Bacteria in heavy metal contaminated soil: diversity, tolerance and use in remediation

The objectives of this project were to determine the bacterial diversity in a heavily contaminated metal region of Portugal. Both traditional and molecular based methods were used to identify tolerant strains and species. The most tolerant species were subsequently identified and utilized for examining the potential for using them to immobilize specific metals from contaminated waste streams by comparing different support materials. Heterotrophic bacterial populations were isolated and characterized from a contaminated industrial area in Northern Portugal. In a first sampling, 278 strains were isolated in different solid media. To assess the diversity of this ecological site and to select representative strains, the isolates were screened by using Random Amplified Polymorphic DNA (RAPD)-PCR profiles. Phenotypic characterization, phylogenetic analysis by sequencing the 16S rRNA genes and metal tolerance tests with zinc (Zn), cadmium (Cd) and arsenic (As) were performed with the selected strains. Recovered gram-positive isolates were related to class Actinobacteria and Bacilli. The majority of the isolates were related to genera Microbacterium and Bacillus. Strains from the genus Arthrobacter were also well represented. 16S rRNA gene sequence similarity of the gram-negative isolates showed that they were related to classes γ-Proteobacteria, ß-Proteobacteria, a-Proteobacteria and Flavobacteria. The most frequently isolated taxa were γ-Proteobacteria, related with the genus Pseudomonas, where a large number of isolates were clustered. These genera are common in metal contaminated environments. Many of the strains (approx. 17) had a high level of tolerance to the heavy metals tested. A total of 13 isolates were not able to grow when metals were present. In a second sampling the soil rhizosphere was screened for bacterial populations, using metal-based selective media for isolation. About 42 strains were recovered when metal supplemented media was used. The gram-positive population were predominantly Bacilli and Actinobacteria members. Bacillus, Microbacterium and Arthrobacter were the most common gram-positive genera. Gram-negative genera were from the same classes as in the first sampling however Sphingobacteria was present. γ-Proteobacteria and ß-Proteobacteria were the most common taxa. The isolates were shown to be very resistant to Zn and As, with about half of the isolates able to grow with Cd present. Interestingly, no strains could grow in the presence of metal mixtures. Despite the number of strains recovered in both samplings the majority of the isolates were clustered within a very small number of genera. During the sampling periods two strains showing low similarity to other bacteria were isolated. These strains were characterized and studied in detail justifying their classification as representing two novel species of the genus Chryseobacterium. The names proposed for these organisms are Chryseobacterium palustre sp. nov. (type strain 3A10[type strain) ) and Chryseobacterium humi sp. nov. (type strain ECP37[type strain] ). Three isolates 1C2, 1ZP4 and EC30 belonging to genera Cupriavidus, Sphingobacterium and Alcaligenes respectively, showing high tolerance to heavy metals, were selected for further study in immobilised systems for Zn and Cd removal. In most cases, matrices (alginate, pectate and a synthetic cross-linked polymer) with immobilised bacteria showed better metal removal. 1C2, a strain belonging to the Cupriavidus genera, was able to increased the removal of Zn; EC30, a bacteria related to Alcaligenes, was the most promising candidate for Cd removal, especially when combined with the synthetic polymer. Removal of metals as single or in binary mixtures was also assessed. Cd removal was most effective when single metal solutions were tested using immobilised bacteria and examining metal matrixes. Based on the strains used and the matrices tested, best results were obtained for removal of Zn from binary mixtures with Cd. Potential exists for further studies to exploit these bacterial strain to develop effective bioremediation approaches for the removal of heavy metals from waste water streams