Open Journal of Soil Science, 2012, 2, 269-274
http://dx.doi.org/10.4236/ojss.2012.23032 Published Online September 2012 (http://www.SciRP.org/journal/ojss) 269
Soil Organic C:N vs. Water-Extractable Organic C:N
Richard L. Haney1*, Alan. J. Franzluebbers2, Virginia. L. Jin3, Mari-Vaughn. Johnson4,
Elizabeth. B. Haney5, Mike. J. White1, Robert. D. Harmel1
1USDA-Agricultural Research Service (ARS), Grassland, Soil & Water Research Laboratory, Temple, USA; 2USDA-ARS, J. Phil
Campbell, Sr. Natural Resource Conservation Center, Watkinsville, USA; 3USDA-ARS, Agroecosystem Management Research Unit,
Lincoln, USA; 4USDA-NRCS, Resources Inventory and Assessment Division, Temple, USA; 5Texas AgriLife Research, Blackland
Research & Extension Center, Temple, USA.
Received May 18th, 2012; revised June 20th, 2012; accepted July 3rd, 2012
ABSTRACT
Traditionally, soil-testing laboratories have used a variety of methods to determine soil organic matter, yet they lack a
practical method to predict potential N mineralization/immobilization from soil organic matter. Soils with high micro-
bial activity may experience N immobilization (or reduced net N mineralization), and this issue remains unresolved in
how to predict these conditions of net mineralization or net immobilization. Prediction may become possible with the
use of a more sensitive method to determine soil C:N ratios stemming from the water-extractable C and N pools that
can be readily adapted by both commercial and university soil testing labs. Soil microbial activity is highly related to
soil organic C and N, as well as to water-extractable organic C (WEOC) and water-extractable organic N (WEON). The
relationship between soil respiration and WEOC and WEON is stronger than between respiration and soil organic C
(SOC) and total organic N (TON). We explored the relationship between soil organic C:N and water-extractable organic
C:N, as well as their relationship to soil micro bial activity as measured by the flush of CO2 following rewetting of dried
soil. In 50 different soils, the relationsh ip between soil microbial activity and water-extractable organic C:N was much
stronger than for soil organic C:N. We concluded that the water-extractable organic C:N was a more sensitive mea-
surement of the soil substrate which drives soil microbial activity. We also suggest that a water-extractable organic C:N
level > 20 be used as a practical threshold to separate those soils that may have immobilized N with high microbial ac-
tivity.
Keywords: Soil Microbial Activity; C:N Ratios; Soil Organic C; N Mineralization; N Immobilization; Soil Testing
1. Introduction
Soil microorganisms are the centerpiece of biogeoche-
mical cycling of nutrients in soil. Soil fertility is directly
related to, and defined by, the heterotrophic activity of
soil microbes as a whole [1]. While shifts in soil micro-
bial community composition and structure are indicators
of altered environmental conditions or management [2-4],
the link between microbial co mposition and soil function
is highly variable and thus limited as a general parameter
for predicting soil activity rates. Thus, the integrated re-
sponse of the soil microbial community is needed to elu-
cidate and predict soil nutrient availability for the man-
agement of one of our most important resources, soil.
The metabolically-active component of soil can be
measured in its simplest form as emission of CO2, which
corresponds to nutrient availability, moisture, and tem-
perature, and can be rapidly quantified [5]. Emission of
CO2 can reveal the broader impacts of management, crop
biodiversity, and climatic changes, but also has predict-
tive capabilities for specifically assessing soil-nutrient
release. Measurement of soil microbial activity, in con-
junction with other soil physical and chemical properties
and processes, can be a valuable tool for developing a
complete profile for soil fertility and may be used to in-
crease the efficiency of fertilizer recommendations.
Microbial mineralization/immobilization of soil N can
be broadly estimated using soil organic C:N [6]. Based
on years of research on conversion rates of decomposa-
ble organic matter by soil fungi and bacteria, soil organic
C:N of 20 is generally considered to be a threshold point
where either net N mineralization or net N immobiliza-
tion occurs [7,8]. In reality, both N mineralization and
immobilization occur simultaneously in soil, making it
difficult to predict the amount of available soil N from
net N mineralization alone. Correlation between the soil
C:N ratio and N immobilization and mineralization is
unclear, partly due to early work that was limited to
measurements of net N rates. Variations of the C:N ra-
*Corresponding a uthor.

