The ferrous iron oxidation kinetics of Thiobacillus ferrooxidans in continuous cultures

The oxidation and growth kinetics of ferrous iron with Thiobacillus ferrooxidans in continuous cultures was examined at several total iron concentrations. On-line off-gas analyses of O₂ and CO₂ were used to measure the oxygen and carbon dioxide consumption rates in the culture. Off-line respiration measurements in a biological oxygen monitor (BOM) were used to measure directly the maximum specific oxygen consumption rate, qO₂,max, of cells grown in continuous culture. It was shown that these reproducibly measured values of qO₂,max vary with the dilution rate. The biomass-specific oxygen consumption rate, qO₂, is dependent on the ratio of the ferric and ferrous iron concentrations in the culture. The oxidation kinetics was accurately described with a rate equation for competitive ferric iron inhibition, using the value of qO₂,max measured in the BOM. Accordingly, only the kinetic constant Ks/K _i needed to be fitted from the measurements. A new method was introduced to determine the steady-state kinetics of a cell suspension in a batch culture that only takes a few hours. The batch culture was set up by terminating the feeding of a continuous culture at its steady state. The kinetic constant K _s/K _i determined in this batch culture agreed with the value determined in continuous cultures at various steady states. Received: 8 February 1999 / Accepted: 17 February 1999     

Ferrous iron oxidation and uranium extraction by Thiobacillus ferrooxidans

The microbiological oxidation of ferrous iron in batch and continuous systems has been investigated in relation to uranium extraction from a low-grade ore by Thiobacillus ferrooxidans. The influence of the parameters, agitation, and aeration on oxygen saturation concentration, rate of oxygen mass transfer, and rate of ferrous iron oxidation was demonstrated. The kinetic values, V_max and K were determined using an adapted Monod equation for different dilution rates and initial concentrations of ferrous iron. The power requirements for initial leaching conditions were also calculated. Uranium extraction as high as 68% has been realized during nine days of treatment. Regrinding the leach residue and its subsequent leaching yielded 87% uranium solubilization.     

Growth Kinetics of Thiobacillus ferrooxidans Isolated from Arsenic Mine Drainage

Thiobacillus ferrooxidans is found in many Alaskan and Canadian drainages contaminated by metals dissolved from placer and lode gold mines. We have examined the iron-limited growth and iron oxidation kinetics of a T. ferrooxidans isolate, AK1, by using batch and continuous cultures. Strain AK1 is an arsenic-tolerant isolate obtained from placer gold mine drainage containing large amounts of dissolved arsenic. The steady-state growth kinetics are described with equations modified for threshold ferrous iron concentrations. The maximal specific growth rate (μ_max) for isolate AK1 at 22.5°C was 0.070 h⁻¹, and the ferrous iron concentration at which the half-maximal growth rate occurred (K_μ) was 0.78 mM. Cell yields varied inversely with growth rate. The iron oxidation kinetics of this organism were dependent on biomass. We found no evidence of ferric inhibition of ferrous iron oxidation for ferrous iron concentrations between 9.0 and 23.3 mM. A supplement to the ferrous medium of 2.67 mM sodium arsenite did not result in an increased steady-state biomass, nor did it appear to affect the steady-state growth kinetics observed in continuous cultures.     

Kinetics of ferrous iron oxidation by batch and continuous cultures of thermoacidophilic Archaea at extremely low pH of 1.1–1.3

The extreme acid conditions required for scorodite (FeAsO₄·2H₂O) biomineralization (pH below 1.3) are suboptimal for growth of most thermoacidophilic Archaea. With the objective to develop a continuous process suitable for biomineral production, this research focuses on growth kinetics of thermoacidophilic Archaea at low pH conditions. Ferrous iron oxidation rates were determined in batch-cultures at pH 1.3 and a temperature of 75°C for Acidianus sulfidivorans, Metallosphaera prunea and a mixed Sulfolobus culture. Ferrous iron and CO₂ in air were added as sole energy and carbon source. The highest growth rate (0.066 h⁻¹) was found with the mixed Sulfolobus culture. Therefore, this culture was selected for further experiments. Growth was not stimulated by increase of the CO₂ concentration or by addition of sulphur as an additional energy source. In a CSTR operated at the suboptimal pH of 1.1, the maximum specific growth rate of the mixed culture was 0.022 h⁻¹, with ferrous iron oxidation rates of 1.5 g L⁻¹ d⁻¹. Compared to pH 1.3, growth rates were strongly reduced but the ferrous iron oxidation rate remained unaffected. Influent ferrous iron concentrations above 6 g L⁻¹ caused instability of Fe²⁺ oxidation, probably due to product (Fe³⁺) inhibition. Ferric-containing, nano-sized precipitates of K-jarosite were found on the cell surface. Continuous cultivation stimulated the formation of an exopolysaccharide-like substance. This indicates that biofilm formation may provide a means of biomass retention. Our findings showed that stable continuous cultivation of a mixed iron-oxidizing culture is feasible at the extreme conditions required for continuous biomineral formation.     

