Effects of low-molecular-weight organic compounds on sulphur immobilization and re-mineralization...

European Journal of Soil Science, April 2010, 61, 287–297 doi: 10.1111/j.1365-2389.2010.01228.x

Effects of low-molecular-weight organic compoundson sulphur immobilization and re-mineralizationand extraction of immobilized sulphur by hot-waterand acid hydrolysis

P . - C . V o n g , S . P i u t t i , S . S l e z a c k - D e s c h a u m e s , E . B e n i z r i & A . G u c k e r tUMR Nancy-Universite-INRA Agronomie et Environnement Nancy-Colmar, 2 Avenue de la Foret de Haye, 54505Vandoeuvre-les-Nancy, France

Summary

We examined the effects of salicylate, glutamate and glucose on sulphur (S) immobilization and re-mineralization in three calcareous soils: from arable, fallow and forest managements. Each of the threesubstrates, at a single rate of carbon (1000 mg C kg−1 soil) was added to the three soils and then incubatedwith Na35

2 SO4 plus Na2SO4 for 1, 2, 6 or 12 weeks prior to analysis. The extraction of the immobilized Swas performed with either hot water (HW-S) or 3 m hydrochloric acid (HCl-S). Except for the forest soil, theaverage amounts of immobilized S increased in the order control < salicylate < glutamate < glucose. Acrossall soils, substrate addition significantly decreased the average value of HCl-S expressed as a percentage ofimmobilized S compared with that of the control. The decreases in HCl-S (58 − 100%) were substrate-and time-independent, suggesting a heterogeneous action of HCl. In contrast, except for the forest soil,we found substantial declines in the average percentage values of HW-S (21 − 75% of the immobilizedS) in the order control > salicylate > glutamate = glucose, suggesting that the hot-water action was substratedependent. In addition, the proportion of HW-S decreased with increasing incubation time, indicating thatthe youngest immobilized S was the most labile. Thus, hot water extracted more homogeneous S compoundsthan HCl. Sulphur immobilization induced by glutamate and glucose produced more recalcitrant S products(small quantities of 35S re-mineralization) than that induced by salicylate. Glutamate tended to generate morerecalcitrant S compounds than glucose. Hot-water extractable S was a valuable and rapid indicator of readilylabile organic S. Consequent microbial S immobilization resulting from glucose or glutamate addition wasfollowed by a small rate of re-mineralization of immobilized S.

Introduction

Sulphur (S) plays a vital role in plant growth and development. Itis an essential constituent of methionine, cysteine, vitamins andseveral co-enzymes. Moreover, S fertilizer application is importantto optimize nitrogen (N) assimilation by plants (Fismes et al.,2000), thereby reducing N losses by leaching or volatilization.While S is crucial for both the yield and the quality of plantproducts, increased incidence of S deficiency in plants has beenreported in Europe (Zhao et al., 1996) and results from thereduction in S emissions in recent decades (Jenkins et al., 1998).

Correspondence: P.-C. Vong. E-mail: [email protected]

Received 22 June 2009; revised version accepted 29 November 2009

Consequently, more attention should be paid to the control ofboth S fertilizer application and S transformations in soil through

immobilization-mineralization processes.Sulphur applied to soil is subject to physical (sorbed onto soil

particles) or biological immobilization. Therefore, the importance

of S immobilization depends on both soil properties, includingsoil organic matter content (SOM), and soil biological activity. Ascarbon (C) availability greatly controls microbial activity, it hasalso been shown to control S immobilization (Knights et al., 2001;

Vong et al., 2008). This dynamic of S immobilization is expectedto be significant in the rhizosphere environment. Low-molecular-weight organic compounds (LMWC), such as glucose, amino acids

and organic acids, stimulate microbial biomass growth and activityin the zone surrounding the roots (see Rovira, 1965; Guckert et al.,1975). While the effects of glucose addition on S immobilization

© 2010 The AuthorsJournal compilation © 2010 British Society of Soil Science 287

288 P.-C. Vong et al.

have been reported (Dedourge et al., 2004; Vong et al., 2007),those of LMWC, such as glutamate and salicylate, have not yetbeen addressed.

In the present study, glucose was chosen as a carbon source,as it is largely abundant in the exudates. The dynamics ofS immobilization and re-mineralization with glucose were thencompared with those with glutamate, as it is representative ofN-containing organic compounds in the exudates (Renella et al.,2007). Glutamate has a small C : N ratio (5:1) that is adequateto promote microbial growth. Moreover, salicylate, a breakdownproduct from lignin and polyphenols, was also included becauseit is a LMWC that is mostly abundant in detritus and does notcontain N. The extraction of immobilized S was performed usingeither hot-water or 3 m HCl hydrolysis. Hot water mainly extractscarbohydrates derived from microbial biomass and plant exudates(Ghani et al., 2003), which reflct the variations of labile soilorganic C and N. Hot-water extractable C is a valuable andrapid indicator of early changes induced by soil management(Leinweber et al., 1995; Gregorich et al., 2003). Additionally,hot-water extractable C is closely correlated to soil microbialbiomass C (Sparling et al., 1998). It is, therefore, expected that thelabile organic S extracted by hot water (HW-S) also reflects thelability of S immobilized by microbial biomass. In addition, theHW-S pool was compared with that from acid hydrolysis with 3 mHCl, as HCl hydrolysis separates labile from resistant organic C(Paul et al., 2006; Silveira et al., 2008). Acid hydrolysis with 3 mHCl is, therefore, also expected to distinguish between labile andrecalcitrant fractions of immobilized S.

The objectives of this study were to investigate over time(i) the impacts of some LMWC that are mostly present inthe rhizosphere on the intensity of S immobilization, (ii) theextractability of immobilized S by hot water in comparison with3 m HCl hydrolysis and (iii) the re-mineralization of immobilized35S. Three calcareous soils (from arable, fallow and forestenvironments) differing in SOM content were chosen. Both thequantity and nature of organic compounds inherent to each soil’sbackground were expected to impact differently upon the pools ofimmobilized S, HW-S and HCl-S derived from both the appliedsulphate S and the sulphate S initially present.

