Advanced Materials Research Vol. 825

Abstract: Bioleaching is the solubilization of metals from ores by microorganisms. This process is more efficient than conventional methods in the metal recovery from low grade ores while decreasing the operating cost and the environmental impact. Among the bioleaching bacteria, the Gram-negative chemolithoautotrophic acidophilic Acidithiobacillus ferrooxidans has the ability to adhere to mineral surface and to form biofilm. The genes involved in the production of this biofilm are controlled by a cellular communication system called Quorum Sensing (QS). The QS occurs mainly through the production of acyl homoserine lactones (AHLs). A functional QS system has been identified in At. ferrooxidans with an acyl synthase (AfeI) and a transcriptional regulator (AfeR). Here, our purpose is to characterize the quorum sensing regulon and particularly the genes involved in biofilm formation in At. ferrooxidans. This has been done by cell adhesion experiments comparing At. ferrooxidans grown with or without Tetrazol, an AHL analogue that activates the cell adhesion to mineral surface. The second step has been to compare the expression of some genes predicted to be involved in the QS regulon in At. ferrooxidans grown in the presence/absence of Tetrazol using real-time PCR. These two different strategies are expected to give insight on the moment at which the At. ferrooxidans QS regulon is switched on. The formation of biofilm could improve the bioleaching operation and therefore the understanding of the QS regulon is of primordial importance.

Abstract: Bioleaching is the solubilization of metals catalyzed by acidophilic microorganisms. Among these, the Gram negative γ-proteobacteria from the genus Acidithiobacillus play a relevant role. Biofilm formation has an impact on bioleaching performance. In Acidithiobacillus sp. the molecular mechanisms involved in biofilm formation are still unknown. However, in many Gram-negative and some Gram-positive bacteria, the intracellular levels of the second messenger cyclic diguanilic acid (c-di-GMP) control the regulation of biofilm formation. These levels depend on the balance between the enzymatic activities of diguanylate cyclases (synthesis) and phosphodiesterases (degradation). Previously, functional c-di-GMP pathways have been characterized in At. ferrooxidans and At. caldus. Here, our main goal was to characterize the presence of a functional c-di-GMP pathway in At. thiooxidans. By bioinformatic analysis, several putative-ORFs encoding DGCs, PDEs and effector proteins have been identified in the genome sequence of At. thiooxidans ATCC 19377. RT-PCR experiments revealed that most of these ORFs are transcribed. In addition, the analysis of nucleotide-enriched fraction extracted from At. thiooxidans cells showed that this bacterium is able to produce c-di-GMP. These results strongly indicate that At. thiooxidans has a functional c-di-GMP pathway.

Abstract: The proteomics of the extracellular proteins (EPs), outer membrane proteins (OMPs) and the periplasmic proteins (PPs) of Acidithiobacillus ferrooxidans ATCC 23270 grown on Fe²⁺ and S⁰ substrates, respectively, were comparatively studied. 39 expression up-regulated proteins (including 13 EPs, 9 OMPs and 17 PPs) were identified and 70% of them contain cysteine residues in sequence. Some of the selected proteins especially the EPs contain abundant of the cysteine residues and one or more-CXXC-functional motifs. The thiol groups on the At. ferrooxidans cell surface were selectively marked by Ca²⁺ and SR-μ-XRF mapping in situ observation revealed that the number of thiols on the surface of the cells grown on S⁰ was about five times as that grown on Fe²⁺ substrate. When 0.01 g/L surfactant Tween-80 was added in the S⁰ culture medium, the adsorption and activation related EPs were down-regulated and the sulfur metabolism related proteins was up-regulated. The same phenomenon was observed when the cells were grown on the more easily adhesion sulfur allotrope μ-S. It indicates that the thiol-rich proteins played important roles in adhesion and sulfur oxidation process of At. ferrooxidans.

Abstract: The leaching heap and acid mine drainage are two key nodes in a bioleaching system. This study aimed to investigate the microbial community structural and functional diversity between the two nodes in bioleaching system from Dexing copper mine in Jiangxi province, China. 16SrRNA gene cloning and metagenomic analysis consistently indicated that there were obvious differences on microbial community structural and functional diversity in the two nodes. In leaching heap, the dominant species was the heterotrophic bacterium Acidiphilium; while the dominant species was the autotrophic bacterium Acidithiobacillus in acid mine drainage. Seven bacteria species were found in both two nodes, while the unique bacteria species in leaching heap and acid mine drainage were eleven and eight, respectively. In relation to the microbial community function aspect, all contigs and singlets were annotated against the non-redundant protein database of NCBI and clustering analyzed with COG database. For the two nodes, the COG clustering results showed that the functional category abundances were different, though the functional categories were similar. And the great majority of ORFs were forecasted as function unknown. All the results meant that the microbial community structural and functional diversity of bioleaching system was not as simple as former thought. This study could provide a new meta-view of theoretical support to bioleaching process.

Abstract: Four well-differentiated clusters have been identified among iron-oxidizing acidithiobacilli. One monophyletic group clusters with At. ferrooxidans^T and another with strains of At. ferrivorans. While At. ferrooxidans and At. ferrivorans share many physiological traits, they differ in some phenotypic characteristics such as motility, pH and temperature minima, and also in terms of genes involved in ferrous iron oxidation. The genome of a strain (CF27) of At. ferrivorans, which is characterized by its marked propensity to form macroscopic growths and biofilms, was sequenced at Genoscope (Evry, France). Comparative genomic studies were carried with all related Acidithiobacillus strains that have been sequenced to date, in order to identify genetic determinants responsible for physiological traits relating to the oxidation of iron and sulfur, and also to extracellular polymeric substances formation.

