A2/O-BCO系統(tǒng)中碳源類型對反硝化除磷及菌群結(jié)構(gòu)的影響

范亞駿,張 淼,季俊杰,程吉林,吳啟超,何成達(dá)*

A2/O-BCO系統(tǒng)中碳源類型對反硝化除磷及菌群結(jié)構(gòu)的影響

范亞駿1,張 淼2,季俊杰2,程吉林1,吳啟超3,何成達(dá)2*

(1.揚(yáng)州大學(xué)水利科學(xué)與工程學(xué)院,江蘇 揚(yáng)州 225127；2.揚(yáng)州大學(xué)環(huán)境科學(xué)與工程學(xué)院,江蘇 揚(yáng)州 225127；3.揚(yáng)州工業(yè)職業(yè)技術(shù)學(xué)院,江蘇 揚(yáng)州 225127)

采用厭氧/缺氧/好氧-生物接觸氧化(A2/O - BCO)工藝處理低碳氮(C/N)比污水,考察單因素碳源(階段I:乙酸鈉;階段II:乙酸鈉+丙酸鈉;階段III:丙酸鈉)對有機(jī)物去除以及同步脫氮除磷的影響,并重點(diǎn)探究乙酸鈉、丙酸鈉混合碳源條件下內(nèi)碳源(PHA、Gly)的轉(zhuǎn)化利用以及反硝化除磷(DPR)機(jī)理,同時(shí)通過高通量測序?qū)Ρ攘瞬煌A段微生物菌群結(jié)構(gòu)的演變規(guī)律.結(jié)果表明:混合碳源提高了有機(jī)物、氮、磷的同步去除效率,厭氧段內(nèi)碳源轉(zhuǎn)化量為226mg/h,釋磷量高達(dá)30.58mg/L,DPR效率穩(wěn)定在90%以上;批次試驗(yàn)表明反硝化聚磷菌(DPAOs)占聚磷菌(PAOs)的比例為72.42%,基本實(shí)現(xiàn)了DPAOs的富集;高通量測序結(jié)果表明混合碳源更有利于形成獨(dú)特的OTUs菌群,PAOs(包括和)和DPAOs (包括和)總量高達(dá)29.13%(>16.18%(階段III)>14.34%(階段I)),有效促進(jìn)了碳源的高效利用以及反硝化除磷效率;BCO反應(yīng)器中氨氧化菌(AOB,包括和)和亞硝酸鹽氧化菌(NOB,以為主)總量從3.89%(N1)增加到23.09%(N2)、37.23%(N3),為反硝化除磷提供充足的電子受體;此外,建立了基于碳源高效利用的運(yùn)行調(diào)控策略,以期為A2/O - BCO工藝的推廣應(yīng)用提供理論參考.

A2/O - BCO工藝；碳源類型；反硝化除磷；內(nèi)碳源轉(zhuǎn)化；高通量測序

氮、磷是引起水體污染的主要元素,也是導(dǎo)致水體富營養(yǎng)化的主要原因[1].目前,城市污水廠主要采用傳統(tǒng)生物脫氮除磷技術(shù),其中硝化菌長污泥齡與聚磷菌短污泥齡的矛盾一定程度上限制了同步脫氮除磷效果[2];此外,實(shí)際生活污水主要特征是進(jìn)水碳氮比(C/N)低,因此碳源不足再一次導(dǎo)致同步脫氮除磷效率難于提高.近年來,污水處理工藝經(jīng)過不斷的發(fā)展與演變,雙污泥反硝化除磷系統(tǒng)應(yīng)運(yùn)而生,通過反硝化除磷(DPR)技術(shù)特有的“一碳兩用”的方式緩解反硝化、除磷過程對碳源的需求矛盾[3-4],通過將硝化菌和聚磷菌分開培養(yǎng)的方法,有效解決了污泥齡的矛盾,被視為高效率、低能耗、可持續(xù)的污水處理技術(shù).

本研究所采用的A2/O-生物接觸氧化(A2/O- BCO)[3]正是一種新型改良雙污泥反硝化除磷工藝,已成功從實(shí)驗(yàn)室小試研究推廣到中試規(guī)模驗(yàn)證[5].碳是微生物細(xì)胞結(jié)構(gòu)組成與生長代謝的基本元素,碳源作為一種能量載體參與脫氮除磷過程[6],是影響工藝運(yùn)行的重要因素之一.目前,針對乙酸鹽或丙酸鹽為單一基質(zhì)進(jìn)行反硝化除磷的研究較多,而將乙酸鈉、丙酸鈉作為混合基質(zhì)的研究較少,且兩者對反硝化除磷效果的影響也存在爭議,如Oehmen等[7]認(rèn)為與乙酸鈉相比,丙酸鈉更有利于反硝化除磷和聚磷菌(PAOs)富集;Pijuan等[8]認(rèn)為乙酸鈉作為最容易利用的碳源組分,有利于提高反硝化除磷潛力和反硝化除磷系統(tǒng)的穩(wěn)定性;而鮑林林等[9]發(fā)現(xiàn)以乙酸鈉、丙酸鈉為碳源的反硝化除磷系統(tǒng)長期運(yùn)行效果差別不大.因此,對于A2/O-BCO系統(tǒng)而言,何種碳源為最優(yōu)碳源尚無定論,且碳源利用對A2/O-BCO系統(tǒng)反硝化除磷的影響機(jī)制還需進(jìn)一步探究.

