Elevated carbon dioxide alters the structure of soil microbial communities Ye Deng1,#, Zhili He1 ...

18 Pages·2012·2.29 MB·English

AEM Accepts, published online ahead of print on 3 February 2012
Appl. Environ. Microbiol. doi:10.1128/AEM.06924-11
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
1 Elevated carbon dioxide alters the structure of soil microbial communities
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3 Ye Deng1,#, Zhili He1,#, Meiying Xu1,2, Yujia Qin1, Joy D. Van Nostrand1, Liyou Wu1, Bruce A.
4 Roe3, Graham Wiley3, Sarah E. Hobbie4, Peter B. Reich5, and Jizhong Zhou1, 6, 7*
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6 1Institute for Environmental Genomics and Department of Botany and Microbiology, University ow
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7 of Oklahoma, Norman, OK 73019, 2Guangdong Provincial Key Lab of Microbial Culture lo
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8 Collection and Application, Guangdong Institute of Microbiology, Guangzhou 510070, China, d
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9 3Advanced Center for Genome Technology and Department of Chemistry and Biochemistry d
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10 University of Oklahoma, Norman, OK 73019, 4Department of Ecology, Evolution, and Behavior, om

11 5Department of Forest Resources, University of Minnesota, St. Paul, MN 55108, 6Earth Sciences h
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12 Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, 7School of :
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13 Environment, Tsinghua University, Beijing 100084, China. e
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17 # Those two authors contributed equally to this work.
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19 *Corresponding author: Dr. Jizhong Zhou ,
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20 Institute for Environmental Genomics (IEG) 1
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21 Department of Botany and Microbiology b
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22 University of Oklahoma g
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23 Norman, OK 73019 s
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24 Phone: [phone]
25 Fax: [phone]
26 E-mail: [email protected]
27
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28 Abstract
29 Pyrosequencing analysis of 16S rRNA genes was used to examine impacts of eCO2 on soil
30 microbial communities from 12 replicates each from ambient CO2 (aCO2) or elevated CO2
31 (eCO2). The results suggest that the soil microbial community composition and structure
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32 significantly altered under eCO2, which was closely associated with soil and plant o
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33 properties. lo
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35 The concentration of atmospheric CO2 is increasing largely due to human activities, and it is
36 projected to reach 700 µmol/mol by the end of this century (15, 17). Although it is well
37 established that eCO2 stimulates plant growth and primary productivity (2, 19, 23), the impact of
38 eCO2 on soil microbial communities remains poorly understood (5, 7, 11-14, 18, 22, 28).
39 Pyrosequencing of 16S rRNA genes has been used to analyze microbial community diversity,
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40 composition, structure and dynamics from different habitats, such as soil (1, 6, 10, 24, 25, 27), w
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41 water (4, 21), fermented foods (16), and human gut (26). In this study, we aimed to: (i) examine a
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42 effects of eCO2 on the taxonomical diversity, composition, and structure of soil microbial d
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43 communities, and (ii) link soil and plant properties with the microbial community composition m

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44 and structure using tag-encoded pyrosequencing of 16S rRNA genes. tt
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45 To address those questions, this study was conducted within the BioCON (Biodiversity, a
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46 CO2 and Nitrogen) experiment site located at the Cedar Creek Ecosystem Science Reserve in .a
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47 Minnesota, USA (23). For this study, 24 soil samples (12 from ambient CO2, 12 from elevated .o
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48 CO2, and all with 16 plant species and without added nitrogen supply) were collected in July o/
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49 2007. eCO2 plots have been treated since 1997 during the plant growing season (May to October) Ap
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50 every year, and each sample was composited from five soil cores at a depth of 0-15 cm. Details il 9
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51 were described in the Supplemental Materials and Methods. 0
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52 The V4-V5 regions of 16S rRNA genes were amplified using a conserved primer pair b
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53 with a unique 6-mer oligonucleotide sequence (barcode) at the 5’-end for each sample (Table u
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54 S1). After preprocessing of all reads, 30,008 and 29,091 high quality sequences were obtained
55 for aCO2 and eCO2 samples, respectively. The numbers of sequences from different samples
56 varied in the range of 1,854 to 3,087 for aCO2 samples, and 1698 to 3,299 for eCO2 samples
57 (Table S2). All sequences were aligned using the RDP Infernal Aligner and the complete linkage
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58 clustering method was used to define OTUs using 97% identity as a cutoff, resulting in 2,527 and
59 2,354 OTUs for aCO2 and eCO2, respectively (Table S2). The average Shannon indices were
60 6.58 and 6.51 for aCO2 and eCO2 samples, respectively (Table S2). However, no significant (p >
61 0.05) differences were seen in the numbers of sequences, OTUs or Shannon diversity index
62 between aCO2 and eCO2 samples. Totally, 3500 OTUs were detected at least two of 12 samples
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63 at aCO2 or eCO2, phylogenetically deriving from one known archaeal phylum and 16 known w
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64 bacterial phyla as well as unclassified phylotypes. Most phylotypes were detected at both aCO2 a
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65 and eCO2 with only few detected at aCO2 or eCO2 (Table S3; Fig. S1). At the phylum level, 811 d
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66 (23.2%) OTUs were derived from Proteobacteria, a phylum with the highest number of m

