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胞外多糖酶解对Anammox颗粒污泥稳定性的影响

2022-03-07杨帆王帅龙曼赵凡郭劲松方芳

土木建筑与环境工程 2022年1期

杨帆 王帅 龙曼 赵凡 郭劲松 方芳

摘 要:为了研究胞外多糖对厌氧氨氧化颗粒污泥稳定性的影响及其机理,采用可酶解多糖的淀粉酶对颗粒污泥进行酶解。结果表明,α-淀粉酶处理组颗粒污泥外边缘出现溶胀,而β-淀粉酶处理组颗粒污泥表面无明显变化,但出现破碎且稳定性明显下降。表面性质及XDLVO理论分析表明,酶解降低了颗粒污泥疏水性,增大了微生物之间的排斥力,从而影响了颗粒污泥的稳定性。傅里叶红外光谱扫描结果表明,酶解后污泥胞外聚合物疏水性官能团含量明显降低。共聚焦扫描发现,α-淀粉酶处理组颗粒污泥外缘α-D-吡喃葡萄糖多糖含量明显下降,而β-淀粉酶处理组β-D-吡喃葡萄糖多糖呈碎片状分布。α-淀粉酶处理组表明,胞外多糖的疏水性作用、通过O—H官能团与阳离子桥接或相互结合作用可以促进微生物之间的聚集。β-淀粉酶处理组表明,胞外多糖长主链之间的缠结以及丰富的结合位点桥接形成骨架,增强了微生物之间的黏附,有利于颗粒污泥的稳定。

关键词:厌氧氨氧化;颗粒污泥;胞外多糖;酶解

中图分类号:X703   文献标志码:A   文章编号:2096-6717(2022)01-0188-09

收稿日期:2021-01-30

基金项目:国家自然科学基金(51878091)

作者简介:杨帆(1997- ),男,主要從事污水处理技术研究,E-mail:youngfan0117@163.com。

方芳(通信作者),女,教授,博士生导师,E-mail:fangfangcq@cqu.edu.cn。

Abstract: In order to study the effect and mechanism of extracellular polysaccharides on the stability of Anammox granular sludge, amylase which can enzymolyze polysaccharides was used to enzymatically hydrolyze granular sludge. The results showed that the outer edge of the granular sludge in the α-amylase treatment group swelled, while the surface of the granular sludge in the β-amylase treatment group did not change significantly, but it was broken and the stability was obviously reduced. The surface properties and XDLVO theoretical analysis showed that enzymatic hydrolysis reduced the hydrophobicity of granular sludge and increased the repulsive force between microorganisms, thereby affecting the stability of granular sludge. The results of Fourier infrared spectroscopy showed that the content of hydrophobic functional groups in extracellular polymers of sludge was significantly reduced after enzymatic hydrolysis. Confocal scanning found that the content of α-D-glucopyranose polysaccharide at the outer edge of the granular sludge in the α-amylase treatment group was significantly decreased, while the β-D-glucopyranose polysaccharide in the β-amylase treatment group was distributed in fragments. Therefore, the α-amylase treatment group showed that the hydrophobic effect of extracellular polysaccharides, binding to each other through O—H functional groups or bridging with cations can promote the aggregation between microorganisms. The β-amylase treatment group showed that the entanglement between the long backbones of extracellular polysaccharides and the bridging of abundant binding sites to form a skeleton enhanced the adhesion between microorganisms and was beneficial to the stability of granular sludge.

Keywords:anaerobic ammonium oxidation; granular sludge; exo-polysaccharide; enzymolysis

厌氧氨氧化(Anaerobic Ammonium Oxidation, Anammox)反应是指厌氧氨氧化菌在厌氧条件下以NO-2-N为电子受体,NH+4-N为电子供体,将NO-2-N和NH+4-N转化为N2的过程[1]。厌氧氨氧化工艺脱氮效率高,且不需要额外的有机碳源,污泥产量低。而厌氧氨氧化颗粒污泥因其结构致密,生物量高,可以促进固液分离、增强污泥保留能力等[1-2],在Anammox工艺中得到了广泛的应用[3-4]。胞外聚合物(Extracellular Polymeric Substances, EPS)是细菌分泌在胞外的高分子聚合物,主要成分为蛋白和多糖[5],可以将微生物细胞黏附起来[6-7],是形成微生物聚集体的关键。因此,从EPS的角度探究影响Anammox颗粒污泥稳定性的因素尤为重要。

有关颗粒污泥EPS的研究比较多[8-10],但多集中在胞外蛋白方面[11-12]。作为EPS中的主要成分之一,胞外多糖对颗粒污泥的影响日益受到重视[13]。Sajjad等[14]认为胞外多糖中带负电的基团可与二价阳离子形成架桥作用,将微生物结合在一起。Lin等[15]认为好氧颗粒污泥中的凝胶对其结构稳定具有重要作用,而多糖则是EPS的主要凝胶成分[16-17]。因此,从胞外多糖的角度深入探讨影响Anammox颗粒污泥稳定性的原因具有重要意义。

