Acquired Thermotolerance and Stressed-Phase Growth of the Extremely Thermoacidophilic Archaeon Metallosphaera sedula in Continuous Culture

The response of an extremely thermoacidophilic archaeon, Metallosphaera sedula (growth temperature range, 50 to 79(deg)C; optimum temperature, 74(deg)C; optimum pH, 2.0), to thermal stress was investigated by using a 10-liter continuous cultivation system. M. sedula, growing at 74(deg)C, pH 2.0, and a dilution rate of 0.04 hr(sup-1), was subjected to both abrupt and gradual temperature shifts in continuous culture to determine the responses of cell density levels and protein synthesis patterns. An abrupt temperature shift from 74 to 79(deg)C resulted in little, if any, changes in cell density and a small increase in total protein per cell. When the culture temperature was shifted further to 80.5(deg)C, cell density dropped to below 5 x 10(sup6) cells/ml from 10(sup8) cells/ml, leading to washout of the culture. Operation at this temperature and slightly higher temperatures, however, could be achieved by exposing the culture to thermal stress more gradually (0.5(deg)C increments). As a result, stable operation could be maintained at temperatures of up to 81(deg)C, and the washout temperature could be increased to 82.5(deg)C. Continuous culture operation at 81(deg)C for 100 h (stressed phase) led to an approximately sevenfold lower steady-state cell density than that observed for operation at or below 79(deg)C. However, sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis (both one and two dimensional) revealed significantly higher levels (sixfold increase) of a 66-kDa stress response protein (MseHSP60), immunologically related to Thermophilic Factor 55 from Sulfolobus shibatae (J. D. Trent, J. Osipiuk, and T. Pinkau, J. Bacteriol. 172:1478-1484, 1990). If the acclimated culture was returned to a lower temperature (i.e., 74(deg)C), the amount of MseHSP60 returned to levels observed prior to thermal acclimation. Furthermore, when the previously acclimated culture (at 81(deg)C) was shifted back from 74 to 81(deg)C, without going through gradual acclimation steps, the result was the immediate onset of washout, suggesting no residual thermotolerance. This study shows that gradual thermal acclimation of M. sedula could only extend the temperature range of stable growth for this organism by 2(deg)C above its maximal growth temperature, albeit at reduced cell densities. Also, this investigation illustrates the utility of continuous culture for characterizing heat shock response and assessing maximum growth temperatures for extremely thermophilic microorganisms.     

Coal Depyritization by the Thermophilic Archaeon Metallosphaera sedula

The kinetics of pyrite oxidation by Metallosphaera sedula were investigated with mineral pyrite and two coals with moderate (Pittsburgh no. 8) and high (New Brunswick, Canada) pyritic sulfur content. M. sedula oxidized mineral pyrite at a greater rate than did another thermophile, Acidianus brierleyi, or a mesophile, Thiobacillus ferrooxidans. Maximum rates of coal depyritization were also greater with M. sedula, although the magnitude of biological stimulation above abiotic rates was notably less than with mineral pyrite. Coal depyritization appears to be limited by the oxidation of pyrite with ferric ions and not by the rate of biotic oxidation of ferrous iron, as evidenced by the maintenance of a high ratio of ferric to ferrous iron in solution by M. sedula. Significant precipitation of hydronium jarosite at elevated temperature occurred only with New Brunswick coal.     

YN23菌株作用下的铁矾沉淀

为更好地了解勤奋金属球菌(Metallosphaera sedula)作用下沉淀的累积进程及沉淀的特性,将一株在我国首次分离鉴定的勤奋金属球菌(YN23)接种在以Fe~(2 )为能源的培养基中,于该菌最佳生长条件(pH1．5,53℃,0．2g·l_(-1)酵母提取物,30g·1_(-1)Fe_2SO_4·7H_2O and 170rpm)下培养。接种25h后,当Fe~(2 )氧化率达到90%时,开始出现沉淀,pH也达到1．92的最高值;到第95h,当沉淀累积到7．9g·1~(-1),时,沉淀反应停止,此时pH达到1．32的最低点。菌群密度随着Fe~(2 )的氧化,前期快速增长;当沉淀出现以后,随着沉淀的累积,逐渐降低。X衍射图谱、红外吸收光谱、能谱和扫描电镜数据揭示,YN23菌株合成的沉淀是黄钾铁矶和黄铵铁矾的混合物,形态特征更接近于后者。     

