設(shè)施菜田土壤pH 和初始 對(duì)反硝化產(chǎn)物比的影響

曹文超，郭景恒，宋 賀，劉 骕，陳吉吉，王敬國(guó)*

（1 中國(guó)農(nóng)業(yè)大學(xué)資源與環(huán)境學(xué)院，北京 100193；2 安徽農(nóng)業(yè)大學(xué)農(nóng)學(xué)院，安徽合肥 230036）

曹文超1，郭景恒1，宋 賀2，劉 骕1，陳吉吉1，王敬國(guó)1*

（1 中國(guó)農(nóng)業(yè)大學(xué)資源與環(huán)境學(xué)院，北京 100193；2 安徽農(nóng)業(yè)大學(xué)農(nóng)學(xué)院，安徽合肥 230036）

【目的】設(shè)施菜田土壤反硝化作用是N2O排放和氮素?fù)p失的重要途徑。本研究通過(guò)室內(nèi)厭氧培養(yǎng)試驗(yàn)，在不同pH和初始條件下，比較設(shè)施菜田土壤反硝化氮素氣體排放及產(chǎn)物比的變化特征。【方法】以設(shè)施菜田土壤為研究對(duì)象，通過(guò)添加一定量低濃度的酸堿溶液調(diào)節(jié)土壤pH分別為酸性、中性和堿性條件，調(diào)節(jié)后的實(shí)測(cè)pH分別為5.63、6.65和7.83；同時(shí)以谷氨酸鈉作為有效性碳，除未添加有效性碳作為對(duì)照處理 (CK)外，其他有效性碳與硝酸鹽的比值分別調(diào)節(jié)為5∶1、15∶1和30∶1，三種pH條件下均設(shè)置 4 個(gè)水平，每個(gè)水平3次重復(fù)。利用自動(dòng)連續(xù)在線(xiàn)培養(yǎng)系統(tǒng) (Robot系統(tǒng))，在厭氧條件下監(jiān)測(cè)不同處理土壤產(chǎn)生的 N2O、NO、N2和CO2濃度的動(dòng)態(tài)變化，通過(guò)計(jì)算N2O/(N2O + NO + N2)指數(shù)估算反硝化過(guò)程N(yùn)2O的產(chǎn)物比。【結(jié)果】增加土壤的pH能顯著減少設(shè)施菜田土壤N2O和NO的產(chǎn)生量，酸性 (pH 5.63) 土壤的N2O、NO產(chǎn)生量峰值在不同初始比下均顯著高于中性 (pH 6.65) 和堿性 (pH 7.83) 土壤 (P＜ 0.05)。中性和堿性土壤在高下有利于減少反硝化過(guò)程N(yùn)2O的產(chǎn)生，而酸性土壤條件下差異并不顯著。中性土壤條件下增加有機(jī)碳含量會(huì)降低NO產(chǎn)生量，而在酸性和堿性土壤上有機(jī)碳的添加對(duì)NO產(chǎn)生量沒(méi)有顯著影響。土壤pH和初始比對(duì)土壤N2O的產(chǎn)生有極顯著的交互效應(yīng) (P＜ 0.001)。酸性和中性土壤上添加有機(jī)碳能夠顯著增加土壤N2的產(chǎn)生速率 (P＜ 0.05)，且與對(duì)照相比，不同pH的土壤添加有機(jī)碳后均顯著促進(jìn)反硝化過(guò)程中N2O向N2的轉(zhuǎn)化。在不同初始下堿性土壤的CO2產(chǎn)生量顯著高于酸性和中性土壤，同時(shí)與對(duì)照相比，添加有機(jī)碳顯著增加了土壤的CO2產(chǎn)生量 (P＜ 0.05)。酸性土壤的N2O產(chǎn)物比在不同初始下均極顯著高于堿性土壤 (P＜ 0.01)，且不同初始下的土壤N2O產(chǎn)物比隨pH的增加顯著下降，二者呈極顯著線(xiàn)性負(fù)相關(guān)關(guān)系(P＜ 0.01)。【結(jié)論】土壤pH降低是設(shè)施菜田土壤N2O和NO排放量較高的重要原因。而且，增加初始土壤有效碳含量促進(jìn)了土壤的反硝化損失，并在中性和堿性土壤中N2O的產(chǎn)生量減少。土壤pH升高和初始增加均降低了產(chǎn)物比，但增加了土壤反硝化作用速率。在利用N2O排放通量和產(chǎn)物比估算土壤反硝化氮素?fù)p失時(shí)，土壤pH和有效碳含量是必須考慮的兩個(gè)重要因素。

