Soil Biology & Biochemistry 55 (2012) 60e69
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Soil Biology & Biochemistry
journal homepage: www.elsevier.com/locate/soilbio
Amino acid, peptide and protein mineralization dynamics in a taiga forest soil
David L. Jones a, *, Knut Kielland b
a
b
Environment Centre Wales, Bangor University, Gwynedd LL57 2UW, UK
Institute of Arctic Biology, University of Alaska Fairbanks, Fairbanks, AK 99775, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 19 January 2012
Received in revised form
29 May 2012
Accepted 2 June 2012
Available online 27 June 2012
The availability of inorganic N has been shown to be one of the major factors limiting primary
productivity in high latitude ecosystems. The factors regulating the rate of transformation of organic N to
nitrate and ammonium, however, remain poorly understood. The aim of this study was to investigate the
nature of the soluble N pool in forest soils and to determine the relative rate of inorganic N production
from high and low molecular weight (MW) dissolved organic nitrogen (DON) compounds in black spruce
forest soils. DON was found to be the dominant N form in soil solution, however, most of this DON was of
high MW of which >75% remained unidentified. Free amino acids constituted less than 5% of the total
þ
DON pool. The concentration of NO
3 and NH4 was low in all soils but significantly greater than the
concentration of free amino acids. Incubations of low MW DON with soil indicated a rapid processing of
þ
amino acids, di- and tri-peptides to NHþ
4 followed by a slower transformation of the NH4 pool to NO3 .
The rate of protein transformation to NHþ
4 was slower than for amino acids and peptides suggesting that
the block in N mineralization in taiga forest soils is the transformation of high MW DON to low MW DON
þ
and not low MW DON to NHþ
4 or NH4 to NO3 . Calculated turnover rates of amino acid-derived C and N
immobilized in the soil microbial biomass were similar with a half-life of approximately 30 d indicating
congruent C and N mineralization.
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Biodegradation
Boreal forest
Coniferous forest
Dissolved organic carbon
Nitrogen mineralization
1. Introduction
Nitrogen (N) availability has been hypothesized to be a key
regulator of net primary productivity and vegetation successional
gradients in taiga forest systems (Vance and Chapin, 2001). The
cycling of the diverse N-containing compounds in these forest soils,
however, remains poorly understood. In particular, the relative
contribution and role of dissolved organic and inorganic N
compounds in plant and microbial nutrition remains controversial
(Owen and Jones, 2001; Schimel and Bennett, 2004). Evidence
suggests that a range of plants may be capable of bypassing the
mineralization step of the nitrogen cycle by directly taking up low
molecular weight (MW) dissolved organic nitrogen compounds
(DON) such as amino acids and peptides thereby obviating the need
for the soil microbial community to process organic N to NHþ
4 and
NO
3 (Chapin et al., 1993; Kielland, 1994; Jones et al., 2005a;
Kielland et al., 2006b; Hill et al., 2011). In many taiga forest systems
the soil microbial community is dominated by mycorrhizal fungi
which can provide roots with an additional mechanism to short
circuit the N cycle. Further, mycorrhizal fungi are capable of
* Corresponding author. Tel.: þ44 1248 382579; fax: þ44 1248 354997.
E-mail address: [email protected] (D.L. Jones).
0038-0717/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.soilbio.2012.06.005
degrading a range of complex soil organic N forms, although the
direct transfer of this N to roots has rarely been demonstrated (e.g.
polyphenol bound protein etc; Jones et al., 2005a; Rains and
Bledsoe, 2007). Competition for DON between the intrinsic
microbial population and roots with associated mycorrhizas can be
expected to be great in the most N limiting ecosystems (Andresen
et al., 2008). To fully appraise the contribution of direct DON
uptake to plant nutrient acquisition in taiga forests, however,
requires a detailed mechanistic understanding of soil N fluxes and
pool sizes and the spatial and seasonal control of these parameters.
Studies have shown that while the size of the amino acid pool in
soil is low, with concentrations typically ranging from 1 to 50 mM,
the flux through this pool can be extremely rapid (Kielland, 1995;
Jones and Kielland, 2002; Jones et al., 2009). Studies with isotopic C
tracers have indicated that the half-life of the amino acid pool in
soil is in the region of 1e6 h indicating that the free amino acid pool
turns over hundreds of times annually in black spruce forests
(Kielland et al., 2007). The impact of this rapid cycling through
the low MW DON pool on subsequent NHþ
4 and NO3 production,
however, remains largely unknown. When adding the N-rich amino
acid arginine to soil, Jones and Kielland (2002) demonstrated
a rapid production and excretion of NHþ
4 into the soil concomitant
with the release of CO2 from the amino acid. This result suggested
that the soil microbial community might be using free amino acids
D.L. Jones, K. Kielland / Soil Biology & Biochemistry 55 (2012) 60e69
not as a source of N but for their energy generating capacity.
Previous work in a wide range of global biomes has indicated that
both these pathways (catabolism and anabolism) operate simultaneously with approximately 30e40% of amino acid-C used in
respiration with the remaining amino acid-C used for cell biomass
production and maintenance (Jones et al., 2005b, 2009). Whether
30e40% of the N associated with the amino acid-C that is respired
gets consistently excreted into the soil, however, remains unknown.
