余浩濤,于莉芳*,李 韌,高 宇,張 瓊,喬冰闖,鄭蘭香,彭黨聰

部分反硝化耦合厭氧氨氧化IFAS系統(tǒng)脫氮性能

余浩濤1,于莉芳1*,李 韌1,高 宇1,張 瓊1,喬冰闖1,鄭蘭香2,3,彭黨聰1

(1.西安建筑科技大學(xué)環(huán)境與市政工程學(xué)院,陜西 西安 710055；2.寧夏大學(xué)生態(tài)環(huán)境學(xué)院,寧夏 銀川 750021；3.中國(guó)葡萄酒產(chǎn)業(yè)技術(shù)研究院,寧夏 銀川 750021)

采用部分反硝化活性污泥耦合厭氧氨氧化生物膜處理低碳氮比廢水(C/TN=1.63),考察生物膜-活性污泥復(fù)合系統(tǒng)(IFAS)進(jìn)行部分反硝化耦合厭氧氨氧化(PD/A)處理低碳氮比廢水的可行性及其耦合后兩相中功能菌活性與菌群分布變化規(guī)律.結(jié)果顯示,系統(tǒng)耦合運(yùn)行期間,出水TN為(5.07±0.2)mg/L,去除率為(90.7±0.1)%,厭氧氨氧化途徑對(duì)TN去除的貢獻(xiàn)率高達(dá)(86.61±3.4)%;固著相對(duì)厭氧氨氧化活性的貢獻(xiàn)率為100%,懸浮相上,(NO3--N)占比為99.32%,(NO2--N)占比為99.22%;與耦合前相比,懸浮相中硝酸鹽還原酶(Nar)活性由(0.43±0.05)μmol/(mg protein·min)增加至(0.49±0.09)μmol/(mg protein·min),亞硝酸鹽轉(zhuǎn)化率明顯升高[(70±2.2)%～(90.01±2.3)%];Illumina MiSeq結(jié)果顯示,固著相上的優(yōu)勢(shì)菌屬為,且耦合前后豐度無明顯變化(33.61%～33.43%),懸浮相上反硝化菌屬,,OLB8豐度增加.以上結(jié)果表明,在IFAS系統(tǒng)中可以實(shí)現(xiàn)穩(wěn)定的PD/A協(xié)同脫氮,耦合后部分反硝化由懸浮相主導(dǎo),厭氧氨氧化由固著相主導(dǎo),厭氧氨氧化菌(AnAOB)與反硝化菌對(duì)NO2---N的競(jìng)爭(zhēng)強(qiáng)化了懸浮相部分反硝化能力.

生物膜-活性污泥復(fù)合工藝；低碳氮比；部分反硝化；厭氧氨氧化；群落結(jié)構(gòu)；高通量測(cè)序

厭氧氨氧化(Anammox)作為新型生物脫氮工藝,具有無需外加有機(jī)碳源、污泥產(chǎn)量低、脫氮效率高、溫室氣體排放少等優(yōu)點(diǎn),在城鎮(zhèn)污水處理廠升級(jí)改造中具有廣闊的應(yīng)用前景[1-2].但其在實(shí)際應(yīng)用中主要受以下因素限制:(1)電子受體NO2--N無法穩(wěn)定供給;(2)Anammox生物量很難富集并保持;(3)有機(jī)物對(duì)厭氧氨氧化菌(AnAOB)的生長(zhǎng)存在一定的抑制作用.已有研究表明,部分反硝化(PD)能在低碳氮比條件和不同溫度下將NO3--N還原為NO2--N, Anammox反應(yīng)所需NO2--N可由PD來提供[1,3-4],且PD利用有機(jī)碳作為電子供體,可以為Anammox解除有機(jī)物的抑制[5].此外,Anammox產(chǎn)生的NO3--N可通過PD繼續(xù)生成NO2--N用于Anammox,提高了脫氮性能,因此部分反硝化耦合厭氧氨氧化(PD/A)工藝成為當(dāng)前低碳氮比廢水處理研究的熱點(diǎn)[4,6-7].

PD/A系統(tǒng)中兩種功能菌存在顯著的生理差異,反硝化菌為異養(yǎng)菌,有較高的產(chǎn)率系數(shù)(0.3gVSS/g NH4+-N)和底物吸收速率,需要良好的傳質(zhì)效率[8];而AnAOB為自養(yǎng)菌,產(chǎn)率系數(shù)低(0.066gVSS/g NH4+-N),需要長(zhǎng)污泥齡來保留和維持穩(wěn)定的生物量[8].而在生物膜-活性污泥復(fù)合工藝(IFAS)中,懸浮相污泥齡調(diào)控靈活,可為生長(zhǎng)較快的微生物提供更好的傳質(zhì)效果,適合反硝化菌的生長(zhǎng);固著相載體能為微生物提供大量的附著點(diǎn)位和生長(zhǎng)空間,也能提供抵御不利環(huán)境的防護(hù)屏障[9],可強(qiáng)化AnAOB在生物膜中的保留[10-11].因此,IFAS系統(tǒng)可成為有效的PD/A耦合形式,但耦合后系統(tǒng)處理低碳氮比廢水的可行性及微生物群落結(jié)構(gòu)變化有待進(jìn)一步研究.

