污泥蚯蚓堆肥過程中胞外聚合物的結(jié)構(gòu)變化

陳 進(jìn),謝佳辰,徐俊杰,夏 慧*,黃 魁,2

污泥蚯蚓堆肥過程中胞外聚合物的結(jié)構(gòu)變化

陳 進(jìn)1,謝佳辰1,徐俊杰1,夏 慧1*,黃 魁1,2

(1.蘭州交通大學(xué)環(huán)境與市政工程學(xué)院,甘肅 蘭州 730070；2.蘭州交通大學(xué),甘肅省黃河水環(huán)境重點(diǎn)實(shí)驗(yàn)室,甘肅 蘭州 730070)

為揭示污泥胞外聚合物(EPS)特征與蚯蚓堆肥產(chǎn)物腐熟的關(guān)系,以無蚯蚓為對(duì)照組,探究蚯蚓堆肥對(duì)污泥EPS結(jié)構(gòu)變化的影響.結(jié)果表明,蚯蚓堆肥顯著加快了蚯蚓堆肥產(chǎn)物有機(jī)質(zhì)的礦化速率,其電導(dǎo)率和硝酸鹽氮含量比對(duì)照組產(chǎn)物分別顯著提升了0.64和0.22倍(<0.05).蚯蚓堆肥產(chǎn)物EPS層中的蛋白質(zhì)和多糖的總含量比對(duì)照組產(chǎn)物分別降低了32.77%和31.54%.三維熒光結(jié)果表明,蚯蚓堆肥過程中蛋白質(zhì)的熒光強(qiáng)度下降速率比對(duì)照組更高,其腐殖質(zhì)在堆肥后期一直處于較高水平.污泥蚯蚓堆肥產(chǎn)物的緊密結(jié)合層(TB-EPS)和松散結(jié)合層(LB-EPS)中的色氨酸含量比對(duì)照組分別減少7.69%和13.62%,而LB-EPS和粘液層(SEPS)中腐殖酸含量比對(duì)照組分別增加了25.1%和7.82%.蚯蚓堆肥期間的硝酸鹽氮含量和電導(dǎo)率均同SEPS、LB-EPS結(jié)構(gòu)中的DOC、蛋白質(zhì)和多糖含量呈顯著正相關(guān)(<0.05),其總DOC含量與EPS各層中的DOC及多糖含量呈顯著相關(guān)性(<0.05).實(shí)驗(yàn)過程中對(duì)照組僅有機(jī)質(zhì)和DOC含量同EPS各層多糖含量呈顯著相關(guān)(<0.05).研究表明,蚯蚓堆肥能夠破壞污泥EPS結(jié)構(gòu)以促進(jìn)有機(jī)物的分解,加快污泥的腐殖化和穩(wěn)定化進(jìn)程,且污泥EPS結(jié)構(gòu)的變化可作為反映污泥蚯蚓堆肥產(chǎn)物腐熟程度的關(guān)鍵指標(biāo).

蚯蚓堆肥；污泥資源化；腐熟；胞外聚合物；蚯蚓糞有機(jī)肥

脫水污泥作為污水處理的終端副產(chǎn)物,具有資源和污染的雙重特征,如何對(duì)其合理的處理處置已成為我國(guó)生態(tài)文明建設(shè)的難題之一[1-2].在碳中和背景之下,污泥資源化已成為水處理行業(yè)的共識(shí).蚯蚓堆肥是一種經(jīng)濟(jì)、綠色并且可持續(xù)的污泥資源化技術(shù)[3].相較于其它的污泥處理方法,該技術(shù)具有能耗低、工藝簡(jiǎn)單、堆肥產(chǎn)物肥效高等優(yōu)點(diǎn)[4].蚯蚓堆肥技術(shù)主要利用蚯蚓腸胃中豐富的功能酶和微生物共同作用,實(shí)現(xiàn)污泥的降解和穩(wěn)定[5-6],同時(shí)將其轉(zhuǎn)化為營(yíng)養(yǎng)豐富的蚯蚓糞肥. 胞外聚合物(EPS)是污泥的重要組成成分,是由微生物在其代謝過程中分泌到胞外的天然高分子聚合物[7].根據(jù)EPS與細(xì)胞之間的緊密程度,可將EPS分為粘液層(SEPS)、松散結(jié)合層(LB-EPS)和緊密結(jié)合層(TB-EPS)[8].EPS有著介導(dǎo)細(xì)胞與外界交互的作用,以此形成一個(gè)巨大的網(wǎng)絡(luò)結(jié)構(gòu),并將自由水、部分結(jié)合水與大部分有機(jī)物質(zhì)儲(chǔ)存其中[9].因此,如何破壞污泥的EPS結(jié)構(gòu),改善其脫水性能,已成為污泥深度脫水問題的關(guān)鍵.且EPS中儲(chǔ)存著污泥中60%～80%的有機(jī)物,其主要由蛋白質(zhì)、多糖、腐殖酸、核酸等構(gòu)成.同時(shí),污泥EPS是大部分溶解性有機(jī)物(DOM)的儲(chǔ)存部位,也是微生物活動(dòng)以及降解有機(jī)物的主要場(chǎng)所[10].可見,把握蚯蚓堆肥進(jìn)程中有機(jī)物變化和EPS結(jié)構(gòu)變化的相互關(guān)系,對(duì)于污泥的資源化與穩(wěn)定化有著重要的意義.

本研究提取蚯蚓堆肥過程中各階段產(chǎn)物的EPS,分析蚯蚓堆肥對(duì)污泥穩(wěn)定化進(jìn)程中堆肥產(chǎn)物EPS結(jié)構(gòu)的影響,旨在為污泥蚯蚓堆肥資源化提供參考.

1 材料和方法

1.1 供試材料

供試污泥來自蘭州市安寧區(qū)七里河污水處理廠,其電導(dǎo)率,含水率,硝酸鹽氮,溶解性有機(jī)碳,蛋白質(zhì)和多糖含量分別為(200.00±0.00)mS/cm,73.02%±(0.00,0.33±0.01)mg/g,(16.09±0.06)mg/g,(17.24±2.24)mg/g和(0.90±0.10)mg/g.蚯蚓品種為赤子愛勝蚓(),體重約為0.4g,且在實(shí)驗(yàn)前經(jīng)脫水污泥飼養(yǎng)馴化.

