Microbial analyses
At the end of the incubation, phospholipid fatty acids in the soils were
measured following standard methods47-48 to
characterize microbial community composition. Phospholipids were
extracted from 3-g freeze-dried soil using 15 mL of a single-phase
chloroform: methanol: citrate buffer (1: 2: 0.8) (0.15 M, pH 4.0), and
measured using a gas chromatograph (Agilent 6890 Series, Agilent
Technologies, Wilmington, DE) with a flame ionization detector.
Nonadecanoic acid, 19:0, was used as an internal standard. Peaks were
identified using the MIDI peak identification software (MIDI, Inc.,
Newark, DE) and a bacterial fatty acid standard49-50.
A total of 34 phospholipid fatty acids were detected in these samples
and assigned to different functional groups according to the
nomenclature in the literature51, including G (+)
bacteria (i14:0, i15:0, i16:0, i17:0, a13:0, a15:0, a16:0 and a17:0);
G(-) bacteria (15:4ɷ3c, 14:0 3OH, 11Me 18:1ɷ7c, cy19:0 ɷ8c, 17:1ɷ8c,
18:1ɷ7c, 16:1ɷ7c, N 16:0, 15:3ɷ3c, 18:1ɷ9c, and 16:1ɷ6c and 15:1G);
fungi (16:1ɷ5c, 18:2ɷ6,9c and 18:3ɷ6c); actinomycetes (10Me16:0, 10Me
17:0, and 10Me20:0) and general bacteria (14:0, 15:0, 16:0, 17:0 and
18:0).
After the incubation, soil amino sugars were also measured to determine
the effects of clay mineralogy on fungal and bacterial
residues52. A sufficient mass of each soil sample was
ground to ensure extraction of approximately 0.3 mg N. Then the samples
were placed in a closed hydrolysis flask (filled with N2gas) and hydrolyzed in 10 mL of 6 M HCl at 105 oC for
8 hours. The solutions were filtered through glass-fiber filters GF6
(Schleicher & Schuell, Germany), and dried at 40 oC
using a rotary evaporator in vacuum. Afterwards, the samples were
re-dissolved in deionized water, their pH adjusted to 6.6-6.8 using 0.4
M KOH and 0.01 M HCl and centrifuged (1000 g ) for 10 mins. Amino
sugars were first recovered from post-incubation soils by freeze-drying
and methanol washing and then extracted with dichloromethane from the
aqueous solution as aldononitrile derivatives. Excess anhydride was
removed with 1 M HCl and deionized water. The amino sugar derivatives
were re-dissolved in a 300-μL mixed solvent of hexane and ethyl acetate
in 1: 1 volume ratio for final analysis after the removal of
dichloromethane by drying under nitrogen gas. The concentrations of
amino sugars were quantified with the internal standard myo-inositol
added before hydrolysis. N-methylglucamine was also added prior to
derivatization to calculate the recovery of amino sugars and the amounts
of amino sugars identified as glucosamine, galactosamine and muramic
acid. The concentration of fungal-derived residues was calculated as the
difference between total glucosamine and twice that of the muramic acid
concentration, while the concentration of bacterial-derived residues was
defined as the sum of bacterial glucosamine plus muramic acid and
galactosamine53-54.
13C solid-state NMR spectroscopy
13C CP/TOSS experiments were conducted at a spinning
speed of 5 kHz and a cross polarization CP time of 1 ms, with1H 90° pulse-length of 4 µs and a recycle delay of 0.8
s using a Bruker AVANCE400 spectrometer at 100 MHz for13C with 4-mm sample rotors. Four-pulse total sideband
suppression was employed before detection, and two-pulse phase-modulated
decoupling was applied for optimum resolution55. The
spectra obtained with cross polarization/total sideband suppression were
assigned to different C functional groups following previous
literature3,56-57. The relative proportions of the
functional groups in the total spectral area were obtained by
integration58 and presented for all the SOM samples in
comparison with those of the original litters in Supplementary Table 2.
The average noise of three chemical shift regions sampled from beyond
0-220 ppm in each spectrum was used as an error to determine the
differences in the functional group distributions between
samples59.