The First Description of the Microbial Diversity in the Amarillo River (La Rioja, Argentina), a Natural Extreme Environment Where the Whole Microbial Community Paints the Landscape Yellow.

The Amarillo River in Famatina, La Rioja, Argentina, is a natural acidic river with distinctive yellow-ochreous iron precipitates along its course. While mining activities have occurred in the area, the river's natural acidity is influenced by environmental factors beyond mineralogy, where microbial species have a crucial role. Although iron-oxidising bacteria have been identified, a comprehensive analysis of the entire microbial community in this extreme environment has not yet been conducted. In this study, we employ high-throughput sequencing to explore the bacterial and fungal diversity in the Amarillo River and Cueva de Pérez terraces, considered prehistoric analogues of the current river basin. Fe(II)-enrichment cultures mimicking different environmental conditions of the river were also analysed to better understand the roles of prokaryotes and fungi in iron oxidation processes. Additionally, we investigate the ecological relationships between bacteria and fungi using co-occurrence and network analysis. Our findings reveal a diverse bacterial community in the river and terraces, including uncultured species affiliated with Acidimicrobiia, part of an uncharacterised universal microbial acidic diversity. Acidophiles such as Acidithiobacillus ferrivorans, the main iron oxidiser of the system, and Acidiphilium, which is unable to catalyse Fe(II) oxidation but has a great metabolic flexibility,, are part of the core of the microbial community, showing significant involvement in intraspecies interactions. Alicyclobacillus, which is the main Fe(II) oxidiser in the enrichment culture at 30 °C and is detected all over the system, highlights its flexibility towards the iron cycle. The prevalence of key microorganisms in both rivers and terraces implies their enduring contribution to the iron cycle as well as in shaping the iconic yellow landscape of the Amarillo River. In conclusion, this study enhances our understanding of microbial involvement in iron mineral precipitation, emphasising the collaborative efforts of bacteria and fungi as fundamental geological agents in the Amarillo River.

Evaluation of the sequential coupling of a bacterial treatment with a physicochemical process for the remediation of wastewater containing Cr and organic pollutants.

A restoration strategy was developed for the treatment of two artificial liquid systems (Minimal Medium, MM, and Water Carbon Nitrogen, WCN) contaminated with Cr(VI), lindane (γ-HCH), phenanthrene (Phe), and reactive black 5 (RB5), through the use of an actinobacteria consortium, coupled with a physicochemical treatment using a column filled with nano-scale zero valent iron particles immobilized on dried Macrocystis pyrifera algae biomass. The Sequential Treatment A (STA: physicochemical followed by biological method) removed the three organic compounds with different effectiveness; however, it was very ineffective for Cr(VI) removal. The Sequential Treatment B (STB: biological followed by the physicochemical method) removed the four compounds with variable efficiencies. The removal of γ-HCH, Phe, and RB5 in both effluents did not present significant differences, regardless of the sequential treatment used. The highest removal of Cr(VI) and total Cr was observed in MM and WCN, respectively. Ecotoxicity tests (L. sativa) of the effluents treated with both methodological couplings demonstrated that the toxicity of WCN only decreased at the end of STA, while that of MM decreased at all stages of both sequential treatments. Therefore, MM would be more appropriate to perform both treatments.

Influence of Extremophiles on the Generation of Acid Mine Drainage at the Abandoned Pan de Azúcar Mine (Argentina).

The risk of generation of acid drainages in the tailings of the Pan de Azúcar mine that closed its activities more than three decades ago, was evaluated through biooxidation studies using iron- and sulfur-oxidizing extremophilic leaching consortia. Most of tailings showed a high potential for generating acid drainage, in agreement with the results from net acid generation (NAG) assays. In addition, molecular analysis of the microbial consortia obtained by enrichment of the samples, demonstrated that native leaching microorganisms are ubiquitous in the area and they seemed to be more efficient in the biooxidation of the tailings than the collection microorganisms. The acid drainages detected at the site and those formed by oxidation of the tailings, produced a significant ecotoxicological effect demonstrated by a bioassay. These drainages, even at high dilutions, could seriously affect a nearby Ramsar site (Laguna de Pozuelos) that is connected to the Pan de Azúcar mine through a hydrological route (Cincel River).

