, 肖美星, 胡姜帅, 樊杰武汉科技大学城市建设学院, 武汉 430065
收稿日期: 2021-05-13; 修回日期: 2021-07-01; 录用日期: 2021-07-01
基金项目: 国家自然科学基金（No.51808416）
作者简介: 龚喜平(1998—), 女, E-mail: 1953800231@ qq.com
通讯作者（责任作者）: 季斌, E-mail: binji@wust.edu.cn
摘要：藻-菌颗粒污泥具有能耗低、温室气体排放量少等优点, 近年来在污水处理领域受到关注.胞外聚合物(EPS)在保护微生物免受恶劣环境影响及促进细胞聚集方面发挥重要作用, 而关于藻-菌颗粒EPS及其表面特性鲜有报道.选取了粒径范围在0.36~0.71、1~1.25、1.6~2.0 mm的3组藻-菌颗粒, 分析了其EPS中多糖和蛋白质的含量, 并进一步地对其组成差别以及官能团特征进行探究, 探讨了EPS提取前后藻-菌聚集体表面电荷的变化.结果表明, 随着粒径的增大, 藻-菌颗粒污泥EPS中多糖和蛋白质的含量均有增加, 而蛋白质与多糖的比值减小.不同粒径的藻-菌颗粒污泥EPS中芳香蛋白与酪氨酸类物质的含量明显增加, 腐殖酸类物质含量亦呈总体增加趋势, 与胞外蛋白相比, 胞外多糖特征峰的波峰强度增加较明显.进一步分析表明, 粒径较大的颗粒可能更能抵御外界环境因素的变化, 以维持颗粒性能.以上结果表明, 粘性和亲水性的多糖、芳香蛋白和酪氨酸类物质可能更有利于藻-菌颗粒的微生物细胞聚集.本文的研究结果进一步地拓展了对藻-菌颗粒污泥的认识, 并为其进一步的工程应用提供基础.
关键词：藻-菌颗粒胞外聚合物(EPS)粒径傅里叶变换红外光谱三维荧光胞外多糖
Study on the extracellular polymeric substances of the microalgal-bacterial granular sludge and its surface characteristics
GONG Xiping, JI Bin
, XIAO Meixing, HU Jiangshuai, FAN JieSchool of Urban Construction, Wuhan University of Science and Technology, Wuhan 430065
Received 13 May 2021; received in revised from 1 July 2021; accepted 1 July 2021
Abstract: Microalgal-bacterial granular sludge has the advantages of low energy consumption and low greenhouse gas emissions, etc. Therefore, it has attracted attention in the field of wastewater treatment in recent years. Extracellular polymeric substances (EPS) play an important role in protecting microorganisms from harsh environments and promoting cell aggregation. However, there are few reports on the EPS and its surface characteristics of microalgal-bacterial granular sludge. In this study, three groups of microalgal-bacterial granular sludge with particle sizes of 0.36~0.71 mm, 1~1.25 mm and 1.6~2.0 mm were applied for study. The contents of polysaccharide and protein in EPS were analyzed, and the composition differences and functional group characteristics were further explored. The changes in the surface charge of microalgal-bacterial aggregates before and after EPS extraction were analyzed. The results showed that with the increase of the particle size, the content of polysaccharide and protein from the EPS of microalgal-bacterial aggregates sludge increased, while the ratio of protein to polysaccharide decreased. The content of aromatic protein and tyrosine substances increased significantly, while the content of humic acid substances also showed an overall increase trend. Compared with extracellular proteins, the peak strength of extracellular polysaccharide characteristic peaks increased more significantly. Further analyses showed that microalgal-bacterial granules with a larger particle size might be more resistant to changes in external environmental factors with a stronger granular stability. The above results showed that sticky and hydrophilic polysaccharides, aromatic proteins and tyrosine-like substances might be more conducive to the accumulation of microbial cells in microalgal-bacterial granular sludge. Results of this study could advance the basic knowledge on microalgal-bacterial granular sludge for its further possible engineering application.
