Christopher G. Bryan

Structure, Function, and Evolution of the Thiomonas spp. Genome

Bacteria of the Thiomonas genus are ubiquitous in extreme environments, such as arsenic-rich acid mine drainage (AMD). The genome of one of these strains, Thiomonas sp. 3As, was sequenced, annotated, and examined, revealing specific adaptations allowing this bacterium to survive and grow in its highly toxic environment. In order to explore genomic diversity as well as genetic evolution in Thiomonas spp., a comparative genomic hybridization (CGH) approach was used on eight different strains of the Thiomonas genus, including five strains of the same species. Our results suggest that the Thiomonas genome has evolved through the gain or loss of genomic islands and that this evolution is influenced by the specific environmental conditions in which the strains live.

Carbon and arsenic metabolism in Thiomonas strains: differences revealed diverse adaptation processes

BackgroundThiomonas strains are ubiquitous in arsenic-contaminated environments. Differences between Thiomonas strains in the way they have adapted and respond to arsenic have never been studied in detail. For this purpose, five Thiomonas strains, that are interesting in terms of arsenic metabolism were selected: T. arsenivorans, Thiomonas spp. WJ68 and 3As are able to oxidise As(III), while Thiomonas sp. Ynys1 and T. perometabolis are not. Moreover, T. arsenivorans and 3As present interesting physiological traits, in particular that these strains are able to use As(III) as an electron donor.ResultsThe metabolism of carbon and arsenic was compared in the five Thiomonas strains belonging to two distinct phylogenetic groups. Greater physiological differences were found between these strains than might have been suggested by 16S rRNA/rpoA gene phylogeny, especially regarding arsenic metabolism. Physiologically, T. perometabolis and Ynys1 were unable to oxidise As(III) and were less arsenic-resistant than the other strains. Genetically, they appeared to lack the aox arsenic-oxidising genes and carried only a single ars arsenic resistance operon. Thiomonas arsenivorans belonged to a distinct phylogenetic group and increased its autotrophic metabolism when arsenic concentration increased. Differential proteomic analysis revealed that in T. arsenivorans, the rbc/cbb genes involved in the assimilation of inorganic carbon were induced in the presence of arsenic, whereas these genes were repressed in Thiomonas sp. 3As.ConclusionTaken together, these results show that these closely related bacteria differ substantially in their response to arsenic, amongst other factors, and suggest different relationships between carbon assimilation and arsenic metabolism.

The effect of CO2 availability on the growth, iron oxidation and CO2‐fixation rates of pure cultures of Leptospirillum ferriphilum and Acidithiobacillus ferrooxidans

Understanding how bioleaching systems respond to the availability of CO2 is essential to developing operating conditions that select for optimum microbial performance. Therefore, the effect of inlet gas and associated dissolved CO2 concentration on the growth, iron oxidation and CO2‐fixation rates of pure cultures of Acidithiobacillus ferrooxidans and Leptospirillum ferriphilum was investigated in a batch stirred tank system. The minimum inlet CO2 concentrations required to promote the growth of At. ferrooxidans and L. ferriphilum were 25 and 70 ppm, respectively, and corresponded to dissolved CO2 concentrations of 0.71 and 1.57 µM (at 30°C and 37°C, respectively). An actively growing culture of L. ferriphilum was able to maintain growth at inlet CO2 concentrations less than 30 ppm (0.31–0.45 µM in solution). The highest total new cell production and maximum specific growth rates from the stationary phase inocula were observed with CO2 inlet concentrations less than that of air. In contrast, the amount of CO2 fixed per new cell produced increased with increasing inlet CO2 concentrations above 100 ppm. Where inlet gas CO2 concentrations were increased above that of air the additional CO2 was consumed by the organisms but did not lead to increased cell production or significantly increase performance in terms of iron oxidation. It is proposed that At. ferrooxidans has two CO2 uptake mechanisms, a high affinity system operating at low available CO2 concentrations, which is subject to substrate inhibition and a low affinity system operating at higher available CO2 concentrations. L. ferriphilum has a single uptake system characterised by a moderate CO2 affinity. At. ferrooxidans performed better than L. ferriphilum at lower CO2 availabilities, and was less affected by CO2 starvation. Finally, the results demonstrate the limitations of using CO2 uptake or ferrous iron oxidation data as indirect measures of cell growth and performance across varying physiological conditions. Biotechnol. Bioeng. 2012; 109:1693–1703.

