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DIVISION S-8—NUTRIENT MANAGEMENT & SOIL & PLANT ANALYSIS

Persulfate Digestion and Simultaneous Colorimetric Analysis of Carbon and Nitrogen in Soil Extracts

Dep. of Ecology, Evolution and Marine Biology, Univ. of California, Santa Barbara, CA 93106

^* Corresponding author (doyle{at}lifesci.ucsb.edu).

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS REFERENCES

 
Persulfate digestions have been used for analyzing dissolved organic carbon (DOC) and nitrogen (DON), but most existing methods do not simultaneously analyze the same digest. Persulfate oxidizes DON to NO^–₃, and DOC to CO₂, which was trapped in the alkaline digest solution. We optimized the persulfate digestion for simultaneous analysis of dissolved CO₂ and NO^–₃ with flow injection analysis (FIA). Dissolved CO₂ was diffused across a Gore-Tex (W.L. Gore & Associates, Inc., Sunnyvale, CA) membrane and analyzed with a pH indicator, while NO^–₃ was analyzed with a standard Griess-Ilosvay reaction after Cd reduction. Digestions of glycine, urea, and yeast extract solutions recovered 97 to 99% of C and 92 to 96% of N, while digestions of lysine and nicotinimide recovered slightly less (93 to 94% C, 87 to 93% N). Glycine N, used as a digested standard to which sample recoveries were scaled, improved calculated recovery 94 to 103%. Dissolved organic C concentrations were not significantly different for persulfate oxidation (PO) and high temperature catalytic oxidation (HTCO) for 13 organic and mineral soil extracts with and without prior chloroform fumigation. Nitrogen recovery was higher for PO than HTCO. The DOC blanks were 0.5 to 1.0 mg C L^–1 and detection limit was 0.5 mg C L^–1, and both could be lowered if needed. The upper limit of analysis was at least 2000 mg C L^–1 with dilution. The digest was performed at temperatures as low as 80°C or as high as 125°C with equivalent results. We recommend the low temperature to reduce leakage. This analytical system is particularly convenient for the simultaneous analysis of microbial biomass C and N with the fumigation-extraction procedure.

Abbreviations: DIC, dissolved inorganic carbon • DOC, dissolved organic carbon • DON, dissolved organic nitrogen • DOP, dissolved organic phosphorus • FIA, flow injection analysis • HTCO, high temperature catalytic oxidation • LTER, long-term ecological research • PO, persulfate oxidation • TDC, total dissolved carbon • TDN, total dissolved nitrogen

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS REFERENCES

 
DISSOLVED ORGANIC C and N play central roles in terrestrial ecology and biogeochemistry. They are important intermediates in decomposition (Chapin et al., 2002). They are important in C movement and storage through the soil profile (Qualls, 2000), influencing pedogenesis (Van Cleve and Powers, 1995). The loss of DON is an important route for N loss in terrestrial ecosystems (Perakis and Hedin, 2002, Vitousek et al., 2002). Finally, DOC and DON are a critical route of organic matter flow from terrestrial into fresh water systems (Aitkenhead and McDowell, 2000; Gomi et al., 2002).

Despite the importance of DOC and DON, their analyses remain difficult and often costly. High temperature catalytic oxidation instruments are fast and accurate, but expensive to purchase and maintain (Merriam et al., 1996). The dichromate digest has been adapted for simultaneous analysis of DOC and DON, but the digest is temperature sensitive, time intensive, and it generates concentrated acid wastes containing heavy metals (Doyle and Schimel, 1998). Persulfate oxidation has been developed for soil extract DON (Cabrera and Beare, 1993) and for fresh water DOC (McDowell et al., 1987). It is conveniently run in an autoclave or drying oven with screw-cap tubes, and yields a nontoxic salt solution. Raimbault et al. (1999) combined PO of seawater and simultaneous analyses of DOC, DON, and dissolved organic phosphorus (DOP) with a segmented-flow analyzer, but this has not been done in soil extracts at high C and N concentrations. For laboratories with nutrient analytical capabilities, PO of DOC and DON could provide a low-cost, high throughput technique.

Accuracy of PO digestion is quite good as well. Recovery of DOC by PO compares well with HTCO even at low concentrations found in seawater and river water (Raimbault et al., 1999; Sharp et al., 1993). Persulfate digestion also gives excellent recovery of DON for a wide range of prepared compounds and natural waters (Nydahl, 1978; Solorzano and Sharp, 1980, Sharp et al., 2002). Merriam et al. (1996) compared DON recovery from prepared solutions as well as soil leachates and found good agreement, though HTCO recovered significantly more DON than PO.

