A/O SBR中聚磷菌合成PHB及影响因子研究
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摘要
本试验以ERP-SBR工艺的逆过程为载体、模拟将来可能采用的含可利用碳源的工业废水作为进水,研究了一株聚磷菌纯培养物在不同A/O SBR培养体系长期驯化的“主体反应器”及短期条件改变的“第二批次试验”下,利用好氧段末端菌体中poly-P作为动力、厌氧条件下PHB储存行为。对起始pH、温度、碳源浓度和营养限制等主要易控的影响因子作用机理进行了分析;并将系统中PO_4~(3-)-p代谢、NaAc吸收利用与PHB合成能力相耦合;为今后以聚磷菌为主体的脱磷剩余污泥资源化利用及PHB规模化生产探索道路。试验主要结果如下:
(1)以模拟废水为碳源、采用实验室规模的ERP-SBR工艺对活性污泥进行为期25天快速诱导驯化,系统对PO_4~(3-)-p的去除率可达80.58%;从该富集聚磷菌的活性污泥里共分离得到36株纯菌株。再结合“异染粒和PHB颗粒染色”定性观察,与“强化吸释磷试验”定量测定,简便快捷地筛选得到一株胞内同时具有PHB颗粒和异染颗粒的高效聚磷菌。结合培养形态观察、生理生化特征分析和16S rDNA分子生物学技术,鉴定该聚磷菌为鲍曼不动杆菌,并命名为Acinetobacter baumannii LB4。
(2)通过综合考察稳定运行的PHB积累系统受起始pH和温度的影响发现:控制起始pH中性偏碱,有利于菌体合成PHB,pH8.0时PHB合成量最大为229.6 mg·L~(-1);调节温度30～35℃,细胞干重和PHB合成量均达到最大值,分别为1264.6 mg·L~(-1)和268.1 mg·L~(-1),且温度不是其限制因素。
不同起始pH长期驯化的试验中,NaAc吸收速率、PO_4~(3-)-p厌氧释放和好氧吸收量及其快速代谢速率、PHB厌氧合成和好氧分解量及其快速代谢速率等7个指标均随pH的升高而增加。其中,厌氧结束时菌体胞内PHB含量由pH6.5的191.6 mg·L~(-1)增加到pH 8.0时的233.5 mg·L~(-1)。初始pH对PHB合成的影响主要作用在PO_4~(3-)-p快速释放速率上。
不同温度长期驯化的试验中,除PO_4~(3-)-p的厌氧释放量和好氧吸收量未随温度呈现规律性变化外,其余5个指标均随温度升高而增加。其中,厌氧结束时菌体胞内PHB含量由20℃的179.3 mg·L~(-1)增加到35℃时的269.2 mg·L~(-1);PHB积累量与吸磷量和释磷量的差值正向相关。温度对PHB合成的影响主要作用在NaAc吸收速率上。分析可知,PO_4~(3-)-p快速释放速率影响NaAc吸收速率,进而引起PHB快速合成速率的差异,最终导致厌氧PHB积累量的不同。
(3)在不同初始碳源浓度长期驯化的试验中,聚磷菌A.baumannii LB4积累PHB量和积累到峰值所用的时间均随初始碳源浓度的提高而增加。分析PHB合成的整个过程,COD_(4000)系统内PHB的浓度最大,为623.6 mg·L~(-1);COD_(500)系统中碳源转化率以及PHB合成速率最高,分别为41.2%和175.2 mg·L~(-1)·h~(-1);而采用COD为2000 mg·L~(-1)的碳源浓度可以驯化出积累PHB质量分数最高的菌株,可占细胞干重27.6%。根据染色观察及现有理论推测得知,本试验中NaAc吸收受阻及PHB合成中断的现象是由于菌体内糖原耗舅鸬摹?
