利用活性污泥混合菌群合成聚羟基烷酸脂的研究
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摘要
聚羟基烷酸脂(PHA)是一类可由微生物在不均衡的营养条件下合成的脂类物质,在微生物代谢中起到积累碳源、平衡细胞内外离子的作用。PHA的物理特性类似于聚丙烯等化学塑料,无毒无害,在自然界可完全降解,是可望替代化学塑料、彻底解决白色污染的良好材料,但是较高的成本阻碍了PHA的大规模应用,使用混合菌种如活性污泥代替纯菌,使用含小分子脂肪酸的废水代替葡萄糖、果糖等底物,可以在降低PHA的生产成本的同时,达到污泥减量、废水资源化等目的。PHA的合成菌种类很多,利用活性污泥作为混合菌来源合成PHA受到了广泛关注,然而目前利用活性污泥合成PHA的工艺方式还比较复杂,往往将富集和驯化污泥合成PHA分成两个阶段或者分别在两个反应器内进行,总工艺时间较长,PHA合成效率还不能令人满意。本课题的研究目的是通过驯化污水厂剩余污泥以小分子脂肪酸为底物合成PHA,寻找简易可行的污泥驯化和PHA的合成工艺,简化操作程序,探索驯化活性污泥合成PHA的影响因素,通过简单的工艺和简单的驯化方式,序批式驯化污泥合成PHA,实现废水和剩余污泥的资源化回收利用和PHA合成的多重目的。
试验以乙酸钠代替乙酸为唯一碳源,接种污水处理厂的剩余污泥,使用序批式反应器合成PHA。通过不同的启动和驯化方式影响PHA合成,研究发现,通过厌氧-好氧运行7-15天恢复污泥的活性,在均衡比例COD:N或COD:P的基础上,以一定的梯度(10%~20%)减少N或P的浓度形成不均衡的营养条件,经过10-15天的驯化,可使活性污泥在厌氧条件下合成PHA;当通过好氧运行7-10天恢复污泥活性,然后在厌氧-好氧交替运行条件下以一定梯度(10%~20%)减少N或P浓度的方式驯化7-10天,可以使活性污泥在好氧条件下合成PHA。通过不同的启动方式实现了对两种不同的PHA优势合成菌的控制和筛选。
试验研究了营养比例(COD:N,COD:P)对PHA合成的影响。研究结果显示, PHA的最大合成量随着碳氮比或碳磷比的提高而提高,营养比例既是刺激微生物合成积累PHA的条件,也影响着细胞内最大PHA产量;厌氧条件下合成PHA时,在COD:N=125:1和COD:P=250:1时最大PHA积累量分别达到细胞干重的57%和26%;好氧条件下合成PHA时,在COD:N=200:1和COD:P=500:1时最大PHA合成量分别达到细胞干重的61%和33%,限制氮源比限制磷源获得的PHA含量更高。同时发现,厌氧条件下合成的PHA由PHB和PHV构成,好氧条件下合成的PHA几乎完全由PHB构成。合成PHA反应器经过约35天持续运行,无论是限制氮源还是限制磷源,均会导致污泥膨胀,其中磷源匮乏的反应器发生了非丝状菌的粘性膨胀,氮源匮乏的反应器发生了丝状菌膨胀,同时PHA合成能力丧失。
试验考察了温度和pH对PHA合成的影响,研究发现在限磷条件下,19℃时PHA合成能力最强;在限氮条件下,16℃时PHA合成能力最强。无论限氮或限磷,结果都显示较低的温度适宜PHA的合成。在不控制pH时(pH介于8~9),PHA的合成情况较好,当pH降低时到7.0时底物利用能力和PHA合成能力下降,pH降至5.0时PHA的合成能力大幅度降低。中性偏碱性的环境有利于PHA的合成。
将含红糖的废水水解酸化以后作为碳源进水合成PHA。研究发现,调整pH后进水,不仅可以使PHA的总量提高,同时也令PHA的单体构成发生变化,PHV的比例获得较大幅度的提高;若含酸废水不经pH调整直接进水,将会对PHA的合成产生冲击,单位基质的PHA的产率和最大PHA积累量分别下降46%和58%,其中PHB受到的影响更大。当调整含酸废水的营养比例使碳源相对过剩时,基质利用效率和PHA的积累量又会提升。
课题对活性污泥中PHA的提取进行了初步研究,使用5%的NaClO对2800mg/L的湿污泥可达到较好的破碎效果。试验设计了一套包括吹扫蒸发、溶剂吸收、尾气过滤三个部分的简易PHA提取装置,使用氯仿溶解萃取PHA,使用NMP吸收氯仿,在一套装置内同时实现了PHA的析出和氯仿溶剂的回收;提取后的PHA粗产品呈白色片状或粉末状,对于PHA含量20%左右的污泥经过提取,获得的PHA纯度可达60-70%以上,同时对材料的表面特性、热稳定性进行分析,并提出了利用剩余污泥驯化、合成、提取PHA的技术策略。
从驯化后的合成PHA的污泥中筛得一株具有较高PHA合成能力的菌,命名为S1,对其进行发酵试验研究表明,该菌在较高的碳磷比下可以大量积累PHA,碳磷比是影响PHA产量的最主要因素;分批发酵实验显示,在COD:P=400时,细胞干重达到了4.78g/L同时PHA的积累量也达到了51.2%。细胞的增长和PHA的积累是同时进行的。16s rDNA测序鉴定表明,S1菌属于β-变形菌纲红环菌目红环菌科陶厄氏菌属。试验同时建立了S1发酵产PHA的动力学模型方程,理论模型与实际试验结果符合较好。
Polyhydroxyalkanoates (PHA) are a class of polymer which can be synthesized by many species of microorganisms under imbalanced nutrient conditions, PHA accumulated in the microbial metabolism plays a carbon source, energy, adjust intracellular ion balance. PHA's physical characteristics are similar to chemical plastics such as polypropylene, and no harm to environment, PHA can be completely degraded in the natural condition, it is expected to replace chemical plastic, and gives good potential alternatives to completely solve the white pollution further. Currently, there are many companies using pure bacteria and pure culture fermentation to produce commercial PHA, but the high cost prevented the large-scale application of PHA.To reduce PHA production costs, a approach is use of mixed bacteria, such as activated sludge instead of