生物質(zhì)炭對土壤N2O消耗的影響及其微生物影響機理*

賀超卉, 董文旭, 胡春勝**, 李佳珍,3

生物質(zhì)炭對土壤N2O消耗的影響及其微生物影響機理*

賀超卉1,2,3, 董文旭2, 胡春勝2**, 李佳珍2,3

(1. 中國科學院大學中丹學院 北京 100049; 2. 中國科學院遺傳與發(fā)育生物學研究所農(nóng)業(yè)資源研究中心/河北省土壤生態(tài)學重點實驗室/中國科學院農(nóng)業(yè)水資源重點實驗室 石家莊 050022; 3. 中國科學院大學 北京 100049)

生物質(zhì)炭在溫室氣體減排方面具有很大的發(fā)展前景, 它不僅能實現(xiàn)固碳, 對于在大氣中停留時間長且增溫潛勢大的N2O也能發(fā)揮積極作用。本研究采用室內(nèi)厭氧培養(yǎng)試驗, 按照生物質(zhì)炭與土壤質(zhì)量比(0、1%和5%)加入一定量生物質(zhì)炭, 土壤重量含水率控制在20%。利用Robotized Incubation平臺實時檢測N2O和N2濃度變化, 通過測定土壤中反硝化功能基因豐度(、、)分析生物質(zhì)炭對N2O消耗的影響及其微生物方面的影響機理。結(jié)果表明: 經(jīng)過20 h厭氧培養(yǎng)后, 0生物質(zhì)炭處理的反硝化功能基因豐度(基因拷貝數(shù)?g-1)分別為6.80×107()、5.59×108()和1.22×108()。與0生物質(zhì)炭處理相比, 1%生物質(zhì)炭處理的基因豐度由最初的2.65×108基因拷貝數(shù)?g-1升至7.43×108基因拷貝數(shù)?g-1,基因豐度則提高了一個數(shù)量級, 由4.82×107基因拷貝數(shù)?g-1升至1.50×108基因拷貝數(shù)?g-1, 然而基因豐度并無明顯變化; 5%生物質(zhì)炭處理的反硝化功能基因豐度并未發(fā)生顯著變化。試驗結(jié)束時, 添加生物質(zhì)炭處理的N2/(N2O+N2)比值也明顯高于0生物質(zhì)炭處理。相關性分析結(jié)果表明,基因豐度和基因豐度均與N2O濃度在0.01水平上顯著相關。試驗末期基因豐度和基因豐度均隨著N2O濃度的降低而升高。因此在本試驗中, 添加1%生物質(zhì)炭可顯著提高和基因型反硝化細菌的豐度, 增大N2/(N2O+N2)比值, 促進N2O徹底還原成N2。生物質(zhì)炭對于N2O主要影響機理是增大了可以還原氧化亞氮的細菌活性, 促進完全反硝化。

生物質(zhì)炭; 溫室氣體減排; 土壤微生物; N2O消耗; 反硝化; 基因豐度

全球溫室氣體中約有8%是由N2O組成的[1], 由于廣泛使用合成氮肥, 農(nóng)業(yè)成為全球N2O排放的主要來源, 農(nóng)業(yè)源溫室氣體排放量占全球溫室氣體排放總量的11%, 已超過2020年的排放目標[2-3]。N2O是一種強效溫室氣體, 在大氣中的停留時間長達114年之久, 以100年計, 單位質(zhì)量的N2O增溫潛勢相當于CO2的298倍[4-5]。并且, 排放過多的N2O到大氣中會造成臭氧層破壞, 當其濃度高到一定程度時還會引發(fā)酸雨, 進而影響人類活動。目前大氣N2O濃度已經(jīng)大幅上升, 從前工業(yè)化時代的270 mol·L-1增加到現(xiàn)在的324 mol·L-1[6]。雖然曾有研究表明N2O可以通過非生物氧化還原過程產(chǎn)生[7-8], 但其產(chǎn)生途徑主要是由微生物利用土壤中的氮, 經(jīng)過一系列反應產(chǎn)生的, 包含以下3個反應過程: 硝化、反硝化作用和硝酸鹽異化還原[9]。土壤中超過2/3的N2O均來自于硝化與反硝化過程[10], 每個過程排放的相對貢獻不僅取決于土壤特征(土壤結(jié)構(gòu)、可用碳源、pH、微生物活性), 還與外部環(huán)境條件密切相關(溫度、降雨量等)。N2O產(chǎn)生途徑的復雜性以及空間和時間的不定性給減少土壤N2O排放帶來了巨大的挑戰(zhàn)[11]。

