CN113005046B - 一种吡啶降解菌剂及其在高盐含吡啶废水处理中的应用 - Google Patents
一种吡啶降解菌剂及其在高盐含吡啶废水处理中的应用 Download PDFInfo
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Abstract
本发明涉及一种吡啶降解菌剂及其在高盐含吡啶废水处理中的应用。由于工业废水往往含有较高的盐分,导致常规微生物对吡啶等难降解有机物的处理效率低。为此,本发明的目的是提高含盐工业有机废水或受吡啶等农药有机污染物污染的土壤中吡啶的微生物降解效率,提供了一种新的、可高效降解高浓度吡啶、且耐盐、环境耐受性好的博德特氏菌(Bordetella sp.PDX03),以期应用于高盐度条件下含吡啶废水的处理及受吡啶污染土壤的微生物修复过程中。根据文献调研,目前由该菌制成的菌剂用于处理高盐条件的吡啶废水尚未见相关报道,该菌已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),保藏编号:CGMCC NO.20075。
Description
技术领域
本发明属于微生物及其降解或修复受吡啶污染的水体和土壤中的应用技术领域。涉及一株具有在高盐度条件下高效降解吡啶的新菌株博德特氏菌的筛选方法、吡啶降解菌剂的制备,吡啶的降解性能以及其在高盐条件下降解吡啶的性能。
背景技术
吡啶的性质及应用介绍:有机化合物,是含有一个氮杂原子的六元杂环化合物。可以看做苯分子中的一个(CH)被N取代的化合物,故又称氮苯,无色或微黄色液体,有恶臭。吡啶及其同系物存在于骨焦油、煤焦油、煤气、页岩油、石油中。吡啶在工业上可用作变性剂、助染剂,以及合成一系列产品(包括药品、消毒剂、染料等)的原料。吡啶在农用化学品、制药工业等方面有更大的应用潜力,因为它是很好的有机溶剂,也是染料、炸药、农药、除草剂等的中间体。
吡啶的危害及微生物方法的优势:吡啶是一种化学和石化工业常见的化学品,易溶于水,容易扩散进入环境,且难以生物降解,是世界范围内的一种严重污染物。由于其有毒、致畸和致癌作用,也被美国环保局列为优先污染物。吡啶会通过吸入、食入、皮吸收等途径侵入人体,有强烈刺激性,能麻醉中枢神经系统。对眼及上呼吸道有刺激作用。长期吸入出现头晕、头痛、失眠、步态不稳及消化道功能紊乱。因此探索一种有效且经济的处理方法处理废水中的吡啶是十分必要的。治理吡啶污染废水的技术有很多,比如高级氧化、物理吸附和膜分离技术等。相较于这些物化方法来说,微生物方法因其低成本和环境友好的优点,被认为是处理吡啶等此类难降解有机污染物的经济、有效而且应用前景广阔的方法。但是生物法的应用会受到环境因素及废水中所含其他物质(如盐度)等的影响;广泛应用吡啶的化工、印染、农药和石油等行业会产生大量的含盐有机工业废水,废水中所含盐类物质为Cl-、Na+、NO3 -、SO4 2-等,虽然这些离子都可为微生物所需要的营养元素,但若浓度过高,会对微生物产生抑制和毒害作用,这导致了含盐和吡啶等有机工业废水的处理更加困难,因此筛选一株耐盐,吡啶降解效率高、环境耐受性好的吡啶降解菌是十分有必要的,对于提高有毒难降解工业有机废水的处理率有较好的应用价值。
发明内容
本发明的目的是提高含盐工业有机废水或受吡啶等农药有机污染物污染的土壤中吡啶的微生物降解效率,提供一种可以高效降解高浓度吡啶、并且耐盐、环境耐受性好的博德特氏菌(Bordetella sp. PDX03),以期应用于高盐度条件下含吡啶废水的处理及受吡啶污染土壤的微生物修复过程中。
为实现上述目的,本发明首先成功培育了一株吡啶高效降解菌株博德特氏菌(Bordetella sp.PDX03),该菌株已于2020年6月12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(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 CGMCC No.20075;该菌株属于博德特氏菌属(Bordetella sp.),命名为 Bordetellasp.PDX03。
本发明提供了所述吡啶降解优势菌在含高浓度吡啶废水中的降解性能。
上述降解性能的测定方法为:将固体培养基上获得的优势菌- 博德特氏菌(Bordetella sp.PDX03)接种于LB富集培养基,培养28h 至对数期后期,经离心机6000r/min离心10min,用无菌水清洗3次,调OD600约为1.0,制成菌悬液。将菌悬液接种至含吡啶浓度为1000 mg/L的废水中,摇床培养,使吡啶充分降解。
上述测定方法中,所述摇床培养过程中,摇床转速为120r/min,控制摇床反应为中温,温度为37±1℃。
上述测定方法中,菌种的接种量为5%-10%(v/v).
