何 琴,李 蕾,瞿 莉,趙小飛,伍 迪,彭緒亞*
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餐廚垃圾干式厭氧消化污泥膨脹微生態(tài)特征
何 琴1,2,李 蕾1,瞿 莉1,趙小飛1,伍 迪1,彭緒亞1*
(1.重慶大學(xué),三峽庫區(qū)生態(tài)環(huán)境教育部重點(diǎn)實(shí)驗(yàn)室,重慶 400045;2.西華師范大學(xué),環(huán)境科學(xué)與工程學(xué)院, 四川 南充 637002)
分別在推流式反應(yīng)器(PFR)(R1)和完全混合式反應(yīng)器(CSTR)(R2)中進(jìn)行餐廚垃圾(KW)中溫干式厭氧消化(AD),并采用MiSeq高通量測序技術(shù)分析反應(yīng)器污泥膨脹前后的微生態(tài)特征.結(jié)果表明,污泥膨脹后古菌群落結(jié)構(gòu)變化不顯著,R1和R2分別以乙酸營養(yǎng)型的和復(fù)合營養(yǎng)型的為優(yōu)勢產(chǎn)甲烷菌;而細(xì)菌群落中某些可能與污泥膨脹有關(guān)的菌屬的相對豐度顯著增大,包括能合成并分泌生物表面活性物質(zhì)的菌屬(如、等)和細(xì)胞壁含分枝菌酸的菌屬(如).反應(yīng)器在污泥膨脹前均經(jīng)歷了揮發(fā)性脂肪酸(VFAs)和氨氮積累,相應(yīng)的,能貢獻(xiàn)系統(tǒng)酸積累(如、和等)和氨氮積累(如、和等)的菌屬也大量增殖.
餐廚垃圾;干式厭氧消化;污泥膨脹;微生物;MiSeq
污泥起泡或膨脹問題是困擾厭氧消化(AD)系統(tǒng)穩(wěn)定運(yùn)行的一大難題[1-3].過量的泡沫或膨脹污泥會引起泵堵塞或反應(yīng)器損壞,以及沼氣產(chǎn)量減小和維護(hù)費(fèi)用增加等問題[1-2].作為典型生化反應(yīng)器的AD反應(yīng)器中,微生物在起泡或膨脹過程中扮演著極其重要的角色[4-5].例如,研究者指出污水污泥中溫濕式AD反應(yīng)器中大量增殖的或sp.是誘導(dǎo)泡沫形成的主要原因[6-7].然而,這些絲狀菌是由污水污泥作為底物或接種污泥所帶入的,并不能解釋其他底物或接種物的AD系統(tǒng)起泡或污泥膨脹.在不同的運(yùn)行條件下,AD系統(tǒng)發(fā)生起泡或污泥膨脹時(shí)的微生物群落結(jié)構(gòu)可能是不同的[4].目前在極易出現(xiàn)污泥膨脹或泡沫的餐廚垃圾(KW)的中溫干式AD系統(tǒng)中,鮮有關(guān)于微生物群落變化以及特征微生物的報(bào)道[8-9].另外,以往研究者們在研究AD起泡反應(yīng)器中微生物的過程中通常采用微生物染色鏡檢、DGGE、qPCR和FISH等常規(guī)分子生物技術(shù)[7,10-12],這些技術(shù)僅能檢測到優(yōu)勢菌種,容易遺漏某些重要的信息,結(jié)果并不全面,而新一代的高通量測序技術(shù)則克服了這一困難.
故本研究分別在推流式反應(yīng)器(PFR)和完全混合式反應(yīng)器(CSTR)中進(jìn)行KW中溫干式AD,并分別通過超負(fù)荷和氨抑制誘導(dǎo)污泥膨脹.隨后結(jié)合運(yùn)行參數(shù)監(jiān)測結(jié)果,采用MiSeq高通量測序技術(shù)研究污泥膨脹的微生態(tài)特征,以期為KW中溫干式AD污泥膨脹機(jī)理研究和及時(shí)消泡提供理論依據(jù).
