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Bioleaching of metal sulfides is performed by diverse microorganisms. The dissolution of metal sulfides occurs via two chemical pathways, either the thiosulfate or the polysulfide pathway. These are determined by the metal sulfides’ mineralogy and their acid solubility. The microbial cell enables metal sulfide dissolution via oxidation of iron(II) ions and inorganic sulfur compounds. Thereby, the metal sulfide attacking agents iron(III) ions and protons are generated. Cells are active either in a planktonic state or attached to the mineral surface, forming biofilms. This review, as an update of the previous one (Vera et al., 2013a), summarizes some recent discoveries relevant to bioleaching microorganisms, contributing to a better understanding of their lifestyle. These comprise phylogeny, chemical pathways, surface science, biochemistry of iron and sulfur metabolism, anaerobic metabolism, cell–cell communication, molecular biology, and biofilm lifestyle. Recent advances from genetic engineering applied to bioleaching microorganisms will allow in the future to better understand important aspects of their physiology, as well as to open new possibilities for synthetic biology applications of leaching microbial consortia.Key points•Leaching of metal sulfides is strongly enhanced by microorganisms•Biofilm formation and extracellular polymer production influences bioleaching•Cell interactions in mixed bioleaching cultures are key for process optimization

Objetivos: Este trabajo es una síntesis que pretende dar cuenta de algunos de los aportes hechos por el Semillero de Investigación “Territorio, Ambiente y Desarrollo”, adscrito al Grupo Interdisciplinario de Estudios sobre el Territorio YUMA-IMA de la Universidad del Tolima, que tiene como objetivo el análisis de la dinámica territorial de una serie de problemas y conflictos ambientales actuales o potenciales, relacionados con proyectos extractivos, su posible incidencia en áreas consideradas de importancia ambiental (ecológica y cultural) y la respuesta social frente a estas situaciones que, en conjunto, integran el Atlas de Conflictos Ambientales del Tolima (ACAT). Materiales y métodos: El ACAT tomó como referencia la experiencia del Atlas de Justicia Ambiental de la Enviromental Justice Organisations, Liabilities and Trade-EJOLT, y fue pensado como una plataforma abierta para facilitar el acceso libre a información sobre proyectos mineros, hidroeléctricos, de hidrocarburos, rellenos sanitarios y agroindustriales a gran escala, en proceso de implementación, con titulación vigente, o con solicitudes de licencias; su construcción se basó en el uso de información espacial de agencias e instituciones estatales, complementada con la georreferenciación a nivel de municipios de las dinámicas de resistencia social. Resultados: Como resultado principal, se evidencia la conflictividad emergente producto del choque entre lo que se puede considerar como ordenamiento ambiental del territorio y las perspectivas del reordenamiento desde la visión hegemónica del extractivismo, que ha tenido respuesta desde el movimiento social que impugna dicha visión, reivindicando la vida y la defensa del territorio.

Extracellular polymeric substances (EPS) produced by microorganisms play a crucial role in various bioprocesses, including bioleaching. The microbial leaching of metal sulfides requires an initial cell attachment, which is facilitated by EPS. These substances are mixtures of polysaccharides, proteins, lipids, and other compounds, and their composition and properties can vary depending on the species, growth conditions, and environmental factors. Despite the significance of iron/sulfur oxidizing species in biomining processes, the knowledge of the interfacial processes between thermoacidophilic archaeal species and mineral surfaces is limited. This study examines the cell surface characteristics and EPS produced by an Acidianus strain. The research was conducted using microscopic techniques, Zeta-potential measurements, spectrophotometric methods, Fourier transform infrared spectroscopy, and fluorescence lectin-binding analysis. The results suggest that non-soluble substrates, such as sulfur or pyrite, induce changes in cell surface structures, including the presence of cell appendages, wider cell envelopes, higher hydrophobicities, and increased EPS production, compared to cells grown with soluble substrates such as tetrathionate or ferrous iron. The EPS mainly consist of proteins and carbohydrates, including glucose, manose, N-acetylgalactosamine, and N-acetylglucosamine residues. This study contributes to a better understanding of the relationship between thermophilic archaea and mineral surfaces in biomining processes.

Bioleaching of metal sulfide ores involves acidophilic microbes that catalyze the chemical dissolution of the metal sulfide bond that is enhanced by attached and planktonic cell mediated oxidation of iron(II)-ions and inorganic sulfur compounds. Leptospirillum spp. often predominate in sulfide mineral-containing environments, including bioheaps for copper recovery from chalcopyrite, as they are effective primary mineral colonizers and oxidize iron(II)-ions efficiently. In this study, we demonstrated a functional diffusible signal factor interspecies quorum sensing signaling mechanism in Leptospirillum ferriphilum and Leptospirillum ferrooxidans that produces (Z)-11-methyl-2-dodecenoic acid when grown with pyrite as energy source. In addition, pure diffusible signal factor and extracts from supernatants of pyrite grown Leptospirillum spp. inhibited biological iron oxidation in various species, and that pyrite grown Leptospirillum cells were less affected than iron grown cells to self inhibition. Finally, transcriptional analyses for the inhibition of iron-grown L. ferriphilum cells due to diffusible signal factor was compared with the response to exposure of cells to N- acyl-homoserine-lactone type quorum sensing signal compounds. The data suggested that Leptospirillum spp. diffusible signal factor production is a strategy for niche protection and defense against other microbes and it is proposed that this may be exploited to inhibit unwanted acidophile species.

