CN107460144B - 一株需氧活性海洋细菌及其脱色絮凝剂的制备方法 - Google Patents
一株需氧活性海洋细菌及其脱色絮凝剂的制备方法 Download PDFInfo
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Abstract
本发明公开了一株需氧活性海洋细菌及其脱色絮凝剂的制备方法。一株需氧活性海洋细菌，从中国浙江舟山朱家尖一处海水养殖场养殖的菲律宾蛤仔吐泥中分离筛选获得，经鉴定为假交替单胞菌（Pseudoalteromonas sp.）。有益效果为：制备的脱色絮凝剂脱色效果很好，对高岭土的絮凝率达到90％以上；对高浓度废水脱色具有较高活性，对亚甲基蓝、孔雀石绿、结晶紫的脱色率达92％以上、99％以上和98％以上；对生物体无潜在毒性，绿色健康。
Description
技术领域
本发明涉及净水技术领域，具体是一株需氧活性海洋细菌及其脱色絮凝剂的制备方法。
背景技术
在工业水、饮用水和生活污水的处理中，絮凝剂的性能至关重要。在水处理的絮凝过程中主要是将水中的纳米级、微米级的悬浮体或胶体杂质颗粒在絮凝剂的作用下，经过凝聚—絮凝反应形成大的絮体颗粒，最后由沉淀、过滤等工艺将其去除。
长期以来，给水和废水处理使用的絮凝剂主要有两大类：以铝盐、铁盐及其水解聚合物为代表的无机絮凝剂和以聚丙烯酰胺及其衍生物为代表的有机高分子絮凝剂。尽管无机和有机絮凝剂因其具有良好的絮凝效果和较低的成本而得到广泛应用，然而它们在使用过程中给环境造成的二次污染不得不引起人们的重视。研究表明，无机絮凝剂中的铝离子易引起老年性痴呆症；铁盐絮凝剂中的铁盐对金属有腐蚀作用，可造成处理水中带有颜色，易形成某些难絮凝沉淀的化合物；而聚丙烯酰胺类絮凝剂的合成单体丙烯酰胺具有累积性神经毒性，已被确定为有基因毒性的致癌物。
现有技术如授权公告号为CN101580550B的中国发明专利，公开了一株好氧罗氏菌属菌株代谢产物胞外多糖、制法和用途。从中国渤海海域野生菲律宾蛤仔黏附污泥中分离获得的好氧菌株ZHT4-13、鉴定为罗氏菌属(Ruthia sp.)。经种子培养、扩大培养，其发酵液经乙醇沉淀离心分离得到细胞外多糖MBF4-13。经测定出该细胞外多糖MBF4-13 具有较高的絮凝活性，对高岭土的FR 值达到80％以上；对六价铬离子去除率达69.3％；对高浓度废水脱色具有较高活性，对亚甲基蓝、孔雀石绿、结晶紫的脱色率达86.11％、99.49％和97.84％；对活性污泥的性能及结构也有明显改善作用。现在能高效产生絮凝剂的菌株种类较少，还需要继续分离筛选出新的高活性菌种。
发明内容
本发明的目的在于提供一株需氧活性海洋细菌及其脱色絮凝剂的制备方法。制备得到的脱色絮凝剂用量少，脱色率高，形成的絮团大，沉降速度快，性能稳定，脱色絮凝效果好；对生物体无潜在毒性，绿色健康。
本发明针对背景技术提到的问题，采用的技术方案为：一株需氧活性海洋细菌，从中国浙江舟山朱家尖一处海水养殖场养殖的菲律宾蛤仔吐泥中分离筛选获得，经鉴定为假交替单胞菌（Pseudoalteromonassp.），于2016年8月29日保藏于中国微生物菌种保藏管理委员会普通微生物中心（CGMCC），保藏号为CGMCC No. 12913。中国微生物菌种保藏管理委员会普通微生物中心（CGMCC）位于北京市朝阳区北辰西路1号院3号。
上述海洋菌株16S rDNA全基因（1277bp）已向美国国家生物技术信息中心（NCBI）的GenBank基因序列数据库提交，登陆号为KX702262。其全序列如下：