A kinetic model incorporating energy spilling for substrate removal in substrate-sufficient batch culture of activated sludge

Batch assays are currently used to study the kinetic behavior of microbial growth. However, it has been shown that the outcome of batch experiments is greatly influenced by the initial ratio of substrate concentration (S _o) to biomass concentration (X _o). Substrate-sufficient batch culture is known to have mechanisms of spilling energy that lead to significant nongrowth-associated substrate consumption, and the Monod equation is no longer appropriate. By incorporating substrate consumption associated with energy spilling into the balance of the substrate oxidation reaction, a kinetic model for the observed specific substrate consumption rate was developed for substrate-sufficient batch culture of activated sludge, and was further verified by experimental data. It was demonstrated that the specific substrate consumption rate increased with the increase of the S _o/X _o ratio, and the majority of substrate was consumed through energy spilling at high S _o/X _o ratios. It appears that the S _o/X _o ratio is a key parameter in regulating metabolic pathways of microorganisms. Received: 18 January 1999 / Received revision: 7 May 1999 / Accepted: 28 May 1999     

Kinetics of synthesis of nitrogenase in batch and continuous culture of Anabaena flos-aquae

Summary Nitrogenase of Anabaena flos-aquae was inactivated by oxygen and recovery of activity was measured in batch and iron, phosphate and urea-limited continuous cultures. In batch culture, canavanine, chloramphenicol, methylamine, proflavine, puromycin and urea inhibited the recovery process. The rate of recovery of nitrogenase activity in continuous cultures was dependent on light intensity, concentration of urea, ammonium salts and nitrate, and independent of growth rate. Oxygen and urea caused an inactivation of nitrogenase in continuous cultures.     

Potential of Thiobacillus ferrooxidans for waste gas purification. Part 1. Kinetics of continuous ferrous iron oxidation

Kinetic data of ferrous iron oxidation by Thionacillus ferrooxidans were determined. The aim was to remove H₂S (<0.5 ppm) from waste gas by a process proposed earlier. Kinetic data necessary for industrial scale-up were investigated in a chemostat airlift reactor (dilution rate 0.02–0.12 h^–1; pH 1.3). Due to the low pH, ferric iron precipitation and wall growth could be avoided. The maximum ferrous iron oxidation rate of submersed bacteria was 0.77 g 1^–1 h^–1, the maximum specific growth rate about 0.12 h^–1 and the yield coefficient was found to be 0.007 g g^–1 Fe²⁺. The specific O₂ demand of an exponentially growing, ironoxidizing batch culture was 1.33 mg O₂ mg^–1 biomass h^–1. The results indicate that a pH of 1.3 has no negative influence on the kinetics of iron oxidation and growth. Correspondence to: W. Schäfer-Treffenfeldt     

Glucose Metabolism in Batch and Continuous Cultures of Gluconacetobacter diazotrophicus PAL 3

Periplasmic glucose oxidation (by way of a pyrrolo-quinoline-quinone [PQQ]–linked glucose dehydrogenase [GDH]) was observed in continuous cultures of Gluconacetobacter diazotrophicus regardless of the carbon source (glucose or gluconate) and the nitrogen source (N₂ or NH₃). Its synthesis was stimulated by conditions of high energetic demand (i.e., N₂-fixation) and/or C-limitation. Under C-excess conditions, PQQ-GDH synthesis increased with the glucose concentration in the culture medium. In batch cultures, PQQ-GDH was actively expressed in very early stages with higher activities under conditions of N₂-fixation. Hexokinase activity was almost absent under any culture condition. Cytoplasmic nicotinamide adenine dinucleotide (NAD)–linked glucose dehydrogenase (GDH) was expressed in continuous cultures under all tested conditions, and its synthesis increased with the glucose concentration. In contrast, low activities of this enzyme were detected in batch cultures. Periplasmic oxidation, by way of PQQ-GDH, seems to be the principal pathway for metabolism of glucose in G. Diazotrophicus, and NAD-GDH is an alternative route under certain environmental conditions.     

