微生物對scCO2-咸水-砂巖體系中礦物反應(yīng)的影響

張鳳君,宋云鵬,鐘 爽,2,樊 凱,李晨陽,2*,張志勇

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微生物對scCO2-咸水-砂巖體系中礦物反應(yīng)的影響

張鳳君1,宋云鵬1,鐘 爽1,2,樊 凱3,李晨陽1,2*,張志勇4

(1.吉林大學(xué)地下水資源與環(huán)境教育部重點實驗室,吉林 長春 130021；2.吉林建筑大學(xué)松遼流域水環(huán)境教育部重點實驗室,吉林 長春 130118；3.陜西省榆林市環(huán)境保護(hù)局,陜西 榆林 719000；4.吉林省松原市前郭縣環(huán)境保護(hù)局,吉林 松原 138000)

利用Illumina MiSeq對scCO2-咸水-砂巖體系中微生物16S rRNA基因V3-V4區(qū)進(jìn)行分析,探究高壓反應(yīng)釜體系中微生物對scCO2的響應(yīng)及微生物對礦物反應(yīng)的作用.結(jié)果顯示,生物量受pH值影響較大,初始pH值為7.02,生物量11.02×106gene/mL,30d時pH值降至5.65,生物量降至0.26×106gene/mL;隨著礦物溶蝕,90d時pH值增至5.87,生物量增至4.61×106gene/mL.群落結(jié)構(gòu)中,(52.60%(30d), 55.34%(90d))與(46.89%(30d), 43.89%(90d))為優(yōu)勢菌門.在屬水平,30,90d時,,與為優(yōu)勢菌屬.產(chǎn)酸菌(,與)促進(jìn)了長石與粘土溶蝕,咸水中K+,Na+,Ca2+,Mg2+,T-Fe濃度高于空白組;鐵還原菌()提高了Fe(II)/Fe(III)比值;微生物膜對Ca2+?Mg2+?Fe2+具有吸附作用.SEM結(jié)果顯示,微生物介導(dǎo)下先于空白組出現(xiàn)菱鐵礦沉淀. scCO2-咸水-砂巖體系中適應(yīng)菌能促進(jìn)礦物溶蝕與碳酸鹽礦物捕獲.

超臨界CO2；土著微生物；礦物溶蝕；礦物捕獲

CO2地質(zhì)封存(GCS)被視為可大規(guī)模減少溫室氣體排放的技術(shù)之一[1].GCS中,CO2常以超臨界狀態(tài)(scCO2)注入陸上深部咸水層、海底咸水層、枯竭油氣田等儲層中進(jìn)行封存[2-3].據(jù)IPCC估算,全球CO2地質(zhì)封存潛力至少為2000億t,其中深部咸水層占封存總量92%以上[4],因此深部咸水層被視為理想儲層[1,5].咸水層封存主要通過水動力捕獲、溶解捕獲、礦物捕獲實現(xiàn)CO2有效儲存[2,6].地球化學(xué)作用中,可逐漸生成方解石?菱鎂礦、菱鐵礦等碳酸鹽礦物以實現(xiàn)CO2礦物捕獲,礦物捕獲被視為CO2最安全捕獲機(jī)制[2,5].而TOUGHREACT等軟件模擬顯示,實現(xiàn)有效CO2礦物捕獲常需上百年甚至上千年時間[2],因此如何加速CO2礦物捕獲成為關(guān)注點.

