技术领域technical field
本发明涉及一种提高活性污泥PHA合成能力的方法，属于活性污泥治理技术领域。The invention relates to a method for improving the PHA synthesis ability of activated sludge, belonging to the technical field of activated sludge treatment.
背景技术Background technique
生物活性污泥法是当前市政及工业废水处理的主要方法，该方法是微生物在不断利用废水中的有机物满足自身生长需求的同时净化污水。但是当活性污泥中的微生物不断增殖使得生物浓度过高时，也会严重影响活性污泥处理污水的效果，因此需要定期排泥以维持反应器中活性污泥的浓度处于合适的水平，排出的这部分污泥称为剩余污泥。剩余污泥作为城市污水处理过程中产生的固体废弃物，其产生量非常大。Biologically activated sludge method is the main method of municipal and industrial wastewater treatment at present. This method is that microorganisms purify sewage while continuously using organic matter in wastewater to meet their own growth needs. However, when the microorganisms in the activated sludge continue to proliferate and the biological concentration is too high, it will also seriously affect the effect of the activated sludge on sewage treatment. Therefore, it is necessary to regularly discharge the sludge to maintain the concentration of the activated sludge in the reactor at an appropriate level. This part of the sludge is called excess sludge. Surplus sludge is a solid waste produced in the process of urban sewage treatment, and its production volume is very large.
剩余污泥成分复杂，有机物含量高达60～70％，同时含有较为丰富的氮、磷等营养元素。未经处理的污泥不但含有大量的微生物，还有许多被活性污泥吸附但是未分解利用的有机物，同时还有许多病原菌、重金属等有毒有害物质，若处置不当，极易造成二次污染。处理剩余污泥所需的投资费用较多，一般污泥处置费用占污水处理厂总费用的25～40％，部分甚至可达65％。The remaining sludge has complex components, with organic matter content as high as 60-70%, and rich nitrogen, phosphorus and other nutrients. Untreated sludge not only contains a large number of microorganisms, but also many organic substances that are adsorbed by activated sludge but not decomposed and utilized. At the same time, there are many toxic and harmful substances such as pathogenic bacteria and heavy metals. If they are not disposed of properly, they will easily cause secondary pollution. The investment cost required to deal with excess sludge is relatively large. Generally, sludge disposal costs account for 25-40% of the total cost of sewage treatment plants, and some even reach 65%.
目前世界上处理活性污泥的方法一般是经过浓缩、消化、脱水等预处理后再进行填埋、焚烧、投海、堆肥处理等方法，传统的剩余污泥处置技术或多或少都会给环境带来不同程度的危害，并且不能将剩余污泥彻底处理掉，只是实现了污泥的减量化处理，现在更多地是考虑将剩余污泥进行资源化的利用。通过使用适当的措施，将剩余污泥中的有机物转化成为具有高附加值的产品，同时对剩余污泥中的微生物资源也加以利用，这样不但可以为剩余污泥的处置提供可持续、无污染的出路，又可以提供相应的资源和产品，具有经济和社会双重效益，符合当今可持续发展的理念。At present, the methods of treating activated sludge in the world are generally pre-treatments such as concentration, digestion, and dehydration, followed by landfill, incineration, throwing into the sea, and composting. The traditional residual sludge disposal technology will more or less harm the environment It brings different degrees of harm, and the excess sludge cannot be completely disposed of. It only realizes the sludge reduction treatment, and now more consideration is given to the resource utilization of the excess sludge. By using appropriate measures, the organic matter in the excess sludge is converted into products with high added value, and the microbial resources in the excess sludge are also utilized, which can not only provide sustainable and pollution-free solutions for the disposal of excess sludge. The way out, and can provide the corresponding resources and products, with economic and social benefits, in line with the concept of sustainable development today.
发明内容Contents of the invention
本发明针对现有技术的不足，提供一种提高活性污泥PHA合成能力的方法。The invention aims at the deficiencies of the prior art, and provides a method for improving the PHA synthesis ability of activated sludge.
