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Extreme acidophiles thrive in environments rich in protons (pH values <3) and often high levels of dissolved heavy metals. They are distributed across the three domains of the Tree of Life including members of the Proteobacteria. The Acidithiobacillia class is formed by the neutrophilic genus Thermithiobacillus along with the extremely acidophilic genera Fervidacidithiobacillus, Igneacidithiobacillus, Ambacidithiobacillus, and Acidithiobacillus. Phylogenomic reconstruction revealed a division in the Acidithiobacillia class correlating with the different pH optima that suggested that the acidophilic genera evolved from an ancestral neutrophile within the Acidithiobacillia. Genes and mechanisms denominated as "first line of defense" were key to explaining the Acidithiobacillia acidophilic lifestyle including preventing proton influx that allows the cell to maintain a near-neutral cytoplasmic pH and differ from the neutrophilic Acidithiobacillia ancestors that lacked these systems. Additional differences between the neutrophilic and acidophilic Acidithiobacillia included the higher number of gene copies in the acidophilic genera coding for "second line of defense" systems that neutralize and/or expel protons from cell. Gain of genes such as hopanoid biosynthesis involved in membrane stabilization at low pH and the functional redundancy for generating an internal positive membrane potential revealed the transition from neutrophilic properties to a new acidophilic lifestyle by shaping the Acidithiobacillaceae genomic structure. The presence of a pool of accessory genes with functional redundancy provides the opportunity to "hedge bet" in rapidly changing acidic environments. Although a core of mechanisms for acid resistance was inherited vertically from an inferred neutrophilic ancestor, the majority of mechanisms, especially those potentially involved in resistance to extremely low pH, were obtained from other extreme acidophiles by horizontal gene transfer (HGT) events.

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This work aimed to remove sulfate and acidity from mine-impacted water (MIW) via electrocoagulation (EC), a technique which stands as an advanced alternative to chemical coagulation in pollutant removal from wastewaters. The multiple electrochemical reactions occurring in the aluminum anode and the stainless steel cathode surfaces can form unstable flakes of metal hydroxysulfate complexes, causing coagulation, flocculation, and floatation; or, adsorption of sulfate on sorbents originated from the electrochemical process can occur, depending on pH value. Batch experiments in the continuous mode of exposition using different current densities (35, 50, and 65 A m-2) were tested, and a statistical difference between their sulfate removals was detected. Furthermore, the intermittent mode of exposure was also tested by performing a 22-factorial design to verify the combination with different current densities, concluding that better efficiencies of sulfate removal were obtained in the continuous mode of exposition, even with lower current densities. After 5 h of electrocoagulation, sulfate could be removed from MIW with a mean efficiency of 70.95% (in continuous mode of exposition and 65 A m-2 current density), and this sulfate removal follows probable third-order decay kinetics in accordance with the quick drop in sulfate concentration until 3 h of exposure time, remaining virtually constant at longer times. Graphical abstract.

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Organisms that thrive in extremely acidic environments (≤pH 3.5) are of widespread importance in industrial applications, environmental issues, and evolutionary studies. Leptospirillum spp. constitute the only extremely acidophilic microbes in the phylogenetically deep-rooted bacterial phylum Nitrospirae. Leptospirilli are Gram-negative, obligatory chemolithoautotrophic, aerobic, ferrous iron oxidizers. This paper predicts genes that Leptospirilli use to survive at low pH and infers their evolutionary trajectory. Phylogenetic and other bioinformatic approaches suggest that these genes can be classified into (i) "first line of defense", involved in the prevention of the entry of protons into the cell, and (ii) neutralization or expulsion of protons that enter the cell. The first line of defense includes potassium transporters, predicted to form an inside positive membrane potential, spermidines, hopanoids, and Slps (starvation-inducible outer membrane proteins). The "second line of defense" includes proton pumps and enzymes that consume protons. Maximum parsimony, clustering methods, and gene alignments are used to infer the evolutionary trajectory that potentially enabled the ancestral Leptospirillum to transition from a postulated circum-neutral pH environment to an extremely acidic one. The hypothesized trajectory includes gene gains/loss events driven extensively by horizontal gene transfer, gene duplications, gene mutations, and genomic rearrangements.