作為污水中最常見的兩種碳源類型,乙酸鈉、丙酸鈉與反硝化除磷特性和微生物菌群結(jié)構(gòu)的研究對實(shí)際低C/N比污水的運(yùn)行具有重要的指導(dǎo)意義.考慮到實(shí)際生活污水水質(zhì)的復(fù)雜性和水量的波動性,本研究采用人工配水,重點(diǎn)考察單因素碳源(乙酸鈉、乙酸鈉+丙酸鈉(等比例混合)、丙酸鈉)對A2/O-BCO系統(tǒng)反硝化除磷的影響,通過批次試驗(yàn)分析內(nèi)碳源轉(zhuǎn)化利用特性和反硝化除磷機(jī)理,并結(jié)合高通量測序?qū)Ρ任⑸锞航Y(jié)構(gòu)演變規(guī)律,采用宏觀對比和微觀機(jī)理相結(jié)合,為加快A2/O-BCO工藝的推廣應(yīng)用提供理論參考.

1 材料與方法

1.1 試驗(yàn)水質(zhì)及接種污泥

為了保證相對穩(wěn)定的進(jìn)水水質(zhì),試驗(yàn)用水為人工配制的低C/N比模擬生活污水,平均C/N比為4.30,平均C/P比為46.58,具體水質(zhì)特征見表1.此外,為了滿足微生物的生長增殖,投加微量元素如下:MgSO4·7H2O 0.45mg/L, FeCl3·6H2O 1.95mg/L, Na2MoO41.05mg/L, ZnSO4·2H2O 0.3mg/L, CuSO40.3mg/L, CoCl2·6H2O 0.3mg/L, CaCl2·2H2O 0.3mg/ L.A2/O反應(yīng)器接種污泥取自揚(yáng)州六圩污水處理廠沉淀池污泥,MLSS在4000mg/L左右,污泥沉降性較好且脫氮除磷性能穩(wěn)定.

表1 A2/O - BCO系統(tǒng)進(jìn)水水質(zhì)特性

1.2 A2/O-BCO反應(yīng)器

本實(shí)驗(yàn)采用的A2/O - BCO系統(tǒng)由原水水箱、A2/O反應(yīng)器、中間沉淀池、中間水箱、BCO反應(yīng)器以及沉淀區(qū)順序連接而成,其中主體反應(yīng)器材質(zhì)均為有機(jī)玻璃,工藝流程見圖1.A2/O反應(yīng)器有效容積24.5L,均分為7個(gè)格室,依次為厭氧區(qū)、缺氧區(qū)和好氧區(qū)(容積分配比1:5:1);進(jìn)水流量48L/d,總水力停留時(shí)間12h; 厭氧區(qū)和缺氧區(qū)設(shè)有攪拌,好氧區(qū)僅占A2/O總?cè)莘e的1/7,停留時(shí)間較短,溶解氧1.0～ 1.5mg/L,吸磷的同時(shí)吹脫反硝化產(chǎn)生的氮?dú)?反應(yīng)器平均污泥濃度(MLSS)為2500～3000mg/L,污泥齡為(10±2) d[10].

A2/O反應(yīng)器出水進(jìn)入中間沉淀池實(shí)現(xiàn)泥水分離,中間沉淀池有效容積15L,沉淀污泥回流到A2/O反應(yīng)器的厭氧區(qū),污泥回流比為100%.中間沉淀池的上清液經(jīng)中間提升泵進(jìn)入BCO反應(yīng)器,總有效容積10.5L,通過3格串聯(lián)(記為N1、N2、N3)逐步實(shí)現(xiàn)氨氮氧化.為了實(shí)現(xiàn)NH4+、微生物、氧氣三者的充分接觸,本試驗(yàn)選擇孔隙率大、比表面積高、密度接近于水且掛膜速度快的聚丙烯懸浮填料(尺寸:5mm×3mm;密度:960～1000kg/m3;孔隙率:98%;比表面積:1500m2/m3)[11];填充率為45%左右以確保所有填料處于流化狀態(tài)[12],進(jìn)而提高傳質(zhì)效果;3格溶解氧(DO)為3.0～4.0mg/L,總氣量為0.18～0.20m3/h,生物膜平均MLSS為1200～1500mg/L,硝化液經(jīng)沉淀區(qū)回流到A2/O反應(yīng)器的缺氧區(qū)為反硝化除磷提供電子受體,硝化液回流比400%.

圖1 A2/O - BCO工藝裝置示意

1.3 試驗(yàn)方案

A2/O-BCO連續(xù)流系統(tǒng)運(yùn)行方案:試驗(yàn)采用連續(xù)進(jìn)水運(yùn)行120d,根據(jù)投加碳源類型的不同,將試驗(yàn)分為3個(gè)階段:階段I(1～40d),乙酸鈉為碳源;階段II (41～80d),乙酸鈉、丙酸鈉等比例混合;階段III(81～ 120d),丙酸鈉為碳源.試驗(yàn)期間水溫在23～26℃范圍內(nèi)波動,污泥回流比、硝化液回流比、A2/O容積分配比、水力停留時(shí)間等運(yùn)行參數(shù)(見1.2)均保持恒定.

