# GRACEnet: Residue quality determination protocols

DRAFT 20-Aug-03; updated September, 2005

Jane Johnson, Don Reicosky

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# Greenhouse gas Reduction through Agricultural Carbon Enhancement network (GRACEnet) is a multi-location research effort within the ARS organization to address scientific and program issues related to agricultural carbon sequestration, greenhouse gas emissions reduction and environmental benefits related to these activities. The emphasis will be on comparing among common management scenarios at each location although the specifics will be location dependent. The soils, crops and condition will be location specific, but consistent methods and detailed record keeping will be used to facilitate cross-location comparison and to ensure quality control. The objective of this document is to provide background and sampling protocols for residue quality parameters.

# Background 

The impact of crop residues on trace gas emissions (CO~2,~ N~2~O) is
dependent upon the quality of the residue (e.g. C:N ratio, N
concentration) and the size of residue. The amount of N~2~O evolved
depended on the type of residue incorporated and the particle size of
the residue (Ambus et al., 2001; Shelp et al., 2000). The incorporation
of crop residues can provide a source of readily available C and N.
Greater emissions of N~2~O follow incorporations of residues with low
C:N ratios, such as legumes of horticultural crops as compared with
cereal straw incorporations (Baggs et al., 2002). Smaller crop residue
particles, allow for increased microbial attack, and thus greater
production of N~2~O (Ambus et al., 2001). Such residues can enhance
metabolic activity and form local anaerobic zones, giving favorable
sites for denitrification and contribute to 'hot spots' of N~2~O
emission (Ball et al., 1999). Homogenous mixing of residue into soil
increased the amount of N~2~O released compared to applying a layer of
residue in soil cylinders (Ambus et al., 2001). The quality of crop
residues can alter the balance of N immobilization and mineralization,
thus indirectly impacting substrate availability for N~2~O formation.

The ratio of C:N is an easy parameter to measure, however it has been
shown that C:N is not sufficient for predicting decomposition (Franck et
al., 1997; Gorissen et al., 1995; Palm and Rowland, 1997; Scholes et
al., 1997). Palm and Rowland, (1997) recommended that lignin, soluble
carbon (soluble sugars, (if %N\> 1.8%) soluble phenolics, total N, total
P, total C, ash-free dry weight be included in a minimum data set of
parameters used to characterizing plant input quality for decomposition
and soil organic matter studies.

Plants fix and assimilate C from CO~2~ into high-energy organic C
compounds (e.g. sugars and starch) during photosynthesis. Roots can also
fix CO~2~ via phospho*enol* pyruvate carboxylase. The impact of C
assimilation in roots ranges from negligible (Farmer and Adams, 1991) to
providing 25 to 33% of the carbon required for N assimilation in nodules
(Vance et al., 1983) or for C exuded as citrate or malate from
phosphorus stressed white lupin (*Lupinus albus* L.) (Johnson et al.,
1996). All of the energy to reduce C to high energy forms directly or
indirectly is derived from photosynthetic C reduction. It has been
estimated that 16 to 33% of the total C assimilated by plants is
translocated and released into the soil, contributing 30-60% of the
organic C pool in soil (Boone, 1994; Heal et al., 1997). Roots
contribute more C to SOM compared to above ground sources (Angers et
al., 1995; Balesdent and Balabane, 1996; Barber, 1979; Flessa et al.,
2000; Gale and Cambardella, 2000; Huggins et al., 1998; Puget and
Drinkwater, 2001).

Organic C products or residue released into the soil can be consumed by
soil fauna (macro and micro), be released as CO~2~, or be converted into
soil organic matter. The balance among these options is dependent upon
both abiotic (temperature, moisture, pH) and biotic factors. Management
such as crop type, fallow frequency, residue management, soil
amendments, use of cover crops, tillage, irrigation, drainage, mulching
and fertilization all can impact the rate of SOM formation, C storage or
release by altering biotic or abiotic factors (Paustian et al., 1997).
The storage of soil C and the release of greenhouse gases (CO~2~, CH~4~
and N~2~O) requires a better understanding of the changes in soil
physical, chemical, and biological processes and properties. One step in
understanding these processes is to identify the most stable forms of C
and the mechanism to increase the input of stable C into the soil. The
system is very complex, involving interactions of the C, N and P cycles
and associated feedback among the plants, animals and soil microbes
(Kennedy, 1999).

# Sampling guidelines

Plant material should be analyzed fresh or freeze-dried, especially if
soluble compounds are to be assayed (Allen, 1989). However, an
acceptable compromise is to dry the material below at or below 45^o^C,
with adequate ventilation to minimize microbial or enzymatic breakdown
(Allen, 1989; NREL, 1996). After drying the material should be ground to
pass through a 1 mm mesh. Determination of equivalent dry-weight at
100^o^C permits results to be expressed on dry-weight basis (Palm and
Rowland, 1997). Biochemical composition varies among species, and
physiological stage (Constantinides and Fownes, 1994; Heal et al.,
1997). Therefore, it is important to include the age or physiological
stage of the material and the organs included.

