Progress in bioleaching: fundamentals and mechanisms of microbial metal sulfide oxidation - part A.

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.

Extracellular Polymeric Substances and Biocorrosion/Biofouling: Recent Advances and Future Perspectives.

Microbial cells secrete extracellular polymeric substances (EPS) to adhere to material surfaces, if they get in contact with solid materials such as metals. After phase equilibrium, microorganisms can adhere firmly to the metal surfaces causing metal dissolution and corrosion. Attachment and adhesion of microorganisms via EPS increase the possibility and the rate of metal corrosion. Many components of EPS are electrochemical and redox active, making them closely related to metal corrosion. Functional groups in EPS have specific adsorption ability, causing them to play a key role in biocorrosion. This review emphasizes EPS properties related to metal corrosion and protection and the underlying microbially influenced corrosion (MIC) mechanisms. Future perspectives regarding a comprehensive study of MIC mechanisms and green methodologies for corrosion protection are provided.

Reverse engineering directed gene regulatory networks from transcriptomics and proteomics data of biomining bacterial communities with approximate Bayesian computation and steady-state signalling simulations.

BACKGROUND: Network inference is an important aim of systems biology. It enables the transformation of OMICs datasets into biological knowledge. It consists of reverse engineering gene regulatory networks from OMICs data, such as RNAseq or mass spectrometry-based proteomics data, through computational methods. This approach allows to identify signalling pathways involved in specific biological functions. The ability to infer causality in gene regulatory networks, in addition to correlation, is crucial for several modelling approaches and allows targeted control in biotechnology applications. METHODS: We performed simulations according to the approximate Bayesian computation method, where the core model consisted of a steady-state simulation algorithm used to study gene regulatory networks in systems for which a limited level of details is available. The simulations outcome was compared to experimentally measured transcriptomics and proteomics data through approximate Bayesian computation. RESULTS: The structure of small gene regulatory networks responsible for the regulation of biological functions involved in biomining were inferred from multi OMICs data of mixed bacterial cultures. Several causal inter- and intraspecies interactions were inferred between genes coding for proteins involved in the biomining process, such as heavy metal transport, DNA damage, replication and repair, and membrane biogenesis. The method also provided indications for the role of several uncharacterized proteins by the inferred connection in their network context. CONCLUSIONS: The combination of fast algorithms with high-performance computing allowed the simulation of a multitude of gene regulatory networks and their comparison to experimentally measured OMICs data through approximate Bayesian computation, enabling the probabilistic inference of causality in gene regulatory networks of a multispecies bacterial system involved in biomining without need of single-cell or multiple perturbation experiments. This information can be used to influence biological functions and control specific processes in biotechnology applications.

Microbial synergy and stoichiometry in heap biooxidation of low-grade porphyry arsenic-bearing gold ore.

Heap biooxidation method was used to evaluate the availability of Paodaoling gold ore in Anhui province, China. 15,000 tons of gold ores (≤ 10 mm in diameter) were bioxidized under mesophilic conditions. Under the synergistic effect of microbial community, arsenic and sulfur were oxidized by 42% and 38% after 80 days. Relatively, leaching of gold was improved from 36 to 78% after heap biooxidation. The sequencing results showed there were 28 operational taxonomic units identified the microbial community in the heap. The main genera were Acidithiobacillus, Ferroplasma, Acidiferrobacter and Nitrospira. According to stoichiometry, the content of microorganisms with various functions tended to be balanced. The biomass production rate was 10 g/s, the CO2 fixation rate was 18 g/s, and the oxygen consumption rate was 60 g/s. This study provides a good basis for the further design and application of heap biooxidation technology.

Correction to: Microbial synergy and stoichiometry in heap biooxidation of low-grade porphyry arsenic-bearing gold ore.

In the original publication the section heading "Classification of microorganisms" appearing above the sub-section "Air and liquid circulation in the heap" in page four is incorrect. The correct section heading should be read as "Results and discussion".

Supported Atomically-Precise Gold Nanoclusters for Enhanced Flow-through Electro-Fenton.

