Francisco Remonsellez

Dynamic of active microorganisms inhabiting a bioleaching industrial heap of low-grade copper sulfide ore monitored by real-time PCR and oligonucleotide prokaryotic acidophile microarray.

The bioleaching of metal sulfide has developed into a very important industrial process and understanding the microbial dynamic is key to advancing commercial bioleaching operations. Here we report the first quantitative description of the dynamic of active communities in an industrial bioleaching heap. Acidithiobacillus ferrooxidans was the most abundant during the first part of the leaching cycle, while the abundance of Leptospirillum ferriphilum and Ferroplasma acidiphilum increased with age of the heap. Acidithiobacillus thiooxidans kept constant throughout the leaching cycle, and Firmicutes group showed a low and a patchy distribution in the heap. The Acidiphilium‐like bacteria reached their highest abundance corresponding to the amount of autotrophs. The active microorganisms in the leaching system were determined using two RNA‐based sensitive techniques. In most cases, the 16S rRNA copy numbers of At. ferrooxidans, L. ferriphilum, At. thiooxidans and F. acidiphilum, was concomitant with the DNA copy numbers, whereas Acidiphilium‐like bacteria and some Firmicutes members did not show a clear correlation between 16S rRNA accumulation and DNA copy numbers. However, the prokaryotic acidophile microarray (PAM) analysis showed active members of Alphaproteobacteria in all samples and of Sulfobacillus genus in older ones. Also, new active groups such as Actinobacteria and Acidobacterium genus were detected by PAM. The results suggest that changes during the leaching cycle in chemical and physical conditions, such as pH and Fe3+/Fe2+ ion rate, are primary factors shaping the microbial dynamic in the heap.

Molecular Characterization of Copper and Cadmium Resistance Determinants in the Biomining Thermoacidophilic Archaeon Sulfolobus metallicus

Sulfolobus metallicus is a thermoacidophilic crenarchaeon used in high-temperature bioleaching processes that is able to grow under stressing conditions such as high concentrations of heavy metals. Nevertheless, the genetic and biochemical mechanisms responsible for heavy metal resistance in S. metallicus remain uncharacterized. Proteomic analysis of S. metallicus cells exposed to 100 mM Cu revealed that 18 out of 30 upregulated proteins are related to the production and conversion of energy, amino acids biosynthesis, and stress responses. Ten of these last proteins were also up-regulated in S. metallicus treated in the presence of 1 mM Cd suggesting that at least in part, a common general response to these two heavy metals. The S. metallicus genome contained two complete cop gene clusters, each encoding a metallochaperone (CopM), a Cu-exporting ATPase (CopA), and a transcriptional regulator (CopT). Transcriptional expression analysis revealed that copM and copA from each cop gene cluster were cotranscribed and their transcript levels increased when S. metallicus was grown either in the presence of Cu or using chalcopyrite (CuFeS2) as oxidizable substrate. This study shows for the first time the presence of a duplicated version of the cop gene cluster in Archaea and characterizes some of the Cu and Cd resistance determinants in a thermophilic archaeon employed for industrial biomining.

The Glycogen-Bound Polyphosphate Kinase from Sulfolobus acidocaldarius Is Actually a Glycogen Synthase