隨著“國際大陸鉆探計劃”等項目實施,發(fā)現(xiàn)每毫升深層地下水微生物量最高可達(dá)108數(shù)量級[7].因此微生物對CO2儲存能力和安全性的影響近年來得到關(guān)注.地下微生物主要有以下作用:代謝產(chǎn)酸促進(jìn)礦物溶蝕[8-11];氧化還原作用影響CO2封存,如鐵還原菌有利于菱鐵礦捕獲[12-14];微生物細(xì)胞外聚合物(EPS)吸附Ca2+?Fe2+?Mg2+等,可提供碳酸鹽成核點促進(jìn)碳酸鹽礦化[15-16].CO2注入地下環(huán)境后pH值?壓力的改變,將會對土著微生物群落結(jié)構(gòu)?生物量產(chǎn)生影響[17-20],同時生物地球化學(xué)作用也將會影響CO2封存過程.當(dāng)前研究中,土著微生物介導(dǎo)下CO2-水-巖實驗較少,且多在非scCO2條件下進(jìn)行.封存場所溫度?壓力?微生物種群特征不同,生物地球化學(xué)作用也將存在差別.因此針對不同場地條件,開展土著微生物介導(dǎo)下scCO2-水-巖體系中的生物地球化學(xué)作用研究具有一定意義.

本文基于我國鄂爾多斯CO2咸水層封存場地情況,開展了土著微生物介導(dǎo)下scCO2-咸水-砂巖相互作用的模擬實驗,采用Illumina MiSeq高通量測序,結(jié)合礦物分析?水化學(xué)分析等技術(shù)手段,探究土著微生物對scCO2注入后的響應(yīng)及微生物對scCO2-咸水-砂巖反應(yīng)體系中礦物溶蝕與捕獲的作用,以期豐富CO2深部咸水層封存中生物地球化學(xué)信息.

1 材料與方法

1.1 實驗材料及儀器設(shè)備

1.1.1 實驗材料 實驗菌由鄂爾多斯盆地深度1909.8~1922.8m咸水離心所得,采用MSM培養(yǎng)基擴(kuò)大培養(yǎng)后進(jìn)行實驗.巖樣為鄂爾多斯石盒子組砂巖巖心,加工成10mm×10mm×1mm切片和2mm粒徑碎屑.按場地咸水測試結(jié)果配置實驗咸水,濃度K+: 76.42mg/L,Na+:2356mg/L,Ca2+:1067.33mg/L,Mg2+: 4.47mg/L,T-Fe:27.47mg/L,Cl-:5339.34mg/L,HCO3-: 520.26mg/L,SO42-:46.28mg/L,NO3-:52.67mg/L,CH3COO-:7.80mg/L,pH值:7.02.所用氣體CO2純度99.5%.

16S rRNA高通量測序及熒光定量PCR (qPCR)主要材料包括:E.Z.N.A. Soil DNA Kit, Qubit2.0DNA檢測試劑盒,Taq DNA Polymerase,TaqDNA聚合酶,Agencourt AMPure XP等.

1.1.2 儀器設(shè)備 采用厭氧培養(yǎng)箱(YQX-II, YUEJIN,上海)進(jìn)行微生物擴(kuò)大培養(yǎng);16S rRNA?qPCR主要儀器包括:凝膠成像系統(tǒng)?Qubit? 2.0熒光計?PCR反應(yīng)擴(kuò)增儀?測序儀?熒光定量PCR儀(ABI Stepone plus)等.

采用原子吸收分光光度計(AA-6300CF)對水溶液陽離子進(jìn)行測試;利用分光光度計對Fe2+測試(菲洛嗪比色法[21]);采用pH值計(STARTER 3100)對取樣后溶液進(jìn)行pH值測試.采用掃描電子顯微鏡(SEM,JSM-6010LA)?能量色散譜儀 (EDS,UK)對礦物形貌進(jìn)行觀察.采用傅里葉紅外光譜儀對礦物表面物質(zhì)進(jìn)行分析.

實驗設(shè)備為250mL高壓反應(yīng)釜,配有恒溫加熱系統(tǒng)?壓力溫度監(jiān)測系統(tǒng)?取樣系統(tǒng)等(圖1).

