陳 浮楊寶丹馬 靜張紹良張媛媛
(1 中國(guó)礦業(yè)大學(xué)環(huán)境與測(cè)繪學(xué)院,江蘇徐州 221116)
(2 中國(guó)礦業(yè)大學(xué)低碳能源研究院,江蘇徐州 221008)
(3 中石化石油工程設(shè)計(jì)有限公司,山東東營(yíng) 257026)
高濃度CO2地下泄漏對(duì)土壤微生物群落結(jié)構(gòu)的影響*
陳 浮1,2楊寶丹1馬 靜2?張紹良2張媛媛3
(1 中國(guó)礦業(yè)大學(xué)環(huán)境與測(cè)繪學(xué)院,江蘇徐州 221116)
(2 中國(guó)礦業(yè)大學(xué)低碳能源研究院,江蘇徐州 221008)
(3 中石化石油工程設(shè)計(jì)有限公司,山東東營(yíng) 257026)
通過(guò)模擬高濃度CO2在農(nóng)田土壤中的地下泄漏,研究了不同濃度CO2泄漏情景下土壤微生物多樣性的變化。實(shí)驗(yàn)設(shè)置了400 g m-2d-1、800 g m-2d-1、1 200 g m-2d-1和2 000 g m-2d-1持續(xù)CO2通氣60 d共計(jì)4個(gè)處理,并與對(duì)照組、恢復(fù)組(2 000 g m-2d-1組停止通氣60 d后)分期采集土壤樣品,分析土壤理化性質(zhì)、土壤閉蓄的氣體濃度、微生物多樣性指數(shù)及主要類群變化規(guī)律。結(jié)果表明,4種處理均提高了土壤中CO2濃度,分別為1.60%、4.80%、10.80%和19.60%。土壤微生物多樣性Chao指數(shù)和Shannon指數(shù)隨CO2通入量增加而減少,降幅分別達(dá)17.00%~27.80%和6.10%~9.50%。相反,非度量多維尺度(NMDS)分析顯示土壤微生物β多樣性在中、低濃度升高,在高、極端濃度表現(xiàn)為降低。擬桿菌屬(Bacteroidales)相對(duì)豐度隨CO2泄漏量增加從3.09%上升至21.20%,可作為高濃度CO2泄漏生態(tài)安全性評(píng)估的敏感性指標(biāo)。基于高通量序列相似度OTU分類的RDA分析表明土壤環(huán)境因子的變化能夠較好地解釋微生物多樣性演替。研究結(jié)果為評(píng)估和監(jiān)測(cè)地下CO2泄漏對(duì)近地表生態(tài)系統(tǒng)環(huán)境風(fēng)險(xiǎn)提供科學(xué)依據(jù)。
碳捕集與封存;CO2泄漏;微生物群落;高通量測(cè)序;多樣性分析
溫室氣體減排已經(jīng)成為各國(guó)政府高度關(guān)注的議題和目標(biāo),CO2捕集與封存(CCS,CO2Capture and Geological Storage)是一項(xiàng)重要的減排技術(shù),因其經(jīng)濟(jì)與環(huán)境雙贏的特點(diǎn)而倍受關(guān)注[1]。盡管CCS工程一直將防止CO2泄漏列為首要條件,但現(xiàn)有的技術(shù)體系還很難保證封存的CO2完全不發(fā)生泄漏,例如,儲(chǔ)運(yùn)、地質(zhì)活動(dòng)或人為操作失誤等均可能導(dǎo)致地下封存的CO2發(fā)生泄漏風(fēng)險(xiǎn)[2]。1986年喀麥隆尼奧斯湖沉積相CO2暴發(fā)導(dǎo)致周邊數(shù)十平方千米范圍內(nèi)2 000多人、數(shù)萬(wàn)牲畜窒息死亡[3]。目前中國(guó)已開(kāi)展了16項(xiàng)CCS試驗(yàn),其中鄂爾多斯咸水層封存和勝利油田驅(qū)油開(kāi)采均為1 Mt級(jí)CO2封存項(xiàng)目,一旦發(fā)現(xiàn)泄漏極可能對(duì)近地表生態(tài)系統(tǒng)產(chǎn)生嚴(yán)重的負(fù)面影響[4]?,F(xiàn)行的CCS工程建設(shè)和安全評(píng)估均側(cè)重于封存選址和CO2地下運(yùn)移[5-8],更多關(guān)注泄漏的環(huán)境后果和評(píng)估。然而,由于CO2地下封存技術(shù)涉及的因素較多,如地質(zhì)環(huán)境、封存材質(zhì)和CO2含量等,很難采用統(tǒng)一的標(biāo)準(zhǔn)進(jìn)行CCS環(huán)境風(fēng)險(xiǎn)評(píng)估,如近幾年歐美國(guó)家已經(jīng)多次出現(xiàn)CCS項(xiàng)目環(huán)境侵權(quán)訴訟[9]。因此,在我國(guó)開(kāi)展高濃度CO2地下泄漏的生態(tài)環(huán)境影響研究,具有重要意義。
已有的研究表明,由于深部高壓和浮力作用,CO2從斷層、裂隙、廢棄通道等逐步向上遷移,改變淺層地下水pH[10],影響礦化物的平衡控制、遷移轉(zhuǎn)化和水環(huán)境[11],并進(jìn)一步對(duì)上層土壤-植物生態(tài)系統(tǒng)產(chǎn)生直接或間接的負(fù)面影響[12]。英國(guó)ASGARD項(xiàng)目組、美國(guó)ZERT研究組、歐盟Geo-Net和中國(guó)農(nóng)業(yè)科學(xué)院等機(jī)構(gòu)已實(shí)施了多項(xiàng)人工控制地下CO2釋放的模擬試驗(yàn),探測(cè)農(nóng)田土壤-植被生態(tài)系統(tǒng)對(duì)地下CO2泄漏的響應(yīng)狀況,但取得的結(jié)論并不一致[13-20]。主要原因可歸于三點(diǎn):一是試驗(yàn)平臺(tái)不一,有盆栽,有小塊實(shí)驗(yàn)田,土壤中CO2擴(kuò)散方式和濃度難以精確測(cè)定;二是持續(xù)暴露的時(shí)間不一,短的約為30 d,最長(zhǎng)的則達(dá)90 d,很難明確土壤微生物群落轉(zhuǎn)變的臨界點(diǎn);三是種植體系不一,不同根系分泌物對(duì)土壤微生物群落影響差異巨大,不同研究之間缺乏可比性。此外,以往的大氣CO2濃度升高對(duì)生態(tài)系統(tǒng)影響的研究很難作為CCS環(huán)境風(fēng)險(xiǎn)評(píng)估的參考。主要原因包括兩點(diǎn),首先是作用途徑不同,大氣CO2濃度倍增實(shí)驗(yàn)中其濃度僅僅8.0?,從地表向土壤中滲透,土壤中CO2濃度一般不高于1.00%,因此,外源CO2對(duì)土壤本身CO2氣體濃度的影響較小,通常變化幅度小于10%,但地下CO2泄漏則由深層土壤向地表逃逸,其濃度可高達(dá)20.00%,導(dǎo)致土壤中CO2濃度增加數(shù)倍甚至數(shù)十倍;二是持續(xù)時(shí)間不同,地表CO2受空氣流動(dòng)影響,極容易發(fā)生擴(kuò)散,因此多采用封閉栽培箱,對(duì)土壤氣的影響相對(duì)小,而地下泄漏CO2在土壤保存時(shí)間很長(zhǎng),其對(duì)土壤性質(zhì)包括微生物多樣性的影響可能更加劇烈。
據(jù)此,本研究在中國(guó)礦業(yè)大學(xué)南湖校區(qū)內(nèi)建立了大田實(shí)驗(yàn)平臺(tái),模擬高濃度CO2地下泄漏情景,研究土壤微生物群落結(jié)構(gòu)對(duì)CO2地下泄漏的響應(yīng),為評(píng)估和監(jiān)測(cè)地下CO2泄漏對(duì)近地表生態(tài)系統(tǒng)環(huán)境風(fēng)險(xiǎn)提供科學(xué)依據(jù)。
1.1 試驗(yàn)場(chǎng)地