反硝化；pH；；N2O；日光溫室

反硝化過(guò)程是土壤氮損失的主要途徑之一，而且該過(guò)程產(chǎn)生的N2O和NO會(huì)給環(huán)境帶來(lái)危害。其中，N2O是重要的溫室氣體，與全球氣候變暖和臭氧層破壞密切相關(guān)[1–2]；而NO是一種具有高度活性且有毒的化合物，會(huì)損耗臭氧[3]和污染空氣[4]，同時(shí)，NO經(jīng)光化學(xué)氧化和水合作用生成HNO2和HNO3，對(duì)土壤和水體系統(tǒng)酸化產(chǎn)生影響[5]。農(nóng)業(yè)活動(dòng)占全球人為活動(dòng)排放N2O和NO總量比例分別約為60%和10%[6]。以我國(guó)農(nóng)田土壤為例，其N(xiāo)2O直接排放量為4.74 × 105t，占全國(guó)N2O排放的55.8%[7]。大量研究表明N2O和NO的排放與氮肥的施用密切相關(guān)，施用氮肥后增加土壤N2O和NO的排放量[8–11]。

我國(guó)設(shè)施蔬菜種植系統(tǒng)中，農(nóng)戶(hù)為了追求高產(chǎn)，過(guò)量施用化學(xué)氮肥。大部分地區(qū)設(shè)施菜田每季化肥氮素投入量通常會(huì)超過(guò)1200 kg/hm2[9]，約為小麥、玉米田的2～7倍[10–12]。除了大量施用化學(xué)氮肥外，有機(jī)肥料的投入量也很大。以壽光設(shè)施菜田為例，有機(jī)肥年均施用量高達(dá)177 t/hm2[13]。此外，該地區(qū)設(shè)施菜田全年灌溉水總量在748～1957 mm之間，平均灌溉量高達(dá)1307 mm[14]。這些都為土壤反硝化微生物提供了大量的底物和適宜的環(huán)境條件，進(jìn)而促進(jìn)反硝化作用的發(fā)生和N2O的大量排放。據(jù)估計(jì)，我國(guó)設(shè)施菜田反硝化年損失量可達(dá)N 45.8 kg/hm2[15]，反硝化過(guò)程對(duì)N2O排放總量的貢獻(xiàn)率可達(dá)22.5%～57.7%[16]。由于方法原因，土壤反硝化氮損失的直接定量測(cè)定比較困難。已有研究表明，利用反硝化產(chǎn)物比和大量的N2O田間觀測(cè)數(shù)據(jù)，可以估算土壤反硝化損失[17–18]。

眾所周知，土壤中每1 mol的NH3-N氧化為NO3–-N即可釋放1 mol質(zhì)子，在沒(méi)有生物利用的情況下，成為導(dǎo)致土壤酸化的重要原因之一。同時(shí)，在土壤根際微域環(huán)境中，根系對(duì)陽(yáng)離子的吸收超過(guò)陰離子時(shí)產(chǎn)生的酸性分泌物也會(huì)造成土壤局部酸化。已有研究表明，設(shè)施菜田每年僅由施肥產(chǎn)生的土壤酸化潛勢(shì)就超過(guò)H+220 kmol/hm2[19]，相同區(qū)域設(shè)施菜田土壤pH值比糧田土壤低0.5個(gè)單位[20]。土壤pH值的降低會(huì)顯著影響反硝化的損失量[21–22]和氣態(tài)產(chǎn)物比的變化[23]。而且，設(shè)施菜田表層土壤的可溶性有機(jī)碳含量顯著高于農(nóng)田土壤[24]，這有利于其形成土壤微域厭氧環(huán)境。設(shè)施菜田通常底施大量的有機(jī)肥和大水漫灌，在生長(zhǎng)季初期 (1到2周內(nèi)) 也會(huì)產(chǎn)生較大范圍的厭氧環(huán)境。有證據(jù)表明，施用有機(jī)肥后提高土壤水分含量，反硝化作用對(duì)N2O的貢獻(xiàn)可在64%～77%之間[25]。

