活性污泥合成PHAs的饥饿丰盛模式耦合磁场作用机理
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摘要
聚羟基脂肪酸酯(Polyhydrocylkanotes，PHAs)不但与化学塑料性质相似，而且具有生物相容性、可完全生物降解，因而作为一种非常有前景的合成塑料替代品而日益受到关注。但较高的生产成本使生物可降解塑料聚羟基脂肪酸酯的大规模使用受到限制。
在所有利用微生物合成PHAs方法中，好氧瞬时供料法由于高的PHAs合成能力及可利用混合菌群(如活性污泥)和有机废弃物合成PHAs等特点，成为目前最具应用前景的技术。利用磁场的生物效应，耦合好氧瞬时供料工艺(饥饿-丰盛模式)，将市政污泥污水处理厂的剩余活性污泥培养成具有高PHAs存储能力的微生物，同时，选用酸化常见产物乙酸、丙酸、丁酸为底物，进行磁场耦合饥饿-丰盛模式条件下的PHAs合成实验，分别考察了磁场强度、底物组成、C：N等因子的影响，并对各因子进行优化实验；采用现代化分析技术，阐述磁场与饥饿丰盛模式耦合作用的机理。
研究结果发现，初始负荷及饥饿-丰盛期比例是驯化高PHAs存储能力微生物的关键因素。初始底物浓度为360mgCOD/1，逐渐增加浓度到6480mgCOD/1，饥饿-丰盛期比例为3：1时，微生物体内PHAs含量从8．74％增加到50．24％，且系统稳定运行。低初始浓度，有利于高存储能力微生物形成优势菌种。磁场耦合饥饿丰盛模式可以进一步提高微生物体内PHAs的含量，可达66．2％。
微生物体内PHAs及HB、HV单体分别在不同磁场强度达到峰值，PHA含量在磁场强度为11mT时最高，在磁场强度为42mT时最低；而HB含量在磁场强度为7mT时达到最高，在磁场强度为42mT时最低；HV含量在磁场强度为21mT时达到最高，在磁场强度为42mT时最低。微生物中PHAs合成率随C：N的升高而上升，在C：N为100：1左右，达到峰值，而活性微生物产率随C：N比的上升呈下降趋势；磁场存在时，微生物中的PHAs合成率随C：N变化趋势没有改变，但合成率均有所提高。根据正交实验结果直观分析，各因素对实验结果的重要性依次为磁场强度、C：N、pH。最优化实验组合为磁场强度取12mT，NH_3-N浓度3．5mg/L(C：N为160：1)、初始pH值7．0。
底物类型及组成比例对微生物中PHAs的合成率、单体HV/HB的比例具有显著的影响。以单一乙酸、丙酸、丁酸为底物时，乙酸和丁酸合成的是HB单体、丙酸合成的是HV单体。用乙酸作底物时，大部分的底物(约55％)是用来进行存储能量，而用于维持微生物生命活动的底物占20％左右，其余用来供应微生物的生长，即合成细胞质，而用丙酸和丁酸做底物时，约有25％左右的底物用来储存，一半左右的底物是用来供应细胞生长，其余用来维持微生物的生命活动。三种酸中，乙酸是最易合成PHAs的底物。
磁场使微生物吸收底物后的能量分配比例发生了变化，磁场强度为7mT，以乙酸或丁酸为底物，微生物PHAs产量Y_(PHA)在磁场强度为7mT时达到峰值，分别为0．70(mgCOD/mgCOD)、0．28(mgCOD/mgCOD)；丙酸为底物，PHA产量Y_(PHA)在磁场强度21mT时达到峰值，为0．31(mgCOD/mgCOD)。磁场对微生物吸收乙酸后的能量分配比例变化影响最大。
底物中乙酸/丙酸与聚合物中HV/HB存在一定的对应关系，经拟合可知两者基本呈线性相关，磁场的存在使各直线的斜率发生变化。磁场为优化PHAs中HV、HB比例，从而为优化PHAs的性能，提供一条新路径。
采用分子生态学技术(PCR-DGGE及16S rDNA鉴定)观察污泥中微生物群落特征的变化，结果表明，磁场强度使微生物群落特征发生了变化，在0mT磁场强度作用下，微生物群落以Leadbetterella byssophila属(屈挠杆菌属)占主导地位；在7mT磁场强度作用下，微生物群落以Unidentified bacterium，(AY34413)占主导地位；在21mT磁场强度作用下，微生物群落以Clostridium属、Alkaliphilus属(梭菌属)占主导地位；在42mT磁场强度作用下，微生物群落以Flavobacterium属(黄杆菌属)占主导地位。通过代谢通量分析可知，磁场影响PHAs合成的根本原因是它增加了碳源(乙酸)在代谢过程中向HB合成路径转化的量，在7mT时乙酰辅酶A进入合成HB途径的通量是进入TCA循环途径的2．7倍；而在42mT时乙酰辅酶A进入合成HB途径的通量是进入TCA循环途径的0．9倍；在0mT时乙酰辅酶A进入合成HB途径的通量是进入TCA循环途径的1．4倍。磁场为提高活性污泥中PHAs的合成率提供了一条新思路。
Polyhydroxyalkanoates (PHAs) have attracted increasing interest as an alternativeto petroleum-derived plastics for its similar mechanical properties to those ofpolypropylene, meanwhile, with the additional advantage of being completelybiodegradable, biocompatible, and produced from renewable resources. But the costof PHAs production is the major obstacle for large-scale commercial exploitation.
