Towards practical cadmium phytoextraction with Noccaea caerulescens.

A series of field trials were conducted to investigate the potential of Noccaea caerulescens F.K. Mey [syn. Thlaspi caerulescens J &C Presl. (see Koch and Al-Shehbaz 2004)] populations (genotypes) derived from southern France to phytoextract localized Cd/Zn contamination in Thailand. Soil treatments included pH variation and fertilization level and application of fungicide. N. caerulescens populations were transplanted to the field plots three months after germination and harvested in May, prior to the onset of seasonal rains. During this period growth was rapid with shoot biomass ranging from 0.93-2.2 g plant(-1) (280-650 kg ha(-1)) DW. Shoot Cd and Zn concentrations for the four populations evaluated ranged from 460-600 and 2600-2900 mg kg(-1) DW respectively. Cadmium and Zn Translocation Factors (shoot/root) for the populations tested ranged from 0.91-1.0 and 1.7-2.1 and Bioaccumulation Factors ranged from 12-15 and 1.2-1.3. We conclude that optimizing the use of fungicidal sprays, acidic soil pH, planting density and increasing the effective cropping period will increase rates of Cd and Zn removal enough to facilitate practical Cd phytoextraction from rice paddy soils in Thailand.

Heavy metal resistance and genotypic analysis of metal resistance genes in gram-positive and gram-negative bacteria present in Ni-rich serpentine soil and in the rhizosphere of Alyssum murale.

Forty-six bacterial cultures, including one culture collection strain, thirty from the rhizosphere of Alyssum murale and fifteen from Ni-rich soil, were tested for their ability to tolerate arsenate, cadmium, chromium, zinc, mercury, lead, cobalt, copper, and nickel in their growth medium. The resistance patterns, expressed as minimum inhibitory concentrations, for all cultures to the nine different metal ions were surveyed by using the agar dilution method. A large number of the cultures were resistant to Ni (100%), Pb (100%), Zn (100%), Cu (98%), and Co (93%). However, 82, 71, 58 and 47% were sensitive to As, Hg, Cd and Cr(VI), respectively. All cultures had multiple metal-resistant, with heptametal resistance as the major pattern (28.8%). Five of the cultures (about of 11.2% of the total), specifically Arthrobacter rhombi AY509239, Clavibacter xyli AY509235, Microbacterium arabinogalactanolyticum AY509226, Rhizobium mongolense AY509209 and Variovorax paradoxus AY512828 were tolerant to nine different metals. The polymerase chain reaction in combination with DNA sequence analysis was used to investigate the genetic mechanism responsible for the metal resistance in some of these gram-positive and gram-negative bacteria that were, highly resistant to Hg, Zn, Cr and Ni. The czc, chr, ncc and mer genes that are responsible for resistance to Zn, Cr, Ni and Hg, respectively, were shown to be present in these bacteria by using PCR. In the case of, M. arabinogalactanolyticum AY509226 these genes were shown to have high homology to the czcD, chrB, nccA, and mer genes of Ralstonia metallidurans CH34. Therefore, Hg, Zn, Cr and Ni resistance genes are widely distributed in both gram-positive and gram-negative isolates obtained from A. murale rhizosphere and Ni-rich soils.

Chromate-tolerant bacteria for enhanced metal uptake by Eichhornia crassipes (Mart.).