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67 detectable OTUs, followed by Actinobacteria (596, 17.0%), Bacteroidetes (448, 12.8%), tt
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68 Acidobacteria (369, 10.5%) (Table 1). a
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69 To examine whether eCO2 affects the taxonomical composition and structure of soil .a
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70 microbial communities, principal coordinate analysis (PCoA) was performed with the relative .
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71 abundance of 454 pyrosequencing data. Two distinct clusters were formed, and aCO2 samples o/
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72 were well separated from eCO2 samples (Fig. 1). Three non-parametric multivariate statistical Ap
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73 tests, ANOSIM (8), Adonis (3), and MRPP (20) analyses showed significant differences (p = il 9
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74 0.001 and δ = 0.481, p = 0.003 and R = 0.209, and p = 0.001 and R2 = 0.082, respectively) based 0
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75 on the abundance of all OTUs detected at aCO2 and eCO2, and such significances (p < 0.05) b
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76 were also observed at the phylum level, including Acidobacteria, Actinobacteria, Bacteroidetes, u
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77 Firmicutes, DP10, Proteobacteria, TM7 and WS3 (Table S4). The results indicated that the
78 overall taxonomic composition and structure of soil microbial communities shifted in response to
79 eCO2.
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80 To further examine effects of eCO2 on microbial communities, we analyzed both
81 significantly changed and unique OTUs. Among 3500 OTUs detected, 1381 were shared by
82 aCO2 and eCO2 samples, and 1146 and 973 unique OTUs were only detected at aCO2 or eCO2,
83 respectively. For those shared OTUs, 27 were significantly (p < 0.05) decreased, and 36 were
84 significantly (p < 0.05) increased at eCO2 (Table S5). For example, 26 significantly changed
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85 OTUs were found in Actinobacteria with seven decreased and 19 increased at eCO2 (Table S5; w
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86 Fig. S2). Also, 11 OTUs were significantly (p < 0.05) changed at eCO2 with nine increased and a
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87 two decreased in Proteobacteria, and five, three, two and one OTUs were derived from α-, β-, δ- d

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88 and γ-Proteobacteria, respectively (Table S5; Fig. 2). In addition, significantly (p < 0.05) m

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89 changed OTUs were observed in other phyla, such as Acidobacteria, Bacteroidetes and tt
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90 Planctomycetes (Table S5; Fig. 2). A large percentages (60.5%) of unique OTUs were detected a
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91 by pyrosequencing with 1146 (32.7 %) from aCO2 and 973 (27.8%) from eCO2, and those OTUs .a
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92 were largely derived from the most abundant phyla (Table S5). For example, 213 and 243 unique .
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93 OTUs were detected at aCO2 and eCO2, respectively for Proteobacteria, and 170 and 150 for o/
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94 Actinobacteria. The analysis of significantly changed and unique OTUs confirmed that the A
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95 taxonomic composition and structure of soil microbial communities significantly changed at il 9
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96 eCO2. 01
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97 To understand whether microbial populations differentially respond to eCO2, detected b
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98 OTUs were mapped to their associated populations at the phylum or lower levels. Based on u
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99 relative abundance of all OTUs in a given phylotype with at least six OTUs detected,
100 significantly changed populations were identified by response ratio (19). At the phylum level,
101 four phyla, including one archaeal phylum (Crenarchaeota) and three bacterial phyla
102 (Proteobacteria, Gennmatimonadetes and DP10) showed significant (p < 0.05) changes in their
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103 relative abundances, but other phyla including Actinobacteria, the most abundant phylum, did
104 not show significant changes in their relative abundances at eCO2 (Fig. 3). A further examination
105 of those significantly changed phyla showed that those changes occurred in some specific
106 microbial populations at the class or lower levels. In Proteobacteria, the relative abundance of
107 some orders, such as Caulobacterales of α-Proteobacteria, Myxococcales of δ-Proteobacteria,
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108 and Xanthomonadales of γ-Proteobacteria significantly (p < 0.05) increased at eCO2 (Fig. S3). w
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109 Also, although significant changes were not seen at eCO2 for the most abundant phyla at the a
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110 phylum level, such significances were detected at class or lower taxonomic levels. For example, d

