Bio-oxidation of iron using Thiobacillus ferrooxidans

The effects of pH, ferrous and ferric ion concentrations on iron oxidation by Thiobacillus ferrooxidans were examined. The initial temperature and bacterial concentration were maintained at 37°C and 2±1×104cells/ml, respectively. The iron oxidation rate increased with increased initial ferrous iron concentration to 4g/l and thereafter decreased. The presence of iron(III) showed a negative effect on the bacterial iron oxidation rate. The increase of pH also showed an increase in the oxidation rate up to pH 1.75. The oxidation rate followed first order kinetics for the parameters studied. A rate equation has been developed.     

Nitrogen and carbon mineralization during incubation of two Bangladesh soils in relation to temperature

Summary At temperatures of 20°, 30°, 40°, 50° and 60°C in a Gangetic alluvial soil (G soil, pH 7.6) N-mineralization and nitrification increased with temperature up to 40°C and mineralized N accumulated entirely as nitrate. At 50° and 60°C mineralized N was relatively low and no nitrification occurred. In the Red soil (R soil, pH 5.2) mineralized N increased with temperature up to 40°C, was somewhat less at 50°C and was at a maximum at 60°C. Nitrification was maximum at 30°C but did not occur at 50° and 60°C. In the G soil C-mineralization increased considerably with temperature, whilst in the R soil there were only small differences due to temperature.     

Characteristics of a moderately thermophilic and acidophilic iron-oxidizing Thiobacillus

Summary Thiobacillus TH1 is an acidophilic chemolithotrophic heterotroph growing at temperatures up to about 50°C on media containing ferrous iron or pyrite when supplemented with yeast extract or glutathione. Virtually no carbon dioxide fixation occurred during growth on iron with yeast extract. Its DNA contains 48 mol % guanine + cytosine. The organism effects the thermophilic leaching of metals from pyrite, chalcopyrite, CuS, and copper concentrates. Oxidation of soluble ferrous iron at pH 1.6 was competitively inhibited by ferric iron and had a K_m of 7.3 mM FeSO₄.     

Effect of temperature and pH onCandida blankii in chemostat culture

The growth characteristics ofCandida blankii as a function of temperature and pH in a simulated bagasse hemicellulose hydrolysate were determined in chemostat culture. The highest maximum specific growth rate of 0.44h^–1 was reached at 38°C and at pH 5.5, with a sharp decrease in growth rate on either side of this temperature. Growth occurred at 46°C but not at 48°C. The protein and cell yields varied little below 40°C and the respective values were 0.22 and 0.5 g/g at 38°C. At the lower pH values, a severe linear decrease in cell and protein yields occurred, whereas a small increase in these yields at decreasing pH values was found when acetic acid was omitted from the medium. In the presence of acetic acid, a very sharp decrease in the growth rate at pH values below pH 4.5 was noted, despite the very low residual acetic acid concentrations, of less than 50 mg/l, in the culture.     

Production and properties of two types of xylanases from alkalophilic thermophilic Bacillus spp.

Summary Four strains (W1, W2, W3, and W4) of alkalophilic thermophilic bacteria which produced xylanase were isolated from soils. They were aerobic, spore-forming, Gram-positive, and rod-shaped bacteria and hence identified as the genus Bacillus. The optimal temperatures for growth of the four strains were between 45° C and 50° C and pH optima were between 9.0 and 10.0. No growth occurred below pH 7.0 or above 55° C. The four strains produced xylanases in medium containing xylan or xylose under these conditions. The optimal pH and temperature for activities of the four xylanases ranged from 6.0 to 7.0 and from 65° C to 70° C, respectively. The four xylanases were stable in the wide pH range from 4.5 to 10.5 at 45° C for 1 h. All xylanases split xylan to yield xylose and xylobiose.     

Characterization of the combined effects of enzyme,pH and temperature treatments for removal of pathogens from sewage sludge

The combined effects of enzyme, pH and temperature treatments for removal of pathogens from sewage sludge were investigated. An adjustment to pH 10 for 24-h at 23 °C or a 3-h hold at 50 °C resulted in a 100% kill of coliforms but a significant count of Salmonella species still survived. However, pH adjustment to 12 with a hold of 48-h at 23 °C or pH adjustment to 10 or 12 and a 3-h hold at 60 °C was required to achieve a 100% kill of Salmonella species. Although protease enzyme treatment at 40 °C with or without alkali treatment eliminated coliforms completely, Salmonella counts were reduced by two to three orders of magnitude. However, complete elimination of pathogens was observed when enzymatic treatment was provided at 50 °C. Application of alkaline protease, through its combined beneficial effects in pathogen reduction, solids reduction and improved solids settling, has potential as an effective procedure for processing of sewage sludge.     

