两种生物除磷系统活性污泥中除磷菌的甄别——淀粉-缺氧/好氧交替与乙酸盐-厌氧/好氧交替系统

周旭红,袁林江*,陈 希,杨 睿,朱 淼,南亚萍,贺向峰,陈 勇

两种生物除磷系统活性污泥中除磷菌的甄别——淀粉-缺氧/好氧交替与乙酸盐-厌氧/好氧交替系统

周旭红1,2,3,袁林江1,2,3*,陈 希4,杨 睿1,2,3,朱 淼1,2,3,南亚萍1,2,3,贺向峰1,2,3,陈 勇1,2,3

(1.西安建筑科技大学环境与市政工程学院,陕西 西安 710055；2.西安建筑科技大学西北水资源与环境生态教育部重点实验室,陕西 西安 710055；3.西安建筑科技大学陕西省环境工程重点实验室,陕西 西安 710055；4.西安工程大学城市规划与市政工程学院,陕西 西安 710048)

为了直接识别出污泥中的聚磷细菌和其种属,本研究采用4',6-二脒基-2-苯基吲哚(DAPI)染色和流式细胞荧光分选技术(FACS)对以淀粉为唯一碳源的缺氧/好氧序批式活性污泥(SBR)系统(R1)的缺氧末期和好氧末期以及以乙酸盐为唯一碳源的厌氧/好氧SBR系统(R2)的好氧末期污泥的聚磷细菌进行了原位分选,并通过16S rRNA高通量测序技术鉴定了分选后细菌的种属.结果表明,在R1中,缺氧期和好氧期均进行生物除磷,且缺氧期吸磷量大于好氧期. R2中发生着厌氧期释磷、好氧期大量吸磷的传统生物除磷.利用FACS在R1和R2污泥中均分选得到106个相对纯度为85%的具有聚磷颗粒的细菌.测序结果表明,在R1系统中,缺氧段优势的聚磷菌属为(37.75%)、unclassified(14.15%)、(6.49%)、unclassified(0.027%)和(0.007%);好氧段优势聚磷菌属为(19.72%)、unclassified(14.62%)、(14.28%)、unclassified(0.046%)、unclassifiedGp3(0.036%)和(0.026%).R1系统中unclassified和仅仅在缺氧条件下具有聚磷功能,而unclassified、unclassifiedGp3和仅在好氧条件下才具有聚磷功能.在R2系统中,优势聚磷菌群为(11.06%)、unclassified(9.29%)、unclassified(7.44%)、unclassified(7.34%)以及(0.31%).这意味着在新型的除磷系统(R1)中,参与除磷过程的细菌包括好氧,缺氧和兼性缺氧聚磷细菌,而在传统的除磷系统(R2)中,参与除磷过程的细菌仅为好氧聚磷细菌.

聚磷菌；SBR；流式细胞荧光分选技术；高通量测序

早在20世纪70年代,人们已经明确认识到在厌氧 /好氧交替运行系统的活性污泥中会发生生物除磷:污水中的磷酸盐浓度显著降低、同时污泥的含磷率不断上升的现象[1];也借助微生物染色观察到污泥中有些细菌在厌氧条件下体内聚-β-羟丁酸(PHB)增加,聚磷颗粒(poly-P)减少,而在好氧条件下,这些细菌体内的PHB和聚磷颗粒含量变化情况恰好相反[2-3].但一直以来对这些聚磷的细菌“身份”没有得到确认,只是概括性地将承担生物除磷的细菌统称为聚磷菌(PAOs),并按照其发生聚磷环境的不同将聚磷菌划分为反硝化聚磷菌(DNPAOs)以及好氧聚磷菌两大类[4-5].