在不同营养条件长期驯化以及短期改变的试验中,发现通过营养平衡的主体反应器驯化得到能够正常生长并积累PHB较多的微生物种群;再采用批式补料方式短期限制营养条件对其进行单独处理,更有利于促进PHB积累量和质量分数的提高,分别可达528.6mg·L~(-1)和32.6%。
(4)通过正交试验对PHB提取参数进行优化,得到最佳操作条件为:萃取温度50℃、萃取时间120 min、NaClO/CHCl_3(V/V)为1/1、CHCl_3为10 mL、干细胞重100 mg;提取的PHB可占细胞干重的33.2%。其中NaClO和CHCl_3体积比对PHB提取量具有显著影响,随NaClO/CHCl_3(WV)降低,PHB提取量增加。
(5)通过紫外吸收光谱、红外光谱和元素分析等手段对提取所得PHB样品的化学结构进行表征,并与标准品比较。结果表明:提取所得样品在紫外吸收光谱215 nm处有巴豆酸共轭羰基的特征峰;在傅利叶红外光谱1723 cm~(-1)具有羰基伸缩振动引起的特征峰;并且样品的C、H元素质量百分比与理论计算值接近。综合上述结果,可鉴定从聚磷菌A.baumannii LB4胞内提取获得的薄膜状物质为聚-β-羟基丁酸。
There has been considerable in the development and production of biodegradable polymer to solve the current problem of pollution caused by the continuous use of synthetic polymer of petroleum origin.Poly-β-hydroxybutyrate(PHB) are known to be accumulated as intracellular inclusion in some bacteria,which is a close substitute for the synthetic plastic.The high cost of PHB production has restricted its application.
This study investigates the PHB storage behavior of a poly-phosphate accumulating organism(PAO) under different growth conditions in detail.Pure culture studies in batch-growth systems were conducted in shake-flasks,using chemically defined growth media with acetate as the sole carbon source.This batch-growth system with anaerobic-aerobic cycles and varying conditions,similar to enhanced biological phosphorus removal processes(EBPR), was also employed.This paper also highly reports the advances of PHB extraction methods from cell and the PHB analytical methods.Therefore,it is a new way for PHB synthesis,which also develops a novel pathway for the reclamation of residual sludge.
The results of this research can be summarized as follows:
(1) Firstly,the enrichment and cultivation of poly-phosphate accumulating activated sludge were studied in a set of sequencing batch reactor(SBR) system,under A/O condition. 30 days later,the both removal rates of ammonium and phosphate were higher than 80%.Then, separation 36 strains were isolated from the activated sludge.One of them with high levels of phosphate uptake and release activities,combined poly-P and PHB granules microscopic staining,was confirmed and named LB4.
Based on the morphological appearances,physiological and biochemical characteristics, together with analysis of 16S rDNA sequence,the activated-sludge bacterium(LB4) were identified as Acinetobacter baumannii.
(2) In this part of the study,the effects of initial pH and temperature on the storage metabolism of A.baumannii,under repeated anaerobic-aerobic cycles on the batch growth, were investigated.
Acinetobacter baumannii LB4 was acclimated for a long term with higher initial pH could store more PHB and release more phosphorus in the anaerobic phase,and degraded more PHB and accumulate more polyphosphate in the following aerobic phase.At the same time,the initial metabolism rate of acetate,phosphorus and poly-β-hydroxybutyrate would be faster.
The acetate-utilization and PHB-storage behavior of Acinetobacter baumannii LB4 acclimated for a long term with higher temperature was similar to those in higher initial pH. Result also shows that,LB4's PHB storage capability was could be described with the PHB content of the cells with respect to was closely related to phosphorus removal amount.
Effects of carbon concentration and nutrient deficiency on the PHB of the batch-growth systems are studied.With 673.6 mg·L~(-1),1355.4 mg·L~(-1),2694.6 mg·L~(-1) and 5393.3 mg·L~(-1) initial sodium acetate,the maximum PHB content of dry biomass were 12.91%,20.83%, 27.60%and 24.91%respectively,while carbon translating ratios were 41.2%,38.5%,33.3% and 22.0%.During the first 120 min of anaerobic phase,PHB forming rate were 175.2 mg·L~(-1)·h~(-1),153.8 mg·L~(-1)·h~(-1),123.9 mg·L~(-1)·h~(-1) and 114.3 mg·L~(-1)·h~(-1).The influence of nutrient deficiency on the storage metabolism of A.baumannii under repeated anaerobic-aerobic cycles on the batch growth was not evident.
