JXB Advance Access originally published online on November 26, 2007
Journal of Experimental Botany 2007 58(15-16):4173-4182; doi:10.1093/jxb/erm274
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© 2007 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
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RESEARCH PAPER |
Effects of bacteria on enhanced metal uptake of the Cd/Zn-hyperaccumulating plant, Sedum alfredii
1Croucher Institute for Environmental Sciences and Department of Biology, Hong Kong Baptist University, Hong Kong SAR, PR China
2State Key Laboratory for Bio-control and School of Life Sciences, Sun Yat-sen (Zhongshan) University, Guangzhou 510275, PR China
* To whom correspondence should be addressed. E-mail: mhwong{at}hkbu.edu.hk
Received 13 July 2007; Revised 1 October 2007 Accepted 15 October 2007
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Abstract |
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To investigate the effects of bacteria (Burkholderia cepacia) on metal uptake by the hyperaccumulating plant, Sedum alfredii, a hydroponic experiment with different concentrations of Cd and Zn was conducted. It was found that inoculation of bacteria on S. alfredii significantly enhanced plant growth (up to 110% with Zn treatment), P (up to 56.1% with Cd treatment), and metal uptake (up to 243% and 96.3% with Cd and Zn treatment, respectively) in shoots, tolerance index (up to 134% with Zn added treatment), and better translocation of metals (up to 296% and 135% with Cd and Zn treatment, respectively) from root to shoot. In the ampicillin added treatment with metal addition, stimulation of organic acid production (up to an increase of 133% of tartaric acid with Cd treatment) by roots of S. alfredii was observed. The secretion of organic acids appears to be a functional metal resistance mechanism that chelates the metal ions extracellularly, reducing their uptake and subsequent impacts on root physiological processes.
Key words: Burkholderia cepacia, metal-tolerant bacteria, organic acid, phytoextraction
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Introduction |
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In order to clean up metal-contaminated soils, heavy metals should be concentrated and extracted by conventional methods for reuse or for proper disposal. A promising option to achieve this is through phytoremediation, the use of plants to remove, destroy, or sequester hazardous substances from the environment (Cunningham et al., 1995). The efficiency of metal accumulation is dependent on two main factors. First, plants must be able to take up and accumulate high concentrations of metals, and secondly they must be able to produce a large biomass. Unfortunately, even the growth of metal-resistant metal-accumulating plants can be severely inhibited when the concentration of available metal in the contaminated soil is very high. This will consequently result in a decrease in plant biomass and, thereby, in the efficiency of phytoremediation.
One way to relieve the toxicity of heavy metals to plants might involve the use of plant growth-promoting bacteria (PGPB), free-living soil bacteria that exert some beneficial effect on plant development when they are either applied to seeds or inoculated into the soil (Glick et al., 1999). Direct mechanisms of plant growth due to PGPB include the provision of bioavailable P for plant uptake, N fixation for plant use, sequestration of iron (Fe) for plants by siderophores, production of plant hormones such as auxins, cytokinins, and gibberellins, and lowering of plant ethylene levels (Glick, 1995; Glick et al., 1999).
Root exudates are plant metabolites that are released to the root surface or into the rhizosphere to enhance nutrient uptake (Curl and Truelove, 1986). They are generally classified into two types, high molecular weight (HMW) and low molecular weight (LMW) materials. The first includes mucilage (mainly polysaccharides and polyuronic acid) and ectoenzymes; the latter mainly consists of organic acids, sugars, phenols, and various amino acids, including non-protein amino acids such as phytosiderophores (Marschner, 1995). The composition and quantity of root exudates vary from plant to plant, with two factors being important. One is a plant's inherent biology, such as plant species, growth and developmental period, and nutrient status. The other is the external environment for plant growth, i.e. soil and its elemental content (Gregory and Atwell, 1991). Included among the various root exudates are organic acids, which are negatively charged anions under a wide range of soil conditions, allowing them to react strongly with metal ions in both soil aqueous and solid phases (Jones and Darrah, 1994; Jones et al., 1996); they might also play a role in weathering processes (Lundström, 1994). Oxalate production has been suggested to have an important role in P solubilization (Knight et al., 1992; Griffiths et al., 1994; Cannon et al., 1995), and citric acid may play a role in K mobilization (Wallander and Wickman, 1999).