Effect of heavy metal-induced stress on two extremophilic microbial communities from Caviahue-Copahue, Argentina.

Metal pollution is a great concern worldwide and the development of new technologies for more sustainable extraction methods as well as for the remediation of polluted sites is essential. Extremophilic microorganisms are attractive for this purpose since they have poly-resistance mechanisms which make them versatile. In this work, we sampled an acidic river and a hot spring of Caviahue-Copahue volcanic environment. The indigenous microbial communities were exposed to five heavy metals (Cd, Co, Cu, Ni and Zn) in batch-cultures favouring different metabolisms of biotechnological interest. Remarkably, high tolerance values were reached in all the cultures, even though most of the metals studied were not present in the environmental sample. Particularly, outstanding tolerances were exhibited by acidophiles, which grew at concentrations as high as 400 mM of Zn and Ni. High-throughput amplicon sequencing of 16S rRNA gene was used to study the indigenous communities and the resistant consortia. We took three approaches for the analysis: phylotypes, OTUs and amplicon sequence variants (ASVs). Interestingly, similar conclusions were drawn in all three cases. Analysing the phylogenetic structure and functional potential of the adapted consortia, we found that the strongest selection was exerted by the culture media. Notably, there was a poor correlation between alpha diversity and metal stress; furthermore, metal stress did not seem to harm the functional potential of the consortia. All these results reveal a great adaptability and versatility. At the end, 25 metal-resistant extremophilic consortia with potential uses in bioremediation, bioleaching or biomonitoring processes were obtained.

Acidophilic microorganisms enhancing geochemical dynamics in an acidic drainage system, Amarillo river in La Rioja, Argentina.

The Amarillo River in La Rioja, Argentina, is a natural acidic environment that is influenced by an abandoned mine. The river is characterized by extremely low pH and high concentrations of metals and metalloids. Fe(III)-bearing neoformed precipitated minerals are widespread along the hydrological basin. This work reports the presence of different species of iron-oxidizing bacteria and demonstrates that their action has a significant role in geochemical processes of the Amarillo River, mainly by catalyzing Fe2+ oxidation and intensifying the Fe(III)-bearing mineral precipitation. Various iron oxidizers (i.e. Acidithiobacillus ferrivorans, Leptospirillum ferrooxidans, Ferrimicrobium acidophilum, Alicyclobacillus cycloheptanicus) were detected in enrichment cultures at different temperatures. Moreover, this is the first report confirming that Acidithiobacillus ferrivorans is able to grow at 4 °C. Other acidophilic bacteria (i.e., Acidiphilium iwatensii) and fungi (e.g., Fodinomyces uranophilus, Coniochaeta fodinicola, Acidea extrema, Penicillium sp. and Cladosporium pseudocladosporioides) were also detected. In vitro laboratory studies recreating natural Fe(III)-bearing mineral formation showed that mineral precipitation rate was higher than 350 mg L-1 day-1 in the presence of microorganisms whereas it was about 15 mg L-1 day-1 under abiotic conditions. Jarosite was the only mineral detected in the precipitates generated by microbial action and it was also identified in the Amarillo River bed sediments. Biological Fe2+ oxidation rates depend on temperature which range from 8 to 32 mM day-1 at 4 and 30 °C, respectively. Finally, a conceptual model recognizing the significant microbial role is proposed to gain a better understanding of the biogeochemistry dynamics of the Amarillo River.

Importance of Initial Interfacial Steps during Chalcopyrite Bioleaching by a Thermoacidophilic Archaeon.