(3) Orthogonal experimental design was applied to optimize the extraction condition PHB from microorganism.The optimized parameters of extracting PHB were as follows:method of extraction strain Acinetobacter baumannii LB4:the extraction temperature 50℃,the extraction time 2 h,the volume ratio of sodium hypochlorite and chloroform 1:1,the liquid volume of chloroform 10 mL,and the dry biomass 100 mg.The extraction yield of PHB under these parameters was 33.2 mg(per 100 mg CDW).
Ultraviolet spectrum,infrared spectrum,and elementary analysis were utilized for the characteristics of PHB structure.
(1)以模拟废水为碳源、采用实验室规模的ERP-SBR工艺对活性污泥进行为期25天快速诱导驯化,系统对PO_4~(3-)-p的去除率可达80.58%;从该富集聚磷菌的活性污泥里共分离得到36株纯菌株。再结合“异染粒和PHB颗粒染色”定性观察,与“强化吸释磷试验”定量测定,简便快捷地筛选得到一株胞内同时具有PHB颗粒和异染颗粒的高效聚磷菌。结合培养形态观察、生理生化特征分析和16S rDNA分子生物学技术,鉴定该聚磷菌为鲍曼不动杆菌,并命名为Acinetobacter baumannii LB4。
(2)通过综合考察稳定运行的PHB积累系统受起始pH和温度的影响发现:控制起始pH中性偏碱,有利于菌体合成PHB,pH8.0时PHB合成量最大为229.6 mg·L~(-1);调节温度30～35℃,细胞干重和PHB合成量均达到最大值,分别为1264.6 mg·L~(-1)和268.1 mg·L~(-1),且温度不是其限制因素。
不同起始pH长期驯化的试验中,NaAc吸收速率、PO_4~(3-)-p厌氧释放和好氧吸收量及其快速代谢速率、PHB厌氧合成和好氧分解量及其快速代谢速率等7个指标均随pH的升高而增加。其中,厌氧结束时菌体胞内PHB含量由pH6.5的191.6 mg·L~(-1)增加到pH 8.0时的233.5 mg·L~(-1)。初始pH对PHB合成的影响主要作用在PO_4~(3-)-p快速释放速率上。
不同温度长期驯化的试验中,除PO_4~(3-)-p的厌氧释放量和好氧吸收量未随温度呈现规律性变化外,其余5个指标均随温度升高而增加。其中,厌氧结束时菌体胞内PHB含量由20℃的179.3 mg·L~(-1)增加到35℃时的269.2 mg·L~(-1);PHB积累量与吸磷量和释磷量的差值正向相关。温度对PHB合成的影响主要作用在NaAc吸收速率上。分析可知,PO_4~(3-)-p快速释放速率影响NaAc吸收速率,进而引起PHB快速合成速率的差异,最终导致厌氧PHB积累量的不同。
(3)在不同初始碳源浓度长期驯化的试验中,聚磷菌A.baumannii LB4积累PHB量和积累到峰值所用的时间均随初始碳源浓度的提高而增加。分析PHB合成的整个过程,COD_(4000)系统内PHB的浓度最大,为623.6 mg·L~(-1);COD_(500)系统中碳源转化率以及PHB合成速率最高,分别为41.2%和175.2 mg·L~(-1)·h~(-1);而采用COD为2000 mg·L~(-1)的碳源浓度可以驯化出积累PHB质量分数最高的菌株,可占细胞干重27.6%。根据染色观察及现有理论推测得知,本试验中NaAc吸收受阻及PHB合成中断的现象是由于菌体内糖原耗舅鸬摹?
在不同营养条件长期驯化以及短期改变的试验中,发现通过营养平衡的主体反应器驯化得到能够正常生长并积累PHB较多的微生物种群;再采用批式补料方式短期限制营养条件对其进行单独处理,更有利于促进PHB积累量和质量分数的提高,分别可达528.6mg·L~(-1)和32.6%。
(4)通过正交试验对PHB提取参数进行优化,得到最佳操作条件为:萃取温度50℃、萃取时间120 min、NaClO/CHCl_3(V/V)为1/1、CHCl_3为10 mL、干细胞重100 mg;提取的PHB可占细胞干重的33.2%。其中NaClO和CHCl_3体积比对PHB提取量具有显著影响,随NaClO/CHCl_3(WV)降低,PHB提取量增加。
(5)通过紫外吸收光谱、红外光谱和元素分析等手段对提取所得PHB样品的化学结构进行表征,并与标准品比较。结果表明:提取所得样品在紫外吸收光谱215 nm处有巴豆酸共轭羰基的特征峰;在傅利叶红外光谱1723 cm~(-1)具有羰基伸缩振动引起的特征峰;并且样品的C、H元素质量百分比与理论计算值接近。综合上述结果,可鉴定从聚磷菌A.baumannii LB4胞内提取获得的薄膜状物质为聚-β-羟基丁酸。
There has been considerable in the development and production of biodegradable polymer to solve the current problem of pollution caused by the continuous use of synthetic polymer of petroleum origin.Poly-β-hydroxybutyrate(PHB) are known to be accumulated as intracellular inclusion in some bacteria,which is a close substitute for the synthetic plastic.The high cost of PHB production has restricted its application.