本試驗(yàn)平臺(tái)在中國(guó)礦業(yè)大學(xué)南湖校區(qū)內(nèi),地理位置為117°08′08″E、34°12′31″N,屬暖溫帶半濕潤(rùn)季風(fēng)氣候,年均溫度14.2 ℃,年均降水量833 mm。土壤類型為潮褐土,陽(yáng)離子交換量約為14.91 ± 3.27 cmol kg-1,質(zhì)地為輕壤,機(jī)械組成(<0.001 mm、0.001~0.005、0.005~0.01、>0.01 mm)為15.12∶22.34∶9.15∶53.39。2012 年9月利用鏟車分0~20 cm、20~90 cm二層取土并分別堆放,由下至上平鋪10 cm粗砂、布設(shè)內(nèi)帶PVC軟管的多孔鋼管、10 cm細(xì)沙和1 cm多孔納米板,最后再分別鋪上50 cm下層土壤和20 cm表土,剩余土鋪設(shè)田間路[21]。盡量保持原土壤構(gòu)型,簡(jiǎn)單平整后放置1.5 a自然壓實(shí)。實(shí)驗(yàn)田總面積約為1 000 m2(40 m×25 m),共建成48個(gè)2.5 m×2.5 m標(biāo)準(zhǔn)樣方(圖1)。
1.2 試驗(yàn)設(shè)計(jì)
試驗(yàn)平臺(tái)包含自動(dòng)控制系統(tǒng)室、地下管路、田間路、水利設(shè)施和田地。本研究針對(duì)15個(gè)樣方開(kāi)展,首先清除雜草和根系,種植玉米。2014年3月7日開(kāi)始持續(xù)通氣60 d,氣源為工業(yè)級(jí)CO2,體積濃度為99.8%。設(shè)計(jì)低濃度(L-400,400 g m-2d-1)、中濃度(M-800,800 g m-2d-1)、高濃度(H-1200,1 200 g m-2d-1)、極端高濃度(E-2000,2 000 g m-2d-1)和對(duì)照組(C-0),各設(shè)3個(gè)重復(fù)。通氣后采3次土樣,分別在14 d、30 d 和60 d。對(duì)照組通氣前(C1)和試驗(yàn)結(jié)束時(shí)(C2)各采1次。恢復(fù)組(R-0)為極端高濃度E組停止CO2通氣60 d后采樣1次。通入量由氣體調(diào)節(jié)閥和流量控制器控制,按下列公式計(jì)算注入速率:
V=FS / ρ (1)
式中,V為流量控制器上CO2注入速率(ml s-1);F為設(shè)計(jì)注入濃度(400 g m-2d-1);S為樣方面積(m-2);ρ為CO2密度(g L-1)。
1.3 樣品采集與分析
采用五點(diǎn)法采集10~20 cm混合土樣。一部分室內(nèi)自然風(fēng)干,過(guò)2 mm篩剔除石礫和植物殘?bào)w等雜物,4 ℃冷藏備用。另一部分無(wú)菌袋密封,存放-20 ℃冰箱,用于微生物測(cè)試。
土壤有機(jī)質(zhì)采用重鉻酸鉀-外加熱法測(cè)定,陽(yáng)離子交換量采用EDTA-銨鹽快速法測(cè)定,機(jī)械分析采用比重計(jì)法測(cè)定[22],NO3--N采用雙波長(zhǎng)分光光度法測(cè)定,土壤pH采用電位法測(cè)定(土:水=1∶2.5)[23]。
土壤中CO2氣體濃度測(cè)量:每個(gè)樣方內(nèi)均垂直埋深20 cm氣體探針(長(zhǎng)40 cm,內(nèi)徑2 cm),底端密封,最下端10 cm管壁上均勻鉆直徑0.3 cm的小孔,外貼防水生物膜,保證氣體自由擴(kuò)散又不積水。氣體探針頂端設(shè)密封閥門,連接到改制的帶自動(dòng)傳輸數(shù)據(jù)功能的便攜式氣體檢測(cè)儀GT901 (TES,臺(tái)灣)上測(cè)量土壤中CO2體積濃度,量程0~30.00%,數(shù)據(jù)傳輸間隔為1 h。
圖1 試驗(yàn)平臺(tái)位置與建設(shè)方案Fig. 1 Location and construction scheme of the experimental platform
DNA提取、擴(kuò)增與測(cè)序:按DNA快速提取試劑盒(Felix bio-tech,美國(guó))上操作說(shuō)明從45個(gè)(每個(gè)0.5 g)土樣中提取總DNA,利用核酸定量?jī)x(NanoDrop ND-1000,美國(guó))檢測(cè)提取的總DNA濃度和純度。采用特異引物338F (5′-ACTCCTACGGGAGGCAGCAG-3′)和806R (5′-GGACTACVSGGGTATCTAAT-3′)對(duì)16S rDNA基因的V4區(qū)進(jìn)行PCR擴(kuò)增(Pyrobest DNA Polymerase,TaKaRa,DR500A)。25 μlPCR體系包含:5 μl 5×Q5 反應(yīng)緩沖劑,5 μl 5×Q5 GC 強(qiáng)化劑,2 μl 2.5 mmol L-1dNTPs,兩個(gè)特異引物各1 μl(10 μmol L-1),0.25 μl Q5 聚合酶,8.75 μl超純無(wú)菌水和2 μl 模板DNA(20 ng μl-1)。PCR擴(kuò)增具體程序如下:98 ℃預(yù)變性5 min,98 ℃變性30 s,50 ℃退火30 s,72 ℃延伸30 s,共持續(xù)25個(gè)循環(huán)周期。最后72 ℃持續(xù)10 min結(jié)束程序。進(jìn)行1%瓊脂糖凝膠電泳檢測(cè),割膠回收目標(biāo)條帶(Axygen Biosciences,Union City,CA,USA),Tris-HCL洗脫。最終利用AxyPrep DNA凝膠抽取儀對(duì)PCR進(jìn)行定量(Axygen Biosciences,Union City,CA,USA)。利用PCR選擇性地富集兩端連有接頭的DNA片段,同時(shí)擴(kuò)增DNA文庫(kù)。利用PicoGreen和熒光分光光度計(jì)方法定量文庫(kù)。此外,使用Agilent 2100對(duì)PCR富集片段進(jìn)行質(zhì)量控制,驗(yàn)證DNA文庫(kù)的片段大小及分布。將所有樣品DNA文庫(kù)均一化至10 nmol L-1后等體積混合。再將混合好的文庫(kù)(10 nmol L-1)逐步稀釋定量至4~5 pmol L-1后進(jìn)行上機(jī)測(cè)序。
1.4 數(shù)據(jù)處理
首先需要去除引物接頭序列,再根據(jù)PE reads之間的overlap關(guān)系,將成對(duì)的reads 拼接(merge)成一條序列,然后按照barcode標(biāo)簽序列識(shí)別并區(qū)分樣品得到各樣本數(shù)據(jù),最后對(duì)各樣本數(shù)據(jù)的質(zhì)量進(jìn)行質(zhì)控過(guò)濾,得到各樣本有效數(shù)據(jù)。利用QIIME(Version 1.7.0,http://qiime.org/)對(duì)原始FASTQ文件進(jìn)行多路復(fù)用剔除和質(zhì)量篩選,調(diào)用uclust對(duì)優(yōu)質(zhì)序列按序列相似度97%進(jìn)行聚類分析,選取每個(gè)類中最長(zhǎng)的序列作代表序列。再將優(yōu)化序列map比對(duì)回OTU代表序列,獲得每個(gè)OTU代表序列的分類學(xué)信息。選取相似度在97%條件下OTU生成預(yù)期的稀釋曲線,利用Chao(The Chao1 estimator,http://www.mothur.org/wiki/Chao)和Shannon(The Shannon index,http://www.mothur. org/ wiki/Shannon)指數(shù)反映單樣本(一個(gè)特定區(qū)域或生態(tài)系統(tǒng))群落的豐富度和均勻性,即α多樣性;利用非度量多維標(biāo)度指數(shù)(NMDS index)計(jì)算各樣本間的進(jìn)化及豐度信息的距離來(lái)反映是否具有顯著的群落差異,即β多樣性;利用SPSS17.0 和Canoco windows for 4.5(http://www.canodraw. com/)軟件進(jìn)行數(shù)據(jù)統(tǒng)計(jì)和冗余分析(RDA)。多重相關(guān)比較顯著性檢驗(yàn)采用最小顯著差法(LSD)。