1.2 試驗(yàn)方法

取6個(gè)污泥蚯蚓堆肥反應(yīng)器,其單個(gè)尺寸為60cm′40cm′30cm,材質(zhì)為塑料.首先向反應(yīng)器中投放10kg的新鮮污泥,為避免堆體缺氧,污泥平均厚度約為50mm.而后向其中3個(gè)反應(yīng)器中各接種600條赤子愛勝蚓,將其作為蚯蚓處理組,剩余3個(gè)反應(yīng)器中不接種蚯蚓并將其作為對(duì)照處理組.所有反應(yīng)器上均覆蓋有黑色帶孔塑料膜,并每隔3d噴灑少量水,保持濕度在60%～70%左右,且均在室溫下進(jìn)行(20～ 25℃).本實(shí)驗(yàn)分為2個(gè)階段,第1階段為持續(xù)20d的蚯蚓堆肥階段,在第20d從反應(yīng)器中取出蚯蚓,第2階段為持續(xù)10d的微生物腐熟階段,且腐熟期間所有反應(yīng)器內(nèi)均不噴灑水.

1.2.1 樣品的采集與保存 每10d取一次樣品,樣品采集前先將堆體充分混勻,之后隨機(jī)取出樣品于無菌塑封袋中.部分新鮮樣品用于提取EPS,部分樣品進(jìn)行風(fēng)干后研磨,冷藏于冰箱中備用.

1.2.2 EPS的提取 參考Hu等[11]方法進(jìn)行,將新鮮樣品與去離子水混勻(干樣:水=1:10;質(zhì)量濃度),在4℃下冷藏,靜置沉淀1.5h后丟棄上清液.沉淀物以4000r/min的速度離心15min,收集上清液為SEPS.將剩余的沉淀物中加入0.9%的生理鹽水,搖勻后超聲處理2min,之后以4000r/min的速度離心20min,收集上清液為L(zhǎng)B-EPS.最后再向沉淀物中加入0.9%的生理鹽水,充分混勻后60℃水浴1h,以4000r/ min的速度離心40min,收集的上清液即為TB-EPS.

1.3 測(cè)定方法

1.3.1 理化性質(zhì)的測(cè)定 將風(fēng)干的樣品與去離子水混勻(干樣:水=1:50;質(zhì)量濃度),磁力攪拌30min后測(cè)定pH值(雷磁PHS-3C,上海)和電導(dǎo)率(雷磁DDS-307,上海).上述混合液稀釋10倍后,使用0.45 μm薄濾膜抽濾,直接采用碳氮分析儀(耶拿MULTI N/C,德國(guó))測(cè)定溶解性有機(jī)碳(DOC).將剩余混合液的DOC濃度稀釋至同一水平后,采用熒光分光光度計(jì)(日立F-7100,日本)測(cè)定可溶性有機(jī)物的三維熒光(3D-EEM),其具體測(cè)定參數(shù)如下:PMT電壓700V,激發(fā)波長(zhǎng)(x)為220～450nm,發(fā)射波長(zhǎng)(m)為220～ 500nm,狹縫寬度為5nm,掃描速度為12000nm/min.硝酸鹽氮的含量采用紫外分光光度法進(jìn)行測(cè)定(HJ/T 346-2007).具體理化測(cè)試參照黃魁等[12]方法進(jìn)行.

1.3.2 多糖和蛋白質(zhì)的測(cè)定 蛋白質(zhì)采用Lowry法測(cè)定,以牛血清蛋白作為校準(zhǔn)進(jìn)行定量.多糖采用蒽酮法測(cè)定,以葡萄糖作為標(biāo)準(zhǔn)進(jìn)行定量.具體測(cè)試參照Fr?lund等[13]方法進(jìn)行.

1.4 統(tǒng)計(jì)方法

使用SPSS 26軟件對(duì)樣品的理化性質(zhì)分別進(jìn)行Spearman相關(guān)性及重復(fù)測(cè)量方差分析.使用Statistica 10.0軟件對(duì)樣品的理化性質(zhì)在各組之間的差異進(jìn)行單因素方差分析(One-way ANOVA).使用HemI 1.0軟件繪制熱圖.使用MATLAB R2019b軟件對(duì)3D-EEM圖譜進(jìn)行體積積分(FRI).在進(jìn)行PARAFAC建模之前,先對(duì)3D-EEM數(shù)據(jù)進(jìn)行預(yù)處理,包括去除一階和二階拉曼散射、拉曼單位標(biāo)準(zhǔn)化等.掃描同一批樣品所使用的去離子水水樣的拉曼單位(在350nm固定激發(fā)波長(zhǎng)下所發(fā)射的390～410nm波長(zhǎng)的拉曼信號(hào)的積分),通過將每個(gè)樣品的3D-EEM除以該積分,使拉曼單位標(biāo)準(zhǔn)化,以減少每個(gè)樣品中不同DOM濃度的影響.使用MATLAB R2019b軟件和DOMFluor工具箱[14]構(gòu)建PARAFAC模型,并采用拆半分析和隨機(jī)初始化檢驗(yàn).最后采用Origin2021對(duì)分析結(jié)果進(jìn)行繪制.

2 結(jié)果與討論

2.1 蚯蚓對(duì)污泥理化性質(zhì)變化的影響

電導(dǎo)率能夠在一定程度上反映堆肥產(chǎn)物的礦化程度,一般認(rèn)為電導(dǎo)率小于4.0mS/cm的腐熟堆肥產(chǎn)物安全可用[15].如圖1(a)所示,第30d時(shí)蚯蚓堆肥產(chǎn)物和對(duì)照組產(chǎn)物中的電導(dǎo)率相比原污泥上升了249.17%和113.17%,且蚯蚓堆肥產(chǎn)物的電導(dǎo)率比對(duì)照產(chǎn)物顯著增長(zhǎng)了0.64倍(<0.05).重復(fù)測(cè)量方差分析表明2實(shí)驗(yàn)組呈現(xiàn)顯著差異(<0.01).在堆肥過程中,2組中的電導(dǎo)率均呈上升趨勢(shì),且蚯蚓堆肥的上升速率更快,可能是蚯蚓與微生物的共同作用加速了污泥中有機(jī)物的礦化速率.

