Rhizosphere microbial community composition affects cadmium and zinc uptake of the metal ...

27 Pages·2015·1.03 MB·English

AEM Accepted Manuscript Posted Online 16 January 2015
Appl. Environ. Microbiol. doi:10.1128/AEM.03359-14
Copyright © 2015, American Society for Microbiology. All Rights Reserved.

1 Rhizosphere microbial community composition affects cadmium
2 and zinc uptake of the metal-hyperaccumulating plant Arabidopsis
3 halleri
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5 E. Marie Muehea, Pascal Weigolda, Irini J. Adaktyloua, Britta Planer-Friedrichb,
6 Ute Kraemerc, Andreas Kapplera, Sebastian Behrensa#
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8 Geomicrobiology, Center for Applied Geosciences, University of Tuebingen, Tuebingen, Germanya; Environmental w
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9 Geochemistry, Bayreuth Center for Ecology and Environmental Research (BayCEER), University of Bayreuth, Bayreuth, lo
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10 Germany b; Department of Plant Physiology, Ruhr University Bochum, Bochum, Germanyc d
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15 Running head: Microbial community affects metal uptake by A. halleri
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17 Keywords: soil remediation, phytoextraction, phytoremediation, microbe-mineral interactions, m
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18 microbe-plant interactions r
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21 # Address correspondence to: r
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22 Sebastian Behrens 3
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23 Geomicrobiology & Microbial Ecology, Center for Applied Geosciences ,

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24 University of Tuebingen, Sigwartstrasse 10, D-72076 Tuebingen, Germany 0
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25 Phone: [phone], Fax: [phone], 9

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26 email: [email protected] y

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28 Future correspondence to s
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29 Department of Civil, Environmental, and Geo- Engineering
30 University of Minnesota
31 500 Pillsbury Drive S.E.
32 Minneapolis, MN 55455-0116, USA
33 email: [email protected]
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34 ABSTRACT
35 The remediation of metal-contaminated soils by phytoextraction depends on plant growth and
36 plant metal accessibility. Soil microorganisms can affect the accumulation of metals by plants
37 by either directly or indirectly stimulating plant growth and activity or by (im)mobilizing
38 and/or complexing metals. Understanding the intricate interplay of metal-accumulating plants
39 with their rhizosphere microbiome is an important step towards the application and
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40 optimization of phytoremediation. We studied the effect of a ‘native’ compared to a strongly
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41 disturbed (gamma-irradiated) soil microbial community on cadmium and zinc accumulation a
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42 by the plant Arabidopsis halleri in soil microcosm experiments. A. halleri accumulated 100%
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43 more cadmium and 15% more zinc when grown on the untreated compared to the gamma-

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44 irradiated soil. Gamma-irradiation neither affected plant growth nor the 1 M HCl-extractable
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45 metal content of the soil. However, it strongly altered soil microbial community composition m
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46 and overall cell numbers. Pyrosequencing of 16S rRNA gene amplicons of DNA extracted s
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47 from rhizosphere samples of A. halleri identified microbial taxa (Lysobacter, Streptomyces, r
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48 Agromyces, Nitrospira, Candidatus Chloracidobacterium) of higher relative sequence n

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49 abundance in the rhizospheres of A. halleri plants grown on untreated compared to gamma- a
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50 irradiated soil leading to hypotheses on their potential effect on plant metal uptake. However, 3
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51 further experimental evidence is required, wherefore we discuss different mechanisms of 2
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52 interaction of A. halleri with its rhizosphere microbiome that might have directly or indirectly
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53 affected plant metal accumulation. Deciphering the complex interactions between A. halleri u
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54 and individual microbial taxa will help to further develop soil metal phytoextraction as t
55 efficient and sustainable remediation strategy.
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59 INTRODUCTION
60 Elevated metal concentrations in agricultural soils decrease the yield and quality of crops.
61 One of these metals is cadmium (Cd), which is present in waste materials and waters of many
62 industries (1), and in phosphate-fertilizers used in agriculture (2, 3). Besides causing a
63 decrease in crop yield (4), Cd is also a risk to human health if larger quantities (>2.5 µg kg-1
64 body weight week-1, EFSA (5)) are consumed (6-8). To preserve crop yield and quality,
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65 sustainable biotechnologies are urgently needed to relieve agricultural soils of metal
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66 contaminants. One promising soil remediation technology is phytoremediation – the use of a
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67 plants for removing inorganic and organic contaminants from soils (9-11). Phytoremediation
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68 is attractive, as it is sustainable and relatively inexpensive compared to other soil clean-up

