Elizabeth Watkin

The resilience and versatility of acidophiles that contribute to the bio‐assisted extraction of metals from mineral sulphides

In this paper, a brief outline is presented on acidic ferric ion oxidation of mineral sulphides for the extraction of metals in both stirred tank reactors for mineral concentrates and heaps for low‐grade ores. The identities and capabilities of the relatively few acidophiles that assist the oxidative processes are summarized and their responses to selected extremes in their growth environments described. Individually, the organisms adapt to the presence of high concentrations of heavy metals and other elements in the bioleaching environment, tolerate a wide range of acidities and can recover from prolonged exposure to temperatures significantly above their preferred temperatures for growth. However, the presence of chloride in their acidic environment presents a significant physiological challenge. Species that exhibit a chemotactic response and attachment to sulphide surfaces, where they can create their own micro‐environments, would be favoured in both heap bioreactors with low availability of energy substrates and physically aggressive, agitated continuous stirred‐tank reactor environments treating concentrates.In this paper, a brief outline is presented on acidic ferric ion oxidation of mineral sulphides for the extraction of metals in both stirred tank reactors for mineral concentrates and heaps for low-grade ores. The identities and capabilities of the relatively few acidophiles that assist the oxidative processes are summarized and their responses to selected extremes in their growth environments described. Individually, the organisms adapt to the presence of high concentrations of heavy metals and other elements in the bioleaching environment, tolerate a wide range of acidities and can recover from prolonged exposure to temperatures significantly above their preferred temperatures for growth. However, the presence of chloride in their acidic environment presents a significant physiological challenge. Species that exhibit a chemotactic response and attachment to sulphide surfaces, where they can create their own micro-environments, would be favoured in both heap bioreactors with low availability of energy substrates and physically aggressive, agitated continuous stirred-tank reactor environments treating concentrates.

Metals tolerance in moderately thermophilic isolates from a spent copper sulfide heap, closely related to Acidithiobacillus caldus, Acidimicrobium ferrooxidans and Sulfobacillus thermosulfidooxidans

Selective enrichments enabled the recovery of moderately thermophilic isolates with copper bioleaching ability from a spent copper sulfide heap. Phylogenetic and physiological characterization revealed that the isolates were closely related to Sulfobacillus thermosulfidooxidans, Acidithiobacillus caldus and Acidimicrobium ferrooxidans. While isolates exhibited similar physiological characteristics to their corresponding type strains, in general they displayed similar or greater tolerance of high copper, zinc, nickel and cobalt concentrations. Considerable variation was found between species and between several strains related to S. thermosulfidooxidans. It is concluded that adaptation to metals present in the bioleaching heap from which they were isolated contributed to but did not entirely explain high metals tolerances. Higher metals tolerance did not confer stronger bioleaching performance, suggesting that a physical, mineralogical or chemical process is rate limiting for a specific ore or concentrate.

Distinct Bradyrhizbium communities nodulate legumes native to temperate and tropical monsoon Australia

Geographic isolation and growing climate aridity played major roles in the evolution of Australian legumes. It is likely that these two factors also impacted on the evolution of their root-nodule bacteria. To investigate this issue, we applied a multilocus sequence analysis (MLSA) approach to examine Bradyrhizobium isolates originating from temperate areas of Western Australia (WA) and the tropical-monsoon area of the Northern Territory (NT). The isolates were mostly collected from the nodules of legumes belonging to tribes, genera and species endemic or native to Australia. Phylogenetic analyses of sequences for the housekeeping atpD, dnaK, glnII, gyrB, recA and 16S rRNA genes and nodulation nodA gene revealed that most isolates belonged to groups that are distinct from non-Australian Bradyrhizobium isolates, which is in line with earlier studies based on 16S rRNA gene sequence analyses. Phylogenetic analysis of the nodA data allowed identification of five major Clades among the WA and NT isolates. All WA isolates grouped in a subgroup I.1 of Clade I with strains originating from temperate eastern Australia. In contrast, the NT isolates formed part of Clades I (subgroup I.2), III (subgroup III.3), IV, V and X. Of these nodA clades, Clade I, Clade IV, Clade X presumably have an Australian origin. Overall, these data demonstrate that the impact of geographic isolation of the Australian landmass is manifested by the presence of numerous unique clusters in housekeeping and nodulation gene trees. In addition, the intrinsic climate characteristics of temperate WA and tropical-monsoon NT were responsible for the formation of distinct legume communities selecting for unrelated Bradyrhizobium groups.

Physicochemical and antimicrobial properties of citral and quercetin incorporated kafirin-based bioactive films

The aim of this study was to determine the physicochemical and antimicrobial properties of kafirin-based bioactive films incorporating the plant essential oil citral and the polyphenol quercetin. The addition of quercetin and citral both imparted a yellowish colour to the films. The tensile strength of films significantly decreased and elongation at break increased when citral was incorporated, whereas addition of quercetin did not alter these two film parameters. The rate of water vapour transmission of the films decreased with citral incorporation but the water vapour permeability was not affected by either citral or quercetin incorporation. Furthermore, incorporation of citral and quercetin significantly lowered the oxygen permeability of the films. Film made of kafirin alone had antimicrobial activity against Listeria monocytogenes, however, films incorporating citral exhibited the highest antimicrobial activity against Campylobacter jejuni, L. monocytogenes, and Pseudomonas fluorescens. These results suggest that kafirin-based films incorporating citral and quercetin have potential as bioactive packaging to improve food safety and quality.