Among the methods of industrial PHAs production, the aerobic dynamic feeding(ADF, feast" /"famine condition) approach is the most promising because of highPHAs accumulation, meanwhile, this method could substantially decreasing the costof PHAs and increasing their market potential for using activated sludge andrenewable sources obtained from waste organic carbon. The objective of this study isto accumulate activated sludge from municipal wastewater treatment plant to themicroorganism with high PHAs storage ability under the co-effect of aerobic dynamicfeeding (ADF, feast" /"famine condition) conditions and magnetic field. Acetate,butyrate and propionate, the common product of acidification, are used as thesubstrate. The impact factors such as magnetic field intensity, composition ofsubstrate and C:N are testified during the experiments. The possible mechanism isexplaned by the morden analytical techniques.
The results show the substrate concentration of start-up and "feast" /"famine" ratioare the important factor for the activated sludge accumulation. The content of PHAsin biomass increased from 8.74% to 50.24% when the concentration increasedgradually to 6.48gCOD/l with the start-up concentration of 0.36gCOD/l and the"famine" /"feast" ratio was 3: 1, meanwhile, the system is stable. The lower substrateconcentration during start-up are benefit to the system with "steady-state" where thestorage microbial consortium will be the dominated one. The co-effect of magneticfield and ADF will enhance the content of PHAs far more. to be 66.2%.
The static magnetic exposure definitely influenced the biosynthesis of PHAs andthe effect depended on the field intensity as following: under intensity of 11mT, themaximum PHAs synthesis occurred; under intensity of 21mT. the maximum HVsynthesis occurred: under intensity of 7mT. the maximum HB synthesis occurred, andunder intensity of 42mT, the minimum HB, HV, PHAs synthesis occurred. PHAs content increase with the ratio of C:N, and reach the maximum at the ratio of 100:1.But the active biomass decreased with the increase of C: N ratio. With the magneticexposure, PHAs content have an enhancement under differ ratio of C: N. According tothe results of orthogonal experiment, the influence order of each factor on PHAsproduction is magnetic field, C:N, intial pH. The best condition for PHAs synthesis ismagnetic intensity 12mT, NH_3-N concentration 3.5mg/L (C: N,160:1 ), initial pHvalue 7.0
The polymer produced from either acetate or butyrate is the homopolymer of PHB.whereas the copolymer P(HB/HV) is formed when propionate is fed only or alongwith either acetate or butyrate. When acetate is fed solely, almost 55% of substrate isfor energy storage, 20% of substrate is for oxidation for energy production and theother is for growth. When propionate or butyrate is fed, almost 25% of substrate is forenergy storage. 50% of substrate is for growth and the other is for oxidation forenergy production. Obviously, acetate rather than butyrate and propionate is thesuitable substrate for energy storage.