Materials and methods

All soil samples were collected from a calcareous soil (RendzicLeptosol: FAO-WRB classification) in the region of Lorraine

in France (48◦51′N, 5◦57′E) in September 2006. For eachmanagement, the sample used for the experiment was a compositesample resulting from five replicates of soil randomly collectedwithin a sub-area of 5000 m2 (50 m × 100 m). The arable soilwas sampled in the Ap horizon (0–20 cm) after spring barleycultivation in a sequential rotation of rape, wheat and springbarley. The fallow soil was shallow (0–20 cm in depth) andhad not been ploughed since 1993. Approximately 70% of itsarea (30% bare) was covered by the following plant species:Taraxacum officinale Weber (17%), Poa trivialis L. (14%),Plantago lanceolata L., Medicago sativa L., Lotus corniculatusL., Dactylis glomerata L. and Arrhenaterum elatius L. (45%);and Achillea millefolium L., Bellis perennis L., Bromus mollis L.,Lotus corniculatus L. and Ranunculis acris L. (24%). The forestsoil was sampled from a broad-leaved forest stand dominated bybeech and oak trees. Prior to sampling, the litter (L) and humuslayer (Oh) were discarded. Samples were collected from the Ahhorizon (0–20 cm). The three areas were adjacent and locatedon the same flat toposequence. The fallow soil is a narrow stripof land (100 m × 1500 m) between the arable and forest lands.At the laboratory, visible roots and recognizable plant residueswere removed and all soils were passed through a 2-mm sieveand dried at room temperature. Table 1 shows the main physicaland chemical characteristics of the studied soils.

An equivalent of 50 g of dry soil from each of the three studiedsoil samples was weighed into separate 200 ml polyethylene pots.For each soil, we prepared 68 pots that corresponded to four treat-ments without (control) or with salicylate, glutamate and glucoseaddition × four incubation times × four replicates (see later),including four pots used for SO4

2−-S determination after 1 weekof soil pre-incubation. The soil was moistened to 50% of water-holding capacity (WHC), and the pots were then pre-incubated for1 week at 25◦C (Chowdhury et al., 2000) to allow partial recoveryof soil microorganisms. At the end of this pre-incubation period,the SO4

2−-S content of the soils (four replicates of each soil)was determined by extraction with 0.009 m Ca(H2PO4)2, whichextracts both free and sorbed forms of SO4

2−-S (Singh, 1984).Briefly, moist soil equivalent to 30 g of dry matter was extractedwith a 0.009 m Ca(H2PO4)2 solution (w/v : 1/5) on an end-over-end shaker for 30 minutes and then filtered through a WhatmanNo. 42 paper disc. Aliquots of 10 ml were dispensed into 20-mlassay tubes and then acidified with 0.2 ml of 2 m HCl. After-wards, 2 ml of a solution containing a mixture of BaCl2·2H2O(100 g l−1) and polyvinylpyrrolidone (20 g l−1) was added to

Table 1 Main physical and chemical characteristics of the soils studied

Clay Silt Sand OMa C N S pH C/N

Soils g / kg−1 soil

Arable soil 357 301 309 39.90 23.22 2.31 0.56 8.1 10.0Fallow soil 345 289 297 67.63 39.32 3.74 0.69 7.9 10.5Forest soil 326 270 278 125.37 72.89 5.78 0.64 7.5 12.6

aOM = organic matter.

© 2010 The AuthorsJournal compilation © 2010 British Society of Soil Science, European Journal of Soil Science, 61, 287–297

Labile organic S in hot water and 3 M HCl 289

each flask. Standards were prepared at the same pH (1.8–2.0)as the samples in 0.009 m Ca(H2PO4)2 (Rabuffetti & Kamprath,1977). The SO4

2−-S precipitated as BaSO4 was then measured tur-bidimetrically at 460 nm. Both organic S and N soil contents weremeasured on a sub-sample of soil residue obtained after remov-ing inorganic S and N by extraction with a 0.01 m CaCl2 solution(instead of 0.009 m Ca(H2PO4)2 solution) as previously described.Soil organic C was measured after elimination of carbonates byacidification using diluted HCl solutions (Harris et al., 2001). Todetermine soil organic S, 1 g of powdered dry soil residue (afterinorganic S removal) was digested in 6 ml of a 1:1 (v/v) mixtureof HNO3 (69.4%) and HClO4 (70.5%) by increasing the temper-ature progressively from 80 to 200◦C, until a clear digest wasobtained. After cooling, the digest was filtered through a What-man No. 42 paper disc into 50 ml volumetric flasks. Aliquots of2 ml were then dispensed into 20 ml assay tubes and adjusted to10 ml with distilled water. To obtain the same pH as the sam-ples (approximately 0.5), standards were prepared in a solutioncontaining a mixture of both concentrated HNO3 (10 ml l−1) andconcentrated HClO4 (10 ml l−1). The SO4

2−-S was then measuredat 460 nm as previously described. The organic C and N soil con-tents were measured by a CN-analyser (Flash EA1112 ThermoFinnigan, Rodano, Italy).

Because SO42−-S content was relatively small (see later) and to

avoid limiting conditions of N and S that would hamper microbialbiomass formation (Chowdhury et al., 2000), the remaining pots(64 for each soil) received a solution containing 80 mg N kg−1

soil as NaNO3 and 20 mg S kg−1 soil as Na2SO4. To achievea homogeneous isotope dilution of 35S with 32S, a solutioncontaining 673.9 kBq kg−1 soil of carrier-free Na35

2 SO4 wasmixed under agitation with that containing unlabelled Na2SO4.The use of 35S allowed us to calculate S in the 0.009 mCa(H2PO4)2 extract (MCP-S), immobilized S, HW-S and HCl-S.In addition, two rates of 0 (control) and 1000 mg C kg−1 soiladded as glucose, glutamate or salicylate were added to thesolution before addition to the pots. The solution containing the35S, 32S, N and C (4.5 ml) was dripped onto the soil in each potand then thoroughly mixed using a spatula. This solution broughtthe final soil moisture up to 80% of WHC. The soil remainedaerobic at this soil-moisture status. All of the pots were weighedand then incubated at 25◦C. During incubation, soil moisturecontent was maintained close to 80% WHC by weighing the potstwice weekly and adding the appropriate amount of distilled water.