A total of 85 chromate-resistant bacteria were isolated from the rhizosphere of water hyacinth grown in Mariout Lake, Egypt, as well as the sediment and water of this habitat. Only 4 (11%), 2 (8%), and 2 (8%) of isolates from each of the environments, respectively, were able to tolerate 200 mg Cr (VI) L(-1). When these eight isolates were tested for their ability to tolerate other metals or to reduce chromate, they were shown to also be resistant to Zn, Mn, and Pb, and to display different degrees of chromate reduction (28% to 95%) under aerobic conditions. The isolates with the higher chromate reduction rates from 42% to 95%, (RA1, RA2, RA3, RA5, RA7, and RA8) were genetically diverse according to RAPD analysis using four differentprimers. Bacterial isolates RA1, RA2, RA3, RAS, and RA8 had 16 S rRNA gene sequences that were most similar to Pseudomonas diminuta, Brevundimonas diminuta, Nitrobacteria irancium, Ochrobactrum anthropi, and Bacillus cereus, respectively. Water hyacinth inoculated with RA5 and RA8 increased Mn accumulation in roots by 2.4- and 1.2-fold, respectively, compared to uninoculated controls. The highest concentrations of Cr (0.4 g kg(-1)) and Zn (0.18 g kg(-1)) were accumulated in aerial portions of water hyacinth inoculated with RA3. Plants inoculated with RA1, RA2, RA3, RA5, RA7, and RA8 had 7-, 11-, 24-, 29-, 35-, and 21-fold, respectively, higher Cr concentrations in roots compared to the control. These bacterial isolates are potential candidates in phytoremediation for chromium removal.

Phenotypic characterization of microbes in the rhizosphere of Alyssum murale.

Metal hyperaccumulator plants like Alyssum murale are used for phytoremediation of Ni contaminated soils. Soil microorganisms are known to play an important role in nutrient acquisition for plants, however, little is known about the rhizosphere microorganisms of hyperaccumulators. Fresh and dry weight, and Ni and Fe concentrations in plant shoots were higher when A. murale was grown in non-sterilized compared to sterilized soils. The analysis of microbial populations in the rhizosphere of A. murale and in bulk soils demonstrated that microbial numbers were affected by the presence of the plant. Significantly higher numbers of culturable actinomycetes, bacteria and fungi were found in the rhizosphere compared to bulk soil. A higher percent of Ni-resistant bacteria were also found in the rhizosphere compared to bulk soil. Percentage of acid producing bacteria was higher among the rhizosphere isolates compared to isolates from bulk soil. However, proportions of siderophore producing and phosphate solubilizing bacteria were not affected by the presence of the plant. We hypothesize that microbes in the rhizosphere of A. murale were capable of reducing soil pH leading to an increase in metal uptake by this hyperaccumulator.

Influence of the zinc hyperaccumulator Thlaspi caerulescens J. & C. Presl. and the nonmetal accumulator Trifolium pratense L. on soil microbial populations.

Metal hyperaccumulator plants like Thlaspi caerulescens J. & C. Presl. are used for phytoremediation of contaminated soils. Since little is known about the rhizosphere of hyperaccumulators, the influence of T. caerulescens was compared with the effects of Trifolium pratense L. on soil microbes. High- and low-metal soils were collected near a zinc smelter in Palmerton, Penn. Soil pH was adjusted to 5.8 and 6.8 by the addition of Ca(OH)2. Liming increased bacterial populations and decreased metal toxicity to levels allowing growth of both plants. The effects of the plants on total (culturable) bacteria, total fungi, as well as cadmium- and zinc-resistant populations were assessed in nonrhizosphere and rhizosphere soil. Both plants increased microbial populations in rhizosphere soil compared with nonrhizosphere soil. Microbial populations were higher in soils planted with T. pratense, but higher ratios of metal-resistant bacteria were found in the presence of T. caerulescens. We hypothesize that T. caerutescens acidifies its rhizosphere. Soil acidification in the rhizosphere of T. caerulescens would affect metal uptake by increasing available metals around the roots and consequently, increase the selection for metal-resistant bacteria. Soil acidification may be part of the hyperaccumulation process enhancing metal uptake from soil.

Intact soil-core microcosms compared with multi-site field releases for pre-release testing of microbes in diverse soils and climates.

