Alkaline Persulfate Oxidation for Determining Total Nitrogen

in Microbial Biomass Extracts

M. L. Cabrera* and M. H. Beare
ABSTRACT in natural waters as an alternative to the acid Kjeldahl
The CHCI., fumigation-extraction method for determining soil mi- digestion. The method uses peroxydisulfate (K2S2O8)
crobial N involves the extraction of CHCI3-fumigated and CHCl3-un- to oxidize N to NOf at elevated temperature in an
fumigated samples with 0.5 M K,SO4, followed by determination of alkaline environment. Under such conditions, K2S2O8
total N in the extracts. Total N is typically determined by Kjeldahl autodecomposes as shown below, generating the O2
digestion (TKN) with the inclusion of NO; and NOj present. The needed for the oxidation of N.
determination of total N by TKN has several procedural constraints
such as an initial concentration step, long digestion times, and con- K2S2O8 + H2O -» 2 KHSO4 1/2 O2
siderable bumping caused by catalyst salts. In contrast, a persulfate
oxidation method (TPN) commonly used for determination of total N D'Elia et al. (1977) found that, for seawater sam-
in sea- and freshwater samples does not require a concentration step ples, TPN yielded similar values to those obtained by
and is much simpler and faster than TKN. The objective of this study adding initial NOj and NOj concentrations to values
was to evaluate and adapt the TPN method to measure total N in 0.5 determined by TKN. Similarly, Nydahl (1978) found
M K2SO4 extracts for determination of soil microbial biomass N. Stud- that, for effluents from sewage sludge plants, TPN
ies were conducted to optimize reagent/sample (R/S) ratio and auto- gave similar results to TKN after reduction of the
claving time, and to evaluate the effects of C and N concentrations.
NOj and NOj present. Solorzano and Sharp (1980)
These studies showed that complete N recovery can be obtained with
a R/S ratio of 1 and an autoclaving time of 30 min in extracts con-
proposed modifications to the method in order to make
taining as much as 250 mg glucose-C L~' at a C/N ratio of 10:1. A
it acceptable for samples from fresh to oceanic waters.
comparison of the optimized TPN method to TKN for extracts of 12 The persulfate oxidation has been used to determine
CHCL3-fumigated soils indicated that the methods gave the same N total N in soil leachates obtained with 0.01 M CaCl2
recoveries. The use of the TPN method in 0.5 M K2SO4 extracts should (Robertson et al., 1988) and water (Quails and Haines,
facilitate more rapid and efficient measurements of soil microbial bi- 1991). Gallardo and Schlesinger (1990) used the pro-
omass N. cedure described by D'Elia (1977) to measure total N
in 0.5 M K2SO4 soil extracts. Ross (1992) adopted a
nonalkaline persulfate oxidation procedure to obtain
complete recovery of inorganic N in 0.5 M K2SO4
T HE CHCL3 FUMIGATION-EXTRACTION
determining soil microbial biomass involves the
extraction of CHCL -fumigated and unfumigated sam-
3
METHOD for extracts of soils with high initial NH^ concentrations,
but acknowledged the potential for incomplete oxi-
dation of organic N forms. Based on these studies, it
ples with 0.5 M K2SO4, followed by determinations appears that TPN is a viable alternative for determin-
of organic C and N in the extracts. Although the analysis ing total N in soil extracts. There have been no stud-
of TOC in these extracts has become relatively routine ies, however, to optimize the method for soil extractants
with the use of automated TOC analyzers (Wu et al., and to compare the recovery of TPN with that of TKN.
1990; Jordan and Beare, 1991), the analysis of total The objective of this study was to evaluate and adapt
N in soil extracts has been limited by a number of the TPN method to measure total N in 0.5 M K2SO4
procedural constraints. Total N has most often been soil extracts for determination of soil microbial bio-
determined by TKN (Brookes et al., 1985; Sparling mass N. We report here on several experiments con-
and West, 1988), which includes an initial sample ducted to optimize the method for these extracts and
concentration step and is limited by the number of on a trial carried out to compare the optimized method
samples that can be retained in a block digester. In to TKN.
addition, either the NO~2 and NOj present have to be
determined separately and added to the amount mea- MATERIALS AND METHODS
sured by TKN or they must be reduced before the acid
digestion. Further, the catalyst salts used in the diges- Preparation of Oxidizing Reagent
tion are susceptible to bumping, which can introduce The oxidizing reagent was prepared as described by Koroleff
considerable analytical error. An alternative method (1983). Briefly, 25 g of low-N K2S2O8 (UN1492, Fisher Sci-
for determining total N in 0.5 M K2SO4 extracts is the entific, Pittsburg, PA) and 15 g of H3BO4 were dissolved in
acid dichromate oxidation, in which N is converted to 50 mL of 3.75 M NaOH, and the volume was made up to 500
NH+4-N and measured by the indophenol colorimetric mL with distilled-deionized water. The reagent can be stored
method. However, Sparling and West (1988) found for up to 1 wk at room temperature in a dark bottle.
interference from Cr3+ in the determination of NHV-
N. General Procedure
Koroleff (1970, 1983) proposed an alkaline persul- Aliquots of sample and oxidizing reagent were pipetted into
fate oxidation method for determining total N (TPN) glass tubes, which were immediately sealed with screw caps
containing Teflon liners. The tubes were weighed and placed
M.L. Cabrera, Dep. of Agronomy and Institute of Ecology, and in an autoclave for 30 min (unless specified differently) at
M.H. Beare, Institute of Ecology, Univ. of Georgia, Athens, GA
30602. Received 6 Aug. 1992. *Corresponding author. Abbreviations: TKN, total Kjeldahl nitrogen; TPN, total persul-
fate nitrogen; TOC, total organic carbon; R/S, reagent to sample
Published in Soil Sci. Soc. Am. J. 57:1007-1012 (1993). ratio; H/L, headspace to liquid volume ratio.