本研究在實(shí)現(xiàn)PD的基礎(chǔ)上,采用IFAS系統(tǒng)進(jìn)行PD/A耦合處理低碳氮比廢水(C/TN=1.63),考察耦合后IFAS系統(tǒng)脫氮性能及氮轉(zhuǎn)化途徑,對(duì)比分析了耦合前后功能微生物代謝活性、酶活性變化情況,采用Illumina MiSeq方法分析了微生物群落結(jié)構(gòu)變化,以期為該工藝的實(shí)際應(yīng)用提供技術(shù)參考.

1 材料與方法

1.1 試驗(yàn)裝置及運(yùn)行條件

試驗(yàn)裝置采用有效容積為3L的SBR反應(yīng)器(圖1),并通過恒溫水浴系統(tǒng)控制反應(yīng)器內(nèi)溫度為25℃,為了充分混合,反應(yīng)器設(shè)置了恒速攪拌系統(tǒng),轉(zhuǎn)速為70r/min.試驗(yàn)所用的接種污泥取自西安市某污水處理廠缺氧池,MLSS為(2323.4±62)mg/L,MLVSS為(1713.6±34)mg/L,Anammox填料取自實(shí)驗(yàn)室已穩(wěn)定運(yùn)行2年的固定床生物膜反應(yīng)器,填料比表面積為500m2/m3,負(fù)荷為0.39mg NH4+-N/(m3·d),最大活性為122.25mg N/(L·d).

試驗(yàn)分為PD培養(yǎng)階段(0～95d)及PD/A耦合階段(96～155d).其中,PD階段污泥齡為15d,換水比恒為50%,前42d以4h/周期運(yùn)行(進(jìn)水5min,攪拌190min,沉降30min,排水10min,閑置5min);43～95d將攪拌時(shí)長(zhǎng)縮短為70min,以2h/周期運(yùn)行;PD/A階段懸浮相污泥齡為15d,生物膜填充率為15%,換水比調(diào)整至33%,將攪拌時(shí)長(zhǎng)重新增加為190min,以4h/周期運(yùn)行.

圖1 PD/A階段反應(yīng)器裝置

1.2 試驗(yàn)用水

本試驗(yàn)用水采用人工模擬低碳氮比廢水,并通過添加NH4Cl、NaNO3和CH3COONa配制進(jìn)水所需的氨氮、硝酸鹽氮及COD.其中,PD階段進(jìn)水水質(zhì)為:60mg/L NO3--N,180mg/L COD, 11.1mg/L KH2PO4, 6mg/L CaCl2·2H2O,3mg/L MgSO4·7H2O; PD/A階段參考西安市某污水廠進(jìn)水,進(jìn)水水質(zhì)為:30mg/L NO3--N,25mg/L NH4+-N,90mg/L COD, 30mg/L KH2PO4, 140mg/L CaCl2·2H2O,140mg/L MgSO4·7H2O;微量元素見文獻(xiàn)[6,12].

1.3 檢測(cè)分析方法

本試驗(yàn)中常規(guī)水質(zhì)(NH4+-N、NO2--N和NO3--N)采用標(biāo)準(zhǔn)方法測(cè)定[12];pH值采用雷磁PHS-3C pH計(jì)測(cè)定;COD采用COD快速分析儀(HACA DRB 200)測(cè)定;PD活性測(cè)定方法見文獻(xiàn)[13],分別用(NO3--N)和(NO2--N)表示NO3--N還原速率和NO2--N積累速率;Anammox活性(NH4+- N)(以氨氮計(jì))測(cè)定方法見文獻(xiàn)[14].

試驗(yàn)采用低溫高壓連續(xù)細(xì)胞破碎儀(JN-02C,廣州聚能納米生物科技有限公司)進(jìn)行粗酶的提取,具體方法參照文獻(xiàn)[15],通過Lowry法測(cè)定蛋白質(zhì)的濃度以表示粗酶的含量.硝酸鹽還原酶(Nar)和亞硝酸鹽還原酶(Nir)活性單位以μmol/(mg protein·min)表示,詳細(xì)測(cè)定方法見文獻(xiàn)[17];肼脫氫酶(HDH)活性測(cè)定體系包括:0.01mol/L磷酸鉀緩沖液(pH=8.0)、50μmol/L馬心細(xì)胞色素C、適量酶液及25μmol/L肼,單位以μmol細(xì)胞色素C/(mg protein·min)表示,具體方法見文獻(xiàn)[16].

1.4 指標(biāo)計(jì)算

(1)PD和PD/A階段NO2--N轉(zhuǎn)化率(NTR)計(jì)算方法分別見公式(1)[12]和公式(2)[6]:

式中:[NO2--N]eff表示出水NO2--N濃度,mg/L; [NO3--N]eff表示出水NO3--N濃度,mg/L;[NO3--N]inf表示進(jìn)水NO3--N濃度,mg/L.

