房福力， 李玉中

(中國農(nóng)業(yè)科學(xué)院農(nóng)業(yè)環(huán)境與可持續(xù)發(fā)展研究所， 北京 100081)

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基于同位素特征的華北平原菜地N2O排放監(jiān)測中取樣時(shí)間的探討

房福力， 李玉中*

(中國農(nóng)業(yè)科學(xué)院農(nóng)業(yè)環(huán)境與可持續(xù)發(fā)展研究所， 北京 100081)

N2O通量； δ15N-N2O； δ18O-N2O； SP； 日變化

菜地N2O排放具有時(shí)間變異性，對(duì)評(píng)價(jià)N2O排放總量和排放系數(shù)具有重要影響。研究N2O同位素和通量日變化對(duì)了解N2O微生物產(chǎn)生過程及減排政策的提出具有指導(dǎo)意義。國內(nèi)研究多關(guān)注N2O通量日變化動(dòng)態(tài)[1-7]，很少關(guān)注N2O同位素日變化動(dòng)態(tài)。采用同位素對(duì)N2O日變化規(guī)律的研究也多集中在草地[8]，濕地[9]和農(nóng)田[10]，在菜地上的研究卻鮮有報(bào)道。

目前研究N2O通量日變化動(dòng)態(tài)主要采用自動(dòng)或半自動(dòng)連續(xù)取樣箱取樣[11-19]，這種取樣方式具有許多優(yōu)點(diǎn)，但同時(shí)也具有取樣量大，影響土壤理化性質(zhì)及植物生長等缺點(diǎn)[14, 20-21]，而這些缺點(diǎn)在監(jiān)測N2O同位素變化并以此為基礎(chǔ)分析N2O微生物來源時(shí)則會(huì)放大[22]。通過氣相色譜—靜態(tài)氣體箱技術(shù)對(duì)草地[8]和農(nóng)田作物[10]的監(jiān)測發(fā)現(xiàn)取樣時(shí)間與土壤類型、 氣候及天氣條件等各種因素有關(guān)，因此取樣時(shí)間的不同會(huì)對(duì)結(jié)果產(chǎn)生影響。了解N2O通量及同位素日變化規(guī)律，對(duì)提高N2O排放量和排放系數(shù)估計(jì)的準(zhǔn)確性，提出有效的減排措施具有重要意義。

本研究通過測定施加尿素處理后菜地土壤釋放N2O的通量及其氮氧同位素值的日變化動(dòng)態(tài)，確定監(jiān)測N2O通量和同位素特征的合適時(shí)間，為長期監(jiān)測N2O通量及同位素變化規(guī)律提供依據(jù)。

1　材料與方法

1.1試驗(yàn)區(qū)概況

試驗(yàn)在中國農(nóng)業(yè)科學(xué)院農(nóng)業(yè)環(huán)境與可持續(xù)發(fā)展研究所順義實(shí)驗(yàn)基地(東經(jīng)40°5′2″，北緯116°55′19″)進(jìn)行，該基地位于華北平原北端，屬于潮白河沖積扇下段，氣候?yàn)榈湫偷呐瘻貛О霛駶櫞箨懶约撅L(fēng)氣候。年平均日照時(shí)數(shù)2684 h，年平均氣溫12.5℃，年平均降水量623.5 mm， 無霜期195 d。供試土壤為潮褐土，土壤有機(jī)質(zhì)含量15.48 g/kg、 全氮0.37 g/kg、 全磷0.61 g/kg、 全鉀20.42 g/kg、有效磷12.69 mg/kg、速效鉀98.10 mg/kg、 pH 8.38。

1.2試驗(yàn)設(shè)計(jì)

試驗(yàn)于2012年4月27日至28日進(jìn)行，為萵苣種植且施肥后第5 d和第6 d。選擇此時(shí)間的原因是根據(jù)本課題之前的研究結(jié)果發(fā)現(xiàn)，在施肥和灌溉后4到7 d，N2O排放量較高且比較平穩(wěn)，不僅能夠代表整個(gè)生長期N2O通量的變化，而且這一時(shí)期土壤環(huán)境較為穩(wěn)定，產(chǎn)生N2O的微生物過程不易發(fā)生較大的轉(zhuǎn)變。以采樣時(shí)間為變量，設(shè)定兩種時(shí)間間隔，分別為2 h和10 min，測定土壤釋放N2O的含量及同位素值，并計(jì)算N2O的通量和SP(位置嗜值，Site preference)，分析N2O通量和同位素特征值(δ15Nbulk-N2O，δ18O-N2O和SP)在24 h內(nèi)的時(shí)間動(dòng)態(tài)變化規(guī)律(2 h)和1 h內(nèi)隨扣箱后密閉時(shí)間(10 min)的變化規(guī)律，以確定具有代表性的取樣時(shí)刻和密閉時(shí)間。供試肥料為尿素，施用量為300 kg/hm2，施肥方式為局部施肥，其他肥料用量和灌溉量按照農(nóng)戶推薦標(biāo)準(zhǔn)。

