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BIOREMEDIATION AND PHYTOREMEDIATION
SUBMITTED TO: Mr. ANAND MOHAN
SUBMITTED BY: GYANENDRA SINGH REG No. 10809904 ROLL NO:ROE107A27 SEC- OE107 B.Tech(Hons)-Biotech
Plants were identified as L. the ßproteobacteria. Bacterial diversity increased upon cadmium addition for both EPA and RCP libraries. each containing about 100 clones from the root surfaces of plants. While concentrations in natural fresh waters are typically around 0. but this technology could beapplied to the remediation of surface waters (38). Cadmium has no knownfunction in biological systems. that is. and even small amounts can be toxic to organisms.Analyses of diversity at the phylotype level showed parallel shifts to more even communities upon cadmium addition. Plants that can accumulate metals at higher concentrations than those found in the environment (for Cd. and lead that pollute surface waters. minor by analysis of the rpl16 chloroplast region. with and without cadmium within the same plant type (EPA or RCP). concentrations in impacted waters can be above several µg/liter (45). Environmental Protection Agency has set the drinking water limit for Cd at 5 µg/liter (46) and has classified Cd as a priority pollutant. indicating that cadmium had little effect. The heavy metal cadmium is well studied in aquatic environments because it is a particularly toxic heavy metal.01 µg/liter.S. Many studies have focused on terrestrial plants and environments (5. Comparative bacterial community studies were based on the analyses of 16S rRNA clone libraries. the use of plants to remove pollutants from the environment. nickel. 49). were small. is an emerging technology that may be used to remediate aquatic systems.ABSTRACT In this study we present the comparative molecular analysis of bacterial communities of the aquatic plantLemna minor from a contaminated site (RCP) and from a laboratory culture (EPA). the effects of cadmium addition on bacterial communities were very noticeable. as well as each of these with the addition of cadmium. Bacterial communities were compared at three phylogenetic levels of resolution. copper. at least 100 mg/kg in dry plant tissues) are termed hyperaccumulators (4). differences in diversity index scores between treatments. Hyperaccumulation may also be based on the . This study is a first step in understanding the role of aquatic plantassociated microbial communities in phytoremediation of heavy metals. zinc. INTRODUCTION Effluent from industrial sources and urban runoff often contains toxic concentrations of heavy metals such ascadmium. differences between unamended andcadmium-amended libraries were much larger. At the level of bacterial divisions. When we compared genera within the most dominant group. indicating that contamination history might be independent of disturbance-induced diversity shifts. The U. Phytoremediation. At finer phylogenetic levels of resolution. percentage changes in diversity indices due to cadmium addition were the same for either plant type.
bacterial increase of metal bioavailability to plants. suggesting that this plant is a hyperaccumulator of Cd. doubling every 2 to 3 days under optimal conditions (28). This plant may be a good choice for remediation projects because. Coler and Gunner observed a rhizosphere effect in the aquatic environment. the amount of metal taken up by the plant compared to the amount in the outside solution (52). due to its rapid growth rate and ease of harvest. Consequently. there have not been studies on the effects of rhizosphere or rhizoplane (root surface) bacteria on metal uptake in plants. which binds the metal. 52). This investigation was based on the hypothesis that plant metal uptake is influenced by the microbial communities associated with aquatic plants. has been well studied for its ability to remove metals from surface waters. This study is the first to address microbial communities associated with L. minor for removal of metals from aquatic environments. especially in relation to heavy metal uptake or sensitivity. a small aquatic floating angiosperm. minor to remove Cd from surface waters for phytoremediation (10. several studies of Lemna and associated bacteria were conducted. keeping the metal from continuous reintroduction into the ecosystem (10).bioconcentration factor. Several possible mechanisms for how bacteria might increase plant metal tolerance and uptake include bacterial stimulation of the production of metal transport proteins in plants. since the bioavailability of metals could be altered by bacterial expression of resistance systems (47). In aquatic systems. Lemna minor. and increase in root biomass by bacteria (12).000 mg/kg Cd (48. Despite the increased interest in L. it can serve as a harvestable accumulator. 