(2)

式中:[NO2--N]表示時(shí)刻的NO2--N濃度,mg/L;[NO2-- N]amx表示Anammox反應(yīng)理論上消耗的NO2--N濃度,mg/L;[NO3--N]inf表示進(jìn)水NO3--N濃度, mg/L; [NO3--N]amx表示Anammox 反應(yīng)理論上產(chǎn)生的NO3-- N濃度,mg/L;[NO3--N]表示時(shí)刻的NO3--N濃度, mg/L.

(2)完全反硝化和厭氧氨氧化是PD/A階段的主要脫氮途徑,忽略細(xì)胞合成消耗的NH4+-N,根據(jù)Anammox反應(yīng)式[13]計(jì)算[6]:

厭氧氨氧化脫氮貢獻(xiàn)率:

完全反硝化脫氮貢獻(xiàn)率:

式中:[NH4+-N]inf表示進(jìn)水NH4+-N濃度,mg/L; [NH4+-N]eff表示出水NH4+-N濃度,mg/L; ?TN表示進(jìn)出水TN變化濃度,mg/L.

(3)部分反硝化代謝活性和反硝化酶活性占比計(jì)算方法分別見公式(5)和公式(6):

1.5 微生物群落分析

微生物群落結(jié)構(gòu)分析采用Illumina MiSeq高通量測(cè)序,取耦合前活性污泥和接種填料及耦合階段運(yùn)行第60d的活性污泥和生物填料進(jìn)行高通量測(cè)序分析.將活性污泥耦合前后樣品分別命名為PD1和PD2,生物填料耦合前后樣品分別命名為A1和A2.樣品由北京奧維森基因科技有限公司提供測(cè)序技術(shù)支持,采用引物序列515a(GTGCC- AGCMGCCGCGGTAA)和806(GGACTACHVGG- GTWTCTAAT)對(duì)樣品16S V4區(qū)進(jìn)行擴(kuò)增,具體方法見文獻(xiàn)[17].

2 結(jié)果與討論

2.1 部分反硝化階段

2.1.1 PD階段運(yùn)行特性 如圖2所示,在1～42d,以4h/周期,HRT=8h運(yùn)行.反應(yīng)器啟動(dòng)第1d, NTRPD僅為26.32%,之后NTRPD快速上升,并在第10d達(dá)到了60.89%,相比生物膜系統(tǒng),本研究采用活性污泥可以更快地實(shí)現(xiàn)部分反硝化[2,13].11～31d,反應(yīng)器穩(wěn)定運(yùn)行,出水NO2--N穩(wěn)定維持在(40±1.4)mg/L,平均NTRPD達(dá)到(63.64±2.8)%;但32～42d出水NO2--N降低, NTRPD降低至(57.14±1.3)%,其原因是停留時(shí)間偏長(zhǎng)時(shí)發(fā)生了內(nèi)碳源反硝化[16].因此,在43～95d,以2h/周期,HRT=4h運(yùn)行,在此期間出水NO2--N恢復(fù)并穩(wěn)定在(42±1.6)mg/L,平均NTRPD恢復(fù)至(70±2.2)%,實(shí)現(xiàn)了PD的穩(wěn)定運(yùn)行,PD階段亞硝酸鹽轉(zhuǎn)化率逐漸升高到趨于穩(wěn)定, 變化規(guī)律與以往研究結(jié)果類似[2].

2.1.2 PD階段典型周期 由圖3可知,0～15min,反硝化菌利用COD快速將NO3--N還原,NO2--N迅速從16.87mg/L積累至36.41mg/L,與生物膜系統(tǒng)相比,本研究中亞硝酸鹽的積累速度更快,達(dá)到峰值所需時(shí)間更短[2].15～120min,由于COD濃度保持在較低水平,反硝化過程受到抑制,NO3--N緩慢降至4.2mg/L,對(duì)應(yīng)NO2--N濃度增加至39.9mg/L,由公式(1)計(jì)算可得,平均NTRPD為(70.7±1.2)%.理論上,部分反硝化(NO3--N→NO2--N)不會(huì)產(chǎn)生堿度,但乙酸鈉的消耗會(huì)導(dǎo)致pH值增加,結(jié)合pH值變化曲線,0～15min,pH值快速上升并到達(dá)峰值,15～120min, pH值逐漸下降,與Gong等[18]典型周期中pH值變化規(guī)律一致.