胞外多糖對Anammox颗粒污泥的形成和稳定有重要作用[18]。有学者指出,胞外多糖通过形成聚合物来促进微生物间的黏附,从而增强颗粒稳定性[13]。此外,多糖交联形成水凝胶也是维持颗粒稳定的重要因素[16-17]。目前,已在好氧颗粒污泥胞外聚合物中鉴定出alginate多糖和granulan多糖两种凝胶多糖[15, 19],它们可以在生物聚集体外形成结构凝胶,有助于聚集体结构的稳定。胞外多糖通过氢键相连接,促进污泥结构稳定[20]。Adav等[21]研究发现,胞外多糖可以形成网格状骨干以维持好氧颗粒污泥的稳定性。Yuan等[22]认为糖是一种具有长链的聚合物,可提供许多结合位点,并通过链缠结来保持颗粒稳定[23]。遗憾的是,这些研究没有直接从多糖链结构角度对胞外多糖的功能进行更全面的认识。

笔者采用可酶解多糖的两种淀粉酶来水解Anammox颗粒污泥胞外多糖,研究酶解前后颗粒污泥的稳定性,结合颗粒污泥表面性质和XDLVO理论分析微生物的聚集,探讨酶解前后污泥EPS的官能团组成和胞外多糖空间分布,以期从胞外多糖的角度理解其对颗粒污泥稳定性的影响及机理。

1 材料与方法

1.1 反应器和废水性质

厌氧氨氧化颗粒污泥取自实验室长期稳定运行的厌氧氨氧化膨胀颗粒污泥床[12]。反应器进水为人工合成废水,NH+4-N和NO-2-N浓度分别约为180、190 mg/L,总氮负荷为2.39 kg/(m3·d),进水pH值为7.8~8.0,每升水添加微量元素1.25 mL,其配方参考Van de Graaf等[24]的研究。反应器温度控制在(32±1) ℃,脱氮性能保持稳定,NH+4 -N、NO-2-N和TN的平均去除率分别为94%、97%和87%左右。

1.2 实验方案

多糖是由糖苷键结合形成的糖链,为研究胞外多糖对颗粒污泥稳定性的影响及机理,采用α-淀粉酶和β-淀粉酶分别水解颗粒污泥。α-淀粉酶可随机水解α-1,4-糖苷键,而β-糖苷键则从非还原末端逐次水解α-1,4-糖苷键,但切断至分支点的α-1,6-糖苷键前则会停止。因此,在α-淀粉酶作用下,多糖糖链被剪切成短链,而经β-淀粉酶水解后仅多糖支链末端被水解,其主链并未被破坏。

将粒径均匀的30 g颗粒污泥均分为3组并分装在50 mL离心管中,设置为对照组、α-淀粉酶处理组和β-淀粉酶处理组,实验设计如表1所示。添加酶解液后,将3组污泥在恒温振荡器中以150 r/min,37 ℃的条件振荡1 h。用去离子水冲洗酶解后的污泥,洗去残留的酶解液,以用于后续分析,对于每个酶处理组和对照组进行3次重复实验。实验所用淀粉酶均购于Coolaber公司,并按Adav等[21]的实验方法配置成相应浓度的溶液。

1.3 Anammox颗粒污泥性能测试

颗粒污泥强度可以用来反映颗粒污泥酶解前后的稳定性,采用超声法测定颗粒污泥的强度[25]。将颗粒污泥放入装有15 mL去离子水的25 mL锥形瓶中,然后将其置于20~25 kHz、65 W的超声浴中,以2.5(开)~3 s(关)为周期进行超声处理。收集超声后的上清液,并用分光光度计在600 nm处测定吸光度,该值可代表上清液的浊度。

颗粒污泥表面疏水性采用基于正十二烷-水系统的微生物黏附烷烃试验测定[26]。将待测污泥研磨制成细胞悬浮液,用去离子水将细胞悬浮液的OD546,0调整到约0.3,取4 mL悬浮液用漩涡振荡器充分混合2 min,随后静置沉降15 min,测定OD546.1。另取4 mL悬浮液与1 mL正十二烷充分混合,使用漩涡振荡器充分混合2 min,随后静置15 min以保证分离,此时部分细胞悬浮液被吸附到烷烃相,而取水相测定OD546,2。测试重复3次,采用式(1)计算相对疏水度。

疏水度(%)=OD546,1-OD546,2OD546,0×100%(1)