Total dissolved carbon and nitrogen (TDC and TDN) are the sum of inorganic and organic constituents. For the purposes of this report, TDC and TDN will be treated as equivalent values to DOC and DON because inorganic components were negligible and not important to digestion chemistry.

Persulfate oxidation depends on peroxydisulfate (K₂S₂O₈) decomposition into the persulfate radical , which is the active oxidizing agent. This decomposition follows an Arrhenius relationship between 50 and 130°C (Kolthoff and Miller, 1951; Goulden and Anthony, 1978); persulfate has a half-life of about 30 s at 130°C and 4 h at 75°C. The decomposition is the rate-limiting step, and further oxidation steps are rapid relative to free radical initiation (Peyton, 1993). Under some conditions, higher temperature may decrease C recovery (Goulden and Anthony, 1978). Thus, high temperature may increase reaction rate but not necessarily completeness. We therefore hypothesized that good results could be achieved by heating in a drying oven overnight (80–90°C) instead of in an autoclave.

Our goal was to use a single persulfate digest for DOC and DON while trapping CO₂ in the digest solution using basic conditions, and to then simultaneously analyze the dissolved inorganic carbon (DIC) and NO^–₃ by FIA. We hypothesized that both DON and DOC could be digested with >95% efficiency in the same digest. Detection of DIC uses the principle of in-line pseudotitration (Ruzicka and Hansen, 1988), where the formation of color depends on CO₂ gas diffusing into a pH indicator. It is not linear with concentration but follows the curve of an acid-base titration. This technique was one of the first developed for continuous flow analyses (Skeggs, 1960), but little work has been done since then. Finally, we hypothesized that reduced digestion temperature would not affect recovery so long as adequate time was allowed for persulfate decomposition.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS REFERENCES

 
Our strategy was to develop FIA colorimetry for DIC in different types of solutions, including persulfate digests, natural waters, and dilute NaOH. We then optimized the concentrations and pH of persulfate digestion using prepared DOC and DON solutions. With this chemistry we compared digestions of soil extracts with HTCO analyses. Finally, we compared digestion efficiency between 75 and 130°C.

Flow Injection Analysis of Dissolved Inorganic Carbon and NO^–₃
Analysis of DIC was developed with a Lachat AE ion analyzer (Milwaukee, WI) and used the analytical manifold shown in Fig. 1 . We used a 60-µL sample loop. Ten percent HCl was added to the sample stream to volatize the dissolved CO₂, which then diffused across a hydrophobic Gore-Tex membrane into a basic solution containing a pH indicator (phenol red, 25 mg L^–1). Phenol red was selected because its pK_a is above the pK_a of H₂CO₃. The CO₂ acidified the indicator slightly and created an acid color peak (440 nm). The identical manifold and diffusion block can also be used for NH₄⁺ analyses, making this chemistry extremely convenient and inexpensive to run. The diffusion block was machined as described in Willason and Johnson (1986), except that the diffusion path was reduced to 16 cm. The membrane was obtained from the original laboratory (R. Petty, 1996, personal communication). A similar membrane is available commercially (Lachat Instruments, Milwaukee, WI). We adjusted the indicator to pH 8.0 and stirred it during analysis. A wick dampened with 1 M NaOH inside the pH indicator bottle scrubbed headspace CO₂, and a soda lime trap on top of the bottle scrubbed replacement air. Sample and indicator flow rates were both 2.3 mL min^–1. Small bubbles sometimes formed in the sample line because of N₂ and O₂ supersaturation after autoclaving, but they were briefly trapped in the pump tube and were not introduced into the sample loops.

View larger version (30K): [in this window] [in a new window]   Fig. 1. Flow injection manifold for detecting carbon dioxide in persulfate digest. Injection valve is shown in the inject position. Reagents, standards, and diffusion block are described in the text. Nominal flow rates are listed; actual flow rates are approximately 2.5 times greater; tubing colors are in parentheses. Manifold tubing is 0.8 mm i.d. (5.2 µL cm–1); 5-cm mixing coil has 70 cm of tubing.