含水率會(huì)影響蚯蚓和微生物的正常生命活動(dòng),從而影響堆肥效率.且含水率30%以下的蚯蚓堆肥產(chǎn)物滿足其作為土壤改良劑或有機(jī)肥料的標(biāo)準(zhǔn)[16].圖1(b)可見,相較于原污泥,對(duì)照組和蚯蚓堆肥產(chǎn)物中的含水率在第20d時(shí)分別下降了9.03%和22.74%.實(shí)驗(yàn)結(jié)束時(shí),蚯蚓堆肥產(chǎn)物的含水率相較于對(duì)照組降低了81.43%,且經(jīng)重復(fù)測(cè)量方差分析,2實(shí)驗(yàn)組表現(xiàn)出顯著性差異(<0.01).蚯蚓堆肥可造成污泥水分的快速損失,主要原因可能是由于蚯蚓的生命活動(dòng)破壞了污泥EPS結(jié)構(gòu),釋放出污泥EPS中包裹的自由水和部分結(jié)合水.

圖1(c)顯示,與原污泥相比,第30d時(shí)對(duì)照組和蚯蚓堆肥產(chǎn)物中的硝酸鹽氮含量分別提升了1.72和2.48倍(<0.01),且蚯蚓堆肥產(chǎn)物比對(duì)照組產(chǎn)物顯著增加了21.8%(<0.01),表明蚯蚓能夠加速堆肥體中的硝化作用.吳穎等[17]研究發(fā)現(xiàn)蚯蚓堆肥可以顯著增加堆肥體中氨氧化古菌和氨氧化細(xì)菌的數(shù)量.同時(shí),蚯蚓活動(dòng)可增加污泥和空氣接觸的比表面積,為硝化作用提供了良好的環(huán)境條件,從而加速污泥中有機(jī)物的降解速率.

圖1 堆肥過程中理化性質(zhì)的變化規(guī)律

如圖1(d)所示,堆肥前10d,兩組中污泥有機(jī)質(zhì)含量均迅速下降.第10～20d內(nèi),對(duì)照組產(chǎn)物的有機(jī)質(zhì)含量無變化,而蚯蚓堆肥產(chǎn)物的有機(jī)質(zhì)含量比對(duì)照組顯著(<0.05)下降了4.18%.但在第30d時(shí),對(duì)照組和蚯蚓堆肥產(chǎn)物中有機(jī)質(zhì)含量分別為266.7g/kg和268.4g/kg.

圖1(e)結(jié)果顯示,與原污泥相比,第20d時(shí)對(duì)照組和蚯蚓堆肥產(chǎn)物中水溶性總氮含量分別下降了27.21%和28.53%.在第30d時(shí)對(duì)照組比原污泥降低了41.13%,而蚯蚓堆肥產(chǎn)物顯著(<0.05)增加了18.53%.在實(shí)驗(yàn)過程中,對(duì)照組中的含量一直呈下降趨勢(shì),但在蚯蚓組中呈現(xiàn)先下降后上升的趨勢(shì).經(jīng)重復(fù)測(cè)量方差分析可知,添加蚯蚓可顯著(<0.01)提高堆肥產(chǎn)物中水溶性總氮的含量.

對(duì)水溶性總磷而言,第20d時(shí)對(duì)照組產(chǎn)物的含量相比原污泥下降了6.67%,而蚯蚓堆肥產(chǎn)物僅下降了0.39%.第30d時(shí)對(duì)照組和蚯蚓堆肥產(chǎn)物相比原污泥分別下降了5.86%和10.92%.在堆肥期間對(duì)照組產(chǎn)物中的水溶性總磷含量呈現(xiàn)先上升后下降的趨勢(shì).蚯蚓堆肥產(chǎn)物中的含量在堆肥期間較為穩(wěn)定,卻在腐熟期間下降明顯.總磷含量的增加可能是由于微生物不斷分泌的有機(jī)酸和磷酸酶的活化導(dǎo)致有機(jī)磷的礦化速率增加[18].Busato等[19]認(rèn)為磷能富集在蚯蚓堆肥產(chǎn)物當(dāng)中,并且有著向可利用的形態(tài)轉(zhuǎn)化的趨勢(shì),這可能是導(dǎo)致蚯蚓堆肥產(chǎn)物中總磷含量呈現(xiàn)此趨勢(shì)的原因.

第20d時(shí)蚯蚓單體重量約為0.6g,蚯蚓數(shù)量約為583條.與投加時(shí)相比,蚯蚓體重增加了50%,堆肥期間蚯蚓生長(zhǎng)情況良好.Rodrigo等[20]發(fā)現(xiàn)蚯蚓的體重在最初的15d內(nèi)增加,在達(dá)到每條600mg之后(初始體重約為每條410mg)會(huì)將營(yíng)養(yǎng)物質(zhì)用于繁殖,這與本實(shí)驗(yàn)的研究結(jié)果一致,表明污泥蚯蚓堆肥期間蚯蚓的生長(zhǎng)狀況良好.

2.2 蚯蚓堆肥對(duì)DOC的含量及結(jié)構(gòu)的影響

2.2.1 DOC含量及其結(jié)構(gòu)的光譜分析 DOC是判斷堆肥腐熟程度的重要標(biāo)準(zhǔn)之一,相關(guān)研究表明[21]堆肥產(chǎn)物當(dāng)中的DOC含量低于4g/kg時(shí)即為腐熟.如圖2所示,在第10d和第20d時(shí)蚯蚓堆肥產(chǎn)物比對(duì)照組分別下降了24.44%和40.89%,表明蚯蚓堆肥可促進(jìn)污泥中DOC的礦化速率.這可能是由于蚯蚓的攝食、破碎等刺激作用促進(jìn)了微生物量的增長(zhǎng),從而加快了微生物對(duì)有機(jī)質(zhì)的分解利用,使蚯蚓堆肥產(chǎn)物的穩(wěn)定化程度更好[22].但在第30d,對(duì)照組和蚯蚓堆肥產(chǎn)物中DOC含量基本一致,可能是由于腐熟期間蚯蚓堆肥中含水率急劇下降導(dǎo)致有機(jī)物降解速率降低.經(jīng)重復(fù)測(cè)量方差分析顯示兩處理組之間在實(shí)驗(yàn)期間表現(xiàn)出顯著性差異(<0.01).