Zeta电位可反映污泥表面带电荷情况,将污泥充分碾磨制成悬浮液,并用0.1 mol/L的NaCl溶液调整悬浮液的OD546约为0.1,采用纳米粒度及Zeta电位分析仪(Zetasizer Nano ZS90,英国)测试,每个样品测试3次,取平均值。

污泥与水、甲酰胺和1-溴萘的接触角被用于分析污泥的表面热力学特性。接触角的测定参照Liu等[27]的方法。用研钵研磨污泥制备污泥悬浮液,以0.45 μm的醋酸纤维素膜过滤。切下一片膜,用接触角测量仪(SDC-100, Shengding, 中国)分别测定膜上污泥与水、甲酰胺和1-溴萘的接触角。每个样品测定10次,取平均值。

1.4 表面热力学和XDLVO理论

根据污泥与水、甲酰胺和1-溴萘的接触角,采用热力学分析方法[28],可计算出Hamaker常数ABLB,界面吸附自由能ΔGadh、范德华作用自由能ΔGLWBL、Lewis酸碱水合作用自由能ΔGABBL等表面热力学计算参数,其中,ΔGadh是ΔGLWBL与ΔGABBL的和。比较這些热力学参数,可以分析酶解对厌氧氨氧化颗粒污泥表面特性的影响。

根据热力学参数和ζ电位,可以获得以距离为变量的微生物间的相互作用总能量WT、压缩双电层能量WR、范德华能量WA和酸碱相互作用能量WAB[28],进而可以从能量的角度解释微生物聚集的机理。WT是WR、WA和WAB的总和,具体计算过程和参数取值在文献[29]中有详细的描述。

1.5 EPS含量及红外光谱分析

采用碱提法提取颗粒污泥EPS[30]。EPS中的胞外蛋白使用BCA蛋白试剂盒(Solarbio,日本)进行测定[31],以牛血清蛋白为参考标准。胞外多糖使用蒽酮硫酸法测定,并以葡萄糖作为参考标准[32]。

傅里叶红外光谱(FTIR)可以有效检测EPS中的官能团[33]。将冷冻干燥的10 mg EPS样品与KBr混合制成压片,采用红外光谱仪(Nicolet iS50, Thermo Fisher Scientific.,美国)在4 000~400 cm-1范围内对EPS样品进行红外光谱扫描。所有FTIR光谱均进行归一化至最大发射1.0后进行数据分析。

1.6 共聚焦激光扫描显微镜

共聚焦激光扫描显微镜(CLSM)可以直接观察多糖的空间分布。参考Adav等[21]和Ni等[18]的方法,分别用20 μL Con A(0.25 g/L)、20 μL CW(0.3 g/L)染色胞外α-D-吡喃葡萄糖多糖和β-D-吡喃葡萄糖多糖。染色后将样品置于共聚焦激光扫描显微镜(TCS SP8 CSU,Leica,德国)下观察,采用LAS X共聚焦软件进行数据分析,并归一化至最大发射1.0后进行数据分析。

2 结果与分析

2.1 颗粒污泥稳定性

图1为酶解后Anammox颗粒污泥聚集体外观形态和局部显微镜图像。对照组的污泥形状规则,呈椭球状,边缘清晰,无碎片。α-淀粉酶处理组的颗粒污泥外缘发生轻微溶胀,出现明显透光现象,但无明显破损。β-淀粉酶处理的颗粒污泥中出现了部分小碎片,但并未发生溶胀,且在显微镜下污泥结构密实,边界清晰可见。

颗粒污泥结构致密,具有一定的强度。采用超声法研究多糖对污泥结构稳定性的影响,A600吸光度的大小可代表污泥经超声处理后上清液的浊度,用于表示污泥解体程度,即间接表明颗粒污泥强度[25],结果如图2所示。β-淀粉酶处理组的颗粒污泥经超声处理后上清液在A600的吸光度迅速上升,表明颗粒发生了明显的解体,颗粒污泥强度明显降低。对照组和α-淀粉酶处理组的颗粒污泥在超声处理下A600吸光度的变化较小。与对照组相比,α-淀粉酶水解后颗粒污泥强度基本不变,但经超声处理20 min后强度略有所下降。

2.2 颗粒污泥表面性质

如表2所示,酶解前后污泥表面Zeta电位相差不大,多糖水解并未改变污泥表面的电负性。与对照组相比,经α-淀粉酶和β-淀粉酶水解后,颗粒污泥的疏水度明显降低。接触角测定结果显示,酶处理组与纯水的接触角明显低于对照组,同样表明酶解降低了Anammox颗粒污泥的表面疏水性。同时,1-溴代萘是一种非极性物质,污泥与其接触角越大则表明污泥亲水性越强,即疏水性减弱。