本发明技术方案如下：Technical scheme of the present invention is as follows:
一种提高活性污泥PHA合成能力的方法，包括如下步骤：A method for improving the synthetic ability of activated sludge PHA, comprising the steps of:
(1)向收集的活性污泥中，按质量比2～5％的比例加入活化的泡囊短波单胞菌(Brevundimonas vesicularis)UJN1，制得混合污泥；(1) Add activated Brevundimonas vesicularis (Brevundimonas vesicularis) UJN1 in a mass ratio of 2 to 5% to the collected activated sludge to prepare mixed sludge;
(2)按20～30g/L的比例将步骤(1)制得的混合污泥加入驯化培养基中，静置培养45～60min，替换45～60％体积的驯化培养基，在溶氧0.04～3.0mg/L、温度28～32℃的条件下，驯化20～22天，每天替换45～60％体积的驯化培养基，制得驯化污泥；(2) Add the mixed sludge prepared in step (1) into the acclimatization medium at a ratio of 20 to 30 g/L, let it stand for 45 to 60 minutes, and replace 45 to 60% of the volume of the acclimatization medium. Under the conditions of ～3.0mg/L and temperature 28～32℃, domesticate for 20～22 days, and replace 45～60% of the volume of the acclimation medium every day to obtain acclimatization sludge;
(3)将步骤(2)制得的驯化污泥按体积百分比45～55％比例加入发酵培养基，在溶氧3.0mg/L～4.5mg/L、温度28～32℃的条件下进行好氧发酵，发酵过程中补加乙酸钠，当乙酸钠不再消耗时，停止发酵，即得。(3) Add the acclimatized sludge prepared in step (2) into the fermentation medium in a proportion of 45-55% by volume, and carry out under the conditions of dissolved oxygen 3.0mg/L-4.5mg/L and temperature 28-32°C. Oxygen fermentation, sodium acetate is added during the fermentation process, when the sodium acetate is no longer consumed, the fermentation is stopped, and the product is obtained.
根据本发明优选的，所述步骤(1)中，泡囊短波单胞菌(Brevundimonasvesicularis)UJN1，2017年11月2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，地址：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：CGMCCNO.14851。Preferably according to the present invention, in the step (1), Brevundimonas vesicularis UJN1 was preserved in the General Microorganism Center of China Committee for the Collection of Microbial Cultures on November 2, 2017, address: Chaoyang District, Beijing Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Courtyard, Beichen West Road, deposit number: CGMCCNO.14851.
根据本发明优选的，所述步骤(1)中，收集的活性污泥由来源于生活污水处理曝气池的污泥，在温度4℃的条件下，6000r/min的条件下离心10min后获得。Preferably according to the present invention, in the step (1), the collected activated sludge is obtained by centrifuging at 6000r/min for 10min at a temperature of 4°C from the sludge from the domestic sewage treatment aeration tank .
根据本发明优选的，所述步骤(1)中，活化步骤如下：Preferably according to the present invention, in said step (1), the activation step is as follows:
将泡囊短波单胞菌(Brevundimonas vesicularis)UJN1接种于牛肉膏蛋白胨液体培养基中，在37℃、160r/min的条件下进行进行菌种活化培养48～60h。Brevundimonas vesicularis UJN1 was inoculated in beef extract peptone liquid medium, and the strain activation culture was carried out at 37°C and 160r/min for 48-60h.
根据本发明优选的，所述步骤(2)中，驯化培养基每升组分如下：Preferably according to the present invention, in the step (2), the components per liter of the acclimatization medium are as follows:
乙酸钠4.0g，硫酸铵0.16g，磷酸二氢钾0.045g，磷酸氢二钾0.121g，微量元素溶液1.0mL，水定容至1L，pH 7.0；Sodium acetate 4.0g, ammonium sulfate 0.16g, potassium dihydrogen phosphate 0.045g, dipotassium hydrogen phosphate 0.121g, trace element solution 1.0mL, water to 1L, pH 7.0;
所述微量元素溶液，每升组分如下：Described trace element solution, every liter of components is as follows:
MnSO4·H2O 1.5g，CuSO4·5H2O 0.2g，CoCl2·6H2O 0.2g，H3BO3 1.0g，ZnSO4·7H2O0.2g，Na2SiO3·9H2O 0.2g，NiSO4·6H2O 0.05g，NaMoO4·2H2O 0.05g，EDTA-2Na 1.0g。MnSO4 ·H2 O 1.5g, CuSO4 ·5H2 O 0.2g, CoCl2 ·6H2 O 0.2g, H3 BO3 1.0g, ZnSO4 ·7H2 O 0.2g, Na2 SiO3 ·9H2 O 0.2g, NiSO4 ·6H2 O 0.05g, NaMoO4 ·2H2 O 0.05g, EDTA-2Na 1.0g.
根据本发明优选的，所述步骤(3)中，发酵培养基每升组分如下：Preferably according to the present invention, in described step (3), every liter of components of fermentation medium is as follows:
乙酸钠6.0g，硫酸铵0.16g，磷酸二氢钾0.045g，磷酸氢二钾0.121g，微量元素溶液1.0mL，水定容至1L，pH 7.0。Sodium acetate 6.0g, ammonium sulfate 0.16g, potassium dihydrogen phosphate 0.045g, dipotassium hydrogen phosphate 0.121g, trace element solution 1.0mL, water to 1L, pH 7.0.
有益效果Beneficial effect
1、本发明首次通过采用泡囊短波单胞菌(Brevundimonas vesicularis)UJN1与活性污泥共同驯化的方法，可以显著提高驯化后活性污泥菌群合成PHA的能力，为低成本大规模生产PHA奠定了基础；1. For the first time, the present invention adopts the method of domesticating Brevundimonas vesicularis (Brevundimonas vesicularis) UJN1 and activated sludge together, which can significantly improve the ability of activated sludge flora to synthesize PHA after domestication, laying the foundation for low-cost large-scale production of PHA foundation;
2、本发明具有结构简单、自动化程度高、造价低廉、易操作等特点，有利于大规模产业化实施。2. The present invention has the characteristics of simple structure, high degree of automation, low cost, easy operation, etc., and is conducive to large-scale industrialization implementation.