After 1, 2, 6 and 12 weeks of incubation, the inorganic Swas extracted as previously indicated. The soil residue wasdried at 37◦C for 1 week and powdered. A sub-sample of 6 gwas extracted with 30 ml distilled water at 70◦C for 18 hours(Sparling et al., 1998). In parallel, a similar soil residue sub-sample was hydrolysed with 3 m HCl at 70◦C for 18 hours. Itis important to note that our goal was to compare the actionsof hot water and 3 m HCl on the extractability of immobilized Safter different incubation times, but not to assess measurements oflabile forms of organic S. Thus, adjustments of HW-S and HCl-Scontents by extraction with water and HCl at room temperature

were not performed. The 35S in the 0.009 m Ca(H2PO4)2,hot-water extract and HCl hydrolysate was determined by liquidscintillation counting of a 1-ml aliquot mixed with 10 ml PerkinElmer scintillation cocktail (Ultima Gold, Rungis, France). Theradioactivity of the sample was measured by a Tri-Carb liquidscintillation analyser (Model 2100 TR, Canberra-Packard, Rungis,France) with an automatic quenching correction to convert countsinto disintegrations. The radioactivity measured was corrected forthe natural decay of 35S (half-life of 88 days) by using the blankradioactive samples prepared at the beginning of the experiment.The 35S in the 0.009 m Ca(H2PO4)2 extract represented the non-immobilized 35S and, thus, the immobilized-35S was calculated asthe difference between the 35S initially applied and the 35S foundin the Ca(H2PO4)2 extract.

Our previous recovery test with these soils using 0.009 mCa(H2PO4)2 showed a 100% recovery of SO4

2−-35S introducedin the samples and extracted immediately after labelled S addi-tion (Vong et al., 2008). Briefly, the test was carried out withsoil samples moistened to 80% of WHC, incubated overnightat 25◦C and autoclaved at 120◦C for 30 minutes on two con-secutive days to kill soil microorganisms (Carter et al., 2007).Consequently, physical immobilization resulting from SO4

2−-35Sadsorption onto soil particles was negligible, and the 35S immo-bilization corresponded exclusively to biological immobiliza-tion. The amounts of MCP-S, immobilized-S, HW-S and HCl-Swere obtained by dividing the corresponding 35S (kBq) foundin each fraction after 1, 2, 6 and 12 weeks by the specificactivity (SA) of the initial SO4

2−-S pool (35S applied/initialSO4

2−-S pool, kBq mg−1 S). The initial SO42−-S pool included

both the applied SO42−-S (20 mg kg−1 soil) and the SO4

2−-Scontent measured after a 1-week soil pre-incubation. At each incu-bation time, the SA values were calculated as the 35S found in the0.009 m Ca(H2PO4)2 extract divided by the corresponding totalSO4

2−-S measured in this extract. A decrease in specific activitywith time indicates an increased re-mineralization of unlabelled Srelative to labelled S. To compare the effects of substrates usedfor the re-mineralization of the immobilized 35S over time, theSA values of treatments in each soil were expressed as percent-age differences of the controls: thus % SA = ((SA treatment - SAcontrol)/SA control) × 100. This calculation takes into accountthe controls, as their SA values were different according to bothsoil management and time interval of soil incubation.

For each soil, data were submitted to a two-way anova usingSAS software V 9.1 (SAS Institute Inc., Cary, NC, USA) withtreatment and time as factors. For the proportion of HCl-S relatedto immobilized S, a one-way anova with time as a factor wasused for each substrate and soil management, as there wereno significant treatment × time interactions in the studied soils.A one-way anova was performed with the cumulated SA valuesexpressed as percentage differences of the controls with treatmentas a factor. For the effects of soil management on immobilized S,as the proportions of HW-S and HCl-S related to immobilized S,a three-way anova with soil, treatment and time as factors was



performed. The mean values were grouped by the Tukey test(P < 0.05) for comparisons.

Results

Immobilized sulphur

The addition of different LMWC substrates progressivelyincreased the amount of immobilized S during soil incubation.Over 12 weeks, the amount of S immobilized increased from3.9 to 11.3 mg S kg−1 soil in the arable soil (Table 2), from 5.0to 12.7 mg S kg−1 soil in the fallow soil (Table 3) and from5.6 to 13.2 mg S kg−1 soil in the forest soil (Table 4). Thesevalues correspond to an increase from 15.2 to 44.2%, 23.0 to58.6% and 24.4 to 57.5% of the amount of SO4

2−-S presentat day 0 (SO4

2−-S applied including SO42−-S present in soil

after 1 week of soil pre-incubation, just before 35S application),

respectively, in the arable, fallow and forest soils. As a whole,the average amount of immobilized S increased in the order con-trol < salicylate < glutamate < glucose, except for the forest soilwhere the effect of glutamate addition did not significantly differfrom that of glucose (Table 4). These results indicate efficient Simmobilization with glucose and glutamate. Across all treatments,the average amount of immobilized S significantly increased(P < 0.0001) in the order arable soil < fallow soil < forest soil,indicating that the largest amount of immobilized S was in theforest soil rich in organic C.