The influence of cultivation methods on Shewanella oneidensis physiology and proteome expression

High-throughput analyses that are central to microbial systems biology and ecophysiology research benefit from highly homogeneous and physiologically well-defined cell cultures. While attention has focused on the technical variation associated with high-throughput technologies, biological variation introduced as a function of cell cultivation methods has been largely overlooked. This study evaluated the impact of cultivation methods, controlled batch or continuous culture in bioreactors versus shake flasks, on the reproducibility of global proteome measurements in Shewanella oneidensis MR-1. Variability in dissolved oxygen concentration and consumption rate, metabolite profiles, and proteome was greater in shake flask than controlled batch or chemostat cultures. Proteins indicative of suboxic and anaerobic growth (e.g., fumarate reductase and decaheme c-type cytochromes) were more abundant in cells from shake flasks compared to bioreactor cultures, a finding consistent with data demonstrating that “aerobic” flask cultures were O₂ deficient due to poor mass transfer kinetics. The work described herein establishes the necessity of controlled cultivation for ensuring highly reproducible and homogenous microbial cultures. By decreasing cell to cell variability, higher quality samples will allow for the interpretive accuracy necessary for drawing conclusions relevant to microbial systems biology research.     

Ethanol production from seaweed extract

Extracts from Laminaria hyperborea could possibly be fermented to ethanol commercially. In particular, seaweed harvested in the autumn contains high levels of easily extractable laminaran and mannitol. Four microorganisms were tested to carry out this fermentation, one bacterium and three yeasts. Only Pichia angophorae was able to utilise both laminaran and mannitol for ethanol production, and its substrate preferences were investigated in batch and continuous cultures. Laminaran and mannitol were consumed simultaneously, but with different relative rates. In batch fermentations, mannitol was the preferred substrate. Its share of the total laminaran and mannitol consumption rate increased with oxygen transfer rate (OTR) and pH. In continuous fermentations, laminaran was the preferred substrate at low OTR, whereas at higher OTR, laminaran and mannitol were consumed at similar rates. Optimisation of ethanol yield required a low OTR, and the best yield of 0.43 g ethanol (g substrate)⁻¹ was achieved in batch culture at pH 4.5 and 5.8 mmol O₂ l⁻¹ h⁻¹. However, industrial production of ethanol from seaweed would require an optimisation of the extraction process to yield a higher ethanol concentration. Journal of Industrial Microbiology & Biotechnology (2000) 25, 249–254. Received 25 February 2000/ Accepted in revised form 05 August 2000     

Environmental and physiological factors affecting the uptake of phosphate by Chlorobium limicola

The uptake of soluble phosphate by the green sulfur bacterium Chlorobium limicola UdG6040 was studied in batch culture and in continuous cultures operating at dilution rates of 0.042 or 0.064 h^–1. At higher dilution rates, washout occurred at phosphate concentrations below 7.1 μM. This concentration was reduced to 5.1 μM when lower dilution rates were used. The saturation constant for growth on phosphate (K _μ) was between 2.8 and 3.7 μM. The specific rates of phosphate uptake in continuous culture were fitted to a hyperbolic saturation model and yielded a maximum rate (Va _max) of 66 nmol P (mg protein)^–1 h^–1 and a saturation constant for transport (K _t) of 1.6 μM. In batch cultures specific rates of phosphate uptake up to 144 nmol P (mg protein)^–1 h^–1 were measured. This indicates a difference between the potential transport of cells and the utilization of soluble phosphate for growth, which results in a significant change in the specific phosphorus content. The phosphorus accumulated within the cells ranged from 0.4 to 1.1 μmol P (mg protein)^–1 depending on the growth conditions and the availability of external phosphate. Transport rates of phosphate increased in response to sudden increases in soluble phosphate, even in exponentially growing cultures. This is interpreted as an advantage that enables Chl. limicola to thrive in changing environments. Received: 9 February 1998 / Accepted: June 1998     

