戴曉虎,于春曉,李 寧,董 濱 (同濟(jì)大學(xué)環(huán)境科學(xué)與工程學(xué)院,污染控制與資源化研究國家重點(diǎn)實(shí)驗(yàn)室,上海200092)

水污染與控制

污泥超高溫(65℃)厭氧消化系統(tǒng)啟動方案

戴曉虎,于春曉,李 寧*,董 濱 (同濟(jì)大學(xué)環(huán)境科學(xué)與工程學(xué)院,污染控制與資源化研究國家重點(diǎn)實(shí)驗(yàn)室,上海200092)

以高溫(55℃)厭氧消化反應(yīng)器的污泥為接種泥,以不同比例的牛糞和脫水污泥為基質(zhì),通過產(chǎn)甲烷潛力測試實(shí)驗(yàn),對污泥超高溫(65℃)厭氧消化系統(tǒng)的啟動策略進(jìn)行了初步的探討.實(shí)驗(yàn)結(jié)果表明:污泥超高溫(65℃)厭氧消化系統(tǒng)具有其可行性;65℃條件下,由于水解酸化過程加快,易發(fā)生 VFAs(尤其是乙酸和丙酸)的累積.同時,與中溫(37℃)和高溫(55℃)污泥厭氧消化系統(tǒng)相比,超高溫(65℃)系統(tǒng)的產(chǎn)氣量雖然較低,但所產(chǎn)沼氣中CH4含量明顯升高,可以達(dá)到79.0%.對系統(tǒng)細(xì)菌和古菌進(jìn)行的多樣性分析結(jié)果表明:超高溫(65℃)條件下,反應(yīng)器中的細(xì)菌以Coprothermobacter、Caldicoprobacter、Ruminiclostridium等極端嗜熱的蛋白質(zhì)水解菌和木質(zhì)纖維素水解菌為主,不同反應(yīng)器之間細(xì)菌種群多樣性的差異是由所投加物料的不同造成的;所有反應(yīng)器的古菌中,嗜熱的氫營養(yǎng)型產(chǎn)甲烷菌 Methanothermabactor成為絕對優(yōu)勢菌群,占古菌的比例均超過96%.在超高溫反應(yīng)器(65℃)的啟動初期,可適當(dāng)提高投加基質(zhì)中牛糞的比例,加快對嗜熱產(chǎn)甲烷菌(氫利用型產(chǎn)甲烷菌)的富集,同時避免系統(tǒng)中的VFAs的積累,保證反應(yīng)器順利啟動.

超高溫；厭氧消化；啟動策略；甲烷嗜熱桿菌

厭氧消化技術(shù)作為一種在實(shí)現(xiàn)污泥穩(wěn)定化同時可產(chǎn)生綠色能源-沼氣的技術(shù),一直被認(rèn)為是污泥處理處置的合適選擇之一[1].厭氧消化系統(tǒng)一般在中溫(37℃)和高溫(55℃)條件下運(yùn)行.然而,中溫厭氧消化系統(tǒng)中由于受到水解步驟的限制,有機(jī)顆粒物的降解率較低,所需的 SRT較長[2].即便在高溫厭氧消化(55℃)系統(tǒng)中,微生物的降解潛能也仍沒有得到充分地釋放,有機(jī)物降解率也僅有 50%左右[3].因此,污泥厭氧消化的產(chǎn)氣潛能還有較大的提高空間[4],其關(guān)鍵是提高污泥中有機(jī)物的水解效率[5].Nielsen等[6]與Scherer等[7]通過污泥厭氧消化的對比研究發(fā)現(xiàn),與普通的中溫(37℃)厭氧消化相比,高溫(55℃)和超高溫(超過 55℃)厭氧消化系統(tǒng)更有利于提高有機(jī)顆粒溶解性及沼氣產(chǎn)量.這主要是由于水解酸化菌生長的最適溫度為 55～70℃[8],當(dāng)厭氧消化系統(tǒng)的溫度從55℃上升到65℃時,污泥中蛋白質(zhì)等難降解有機(jī)物的溶解性會顯著提高,傳質(zhì)速率加快[2].同時,嗜高溫的氫利用型產(chǎn)甲烷菌的最佳生存溫度是 55～70℃[9],如若將厭氧消化的溫度提升到65℃,氫利用型產(chǎn)甲烷途徑的效率將有可能達(dá)到最大[10].

污泥熱水解是一種能夠加快污泥水解速率,提高甲烷產(chǎn)率的前處理技術(shù),近年來在實(shí)際工程中得到了廣泛的應(yīng)用[11-12].熱水解后污泥溫度較高,利用熱水解污泥中的剩余熱量,進(jìn)行超高溫厭氧消化,能提高顆粒狀污泥的溶解性和降解速率以及嗜高溫的水解酸化微生物活性,并富集嗜高溫的氫利用型產(chǎn)甲烷菌以提高產(chǎn)氣效率[4].而且,氫利用型產(chǎn)甲烷途徑的充分利用,能夠減少 CO2排放,節(jié)省以乙酸等短鏈脂肪酸形式存在的碳源,方便對碳源進(jìn)行后續(xù)的綜合利用.此外,當(dāng)溫度上升至60℃以上時,污泥中的膠體物質(zhì)降解率提高,絲狀菌被殺死,能夠明顯提高污泥的脫水性[13];同時,污泥中的致病菌也能夠被殺滅,初步滿足污泥處理處置無害化的要求[14].