pure bacteria, using organic waste, food waste instead of glucose, fructose, etc. as the substrate, can greatly reduce the cost of PHA production, but research shows that, PHA could synthesized by too many microorganisms, not all strains are suitable for mixed culture product PHA , some PHA synthesis process is relatively complicated, not easy to operate and control the conditions, the domestication time and efficiency was not satisfactory. The purpose of this research topic is to acclimate excess sludge synthesize PHA by low molecular fatty acid, find a simple and feasible for PHA synthesis methods, simplify procedures and steps, while maximizing the yield of PHA.
With acetate as sole carbon source, sludge or excess sludge from urban sewage treatment plants secondary settling tank is inoculated to reactor, study the way of different start and acclimated sludge to synthesize PHA. The results found that, running first by anaerobic - aerobic for 7-15 days, the activity of the sludge was restored, then the ratio of COD: N or COD: P, was gradually increased, N or P element concentration reduced on a certain gradient (10% -20%), after 10-15 days of acclimation, PAOs or GAOs which Synthesize PHA under anaerobic conditions were gained. when the first starting phase changed to complete aerobic condition for 7-10 days,and then anaerobic anoxic condition for acclimation, the concentration of N or P was reduced meantime, after 7-10 days, PHA synthesized under aerobic conditions in activated sludge was gained. In terms of a single carbon source, through a different start, domestication of control was achieved; PHA synthesis of two different strains could be control and selection.
In the basis of domestication, using acetate as sole carbon source, the ratio of nutrients (COD:N, COD:P) on PHA synthesis were adjusted, the results show that with carbon-nitrogen or carbon-phosphorus ratios increased, PHA yield increased, too. The proportion of both nutrients stimulate microbial synthesis of PHA, and also affects the maximum PHA yield of cells; for the synthesis of PHA under anaerobic conditions in the sludge,of the COD:N=125:1 and COD:P=250:1, the maximum PHA accumulations in cell dry weight were 57% and 26% respectively; PHA synthesized under aerobic conditions, the maximum amount of PHA synthesis of cell dry weight in COD:N=200:1 and COD:P=500 were 61% and 33% respectively. Limiting nitrogen source was batter to the PHA yield than limiting the phosphorus content. The experiment also found that under anaerobic conditions the constituted of the PHA are PHB and PHV, PHV PHA proportion accounted for 10% to 20%; PHA synthesized under aerobic conditions is almost PHB. Both PHA production reactors appears sludge bulking after about 35 days of operation, when phosphorus is restricted, non-filamentous sludge bulking was founded, when nitrogen source is restricted, filamentous sludge bulking occurred, then the PHA synthesis capacity lost.
