朱俊兆,卓 楊,華飛虎,周夢雨,王雪娜,劉金洋,韓 蕓

提高污泥含固率對(duì)高溫厭氧消化互營產(chǎn)甲烷影響

朱俊兆,卓 楊,華飛虎,周夢雨,王雪娜,劉金洋,韓 蕓*

(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)

以常規(guī)含固率(2%)剩余污泥高溫厭氧消化排泥為接種污泥,分析接種污泥在不同氨濃度下厭氧消化各步驟的動(dòng)力學(xué)速率變化,并以熱水解預(yù)處理的高含固污泥(10%)為基質(zhì)進(jìn)行連續(xù)試驗(yàn),探討高溫厭氧消化條件下基質(zhì)由常規(guī)含固率快速切換至高含固率的產(chǎn)甲烷性能變化.結(jié)果表明,隨著氨濃度上升,接種污泥對(duì)乙酸、丙酸、丁酸和熱水解污泥的比產(chǎn)甲烷活性均有所下降,但其氫利用速率和互營乙酸氧化速率未受到顯著影響.高溫厭氧消化基質(zhì)含固率由2%切換至10%連續(xù)運(yùn)行試驗(yàn)結(jié)果表明,在有機(jī)負(fù)荷高達(dá)14g COD/(L·d)時(shí),雖然系統(tǒng)COD去除率不足(27.99±3.66)%,且存在VFAs積累(10.41±2.25)g COD/L,但pH值仍可穩(wěn)定在(7.74±0.09),說明該切換策略具有可行性.穩(wěn)定運(yùn)行系統(tǒng)中產(chǎn)氫產(chǎn)乙酸和互營乙酸氧化功能菌屬以(15.29%)、(8.89%)(17.99%)和(1.60%)為主,切換后乙酸營養(yǎng)型產(chǎn)甲烷菌被淘汰,而參與互營乙酸氧化過程的和相對(duì)豐度顯著提高,說明體系通過構(gòu)建互營乙酸氧化產(chǎn)甲烷途徑來抵御高溫高氨的環(huán)境脅迫.因此,以常規(guī)含固率高溫厭氧消化污泥作為接種泥是實(shí)現(xiàn)由常規(guī)含固率切換至高含固熱水解-高溫厭氧消化的有效策略,并且接種污泥的互營乙酸氧化活性是切換成功的關(guān)鍵因素.

高含固污泥；高溫厭氧消化；熱水解預(yù)處理；互營乙酸氧化；氨抑制

厭氧消化作為廣泛應(yīng)用的污泥穩(wěn)定化和資源化技術(shù),可將污泥中的生物質(zhì)能以甲烷形式回收[1].近年來,污泥熱水解預(yù)處理技術(shù)的發(fā)展將厭氧消化污泥含固率提升至10%以上,可有效解決高含固污泥在厭氧消化時(shí)傳質(zhì)受阻和中間產(chǎn)物積累的問題[2].高含固污泥熱水解厭氧消化工藝具有機(jī)負(fù)荷高和投資低等優(yōu)勢,成為城市污水處理廠污泥厭氧消化系統(tǒng)改進(jìn)工藝的主推方向之一[3].高溫厭氧消化是高含固熱水解預(yù)處理技術(shù)理想的耦合工藝,厭氧消化在高溫條件下不僅擁有更快的產(chǎn)甲烷速率,而且有利于熱水解換熱過程中的能量回收[4].然而,高含固熱水解-高溫厭氧消化(THP-TAD)具有高氨和高pH值的特點(diǎn),并且在高溫下大幅上升的游離氨將對(duì)厭氧消化過程產(chǎn)生更強(qiáng)的抑制作用[5].除此之外,有機(jī)負(fù)荷和高溫條件極易致使產(chǎn)甲烷菌受到抑制作用,進(jìn)而導(dǎo)致氫分壓上升和揮發(fā)性脂肪酸(VFAs)過量積累,最終導(dǎo)致反應(yīng)器的酸化甚至崩潰.因此,在高氨氮環(huán)境下維持穩(wěn)定的產(chǎn)甲烷性能是THP-TAD需要解決的首要問題.

互營乙酸氧化菌(SAOB)是與產(chǎn)甲烷作用相關(guān)的核心菌群[6].SAOB通常適宜在高溫下將乙酸氧化為H2與CO2,繼而被氫營養(yǎng)型產(chǎn)甲烷菌利用[7],即互營乙酸氧化產(chǎn)甲烷過程(SAO-HM).在不利于乙酸營養(yǎng)型產(chǎn)甲烷菌生長的厭氧環(huán)境下,SAOB通常會(huì)調(diào)節(jié)乙酸降解動(dòng)力學(xué)[8].例如在高氨[9]和高VFAs[10]環(huán)境中,互營乙酸氧化產(chǎn)甲烷是降解乙酸的主要途徑.因此,在厭氧體系中建立SAO-HM是實(shí)現(xiàn)THP-TAD的關(guān)鍵因素,已有研究證實(shí)可通過氨誘導(dǎo)和酸誘導(dǎo)逐步建立SAO-HM[11-12].在常規(guī)高溫厭氧消化切換至高含固熱水解-高溫厭氧消化過程中,低氨低VFAs環(huán)境會(huì)逐漸轉(zhuǎn)向高氨高VFAs環(huán)境,更有利于SAOB與氫營養(yǎng)型產(chǎn)甲烷菌建立互營關(guān)系.此種切換方式是在THP-TAD體系下實(shí)現(xiàn)SAO-HM途徑的潛在策略,而目前對(duì)其可行性及運(yùn)行特性尚缺乏研究.