目前,國內(nèi)外關(guān)于超高溫厭氧消化的研究主要集中于牛糞或餐廚垃圾的可行性探索,對于系統(tǒng)中酸化和甲烷化過程的基礎(chǔ)研究較少[15],純污泥體系的超高溫厭氧消化系統(tǒng)的相關(guān)研究更是幾近空白.本研究主要通過產(chǎn)甲烷潛力測試實(shí)驗(yàn)(BMP),對超高溫條件下反應(yīng)器不同啟動方案中的產(chǎn)氣性能和各項(xiàng)指標(biāo)進(jìn)行分析對比,以確定超高溫厭氧消化反應(yīng)器的最佳啟動條件,為污泥超高溫厭氧消化反應(yīng)器的啟動提供理論依據(jù).

1 材料與方法

1.1 實(shí)驗(yàn)材料

接種牛糞取自上海某生態(tài)園,取回后為了保證微生物的活性在 35℃下保存,并于 2d內(nèi)完成接種;脫水污泥取自上海某污水處理廠,于 4℃下保存;所使用的接種泥取自本實(shí)驗(yàn)室穩(wěn)定運(yùn)行的高溫(55℃)厭氧消化反應(yīng)器.上述物料的基本參數(shù)如表1所示.使用前用去離子水分別將牛糞和脫水污泥稀釋至含固率(TS)為8%.

表1 牛糞、脫水污泥和接種泥的相關(guān)參數(shù)Table 1 Characteristics of cow dung, dewatered sludge and inoculum

1.2 BMP實(shí)驗(yàn)設(shè)計(jì)

實(shí)驗(yàn)分2個批次進(jìn)行.BMP實(shí)驗(yàn)通過全自動產(chǎn)甲烷潛力測試儀(AMPTS II,bioprocess control,瑞典)完成.第一批次的BMP實(shí)驗(yàn)主要為了完成超高溫甲烷菌的富集并初步探索超高溫厭氧消化反應(yīng)器啟動的可行性,第二批實(shí)驗(yàn)在第一批BMP實(shí)驗(yàn)的基礎(chǔ)上,對超高溫厭氧消化反應(yīng)器的啟動過程的穩(wěn)定性和微生物種群分布特征進(jìn)行了研究.

表2 實(shí)驗(yàn)設(shè)計(jì)參數(shù)Table 2 Designed experimental parameters

第一批次的實(shí)驗(yàn)以穩(wěn)定運(yùn)行的污泥高溫(55℃)厭氧消化反應(yīng)器的出料為接種泥.第二批次的接種泥為第一批次超高溫(65℃)厭氧消化反應(yīng)器的出料.第一批次實(shí)驗(yàn)的基質(zhì)為牛糞和污泥的混合物,接種比(接種泥:基質(zhì)(VS:VS))為1:1,反應(yīng)器為有效容積600mL的雙孔塞玻璃瓶.實(shí)驗(yàn)設(shè)置空白對照組和實(shí)驗(yàn)組,空白對照組的反應(yīng)器中僅添加接種泥,實(shí)驗(yàn)組所投加基質(zhì)設(shè)置為不同比例的牛糞和污泥,具體參數(shù)如表2所示.

反應(yīng)器用雙孔塞和止水夾密封,啟動前用真空泵抽真空后,用氮?dú)獯得?min以進(jìn)一步去除上層空間和溶液中的空氣.實(shí)驗(yàn)設(shè)3組平行,結(jié)果分析取其平均值.反應(yīng)器置于 65℃水浴鍋中,攪拌設(shè)置為5min工作,5min停止,在反應(yīng)器運(yùn)行過程中,采集累積甲烷產(chǎn)量和產(chǎn)甲烷速率的數(shù)據(jù).

1.3 測試方法

全自動產(chǎn)甲烷潛力測試儀能測定并記錄累積甲烷產(chǎn)量和產(chǎn)甲烷速率.實(shí)驗(yàn)過程中以集氣袋收集氣體,通過氣相色譜(GC112A,上海儀電,中國)測定氣體成分.實(shí)驗(yàn)結(jié)束后反應(yīng)器中樣品的pH 值通過pH計(jì)(S210,梅特勒,瑞士)測定.樣品取出并稀釋離心后,上清液經(jīng)0.45 μm濾膜過濾,測定其中的揮發(fā)性脂肪酸 (Volatile Fatty Acids, VFAs) (GC 2010plus,島津,日本)和堿度(自動電位滴定儀 G20,梅特勒,瑞士).氨氮含量通過使用凱氏定氮儀(9860,海能,中國)由滴定法測定.

1.4 微生物種群結(jié)構(gòu)分析

反應(yīng)器出料于 2mL凍存管中,于-80℃冷凍保存.DNA 樣品使用土壤 DNA 提取試劑盒(PowerSoil,MO BIO, USA)提取.提取出的 DNA經(jīng)過 PCR 擴(kuò)增,采用高通量測序(Miseq4000, Illumina)分析其中細(xì)菌與古菌的種群結(jié)構(gòu).PCR產(chǎn)物先利用QuantiFLuorTM系統(tǒng)(Promega)測定濃度,隨后依據(jù)AxyPrep DNA 試劑盒(AXYGEN, USA)的凝膠回收的方法進(jìn)行提純[16].提純的PCR 產(chǎn)物的質(zhì)量通過凝膠電泳確定.引物設(shè)計(jì)根據(jù)Illumina 公司(San Diego, California, USA)的操作手冊,針對細(xì)菌和古菌的 16s rRNA基因的通用擴(kuò)增引物對分別為 338f(5'-ACTCCTACGGGAGGCAGCA-3')/806r(5'-GGACTACHV GGGTWTCTAAT-3')[17]和 524f(5'-TGYCAGCCGCCGCGGTAA-3')/958r(5'-YCCGGCGTTGAVTCCAATT-3')[18].

