王 攀,楊鑫玉,鄭 義,任連海

廚余垃圾厭氧發(fā)酵失穩(wěn)調(diào)控及微生物群落分析

王 攀,楊鑫玉,鄭 義,任連海*

(北京工商大學(xué)生態(tài)環(huán)境學(xué)院,國(guó)家環(huán)境保護(hù)食品鏈污染防治重點(diǎn)實(shí)驗(yàn)室,北京 100048)

針對(duì)廚余垃圾厭氧發(fā)酵過(guò)程中容易積累丙酸和丁酸導(dǎo)致反應(yīng)體系酸化失穩(wěn)的問(wèn)題,馴化了富集耐丙酸和耐丁酸厭氧發(fā)酵菌群的接種菌泥,探究利用其對(duì)廚余垃圾干式厭氧發(fā)酵酸化失穩(wěn)體系進(jìn)行調(diào)控后對(duì)甲烷產(chǎn)量和微生物群落的影響.酸化失穩(wěn)厭氧體系中添加耐丙酸菌泥調(diào)控后,與空白對(duì)照組相比反應(yīng)體系中丙酸濃度削減6900.81mg/L,累積甲烷產(chǎn)量提升了259%;添加耐丁酸菌泥調(diào)控后,反應(yīng)體系中丁酸濃度削減5371.56mg/L,累積甲烷產(chǎn)量提高了210%.微生物多樣性分析表明,利用耐丙酸和耐丁酸菌群調(diào)控后,細(xì)菌種群豐富度明顯提高.細(xì)菌屬水平分析表明,投加耐丙酸馴化菌群后有利于和等揮發(fā)性脂肪酸降解菌相對(duì)豐度增加;投加耐丁酸馴化菌群后,與乙酸型產(chǎn)甲烷菌有協(xié)同作用的相對(duì)豐度有所增長(zhǎng).古菌屬水平分析表明耐丙酸和丁酸菌調(diào)控后,氫型產(chǎn)甲烷菌和乙酸型產(chǎn)甲烷菌的相對(duì)豐度明顯提高.

廚余垃圾；干式厭氧發(fā)酵；酸化失穩(wěn)調(diào)控；微生物群落

廚余垃圾是城市生活垃圾的重要組成部分[1],隨著全國(guó)垃圾分類(lèi)工作的不斷推進(jìn),越來(lái)越多的廚余垃圾被分離出來(lái)需要妥善處理.廚余垃圾富含有機(jī)質(zhì),易被微生物降解,厭氧發(fā)酵技術(shù)逐漸成為處理處置廚余垃圾的主流技術(shù)[1].厭氧發(fā)酵包括濕式和干式發(fā)酵[2].干式厭氧發(fā)酵技術(shù)對(duì)原料預(yù)處理要求低、沼液產(chǎn)量低、耗能少、產(chǎn)氣率較高[3],但由于其高有機(jī)負(fù)荷運(yùn)行,容易發(fā)生底物濃度過(guò)高,水解酸化速率過(guò)快,揮發(fā)性脂肪酸(VFAs)迅速積累,系統(tǒng)pH值降低,從而抑制產(chǎn)甲烷菌活性,導(dǎo)致厭氧發(fā)酵體系酸化失穩(wěn)等問(wèn)題[4].

在酸化失穩(wěn)的厭氧發(fā)酵體系中,快速積累的VFAs主要有乙酸、丙酸、丁酸等,由于酸積累抑制微生物生長(zhǎng),從而影響系統(tǒng)穩(wěn)定性.丙酸、丁酸降解過(guò)程中所需的吉布斯自由能較高,不能自發(fā)進(jìn)行[5-6].在目前的許多研究中,丙酸和丁酸被認(rèn)為是引發(fā)酸抑制產(chǎn)生的重要原因[7-8].研究發(fā)現(xiàn)丙酸在非常低的濃度下會(huì)抑制厭氧發(fā)酵過(guò)程中甲烷生成,并且丙酸降解過(guò)程較為緩慢[8-9].此外,研究發(fā)現(xiàn)在厭氧發(fā)酵系統(tǒng)出現(xiàn)酸抑制現(xiàn)象時(shí),VFAs主要由丁酸組成,占總VFAs的30%～40%[10].對(duì)于厭氧發(fā)酵酸化失穩(wěn)體系的調(diào)控手段主要有預(yù)處理[11]、酸堿化學(xué)調(diào)控[12]和生物調(diào)控的方法.有研究表明向有機(jī)負(fù)荷過(guò)載的酸抑制發(fā)酵體系連續(xù)投加丙酸降解菌群能快速降解積累的丙酸并提高產(chǎn)甲烷效率[13].Li等[14]利用耐酸的產(chǎn)甲烷菌對(duì)厭氧發(fā)酵體系進(jìn)行生物調(diào)控可恢復(fù)了過(guò)載的厭氧發(fā)酵體系,并且加速了累積的VFAs尤其是乙酸和丁酸的降解,該方法增加了和的數(shù)量從而重建了厭氧發(fā)酵系統(tǒng)的產(chǎn)甲烷菌群.由于厭氧發(fā)酵菌群體系的復(fù)雜性,耐丙酸微生物菌群和耐丁酸微生物菌群調(diào)控改善厭氧酸化失穩(wěn)體系的微生物學(xué)機(jī)理需進(jìn)一步深入探討.