Studies of thermophilic microorganisms have shown that they have a considerable biotechnological potential due to their optimum growth and metabolism at high temperatures. Thermophilic archaea have unique characteristics with important biotechnological applications; many of these species could be used in bioleaching processes to recover valuable metals from mineral ores. Particularly, bioleaching at high temperatures using thermoacidophilic microorganisms can greatly improve metal solubilization from refractory mineral species such as chalcopyrite (CuFeS2), one of the most abundant and widespread copper-bearing minerals. Interfacial processes such as early cell adhesion, biofilm development, and the formation of passive layers on the mineral surface play important roles in the initial steps of bioleaching processes. The present work focused on the investigation of different bioleaching conditions using the thermoacidophilic archaeon Acidianus copahuensis DSM 29038 to elucidate which steps are pivotal during the chalcopyrite bioleaching. Fluorescent in situ hybridization (FISH) and confocal laser scanning microscopy (CLSM) were used to visualize the microorganism-mineral interaction. Results showed that up to 85% of copper recovery from chalcopyrite could be achieved using A. copahuensis. Improvements in these yields are intimately related to an early contact between cells and the mineral surface. On the other hand, surface coverage by inactivated cells as well as precipitates significantly reduced copper recoveries.

Meta-Analysis of Microbial Communities in Hot Springs: Recurrent Taxa and Complex Shaping Factors beyond pH and Temperature.

The study of microbial communities from extreme environments is a fascinating topic. With every study, biologists and ecologists reveal interesting facts and questions that dispel the old belief that these are inhospitable environments. In this work, we assess the microbial diversity of three hot springs from Neuquén, Argentina, using high-throughput amplicon sequencing. We predicted a distinct metabolic profile in the acidic and the circumneutral samples, with the first ones being dominated by chemolithotrophs and the second ones by chemoheterotrophs. Then, we collected data of the microbial communities of hot springs around the world in an effort to comprehend the roles of pH and temperature as shaping factors. Interestingly, there was a covariation between both parameters and the phylogenetic distance between communities; however, neither of them could explain much of the microbial profile in an ordination model. Moreover, there was no correlation between alpha diversity and these parameters. Therefore, the microbial communities' profile seemed to have complex shaping factors beyond pH and temperature. Lastly, we looked for taxa associated with different environmental conditions. Several such taxa were found. For example, Hydrogenobaculum was frequently present in acidic springs, as was the Sulfolobaceae family; on the other hand, Candidatus Hydrothermae phylum was strongly associated with circumneutral conditions. Interestingly, some singularities related to sites featuring certain taxa were also observed.

Bacillus cytotoxicus Isolated from a Pristine Natural Geothermal Area Reveals High Keratinolytic Activity.

Geothermal areas are the niches of a rich microbial diversity that is not only part of the intangible patrimony of a country but also the source of many microbial species with potential biotechnological applications. Particularly, microbial species in geothermal areas in Argentina have been scarcely explored regarding their possible biotechnological uses. The purpose of this work was to explore the proteolytic and keratinolytic enzymatic potential of microorganisms that inhabit in the Domuyo geothermal area in the Neuquén Province. To this end, we did enrichment cultures from two high-temperature natural samples in mineral media only supplemented with whole chicken feathers. After the isolation and the phylogenetic and morphologic characterization of different colonies, we obtained a collection of Bacillus cytotoxicus isolates, a species with no previous report of keratinolytic activity and only reported in rehydrated meals connected with food poisoning outbreaks. Its natural habitat has been unknown up to now. We characterized the proteolytic and keratinolytic capacities of the B. cytotoxicus isolates in different conditions, which proved to be remarkably high compared with those of other similar species. Thus, our work represents the first report of the isolation as well as the keratinolytic capacity characterization of strains of B. cytotixicus obtained from a natural environment.

Enrichment and isolation of acid-tolerant sulfate-reducing microorganisms in the anoxic, acidic hot spring sediments from Copahue volcano, Argentina.

The geothermal Copahue-Caviahue (GCC) system (Argentina) is an extreme acidic environment, dominated by the activity of Copahue volcano. Environments characterised by low pH values, such as volcanic areas, are of particular interest for the search of acidophilic microorganisms with application in biotechnological processes. In this work, sulfate-reducing microorganisms were investigated in geothermal acidic, anaerobic zones from GCC system. Sediment samples from Agua del Limón (AL1), Las Máquinas (LMa2), Las Maquinitas (LMi) and Baño 9 (B9-2, B9-3) were found to be acidic (pH values 2.1-3.0) to moderate acidic (5.1-5.2), containing small total organic carbon values, and ferric iron precipitates. The organic electron donor added to the enrichment was completely oxidised to CO2. Bacteria related to 'Desulfobacillus acidavidus' strain CL4 were found to be dominant (67-83% of the total number of clones) in the enrichment cultures, and their presence was confirmed by their isolation on overlay plates. Other bacteria were also detected with lower abundance (6-20% of the total number of clones), with representatives of the genera Acidithiobacillus, Sulfobacillus, Alicyclobacillus and Athalassotoga/Mesoaciditoga. These enrichment and isolates found at low pH confirm the presence of anaerobic activities in the acidic sediments from the geothermal Copahue-Caviahue system.