圖1 實驗裝置

1.2 實驗方法

1.2.1 高壓反應(yīng)釜模擬實驗 取50mL菌液離心后去上清液,在厭氧培養(yǎng)箱中將沉淀物加入到180mL配置咸水中混合均勻.將180mL混合液,20g巖樣(碎屑、切片)加入反應(yīng)釜擰緊,通過加壓泵注入CO2至12MPa.空白組直接將180mL地層咸水?20g巖樣加入反應(yīng)釜,注入CO2至12MPa,55°C運(yùn)行90d.每15d對溶液取樣分析;對90d礦物進(jìn)行分析;30d樣品進(jìn)行生物分析,以研究scCO2注入短期內(nèi)生物的響應(yīng),對90d樣品進(jìn)行生物分析,以研究scCO2注入后較長時期內(nèi)生物的響應(yīng).

1.2.2 DNA提取與PCR擴(kuò)增 細(xì)菌基因組中編碼16S rRNA的rDNA 基因具有良好的進(jìn)化保守性?適宜分析的長度(約為1540bp)以及與進(jìn)化距離相匹配的良好變異性,所以成為細(xì)菌分子鑒定的標(biāo)準(zhǔn)標(biāo)識序列.16S rDNA序列包含9或10個可變區(qū)和11個恒定區(qū).保守序列區(qū)域反映了生物物種間的親緣關(guān)系,而高變序列區(qū)域則能體現(xiàn)物種間的差異.目前細(xì)菌16S測序的引物有:V3-V4區(qū)?V1-V3區(qū)?V3區(qū)?V4區(qū)?V4-V5區(qū)?V6-V8區(qū).細(xì)菌方面一般做V3-V4區(qū)較多,因為其有較好的覆蓋度.并且Li等人[21]及Nicot等[22]在微生物群落中不同菌的鑒定中均選用了V3-V4區(qū).

利用E.Z.N.A. Soil DNA Kit (OMEGA) 試劑盒提取水樣中基因組DNA (Promega, Madison, WI, USA),用Qubit2.0DNA試劑盒精確定量所提取的DNA.采用341F和805R引物對細(xì)菌總DNA的V3-V4區(qū)進(jìn)行擴(kuò)增.341F引物: 5′-CCCTACAC- GACGCTC TTCCGATCTG-3′;805R引物: 5′- GACTGGAGTTCCTT GGCACCCGAGAATTCCA-3′.擴(kuò)增采用20μL反應(yīng)體系:4μL的5×FastPfu Buffer, 2μL的2.5mmol·L dNTPs,引物341F/805R各0.4μL, 0.4μL的Taq酶,1.0μL的模板,12.2μL的ddH2O.PCR擴(kuò)增: 95℃預(yù)變性3min,95℃變性30s,45℃退火30s, 72℃延伸30s,重復(fù)5個循環(huán); 95℃變性30s,55℃退火30s,72℃延伸30s, 72℃修復(fù)延伸5min,重復(fù)20個循環(huán).PCR產(chǎn)物經(jīng)2%瓊脂糖凝膠電泳檢測后用瓊脂糖回收試劑盒SK8131回收.采用Tris-HCl洗脫后連接“Y”接頭,使用磁珠篩選去除接頭自連片段,按照每個樣品測一萬條序列加入1ng PCR產(chǎn)物的標(biāo)準(zhǔn)富集PCR產(chǎn)物,然后用0.1mol/L的NaOH溶液變性,獲得單鏈DNA片段.

1.2.3 熒光定量PCR(qPCR) 通過qPCR對水樣基因組DNA拷貝數(shù)進(jìn)行測試.用StepOne型熒光定量PCR儀(ABI)?SMA4000微量分光光度計(merinton)進(jìn)行濃度測定.依據(jù)其在260nm和280nm之間吸光率比值(1.8~2..0之間)及在260nm和230nm吸光率比值(高于1.70)進(jìn)行判斷.