本研究針對(duì)廚余垃圾干式厭氧發(fā)酵體系中易積累造成體系酸化失穩(wěn)問(wèn)題的丙酸和丁酸,馴化耐丙酸和耐丁酸厭氧發(fā)酵菌群,研究其對(duì)廚余垃圾厭氧發(fā)酵酸化失穩(wěn)的調(diào)控能力,利用高通量測(cè)序技術(shù)分析調(diào)控過(guò)程中微生物群落結(jié)構(gòu)的變化,探討微生物機(jī)理,以期為廚余垃圾干式厭氧發(fā)酵酸化失穩(wěn)的調(diào)控提供理論基礎(chǔ).

1 材料與方法

1.1 材料

廚余垃圾采集于北京市海淀區(qū)某居民小區(qū)(廚余垃圾1#)和北京工商大學(xué)食堂(廚余垃圾2#),分別確定為1#和2#采樣點(diǎn).為保證廚余垃圾成分的均一性和穩(wěn)定性,廚余垃圾連續(xù)多日取樣并在取樣前混合均勻;調(diào)控所使用的馴化菌泥取自山東省某工廠污水處理厭氧工藝段厭氧顆粒污泥.實(shí)驗(yàn)底物的基本理化性質(zhì)見(jiàn)表1.

表1 實(shí)驗(yàn)底物理化參數(shù)

1.2 實(shí)驗(yàn)裝置及運(yùn)行過(guò)程

首先采用序批式厭氧發(fā)酵反應(yīng)裝置進(jìn)行耐丙酸和耐丁酸的產(chǎn)甲烷菌泥馴化實(shí)驗(yàn).分別將700mL厭氧顆粒污泥添加在2個(gè)1L的厭氧發(fā)酵瓶中.每隔24h加入含丙酸鈉或丁酸鈉的碳源營(yíng)養(yǎng)液.

厭氧發(fā)酵酸化失穩(wěn)調(diào)控實(shí)驗(yàn)采用序批式厭氧發(fā)酵反應(yīng)裝置.發(fā)酵底物物料配比為廚余垃圾1# : 廚余垃圾2# = 1:3(按TS計(jì)).發(fā)酵前在120℃ 下加熱80min、6000r/min將廚余垃圾脫油,將脫油處理后的廚余垃圾混合物與厭氧污泥顆粒以1:1(按VS計(jì))配比混合,實(shí)驗(yàn)設(shè)計(jì)分組如表2,每組設(shè)置2平行實(shí)驗(yàn).

厭氧發(fā)酵實(shí)驗(yàn)在中溫條件(35℃)下進(jìn)行,每隔24h采集少量發(fā)酵底物檢測(cè)相關(guān)性能指標(biāo),利用排水法測(cè)定沼氣的產(chǎn)氣量.每日定時(shí)采集沼氣進(jìn)行檢測(cè),當(dāng)觀察到甲烷含量持續(xù)下降時(shí)(即酸化失穩(wěn)),按體積比20%將馴化體系中最佳產(chǎn)氣時(shí)刻的菌泥加入到酸化失穩(wěn)的廚余垃圾干式厭氧發(fā)酵體系中.連續(xù)觀察耐丙酸產(chǎn)甲烷菌群調(diào)控(PAMR)和耐丁酸產(chǎn)甲烷菌群調(diào)控(BAMR)的調(diào)控效果和發(fā)酵性能.

表2 實(shí)驗(yàn)設(shè)計(jì)分組

1.3 分析方法

含水率采用烘干法計(jì)算;灰分采用灼燒法測(cè)定; pH值采用pH電極法測(cè)定,以上方法均參考《水和廢水監(jiān)測(cè)分析方法》[15].化學(xué)需氧量(COD)采用消解分光光度法[16];氨氮采用納氏分光光度法[17];粗脂肪采用索氏提取法[18];粗蛋白采用凱氏定氮法[19];粗纖維采用范式洗滌法[20];碳氮比(C/N)采用元素分析儀(Vario EL/micro cube)測(cè)定.

