CN116855412A - 一种短程硝化混合菌培养及冷冻干燥保存与快速恢复活性的方法 - Google Patents
一种短程硝化混合菌培养及冷冻干燥保存与快速恢复活性的方法 Download PDFInfo
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Abstract
本发明公开了一种短程硝化混合菌培养及冷冻干燥保存与快速恢复活性的方法,本发明采用甘油作为短程硝化混合菌冻干保存法中的保护剂,以消除现有常见真空冷冻干燥方法的不利影响,提供短程硝化混合菌冷干保存后的快速恢复活性的方法,可以达到3天左右恢复活性,活化后亚硝转化率可达90%以上。
Description
技术领域
本发明涉及一种短程硝化混合菌培养及冷冻干燥保存与快速恢复活性的方法。
背景技术
氮素在水体中的过度积累造成了水体富营养化现象,严重危害生态系统安全。一般采用生物法进行废水脱氮。随着低碳高氮废水排放量的急剧增加,传统生物脱氮工艺已无法满足实用要求,研发新型的生物脱氮工艺迫在眉梢。短程硝化-反硝化工艺(SBNR)是在此背景下应运而生的一种新型生物脱氮工艺,其将氨氮只进行亚硝化过程,控制在亚硝酸盐阶段,然后进行反硝化作用转化为氮气。该工艺较传统的完全硝化反硝化工艺,缩短了氮素转化历程,节省了25%的需氧量,降低了40%的反硝化阶段所需碳源,同时可与厌氧氨氧化工艺等组合,开发其它新型生物脱氮工艺。
硝化细菌在启动及运行短程硝化工艺中起着至关重要的作用,它们是短程硝化的承载者,其数量和活性决定了硝化作用的潜力。硝化细菌存在生长速度极慢、细胞产率低的特点,导致短程硝化-反硝化工艺难以启动,同时硝化细菌对氧气、pH、温度等环境条件较敏感,致使短程硝化-反硝化工艺启动后容易失去稳定。随着废水生物脱氮工程的普及,对高效硝化菌种的需求日益增大,如何经济有效地扩增和保存高效硝化菌种已成了人们关注的焦点,硝化细菌属中温菌,低温可降低硝化细菌的代谢活性,目前对于硝化细菌的保存主要是在低温下进行,且通常用于纯种保存,关于短程硝化混合菌种的保存鲜有报道,纯种菌种保存条件较为苛刻,保存费用较高,不利于硝化细菌的大规模使用和商业推广。同时常温下的短程硝化污泥经长时间储存后(>6个月),好氧污泥的活性恢复时间较长,大约需要一个月左右,且恢复后结构不稳定,对氨氮去除率较低,不能够达到理想要求。
发明内容
本发明是为了解决上述现有技术存在的问题而提供一种短程硝化混合菌培养及冷冻干燥保存与快速恢复活性的方法。
本发明所采用的技术方案有:
使用NH4Cl-0.4g,KH2PO4-0.08g,NaHCO3-1g,MgSO4·7H2O-0.02g,CaCl2·2H2O-0.01g、微量元素溶液1mL以及1000mL超纯水作为筛选培养基,调节pH7.8-8.5,添加污泥50mL培养,经过4个周期总共20d后,即得到短程硝化混合菌,所述短程硝化混合菌的氨氮降解率稳定在90%以上,亚硝酸盐氮转化率稳定在90%以上,同时菌液浓度达到1*108个/mL;
2)保存前测定短程硝化混合菌的短程硝化能力;
前期筛选的短程硝化混合菌以NH4Cl作为唯一氮源,氨氮浓度为300-400mg/L,磷酸盐浓度为60-80mg/L,镁离子浓度20mg/L,钙离子浓度10mg/L,碳酸氢钠浓度100mg/L,pH7.8,曝气溶氧控制在4-6mg/L,将短程硝化混合菌培养至亚硝转化率达90%以上,并收集菌体;
3)短程硝化混合菌的冷冻干燥保存与活性恢复;
冷冻干燥保存:取适量清洗后的短程硝化混合菌置于50mL离心管中,离心(4000r/min,10min)后弃置上清液,之后向离心管中加入适量0.2mol/L磷酸盐缓冲液,旋转混合后再离心(4000r/min,10min)弃置上清液,此过程进行三次,之后再用基质溶液清洗离心一次,混匀之后向培养皿中加入适量清洗离心后的湿污泥,加入保护剂,然后放入-20℃冰箱24h进行预冻;
预冻完成后将各表面皿放入Flexi-Dry真空冷冻干燥机中冷冻干燥24h,冻干完成后,将获得的冻干活性污泥粉储存在真空袋中,在4℃、遮光条件下保藏6个月;
活性恢复:保藏期结束后,先将保存菌粉置于室温,之后各菌粉加入适量0.2mol/L磷酸盐缓冲液清洗离心三次,离心清洗完成后,弃置上清液并将污泥转移至含有培养底物的培养瓶中,放入4℃冰箱中使细菌细胞重新补充水分;24h后弃置培养瓶上清液并再次对各瓶内污泥清洗离心,之后重新加入基质溶液并曝氧气5min后放入恒温摇床中开始活化过程,活化过程定期监测氨氮、亚硝态氮浓度。