標(biāo)準(zhǔn)品制備過程包括PCR反應(yīng)體系建立?PCR電泳?PCR回收?連接產(chǎn)物轉(zhuǎn)化?質(zhì)粒提取?定量質(zhì)粒信息?標(biāo)準(zhǔn)曲線樣品制備,10倍梯度稀釋構(gòu)建好的各質(zhì)粒,90μL稀釋液+10μL 質(zhì)粒,一般做4~6個點,通過預(yù)實驗選取合適標(biāo)準(zhǔn)品用于制備標(biāo)準(zhǔn)曲線(qPCR具體操作過程詳見http://www.Sangon.com/ services_geneanalysis.html).

將樣品稀釋10倍上機(jī),qPCR主混液20μL:10μL SybrGreen qPCR Master Mix(2X),0.4μL引物F (10μM),0.4μL引物R (10μM),7.2μL ddH2O,2μL 模板(DNA).在熒光定量PCR儀中進(jìn)行反應(yīng).qPCR循環(huán): 95℃預(yù)變性3min;95℃變性15s,45℃退火20s,72℃延伸30s, 72℃修復(fù)延伸8min,重復(fù)45個循環(huán).根據(jù)相應(yīng)的標(biāo)準(zhǔn)曲線,計算在樣品中目的種群16S rRNA 基因的濃度.

1.2.4 建庫測序及序列處理 PCR擴(kuò)增產(chǎn)物純化后,利用Qubit 2.0DNA檢測試劑盒對DNA精確定量,按照1:1等量混合后,利用Illumina MiSeq platform (Illumina, San Diego, CA, USA) 測序平臺進(jìn)行測序[生工生物工程(上海)股份有限公司].最終上機(jī)測序濃度為20pmol.

使用Trimmomatic軟件對原始測序序列進(jìn)行質(zhì)量控制,參數(shù)為堿基最小質(zhì)量Q30,移動檢測區(qū)間6bp.后用Flash軟件對雙端測序序列進(jìn)行拼接,保證長度在200bp以上并且N堿基小于6的序列.利用QIIME軟件將序列與Greengene的16S rRNA數(shù)據(jù)庫進(jìn)行對比,以97%相似性閥值進(jìn)行OTUs聚類.嵌合體OTUs使用QIIME內(nèi)置的UCHIME程序進(jìn)行刪除,之后每個OUT的代表序列以RDP classfier進(jìn)行物種歸類(80%置信空間),進(jìn)而計算出樣品總體群落的物種組成.利用PICRUSt軟件推演微生物群落功能.

2 結(jié)果與討論

2.1 微生物對scCO2的響應(yīng)

2.1.1 微生物量變化 scCO2注入后,樣品16S rRNA基因拷貝數(shù)在90d內(nèi)先降低后增加(圖2a),初始?30d?90d樣品中16S rRNA基因含量分別為11.02×106、0.26×106、4.61×106gene/mL.由于基因含量與生物量成正比,因此16S rRNA基因拷貝數(shù)表明scCO2注入后微生物量在90d內(nèi)先降低后增加.

體系pH值在90d內(nèi)也先降低后增加(圖2b),5d時微生物組pH值由7.02降至5.16,對照組降至5.19 ,說明scCO2注入后體系改變?yōu)楦邏核嵝原h(huán)境,pH值降低主要是由于scCO2溶于水后碳酸的水解.而5d后兩組實驗pH值均逐漸升高,90d時生物組pH值升至5.87,對照組升至6.04,說明礦物的溶蝕過程會不斷消耗H+使得pH得到緩沖.pH值變化與生物量變化趨勢相同,均在90d內(nèi)先降低后增加.因此scCO2注入后pH值變化可能是影響生物量的主要因素,且Gulliver等人[19]發(fā)現(xiàn)CO2注入后pH值變化對微生物影響顯著.另外,微生物組pH值始終低于對照組,且隨著生物量在30d后的增加,兩組實驗pH值差值也逐漸增大.因此scCO2注入后,某些適應(yīng)高壓酸性環(huán)境的微生物可能具有產(chǎn)酸作用.

