerans)的相似性较高,确定分离到的菌株为耐辐射甲基杆菌,编号为JB18,序列已提交NCBI的GENBANK数据库,菌株基因序列登录号为MZ723900。GGCTCAGAGCGAACGCTGGCGGCAGGCTTAACACATGCAAGTCGAGCGGGCCCTTCGGGGTCAGCGGCGGACGGGTGAGTAACGCGTGGGAACGTGCCTTCTGGTTCGGAATAACCCTGGGAAACTAGGGCTAATACCGGATACGCCCTTTTGGGGAAAGGTTACTGCCGGAAGATCGGCCCGCGTCTGATTAGCTAG TTGGTGGGGTAACGGCCTACCAAGGCGACGATCAG TAGCTGGTCTGAGAGGATGATCAGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGCAAGCCTGATCCAGCCATGCCG CGTGAGTGATGAAGGCCTTAGGGTTGTAAAGCTTTTATCCGGGACGATAAT GACGGTA CCGGAGGAATAAGCCCCGGCTAACTTCGTGCCAGCAGCCGCGGTAATACGAAGGGGGGC TAGCGTTGCTCGGAATCACTGGGCGTAAAGGGCGCGTAGGCGGCGTTTTAAGTCGGGGG TGAAAGCCTGTGGCTCAACCACAGAATGGCCTTCGATACTGGGACGCTTGAGTATGGTA GAGGTTGGTGGAACTGC GAGTGTAGAGGTGAAATTCGTAGATATTCGCAAGAACACCGG TGGCGAAGGCGGCCAACTGGACCATTACTGACGCTGAGGCGCGAAAGCGTGGGGAGCA AACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAATGCCAGCTGTTGGGGT GCTTGCACCGCAGTAGCGCAGCTAACGCTTTGAGCATTCCGCCTGGG GAGTACGGTCGC AAGATTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTT AATTCGAAGCAACGCGCAGAACCTTACCATCCTTTGACATGGCGTGTTACCCCAGAGAGA TCTGGGGTCCCCTTCGGGGGCGCACACAGGTGCTGCATGGCTGTCGTCAGCTCGTGT CGTGAGATGTTGGGTTA AGTCCCGCAACGAGCGCAACCCACGTCCTTAGTTGCCATCAT TCAGTTGGGCACTCTAGGGAGACTGCCGGTGATAAGCCGCGAGGAAGGTGTGGATGACGTCCAAGTCCTCATGGCCCTTACGGGATGGGCTACACACGTGCTACAATGGCGGTGACAG TGGGAGGCGAAGGAGCGATCTGGAGCAATCCCCAAAAGCCGTCTCAGTTCG GATTGC ACTCTGCAACTCGAGTGCATGAAGGCGGAATCGCTAGTAATCGTGGATCAGCATGCCACGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTTGGTCTTACCCGACGGCGCTGCGCCAACCGCAAGGAGGCAGGCGACCACGGTAGGGTCAGCGACTGGGGTGAAGTCGTAA. The present invention utilizes said ITS sequence and the existing ITS sequence in GenBank to carry out BLAST homology analysis, utilizes software MEGA X to construct evolutionary tree, as shown in Figure 1, this bacterial strain is similar to the Methylobacterium radiotolerans (Methylobacterium radiotolerans) The isolated strain was determined to be Methylobacterium radiodurans, numbered JB18, the sequence has been submitted to NCBI's GENBANK database, and the accession number of the strain gene sequence is MZ723900.