After the alkaline sample digests were opened, they slowly exchanged DIC with air. Samples with high concentrations of DIC decreased in concentration, while blanks increased. Digests left capped overnight lost DIC as well. Several methods are described to eliminate sample DIC exchange, including mineral oil films, Saran Wrap (Dow Chemical Co., Midland, MI), and tipping floating disks (Furman, 1976). We chose to analyze digests within 6 h after digestion and to minimize sample exposure to air. Digests were opened <10 min before analysis and the tubes were placed directly on the autosampler. A 3-h test with digest solution showed no significant DIC loss or absorption.

The primary C standard (7.00 g NaHCO₃ L^–1) was dissolved in 0.5 M K₂SO₄ then immediately poured into glass scintillation vials and capped with no head space. A new vial was opened each day and diluted to 0, 5, 10, 20, 50, and 100 mg C L^–1 in digested 50:50 mixture of 0.5 M K₂SO₄ and persulfate reagent. Because of the titration nature of this colorimetry, calibrations were often curved, depending on subtle changes in the diffusion membrane or pH of the indicator and carrier. Third-order polynomial regressions of standards were used to calculate sample concentrations. One calibration standard was analyzed after every 10 samples to check for drift, which typically did not exceed 5% during the course of an analytical day. This colorimetry was also adapted to analyze DIC in natural waters and dilute NaOH solutions (0.05 M) in our laboratory. The latter were CO₂ traps used in soil respiration assays, and they were analyzed colorimetrically in lieu of titration.

Organic Carbon and Nitrogen Solutions
Prepared DOC and DON solutions varied from very easily digested (glycine), to progressively more difficult and complex compounds (lysine, urea, nicotinamide, and yeast extract). Solutions of purchased chemicals were mixed to contain 200 mg N L^–1 according to label contents. Yeast extract C and N content was measured with a CN analyzer (Fisons, Milan, Italy). One-half to 1 mL was digested during accuracy tests and to optimize the oxidation conditions. These solutions also were added in successively higher volumes to determine upper limits of oxidation. Recovery equaled measured DOC and DON divided by known values.

We also tested digestion of soil extracts. The soils were selected from a variety of forest, tundra, and grassland sites (Table 1). They included forest floor samples from mixed hardwood and pine forests [Harvard Forest long-term ecological research (LTER) site, 42°30' N, 72°10' W], five Alaskan taiga stands {birch (Betula spp.), white spruce [Picea glauca (Moench) Voss], black spruce [Picea mariana (P. Mill.) Britton et al.], alder [Alnus incana (L.) Moench], and poplar (Populus spp.), Bonanza Creek LTER site, 65°45' N, 148°15' W}, Alaskan tussock tundra (acidic and nonacidic soils, Toolik Lake LTER site, 68°38' N, 149°38' W), and grassland soils of the California coast (Sedgwick Reserve, 34°42' N, 120°3' W). Soil C and N contents were determined on the combustion analyzer described above, while pH was determined on 5:1 water-to-soil slurries with a pH electrode.

Digest Chemistry
We used the same persulfate reagent mixture as Cabrera and Beare (1993), but we increased the NaOH concentration to 0.42 M (50 g K₂S₂O₈, 16.8 g NaOH, 30 g H₃BO₄ L^–1). This neutralized acidity generated by persulfate decomposition and gave a moderately basic final pH (7.8) that retained DIC for at least 1 to 2 h after opening tubes. Higher base concentrations (0.5 M) were tested with good accuracy, but they had elevated blanks because of increased CO₂ trapping. We purchased low-N K₂S₂O₈ and did not recrystalize it. Five milliliters of 0.5 M K₂SO₄ extract was mixed with 5 mL persulfate reagent. After mixing with reagent and sealing in tubes, samples may be stored for 1 to 2 d before heating. Each solution was digested with five replicates within a trial. A trial was considered to be a batch of replicate digests heated at the same time. At the highest temperature, four to five trials were made for each solution on separate analytical days, which made 20 to 25 replicate determinations. Digestion was performed in an autoclave (125°C), water baths (100, 80, or 75°C), or a drying oven (75°C). Digestion time was at least seven times the half-life of persulfate obtained from the Arrhenius relationship of persulfate decomposition (34 s, 10 min, 2 h, and 4 h, respectively; Peyton, 1993).