The static magnetic exposure and ADF has definitely infuenced the energydistrubition of substrate in microorganism. The PHAs production yield of acetate orbutyrate reach the maximum value of 0.7(mgCOD/mgCOD) or 0.28(mgCOD/mgCOD) under magnetic intensity of 7mT. And the PHAs production yieldof propionate reach the maximum value of 0.31(mgCOD/mgCOD) under themagnetic intensity of 21 mT.
The ratio of acetate/propionate has a linear relationship with the ratio of HV/HB.The magnetic field change the slope of line and it sounds the magnetic field provide anew optimized way to PHV/PHB and the characteristics of PHAs.
Based on PCR-DGGE and 16S rDNA analysis, there have a change of microbialcommunity under different magnetic field. The microbial community are dominatedby Leadbetterella byssophila under no magnetic intensity, dominated by Unidentifiedbacterium, (AY34413 ) under magnetic intensity of 7mT, dominated by Clostridium,Alkaliphilus under magnetic intensity of 21mT. dominated by Flavobacterium undermagnetic intensity of 42mT. Based on metabolic flux analysis, we conclude that thereason of PHAs content in biomass improved by magnetic field is that the carbonsource (acetate) is in a state of flux during the metabolic process. There is 2.7times ofacetyl-CoA into PHB synthesis compared to TCA cycle under magnetic intensity of 7mT, and 0.9 times of acetyl-CoA into PHB synthesis compared to TCA cycle undermagnetic intensity of 42mT, and 1.4 times of acetyl-CoA into PHB synthesiscompared to TCA cycle under magnetic intensity of 0mT. The co-effect of staticmagnetic and "famine" /"feast" condition provide us a new method to improve thePHAs synthesis by activated sludge.
在所有利用微生物合成PHAs方法中，好氧瞬时供料法由于高的PHAs合成能力及可利用混合菌群(如活性污泥)和有机废弃物合成PHAs等特点，成为目前最具应用前景的技术。利用磁场的生物效应，耦合好氧瞬时供料工艺(饥饿-丰盛模式)，将市政污泥污水处理厂的剩余活性污泥培养成具有高PHAs存储能力的微生物，同时，选用酸化常见产物乙酸、丙酸、丁酸为底物，进行磁场耦合饥饿-丰盛模式条件下的PHAs合成实验，分别考察了磁场强度、底物组成、C：N等因子的影响，并对各因子进行优化实验；采用现代化分析技术，阐述磁场与饥饿丰盛模式耦合作用的机理。
研究结果发现，初始负荷及饥饿-丰盛期比例是驯化高PHAs存储能力微生物的关键因素。初始底物浓度为360mgCOD/1，逐渐增加浓度到6480mgCOD/1，饥饿-丰盛期比例为3：1时，微生物体内PHAs含量从8．74％增加到50．24％，且系统稳定运行。低初始浓度，有利于高存储能力微生物形成优势菌种。磁场耦合饥饿丰盛模式可以进一步提高微生物体内PHAs的含量，可达66．2％。
微生物体内PHAs及HB、HV单体分别在不同磁场强度达到峰值，PHA含量在磁场强度为11mT时最高，在磁场强度为42mT时最低；而HB含量在磁场强度为7mT时达到最高，在磁场强度为42mT时最低；HV含量在磁场强度为21mT时达到最高，在磁场强度为42mT时最低。微生物中PHAs合成率随C：N的升高而上升，在C：N为100：1左右，达到峰值，而活性微生物产率随C：N比的上升呈下降趋势；磁场存在时，微生物中的PHAs合成率随C：N变化趋势没有改变，但合成率均有所提高。根据正交实验结果直观分析，各因素对实验结果的重要性依次为磁场强度、C：N、pH。最优化实验组合为磁场强度取12mT，NH_3-N浓度3．5mg/L(C：N为160：1)、初始pH值7．0。