圖2 PD階段進(jìn)出水氮濃度及NTRPD變化

圖3 PD階段典型周期內(nèi)氮素、COD和pH值變化(第70d)

2.2 部分反硝化耦合厭氧氨氧化階段

2.2.1 PD/A階段運(yùn)行特性 由圖4可知,反應(yīng)器在加入?yún)捬醢毖趸锬ず?快速實(shí)現(xiàn)了PD/A協(xié)同脫氮,耦合后第一天(96d),NH4+-N由進(jìn)水的22.15mg/L降至2.29mg/L(去除率為89.66%),NO3--N由進(jìn)水32.39mg/L降至5.6mg/L(去除率為82.4%),出水TN為7.95mg/L,去除率為85.42%;之后反應(yīng)器運(yùn)行穩(wěn)定,出水NH4+-N降至(1.1±0.3)mg/L,出水NO3--N降至(3.1±0.4)mg/L,出水TN為(5.07±0.2)mg/L,去除率為(90.7±0.1)%.由公式(2)計(jì)算,耦合階段運(yùn)行穩(wěn)定期間平均NTRPD/A為(90.01±2.3)%,顯著高于PD階段中的NTRPD(70±2.2)%,這與Jiang等[19]實(shí)驗(yàn)得出耦合后NTRPD/A高于耦合前的變化規(guī)律一致,這可能是因?yàn)锳nammox對(duì)NO2?-N(PD過程產(chǎn)物)的快速消耗強(qiáng)化了系統(tǒng)的部分反硝化能力,而PD過程又可以為Anammox過程提供更多的電子受體.由此可見,IFAS系統(tǒng)內(nèi),部分反硝化和厭氧氨氧化可以相互促進(jìn),可有效實(shí)現(xiàn)PD/A協(xié)同脫氮[2].

圖4 PD/A階段反應(yīng)器脫氮性能

(a)進(jìn)出水氮濃度(b)TN及其去除率(c)兩種脫氮途徑對(duì)TN去除貢獻(xiàn)率

如圖4c所示,厭氧氨氧化途徑對(duì)TN去除的平均貢獻(xiàn)率為(86.61±3.4)%,而完全反硝化途徑對(duì)TN去除的平均貢獻(xiàn)率僅為(13.57±2.4)%.顯然,厭氧氨氧化途徑在脫氮過程中起主導(dǎo)作用,這說明AnAOB對(duì)NO2--N的競(jìng)爭(zhēng)能力強(qiáng)于反硝化菌,這對(duì)NH4+-N和NO2--N的穩(wěn)定去除起著關(guān)鍵作用[3]. Cao等[20]接種具有較高NTRPD(80%)的PD活性污泥進(jìn)行PD/A,在C/NO3--N=3條件下,厭氧氨氧化途徑對(duì)TN去除貢獻(xiàn)率為90%,TN去除率達(dá)到了97%,這可能是由于高NTRPD條件下,更多的NO2--N通過厭氧氨氧化途徑而不是完全反硝化途徑生成N2,進(jìn)一步表明高NTRPD對(duì)PD/A實(shí)現(xiàn)高效穩(wěn)定的脫氮性能起關(guān)鍵作用.

圖5 PD/A階段典型周期內(nèi)各氮素轉(zhuǎn)化、COD和pH值變化 (第136d)

2.2.2 PD/A階段典型周期 如圖5所示,PD/A工藝可以清晰地劃分為PD和Anammox兩個(gè)階段. 0～21min,PD過程所需底物NO3--N和COD充足,PD利用有機(jī)物快速將NO3--N還原,NO2--N濃度迅速積累至9.67mg/L,反應(yīng)前期PD過程占主導(dǎo).而由于有機(jī)物對(duì)Anammox活性有抑制作用,有機(jī)物的消耗通常在Anammox反應(yīng)之前進(jìn)行[5].21～240min,觀察到NO2--N和NH4+-N同時(shí)被消耗,NO3--N濃度開始緩慢上升,這個(gè)階段COD濃度較低,反硝化過程受限,使得NO2--N通過Anammox途徑去除.周期內(nèi)NTRPD/A(91.23%)顯著高于PD階段中的NTRPD((70.7±1.2)%),說明耦合后厭氧氨氧化競(jìng)爭(zhēng)NO2--N提高了NTR.經(jīng)計(jì)算,耦合階段中?NO2--N/?NH4+- N=1.22,略大于Anammox反應(yīng)式中的?NO2--N/ ?NH4+-N=1.146,說明系統(tǒng)中存在完全反硝化過程,但反應(yīng)后期對(duì)氮的去除主要依靠Anammox途徑,因此反應(yīng)后期Anammox占主導(dǎo).

2.2.3 不同生物相代謝活性變化 如圖6a所示,在PD階段,隨著培養(yǎng)時(shí)間的增加,(NO3--N)和(NO2-- N)均逐漸上升并趨于穩(wěn)定.耦合前,(NO3--N)穩(wěn)定在(4664.14±51)mgN/(L·d),(NO2--N)穩(wěn)定在(4468.22±32)mgN/(L·d),(NO2--N)/(NO3--N)為(0.95±0.08),(NO3--N)和(NO2--N)相近,可以保證NO2--N的穩(wěn)定積累,部分反硝化活性的增加使得PD階段NTRPD從26.32%增長(zhǎng)到(70±2.2)%;耦合后,(NO3--N)和(NO2--N)低于耦合前,隨著運(yùn)行時(shí)間的增加,活性有恢復(fù)趨勢(shì),在136d,(NO3--N)恢復(fù)至3531.11mgN/(L·d),(NO2--N)恢復(fù)至3267.53mgN/ (L·d).由公式(5),(6)計(jì)算,懸浮相上,(NO3--N)占比為99.32%,(NO2--N)占比為99.22%.在懸浮相上沒有檢測(cè)到Anammox活性,這可能是AnAOB在固著相上沒有脫落或者脫落的生物量在懸浮相中活性較低導(dǎo)致.

