FNA對短程硝化污泥菌群結(jié)構(gòu)的影響

高春娣,趙 楠,安 冉,韓 徽,張 娜,彭永臻

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FNA對短程硝化污泥菌群結(jié)構(gòu)的影響

高春娣*,趙 楠,安 冉,韓 徽,張 娜,彭永臻

(北京工業(yè)大學(xué)環(huán)境與能源工程學(xué)院,城鎮(zhèn)污水深度處理與資源化利用技術(shù)國家工程實(shí)驗(yàn)室,北京 100124)

在SBR反應(yīng)器增加游離亞硝酸(FNA)預(yù)處理單元,投加濃度為1.2mgHNO2-N/L的FNA進(jìn)行缺氧攪拌4.5h,連續(xù)處理3d,考察短程硝化污泥中FNA對氨氧化菌(AOB),絲狀菌和微生物菌群結(jié)構(gòu)的影響.研究表明,FNA對AOB有短時(shí)抑制作用,并能夠抑制優(yōu)勢絲狀菌(微絲菌屬)和(噬纖維菌)的增殖,分別由5.1%和1.1%下降到0.78%和幾乎不可見.SVI從281mL/g降低到100mL/g左右.NAR能夠維持在90%左右,短程硝化不受到破壞.高通量結(jié)果顯示,FNA處理后微生物菌群結(jié)構(gòu)多樣性與豐度出現(xiàn)下降,但(陶厄氏菌屬)和出現(xiàn)了增殖,分別增加到5.58%和7.82%,同步硝化反硝化(SND)作用明顯,這使得即便只有短程硝化,總氮去除率依然能達(dá)到60%以上.

短程硝化；污泥膨脹；污泥沉降性能；絲狀菌；AOB；微生物菌群結(jié)構(gòu)；SND

短程硝化反硝化存在節(jié)省25%的曝氣能耗、減少40%的碳源投加量以及減少污泥產(chǎn)量等優(yōu)點(diǎn)[1-2],因此被廣泛地應(yīng)用于實(shí)際生活污水處理[3-6].短程硝化反硝化技術(shù)是將硝化反應(yīng)控制并維持在亞硝酸鹽階段,不進(jìn)行亞硝酸鹽至硝酸鹽的轉(zhuǎn)化[7].在這一過程中,亞硝酸鹽是必不可少的中間產(chǎn)物,積累率最高可達(dá)90%以上[2].當(dāng)前關(guān)于亞硝酸鹽對硝化作用的影響多圍繞對硝化菌,也就是氨氧化菌(AOB)和亞硝酸鹽氧化菌(NOB)的影響展開.有研究表明,超過一定濃度的亞硝酸鹽對微生物以及污泥沉降性能具有一定的影響[8-13],也有研究表明是亞硝酸鹽的質(zhì)子化產(chǎn)物游離亞硝酸(FNA)對微生物種群具有抑制作用,而非亞硝酸鹽[9,14].研究發(fā)現(xiàn)FNA對AOB,NOB,反硝化菌和厭氧氨氧化菌等均有影響[14-19],并且FNA對微生物的抑制作用還會影響到污泥中微生物菌群結(jié)構(gòu)的變化[20],利用這種抑制特性能夠?qū)崿F(xiàn)短程硝化[21-23].

隨著短程硝化反硝化研究和應(yīng)用的不斷深入,隨之而來的污泥膨脹問題也越來越突出.由于亞硝酸鹽的積累會使污泥的沉降性能惡化[11],這就導(dǎo)致無論是在實(shí)驗(yàn)室規(guī)模研究,還是在實(shí)際應(yīng)用中,污泥膨脹都時(shí)有發(fā)生.而現(xiàn)有研究中又缺乏FNA對絲狀菌影響的相關(guān)報(bào)道,并且關(guān)于活性污泥系統(tǒng)中微生物對FNA影響適應(yīng)性的研究也較少[14].針對這一現(xiàn)狀,本研究采用增加FNA預(yù)處理單元的方法,專門考察短程硝化污泥中FNA對絲狀菌和硝化菌等微生物菌群的抑制作用以及對這種抑制作用的適應(yīng)性.

1 材料與方法

1.1 試驗(yàn)裝置

試驗(yàn)裝置為SBR反應(yīng)器,由圓柱形有機(jī)玻璃制成,有效容積8L.反應(yīng)器上部固定電動攪拌器,底部裝有曝氣盤并連接轉(zhuǎn)子流量計(jì)和空氣壓縮機(jī),可直接調(diào)控溶解氧(DO)量,進(jìn)、排水口連接蠕動泵自動進(jìn)水、電動閥自動排水.溶氧儀裝有DO和pH值探頭,實(shí)時(shí)監(jiān)測反應(yīng)過程DO和pH值的變化.試驗(yàn)裝置見圖1.