Arsenic-tolerant microbial consortia from sediments of Copahue geothermal system with potential applications in bioremediation.

Although arsenic (As) is recognized as a toxic element for living species, some microorganisms have the ability to tolerate and transform it; recent studies have proposed to take advantage of such capacity to develop sustainable bioremediation strategies. In this study, we evaluated the adaptation to increasing concentrations of As(III) and As(V) of three metabolically different microbial cultures (heterotrophic, autotrophic-acidophilic, and anaerobic) obtained from a sample with low-soluble As content from the Copahue geothermal system. At the end of the adaptation process, the heterotrophic culture was able to grow at 20 mM and 450 mM of As(III) and As(V), respectively; the autotrophic-acidophilic culture showed tolerance to 15 mM of As(III) and 150 mM of As(V), whereas the anaerobic culture only developed in As(V) at concentrations up to 50 mM. The most tolerant consortia were characterized by their growth performance, complexity, and the presence of genes related to As metabolism and resistance. Regarding the consortia complexity, the predominant genera identified were: Paenibacillus in both heterotrophic consortia, Acidithiobacillus in the autotrophic-acidophilic consortium tolerant to As(III), Acidiphilium in the autotrophic-acidophilic consortium tolerant to As(V), and Thiomonas and Clostridium in the anaerobic consortium. This study is the first report of As tolerance microorganisms obtained from Copahue and reasserts the versatility and flexibility of the community of this natural extreme environment; also, it opens the door to the study of possible uses of these consortia in the design of biotechnological processes where the As concentration may fluctuate.

Domestication of Local Microbial Consortia for Efficient Recovery of Gold Through Top-Down Selection in Airlift Bioreactors.

Extreme acidophiles play central roles in the geochemical cycling of diverse elements in low pH environments. This has been harnessed in biotechnologies such as biomining, where microorganisms facilitate the recovery of economically important metals such as gold. By generating both extreme acidity and a chemical oxidant (ferric iron) many species of prokaryotes that thrive in low pH environments not only catalyze mineral dissolution but also trigger both community and individual level adaptive changes. These changes vary in extent and direction depending on the ore mineralogy, water availability and local climate. The use of indigenous versus introduced microbial consortia in biomining practices is still a matter of debate. Yet, indigenous microbial consortia colonizing sulfidic ores that have been domesticated, i.e., selected for their ability to survive under specific polyextreme conditions, are claimed to outperform un-adapted foreign consortia. Despite this, little is known on the domestication of acidic microbial communities and the changes elicited in their members. In this study, high resolution targeted metagenomic techniques were used to analyze the changes occurring in the community structure of local microbial consortia acclimated to growing under extreme acidic conditions and adapted to endure the conditions imposed by the target mineral during biooxidation of a gold concentrate in an airlift reactor over a period of 2 years. The results indicated that operative conditions evolving through biooxidation of the mineral concentrate exerted strong selective pressures that, early on, purge biodiversity in favor of a few Acidithiobacillus spp. over other iron oxidizing acidophiles. Metagenomic analysis of the domesticated consortium present at the end of the adaptation experiment enabled reconstruction of the RVS1-MAG, a novel representative of Acidithiobacillus ferrooxidans from the Andacollo gold mineral district. Comparative genomic analysis performed with this genome draft revealed a net enrichment of gene functions related to heavy metal transport and stress management that are likely to play a significant role in adaptation and survival to adverse conditions experienced by these acidophiles during growth in presence of gold concentrates.