The family Acidiferrobacteraceae (order Acidiferrobacterales) currently contains Gram negative, neutrophilic sulfur oxidizers such as Sulfuricaulis and Sulfurifustis, as well as acidophilic iron and sulfur oxidizers belonging to the Acidiferrobacter genus. The diversity and taxonomy of the genus Acidiferrobacter has remained poorly explored. Although several metagenome and bioleaching studies have identified its presence worldwide, only two strains, namely Acidiferrobacter thiooxydans DSM 2932T, and Acidiferrobacter spp. SP3/III have been isolated and made publically available. Using 16S rRNA sequence data publically available for the Acidiferrobacteraceae, we herein shed light into the molecular taxonomy of this family. Results obtained support the presence of three clades Acidiferrobacter, Sulfuricaulis and Sulfurifustis. Genomic analyses of the genome sequences of A. thiooxydansT and Acidiferrobacter spp. SP3/III indicate that ANI relatedness between the SPIII/3 strain and A. thiooxydansT is below 95–96%, supporting the classification of strain SP3/III as a new species within this genus. In addition, approximately 70% of Acidiferrobacter sp. SPIII/3 predicted genes have a conserved ortholog in A. thiooxydans strains. A comparative analysis of iron, sulfur oxidation pathways, genome plasticity and cell-cell communication mechanisms of Acidiferrobacter spp. are also discussed.

Acidithiobacillus species are fundamental players in biofilm formation by acidophile bioleaching communities. It has been previously reported that Acidithiobacillus ferrooxidans possesses a functional quorum sensing mediated by acyl-homoserine lactones (AHL), involved in biofilm formation, and AHLs naturally produced by Acidithiobacillus species also induce biofilm formation in Acidithiobacillus thiooxidans. A c-di-GMP pathway has been characterized in Acidithiobacillus species but it has been pointed out that the c-di-GMP effector PelD and pel-like operon are only present in the sulfur oxidizers such as A. thiooxidans. PEL exopolysaccharide has been recently involved in biofilm formation in this Acidithiobacillus species. Here, by comparing wild type and &Delta, pelD strains through mechanical analysis of biofilm-cells detachment, fluorescence microscopy and qPCR experiments, the structural role of PEL exopolysaccharide and the molecular network involved for its biosynthesis by A. thiooxidans were tackled. Besides, the effect of AHLs on PEL exopolysaccharide production was assessed. Mechanical resistance experiments indicated that the loss of PEL exopolysaccharide produces fragile A. thiooxidans biofilms. qRT-PCR analysis established that AHLs induce the transcription of pelA and pelD genes while epifluorescence microscopy studies revealed that PEL exopolysaccharide was required for the development of AHL-induced biofilms. Altogether these results reveal for the first time that AHLs positively regulate pel genes and participate in the molecular network for PEL exopolysaccharide biosynthesis by A. thiooxidans.

Acidithiobacillus species are fundamental players in biofilm formation by acidophile bioleaching communities. It has been previously reported that Acidithiobacillus ferrooxidans possesses a functional quorum sensing mediated by acyl-homoserine lactones (AHL), involved in biofilm formation, and AHLs naturally produced by Acidithiobacillus species also induce biofilm formation in Acidithiobacillus thiooxidans. A c-di-GMP pathway has been characterized in Acidithiobacillus species but it has been pointed out that the c-di-GMP effector PelD and pel-like operon are only present in the sulfur oxidizers such as A. thiooxidans. PEL exopolysaccharide has been recently involved in biofilm formation in this Acidithiobacillus species. Here, by comparing wild type and ΔpelD strains through mechanical analysis of biofilm-cells detachment, fluorescence microscopy and qPCR experiments, the structural role of PEL exopolysaccharide and the molecular network involved for its biosynthesis by A. thiooxidans were tackled. Besides, the effect of AHLs on PEL exopolysaccharide production was assessed. Mechanical resistance experiments indicated that the loss of PEL exopolysaccharide produces fragile A. thiooxidans biofilms. qRT-PCR analysis established that AHLs induce the transcription of pelA and pelD genes while epifluorescence microscopy studies revealed that PEL exopolysaccharide was required for the development of AHL-induced biofilms. Altogether these results reveal for the first time that AHLs positively regulate pel genes and participate in the molecular network for PEL exopolysaccharide biosynthesis by A. thiooxidans.

En este trabajo nos proponemos abordar las Consultas Populares celebradas en casos de conflictos sociales y ambientales en Colombia como una posibilidad de acción participativa de las comunidades en la lucha por la justicia socioambiental. Presentamos acá la confluencia de la Educación Ambiental de Base Comunitaria en diálogo con el campo de la Ecología Política. Analizaremos los casos en que se aplicó la Consulta Popular en las ciudades de Ibagué y Cajamarca, departamento del Tolima, Colombia, referente a la actuación del movimiento ambiental a frente al conflicto por el proyecto minero La Colosa de la multinacional Anglo Gold Ashanti. Buscamos comprender cómo la lucha social y ambiental puede ingresar al campo político y convertirse en un instrumento de acción comunitaria. Identificamos en las luchas los sujetos involucrados, tanto el movimiento ambientalista junto a la Universidad del Tolima, como los ciudadanos que lo apoyan. Pensar nuevas formas de educación ambiental desde los saberes de los pueblos y sus territorios en disputa se pone en marcha con la práctica para ampliar los horizontes acerca de la resistencia con las empresas neoextrativistas en la América Latina y hacer complejo el tema de los conflictos ambientales es lo que motiva a los autores en dicha praxis.