厭氧-缺氧/好氧批次試驗(yàn)運(yùn)行方案[13]:取一定量A2/O反應(yīng)器好氧末污泥,采用轉(zhuǎn)速3000r/min離心10min,再用去離子水沖洗3次,消除COD以及其他物質(zhì)對實(shí)驗(yàn)的影響,隨后將污泥置于有效容積為2L的密閉容器,加水定容使MLSS濃度在3000mg/L左右.試驗(yàn)用水仍為人工配水,碳源類型與連續(xù)流系統(tǒng)一致,初始COD濃度300mg/L左右,置于磁力攪拌器上進(jìn)行厭氧攪拌(120min),消耗外碳源的同時(shí)完成磷釋放;厭氧結(jié)束將混合液等分為2份,分別進(jìn)行缺氧、好氧反應(yīng)(210min),其中缺氧階段通過投加NaNO3保證初始NO3--N濃度為25mg/L,好氧階段DO濃度為3.0 ～ 4.0mg/L,每隔一定的時(shí)間取樣,測定水樣中COD、PO43--P、NO3--N、NO2--N以及泥樣中聚羥基脂肪酸酯(PHA)、糖原(Gly)等相關(guān)指標(biāo).

1.4 檢測分析方法

COD測定采用COD快速消解儀(北京連華科技);PO43--P、NO3--N、NO2--N等采用標(biāo)準(zhǔn)方法[14]測定;DO濃度測定采用便攜式溶氧儀(HQ30D型,美國哈希);污泥樣品(接種污泥記為D0,階段I記為D40,階段II記為D80,階段III記為D120)和生物膜樣品(N1、N2、N3,階段II末80d取樣)采用真空冷凍干燥機(jī)(FD-1A-50型)進(jìn)行預(yù)處理,然后送至上海美吉生物醫(yī)藥科技有限公司進(jìn)行高通量測序(SRP223205).

1.5 分析計(jì)算方法

A2/O反應(yīng)器厭氧區(qū)內(nèi)碳源轉(zhuǎn)化率(CODINTRA)計(jì)算方法[15]如下式:

CODINTRA=[(CODIN,AN+ CODW-2CODEF,AN)-1.71

(NO2-,IN,AN- NO2-,EF,AN) - 2.86(NO3-,IN,AN-

NO3-,EF,AN)] (1)

(CODINTRA) = CODINTRA/(CODIN,AN–

CODEF,AN)×100% (2)

式中:CODINTRA——A2/O厭氧內(nèi)碳源轉(zhuǎn)化量, mg/h;——進(jìn)水流量, L/h;CODIN,AN、CODEF,AN——A2/O厭氧進(jìn)水、出水COD濃度, mg/L;NO2,-IN,AN、NO2-,EF,AN——A2/O厭氧進(jìn)水、出水NO2--N濃度, mg/L;NO3-,IN,AN、NO3-,EF,AN——A2/O厭氧進(jìn)水、出水NO3--N濃度, mg/L;CODW——回流污泥COD濃度, mg/L.

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BCO反應(yīng)器中同步硝化反硝化(SND)氮損失計(jì)算方法[16]如下式:

SNDBCO= (1 - (ΔNO3-,BCO–ΔNO2-,BCO)/

ΔNH4+,BCO)×100% (3)

式中:SNDBCO——BCO反應(yīng)器中的氮損失率, %; ΔNO3-,BCO——BCO反應(yīng)器中NO3--N的變化量, mg/L;ΔNO2-,BCO——BCO反應(yīng)器中NO2--N的變化量, mg/L;ΔNH4+,BCO——BCO反應(yīng)器中NH4+-N的變化量, mg/L.

A2/O反應(yīng)器反硝化除磷效率((DPR))計(jì)算方法如下式:

(DPR)=(2×PO43-,AN+3×PO43-,EF-5×PO43-,EF,A)/

(2×PO43-,AN+ 3×PO43-,EF- 5×PO43-,EF,O) ×100% (4)

式中:PO43-,AN——A2/O厭氧出水PO43--P濃度, mg/L; PO43-,EF——BCO反應(yīng)器最終出水PO43--P濃度, mg/L; PO43-,EF,A——A2/O缺氧末出水PO43--P濃度, mg/L; PO43-,EF,O——A2/O好氧末出水PO43--P濃度, mg/L