為進(jìn)一步揭示蚯蚓堆肥產(chǎn)物中不同階段的DOC結(jié)構(gòu)變化,將3D-EEM圖譜劃分為5個(gè)區(qū)域,并對(duì)各區(qū)域進(jìn)行FRI分析.其中區(qū)域Ⅰ和Ⅱ分別代表酪氨酸和色氨酸等類芳香族類蛋白質(zhì),區(qū)域Ⅲ代表富里酸類物質(zhì),區(qū)域Ⅳ代表可溶性微生物副產(chǎn)物,區(qū)域Ⅴ代表腐殖酸類物質(zhì)[23].如圖2(b)、(c)所示,與原始污泥相比,蚯蚓糞和對(duì)照組產(chǎn)物中腐殖酸的含量分別增加了28.62%和32.24%,富里酸含量也分別增加了11.31%和1.08%.然而,蚯蚓糞中蛋白質(zhì)的含量相比原始污泥下降了10.4%,而對(duì)照組僅下降了1.1%.以上結(jié)果表明蚯蚓能夠促進(jìn)污泥中蛋白質(zhì)的降解,并將其轉(zhuǎn)化為富里酸和腐殖酸,從而提高堆肥產(chǎn)物的芳香化和腐殖化程度.先前研究[24-25]發(fā)現(xiàn),在有機(jī)物含量較高的基質(zhì)堆肥過程中,蛋白類物質(zhì)不斷減少,而富里酸和腐殖酸物質(zhì)呈現(xiàn)上升的趨勢(shì),這與本實(shí)驗(yàn)的研究結(jié)果一致.

2.2.2 3D-EEM平行因子分析 PARAFAC模型可以將給定三維熒光數(shù)據(jù)集分離為獨(dú)立且不同的熒光組分,并且能夠?qū)埐钭钚』痆26-28].如圖3(a)～(c)所示,污泥及各層EPS結(jié)構(gòu)中共鑒定出3種熒光組分:組分Ⅰ代表一種蛋白質(zhì),有著和類色氨酸相似的熒光[29];組分Ⅱ被認(rèn)為具有類似腐殖質(zhì)的熒光[30];組分Ⅲ是一種蛋白質(zhì),通常在人為影響因素較大的地區(qū)發(fā)現(xiàn),也與木質(zhì)素的分解有關(guān)[31].

如圖3(d)～(f)所示,為3種熒光組分在不同堆肥階段的變化規(guī)律.在前10d蚯蚓堆肥和對(duì)照組中產(chǎn)物的可溶性微生物副產(chǎn)物的熒光強(qiáng)度均從初始值下降至0,且在蚯蚓堆肥產(chǎn)物中的下降速率更快,該情況在組分Ⅲ的變化中也有發(fā)生,表明蚯蚓可以促進(jìn)微生物活動(dòng)對(duì)色氨酸等類蛋白質(zhì)的強(qiáng)烈生物氧化作用[32].第20～30d期間,蚯蚓堆肥產(chǎn)物中組分Ⅲ的max下降程度明顯高于對(duì)照組,表明蚯蚓可以促進(jìn)堆肥過程中蛋白質(zhì)等物質(zhì)的礦化速率,這與理化性質(zhì)的分析結(jié)果一致.在第10d至堆肥結(jié)束,蚯蚓堆肥產(chǎn)物中組分Ⅱ的max變化較小,一直維持在高于對(duì)照組的水平,證明其比對(duì)照組的腐殖化程度更高,且具有更好的腐熟效果.

圖2 堆肥過程中DOC含量及三維熒光光譜區(qū)域體積百分比變化情況

2.3 蚯蚓堆肥對(duì)EPS結(jié)構(gòu)的影響

2.3.1 EPS結(jié)構(gòu)中蛋白質(zhì)含量的變化 由圖4可知,污泥中的蛋白質(zhì)主要儲(chǔ)存于污泥的TB-EPS中.前10d內(nèi),對(duì)照組和蚯蚓堆肥產(chǎn)物TB-EPS中的蛋白質(zhì)分別顯著下降了23.87%和32.21%(<0.01).且第30d時(shí)相比原始污泥,對(duì)照組產(chǎn)物和蚯蚓堆肥產(chǎn)物TB-EPS中的蛋白質(zhì)含量分別下降了57.83%和59.72%.但在第20d,對(duì)照組產(chǎn)物的SEPS、LB-EPS和TB-EPS中蛋白質(zhì)含量相比第10d分別增加了6471.28、107.24和1.21倍,而蚯蚓堆肥產(chǎn)物分別增加了1841.29、98.23和0.51倍.在堆肥前10d蚯蚓和微生物劇烈消耗蛋白質(zhì)后,其含量的升高可能與EPS結(jié)構(gòu)在逐漸恢復(fù)有關(guān)[33].蚯蚓堆肥產(chǎn)物的蛋白質(zhì)含量的增加程度較少可能是由于蚯蚓破壞了污泥EPS結(jié)構(gòu),并且結(jié)合3D-EEM圖譜的FRI分析結(jié)果,蚯蚓能夠促進(jìn)污泥中蛋白質(zhì)的降解,從而導(dǎo)致EPS結(jié)構(gòu)的恢復(fù)程度較低.在第30d時(shí),相比第20d對(duì)照組產(chǎn)物各層EPS中的蛋白質(zhì)含量分別下降了98.6%、79.8%和47.25%,蚯蚓堆肥產(chǎn)物分別下降了95.38%、93.04%和39.48%.在腐熟期間EPS各層中的蛋白質(zhì)含量均呈現(xiàn)下降趨勢(shì).