# Analytical methods

There are two extraction approaches for characterizing residue quality.
One is to use a sequential extraction scheme (Figure 1). Sequential
extraction allows isolation of more specific components with a limited
amount of plant material however they it is time consuming, expensive
and has more potential for experimental error (Palm and Rowland, 1997).
The second method is to do separate extractions of a limited number of
components (Figure 2). An advantage of using the separate extraction
method is that for example, lignin could be extracted without having to
first extract starch. Also, separate extraction tends to reduce the
experimental error (Palm and Rowland, 1997).

Methods for determining extractives, starch, total carbohydrate by HPLC,
acid-soluble lignin, acid-insoluble lignin and ash have been developed
by the National Renewable Energy Laboratory (NREL) at Golden, CO and
have been accepted by the ASTM as ASTM standard test methods. These
methods are available at the NREL web site
http://www.eere.energy.gov/biomass/analytical_procedures.html as
standard biomass analytical procedures (Table 1). The methods at the
NREL site have the advantage of being very detailed, complete with
background references, step-by-step protocols and sample calculation.
Currently, the methods can be downloaded free of charge. In addition
this web site has a biomass feedstock composition and property database,
which has information on agricultural residues, wood, herbaceous energy
crops and other potential biofuel sources.

Table 1. A partial list of protocols available at NREL for
characterizing residue quality.

  ---------------- ---------- ---------------------------------------------------------------
  Component        NREL       Link:
                   protocol   http://www.eere.energy.gov/biomass/analytical_procedures.html

  Total solids     LAP-001    
  (biomass)                   

  Extractives      LAP-010    

  Starch           LAP-016    

  Carbohydrates    LAP-002    

  Acid-insoluble   LAP-003    
  lignin                      

  Acid-soluble     LAP-004    
  lignin                      

  Ash              LAP-005    

                              
  ---------------- ---------- ---------------------------------------------------------------

Table 2. Additional methods for characterizing residue quality.

  ------------------------------- ---------------------------------------
  Component                       Citation

  Nonstructural Carbohydrates     (Hendrix, 1993)

  Soluble sugars                  (Dubois et al., 1956)

  Soluble C and N                 (Anderson and Ingram, 1993)

  Soluble phenolics               (Waterman and Mole, 1994)

                                  

  Alkaline extractable phenolics  (Martens, 2002)
  ------------------------------- ---------------------------------------

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References

Allen, S.E., (ed.) 1989. Chemical Analysis of Ecological Material 2nd
edition, pp. 1-368. Blackwell, Oxford, UK.

Ambus, P., E.S. Jensen, and G.P. Robertson. 2001. Nitrous oxide and
N-leaching losses from agricultural soil: Influence of crop residue
particle size, quality and placement. Phyton-Annales REI Botanicae
41:7-15.

Anderson, J.M., and J.S.I. Ingram. 1993. Tropical Soil Biology and
Fertility: A Handbook of Methods. 2nd ed. CAB International,
Wallingford, UK.

Angers, D.A., R.P. Voroney, and D. Cote. 1995. Dynamics of soil organic
matter and corn residues affected by tillage practices. Soil Sci. Soc.
Am. J. 59:1311-1315.

Baggs, E.M., R.M. Rees, K. Castle, A. Scott, K.A. Smith, and A.J.A.
Vinten. 2002. Nitrous oxide release from soils receiving N-rich crop
residues and paper mill sludge in eastern Scotland. Agric. Ecosyst.
Environ. 90:109-123.

Balesdent, J., and M. Balabane. 1996. Major contribution of roots to
soil carbon storage inferred from maize cultivated soils. Soil Biol.
Biochem. 28:1261-1263.

Ball, B.C., J.P. Parker, and A. Scott. 1999. Soil and residue management
effects on cropping conditions and nitrous oxide fluxes under controlled
traffic in Scotland 2. Nitrous oxide, soil N status and weather. Soil
Till. Res. 52:191-201.

Barber, S.A. 1979. Corn residue management and soil organic matter.
Agron. J. 71:625-627.

Boone, R.D. 1994. Light fraction soil organic matter: origin and
contribution to net nitrogen mineralizaton. Soil Biol. Biochem.
26:1159-1168.

Constantinides, M., and J.H. Fownes. 1994. Nitrogen mineralization from
leaves and litter of tropical plants: Relationship to nitrogen, lignin
and soluble polyphenol concentrations. Soil Biol. Biochem. 26:49-55.