Stable carbon isotope fractionations of the hyperthermophilic crenarchaeon Metallosphaera sedula

The stable carbon isotopic compositions of the inorganic carbon source, bulk cell material, and isoprenoid lipids of the hyperthermophilic crenarchaeon Metallosphaera sedula, which uses a 3-hydroxypropionate-like pathway for autotrophic carbon fixation, have been measured. Bulk cell material was approximately 3 per thousand enriched in 13C relative to the dissolved inorganic carbon, and 2 per thousand depleted in 13C relative to isoprenoid membrane lipids. The isotope data suggested that M. sedula uses mainly bicarbonate rather than CO(2) as inorganic carbon source, which is in accordance with a 3-hydroxypropionate-like carbon fixation pathway. To the best of our knowledge this is the first report of 13C fractionation effects of such a hyperthermophilic crenarchaeon.     

Increased chalcopyrite bioleaching capabilities of extremely thermoacidophilic Metallosphaera sedula inocula by mixotrophic propagation

Journal of Industrial Microbiology & Biotechnology - Extremely thermoacidophilic Crenarchaeota belonging to the order Sulfolobales, such as Metallosphaera sedula, are metabolically versatile...     

Labeling and enzyme studies of the central carbon metabolism in Metallosphaera sedula

Metallosphaera sedula (Sulfolobales, Crenarchaeota) uses the 3-hydroxypropionate/4-hydroxybutyrate cycle for autotrophic carbon fixation. In this pathway, acetyl-coenzyme A (CoA) and succinyl-CoA are the only intermediates that can be considered common to the central carbon metabolism. We addressed the question of which intermediate of the cycle most biosynthetic routes branch off. We labeled autotrophically growing cells by using 4-hydroxy[1-¹⁴C]butyrate and [1,4-¹³C₁]succinate, respectively, as precursors for biosynthesis. The labeling patterns of protein-derived amino acids verified the operation of the proposed carbon fixation cycle, in which 4-hydroxybutyrate is converted to two molecules of acetyl-CoA. The results also showed that major biosynthetic flux does not occur via acetyl-CoA, except for the formation of building blocks that are directly derived from acetyl-CoA. Notably, acetyl-CoA is not assimilated via reductive carboxylation to pyruvate. Rather, our data suggest that the majority of anabolic precursors are derived from succinyl-CoA, which is removed from the cycle via oxidation to malate and oxaloacetate. These C₄ intermediates yield pyruvate and phosphoenolpyruvate (PEP). Enzyme activities that are required for forming intermediates from succinyl-CoA were detected, including enzymes catalyzing gluconeogenesis from PEP. This study completes the picture of the central carbon metabolism in autotrophic Sulfolobales by connecting the autotrophic carbon fixation cycle to the formation of central carbon precursor metabolites.Sulfolobales (Crenarchaeota) comprise extreme thermoacidophiles from volcanic areas that grow best at a pH of around 2 and a temperature of 60 to 90°C (32, 33). Most Sulfolobales can grow chemoautotrophically on sulfur, pyrite, or H₂ under microaerobic conditions, which also applies to Metallosphaera sedula (31), the organism studied here. Its genome has been sequenced (2). Some species of the Sulfolobales secondarily returned to a facultative anaerobic or even strictly anaerobic life style (33), and some laboratory strains appear to have lost their ability to grow autotrophically (8). Autotrophic representatives of the Sulfolobales use a 3-hydroxypropionate/4-hydroxybutyrate cycle (in short, hydroxypropionate/hydroxybutyrate cycle) for autotrophic carbon fixation (Fig. ​(Fig.1)1) (6-8, 38). The enzymes of this cycle are oxygen tolerant, which predestines the cycle for the lifestyle of the aerobic Crenarchaeota (8). The presence of genes coding for key enzymes of the hydroxypropionate/hydroxybutyrate cycle in the mesophilic aerobic “marine group I” Crenarchaeota suggests that these abundant marine archaea use a similar autotrophic carbon fixation mechanism (6, 24, 68) (for a review of autotrophic carbon fixation in Archaea, see reference 7).Open in a separate windowFIG. 