Lack of Microbial Diversity in an Extreme Mars Analog Setting: Poás Volcano, Costa Rica.

The Poás volcano in Costa Rica has been studied as a Mars geochemical analog environment, since both the style of hydrothermal alteration present and the alteration mineralogy are consistent with Mars' relict hydrothermal systems. The site hosts an active volcano, with high-temperature fumaroles (up to 980°C) and an ultra-acidic lake. This lake, Laguna Caliente, is one of the most dynamic environments on Earth, with frequent phreatic eruptions, temperatures ranging from near-ambient to almost boiling, a pH range of -1 to 1.5, and a wide range of chemistries and redox potential. Martian acid-sulfate hydrothermal systems were likely similarly dynamic and equally challenging to life. The microbiology existing within Laguna Caliente was characterized for the first time, with sampling taking place in November, 2013. The diversity of the microbial community was surveyed via extraction of environmental DNA from fluid and sediment samples followed by Illumina sequencing of the 16S rRNA gene. The microbial diversity was limited to a single species of the bacterial genus Acidiphilium. This organism likely gets its energy from oxidation of reduced sulfur in the lake, including elemental sulfur. Given Mars' propensity for sulfur and acid-sulfate environments, this type of organism is of significant interest to the search for past or present life on the Red Planet. Key Words: Mars astrobiology-Acid-sulfate hydrothermal systems-Extremophiles-Acidic-High temperature-Acidiphilium bacteria. Astrobiology 18, 923-933.

Sticking together: inter-species aggregation of bacteria isolated from iron snow is controlled by chemical signaling.

Marine and lake snow is a continuous shower of mixed organic and inorganic aggregates falling from the upper water where primary production is substantial. These pelagic aggregates provide a niche for microbes that can exploit these physical structures and resources for growth, thus are local hot spots for microbial activity. However, processes underlying their formation remain unknown. Here, we investigated the role of chemical signaling between two co-occurring bacteria that each make up more than 10% of the community in iron-rich lakes aggregates (iron snow). The filamentous iron-oxidizing Acidithrix strain showed increased rates of Fe(II) oxidation when incubated with cell-free supernatant of the heterotrophic iron-reducing Acidiphilium strain. Amendment of Acidithrix supernatant to motile cells of Acidiphilium triggered formation of cell aggregates displaying similar morphology to those of iron snow. Comparative metabolomics enabled the identification of the aggregation-inducing signal, 2-phenethylamine, which also induced faster growth of Acidiphilium. We propose a model that shows rapid iron snow formation, and ultimately energy transfer from the photic zone to deeper water layers, is controlled via a chemically mediated interplay.

Generation of acid mine drainage around the Karaerik copper mine (Espiye, Giresun, NE Turkey): implications from the bacterial population in the Acisu effluent.

The Karaerik Cu mine is a worked-out deposit with large volumes of tailings and slags which were left around the mine site without any protection. Natural feeding of these material and run-off water from the mineralised zones into the Acisu effluent causes a serious environmental degradation and creation of acid mine drainage (AMD) along its entire length. This research aims at modelling the formation of AMD with a specific attempt on the characterisation of the bacterial population in association with AMD and their role on its occurrence. Based on 16SrRNA analyses of the clones obtained from a composite water sample, the bacterial community was determined to consist of Acidithiobacillus ferrivorans, Ferrovum myxofaciens, Leptospirillum ferrooxidans and Acidithiobacillus ferrooxidans as iron-oxidising bacteria, Acidocella facilis, Acidocella aluminiidurans, Acidiphilium cryptum and Acidiphilium multivorum as iron-reducing bacteria, and Acidithiobacillus ferrivorans, Acidithiobacillus ferrooxidans, Acidithiobacillus thiooxidans and Acidiphilium cryptum as sulphur-oxidising bacteria. This association of bacteria with varying roles was interpreted as evidence of a concomitant occurrence of sulphur and iron cycles during the generation of AMD along the Acisu effluent draining the Karaerik mine.

Comparative metagenomics reveals microbial community differentiation in a biological heap leaching system.

The microbial community in a biological heap leaching (BHL) system is crucial for the decomposition of ores. However, the microbial community structure and functional differentiation in different parts of a biological heap leaching system are still unknown. In this study, metagenomic sequencing was used to fully illuminate the microbial community differentiation in the pregnant leach solution (PLS) and leaching heap (LH) of a BHL system. Long-read sequences (1.3 million) were obtained for the two samples, and the MG_RAST server was used to perform further analysis. The taxa analysis results indicated that the dominant genera of PLS is autotrophic bacterium Acidithiobacillus, but heterotrophic bacterium Acidiphilium is predominant in LH. Furthermore, functional annotation and hierarchical comparison with different reference samples showed that the abundant presence of genes was involved in transposition, DNA repair and heavy metal transport. The sequences related to transposase, which is important for the survival of the organism in the hostile environment, were both mainly classified into Acidiphilium for PLS and LH. These results indicated that not only autotrophic bacteria such as Acidithiobacillus, but also heterotrophic bacteria such as Acidiphilium, were essential participants in the bioleaching process. This new meta-view research will further facilitate the effective application of bioleaching.