底物类型及组成比例对微生物中PHAs的合成率、单体HV/HB的比例具有显著的影响。以单一乙酸、丙酸、丁酸为底物时，乙酸和丁酸合成的是HB单体、丙酸合成的是HV单体。用乙酸作底物时，大部分的底物(约55％)是用来进行存储能量，而用于维持微生物生命活动的底物占20％左右，其余用来供应微生物的生长，即合成细胞质，而用丙酸和丁酸做底物时，约有25％左右的底物用来储存，一半左右的底物是用来供应细胞生长，其余用来维持微生物的生命活动。三种酸中，乙酸是最易合成PHAs的底物。
磁场使微生物吸收底物后的能量分配比例发生了变化，磁场强度为7mT，以乙酸或丁酸为底物，微生物PHAs产量Y_(PHA)在磁场强度为7mT时达到峰值，分别为0．70(mgCOD/mgCOD)、0．28(mgCOD/mgCOD)；丙酸为底物，PHA产量Y_(PHA)在磁场强度21mT时达到峰值，为0．31(mgCOD/mgCOD)。磁场对微生物吸收乙酸后的能量分配比例变化影响最大。
底物中乙酸/丙酸与聚合物中HV/HB存在一定的对应关系，经拟合可知两者基本呈线性相关，磁场的存在使各直线的斜率发生变化。磁场为优化PHAs中HV、HB比例，从而为优化PHAs的性能，提供一条新路径。
采用分子生态学技术(PCR-DGGE及16S rDNA鉴定)观察污泥中微生物群落特征的变化，结果表明，磁场强度使微生物群落特征发生了变化，在0mT磁场强度作用下，微生物群落以Leadbetterella byssophila属(屈挠杆菌属)占主导地位；在7mT磁场强度作用下，微生物群落以Unidentified bacterium，(AY34413)占主导地位；在21mT磁场强度作用下，微生物群落以Clostridium属、Alkaliphilus属(梭菌属)占主导地位；在42mT磁场强度作用下，微生物群落以Flavobacterium属(黄杆菌属)占主导地位。通过代谢通量分析可知，磁场影响PHAs合成的根本原因是它增加了碳源(乙酸)在代谢过程中向HB合成路径转化的量，在7mT时乙酰辅酶A进入合成HB途径的通量是进入TCA循环途径的2．7倍；而在42mT时乙酰辅酶A进入合成HB途径的通量是进入TCA循环途径的0．9倍；在0mT时乙酰辅酶A进入合成HB途径的通量是进入TCA循环途径的1．4倍。磁场为提高活性污泥中PHAs的合成率提供了一条新思路。
Polyhydroxyalkanoates (PHAs) have attracted increasing interest as an alternativeto petroleum-derived plastics for its similar mechanical properties to those ofpolypropylene, meanwhile, with the additional advantage of being completelybiodegradable, biocompatible, and produced from renewable resources. But the costof PHAs production is the major obstacle for large-scale commercial exploitation.
Among the methods of industrial PHAs production, the aerobic dynamic feeding(ADF, feast" /"famine condition) approach is the most promising because of highPHAs accumulation, meanwhile, this method could substantially decreasing the costof PHAs and increasing their market potential for using activated sludge andrenewable sources obtained from waste organic carbon. The objective of this study isto accumulate activated sludge from municipal wastewater treatment plant to themicroorganism with high PHAs storage ability under the co-effect of aerobic dynamicfeeding (ADF, feast" /"famine condition) conditions and magnetic field. Acetate,butyrate and propionate, the common product of acidification, are used as thesubstrate. The impact factors such as magnetic field intensity, composition ofsubstrate and C:N are testified during the experiments. The possible mechanism isexplaned by the morden analytical techniques.