2 結(jié)果與討論

2.1 厭氧消化產(chǎn)氣性能

2.1.1 累積甲烷產(chǎn)量 圖1為第一批次BMP實(shí)驗(yàn)中各反應(yīng)器的產(chǎn)甲烷情況,從圖1可以看出:各反應(yīng)器的產(chǎn)甲烷過程均順利進(jìn)行,在第14～16d達(dá)到了累積甲烷產(chǎn)量的最大值,說明超高溫(65℃)厭氧消化反應(yīng)器的啟動是可行的.且牛糞投加比例高的反應(yīng)器的啟動速度和累積甲烷產(chǎn)量明顯高于污泥投加比例高的反應(yīng)器,這主要由于牛糞中所存在的嗜熱甲烷菌(氫利用型產(chǎn)甲烷菌)能夠快速適應(yīng)65℃環(huán)境[19],保證了厭氧消化產(chǎn)甲烷過程的順利啟動和進(jìn)行.因此,在反應(yīng)器啟動初期,適當(dāng)增加牛糞的比例能夠加快嗜熱產(chǎn)甲烷菌的富集,保證反應(yīng)器快速穩(wěn)定啟動.

圖1 第一批次實(shí)驗(yàn)累積甲烷產(chǎn)量Fig.1 Cumulative methane production of the first batch experiments

圖2為第二批次BMP實(shí)驗(yàn)中各反應(yīng)器的累積產(chǎn)甲烷情況.可以看出,和第一批次相比,各實(shí)驗(yàn)組的產(chǎn)甲烷速率和累積甲烷產(chǎn)量均明顯上升,說明第一批次BMP實(shí)驗(yàn)有效地實(shí)現(xiàn)了嗜熱產(chǎn)甲烷菌的初次富集,使體系中嗜熱產(chǎn)甲烷菌的含量上升,再次確認(rèn)了超高溫厭氧消化反應(yīng)系統(tǒng)啟動的可行性.但是,即使在第二批次BMP實(shí)驗(yàn)中,各反應(yīng)器的累積甲烷產(chǎn)量也只達(dá)到了600～800mL,低于相同條件下的中溫和高溫厭氧消化甲烷產(chǎn)量(約1200～1400mL).

2.1.2 氣體成分 在 65℃條件下,厭氧消化所產(chǎn)沼氣中,CH4含量在60%～80%之間(圖3),明顯高于中溫(37℃)和高溫(55℃)條件下的 CH4含量(48%～65%)[20].沼氣中的 CO2含量在 20%～26%,低于中溫(37℃)和高溫(55℃)條件下的CO2含量(36%～41%)[20],這證明:氫利用型產(chǎn)甲烷途徑在65℃條件下得到了強(qiáng)化,成為主要的產(chǎn)甲烷途徑.另外,雖然隨著投加物料中污泥比例的升高,產(chǎn)甲烷速率在一定程度上減慢,但CH4含量逐漸升高,純污泥作為基質(zhì)的反應(yīng)器中,CH4含量甚至可以達(dá)到 79.0%,表明超高溫條件下氣體成分的差異與牛糞和污泥的物質(zhì)組成相關(guān).

圖2 第二批次實(shí)驗(yàn)累積甲烷產(chǎn)量Fig.2 Cumulative methane production of the second batch experiments

Fig.3 第二批次實(shí)驗(yàn)各反應(yīng)器產(chǎn)氣中CH4和CO2含量Fig.3 Methane and carbon dioxide content in biogas of each reactor of the second batch experiments

2.2 液相指標(biāo)

2.2.1 VFAs 圖4為第一批次各樣品中VFAs的濃度和組成情況,可以看出,各反應(yīng)器均發(fā)生了較嚴(yán)重的VFAs累積,達(dá)到了1900～4000mg/L.當(dāng)溫度上升至 65℃時,達(dá)到水解酸化菌的最適溫度(55～75℃)[6],水解酸化過程加快,VFAs快速產(chǎn)生,但是,在此溫度條件下,乙酸利用型產(chǎn)甲烷菌的活性降低甚至喪失,導(dǎo)致乙酸向甲烷的轉(zhuǎn)化過程被抑制,造成了VFA快速累積[14].

圖4 第一批次各反應(yīng)器中的VFAs濃度及組成(產(chǎn)氣終止時取樣)Fig.4 The concentrations and compositions of VFAs in each reactor of the first batch experiments (sampled when gas production stopped)

如圖5所示,與第一批次實(shí)驗(yàn)中的反應(yīng)器相比,第二批次實(shí)驗(yàn)的反應(yīng)器中 VFAs累積更加明顯(表3),說明反應(yīng)器中的水解酸化過程繼續(xù)加強(qiáng),其中丙酸的累積最明顯,且隨著污泥比例的升高,累積程度也相應(yīng)略微升高.丙酸的嚴(yán)重累積可能是由于:在 65℃條件下,微生物種群結(jié)構(gòu)發(fā)生改變,乙酸利用型產(chǎn)甲烷菌失活.與此同時,嗜熱的氫利用型產(chǎn)甲烷菌成為優(yōu)勢菌群.在氫利用型產(chǎn)甲烷途徑為主的情況下,乙酸氧化菌將乙酸轉(zhuǎn)化為H2/CO2,在氫利用型產(chǎn)甲烷菌的作用下轉(zhuǎn)化為甲烷[21].乙酸氧化也會導(dǎo)致氫分壓上升,使得丙酸氧化微生物活性下降,從而導(dǎo)致丙酸降解過程被抑制[22].而且,乙酸是丙酸的降解產(chǎn)物[23],反應(yīng)器中乙酸的累積也會影響丙酸的降解速率[24-25],并進(jìn)一步出現(xiàn)丙酸累積抑制甲烷菌生長的情況[26].由于牛糞和污泥的組成成分不同,牛糞中含有較多難生物降解的木質(zhì)纖維素類物質(zhì),升溫后仍不易降解,但污泥的主要成分是蛋白質(zhì),隨著溫度的上升,其溶解性和降解率提高[3],因此,投加了污泥的反應(yīng)器(#2和#3)中,VFAs 累積更加嚴(yán)重,反應(yīng)器的啟動速率慢,甲烷產(chǎn)量低.