圖6 懸浮相(a)和固著相(b)上代謝活性變化

如圖6b所示,耦合前(95d),(NH4+-N)為122.25mgN/(L·d),耦合后活性低于耦合前,這是由于PD/A階段運(yùn)行初期,有機(jī)物存在時(shí)反硝化菌會(huì)與AnAOB爭(zhēng)奪底物NO2--N,對(duì)Anammox活性造成了影響[21],隨著微生物對(duì)運(yùn)行條件的適應(yīng),(NH4+-N)有恢復(fù)趨勢(shì),在第136d,(NH4+-N)逐漸恢復(fù)至104.97mg N/(L·d),這與Du等[12]實(shí)驗(yàn)得出耦合后Anammox活性先下降后上升變化趨勢(shì)一致.值得注意的是,恢復(fù)后活性數(shù)值仍低于耦合前,這是由于除了有機(jī)物對(duì)AnAOB的抑制作用外,耦合前Anammox生物膜培養(yǎng)環(huán)境從高負(fù)荷(0.39mg NH4+- N/(m3·d))、35℃變?yōu)榈拓?fù)荷(0.05mg NH4+-N/ (m3·d))、25℃也對(duì)厭氧氨氧化活性造成了影響.在固著相上檢測(cè)到了反硝化活性,但占比極低,固著相上對(duì)Anammox活性貢獻(xiàn)率為100%.而Anammox活性數(shù)值較低主要與厭氧氨氧化菌的生長(zhǎng)速率低于反硝化菌有關(guān)[3].雖然Anammox活性下降,但并未影響反應(yīng)器運(yùn)行狀況.因此,NO2--N的穩(wěn)定積累是PD/A階段實(shí)現(xiàn)穩(wěn)定高效脫氮的關(guān)鍵[2].

2.2.4 不同生物相酶活性變化 如圖7a所示,PD階段,Nar活性逐漸上升并穩(wěn)定在(0.43±0.05) μmol/(mg protein·min),Nir活性逐漸降至較低水平(0.05±0.01)μmol/(mg protein·min),Nar/Nir為(8.47±0.14),顯然,Nar在PD階段占據(jù)主導(dǎo)地位,并與PD階段中反硝化活性和NTRPD逐漸上升相符,這也從酶活性方面說明,保持Nar與Nir的巨大差異對(duì)PD的實(shí)現(xiàn)至關(guān)重要[5].耦合后,Nar活性較耦合前有所上升(0.49±0.09)μmol/(mg protein·min),Nir則維持較低活性,Nar/Nir為8.76,說明AnAOB與反硝化菌競(jìng)爭(zhēng)NO2--N強(qiáng)化了PD過程,符合PD/A階段中NTRPD/A高于PD階段的結(jié)果.由公式(7),(8)計(jì)算,懸浮相上,Nar占比為97.34%,Nir占比為65.89%.在懸浮相中沒有檢測(cè)到HDH活性,這與活性結(jié)果一致.

如圖7b所示,耦合前,HDH活性為0.10 μmol細(xì)胞色素C/(mg protein·min).耦合后,HDH活性出現(xiàn)降低,隨著Anammox菌對(duì)反應(yīng)器運(yùn)行條件的適應(yīng),第155d,HDH活性恢復(fù)至0.075μmol細(xì)胞色素C/(mg protein·min).耦合后HDH活性與Anammox活性變化規(guī)律一致,分析原因與進(jìn)水中有機(jī)物和Anammox生物膜在耦合前后的運(yùn)行條件變化有關(guān).在生物膜上檢測(cè)到了Nar和Nir存在,但占比很低,與反硝化活性在懸浮相和固著相上差異巨大的結(jié)果一致.

圖7 懸浮相(a)和固著相(b)上酶活性變化

2.3 微生物群落結(jié)構(gòu)變化

如圖8a所示,懸浮相上,耦合后Bacteroidota (45.3%～41.1%)和Proteobacteria(27.9%～16.9%)豐度降低,而Verrucomicrobiota豐度增加(11.61%～ 21.63%),這3種菌門都包含反硝化菌屬,其豐度變化可能與反硝化菌屬豐度的變化有關(guān).固著相上,包含AnAOB菌屬的Planctomycetota(38.75%～36.21%)[1]與和反硝化菌屬有關(guān)的Proteobacteria(20.97%～ 21.39%)和Bacteroidota(7.4%～7.1%)豐度均無明顯變化.