ABSTRACT Inorganic polyphosphate (polyP) is obtained by the polymerization of the terminal phosphate of ATP through the action of the enzyme polyphosphate kinase (PPK). Despite the presence of polyP in every living cell, a gene homologous to that of known PPKs is missing from the currently sequenced genomes of Eukarya,Archaea, and several bacteria. To further study the metabolism of polyP in Archaea, we followed the previously published purification procedure for a glycogen-bound protein of 57 kDa with PPK as well as glycosyl transferase (GT) activities from Sulfolobus acidocaldarius (R. Skórko, J. Osipiuk, and K. O. Stetter, J. Bacteriol. 171:5162–5164, 1989). In spite of using recently developed specific enzymatic methods to analyze polyP, we could not reproduce the reported PPK activity for the 57-kDa protein and the polyP presumed to be the product of the reaction most likely corresponded to glycogen-bound ATP under our experimental conditions. Furthermore, no PPK activity was found associated to any of the proteins bound to the glycogen-protein complex. We cloned the gene corresponding to the 57-kDa protein by using reverse genetics and functionally characterized it. The predicted product of the gene did not show similarity to any described PPK but to archaeal and bacterial glycogen synthases instead. In agreement with these results, the recombinant protein showed only GT activity. Interestingly, the GT from S. acidocaldarius was phosphorylated in vivo. In conclusion, our results convincingly demonstrate that the glycogen-protein complex of S. acidocaldarius does not contain a PPK activity and that what was previously reported as being glycogen-bound PPK is a bacterial enzyme-like thermostable glycogen synthase.

Isolation and characterization of a novel Acidithiobacillus ferrivorans strain from the Chilean Altiplano: attachment and biofilm formation on pyrite at low temperature

Microorganisms are used to aid the extraction of valuable metals from low-grade sulfide ores in mines worldwide, but relatively little is known about this process in cold environments. This study comprises a preliminary analysis of the bacterial diversity of the polyextremophilic acid River Aroma located in the Chilean Altiplano, and revealed that Betaproteobacteria was the most dominant bacterial group (Gallionella-like and Thiobacillus-like). Taxa characteristic of leaching environments, such Acidithiobacillus and Leptospirillum, were detected at low abundances. Also, bacteria not associated with extremely acidic, metal-rich environments were found. After enrichment in iron- and sulfur-oxidizing media, we isolated and identified a novel psychrotolerant Acidithiobacillus ferrivorans strain ACH. This strain can grow using ferrous iron, sulfur, thiosulfate, tetrathionate and pyrite, as energy sources. Optimal growth was observed in the presence of pyrite, where cultures reached a cell number of 6.5 · 10(7) cells mL(-1). Planktonic cells grown with pyrite showed the presence of extracellular polymeric substances (10 °C and 28 °C), and a high density of cells attached to pyrite grains were observed at 10 °C by electron microscopy. The attachment of cells to pyrite coupons and the presence of capsular polysaccharides were visualized by using epifluorescence microscopy, through nucleic acid and lectin staining with Syto(®)9 and TRITC-Con A, respectively. Interestingly, we observed high cell adhesion including the formation of microcolonies within 21 days of incubation at 4 °C, which was correlated with a clear induction of capsular polysaccharides production. Our data suggests that attachment to pyrite is not temperature-dependent in At. ferrivorans ACH. The results of this study highlight the potential of this novel psychrotolerant strain in oxidation and attachment to minerals under low-temperature conditions.

Sulfur Oxygenase Reductase (Sor) in the Moderately Thermoacidophilic Leaching Bacteria: Studies in Sulfobacillus thermosulfidooxidans and Acidithiobacillus caldus.

The sulfur oxygenase reductase (Sor) catalyzes the oxygen dependent disproportionation of elemental sulfur, producing sulfite, thiosulfate and sulfide. Being considered an “archaeal like” enzyme, it is also encoded in the genomes of some acidophilic leaching bacteria such as Acidithiobacillus caldus, Acidithiobacillus thiooxidans, Acidithiobacillus ferrivorans and Sulfobacillus thermosulfidooxidans, among others. We measured Sor activity in crude extracts from Sb. thermosulfidooxidans DSM 9293T. The optimum temperature for its oxygenase activity was achieved at 75 °C, confirming the “thermophilic” nature of this enzyme. Additionally, a search for genes probably involved in sulfur metabolism in the genome sequence of Sb. thermosulfidooxidans DSM 9293T was done. Interestingly, no sox genes were found. Two sor genes, a complete heterodisulfidereductase (hdr) gene cluster, three tetrathionate hydrolase (tth) genes, three sulfide quinonereductase (sqr), as well as the doxD component of a thiosulfate quinonereductase (tqo) were found. Seven At. caldus strains were tested for Sor activity, which was not detected in any of them. We provide evidence that an earlier reported Sor activity from At. caldus S1 and S2 strains most likely was due to the presence of a Sulfobacillus contaminant.