Gold (Au) has been considered catalytically inert for decades, but recent reports have described the ability of Au nanoparticles to catalyze H2O2 decomposition in the Haber-Weiss cycle. Herein, the design and demonstration of a flow-through electro-Fenton system based on an electrochemical carbon nanotube (CNT) filter functionalized with atomically precise Au nanoclusters (AuNCs) is described. The functionality of the device was then tested for its ability to catalyze antibiotic tetracycline degradation. In the functional filters, the Au core of AuNCs served as a high-performance Fenton catalyst; while the AuNCs ligand shells enabled CNT dispersion in aqueous solution for easy processing. The hybrid filter enabled in situ H2O2 production and catalyzed the subsequent H2O2 decomposition to HO·. The catalytic function of AuNCs lies in their ability to undergo redox cycling of Au+/Au0 under an electric field. The atomically precise AuNCs catalysts demonstrated superior catalytic activity to larger nanoparticles; while the flow-through design provided convection-enhanced mass transport, which yielded a superior performance compared to a conventional batch reactor. The adsorption behavior and decomposition pathway of H2O2 on the filter surfaces were simulated by density functional theory calculations. The research outcomes provided atomic-level mechanistic insights into the Au-mediated Fenton reaction.

Methanogenic Degradation of Long n-Alkanes Requires Fumarate-Dependent Activation.

Methanogenic degradation of n-alkanes is prevalent in n-alkane-impacted anoxic oil reservoirs and oil-polluted sites. However, little is known about the initial activation mechanism of the substrate, especially n-alkanes with a chain length above C16 Here, a methanogenic C16 to C20n-alkane-degrading enrichment culture was established from production water of a low-temperature oil reservoir. At the end of the incubation (364 days), C16 to C20 (1-methylalkyl)succinates were detected in the n-alkane-amended enrichment culture, suggesting that fumarate addition had occurred in the degradation process. This evidence is supported further by the positive amplification of the assA gene encoding the alpha subunit of alkylsuccinate synthase. A phylogenetic analysis shows these assA amplicons to be affiliated with Smithella and Desulfatibacillum clades. Together with the high abundance of these clades in the bacterial community, these two species are postulated to be the key players in the degradation of C16 to C20n-alkanes in the present study. Our results provide evidence that long n-alkanes are activated via a fumarate addition mechanism under methanogenic conditions.IMPORTANCE Methanogenic hydrocarbon degradation is the major process for oil degradation in subsurface oil reservoirs and is blamed for the formation of heavy oil and oil sands. Addition of n-alkanes to fumarate yielding alkyl-substituted succinates is a well-characterized anaerobic activation mechanism for hydrocarbons and is the most common activation mechanism in the anaerobic biodegradation of n-alkanes with chain lengths less than C16 However, the activation mechanism involved in the methanogenic biodegradation of n-alkanes longer than C16 is still uncertain. In this study, we analyzed a methanogenic enrichment culture amended with a mixture of C16 to C20n-alkanes. These n-alkanes can be activated via fumarate addition by mixed cultures containing Smithella and Desulfatibacillum species under methanogenic conditions. These observations provide a fundamental understanding of long-n-alkane metabolism under methanogenic conditions and have important applications for the remediation of oil-contaminated sites and for energy recovery from oil reservoirs.

Electroactive Modified Carbon Nanotube Filter for Simultaneous Detoxification and Sequestration of Sb(III).