2.1 高濃度CO2地下泄漏對(duì)土壤理化性質(zhì)的影響
從表1可以看出,持續(xù)通氣60 d后土壤化學(xué)特性發(fā)生顯著變化。土壤pH、電導(dǎo)率和NO3--N隨CO2泄漏量增加而下降,pH和NO3--N尤為顯著。停止通氣后pH則恢復(fù)明顯,NO3--N略有回升,但電導(dǎo)率則繼續(xù)下降,有機(jī)質(zhì)含量卻隨CO2泄漏量呈上升趨勢(shì),停止通入后明確回落。統(tǒng)計(jì)分析發(fā)現(xiàn)CO2泄漏量與主要土壤因子變化存在顯著性相關(guān)關(guān)系(表1)。
2.2 泄漏對(duì)土壤中CO2濃度的影響
根據(jù)上述公式計(jì)算,L-400、M-800、H-1200、E-2000對(duì)應(yīng)的注入速率分別為16.64 ml s-1、29.27 ml s-1、43.91 ml s-1和73.18 ml s-1。樣品主要采集10~20 cm表層土壤,因此CO2傳感器探針設(shè)置監(jiān)測(cè)位置為埋深10~20 cm處。從圖2可以看出,E-2000組通氣6 h后土壤中CO2濃度趨向穩(wěn)定,約為20.00%,H-1200組通氣8 h后趨向穩(wěn)定,約為10.06%,24 h內(nèi)各組CO2濃度均呈現(xiàn)飽和,C-0組一直保持約0.60%,組內(nèi)差異不顯著。其中的主要原因是,土壤中CO2濃度受通氣量、氣體來(lái)源、地表氣候因子、通氣時(shí)間、土壤孔隙度、土壤含水率和pH等多因素的影響,本模擬平臺(tái)設(shè)置的CO2泄漏呈近似均勻擴(kuò)散,與先前模擬試驗(yàn)呈同心圓點(diǎn)源擴(kuò)散完全不同,保證土壤中CO2濃度只受土壤自身狀況的影響[24]。當(dāng)通氣一段時(shí)間后土壤中CO2濃度達(dá)到飽和,持續(xù)通氣則向地表逸散,并不能持續(xù)增加土壤中CO2濃度。
表1 不同處理各時(shí)期土壤理化特征變化Table 1 Soil physicochemical properties relative to treatment and timing of sampling
2.3 土壤微生物群落多樣性和豐富度變化
全部45個(gè)樣品采用Illumina Miseq測(cè)序技術(shù)共獲取了2 121 986個(gè)序列,所有樣品的稀釋曲線均傾向于飽和平穩(wěn)狀態(tài),表明測(cè)序數(shù)據(jù)量合理。圖3a顯示Chao和Shannon指數(shù)隨CO2泄漏量和時(shí)間的變化,2個(gè)指數(shù)均隨CO2泄漏量增加而減小,L-400、M-800組與C-0對(duì)照組變化小,說(shuō)明中、低濃度CO2通入對(duì)土壤微生物影響較小。E-2000組與C-0對(duì)照組相比二個(gè)指數(shù)最大分別下降了17.00%~27.80%和6.10%~9.50%,說(shuō)明高、極高濃度CO2通入對(duì)微生物影響顯著。CO2泄漏量800 g m-2d-1可能是微生物的一個(gè)重要閾值,更高的濃度可能徹底改變了微生物對(duì)土壤的環(huán)境適應(yīng)性。與此同時(shí),停止CO2通入60 d,微生物群落α多樣性和豐富度均呈現(xiàn)大幅度反彈,說(shuō)明土壤微生物對(duì)CO2脅迫具備較強(qiáng)的耐受性。
圖2 土壤中CO2濃度隨通氣時(shí)間的變化Fig. 2 Variation of CO2concentration in the soil with aeration going on
在O T U分類基礎(chǔ)上采用非度量多維尺度(NMDS)指數(shù)作β多樣性分析。NMDS指數(shù)顯示C-0、L-400、和R-0樣品和其他樣品群落區(qū)分開(kāi),第2軸又將M-800和H-1200、E-2000組樣品分開(kāi)(圖3b)。NMDS指數(shù)更好地解釋了H-1200、 E-2000組高濃度下“抑制狀態(tài)”與L、M組低濃度下“促進(jìn)狀態(tài)”下土壤微生物群落結(jié)構(gòu)的變化。
2.4 土壤微生物結(jié)構(gòu)與組成變化
所有樣品中95%以上的序列可以明確分類至不同類群,包括酸桿菌門(Acidobacteria)、放線菌門(Actinobacteria)、擬桿菌門(Bacteroidetes)、綠彎菌門(Chloroflexi)、厚壁菌門(Firmicutes)、浮霉菌門(Planctomycetes)、變形菌門(Proteobacteria)、芽單胞菌門(Gemmatimonadetes)和疣微菌門(Verrucomicrobia)等9大菌門相對(duì)豐度占90%以上,其中放線菌門(Actinobacteria)最為豐富,約占35.51%(圖4)。C O2泄漏造成了厭氧環(huán)境,酸桿菌門(Acidobacteria)、綠彎菌門(Chloroflexi)和芽單胞菌門(Gemmatimonadetes)等好氧菌門相對(duì)豐度隨C O2泄漏量呈下降趨勢(shì),擬桿菌門(Bacteroidetes)和厚壁菌門(Firmicutes)等厭氧菌門則呈相反的趨勢(shì)。其中擬桿菌屬(Bacteroidales)變化最為顯著,隨CO2通入從C-0處理開(kāi)始時(shí)相對(duì)豐度3.0 9%上升至E-2000處理60 d后相對(duì)豐度21.20%,乳酸菌屬(Lactobacillus)次之,從C-0處理開(kāi)始時(shí)相對(duì)豐度1.10%增加至E-2000處理60 d后相對(duì)豐度6.95%。熱微菌屬(Thermomicrobia)則隨CO2通入從C-0處理開(kāi)始時(shí)相對(duì)豐度16.70%下降至E-2000處理60 d后相對(duì)豐度3.20%。
圖3 不同處理微生物群落α多樣性(a)與β多樣性(b)分析Fig. 3 Alpha diversity(a)and beta diversity(b)analysis of microbial community relative to treatment
圖4 不同處理的土壤微生物菌門相對(duì)豐度變化Fig. 4 Phylum composition of the soil microbes relative to treatment
為進(jìn)一步檢測(cè)土壤環(huán)境因子與微生物群落變化的對(duì)應(yīng)關(guān)系,引入了RDA分析。從圖5可以看出:不同處理各時(shí)期土壤微生物群落與pH、EC值和NO3--N等環(huán)境因子呈現(xiàn)顯著相關(guān)(p<0.005),在第1軸上解釋了C-0、L-400組與M-800、H-1200、E-2000組之間的差異,pH是最長(zhǎng)的箭頭,表示其是引起群落分異的最重要因子。有機(jī)質(zhì)在第2軸上較好地解釋了L-400、M-800組與H-1200、E-2000組之間群落差異。