Acidithiobacillus ferrooxidans is an acidophilic bacterium able to grow in environments with high concentrations of metals. It is a chemolithoautotroph able to form biofilms on the surface of solid minerals to obtain its energy. The response of both planktonic and sessile cells of A. ferrooxidans ATCC 23270 grown in elemental sulfur and adapted to high copper concentration was analyzed by quantitative proteomics. It was found that 137 proteins varied their abundance when comparing both lifestyles. Copper effllux proteins, some subunits of the ATP synthase complex, porins, and proteins involved in cell wall modification increased their abundance in copper-adapted sessile lifestyle cells. On the other hand, planktonic copper-adapted cells showed increased levels of proteins such as: cupreredoxins involved in copper cell sequestration, some proteins related to sulfur metabolism, those involved in biosynthesis and transport of lipopolysaccharides, and in assembly of type IV pili. During copper adaptation a decreased formation of biofilms was measured as determined by epifluorescence microscopy. This was apparently due not only to a diminished number of sessile cells but also to their exopolysaccharides production. This is the first study showing that copper, a prevalent metal in biomining environments causes dispersion of A. ferrooxidans biofilms., Supported by FONDECYT Chile, (Science and Technology Chilean Fund) grant n° 1150791 to C.A. Jerez. The Proteomics Unit of the Spanish National Biotechnology Centre belongs to ProteoRed (PRB2 -ISCIII) and is supported by FIS grant PT13/0001.

Industrial biomining processes are currently focused on metal sulfides and their dissolution, which is catalyzed by acidophilic iron(II)- and/or sulfur-oxidizing microorganisms. Cell attachment on metal sulfides is important for this process. Biofilm formation is necessary for seeding and persistence of the active microbial community in industrial biomining heaps and tank reactors, and it enhances metal release. In this study, we used a method for direct quantification of the mineral-attached cell population on pyrite or chalcopyrite particles in bioleaching experiments by coupling high-throughput, automated epifluorescence microscopy imaging of mineral particles with algorithms for image analysis and cell quantification, thus avoiding human bias in cell counting. The method was validated by quantifying cell attachment on pyrite and chalcopyrite surfaces with axenic cultures of Acidithiobacillus caldus, Leptospirillum ferriphilum, and Sulfobacillus thermosulfidooxidans. The method confirmed the high affinity of L. ferriphilum cells to colonize pyrite and chalcopyrite surfaces and indicated that biofilm dispersal occurs in mature pyrite batch cultures of this species. Deep neural networks were also applied to analyze biofilms of different microbial consortia. Recent analysis of the L. ferriphilum genome revealed the presence of a diffusible soluble factor (DSF) family quorum sensing system. The respective signal compounds are known as biofilm dispersal agents. Biofilm dispersal was confirmed to occur in batch cultures of L. ferriphilum and S. thermosulfidooxidans upon the addition of DSF family signal compounds. IMPORTANCE The presented method for the assessment of mineral colonization allows accurate relative comparisons of the microbial colonization of metal sulfide concentrate particles in a time-resolved manner. Quantitative assessment of the mineral colonization development is important for the compilation of improved mathematical models for metal sulfide dissolution. In addition, deep-learning algorithms proved that axenic or mixed cultures of the three species exhibited characteristic biofilm patterns and predicted the biofilm species composition. The method may be extended to the assessment of microbial colonization on other solid particles and may serve in the optimization of bioleaching processes in laboratory scale experiments with industrially relevant metal sulfide concentrates. Furthermore, the method was used to demonstrate that DSF quorum sensing signals directly influence colonization and dissolution of metal sulfides by mineral-oxidizing bacteria, such as L. ferriphilum and S. thermosulfidooxidans.

The oxidation of arsenic (As) is a key step in its removal from water, and biological oxidation may provide a cost-effective and sustainable method. The biofilm-formation ability of Ancylobacter sp. TS-1, a novel chemolithoautotrophic As oxidizer, was studied for four materials: polypropylene, graphite, sand, and zeolite. After seven days under batch mixotrophic conditions, with high concentrations of As(III) (225 mg·L−1), biofilm formation was detected on all materials except for polypropylene. The results demonstrate As(III)-oxidation of TS-1 biofilms and suggest that the number of active cells was similar for graphite, sand, and zeolite. However, the biofilm biomass follows the specific surface area of each material: 7.0, 2.4, and 0.4 mg VSS·cm−3 for zeolite, sand, and graphite, respectively. Therefore, the observed biofilm-biomass differences were probably associated with different amounts of EPS and inert biomass. Lastly, As(III)-oxidation kinetics were assessed for the biofilms formed on graphite and zeolite under chemolithoautotrophic conditions. The normalized oxidation rate for biofilms formed on these materials was 3.6 and 1.0 mg·L−1·h−1·cm−3, resulting among the highest reported values for As(III)-oxidizing biofilms operated at high-As(III) concentrations. Our findings suggest that biofilm reactors based on Ancylobacter sp. TS-1 are highly promising for their utilization in As(III)-oxidation pre-treatment of high-As(III) polluted waters.