The ecological coherence of temperature and salinity tolerance interaction and pigmentation in a non-marine vibrio isolated from Salar de Atacama

The occurrence of microorganisms from the Vibrio genus in saline lakes from northern Chile had been evidenced using Numerical Taxonomy decades before and, more recently, by phylogenetic analyses of environmental samples and isolates. Most of the knowledge about this genus came from marine isolates and showed temperature and salinity to be integral agents in shaping the niche of the Vibrio populations. The stress tolerance phenotypes of Vibrio sp. Teb5a1 isolated from Salar de Atacama was investigated. It was able to grow without NaCl and tolerated up to 100 g/L of the salt. Furthermore, it grew between 17° and 49°C (optimum 30°C) in the absence of NaCl, and the range was expanded into cold temperature (4–49°C) in the presence of the salt. Other additional adaptive strategies were observed in response to the osmotic stress: pigment production, identified as the known antibacterial prodigiosin, swimming and swarming motility and synthesis of a polar flagellum. It is possible to infer that environmental congruence might explain the cellular phenotypes observed in Vibrio sp. considering that coupling between temperature and salinity tolerance, the production of antibacterial agents at higher temperatures, flagellation and motility increase the chance of Vibrio sp. to survive in salty environments with high daily temperature swings and UV radiation.

Normalization of Quantitative Real-Time PCR Data of Identified Genes from an Industrial Bioleaching Operation

A strategy for the monitoring and control of genetic expression in an industrial bioleaching process of copper sulphide minerals is developed in order to understand more fully this process and investigate optimization possibilities. The aim of this research is to find a group of housekeeping genes to normalize the genetic expression data associated to the metabolic functions from industrial bioleaching samples obtained through specific real-time PCR. The data includes the quantification of the previously identified genes in the industrial process in samples from different strips (heap sections) and times. Two studies were carried out, one with the gene expression data associated to each species and the other with the expression data from all the genes detected in the industrial process, during a period of 18 months, using the VBA applet geNorm [1]. The first analysis showed that for the archaea Ferroplasma acidiphilum, the gene with the most stable expression codifies to the ribosomal protein S4 (rpS4) and secondly the mercury reductase gene (merA). In the case of Acidithiobacillus ferrooxidans DM and D2 strains, genes merA and pyruvate dehydrogenase (pdhA) presented similar factors of stability. In the community analysis it was concluded that the gene with the most stable expression was pdhA of A.ferrooxidans. A new analysis was done including other genes using geNorm to define the most stable genes for the whole community. In the community analysis it was concluded that the gene with the most stable expression was pdhA of A.ferrooxidans and merA of F.acidiphilum.

Identification and Characterization of a Psychrotolerant Acidithiobacillus Strain from Chilean Altiplano

Mesophilic iron and sulfur-oxidizing acidophiles are commonly used for the extraction of base metals from low-grade sulfide ores in some copper Chilean mines. However, relatively little is known about their activities in cold environments. Some natural ecosystems present in the Andes Mountains, such as the Chilean Altiplano, meet environmental conditions for the growth of psychrotolerant leaching microorganisms. In this work, we obtained enrichment cultures of iron- and sulfur-oxidizing microorganisms from an acid river in the Chilean Altiplano. Molecular identification was performed using PCR products of bacterial 16S rRNA clone libraries, and the sequences analysis revealed the presence of a microorganism related to the recently described psychrotolerant Acidithiobacillus ferrivorans. The Acidithiobacillus strain was able to grow at temperatures ranging between 4 and 30°C, and pH values ranged between 1.7 and 2.5. According to the energy sources, this microorganism was able to grow using ferric iron, sulfur, thiosulfate and tetrathionate. Optimal growth was observed in presence of ferric ion, where the culture reached a potential redox value of 600 mV and a cellular number of 3×107 cells/mL. Molecular analysis of variants of gene encoding for rusticyanin showed that rusB gene was amplified from A. ferrivorans strain and no PCR product was obtained for the rusA gene. Our description is consistent with data previously reported for A. ferrivorans strains. Finally, results of this study highlight the importance and potential of novel native bacterial species proficient in mineral oxidation under low-temperature conditions.