2 結(jié)果與討論

2.1 各階段C、N、P去除效果分析

如圖2A所示,在A2/O - BCO系統(tǒng)中,COD的去除主要發(fā)生在厭氧區(qū),各階段厭氧區(qū)平均出水COD濃度分別為98.15, 40.03和68.82mg/L,其中階段I厭氧出水濃度最高,是階段II的2.45倍.由于進(jìn)水中幾乎不存在NO2--N和NO3--N,CODINTRA效率高達(dá)93.44% ～ 98%,與He等[17]采用的SNADPR (同步硝化內(nèi)源反硝化除磷)工藝(87.46% ～ 98.56%)較為接近,但遠(yuǎn)高于Zhao等[15]采用的SBR工藝(71.2%),這表明系統(tǒng)中較高的內(nèi)碳源轉(zhuǎn)化率可為反硝化除磷過程提供充足的電子供體.此外,當(dāng)進(jìn)水碳源為乙酸鈉、丙酸鈉的混合碳源時(shí)(階段II),內(nèi)碳源轉(zhuǎn)化量相對較高,平均值在226mg/h,高于單一碳源乙酸(階段I, 152mg/h)和丙酸(階段III, 198mg/h),這與文獻(xiàn)[18-19]的結(jié)果類似,同時(shí)也表明碳源種類與內(nèi)碳源的轉(zhuǎn)化息息相關(guān).因此,階段II 中A2/O反應(yīng)器厭氧段與最終出水COD濃度相差不大,不僅實(shí)現(xiàn)了碳源的高效利用而且為BCO反應(yīng)器中自養(yǎng)型硝化菌的富集提供了有利條件[20].

A:COD和CODINTRA; B: PO43--P; C: NH4+-N、NO3--N、NO2--N和SND; D:TN、PO43--P去除率和DPR

與內(nèi)碳源轉(zhuǎn)化的變化趨勢相似,如圖2B所示,階段I中平均厭氧釋磷量為9.27mg/L,階段II厭氧釋磷量急劇上升,最高值為30.58mg/L;階段III釋磷量呈下降趨勢,平均值為13.55mg/L.就整個(gè)試驗(yàn)過程而言,除磷效果并不理想,其中60～80d平均去除率為92.14%,出水PO43--P濃度穩(wěn)定低于0.5mg/L,其余時(shí)段出水PO43--P濃度大都在2～3mg/L,去除率僅為50%.猜測其主要原因如下:①系統(tǒng)中存在其他異養(yǎng)菌群,如反硝化聚糖菌(DGAOs),可以利用降解Gly產(chǎn)生的能量及還原力吸收有機(jī)物但不釋磷[21],容易出現(xiàn)厭氧段內(nèi)碳源轉(zhuǎn)化量大但釋磷量卻很少的現(xiàn)象.②碳源類型會直接影響內(nèi)碳源的種類(如PHB、PHV等),PO43--P的釋放主要受PHB轉(zhuǎn)化的影響,而除磷效率主要依賴于PHV的合成與降解,研究表明投加適當(dāng)?shù)谋徕c有利于PHV的合成,進(jìn)而提高磷的去除效果[22-24];③電子受體不足導(dǎo)致缺氧吸磷不充分.

A2/O - BCO是一個(gè)雙污泥系統(tǒng),氨氮的氧化主要在BCO中進(jìn)行,由于A2/O反應(yīng)器對碳源的高效利用使得進(jìn)入到BCO的有機(jī)物幾乎為難降解有機(jī)物,且硝化過程的實(shí)現(xiàn)主要通過化能自養(yǎng)型微生物[12],因此NH4+-N去除效果幾乎不受碳源類型的影響.如圖2C所示,出水NH4+-N濃度均低于5mg/L,穩(wěn)定達(dá)一級A排放標(biāo)準(zhǔn),且出水中以NO3--N為主,幾乎無NO2--N產(chǎn)生.值得一提的是,3個(gè)階段,BCO反應(yīng)器出現(xiàn)了不同程度的氮損失,盡管SND效率波動范圍較大(10%～80%),但表明了SND現(xiàn)象的存在,一方面隨著生物膜厚的變化,生物膜內(nèi)產(chǎn)生溶解氧梯度,形成了局部缺氧區(qū)[25];另一方面可能由于BCO反應(yīng)器中沒能達(dá)到完全混合狀態(tài),易受曝氣量[26]、基質(zhì)濃度[27]等多種因素的干擾,具體原因還需進(jìn)一步探討.

除此之外,結(jié)合圖2C中NO3--N變化和圖2D中反硝化除磷效率可知,硝化液中較高的NO3--N濃度有利于提高反硝化除磷效果,階段II中60～80d, DPR效率穩(wěn)定在90%以上,這一現(xiàn)象再次表明電子受體濃度會直接影響反硝化除磷效果.盡管如此,TN、PO43--P去除率呈現(xiàn)出此消彼長的規(guī)律,尤其在階段III, TN去除率維持在80%左右,但PO43--P去除率不超過60%.一方面是反硝化除磷過程還伴隨著外源反硝化的發(fā)生,與內(nèi)源反硝化相比,外源反硝化速率更快[28],導(dǎo)致TN去除率相對更穩(wěn)定;另一方面,反硝化除磷效率與內(nèi)碳源轉(zhuǎn)化、釋磷量密切相關(guān),階段III中較少的內(nèi)碳源轉(zhuǎn)化量(圖2A)導(dǎo)致反硝化除磷動力不足,但剩余的有機(jī)物促進(jìn)了外源反硝化,致使較低的出水NO3--N濃度(圖2C)和較穩(wěn)定的TN去除率(圖2D).

