Appl Microbiol Biotechnol (2014) 98:23092319

DOI 10.1007/s00253-013-5179-0

ENVIRONMENTAL BIOTECHNOLOGY

Changes in bacterial CO2 fixation with depth

in agricultural soils
Xiaohong Wu & Tida Ge & Hongzhao Yuan & Baozhen Li &
Hanhua Zhu & Ping Zhou & Fanggong Sui &
Anthony G. ODonnell & Jinshui Wu

Received: 5 June 2013 / Revised: 4 August 2013 / Accepted: 6 August 2013 / Published online: 30 August 2013
# Springer-Verlag Berlin Heidelberg 2013

Abstract Soils were incubated continuously in an atmosphere of 14CO2 and the distribution of labeled C into soil
organic carbon (14C-SOC) was determined at 01, 15, and 5
17 cm down the profile. Significant amounts of 14C-SOC were
measured in paddy soils with a mean of 1,180.6
105.2 mg kg1 at 01 cm and 135.347.1 mg kg1 at 1
5 cm. This accounted for 5.90.7 % and 0.70.2 %, respectively, of the total soil organic carbon at these depths. In the
upland soils, the mean 14C-SOC concentrations were 43 times
(01 cm) and 11 times (15 cm) lower, respectively, than
those in the paddy soils. The amounts of 14C incorporated
into the microbial biomass (MBC) were also much lower in
upland soils (5.03.6 % and 2.91.9 % at 01 and 15 cm,
respectively) than in paddy soils (34.112.4 % and 10.2
2.1 % at 01 and 15 cm, respectively). Similarly, the amount
of 14C incorporated into the dissolved organic carbon (DOC)
X. Wu : T. Ge : H. Yuan : B. Li : H. Zhu : P. Zhou : J. Wu
Changsha Research Station for Agricultural and Environmental
Monitoring and Key Laboratory of Agro-ecological Processes in
Subtropical Region, Institute of Subtropical Agriculture, Chinese
Academy of Sciences, Changsha, Hunan 410125, China
X. Wu : T. Ge : B. Li : P. Zhou : A. G. ODonnell : J. Wu
ISA-CAS and UWA Joint Laboratory for Soil Systems Biology,
Hunan 410125, China
F. Sui
School of Resource and Environment, Qingdao Agricultural
University, Qingdao 266109, China
A. G. ODonnell
Institute of Agriculture, Faculty of Science, University of Western
Australia, Crawley, WA 6009, Australia
T. Ge (*) : J. Wu (*)
Institute of Subtropical Agriculture, Chinese Academy of Sciences,
Changsha, Hunan 410125, China
e-mail: gtd@isa.ac.cn
e-mail: jswu@isa.ac.cn

was considerably higher in paddy soils (26.16.9 % and 6.9

1.3 % at 01 and 15 cm, respectively) than in upland soils
(6.02.7 % and 4.32.2 %, respectively). The observation
that the majority of the fixed 14C-SOC, RubisCO activity and
cbbL gene abundance were concentrated at 01 cm depth and
the fact that light is restricted to the top few millimeters of the
soil profiles highlighted the importance of phototrophs in CO2
fixation in surface soils. Phylogenetic analysis of the cbbL
genes showed that the potential for CO2 fixation was evident
throughout the profile and distributed between both photoautotrophic and chemoautotrophic bacteria such as
Rhodopseudomonas palustris , Bradyrhizobium japonicum,
Rubrivivax gelatinosus and Ralstonia eutropha.
Keywords Autotrophic bacteria . Atmospheric CO2 fixation .
RubisCO . cbbL genes . 14C continuous labeling . Soil organic
carbon (SOC) . Soil depth

Introduction
Increased atmospheric carbon dioxide (CO2) concentration
resulting from growing industrialization remains a major environmental and political issue (Pachauri and Reisinger 2007;
World Meteorological Organization 2009). It is widely acknowledged that rising CO2 emissions can, at least in part,
be mitigated by C sequestration in terrestrial ecosystems
(Lacis et al. 2010). Autotrophic bacteria able to assimilate
CO2 via the CalvinBensonBassham (CBB) cycle have been
shown to contribute significantly to the net uptake of atmospheric CO2 in oceans and wetlands (Cannon et al. 2001;
Stanley et al. 2003), and we have recently shown that soils
exhibit a similar potential and can account for up to 0.36 % of
the total C fixed in rice paddy soils and 0.19 % in upland soils
over an 80-day incubation period (Yuan et al. 2012a). How