關于生物質(zhì)炭的研究早在19世紀就已經(jīng)開展, 最初是亞馬遜河流域的印第安人在“Terra Preta”上種植農(nóng)作物, 發(fā)現(xiàn)這種土壤可以提高糧食產(chǎn)量。經(jīng)后面研究證實, 這種黑色土壤富含穩(wěn)定的生物質(zhì)炭, 是增加土壤肥力和糧食作物增產(chǎn)的主要原因[12-14]。隨著人們對生物質(zhì)炭的認識不斷深入, 它也逐漸被應用在各個領域的研究當中。生物質(zhì)炭是生物質(zhì)在厭氧或無氧的密閉環(huán)境中高溫熱解(<700 ℃)生成的孔隙豐富、性質(zhì)穩(wěn)定、富含碳素并具有不同程度芳香化的固態(tài)物質(zhì)[15-16]。生物質(zhì)炭能將植物光合作用所固定的有機碳轉(zhuǎn)化為穩(wěn)定的惰性碳, 使其不被微生物迅速礦化, 從而實現(xiàn)固碳減排。因此, 生物質(zhì)炭對緩解全球變暖意義重大。

向土壤中添加生物質(zhì)炭是目前控制土壤N2O排放的重要措施, 首次關于生物質(zhì)炭可減少土壤N2O排放的報道是溫室試驗, 研究發(fā)現(xiàn), 向種有黃豆()的土壤使用生物質(zhì)炭后, N2O排放可減少50%, 而對于腐殖生臂形草()草地, 減排效率則高達80%[17]。此后, 利用生物質(zhì)炭減少土壤N2O排放成為研究的熱點, 并且眾多研究者們也根據(jù)試驗結(jié)果提出了不同的假設來解釋這一現(xiàn)象。比如, 生物質(zhì)炭可加強土壤的通氣性, 增大土壤pH, 有利于土壤固氮, 可與土壤中的有機碳和氮反應, 改良酶活性等。然而, 各種機理都存在一定的爭議性。迄今為止, 關于生物質(zhì)炭抑制農(nóng)田N2O排放的報道及相關研究日漸增加, 由于試驗環(huán)境、土壤特性和生物質(zhì)炭的制作條件不盡相同, 因此得出的結(jié)論也存在很大的差異。并且眾多研究中的關注點都是N2O排放, 少有針對N2O從土壤排放后的消耗進行深入研究。所以本研究探究了添加不同量生物質(zhì)炭對N2O排放的影響, 同時通過檢測不同處理中土壤反硝化功能基因、和的豐度以分析生物質(zhì)炭的微生物作用機理。旨在研究生物質(zhì)炭影響土壤N2O排放的基礎上進一步探討排放之后的N2O氣體在生物質(zhì)炭改良后的土壤中的微生物消耗機理, 以此從機理層面驗證生物質(zhì)炭對N2O減排的積極作用及其環(huán)境效益。

1 材料與方法

1.1 供試土壤與生物質(zhì)炭

選用表層0~10 cm的潮褐土(中國科學院欒城農(nóng)業(yè)生態(tài)系統(tǒng)試驗站, 37°53′N, 114°41′E), 自然風干后挑選出土壤中的植物殘渣和石頭等雜物, 過2 mm篩后避光保存以備用。試驗所用生物質(zhì)炭購買自陜西億鑫生物能源科技開發(fā)有限公司, 最高熱解溫度(HTT)為520 ℃, 粒徑≤2 mm, 以便與土壤充分混勻。