2.3.2 EPS結(jié)構(gòu)中多糖含量的變化 圖5多糖含量發(fā)現(xiàn),該成分主要儲(chǔ)存于TB-EPS中.第0～10d期間,兩組實(shí)驗(yàn)中污泥的TB-EPS中多糖含量呈現(xiàn)下降趨勢(shì),蚯蚓堆肥產(chǎn)物和對(duì)照組分別下降了33.33%和24.24%,多糖物質(zhì)主要被TB-EPS緊密包裹的微生物消耗.在第10～20d,蚯蚓堆肥產(chǎn)物和對(duì)照組TB-EPS中的多糖含量相比第10d分別增加了9.1%和12%,且在此期間樣品各層EPS中的多糖含量均有所增加.腐熟結(jié)束時(shí),對(duì)照組和蚯蚓堆肥產(chǎn)物中TB-EPS的多糖含量相較于原污泥分別降低了38.6%和47.23%.第0～30d期間,對(duì)照組和蚯蚓堆肥產(chǎn)物的SEPS中的多糖含量分別增加了21倍和10倍,且LB-EPS中的多糖含量分別增加了9倍和7倍.

圖4 EPS各層中蛋白質(zhì)含量的變化

有研究發(fā)現(xiàn),多糖的生成可為EPS結(jié)構(gòu)提供一定的穩(wěn)定性[34].結(jié)合圖4c和圖5c,觀察到堆肥過程中兩實(shí)驗(yàn)組TB-EPS中的蛋白質(zhì)含量和多糖含量變化趨勢(shì)相似.說明在第10～20d期間微生物在積極分泌蛋白質(zhì)和多糖以恢復(fù)EPS結(jié)構(gòu),但蚯蚓促進(jìn)污泥中蛋白質(zhì)及多糖的分解[35],阻礙了EPS結(jié)構(gòu)的恢復(fù),導(dǎo)致第30d時(shí)蚯蚓堆肥產(chǎn)物中EPS各層中的蛋白質(zhì)和多糖含量均低于對(duì)照組.

圖5 EPS各層中多糖含量的變化

2.3.3 蚯蚓堆肥對(duì)EPS中DOC含量及結(jié)構(gòu)的影響 由圖6可見,污泥中的DOC和蛋白質(zhì)及多糖的分布情況相似,同樣主要分布于TB-EPS中.第30d時(shí)對(duì)照組產(chǎn)物中SEPS和LB-EPS的DOC含量對(duì)比原污泥分別增加了3.86、0.86倍,蚯蚓堆肥產(chǎn)物中分別增加了2.35、0.63倍.表明隨著堆肥時(shí)間的不斷增加,SEPS和LB-EPS中的DOC含量均呈現(xiàn)上升趨勢(shì),但蚯蚓堆肥中的上升速率較低.在第10d時(shí),對(duì)照組和蚯蚓堆肥過程中TB-EPS的DOC含量分別下降了16.37%和25.15%.至堆肥結(jié)束時(shí),對(duì)照組產(chǎn)物和蚯蚓堆肥產(chǎn)物中DOC含量相比原始污泥分別下降了36.75%和40.92%.而在第20d時(shí)TB-EPS層中DOC含量出現(xiàn)上升,對(duì)照組和蚯蚓堆肥產(chǎn)物中比第10d時(shí)分別上升了21.48%和7.97%.對(duì)比圖4(c)、5(c)和圖6(g)可發(fā)現(xiàn)在堆肥的整個(gè)過程中, TB-EPS中的蛋白質(zhì)、多糖及DOC含量的變化趨勢(shì)相似.

綜合EPS各層蛋白質(zhì)、多糖和DOC含量的變化趨勢(shì)分析,在第0～10d期間,通過蚯蚓對(duì)微生物的刺激作用[22],其活性瞬間增加,加快了微生物對(duì)TB-EPS中DOC的利用,以及這部分DOC有著較快的礦化速率[36],導(dǎo)致了其含量的快速降低.這部分有機(jī)物主要由來自植物和微生物的多糖以及蛋白質(zhì)、肽和氨基糖聚合物的多糖和寡糖組成[37],同時(shí)也是微生物可直接利用的碳源和氮源.在第10～20d期間3種成分在EPS各層均呈現(xiàn)上升趨勢(shì),但蚯蚓堆肥產(chǎn)物中的上升程度均低于對(duì)照組,可能是由于在此階段微生物的活性最高.微生物在消耗大量的TB-EPS中的DOC后,開始通過EPS向外界捕獲較易溶解的有機(jī)物和部分難降解的有機(jī)物以維持生命活動(dòng)[36],導(dǎo)致TB-EPS中DOC含量有所上升.同時(shí)蚯蚓促進(jìn)EPS結(jié)構(gòu)中蛋白質(zhì)和多糖的分解使EPS結(jié)構(gòu)穩(wěn)定性有所下降,TB-EPS所包裹的DOM從而出現(xiàn)遷移至SEPS和LB-EPS的現(xiàn)象.在堆肥的腐熟階段,由于蚯蚓已經(jīng)破壞了污泥的EPS結(jié)構(gòu),并且其恢復(fù)程度較低,導(dǎo)致DOC持續(xù)向外層EPS遷移.

圖6 EPS各層中DOC含量及三維熒光光譜區(qū)域體積百分比變化情況

圖6(b)、(e)、(h)和6(c)、(f)、(i)分別為對(duì)照組與蚯蚓堆肥產(chǎn)物EPS各層的3D-EEM區(qū)域體積百分比隨時(shí)間的變化情況.相較于原始污泥,蚯蚓堆肥產(chǎn)物SEPS層中酪氨酸、富里酸和腐殖酸含量分別降低了8.22%、7.2%和9.6%.而色氨酸和可溶性微生物副產(chǎn)物分別增加了6.03%和4.63%.蚯蚓堆肥產(chǎn)物L(fēng)B-EPS中酪氨酸和富里酸含量相比原始污泥分別降低了11.75%和4.84%,而色氨酸、可溶性微生物副產(chǎn)物和腐殖酸含量較原始污泥分別增加了6.15%、1.86%和31.81%.TB-EPS中酪氨酸和富里酸含量比原始污泥分別增加了8.95%和8.54%,色氨酸和腐殖酸的含量比原始污泥分別減少了5.18%和15.62%.