1.Proposed 3-hydroxypropionate/4-hydroxybutyrate cycle functioning in autotrophic carbon fixation in Sulfolobales and its relation to the central carbon metabolism, as studied in this work for Metallosphaera sedula. The situation may be similar in other Sulfolobales and possibly in autotrophic marine Crenarchaeota. Enzymes: 1, acetyl-CoA/propionyl-CoA carboxylase; 2, malonyl-CoA reductase (NADPH); 3, malonic semialdehyde reductase (NADPH); 4, 3-hydroxypropionate-CoA ligase (AMP forming); 5, 3-hydroxypropionyl-CoA dehydratase; 6, acryloyl-CoA reductase (NADPH); 7, acetyl-CoA/propionyl-CoA carboxylase; 8, methylmalonyl-CoA epimerase; 9, methylmalonyl-CoA mutase; 10, succinyl-CoA reductase (NADPH); 11, succinic semialdehyde reductase (NADPH); 12, 4-hydroxybutyrate-CoA ligase (AMP forming); 13, 4-hydroxybutyryl-CoA dehydratase; 14 and 15, crotonyl-CoA hydratase/(S)-3-hydroxybutyryl-CoA dehydrogenase (NAD⁺); 16, acetoacetyl-CoA β-ketothiolase; 17, succinyl-CoA synthetase (ADP forming); 18, succinic semialdehyde dehydrogenase; 19, succinate dehydrogenase (natural electron acceptor unknown); 20, fumarate hydratase; 21, malate dehydrogenase; 22, malic enzyme; 23, PEP carboxykinase (GTP); 24, pyruvate:water dikinase (ATP); 25, enolase; 26, phosphoglycerate mutase; 27, phosphoglycerate kinase; 28, glyceraldehyde 3-phosphate dehydrogenase; 29, triosephosphate isomerase; 30, fructose 1,6-bisphosphate aldolase/phosphatase; 31, (si)-citrate synthase; 32, aconitase; 33, isocitrate dehydrogenase.In the cycle, one molecule of acetyl-coenzyme A (CoA) is formed from two molecules of bicarbonate. The key carboxylating enzyme is a bifunctional biotin-dependent acetyl-CoA/propionyl-CoA carboxylase (10, 11, 36, 38, 48, 49). In Bacteria and Eukarya, acetyl-CoA carboxylase catalyzes the first step in fatty acid biosynthesis. However, archaea do not contain fatty acids, and therefore acetyl-CoA carboxylase obviously plays a different metabolic role. The hydroxypropionate/hydroxybutyrate cycle can be divided into two parts. The first transforms acetyl-CoA and two bicarbonate molecules via 3-hydroxypropionate to succinyl-CoA, and the second converts succinyl-CoA via 4-hydroxybutyrate to two acetyl-CoA molecules. In brief, the product of the acetyl-CoA carboxylase reaction, malonyl-CoA, is reduced via malonic semialdehyde to 3-hydroxypropionate, which is further reductively converted to propionyl-CoA. Propionyl-CoA is carboxylated to (S)-methylmalonyl-CoA by the same carboxylase as that that carboxylates acetyl-CoA (11, 36). (S)-Methylmalonyl-CoA is isomerized to (R)-methylmalonyl-CoA, followed by carbon rearrangement to succinyl-CoA catalyzed by coenzyme B₁₂-dependent methylmalonyl-CoA mutase.Succinyl-CoA then is converted into two molecules of acetyl-CoA via succinic semialdehyde, 4-hydroxybutyrate, 4-hydroxybutyryl-CoA, crotonyl-CoA, 3-hydroxyacetyl-CoA, and acetoacetyl-CoA. This reaction sequence apparently is common to the autotrophic Crenarchaeota, as it also is used by autotrophic Crenarchaeota of the orders Thermoproteales and Desulfurococcales, which use a dicarboxylate/4-hydroxybutyrate cycle for autotrophic carbon fixation (8, 34, 55, 56) (also see the accompanying work [57]).From the list of intermediates of the hydroxypropionate/hydroxybutyrate cycle, acetyl-CoA and succinyl-CoA are the only intermediates considered common to the central carbon metabolism. In this work, we addressed the question of which intermediate of the cycle most biosynthetic routes branch off, and we came to the conclusion that succinyl-CoA serves as the main precursor for cellular carbon. This requires one turn of the cycle to regenerate the CO₂ acceptor and to generate one extra molecule of acetyl-CoA from two molecules of bicarbonate. Acetyl-CoA plus another two bicarbonate molecules are converted by an additional half turn of the cycle to succinyl-CoA. This strategy differs from that of the anaerobic pathways, in which acetyl-CoA is reductively carboxylated to pyruvate, and from there the other precursors for building blocks ultimately are derived (discussed in reference 7).     

