袁　悅，彭永臻，劉　曄，王淑瑩

(北京工業(yè)大學(xué) 北京市水質(zhì)科學(xué)與水環(huán)境恢復(fù)工程重點(diǎn)實(shí)驗(yàn)室，北京市污水脫氮除磷處理與過程控制工程技術(shù)研究中心，北京100124)

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發(fā)酵種泥的投加對新鮮剩余污泥發(fā)酵產(chǎn)酸的影響

袁悅，彭永臻，劉曄，王淑瑩

(北京工業(yè)大學(xué) 北京市水質(zhì)科學(xué)與水環(huán)境恢復(fù)工程重點(diǎn)實(shí)驗(yàn)室，北京市污水脫氮除磷處理與過程控制工程技術(shù)研究中心，北京100124)

摘要:為進(jìn)一步提高剩余污泥厭氧發(fā)酵產(chǎn)短鏈脂肪酸(SCFAs)的效率,將pH=10下長期(90 d)厭氧發(fā)酵的種泥接入至新鮮剩余污泥考察其對新鮮剩余污泥厭氧發(fā)酵產(chǎn)酸的影響.實(shí)驗(yàn)在2種pH條件下進(jìn)行:不控制pH (pH=7.4±0.2),一組反應(yīng)器中投加新鮮剩余污泥,并接種厭氧發(fā)酵種泥,另一組反應(yīng)器只投加新鮮剩余污泥;控制pH為堿性 (pH=10±0.2),一組反應(yīng)器中投加新鮮剩余污泥,并接種厭氧發(fā)酵種泥,另一組反應(yīng)器只投加新鮮剩余污泥.結(jié)果表明:相同pH條件下,接種厭氧發(fā)酵種泥反應(yīng)器中的最大產(chǎn)酸量較不接種分別提高4.9(pH=7.4±0.2) 和16.4 mg /g VSS(以COD計(jì)) (pH=10±0.2);同時(shí)接種發(fā)酵污泥、剩余污泥在pH=10±0.2下的最大產(chǎn)酸量較pH=7.4±0.2下的最大產(chǎn)酸量高146.2 mg/g VSS;堿性條件、不接種發(fā)酵種泥的產(chǎn)酸量較不控制pH、接種發(fā)酵種泥的高129.8 mg/g VSS.投加發(fā)酵種泥可提高新鮮剩余污泥厭氧發(fā)酵過程中SCFAs的產(chǎn)生效率;堿性條件與厭氧發(fā)酵種泥對新鮮剩余污泥發(fā)酵產(chǎn)酸具有協(xié)同促進(jìn)作用;與接種發(fā)酵種泥相比,堿性條件對新鮮剩余污泥厭氧發(fā)酵產(chǎn)酸促進(jìn)能力高.

關(guān)鍵詞:發(fā)酵種泥; 剩余污泥; 厭氧發(fā)酵; 短鏈脂肪酸; pH值

目前,城市污水處理廠多采用活性污泥法去除生活污水中的有機(jī)物、氮和磷,然而，傳統(tǒng)活性污泥法會產(chǎn)生大量的污泥,而處理這些污泥的費(fèi)用占水廠總運(yùn)行費(fèi)用的60%[1].因此，為降低污泥處理成本,需對污泥進(jìn)行減量化.污泥減量化處理常用的方法有厭氧消化和好氧消化[2-4].其中厭氧消化又以能產(chǎn)生甲烷氣體、氫氣,活性污泥法脫氮除磷的重要有機(jī)碳源——短鏈脂肪酸(SCFAs)等能源質(zhì)引起廣泛關(guān)注[5-9].城市生活污水的碳氮比、碳磷比較低,污水處理廠為了提高脫氮除磷效果,需要外加碳源,方法之一是直接加入化學(xué)制成的碳源,如乙酸鈉、乙醇等，該方法無疑增加了污水處理廠的處理成本.近年來，有文獻(xiàn)報(bào)道污泥厭氧發(fā)酵不僅可以達(dá)到污泥減量,還可以獲得大量的易生物降解碳源——SCFAs[10-11]. 污泥厭氧消化包括3個階段:水解、酸化和產(chǎn)甲烷.其中水解是整個消化過程的限制性過程[12-13].從產(chǎn)酸的角度出發(fā),提高水解過程并且抑制產(chǎn)甲烷過程才能使得SCFAs積累.研究表明,污泥在堿性條件下發(fā)酵產(chǎn)酸量比酸性或中性時(shí)高得多[14-15],但是達(dá)到最大SCFAs產(chǎn)量的發(fā)酵時(shí)間仍需要8～10 d.

