CN106635866A - 一种排硫硫杆菌及高活性培养的方法 - Google Patents
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- QXNVGIXVLWOKEQ-UHFFFAOYSA-N Disodium Chemical compound [Na][Na] QXNVGIXVLWOKEQ-UHFFFAOYSA-N 0.000 claims description 8
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- JZCCFEFSEZPSOG-UHFFFAOYSA-L copper(II) sulfate pentahydrate Chemical compound O.O.O.O.O.[Cu+2].[O-]S([O-])(=O)=O JZCCFEFSEZPSOG-UHFFFAOYSA-L 0.000 claims description 8
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- TZBAVQKIEKDGFH-UHFFFAOYSA-N n-[2-(diethylamino)ethyl]-1-benzothiophene-2-carboxamide;hydrochloride Chemical compound [Cl-].C1=CC=C2SC(C(=O)NCC[NH+](CC)CC)=CC2=C1 TZBAVQKIEKDGFH-UHFFFAOYSA-N 0.000 description 1
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Abstract
本发明公开了一种排硫硫杆菌及高活性培养的方法,其分类名为排硫硫杆菌,菌种的拉丁学名为Thiobacillus thioparus,保藏日期是2016年7月11日,保藏中心登记入册编号是CGMCC No.12756。将自养培养基和保护剂于容器中配制混合培养基,混合培养基的装液量为容器体积容量的10%~20%;对培养基灭菌;按混合培养基体积比的2~5%接种排硫杆菌;控制温度28~30℃,摇床转速为160~180rpm/min,培养48h~72h。采用该方法培养排硫硫杆菌,能有效避免化能自养培养基中无机盐对排硫硫杆菌细胞膜和细胞壁产生的蛋白质盐溶现象,降低菌体死亡率提高菌体活性。此外,该发明具有操作简单、成本低廉、效果明显等特点。
Description
技术领域
本发明涉及生物化工领域,具体涉及一种排硫硫杆菌及高活性培养的方法,采用本方法培养排硫硫杆菌能极大降低菌体死亡率,明显提高单位菌液中活菌数。
背景技术
传统的排硫硫杆菌培养方法是依据化能自养细菌生长需求,以硫代硫酸盐等简单无机盐作为碳源、氮源。排硫硫杆菌在这类培养基下生长缓慢,至少需要5天才会在培养基中看见直径小于1毫米的圆形菌落。排硫硫杆菌在这一类培养基中与游离硫形成粘膜,长时间静置,代谢产生的硫会在培养基中形成白色或淡黄色沉淀。这类由NH4Cl、Fe2+等构成的培养基有较强盐腐蚀性,即增加蛋白质分子表面电荷,增强蛋白质分子和水分子的作用,使蛋白质在水中溶解度增大。对排硫硫杆菌的细胞膜和细胞壁产生蛋白质盐溶现象。在菌体培养过程中,一部分排硫硫杆菌因为蛋白质盐溶而造成细胞壁破裂而死亡。最终导致菌体培养周期长,活菌数低等现象。
随着利用微生物代谢机制净化含硫化物的废气、废水研究在学术圈逐渐展开,排硫硫杆菌培养周期长、活菌数低等问题使得该菌难以适应工业化的效率高、周期短、成本低的特性。如何避免排硫硫杆菌在含硫溶液中发生盐溶现象,提高活菌数和菌体活力成为将微生物应用到工业的技术关键。
发明内容