Studies on the growth ofThiobacillus ferrooxidans

Summary Uranyl sulphate (0.2–0.9 mM) inhibited ferrous iron oxidation by growing cultures ofThiobacillus ferrooxidans. The addition of 5–100 mM uranium to the cultures caused immediate cessation of carbon dioxide fixation, rapid loss of viability and gradual depression of ferrous iron oxidation. Virtually no uranium was found in washed cells grown in the presence of subtoxic to toxic amounts of uranyl sulphate. Uranium-poisoned organisms appeared plasmolyzed in electron micrographs. Cultures tolerant to 5 mM UO₂ ²⁺ were develoepd by successive subculturing in increased uranium concentrations. The tolerance was maintained during subculturing in uranium-free medium. Frequency of mutants resistant to 1.0 and 1.5 mM UO₂ ²⁺ was 1 per 1.3×10⁶ and 1 per 9.0×10⁸, respectively. The frequency was increased in the presence of 15–150 mM nickel, zinc and manganese. In liquid cultures, bivalent cations and EDTA alleviated the toxicity of 2 mM uranyl sulphate.     

Effect of ferrous iron concentration on the catalytic activity of immobilized cells of Thiobacillus ferrooxidans

 The kinetics of continuous oxidation of ferrous iron by immobilized cells of Thiobacillus ferrooxidans was studied in a packed-bed bioreactor. Polyurethane foam biomass support particles were used as carriers for cell immobilization. Effects of ferrous iron concentration and its volumetric loading on the kinetics of the reaction were investigated. Media containing different concentrations of ferrous iron in the range 5–20 kg m^-3 were tested. For each medium the kinetics of the reaction at different volumetric loadings of ferrous iron, at a constant temperature of 30^°C, were determined. With media containing 5 kg m^-3 and 10 kg m^-3 Fe²⁺, the fastest oxidation rates of 34.25 kg m^-3 h^-1 and 32 kg m^-3 h^-1 were achieved at a dilution rate of around 6 h^-1, which represents a residence time of 10 min. Employing a higher concentration of ferrous iron (20 kg m^-3) in the medium resulted in lower oxidation rates, with a maximum value of 10 kg m^-3 h^-1, indicating an inhibitory effect of ferrous iron on growth and activity of T. ferrooxidans. The reliable performance of the bioreactor during the course of the experiments confirmed the suitability of polyurethane foam biomass support particles as carriers for T. ferrooxidans immobilization. Received: 5 December 1995/Received revision: 21 April 1996/Accepted: 29 April 1996     

Ferrous sulphate oxidation using Thiobacillus ferrooxidans cells immobilised in polyurethane foam support particles

Summary Oxidation of ferrous iron by Thiobacillus ferrooxidans cells passively immobilised in polyurethane foam particles, using both repeated batches and continuous operation, was studied in a laboratory-scale reactor. Repeated batches yielded complete oxidation at higher rates than single batches, providing resident inocula for subsequent batches. In continuous operation maximum ferric iron productivities were achieved at dilution rates well above theoretical washout values. At a dilution rate of 0.31 h^–1 [approximately three times the maximum specific growth rate (_max)], a productivity of 1.56 kg m^–3 h^–1, based on total ferric iron, or 1.0 kg m^–3 h^–1 based on dissolved ferric iron, was achieved. In addition, cells immobilised in the foam particles retained their oxidative ability for periods of up to 6 weeks when stored in the open air and could be reused immediately.     

Cell growth and oxygen transfer in Methylosinus trichosporium OB3b cultures

Summary The steady-state concentration of M. trichosporium OB3b increased about two-fold in the continuous culture when the feeding medium was supplemented by ferrous sulphate (50mg/L) and citric acid (100mg/L) at a steady state. In batch and continuous cultures, the cell growth was significantly inhibited by excess N-sources (NH₄OH, NH₄Cl, NH₄NO₃, HNO₃, and NaNO₃) and ammonium N-sources were more inhibitory. Both volumetric O₂ transfer coefficient and specific O₂ uptake rate increased monotonously in an extensive range of air flow rate (0.1–7 vvm) and the methane interfered with the O₂ transfer even at very low flow rates (0.01–0.1 vvm).     