KW取自重慶大學(xué)某學(xué)生食堂.按本實(shí)驗(yàn)室以往報(bào)道[3,13]的方式進(jìn)行采集、預(yù)處理及存儲.接種污泥取自常溫下運(yùn)行的某戶農(nóng)家沼氣池,在接種前過10目篩,去除其中的無機(jī)混雜物.隨后離心沉淀以滿足干式運(yùn)行總含固率(TS)要求(20%~40%)[14].并在(37±1)℃預(yù)孵化2周,以去除其中殘留的原有機(jī)底物.而R2在預(yù)孵化完成進(jìn)行馴化過程中因反應(yīng)器底部出料口漏水而停止馴化,待反應(yīng)器維修好后重新馴化.KW和接種污泥的主要理化性質(zhì)見表1.
表1 KW和接種污泥的理化特征
注: S1和S2分別代表R1和R2的接種污泥.
R1為總?cè)莘e30L,工作容積18L的PFR,通過與循環(huán)加熱水箱相連的容器夾套維持消化污泥溫度為(37 ± 1)℃.半連續(xù)方式運(yùn)行,回流比5:1.初始有機(jī)負(fù)荷(OLR) 3.0kgVS/(m3·d),以一定的梯度逐步提升OLR,分別在3.0、4.1、5.5、6.8和8.5kgVS/(m3·d)運(yùn)行1、1、2、3、2個(gè)水力停留時(shí)間(HRT),各個(gè)負(fù)荷的HRT分別為45d、35d、25d、20d和15d.R2為總?cè)莘e50L,工作容積30L的CSTR,溫度(37±1)℃,溫控方式同R1.攪拌轉(zhuǎn)速為45r/min,采用攪拌5min,停歇55min的間歇攪拌方式.半連續(xù)方式運(yùn)行,以3.0kgVS/(m3·d)負(fù)荷啟動并在該負(fù)荷下長期運(yùn)行.
在每日進(jìn)料前測定理化參數(shù),如產(chǎn)氣量、氣體成分、pH值、TS、VS、總堿度(TA)、總氨氮(TAN)、VFAs,測定方法參考以往報(bào)道[3,5,13].沼氣量換算成標(biāo)準(zhǔn)狀態(tài)(0℃,101.325kPa)下的氣體體積.比甲烷產(chǎn)率(SMP)的計(jì)算參考本實(shí)驗(yàn)室以往報(bào)道[3,5].
分別在R1、R2污泥膨脹前(第IV階段,見圖1)和膨脹后(第V階段)各采集3個(gè)污泥樣品.使用E.Z.N.A Soil DNA試劑盒(Omega,美國)參照操作說明進(jìn)行基因組DNA抽提.對所提取的3份DNA進(jìn)行純化并混合.使用細(xì)菌引物338F (5’-ACTCC TACGG GAGGC AGCAG-3’)和806R (5’-GGACT ACCAG GGTAT CTAAT -3’)[15],古菌引物Arch344F (5’-ACGGG GYGCA GCAGG CGCGA-3’)和Arch915R (5’-GTGCT CCCCC GCCAA TTCCT-3’)[13]對16S rRNA基因片段進(jìn)行PCR擴(kuò)增(GeneAmp? 9700型,ABI,美國).擴(kuò)增產(chǎn)物用2%瓊脂糖凝膠電泳檢測,經(jīng)純化后送往上海美吉生物技術(shù)有限公司進(jìn)行MiSeq高通量測序.參考文獻(xiàn)[16]對原始數(shù)據(jù)進(jìn)行質(zhì)量控制和OTU聚類分析.采用Mothur軟件(v.1.30.1)對OTU進(jìn)行多樣性指數(shù)分析.
使用PASW Statistic軟件(v 18.0)分析各個(gè)理化指標(biāo)的均值和標(biāo)準(zhǔn)偏差.理化數(shù)據(jù)之間的差異顯著性采用t檢驗(yàn)分析(置信水平為5%).污泥膨脹前后微生物相對豐度差異通過2檢驗(yàn)計(jì)算[17].