1007
1008 SOIL SCI. SOC. AM. J., VOL. 57, JULY-AUGUST 1993

120 °C. After the autoclaving, the tubes were reweighed to Table 1. Some chemical and physical properties of the soils
determine moisture losses. The relatively small water losses used.
(mean = 1.2%; SD = 1.8%) were used to correct the final Cation-
NOj concentration in the solutions. Nitrate was determined exchange
using the Gries-Ilosvay procedure (Keeney and Nelson, 1982) Soil series C N capacity Silt Clay
with an Alpkem RFA-300 Autoanalyzer (Alpkem Corp.,
Clackamas, OR). — g kg-' — cmolc kg-' —— g lkg-' ——
Cecil 5.4 0.45 1.54 101 36
Dothan 4.0 0.29 1.65 130 29
Optimization Studies Eudora 8.2 0.83 10.23 653 107
Greenville 1 9.4 0.76 5.15 151 394
For the method to be useful for total N determinations, it Greenville 2 6.3 0.50 3.46 137 225
should digest all organic N compounds and recover primary Hiwassee 1 11.8 1.11 5.50 190 131
forms of inorganic N, namely NH^ and NO 5. The main ob- Hiwassee 2 20.2 1.91 6.66 201 112
Irwin 41.5 3.23 27.34 552 266
jective of the optimization studies was to adapt the method to Kahola 14.3 1.25 14.85 742 190
ensure complete oxidation and recovery of NH;J, as well as Pacolet 14.6 1.33 6.91 193 274
complete recovery of NOj. Urea and L-glutamic acid were Tifton 1 7.5 0.54 2.25 159 45
also used in some of these studies (to assess the oxidation and Tifton2 16.1 1.42 5.08 111 52
recovery of organic N), but the main test of the method's
capacity to oxidize organic N was evaluated after the optimi-
zation for recovery of inorganic N. As described above, the
method oxidizes NH^ with the O2 generated by the autode- soils have been recently fertilized with ammoniacal fertilizers,
composition of K2S2O8. Because the organic C present in the soil microbial biomass extracts may have a relatively high con-
samples is also oxidized by the O2 generated in the autode- centration of NHf and a low concentration of organic C. We
composition, organic C may compete with NH^ for the O2 conducted this study to determine if, in those cases, a low R/
released. For this reason, most of the simulated extracts used S ratio may be sufficient to oxidize all forms of N present. 1
in the optimization studies were amended with organic C in Solutions of 0.5 M K2SO4 containing 20 mg NH 4-N L- and
the form of glucose. C contents of 0, 25, 50,100, and 200 mg C L-1 were prepared
Effect of Reagent to Sample Ratio and Nitrogen Content with reagent-grade NH4C1 and glucose. A 5-mL aliquot of each
on Ammonium Oxidation. Because the C/N ratio of soil mi- solution was placed in a 20-mL glass tube and received 1, 2,
crobial biomass extracts usually varies between 5 and 10, we 3, or 5 mL of oxidizing reagent (R/S = 0.2, 0.4, 0.6, and 1,
conducted this study at a fixed C/N ratio = 10 in order to respectively). All treatments were replicated three times and
achieve the highest expected amount of C for a given amount blanks were prepared at each R/S ratio by mixing the proper
of N in the extract. Solutions of 0.5 M K2SO4 containing 2.5, amount of oxidizing reagent with 5 mL of 0.5 M K2S04.
5, 10, 25, 35, and 50 mg N L~* and the required C to achieve Effect of Sodium Hydroxide Concentration in Oxidizing
a C/N ratio = 10 were prepared with reagent-grade NH4C1 Reagent on Nitrogen Recovery. According to Solorzano and
and glucose. A 15-mL aliquot of each solution was placed in Sharp (1980), the persulfate oxidation must be carried out at
a 50-mL glass tube and received 3 or 9 mL of oxidizing reagent a pH of 12.6 to 13.2. Because preliminary measurements in-
(R/S = 0.2 and 0.6, respectively). All treatments were rep- dicated an initial pH of 9.9 in the samples prepared with the
licated four times and blanks were prepared at each R/S ratio reagent described above (0.375 M NaOH) at an R/S = 1, we