蚯蚓堆肥產(chǎn)物中TB-EPS及LB-EPS的色氨酸含量相比對(duì)照組分別減少7.69%和13.62%.同時(shí)其LB-EPS和SEPS中腐殖酸含量比對(duì)照組分別增加了25.1%和7.82%,表明微生物正在將蛋白質(zhì)轉(zhuǎn)化為腐殖酸.非腐殖質(zhì)類物質(zhì)(例如多糖、蛋白質(zhì)等)會(huì)在蚯蚓堆肥的過程中轉(zhuǎn)化為腐殖質(zhì),從而增加了堆肥產(chǎn)物的腐殖化和穩(wěn)定化,這與Huang等[38]的研究結(jié)果一致.

2.3.4 EPS結(jié)構(gòu)與污泥蚯蚓堆肥腐熟的關(guān)系 研究表明,電導(dǎo)率、DOC和硝酸鹽氮含量均可反映污泥蚯蚓堆肥產(chǎn)物的腐熟程度[39].本研究采用Spearman相關(guān)系數(shù)檢驗(yàn)確定蚯蚓堆肥進(jìn)程和EPS結(jié)構(gòu)變化的相關(guān)性.

如表(1),對(duì)照組產(chǎn)物SEPS層、LB-EPS和TB-EPS層中多糖含量與其有機(jī)質(zhì)含量的相關(guān)系數(shù)分別為-0.958、-0.713和0.678(<0.05).對(duì)照組產(chǎn)物中的總DOC含量和SEPS層、LB-EPS與TB-EPS層中多糖含量的相關(guān)系數(shù)分別為-0.916、-0.762和0.643(<0.05).

如表1所示,蚯蚓堆肥產(chǎn)物中其中電導(dǎo)率和硝酸鹽氮含量均與DOC含量呈現(xiàn)顯著負(fù)相關(guān)(= -0.749、-0.769,<0.01),但電導(dǎo)率和硝酸鹽氮含量呈現(xiàn)顯著正相關(guān)(=0.958,<0.01).該結(jié)果表明本研究中電導(dǎo)率、DOC和硝酸鹽氮含量均可以作為污泥蚯蚓堆肥腐熟度的關(guān)鍵指標(biāo).同時(shí),蚯蚓堆肥產(chǎn)物的SEPS、LB-EPS中的DOC、蛋白質(zhì)及多糖含量與其電導(dǎo)率都呈現(xiàn)顯著正相關(guān)性,相關(guān)系數(shù)分別為0.848、0.650、0.788、0.770、0.767和0.827(<0.05). 其DOC含量同SEPS、LB-EPS和TB-EPS各層中的DOC含量及多糖含量呈顯著相關(guān)性,相關(guān)系數(shù)分別為-0.877,-0.622,-0.762,-0.951, -0.853和0.783(<0.05).硝酸鹽氮含量和SEPS、LB-EPS層中的DOC、蛋白質(zhì)及多糖含量呈現(xiàn)出顯著正相關(guān),相關(guān)系數(shù)分別為0.888、0.643、0.741、0.769、0.727和0.839(<0.05).因此,上述相關(guān)性表明EPS各層結(jié)構(gòu)中的DOC及多糖含量,和SEPS、LB-EPS中的蛋白質(zhì)含量變化可以有效地表征蚯蚓促進(jìn)堆肥穩(wěn)定化和腐殖化的進(jìn)程.

表1 對(duì)照組和蚯蚓堆肥產(chǎn)物的EPS結(jié)構(gòu)與其理化性質(zhì)參數(shù)的相關(guān)性分析

注:*<0.05, **<0.01.

研究結(jié)果顯示,蚯蚓可通過調(diào)節(jié)基質(zhì)特性和改變微生物活性,有效的促進(jìn)有機(jī)物的降解[40].在本研究中.蚯蚓可以通過破壞污泥的EPS結(jié)構(gòu),釋放其中包含的水和有機(jī)物,從而加速污泥的腐殖化和穩(wěn)定化進(jìn)程,所以污泥EPS的結(jié)構(gòu)特征變化可有效地表征蚯蚓糞的穩(wěn)定性和腐熟程度.

3 結(jié)論

3.1 蚯蚓堆肥能夠促進(jìn)污泥EPS結(jié)構(gòu)中的蛋白質(zhì)和多糖向腐殖質(zhì)類物質(zhì)的轉(zhuǎn)化,從而提高堆肥產(chǎn)物的穩(wěn)定化和腐殖化程度.

3.2 堆肥過程中,蚯蚓堆肥EPS結(jié)構(gòu)中蛋白質(zhì)和多糖的總量分別降低32.77%和31.54%,破壞了其穩(wěn)定性,導(dǎo)致EPS結(jié)構(gòu)中的DOM呈現(xiàn)由內(nèi)向外遷移的趨勢(shì).

3.3 污泥蚯蚓堆肥產(chǎn)物EPS各層中的DOC和多糖含量,及SEPS、LB-EPS中的蛋白質(zhì)含量變化可以有效地表征蚯蚓堆肥的穩(wěn)定化和腐殖化進(jìn)程(< 0.05).

[1] 戴曉虎.我國(guó)污泥處理處置現(xiàn)狀及發(fā)展趨勢(shì) [J]. 科學(xué), 2020,72(6): 30-34.

Dai X H. Applications and Perspectives of Sludge Treatment and Disposal in China [J]. Science, 2020,72(6):30-34.

[2] 薛重華,孔祥娟,王 勝,等.我國(guó)城鎮(zhèn)污泥處理處置產(chǎn)業(yè)化現(xiàn)狀、發(fā)展及激勵(lì)政策需求 [J]. 凈水技術(shù), 2018,37(12):33-39.

Xue C H, Kong X J, Wang S, et al. Industrialization status, development analysis and incentive policy demands of municipal sludge treatment and disposal industry in China [J]. Water Purification Technology, 2018,37(12):33-39.