Acidiphilium iwatense sp. nov., isolated from an acid mine drainage treatment plant, and emendation of the genus Acidiphilium.

Several strains of aerobic, acidophilic, chemo-organotrophic bacteria belonging to the genus Acidiphilium were isolated from an acid mine drainage (AMD) (pH 2.2) treatment plant. 16S rRNA gene sequence comparisons showed that most of the novel isolates formed a phylogenetically coherent group (designated Group Ia) distinguishable from any of the previously established species of the genus Acidiphilium at <98% similarity. This was supported by genomic DNA-DNA hybridization assays. The Group Ia isolates were characterized phenotypically by an oval cell morphology, non-motility, growth in the range pH 2.0-5.5 (optimum pH 3.5), lack of photosynthetic pigment and the presence of C19:0 cyclo ω8c as the main component of the cellular fatty acids and ubiquinone-10 as the major quinone. On the basis of these data, the name Acidiphilium iwatense sp. nov. is proposed to accommodate the Group Ia isolates, and the description of the genus Acidiphilium is emended. The type strain of Acidiphilium iwatense sp. nov. is MS8(T) ( =NBRC 107608(T)=KCTC 23505(T)).

The role of Acidithiobacillus ferrooxidans in alleviating the inhibitory effect of thiosulfate on the growth of acidophilic Acidiphilium species isolated from acid mine drainage samples from Garubathan, India.

Several acidophilic chemolithoautotrophic and heterotrophic strains were isolated from acid mine drainage samples from Garubathan, West Bengal, India. The strains, chemolithoautotrophic DK6.1 and heterotrophic DKAP1.1, used in this study were assigned to the species Acidithiobacillus ferrooxidans and Acidiphilium cryptum, respectively. Unamended filtered and subsequently autoclaved elemental sulfur spent medium of A. ferrooxidans was used as the medium to study heterotrophic growth of A. cryptum DKAP1.1. While characterizing the heterotrophic strain, an inhibitory effect of thiosulfate on A. cryptum DKAP1.1 was identified. The lethality of thiosulfate broth was directly related to the concentration of thiosulfate in the medium. Nonviability of A. cryptum DKAP1.1 in the presence of thiosulfate was alleviated by A. ferrooxidans DK6.1 in co-culture. Microbiological data on a positive growth effect for A. ferrooxidans DK6.1 caused by co-culturing in solid media in the presence of A. cryptum DKAP1.1 is also presented.

Preferential use of an anode as an electron acceptor by an acidophilic bacterium in the presence of oxygen.

Several anaerobic metal-reducing bacteria have been shown to be able to donate electrons directly to an electrode. This property is of great interest for microbial fuel cell development. To date, microbial fuel cell design requires avoiding O(2) diffusion from the cathodic compartment to the sensitive anodic compartment. Here, we show that Acidiphilium sp. strain 3.2 Sup 5 cells that were isolated from an extreme acidic environment are able to colonize graphite felt electrodes. These bacterial electrodes were able to produce high-density electrocatalytic currents, up to 3 A/m(2) at a poised potential of +0.15 V (compared to the value for the reference standard calomel electrode) in the absence of redox mediators, by oxidizing glucose even at saturating air concentrations and very low pHs.

[Isolation and characterization of Acidiphilium strain teng-A and its metabolism of fe (III) during pure- and mixed cultivation].