本发明还提供了该菌在高盐条件下的吡啶降解性能。
上述的影响,所述的测定方法为将菌悬液加入至含盐的无机盐培养基中,摇床培养,使吡啶完全降解。
上述测定方法中,所述摇床培养过程中,摇床转速为120r/min,摇床培养的温度为37±1℃左右。
上述方法中无机盐培养基的吡啶理论浓度为1000mg/L。
上述方法中菌悬液的接种量为5%-10%(v/v)。
上述废水中,NaCl的浓度分别为0g/L-50g/L;NaNO3的浓度分别为0g/L-60g/L;NaSO4的浓度分别为0mg/L-50g/L。
本发明的优点和积极效果:
本发明与现有处理方法或技术相比有以下显著优点和有益效果:
(1)本发明采用高浓度吡啶作为唯一碳源对污泥进行驯化、筛选后,获得了一株能以吡啶作为唯一碳源生长的高效吡啶降解菌 Bordetella sp.PDX03。首次发现博德特氏菌(Bordetella sp.PDX03)能以吡啶为唯一碳源,对吡啶具有较好的降解能力。在54h内吡啶降解率可达100%,矿化率可达94.87%。
(2)本发明还发现该菌株可用于高盐环境下降解废水中高浓度的吡啶,不会产生二次污染,且对环境条件要求低,即使在高盐度环境中,仍可以将吡啶完全降解,可用于高盐度吡啶污染的废水处理或土壤修复中。
(3)鉴于吡啶的毒性及持久性,以及当前耐盐吡啶降解微生物降解研究的缺乏,利用高效吡啶降解菌博德特氏菌进行高盐度环境中的吡啶降解具有非常重要的现实意义和应用价值。
附图说明
图1为博德特氏菌PDX03在固体培养基上的形态图。
图2为博德特氏菌PDX03在电子显微镜下的形态图。
图3为博德特氏菌PDX03扫描电镜图。
图4为博德特氏菌PDX03的菌种鉴定结果。
图5为博德特氏菌PDX03在LB培养基中的生长曲线。
图6为博德特氏菌PDX03的吡啶降解实验结果。
图7为菌PDX03在不同浓度的氯化钠条件下的吡啶降解特性。
图8为菌PDX03在不同浓度的硝酸钠条件下的吡啶降解特性。
图9为菌PDX03在不同浓度的硫酸钠条件下的吡啶降解特性。
具体实施方式
以下结合说明书附图和具体优选的实施例对本发明作进一步的描述,但并不因此限制本发明的保护范围。
以下实施例中所采用的材料和仪器均为市售。
实施例1:吡啶降解菌的筛选和鉴定
一株吡啶高效降解菌株博德特氏菌(Bordetella sp.PDX03),该菌株已于2020年06月12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(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CGMCC No.20075,该菌株属于博德特氏菌属 (Bordetella sp.),命名为Bordetella sp.PDX03。
本发明是从湖南某农药化工企业废水处理站剩余污泥中取得种源,经过驯化、筛选、分离得到。
具体的筛选方法为:向100mL含有1000mg/L吡啶溶液的无机盐培养基中加入10mL泥水混合液,在37±1℃的恒温培养箱中以 120r/min的转速恒温培养7天,然后取10mL转接到含1250mg/L 吡啶的无机盐培养基中,继续培养使吡啶的最终浓度达到2000mg/L;将最后一次的培养液用无菌水进行稀释为10-1、10-2、10-3、10-4、10-5五个浓度梯度,分别取其中的两滴滴于吡啶浓度为1500mg/L的无机盐固体培养基上,用涂布器将其涂均匀,于37±1℃的恒温培养箱中恒温培养至菌落长出。用接种环挑选其中长势较好、数量较多、且呈单个分布的菌落,采用梯度平板划线法划线于无机盐培养基,待菌长出重复该步骤至菌纯化为纯菌。将纯化的菌接种到液体LB培养基中扩大培养后,提取该菌株DNA,PCR扩增后,进行16SrDNA测序,将所测序列提交GenBank进行分析,鉴定该菌株为博德特氏菌 (Bordetella sp.),将其命名为Bordetella sp.PDX03。
其中,所述的无机盐培养基的组分包括:K2HPO4·3H2O:0.262 g/L,KH2PO4:0.5g/L,MgSO4·7H2O:0.4g/L,FeSO4·7H2O:0.002g/L, (NH4)2SO4:0.2g/L,CaSO4·2H2O:0.08g/L,该无机盐培养基的pH为6.5。
所述的LB培养基组成包括:胰蛋白胨:10g/L,氯化钠:10g/L, 酵母浸粉:5g/L。