厭氧條件下污泥的水解、酸化和產(chǎn)甲烷各過程是通過大量的細(xì)菌或者古菌實(shí)現(xiàn)的.因此,污泥厭氧發(fā)酵過程中微生物起著很重要的作用[16-18].該過程引進(jìn)水解菌、發(fā)酵菌或許會進(jìn)一步提高剩余污泥產(chǎn)酸量,縮短發(fā)酵時(shí)間.接偉光等[19]通過分離剩余污泥中的耐堿性菌株HIT-01和HIT-02,并將2種細(xì)菌單獨(dú)或者同時(shí)投加到新鮮污泥中,觀察其在堿性條件下的產(chǎn)酸量情況.結(jié)果表明，接種耐堿性菌株可以增加剩余污泥發(fā)酵產(chǎn)酸,同時(shí)接種2種菌株時(shí)SCFAs積累量最多.然而該研究依然控制整個發(fā)酵過程為堿性條件,未考查不控制pH(即中性)的情況,并且投加的菌種是通過分離純培養(yǎng)得到的,操作復(fù)雜. 基于以上研究,將pH=10下長期發(fā)酵的污泥作為種泥接至新鮮剩余污泥中,在不同條件下(堿性條件、不控制pH條件)考察其對新鮮剩余污泥厭氧發(fā)酵過程中產(chǎn)生SCFAs的影響,并進(jìn)一步從產(chǎn)酸所需的底物(蛋白質(zhì)和多糖)方面分析不同條件下影響產(chǎn)SCFAs的原因.

1實(shí)驗(yàn)

1.1剩余污泥

實(shí)驗(yàn)所用新鮮剩余污泥均取自以實(shí)際生活污水為處理對象的中試SBR (有效體積6 m3).此SBR 的1個運(yùn)行周期為12 h,運(yùn)行方式為:進(jìn)水(0.25 h)、好氧(6 h)、缺氧(1 h)、厭氧(2 h)、沉淀(2.25 h)、排水(0. 5 h).其中好氧段主要是短程硝化,缺氧段則是短程反硝化,剩余污泥在厭氧末端排出.排出的污泥在4 ℃下沉淀濃縮,濃縮后污泥的主要成分如表1所示.

表1　實(shí)驗(yàn)用剩余污泥性質(zhì)

注:TSS為污泥中懸浮物固體濃度, VSS為污泥中揮發(fā)性懸浮物固體濃度, TCOD為剩余污泥總COD, SCOD為可溶性COD, SCFAs為短鏈脂肪酸.

1.2實(shí)驗(yàn)方法

1.2.1厭氧發(fā)酵種泥的馴化

采用5 L的半連續(xù)厭氧發(fā)酵反應(yīng)器(SAFR).控制污泥發(fā)酵的pH在10±0.2,溫度為(30±2)℃,污泥停留時(shí)間SRT為6 d,即每天排833 mL發(fā)酵污泥,并加入等體積(833 mL)的新鮮剩余污泥至SAFR中.該反應(yīng)器的污泥來源為上述新鮮剩余污泥,運(yùn)行30 d后監(jiān)測其產(chǎn)酸量,在90 d時(shí)測定發(fā)酵污泥的微生物菌群結(jié)構(gòu).

