内源酶生物预处理强化污泥厌氧消化效能的研究
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摘要
污泥厌氧消化是目前应用最为广泛的污泥处理方法，但其处理周期长，需要大量的基建投资和占地面积。基于污泥胞外聚合物(EPS)的化学组成，在处置污泥中加入水解酶进行预处理，不仅能够提高污泥的厌氧可生化性，缩短消化时间，而且具有容易控制、不产生二次污染的优点。本文以内源酶产生菌的分离与筛选为基础，开展了环境因素对其生长及产酶特性的影响、菌株的发酵动力学特征及发酵液的酶学特性等基础研究。并利用菌株发酵液作为内源性水解酶处理污泥，考察了不同处理方式和条件对污泥水解酸化的影响。将预处理后污泥进行厌氧消化，通过BMP(Biochemical Methane Potential)实验，考察预处理对污泥产气、有机物去除效果和微生物群落的影响，并对内源酶预处理污泥强化厌氧消化效能的机制进行了探讨。
经富集、分离和筛选，从污泥中优选得到4株高效产酶菌株，且能在灭菌污泥中生长产酶。其中，菌株WYDF和WYZZ分别属于枯草芽孢杆菌(Bacillus subtilis)和地衣芽孢杆菌(Bacillus licheniformis)，主要产生淀粉酶；菌株WYC和WYE均属嗜水气单胞菌(Aeromonas hydrophila)，主要产生蛋白酶。培养温度、碳源、氮源、培养液初始pH都可以影响菌株的生长和酶的产生。在优化培养条件下，经34h的发酵，菌株WYDF、WYZZ、WYC和WYE的蛋白酶活性可分别达到121、158、671、和552U/mL；WYDF和WYZZ的淀粉酶活性分别达到162和158U/mL。胞外酶分子量分析结果表明，该4株菌所产生的淀粉酶和蛋白酶具有多样性，可用于组成复杂的胞外聚合物和细胞溶出物的水解。
以分离得到的菌株发酵培养液作为内源性水解酶预处理污泥，考察单一的内源酶或任意两种内源酶等量复配后，处理温度、时间、不同内源性水解酶复配比例对预处理效果的影响。结果表明，在中温范围内，内源酶预处理污泥的最佳温度为37℃，在此温度下，发酵7h即可达到较为理想的污泥水解效果。菌株WYDF产生的内源水解酶37℃下处理污泥28h时SCOD和VFAs增量分别为3152和1803mg/L，此时SCOD/TCOD达到最大值11.1%，与未处理污泥相比SCOD和VFAs含量分别提高了142%和219%；使粒径在2001000m固体颗粒减少，粒径在3070m的絮体量增加。随着预处理时间的延长，各试样中VFAs的乙酸含量逐渐降低。优化内源淀粉酶和蛋白酶配比，研究不同配比对预处理效果的影响，结果发现利于有机物的溶出和VFAs产生的比例均在1:3时最佳。
通过生物化学甲烷势的研究发现，37℃下内源水解酶预处理，促使污泥产气量增加、产气稳定性增强；例如WYDF内源酶单独处理后，其厌氧消化25d产气量由原污泥633mL提升至702mL，VS去除率由36.02%增至39.14%，甲烷产率由137mL CH4/g VSadded提升至161mL CH4/g VSadded。与单一内源酶预处理相比较，两种内源酶复配后更能有效的提高污泥厌氧消化性能；不同内源酶配比存在最优比例。
通过污泥粒径、表面特征、内源酶的稳定性和微生物群落分析，内源酶预处理污泥强化厌氧消化效能的主要机理包括：内源酶水解胞外聚合物为厌氧消化菌群提供了可直接利用的碳源和能源，促使产酸发酵菌迅速繁殖，进而促进产甲烷菌群的滋生与壮大；胞外聚合物的消失使细菌细胞壁得以暴露加速了其在厌氧消化过程中的溶胞速度；内源酶可持续作用于细菌细胞释放的碳水化合物和蛋白质加速了厌氧消化进程。
Anaerobic digestion has been widely adopted for the treatment of municipal sludge before final diposal; it is considered a major and essential part of a modern WWTP. In terms of the composition of Extracellular Polymeric Substance, adding hydrolase may be another pretreatment way to enhance sludge biogegradability and reduce hydraulic retention time.
In this work, hydrolyse producing bacteria were isolated from waste activated sludge; effects of cultivation condition and nutritional factors on enzyme producting capability, the basic property of the model of batch liquid fermentation kinetics and enzymatic characteristics of stains were investigated; the fementation liquid which containing hydrolase was adding to waste activated sludge as endogenouse hydrolase to strengthen the sludge hydrolysis efficiency, and effect of different temperatures, time, enzyme on hydrolytic and acidification. Biochemical Methane Potential (BMP)tests were carried out to assess sludge biodegradability, in the same time, mechanism of endogenouse hydrolase strengthen anaerobic digestion of sludge was discussed.
