Journal of

Plant Ecology Changes in soil microbial biomass

VOLUME 3, NUMBER 3,
PAGES 209–217 and community structure with
SEPTEMBER 2010

doi: 10.1093/jpe/rtq001 addition of contrasting types of

plant litter in a semiarid grassland
Advanced Access published
on 25 February 2010

available online at
www.jpe.oxfordjournals.org
ecosystem
Hongmei Jin1, 2, 3, Osbert Jianxin Sun1, * and Jianfeng Liu2, 4
1
MOE Key Laboratory for Silviculture and Conservation and Institute of Forestry and Climate Change Research, Beijing
Forestry University, Beijing 100083, China
2
State Key Laboratory of Vegetation and Environmental Change, Institute of Botany, Chinese Academy of Sciences, Beijing
100093, China
3
Institute of Agricultural Resources and Environment, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China
4
Institute of Forestry, Chinese Academy of Forestry, Beijing 100091, China
*Correspondence address. College of Forest Science, Beijing Forestry University, 35 Qinghua East Road, Haidian
District, Beijing 100083, China. Tel: +86-10-62337095; E-mail: sunjianx@bjfu.edu.cn

Abstract

Aims Important Findings

Elevated atmospheric CO2 has the potential to enhance the net pri-
mary productivity of terrestrial ecosystems. However, the role of soil
microorganisms on soil C cycling following this increased available
C remains ambiguous. This study was conducted to determine how
quality and quantity of plant litter inputs would affect soil microor-
ganisms and consequently C turnover.
Methods
Soil microbial biomass and community structure, bacterial com-
munity-level physiological profile, and CO2 emission caused by
different substrate C decomposition were investigated using tech-
niques of biological measurements, chemical and stable C isotope
analysis, and BIOLOG-ECO microplates in a semiarid grassland
ecosystem of northern China in 2006 and 2007 by mixing three
contrasting types of plant materials, C3 shoot litter (SC3), C3 root
litter (RC3), and C4 shoot litter (SC4), into the 10- to 20-cm
soil layer at rates equivalent to 0 (C0), 60 (C60), 120 (C120) and
240 g C m
2 (C240).
Litter addition significantly enriched soil microbial biomass C and N
and resulted in changes in microbial structure. Principal component
analysis of microbial structure clearly differentiated among zero ad-
dition, C3-plant-derived litter, and C4-plant-derived litter and among
shoot- and root-derived litter of C3 plants; soil microorganisms mainly
utilized carbohydrates without litter addition, carboxylic acids with
C3-plant-derived litter addition and amino acids with C4-plant-derived
litter addition. We also detected stimulated decomposition of older
substrate with C4-plant-derived litter inputs. Our results show that both
quality and quantity of belowground litter are involved in affecting soil
microbial community structure in semiarid grassland ecosystem.
Keywords: belowground process d decomposition d plant
litter d microbial community d priming effect d semiarid
grassland
Received: 19 November 2009 Revised: 24 January 2010 Accepted:
27 January 2010

of soil microorganisms through modification of the quantity

INTRODUCTION
Soil microorganisms play a fundamental role in driving C turn-
over and nutrient cycling in all terrestrial ecosystems. Changes
in land use and cover, management and plant productivity
may influence the biomass, structure, and functional processes
and types of organic matter inputs (Calderón et al. 2000;
(Steenwerth et al. 2003). Many studies in recent years
have shown that soil C inputs in a variety of forms can signif-
icantly impact soil microbial biomass, composition, and
activities (Bardgett et al. 1999; Bossio and Scow 1998; Brant

Ó The Author 2010. Published by Oxford University Press on behalf of the Institute of Botany, Chinese Academy of Sciences and the Botanical Society of China.
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210 Journal of Plant Ecology

et al. 2006; Griffiths et al. 1999; Tu et al. 2006) and that shifts
in soil microbial community structure may occur with
changes in substrate types (Bossio and Scow 1998; Fontaine
et al. 2004).
It is generally accepted that organic matter inputs can
increase soil microbial biomass and activity due to an increase
in energy availability (Bossio and Scow 1998; Fontaine et al.
2004; Tu et al. 2006; Xiao et al. 2007). Different types of soil
microorganisms differ in their substrate specificity such that
microbial community structure may be modified with chang-
ing organic matter input. For example, Brant et al. (2006)
found that alteration of plant litter inputs changed soil micro-
bial utilization of C compounds; studies of Williams et al.
(2006) and Moore-Kucera and Dick (2008) showed that
organic matter quality could influence C flow from added
materials into the soil microbial community. Competition
for energy and nutrients between microorganisms specialized
in the available organic matter and those feeding on polymer-
ized soil organic matter have been found to result in the
decomposition of old soil organic C (SOC) (Fontaine et al.
2004, 2007). Studies of Hamer et al. (2004) and Hamer and
Marschner (2005) showed that the mineralization of seem-
ingly recalcitrant organic matters was greatly enhanced with
MATERIALS AND METHODS
Study site and experimental setup
This study was conducted in East Ujimuqin of Inner Mongolia,
northern China. Land cover of the area is typical steppe inter-
spersed with meadows. Climate is of continental and monsoon
type with long-term (1960–2005) mean of air temperature at
14.1°C and rainfall at 396 mm for the period May–October.
The daily precipitation from May 2006 to October 2007 is
showed in Fig. 1. Base soils in this region are of Calcic–orthic
Aridisol.
The study site is in an Achnatherum splendens meadow (lat-
itude 45°29#N, longitude 116°57#E and altitude 822 m above
sea level). The land was used for growing alfalfa between 1990
and 1998 and subsequently abandoned and subjected to on-
and-off grazing activities. Between 12 April and 6 May
2006, forty-eight 2 3 1.5 m plots were laid out, with 0.5-m
spacing between adjacent plots, and treated with subsoil inputs
of C3 shoot litter (SC3), C3 root litter (RC3), and C4 shoot litter
(SC4) at rates equivalent to 0 (C0), 60 (C60), 120 (C120), and
240 g C m
2 (C240) and investigated for changes in soil
Daily pr e ci pita t ion (mm)