圖2 scCO2注入后16S rRNA基因拷貝數(shù)與pH值變化

(a) 16S rRNA基因拷貝數(shù);(b) pH值

圖3 微生物群落中門水平豐富度變化

豐度小于1%的歸屬于others

2.1.2 微生物群落結(jié)構(gòu)變化 物種注釋結(jié)果表明,初始樣本門水平菌群歸為硝化螺旋菌門(phylum Nitrospirae)、厚壁菌門(phylum Firmicutes)、變形菌門(phylum Proteobacteria),占比分別為71.24%, 24.18%,3.18%(圖3).30d時,厚壁菌門?變形菌門成為優(yōu)勢菌門,占比分別為46.89%,52.60%,而硝化螺旋菌門基本消失.90d時厚壁菌門?變形菌門仍為優(yōu)勢菌門,占比分別為43.89%,55.34%.因此scCO2注入后,厚壁菌門與變形菌門對極端酸性?高壓環(huán)境的耐受性強(qiáng)于硝化螺旋菌門.

微生物屬水平注釋結(jié)果顯示,scCO2注入后優(yōu)勢菌屬豐富度發(fā)生變化(圖4).初始樣品中有熱脫硫弧菌屬(,72.11%),高溫厭氧桿菌屬(,19.91%),微小桿菌屬(,2.31%),檸檬酸桿菌屬(,1.62%),不動桿菌屬(, 0.98%)與假單胞菌屬(,0.57%).30d時,微小桿菌屬,檸檬酸桿菌屬,不動桿菌屬,假單胞菌屬演變?yōu)閮?yōu)勢菌屬,占比分別為:43.62%,25.28%, 13.68%,7.29%;90d時,此4類菌屬依舊為優(yōu)勢菌屬,占比分別為:40.36%,24.38%,14.47%,9.85%.據(jù)文獻(xiàn)報道,熱脫硫弧菌屬適宜pH值為6.5~8.5[24],高溫厭氧桿菌屬適宜pH值為7~8[26],而微小桿菌屬,檸檬酸桿菌屬,不動桿菌屬與假單胞菌屬在pH值為5~6均具有適應(yīng)性[27-31].微生物組5d時pH值由初始7.02降至5.16,90d時緩沖至5.87,因此在scCO2注入后熱脫硫弧菌屬與高溫厭氧桿菌屬生長受到抑制并逐漸消失,微小桿菌屬,檸檬酸桿菌屬,不動桿菌屬,假單胞菌屬因具有適應(yīng)性而演化為優(yōu)勢菌屬.

圖4 微生物群落中屬水平豐度變化

豐度小于1%的歸屬于others

圖5 90d時礦物溶蝕SEM圖與EDS能譜分析

(a)微生物介導(dǎo)下礦物溶蝕;(b)無微生物介導(dǎo)下礦物溶蝕;(c)粘土礦物EDS能譜; (d)鉀長石EDS能譜

2.2 礦物溶蝕

初始巖心中,石英占51%,長石占19%,粘土礦物占16%,巖屑占10%,其它4%.由于石英的化學(xué)性質(zhì)較為穩(wěn)定,在水-巖-CO2體系中通常不參與反應(yīng),因此對占比第二,第三高的長石類礦物與粘土礦物溶蝕進(jìn)行分析.根據(jù)SEM?EDS分析發(fā)現(xiàn),微生物能夠以生物膜形式附著于礦物表面,而且在微生物膜附近長石類礦物、粘土礦物發(fā)生溶蝕(圖5a).無微生物介導(dǎo)時,scCO2溶于水后雖然也使得長石?粘土礦物發(fā)生溶蝕(圖5b),但其溶蝕程度卻弱于微生物組.