沼氣組分和揮發(fā)性脂肪酸(VFAs)采用氣相色譜法進(jìn)行分析.沼氣采用氣相色譜法(填充柱,柱溫80℃,進(jìn)樣口溫度150℃,檢測(cè)器溫度150℃,載氣為高純氬氣);揮發(fā)性脂肪酸(VFAs)采用氣相色譜法(FFAP毛細(xì)管柱,柱溫160℃,檢測(cè)器溫度250℃,載氣為高純氮?dú)?.

1.4 DNA提取及測(cè)序

采用FastDNA?土壤DNA提取試劑盒(MP Biomedicals, America)提取DNA.提取后DNA在-20℃下保存,然后進(jìn)行聚合酶鏈反應(yīng)(PCR)擴(kuò)增,本研究使用Ion Plus Fragment library Kit 48rxns(16S V4)和TruSeq?DNA PCR-Free Sample Preparation Kit(Archaea)構(gòu)建文庫(kù),并通過(guò)Qubit和Q-PCR進(jìn)行定量.利用Thermofisher Ion S5TMXL and HiSeq2500PE250對(duì)定量文庫(kù)進(jìn)行測(cè)序.

1.5 數(shù)據(jù)分析方法

采用Origin和Excel軟件對(duì)數(shù)據(jù)進(jìn)行分析處理.

2 結(jié)果與分析

2.1 耐酸產(chǎn)甲烷菌群馴化過(guò)程中厭氧發(fā)酵特征

向厭氧發(fā)酵顆粒污泥中連續(xù)投加丙酸鈉馴化耐丙酸產(chǎn)甲烷菌群.如圖1(a)所示,在發(fā)酵的第1～4d丙酸鈉投加濃度為500mg/L,發(fā)酵第5～8d為1000mg/L,發(fā)酵第9～29d每隔2d提高500mg/L.在馴化過(guò)程中,耐丙酸產(chǎn)甲烷菌泥在發(fā)酵的第18d日甲烷產(chǎn)量達(dá)到最高(893.78mL),此時(shí)其pH值為7.76.取用這一時(shí)刻的耐丙酸馴化菌泥作為厭氧發(fā)酵酸化失穩(wěn)體系接種菌泥.在馴化的第24d日甲烷產(chǎn)量有所上升,為736.44mL,隨后日甲烷產(chǎn)量迅速下降.在此馴化體系中,耐丙酸產(chǎn)甲烷菌群的最高丙酸耐受濃度為35320mg/L,遠(yuǎn)高于之前研究中4000mg/L的抑制濃度[21].由此,選用第18d、第24d和第30d(結(jié)束時(shí))的菌泥(MP18、MP24、MP30)進(jìn)行高通量測(cè)序檢測(cè)微生物群落分析.

馴化耐丁酸產(chǎn)甲烷菌群采用丁酸鈉為碳源,如圖1(b)所示,馴化過(guò)程的第2～5d投加丁酸鈉500mg/ L,第6d開(kāi)始每隔兩日提高500mg/L的丁酸鈉.在發(fā)酵的第24d達(dá)到最高日甲烷產(chǎn)量(1535.02mL),此時(shí)耐丁酸產(chǎn)甲烷菌泥的pH值為7.70.選用第24d的耐丙酸馴化菌泥,作為厭氧發(fā)酵酸化失穩(wěn)體系接種菌泥.在馴化第30d日甲烷產(chǎn)量出現(xiàn)產(chǎn)氣小高峰,為1320.83mL,丁酸濃度為14455.35mg/L.在此馴化體系中,耐丁酸產(chǎn)甲烷菌群的最高丙酸耐受濃度為17110.39mg/L.由此,選第24d、第30d和第36d(結(jié)束時(shí))的菌泥(MB24、MB30、MB36)進(jìn)行高通量測(cè)序檢測(cè)微生物群落,探究丁酸產(chǎn)甲烷菌群的馴化演替規(guī)律.

圖1 耐酸產(chǎn)甲烷菌群馴化過(guò)程中酸投加量、酸濃度及日甲烷產(chǎn)量變化

2.2 酸化失穩(wěn)體系調(diào)控過(guò)程中厭氧發(fā)酵特征

如圖2(a)所示,廚余垃圾干式厭氧發(fā)酵過(guò)程中日甲烷產(chǎn)量呈降低趨勢(shì),在發(fā)酵第10d幾乎不產(chǎn)氣,丙酸濃度達(dá)到7112.14mg/L.此時(shí)體系 pH值為5.56,因此認(rèn)為發(fā)酵第10d時(shí)出現(xiàn)酸化失穩(wěn).將耐丙酸產(chǎn)甲烷菌泥加入到此失穩(wěn)體系中,投加后體系的pH值為5.68,投加馴化耐丙酸菌泥對(duì)酸化失穩(wěn)體系pH值影響較小.隨著調(diào)控反應(yīng)的進(jìn)行,耐丙酸菌群對(duì)于酸化失穩(wěn)體系具有良好的調(diào)控效果, 丙酸濃度在第10～15d下降明顯,發(fā)酵結(jié)束時(shí),與空白對(duì)照組相比調(diào)控組丙酸濃度削減了6900.81mg/L.同時(shí),日甲烷產(chǎn)量明顯升高,在發(fā)酵第11d,調(diào)控組日甲烷產(chǎn)量(17.0± 0.8mL/g VS),比空白對(duì)照組(2.8±1.1mL/g VS)高了507%,經(jīng)過(guò)計(jì)算得出,在整個(gè)調(diào)控階段(第10～15日)調(diào)控組累積甲烷產(chǎn)量為46.0mL/g VS,比空白對(duì)照組(12.8mL/g VS)提高259%.