Microbiological Oxidation of Ferrous Iron at Low Temperatures

Acidophilic iron-oxidizing bacteria were enriched from mine water samples with ferrous sulfate as the substrate at incubation temperatures in the range of 4 to 46°C. After several subcultures at each test temperature except 46°C, which was prohibitive to growth, the rates of iron oxidation were determined in batch cultures. The results yielded linear rates in a semilogarithmic scale. The rate constants of iron oxidation by growing cultures were fitted into the Arrhenius equation, which displayed linearity in the 4 to 28°C range and yielded an activation energy value of 83 ± 3 kJ/mol.     

Oxygen transfer rate,respiration and yields in batch and chemostat cultures ofKlebsiella aerogenes

The effect was studied of oxygen supply on the changes in total and specific rate of oxygen consumption by the cells, oxygen transfer rate, saturation concentrations of dissolved oxygen and the yields of batch and continuous cultivations. Experiments were done on the microorganismKlebsiella aerogenes CCM 2318 growing on synthetic glucose medium. Continuous cultivations were carried out at dilution rates of 0.96 and 0.178 h⁻¹. The rate of oxygen transfer was determined by the sulphite method and the coefficient K_La was assessed using the dynamic method with a correction for changes in the saturations of dissolved oxygen. A lowered oxygen supply in batch cultivations caused deformations in the course of cell respiration. Comparison of results of batch and continuous cultivations showed that the highest yields Y_x/s and Y_x/o are attained at low dilution rates without oxygen limitation. Batch cultivations, on the other hand, exhibit the lowest yields and the highest cell respiration levels. In both types of cultivations, a respiration peak was ascertained under the conditions of growth limitation by oxygen.     

Ferrous Iron Oxidation by Thiobacillus ferrooxidans: Inhibition with Benzoic Acid, Sorbic Acid, and Sodium Lauryl Sulfate

Benzoic acid, sorbic acid, and sodium lauryl sulfate at low concentrations (5 to 10 mg/liter) each effectively inhibited bacterial oxidation of ferrous iron in batch cultures of Thiobacillus ferrooxidans. The rate of chemical oxidation of ferrous iron in low-pH, sterile batch reactors was not substantially affected at the tested concentrations (5 to 50 mg/liter) of any of the compounds.     

An elevation of the molar growth yield of Zymomonas mobilis during aerobic exponential growth

Elevated values of molar growth yield (Y_x/s = 14–26 g mol^–1) were obtained during exponential growth (μ > 0.4 h^–1) of Zymomonas mobilis ATCC 29191 by using reduced concentrations of glucose (6.25–100 mM) and increased oxygen supply (E _h > 300 mV) in the growth medium, as compared to the Y_x/s of anaerobic exponential growth (8–10 g mol^–1). Aerobically grown cells showed an increased maximum growth rate (μ_max), and a reduced specific glucose consumption rate (q_s), and specific ethanol formation rate (q_p), thus demonstrating a more pronounced energy-coupling growth under oxic conditions. These results can be neither explained by the concept of a solely operating Entner-Doudoroff pathway as an ATP source in aerobically growing cultures of Z. mobilis nor considered to be consistent with existing data on the lack of the Pasteur effect in this bacterium. Therefore, the results rather give evidence for the essential contribution of aerobic ATP generation under the reported conditions. Received: 24 September 1996 / Accepted: 9 December 1996     

Evaluation of the maximum specific growth rate of a yeast indicating non-linear growth trends in batch culture

The maximum specific growth rate (μ_max) of an ethanolic D-xylose-fermenting yeast, Pichia stipitis, showing non-linear growth trends in batch culture, was calculated using the rate equation μ₂ = (1/Δt) ln(x ₂/x ₁). The absolute error Δμ, affecting μ₂, was derived using an equation given by Borzani (1994). Based on the assumption of linearity of growth curves between two closest time points, the relation between the two rate formulae, μ₁ = (1/xˉ)dx _t /dt and μ₂ = (1/Δt) ln(x ₂/x ₁) was established. In a particular condition, when μ₁ = μ₂, an equation has been developed, the roots of which are the specific growth rates at different time points. This revised version was published online in July 2006 with corrections to the Cover Date.