本发明所述JB18优选分离筛选自黑龙江省齐齐哈尔市齐齐哈尔大学生命科学与农林学院环境修复实验室的铅镉污染土壤,本发明对所述分离筛选的方法并没有特殊限定,优选包括:将10-5g/mL、10-6g/mL、10-7g/mL浓度土壤悬液取0.1mL接种到铅浓度为200mg/L、镉浓度为20mg/L的LB培养基平板上,在恒温培养箱中30℃,培养3~5天,观察是否有菌落长出,若有菌株长出,则继续增加LB培养基中铅、镉浓度,从而确定铅、镉离子的最低抑制浓度;因复合铅镉浓度的胁迫比单一铅、镉浓度大,故再通过含复合铅镉浓度的LB培养基进行筛选。本发明所述重金属优选包括铅和/或镉。在本发明中,利用所述JB18修复铅污染的土壤时,所述土壤的溶液中铅的浓度优选不高于200mg/L;利用所述JB18修复镉污染的土壤时,所述土壤的溶液中镉的浓度优选不高于400mg/L;利用所述JB18修复铅和镉污染的土壤时,所述土壤的溶液中铅的浓度优选不高于200mg/L,镉的浓度优选不高于20 mg/L。The JB18 described in the present invention is preferably isolated and screened from lead and cadmium contaminated soil in the Environmental Remediation Laboratory of the School of Life Sciences and Agriculture and Forestry, Qiqihar University, Qiqihar City, Heilongjiang Province. The method for the separation and screening in the present invention is not particularly limited, and preferably includes: 10 - Take 0.1 mL of the soil suspension at 5 g/mL, 10 -6 g/mL, and 10 -7 g/mL concentration and inoculate it on an LB medium plate with a lead concentration of 200 mg/L and a cadmium concentration of 20 mg/L. In the box at 30°C, cultivate for 3 to 5 days, observe whether there are colonies growing, if there are strains growing, continue to increase the concentration of lead and cadmium in the LB medium, so as to determine the minimum inhibitory concentration of lead and cadmium ions; The stress of cadmium concentration is greater than that of single lead and cadmium concentration, so the LB medium containing compound concentration of lead and cadmium is used for screening. The heavy metals in the present invention preferably include lead and/or cadmium. In the present invention, when using the JB18 to repair lead-contaminated soil, the concentration of lead in the soil solution is preferably not higher than 200mg/L; when using the JB18 to repair cadmium-contaminated soil, the solution of the soil The concentration of cadmium is preferably not higher than 400mg/L; when using the JB18 to restore lead and cadmium-contaminated soil, the concentration of lead in the soil solution is preferably not higher than 200mg/L, and the concentration of cadmium is preferably not higher than 20 mg /L.
本发明还提供了一种修复铅和/或镉污染的土壤的菌剂,所述菌剂的活性成分包括所述 JB18。The present invention also provides a bacterial agent for remediating soil polluted by lead and/or cadmium, the active ingredient of the bacterial agent includes the JB18.
下面结合实施例对本发明提供的耐辐射甲基杆菌JB18在修复重金属污染土壤中的应用进行详细的说明,但是不能把它们理解为对本发明保护范围的限定。The application of Methylobacterium radioduran JB18 provided by the present invention in repairing heavy metal-contaminated soil will be described in detail below in conjunction with the examples, but they should not be understood as limiting the protection scope of the present invention.
实施例1Example 1
耐辐射甲基杆菌(Methylobacterium radiotolerans)的分离和鉴定Isolation and Identification of Methylobacterium radiotolerans
一、JB18是从黑龙江省齐齐哈尔市齐齐哈尔大学生命科学与农林学院环境修复实验室的铅镉污染土壤内筛选出的耐性菌株,具体包括如下的步骤:1. JB18 is a resistant strain screened from lead and cadmium contaminated soil in the Environmental Remediation Laboratory of the School of Life Sciences and Agriculture and Forestry, Qiqihar University, Qiqihar City, Heilongjiang Province. The specific steps are as follows:
1.配制培养基1. Preparation of culture medium
LB培养基:牛肉膏3g,蛋白胨10g,琼脂20g,蒸馏水1000mL,氯化钠5g,pH值 7.4。LB培养基具体配制过程:将3g牛肉膏、10g蛋白胨和5g氯化钠放入烧杯中并加入少量蒸馏水,用玻璃棒搅拌溶解后加入20g琼脂粉搅拌均匀并补水到1000mL,分装于250mL 的三角瓶中,灭菌锅设置为121℃、20min。LB medium: beef extract 3g, peptone 10g, agar 20g, distilled water 1000mL, sodium chloride 5g, pH 7.4. The specific preparation process of LB medium: put 3g beef extract, 10g peptone and 5g sodium chloride into a beaker and add a small amount of distilled water, stir and dissolve with a glass rod, add 20g agar powder, stir evenly and replenish water to 1000mL, and distribute in 250mL In the Erlenmeyer flask, the autoclave was set at 121°C for 20 minutes.