A Deeper Look into the Biodiversity of the Extremely Acidic Copahue volcano-Río Agrio System in Neuquén, Argentina.

The Copahue volcano-Río Agrio system, on Patagonia Argentina, comprises the naturally acidic river Río Agrio, that runs from a few meters down the Copahue volcano crater to more than 40 km maintaining low pH waters, and the acidic lagoon that sporadically forms on the crater of the volcano, which is studied for the first time in this work. We used next-generation sequencing of the 16S rRNA gene of the entire prokaryotic community to study the biodiversity of this poorly explored extreme environment. The correlation of the operational taxonomic units (OTUs)s presence with physicochemical variables showed that the system contains three distinct environments: the crater lagoon, the Upper Río Agrio, and the Salto del Agrio waterfall, a point located approximately 12 km down the origin of the river, after it emerges from the Caviahue lake. The prokaryotic community of the Copahue Volcano-Río Agrio system is mainly formed by acidic bacteria and archaea, such as Acidithiobacillus, Ferroplasma, and Leptospirillum, which have been isolated from similar environments around the world. These results support the idea of a ubiquitous acidic biodiversity; however, this highly interesting extreme environment also has apparently autochthonous species such as Sulfuriferula, Acidianus copahuensis, and strains of Acidibacillus and Alicyclobacillus.

Combined strategy for removal of Reactive Black 5 by biomass sorption on Macrocystis pyrifera and zerovalent iron nanoparticles.

Reactive Black 5, RB5, has been used as a model azo dye to evaluate the removal efficiency of sorption on Macrocystis pyrifera biomass (Mpyr) and commercial zerovalent iron nanoparticles (nZVI) in individual and combined treatments. The best conditions for the treatment with the isolated materials were first determined, and then, in series and combined treatments were performed under these conditions, achieving removal efficiencies higher than 80% of the initial dye concentration. Strengths and weaknesses of all removal strategies (individual, in series and combined) are analyzed regarding the application on real effluents. Mpyr efficiently adsorbed RB5, but also increased the total organic content by partial dissolution of components of the algal biomass. Removal experiments with commercial nZVI were also efficient but liberated Fe to the solution, and sulfanilic acid was observed after the treatment as a product of RB5 degradation. In contrast, after the Mpyr treatment, no sulfanilic acid was detected, suggesting that sulfanilic acid is efficiently adsorbed by the biomass. The best condition was the integrated use of Mpyr and nZVI, with a remarkable removal efficiency (69-80%) obtained after only 1 h of treatment. Finally, nZVI were successfully immobilized in Mpyr, and the hybrid material was used to remove RB5 in continuous flow experiments at pH 3, obtaining a removal capacity of 39.9 mg RB5 g-1 after a total processed volume of 630 mL of [RB5]0 = 100 mg L-1.

Effects of Physiochemical Factors on Prokaryotic Biodiversity in Malaysian Circumneutral Hot Springs.

Malaysia has a great number of hot springs, especially along the flank of the Banjaran Titiwangsa mountain range. Biological studies of the Malaysian hot springs are rare because of the lack of comprehensive information on their microbial communities. In this study, we report a cultivation-independent census to describe microbial communities in six hot springs. The Ulu Slim (US), Sungai Klah (SK), Dusun Tua (DT), Sungai Serai (SS), Semenyih (SE), and Ayer Hangat (AH) hot springs exhibit circumneutral pH with temperatures ranging from 43°C to 90°C. Genomic DNA was extracted from environmental samples and the V3-V4 hypervariable regions of 16S rRNA genes were amplified, sequenced, and analyzed. High-throughput sequencing analysis showed that microbial richness was high in all samples as indicated by the detection of 6,334-26,244 operational taxonomy units. In total, 59, 61, 72, 73, 65, and 52 bacterial phyla were identified in the US, SK, DT, SS, SE, and AH hot springs, respectively. Generally, Firmicutes and Proteobacteria dominated the bacterial communities in all hot springs. Archaeal communities mainly consisted of Crenarchaeota, Euryarchaeota, and Parvarchaeota. In beta diversity analysis, the hot spring microbial memberships were clustered primarily on the basis of temperature and salinity. Canonical correlation analysis to assess the relationship between the microbial communities and physicochemical variables revealed that diversity patterns were best explained by a combination of physicochemical variables, rather than by individual abiotic variables such as temperature and salinity.