1.2.2厭氧發(fā)酵種泥對剩余污泥發(fā)酵產(chǎn)酸的影響

取SAFR中的厭氧發(fā)酵種泥1 200 mL,離心棄上清液,再用蒸餾水清洗離心2次.本實(shí)驗(yàn) 共有4組反應(yīng)器:1)不控制pH(pH=7.4±0.2),投加新鮮剩余污泥(1#);2)不控制pH(pH=7.4±0.2),投加新鮮剩余污泥,并接種厭氧發(fā)酵種泥(2#);3)控制pH(pH=10±0.2),投加新鮮剩余污泥(3#);4)控制pH(pH=10±0.2),投加新鮮剩余污泥,并接種厭氧發(fā)酵種泥(4#).每組反應(yīng)器設(shè)有2個平行實(shí)驗(yàn) ,將4組反應(yīng)器置于磁力攪拌器上,轉(zhuǎn)速為120 r/min,溫度θ在(30±2) ℃.每組反應(yīng)器吹氮?dú)? min,去除反應(yīng)器中的溶解氧,保持厭氧環(huán)境.反應(yīng)期間,為了保證各反應(yīng)器的pH在10±0.2,用2 mol·L-1的NaOH和2 mol·L-1HCl調(diào)節(jié).取樣周期為1 d,取出的水樣在轉(zhuǎn)速為4 000 r/min的離心機(jī)中離心10 min,然后用0.45 μm的濾膜抽濾,濾液用來分析各項(xiàng)指標(biāo),過濾后的水樣立即測定,最終數(shù)據(jù)為每組平行實(shí)驗(yàn)的平均值.

1.3DNA提取、PCR擴(kuò)增和Miseq測序

在SAFR運(yùn)行到第90 天并且投加新鮮剩余污泥前取發(fā)酵污泥.首先,泥樣在轉(zhuǎn)速為4 000 r/min的離心機(jī)中離心5 min,棄上清液,然后將泥樣放置冷凍干燥機(jī)進(jìn)行冷凍干燥.稱取0.034 6 g干污泥,用DNA試劑盒(FastDNA Spin Kit for Soil)提取DNA.設(shè)計(jì)的引物用于擴(kuò)增16S rRNA基因的V3+V4區(qū)[20],引物系列見表2. PCR反應(yīng)體系(20 μL)如下:5×FastPfu Buffer，4 μL;2.5 mmol/L dNTPs,2 μL;Forward Primer (5 μmol/L),0.4 μL;Reverse Primer (5 μmol/L),0.4 μL; FastPfu Polymerase,0.4 μL;Template DNA,10 ng;ddH2O,20 μL.

PCR擴(kuò)增條件如下:95 ℃預(yù)變性2 min;然后,95 ℃變性30 s,55 ℃退火30 s,72 ℃延伸45 s,共25個循環(huán);72 ℃最終延伸10 min,最終4 ℃保存.Illumina Miseq測序平臺為PE300,測得的原始數(shù)據(jù)可在NCBI數(shù)據(jù)庫通過檢索號SRA230879獲得,序列的基本信息見表3.

表2　PCR擴(kuò)增所用引物

表3　Illumina Miseq測序序列信息

注:OUT為Operational taxonomic units, 相似水平為0.97.

1.4分析方法

TSS、VSS采用稱重法測定.TCOD、SCOD采用5B-3(B)型COD快速測儀,碳水化合物采用苯酚-硫酸比色法;蛋白質(zhì)采用Lowry-Folin試劑.SCFAs采用Agilent7890A氣相色譜儀測定,FID檢測器,色譜柱型號及尺寸:AgilentdB-WAXetr, 30 m×0.53 mm×0.001 mm,N2為載氣,載氣流量為20 mL·min-1,進(jìn)樣口和檢測器分別維持在220和250 ℃,烘箱起始溫度為80 ℃,最后升溫至240 ℃,進(jìn)樣體積2 μL.實(shí)驗(yàn) 中檢測到的SCFAs包括乙酸、丙酸、正丁酸、異丁酸、正戊酸和異戊酸,將其濃度分別乘以比例系數(shù)1.07、1. 51、1. 82、1.82、2.04、2.04轉(zhuǎn)化為COD 濃度[21]后的加合計(jì)為總SCFAs濃度.采用WTWpH/Oxi340i測定儀測定pH.