Herein, we rationally designed a dual-functional electroactive filter system for simultaneous detoxification and sequestration of Sb(III). Binder-free and nanoscale TiO2-modified carbon nanotube (CNT) filters were fabricated. Upon application of an external electrical field, in situ transformation of Sb(III) to less toxic Sb(V) can be achieved, which is further sequestered by TiO2. Sb(III) removal kinetics and capacity increase with applied voltage and flow rate. This can be explained by the synergistic effects of the filter's flow-through design, electrochemical reactivity, small pore size, and increased number of exposed sorption sites. STEM characterization confirms that Sb were mainly sequestered by TiO2. XPS, AFS, and XAFS results verify that the Sb(III) conversion process was accelerated by the electrical field. The proposed electroactive filter technology works effectively across a wide pH range. The presence of sulfate, chloride, and carbonate ions negligibly inhibited Sb(III) removal. Exhausted TiO2-CNT filters can be effectively regenerated using NaOH solution. At 2 V, 100 µg/L Sb(III)-spiked tap water generated ∼1600 bed volumes of effluent with >90% efficiency. Density functional theory calculations suggest that the adsorption energy of Sb(III) onto TiO2 increases (from -3.81 eV to -4.18 eV) and Sb(III) becomes more positively charged upon application of an electrical field.

Sugar sources as Co-substrates promoting the degradation of refractory dye: A comparative study.

Four sugar sources were used as co-substrates to promote the degradation of a selected refractory dye reactive black 5 (RB5) by the natural bacterial flora DDMZ1. The boosting performance of the four sugar sources on RB5 decolorization ranked as: fructose > sucrose > glucose > glucose + fructose. Kinetic results of these four co-metabolism systems agreed well with a first-order kinetic model. Four sugar sources stimulated the extracellular azoreductase secretion causing enhanced enzyme activity. An increased formation of low molecular weight intermediates was caused by the addition of sugar sources. The toxicity of RB5 degradation products was significantly reduced in the presence of sugar sources. The bacterial community structure differed remarkably as a result of sugar sources addition. For a fructose addition, a considerably enriched population of the functional species Burkholderia-Paraburkholderia and Klebsiella was noted. The results enlarge our knowledge of the microkinetic and microbiological mechanisms of co-metabolic degradation of refractory pollutants.

Automated Microscopic Analysis of Metal Sulfide Colonization by Acidophilic Microorganisms.

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.

Multi-omics Reveals the Lifestyle of the Acidophilic, Mineral-Oxidizing Model Species Leptospirillum ferriphilumT.

Leptospirillum ferriphilum plays a major role in acidic, metal-rich environments, where it represents one of the most prevalent iron oxidizers. These milieus include acid rock and mine drainage as well as biomining operations. Despite its perceived importance, no complete genome sequence of the type strain of this model species is available, limiting the possibilities to investigate the strategies and adaptations that Leptospirillum ferriphilum DSM 14647T (here referred to as Leptospirillum ferriphilumT) applies to survive and compete in its niche. This study presents a complete, circular genome of Leptospirillum ferriphilumT obtained by PacBio single-molecule real-time (SMRT) long-read sequencing for use as a high-quality reference. Analysis of the functionally annotated genome, mRNA transcripts, and protein concentrations revealed a previously undiscovered nitrogenase cluster for atmospheric nitrogen fixation and elucidated metabolic systems taking part in energy conservation, carbon fixation, pH homeostasis, heavy metal tolerance, the oxidative stress response, chemotaxis and motility, quorum sensing, and biofilm formation. Additionally, mRNA transcript counts and protein concentrations were compared between cells grown in continuous culture using ferrous iron as the substrate and those grown in bioleaching cultures containing chalcopyrite (CuFeS2). Adaptations of Leptospirillum ferriphilumT to growth on chalcopyrite included the possibly enhanced production of reducing power, reduced carbon dioxide fixation, as well as elevated levels of RNA transcripts and proteins involved in heavy metal resistance, with special emphasis on copper efflux systems. Finally, the expression and translation of genes responsible for chemotaxis and motility were enhanced.IMPORTANCELeptospirillum ferriphilum is one of the most important iron oxidizers in the context of acidic and metal-rich environments during moderately thermophilic biomining. A high-quality circular genome of Leptospirillum ferriphilumT coupled with functional omics data provides new insights into its metabolic properties, such as the novel identification of genes for atmospheric nitrogen fixation, and represents an essential step for further accurate proteomic and transcriptomic investigation of this acidophile model species in the future. Additionally, light is shed on adaptation strategies of Leptospirillum ferriphilumT for growth on the copper mineral chalcopyrite. These data can be applied to deepen our understanding and optimization of bioleaching and biooxidation, techniques that present sustainable and environmentally friendly alternatives to many traditional methods for metal extraction.