因此,在超高溫厭氧消化反應(yīng)器的啟動初期,為避免 VFAs尤其是丙酸的大量累積,盡快完成嗜熱產(chǎn)甲烷菌的富集,保證反應(yīng)器順利啟動,宜采用較高比例的牛糞作為進(jìn)料.污泥:牛糞(VS: VS)=1:1作為啟動初期的投加方案是具有其可行性的.

表3 各反應(yīng)器運(yùn)行性能參數(shù) (產(chǎn)氣終止時取樣)Table 3 The performance characteristics in each digester (sampled when gas production stopped)

圖5 第二批次各反應(yīng)器中的VFAs濃度及組成(產(chǎn)氣終止時取樣)Fig.5 The concentrations and compositions of VFAs in each digester of the second batch experiments (sampled when gas production stopped)

2.2.2 氨氮在本實(shí)驗(yàn)中,各反應(yīng)器中的氨氮濃度均在 2000～2500mg/之間,遠(yuǎn)低于氨抑制濃度(5880～6000mg/L),因此,在所實(shí)驗(yàn)的 TS和溫度(65℃)條件下,氨抑制情況并不容易發(fā)生[27].而且,由于牛糞中木質(zhì)纖維素的含量較高[3],牛糞投加比例較高的反應(yīng)器中物料碳氮比較高,氨氮濃度相對較低.

2.2.3 pH值和堿度 兩個批次BMP實(shí)驗(yàn)中的反應(yīng)器的pH值分別為7.7～7.9和7.6～7.7,略高于產(chǎn)甲烷菌的最佳pH值范圍(6.8～7.2)[20].

堿度能夠反映厭氧消化系統(tǒng)的緩沖能力[28].如表3所示，BMP實(shí)驗(yàn)中各組反應(yīng)器中的堿度均在 4000～6000mg/L(以 CaCO3計(jì))之間,且隨著污泥比例的增加,系統(tǒng)堿度(尤其是第一批 BMP實(shí)驗(yàn)中)基本呈現(xiàn)上升趨勢.這主要由于與牛糞相比,污泥中的蛋白質(zhì)含量較高,蛋白質(zhì)降解過程中會產(chǎn)生較高濃度的游離氨,提高系統(tǒng)堿度[20].較強(qiáng)的緩沖性能使得各反應(yīng)器在較高濃度的VFAs累積的情況下,沒有發(fā)生pH值明顯降低的現(xiàn)象.

VFAs/堿度是表征厭氧消化系統(tǒng)穩(wěn)定性的參數(shù):當(dāng) VFAs/堿度值小于 0.4時,系統(tǒng)穩(wěn)定,當(dāng)VFAs/堿度值在0.4～0.8之間時,系統(tǒng)可能會發(fā)生失穩(wěn)現(xiàn)象;當(dāng)VFA/堿度值大于0.8時,系統(tǒng)嚴(yán)重失穩(wěn)[29-30].兩批BMP實(shí)驗(yàn)中各反應(yīng)器的VFAs/堿度值均在0.4～0.8之間,這說明,在目前的TS和實(shí)驗(yàn)條件下,反應(yīng)器是不穩(wěn)定的,且隨著污泥投加量的增加,反應(yīng)失穩(wěn)的可能性增大.與第一批次 BMP實(shí)驗(yàn)相比,第二批次實(shí)驗(yàn)中,各反應(yīng)器的穩(wěn)定性有提高,這說明,對嗜熱微生物的富集是有效的,反應(yīng)器啟動初期增加牛糞的投加量和富集次數(shù),能夠提高反應(yīng)器的穩(wěn)定性,保證反應(yīng)器穩(wěn)定啟動.

2.3 微生物種群結(jié)構(gòu)分析

2.3.1 細(xì)菌種群結(jié)構(gòu) 圖 6為各個反應(yīng)器中細(xì)菌多樣性分析結(jié)果:在各反應(yīng)器中,細(xì)菌種群均呈現(xiàn)明顯的多樣性,但各反應(yīng)器中細(xì)菌種群的分布差異比較明顯.#1反應(yīng)器中,Caldicoprobacter、Ruminiclostridium、 OPB54_norank、 D8A-2_norank和Coprothermobacter五種細(xì)菌含量均較高,分別達(dá)到了細(xì)菌總量的16.93%、13.90%、13.10%、9.90%和 9.62%.#2 反應(yīng)器的優(yōu)勢菌種為Caldicoprobacte和Coprothermobacter,分別占細(xì)菌的 18.74%、17.51%.#3 反應(yīng)器中, Coprothermobacter成為了最主要的優(yōu)勢菌種,所占比例達(dá)到了 21.85%.Coprothermobacter、Caldicoprobacter、Ruminiclostridium都是極端嗜熱的水解菌[15,31-32],而多存在于中溫和高溫反應(yīng)器中的水解酸化菌,如:Bacteroidetes 和Proteobacteria[33]在超高溫反應(yīng)器中沒有檢測到,Firmicutes所占的比例很低,僅1.5%～3.5%,這說明,與中溫和高溫反應(yīng)器相比,超高溫條件下,細(xì)菌的種群結(jié)構(gòu)發(fā)生了明顯的改變,極端嗜熱的細(xì)菌成為優(yōu)勢菌種.隨著反應(yīng)器中所投加污泥比例的升高,Coprothermobacter的種群優(yōu)勢逐漸增大,而其比例的變化與牛糞和污泥的組成成分相關(guān).Coprothermobacter是一種極端嗜熱的蛋白質(zhì)水解菌,在厭氧消化過程中主要參與蛋白質(zhì)和多肽的降解,在70℃仍能存活[15,34-35],在牛糞厭氧消化反應(yīng)器[36]和污泥高溫厭氧消化反應(yīng)器[37-38]中均有發(fā)現(xiàn),牛糞厭氧消化反應(yīng)器中Coprothermobacter的含量較低[39].與牛糞相比,污泥中蛋白質(zhì)含量較高,因此,在#3反應(yīng)器中,Coprothermobacter的所占細(xì)菌的比例要明顯高于其在#1號反應(yīng)器中的比例.

