Abstract: The highly conserved operon iron–sulfur cluster (iscSUA) is essential for the general
biogenesis and transfer of iron–sulfur proteins in bacteria. In this study, expression, purification and
characterization of the proteins of the isc operon (iscSUA) of Acidithiobacillus ferrooxidans ATCC
23270 was studied. Assembly and transfer of [Fe4S4] in vitro during the isc proteins and other iron
sulfur proteins was studied in order to detect the pathway and mechanism of [Fe4S4] assembly and
transfer in vivo. The [Fe4S4] cluster was successfully assembled in iron-sulfur proteins in vitro in the
presence of Fe2+ and sulfide, and it was successfully transferred from IscA or IscU to iron- sulfur
proteins. Our results support and extend certain models of iron-sulfur clusters assembly and
Abstract: This study investigated the effect of sulphate concentration and residence time on the
performance of anaerobic sulphate reduction by a mixed sulphate reducing bacteria (SRB) culture
using lactate as the sole carbon source and electron donor. The process performance is related to the
population structure of the microbial consortia and dominant metabolic reactions. Laboratory scale
chemostat cultures at different residence times (1-4 d) and sulphate concentrations (1.0-10.0 g/L)
were employed. Lactate oxidation was prevalent at feed sulphate concentrations of 1.0 to 5.0 g/L. A
corresponding increase in the volumetric sulphate reduction rate with increasing volumetric loading
rate was also observed at this range. However, at the higher feed sulphate concentration range
(10.0-15.0 g/L), sulphate inhibition, lactate fermentation and an increased microbial diversity were
evident. At each feed concentration of sulphate in the range 5.0 to 15.0 g/L, varying dilution rates
resulted in significant shifts in dominant metabolic reactions. Sulphate concentration and residence
time have significant effects on both the structure of the microbial population and kinetics of
biological sulphate reduction.
Abstract: A collection of highly nickel and cobalt-resistant enterobacteria were isolated from the
Punta Gorda serpentine deposit (Moa, Cuba). The most nickel and cobalt resistant strain (termed C-
1) was assigned to Serratia marcescens by 16S rRNA analysis and DNA/DNA hybridization and
the molecular mechanisms underlying its inducible cobalt and nickel resistance was investigated.
Genes involved in metal resistance were identified by transposon mutagenesis followed by selection
for Co- and Ni-sensitive derivatives. The transposon insertion causing the highest decrease in metal
resistance was located in the ncrABC determinant. The three ORFs (ncrA, ncrB and ncrC) were
cloned in E. coli. The predicted NcrA product was an NreB ortholog of the major facilitator protein
superfamily and was central for Co/Ni resistance in S. marcescens strain C-1. NcrA also mediated
metal resistance in E. coli and caused decreased accumulation of Co and Ni in this heterologous
host. NcrB may be a regulatory protein. NcrC was a protein of the Ni–Co transport (NiCoT) protein
family and necessary for full metal resistance in E. coli, but only when NcrA was also present.
Without NcrA, NcrC caused a slight decrease in metal resistance and mediated increased
accumulation of Ni and Co. As the cytoplasmic metal concentration can be assumed to be the result
of a flow equilibrium of uptake and efflux processes, this interplay between metal uptake system
NcrC and metal efflux system NcrA may contribute to nickel and cobalt resistance in this
Abstract: The microbial community of a pilot plant for the production of iron hydroxysulfates by
biological oxidation of ferrous iron was studied using molecular techniques. The 16S rRNA gene
libraries were dominated by one sequence type, which can be classified phylogenetically to the
class of Betaproteobacteria. These bacteria have no close cultivated relatives and were also proved
as the dominating species in other mine waters.
The microbial community was observed over a period of 18 months using TRFLP to investigate
the influence of different process parameters on the composition of the microbial diversity. These
studies verified the dominance of the Betaproteobacteria in the pilot plant.
Abstract: An understanding of the physiology and metabolic complexity of microbial consortia
involved in metal solubilization is a prerequisite for the rational improvement of bioleaching
technologies. Among the most challenging aspects that remain to be addressed is how aerobic
acidophiles, especially Fe(II)-oxidizers, contend with the paradoxical hazards of iron overload and
iron deficiency, each with deleterious consequences for growth. Homeostatic mechanisms
regulating the acquisition, utilization/oxidation, storage and intracellular mobilization of cellular
iron are deemed to be critical for fitness and survival of bioleaching microbes. In an attempt to
contribute to the comprehensive understanding of the biology and ecology of the microbial
communities in bioleaching econiches, we have used comparative genomics and other bioinformatic
tools to reconstruct the iron management strategies in newly sequenced acidithiobacilli and other
biomining genomes available in public databases.
