Heavy-Metal Phytoextraction Potential of Spinach and Mustard Grown in Contaminated Calcareous Soils

Laboratory batch and greenhouse pot experiments were conducted to determine the extraction efficiency of ethylenediaminetetraacetic acid (EDTA) for solubilizing lead (Pb) and cadmium (Cd) and to explore the natural and chemically induced Pb and Cd phytoextraction efficiencies of spinach and mustard after EDTA application. The EDTA was applied at 0, 1.25, 2.5, and 5.0 mM kg^?1 soil in three replicates. Addition of EDTA increased significantly the soluble fraction Pb and Cd over the control and maximum increases for Pb (1.42- and 1.96-fold) and Cd (1.45- and 1.38-fold) were observed with the addition of 5.0 mM EDTA kg^?1 in Gujranwala and Pacca soils, respectively. Similarly, addition of EDTA increased significantly the Pb and Cd concentrations in the plant shoots, soil solution, bioconcentration factor, and phytoextraction rate. Mustard exhibited better results than spinach when extracting Pb and Cd from both contaminated soils.     

Phytoextraction Potential of Indian Mustard at Various Levels of Zinc Exposure

ABSTRACT

Indian mustard (Brassica juncea Czern.) has the potential to extract zinc (Zn) and other metals from contaminated soils, but the potential to accumulate metals at different levels of exposure is not well documented. The objectives of this research were to assess plant growth and Zn accumulation for different metal-accumulating accessions of Indian mustard grown with various Zn concentrations. In the experiment, three accessions of Indian mustard (426308, 182921, and 211000) were supplied with 12 levels of Zn (ranging from 0.0 to 7.0 mg L^?1) for three weeks in solution culture. Accession 426308 had a greater capacity for dry-mass accumulation than the others, but differences among accessions lessened as the concentration of Zn in solution increased. Accessions did not differ in Zn concentrations in shoots, but accession 426308 had a greater potential to accumulate Zn than the other accessions. Elevating the Zn supply in solutions had a limited effect on increasing the total Zn accumulation of shoots. Plants suffered Zn-induced iron (Fe) deficiency if the Zn concentration in solution exceeded 2.0 mg Zn L^?1. The level of Zn tolerance of Indian mustard accessions was: 211000 > 182921 > 426308. Maximum Zn accumulation in shoots was approximately 5.0 mg Zn per plant. The phytoextraction potential of Indian mustard may be limited under Zn-contaminated conditions by nutrient disorders and toxic effects of Zn that suppress growth.     

Effect of Coal Combustion By-products on Phosphorus Runoff from a Coastal Plain Soil

Coal combustion by-products can lower soil phosphorus (P) solubility, but few studies have assessed their effect on runoff P. A soil with elevated P content was amended with fluidized bed combustion ash, flue gas desulfurization gypsum, and anthracite refuse ash at rates of 0–40 g kg^?1 soil, and runoff from small plots was monitored over 3 years. In the first year, by-products lowered dissolved P in runoff by up to 47% below the untreated control; however, effects did not persist into the remaining years of the study. Total P losses were not significantly affected by coal combustion by-products, likely because of elevated particulate P losses. Water-extractable P was up to 40% less in treated soils than in untreated soils across the 3 years. Results demonstrate that although coal combustion by-products readily lower P solubility in soils, their impact on P losses in runoff can be undermined by erosional processes.     

Evaluation of functional diversity in rhizobacterial taxa of a wild grass (Saccharum ravennae) colonizing abandoned fly ash dumps in Delhi urban ecosystem