Caldicoprobacter是一種嚴(yán)格厭氧的嗜熱菌,最適生長溫度為 65℃(55～75℃),最適生長的 pH值為6.9(6.2～8.3),是污泥高溫厭氧消化反應(yīng)器重要的水解菌[31,40].Caldicoprobacter能夠產(chǎn)生耐高溫的木聚糖酶,一般參與半纖維素(木聚糖等)的降解[41].Ruminiclostridium是一種嗜熱厭氧菌,最佳的生長溫度為55 ～60℃,pH值為 7.3～7.5,能夠合成纖維素降解酶,降解木質(zhì)纖維素類物質(zhì)(纖維素、半纖維素和植物細(xì)胞壁多糖等),普遍存在于纖維素類物質(zhì)的堆肥中[32,42-44].牛糞中纖維素和半纖維素等物質(zhì)的含量較高,因此,在投加了牛糞的#1和#2反應(yīng)器中,Caldicoprobacter、Ruminiclostridium所占比例均較高.

由于Coprothermobacter、Caldicoprobacter、Ruminiclostridium的水解終產(chǎn)物均為乙酸、H2和CO2等,這些的細(xì)菌的存在有利于促進(jìn)氫利用型產(chǎn)甲烷菌的活性,從而促進(jìn) CO2/H2產(chǎn)甲烷途徑[36,40,44].

另外,Intrasporangiaceae、Tepidimicrobium、Proteiniphilum等均存在于3個反應(yīng)器中,這些細(xì)菌大多是嗜熱的蛋白質(zhì)和碳水化合物水解菌[45-47],能利用多種碳源,因此這些細(xì)菌的含量在各個反應(yīng)器中的差別并不明顯.

圖6 各反應(yīng)器中的細(xì)菌相對豐度對比Fig.6 the relative abundance of bacteria in each digester

綜上所述,在超高溫(65℃)條件下,3個反應(yīng)器中細(xì)菌主要為極端嗜熱的蛋白質(zhì)和木質(zhì)纖維素水解菌,不同反應(yīng)器中細(xì)菌種群多樣性的差異部分來源于牛糞和污泥中本身所含細(xì)菌種類的不同,本質(zhì)上是由牛糞和污泥的組成成分的不同決定的.

2.3.2 古菌種群結(jié)構(gòu) 圖 7為第二批次實(shí)驗(yàn)中各反應(yīng)中的古菌多樣性分析結(jié)果.可以看出,與細(xì)菌相比,各反應(yīng)器中古菌的種類較少,且分布情況相近,經(jīng)過二次富集后,各反應(yīng)器中的古菌均以嗜熱產(chǎn)甲烷菌Methanothermabactor為主,所占比例超過 96%.這說明,Methanothermabactor在牛糞和高溫厭氧消化污泥中都存在,這與Chachkhiani[48]和Zeikus[49]等的結(jié)論一致.且在超高溫條件(65℃)下,Methanothermabactor能夠被富集,最終成為優(yōu)勢菌群.Methanothermabactor是一種氫利用性產(chǎn)甲烷菌,利用H2和CO2途徑產(chǎn)甲烷,最佳pH值范圍為7.2～7.6,最適溫度范圍在65～70℃[49-50].各反應(yīng)器終止時的 pH 值接近Methanothermabactor的最適 pH值范圍,均在7.6～7.7之間,保證了過程中Methanothermabactor的生長.而中溫和高溫厭氧消化反應(yīng)器中普遍存在的乙酸利用型產(chǎn)甲烷菌 Methanosaeta[51-52]所占的比例很低,分別只占 0.38%、1.86%和2.61%,3個反應(yīng)器中均未檢測到Methanosarcina.這為反應(yīng)器中以氫利用型產(chǎn)甲烷過程為主的甲烷化過程提供了直接證據(jù),而乙酸利用型產(chǎn)甲烷途徑幾乎終止,從而導(dǎo)致乙酸等揮發(fā)性脂肪酸大量累積.另外,與投加生污泥的反應(yīng)器相比,投加牛糞的反應(yīng)器中Methanosaeta的含量更低. Methanothermabactor的成功富集保證了反應(yīng)器中產(chǎn)甲烷過程的順利進(jìn)行,為超高溫厭氧消化系統(tǒng)順利啟動提供了條件.

圖7 各反應(yīng)器中的古菌相對豐度對比Fig.7 The abundance of archaea in each digester

在污泥超高溫(65℃)厭氧消化系統(tǒng)中,CO2/ H2產(chǎn)甲烷途徑成為主要的產(chǎn)甲烷途徑,乙酸產(chǎn)甲烷途徑幾乎不再進(jìn)行,在實(shí)現(xiàn)CO2減排的同時,能夠節(jié)省以短鏈脂肪酸為代表的有機(jī)碳源.這些富含短鏈脂肪酸的厭氧消化沼液可以作為碳源,補(bǔ)充到污水處理的反硝化池中,促進(jìn)脫氮除磷,節(jié)省污水處理的成本,提高污水處理效果,實(shí)現(xiàn)碳源的綜合利用.