經(jīng)過餐廚垃圾AD馴化后,R1在3.0~ 6.8kgVS/(m3·d)高效穩(wěn)定地運(yùn)行(圖1).在OLR繼續(xù)提升至8.0kgVS/(m3·d)并運(yùn)行1個(gè)HRT后(第204d),污泥體積開始膨脹.直至第214d膨脹的污泥堵塞出氣口,反應(yīng)器內(nèi)部壓力持續(xù)增大最終導(dǎo)致反應(yīng)器頂蓋炸裂,結(jié)束運(yùn)行.R2經(jīng)長期運(yùn)行后,系統(tǒng)中開始逐漸反復(fù)出現(xiàn)氨抑制現(xiàn)象,最后系統(tǒng)失穩(wěn)(圖1),并在運(yùn)行第220d開始出現(xiàn)污泥膨脹,直至233d膨脹的污泥占據(jù)反應(yīng)器的整個(gè)頂空體積,迫使運(yùn)行停止.
圖1 運(yùn)行期間反應(yīng)器性能參數(shù)變化情況
R1的I-V運(yùn)行階段分別代表OLR為3.0、4.1、5.5、6.8和8.5kgVS/(m3·d)的階段;R2的I-V運(yùn)行階段分別代表馴化期、穩(wěn)定期、臨界失穩(wěn)期、失穩(wěn)期、污泥膨脹期
本文所述的AD污泥膨脹或起泡現(xiàn)象描述的都是沼氣氣體在反應(yīng)器內(nèi)被截留的現(xiàn)象;區(qū)別在于兩個(gè)現(xiàn)象所處的反應(yīng)器含固率不同.起泡現(xiàn)象出現(xiàn)在濕式AD反應(yīng)器中,沼氣氣泡上升過程中攜帶固體顆粒一起上升,最后在反應(yīng)器上部累積,反應(yīng)器內(nèi)含物出現(xiàn)明顯的分層現(xiàn)象,且上部形成的泡沫層的含固率明顯高于下部的消化液.而在高含固率的干式AD反應(yīng)器中,分布在消化污泥中的產(chǎn)甲烷菌所產(chǎn)生的氣體被表面活性物質(zhì)以及污泥固體顆粒截留,無法順利上升或溢出污泥,使得氣體均勻分布在污泥體中,形成富含細(xì)小沼氣氣泡的污泥膨脹體,即整體體積膨脹.
而類似于濕式AD的泡沫現(xiàn)象,本研究的R1和R2在污泥膨脹前均監(jiān)測到VFAs和TAN的大量積累(圖1b,c).Zhang等[9]在餐廚垃圾AD系統(tǒng)中觀察到總VFAs迅速積累至20.00g/L的同時(shí),系統(tǒng)中出現(xiàn)了嚴(yán)重的泡沫事件.另外,由于產(chǎn)甲烷菌對抑制物的敏感性,氨抑制最后也往往表現(xiàn)為VFAs積累[18-19].R2正是由于逐漸積累的TAN導(dǎo)致VFAs快速積累的;隨后pH值的下降,使得TAN平衡從游離態(tài)(NH3)轉(zhuǎn)向毒性更低的離子形態(tài)(NH4+),雖然減弱了對產(chǎn)甲烷菌活性的抑制作用,但VFAs仍繼續(xù)積累(圖1b,c). Lv等[20]也在雞糞和青貯AD起泡前觀察到了TAN和VFAs先后大量積累.
系統(tǒng)中積累的VFAs和NH4+可能對污泥膨脹有貢獻(xiàn).因?yàn)閂FAs的羧基端具有表面活性性質(zhì),而NH4+也可以與長鏈脂肪酸作用形成同時(shí)具有疏水和親水端的離子化合物,它們趨于附著在微小的沼氣氣泡的氣液界面并積累,減小消化液的表面張力,從而防止氣泡破滅,促進(jìn)污泥膨脹[6,21].
利用MiSeq高通量測序技術(shù)分析各個(gè)反應(yīng)器在污泥膨脹前后的微生物群落組成,其統(tǒng)計(jì)學(xué)分析結(jié)果見表2.
各個(gè)樣品細(xì)菌和古菌群落良好的文庫覆蓋率表明所測的序列量已基本上可完整反映該樣品實(shí)際序列信息.豐富度指數(shù)(ACE和Chao1)指數(shù)和多樣指數(shù)(Shannon和Simpson)結(jié)果表明,R1中,相比于污泥膨脹發(fā)生當(dāng)天所取樣品R1-2,負(fù)荷6.8kgVS/(m3·d)穩(wěn)定運(yùn)行期的樣品R1-1的細(xì)菌和古菌群落的豐富度和多樣性均最高.R2中,與污泥膨脹發(fā)生前的失穩(wěn)期樣品R2-1相比較,污泥膨脹期樣品R2-2的細(xì)菌群落豐富度更高,而多樣性則較低;而古菌群落的豐富度和多樣性則是在污泥膨脹發(fā)生前更高.
