班巧英,張 瑞,張立國,李建政,梅 芝

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熒光定量PCR解析酸性條件下丙酸氧化菌的演替

班巧英1*,張 瑞1,張立國1,李建政2,梅 芝1

(1.山西大學(xué)環(huán)境與資源學(xué)院,山西太原 030006；2.哈爾濱工業(yè)大學(xué)市政與環(huán)境工程學(xué)院,黑龍江哈爾濱 150090)

采用實(shí)時(shí)熒光定量PCR技術(shù)考察了UASB酸性條件下丙酸氧化菌的演替規(guī)律.結(jié)果表明,至少有3種已鑒定的丙酸氧化菌(,和)存在于UASB反應(yīng)器中.在pH值為7.5~7.1時(shí),是主要的丙酸氧化菌群,其數(shù)量為(5.0~5.8)×10316S rRNA 基因拷貝數(shù)/ng DNA,約占檢測(cè)到丙酸氧化菌總數(shù)的90.6%以上. pH從7.1降到6.8導(dǎo)致數(shù)量顯著增加,和成為優(yōu)勢(shì)菌群,其數(shù)量占丙酸氧化菌總數(shù)的88.9%.在pH6.5條件下,(74.8%)成為優(yōu)勢(shì)丙酸氧化菌群.當(dāng)pH￡6.0時(shí),.和演替為優(yōu)勢(shì)丙酸氧化菌,表明這兩種丙酸氧化菌具有較強(qiáng)的耐酸性.當(dāng)pH￡6.0時(shí),丙酸氧化菌總數(shù)隨pH降低而明顯減少,這可能是厭氧反應(yīng)器在酸性條件下發(fā)生丙酸積累的根本原因之一.

UASB；丙酸氧化菌；酸性條件；熒光定量PCR

厭氧生物處理技術(shù)廣泛用于處理各種有機(jī)廢棄物,同時(shí)回收甲烷作為清潔能源[1-3].有機(jī)物厭氧發(fā)酵可產(chǎn)生大量的中間代謝產(chǎn)物,包括丙酸[4].從熱力學(xué)的角度看,丙酸降解是一個(gè)高度吸能的過程,很難自發(fā)進(jìn)行.所以,當(dāng)系統(tǒng)遭受溫度、有機(jī)負(fù)荷率、毒性物質(zhì)等沖擊時(shí),丙酸降解受到抑制,并在厭氧消化系統(tǒng)內(nèi)過量積累,進(jìn)而導(dǎo)致反應(yīng)器“酸化”、運(yùn)行失敗[5-6].因此,強(qiáng)化厭氧消化系統(tǒng)中丙酸氧化菌群的代謝活性對(duì)于提高厭氧反應(yīng)器運(yùn)行效能及運(yùn)行穩(wěn)定性具有重要意義.

在厭氧生境中,丙酸的降解是由丙酸氧化菌來完成的,其在有機(jī)物厭氧降解過程中起著不可替代的作用[7].由于丙酸氧化菌的生長需要耗氫菌的協(xié)同作用而導(dǎo)致其分離培養(yǎng)十分困難.迄今為止,只有一些生長緩慢的丙酸氧化菌被分離和鑒定[5].已鑒定的丙酸氧化菌在分類學(xué)上屬于屬,屬,屬和屬[5].它們廣泛分布于厭氧顆粒污泥、沼澤、污泥消化器等厭氧生境中[8-10].厭氧反應(yīng)器運(yùn)行的效能與微生物群落結(jié)構(gòu)密切相關(guān).DGGE(變性梯度凝膠電泳)、FISH(熒光原位雜交)、qPCR(定量PCR)等分子生物學(xué)技術(shù)常用于解析有機(jī)物厭氧降解過程中的微生物群落結(jié)構(gòu)[11].丙酸氧化菌的組成和分布受到諸多因素的影響,例如:溫度、水力停留時(shí)間、pH、氫分壓等[5].盡管關(guān)于中性條件下丙酸氧化菌定性和定量分析的研究已有報(bào)道[12-13].然而,酸性環(huán)境中丙酸氧化菌定量分析的相關(guān)報(bào)道仍然較少.前期研究發(fā)現(xiàn)和是UASB(升流式厭氧污泥床)反應(yīng)器中主要的丙酸氧化菌[14].因此,本研究針對(duì)這兩個(gè)屬中已鑒定的丙酸氧化菌,采用實(shí)時(shí)熒光定量PCR技術(shù)分析其在不同pH值條件下的數(shù)量差異,為優(yōu)化厭氧反應(yīng)器運(yùn)行參數(shù)、強(qiáng)化反應(yīng)器運(yùn)行效能及運(yùn)行穩(wěn)定性提供理論基礎(chǔ).

