The roles of microorganisms in litter decomposition and soil formation

The roles of microorganisms in litter decomposition and soilformation

Satoru Hobara • Takashi Osono • Dai Hirose •

Kenta Noro • Mitsuru Hirota • Ronald Benner

Received: 1 August 2012 / Accepted: 15 September 2013 / Published online: 9 October 2013

� Springer Science+Business Media Dordrecht 2013

Abstract Much has been learned about the micro-

bial decomposition of plant litter, but relatively little is

known about microbial contributions to litter and soil

chemistry. We conducted a 3-year litterbag experi-

ment and measured hydrolyzable amino acids (AA)

and amino sugars (AS) to gain insights about micro-

bial contributions to the chemical characteristics of

decomposing litter and soil. Microscopic observations

of hyphae were used to estimate fungal contributions

to litter. The carbon (C)-normalized yields of AA and

AS increased during decomposition along with nitro-

gen (N), indicating a shift in chemical characteristics

from C-rich plant-derived biopolymers to N-rich,

microbially-derived biochemicals. The contributions

of fungal biomass to C and N were minor, but

necromass of fungi as melanized and clamp-bearing

hyphae increased during litter decomposition. Yields

of glucosamine and galactosamine in litter approached

those in microorganisms, particularly bacteria, sug-

gesting major contributions of bacterial residues to

litter during decomposition. The microbial contribu-

tions to decomposing litter were consistent with those

observed in organic and mineral soils. Microorgan-

isms play important roles in the organization and

stabilization of soil organic matter as well as N

immobilization and organic C preservation.

Keywords Litter decomposition � Carbon and

nitrogen dynamics �Amino sugars �Amino acids �Fungi � Bacteria

Responsible Editor: Kathleen Lohse

Electronic supplementary material The online version ofthis article (doi:10.1007/s10533-013-9912-7) contains supple-mentary material, which is available to authorized users.

S. Hobara (&) � K. Noro

Department of Environmental and Symbiotic Science,

Rakuno Gakuen University, 582 Midorimachi,

Bunkyodai, Ebetsu 069-8501, Japan

e-mail: [email protected]

S. Hobara � R. Benner

Department of Biological Sciences, University of South

Carolina, Columbia, SC 29208, USA

T. Osono

Center for Ecological Research, Kyoto University,

Otsu 520-2113, Japan

D. Hirose

College of Pharmacy, Nihon University, Funabashi,

Chiba 274-8555, Japan

M. Hirota

Graduate School of Life and Environmental Sciences,

University of Tsukuba, Tsukuba 305-8572, Japan

123

Biogeochemistry (2014) 118:471–486

DOI 10.1007/s10533-013-9912-7

http://dx.doi.org/10.1007/s10533-013-9912-7

Abbreviations

AA Amino acid

AS Amino sugar

THAA Total hydrolyzable amino acid

THAS Total hydrolyzable amino sugar

GlcN Glucosamine

GalN Galactosamine

OC Organic carbon

Introduction

Litter decomposition in terrestrial ecosystems is an

important process for nutrient regeneration, maintaining

productivity and influencing ecosystem structure (Swift

et al. 1979). The process of litter decomposition is

closely associated with microbial activities, which alter

the chemical composition of litter and control carbon

(C) and nitrogen (N) dynamics in soil (Berg and

McClaugherty 2008). Hence, investigations of microbial

contributions to litter chemistry during decomposition

are essential for fully understanding the mechanisms of

litter decomposition and C and N dynamics in soil.

The enzymatic activities of microorganisms play a

major role in the decomposition of plant litter and soil

formation (Aber and Melillo 2001; Berg and McClaugh-

erty 2008), whereas much less is known about microbial

contributions to litter chemistry. Microbial growth and

the resulting biomass and necromass significantly alter

the chemical characteristics of soil organic matter, as

observed in nutrient immobilization (Hart et al. 1994;

Bengtsson et al. 2003; Tremblay and Benner 2006;

Simpson et al.2007; Wanek et al. 2010; Liang and Balser

2011; Cotrufo et al. 2013). In addition, the transforma-

tions and fates of microbial biomass and necromass

affect soil formation, stabilization, and fertility (Six et al.

2006; Fontaine et al. 2011; Miltner et al. 2011).

The effects of microbial growth and decomposition

processes can be traced through measurements of

amino acids (AA) and amino sugars (AS), biomole-

cules that are much more abundant in microbes than in

plant litter (Amelung et al. 2001; Tremblay and

Benner 2006). The composition and yields of AA and

AS in litter changes with sources and decomposition

processes, and some of these biomolecules, such as

muramic acid (MA), are uniquely found in specific

microorganisms (Parsons 1981; Benner and Kaiser

2003; Amelung et al. 2008). Furthermore, AA and AS

are important components of N immobilization

(Marumoto et al. 1982; Mengel 1996; Tremblay and

Benner 2006; He et al. 2011a, b) and C sequestration

(Glaser et al. 2006; Liang et al. 2007; Knicker 2011) in

soils. Therefore, the characterization of AA and AS in

decomposing litter provides important information

about microbial contributions to litter chemistry and

the biogeochemical processes leading to soil forma-

tion. However, the characterization of AA and AS has

been performed mostly for bulk litter and/or soil

samples (Solomon et al. 2002; Ding et al. 2010), and

few studies have examined relationships between litter

and microbial characteristics during decomposition

processes (Guggenberger et al. 1999).

We hypothesize that microorganisms play major

roles in both litter decomposition and soil formation.

In the present study, we measured total hydrolyzable

AA and AS in decomposing plant litter from three

plant species, and compared them with analyses of

soils (organic and mineral) and microorganisms (fungi

and bacteria) to investigate the origins and dynamics

of these major biochemicals during the decomposition

process. Fungi typically dominate soil microbial

biomass during the early stages of litter decomposition

(Berg and McClaugherty 2008). Thus, we also

estimated the biomass and necromass of fungi to

further elucidate their contributions to litter chemistry.