表2 微生物序列統(tǒng)計(jì)學(xué)分析結(jié)果
注: R1-1和R1-2、R2-1和R2-2分別代表R1、R2污泥膨脹前后的微生物樣品,分別取自R1、R2的第IV和V階段.
污泥膨脹前后真細(xì)菌和古菌群落結(jié)構(gòu)組成分別見圖2a和圖2b.
圖2 不同樣品中真細(xì)菌(a)和古菌(b)的群落結(jié)構(gòu)
僅顯示至少在一個(gè)樣品中的比例大于1%的古菌屬或細(xì)菌門
污泥膨脹前,Firmicutes和Bacteroidetes是兩個(gè)反應(yīng)器共同的優(yōu)勢細(xì)菌門,兩者相對豐度總和高達(dá)56.2%~89.0%(圖2a).它們主要負(fù)責(zé)有機(jī)底物的水解和酸化,也是其他KW的AD系統(tǒng)中常見的優(yōu)勢細(xì)菌門[4,22].污泥膨脹后,兩個(gè)反應(yīng)器中的Firmicutes門比例均明顯減小,尤其是R2中減小幅度更大;另一個(gè)優(yōu)勢菌門Bacteroidetes除在R1中減小約20%外,在R2中無明顯變化.而Actinobacteria的相對豐度在污泥膨脹后的兩個(gè)反應(yīng)器中明顯增大,分別增大為26.03%和16.39%.Actinobacteria的成員大多具有絲狀結(jié)構(gòu),也主要負(fù)責(zé)水解和酸化過程[23].而R2中Actinobacteria的相對豐度低于R1,可能是因?yàn)镽2的攪拌剪切作用不利于絲狀菌生長.另外,R2中的Synergistetes增大了106%,其成員主要是厭氧氨基酸降解菌以及可與產(chǎn)甲烷菌共養(yǎng)的互養(yǎng)乙酸氧化細(xì)菌[24-25].為明晰這些變化與污泥膨脹現(xiàn)象之間的相關(guān)性,找出與之相關(guān)的特征微生物,進(jìn)一步從屬水平上分析污泥膨脹后變化顯著的細(xì)菌屬,發(fā)現(xiàn)許多菌屬的比例在污泥膨脹后顯著增大(圖3和圖4).
在污泥膨脹后的R1中,屬于Actinobacteria門的屬的增幅高達(dá)1334%(圖3).眾所周知,屬是絲狀菌,且細(xì)胞壁上存在分枝菌酸,極其疏水[6,26].而細(xì)胞的疏水性質(zhì)驅(qū)使絲狀菌附著在氣泡上,并在氣泡界面上積累,同時(shí)分泌大量具有表面活性性質(zhì)的用于有機(jī)物降解的胞外酶,降低污泥表面張力,促進(jìn)了污泥膨脹[27].可見該微生物的增殖也許與污泥膨脹相關(guān).Actinobacteria門屬的增幅超過55%,其成員的細(xì)胞壁上含分枝菌酸,且能產(chǎn)生生物表面活性物質(zhì)(如磷脂)[26],其大量繁殖也可能與系統(tǒng)污泥膨脹有關(guān).
另外,大量產(chǎn)酸菌在R1污泥膨脹后也顯著增殖,如Fastidiosipila、Petrimonas、Anaerosalibacter、Defluviitalea、Mobilitalea、Ruminococcaceae_ NK4A214、Guggenheimella、Caldicoprobacter[28-35](圖3).其中,Fastidiosipila污泥膨脹后的增幅為34%,以乙酸和丁酸為主要的末端產(chǎn)物[28]. Petrimonas增幅為75%,在元素硫存在時(shí)可水解多種碳水化合物和有機(jī)酸,并產(chǎn)生大量的乙酸[29].耐鹽耐熱菌屬Anaerosalibacter增幅高達(dá)235%,并以乙酸、丁酸和H2為主要的發(fā)酵產(chǎn)物[30].而可發(fā)酵多種糖類并以以乳酸、乙酸等有機(jī)酸作為最終產(chǎn)物的Caldicoprobacter屬增幅為320%[35].