Sulphur in hot-water extracts and immobilized sulphur

The HW-S content varied narrowly between 2.9 and 4.2 (Table 2),2.6 and 3.5 (Table 3) and 2.7 and 3.9 mg S kg−1 soil in the arable,fallow and forest soils, respectively (Table 4). The HW-S con-tent remained relatively constant because that of immobilized S

Table 2 Sulphur immobilization (Imm-S), S in hot-water extract (HW-S), in 3 m HCl hydrolysis (HCl-S) and in 0.009 m Ca(H2PO4)2 extract (MCP-S),total sulphate S (SO4

2−-S) and specific activity (SA) value of total sulphate S in the arable soil incubated without (control) or with salicylate, glutamateand glucose after 1, 2, 6 and 12 weeks. Sulphur immobilization (Imm-S) represents initial SO4

2−-S (SO42−-S applied plus SO4

2−-S present in soil before35S application) minus MCP-S

Time Imm-S HW-S HCl-S MCP-S SO42−-S HW-S HCl-S SA

Treatment Week / mg kg−1 soil / % imm-S / kBq mg−1 S

Control 1 3.9 (0.2) 2.9 (0.1) 3.9 (0.2) 21.7 (0.2) 45 (1) 75 (5) 100 (1) 12.8 (0.2)2 4.9 (0.5) 2.9 (0.2) 4.8 (0.4) 20.6 (0.5) 39 (1) 59 (9) 98 (2) 13.8 (0.4)

6 6.4 (0.8) 3.5 (0.3) 6.1 (0.4) 19.1 (0.8) 43 (1) 55 (8) 95 (8) 11.8 (0.7)

12 7.6 (0.5) 3.3 (0.1) 7.4 (0.4) 17.9 (0.5) 38 (2) 43 (3) 97 (3) 12.3 (0.4)Mean 5.7d 3.1a 5.5c 19.8a 41a 58a 98a 12.7a

Salicylate 1 5.0 (0.2) 3.1 (0.4) 4.6 (0.2) 20.5 (0.2) 41 (2) 62 (11) 92 (5) 13.0 (0.3)2 5.9 (0.3) 3.1 (0.1) 5.7 (0.2) 19.6 (0.3) 34 (1) 53 (3) 97 (2) 15.2 (0.4)6 7.6 (0.5) 3.2 (0.1) 6.0 (0.1) 17.9 (0.5) 43 (2) 42 (2) 79 (4) 11.1 (0.5)

12 9.0 (0.1) 3.5 (0.7) 8.1 (0.6) 16.5 (0.1) 39 (1) 39 (7) 89 (6) 11.2 (0.3)Mean 6.9c 3.2a 6.1b 18.6b 39b 49b 89b 12.6a

Glutamate 1 6.7 (0.3) 3.3 (0.2) 6.0 (0.5) 18.9 (0.3) 43 (1) 50 (5) 90 (10) 11.7 (0.2)2 7.4 (0.3) 3.4 (0.2) 7.0 (0.6) 18.1 (0.3) 37 (1) 45 (1) 95 (7) 13.0 (0.3)

6 8.8 (0.4) 3.2 (0.1) 7.1 (0.4) 16.7 (0.4) 43 (1) 36 (2) 80 (3) 10.3 (0.1)12 10.1 (0.4) 3.2 (0.1) 8.8 (0.2) 15.4 (0.4) 36 (1) 32 (1) 87 (2) 11.2 (0.4)Mean 8.3b 3.3a 7.2a 17.3c 40b 41c 88bc 11.5b

Glucose 1 6.6 (0.3) 2.9 (0.1) 6.0 (0.2) 19.0 (0.3) 41 (1) 44 (3) 92 (5) 12.3 (0.1)

2 8.1 (0.2) 3.0 (0.1) 7.0 (0.2) 17.5 (0.2) 34 (3) 37 (1) 87 (4) 13.8 (1.2)

6 9.6 (0.1) 4.2 (0.2) 7.5 (0.2) 15.9 (0.1) 40 (1) 43 (2) 78 (3) 10.5 (0.3)12 11.3 (0.4) 3.1 (0.2) 9.4 (0.4) 14.2 (0.4) 35 (1) 28 (2) 83 (5) 10.7 (0.4)Mean 8.9a 3.3a 7.5a 16.7d 37c 38c 85c 11.8b

EffectTreatment ∗∗∗ NS ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗

Time ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗

Treatment × time NS ∗∗∗ NS NS ∗∗∗ ∗ NS ∗∗∗

∗, ∗∗and ∗∗∗indicate significance at P < 0.05, 0.01 and 0.001, respectively; NS = not significant.Values in bold italic, especially illustrated for the sulphate S specific activity (SA), show significant differences from the corresponding controls.Total sulphate S represents SO4

2−-S derived from soil plus MCP-S. In each column, bold letters denote significant statistical differences at P < 0.05(Tukey test). Values in parentheses are standard errors corresponding to means of four replicates.




2−-S) and specific activity (SA) value of total sulphate S in the fallow soil incubated without (control) or with salicylate, glutamateand glucose after 1, 2, 6 and 12 weeks



Control 1 5.0 (0.3) 3.1 (0.3) 5.0 (0.3) 16.7 (0.3) 29 (1) 61 (3) 100 (1) 17.6 (0.4)

2 5.5 (0.4) 2.9 (0.1) 5.4 (0.4) 16.2 (0.4) 31 (1) 54 (3) 98 (2) 16.0 (0.5)

6 8.2 (0.1) 3.3 (0.3) 7.5 (0.3) 13.5 (0.1) 27 (1) 40 (3) 92 (5) 15.4 (0.7)12 8.7 (0.3) 2.9 (0.2) 8.5 (0.7) 12.9 (0.3) 33 (1) 33 (1) 98 (7) 12.4 (0.4)

Mean 6.8d 3.0b 6.6d 14.8a 30a 47a 97a 15.3a

Salicylate 1 7.3 (0.5) 3.2 (0.2) 6.7 (0.5) 14.4 (0.5) 28 (1) 45 (5) 93 (9) 15.8 (0.3)2 7.4 (0.1) 3.2 (0.2) 6.8 (0.7) 14.2 (0.1) 28 (2) 43 (2) 92 (8) 16.0 (1.3)6 9.7 (0.1) 3.3 (0.1) 8.2 (0.2) 12.0 (0.1) 27 (1) 34 (2) 85 (1) 13.8 (0.2)