by mixing the proper amount of oxidizing reagent with 15 mL conducted a study to compare the effect of 0.375, 0.475, and
0.5 M K2SO4. The amount of K2S2Og required for the oxida- 0.575 M NaOH in the oxidizing reagent. The three oxidizing
tion, expressed as a percentage of the amount added, was cal- reagents were tested with solutions of 0.5 M K2SO4 containing
culated assuming that C and N require 1 and 1.5 mol of O2 5, 10, 20, 30, and 40 mg N L"1 (as urea) and corresponding
per mol of element, respectively. amounts of glucose to obtain a constant C/N = 10. A 15-mL
Effect of Headspace to Liquid Volume Ratio on Am- aliquot of each solution was placed into a 50-mL tube and 15
monium Oxidation. We were interested in studying the effect mL of oxidizing reagent added. Each treatment was replicated
of H/L ratio because a differential accumulation of CO2 in the five times and blanks were prepared by digesting 15 mL of
headspace may affect the efficiency of the oxidation process. 0.5 M K2SO4 with 15 mL of the corresponding oxidizing re-
The test solution was prepared in 0.5 M K2SO4 and contained agent. Initial pH values were 9.9, 10.9, and 12.6 for 0.375,
25 mg N L-1 (as NH4C1) and 250 mg C L-1 (as glucose). To 0.475, and 0.575 M NaOH in the oxidizing reagent, respec-
obtain different H/L ratios at a constant R/S = 0.6, volumes tively.
of 3.75, 7.50,10,15, and 20 mL of the solution were pipetted Effect of Autoclaving Time on Urea Oxidation. Nydahl
into 50-mL glass tubes and amended with oxidizing reagent in (1978) found that autoclaving samples for 10 min at 120 °C
volumes of 2.25, 4.5, 6, 9, and 12 mL, respectively. This was sufficient to decompose all the K2S2O8 added in 10 mL
yielded H/L ratios of 0.56, 1.08, 2.13, 3.17, and 7.33. All of oxidizing reagent with a low concentration of K2S2O8 (0.05
treatments were replicated four times. M). Koroleff (1983), on the other hand, recommended 30 min
Effect of Reagent to Sample Ratio and Nitrogen Source of autoclaving for the higher concentration of K2S2O8 (0.185
on Nitrogen Oxidation. This study was conducted to evaluate M) used in this work. In order to study the effect of autoclaving
the effect of R/S on the oxidation of N from organic sources time under our experimental conditions, we evaluated N re-
and from NH4C1. Solutions containing 20 mg 1N L-1 (as urea, covery at 10, 20, 30, and 40 min of autoclaving. The test
NH4C1, or L-glutamic acid) and 200 mg C L- (adjusted with solutions were prepared in 0.5 M K2S04 or 1 M KC1 and
glucose) were prepared in a 0.5 M K2SO4 matrix. Fifteen milli- contained 20 mg urea-N L-1 and 200 mg C L-1 (adjusted with
liters of each solution were pipetted into 50-mL glass tubes, glucose). The KC1 solution was included because it is a com-
which received 3, 6, 9, or 15 mL of oxidizing reagent (R/S mon soil extractant and there may be interest in determining
= 0.2, 0.4, 0.6, and 1.0, respectively). Blanks were prepared its total N content. We used six replicates of each treatment
by adding the corresponding amounts of oxidizing reagent to and an R/S = 1.
tubes containing 15 mL of a glucose solution (200 mg C L-1) Recovery of Nitrate. Solutions of 0.5 M K2SO4 containing
prepared in 0.5 M K2SO4. All treatments were replicated six 10 mg N L-1 (as KNO3 or urea) and 100 mg C L-1 (adjusted
times. with glucose) were used to evaluate the recovery of NOf and
Effect of Reagent to Sample Ratio and Carbon Content urea-N at an R/S = 1. Urea was included to check the per-
on Ammonium Oxidation. In certain cases, particularly when formance of the method during the evaluation of NOj recov-
 CABRERA & BEARE: ALKALINE PERSULFATE OXIDATION FOR DETERMINING MICROBIAL NITROGEN 1009