附图说明Description of drawings
图1是活性污泥驯化装置的结构示意图；Fig. 1 is the structural representation of activated sludge domestication device;
图2是活性污泥驯化过程中乙酸钠消耗情况曲线图；Fig. 2 is a curve diagram of sodium acetate consumption in the activated sludge domestication process;
图3是活性污泥发酵过程中乙酸钠消耗情况曲线图；Fig. 3 is a curve diagram of sodium acetate consumption in the activated sludge fermentation process;
图4是活性污泥发酵过程中PHA的产率情况曲线图；Fig. 4 is the productive rate situation curve figure of PHA in the activated sludge fermentation process;
具体实施方式Detailed ways
下面将结合实施例及附图，对本发明的技术方案作进一步阐述，但本发明所保护范围不限于此。The technical solutions of the present invention will be further described below in conjunction with the embodiments and accompanying drawings, but the protection scope of the present invention is not limited thereto.
实施例中所使用活性污泥驯化装置为现有市售装置，结构如图1所示，由搅拌机、多气孔管道、气泵、进水泵、排水泵、pH电极、T电极、DO电极、敞口箱、废液桶、反应桶(容积为50L)及插排等组成。The activated sludge acclimation device used in the embodiment is an existing commercially available device, and its structure is as shown in Figure 1. Box, waste liquid barrel, reaction barrel (volume 50L) and plug-in row, etc.
生物材料来源source of biological material
泡囊短波单胞菌(Brevundimonasvesicularis)UJN1，2017年11月2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，地址：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.14851。Brevundimonas vesicularis (Brevundimonas vesicularis) UJN1, preserved in the General Microbiology Center of China Committee for the Collection of Microbial Cultures on November 2, 2017, address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Deposit number: CGMCC NO.14851.
检测方法Detection method
MLSS(混合液悬浮固体浓度)：是指单位体积混合液内含活性污泥固体物质的总量，单位是mg/L。测MLSS需要：定量滤纸、电子分析天平、烘箱、干燥器等。测定方法为：取100mL混合液用滤纸过滤，待烘箱中温度升到105℃后，将滤干后的滤纸放入烘箱烘2小时，取出置于干燥器中放置操作半小时。称量后减去滤纸重量，并且测滤纸的重量也要采用上述同样的步骤。该实验必须严格按照上述操作，否则会有偏差。MLSS (mixed liquor suspended solids concentration): refers to the total amount of activated sludge solids contained in a unit volume of mixed liquor, in mg/L. Measuring MLSS requires: quantitative filter paper, electronic analytical balance, oven, dryer, etc. The measurement method is: take 100mL of the mixed solution and filter it with filter paper. After the temperature in the oven rises to 105°C, put the filtered filter paper in the oven for 2 hours, take it out and place it in a desiccator for half an hour. Subtract the weight of the filter paper after weighing, and use the same steps above to measure the weight of the filter paper. The experiment must be strictly in accordance with the above operation, otherwise there will be deviation.
VSS(挥发性悬浮固体量)：是指在600℃条件下能燃烧，并以气体形式逸出的那部分固体。它通常用于表示污泥中的有机物的含量，常用mg/L表示，有时也用重量百分数表示。VSS测定方法为：取定量污泥，6000rpm离心10min后，将离心后固体部分完全转移至坩埚内，放入烘箱内于100℃下烘至恒重，重量记为m1。恒重污泥放入马弗炉内，在600℃下灼烧1小时，重量记为m2。VSS＝(m1-m2)/0.1。VSS (Volatile Suspended Solids): Refers to the part of solids that can burn at 600°C and escape in the form of gas. It is usually used to indicate the content of organic matter in sludge, usually expressed in mg/L, and sometimes expressed in weight percent. The VSS measurement method is as follows: take quantitative sludge, centrifuge at 6000rpm for 10min, transfer the solid part after centrifugation to a crucible, put it in an oven and dry it at 100°C to constant weight, and record the weight as m1. The sludge with constant weight was put into the muffle furnace and burned at 600°C for 1 hour, and the weight was recorded as m2. VSS=(m1-m2)/0.1.