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111 in Acidobacteria, significant (p < 0.05) changes were observed in the relative abundance in the m

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112 classes of Gp1, Gp2 and Gp3 but not in other classes (Fig. S4A); in Verrucomicrobia, all tt
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113 significant (p < 0.05) changes appeared to be decreased in Spartobacteria but not in other classes a
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114 (Fig. S4B); in Firmicutes, the relative abundances of unclassified phylotypes were significantly .a
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115 (p < 0.05) decreased (Fig. S4C). These results indicated that eCO2 might differentially affect .o
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116 some specific microbial populations at different taxonomic levels such as phylum, class, and /
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117 order with decreased or increased relative abundances at eCO2. Recently, a study using a Ap
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118 comprehensive functional gene array, GeoChip 3.0 (12) demonstrated that the functional il 9
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119 composition and structure of soil microbial communities were significantly altered at eCO2 in 01
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120 BioCON, which may be due to eCO2-induced shifts in microbial populations. However, the b
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121 linkage between taxonomical populations and their functions of soil microbial communities u
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122 needs further investigations.
123 To link the microbial community structure with soil and plant properties, Mantel tests
124 were performed. First, using the BioENV procedure (9), four plant variables, including
125 belowground carbon % (BPC), aboveground carbon % (APC), aboveground total biomass
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126 (ATB), and total biomass (TB), and four soil variables, including proportion soil moisture at the
127 depth of 0-17 cm (PSM0-17), %C at the depth of 0-10 cm (C0-10), %N at the depth of 0-10 cm
128 (N0-10), and net N mineralization, were selected for partial Mantel tests (Table S6). Second,
129 partial Mantel tests were performed to correlate the microbial community measured by the
130 relative abundance of all detected 3500 OTUs with the above selected sets of plant and soil
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131 variables. Although the microbial community was not significantly (p > 0.05) correlated with w
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132 plant variables or soil variables (Table S6) at the community level, significant correlations were a
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133 observed with specific microbial populations at different taxonomic levels (phylum, class, order, d

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134 family and genus). At the phylum level, five phyla (Firmicutes, Proteobacteria, Chlamydiae, m

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135 Gemmatimonadetes, and Nitrospirae) were significantly (p < 0.05) correlated with the selected tt
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136 plant properties (Table S6). At the class level, nine classes were significantly (p < 0.05) a
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137 correlated with soil or plant characteristic. Bacilli, Clostridia, α-Proteobacteria, Chlamydiae, .a
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138 Nitrospirae, and an unclassified phylotype from the TM7 phylum were significantly (p < 0.05) .
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139 correlated with the selected plant variables, Subdivision 3 of Verrucomicrobiae (p = 0.05) with /
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140 soil variables, and γ-Proteobacteria (p < 0.05) with both soil and plant variables (Table 2). A
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141 Similarly, significant (p < 0.05) correlations between the microbial community and the selected il 9
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142 plant and/or soil properties were detected in 12 orders (Table S7), 23 families (Table S8), and 48 0
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143 genera (Table S9). In addition, many unclassified phylotypes were significantly (p < 0.05) b
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144 correlated with the selected soil or/and plant variables, suggesting that soil and plant factors may u
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145 also shape taxonomically uncharacterized microbial communities (Table 2; Table S6, S7, S8,
146 S9). The above results suggest that the variables selected greatly influenced the taxonomic
147 composition and structure of microbial communities in this grassland ecosystem.
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148 In summary, this study used pyrosequencing technologies to examine the taxonomical
149 diversity, composition, and structure of soil microbial communities in a grassland ecosystem,
150 and their responses to eCO2. The microbial community composition and structure significantly
151 shifted at eCO2 potentially due to differential responses of specific microbial populations to
152 eCO2. Plant and soil properties, such as plant biomass, soil moisture, and soil C and N contents
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153 may greatly shape the microbial community composition and structure, and regulate ecosystem w
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154 functioning in this grassland. a
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155 d