Abstract: Ferrovum is a recently found new genus of acidophilic iron-oxidizing bacteria. Ferrovum spp. dominate the microbial community of a pilot plant for biological mine-water treatment and together with some Gallionella relatives appear to be key players of the process. Isolation of Ferrovum strains should greatly be facilitated by a new APPW medium. Sequencing of the genome of Ferrovum sp. JA12 so far did not point to any alternative electron donors and also did not reveal genes for nitrogen fixation.

Abstract: Here we describe the potential uptake and assimilation pathways for the essential nutrients C, N, P, and S in the acidophilic iron oxidiser Ferrovum strain JA12, a member of a novel genus among the Betaproteobacteria. Comparative genomics proved to be a powerful approach to give first insights into the biochemical potential of this novel genus and to understand the reasons for the dominating abundance of Ferrovum spp. in a pilot plant to remediate acid mine drainage.

Abstract: Acidithiobacillus ferrooxidans strain D2 was isolated from a copper bioleaching operation in Atacama Desert, Chile. Copper is widely used as cofactor in proteins but high concentrations of copper are toxic. Cells require certain mechanisms to maintain the copper homeostasis and avoid toxic effects of high intracellular concentration. The molecular response of A. ferrooxidans strain D2 grown in the presence/absence of copper was examined using a A. ferrooxidans whole-genome DNA microarrays. Roughly 23% of 3,147 genes represented on the microarray were differentially expressed; about 9% of them were upregulated in the presence of copper. Among the upregulated genes, those encoding for the copper efflux protein (CusA) and for the copper-translocating P-type ATPase (CopA) were upregulated. The expression of genes encoding proteins related to iron transport was repressed. Similarly, genes related with assimilative metabolism of sulfur (L-cysteine biosynthesis) cysB, cysJ, cysI, CysD-2 and cysN were upregulated. Our results show that when A. ferrooxidans strain D2 was challenged with high copper concentrations, genes related to copper stress response were upregulated as well as others that have not been reported to be related to that mechanism. In addition, some genes related to other metabolic pathways were repressed, probably because of the energy cost of the stress response.

Abstract: The structure of the microbial community inhabiting the copper bioleaching heap at Escondida mine has been systematically monitored since the operation was started up (2006), using biomolecular and microbiological analyses. Recent molecular analyses showed that L. ferriphilum was one of the most abundant organisms in the process during year 2012. In order to study the biological dynamics of carbon and nitrogen in the process, the expression levels of thirteen genes of L. ferriphilum were analyzed by RT-qPCR. The 16S rRNA and alaS genes were used as reference, and two relative quantification methods (ΔΔct and and Pffafl) were applied to estimate the relative expression levels of metabolic genes. On the day 110 of operation, a significant increment in the expression level of one gene involved in the cycle of tricarboxilic acids (2-oxoglutarate-acceptor oxidoreductase, oorA) was detected. By other hand, the expression level of two genes involved in carbohydrate metabolism (glgP, Glycogen phosphorilase, and glgA, Glycogen synthase) gradually increased, as the operation time progressed. The expression levels of genes involved in the fixation and assimilation of nitrogen increased at later stages of the process. A significant increase of the expression level of the gene annotated for Nitrogenase iron protein (nifH) was detected on the day 185 of operation. The opposite trend was observed for the gene annotated as Ammonium transporter protein (amt), as an elevated expression level was observed in earlier stages to suddenly decrease on the day 185 of operation, suggesting a change of the nitrogen source. In agreement with molecular quantitative analyses, this work confirmed that L. ferriphilum was an active member of the community during the period studied. This work gives new insights into biological dynamics of carbon and nitrogen, and suggests the potential guidelines to enhance the efficiency of biological components in industrial heap bioleaching processes.

Abstract: Acidithiobacillus ferrooxidans is a chemiolithoautotrophic Gram-negative bacterium widely spread in ambient temperature bioleaching processes. Several strains of At. ferrooxidans were isolated and studied and, some time later, questions arise about whether it was a species with a wide metabolic variation or a group of closely related species. Advances in molecular biology, phylogeny and genomics have shed some light on At. ferrooxidans strains and allows their grouping according to their relations. However, significant challenges remains to be met, such as understanding how a particular strain faces environmental challenges and how a particular kind of adaptive response affects the growth and activity of the strain. The purpose of this study was to identify differential expression signals between At. ferrooxidans strains −with different abundances and dynamics− present in the bioleaching system at Escondida mine. Culture characterization and DNA macroarrays techniques provided some answers. Analysis of growth curves showed that IESL 32 had the highest anaerobic growth rate, while aerobic growth was similar for all strains. It was shown that though the phylogenetic analysis based on 16S rRNA sequences suggested a close relation between IESL 32 and the type strain ATCC 23270, the growth curves and the expression profile showed that the type strain and strain D2 had the closest similarity. Growth experiments under different conditions, together with the comparative analysis of gene expressions in At. ferrooxidans, could be a springboard for future investigations of strain characterization to broaden our knowledge about adaptation, metabolic strategies, regulation and microbial diversity in industrial processes.