12 10.3 (0.4) 3.4 (0.3) 9.4 (0.5) 11.3 (0.4) 32 (1) 33 (3) 91 (5) 10.9 (0.4)Mean 8.7c 3.3a 7.8c 13.0b 29b 39b 90b 14.1b

Glutamate 1 8.7 (0.1) 2.9 (0.2) 7.0 (1.1) 12.9 (0.1) 26 (1) 33 (1) 81 (12) 15.7 (0.4)2 8.9 (0.2) 2.6 (0.2) 7.7 (0.4) 12.8 (0.2) 28 (1) 29 (3) 86 (4) 14.0 (0.6)6 10.7 (0.1) 3.1 (0.1) 8.0 (0.3) 11.0 (0.1) 25 (1) 29 (1) 75 (3) 13.9 (0.7)

12 11.8 (0.2) 2.8 (0.1) 10.4 (0.3) 9.8 (0.2) 29 (1) 24 (1) 87 (4) 10.5 (0.4)Mean 10.0b 2.9c 8.3b 11.6c 27c 29c 82c 13.5c

Glucose 1 7.9 (0.3) 2.9 (0.3) 7.4 (0.7) 13.7 (0.3) 25 (1) 36 (2) 93 (9) 16.7 (0.6)2 9.3 (0.1) 2.6 (0.1) 7.9 (0.7) 12.4 (0.1) 26 (1) 28 (2) 85 (9) 15.0 (0.5)6 11.2 (0.2) 3.5 (0.4) 9.9 (0.2) 10.5 (0.2) 23 (1) 31 (4) 89 (3) 14.1 (0.5)

12 12.7 (0.1) 2.7 (0.1) 11.4 (0.5) 9.0 (0.1) 28 (1) 21 (1) 90 (4) 10.0 (0.4)Mean 10.3a 2.9bc 9.2a 11.4d 26d 29c 89b 13.9bc

EffectTreatment ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗

Time ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ ∗ ∗∗∗

Treatment × time ∗∗∗ ∗ ∗ ∗∗∗ ∗∗ ∗∗∗ NS ∗∗

∗, ∗∗and ∗∗∗indicate significance at P < 0.05, 0.01 and 0.001, respectively; NS = not significant.Values in bold italic, especially illustrated for the sulphate S specific activity (SA), show significant differences from the corresponding controls.Sulphur immobilization (Imm-S) represents initial SO4


2−-S present in soil before 35S application) minus MCP-S. Totalsulphate S represents SO4

2−-S derived from soil plus MCP-S. In each column, bold letters denote significant statistical differences at P < 0.05 (Tukeytest). Values in parentheses are standard errors corresponding to means of four replicates.

progressively increased over time. Therefore, no significant corre-lations were found between HW-S and immobilized S (r = 0.46,

P > 0.05 in the arable soil; r = −0.06, P > 0.05 in the fallowsoil; r = 0.30, P > 0.05 in the forest soil) (Figure 1). Expressedas percentages of immobilized S, the values between treatments

ranged from 28 to 75%, 21 to 61% and 22 to 49% in thearable, fallow and forest soils, respectively. Thus, across all treat-

ments, the average proportion of HW-S decreased significantly(P < 0.0001) in the order arable soil > fallow soil > forest soil.

On average, the percentage values of HW-S decreased in theorder control > salicylate> glutamate = glucose in the arable andfallow soils, and control = salicylate > glucose > glutamate in

the forest soil. The proportions of HW-S declined progressivelyand significantly over time, indicating enhanced stabilities of the

immobilized S with increasing incubation times. This observa-tion implies that there is a homogeneous action of hot water, and

indicates that newly immobilized S was more labile than the olderimmobilized S.

Sulphur after HCl hydrolysis and immobilized sulphur

Within the treatments, HCl-S content significantly increasedfrom 3.9 to 9.4, 5.0 to 11.4 and 5.1 to 10.0 mg S kg−1 soil,respectively, in the arable, fallow and forest soils (Tables 2–4).Consequently, we found strong positive correlations of HCl-S content with that of immobilized-S (r = 0.97, P < 0.001 inthe arable soil; r = 0.96, P < 0.001 in the fallow soil andr = 0.87, P < 0.001 in the forest soil) (Figure 1). Expressedas percentages of immobilized-S, the values between treatmentsvaried from 78 to 100% in the arable soil, 75 to 100% inthe fallow soil and 58 to 96% in the forest soil. Therefore,across all treatments, the average proportion of HCl-S decreasedsignificantly (P < 0.0001) in the order arable soil = fallow




2−-S) and specific activity (SA) value of total sulphate S in the forest soil incubated without (control) or with salicylate, glutamateand glucose after 1, 2, 6 and 12 weeks



Control 1 5.6 (0.5) 2.7 (0.1) 5.1 (0.3) 17.3 (0.5) 38 (1) 49 (6) 91 (10) 13.3 (0.5)

2 6.9 (0.4) 2.8 (0.1) 6.6 (0.4) 16.0 (0.4) 41 (3) 40 (3) 96 (8) 11.3 (0.6)

6 9.5 (0.3) 3.2 (0.2) 6.6 (0.4) 13.4 (0.3) 35 (2) 34 (3) 69 (6) 11.4 (0.5)12 11.5 (0.2) 3.2 (0.1) 9.3 (0.5) 11.4 (0.2) 38 (1) 28 (1) 81 (5) 8.8 (0.2)

Mean 8.4c 3.0b 6.9b 14.6a 38a 38a 84a 11.2a

Salicylate 1 7.3 (0.3) 3.4 (0.2) 5.5 (0.3) 15.7 (0.3) 37 (1) 46 (5) 76 (1) 12.5 (0.1)2 7.8 (0.2) 3.0 (0.2) 6.4 (0.3) 15.1 (0.2) 37 (2) 39 (3) 82 (6) 12.1 (0.7)6 10.9 (0.2) 3.3 (0.3) 6.3 (0.3) 12.1 (0.2) 33 (1) 30 (2) 58 (2) 10.8 (0.3)