尽管生物聚磷现象的发现至今已有半个多世纪,生物除磷也已被广泛应用于城市污水处理中,但生物除磷活性污泥中聚磷菌到底是哪些种(属)的细菌,仍尚无定论[6-7].1975年Fuhs等[8]最先从污水处理厂聚磷污泥中用平板划线法分离出不动杆菌(),发现其能够在生长培养基中大量吸收磷酸盐,因而认定是污水处理厂污泥中主要聚磷菌,而Wagner等[9]认为不动杆菌并不是主要聚磷菌,因为分离得到的并没有充分吸磷和释磷:另有研究采用荧光抗体[10]或API系统[11]方法专门对实际污水处理厂污泥中的不动杆菌数量进行了追踪,表明不动杆菌数量并不随着系统除磷酸盐能力提高而增加、两者之间并没有关联性,不支持不动杆菌是主要聚磷菌的认识.1991年Nakamura等[12]从含磷率达到6%以上的污泥中划线分离获得了微球菌属积磷小月菌(),该菌在厌氧条件下释放磷酸盐并吸收葡萄糖,与在活性污泥聚磷相似,但Santos等[13]研究发现并不储存聚羟链烷酸(PHAs).1997年Stante等[14]在强化生物除磷系统(EBPR)中分离出俊片菌属(),在培养基中具有厌氧释磷和好氧吸磷行为,确定其为一种典型的聚磷细菌,但任世英等[15]研究发现只储存少量磷酸盐多聚物,且速度很慢.2007年Cai等[16]在实验室活性污泥中纯化分离出新型菌株GM6,该菌株在纯化培养基具有较高的聚磷能力,但在厌氧/好氧交替环境中并没有表现出典型的聚磷行为.2016年Terashima等[17]证实了日本氧化沟污水处理厂中脱氯单胞菌()为优势菌属,且分离得到纯在培养时细胞内有多聚磷酸盐.通过传统平板划线分离获得纯菌能够在培养基中具有吸磷的菌还有:芽孢杆菌()[18-19]、葡萄球菌()[19],气单胞菌()[20]等.但这些从聚磷污泥中分离得到的纯菌大多没有典型聚磷菌的代谢行为.有研究认为[21-22]聚磷菌要在有发酵产酸菌存在且与其它竞争水中挥发性脂肪酸(VFAs)的异养菌(如聚糖菌)共存下才表现出的适应性行为,因此分离纯化后再鉴定可能无法通过其代谢行为来确定其聚磷菌身份.传统平板划线分离-培养法是对生物除磷活性污泥中细菌先进行分离鉴定,然后通过纯菌的再培养条件下的代谢情况来判定某一个菌是否有聚磷菌的行为[23].但由于纯培养所提供的条件与活性污泥混合菌群条件不同,因此分离得到的纯菌的代谢过程和活性污泥中的细菌在实际环境中的代谢过程也可能会有所不同.这样难免出现原本推测在活性污泥中可能具备聚磷能力的细菌却在厌氧-好氧交替的纯培养条件下不能呈现出释磷-超量聚磷能力[17,24],同样也会将某些随着污泥聚磷能力增强,数量也随之增加的细菌被推测为聚磷菌,然而这些细菌在经过分离培养后,很多是被否定的[25-26].

Kong等[27]通过荧光原位杂交(FISH)结合微自放射照相技术(MAR)显示四球虫菌()参与磷的摄取,被证实它是一种PAOs.Crocetti等[28]采用同样方法确定了污泥中是污泥中的PAOs之一,开创了PAOs原位鉴定技术.但MAR-FISH技术原位鉴定技术步骤繁杂条件要求高.

FACS可将不同荧光标记细菌从混合细菌种直接“挑”出来,高效准确地实现目标细胞原位直接分离[29].2019年Wang等[30]应用流式细胞术细胞分选结合16S rRNA基因高通量测序甄别出生物除硫除磷系统中一些功能菌.该技术也可能成为PAOs原位分离的新技术.