Experiment with different temperatures and different pH conditions was studied, the synthesis of PHA under the conditions of phosphorus limited shows a maximum yield in 19℃; while in N-limited conditions, the PHA yielded max at 16℃. No matter what nutrients are limited, it indicated that a lower temperature is suitable for PHA synthesis. In pH affect study, without control of pH conditions, pH ranged of 8 ~ 9, PHA synthesis is better, when pH is lowered to 7.0 substrate utilization capacity and PHA synthesis decreased, when pH is lowered to 5.0, the synthesis of PHA capacity significantly reduced. Neutral and alkaline environment is conducive to PHA synthesis.
In order to simulate the real wastewater contained molecule organic acid, use brown sugar acidification wastewater as a carbon source to synthesize PHA, adjust the pH of fatty acid wastewater and then import to reactor, the experiment showed that not only can the rich carbon source to increase the total amount of PHA, but also makes the PHA monomer composition changes, PHV ratio was dramatically increased; if the water without pH adjustment import into the PHA synthesis reactor directly, it will have some impact on the synthesis of Panther substrate utilization rate and the maximum PHA production rate decreased by 46% and 58%, respectively; analysis from the PHA monomer product, PHB yield is affected greater, the efficiency in the use of substrate is decreased, either. When adjusting the proportion of nutrient ratio of fatty acid wastewater to make a condition that carbon is excess to nitrogen or phosphorus, PHA accumulation will get a promotion, the proportion of substrate for PHA synthesis increased by more than 100%. An unbalanced diet is to stimulate PHA accumulation ratio is still the best external factors, partially offset by the adverse effects of lower pH.
Base on the PHA synthesis study, using 5% NaClO on 2800mg/L of sludge can be broken to achieve better results. Designed a set including the purge evaporation, solvent absorption, emission security under laboratory conditions bottle PHA extraction apparatus, using chloroform dissolved PHA, using NMP absorbed chloroform, synchronization of the PHA precipitation and chloroform solvent recovery; The PHA extraction was white flakes or powder, the impurities was residual fragments of the activated sludge, for about 20% dry cell weight of the PHA in activated sludge, after extraction, the product obtained over 60-70% in purity.
After the pilot production of domesticated PHA, screening a strain with a high PHA synthesis, named as S1, its fermentation experiments showed that the strain could accumulated PHA at a higher ratio of carbon to phosphorus, carbon-phosphorus ratio is the main factors affecting the PHA yield, which is consistent with reactor acclimation results; batch fermentation experiments showed that, in the COD:P=400, the cell dry weight reached 4.78g/L at the same time the accumulation of PHA also reached to 51.2%. Cell growth and PHA accumulation was simultaneous. 16s rDNA sequencing showed that, S1 bacteria was closely related to Thauera sp.
试验以乙酸钠代替乙酸为唯一碳源,接种污水处理厂的剩余污泥,使用序批式反应器合成PHA。通过不同的启动和驯化方式影响PHA合成,研究发现,通过厌氧-好氧运行7-15天恢复污泥的活性,在均衡比例COD:N或COD:P的基础上,以一定的梯度(10%~20%)减少N或P的浓度形成不均衡的营养条件,经过10-15天的驯化,可使活性污泥在厌氧条件下合成PHA;当通过好氧运行7-10天恢复污泥活性,然后在厌氧-好氧交替运行条件下以一定梯度(10%~20%)减少N或P浓度的方式驯化7-10天,可以使活性污泥在好氧条件下合成PHA。通过不同的启动方式实现了对两种不同的PHA优势合成菌的控制和筛选。
试验研究了营养比例(COD:N,COD:P)对PHA合成的影响。研究结果显示, PHA的最大合成量随着碳氮比或碳磷比的提高而提高,营养比例既是刺激微生物合成积累PHA的条件,也影响着细胞内最大PHA产量;厌氧条件下合成PHA时,在COD:N=125:1和COD:P=250:1时最大PHA积累量分别达到细胞干重的57%和26%;好氧条件下合成PHA时,在COD:N=200:1和COD:P=500:1时最大PHA合成量分别达到细胞干重的61%和33%,限制氮源比限制磷源获得的PHA含量更高。同时发现,厌氧条件下合成的PHA由PHB和PHV构成,好氧条件下合成的PHA几乎完全由PHB构成。合成PHA反应器经过约35天持续运行,无论是限制氮源还是限制磷源,均会导致污泥膨胀,其中磷源匮乏的反应器发生了非丝状菌的粘性膨胀,氮源匮乏的反应器发生了丝状菌膨胀,同时PHA合成能力丧失。