圖5 土壤微生物群落結(jié)構(gòu)與土壤因子RDA分析Fig. 5 RDA analysis of soil microbial community with soil factors
CO2濃度、根系分泌物和時(shí)間通常被認(rèn)為是影響土壤微生物多樣性的3個(gè)重要變量[25],本研究在考慮上述變量基礎(chǔ)上設(shè)計(jì)了4個(gè)通氣梯度研究高濃度CO2地下泄漏對(duì)土壤微生物的脅迫,結(jié)果發(fā)現(xiàn)多樣性、豐富度和群落結(jié)構(gòu)均發(fā)生了變化,這與前人CO2濃度倍增實(shí)驗(yàn)取得的結(jié)論相對(duì)一致[26-29],但變化特征卻具有明顯的差異。理論而言,地下CO2泄漏對(duì)土壤和農(nóng)作物的影響完全不同于以往的大氣CO2濃度倍增實(shí)驗(yàn),前者首先影響土壤環(huán)境,土壤CO2濃度飽和后再逸散入大氣,同時(shí)在近地表受氣流影響很難形成高濃度的聚集[30]。因此,實(shí)際狀況下地下CO2泄漏對(duì)農(nóng)作物幾乎不可能產(chǎn)生“施肥”效應(yīng),為此精準(zhǔn)的實(shí)驗(yàn)?zāi)M平臺(tái)至關(guān)重要。以往模擬試驗(yàn)均采用點(diǎn)源,尤其是盆栽實(shí)驗(yàn)幾乎不考慮泄漏與土壤中CO2濃度的關(guān)系,一般情況下點(diǎn)源擴(kuò)散在土壤中CO2濃度呈同心圓狀[16-18],但環(huán)狀條帶極不穩(wěn)定,易造成多重濃度反復(fù)交叉干擾。這與地下CO2泄漏以及大氣CO2濃度倍增實(shí)驗(yàn)緩慢向上或向下均勻的影響特征完全不同,每次采樣均需要重復(fù)測(cè)定土壤氣濃度,耗時(shí)費(fèi)力精度差。后續(xù)植被實(shí)驗(yàn)更難,往往一個(gè)濃度條帶上僅能生長(zhǎng)一株玉米,樣品難以滿足實(shí)驗(yàn)要求。本模擬實(shí)驗(yàn)平臺(tái)解決了泄漏與土壤中CO2濃度的關(guān)聯(lián)性,獲取均勻態(tài)擴(kuò)散,極大地方便了研究[24]。CO2注入加大了土壤中CO2濃度,可能改變了土壤物理化學(xué)性質(zhì),導(dǎo)致微生物群落結(jié)構(gòu)的變化。一般認(rèn)為CO2等酸氣對(duì)土壤pH屬性影響最為明顯影響,本實(shí)驗(yàn)結(jié)果顯示土壤pH隨CO2通入濃度梯度降低而逐漸下降,這與Beaubien[26]和Oppermann[31]等關(guān)于泄漏點(diǎn)pH呈下降趨勢(shì)的結(jié)論一致,但McFarland等[32]并未發(fā)現(xiàn)極端CO2濃度變化會(huì)引起pH的明顯變化。造成這一差異的原因可能來(lái)自兩個(gè)方面:一是與土壤本底pH有關(guān),CO2泄漏不可能造成土壤無(wú)限的酸化;二是當(dāng)土壤中CO2達(dá)到飽和后,大量CO2外排并逸散入大氣,換言之,大氣中CO2濃度即使再高,也不可能無(wú)限擴(kuò)大土壤中CO2濃度,這與地下泄漏直接作用于土壤環(huán)境的影響完全不同。此外,本實(shí)驗(yàn)證實(shí)了Luis等[33]關(guān)于土壤有機(jī)質(zhì)隨CO2通入呈上升的趨勢(shì),表明60 d高濃度CO2泄漏能改變土壤有機(jī)碳庫(kù),這可能與還原條件下有機(jī)質(zhì)轉(zhuǎn)化率更低密切相關(guān)。同時(shí)發(fā)現(xiàn)NO3--N也是隨著CO2泄漏量急劇下降的,可能是CO2通入促進(jìn)反硝化菌生長(zhǎng),利用硝態(tài)氮參加反硝化反應(yīng)有關(guān)[34],然而,已知的絕大多數(shù)反硝化菌均為異養(yǎng)微生物,利用有機(jī)碳而非CO2生長(zhǎng),因此,也有可能是其他土壤性質(zhì)的變化,例如土壤酸化導(dǎo)致大量有機(jī)碳溶出,進(jìn)而刺激了反硝化微生物生長(zhǎng),或者是土壤理化性質(zhì)的變化,導(dǎo)致土壤硝態(tài)氮快速淋溶至深層土壤。但也有相反的研究結(jié)果,如McFarland等[32]認(rèn)為NO3--N會(huì)隨CO2增加而增大,其原因可能是CO2通入可能會(huì)抑制生物的生長(zhǎng),減少植被和土壤微生物對(duì)氮的需求,但具體的機(jī)制仍需進(jìn)一步研究,并在不同CO2封存條件下具有不同的響應(yīng)規(guī)律。
根系分泌物是影響微生物群落結(jié)構(gòu)及多樣性的重要因子[27,35],但相互作用關(guān)系極為復(fù)雜。本實(shí)驗(yàn)前剔除了雜草和根系,再統(tǒng)一種植玉米,目的即是消除不同植物根系分泌物對(duì)微生物影響的不確定性,同一植物根系且初始土壤因子基本無(wú)差異,CO2注入則成為影響微生物的單一因子。已有報(bào)道表明高濃度CO2泄漏會(huì)降低土壤微生物多樣性和豐富度,本研究也得到了類似結(jié)果[3,25,36]。不同處理各時(shí)期群落特征存在差異,說(shuō)明CO2泄漏已經(jīng)對(duì)土壤微生物多樣性產(chǎn)生了重要影響。2014年5月勝利油田G89-1驅(qū)油開(kāi)采區(qū)生態(tài)本底值調(diào)查時(shí)發(fā)現(xiàn)了數(shù)處地下CO2泄漏點(diǎn),連續(xù)觀察了3 a,最高一處近地表CO2濃度高達(dá)0.20%,是正常大氣CO2濃度值的5倍,泄漏點(diǎn)周邊1.5 m范圍內(nèi)植物均無(wú)法成活。與天然CO2通道周邊觀察結(jié)果極為相似[26],影響范圍小主要原因是CO2在風(fēng)速大于1 m s-1時(shí)就能迅速擴(kuò)散[30],一旦發(fā)生大量泄漏則破壞力極大[3]。其外,本研究發(fā)現(xiàn)玉米根系下擬桿菌目(Bacteroidales)相對(duì)豐度隨CO2通氣量持續(xù)顯著增加,但這類群的新陳代謝及功能多樣性還需要更充分的信息加以分析,這是未來(lái)研究的一個(gè)重要方向。
盡管很難解釋高濃度CO2泄漏條件下一些微生物的增加或減少,但高通量測(cè)序技術(shù)提供了不同類群微生物的動(dòng)態(tài)變化趨勢(shì)[18,37],可在一定程度較為明確地解釋與CO2通入量相關(guān)的土壤環(huán)境下微生物群落的差異性。此外,不同處理組類群中序列分布是不同的,它表明時(shí)間和CO2通入量均產(chǎn)生了顯著的影響,R-0組群落α多樣性和豐富度更為接近C-0對(duì)照組,但反映群落結(jié)構(gòu)和組成的β多樣性則更接近H-1200組和E-2000組,它顯示土壤微生物盡管可以適應(yīng)新的環(huán)境體系,但其功能的恢復(fù)需要更長(zhǎng)的時(shí)間,不同于α多樣性和豐富度的恢復(fù)。這些研究結(jié)果表明,高濃度CO2地下泄漏極可能對(duì)土壤微生物群落及其功能產(chǎn)生重要影響,未來(lái)仍需對(duì)土壤微生物群落的底物利用方式,生長(zhǎng)代謝速率及其恢復(fù)力對(duì)CO2泄漏的適應(yīng)規(guī)律進(jìn)一步研究。