圖1 試驗(yàn)裝置示意

1.貯水箱;2.蠕動泵;3.電動水閥;4.攪拌器;5.空氣泵;6.曝氣盤;7.WTW 溶氧儀;8.pH值探頭;9.DO 探頭;10.加熱棒;11.空氣轉(zhuǎn)子流量計(jì);12.FNA預(yù)處理單元

1.2 試驗(yàn)水質(zhì)及接種污泥

試驗(yàn)種泥為馴化良好的短程硝化污泥,沉降性能良好,SV在40%左右.進(jìn)水水質(zhì)為某高校家屬區(qū)實(shí)際生活污水,平均C/N<3.0為低碳氮比生活污水.水質(zhì)參數(shù)如表1所示.

表1 實(shí)際生活污水的水質(zhì)參數(shù)

1.3 試驗(yàn)運(yùn)行方案

第I 階段:污泥膨脹的誘發(fā)(1~40d)

將短程硝化污泥投加到SBR反應(yīng)器中,污泥濃度維持在2500~3000mg/L,排水比為50%,維持溫度在24℃左右.通過低DO(0.5~1.0mg/L),低負(fù)荷運(yùn)行條件來誘發(fā)污泥膨脹.運(yùn)行方式為進(jìn)水,好氧攪拌,沉淀30min,排水,每天運(yùn)行4個周期.運(yùn)行過程中,pH值隨著亞硝化反應(yīng)的進(jìn)行不斷下降,當(dāng)亞硝化過程完成后,曝氣作用將水中的CO2吹脫導(dǎo)致pH值上升,出現(xiàn)pH值的突變點(diǎn),此突變點(diǎn)即為氨谷點(diǎn).通過實(shí)時(shí)監(jiān)測pH值變化,來控制好氧曝氣時(shí)長,從而實(shí)現(xiàn)對氨谷點(diǎn)的實(shí)時(shí)控制.

第II階段:FNA預(yù)處理(41~43d)

從發(fā)生膨脹的SBR反應(yīng)器中取出全部泥水混合物,靜沉后去除上清液,污泥用去離子水離心(4000r/min,5min)洗絳3次消除污泥中NH4+和NO2－等干擾,用去離子水定容至1.0L,控制溫度為24℃,一次性投加NaNO2儲備液使NO2－濃度為5.1g/L,FNA濃度為1.2mgHNO2-N/L,缺氧攪拌4.5h,反應(yīng)進(jìn)行中通過投加0.1mol/L的HCl和NaOH控制pH值在7.0±0.05.反應(yīng)結(jié)束后,用去離子水離心洗泥3次.洗泥結(jié)束后投加泥到SBR反應(yīng)器中,運(yùn)行方式同第I階段.每天進(jìn)行一次,共運(yùn)行3d.FNA濃度根據(jù)公式(1)計(jì)算[24]:

(2)

式中:NO2－為亞硝酸鈉的濃度,mg/L;為反應(yīng)器內(nèi)的溫度,℃.

第III階段:反應(yīng)器正常運(yùn)行階段(44~72d)

溫度為室溫(20~21℃),與第I階段運(yùn)行方式相同,每天運(yùn)行4個周期,共運(yùn)行29d.

1.4 試驗(yàn)分析指標(biāo)及方法

水樣分析項(xiàng)目中NH4+,NO2-,NO3-使用Lachat QuikChem8000流動注射自動測定儀(Lachat Instruments, Milwaukee,USA).MLSS按國家標(biāo)準(zhǔn)方法測定[25].DO和pH值采用WTW溶解氧測定儀(Multi340i型)測定.絲狀菌普通鏡檢通過革蘭式染色法,所用儀器為OLYMPUS-BX61顯微鏡,并通過Image-Pro Plus軟件分析細(xì)菌的大小和形態(tài).采用Fast DNASpin Kit for Soil(QBIOgen Inc,Carlsba,CA,美國)DNA提取試劑盒提取反應(yīng)器活性污泥樣品的總DNA.MiSeq高通量測序?qū)嶒?yàn)流程包括:完成基因組DNA 提取,進(jìn)行PCR 擴(kuò)增,并將PCR 產(chǎn)物進(jìn)行檢測定量,構(gòu)建MiSeq文庫,最終進(jìn)行MiSeq測序并進(jìn)行微生物菌群結(jié)構(gòu)分析.