Cakmakci et al (2001) showed that the inoculation of the PGPB, Burkholderia cepacia, significantly increased the root yield (from 6.1% to 13.0%) and sugar yield (from 2.3% to 7.8%) of sugar beet. It has been claimed that the rhizosphere microbes can increase the tolerance of their host plants to heavy metals when present at toxic levels (H
flich and Metz, 1997). Furthermore, the rhizosphere microbes can protect plants by facilitating the uptake of Fe3+ against the toxic effects of Ni (Burd et al., 1998). Only a few attempts have been made to study the rhizosphere bacteria of hyperaccumulating plants and their role in the tolerance to and uptake of heavy metals, though many soil bacteria have been found to be tolerant to heavy metals and play important roles in mobilization or immobilization of heavy metals (Gadd, 1990). The present study was therefore designed to investigate (i) the effects of metal-tolerant bacteria on the growth of Sedum alfredii; (ii) the effects of bacteria on P, Cd, and Zn uptake and accumulation under five different concentrations of Cd or Zn in hydroponic solution; and (iii) the effects of bacteria on metal tolerance of S. alfredii by root exudation.
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Materials and methods |
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Plants
The effects of microbes on Cd and Zn uptake by plants were investigated using S. alfredii, a Cd/Zn-hyperaccumulating plant native to China. Mature plants collected from an ancient Pb/Zn mine (118°56'N 29°17'E) located in Qu Zhou City, Zhejinag Province, were clipped
5 cm long from the tips, grown on potting soil, and propagated in a greenhouse for 1 month.
Bacterial culture
Pure culture of the metal-tolerant bacterium B. cepacia isolated from the rhizosphere of S. alfredii in the Pb/Zn mine (identified using sequence analysis of the small ribosomal subunit, 16S r DNA, accession number: AB051408) was used in this study to determine its influence on metal uptake by plants. The bacteria were grown in LB medium for 24 h on a shaker at 200 rpm (model: 462801, Lab-Line, Barnstead, NH, USA). The cultures were centrifuged at 8000 rpm for 10 min (Beckman Avanti J25 I, Fullerton, CA, USA), then washed with 0.85% sterilized NaCl twice and resuspended in deionized water.
Hydroponic experiment
New shoots (1 month old) were cut and selected for the hydroponic test. The new shoots were first grown in 1/10 Hoagland solution (Hoagland and Arnon, 1950) for 2 weeks for initiation of new roots. Then roots of each plant surface were sterilized by shaking in 70% ethanol for 30 s, followed by shaking in 10% hypochlorite for 1 min, and then five 5 min washes with sterilized double-distilled water. In Zn treatments, Zn [zinc(II) sulphate] was added at 10, 20, 40, and 80 mg l–1, while for Cd treatments, Cd [cadmium(II) nitrate] was added at 1, 2, 4, and 8 mg l–1. There were six replicates for each treatment. During the period of metal treatment, solutions were adjusted daily with diluted HCl and NaOH to pH 5.8, aerated every day, and renewed once every 3 d. Washed bacterial suspension (1 ml) was added to a hydroponic solution in which the final population size of bacteria was 3x108 cfu ml–1. At the same time, 0.1 mg ml–1 ampicillin (to inhibit both Gram-positive and Gram-negative bacteria; de Souza et al., 1999) and washed bacterial suspension (1 ml) were added to the solution as an ampicillin added treatment.
Plant harvest, root exudate collection, and analysis
After 4 weeks exposure to metals, plant samples were washed thoroughly with deionized water, and the roots of intact plants were immersed in 20 mmol l–1 Na2-EDTA for 15 min to remove the Cd2+ and Zn2+ adhering to the root surfaces (Yang et al., 1996). The plant roots were then washed with tap water, and soaked in 30 mg l–1 chloramphenicol for 2 h to minimize microbial growth (Subbarao et al., 1997), followed by washing with tap water and then by sterilized distilled water before collection of root exudates. Each plant was placed into 30 ml of sterilized, distilled water for 6 h prior to collection of root exudates. The root exudate solution was immediately filtered through a 0.45 µm membrane filter. The solution containing root exudates was immediately stored at –20 °C for HPLC analysis (for lactic, formic, acetic, tartaric, malic, oxalic, succinic, and citric acids) according to the method described by Subbarao et al. (1997). pH, electrical conductivity (EC; Orion meter), and dissolved organic carbon (DOC; Total Organic Carbon analyzer, TOC-5000A, Shimadzu, Japan) of the root exudate solution collected were also measured. The plants were then separated into roots and shoots, oven-dried (70 °C) to a constant weight, followed by digestion with concentrated HNO3. Concentrations of Cd or Zn in the digests were determined by inductively coupled plasma-atomic emission spectrometry (ICP-AES). Blanks and standard material (NIST 1570a, spinach leaves) were used for quality control, and the recovery rates were within 100±10%. Shoot and root P were measured in digests using the molybdenum blue method (Page et al., 1982).