Sin resumen

After 24h of exposure to acidic media, pyrite generates reactive oxygen species (ROS). Freshly-crushed pyrite with grain sizes between 50-100 μm at a 5 % (w/v), pulp density generated 0.17 ± 0.01 mM H2O2, while 10% pyrite generated 0.29 ± 0.01 mM and 30 % pyrite generated approximately 0.83 ± 0.06 mM. These levels of H2O2 inhibit iron oxidation in iron-grown cells of AcidithiobacillusferrooxidansT but not in pyrite-grown cells. ROS originating from pyrite, which was incubated for 24 h in acidic medium, prohibited pyrite dissolution by iron-grown cells, while pyrite-grown cells were adapted to these concentrations of ROS. Periodical addition of 100 μM H2O2 to pyrite cultures inoculated with pyrite-grown cells did not lower iron dissolution as it was observed with iron-grown cells. By high throughput proteomics analysis, an increased expression of proteins related to oxidative stress management, iron-and sulfur oxidation systems, carbon fixation and biofilm formation was observed in biofilm cells grown on pyrite compared to iron-grown cells.

The family Acidiferrobacteraceae (order Acidiferrobacterales) currently contains three genera of chemolithoautotrophs: Sulfuricaulis (2016), Sulfurifustis (2015) and Acidiferrobacter (2011). While the two former are neutrophilic sulfur oxidizers isolated from lake sediments in Japan, the latter is an extremely acidophilic, moderately osmophilic, thermotolerant iron/sulfur oxidizer known to occur in macroscopic streamers in Rio Tinto, Spain and in acid waters worldwide. The type strains of both Sulfuricaulis limnicola (HA5T) and Sulfurifustis variabilis (skN76T) have been sequenced, and the draft genome of the ZJ isolate of Acidiferrobacter thiooxydans (MDCF01) has recently been deposited in public databases. Despite this fact, little evidence on the genomic diversity and evolution of this group has been presented so far. Using comparative genomic analyses and phylogenetic reconstruction strategies, we explored the evolutionary information contained in the available genome sequences to shed light on the taxonomic status of a novel isolate of the genus Acidiferrobacter (SP-III/3; DSM 27195).

We studied the surface properties of F. acidiphilum DSM 28986 by attenuated total reflection-Fourier transformed infra-red (ATR-FTIR) spectroscopy and microbial adhesion to hydrocarbon (MATH) techniques. In addition, extracellular polymeric substances (EPS) were extracted and characterized by conventional colorimetric analysis and fluorescence lectin-binding analysis (FLBA). Results showed that: 1) cells selectively adhered to mineral surfaces and showed maximum attachment to pyrite of approx. 50% within 30 min; 2) EPS synthesis by F. acidiphilum DSM 28986 was influenced by growth substrates; and 3) tightly-bound EPS (capsular EPS) were composed of carbohydrates and proteins. In contrast, loosely-bound EPS (colloidal EPS) were mainly characterized as carbohydrates. Monosaccharides like glucose, fucose, arabinose, galactose, mannose, and sialic acid were detected in the EPS of F. acidiphilum DSM 28986. This study provides first insight into surface characterization of the cell wall-lacking archaeon F. acidiphilum and facilitates the understanding of interactions of this organism with other acidophiles and metal sulfides.

La gestión del riesgo es un componente fundamental para la ordenación de cuencas hidrográficas y, en general, para la gestión integral del territorio; sin embargo, los ejercicios de gestión del riesgo han sido hegemonizados desde las visiones parciales de las ciencias naturales y aplicadas, por lo que se han centrado principalmente en el análisis de amenazas, y se ha dado menor importancia a los componentes de la vulnerabilidad. Este trabajo esboza una propuesta metodológica para el análisis y la espacialización de la vulnerabilidad ante amenazas de inundación, remoción en masa y flujos torrenciales, soportada en un marco teórico holístico y en el uso de sistemas de información geográfica. Dicha metodología se aplicó a una microcuenca del río Combeima, ubicada en el departamento del Tolima. Los resultados obtenidos permiten verificar su viabilidad práctica, siendo además susceptible de generalización.