Soil Organic C:N vs. Water-Extractable Organic C:N 270
tios of different pools of organic matter and variations of
the C and N assimilation efficiency of the microbial bio-
mass may also confound the usefulness of the soil C:N
ratio to predict gross N transformation rates [8].
Current literature suggests that soil respiration rates can
be used to predict soil N mineralization/immobilization
rates and provide an estimate of soil N mineralization
potential [9,10]. While soil CO2-C respiration rates have
been shown to be correlated to N mineralization [8,11],
respiration alone is not an indicator of N immobilization
and may not accurately predict net N mineralization/
immobilization. As a result, a modified approach couples
soil organic C:N with the soil respiration rate for mode-
ling net N mineralization rates in soils [12,13]. High soil
microbial activity does not always lead to high N miner-
alization due to immobilization that can occur; however,
determining the C:N ratio from a much smaller more
active pool of C and N to soil microbial activity could
increase the accuracy of predicting the net mineralize-
tion/immobilization. A more sensitive and effective ap-
proach may be to assess the much smaller fractions of
water-extractable organic C and N, which are highly re-
lated to soil microbial activity [14]. Results of a review
paper on N cycling focus the importance of both sub-
strate quantity (as C and N concentration) and qu ality (as
C:N ratio) for N cycling rates [9].
In this study, we wanted to explore the link between
the release of CO2 following rewetting of dried soil and
soil organic C:N vs. water-extractable organic C:N. We
hypothesized: 1) soil microbial activity may be more
strongly correlated with water-extractable organic C and
N concentrations than soil organic C and N concentra-
tions and 2) C:N ratio calculated from the water-extra-
ctable organic fraction may be an additional tool in con-
junction with the flush of CO2 following rewetting of
dried soil to better predict plant available N and N im-
mobilization.
2. Materials and Methods
Soil was collected from agricultural fields in Idaho,
Georgia, Maine, Mississippi Oklahoma, Texas, and
Wyoming. Crop management varied: (till/no-till, con-
tinuously cropped/crop rotation) along with soil type.
Soils had clay content from 10 to 55% (data not shown),
pH from 5.56 to 8.02 (Figure 1), and soil organic C from
3.63 to 41.31 g·C·kg–1 soil (Figure 2).
All samples were dried overnight at 50˚C and ground
to pass a 2-mm sieve. Soil organic C (SOC) and total N
(TN) were determined on 2 g subsamples using dry com-
bustion (Elementar; Hanau, Germany). Water-extractable
organic C (WEOC) and water-extractable N (WEN) were
determined from 4 g of dry soil with 40 mL of deionized
water and shaking for 10 minutes on a mechanical shaker
(Eberbach, Ann Arbor, MI). Samples were then centri-
Figure 1. Soil pH range.
Figure 2. Range in soil organic C.
fuged for 5 minutes at 3500 rpm, filtered through What-
man 2 V paper, and analyzed for WEOC and WEN
(Apollo 9000, Teledyne-Tekmar; Mason, Ohio). Inorganic
NH4-N, and NO3-N concentrations were also determined
(Flow Solution IV, OI Analytical; College Station, TX).
Soil organic N (SON) was calculated by subtracting inor-
ganic N content (NH4-N and NO3-N) from total N. Water-
extractable organic N (WEON) was calculated by sub-
tracting inorganic N content (NH4-N and NO3-N) from
WEN.
The release of CO2 from 24 hour incubation after re-
wetting dried soil is directly related to the fertility of a
given soil; the method is designed to mimic the natural
soil drying and rewetting cycle and is designed to be
readily adopted by soil testing labs.The flush of CO2 in
24 hours (1-day) following rewetting of dried soil was
determined from 40 g subsamples in 50 ml polypropylene
disposable beakers (Fisherbrand Cat. No. 01-291-10)
with four to five 6.35-mm holes drilled in the bottom. A
Whatman GF/D 4.25-cm glass microfiber filter (Cat No.