2310

this fixed 14C is distributed down the soil profile and its
distribution between C pools has yet to be determined.
The enzyme responsible for CO2 fixation via the CBB
cycle, Ribulose-1,5-bisphospahate carboxylase/oxygenase
(RubisCO), has four phylogenetically related forms, that vary
in structure, catalytic properties, and substrate specificity
(Andersson and Backlund 2008). Forms I, II, and III are
known to exhibit RuBP-dependent CO2 fixation ability, while
Form IV has been designated as a "RubisCO-like" protein that
is not involved in the CBB cycle (Hanson and Tabita 2001;
Ashida et al. 2005). Form I, which is encoded by the cbbL
gene (Kusian and Bowien 1997), is a well-recognized, autotrophic CO2-fixing enzyme that occurs in green plants, green
algae, cyanobacteria, and photo- and chemoautotrophic bacteria (Tolli and King 2005; Selesi et al. 2005; Yuan et al.
2012b). Form I RubisCOs (IA to ID) are thought to predominate in soils and are found in plants, algae, cyanobacteria, and
autotrophic bacteria (Tcherkez et al. 2006). Phylogenetic studies on the cbbL gene sequences have shown that cbbL genes
encoding form I RubisCO can be further assigned to one of
four clades, IA to ID (Yuan et al. 2012a). These clades
encompass the obligate autotrophic bacteria, including some
marine cyanobacteria (Synechococcus and Prochlorococcus)
(form IA RubisCO), the facultative autotrophic bacteria (form
IC), green plants, green algae, and some cyanobacterial sequences (form IB), and the chromophytic algae (form ID)
(Tabita 1999). Photoautotrophs can access CO2 from the
atmosphere for growth in the presence of light, whereas
chemoautotrophs usually derive energy by oxidizing a wide
range of inorganic compounds (e.g., H2, NH4+, NO2, S2O32,
H2S, Fe2+, Mn2+, CO, CH4, CH3OH) as electron donors for
biosynthesis reactions via CO2 assimilation (Shively et al.
1998).
Soils are complex systems that differ in their physical and
chemical properties. The changes in physical and chemical
conditions associated with soil depth, such as light, substrate
and nutrient availability, are considered to provide diverse
niches for the colonization of autotrophic bacteria (Selesi
et al. 2007). Therefore, a better understanding of bacterial
autotrophy and how it changes down the profile is needed.
Paddy soils, covering an area of 26 % that of China's total
croplands, are considered to be an important C sink with a
carbon sequestration potential of 0.7 Pg (Pan et al. 2003). Rice
paddy soils, accounting for 80 % of the rice fields in China
(Lal 2002), are managed differently from upland soils, where
regular flooding, intermittent irrigation, ploughing and puddling are routinely practiced (Kimura et al. 2004). These
management-induced changes in oxic and anoxic conditions
can lead to the accumulation of organic matter (KgelKnabner et al. 2010) such that soil management (flooding)
has been used to increase the carbon stock in paddy soils by
between 12 % and 58 % above that found in upland soils (Guo
and Lin 2001). In the work reported in this paper, 14CO2

Appl Microbiol Biotechnol (2014) 98:23092319

continuous labeling and culture-independent molecular techniques were combined to investigate the distribution patterns
of assimilated 14C (as 14C-SOC) and the abundance and
diversity of CO2 fixing bacteria at different depths (01, 1
5, 517 cm). By quantifying 14C-CO2 incorporation into 14CSOC, this study provides important new information on the
distribution of autotrophic bacterial CO2 assimilation with
depth in paddy and upland soils.

Material and methods

Site description and sample preparation
Three paddy soils (P1, P2, P3) and three upland soils (U1, U2,
U3) subject to different cultivation methods were collected
from the Ap horizon (020 cm) at different geographical
regions in China (Table 1). The sampling sites cover an area
with annual temperatures ranging from approximately 8.1 C
to 16.8 C and annual rainfall of approximately 721 to
1,400 mm. Site details and soil physiochemical properties
are as shown in Table 1. After removing visible plant residues,
soil samples were passed through a 5-mm mesh. The paddy
soils were flooded with distilled water, and the upland soils
were adjusted to 45 % water-holding capacity (WHC) (Priha
and Smolander 1999). All soils were pre-incubated for 2 weeks
at 25 C prior to incubation and after flooding or rewetting.
Incubation experiment with 14CO2
Since our previous studies on these soils had shown that there
was no 14CO2 incorporation into SOC when soils were incubated in the dark (Yuan et al. 2012a), all soils were incubated
in the light. For each soil, four replicates were prepared by
adding moist soil (dry soil equivalent) into the PVC containers
(10 cm diameter, 20 cm height) to a depth of 17 cm. The
containers were then incubated for 110 days in an airtight,
glass chamber (80250 cm, height 120 cm), as described
previously (Ge et al. 2012; Yuan et al. 2012a). This chamber
was capable of generating 14CO2 (concentration maintained at
~350 l l1) through a reaction between Na214CO3 (at a
radioactivity level of 1.65104 Bq ml1) and HCl (2 M).
Soils were illuminated for 12 h each day (8:00 AM to 8:00
2 1
PM ) at an artificial light intensity of 500 mmol photons m s
PAR. Relative humidity was maintained at 8090 % with day/
night temperatures of 311 C and 241 C, respectively,
throughout the incubation period. Following incubation, soils
from 01, 15, and 517 cm depths were each divided into
two sub-samples. One sub-sample was stored at 4 C for
biochemical analysis, whilst the other was wrapped tightly
in aluminium foil, immersed immediately into liquid nitrogen,
and frozen at 70 C for molecular analysis. Soil water

Appl Microbiol Biotechnol (2014) 98:23092319

2311

Table 1 Characteristics of the paddy and upland soils used

Soil

Land Use

pH

SOC (g kg1)

TN (g kg1)

TP (g kg1)

CEC (cmol kg1)

Clay content (%)

P1
P2
P3
U1
U2
U3

Rice cultivation
Rice cultivation
Rice cultivation
Maize cultivation
Maize cultivation
Maize cultivation