ATGCTTGGGAACATGCCTTGAGGTGGGGGACAACAGTTGGAAACGACTGCTAATACCGCATAATGTCTACGGACCAAAGGGGGCTTCGGCTCTCGCCTTTAGATTGGCCCAAGTGGGATTAGCTAGTTGGTGAGGTAATGGCTCACCAAGGCGACGATCCCTAGCTGGTTTGAGAGGATGATCAGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCCATGCCGCGTGTGTGAAGAAGGCCTTCGGGTTGTAAAGCACTTTCAGTCAGGAGGAAAGGTTAGTAGTTAATACCTGCTATCTGTGACGTTACTGACAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCGAGCGTTAATCGGAATTACTGGGCGTAAAGCGTACGCAGGCGGTTTGTTAAGCGAGATGTGAAAGCCCCGGGCTCAACCTGGGAACTGCATTTCGAACTGGCAAACTAGAGTGTGATAGAGGGTGGTAGAATTTCAGGTGTAGCGGTGAAATGCGTAGAGATCTGAAGGAATACCGATGGCGAAGGCAGCCACCTGGGTCAACACTGACGCTCATGTACGAAAGCGTGGGGAGCAAACGGGATTAGATACCCCGGTAGTCCACGCCGTAAACGATGTCTACTAGAAGCTCGGAGCCTCGGTTCTGTTTTTCAAAGCTAACGCATTAAGTAGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTACACTTGACATACAGAGAACTTACCAGAGATGGTTTGGTGCCTTCGGGAACTCTGATACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCCTATCCTTAGTTGCTAGCAGGTAATGCTGAGAACTCTAAGGAGACTGCCGGTGATAAACCGGAGGAAGGTGGGGACGACGTCAAGTCATCATGGCCCTTACGTGTAGGGCTACACACGTGCTACAATGGCGCATACAGAGTGCTGCGAACTCGCGAGAGTAAGCGAATCACTTAAAGTGCGTCGTAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGCGTATCAGAATGACGCGGTGAATACGTTCCCGGGCCTTGTACACACCGC。
一株需氧活性海洋细菌的筛选方法为：采集新鲜菲律宾蛤仔，放入无菌水中吐泥，取1ml泥，采用梯度稀释倒平板分离和划线纯化，经活性测试筛选出具有高絮凝和脱色活性的菌株Pseudoalteromonassp. GHS18。
一株需氧活性海洋细菌的脱色絮凝剂的制备方法，包括以下步骤：
种子液培养：先在斜面培养基上接种保藏号为CGMCC No. 12913的海洋细菌Pseudoalteromonassp. GHS18，并于22-27℃恒温培养24-72h，将菌种活化，再加入培养基体积0.7-1.3倍的无菌水，振荡制得种子液。上述种子液的培养可将本发明海洋细菌从保藏状态恢复到新陈代谢旺盛的状态，生长繁殖速度快，得到数量充足的菌种；
扩大发酵培养：每100ml液体发酵培养基接入0.2-0.5ml种子液，于22-27℃恒温摇床发酵培养；摇床速率为100-140r/min，发酵培养时间为3-4d。在发酵培养期间，本发明的海洋细菌可充分利用培养基中的碳源、氮源及生长因子，进行旺盛的新陈代谢，产生大量的代谢产物---胞外多糖；
胞外多糖提取：先将发酵完成的菌液高速离心，取上清液，再在上清液中加入4-6倍体积的乙醇，并于2-5℃下静置10-18h，离心取沉淀，真空干燥得胞外多糖粗制品。亲水的多糖水化层被乙醇破坏，多糖在水中的溶解度大幅下降，以沉淀的形式从溶液中析出，同时多糖的活性不会被破坏；
絮凝剂的制备：在胞外多糖粗制品中加水，振荡后加入乙醇，静置后离心取沉淀，真空干燥得到絮凝剂。水为粗制品体积的1-2倍，乙醇体积为水溶液体积的4-6倍，乙醇温度为2-5℃。制备得到的脱色絮凝剂用量少，脱色率高，形成的絮团大，沉降速度快，性能稳定，脱色絮凝效果好；对生物体无潜在毒性，绿色健康。