2.3 XDLVO理论分析

以接触角和Zeta电位值计算酶解前后Anammox颗粒污泥表面热力学参数,结果如表3所示。

3 讨论

颗粒污泥被认为具有水凝胶的性质[17],水凝胶可通过氢键、疏水性作用和链缠结等保持颗粒污泥的稳定[23]。多糖是EPS的主要凝胶成分[15],可以通过氢键与Ca2+形成稳定的链间交联和桥接,形成将细胞结合在一起的“蛋盒模型”[16]。此外,多糖是一种两亲性聚合物,可以将疏水性基团插入亲水性基团中,并通过这种有序的结构提高颗粒的稳定性。因此,采用对Anammox颗粒污泥进行酶解以破坏多糖的结构来探究多糖对Anammox颗粒污泥稳定性的影响。

颗粒污泥表面特性及热力学分析表明,胞外多糖的酶解阻碍了颗粒污泥微生物的聚集,从而影响了颗粒污泥的稳定性。疏水性作用对微生物的聚集非常重要[34],且疏水性越强颗粒污泥越稳定[40]。研究中,酶解导致颗粒污泥疏水度和稳定性明显下降,基于XDLVO理论分析进一步表明,酶解提高了污泥聚集的能量势垒,阻碍了微生物之间的聚集[41],从而影响了颗粒污泥内部的稳定性。因此,多糖可以在疏水性作用下降低微生物聚集的排斥力,促进微生物之间的聚集,从而影响颗粒稳定性。

α-淀粉酶处理组颗粒污泥被酶解部分发生明显溶胀,β-淀粉酶处理组则出现颗粒污泥破碎且颗粒强度下降的现象,这表明多糖之间的交联结合以及链结构对维持颗粒稳定性也有着重要作用。Caudan等[20]对颗粒污泥进行染色以及淀粉酶水解的研究发现,多糖会在细菌群落之间交联形成水凝胶的屏障,为聚集体提供凝聚力。此外,多糖链上丰富的官能团可以提供足够的结合位点[42],而其较长的碳主链也可以确保细胞间相互作用[22]。α-淀粉酶处理组颗粒污泥外缘被酶解,由于对多糖主链以及支链的同时破坏,EPS疏水性官能团和羟基含量明显降低,影响了多糖之间的交联结合,从而导致α-淀粉酶水解后污泥外缘发生明显溶胀。而β-淀粉酶仅破坏多糖支链末端,因此,β-淀粉酶处理组颗粒污泥外观并无明显变化,这表明多糖主链之间的缠结可以维持颗粒的形态稳定,但颗粒稳定性明显下降,说明多糖支链末端的结合位点之间的交联也有助于维持颗粒污泥稳定。因此,多糖丰富的官能团以及长主链可通过交联结合以及链缠结等作用促进颗粒污泥的稳定性。

通過对颗粒污泥胞外多糖的酶解可以了解到多糖性质如何影响聚集体。胞外多糖的疏水性,骨架作用以及形成水凝胶等都会影响微生物的聚集过程,进而影响聚集体的形态。不同的反应器运行工况往往会影响胞外多糖的性质。Yin等[43]研究发现,在氮不足的情况下,AGS的颗粒结构不规则且疏松,而且胞外多糖的显著增加被认为是影响颗粒稳定性的原因。Liu等[44]研究发现,更大的剪切力会刺激胞外多糖的产生,并促进造粒。因而可以通过调节反应器工况以影响胞外多糖的性质,从而影响聚集体形成、形态以及保持聚集体的稳定。

4 结论

对Anammox颗粒污泥进行酶解的结果表明,多糖对颗粒污泥的稳定性有着重要作用。经α-淀粉酶处理后,颗粒污泥外边缘出现溶胀,但污泥稳定性并无明显变化,而经β-淀粉酶处理后,颗粒污泥表面并无变化,但污泥出现破碎且稳定性明显下降。

对酶解前后污泥表面性质的表征发现,酶解降低了污泥疏水性,但未改变污泥表面电负性。XDLVO理论的分析表明,Anammox颗粒污泥经淀粉酶处理后,污泥微生物聚集的能量势垒被提高,即多糖可以促进微生物间的聚集从而促进颗粒污泥稳定性。FTIR结果表明,经淀粉酶水解后,污泥EPS中疏水性官能团和羟基含量明显降低。共聚焦扫描显微镜发现,α-淀粉酶处理组颗粒污泥外缘α-D-吡喃葡萄糖多糖含量明显下降,而β-淀粉酶处理组β-D-吡喃葡萄糖多糖则呈碎片状分布。

α-淀粉酶处理组表明胞外多糖的疏水性作用、通过O—H官能团与阳离子桥接或相互结合作用可以促进微生物之间的聚集。β-淀粉酶处理组表明胞外多糖长主链之间的缠结以及支链末端丰富的结合位点桥接形成骨架增强了微生物之间的黏附,有利于颗粒污泥的稳定。参考文献:

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(編辑 黄廷)