Biological denitrification typically requires the addition of a supplemental electron donor, which can add a significant operating expense to wastewater treatment facilities. Most common electron donors are organic, but reduced inorganic sulfur compounds (RISCs), such as sulfide (HS−) and elemental sulfur (S0), may be more cost-effective. S0 is an inexpensive and well characterized electron donor, but it provides slow denitrification rates due to its low solubility. A lesser-known RISC is sulfite ( $${\text{SO}}_{3}^{2 - }$$ ), which can be easily produced from S0 by a simple combustion process. Unlike S0, $${\text{SO}}_{3}^{2 - }$$ is highly soluble, and therefore may provide higher denitrification rates. However, very little is known about microbial denitrification with $${\text{SO}}_{3}^{2 - }$$ . Also, $${\text{SO}}_{3}^{2 - }$$ is a strong reductant that reacts abiotically with oxygen and has toxic effects on microorganisms. This paper reviews $${\text{SO}}_{3}^{2 - }$$ in the environment, $${\text{SO}}_{3}^{2 - }$$ chemistry, microbiology, toxicity, and its potential use for denitrification. Since $${\text{SO}}_{3}^{2 - }$$ is an intermediate in the sulfur oxidation pathway of most sulfur-oxidizing microorganisms, it is an energetic electron donor and it should select for a $${\text{SO}}_{3}^{2 - }$$ -oxidizing community. Our review of the literature, as well as our own lab experience, suggests that $${\text{SO}}_{3}^{2 - }$$ can effectively serve as an electron donor for denitrification. Further research is needed to determine the kinetics of $${\text{SO}}_{3}^{2 - }$$ -based denitrification, its toxic threshold for sulfur-oxidizing microorganisms, and its potential inhibition of sensitive species such as nitrifying microorganisms and potential formation of nitrous oxide. Its effect on sludge settling efficiency also should be explored.

The understanding of biofilm formation by bioleaching microorganisms is of great importance for influencing mineral dissolution rates and to prevent acid mine drainage (AMD). Thermo-acidophilic archaea such as Acidianus, Sulfolobus and Metallosphaera are of special interest due to their ability to perform leaching at high temperatures, thereby enhancing leaching rates. In this work, leaching experiments and visualization by microscopy of cell attachment and biofilm formation patterns of the crenarchaeotes Sulfolobus metallicus DSM 6482(T) and the Acidianus isolates DSM 29038 and DSM 29099 in pure and mixed cultures on sulfur or pyrite were studied. Confocal laser scanning microscopy (CLSM) combined with fluorescent dyes as well as fluorescently labeled lectins were used to visualize different components (e.g. DNA, proteins or glycoconjugates) of the aforementioned species. The data indicate that cell attachment and the subsequently formed biofilms were species- and substrate-dependent. Pyrite leaching experiments coupled with pre-colonization and further inoculation with a second species suggest that both species may negatively influence each other during pyrite leaching with respect to initial attachment and pyrite dissolution rates. In addition, the investigation of binary biofilms on pyrite showed that both species were heterogeneously distributed on pyrite surfaces in the form of individual cells or microcolonies. Physical contact between the two species seems to occur, as revealed by specific lectins able to specifically bind single species within mixed cultures.

Reactive oxygen species (ROS) cause oxidative stress and growth inhibition by inactivation of essential enzymes, DNA and lipid damage in microbial cells. Acid mine drainage (AMD) ecosystems are characterized by low pH values, enhanced levels of metal ions and low species abundance. Furthermore, metal sulfides, such as pyrite and chalcopyrite, generate extracellular ROS upon exposure to acidic water. Consequently, oxidative stress management is especially important in acidophilic leaching microorganisms present in industrial biomining operations, especially when forming biofilms on metal sulfides. Several adaptive mechanisms have been described, but the molecular repertoire of responses upon exposure to pyrite and the presence of ROS are not thoroughly understood in acidophiles. In this study the impact of the addition of H2O2 on iron oxidation activity in Acidithiobacillus ferrooxidans DSM 14882T was investigated. Iron(II)- or sulfur-grown cells showed a higher sensitivity toward H2O2 than pyrite-grown ones. In order to elucidate which molecular responses may be involved, we used shot-gun proteomics and compared proteomes of cells grown with iron(II)-ions against biofilm cells, grown for 5 days in presence of pyrite as sole energy source. In total 1157 proteins were identified. 213 and 207 ones were found to have increased levels in iron(II) ion-grown or pyrite-biofilm cells, respectively. Proteins associated with inorganic sulfur compound (ISC) oxidation were among the latter. In total, 80 proteins involved in ROS degradation, thiol redox regulation, macromolecule repair mechanisms, biosynthesis of antioxidants, as well as metal and oxygen homeostasis were found. 42 of these proteins had no significant changes in abundance, while 30 proteins had increased levels in pyrite-biofilm cells. New insights in ROS mitigation strategies, such as importance of globins for oxygen homeostasis and prevention of unspecific reactions of free oxygen that generate ROS are presented for A. ferrooxidans biofilm cells. Furthermore, proteomic analyses provide insights in adaptations of carbon fixation and oxidative phosphorylation pathways under these two growth conditions.