2結(jié)果與討論

2.1SAFR中的SCFAs產(chǎn)量及微生物菌群

如圖1所示,SAFR反應(yīng)器在運(yùn)行30 d后,經(jīng)過60 d的穩(wěn)定運(yùn)行,其平均產(chǎn)酸量為(1 721.4±91.2) mg/L. Illumina Miseq測序表明:在phylum水平上分析可得到10種細(xì)菌(圖2),其中Chloroflexi, Proteobacteria, Bacteroidetes和Firmicute是主要的菌屬,Chloroflexi為SAFR中最豐富的菌屬,占30.1%[22].為了進(jìn)一步確定檢測的細(xì)菌在污泥發(fā)酵過程所起的作用,又從genus水平上對Illumina Miseq測序結(jié)果進(jìn)行分析,見表4.水解細(xì)菌——Alcaligenes(3.47%)和Anaerolinea(3.34%)、產(chǎn)酸細(xì)菌——Erysipelothrix(7.31%),Paludibacter(4.96%)和Tissierella(3.59%)分別得到富集[22].

因此,綜合SAFR反應(yīng)器穩(wěn)定的產(chǎn)酸性及富有水解菌和產(chǎn)酸菌,其發(fā)酵污泥可用作種泥.

圖1　SAFR中隨時(shí)間變化的產(chǎn)酸量

Fig.1SCFAs production from excess sludge with the fermentation time

圖2　SAFR中微生物在門水平上的相對豐度

表4SAFR中16 s rRNA基因序列的系統(tǒng)分類(屬的水平, 相對豐度>1%)

Tab.4Phylogenetic classification of the 16 s rRNA gene sequences (relative abundance>1% at genus level) at SAFR

PhylumGenusSAFR/%FirmicutesErysipelothrix7.31BacteroidetesPaludibacter4.96FirmicutesTissierella3.59VerrucomicrobiaProsthecobacter3.57ProteobacteriaAlcaligenes3.47ChloroflexiAnaerolinea3.34ChloroflexiLeptolinea1.08ProteobacteriaThauera8.97

2.2剩余污泥厭氧發(fā)酵產(chǎn)生的SCFAs

圖3表明新鮮剩余污泥在不同條件下產(chǎn)短鏈脂肪酸(SCFAs)的情況.發(fā)酵前期,1#(1～9 d)、2#(1～9 d)、3#(1～10 d)、4#(1～6 d)反應(yīng)器的產(chǎn)酸量均呈現(xiàn)增長的趨勢.而反應(yīng)后期,1#、2#、4#呈下降趨勢,3#則趨于穩(wěn)定.這是因?yàn)?#、2#反應(yīng)器的pH均是中性,隨著發(fā)酵時(shí)間的進(jìn)行,產(chǎn)甲烷菌出現(xiàn),消耗了SCFAs.而3#反應(yīng)器的堿性條件短時(shí)間內(nèi)抑制了產(chǎn)甲烷菌的生長,4#反應(yīng)器雖然是堿性條件,但是接種的厭氧發(fā)酵種泥中含有極少量的產(chǎn)甲烷菌[17、22],待適應(yīng)了厭氧發(fā)酵環(huán)境后便消耗SCFAs.因此,為了獲取不同條件下的最大產(chǎn)酸量,需要控制各自的SRT,即1#～9 d、2#～9 d、3#～10 d、4#～6 d.