土壤pH用電位法測定。稱取10 g風干土樣于50 mL高型燒杯中, 加入25 mL蒸餾水, 用玻璃棒攪拌1~2 min, 靜置30 min, 然后用便攜式pH計(METTLER TOLEDO)測定上層清液的pH。土壤總碳和總氮含量采用元素分析儀(vario MACRO cube; Elementar, Germany)測定。土壤有機質(zhì)的測定采用重鉻酸鉀容量法-稀釋熱法。土壤容重采用環(huán)刀法測定。土壤孔隙度(t)計算如下[18]:

t=1-b/d(1)

式中:b為土壤容重,d為土壤比重。

根據(jù)上述方法, 本試驗用土pH 為7.61, 土壤有機碳含量為9.3 g?kg-1, 全碳和全氮分別為14.9 g?kg-1和1.0 g?kg-1, 碳氮比是9.30。土壤容重為1.30 g?cm-3, 孔隙度為50.94%。

1.2 試驗設計

本試驗為室內(nèi)厭氧培養(yǎng)(含氧量為0), 試驗采用120 mL培養(yǎng)瓶, 所加土壤質(zhì)量為10 g, 按照生物質(zhì)炭與土壤質(zhì)量比加入一定量生物質(zhì)炭[不添加生物質(zhì)炭(0BC)、添加1%生物質(zhì)炭(1%BC)和添加5%生物質(zhì)炭(5%BC)], 并將土壤含水率調(diào)節(jié)為20%, 每個處理設置3個重復。為防止小瓶漏氣, 準備工作結(jié)束后蓋上橡膠蓋, 并用鋁蓋壓緊密封。隨后用真空抽氣泵系統(tǒng)將每個小瓶中的空氣置換為氦氣, 制造厭氧環(huán)境。將小瓶內(nèi)部氣壓與大氣壓平衡后, 使用注射器向培養(yǎng)瓶內(nèi)注入1 mL的純N2O氣體(99.8%), 利用Robotized Incubation平臺實時測定培養(yǎng)瓶內(nèi)的N2O和N2濃度變化。為了比較滅菌與不滅菌之間的效果差異, 另外設置了兩組滅菌試驗, 滅菌溫度為130 ℃, 時長為1 h, 其生物質(zhì)炭添加量為0和5%, 其他條件和步驟均與不滅菌處理保持一致。試驗共進行20 h, 試驗結(jié)束后取各重復的土壤樣品, 用于后續(xù)的測定。

1.3 土壤NH4+-N和NO3–-N測定

試驗結(jié)束后, 準確稱取每個重復的10.00 g土樣, 加入50 mL 2 mol?L-1KCl溶液浸提, 在震蕩機上振蕩1 h, 取出靜置并過濾。浸提液中的NO3–-N使用紫外分光光度計(UV-2450, Shimadzu, Japan)測定, NH4+-N使用全自動化學分析儀(SmartChem 140, AMS Alliace, France)測定。

1.4 土壤微生物總DNA提取

為了探究生物質(zhì)炭對土壤N2O消耗的影響及其微生物方面的影響機制, 分別提取了培養(yǎng)前的干土和培養(yǎng)后各處理的土壤樣品的DNA, 提取方法按照FastDNA Spin Kit for Soil (MP biomedicals, USA)試劑盒的操作手冊進行。提取后用微量紫外-可見光分光光度計(NanoDrop ND-2000c Technologies, Wilmington, DE)測定其濃度, 初步判斷土壤微生物總DNA提取效果。隨后將提取成功的土壤DNA保存至-20 ℃條件下, 待定量PCR擴增時再取出依次將濃度稀釋至20 ng?μL-1左右。