Dubois, M., K.A. Gilles, J.K. Hamilton, P.A. Rebers, and F. Smith. 1956.
Colorimetric method for determination of sugars and related substances.
Analytical Chemistry 28:350-358.

Farmer, A.M., and M.S. Adams. 1991. Carbon uptake by roots, p. 627-637,
*In* Y. Waisel, et al., eds. Plant Roots: The Hidden Half. Marcel
Dekker, New York.

Flessa, H., B. Ludwig, B. Heil, and W. Merbach. 2000. The origin of soil
organic C, dissolved organic C and respiration in a long-term experiment
in Halle, Germany, determined by ^13^C natural abundance. J Plant
Nutrit. Soil Sci. 163:157-163.

Franck, W.M., B.A. Hungate, F.S. Chapin, and C.B. Field. 1997.
Decomposition of litter produced under elevated CO~2~ - dependence on
plant species and nutrient supply. Biogeochemistry 36:223-237.

Gale, W.J., and C.A. Cambardella. 2000. Carbon dynamics of surface
residue- and root-derived organic matter under simulated no-till. Soil
Sci. Soc. Am. J. 64:190-195.

Gorissen, A., J.H. van Ginkel, J.J.B. Keurentjes, and J.A. Van Veen.
1995. Grass root decomposition is retarded when grass has been grown
under elevated CO~2~. Soil Biol. Biochem. 27:117-120.

Heal, O.W., J.M. Anderson, and M.J. Swift. 1997. Plant litter quality
and decomposition: an historical overview., p. 47-66, *In* G. Cadish and
K. E. Giller, eds. Driven by Nature, Plant Litter Quality and
Decomposition. CAB International, Wallingford.

Hendrix, D.L. 1993. Rapid extraction and analysis of nonstructural
carbohyrates in plant tissue. Crop Sci. 33:1306-1311.

Huggins, D.R., G.A. Buyanovky, G.H. Wagner, J.R. Brown, R.G. Darmody,
T.R. Peck, G.W. Lesoing, M.B. Vanotti, and L.G. Bundy. 1998. Soil
organic C in the tallgrass prairie-derived region of the corn belt:
effects of long-term crop management. Soil Till. Res. 47:219-234.

Johnson, J.F., D.L. Allan, C.P. Vance, and G. Weiblen. 1996. Root carbon
dioxide fixation by phosphorus-deficient *Lupinus albus*, Contribution
to organic acid exudation by proteoid roots. Plant Physiol. 112:19-30.

Kennedy, A.C. 1999. Bacterial diversity in agroecosystems. Agric.
Ecosyst. Environ 74:65-76.

Martens, D.A. 2002. Identification of Phenolic Acid Composition of
Alkali-extracted Plants and Soils. Soil Sci. Soc. Am. J. 66:1240-1248.

NREL. 1996. Determination of Carbohydrates in Biomass by High
Performance Liquid Chromatography LAP-002

Standard Biomass Analytical Methods, NREL Laboratory Analytical
Procedures \[Online\]. Available by National Renewable Energy
Laboratory, Golden, CO
http://www.ott.doe.gov/biofuels/analytical_methods.html (posted Last
updated: 11/22/02; verified 3/24/2003).

Palm, C.A., and A.P. Rowland. 1997. A minimum dataset for
characterization of plant quality for decomposition, p. 379-392, *In* G.
Cadisch and K. E. Giller, eds. Driven by Nature: Plant litter quality
and decomposition. CAB International, Cambridge, UK.

Paustian, K., H.P. Collins, and E.A. Paul. 1997. Management controls on
soil carbon, p. 15-49, *In* E. A. Paul, et al., eds. Soil Organic matter
in Temperate Agroecosystems: Long-Term Experiments in North America. CRC
Press, Boca Raton, FL.

Puget, P., and L.E. Drinkwater. 2001. Short-term dynamics of root- and
shoot-derived carbon from a leguminous green manure. Soil Sci. Soc. Am.
J. 65:771-779.

Scholes, M.C., D. Powlson, and G.L. Tian. 1997. Input control of organic
matter dynamics. Geoderma 79:25-47.

Shelp, M.L., E.G. Beauchamp, and G.W. Thurtell. 2000. Nitrous oxide
emissions from soil amended with glucose, alfalfa, or corn residues.
Commun. Soil Sci. Plant Anal. 31:877-892.

Vance, C.P., S. Stade, and C.A. Maxwell. 1983. Alfalfa root nodule
carbon dioxide fixation: I. Association with nitrogen fixation and
incorporation into amino acids. Plant Physiol. 72:469-473.

Waterman, P., and S. Mole. 1994. Analysis of phenolic plant metabolites
Blackwell Scientific Publications, London, UK.