1 材料與方法

1.1 反應(yīng)器運(yùn)行控制

試驗(yàn)裝置為UASB反應(yīng)器,其有效容積為4L.試驗(yàn)廢水為合成廢水,丙酸為唯一碳源,并補(bǔ)充其他微生物生長所必須的大量、微量元素及維生素等[14].在溫度(35±1)℃,水力停留時(shí)間6h,進(jìn)水丙酸濃度為2000mg/L,污泥床pH7.5條件下啟動(dòng)UASB反應(yīng)器,待運(yùn)行穩(wěn)定后,將UASB反應(yīng)器的污泥床pH逐漸降低至7.1,6.8,6.5,6.0,5.5,5.0, 4.5,4.0.每次改變pH值均在前一次運(yùn)行達(dá)到穩(wěn)定時(shí)進(jìn)行.每次運(yùn)行達(dá)到穩(wěn)定后從污泥床取樣,保存至-20℃?zhèn)溆?

1.2 分析項(xiàng)目及方法

pH值和生物量采用標(biāo)準(zhǔn)方法測(cè)定[15],產(chǎn)氣量通過濕式氣體流量計(jì)(LML-1,長春汽車濾清器有限責(zé)任公司)測(cè)定,氣體組成和揮發(fā)酸組成采用氣相色譜儀測(cè)定(SP-6800A和SP6890,山東魯南瑞虹化工儀器有限公司)[14].

1.3 DNA提取及qPCR

表1 qPCR中所用引物序列

稱取0.15g污泥(濕重),采用DNA提取試劑盒(MO Bio Laboratories,Inc,Carlsbad,CA,USA)提取厭氧活性污泥總DNA.DNA濃度用分光光度計(jì)測(cè)定(Thermo Fisher Scientific Inc.USA).

根據(jù)和中已鑒定丙酸氧化菌的16S rRNA基因序列設(shè)計(jì)特異引物,引物序列見表1[16].本研究中實(shí)時(shí)熒光定量PCR分析采用絕對(duì)定量方式.標(biāo)準(zhǔn)樣品為包含目的基因片段的融合質(zhì)粒,用超純水進(jìn)行10倍梯度稀釋(101~107).標(biāo)準(zhǔn)樣品和待測(cè)樣品同時(shí)進(jìn)行熒光定量PCR擴(kuò)增.qPCR在熒光定量PCR系統(tǒng)(Model 7500,ABI,USA)中完成.所用熒光染料為SYBR green I.反應(yīng)體系為:SYBR Green PCR Master Mix (Toyobo Co.,LTD.,Japan)10μL,DNA樣品3μL,引物各1μL,ROX 0.04μL,4.96μL H2O.反應(yīng)程序: 94℃預(yù)變性1min,94℃ 15s,58℃ 30s,72℃ 35s,40循環(huán).每個(gè)樣品設(shè)置3個(gè)重復(fù).以達(dá)到對(duì)數(shù)增長時(shí)的循環(huán)數(shù)(值)為橫坐標(biāo),標(biāo)準(zhǔn)質(zhì)粒數(shù)目的對(duì)數(shù)值為縱坐標(biāo)繪制標(biāo)準(zhǔn)曲線(圖1).