目前鉴定出来除了在传统厌氧/好氧交替环境中利用VFAs为碳源进行聚磷的微生物外,近年来还报道了在缺氧/好氧交替环境中利用淀粉为碳源聚磷的微生物.但对这种系统中聚磷菌的“身份”尚不清楚[31].因此,本文借助DAPI标记聚磷微生物并通过FACS对以淀粉为唯一碳源的缺氧/好氧生物除磷SBR中缺氧末端和好氧末端参与除磷的微生物,以及传统以乙酸钠为唯一碳源的SBR系统中聚磷菌进行了分选,对分选得到的所有聚磷菌经过高通量核酸测序后,确定了优势聚磷菌的种类,为丰富聚磷菌的认识、探明生物除磷菌奠定了基础.

1 材料与方法

1.1 反应器及运行条件

采用塑料材质的圆柱形反应器,柱体内径18.7cm,柱高20cm,总有效容积4L.反应装置如图1所示,采用微孔曝气器进行曝气,曝气量可由空气流量计进行调节,气体流速为200mL/min,自控装置控制厌氧、缺氧、好氧等操作过程,厌氧和缺氧搅拌器转速为300r/min.

图1 SBR系统实验装置

1.空气泵;2.流量计;3.液位控制计;4.搅拌器;5.pH控制仪;6.DO测量仪;7.微孔曝气头;8.SBR反应器;9.出水泵;10.进水泵;11.进水箱;12.出水箱;13.可编程控制器

反应器接种污泥取自西安市某废水处理厂厌氧/好氧(A/O)系统,取回的污泥过筛(50目)后直接加入R1和R2系统中驯化培养.R1系统每天运行4个周期,每周期6h.其中,进水8min,缺氧90min,好氧210min,沉淀40min,排水加闲置12min.R2系统运行周期和时间安排与R1系统一致,其中,厌氧90min. R1、R2两系统水力停留时间均为12h,污泥停留时间均控制在15d左右,均保持污泥浓度在3000～ 3500mg/L之间,好氧期溶解氧(DO)也均保持在2mg/L左右.

1.2 进水水质

采用人工配水模拟生活废水,R1反应器配方如下(g/L): MgSO4·7H2O:0.1,无水 CaCl2:0.01, NaNO3: 0.122,淀粉:0.4,NH4Cl:0.038;R2反应器配方如下(g/L): 无水CaCl2:0.01,NaAc:0.51, MgSO4·7H2O:0.1, NH4Cl:0.0608,其中每升配水加入微量元素浓缩液0.5mL,微量元素浓缩液组成如下(g/L): Na2MoO4·

H2O:0.06,FeSO4·7H2O:1.54,CuSO4·5H2O:0.03,CoCl2·7H2O:0.15,KI:0.18,H3BO3:0.15,MnCl2·4H2O:0.12,

ZnSO4·7H2O:0.12,EDTA:10.进水的pH值采用盐酸和氢氧化钠调节,控制范围为7.3～7.5.

1.3 水质参数分析

试验过程中按照每个周期的设定时间定期取样,从反应器最上部取混合液,取出的样品用滤纸过滤后用于测定水质指标.COD、TP和PO43--P采用标准方法测定[32],采用PE680型气相色谱仪测定PHB和聚β-羟戊酸(PHV)[33],测定VFAs和乳酸采用安捷伦气相色谱仪(hp 6890N,Amercian)型,DO和pH值的测定分别采用HACH-HQ40d型溶解氧仪和PB-20型pH计,微生物特征分析采用奥林巴斯(Olympus)型荧光显微镜进行观察.

1.4 活性污泥DAPI染色

分别取R1、R2反应器不同阶段的活性污泥用DAPI进行染色.染色具体步骤同文献[34-35]采用的方法.通过染色发现R1缺氧阶段与好氧阶段都有聚磷颗粒形成,而R2系统厌氧末端并未发现聚磷颗粒的存在,但好氧末端有大量聚磷颗粒的合成.因此认为R1中缺氧阶段和好氧阶段具有微生物聚磷,参与R2除磷的微生物仅在好氧期吸磷.选取染色后有聚磷颗粒的样本将其制备成菌悬液并分选其中能够聚磷的微生物.