Table 2. Effect of reagent/sample (R/S) ratio (0.2 or 0.6) and Table 3. Effect of reagent/sample (R/S) ratio and C content
N content on NH^ recovery. on NH4-N recovery.
Persulfate NH4-N recovery
Sample N recovery requiredt
Glucose 0.2 0.4 0.6 1.0
N C 0.2 0.6 0.2 0.6
mg C L-1 ofapjdied ———
— mgL ' — 0 62.5 89.4 100.7 99.1
2.5 25 99.8 98.9 12.8 4.2 25 71.1 87.6 93.1 100.1
5 50 102.5 102.0 25.4 8.5 50 76.9 92.3 94.4 101.9
10 100 98.3 104.4 50.8 16.9 100 72.2 83.5 93.7 102.5
25 250 0.6 101.3 127.1 42.5 200 44.8 84.4 96.0 102.2
35 350 0.0 95.2 178.0 59.2 LSD(O.OS) 2.6
50 500 0.0 82.5 254.0 84.7
LSD(O.OS) 1.3 Source of variation
R/S **
Source of variation C content **
N content ** Replicate NS
R/S ** R/S x C **
Replicate NS
N x R/S ** ** Significant at the 0.01 probability level; NS indicates not significant
at the 0.05 probability level.
** Significant at the 0.01 probability level; NS indicates not significant
at a 0.05 probability level.
t Persulfate required to oxidize all glucose-C and NHJ present in the
sample. wetted to 60% water-holding capacity with a N-free nutrient
solution (Cabrera and Kissel, 1988) and incubated in a plastic
bag for 15 d at 30 °C. Water-holding capacity was measured
ery. Each treatment was replicated nine times and blanks were as the water content of the soil after saturation followed by 24
carried out using a solution of 0.5 M K2SO4 containing 100 h of free drainage. Following incubation, the soil was leached
mg C L~' as glucose. with 600 mL (about four pore volumes) of N-free nutrient
solution, returned to the bag, and refrigerated at 3 °C. Four
Comparison of Total Persulfate Nitrogen to Total subsamples of each soil (=60 g dry weight equivalent) were
Kjeldahl Nitrogen weighed into 500-mL flasks and fumigated in a desiccator (3
d) with alcohol-free CHC13. After fumigation, the CHC13 was
To determine the efficacy of the persulfate oxidation pro- removed and the desiccator repeatedly evacuated to remove
cedure, TPN results were compared with measures of TKN residual CHC13. Two of the subsamples were extracted with
plus (NOj + NOj )-N in CHCl3-fumigated extracts of 12 0.5 M K2SO4 (300 mL) and two were extracted with 1 M KC1
different soils. Samples were collected from the upper 15 cm (300 mL). After 30 min of rotary shaking, extracts were fil-
of areas mapped as Cecil loamy sand (clayey, kaolinitic, thermic tered (preleached no. 42 Whatman) and the paired filtrates
Typic Kanhapludult), Dothan loamy sand (fine-loamy, sili- combined. Comparisons of TPN and TKN recoveries were
ceous, thermic Plinthic Kandiudult), Greenville sandy clay loam made from five replicate analyses of each K2SO4 and KC1
and Greenville clay loam (clayey, kaolinitic, thermic Rhodic extract. For TPN analyses, extracts were combined with an
Kandiudults), Hiwassee fine sandy loam (clayey, kaolinitic, oxidizing reagent (R/S = 1) in capped tubes and autoclaved
thermic Rhodic Kanhapludult), Pace-let sandy clay loam (clayey, at 120 °C for 30 min. The digests were analyzed for NQ-3-N
kaolinitic, thermic Typic Kanhapludult), and Tifton loamy sand as described above and the concentrations corrected for water
(fine-loamy, siliceous, thermic Plinthic Kandiudult) in Geor- loss. For the TKN analyses, extracts (15 mL) were acidified
gia, and from areas mapped as Eudora silt loam (coarse-silty, with 0.25 mL of concentrated H2SO4 and heated in an alu-
mixed, mesic Fluventic Hapludoll), Kahola silt loam (fine- minum block (110-120 °C) to reduce the volume to 1 to 2 mL.
silty, mixed, mesic Cumulic Hapludoll), and Irwin silt loam After cooling, the tubes were amended with 3 mL of H2SO4,
(fine, mixed, mesic Pachic Argiustoll) in Kansas. The soils 1 g of Na2SO4, and 0.1 g of CuSO4 and heated in the block
were selected to represent a range of conditions, including digester for 3 h at 340 °C (Sparling and West, 1988). The
differences in texture and total N content (Table 1). For these tubes were brought to volume (50 mL) with deionized water
comparisons, =310 g of air-dried, sieved (2.0-mm) soil was and the NH v-N was determined by the salicylate method (Kee-