巖心礦物中,長石類礦物主要包括鉀長石?鈉長石?鈣長石,粘土礦物主要成分為含有鐵?鎂離子的綠泥石、伊利石等.因此對水溶液K+,Na+,Mg2+, T-Fe,Ca2+濃度進(jìn)行分析,探究微生物對礦物溶蝕的影響.在微生物介導(dǎo)下,K+,Na+, Mg2+,T-Fe,Ca2+溶出量均高于對照組(圖6).90d時,生物組K+,Na+,Mg2+, T-Fe,Ca2+分別增至121.13, 2850.10,56.30,183.91, 1798.10mg/L,而對照組K+, Na+,Mg2+,T-Fe,Ca2+增至為105.10,2728.00,38.61, 126.30,1625.05mg/L.由此可見體系中適應(yīng)菌群促進(jìn)了長石?粘土礦物溶蝕.再結(jié)合圖2生物組pH值始終低于對照組,說明優(yōu)勢菌群具有產(chǎn)酸作用.

圖6 咸水中K+?Na+?Mg2+?T-Fe?Ca2+濃度變化

(a)ρ(K+);(b)ρ(Na+);(c)ρ(Mg2+);(d)ρ(T-Fe);(e) ρ(Ca2+)

2.3 礦物捕獲

CO2的碳酸鹽礦化被視為最安全的捕獲方式.SEM圖發(fā)現(xiàn),微生物組有少許菱形礦物產(chǎn)生(圖7a),而對照組未找到菱形次生礦物.EDS能譜顯示(圖7b),菱形礦物主要有Fe,Al,K,Si,C,O組分.由于巖心含有石英和長石,能譜分析需考慮背景礦物影響,其中Al,Si,K應(yīng)為背景礦物成分,因此推斷菱形礦物是菱鐵礦(FeCO3).王博強(qiáng)等人[18]在3MPa非scCO2壓力下微生物-CO2-咸水-砂巖反應(yīng)體系中也發(fā)現(xiàn)了次生菱鐵礦生成.

菱鐵礦發(fā)生沉淀與其飽和指數(shù)相關(guān),90d時微生物組觀察到次生菱鐵礦生成可能與體系中適應(yīng)菌群的生物行為有利于提高菱鐵礦飽和度有關(guān).在微生物介導(dǎo)下,綠泥石、伊利石中鐵的溶出量明顯高于空白組(圖6d),更高的鐵溶出量有利于提升菱鐵礦飽和度促進(jìn)其沉淀,且田海龍[32]的水-巖-氣模擬實驗發(fā)現(xiàn)更高的Fe溶出量易于生成菱鐵礦.除此之外, Gulliver[19]等研究表明微生物鐵還原作用有利于提高Fe2+濃度以促進(jìn)菱鐵礦沉淀,90d時微生物組發(fā)現(xiàn)次生菱鐵礦生成也可能與生物組中適應(yīng)菌群有鐵還原作用有關(guān).

圖7 次生碳酸鹽礦物SEM圖與EDS能譜

(a)SEM圖;(b)EDS能譜分析

2.4 微生物的介導(dǎo)作用

30,90d時,微小桿菌屬(),檸檬酸桿菌屬(),不動桿菌屬()和假單胞菌屬()為優(yōu)勢菌屬.利用Blastn將其OTU序列與對應(yīng)數(shù)據(jù)庫比對,篩選出最佳比對結(jié)果,并分析功能(表1).

2.4.1 微生物產(chǎn)酸作用 微生物組比對照組pH值值低(圖2b)以及離子溶出量更高(圖6),驗證了微生物的產(chǎn)酸作用.據(jù)文獻(xiàn)報道微小桿菌屬[27-30,36],不動桿菌屬[22,36-37]和假單胞菌屬[31,37-38]均具有產(chǎn)酸作用.在營養(yǎng)源不足時許多微生物也可利用乙酸鈉?氨基酸?礦物中腐殖質(zhì)等為碳源進(jìn)行發(fā)酵產(chǎn)酸[30],其分泌的可溶性EPS(如檸檬酸,草酸,醋酸,乳酸,琥珀酸等有機(jī)酸)通過水解產(chǎn)H+可使pH值降低[40],可促進(jìn)礦物溶蝕釋放更多Ca2+,Mg2+,Fe2+到溶液中.而溶液中更高的Ca2+,Mg2+,Fe2+濃度將有利于提高碳酸鹽礦物飽和度,促進(jìn)CO2礦化捕獲.