圖2 厭氧發(fā)酵酸化失穩(wěn)體系調(diào)控過(guò)程中酸含量及日甲烷產(chǎn)量的變化

如圖2(b)所示,廚余垃圾厭氧發(fā)酵第10d時(shí)酸化失穩(wěn),此時(shí)丁酸濃度為5009.07mg/L, pH值為5.48.將耐丁酸產(chǎn)甲烷菌泥加入到此失穩(wěn)體系中,投加后體系的pH值為5.57.隨著調(diào)控的進(jìn)行,酸化失穩(wěn)發(fā)酵體系中丁酸濃度明顯下降,發(fā)酵結(jié)束時(shí),與空白對(duì)照組相比丁酸濃度削減了5371.56mg/L.在發(fā)酵第11d,調(diào)控組日甲烷產(chǎn)量(17.1±2.1mL/g VS)與空白對(duì)照實(shí)驗(yàn)組(2.7±2.1mL/g VS)相比提高了533%(195.76± 9.36mL/g VS),經(jīng)過(guò)計(jì)算得出,在整個(gè)調(diào)控階段(第10～15d)累積甲烷產(chǎn)量(40.0mL/g VS)比空白對(duì)照組(12.9mL/g VS)提高了210%.

2.3 微生物群落特征

2.3.1 微生物群落多樣性和豐富度 Alpha Diversity常被用于分析微生物群落多樣性和豐富度,利用Observed_Species、Shannon、Simpson、Chao1和ACE等指數(shù),在97%一致性閾值下對(duì)不同樣本的微生物群落豐富度進(jìn)行比較分析,觀察馴化體系內(nèi)的種群豐富度變化.表3中為耐丙酸菌群馴化階段(MP)、耐丙酸菌群調(diào)控階段(mP)、耐丁酸菌群馴化階段(MB)和耐丁酸菌群調(diào)控階段(mB)的Alpha Diversity指數(shù).

Observed_Species指數(shù)數(shù)值越高,樣品內(nèi)微生物種群豐富度越高.由表3可以看出,在馴化階段,隨著丙酸和丁酸濃度的升高,無(wú)論是細(xì)菌還是古菌,其Observed_Species指數(shù)都呈現(xiàn)出逐漸降低的趨勢(shì),說(shuō)明體系內(nèi)微生物種群豐富度明顯下降,這證實(shí)了在趨于酸化的干式厭氧發(fā)酵體系中,揮發(fā)性脂肪酸累積使厭氧發(fā)酵菌群的豐富度遭到破壞.

由表3可以看出,在耐丙酸酸化失穩(wěn)調(diào)控階段,細(xì)菌種群豐富度明顯提高,體系中的Observed_ Species指數(shù)由第10d的1171(mP10)提高到第15d的1224(mP15),而在耐丁酸酸化失穩(wěn)調(diào)控階段,體系中Observed_Species指數(shù)先降低后上升,在第15d恢復(fù)到了824(mB15),說(shuō)明細(xì)菌種群豐富度雖然有波動(dòng),但整體呈現(xiàn)為波動(dòng)上升.

表3所示,在耐丙酸和耐丁酸產(chǎn)甲烷菌群調(diào)控階段,古菌的Observed_Species指數(shù)均呈現(xiàn)出先降低后上升的趨勢(shì),說(shuō)明添加調(diào)控菌泥后,系統(tǒng)中古菌的種群豐富度發(fā)生了變化,但隨著酸抑制的逐漸緩解,體系中古菌豐富度也逐漸增加,并且根據(jù)之前的分析由圖2可以看出,甲烷產(chǎn)率也隨之逐漸增長(zhǎng),間接說(shuō)明古菌豐富度的高低與甲烷產(chǎn)量之間具有一定的相關(guān)性.

