dc.description.abstract |
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. |
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