the addition of easily degradable substrates, such as fructose 0 60 120 180 240 300 360 420 480 540
or alanine. 25
Days after litter addition
Elevated atmospheric CO2 and global warming both have 20
the potential to enhance the net primary productivity of ter- 15
restrial ecosystems and result in land cover change. Changes in 10
ecosystem productivity and vegetation type may alter the
5
amount and types of plant-derived SOC inputs, which in turn
0
affect the structure and functional processes of soil microor-
12
ganisms (Hooper and Vitousek 1998; Jones 1998). One of SC3
the key questions that has been called for clarification is 9
how an increase in plant-derived C inputs would affect soil C0
6
C stores (Davidson and Janssens 2006). Research to date C60
3 C120
has demonstrated different patterns of responses ranging from
Soil water content (%)

C240
enhanced C sequestration (e.g. Gifford 1994; Jastrow et al. 12
2005) to stimulated decomposition of older soil C stock due RC3
9
to ‘priming effect’ (e.g. review by Kuzyakov et al. 2000).
The study of Xiao et al. (2007) shows that an increase in below- 6
ground organic C inputs is more likely to accelerate SOC cy-
3
cling than to result in enhanced SOC stores in semiarid
grassland ecosystems of northern China because of increased 12
SC4
microbial biomass and activities. However, it is yet unclear 9
whether the observed microbial responses are merely quanti-
6
tative or are there also qualitative traits involved.
In this study, we investigated changes in soil microbial bio- 3
mass and community structure and SOC, in 2006 and 2007, 0
following addition of three contrasting types of plant litter: -06 06 06 7 7 7
y-0 uly-0 ust-0 ber-0
7
C3 shoot litter, C3 root litter and C4 shoot litter, at rates equiv- u ne July- gust- a
J M J g o
Au Au Oct
alent to 0, 60, 120 and 240 g C m
2. Litter treatments were
applied to subsoil layer on a grassland site of Inner Mongolia,
Figure 1: daily precipitation and soil water content in the 0- to 20-cm
northern China. Our overall objective was to determine how soil layer for treatment plots with different types and rates of plant litter
quality and quantity of plant litter inputs would affect soil inputs during the growing season in 2006 and 2007. Vertical bars
microorganisms and consequently C turnover. illustrate standard errors of means (n = 4).
Jin et al. | Soil microbial biomass and community structure
microorganisms and organic C. The treatments were applied in
a randomized complete block design, with four replicates of
12 litter type 3 input rate combinations. The SC3 and RC3 litter
were recently senescent shoots/leaves and roots of local
C3-herbage in mixture (predominantly Stipa grandis P. Smirn.
and Leymus chinensis (Trin.) Tzvel.), and the SC4 litter was har-
vested forage corn straws produced locally (Table 1). Litter was
fragmented into <2-mm particles and added to the 10- to 20-
cm subsoil layer as described in Xiao et al. (2007). Because
>65% of the roots are distributed in the upper 10 cm in this
grassland ecosystem (Zhou et al. 2007), our treatments caused
very little damage to existing vegetation when lifting and
replacing the top 10-cm soil blocks. The treatments were com-
pleted before the regreening stage of local herbages, allowing
the disturbed soil and vegetation to recover. SOC concentra-
tion prior to adding litter was on average 2.06 kg m
2 in
the 10- to 20-cm soil layer, and the four addition rates repre-
sented an increase in SOC by 0%, 3%, 6%, and 12%, respec-
tively, in the 10- to 20-cm soil layer. Basic information on soil
property before and after the study is listed in Table 2.
Soil samples were taken with a soil sampler (3 cm inner di-
ameter, 10 cm length) from the 0- to 20-cm soil layer at three
triangularly distributed locations in each plot in June, August
and September in 2006 and May, July, August and October in
2007. The three soil samples per plot were combined at
the time of collection and analyzed for pH, C, N and microbial
variables.
Aboveground living tissues in each plot were clip-harvested
from a 0.5 3 0.5 m subplot, and roots were collected to 20-cm
depth within the same subplot with a soil core sampler (8 cm
inner diameter), both in August 2007. The biomass of above-
ground living tissues and roots was determined by oven-drying
at 65°C to constant mass.
Measurements of lignin content of plant litter
The three types of plant litter were analyzed for acid-insoluble
(Klason) lignin. Briefly, five well-mixed samples for each
litter type were oven-dried at 65°C to constant mass and
ground to pass 1-mm sieve, and then 1 g (dry weight) of
the ground material from each sample was taken and added
with 25 ml of 72% H2SO4 to form an analytical sample, which
was maintained at 25°C for 2 h for pre-hydrolysis. After this
pre-hydrolysis, each analytical sample was added with
Table 1: chemical properties and input rates of plant litter added to 10- to 20-cm soil layer of treatment plots
Litter mass added to subsoil layer (g m
2)
Litter C content N content C:N Lignin
type (%) (%) ratio (%) C60
SC3 36.8 (6.0) 0.29 (0.02) 125 (23) 20.1 (2.5) 163
RC3 41.0 (5.3) 0.56 (0.02) 73 (14) 29.1 (4.2) 146
SC4 41.2 (2.2) 0.58 (0.02) 71 (18) 15.1 (1.8) 145
Values in parentheses show standard errors (n = 5). Symbols: SC3, C3 shoot litter; RC3, C3 root litter and SC4, C4 shoot litter; C60, C120 and C240 are
treatments with litter input rates of 60, 120, and 240 g C equivalent, respectively.
211
50 ml distilled water before being placed in a boiling water bath
for 3 h to continue with the hydrolysis. All analytical samples
were shaken at a half-hourly interval and filtered over glass
filters G4 upon completion of the hydrolysis process. The resid-
uals, i.e. acid-insoluble lignin, were washed free of acid and
dried at 105°C for 24 h before being weighed.
Measurements of soil and microorganisms
Soil pH and electric conductivity (EC) (soil:water (w/v) ratio =
1:2.5) were determined with the combination glass electrode of
Delta 320 and Delta 326 (Mettler Toledo Instruments Ltd, Lan-
gacher, Switzerland), respectively. Particle-size distribution
was measured with a particulate size description analyzer
(Mastersizer 2000; Malvern Instruments Ltd, Worcester,
UK). The SOC was determined with dichromate oxidation
method (Nelson and Sommers 1982). Dissolved organic C
(DOC) and dissolved organic N (DON) were determined with
extracted organic C and N in 0.5 mol l
1 K2SO4 solution and
measured with a total organic carbon analyzer (Phoenix 8000;
Tekmar-Dohrmann, Cincinnati, OH, USA) for DOC and semi-
micro Kjeldahl method for DON.
Soil microbial biomass C (MBC) and microbial biomass N
(MBN) were measured using the chloroform-fumigation
extraction method (Vance et al. 1987). Dichromate oxidation
method and semi-micro Kjeldahl method were used to deter-
mine C and N in the extracts. The MBC was calculated as
follows:


MBC mg Cmic g
1 dry soilÞ = ðCF
CNF kec ; ð1Þ
where, CF is the extracted C in fumigated soil, CNF the
extracted C in non-fumigated soil and kec = 0.38, which is
the factor to convert the extracted organic C to MBC. The
MBN was calculated as follows:


MBN mg Nmic g
1 dry soilÞ = ðNF
NNF ken ; ð2Þ
where, NF is the extracted N in fumigated soil, NNF the
extracted N in non-fumigated soil and ken = 0.45, which is
the factor to convert the extractable N to MBN.
Soil microbial (MR) was measured by determining CO2 evo-
lution over a 7-day incubation. Specifically, 30 g (dry weight
equivalent) of each soil sample was adjusted to 50% of water
holding capacity and incubated at 28°C for 7 days in the dark.
Respired CO2 was absorbed in 5-ml 0.1 M NaOH solution
N equivalent of litter inputs (g m
2)
C120 C240 C60 C120 C240
326 652 0.47 0.95 1.89
292 584 0.82 1.64 3.27
290 580 0.84 1.68 3.36
212 Journal of Plant Ecology

Table 2: information on soil properties in 0- to 20-cm soil layer before and after litter addition treatments

After treatment (n = 12)

Before treatment (n = 5) SC3 RC3 SC4

3
Bulk density (g cm ) 1.47 (0.30) — — —
Total N (g kg
1) 1.184 (0.020) 1.228 (0.076) 1.170 (0.076) 1.177 (0.076)
Organic C (g kg
1) 14.08 (0.53) 14.15 (0.24) 14.09 (0.19) 13.79 (0.64)
C:N ratio 12.42 (2.59) 12.03 (0.81) 12.75 (0.89) 12.54 (1.00)
pH 8.69 (0.70) 8.46 (0.07) 8.72 (0.04) 8.56 (0.06)

1
EC (mS m ) 172 (28) 180 (7) 145 (5) 195 (9)
Particle-size distribution (%)

<0.001 mm 0.57 (0.31) 0.38 (0.19) 0.38 (0.09) 0.36 (0.05)

0.001–0.005 mm 6.46 (3.09) 4.63 (1.69) 4.83 (0.95) 4.64 (0.62)
0.005–0.01 mm 5.99 (1.57) 4.28 (1.36) 4.47 (0.69) 4.15 (0.59)
0.01–0.05 mm 53.57 (10.43) 45.48 (5.54) 48.71 (4.06) 46.31 (3.35)
0.05–0.2 mm 16.07 (7.11) 25.66 (6.04) 23.85 (3.95) 24.20 (2.80)
0.2–2.0 mm 17.39 (4.42) 19.57 (2.72) 17.76 (1.97) 20.33 (2.13)

Values in parentheses show standard errors of means. Symbols: SC3, C3 shoot litter; RC3, C3 root litter and SC4, C4 shoot litter.