5.70.01
6.70.01
5.10.00
8.40.01
7.40.00
6.00.04

20.90.72
17.40.40
17.00.16
7.70.33
20.60.65
11.10.28

2.80.00
2.20.01
2.50.03
1.40.01
2.20.01
1.60.01

0.70.00
0.90.01
0.60.03
1.10.02
0.70.02
1.30.09

13.20.23
15.90.24
5.60.05
9.50.04
13.60.10
12.10.41

33.20.43
24.50.39
22.10.55
15.54.36
17.40.18
21.00.56

Soils under rice or maize cultivation. Values are meansSD (n =4)

content was measured by oven drying the fresh soil at 105 to

a constant weight for 12 h after the sampling immediately.
Soil characteristics
Soil clay content was measured using the sedimentation pipette method (Mller and Hper 2004) and USA classification
(Kellogg 1993). Soil pH was determined in 1:2.5 (w/v) soilto-H 2 O with a pH meter (Delta 320, Mettler-Toledo
Instruments Ltd., China). Soil organic C (SOC) and total N
(TN) were determined by dry combustion with a macro elemental analyser (Vario MAX C/N, Elementar, Germany).
Cation exchange capacity (CEC) was determined from the
sum of exchangeable Ca, Mg, K, Na, and H cations (Kellogg
1993).
14
C-labelled organic C (14C-SOC) in soil was measured
according to Wu and O'Donnell (1997). Briefly, a portion
(1.50 g) of air-dry soil sample (particle size <0.149 mm) was
digested at 165 C for 8 min in a double-necked flask containing 20 ml of 0.2 M K2Cr2O7 and 30 ml of concentrated
H 2 SO 4 H 3 PO 4 (5:1; v/v), with pure O 2 continually
replenishing during the digestion and for 10 min thereafter.
The evolved CO2 was trapped in 40 ml 0.4 M NaOH in a
100 ml vial connected to the flask. The 14C radioactivity of
CO2-C trapped was determined by mixing 1 ml of NaOH with
9 ml of RIA cocktail (Beckman Coulter, Brea, CA, USA)
using an automated liquid scintillation counter (LS-6500;
Beckman, Germany) for 5 min. The counting efficiency was
determined as 9598 %. Counts (CMP) were automatically
corrected with counting efficiencies using a stored quench
curve to give the values of 14C radioactivity (DPM or Bq) in
the trap.
The amount of 14C-SOC (mg C Kg1 soil) was calculated
as the following formula:
.
14
CSOC F1 Rs Rp W

where F 1 represents the factor to convert the counting volume

(1 ml from 40 ml plus soil water volume in ml), R s and R p
denote the radioactivity (Bq ml1; blank counts omitted) for

the trap solution and that for Na214CO3 (Bq mg1 C l1) used
to produce 14C-CO2 in the growth chamber, and W is the
weight (kg) of digested soil on an oven-dry basis, respectively.
14
C-labelled microbial biomass C ( 14 C-MBC) was
analysed using fumigation-extraction (Wu et al. 1990) and
determined as above. The amount of 14C-labelled dissolved
organic C (14C-DOC) was measured in the K2SO4 extracts of
non-fumigated soil (Ge et al. 2013). The effectiveness of total
C was investigated using standards made from potassium
hydrogen phthalate according to Wu and O'Donnell (1997).
cbbL gene amplification and clone library construction
DNA was extracted from 500 mg of soil using the FastDNA
Spin Kit (MPbio, USA) according to the manufacturer's protocol. To construct the clone library, the cbbL genes of soils
P1 and U1 were amplified separately in 25 l reaction mixtures using an Eppendorf Mastercycler (Eppendorf Model5333, Germany). The total 25 l reaction mix contained
12.5 l 2 PCR MasterMix (Tiangen, China), approximately
50 ng soil DNA, and 100 pmol of each cbbL primer as
described by Nanba et al. (2004). Cycle parameters for the
PCR started with a 3-min denaturation at 95 C, followed by
5 cycles of 30 s denaturation at 95 C, 50 s annealing temperature decreased from 66 C to 62 C and an extension at 72 C
for 90 s. In addition, another 29 cycles at 95 C for 45 s, 62 C
for 50 s, and 72 C for 90 s were conducted, ending with a
final extension at 72 C for 10 min. The PCR products were
loaded onto 1 % (w/v) agarose gels and visualized by UV
excitation after staining with ethidium bromide. The expected
bands, confirmed by comparison with a 2,000-bp DNA marker, were excised and purified for cloning with an agarose gel
DNA purification kit (Tiangen, China).
Purified PCR products were cloned into Escherichia coli
DH5-competent cells using the pGEM-T Easy Vector
System (Promega, Mannheim, Germany) according to the
manufacturer's instructions. White transformed clones were
arbitrarily selected and grown overnight in LuriaBertani
broth prior to screening for the presence of positive inserts
by PCR with primers SP6 and T7. The gene diversity of 636
clones with the correct size inserts derived from the two soils

2312

Appl Microbiol Biotechnol (2014) 98:23092319

(106 clones for different depths of each soil) was sequenced

by BGI (Wuhan, China). The cbbL clone sequences were
aligned using Clustal W (http://www.ebi.ac.uk/clustalw), and
sequences with identities of 97 % were defined as an
operational taxonomic unit (OTU). Based on the representative sequence of each OTU and closely related cbbL sequences in GenBank, the phylogenetic tree was constructed
using Clustal X 1.83 and MEGA 4.0 using a neighbor-joining
algorithm (Tamura et al. 2007). Bootstrap analysis of 1,000
replicates was used to estimate the stability of the tree
topologies.

Prior to use, the resultant pellets were dissolved in 60 l of the

TrisHCl/DTT extractant.
RubisCO activity was measured at 30 C in 1.5 ml reaction
mixtures containing the buffer system, substrates, cofactor,
and coupling enzymes and monitored using spectrophotometry (UV-2450; Shimadzu, Japan) according to Takai et al.
(2005) and Yuan et al. (2012a). Reactions in the absence of
ribulose bisphosphate (RuBP) and in the presence of heatdenatured protein extracts were used as negative controls.
RubisCO activity was calculated as described by Ezaki et al.
(1999).

Nucleotide sequence-accession numbers

Statistical analysis

Clone sequences have been deposited in GenBank with accession numbers JQ836201JQ836264, JQ836293
JQ836358, JQ836364JQ836491, JQ836493JQ836424,
JQ836455JQ836469, JQ964821JQ965000.