表1 微生物群落中優(yōu)勢菌屬特性

2.4.2 微生物Fe(III)還原作用 菱鐵礦Fe為二價,因此溶液中Fe(II)濃度對于菱鐵礦生成至關(guān)重要.若微生物對Fe(III)具有還原作用,將有利于次生菱鐵礦生成.

初始巖心Fe(II)/Fe(III)比例一定,因此溶液中Fe(II)/Fe(III)比例的變化可反應(yīng)微生物是否有Fe(III)還原作用.通過Fe(II)/Fe(III)比值測試與計算(表2),發(fā)現(xiàn)對照組中Fe(II)/Fe(III)基本維持在0.97~1.11間,而微生物組中的Fe(II)/Fe(III)的比值在90d內(nèi)持續(xù)增大,15d時為1.02,90d時增至1.38.說明土著微生物中某些菌種具有鐵還原的作用.

通過文獻(xiàn)查閱,優(yōu)勢菌屬中檸檬酸桿菌屬()具有Fe(III)還原作用,可通過酶促作用以Fe(III)為終端電子受體,以H2?小分子有機(jī)酸,脂肪酸或葡萄糖等為電子供體,實現(xiàn)Fe(III)還原為Fe(II)的鐵呼吸代謝[35].產(chǎn)酸菌可促進(jìn)T-Fe溶出,鐵還原菌如檸檬酸桿菌屬又可通過自身代謝提高溶液中Fe(II)/Fe(III)比例,誘導(dǎo)菱鐵礦固碳礦物生成,這也是微生物組比對照組提前觀察到菱鐵礦生成的原因之一.

表2 咸水中Fe(II)/Fe(III)變化

2.4.3 微生物吸附作用 SEM發(fā)現(xiàn)礦物表面有大量桿狀菌和少許球形菌存在,且以生物膜形式附著于礦物表面(圖5a,8a),而資料顯示優(yōu)勢菌屬微小桿菌屬,檸檬酸桿菌屬,不動桿菌屬,假單胞菌屬為桿狀菌[31-36],且微生物以膜形式存在有利于增強(qiáng)微生物群落對極端環(huán)境的抵抗性[38].

圖8 微生物膜SEM圖與EDS能譜分析

(a)SEM圖;(b)EDS能譜分析

EDS分析發(fā)現(xiàn)微生物膜吸附有Ca2+、Mg2+、Fe2+離子(圖8b).據(jù)文獻(xiàn)報道某些細(xì)菌能利用其產(chǎn)生的胞外多糖?蛋白質(zhì)和核酸等吸附Ca2+、Mg2+、Fe2+可溶性金屬離子[39],使生物膜附近碳酸鹽礦物處于飽和態(tài),提供碳酸鹽成核點.

FT-IR分析顯示微生物組礦物表面物質(zhì)吸收峰多于對照組(圖9).2360cm-1處吸收峰主要是-N=C= O[40];1535cm-1處吸收峰主要是-NH2, C=O,C=N之間伸縮振動峰,可能與蛋白質(zhì)有關(guān)[41];1400cm-1處吸收峰主要為CH3COO-[42];1644cm-1處吸收峰主要為-C=O[32].這些振動峰的出現(xiàn)可能與EPS中蛋白質(zhì)、多糖、核酸等有機(jī)物成分有關(guān),也說明微生物膜附著于礦物表面.因此微生物膜吸附飽和作用也是90d時生物組提前觀察到菱鐵礦生成的原因之一.