3 結(jié)論

3.1 研究證實(shí)了污泥超高溫(65℃)厭氧消化反應(yīng)器啟動的可行性,在進(jìn)料中混合投加含有嗜熱產(chǎn)甲烷菌(氫利用型產(chǎn)甲烷菌)的牛糞能夠加快反應(yīng)器的啟動速率.

3.2 65℃條件下,污泥中蛋白質(zhì)等有機(jī)顆粒的溶解性提高,水解酸化菌的活性提高,易造成VFAs累積,反應(yīng)器啟動初期,宜通過逐步降低牛糞比例的方式來保證反應(yīng)器啟動過程的穩(wěn)定性,研究中所采用的方案-牛糞:污泥(VS:VS)=1:1可以作為啟動的初始投加方案.

3.3 與中溫(37℃)和高溫(55℃)污泥厭氧消化相比,超高溫(65℃)的產(chǎn)氣量較低,但所產(chǎn)生物氣中CH4含量高79.0%,CO2含量低(20.0%).

3.4 超高溫(65℃)條件下,各反應(yīng)器中的細(xì)菌以嗜熱的木質(zhì)纖維素和蛋白質(zhì)的水解菌為主,包括 Coprothermobacter、Caldicoprobacter、Ruminiclostridium,不同反應(yīng)器之間細(xì)菌種群多樣性的差異主要由所投加物料的不同造成.氫利用型產(chǎn)甲烷菌Methanothermabactor成為主要的產(chǎn)甲烷菌,占各反應(yīng)器的甲烷菌的總量中占 96%以上,而乙酸利用型產(chǎn)甲烷菌 Methanosaeta、Methanosarcina等已經(jīng)幾乎不存在了.

[1] 李建華,劉文靜,李 寧.沼液中溶解游離氨基酸的測定——柱前衍生-反相高效液相色譜法 [J]. 中國環(huán)境科學(xué), 2016,36(8): 2355-2363.

[2] Lee M Y, Cheon J H, Hidaka T, et al. The performance and microbial diversity of temperature- phased hyperthermophilic and thermophilic anaerobic digestion system fed with organic waste [J]. Water Science and Technology, 2008:283-289.

[3] Nielsen H B, Mladenovska Z, Westermann P, et al. Comparison of two-stage thermophilic (68℃/55 ℃) anaerobic digestion with one-stage thermophilic (55℃) digestion of cattle manure [J]. Biotechnology and Bioengineering, 2004,86(3):291-300.

[4] Lu J, Gavala H N, Skiadas I V, et al. Improving anaerobic sewage sludge digestion by implementation of a hyper-thermophilic prehydrolysis step [J]. Journal of Environmental Management, 2008,88(4):881-889.

[5] Frolund B, Griebe T, Nielsen P H. Enzymatic activity in the activated-sludge floc matrix [J]. Applied Microbiology and Biotechnology, 1995,43(4):755-761.

[6] Nielsen B, Petersen G. Thermophilic anaerobic digestion and pasteurisation. Practical experience from Danish wastewater treatment plants [J]. Water Science and Technology, 2000,42(9): 65-72.

[7] Scherer P A, Vollmer G R, Fakhouri T, et al. Development of a methanogenic process to degrade exhaustively the organic fraction of municipal “grey waste” under thermophilic and hyperthermophilic conditions [J]. Water Science and Technology,2000,41(3):83-91.

[8] Wiegel J. Temperature spans for growth: hypothesis and discussion [J]. FEMS Microbiology Reviews, 1990,6(2/3):155-169.

[9] Wasserfallen A, N?lling J, Pfister P, et al. Phylogenetic analysis of 18 thermophilic Methanobacterium isolates supports the proposals to create a new genus, Methanothermobacter gen. nov., and to reclassify several isolates in three species, Methanothermobacter ther mautotrophicus comb. nov., Methanothermobacterwolfeii comb. nov., and Methanothermobacter marburgensis sp. nov [J]. International Journal of Systematic and Evolutionary Microbiology, 2000,50(1):43-53.

[10] Ahring B K. Status on science and application of thermophilic anaerobic digestion [J]. Water Science and Technology, 1994, 30(12):241-249.

[11] Souza T S O, Ferreira L C, Sapkaite I, et al. Thermal pretreatment and hydraulic retention time in continuous digesters fed with sewage sludge: assessment using the ADM1 [J]. Bioresource Technology, 2013,148:317-324.

[12] Xue Y, Liu H, Chen S, et al. Effects of thermal hydrolysis on organic matter solubilization and anaerobic digestion of high solid sludge [J]. Chemical Engineering Journal, 2015,264:174-180.

[13] Wang F, Hidaka T, Tsuno H, et al. Co-digestion of polylactide and kitchen garbage in hyperthermophilic and thermophilic continuous anaerobic process [J]. BioresourceTechnology, 2012, 112:67-74.

[14] Ahring B K, Ibrahim A A, Mladenovska Z. Effect of temperature increase from 55 to 65℃ on performance and microbial population dynamics of an anaerobic reactor treating cattle manure [J]. Water Research, 2001,35(10):2446-2452.

[15] Lee M, Hidaka T, Hagiwara W, et al. Comparative performance and microbial diversity of hyperthermophilic and thermophilic co-digestion of kitchen garbage and excess sludge [J]. BioresourceTechnology, 2009,100(2):578-585.