本研究以常規(guī)高溫厭氧消化污泥為接種泥開展THP-TAD切換試驗(yàn),通過分析氨抑制條件下常規(guī)高溫厭氧污泥代謝典型基質(zhì)的產(chǎn)甲烷特性,特別是不同氨濃度下的氫利用速率以及互營乙酸氧化速率以探討該策略的可行性.通過監(jiān)測連續(xù)運(yùn)行反應(yīng)器中指標(biāo)以評(píng)估該策略的適用性,并探討切換后的核心菌群和產(chǎn)甲烷菌之間的代謝關(guān)系,為高含固熱水解-高溫厭氧消化系統(tǒng)啟動(dòng)和工藝優(yōu)化提供技術(shù)支撐.

1 材料與方法

1.1 接種污泥與基質(zhì)

接種污泥取自實(shí)驗(yàn)室穩(wěn)定運(yùn)行280d以上的常規(guī)高溫厭氧消化反應(yīng)器(55 ± 1)℃,接種污泥基本指標(biāo)如表1所示.該反應(yīng)器有效容積5L,基質(zhì)為剩余污泥,COD去除率為(34.39 ± 2.83)%,水力停留時(shí)間(HRT)20d.連續(xù)運(yùn)行試驗(yàn)所用消化基質(zhì)為熱水解預(yù)處理的剩余污泥,剩余污泥取自西安市某污水處理廠(A2/O工藝),脫水后調(diào)整含固率至10%,后置于熱水解反應(yīng)釜(KCF-5,北京世紀(jì)森朗),熱水解條件為165 ℃、30min.消化基質(zhì)于4 ℃冰箱保存待用.樣品預(yù)處理方法:將污泥樣品于10000r/min離心10min后(5804R,Eppendorf),取上清液測定溶解態(tài)指標(biāo),將污泥樣品在超聲破碎機(jī)中破胞進(jìn)行總化學(xué)需氧量測定.接種污泥和消化基質(zhì)指標(biāo)見表1.

表1 接種污泥和消化基質(zhì)基本指標(biāo)

注:“/”表示該指標(biāo)無單位.

1.2 試驗(yàn)設(shè)置

1.2.1 比產(chǎn)甲烷活性測定 為探究氨氮濃度對(duì)接種污泥底物代謝的影響,試驗(yàn)測定了不同氨氮濃度下接種污泥對(duì)典型基質(zhì)的比產(chǎn)甲烷活性(SMA),采用有效容積120mL厭氧瓶一式3份進(jìn)行,操作流程如圖1所示:

各試驗(yàn)組氨氮濃度為接種泥所含有的氨氮濃度及氨氮投加濃度之和,未包含熱水解濾液投加導(dǎo)致的氨氮濃度增加(約28.88mg/L).產(chǎn)生的氣體用玻璃注射器測量體積后用排水集氣法收集,并利用氣相色譜儀測量氣體組分.計(jì)算公式為:

式中:SMA為比產(chǎn)甲烷活性,mLCH4/(gVSS·d);CH4為累計(jì)甲烷產(chǎn)量,mL;R為厭氧瓶中添加的污泥量,L;VSS為所用污泥的揮發(fā)性懸浮固體含量,g/L;為時(shí)間,d.

圖1 比產(chǎn)甲烷活性測定流程

Fig.1 The measurement process of specific methanogenic activity

1.2.2 氫利用速率和互營乙酸氧化速率 為表征其代謝速率,設(shè)計(jì)測定裝置如下:

(1)氫利用速率測定使用文獻(xiàn)[13]所示的方法,計(jì)算公式為:

式中:HUR為氫利用速率,mLH2/(gVSS·h);H2為累計(jì)氫氣消耗量,mL;R為厭氧瓶中添加的污泥量,L;VSS為所用污泥的揮發(fā)性懸浮固體含量,g/L;為時(shí)間,h.

同型產(chǎn)乙酸速率測定:殺滅產(chǎn)甲烷菌后,測定方法和計(jì)算方法與氫利用速率相同.

(2)互營乙酸氧化速率測定裝置:如圖所示,將一定體積已殺滅產(chǎn)甲烷菌的厭氧污泥置于厭氧瓶,投加適量乙酸鈉作為消化基質(zhì),厭氧瓶頂空與氣袋相連接以擴(kuò)大頂空體積,設(shè)置蠕動(dòng)泵加速氣體循環(huán),每隔一定時(shí)間在厭氧瓶中取樣,測定液體樣品中的乙酸濃度,在氣袋中采樣測定氣體中的氫氣含量.計(jì)算公式為:

式中:SAO為互營乙酸氧化速率,mgHAc/(gVSS·h);HAc為乙酸消耗量,mg;R為厭氧瓶中添加的污泥量,L;VSS為所用污泥的揮發(fā)性懸浮固體含量,g/L;為時(shí)間,h.

1.2.3 連續(xù)運(yùn)行試驗(yàn)設(shè)置 實(shí)驗(yàn)設(shè)置4個(gè)連續(xù)運(yùn)行的完全混合厭氧反應(yīng)器,反應(yīng)器有效容積均為1.2L,HRT分別為20、15、10和5d,依次編號(hào)R1～R4,反應(yīng)器接種常規(guī)高溫厭氧消化污泥800mL,并以熱水解污泥為基質(zhì)進(jìn)行連續(xù)運(yùn)行試驗(yàn),采用集氣袋收集氣體,每日手動(dòng)進(jìn)排污泥.在連續(xù)運(yùn)行期間監(jiān)測VFAs、TAN、COD、容積產(chǎn)氣率和堿度等指標(biāo).