suspended inside a Mason jar (Hu and van Bruggen 1997). The tem (LI-COR, Lincoln, NE, USA). To determine d13C in respired
NaOH solution containing dissolved CO2 was then titrated CO2, gas samples were collected with a syringe from closed cy-
with 0.05 M HCl solution to determine the amount of CO2 lindrical polyvinyl chloride chambers (20 cm inner diameter and
evolved. 30 cm height) after reaching steady state and stored in 50-ml
The metabolic quotient, qCO2, was calculated as follows: Tedlar gas vacuum containers (TPV/L-005, 3.0 kPa; Delin Co.,
MR Ltd, Shanghai, China) prior to analysis. The gas samples were
qCO2 = : ð3Þ measured on an isotope ratio mass spectrometer (Thermo
MBC
Finnigan MAT DELTAplus XP, Flash EA 1112 series, Bremen,
Soil bacterial community-level physiological profile
Germany) as described in Högberg and Ekblad (1996). In our
(CLPP), as indicated by substrate utilization patterns, was de-
study, soil CO2 emission on plots of zero plant litter inputs
termined using BIOLOG-ECO microplates (Biolog Inc.,
was defined as basal respiration, CO2basal, consisting of CO2 de-
Hayward, CA, USA) for subsamples collected in July 2007.
rived from the current substrate (CO2substrate; inclusive of micro-
The suspensions for inoculation were prepared as described
bial and root respiration) and that in the carrier gas (CO2gas):
in Buyer and Drinkwater (1997). Each well of BIOLOG-
ECO microplate was inoculated with 125 ll of the diluted soil dCO2basal dCO2substrate dCO2gas
= + : ð4Þ
extracts and incubated at 25°C in the dark. The absorbency at dt dt dt
590 nm (optical density) was measured every 24 h for a week d13 Csubstrate was calculated as follows:
using the Microplate Autoreader (Bio-Tek Instruments Inc., dCO2gas
Winooski, VT, USA). Data were corrected as in Buyer and d13 Cbasal dCOdt2basal
d13 Cgas
d13 Csubstrate = dCO2basal dCO2gas
dt
; ð5Þ
Drinkwater (1997). In order to minimize the effects of differ- dt
dt
ent inoculation densities, data from the 168-h reading were dCO
where, dt2gas is the CO2 concentration in the carrier gas up-
normalized by dividing the absorbency of each well with the
stream the gas-tight chamber multiplied by the gas flow rate
average absorbency for the whole plate (average well color
(2.5 l min
1) and d13Cgas =
7.7&.
development).
The soil CO2 emission on plots with SC4 treatment was
13
defined as substrate-induced respiration, CO2SIR, con-
Measurements of soil CO2 emission and C sisting of CO2 derived from the newly added substrate
abundance (CO#2substrate ; inclusive of microbial and root respiration),
Soil CO2 emission and 13C abundance in respired CO2 were mea- C4-litter (CO2litter), and in the carrier gas (CO2gas):
sured on the SC4 and zero litter addition treatment plots for iden-
dCO2SIR dCO#2substrate dCO2litter dCO2gas
tification of sources of CO2 emission in July 2007. Measurements = + + : ð6Þ
dt dt dt dt
of soil CO2 emission were made at three triangularly distributed
points on each plot with an LI-8100 automated soil CO2 flux sys-
Jin et al. | Soil microbial biomass and community structure 213

CO2litter was calculated as follows:

! CO2SIR  13 13

CO2gas

13 13

CO2litter dt d CSIR
d C#substrate + dt CO 2gas d C#substrate
d Cgas
mg m
2 min
1 = ;
dt d13 Clitter
d13 C#substrate
ð7Þ

where d13Clitter is the value for SC4 (
12.547 6 0.201&;

mean 6 standard error; n = 5).
CO2 derived from the newly added substrate following SC4
inputs was calculated as follows:
CO#2 ðmg m
2 min
1 Þ = CO2SIR
CO2litter ;
assuming that the changes in d13C of microbial respired CO2
and root respired CO2, dC#substrate , were negligible as our
results indicated that litter addition had little effect on d13Csoil
(Table 3).
Statistical analyses
Repeated measures analysis of variance was used for evaluating
the effects of litter type and addition rate on MBC, MBN, micro-
bial C:N ratio and qCO2 in the 0- to 20-cm soil layer during the
growing season in 2006 (four replicates 3 three sampling
times) and 2007 (four replicates 3 four sampling times), re-
spectively. Student’s t-test was used for assessing the interan-
nual variation of microbial variables. These statistical analyses
were performed using SPSS (v. 13.0). Multiple means were
compared with least square difference test at P = 0.05.
The utilization profiles for 31 types of C sources in the 0- to
20-cm soil layer in 2007 were examined with principal com-
ponent analysis (PCA). Canonical correspondence analysis
was used for assessing the bacterial CLPP with biological
and soil factors. Both types of data analysis were performed
with CANOCO (v. 4.5).
RESULTS
The three types of plant litter differed markedly in chemical
properties (Table 1): N content in the SC3 was only about half
ð8Þ that in the RC3 and SC4; lignin content ranked in the order of
RC3 > SC3 > SC4, while C content was similar between RC3 and
SC4 but slightly lower in the SC3.
Litter addition treatments significantly (P < 0.05) increased
the fraction of sand (0.05–2 mm) and decreased the fractions of
silt (0.001–0.05 mm) and clay (<0.001 mm) of the 0- to 20-cm
soil layer (Table 2). The effects of litter type and addition rate
on total nitrogen, SOC, soil C:N ratio, pH and EC were all mar-
ginal. Water content in the 0- to 20-cm soil layer was mainly
affected by rainfall (Fig. 1).
MBC and MBN in the 0- to 20-cm soil layer significantly (P <
0.001) increased with the rate of plant litter addition, regard-
less of litter types; both variables had weaker response in 2007
than in 2006 for all treatments except SC4, which showed
a slightly stronger response of MBC in 2007 than in 2006
(Fig. 2). Microbial C:N ratio significantly declined with the rate
of plant litter addition and was mostly greater in 2007 than in
2006 (P < 0.05; Fig. 2).
Assessment of the substrate utilization patterns of soil mi-
crobial community in 2007 revealed distinct grouping for
treatments of zero addition, C3-litter and C4-litter (Figs. 3
and 4). Soil microbial community mainly utilized carbohy-
drate (C) substrates in the C0 treatment, amino acid (AA)