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69 strategies. So far it has rarely been employed on field scale (12) due to its low efficiency,
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70 slow progress, and the need to develop individual implementation strategies depending on m
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71 field site conditions and plant type. s
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72 Arabidopsis halleri has been studied as model plant for Cd as well as zinc (Zn) r
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73 hyperaccumulation in order to apply the plant for phytoremediation of Cd- and Zn- n

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74 contaminated soils (10, 13). Wild A. halleri plants have been shown to accumulate up to a
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75 354 µg Cd (14) and 21.5 mg Zn (15) g-1 dry above-ground biomass. A. halleri’s efficiency to 3
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76 take up Cd and Zn from soil is limited by (i) the plant’s overall low biomass and slow growth, 2
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77 (ii) a restricted radius and expansion of the plant’s root system (10), and (iii) the general
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78 bioavailability of metals in soil for plant root uptake (11, 16). Recent research aiming at u
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79 optimizing plant metal accumulation focused on plant growth-promoting substances and/or t
80 processes that provide metals to the plant, e.g. metal mobilization from minerals or enhanced
81 metal mobility by metal complexation with organic ligands and chelators (11, 12). As soil
82 microorganisms are known to affect plant growth (17) and the mobility of nutrients, metals,
83 and contaminants in soils (18), phytoremediation research increasingly focusses on the
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84 complex interplay of the plant with the soil microbial community (17, 19). Several studies
85 have previously shown that soil microorganisms can promote the accumulation of various
86 metals in different plant species (20-29). Most of these studies focused on specific groups of
87 microorganisms or were performed with soil extracts in artificial cultivation systems under
88 laboratory conditions (hydroponics cultures). Currently not much is known about the impact
89 of the soil microbial community at heavy metal contaminated sites on Cd and Zn
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90 accumulation by A. halleri. We therefore ask the questions: Will Cd and Zn accumulation by
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91 A. halleri be affected when the plant is grown on soil from a heavy metal contaminated field a
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92 site that either contains the ’native’ soil microbial community or a microbial community that
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93 has been strongly altered in species richness and evenness? Can the comparison of the two

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94 soil microbial communities help to identify microbial taxa living in the rhizosphere of
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95 A. halleri that might promote metal accumulation by A. halleri? m
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96 We setup plant microcosms (Fig. S1A) using Cd/Zn-contaminated soil that was either not s
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97 treated or gamma-irradiated. Plant Cd and Zn accumulation, soil geochemical parameters, and r
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98 bacterial cell numbers were quantified. Pyrosequencing of 16S rRNA gene amplicons was n

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99 used to analyze differences in microbial diversity and the relative abundance of individual a
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100 taxa in the rhizospheres of A. halleri plants grown in the untreated and gamma-irradiated soil 3
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101 microcosms 2
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102 MATERIAL AND METHODS
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103 Plant and soil characterization. Arabidopsis halleri ssp. halleri (L.) O’Kane & Al-Shehbaz u
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104 (accession Langelsheim Lan 3.1, Germany) plants were maintained in the greenhouse of the t
105 Department of Plant Physiology, Ruhr University Bochum, Germany. For experiments,
106 offshoot clones of similar size and comparable number of leaves were separated from well-
107 grown mother plants just before the experiment. Cadmium contaminated soil was collected at
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108 Langelsheim in the Harz Mountains, Germany (N51.9429, E10.3489) in October 2009 and
109 July 2010. The soil was sieved (2 mm) and stored in the dark at 4°C.
110 Experimental setup. For microcosm setup, regular quartz sand (Hornbach gardening center,
111 Germany) was cleaned with 6 M HCl for 48 h, neutralized to pH 5.5 by washing with water,
112 dried at 25°C, and baked at 180°C for 4.25 h. The sand was mixed with soil at a ratio of 1:3
113 (w/w), in order to allow the plant to grow more easily on the soil (Fig. S1B). Half of the soil-
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114 sand mix was sterilized by gamma radiation at 35 kGy at the Beta-Gamma-Service (BGS),
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115 Wiehl, Germany. For initial wetting of the dry soil to a water content of approximately 35% a
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116 (w/w), a soil extract instead of pure water was used in order to avoid shifts in ionic strength
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117 and mobility of mineral-bound ions. The soil extract was prepared by mixing the soil with