ui 100 100
< O
DC Urea UJ
80 80 s Q
UJ 90
Ammonium Chloride. V) DC NaOH
V)
60 L-Glutamic Acid 60
UJ
<
Persulfate o O
0.375 M
a o o 80 0.475 M
UJ UJ 0.575 M
DC 40 40 DC
UJ I LSD (0.05)
O 70
o 20 20 Ul
UJ DC
DC DC
Ul
D. 60
0.2 0.4 0.6 0.8 1.0 1.2 0 5 10 15 20 25 30 35 40 45
REAGENT/SAMPLE RATIO (R/S) INITIAL UREA CONCENTRATION (mg N I
Fig. 1. Nitrogen recovery as affected by reagent/sample ratio Fig. 2. Urea-N recovery as affected by initial urea and NaOH
and N source. concentrations.
1010 SOIL SCI. SOC. AM. J., VOL. 57, JULY-AUGUST 1993

ney and Nelson, 1982) with an Alpkem Rapid Flow Analyzer

(Alpkem Corp., Clackamas, OR). Initial (NOj + NQ-3)-N a. K 2 SO 4 Extracts
concentrations in the extracts were added to the NHV-N de- . — TPN = 0.405 + 0.975 (TKN + NOg)
termined by TKN for comparison with TPN results. Total or- R2 = 0.98, P<0.01
ganic C in the extracts was measured with a combustion- ••-• TPN = TKN + NO7
nondispersive infrared gas analysis method on a Shimadzu TOC-
500 analyzer (Shimadzu Corp., Kyoto, Japan).
Statistical Analyses
Analyses of variance and regressions were carried out with
procedures available in SAS (SAS Institute, 1985).

