强还原处理中pH对硫酸根去除效果及产物的影响①

孟天竹，朱同彬，张金波,2,3，蔡祖聪,2,3*

(1 南京师范大学地理科学学院，南京 210023；2 江苏省物质循环与污染控制重点实验室，南京 210023；3 江苏省地理信息资源开发与利用协同创新中心，南京 210023)



强还原处理中pH对硫酸根去除效果及产物的影响①

孟天竹1，朱同彬1，张金波1,2,3，蔡祖聪1,2,3*

(1 南京师范大学地理科学学院，南京 210023；2 江苏省物质循环与污染控制重点实验室，南京 210023；3 江苏省地理信息资源开发与利用协同创新中心，南京 210023)

摘 要：强还原灭菌法(reductive soil disinfestation,RSD)，即淹水加有机物料创造强还原条件，可以有效修复退化土壤，但对硫酸根去除效率低。为探索RSD处理时pH对去除效果的影响，本试验选取积累严重的退化蔬菜地土壤设置5个处理：淹水(CK)；淹水+紫花苜蓿(RSD0)；淹水+紫花苜蓿+石灰，调节土壤至不同pH(分别标记为RSD1、RSD2和RSD3)。培养结束后CK处理中含量仍高达S 691 mg/kg，显著高于RSD处理中含量。在RSD处理中，含量随着pH的提高而下降，但其他形态硫含量显著升高，且施加石灰处理的土壤中总硫含量高于不加石灰的处理。由此说明，在强还原处理时施用石灰生成硫酸钙沉淀，可有效降低含量，但不利于降低总硫含量。

关键词：退化土壤；RSD；石灰；

因复种指数高、受季节影响小等优点，近30年来，我国的设施蔬菜栽培种植面积持续增长，至2013年已达到165万hm2，占农田面积的14.5%[1]。值得注意的是，设施蔬菜种植过程中施用大量化肥，易引起土壤酸化、盐渍化、硝态氮及硫酸根大量积累和土传病害的发生[2-5]，导致土壤退化，影响设施蔬菜种植的可持续发展。

近年来，强还原土壤灭菌法(reductive soil disinfestation,RSD)作为一种环境友好型修复退化土壤的方法在美国、荷兰和日本被广泛采用[4,6-7]，该方法包括以下步骤：①向土壤中添加易降解有机物料，如：植物秸秆和绿肥等；②淹水或灌溉至土壤水分饱和；③覆膜以隔绝土壤与大气的气体交换。RSD方法创造的强还原条件可以杀灭土壤中的土传病原菌和根结线虫[7-8]，并可有效改善土壤结构[9-10]，去除土壤中累积的硝态氮(NO3-)，降低土壤电导率(EC)，提高酸化土壤的pH[5]，因而不仅是有效的土壤灭菌方法，而且也是有效地改良退化土壤理化性质的方法[11-12]。

但是，有研究指出硫酸还原菌在pH为中性(pH = 6～8)的条件下生长速度最快且浓度显著下降[16-19]，而在酸性条件下硫酸还原菌生长几乎可以忽略不计[20-21]。一般情况下，退化设施蔬菜地土壤酸化严重(pH = 4～5)，因此，调节土壤pH至中性，可能会促进硫酸还原菌的活性，提高的去除效率。工业废水(如矿业、食品加工业和造纸业等)处理过程中，为提高硫酸根还原菌对的去除效率，通常会施加NaOH、Ca(OH)2或CaO提高酸性废水pH[16,22]。施撒石灰是一种常见的提高土壤pH的农业措施，本研究选取累积的退化设施蔬菜地土壤，采用RSD处理，同时添加石灰调节土壤至不同pH，研究pH对去除效率及其产物的影响。

1 材料与方法

1.1 试验材料

供试土壤采自安徽省乌江镇和县郊区(31°51′N,118°45′E)退化严重的设施蔬菜地。该地已种植大棚作物近10年，每年种植2～3茬(辣椒、西红柿、茄子、甜瓜和四季豆)。土壤类型为普通简育水耕人为土。在大棚内随机选取10个样地采集土样，采样深度为0～20 cm。剔除土壤石块和植物根系后，将新鲜土壤混合均匀，过2 mm筛，用塑封袋密封，于4℃下保存。供试土壤pH 4.4，电导率0.74 mS/cm，总碳15.9 g/kg，总氮2.08 g/kg，总硫1.02 g/kg，28.9 mg/kg，147 mg/kg939 mg/kg，土壤体积质量 1.07 g/cm3。