Bioenergetic Response of the Extreme Thermoacidophile Metallosphaera sedula to Thermal and Nutritional Stresses

The bioenergetic response of the extremely thermoacidophilic archaeon Metallosphaera sedula to thermal and nutritional stresses was examined. Continuous cultures (pH 2.0, 70(deg)C, and dilution rate of 0.05 h(sup-1)) in which the levels of Casamino Acids and ferrous iron in growth media were reduced by a step change of 25 to 50% resulted in higher levels of several proteins, including a 62-kDa protein immunologically related to the molecular chaperone designated thermophilic factor 55 in Sulfolobus shibatae (J. D. Trent, J. Osipiuk, and T. Pinkau, J. Bacteriol. 172:1478-1484, 1990), on sodium dodecyl sulfate-polyacrylamide gels. The 62-kDa protein was also noted at elevated levels in cells that had been shifted from 70 to either 80 or 85(deg)C. The proton motive force ((Delta)p), transmembrane pH ((Delta)pH), and membrane potential ((Delta)(psi)) were determined for samples obtained from continuous cultures (pH 2.0, 70(deg)C, and dilution rate of 0.05 h(sup-1)) and incubated under nutritionally and/or thermally stressed and unstressed conditions. At 70(deg)C under optimal growth conditions, M. sedula was typically found to have a (Delta)p of approximately -190 to -200 mV, the result of an intracellular pH of 5.4 (extracellular pH, 2.0) and a (Delta)(psi) of +40 to +50 mV (positive inside). After cells had been shifted to either 80 or 85(deg)C, (Delta)(psi) decreased to nearly 0 mV and internal pH approached 4.0 within 4 h of the shift; respiratory activity, as evidenced by iron speciation in parallel temperature-shifted cultures on iron pyrite, had ceased by this point. If cultures shifted from 70 to 80(deg)C were shifted back to 70(deg)C after 4 h, cells were able to regain pyrite oxidation capacity and internal pH increased to nearly normal levels after 13 h. However, (Delta)(psi) remained close to 0 mV, possibly the result of enhanced ionic exchange with media upon thermal damage to cell membranes. Further, when M. sedula was subjected to an intermediate temperature shift from 73 to 79(deg)C, an increase in pyrite dissolution (ferric iron levels doubled) over that of the unshifted control at 73(deg)C was noted. The improvement in leaching was attributed to the synergistic effect of chemical and biological factors. As such, periodic exposure to higher temperatures, followed by a suitable recovery period, may provide a basis for improving bioleaching rates of acidophilic chemolithotrophs.     