Table 3: soil CO2 emission from substrate and litter in SC4 treatments in July 2007

Measured Calculated
Gas flow Chamber CO2
rate d13Csample concentration CO2 d13Csubstrate CO2litter CO#2 (CO#2 – CO2basal)/
d13Csoil (&) (l min
1) (&) (lmol mol
1) (mg m
2 min
1) (&) (mg m
2 min
1) (mg m
2 min
1) CO2basal (%)

Basal respiration
C0
21.640 2.5
9.563 429.47 2.94
0.0305 — — —
(0.006) (0.173) (0.56) (0.49) (0.0004)
Substrate-induced respiration (SIR)
C60
21.699 2.5
9.659 430.82 2.57
0.0305 0.609 1.96
33.3
(0.045) (0.089) (1.30) (0.68) (0.0003) (0.003) (0.68) (23.1)
C120
21.473 2.5
10.145 430.23 3.71
0.0317 0.628 3.08 4.8
(0.241) (0.305) (0.30) (0.57) (0.0007) (0.012) (0.56) (19.2)
C240
21.735 2.5
10.194 430.87 3.86
0.0317 0.630 3.23 9.8
(0.171) (0.294) (0.52) (0.62) (0.0006) (0.011) (0.62) (21.0)

Values in parentheses show standard errors of means (n = 4). C60, C120, and C240 are treatments with litter input rates of 60, 120, and 240 g C
equivalent, respectively. d13Csubstrate, CO2litter and CO#2 were calculated by Equations (4–8).
214 Journal of Plant Ecology

SC3 RC3 SC4

400
M B C ( µ g C m ic g -1 d r y s o i l)

2006
** *
* NS
300 2007 ** **
NS NS
NS
200 NS NS NS

100

50
M B N ( µ g N m ic g -1 d r y s o il )

40 2006 ** ***
2007
** **
30 * *
* * NS
20
NS NS NS
10

20
M ic r o b ia l C :N r a tio

2006 NS NS
16 NS
2007
* NS NS * NS ***
12 * * ***
8

0
C0 C60 C120 C240 C0 C60 C120 C240 C0 C60 C120 C240
-2
Addition rate (g C m ) Addition rate (g C m-2) Addition rate (g C m )
-2

Figure 2: soil MBC, MBN and C:N ratio in the 0- to 20-cm soil layer under treatments of C3 shoot litter (SC3) inputs, C3 root litter (RC3) inputs and
C4 shoot litter (SC4) inputs during the growing season in 2006 and 2007, respectively. Vertical bars illustrate standard errors of means (n = 4), and
asterisks indicate significance level of the difference between the measurements in 2006 and 2007 (*P < 0.05; **P < 0.01; ***P < 0.001).

substrates in the SC3 and RC3 treatments and carboxylic acid
(CA) substrates in the SC4 treatment irrespective of the addi-
tion rate (Fig. 3). The factors influencing the substrate utiliza-
tion pattern of soil microbial community were microbial
biomass (i.e. MBC, MBN) and activity (i.e. MR), energy avail-
ability (i.e. DOC) and plant root biomass in the litter addition
treatments and microbial C:N ratio in the zero litter addition
treatment (i.e. C0 plots; Fig. 4). Soil water content had no sig-
nificant impact on microbial variables (i.e. MBC, MBN, micro-
bial C:N ratio, MR and qCO2) in the 0- to 20-cm soil layer.
The metabolic quotient, qCO2, significantly increased with
the rate of RC3 addition in 2006 (P < 0.001) and mostly de-
clined (P < 0.05) with the addition rate for all three plant litter
types in 2007 (Fig. 5). There were significant reductions (P <
0.05 ; 0.001) in qCO2 from 2006 to 2007 for the SC3 treatment
at an addition rate of 240 g C m
2 and the RC3 treatment at 120
and 240 g C m
2, respectively (Fig. 5).
In the SC4 treatment, CO2 emission out of added plant lit-
ter accounted for 34%, 18%, and 17% of the observed soil
respiration at addition rates of 60, 120 and 240 g C m
2,
respectively. Discounting this fraction of CO2 emission, the
rates of soil respiration were on average 33% smaller at
60 g C m
2 and 5% and 10% greater at 120 and 240 g
C m
2, respectively, than the basal respiration at zero litter
addition (Table 3).
DISCUSSION
In this study, we found that an increase in plant litter inputs
resulted in changes in microbial production and activity asso-
ciated with possible alteration in microbial community struc-
ture and that there was some indication of priming effect of
older soil C by added plant litter.
Soil microorganisms play a key role in grassland ecosystems
by regulating the dynamics of organic matter decomposition
and plant nutrient availability (Bardgett et al. 1999). In this
study, plant litter addition to subsoil layer enhanced soil mi-
crobial biomass over 2 years with diminishing temporal effects.
The study of Xiao et al. (2007) suggests that soil microorgan-
isms are limited by energy in temperate grasslands of northern
China. In such oligotrophic soil environments, lack of available
C (i.e. energy substance) in substrates is a primary constraint to
soil microbial growth and activity, hence restricting decompo-
sition of the old SOC (Fontaine et al. 2007; Griffiths et al. 1999).
Given that microbial biomass and activity significantly cor-
related with DOC (r = 0.491; P < 0.01) and DON (r = 0.257;
Jin et al. | Soil microbial biomass and community structure 215