不同CO2泄漏情景下農(nóng)田土壤微生物群落結(jié)構(gòu)、多樣性和豐富度存在顯著性差異。Chao指數(shù)和Shannon指數(shù)隨CO2泄漏量增加而減少,降幅分別達(dá)17.00%~27.80%和6.10%~9.50%。同時(shí),微生物類群隨CO2泄漏梯度增加發(fā)生了明顯的異化。β多樣性分析表明不同CO2泄漏情景影響了農(nóng)田土壤微生物群落組成,土壤pH和CO2通入量可能是影響微生物多樣性的最重要環(huán)境因子。擬桿菌屬(Bacteroidales)相對(duì)豐度隨CO2泄漏量增加從3.09%上升至21.20%,可作為監(jiān)測(cè)和評(píng)估CO2地下泄漏生態(tài)環(huán)境風(fēng)險(xiǎn)的敏感性指標(biāo)。盡管60 d恢復(fù)實(shí)驗(yàn)表明土壤微生物群落多樣性和豐富度恢復(fù)較好,但其功能恢復(fù)力尚未可知,未來(lái)仍需重點(diǎn)研究高濃度CO2地下泄漏對(duì)土壤生態(tài)系統(tǒng)功能的潛在風(fēng)險(xiǎn)。
參 考 文 獻(xiàn)
[1]Dai Z X,Stauffer P H,Carey J W,et al. Presite characterization risk analysis for commercialscale carbon sequestration. Environmental Science & Technology,2014,48(7):3908—3915
[2]任韶然,李德祥,張亮,等. 地質(zhì)封存過(guò)程中泄漏途徑及風(fēng)險(xiǎn)分析.石油學(xué)報(bào),2014,35(3):591—601
Ren S R,Li D X,Zhang L,et al. Leakage pathways and risk analysis of carbon dioxide in geological storage (In Chinese). Acta Petrolei Sinica,2014,35 (3):591—601
[3]Patil R H,Colls J J,Steven M D. Effects of CO2gas as leaks from geological storage sites on agro-ecosystems. Energy,2010,35(12):4587—4591
[4]IPCC. Climate change 2014:Mitigation of climate change//Contribution of working group III to the fifth assessment report of the intergovernmental panel on climate change. Cambridge,United Kingdom and New York,USA:Cambridge University Press,2014:18—28
[5]Bachu S,Bonijoly D,Bradshaw J,et al. CO2storage capacity estimation:Methodology and gaps. International Journal of Greenhouse Gas Control,2007,1(4):430—443
[6]Gibson-Pool C M,Svendsen L,Underschultz J,et al. Site characterization of a basin-scale CO2geological storage system:Gippsland basin,Southeast Australia. Environmental Geology,2008,54(1):1583—1606
[7]Okwen R T,Stewart M T,Cunninghan J A. Analytical model for screening potential CO2repositories. Computational Geosciences,2011,15(4):755—770
[8]Wielopolski L,Mitra S. Near-surface soil carbon detection for monitoring CO2seepage from a geological reservoir. Environmental Earth Sciences,2010,60 (2):307—312
[9]Wilson E J,Mark A,F(xiàn)igueiredo D. Geologic carbon dioxide sequestration:An analysis of subsurface property law. Journal of Environmental Law Report,2006,36:l14—121
[10]Lemieux J M. Review:The potential impact of underground geological storage of carbon dioxide in deep saline aquifers on shallow groundwater resources. Hydrogeology Journal,2011,19(4):757—778
[11]Kharaka Y K,Thordsen J J,Kakouros E,et al. Changes in the chemistry of shallow groundwater related to the 2008 injection of CO2at the ZERT field site,Bozeman,Montana. Environmental Earth Sciences,2010,60(2):273—284
[12]李琦,劉桂臻,張建,等. 二氧化碳地質(zhì)封存環(huán)境監(jiān)測(cè)現(xiàn)狀及建議. 地球科學(xué)進(jìn)展,2013,28(6):718—727 Li Q,Liu G Z,Zhang J,et al. Status and suggestion of environmental monitoring for CO2geological storage (In Chinese). Advances in Earth Science,2013,28 (6):718—727
[13]West J M,Jones D G,Annunziatellis A,et al. Comparison of the impacts of elevated CO2soil gas concentrations on selected European terrestrial environments. International Journal of Greenhouse Gas Control,2015,42:357—371
[14]Fessen J E,Clegg S M,Rahn T A,et al. Novel MVA tools to track CO2seepage tested at the ZERT controlled release site in Bozeman,MT. Environmental Earth Sciences,2010,60(2):325—334