Statistical differences were tested by one-way ANOVA

followed by a Duncan test (p <0.05) using the SPSS 16.0
statistical package. Correlation analysis was conducted in
SPSS 16.0 using the Pearson correlation method with
significance defined at the 95 % or 99 % confidence
level.

Quantification of cbbL gene

Quantification of cbbL genes was performed for all soils using
the TaqMan system and the cbbL primers, as described above.
Real-time PCR was carried out in a total volume of 10 l
reaction mix containing 5 l 1 SYBR Premix ExTaq (Takara
Bio Inc, Shiga, Japan), 0.2 M of each primer (Invitrogen,
China), 0.2 l ROX (provided with SYBR Premix ExTaq),
1 l soil DNA template (approximately 5 ng l1), and 3.4 l
sterilized water. Amplifications were done using an ABI
Prism 9700 Real-Time PCR System (PerkinElmer, Applied
Biosystems, USA) and the protocol described above. Each
sample was run in triplicate. A standard curve was established
from 10-fold serial dilutions of a known copy number of
plasmids containing the cbbL gene ranging from 3.7102 to
3.7108 cbbL copies l1. Standard curve samples were
conducted in parallel with the amplification of the soil cbbL
gene. Data analysis was done automatically using the SDS 2.3
software included with the Real-Time PCR System. The amplification efficiency was 95 %.
RubisCO enzyme activity analysis
Total protein was extracted as described by Yuan et al.
(2012a). Briefly, 2.0 g soil (four replicates) was suspended
in 6 ml protein extractant containing TrisHCl (100 mM,
pH 7.8) and Dithioreitol (DTT, 1 mM). The suspension was
then thoroughly disrupted by ultrasonication (JY92-II Scientz,
China) in an ice bath and centrifuged at 20,000g for 20 min
at 4 C. Resultant pellets used for determining RubisCO
activity were obtained by adding solid ammonium sulfate (to
reach 80 % saturation) to the supernatant, followed by stirring
for 30 min and centrifugation at 4 C (20,000g, 20 min).

Results
14

CO2 incorporation into different carbon pools with soil

depth
After 110 days of incubation, the radioactivity at different
depths over a 017 cm distance from the soil surface was
measured. These measurements indicated that labeled C incorporation into soil organic carbon (14C-SOC), microbial
biomass (14C-MBC) and dissolved organic matter (14CDOC) was strongly dependent on soil type, with higher levels
found in paddy soils than in upland soils, irrespective of soil
depth.
The amounts of 14C-SOC were characterized by higher
incorporation of the label at the soil surface (01 cm) followed
by a sharp decrease in radioactivity with depth (Fig. 1a). Mean
concentrations of 14C-SOC in paddy soils were 1,180.6
105.2 mg kg1 at 01 cm and 135.347.1 mg kg1 at 1
5 cm, respectively (Fig. 1a); incorporation below 5 cm in the
paddy soils was variable and barely measurable (40.4
3.2 mg kg1 in P2, 15.31.2 mg kg1 in P3; Fig. 1a). The
amounts of 14C incorporated into the upland soils was 43
times lower (27.35.1 mg kg1; Fig. 1a) to 1 cm depth and
11 times lower than in paddy soils between 1 and 5 cm (11.9
3.3 mg kg1; Fig. 1a). As with the paddy soils, the 14C-SOC
content was largely undetectable between 5 and 17 cm (5.7
1.0 mg kg1 in U1; Fig. 1a) in upland soils.
The amount of 14C-MBC and 14C-DOC varied markedly
amongst soils and was significantly correlated with 14C-SOC
content after 110 days of incubation (p <0.01, Table 3). 14CMBC content decreased in both soils between the surface (top

Appl Microbiol Biotechnol (2014) 98:23092319

soils) and was largely undetectable between 517 cm, except

in paddy soil P1 (7.2 mg kg1; Fig. 1b). The amounts of 14CDOC showed similar trends as with the 14C-SOC and 14CMBC and decreased significantly with depth (Fig. 1c).

1500

0-1 cm
1-5 cm
5-17 cm

a
1000

14

-1

C-SOC (mg kg )

2313

Contributions of microbial-assimilated C to different soil

carbon pools

500

40

a
a

a
c

20

nd

0
P1

400

b
b

nd

nd
P2

P3

U1

U2

U3

14

-1

C-MBC (mg kg )

a
200

b
b

a
b

P1

nd a nd nd

nd

0
P2

P3

U1

b
nd

U2

nd
U3

50

a
a
a

Phylogenetic analysis of cbbL gene clones from soil

-1

C-DOC (mg kg )

40

The amount of 14C-SOC as a percentage of total SOC

(14C-SOC/SOC) was considered to represent the contribution of assimilated 14C to the soil organic carbon
pool. In paddy soils, this percentage averaged between
5.90.7 % at the surface and between 0.70.2 % at 1
5 cm depths. These values were considerably higher
than those for the corresponding depths in the upland
soils. In upland soils the mean percentage incorporation
was 0.30.1 % at the surface and 0.10.1 % between 1
and 5 cm. The percentage of 14C-SOC to SOC decreased with depth in both paddy soils and upland soils
(Fig. 1a).
Similarly, the 14C-MBC/MBC levels were much lower in
upland soils (5.03.6 % and 2.91.9 % at 01 and 15 cm,
respectively) than in paddy soils (34.112.4 % and 10.2
2.1 % at 01 cm and 15 cm, respectively). The 14C-DOC/
DOC showed similar trends, with much higher levels in paddy
soils (26.16.9 % and 6.91.3 % at 01 and 15 cm, respectively) compared to those in upland soils (6.02.7 % and 4.3
2.2 % at 01 and 15 cm, respectively) despite the difference
in soil depth (Table 2). The percentage of 14C-MBC recovered
in MBC decreased with depth in both soils, while the 14CDOC/DOC levels decreased from the surface to 15 cm but
increased slightly at 517 cm (Table 2).