1823-042) was placed in the bottom of each beaker to
prevent soil loss. The beaker and Solvita® gel paddle
were placed in a gas-tight 250-mL glass jar filled with 25
mL of water and sealed with a screw-top lid; the jar had a

Soil Organic C:N vs. Water-Extractable Organic C:N 271
convex bottom to allow for drainage. Capillary action
was used to rewet soil according to its water holding cap-
ability [15]. Soils were incubated at 25˚C, and respired
CO2 was trapped during 24 h. The quantity of 1-day
CO2-C released was determined using a digital-color
reader (DCR) (www.solvita.com). The Solvita system for
esti- mating the flush of 1-day CO2 following rewetting
of dried soil has been shown to be highly correlated with
the commonly used titration method and 1-day CO2-C
IRGA method [5]. SigmaStat imbedded in SigmaPlot ver.
12.1 was used for linear regression analysis.
3. Results and Discussion
Soil organic C and SON were highly related (r2 = 0.93),
as were WEOC and WEON (r2 = 0.84). Interestingly,
ratios of SOC to SON an d WEOC to WEON were nearly
similar (10.8 and 10.9, respectively), suggesting that
WEOC and WEON are a subset of the much larger SOC
and SON pools (Figures 3 and 4). Some soil test labs
currently use SOC data determined from various methods
(loss on ignition, titration, and combustion) to estimate
potential N release, while few if any use SOC:SON to
estimate potential N mineralization. Since both substrate
availability and SOC:SON have a strong influence on N
mineralization rates [9], it is conceivable that WEOC:
WEON could be a better method for determining the
state of potential N mineralization/immobilization as an
alternative to SOC:SON. However, our data indicate that
SOC:SON and WEOC:WEON were poorly related
(Figure 5). This weak relationship may be attributed to
the fact that SOC and SON were roughly 40 times larger
than WEOC and WEON fractions (Figures 3 and 4). The
water-extractable organic fraction, though significantly
smaller than total SOC and SON, is a critical component
used by soil microorganisms that drive the nutrient
cycling system [16-19].
We found that soil microbial activity measured as the
flush of 1-day CO2 following rewetting of dried soil was
significantly correlated to SOC, SON, WEOC, and
WEON. Water-extractable organic C and WEON, how-
ever, had a stronger relationship with the flush of 1-day-
CO2 than SO C and SON (Table 1).
This finding is consistent with documented findings
[16-19] and the authors’ theory that the water-extractable
C and N pool is the more readily available energy pool
for soil microbes as compared to the total SOC and TON
pools. In this data set, organic N accounted for 97.4% of
the total soil N, while organic N accou nts for 53% of the
water extractable total N. While SOC:SON may be a
useful indicator of soil quality/fertility, it may not be
sensitive enough to reliably quantify the quality of
organic matter that soil microbes are actively mineraliz-
ing due to the sheer size of the C and N pools compared
to the water extractable C and N pools. Therefore,
Figure 3. Soil organic N vs. soil organic C.
Figure 4. Water extractable organic N vs. water extractable
organic C.
Figure 5. Soil C:N ratio vs. water extractable soil C:N ratio.
WEOC:WEON could be considered a more accurate
property to predict v ariations in mineralization/immobili-
zation potential of a given soil as compared with SOC:
SON.
It may be possible to combine the flush of CO2
following rewetting of dried soil with WEOC:WEON
and develop a potential N mineralization/ immobilization

Soil Organic C:N vs. Water-Extractable Organic C:N 272
Table 1. Correlation table showing correlation coefficients
(r) comparing 1-day CO2-C (mg·C·kg-1 soil) and total and
water-extractable organic C and N concentrations (mg·C·kg-1
soil; mg·N·kg-1 soil). All correlations were significant (P <
0.0001; n = 50).