Genome analysis of the thermoacidophilic archaeon Acidianus copahuensis focusing on the metabolisms associated to biomining activities.

BACKGROUND: Several archaeal species from the order Sulfolobales are interesting from the biotechnological point of view due to their biomining capacities. Within this group, the genus Acidianus contains four biomining species (from ten known Acidianus species), but none of these have their genome sequenced. To get insights into the genetic potential and metabolic pathways involved in the biomining activity of this group, we sequenced the genome of Acidianus copahuensis ALE1 strain, a novel thermoacidophilic crenarchaeon (optimum growth: 75 °C, pH 3) isolated from the volcanic geothermal area of Copahue at Neuquén province in Argentina. Previous experimental characterization of A. copahuensis revealed a high biomining potential, exhibited as high oxidation activity of sulfur and sulfur compounds, ferrous iron and sulfide minerals (e.g.: pyrite). This strain is also autotrophic and tolerant to heavy metals, thus, it can grow under adverse conditions for most forms of life with a low nutrient demand, conditions that are commonly found in mining environments. RESULTS: In this work we analyzed the genome of Acidianus copahuensis and describe the genetic pathways involved in biomining processes. We identified the enzymes that are most likely involved in growth on sulfur and ferrous iron oxidation as well as those involved in autotrophic carbon fixation. We also found that A. copahuensis genome gathers different features that are only present in particular lineages or species from the order Sulfolobales, some of which are involved in biomining. We found that although most of its genes (81%) were found in at least one other Sulfolobales species, it is not specifically closer to any particular species (60-70% of proteins shared with each of them). Although almost one fifth of A. copahuensis proteins are not found in any other Sulfolobales species, most of them corresponded to hypothetical proteins from uncharacterized metabolisms. CONCLUSION: In this work we identified the genes responsible for the biomining metabolisms that we have previously observed experimentally. We provide a landscape of the metabolic potentials of this strain in the context of Sulfolobales and propose various pathways and cellular processes not yet fully understood that can use A. copahuensis as an experimental model to further understand the fascinating biology of thermoacidophilic biomining archaea.

Thermophilic microorganisms in biomining.

Biomining is an applied biotechnology for mineral processing and metal extraction from ores and concentrates. This alternative technology for recovering metals involves the hydrometallurgical processes known as bioleaching and biooxidation where the metal is directly solubilized or released from the matrix for further solubilization, respectively. Several commercial applications of biomining can be found around the world to recover mainly copper and gold but also other metals; most of them are operating at temperatures below 40-50 °C using mesophilic and moderate thermophilic microorganisms. Although biomining offers an economically viable and cleaner option, its share of the world´s production of metals has not grown as much as it was expected, mainly considering that due to environmental restrictions in many countries smelting and roasting technologies are being eliminated. The slow rate of biomining processes is for sure the main reason of their poor implementation. In this scenario the use of thermophiles could be advantageous because higher operational temperature would increase the rate of the process and in addition it would eliminate the energy input for cooling the system (bioleaching reactions are exothermic causing a serious temperature increase in bioreactors and inside heaps that adversely affects most of the mesophilic microorganisms) and it would decrease the passivation of mineral surfaces. In the last few years many thermophilic bacteria and archaea have been isolated, characterized, and even used for extracting metals. This paper reviews the current status of biomining using thermophiles, describes the main characteristics of thermophilic biominers and discusses the future for this biotechnology.

Draft Genome Sequence of the Sulfate-Reducing Bacterium Desulfotomaculum copahuensis Strain CINDEFI1 Isolated from the Geothermal Copahue System, Neuquén, Argentina.

Desulfotomaculum copahuensis strain CINDEFI1 is a novel spore-forming sulfate-reducing bacterium isolated from the Copahue volcano area, Argentina. Here, we present its draft genome in which we found genes related with the anaerobic respiration of sulfur compounds similar to those present in the Copahue environment.