如圖8b所示,懸浮相上,、、、、OLB8是已被報(bào)道的反硝化菌屬[22-24],其中和菌屬可以將NO3--N還原為NO2--N,在PD中發(fā)揮關(guān)鍵作用[6,25];、和是反硝化菌,能夠進(jìn)行完全反硝化過程(NO3--N→N2)[22-24].屬于Verrucomicrobia菌門,其豐度的增加導(dǎo)致了耦合后Verrucomicrobia菌門豐度增加[24];以上反硝化菌屬總豐度從耦合前的13.38%增加到了耦合后的51%.懸浮相上檢測(cè)到了菌屬豐度的增加,但豐度僅為1.01%,表明AnAOB基本沒有脫落,與懸浮相中沒有檢測(cè)到生物代謝活性和酶活性結(jié)果一致.固著相上,豐度最高的是與Anammox相關(guān)的菌屬,且耦合前(33.61%)和耦合后(33.43%)的豐度并未發(fā)生明顯變化,耦合后部分反硝化菌屬和豐度有所增加.

部分反硝化實(shí)現(xiàn)亞硝酸鹽積累的兩個(gè)途徑可以分為:(1)富集培養(yǎng)具有部分反硝化能力的菌群[26];(2)利用反硝化菌中Nar和Nir對(duì)電子供體的競(jìng)爭(zhēng)能力差異導(dǎo)致亞硝酸鹽積累[27],在可進(jìn)行完全反硝化過程的菌屬占優(yōu)勢(shì)時(shí),NO3--N殘留濃度(>2mg/L)是保證PD的關(guān)鍵,NO3--N的存在會(huì)阻礙Nir接受細(xì)胞色素C傳遞的電子從而抑制NO2--N的還原[27].本研究中,IFAS系統(tǒng)的Nar和Nir酶活性主要來源于懸浮相 (圖7),耦合后懸浮相的可進(jìn)行完全反硝化過程的菌屬顯著增加并占絕對(duì)優(yōu)勢(shì)(圖8b);而典型周期內(nèi)NO3--N最低濃度為2.2mg/L(圖5).因此,可以看出IFAS系統(tǒng)中,耦合后Anammox過程通過上述第(2)種途徑強(qiáng)化懸浮相部分反硝化能力.本研究中生物膜上的AnAOB幾乎沒有脫落,而且其上生長(zhǎng)的少量反硝化菌屬?zèng)]有明顯的活性貢獻(xiàn),與以往耦合研究中反硝化菌屬豐度增加會(huì)引起厭氧氨氧化豐度降低[4]有所區(qū)別,部分反硝化由懸浮相主導(dǎo),厭氧氨氧化由固著相主導(dǎo).說明IFAS系統(tǒng)中生物膜對(duì)AnAOB有很好的持留性能,懸浮相反硝化菌對(duì)碳源有較高的利用效率,采用IFAS系統(tǒng)進(jìn)行PD/A耦合可以同時(shí)為兩種功能微生物提供良好的生長(zhǎng)環(huán)境.

圖8 懸浮相和固著相微生物群落相對(duì)豐度

(a)門水平(b)屬水平

3 結(jié)論

3.1 PD/A在IFAS系統(tǒng)中可實(shí)現(xiàn)協(xié)同脫氮.出水TN為(5.07±0.2) mg/L,厭氧氨氧化途徑對(duì)TN去除的平均貢獻(xiàn)率為(86.61±3.4)%,厭氧氨氧化對(duì)TN去除的貢獻(xiàn)占主導(dǎo)地位.

3.2 固著相對(duì)厭氧氨氧化活性的貢獻(xiàn)率為100%,懸浮相上,(NO3--N)占比為99.32%,(NO2--N)占比為99.22%.耦合后IFAS系統(tǒng)中部分反硝化由懸浮相主導(dǎo),厭氧氨氧化由固著相主導(dǎo),耦合后NTRPD/A和Nar的上升說明AnAOB與反硝化菌競(jìng)爭(zhēng)NO2--N強(qiáng)化了部分反硝化能力.

3.3 耦合后固著相上優(yōu)勢(shì)菌為,相對(duì)豐度為33.43%;懸浮相中完全反硝化菌屬豐度占絕對(duì)優(yōu)勢(shì),殘留NO3--N的存在,抑制了NO2--N還原,保證了高NO2--N積累特性. IFAS系統(tǒng)中生物膜對(duì)AnAOB有很好的持留性能,可以同時(shí)為兩種功能微生物提供良好的生長(zhǎng)環(huán)境.

[1] Wang Z, Bin L, Xiao L, et al. Microbial diversity reveals the partial denitrification-anammox process serves as a new pathway in the first mainstream anammox plant [J]. Science of the Total Environment, 2021,764:142917.

[2] Chen K, Zhang L, Sun S, et al. In situ enrichment of anammox bacteria in anoxic biofilms are possible due to the stable and long- term accumulation of nitrite during denitrification [J]. Bioresource Technology, 2020,300:122668.