1.5 實時熒光定量PCR

本試驗主要分析的基因是土壤中亞硝酸鹽還原酶編碼基因()和N2O還原酶編碼基因(), 基因豐度以每種基因的拷貝數(shù)?g-1(干土)表示。反硝化功能基因熒光定量PCR反應體系為20 μL, 包含10 μL 2 × TB Green Premix Ex Taq (Takara Biotech, Dalian, China)、各0.5 μL的上游引物和下游引物(10 μmol?L-1)、8 μL超純水和1 μL稀釋的DNA模板。每種基因?qū)囊锓謩e是F1aCu:R3Cu ()[19], cd3aF:R3cd ()[20-21], nosZ- F:nosZ-1622R ()[20,22]。分別以含有亞硝酸鹽還原酶基因()和一氧化二氮還原酶基因()的重組pGEM?-T裁體作為標準質(zhì)粒, 然后計算出標準質(zhì)粒的拷貝數(shù), 按照10倍濃度梯度進行稀釋, 并以108~102濃度梯度的標準質(zhì)粒作為模板, 同時設置3個陰性對照, 和DNA模板同時在熒光定量PCR儀(CFX Connect?, Bio-Rad, USA)進行定量PCR擴增。擴增程序為: 95 ℃預變性2 min, 95 ℃變性30 s, 57 ℃()、56.8 ℃()、59 ℃()退火40 s, 72 ℃延伸30 s, 40個循環(huán)。

1.6 數(shù)據(jù)處理與分析

所有數(shù)據(jù)均使用EXCEL 2016和IBM SPSS Statistics 19.0 (SPSS Inc., USA)進行處理與分析, 在SPSS中采用單因素方差分析, 處理間差異用Duncan法進行多重比較, 相關性分析使用Pearson法。繪圖所用軟件為EXCEL 2016和OriginPro 9.0。

2 結(jié)果與分析

2.1 N2O和N2濃度變化

在厭氧條件下經(jīng)過20 h培養(yǎng)后, 滅菌處理后的土壤中N2O濃度基本保持不變, 顯著高于未經(jīng)滅菌的土壤(圖1), 表明微生物在N2O和N2的轉(zhuǎn)換過程充當重要角色。添加生物質(zhì)炭時, N2O濃度的下降速率和N2的生成速率大于0BC處理, 而且在室內(nèi)培養(yǎng)20 h后, 添加1%和5%生物質(zhì)炭處理的N2O濃度由最初的近200 μmol?L-1基本降為零, 表明生物質(zhì)炭可以促進土壤N2O消耗過程。在試驗末1%和5%生物質(zhì)炭處理的N2濃度稍高于0BC處理, 不過3個處理間的差異不大。由圖1c可知, N2/(N2O+N2)及N2O/ (N2O+N2)變化趨勢基本一致, 意味著在本厭氧試驗中, 注入的N2O主要是被微生物通過反硝化過程轉(zhuǎn)化為N2。并且隨著培養(yǎng)時間的延長, 不同處理之間的差異也逐漸變大。雖然滅菌處理后N2濃度仍有小幅度上升, 這可能是土壤及土壤與生物質(zhì)炭的混合物中存在某些化學還原過程, 將少量的N2O還原成了N2。

圖1 厭氧條件下添加生物質(zhì)炭對土壤N2O(a)、N2(b)濃度及其所占比例(c)的影響[圖c中, 實線為N2/(N2O+N2)的比值, 虛線為N2O/(N2O+N2)的比值]

0BC: 0生物質(zhì)炭處理; 1%BC: 1%生物質(zhì)炭處理; 5%BC: 5%生物質(zhì)炭處理; 0BCS: 0生物質(zhì)炭+高壓蒸汽滅菌處理; 5%BCS: 5%生物質(zhì)炭+高壓蒸汽滅菌處理。0BC: 0 biochar application; 1%BC: 1% biochar application; 5%BC: 5% biochar application; 0BCS: 0 biochar application and autoclaving; 5%BCS: 5% biochar application and autoclaving.