本发明的目的是提供一种排硫硫杆菌,本发明的另一目的是提供一种利用上述排硫硫杆菌进行高活性培养的方法。
本发明的技术方案为:一种硫杆菌,其分类名为排硫硫杆菌,菌种的拉丁学名为Thiobacillus thioparus,保藏日期是2016年7月11日,保藏中心登记入册编号是CGMCCNo.12756。
本发明还提供了一种利用上述的硫杆菌进行高活性培养的方法,其具体步骤如下:将自养培养基和保护剂于容器中配制混合培养基,混合培养基的装液量为容器体积容量的10%~20%;对培养基灭菌;按混合培养基体积比的2~5%接种排硫杆菌;控制温度28~30℃,摇床转速为160~180rpm/min,培养48h~72h,可清楚看见白黄色菌落。
优选混合培养基中自养培养基组分为:Na2S2O3 9~10g/L、KH2PO43~5g/L、K2HPO44~5g/L、MgSO4.7H2O 0.7~0.9g/L、NH4Cl 0.4~0.6g/L、乙二酸四乙酸二钠0.5~0.6g/L、ZnSO4.7H2O 0.22~0.32g/L、CaCl20.05~0.08g/L、MnCl2.4H2O0.05~0.08g/L、FeSO4.7H2O0.05~0.08g/L、(NH4)6Mo7O240.01~0.03g/L、CuSO4.5H2O 0.01~0.03g/L和CoCl2.6H2O0.01~0.03g/L。
优选混合培养基中保护剂的组分及各组分占自养培养基的质量百分比分别为:2~4%海藻糖,0.1~0.3%聚乙二醇4000(PEG),0.3~0.5%蔗糖,0.2~0.4%NaCl。
优选混合培养基的pH值为6.5~7.0。
对培养基灭菌,一般采用高压蒸汽灭菌,其中Na2S2O3和保护剂不可采用高温灭菌法,可采用膜过滤或紫外灭菌法。
CGMCC No.12756菌株具有以下性质:
1、培养特征:
在固体LB培养基上培养,呈白色或淡黄色、微透明小圆点状,有光泽,边缘光滑整齐。
2、菌体形态特征:
在固体LB培养基上呈白色或淡黄色球形或椭圆型,直径0.5~1.7微米,单细胞外通常有一层荚膜。以极生鞭毛运动。
3、生理生化性质:
表1 CGMCC No.12756菌株生理生化性质
+:为可利用;-:为不可利用。
有益效果:
本发明主要贡献在于抗盐溶菌体保护剂的研发。保护剂能够在细菌外表面形成一层保护膜,在不影响离子通道的同时降低菌体内外渗透压差,保护菌体免受盐溶侵蚀,从而提高单位体积内活菌数。解决了细菌在高含盐环境中细胞壁因蛋白质盐溶而造成的破裂及失活现象。本发明具有操作简单、成本低廉、效果明显等特点。
保藏信息
上述硫杆菌其分类名为排硫硫杆菌,菌种的拉丁学名为Thiobacillusthioparus,参据的微生物(株)为:NJYH5327,该菌株是本课题组自主筛选并保藏于中国微生物菌种保藏管理委员会普通微生物中心(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),其简称为CGMCC,保藏日期是2016年7月11日,登记入册的编号是CGMCCNo.12756。
具体实施方式
以下结合实例,对本发明的具体实施方式作进一步描述。实例中所用脱硫杆菌为实验室自主筛选获得。
实例1
按上诉排硫硫杆菌培养基成分分别配制A1、B1两个培养基,A1:Na2S2O3 9g/L、KH2PO43g/L、K2HPO44g/L、MgSO4.7H2O 0.7g/L、NH4Cl 0.4g/L、乙二酸四乙酸二钠0.5g/L、ZnSO4.7H2O 0.22g/L、CaCl20.05g/L、MnCl2.4H2O 0.05~g/L、FeSO4.7H2O 0.05g/L、(NH4)6Mo7O240.01g/L、CuSO4.5H2O 