Four strains with high and steady amylase or protease activity were isolated from sludge; furthermore, the strains can be cultured in sterilized sludge, retaining partial enzymatic activity. Physiological and biochemical analysis showed that the strain was Bacillus subtilis, cillus licheniformis, Aeromonas hydrophilaand and Aeromonas hydrophila respectively, and we named them WYDF、WYZZ、WYC and WYE. WYDF and WYZZ produce amylase, mainly; WYC and WYE produce protease. Bacteria growth mass and enzyme producing activity were effected by carbon source, nitrogen source and initial pH; the growth and enzyme producing is not simultaneous, and both of them kinetics were established. The crude enzyme fementation of the four strains were separation and purification, the results show that each of each crude enzyme is different and the mixture of several kinds enzyme.
The effects of temperatures, time and mixed proportion of two kinds of endogenous enzyme on sludge pretreatment efficiency were investigated. The results showed that temperature of pretreatment play a critical role in the enzyme hydrolysis sludge progress; at the middle temperature,37℃is suitable for endogenous enzyme pretreatment，and7h is enough for a efficient sludge solubilization. Endogenous which procuded by strain WYDF presents the strongest ability to hydrolyze and acidize the sludge. The concentration of SCOD and VFAs increased by as much as142%and219%when the sludge was treated for28h, at37℃compared to a control (without adding of endogenous hydrolysis); SCOD/TCOD reach up to11.1%; solid particle ranged from200to1000m was reduced, the number of3070m increased. All samples, the acetic acid content increased with respect to pretreatment duration.
Effect of proportion between amylase and protease on pretreatment efficiency was studied. The result showed that the ratio was1:3which was beneficial to produce VFAs and organic dissolution.
Biochemical Methane Potential tests were carried out to evaluate the effect of different enzyme pretreatment condition on sludge subsequent anaerobic digestion. It shows that pretreated sludge by endogenous no matter which strain the anaerobic digestion efficiency was impoved. For the sludge pretreated by WYDF, volatile solids removal rate, VS removal rate and methane productivity were improved from633to702mL,36.02%to39.14,137to161mL CH4/g VSadded, respectively. It was compared to untreated sludge, at the end of the test (25d). Mixed enzyme improved sludge anaerobic degradation compared with hydrolase used alone; optimal proportion exists in different endogenous hydrolase for sludge pretreatment.
The change of sludge surface configurationin were observed before and after enzymatic treat by scanning electron microscope, and the change of particle size was messured, in order to provide some insights into sludge disintegration process. Results indicate that endogenous enzyme disintegrate sludge, improve the bioavailbility, reduce the number of larger size solid by hydrolysis EPS and destroy the structure of filamentous bacteria. In addition, enzyme will not only act on sludge in the process of pretreatment, but it will also continue to play a role in the subsequent anaerobic digestion.
Microbial community was detected by DGGE technology; the figure demonstrated that pretreated sludge results in an increase in microorganism species in the earlier stage of anaerobic digestion, compared to control; it observed no change with further digestion.
经富集、分离和筛选，从污泥中优选得到4株高效产酶菌株，且能在灭菌污泥中生长产酶。其中，菌株WYDF和WYZZ分别属于枯草芽孢杆菌(Bacillus subtilis)和地衣芽孢杆菌(Bacillus licheniformis)，主要产生淀粉酶；菌株WYC和WYE均属嗜水气单胞菌(Aeromonas hydrophila)，主要产生蛋白酶。培养温度、碳源、氮源、培养液初始pH都可以影响菌株的生长和酶的产生。在优化培养条件下，经34h的发酵，菌株WYDF、WYZZ、WYC和WYE的蛋白酶活性可分别达到121、158、671、和552U/mL；WYDF和WYZZ的淀粉酶活性分别达到162和158U/mL。胞外酶分子量分析结果表明，该4株菌所产生的淀粉酶和蛋白酶具有多样性，可用于组成复杂的胞外聚合物和细胞溶出物的水解。
以分离得到的菌株发酵培养液作为内源性水解酶预处理污泥，考察单一的内源酶或任意两种内源酶等量复配后，处理温度、时间、不同内源性水解酶复配比例对预处理效果的影响。结果表明，在中温范围内，内源酶预处理污泥的最佳温度为37℃，在此温度下，发酵7h即可达到较为理想的污泥水解效果。菌株WYDF产生的内源水解酶37℃下处理污泥28h时SCOD和VFAs增量分别为3152和1803mg/L，此时SCOD/TCOD达到最大值11.1%，与未处理污泥相比SCOD和VFAs含量分别提高了142%和219%；使粒径在2001000m固体颗粒减少，粒径在3070m的絮体量增加。随着预处理时间的延长，各试样中VFAs的乙酸含量逐渐降低。优化内源淀粉酶和蛋白酶配比，研究不同配比对预处理效果的影响，结果发现利于有机物的溶出和VFAs产生的比例均在1:3时最佳。
通过生物化学甲烷势的研究发现，37℃下内源水解酶预处理，促使污泥产气量增加、产气稳定性增强；例如WYDF内源酶单独处理后，其厌氧消化25d产气量由原污泥633mL提升至702mL，VS去除率由36.02%增至39.14%，甲烷产率由137mL CH4/g VSadded提升至161mL CH4/g VSadded。与单一内源酶预处理相比较，两种内源酶复配后更能有效的提高污泥厌氧消化性能；不同内源酶配比存在最优比例。
通过污泥粒径、表面特征、内源酶的稳定性和微生物群落分析，内源酶预处理污泥强化厌氧消化效能的主要机理包括：内源酶水解胞外聚合物为厌氧消化菌群提供了可直接利用的碳源和能源，促使产酸发酵菌迅速繁殖，进而促进产甲烷菌群的滋生与壮大；胞外聚合物的消失使细菌细胞壁得以暴露加速了其在厌氧消化过程中的溶胞速度；内源酶可持续作用于细菌细胞释放的碳水化合物和蛋白质加速了厌氧消化进程。
Anaerobic digestion has been widely adopted for the treatment of municipal sludge before final diposal; it is considered a major and essential part of a modern WWTP. In terms of the composition of Extracellular Polymeric Substance, adding hydrolase may be another pretreatment way to enhance sludge biogegradability and reduce hydraulic retention time.