Current evidence suggests that while amino acid turnover is
rapid in taiga forests soils, the levels of inorganic N are extremely
low with very little accumulation of NO
3 (Schimel et al., 1996;
Kielland et al., 2006a). This lack of NO
3 accumulation has led to the
hypothesis that there is a nitrification block in black spruce soils
possibly induced by the low pH of these ecosystems which
suppresses nitrifying bacteria. An alternative explanation, however,
is that in these N limiting environments NO
3 fails to accumulate
due to the lack of nitrification precursors due to rapid removal of
NHþ
4 by plants and microorganisms. Similarly, nitrification could be
rapid but continual removal of NO
3 by plants and microorganisms
would also prevent accumulation. The aim of this study was
therefore to evaluate the points in the breakdown pathway of DON
that limit inorganic N production in black spruce soils.
2. Materials and methods
2.1. Site characteristics
Soil was collected from the Bonanza Creek Taiga LTER black
spruce (Picea mariana L.) sites which are located approximately
20 km SW of Fairbanks, Alaska (65 450 N, 148 150 W) on the Tanana
River floodplain. This vegetation was essentially a black spruce
monoculture with moss understory and represents the terminal
vegetational successional stage at this location (occurs at >500
years; Viereck et al., 1993a). The mean annual temperature
is 3.3 C, and mean annual precipitation 269 mm y1, of which
approximately 37% falls as snow (Viereck et al., 1993b). Soil degree
days (above 0 C) average 640 (equivalent to 100 growing days or
less per year) and annual productivity is approximately
110 g m2 y1 (Viereck et al., 1983) with a standing above-ground
biomass ranging from 2 to 11 kg m2 y1 (Van Cleve et al., 1991).
The soils have a coarse loamy texture and are classified as Histic
Pergelic Cryaquepts, derived from a loess parent material (Viereck
et al., 1983, 1993a). The site is characterized by poor drainage
with soils of high organic matter, relatively acidic, and underlain by
permafrost (Maximum active layer thaw depth 112 23 cm). The
forest floor chemistry is characterized by a low bulk density
(0e20 cm ¼ 0.22 0.02 g cm3), high lignin content and high C:N
ratios (Total C ¼ 21.3 2.8%; Total N ¼ 0.73 0.12%). These forests
exhibit very low rates of decomposition and N mineralization (Fox
and Van Cleve, 1983; Van Cleve et al., 1993a,b; Klingensmith and
Van Cleve, 1993; Kielland et al., 2006a).
Soil samples were collected with a 6.5 i.d. cm stainless steel
corer to a depth of 20 cm. Ten samples were randomly collected
from each of three independent forest stands spread along a 20 km
transect along the Tanana River. Soil cores were subsequently subdivided based on horizon (L and O1), sieved to pass 2.5 cm and
stored in sealed, gas permeable plastic bags at 2 1 C for 10 d prior
to the start of the incubations. The L horizon was composed largely
of moss and pine needles in the early stages of decomposition (i.e.
tissues still recognisable) whilst the O1 horizon contained well
decomposed organic residues derived from moss, needles and
roots. Previous studies at this site and others have shown that over
this storage period, basal soil respiration or NHþ
4 /NO3 contents do
not change significantly from those analyzed immediately after
collection (Jones and Kielland, 2002). Soil solution was extracted
61
from a separate set of intact soil cores collected as described above.
The initial soil water content of the cores was 942 169 g kg1
expressed on a dry weight basis.
2.2. Soil characterization
Soil solution was extracted by the centrifugation-drainage
technique described by Giesler and Lundström (1993). Briefly,
after placing approximately 80e100 g of intact field-moist soil (L or
O1 horizon) into the PTFE centrifugal extraction cups, the soil was
centrifuged for 30 min at a speed of 6000 g at 4 C. Typically,
15e20 ml of soil solution was extracted from each soil sample
equating to 40e60 % of the total soil water in the sample. The
collected soil solutions were stored frozen at 20 C to await
analysis. Only three soil solution extractions were performed for
each horizon from each of the three forest stands (i.e. 9 samples in
total for each horizon).
2.3. Nitrogen mineralization
Within 10 d of collection, 10 g samples of soil were transferred to
50 cm3 polypropylene tubes followed by storage in a dark climatecontrolled chamber at 10.0 0.2 C overnight. The following day,
1 ml of either distilled water or a DON solution was added to the soil
in a drop-wise fashion (drop volume ¼ 16 3 ml) and the tubes
agitated slightly to ensure good mixing and the tubes loosely capped to ensure adequate soil aeration. The DON solutions contained
either protein [bovine serum albumin (BSA) or casein], di- and tripeptides (L-Ala-Ala, L-Ala-Ala-Ala or L-Ala-Gln where Ala ¼ alanine
and Gln ¼ glutamine), a single amino acid (L-Ala) or a mixture of
amino acids. BSA was composed of a single polypeptide of
66,340 MW whilst casein contained four different polypeptide
subunits with an average MW of 30,000. All chemicals were
obtained at purities >97% (SigmaeAldrich, Poole, Dorset, UK). The
rate of N addition in all the DON solutions was 2 mmol N kg1. The
amino acid mixture contained the following L isomeric amino acids
in equimolar proportions: alanine, arginine, aspartate acid, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, phenylalanine, proline, serine, threonine, tyrosine and valine. The N addition
rate was chosen based upon the total amount of amino acid in plant
cells (5e20 mM; Jones et al., 2005a) and the likely concentration
present in the soil after lysis of a root epidermal cell. The tubes were
then incubated at 10.0 0.2 C for 60 d. Water loss from the tubes
was negligible and represented <2% of the total soil weight and the
loss was therefore not corrected for.