采用高效液相色谱法(HPLC)测定乙酸钠浓度，色谱分析条件如下：检测器为UltiMate3000紫外检测器，色谱柱为Aminex HPX-87H有机酸柱(300mm×7.8mm，9μm)，柱温45℃，检测波长214nm，流动相为5mM H2SO4，流速0.6mL/min，进样体积10μL。样品经离心分离、0.22μm孔径的滤膜过滤处理后进样检测分析。Adopt high-performance liquid chromatography (HPLC) to measure sodium acetate concentration, and chromatographic analysis condition is as follows: detector is UltiMate3000 ultraviolet detector, and chromatographic column is Aminex HPX-87H organic acid column (300mm * 7.8mm, 9 μ m), column temperature 45 ℃ , the detection wavelength is 214nm, the mobile phase is 5mM H2 SO4 , the flow rate is 0.6mL/min, and the injection volume is 10μL. The samples were centrifuged and filtered through a filter membrane with a pore size of 0.22 μm, and then injected for detection and analysis.
PHA的测定Determination of PHA
PHA标准品的检测：实验室前期研究结果显示，PHA由两种单体组成，即3HB(3-羟基丁酸)和3HV(3-羟基戊酸)。购买SIGMA公司的标准品3HBME(3-羟基丁酸甲酯)及3HVME(3-羟基戊酸甲酯)，取一定量溶解于1mL氯仿中，配制梯度溶液于1.5mL样品瓶中进行GC检测，绘制标准曲线。Detection of PHA standard products: The results of preliminary laboratory research show that PHA is composed of two monomers, namely 3HB (3-hydroxybutyric acid) and 3HV (3-hydroxyvaleric acid). Purchase the standard products 3HBME (3-hydroxybutyrate methyl ester) and 3HVME (3-hydroxyvalerate methyl ester) from SIGMA Company, take a certain amount and dissolve them in 1mL chloroform, prepare a gradient solution and carry out GC detection in a 1.5mL sample bottle. Draw a standard curve.
活性污泥样品预处理：取活性污泥样品100mL，温度4℃转速6000rpm的条件下离心10min，收集活性污泥，加入5％的次氯酸钠溶液终止反应进行。活性污泥样品在真空冷冻干燥机中进行真空冷冻干燥。称取50mg干燥的样品于酯化瓶中，加入2mL氯仿，1mL酯化液(850μL的甲醇和150μL的浓硫酸)，旋紧酯化瓶盖，在100℃沸水中进行3h的酯化反应，然后取出酯化瓶，充分冷却达室温后，再向酯化瓶中加入1mL去离子水并且剧烈震荡，静置分层后，取下层有机相进行气相色谱分析。气相色谱条件：Activated sludge sample pretreatment: Take 100 mL of activated sludge sample, centrifuge at 4°C and rotate at 6000 rpm for 10 minutes, collect activated sludge, and add 5% sodium hypochlorite solution to terminate the reaction. Activated sludge samples were vacuum freeze-dried in a vacuum freeze dryer. Weigh 50 mg of dried sample into the esterification bottle, add 2 mL of chloroform, 1 mL of esterification solution (850 μL of methanol and 150 μL of concentrated sulfuric acid), tighten the cap of the esterification bottle, and carry out the esterification reaction in boiling water at 100 ° C for 3 h. Then take out the esterification bottle, fully cool down to room temperature, then add 1 mL of deionized water into the esterification bottle and shake vigorously, after standing to separate layers, take the lower organic phase for gas chromatographic analysis. Gas chromatography conditions:
毛细管柱：rtx-5型石英毛细管柱；检验器：氢焰离子检验器(FID)；进样器温度：200℃；检测器温度：275℃；载气：N2：40.0mL/min，H2：60.0mL/min，空气：400.0mL/min；程序升温：80℃(1min)→10℃/min→120℃→45℃/min→160℃(5min)；进样：分流比，30:1；进样量：1.0μL。Capillary column: rtx-5 quartz capillary column; detector: hydrogen flame ion detector (FID); injector temperature: 200°C; detector temperature: 275°C; carrier gas: N2 : 40.0mL/min, H2 : 60.0mL/min, air: 400.0mL/min; temperature program: 80°C (1min) → 10°C/min → 120°C → 45°C/min → 160°C (5min); injection: split ratio, 30: 1; Injection volume: 1.0 μL.