1.5 流式细胞术收集含poly-P的细胞

DAPI染色方法和FACS分选方法在文献[36-37]的方法上进行改进.具体为:

(I)菌悬液制备与染色:取运行稳定的R1反应器缺氧末端和好氧末端活性污泥和R2反应器好氧末端的活性污泥各5mL于50mL离心管中,先离心5min(4℃, 10000r/min),倾去上清液后在离心管中加入10mL 0.1´磷酸盐缓冲溶液(PBS),重复离心,弃上清3次,去除活性污泥表面杂质.最后加0.1×PBS缓冲液至30mL,用超声波细胞粉碎机(型号:BILON92-II)将细胞混合液离散(单位体积输入能量:72.5J/mL).所制备的菌悬液中值粒径为(30±1.27)µm,吸光度(OD)值在0.4～0.5之间,脱氧核糖核酸(DNA)含量仅为3.44mg/(gVSS).制好的菌悬液每毫升加入30µL的甲酸,将pH值调至6.5以下,以抑制微生物代谢.用于菌悬液染色的DAPI浓度为10µg/mL,每毫升菌悬液加入10μL的DAPI染色液,37℃避光震荡染色30min.流式细胞分选前需用细菌涡旋仪(UVS-1)将菌悬液涡旋30s.

(Ⅱ)流式细胞仪分选细菌:采用北京易科生物科技攀博有限公司的流式细胞分选仪(BD AriaⅢ Special Order)进行细菌分离.DAPI用355nm(UV)激光器激发,并用460/50nm带通滤光片收集标准DAPI-DNA发射光;用585/15nm带通滤波片收集了黄色的DAPI-poly-P发射光.DAPI-DNA和DAPI- poly-P的测量值以对数标度进行采集,并使用Flow Jo软件7.6.5版进行采集后分析.分选速率为2000个/s,设置四路分选中的左二通道电压使液滴偏转到载玻片上,用于荧光显微镜观察分选纯度,其余偏转至无菌离心管中.通过荧光显微镜观察后分选目标菌群的纯度高达85%以上.

1.6 流式细胞术后分选得到的菌16S rDNA基因测序鉴定

取R1反应器污泥混合液以及两系统经过流式细胞仪分选后的所有细菌菌群进行16S rDNA高通量测序(上海生物工程公司).所测序菌群的扩增引物选择Illumina Miseq测序平台所提供的V3-V4区域的通用引物341F(5'-CCTACGGGNGGCWGCAG-3')和805R(5'-GACTACHVGGGTATCTAATCC-3').

2 结果与分析

2.1 SBR系统的除磷特性

如表1、2所示,在R1中,整个周期内无释磷现象发生,淀粉的主要水解产物为乳酸,系统缺氧期中基本没有PHB和PHV的合成,微生物合成内碳源主要为糖原.而R2系统相比R1有厌氧末端合成较多PHB和PHV,表现为典型传统厌氧释磷、好氧吸磷的除磷模式.总之,R1存在着不同于传统除磷的过程,推测参与R1系统除磷微生物可能也不同于传统除磷体系,此结论与Luo等[31]研究相符.

表1 R1反应器运行中系统周期内各物质浓度变化

表2 R2反应器运行中系统周期内各物质浓度变化

2.2 流式细胞仪分选系统中的PAOs

借助FACS分选R1系统同周期内缺氧末端、好氧末端以及R2好氧末端的除磷微生物.如图2所示,首先排除细菌中的杂质和细菌碎片,接着除去细菌中的粘结细菌后剩余游离细菌,游离细菌被DAPI染料染色后,选取能够发出DAPI-DNA荧光的细菌,最后在所有具备DAPI-DNA荧光的细菌中选取出能够发出DAPI-poly-P荧光的细菌,即为目标细菌.

为了获得用于产生16S rRNA基因克隆文库的足够的DNA,所取每个样品各准确分选106个目标细菌.R1缺氧末端和好氧末端用于分选的细菌总数分别为157435053和8426607,而R2好氧末端所用细菌总数为6429640,从分选所用细菌数量占比可以看出具备聚磷功能的微生物在R1、R2两个除磷系统中均为少部分.