1.3樣品采集與分析

N2O氣體采樣采用靜態(tài)氣體箱。取樣間隔為兩種: 1)每2 h取一次樣，扣箱1 h后取樣，1 h后打開箱體，一天取樣12次。2)確定具有代表性的取樣時(shí)候后，對(duì)于一次取樣，從開始扣箱的時(shí)刻計(jì)時(shí)，分別在0、 10、 20、 30、 40、 50、 60 min取樣。兩種時(shí)間間隔每次取三瓶樣品作為重復(fù)，取樣量分別為20、 120、 120 mL，用注射器抽取箱內(nèi)氣體，注入相應(yīng)體積的真空玻璃瓶中，分別測定N2O含量和δ15Nbulk-N2O和δ15Nα-N2O。每次開箱后左右晃動(dòng)箱體以置換箱內(nèi)氣體，在取氣之前都要用注射器連接靜態(tài)氣體箱后來回抽動(dòng)注射器以混勻箱中氣體。土壤溫度、 空氣溫度通過站內(nèi)氣象站測定，箱內(nèi)溫度采用溫度計(jì)測定。

N2O通量計(jì)算公式為:

(1)

式中，F(xiàn)—?dú)怏w排放通量，為單位時(shí)間單位面積土壤表面釋放的N2O[μg/(m2·h)]； A—取樣箱的底面積(m2)； V—取樣箱的體積(m3)； T1、 T2—分別為扣箱前和扣箱后箱體內(nèi)的溫度(℃)； t1、 t2—分別為扣箱和開箱時(shí)間(h)； M0—?dú)怏w的摩爾質(zhì)量(g/moL)； m1、 m2—分別為扣箱前和扣箱后箱內(nèi)氣體的質(zhì)量(μg)； c1、 c2—分別為扣箱前和扣箱后箱內(nèi)氣體的體積濃度(μL/L)。

N2O同位素比率采用痕量氣體(Isotope Trace Gas, UK)，用穩(wěn)定同位素儀(Isoprime 100, UK)測定，具體方法參照[25]。N2O同位素組成的δ表示方法為:

(2)

式中，R=15N/14N和18O/16O，下標(biāo)sample和standard分別表示樣品和標(biāo)準(zhǔn)物。其中氮和氧的標(biāo)準(zhǔn)物分別為標(biāo)準(zhǔn)大氣中的氮?dú)?N2-atm)和位于維也納(Vienna)國際原子能組織同位素實(shí)驗(yàn)室配制的平均海洋水(standard mean ocean water，SMOW)，即V-SMOW。

除此之外，SP和δ15Nβ來源于以下公式:

SP=δ15Nα-δ15Νβ

(3)

(4)

δ15Nbulk-N2O、δ15Nα-N2O、 δ18O和SP的精度分別為0.2‰、 1‰、 0.5‰和1.3‰。

靜態(tài)氣體箱中的氣體是空氣和土壤釋放出來的氣體的混合物[10]，且服從公式

(5)

式中， δmeasCmeas、δatmCatm和δSDCSD分別表示來自于測定的(measure)，空氣的(atmosphere)和土壤驅(qū)動(dòng)排放(Soil-Derived)的N2O的同位素特征值和含量。空氣中N2O含量，δ15Nbulk、δ15Nα、 δ15Nβ、 δ18O和SP分別為324 μg/L、 7.0‰、 16.9‰、 -1.8‰、 43.7‰和18.7‰[27]。CSD為靜態(tài)氣體箱中測定N2O的含量(Cmeas)和空氣中N2O(Catm)的差值。根據(jù)方程(5)，當(dāng)CSD/Catm<0.3時(shí)誤差增大，計(jì)算得來的同位素比率不適合使用[29]。由于土壤釋放的N2O具有顯著的時(shí)空變異性，為了表示土壤釋放N2O的同位素特征，本研究用通量加權(quán)平均值來表示整個(gè)生長季N2O特征，公式為

(6)