TOC TON WEOC WEON
1-Day CO2-C 0.704 0.682 0.874 0.869
TOC . 0.965 0.755 0.737
TON . . 0.725 0.736
WEOC . . . 0.919
indicator (Figure 6). Soil organic C:N > 20 reflects
reduced N availability due to greater immobilizatio n o f N
[20,21], although the breakpoint can be as high as 30,
depending on the [22]. Using a threshold SOC to SON
value of 20, above which no net N mineralization would
occur, our results indicate N immobilization in 4 of 50
(8%) soil samples, whereas when using WEOC to
WEON, 16 of 50 (30%) soil samples immobilized N
(Figure 6). This information suggests that the WEO C:N
ratio may be more indicative of th e quality (water extrac-
table organic C) of the organic C as opposed to quantity
(soil organic C) of substrate available for soil microbial
activity, which is an important point since easily decom-
posable substrate should translate into N mineralization
release. The importance of characterizing the quality vs.
the quantity of organic C is a furtherance of the sen-
sitivity that is revealed by a nutrient cycling system that
is driven by C. We define the active soil C pool (WEOC)
as one that is easily mineralizable and therefore a direct
driver of microbial activity (1-day CO2) responsible for
providing N and P mineralization in soil. Utilizing these
three poo ls (WEOC, WEON, 1 -day CO2) in a soil testin g
environment could increase our awareness of soil micro-
bial activity and possibly help us account for an often
overlooked source of N that can be easily subtracted
from fertilizer recommendations. This will have a two-
fold effect: 1) Save producers input costs in terms of
reduced fertilizer input and 2) reduce the N and P loading
in soil which is subject to loss from erosion, leaching,
denitrification, and leaching that will ultimately affect
the quality of our drinking water and water bodies, both
freshwater and saltwater.
A more sensitive C:N indicator combined with a rapid
method for soil microbial activity would help soil test
labs offer reliable estimates of the mineralization/immo-
bilization state of soil. Soil samples are usually taken
prior to planting, which is a time when this information
would be most needed for fertilizer decisions by pro-
ducers. Spring sampling would be at a time of the year
when the soil microbial community would have stabi-
lized over winter following decomposition of the pre-
vious crop.
Currently there are no standardized universal soil test
Figure 6. 1-day CO2-C vs. soil organic C:N and water
extractable organic C:N.
methods for quantifying the active portion of soil. Con-
sequently the majority of soil test labs ignore the active
soil C pools. In terms of N, soil labs often test for total
and inorganic N, while only a few test for ammonium.
On the other hand, some labs do not so il test for N at all.
Because the cost for fertilizer usually accounts for
roughly 30% - 40% of the total budget of a modern
farming system, it is a shame that all available N re-
sources are not measured and accounted for. Develop-
ing soil test methods to account for soil C and N pools
that contribute to plant-available nutrients is a necessary
step towards improving resource efficiency and reducing
input cost. In addition to using WEOC:WEON instead of
SOC:SON, in future research we intend to investigate the
possibility of using a sliding scale for WEOC: WEON
that can be developed and compared to yield in
unfertilized and unamended plots. The sliding scale would
begin with a linear response and represent increased N
mineralization potential as WEOC:WEON declin es. This
sliding scale could be quickly evaluated by geographic
area using WEOC:N and CO2-C respiration data and test
plots for a variety of crops and forages. For example,
higher C:N ratios would predict less N mineralization as
compared to low C:N ratios. Setting a break-point of
20:1 above which no potential N mineralization occurs
could reduce the possibility of under-fertilizing based on
recommendations from soil microbial activity alone. This

Soil Organic C:N vs. Water-Extractable Organic C:N 273
would represent a safety factor to guard against high soil
microbial activity (CO2 respiration) with little N released
when N is immobilized due to high WEOC:WEON.
In preliminary investigations, we found that soils with
low WEOC:N ratios released more N than soils with high
WEOC:N ratios. We experienced this in fields here in
Texas that had legume cover crops vs. no cover crops for
two years. The C:N ratio from a water extraction ave-
raged 10:1 in cov er crop soils vs. 16:1 in non- cover crop
soils. Both of these fields were in a replicated corn-wheat
rotation. Wheat yields increased 10 bu and corn yields
increased 20 bu in the cover crop vs. no cover-crop f ield s,
which corresponds to the WEO C:N ratios from the two
treatments (data not shown). The sliding scale WEOC:N
ratio could be developed by geographic areas based on
local climatic conditions which would require crop or
forage yield, a total C:N analyzer for water extraction,
Solvita DCR and paddle kit, and inorganic N analyzer
(both NH4-N and NO3-N). Overall this future research
could provide insight into the application of the pre-
viously described methods and their application to both
conventional and organic farming systems.
