CN113684142A - 四株湖泊沉积物高效解磷菌及其解磷有机酸谱 - Google Patents
四株湖泊沉积物高效解磷菌及其解磷有机酸谱 Download PDFInfo
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- 239000010452 phosphate Substances 0.000 title claims abstract description 40
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Abstract
本发明公开了四株湖泊沉积物高效解磷菌(一株解磷不动杆菌和三株解磷假单胞菌)及其解磷有机酸谱。这些菌株能够将难溶性无机磷酸盐溶解为可溶性磷酸盐，在分别以磷酸三钙和羟基磷灰石为唯一磷源的培养液中，正磷酸盐积累量最多可达291.64mg/L。进行培养物滤液的HPLC分析，发现葡萄糖酸和丙酸在解磷过程中占据主要地位，为进一步研究有机酸溶磷途径提供线索，有助于后续对湖泊沉积物中解磷微生物影响富营养化水平的研究。
Description
技术领域：
本发明属于微生物技术领域，具体涉及一株高效解磷不动杆菌和三株高效解磷假单胞菌及其在溶磷过程中的有机酸谱。
背景技术：
湖泊水体富营养化主要表现为营养盐即磷的积累，包括外源磷的输入和内源磷的释放。在评估有效控制富营养化水体中的外源磷输入时，沉积物中内源磷的释放在湖泊上覆水中磷浓度升高时起着重要作用。因此，研究湖泊底泥中内源磷的释放能力及其影响因素显得尤为重要，之前对于内源磷释放的影响因素研究主要集中在温度、氧化还原电位、pH以及再悬浮等物理化学性质的改变。但是，与物理和化学效应相比，微生物对内源磷的释放有显著影响。现有的研究已经对海洋、河流和湖泊环境中解磷菌的数量、类型和分布进行了说明。然而，在以磷为限制因子、以磷酸钙为主要磷素形态的富营养化水生生态系统中，对解磷菌株特性及其对沉积物磷释放潜力的研究非常有限。
许多报告表明，pH值降低与解磷菌溶解难溶性磷酸盐之间存在正相关关系。之前的研究指出微生物在矿化过程中产生的有机酸导致pH值的降低，最常报道的有机酸有柠檬酸、甲酸、富马酸、葡萄糖酸、琥珀酸、丙酸、酒石酸、乳酸和异戊酸等。但没有确定具体是哪些有机酸在发挥作用，以及解磷菌在面对不同底物时分泌的有机酸种类是否会改变，本研究尝试通过不同磷源探索产生的有机酸共性，即用高效液相(HPLC)法对培养液中的有机酸进行定性和定量分析。
发明内容：
本发明的目的是基于以上的问题述求，提供一株高效解磷不动杆菌和三株高效解磷假单胞菌，申请人将其分别命名为IPB0、IPB1、IPB2、IPB3。证实了这些菌株的高效解磷能力以及观察到培养液中pH值降低的现象后，使用高效液相(HPLC)法对培养液中的有机酸进行定性和定量分析，探索出解磷菌在溶解难溶性磷酸盐过程中所表现出来的有机酸共性，发现葡萄糖酸和丙酸占据主要地位，为进一步探索解磷菌溶解机制奠定基础。
本发明的目的是采用以下技术方案实现的：分别以磷酸三钙(TCP)和羟基磷灰石(HAP)作为控制磷源，采用平板筛选法从太湖沉积物中分离出八株具有解磷功能的细菌，再通过摇瓶法复筛挑选出四株高效解磷的细菌。
在唯一磷源分别为磷酸三钙或羟基磷灰石的难溶性磷液体培养基中，磷浓度表示为液体培养基中正磷酸盐的积累量。菌株IPB1在四个解无机磷菌中增长最快，但是磷积累最多是IPB3，比对照组提高了243.88mg/L。
随着液体培养基中难溶性无机磷的溶解，与对照组相比所有实验组中的pH值最终都下降了，表明解无机磷菌在培养过程中有酸性物质产生。进行培养物滤液的HPLC分析，以鉴定和定量四种解无机磷菌在溶解磷酸三钙和羟基磷灰石期间产生的有机酸。综合共有的有机酸种类以及含量变化可得出，葡萄糖酸和丙酸在增溶过程中占据主要地位。
本文所提供的菌株丰富了湖泊沉积物解磷菌菌种资源，在解磷微生物对湖泊富营养化机制影响的研究中作出贡献。
附图说明：