1.3 理化指標(biāo)分析

TS和VSS均采用重量法測定;COD采用重鉻酸鉀法測定;pH值采用上海精科PHS-3CpH計(jì)測定;堿度采用滴定法測定,總堿度的滴定終點(diǎn)為3.8[14];氨氮采用納氏試劑分光光度法測定;VFAs測定采用氣相色譜法(BEIFEN Corp.3420A、FID檢測器),色譜柱為DB-FFAP毛細(xì)柱(50m × 0. 32mm × 0.50μm),測定條件為初始柱箱溫度120℃,保持初始柱箱溫度1min后進(jìn)入升溫程序1,升溫程序1以10℃/min速率升溫,最終溫度為180℃,最終保持時(shí)間5min,然后進(jìn)入程序2,程序以10℃/min速率升溫,最終溫度為220℃,最終保持時(shí)間5min.進(jìn)樣口溫度180℃、檢測器溫度250℃;氣體組分包括CH4、CO2、H2和N2,采用氣相色譜法(BEIFEN Corp.3420A、TCD檢測器),色譜柱為蘭化TDX-01(3mm×2m),測定條件為進(jìn)樣口溫度80℃、柱箱溫度100℃、檢測器溫度100℃.

游離氨(FAN)濃度依據(jù)文獻(xiàn)[15]中的公式計(jì)算:

式中:FAN為游離氨,mg/L;TAN為總氨氮,mg/L;為厭氧消化熱力學(xué)溫度,K.

Gompertz方程廣泛應(yīng)用于估算甲烷產(chǎn)量和氫氣消耗量,利用修正的Gompertz方程擬合產(chǎn)甲烷和耗氫的動(dòng)力學(xué)曲線,得到相關(guān)參數(shù)并評(píng)估了參數(shù)與模型的擬合度,其方程如下式所示:

式中:()為時(shí)間時(shí)刻累計(jì)甲烷產(chǎn)量或耗氫量,mL;max為最大累計(jì)甲烷產(chǎn)量或耗氫量,mL;max為最大甲烷產(chǎn)率或耗氫速率,mL/d或mL/h;為滯后時(shí)間;為消化時(shí)間;e為自然指數(shù)常數(shù).

1.4 微生物群落分析

為探討切換前后微生物群落結(jié)構(gòu)變化,在穩(wěn)定運(yùn)行的常規(guī)高溫反應(yīng)器C1和切換完成后的R1～R4高含固熱水解高溫反應(yīng)器中采集微生物樣本.使用E.Z.N.ATMMag-Bind Soil DNA Kit (OMEGA, M5635-02)對(duì)樣本進(jìn)行DNA抽提.利用Qubit3.0DNA檢測試劑盒對(duì)基因組DNA精確定量后,以確定PCR反應(yīng)加入的DNA量.細(xì)菌PCR第一輪擴(kuò)增所用引物為341F(5'-CCTACGGGNGG- CWGCAG-3')和805R(5'-GACTACHVGGGTATC- TAATCC-3').第二輪擴(kuò)增引入Illumina橋式PCR兼容引物.古菌引用槽式PCR擴(kuò)增有三輪,第一輪使用340F(5'-CCCTAYGGGGYGCASCAG-3')和1000R (5'-GGCCATGCACYWCYTCTC-3')進(jìn)行引物擴(kuò)增.第二輪使用第一輪PCR產(chǎn)物進(jìn)行擴(kuò)增,349F(5'- GYGCASCAGKCGMGAAW-3')和806R(5'-GGA- CTACVSGGGTATCTAAT-3'),第三輪擴(kuò)增引入Illumina橋式PCR兼容引物.細(xì)菌和古菌PCR所用引物均融合了測序平臺(tái)的V3～V4通用引物.通過2%瓊脂糖凝膠電泳檢測文庫大小,使用Qubit3.0熒光定量儀進(jìn)行文庫濃度測定,最后在Illumina平臺(tái)進(jìn)行高通量測序.數(shù)據(jù)處理由上海生工生物工程股份有限公司完成.

2 結(jié)果與討論

2.1 不同氨濃度下常規(guī)含固率高溫厭氧污泥的產(chǎn)甲烷特性

圖2 不同氨濃度接種污泥對(duì)典型基質(zhì)的產(chǎn)甲烷特性

為探討常規(guī)含固率高溫厭氧消化污泥作為接種污泥實(shí)現(xiàn)高含固熱水解-高溫厭氧消化的可行性,對(duì)常規(guī)高溫厭氧污泥在不同氨濃度下代謝典型基質(zhì)的產(chǎn)甲烷性能進(jìn)行測定,結(jié)果如圖2所示,在0.5和2.0g/LNH4+-N時(shí),乙酸、丙酸和丁酸的累計(jì)產(chǎn)甲烷量基本相同,分別為(62.21±5.82)和(44.01± 12.61) mLCH4,而熱水解污泥的累計(jì)產(chǎn)甲烷量明顯低于其他組,僅為(23.99±2.52)和(16.70±0.90)mLCH4,這是由于熱水解污泥含有的抑制物會(huì)影響產(chǎn)甲烷性能[16].在2g/LNH4+-N時(shí),丙酸組累計(jì)產(chǎn)甲烷量略高于乙酸和丁酸,這可能是不同VFAs的產(chǎn)氫產(chǎn)乙酸反應(yīng)過程差異及接種泥的乙酸裂解、氧化反應(yīng)在不同氨濃度下的抑制性導(dǎo)致.丙酸組具有最長的滯后期,為2.74d(表2),丙酸是高溫厭氧消化系統(tǒng)中最易積累的VFAs之一,與其他VFAs相比其對(duì)產(chǎn)甲烷菌的毒性最大,故而代謝緩慢[17].產(chǎn)甲烷菌可以直接利用乙酸產(chǎn)生甲烷,而代謝丁酸微生物的比生長速率較大,尤其是在高溫條件下可達(dá)到0.77d-1,故丁酸作為底物時(shí)也極易降解,通常難以發(fā)生積累[18].圖2中空白組最大產(chǎn)甲烷速度早于部分基質(zhì)組,因此對(duì)最大甲烷產(chǎn)量及最大產(chǎn)甲烷速率進(jìn)行數(shù)據(jù)擬合,隨后將最大甲烷產(chǎn)量及甲烷產(chǎn)率減去空白組,其結(jié)果見表2.SMA由最大產(chǎn)甲烷速率折算至每克揮發(fā)性懸浮固體.SMA在0.5g/LNH4+-N下最大.隨著氨氮濃度增加到2.0g/LNH4+-N,各組SMA分別下降了28.05%,57.89%,61.27%和13.33%.其中,乙酸和熱水解污泥的下降程度較小,這說明乙酸裂解型產(chǎn)甲烷依然貢獻(xiàn)度較大,且微生物對(duì)熱水解污泥的適應(yīng)性較強(qiáng).