Digestions were performed in acid-washed 15-mL glass Pyrex (Corning Glass Works, Corning, NY) tubes with Teflon-lined caps (Kimball 45066a-16125, Corning 9826-16x; Teflon is manufactured by E.I. DuPont de Nemours & Co. Inc., Wilmington, DE). We inverted tubes during digestion and until analysis to create a liquid seal at the tube cap. This lowered blanks and also revealed leaking tubes, which arose after three to five uses of the caps. We recommend using caps only twice. Sample loss was qualitatively determined by observing sample height in tubes, and no corrections for loss were made because leakage was homogenous. Caution should be used when tightening and removing tube caps because the tubes sometimes shatter. A 5-cm x 10-cm x 3-mm rubber sheet was used to grasp the tubes and protect fingers from sharp glass.

When sample DOC concentrations were expected to exceed the method's range (100 mg C L^–1 in the digest), a <5 mL sample was added, and 0.5 M K₂SO₄ (adjusted to pH 4 to reduce DIC) was added to maintain total sample volume of 5 mL. DOC and DON concentrations in extracts were corrected for dilution. Recovery of DON was precise within digest batches, but varied between trials. We corrected DON results for glycine standard recovery in each trial, but this was not necessary for DOC recovery. We assumed that dilution errors and minor oxidation artifacts would be consistent with glycine and other compounds. Blanks and detection limits were defined in terms of concentration in the final digest. We used a spreadsheet that corrected for blanks, dilution, DIC calibration nonlinearity, instrument drift, and digestion efficiency. It is available on the Santa Barbara Channel Long-Term Ecological Research web site, http://sbc.lternet.edu/external/Land/presentations/persulfate_digest_template.xls (verified 26 Nov. 2003).

We compared persulfate-DOC and DON with HTCO- DOC and DON as measured on a Shimadzu 5000 (Kyoto, Japan) TOC analyzer fitted with a N detector (W. McDowell, 1998, personal communication). This laboratory was selected because of its experience with HTCO (Merriam et al., 1996), and the results were taken as exemplary of typical HTCO methods. All solutions were measured on two separate occasions. In the first run, the prepared solutions and initial soil extract DON values agreed closely with PO (95–106%), but values for soil extracts at the end of the run were lost because of instrument problems. We therefore averaged values from both runs for prepared solutions, but had only a single HTCO determination for soil extracts. While HTCO determinations had their own experimental errors, we considered them to be accurate as a standard method, and we regressed our values against them and compared slopes to unity. Thus, recovery of DOC and DON was defined as the values determined by persulfate digestion followed by colorimetric analysis relative to those measured by HTCO.

No active degassing measures were taken, and FIA of undigested extracts showed negligible DIC relative to DOC (<1 mg C L^–1). Some digests turned slightly pink from Mn compounds, but this does not affect oxidation efficiency (Williams et al., 1995) or analysis by FIA. We measured interference of Mn with NO^–₃ at 520 nm with KMnO₄ solutions. One milligram Mn L^–1 gave absorbance equivalent to 0.013 mg N L^–1. Soil extract digests rarely were as pink as this concentration, and N values were >5 mg N L^–1, so MnO₄ interference was negligible.

Statistical analyses were performed by Systat v. 10 (Systat Software, Inc., Richmond, CA). Significance level was 0.05 unless otherwise specified. Regression slopes were all significantly different from zero (p < 0.01).

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS REFERENCES

 
Flow Injection Analysis of Dissolved Inorganic Carbon in Solution
Flow injection analysis of DIC provided results for solutions with 2 to 100 mg C L^–1 with excellent precision. Replicate analyses of 50 mg C L^–1 NaHCO₃ solutions had CVs of 1.7 to 3% (n = 10). Detection limit was calculated by multiplying the standard deviation of repeated low samples by the t value for the 95% confidence interval (n = 5). This gave a FIA detection limit of approximately 0.5 mg C L^–1. This could be lowered by a factor of 5 to 10 after lengthening the FIA sample loop. This method of DIC analysis appeared to be faster and simpler than either titration (Snyder and Trofymow, 1984) or infrared gas analysis (McDowell et al., 1987). Flow injection analysis with a diffusion block analyzed samples in about one-third the time needed by bubble-segmented flow as used by Raimbault et al. (1999).

Organic Carbon Recovery, Prepared Solutions
Blanks averaged about 0.6 ± 0.9 mg C L^–1 across all trials. They were very precise within trials (SD 0.15 mg C L^–1, n = 5). Between-trial variability (±1 mg C L^–1) of blanks was small relative to the high range of this method (5–100 mg C L^–1). This result is about the same as achieved by McKenna and Doering (1995) with an automated digester, and about 10-fold higher than McDowell et al. (1987) achieved with extensive purification and sparging.