实施例1Example 1
一种提高活性污泥PHA合成能力的方法，包括如下步骤：A method for improving the synthetic ability of activated sludge PHA, comprising the steps of:
(1)向收集的活性污泥中，按质量比2％的比例加入活化的泡囊短波单胞菌(Brevundimonas vesicularis)UJN1，制得混合污泥；(1) To the collected activated sludge, add activated Brevundimonas vesicularis (Brevundimonas vesicularis) UJN1 in a ratio of 2% by mass to prepare mixed sludge;
所述收集的活性污泥由来源于取自济南光大水务二厂生活污水处理曝气池的污泥，在温度4℃的条件下，6000r/min的条件下离心10min后获得；The collected activated sludge is derived from the sludge taken from the domestic sewage treatment aeration tank of Jinan Everbright Water Plant No. 2, and obtained after centrifugation at 6000r/min for 10min at a temperature of 4°C;
所述活化步骤如下：The activation steps are as follows:
将泡囊短波单胞菌(Brevundimonas vesicularis)UJN1接种于牛肉膏蛋白胨液体培养基中，在37℃、160r/min的条件下进行进行菌种活化培养48h；Brevundimonas vesicularis UJN1 was inoculated in beef extract peptone liquid medium, and the strain activation culture was carried out at 37°C and 160r/min for 48h;
(2)按30g/L的比例将步骤(1)制得的混合污泥加入驯化培养基中，静置培养60min，替换60％体积的驯化培养基，在溶氧3.0mg/L、温度32℃的条件下，驯化21天，每天替换60％体积的驯化培养基，制得驯化污泥；(2) Add the mixed sludge prepared in step (1) into the acclimatization medium at a ratio of 30g/L, let it stand for 60min, and replace 60% of the volume of the acclimatization medium. Under the condition of ℃, domesticate for 21 days, and replace 60% of the volume of the domestication medium every day to obtain the domestication sludge;
所述步骤(2)中，驯化培养基每升组分如下：In the step (2), the components per liter of the acclimatization medium are as follows:
乙酸钠4.0g，硫酸铵0.16g，磷酸二氢钾0.045g，磷酸氢二钾0.121g，微量元素溶液1.0mL，水定容至1L，pH 7.0；Sodium acetate 4.0g, ammonium sulfate 0.16g, potassium dihydrogen phosphate 0.045g, dipotassium hydrogen phosphate 0.121g, trace element solution 1.0mL, water to 1L, pH 7.0;
所述微量元素溶液，每升组分如下：Described trace element solution, every liter of components is as follows:
MnSO4·H2O 1.5g，CuSO4·5H2O 0.2g，CoCl2·6H2O 0.2g，H3BO3 1.0g，ZnSO4·7H2O0.2g，Na2SiO3·9H2O 0.2g，NiSO4·6H2O 0.05g，NaMoO4·2H2O 0.05g，EDTA-2Na 1.0g；MnSO4 ·H2 O 1.5g, CuSO4 ·5H2 O 0.2g, CoCl2 ·6H2 O 0.2g, H3 BO3 1.0g, ZnSO4 ·7H2 O 0.2g, Na2 SiO3 ·9H2 O 0.2g, NiSO4 6H2 O 0.05g, NaMoO4 2H2 O 0.05g, EDTA-2Na 1.0g;
在对活性污泥微生物的驯化过程中，以乙酸钠作为单一碳源，并且投加少量的氮源和磷源，适当提高培养基中的碳氮比，保证复合菌群处于碳源时而丰富时而匮乏的不平衡生长状态，每天不断地排出静止沉淀后的上清和补充新鲜的培养基。驯化前10天为微生物的适应期，菌群在不断地适应碳氮比不平衡状态，并且逐渐适应以乙酸钠作为单一碳源的生长状态，不断地进行选择淘汰，更适应于这种驯化条件能积累PHA的菌群不断地得到富集。从第11天开始复合菌群可以完全消耗掉投加的乙酸钠，并且随着驯化时间的延长，消耗乙酸钠的速度越来越快。通过为期21天的驯化后，得到一个积累PHA能力提高的混合菌群。In the domestication process of activated sludge microorganisms, sodium acetate is used as a single carbon source, and a small amount of nitrogen and phosphorus sources are added to properly increase the carbon-nitrogen ratio in the medium to ensure that the complex bacteria are sometimes rich in carbon sources. In the state of lack of unbalanced growth, the supernatant after static precipitation was continuously discharged and fresh medium was replenished every day. The 10 days before domestication is the adaptation period of microorganisms. The flora is constantly adapting to the imbalanced state of carbon-nitrogen ratio, and gradually adapting to the growth state of sodium acetate as the single carbon source. Continuous selection and elimination are more suitable for this domestication condition. Bacteria that can accumulate PHA were continuously enriched. From the 11th day, the complex flora could completely consume the added sodium acetate, and with the prolongation of the acclimatization time, the rate of consumption of sodium acetate became faster and faster. After 21 days of acclimatization, a mixed flora with improved ability to accumulate PHA was obtained.
(3)将步骤(2)制得的驯化污泥按体积百分比50％比例加入发酵培养基，在溶氧4.5mg/L、温度28～32℃的条件下进行好氧发酵，发酵过程中补加乙酸钠，当乙酸钠不再消耗时，停止发酵，即得；(3) Add the domesticated sludge obtained in step (2) into the fermentation medium in a proportion of 50% by volume, and carry out aerobic fermentation under the conditions of dissolved oxygen 4.5mg/L and temperature 28～32°C. Add sodium acetate, when the sodium acetate is no longer consumed, stop the fermentation, that is;
所述发酵培养基每升组分如下：Described fermented medium per liter composition is as follows:
乙酸钠6.0g，硫酸铵0.16g，磷酸二氢钾0.045g，磷酸氢二钾0.121g，微量元素溶液1.0mL，水定容至1L，pH 7.0；Sodium acetate 6.0g, ammonium sulfate 0.16g, potassium dihydrogen phosphate 0.045g, dipotassium hydrogen phosphate 0.121g, trace element solution 1.0mL, water to 1L, pH 7.0;
在发酵开始阶段添加6g/L乙酸钠，并实时检测乙酸钠的消耗情况。在乙酸钠消耗完毕后，再次补加乙酸钠，连续三次。大约在发酵24小时后，活性污泥不再消耗乙酸钠，停止发酵，收集菌体。Add 6g/L sodium acetate at the beginning of fermentation, and detect the consumption of sodium acetate in real time. After the sodium acetate was consumed, sodium acetate was added again for three consecutive times. After about 24 hours of fermentation, the activated sludge no longer consumes sodium acetate, stops fermentation, and collects bacteria.