[3] Xia H, Wu Y, Chen X M, et al. Effects of antibiotic residuals in dewatered sludge on the behavior of ammonia oxidizers during vermicomposting maturation process [J]. Chemosphere, 2019,218: 810-817.

[4] Lirikum, Kakati L N, Thyug L, et al. Vermicomposting: an eco- friendly approach for waste management and nutrient enhancement [J]. Tropical Ecology, 2022,63:325-337.

[5] Deka H, Deka S, Baruah C K, et al. Vermicomposting of distillation waste of citronella plant (Jowitt.) employing[J]. Bioresource Technology, 2011,102(13):6944- 6950.

[6] Dominguez J. 20 state-of-the-art and new perspectives on vermicomposting research [M]. Earthworm Ecology Boca Raton: CRC press, 2004:401-424

[7] Staudt C, Horn H, Hempel D C, et al. Volumetric measurements of bacterial cells and extracellular polymeric substance glycoconjugates in biofilms [J]. Biotechnology and Bioengineering, 2004,88(5):585- 592.

[8] Zhang W J, Cao B D, Wang D S, et al. Influence of wastewater sludge treatment using combined peroxyacetic acid oxidation and inorganic coagulants re-flocculation on characteristics of extracellular polymeric substances (EPS) [J]. Water Research, 2016,88:728-739.

[9] 陳丹丹,竇昱昊,盧 平,等.污泥深度脫水技術(shù)研究進(jìn)展 [J]. 化工進(jìn)展, 2019,38(10):4722-4746.

Chen D D, Dou Y H, Lu P, et al. A review on sludge deep dewatering technology [J]. Chemical Industry and Engineering Progress, 2019, 38(10):4722-4746.

[10] Sheng G P, Yu H Q, Li X Y. Extracellular polymeric substances (EPS) of microbial aggregates in biological wastewater treatment systems: a review [J]. Biotechnology Advances, 2010,28(6):882-894.

[11] Hu X J, Kang F X, Yang B, et al. Extracellular polymeric substances acting as a permeable barrier hinder the lateral transfer of antibiotic resistance genes [J]. Frontiers in Microbiology, 2019,10:736.

[12] 黃 魁,夏 慧,陳景陽(yáng),等.蚯蚓對(duì)城市污泥蚯蚓堆肥過程中微生物特征變化的影響 [J]. 環(huán)境科學(xué)學(xué)報(bào), 2018,38(8):3146-3152.

Huang K, Xia H, Chen J Y, et al. Effects of earthworms on changes of microbial feature during vermicomposting of municipal sludge [J]. Acta Scientiae Circumstantiae, 2018,38(8):3146-3152.

[13] Fr?lund B, Palmgren R, Keiding K, et al. Extraction of extracellular polymers from activated sludge using a cation exchange resin [J]. Water Research, 1996,30(8):1749-1758.

[14] Stedmon C A, Mark S, Bro R. Tracing dissolved organic matter in aquatic environments using a new approach to fluorescence spectroscopy [J]. Marine Chemistry, 2003,82(3/4):239-254.

[15] 段曼莉,鄢入泮,周蓓蓓,等.去電子水對(duì)牛糞秸稈好氧堆肥進(jìn)程及細(xì)菌群落的影響 [J]. 環(huán)境科學(xué)學(xué)報(bào), 2022:42(2):249-257.

Dan M L, Yan R P, Zhou B B, et al. Effect of de-electron water on maturation process and bacterial community during aerobic composting of cow manure and straw [J]. Acta Scientiae Circumstantiae, 2022:42(2):249-257.

[16] 李 輝,吳曉芙,蔣龍波,等.城市污泥脫水干化技術(shù)進(jìn)展 [J]. 環(huán)境工程, 2014,32(11):102-107,101.

Li H, Wu X F, Jiang L B, et al. Process on the dewatering and drying technology of municipal sludge [J]. Environmental Engineering, 2014, 32(11):102-107,101.

[17] 吳 穎,黃 魁,夏 慧,等.污泥四環(huán)素含量對(duì)蚯蚓堆肥中氨氧化菌群的影響 [J]. 環(huán)境科學(xué), 2019,40(6):2954-2960.

Wu Y, Huang K, Xia H, et al. Effects of different concentrations of tetracycline in sludge on ammonia oxidizers during vermicomposting [J]. Environmental Science, 2019,40(6):2954-2960.

[18] Gaume A, M?chler F, Frossard E. Aluminum resistance in two cultivars of Zea mays L.: root exudation of organic acids and influence of phosphorus nutrition [J]. Plant and Soil, 2001,234(1):73-81.

[19] Busato J G, Lima L S, Aguiar N O, et al. Changes in labile phosphorus forms during maturation of vermicompost enriched with phosphorus- solubilizing and diazotrophic bacteria [J]. Bioresource technology, 2012,110:390-395.

[20] Ramos R F, Santana N A, Andrade N, et al. Vermicomposting of cow manure: Effect of time on earthworm biomass and chemical, physical, and biological properties of vermicompost [J]. Bioresource Technology, 2022,345:126572.

[21] Bernai M P, Paredes C, Sánchez-Monedero M A, et al. Maturity and stability parameters of composts prepared with a wide range of organic wastes [J]. Bioresource Technology, 1998,63(1):91-99.

[22] Aira M, Monroy F, Domínguez J. Earthworms strongly modify microbial biomass and activity triggering enzymatic activities during vermicomposting independently of the application rates of pig slurry [J]. Science of the total Environment, 2007,385(1-3):252-261.

[23] Chen W, Westerhoff P, Leenheer J A, et al. Fluorescence excitation- emission matrix regional integration to quantify spectra for dissolved organic matter [J]. Environmental Science & Technology, 2003,37(24): 5701-5710.

[24] Marhuenda-Egea F C, Martinez-Sabater E, Jordá J, et al. Dissolved organic matter fractions formed during composting of winery and distillery residues: evaluation of the process by fluorescence excitation–emission matrix [J]. Chemosphere, 2007,68(2):301-309.

[25] Huang K, Chen J Y, Guan M X, et al. Effects of biochars on the fate of antibiotics and their resistance genes during vermicomposting of dewatered sludge [J]. Journal of Hazardous Materials, 2020,397: 122767.