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118 sterile water (ratio of 1:1.4) (Fig. S1C). After incubation for six hours the soil extract was
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119 obtained by collecting the supernatant after sedimentation for 5 min. The soil extract was m
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120 gamma-radiation sterilized before use. 900 g of sterile and non-sterile soil-sand mixes per s
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121 plant microcosm were amended with 33 mL of sterile soil extract and homogenized using a r
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122 sterile spatula. The bottom of each plant microcosm was covered with the soil-sand mix. n

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123 Three root bags (polyester mesh, 50.0 μm pore size, Ø 4 cm, length 10 cm) were filled with a
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124 the prepared soil and evenly distributed in each microcosm. The remaining soil was filled in 3
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125 between the root bags (Fig. S1D). A. halleri offshoot clones were washed three times in sterile 2
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126 water and placed into the root bags (Fig. S1E). 10 mL of sterile water were added to each
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127 plant directly and an additional 50 mL to the whole pot. For each soil, three microcosms with u
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128 three plants each were set up. Microcosm with the same soil were placed onto the same trays t
129 and covered with clear plastic hoods to prevent cross-contamination (Fig. S1A). Microcosms
130 were irrigated with approximately 15 mL of sterile water once a week. Microcosms were
131 moved randomly within their trays. Trays were also moved once per week in order to ensure
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132 unbiased light conditions in the greenhouse. Plants were grown for four weeks at 20 to 26°C
133 with a 16 h/8 h day/night cycle. The experiment was repeated three times.
134 Sampling. Samples for geochemical and microbiological characterization of the soil and for
135 plant metal uptake were taken in the beginning of the experiment and after plant microcosm
136 incubation. Plant clones were weighed at the start of the experiment in order to use plants of
137 similar weight in the experiment. This also allowed for calculation of the increase in plant
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138 biomass after incubation. The total element (Cd, Zn) content of plant clones was determined
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139 at the start of the experiment using ICP-OES (see supplementary information) by washing a
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140 plants in water and drying at 60°C. As no additional rooting agent was added to the soil, some
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141 plants did not grow well within the four weeks of incubation, independent of the treatment.

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142 Only plants that developed a root system were considered for analysis. The above-ground
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143 material was sampled, washed in water and dried with tissue paper. The green plant tissue m
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144 was weighed and ground in liquid nitrogen. The homogenized material was dried at 60°C for s
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145 total elemental (Cd, Zn) analysis with ICP-OES. Thin roots could not be harvested for r
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146 element analysis because it was not possible to completely separate them from the soil. n

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147 For water content and pH measurements, soil samples were taken at the start and end of a
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148 incubation of the plant microcosms and stored at 4°C until analysis (see supplementary 3
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149 information). Total and 0.1 M HCl-extractable Cd and Zn were quantified in bulk soil 2
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150 sampled at the start and in bulk soil and rhizosphere soil at the end of incubation (see
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151 supplementary information). Soil samples for molecular analysis were stored at -20°C to u
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152 determine total bacterial 16S rRNA gene copy numbers and the analysis of the microbial t
153 community composition by pyrosequencing. Start bulk soil samples were taken from the
154 initially wetted sand-soil mix. End bulk soil samples were taken from 4 cm depth in between
155 two plants. End rhizosphere samples were taken from root washes, by first shaking soil of
156 roots (this soil was used for metal extraction) and followed by incubating roots in 20 mL of
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157 sterile water for 30 min. Roots were removed from the suspension, which was centrifuged for
158 10 min at 7500 g. The supernatant was filtered through 0.22 µm filters (Durapore® membrane
159 filters, GVWP02500, Millipore). Soil suspension pellet and filter were combined, frozen at -
160 20°C (see supplementary information).
161 Geochemical analyses and molecular biology techniques. All geochemical and molecular
162 analyses are described in detail in the supplement. Sequencing reads are available at the
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163 NCBI Sequence Read Archive (SRA) database under accession no. PRJEB8016.
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164 RESULTS a
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165 Geochemical parameters in the soil microcosms. The pH of the Langelsheim soil/sand
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166 mixture was 5.9 (Table S1) and did not change during microcosm incubation. Langelsheim