在发酵过程中，每隔两个小时取样一次，结束发酵后，干燥样品进行酯化反应，GC检测PHA的含量。在发酵的前22h内，PHA含量在不断地上升，说明复合菌群在不断地利用乙酸钠合成PHA，最大积累量可以达到污泥干重的22％。分别测定发酵过程中活性污泥的总悬浮固体量(MLSS)和挥发性悬浮固体量(VSS)，在PHA产量最高点时，VSS与MLSS的百分比为52％，PHA最大产量达到细胞干重的42.3％。During the fermentation process, samples were taken every two hours. After the fermentation was finished, the samples were dried for esterification, and the content of PHA was detected by GC. In the first 22 hours of fermentation, the content of PHA was constantly increasing, which indicated that the composite flora was constantly using sodium acetate to synthesize PHA, and the maximum accumulation amount could reach 22% of the dry weight of the sludge. The total suspended solids (MLSS) and volatile suspended solids (VSS) of the activated sludge were measured respectively during the fermentation process. When the PHA production was at its highest point, the percentage of VSS and MLSS was 52%, and the maximum production of PHA reached 50% of the dry cell weight. 42.3%.
通过上述方法可知采用微好氧-好氧模式对活性污泥驯化后，活性污泥菌群合成PHA的能力得到显著提高。Through the above method, it can be known that the ability of the activated sludge flora to synthesize PHA is significantly improved after the activated sludge is domesticated by using the microaerobic-aerobic mode.
实施例2Example 2
一种提高活性污泥PHA合成能力的方法，包括如下步骤：A method for improving the synthetic ability of activated sludge PHA, comprising the steps of:
(1)向收集的活性污泥中，按质量比5％的比例加入活化的泡囊短波单胞菌(Brevundimonas vesicularis)UJN1，制得混合污泥；(1) To the collected activated sludge, add activated Brevundimonas vesicularis (Brevundimonas vesicularis) UJN1 in a ratio of 5% by mass to prepare mixed sludge;
所述收集的活性污泥由来源于取自山东滨化集团污水处理厂的环氧丙烷皂化废水处理曝气池的污泥，在温度4℃的条件下，6000r/min的条件下离心10min后获得；The collected activated sludge is derived from the sludge from the propylene oxide saponification wastewater treatment aeration tank of Shandong Binhua Group Wastewater Treatment Plant, and centrifuged at 6000r/min for 10min at a temperature of 4°C. get;
所述活化步骤如下：The activation steps are as follows:
将泡囊短波单胞菌(Brevundimonas vesicularis)UJN1接种于牛肉膏蛋白胨液体培养基中，在37℃、160r/min的条件下进行进行菌种活化培养60h；Brevundimonas vesicularis UJN1 was inoculated in beef extract peptone liquid medium, and the strain activation culture was carried out at 37°C and 160r/min for 60h;
(2)按20g/L的比例将步骤(1)制得的混合污泥加入驯化培养基中，静置培养45min，替换45％体积的驯化培养基，在溶氧0.04mg/L、温度28～32℃的条件下，驯化21天，每天替换45％体积的驯化培养基，制得驯化污泥；(2) Add the mixed sludge prepared in step (1) into the acclimatization medium in a ratio of 20g/L, let it stand for 45min, replace the acclimatization medium of 45% volume, in dissolved oxygen 0.04mg/L, temperature 28 Under the condition of ~32°C, domesticate for 21 days, replace 45% of the volume of the domestication medium every day, and prepare the domestication sludge;
所述步骤(2)中，驯化培养基每升组分如下：In the step (2), the components per liter of the acclimatization medium are as follows:
乙酸钠4.0g，硫酸铵0.16g，磷酸二氢钾0.045g，磷酸氢二钾0.121g，微量元素溶液1.0mL，水定容至1L，pH 7.0；Sodium acetate 4.0g, ammonium sulfate 0.16g, potassium dihydrogen phosphate 0.045g, dipotassium hydrogen phosphate 0.121g, trace element solution 1.0mL, water to 1L, pH 7.0;
所述微量元素溶液，每升组分如下：Described trace element solution, every liter of components is as follows:
MnSO4·H2O 1.5g，CuSO4·5H2O 0.2g，CoCl2·6H2O 0.2g，H3BO3 1.0g，ZnSO4·7H2O0.2g，Na2SiO3·9H2O 0.2g，NiSO4·6H2O 0.05g，NaMoO4·2H2O 0.05g，EDTA-2Na 1.0g；MnSO4 ·H2 O 1.5g, CuSO4 ·5H2 O 0.2g, CoCl2 ·6H2 O 0.2g, H3 BO3 1.0g, ZnSO4 ·7H2 O 0.2g, Na2 SiO3 ·9H2 O 0.2g, NiSO4 6H2 O 0.05g, NaMoO4 2H2 O 0.05g, EDTA-2Na 1.0g;
在对活性污泥微生物的驯化过程中，以乙酸钠作为单一碳源，并且投加少量的氮源和磷源，适当提高培养基中的碳氮比，保证复合菌群处于碳源时而丰富时而匮乏的不平衡生长状态，每天不断地排出静止沉淀后的上清和补充新鲜的培养基。由图2的结果可以看出，驯化前10天为微生物的适应期，菌群在不断地适应碳氮比不平衡状态，并且逐渐适应以乙酸钠作为单一碳源的生长状态，不断地进行选择淘汰，更适应于这种驯化条件能积累PHA的菌群不断地得到富集。从第11天开始复合菌群可以完全消耗掉投加的乙酸钠，并且随着驯化时间的延长，消耗乙酸钠的速度越来越快。通过为期21天的驯化后，得到一个积累PHA能力提高的混合菌群。In the domestication process of activated sludge microorganisms, sodium acetate is used as a single carbon source, and a small amount of nitrogen and phosphorus sources are added to properly increase the carbon-nitrogen ratio in the medium to ensure that the complex bacteria are sometimes rich in carbon sources. In the state of lack of unbalanced growth, the supernatant after static precipitation was continuously discharged and fresh medium was replenished every day. From the results in Figure 2, it can be seen that the 10 days before domestication is the adaptation period of microorganisms, and the flora is continuously adapting to the state of carbon-nitrogen ratio imbalance, and gradually adapting to the growth state with sodium acetate as the single carbon source, and constantly selecting Elimination, the bacteria that are more suitable for this domestication condition and can accumulate PHA are continuously enriched. From the 11th day, the complex flora could completely consume the added sodium acetate, and with the prolongation of the acclimatization time, the rate of consumption of sodium acetate became faster and faster. After 21 days of acclimatization, a mixed flora with improved ability to accumulate PHA was obtained.
(3)将步骤(2)制得的驯化污泥按体积百分比50％比例加入发酵培养基，在溶氧4.5mg/L、温度28～32℃的条件下进行好氧发酵，发酵过程中补加乙酸钠，当乙酸钠不再消耗时，停止发酵，即得；(3) Add the domesticated sludge obtained in step (2) into the fermentation medium in a proportion of 50% by volume, and carry out aerobic fermentation under the conditions of dissolved oxygen 4.5mg/L and temperature 28～32°C. Add sodium acetate, when the sodium acetate is no longer consumed, stop the fermentation, that is;
所述发酵培养基每升组分如下：Described fermentation medium per liter composition is as follows:
乙酸钠6.0g，硫酸铵0.16g，磷酸二氢钾0.045g，磷酸氢二钾0.121g，微量元素溶液1.0mL，水定容至1L，pH 7.0；Sodium acetate 6.0g, ammonium sulfate 0.16g, potassium dihydrogen phosphate 0.045g, dipotassium hydrogen phosphate 0.121g, trace element solution 1.0mL, water to 1L, pH 7.0;
发酵过程中乙酸钠消耗情况如图3所示：Sodium acetate consumption in the fermentation process is shown in Figure 3:
在发酵开始阶段添加6g/L乙酸钠，并实时检测乙酸钠的消耗情况。在乙酸钠消耗完毕后，补加乙酸钠，连续补加三次。发酵24小时后，活性污泥不再消耗乙酸钠，停止发酵，收集菌体。Add 6g/L sodium acetate at the beginning of fermentation, and detect the consumption of sodium acetate in real time. After the sodium acetate is consumed, add sodium acetate for three consecutive additions. After 24 hours of fermentation, the activated sludge no longer consumes sodium acetate, so the fermentation is stopped and the bacteria are collected.