2 結(jié)果與分析

2.1 UASB運(yùn)行效能

在35℃、有機(jī)負(fù)荷率12kg COD/(m3·d)條件下,考察了pH值降低對(duì)UASB中丙酸降解的影響.結(jié)果表明,當(dāng)污泥床pH值為7.5~6.8時(shí),丙酸去除率高達(dá)93.6%以上,且大部分丙酸(81.5%~90%)是在污泥床去除的(表2).當(dāng)污泥床pH值分階段從6.8逐漸降低至4.5時(shí),污泥床對(duì)丙酸的去除率減少了17.1%~84.9%.當(dāng)pH值降低至4.0時(shí),丙酸幾乎不能分解.在整個(gè)運(yùn)行期間,氫氣含量低于色譜檢測(cè)限,出水乙酸濃度總是低于70mg/L,比產(chǎn)甲烷速率為190.7~346.9LCH4/(kg CODremoved·d).

2.2 丙酸氧化菌的qPCR分析

厭氧生物處理反應(yīng)器的運(yùn)行效能與系統(tǒng)中的微生物群落結(jié)構(gòu)密切相關(guān).前期研究發(fā)現(xiàn)該UASB中的主要丙酸氧化菌在分類學(xué)上屬于屬和屬[14].因此,本研究針對(duì)上述兩個(gè)屬中已鑒定的丙酸氧化菌,通過qPCR檢測(cè)技術(shù)探討了UASB反應(yīng)器污泥床在pH7.5、pH7.1、pH6.8、pH6.5、pH6.0、pH5.5、pH5.0、pH4.5、pH4.0條件下丙酸氧化菌的數(shù)量.結(jié)果表明,有3種已鑒定丙酸氧化菌存在于所有檢測(cè)的樣品中,分別是、和.其中,和是兩個(gè)專性互營菌,只有與產(chǎn)甲烷菌共培養(yǎng)時(shí)才能生長[5].而.除了能與產(chǎn)甲烷菌共培養(yǎng)外,還能在一些簡單底物上進(jìn)行純培養(yǎng)[5].spp.通過甲基丙二酰-輔酶A途徑(MMC)降解丙酸,而.先將兩分子丙酸整合轉(zhuǎn)化為1分子乙酸和1分子丁酸,然后丁酸再通過-氧化分解成2分子乙酸[5].和生長的pH 范圍分別是6.5~7.5和6.3~7.8,而生長的pH范圍仍然是未知的[17-19].

丙酸氧化菌的qPCR分析表明,在中性條件下(pH 7.5和pH 7.1),是UASB反應(yīng)器中優(yōu)勢(shì)丙酸氧化菌,其含量為5.0~5.8×10316S rRNA基因拷貝數(shù)/ng DNA,約占檢測(cè)到丙酸氧化菌總數(shù)的90.6%以上(圖2和圖3).這是由于有更高的比生長速率和丙酸利用率,在適宜的條件下其生長和代謝更快[19].類似地,在一個(gè)處理生物垃圾-丙酸合物的中溫厭氧消化器中(pH7.0),spp.是優(yōu)勢(shì)丙酸氧化菌[20].Shigematsu等[21]的研究也發(fā)現(xiàn),在一個(gè)以丙酸為唯一碳源的恒化器中,高稀釋率促進(jìn)spp.生長,而低稀釋率有利于spp.的生長.與此相反,Worm等發(fā)現(xiàn)中性條件下UASB處理丙酸合成廢水時(shí),spp.長期占據(jù)優(yōu)勢(shì)地位.但是當(dāng)反應(yīng)器中長期缺乏鉬、鎢、硒時(shí),spp.取代了spp.[13].另外,也有研究發(fā)現(xiàn),在處理全脂奶粉,蔗糖,混合酸(丁酸、丙酸、乙酸)廢水時(shí),或是優(yōu)勢(shì)丙酸氧化菌[12,22].這些研究暗示了底物組成對(duì)丙酸氧化菌的組成也有一定影響.從UASB反應(yīng)器中丙酸氧化菌數(shù)量上看,盡管丙酸氧化菌的數(shù)量在pH7.5~7.1時(shí)較低,但由于具有較高的代謝活性,使得丙酸去除率高達(dá)95%以上(圖4和表2).