An acidophilic, aerobic and chemoheterotrophic bacterial strain Teng-A was isolated from acidic environmental samples collected at sulfidic hot springs of Tengchong County, Yunnan Province, China. Cells of strain Teng-A was rod-shaped (0.6-0.8 microm x 1.0 - 1.5 microm), Gram-negative, motile with flagella. Strain Teng-A grew well at temperature of 29-33 degrees C and at pH of 3.0-4.0. It used a wide variety of organic compounds for growth, but did not use ferrous iron, elemental sulfur, thiosulfate and tetrathionate as the sole energy source. Its G + C content was determined to be 69.6 mol%. Phylogenetic analysis based on 16S rRNA gene sequence demonstrated that it was closely related to species of Acidiphilium. Under anoxic conditions, the strain Teng-A reduced Fe(III) to Fe(II) with glucose or hy drogen as electron donor (reduction rate is 11.56 mg/L day and 15.34 mg/L x day, respectively). Metabolisms/Oxidation of ferrous iron by Acidithiobacillus ferrooxidans LJ-1 and Leptospirilum ferriphilum LJ-2, in the presence and absence of strain Teng-A were studied. When incubated with strain Teng-A, the oxidation rates of Fe(II) was slightly decreased at the first 3 days (0.44 g/L x day and 0.4 g/L x day respectively) compared to pure culture of At ferrooxidans and L. ferriphilum, but all Fe(II) was completely oxidized after 5 days. It was found that the morphologies of precipitates of Fe (III) produced during pure and mixed cultivation were different. The potential application of Acidiphilium in bioleaching and its potential role during formation of precipitated ores were discussed.

Immunobiological activities of a new nontoxic lipopolysaccharide from Acidiphilium GS18h/ATCC55963, a soil isolate from an Indian copper mine.

A novel nontoxic lipopolysaccharide (LPS) was purified from Acidiphilium strain GS18h/ATCC55963. The chemical composition of the lipid A part of this LPS is distinctly different from that of known lipid A molecules. The LPS was investigated to determine its capacity to provide protection against toxic LPS or endotoxic shock, as has been reported for other nontoxic LPSs (Rhodobacter sphaeroides and Rhodobacter capsulatus), and also the extent and type of immunomodulatory response in terms of tumor necrosis factor alpha (TNF-alpha), interleukin-1beta (IL-beta), and IL-6 release as well as NO secretion by stimulated monocyte-macrophage systems. This study demonstrates clearly that mice immunized or primed with this LPS are fully protected against challenge with toxic Escherichia coli LPS. Unlike most of the extensively studied nontoxic LPSs, this LPS induced reactive nitrogen intermediates and released TNF-alpha, IL-beta and IL-6 in both mouse and human monocyte-macrophage systems. However, the extent of the cytokine and lymphokine releasing response was well below the range of the toxic LPS, for example that of E. coli. Owing to its capacity to provide immunostimulation of the host without causing any lethality to ensure protection against endotoxic shock, this LPS appears to have potential therapeutic value.

Acidophilic microbial communities associated with a natural, biodegraded hydrocarbon seepage.

Characterization of microbial communities present in a surface petroleum seep in which hydrocarbons have been biodegraded for thousands of years in order to improve the understanding on natural petroleum biodegradation. DNA was extracted from a natural, surface petroleum seep and subjected to culture independent analysis (rRNA gene-based denaturing gradient gel electrophoresis and phylogenetic analysis of clone libraries). Molecular analysis suggested dominance by acidophilic bacteria, especially Alphaproteobacteria (mainly bacteria related to Acidiphilium and Acidocella). Archaea were not detected, but fungi were present. pH of the samples was around 3.5. Acidophilic microbial communities are associated with an acidic petroleum seep. Microbial community structure analysis gives information on the environmental conditions under which petroleum biodegradation occurs. This knowledge could be applied to define conditions for specific cultivation or activity measurements. The activity of acidophilic micro-organisms deserves more attention with respect to their involvement in natural petroleum degradation. This knowledge will contribute to the design of oil bioremediation strategies for polluted acidic settings.

Isolation and characterisation of the lipopolysaccharide from Acidiphilium strain GS18h/ATCC55963, a soil isolate of Indian copper mine.

The lipopolysaccharide (LPS) of the Gram-negative Acidiphilium strain GS18h/ATCC55963, a new soil isolate, exhibited very low endotoxic activity as determined by Limulus gelation activity, lethal toxicity in galactosamine (GalN) sensitised mice, and level of tumor necrosis factor alpha (TNFalpha) in the blood serum of BALB/c mice. Analysis of the LPS, specially of lipid A which usually accounts for the toxicity, revealed the latter to contain glucosamine and phosphate besides fatty acids, of which 14:0(3-OH), 18:0(3-OH), 18:1 and 19:0(cyclo) are the major components, while 12:0, 16:0, 19:1, 20:0(3-OH) and 20:1(3-OH) are present in small amounts. The 14:0(3-OH) and 18:0(3-OH) fatty acids are amide-linked, whereas the rest are ester bound. Glucose, galactose, mannose, rhamnose, heptose, galacturonic acid and 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) were present in the polysaccharide part of this LPS. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the LPS showed a macromolecular heterogeneity distinctly different from those of Escherichia coli or Salmonella. The toxicity of this LPS being extremely low attributed to fatty acid composition of its lipid A, promises potential therapeutic application.