Shannon指數(shù)越高,說(shuō)明微生物群落豐富度越高,多樣性越高,物種分布越均勻.由表3可以看出,在馴化階段,耐丙酸產(chǎn)甲烷菌在第18d時(shí)Shannon指數(shù)最高,為6.785(MP18),耐丁酸產(chǎn)甲烷菌在第24d時(shí)Shannon指數(shù)最高,為6.404(MB24),說(shuō)明此時(shí)系統(tǒng)內(nèi)細(xì)菌種群分布較均勻,根據(jù)Observed_Species指數(shù)分析,其細(xì)菌種群豐富度也較高,說(shuō)明此時(shí)的兩種馴化菌泥都可分別添加到酸化失穩(wěn)體系中進(jìn)行調(diào)控;在調(diào)控階段,酸化失穩(wěn)體系在第15d時(shí)Shannon指數(shù)最高,為7.815(mP15),說(shuō)明經(jīng)過(guò)調(diào)控后,酸化失穩(wěn)體系中細(xì)菌種群的豐富度和多樣性都得到了恢復(fù),且細(xì)菌種群的分布較為均勻.耐丁酸產(chǎn)甲烷菌群調(diào)控的酸化體系在第10d時(shí)的Shannon指數(shù)最高,為6.455(mB10),說(shuō)明加入調(diào)控菌泥后,瞬時(shí)提高了體系中細(xì)菌豐富度和多樣性.

由表3還可以看出,在調(diào)控階段,體系內(nèi)古菌的Shannon指數(shù)在第10d最高,為3.084(mP10),說(shuō)明在調(diào)控當(dāng)天,添加耐丙酸產(chǎn)甲烷菌泥迅速提高了酸化失穩(wěn)體系中古菌種群的豐富度和多樣性.表格中各個(gè)指數(shù)的變化以及圖2中沼氣對(duì)比變化說(shuō)明耐丙酸、耐丁酸產(chǎn)甲烷菌群調(diào)控都有助于恢復(fù)酸化條件下菌群的多樣性和豐富度,從而促進(jìn)恢復(fù)厭氧干式發(fā)酵體系進(jìn)而提高甲烷產(chǎn)量.

表3 Alpha Diversity指數(shù)

2.3.2 微生物細(xì)菌門(mén)水平分析 由圖3(a)可以看出,在耐丙酸菌和耐丁酸菌的馴化過(guò)程中,、和等水解酸化菌門(mén)占據(jù)優(yōu)勢(shì)地位.之前有研究表明[22],和菌門(mén)是厭氧系統(tǒng)中最常見(jiàn)的細(xì)菌.菌門(mén)包括多種梭狀芽胞桿菌,是促進(jìn)復(fù)雜有機(jī)物降解的關(guān)鍵細(xì)菌,可以水解纖維素等大分子有機(jī)底物并產(chǎn)生VFAs,例如乙酸,是乙酸異養(yǎng)型微生物產(chǎn)甲烷過(guò)程中的主要前體物.菌門(mén)在生長(zhǎng)過(guò)程中可以產(chǎn)生各種裂解酶,包括可以降解復(fù)雜有機(jī)物的水解酶和脂肪酶,從而降解多糖等大分子有機(jī)物[23].可以降解有機(jī)化合物,并且該菌門(mén)中有一些細(xì)菌,如和可以通過(guò)中間電子轉(zhuǎn)移促進(jìn)產(chǎn)甲烷菌的生長(zhǎng)[24].

在耐丙酸菌的馴化過(guò)程中,、和菌門(mén)的相對(duì)豐度逐漸增長(zhǎng),這些細(xì)菌通常與厭氧發(fā)酵初級(jí)階段即水解酸化階段相關(guān).有研究表明菌門(mén)可以利用乙酸進(jìn)行異養(yǎng)生長(zhǎng)[25],可以降解各種碳水化合物和氨基酸[24].然而隨著丙酸負(fù)荷的增加,菌門(mén)的相對(duì)豐度逐漸降低由70.17% (MP18)降低至19.13%(MP30).Li0等人也研究發(fā)現(xiàn)經(jīng)丙酸鈉馴化后的污泥比未經(jīng)馴化的污泥中菌門(mén)的豐度低,說(shuō)明丙酸負(fù)荷的增加擾動(dòng)了系統(tǒng)的穩(wěn)定性,導(dǎo)致主要的水解酸化菌的相對(duì)豐度開(kāi)始下降,并且從圖1(a)可以看出,在馴化第18d后相應(yīng)體系內(nèi)日甲烷產(chǎn)量也有所降低.因此,選取耐丙酸產(chǎn)甲烷菌群馴化體系第18d的馴化菌泥投加到廚余垃圾干式厭氧發(fā)酵酸化失穩(wěn)第10d的體系中進(jìn)行調(diào)控,在調(diào)控過(guò)程中,菌門(mén)的相對(duì)豐度先上升后下降,由45.85%(mP10)上升至63.23% (mP13)隨后又降低至59.50%(mP15),菌門(mén)的相對(duì)豐度開(kāi)始逐漸增加,由4.30%(mP10)增長(zhǎng)至8.73%(mP15),同時(shí)菌門(mén)的相對(duì)豐度逐漸降低由28.86%(mP10)降低至14.22% (mP15).由圖2(a)可以看出,調(diào)控后體系內(nèi)丙酸濃度得到了削減,體系的酸化失穩(wěn)狀態(tài)逐漸解除,菌群的變化同時(shí)表明,加入耐丙酸產(chǎn)甲烷菌泥后,緩解了體系中的酸積累,酸化失穩(wěn)體系水解酸化微生物開(kāi)始逐漸恢復(fù).