Prepared C standards were analyzed on five separate analytical days (Table 2). Bicarbonate standard recovery was complete and precise (99.9 ± 2.6%). Glycine, urea, and yeast extracts were completely oxidized to DIC (97–99.3%) while lysine and nicotinamide were not as completely digested (93 and 94% recovery, respectively). The CVs for each solution when taken across all trials (n = 18–20) was 4%. Analysis of variance of these results showed that the compounds were significant sources of variance (p = 0.001), but oxidation method was not a significant source when comparing all compounds. When oxidation methods were compared by compound, yeast extract DOC recovery was significantly less for HTCO (92.2%) than for PO (99.3%), suggesting that PO outperforms HTCO for difficult to digest solutions. Digestion trial was not a significant source of variance, so results from all five trials were pooled when calculating means.

View larger version (22K): [in this window] [in a new window]   Fig. 2. Total dissolved organic (a) C and (b) N recoveries from fumigated and unfumigated soil extracts by persulfate digestion vs. high temperature catalytic oxidation (HTCO). Persulfate values are averages of three separate persulfate trials, three to five replicates per trial (n = 12–15), while HTCO values are single determinations. Vertical error bars are the standard deviations of all replicates taken together. Total observations = 25.

Organic Nitrogen Recovery, Soil Extracts
Regression analyses of DON determinations did not include prepared DON solutions because they were much higher in DON (200 mg N L^–1) than the soil extracts and would have statistically weighted the results and given artificially high correlation coefficients (Fig. 2b). Persulfate oxidation recovered 95% of DON vs. HTCO, and the two methods were highly correlated (r² = 0.98). These results indicate that PO works as well as HTCO for complex soil organic solutions.

Soil DON studies often include isotope determinations, but the quantity of N in 10 mL of digest is sometimes not sufficient for ¹⁵N analysis. We scaled up the digest to 50 mL of total volume in screw cap tubes (Pyrex 9825 with polyseal cone caps) and digested them in a drying oven at 80°C. We programmed our autosampler to collect solution directly from these tubes. Analyses of C and N in large tubes vs. small tubes were not significantly different (ANOVA, data not shown), thus this procedure may be successfully increased in volume for greater total N quantity.

The range and detection limits of DOC and DON analyses could be lowered through scrupulous work with muffled glassware, acidified and sparged samples, and septa-sealed digestion tubes that would lower C blanks. Thus, the effective range of this method could possibly be lowered to that of marine and fresh waters (Raimbault et al., 1999).

Temperature Effects
Digestion of glycine and lysine at 70, 80, 100, and 125°C gave high recovery of C (100–107% C) that did not vary significantly with temperature (Table 4). Absolute recovery of N was generally <100% (88–96%), as described previously, and there was no variation due to digestion temperature for lysine. Glycine N recovery vs. temperature was not significantly different between 80 and 125°C, but was less at 70°C (ANOVA, p = 0.049). We also digested the soil extracts at 80°C (data not shown). Dissolved organic carbon values were indistinguishable from earlier autoclaved digestions. Because of occasional CO₂ leakage while autoclaving, however, we recommend heating in a drying oven overnight because it generates less pressure in the tubes. Recovery of DON from the same trial was not significantly different at either temperature from previous digests and was highly correlated (r² = 0.995). These tests confirm that oxidation efficiency was not temperature sensitive as long as adequate time was allowed for persulfate radical generation. For overnight digestion, 80°C would be a safe minimum temperature.

Recovery of DON from glycine and lysine dropped off at a slightly lower concentration than the upper limit of detection of DOC (90–95 mg C L^–1), while N recovery from yeast extract was complete until digests contained 120 mg C L^–1, (data not shown). The low recovery of N from amino acids is because of their low C:N ratio and the extra oxidant needed to oxidize N.

While the oxidation chemistry of persulfate radical is not stoichiometrically fixed or strictly related to oxidation states of the reduced compounds, the chemical equivalents (eq) needed for DOC and DON oxidation can be estimated by examining their initial oxidation states. Organic C has an average valence close to 0 (Harris and Adams, 1979), while amino N has an average valence close to –3. Organic C must be oxidized four valences to +4, while eight electrons must be removed from amino-N to reach oxidation state +5. Free radical oxidation proceeds with several free radical collisions needed to fully oxidize each atom (Peyton, 1993). Thus, N oxidation could take up to twice as many free radical interactions as C oxidation. Oxidation equivalents of a solution with organic C and N in it may be calculated as

With this expression, the digestion upper limit for glycine was calculated. Oxidation efficiency for glycine fell below 95% once concentrations exceeded 100 µeq. Some authors determine PO upper limits for DON oxidation using compounds unlike their environmental samples without considering cumulative reduction capacity of C and N (Ebina et al., 1982). This could lead to under- or overestimates of the upper limit of detection, depending on the C:N ratios of their standards and samples. Because of its low C:N ratio, glycine would be recommended as a conservative DOC/DON upper limit standard.