圖9 礦物表面物質(zhì)FT-IR圖譜

(a)有微生物組;(b)無微生物組

3 結(jié)論

3.1 微生物對scCO2注入后的響應(yīng)表明:初期pH的降低使生物量急劇降低,隨著礦物溶蝕pH值的升高,生物量又逐漸增加;變形菌門與厚壁菌門對高壓酸性環(huán)境的耐受性強(qiáng)于硝化螺旋菌門;熱脫硫弧菌屬、高溫厭氧桿菌屬對高壓酸性環(huán)境不適應(yīng),微小桿菌屬、檸檬酸桿菌屬、不動桿菌屬和假單胞菌屬具有適應(yīng)性而逐漸演化為優(yōu)勢菌屬.

3.2 微生物介導(dǎo)作用研究表明:產(chǎn)酸菌(微小桿菌屬,不動桿菌屬和假單胞菌屬)可促進(jìn)長石、粘土礦物溶蝕,提高K+,Na+,Ca2+,Mg2+,T-Fe的釋放;鐵還原菌(檸檬酸桿菌屬)可提高Fe(II)/Fe(III)比值,促進(jìn)菱鐵礦捕獲;微生物膜中EPS對Ca2+,Mg2+,Fe2+的吸附作用可改變局部碳酸鹽飽和度,提供碳酸鹽成核點,促進(jìn)碳酸鹽礦物生成.因此scCO2注入后,適應(yīng)菌能促進(jìn)礦物溶蝕與碳酸鹽礦物捕獲.

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Impact of microorganisms on the mineral interaction in scCO2-saline-sandstone system.

ZHANG Feng-jun1, SONG Yun-peng1, ZHONG Shuang1,2, FAN Kai3, LI Chen-yang1,2*, ZHANG Zhi-yong4

(1.Key Laboratory of Groundwater Resources and Environment, Ministry of Education, Jilin University, Changchun 130021, China；2.Key Laboratory of Songliao Aquatic Environment, Ministry of Education, Jilin Jianzhu University, Changchun 130118, China；3.Yulin Environmental Protection Bureau, Shanxi Province, Yulin 719000, China;4.Qianguo County Environmental Protection Bureau, Songyuan City, Jilin Province, Songyuan 138000, China)., 2019,39(1)：281~289

Through the Illumina MiSeq analysis within V3-V4region of 16S rRNA gene, the response of microorganisms to scCO2and its feedback on the mineral interaction in scCO2-saline-sandstone system was investigated. The results showed that biomass was affected by pH value. The initial pH was 7.02 and biomass was 11.02×106gene/mL. And the pH dropped to 5.65 and biomass decreased to 0.26×106gene/mL at 30th-day. However with the dissolution of minerals, the pH increased to 5.87 and biomass increased to 4.61×106gene/mL at 90th-day. In the community structure,(52.60% (30d), 55.34% (90d)) and(46.89% (30d), 43.89% (90d)) were dominant phylum. At the genus level,,,andwere dominant genus at 30thand 90th-day. Acid-producing bacteria (,a and) promoted the dissolution of feldspar and clay, and the concentrations of K+, Na+, Ca2+, Mg2+and T-Fe were higher than those in blank group. Iron-reducing bacteria () increased the ratio of Fe(II)/Fe(III). Biofilm showed an adsorption function of Ca2+, Mg2+and Fe2+. At last, the SEM results showed that the micron-mediated precipitation of siderite appeared before the blank group. Therefore, the adaptable bacteria in the scCO2-salt-sandstone system could promote the mineral dissolution and carbonates capture.

supercritical CO2；indigenous microorganisms；mineral corrosion；mineral trapping

X172

A

1000-6923(2019)01-0281-09

張鳳君(1957-),男,吉林長春人,教授,博士,主要從事微生物介導(dǎo)下CO2咸水層封存過程生物地球化學(xué)行為研究以及水處理技術(shù)等.發(fā)表論文50余篇.

2018-05-21

國家自然科學(xué)基金資助項目(41472214)

* 責(zé)任作者, 講師, lichenyang0331@126.com