RESULTS AND DISCUSSION 10

-1.
TKN + MO3 (mg M L )
Optimization Studies
Effect of Reagent to Sample Ratio and Nitrogen
Content on Ammonium Recovery. Almost complete —— TPN = 1.132 + 0.711 (TKN + NOs)
recovery of the NH^ added was obtained with N con-
centrations as high as 10 mg N L"1 at an R/S =1 0.2,
and with N concentrations as high as 25 mg N L" at an
R/S = 0.6 (Table 2). At an R/S = 0.2, the recoveries
decreased to zero above 10 mg N L-1 because the O2
generated by the persulfate was completely consumed in
the oxidation of C from the glucose added. The same
effect can explain the reduction of recoveries above 25
mg N L-1 at an R/S = 0.6. The recovery was 95.2%
when the percentage of K2S2O8 consumed was 59.2%,
and decreased to 82.5% when the consumption of K2S208
increased to 84.7%. These results agree with those of TKN + NO3 (mg N L )
Nydahl (1978), who found an NH^ recovery of 97.5% Fig. 3. Comparison of values measured by total persulfate N
when 50% of the K2S2O8 was consumed, and a recovery (TPN) to those obtained by total Kjeldahl N (TKN; including
of 80.6% with a persulfate consumption of 100%. We initial NOf and NOf ) for (a) 0.5 M K2SO4 and (b) 1 M
should point out, however, that the effect of the propor- KC1 extracts.
tion of persulfate consumed is expected to vary depend-
ing on the N and C sources being oxidized. For example,
Nydahl (1978) recovered only 58% of the N added at a Ammonium Recovery. Studying the effect of H/L ratio
consumption level of 50% when the N and C sources is important because the organic C oxidized during the
used were acetyl glycine and sucrose instead of NH4C1 digestion is converted to CO2, which could be evolved
and glucose. With acetyl glycine and sucrose, he ob- and accumulate in the headspace. In theory, smaller H/
served reduced recoveries at persulfate consumption lev- L ratios would lead to higher pressures in the headspace,
els as low as 10%. Koroleff (1983) recommends a which could affect the oxidation reaction. Our results
maximum persulfate consumption of 10% in order to showed that all the H/L ratios yielded near-100% recov-
obtain complete oxidation of organic compounds. eries with no significant differences (P < 0.05) between
Effect of Headspace to Liquid Volume Ratio on them (data not shown). The significance of this finding
is a considerable flexibility in the size of the tubes that
may be used for the oxidation.
Table 4. Effect of extractant and autoclaving time on urea-N Effect of Reagent to Sample Ratio and Nitrogen
recovery. Source. The N recovery from all sources was almost
Extractant Autoclave time N recovered complete at R/S = 1.0 (101.8, 98.7, and 97.8% for
min %
urea, NH4C1, and L-glutamic acid, respectively) and sig-
nificantly decreased at lower R/S ratios (Fig. 1). In gen-
0.5 M K2SO4 10 97.9 (1.4)t eral, the recoveries were significantly higher for urea
20 99.5 (1.1)
30 97.6 (1.1) than for any of the other two sources. The lower recovery
40 98.5 (1.1) obtained with NH4C1 at an R/S = 0.2 was probably due
1MKC1 10 92.8 (1.1) to a more rapid NH3 volatilization than NH^ oxidation.
20 92.8 (0.8) Ross (1992) reported 10 to 20% lower recoveries of ex-
30 92.7 (1.3)
40 92.6 (0.7) tractable N using alkaline (0.2 M NaOH) vs. nonalkaline
LSD(0.05) 1.3 persulfate oxidizing reagents in soils with high initial
Source of variation
NHJ concentrations (56 and 64 mg N kg"1)- Apparently,
#* NH3 volatilization may result in incomplete N recoveries
Extractant when the concentration of oxidizing reagent is not suf-
Time NS
Extractant x time NS ficient to ensure a fast oxidation reaction. The results of
this study, however, show that quantitative recovery of
** Significant at the 0.01 probability level; NS indicates not significant
at a 0.05 probability level. NH^ can be obtained with appropriate R/S ratios. In
t Values in parentheses are standard deviations. addition, the results indicate that an R/S = 1 would be
 CABRERA & BEARE: ALKALINE PERSULFATE OXIDATION FOR DETERMINING MICROBIAL NITROGEN 1011

Table 5. Comparison of total persulfate N (TPN) to total Kjeldahl N (TKN) for 0.5 M K2SO4 extracts.
Series Total organic C TKN NO3-Nt TKN + NO3-N TPN
———— mg L-' ————
Cecil 17.5 (0.7)* 2.9 (0.08) 0.06 (0.004) 2.9 (0.07) 2.9 (0.04)
Dothan 10.8 (0.3) 1.7 (0.18) 0.01 (0.000) 1.7 (0.18) 1.7 (0.49)
Eudora 16.1 (0.5) 3.1 (0.14) 0.00 (0.000) 3.1 (0.14) 3.5 (0.15)
Greenville 1 36.0 (0.5) 4.3 (0.31) 0.37 (0.019) 4.7 (0.30) 5.4 (0.12)
Greenville 2 39.1 (0.5) 4.6 (0.15) 0.12 (0.000) 4.7 (0.15) 4.8 (0.05)
Hiwassee 1 41.7 (0.6) 7.0 (0.12) 0.01 (0.007) 7.0 (0.12) 7.0 (0.11)
Hiwassee 2 59.0 (0.5) 8.8 (0.87) 0.01 (0.000) 8.8 (0.87) 8.9 (0.24)
Irwin 107.8 (0.3) 17.4 (0.43) 0.09 (0.005) 17.5 (0.43) 16.0 (0.17)
Kahola 22.1 (0.9) 3.3 (0.20) 0.00 (0.000) 3.3 (0.30) 3.8 (0.11)
Pacolet 57.4 (1.3) 8.3 (0.12) 0.01 (0.004) 8.3 (0.07) 8.2 (0.17)
Tifton 1 13.7 (0.2) 2.0 (0.08) 0.05 (0.009) 2.1 (0.12) 2.7 (0.34)
Tifton 2 38.5 (1.0) 6.9 (1.63) 0.03 (0.017) 6.9 (1.64) 7.7 (0.20)
t NO3-N includes NO3 + NO2.
i Values in parentheses are standard deviations.