2.2 土壤NH4+-N和NO3–-N含量變化

與試驗初始含量相比, 所有處理的土壤NH4+-N含量都顯著升高, 由最初的8.12 mg·kg-1左右升至19.69 mg·kg-1(0BC)、18.72 mg·kg-1(1%BC)和13.97 mg·kg-1(5%BC), 而NO3–-N含量則由6.11 mg·kg-1降至0.1 mg·kg-1左右(表1)。很明顯, 此時占主導地位的是反硝化過程, 試驗末反硝化過程的底物NO3–-N基本被完全消耗了。隨著生物質(zhì)炭添加量的增加, 試驗末NH4+-N含量呈現(xiàn)由高到低的趨勢, NO3–-N含量大幅度下降, 這可能是在厭氧條件下, 反硝化作用強于硝化過程, 但是生物質(zhì)炭本身經(jīng)過高溫裂解后可能含有某些有毒有機物, 會抑制微生物生長繁殖, 從而減弱反硝化作用, 使得最后1%和5%生物質(zhì)炭處理的NO3–-N含量稍高于0BC處理。此外, 土壤礦化作用及硝酸鹽異化還原成銨(DNRA)的過程也會分別增加NH4+-N和減少NO3–-N含量。

表1 厭氧條件下添加生物質(zhì)炭對試驗前和試驗末土壤NH4+-N和NO3–-N含量的影響

Table 1 Impact of biochar on initial and final soil NH4+-N and NO3–-N contents under anaerobic condition mg?kg-1

0BC: 0生物質(zhì)炭處理; 1%BC: 1%生物質(zhì)炭處理; 5%BC: 5%生物質(zhì)炭處理。數(shù)據(jù)為3次重復的平均值加減標準誤。同一行內(nèi)不同字母表示在0.05水平下差異顯著。0BC: 0 biochar application; 1%BC: 1% biochar application; 5%BC: 5% biochar application. Values are means ± S.E. (= 3). Different letters within a row indicate significant differences at0.05.

2.3 反硝化功能基因豐度

培養(yǎng)前后每克干土中反硝化功能基因的拷貝數(shù)變化如圖2所示。培養(yǎng)試驗開始前, 3種反硝化功能基因豐度(基因拷貝數(shù)?g-1)分別為5.59×107()、2.65×108()和4.82×107()。試驗開始時基因豐度比及大一個數(shù)量級, 試驗前后土壤中的基因豐度變化也最為顯著。經(jīng)過20 h厭氧培養(yǎng)后, 0、1%和5%生物質(zhì)炭處理的基因豐度變化不大, 與0BC處理相比, 1%BC和5%BC處理的基因豐度稍有下降, 但是統(tǒng)計學上變化并不顯著。而試驗前后及基因豐度均顯著提高一倍以上, 其中以添加1%生物質(zhì)炭時基因豐度變化最明顯。在1%BC處理中,基因豐度由最初的5.59×107基因拷貝數(shù)?g-1提高至6.24×107基因拷貝數(shù)?g-1,基因豐度則由2.65×108基因拷貝數(shù)?g-1升至7.43×108拷貝數(shù)?g-1,基因豐度提高了一個數(shù)量級, 由4.82×107基因拷貝數(shù)?g-1升至1.50×108基因拷貝數(shù)?g-1。

圖2 厭氧條件下添加生物質(zhì)炭對土壤反硝化功能基因豐度的影響

dry soil: 培養(yǎng)前土壤; 0BC: 0生物質(zhì)炭處理; 1%BC: 1%生物質(zhì)炭處理; 5%BC: 5%生物質(zhì)炭處理。不同字母表示在<0.05水平下差異顯著。dry soil: soil before the experiment; 0BC: 0 biochar application; 1%BC: 1% biochar application; 5%BC: 5% biochar application. Different letters indicate significant differences at0.05.