從圖3還可以看出,在發(fā)酵第6 天,4#反應(yīng)器(控制pH=10±0.2,并接種厭氧發(fā)酵種泥)產(chǎn)酸量達(dá)187.2 mg /g VSS,而1#、2#、3#反應(yīng)器的最大產(chǎn)酸量分別是36.1 (9 d)、41.0 (9 d)和170.8 mg /g VSS(10 d).這是因?yàn)閴A性條件利于剩余污泥發(fā)酵產(chǎn)酸[14-15],其次接種的厭氧發(fā)酵種泥引入了產(chǎn)酸菌, 提高了產(chǎn)酸效率, 將最大產(chǎn)酸量的時(shí)間提前了3～4 d.另外,在pH不控的條件下,投加種泥的最大產(chǎn)酸量(2#)較不投加種泥的最大產(chǎn)酸量(1#)提高了4.9 mg/g VSS(顯著性P<0.05);在堿性條件下,投加種泥(4#)較不投加種泥的最大產(chǎn)酸量(3#)提高了16.4 mg/g VSS.這說明投加發(fā)酵種泥可提高新鮮剩余污泥厭氧發(fā)酵過程中SCFAs的產(chǎn)生效率,并且堿性條件及厭氧發(fā)酵種泥對剩余污泥厭氧發(fā)酵產(chǎn)酸具有協(xié)同促進(jìn)作用.由于堿性條件利于剩余污泥發(fā)酵產(chǎn)酸[14-15],均接種發(fā)酵污泥的剩余污泥在pH=10±0.2下的最大產(chǎn)酸量較pH=7.4±0.2的高146.2 mg /g VSS.此外,相對于2#反應(yīng)器,3#反應(yīng)器的最大產(chǎn)酸量提高了129.8 mg/g VSS,這表明堿性條件較厭氧發(fā)酵種泥對新鮮剩余污泥厭氧發(fā)酵產(chǎn)酸更具促進(jìn)作用.

圖3　4組反應(yīng)器中短鏈脂肪酸產(chǎn)量

2.3剩余污泥厭氧發(fā)酵產(chǎn)生的溶解性蛋白質(zhì)和多糖

污泥胞外聚合物(extracellular polymeric substances, EPS)是附著于微生物細(xì)胞壁上的大分子有機(jī)多聚物,主要來源為進(jìn)水基質(zhì)、微生物新陳代謝和細(xì)胞自溶,是產(chǎn)生SCFAs所需要的主要基質(zhì).EPS具有復(fù)雜的化學(xué)組成,占總量70%～80%的蛋白質(zhì)和多糖是最主要的2種成分,而核酸、腐殖質(zhì)、糖醛酸、脂類和氨基酸等的含量相對較低[23].因此,本文主要研究了溶解性蛋白質(zhì)和多糖,從產(chǎn)酸所需的基質(zhì)進(jìn)一步分析剩余污泥在不同條件下產(chǎn)酸量不同的原因.

圖4、5表明,溶解性的多糖濃度低于蛋白質(zhì)的濃度,這與污泥中蛋白質(zhì)含量高于多糖有關(guān).另外,溶解性的多糖濃度變化較蛋白質(zhì)的波動大.相對于中性條件,溶解性蛋白質(zhì)在堿性條件下的溶出量較高,這是因?yàn)閴A性導(dǎo)致EPS中的酸性基團(tuán)分離，使得帶負(fù)電的EPS相互排斥,進(jìn)而增加蛋白質(zhì)和多糖的溶解性[24].然而接種了厭氧發(fā)酵種泥的4#反應(yīng)器又比3#的溶解性蛋白質(zhì)高,這是因?yàn)榻臃N種泥引入了水解菌,這些細(xì)菌會釋放胞外酶[23],進(jìn)而促進(jìn)了蛋白質(zhì)、多糖的釋放.