Keywords: microalgal-bacterial granular sludgeextracellular polymeric substances(EPS)particle sizeFTIR3D-EEMexopolysaccharide
1 引言(Introduction)藻-菌颗粒污泥工艺具有能耗低、CO2排放量少等优势, 作为一种有潜力的废水处理技术近几年被广泛关注(Mu?oz et al., 2005; Su et al., 2011; Zhang et al., 2021).其可基于藻-菌间的互利共生关系, 无需外部曝气, 同时减少二氧化碳排放(Ji et al., 2020a; Guo et al., 2021).据报道, 藻-菌颗粒污泥工艺的污染物去除效果可优于好氧颗粒污泥工艺(Liu et al., 2018; Guo et al., 2021).目前关于藻-菌颗粒污泥处理市政污水的研究主要集中在光照、温度等环境因素(Meng et al., 2019; Ji et al., 2021), 四环素等新型污染物(Wang et al., 2020; Wang et al., 2021), 以及污染物去除机理(Ji et al., 2020b)等, 关于藻-菌颗粒污泥胞外聚合物(EPS)及其表面特性的研究鲜有报道.
胞外聚合物(EPS)是微生物分泌的一类复杂的高分子聚合物, 主要由胞外蛋白(PN)和胞外多糖(PS)等组成(Yan et al., 2015; Xiao et al., 2016).EPS显著影响微生物聚集体的物理化学性质, 特别是表面电荷、疏水性、粘附性、结构和沉降特性(Lin et al., 2014; Xiao et al., 2016).胞外多糖中的阿拉伯糖、岩藻糖、鼠李糖等已被证实可促进微藻类细胞聚集、影响细胞与基质的粘附(Bahat-Samet et al., 2004; Willis et al., 2013).Su等(2020)的研究发现, EPS可通过改变微生物细胞的表面电荷和/或疏水性并形成聚合物机制来促进细胞聚集.此外, EPS基质可为细胞提供一层保护层, 使其免受环境的不利影响(Lin et al., 2014; 樊鹏超等, 2017).
如上所述, EPS可在微生物聚集体的细胞聚集及颗粒特性维持中起到重要作用, 阐明藻-菌颗粒污泥EPS及其表面特性, 有助于进一步认识藻-菌颗粒的特质.本研究拟通过三维荧光(3D-EEM)和傅里叶变换红外光谱(FTIR)等表征方法, 比较不同粒径藻-菌颗粒污泥EPS含量和组成差别及官能团特征, 探讨EPS提取前后藻-菌颗粒表面电荷的变化, 阐明藻-菌颗粒的EPS表面特性及其维持藻-菌颗粒特性的关键物质.本研究可为藻-菌颗粒污泥的进一步工程应用提供理论基础.
2 材料与方法(Materials and methods)2.1 藻-菌颗粒的培养条件本研究选取的藻-菌颗粒在合成废水中培养而成, 预先将活性污泥培养成好氧颗粒污泥, 再以LED为光源, 光强为200 mmol·m-2·s-1, 经过3个月的运行(即曝气培养1个月和静置培养2个月), 得到成熟的藻-菌颗粒污泥, 并用于后续的实验.合成废水主要成分如下：571.5 mg·L-1 CH3COONa、114.6 mg·L-1 NH4Cl、22.0 mg·L-1 KH2PO4、10.0 mg·L-1 FeSO4·7H2O、20.0 mg·L-1 NaHCO3、10.0 mg·L-1 CaCl2、50.0 mg·L-1 MgSO4·7H2O和1.0 ml·L-1微量元素溶液.微量元素溶液包括10 g·L-1 EDTA、150 mg·L-1 H3BO3、100 mg·L-1 MnSO4·H2O、30 mg·L-1 CuSO4·5H2O、120 mg·L-1 ZnSO4·7H2O、60 mg·L-1 Na2MoO4·2H2O、180 mg·L-1 KI和150 mg·L-1 CoCl2·6H2O.初始pH值控制在7左右.
采用筛孔分别为0.36、0.71、1.0、1.25、1.6、2.0 mm的系列筛网对不同粒径的藻-菌颗粒污泥进行分离, 筛选出3段粒径范围大小的颗粒污泥, 其粒径分别为0.36~0.71、1~1.25、1.6~2.0 mm.