12 12.3 (0.4) 3.2 (0.1) 7.8 (0.2) 10.7 (0.4) 36 (1) 26 (1) 64 (2) 8.6 (0.3)

Mean 9.6b 3.2a 6.5c 13.4b 36b 35a 70c 11.0a

Glutamate 1 9.0 (0.3) 2.8 (0.2) 6.9 (0.1) 14.0 (0.3) 37 (1) 31 (2) 76 (2) 11.0 (0.4)2 9.1 (0.3) 2.7 (0.4) 7.8 (0.2) 13.8 (0.3) 34 (1) 29 (5) 86 (3) 12.0 (0.5)6 11.8 (0.2) 2.9 (0.4) 7.8 (0.4) 11.2 (0.2) 34 (1) 25 (3) 66 (3) 9.8 (0.3)

12 12.8 (0.1) 3.0 (0.1) 9.0 (1.3) 10.2 (0.1) 35 (2) 23 (1) 70 (11) 8.6 (0.5)

Mean 10.7a 2.8b 7.9a 12.3c 35bc 27c 75b 10.3b

Glucose 1 8.0 (0.3) 3.2 (0.5) 6.6 (0.5) 14.9 (0.3) 36 (1) 40 (6) 82 (5) 12.2 (0.4)2 9.2 (0.1) 2.8 (0.1) 8.1 (0.2) 13.7 (0.1) 36 (3) 31 (1) 88 (3) 11.3 (1.1)6 11.6 (0.2) 3.9 (0.4) 8.2 (0.5) 11.3 (0.3) 32 (1) 34 (3) 71 (3) 10.4 (0.3)

12 13.2 (0.1) 2.9 (0.1) 10.0 (0.9) 9.7 (0.1) 34 (2) 22 (1) 75 (6) 8.5 (0.6)

Mean 10.5a 3.2a 8.2a 12.4c 34c 32b 79b 10.6b

EffectTreatment ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗

Time ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗

Treatment × time ∗∗∗ ∗∗ ∗ ∗∗∗ ∗∗ ∗∗ NS ∗∗∗

∗, ∗∗and ∗∗∗indicate significance at P < 0.05, 0.01 and 0.001, respectively; NS = not significant.Values in bold italic, especially illustrated for the sulphate S specific activity (SA), show significant differences from the corresponding controls.Sulphur immobilization (Imm-S) represents initial SO4


2−-S present in soil before 35S application) minus MCP-S. Totalsulphate S represents SO4

2−-S derived from soil plus MCP-S. In each column, bold letters denote significant statistical differences at P < 0.05 (Tukeytest). Values in parentheses are standard errors corresponding to means of four replicates.

soil > forest soil. Substrate addition decreased significantly theaverage proportion of HCl-S in all the soils compared with thecontrols. In contrast to HW-S, these decreases were independentof the nature of the substrate, the soil management and the periodof incubation. Consequently, with the exception of the arablesoil enriched with glucose (Table 2) and the forest soil enrichedwith salicylate (Table 4), the proportion of HCl-S at week 12did not significantly differ from that at week 1. This indicatesa heterogeneous action of HCl that did not distinguish betweennew and old immobilized S.

Sulphate sulphur specific activity

To detect the re-mineralization of immobilized 35S over time, weexamined the SA values of the sulphate S pool. After a 1-weekpre-incubation, soil SO4

2−-S content was relatively small and

amounted to 5.5, 1.7 and 2.9 mg kg−1 soil, respectively, in thearable, fallow and forest soils. Thus, the initial unlabelled sul-phate S pools at day 0 were 25.5, 21.7 and 22.9 mg kg−1 soil inthe arable, fallow and forest soils, respectively. Accordingly, theSA values of the sulphate S pool at day 0 corresponded to 26.4,31.1 and 29.4 kBq mg−1 S in the arable, fallow and forest soils,respectively. At week 1, the SA values of all treatments substan-tially decreased. This decrease results from a dilution of labelled Swith unlabelled sulphate S derived from soil (results not shown).At each incubation time, the sulphate-S content derived fromsoil corresponded to the difference between the total SO4

2−-Sand the MCP-S content (Tables 2–4). In addition, the immobi-lization of labelled S (results not shown) partly contributed tothe decline of the SA values. Across all treatments, sulphate SAvalues varied between 10.3 and 15.2 (Table 2), 10.0 and 17.6(Table 3) and 8.5 and 13.3 (Table 4) kBq mg−1 S in the arable,



y = 2.99x - 2.21 R2 = 0.214

y = 1.34x - 1.36 R2 = 0.936

0

2 a b

a b

a b

4

6

8

10

12

14

16a

b

c

y = -0.50x + 10.48 R2 = 0.004

y = 1.19x - 0.55 R2 = 0.914

0

2

4

6

8

10

12

14

16

Imm

obili

zed-

S /

mg

kg–1

soi

l

y = 2.21x + 3.0 R2 = 0.093

y = 1.41x - 0.65 R2 = 0.701

0

2

4

6

8

10

12

14

16

0 2 4 6 8 10 12

S in hot-water extract or in 3 M HCl hydrolyzis / mg k g–1 soil

Control Salicylate GlucoseGlutamate

Figure 1 Correlations of immobilized-S with S in hot-water extract(a) or S in 3 m HCl hydrolysis (b) in the arable (a), fallow(b) and forest (c) soils incubated without (control) or with salicylate,glutamate and glucose after 1, 2, 6 and 12 weeks (from open to fullyclosed black symbols, respectively).

fallow and forest soils, respectively. In the arable soil, we founda non-significant positive SA value with salicylate (2.0% of thecontrol) at week 1 and a significant positive SA value (10.2%of the control) at week 2 (Figure 2). Similarly, in the forest soil,non-significant positive values were noted with salicylate and glu-tamate (6.4 and 5.8% of the control, respectively) at week 2. Withrespect to the fallow soil, a negligible positive SA value (0.4% ofthe control) was noted with salicylate at week 2. Negative valuesof SA were then recorded for all substrates in all the soils. Asa whole, the SA values of the sulphate S pool decreased moremarkedly in the arable and fallow soils than in the forest soilat week 12. In the arable soil, we found significant cumulatedcontents of SA in the decreasing order salicylate > glucose =glutamate (Figure 2). In contrast, no significant cumulated SAcontents were found in the fallow soil, but a noticeable, butnon-significant (P = 0.061), difference in cumulated SA valuesbetween treatments was obtained in the forest soil. Across allsoils, we noted the same trend in cumulated SA contents valuesin the order salicylate > glucose > glutamate treatments.