The tolerance index (TI) was calculated by the following formula (Wilkins, 1978):
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Organic acid analysis
Analysis of free organic acids was carried out using an HPLC system (Agilent 1100 Series, Agilent Technologies, Santa Clara, CA, USA), with a diode-array detector (DAD) and a multiwavelength fluorescence detector. Orthophosphoric acid (25 mM) was used to elute fractions from a PrevailTM organic acid column (150 mmx4.6 mm, 5 µm) with a PrevailTM organic acid guard column (7.5 mmx4.6 mm). To remove free metal species, samples were treated with ion-exchange resin (AG 50W-X8 100–200 hydrogen form resin, Bio-Rad, Hercules, CA, USA). Both samples and eluant were pre-filtered through 0.45 µm membrane filters, and 20 µl of triplicate samples were run for 10 min.
Statistical analyses
To compare the effects of Cd and Zn concentrations and bacteria on biomass and metal concentrations of plants, two-way analyses of variance (ANOVAs) were used. The translocation factor (TF) for metals within a plant was expressed by the ratio [metal]shoot/[metal]root, which showed the metal translocation properties from roots to above-ground parts (Stoltz and Greger, 2002).
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Results |
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Effect of ampicillin on bacteria
Ampicillin inhibits both Gram-positive and Gram-negative bacteria by interfering with bacterial cell wall synthesis; however, it had a minimal effect on plant physiology (de Souza et al., 1999). In the hydroponic experiment, no bacterial isolates were detected in the ampicillin treatment with and without metal addition (data not shown). Therefore, it is suggested that ampicillin inhibits the activity of bacteria when S. alfredii were transferred to the nutrient solution containing the heavy metals Cd and Zn.
Plant biomass
The shoot and root biomasses of S. alfredii on a dry weight basis are shown in Fig. 1. Addition of Cd and Zn to the nutrient solution had no significant effect on the plant biomass, while the shoot weight decreased by 28.0, 14.4, and 28.8% under Cd treatments of 2, 4, and 8 mg l–1 respectively, when compared with control solution. In contrast, the root weight under Cd treatments of 1 mg l–1 and 2 mg l–1 increased by 74.9% and 275%, respectively. With Zn treatment, the root weight increased by 17.0, 203, 124, and 22% with 10, 20, 40, and 80 mg l–1 Zn in the nutrient solution, respectively. Bacterial inoculation significantly (P <0.001) increased the biomass of shoots under Zn treatment by 0.8–110%, and that of roots under Cd and Zn treatments by 43.6–139% and 20.9–243%, respectively.
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Plant P uptake
Table 1 shows the P uptake by S. alfredii in shoots and roots under different concentrations of metals in the presence and absence of bacteria. In general, metal additions had no effect on P uptake by plants, while P uptake in shoots under Zn treatment was significantly (P <0.05) increased by 9.9–57%. Inoculation with B. cepacia significantly enhanced P uptake of shoots and roots under Cd treatment by 1.7–56.1% and 10.1–199%, respectively. Conversely, bacterial inoculation significantly (P <0.001) decreased the P uptake in roots under Zn treatment by 41.6–69.9%, while total P uptake (expressed in mg per root) under Zn treatment increased significantly due to bacterial inoculation.
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Metal uptake, tolerance index, and translocation factor of S. alfredii
Uptakes of Cd and Zn in shoots and roots of S. alfredii are shown in Table 2. Addition of Cd and Zn to nutrient solution significantly (P <0.001) enhanced metal uptake in shoots and roots. The Cd concentration in shoots and roots reached 1210 mg kg–1 and 10 850 mg kg–1, respectively, while Zn in shoots and roots reached 23 250 mg kg–1 and 29 310 mg kg–1, respectively. Inoculation with B. cepacia significantly enhanced Cd and Zn uptake in shoots by 36.5–243% and 12.2–96.3%, respectively. Nevertheless, bacterial inoculation had a significant effect on the concentrations of Cd and Zn in roots, causing a reduction of 0.2–50.0% and 14.7–65.5%, respectively. The results indicated that the rhizosphere bacteria played an important role in metal uptake in shoots by the plants. Furthermore, it is evident that ampicillin inhibited the growth of bacteria and resulted in a decrease of metal uptake and accumulation by S. alfredii.