Materials and methods

Study sites, litterbag experiment, and sample

collections

Litterbag experiments were carried out in grasslands

and forests of Sugadaira Montane Research Center,

University of Tsukuba, Nagano, Japan (368300N,

1288200E). The soil was originally derived from

volcanic ash, classified as Typic Melanudand (Iimura

et al. 2010). Three sites that were adjacent to each

other but dominated by different plant species were

selected for the litterbag experiments: a grassland

dominated by Miscanthus sinensis, a coniferous

evergreen forest dominated by Pinus densiflora, and

a deciduous broad-leaf forest dominated by Quercus

crispula. We conducted the litterbag experiments at

these sites using litter of the dominant species. These

different plant species were chosen to gain a more

comprehensive understanding of biogeochemical pro-

cesses during litter decomposition and soil formation.

472 Biogeochemistry (2014) 118:471–486

123

Further detailed description of the study site and the

soils is provided in (Kato and Hayasi (2006) and

Iimura et al. (2010).

Freshly fallen leaves of the dominant plant species

(Miscanthus sinensis, Pinus densiflora, or Quercus

crispula) were collected from the soil surface at each

site during November 2003. The leaves were dried in

an oven at 40 �C for 1 week. Litter (*2 g) was placed

in litterbags (18 cm 9 18 cm) made of polypropylene

shade cloth with a mesh size of approximately 2 mm. A

total of 420 bags were prepared. Approximately 20 g

of each litter sample was stored for chemical analyses.

The decomposition experiment covered a 36-month

period from November 2003 to November 2006. A

transect was established at each site, and 10 subplots

(2 m 9 2 m) were located at 1 m intervals along the

transect, making a total of 30 subplots for the three

sites. Litterbags were place on the litter layer in

November 2003, with 14 bags per subplot. The

litterbags were fixed to the litter layer using metal

pins to prevent movement and to ensure good contact

between the bags and the litter layer. Sampling of the

bags took place 7 times: 6, 9, 12, 18, 21, 24, and

36 months after initiation of the experiment. On each

sampling occasion, 20 bags were collected for each

plant species from 10 subplots (2 bags each) and

transported them to the laboratory.

One bag from each subplot was used for estimation

of hyphal mass (see below), and another bag was used

for mass determination and chemical analyses. Sam-

ples were dried to a constant mass at 40 �C, and mean

values of mass loss were calculated for each sampling.

The subsamples for ash and chemical analyses were

ground in a laboratory mill to pass a 0.5-mm screen

and combined prior to analyses.

Soil and fungi samples were also analyzed to

compare their chemical compositions with those of

decomposing litter. Soils were sampled from the

organic (Oe?Oa) horizon and the mineral soil A

horizon in each site. After sieving with a 2 mm mesh

screen, soils were dried at 40 �C for 1 week, and

ground for analyses. One of the dominant fungal

species at all sites, Nigrospora sphaerica (Osono

2010), was grown in the laboratory for chemical

analyses. Nigrospora sphaerica is the dominant fungal

species colonizing decomposing leaves. We chose this

species because this was the only fungi observed in all

study sites throughout the experiment (T. Osono

unpublished data). The frequency of occurrence of

this species in litter was 75–90 % for Miscanthus

sinensis (Osono 2010), 20–25 % for Pinus densiflora,

and 25–45 % for Quercus crispula (T. Osono unpub-

lished data). Three isolates of N. sphaerica were

obtained from Miscanthus sinensis leaves, and grown

as a pure culture in Czapek medium (NaNO3 0.2 %,

K2HPO4 0.1 %, MgSO4�7H2O 0.05 %, KCl 0.05 %,

FeSO4 0.001 %, and sucrose 3 %). We used chemi-

cally defined, synthetic Czapek medium, instead of

natural medium, to incubate N. sphaerica isolates to

avoid the contamination of amino acids in the

medium. The cultured fungal biomass was harvested,

washed with sterile distilled water, dried at 40 �C, and

ground for analyses. We also analyzed a natural fungal

community, Cortinarius armillathus, and cultures of

Micrococcus lysodeikticus ATCC 4698 and cultures of

Aerobacter aerogenes and Azobacter vinelandii.

These bacterial cultures were obtained Sigma

Chemicals.

Hyphal length

Hyphal length in litter was measured by the agar-film

method in litter samples collected at 12, 24 and

36 months (Osono and Takeda 2001). Briefly, a

subsample (1 g) from each litterbag was homogenized

in a blender at 10,000 rev/min in 49 ml of deionized

water for 3 min. The suspension (20 ml) was diluted

with 20 ml of molten agar solution (final concentra-

tion 1.5 % (w/v)) and mixed at low speed on a

magnetic stirring plate. Three agar films were prepared

for each suspension in a haemocytometer (0.1 mm

depth), transferred to a glass slide, and dried for 24 h.

The films were dual-stained with fluorescent bright-

ener (FB) and acridine orange (AO) each for 1 h.

The stained films were mounted on slides with one

drop of immersion oil and examined with an epiflu-

orescent microscope (Nikon Microphot-SA, Nikon,

Tokyo). Darkly pigmented hyphae that were not

stained with FB were observed by bright-field micros-

copy. Microscope fields were selected randomly and

25 fields were observed for each slide. Total hyphal

length was calculated as the sum of FB-stained and

darkly pigmented hyphal lengths. We used following

definitions for fungal hyphae in this study: AO-stained

hyphae as living hyphae (Ono 1998); darkly-pig-

mented hyphae as melanized hyphae; clamp-bearing

hyphae as basidiomycetous hyphae. The Basidiomy-

cota includes such functional groups as lignin

Biogeochemistry (2014) 118:471–486 473

123

decomposing and mycorrhizal fungi. Hence, the

amount of clamp-bearing hyphae can represent

potential functional capabilities of fungal communi-

ties. It is recognized, however, that the fungal biomass

of the Basidiomycota may have been underestimated

because the frequency of clamp formation varies

between species.