0.8
D-Malic Acid (CA)
-D-Lactose (C)
L-Asparagine (AA) D-Cellobiose (C)
L-Threonine (AA) -Methyl-D-Glucoside (C)

Axis 2 (13.3%)
4-Hydroxy Benzoic Acid (AC)
D-Mannitol (C)
Tween 40 (P) D-Xylose (C)
Glycyl-L-Gutamic Aid (AA)
Tween 80 (P)
i-Erythritol (C)
Glycogen (P)
-Ketobutyric Acid (CA)
N-Acetyl-D-Glucosamine (C)
L-Phenylalanine (AA)
Pyruvic Acid Methyl Ester (CA)
L-Arginine (AA)
Glucose-1-phosphate (C) L-Serine (AA)
Itaconic Acid (CA)
-Hydroxybutyric Acid (CA) D-Galacturonic Acid (CA)
Phenylethylamine (A) Putrescine (A)

D,L- -Glycerol Phosphate (CA)

2-Hydroxy Benzoic Acid (AC)
D-Galactonic Acid -Lactone (CA)

D-Glucosaminic Acid (CA)

-Cyclodextrin (P)
-1.0

-1.0 Axis 1 (50.2%) 1.0

Figure 3: utilization profiles for 31 types of C sources in the 0- to 20-cm soil layer under treatments with different types and rates of plant litter
inputs in 2007. Arrow lines indicate the types of specific C sources and their relative effects on bacterial CLPP (capital letters in parentheses are C,
carbohydrates; P, polymers; CA, carboxylic acids; AC, aromatic compounds; AA, amino acids and A, amines; the longer the arrow line, the stronger
the effect); symbols designate types of plant litter input treatments (open circle, C0; open square, SC3C60; open diamond, SC3C120; plus symbol,
SC3C240; times symbol, RC3C60; open triangle, RC3C120; inverted open triangle, RC3C240; filled circle, SC4C60; filled square, SC4C120; filled di-
amond, SC4C240), with each symbol corresponding to one treatment plot. The distance between symbols reflects their dissimilarity, while
the relative position of a symbol to an arrow line in perpendicular distance is a function of the influence of the specified C source on bacterial
CLPP of the treatment.

P < 0.01), the temporal differences in microbial variables are
likely caused by a decline in available energy (i.e. DOC) and
nutrients (i.e. DON) with time. Entry of C-rich substrate (such
as plant litters and fertilizers) into the soil is therefore a key fac-
tor which governs the size and activity of the microbial biomass.
Bacterial communities respond quickly to environmental
changes because of their high growth rate and short life span
(Øvreås 2000). Changes in soil microbial biomass are associ-
ated with shifts in the microbial community structure, in par-
ticular the ratio of bacteria to fungi (Bardgett et al. 1999; Lovell
et al. 1995). In this study, we found that with increasing rate of
plant litter addition, the microbial C:N ratio was mostly re-
duced, especially in the first year of treatment, demonstrating
the stronger effects of extraneous SOC inputs on bacterial com-
munities than on fungi. However, we also found significantly
higher soil microbial C:N ratio in 2007 than in 2006, suggesting
that fungi became relatively more abundant with time follow-
ing plant litter addition.
In addition to the temporal changes following plant litter
addition, our results also show contrasting effects of different
plant litter types on soil microbial community structure. PCA
analysis of microbial communities assessed by BIOLOG-ECO
microplates clearly differentiated among zero addition, C3-
plant-derived litter and C4-plant-derived litter and among
shoot- and root-derived litter of C3 plants; soil microorganisms
mainly utilized carbohydrates without litter addition, CA with
C3-litter addition, and AA with C4-litter addition, indicating
different impacts between C3- and C4-plant litter input. Most
recent studies by Auerswald et al. (2009) and Wittmer et al.
(2010) show that Inner Mongolia grassland has experienced
changes in plant community structure with increasing abun-
dance of C4 plants, possibly relating to climate warming of the
region. Associated with this increasing abundance of C4 plants
could likely be a change in soil microbial community. This may
lead to modification of the soil environment and produce
a feedback effect on plant community structure.
In semiarid grassland ecosystem, water availability can have
a strong effect on microbial processes, perhaps even more pro-
nounced than changes in C inputs. In this study, however, lit-
ter addition treatment did not result in significant changes in
soil water content. Moreover, we found that soil water content
had no significant impacts on microbial variables such as MBC,
MBN, microbial C:N ratio, MR and qCO2. Therefore, changes
in microbial biomass and community structure with plant litter
addition in our study are unlikely related to changes in soil wa-
ter content as an artifact of plant litter addition treatments.
216 Journal of Plant Ecology