當(dāng)氨氮濃度增加到3.0g/LNH4+-N時(shí),產(chǎn)甲烷過程明顯呈現(xiàn)兩段式產(chǎn)氣,見圖2.在第一階段,各組SMA均受到嚴(yán)重抑制,相較于2.0g/LNH4+-N分別下降了97.12%,94.74%,77.27%和80.17%.接種污泥體系中的產(chǎn)甲烷途徑為乙酸裂解型產(chǎn)甲烷,但乙酸營養(yǎng)型產(chǎn)甲烷菌對(duì)氨耐受能力較差,導(dǎo)致產(chǎn)甲烷過程近乎停滯[19].研究表明,互營乙酸氧化產(chǎn)甲烷途徑占比在大于3.0g/LNH4+-N時(shí)會(huì)顯著增加[20],而接種污泥的氫營養(yǎng)型產(chǎn)甲烷菌相對(duì)豐度較低,這可能是導(dǎo)致氨氮濃度提升至3000mg/L后SMA降幅較大的原因.在第二階段時(shí),除熱水解污泥組下降外,其余各組的SMA分別提升至76.67%,83.33%和44.44%,這可能是由于參與SAO-HM過程的微生物對(duì)氨氮具有更高的耐受能力,在高氨氮環(huán)境中對(duì)乙酸代謝起重要作用.同時(shí),第二階段中熱水解污泥組SMA下降,可能是高氨氮環(huán)境與熱水解污泥攜帶的抑制物協(xié)同作用,對(duì)微生物產(chǎn)生抑制[21].

表2 不同氨濃度下接種污泥對(duì)典型基質(zhì)代謝的動(dòng)力學(xué)參數(shù)

注:由于3g/LNH4+-N時(shí)為兩段式產(chǎn)氣,故分別進(jìn)行擬合.

2.2 氫利用速率及互營乙酸氧化速率

氫利用速率和同型產(chǎn)乙酸速率大小能夠反映接種污泥對(duì)厭氧系統(tǒng)氫分壓的調(diào)控潛能.對(duì)不同氨濃度下常規(guī)高溫厭氧污泥的氫利用速率、同型產(chǎn)乙酸速率和互營乙酸氧化速率進(jìn)行測定,由圖3(a)和(b)可知,氫營養(yǎng)型產(chǎn)甲烷菌和同型產(chǎn)乙酸菌能快速適應(yīng)高氫分壓環(huán)境.

在測定開始時(shí)其耗氫量便快速增長,此時(shí)裝置中的氫氣基質(zhì)充足,反應(yīng)不受限制,在經(jīng)過3～4h后,由于氫分壓降低,反應(yīng)受基質(zhì)濃度限制速率逐步減緩,其總氫利用速率和同型產(chǎn)乙酸速率在2.0g/ LNH4+-N時(shí)達(dá)到最大,分別為(6.98±0.86)和(6.65± 0.41)mLH2/(gVSS·h),而在3.0g/LNH4+-N時(shí),氫利用速率為(5.26±0.28)mLH2/(gVSS·h),與2.0g/LNH4+-N相比僅下降24.79%.接種的常規(guī)高溫厭氧污泥以乙酸營養(yǎng)型產(chǎn)甲烷菌為主,在0.5g/ LNH4+-N時(shí)其乙酸的SMA為(40.96±0.16)mLCH4/(gVSS·d),而在3.0g/ LNH4+-N時(shí)乙酸SMA僅為(3.65±0.48)mLCH4/ (gVSS·d),與2.0g/LNH4+-N相比下降87.76%,氨氮濃度上升對(duì)乙酸SMA影響較大.對(duì)氫利用速率進(jìn)行單因素ANOVA檢驗(yàn),顯著性為0.252(>0.05),這說明氨氮濃度對(duì)氫利用速率影響不顯著.氫營養(yǎng)型產(chǎn)甲烷菌是高氨氮環(huán)境下主導(dǎo)產(chǎn)甲烷過程的功能菌群[22],彭韻等[23]發(fā)現(xiàn)在高氨氮環(huán)境中,乙酸型產(chǎn)甲烷途徑會(huì)發(fā)生轉(zhuǎn)變,而氫型產(chǎn)甲烷途徑相關(guān)基因依然保持穩(wěn)定.上述結(jié)果為高氨氮環(huán)境下實(shí)現(xiàn)互營乙酸氧化產(chǎn)甲烷奠定了基礎(chǔ).