綜上所述，土壤pH和有機(jī)碳是影響反硝化過(guò)程與N2O排放的重要環(huán)境因子，它們之間存在著交互作用，但目前相關(guān)的研究較少、亟待深入。本研究選擇pH下降明顯、有機(jī)肥施用量很高的設(shè)施蔬菜集中種植區(qū)代表性土壤，通過(guò)在線(xiàn)連續(xù)監(jiān)測(cè)的自動(dòng)培養(yǎng)系統(tǒng)，研究外源碳加入和pH對(duì)反硝化產(chǎn)物比的影響，從而為探討通過(guò)降低產(chǎn)物比以實(shí)現(xiàn)N2O減排的措施提供理論依據(jù)。對(duì)土壤反硝化產(chǎn)物比及其影響因素的深入了解，又可為利用N2O的田間觀測(cè)數(shù)據(jù)準(zhǔn)確估算土壤反硝化氮損失提供參考。

1 材料與方法

1.1 采樣點(diǎn)概況及土壤樣品采集

供試土壤采自中國(guó)農(nóng)業(yè)大學(xué)山東省壽光市羅家村 (36°55′N(xiāo)，118°35′E) 設(shè)施菜田長(zhǎng)期定位試驗(yàn)點(diǎn)。該地區(qū)年平均溫度和降雨分別為12.4℃和558 mm。試驗(yàn)始于2004年2月，每年種植兩季番茄，即每年2～6月為冬春季，8月至次年1月為秋冬季，7月份休閑。試驗(yàn)初始0—10 cm土層土壤理化性質(zhì)為全氮1.37 g/kg，有機(jī)質(zhì)18.3 g/kg，pH 5.7 (0.05 mol/L CaCl2)，有效磷和速效鉀含量均為299 mg/kg，有關(guān)試驗(yàn)點(diǎn)的更多信息可參見(jiàn)He等[26]的研究。

在冬春季和秋冬季番茄移栽前，分別基施風(fēng)干雞糞 8 t/hm2和 10 t/hm2(約為 N 146 kg/hm2和 211 kg/hm2)，尿素作為追肥隨灌溉水施用，每次用量為N 120 kg/hm2，冬春季為6次，秋冬季共5次。本試驗(yàn)以農(nóng)戶(hù)習(xí)慣施肥土壤作為供試樣品，為避免雞糞施用的影響，于2015年6月番茄最后收獲期采集0—20 cm土壤樣品，充分混合后用冰盒運(yùn)回實(shí)驗(yàn)室，在4℃下保存?zhèn)溆谩?/p>

1.2 試驗(yàn)設(shè)計(jì)與方法

分別稱(chēng)取相當(dāng)于10.0 g烘干土重的新鮮土樣于120 mL血清瓶中，根據(jù)已知的土壤硝酸鹽含量和谷氨酸鈉添加量配置一定濃度的溶液，將溶液用注射器加入至土壤中后，調(diào)節(jié)土壤質(zhì)量含水量為25%。用橡膠塞和鋁蓋密封，并利用抽真空–充氦氣系統(tǒng)(北京帥恩科技有限公司) 反復(fù)用He (99.999%) 氣沖洗血清瓶3次，最后充上He氣。將所有血清瓶置于20℃恒溫水浴槽中培養(yǎng)，平衡血清瓶?jī)?nèi)氣壓后，利用Robot自動(dòng)培養(yǎng)系統(tǒng)每間隔8 h在線(xiàn)連續(xù)監(jiān)測(cè)血清瓶?jī)?nèi)N2O、NO、N2和CO2的濃度變化。每個(gè)處理均重復(fù)3次。

