聚磷菌的筛选及其体内聚磷酸盐推动PHB积累的研究
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摘要
PHB是细菌在不利于生长和营养失衡的条件下胞内大量积累的聚羟基酯。其作为合成聚合物的替代品具有很多优点,它不仅具有可生物降解性和生物相容性,而且还能以可再生资源作为生产原料。
本研究以污水处理厂的活性污泥为样品,从中筛选出三株高效聚磷菌。对此三株菌从形态特征、生理生化指标、16SrDNA三方面进行鉴定,确认此三株聚磷菌分别为鲍氏不动杆菌(Acinetobacter baumannii)、Pseudochrobactrum saccharolyticum、荧光假单胞菌(Pseudomonas fluorescens),并将三株菌分别编号为YPS-1、YPS-2和YPS-3。
选取聚磷菌YPS-2进行聚磷培养基及培养条件的优化。本试验设计好氧/厌氧两段式工艺进行微生物胞内合成与积累聚-β-羟基丁酸(PHB)。以菌体生长和聚磷能力为指标,通过摇瓶培养发酵,对培养基中碳源、氮源和磷源进行优化,试验确定的碳源为CH_3COONa、氮源为(NH_4)_2SO_4、磷源为KH_2PO_4。通过单因子试验、正交试验对好氧阶段菌体生长及聚/释磷条件进行优化,得到对聚磷菌YPS-2聚磷的最适培养基成分及其浓度为:碳源浓度1%、氮源浓度0.1%、磷源浓度100 mg/L;最佳条件为:培养基初始pH值为7、培养温度为30℃、接种量为10%、培养时间为72h。优化的聚磷条件为典型周期的研究提供了理论基础。
厌氧培养营养成分对菌体释磷及PHB的积累有重要影响,通过对四种营养条件(缺N、缺P、缺N、P、全营养)下,菌体生长、释磷率、PHB积累情况的研究,发现营养均衡有利于菌体生长,但缺少氮源和磷源的培养有利于菌体释磷和PHB的积累,充分说明PHB需在不平衡生长条件下得以积累。
好氧/厌氧典型周期中菌体在好氧阶段生长旺盛,厌氧阶段基本不生长;聚磷量和释磷量则随时间的增加而增加,但厌氧释磷需时较长。聚磷量、释磷量、单位质量干细胞中PHB所占比重与时间均存在一定的线形关系,随时间的增加而增加,但其增加幅度各有不同。在最佳发酵条件下,经好氧/厌氧交替培养后菌体聚磷量超过70mg/L、释磷量超过45mg/L、单位质量干细胞中PHB所占比重达55%以上。较好地实现了生物除磷与PHB的积累。
为了提高PHB产率,对收集的菌体进行预处理发现SDS/EDTA混合液具有较好的破壁效果。通过正交试验优化PHB提取条件,优化结果为:NaClO浓度为30%、NaClO/CCl_4(V/V)=3:1、温度为40℃、时间为24h。与未优化前的PHB产率相比,优化后PHB大幅提高,大约为优化前的2倍。样品与标准品的红外分析光谱表明,样品中含有PHB,为进一步PHB的提取与定性分析提供了参考。
Poly-β-hydroxybutyrate(PHB) is a group of polyesters produced and naturally accumulated by a large number of bacteria as intracellular granules in response to unfavorable growth conditions and nutrient imbalance.Poly-β-hydroxybutyrate represent an interesting alternative to synthetic polymers due to many advantages.Not only they are biodegradable and biocompatible,but they can also be produced from renewable resources.
This study was conducted by using the activated sludge from sewage treatment plant as a simple,three effective phosphate-accumulating organisms(PAOs) were screened.The identification of these PAOs according to their physiological,biochemical properties and 16SrDNA gene sequence,the result showed that they are Acinetobacter baumannii, Pseudochrobactrum saccharolyticum,Pseudomonas fluorescens,the strains number are YPS-1, YPS-2,YPS-3.