Bioleaching of metal sulfides represents an interfacial process where biofilm formation is important in the initial steps of this process. In technical applications of bioleaching, such as reactor leaching in the temperature range of 50 up to 90 °C and also in (self-heating) heaps, thermophilic archaea play an important role and often are the leaching organisms of choice. Nevertheless, to date there is little information available on the interactions between thermoacidophilic archaea and their natural mineral substrates such as pyrite. Especially for extracellular polymeric substances (EPS) of archaea and their biofilms in bioleaching environments information is rather limited. The present work focused on investigations of biofilm dynamics and EPS production of the thermoacidophilic archaeon Acidianus sp. DSM 29099 under bioleaching conditions. The results show that biofilms are dispersed non-homogeneously on pyrite. Large parts of the pyrite surfaces remain free of cells. Cell detachment from pyrite results in microbial “footprints” which, based on lectin binding assays, consist of mannose, glucose and fucose containing compounds. A monolayer biofilm develops on pyrite after 2–4 days of incubation. In addition, the pyrite surface is covered with a layer of organic compounds. EPS analysis indicates the presence of proteins, polysaccharides and uronic acids, the composition of which varies according to substrate and lifestyle (i.e. planktonic, biofilm cells). This report provides insight into EPS and biofilm characteristics of thermophilic archaea and improves understanding of the mineral-microbial-biofilm interfacial interactions in extreme environments. Moreover, the results on interaction dynamics of archaeal microbial consortia will facilitate the understanding of thermophilic bioleaching.

Deep continental subsurface is defined as oligotrophic environments where microorganisms present a very low metabolic rate. To date, due to the energetic cost of production and maintenance of biofilms, their existence has not been considered in poor porous subsurface rocks. We applied fluorescence in situ hybridization techniques and confocal laser scanning microscopy in samples from a continental deep drilling project to analyze the prokaryotic diversity and distribution and the possible existence of biofilms. Our results show the existence of natural microbial biofilms at all checked depths of the Iberian Pyrite Belt (IPB) subsurface and the co-occurrence of bacteria and archaea in this environment. This observation suggests that multi-species biofilms may be a common and widespread lifestyle in subsurface environments., MINECO

Industrial biomining processes are currently focused on metal sulfides and their dissolution, which is catalyzed by acidophilic iron(II)- and/or sulfur-oxidizing microorganisms. Cell attachment on metal sulfides is important for this process. Biofilm formation is necessary for seeding and persistence of the active microbial community in industrial biomining heaps and tank reactors, and it enhances metal release. In this study, we used a method for direct quantification of the mineral-attached cell population on pyrite or chalcopyrite particles in bioleaching experiments by coupling high-throughput, automated epifluorescence microscopy imaging of mineral particles with algorithms for image analysis and cell quantification, thus avoiding human bias in cell counting. The method was validated by quantifying cell attachment on pyrite and chalcopyrite surfaces with axenic cultures of Acidithiobacillus caldus, Leptospirillum ferriphilum, and Sulfobacillus thermosulfidooxidans. The method confirmed the high affinity of L. ferriphilum cells to colonize pyrite and chalcopyrite surfaces and indicated that biofilm dispersal occurs in mature pyrite batch cultures of this species. Deep neural networks were also applied to analyze biofilms of different microbial consortia. Recent analysis of the L. ferriphilum genome revealed the presence of a diffusible soluble factor (DSF) family quorum sensing system. The respective signal compounds are known as biofilm dispersal agents. Biofilm dispersal was confirmed to occur in batch cultures of L. ferriphilum and S. thermosulfidooxidans upon the addition of DSF family signal compounds.IMPORTANCE The presented method for the assessment of mineral colonization allows accurate relative comparisons of the microbial colonization of metal sulfide concentrate particles in a time-resolved manner. Quantitative assessment of the mineral colonization development is important for the compilation of improved mathematical models for metal sulfide dissolution. In addition, deep-learning algorithms proved that axenic or mixed cultures of the three species exhibited characteristic biofilm patterns and predicted the biofilm species composition. The method may be extended to the assessment of microbial colonization on other solid particles and may serve in the optimization of bioleaching processes in laboratory scale experiments with industrially relevant metal sulfide concentrates. Furthermore, the method was used to demonstrate that DSF quorum sensing signals directly influence colonization and dissolution of metal sulfides by mineral-oxidizing bacteria, such as L. ferriphilum and S. thermosulfidooxidans.

This study describes the feeding ecology of white croaker (Micropogonias furnieri) and rough scad (Trachurus lathami) larvae in the Rio de la Plata Estuary (RPE) during a breeding season. The working hypotheses were that the coexistence of the species under study is favored by spatial segregation and divergence in mouth gape size, which mirrors their diet composition and trophic niche breadth (TNBs), based on prey size. Micropogonias furnieri was more abundant at the innermost stations, while T. lathami was most abundant at outermost stations of the estuary. However, their abundances were uncorrelated to environmental salinity and temperature. Micropogonias furnieri and T. lathami larvae showed differences in their gape growth patterns and in the type of preys ingested, which resulted in low dietary overlap. This did lead to differences in the TNBS and supported the prediction of the trophic differentiation hypothesis and could be interpreted as a mechanism to minimize food competition in the RPE ecosystem.

This study focused on colonization and biofilm formation of a new crenarchaeoteAcidianussp. DSM 29099 on pyrite and chalcopyrite. Confocal laser scanning microscopy (CLSM) in combination with several fluorescent stains was applied to examine spatial distribution of cells and biofilms, as well as extracellular polymeric substances (EPS) production on the substrates. Around 60% and 35% of the inoculum adhered to pyrite and chalcopyrite within 2 h, respectively. Cells ofAcidianussp. DSM 29099 were heterogeneously distributed on both pyrite and chalcopyrite surfaces, while large mineral surfaces remained uncolonized. Biofilm cells on pyrite were often found to be embeded in EPS. EPS residues like mannose and glucose were possibly involved in intial attachment to pyrite. A mature biofilm on pyrite was developed after 2-4 days of incubation.