RSD处理以紫花苜蓿为有机物料。供试紫花苜蓿购于山东省滨州市无棣县，60oC 烘干后粉碎，过0.25 mm筛。紫花苜蓿总碳含量为549 g/kg，总氮20.3 g/kg，总硫1.63 g/kg。

1.2 试验设计

试验设置5个处理：对照(淹水，CK)；淹水+紫花苜蓿(RSD0)；淹水+紫花苜蓿+石灰，石灰添加量分别为1.88、3.58和5.28 g/kg干土，调节土壤pH至6.7、7.7和8.4，分别标记为RSD1、RSD2和RSD3。紫花苜蓿添加量为4.67 g/kg干土，折合大田施用量为9.8 t/hm2，随紫花苜蓿添加到土壤的硫为10.9 mg/kg。

称取相当于210 g干土重的新鲜土壤与紫花苜蓿和不同用量的石灰充分混匀后装入PVC柱中(直径5 cm，高15 cm)。土层厚度为10 cm，体积质量1.07 g/cm3。按水土比1︰1(质量比)加入蒸馏水，形成厚约为1 cm的水层。35℃ 恒温培养箱中培养360 h。分别在第24、48、72、120、240和360 h测定土壤氧化还原电位和pH，培养360 h后破坏性采样。采样时先打开PVC柱底的阀门将柱中的自由水排出，待水排干后(约10 min)关上阀门，将柱中土壤充分混匀。其中，一部分土样用于土壤含水量测定，剩下的土样测pH，EC，和总硫含量。定量淋溶液的体积后，过滤，分析和浓度。

1.3 测定方法

土壤pH(水土比2.5︰1)采用pH计(Mettler S220，瑞士)测定，土壤Eh用ORP检测器(Mettler S220，瑞士)测定，土壤EC值(水土比5︰1)采用电导率仪(KangYi Corp.，中国)测定，土壤总碳和总氮采用SerCon 20-22同位素质谱仪(SerCon Ltd，Crewe，美国)测定。土壤用2 mol/L KCl(水土比5︰1)浸提，25℃、300 r/min下震荡1 h，定量滤纸过滤，采用流动分析仪测定浸提液中的含量(Skalar，Breda，荷兰)。土壤用0.016 mol/L KH2PO4(水土比5︰1)浸提，25℃、300 r/min下震荡1 h，8 000 r/min离心10 min，取上清液过0.45 μm 滤膜后用液相色谱测定滤液中浓度(Thermo Dionex ICS- 1100，美国)。土壤在60℃下烘干研磨过100目筛，采用元素分析仪(Elementar，Vario MAX CNS，德国)测定总硫含量。

1.4 数据处理

C(N)= V × L(N)/ w + C(N)R

采用SPSS 17.0软件中Pearson相关系数进行相关性分析，采用方差分析和Duncan法进行处理间差异显著性检验(α = 0.05)。

2 结果与分析

2.1 土壤pH、EC 和Eh变化

培养结束后，各处理土壤的pH均显著高于土壤初始pH(4.38)(P＜0.05，表1)。不同处理的土壤pH在处理过程中的变化趋势不同(图1)。CK和RSD0处理的土壤pH分别升高至4.91和5.85，但RSD1、RSD2和RSD3处理pH分别降至6.55、7.07和7.43，各处理间pH差异显著(P＜0.05)。同时，各处理土壤EC值显著下降，CK、RSD0、RSD1、RSD2和RSD3处理EC值由初始的0.74 mS/cm分别降至0.53、0.39、0.31、0.29和0.27 mS/cm。

在培养过程中各处理的土壤Eh均下降，但下降模式和速率不同(图2)，表现为pH越高，Eh下降速率越快。CK处理的土壤Eh缓慢下降，培养结束时仅从初期的412 mV下降到340 mV，RSD0处理的土壤Eh下降到 -100 mV以下需要240 h，而RSD+石灰处理仅需48 h，而后基本维持在 -100 mV以下(图 2)。培养结束时的土壤Eh与土壤pH呈显著负相关关系(r = -0.85，P＜0.01)，与EC呈显著正相关关系(r = 0.93，P＜0.01，表 2)。

表1　培养结束后不同处理的土壤理化性质Table 1 Soil properties under different treatments at the end of incubation

图1　淹水条件下不同处理土壤pH的变化Fig.1 Changes in soil pH values under different treatments during 360 h incubation period

图2　淹水条件下不同处理土壤Eh的变化Fig.2 Changes in soil Eh values under different treatments during 360 h incubation period