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.     

Studies on the alkalophilicStreptomyces with extracellular xylanolytic activity

Summary An alkalophilicStreptomyces which produced xylanase, isolated from soil, grew in a temperature range of 15–37°C. The pH optimum for growth was 10 and no growth occurred at pH 7. On a simple wheat bran medium the microorganism exhibited maximum enzyme secretion of 12 U/ml at pH 10. The enzyme had a broad pH optimum of 4.8–10 and the optimum temperature of 50°C. It was completely inactivated at 60°C in 2 h. The enzyme hydrolyzed xylan to a mixture of oligomeric products indicating that the main activity was of the endoxylanase type. The culture filtrate had no cellulase activity.     

Isolation and characterization of Acidicaldus organivorus, gen. nov., sp. nov.: a novel sulfur-oxidizing, ferric iron-reducing thermo-acidophilic heterotrophic Proteobacterium

Thermo-acidophilic prokaryotes isolated from geothermal sites in Yellowstone National Park were identified as novel α-Proteobacteria, distantly related (~93% 16S rRNA gene identity) to the mesophilic acidophile Acidisphaera rubrifaciens. One of these isolates (Y008) was shown to be more thermophilic than all previously characterized acidophilic proteobacteria, with a temperature optimum for growth between 50 and 55°C and a temperature maximum of 65°C. Growth was observed in media maintained at pH between 1.75 and 3.0 and was fastest at pH between 2.5 and 3.0. The G + C content of Y008 was 71.8±0.9 mol%. The acidophile was able to grow heterotrophically on a range of organic substrates, including various monosaccharides, alcohols and amino acids and phenol, though growth on single organic compounds required the provision of one or more growth factors. The isolate oxidized sulfur to sulfuric acid in media containing yeast extract, but was not capable of autotrophic growth with sulfur as energy source. Growth occurred under aerobic conditions and also in the absence of oxygen via anaerobic respiration using ferric iron as terminal electron acceptor. Based on these genotypic and phenotypic traits, it is proposed that Y008 represents the type species of Acidicaldus organivorus, gen. nov., sp. nov.     

Aggregation of recombinant bovine granulocyte colony stimulating factor in solution

Aggregation of recombinant bovine granulocyte colony-stimulating factor (rbG-CSF) was examined by the techniques of size exclusion chromatography (SEC), multiangle laser light scattering (MALS), and SDS-PAGE. Solutions of rbG-CSF in different buffers and pH were exposed to an elevated temperature of 50°C to induce aggregation. The formation of noncovalent soluble aggregates with molecular weight in the millions of Daltons was observed when a solution of rbG-CSF at pH 2.9 was exposed to 50°C. Precipitated protein was the main product of rbG-CSF aggregation in citrate and phosphate buffers at a pH greater than 4. It was demonstrated that precipitant was a mixture of covalent and noncovalent aggregates. The ratio of covalent to noncovalent binding increased with increase in pH of the protein solution. The covalent binding that occurred was primarily due to disulfide linkages via intermolecular disulfide scrambling as demonstrated by SDS-PAGE.     

Anaerobic and aerobic oxidation of ferrous iron at neutral pH by chemoheterotrophic nitrate-reducing bacteria

Nine out of ten anaerobic enrichment cultures inoculated with sediment samples from various freshwater, brackish-water, and marine sediments exhibited ferrous iron oxidation in mineral media with nitrate and an organic cosubstrate at pH 7.2 and 30° C. Anaerobic nitrate-dependent ferrous iron oxidation was a biological process. One strain isolated from brackish-water sediment (strain HidR2, a motile, nonsporeforming, gram-negative rod) was chosen for further investigation of ferrous iron oxidation in the presence of acetate as cosubstrate. Strain HidR2 oxidized between 0.7 and 4.9 mM ferrous iron aerobically and anaerobically at pH 7.2 and 30° C in the presence of small amounts of acetate (between 0.2 and 1.1 mM). The strain gained energy for growth from anaerobic ferrous iron oxidation with nitrate, and the ratio of iron oxidized to acetate provided was constant at limiting acetate supply. The ability to oxidize ferrous iron anaerobically with nitrate at approximately pH 7 appears to be a widespread capacity among mesophilic denitrifying bacteria. Since nitrate-dependent iron oxidation closes the iron cycle within the anoxic zone of sediments and aerobic iron oxidation enhances the reoxidation of ferrous to ferric iron in the oxic zone, both processes increase the importance of iron as a transient electron carrier in the turnover of organic matter in natural sediments. Received: 24 April 1997 / Accepted: 22 September 1997     

Reduced free radical generation during reperfusion of hypothermically arrested hearts