Metal rich fly ash dumps may serve as repository of ecologically useful multi-functional rhizobacteria having potential use in the development of vegetation at the dumps. Therefore, in the present study bacteria from the rhizosphere of a wild perennial grass colonizing Indraprastha and Badarpur fly ash dumps of Delhi region were purified, identified and functionally characterized. The fly ash had low levels of nutrients, moisture and organic matter coupled with toxic levels of heavy metals (As, Cd, Cr, Cu, Hg, Ni, Pb and Zn). Both the dumps were mostly barren except for a few patches of Saccharum ravennae and some weedy species. Sixty five dominant, morphologically distinct rhizobacteria were purified, which belonged to 18 genera and 38 species. Gram-positive bacteria were dominating in the fly ash environment. Bacillus spp. and Paenibacillus spp. were common at both the dumps. Multi-metal tolerance was shown by diverse bacterial taxa. The minimum inhibitory concentration (MIC) was highest for As (12.5-20.0 mM) and Pb (7.5-10.0 mM), although many rhizobacteria also possessed significant tolerance to Cr, Zn, Ni, Cu, Co and Cd. The tolerance profiles of rhizobacteria to different metals may be ranked in the decreasing order as As > Pb > Cr > Zn > Ni > Cu > Co > Cd > Hg. Majority of rhizobacteria showed good siderophore activity. Multiple-metal tolerance was also coupled with high siderophore production in some of the isolates (Microbacterium barkeri IPSr74, Serratia marcescens IPSr90 and IPSr82, Enterococcus casseliflavus BPSr32, Bacillus sp. IPSr80, Pseudomonas aeruginosa BPSr43 and Brochothrix campestris BPSr3). Most of the bacteria could grow on nitrogen-deficient medium. However, the dominant nitrogen-fixers reported from the rhizosphere of other Saccharum species were not detected. S. marcescens IPSr90 was the only rhizobacterium, which showed ACC-deaminase (ACCD) activity. Proportion of phosphate-solubilizing bacteria was high. Considerable improvement in the seedling establishment, plant weight and shoot length in rhizobacterial inoculated plants of S. ravennae in fly ash environment indicated the significance of rhizobacteria in its colonization and spread to the dumps. Representative rhizobacteria, with high MIC (for most of the metals) and good plant growth promoting (PGP) traits comparable to commercially useful bacterial inoculants were identified as S. marcescens IPSr82 and IPSr90, P. aeruginosa BPSr43, Paenibacillus larvae BPSr106, Arthrobacter ureafaciens BPSr55, Paenibacillus azotofixans BPSr107 and E. casseliflavus BPSr32. S. ravennae and some of these rhizobacteria may be potentially useful for the development of inoculation technologies for conversion of barren fly ash dumps into ecologically and economically productive habitats.     

Roles of Bacillus megaterium in Remediation of Boron,Lead, and Cadmium from Contaminated Soil

Phytoremediation is an attractive, economical alternative to soil removal and burial methods to remediate contaminated soil. The objective of this study was to investigate the effects of adding different rates of Bacillus megaterium on the capacity of Brassica napus plants to take up boron (B), lead (Pb), and cadmium (Cd) from polluted soils under field conditions. Field experiments were conducted using a randomized complete block design with control (without pollution and B. megaterium application) and B, Pb, and Cd in two doses (0 and 100 mg kg^?1), B. megaterium with four doses (no application and 10⁸ cfu B. megaterium ml^?1 sprayed at 50 ml plot^?1, 100 ml plot^?1, 150 ml plot^?1). Results indicated that soil pollution treatments significantly decreased seed (SDMY), shoot (SHDMY), root (RDMY), and total dry-matter yield (TDMY) of plants at 42.9, 3.8, 62.6, and 23.4% for B-polluted treatment; 25.8, 8.7, 17.6, and 14.2% for Pb-polluted treatment; and 33.2, 7.0, 14.0, and 16.4% for Cd-treatment without B. megaterium application, respectively. However, the application of B. megaterium ameliorated the negative effects of B, Pb, and Cd at 41.4, 52.7, and 10.9% for B; 24.4, 21.6, and 4.9% for Pb; and 22.8, 22.0, and 3.3% for Cd, respectively. The potentially bioavailable and relatively available fraction of soil B, Pb, and Cd increased with increases in the B. megaterium application but total fraction and stable fraction decreased. It is concluded that the seed and shoot parts of B. napus can be used as hyperaccumulators for plant B, Pb, and Cd remediation according to remediation factors but the shoot is the biggest part of the plant, and thus an important portion of the plant to remove B, Pb, and Cd from the B-, Pb-, and Cd-contaminated soils. To decrease desired concentration for 8 mg B kg^?1, 4 mg Pb kg^?1, and 3 mg Cd kg^?1 in the active rooting zone of soil, approximately 2, 6, and 21 years would be necessary with only 150 ml plot^?1 B. megaterium–sprayed soil cultivated with B. napus, respectively.     