[26] He W, Hur J. Conservative behavior of fluorescence EEM-PARAFAC components in resin fractionation processes and its applicability for characterizing dissolved organic matter [J]. Water Research, 2015,83: 217-226.

[27] Yu H B, Song Y H, Du E, et al. Comparison of PARAFAC components of fluorescent dissolved and particular organic matter from two urbanized rivers [J]. Environmental Science and Pollution Research, 2016,23(11):10644-10655.

[28] Zhao Y, Song K S, Li S J, et al. Characterization of CDOM from urban waters in Northern-Northeastern China using excitation-emission matrix fluorescence and parallel factor analysis [J]. Environmental Science and Pollution Research, 2016,23(15):15381-15394.

[29] Murphy K R, Hambly A, Singh S, et al. Organic matter fluorescence in municipal water recycling schemes: toward a unified PARAFAC model [J]. Environmental Science & Technology, 2011,45(7):2909- 2916.

[30] Limberger R, Birtel J, Peter H, et al. Predator-induced changes in dissolved organic carbon dynamics [J]. Oikos, 2019,128(3):430-440.

[31] Hernes P J, Bergamaschi B A, Eckard R S, et al. Fluorescence‐based proxies for lignin in freshwater dissolved organic matter [J]. Journal of Geophysical Research: Biogeosciences, 2009,114(G4).

[32] He X S, Xi B D, Jiang Y-H, et al. Structural transformation study of water-extractable organic matter during the industrial composting of cattle manure [J]. Microchemical Journal, 2013,106:160-166.

[33] Tang Y F, Dai X H, Dong B, et al. Humification in extracellular polymeric substances (EPS) dominates methane release and EPS reconstruction during the sludge stabilization of high-solid anaerobic digestion [J]. Water Research, 2020,175:115686.

[34] Park C, Novak J T. Characterization of lectins and bacterial adhesins in activated sludge flocs [J]. Water Environment Research, 2009, 81(8):755-764.

[35] Yang J, Lv B Y, Zhang J, et al. Insight into the roles of earthworm in vermicomposting of sewage sludge by determining the water-extracts through chemical and spectroscopic methods [J]. Bioresource Technology, 2014,154:94-100.

[36] Said-Pullicino D, Erriquens F G, Giovanni G. Changes in the chemical characteristics of water-extractable organic matter during composting and their influence on compost stability and maturity [J]. Bioresource Technology, 2007,98(9):1822-1831.

[37] Gigliotti G, Kaiser K, Guggenberger G, et al. Differences in the chemical composition of dissolved organic matter from waste material of different sources [J]. Biology and Fertility of Soils, 2002,36(5):321- 329.

[38] Hanc A, Enev V, Hrebeckova T, et al. Characterization of humic acids in a continuous-feeding vermicomposting system with horse manure [J]. Waste Management, 2019,99:1-11.

[39] Gómez-Brandón M, Lazcano C, Lores M, et al. Short-term stabilization of grape marc through earthworms [J]. Journal of Hazardous Materials, 2011,187(1-3):291-295.

[40] Ali U, Sajid N, Khalid A, et al. A review on vermicomposting of organic wastes [J]. Environmental Progress & Sustainable Energy, 2015,34(4):1050-1062.

Changes in the structure of extracellular polymeric substances during sludge vermicomposting.

CHEN Jin1, XIE Jia-chen1, XU Jun-jie1, XIA Hui1*, HUANG Kui1,2

(1.School of Environmental and Municipal Engineering, Lanzhou Jiaotong University, Lanzhou 730070, China；2.Key Laboratory of Yellow River Water Environment in Gansu Province, Lanzhou Jiaotong University, Lanzhou 730070, China)., 2022,42(11)：5188～5197

Extracellular polymer substance (EPS) is an important factor affecting the stabilization of excess sludge. To reveal the relationship between EPS characteristics of sludge and its decomposition of vermicomposting, this study investigated the effect of vermicomposting on structural changes of sludge EPS, by comparing with the control treatment without earthworms. The results showed that the electric conductivity and nitrate nitrogen content of vermicompost significantly increased by 0.64 and 0.22 fold, respectively, compared with control treatment (<0.05), suggesting that vermicomposting can significantly accelerate the mineralization rate of organic matter in sludge. In addition, total protein, and polysaccharide contents of EPS in each layer of vermicomposting decreased by 32.77% and 31.54% compared with control treatment, respectively. Fluorescence intensity of protein reduced more rapid in vermicompost than control, while humus of vermicompost remained at a higher level in later stages. Compared to control, the tryptophan contents in tightly-bound EPS (TB-EPS) and loosely-bound EPS (LB-EPS) in vermicompost decreased by 7.69% and 13.62%, respectively, while the humic acid content in LB-EPS and soluble EPS (SEPS) increased by 25.1% and 7.82%, respectively. The nitrate nitrogen content and electric conductivity during vermicomposting were significantly and positively correlated with DOC, protein, and polysaccharide contents in the structure of SEPS and LB-EPS (<0.05), and their total DOC contents was significantly correlated with DOC and polysaccharide contents in each layer of EPS (<0.05). However, only organic matter and DOC contents were significantly correlated with the polysaccharide contents of EPS layers in control during the experiment (<0.05). The study indicated that earthworms could destroy the EPS structure of sludge to promote the decomposition of organic matter, thus accelerating the humification and stabilization process of vermicomposting, and the change of EPS structure of sludge could be a key indicator to assess the degree of sludge vermicomposting.

vermicomposting；sludge recycling；maturity；extracellular polymer substance；vermicompost fertilizer

X705

A

1000-6923(2022)11-5188-10

陳 進(jìn)(1998-),男,甘肅蘭州人,蘭州交通大學(xué)碩士研究生,主要研究方向?yàn)槲勰噘Y源化.發(fā)表論文2篇.

2022-04-14

國(guó)家自然科學(xué)基金項(xiàng)目(51868036;52000095);甘肅省青年博士基金項(xiàng)目(2021-QB051);甘肅省科技計(jì)劃資助項(xiàng)目(20JR2RA002)

* 責(zé)任作者, 副教授, xiahui@mail.lzjtu.cn