Estimation of hyphal mass and AA, GlcN,

and GalN as hyphae

We used a conversion from hyphal length to hyphal

mass for estimating the AA, glucosamine (GlcN), and

galactosamine (GalN) contents of hyphae. We con-

verted hyphal length to hyphal mass according to the

equation in Paul and Clark (1989):

F ¼ pr2L D S� 102

where F is hyphal mass (g g-1 (w/w)), r is average

hyphal radius (cm) (0.00011 for Miscanthus sinensis

litter, 0.000131 for Pinus densiflora litter, 0.000135

for Quercus crispula litter), L is hyphal length

(m g-1), D is hyphal density (g cm-3) (1.1, from

Saito (1955)), and S is solid content (0.15, from (Baath

and Soderstrom (1977)). Total hydrolyzable AA,

GlcN, and GalN yields as hyphae in litter were

calculated multiplying the hyphal mass in litter by AA,

GlcN, and GalN yields in the Nigrospora culture,

respectively. In this study, we defined living hyphal

mass as fungal biomass and the remnant hyphae (total

minus living hyphae) as fungal necromass.

Ash and chemical analyses

Ash determinations were made by combusting subs-

amples from each litterbag at 800 �C for 4 h.

Elemental C and N contents were measured using a

CHN analyzer (Sumigraph NC-22, Sumitomo Chem-

ical Co., Osaka, Japan). Weight losses and elemental

compositions are reported on an ash-free dry weight

(AFDW) basis.

Total hydrolyzable amino acids (THAA) were

analyzed in dried samples (*5 mg; decomposing

litter, soils, fungal body, and references of other

microorganisms) that were placed in a sealed vacuum-

reaction-tube with 6 M HCl at 110 �C for 20 h.

Following hydrolysis, amino acids were analyzed

using the HPLC AccQ method (Reverter et al. 1997).

Separations were carried out on a reversed-phase

column (AccQ Tag C18, Waters Japan, Tokyo) heated

to 40 �C with a flow rate of 1.0 ml min-1 and eluted

with mobile phase A (0.04 M KH2PO4) and mobile

phase B (acetonitrile/water = 60/40 (v/v)) using a

linear gradient program. The following amino acids

were analyzed: alanine, arginine, aspartic acid, glu-

tamic acid, glycine, histidine, isoleucine, leucine,

lysine, methionine, phenylalanine, proline, serine,

threonine, tyrosine, and valine.

Total hydrolyzable amino sugars (THAS) were

analyzed in dried samples (*5 mg) placed in sealed

ampules with 3M HCl at 100 �C for 5 h (Kaiser and

Benner 2000). Acetyl groups are removed from amino

sugars during acid hydrolysis, so concentrations of the

deacetylated forms are presented. Hydrolyzed samples

were centrifuged, and the supernatant was neutralized

with the self-absorbed AG 11 A8 resin (Bio-Rad).

After desalting using the cation-exchanger AG50 X8

resin (Na?-form, Bio-Rad), concentrations of GalN,

GlcN, and mannosamine (ManN) were measured

using high-performance anion exchange chromatog-

raphy (HPAEC) with pulsed amperometric detection

(PAD). Although GalN and MA are assumed to

originate mostly from bacteria (Parsons 1981), we

used GalN as a tracer of bacteria in this study because

it is more abundant and persistant in litter and soil than

MA (Amelung 2003, Tremblay and Benner 2006,

Engelking et al. 2007). A Dionex ion chromatography

system with a CarboPac PA1 column with guard

column and PAD detector were used for all analyses.

Separation was performed under isocratic conditions

with a 12 mM NaOH mobile phase at a flow rate of

1 ml min-1. In the present paper, we do not report

ManN concentrations because they were low and often

below the level of quantification. Although GlcN/

GalN ratio has been used for evaluating the influence

of fungi, we used GlcN/GalN ratio as a diagenetic

indicator in this study because this ratio decreases with

decomposition in both soil (Amelung et al. 2001) and

water (Tremblay and Benner 2006), irrespective of the

presence of fungi.

The C- and N-normalized yields of AA and AS was

used in this study because they trace the fraction of

total C and N in litter and soil as these biochemicals. A

degradation index (DI) based on the composition of

protein amino acids was used to investigate the extent

of diagenetic alteration in decomposing litter and soils

(Dauwe et al. 1999).


123

The percentage of detrital N in litter and soils

derived from microorganisms were quantified using

the following equation (Tremblay and Benner 2006):

%Nimmob ¼ 100 Nsample � Nplant

� �=Nsample;

where Nimmob is the fraction of N immobilized by

microbes, Nsample is the total N content of the sample,

and Nplant is the total N content in the plant litter. The

Nplant from the remaining at time t was determined

assuming plant N had the same reactivity as plant C

(indicated from litter C content at time t (Ct) and fresh

litter C content (C0)) during decomposition:

Nplant;t ¼ Nplant;0 Ct=C0ð Þ:

One-way analysis of variance (ANOVA) was used

to compare the yields of AA and AS (average of all

sites) between fresh (initial) and decomposed

(36 months) litter. Correlation and regression analyses

were performed using JMP 8.0.2 software (SAS

Institute Japan).

Results

Bulk parameters

Changes in bulk parameters during the experiments

were variable among plant species (Table 1). Rates of

mass loss (AFDW) from litterbags were lower during

winter, but seasonal effects on mass loss were

relatively minor. Quercus litter showed higher losses

(89 %) during 36 months than Miscanthus (71 %) and

Pinus litter (67 %). The carbon content of litter

decreased with time for Miscanthus (*7 % during

36 months) and Quercus (*2 % during 36 months)

litter, but remained fairly constant for Pinus litter. The

losses of C from decomposing litter ranged from 67 to

90 % (Fig. 1A).

The N content of litter increased up to four fold

during the experiments (Table 1), leading to net

increases in the % initial N, especially during the first

year (Fig. 1B). Pinus litter N increased to 152 % of the

initial N in the first year and was 121 % at the end of

the experiment. The % initial N for Quercus litter also

increased during the first 12 months of decomposition,

but decreased rapidly to 21 % at the end of the study.

The pattern of N dynamics in the Miscanthus litter was

quite different and steadily declined during the study

to 60 % of the initial N content after 36 months

(Table 1).