2.2 反硝化除磷機(jī)理分析

為了更深入地分析反硝化除磷機(jī)理,在階段II,取運(yùn)行80d的活性污泥進(jìn)行厭氧/缺氧-好氧批次實(shí)驗(yàn),分析典型周期內(nèi)氮、磷、胞內(nèi)和胞外碳源的變化.厭氧段,聚磷菌分解體內(nèi)的ATP和聚磷酸鹽,釋放無機(jī)磷,同時(shí)攝取污水中的有機(jī)物,利用NADH2和產(chǎn)生的能量合成PHA儲存于細(xì)胞體內(nèi)[29].如圖3所示,COD濃度在30min內(nèi)從316.14mg/L變成192.60mg/L,COD被快速消耗的同時(shí)伴隨著磷酸鹽的釋放, PO43--P濃度從0.2mg/L上升到12.68mg/L,占整個(gè)厭氧階段總釋磷量的52.83%;厭氧結(jié)束時(shí)(120min),COD濃度降為52.67mg/L,PO43--P濃度達(dá)到24.02mg/L.厭氧前30min為快速釋磷階段,釋磷速率為25.26mg PO43--P/h,厭氧后60min(30 ～ 90min)為慢速釋磷階段,釋磷速率僅為11.34mg PO43--P/h.圖3A中PHA含量從49.68mgCOD/gVSS增加到97.25mgCOD/gVSS,Gly從158.25mgCOD/gVSS減少為128.84mgCOD/gVSS,其中前30min PHA增長量為23.15mgCOD/gVSS,Gly減小量為23.23mgCOD/gVSS,PHA、Gly與COD、PO43--P的變化規(guī)律具有一致性.

A: COD、PHA和Gly; B: PO43--P、NO3--N和NO2--N

缺氧段和好氧段,聚磷菌不斷氧化分解體內(nèi)存儲的PHA并釋放能量,將污水中的無機(jī)磷攝入體中,一部分合成ATP,一部分合成聚磷酸鹽存于細(xì)胞內(nèi).兩者除磷機(jī)理類似,不同的是前者的電子受體為硝酸鹽,后者為氧氣[30].反應(yīng)時(shí)間150min時(shí),缺氧段和好氧段PHA的消耗量分別為55.80和70.29mgCOD/ gVSS,殘留PO43--P濃度分別為4.68和0.06mg/L.相比之下,缺氧過程出水PO43--P濃度相對較高.結(jié)合NO3--N的變化規(guī)律可知,在反應(yīng)時(shí)間270min,電子受體硝酸鹽已經(jīng)基本消耗完,存儲于反硝化聚磷菌(DPAOs)細(xì)胞內(nèi)的PHA由于缺乏電子受體而不能繼續(xù)氧化,導(dǎo)致缺氧吸磷停止.李勇智等[31]在試驗(yàn)中也有相似現(xiàn)象,當(dāng)投加硝酸鹽氮濃度為30mg/L時(shí),缺氧出水PO43--P濃度偏高.再一次表明階段III中,導(dǎo)致除磷效果不理想的一個(gè)重要原因可能是電子受體硝酸鹽氮的缺乏.根據(jù)Wachtmeister等[13]推薦的方法得出DPAOs占PAOs的比例約72.42%,與前期的研究結(jié)論(73.80%)較為接近[3],但遠(yuǎn)高于接種污泥的13.26%,表明系統(tǒng)經(jīng)過長期運(yùn)行,已基本實(shí)現(xiàn)了功能菌的富集.

此外,缺氧反硝化除磷過程還出現(xiàn)了NO2--N積累的現(xiàn)象(圖3B),在反應(yīng)時(shí)間200min,NO2--N濃度達(dá)到峰值7.8mg/L,在隨后70min內(nèi)被逐步反硝化.研究表明,DPAOs依次攝取不同類型的PHA,其中PHB的降解速率比PHV的降解速率更快[32],即PHB攝取先于PHV;但PHV與氮和磷的生物降解更相關(guān),由于PHV的降解速率受限,出現(xiàn)了短暫的亞硝酸鹽積累.因此,有關(guān)碳源類型對內(nèi)碳源種類的影響還有待深入研究.

2.3 碳源類型對微生物結(jié)構(gòu)的影響

2.3.1 微生物多樣性分析 表2對比了不同碳源類型條件下,微生物的物種豐富度和多樣性.7個(gè)樣品(4個(gè)污泥樣品和3個(gè)生物膜樣品)檢索到的序列數(shù)在32923～57737范圍內(nèi)波動,將有效序列在97%的相似性類聚,獲得了454～1151個(gè)OTUs;所有樣品的覆蓋度(coverage)都在99%以上,表明所取樣品具有足夠的取樣深度[33],可反映微生物的真實(shí)情況.整體上,相對于接種污泥D0而言,3個(gè)污泥樣品的OTUs、Ace和Chao指數(shù)均有明顯下降,表明隨著碳源類型的改變,細(xì)菌群落的多樣性下降;而shannon和simpson指數(shù)的變化表明物種的豐富度下降,意味著碳源類型對微生物菌群結(jié)構(gòu)有較為明顯的影響.N1～N3樣品為BCO反應(yīng)器中的生物膜,由于只進(jìn)行硝化反應(yīng),功能單一且針對性強(qiáng),此階段多為難降解有機(jī)物,不易被微生物所利用,因而微生物的多樣性以及豐富度較A2/O污泥而言低得多,該現(xiàn)象與前期研究較為相似[34].