[15]Wells A,Strazisar B,Diehl J R,et al. Atmospheric tracer monitoring and surface plume development at the ZERT pilot test in Bozeman,Montana,USA. Environmental Earth Sciences,2010,60(2):299—305
[16]Oldenburg C M,Lewicki J L,Pan L,et al. Origin of the patchy emission pattern at the ZERT CO2release test. Environmental Earth Sciences,2010,60(2):241—250
[17]伍洋,馬欣,李玉娥,等. 地質(zhì)封存CO2泄漏對(duì)農(nóng)田生態(tài)系統(tǒng)的影響評(píng)估及耐受閾值.農(nóng)業(yè)工程學(xué)報(bào),2012,28(2):196—205
Wu Y,Ma X,Li Y E,et al. Impact assessment and tolerable threshold value of CO2leakage from geological storage on agro-ecosystem(In Chinese). Transactions of the CSAE,2012,28(2):196—205
[18]田地,馬欣,李玉娥,等.利用高通量測(cè)序?qū)Ψ獯鍯O2泄漏情景下土壤微生物的研究. 環(huán)境科學(xué),2013,34 (10):4096—4104
Tian D,Ma X,Li Y E,et al. Research on soil bacteria under the impact of sealed CO2leakage by high-throughput sequencing technology(In Chinese). Environmental Science,2013,34(10):4096—4104
[19]蔣金豹,Michael D S,何汝艷,等. 利用大豆光譜特征判定地下封存CO2泄漏. 農(nóng)業(yè)工程學(xué)報(bào),2013,29 (12):163—169
Jiang J B,Michael D S,He R Y,et al. Judgment of CO2leaking in underground storage using spectral characteristics of soybean(In Chinese). Transactions of the CSAE,2013,29(12):163—169
[20]聶莉娟,馬俊杰,趙雪峰,等. 模擬CCS技術(shù)CO2泄漏對(duì)C3、C4作物土壤化學(xué)性質(zhì)的影響. 水土保持學(xué)報(bào),2015,29(5):200—205Nie L J,Ma J J,Zhao X F,et al. Effects of simulation CO2leakage of CCS on soil chemical properties under C3 and C4 crops(In Chinese). Journal of Soil and Water Conservation,2015,29(5):200—205
[21]Chen F,Zhang S L,Ma J,et al. A set of efficient device simulating the potential impact of CO2leakage from CO2enhanced oil recovery on farmland ecosystem// The 14th annual carbon capture,utilization & storage conference. Pittsburgh:Access Intelligence Press,2015:81
[22]張紅,呂家瓏,曹瑩菲,等. 不同植物秸稈腐解特性與土壤微生物功能多樣性研究. 土壤學(xué)報(bào),2014,51 (4):743—752
Zhang H,Lü J L,Cao Y F,et al. Decomposition characteristics of different plant straws and soil microbial functional diversity(In Chinese). Acta Pedologica Sinica,2014,51(4):743—752
[23]楊帆,黃來(lái)明,李德成,等. 高寒山區(qū)地形序列土壤有機(jī)碳和無(wú)機(jī)碳垂直分布特征及其影響因素. 土壤學(xué)報(bào),2015,52(6):1226—1236
Yang F,Huang L M,Li D C,et al. Vertical distributions of soil organic and inorganic carbon and their controls along toposequences in an alpine region (In Chinese). Acta Pedologica Sinica,2015,52 (6):1226—1236
[24]陳浮,譚敏,馬靜,等. 一種模擬CO2在土壤中均勻擴(kuò)散的試驗(yàn)裝置與方法.中國(guó)專利:201510375957.9. 2015-08-06
Chen F,Tan M,Ma J,et al. A Set of devices and methods simulating CO2homogeneous diffusion in soil (In Chinese). China Patent:201510375957.9. 2015-08-06
[25]Fernández M I,Touceda M,Pedescoll A,et al. Shortterm effects of simulated below-ground carbon dioxide leakage on a soil microbial community. International Journal of Greenhouse Gas Control,2015,36:51—59
[26]Beaubien S E,Ciotoli G,Coombs P,et al. The impact of a naturally occurring CO2gas vent on the shallow ecosystem and soil chemistry of a Mediterranean pasture (Latera,Italy). International Journal of Greenhouse Gas Control,2008,3(2):373—387
[27]West J M,Pearce J M,Coombs P,et al. The impact of controlled injection of CO2on the soil ecosystem and chemistry of an English lowland pasture. Energy Procedia,2009,1(1):1863—1870
[28]Antruck H,Imek M. Effect of soil CO2concentration on microbial biomass. Biology and Fertility of Soils,1997,25(3):269—273