1.5 數(shù)據(jù)分析方法

本試驗(yàn)中亞硝酸鹽積累率的計(jì)算如式(3):

式中:NAR為亞酸鹽積累率,%;NO-為氮氧化物的濃度,mg/L;NO2-為亞硝酸鹽的濃度,mg/L;NO3-為硝酸鹽的濃度,mg/L.

2 結(jié)果與討論

2.1 FNA對污泥沉降性能的影響

圖2 FNA預(yù)處理前、后SV和SVI的變化情況

圖3 革蘭氏染色圖片

a)接種污泥革蘭氏染色圖片;b)膨脹污泥SVI值為280的革蘭氏染色圖片;c)停止FNA預(yù)處理,系統(tǒng)穩(wěn)定運(yùn)行SVI值為100的革蘭氏圖片

在第I階段短程硝化污泥接種初期,系統(tǒng)內(nèi)污泥的SVI為138mL/g左右(如圖2所示),由于采用低DO運(yùn)行結(jié)合低碳氮比進(jìn)水,絲狀菌在與菌膠團(tuán)細(xì)菌對營養(yǎng)物質(zhì)的競爭中處于優(yōu)勢而大量繁殖,在試驗(yàn)第15~40d,SVI逐漸上升,第40d達(dá)到281mL/g.圖3絲狀菌鏡檢可看出,種泥(圖3a)中只有少量的絲狀菌,菌膠團(tuán)結(jié)構(gòu)密實(shí),發(fā)生膨脹后(圖3b)大量絲狀菌增殖,菌絲從菌膠團(tuán)中伸出使菌膠團(tuán)結(jié)構(gòu)松散.由于絲狀菌的大量繁殖,活性污泥的沉降性能惡化導(dǎo)致污泥流失.第II階段的第1d,SVI就下降近50%,達(dá)到115mL/g,并在第III階段SVI穩(wěn)定維持在100mL/g左右.圖3c,較膨脹階段的污泥,第III階段污泥中的菌絲大量減少,污泥沉降性能良好,盡管采用與第I階段相同的運(yùn)行方式,污泥的沉降性能也沒有惡化.分析原因,認(rèn)為是FNA對優(yōu)勢絲狀菌與菌膠團(tuán)活性的抑制具有差異性,其中優(yōu)勢絲狀菌對FNA敏感度要高于菌膠團(tuán),所以當(dāng)優(yōu)勢絲狀菌活性受到FNA抑制時(shí),菌膠團(tuán)在營養(yǎng)物質(zhì)的攝取過程中具有優(yōu)勢而加快增殖,從而使污泥的沉降性能能夠得到改善.由此可見FNA對優(yōu)勢絲狀菌的抑制具有長期不可恢復(fù)性,并能夠有效改善污泥沉降性能惡化的問題.

2.2 FNA對NH4+-N、氮氧化物去除效果的影響

反應(yīng)器始終在低DO條件下運(yùn)行,由于AOB對氧的親和力較強(qiáng)[26-27],加之種泥短程硝化性能良好,所以從圖4可看出,第I階段氨氮去除率穩(wěn)定上升,達(dá)90%以上,NH4+-N平均出水在5mg/L左右, NO2--N積累率(NAR)維持在90%以上,短程硝化效果良好.第II階段中的第1d,AOB活性受到抑制,出水NH4+-N為13.79mg/L,相較于階段I增加了8.79mg/L.相關(guān)文獻(xiàn)表明,AOB的FNA抑制濃度為0.50~0.63mgHNO2-N/L[28-29],試驗(yàn)中FNA濃度為1.2mgHNO2-N/L,而從第III階段的第2d,AOB的活性開始逐漸恢復(fù),出水NH4+-N達(dá)到2~5mg/L,并在之后的運(yùn)行中穩(wěn)定維持在0~4mg/L,說明FNA對AOB活性的抑制具有短時(shí)性,當(dāng)停止FNA抑制后,AOB的活性能夠得以恢復(fù).其次種泥是短程硝化污泥,即污泥硝化菌AOB占有優(yōu)勢,而NOB含量低,且FNA對NOB抑制作用要強(qiáng)于AOB[28],加之低DO條件運(yùn)行,使得污泥中的NOB含量逐漸降低,變得微乎其微,所以在第III階段中,出水NO3-接近于0.06mg/L左右,從而污泥NAR能夠維持在95% 以上,并穩(wěn)定維持短程硝化,使得受到FNA抑制的污泥仍能夠保持良好的短程硝化的性能.