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Table 3 shows the TI and TF of S. alfredii under different concentrations of metals in the presence and absence of bacteria. Increasing concentrations of Cd and Zn had no significant effect on the TI, while the TI of shoots decreased with the increasing trend of Cd and Zn in most cases. In contrast, the TIs of roots increased significantly (P <0.01) with increasing Zn and reached 700% with 80 mg l–1 Zn in the nutrient solution. In general, bacterial inoculation increased the metal TI in shoots and roots of plants; the inoculation significantly (P <0.001) increased the TI of shoots with increasing Zn by 5.70–134%. According to Table 3, increasing the Cd and Zn concentrations had no significant effect on the TF of S. alfredii. In many studies, the TF of hyperaccumulating plants was >1 (Ma et al., 2001; Yang et al., 2002). Conversely, the TFs reported in this study were smaller than that in the treatment without bacterial inoculation, as most of the metals were accumulated in roots (Table 3). Nevertheless, bacterial inoculation significantly (P <0.001) increased the TF of Cd and Zn by 0.91 and 1.53, respectively.
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pH, EC, and DOC of root exudates
Table 4 shows values of the pH, EC, and DOC of root exudates collected over the 6 h period after the 4 weeks of metal exposure. Addition of Cd or Zn to the nutrient solution had a significant effect on the pH of root exudates, with pH increasing according to the increase in Cd, but decreasing according to the increase in Zn. Inoculation with B. cepacia significantly lowered the pH of root exudates by 0.13–0.48 and 0.05–0.29 in the treatments with Cd and Zn, respectively. The present results showed that the EC of root exudates was not affected by the addition of Cd, while it was significantly increased by the addition of Zn when compared with the control. In addition, the inoculation with B. cepacia did not have a significant effect on the EC of root exudates of S. alfredii. The mean DOC (dry weight basis in root) in the root exudates was significantly higher (P <0.01 for Cd and P <0.05 for Zn) in ampicillin added treatment for both Cd and Zn treatments.
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Contents of root exudates
Oxalic acid and tartaric acid were the main constituents detected in the root exudates of S. alfredii over the 6 h period (Table 5). Metal addition had a significant effect (P <0.05) on the concentration of organic acids secreted by roots. In general, inoculation with B. cepacia lowered the secretion of organic acids. Bacterial inoculation significantly lowered the secretion of tartaric acid by 21.4–55.9% under Cd treatment. The present results indicated that the secretion of organic acids might be involved in the detoxification of metals and metal tolerance of S. alfredii without bacterial inoculation.
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Discussion |
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Effects of bacteria on plant biomass
The present study showed that the addition of metals (especially the highest concentration) to the nutrient solution substantially inhibited the growth of S. alfredii, which resulted in a 28.8% and 7.5% reduction in shoot weight under Cd and Zn treatments, respectively. Conversely, inoculation with B. cepacia alleviated the inhibitory effects caused by metals on plant growth and the TI, and even stimulated the growth of S. alfredii. Bacteria in the rhizosphere interact in numerous ways with plants to improve their growth (Kapulnik, 1996). Aside from the well-characterized N-fixing legume symbioses and plant–pathogen host interactions, rhizosphere bacteria can stimulate plant growth by producing phytohormones (Fallik et al., 1994), enhancing mineral and water uptake (Lin et al., 1983), producing antibiotics to inhibit pathogens (Lesinger and Margraff, 1979), and altering root morphology (Lin et al., 1983; Kapulnik, 1996).
Plant growth promotion by rhizosphere bacteria is possibly due to the utilization of 1-aminocyclopropane-1-carboxylic acid (ACC), synthesis of phytohormones, and solubilization of minerals (Glick et al., 1998; Gupta et al., 2002). Gupta et al. (2002) showed that bacterial inoculation protected the plants against the inhibitory effects of heavy metals. It is likely that the siderophore-producing and P-solubilizing isolates might have helped plant root proliferation and enhanced the uptake of soil minerals such as Fe and P by the host plant. The other possible mechanism of plant growth promotion is the microbial production of indole acetic acid (IAA). The IAA produced by bacteria promotes root growth by directly stimulating plant cell elongation or cell division (Glick et al., 1998). The present study showed that bacteria appear to increase the plant's potential for Cd/Zn phytoremediation because they protect the plant against metal inhibition and facilitate Cd and Zn accumulation.