Species-specific genes have been identified with distinctive functional roles in iron
management as well as genes shared by several species in biomining consortia. Their analysis
contributes to our understanding of the general survival strategies in acidic and iron loaded
environments and suggests functions for genes with currently unknown functions that might reveal
novel aspects of iron response in acidophiles. Comprehensive examination of the occurrence and
conservation of regulatory functions and regulatory sites also allowed the prediction of the metal
regulatory networks for these biomining microbes.
Abstract: Small regulatory RNAs (srRNAs) control gene expression in Bacteria, usually at the posttranscriptional
level, by acting as antisense RNAs that bind targeted mRNAs or by interacting with
regulatory proteins. srRNAs are involved in the regulation of a large variety of processes such as
plasmid replication, transposition and global genetic circuits that respond to environmental changes.
Since their discovery a few years ago, it has become apparent that they are prolific and widespread. In
this study, we describe bioinformatic approaches to srRNA discovery in the biomining microorganisms
Acidithiobacillus ferrooxidans, A. caldus and A. thiooxidans. Intergenic regions of the annotated
genomes were extracted and computationally searched for srRNAs. Candidate srRNAs that were
associated with predicted sigma 70 promoters and/or rho-independent terminators were chosen for
further study. The resulting potential srRNAs include known examples from other microorganisms and
some novel candidates and reveal interesting underlying biology of the Acidithiobacillus genus.
Abstract: Microbial heap bioleaching is being used as an industrial process to recover copper from
low grade ores. It is known that a consortium of different microorganisms participates in this
process. Therefore identification and quantification of communities inhabiting heap bioleaching
operations is a key step for understanding the dynamics and role of these microorganisms in the
process. A quantitative real-time PCR approach was used to investigate the microbial dynamics in
this process. To study the microbial population inhabiting a low-grade copper sulphide ore
bioleaching industrial heap process at Escondida Mine in Chile, 16S rRNA genetic libraries were
constructed using bacterial and archaeal universal primers. Phylogenetic analyses of sequences
retrieved from genetic libraries showed that the community is mainly composed by microoganisms
related to Acidithiobacillus ferrooxidans (2 strains), Acidithiobacillus thiooxidans, Leptospirillum
ferrooxidans, Leptospirillum ferriphilum and the archaea Ferroplasma.
Specific primers for real-time PCR determination were designed and tested to amplify each of
the sequences obtained by cloning. Standard curves for real time PCR were performed using
plasmid DNA from selected clones. This methodology is actually being used to monitor relevant
microorganisms inhabiting this low-grade copper sulphide ore bioleaching industrial heap.
Abstract: The majority of mining companies in the north of Chile that use biohydrometallurgical
processes to leach low-grade copper sulphide ore are located in the high Andes. This makes the
study of the effect of low temperature on bacterial activity important for the design and
optimization of the bioleaching process. In this study bacterial activity of iron and sulphur oxidizing
microorganisms was analyzed in the laboratory at various temperatures.
Flask tests were carried out at a range of temperatures to determine the bacterial activity in
pregnant leaching solution (PLS) from a pilot plant at the Escondida Mine. Ratkowsky curves were
constructed by monitoring the concentration of ferrous iron and acid production during the tests.
Also leaching tests were carried out at ~10 and ~20°C on a 1 m column using a copper ore sample.
In an industrial solution in flask tests, iron oxidizing activity was observed until the temperature
was reduced to 20°C. In the same culture, after an adaptation process, iron oxidizing activity was
observed down to 5°C.
In the column tests, 41.2% of copper was recovered after 91 days of leaching at 10 °C and 57.2
% at ~20°C. Predominant 16S rRNA gene sequences were related to Acidithiobacillus ferrooxidans,
Acidithiobacillus thiooxidans and Leptospirillum ferriphilum.
Abstract: Acid mine drainage (AMD) waters are highly acidic (pH < 4), contain high
concentrations of sulfate and dissolved metals, and are very toxic to many living organisms. The
development of technologies to treat sulfate contaminated wastewaters by using sulfate-reducing
bacteria (SRB) has produced a cost-effective route to treat AMD. Notwithstanding, the SRB
sensitivity to acid limits their use in AMD remediation. In the current study, acidophilic strains of
SRB were isolated from an AMD followed by their molecular characterization. One SRB-culture
was able to grow at pH 4.5 in Postgate C modified medium containing ethanol as carbon source,
indicating that such bacterium has the potential for the bioremediation of acidic waters. Following,
the strains were characterized by molecular biology techniques. The characterization was done by
PCR amplification, cloning and sequencing of the genes coding for parts of the alpha and beta
subunits of dissimilatory sulfite reductase (dsrAB) and hydrogenase (hyd), which encode key
enzymes of the SRB energy metabolism. Phylogenetic analysis suggested a line of SRB descent
from the delta-Proteobacteria among the strains identified as Desulfovibrio fructosovorans.