18). These included microscopic observations and enumeration of bacteria on plant surfaces as well as several culture-dependent studies (for review. Metal-tolerant bacteria associated with Brassica juncea increased heavy metal uptake in the plants. Others have related the basis of this plant-microbe interaction to bacterial metal resistance. From the 1960s to the 1980s. we have used 16S rRNA analysis to describe bacterial communities associated with the roots of Lemna minor plants from an environment with low- . Several studies of phytoremediation in terrestrial systems have shown that rhizosphere bacteria may contribute to plant metaltolerance and increase metal uptake. L. minor in the presence of a heavy metal. there have been few recent studies of microbial communities associated with the plant. see reference 27). minor has been shown to accumulate as much as 1. showing that microbial activity around the roots ofLemna minor was greater than that around inert surfaces (8). possibly due to increased root biomass stimulated by bacteria (37). it is important to address the influence of Cd exposure on the indigenous bacterial communities of plants from aquatic environments. In this study. perhaps making the microenvironment around the plant less toxic (13). Because plantassociated microbial communities have previously been shown to be involved in controlling plant uptake of metals from their surrounding environment. While the definitions of the rhizosphere may differ from terrestrial to aquatic systems due to high diffusion rates of root exudates in water. Some bacteria expressing Cd resistance produce more extracellular polymeric substances. This plant rarely flowers in nature and most often grows clonally.300 times more Cd than concentrations present in the surrounding water or as much as 14. a number of studies in the past two decades have focused on the ability of L.
St.25 mM each deoxynucleoside triphosphate. with an initial denaturation at 95°C for 5 min and a final extension at 72°C for 5 min (31). CA). personal communication). MO). PCR amplifications were prepared with 1x buffer. Valencia. WI) followed by PCR with primers F71 (22) and R622 (31). we identified plants isolated from RCP as L.5 mM MgCl2 (Sigma. obtained from the U. and directly sequenced. minor used by the U. Madison. 16 h light. Plant identification Because the genus Lemna has few distinguishing characteristics. and 72°C for 45 seconds. OH (EPA). contamination originates mostly from urban runoff (41). Coralville. Frederick. 1 U/10 µl Taq polymerase (all from Promega. 0. 0.S.level surface water pollution as well as from the sensitive toxicity test strain of L. quantified by comparison to PCR ladder V (PGC Scientifics. Foster City. IA). Sediments from this site have Cd concentrations in the mg/kg range and variable surface water concentrations in the range of 1 to 100 µg/liter (30. Waltham. Environmental Protection Agency. MATERIALS AND METHODS Plant collections and culture. Massachusetts. Plants were kept in culture with water from Rice City Pond. Both plant types were grown under constant conditions (20°C. CA). was grown in culture containing autoclaved distilled water amended with a nutrient solution defined by the American Society for Testing and Materials (here termed EPA water) (2). PCR products were purified with the QIAquick PCR purification kit (QIAGEN. Samples of Lemna minor were collected from Rice City Pond (RCP).5 µM each primer (Integrated DNA Technologies. Upland. Dye terminator cycle sequencing reactions were prepared according to manufacturer's instructions with the primer F71 and were . Environmental Protection Agency. 1. WI). today. We compared bacterial communities based on types of plants or Cd concentrations in water and also determined how bacterial communities on the plant surfaces change when plants are exposed to Cd. MA) andconsisted of 35 cycles of 95°C for 45 seconds. Louis. 8 h dark). Lanza. CA). an impoundment of the Blackstone River near Uxbridge. 52°C for 45 seconds. Historically. minor by molecular analysis of the chloroplast rpl16 intron region (22. and 40 ng DNA template in a final volume of 30 µl. Madison. 41. DNA was extracted from plants using the Wizard Genomic DNA purification kit (Promega. Sequencing reactions were prepared using the ABI Big Dye 3. G. In addition. a Cd-sensitive toxicity test strain of Lemna minor. one marker that has been previously used for plant classification. Reactions were performed in a PTC-200 Peltier thermal cycler (MJ Research.1 kit (Applied Biosystems. 31). Cincinnati.S. mill wastes contributed to the heavy metal pollution at this site. MD) using a digital imaging system with LabWorks software (UVP.