Intact soil-core microcosms were used to compare persistence of Pseudomonas chlororaphis 3732RN-L11 in fallow soil and on wheat roots with field releases at diverse sites. Parallel field and microcosm releases at four sites in 1996 were repeated with addition of one site in 1997. Microcosms were obtained fresh and maintained at 60% soil water holding capacity in a growth chamber at 70% relative humidity, a 12-hour photoperiod, and constant temperature. Persistence of 3732RN-L11 was measured at each site in field plots and microcosms at 7-21 day intervals, and in duplicate microcosms sampled at an independent laboratory. Linear regression slopes of field plot and microcosm persistence were compared for each site, and between identical microcosms sampled at different sites, using log10 transformed plate counts. Microcosm persistence closely matched field plots for wheat roots, but persistence in fallow soil differed significantly in several instances where persistence in field plots was lower than in microcosms. Analysis of weather variations at each site indicated that rainfall events of 30-40 mm caused decreased persistence in fallow soil. Cooler temperatures enhanced persistence in field plots at later time points. Inter-laboratory comparison of regression slopes showed good agreement for data generated at different sites, though in two instances, longer sampling periods at one site caused significant differences between the sites. Soil characteristics were compared and it was found that fertility, namely the carbon to nitrogen ratio, and the presence of expanding clays, were related to persistence. These microcosm protocols produced reliable data at low cost, and were useable for pre-release risk analyses for microorganisms.

Phytoremediation of soil metals.

The phytoremediation of metal-contaminated soils offers a low-cost method for soil remediation and some extracted metals may be recycled for value. Both the phytoextraction of metals and the phytovolatilization of Se or Hg by plants offer great promise for commercial development. Natural metal hyperaccumulator phenotype is much more important than high-yield ability when using plants to remove metals from contaminated soils. The hypertolerance of metals is the key plant characteristic required for hyperaccumulation; vacuolar compartmentalization appears to be the source of hypertolerance of natural hyperaccumulator plants. Alternatively, soil Pb and Cr6+ may be inactivated in the soil by plants and soil amendments (phytostabilization). Little molecular understanding of plant activities critical to phytoremediation has been achieved, but recent progress in characterizing Fe, Cd and Zn uptake by Arabidopsis and yeast mutants indicates strategies for developing transgenic improved phytoremediation cultivars for commercial use.

Validation of Microcosms for Examining the Survival of Pseudomonas aureofaciens (lacZY) in Soil.

Evaluating the safety and efficacy of a recombinant bacterium prior to its release into the terrestrial environment requires that risk assessment data be collected in the laboratory. Much of this information is obtained with the use of microcosms. The design of the microcosm significantly affects the ability of the recombinant microorganism to survive in soil and, thus, complicates the risk assessment process. To standardize microcosms for future use, we evaluated the survival of Pseudomonas aureofaciens 3732 RN-L11 (lacZY Rif(supr) Nal(supr)) in intact soil cores (5.0 by 15 cm; polyvinyl chloride core) and disturbed soil microcosms (50 g of fresh, sieved soil). Survival data were compared with those obtained during a field release. The intact soil core microcosm was shown to closely simulate results obtained in the field. The intact soil core microcosm closely predicts survival in bulk soil and in the rhizosphere of wheat. Data obtained with the microcosm were also similar when evaluated in separate studies in two different years. In 1993, P. aureofaciens survived for approximately 63 days in bulk soil and 96 days in the rhizosphere. The disturbed soil microcosm exhibited a much more rapid decline in population size (34 days to zero) than the intact core microcosm. We speculate that drying and sieving of soil for the disturbed soil microcosm affected the ability of the soil to support the survival of P. aureofaciens. These results demonstrate that a small, inexpensive, and simple intact soil core microcosm may be appropriate for risk assessment.

Pseudomonas aureofaciens in soil: survival and recovery efficiency.