作为优选，斜面培养基成分及其浓度为葡萄糖15-25g/L、尿素0.44-0.52g/L、硫酸铵0.19-0.24g/L、酵母膏0.4-0.6g/L、磷酸氢二钾1.5-5 g/L、磷酸二氢钾0.4-2g/L、琼脂12-17g/L和余量陈化海水或人工海水；调节pH为7.2-7.4，115-121℃高压灭菌20-35min。其中，人工海水配方为：氯化钠25.0 g/L、氯化镁1.9g/L、硫酸镁3.1 g/L、氯化钙1.3 g/L、碳酸氢钠0.2 g/L、氯化钾0.68g/L、溴化钠0.06 g/L、硼酸0.058 g/L、硅酸钠0.0024 g/L、磷酸0.002 g/L、六氯化二铝 0.014 g/L、氨水 0.002 g/L、硝酸锂 0.0014g/L、蒸馏水余量。上述培养基不仅能提供适合本发明海洋细菌适宜的环境，还能提供菌种生长繁殖所需的碳源、氮源及生长因子，菌种的繁殖速度快，并且能诱导菌种分泌产生大量的胞外多糖，胞外多糖产量提高。液体发酵培养基为斜面培养基成分中不添加琼脂制得。
与现有技术相比，本发明的优点在于：本发明从菲律宾蛤仔黏附淤泥中分离纯化并筛选出一株需氧活性海洋细菌，鉴定为假交替单胞菌（Pseudoalteromonassp.），并提取胞外多糖制得脱色絮凝剂。本发明制备的脱色絮凝剂脱色效果很好，对高岭土的絮凝率达到90％以上；对高浓度废水脱色具有较高活性，对亚甲基蓝、孔雀石绿、结晶紫的脱色率达92％以上、99％以上和98％以上；絮凝剂性质稳定，制备步骤简单，原料成本低廉，经济效益高，可大规模生产。
具体实施例
下面通过实施例对本发明方案作进一步说明：
实施例1：
一株需氧活性海洋细菌，从中国浙江舟山朱家尖一处海水养殖场养殖的菲律宾蛤仔吐泥中分离筛选获得，经鉴定为假交替单胞菌（Pseudoalteromonassp.），于2016年8月29日保藏于中国微生物菌种保藏管理委员会普通微生物中心（CGMCC），保藏号为CGMCC No.12913。中国微生物菌种保藏管理委员会普通微生物中心（CGMCC）位于北京市朝阳区北辰西路1号院3号。
脱色絮凝剂的制备方法包括以下步骤：
1）种子液培养：将保藏号为CGMCC No. 12913的海洋细菌接种于斜面培养基，于25℃恒温培养36h。加入等体积无菌水，振荡制得种子液。上述种子液的培养可将海洋细菌Pseudoalteromonassp. GHS18从保藏状态恢复到新陈代谢旺盛的状态，生长繁殖速度快，得到数量充足的菌种；
2）扩大发酵培养：在每100ml液体发酵培养基接入0.4ml种子液，于25℃恒温摇床发酵培养3.6d，摇床速率为140r/min。在发酵培养期间，海洋细菌Pseudoalteromonassp.GHS18可充分利用培养基中的碳源、氮源及生长因子，进行旺盛的新陈代谢，产生大量的代谢产物---胞外多糖；
3）胞外多糖提取：将发酵完成的菌液离心，取上清液，在上清液中加入5倍体积的乙醇。在4℃下静置15h，离心取沉淀，真空干燥得胞外多糖粗制品。亲水的多糖水化层被乙醇破坏，多糖在水中的溶解度大幅下降，以沉淀形式与其他成分分离，以沉淀的形式从溶液中析出，同时多糖的活性不会被破坏；
4）絮凝剂的制备：在胞外多糖粗制品中加1.7倍体积的水，振荡后加入5倍体积的乙醇，乙醇温度为4℃。静置后离心取沉淀，真空干燥得到脱色絮凝剂。制备得到的脱色絮凝剂用量少，脱色率高，形成的絮团大，沉降速度快，性能稳定，脱色絮凝效果好；对生物体无潜在毒性，绿色健康。脱色效果很好，对高岭土的絮凝率达到90％以上；对高浓度废水脱色具有较高活性，对亚甲基蓝、孔雀石绿、结晶紫的脱色率达92％以上、99％以上和98％以上；絮凝剂性质稳定，制备步骤简单，原料成本低廉，经济效益高，可大规模生产。