At time periods of 0, 1, 5, 10, 20, 30 and 60 d after DON addition,
individual tubes were removed and soluble and exchangeable
inorganic N extracted by shaking the soil with 30 ml of 1 M KCl on
a reciprocating shaker (Unimax 2010; Heidolph Elektro GmbH,
Kelheim, Germany; 250 rev min1) for 30 min. After shaking, the
soil suspensions were centrifuged (15 min; 14,000 g) and the
supernatant recovered and stored at 20 C to await analysis.
2.4. Chemical analysis
Total dissolved N (TDN) in soil solutions was determined with
a Shimadzu TOCV-TNM1 analyzer (Shimadzu Corp., Kyoto, Japan).
NHþ
4 in soil solutions and extracts was determined colorimetrically
by the salicylate-nitroprusside method of Mulvaney (1996) on
a Skalar autoanalyzer (Skalar UK Ltd., York, UK). NO
3 was determined colorimetrically using the same Skalar autoanalyzer in
which NO
3 was first reduced to NO2 with a CdeCu column fol
lowed by the reaction of the NO2 produced with N-1-napthylethylenediamine to produce a chromophore. DON was calculated as
the difference between the TDN reading and the combined NHþ
4
62
D.L. Jones, K. Kielland / Soil Biology & Biochemistry 55 (2012) 60e69
and NO
3 reading (DIN). Total free amino acids were determined on
a Cary Eclipse microplate fluorometer (Varian Australia Pty Ltd.,
Mulgrave, Victoria, Australia) using the fluorometric o-phthaldialdehyde-b-mercaptoethanol procedure of Jones et al. (2002) and
assuming an amino acid N content of 168 g kg1. Proteins in soil
solution were estimated by the method of Bradford (1976) using
BSA as a standard and assuming a protein N content of 151 g kg1.
Protein values were corrected for interference from humic/fulvic
substances, which cross-react with the Coomassie Brilliant Blue
stain (Roberts and Jones, 2008). Briefly, the protein assay was
undertaken with humic acid in place of protein and a standard
curve produced. Secondly, the absorbance of the unamended
humic acid standards was determined at 400 nm as described
below. Next the absorbance of the soil solutions was determined at
400 nm allowing the degree of interference to be calculated from
the humic acid standards. The average interference of the humic/
fulvic substances was 26 3% of the total uncorrected protein
reading. Coloured compounds in the soil solution (comprised of
humic and fulvic acid-like substances; Kane et al., 2006) were
measured by determining the absorbance of the soil solutions at
400 and 600 nm and comparison against a commercial humic acid
standard (humic acid, sodium salt; H16752; SigmaeAldrich Co.;
Claret et al., 2008; Tanaka, 2012). Total soluble phenols and tannins
were determined by the method of Swain and Hillis (1959) modified for use with a microplate reader. Low MW DON and DOC were
determined after passage through a pre-rinsed cellulose triacetate
nominal 5000 MW cut-off ultrafiltration membrane (VectraSpin;
Whatman Ltd, Maidstone, UK).
þ
plus the NO
3 produced from NH4 . We assumed that any NO3
production in the soil originated from NHþ
produced
from
the
4
added DON. The rate of NO
3 production from DON is therefore
intrinsically dependent upon the concentration of NHþ
4 in soil.
Consequently, after determination of kNHþ , this value was used in
4
a second subroutine to determine the first order kinetic rate
constant for NO3 production (kNO3 ). A second approach was also
used to calculate kNHþ in which a linear form of the first order
4
kinetic equation
lnðCt =C0 Þ ¼ kNHþ t
4
where Ct is the concentration at time t and C0 is the initial substrate
concentration, was fitted to the experimental data. Regression
analysis in Sigmaplot v12 was used to determine kNHþ .
4
3. Results
3.1. Soil solution characteristics
Visual inspection of the soil revealed that most of the roots were
present in the L and O horizons of the soil rather than in the loess
dominated mineral subsoil. The characteristics of the soil solutions
extracted from the surface horizons of three independent forest
stands are shown in Tables 1 and 2. Significantly greater acidity and
electrical conductivity were observed in the soil solutions of the L
horizons in comparison to the O horizons. The soil solutions
extracted from both the L and O horizons exhibited a strong brown
colouration indicating the presence of high amounts of dissolved
organic carbon (DOC) and high MW aromatic compounds. A
significantly greater amount of these substances were detected in
the L horizon in comparison to the O horizon. After comparison of
the colour of the soil solutions with a humic acid standard we
calculated that these coloured compounds (humic and fulvic acidlike substances) represented approximately 6.8 1.2% of the total
DOC. Similarly, the total amount of DOC attributable to phenolic
substances was estimated to be 5.8 1.0% of the total DOC. The
colour of the soil solutions was closely correlated with their
phenolic content indicating that solution colour can be used as
a simple surrogate for phenolic containing humic/fulvic substances
(Supplementary Information Figure S1; r2 ¼ 0.93). Passage of the
soil solutions through a 5000 MW cut-off filter indicated that the
majority of this DOC was of a high molecular weight nature in both
horizons (76 3% of the total DOC; Table 1).