由圖3(b)可以看出,在耐丁酸產(chǎn)甲烷菌的馴化過(guò)程中,其中和菌門(mén)的相對(duì)豐度有所增長(zhǎng),隨著丁酸鈉投加量的增加,菌門(mén)的相對(duì)豐度由33.63% (MB23)增長(zhǎng)至44.07%(MB35),菌門(mén)的相對(duì)豐度由3.95%(MB23)增長(zhǎng)至14.75%(MB35),和菌門(mén)可以降解纖維素和半纖維素,也可以降解蛋白質(zhì)[26].與耐丙酸產(chǎn)甲烷菌馴化過(guò)程相比,菌門(mén)的增長(zhǎng)可能是因?yàn)槠鋵?duì)丁酸耐受性較強(qiáng)[26].同時(shí),和菌門(mén)的相對(duì)豐度逐漸降低,菌門(mén)的相對(duì)豐度由32.54%(MB23)降低至17.69% (MB35),菌門(mén)的相對(duì)豐度由14.32% (MB23)降低至5.06% (MB35).和菌門(mén)都是厭氧過(guò)程中主要的水解酸化菌,其相對(duì)豐度的降低說(shuō)明由于丁酸負(fù)荷的增加,系統(tǒng)穩(wěn)定性開(kāi)始降低,相應(yīng)地在圖1(b)中可以看出,日甲烷產(chǎn)量從馴化第23d后開(kāi)始下降.因此,將耐丁酸產(chǎn)甲烷菌群馴化體系第23d的菌泥投加到廚余垃圾干式厭氧發(fā)酵的酸化失穩(wěn)體系中進(jìn)行調(diào)控.在細(xì)菌門(mén)水平分析,菌門(mén)的相對(duì)豐度在調(diào)控過(guò)程中逐漸增長(zhǎng)由40.41% (mB10)增長(zhǎng)至60.38%(mB15),有研究發(fā)現(xiàn),菌門(mén)在丁酸的降解中起著重要的作用[27],因此,在耐丁酸產(chǎn)甲烷菌調(diào)控過(guò)程中逐漸成為優(yōu)勢(shì)菌門(mén),并且由圖2(b)可以看出,調(diào)控體系中丁酸濃度有所削減,說(shuō)明加入調(diào)控菌群后,失穩(wěn)體系中降解丁酸微生物逐漸增加,逐漸緩解了系統(tǒng)中丁酸積累.

圖3 細(xì)菌門(mén)水平相對(duì)豐度

2.3.3 微生物屬水平分析 由圖4(a)可以看出,在耐丙酸菌群的馴化過(guò)程中,隨著丙酸負(fù)荷的增加菌屬的相對(duì)豐度逐漸降低,由57.48% (MP18)降低至2.68%(MP30),說(shuō)明菌屬對(duì)丙酸的耐受度較低,菌屬是乳酸菌的一種,通常在厭氧消化過(guò)程中與嗜氫營(yíng)養(yǎng)型古菌共生,將有機(jī)酸分解為乙酸和氫氣[28],同時(shí)由圖1(a)可以看出,在馴化第18d后,體系日甲烷產(chǎn)量逐漸降低,與菌屬的降低趨勢(shì)相同.

酸化失穩(wěn)體系投加耐丙酸馴化菌泥調(diào)控過(guò)程中菌屬的相對(duì)豐度有所增加,由1.36%(mP10)增加至40.11%(mP15).同時(shí),水解酸化菌屬在調(diào)控過(guò)程中得到恢復(fù)和富集,投加丙酸馴化菌泥中該菌屬的相對(duì)豐度為3.05%(MP18),發(fā)酵酸化體系中的菌群相對(duì)豐度由2.77%(mP10)提高至5.89%(mP15).有研究表明和能夠加速 VFAs 向乙酸鹽的轉(zhuǎn)化,有助于加速厭氧消化產(chǎn)氣進(jìn)程[29].此外還觀察到,菌屬的相對(duì)豐度在調(diào)控過(guò)程中逐漸升高,由0.28%(mP10)提高至2.41% (mP15),菌屬在調(diào)控過(guò)程中得到恢復(fù)和富集,菌屬能夠降解丙酸和丁酸轉(zhuǎn)化為乙酸和氫氣[30],同時(shí)在圖2(a)中可以觀察到,添加耐丙酸產(chǎn)甲烷菌泥后,體系中丙酸濃度開(kāi)始下降,說(shuō)明通過(guò)添加耐丙酸產(chǎn)甲烷菌群可以增加體系內(nèi)降解丙酸的微生物,逐漸緩解了酸化失穩(wěn)體系中丙酸積累的脅迫效應(yīng).