图5说明:在37±1℃、120r/min条件下PDX03的生长规律为 8h进入对数生长期,28h进入稳定期。
实施例2:
吡啶降解菌在含吡啶废水中降解吡啶的性能,其步骤为:将菌PDX03的菌悬液接种到含吡啶浓度为1000mg/L的废水中,摇床转速120r/min、温度37±1℃条件下处理60h,分光光度计测定菌的 OD600,高效液相法测定废水中吡啶含量,总有机碳测定仪测定废水的总有机碳含量。
图6说明:在37±1℃,120rmp条件下,在以吡啶为唯一碳源的无机盐培养基中,菌PDX03的生长速度小于在LB培养基中,且在48h后停止生长,原因可能是可利用碳源的缺乏。同时,在54h 时,吡啶完全降解。其次在吡啶降解过程中可发现pH的升高,无机盐培养环境由弱酸性转为弱碱性。最后该菌处理54h后对废水中吡啶的矿化率可以达到94.87%。
实施例3:
考察不同浓度的氯化钠、硝酸钠、硫酸钠对菌PDX03降解吡啶的影响:将菌PDX03的菌悬液(OD600约为1.0)接种到含吡啶浓度为 1000mg/L的液体无机盐培养基中,该培养基中NaCl浓度分别为10 mg/L、20mg/L、30mg/L、50mg/L,NaNO3的浓度分别为5mg/L、 10mg/L、20mg/L、40mg/L、60mg/L;NaSO4的浓度分别为5mg/L、 10mg/L、20mg/L、30mg/L、40mg/L、50mg/L。摇床转速120r/min、温度37±1℃条件下培养60小时,分光光度计测定菌PDX03的生长情况,高效液相法测定吡啶含量。
由图7可知,在氯化钠浓度在10mg/L以上时,随着氯化钠浓度的升高,菌PDX03的生长速率降低,同时降解率也相应的降低。氯化钠浓度为10mg/L时,在48h时吡啶可以完全降解,与其最短的降解时间相同,可以认为氯化钠浓度为10mg/L对该菌对吡啶的降解几乎无影响。在氯化钠浓度为50mg/L时,吡啶也可以在56h内被完全降解。因此可以认为菌PDX03对氯化钠有较强的耐盐性。
图8说明:由图可知,向培养基中添加硝酸钠时,菌种的适应时间加长,32h才开始生长,相应的吡啶开始降解的时间也后移,因此加入硝酸钠之后,吡啶完全降解的时间加长。适应期过后,菌的生长速度和吡啶的降解速度都较快。随着硝酸钠浓度的升高,对菌PDX03的生长和吡啶降解的影响依次增大。当硝酸钠的浓度为5 mg/L时,降解最快,在64h时吡啶可以被完全降解,当硝酸钠的浓度为60mg/L时,在72h时吡啶可以被完全降解。尽管该菌对硝酸钠的环境需要一定的适应时间,但是在适应期过后,硝酸钠对该菌降解吡啶的影响不大。
图9说明:培养基中加入硫酸钠时,菌的生长速率及吡啶的降解速率较未加时低。但是硫酸钠对菌生长及降解吡啶的影响规律不同于氯化钠和硝酸钠的影响规律。从5㎎/L至50㎎/L,随着浓度的升高,硫酸钠对菌PDX03的生长和降解吡啶的影响越小。即菌PDX03比较适合高浓度硫酸钠降解含高浓度的吡啶废水。
本发明提供的一种在高盐条件下耐受和降解高浓度吡啶菌株的驯化和筛选方法,该菌株除了对高浓度吡啶具有高效降解能力外,还对高盐环境有很强的耐受性,可以在高盐条件下有效去除吡啶,不产生二次污染,使用安全。可提升高盐度环境中含吡啶废水的生物处理效果,具有非常重要的现实意义和应用价值。
最后应说明的是:以上所述仅为本发明的优选实施例而已,并不用于限制本发明,尽管参照前述实施例对本发明进行了详细的说明,对于本领域的技术人员来说,其依然可以对前述各实施例所记载的技术方案进行修改,或者对其中部分技术特征进行等同替换,凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (3)
1.一株能在高盐度条件下降解吡啶的优势菌株,已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),保藏编号:CGMCC NO.20075,所述菌株属于博德特氏菌属,命名为Bordetella sp.PDX03。
2.一种在高盐条件下降解吡啶的生物降解菌剂,其特征在于,所述生物降解菌剂由权利要求1所述的博德特氏菌经活化、培养制备得到。
3.一种权利要求2所述的菌剂的用途,其特征在于:在高盐条件下生物处理含吡啶的有机难降解废水或土壤修复。
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