Soil Organic Matter Management: The Roles of Residue Quality in C Sequestration and N Supply - Sustainable Management of Soil Organic Matter - page 200

Materials and Methods

Soil samples were collected from the French Pyrenean piedmont, from a
Pinus pinaster forest and from adjacent agricultural sites, converted to
intensive maize cropping for 7 and 35 years. Soils are thick humic acid
loamy soils, developed from Quaternary silty alluvial deposits, classified as
vermic Haplumbrepts in the soil taxonomy (Arrouays, 1992). Samples were
collected from the ploughed layer or from an equivalent depth in the forest
soil (Balesdent et al ., 1998). Soils were air dried and clods were broken
apart to < 4 mm.
Particle size fractionation of the soil was based on mechanical
dispersion of the soil and is described in detail by Balesdent et al . (1998).
Briefly, in a first step, soil was dispersed by agitation with glass beads.
The > 50 m particle size fraction was discarded by sieving, sonicated

to disrupt microaggregates, and particle size fractions were separated by
sedimentation. The < 2

µ

m fractions were recovered after flocculation with
CaCl 2 and then freeze dried. Aliquots were kept for transmission electron
microscopy without flocculation and freeze drying. All separations were
performed in triplicate. C and N contents were determined by dry
combustion. Results are expressed on an oven dry mass basis. Mineralogy
of the < 2

µ

m fractions was determined with an X-ray diffractometer after
OM oxidation with H 2 O 2 .
For transmission electron microscopy, the < 2

µ

m suspensions were
first concentrated by centrifugation and equilibrated with 0.32 kPa.
Millimetre sub-samples were fixed with glutaraldehyde and osmium, then
exchanged with methanol, propylene oxide and embedded in a Spurr resin
(Chenu and Jaunet, 1992). Two samples were embedded for each soil
situation, each coming from different replicate fractionations. Ultrathin
sections (80 nm) were made and deposited on metal grids. The sections
were stained with uranyl acetate, lead citrate (Lewis and Knight, 1976) or
silver proteinate (Thierry, 1967) to contrast specific functional groups of
the organic matter and were observed on a Philips EM 420 TEM coupled
with a AN 10000 EDS elemental X-ray analyser. The organic constituents
were identified on the basis of their morphology and reaction with stains.
We used the heavy elements staining the ultrathin sections, which were eas-
ily detected with EDS, as probes for organic matter. On five photographs
taken at random at the same magnification for each sample (

µ

× 37,000), each

representing a surface of 16 m 2 , we identified on morphological criteria

(i) organic particles; (ii) organomineral aggregates; and (iii) mineral
particles. We classified as mineral particles those in which organic features
were not detectable on paper prints of the photographs at the working mag-
nification. The contours of particles and aggregates were traced manually
on the photographs, scanned and we measured their numbers, dimensions
and surfaces using the NIH-Image program for Macintosh computers.

µ