Biofilm formation by new isolates, Leptospirillum sp. strain ZC, Acidithiobacillus sp. strain 61 and Sulfobacillus sp. strain 6 isolated from different biotopes of sulfide ores in Armenia on pyrite was studied by atomic force microscopy (AFM) combined with epifluorescence microscopy (EFM). It was revealed that all the tested species formed monolayer biofilms The results showed that in contrast to Acidithiobacillus sp. strain 61 and Sulfobacillus sp. strain 6, cells of Leptospirillum sp. strain ZC were often observed by clusters and aggregates.The composition of capsular and colloidal EPS formed by isolated Leptospirillum sp. strain ZC, Acidithiobacillus sp. strain 61 and Sulfobacillus sp. strain 6 was studied. It was revealed that the EPS of all three species grown on iron (II) mainly consisted of carbohydrates, proteins as well as uronic acids.

Biofilm formation of microorganisms on relevant surfaces is of great importance for biomining and acid mine drainage (AMD). Thermo-acidophilic archaea like Acidianus, Sulfolobus and Metallosphaera are of special interest due to their ability to enhance leaching rates. Visualization and investigation of microbial attachment and biofilm formation of metal-oxidizing organisms up to now has been done mostly with mesophilic or moderately thermophilic bacteria. In this study, attachment and biofilms by the crenarchaeota Sulfolobus metallicus DSM 6482T and a new isolate Acidianus sp. DSM 29099 on sulfur or pyrite were analyzed. Confocal laser scanning microscopy (CLSM) combined with fluorescent dyes specific for nucleic acids or glycoconjugates were used to monitor biofilm formation on surfaces. The data indicate that cell attachment and the subsequently formed biofilm structures were species and substrate dependent. The investigation of binary biofilms on pyrite showed that both species were heterogeneously distributed on pyrite surfaces in the form of individual cells or microcolonies. In addition, physical contact between the two species was visible, as revealed by specific lectins able to distinguish single species.

Acidithiobacillus ferrooxidansandAcidithiobacillus ferrivoransare used in bioleaching to recover metals such as copper or gold from low-grade ores.At. ferrooxidansis one of the best studied bioleaching microorganisms.At. ferrivoransis known for its ability to leach ores at low temperature. Both microorganisms are involved in acid mine drainage (AMD) formation. The cells are embedded in extracellular polymeric substances (EPS), which play an important role in the attachment to metal-sulfide-surfaces and in catalysis of their dissolution. EPS amounts and compositions differ, depending on the energy source, as it was shown forAt. ferrooxidansR1. Recently,At. ferrooxidansstrains were reclassified into four different subgroups, which are considered to correspond to four different species. As well, a biofilm study, using three of these closely related iron-oxidizingAcidithiobacillusspecies, demonstrated differences regarding attachment to pyrite among them. Consequently, this study focuses on the investigation ofAt. ferrooxidansandAt. ferrivoransspecies and their EPS composition with regard to varying cultivation conditions. Also different energy sources like, elemental sulfur or pyrite were tested. EPS were extracted by the use of a cation exchange resin (DOWEX). Obtained EPS were analyzed for their total amount of proteins, extracellular DNA (eDNA) and carbohydrates.

Reactive oxygen species (ROS), such as hydrogen peroxide (H2O2), superoxide (O2-) and hydroxyl radicals (OH.) are known to be formed on the surface of metal sulfides in aqueous solution under oxic and anoxic conditions. Consequently bacteria which have not been adapted to their presence are metabolically inhibited [1], presumably due to the presence of these ROS. Pyrite-grown cells ofAcidithiobacillus ferrooxidansT, in contrast to iron (II)-grown cells, were able to oxidize iron (II)-ions or pyrite after 24 h starvation and contact with 1 mM externally added H2O2. In this study, similar results were obtained withAcidiferrobactersp. SPIII/3. However,Acidithiobacillus ferrivoransSS3 showed the highest tolerance towards contact with H2O2, whileLeptospirillum ferrooxidansDSM 2391 was most sensitive. Similar results were obtained after exposure to defined doses of gamma radiation, which cleaves water molecules and generates ROS. In this study members of the three aforementioned genera of mineral-oxidizing bacteria were compared regarding their ability to survive, colonize pyrite and to oxidize iron (II)-ions after exposure to different concentrations of H2O2. Pyrite colonization was studied after exposure to endogenous ROS formed on pyrite or after external addition of H2O2using confocal laser scanning microscopy (CLSM).

