CN107502569B - 用于脱色絮凝的复合菌剂及其絮凝剂的制备方法 - Google Patents
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Abstract
本发明公开了用于脱色絮凝的复合菌剂及其絮凝剂的制备方法,复合菌剂包括保藏号为CGMCC No.12913的假交替单胞菌Pseudoalteromonassp.GHS18和保藏号为CGMCC No.12914的交替假单胞菌Pseudoalteromonassp.JP。由复合菌剂协同发酵得到的胞外多糖制得絮凝剂。有益效果为:制得的脱色絮凝剂脱色效果很好,对高岭土的絮凝率达到92%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95%以上、99.6%以上和99%以上,对生物体无直接或潜在毒性,可自然降解,绿色健康,无污染。
Description
技术领域
本发明涉及净水技术领域,具体是用于脱色絮凝的复合菌剂及其絮凝剂的制备方法。
背景技术
絮凝技术被广泛应用在给水、废水絮凝净化、发酵工业、固液分离等方面,絮凝效果取决于选取的絮凝剂的种类。 目前,用于水处理行业的絮凝剂主要有两大类 :以铝系、铁系及其聚合物为代表的无机絮凝剂和以PAM及其衍生物为代表的有机高分子絮凝剂。它们凭借其在使用过程中絮凝效果好且成本低得到广泛的应用,但是它们对环境存在不安全性已引起人们足够的重视。 铝系絮凝剂中铝离子易引起老年性痴呆症 ;铁系絮凝剂中铁离子对金属有腐蚀作用,容易引起处理后水中带有颜色,增加污泥处理负荷;PAM的单体有很强的神经毒性,有很强的致癌性。
现有技术如授权公告号为CN 101580550 B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、墨水蓝、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菌种。
发明内容
本发明的目的在于提供一种用于脱色絮凝的复合菌剂及其絮凝剂的制备方法,假交替单胞菌Pseudoalteromonas sp. GHS18和交替假单胞菌Pseudoalteromonas sp. JP协同发酵得到的胞外多糖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,采取的技术方案为:假交替单胞菌Pseudoalteromonas sp. GHS18和交替假单胞菌Pseudoalteromonas sp. JP协同发酵得到的胞外多糖制得絮凝剂。其中,假交替单胞菌Pseudoalteromonas sp. GHS18的分类命名为假交替单胞菌(Pseudoalteromonas sp.),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。交替假单胞菌Pseudoalteromonas sp. JP的分类命名为交替假单胞菌(Pseudoalteromonas sp.),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. 12914。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。上述两株海洋细菌均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。
海洋菌株GHS18的16S rDNA全基因(1277 bp)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。
海洋菌株JP的16S rDNA全基因(1279 bp)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KX702268。其全序列如下:
AATGCTTGGGAACATGCCTTGAGGTGGGGGACAACAGTTGGAAACGACTGCTAATACCGCATAATGTCTACGGACCAAAGGGGGCTTCGGCTCTCGCCTTTAGATTGGCCCAAGTGGGATTAGCTAGTTGGTGAGGTAATGGCTCACCAAGGCGACGATCCCTAGCTGGTTTGAGAGGATGATCAGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCCATGCCGCGTGTGTGAAGAAGGCCTTCGGGTTGTAAAGCACTTTCAGTCAGGAGGAAAGGTTAGTAGTTAATACCTGCTAGCTGTGACGTTACTGACAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCGAGCGTTAATCGGAATTACTGGGCGTAAAGCGTACGCAGGCGGTTTGTTAAGCGAGATGTGAAAGCCCCGGGCTCAACCTGGGAACTGCATTTCGAACTGGCAAACTAGAGTGTGATAGAGGGTGGTAGAATTTCAGGTGTAGCGGTGAAATGCGTAGAGATCTGAAGGAATACCGATGGCGAAGGCAGCCACCTGGGTCAACACTGACGCTCATGTACGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCTACTAGAAGCTCGGAACCTCGGTTCTGTTTTTCAAAGCTAACGCATTAAGTAGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTACACTTGACATACAGAGAACTTACCAGAGATGGTTTGGTGCCTTCGGGAACTCTGATACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCCTATCCTTAGTTGCTAGCAGGTAATGCTGAGAACTCTAAGGAGACTGCCGGTGATAAACCGGAGGAAGGTGGGGACGACGTCAAGTCATCATGGCCCTTACGTGTAGGGCTACACACGTGCTACAATGGCGCATACAGAGTGCTGCGAACCTGCGAAGGTAAGCGAATCACTTAAAGTGCGTCGTAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGCGTATCAGAATGACGCGGTGAATACGTTCCCGGGCCTTGTACA CACCGCC。
絮凝剂的制备方法,包括以下步骤:
种子液培养:将复合菌剂接种于斜面培养基,于22-27℃恒温培养24-72h后,加入培养基体积0.7-1.3倍的无菌水,振荡制得种子液;其中,复合菌剂中假交替单胞菌Pseudoalteromonas sp. GHS18和交替假单胞菌Pseudoalteromonas sp. JP的质量比为0.5-2.5:0.5-2.5。上述种子液的培养可将菌种从保藏状态恢复到活力旺盛的状态,并且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3-4d,摇床速率为100-140r/min。在发酵培养期间,两株海洋细菌之间互利共栖协同作用,菌种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:将发酵完成的菌液离心,取上清液,在上清液中加入上清液体积4-6倍的乙醇,在2-5℃下静置10-18h,离心取沉淀,真空干燥得胞外多糖粗制品。亲水的多糖的水化层被乙醇破坏,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,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℃。假交替单胞菌Pseudoalteromonas sp. GHS18和交替假单胞菌Pseudoalteromonas sp. JP之间的生长繁殖有明显的协同作用,制备得到的絮凝剂脱色絮凝效果均优于单一菌种发酵得到的絮凝剂,具有明显进步。制得的脱色絮凝剂脱色效果很好,对高岭土的絮凝率达到92%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95%以上、99.6%以上和99%以上。
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。
与现有技术相比,本发明的优点在于:假交替单胞菌Pseudoalteromonas sp.GHS18和交替假单胞菌Pseudoalteromonas sp. JP之间的生长繁殖有明显的协同作用,制备得到的絮凝剂脱色絮凝效果均优于单一菌种发酵得到的絮凝剂,具有明显进步。制得的脱色絮凝剂脱色效果很好,对高岭土的絮凝率达到92%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95%以上、99.6%以上和99%以上;絮凝剂对生物体无直接或潜在毒性,可自然降解,绿色健康,无污染;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。
具体实施方式
下面通过实施例对本发明方案作进一步说明:
实施例1:
假交替单胞菌Pseudoalteromonas sp. GHS18和交替假单胞菌Pseudoalteromonas sp. JP协同发酵得到的胞外多糖制得的絮凝剂。其中,假交替单胞菌Pseudoalteromonas sp. GHS18的分类命名为假交替单胞菌(Pseudoalteromonas sp.),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。交替假单胞菌Pseudoalteromonas sp. JP的分类命名为交替假单胞菌(Pseudoalteromonas sp.),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. 12914。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。上述两株海洋细菌均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。
絮凝剂的最优选制备方法,包括以下步骤:
1)种子液培养:将复合菌剂接种于斜面培养基,于25℃恒温培养36h后,加入培养基体积1.2倍的无菌水,振荡制得种子液;其中,复合菌剂中假交替单胞菌Pseudoalteromonas sp. GHS18和交替假单胞菌Pseudoalteromonas sp. JP的质量比为1:1。上述种子液的培养可将菌种从保藏状态恢复到活力旺盛的状态,并且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,两株海洋细菌之间互利共栖协同作用,菌种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。亲水的多糖的水化层被乙醇破坏,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,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,真空干燥得到絮凝剂。假交替单胞菌Pseudoalteromonas sp. GHS18和交替假单胞菌Pseudoalteromonas sp. JP之间的生长繁殖有明显的协同作用,制备得到的絮凝剂脱色絮凝效果均优于单一菌种发酵得到的絮凝剂,具有明显进步。制得的脱色絮凝剂脱色效果很好,对高岭土的絮凝率达到92%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95%以上、99.6%以上和99%以上。
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。上述培养基不仅能提供适合复合菌剂适宜的环境,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:
絮凝剂的制备方法,包括以下步骤:
1)种子液培养:将复合菌剂接种于斜面培养基,于23℃恒温培养72h后,加入培养基体积1.3倍的无菌水,振荡制得种子液;其中,复合菌剂中假交替单胞菌Pseudoalteromonas sp. GHS18和交替假单胞菌Pseudoalteromonas sp. JP的质量比为1:2。斜面培养基成分及其浓度为葡萄糖18g/L、尿素0.51g/L、硫酸铵0.24g/L、酵母膏0.4g/L、磷酸氢二钾3.5g/L、磷酸二氢钾1.7g/L、琼脂15g/L和余量陈化海水或人工海水;调节pH为7.4,121℃高压灭菌30min。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0.058g/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;
2)扩大发酵培养:在每100ml液体发酵培养基接入0.2ml种子液,于23℃恒温摇床发酵培养4d,摇床速率为120r/min。液体发酵培养基成分及其浓度为葡萄糖18g/L、尿素0.51g/L、硫酸铵0.24g/L、酵母膏0.4g/L、磷酸氢二钾3.5g/L、磷酸二氢钾1.7g/L和余量陈化海水或人工海水;调节pH为7.4,121℃高压灭菌30min。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;
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6倍体积的乙醇。在3℃下静置18h,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1.1倍水。振荡后加入6倍体积乙醇,乙醇温度为3℃。静置后离心取沉淀,真空干燥得到胞外多糖即微生物絮凝剂。假交替单胞菌Pseudoalteromonas sp. GHS18和交替假单胞菌Pseudoalteromonas sp. JP之间的生长繁殖有明显的协同作用,制备得到的絮凝剂脱色絮凝效果均优于单一菌种发酵得到的絮凝剂,具有明显进步。制得的脱色絮凝剂脱色效果很好,对高岭土的絮凝率达到92%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95%以上、99.6%以上和99%以上。
实施例3:絮凝活性的测定
采用对高岭土悬浊液的絮凝活性测试法 (R.Kurane,K.Takeda,T.Suzuki.Screening and Characteristeristics of Microbial Flocculants.Agric.Biol.Chem.1986 :2301-2307)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 93.72%。
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 95.80%、99.87%和 99.32%。
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82%。
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> 用于脱色絮凝的复合菌剂及其絮凝剂的制备方法
<130> 2
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 1277
<212> DNA
<213> 海洋细菌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 aatgctgaga 1020
actctaagga gactgccggt gataaaccgg aggaaggtgg ggacgacgtc aagtcatcat 1080
ggcccttacg tgtagggcta cacacgtgct acaatggcgc atacagagtg ctgcgaactc 1140
gcgagagtaa gcgaatcact taaagtgcgt cgtagtccgg attggagtct gcaactcgac 1200
tccatgaagt cggaatcgct agtaatcgcg tatcagaatg acgcggtgaa tacgttcccg 1260
ggccttgtac acaccgc 1277
<210> 2
<211> 1279
<212> DNA