The results show the substrate concentration of start-up and "feast" /"famine" ratioare the important factor for the activated sludge accumulation. The content of PHAsin biomass increased from 8.74% to 50.24% when the concentration increasedgradually to 6.48gCOD/l with the start-up concentration of 0.36gCOD/l and the"famine" /"feast" ratio was 3: 1, meanwhile, the system is stable. The lower substrateconcentration during start-up are benefit to the system with "steady-state" where thestorage microbial consortium will be the dominated one. The co-effect of magneticfield and ADF will enhance the content of PHAs far more. to be 66.2%.
The static magnetic exposure definitely influenced the biosynthesis of PHAs andthe effect depended on the field intensity as following: under intensity of 11mT, themaximum PHAs synthesis occurred; under intensity of 21mT. the maximum HVsynthesis occurred: under intensity of 7mT. the maximum HB synthesis occurred, andunder intensity of 42mT, the minimum HB, HV, PHAs synthesis occurred. PHAs content increase with the ratio of C:N, and reach the maximum at the ratio of 100:1.But the active biomass decreased with the increase of C: N ratio. With the magneticexposure, PHAs content have an enhancement under differ ratio of C: N. According tothe results of orthogonal experiment, the influence order of each factor on PHAsproduction is magnetic field, C:N, intial pH. The best condition for PHAs synthesis ismagnetic intensity 12mT, NH_3-N concentration 3.5mg/L (C: N,160:1 ), initial pHvalue 7.0
The polymer produced from either acetate or butyrate is the homopolymer of PHB.whereas the copolymer P(HB/HV) is formed when propionate is fed only or alongwith either acetate or butyrate. When acetate is fed solely, almost 55% of substrate isfor energy storage, 20% of substrate is for oxidation for energy production and theother is for growth. When propionate or butyrate is fed, almost 25% of substrate is forenergy storage. 50% of substrate is for growth and the other is for oxidation forenergy production. Obviously, acetate rather than butyrate and propionate is thesuitable substrate for energy storage.
The static magnetic exposure and ADF has definitely infuenced the energydistrubition of substrate in microorganism. The PHAs production yield of acetate orbutyrate reach the maximum value of 0.7(mgCOD/mgCOD) or 0.28(mgCOD/mgCOD) under magnetic intensity of 7mT. And the PHAs production yieldof propionate reach the maximum value of 0.31(mgCOD/mgCOD) under themagnetic intensity of 21 mT.
The ratio of acetate/propionate has a linear relationship with the ratio of HV/HB.The magnetic field change the slope of line and it sounds the magnetic field provide anew optimized way to PHV/PHB and the characteristics of PHAs.
Based on PCR-DGGE and 16S rDNA analysis, there have a change of microbialcommunity under different magnetic field. The microbial community are dominatedby Leadbetterella byssophila under no magnetic intensity, dominated by Unidentifiedbacterium, (AY34413 ) under magnetic intensity of 7mT, dominated by Clostridium,Alkaliphilus under magnetic intensity of 21mT. dominated by Flavobacterium undermagnetic intensity of 42mT. Based on metabolic flux analysis, we conclude that thereason of PHAs content in biomass improved by magnetic field is that the carbonsource (acetate) is in a state of flux during the metabolic process. There is 2.7times ofacetyl-CoA into PHB synthesis compared to TCA cycle under magnetic intensity of 7mT, and 0.9 times of acetyl-CoA into PHB synthesis compared to TCA cycle undermagnetic intensity of 42mT, and 1.4 times of acetyl-CoA into PHB synthesiscompared to TCA cycle under magnetic intensity of 0mT. The co-effect of staticmagnetic and "famine" /"feast" condition provide us a new method to improve thePHAs synthesis by activated sludge.
引文
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