[29]Morales S E,Holben W E. Simulated geologic carbon storage leak reduces bacterial richness and alters bacterial community composition in surface soil. Soil Biology & Biochemistry,2014,76:286—296
[30]Xu Y Q,Zhang S L,Hou H P,et al. Influence of CO2leakage from oil-producing wells on crop growth based on improved CASA model. International Journal of Remote Sensing,2016,37(2):290—308
[31]Oppermann B I,Michaelis W,Blumenberg M,et al. Soil microbial community changes as a result of longterm exposure to a natural CO2vent. Geochimica et Cosmochimica Acta,2010,74(9):2697—2716
[32]McFarland J W,Waldrop M P,Haw M. Extreme CO2disturbance and the resilience of soil microbial communities. Soil Biology & Biochemistry,2013,65:274—286
[33]Luis E S M,Paula A,Estanislao L C,et al. Highthroughput sequencing of 16S RNA genes of soil bacterial communities from a naturally occurring CO2gas vent. International Journal of Greenhouse Gas Control,2014,29:176—184
[34]周漢昌,劉文釗,劉毅,等. 土壤團(tuán)聚體N2O釋放與反硝化微生物豐度和組成的關(guān)系.土壤學(xué)報(bào),2015,52 (5):1144—1152
Zhou H C,Liu W Z,Liu Y,et al. Relationships of N2O emission with abundance and composition of denitrifying microorganisms in soil aggregates(In Chinese). Acta Pedologica Sinica,2015,52(5):1144—1152
[35]Shen C C,Xiong J B,Zhang H Y,et al. Soil pH drives the spatial distribution of bacterial communities along elevation on Changbai Mountain. Soil Biology & Biochemistry,2013,57(2):204—211
[36]Liu Y,Zhou H M,Pan G X,et al. Short-term response of nitrifier communities and potential nitrification activity to elevated CO2and temperature interaction in a Chinese paddy field. Applied Soil Ecology,2015,96:88—98
[37]Singh K M,Shah T,Deshpange S,et al. High throughput 16S rRNA gene-based pyrosequencing analysis of the fecal microbiota of high FCR and low FCR broiler growers. Molecular Biology Reports,2012,39(12):10595—10602
Effects of Underground Leakage of High Concentration CO2on Soil Microbial Community Structure
CHEN Fu1,2YANG Baodan1MA Jing2?ZHANG Shaoliang2ZHANG Yuanyuan3
(1 School of Environment Science and Spatial Informatics,China University of Mining and Technology,Xuzhou,Jiangsu 221116,China)
(2 Low Carbon Energy Institute,China University of Mining and Technology,Xuzhou,Jiangsu 221008,China)
(3 Sinopec Petroleum Engineering Design Company Limited,Dongying,Shandong 257026,China)
【Objective】 To cope with the trend of global warming,CO2capture and storage (CCS)is one of the major technologies for reduction of CO2emission. And then the captured CO2,nil in commercial value,is injected underground to raise the output of petroleum or coalbed methane,so as to maximize its economic profit. However,the prevailing technologies are far from being capable of guaranteeing zero leakage of the stored CO2during the processes of CO2storage and transportation,geological activities and human misoperation. Once the risk of potential CO2leakage becomes real,the leakage will pose an enormous threat to the near surface ecosystem. It is,therefore,essential to explore in depth effects of underground CO2leakage on farmland soil environment,especially tolerance and sensitivity of soil microbial communities to different concentrations of CO2. So the study was conducted. 