Osmotic response in Leptospirillum ferriphilum isolated from an industrial copper bioleaching environment to sulfate.

Iron and sulfur-oxidizing microorganisms play important roles in several natural and industrial processes. Leptospirillum (L.) ferriphilum, is an iron-oxidizing microorganism with a remarkable adaptability to thrive in extreme acidic environments, including heap bioleaching processes, acid mine drainage (AMD) and natural acidic water. A strain of L. ferriphilum (IESL25) was isolated from an industrial bioleaching process in northern Chile. This strain was challenged to grow at increasing concentrations of sulfate in order to assess changes in protein expression profiles, cells shape and to determine potential compatible solute molecules. The results unveiled changes in three proteins: succinyl CoA (SCoA) synthetase, isocitrate dehydrogenase (IDH) and aspartate semialdehyde dehydrogenase (ASD); which were notably overexpressed when the strain grew at elevated concentrations of sulfate. ASD plays a pivotal role in the synthesis of the compatible solute ectoine, which was identified along with hydroxyectoine by using matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF). The relationship between IDH, SCoA, and ectoine production could be due to the TCA cycle, in which both enzymes produce metabolites that can be utilized as precursors or intermediates in the biosynthesis of ectoine. In addition, distinct filamentous cellular morphology in L. ferriphilum IESL25 was observed when growing under sulfate stress conditions. This study highlights a new insight into the possible cellular responses of L. ferriphilum under the presence of high sulfate levels, commonly found in bioleaching of sulfide minerals or AMD environments.

Validation of Genetic Markers Associated to Oxygen Availability in Low-Grade Copper Bioleaching Systems: An Industrial Application.

Forced aeration is one of the major energy consumption factors of the bioleaching process of run-of-mine ore. The effect of aeration in the microbial community has scarcely been studied at industrial level. Leptospirillum ferriphilum is one of the most representative species of the Fe3+ producing population in this kind of systems. We analyzed the effect of oxygen availability on L. ferriphilum by growth activity and transcriptional dynamics of its two terminal oxidases (cbb3 and bd complexes) under different experimental test: culture reactor, bioleaching column, and industrial heap tests. Relatively low O2 availability triggered important changes in the microbial community composition, cell growth, microbial activity and cydAB genes transcription in all cases of study. We assessed the potential role of the terminal oxidases on the adaptation to variable aeration conditions in different lifestyles of L. ferriphilum and identified transcriptional markers associated to oxygen metabolism in an industrial system. An interesting hypothesis about the possible role of the cbb3 complex in the response to oxidative stress as well as their role as a high oxygen-affinity oxidase in L. ferriphilum is proposed and discussed. This study successfully proves the function of the cydAB genes as valid genetic markers for low-grade copper industrial bioleaching systems.

Identification of trehalose as a compatible solute in different species of acidophilic bacteria.

The major industrial heap bioleaching processes are located in desert regions (mainly Chile and Australia) where fresh water is scarce and the use of resources with low water activity becomes an attractive alternative. However, in spite of the importance of the microbial populations involved in these processes, little is known about their response or adaptation to osmotic stress. In order to investigate the response to osmotic stress in these microorganisms, six species of acidophilic bacteria were grown at elevated osmotic strength in liquid media, and the compatible solutes synthesised were identified using ion chromatography and MALDI-TOF mass spectrometry. Trehalose was identified as one of, or the sole, compatible solute in all species and strains, apart from Acidithiobacillus thiooxidans where glucose and proline levels increased at elevated osmotic potentials. Several other potential compatible solutes were tentatively identified by MALDITOF analysis. The same compatible solutes were produced by these bacteria regardless of the salt used to produce the osmotic stress. The results correlate with data from sequenced genomes which confirm that many chemolithotrophic and heterotrophic acidophiles possess genes for trehalose synthesis. This is the first report to identify and quantify compatible solutes in acidophilic bacteria that have important roles in biomining technologies.

Draft genome sequence of Acidithiobacillus thiooxidans CLST isolated from the acidic hypersaline Gorbea salt flat in northern Chile.