2.2 EPS提取与分析采用一种改进的热提取方法从不同粒径的藻-菌颗粒污泥中提取胞外聚合物(EPS).将不同粒径的藻-菌颗粒污泥依次用0.9%NaCl溶液、0.45%NaCl溶液、去离子水清洗3次, 浓度连续降低的NaCl溶液可以使细胞进行渐进的渗透修饰, 从而防止细胞裂解(Phélippé et al., 2019).将剩余颗粒重新悬浮于30 mL去离子水中, 在80 ℃下加热30 min.将提取液以4500 r·min-1的速度离心20 min, 上清液通过0.45 μm滤膜过滤, 收集的滤液即提取的藻-菌颗粒污泥的EPS, 用于后续蛋白质(PN)、多糖(PS)和三维荧光(3D-EEM)分析.EPS中PS的含量以葡萄糖为标准品, 用硫酸蒽酮法测定.PN的含量以牛血清白蛋白为标准品, 采用快速Lowry法蛋白质试剂盒测定(Zhang et al., 2016).挥发性悬浮固体浓度(MLVSS)采用标准重量法(水和废水监测分析方法指南编委会, 2002)测定, EPS中PN和PS以每克VSS中的蛋白和多糖计.
2.3 红外光谱和三维荧光光谱采用溴化钾压片测试方法, 取1~2 mg在-70 ℃冷冻干燥的EPS粉末与200 mg KBr研细均匀, 置于模具中, 在油压机上压成透明薄片, 将样片放入红外光谱仪(Thermo Nicolet 6700)中测试, 波数范围4000~400 cm-1, 扫描次数为32, 分辨率为4 cm-1.
取3 mL左右的EPS样品, 使用荧光光度计(F-7100, 日本日立)测定EPS荧光光谱, 用去离子水作为空白样, 并从单个样品光谱中去除.以5 nm为采样间隔, 改变200~400 nm间的激发波长; 以1 nm为增量, 连续扫描200~500 nm发射光谱, 扫描速度为1200 nm·min-1(He et al., 2018b).采用Origin 2018绘制三维荧光光谱图.
2.4 其他分析方法分别将提取EPS前后不同粒径的藻-菌颗粒污泥碾碎, 匀浆成污泥悬浮液备用.污泥Zeta电位采用布鲁克海文(Brookhaven)多角度粒度分析仪测定.所有数据通过计算3个平行样品的平均值获得.
通过全自动比表面及孔隙度分析仪(麦克ASAP2460)测定藻-菌颗粒污泥平均孔径和孔容体积.
3 结果与讨论(Results and discussion)3.1 藻-菌颗粒污泥特征根据藻-菌颗粒污泥大小的不同, 将其分为3组.分组后的藻-菌颗粒污泥具有明显不同的形态特征.不同粒径的藻-菌颗粒污泥表观图如图 1所示, 粒径较大的颗粒形状相对规则, 呈带刺球状, 透光性差, 颜色呈深绿色, 较小的颗粒形状不规则, 颜色较浅.
图 1(Fig. 1)
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| 图 1 不同粒径藻-菌颗粒表观图 (a.0.36~0.71 mm, b.1~1.25 mm, c. 1.6~2.0 mm) Fig. 1The appearance of microalgal-bacterial granular sludge with different particle sizes (a. 0.36~0.71 mm, b. 1~1.25 mm, c. 1.6~2.0 mm) |
由表 1可知, 随着粒径的增大, 藻-菌颗粒污泥的平均孔径和孔容体积呈减小的趋势.表明在本研究的颗粒粒径范围内, 粒径越大的藻-菌颗粒污泥, 结构越趋近致密, 与Liu等和He等得到的研究结果相似(Liu et al., 2017; He et al., 2018a).