表2　培养结束时不同土壤理化性质之间的相关系数Table 2 Correlation coefficients between different soil properties at the end of incubation

2.2 土壤氮、硫含量的变化

淹水添加紫花苜蓿可以有效去除土壤中累积的硝态氮。培养360 h后，CK处理中含量从初始的147 mg/kg下降至30.3 mg/kg，而RSD处理均降至1 mg/kg以下(表1)。含量变化与相反，培养结束后CK、RSD0、RSD1、RSD2和RSD3处理中含量分别升至56.5、50.4、55.9、72.4和81.0 mg/kg，比初始值28.9 mg/kg均有显著提高(P＜0.05，表1)。

3 讨论

与过去的研究结果相一致[5]，本试验中RSD处理快速降低土壤Eh且完全消除了土壤中累积的单独淹水处理(CK)土壤Eh下降非常缓慢(图1)，硝态氮也未能完全去除(表1)，可能与供试土壤易降解有机碳不足有关，因而不能有效还原硝态氮及其他氧化物质。淹水条件下添加苜蓿，提供了大量的易降解有机物，刺激了土壤微生物活性，造成了Eh的快速下降和有效去除。由于和其他氧化物质还原消耗大量H+，使土壤pH显著上升(表1)，因此RSD可以有效地提高酸化土壤的pH。在RSD处理过程中，由于有机氮的矿化和硝态氮异化还原为铵[23-24]，土壤中含量显著升高(表1)。文献资料表明，导致设施蔬菜地土壤次生盐渍化的盐主要为硫酸盐和硝酸盐[3]。RSD处理几乎全部去除了硝酸根，大幅度降低了硫酸根含量，所以，经RSD处理后土壤的次生盐渍化程度下降，表现为EC值的降低(表1)。

RSD处理后，土壤回复到落干状态，厌氧条件下转化生成的有机硫和硫化物可能会再次矿化或氧化生成土壤再次酸化后硫酸钙会解离成所以，RSD处理时，添加石灰对的转化产物的长期影响还需要进一步试验验证。添加石灰有利于增加和提高微生物的数量和活性，从而消耗更多的氧气，促进了RSD处理土壤Eh的下降(图2)。快速强还原条件可以有效杀灭病原菌菌丝及孢子，有利于增强RSD的杀菌效果[34-36]。此外，RSD处理时添加石灰提高了处理后的pH，为有益微生物的生长提供了更好的环境条件，不利于偏好酸性环境的病原菌的生长。然而，有机物料厌氧降解产生有机酸是RSD方法杀灭病原菌的一个重要机制[37-39]，只有未电离状态的有机酸可穿透细胞膜，具有杀菌作用，而未电离状态的有机酸含量和土壤pH呈负相关关系[40]，施加石灰会降低未电离状态的有机酸。因此，施加石灰是否可以增强RSD方法的灭菌效果需要进一步研究。由于添加石灰的正反效应可能同时发生，RSD处理时添加石灰的实际效果需要田间试验的验证。

4 结论

RSD处理时施加石灰进一步地改善土壤酸化和盐渍化。同时，施加石灰可以有效降低土壤浓度，但减少的大部分并未从土壤中脱除而是转化为其他硫形态继续残留在土壤中。施加石灰对强还原方法修复退化土壤的实际效果需要田间试验的验证。

参考文献：

[1]Food and Agriculture Organization(FAO).United Nations(2013)FAO statistical databases[R].2013.http://faostat.fao.org/default.aspx.

[2]Blok W J,Lamers J G,Termorshuizen A J,et al.Control of soilborne plant pathogens by incorporating fresh organic amendments followed by tarping[J].The American Phytopathological Society,1999,90:253-259

[3]Cao Z H,Huang J F,Zhang C S,et al.Soil quality evolution after land use change from paddy soil to vegetable land[J].Environmental Geochemistry and Health,2004,26:97-103

[4]Messiha N A S,Diepeningen A D,Wenneker M,et al.Biological soil disinfestation(RSD),a new control method for potato brown rot,caused by Ralstonia solanacearum race 3 biovar 2[J].European Journal of Plant Pathology,2007,117:403-415

[5]朱同彬,张金波,蔡祖聪.淹水条件下添加有机物料对蔬菜地土壤硝态氮及氮素气体排放的影响[J].应用生态学报,2012,23:109-114

[6]Momma N.Biological soil disinfestation(BSD)of soilborne pathogens and its possible mechanisms[J].Japan International Research Center for Agricultural Sciences,2008,42:7-12