4. Conclusion
Our data suggests the C:N ratios determined from soil
water extractions are likely to be more sensitive than
total soil C:N to microbial activity an d therefore can be a
better measurement of the impact of management inputs.
Determining the quality as opposed to the quantity of
organic C could enhance our ability to rapidly measure
impacts on soil microbial activity which is an active re-
flection of soil health. This informatio n can be applied to
track the relative impact of management decisions on soil
fertility ultimately improving the efficiency of manage-
ment decisions. Combining WEO C:N ratios with micro-
bial activity could be used as a rapid soil biology indica-
tor with healthy soil having a WEO C:N ratio below 20:
1 and microbial activity between 30 - 50 ppm C. Track-
ing soil biology improvement or degradation is currently
difficult due to the lack of a proper tool and rapid, accu-
rate analytical methods. Introducing management schemes
to improve the C:N ratio and increase microbial activity
should result in increased soil fertility/soil biology and
highly productive and sustainable systems. Using soil
testing labs to measure soil biology and tracking man-
agement inputs based on soil biology/soil fertility im-
provements would be a step towards true sustainability
that can be easily acquired through adoption of both the
water extraction method for determining organic C and N
and the Solvita soil respiration method.
REFERENCES
[1] K. Alef, T. H. Beck, L. Zelles and D. Kleiner, “A Com-
parison of Methodsto Estimate Microbial Biomass and
N-Mineralization in Agricultural and Grassland Soils,”
Soil Biology and Biochemistry, Vol. 20, No. 4, 1988, pp.
561-565. doi:10.1016/0038-0717(88)90073-9
[2] K. P. Dierksen, G. W. Whittaker, G. M. Banowetz, M. D.
Azevedo, A. C. Kennedy, J. J. Stein and S. M. Griffith,
“High Resolution Characterization of Soil Biological
Communities by Nucleic Acid and Fatty Acid Analyses,”
Soil Biology and Biochemistry, Vol. 34, No. 12, 2002, pp.
1853-1860. doi:10.1016/S0038-0717(02)00198-0
[3] W. R. Cookson, D. A. Abaye, P. Marschner, D. V. Mur-
phy, E. A. Stockdale and K. W. T. Goulding, “The Con-
tribution of Soil Organic Matter Fractions to Carbon and
Nitrogen Mineralization and Microbial Community Size
and Structure,” Soil Biology and Biochemistry, Vol. 37,
No. 9, 2005, pp. 1726-1737.
doi:10.1016/j.soilbio.2005.02.007
[4] B. Drigo, G. A. Kowalchuck and J. A. van Veen, “Cli-
mate Change Goes Underground: Effects of Elevated
Atmospheric CO2 on Microbial Community Structure and
Activities in the Rhizosphere,” Soil Biology and Bio-
chemistry, Vol. 44, No. 5, 2008, pp. 667-679.
doi:10.1007/s00374-008-0277-3
[5] R. L. Haney, W. F. Brinton and E. Evans, “Soil CO2 Res-
piration: Comparison of Chemical Titration, CO2 IRGA
Analysisand the Solvita Gel System,” Renewable Agri-
culture and Food Systems, Vol. 23, No. 2, 2008, pp.
171-176. doi:10.1017/S174217050800224X
[6] E. A. Paul and N. G. Juma, “Mineralisation and Immobi-
lization of Soil Nitrogen by Microorganisms,” In: F. E.
Clark and T. Rosswall, Eds., Ecological Bulletin, Vol. 33,
Stockholm, 1981, pp. 197-204.
[7] R. L. Tate, “Soil Microbiology,” Wiley, New York, 1995.