Efficient methods for the recovery of genetically engineered organisms (GEM) added to soil are critical if the safety of potential releases is to be evaluated and the actual release is to be monitored. Pseudomonas aureofaciens strain 3732 RN-L11 (lacZY) was added to 10 g sieved soil microcosms and incubated for 5 and 28 days. Various diluents, shaking methods, and settling of soil were examined to determine the optimum method for recovery of the GEM from the soil. Of the diluents examined, 0.1% agar gave significantly lower numbers than distilled water, 1.0% sodium metaphosphate, 1% peptone, and phosphate-buffered water. After 5 days of incubation, shaking for 10 min with glass beads and shaking for 30 min without glass beads resulted in the highest recovery of the GEM from soil, while sonification resulted in the lowest recovery. After 28 days of incubation, sonification produced significantly lower numbers than any of the other treatments. The addition of 1% CaCl2 to enhance settling significantly increased recovery efficiency. Although the use of CaCl2 in distilled water and shaking for 10 min was an effective method for recovering P. aureofaciens from a Maryland soil, when the same extraction procedure was compared with a standard technique (dd H2O, shaking for 10 min) for eight divergent soils, neither extraction method was consistently better than the other. Statistical analysis of the data showed the need for log transformation of the raw data. Four microcosm and two plate replicates for each dilution provided the greatest ability to detect differences between treatment means while maximizing experimental efficiency.

Genotypic Diversity among Strains of Bradyrhizobium japonicum Belonging to Serogroup 110.

Thirty-three strains of Bradyrhizobium japonicum within serogroup 110 were examined for genotypic diversity by using DNA-DNA hybridization analyses. The analysis of the DNA from 15 hydrogen-uptake-negative strains with the bradyrhizobial uptake hydrogenase probe pHU52 showed variation in degree of homology and restriction fragment length polymorphism of EcoRI-restricted DNA. Clustering analysis of the 33 strains on the basis of DNA-DNA hybridization analysis with four restriction enzymes and with the bradyrhizobial nodulation locus, pRJUT10, as probe indicated the existence of four groups of strains, which were less than 70% similar. Restriction digestion of genomic DNA with BamHI and DNA-DNA hybridization with pRJUT10 permitted classification of each of the strains according to a specific fingerprint pattern.

In vivo transfer of pR68.45 from Pseudomonas aeruginosa into indigenous soil bacteria.

The release of genetically engineered organisms (GEMs) into the environment could result in novel gene sequences becoming transferred to, and established in, the indigenous soil biota. The potential for recombination in nonsterile soil is difficult to determine due to problems isolating transconjugants of indigenous microbes, while concurrently suppressing introduced donors. We have developed a system that allows us to detect the transfer of the plasmid R68.45 from Pseudomonas aeruginosa strain PA025 into the indigenous soil bacterial population. Transconjugants were selected by plating on minimal media containing antibiotics and were verified by DNA-DNA hybridization. The observed maximum transfer frequency was approximately 10(-6). Fatty acid analysis of transconjugants showed that intergeneric transfer was occurring between the introduced organism and genetically dissimilar species.

New culture medium containing ionic concentrations of nutrients similar to concentrations found in the soil solution.

A new growth medium which closely approximates the composition of the soil solution is presented. This soil solution equivalent (SSE) medium contains the following components (millimolar): NO(3), 2.5; NH(4), 2.5; HPO(4), 0.005; Na, 2.5; Ca, 4.0; Mg, 2.0; K, 0.503; Cl, 4.0; SO(4), 5.0; ethylenediamine-di(o-hydroxyphenylacetic acid), 0.02; and MES [2-(N-morpholino)ethanesulfonic acid] (to maintain the pH at 6.0), 10, plus 0.1% arabinose. The advantages of the SSE medium are discussed.

A reevaluation of the Fe(II), Ca(II), Zn(II), and proton formation constants of 4,7-diphenyl-1,10-phenanthrolinedisulfonate.