In the black spruce forest stands, DON represented the dominant N pool in soil solution representing over 90% of the soluble
2.5. Statistical and data analysis
Linear regression and t-tests analyses of the soil solution data
were performed using MS Excel 9.0 (Microsoft Corp., Redwood, CA),
Minitab v16 (Minitab Inc., State College, PA) and Sigmaplot v12
(SPSS Inc., Chicago, IL). The net amount of NHþ
4 and NO3 produced
from the DON substrates added to the soil was calculated after
subtraction of the amount of NHþ
4 and NO3 present in the control
treatment (zero DON, distilled water addition). Rate constants for
the production of NHþ
4 and NO3 from the different DON sources
were determined using a least sum of squares optimisation routine
using Excel 9.0. This routine minimized the difference between the
experimental data and a first order kinetic model in which the first
order constants were varied. The time step in the iteration model
was 1 h. The first subroutine determined the first order kinetic rate
constant for NHþ
) from the DON substrates
4 production (kNHþ
4
þ
assuming that cumulative NHþ
4 production was the sum of NH4
Table 1
Chemical characteristics of soil solutions from the surface horizons (L or O) of three black spruce forest stands. Values represent means SEM. The P value describes statistical
differences between the L and O horizon data with * and *** indicating significant differences at the P < 0.05 and P < 0.001 level respectively and NS indicating no significant
difference between the two horizons (P > 0.05). Low molecular weight (MW) DOC is defined as being < 5000 MW while the high MW fraction is defined as being > 5000 MW.
To approximate the values in mg C kg1, multiply the mg C l1 values by a factor of 0.94.
pH
L horizon
Stand 1
Stand 2
Stand 3
Mean SEM
O horizon
Stand 1
Stand 2
Stand 3
Mean SEM
P value
Electrical
conductivity
(mS cm1)
Humic substances
(mg C l1)
Total phenols
(mg C l1)
Total phenols
(% of DOC)
DOC (mg C l1)
Low MW
DOC (mg C l1)
DOC-to-DON
ratio
Low
MW DOC-to Low
MW DON ratio
4.31
4.38
3.59
4.09
0.25
0.34
0.08
0.25
193
120
306
206
28
2
21
54
2.55
2.33
4.31
3.07
0.25
0.37
0.35
0.63
17.2
15.6
30.3
21.0
2.7
0.8
1.7
4.7
1.8
7.3
9.3
6.2
0.5
0.6
2.6
2.2
990
215
387
530
98
16
113
234
283
74
65
140
66
27
16
71
56
37
46
47
3
3
2
5
34
33
29
32
5.91
4.79
5.69
5.46
*
0.16
0.05
0.19
0.34
95
90
71
85
14
3
6
7
1.29
1.83
0.76
1.29
*
0.13
0.08
0.05
0.31
5.5
10.8
4.0
6.8
*
0.6
0.8
0.7
2.0
6.5
6.3
3.6
5.5
NS
1.8
0.3
1.0
0.9
93
171
118
127
NS
14
5
16
23
25
27
23
25
NS
9
3
3
1
40 36 35 37 NS
3
4
1
1
20 17 16 18 ***
*
3
12
2
2
4
1
3
1
D.L. Jones, K. Kielland / Soil Biology & Biochemistry 55 (2012) 60e69
63
Table 2
Chemical characteristics of soil solutions from the surface horizons (L or O) of three black spruce forest stands. Values represent means SEM. The P value describes the
statistical significance of the L and O horizon data with NS indicating no significant difference between the two horizons. Low molecular weight (MW) DOC is defined as
being < 5000 MW while the high MW fraction is defined as being > 5000 MW. To convert from mg l1 to mg kg1 multiply the table values by a factor of 0.94.
NO
3
(mg N l1)
L horizon
Stand 1
Stand 2
Stand 3
Mean SEM
O horizon
Stand 1
Stand 2
Stand 3
Mean SEM
P value
NHþ
4
(mg N l1)
DIN
(mg N l1)
DON
(mg N l1)
DON
(% of
total N)
Low MW
DON
(mg
N l1)
Low MW
DON (% of DON)
0.21
0.17
0.15
0.17
0.02
0.13
0.10
0.02
1.06
0.50
0.39
0.65
0.06
0.13
0.18
0.21
1.3
0.7
0.5
0.8
0.1
0.1
0.2
0.2
17.5
5.8
8.6
10.6
0.9
0.5
2.6
3.5
93
90
95
92
1
2
1
2
8.0
2.3
2.2
4.1
1.3
0.3
0.4
1.9
45
40
28
38
0.43
0.06
0.32
0.27
NS
0.09
0.01
0.14
0.11
0.26
0.46
0.29
0.34
NS
0.01
0.20
0.16
0.06
0.7
0.5
0.6
0.6
NS
0.1
0.2
0.3
0.1
2.3
4.9
3.4
3.5
NS
0.3
0.7
0.4
0.8
77 90 85 84 NS
4
2
6
4
1.2
1.6
1.5
1.4
NS
0.2
0.3
0.2
0.1
53 35 46 45 NS
Protein
(mg N l1)
Protein
(% of DON)
Amino acids
(mg N l1)
Amino acids
(% of DON)
5
4
5
5
1.1
1.4
0.8
1.1
0.2
0.1
0.2
0.2
7
24
14
15
1
1
8
5
0.92
0.17
0.12
0.40
0.08
0.05
0.04
0.26
5
3
1
3
1
1
0
1
8
9
10
5
0.8
0.8
0.6
0.8
NS
0.1
0.1
0.2
0.1
37 17 20 24 NS
7
1
6
6
0.20
0.05
0.03
0.09
NS
0.09
0.01
0.01
0.05
10 1
1
4
NS
4
0
0
3
concentration than the free amino acid pool (P < 0.05). The
concentration of NHþ
4 was about two fold greater than for NO3
þ
(P < 0.05). The concentration of NH4 was significantly linearly
correlated with DON (r2 ¼ 0.84; P < 0.001) whilst no significant
relationship was observed between NO
3 and DON (P > 0.05).