Several studies indicate the presence of hydroxyl radical (OH·) as well as its involvement in the myocardial reperfusion injury. A transition metal-like iron is necessary for the conversion of superoxide anion (O₂ ^–) to a highly reactive and cytotoxic hydroxyl radical (OH·). In the present study, we have examined the generation of OH· and free iron in reperfused hearts following either normothermic (37°C) or hypothermic ischemia (5°C). Employing the Langendorff technique, isolated rat hearts were subjected to global ischemia for 30 min at 37°C or 5°C and were then reperfused for 15 min at 37°C. The results of the study suggest that both the OH· generation in myocardium and free iron release into perfusate were significantly lower in hearts made ischemic at 5°C as compared to 37°C. Release of myoglobin and lactic acid dehydrogenase into perfusate also followed a similar pattern. Furthermore, in in vitro studies, chemically generated O₂ ^– at 5°C caused a significantly lower rate of oxidation of oxymyoglobin as well as generation of OH° and free iron as compared to 37°C. These results suggest that (1) reperfusion of hypothermic ischemic heart is associated with a reduction in the generation of OH· and cellular damage compared to that of normothermic ischemic heart, and (2) myoglobin, an intracellular protein, is a source of free iron and plays a role in the reperfusion injury mediated by free radicals.Abbreviations OH· hydroxyl radical - O₂ ^– superoxide anion - ODFR oxygen-derived free radicals - KHB Krebs-Henseleit buffer - LDH lactate hydrogenase - SOD superoxide dismutase     

Prebiotic polymerization: Oxidative polymerization of 2,3-dimercapto-l-propanol on the surface of iron(III) hydroxide oxide

The oxidation of 2,3-dimercapto-l-propanol by ferric ions on the surface of iron(III) hydroxide oxide (Fe(OH)O) yielded polydisulfide oligomers. This polymerization occurred readily at low dithiol concentration under mild aqueous conditions. Polydisulfide polymers up to the 15-mer were synthesized from 1 mM dithiol in 5 ml water reacted with iron(III) hydroxide oxide (20 mg, 160 µ mole Fe) for 3 days under anaerobic conditions at 40 °C and pH 4. About 91% of the dithiol was converted to short soluble oligomers and 9% to insoluble larger oligomers that were isolated with the Fe (OH)O phase. Reactions carried out at the same ratio of dithiol to Fe(OH)O but at higher dithiol concentrations gave higher yields of the larger insoluble oligomers. The relationship of these results to prebiotic polymer synthesis is discussed.     

Optimal operational factors for nitrite accumulation in batch reactors

The environmental factors that affected the accumulation of nitrite in nitrifying reactors were investigated using a mixed culture. A batch reactor with 50 mg-N/l of ammonia was used. The pH, temperature and dissolved oxygen concentration were varied. The concentration of unionized free ammonia also changed with the oxidation of ammonia and the variation of pH and temperature. The accumulation of nitrite was affected sensitively by pH and temperature. A higher nitrite concentration was observed at pH 8-9 or temperature around 30 °C. The dissolved oxygen also affected, giving the highest nitrite accumulation at around 1.5 mg/l. These were the favoredconditions for nitrite production. The free ammonia concentration influenced thenitrite accumulation also, by inhibiting nitrite oxidation. The inhibition becameapparent at a concentration of approximately 4 mg/l or above, but insignificant atbelow 1 mg/l. Thus, simultaneously high free ammonia concentration and maximumspecific ammonia-oxidation rate (above 15 × 10^-3 mg-N/mg-VSSh)were needed for a significant nitrite accumulation. When the two conditions were met, thenthe highest accumulation was observed when the ratio of the maximum specific oxidationrate of ammonia to the maximum specific oxidation rate of nitrite (k_a/k_n) was highest.Under the optimal operating conditions of pH 8, 30 °C and 1.5 mg/l of dissolvedoxygen, as much as 77% of the removed ammonia accumulated in nitrite.     

Xylanolytic activities ofSpirochaeta thermophila

Spirochetes capable of degrading xylan or cellulose have not been commonly isolated, nor have their polysaccharolytic activities been characterized.Spirochaeta thermophila strain RI 19.B1 is xylanolytic and grows well at 65°C with oatspelt (OX), birchwood (BX), corncob (CCX-A) xylans, or glucuronoxylan (MGX) as the energy source. All xylans were extensively degraded and utilized during growth. About 72–82% of the initial hexuronic acids and 57–79% of initial pentoses disappeared during growth.S. thermophila possessed xylanase, xylosidase, and arabinofuranosidase enzyme activities. Low levels of these activities were detected with growth on glucose, but high expression of these activities occurred during growth on OX. All three activities were cell-associated and were more stable in cells than cell extracts. Xylan-degrading activities were measured with cells or cell extracts exposed (60 min) to a variety of temperatures (65°–85°C) and pHs (5.0–8.0). More than 50% loss of activities occurred at temperatures above 75°C. Although pH stability was affected by buffer, the optimal range was pH 6.5–7.5. These temperature and pH profiles for xylan-degrading activities coincide with those found for the growth ofS. thermophila.     