Molar ratios of C/N in the three litter types

decreased gradually with time (Table 1). Variability

among C/N ratios of the initial litter samples ranged

from 58.9 to 148.8 and decreased to 23.7–40.6 by the

end of the experiment. The C/N values for the highly

decomposed litter samples (14.6–32.8) were

approaching those in organic and mineral soils. Fungi

had C/N ratios of 14.2–21.0, which were similar to

those in litter and soil samples, whereas bacteria had

much lower C/N ratios (3.7–4.7) (Table 2).

Amino acids and amino sugars yields in litter, soil

and fungi

The C-normalized yields of THAA were 12- to 57-fold

higher than those of THAS for litter and soil samples

(Table 1). Pinus litter had lower THAA and THAS

yields than Miscanthus and Quercus. The percentage

of C as AA and AS in litter samples ranged from 1.73

to 9.00 % and from 0.04 to 0.70 %, respectively, and

the percentage of N as AA and AS ranged from 46.5 to

88.8 % and from 0.91 to 2.77 %, respectively. The

yields of THAA and THAS in litter increased during

the experiment, and appeared to approach those of

soils during decomposition. Significant increases

(P \ 0.05 by ANOVA) in yields of THAA and THAS

were observed during litter decomposition. The

increase was greater in THAS (4- to 9-fold) than

THAA (2- to 3-fold) and was particularly high in

Pinus litter. The yields of THAA and THAS in soils

were similar to those of more highly decomposed litter

and were higher in the mineral soil layer than the organic

layer. Nigrospore hyphae, the most abundant fungal

hyphae at the study site, exhibited 1.7- to 9.0-fold higher

THAS yields than those in litter (Tables 1 and 2). In

contrast, the THAA yield in Nigrospore hyphae

(970 nmol [mg C]-1) was in the middle of the range

of values in litter (288–1517 nmol [mg C]-1) (Table 2).

Bacteria had 8- to 43-fold higher THAA yields than

those in litter and were generally higher than THAS

yields in litter. The THAA yields of bacteria were also 2-

to 9-fold higher than those of fungi.

The percentages of C as AA and AS in litter

increased with decomposition, approaching those of

soils and microorganisms, especially fungi (Fig. 2).

The percentage of N as AA generally fluctuated


123

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soil

45.8

1.8

125.3

1358

57.8

36.0

21.8

1.7

7.3

257.4

0.4

22.0

51.6

5

Min

eral

soil

20

.31

.22

16

.61

51

88

6.7

47

.63

8.9

1.2

7.6

84

1.2

0.6

22

.02

1.2

2

Qu

ercu

scr

ispu

lafo

rest

Fre

shli

tter

10

04

7.5

0.8

76

3.7

68

11

2.0

11

.01

.01

1.1

4.1

17

1.8

0.0

90

.91

1.5

2

6m

on

ths

litt

er8

2.6

45

.41

.11

47

.96

30

18

.81

5.7

3.1

5.0

3.7

75

0.2

0.1

41

.08

1.2

6

9m

on

ths

lite

r7

3.4

45

.51

.36

39

.11

20

83

1.1

25

.16

.04

.27

.18

78

.80

.22

1.4

61

.07

12

mo

nth

sli

tter

62

.04

5.3

1.6

43

2.2

13

57

46

.43

7.1

9.3

4.0

8.0

77

3.2

0.3

31

.80

1.0

4

18

mo

nth

sli

tter

53

.04

4.5

1.6

33

1.8

10

99

45

.73

3.0

12

.72

.66

.53

58

.80

.33

1.7

51

.02

21

mo

nth

sli

tter

25

.34

5.7

1.5

63

4.1

13

21

42

.82

9.0

13

.82

.17

.87

75

.40

.31

1.7

51

.10

24

mo

nth

sli

tter

28

.44

5.3

1.7

72

9.8

15

17

50

.73

5.7

14

.92

.49

.00

75

.70

.36

1.8

10

.97


123

between 50 and 70 % during litter decomposition,

while the %N as AS constantly increased by 1–2 %.

Nitrogen yields as AA plus AS were correlated with

total N content in decomposing litter from the three

plant species (R = 0.898, P \ 0.001) (Fig. 3). This

strong relationship indicated changes in total N

content of litter reflected those in AA and AS

throughout decomposition. In addition, the slope of

the line indicated AA and AS accounted for about

60 % of total N in the decomposing litter.

The % initial yields of THAA and THAS typically

increased during the early stages of decomposition and

decreased during the latter stages (Figs. 1C, D). The %

initial yield of THAS increased to a greater extent than

did THAA yield. Net increases in % initial yields were

observed for THAA of Pinus and THAS of Miscan-

thus and Pinus. Changing patterns in % initial yields of

THAA and THAS tracked those of % initial N,

especially for THAS.

The yields of GlcN and GalN increased in all litter

samples during the experiment. Yields increased 3- to

8-fold for GlcN and 10- to 17-fold for GalN,

approaching those in soils (Table 1, Fig. 4). The

yields of GlcN and GalN in decomposing litter were

more similar to those of bacteria rather than fungi.

Glucosamine was the most abundant AS in fresh litter,

with 3- to 11-fold higher yields than GalN, whereas the

increases in yields were greater for GalN than GlcN

(Fig. 5). In the first year, the % initial of both GlcN and

GalN increased in litter for all plant species. Greater

increases in %initial GalN than GlcN were observed

for Miscanthus and Quercus litter in the first year,

while Pinus litter showed similar increases in %initial

GlcN and GalN. In contrast, in the second year, greater

increases in %initial GalN than GlcN was observed

for Pinus litter, but not for Miscanthus and Quercus

litter. The GlcN/GalN ratio of litter showed clear

patterns in all plant species, decreasing (3.0–11.1 to

1.3–2.4) to values similar to those in soils (Table 1).