2.筛选培养2. Screening culture
称取土样10g置于锥形瓶中,加入无菌水90mL制成土壤悬液,放入适量玻璃珠后封口移入恒温摇床中,180r/min、30℃处理2h,静置30min。取土壤悬液的1mL加入到9mL 无菌水得到10-1的土壤菌悬液,继续稀释,依次得到10-2、10-3、10-4、10-5、10-6、10-7和10-8的浓度土壤菌悬液。将10-5、10-6、10-7浓度菌悬液取0.1mL接种到铅浓度为200mg/L、镉浓度为20mg/L的LB培养基平板上,在恒温培养箱中30℃培养3~5天,观察是否有菌落长出,若有菌株长出,则继续增加LB培养基中铅、镉浓度,从而确定铅、镉离子的最低抑制浓度。因复合铅镉浓度的胁迫比单一铅、镉浓度大,故再通过含复合铅镉浓度的LB培养基进行筛选。确定铅离子的最低抑制浓度为200mg/L;确定镉离子的最低抑制浓度为400mg/L,铅镉复合浓度为200/20mg/L时,JB18仍能存活。Weigh 10g of soil sample and place it in a Erlenmeyer flask, add 90mL of sterile water to make a soil suspension, put in an appropriate amount of glass beads, seal it and move it into a constant temperature shaker, treat it at 180r/min, 30°C for 2h, and let it stand for 30min. Take 1mL of the soil suspension and add it to 9mL sterile water to obtain a 10 -1 soil bacterial suspension, and continue to dilute to obtain 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 And the concentration of 10 -8 soil bacteria suspension. Inoculate 0.1 mL of the 10 -5 , 10 -6 , and 10 -7 concentration bacterial suspension onto an LB medium plate with a lead concentration of 200 mg/L and a cadmium concentration of 20 mg/L, and incubate in a constant temperature incubator at 30°C for 3 After ~5 days, observe whether there are bacterial colonies to grow, if there are bacterial strains to grow, then continue to increase the concentration of lead and cadmium in the LB medium, so as to determine the minimum inhibitory concentration of lead and cadmium ions. Because the stress of compound lead and cadmium concentration is greater than that of single lead and cadmium concentration, the LB medium containing compound lead and cadmium concentration is used for screening. The minimum inhibitory concentration of lead ion was determined to be 200mg/L; the minimum inhibitory concentration of cadmium ion was determined to be 400mg/L, and JB18 could still survive when the combined concentration of lead and cadmium was 200/20mg/L.
二、采用ITS基因测序法对JB18进行分类鉴定2. Classification and identification of JB18 by ITS gene sequencing method
1.ITS基因PCR扩增引物为:1. ITS gene PCR amplification primers are:
ITS1(7F):5‘-CAGAGTTTGATCCTGGCT-3’;ITS1(7F): 5'-CAGAGTTTGATCCTGGCT-3';
ITS2(1540R):5‘-AGGAGGTGATCCAGCCGCA-3’。ITS2(1540R): 5'-AGGAGGTGATCCAGCCGCA-3'.
ITS3(27F):5‘-AGTTTGATCMTGGCTCAG-3’;ITS3(27F): 5'-AGTTTGATCMTGGCTCAG-3';
ITS4(1492R):5‘-GGTTACCTTGTTACGACTT-3’。ITS4(1492R): 5'-GGTTACCTTGTTACGACTT-3'.
2.扩增程序为:预变性95℃300s;变性94℃30s,退火57℃30s,延伸72℃90s,重复30次;修复延伸72℃600s。2. The amplification program is: pre-denaturation at 95°C for 300s; denaturation at 94°C for 30s, annealing at 57°C for 30s, extension at 72°C for 90s, repeated 30 times; repair extension at 72°C for 600s.
基因扩增产物纯化后经生工公司测序,将所得序列与GenBank中已有的ITS序列进行 BLAST同源性分析,利用软件MEGA X构建进化树。结果如图1所示,该菌株与耐辐射甲基杆菌(Methylobacterium radiotolerans)的相似性较高,确定分离到的菌株为耐辐射甲基杆菌,编号为JB18,序列已提交NCBI的GenBank数据库,菌株基因序列登录号为MZ723900。After the gene amplification product was purified, it was sequenced by Sangon Company, and the resulting sequence was subjected to BLAST homology analysis with the existing ITS sequence in GenBank, and the phylogenetic tree was constructed using the software MEGA X. As a result, as shown in Figure 1, the strain has a high similarity with Methylobacterium radiotolerans, and it is determined that the isolated strain is Methylobacterium radiotolerans, numbered JB18, and the sequence has been submitted to the GenBank database of NCBI. The gene sequence accession number is MZ723900.
三、JB18的生理生化试验3. Physiological and biochemical tests of JB18
1.甲基红试验1. Methyl red test
(1)葡萄糖蛋白胨培养基:7.5g蛋白胨,7.5g葡萄糖,7.5g磷酸氢二钾,定容到1500mL,pH为7。(1) Glucose-peptone medium: 7.5g peptone, 7.5g glucose, 7.5g dipotassium hydrogen phosphate, dilute to 1500mL, pH 7.
(2)步骤:将JB18接种到葡萄糖蛋白胨培养基中,灭菌棉塞封口,培养温度30℃,24h后,向葡萄糖蛋白胨培养基中滴加1~2滴甲基红试剂,观察颜色变化。若变为红色表示培养基中含有酸性物质则为阳性,用“+”表示;若变为黄色表示培养基中不含有酸性物质则为阴性,用“-”表示。(2) Steps: Inoculate JB18 into the glucose-peptone medium, seal with sterilized cotton plug, cultivate at 30°C, after 24 hours, add 1-2 drops of methyl red reagent to the glucose-peptone medium, and observe the color change. If it turns red, it means that the medium contains acidic substances, it is positive, and it is represented by "+"; if it turns yellow, it means that the medium does not contain acidic substances, it is negative, and it is represented by "-".