表2 微生物菌群豐富度和多樣性

圖4 Venn圖

Fig.4 Venn diagram

A:活性污泥樣品; B:生物膜樣品

圖4A顯示4個(gè)污泥樣品共有的OTUs數(shù)為408,而D40、D80、D120單獨(dú)所有的OTUs數(shù)從接種污泥D0時(shí)的100降為40、92、20,意味著階段II具有獨(dú)特功能的微生物種類更為多樣,初步驗(yàn)證了圖2中混合碳源在污染物去除性能上的優(yōu)越性.類似地,圖4B顯示的BCO樣品中,N1所獨(dú)有的OTUs數(shù)高達(dá)172,遠(yuǎn)遠(yuǎn)高于N2(21)、N3(52),這是因?yàn)镹1進(jìn)水水質(zhì)最為復(fù)雜,微生物菌群種類較多,而N1到N3推流過程中,功能菌群被不斷篩選和淘洗[35],但整體上N1、N2、N3都具有獨(dú)特的OTUs,初步表明各階段含有特定菌群且具有不同的處理功能.

2.3.2 功能菌群結(jié)構(gòu)分析 圖5分別從門和屬水平上對污泥、生物膜樣品的微生物菌群結(jié)構(gòu)和功能菌演變規(guī)律進(jìn)行了對比分析.在污泥樣品中(圖5A),Proteobacteria(21.92%～42.62%)、Chloroflexi (15.95%～26.61%)和Bacteroidetes(2.12%～21.97%)是最主要的三大門,已被廣泛證實(shí)含有PAOs和DPAOs[10];Actinobacteria、Saccharibacteria進(jìn)行有機(jī)物降解、反硝化[36],總量從24.20%(D0)降至21.00% (D40)、3.96% (D80)、4.20%(D120),證實(shí)了碳源類型對COD去除以及內(nèi)碳源轉(zhuǎn)化的內(nèi)在影響(圖2(A)); Parcubacteria在厭氧/缺氧代謝過程可利用NO3-代替O2作為電子受體[37],在階段II占據(jù)14.96%的比例(階段I占0.6%,階段II占3.65%),有效促進(jìn)了DPR性能的提升(圖2D);此外,階段II中與Bacteroidetes同門的Chlorobi占6.11%,遠(yuǎn)高于其他樣品(0 ～ 1%).

圖5 微生物菌群分布

A、B:門水平; C、D:屬水平

在屬水平上(圖5C), PAOs中(屬于Proteobacteria)從0.39%(D0)升高至8.49%(D40)、18.72%(D80),但在階段III(120d)下降到10.23%,同樣屬于PAOs的[38]從1.06%增加到1.35%～2.98%.階段II,乙酸鈉、丙酸鈉共存時(shí),屬于DPAOs的(4.78%)、(3.27%)[39]達(dá)到峰值;由于反硝化除磷效果的增強(qiáng),、、、這4類菌群總量分別占14.34%(D40)、29.13%(D80)和16.18%(D120),而接種污泥中僅占1.70%.然而,、作為GAOs中2大優(yōu)勢菌屬[40],其總量從1.85%(D0)增加到5.27%(D40)、20.28%(D80)、27.98%(D120).導(dǎo)致上述菌群分布變化的主要原因在于PAOs和GAOs對乙酸、丙酸的不同親和力以及對電子受體、電子供體的不同響應(yīng)規(guī)律[41].PAOs可同時(shí)利用乙酸、丙酸來還原NO3-和NO2-,而GAOs特別是通常更容易利用丙酸來還原NO3-[42],換言之,丙酸的存在提高了GAOs在DPR過程的競爭優(yōu)勢,促進(jìn)了功能菌群的多樣性和系統(tǒng)運(yùn)行的穩(wěn)定性(圖2).而碳源類型導(dǎo)致的不同電子供體PHB、PHV,直接影響內(nèi)碳源轉(zhuǎn)化的化學(xué)計(jì)量參數(shù)和氮、磷生物降解特性[10],進(jìn)而改變了DPR過程功能菌演變.

此外,與接種污泥相比,階段I ～ III中包括(0.25%～2.39%)、(1.39%～ 3.03%)、(0.07%～0.19%)、(0.02% ～0.05%)等在內(nèi)的普通異養(yǎng)菌(OHOs)含量均有所降低,表明外源反硝化作用受到了抑制;而在顆粒污泥形成過程中發(fā)揮重要作用的絲狀菌屬、[2],分別從1.13%、0.94%增加至1.49%～11.87%、2.98%～4.31%.此外,由于A2/O反應(yīng)器不進(jìn)行硝化,AOB(包括和)含量從4.03%降至0.83%(D40)、0.33%(D80)、0.25%(D120),而NOB(以為主)從3.01%下降至0.10%～0.48%,表明硝化菌已被逐步淘洗出去.