The GlcN/GalN ratio decreased in Pinus and Quercus

litter throughout the study period, whereas the GlcN/

GalN ratio in Miscanthus litter decreased only during

the early stages of decomposition. Significant varia-

tion in the GlcN/GalN ratio was a function of the

C-normalized yield of GalN (Fig. 6; R = 0.636,

P \ 0.05). Soils exhibited only minor variations in

AS composition among sites. Although fungi and

bacteria both had higher yields of GlcN than GalN, the

yield of GalN was considerably high in some bacteriaTa

ble

1co

nti

nu

ed

Sam

ple

%R

emai

nin

g

(AF

DW

)

C (wt%

)

N (wt%

)

C/N

(mo

le)

TH

AA

TH

AS

(nm

ol

[mg

C]-

1)

Glc

N

(nm

ol

[mg

C]-

1)

GaI

N

(nm

ol

[mg

C]-

1)

Glc

N/

GaI

N

%C

as

TH

AA

%N

as

TH

AA

%C

as

TH

AS

%N

as

TH

AS

Dl

36

mo

nth

sli

tter

10

.54

5.5

1.7

53

0.4

14

32

51

.73

5.4

16

.32

.28

.50

72

.90

.37

1.8

91

.02

Org

anic

soil

46.0

1.4

032.8

1043

34.7

22.9

11.8

1.9

5.7

257.5

0.2

51.6

01.9

3

Min

eral

soil

23

.71

.35

17

.51

58

17

7.1

40

.93

6.2

1.1

8.1

34

5.4

0.5

61

.89

1.1

3

Av

erag

eo

fal

lsi

tes

Fre

shli

tter

46

.90

.70

90

.55

39

12

.09

.72

.16

.53

.25

69

.40

.09

1.0

91

.27

36

mo

nth

sli

tter

44

.11

.65

31

.61

18

36

8.3

43

.02

5.3

1.9

6.9

86

1.7

0.4

92

.46

0.7

7

Org

anic

soil

43.1

1.3

147.2

1055

40.9

25.3

15.5

3.2

5.7

960.3

0.2

91.6

81.4

2

Min

eral

soil

19

.81

.21

16

.21

61

99

7.2

54

.04

2.6

1.3

8.1

34

2.7

0.7

02

.17

1.2

5

Sta

nd

ard

dev

iati

on

of

all

site

Fre

shli

tter

4.6

0.2

65

0.6

21

85

.84

.01

.94

.21

.32

4.5

0.0

40

.17

0.2

4

36

mo

nth

sli

tter

8.5

0.1

48

.52

38

25

.29

.91

5.4

0.6

1.4

41

3.7

0.1

80

.50

0.2

2

Org

anic

soil

5.3

0.2

34.2

162

12.1

6.6

6.0

0.3

0.8

21.9

0.0

90.2

30.3

0

Min

eral

soil

4.1

0.1

41

.51

24

27

.01

7.2

8.9

0.1

0.5

32

.40

.19

0.3

80

.13


123

(Table 2). Fungi exhibited much lower yields of GalN

than highly decomposed litter. The AA degradation

index decreased with decomposition (Table 1) and

showed a significant relationship with the GlcN/GalN

ratio (Fig. 7, R = 0.646, P \ 0.05).

The mol% of individual AA was relatively constant

during litter decomposition (Fig. 7, Supplementary

Fig. 1). The mol% Gly increased during decomposi-

tion by 1.6–2.2 % among the three plant types,

whereas the mol % Leu decreased by 0.8–1.3 %.

Organic soils mostly showed similar AA compositions

to those of well-decomposed litter. Mineral soils had

appreciably different AA compositions from litter and

organic soils, especially in mol% Asp, Leu, and Gly.

Microorganisms exhibited higher mol% Glu and Ala

and lower mol% Pro and Ser than litter and organic

and mineral soils.

Length and mass of hyphae

Observations of hyphae revealed that average hyphal

length varied significantly among the plant litter types

(Table 3). Pinus litter had the highest hyphal lengths

among species and no substantial changes in hyphal

length throughout the experiment. Miscanthus and

Quercus litter had 37–80 % shorter hyphal lengths

than those in Pinus litter, and decreased (40–70 %) in

hyphal length from 12 to 24 months. Hyphal mass

(wt%) was greatest in Pinus litter and accounted for

\1 % of total weight. Temporal patterns of hyphal

mass were similar to those of hyphal length.

Length of specific types of melanized hyphae in

litter was relatively long (319–2233 m g-1) compared

with those of living (29–711 m g-1) and clamp-

bearing hyphae (99–1491 m g-1). Living hyphal

lengths decreased, especially from 12 to 24 months,

in all litter samples. In contrast, lengths of melanized

and clamp-bearing hyphae increased with time, espe-

cially for Miscanthus and Pinus litter. Lengths of

melanized and clamp-bearing hyphae were the longest

for Pinus litter, whereas Quercus litter had the longest

lengths of living hyphae throughout the experiment.

Specific hyphal types accounted for a minor portion

of total hyphae. Living hyphae accounted for 1–11 %,

melanized hyphae accounted for 7–23 %, and clamp-

bearing hyphae accounted for 3–19 % of total hyphal.

0

20

40

60

80

100

120

140

160

0 5 10 15 20 25 30 35 40

N(%

init

ial)

Months

0 20 40 60 80

100 120 140 160 180 200

0 5 10 15 20 25 30 35 40

TH

AA

(%in

itia

l)

Months

0

50

100

150

200

250

300

350

0 5 10 15 20 25 30 35 40

TH

AS

(%in

itia

l)

Months

A B

C D

Fig. 1 Yields of C (A), N (B), total hydrolyzable amino acids

(THAA) (C), and total hydrolyzable amino sugars (THAS)

(D) through litterbag experiment, given as % of initial contents.

Circle, triangle, and square indicate Miscanthus, Pinus, and

Quercus litter, respectively


123

Living hyphae decreased in Pinus and Quercus litter,

whereas melanized and clamp-bearing hyphae

increased during the decomposition. In particular,

the length of melanized hyphae increased by 1.5- to

2-fold.