圖4 系統(tǒng)運(yùn)行不同階段NH4+-N,NO2－-N,NO3－-N濃度和NAR的變化

好氧運(yùn)行模式下,理論上只進(jìn)行到硝化階段無法進(jìn)行TN的去除.從圖4、5中可看出,第I階段TN去除率達(dá)到80%左右,是因?yàn)槲勰嘣诘虳O條件下,系統(tǒng)中存在好氧反硝化菌,發(fā)生了同步硝化反硝化(SND)[30],使TN得以去除.第III階段的第1d,TN去除率為0,之后開始升高,并在第15d之后最高達(dá)到60%左右,但始終低于第I階段.原因可能是FNA對好氧反硝化菌也具有短時(shí)抑制作用且活性無法得到完全恢復(fù)或污泥中出現(xiàn)能夠適應(yīng)高濃度FNA的好氧反硝化菌,但其反硝化能力要弱于第I階段的好氧反硝化菌.

圖5 系統(tǒng)運(yùn)行不同階段TN濃度和去除率的變化

2.3 活性污泥種群結(jié)構(gòu)的分析

為了探究污泥在3個階段微生物種群結(jié)構(gòu)的變化情況,分別取第0d種泥,第40d膨脹污泥和第72d污泥樣品,分別記為1,2,3,進(jìn)行MiSeq高通量測序技術(shù)分析.

從3個污泥樣品中分別獲得了30242,52902, 33531條優(yōu)化序列,有效序列平均長度為441,符合MiSeq高通量測序技術(shù)要求.3個泥樣分別為970, 1084,1102個OTUs.如表2所示,Ace,Chao,Sobs代表微生物菌群豐度指數(shù),可看出,2 號泥樣中微生物種群豐度有所上升,這有可能是絲狀菌的大量增殖或膨脹系統(tǒng)中出現(xiàn)了新微生物菌種導(dǎo)致.而3號泥樣豐度下降,也再次證明了FNA對微生物具有一定的抑制作用.相關(guān)文獻(xiàn)報(bào)道[22,31],微生物種群Shannon多樣性指數(shù),可用來估算污泥中微生物多樣性和均一性.2號泥樣的Shannon指數(shù)與種泥相比僅有輕微的下降,說明發(fā)生膨脹后,其微生物的多樣性與均一性變化可忽略不計(jì).但3號泥樣中微生物菌群的多樣性、均一性均下降,說明FNA對微生物菌群的多樣性與均一性產(chǎn)生抑制作用.

含有絲狀菌菌屬的3個菌門Bacteroidetes(擬桿菌門),Actinobacteria(放線菌門)和Chloroflexi(綠彎菌門),在污泥膨脹后分別由24.27%,5.5%,5.29%上升至27.60%,8.52%和12.2%.而第III階段Bacteroidetes下降幅度最大,由27.6%下降至7.67%, Chloroflexi也下降為原來的50%左右.圖6中,污泥膨脹后優(yōu)勢菌(微絲菌屬)和(噬纖維菌)分別由3.2% 上升到5.1%,由幾乎不可見增長到了1.1%,而第III階段,分別下降至0.78%和幾乎不可見.但絲狀菌(鏈球菌)在前2個階段中幾乎沒有,當(dāng)FNA處理后增長至3.1%.說明FNA對部分絲狀菌具有抑制作用尤其是優(yōu)勢菌和.

表2 不同階段反應(yīng)中污泥樣品多樣性指數(shù)統(tǒng)計(jì)

如圖7可知,Proteobacteria(變形菌門)在3個階段中均占有很大的優(yōu)勢,分別為39.44%,30.63%, 67.86%.Proteobacteria包括β綱,α綱,δ綱,γ綱,這4種綱中含有能夠進(jìn)行硝化反硝化菌屬,其中β綱,γ綱包括氨氧化菌屬,也正因如此3個階段中污泥具有良好的去除氮物質(zhì)的性能.污泥膨脹后Proteobacteria含量下降,主要因?yàn)榇藭r(shí)絲狀菌處于優(yōu)勢,使Proteobacteria在競爭底物中處于劣勢,導(dǎo)致其含量下降.第III階段Proteobacteria含量上升,因?yàn)镕NA對微生物具有抑制作用,使能夠適應(yīng)高濃度FNA的Proteobacteria菌屬占據(jù)優(yōu)勢并得以增殖.