Society’s demand for metals is ever increasing while stocks of high-grade minerals are being depleted. Biomining, for example of chalcopyrite for copper recovery, is a more sustainable biotechnological process that exploits the capacity of acidophilic microbes to catalyze solid metal sulfide dissolution to soluble metal sulfates. A key early stage in biomining is cell attachment and biofilm formation on the mineral surface that results in elevated mineral oxidation rates. Industrial biomining of chalcopyrite is typically carried out in large scale heaps that suffer from the downsides of slow and poor metal recoveries. In an effort to mitigate these drawbacks, this study investigated planktonic and biofilm cells of acidophilic (optimal growth pH, Measurement(s)biofilm formation • Proteome • protein expression data • RNA • transcriptomeTechnology Type(s)fluorescence microscopy • mass spectrometry • RNA sequencingSample Characteristic - OrganismLeptospirillum ferriphilum • Sulfobacillus thermosulfidooxidans • Acidithiobacillus caldusSample Characteristic - Environmentmine Machine-accessible metadata file describing the reported data: 10.6084/m9.figshare.12173637

In this study, initial attachment to and biofilm formation of Sulfobacillusthermosulfidooxidans DSM 9293T on pyrite in the presence of Leptospirillumferriphilum DSM 14647T were investigated. Interactions of S.thermosulfidooxidansT and L.ferriphilumT were studied by means of monitoring attachment behavior and biofilm formation on pyrite. Our preliminary results showed that 1): Pre-established biofilms of L.ferriphilumT had effects on attachment of S. thermosulfidooxidansT to pyrite; 2): physical contact between cells of L. ferriphilumT and S. thermosulfidooxidansT on pyrite were visible 3): Pyrite leaching by cells of S. thermosulfidooxidansT was inhibited by the presence of inactive cells of L.ferriphilumT.

Bioleaching corresponds to the microbial-catalyzed process of conversion of insoluble metals into soluble forms. As an applied biotechnology globally used, it represents an extremely interesting field of research where omics techniques can be applied in terms of knowledge development, but moreover in terms of process design, control, and optimization. In this mini-review, the current state of genomics, proteomics, and metabolomics of bioleaching and the major impacts of these analytical methods at industrial scale are highlighted. In summary, genomics has been essential in the determination of the biodiversity of leaching processes and for development of conceptual and functional metabolic models. Proteomic impacts are mostly related to microbe-mineral interaction analysis, including copper resistance and biofilm formation. Early steps of metabolomics in the field of bioleaching have shown a significant potential for the use of metabolites as industrial biomarkers. Development directions are given in order to enhance the future impacts of the omics in biohydrometallurgy.

Biofilms are surface-associated colonies of microorganisms embedded in a matrix of extracellular polymeric substances (EPS). As EPS mediate the contact between cells and surfaces, an understanding of their composition and production is of particular interest. In this study, the EPS components of Sulfolobus metallicus DSM 6482(T) forming biofilms on elemental sulfur (S(0)) were investigated by confocal laser scanning microscopy (CLSM). In order to visualize cell and EPS distributions, biofilm cells were stained with various dyes specific for glycoconjugates, proteins, nucleic acids and lipids. Biofilm cells on S(0) were heterogeneously distributed and characterized as individual cells, microcolonies, and large clusters up to a hundred micrometers in diameter. The glycoconjugates in biofilms were detected by fluorescence lectin-binding analysis (FLBA). Screening of 72 commercially available lectins resulted in the selection of 21 lectins useful for staining biofilms of S. metallicus (T). Capsular EPS from planktonic cells were mainly composed of carbohydrates and proteins. In contrast, colloidal EPS from planktonic cells were dominated by carbohydrates. Proteins were found to be major components in EPS from biofilms on S(0). Using specific probes combined with CLSM, we showed that extracellular proteins and nucleic acids were present in the EPS matrix. Finally, we showed that S. metallicus (T) cells were embedded in a flexible EPS matrix. This study provides new insights into archaeal biofilms and EPS composition and properties with respect to their interactions with S(0).

Special issue in honor of Bernard Tribollet Edited by Nadine Pébère, Oscar Rosa Mattos, Marco Musiani, Mark E. Orazem and Vincent Vivier; International audience; Copper alloys, often used in cooling circuits of industrial plants using seawater as coolant, can be affectedby biocorrosion induced by biofilm formation. Extracellular polymeric substances (EPS) produced by bacteriaplay a fundamental role in the different stages of biofilm formation, maturation and maintenance.The influence of loosely bound (LB) and tightly bound (TB) EPS, extracted from marine PseudomonasNCIMB 2021, on the electrochemical behaviour of 70Cu–30Ni (wt.%) alloy in static artificial seawater(ASW) and on the chemical composition of oxide layers was studied by combined electrochemical measurements(polarization curves, EIS) and surface analysis (XPS, ToF-SIMS). Results were compared withthose obtained in the presence of bovine serum albumin (BSA), a model protein. Compared to 70Cu–30Ni alloy in static ASW without biomolecules, for which a thick duplex oxide layer (outer redepositedCu2O layer and inner oxidized nickel layer) is shown, the presence of BSA, TB EPS and LB EPS leads toa mixed oxide layer (oxidized copper and nickel) with a lower thickness. In the biomolecules-containingsolutions, this oxide layer is covered by an adsorbed organic layer, mainly composed of proteins. A modelis proposed to analyse impedance data obtained at the corrosion potential. The fitting procedure ofimpedance diagrams allows extracting the anodic charge transfer resistance, from which the corrosioncurrent density can be calculated. The results show a slow-down of the anodic reaction in the presenceof biomolecules (BSA, TB EPS and LB EPS), and a corrosion inhibition effect by LB EPS and to a lesser extentby BSA. No detrimental effect is evidenced with TB EPS.