Hyphal contributions to AA and AS in litter

The calculated percentages of total AA and AS in

hyphae was highest at 12 months, accounting for

0.85–2.55 % of THAA, 1.82–4.36 % for GlcN, and

0.07–0.57 % for GalN, and decreased during further

decomposition (Table 3). The decreases in hyphal

contributions to THAA, GlcN, and GalN were most

dramatic from 12 to 24 months. Galactosamine as

hyphae decreased by 93–98 % from 12 to 36 months,

which is much greater than observed for AA

(37–65 %) and GlcN (44–63 %). Hyphae in Pinus

litter had the greatest contributions of these biomol-

ecules. No significant relationship was found between

total hyphal length (or total hyphal mass) and THAA,

GlcN, or GalN yields in litter. The yields of GlcN as

hyphae were less than 5 % and decreased more rapidly

than hyphal length. Specific types of hyphae

accounted for a very low fraction of these biomole-

cules, \0.2 % for living hyphae, \0.7 % for melan-

ized hyphae, and\0.5 % for clamp-bearing hyphae.

Proportions of plant-derived N and immobilized N

The percentage of plant N in litter quantified using the

C and N results of litter indicated that plant N

decreased with time, in contrast to total N (Fig. 8).

Immobilized N (total N minus plant N) was[50 % of

total N in all litter samples at the end of the study.

Table 2 Molar C to N ratios, C-normalized yields of total hydrolyzable amino acids (THAA), total hydrolyzable amino (THAS),

glucosamine (GlcN), and galactosamine (GaIN), GlcN/GaIN ratio, and percentages of C and N as THAA and THAS of fungi and

bacteria

Sample C/N (mole) THAA

(nmol

[mg C]-1)

THAS

(nmol

[mg C]-1)

GlcN

(nmol

[mg C]-1)

GaIN

(nmol

[mg C]-1)

GlcN/

GaIN

%C as

THAA

%N as

THAA

%C as

THAS

%N as

THAS

Fungi

Nigrospora

sphaerica

19.8 970 161.3 157.6 0.8 188 5.0 31.6 1.2 4.5

Cortinarius

armillathus

(stalk)

21.0 1309 344.6 341.2 3.3 104 7.3 35.8 2.5 8.5

Cortinarius

armillathus

(pileus)

14.2 3436 138.2 137.8 0.4 369 19.3 63.5 1.0 2.4

Average 18.3 1905 214.7 212.2 1.5 220 10.5 43.6 1.5 5.1

Standard

deviation

3.6 1337 113.1 112.2 1.6 135 7.7 17.3 0.8 3.1

Bacteria

Micrococcus

lysodeikticus

4.7 10608 197.5 196.2 1.2 164 56.4 66.5 1.4 1.1

Bacillus subtilisa 4.5 10190 286.4 251.2 30.5 8 56.9 61.2 3.9 2.4

Aerobacter

aerogenes

3.7 10004 54.1 53.6 0.5 100 56.0 48.9 0.4 0.2

Azobacter

vinelandii

3.8 11641 68.1 51.4 14.7 3 64.3 57.9 0.5 0.3

Pseudomonas

fluorescensa4.1 8827 128.3 97.4 30.9 3 48.1 47.3 1.4 0.9

Average 4.2 10254 146.9 130.0 15.6 56 56.3 56.4 1.5 1.0

Standard deviation 0.4 1019 96.4 89.6 14.9 73 5.8 8.2 1.4 0.9

a Cited from Benner and Kaiser (2003) and Kaiser and Benner (2008)


123

Pinus litter exhibited the highest percentage (73 %) of

immobilized N.

Discussion

Influences of microorganisms on litter

decomposition and soil formation

Microorganisms had a major impact on the chemical

composition of decomposing litter. The net increases

of N in decomposing litter were in sharp contrast to the

net losses of C. Likewise, net increases of AA and AS

were also observed, and these biochemicals comprised

about 60 % of total N. The yields of AA and AS in

decomposed litter approached those found in micro-

organisms. The N immobilized during litter decom-

position was estimated to be greater than the

remaining plant N at the end of the study. These

observations indicated C-rich plant litter was gradu-

ally transformed to N-rich litter by microorganisms.

Microbes contributed newly synthesized ‘‘immobi-

lized’’ forms of N, such as AA and AS, to decompos-

ing litter in addition to degrading biomolecules of

plant origin (Tremblay and Benner 2006; Miltner et al.

2011). Furthermore, the observed transformation of

AA and AS yields from plant litter to soils, indicates

microorganisms play an important role in soil

formation.

The data on fungal hyphae in decomposing plant

litter enable us to further explore microbial contribu-

tions to AA and AS. Hyphae accounted for \5 % of

THAA, GlcN, and GalN in the decomposing litter, and

living hyphae accounted for \0.2 %, suggesting

identifiable hyphae contributed a minor fraction of

these N-containing biomolecules. It appears most of

the immobilized AA and AS were derived primarily

from bacteria (biomass and necromass) and physically

Fig. 2 Relationship between THAA and THAS contents

(%OC) for litter, soils, and microorganisms. Bars are standard

deviations (n = 3–5). Arrow indicates increasing diagenetic

alteration from litter to soil

Total N (µmol / gC)

AA

+ A

S N

(µ

mo

l / g

C)

Fig. 3 Total nitrogen yield versus sum of nitrogen yields from

total hydrolyzable amino acids (AA) plus total hydrolyzable

amino sugars (AS) of decomposing litter. Circle, triangle, and

square indicate Miscanthus, Pinus, and Quercus, respectively.

A linear regression of the data is shown (y = 0.60x ? 0.14,

R = 0.898, P \ 0.001)

Fig. 4 Relationship between GlcN and GalN contents (%OC)

for litter, soils, and microorganisms. Bars are standard

deviations (n = 3–5). Arrow indicates increasing diagenetic

alteration from litter to soil


123

unidentified residues from fungi. Although the contri-

bution of bacterial mass to the biomolecules was not

evaluated in this study, it would likely account for a

similar or smaller contribution as was observed for

fungal hyphae (Guggenberger et al. 1999; Joergensen

and Wichern 2008). Thus, decomposed microbial

remnants are likely to be major contributors to

immobilized AA and AS in decomposing litter.