表 1(Table 1)
| 表 1 不同粒径藻-菌颗粒污泥BET特性 Table 1 BET characteristics of microalgal-bacterial granular sludge with different particle size | ||||||||||||
表 1 不同粒径藻-菌颗粒污泥BET特性 Table 1 BET characteristics of microalgal-bacterial granular sludge with different particle size
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3.2 藻-菌颗粒污泥EPS组分及含量分析长期以来, EPS被认为有助于细菌和藻-菌联合体的颗粒化(Huang et al., 2015).图 2显示了PN、PS、PN+PS含量以及PN/PS比值的变化.随着粒径的增加, PN、PS含量均呈增加的趋势, PS的含量由(31.39±1.39) mg·g-1增加至(55.30±1.25) mg·g-1, 增加了0.76倍；PN含量由(101.39±0.95) mg·g-1增加至(150.81±0.16) mg·g-1, 增加了0.49倍；EPS总量从(132.78±2.34) mg·g-1增加到(206.11±1.41) mg·g-1, 而PN/PS从(3.23±0.11)减小到(2.73±0.06).
图 2(Fig. 2)
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| 图 2 不同粒径藻-菌颗粒污泥EPS组分及含量 Fig. 2Composition and content of EPS of microalgal-bacterial granular sludge with different particle size |
从结果可知, EPS的含量与粒径大小呈正相关关系, 表明粒径较大的藻-菌颗粒污泥能分泌更多的EPS, EPS含量的增加有利于藻-菌联合体的颗粒化(Huang et al., 2015).PN为藻-菌颗粒污泥EPS中的主要物质, 占EPS含量的70%以上.但随着藻-菌颗粒污泥粒径的增加, PN在EPS中占比呈减少趋势, 由76.4%减小到73.2%, PS含量的增速远大于PN含量的增速.事实上, 粘性和亲水性的PS更有利于细胞聚集和造粒(Hou et al., 2015), 其能够促使生物群体通过桥联作用形成交叉的网状结构(Yan et al., 2015), 从而促进颗粒状结构的形成.
通过三维荧光光谱以表征EPS中的有机物质(图 3).分别在不同粒径的藻-菌颗粒污泥的EPS中观察到4个明显的峰值A(Ex/Em 275/340~350), B (Ex/Em 225/300~320), C (Ex/Em 225/330~345)和D (Ex/Em 275/420~430), 分别代表可溶解性微生物副产物、芳香蛋白酪氨酸类物质、芳香蛋白色氨酸类物质和腐殖酸类物质(Chen et al., 2003；王硕等, 2015).由图 3可知, 随着藻-菌颗粒污泥粒径的增大, 芳香蛋白酪氨酸类物质的含量明显增加, 腐殖酸类物质含量亦呈总体增加趋势, 芳香蛋白色氨酸类物质和可溶解性微生物副产物含量变化不大.此外, 根据发射波长尺度的不同, 峰A、D发生红移, 峰B、C发生蓝移.三维荧光光谱峰值的红移与羰基、羟基、氨基或羧基等官能团的存在有关, 而蓝移意味着芳香性物质、芳环数量和共轭键的丰度减少(Chen et al., 2003).通过三维荧光光谱表征的EPS中蛋白质含量的变化趋势和采用Lowry法测定的蛋白质含量变化趋势一致.芳香蛋白酪氨酸类物质含量的明显增加, 说明其在藻-菌颗粒污泥造粒和促进颗粒絮凝方面可能起到重要作用.如其带来的苯环间的Π-Π堆积作用(李旖瑜等, 2017)在促进藻-菌聚集体造粒方面可能起到重要作用.