Microbiological Redox Potential Control to Improve the Efficiency of Chalcopyrite Bioleaching

The effect of controlling the redox potential (E_h) on chalcopyrite bioleaching kinetics was studied as a new aspect of redox control during chalcopyrite bioleaching, and its mechanism was investigated by employing the “normalized” solution redox potential (E_normal) and the reaction kinetics model. Different E_h ranges were established by use of different acidophiles (Sulfobacillus acidophilus YTF1; Sulfobacillus sibiricus N1; Acidimicrobium ferrooxidans ICP; Acidiplasma sp. Fv-AP). Cu dissolution was very susceptible to real-time change in E_h during the reaction. It was found that efficiency of bioleaching of chalcopyrite can be effectively evaluated on the basis of E_normal, since it is normalized for real-time fluctuations of concentrations of major metal solutes during bioleaching. For steady Cu solubilization during bioleaching at a maximum rate, it was important to maintain a redox potential range of 0 ≤ E_normal ≤ 1 (?0.35 mV optimal) at the mineral surface by employing a “weak” ion-oxidizer. This led to a copper recovery of > 75%. At higher E_normal levels (E_normal > 1 by “strong” microbial Fe²⁺ oxidation), Cu solubilization was slowed by diffusion through the product film at the mineral surface (< 50% Cu recovery) caused by low reactivity of the chalcopyrite and by secondary passivation of the chalcopyrite surface, mainly by jarosite.     

Effect of Activated Carbon Addition on the Conventional and Electrochemical Bioleaching of Chalcopyrite Concentrates

The effect of activated carbon addition on the rate and efficiency of copper mobilization from Sarcheshmeh chalcopyrite concentrate was studied in the presence and absence of a mixed culture of moderately thermophilic microorganisms. Conventional leaching at a 10% (w/v) pulp density in 500-ml Erlenmeyer flasks on a rotary shaker at 150 rpm, and electrochemical bioleaching in a stirred bioreactor at an ORP (oxidation-reduction potential) range of 400 to 430 mV measured against a Ag/AgCl reference electrode. The bioreactor contained ore concentrate at a pulp density of 20%, which was stirred at 600 rpm. All experiments were conducted in the presence and absence of 3 g/L activated carbon, at initial pH 1.5, temperature 50°C, in Norris's nutrient medium with an addition of 0.02% (w/v) yeast extract. The results showed that the addition of activated carbon increased the rate and yield of copper extraction from the concentrate especially in the presence of bacteria. Final recovery after 20 days was 52% and 44% in the shake flask experiments with and without carbon addition, respectively. Enhanced rates of copper mobilization were achieved in the electrochemical bioleaching experiments in which copper was leached selectively relative to iron. Final copper recovery after 10 days was 85% and 77% in the presence and absence of activated carbon, respectively. The positive effect of activated carbon on copper extraction could be related to the galvanic interaction between the inert carbon as cathode and chalcopyrite as anode. The bacterial elimination of sulfur produced on the sulfide minerals during chemical leaching is assumed to intensify the galvanic interaction. It seems that maintaining the ORP at a low potential and efficient mixing improves the bacterial and chemical subsystems in the electro-bioreactor that accelerates the rate of copper mobilization from the concentrate.     

Draft Genome Sequence of the Extremely Halophilic Archaeon Halogranum salarium B-1T

Halogranum salarium is an extremely halophilic archaeon isolated from evaporitic salt crystals and belongs to the family Halobacteriaceae. Here, we present the 4.5-Mb draft genome sequence of the type strain (B-1^T) of H. salarium. This is the first report of the draft genome sequence of a haloarchaeon in the genus Halogranum.     

Characterization of acetyl-CoA/propionyl-CoA carboxylase in Metallosphaera sedula. Carboxylating enzyme in the 3-hydroxypropionate cycle for autotrophic carbon fixation.