0.8 BGB 5
2006 SC3

qCO2 (mg CO2-C g Cmic h )

MR

-1
DOC 2007
MBC 4
MBN
Axis 2 (14.2%)

qCO 2 NS NS NS *

-1
ANPP 3
DON pH

Microbial C:N ratio

1
-0.8

0
5
-1.0 1.0 RC3
Axis 1 (65.0%)

qCO2 (mg CO2-C g Cmic h )

-1
4 NS
***
Figure 4: canonical correspondence analysis ordination diagram of
bacterial CLPP with biological and soil factors in the 0- to 20-cm soil NS **

-1
layer of treatment plots with different types and rates of plant litter 3
inputs in 2007. Symbols designate types of plant litter input treatments
(open circle, C0; open square, SC3C60; open diamond, SC3C120; plus 2
symbol, SC3C240; times symbol, RC3C60; open triangle, RC3C120;
inverted open triangle, RC3C240; filled circle, SC4C60; filled square,
SC4C120; filled diamond, SC4C240; each symbol corresponds to one 1
treatment plot), and the arrow lines indicate the types of biological
and soil factors and their relative effects on bacterial CLPP. The distance 0
between symbols reflect their dissimilarity, and the relative position of
5
a symbol to an arrow line in perpendicular distance is a function of the SC4
qCO2 (mg CO2-C g Cmic h )

influence of the specified biological or soil factor on bacterial CLPP of

-1

the treatment. ANPP, aboveground net primary productivity; BGB, be- 4

lowground biomass; MR, microbial activity; microbial C:N ratio, micro- NS
NS
bial biomass C:N ratio; pH, soil pH; qCO2, microbial metabolic quotient. NS
-1