Persulfate oxidation also may be influenced by factors such as molecular structure or quenching molecules (Peyton, 1993). Goulden and Anthony (1978) found that varied DOC compounds were oxidized at essentially the same rate, indicating that the original C-C bonds did not primarily control oxidation kinetics. Another potential interferent is HCO₃, especially in basic solution (Peyton, 1993). We tested this with increasing additions of HCO₃ up to 500 mg C L^–1, with no effect on the oxidation of NH₄⁺, lysine, or yeast (data not shown). Peyton (1993) also considered that oxygen may be limiting in this oxidation. We added H₂O₂ to digests, but this did not improve oxidation efficiency (data not shown).

pH Effects and Mechanistic Concerns
We achieved complete recovery of C and N at the pH used in this study (9–10), but the ideal pH for PO is not clear. Also, some reports in the literature are contradictory (Nydahl, 1978; Solorzano and Sharp, 1980; Ebina et al., 1982; McDowell et al., 1987; Cabrera and Beare, 1993; Williams et al., 1995; Raimbault et al., 1999; Sharp et al., 2002). This may be because of differing pH optima for different stages of oxidation and analysis: persulfate decomposition, oxidation by the radical, and analytical conditions for the end products (CO₂, NO^–₃, PO₄^–3). Decomposition is effectively pH-independent above 2 (Peyton, 1993). Oxidation of DOC has a broad optimum at pH 5, as determined by evolution of CO₂ from nicotinic acid (Goulden and Anthony, 1978), but optimal oxidation pH's for DON and DOP have not been as carefully studied.

Raimbault et al. (1999) analyzed seawater DOC, DON, and DOP under alkaline conditions, but this may not work for solutions with PO₄^–3 concentrations >10 µM where aluminum and calcium minerals precipitate.

Microbial Flush Dissolved Organic Carbon and Nitrogen
Microbial flush C and N are indicators of total biomass in soil, and are calculated by subtracting unfumigated extract DOC and DON from values for fumigated soils. It is possible that fumigated and unfumigated organic matter could be qualitatively different, so we compared flush C and N when analyzed by PO vs. HTCO (Fig. 3a,b) . Precision for PO-C and PO-N were acceptable for this type of analysis (CV = 5–10%), and both methods were highly correlated for C and N with flush values determined by HTCO (r² = 0.949 and 0.924, respectively). The slopes of the relationships are close to 1, and vary slightly depending on whether a single high value is included or excluded from the regression. If that point is excluded, it appears that for C, the PO method may slightly underestimate flush relative to HTCO, while for N, the methods give values within 10% of each other. Our results indicate that PO is adequate for microbial biomass measurements. We measured negative flush values by both methods for two soils, and these were excluded from this analysis. As both oxidation methods gave negative values, any problem was due to the fumigation-extraction technique, not the oxidation method.

View larger version (22K): [in this window] [in a new window]   Fig. 3. Microbial flush (a) C and (b) N values determined by persulfate oxidation vs. high temperature catalytic oxidation (HTCO) from fumigated and unfumigated soils. Persulfate values are averages of three separate trials (n = 6–10). Error bars represent one standard deviation. Solid lines include all data while dashed lines exclude the highest point. Total observations = 10.

	CONCLUSIONS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS REFERENCES

	ACKNOWLEDGMENTS

 
This work was funded by the National Science Foundation through the Arctic Transitions in the Land-Atmosphere System (ATLAS, grant No. OPP 9731999), Microbial Observatories (grant No. MCB 9977874), Bonanza Creek Long Term Ecological Research (grant No. DEB 0080609), and Terrestrial Ecosystems and Global Change programs (grant No. DEB 9523427). We would like to thank Robert Petty for the Gore-Tex membrane and Gary Peyton for valuable consultation.

Received for publication October 2, 2002.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION CONCLUSIONS REFERENCES

 

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