N in the L horizon and over 75% in the O horizon (Table 2). In the L
horizon, DON represented 0.14 0.05% of the total N whilst in the O
horizon it represented 0.05 0.01% of the total N. Fractionation
using a 5000 MW cut-off filter indicated that most of the DON was
of a high molecular weight nature in both soil horizons (59 4% of
total DON). The DOC-to-DON ratio of the soil solutions was high in
both soil horizons (41 3) with the value being significantly greater
in the high MW fraction (56 5) in comparison to the low MW
fraction (25 3; P < 0.001; Fig. 1).
Free amino acids constituted only a small proportion of the total
DON representing 4 1% of the total DON pool and 8 3% of the
low MW DON pool. Proteins in soil solution were present at higher
concentrations and represented 20 4% of the total DON pool and
37 10% of the high MW DON pool. Calculations indicated that
most (>75%) of the DON in soil solution remained unidentified.
The concentration of KCl-extractable inorganic N in soil solution
was low and significantly less than that of DON in both soil horizons
(P < 0.01). The total DIN pool, however, was significantly greater in
3.2. Nitrogen mineralization dynamics
The amount of background N in the non-DON amended soil over
the 60 d incubation period remained low in both the L and O
horizon (Fig. 2). In the L horizon, a net immobilization of NHþ
4 was
observed over the first 10 d after transfer of the soils from 2 C (0 d;
soil storage temperature) to 10 C (temperature of the mineralization experiment). The concentration of NO
3 gradually increased
over the 60 d period in the L horizon but still remained very low. In
contrast, no net NHþ
4 immobilization was observed in the O horizon
soil where the amount of NHþ
4 remained relatively constant
throughout the incubation period. In this soil horizon the
1000
-1
Dissolved organic carbon (mg L )
1500
800
B
A
1000
500
600
0
0
5
10
15
20
400
200
< 5000 MW fraction
> 5000 MW fraction
0
0
2
4
6
8
10
12
Dissolved organic nitrogen (mg L-1)
Fig. 1. Relationship between DOC and DON in soil solutions extracted from the surface horizon of a black spruce forest soil. Panel A (inset) shows the relationship between total DOC
and total DON in soil solution whilst Panel B shows the relationship between DOC and DON in the low (<5000 MW) and high (>5000 MW) molecular weight fraction of soil
solution. The axes legends are the same for both panels.
64
D.L. Jones, K. Kielland / Soil Biology & Biochemistry 55 (2012) 60e69
L horizon
NO3NH4+
0.8
-1
Soluble N (mmol kg )
1.0
0.6
0.4
0.2
0.0
-1
Soluble N (mmol kg )
O horizon
0.8
A first order kinetic model was fitted to the experimental data to
mathematically describe the N mineralization rate. This model
relied on the assumption that the DON added to soil would be
converted either directly (external deamination or internal deamination and excretion) or indirectly (biomass immobilization and
turnover) to NHþ
4 . It was further assumed that the soil was at steady
state over the 60 d experimental period and that the kNHþ and kNHþ
4
4
values remained constant over this period. It was also assumed that
this NO
3 would not be taken up by soil microorganisms and
therefore this end product would gradually accumulate (Hill et al.,
2011). The least squares iteration model provided an excellent fit to
the experimentally derived NO
3 data, however, it tended to
underestimate the rate of NHþ
4 production particularly for the
peptide AlaeGln. The model results are summarized in Table 3 and
show that the rate of NHþ
4 production from amino acids and
peptides was very similar (P > 0.05). In contrast, the rate of NHþ
4
production from protein was approximately two-fold lower than
for peptides and amino acids. The rate of NO
3 production was
statistically similar for all treatments (P > 0.05).
0.6
4. Discussion
0.4
4.1. Soil solution chemistry
0.2
0.0
0
10
20
30
40
50
60
Time (days)
þ
Fig. 2. Concentration of NO
3 and NH4 in the L (upper panel) and O (lower panel)
horizon of a black spruce forest during incubation at 10 C without DON amendment.
All values represent means SEM (n ¼ 3) where each replicate represents soil from an
independent forest stand. The legend is the same for both panels.
concentration of NO
3 tended to increase over the duration of the
experiment with a three-fold increase observed over the 60 d
period. The low degree of variability in DIN concentrations within
and between the three forest stands allowed the net amount of N
originating from the added DON to be calculated with a high degree
of certainty.
In the L horizon, no significant net N production from the added
DON substrates could be detected and N levels in the soil were not
statistically different (P > 0.05; paired t-test) from the control
treatment to which only distilled water has been added (Fig. 3).