[3] Du R, Cao S, Li B, et al. Synergy of partial-denitrification and anammox in continuously fed upflow sludge blanket reactor for simultaneous nitrate and ammonia removal at room temperature [J]. Bioresource Technology, 2019,274:386-394.

[4] Gao R, Peng Y, Li J, et al. Nitrogen removal from low COD/TIN real municipal sewage by coupling partial denitrification with anammox in mainstream [J]. Chemical Engineering Journal, 2021,410:128221.

[5] Du R, Peng Y, Ji J, et al. Partial denitrification providing nitrite: Opportunities of extending application for anammox [J]. Environment International, 2019,131:105001.

[6] Du R, Cao S, Li B,et al. Performance and microbial community analysis of a novel DEAMOX based on partial-denitrification and anammox treating ammonia and nitrate wastewaters [J]. Water Research, 2017,108:46-56.

[7] Li W, Shan X Y, Wang Z Y, et al. Effect of self-alkalization on nitrite accumulation in a high-rate denitrification system: Performance, microflora and enzymatic activities [J]. Water Research, 2016,88:758-765.

[8] Wang L, Hong Y, Gu J-D, et alInfluence of critical factors on nitrogen removal contribution by anammox and denitrification in an anammox-inoculated wastewater treatment system [J]. Journal of Water Process Engineering, 2021,40:101868.

[9] Mehrdadi N, Azimi A A, Bidhendi G R N, et al. Determination of design criteria for comparison of h-ifas reactor and extended aeration activated sludge process [J]. Iranian Journal of Environmental Health Science and Engineering, 2007,3(1):53-64.

[10] 劉國(guó)華,李欽渝,徐相龍,等.IFAS污水處理工藝的研究進(jìn)展[J]. 環(huán)境工程, 2022,40(2):214-224.

Liu G H, Li Q Y, Xu X L, et al. Research progress of IFAS process for wastewater treatment [J]. Environmental Engineering, 2022,40(2): 214-224.

[11] Yang S, Peng Y, Zhang L, et al. Autotrophic nitrogen removal in an integrated fixed-biofilm activated sludge (IFAS) reactor: Anammox bacteria enriched in the flocs have been overlooked [J]. Bioresource Technology, 2019,288:121512.

[12] Du R, Peng Y, Cao S, et al. Mechanisms and microbial structure of partial denitrification with high nitrite accumulation [J]. Applied Microbiology and Biotechnology, 2016,100(4):2011-2021.

[13] 于莉芳,張興秀,張 瓊,等.生物膜系統(tǒng)中部分反硝化實(shí)現(xiàn)特性[J]. 環(huán)境科學(xué), 2021,42(9):4390-4398.

Yu L F, Zhang X X, Zhang Q, et al. Characteristics of partial denitrification in biofilm system [J]. Environmental Science, 2021, 42(9):4390-4398.

[14] 呂 愷,王康舟,姚雪薇,等.基于氨氮,硝氮及乙酸條件下Anammox菌的培養(yǎng)[J]. 中國(guó)環(huán)境科學(xué), 2020,40(10):4348-4353.

Lv K, Wang K Z, Yao X W, et al. Enrichment of Anammox under conditions of ammonium, nitrate and acetic acid as substrates [J].China Environmental Science, 2020,40(10):4348-4353.

[15] Yu L, Li R, Mo P, et al. Stable partial nitrification at low temperature via selective inactivation of enzymes by intermittent thermal treatment of thickened sludge [J]. Chemical Engineering Journal, 2021,418(3): 129471.

[16] Shimamura M, Nishiyama T, Shigetomo H, et al. Isolation of a multiheme protein with features of a hydrazine-oxidizing enzyme from an anaerobic ammonium-oxidizing enrichment culture [J]. Applied and Environmental Microbiology, 2007,73(4):1065-1072.

[17] 于莉芳,汪 宇,滑思思,等.城市污水處理廠進(jìn)水氨氧化菌對(duì)活性污泥系統(tǒng)的季節(jié)性影響[J]. 環(huán)境科學(xué), 2021,42(4):1923-1929.

Yu L F, Wang Y, Hua S S, et al. Seasonal effects of influent ammonia oxidizing bacteria of municipal wastewater treatment plants on activated sludge system [J]. Environmental Science,2021,42(4):1923-1929.

[18] Gong L, Huo M, Yang Q, et al. Performance of heterotrophic partial denitrification under feast-famine condition of electron donor: a case study using acetate as external carbon source [J]. Bioresource Technology, 2013,133:263-269.

[19] Jiang H, Wang Z, Ren S, et al. Enrichment and retention of key functional bacteria of partial denitrification-Anammox (PD/A) process via cell immobilization: A novel strategy for fast PD/A application [J]. Bioresource Technology, 2021,326:124744.