[16] 戴曉虎,何 進(jìn),嚴(yán) 寒,等.游離氨調(diào)控對污泥高含固厭氧消化反應(yīng)器性能的影響 [J]. 環(huán)境科學(xué), 2017,38(2):679-687.

[17] Lee C K, Barbier B A, Bottos E M, et al. The Inter-Valley soil comparative survey: the ecology of dry valley edaphic microbial communities [J]. Isme Journal, 2012,6(5):1046-57.

[18] Pires A C C, Cleary D F R, Almeida A, et al. Denaturing gradient gel electrophoresis and barcoded pyrosequencing reveal unprecedented archaeal diversity in mangrove sediment and rhizosphere samples [J]. Applied and Environmental Microbiology, 2012, 78 (16):5520-5528.

[19] Krakat N, Westphal A, Satke K, et al. The microcosm of a biogas fermenter: comparison of moderate hyperthermophilic (60℃) with thermophilic (55℃) conditions [J]. Engineering in Life Sciences, 2010,10(6):520-527.

[20] Khalid A, Arshad M, Anjum M, et al. The anaerobic digestion of solid organic waste [J]. Waste Management, 2011,31(8):1737-1744.

[21] Lepist? S S, Rintala J A. Thermophilic anaerobic digestion of the organic fraction of municipal solid waste: start-up with digested material from a mesophilic process [J]. Environmental Technology, 1995,16(2):157-164.

[22] Fukuzaki S, Nishio N, Shobayashi M, et al. Inhibition of the fermentation of propionate to methane by hydrogen, acetate, and propionate [J]. Applied and Environmental Microbiology, 1990, 56(3):719-723.18.

[23] Wu W M, Bhatnagar L, Zeikus J G. Performance of anaerobic granules for degradation of pentachlorophenol [J]. Applied and Environmental Microbiology, 1993,59(2):389-397.

[24] Ahring B K, Westermann P. Product inhibition of butyrate metabolism by acetate and hydrogen in a thermophilic coculture [J]. Applied and Environmental Microbiology, 1988,54(10):2393-2397.

[25] Van Lier J B, Grolle K C, Frijters C T, et al. Effects of acetate, propionate, and butyrate on the thermophilic anaerobic degradation of propionate by methanogenic sludge and defined cultures [J]. Applied and Environmental Microbiology, 1993, 59(4):1003-1011.

[26] Park Y J, Tsuno H, Hidaka T, et al. Evaluation of operational parameters in thermophilic acid fermentation of kitchen waste [J]. Journal of Material Cycles and Waste Management, 2008,10(1): 46-52.

[27] Ahring B K, Angelidaki I, Johansen K. Anaerobic treatment of manure together with industrial waste [J]. Water Science and Technology, 1992,25(7):311-318.

[28] Gao S, Zhao M, Chen Y, et al. Tolerance response to in situ ammonia stress in a pilot-scale anaerobic digestion reactor for alleviating ammonia inhibition [J]. BioresourceTechnology, 2015, 198:372-379.

[29] Borja R, Rincon B, Raposo F, et al. Mesophilic anaerobic digestion in a fluidised-bed reactor of wastewater from the production of protein isolates from chickpea flour [J]. Process Biochem., 2004,39:1913–1921.

[30] Raposo F, Borja R, Martín M A, et al. Influence of inoculum–substrate ratio on the anaerobic digestion of sunflower oil cake in batch mode: process stability and kinetic evaluation [J]. Chem. Eng. J. 2009,149:70–77.

[31] Yokoyama H, Wagner I D, Wiegel J. Caldicoprobacteroshimai gen. nov., sp. nov., an anaerobic, xylanolytic, extremely thermophilic bacterium isolated from sheep faeces, and proposal of Caldicoprobacteraceae fam. nov [J]. International Journal ofSystematic and Evolutionary Microbiology, 2010,60(1):67-71.

[32] Koeck D E, Wibberg D, Maus I, et al. Complete genome sequence of the cellulolytic thermophile Ruminoclostridiumcellulosi wild-type strain DG5isolated from a thermophilic biogas plant [J]. Journal of Biotechnology, 2014,188:136-137,23.

[33] Sun W, Yu G, Louie T, et al. From mesophilic to thermophilic digestion: the transitions of anaerobic bacterial, archaeal, and fungal community structures in sludge and manure samples [J]. Applied Microbiology and Biotechnology, 2015,99(23):10271-10282.

[34] Etchebehere C, Pavan M E, Zorzopulos J, et al. Coprothermobacter platensis sp. nov., a new anaerobic proteolytic thermophilic bacterium isolated from an anaerobic mesophilic sludge [J]. International Journal of Systematic and Evolutionary Microbiology, 1998,48(4):1297-1304.

[35] Cai H, Gu J, Wang Y. Protease complement of the thermophilic bacterium Coprothermobacter proteolyticus [J]. Proceeding of the International Conference on Bioinformatics and Computational Biology BIOCOMP, 2011,11:18-21.

[36] Liao W, Liu Y, Wen Z, et al. Studying the effects of reaction conditions on components of dairy manure and cellulose accumulation using dilute acid treatment [J]. Bioresource Technology, 2007,98(10):1992-1999.

[37] Kobayashi T, Li Y Y, Harada H. Analysis of microbial community structure and diversity in the thermophilic anaerobic digestion of waste activated sludge [J]. Water Science and Technology, 2008,57(8):1199-1205.

[38] Cheon J H, Hidaka T, Mori S, et al. Applicability of random cloning method to analyze microbial community in full-scale anaerobic digesters [J]. Journal of Bioscience and Bioengineering, 2008,106(2):134-140.