Effects of bacteria on metal uptake
Inoculation with B. cepacia significantly enhanced Cd and Zn accumulation in shoots, while less Cd and Zn were accumulated in roots when compared with ampicillin added treatment. A better translocation of metals from root to shoot might be involved and facilitated by bacteria. The present study was in good agreement with that of Höflich and Metz (1997) who revealed that some PGPB were able to stimulate plant growth and metal uptake when the plants were grown in metal-contaminated soils. A high proportion of metal-resistant bacteria remain in the rhizosphere of the hyperaccumulators Thalaspi caerulescens (Delorme et al., 2001) and Alyssum bertolonii (Mengoni et al., 2001) or Alyssum murale (Abou-Shanab et al., 2003a) grown in soil contaminated with Zn and Ni, or Ni, respectively. The presence of rhizosphere bacteria increased the concentration of Zn (Whiting et al., 2001), Ni (Abou-Shanab et al., 2003b), and Se (De Souza et al., 1999) in T. caerulescens, A. murale, and Brassica juncea, respectively. Inoculation of rape (canola, Brassica napus) with metal-resistant PGPB containing ACC deaminase stimulated growth of plants cultivated in Cd-contaminated soil (Belimov et al., 2001). In addition, various N2-fixing and auxin-producing PGPB immobilized Cd and promoted growth and nutrient uptake by barley plants in the presence of toxic Cd concentrations (Belimov and Dietz, 2000; Pishchik et al., 2002).
Effects of bacteria on metal tolerance
Table 3 shows the influence of bacteria on the TI of S. alfredii under Cd and Zn. In ampicillin added treatment, the TI decreased in shoots with increasing concentrations of metals in nutrient solution, while the TI in roots increased, especially under Zn treatment. However, both shoot and root tolerance to Cd and Zn was increased in treatments with bacterial inoculation, for shoots and roots under Cd treatment by 15.0–33.2% and 20.0–25.4%, respectively, and for shoots and roots under Zn treatment by 5.8–134% and 37.2–97.1%, respectively. The present study showed that bacterial inoculation could enhance the metal tolerance of S. alfredii, which might be explained by the production of siderophores by bacteria which contain the enzyme ACC deaminase, protecting the plant against Zn toxicity by decreasing the level of ethylene stress (Burd et al., 1998).
Interaction between plant P and metal uptake
A positive correlation coefficient was obtained between plant P and metal uptake (Table 6). The present results revealed that P and metals in nutrient solution may be co-transported by plants, but most P was accumulated in the roots. Plant P uptake is facilitated by P carriers located in the root plasma membranes (Marschner, 1995). Since metal ions and P are not chemical analogues, therefore, they would be taken up and transported by plants without any antagonistic effect.
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Interaction between root exudates and metal tolerance and uptake
A negative correlation coefficient between organic acid secretion and metal uptake by plants was found (Table 7). Both oxalic and tartaric acids were negatively correlated to Cd and Zn uptake in shoots and roots of S. alfredii. The present results show that the secretion of organic acids might play an important role in heavy metal tolerance in the absence of bacteria, as more organic acid was secreted in the ampicillin added treatment (Table 5). As discussed in the previous section, bacteria might impose metal tolerance and inhibit the toxic effects derived from increasing metal concentrations in the nutrient solution. It has been suggested that organic acids play an important role in heavy metal tolerance, for example, citrate for Zn tolerance (Godbold et al., 1984) and malate for Ni tolerance (Yang et al., 1997). Jones and Darrah (1994) indicated that citric acid secreted by plant roots is an effective Al-chelator for decreasing Al toxicity. Al-induced organic acid exudation has been associated with Al resistance in higher plants (Delhaize and Ryan, 1995; Kochian, 1995; Ma, 2000; Matsumoto, 2000). Al enhances the exudation of malate in wheat (Delhaize et al., 1993), oxalate in buckwheat (Zheng et al., 1998), and citrate in corn (Pellet et al., 1995), snapbean (Miyasaka et al., 1991), and soybean (Silva et al., 2001). The role of malic acid in Al tolerance is to prevent the blocking of the Ca2+ channel in the root cell plasma membrane (Huang et al., 1996). Therefore, the present results suggested that secretion of organic acids appears to be a functional metal resistance mechanism that chelates the metal ions extracellularly, and reduces metal uptake and subsequent stresses on roots.
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Conclusion |
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The present study demonstrated that inoculation of S. alfredii with B. cepacia, a metal-tolerant bacterium, significantly enhanced plant growth, metal, and P uptake in shoots, and also resulted in a better translocation of metals from root to shoot. In addition, metal tolerance to Cd and Zn was also increased by protecting the plants against the inhibitory effects of metals. In addition, stimulation of organic acid production by Cd and Zn in S. alfredii was found, with more organic acids secreted in the ampicillin added treatment. However, the possible role of organic acids in alleviating metal toxicity should be tested further. The microbial enhancement on Cd and Zn removal may therefore provide a new technique for the decontamination of metal-polluted soils and may have potential for phytoremediation and phytomining of metals from soils.
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Acknowledgements |
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Financial support from the Research Grants Council of the University Grants Committee of Hong Kong (CERG: HKBU 2181/03M; and Area of Excellence: CityU/AoE/03-04/02) is gratefully acknowledged.
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