试验考察了温度和pH对PHA合成的影响,研究发现在限磷条件下,19℃时PHA合成能力最强;在限氮条件下,16℃时PHA合成能力最强。无论限氮或限磷,结果都显示较低的温度适宜PHA的合成。在不控制pH时(pH介于8~9),PHA的合成情况较好,当pH降低时到7.0时底物利用能力和PHA合成能力下降,pH降至5.0时PHA的合成能力大幅度降低。中性偏碱性的环境有利于PHA的合成。
将含红糖的废水水解酸化以后作为碳源进水合成PHA。研究发现,调整pH后进水,不仅可以使PHA的总量提高,同时也令PHA的单体构成发生变化,PHV的比例获得较大幅度的提高;若含酸废水不经pH调整直接进水,将会对PHA的合成产生冲击,单位基质的PHA的产率和最大PHA积累量分别下降46%和58%,其中PHB受到的影响更大。当调整含酸废水的营养比例使碳源相对过剩时,基质利用效率和PHA的积累量又会提升。
课题对活性污泥中PHA的提取进行了初步研究,使用5%的NaClO对2800mg/L的湿污泥可达到较好的破碎效果。试验设计了一套包括吹扫蒸发、溶剂吸收、尾气过滤三个部分的简易PHA提取装置,使用氯仿溶解萃取PHA,使用NMP吸收氯仿,在一套装置内同时实现了PHA的析出和氯仿溶剂的回收;提取后的PHA粗产品呈白色片状或粉末状,对于PHA含量20%左右的污泥经过提取,获得的PHA纯度可达60-70%以上,同时对材料的表面特性、热稳定性进行分析,并提出了利用剩余污泥驯化、合成、提取PHA的技术策略。
从驯化后的合成PHA的污泥中筛得一株具有较高PHA合成能力的菌,命名为S1,对其进行发酵试验研究表明,该菌在较高的碳磷比下可以大量积累PHA,碳磷比是影响PHA产量的最主要因素;分批发酵实验显示,在COD:P=400时,细胞干重达到了4.78g/L同时PHA的积累量也达到了51.2%。细胞的增长和PHA的积累是同时进行的。16s rDNA测序鉴定表明,S1菌属于β-变形菌纲红环菌目红环菌科陶厄氏菌属。试验同时建立了S1发酵产PHA的动力学模型方程,理论模型与实际试验结果符合较好。
Polyhydroxyalkanoates (PHA) are a class of polymer which can be synthesized by many species of microorganisms under imbalanced nutrient conditions, PHA accumulated in the microbial metabolism plays a carbon source, energy, adjust intracellular ion balance. PHA's physical characteristics are similar to chemical plastics such as polypropylene, and no harm to environment, PHA can be completely degraded in the natural condition, it is expected to replace chemical plastic, and gives good potential alternatives to completely solve the white pollution further. Currently, there are many companies using pure bacteria and pure culture fermentation to produce commercial PHA, but the high cost prevented the large-scale application of PHA.To reduce PHA production costs, a approach is use of mixed bacteria, such as activated sludge instead of pure bacteria, using organic waste, food waste instead of glucose, fructose, etc. as the substrate, can greatly reduce the cost of PHA production, but research shows that, PHA could synthesized by too many microorganisms, not all strains are suitable for mixed culture product PHA , some PHA synthesis process is relatively complicated, not easy to operate and control the conditions, the domestication time and efficiency was not satisfactory. The purpose of this research topic is to acclimate excess sludge synthesize PHA by low molecular fatty acid, find a simple and feasible for PHA synthesis methods, simplify procedures and steps, while maximizing the yield of PHA.
With acetate as sole carbon source, sludge or excess sludge from urban sewage treatment plants secondary settling tank is inoculated to reactor, study the way of different start and acclimated sludge to synthesize PHA. The results found that, running first by anaerobic - aerobic for 7-15 days, the activity of the sludge was restored, then the ratio of COD: N or COD: P, was gradually increased, N or P element concentration reduced on a certain gradient (10% -20%), after 10-15 days of acclimation, PAOs or GAOs which Synthesize PHA under anaerobic conditions were gained. when the first starting phase changed to complete aerobic condition for 7-10 days,and then anaerobic anoxic condition for acclimation, the concentration of N or P was reduced meantime, after 7-10 days, PHA synthesized under aerobic conditions in activated sludge was gained. In terms of a single carbon source, through a different start, domestication of control was achieved; PHA synthesis of two different strains could be control and selection.