表2 不同pH條件下UASB反應(yīng)器運(yùn)行特征

pH從7.1降低至6.8導(dǎo)致的數(shù)量增加了2.1倍,但是它的相對(duì)豐度卻降低至38.2%.相反,的含量顯著增加,占丙酸氧化菌總數(shù)的50.6%.和成為pH6.8條件下的主要丙酸降解菌群,相對(duì)豐度為88.9%.同時(shí),UASB的生物量較之前增加了16.0%增加量,使得污泥床的丙酸去除率達(dá)到81.5%(表2).

圖3 不同pH條件下丙酸氧化菌的相對(duì)豐度

當(dāng)pH由6.8降低至6.5時(shí),S.propionica的數(shù)量達(dá)到最大值(4.6×10416S rRNA基因拷貝數(shù)/ng DNA),成為優(yōu)勢(shì)丙酸氧化菌群(圖2).當(dāng)pH從6.5降低至6.0,導(dǎo)致P.propionicum的數(shù)量增加了1倍達(dá)到最大值.相反,S.propionica的數(shù)量減少了43.9%.二者總數(shù)為4.1×104 16S rRNA基因拷貝數(shù)/ ng DNA,占丙酸氧化菌總數(shù)的90.9%,共同成為優(yōu)勢(shì)丙酸氧化菌群.當(dāng)pH 進(jìn)一步降低至5.5,5.0,4.5以及 4.0時(shí),所有檢測(cè)到的丙酸氧化菌逐漸減少(圖4).由圖3可知,在pH6.0~4.5條件下,P.propionicum和S.propionica依然是UASB反應(yīng)器中優(yōu)勢(shì)丙酸氧化菌群,占丙酸氧化菌總數(shù)的81.4%~89.5%.分析認(rèn)為, S.propionica能在低pH條件下生存可能與其代謝途徑有關(guān)[17]. S.propionica降解丙酸時(shí)可產(chǎn)生中間代謝產(chǎn)物丁酸,丁酸的降解在熱動(dòng)力學(xué)上比丙酸更容易,以至于產(chǎn)物抑制容易解除,可促進(jìn)丙酸降解.事實(shí)上,前期研究的確發(fā)現(xiàn)該UASB系統(tǒng)中存在降解丁酸的微生物—Syntrophomonas spp.[14]. S.propionica獨(dú)特的代謝途徑使其能在酸性環(huán)境下生長.然而,P.propionicum能在低pH 條件下生存的原因有待進(jìn)一步研究.

另外,盡管丙酸是該UASB反應(yīng)器中唯一的碳源,但是檢測(cè)到的丙酸氧化菌僅占細(xì)菌總數(shù)的0.55%~3.65%,說明該UASB反應(yīng)器中可能存在著未知的丙酸氧化菌.

以上結(jié)果表明,pH降低使得丙酸氧化菌群發(fā)生了明顯的演替.在中性條件下,是UASB反應(yīng)器中的優(yōu)勢(shì)丙酸氧化菌,隨著pH降低逐漸演替為和(pH6.8)、(pH6.5)、和(pH6.0~4.0).這可能是弱酸性環(huán)境中(pH6.5~5.0),丙酸去除率得到一定程度恢復(fù)的主要原因.

3 結(jié)論

3.1 pH降低對(duì)丙酸氧化菌的生長有明顯的抑制作用.在pH6.5條件下達(dá)到最大值6.2×10416S rRNA基因拷貝數(shù)/ngDNA.當(dāng)pH由6.5分階段降低至4.0時(shí),丙酸氧化菌總數(shù)逐漸減少,這可能就是厭氧生物處理反應(yīng)器在酸性條件下發(fā)生丙酸積累的根本原因.