Additionally, no net N immobilization was observed. In contrast, N
accumulation from the added DON substrates was readily observed
in the O horizon soil (Fig. 4). For all the added low MW DON
substrates (amino acids and peptides) a significant increase in soil
NHþ
4 concentration was observed within 1 d. In the case of the high
MW substrates, a significant increase in the amount of soil NHþ
4 was
only observed after 5 d. In all DON treatments the concentration of
NHþ
4 gradually increased over time with concentrations being
maximal at 10 d for the low MW DON compounds and 20 d for the
high MW compounds (Fig. 4). After this peak in NHþ
4 concentration,
the levels in soil gradually decreased to close to background levels
(non-DON amended soil) by 60 d. In contrast to NHþ
4 , a significant
increase in soil NO
3 concentrations was only observed after 5 d for
the low MW substrates and 10e20 d for the high MW substrates.
This NO
3 continued to increase over the 60 d period in most
treatments. By the end of the 60 d incubation period, 28.9 1.0% of
the protein-N, 56.6 4.9% of the peptide-N and 72.3 12.8% of the
amino acid-N had been mineralized to NHþ
4 and NO3 .
Estimates of soluble N concentrations in soil are strongly
influenced by the method used to recover soil water and the
specificity of the analytical procedures used (Jones and Willett,
2006; Whiffen et al., 2007; Roberts and Jones, 2008). The results
presented here showing that DON represents the dominant form of
N available in black spruce soil solution are in agreement with
previous studies using chemical extracts to quantify DON both in
solution and sorbed to the solid phase (Jones and Kielland, 2002).
Of the DON recovered, only 25% could be identified as amino acids
and proteins with the remaining 75% remaining unidentified. This
problem of compound identification is longstanding and is severely
limiting our ability to predict the impacts and behaviour of DOC and
DON in soil (Kalbitz et al., 2000; Chantigny, 2003; Bolan et al.,
2011). It is likely that this fraction includes N largely held within
or bound to the surface of humic substances, many of which form
abiotically in solution (Collins et al., 1992; Gonzalez and Laird,
2004). Typically, it has been thought that most of this DON is of
high MW, is largely recalcitrant and represents the dominant DON
lost from forest catchments (Perakis and Hedin, 2002; Schmidt
et al., 2011). However, the results presented here and in
Smolander and Kitunen (2002) suggest that although only a small
proportion of the DON was present as low MW amino acids
(<200 MW), a large proportion (38e45%) of the DON was present
in the <5000 MW fraction. This fraction would include peptides
2e25 subunits long and within the range taken up directly by soil
microorganisms without the need for extracellular cleavage (Hill
et al., 2011). Further work is required to develop analytical
methods to determine the chemical nature of this fraction, and in
particular its peptide content.
Soil C-to-N ratio is a strong predictor of N mineralization.
Similarly, the soil DOC-to-DON ratio is frequently used as an indicator of DOM quality (McDowell et al., 2004; Bernal et al., 2005;
McGroddy et al., 2008). The fractionation results presented here
showed, however, that while there was no difference in the bulk
DOC-to-DON ratio between the soil L and O horizons, there was
a significant difference in this parameter between horizons in the
low MW fraction. Based upon our N mineralization results, it
suggests that the bulk DOC-to-DON ratio is a rather insensitive
predictor of N availability.
Our results showed that these soils contain a large amount of
soluble phenolic substances relative to the amount of DON.
D.L. Jones, K. Kielland / Soil Biology & Biochemistry 55 (2012) 60e69
65
-1
Soluble N (mmol kg )
2.0
Protein (casein)
NO3-
1.5
NH4+
NO3-
Protein (BSA)
NH4+
1.0
0.5
0.0
-0.5
-1
Soluble N (mmol kg )
Peptide (Ala-Ala-Ala)
Peptide (Ala-Gln)
1.5
1.0
0.5
0.0
-0.5
-1
Soluble N (mmol kg )
Peptide (Ala-Ala)
Amino acid mix
1.5
1.0
0.5
0.0
-0.5
-1
Soluble N (mmol kg )
Amino acid (Ala)
1.5
1.0
0.5
0.0
-0.5
0
10
20
30
40
50
60
Time (days)
NO
3
NHþ
4
Fig. 3. Time-dependent concentration of
and
in the L horizon of a black spruce forest amended with dissolved organic N compounds (proteins, peptides and amino acids)
and incubated over a 60 d period at 10 C. All values represent means SEM (n ¼ 3) where each replicate represents soil from an independent forest stand. The initial amount of
DON added to each soil was 2 mmol N kg1.
Polyphenols have been hypothesized to represent a significant
block to protein breakdown by not only binding to the substrate but
also by binding to proteases causing steric hindrance and denaturation (Ladd and Butler, 1971; Wurzburger and Hendrick, 2006).
Butler and Ladd (1971) also showed that the high MW DOC fraction
was particularly inhibitory against proteases. We surmise that the
higher concentrations of high MW polyphenols observed in the L
horizon may have been one reason why N mineralization was
inhibited relative to that in the O horizon. Our results also support
the findings of Kane et al. (2006) which suggested that black spruce
stand productivity and soil C turnover was inversely correlated
with high MW humic substances. Further work is therefore
required to identify differences in phenolic composition and
behaviour (i.e. protein binding) in relation to soil depth to determine if humic substances are more inhibitory from fresh litter in
comparison to well humified soil organic matter.