為進一步了解試驗前后反硝化細菌與N2O濃度之間的關系, 對所有處理中的基因豐度與N2O濃度數(shù)據(jù)匯總并進行了相關性分析, 匯總結(jié)果如圖3所示。字母“b”表示試驗前, 字母“a”表示試驗后。試驗初期由于注入了1 mL 99.8%的N2O氣體, 培養(yǎng)瓶內(nèi)N2O濃度較大, 在試驗結(jié)束時, 3個生物質(zhì)炭處理的N2O濃度都明顯下降, 具體趨勢如圖1a所示。而隨著N2O濃度的下降各種反硝化菌基因豐度也出現(xiàn)不同程度的上升, 其中基因豐度變化最為明顯。相關性分析顯示,基因豐度和基因豐度均與N2O濃度在0.01水平上顯著相關, 但是與基因豐度之間無顯著相關性。

3 討論

亞硝酸鹽還原酶(Nir)和氧化亞氮還原酶(Nos)是反硝化過程的關鍵酶, 其中亞硝酸鹽還原酶共有兩種類型: 一種是由基因編碼的[23], 還有一種則是由基因編碼的[24],和也是反硝化功能基因中被研究最多的基因[25]。由基因編碼的氧化亞氮還原酶是反硝化作用的最后一步, 將N2O催化還原為N2。因為在土壤pH≤6.1時, 微生物很難產(chǎn)生氧化亞氮還原酶[26], 且對O2十分敏感[27-28]。故本試驗所用土壤pH為7.61, 培養(yǎng)環(huán)境為厭氧(充滿氦氣), 排除pH和O2對生物質(zhì)炭作用的影響。

圖3 厭氧條件下土壤反硝化N2O濃度和反硝化功能基因豐度關系圖

空心符號表示試驗前(a)的基因豐度, 實心符號表示試驗后(b)的基因豐度。Hollow symbols indicate gene abundance before the trail (a), filled symbols indicate gene abundance after the trial (b).

本試驗結(jié)果顯示向土壤中施加少量生物質(zhì)炭可促進N2O向N2的轉(zhuǎn)化過程, 增大和反硝化基因豐度。經(jīng)過20 h厭氧培養(yǎng)后, 3種不同生物質(zhì)炭處理的及基因拷貝數(shù)均顯著提高一倍以上, 然而基因豐度稍有上升, 但是統(tǒng)計學上變化并不顯著。在添加1%生物質(zhì)炭處理中,基因豐度變化不明顯,基因豐度則提高了一個數(shù)量級。與0BC處理相比, 添加生物質(zhì)炭處理可抑制N2O濃度, 并且反硝化功能基因豐度(和)均高于0BC處理。除此之外, 生物質(zhì)炭對N2O的抑制作用在很多學術報道中都有所體現(xiàn)[18,29-32]。Anderson等[33]研究表明, 向土壤中施加松木生物質(zhì)炭后可增大反硝化菌(如)的數(shù)量; Chen等[34]發(fā)現(xiàn)在水稻()田里施加生物質(zhì)炭同樣可以促進的生長; 但是也有研究者提出添加生物質(zhì)炭并不會影響基因豐度[35-36]。不過, 生物質(zhì)炭對N2O的具體影響還取決于試驗所處的環(huán)境及供試土壤和生物質(zhì)炭類型, 試驗條件有利于硝化過程的進行或者使用糞肥生產(chǎn)的生物質(zhì)炭則對N2O排放無抑制作用。在本厭氧培養(yǎng)試驗中, 向土壤中添加生物質(zhì)炭可促進N2O轉(zhuǎn)化為N2, 顯著提高和基因型反硝化細菌的豐度, 但是卻降低了基因豐度, 雖然影響并不顯著。這說明生物質(zhì)炭主要是通過提高基因豐度促進完全反硝化過程, 通過微生物消耗途徑使得注入培養(yǎng)瓶內(nèi)的N2O被還原成N2。有研究表明, 在堆肥過程中添加生物質(zhì)炭可顯著增大和基因拷貝數(shù), 降低基因豐度從而抑制N2O排放, 且在控制N2O排放中起關鍵作用的是和基因[37], 這與本試驗相關性分析得出的結(jié)果相吻合,基因豐度和基因豐度均與N2O濃度在0.01水平上顯著相關。在試驗后期, 隨著N2O濃度的降低,基因豐度和基因豐度顯著升高, 以基因豐度變化最為明顯。此外, 在砂土(Tenosol)[38]和粉黏壤土[39]中施加生物質(zhì)炭也可提高基因豐度。Castaldi等[40]和Xu等[41]通過定量研究反硝化酶活性(DEA), 指出生物質(zhì)炭可大幅度提升DEA速率。Cayuela等[42]通過研究15種農(nóng)田土壤發(fā)現(xiàn)與本試驗相近的結(jié)果, 即添加生物質(zhì)炭后每種土壤的N2O/(N2O+N2)比值都有所下降; 作者提出生物質(zhì)炭在增大基因豐度的同時, 還可以作為傳遞電子的介質(zhì), 有利于電子與反硝化微生物之間的傳遞, 促進N2O還原為N2。不過, 生物質(zhì)炭對N2O的抑制機理和整個微生物過程還不是很明確, 因為它本身會釋放出多環(huán)芳烴(PAHs)等有害物質(zhì), 抑制硝化和反硝化過程[43-44]。但是Alburquerque等[45]研究結(jié)果卻又與此相悖, 作者提出高濃度的PAHs(萘、菲和芘等)并不會減弱生物質(zhì)炭對N2O排放的抑制作用。