如圖5B所示,BCO反應(yīng)器中檢測到Proteobacteria占77.60%～81.79%,負(fù)責(zé)硝化的Planctomycetes、Nitrospira[43]總量從2.70% (N1)增加至11.70% (N2)和21.91%(N3);由于從N1到N3過程COD含量逐漸減小,具有分解代謝有機(jī)物功能的Actinobacteria(屬于絲狀菌)[44]從4.67%下降到2.97%,2.28%;其他3種門Chloroflexi(1.61%～ 2.78%)、Bacteroidetes(1.66%～3.65%)、Chlamydiae (1.25%～2.31%)含量相近.在屬水平上(圖5D),、兩種典型的AOB菌屬從2.33%(N1)增加到16.03%(N2)、21.80% (N3),而作為NOB菌屬從1.56%(N1)增加到7.06%(N2)、15.43%(N3).由于三段串聯(lián)的運(yùn)行模式,AOB、NOB總量從3.89%增加到23.09%、37.23%,遠(yuǎn)遠(yuǎn)高于某中試硝化MBBR中AOB(6.5%～7.0%)和NOB(2.3%～3.8%)的相對豐度[45].然而,盡管進(jìn)入BCO反應(yīng)器的COD濃度不高(圖2A),OHOs仍然占據(jù)了較大比例,如與反硝化相關(guān)的[46]在N1中占40.14%,N2和N3分別下降至12.22%和2.65%;作為生物膜附著骨架的、,百分比含量在0.74%～1.05%、6.78%～ 8.03%范圍內(nèi)波動.此外,具有一定同步硝化反硝化能力的[47]菌屬占0.84%～2.19%,與BCO反應(yīng)器中存在氮損失的現(xiàn)象相吻合(圖2C).門、屬水平上菌群結(jié)構(gòu)的巨大差異表明A2/O- BCO系統(tǒng)實(shí)現(xiàn)了功能菌群的分離,體現(xiàn)了雙污泥系統(tǒng)的本質(zhì).

2.4 基于碳源高效利用的運(yùn)行調(diào)控策略

總體而言,A2/O-BCO系統(tǒng)基于反硝化除磷可以實(shí)現(xiàn)碳源的高效利用,尤其是采用混合碳源時(shí)(階段II),在污染物去除、內(nèi)碳源轉(zhuǎn)化利用以及微生物菌群結(jié)構(gòu)方面具有明顯的優(yōu)勢,為污水處理廠的運(yùn)行和管理提供了一種可靠的運(yùn)行控制策略.一方面,根據(jù)PAOs-GAOs競爭理論,與單一碳源相比,混合碳源更有利于DPR系統(tǒng)穩(wěn)定性和微生物多樣性;另一方面,碳源類型決定了內(nèi)碳源的組成和轉(zhuǎn)化(包括PHA和Gly),尤其是PHA類型與污染物性能密切相關(guān),乙酸鈉-丙酸鈉共存促進(jìn)了PHB和PHV的平衡,并加速了功能菌富集.

據(jù)報(bào)道,實(shí)際污水處理廠進(jìn)水中乙酸、丙酸組分占原水總量的49%～71%和24%～33%[48],因此,本研究對污水廠的碳源高效利用及外碳源投加具有重要的參考價(jià)值:①碳源類型會影響污染物去除效果,當(dāng)然有機(jī)物濃度也不容忽視[10];②內(nèi)碳源轉(zhuǎn)化利用有助于深入理解污染物代謝途徑和反硝化除磷機(jī)理,這也為其他反硝化除磷系統(tǒng)的機(jī)理分析提供了參考;③雖然PAOs和GAOs是反硝化除磷過程的兩大主要競爭菌群,但某些特殊菌屬(如、、、等)對系統(tǒng)的穩(wěn)定運(yùn)行發(fā)揮重要作用.當(dāng)然,考慮到實(shí)際污水的復(fù)雜性和多變性,為了更好地應(yīng)用于工程實(shí)踐,需要在經(jīng)濟(jì)運(yùn)行(如碳源、曝氣能耗和污泥產(chǎn)量)的前提下[49],對實(shí)際生活污水處理過程中碳源的高效利用進(jìn)行長期優(yōu)化.

除此之外,A2/O-BCO系統(tǒng)還存在諸多不足:①缺氧段反硝化除磷效果需進(jìn)一步強(qiáng)化,其中電子受體不足的問題亟待解決,而解決這一問題的關(guān)鍵在于保證BCO反應(yīng)器穩(wěn)定高效的硝化效果,進(jìn)而通過硝化液回流比的優(yōu)化為缺氧區(qū)提供充足的硝化液;②BCO反應(yīng)器出水的NO--N濃度偏高,一方面可在反應(yīng)器之后增加尾水處理,確保出水TN的高標(biāo)準(zhǔn)排放,另一方面可強(qiáng)化短程硝化,實(shí)現(xiàn)短程反硝化除磷耦合技術(shù),更大程度上節(jié)省碳源和曝氣量.