66
D.L. Jones, K. Kielland / Soil Biology & Biochemistry 55 (2012) 60e69
2.0
-1
Soluble N (mmol kg )
Protein (casein)
NO3-
1.5
NH4+
NO3-
Protein (BSA)
NH4+
1.0
0.5
0.0
-0.5
Peptide (Ala-Ala-Ala)
Peptide (Ala-Gln)
-1
Soluble N (mmol kg )
1.5
1.0
0.5
0.0
-0.5
Peptide (Ala-Ala)
Amino acid mix
-1
Soluble N (mmol kg )
1.5
1.0
0.5
0.0
-0.5
Amino acid (Ala)
-1
Soluble N (mmol kg )
1.5
1.0
0.5
0.0
-0.5
0
10
20
30
40
50
60
Time (days)
NO
3
NHþ
4
Fig. 4. Time-dependent concentration of
and
in the O horizon of a black spruce forest amended with dissolved organic N compounds (proteins, peptides and amino acids)
and incubated over a 60 d period at 10 C. All values represent means SEM (n ¼ 3) where each replicate represents soil from an independent forest stand. The initial amount of
DON added to each soil was 2 mmol N kg1.
4.2. Nitrogen dynamics
Table 3
First order kinetic rate constants for the production of NHþ
) and NO
)
4 (kNHþ
3 (kNO
3
4
from either proteins, peptides or amino acids added to the O horizon of a black
spruce forest soil. The half-life of this first order kinetic reaction is also provided.
Values represent means SEM (n ¼ 3).
Ammonium
kNHþ
4
Proteins
Peptides
Amino acids
Mean SEM
0.010
0.024
0.025
0.020
0.001
0.003
0.004
0.003
Nitrate
Half-life (d)
kNO3
0.022
0.020
0.022
0.021
73.1
30.3
28.9
42.1
6.5
4.2
5.0
8.4
Half-life (d)
0.001
0.003
0.002
0.001
31.1
36.6
31.3
33.5
1.
5.4
2.4
2.4
The results presented here clearly show that the rate of protein
breakdown is the limiting step in the transformation of soil organic
N to NO
3 . This view is also consistent with the N cycling paradigm
proposed by Schimel and Bennett (2004) and similar work in
temperate agroecosystems (Jan et al., 2009). The current view is
that proteins, which are largely immobilized on soil particles, must
be extracellularly cleaved to shorter chain units as these have less
potential for sorption and can therefore diffuse faster in soil. Due to
the vast array of potential enzymes which occur in soil microorganisms (e.g. endo- and exoproteases, peptidases, deaminases), it is
D.L. Jones, K. Kielland / Soil Biology & Biochemistry 55 (2012) 60e69
likely that a whole array of protein degradation products could be
produced from NHþ
4 through to relatively long peptides (6e10
residues). Based on a range of mineralization studies on DON and
amino acids, including those presented here, it appears that
microbial uptake of DON occurs mainly to obtain the C contained
within the DON rather than the N per se (Barraclough, 1997; Jones
et al., 2005b; Schmidt et al., 2011). This suggests that direct
deamination is not likely to be a significant breakdown pathway. It
is more likely therefore that free amino acids and peptides are the
primary products released during proteolysis although the relative
contribution of each pathway (i.e. exo- vs endo-protease) remains
unknown. Based on our results showing N mineralization from
amino acids and peptides was very similar, we conclude that either
(1) the rate of peptidase activity is very fast in soil, or (2) that soil
microorganisms take up both amino acids and peptides directly
from soil solution. Both of these scenarios are probably operating.
Evidence to support the former is consistent with Kielland et al.
(2007) who found relatively high levels of peptidase activity (ca.
11 mg kg1 h1). Evidence to support the latter is provided by
microbes grown in pure cultures and soil N competition studies in
which it has been shown that it is more energetically favourable to
take up peptides in comparison to amino acids (Payne, 1976).
Recent work in soil and solution culture has also demonstrated that
plant roots can directly take up a range of short chain peptides
without the need for prior cleavage to amino acids (Hill et al., 2011;
Soper et al., 2011). This implies that competition for N resources in
oligotrophic habitats, such as those studied here, may be occurring
at a much higher point in the N cycle than previously considered. It
is unfortunate therefore that analytical techniques are not available
to characterise the low MW DON pool (<5000 MW) in soil to
quantify the concentration of individual peptides.
The extractant used in our incubation studies (1 M KCl) was
chosen to recover both the soluble and exchangeable inorganic N
pools. However, sorption to the solid phase can significantly lower
solution substrate concentrations and potentially limit their availability to the soil microbial community. Based on the comprehensive survey of Alaskan Black Spruce soils by Ping et al. (2010), we
estimate that the cation exchange capacity (CEC) of our soils was in
the region 70e90 cmol kg1 and that the CEC is likely to be greater
in the O horizon than in the L horizon. The extent to which sorption
of C and N substrates will affect microbial activity is dependent
upon a range of factors, however, substrate charge is a key determinant. While Jones and Edwards (1998) showed that sorption of
highly charged substrates can significantly repress microbial
bioavailability, a recent study with weakly charged amino acids
showed that sorption did not greatly affect microbial uptake
(Fischer et al., 2010). Similarly, sorption of NHþ
4 to cation exchange
surfaces can also cause both increases and decreases in rates of
nitrification (Jiang et al., 2011). Our results suggest that the sorption
of amino acids and peptides to the solid phase were similar as no
differences in rates of downstream N cycling were seen between
the two substrate types. Differences in sorption are also unlikely to
explain the differences observed between the two horizons (Ping
et al., 2010).