YPS-2 was selected to continue the experiments of optimization.Aerobic/anaerobic twostage craft were designed for PHB compound and accumulation.Take cell growth and phosphorus accumulate rate as indicators,the experiment was advanced through the shake flask fermentation,the optimal results were as follows:carbon source is CH_3COONa,nitrogen source is(NH_4)_2SO_4,phosphorus source is KH_2PO_4.Under the aerobic stage,cell growth and phosphorus accumulate/release rate were investigated using the single-factor experiment and the orthogonal design experiment method.The optimal medium concentration was as follows: carbon source is 1%,nitrogen source is 0.1%,phosphorus source is 100 mg/L.The optimal culture conditions were as follows:medium pH value is 7,the temperature is 30℃,for 10%of volume,incubation time is 72h.The result of phosphorus accumulate conditions provides a theoretical basis for research of typical cycle.
It is important for phosphate release and PHB in anaerobic culture.The study of cell growth,phosphorus release rate and PHB accumulation under four nutritional conditions(limit N,limit P,limit N and P,complete nutrition),found that the nutritional balance in favor of cell growth,but the condition,lack of nitrogen and phosphorus is conducive to phosphate release quantity and the accumulation of PHB.This shows that,PHB needs to be accumulated in the unbalanced growth conditions.
During the aerobic/anaerobic bacterial growth with a typical cycle,YPS-2 grows strongly, while it has almost no growth in anaerobic stage.Phosphorus accumulation and phosphorus release increased with time,but the anaerobic phosphorus release will take longer.There are certain linear relations among phosphorus,PHB and time,they are increased with time,but the slopes of them were not same.Under the aerobic/anaerobic alternating optimum conditions, accumulation of phosphorus more than 70mg/L,phosphorus release more than 45mg/L,the quality of stem cell units in the proportion of PHB more than 55%.It better achieved biological phosphorus removal and accumulation of PHB.
In order to improve the production of PHB,make the cells pre-treatment,found that SDS/EDTA mixture more effective than others.Conditions of PHB extraction were investigated using the orthogonal design experiment method.The optimal conditions were as follows:concentration of NaClO is 30%,NaClO/CCl_4(V/V)=3:1,temperature is 40℃,time is 24h.Compared with the non-optimized,the production of PHB increased significantly,it is two times the size of former.Infrared spectrum analysis for Samples and standards show that samples containing PHB,the result can be used to further qualitative analysis and improve the yield of PHB.
本研究以污水处理厂的活性污泥为样品,从中筛选出三株高效聚磷菌。对此三株菌从形态特征、生理生化指标、16SrDNA三方面进行鉴定,确认此三株聚磷菌分别为鲍氏不动杆菌(Acinetobacter baumannii)、Pseudochrobactrum saccharolyticum、荧光假单胞菌(Pseudomonas fluorescens),并将三株菌分别编号为YPS-1、YPS-2和YPS-3。
选取聚磷菌YPS-2进行聚磷培养基及培养条件的优化。本试验设计好氧/厌氧两段式工艺进行微生物胞内合成与积累聚-β-羟基丁酸(PHB)。以菌体生长和聚磷能力为指标,通过摇瓶培养发酵,对培养基中碳源、氮源和磷源进行优化,试验确定的碳源为CH_3COONa、氮源为(NH_4)_2SO_4、磷源为KH_2PO_4。通过单因子试验、正交试验对好氧阶段菌体生长及聚/释磷条件进行优化,得到对聚磷菌YPS-2聚磷的最适培养基成分及其浓度为:碳源浓度1%、氮源浓度0.1%、磷源浓度100 mg/L;最佳条件为:培养基初始pH值为7、培养温度为30℃、接种量为10%、培养时间为72h。优化的聚磷条件为典型周期的研究提供了理论基础。
厌氧培养营养成分对菌体释磷及PHB的积累有重要影响,通过对四种营养条件(缺N、缺P、缺N、P、全营养)下,菌体生长、释磷率、PHB积累情况的研究,发现营养均衡有利于菌体生长,但缺少氮源和磷源的培养有利于菌体释磷和PHB的积累,充分说明PHB需在不平衡生长条件下得以积累。
好氧/厌氧典型周期中菌体在好氧阶段生长旺盛,厌氧阶段基本不生长;聚磷量和释磷量则随时间的增加而增加,但厌氧释磷需时较长。聚磷量、释磷量、单位质量干细胞中PHB所占比重与时间均存在一定的线形关系,随时间的增加而增加,但其增加幅度各有不同。在最佳发酵条件下,经好氧/厌氧交替培养后菌体聚磷量超过70mg/L、释磷量超过45mg/L、单位质量干细胞中PHB所占比重达55%以上。较好地实现了生物除磷与PHB的积累。
为了提高PHB产率,对收集的菌体进行预处理发现SDS/EDTA混合液具有较好的破壁效果。通过正交试验优化PHB提取条件,优化结果为:NaClO浓度为30%、NaClO/CCl_4(V/V)=3:1、温度为40℃、时间为24h。与未优化前的PHB产率相比,优化后PHB大幅提高,大约为优化前的2倍。样品与标准品的红外分析光谱表明,样品中含有PHB,为进一步PHB的提取与定性分析提供了参考。