a
30

14

b
3

bb

b
b

a
b

b
nd

nd

0
P1

P2

P3

U1

U2

b nd
U3

Soils

Fig. 1 The amount of 14C-SOC, 14C-MBC, 14C-DOC at different depths

in paddy and upland soils incubated continuously in 14CO2 for 110 days.
Error bars indicate the standard error of the mean (n =4). Means with the
same letter are not significantly different at the 0.05 level; nd not
detectable

1 cm, mean 303.949.7 mg kg1 in paddy soils and 4.9

2.8 mg kg1 in upland soils) and 15 cm depth (mean 48.2
27.9 mg kg1 in paddy soils and 3.20.3 mg kg1 in upland

Analysis of the 636 clone sequences resulted in the recovery of

59 OTUs in which the sequence similarity was greater than
97 % to known cbbL sequences. Phylogenetic analysis revealed that the majority of these OTUs (72 %) could be
grouped into nine clusters whilst 28 % of the sequences could
not be assigned to known taxa. One of the OTUs (JQ836206)
was closely related to Rubrivivax gelatinosus, a purple nonsulfur phototrophic bacterium with up to 99 % homology
(Fig. 2). However, most of the retrieved cbbL sequences were
only distantly affiliated with known autotrophs, suggesting that
these soils may harbor numerous, novel cbbL-carrying bacteria. Phylogenetic analysis also showed that the cbbL sequences
were not randomly distributed but formed distinct assemblages
according to soil depth. Phylotpes from 0 to 1 cm soils were
mainly assigned to clusters, I, II, V, VI, VII, and affiliated with
cultured bacteria such as Rhodopseudomonas palustris ,
Rhodospirillum centenum, Azospirillum lipoferum, Azoarcus
sp., R. gelatinosus, Bradyhizobium japonicum, Aminobacter

2314

Appl Microbiol Biotechnol (2014) 98:23092319

Table 2 Changes in 14C-soil carbon pools with depth in paddy and upland soils incubated for 110 days in 14C-CO2
Soils

01 cm

15 cm

14

C-SOC/
SOC (%)

14

C-MBC/
MBC (%)

14

C-DOC/
DOC (%)

14

P1

5.010.58b

24.981.53b

24.580.66b

P2
P3
Average
U1
U2
U3
Average

6.890.36a
5.720.43ab
5.870.67
0.260.01c
0.140.01c
0.440.04c
0.280.11

54.370.83a
22.901.26c
34.0912.44
0.580.07d
10.610.94c
3.670.58d
4.953.63

36.444.34a
17.200.73c
26.086.86
10.311.30d
3.030.31e
4.620.60e
5.992.71

517 cm
14

C-MBC/
MBC (%)

14

C-DOC/
DOC (%)

14

1.060.11a

11.351.10a

8.200.87a

nd

1.040.10a

4.970.76a

0.400.02c
0.660.04b
0.710.24
0.120.00e
0.040.00f
0.210.01d
0.120.06

12.471.64a
6.800.31b
10.212.12
0.000.00b
5.231.06b
4.231.10b
2.851.87

4.850.50bc
7.531.44ab
6.861.25
7.832.37b
2.680.66c
2.400.23c
4.302.16

0.220.01a
0.090.00b
nd
0.070.00b
nd
0.040.00c
nd

nd
nd
nd
nd
1.850.82a
2.410.97a
nd

6.891.37a
nd
nd
5.391.04a
nd
nd
nd

C-SOC/
SOC (%)

C-SOC/
SOC (%)

14

C-MBC/
MBC (%)

14

C-DOC/
DOC (%)

14

C-SOC, 14 C-MBC, 14 C-DOC, radiolabeled soil organic carbon, microbial biomass carbon and dissolved organic carbon, respectively. Means (SD,
where n =4) in the same column with the same letter are not significantly different at the 0.05 level

nd not detectable

sp., Ralstonia eutropha , and Thermomonospora curvata .

Further down the profile, 15 cm depth, most of the OTUs
were grouped into clusters I, III, VI, VII, and IX, and constituted branching lineages originating from ammonium, carbon
monoxide, sulfur or methane oxidizers, such as Thiobacillus
denitrificans and Methylococcus capsulatus. The remaining
OTUs from 5 to 17 cm mostly formed distinct monophyletic
clusters in groups I, II, III, V, VI, and VII, and displayed
sequence similarity with facultative lithotrophs.
Abundance of cbbL gene
In the paddy soils, the number of cbbL gene copies ranged from
3.8108 1.1108 to 7.1108 1.1108 copies g dry soil1 at
the soil surface. A significant decrease in cbbL gene abundance
was observed at both depths of 15 and 517 cm except in soil
P1, where cbbL gene copy numbers were not significant at 5
17 cm depth (Fig. 3a). However, in the upland soils, the number
of cbbL gene copies was evenly distributed across depths, with
means of 1.1108 2.2107 copies g dry soil1 at 01 cm,
1.1108 2. 1107 copies g dry soil1 at 15 cm, and 9.7107
2.7107 copies g dry soil1 at 517 cm, respectively (Fig. 3a).
Soil type was shown to influence the abundance of cbbL
copies, with paddy soils harboring significantly greater numbers of cbbL genes than upland soils, irrespective of the distance to the soil surface. Correlation analysis showed that cbbL
gene abundance was significantly related to 14C-SOC content
(p <0.01) at 01 cm depths (Table 3).
RubisCO enzyme activity
Activities of RubisCO, the key enzyme in the CBB cycle,
were monitored throughout the 110-day incubation. Soil type