Multiple osmotic stress responses in acidihalobacter prosperus result in tolerance to chloride ions

Extremely acidophilic microorganisms (pH optima for growth of ≤3) are utilized for the extraction of metals from sulfide minerals in the industrial biotechnology of “biomining.” A long term goal for biomining has been development of microbial consortia able to withstand increased chloride concentrations for use in regions where freshwater is scarce. However, when challenged by elevated salt, acidophiles experience both osmotic stress and an acidification of the cytoplasm due to a collapse of the inside positive membrane potential, leading to an influx of protons. In this study, we tested the ability of the halotolerant acidophile Acidihalobacter prosperus to grow and catalyze sulfide mineral dissolution in elevated concentrations of salt and identified chloride tolerance mechanisms in Ac. prosperus as well as the chloride susceptible species, Acidithiobacillus ferrooxidans. Ac. prosperus had optimum iron oxidation at 20 g L−1 NaCl while At. ferrooxidans iron oxidation was inhibited in the presence of 6 g L−1 NaCl. The tolerance to chloride in Ac. prosperus was consistent with electron microscopy, determination of cell viability, and bioleaching capability. The Ac. prosperus proteomic response to elevated chloride concentrations included the production of osmotic stress regulators that potentially induced production of the compatible solute, ectoine uptake protein, and increased iron oxidation resulting in heightened electron flow to drive proton export by the F0F1 ATPase. In contrast, At. ferrooxidans responded to low levels of Cl− with a generalized stress response, decreased iron oxidation, and an increase in central carbon metabolism. One potential adaptation to high chloride in the Ac. prosperus Rus protein involved in ferrous iron oxidation was an increase in the negativity of the surface potential of Rus Form I (and Form II) that could help explain how it can be active under elevated chloride concentrations. These data have been used to create a model of chloride tolerance in the salt tolerant and susceptible species Ac. prosperus and At. ferrooxidans, respectively.

Dissimilatory ferrous iron oxidation at a low pH: a novel trait identified in the bacterial subclass Rubrobacteridae

A novel iron-oxidizing acidophilic actinobacterium was isolated from spoil material at an abandoned copper mine. Phylogenetic analysis placed the isolate within the Rubrobacteridae subclass of the Actinobacteria. Its optimum temperature and pH for growth are 30-35 degrees C and pH 3.0, respectively. Although it could catalyze the dissimilatory oxidation of ferrous iron, growth yields declined progressively in media containing ferrous iron concentrations >100 microM. The isolate, Pa33, did not grow or oxidize iron in the absence of organic carbon, and appeared to be an obligate heterotroph. Specific rates of iron oxidation were much smaller than those determined for the autotrophic iron-oxidizing proteobacterium Acidithiobacillus ferrooxidans and the heterotrophic iron-oxidizing actinobacterium Ferrimicrobium acidiphilum. Iron oxidation by isolate Pa33 appears to be a defensive mechanism, in which iron oxidation converts a soluble species to which the bacterium is sensitive to an oxidized species (ferric iron) that is highly insoluble in the spoil from which it was isolated. This is the first report of acidophily or dissimilatory iron oxidation within the Rubrobacteridae subclass and one of very few within the Actinobacteria phylum as a whole.

Adaptation and Evolution of Microbial Consortia in a Stirred Tank Reactor Bioleaching System: Indigenous Population versus a Defined Consortium

To participate in the investigation concerning these key questions, the bioleaching of a cobaltiferous pyrite by two different microbial consortia was studied. The first was an indigenous population taken from industrial bioreactors used to treat a cobaltiferous pyrite at the Kasese Cobalt Company site in Uganda [2]. The second was a defined consortium comprising four organisms which had been found to dominate the indigenous population. These organisms had been maintained in synthetic media as pure cultures in order to observe whether they would loose any adaptational advantages accrued during the period spent in the bioreactor system.

Insights into Heap Bioleaching at the Agglomerate-Scale

Previous agglomerate-scale heap bioleaching studies have outlined the variations in cell numbers of the liquid and attached phases during colonisation of sterilised ore by a pure culture. In this study, a mixed mesophilic culture was used in agglomerate-scale columns containing non-sterilised low-grade copper ore. Over a six - month period, columns were harvested at various intervals to provide snapshots of the metal distribution and the quantity, location, and ecological variations of mineral-oxidizing microbes within the ore bed. The initial colonisation period in this experiment was dissimilar to previous work, as the indigenous community was retained within the ore-bed throughout acid agglomeration. The overall colonisation phase lasted for approximately 1,000 hours until cell concentrations stabilised. In each column, less than 0.05% of the total cells were found in the leachate, 15-20% in the interstitial phase and the remaining ~80% were attached to the mineral surface. Once cell numbers had stabilised, interstitial cell concentrations were approximately 2,000× greater than those in the leachate. This difference persisted for the duration of the experiment. Copper concentrations in the two liquid phases generally decreased over time, but were on average 50× higher in the interstitial phase. Iron concentrations were more stable, but again were 30× higher in the interstitial phase. This demonstrates that that the difference in cell concentration between the leachate and interstitial phases cannot be explained through diffusion gradients within the system as it is much greater than those observed for the dissolved metals. It also shows that the specific environmental conditions of the interstitial and attached cells are very different to those inferred through analysis of leachates alone.