Calcium and acid stress interact to affect the growth of Rhizobium leguminosarum bv. trifolii

Abstract The effect of acidity on the growth and survival of six strains of Rhizobium leguminosarum bv. trifolii (WU95, NA3001, WSM409, TA1, NA3025 and NA3039) was studied. Acid conditions reduced the growth rate of all strains; mean generation times were 50–60% slower at pH 5.0 than at pH 7.0. The critical pH for growth on solid media was in the range of 4.3–4.6. This is consistent with growth of the strains at different pHs in liquid culture, with NA3001 being the only strain to exhibit a normal growth pattern at pH 4.5. The interaction of acidity and calcium on the growth and survival of three of the strains (WU95, WSM409 and TA1) was studied in the presence of high (300 μ m ) and low (20 μ m ) concentrations of phosphate. A region of “acid-stress” somewhere below pH 5.0 was observed where growth rate slowed rapidly over 0.2–0.3 of a pH unit. The presence of 300 μ m phosphate did not affect the critical pH for growth or growth rate within the “acid-stress” zone, but did reduce the mean generation time of all strains studied at pH above the “acid-stress” zone. At pH 7.0, increasing calcium from 300 μ m to 3000 μ m had little effect on growth rate, but high calcium increased growth rate within the “acid-stress” zone and enabled growth at a lower pH than that observed with the low calcium concentrations. A four-zone model for the response of root nodule bacteria to acidity is proposed.

Medicago sativa and Medicago murex differ in the nodulation response to soil acidity

Nodulation of Medicago sativa by Sinorhizobium meliloti is challenged by acidity, but the ability of M. murex to nodulate in acid soils provided the opportunity to compare the symbiotic development between the two species in an acid sandy soil. Soil acidity had different effects on the nodulation of Medicago spp. In soil of pH 4.3, M. murex produced fewer nodules than plants grown in soil of pH 7.0, but these nodules developed at a similar rate to those on plants grown in soil of neutral pH. The uppermost nodule on M. murex formed lower down the tap-root of plants grown in soil of pH 4.3. In identical soils, M. sativa produced fewer nodules when grown in the acidic soil and nodules appeared later compared to those on plants grown in soil of pH 7.0. However, the location of the uppermost nodule was the same in plants grown in soils of pH 4.3 and 7.0. It is suggested that M. murex formed the first nodules near the actively growing root tip, while M. sativa formed nodules later at the more mature region of the root, above the root tip.

Identification of tolerance to soil acidity in inoculant strains of Rhizobium leguminosarum bv. trifolii

The acid-soil tolerance of six strains (WU95, NA3001, WSM409, TA1, NA3025 and NA3039) of Rhizobium leguminosarum bv. trifolii was assessed in a three-year cross-row field experiment in an acid sandy soil of pH 4.2. Strains WSM409, NA3039 and WU95 were more acid-soil tolerant than strains NA3025, TA1 and NA3001. Strains WSM409 and NA3039 colonised and persisted in acid-soil to a greater degree than strains TA1 and NA3001. The data from this study clearly identified strain WSM409 as a strain with outstanding potential for improving the production of clovers on acid soils.

RNA transcript sequencing reveals inorganic sulfur compound oxidation pathways in the acidophile Acidithiobacillus ferrivorans

Acidithiobacillus ferrivorans is an acidophile implicated in low-temperature biomining for the recovery of metals from sulfide minerals. Acidithiobacillus ferrivorans obtains its energy from the oxidation of inorganic sulfur compounds, and genes encoding several alternative pathways have been identified. Next-generation sequencing of At. ferrivorans RNA transcripts identified the genes coding for metabolic and electron transport proteins for energy conservation from tetrathionate as electron donor. RNA transcripts suggested that tetrathionate was hydrolyzed by the tetH1 gene product to form thiosulfate, elemental sulfur and sulfate. Despite two of the genes being truncated, RNA transcripts for the SoxXYZAB complex had higher levels than for thiosulfate quinone oxidoreductase (doxDAgenes). However, a lack of heme-binding sites in soxX suggested that DoxDA was responsible for thiosulfate metabolism. Higher RNA transcript counts also suggested that elemental sulfur was metabolized by heterodisulfide reductase (hdrgenes) rather than sulfur oxygenase reductase (sor). The sulfite produced as a product of heterodisulfide reductase was suggested to be oxidized by a pathway involving the sat gene product or abiotically react with elemental sulfur to form thiosulfate. Finally, several electron transport complexes were involved in energy conservation. This study has elucidated the previously unknown At. ferrivorans tetrathionate metabolic pathway that is important in biomining.

Physiological responses to acid stress of an acid-soil tolerant and an acid-soil sensitive strain of Rhizobium leguminosarum biovar trifolii

Physiological responses to acid stress in two strains of Rhizobium leguminosarum bv trifolii of differing acid-soil tolerance were compared. Acidity affected the size and morphology of the acid-tolerant strain, WSM409, but not of the acid-sensitive strain, TA1. Acid grown cells of WSM409 and TA1 had less cell-associated Ca and Mg and more P than cells grown at pH 7.0. Potassium content was lower in acid grown cells; WSM409 was less affected by pH than that in TA1. WSM409 was more tolerant of pH shock at pH 3.5 when grown at pH 4.8 than when grown at pH 7.0. TA1 was more sensitive to pH shock when grown at pH 4.8 than when grown at pH 7.0. WSM409 shows a characteristic adaptive acid tolerance response, whereas TA1 shows an acid sensitive response.

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.