图2 流式细胞仪分选R1中缺氧末端、好氧末端和R2中好氧末端具备聚磷颗粒细菌的检测结果

a. R1缺氧末;b. R1好氧末端;c. R2好氧末端;不同点在相应区域分布的密集情况代表不同细菌于相应区域分布的密集情况

2.3 微生物菌群分析

如图3所示,在R1系统中,分选前优势菌属为(21.56%)和(15.27%).经过FACS分选后,缺氧末端和好氧末端活性污泥中均没有检测到,说明此细菌在R1中并没有参与聚磷,因此体内没有聚磷颗粒形成.在分选后的缺氧期聚磷菌和好氧期聚磷菌中占比分别为6.49%和14.28%,显然,在R1系统中,在缺氧期和好氧期均发挥了一定的聚磷作用.2008年任世英等[38]从海洋中分离得到,并证明其除磷能力,且能够水解淀粉.本研究的缺氧/好氧系统中,分选前仅占0.019%,在缺氧末端和好氧末端聚磷菌中此菌占比分别高达37.75%和19.72%.说明在以淀粉为唯一碳源的缺氧/好氧系统中,主要起聚磷作用的优势菌群为.此外unclassified分选前丰度仅为0.08%,经过分选后,在R1缺氧末端和好氧末端除磷菌中,unclassified丰度分别为14.15%和14.62%,可见unclassified在缺氧期和好氧期均发挥了重要的聚磷作用.(0.007%)以及unclassified(0.027%)等细菌仅在缺氧期具备聚磷能力,而unclassified0.046%)、unclassified(0.036%)和(0.026%)等细菌仅在好氧期具有聚磷能力.这说明细菌除磷与环境息息相关,聚磷只有在适宜的条件下才能进行,缺氧期与好氧期除磷机制不同[31,34],发挥除磷作用的细菌也不同,因此具有聚磷颗粒的细菌也不同.

在R2系统中,(11.06%)、unclassified(9.29%)和unclassified(7.44%) unclassified(7.34%)等为主要参与除磷的微生物.其中是最近两年新确认的同一样重要的聚磷菌,与也有一些共同的特征,并且生态生理学与非常相似[17].

鉴定结果表明,unclassified为R1、R2两个系统共有的聚磷菌.在R1系统中,unclassified在分选前后占比均不到0.35%,这说明在此系统中并不起主导除磷的作用.而此菌在分选后的R2系统中占比为3.69%,可见unclassified在R2系统中的占比高于R1,这是由于unclassified更倾向于在以短链脂肪酸乙酸钠为碳源的系统中聚磷[39].

图3 R1系统分选前后及R2系统分选后微生物属水平优势菌属的相对丰度

X2、X3的优势菌群中已排除X1中不存在的细菌;X1:R1分选前;X2:R1缺氧末分选后;X3:R1好氧末分选后;X4:R2好氧末分选后

相比两个系统中主要的除磷优势菌群鉴定结果,虽然两种系统的反应时间相同,但由于运行条件和基质不同,主要参与除磷的微生物的种类和数量也存在较大差异.此外既能够在缺氧的环境中聚磷,也能在好氧环境中聚磷,目前尚未有人报道该菌属在反应器活性污泥中的聚磷能力,研究通过DAPI标记聚磷颗粒细菌后利用FACS技术分选发现其在活性污泥中的聚磷能力为丰富聚磷菌的认识、探明生物除磷菌奠定了基础.

3 结论

3.1 在以淀粉为唯一碳源的缺氧/好氧系统中,实现了缺氧期和好氧期均吸磷,并且缺氧期的吸磷量大于好氧期吸磷量,而以乙酸钠为唯一碳源的系统中为传统除磷过程.