Multiple Metal Resistant Transferable Phenotypes in Bacteria as Indicators of Soil Contamination with Heavy Metals (6 pp)

Background, Aim and Scope   Environmental contamination by heavy metals affects microbial communities. The number of single and multiple heavy metal resistant bacteria may be an indictor of the level of contamination. This paper details the isolation and characterisation of metal resistant microorganisms isolated from rhizosphere/soil samples obtained from an abandoned zinc, lead and copper mine and a local unaffected site. This data was compared to the level of heavy metal in the soils to establish the effect of metals on the microbial community and to determine the relationship between pollutant levels and resistant strains. This paper outlines the diversity of transferable resistance determinants between both sites and details the levels of heavy metal resistant bacteria and those expressing transferable multiple heavy metal tolerance.Methods   The sample sites were located in Co. Galway, Ireland. The first sample site (site A) was a former lead, zinc and copper mine, which was closed in 1961 due to exhaustion of ore. The second site (site B) was located two and a half kilometres from the mining site and was not affected by the mining operations. Composite soil samples were characterised for general soil matrix composition, organic content, pH and general chemical parameters. The soil was also enumerated for the total viable heterotrophic counts and tested on Pseudomonas selective agar (PSA) for total Pseudomonas counts and Sucrose Asparagine (SA), which is semi-selective for fluorescent Pseudomonas.Results and Discussion   Samples from both site A and site B were analysed by atomic absorption spectrophotometry for the presence of heavy metals. In the case of copper, which has a Dutch list recommended minimum permissible level of 190 µg/Kg dry weight, the levels detected at site A were 1270 µg/Kg dry weight while site B was detected at 36 µg/Kg. The arsenic levels detected at site A were eight times the permissible level (416 µg/Kg) while only half the permissible level was found at site B (13 µg/Kg). Zinc concentrations were also high at site A (4460 µg/Kg) while at site B (553 µg/Kg) they were well below the Dutchlist guidelines (720 µg/Kg). A large number of heavy metal tolerant strains were isolated from both sites. 270 isolates (site A (170) and site B (100)) were screened against 8 metals to examine the extent of multiple resistance. 82% of the strains from site A were found to be resistant to 5 metals. A total of 18% showed resistance to all 8 metals and of those examined only 4% were resistant to only one metal. In contrast isolates from site B showed no multiple resistance to more than 5 metals, while 62% showed resistance to individual metals only. Site A had a higher level of multiple heavy metal resistance strains. Stains isolated from site A had 23 (14%) isolates resistant to zinc, copper, nickel, arsenic and cobalt and site B had no bacteria resistant to all five of these selected metals. The transferability of heavy metal resistance was investigated in the case of 60 multiple heavy metal resistant isolates taken from site A and 50 multiple resistance isolates from site B. Transfer was only detected in isolates from site A, 13% showed transfer and expression of copper, zinc and arsenic resistance determinants. In most cases the transconjugants only expressed resistance to copper, zinc and arsenic, which were the metals, used for selection. This co-transfer of all three determinants suggests a genetic link between these resistance determinants.Conclusion   Heavy metal resistant bacteria are present in both sites, however, the number and presence of multiple transferable resistance phenotypes are confined to the isolates from the heavy metal contaminated site. The presence of high levels of heavy metals selects these multiple resistance phenotypes. Within these communities there seems to be little diversity between the microorganisms, which provides a hugely preferable environment for gene transfer of such metal resistant determinants.Recommendation and Perspective   The experiments have shown a microbes ability to mobilize heavy metal determinants and a relationship between heavy metal resistance and metal contamination has been identified These multiple heavy metal resistant bacteria could eventually be used for detection and qualification of the level of heavy metal-polluted soil/water environments.     