This study investigates the PHB storage behavior of a poly-phosphate accumulating organism(PAO) under different growth conditions in detail.Pure culture studies in batch-growth systems were conducted in shake-flasks,using chemically defined growth media with acetate as the sole carbon source.This batch-growth system with anaerobic-aerobic cycles and varying conditions,similar to enhanced biological phosphorus removal processes(EBPR), was also employed.This paper also highly reports the advances of PHB extraction methods from cell and the PHB analytical methods.Therefore,it is a new way for PHB synthesis,which also develops a novel pathway for the reclamation of residual sludge.
The results of this research can be summarized as follows:
(1) Firstly,the enrichment and cultivation of poly-phosphate accumulating activated sludge were studied in a set of sequencing batch reactor(SBR) system,under A/O condition. 30 days later,the both removal rates of ammonium and phosphate were higher than 80%.Then, separation 36 strains were isolated from the activated sludge.One of them with high levels of phosphate uptake and release activities,combined poly-P and PHB granules microscopic staining,was confirmed and named LB4.
Based on the morphological appearances,physiological and biochemical characteristics, together with analysis of 16S rDNA sequence,the activated-sludge bacterium(LB4) were identified as Acinetobacter baumannii.
(2) In this part of the study,the effects of initial pH and temperature on the storage metabolism of A.baumannii,under repeated anaerobic-aerobic cycles on the batch growth, were investigated.
Acinetobacter baumannii LB4 was acclimated for a long term with higher initial pH could store more PHB and release more phosphorus in the anaerobic phase,and degraded more PHB and accumulate more polyphosphate in the following aerobic phase.At the same time,the initial metabolism rate of acetate,phosphorus and poly-β-hydroxybutyrate would be faster.
The acetate-utilization and PHB-storage behavior of Acinetobacter baumannii LB4 acclimated for a long term with higher temperature was similar to those in higher initial pH. Result also shows that,LB4's PHB storage capability was could be described with the PHB content of the cells with respect to was closely related to phosphorus removal amount.
Effects of carbon concentration and nutrient deficiency on the PHB of the batch-growth systems are studied.With 673.6 mg·L~(-1),1355.4 mg·L~(-1),2694.6 mg·L~(-1) and 5393.3 mg·L~(-1) initial sodium acetate,the maximum PHB content of dry biomass were 12.91%,20.83%, 27.60%and 24.91%respectively,while carbon translating ratios were 41.2%,38.5%,33.3% and 22.0%.During the first 120 min of anaerobic phase,PHB forming rate were 175.2 mg·L~(-1)·h~(-1),153.8 mg·L~(-1)·h~(-1),123.9 mg·L~(-1)·h~(-1) and 114.3 mg·L~(-1)·h~(-1).The influence of nutrient deficiency on the storage metabolism of A.baumannii under repeated anaerobic-aerobic cycles on the batch growth was not evident.
(3) Orthogonal experimental design was applied to optimize the extraction condition PHB from microorganism.The optimized parameters of extracting PHB were as follows:method of extraction strain Acinetobacter baumannii LB4:the extraction temperature 50℃,the extraction time 2 h,the volume ratio of sodium hypochlorite and chloroform 1:1,the liquid volume of chloroform 10 mL,and the dry biomass 100 mg.The extraction yield of PHB under these parameters was 33.2 mg(per 100 mg CDW).
Ultraviolet spectrum,infrared spectrum,and elementary analysis were utilized for the characteristics of PHB structure.
引文
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