Robot自動(dòng)培養(yǎng)系統(tǒng)包括自動(dòng)進(jìn)樣和氣體分析模塊。其中自動(dòng)進(jìn)樣模塊包括雙向旋轉(zhuǎn)的蠕動(dòng)泵(Gilson Model 222，Gilson，法國(guó)) 和頂空自動(dòng)采樣器(CTC GC-Pal)。氣體分析模塊由氣相色譜 (Agilent 7890A) 和氮氧化物分析儀 (Model 200E，美國(guó)) 組成。而氣相色譜中包含熱導(dǎo)檢測(cè)器 (TCD)、電子捕獲檢測(cè)器 (ECD) 和火焰離子化檢測(cè)器 (FID) 3個(gè)檢測(cè)器，能夠監(jiān)測(cè)N2O (ECD、TCD)，N2(TCD)，CO2(TCD) 和O2(TCD) 氣體濃度的變化。氮氧化物分析儀用來(lái)定量監(jiān)測(cè)NO氣體濃度。有關(guān)Robot自動(dòng)培養(yǎng)系統(tǒng)的運(yùn)行模塊細(xì)節(jié)可參見(jiàn)Molstad等[28]和McMillan 等[29]的研究。

表1 不同土壤pH下初始理化性質(zhì)Table 1 Initial soil physical and chemical properties with different soil pH

1.3 土壤基礎(chǔ)參數(shù)測(cè)定

1.4 數(shù)據(jù)處理

氣體數(shù)據(jù)計(jì)算根據(jù)在線(xiàn)監(jiān)測(cè)的血清瓶中氣體數(shù)值和已知標(biāo)準(zhǔn)氣體濃度，計(jì)算相應(yīng)氣體每克干土的產(chǎn)生量。研究厭氧培養(yǎng)下反硝化產(chǎn)物比N2O/(N2O +NO + N2) 的變化，以計(jì)算N2O的產(chǎn)生量指數(shù)IN2O，計(jì)算公式如下：

其中：N2O (t)、N2(t)、NO (t) 均為培養(yǎng)時(shí)間t下的累積產(chǎn)生量。時(shí)間t的選擇依據(jù)是以N2O產(chǎn)生速率為正值或N2O產(chǎn)生量達(dá)最大值時(shí)作為終止時(shí)間基準(zhǔn)，即利用從初始培養(yǎng)至達(dá)到N2O最大累積產(chǎn)生量時(shí)的反硝化產(chǎn)物比均值進(jìn)行比較。

試驗(yàn)數(shù)據(jù)采用SigmaPlot 12.5作圖，利用SPSS 20.0分析軟件進(jìn)行單、雙因素方差分析。

2 結(jié)果與分析

圖1 不同pH和初始 下土壤N2O和NO產(chǎn)生量的動(dòng)態(tài)變化Fig. 1 Dynamics of N2O and NO production during the anaerobic incubation from soil under different pH(acidic, neutral and alkaline) and levels

由圖1整體來(lái)看，連續(xù)培養(yǎng)過(guò)程中土壤反硝化N2O產(chǎn)生量變化明顯，且隨土壤pH的增加N2O產(chǎn)生量顯著減少。除未添加谷氨酸鈉的對(duì)照處理 (CK)外，其它3個(gè)不同初始C/NO3–處理中N2O產(chǎn)生量的動(dòng)態(tài)變化過(guò)程相類(lèi)似，均呈現(xiàn)先增加達(dá)到峰值后再減少的趨勢(shì)。在pH 5.63條件下，初始C/NO3–比為5∶1、15∶1和30∶1的處理中，N2O產(chǎn)生量分別在144、120和128 h時(shí)達(dá)到峰值，且三者均差異不顯著 (圖1)。在中性條件下 (pH 6.65)，初始比為5∶1、15∶1和30∶1的處理中，N2O產(chǎn)生量分別在72、48和40 h時(shí)達(dá)到峰值，分別為N 1.05、0.71 和 0.43 μmol/g，三者差異極顯著 (P＜ 0.01)。單從峰值來(lái)看，在土壤pH為7.83時(shí)，添加谷氨酸鈉的3個(gè)處理均在連續(xù)培養(yǎng)32 h時(shí)達(dá)到峰值，且初始C/NO3–為30∶1的處理顯著降低了N2O的排放 (P＜0.05)。可見(jiàn)，中性和堿性土壤中添加有效性碳有利于減少反硝化過(guò)程N(yùn)2O的產(chǎn)生，而酸性條件下添加有效性碳并不能降低N2O產(chǎn)生量。