进一步地,所述微量元素溶液,其配方为:氯化锌80mg,无水硫酸铜20mg,硼酸20mg,七水硫酸亚铁100mg、水1000mL。
进一步地,所述基质溶液的配方为:100mg/L氨氮,20mg/L无机磷,20mg/L MgSO4·H2O,10mg/L CaCl·H2O,100mg/L NaHCO3。
进一步地,所述保护剂为甘油,加入保护剂的质量为污泥、基质溶液和保护剂三者总质量的3%。
进一步地,所述培养底物包括:氨氮100mg/L,无机磷20mg/L,MgSO4·H2O 20mg/L,CaCl·H2O 10mg/L以及碳酸氢钠100mg/L。
本发明具有如下有益效果:
本发明采用甘油作为短程硝化混合菌冻干保存法中的保护剂,以消除现有常见真空冷冻干燥方法的不利影响。本发明可以达到3天左右恢复活性,活化后亚硝转化率可达90%以上。
附图说明
图1为本发明真空冷冻保存前后样品形态;
图2为短程硝化混合菌的冻干后在较高氨氮废水中短程硝化活性恢复实验中氨氮、亚硝态氮浓度变化曲线;
图3为短程硝化混合菌的冻干粉在实际工业废水中的应用中硝态氮、亚硝态氮变化曲线。
具体实施方式
下面结合附图对本发明作进一步的说明。
如图1至图3,本发明实施例中氨氮的测定方法为:纳氏试剂光度法;亚硝态氮的测定方法:紫外分光光度法。
短程硝化混合菌的培养:
利用合适的培养液,培养液成分:NH4Cl 0.4g、KH2PO4 0.08g、NaHCO31g、MgSO4·7H2O 0.02g、CaCl2·2H2O 0.01g、微量元素溶液1mL/L(氯化锌80mg,无水硫酸铜20mg,硼酸20mg,七水硫酸亚铁100mg)、水1000mL,pH7.8-8.5,作为筛选培养基,添加污泥50mL保存,经过4个周期总共20d后(每个周期要换水、补充营养元素),即得到短程硝化混合菌,所述短程硝化混合菌的氨氮降解率稳定在90%以上,亚硝酸盐氮转化率稳定在90%以上,同时菌液浓度达到1*108个/mL。
保存前测定短程硝化混合菌的短程硝化能力;
前期筛选的短程硝化混合菌以NH4Cl作为唯一氮源,氨氮浓度为300-400mg/L,磷酸盐浓度为60-80mg/L,镁离子浓度20mg/L,钙离子浓度10mg/L,碳酸氢钠浓度100mg/L,pH7.8,曝气溶氧控制在4-6mg/L,将短程硝化混合菌培养至亚硝转化率达90%以上,并收集菌体;
短程硝化混合菌的真空冷冻干燥保存
(1)预处理:取适量清洗后的氨氧化细菌混培液置于50mL离心管中,离心(4000r/min,10min)后弃置上清液,之后向离心管中加入适量0.2mol/L磷酸盐缓冲液(PBS),旋转混合后再离心(4000r/min,10min)弃置上清液,此过程进行三次。之后再用基质溶液(基质溶液的配方为:100mg/L氨氮,20mg/L无机磷,20mg/L MgSO4·H2O,10mg/L CaCl·H2O,100mg/LNaHCO3)清洗离心一次,混匀之后向培养皿中均加入适量清洗离心后的湿污泥,加入甘油(甘油为保护剂,加入甘油的质量为污泥、基质溶液和保护剂三者总质量的3%)。
(2)放入-20℃冰箱24h进行预冻。
(3)预冻完成后将各表面皿放入Flexi-Dry真空冷冻干燥机(温度-92℃,真空度5Pa)中冷冻干燥24h。冻干完成后,将获得的冻干活性污泥粉储存在真空袋中(有利于菌种储存、运输),在4℃、遮光条件下保藏6个月以上。
短程硝化混合菌的冻干后活性恢复。
本实施例采用自配氨氮废水,在500mL自来水中,投加0.382g NH4Cl,使得氨氮初始浓度为200mg/L,另需投加短程硝化混合菌所需的其他营养元素:磷酸盐浓度为60-80mg/L,镁离子浓度20mg/L,钙离子浓度10mg/L,碳酸氢钠浓度100mg/L,使用10%NaHCO3溶液调节废水pH至7.8,调节温度33℃。保藏期结束后,先将保存菌粉置于室温,之后各菌粉加入适量0.2mol/L磷酸盐缓冲液(PBS)清洗离心三次。离心清洗完成后,弃置上清液并将污泥转移至培养底物(培养底物包括:氨氮100mg/L,无机磷20mg/L,MgSO4·H2O 20mg/L,CaCl·H2O10mg/L以及碳酸氢钠100mg/L)的培养瓶中,放入4℃冰箱中使细菌细胞重新补充水分。