True Growth Rate Kinetics: An Account of the Colonisation and Transport of Microorganisms on Whole Low Grade Ore, at the Agglomerate Scale

The quantification of microbial colonisation and growth rate kinetics is essential in the characterisation of bioleaching systems for modelling heap performance. An experimental system, designed to simulate heap bioleaching conditions at the agglomerate scale, was used to quantify the microbial growth kinetics of a pure culture of Acidithiobacillus ferrooxidans in the PLS, the interstitial phase and attached to the mineral ore. Conventional methods for the quantification of microbial growth rate kinetics associated with the ore and in the flowing PLS have been found to be inadequate in the characterisation of whole ore systems owing to microbial transport between these regions. Growth within the whole ore system was dominated by the microbial communities associated with the ore. Two models were used to estimate the true growth rate kinetics of Acidithiobacillus ferrooxidans on whole low grade ore as a function of growth and transport between the identified phases. The “hydrodynamics” model assumed that microbial transport was promoted by fluid flow dynamics whilst the “biomass balance” model assumed that the microbial concentration gradient across identified phases was the driving force for transport.

Comparison of Microbiological Populations of Mineral Heaps and Mine Wastes of Differing Ages in Active and Abandoned Copper Mines

Sulfide mineral-oxidising microorganisms play a fundamental role in global mineral cycles. Currently, the most common route for the exposure of sulfide minerals is as a result of extractive industries. However, there is a dearth of knowledge about the diversity of microbial populations of waste mineral heaps and biomining operations. Increased understanding of these populations, how they interact and the way in which they change over time has important implications for biomining, but also, crucially, in understanding the longevity of pollution genesis within mineral heaps. To this end, the microbial populations of heaps and mine wastes at three copper mines were investigated using biomolecular and classical techniques. The materials studied were: (i) an 11 year old biomining heap at the Bingham Canyon mine, Salt Lake City, U.S.A.; (ii) two mineral waste heaps deposited around 50 years ago at the São Domingos mine in southern Portugal; (iii) well-weathered mine spoil deposited at least 120 years ago at the Mynydd Parys mine, North Wales, U.K.. The Portuguese samples were found to be the most reactive, in terms of both acidity and concentrations of readily-extractable metals, while the Bingham Canyon heap was somewhat less so. T-RFLP, clone library and culture data implied that both of these sites were dominated by extremely acidophilic mineral-oxidising “Firmicutes”. The population of the São Domingos heaps was very simple, while that of the biomining heap was slightly more complex. In contrast, the spoil from Mynydd Parys was much more weathered, and thus less extreme. While extremely acidophilic mineral-oxidising Bacteria were isolated, the cultured mineral-oxidising population was dominated by moderately acidophilic, heterotrophic Gammaproteobacteria, though the total microbial population was dominated by uncultured Actinobacteria and unclassified Bacteria. The data imply that biodiversity may not be linked to heap age per se, but rather heap pH and possibly readily extractable metal concentrations. As sulfide minerals become depleted as heaps age, the microbial population becomes less dependent on mineral oxidation, and more ‘heterotrophically-inclined’. However, the potential for mineral-oxidation, and thus pollution genesis, remains. Advanced Materials Research Online: 2007-07-15 ISSN: 1662-8985, Vols. 20-21, pp 585-585 doi:10.4028/www.scientific.net/AMR.20-21.585

Anthropogenic remediation of heavy metals selects against natural microbial remediation

In an era of unprecedented environmental change, there have been increasing ecological and global public health concerns associated with exposure to anthropogenic pollutants. While there is a pressing need to remediate polluted ecosystems, human intervention strategies might unwittingly oppose selection for natural detoxification, which is primarily carried out by microbes. We test this possibility in the context of a ubiquitous chemical remediation strategy aimed at targeting toxic metal pollution: the addition of lime-containing materials. Here we show that raising pH by liming decreased the availability of toxic metals in acidic mine-degraded soils, but as a consequence selected against microbial taxa that naturally remediate soil through the production of metal-scavenging siderophores. Understanding the ecological and evolutionary consequences of human intervention on key traits is crucial for the engineering of evolutionary resilient microbial communities, having important implications for human health and biotechnology.