2.明胶液化试验2. Gelatin liquefaction test
(1)明胶液化培养基:蛋白胨5g,明胶200g,葡萄糖20g,蒸馏水1000mL。(1) Gelatin liquefaction medium: peptone 5g, gelatin 200g, glucose 20g, distilled water 1000mL.
(2)步骤:将JB18接种于明胶表面,28℃下暗培养,每隔5d观察一次。先将试管低温冷却,待未接种的CK试管中明胶凝固后观察记录。若冷却后明胶不液化则无蛋白酶水解作用,反之则表明有蛋白酶水解作用。水解为阳性用“+”表示,不水解为阴性用“-”表示。(2) Step: Inoculate JB18 on the surface of gelatin, culture in dark at 28°C, and observe every 5 days. Cool the test tube at low temperature first, and observe and record after the gelatin in the uninoculated CK test tube solidifies. If the gelatin does not liquefy after cooling, there is no protease hydrolysis, otherwise, it indicates that there is protease hydrolysis. The hydrolysis is positive with "+", and the non-hydrolysis is negative with "-".
3.淀粉水解试验3. Starch hydrolysis test
(1)淀粉水解培养基:磷酸氢二钾10g,磷酸二氢钾0.3g,氯化钠0.5g,碳酸镁1g,硝酸钾1g,可溶性淀粉2.0g,琼脂15g,pH为7.2,蒸馏水1000mL。(1) Starch hydrolysis medium: 10 g dipotassium hydrogen phosphate, 0.3 g potassium dihydrogen phosphate, 0.5 g sodium chloride, 1 g magnesium carbonate, 1 g potassium nitrate, 2.0 g soluble starch, 15 g agar, pH 7.2, 1000 mL distilled water.
(2)步骤:将培养基倒入培养皿中,待培养基凝固后采用点接法接种,28℃下暗培养 10~20d后,将配制好的碘液倒入培养基表面。如有淀粉酶产生,则菌落的周围不会变为蓝色,反之则变成蓝色,表明有淀粉酶产生。利用形成的透明圈来检测是否产生淀粉酶。淀粉酶水解为阳性用“+”来表示,淀粉酶不水解为阴性用“-”来表示。(2) Steps: Pour the culture medium into a petri dish, inoculate with the spot method after the culture medium is solidified, cultivate in dark at 28°C for 10-20 days, and then pour the prepared iodine solution onto the surface of the culture medium. If amylase is produced, the surrounding of the colony will not turn blue, otherwise it will turn blue, indicating that amylase is produced. Use the transparent circle formed to detect amylase production. Amylase hydrolysis is positive with "+", and amylase non-hydrolysis is negative with "-".
4.纤维素水解试验4. Cellulose hydrolysis test
(1)纤维素水解培养基:硫酸镁0.5g,磷酸氢二钾0.5g,氯化钠0.5g,硝酸钾1g,滤纸条,蒸馏水1000mL。(1) Cellulose hydrolysis medium: 0.5 g of magnesium sulfate, 0.5 g of dipotassium hydrogen phosphate, 0.5 g of sodium chloride, 1 g of potassium nitrate, filter paper strips, and 1000 mL of distilled water.
(2)步骤:将滤纸条放置在试管的培养基中,一半浸入培养基中一半在培养基上,将待测菌株接种于已灭菌的试管中的滤纸条上,28℃培养30d后,观察其是否能在滤纸条上生长。纤维素水解为阳性用“+”表示,纤维素不水解为阴性用“-”表示。(2) Step: Place the filter paper strip in the culture medium of the test tube, half of which is immersed in the culture medium and half is on the culture medium, inoculate the strain to be tested on the filter paper strip in the sterilized test tube, and incubate at 28°C for 30 days Finally, observe whether it can grow on the filter paper strip. Cellulose hydrolysis is positive with "+", and cellulose is not hydrolyzed is negative with "-".
5.麦芽糖利用测定5. Maltose Utilization Assay
(1)土豆200g,麦芽糖20g,琼脂20g,蒸馏水1000mL。(1) 200g potatoes, 20g maltose, 20g agar, 1000mL distilled water.
(2)观察菌株能否正常生长。能正常生长则为阳性用“+”表示,不能正常生长用“-”表示。(2) Observe whether the strain can grow normally. If it can grow normally, it is positive with "+", and if it cannot grow normally, it is indicated with "-".
6.试验结果如表1所示6. The test results are shown in Table 1
表1 JB18的生理生化性质Table 1 Physiological and biochemical properties of JB18
以上所述仅是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。The above is only a preferred embodiment of the present invention, it should be pointed out that, for those of ordinary skill in the art, without departing from the principle of the present invention, some improvements and modifications can also be made, and these improvements and modifications can also be made. It should be regarded as the protection scope of the present invention.
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