Total DNA extraction. (31) and were aligned with those in the database using the Clustal interface in the software package BioEdit (17). MgCl2. CA). pGEMF (5'-GCAAGGCGATTAAGTTGGG-3') and pGEMR (5'-ATGACCATGATTACGCCAAG-3').5%) to facilitate cell lysis. and 72°C for 30 seconds with an initial denaturation at 95°C for 5 min and a final extension at 72°C for 5 min. Ligation into the vector at an insert to vector ratio of 1:1 and transformation of E. One hundred positive clones were chosen randomly for community analysis. Amplification used 30 cycles of 95°C for 30 seconds. After 14-day treatments. (3). Madison. DNA extraction was followed by PCR using bacterium-specific primers 338F (1) (the complement of EUB338) and 1492R (29) (E. Sequences were compared to those previously submitted to GenBank by Les et al. . Plants were rinsed in sterile distilled water and roots were separated from fronds. The protocol for mini preparation of genomic DNA from bacteria followed that from the work of Ausubel et al. EPA plants were grown in EPA water and RCP plants were grown in RCP water. three separate reactions were run for each sample and then pooled together before DNA quantification. and DNA template in 30-µl volumes at the same concentrations described above. Experimental setup. Products inserted randomly into the vector in a forward (sequence beginning with 338F) or reverse (sequence beginning with 1492R) direction relative to the forward vector primer site. Taq polymerase. 56°C for 30 seconds. 18). coli JM109 cells followed manufacturer's instructions. Experiments were run for 14 days because previous studies showed that longer periods of incubation may lead to metal desorption from plant tissue (10. PCR amplification and cloning of 16S rRNA genes. colinumbering). Cloning of mixed community 16S rRNA gene fragments was performed using the pGEM T-easy vector (Promega. One hundred µg/liter was chosen as a test concentration based on literature reports and our previous test series that showed this as the highest sublethal concentration of Cd to both plant types over two weeks (21. Roots were removed from buffer and cell material was collected by centrifugation. We designed PCR primers specific for the vector sites flanking the insert. WI). Foster City. and RNase A (25 µg/ml) was added prior to proteinase K addition to remove any coextracted RNA. EPA plants and RCP plants were used for 14-day treatment experiments and were grown either with or without 100 µg/liter CdSO4.sequenced with a model 3730xl DNA Analyzer (Applied Biosystems. with the following modifications: lysozyme (300 µg/ml) was added followed by sodium dodecyl sulfate (0. Reaction mixtures were prepared with PCR buffer. about 20 plant roots per sample were harvested. BioEdit was also used to create similarity matrices for Lemnaceae sequences and phylogenetic trees were created with the software MEGA 2 (26). Roots from each sample were pooled in 0. to amplify 16S rRNA gene products from the vector of individual clone colonies. primers. deoxynucleoside triphosphates.1 M potassium phosphate buffer at pH 7 and were shaken for 5 min to remove bacteria from the root surface. In order to minimize PCR bias in subsequent cloning steps. data not shown). Quantification of PCR products was performed as previously described.