與N2O類(lèi)似，土壤反硝化NO的產(chǎn)生量也隨土壤pH的增加而減少。在酸性條件下 (pH 5.63)，添加谷氨酸鈉的處理均在培養(yǎng)96 h后達(dá)到峰值，且差異不顯著 (圖1)。未添加谷氨酸酸鈉的處理 (CK)，NO的產(chǎn)生量較少且下降緩慢，導(dǎo)致其累積排放時(shí)間較長(zhǎng)，這可能與土壤中可利用性碳不足導(dǎo)致缺乏電子供體有關(guān)。在pH為6.65的中性土壤條件下，初始為5∶1的處理在培養(yǎng)64 h時(shí)NO產(chǎn)生量達(dá)到峰值，為N 0.81 μmol/g，分別是初始為15∶1和30∶1處理峰值的1.74和1.55倍 (圖1)。而在pH為7.83的堿性土壤中，NO的產(chǎn)生量均在培養(yǎng)24 h時(shí)達(dá)到峰值，且初始為 5∶1、15∶1 和30∶1的處理間NO產(chǎn)生量差異不顯著。可見(jiàn)，酸性和堿性土壤中有機(jī)碳的添加對(duì)NO產(chǎn)生量不會(huì)產(chǎn)生顯著影響，而在中性條件下有機(jī)碳含量會(huì)降低NO產(chǎn)生量。

由圖2可知，隨著培養(yǎng)時(shí)間的延長(zhǎng)不同土壤pH下各處理的N2產(chǎn)生量均呈上升趨勢(shì)，且添加谷氨酸鈉后N2產(chǎn)生量均達(dá)到穩(wěn)定狀態(tài)。由其平衡的時(shí)間來(lái)看，隨著土壤pH的增加N2O還原為N2的速率顯著加快。除pH值為7.83的堿性土壤外，N2產(chǎn)生速率在pH為5.63的酸性土壤和pH為6.65的中性土壤中均隨初始土壤C/NO3–的增加而增大。可見(jiàn)，在一定程度上增加土壤pH值并同時(shí)向土壤中添加可利用性碳可促進(jìn)反硝化過(guò)程中N2O向N2的轉(zhuǎn)化，有利于減少溫室氣體N2O的排放。

圖2 不同pH和初始下土壤N2和CO2產(chǎn)生量的動(dòng)態(tài)變化Fig. 2 Dynamics of N2 and CO2 production from three pH soils (acidic, neutral and alkaline) during the anaerobic incubation with different levels

碳源為反硝化微生物提供反應(yīng)所需的電子供體和能源，CO2的產(chǎn)生量可間接反映微生物的活性。由圖2可知，各pH土壤在添加有效性碳后，CO2產(chǎn)生量均顯著高于對(duì)照處理 (CK)，且在相同培養(yǎng)時(shí)間和初始下，CO2產(chǎn)生量隨土壤pH的增加而顯著增大 (P＜ 0.01)。酸性土壤中，連續(xù)培養(yǎng)256 h后對(duì)照處理CO2產(chǎn)生量為2.14 μmol/g，初始為5∶1、15∶1和30∶1處理的CO2產(chǎn)生量分別是對(duì)照處理的3.55、3.93和3.92倍 (圖2)。在土壤pH為6.65條件下，連續(xù)培養(yǎng)136 h后，初始為5∶1處理的CO2產(chǎn)生量為5.69 μmol/g (圖2)，且與初始為15∶1和30∶1的處理均差異極顯著 (P＜ 0.01)。堿性土壤中，在土壤連續(xù)培養(yǎng)112 h后，CO2產(chǎn)生量在添加有效性碳的不同處理間差異顯著 (P＜ 0.05)。此外，隨著pH增加，土壤的平均呼吸速率也明顯增加。

由表3可知，當(dāng)N2O達(dá)最大累積產(chǎn)生量時(shí)，土壤pH和分別對(duì)N2O產(chǎn)生量有極顯著作用(P＜ 0.001)，且二者交互效應(yīng)極顯著 (P＜ 0.001)。同時(shí)，整體來(lái)看，在不同初始下各土壤 pH 對(duì)N2O累積產(chǎn)生量影響顯著 (P＜ 0.05)。而對(duì)于不同初始C/NO3–來(lái)說(shuō)，與對(duì)照處理 (CK) 相比，添加有效性碳后，顯著增加了土壤N2O的產(chǎn)生，但高比值 (30∶1) 的N2O最大累積量顯著低于其他兩個(gè)水平處理 (P＜ 0.05)。