Autotrophic Archaea of the family Sulfolobaceae (Crenarchaeota) use a modified 3-hydroxypropionate cycle for carbon dioxide assimilation. In this cycle the ATP-dependent carboxylations of acetyl-CoA and propionyl-CoA to malonyl-CoA and methylmalonyl-CoA, respectively, represent the key CO2 fixation reactions. These reactions were studied in the thermophilic and acidophilic Metallosphaera sedula and are shown to be catalyzed by one single large enzyme, which acts equally well on acetyl-CoA and propionyl-CoA. The carboxylase was purified and characterized and the genes were cloned and sequenced. In contrast to the carboxylase of most other organisms, acetyl-CoA/propionyl-CoA carboxylase from M. sedula is active at 75 degrees C and is isolated as a stabile functional protein complex of 560 +/- 50 kDa. The enzyme consists of two large subunits of 57 kDa each representing biotin carboxylase (alpha) and carboxytransferase (gamma), respectively, and a small 18.6 kDa biotin carrier protein (beta). These subunits probably form an (alpha beta gamma)4 holoenzyme. It has a catalytic number of 28 s-1 at 65 degrees C and at the optimal pH of 7.5. The apparent Km values were 0.06 mm for acetyl-CoA, 0.07 mm for propionyl-CoA, 0.04 mm for ATP and 0.3 mm for bicarbonate. Acetyl-CoA/propionyl-CoA carboxylase is considered the main CO2 fixation enzyme of autotrophic members of Sulfolobaceae and the sequenced genomes of these Archaea contain the respective genes. Due to its stability the archaeal carboxylase may prove an ideal subject for further structural studies.     

The Community Dynamics of Major Bioleaching Microorganisms During Chalcopyrite Leaching Under the Effect of Organics

To determine the effect of organics (yeast extract) on microbial community during chalcopyrite bioleaching at different temperature, real-time polymerase chain reaction (PCR) was employed to analyze community dynamics of major bacteria applied in bioleaching. The results showed that yeast extract exerted great impact on microbial community, and therefore influencing bioleaching rate. To be specific, yeast extract was adverse to this bioleaching process at 30°C due to decreased proportion of important chemolithotrophs such as Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans. However, yeast extract could promote bioleaching rate at 40°C on account of the increased number and enhanced work of Ferroplasma thermophilum, a kind of facultative bacteria. Similarly, bioleaching rate was enhanced under the effect of yeast extract at 50°C owing to the work of Acidianus brierleyi. At 60°C, bioleaching rate was close to 100% and temperature was the dominant factor determining bioleaching rate. Interestingly, the existence of yeast extract greatly enhanced the relative competitiveness of Ferroplasma thermophilum in this complex bioleaching microbial community.     

两株不同来源的嗜酸氧化亚铁硫杆菌对黄铜矿浸出的研究

为了比较两株不同来源的嗜酸氧化亚铁硫杆菌菌株在不同培养基中的亚铁氧化活性和黄铜矿浸出能力,本研究采用了分离自广东梅山酸性矿坑水中的菌株M1和标准菌株ATCC 23270,对其在9K培养基中的亚铁氧化活性和矿物培养基中氧化还原电位以及浸矿效率进行了测定,该矿物培养基中黄铜矿来自广东梅山.研究结果表明,菌株M1在9K培养基中需5天才能将亚铁完全氧化.而ATCC 23270只需4天,但是菌株MI的铜离子浸出效率(38%)却高于ATCC 23270(31%),浸出30天后,菌株M1浸矿体系的氧化还原电位从最初348 mV上升到520 mV,而ATCC 23270上升较小,仅从最初350 mV上升到491 mV.氧化还原电位的变化说明从广东梅山分离得到的菌株M1在浸矿体系中亚铁氧化活性比ATCC 23270更高.菌株M1比长期实验室培养的标准菌株ATCC 23270更适合当地矿物的微生物浸出,因而在生物浸出工艺中,应考虑采用分离或富集当地原生菌株来进行浸矿.