LIBSHUFF was used to determine whether clone libraries from plants of various metal treatments (with and without Cd) were significantly different (P < 0. both take richness and evenness into account (32). Phylogenetic trees were created with MEGA 2. AY707525 to AY707602. Further analyses of libraries at the phylotype level included nucleotide diversity and using the software package Arlequin (39). or relative abundance patterns. is the average sequence divergence or an estimate of the total genetic variation in a sample. coverage estimators that determine the number of probablephylotypes in the environment compared to the numbers observed. of the community based on sequences were created (35). the number of phylotypes. genus. . WI) and were searched against the Ribosomal Database Project (RDP II) (7) to determine nearest matches. Possible chimeras were detected using CHIMERA_CHECK in the RDP II and also with the software Bellerophon (20). PCR products from clone libraries were sequenced as previously described using the forward vector primer pGEMF. RESULTS Plant identification. The Shannon-Weiner index [H = . Sequences were divided into two groups. and phylotype levels. coli numbering) were deposited in GenBank with the accession numbers AY707525-AY707602 (forward sequences) and AY803199-AY803235 (reverse sequences). Diversity profiles.05) from one another by comparing coverage curves of libraries to each other first in an X/Y fashion and then the reverse. Dividing over the length of sequences equals the average nucleotide diversity. Simpson's reciprocal index (1/D). Partial sequences of 600 bp in length.(pi) (ln pi)] and Simpson's reciprocal index. Y/X (40).Rarefaction curves were created using the software aRarefactWin (19) to determine whether environments were exhaustively sampled. The Shannon-Weiner index(H). and AY803199to AY803235. 1/D. Nucleotide sequence accession numbers. richness (S). Diversity indices and statistics. the probability that randomly chosen bases differ at a given position (33). and the distribution of phylotype abundance were calculated at the division. Sequences were first edited manually using the program SeqEd (Genetics Computer Group. based on whether orientation in the vector was in the forward or reverse direction. evenness (EH = H/ln S). BioEdit was used to create similarity matrices for all sequences and to determine phylotypes (based on 97% similarity). Madison. Multiple sequence alignments were created using ClustalX (43) or the Clustal interface within the software package BioEdit.Sequencing and phylogenetic analysis. Sequences were submitted to GenBank with the accession numbers AY714543 to AY714544. from positions 338 to 940 or 940 to 1492 (E. where D equals (pi)2 and where pi is the proportion of phylotypes (or groups) i relative to the total number of phylotypes.The software EstimateS 6 was used to calculate SACE and SChao1 (9).
was L.000 bootstrap replicates. 2) down to levels of genera (Fig. FIG. 1). Results were found to be similar. and only the results of forward analyses are shown. Results from forward and reverse analyses were combined where possible. Abundance of clones from bacterial divisions present in clone libraries of (A) EPA plants (black bars) or EPA plants plus Cd (gray bars). genetic diversity. forward and reverse data sets were both analyzed separately. 2. Where results were not combined (LIBSHUFF analysis. and phylogenetic tree construction) (Fig. and phylotypes. The scale bar indicates 0. Based on the region analyzed. 4).02 nucleotide substitution. EPA plants and RCP plants group with the Lemna minor rpl16 gene. EPA plants are identical to the strain in the database and RCP plants are slightly less closely related but still within the Lemna minor cluster. and RCP plants in Cd. EPA plants in Cd. FIG. The number of clones used for each clone library analysis is indicated. with similarity >98%. Bacterial communities were compared at the level of bacterial divisions (Fig. View larger version (19K): .Phylogenetic analysis of the rpl16 chloroplast intron region of EPA and RCP plants revealed that RCP plants and EPA plants clustered well with Lemna minor (Fig. Phylogenetic analysis of the rpl16 chloroplast intron region was used for identification of members of the family Lemnaceae. The rpl16 region fromPistia stratiotes was used as an outgroup. 3a to d). View larger version (27K): Bacterial community analysis. RCP clone libraries are shown in panel B for RCP plants (black bars) or RCP plants plus Cd (gray bars). Phylogenetic identification of the plants used in this study. The phylogenetic tree was constructed using neighbor-joining analysis with 1. 1. Based on a similarity matrix of all Lemnaceae rpl16 sequences used in the construction of the phylogenetic tree. RCP plants. while other species of Lemna form separate clusters. 16S rRNA gene analysis was completed for clone libraries of bacteria from roots of EPA plants. the nearest match for both EPA and RCP plants. Bootstrap values are indicated at nodes. minor. Eight putative chimeras out of 365 sequences were detected and removed from the analysis.
