Advanced Materials Research Vols. 20-21

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 transfer.

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 bacterium.

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.