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156 Acknowledgements m

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157 This work is supported by the United States Department of Agriculture (Project 2007-35319- tt
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158 18305) through NSF-USDA Microbial Observatories Program, by the Department of Energy a
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159 under the contract DE-SC0004601 through Genomics: GTL Foundational Science, Office of .a
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160 Biological and Environmental Research, and by the National Science Foundation under Grant .
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161 Numbers DEB-0716587 and DEB-0620652 as well as the DEB-0322057, DEB-0080382 (the /
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162 Cedar Creek Long Term Ecological Research project), DEB-0218039 DEB-0219104, DEB- A
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163 0217631, DEB-0716587 BioComplexity, LTER and LTREB projects, the DOE Program for il
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164 Ecosystem Research, and the Minnesota Environment and Natural Resources Trust Fund. We 0
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165 also acknowledge members of the Oklahoma University’s Advanced Center for Genome b
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166 Technology, in particular, Chunmei Qu and Yanbo Xing, for sample manipulations for the u
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167 454/Roche GS-FLX Titanium pyrosequencing. t
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169 Table 1. Numbers of OTUs detected at aCO2 and eCO2 conditions.
No. of OTUs
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Domain Phylum Total OTUsa Average OTUsb Average OTUsb
(unpaired
(%) (aCO) (eCO)
2 2 t-test)
Crenarchaeota 10 (0.28) 4.42±1.73 3.17±1.59 0.08
Archaea
Unclassified 1 (0.03) / / /
Acidobacteria 369 (10.50) 112.33±20.08 91.08±19.11 0.01
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Actinobacteria 596 (17.00) 179.83±26.18 179.25±25.52 0.96 o
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Bacteroidetes 448 (12.80) 105.58±24.54 100.17±21.07 0.57
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BRC1 2 (0.57) 0.17±0.39 0.25±0.45 0.63 lo
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Chlamydiae 36 (1.03) 3.08±2.39 3.92±3.00 0.46 d
Chloroflexi 94 (2.69) 17.33±4.23 14.92±6.04 0.27 e
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Cyanobacteria 3 (0.86) 0.75±0.87 0.17±0.39 0.04 f
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Firmicutes 118 (3.4) 28.58±5.65 24.33±5.99 0.09 m
Bacteria Gemmatimonadetes 86 (2.46) 22.83±5.81 17.92±5.52 0.05
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Nitrospirae 6 (0.17) 3.00±1.48 2.00±1.04 0.07 tt
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OP10 8 (0.23) 1.42±1.51 0 0.00 :/
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Planctomycetes 251 (7.17) 42.00±8.82 39.67±12.37 0.60 e
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Proteobacteria 811 (23.2) 207.08±30.79 220.33±30.22 0.30
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TM7 52 (1.49) 7.83±3.79 6.25±4.11 0.34 a
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Verrucomicrobia 127 (3.63) 29.33±8.32 22.92±8.01 0.07 m
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WS3 6 (0.17) 1.00±0.74 0.83±1.19 0.68 o
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Unclassified 454 (13.00) / / /
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Unclassified 22 (0.63) / / /
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Total 3500 (100) 846.75±108.71 800.5±122.03 0.34 A
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170 a Total number of OTUs detected by pyrosequencing across all 24 samples. ril
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171 bThe average number of detected OTUs by 12 samples under aCO2 or eCO2. , 2
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173 Table 2. Partial Mantel analysis of the relationship between the relative abundance of class and soil or
174 plant properties. Only significantly (p < 0.05) changed phytotypes are shown.
Soila Plantb
In association with: controlling Plantb Soila
Phylum Class r p r p
All detected OTUs -0.018 0.508 0.266 0.065
Bacilli 0.184 0.118 0.379 0.020
Firmicutes
Clostridia 0.040 0.380 0.316 0.014 D
α-Proteobacteria 0.137 0.139 0.300 0.012 o
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Proteobacteria β-Proteobacteria 0.220 0.077 0.470 0.003 n
γ-Proteobacteria 0.317 0.023 0.353 0.021 lo
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TM7 Unclassified -0.065 0.621 0.415 0.010 d
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Chlamydiae Chlamydiae 0.192 0.157 0.356 0.026 d

Nitrospira Nitrospira -0.033 0.520 0.404 0.013 fr
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Verrucomicrobia Subdivision3 0.289 0.049 -0.193 0.922 m

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