high temperature catalytic oxidation

Analytica Chimica Acta 445 (2001) 107115

High-temperature catalytic oxidation method for measuringtotal dissolved nitrogen in K2SO4 soil extracts

G. Alavoine, B. NicolardotINRA Unit dAgronomie de Chlons-Reims, Centre de Recherche Agronomique,

2 Esplanade Roland Garros, BP 224, 51686 Reims Cedex 2, France

Received 16 March 2001; accepted 28 June 2001

Abstract

A high-temperature catalytic oxidation (HTCO) method was evaluated and compared with the Kjeldahl method for de-termining total dissolved N (TDN) in 0.025 M K2SO4 soil extracts used to assess soil microbial biomass nitrogen. First theHTCO method was evaluated for routine analysis for TDN in K2SO4 solutions. Nitrogen recoveries measured by HTCOexceeded 98% for compounds containing organic and inorganic N and for concentrations up to 10 mg N l1. The proposedindirect injection of sample into the furnace safeguards the catalyst and quartz combustion tube. In these conditions, the drift ofsignal sensitivity during sequence analysis was negligible implying that HTCO can be used for the routine analysis of samplesin a 0.025 M K2SO4 matrix. The HTCO and Kjeldahl methods were then compared for determining soil microbial biomassnitrogen in different types of soil using the fumigation-extraction method. The TDN concentrations in K2SO4 soil extractsmeasured with both methods were well correlated. Some discrepancies however, were, observed for TDN concentrations>10 mg N l1 due to a probable underestimation of N in the soil extracts with the Kjeldahl method. The calculated extractablemicrobial biomass values resulting from the difference in TDN between control and fumigated soils were in good agreementfor both methods. HTCO thus provides a very rapid and reliable method for measuring TDN in K2SO4 soil extracts used todetermine soil microbial biomass nitrogen. 2001 Elsevier Science B.V. All rights reserved.

Keywords: Microbial biomass nitrogen; Total dissolved nitrogen; High-temperature catalytic oxidation; Kjeldahl method

1. Introduction

CHCl3 fumigation-extraction (FE) is commonlyused to determine soil microbial biomass from mea-surement of the organic C and total nitrogen in 0.5 MK2SO4 extracts [13]. Measuring the organic C inaqueous solutions is, at present, also easy, rapid andaccurate thanks to the use of automated infrared anal-ysers after oxidation of the organic carbon to CO2[4]. Elsewhere, the methods generally used to de-

Corresponding author. Tel.: +33-3-26-77-35-80;fax: +33-3-26-77-35-91.E-mail address: [email protected] (G. Alavoine).

termine total nitrogen in the microbial biomass, arethe total Kjeldahl nitrogen (TKN) methods [57] andthe alkaline peroxidisulfate oxidation method [38].Although the digestion step for the peroxidisulfate ox-idation method is much simpler than for the Kjeldahlmethod, both methods require an additional step tomeasure inorganic N after the digestion, and thus aretime-consuming. The TKN method is the referencemethod, but the numerous adaptations [9] precludeit from being a totally universal method, especiallywhen the soil extracts contain a lot of nitrate, in whichcase the measurement of total N may be inaccurateand unreliable [10]. On the other hand, the numerousmanual analytical procedures cited in the literature

0003-2670/01/$ see front matter 2001 Elsevier Science B.V. All rights reserved.PII: S 0003 -2670 (01 )01239 -9

108 G. Alavoine, B. Nicolardot / Analytica Chimica Acta 445 (2001) 107115

for the measurement of N in soils extracts can lead tohighly variable biomass N results, because such meth-ods are not always properly calibrated or applied [4].

Various authors have already chosen oxidative com-bustion methods [11] to determine total dissolved N(TDN) in soil solution and extracts, for concentrationsranging from 0.5 to 125 mg N l1 in a non-saline ma-trix and by using a catalytic oxidation. The methodconsisted of a catalysed combustion of the sample in avertical furnace at 680C, followed by detection of Ngases generated during the combustion. Suzuki et al.[12] also used high-temperature catalytic oxidation(HTCO) at the same temperature to determine total Nin a seawater matrix, but for a range of very low con-centrations (0.070.7 mg N l1). Finally, Walsh [13]also determined total N in seawater but used an oxida-tive combustion method at 1100C without catalyst.In the latter, the direct liquid injection was replacedwith indirect injection into a quartz boat before intro-duction into the furnace.

The aim of our work was to evaluate HTCO methodfor measuring total dissolved N in K2SO4 soil ex-tracts following the procedure proposed in the originalmethod [1,2] and commonly used by numerous au-thors [3,4,816], except that 0.5 M K2SO4 extractantwas replaced by 0.025 M K2SO4. The modification ofthe original procedure was justified by the work ofChaussod et al. [17] who have clearly showed that theuse of a K2SO4 concentration

G. Alavoine, B. Nicolardot / Analytica Chimica Acta 445 (2001) 107115 109

Table 2Main analytical characteristics of soils

Soilno.