SAOB是高氨氮環(huán)境中代謝乙酸的核心菌群,其活性大小能間接表征厭氧微生物通過互營乙酸氧化產(chǎn)甲烷的代謝速率.由圖3(c)可知,在2.0g/ LNH4+-N時(shí),其互營乙酸氧化速率和同型產(chǎn)乙酸速率均最高,分別為(5.16±0.51)和(6.65±0.41)mLH2/ (gVSS·h).同型產(chǎn)乙酸和互營乙酸氧化速率同時(shí)偏高的原因可能是由于它們是同一種SAOB介導(dǎo),因?yàn)椴糠諷AOB同時(shí)具有互營乙酸氧化功能和同型產(chǎn)乙酸功能[24,25],接種污泥的互營乙酸氧化速率在0.5和3.0g/LNH4+-N時(shí)分別為(4.74±0.33)和(4.40± 0.30)mgHAc/(gVSS·h).對(duì)互營乙酸氧化速率進(jìn)行單因素ANOVA檢驗(yàn),顯著性為0.534(p>0.05),此結(jié)果說明常規(guī)含固率高溫厭氧消化污泥在高氨氮或低氨氮環(huán)境中,即便乙酸營養(yǎng)型產(chǎn)甲烷菌被抑制也依然保持代謝乙酸的能力,說明采用該污泥作為接種泥強(qiáng)化SAO-HM途徑實(shí)現(xiàn)高含固熱水解-高溫厭氧消化具備可能性.

2.3 切換過程中高含固熱水解高溫厭氧消化的運(yùn)行特性

2.3.1 反應(yīng)器運(yùn)行性能 以2%含固率高溫厭氧消化污泥為接種污泥,經(jīng)熱水解預(yù)處理的10%高含固污泥為基質(zhì),采用小試CSTR系統(tǒng)開展連續(xù)運(yùn)行實(shí)驗(yàn).各反應(yīng)器共運(yùn)行69d,CSTR系統(tǒng)運(yùn)行性能和OLR如圖4所示.系統(tǒng)在啟動(dòng)大約12d后產(chǎn)氣量達(dá)到穩(wěn)定,容積產(chǎn)氣率分別穩(wěn)定在 (0.40±0.09),(0.61±0.12), (0.95±0.17)和(1.79±0.22)L/(L·d),且在較短的HRT也可以有效轉(zhuǎn)化有機(jī)物.在高OLR下,系統(tǒng)仍可維持一定的有機(jī)物去除率.在穩(wěn)定期時(shí)COD去除率分別為(37.76±3.11)%(HRT為20d),(33.04±3.28)%(HRT為15d),(30.62±3.71)%(HRT為10d)和(27.99±3.66)% (HRT為5d).OLR最高時(shí)的COD去除率相比最低時(shí)下降25.87%,表明更高的OLR可能會(huì)使微生物承受的負(fù)荷更大,進(jìn)而導(dǎo)致COD轉(zhuǎn)化率降低[26].同時(shí),VS去除率分別為(35.65±2.68)%,(30.57±3.04)%, (28.47 ±2.57)%和(26.13±2.34)%.Wu等[27]分析了水熱預(yù)處理后的高含固污泥高溫厭氧消化去除率,熱水解后的污泥TCOD為83.6g/L,VS/TS為0.55,高溫條件下反應(yīng)器的COD去除率為37.8%,與本研究的去除率接近.說明在高氨氮、高酸以及高負(fù)荷沖擊環(huán)境下,系統(tǒng)仍然能保持正常的COD去除率.因此,提高含固率是實(shí)現(xiàn)高含固熱水解-高溫厭氧消化的合理策略.

圖4 切換過程中高含固熱水解高溫厭氧消化的運(yùn)行特性

氨抑制是影響高溫厭氧消化過程穩(wěn)定性的重要因素[28],在本實(shí)驗(yàn)切換過程中卻未出現(xiàn)明顯的氨抑制現(xiàn)象.常規(guī)高溫厭氧污泥的總氨氮濃度為(599.98± 90.12)mg/L,在基質(zhì)改變?yōu)闊崴馕勰嗪?各反應(yīng)器的總氨氮濃度逐漸上升,在穩(wěn)定期時(shí),各反應(yīng)器總氨氮濃度達(dá)到(2349.37±110.22)mg/L(見圖4).游離氨可對(duì)微生物表現(xiàn)出更強(qiáng)的毒性,但也存在高游離氨濃度環(huán)境下系統(tǒng)穩(wěn)定運(yùn)行的現(xiàn)象,Kim等[29]發(fā)現(xiàn)游離氨濃度達(dá)到700mg/L時(shí)系統(tǒng)仍能穩(wěn)定運(yùn)行不受抑制.Calli等[30]將配水運(yùn)行的厭氧消化系統(tǒng)游離氨濃度提升至800mg/L,但COD降解率仍能維持在78%～96%.戴曉虎等[31]認(rèn)為厭氧消化氨抑制研究的重點(diǎn)不僅在于獲得抑制閾值濃度,更應(yīng)該傾向于解析游離氨濃度帶來的微生物種群結(jié)構(gòu)和代謝途徑的變化.在高溫厭氧消化由常規(guī)含固率提升至高含固率過程中,游離氨濃度由接種污泥的(87.80±7.60)mg/L增加至切換完成后的(665.65±121.29)mg/L,在此過程中,游離氨上升很可能會(huì)造成微生物種群結(jié)構(gòu)的變化,進(jìn)而導(dǎo)致產(chǎn)甲烷途徑的改變.在高氨氮厭氧消化環(huán)境中,互營乙酸氧化產(chǎn)甲烷途徑占甲烷生成的68%～75%[32],該途徑的轉(zhuǎn)變有利于體系中的乙酸降解乃至其余VFAs氧化反應(yīng).因此,氨氮濃度提升對(duì)于切換過程中產(chǎn)甲烷途徑的轉(zhuǎn)變至關(guān)重要.