【Method】 An experimental platform to simulate underground CO2leakage was constructed in an idle farmland,in the South Lake Campus of the China University of Mining and Technology. The platform was used to simulate leakage of CO2varying in intensity,i.e. 400 g m-2d-1,800 g m-2d-1,1 200 g m-2d-1and 2 000 g m-2d-1for 60 days,thus forming four treatments,i.e. L-400,M-800,H-1200 and E-2000. Besides,the experiment also had a control group and a recovery group. Soil samples were collected from the four treatments and their 3 replicates on the 14th,30th and 60th day after the start of simulated leakage,from the control group on the day before the start of leakage (C1)and at the end of the experiment(C2),and from the recovery group,which was actually Treatment E2000,60 days after the stop of the leakage. The soil samples were analyzed for soil physical and chemical properties,concentration of occluded soil,structure and α and β diversities of soil microbial community with the conventional physicochemical analysis method and the Illumna second generation gene sequencing method based on the Miseq platform.【Result】 Results show that CO2leakage decreased soil pH,electrical conductivity and nitrate nitrogen content and the effect was enhanced with rising CO2concentration whereas it had an opposite effect on soil organic matter content. In all the four treatments. Soil CO2concentration increased till it reached saturation in 24 h,when soil CO2gas concentration leveled off at 1.60%,4.80%,10.80% and 19.60%,respectively. Along with increasing CO2flux,soil microbial community decreased in diversity,Chao index and Shannon index,by 17.00%~27.80% and 6.10%~9.50%,respectively. In contrast,soil microbial community increased in β diversity(NMDS index)in Treatments L-400 and M-800(low CO2concentration)but decreased in Treatments H-1200 and E-2000. Some bacteria,like Bacteroidales,varied extremely,either rising up or falling down in relative abundance with increasing CO2leakage. 【Conclusion】 The structure,diversity and abundance of soil microbial community varied significantly from treatment to treatment. Soil pH and CO2flux were the two most important environmental factors affecting soil microbial diversity. Bacteroidales was very sensitive to CO2stress so that it can beused as a key indicator in monitoring and evaluating ecological risk of underground CO2leakage. The 60 d short term recovery experiment indicates that the soil microbial community recovered well in diversity and richness,but it is still unclear whether it did in function. Therefore,in future studies,focuses should be laid on impacts of underground high concentration CO2leakage on functions of the soil ecosystem.
Carbon capture and storage;CO2leakage;Microbial community;High-throughput sequencing;Diversity analysis
X171
A
10.11766/trxb201607110265
(責(zé)任編輯:盧 萍)
* 國(guó)家科技支撐計(jì)劃項(xiàng)目(2012BAC24B05)和江蘇省煤基CO2捕集與地質(zhì)儲(chǔ)存重點(diǎn)實(shí)驗(yàn)室開(kāi)放基金項(xiàng)目(2015A01、2015B02)資助 Supported by the National Key Technology R&D Program of China(No. 2012BAC24B05)and the Project of Key Laboratory of Coal-based CO2Capture and Geological Storage,Jiangsu Province(Nos. 2015A01,2015B02)
? 通訊作者 Corresponding author,E-mail:jingma2013@cumt.edu.cn
陳 ?。?974—),男,江蘇射陽(yáng)人,博士,教授,主要從事生態(tài)安全監(jiān)測(cè)、評(píng)價(jià)與修復(fù)研究。E-mail:chenfu@cumt.edu.cn
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2016-09-26;優(yōu)先數(shù)字出版日期(www.cnki.net):2016-10-20