圖4 細(xì)菌屬水平相對(duì)豐度

由圖4(b)可以看出,在耐丁酸抑制產(chǎn)甲烷菌群馴化體系中,菌群的相對(duì)豐度先增長(zhǎng)后下降,在馴化第30d,其相對(duì)豐度到達(dá)最高27.60% (MB30),由圖1 (b)可以看出,在馴化第30d,出現(xiàn)產(chǎn)氣小高峰,菌群的變化同產(chǎn)氣情況相似,和菌屬的相對(duì)豐度隨著丁酸負(fù)荷的增加而減少,菌屬的相對(duì)豐度由0.69%(MB23)降至0.12%(MB35),菌屬的相對(duì)豐度由0.45%(MB23)降至0.09%(MB35)說(shuō)明丁酸脅迫會(huì)影響和菌屬的相對(duì)豐度.同時(shí),也觀察到菌屬的相對(duì)豐度隨著丁酸負(fù)荷的增加而增加,由0.04%(MB23)增加至1.07%(MB35).菌屬是一類(lèi)蛋白質(zhì)水解菌,也是一類(lèi)產(chǎn)酸菌,通常它的出現(xiàn)及富集表明系統(tǒng)開(kāi)始出現(xiàn)酸積累[31].

選取第24d的耐丁酸馴化菌泥投加到酸化失穩(wěn)體系第10d中進(jìn)行調(diào)控,由圖4(b)可以看出,的相對(duì)豐度逐漸增加,由16.25% (mB10)增長(zhǎng)至36.83%(mB15),說(shuō)明通過(guò)耐丁酸產(chǎn)甲烷菌群調(diào)控,酸化失穩(wěn)體系內(nèi)得到富集和恢復(fù).同時(shí)也觀察到在調(diào)控過(guò)程中的相對(duì)豐度維持穩(wěn)定,中存在互養(yǎng)代謝碳原子數(shù)在4～18的脂肪酸的嗜中溫菌屬,并且可以與產(chǎn)甲烷菌協(xié)同作用降解丁酸[32].

由圖5(a)可以看出,在耐丙酸菌群的馴化過(guò)程中,和菌屬占據(jù)優(yōu)勢(shì)地位.菌屬為氫型產(chǎn)甲烷古菌[28].是一種嚴(yán)格的嗜乙酸產(chǎn)甲烷菌[33].馴化階段日甲烷產(chǎn)量最大時(shí),體系中占據(jù)著優(yōu)勢(shì)地位,達(dá)到了70.05%(MP18),說(shuō)明在馴化體系內(nèi)主要為氫型產(chǎn)甲烷菌.在酸化失穩(wěn)體系中投加耐丙酸馴化菌泥后,體系中氫型產(chǎn)甲烷古菌的相對(duì)豐度明顯得到提高,由15.76%(mP10)增長(zhǎng)至76.02% (mP15),同時(shí)菌屬的相對(duì)豐度在調(diào)控過(guò)程中逐漸降低,由76.25%(mP10)降低至22.90% (mP15),說(shuō)明在耐丙酸產(chǎn)甲烷菌群的調(diào)控過(guò)程中,氫型產(chǎn)甲烷菌逐漸演替為優(yōu)勢(shì)菌.

圖5 古菌屬水平相對(duì)豐度

由圖5(b)可以看出,古菌屬水平上,在耐丁酸菌群馴化過(guò)程中和仍然占據(jù)優(yōu)勢(shì)地位,其相對(duì)豐度之和占古菌總數(shù)的90%以上.在日甲烷產(chǎn)量最高以及產(chǎn)氣小高峰時(shí),菌群的相對(duì)豐度達(dá)到75.80%(MB24)和89.03%(MB30),此時(shí)系統(tǒng)主要以乙酸型產(chǎn)甲烷為主,甲烷的增長(zhǎng)可能與菌群的相對(duì)豐度相關(guān).將耐丁酸馴化菌泥投加到酸化失穩(wěn)的體系中,菌屬的相對(duì)豐度提高到了86.95% (mB10),通過(guò)調(diào)控瞬時(shí)補(bǔ)充了酸化失穩(wěn)體系中的乙酸型產(chǎn)甲烷菌,并且根據(jù)圖2(b)可以看出系統(tǒng)內(nèi)日甲烷產(chǎn)量也在第10d調(diào)控后迅速上升,說(shuō)明菌屬可以提高體系中的產(chǎn)甲烷量.有研究表明菌屬在高濃度的VFAs環(huán)境成為優(yōu)勢(shì)菌屬,此時(shí)體系正處于酸積累狀態(tài),VFAs濃度較高[34].在調(diào)控結(jié)束時(shí),菌屬相對(duì)豐度為95.71%(mB15).由圖4(b)中可以看出,在調(diào)控階段菌屬的相對(duì)豐度逐漸增加,由16.25%(mB10)增長(zhǎng)到了36.83%(mB15),Kurade等[35]發(fā)現(xiàn)菌屬在產(chǎn)乙酸中具有重要的作用,此時(shí)體系中乙酸型產(chǎn)甲烷菌占據(jù)主導(dǎo)作用,說(shuō)明此時(shí)可能存在菌屬與菌屬的協(xié)作共生關(guān)系,共同促進(jìn)了甲烷生成0.