The compound 4,7-diphenyl-1,10-phenanthrolinedisulfonic acid (BPDS) has been found to be very useful in studying Fe uptake by plants, because it forms a large charged complex that is not absorbed. The quantity of BPDS bound to metals in hydroponic solutions can be estimated from calculations using formation constants of BPDS complexes. These formation constants were used in an earlier experiment to predict the availability of Cu to corn plants. In the experiment, bioassays indicated that Cu was not as phytoavailable in the BPDS-added solutions as predicted by chemical equilibrium calculations. To determine sources of error in this prediction, metal and proton BPDS formation constants were reevaluated at 25 degrees C and 0.10M ionic strength. The CaBPDS formation constant was determined by direct measurement of CaBPDS3 formation and was shown to be approximately 1.0; a value much less than that reported before. Formation constants for the HBPDS, H(BPDS)2, and H(BPDS)3 beta 1, beta 2, and beta 3 complexes were, respectively, 5.05 +/- 0.044, 7.44 +/- 0.019, and 9.33 +/- 0.28. The BPDS sulfonic acid group pKs were less than 1.0, not 2.8 as has been reported. The FeBPDS3 complex determined by ligand competition with EDTA (ethylenediaminetetraacetate) was 20.24 +/- 0.08. Copper and Zn constants were determined using the method of corresponding solutions. The CuBPDS, CuBPDS2, and CuBPDS3 beta 1, beta 2, and beta 3 constants were, respectively, 9.76 +/- 0.08, 15.9, and 20.9. The ZnBPDS, ZnBPDS2, and ZnBPDS3 beta 1, beta 2, and beta 3 constants were, respectively, 6.43 +/- 0.07, 10.7 +/- 5.4, and 17.3 +/- 0.8. Results indicated that BPDS affinity to metals was similar to that of its parent compound, phenanthroline, and that errors in published formation constants caused erroneous predictions of Cu phytoavailability used in an earlier experiment.

Phenotypic Diversity among Strains of Bradyrhizobium japonicum Belonging to Serogroup 110.

Thirty-four strains of Bradyrhizobium japonicum within serogroup 110 were examined for phenotypic diversity. The strains differed in their abilities to nodulate and fix dinitrogen with Glycine max (L.) Merr. cv. Williams. Thirteen strains expressed uptake hydrogenase activity when induced as free-living cultures in the presence of 2% hydrogen and oxygen. Six bacteriophage susceptibility reactions were observed. Each of the strains produced either a large, mucoid or a small, dry colony morphology, but colony type was not related to effectiveness for nitrogen fixation.

Cadmium Resistance Screening in Nitrilotriacetate-Buffered Minimal Media.

Media used to determine the MICs of heavy metals for bacteria are unreliable because organic components in the media bind or chelate most of the metal being studied. To define specific metal activity in media and to maintain metal activity at a constant level, HEPES-MES [N-2-hydroxyethylpiperazine-N' -2-ethanesulfonic acid-2-(N-morpholine)ethanesulfonic acid] salts medium with arabinose medium was modified, and the modified medium was used to examine the MIC of cadmium for Rhizobium fredii USDA 201. Arabinose-HEPES-MES was modified by addition of the chelator nitrilotriacetate to buffer the supply of free Cd ion to maintain a constant Cd activity and by the use of only MES to buffer pH (buffered arabinose-MES medium [BAM]). Ca and Mg were supplied at the normal levels for soil solutions, and other trace elements were supplied at the levels required for normal growth of plants. The concentration of free Cd ion was calculated by using the computer program GEOCHEM-PC with a corrected data base. The Cd MIC in BAM was 14.0 muM, while that in a tryptone-yeast extract medium was 107 muM. The results indicate that substantial free Cd is removed from solution in most standard media, resulting in falsely high MICs. The new BAM medium allows for the precise determination of MICs, thus avoiding the uncertainties associated with other media.

Influence of soil variables on in situ plasmid transfer from Escherichia coli to Rhizobium fredii.