2.3.2 pH值、VFAs、堿度及氫分壓 pH值、VFAs、堿度是影響體系穩(wěn)定性的主要因素,能反映厭氧體系的酸堿平衡和緩沖能力[33].在切換過程中,所有反應(yīng)器的pH值在穩(wěn)定期均保持在7.6～8.2之間,在啟動(dòng)期(第1～33d)pH值均在不斷升高,這是由于基質(zhì)中蛋白質(zhì)含量隨著含固率升高.同時(shí),這也導(dǎo)致各反應(yīng)器在切換過程中總堿度在不斷上升,最終每臺(tái)反應(yīng)器的總堿度維持在(7.41±0.08)g/L(見圖4),表明系統(tǒng)的緩沖能力得到極大提升.

啟動(dòng)初期的總VFAs濃度較低,僅為(0.60± 0.27)gCOD/L,且各反應(yīng)器均以乙酸和丙酸為主(圖5).在穩(wěn)定期時(shí)各反應(yīng)器總VFAs分別增加到(4.12± 0.71),(7.17±1.39),(6.20±1.01)和(10.41±2.25)g COD/ L(圖4),丙酸逐漸取代乙酸成為VFAs的主要成分,分別占總組份的44.41%,66.63%,61.15%和55.39%.高負(fù)荷厭氧體系的丙酸易發(fā)生積累[34],較高的OLR容易導(dǎo)致產(chǎn)酸和產(chǎn)甲烷過程代謝失衡[35],VFAs積累最終會(huì)導(dǎo)致系統(tǒng)pH值急劇下降.本研究中,隨著OLR的增加體系中丁酸和戊酸的比例隨之增加,丁酸易在高有機(jī)負(fù)荷下積累[36],而由于戊酸氧化的主要中間產(chǎn)物丙酸較難降解,戊酸的產(chǎn)氫產(chǎn)乙酸過程也被限制.在啟動(dòng)初期,體系中氨濃度和VFAs濃度逐漸上升,產(chǎn)甲烷過程被抑制,氫分壓增加導(dǎo)致VFAs積累[37],雖然VFAs濃度上升,但同時(shí)總堿度也在逐步增加,這可能減緩了VFAs積累,使得水解產(chǎn)酸菌和產(chǎn)甲烷菌達(dá)到代謝平衡.在穩(wěn)定期,SAOB和氫營養(yǎng)型產(chǎn)甲烷菌逐漸取代乙酸營養(yǎng)型產(chǎn)甲烷菌成為優(yōu)勢菌群,使得產(chǎn)甲烷途徑轉(zhuǎn)變?yōu)镾AO-HM, VFAs濃度逐漸趨緩并保持穩(wěn)定.

圖5 連續(xù)運(yùn)行反應(yīng)器中VFAs濃度(a)R1;(b)R2;(c)R3;(d)R4

氫分壓影響厭氧消化過程中的熱力學(xué)和降解途徑,高氫分壓會(huì)抑制VFAs降解導(dǎo)致其積累.在本文中,各反應(yīng)器的氫分壓均保持在極低的范圍(圖5),分別為(3.04±1.69),(4.25±4.44),(6.25±4.79)和(13.60±6.66)Pa,這說明系統(tǒng)消耗氫的能力逐漸提升.隨著SAO-HM體系的建立,氫營養(yǎng)型產(chǎn)甲烷菌對(duì)氫氣的消耗促使系統(tǒng)維持低氫分壓,保證SAO-HM過程能夠順利進(jìn)行,故而氫分壓并未影響VFAs氧化反應(yīng).

2.3.3 最大產(chǎn)甲烷活性和氫利用速率 為探討切換過程中產(chǎn)甲烷途徑的變化,將切換完成后各反應(yīng)器的排泥用無氧水淘洗并低速離心,控制氨氮濃度至(0.52±0.02)g/LNH4+-N測定氫利用速率和乙酸SMA.其結(jié)果由圖6所示,HRT為5d時(shí)其氫利用速率達(dá)到最大,為(14.84±0.57)mLH2/(gVSS·h),在HRT提升后,其氫利用速率分別為(11.46±0.71),(7.95±0.84)和(10.89±0.79)mLH2/(gVSS·h),與常規(guī)高溫厭氧污泥在0.5g/LNH4+-N下的氫利用速率(5.89± 0.43) mLH2/(gVSS·h)相比分別提升了151.95%,94.57%, 34.97%和84.89%;另一方面,常規(guī)高溫厭氧消化污泥在0.5g/LNH4+-N下乙酸的SMA為(40.96±0.16) mLCH4/(gVSS·d),切換后乙酸SMA分別變化至(15.01±1.98),(35.71±4.23),(16.79±1.25)和(39.47± 2.56)mLCH4/(gVSS·d).切換完成后,氫利用速率均有不同程度的提升,而乙酸SMA呈現(xiàn)下降趨勢.乙酸是產(chǎn)甲烷菌最易利用的基質(zhì)之一,但乙酸SMA下降,這說明關(guān)于乙酸代謝的功能微生物出現(xiàn)抑制或轉(zhuǎn)化路徑發(fā)生改變.SAO-HM途徑相比乙酸裂解途徑的乙酸降解速率較慢[38],這可能是導(dǎo)致乙酸SMA下降的重要原因.氫利用速率提升說明產(chǎn)甲烷菌群從乙酸營養(yǎng)型轉(zhuǎn)變?yōu)闅錉I養(yǎng)型,產(chǎn)甲烷途徑已經(jīng)轉(zhuǎn)變?yōu)镾AO-HM.