Discussion

Sulphur immobilization

In the present study, we used a 0.009 m Ca(H2PO4)2 solution thatextracts both sorbed and free sulphate-S present in the soil solution(Singh, 1984), as confirmed by the recovery test, to show that therewas no physical immobilization of SO4

2−-35S applied. In addition,all SO4

2− ions in soil solutions with a pH > 6 are not sorbed ontosoil particles (Eriksen et al., 1998). Consequently, we can assumethat the measured immobilized S corresponded mainly to labelledmicrobial biomass S. This fraction may also include labelled deadmicrobial cells and microbial metabolites. The rate of C added was1000 mg kg−1 soil, which is approximately that (750 μg g−1 soil)found in the rhizosphere (Bremer & Kuikman, 1994). With theexception of the forest soil (Table 4), the significantly smalleraverage extent of S immobilization obtained with glutamate thanwith glucose suggests that microorganisms utilized glucose morerapidly and efficiently than glutamate (Tables 2–4). Hence, theaddition of a readily metabolizable compound, such as glucose,



-70

-60

-50

-40

-30

-20

-10

0

10

20

Sul

phat

e S

A v

alue

/ %

diff

eren

ce o

f the

con

trol

-70

-60

-50

-40

-30

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-10

0

10

20

-70

-60

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-40

-30

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-10

0

10

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1 2 6 12

Weeks

Salicylate Glutamate Glucose

*

*

* * * **

a

b

b

* * *

* * * **

* *

a

a

a

a*

*

*

*

a

a

a

b

c

Cumulated values

Figure 2 Sulphate specific activity (SA) values expressed aspercentage differences of the controls (without substrate) in thearable (a), fallow (b) and forest soils (c) incubated with salicylate,glutamate and glucose after 1, 2, 6 and 12 weeks. Different lettersdenote significant statistical differences at P < 0.05 (Tukey test).*Indicates significant difference from the control (see Tables 2–4):vertical bars are SE, n = 4.



stimulated microbial-35S removal (Dedourge et al., 2004) andmicrobial activity (Pennanen et al., 2004; Vong et al., 2008).We also recorded significantly smaller average amounts of Simmobilization with salicylate than with glutamate and glucose.

Schutter & Dick (2001) showed that sugars were the majorprimary substrates used by microbial communities in the earlycomposting process, and that these communities were adapted toa simple rather than a more complex C source, such as salicylate.Similarly, Bittman et al. (2005) stressed that soil bacteria growfaster on labile organic substrates such as simple carbohydratesand amino acids. Moreover, the intensity of S immobilizationprogressively increased over time (Tables 2–4). This increase isprobably because of microbial growth and dynamic cycling oforganic compounds through a build-up effect that occurred dur-ing soil incubation. In fact, in the closed system without plants,new compounds that are formed are subsequently re-used bymicroorganisms (Gruber et al., 2006), which contributes to theaccumulation of immobilized S. The observed greater level of Simmobilization in the forest soil, followed by the fallow soil andthe arable soil, could be attributed to the initial organic C contentin these soils (Table 1). The overall results clearly indicate theimportant role of LMWC such as glucose and glutamate, compo-nents most commonly found in root exudates, as energy sourcesfor microbial growth and activity. They then can efficiently stim-ulate microbial activity with consequent S immobilization.

Relationships between HW-S and immobilized sulphur

The HW-S pool corresponds to labile organic S. Over time, therelatively stable HW-S content (Tables 2–4), as well as the non-significant correlations observed between HW-S and immobilizedS (Figure 1), suggest a homogeneous action of hot water inextracting the immobilized S by releasing a homogeneouslyconstant pool of labile organic S. As a whole, the proportion ofhot-water extractable S, which varied between 21 and 75% of the Simmobilized, was dependent on the intensity of the immobilized S,the nature of the substrate added, as well as the soil management.These results suggest that hot-water extractable S can be usefulfor assessing the active pool of organic S.

In the present study, the amount of the immobilized Sprogressively increased with the length of time of soil incubation.Similarly, the application of a C source, such as farmyard manure,results in an accumulation of carbon-bonded S compared withester S (Yang et al., 2007). Most importantly, carbon-bonded Sis firmly integrated into the structure of humic acids (Bettanyet al., 1979). These observations help to explain why hot waterdid not substantially extract immobilized S as the incubation timeincreased. HW-S differs from HW-C, which has been found to bepositively correlated with microbial biomass C (Sparling et al.,1998; Chowdhury et al., 2000). These results are consistent withthose of our previous work showing that HW-35S was not wellcorrelated with 35S immobilized in microorganisms, because theaddition of C did not increase HW-35S (Vong et al., 2007).

The decline in the proportion of HW-S with increasingtime demonstrates a difference in the composition and structurebetween young and old organic S, which results in differentS labilities. In agreement with Ghani et al. (1993), the newly-immobilized S was more labile than the older immobilized S.These, and our, results indicate the usefulness of hot-waterextractable S as an indicator of labile soil organic S at an earlystage, as, proportionately, the young immobilized S was the mostextractable. The proportion of HW-S related to immobilized Swas substrate and soil dependent. In addition, our previous studiesusing calcareous arable soil sampled from the same experimen-tal site, showed that plants requiring large amounts of S, such asrape and ryegrass, are able to deplete S from the HW-S pool whenthe soil solution is sulphate-S-deficient (Vong et al., 2002, 2004,2007). The results show that hot-water extractable S is an activepool that is indicative of labile soil organic S readily available forplants.