10.1601/nm.2199 CLST is an extremely acidophilic gamma-proteobacteria that was isolated from the Gorbea salt flat, an acidic hypersaline environment in northern Chile. This kind of environment is considered a terrestrial analog of ancient Martian terrains and a source of new material for biotechnological applications. 10.1601/nm.2199 plays a key role in industrial bioleaching; it has the capacity of generating and maintaining acidic conditions by producing sulfuric acid and it can also remove sulfur layers from the surface of minerals, which are detrimental for their dissolution. CLST is a strain of 10.1601/nm.2199 able to tolerate moderate chloride concentrations (up to 15 g L-1 Cl-), a feature that is quite unusual in extreme acidophilic microorganisms. Basic microbiological features and genomic properties of this biotechnologically relevant strain are described in this work. The 3,974,949 bp draft genome is arranged into 40 scaffolds of 389 contigs containing 3866 protein-coding genes and 75 RNAs encoding genes. This is the first draft genome of a halotolerant 10.1601/nm.2199 strain. The release of the genome sequence of this strain improves representation of these extreme acidophilic Gram negative bacteria in public databases and strengthens the framework for further investigation of the physiological diversity and ecological function of 10.1601/nm.2199 populations.

Draft genome sequence of chloride-tolerant Leptospirillum ferriphilum Sp-Cl from industrial bioleaching operations in northern Chile.

Leptospirillum ferriphilum Sp-Cl is a Gram negative, thermotolerant, curved, rod-shaped bacterium, isolated from an industrial bioleaching operation in northern Chile, where chalcocite is the major copper mineral and copper hydroxychloride atacamite is present in variable proportions in the ore. This strain has unique features as compared to the other members of the species, namely resistance to elevated concentrations of chloride, sulfate and metals. Basic microbiological features and genomic properties of this biotechnologically relevant strain are described in this work. The 2,475,669 bp draft genome is arranged into 74 scaffolds of 74 contigs. A total of 48 RNA genes and 2,834 protein coding genes were predicted from its annotation; 55 % of these were assigned a putative function. Release of the genome sequence of this strain will provide further understanding of the mechanisms used by acidophilic bacteria to endure high osmotic stress and high chloride levels and of the role of chloride-tolerant iron-oxidizers in industrial bioleaching operations.

Distribution of prokaryotic genetic diversity in athalassohaline lakes of the Atacama Desert, Northern Chile.

Athalassohaline lakes are inland saline aquatic environments with ionic proportions quite different from the dissolved salts in seawater. Prokaryotes inhabiting athalassohaline environments are poorly known and very few of such places have been surveyed for microbial diversity studies around the world. We analyzed the planktonic bacterial and archaeal assemblages inhabiting several of these evaporitic basins in a remote and vast area in northern Chile by PCR-denaturing gradient gel electrophoresis (DGGE) and sequencing of 16S rRNA gene fragments. Most systems were springs and athalassohaline ponds in different saltflats of the Atacama Desert region, including Salar de Llamará (in the Central Depression), Salar de Atacama (in the Pre-Andean Depression) and Salar de Ascotán (in the Altiplano). Overall, we analyzed more than 25 samples from 19 different environments with strong gradients of altitude, qualitative ionic compositions and UV influence. Between 4 and 25 well-defined DGGE bands were detected for Bacteria in each sample, whereas Archaea ranged between 1 and 5. Predominant DGGE bands (defined by intensity and frequency of appearance) were excised from the gel and sequenced. Bacterial assemblages were dominated by the Cytophaga-Flavobacterium-Bacteroides (CFB) phylum and a few Proteobacteria. There was a tendency for increasing contribution of CFB with higher salinities and altitude. Thus, CFB accounted for the major fraction of band intensity in the Ascotán samples and for lower percentages in Atacama and Llamará. When the distribution of particular CFB sequences was examined, there were several relatives of Psychroflexus torquis substituting each other as salinity changed in Ascotán. Another set of CFB sequences, very distantly related to Cytophaga marinovorus, was abundant in both Llamará and Atacama at salinities lower than 7%. Archaeal assemblages were dominated by uncultured haloarchaea distantly related to cultured strains mostly obtained from thalassohaline environments. Most of the archaeal sequences did not have a close match with environmental 16S rRNA genes deposited in the database either. Therefore, athalassohaline environments are excellent sources of new microorganisms different from their counterparts in thalassohaline sites and useful tools to relate microbial genetic diversity and environmental characteristics such as changes in salinity (both qualitative and quantitative) and altitude.