图 3(Fig. 3)
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| 图 3 不同粒径藻-菌颗粒污泥EPS的三维荧光光谱 (a.0.36~0.71 mm, b.1~1.25 mm, c.1.6~2.0 mm) Fig. 3The three-dimensional fluorescence spectrum of the EPS of microalgal-bacterial granule sludge with different particle size (a. 0.36~0.71 mm, b. 1~1.25 mm, c. 1.6~2.0 mm) |
通过FTIR进一步检测藻-菌颗粒污泥EPS随粒径增加而发生的变化.如图 4所示, 3种粒径EPS的FTIR峰值和数目相当接近, 表明化学基团的类型是相似的.然而, 对比各个峰的强度表明, 化学基团的相对含量存在差异.3424 cm-1处的吸收峰, 是由—OH和—NH2的伸缩振动引起的(Yan et al., 2015), 2933 cm-1附近出现的不对称吸收峰属于烷烃类有机物和大分子多糖中—CH2的不对称伸缩振动(李莹等, 2020).1645 cm-1和1252 cm-1附近的吸收峰为蛋白质二级结构所特有, 分别为酰胺Ⅰ带中β-折叠片状蛋白质结构的C＝C和C＝O伸缩振动(Hou et al., 2015)和酰胺Ⅲ带中—CN的伸缩振动(Yan et al., 2015).1406 cm-1处的峰属于与氨基酸相关的—COO中C＝O的对称拉伸(Hou et al., 2015), 1058 cm-1附近的峰是由于多糖或类多糖物质中的对称和不对称C＝O伸缩振动所致(李莹等, 2020).吸收峰小于1000 cm-1属于指纹区域, 出现了氨基酸和核酸的特征伸缩振动峰(Yan et al., 2015).
图 4(Fig. 4)
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| 图 4 不同粒径藻-菌颗粒污泥EPS红外光谱 Fig. 4Infrared spectra of EPS from microalgal-bacterial granular sludge with different particle size |
上述表明, 不同粒径的藻-菌颗粒污泥EPS中均存在蛋白质、多糖、醇类、核酸等物质.随着藻-菌颗粒污泥粒径的增大, 3424、2933、1645和1252 cm-1处的波峰强度逐渐增大, 反映PN特征峰的波峰强度增值明显小于PS特征峰的波峰强度增值, 这与PN/PS的减小相一致.
3.3 藻-菌颗粒表面特性分析EPS覆盖在微生物细胞的表面, 其组成可以改变颗粒污泥的表面性质, 如疏水性和表面电荷等, 促进细胞之间的聚集和颗粒结构稳定性(Yan et al., 2015).为进一步研究藻-菌颗粒表面特性, 测量并记录了提取EPS前后藻-菌颗粒污泥在pH为8.5的去离子水中的Zeta电位(图 5).结果表明, 在相同条件下, 提取EPS前, 3组藻-菌颗粒污泥的Zeta电位分别为(-20.15±0.59)、(-21.28±0.30)、(-23.18±0.44) mV, 粒径越小的藻-菌颗粒污泥Zeta电位绝对值越小.因此, 颗粒污泥的粒径越小, 颗粒间的排斥力越小, 从而有利于颗粒间的聚集.而提取EPS后, 3组藻-菌颗粒污泥的Zeta电位分别为(-33.00±0.55)、(-32.54±0.19)、(-30.39±0.05) mV, 粒径越小的藻-菌颗粒污泥Zeta电位绝对值越大, 颗粒表面的负电荷量明显上升.提取EPS前后不同粒径的藻-菌颗粒污泥Zeta电位差值分别为(-12.85±0.04)、(-11.26±0.11)、(-7.21±0.39) mV, 粒径越大的藻-菌颗粒污泥在提取EPS后, 表面电荷变化量越小, 表明粒径较大的颗粒可能更能抵御外界环境因素的变化, 维持颗粒性能.
图 5(Fig. 5)
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| 图 5 提取EPS前后不同粒径藻-菌颗粒污泥Zeta电位 Fig. 5Zeta potential of microalgal-bacterial granular sludge with different particle size before and after EPS extraction |
4 结论(Conclusions)1) 随着藻-菌颗粒污泥粒径的增大, EPS中PN和PS的含量随着颗粒粒径的增加而增加, PN是该藻-菌颗粒污泥EPS中的主要成分, 占其含量的70%以上, 而PN/PS随着粒径的增加而减小, 表明PS在藻-菌聚集体颗粒化过程中可能起到更重要的作用.
2) 由三维荧光和FTIR表征结果, 随着藻-菌颗粒污泥粒径的增大, 芳香蛋白酪氨酸类物质含量增加明显, 说明芳香蛋白酪氨酸类物质可能更有利于维持颗粒结构, 促进颗粒的增长.
3) 由Zeta电位表征结果可知, 粒径较大的颗粒可能更能抵御外界环境因素的变化, 维持颗粒性能.
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