在前人的研究中就生物質(zhì)炭的作用機理也做出了很多假設: 降低反硝化速率[18,46], 促進完全反硝化[41,47], 或二者結(jié)合[48-49]。降低反硝化速率主要是因為生物質(zhì)炭可改善土壤通氣性或減少無機氮等底物, 從而抑制反硝化過程。本試驗結(jié)果與van Zwieten等[5]和Harter等[47]在石灰性土壤中施加生物質(zhì)炭的研究一致, 作者提出的機理主要是因為生物質(zhì)炭提高了土壤pH進而增大了還原N2O的細菌活性和基因表達, 促進完全反硝化。但是生物質(zhì)炭增大土壤pH從而降低N2O排放并不是唯一機理[50]。有研究發(fā)現(xiàn), pH增大后甚至還可能促進硝化作用和N2O排放[51]。Shan等[52]分別向酸性土和堿性土中加入生物質(zhì)炭, 發(fā)現(xiàn)兩種土壤的pH都有所上升, 且增大了堿性土中的基因豐度, 但是對酸性土中的基因并無影響。生物質(zhì)炭含有的易分解有機碳(如可溶性有機碳等)含量較高時, 也可增強基因型的微生物活性, 促進N2O生物消耗過程[53]。向土壤中添加生物質(zhì)炭后, 它本身含有的碳元素也會影響土著微生物群落結(jié)構(gòu)。而且基因型微生物對外源加入的碳十分敏感, 極易受到影響[54]。值得一提的是, 本研究中添加1%生物質(zhì)炭處理對功能基因的影響最為明顯, 而添加5%生物質(zhì)炭處理與不添加對照間無顯著差異。這可能與生物質(zhì)炭含有的某些化學物質(zhì)有關: 低添加量時, 可促進反硝化菌生長; 高劑量時則表現(xiàn)出一定的毒物效應, 抑制反硝化基因表達。這種雙向反應也被稱為hormesis效應[55], 并且生物質(zhì)炭對微生物的影響并不是簡單的線性關系, 而存在一個最優(yōu)劑量[56]。在本試驗中, 生物質(zhì)炭的最佳添加量為土壤質(zhì)量的1%。

4 結(jié)論

在厭氧培養(yǎng)試驗中, 褐土中添加生物質(zhì)炭顯著提高了和基因型反硝化細菌的豐度, 促進N2O徹底還原成N2。生物質(zhì)炭對于N2O主要影響機理是增大了還原氧化亞氮的細菌活性, 促進完全反硝化。與添加1%生物質(zhì)炭相比, 添加5%生物質(zhì)炭對N2O的影響并不明顯, 原因可能是生物質(zhì)炭含有的某些化學物質(zhì)在高濃度時具有一定的生物毒性。