Soil description Clay(g kg1)

Silt(g kg1)

Sand(g kg1)

CaCO3(g kg1)

C(g kg1)

N(g kg1)

pH

1 Hypercalcareous rendosol 99 110 8 783 18.1 1.7 8.32 Redoxisol brunisol desaturated 232 509 259 0 19.0 1.9 6.63 Calcareous clayey soil on calcareous rock 263 287 30 420 12.9 1.2 8.54 Calcareous clayey soil 369 175 109 347 13.1 1.3 8.55 Deep brown soil 313 572 113 2 10.4 1.1 7.4

2.1.2. K2SO4 soil extractsSoil microbial biomass was determined for five

different agricultural soils (Table 2). Soil 1 was a cul-tivated soil (Champagne, France), soil 2, a grasslandsoil (Brittany, France), and soils 35 were vineyardsoils (Burgundy, France). For these different soils, var-ious management strategies (N fertilisation, organicamendments) have been taken into account in orderto measure a wide range of TDN in soil extracts. Themicrobial biomass nitrogen was determined on sieved(


Fig. 1. Scheme of the indirect injection system.

a programmable controller. Samples (40 ml) were in-troduced into the digestion tubes to ensure sufficientsensitivity.

Before the digestion, any nitrate present in the sam-ples was reduced for 48 h at 120C in the presenceof 5 M H2SO4 (addition of 11 ml of 96% H2SO4 persample) and reduced iron (1 g per sample) [10]. Forthe digestion, 3 ml of 96% H2SO4 was introduced intothe tubes with a one-half Kjeldahl tablet (Merck ref.15348). To avoid liquid ejection, the digestion tem-perature was then slowly increased (50C h1) from150C to a final temperature of 375C which wasmaintained for 2 h. The ammonium nitrogen presentin the digests was then steam distilled after addition ofan excess of 12 M NaOH [22]. The NH3 was trappedby 0.05 M H2SO4 and determined by back titrationusing a 719 titrator (Metrohm, Herisau, Switzerland).When the TDN concentrations in the samples were98%(Table 1) and in agreement with other published data:for lower concentrations, 0.56 mg N l1 dissolved inseawater, and measured by high-temperature oxidation(HTO), Walsh [13] reported almost 100% N recoveryfor NH4Cl and KNO3, 99.5% for leucine and 101.7%for serine but slightly more than 102% for urea. Mer-riam et al. [11], using a HTCO method at 680C,obtained average recoveries for N compounds dis-solved in doubly deionised water, of up to 90% exceptfor urea that gave an average of 88.4% for concentra-tions between 0.5 and 10 mg N l1, and with a slightdecline in percentage recovery as the concentrationincreased. Recovery values exceeding 100% were ob-tained with our HTCO method for urea, atropine andsodium nitrite. This might be explained by a better


Fig. 2. Theoretical and measured total dissolved N obtained with HTCO and Kjeldahl (TKN) methods (line corresponds to y = x; barscorrespond to confidence intervals, P = 0.05).

oxidation or conversion to nitric oxide (NO) withthese compounds than with the KNO3 and (NH4)2SO4standards used to calibrate the analyser. Elsewhere,the N recovery obtained with the TKN method, forthe NO3 solution, was 100% and in good agreementwith the results published by Guiraud and Fardeau[10].

Table 3Drift of sensitivity of standard (16 mg N l1) put in different positions in analysis sequencesa

Sequence number Number of samples analysed before the analysis of the three replicates of standard (mg N l1)5 10 15 20 25 30 35

1 15.94 (0.28) 15.57 (0.13) 15.69 (0.08) 16.01 (0.19) 15.96 (0.18) 16.05 (0.32) 16.17 (0.11)2 16.07 (0.14) 16.08 (0.06) 16.02 (0.08) 16.01 (0.11) 16.03 (0.06) 15.98 (0.10) 16.08 (0.15)3 16.14 (0.05) 16.26 (0.14) 16.19 (0.12) 16.11 (0.10) 16.21 (0.14) 16.27 (0.08) 16.34 (0.26)4 15.98 (0.11) 16.03 (0.10) 16.07 (0.12) 15.97 (0.10) 16.02 (0.14) 15.91 (0.08) 15.88 (0.16)

a Values in parentheses are standard error (n = 3).

The results obtained for analysis of the same stan-dard (16 mg N l1) placed at different positions in ananalytical sequence are presented in Table 3. Underour analytical conditions using indirect injection, therewas generally not much sensitivity drift of the signalduring a sequence analysis, the mean R.S.D. betweenthe three replicates often being


difference between the theoretical value and the mea-sured value was 2.7%. Direct injection of the salt so-lutions into the furnace, as described by Alavoine andNicolardot [20], cannot be used for the routine analysisof samples in a K2SO4 matrix due to the poor repro-ducibility and high drift of sensitivity, a 25% decreaseof the signal, compared to its initial value, was ob-tained when a standard was analysed after the first 25samples in the analysis sequence (results not shown).This drift of sensitivity with direct injection may beexplained by a rapid decrease of the catalyst efficiencywhich was certainly due to the deposit of salt on thePt/Al2O3 pellets.