圖3 不同初始下反硝化產(chǎn)物比和土壤pH的關(guān)系Fig. 3 Relationship between the product ratio of denitrification and soil pH with different initial levels

表2 不同pH和初始下厭氧培養(yǎng)土壤反硝化產(chǎn)物比 比較分析Table 2 Compared analysis for the product ratio of denitrification in soils under the anaerobic incubation with different pH and initial levels

表2 不同pH和初始下厭氧培養(yǎng)土壤反硝化產(chǎn)物比 比較分析Table 2 Compared analysis for the product ratio of denitrification in soils under the anaerobic incubation with different pH and initial levels

注（Note）：同行數(shù)據(jù)后不同小寫(xiě)字母表示 間差異顯著 (P ＜ 0.05)，同列數(shù)據(jù)后不同大寫(xiě)字母表示 pH 間差異極顯著 (P ＜0.01); 表中數(shù)值為平均值 ± 標(biāo)準(zhǔn)差 (n = 3)。 Values followed by different lowercase letters in a row indicate significant differences (P ＜0.05) among different ratios and capital letters in a column indicate significant differences (P ＜ 0.01) among different pH; Values are mean ± SD (n = 3).

C/NO3–CK 5∶1 15∶1 30∶1 5.630.58 ± 0.06 aA0.41 ± 0.05 bA0.41 ± 0.05 bA0.42 ± 0.06 bA 6.650.55 ± 0.07 aA0.34 ± 0.05 bB0.31 ± 0.02 bcB0.26 ± 0.007 cB 7.830.34 ± 0.01 aB 0.17 ± 0.01 bC0.17 ± 0.01 bC 0.16 ± 0.004 bB pH

表3 土壤pH和初始對(duì)N2O產(chǎn)生量的因子分析Table 3 Multivariate analysis of variance for the effects of pH and initial on the N2O production

表3 土壤pH和初始對(duì)N2O產(chǎn)生量的因子分析Table 3 Multivariate analysis of variance for the effects of pH and initial on the N2O production

P 值P value pH 2 24.6 15960 ＜0.001 C/NO3– 3 1.53 992 ＜0.001 pH × (C/NO3–) 6 0.743 482 ＜0.001因子Factor自由度df均方Mean square F 值F value

3 討論

土壤pH值是影響反硝化作用特征的重要因素。在低pH值條件下 (pH 5.63)，對(duì)照處理 (CK) 的氮素氣態(tài)產(chǎn)物以N2O和NO為主，這是由于低pH阻礙了N2O還原酶的形成[30]。同時(shí)，基于在Paracoccus denitrificans上研究的結(jié)果，低pH也會(huì)干擾周質(zhì)空間下的蛋白組裝[31]。本研究發(fā)現(xiàn)，不同初始下土壤N2O產(chǎn)生量指數(shù)隨pH的增加顯著下降，且二者呈極顯著線(xiàn)性負(fù)相關(guān)關(guān)系 (圖3)，這與Liu等[32]和Qu等[23]的研究結(jié)果類(lèi)似。同時(shí)，在堿性土壤和中性土壤中添加有效碳后，反硝化作用N2O產(chǎn)物比值 (0.16～0.34) 均低于Schlesinger等[21]匯總的農(nóng)田土壤反硝化產(chǎn)物比結(jié)果 (0.37)，而在酸性土壤中有無(wú)添加有效性碳N2O產(chǎn)物比均較高 (0.41～0.58)。由于土壤pH對(duì)反硝化N2O產(chǎn)物比的影響不可忽視，因而，對(duì)不同pH的土壤僅用單一的產(chǎn)物比并結(jié)合N2O排放通量估算土壤反硝化的總損失量存在較大的誤差。