最后,在R1的膨脹污泥樣品中也檢測到了蛋白類物質(zhì)代謝菌屬的大量增殖,如、、、[25,29,34,36].其中,屬是典型厭氧氨基酸降解細(xì)菌,當(dāng)與耗氫的產(chǎn)甲烷菌聯(lián)合培養(yǎng)時(shí)可發(fā)酵多種氨基酸(如丙氨酸、絲氨酸、半胱氨酸等)[25],增幅高達(dá)696%.專性厭氧的蛋白質(zhì)水解菌增幅約為71%,可水解發(fā)酵酵母提取物、蛋白胨、丙酮酸、甘氨酸和精氨酸等,并以乙酸和NH3為其主要的水解產(chǎn)物[29].可代謝多種蛋白類物質(zhì)鐵還原細(xì)菌增幅為433%[37].
圖3 R1污泥膨脹前后細(xì)菌相對豐度差異性比較
僅顯示相對豐度高于0.5%且在污泥污泥膨脹后顯著增大的細(xì)菌屬
圖4 R2污泥膨脹前后細(xì)菌相對豐度差異性比較
僅顯示相對豐度高于0.5%且在污泥污泥膨脹后顯著增大的細(xì)菌屬
污泥膨脹后的R2中,可發(fā)酵多種碳水化合物并以乳酸為主要的發(fā)酵產(chǎn)物的屬增幅高達(dá)1189%[34](圖4).而乳酸被廣泛地應(yīng)用于食品行業(yè)作為發(fā)泡劑[27],且在發(fā)酵過程中還會產(chǎn)生具有發(fā)泡性質(zhì)的生物表面活性劑(主要分為蛋白類、糖脂、糖蛋白、糖脂肽四大類)[37].Kougias等也在其泡沫反應(yīng)器中也檢測到了該菌屬的大量增殖[27].另外,降解纖維素或半纖維產(chǎn)甲酸、乙酸等VFAs的屬在污泥膨脹后的R2中大幅增殖,增幅高達(dá)1021%[38].可發(fā)酵糖類和淀粉等碳水化合物產(chǎn)乙酸、甲酸和乙醇的屬增幅110%[39].最后,在污泥膨脹后的R2樣品中,也檢測到了曾在R1中大量增殖的絲狀菌屬,產(chǎn)酸菌屬、和_NK4A214,以及產(chǎn)NH4+菌屬、和的大量增殖.
從圖2b可看出,兩個(gè)反應(yīng)器有著不同的優(yōu)勢產(chǎn)甲烷菌屬,且污泥膨脹前后無明顯變化,分別為乙酸型產(chǎn)甲烷菌,以及復(fù)合營養(yǎng)型的.此差異有可能是由系統(tǒng)內(nèi)抑制物質(zhì)濃度或反應(yīng)器構(gòu)型的不同造成的.研究表明,在高TAN反應(yīng)器中的乙酸裂解途徑,轉(zhuǎn)變?yōu)橐宜崾紫冉?jīng)互營乙酸氧化生成H2和CO2,隨后再被耗氫的產(chǎn)甲烷菌利用生成甲烷的途徑[40-41].故R2運(yùn)行初期較高的TAN使可利用H2/CO2的得到了一定程度的富集;加之R2運(yùn)行時(shí)的攪拌剪切作用不利于絲狀結(jié)構(gòu)的形成,其含量逐漸減少,最終變?yōu)橹鲗?dǎo)產(chǎn)甲烷菌[19,22,42].而由于R1的推流方式有利于形成多細(xì)胞聚集結(jié)構(gòu),用于抵抗環(huán)境中高VFAs和TAN抑制[22],故并未因污泥膨脹期系統(tǒng)高VFAs和TAN而淘汰.
另外,反應(yīng)器中還存在少量典型的氫營養(yǎng)型產(chǎn)甲烷菌,包括以及,它們可不斷消耗系統(tǒng)中的H2,從而避免氫分壓過高而產(chǎn)生抑制[40-41].