圖4　4組反應(yīng)器中溶解性蛋白質(zhì)的釋放量

圖5　4組反應(yīng)器中溶解性多糖的釋放量

短鏈脂肪酸是產(chǎn)酸菌利用溶解性蛋白質(zhì)和多糖的水解產(chǎn)物(氨基酸、單糖)生成的,而蛋白質(zhì)的水解伴隨有NH4+-N的生成.因此，發(fā)酵液中NH4+-N的釋放可以表征溶解性蛋白質(zhì)的水解作用[25].由圖6可以看出,隨著發(fā)酵時(shí)間不斷增加,4組反應(yīng)器發(fā)酵液中的NH4+-N含量逐步升高,這表明溶解性有機(jī)物不斷水解生成小分子有機(jī)物,為產(chǎn)酸菌發(fā)酵產(chǎn)酸提供了所需的基質(zhì).4組反應(yīng)器的NH4+-N含量高低順序?yàn)?#>3#>2#>1#.顯然4#反應(yīng)器的產(chǎn)酸基質(zhì)最充足,這與產(chǎn)酸量最多相吻合.另外,堿性條件抑制產(chǎn)甲烷菌的活性,也造就了SCFAs的積累.因此,接入的產(chǎn)酸菌、足夠的發(fā)酵產(chǎn)酸所需基質(zhì)以及產(chǎn)甲烷過程受抑制是4#反應(yīng)器產(chǎn)酸效率高的原因.

圖6　4組反應(yīng)器中氨氮(NH4+-N)的釋放量

3結(jié)論

1)投加厭氧發(fā)酵種泥提高了剩余污泥厭氧發(fā)酵產(chǎn)短鏈脂肪酸的效率.在pH不控的條件下,投加種泥的產(chǎn)酸量較不投加提高了4.9 mg/g VSS;在堿性pH條件下,投加種泥較不投加種泥的產(chǎn)酸量提高了16.4 mg /g VSS. 接入的產(chǎn)酸菌以及足夠的發(fā)酵產(chǎn)酸所需基質(zhì)是產(chǎn)酸效率高的原因.

2)種泥的投加與堿性條件對剩余污泥發(fā)酵產(chǎn)酸具有協(xié)同促進(jìn)作用.

3)堿性條件比投加種泥對剩余污泥發(fā)酵產(chǎn)酸更具促進(jìn)作用.

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(編輯劉彤)

doi：10.11918/j.issn.0367-6234.2016.08.006

收稿日期:2016-02-18

基金項(xiàng)目:國家自然科學(xué)基金(51578014); 北京市教委資助項(xiàng)目

作者簡介:袁悅(1988—), 女, 博士;

通信作者:彭永臻, pyz@bjut.edu.cn

中圖分類號:X703

文獻(xiàn)標(biāo)志碼:A

文章編號:0367-6234(2016)08-0037-05

Effect of fermented sludge on short-chain fatty acids (SCFAs) production from excess sludge

YUAN Yue, PENG Yongzhen, LIU Ye, WANG Shuying

(Key Laboratory of Beijing for Water Quality Science and Water Environment Recovery Engineering,Engineering Research Center of Beijing, Beijing University of Technology, Beijing 100124, China)

Abstract:To enhance the production efficiency of short chain fatty acids (SCFAs) in anaerobic sludge fermentation reactor, sludge from another stabilized fermentation reactor (over 90 days) under pH 10 was seeded into the reactors to examine its effect on SCFAs production. Two sets of experiment were designed to investigate the SCFAs production with/without seeding sludge under pH=7.4±0.2 and pH=10±0.2, respectively. Experimental results showed that (1) under same pH condition, the maximum SCFAs accumulation with seeding sludge additive increased by 4.9 mg COD/g VSS (pH=7.4±0.2) and 16.4 mg COD/g VSS (pH=10±0.2) respectively, in comparison with the control reactors; (2) with two reactors with seeding sludge, the maximum SCFAs accumulation at pH 10±0.2 increased by 146.2 mg COD/g VSS as compared with the condition of pH=7.4±0.2; (3) the maximum SCFAs accumulation at pH 10±0.2 without seeding sludge was 129.8 mg COD/g VSS higher than that of pH 7.4±0.2 with seeding sludge. Overall, seeding sludge improved SCFAs production efficiency, meanwhile, seeding sludge under alkaline pH could play synergetic role in enhancing the SCFAs accumulation.

Keywords:fermented sludge; excess sludge; anaerobic fermentation; short-chain fatty acids; pH value

彭永臻(1949—), 男, 博士生導(dǎo)師，中國工程院院士