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Biochar’s effect on soil N2O consumption and the microbial mechanism*

HE Chaohui1,2,3, DONG Wenxu2, HU Chunsheng2**, LI Jiazhen2,3

(1. Sino-Danish College of University of Chinese Academy of Sciences, Beijing 100049, China; 2. Center for Agricultural Resources Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences / Hebei Laboratory of Soil Ecology / Key Laboratory of Agricultural Water Resources, Chinese Academy of Sciences, Shijiazhuang050022, China; 3. University of Chinese Academy of Sciences, Beijing 100049, China)

Biochar is a promising material for mitigating greenhouse gas emissions. In addition to carbon sequestration, it has positive effect on the ozone-depleting gas nitrous oxide (N2O), which is with long residence time and strong warming potential. In this research effort, an anaerobic incubation experiment was conducted. Three treatments with different biochar application rates were set, taking account of biochar to soil ratio (/): 0 (0BC), 1% (1%BC) and 5% (5%BC). Soil gravimetric water content was controlled at 20%. According to the robotized incubation platform providing real-time determination of N2O and N2concentrations and soil denitrification functional gene abundance measurement, we analyzed the impact of biochar on N2O consumption and biological mechanisms. The main results indicated that after a 20-hour anaerobic incubation, the denitrification functional gene abundance of 0BC treatment was 6.80×107(), 5.59×108(), 1.22×108() gene copies per gram soil, respectively. Compared with 0BC treatment, thegene abundance of 1%BC treatment increased from the initial 2.65×108to 7.43×108gene copies per gram soil, while, thegene abundance increased by an order of magnitude from 4.82×107to 1.50×108gene copies per gram soil. However, there was no significant change ingene abundance. And the denitrification functional gene abundance of 5%BC treatment did not show marked variations. In conclusion, the N2/(N2O+N2) ratio of treatments with biochar application was clearly higher than 0BC treatment. The results of correlation analysis showed thatandgene abundance was significantly correlated with the N2O concentration at 0.01 level, and the abundance ofandgenes all increased as N2O concentration declined at the end of the experiment. Therefore, in the present trial, a 1% biochar addition significantly increased the abundance of denitrifying bacteria withandgenotypes and N2/(N2O+N2) ratio, and promoted the complete reduction of N2O to N2. The main mechanism of the biochar effect on N2O emission was the enhanced reduction activities and gene expression of-containing microorganisms, resulting in complete denitrification.

Biochar; Greenhouse gases emission reduction; Soil microbe; N2O consumption; Denitrification; Gene abundance

, E-mail: cshu@sjziam.ac.cn

Mar. 8, 2019;

Apr. 20, 2019

S154.1; S154.36

2096-6237(2019)09-1301-08

10.13930/j.cnki.cjea.190175

* 國家重點研發(fā)計劃項目(2017YFD0800601)和中國科學院重點項目(ZDRW-ZS-2016-5-1)資助

胡春勝, 主要研究方向為農(nóng)田生態(tài)系統(tǒng)碳、氮、水循環(huán)及土壤生態(tài)過程。E-mail: cshu@sjziam.ac.cn 賀超卉, 主要研究方向為土壤氮循環(huán)過程。E-mail: Chaohui_He@outlook.com

2019-03-08

2019-04-20

* This study was supported by the National Key Research and Development Project of China (2017YFD0800601) and the Key Program of Chinese Academy of Sciences (ZDRW-ZS-2016-5-1).

賀超卉, 董文旭, 胡春勝, 李佳珍. 生物質(zhì)炭對土壤N2O消耗的影響及其微生物影響機理[J]. 中國生態(tài)農(nóng)業(yè)學報(中英文), 2019, 27(9): 1301-1308

HE C H, DONG W X, HU C S, LI J Z.Biochar’s effect on soil N2O consumption and the microbial mechanism[J]. Chinese Journal of Eco-Agriculture, 2019, 27(9): 1301-1308