there was a decrease of Leptothrix to 23%. Genera listed in bold are dominant groups within each library. but in the presence of Cd. Phylogenetic relationships of bacteria detected on EPA plants (EPA libraries A and B. (C) RCP plants in RCP water. 3). at the same percentages (Table 1). 3. The Shannon and Simpson's reciprocal indices showed an increase in diversity for Cd-amended libraries over unamended libraries for both RCP and EPA plants. Genera are assigned based on closest RDP II matches. Staphylococcus aureus is included as an outgroup for each tree. View larger version (34K): Bacterial diversity at the division level. the relative . The scale bar represents nucleotide substitutions. Relative abundance of genera within the dominant group. left) and on RCP plants (RCP libraries C and D. Numbers in parentheses represent the number of clones represented by each phylotype ( 97% similarity). (B) EPA plants in EPA water amended with Cd. and phylotype level Bacterial diversity at the genus level. For each tree. ß-proteobacteria. in plant-associated communities. represents no Cd added and represents the addition of Cd. with cyanobacteria. Also. Trees were constructed using the neighbor-joining algorithm with 1. Shannon-Weiner and Simpson's reciprocal indices calculated at the division level showed that shifts in diversity between Cd treatments were rather small. View larger version (50K): FIG. an increase in diversity was observed with Cd addition in both the EPA and RCP clone libraries. While Sterolibacterium decreased to only 19% of the total community. Percentages are based on the number of clones (n) of ß-proteobacteria in each library. The dominant bacterial division in all libraries was the ß-proteobacteria. and (D) RCP plants in RCP water amended with Cd. The influence of cadmium addition is shown for (A) EPA plants in EPA water. In the case of RCP plants. less than 10% for the Shannon index and about 15% for the Simpson'sindex (Table 1). -proteobacteria and sphingobacteria also represented in all libraries. -proteobacteria. genus level (within ß-proteobacteria). There was also an increase in the abundance of ß-proteobacteria that matched most closely with unclassified environmental clones. Diversity indices for clone libraries at the division level. When comparing genera within the ß-proteobacteria. or abundance distribution of genera. mostly belonging to the order Burkholderiales (Fig. there was a shift from clear dominance by Sterolibacterium (70%) in the unamended library to a more even community distribution in the presence of Cd. 4. For the EPA plants. right) inferred from 16S rRNA sequence analysis. TABLE 1.Hydrogenophaga rose to 27% and Acidovorax appeared at 12%. Cd addition also caused an increase in evenness. Leptothrix clearly dominated at 73% in the unamended library.FIG.000 bootstrap replicates.
Coverage estimators suggest that 45 to 75% of the diversity present in our samples was covered. In EPA libraries. with the highest level of coverage occurring in the EPA plus Cd library (Table 3). Coverage estimators provide an estimate of the number of phylotypes expected in the sample source and allow us to determine how well the sample represents that environment.205). Shannon and Simpson's reciprocal indices also showed an increase in bacterial diversity from unamended to Cd-amended libraries. Phylotypes were determined by 97% similarity.abundance of Rhodoferax more than tripled from 10% without Cd to 36% with Cd. When coverage curves of EPA plants grown without Cd were compared to EPA plants plus Cd (X/Y). there was a significant difference (P = 0. At the phylotype level. Rarefaction curves for clone libraries from EPA ( ). TABLE 2. but the appearance of Zoogloea added Rhodocyclales to the RCP plus Cd library (Table 2). rarefaction curves began to level off only asymptotically. meaning that the full level of sampling effort had not been reached (Fig. Rarefaction curves were used to determine whether the level of sampling effort was sufficient to represent the possible number of phylotypes in samples. EPA plus Cd ( ). but the relative increase was smaller than that seen with other diversity indices (Table 1).Rhodocyclales were absent from the RCP without Cd library. determined by asymptotic leveling off of curves. 5). when EPA plants plus Cd were compared to EPA plants without Cd (Y/X). FIG. indicating that the RCP and RCP plus Cd libraries have fewer common genera than the EPA and EPA plus Cd libraries. however. while Zoogloea appeared only in the RCP plus Cd library at 20% (Fig. LIBSHUFF analysis showed that RCP plants without Cd compared to RCP plants with Cd had significantly different bacterial communities in both X/Y and Y/X comparisons (P = 0. The EPA without Cd library contains most sequences found in the EPA plus Cd library. Nucleotide diversity and increased following the addition of Cd. but the EPA plus Cd library contains new sequences not found in the EPA without Cd library. RCP ( ). In RCP libraries members of the order Burkholderiales dominated libraries plus or without Cd. Relative abundance of orders of bacteria within the ß-proteobacteria Bacterial diversity at the phylotype level. the shift from dominance by Sterolibacterium caused a sharp decrease in the order Rhodocyclales and an increase in the order Burkholderiales upon Cd addition. 3). For all samples.001). and RCP plus Cd (•). the difference was not significant (P = 0.001). 5. View larger version (15K): .