24h后弃置培养瓶上清液并再次对各瓶内污泥清洗离心,之后重新加入基质溶液并曝氧气5min后放入恒温摇床中开始活化过程,在活化时间6h、12h、24h、48h、72h分别测定出水氨氮、亚硝态氮浓度,数据如附图2所示。在运行24h后,完成短程硝化的快速启动,在运行72h后,氨氮降解率达91.6%,亚硝态氮转化率达95.9%,该结果可初步证实冻干后的短程硝化混合菌活化成功,并能够实现短程硝化。
如图2和图3,为进一步证实短程硝化混合菌冻干后的活性恢复情况及短程硝化启动的效果,采取江苏盐城某养殖场厂养殖废水进行短程硝化快速启动实验,其主要污染物为氨氮类,经过测定废水中氨氮浓度为300mg/L左右,将短程硝化混合菌冻干粉经过补充水分等处理后,投入到含氨氮的养殖废水中,适量补充磷源KH2PO4和微量元素并调节体系pH控制在7.0-7.8之间,调节温度33℃,按0h、6h、12h、24h、48h、72h定时取样监测体系中氨氮、亚硝态氮的变化,数据如附图3所示,结果表明:废水中氨氮在6h之内开始降解,经过24h,氨氮浓度已下降到150mg/L,降解率实现50%。72h后,氨氮浓度已降到34mg/L,降解率高达82.3%,亚硝转化率达95.6%。上述氨氮的降解基本以转换为亚硝态氮的形式发生。
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作出若干改进,这些改进也应视为本发明的保护范围。
Claims (5)
1.一种短程硝化混合菌培养及冷冻干燥保存与快速恢复活性的方法,其特征在于:包括以下步骤:
1)短程硝化混合菌的培养:
使用NH4Cl-0.4g,KH2PO4-0.08g,NaHCO3-1g,MgSO4·7H2O-0.02g,CaCl2·2H2O-0.01g,微量元素溶液1mL以及1000mL超纯水作为筛选培养基,调节pH7.8-8.5,添加污泥50mL培养,经过4个周期总共20d后,即得到短程硝化混合菌,所述短程硝化混合菌的氨氮降解率稳定在90%以上,亚硝酸盐氮转化率稳定在90%以上,同时菌液浓度达到1*108个/mL;
2)保存前测定短程硝化混合菌的短程硝化能力;
前期筛选的短程硝化混合菌以NH4Cl作为唯一氮源,氨氮浓度为300-400mg/L,磷酸盐浓度为60-80mg/L,镁离子浓度20mg/L,钙离子浓度10mg/L,碳酸氢钠浓度100mg/L,pH7.8,曝气溶氧控制在4-6mg/L,将短程硝化混合菌培养至亚硝转化率达90%以上,并收集菌体;
3)短程硝化混合菌的冷冻干燥保存与活性恢复;
冷冻干燥保存:取适量清洗后的短程硝化混合菌置于50mL离心管中,离心后弃置上清液,之后向离心管中加入适量0.2mol/L磷酸盐缓冲液,旋转混合后再离心弃置上清液,此过程进行三次,之后再用基质溶液清洗离心一次,混匀之后向培养皿中加入适量清洗离心后的湿污泥,加入保护剂,然后放入-20℃冰箱24h进行预冻;
预冻完成后将各表面皿放入Flexi-Dry真空冷冻干燥机中冷冻干燥24h,冻干完成后,将获得的冻干活性污泥粉储存在真空袋中,在4℃、遮光条件下保藏6个月;
活性恢复:保藏期结束后,先将保存菌粉置于室温,之后各菌粉加入适量0.2mol/L磷酸盐缓冲液清洗离心三次,离心清洗完成后,弃置上清液并将污泥转移至含有培养底物的培养瓶中,放入4℃冰箱中使细菌细胞重新补充水分;24h后弃置培养瓶上清液并再次对各瓶内污泥清洗离心,之后重新加入基质溶液并曝氧气5min后放入恒温摇床中开始活化过程,活化过程定期监测氨氮、亚硝态氮浓度。
2.如权利要求1所述的短程硝化混合菌培养及冷冻干燥保存与快速恢复活性的方法,其特征在于:所述微量元素溶液,其配方为:氯化锌80mg,无水硫酸铜20mg,硼酸20mg,七水硫酸亚铁100mg、水1000mL。
3.如权利要求1所述的短程硝化混合菌培养及冷冻干燥保存与快速恢复活性的方法,其特征在于:所述基质溶液的配方为:100mg/L氨氮,20mg/L无机磷,20mg/L MgSO4·H2O,10mg/L CaCl·H2O,100mg/L NaHCO3。
4.如权利要求1所述的短程硝化混合菌培养及冷冻干燥保存与快速恢复活性的方法,其特征在于:所述保护剂为甘油,加入保护剂的质量为污泥、基质溶液和保护剂三者总质量的3%。
5.如权利要求1所述的短程硝化混合菌培养及冷冻干燥保存与快速恢复活性的方法,其特征在于:所述培养底物包括:氨氮100mg/L,无机磷20mg/L,MgSO4·H2O 20mg/L,CaCl·H2O 10mg/L以及碳酸氢钠100mg/L。
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