3.2 隨著pH逐漸降低,丙酸氧化菌群發(fā)生了明顯的生態(tài)演替.在pH7.5~7.1時(shí),是優(yōu)勢(shì)丙酸氧化菌;然后逐漸演替為和(pH6.8)、(pH6.5),和(pH6.0~4.0).

[1] 何 強(qiáng),孫興福,艾海男,等.兩相一體式污泥濃縮消化反應(yīng)器運(yùn)行效能及其微生物特性[J]. 中國環(huán)境科學(xué), 2012,32(11): 2039-2046.

[2] 陳廣銀,鄭 正,常志州,等.不同氮源對(duì)麥稈厭氧消化過程的影響[J]. 中國環(huán)境科學(xué), 2011,31(1):73-77.

[3] 宋佳秀,任南琪,錢東旭,等.醌呼吸影響厭氧消化產(chǎn)CO2/CH4及轉(zhuǎn)化有毒物質(zhì)的研究[J]. 中國環(huán)境科學(xué), 2014,34(5):1236- 1241.

[4] Meng X, Zhang Y, Li Q, et al. Adding Fe0powder to enhance the anaerobic conversion of propionate to acetate [J]. Biochemical Engineering Journal, 2013,73:80–85.

[5] Li J, Ban Q, Zhang L, et al. Syntrophic propionate degradation in anaerobic digestion: A review [J]. International Journal of Agriculture and Biology, 2012,5:843–850.

[6] Narihiro T, Terada T, Ohashi A, et al. Quantitative detection of previously characterized syntrophic bacteria in anaerobic wastewater treatment systems by sequence-specific rRNA cleavage method [J]. Water Research, 2012,46:2167–2175.

[7] Boone D R, Bryant M P. Propionate-degrading bacterium, Syntrophobacter wolinii sp. nov. gen. nov., from methanogenic ecosystems [J]. Applied and Environmental Microbiology, 1980, 40:626–632.

[8] Sekiguchi Y, Kamagata Y, Nakamura K, et al. Fluorescence in situ hybridization using 16S rRNA- targeted oligonucleotides reveals localization of methanogens and selected uncultured bacteria in mesophilic and thermophilic sludge granules [J]. Applied and Environmental Microbiology, 1999,65:1280–1288.

[9] Lueders T, Pommerenke B, Friedrich M W. Stable-isotope probing of microorganisms thriving at thermodynamic limits: syntrophic propionate oxidation in flooded soil [J]. Applied and Environmental Microbiology, 2004,70:5778–5786.

[10] Harmsen H J M, Kengen H M P, Akkermans A D L, et al. Detection and localization of syntrophic propionate-oxidizing bacteria in granular sludge by in situ hybridization using 16S rRNA-based oligonucleotide probes [J]. Applied and Environmental Microbiology, 1996,62:1656–1663.

[11] Kundu K, Bergmann I, Hahnke S, et al. Carbon source-a strong determinant of microbial community structure and performance of an anaerobic reactor [J]. Journal of Biotechnology, 2013,168: 616–624.

[12] Ariesyady H D, Ito T, Okabe S. Functional bacterial and archaeal community structures of major trophic groups in a full-scale anaerobic sludge [J]. Water Research, 2007,41:1554–1568.

[13] Worm P, Fermoso F G, Lens P N L, et al. Decreased activity of a propionate degrading community in a UASB reactor fed with synthetic medium without molybdenum, tungsten and selenium [J]. Enzyme and Microbiology Technology, 2009,45:139–145.

[14] Zhang L, Ban Q, Li J, et al. Response of syntrophic propionate degradation to pH decrease and microbial community shifts in an UASB reactor [J]. Journal of Microbiology and Biotechnology, 2016,26:1409–1419.

[15] 國家環(huán)保局.水和廢水監(jiān)測(cè)分析方法[M]. 北京:中國環(huán)境科學(xué)出版社, 2002.