such as environment. such as the dominance ofSterolibacterium in EPA libraries and Leptothrix in RCP libraries. These parameters showed changes above 30% between EPA and RCP plant groups. Estimators of phylotype abundance in clone libraries In order to exhaustively sample and fully cover all of the diversity from aquatic and other environments. Other factors besidesgenetic variation may influence plant-associated bacterial communities. . parallel shifts in diversity were observed from unamended to Cd-amended libraries for EPA and RCP. we chose to focus on the effect of additional Cd. were made with plants from the same species. minor. The difference in diversity scores between EPA and RCP plants was in most cases less than half of the change in diversity observed between unamended and Cd-amended treatments within the same plant groups (Table 1).TABLE 3. valid comparisons can be made (23). nucleotide diversity and . extremely large (>1. biased estimates of phylotype richness if libraries are too small. which for many studies is not feasible.html (24). while distinct differences between genera from EPA or RCP were evident. if estimates are stable and unbiased. as the effects of placing the plants in new types of water might result in additional stress to the plants and associated microbes. Because the two types of plants in our study had been originally cultured in different types of water. As at the genus level. these scores showed the same percentage increase between unamended and Cd-amended libraries for both EPA and RCP plants (Table 1). Shannon and Simpson's index scores showed that there was littledifference in diversity between EPA and RCP libraries.org/lomethods/free/2004/0114a. The exceptions were the genetic diversity measures. We determined that all of our libraries were most likely large enough to produce stable coverage estimates by using a program available athttp://www. Our comparisons. however. Analysis at the phylotype level showed that the RCP libraries had less diverse microbial communities than their EPA counterparts.000 clones) libraries may need to be constructed. causing different growth patterns. or RCP plants grown in RCP water) with the addition of Cd as the only added stress to either system. we determined that the field isolate of Lemna from Rice City Pond was part of the species L.aslo. DISCUSSION Based on chloroplast intron analyses. therefore. Coverage estimators can give unstable. Data shown here compared plantsin their own types of water (EPA plants grown in EPA water. Within the ß-proteobacteria. there weresome noteworthy patterns that appeared when looking across plant ecotypes (EPA compared to RCP). Interestingly. although there is slight genetic variation between the field isolate and other strains. While the main focus of this study was to study shifts in plantassociated bacterial communities with and without added Cd. Comparisons between plant groups (EPA versus RCP). The type of water in which the plants are grown may provide different nutrients to plants.
Several genera within the ß-proteobacteria are known for high tolerance to heavy metals (47). as opposed to plant type. 16). when Cd was added. found in the RCP libraries. 44). The dominant member of the EPA library was most closely related to Sterolibacterium denitrificans. As the EPA library was the least exposed to Cd. comparisons between Cd-amended and unamended libraries within the same plant type showed very similar diversityindex scores. The Burkholderiales. a low-level contamination concentration. we saw that upon addition of 100 µg/liter CdSO4. Community shifts between unamended and Cd-amended libraries for both EPA and RCP plants followed the same proportional changes. We may expect that at higher concentrations of Cd. This could be tested with a range of Cd concentrations. and adding moderate stress may decrease competitive exclusion. RCP. Sterolibacterium. the genus Zoogloea appeared in the clone library. communities will contain the most species (14. dominated the EPA plus Cd. At the division level. there was an increase in diversity at finer phylogenetic levels after two weeks. we may expect that there are fewer Cd-tolerant bacteria present here. This adsorption could keep levels of Cd entering the plant at lower. and RCP plus Cd libraries. In RCP plants.The intermediate disturbance hypothesis states that when a disturbance (such as Cd addition) is neither too high nor too low. we will see an increase in the number of metal-tolerant bacteria. Zoogloea. Giller et al. it may offer an explanation for the increases in diversity upon the addition of heavy metal. While there are no reports of Cd tolerance in Rhodoferax. these Burkholderiales genera did increase in relative abundance upon Cd addition. and there was less evenness in the RCP libraries due to higher dominance of ß-proteobacteria. or Methylophilus. the order within the ß-proteobacteria containing known