反應(yīng)器的古菌群落結(jié)構(gòu)在污泥膨脹前后均無明顯變化,故推測產(chǎn)甲烷古菌與污泥膨脹之間無明顯相關(guān)性[27].
本研究分別通過超負(fù)荷或氨抑制在兩個(gè)餐廚垃圾厭氧消化反應(yīng)器中誘導(dǎo)了污泥膨脹,隨后在兩個(gè)污泥膨脹反應(yīng)器中均檢測到了細(xì)胞壁含有分枝菌酸的的大量增殖.另外,還檢測到可產(chǎn)生具有表面活性的物質(zhì)的菌屬大量增殖,如可產(chǎn)生生物表面活性劑磷脂且細(xì)胞壁也含有分枝菌酸的(R1);分泌發(fā)泡物質(zhì)乳酸以及大量生物表面活性物質(zhì)的(R2).這些菌屬所具有的極其輸水的絲狀結(jié)構(gòu),或產(chǎn)生的具有表面活性性質(zhì)的物質(zhì),促進(jìn)了污泥膨脹.另外,大量產(chǎn)酸菌(、和等)和產(chǎn)NH4+的菌屬(、和等)的比例在污泥膨脹后大幅增加,這解釋了系統(tǒng)在污泥膨脹前出現(xiàn)的VFAs快速積累和NH4+大量富集的現(xiàn)象.然而,如前所述,高濃度的VFAs和NH4+可促進(jìn)污泥膨脹.故推測這些產(chǎn)酸和產(chǎn)NH4+菌屬的大幅增殖現(xiàn)象可能也對系統(tǒng)污泥膨脹有一定的潛在貢獻(xiàn).
3.1 污泥膨脹前后古菌群落結(jié)構(gòu)變化不顯著,分別以乙酸營養(yǎng)型的和復(fù)合營養(yǎng)型的為優(yōu)勢產(chǎn)甲烷菌.
3.2 某些特征微生物的大量繁殖以及表面活性物質(zhì)的大量積累共同導(dǎo)致了AD污泥污泥膨脹.
3.3 這些特征微生物包括細(xì)胞壁含分枝菌酸的菌屬(如、)和能合成并分泌生物表面活性物質(zhì)的菌屬(如、等).
3.4 可貢獻(xiàn)系統(tǒng)VFAs積累(如、和等)和TAN積累(如、和等)的菌屬也大量增殖.
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Microbial characteristics of bulking sludge in high-solids anaerobic digestion of kitchen waste.
HE Qin1,2, LI Lei1, QU Li1, ZHAO Xiao-fei1, WU Di1, PENG Xu-ya1*
(1.Key Laboratory of Three Gorges Reservoir Region’s Eco-Environment, Ministry of Education, Chongqing University, Chongqing 400045, China;2.Department of Environmental Science and Engineering, China West Normal University, Nanchong 637002, China)., 2018,38(3):1010~1017
A plug-flow reactor (PFR) (R1) and a completely-stirred tank reactor (CSTR) (R2) were operated under mesophilic temperature (37±1℃) for high-solids digestion of kitchen waste to investigate the microbial community characteristics before and after sludge-bulking using MiSeq high-throughput sequencing technology. The results showed that the archaeal community structure changed little after sludge-bulking, and acetoclastic methanogenand mixotrophic methanogendominated in both reactors. There was a marked increase in the relative abundance of bacteria genera that might be related to sludge bulking. Those proliferated bacteria genera are those capable of producing biosurfactants (,, etc.) and those containing mycolic acids in cell walls (,, etc.). In addition, volatile fatty acids (VFAs) and total ammonia nitrogen (TAN) were accumulated in these reactors before sludge-bulking. Accordingly, bacteria that can contribute to the accumulation of VFAs (,and, etc.) and TAN (and, etc.) were observed to proliferate.
food waste;high-solid anaerobic digestion;sludge bulking;microorganisms;MiSeq
X705
A
1000-6923(2018)03-1010-08
何 琴(1988-),女,四川遂寧人,講師,博士,主要從事固體廢物污染控制與資源化研究.發(fā)表論文14篇.
2017-08-12
中央高?;究蒲袠I(yè)務(wù)費(fèi)項(xiàng)目(106112017CDJXY 210006)
* 責(zé)任作者, 教授, xypeng33@126.com