and depth had a noticeable effect on RubisCO and enzyme

activity decreased with increasing depth (63.51.0 and 31.0
4.4 nmol CO2 g1 soil min1 at 01 cm, 45.77.8 and 22.8
1.1 nmol CO2 g1 soil min1 at 15 cm, 42.97.9 and 21.1
1.7 nmol CO2 g1 soil min1 at 517 cm in paddy and upland
soils, respectively; Fig. 3b). In general, RubisCO activities
decreased with increasing distance from the soil surface over
the 110 days and were higher in paddy soils than in upland
soils, irrespective of the distance from the soil surface.
Correlation analysis showed that RubisCO activity was positively related to the abundance of the cbbL gene (p <0.01) at
01 cm, while no significant correlation was found at greater
depths (Table 3). Significant correlations were also evident
between RubisCO activity and 14C-SOC concentration (p <
0.01) at 01 and 15 cm depths (Table 3).

Discussion
Dynamics of microbial-assimilated 14C
The work reported here examines the extent of microbial
autotrophy at depth and how this fixed C is subsequently
distributed between the SOC, MBC and DOC fractions.
Fig. 2 Phylogenetic tree of cbbL sequences obtained from different
depths in paddy (P) and upland (U) soils. The tree was constructed using
neighbor-joining analysis based on bootstrap resampling of the data
(1,000). Bootstrap values higher than 50 % are shown at the branch
points. At the tip labels, the numbers directly preceding P or U indicate
the number of clones recovered and the numbers 1, 5, and 17 following P
and U refer to the 01, 15, and 517 cm sampling depths. Form 1A and
form IC refer to cbbL gene sequences from obligate autotrophic bacteria,
including some marine cyanobacteria, and facultatively autotrophic bacteria, respectively

Appl Microbiol Biotechnol (2014) 98:23092319

2315

92
94
67

JQ836347 8P1+6P5+29P17+U1
JQ836226 P1+P5+P17+U1
JQ836261 P1+U17

61

Bradyrhizobium japonicum USDA 6

Rhodopseudomonas palustris BisB

55

AP012206
CP000301

Aminobacter sp. COX

JQ964825

71

AY422046

P1+3P5+2P17+5U1+4U5

Rhodospirillum centenum SW CP000613

Azospirillum lipoferum 4B FQ311869

97
99

JQ836303 9P1+17P5+14P17+7U17
JQ836372 2P17+5U1+5U5+4U17
JQ964851 U1
JQ836382 3P17+U17
JQ836217 P1+P5+P17
JQ836215 P1+2U1+2U17
JQ836202 P1+U1
JQ836345 P1+2P5+P17+U5
JQ836385 3P1+4P5+4P17
99 JQ836385
U5
JQ964906 U5

99

90

Cluster

Mesorhizobium ciceri biovar biserrulae WSM 1271

CP002447

Nitrosospira sp. AF

AF426415

JQ836220 4P1+P17+2U1+4U5+U17
JQ964873 U1+U17

68

Acidiphilium multivorum AIU 301

70

AP012035

JQ964915 U1+3U5+3U17
JQ836319 P5
JQ964941 3U1+2U17
JQ836243 18P1+5P5+3P17+4U1+U5+4U17
JQ836208 3P1+5P5+7P17+2U1+U5+U17

58
99

Azoarcus sp. KH32C

99

Rubrivivax gelatinosus strain DSM 149

Ralstonia eutropha M17744

99
79

Cluster
HQ877082

CP001738

JQ964976 3U3
JQ836370 3P1+3P5+4P17+9U1+15U5+18U17
JQ836389 P17+32U1+35U5+14U17
JQ836390 3P17+U17
JQ836234
4P1+P17+U1+U5+U17
99
JQ836331
2P1+3P5+2P17+8U1+3U5+4U17
72
JQ964993
2U1+U17

90

54
63

Synechococcus sp. PCC700

JQ836212

Cluster

AM701777
Cluster

P1

Thiohalospira alkaliphila strain ALgr 6sp

68

Cluster

JQ836227 2P5
JQ836219 5P1+2P5+2P17+2U1
JQ964888 U5
JQ836236 2P1
JQ964837 4U1+U5+3U17

Thermomonospora curvata DSM 43183

61

Cluster

AP012305

JQ836245 P1
JQ964964 U3
JQ836348 13P1+4P5+3U1+U5+3U17
JQ836353 P5
JQ964911 U5
JQ964896 3U1+6U5+8U17
JQ836206 10P1+18P5+12P17+U1

99

Cluster

Form IC

JQ964887 3P1+3P5+8P17+9U1+16U5+15U17
JQ836400 P17

75
56

63

Cluster

JQ964835 2U1+3U5+5U17
JQ836357 P5
JQ964949 U17

98
58

GQ888604

99

Acidithiobacillus caldus strain DSM 8584

60
97
99

JQ836323
71
83
80

GQ409763

JQ836401 P17
JQ836380 2P1+P5+P17
JQ836374 2P5+2P17+2U1
JQ836332 P5
JQ964975 U17
Methylococcus capsulatus