斜面培养基成分及其浓度为葡萄糖22g/L、尿素0.47g/L、硫酸铵0.2g/L、酵母膏0.5g/L、磷酸氢二钾2.7g/L、磷酸二氢钾1.1g/L、琼脂13g/L和余量陈化海水或人工海水；调节pH为7.3，121℃高压灭菌25min。其中，人工海水配方为：氯化钠25.0 g/L、氯化镁1.9g/L、硫酸镁3.1 g/L、氯化钙1.3 g/L、碳酸氢钠0.2 g/L、氯化钾0.68g/L、溴化钠0.06 g/L、硼酸0.058 g/L、硅酸钠0.0024 g/L、磷酸0.002 g/L、六氯化二铝 0.014 g/L、氨水 0.002 g/L、硝酸锂 0.0014g/L、蒸馏水余量。液体发酵培养基为斜面培养基成分中不添加琼脂制得。上述培养基不仅能提供适合海洋细菌GHS18适宜的环境，还能提供菌种生长繁殖所需的碳源、氮源及生长因子，菌种的繁殖速度快，并且能诱导菌种分泌产生大量的胞外多糖，胞外多糖产量提高。
实施例2：
脱色絮凝剂的最优选制备方法，包括以下步骤：
1）种子液培养：将保藏号为CGMCC No. 12913的海洋细菌接种于斜面培养基，于25℃恒温培养36h。加入等体积无菌水，振荡制得种子液。斜面培养基成分及其浓度为葡萄糖22g/L、尿素0.47g/L、硫酸铵0.2g/L、酵母膏0.5g/L、磷酸氢二钾2.7g/L、磷酸二氢钾1.1g/L、琼脂13g/L和余量陈化海水或人工海水；调节pH为7.3，121℃高压灭菌25min。其中，人工海水配方为：氯化钠25.0 g/L、氯化镁1.9g/L、硫酸镁3.1 g/L、氯化钙1.3 g/L、碳酸氢钠0.2 g/L、氯化钾0.68g/L、溴化钠0.06 g/L、硼酸0.058 g/L、硅酸钠0.0024 g/L、磷酸0.002g/L、六氯化二铝 0.014 g/L、氨水 0.002 g/L、硝酸锂 0.0014g/L、蒸馏水余量；
2）扩大发酵培养：在每100ml液体发酵培养基接入0.4ml种子液，于25℃恒温摇床发酵培养3.6d，摇床速率为140r/min。液体发酵培养基成分及其浓度为葡萄糖22g/L、尿素0.47g/L、硫酸铵0.2g/L、酵母膏0.5g/L、磷酸氢二钾2.7g/L、磷酸二氢钾1.1g/L和余量陈化海水或人工海水；调节pH为7.3，121℃高压灭菌25min。其中，人工海水配方为：氯化钠25.0g/L、氯化镁1.9g/L、硫酸镁3.1 g/L、氯化钙1.3 g/L、碳酸氢钠0.2 g/L、氯化钾0.68g/L、溴化钠0.06 g/L、硼酸0.058 g/L、硅酸钠0.0024 g/L、磷酸0.002 g/L、六氯化二铝 0.014 g/L、氨水 0.002 g/L、硝酸锂 0.0014g/L、蒸馏水余量；
3）胞外多糖提取：将发酵完成的菌液离心，取上清液，在上清液中加入5倍体积的乙醇。在4℃下静置15h，离心取沉淀，真空干燥得胞外多糖粗制品；
4）絮凝剂的制备：在胞外多糖粗制品中加1.6倍水。振荡后加入5倍体积乙醇，乙醇温度为4℃。静置后离心取沉淀，真空干燥得到脱色絮凝剂。上述脱色絮凝剂脱色效果很好，对高岭土的絮凝率达到90％以上；对高浓度废水脱色具有较高活性，对亚甲基蓝、孔雀石绿、结晶紫的脱色率达92％以上、99％以上和98％以上；絮凝剂性质稳定，制备步骤简单，原料成本低廉，经济效益高，可大规模生产。
实施例3：絮凝活性的测定
采用絮凝活性测试法 (R.Kurane，K.Takeda，T.Suzuki.Screening andCharacteristeristics of Microbial Flocculants.Agric.Biol.Chem.1986 ：2301-2307) 对高岭土悬浊液的絮凝活性进行测定，取5g/L的高岭土悬浊液93ml，加入5ml 1％(m/v)CaCl2做助凝剂，加入 2ml 实施例2样品，调节 pH 值为 9，混合快速搅拌 1min，再慢速搅拌 5min，静置 10min，取水平面下 1cm 处混合液，可见分光光度计测 550nm 下吸光度 A，同时以 2ml 蒸馏水代替样品作为对照，测吸光度 B，计算絮凝率，FR(％ ) ＝ (A-B)/A×100％。则海洋细菌脱色絮凝剂对高岭土悬浊液的絮凝率为 91.41％。