Comparison of the chemical characteristics of plant

litter, soils, and microorganisms provides additional

insights about the origins of AA and AS in litter. The

yields of AA and AS in fresh litter were much lower

than those in microorganisms and soils, and they

increased during litter decomposition. The yields of

AA and AS in litter approached to those of fungi,

whereas the ratios of GlcN:GalN indicated bacterial

contributions. THAA yields in Pinus litter increased

about four fold and GlcN yields increased about eight

fold during decomposition, which were greater than

those observed in Miscanthus and Quercus litter. This

could indicate the contribution of basidiomycetous

fungi to decomposing litter. In contrast to the differ-

ences in the increases, the increase in GalN yield

during decomposition was similar between Pinus and

other species. In addition, a net increase in %initial

GalN was observed in Pinus litter during the second

year, when hyphal increases were absent. These results

suggest an increasing role of bacteria in N-immobi-

lization during later stages of decomposition. Most

GalN in soil is thought to be of bacterial origin

(Parsons 1981; Amelung 2001; Amelung et al. 2008),

and results from the present study are consistent with a

large contribution of GalN from bacteria. Further-

more, the GlcN/GalN ratios in litter and soils in the

0

100

200

300

400

500

600

700

0 5 10 15 20 25 30 35 40

AS

(%in

itia

l)

0

50

100

150

200

250

300

350

0 5 10 15 20 25 30 35 40

AS

(%in

itia

l)

Months

0

100

200

300

400

500

600

0 5 10 15 20 25 30 35 40

AS

(%in

itia

l)

A

B

C

GalN

GlcN

Months

Months

Fig. 5 Percent initial yields of GlcN (square) and GalN (circle)

for Miscanthus (A), Pinus (B), and Quercus (C) litter

0

2

4

6

8

10

12

0.00 0.10 0.20 0.30 0.40

Glc

N/G

a lN

rati

o

GalN (%OC)

Initial litter

Decomposed litter

Organic soil

Mineral soil

Fig. 6 Relationship between the GalN yield (%OC) and GlcN/

GalN ratio for litter and soils. A regression curve was fitted to

the relationship (y = 0.7x-0.471, R = 0.947, P \ 0.001). Arrow

indicates increasing diagenetic alteration from litter to soil

0

2

4

6

8

10

12

-1.0 -0.5 0.0 0.5 1.0 1.5 2.0

Glc

N/G

alN

r ati

o

DI

Initial litter

Decomposed litter

Organic soil

Mineral soil

Fig. 7 Relationship between the amino acid Degradation Index

(DI) and GlcN/GalN ratios for litter and soils. Arrow indicates

increasing diagenetic alteration from litter to soil


123

present study approached those of bacteria, which also

suggests bacteria could be an important source of these

AS in the decomposed litter and soils.

Several studies of microbial contributions to

decomposed litter and soil organic matter have used

GlcN as a fungal tracer and muramic acid (MA) as a

bacterial tracer (as reviewed by Joergensen and

Wichern 2008). Using this approach, these studies

concluded fungal contributions to organic N were

much greater than bacterial contributions. Our results

suggest bacteria are more important sources of organic

N in decomposed litter and soils than previously

recognized. Biodegradation of the molecular sources

of MA in litter and soil is more rapid than that of the

molecular sources of GlcN and GalN (Amelung 2003;

Tremblay and Benner 2006; Engelking et al. 2007). In

addition, GalN occurs in various bacterial cell wall

and membrane complexes and in archaeal glycopro-

teins (Parsons 1981; Amelung 2001; Schaffer and

Messner 2001; Giroldo and Vieira 2002), but GalN

does not occur in bacterial peptidoglycan, the only

known molecular source of MA. Thus, differences in

the relative reactivities of these biochemical compo-

nents of bacteria could lead to the observed differences

among the yields of these tracers (Kawasaki and

Benner 2006; Tremblay and Benner 2006).

Microbial contributions to N immobilization

in decomposing litter and soil

The sum of AA-N plus AS-N in decomposing litter

accounted for a fairly constant and large fraction of

total N (about 60 %) throughout the period, as has

been observed in other litterbag experiments (Tremb-

lay and Benner 2006; Rovira et al. 2008). The %

initial yields of AA and AS during the experiment

tracked those of N, indicating they were dominant

components of N throughout the study. In addition, the

dramatic increases in AA and AS yields during

decomposition and from organic soil to mineral soil,

indicate a major contribution of microbes to N

immobilization in the decomposing litter and soil. A

greater increase in GalN compared with GlcN in the

decomposing litter and soil likely reflected substantial

contributions from bacteria to N immobilization.

Fungal contributions to N immobilization were evi-

dent from the rapid increase in living fungal hyphae

and the contributions of hyphae to AA and AS in the

early stages of litter decomposition. The observations

that N immobilization was greatest in Pinus litter,

which also had the highest fungal infection, and that

both N immobilization and fungal infection in Mi-

scanthus litter were relatively low during the early

Table 3 Length and mass of hyphae and percentages of THAA, GlcN, and GaIN as hyphae to total yields in decaying litters

Sample Hyphal

length

Hyphal

mass

THAA as

hyphae

GlcN as

hyphae

GaIN as

hyphae

Length of specific hyphae (m g-1)

(m g-1) (wt%) (%total

THAA)

(%total

GlcN)

(%total

GaIN)

Living Melanized Clamp-bearing

Miscanthus sinensis

12 months litter 5867 0.40 0.85 1.82 0.07 66 (1) 406 (7) 149 (3)

24 months litter 3470 0.24 0.54 0.78 0.01 29 (1) 355 (10) 150 (4)

36 months litter 3274 0.22 0.53 0.68 0.00 96 (3) 431 (13) 170 (5)

Pinus densiflora

12 months litter 10594 0.94 2.55 4.36 0.57 378 (4) 1352 (13) 1397 (13)

24 months litter 10424 0.93 2.05 2.98 0.04 56 (1) 2212 (21) 1491 (14)

36 months litter 9801 0.87 1.70 2.46 0.03 137 (1) 2233 (23) 1473 (15)

Quercus crispula

12 months litter 6662 0.63 0.99 2.15 0.41 711 (11) 710 (11) 412 (6)

24 months litter 2037 0.19 0.27 0.68 0.01 196 (10) 319 (16) 99 (5)

36 months litter 2497 0.24 0.35 0.84 0.01 146 (6) 436 (17) 465 (19)

The percentages of each length to total hyphal length are shown in parentheses


123

stage of decomposition suggest fungi were important

for N immobilization. The major contributions of AA

and AS from microbes during litter decomposition is

consistent with the prevalence of amide groups in soil

N, which often account for more than 60 % of soil N

(Knicker et al. 1993; Schmidt et al. 1997; Knicker

et al. 2000; Martens and Loeffelmann 2003; Smernik

and Baldock 2005).