In the basis of domestication, using acetate as sole carbon source, the ratio of nutrients (COD:N, COD:P) on PHA synthesis were adjusted, the results show that with carbon-nitrogen or carbon-phosphorus ratios increased, PHA yield increased, too. The proportion of both nutrients stimulate microbial synthesis of PHA, and also affects the maximum PHA yield of cells; for the synthesis of PHA under anaerobic conditions in the sludge,of the COD:N=125:1 and COD:P=250:1, the maximum PHA accumulations in cell dry weight were 57% and 26% respectively; PHA synthesized under aerobic conditions, the maximum amount of PHA synthesis of cell dry weight in COD:N=200:1 and COD:P=500 were 61% and 33% respectively. Limiting nitrogen source was batter to the PHA yield than limiting the phosphorus content. The experiment also found that under anaerobic conditions the constituted of the PHA are PHB and PHV, PHV PHA proportion accounted for 10% to 20%; PHA synthesized under aerobic conditions is almost PHB. Both PHA production reactors appears sludge bulking after about 35 days of operation, when phosphorus is restricted, non-filamentous sludge bulking was founded, when nitrogen source is restricted, filamentous sludge bulking occurred, then the PHA synthesis capacity lost.
Experiment with different temperatures and different pH conditions was studied, the synthesis of PHA under the conditions of phosphorus limited shows a maximum yield in 19℃; while in N-limited conditions, the PHA yielded max at 16℃. No matter what nutrients are limited, it indicated that a lower temperature is suitable for PHA synthesis. In pH affect study, without control of pH conditions, pH ranged of 8 ~ 9, PHA synthesis is better, when pH is lowered to 7.0 substrate utilization capacity and PHA synthesis decreased, when pH is lowered to 5.0, the synthesis of PHA capacity significantly reduced. Neutral and alkaline environment is conducive to PHA synthesis.
In order to simulate the real wastewater contained molecule organic acid, use brown sugar acidification wastewater as a carbon source to synthesize PHA, adjust the pH of fatty acid wastewater and then import to reactor, the experiment showed that not only can the rich carbon source to increase the total amount of PHA, but also makes the PHA monomer composition changes, PHV ratio was dramatically increased; if the water without pH adjustment import into the PHA synthesis reactor directly, it will have some impact on the synthesis of Panther substrate utilization rate and the maximum PHA production rate decreased by 46% and 58%, respectively; analysis from the PHA monomer product, PHB yield is affected greater, the efficiency in the use of substrate is decreased, either. When adjusting the proportion of nutrient ratio of fatty acid wastewater to make a condition that carbon is excess to nitrogen or phosphorus, PHA accumulation will get a promotion, the proportion of substrate for PHA synthesis increased by more than 100%. An unbalanced diet is to stimulate PHA accumulation ratio is still the best external factors, partially offset by the adverse effects of lower pH.
Base on the PHA synthesis study, using 5% NaClO on 2800mg/L of sludge can be broken to achieve better results. Designed a set including the purge evaporation, solvent absorption, emission security under laboratory conditions bottle PHA extraction apparatus, using chloroform dissolved PHA, using NMP absorbed chloroform, synchronization of the PHA precipitation and chloroform solvent recovery; The PHA extraction was white flakes or powder, the impurities was residual fragments of the activated sludge, for about 20% dry cell weight of the PHA in activated sludge, after extraction, the product obtained over 60-70% in purity.
After the pilot production of domesticated PHA, screening a strain with a high PHA synthesis, named as S1, its fermentation experiments showed that the strain could accumulated PHA at a higher ratio of carbon to phosphorus, carbon-phosphorus ratio is the main factors affecting the PHA yield, which is consistent with reactor acclimation results; batch fermentation experiments showed that, in the COD:P=400, the cell dry weight reached 4.78g/L at the same time the accumulation of PHA also reached to 51.2%. Cell growth and PHA accumulation was simultaneous. 16s rDNA sequencing showed that, S1 bacteria was closely related to Thauera sp.
引文
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