3 結(jié)論

3.1 經(jīng)耐丙酸產(chǎn)甲烷菌群調(diào)控后,廚余垃圾厭氧發(fā)酵失穩(wěn)體系得到恢復(fù),丙酸削減濃度達(dá)到了6900.81mg/L,與空白對(duì)照組相比累積甲烷產(chǎn)量提高了259%;

3.2 經(jīng)耐丁酸產(chǎn)甲烷菌群調(diào)控后,廚余垃圾厭氧發(fā)酵失穩(wěn)體系得到恢復(fù),丁酸削減濃度達(dá)到了5371.56mg/L,與空白組相比累積甲烷產(chǎn)量提高了210%;

3.3 耐丙酸、耐丁酸產(chǎn)甲烷菌群調(diào)控可改變酸化體系微生物群落結(jié)構(gòu),從而使酸化失穩(wěn)體系得到恢復(fù).經(jīng)調(diào)控后酸化失穩(wěn)體系細(xì)菌和古菌的多樣性和豐富度明顯提高.在屬水平上耐丙酸產(chǎn)甲烷菌群的投加使有利于加速VFAs降解的和相對(duì)豐度增長(zhǎng),氫型產(chǎn)甲烷菌為優(yōu)勢(shì)菌群;耐丁酸產(chǎn)甲烷菌群的投加使有利于降解丁酸的和相對(duì)豐度增長(zhǎng),乙酸型產(chǎn)甲烷菌為優(yōu)勢(shì)菌群.

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Regulation of acidified dry anaerobic digestion of kitchen waste and microbial community analysis.

WANG Pan, YANG Xin-yu, ZHENG Yi, REN Lian-hai*

(State Environmental Protection Key Laboratory of Food Chain Pollution Control, School of Ecology and Environment, Beijing Technology and Business University, Beijing 10048, China)., 2022,42(4)：1770～1779

Aiming at the problems caused by the accumulation of propionic acid and butyric acid during anaerobic digestion, the sludge with propionic acid and butyric acid tolerant microorganism were domesticated, and they were added into the acidified systems of dry anaerobic digestion of kitchen waste to explore the effects on methane production and microbial communities. After treated by the sludge with propionic acid and butyric acid tolerant bacteria, the propionic acid concentration reduced by 6900.81mg/L, and the cumulative methane production increased by 259%, while the butyric acid concentration reduced by 5371.56mg/L, and the cumulative methane production increased by 210%, respectively. The richness of bacteria in the acidified systems was significantly improved after the regulation with propionic acid and butyric acid tolerant bacteria. The analysis of microbial community structure at genus level showed that the relative abundance ofand, having the ability of degrading volatile fatty acids, began to increase in the reactors treated by the sludge with propionic acid tolerant bacteria. After adding the sludge with butyric acid tolerant bacteria, the relative abundance ofincreased.have a synergistic effect with acetic acid methanogens. According to the analysis of archaea, the relative abundance of hydro methanogens and acetic acid methanogens increased in the groups treated with propionic acid and butyric acid tolerant bacteria, respectively.

kitchen waste；dry anaerobic fermentation；regulation of acidified anaerobic fermentation system；microbial community

X705,X703.5

A

1000-6923(2022)04-1770-10

王 攀(1983-),女,河北石家莊人,教授,博士,主要從事固體廢棄物資源化方向.發(fā)表論文50余篇.

2021-09-13

國(guó)家重點(diǎn)研發(fā)計(jì)劃重點(diǎn)專(zhuān)項(xiàng)(2019YFC1906303);國(guó)家自然基金資助項(xiàng)目(42007350);北京市自然基金資助項(xiàng)目(8202010);2021年研究生科研能力提升計(jì)劃項(xiàng)目(202105)

*責(zé)任作者, 教授, renlh@th.btbu.edu.cn