<213> 海洋细菌Pseudoalteromonas sp. JP
<400> 2
aatgcttggg aacatgcctt gaggtggggg acaacagttg gaaacgactg ctaataccgc 60
ataatgtcta cggaccaaag ggggcttcgg ctctcgcctt tagattggcc caagtgggat 120
tagctagttg gtgaggtaat ggctcaccaa ggcgacgatc cctagctggt ttgagaggat 180
gatcagccac actgggactg agacacggcc cagactccta cgggaggcag cagtggggaa 240
tattgcacaa tgggcgcaag cctgatgcag ccatgccgcg tgtgtgaaga aggccttcgg 300
gttgtaaagc actttcagtc aggaggaaag gttagtagtt aatacctgct agctgtgacg 360
ttactgacag aagaagcacc ggctaactcc gtgccagcag ccgcggtaat acggagggtg 420
cgagcgttaa tcggaattac tgggcgtaaa gcgtacgcag gcggtttgtt aagcgagatg 480
tgaaagcccc gggctcaacc tgggaactgc atttcgaact ggcaaactag agtgtgatag 540
agggtggtag aatttcaggt gtagcggtga aatgcgtaga gatctgaagg aataccgatg 600
gcgaaggcag ccacctgggt caacactgac gctcatgtac gaaagcgtgg ggagcaaaca 660
ggattagata ccctggtagt ccacgccgta aacgatgtct actagaagct cggaacctcg 720
gttctgtttt tcaaagctaa cgcattaagt agaccgcctg gggagtacgg ccgcaaggtt 780
aaaactcaaa tgaattgacg ggggcccgca caagcggtgg agcatgtggt ttaattcgat 840
gcaacgcgaa gaaccttacc tacacttgac atacagagaa cttaccagag atggtttggt 900
gccttcggga actctgatac aggtgctgca tggctgtcgt cagctcgtgt tgtgagatgt 960
tgggttaagt cccgcaacga gcgcaacccc tatccttagt tgctagcagg taatgctgag 1020
aactctaagg agactgccgg tgataaaccg gaggaaggtg gggacgacgt caagtcatca 1080
tggcccttac gtgtagggct acacacgtgc tacaatggcg catacagagt gctgcgaacc 1140
tgcgaaggta agcgaatcac ttaaagtgcg tcgtagtccg gattggagtc tgcaactcga 1200
ctccatgaag tcggaatcgc tagtaatcgc gtatcagaat gacgcggtga atacgttccc 1260
gggccttgta cacaccgcc 1279
Claims (6)
1.用于脱色絮凝的复合菌剂,其特征在于:
所述的复合菌剂由保藏号为CGMCC No. 12913的假交替单胞菌Pseudoalteromonassp. GHS18和保藏号为CGMCC No. 12914的交替假单胞菌Pseudoalteromonas sp. JP组成。
2.一种用于脱色絮凝的复合菌剂絮凝剂的制备方法,其特征在于:所述的絮凝剂的制备方法包括将权利要求1所述复合菌剂的种子液培养、扩大发酵培养、胞外多糖提取和絮凝剂的制备。
3. 根据权利要求2所述的用于脱色絮凝的复合菌剂絮凝剂的制备方法,其特征在于:所述的种子液培养步骤为:将复合菌剂接种于斜面培养基,于22-27℃恒温培养24-72h后,加入培养基体积0.7-1.3倍的无菌水,振荡制得种子液;其中,复合菌剂中假交替单胞菌Pseudoalteromonas sp. GHS18和交替假单胞菌Pseudoalteromonas sp. JP的质量比为0.5-2.5:0.5-2.5。
4.根据权利要求2所述的用于脱色絮凝的复合菌剂絮凝剂的制备方法,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3-4d,摇床速率为100-140r/min。
5.根据权利要求2所述的用于脱色絮凝的复合菌剂絮凝剂的制备方法,其特征在于:所述的胞外多糖提取步骤为:将发酵完成的菌液离心,取上清液,在上清液中加入上清液体积4-6倍的乙醇,在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2所述的用于脱色絮凝的复合菌剂絮凝剂的制备方法,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,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℃,得到絮凝剂。
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