[39] Tandishabo K, Nakamura K, Umetsu K, et al. Distribution and role of Coprothermobacter spp. in anaerobic digesters [J]. Journal of Bioscience and Bioengineering, 2012,114(5):518-520.

[40] Bouanane-Darenfed A, Fardeau M L, Grégoire P, et al. Caldicoprobacter algeriensis sp. nov., a new thermophilic anaerobic, xylanolytic bacterium isolated from an algerian hot spring [J]. Current Microbiology, 2011,62(3):826-832.

[41] Bouacem K, Bouanane-Darenfed A, Boucherba N, et al. Partial characterization of xylanase produced by Caldicoprobacter algeriensis, a new thermophilic anaerobic bacterium isolated from an algerian hot spring [J]. Applied Biochemistry and Biotechnology, 2014,174(5): 1969-1981.

[42] Ravachol J, Borne R, Meynial-Salles I, et al. Combining free and aggregated cellulolytic systems in the cellulosome-producing bacterium Ruminiclostridiumcellulolyticum [J]. Biotechnology for Biofuels, 2015,8(1):1.

[43] Dumitrache A, Akinosho H, Rodriguez M, et al. Consolidated bioprocessing of Populus using Clostridium (Ruminiclostridium) thermocellum: a case study on the impact of lignin composition and structure [J]. Biotechnology for Biofuels, 2016,9(1):1.

[44] Ravachol J, de Philip P, Borne R, et al. Mechanisms involved in xyloglucan catabolism by the cellulosome-producing bacterium Ruminiclostridiumcellulolyticum [J]. Scientific Reports, 2016,6.

[45] Jung S Y, Kim H S, Song J J, et al. Kribbiadieselivorans gen. nov., sp. nov., a novel member of the family Intrasporangiaceae [J]. International Journal of Systematic and Evolutionary microbiology, 2006,56(10):2427-2432.

[46] Lee S D, Lee D W. Lapillicoccusjejuensis gen. nov., sp. nov., a novel actinobacterium of the family Intrasporangiaceae, isolated from stone [J]. International Journal of Systematic and Evolutionary Microbiology, 2007,57(12):2794-2798.

[47] Phitsuwan P, Tachaapaikoon C, Kosugi A, et al. A cellulolytic and xylanolytic enzyme complex from an alkalothermoanaerobacterium, Tepidimicrobium xylanilyticum BT14 [J]. Journal of Microbiology and Biotechnology, 2010, 20(5):893-903.

[48] Chachkhiani M, Dabert P, Abzianidze T, et al. 16S rDNA characterisation of bacterial and archaeal communities during start-up of anaerobic thermophilic digestion of cattle manure [J]. Bioresource Technology, 2004,93(3):227-232.

[49] Zeikus J G, Wolee R S. Methanobacterium thermoautotrophicus sp. n., an anaerobic, autotrophic, extreme thermophile [J]. Journal of Bacteriology, 1972,109(2):707-713.

[50] Kaster A K, Goenrich M, Seedorf H, et al. More than 200genes required for methane formation from H2and CO2and energy conservation are present in Methanothermobacter marburgensis and Methanothermobacter Thermautotrophicus [J]. Archaea, 2011,2011.

[51] Stams A J M. Metabolic interactions between anaerobic bacteria in methanogenic environments [J]. Antonievan Leeuwenhoek, 1994,66(1-3):271-294.

[52] Smith K S, Ingram-Smith C. Methanosaeta, the forgotten methanogen? [J]. Trends in Microbiology, 2007,15(4):150-155.

Start-up strategy for hyperthermophilic anaerobic digestion system of sewage sludge.

DAI Xiao-hu, YU Chun-xiao, LI Ning*, DONG Bin (State Key Laboratory of Pollution Control and Resource Reuse, College of Environmental Science and Engineering, Tongji University, Shanghai 20092, China). China Environmental science, 2017,37(7)：2527～2535

The feasibility of hyperthermophilic anaerobic digestion system of sewage sludge is evaluated in the study, which is conducted with biochemical methane potential experiments using thermophilic anaerobic digestate as inoculum and cow dung and dewatered sludge as substrate. The results show that:①the start-up of hyperthermophilic sludge anaerobic digestion system is practicable;②the accumulation of VFAs (particularly propionic acid) is easy to occur due to the acceleration of hydrolysis and acidification process under 65℃;③compared with mesophilic(37℃) and thermophilic (55℃) anaerobic digestion systems, the total gas production of hyperthermophilic (65℃) system is relatively lower, while the methane content is elevated significantly, even reaching 79.0%;④under hyperthermophilic (65℃) condition, the extremely thermophilic bacteria responsible for lignocellulose and protein degradation such as Coprothermobacter、Caldicoprobacter、Ruminiclostridiumwere dominant, the difference of which is due to the different substrate addition; hydrogenotrophic methanogens-Methanothermobacter accounted for nearly 96% of archaea in all the digesters. Thus, in the start-up period, the addition of cow dung can not only accelerate the accumulation of hyperthermophilic methanogens (Hydrogenotrophic methanogens), but also avoid the accumulation of VFAs, particularly the accumulation of propionic acids, ensuring successful start-up of the system.

hyperthermophilic；anaerobic digestion；start-up strategy；Methanothermobacter

X703.1

A

1000-6923(2017)07-2527-09

戴曉虎(1962-),男,江蘇鎮(zhèn)江人,教授,博士,主要研究方向?yàn)槌鞘杏袡C(jī)廢棄物的處理與處置.發(fā)表論文120余篇.

2016-11-31

國家自然科學(xué)基金項(xiàng)目(51678429,51308402,51538008)

* 責(zé)任作者, 博士, lining@#edu.cn