[7]Butler D M,Rosskopf E N,Kokalis-Burelle N,et al.Exploring warm-season cover crops as carbon sources for anaerobic soil disinfestation(ASD)[J].Plant and Soil,2011,355:149-165

[8]Lamers J G,Wanten P J,Blok W J,et al.Biological soil disinfestation:A safe and effective approach for controlling soilborne pests and diseases[J].Agroindustria,2004,3:289-291

[9]Akhtara M,Malik A.Roles of organic soil amendments and soil organisms in the biological control of plant-parasitic nematodes:A review[J].Bioresource Technology,2000,74:35-47

[10]Oka Y.Mechanisms of nematode suppression by organic soil amendments—A review[J].Applied Soil Ecology,2010,44:101-115

[11]蔡祖聪,张金波,黄新琦,等.强还原土壤灭菌防控作物土传病的应用研究[J].土壤学报,2015,52(3):469-476

[12]黄新琦,温腾,孟磊,等.土壤厌氧还原消毒对尖孢镰刀菌的抑制研究[J].土壤,2014,46(5):851-855

[13]Koike S,Subbarao K,Davis R M,et al.Vegetable Diseases Caused by Soilborne Pathogens[M].Oakland,California:Univeristy of California Publication,2003

[14]Yoda M,Kitagawa M,Miyaji Y.Long term competition between sulfate-reducing and methane-producing bacteria for acetate in anaerobic biofilm[J].Water Research,1987,21:1 547-1 556.

[15]Herlihy A T,Mills A L,Hornberger G M,et al.The importance of sediment sulfate reduction to the sulfate budget of an impoundment receiving acid mine drainage[J].Water Resources Research,1987,23(2):287-292

[16]Elliott P,Ragusa S,Catcheside D.Growth of sulfatereducing bacteria under acidic conditions in an upflow anaerobic bioreactor as a treatment system for acid mine drainage[J].Water Reasearch,1998,32(12):3 724-3 730

[17]Liang F,Xiao Y,Zhao F.Effect of pH on sulfate removal from wasterwater using a bioelectrochemical system[J].Chemical Engineering Journal,2013,218:147-153

[18]Widdel F.Microbiology and Ecology of Sulfate- and Sulfur-reducing Bacteria[M]// Zehnder A J B.Boilogy of anaeraboc microorganisms.New York:Wiley & Sons Interscience,1988:469-585

[19]Zhao C,Yang Q,Chen W,et al.Isolation of a sulfate reducing bacterium and its application in sulfate removal from tannery wasterwater[J].African Journal of Biotechnol,2010,10:11 966-11 971

[20]Fortin D,Davis B,Beveridge T J.Role of Thiobacillus and sulfate-reducing bacteria in iron bicycling in oxic and acidic mine tailings[J].Microbiology Ecology,1996,21:11-24

[21]O’Flaherty V,Mahony T S,O'Kennedy R,et al.Effect of pH on growth kinetics and sulphide toxicity thresholds of a range of methanogenic,syntrophic and sulphate-reducing bacteria[J].Process Biochemistry,1998,33:555-569

[22]Lens P,Hulshoff Pol L.Environmental Technologies to Treat Sulfur Pollution:Principles and Engineering[M].London:International Association on Water Quality,2000[23]Burgin A J,Hamilton S K.Have we overemphasized the role of denitrification in aquatic ecosystems:A review of nitrate removal pathways[J].Frontiers in Ecology and the Environment,2005,5:89-96

[24]Meng T Z,Zhu T B,Zhang J B,et al.Liming accelerates theremoval and reduces N2O emission in degraded vegetable soil treated by reductive soil disinfestation(RSD)[J].Journal of Soils and Sediments,2015,15(9):1 968-1 976

[25]Goh,K,Gregg,P.Field studies on the fate of radioactive sulphur fertilizer applied to pastures[J].Fertilizer Research,1982,3(4):337-351

[26]Wu J,O'Donnell A G,Syers J K.Influences of glucose,nitrogen and plant residues on the immobilization of sulphate-S in soil[J].Soil Biology and Biochemistry,1995,27(11):1 363-1 370

[27]Curtin D,Campbell C A,Jalil A.Effects of acidity on mineralization:426 pH-dependence of organic matter mineralization in weakly acidic soils[J].Soil Biology and Biochemistry,1998,30:57-64

[28]Nyborg M,Hoyt P B.Effects of soil acidity and liming on minerazation of soil nitrogen[J].Canadian Journal of Soil Science,1978,58:331-338