圖6 切換完成后的最大乙酸SMA和最大氫利用速率

Fig 6 The maximum specific acetate methanogenic activity and hydrogen utilization rate after switching completion

2.4 微生物群落結(jié)構(gòu)變化

在厭氧系統(tǒng)中,細(xì)菌直接參與底物降解過程,其中間產(chǎn)物會(huì)被產(chǎn)甲烷菌利用,因此細(xì)菌的群落結(jié)構(gòu)會(huì)最先受到影響.細(xì)菌屬水平的相對(duì)豐度如圖7(a)所示,屬為嗜熱厭氧菌,其相對(duì)豐度由7.65%上升至15.29%.已有研究證實(shí),是與氫營養(yǎng)型產(chǎn)甲烷菌共同進(jìn)行互營氧化過程中降解乙酸鹽的主要菌屬[21].的相對(duì)豐度由0.19%提高至3.29%,該類菌屬主要是在高溫條件下對(duì)大分子有機(jī)物(如蛋白質(zhì))降解過程起重要作用[39].和相對(duì)豐度分別由0.46%和0.05%顯著提升至8.89%和17.99%,二者主要將糖轉(zhuǎn)化為乙酸、H2和CO2[40-41],可能與氫營養(yǎng)型產(chǎn)甲烷菌存在互營關(guān)系.此外,和對(duì)高氨氮和高VFAs環(huán)境也表現(xiàn)出極強(qiáng)的耐受性,研究也發(fā)現(xiàn)這些菌屬只在高氨氮反應(yīng)體系中出現(xiàn)[42].互營單胞菌屬相對(duì)豐度由0.09%提高至1.60%,其主要功能就是將有機(jī)酸降解為乙酸和氫氣供產(chǎn)甲烷菌利用,參與互營乙酸氧化代謝過程[43].因此以上結(jié)果說明,在高溫厭氧消化由常規(guī)含固率切換至高含固率的過程中,環(huán)境變化導(dǎo)致、和等菌屬的相對(duì)豐度增加,促進(jìn)SAO-HM途徑的構(gòu)建.此類細(xì)菌對(duì)極端環(huán)境的強(qiáng)耐受性保障了高含固熱水解-高溫厭氧消化體系下水解酸化、產(chǎn)氫產(chǎn)乙酸和互營乙酸氧化階段的順利進(jìn)行,這可能也是在高溫高氨環(huán)境下系統(tǒng)切換過程保持穩(wěn)定的重要原因.

由圖7(b)中可知,甲烷八疊球菌()、甲烷絲菌()和甲烷熱桿菌()是系統(tǒng)的優(yōu)勢古菌屬.在切換完成后,其相對(duì)豐度從接種污泥的16.52%分別上升至49.78%,53.48%, 67.01%和64.73%.除可利用乙酸外,可利用氫氣產(chǎn)生甲烷,并且已有研究證實(shí)其可以促進(jìn)SAOB的生長[44].另外,因細(xì)胞表面有較高的卷起率,游離氨會(huì)更少擴(kuò)散進(jìn)其細(xì)胞內(nèi)[6].因此推測在高含固厭氧消化系統(tǒng)中,游離氨濃度上升對(duì)相對(duì)豐度影響不顯著[31].常規(guī)含固率高溫接種污泥以乙酸營養(yǎng)型的甲烷絲菌主導(dǎo),其相對(duì)豐度達(dá)到62.41%,切換后相對(duì)豐度由62.41%分別下降至0.47%,0.65%,0.69%和0.74%,這可能是切換過程中體系中的氨氮濃度在不斷增加所導(dǎo)致的.研究表明,氨氮高于1700mg/L時(shí),會(huì)顯著抑制的活性,但的氨氮耐受閾值高達(dá)7000mg/L[45].因此,在高VFAs和高氨氮濃度體系下,而非會(huì)成為優(yōu)勢菌屬,這與之前的研究結(jié)果一致[46–48].氫營養(yǎng)型甲烷熱桿菌在接種污泥中相對(duì)豐度僅占2.69%.在55℃以上的消化溫度,被認(rèn)為是產(chǎn)甲烷的核心功能微生物,能利用H2與CO2合成甲烷[49],并且已證實(shí)其是能夠參與SAO- HM途徑的產(chǎn)甲烷古菌[50].相對(duì)豐度在切換后從2.69%分別上升至48.71%, 37.79%,28.89%和33.34%,這可能是切換后系統(tǒng)氫利用速率大幅提升的重要原因.

綜上所述,在切換過程中SAOB和氫營養(yǎng)型產(chǎn)甲烷菌強(qiáng)化的互營作用抵御了高溫下的氨脅迫和酸脅迫.因此,在厭氧體系中保持高豐度的氫營養(yǎng)型產(chǎn)甲烷菌有利于體系保持低氫分壓,從而更易于保持高負(fù)荷體系下乙酸化和甲烷化的平衡,促進(jìn)產(chǎn)甲烷菌與互營氧化細(xì)菌之間的物質(zhì)及能量交換過程.切換完成后,體系的產(chǎn)甲烷途徑轉(zhuǎn)變?yōu)榛I乙酸氧化產(chǎn)甲烷,證實(shí)以常規(guī)高溫厭氧消化污泥為接種泥切換至高含固熱水解-高溫厭氧消化是一種有效策略.