Microbial contributions to C preservation

in decomposing litter and soil

The increasing microbial contributions to AA and AS

yields in decomposing litter demonstrate a net input of

microbial C as well as N. Altered forms of peptides

and proteinaceous materials appear to be important

constituents of recalcitrant soil organic matter frac-

tions (Knicker 2004, 2011). The estimation of plant N

indicates more than half of the total N in decomposed

litter was of microbial origin. Percentages of total N as

AA?AS were relatively constant during decomposi-

tion. The C:N ratios of AA and AS are typically in the

range of 3–8, indicating significant contributions of

microbial C exist in decomposed litter and soil.

Proteins have been suggested to be resistant to

decomposition due to complexation with humic sub-

stances (Sutton and Sposito 2005; Hsu and Hatcher

2006). It has also been suggested that peptides and

proteins are resistant to decomposition due to adsorp-

tion onto mineral surfaces, such as aluminum-oxides

(Ensminger and Gieseking 1942; Leinweber and

Schulten 2000). These mechanisms could be linked

because humic substances are also adsorbed to mineral

surfaces (Leinweber and Schulten 2000). Soils with

older radiocarbon ages have a relatively larger fraction

of proteinous components bound to minerals (Mikutta

et al. 2010), suggesting the importance of minerals for

the preservation of peptides in soil.

Most plant carbohydrates in litter are thought to be

rapidly degraded, whereas microbial cell wall frag-

ments appear to remain in soil for long time (Tisdall

and Oades 1980; Foster et al. 1983; Angers 1992;

Chantigny et al. 1997). Our results demonstrated a

clear decline in GlcN/GalN ratios during litter decom-

position to values \2.5. Declining GlcN/GalN ratios

with increasing diagenetic alterations could be due to

selective removal of GlcN or increasing microbial

contributions of GalN as decomposition progresses.

An increasing relative role of bacteria in N-immobi-

lization with time was observed in decomposing litter,

and bacteria have higher GalN yields than fungi,

indicating bacterial contributions to organic matter

preservation. Lower GlcN/GalN ratios (0.5–0.7) have

been observed in deep soil layers that have been buried

for long time ([6,200 years) (Calderoni and Schnitzer

1984), indicating GalN is less reactive than GlcN. In

addition, the GalN in bacterial cells has a significantly

lower decay coefficient than GlcN and several other

Fig. 8 Estimated changes in total N and plant N in litterbag of

Miscanthus (A), Pinus (B), and Quercus (C) litter during

experiment. Circle and triangle indicate total N and plant N,

respectively


123

bacterial tracers, including D-amino acids and MA,

which are found in various cell wall components

(Kawasaki and Benner 2006).

Relatively high GlcN/GalN ratios in organisms and

its gradual decrease to values \2 during decomposi-

tion is consistently observed in litterbag studies

conducted in soil (Amelung et al. 2001) and water

(Tremblay and Benner 2006). In addition, highly

decomposed marine organic matter typically exhibits

low GlcN/GalN ratios (Ogawa et al. 2001; Benner and

Kaiser 2003; Kaiser and Benner 2009). The differ-

ences in reactivity between GlcN and GalN in

decomposing bacteria and the decreasing GlcN/GalN

ratio with increasing decomposition indicates GlcN/

GalN ratios are useful indicators of the extent of

diagenetic alteration of natural organic matter in a

wide ranges of systems. Likewise, yields of common

biochemicals, such as AA, are also useful indicators of

the diagenetic state of organic matter (Cowie and

Hedges 1994).

Although GlcN appears to be less resistant to

decomposition than GalN, the substantial (3- to 8-fold)

increases in GlcN yields indicate it occurs in resistant

forms of organic matter. Chitin, a polymer of acety-

lated GlcN subunits, is rapidly degraded when added

to soil, whereas GlcN yields in soil are relatively high,

indicating that GlcN in soil could be in molecular

complexes that are stabilized against microbial attack

(Bondietti et al. 1972; Parsons 1981). The higher

percentage of melanized and clamp-bearing hyphae to

total hyphae in the latter stages of the decomposition,

suggests the melanization process increases the resis-

tance of basidiomycetous hyphae to decomposition.

Melanins are assumed to be generated by the conden-

sation of phenolic precursors present in fungal cell

walls and to increase the resistance of hyphae to

decompose (Linhares and Martin 1978; Martin and

Haider 1986; Chenu and Cosentino 2011).

Organic matter derived from microorganisms plays

a critical role in the formation and preservation of soil

organic matter (Liang et al. 2011). The molecular

signatures of heterotrophic microbes are evident in

decomposing litter and soils, particularly in amino

acids and amino sugars. The yields of these microbial

biomolecules increase rapidly during litter decompo-

sition and appear to be important for shaping the

chemical composition and long-term stabilization of

soil organic matter.

Acknowledgments We thank Karl Kaiser and Mike Philben

for analyzing C, N, and AA in several microorganisms and for

assistance with AA and AS analyses. We also thank Yoko

Morimoto, Ruth Flerus, Tomohiro Kasuga, Seiya Shiratori,

Marin Otomichi, Hiroki Inoue, Hidetomo Iwano, Isao Kato,

Hiroshi Yokota, and Teruo Matsunaka for support in the

laboratory. This research was supported by grant from the

Japanese Society for the Promotion of Science (No. 21710014)

and by Grants-in-Aid to Cooperative Research from Rakuno

Gakuen University (2008). Ronald Benner acknowledges

support from NSF grant 0843417.

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The roles of microorganisms in litter decomposition and soil formation