[29]Al-Zuhair S,El-Naas M H,Al-Hassani H.Sulfate inhibition effect on sulfate reducing bacteria[J].Journal of Biochemical Technology,2008,1:39-44

[30]Perry R H,Green D W,Maloney J O.Perry’s Chemical Engineer’S Handbook[M].Singapore:McGraw-Hill,1984[31]McLaren R G,Cameron K C,Fraser P M.A comparison of the effects of subsoiling on plant uptake and leaching losses of sulphur and nitrogen from a simulated urine patch[J].Developments in Plant and Soil Sciences,1993,54:495-498

[32]Eriksen J,Murphy M D,Schnug E.The soil sulphur cycle[J].Nutrients in Ecosystems,1998,2:39-73

[33]Devai I,DeLaune R D.Formation of volatile sulfur compounds in salt marsh sediment as influenced by soil redox condition[J].Organic Geochemistry,1995,23(4):283-287

[34]Huang X,Wen T,Zhang J,et al.Toxic organic acids produced in biological soil disinfestation mainly caused the suppression of Fusarium oxysporum f.sp.cubense[J].Biocontrol,2015,60(1):113-124

[35]Lamers J G,Wanten P J,Blok W J.Biological soil disinfestation:A safe and effective approach for controlling soilborne pests and diseases[J].Agroindustria,2004,3:289-291

[36]Butler D M,Rosskopf E N,Kokalis-Burelle N,et al.Exploring warm-season cover crops as carbon sources for anaerobic soil disinfestation(ASD)[J].Plant and Soil,2012,355:149-165

[37]Oka Y.Mechanisms of nematode suppression by organic soil amendments—A review[J].Applied Soil Ecology,2010,44:101-115

[38]Momma N,Kobara Y,Uematsu S,et al.Development of biological soil disinfestations in Japan[J].Applied Microbiology and Biotechnology,2013,97(9):3 801-3 809

[39]Mowlick S,Inoue T,Takehara T,et al.Changes and recovery of soil bacterial communities influenced by biological soil disinfestation as compared with chloropicrintreatment[J].AMB Express,2013,3:46-58

[40]McElderry C F,Browning M,Amador J A.Effect of shortchain fatty acids and soil atmosphere on Tylenchorhynchus[J].Journal of Nematology,2005,37:71-77

Effects of Liming on Sulfate Removal and Transformation in Degraded Vegetable Soil Treated by Reductive Soil Disinfestation(RSD)

MENG Tianzhu1,ZHU Tongbin1,ZHANG Jinbo1,2,3,CAI Zucong1,2,3*
(1 School of Geography Sciences,Nanjing Normal University,Nanjing 210023,China; 2 Jiangsu Provincial Key Laboratory of Materials Cycling and Pollution Control,Nanjing 210023,China; 3 Jiangsu Center for Collaborative Innovation in Geographical Information Resource Development and Application,Nanjing 210023,China)

Abstract:Reductive soil disinfestations(RSD),namely amending organic materials and mulching or flooding to create strong reductive status,has been widely applied to improve degraded soils,but the removal efficiency of sulfatein RSD is low.To investigate the effect of liming onremoval during RSD treatment,aaccumulated(939 mg/kg)vegetable soil was treated by five treatments:control(flooding only,noted as CK),flooding + alfalfa(RSD0)and RSD0+ lime at three application rates,which adjusted soil pH to 6.7,7.7 and 8.4,noted as RSD1,RSD2and RSD3,respectively.The results showed thatcontent in CK treatment was as high as 691 mg/kg,and was significant higher than those in RSD treatments after the incubation.In RSD treatments,contents decreased with the increase of pH.But,other sulfur(S)forms increased significantly,which indicated that disappearedwas mainly transformed into other S forms.Total S contents in RSD+liming treatments were higher than that in RSD0treatment.The results indicates that liming stimulates the conversion ofinto calcium sulfate,which effectively decreasescontent but cannot decrease total S content.

Key words:Degraded soil; RSD; Lime;

作者简介：孟天竹(1988—)，女，江苏南京人，博士研究生，主要从事修复退化土壤方面的研究。E-mail:zmeng09@163.com

* 通讯作者(zccai@njnu.edu.cn)

基金项目：①江苏省自然科学基金项目(BK20140062)、国家自然科学基金项目(41301313，41330744)和江苏高校优势学科建设工程项目(PAPD，164320H116)资助。

DOI:10.13758/j.cnki.tr.2016.01.018

中图分类号：S156