Enhanced establishment and growth of giant cardon cactus in an eroded field in the Sonoran Desert using native legume trees as nurse plants aided by plant growth-promoting microorganisms and compost

To evaluate the feasibility of long-term desert reforestation technology of mixed vegetation, cardon cactus (Pachycereus pringlei) seedlings from indoor and outdoor nurseries were planted in the field adjacent to one seedling of potential legume nurse trees: mesquite amargo (Prosopis articulata), yellow palo verde (Parkinsonia microphylla), and blue palo verde (Parkinsonia florida). Some of the planting holes were also supplemented with common dairy compost. Additionally, the combinations of legume tree–cactus were inoculated with either a consortium of desert arbuscular mycorrhizal (AM) fungi, plant growth promoting bacteria (PGPB; the diazotroph Azospirillum brasilense Cd, and the phosphate solubilizer Paenibacillus sp.), or a mixture of all. The field experiments were evaluated periodically during 30 months for survival and growth. Cardons reared in an outdoor screen house survived better in the field than those reared in a controlled growth chamber and hardened later outdoors. Association with any legume nurse tree increased survival and enhanced growth of untreated cardons. For cardons growing alone, application of either compost, AM fungi, and all the treatments combined increased survival. For these plants, no treatment affected plant growth during the first 3 months after transplanting. Later, all treatments, except for AM fungi, enhanced plant growth. However, only 2 years after transplanting the enhanced growth effect of AM fungi was also significant. In the presence of the legume nurse trees, transient positive effects on cardon growth were recorded. General evaluation after 30 months of cultivation showed that the treatments positively affected cardon growth when growing alone or in combination only with mesquite amargo but not with the other two legume trees. This study proposes that young legume trees have the capacity to enhance survival and growth of cardon cactus, depending on the legume cactus combination. Additional treatments such as compost or PGPB can either amplify the effect or else attenuate it.     

Molecular Monitoring of SRB Community Structure and Dynamics in Batch Experiments to Examine the Applicability of in situ Precipitation of Heavy Metals for Groundwater Remediation (15 pp)

Background, Aims and Scope   Sulfate-reducing bacteria (SRB) are known for their capacity to reduce and precipitate heavy metals (HM) as metal sulfides, offering the opportunity to create an in situ reactive zone for the treatment of heavy metal-contaminated groundwater, a process called in situ metal precipitation (ISMP). The applicability of the ISMP technology first has to be investigated at a laboratory scale before going into an on site application. The evaluation and optimization of the ISMP process is facilitated when physical/chemical analysis techniques are combined with molecular tools that specifically monitor the abundance, diversity and dynamics of the indigenous sulfate reducing microbial community. In this study, batch experiments were conducted in order to investigate the feasibility of ISMP as a groundwater remediation strategy for an industrial site contaminated with elevated levels of Zn, Cd, Co and Ni. Methods   The potential of different types of carbon source/ electron donor (lactate, acetate, methanol, ethanol, Hydrogen Release Compound?, molasses) to stimulate the sulfate reduction and metal precipitation activity of the naturally present (or indigenous) SRB community was explored. In addition, the effect of amending vitamin B12 and yeast extract was evaluated. The ISMP process was monitored by combining analytical analyzes of process parameters (SO42-concentration, heavy metal concentrations, pH, Eh) with molecular tools such as SRB subgroup and genus specific PCR, denaturing gradient gel electrophoresis (DGGE), and phylogenetic analysis of clone sequences, based on either the 16S rRNA or the dsr (dissimilatory sulfite reductase) gene. Results and Discussion   The efficiency of different carbon-sources to stimulate the ISMP process followed the order HRC 〉 molasses 〉 methanol 〉 lactate 〉 ethanol 〉 acetate. Within 10 weeks, the highest sulfate and metal removal efficiencies ranged from 85% to 99%. Addition of yeast extract boosted the ISMP process, whereas vitamin B12 negligibly affected SRB activity. Analysis of the sulfate reducing population by SRB subgroup and genus specific PCR demonstrated that members of the genus Desulfosporosinus dominated in all batch tests, while 16S rDNA DGGE profiles additionally revealed the presence in the microbial communities of non-sulfate reducing bacteria within the family Clostridium and the -proteobacteria. The dsrB-based DGGE profiles allowed us to assess the diversity and dynamics of the sulfate reducing community and added to a better understanding of the effects of different batch conditions on the ISMP process. Remarkably, all dsrB sequences affiliated with the dsrB gene sequence cluster found in Desulfotomaculum, which received their xenologous dsrB gene from the -proteobacteria. Conclusions   The batch experiments, which aimed at stimulating the activities of the indigenous SRB communities, demonstrated that these communities were present and that their activities could be used to obtain efficient in situ precipitation of the contaminating heavy metals. This opens the possibility to test this concept in the future as an on site demonstration as part of the groundwater strategy for the heavy metal contaminated site. Although batch setups are suitable for preliminary feasibility studies for ISMP, they do not reflect the in situ situation where sulfate and heavy metal and metalloid polluted groundwater are supplied continuously. A sulfate reducing strain JG32A was isolated from whose 16S rRNA gene affiliated with the genus Desulfosporosinus, while its dsrB gene sequence clustered with Desulfotomaculum dsrB gene sequences, which received their xenologous dsr genes from -proteobacteria. Therefore we hypothesize that the batch experiments enrich members of the Desulfosporosinus genus that possess a non-orthologous dsrB gene. Recommendation and Perspective   The next step towards an on site pilot test for ISMP will be the setup of a series of column experiments, with process conditions that are selected based on the above mentioned results. This will allow to define optimal ISMP process conditions and to test its long-term efficacy and sustainability before going into an on site bioremediation application. By applying the described molecular tools together with physical-chemical analyzes, it can be investigated whether the same SRB community is enriched and which type of C-source is most effective in promoting and sustaining its growth and sulfate-reduction activity.     