3 結(jié)論

3.1 A2/O - BCO系統(tǒng)中碳源類型對厭氧段內(nèi)碳源轉(zhuǎn)化的影響較為顯著,但BCO反應(yīng)器的硝化效果幾乎不受碳源影響;反硝化除磷過程氮和磷的去除效果呈現(xiàn)此消彼長的趨勢,且當(dāng)乙酸鈉、丙酸鈉等比例混合時(shí),C、N、P達(dá)到最優(yōu)的處理效果.

3.2 反硝化除磷批次試驗(yàn)表明,階段II中DPAOs占PAOs的比例為72.42%,基本實(shí)現(xiàn)了DPAOs的富集,內(nèi)碳源PHA、Gly與COD、PO43--P的變化規(guī)律呈現(xiàn)一致性,而電子受體不足是反硝化除磷效果不理想的一個(gè)重要因素.

3.3 與接種污泥相比,3個(gè)污泥樣品的菌群結(jié)構(gòu)多樣性和豐富度均有所下降,而D80特有的菌群OTUs數(shù)最多;BCO反應(yīng)器3個(gè)生物膜樣品的多樣性和豐富度比污泥樣品低,但3個(gè)格室在推流過程中均呈現(xiàn)出特有的OTUs菌群.

3.4 污泥和生物膜樣品在門、屬水平上均呈現(xiàn)巨大差異,表明A2/O - BCO系統(tǒng)實(shí)現(xiàn)了功能菌群的分離,其中A2/O反應(yīng)器中Accumulibacter、Acinetobacter、Dechloromonas、Pseudomonas等大量富集,是反硝化除磷效果較好的重要原因;BCO反應(yīng)器中Nitrosomonas、Nitrosomonadaceae、Nitrospira是進(jìn)行硝化的主要功能菌群,而反硝化菌Thermomonas、Anaerolineaceae、Zoogloea、Pseudomonas等的存在促進(jìn)了氮損失.

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Effect of carbon source types on denitrifying phosphorus removal and microbial community in the A2/O - BCO process.

FAN Ya-jun1, ZHANG Miao2, JI Jun-jie2, CHENG Ji-lin1, WU Qi-chao3, HE Cheng-da2*

(1.College of Hydraulic Science and Engineering, Yangzhou University, Yangzhou 225127, China；2.College of Environmental Science and Engineering, Yangzhou University, Yangzhou 225127, China；3.Yangzhou Polytechnic Institute, Yangzhou 225127, China)., 2022,42(1)：172～182

The anaerobic/anoxic/aerobic-biological contact oxidation (A2/O-BCO) process was used (1) to treat low carbon/nitrogen ratio (C/N) wastewater, (2) to monitor the effect of single factor of carbon source (Phase I: sodium acetate; Phase II: Sodium acetate + sodium propionate; Phase III: sodium propionate) on organic matter, and (3) to investigate the simultaneous nitrogen and phosphorus removals were investigated. The research explored the transformation and utilization of internal carbon sources (PHA and Gly) and the mechanism of denitrifying phosphorus removal (DPR) under the mixed carbon source conditions of sodium acetate and sodium propionate. Meanwhile, the evolution rules of microbial community structure at different phases were compared through high-throughput sequencing. The results showed that the mixed carbon source improved the simultaneous removal efficiency of organic matter, nitrogen and phosphorus, and the transformation amount of internal carbon source was 226mg/h with the phosphorus release amount of 30.58mg/L in the anaerobic section, and the DPR efficiency was above 90%. The batch test showed that the proportion of denitrifying phosphorous accumulating bacteria (DPAOs) to phosphorous accumulating bacteria (PAOs) was 72.42%, which basically realized the enrichment of DPAOs. The results of high-throughput sequencing showed that the mixed carbon sources were more conducive to the formation of unique OTUsbacterial genus, and the total amount of PAOs (includingand) and DPAOs (includingand) was up to 29.13%(>16.18%(Phase III)>14.34%(Phase I)), thus efficiently promoting the utilization of carbon source and efficiently denitrifying phosphorus removal. In the BCO reactor, the total amount of ammonia-oxidizing bacteria (AOB, includingand) and nitrite-oxidizing bacteria (NOB, mainly) increased from 3.89%(N1) to 23.09%(N2) and 37.23%(N3), which provided sufficient electron receptors for denitrifying phosphorus removal. In addition, the operation regulation strategy based on the efficient utilization of carbon source was established, in order to provide theoretical reference for the application of A2/O-BCO process.

A2/O-BCO process；carbon source type；denitrifying phosphorus removal；internal carbon source transformation；high-throughput sequencing

X703.5

A

1000-6923(2022)01-0172-11

范亞駿(1990-),男,江蘇靖江人,揚(yáng)州大學(xué)博士研究生,主要從事水污染控制方向.發(fā)表論文4篇.

2021-06-15

國家自然科學(xué)基金資助項(xiàng)目(51808482);博士后科學(xué)基金資助面上項(xiàng)目(2018M632392);江蘇省水環(huán)境保護(hù)技術(shù)與裝備工程實(shí)驗(yàn)室開放課題(W1904)

* 責(zé)任作者, 教授, hcd@yzu.edu.cn