Characterization and Salt Response in Recurrent Halotolerant Exiguobacterium sp. SH31 Isolated From Sediments of Salar de Huasco, Chilean Altiplano

Poly-extremophiles microorganisms have the capacity to inhabit hostile environments and can survive several adverse conditions that include as variations in temperature, pH, and salinity, high levels UV light and atmospheric pressure, and even the presence of toxic compounds and the formation of reactive oxygen species (ROS). A halotolerant Exiguobacterium strain was isolated from Salar de Huasco (Chilean Altiplano), a well-known shallow lake area with variable salinity levels, little human intervention, and extreme environmental conditions, which makes it ideal for the study of resistant mechanisms and the evolution of adaptations. This bacterial genus has not been extensively studied, although its cosmopolitan location indicates that it has high levels of plasticity and adaptive capacity. However, to date, there are no studies regarding the tolerance and resistance to salinity and osmotic pressure. We set out to characterize the Exiguobacterium sp. SH31 strain and describe its phenotypical and genotypical response to osmotic stress. In this context, as a first step to characterize the response to the SH31 strain to salinity and to establish the bases for a molecular study, we proposed to compare its response under three salt conditions (0, 25, and 50 g/l NaCl). Using different physiology, genomic, and transcriptomic approaches, we determined that the bacterium is able to grow properly in a NaCl concentration of up to 50 g/l; however, the best growth rate was observed at 25 g/l. Although the presence of flagella is not affected by salinity, motility was diminished at 25 g/l NaCl and abolished at 50 g/l. Biofilm formation was induced proportionally with increases in salinity, which was expected. These phenotypic results correlated with the expression of related genes: fliG and fliS Motility); opuBA and putP (transport); glnA, proC, gltA, and gbsA (compatible solutes); ywqC, bdlA, luxS y pgaC (biofilm and stress response); and therefore, we conclude that this strain effectively modifies gene expression and physiology in a differential manner when faced with different concentrations of NaCl and these modifications aid survival.

Diversity of Thermophilic Iron-Pyrite-Oxidizing Enrichments from Solfataric Hot Springs in the Chilean Altiplano

Relatively little is known about the microbial communities present in natural environments that meet physico-chemical conditions for the development of potential leaching microorganisms such as thermal ecosystems from the Chilean Altiplano. Thermophilic leaching enrichments were obtained and identified from a high altitude solfataric pound in Lirima hot springs in the Chilean Altiplano. This ecosystem is characterized by hot underground freshwaters, enriched in sulfur compounds showing pH from neutral to acidic. Microbial diversity has been scarcely explored here, and preliminary results demonstrate that hydrothermal pounds are represented by thermophilic anaerobic and acidophilic taxa. Thermophilic leaching cultures in shake flasks were obtained using ferrous iron and pyrite as energy source. The presence of Bacteria and Archaea in oxidizing enrichments was determined by PCR amplification of 16S rRNA genes. A preliminary analysis of microbial diversity using massive sequencing revealed that Bacteria were more abundant than Archaea in both enrichments. Specifically for the iron-oxidizing culture, the majority of the sequences clustered within the Proteobacteria phylum (79%). Among Proteobacteria, the proportion of Betaproteobacteria (42.2%) and Gammaproteobacteria (21.27%) was much higher than that of Alphaproteobacteria (15.5%). Within the Betaproteobacteria class, the most frequent genus was Leptothrix-like. Similar results were obtained for the pyrite oxidizing culture. Interestingly, this study shows the presence of microorganisms close to the Leptothrix genus under low pH conditions (1.7-2.8) and their capacity to grow at high temperatures with ferrous iron or pyrite as sole energy source