Nitrite production by Nitrosomonas europaea and Nitrosospira sp. AV in soils at different solution concentrations of ammonium

Although it remains unclear why NH₃-oxidizing bacteria (AOB) of the genus Nitrosospira dominate soil environments, and why Nitrosomonas spp. are less common, virtually no studies have compared their behavior in soil. In this study, the NH₃ oxidation rates of Nitrosomonas europaea (ATCC 19718) and Nitrosospira sp. AV were compared in three differently textured soils containing a range of extractable contents (2-11 μg soil). Soils were adjusted to pH 7.0-7.4 with CaCO₃ and sterilized with γ-radiation. Cell suspensions of each bacterium were inoculated into the soils to bring them to two-third of water-holding capacity and cell densities ∼2.5×10⁶ g⁻¹ soil. In virtually all cases, rates of production for both N. europaea and Nitrosospira sp. AV were linear over 48 h, and represented between 13 and 75%, respectively, of the maximum rates achieved in soil-free bacterial suspensions. Soil solution concentrations that supported these rates ranged between 0.2 and 1.5 mM. Addition of 21-36 μg soil raised soil solution levels to 1.8-2.5 mM and stimulated production to a greater extent in N. europaea (3.3-6.6-fold) than in Nitrosospira sp. AV (1-2.1-fold). Maximum rates of production were obtained by raising soil solution levels to 3-4 mM with a supplement of ∼80-90 μg soil. K_s values in soil for Nitrosospira sp. AV and N. europaea were estimated as 0.14 and 1.9 mM , respectively, and estimates of V_max were about 3.5-times higher for N. europaea (0.007 pmol h⁻¹ cell⁻¹) than for Nitrosospira sp. AV (0.002 pmol h⁻¹ cell⁻¹). The cell density of N. europaea increased in sterile Steiwer soil independent of supplemental . In the case of treatments receiving supplemental , growth yields of N. europaea calculated from either produced or consumed were similar to those reported in literature (3.5×10⁶-6×10⁶ cells μmol⁻¹). A higher growth yield was measured in the case of zero added (2.7×10⁷ cells μmol⁻¹), indicating that use of organic carbon compounds might have occurred and resulted in some energy sparing. Our results suggest that Nitrosospira spp. with a K_s similar to Nitrosospira sp. AV may have an advantage for survival in soil environments where soil solution levels are less than 1 mM. However, it is apparent that AOB like N. europaea are poised to take advantages of modest increases in extractable that raise soil solution levels to about 2.0-2.5 mM.