68

Form IA

JQ836328 2P1+4P5+P17
JQ836358 3P5+U5
JQ836384 P17
JQ836310 P5

99

Cluster

AF447860

2P5

Thiobacillus denitrificans ATCC 25259

CP000116

JQ836344 P1+6P5
JQ836349 6P5+P17
Thiohalospira halophila

GQ888618

0.05

These data are needed to understand better the mechanisms

underpinning C fixation and its potential role in the

sequestration of soil carbon. As with other C inputs to soil,

the 14C fixed by autotrophic bacteria can be partitioned into

2316

Appl Microbiol Biotechnol (2014) 98:23092319

action of syntrophic organisms, such as hydrogenotrophic

methanogens (Lee et al. 2012), to 14CH4. Some of the fixed
14
C will eventually be incorporated into the soil organic
matter, microbial biomass and dissolved organic carbon
(Kuzyakov and Schneckenberger 2004). Though critical to
understanding C sequestration in soils, the mechanisms by
which such partitioning occurs or how these processes are
regulated have yet to be fully elucidated.
Previous studies have reported that over an 80-day incubation period with continuous labeling, the 14C-SOC/SOC levels
range between 0.1 % and 0.4 % in unplanted soils (Ge et al.
2012). Here, different sampling methods, soil characteristics
and the extended labeling period (110 days instead of 80 days)
may explain why the relative percentage of 14C-SOC to SOC
was higher with an average value of 5.9 % between 0 and 1 cm
in these unplanted paddy soils (Table 2). Since we were not
able to quantify re-mineralization and did not measure methane efflux of labeled C following incorporation, total 14C
incorporation into the SOC fractions is likely to have been
even higher.
The transformation of fixed 14C is expected to contribute
significantly to the labile pools of SOC, especially for MBC
and DOC (Lu et al. 2004a; Ge et al. 2012). Ge et al. (2012)
reported that the proportion of plant-derived C recovered in the
MBC was between 1.77.7 % in paddy soils whilst 0.83.2 %
has been reported for upland soils (Kuzyakov et al. 2001).
Here, the amount of microbially assimilated label incorporated
into the MBC and the DOC was relatively high. Previous
studies have indicated that the microbial communities
accessing rice derived C (Lu et al. 2004b) may be quite distinct
from those utilizing other plant derived residue C (Williams
et al. 2006). It has also been shown that the response of the
microbial community to plant residue carbon varies with plant
age (Butler et al. 2003; Lu et al. 2004a). Thus, it is possible that
the utilization and therefore the incorporation and stabilization
of microbially assimilated 14CO2 may be quite distinct from
that of plant assimilated C due to selective turnover and

0-1 cm
1-5 cm
5-17 cm

a
8

-1

cbbLabundance (10 copies g dry soil)

10

a
c

bb

a a a

a a a

aa

0
P1

P3

U1

U2

U3

80

-1

RubisCO (nmol CO2 g (soil) min )

P2

a
b

60

-1

b
b
40

a
bb

bb

20

0
P1

P2

P3

U1

U2

U3

Soils
Fig. 3 The cbbL gene copy number and RubisCO activities at different
depths in paddy and upland soils incubated in 14CO2 for 110 days. Error
bars indicate the standard error of the mean (n =4). Means with the same
letter are not significantly different at the 0.05 level

several fractions. Some of the fixed C may be mineralized and

lost to the atmosphere as 14CO2 or can be reduced by the

Table 3 Relationship between 14C-SOC, 14C-MBC, 14C-DOC, cbbL abundance, and RubisCO activity in soils incubated with 14CO2 for 110 days
14

14

14

cbbL
abundance

RubisCO

Water
content

C-SOC
C-MBC
14
C-DOC

1.000
0.971a
0.926a

0.965a
1.000
0.908b

0.936a
0.944a
1.000

0.945a
0.830b
0.816 b

0.970a
0.938a
0.951a

0.983a
0.989a
0.973a

cbbL abundance
RubisCO
Water content

0.194
0.841b
0.954a

0.121
0.766
0.861b

0.766
0.761
0.889b

1.000
0.684
0.250

0.903a
1.000
0.862b

0.872b
0.972a
1.000

C-SOC

14

14

C-MBC

C-DOC

The right, upper part and the left, lower part represent coefficients in 01 cm and 15 cm soil depth, respectively. For data on 14 C-SOC, 14 C-MBC, 14 CDOC, cbbL abundance, and RubisCO activity, see Figs. 1 and 3
a

Correlation is significant at the 0.01 level (two-tailed)

Correlation is significant at the 0.05 level (two-tailed)

Appl Microbiol Biotechnol (2014) 98:23092319

mineralisation by different microbial communities. The decrease in percent 14C-MBC relative to 14C-SOC recovered
with depth suggests a decrease in mineralization or in the
utilization efficiency of the assimilated C (14C-SOC).
DOC is a product of the physical, chemical and biological
breakdown of soil organic matter (Fang and Moncrieff 2005).
The significant linear relationship between 14C-DOC and 14CSOC (Table 3) suggests that the 14C-DOC was released from
organic materials that originated in microorganisms following
their assimilation of atmospheric CO2. 14C-DOC is a readily
degraded labile carbon pool that can serve as substrate for
microbial activity (Watanabe et al. 2004). This is evidenced
here by the significant correlation between 14C-DOC and 14CMBC (Table 3). The relative proportion of assimilated 14C in
DOC due to autotrophic bacteria (Table 2) are comparable
with those from plant-derived C; 6.714.6 % in paddy soils
and 0.61.5 % in upland soils (Liang et al. 2002; Ge et al.
2012). These data support the hypothesis that significant
amounts of microbially assimilated C are released following
biomass turnover. By providing inputs of dissolved, readily
available C, microbial assimilation is expected to play an
important role in soil biogeochemical cycling generally.
Thus, although the importance of photoautotrophy is well
established for cyanobacteria and green algae in barren or
marginal soils (Freeman et al. 2009), the work reported here
suggests that even for soils where SOC levels are unlikely
to limit microbial activity (Table 1), bacterial CO2 fixation
by regulating the available C, plays a key role in driving
soil processes. Furthermore, the ratios of 14C-MBC/MBC
and 14C-DOC/DOC were much larger than those of 14CSOC in both paddy and upland soils, indicating that the
turnover rate of 14C-MBC and 14C-DOC is faster than that
of 14C-SOC. This is supported by previous studies (Ge
et al. 2012).
Autotrophic bacteria contribute to 14CO2 fixation along soil
profiles
Statistically significant correlations between 14C-SOC content, cbbL gene abundance and RubisCO activity were evident in the top 1 cm of soil (Table 3). This indicates that
surface fixed 14C was mainly derived from autotrophic processes. In natural soils, photoautotrophic processes are restricted to the top few millimeters of the soil profiles. The
observation that the majority of the fixed 14C-SOC was concentrated at 01 cm depth with only trace or no amounts of
14
C-SOC detected between 5 and 17 cm, reinforces the importance of phototrophs in CO2 fixation in surface soils. This
is supported by the phylogenetic analysis which showed that
bacterial cbbL sequences from facultatively bacteria such as
Rhodopseudomonas palustris , Azoarcus sp. KH32C ,
Rhodospirillum centenum and Azospirillum lipoferum (form
IC RubisCO, Fig. 2) could be recovered from the top 1 cm of