3.2 在R1系统中,缺氧期和好氧期优势聚磷菌为、和unclassified等,其中、和仅在缺氧期具备聚磷功能,unclassifiedGp3、和也仅在好氧期才会发挥聚磷能力.在R2系统中优势聚磷菌为unclassified、unclassified、unclassified以及.

3.3 在新型的除磷系统(R1)中,参与除磷过程的细菌包括缺氧、好氧和兼性缺氧聚磷细菌,表明了该系统除磷由多种不同类型的除磷菌承担,并在多种环境下都出现磷的摄取;而在传统除磷系统(R2)中,参与除磷过程的细菌仅有好氧聚磷细菌.

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Screening of phosphorus removing bacteria from activated sludge for biological phosphorus removal: starch-anoxic/aerobic alternation and acetate-anaerobic/aerobic alternation system.

ZHOU Xu-hong1,2,3, YUAN Lin-jiang1,2,3*, CHEN Xi4, YANG Rui1,2,3, ZHU Miao1,2,3, NAN Ya-ping1,2,3, HE Xiang-feng1,2,3, CHEN Yong1,2,3

(1.School of Environmental and Municipal Engineering, Xi’an University of Architecture and Technology, Xi’an 710055, China；2.Key Laboratory of Northwest Water Resource, Environment and Ecology, Ministry of Education, Xi’an University of Architecture and Technology, Xi’an 710055, China；3.Shaanxi Key Laboratory of Environmental Engineering, Xi’an University of Architecture and Technology, Xi’an 710055, China；4.School of Urban Planning and Municipal Engineering, Xi'an University of Engineering, Xi'an 710048, China)., 2022.42(9)：4166～4173

In order to identify phosphorus accumulating organisms(PAOs) in situ from activated sludge. 4', 6-diamidino-2- phenylindole (DAPI) staining and flow cytometry fluorescence sorting (FACS) were used to sceen the PAOs from the two sludge at the anoxic/aerobic SBR system (R1) with starch as the only carbon source and the anaerobic/aerobic system (R2) with acetate as the only carbon source. The species of the sorted bacteria were identified by 16S rRNA high-throughput sequencing. The results showed that in the R1, biological phosphorus removal was carried out in both the anoxic periods and aerobic periods. The phosphorus uptake in the anoxic period was greater than that in the aerobic period. In the R2, the phosphorus release in the anaerobic period and a large amount of phosphorus absorption in the aerobic period occurs. Results of the in situ fluorescence staining showed that 106bacteria with a relative purity of 85% with phosphorus-accumlating cell were sorted from the R1 and the R2. The sequencing results showed that in the R1, the dominant genera of PAOs in the anoxic periods were(37.75%), unclassified(14.15%),(6.49%), unclassified(0.027%) and(0.007%). The dominant PAOs in the aerobic periods were(19.72%), unclassified(14.62%),(14.28%), unclassified(0.046%), unclassifiedGp3 (0.036%) and(0.026%). In the R1, unclassifiedandonly had phosphorus accumulation function under anoxic periods, while unclassified, unclassifiedGp3 andonly had phosphorus accumulation function under aerobic periods. In the R2, the dominant PAOs were(11.06%), unclassified(9.29%), unclassified(7.44%), unclassified(7.34%) and(0.31%). This means that in the R1, the bacteria involved in the phosphorus removal process include aerobic, anoxic and both aerobic and anoxic phosphorate accumulating bacteria three types. In the R2, the bacteria involved in the phosphorus removal process are only aerobic phosphorus accumulating bacteria.

phosphorus accumulating bacteria；SBR；flow cytometry fluorescence sorting technology；high-throughput sequencing

X703.5

A

1000-6923(2022)09-4166-08

2022-02-10

国家自然科学基金资助项目(51078304,51278406);陕西省自然科学基金资助项目(2021JQ-690)

*责任作者, 教授, yuanlinjiang@xauat.edu.cn

周旭红(1996-),女,甘肃定西人,西安建筑科技大学硕士研究生,主要从事废水生物处理理论与技术研究.