式中，ci和δi分別表示在i時(shí)刻測定的含量和同位素值；δcw是某一天土壤釋放N2O的通量加權(quán)值，此時(shí)n=3，即為重復(fù)數(shù)。

1.4數(shù)據(jù)處理

試驗(yàn)數(shù)據(jù)用Microsoft Excel 2010 軟件進(jìn)行處理； 應(yīng)用SAS8.1軟件進(jìn)行統(tǒng)計(jì)分析，采用相關(guān)分析法分析土壤水分、 土壤溫度、 土壤N2O通量、 δ15Nbulk-N2O、 δ18O-N2O和SP之間的相關(guān)性； LSD法進(jìn)行多重比較和顯著性顯驗(yàn)(P<0.05)。

2　結(jié)果與分析

圖1　靜態(tài)氣體箱覆蓋下土壤溫度及土壤充水孔隙度日變化動(dòng)態(tài) Fig.1　Diurnal temperature and water filled-pore space of soil under stable flux chamber

2.1靜態(tài)氣體箱中N2O含量和通量日變化動(dòng)態(tài)

圖2　靜態(tài)氣體箱中土壤釋放N2O含量及相應(yīng)通量隨時(shí)間變化Fig.2　Change of N2O concentrations and soil-derived flux of N2O within the chamber headspace with time

2.2N2O含量和同位素特征值日變化動(dòng)態(tài)

表1　華北平原菜地土壤N2O通量及同位素日變化

注(Note): 同列數(shù)據(jù)后不同字母表示處理間差異達(dá)5%顯著水平 Values followed by different letters in a column are significant among treatments at the 5% level.

2.3扣箱后1 h內(nèi)N2O含量和同位素特征值變化動(dòng)態(tài)

圖3　N2O含量、 δ15N-N2O、 δ18O-N2O和SP在扣箱1 h內(nèi)的變化趨勢Fig.3　Changes in the concentration of N2O, δ15N-N2O, δ18O-N2O and SP within the headspace of flux chamber during 1h chamber closing[注(Note): 9: 00為扣箱0時(shí)，取空氣值為樣品，其含量為324 μg/L[39]，δ15N-N2O為7‰，δ18O-N2O為43.7‰，SP為18.7‰[26] Solid circles at time 9 am indicate previously reported values for the concentrations of N2O[39], δ15N-N2O, δ18O-N2O and SP of N2O in the atmosphere were 324 μg/L, 7‰, 43.7‰ and 18.7‰[26], respectively.]

3　討論

3.1根據(jù)通量和同位素變化選取監(jiān)測時(shí)刻

由于N2O通量日變化較大，為了方便進(jìn)行長期觀測，目前大部分研究都以某一時(shí)間段的通量代表全天的通量，并以此計(jì)算累計(jì)N2O排放量和排放系數(shù)[1,3,6-7]。本研究結(jié)果表明(表1)，在11: 00至17: 00，N2O通量較高且基本保持穩(wěn)定，但是采用此時(shí)間段任意一個(gè)值表示全天平均通量都會(huì)使對(duì)N2O估計(jì)排放量偏高，如果用9: 00的N2O的通量83.66 μg/(m2·h)表示全天通量平均值82.82 μg/(m2·h)(表1)，則誤差較小。但是問題在于不同土壤環(huán)境條件和取樣時(shí)間，N2O通量受各種因素影響，如溫度、 土壤含水量及微生物過程，而這些參數(shù)在一天之內(nèi)也會(huì)隨時(shí)間的變化而改變，因此N2O通量也會(huì)隨之變化，對(duì)于某一特定土壤和環(huán)境，哪個(gè)時(shí)刻能夠代表N2O全天通量平均值也會(huì)不一樣。

3.2單次取樣密閉時(shí)間的選取

3.3采用SP區(qū)別N2O來源

假如N2O完全來自于土壤硝化作用和反硝化作用，那么同位素混合模型[46]，

fD=N2OD/N2Ototal

(7)

(8)

fD=1-fN

(9)

式中, SPD和SPN表示純培養(yǎng)試驗(yàn)中N2OD和N2ON得到的SP值(37‰和0‰)； SPtotal為環(huán)境樣品的N2Ototal的位置嗜值，fD和fN分別表示產(chǎn)生的N2O來自反硝化作用和硝化作用的比例。

4　結(jié)論

3)扣箱40 min后，N2O含量、 δ15N-N2O和SP值達(dá)到穩(wěn)定，因此選取40 min作為觀測N2O含量和同位素變化的密閉時(shí)間。