A model system was established to determine whether intergeneric plasmid transfer occurs in soil and how various soil variables affect the rate of plasmid transfer. The donor bacterium, Escherichia coli HB101 carrying plasmid pBLK1-2 (pRK2073::Tn5), and the recipient bacterium, Rhizobium fredii USDA 201, were inoculated into a sterile Adelphia fine-sandy-loam soil. Transconjugants were enumerated by direct plating on antibiotic-amended HM [N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid; 2-(N-morpholino) ethanesulfonic acid] salts medium. Randomly chosen transconjugants were verified by serological typing and Southern hybridization with a Tn5 gene probe. The maximum transfer frequency was observed after 5 days of incubation (1.8 x 10(-4) per recipient). The influences of clay (0 to 50% addition), organic matter (0 to 15% addition), soil pH (4.3 to 7.25), soil moisture (2 to 40%), and soil incubation temperature (5 to 40 degrees C) on plasmid transfer were examined. Maximum transfer frequencies were noted at a clay addition of 15%, an organic matter addition of 5%, a soil pH of 7.25, a soil moisture content of 8%, and a soil incubation temperature of 28 degrees C. These results indicate that intergeneric plasmid transfer may occur in soil and that soil variables may significantly affect the rate of transfer.

Long-Term Effects of Metal-Rich Sewage Sludge Application on Soil Populations of Bradyrhizobium japonicum.

The application of sewage sludge to land may increase the concentration of heavy metals in soil. Of considerable concern is the effect of heavy metals on soil microorganisms, especially those involved in the biocycling of elements important to soil productivity. Bradyrhizobium japonicum is a soil bacterium involved in symbiotic nitrogen fixation with Glycine max, the common soybean. To examine the effect of metal-rich sludge application on B. japonicum, the MICs for Pb, Cu, Al, Fe, Ni, Zn, Cd, and Hg were determined in minimal media by using laboratory reference strains representing 11 common serogroups of B. japonicum. Marked differences were found among the B. japonicum strains for sensitivity to Cu, Cd, Zn, and Ni. Strain USDA 123 was most sensitive to these metals, whereas strain USDA 122 was most resistant. In field studies, a silt loam soil amended 11 years ago with 0, 56, or 112 Mg of digested sludge per ha was examined for total numbers of B. japonicum by using the most probable number method. Nodule isolates from soybean nodules grown on this soil were serologically typed, and their metal sensitivity was determined. The number of soybean rhizobia in the sludge-amended soils was found to increase with increasing rates of sludge. Soybean rhizobia strains from 11 serogroups were identified in the soils; however, no differences in serogroup distribution or proportion of resistant strains were found between the soils. Thus, the application of heavy metal-containing sewage sludge did not have a long-term detrimental effect on soil rhizobial numbers, nor did it result in a shift in nodule serogroup distribution.

Aflatoxin decomposition in various soils.

The persistence of aflatoxin in the soil environment could potentially result in a number of adverse environmental consequences. To determine the persistence of aflatoxin in soil, 14C-labeled aflatoxin B1, was added to silt loam, sandy loam, and silty clay loam soils and the subsequent release of 14CO2 was determined. After 120 days of incubation, 8.1% of the original aflatoxin added to the silt loam soil was released as CO2. Aflatoxin decomposition in the sandy loam soil proceeded more quickly than the other two soils for the first 20 days of incubation. After this time, the decomposition rate declined and by the end of the study, 4.9% of the aflatoxin was released as CO2. Aflatoxin decomposition proceeded most slowly in the silty clay loam soil. Only 1.4% of aflatoxin added to the soil was released as CO2 after 120 days incubation. To determine whether aflatoxin was bound to the silty clay loam soil, aflatoxin B1 was added to this soil and incubated for 20 days. The soil was periodically extracted and the aflatoxin species present were determined using thin layer chromatographic (TLC) procedures. After one day of incubation, the degradation products, aflatoxins B2 and G2, were observed. It was also found that much of the aflatoxin extracted from the soil was not mobile with the TLC solvent system used. This indicated that a conjugate may have formed and thus may be responsible for the lack of aflatoxin decomposition.