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167 soil contained 12.2±1.9 µg Cd and 3.3±0.5 mg Zn g-1 dry soil (Table S1). Further information p:
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168 on soil metal and carbon content are available in the supplement (Table S1). m
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169 Cd and Zn in the soil. 0.1 M HCl-extractable Cd and Zn concentrations were not affected by m
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170 gamma-irradiation (Fig. 1A). The initial 0.1 M HCl-extractable Cd content was 6.7±0.4 µg Cd rg
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171 g-1 dry soil. After one month of incubation the 0.1 M HCl-extractable Cd content decreased to n

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172 5.7±0.5 and 5.5±0.3 µg Cd g-1 dry soil in the untreated bulk and rhizosphere soil, respectively. r
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173 In the gamma-irradiated bulk and rhizosphere soil the 0.1 M HCl-extractable Cd content
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174 decreased to 6.0±0.5 and 5.9±0.5 µg Cd g-1 dry soil, respectively. The initial 0.1 M HCl- 0
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175 extractable Zn content was 2047.9±123.8 µg Zn g-1 dry soil. After one month of incubation, b
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176 the 0.1 M HCl-extractable Zn content decreased to 1677.9±152.1 and 1637.6±158.7 µg Zn g-1 u
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177 dry soil in the untreated bulk and rhizosphere soil, respectively. In the gamma-irradiated bulk
178 and rhizosphere soil Zn contents decreased to 1761.5±166.0 and 1738.5±230.4 µg Zn g-1 dry
179 soil, respectively. Aqua regia extractions did not reveal significant changes in total Cd and Zn
180 contents in the soil before and after incubation (Fig. S2).
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181 Cd and Zn accumulation by A. halleri. The initial average above-ground weight of
182 A. halleri offshoot clones was 0.47±0.23 g, which increased by 60% during four weeks of
183 incubation (Fig. S1E and F show the different sizes of plants before and after incubation).
184 Plant growth was unaffected by soil gamma-irradiation as a similar plant biomass increase
185 was observed for plants grown on untreated and gamma-irradiated soil (data not shown).
186 Initially A. halleri clones did not contain more than 3 µg of Cd g-1 dry above-ground biomass
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187 (Fig. 1B). After cultivation, A. halleri plants accumulated 61.7±12.8 µg and 33.2±8.4 µg Cd
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188 g-1 dry plant material on untreated or gamma-irradiated soil, respectively, which are a
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189 significantly different at a 95% confidence interval. A. halleri clones contained initially
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190 approximately 0.4 mg of Zn g-1 dry above-ground biomass (Fig. 1D). After four weeks of

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191 cultivation, A. halleri accumulated 12.2±1.1 and 10.7±1.7 mg of Zn g-1 dry above-ground p
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192 biomass on untreated and gamma-irradiated soil, respectively, which are not significantly m
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193 different at a 95% confidence interval. However, at a confidence interval of 94% the s
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194 difference in Zn concentrations comparing the green biomass of plants grown on untreated r
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195 and gamma-irradiated soil becomes significant. n

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196 Microbial community composition. Bacterial 16S rRNA gene copy numbers. Quantitative a
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197 PCR analysis targeting the 16S rRNA gene was used to approximate total bacterial cell 3
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198 numbers in the soil (Fig. S3). Initial bacterial 16S rRNA gene copy numbers in the native, 2
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199 untreated soil were 1.2×108±2.0×107 16S rRNA gene copies g-1 dry soil, which remained
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200 unchanged in A. halleri rhizospheres after one month of incubation. Total bacterial 16S rRNA u
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201 gene copy numbers in the gamma-irradiated soil were initially significantly lower compared t
202 to the untreated soil (2.1×107±5.3×105 16S rRNA gene copies g-1 dry soil). Bacterial
203 16S rRNA gene copy numbers in the rhizosphere of A. halleri increased during incubation on
204 gamma-irradiated soil to 1.3×108±3.0×107 16S rRNA gene copies g-1 dry soil.
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205 Microbial community richness and diversity. Pyrosequencing of bacterial 16S rRNA gene
206 amplicons was used to compare the composition of the microbial communities present in the
207 initial native bulk soil and in the rhizosphere of A. halleri after one month of plant growth in
208 the untreated and gamma-irradiated soil microcosms (Fig. 2 and S4, Tables S2 and S3). For
209 the initial bulk soil 7461 sequence reads were obtained after quality-processing of the raw
210 data. For the A. halleri rhizosphere samples collected after incubation a total of 10124 and
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211 11305 sequence reads were obtained after quality-processing for the untreated and gamma-
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212 irradiated microcosms, respectively. Archaeal 16S rRNA gene PCR products could only be a
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213 generated for the untreated soil samples. After gamma-irradiation no archaeal 16S rRNA
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214 genes could be amplified (data not shown). Unfortunately, this precluded further comparative