metal-resistant bacteria such as Ralstonia (7. In our study. While the intermediate disturbance hypothesis has been applied to few microbial systems (11). (15) describe how stable environments may contain high numbers of competitive species. As stress increases. at which point diversity will decrease. showing that while the genera within communities of the two plant types were different. Leptothrix. there will be an increase of species until the stress becomes too great. while the EPA library was dominated by the Rhodocyclales. Diversity scores were lower for RCP plants than EPA plants. the addition of Cd caused a parallel shift in the overall diversity of genera detected. and larger changes were observed when comparing between plant types than between Cd treatments for the same plant type. while not of the order Burkholderiales but of the order Rhodocyclales. Hydrogenophaga. the largest changes in diversity were related to Cd addition. Diversity index calculations for genera within the ß-proteobacteria showed that at this level. This indicated that Cd had little influence on community diversity at the division level. for instance) will survive. less toxic concentrations. is also known for its ability to bind heavy metals in its polysaccharide matrix and has been proposed as a bioremediation agent itself (25). Other members of this genus that cluster well with those found in the EPA samples include uncultured clones found in biofilms on . and its close relative Sphaerotilus are able to adsorb heavy metal cations to their outer sheaths (36). Organisms that are adapted to the stress (tolerance to higher concentrations of Cd. which is a cholesteroloxidizingbacterium (42).
M. while showing similar results to those seen at the genus level. Soc.oxygen transfer membranes.org). Investigating the response of the microbial community to heavy metal amendments will lead to a better understanding of the major factors that link microbial activity to the metal toxicity and uptake behavior of plants. Despite biases associated with molecular studies (51). . also allowed comparisons of genetic diversity and comparison of libraries using LIBSHUFF. Future isolation of bacteria from Lemna roots will allow us to characterize directly metal tolerance or resistance of plant-associated bacteria (L. Anand Mohan for helpful discussions and we also grateful to American Society for Mirobiology (asm. it is apparent that despite compositional differences. this is one of the first studies describing bacterial communities associated with aquatic plants. Cd causes the communities to shift similarly. Coupled with diversity index data. Leptothrix was dominant in the RCP libraries and has previously been reported in waters of Lemna-covered ponds (27). this is the first detailed molecular study of the bacterial communities associated with Lemna minor and the first report to show Lemna community changes in response to the addition of a heavy metal. Combining molecular analyses with culturing methods will allow for a deeper understanding of community composition and function. Genetic diversity parameters revealed that libraries within plant types with or without Cd were closely related. Nüsslein. M. T. as opposed to EPA plants. 2003) and their potential effects on plant metal uptake or plant metaltolerance. or in drinking water systems (6. 103rd Gen. Molecular analyses give more description of the diversity of a particular environment since culture-based studies may detect only a small fraction of the bacteria present.. It is reasonable to expect Sterolibacterium to be associated with plant roots since certain root exudates are sterol compounds. Because changes in diversity with Cd addition were the same for either plant type. such as sequestration. 50). Rothfeder. In relation to plants and heavy metals in general. To our knowledge. while those from different plant types were dissimilar. and K. which have been grown in EPA water without any Cd input. Phylotype analyses. Meet. these currently offer the most complete picture of community composition and thus are an ideal starting point for community studies. efflux pump mechanisms. abstr. ACKNOWLEDGMENTS We gratefully acknowledge Mr. Abstr. Studies of Cd resistance systems. RCP plants are exposed to low concentrations of Cd both at the site and in laboratory cultures when grown with RCP water. Microbiol. N276. or adsorption to extracellular polymericsubstances may elucidate how the plant-microbe interaction might support plant heavy metal tolerance. Am. Because aquatic plants may be used in phytoremediation projects. knowledge of what types of bacteria are present and whether these bacteria may facilitate metal uptake will be important for controlling and improvingphytoremediation efficiency. Stout. history of preexposure may be independent from the observed disturbance-induced diversity shifts.
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