[8] G. Bengtston, P. Bengtson and K. F. Mansson, “Gross
Nitrogen Mineralization-, Immobilization-, and Nitrifica-
tion Rates as a Function of Soil C:N Ratio and Microbial
Activity,” Soil Biology and Biochemistry, Vol. 35, No. 1,
2003, pp. 143-154. doi:10.1016/S0038-0717(02)00248-1
[9] M. S. Booth, J. S. Stark and E. Rastetter, “Controls on
Nitrogen Cycling in Terrestrial Ecosystems: A Synthetic
Analysis of Literature Data,” Ecological Monographs,
Vol. 75, No. 2, 2005, pp. 139-157. doi:10.1890/04-0988
[10] J. Luxhøi, S. Bruun, B. Stenberg, T. A. Breland and L. S.
Jensen, “Prediction of Gross and Net N Mineralization-
Immobilization-Turnover from Respiration,” Soil Science
Society of America Journal, Vol. 70, No. 4, 2006, pp.
1121-1128. doi:10.2136/sssaj2005.0133
[11] J. E. Barrett and I. C. Burke, “Potential N Immobilization
in grassland Soils across a Soil Organic Matter Gradient,”
Soil Biology and Biochemistry, Vol. 32, No. 11-12, 2000,
pp. 1707-1716. doi:10.1016/S0038-0717(00)00089-4
[12] J. A. van Veen, J. N. Ladd and M. J. Frissel, “Modelling
C and N Turnover through the Microbial Biomass in
Soil,” Plant and Soil, Vol. 76, No. 1-3, 1984, pp. 257-274.
doi:10.1007/BF02205585
[13] B. H. Janssen, “Nitrogen Mineralization in Relation to
C:N Ratio and Decomposability of Organic Materials,”
Plant and Soil, Vol. 181, No. 1, 1996, pp. 39-45.

Soil Organic C:N vs. Water-Extractable Organic C:N 274
doi:10.1007/BF00011290
[14] R. G. Woodmansee and D. A. Duncan, “Nitrogen and
Phosphorus Dynamics and Budgets in Annual Grass-
lands,” Ecology, Vol. 61, No. 4, 1980, pp. 893-904.
doi:10.2307/1936759
[15] R. L. Haney and E. B. Haney, “Simple and Rapid Labo-
ratory Method for Rewetting Dry Soil for Incubations,”
Communications in Soil Science and Plant Analysis, Vol.
41, No. 12, 2010, pp. 1493-1501.
doi:10.1080/00103624.2010.482171
[16] J. R. Burford and J. M. Bremner, “Relationships between
the Denitrification Capacities of Soils and Total, Wa-
ter-Soluble and Readily Decomposable Soil Organic
Matter,” Soil Biology and Biochemistry, Vol. 7, No. 6,
1975, pp. 389-394. doi:10.1016/0038-0717(75)90055-3
[17] E. A. Davidson, L. F. Galloway and M. K. Strand, “As-
sessing Available Carbon: Comparison of Techniques
across Selected Forest Soils,” Communications in Soil
Science and Plant Analysis, Vol. 18, No. 1, 1987, pp.
45-64. doi:10.1080/00103628709367802
[18] R. G. Qualls and B. L. Haines, “Biodegradability of Dis-
solved Organic Matter in Forest Throughfall, Soil Solu-
tion and Stream Water,” Soil Science Society of America
Journal—Abstract, Vol. 56, No. 2, 1992, pp. 578-586.
doi:10.2136/sssaj1992.03615995005600020038x
[19] T. DeLuca and D. R. Keeney, “Soluble Organics and
Extractable Nitrogen in Paired Prairie and Cultivated
Soils of Central Iowa,” Soil Science, Vol. 155, No. 3,
1993, pp. 219-228.
doi:10.1097/00010694-199303000-00008
[20] Alexander and Martin, “Introduction to Soil Microbiol-
ogy,” 2nd Edition, Wiley, New York, 1977.
[21] D. M. Sylvia, J. J. Fuhrmann, P. G. Hartel and D. A.
Zuberer, “Principles and Applications of Soil Microbiol-
ogy,” Prentice Hall, Upper Saddle River, 1988.
[22] L. E. Jackson, J. P. Schimel and M. K. Firestone, “Short-
Term Partitioning of Ammonium and Nitrate between
Plants and Microorganisms in an Annual Grassland,” Soil
Biology and Biochemistry, Vol. 21, No. 3, 1989, pp. 409-
416. doi:10.1016/0038-0717(89)90152-1