0.01g/L、CoCl2.6H2O0.01g/L,pH6.8;B1含有本发明的菌体保护剂,其中自养培养基组分为:Na2S2O39g/L、KH2PO43g/L、K2HPO44g/L、MgSO4.7H2O 0.7g/L、NH4Cl 0.4g/L、乙二酸四乙酸二钠0.5g/L、ZnSO4.7H2O 0.22g/L、CaCl20.05g/L、MnCl2.4H2O0.05~g/L、FeSO4.7H2O 0.05g/L、(NH4)6Mo7O240.01g/L、CuSO4.5H2O 0.01g/L、CoCl2.6H2O0.01g/L;再加入占上述自养培养基质量百分比为:2wt%海藻糖+0.1wt%聚乙二醇4000(PEG)+0.3wt%蔗糖+0.2wt%NaCl;得混合培养基,其pH6.8。将排硫硫杆菌按培养基容积的2%分别接种于A1、B1两个培养基,摇床转速160rpm/min,温度28℃条件下培养48h。将A1、B1两个培养基所培养菌液分别吸取1ml,稀释100倍后吸取100μl菌悬液涂布于直径为9cm的无菌空白培养基A1、B1上,在最适条件下继续培养5天计算菌落数从而计算活菌数。硫离子含量采用国家环境保护总局标准,《水质硫化物的测定碘量法HJ/T60—2000》。
没有添加本发明的培养基A1活菌数为3.754×109cfu/ml,添加有本发明的培养基B1活菌数为5.920×109cfu/ml,培养条件B1较培养条件A1活菌数提高了157.69%。对培养前后硫化物含量检测表明,相同体积的菌液,培养条件B1较培养条件A1对硫化物代谢能力提高19.4%。
实例2
分别配制A2、B2两个培养基,A2:Na2S2O3 10g/L、KH2PO44g/L、K2HPO45g/L、MgSO4.7H2O0.8g/L、NH4Cl 0.5g/L、乙二酸四乙酸二钠0.6g/L、ZnSO4.7H2O0.32g/L、CaCl20.07g/L、MnCl2.4H2O 0.06g/L、FeSO4.7H2O 0.06g/L、(NH4)6Mo7O240.02g/L、CuSO4.5H2O 0.02g/L、CoCl2.6H2O 0.02g/L,pH6.9,B2含有本发明的菌体保护剂,其中自养培养基组分为:Na2S2O310g/L、KH2PO44g/L、K2HPO45g/L、MgSO4.7H2O 0.8g/L、NH4Cl 0.5g/L、乙二酸四乙酸二钠0.6g/L、ZnSO4.7H2O 0.32g/L、CaCl20.07g/L、MnCl2.4H2O 0.06g/L、FeSO4.7H2O 0.06g/L、(NH4)6Mo7O240.02g/L、CuSO4.5H2O 0.02g/L、CoCl2.6H2O 0.02g/L;再加入占上述自养培养基质量百分比为3wt%海藻糖+0.2wt%聚乙二醇4000(PEG)+0.4wt%蔗糖+0.3wt%NaCl;得混合培养基,其pH6.9。将排硫硫杆菌按培养基容积的3%分别接种于A2、B2两个培养基,摇床转速为170rpm/min,温度29℃条件下培养60h。将A2、B2两个培养基所培养菌液分别吸取1ml,稀释100倍后吸取100μl菌悬液涂布于直径为9cm的无菌空白培养基A2、B2上,在最适条件下继续培养5天计算菌落数从而计算活菌数。硫离子含量采用国家环境保护总局标准,《水质硫化物的测定碘量法HJ/T60—2000》。
没有添加本发明的培养基A2活菌数为3.533×109cfu/ml,添加有本发明的培养基B2活菌数为7.458×109cfu/ml,培养条件B2较培养条件A2活菌数提高了211.09%。对培养前后硫化物含量检测表明,相同体积的菌液,培养条件B2较培养条件A2对硫化物代谢能力提高32.8%。
实例3