NO是反硝化作用過(guò)程的中間產(chǎn)物，同時(shí)也是編碼亞硝酸鹽還原酶 (NIR) 和一氧化氮還原酶 (NOR)轉(zhuǎn)錄的誘導(dǎo)因子[33]。Bergaust等[33]發(fā)現(xiàn)，在模式菌株P(guān). denitrificans上NO還可以誘導(dǎo)nosZ基因的轉(zhuǎn)錄。本研究結(jié)果表明，NO的最大累積量在低pH土壤 (pH 5.63) 中顯著高于其他pH處理 (圖1)，且在土壤溶液中NO濃度的最大值為6.2 μmol/L (數(shù)據(jù)未顯示)。這是由于NO在低pH條件下的產(chǎn)生速率大于其消耗速率，還可能與低pH下的微生物會(huì)優(yōu)先將硝酸鹽完全還原為亞硝酸鹽，而不是還原亞硝酸鹽有關(guān)。此外，在低pH土壤條件下，尤其在土壤亞硝態(tài)氮出現(xiàn)累積時(shí)，化學(xué)分解反應(yīng)等非生物因素對(duì)NO的排放貢獻(xiàn)也不容忽視[34]，還需進(jìn)一步探究。

與對(duì)照相比 (CK)，各pH土壤在添加谷氨酸鈉后均顯著增加了反硝化速率，且土壤反硝化速率的最大值也隨有效性碳含量的增加而增大，這與Jahangir等[35]的研究結(jié)果相一致。原因可能是有效性碳增加了土壤nir和nosZ的基因拷貝濃度[36]，促進(jìn)了反硝化微生物酶的活性[37]。同時(shí)，可利用性碳源的添加也會(huì)增加細(xì)胞的呼吸作用，顯著促進(jìn)CO2的排放，且隨土壤pH值的增加呈上升趨勢(shì) (圖2)。在同一初始比土壤下，土壤pH的調(diào)節(jié)對(duì)N2O和CO2兩種溫室氣體的排放存在某種程度上的“此消彼長(zhǎng)”效應(yīng) (trade-off effect)。增加土壤pH后，N2O產(chǎn)生量明顯減少的同時(shí)CO2的產(chǎn)生量也會(huì)顯著增加，這可能與提高土壤pH后促進(jìn)了土壤碳、氮的礦化有關(guān)[38]。

雖然在低pH條件下，有效碳的添加對(duì)反硝化N2O的產(chǎn)物比沒(méi)有影響，但在中性和石灰性土壤上，碳的有效性顯著影響該產(chǎn)物比。因此，在利用N2O排放通量來(lái)估算土壤反硝化損失時(shí)，活性有機(jī)碳含量是應(yīng)該予以重視的一個(gè)重要因素。

表4 培養(yǎng)結(jié)束后不同pH和初始下銨態(tài)氮及硝態(tài)氮含量及氣態(tài)氮素?fù)p失總量Table 4 Concentrations of ammonium and nitrate and the total N gaseous loss in the end of the incubation

表4 培養(yǎng)結(jié)束后不同pH和初始下銨態(tài)氮及硝態(tài)氮含量及氣態(tài)氮素?fù)p失總量Table 4 Concentrations of ammonium and nitrate and the total N gaseous loss in the end of the incubation

注（Note）：同列數(shù)據(jù)后不同小寫(xiě)字母表示相同 pH 值不同間差異顯著 (P ＜ 0.05) Values followed by different lowercase letters in a column indicate significant differences (P ＜ 0.05) among different ratios; 表中數(shù)值為平均值 ± 標(biāo)準(zhǔn)差 (n = 3) Values are mean ± SD (n = 3).

氣態(tài)氮損失總量 (mg/kg)Total gaseous loss 5.63 256 CK 28.9 ± 1.6 a 11.4 ± 1.0 d 27.4 ± 0.36 c 5∶1 0 b 28.2 ± 12 c 57.5 ± 0.36 a 15∶1 0 b 47.7 ± 0.7 b 56.5 ± 0.71 ab 30∶1 0 b 110 ± 8.3 a 55.9 ± 0.43 b 6.65 136 CK 34.0 ± 1.4 a 6.0 ± 0.6 d 13.4 ± 0.53 b 5∶1 0 b 20.1 ± 3.6 c 49.3 ± 0.52 a 15∶1 0 b 94.8 ± 2.6 b 48.9 ± 0.54 a 30∶1 0 b 157 ± 1.0 a 49.4 ± 0.43 a 7.83 184 CK 20.5 ± 7.6 a 3.4 ± 0.8 d 18.0 ± 0.19 b 5∶1 0 b 17.2 ± 3.6 c 60.0 ± 0.86 a 15∶1 0 b 106 ± 7.8 b 60.2 ± 0.86 a 30∶1 0 b 235 ± 8.1 a 58.6 ± 0.13 a pH 培養(yǎng)時(shí)間 (h)Incubation time C/NO3– NO3–-N(mg/kg)NH4+-N(mg/kg)