the most appropriate for soil extracts because these ex- concentration of 0.375 M NaOH in the oxidizing reagent
tracts may contain organic N compounds more difficult appears to be the most adequate of the concentrations
to oxidize than L-glutamic acid. An R/S = 1 would tested.
ensure that <20% of the O2 generated by persulfate would Effect of Autoclaving Time on Urea Oxidation.
be required for the oxidation because, in general, the 0.5 Varying the autoclaving time from 10 to 40 min did not
M K2SO4 extracts of CHCl3-fumigated soils contain <200 have any significant effect on the amounts of urea-N
mg C L-1. recovered in either of the two matrices used (Table 4).
Effect of Reagent to Sample Ratio and Carbon Even though 10 min appears to achieve maximum oxi-
Content on Ammonium Recovery. There was a sig- dation of urea, we maintain an autoclaving time of 30
nificant interaction between R/S and C content because, min in routine analyses to ensure the complete decom-
as expected, increasing R/S from 0.2 to 1 resulted in a position of K2S2O8. This is necessary to prevent oxida-
greater change in N recovery at 200 mg C L"1 than at tion of the Cd column (used for NOf reduction) by
lower C contents (Table 3). It is interesting to note that, undecomposed K2S2O8. Also, in cases in which a buffer
at R/S = 0.2 and low C concentrations (0 and 25 mg C containing N (i.e., NH4C1) is used in the reduction of
L"1), the oxidation of NH? was low even though the NOj to NO~2, any undecomposed K2S2O8 could oxidize
estimated amounts of K2S2O8 required for the oxidation N in the buffer, causing a high blank (Nydahl, 1978).
were lower than 22.8% of the amounts applied. These There was a significant difference between matrices
results suggest that an R/S = 1 is required to oxidize (P < 0.01) in the average percentage of N recovered.
and recover all the NH^ in the extract, even when low The average recovery was 98.4% (SD = 1.31) with 0.5
concentrations of C are present. M K2SO4 and 92.7% (SD = 0.93) with 1 M KC1. The
Effect of Sodium Hydroxide Concentration in Ox- lower recovery with 1 M KC1 could have been caused
idizing Reagent on Nitrogen Recovery. In general, in- by incomplete decomposition of the urea, incomplete
creasing the concentration of NaOH decreased N oxidation of the NHJ released, or volatilization of NH3
recoveries (Fig. 2). There was a significant interaction from the sample. Incomplete decomposition of the urea
between NaOH concentration and N content of the sam- or incomplete oxidation may have been caused by the
ple, which resulted from the fact that increasing NaOH presence of Cl (0.5 M KC1) in the final sample plus
concentration from 0.375 to 0.575 M reduced N recov- reagent mixture, as suggested by Quails and Haines
eries more at high N concentrations than at low N con- (1991). Alternatively, Nydahl (1978) did not observe in-
centrations. The low recoveries observed at 0.575 M terference by 0.33 M NaCl in the oxidation of 2 /unol
NaOH were probably the result of increased NH3 vola- of NH4C1 by 0.5 mmol K2S2O8, but on a molar basis his
tilization at high pH values. Based on these results, a samples contained about a twofold higher K2S2O8 to N