Recent advances in anaerobic biological processes for textile printing and dyeing wastewater treatment: a mini-review.

Textile printing and dyeing wastewater is usually characterized by high pH, high turbidity, poor bio-degradability, complex composition, and high chrominance, and is discharged in large amounts. It has been regarded as one of the hardest to treat forms of industrial wastewater. Conventional physicochemical technologies can remove these contaminants from water bodies, but at the expense of high energy consumption and high cost. Alternatively, biological processes with limited energy consumption, low cost and high efficiency are considered as promising technologies. Among them, the anaerobic biological processes have been proven to be effective for the treatment of high-concentration textile printing and dyeing wastewater. In this mini-review, recent advances on high-rate anaerobic technologies for such purposes are reviewed. Current limitations of these technologies are summarized, and future research directions are indicated.

Proteins dominate in the surface layers formed on materials exposed to extracellular polymeric substances from bacterial cultures.

The chemical compositions of the surface conditioning layers formed by different types of solutions (from isolated EPS to whole culture media), involving different bacterial strains relevant for biocorrosion were compared, as they may influence the initial step in biofilm formation. Different substrata (polystyrene, glass, steel) were conditioned and analyzed by X-ray photoelectron spectroscopy. Peak decomposition and assignment were validated by correlations between independent spectral data and the ubiquitous presence of organic contaminants on inorganic substrata was taken into account. Proteins or peptides were found to be a major constituent of all conditioning layers and polysaccharides were not present in appreciable concentrations; the proportion of nitrogen which may be due to DNA was lower than 15%. There was no significant difference between the compositions of the adlayers formed from different conditioning solutions, except for the adlayers produced with tightly bound EPS extracted from D. alaskensis.

Manipulation of pyrite colonization and leaching by iron-oxidizing Acidithiobacillus species.

In this study, the process of pyrite colonization and leaching by three iron-oxidizing Acidithiobacillus species was investigated by fluorescence microscopy, bacterial attachment, and leaching assays. Within the first 4-5 days, only the biofilm subpopulation was responsible for pyrite dissolution. Pyrite-grown cells, in contrast to iron-grown cells, were able to oxidize iron(II) ions or pyrite after 24 h iron starvation and incubation with 1 mM H2O2, indicating that these cells were adapted to the presence of enhanced levels of reactive oxygen species (ROS), which are generated on metal sulfide surfaces. Acidithiobacillus ferrivorans SS3 and Acidithiobacillus ferrooxidans R1 showed enhanced pyrite colonization and biofilm formation compared to A. ferrooxidans (T). A broad range of factors influencing the biofilm formation on pyrite were also identified, some of them were strain-specific. Cultivation at non-optimum growth temperatures or increased ionic strength led to a decreased colonization of pyrite. The presence of iron(III) ions increased pyrite colonization, especially when pyrite-grown cells were used, while the addition of 20 mM copper(II) ions resulted in reduced biofilm formation on pyrite. This observation correlated with a different extracellular polymeric substance (EPS) composition of copper-exposed cells. Interestingly, the addition of 1 mM sodium glucuronate in combination with iron(III) ions led to a 5-fold and 7-fold increased cell attachment after 1 and 8 days of incubation, respectively, in A. ferrooxidans (T). In addition, sodium glucuronate addition enhanced pyrite dissolution by 25%.

Visualization and analysis of EPS glycoconjugates of the thermoacidophilic archaeon Sulfolobus metallicus.

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).

Methanobacterium movilense sp. nov., a hydrogenotrophic, secondary-alcohol-utilizing methanogen from the anoxic sediment of a subsurface lake.