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RESUMO: Rejeitos de carvão contêm vários minerais, entre os quais a pirita, que se oxida e gera a drenagem ácida de minas (DAM), um efluente ácido com alto poder poluidor. Atualmente, o setor de mineração de carvão brasileiro controla a DAM por meio do tratamento pelo método de neutralização/precipitação de metais, que consome substanciais quantidades de reagentes e gera um grande volume de lodo. O objetivo deste trabalho foi estudar a minimização da geração da DAM por outra abordagem, o método preventivo de isolamento e exclusão de sulfetos. O processo consiste na remoção da pirita da massa de resíduos, reduzindo a geração de acidez e minimizando a carga de metais nas estações de tratamento. A metodologia do trabalho consistiu na separação gravimétrica por meio denso de uma amostra típica de rejeito de carvão, obtendo-se três frações densimétricas: inferior a 2,2, entre 2,2 e 2,7 e superior a 2,7. Cada fração foi submetida às análises imediata e elementar. Também foram realizados ensaios estáticos e cinéticos em células úmidas para avaliação da geração de acidez no rejeito bruto e na fração com densidade intermediária. Os resultados demonstraram que, por intermédio do beneficiamento gravimétrico dos rejeitos, é possível reduzir em 30% o volume de material descartado. Esse material apresenta um potencial de geração de acidez líquido de 80 a 90% inferior ao do rejeito de carvão bruto. Além disso, estima-se que os custos com reagentes no tratamento de águas ácidas produzidas da mineração de carvão possam ser reduzidos em aproximadamente 85%.

ABSTRACT: Coal wastes contain many minerals, including the pyrite, which oxidize and generates the acid mine drainage (AMD), a highly polluting effluent. Currently, the Brazilian coal mining operations emphasise AMD control using waste water treatment systems which consume substantial amounts of reagents and generate large amounts of sludge. The aim of this work was to study the acid mine drainage control by another approach, the sulphide exclusion preventive method. The method comprises in removing the pyrite form coal tailings, reducing the acid generation and minimizing the metals load in wastewater treatment plants. Accordingly, a typical coal waste sample was submitted to a density separation process to divide the sample in the following relative density ranges: below 2.2, from 2.2 to 2.7 and above 2.7. All density fractions were submitted to ultimate and proximate analysis. Static and kinetic tests were carried with the raw waste and the pyrite-free fraction. The results showed that, by gravity processing of coal tailings, it is possible to decrease 30% the volume of wastes. The remaining material presents an acid generation potential of about 80-90% lower than the raw waste. Furthermore, it is expected a reduction in costs of reagents of approximately 85% in acid mine treatment plants.

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Biochemical passive treatment represents a promising option for the remediation of acid mine drainage. This study determined the effect of three hydraulic retention times (1, 2, and 4 days) on changes in system efficiency, reactive mixture, and microbial activity in bioreactors under upward flow conditions. Bioreactors were sacrificed in the weeks 8, 17 and 36, and the reactive mixture was sampled at the bottom, middle, and top layers. Physicochemical analyses were performed on reactive mixture post-treatment and correlated with sulfate-reducing bacteria and cellulolytic and dehydrogenase activity. All hydraulic retention times were efficient at increasing pH and alkalinity and removing sulfate (>60%) and metals (85-99% for Fe(2+) and 70-100% for Zn(2+)), except for Mn(2+). The longest hydraulic retention time (4 days) increased residual sulfides, deteriorated the quality of treated effluent and negatively impacted sulfate-reducing bacteria. Shortest hydraulic retention time (1 day) washed out biomass and increased input of dissolved oxygen in the reactors, leading to higher redox potential and decreasing metal removal efficiency. Concentrations of iron, zinc and metal sulfides were high in the bottom layer, especially with 2 day of hydraulic retention time. Sulfate-reducing bacteria, cellulolytic and dehydrogenase activity were higher in the middle layer at 4 days of hydraulic retention time. Hydraulic retention time had a strong influence on overall performance of passive reactors.

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Using phylogenomic and gene compositional analyses, five highly conserved gene families have been detected in the core genome of the phylogenetically coherent genus Acidithiobacillus of the class Acidithiobacillia. These core gene families are absent in the closest extant genus Thermithiobacillus tepidarius that subtends the Acidithiobacillus genus and roots the deepest in this class. The predicted proteins encoded by these core gene families are not detected by a BLAST search in the NCBI non-redundant database of more than 90 million proteins using a relaxed cut-off of 1.0e-5. None of the five families has a clear functional prediction. However, bioinformatic scrutiny, using pI prediction, motif/domain searches, cellular location predictions, genomic context analyses, and chromosome topology studies together with previously published transcriptomic and proteomic data, suggests that some may have functions associated with membrane remodeling during cell division perhaps in response to pH stress. Despite the high level of amino acid sequence conservation within each family, there is sufficient nucleotide variation of the respective genes to permit the use of the DNA sequences to distinguish different species of Acidithiobacillus, making them useful additions to the armamentarium of tools for phylogenetic analysis. Since the protein families are unique to the Acidithiobacillus genus, they can also be leveraged as probes to detect the genus in environmental metagenomes and metatranscriptomes, including industrial biomining operations, and acid mine drainage (AMD).