Table 6. Comparison of total persulfate N (TPN) to total Kjeldahl N (TKN) for 1 M KC1 extracts.
Series Total organic C TKN NO3-Nt TKN + NO3-N TPN
———— mg L-' ———
Cecil 14.8 (O.l)t 2.6 (0.09) 0.13 (0.005) 2.7 (0.09) 3.8 (0.06)
Dothan 6.9 (0.1) 1.1 (0.09) 0.01 (0.009) 1.1 (0.10) 1.3 (0.10)
Eudora 14.4 (0.1) 2.8 (0.18) 0.04 (0.005) 2.8 (0.18) 3.0 (0.15)
Greenville 1 11.9 (0.3) 2.2 (0.07) 0.34 (0.009) 2.5 (0.06) 3.0 (0.22)
Greenville 2 16.1 (0.1) 2.2 (0.07) 0.12 (0.005) 2.3 (0.07) 3.2 (0.05)
Hiwassee 1 29.2 (0.3) 5.4 (0.09) 0.02 (0.024) 5.4 (0.08) 5.3 (0.10)
Hiwassee 2 46.4 (1.1) 8.0 (0.14) 0.05 (0.005) 8.0 (0.14) 6.3 (0.13)
Irwin 99.5 (1.3) 15.6 (0.90) 0.05 (0.007) 15.6 (0.50) 14.7 (0.15)
Kahola 20.6 (0.9) 3.3 (0.14) 0.05 (0.025) 3.3 (0.12) 3.0 (0.20)
Pacolet 33.8 (0.6) 6.0 (0.08) 0.01 (0.005) 6.0 (0.08) 5.4 (0.14)
Tifton 1 11.7 (0.1) 1.9 (0.08) 0.08 (0.000) 1.9 (0.08) 2.4 (0.07)
Tifton 2 36.9 (0.2) 8.1 (1.17) 0.00 (0.000) 8.1 (1.17) 7.1 (0.11)
t NO3-N includes NO3 + NO2.
t Values in parentheses are standard deviations.
1012 SOIL SCI. SOC. AM. J., VOL. 57, JULY-AUGUST 1993

ratio (250) than ours (129.5). The higher Cl concentra- can be used to digest at least 150 to 200 samples per
tion in our samples associated with a lower K2S2O8 to N autoclave cycle. The use of this method in 0.5 M K2SO4
ratio may have decreased the decomposition of the urea extracts should facilitate more rapid and efficient mea-
or the oxidation of the NH| released. Incomplete de- surements of soil microbial biomass N.
composition of urea or incomplete oxidation of the
NHJ" released may also have been caused by the lower ACKNOWLEDGMENTS
initial pH observed in the sample plus reagent mixtures
of solutions prepared with 1 M KC1 (9.79) than in those We thank Mary E. Weise, Bert Lankester, and Sarah Tyson
of samples prepared with 0.5 M K2SO4 (9.90). On the for laboratory and technical assistance, David C. Coleman and
other hand, another possible explanation for the lower Weixin Cheng for constructive comments on the manuscript,
recovery observed with 1 M KC1 may be a larger vola- and Jessica Davis-Carter and Fernando Garcia for help with
tilization of NH3 prior to complete oxidation of NH^ to soil sample collection. The research was supported by grants
from NSF (BSR 8818302) and USDA-NRI (91-37102-6785)
NO~3, although the reasons for this are not clear. Evi- to UGA Research Foundation.
dently, more research would be needed to adapt the method
to 1 M KC1 extracts.
Recovery of Nitrate. The recoveries of NOj (97.8%,
SD = 1.24) and urea (99.1%, SD = 1.04) were com-
plete, which indicates that there are no significant losses
of NOj when using an R/S = 1 and an autoclaving time
of 30 min.

Comparison of Total Persulfate Nitrogen and Total

Kjeldahl Nitrogen
The TPN yielded similar concentrations to TKN in the
0.5 M K2SO4 extracts (Table 5, Fig. 3a). The regression
of TPN values on TKN values yielded a straight line
with intercept (0.405) and slope (0.975) not significantly
different from O and 1, respectively (P < 0.05). We
should point out, however, that in extracts from Irwin
soil TPN significantly underestimated TKN by 8.5%.
We did not include this extract in the overall regression
because its N concentration (17.4 mg N L-1 by TKN)
is almost twice as high as the highest concentration mea-
sured in the other extracts and therefore would bias the
regression considerably. Based on these results, it ap-
pears that 0.5 M K2SO4 extracts with >9 mg N Ir1
should be diluted in order to ensure complete oxidation
of the organic N compounds.
In the 1 M KC1 extracts, TPN overestimated TKN +
NO-3-N at TKN + NO^-N concentrations lower than
3 mg N L"1, and underestimated it at higher concentra-
tions (Table 6, Fig. 3b). These results agree with those
of the previously described study in which we recovered
only 92.7% of the urea-N present in a 1 M KC1 extract
containing 20 mg N L"1. These low recoveries at high
N concentrations in 1M KC1 extracts are not well under-
stood and need further study.

CONCLUSIONS
The alkaline persulfate oxidation described here al-
lows complete recovery of the total N present in 0.5 M
K2SO4 soil extracts when using an R/S = 1 and an
autoclaving time of 30 min. The method is not affected
by H/L ratios varying from 0.56 to 7.33, which provides
flexibility in the size of the tubes used for the digestion
in the autoclave. Because three or four racks containing
40, 50-mL tubes easily fit in an autoclave, the method