Poly-β-hydroxybutyrate(PHB) is a group of polyesters produced and naturally accumulated by a large number of bacteria as intracellular granules in response to unfavorable growth conditions and nutrient imbalance.Poly-β-hydroxybutyrate represent an interesting alternative to synthetic polymers due to many advantages.Not only they are biodegradable and biocompatible,but they can also be produced from renewable resources.
This study was conducted by using the activated sludge from sewage treatment plant as a simple,three effective phosphate-accumulating organisms(PAOs) were screened.The identification of these PAOs according to their physiological,biochemical properties and 16SrDNA gene sequence,the result showed that they are Acinetobacter baumannii, Pseudochrobactrum saccharolyticum,Pseudomonas fluorescens,the strains number are YPS-1, YPS-2,YPS-3.
YPS-2 was selected to continue the experiments of optimization.Aerobic/anaerobic twostage craft were designed for PHB compound and accumulation.Take cell growth and phosphorus accumulate rate as indicators,the experiment was advanced through the shake flask fermentation,the optimal results were as follows:carbon source is CH_3COONa,nitrogen source is(NH_4)_2SO_4,phosphorus source is KH_2PO_4.Under the aerobic stage,cell growth and phosphorus accumulate/release rate were investigated using the single-factor experiment and the orthogonal design experiment method.The optimal medium concentration was as follows: carbon source is 1%,nitrogen source is 0.1%,phosphorus source is 100 mg/L.The optimal culture conditions were as follows:medium pH value is 7,the temperature is 30℃,for 10%of volume,incubation time is 72h.The result of phosphorus accumulate conditions provides a theoretical basis for research of typical cycle.
It is important for phosphate release and PHB in anaerobic culture.The study of cell growth,phosphorus release rate and PHB accumulation under four nutritional conditions(limit N,limit P,limit N and P,complete nutrition),found that the nutritional balance in favor of cell growth,but the condition,lack of nitrogen and phosphorus is conducive to phosphate release quantity and the accumulation of PHB.This shows that,PHB needs to be accumulated in the unbalanced growth conditions.
During the aerobic/anaerobic bacterial growth with a typical cycle,YPS-2 grows strongly, while it has almost no growth in anaerobic stage.Phosphorus accumulation and phosphorus release increased with time,but the anaerobic phosphorus release will take longer.There are certain linear relations among phosphorus,PHB and time,they are increased with time,but the slopes of them were not same.Under the aerobic/anaerobic alternating optimum conditions, accumulation of phosphorus more than 70mg/L,phosphorus release more than 45mg/L,the quality of stem cell units in the proportion of PHB more than 55%.It better achieved biological phosphorus removal and accumulation of PHB.
In order to improve the production of PHB,make the cells pre-treatment,found that SDS/EDTA mixture more effective than others.Conditions of PHB extraction were investigated using the orthogonal design experiment method.The optimal conditions were as follows:concentration of NaClO is 30%,NaClO/CCl_4(V/V)=3:1,temperature is 40℃,time is 24h.Compared with the non-optimized,the production of PHB increased significantly,it is two times the size of former.Infrared spectrum analysis for Samples and standards show that samples containing PHB,the result can be used to further qualitative analysis and improve the yield of PHB.
引文
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