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215 analysis of the archaeal communities from untreated and gamma-irradiated microcosms.
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216 Calculation of the Chao1 and ACE richness estimators for 97% OTU clusters after random m
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217 subsampling revealed that our sequencing approach covered 36-52% and 38-53% of the s
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218 bacterial OTU richness for the untreated soil samples before and after incubation, r
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219 respectively. For the gamma-irradiated soil richness coverage for 97% OTU clusters as n

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220 estimated by Chao1 and ACE ranged from 45-55% (Table S2). Rarefaction curves calculated a
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221 for 97% OTU clusters did not reach saturation for any of the three sequenced soil samples 3
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222 (Fig. S4A). Both bacterial richness estimators (Chao1/ACE) and diversity indexes 2
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223 (Shannon/Simpson) calculated for 97% OTU clusters indicated a higher richness and diversity
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224 in the initial bulk soil and in rhizospheres from plants grown on untreated soil compared to u
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225 rhizospheres from plants grown on gamma-irradiated soils (Table S2). t
226 Microbial community shifts with time. Cluster analysis based on Bray-Curtis dissimilarity
227 showed that after one month of incubation the rhizosphere microbial community of the
228 untreated soil was more similar to the initial, native bulk soil microbial community than the
229 rhizosphere microbial community of the gamma-irradiated microcosms (Fig. 2A). After plant
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230 cultivation, the relative abundance of sequences affiliated with the phyla Proteobacteria and
231 Bacteroidetes increased under both microcosm conditions, although a much stronger increase
232 of the relative abundance of both taxa was observed in the rhizosphere of A. halleri grown on
233 gamma-irradiated soil (Fig. 2B). Lists of the most abundant taxa (at the family level) found in
234 the ‘native’ bulk soil and the rhizosphere of plants grown on untreated or gamma-irradiated
235 soil are given in Table S3 in the supplementary information.
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236 Microbial community shifts in the rhizosphere. The microbial community composition of
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237 rhizospheres of A. halleri grown on untreated, ‘native’ soil and gamma-irradiated soil were a
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238 compared. 16S rRNA gene sequence reads obtained from the rhizosphere of plants grown on
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239 untreated soil could be clustered into 1449 OTUs using a 97% sequence similarity cutoff

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240 (Fig. S4B). The five most abundant OTUs with relative sequence abundances ranging
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241 between 0.9 and 3% in the untreated soil were classified as Candidatus Chloracidobacterium m
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242 (3.0%), Nitrospira (2.2%), Acidovorax (1.3%), Gemmatimonas (1.0%), and Candidatus s
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243 Solibacter (0.9%) (table 1). 1191 out of 1449 OTUs were found only in the untreated soil r
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244 samples but not in the gamma-irradiated soil. Among these OTUs specific to untreated soil, n

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245 the five most abundant OTUs were classified as Lysobacter (0.7%), Streptomyces (0.5%), a
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246 Nocardioides (0.3%), Kineosporia (0.3%), and Steroidobacter (0.3%) (table 1). After gamma- 3
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247 irradiation the microbial community richness in the rhizosphere of A. halleri was strongly 2
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248 diminished. The total sequence reads obtained for the gamma-irradiated soil could be
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249 clustered into 406 OTUs. The five most abundant OTUs belonged to the genera Acidovorax u
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250 (8.0%), Sediminibacterium (4.5%), Sphingopyxis (2.9%), Sphingobium (2.7%), and t
251 Phenylobacterium (2.6%) (Table 1). Also the gamma-irradiated rhizosphere of A. halleri
252 contained specific OTUs (148 OTUs), not found in the untreated soil. The five most abundant
253 OTUs found only in the rhizosphere of plants grown on gamma-irradiated soil were classified
254 as Sphingopyxis (2.9%), Rhizobium (0.9%), Microbacterium (0.5%), Bosea (0.3%), and
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