4)4月27日至4月28日的24 h之內(nèi)，SP值的通量加權(quán)平均值為22.54‰，根據(jù)國際上的相關(guān)研究結(jié)論，即細(xì)菌硝化作用產(chǎn)生N2O的SP值為37‰，反硝化作用產(chǎn)生N2O的SP值為0‰，硝化作用對(duì)N2O的產(chǎn)生起主導(dǎo)作用，大約60.92%的N2O由硝化作用產(chǎn)生，39.08%的N2O由反硝化作用產(chǎn)生。

5)在華北平原測定菜地N2O氣體通量和同位素特征值變化時(shí)，選取9: 00作為具有代表性的觀測點(diǎn)，選取40 min作為密閉時(shí)間比較適宜。

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Preliminary research on N2O sampling time based on isotopomer signature measurement of vegetable fields in the North China Plain

FANG Fu-li, LI Yu-zhong*

(InstituteofEnvironmentandSustainableDevelopmentinAgriculture,ChineseAcademyofAgriculturalSciences,Beijing100081,China)

【Objectives】 Emissions of N2O from vegetable soils are extremely variable in time and therefore there are considerable uncertainties in calculating emission of N2O flux and emission coefficients. It is thus important to investigate the optional sampling time and characteristic of diurnal pattern for N2O. More insights into N2O diurnal variation would also be helpful for long term monitoring of N2O release and for understanding the associated microbiological mechanisms.【Methods】 Field experiment in situ was conducted to study N2O emission after urine amendment and subsequent irrigation for a lettuce production system in North China Plain in 2012. During periods of high N2O fluxes in 5-6 days, N2O emission was collected manually by a closed static flux chamber at two intervals, two hours for diurnal variation and 10 minutes for appropriate enclosure period. N2O concentrations were measured by a gas chromatograph equipped with an electron capture detector (GC-ECD), and isotopomers such as δ15N-N2O, δ18O-N2O and site preference (SP) (difference between the center and the end N atom of the N2O asymmetric molecule) were analyzed and obtained by an elemental analyzer-isotope ratio mass spectrometer(EA-IRMS) system.【Results】 1) During the period of high N2O emission，there was a strong diurnal variation in N2O fluxes ranging from 20.41 to 130.45 μg/(m2·h), with the maximum and minimum values being observed at 1 pm in Apr. 27thand 5 am in Apr. 28th, respectively. Flux measured at 9 am could be used to represent the daily average of the whole day. The fluxes of N2O emitted from the soil were correlated significantly(R2=0.82,P<0.01) with the soil temperature at 5 cm, varied markedly across time, but insignificantly with water filled-pore space (WFPS). 2) Diurnally, the values of δ15Nbulk-N2O and δ18O-N2O decreased first and then increased gradually with time while the values of SP had an opposite trend, suggesting that production processes of N2O were associated with different microbial pathways. Conversely, the stable values of SP of N2O from 9 am through 5 pm indicated that nitrification was the dominant pathway for N2O production. 3) In measuring N2O emission, appropriate enclosure period in the static chamber should be 40 minutes, which was long enough to allow detection of N2O concentration, δ15N-N2O and SP and also short enough to avoid feedback of N2O concentration and microbial pathway change. 4) The calculated emission-weighted site preference was 22.54‰. The N2O emission in this study was dominantly produced from nitrification, contributing 60.9% of the daily totals of N2O emission.【Conclusions】 There were large diurnal variations of N2O concentration and isotopomers from lettuce production system in North China Plain. Considering both values of N2O flux and isotopomers, it was suggested that 9 am was an appropriate time to measure the flux and isotopomer signature of N2O and that enclosure period for one sample event should be 40 minutes.

N2O flux; δ15N-N2O; δ18O-N2O; SP; diurnal variation

2015-03-16接受日期: 2015-06-14網(wǎng)絡(luò)出版日期: 2015-06-29

國家自然科學(xué)基金(41473004)； 國家科技支撐計(jì)劃項(xiàng)目(2011BAD32B03)； 國家自然科學(xué)基金項(xiàng)目(41301553)； 中央級(jí)公益性科研院所基本業(yè)務(wù)費(fèi)項(xiàng)目(BSRF201304)資助。

房福力(1985—)， 男， 山東青島人， 博士， 主要從事植物營養(yǎng)與環(huán)境研究。 E-mail: fulifangfu@163.com

E-mail: liyuzhong@caas.cn

S153.6+1

A

1008-505X(2016)04-0978-10