[16] Ban Q, Zhang L, Li J. Shift ofpropionate-oxidizing bacteria with HRT decrease in an UASB reactor containing propionate as a sole carbon source [J]. Applied Biochemistry and Biotechnology, 2015,175:274–286.

[17] Liu Y, Balkwill D L, Aldrich H C, et al. Characterization of the anaerobic propionate-degrading syntrophs Smithella propionica gen. nov., sp. nov. and Syntrophobacter wolinii [J]. International Journal Systematic Bacteriology, 1999,49:545–556.

[18] Imachi H S, Sakai Ohashi A, Harada H, et al.sp. nov., an, mesophilic, obligately syntrophic, propionate- oxidizing bacterium [J]. International Journal of Systematic and Evolutionary Microbiology, 2007,57:1487–1492.

[19] de Bok F A M, Harmsen H J M, Plugge C M, et al. The first true obligately syntrophic propionate-oxidizing bacterium,sp. nov., co-cultured with, and emended description of the genus Pelotomaculum [J]. International Journal of Systematic and Evolutionary Microbiology, 2005,55:1697–1703.

[20] Li C, Moertelmaier C, Winter J, et al. Microbial community shifts during biogas production from biowaste and/or propionate [J]. Bioengineering, 2015,2:35–53.

[21] Shigematsu T, Era S, Mizuno Y, et al. Microbial community of a mesophilic propionate-degrading methanogenic consortium in chemostat cultivation analyzed based on 16S rRNA and acetate kinase genes [J]. Applied and Environmental Microbiology, 2006,72:401–415.

[22] Harmsen H J M, Kengen H M P, Akkermans A D L, et al. Detection and localization of syntrophic propionate-oxidizing bacteria in granular sludge by in situ hybridization using 16S rRNA-based oligonucleotide probes [J]. Applied and Environmental Microbiology, 1996,62:1656–1663.

The succession of propionate-oxidizing bacteria at acidic conditions was analyzed byRT-PCR.

BAN Qiao-ying1*, ZHANG Rui1, ZHANG Li-guo1, LI Jian-zheng2, MEI Zhi

(1.College of Environment and Resource, Shanxi University, Taiyuan 030006, China；2.School of Municipal and Environmental Engineering, Harbin Institute of Technology, Harbin 150090, China)., 2017,37(10)：3861~3866

Succession of propionate-oxidizing bacteria at acidic conditions was investigated in an upflow anaerobic sludge bed (UASB) reactor by quantitative real-time fluorescence polymerase chain reaction (qPCR). The results showed that at least three identified species of propionate-oxidizing bacteria (,, and) existed in the UASB system.was dominated at pH7.5~7.1 and its quantity was (5.0~5.8)×10316S rDNA copies per ng DNA, accounting for above 90.6% of the total detectable propionate-oxidizing bacteria. pH decreased from 7.1 to 6.8 resulted inwas significantly increased.andbecame dominant propionate-oxidizing bacteria and occupied 88.9%.accounted for 74.8% in total detectable propionate-oxidizing bacteria at pH 6.5, whereasandsucceed to dominant propionate-oxidizing bacteria at pH￡6.0, indicating thatandwere more acid resistant. In addition, the total number of propionate-oxidizing bacteria was gradually reduced at pH￡6.0. It might be one of the reasons for propionate accumulation at acidic conditions in anaerobic reactors.

UASB；propionate-oxidizing bacteria；acidic conditions；qPCR

X703.5

A

1000-6923(2017)10-3861-06

班巧英(1982-),女,山西忻州人,講師,博士,主要從事廢水厭氧生物處理研究.發(fā)表論文30余篇.

2017-03-13

國家自然科學(xué)基金資助項(xiàng)目(51708341);天津市水質(zhì)科學(xué)與技術(shù)重點(diǎn)實(shí)驗(yàn)室開放研究基金資助項(xiàng)目 (TJKLAST-ZD- 2016-05);山西省基礎(chǔ)研究項(xiàng)目(2015021136, 2015021134)

* 責(zé)任作者, 講師, banqiaoying@163.com