3.2. Comparison of N concentrations measured withTKN and HTCO methods

The total dissolved nitrogen (TDN) values ofK2SO4 soil extracts measured with both methods

Fig. 3. Comparison of TDN concentrations measured in K2SO4 extracts, using HTCO and Kjeldahl (TKN) methods (line corresponds toy = x; bars correspond to confidence intervals, P = 0.05).

(Fig. 3) were well correlated (r = 0.98; P = 0.01).Nevertheless, different concentrations were obtainedby the HTCO and TKN methods for some extracts. Forhigh N concentration extracts (TDN >10 mg N l1),the TKN method tended to give lower concentrationsthan the HTCO method. This could be due to an un-derestimation of TDN by the TKN method since the Nrecovery by HTCO method was close to 1002%, theHTCO values then being considered as more reliable.This underestimation cannot occur during the nitratereduction procedure, which is considered to be com-plete [10]. Despite a progressive increase in tempera-ture, some irregular losses by ejection of liquid and Noutside the digestion tubes may occur during the vol-ume reduction step, just before digestion of the extract.In addition, some errors may be due to an inaccuratemeasurement of the blanks for some analytical sets.

Precautions must also be taken to avoid possible bi-ological evolution of the N concentration in filtered


Fig. 4. Evolution of the total dissolved N concentration (TDN) measured by HTCO in K2SO4 soil extracts. (bars correspond to confidenceintervals, P = 0.05).

extracts before the analysis, as is shown in Fig. 4.These results indicate that the measured concentrationcan decrease significantly when the extracts remain atlaboratory temperature for several hours before beinganalysed. In addition, the evolution of the concentra-tion depends on the sample treatment sample evolu-tion being much greater with fumigated soil extracts.It thus, seems very important to analyse the extractsquickly after defrosting or just after soil extraction, aschemicals (e.g. HCl or Hg2Cl2) cannot be used for bi-ological stabilisation of the soil extracts due to theirdestructive effect on the catalyst and materials of theanalyser materials.

The extractable microbial biomass nitrogen values(EN = TDN in K2SO4 fumigated soil extractTDNin K2SO4 control soil extract) obtained with bothmethods (Fig. 5) were well correlated (r = 0.98;

P = 0.01). The R.S.D. values were higher for theTKN method, the average being 6.5 and 4.4% for anEN value higher than 40 and less than 20 mg N kg1soil, respectively, compared to 2.8 and 1.4% forR.S.D. values of EN measurements obtained withthe HTCO method. The R.S.D. values for the TKNmethod were similar to those indicated by Joer-gensen and Olfs [4] who compared several methodsof Kjeldahl digestion (mean R.S.D. = 6.7%) andUV-peroxodisulfate oxidation/colorimetric detection(UV/COL) (mean R.S.D. = 14.3%). Finally, ourresults tend to show that the semi-micro Kjeldahlused here was not sensitive enough to give reli-able results within the range of concentration of060 mg N kg1 soil which includes the N concentra-tions generally obtained for fumigated or control soilextracts.


Fig. 5. Relationship between extractable microbial biomass nitrogen values (EN) obtained with HTCO and TKN methods (line correspondsto y = x; bars correspond to confidence intervals, P = 0.05).

4. Conclusions

Until now, the HTCO method has not been usedto determine TDN in K2SO4 soils extracts. This maybe explained by the difficulty of performing routinedeterminations of TDN in a K2SO4 matrix using ahigh-temperature combustion method with direct in-jection of sample into the furnace. The TDN can bemeasured in such soil extracts if an indirect methodof sample injection is used.

Although further investigations would be necessaryto precisely identify the origin of the differences no-ticed between TDN concentration values measured byeach method, this work has clearly shown that soil ex-tractable microbial biomass nitrogen values obtainedwith HTCO and TKN methods were well correlated.Thus, the automated HTCO method appears to be avery rapid and reliable method for measuring TDN

in these 0.025 M soil extracts. In fact, and taking intoaccount the procedures used in the quality controlsof the analysis, more than 48 samples can be anal-ysed per day using this modified HTCO method. Byusing the efficient salt trap, it should be possible toanalyse more than 1500 K2SO4 soil extracts withoutdestroying the quartz combustion tube or the solidmodule.

The determination of TDN in a K2SO4 ma-trix >0.025 M using HTCO was not carried outin our work. However, for higher K2SO4 con-centrations, the determination will be possiblewith the HTCO method but will imply some con-straints, particularly for a 0.5 M K2SO4 matrix.Indeed, the trap for the salts, and probably the cat-alyst, may have to be replaced more often and thelife of the quartz injection module could also beshorter.


Acknowledgements

We thank the Euroglas Analytical Instrument Com-pany and especially Mr. P. Veth and Mr. H. Kampertfor providing valuable advice and help in perfectingthe HTCO method.

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high temperature catalytic oxidation

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