In this work, hydrolyse producing bacteria were isolated from waste activated sludge; effects of cultivation condition and nutritional factors on enzyme producting capability, the basic property of the model of batch liquid fermentation kinetics and enzymatic characteristics of stains were investigated; the fementation liquid which containing hydrolase was adding to waste activated sludge as endogenouse hydrolase to strengthen the sludge hydrolysis efficiency, and effect of different temperatures, time, enzyme on hydrolytic and acidification. Biochemical Methane Potential (BMP)tests were carried out to assess sludge biodegradability, in the same time, mechanism of endogenouse hydrolase strengthen anaerobic digestion of sludge was discussed.
Four strains with high and steady amylase or protease activity were isolated from sludge; furthermore, the strains can be cultured in sterilized sludge, retaining partial enzymatic activity. Physiological and biochemical analysis showed that the strain was Bacillus subtilis, cillus licheniformis, Aeromonas hydrophilaand and Aeromonas hydrophila respectively, and we named them WYDF、WYZZ、WYC and WYE. WYDF and WYZZ produce amylase, mainly; WYC and WYE produce protease. Bacteria growth mass and enzyme producing activity were effected by carbon source, nitrogen source and initial pH; the growth and enzyme producing is not simultaneous, and both of them kinetics were established. The crude enzyme fementation of the four strains were separation and purification, the results show that each of each crude enzyme is different and the mixture of several kinds enzyme.
The effects of temperatures, time and mixed proportion of two kinds of endogenous enzyme on sludge pretreatment efficiency were investigated. The results showed that temperature of pretreatment play a critical role in the enzyme hydrolysis sludge progress; at the middle temperature,37℃is suitable for endogenous enzyme pretreatment，and7h is enough for a efficient sludge solubilization. Endogenous which procuded by strain WYDF presents the strongest ability to hydrolyze and acidize the sludge. The concentration of SCOD and VFAs increased by as much as142%and219%when the sludge was treated for28h, at37℃compared to a control (without adding of endogenous hydrolysis); SCOD/TCOD reach up to11.1%; solid particle ranged from200to1000m was reduced, the number of3070m increased. All samples, the acetic acid content increased with respect to pretreatment duration.
Effect of proportion between amylase and protease on pretreatment efficiency was studied. The result showed that the ratio was1:3which was beneficial to produce VFAs and organic dissolution.
Biochemical Methane Potential tests were carried out to evaluate the effect of different enzyme pretreatment condition on sludge subsequent anaerobic digestion. It shows that pretreated sludge by endogenous no matter which strain the anaerobic digestion efficiency was impoved. For the sludge pretreated by WYDF, volatile solids removal rate, VS removal rate and methane productivity were improved from633to702mL,36.02%to39.14,137to161mL CH4/g VSadded, respectively. It was compared to untreated sludge, at the end of the test (25d). Mixed enzyme improved sludge anaerobic degradation compared with hydrolase used alone; optimal proportion exists in different endogenous hydrolase for sludge pretreatment.
The change of sludge surface configurationin were observed before and after enzymatic treat by scanning electron microscope, and the change of particle size was messured, in order to provide some insights into sludge disintegration process. Results indicate that endogenous enzyme disintegrate sludge, improve the bioavailbility, reduce the number of larger size solid by hydrolysis EPS and destroy the structure of filamentous bacteria. In addition, enzyme will not only act on sludge in the process of pretreatment, but it will also continue to play a role in the subsequent anaerobic digestion.
Microbial community was detected by DGGE technology; the figure demonstrated that pretreated sludge results in an increase in microorganism species in the earlier stage of anaerobic digestion, compared to control; it observed no change with further digestion.
引文
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