图1为四株解磷菌在难溶性磷液体培养基(唯一磷源分别为磷酸三钙或羟基磷灰石)中的解磷能力。
图2为四株解磷菌在难溶性磷液体培养基(唯一磷源分别为磷酸三钙或羟基磷灰石)中的pH变化。
具体实施方式：
实施例1：四株高效解磷菌的分离、纯化及鉴定
取5g新鲜沉积物样品加入100mL无菌水，再在三角瓶中加入约10g玻璃珠(直径1mm)帮助混匀。将三角瓶在28℃，120rpm条件下振荡过夜，即得沉积物稀释母液(已稀释20倍)。涂布平板之前需再稀释成不同的浓度梯度，取沉积物稀释母液1mL加入9mL无菌水，即得10-1稀释样品；再取10-1稀释样品1mL加入9mL无菌水，即得10-2稀释样品，以此类推，可得到10-3、10-4、10-5等浓度梯度。
难溶性磷液体培养基：葡萄糖10.0g；(NH4)2SO40.5g，NaCl 0.3g，KCl 0.3g，FeSO4.7H2O 0.03g，MnSO4.4H2O 0.03g，MgSO4.7H2O 0.3g，pH为7.2，蒸馏水1000mL，用磷酸钙(TCP，10g/L)或羟基磷灰石(HAP，10g/L)作为唯一的磷来源。相应的固体培养基在液体培养基的基础上添加琼脂粉15-20g/L，磷酸钙或羟基磷灰石单独灭菌，倒平板前再加入摇匀。
取各浓度梯度的沉积物稀释液100μL涂布难溶性磷平板，每个深度的每个浓度均设置3个重复。将平板孵育3-7天(28℃，黑暗，有氧)，每天观察平板上菌落生长状况，直到没有新的菌斑长出，挑选和纯化固体平板上具有独特形态即明显溶磷圈的菌落，反复多次划线进行纯化。纯化后的菌株在液体培养基中培养，然后取适量菌液用0.45μm滤膜过滤去除难溶性磷，送去上海杰李生物技术有限公司进行一代测序，将序列结果与NCBI数据库中的其他序列比较，找出最相似的菌株，使用Mega软件进行系统发育树的分析，鉴定结果如下表。
实施例2：四株高效解磷菌的解磷能力测定
将分离纯化后的细菌接种至难溶性磷液体培养基(分别用磷酸三钙和羟基磷灰石作为唯一磷源)中，定量评估磷释放能力。在28℃下培养预定的时间后，将培养液摇匀以释放附着的细菌，然后静置10-15分钟以沉淀磷酸三钙或羟基磷灰石，然后以12,000rpm离心10分钟以去除菌体。抗败血酸-钼酸铵比色法测定上清液中的正磷酸盐含量，培养液中所积累的正磷酸盐的浓度来表达磷释放能力。
实施例3：培养液中pH值的变化以及有机酸谱的测定
将四株解磷菌接在相应的液体培养基后，于37℃的培养箱摇床中以150rpm的速度培养24h，然后取2mL培养液以12,000rpm离心10min，用0.22μm尼龙过滤器过滤收集上清。通过配备2998PDA检测器和自动进样器的高效液相色谱(HPLC)(Waters)，使用RP-18色谱柱(250mm×4.6mm)分析有机酸。洗脱液的检测在210nm处进行，并且基于各有机酸的保留时间来鉴定有机酸，同时以柠檬酸，甲酸，富马酸，葡萄糖酸，琥珀酸，丙酸和酒石酸标准品获得的峰面积作为定量上清液中有机酸含量的参考。结果如下表所示。
综上所述，所提供的高效解磷菌株对于富营养化浅水湖泊水-沉积物界面内源磷素的释放具有重要意义，此外有机酸谱显示出葡萄糖酸在溶解过程中的主导地位，为后续深入研究葡萄糖酸的产生途径提供支持。
Claims (6)
1.一株高效解磷不动杆菌和三株高效解磷假单胞菌，其特征是：分离自浅水富营养化湖泊-太湖沉积物。
2.权利要求1所述的四株高效解磷菌在湖泊水-沉积物界面均可将难溶性磷酸盐转化为可溶性正磷酸盐的应用。
3.根据权利要求2所述应用，其中的难溶性磷酸盐指磷酸三钙和羟基磷灰石。
4.权利要求1所述的四株高效解磷菌在溶磷过程中，可检测出其培养液pH值降低的现象。
5.权利要求4所述现象是由权利要求1所述的四株解磷菌在溶磷过程中分泌小分子有机酸造成的。
6.使用高效液相(HPLC)法对权利要求5所述的小分子有机酸进行定量分析。
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