The lack of significant net N mineralization in the L horizon
relative to the O horizon was surprising and suggests vertical
stratification in N availability in these black spruce topsoils. The
reasons for these differences could include: (1) greater polyphenol
inhibition of protease activity in the L horizon, (2) greater binding
of polyphenols to amino acids, peptides and proteins in the L
horizon preventing uptake by microbial membrane transporters,
(3) N limitation (in addition to C) in the L horizon, (4) less microbial
turnover in the L horizon relative to the O horizon, (5) pH induced
shifts in community composition between the horizons, or (6)
nutrient limitation preventing use of the N substrates. The first
67
argument we discount as a lack of N mineralization was also seen in
the amino acids and peptide substrates. The second argument is
possible although we would have expected this to have differentially affected proteins more. The third argument has some credibility as very low rates of inorganic N were present in the L horizon
throughout the 60 d incubation. The fourth hypothesis is also
potentially possible although we have no direct data to support this
either way. Low pH linked to the fifth hypothesis may explain the
lack of NO
3 production which is known to be limited in acid soils
due to a low nitrifier population, however, it fails to explain the lack
of NHþ
4 production and is thus discounted. In this situation it is
likely that low pH would lead to a more fungal based community
with a higher C:N ratio from which more N mineralization should
occur which is also not the case. The sixth hypothesis can also be
discounted as microbial amino acid use in these soils has been
shown previously to be very rapid in both the laboratory and field
(Jones and Kielland, 2002; Kielland et al., 2007; McFarland et al.,
2010). In summary, there are multiple reasons to explain why
there is a net immobilization of N in the L horizon, linked both to
the C:N ratio of the microbial biomass and the reaction of polyphenolics with the available substrates.
4.3. Carbon and nitrogen turnover in the microbial biomass
The results presented in Table 3 show that the half-life for the
production of NHþ
4 and NO3 from the added N substrates is
approximately 1 month. This contrasts strongly with rates of amino
acid-C mineralization by the soil microbial community which
typically possess half-lives less than 12 h in these soils (Jones and
Kielland, 2002; Kielland et al., 2007). It should be noted, however,
that the half-lives reported for 13C or 14C labelled amino acids
typically refer to the initial fast phase of mineralization which is
ascribed to the use of added C in anabolic processes. This first
mineralization phase typically only accounts for ca. 20e25% of the
total C added and is chosen to reflect the depletion of amino acids
from the soil solution (Jones and Kielland, 2002; Jones et al., 2009).
The remaining 75e80% of the added C is subsequently immobilized
in the biomass and turns over much slower (see Fig. S2 Supplementary On-line Material). Based on a re-analysis of the data for the
Alaskan black spruce soils presented in Jones et al. (2009), we
calculate that the half-life for this second slower phase of C derived
from the added amino acids is 44 1 d. If a NewtoneRaphson
transformation is applied to the complete dataset then the
average half-life for the added amino acid-C (i.e. average of both
pools together) can be calculated as 32 2 d, remarkably similar to
that reported for NHþ
4 here. We conclude therefore that N and C
mineralization occur congruently, primarily as a result of the
progressive turnover of the microbial biomass.
4.4. Ecological implications
One of the key findings of our experiments was evidence for
clear stratification within the soil profile with adjacent horizons,
both containing abundant roots, behaving in highly contrasting
ways with respect to their N cycling characteristics. Similar observations within topsoils have also been made in other spruce forest
types although the exact spatial patterns were different (Jones
et al., 2008; Matejek et al., 2010; Razgulin, 2010). This would also
imply that the mechanism for plant N retrieval from these distinct
horizons is also different. In the L horizon, it is clear that
microbially-mediated N release is a very slow process and that
plant reliance on this process to cycle sufficient N from litter would
be inefficient. Therefore we speculate that in the L horizon N
acquisition by plants occurs predominantly through mycorrhizal
networks which are more adapted to access polyphenol bound N
68
D.L. Jones, K. Kielland / Soil Biology & Biochemistry 55 (2012) 60e69
than tree roots directly (Brzostek and Finzi, 2011). The use of
mycorrhizas may also reduce plant competition, particularly if the
mycorrhizas are specifically selected to breakdown litter originating from black spruce (Talbot and Finzi, 2008; Aponte et al.,
2011). By contrast, in the O horizon where N mineralization is
much more rapid, we speculate that N can be accessed by roots
alone without the need for mycorrhizas. This is supported by
studies at the same location which have shown that fine root
production by black spruce is much greater in the O horizon than
the L horizon (Ruess et al., 2003).
Acknowledgements
We would like to thank Robert Leatherday, John Farrar and Karl
Olsen for technical assistance and academic input to the study. This
work was funded by the UK Natural Environment Research Council
and the US National Science Foundation.
Appendix A. Supplementary information
Supplementary information related to this article can be found
online at http://dx.doi.org/10.1016/j.soilbio.2012.06.005.
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