A novel strain of methanogenic archaea, designated MC-20(T), was isolated from the anoxic sediment of a subsurface lake in Movile Cave, Mangalia, Romania. Cells were non-motile, Gram-stain-negative rods 3.5-4.0 µm in length and 0.6-0.7 µm in width, and occurred either singly or in short chains. Strain MC-20(T) was able to utilize H2/CO2, formate, 2-propanol and 2-butanol as substrate, but not acetate, methanol, ethanol, dimethyl sulfide, monomethylamine, dimethylamine or trimethylamine. Neither trypticase peptone nor yeast extract was required for growth. The major membrane lipids of strain MC-20(T) were archaeol phosphatidylethanolamine and diglycosyl archaeol, while archaeol phosphatidylinositol and glycosyl archaeol were present only in minor amounts. Optimal growth was observed at 33 °C, pH 7.4 and 0.08 M NaCl. Based on phylogenetic analysis of 16S rRNA gene sequences, strain MC-20(T) was closely affiliated with Methanobacterium oryzae FPi(T) (similarity 97.1%) and Methanobacterium lacus 17A1(T) (97.0%). The G+C content of the genomic DNA was 33.0 mol%. Based on phenotypic and genotypic differences, strain MC-20(T) was assigned to a novel species of the genus Methanobacterium for which the name Methanobacterium movilense sp. nov. is proposed. The type strain is MC-20(T) ( = DSM 26032(T) = JCM 18470(T)).

Methanosarcina spelaei sp. nov., a methanogenic archaeon isolated from a floating biofilm of a subsurface sulphurous lake.

A novel methanogenic archaeon, strain MC-15(T), was isolated from a floating biofilm on a sulphurous subsurface lake in Movile Cave (Mangalia, Romania). Cells were non-motile sarcina-like cocci with a diameter of 2-4 µm, occurring in aggregates. The strain was able to grow autotrophically on H2/CO2. Additionally, acetate, methanol, monomethylamine, dimethylamine and trimethylamine were utilized, but not formate or dimethyl sulfide. Trypticase peptone and yeast extract were not required for growth. Optimal growth was observed at 33 °C, pH 6.5 and a salt concentration of 0.05 M NaCl. The predominant membrane lipids of MC-15(T) were archaeol and hydroxyarchaeol phosphatidylethanolamine, phosphatidylglycerol, and phosphatidylinositol as well as hydroxyarchaeol phosphatidylserine and archaeol glycosaminyl phosphatidylinositol. The closely related species, Methanosarcina vacuolata and Methanosarcina horonobensis, had a similar composition of major membrane lipids to strain MC-15(T). The 16S rRNA gene sequence of strain MC-15(T) was similar to those of Methanosarcina vacuolata DSM 1232(T) (sequence similarity 99.3%), Methanosarcina horonobensis HB-1(T) (98.8%), Methanosarcina barkeri DSM 800(T) (98.7%) and Methanosarcina siciliae T4/M(T) (98.4%). DNA-DNA hybridization revealed 43.3% relatedness between strain MC-15(T) and Methanosarcina vacuolata DSM 1232(T). The G+C content of the genomic DNA was 39.0 mol%. Based on physiological, phenotypic and genotypic differences, strain MC-15(T) represents a novel species of the genus Methanosarcina, for which the name Methanosarcina spelaei sp. nov. is proposed. The type strain is MC-15(T) ( = DSM 26047(T) = JCM 18469(T)).

Characterization of exopolymeric substances (EPS) produced by Aeromonas hydrophila under reducing conditions.

The aim of this work was to investigate the production of extracellular polymeric substances (EPS) by Aeromonas hydrophila grown under anaerobic conditions. EPS composition was studied for planktonic cells, cells attached to carbon fibre supports using a soluble ferric iron source and cells grown with a solid ferric iron mineral (gossan). Conventional spectrophotometric methods, Fourier transform infrared (FTIR) and confocal laser scanning microscopy (CLSM) were used to determine the main components in the biofilm extracted from the cultures. The key EPS components were proteins, indicating their importance for electron transfer reactions. Carbohydrates were observed mostly on the mineral and contained terminal mannosyl and/or terminal glucose, fucose and N-acetylgalactosamine residues.