实施例4：脱色活性测定
取10ml亚甲基蓝和孔雀石绿(10mg/L)以及10mL结晶紫(20mg/L)于大试管中，加入1ml 浓度为 2g/L 的实施例2样品，漩涡振荡 1min，调节溶液 pH 值为9，静置 30min ，取水平面下 1cm 处的溶液，测定相应染料在最大吸收波长处的吸光度，脱色前溶液的吸光度为A，脱色后溶液的吸光度为A0，计算脱色率，DR(％) ＝ (A-A0)/A×100％。则海洋细菌脱色絮凝剂对亚甲基蓝、孔雀石绿和结晶紫的脱色率分别为 92.21％、99.40％和 98.37％。
实施例5：沉降实验
在培养进入生长稳定期的100ml新鲜小球藻藻液中加入1ml浓度为2g/L的实施例2样品，混合快速搅拌1min，再慢速搅拌5min，静置30min，小球藻沉降率75%。
本发明的操作步骤中的常规操作为本领域技术人员所熟知，在此不进行赘述。
以上所述的实施例对本发明的技术方案进行了详细说明，应理解的是以上所述仅为本发明的具体实施例，并不用于限制本发明，凡在本发明的原则范围内所做的任何修改、补充或类似方式替代等，均应包含在本发明的保护范围之内。
SEQUENCE LISTING
<110> 浙江海洋大学
<120> 一株需氧活性海洋细菌及其脱色絮凝剂的制备方法
<130> 1
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 1277
<212> DNA
<213> 海洋菌株Pseudoalteromonas sp. GHS18
<400> 1
atgcttggga acatgccttg aggtggggga caacagttgg aaacgactgc taataccgca 60
taatgtctac ggaccaaagg gggcttcggc tctcgccttt agattggccc aagtgggatt 120
agctagttgg tgaggtaatg gctcaccaag gcgacgatcc ctagctggtt tgagaggatg 180
atcagccaca ctgggactga gacacggccc agactcctac gggaggcagc agtggggaat 240
attgcacaat gggcgcaagc ctgatgcagc catgccgcgt gtgtgaagaa ggccttcggg 300
ttgtaaagca ctttcagtca ggaggaaagg ttagtagtta atacctgcta tctgtgacgt 360
tactgacaga agaagcaccg gctaactccg tgccagcagc cgcggtaata cggagggtgc 420
gagcgttaat cggaattact gggcgtaaag cgtacgcagg cggtttgtta agcgagatgt 480
gaaagccccg ggctcaacct gggaactgca tttcgaactg gcaaactaga gtgtgataga 540
gggtggtaga atttcaggtg tagcggtgaa atgcgtagag atctgaagga ataccgatgg 600
cgaaggcagc cacctgggtc aacactgacg ctcatgtacg aaagcgtggg gagcaaacgg 660
gattagatac cccggtagtc cacgccgtaa acgatgtcta ctagaagctc ggagcctcgg 720
ttctgttttt caaagctaac gcattaagta gaccgcctgg ggagtacggc cgcaaggtta 780