3 NS

Currently, there are considerable interests in the effects of

2
changes in mineralization rates on feedbacks within the global
C cycle (Cox et al. 2000; Lal 2004). Soil microorganisms min-
eralize humified soil organic matter at a remarkably constant 1
rate over long periods, even when fresh substrate is not sup-
plied (Joergensen et al. 1990). Addition of easily degradable 0
C0 C60 C120 C240
substrates may enhance the mineralization of recalcitrant
-2
organic matters (Hamer and Marschner 2005; Hamer et al. Addition rate (g C m )
2004). Thus, the responses of soil microorganisms to gains
in plant productivity could complicate the process in soil C Figure 5: soil microbial metabolic quotient (qCO2) in the 0- to 20-cm
soil layer for treatment plots with different types and rates of plant litter
turnover. Our measurements of soil CO2 emission rate, 13C
inputs during the growing season in 2006 and 2007. Vertical bars il-
natural abundance of respired CO2 from soil and changes in lustrate standard errors of means (n = 4), and asterisks indicate signif-
SOC content indicated that there was likely a priming effect icance level of the difference between the measurements in 2006 and
of older soil organic matter by plant litter addition. 2007 (*P < 0.05; **P < 0.01; ***P < 0.001).
Results from this study indicate that the effects of plant-de-
rived SOC inputs on soil C stores involve more complex processes insight in the feedback of soil microbial dynamics to vegetation
than simply changes in soil microbial production and activity. structure and function in the context of global climate change.
Both quality and quantity of belowground litter are involved
in affecting soil microbial community structure. Therefore, FUNDING
changes in productivity and vegetation type as a result of global National Natural Science Foundation of China (40741006,
climate change are likely to have profound impact on soil micro- 30521002, 30821062).
bial communities, which may in turn cause changes in plant
community structure because of differential utilization of soil or-
ganic substrate by different microbial functional groups. Long-
ACKNOWLEDGEMENTS
term experiments that incorporate studies of both plant commu- We thank J. Lin for help with setting up the experiment and the Grass-
nity and soil microbial dynamics may help with gaining further land Work Station of East Ujimuqin Banner of Inner Mongolia for
Jin et al. | Soil microbial biomass and community structure
logistic assistance. Critical reviews and constructive comments by the
Associate Editor, Dr Philip Fay, and two anonymous referees are grate-
fully acknowledged.
REFERENCES
Auerswald K, Wittmer MHOM, Männel TT, et al. (2009) Large re-
gional-scale variation in C3/C4 distribution patterns of Inner Mon-
golia steppe is revealed by grazer wool carbon isotope composition.
Biogeosci 6:795–805.
Bardgett RD, Lovell DL, Hobbs PJ, et al. (1999) Seasonal changes in soil
microbial communities along a fertility gradient of temperate grass-
lands. Soil Biol Biochem 31:1021–30.
Bossio DA, Scow KM (1998) Impacts of carbon and flooding on soil
microbial communities: phospholipid fatty acid profiles and sub-
strate utilization patterns. Microb Ecol 35:265–78.
Brant JB, Sulzman EW, Myrold DD (2006) Microbial community uti-
lization of added C substrates in response to long-term C input ma-
nipulation. Soil Biol Biochem 38:2219–32.
Buyer JS, Drinkwater LE (1997) Comparison of substrate utilization
assay and fatty acid analysis of soil microbial communities. J Micro-
biol Methods 30:3–11.
Calderón FJ, Jackson LE, Scow KM, et al. (2000) Microbial responses to
simulated tillage in cultivated and uncultivated soils. Soil Biol Bio-
chem 32:1547–59.
Cox PM, Betts RA, Jones CD, et al. (2000) Acceleration of global warm-
ing due to C-cycle feedbacks in a coupled climate model. Nature
408:184–7.
Davidson EA, Janssens IA (2006) Temperature sensitivity of soil car-
bon decomposition and feedbacks to climate change. Nature
440:165–173.
Fontaine S, Bardoux G, Abbadie L, et al. (2004) Carbon input to soil
may decrease soil C content. Ecol Lett 7:314–20.
Fontaine S, Barot S, Barré P, et al. (2007) Stability of organic carbon in
deep soil layers controlled by fresh carbon supply. Nature 450:277–81.
Gifford RM (1994) The global carbon cycle: a viewpoint on the missing
sink. Aust J Plant Physiol 21:1–15.
Griffiths BS, Ritz K, Ebblewhite N, et al. (1999) Soil microbial commu-
nity structure: effects of substrate loading rates. Soil Biol Biochem
31:145–53.
Hamer U, Marschner B (2005) Priming effects in soils after combined
and repeated substrate additions. Geoderma 128:38–51.
Hamer U, Marschner B, Brodowski S, et al. (2004) Interactive priming
of black carbon and glucose mineralisation. Org Geochem
35:823–30.
Högberg P, Ekblad A (1996) Substrate-induced respiration measured in
situ in a C3-plant ecosystem using additions of C4-sucrose. Soil Biol
Biochem 28:1131–8.
Hooper DU, Vitousek PM (1998) Effects of plant composition and di-
versity on nutrient cycling. Ecol Monogr 68:121–49.
217
Hu S, van Bruggen AHC (1997) Microbial dynamics associated with
multiphasic decomposition of 14C-labeled cellulose in soil. Microb
Ecol 33:134–43.
Jastrow JD, Miller RM, Matamala R, et al. (2005) Elevated atmo-
spheric carbon dioxide increases soil carbon. Glob Change Biol
11:2057–64.
Joergensen RG, Brookes PC, Jenkinson DS (1990) Survival of the soil
microbial biomass at elevated temperatures. Soil Biol Biochem
22:1129–36.
Jones DL (1998) Organic acids in the rhizosphere—a critical review.
Plant Soil 205:25–44.
Kuzyakov Y, Friedel JK, Stahr K (2000) Review of mechanisms and
quantification of priming effects. Soil Biol Biochem 32:1485–98.
Lal R (2004) Soil C sequestration impacts on global climatic change and
food security. Science 304:1623–27.
Lovell RD, Jarvis SC, Bardgett RD (1995) Soil microbial biomass and
activity in long-term grassland: effects of management changes. Soil
Biol Biochem 27:969–75.
Moore-Kucera J, Dick RP (2008) Application of 13C-labeled litter and
root materials for in situ decomposition studies using phospholipid
fatty acids. Soil Biol Biochem 40:2485–93.
Nelson DW, Sommers LE (1982) Total carbon, organic carbon, and or-
ganic matter. In: Page AL, Miller RH, Keeney DR (eds). Methods of
Soil Analysis. Madison, WI: American Society of Agronomy and Soil
Science Society of American, 101–29.
Øvreås L (2000) Population and community level approaches for an-
alyzing microbial diversity in natural environments. Ecol Lett
3:236–51.
Steenwerth KL, Jackson LE, Calderón FJ, et al. (2003) Soil microbial
community composition and land use history in cultivated and
grassland ecosystems of coastal California. Soi^image_type=^[taginfo
"jpertq001f01_lw", rg_type]l Biol Biochem 35:489–500.
Tu C, Ristaino JB, Hu S (2006) Soil microbial biomass and activity in
organic tomato farming systems: effects of organic inputs and straw
mulching. Soil Biol Biochem 38:247–55.
Vance ED, Brookes PC, Jenkinson DS (1987) An extraction method
for measuring soil microbial biomass C. Soil Biol Biochem 19:
703–7.
Williams MA, Myrold DD, Bottomley PJ (2006) Carbon flow from 13C-
labeled straw and root residues into the phospholipid fatty acids of
a soil microbial community under field conditions. Soil Biol Biochem
38:759–68.
Wittmer MHOM, Auerwald K, Bai YF, et al. (2010) Changes in the
abundance of C3/C4 species of Inner Mongolia grassland: evidence
from isotopic composition of soil and vegetation. Glob Change Biol
16:605–16.
Xiao CW, Janssens IA, Liu P, et al. (2007) Irrigation and enhanced soil
carbon input effects on below-ground carbon cycling in semiarid
temperate grasslands. New Phytol 174:835–46.
Zhou Z, Sun OJ, Huang J, et al. (2007) Soil carbon and nitrogen stores
and storage potential as affected by land-use in an agro-pastoral eco-
tone of northern China. Biogeochem 82:127–38.