发酵过程中PHA合成情况如图4所示：The synthesis situation of PHA in the fermentation process is as shown in Figure 4:
在发酵过程中，每隔两个小时取样一次，结束发酵后，干燥样品进行酯化反应，GC检测PHA的含量。在发酵的前22h内，PHA含量在不断地上升，说明复合菌群在不断地利用乙酸钠合成PHA，最大积累量可以达到污泥干重的25.52％。分别测定发酵过程中活性污泥的总悬浮固体量(MLSS)和挥发性悬浮固体量(VSS)，在PHA产量最高点时，VSS与MLSS的百分比为52.45％，PHA最大产量可以达到细胞干重的48.66％。During the fermentation process, samples were taken every two hours. After the fermentation was finished, the samples were dried for esterification, and the content of PHA was detected by GC. In the first 22 hours of fermentation, the content of PHA was constantly increasing, which indicated that the composite flora was constantly using sodium acetate to synthesize PHA, and the maximum accumulation amount could reach 25.52% of the dry weight of the sludge. The total suspended solids (MLSS) and volatile suspended solids (VSS) of the activated sludge were measured respectively during the fermentation process. At the peak of PHA production, the percentage of VSS and MLSS was 52.45%, and the maximum production of PHA could reach the dry weight of cells. 48.66%.
通过上述方法可知采用微好氧-好氧模式对活性污泥驯化后，活性污泥菌群合成PHA的能力得到显著提高。Through the above method, it can be known that the ability of the activated sludge flora to synthesize PHA is significantly improved after the activated sludge is domesticated by using the microaerobic-aerobic mode.
对比例1Comparative example 1
如实施例1所述的提高活性污泥PHA合成能力的方法，不同之处在于，采用假单胞菌(Pseudomonas sp.)QL212，该菌保藏于中国微生物菌种保藏管理委员会普通微生物中心，地址：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：CGMCCNO.9651。The method for improving the PHA synthesis ability of activated sludge as described in Example 1, the difference is that Pseudomonas sp. (Pseudomonas sp.) QL212 is used, which is preserved in the General Microorganism Center of China Microbiological Culture Collection Management Committee, address : Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard No. 1, Beichen West Road, Chaoyang District, Beijing, deposit number: CGMCCNO.9651.
将假单胞菌(Pseudomonas sp.)QL212回注活性污泥，采用相同条件驯化来源相同的环氧丙烷皂化废水活性污泥，驯化21天后进行好氧发酵，PHA最大产量达到细胞干重的38.76％。Pseudomonas sp. (Pseudomonas sp.) QL212 was reinjected into the activated sludge, and the same conditions were used to domesticate the activated sludge of propylene oxide saponification wastewater from the same source. After 21 days of domestication, aerobic fermentation was carried out, and the maximum PHA production reached 38.76% of the dry cell weight. %.
结果分析Result analysis
将泡囊短波单胞菌(Brevundimonas vesicularis)UJN1菌株回注活性污泥进行驯化其PHA最大产量达到细胞干重的48.66％，较假单胞菌(Pseudomonas sp.)QL212回注污泥进行驯化，其合成PHA的能力提高了23.84％。Brevundimonas vesicularis (Brevundimonas vesicularis) UJN1 strain was reinjected into activated sludge for domestication, and its maximum PHA production reached 48.66% of dry cell weight, which was higher than that of Pseudomonas sp. QL212 for reinjection of sludge. Its ability to synthesize PHA has increased by 23.84%.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111548952A (en)* | 2020-04-16 | 2020-08-18 | 广东省农业科学院农业资源与环境研究所 | Method for domesticating microbial flora for degrading efficient sulfur-series malodorous substances |
| CN111849808A (en)* | 2020-07-06 | 2020-10-30 | 北京大学深圳研究院 | Methods of enrichment culture, inoculation and PCR amplification of PHA-producing bacteria from marine sources |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106754471A (en)* | 2016-11-21 | 2017-05-31 | 天津大学 | A kind of method for producing PHA pseudomonad Primary Studies in activated sludge |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106754471A (en)* | 2016-11-21 | 2017-05-31 | 天津大学 | A kind of method for producing PHA pseudomonad Primary Studies in activated sludge |
| Title |
|---|
| JOHANNA A.SILVA等: "Biosynthesis of poly-β-hydroxyalkanoate by Brevundimonas vesicularis LMG P-23615 and Sphingopyxis macrogoltabida LMG 17324 using acid-hydrolyzed sawdust as carbon source", 《JOURNAL OF BIOSCIENCE AND BIOENGINEERING》* |
| 仇少静等: "高PHAs合成性能菌株的筛选及其条件优化", 《环境工程学报》* |
| 郑裕东等: "厌氧-好氧驯化活性污泥生物合成PHA的研究", 《环境科学研究》* |
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111548952A (en)* | 2020-04-16 | 2020-08-18 | 广东省农业科学院农业资源与环境研究所 | Method for domesticating microbial flora for degrading efficient sulfur-series malodorous substances |
| CN111548952B (en)* | 2020-04-16 | 2022-07-12 | 广东省农业科学院农业资源与环境研究所 | Method for domesticating microbial flora for degrading efficient sulfur-series malodorous substances |
| CN111849808A (en)* | 2020-07-06 | 2020-10-30 | 北京大学深圳研究院 | Methods of enrichment culture, inoculation and PCR amplification of PHA-producing bacteria from marine sources |
| Publication number | Publication date |
|---|---|
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| Publication | Publication Date | Title |
|---|---|---|
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