2317

these soils. Whilst these results do not exclude the possibility

of chemoautotrophic CO2 fixation our previous work showed
that there was little or no CO2 fixation when soils were
incubated in the dark. Thus, chemoautotrophy is unlikely to
be quantitatively significant in the incorporation of atmospheric C in these soils. Further work using radioisotope
probing (Jenkins et al. 2010; O'Donnell et al. 2012) would
help elucidate the routes by which 14CO2 is incorporated into
soil microorganisms. Recently, Hart et al. (2013) have shown
that chemoautotrophic bacteria are capable of assimilating
4.520.05 g CO2 kg1 dry soil when provided with S2O32
and incubated for 40 h in the dark in an atmosphere of 1,000
ppmv CO2. Miltner et al. (2004, 2005) also have observed
high amounts of fixed 14CO2 (corresponding to 0.05 % of the
total organic carbon) by chemoautotrophs and heterotrophs in
extremely high SOC content (319 mg g1) soils or those with
readily available substrates addition. Although soils in our
current study were incubated under ambient atmospheric
CO2 concentration (350 ppmv) in the absence of added C or
electron donors, photoautotrophs may impact indirectly on
chemoautophic C fixation by providing reduced organic and
inorganic compounds (Shively et al. 1998). For example,
during growth of Ralstonia eutropha on fructose, glycerol or
gluconate, RubisCO activities increased to intermediate or
high levels (Friedrich et al. 1981). It would be valuable to
investigate further the role of reduced forms of fertilizer such
as NH4+ and S0 on chemoautotrophic C fixation especially
given the importance of N and S fertilizers in paddy rice
production systems (Alfreider et al. 2011; Kellermann et al.
2012).
The detection of 14C-OC further down the profile probably
results from the movement of microbially assimilated C from
the soil surface to the subsoil. Thus, the availability of readily
degradable carbon substrates such as 14C-MBC and 14C-DOC
could have facilitated chemoautotrophy either through the
provision of reduced substrates or by lowering local redox
conditions. In addition, 36 % of the assimilated 14C was
emitted to the atmosphere as 14CH in rice soils (Dannenberg
and Conrad 1999), which could provide abundant electron
donors (methane) for the obligate methanotrophs to fix CO2
(Kleiveland et al. 2012). Although this requires further investigation, some support for chemoautotrophy at depth is seen
from the increases in clone numbers (27 clones from 1 to 5 cm
and eight clones from 5 to 17 cm) that clustered with known
methane and sulfur oxidizing bacteria such as Metylococcus
capsulatus and Thiobacillus denitrificans, with 83 % and
88 % sequence similarities, respectively.
Paddy rice systems provide a physicochemical environment that has the potential to retain and stabilize newly formed
14
C-SOC through complexation reactions with active iron
oxides and the formation of soil aggregates (0.252 mm).
However, opportunities for stabilization are much more limited in upland soils where the amount of active iron and soil

2318

aggregation are lower (Pan et al. 2008; Ge et al. 2012).

Understanding and managing these stabilization reactions
are integral to the management of C sequestration and to the
mitigation of greenhouse gas emissions. Additional studies
are needed to understand fully the mechanisms regulating the
transformation, translocation and stabilization of microbially
assimilated C in soils.
Taken together, the results presented in this paper indicate
that the extent of C fixation at the surface of paddy and upland
soil systems is significant and could be capable of driving soil
C cycling by supplying labile carbon in ways equivalent to
plant residue mineralization. Whilst more extensive surveys
are needed, the data suggest that paddy soils have the potential
to enhance carbon sequestration through increased numbers
and activities of autotrophic bacteria. The results show that
although the fixed 14C-SOC was mainly concentrated at the
soil surface and microbially assimilated C can move through
the profile to the subsoil with the extent of translocation
dependent on soil type and soil properties. The distribution
of cbbL genes down the profile suggests that chemoautotrophic activities may be stimulated by the availability of organic
substrates derived from the photosynthesis of autotrophic
bacteria at the soil surface.

Acknowledgments This study was supported financially by the National Natural Science Foundation of China (41271279; 41090283;
41301275), International cooperation and regional science and technology of Hunan Province (2013WK4009), the Scientific Research Foundation for the Returned Overseas Chinese Scholars, State Education Ministry, the CAS/SAFEA International Partnership Program for Creative
Research Teams (KZCX2-YW-T07; 201004910058).

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