[20] Cao S, Peng Y, Du R, et al. Feasibility of enhancing the denitrifying ammonium oxidation (DEAMOX) process for nitrogen removal by seeding partial denitrification sludge [J]. Chemosphere, 2016,148:403-407.

[21] Deng S, Peng Y, Zhang L, et al. Advanced nitrogen removal from municipal wastewater via two-stage partial nitrification-simultaneous anammox and denitrification (PN-SAD) process [J]. Bioresource Technology, 2020,304:122955.

[22] Roy D, Lemay J F, Drogui P, et alIdentifying the link between MBRs' key operating parameters and bacterial community: A step towards optimized leachate treatment [J]. Water Research, 2020,172: 115509.

[23] Liang B, Kang F, Yao S,et alExploration and verification of the feasibility of the sulfur-based autotrophic denitrification integrated biomass-based heterotrophic denitrification systems for wastewater treatment: From feasibility to application [J]. Chemosphere, 2022,287: 131998.

[24] Gong Q, Wang B, Gong X, et al. Anammox bacteria enrich naturally in suspended sludge system during partial nitrification of domestic sewage and contribute to nitrogen removal [J]. Science of the Total Environment, 2021,787:147658.

[25] Huang X, Mi W, Ito H, et al. Unclassified Anammox bacterium responds to robust nitrogen removal in a sequencing batch reactor fed with landfill leachate [J]. Bioresource Technology, 2020,316:123959.

[26] Le T, Peng B, Su C, et al. Nitrate residual as a key parameter to efficiently control partial denitrification coupling with anammox [J]. Water Environment Research, 2019,91(11):1455-1465.

[27] Rijn J V, Tal Y, Barak Y. Influence of volatile fatty acids on nitrite accumulation by a pseudomonas stutzeri strain isolated from a denitrifying fluidized bed reactor [J]. Applied and Environmental Microbiology, 1996,62(7):2615-2620.

[28] Zhang H, Du R, Cao S, et al. Mechanisms and characteristics of biofilm formation via novel DEAMOX system based on sequencing biofilm batch reactor [J]. Journal of Bioscience and Bioengineering, 2019,127(2):206-212.

Performance and microbial community of the partial denitrification coupled with anaerobic ammonia oxidation in an IFAS system.

YU Hao-tao1, YU Li-fang1*, LI Ren1, Gao Yu1, ZHANG Qiong1, QIAO Bing-chuang1, ZHENG Lan-xiang2,3, PENG Dang-cong1

(1.School of Municipal and Environmental Engineering, Xi'an University of Architecture and Technology, Xi'an 710055, China；2.School of Ecological Environment, Ningxia University, Yinchuan 750021, China；3.China Wine Industry Technology Institute, Yinchuan 750021, China)., 2022,42(9)：4107～4114

In this study, an integrated fixed-film activated sludge (IFAS) system was established to explore the feasibility of treating low carbon/nitrogen (C/TN=1.63) wastewater by coupling partial denitrification (PD) suspended sludge and anaerobic ammonia oxidation (Anammox) biofilm. During the experiment, As a result, the TN removal efficiency could be up to (90.7±0.1)% with the effluent TN of (5.07±0.2) mg/L, and the Anammox pathway contributed to TN removal reached (86.61±3.4)%. And the contribution of biofilm to anammox activity was 100%,(NO3--N) accounted for 99.32%,(NO2--N) accounted for 99.22% in the suspended sludge; indicating that partial denitrification process was dominated in the suspended sludge and Anammox process in the biofilm. Compared with that before coupling, the nitrate reductase (Nar) in the suspended sludge increased from (0.43±0.05) to (0.49±0.09) μmol/(mg protein·min). The nitrite transformation ratio significantly increased from (70±2.2)% to (90.01±2.3)%. In other words, the competition of nitrite between denitrifying bacteria and anaerobic ammonia oxidation bacteria (AnAOB) partially strengthened the partial denitrification process in the suspended sludge. Illumina MiSeq sequencing results show that the abundance of, the dominant bacteria in the biofilm (33.61～33.43)%, had no obvious change before and after coupling. In the phase of suspended sludge, the abundance of,, and OLB8tended to increase. Evidently, the PD/A technology in the IFAS system will provide technical guidance for its practical application.

integrated fixed-film activated sludge (IFAS)；low carbon/nitrogen wastewater；partial denitrification；anaerobic ammonia oxidation；community structure；high-throughput sequencing

X703.5

A

1000-6923(2022)09-4107-08

2022-02-08

陜西省重點(diǎn)研發(fā)計(jì)劃項(xiàng)目(2019ZDLSF0605);國(guó)家重點(diǎn)研發(fā)計(jì)劃項(xiàng)目(2019YFD1002500);西安建筑科技大學(xué)基金項(xiàng)目(ZR20020)

*責(zé)任作者, 副教授, yulifang@xauat.edu.cn

余浩濤(1994-),男,山西陽泉人,西安建筑科技大學(xué)碩士研究生,主要研究方向?yàn)槲鬯锾幚砝碚撆c技術(shù).發(fā)表論文1篇.