Relationships between HCl-S and immobilized-S

As expected, a greater yield of S extraction was obtainedwith 3 m HCl than with hot water (Tables 2–4). In contrastto hot water, HCl hydrolysed recalcitrant S compounds, asevidenced by the observed strong positive correlations betweenHCl-S and immobilized S (Figure 1). Indeed, HCl can removecomponents such as carbohydrates, proteins, amino acids andnucleic acids, which are strongly integrated into the structureof humic compounds (Paul et al., 2006; Silveira et al., 2008).The smallest proportions of HCl-S were observed in the forestsoil, which was rich in organic C content (Table 1). In this soil,S would be strongly bound in complex humic macromoleculesand would, thus, be less accessible to HCl hydrolytic action(Table 4). This clearly indicates an enhanced chemical stabilityof the immobilized S in the forest soil. Therefore, in contrast toHW-S, the turnover rates of HCl-S will differ over time.

Remineralization of the immobilized-35S

The 0.009 m Ca(H2PO4)2 solution used in this study may co-extract soluble organic S. However, soluble organic S is rapidlyturned over in well-aerated soils (Janzen & Bettany, 1987), andthe presence of this fraction in a calcareous soil with a grumulousstructure such as the studied soils, would be negligible. Thus,soluble organic S would not affect the SA values calculated asratios between labelled and unlabelled sulphate S measured inthe 0.009 m Ca(H2PO4)2 solution (Tables 2–4). Immobilizationand re-mineralization of S occur concurrently in soil and anumber of short-term measurements would be needed to correctlydetect these dynamic processes over time. However, our resultsshowed an early re-mineralization of salicylate in these threesoils (Tables 2–4 and Figure 2). Indeed, an increase in SA valuesindicates that immobilized-35S is re-mineralized (Eriksen, 2005).With salicylate, a complex molecule not immediately available formicroorganism growth, the maximum amount of immobilized-35S



induced by labile C present in the studied soils was reachedquickly (within 1 week in the arable soil and 2 weeks in thefallow and forest soils richer in organic C). Consequently, earlyre-mineralization of immobilized-35S took place thereafter, asit has been found that freshly immobilized S is more readilymobilized than the bulk of organic S (Ghani et al., 1993).Similarly, in the forest soil amended with glutamate, the early andsignificantly greater level of immobilization of 35S noted at week 1(17.2% more than the control) was followed by a re-mineralizationof immobilized-35S observed at week 2. No positive increasein SA values (indicating 35S re-mineralization) was found withglucose. This observation may be because re-mineralization ofimmobilized-35S with glucose may go undetected for the timeintervals studied. In the arable soil and especially in the fallowsoil, the SA values decreased more markedly than in the forest soilat week 12, suggesting a difference in the nature and compositionof the microorganisms in these soils. Thus, the extent of 35S re-mineralization would differ according to soil management.

In the arable soil, the significant cumulated SA values werein the order salicylate > glutamate = glucose and this suggeststhat glutamate and glucose addition produced more recalcitrantS compounds than salicylate. Most importantly, although statis-tically non-significant, the results collectively showed the sametrend of changes in cumulated SA values in the order salicy-late > glucose > glutamate in the three soils. These results indi-cate that glutamate enrichment induced more recalcitrant S com-pounds than glucose. Furthermore, both bacteria and fungi areable to use glutamate (C/N = 5) because it is easily decomposedand assimilated (Lucas et al., 2007). Our results support those ofOgawa et al. (2001), which showed that 7% of the initial glucoseC was converted by bacteria to recalcitrant particulate organic Ccompared with 22% of the initial glutamate C after 48 hours.According to these researchers, these new organic compoundsremain recalcitrant at least for 1 year. Similarly, Gruber et al.(2006) indicated that bacteria rapidly utilized glucose C to pro-duce new dissolved organic compounds of high molecular weightwithin 2 days. These new organic compounds, representing 3–5%of the glucose C added, were recalcitrant and persisted over amonth. As a whole, the steady decrease in SA values, suggestingan immobilization of 35S over 35S re-mineralization, was morepronounced in the fallow soil than in the arable soil. These resultscould be attributed to the greater biological activity observed in thefallow soil compared with the arable soil (Vong et al., 2008). Inthe forest soil, the autochthonous microbial communities that arealready adapted to use complex litters and recalcitrant humic com-pounds (Osono & Takeda, 2001) may easily break down the addedsubstrates. This observation may explain the relatively faster 35Sre-mineralization noted in the forest soil (especially at week 12)compared with the fallow soil. Finally, glutamate, which hasa small C : N ratio (5:1), promoted more recalcitrant microbialS compounds than glucose. Glutamate contributed to microbialS-containing amino acid synthesis. In agreement with Bettanyet al. (1979), these amino acids provided the organic C-bondedS that is firmly integrated into the structure of humic acids.

Conclusions

The LMWC largely present in the rhizosphere, such as glucoseand glutamate, efficiently stimulated microbial biomass growthand activity with consequent S immobilization. The action of 3 mHCl was drastic, resulting in the release of more heterogeneousmaterials, including recalcitrant S components, than hot water.By using hot water as an extractant, we demonstrated that newlyimmobilized-S was more labile than the older immobilized-S. Fur-thermore, glutamate and glucose addition yielded more recalcitrantorganic S compounds than salicylate. Apparently, glutamate pro-duced more recalcitrant S compounds than glucose. Finally, on thebasis of our previous findings, which showed that HW-S is readilyavailable for plants, we concluded that HW-S is a homogeneouspool in terms of turnover rate. Thus, measurement of HW-S mayaid S modelling. Further chemical characterization of HW-S couldimprove the usefulness of this fraction in understanding sulphurdynamics in the soil-plant system or in different agro-ecosystems.Efficient microbial S immobilization was followed by reducedre-mineralization of the immobilized S, at least in the short-term.This evidence should be taken into account when synchronizingS application with S requirement by plants.

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