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Effects of pH and initial labileratio on denitrification in a solar greenhouse vegetable soil

CAO Wen-chao1, GUO Jing-heng1, SONG He2, LIU Su1, CHEN Ji-ji1, WANG Jing-guo1*
(1 College of Resource and Environment, China Agricultural University, Beijing 100193, China;2 College of Agronomy, Anhui Agricultural University, Hefei, Anhui 230036, China)

【Objectives】Denitrification is one of predominant process for N2O emission from solar greenhouse vegetable soils. An anoxic incubation experiment was conducted to explore effects of soil pH and initial labileratio on nitrogen gaseous emissions (i.e. N2O, NO and N2) and the N2O product during the denitrification in a solar greenhouse vegetable soil.【Methods】A typical greenhouse vegetable soil in Shouguang City was selected for the study, and soil pH was adjusted to acidic, neutral and alkaline by adding a certain amount of lowconcentration of acid (0.1 mol/L HCl) or alkaline (0.1 mol/L NaOH) solution. The final soil pH values were 5.63,6.65 and 7.83 (adjusted), respectively. Sodium glutamate (C5H8NO4Na) was added as a labile organic carbon source, and the ratios of initial labile C towere adjusted to 5∶1, 15∶1 and 30∶1 by adding different amounts of sodium glutamate and no sodium glutamate addition in the control (CK). Four levels of initial labile C/NO3–ratios were set up in the three soils with different pH, and there were three replicates in each level. A robotized incubation system was used to monitor the kinetics of gaseous products (N2O, NO, N2, and CO2) of denitrification under anoxic conditions and to estimate the N2O product ratio of denitrification by calculating Index N2O/(N2O + NO + N2).【Results】The increase in soil pH significantly reduced N2O and NO production in the soil, and the peak values of N2O and NO production in the acidic soil were significantly higher than those of the alkaline and neutral soils with different initial C/NO3–ratios (P＜ 0.05). With the increase of initial labile C, the production of N2O was reduced in the neutral and alkaline soils, but kept unchanged in the acidic soil. The addition of sodium glutamate reduced NO emission in the neutral soil. However, there were no differences in NO production in other pH levels. A significant interaction on N2O production was observed between soil pH and initial labileratio (P＜ 0.001). The N2production rate significantly increased with sodium glutamate addition in the acidic and neutral soils (P＜ 0.05). The addition of sodium glutamate could promote the reduction of N2O to N2in denitrification process at different soil pH. The CO2production was significantly higher in the alkaline soil than those in the acidic and neutral soils. As compared with CK, the CO2production increased significantly with the sodium glutamate addition at different soil pH (P＜ 0.05). The N2O product ratio in the acidic soil was significantly higher than that in the alkaline soil under different initial labile(P＜ 0.01), the N2O product ratio was significantly decreased with the increase of soil pH, and a significant linear negative correlation relationship was observed between soil pH and the Index N2O (P＜ 0.01).【Conclusions】High N2O and NO emissions were usually found in greenhouse vegetable soils, primarily due to the decline of soil pH.Furthermore, increase in labile C availability promoted the N loss of denitrification, and decreased N2O production in neutral and alkaline soils. With the increase of both soil pH and labile, the N2O product ratio reduced,but the denitrification activity increased. Both soil pH and carbon availability were crucial factors for estimation of N loss of denitrification by multiplying N2O flux and N2O product ratio.

denitrification; pH;nitrous oxide; solar greenhouse

2017–02–27 接受日期：2017–04–04

國(guó)家自然科學(xué)基金項(xiàng)目（41230856，41301258）資助。

曹文超（1986—），男，山東鄆城人，博士研究生，主要從事土

壤氮素轉(zhuǎn)化及其環(huán)境效應(yīng)方面的研究。E-mail：caochaoqun66@163.com。 * 通信作者 E-mail：wangjg@cau.edu.cn