3 結(jié)論

3.1 乙酸、丙酸、丁酸和熱水解污泥的SMA隨著氨濃度的上升均受到顯著抑制,而氫利用速率和互營乙酸氧化速率卻未受到明顯影響,表明通過建立互營乙酸氧化產(chǎn)甲烷途徑實(shí)現(xiàn)高含固熱水解-高溫厭氧消化具備可行性.

3.2 連續(xù)運(yùn)行試驗(yàn)顯示不同HRT下的高含固熱水解-高溫厭氧消化反應(yīng)器均能穩(wěn)定運(yùn)行,容積產(chǎn)氣率分別為(0.40±0.09),(0.61±0.12),(0.95±0.17)和(1.79± 0.22)L/(L·d).游離氨達(dá)到(665.65±121.29)mg/L時(shí),系統(tǒng)未有明顯的氨抑制和酸積累現(xiàn)象.在切換過程中,微生物通過改變產(chǎn)甲烷途徑來響應(yīng)游離氨濃度上升產(chǎn)生的環(huán)境脅迫.

3.3 切換后各反應(yīng)器污泥最大乙酸SMA均有不同程度的降低,而最大氫利用速率顯著提升.同時(shí)產(chǎn)氫產(chǎn)乙酸和互營乙酸氧化功能微生物主要為、、和,其相對(duì)豐度均大幅提升,促進(jìn)了互營乙酸氧化產(chǎn)甲烷途徑的構(gòu)建.切換后乙酸營養(yǎng)型產(chǎn)甲烷菌被淘汰,參與互營乙酸氧化過程的和相對(duì)豐度顯著提高.切換過程成功將產(chǎn)甲烷途徑由乙酸裂解產(chǎn)甲烷轉(zhuǎn)變?yōu)榛I乙酸氧化產(chǎn)甲烷,從而證實(shí)高溫厭氧消化由常規(guī)含固率切換至高含固率是實(shí)現(xiàn)高含固熱水解-高溫厭氧消化的有效策略.

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Effects of the increased solid content of waste activated sludge on syntrophic acetate oxidation for methane production through thermophilic anaerobic digestion.

ZHUJun-zhao, ZHUO Yang, HUA Fei-hu, ZHOU Meng-yu, WANG Xue-na, LIU Jin-yang, HAN Yun*

(School of Environmental and Municipal Engineering, Xi'an University of Architecture and Technology, Xi'an 710055, China)., 2023,43(9)：4697～4707

This study investigated the kinetic rate variations of each stage involved in anaerobic digestion under different ammonia loadings, in this process, the inoculated sludge was obtained from a thermophilic anaerobic digestion system with solid content of 2%. Furthermore, effects of thequick switch between low solid content (2%) and high solid content (10%) on methane production potentials were explored through feeding thermal hydrolyzed sludge to a continuous flow model. Results showed that with ammonia loading increasing, the specific methanogenic activities of typical substrates like acetic acid, propionic acid, butyric acid, and thermal hydrolyzed sludge were all decreased, while the hydrogen utilization rates (HUR) and syntrophic acetate oxidation (SAO) rates were not significantly affected. With continuous flow model, although the chemical oxygen demand (COD) removal ratio was insufficient (27.99±3.66)% and the VFAs accumulation (10.41±2.25) g COD/L was observed when the organic loading reached 14g COD/(L·d), the pH values remained stable at (7.74±0.09). This result suggested that this switching strategy was feasible in achieving stable operating condition. The predominant hydrogen-producing acetogen and syntrophic acetate oxidizing functional genera were(15.29%),(8.89%),(17.99%), and(1.60%) at stable stage. Moreover,, acetoclastic methanogen, was eliminated, whileand, being involved in the syntrophic acetate oxidation process, was significantly enriched. It can be concluded that the above anaerobic digestion system resists the environmental stress of high temperature and ammonia through establishing syntrophic acetate oxidation coupled with hydrogenotrophic methanogenesis (SAO-HM) pathway. Overall, using conventional thermophilic anaerobic digestion sludge as the inoculated sludge is an effective strategy to achieveswitching from conventional solid content to high solid thermal hydrolysis pretreatment following thermophilic anaerobic digestion (THP-TAD), and the syntrophic acetate oxidation activity of the inoculated sludge is the key factor for this successful switch.

high solid sludge；thermophilic anaerobic digestion；thermal hydrolysis pretreatment；syntrophic acetate oxidation；ammonia inhibition

X703

A

1000-6923(2023)09-4697-11

朱俊兆(1998-),男,陜西西安人,西安建筑科技大學(xué)碩士研究生,主要研究方向?yàn)槌鞘形鬯畯S污泥處理與處置.17342947626@163.com

朱俊兆,卓 楊,華飛虎,等.提高污泥含固率對(duì)高溫厭氧消化互營產(chǎn)甲烷影響 [J]. 中國環(huán)境科學(xué), 2023,43(9):4697-4707.

ZhuJ Z, Zhuo Y, Hua F H, et al. Effects of the increased solid content of waste activated sludge on syntrophic acetate oxidation for methane production through thermophilic anaerobic digestion [J]. China Environmental Science, 2023,43(9):4697-4707.

2023-02-19

國家自然科學(xué)基金面上項(xiàng)目(52070153);國家自然科學(xué)基金青年科學(xué)基金項(xiàng)目(52200175);陜西省自然科學(xué)基礎(chǔ)研究計(jì)劃資助項(xiàng)目(項(xiàng)目編號(hào)2022JQ-445)

* 責(zé)任作者, 教授, hanyun@ xauat.edu.cn