分别配制A3、B3两个培养基,A3:Na2S2O3 10g/L、KH2PO45g/L、K2HPO45g/L、MgSO4.7H2O0.9g/L、NH4Cl 0.6g/L、乙二酸四乙酸二钠0.6g/L、ZnSO4.7H2O0.32g/L、CaCl20.08g/L、MnCl2.4H2O 0.08g/L、FeSO4.7H2O 0.08g/L、(NH4)6Mo7O240.03g/L、CuSO4.5H2O 0.03g/L、CoCl2.6H2O 0.03g/L,pH7.0,B3含有本发明的菌体保护剂,其中自养培养基组分为:Na2S2O310g/L、KH2PO45g/L、K2HPO45g/L、MgSO4.7H2O 0.9g/L、NH4Cl 0.6g/L、乙二酸四乙酸二钠0.6g/L、ZnSO4.7H2O 0.32g/L、CaCl20.08g/L、MnCl2.4H2O 0.08g/L、FeSO4.7H2O 0.08g/L、(NH4)6Mo7O240.03g/L、CuSO4.5H2O 0.03g/L、CoCl2.6H2O 0.03g/L;再加入占上述自养培养基质量百分比为:4wt%海藻糖+0.3wt%聚乙二醇4000(PEG)+0.5wt%蔗糖+0.4wt%NaCl;得混合培养基,其pH7.0。将排硫硫杆菌按5%分别接种于A3、B3两个培养基,摇床转速180rpm/min,温度30℃条件下培养72h。将A3、B3两个培养基所培养菌液分别吸取1ml,稀释100倍后吸取100μl菌悬液涂布于直径为9cm的无菌空白培养基A3、B3上,在最适条件下继续培养5天计算菌落数从而计算活菌数。硫离子含量采用国家环境保护总局标准,《水质硫化物的测定碘量法HJ/T60—2000》。
没有添加本发明的培养基A3活菌数为3.607×109cfu/ml,添加有本发明的培养基B3活菌数为6.273×109cfu/ml,培养条件B3较培养条件A3活菌数提高了177.55%。对培养前后硫化物含量检测表明,相同体积的菌液,培养条件B3较培养条件A3对硫化物代谢能力提高24.8%。
最终结果表明,该排硫硫杆菌培养方法具有明显可行性,该发明具有操作简单、成本低廉、效果明显等特点。
Claims (5)
1.一种硫杆菌,其分类名为排硫硫杆菌,菌种的拉丁学名为Thiobacillus thioparus,保藏日期是2016年7月11日,保藏中心登记入册编号是CGMCC No.12756。
2.一种利用如权利要求1所述的硫杆菌进行高活性培养的方法,其具体步骤如下:将自养培养基和保护剂于容器中配制混合培养基,混合培养基的装液量为容器体积容量的10%~20%;对培养基灭菌;按混合培养基体积比的2~5%接种排硫杆菌;控制温度28~30℃,摇床转速为160~180rpm/min,培养48h~72h。
3.根据权利要求2所述的方法,其特征在于混合培养基中自养培养基组分为:Na2S2O3 9~10g/L、KH2PO4 3~5g/L、K2HPO4 4~5g/L、MgSO4.7H2O 0.7~0.9g/L、NH4Cl 0.4~0.6g/L、乙二酸四乙酸二钠0.5~0.6g/L、ZnSO4.7H2O 0.22~0.32g/L、CaCl2 0.05~0.08g/L、MnCl2.4H2O 0.05~0.08g/L、FeSO4.7H2O 0.05~0.08g/L、(NH4)6Mo7O24 0.01~0.03g/L、CuSO4.5H2O 0.01~0.03g/L和CoCl2.6H2O 0.01~0.03g/L。
4.根据权利要求2所述的方法,其特征在于混合培养基中保护剂的组分及各组分占自养培养基的质量百分比分别为:2~4%海藻糖,0.1~0.3%聚乙二醇4000,0.3~0.5%蔗糖,0.2~0.4%NaCl。
5.根据权利要求2所述的方法,其特征在于混合培养基的pH值为6.5~7.0。
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