Studies on cellulase production by a Bacillus subtilis

Bacillus subtilis AU-1 was found to produce carboxymethylcellulase (CMCase) and Avicelase activities in the culture supernatant when grown on a variety of carbohydrates as major carbon source. Maximum CMCase production was obtained in a liquid medium containing 0.2% D (+) raffinose as inducer, 0.5% each of yeast extract, casamino acids and proteose peptone at 50 °C and at an initial pH of 6.0. CMCase activity was detected at early log phase of growth, and reached the maximum level at early stationary phase of growth which occurred at the 10th hour of cultivation. The optimal temperature for CMCase activity was 65 °C, and the enzyme was highly stable up to 60 °C. CMCase synthesis was subjected to catabolite repression by glucose and cellobiose.     

Isolation and characterization of an extremely thermophilic,cellulolytic, anaerobic bacterium

Summary A cellulolyticm obligately anaerobic, extreme thermophile (strain NA10) was isolated from an alkaline hot spring in Nagano Prefecture, Japan. The microorganism was a non-spore-forming, flagellated rod which had a negative reaction to Gram stain, and occurred singly or in pairs. The growth temperature was between 50° C and 85° C with the optimum at 75° C, and the growth pH was between 6.0 and 9.5 with the optimum at 8.1. The anaerobe characteristically fermented cellulose, and produced acetic acid, H₂, CO₂ (main products) and lactic acid (minor product). The DNA had a base composition of 37.7 mol% guanine+cytosine content.     

The effect of temperature on growth and cellulase (β-1,4-endoglucanase) production in the compost fungusChaetomium thermophile var.dissitum

Chaetomium thermophile var.dissitum, isolated from an experimental urban refuse compost, had the following growth characteristics: Minimum temperature, 27±1°C; optimum, 45–50°C; maximum, 57±1°C; pH optimum 5.5–6.0.A number of carbohydrates could be used for growth, but cellulase formation measured with carboxymethylcellulose as substrate was initiated only on cellulose or xylan. With cellulose as the carbon source, cellulase accumulation in the culture filtrate followed closely that of growth, when the temperature was varied. pH optimum for the cellulase system was 5.0.The optimum temperature for cellulase activity with carboxymethylcellulose as substrate varied between 77°C with 1/2 h incubation time and 58°C with 10 h incubation time.With cotton as substrate, the optimum temperature was 58°C regardless of incubation time. Carboxymethylcellulose had a higher stabilizing effect on the enzyme than cotton. The temperature stability of the cellulase was highest at pH 6.0.     

CMCase production by Spicellum roseum in liquid and solid culture

Summary CMCase was produced by 7 strains of Spicellum roseum in both liquid and wheat bran solid substrate cultures. No growth occurred above 35°C. Maximum enzyme production occurred at 30°C, whereas best enzyme activity occurred at pH 5.0 and 50°C. In liquid cultures of S. roseum, NRRL strains 13103, 13104, and 13106 produced activities of ca. 1.1, 1.5, and 1.5 mg glucose per hr/ml culture supernate at 1 week and 2.9, 1.5, and 2.1, respectively at 3 weeks compared to Trichoderma reesei NRRL 11236 (MCG77), which produced activities of 2.8 and 1.3 at 1 and 3 weeks.The mention of firm names or trade products does not imply that they are endorsed or recommended by the U.S. Department of Agriculture over other firms or similar products not mentioned.     

Leptospirillum-Like Bacteria and Evaluation of Their Role in Pyrite Oxidation

Two strains of Leptospirillum-like bacteria isolated from dumps of Alaverdi and Akhtala sulfide ore deposits in Armenia were studied. The optimum and maximum temperatures for the growth of both strains were 37 and 40°C, respectively. The pH optimum was 2.0–2.3. Bacterial growth and ferrous iron oxidation were inhibited by yeast extract. The pyrite-leaching activity of the Leptospirillum-like bacteria under mesophilic conditions was close to that of Acidithiobacillus ferrooxidans and exceeded by 2.0–2.7 times the activity of these moderately thermophilic bacteria at 37°C. The leaching of pyrite by Leptospirillum-like bacteria increased in the presence of sulfur-oxidizing bacteria, particularly, in their association with a thermotolerant sulfur-oxidizing bacterium.