圖6 MiSeq高通量測序序列在屬水平上的分布

圖7 MiSeq高通量測序序列在門水平上的分布

圖6中(陶厄氏菌屬)是β綱下能夠利用[32-34].污泥膨脹后由8.57%減少至0.52%,第III階段上升至5.58%.具有反硝化功能的兼性厭氧菌[35-36],第I階段和第III階段中分別為4.5%和7.82%,而第II階段僅占0.6%,即經(jīng)過FNA處理后,污泥中含量增加了7.22%.好氧反硝化菌(海生桿菌屬)[37]在種泥和第III階段中均幾乎不可見,而發(fā)生膨脹后,其含量增長至6.8%.由此可見,高濃度FNA對增長具有抑制作用且不可恢復(fù),而具有好氧反硝化功能的和能夠適應(yīng)高濃度FNA,并在第III階段中含量上升,這也是在第III階段中仍能發(fā)生SND的主要原因之一.但FNA對微生物種群結(jié)構(gòu)具有影響,以及和反硝化能力要弱于的原因,導(dǎo)致了第III階段中TN去除率要明顯低于第I階段的80%,僅維持在60%左右.

3 結(jié)論

3.1 對于短程硝化過程中發(fā)生的絲狀菌污泥膨脹,FNA對絲狀菌增殖有顯著的抑制作用.短程硝化污泥經(jīng)過FNA濃度為1.2mgHNO2-N/L,4.5h的預(yù)處理后,優(yōu)勢絲狀菌由5.1%下降至0.78%,由1.1%減少到幾乎不可見.SVI迅速從281mL/g降低到100mL/g左右,且NAR能夠維持在90%左右,短程硝化不受到破壞.

3.2 FNA對AOB和好氧反硝化菌均有抑制作用,當(dāng)FNA為1.2mgHNO2-N/L時(shí),對AOB的抑制作用是短時(shí)且可恢復(fù)的,但對的抑制作用具有不可恢復(fù)性.

3.3 FNA能夠影響微生物的種群結(jié)構(gòu),使得污泥中微生物的多樣性、均一性以及物種豐度下降.對高濃度FNA的抑制具有較強(qiáng)的適應(yīng)能力,在FNA處理結(jié)束之后,污泥中占微生物菌群的67.86%,污泥中具有反硝化功能的和含量上升,使在第III階段中仍能夠發(fā)生SND,保證了良好的TN去除效果.

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Effect of FNA on microorganism community structures of partial nitrification sludge.

GAO Chun-di*, ZHAO Nan, AN Ran, HAN Hui, ZHANG Na, PENG Yong-zhen

(National Engineering Laboratory for Advanced Municipal Wastewater Treatment and Reuse Technology, College of Environmental and Energy Engineering, Beijing University of Technology, Beijing 100124, China)., 2019,39(5)：1977~1984

The long-term effects on ammonia-oxidizing bacteria (AOB), filamentous bacteria and microorganism community structures in partial nitrification sludge was investigated by adding a pretreatment unit of free nitrous acid (FNA) in the sequence bath reactor (SBR) for three days at 1.2mg HNO2-N/Lfor 4.5. The results showed that FNA had the short-time effect on AOB,andof the dominant filamentous bacteria were also decreased from 5.1% and 1.1% to 0.78% and almost invisible. Sludge volume index (SVI) maintained at 110mL/g dropping from 281mL/g, and the nitrite accumulation rate (NAR) was kept at around 90%, indicating the partial nitrification was not undermined. Furthermore, High-throughput sequencing results showed that the diversity and uniformity of microorganism community decreased. However, the proliferation ofandincreased to 5.58% and 7.82%. Simultaneous nitrification and denitrification (SND) had significant effects, and the total nitrogen removal rate was nevertheless maintained more than 60% even only with partial nitrification.

partial nitrification；sludge bulking；sludge settleability；filamentous bacteria；AOB；microorganism community structures；SND

X172

A

1000-6923(2019)05-1977-08

高春娣(1973-),女,河北唐山人,教授,博士,主要研究方向?yàn)槌擎?zhèn)污水深度處理與資源化利用,絲狀菌污泥膨脹機(jī)制與控制.發(fā)表論文38篇.

2018-10-15

國家自然科學(xué)基金資助項(xiàng)目(51478012);北京市科技重大專項(xiàng)(Z181100005318001)

*責(zé)任作者, 教授, gaochundi@bjut.edu.cn