aaactcaaat gaattgacgg gggcccgcac aagcggtgga gcatgtggtt taattcgatg 840
caacgcgaag aaccttacct acacttgaca tacagagaac ttaccagaga tggtttggtg 900
ccttcgggaa ctctgataca ggtgctgcat ggctgtcgtc agctcgtgtt gtgagatgtt 960
gggttaagtc ccgcaacgag cgcaacccct atccttagtt gctagcaggt aatgctgaga1020
actctaagga gactgccggt gataaaccgg aggaaggtgg ggacgacgtc aagtcatcat1080
ggcccttacg tgtagggcta cacacgtgct acaatggcgc atacagagtg ctgcgaactc1140
gcgagagtaa gcgaatcact taaagtgcgt cgtagtccgg attggagtct gcaactcgac1200
tccatgaagt cggaatcgct agtaatcgcg tatcagaatg acgcggtgaa tacgttcccg1260
ggccttgtac acaccgc 1277
Claims (6)
1.一株需氧活性海洋细菌，其特征在于：所述的海洋细菌菌株为：Pseudoalteromonassp.GHS18；保藏号为：CGMCC No.12913。
2.根据权利要求1所述的一株需氧活性海洋细菌，其特征在于：所述的海洋细菌Pseudoalteromonassp.GHS18菌株的斜面培养基成分及其浓度为：葡萄糖10-25g/L、尿素0.43-0.52g/L、硫酸铵0.18-0.24g/L、酵母膏0.4-0.6g/L、磷酸氢二钾1.25-5g/L、磷酸二氢钾0.5-2g/L、琼脂12-16g/L和余量陈化海水或人工海水；调节pH为7.2-7.4，115-121℃高压灭菌20-30min，其中，所述人工海水配方为：氯化钠25.0g/L、氯化镁1.9g/L、硫酸镁3.1g/L、氯化钙1.3g/L、碳酸氢钠0.2g/L、氯化钾0.68g/L、溴化钠0.06g/L、硼酸0.058g/L、硅酸钠0.0024g/L、磷酸0.002g/L、六氯化二铝0.014g/L、氨水0.002g/L、硝酸锂0.0014g/L、蒸馏水余量。
3.一株权利要求2所述的需氧活性海洋细菌的脱色絮凝剂的制备方法，其特征在于：包括种子液培养、扩大发酵培养、胞外多糖提取和絮凝剂的制备，所述的种子液培养步骤为：先在权利要求2所述的斜面培养基上接种海洋细菌Pseudoalteromonassp.GHS18，并于22-27℃恒温培养24-72h，将菌种活化，再加入培养基体积0.7-1.3倍的无菌水，振荡制得种子液。
4.根据权利要求3所述的一株需氧活性海洋细菌的脱色絮凝剂的制备方法，其特征在于：所述的扩大发酵培养步骤为：每100ml液体发酵培养基接入0.2-0.5ml种子液，于22-27℃恒温摇床发酵培养；摇床速率为100-140r/min，发酵培养时间为3-4d；所述的液体发酵培养为权利要求2所述的斜面培养基成分中不添加琼脂制得。
5.根据权利要求3所述的一株需氧活性海洋细菌的脱色絮凝剂的制备方法，其特征在于：所述的胞外多糖提取步骤为：先将发酵完成的菌液高速离心，取上清液，再在上清液中加入4-6倍体积的乙醇，并于2-5℃下静置10-18h，离心取沉淀，真空干燥得胞外多糖粗制品。
6.根据权利要求3所述的一株需氧活性海洋细菌的脱色絮凝剂的制备方法，其特征在于：所述的絮凝剂的制备步骤为：在胞外多糖粗制品中加水溶解，振荡后加入4-6倍体积的乙醇，静置后离心取沉淀，真空干燥得到脱色絮凝剂；水为粗制品体积的1-2倍，乙醇温度为2-5℃。
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