Formation and growth of heterotrophic aerobic biofilms on small suspended particles in airlift reactors

In this article, the conditions for aerobic biofilm formation on suspended particles, the dynamics of biofilm formation, and the biomass production during the start-up of a Biofilm Airlift Suspension reactor (BAS reactor) have been studied. The dynamics of biofilm formation during start up in the biofilm airlift suspension reactor follows three consecutive stages: bare carrier, microcolonies or patchy biofilms on the carrier, and biofilms completely covering the carrier. The effect of hydraulic retention time and of substrate loading rate on the formation of biofilms were investigated. To obtain in a BAS reactor a high biomass concentration and predominantly continuous biofilms, which completely surround the carrier, the hydraulic retention time must be shorter than the inverse of the maximum growth rate of the suspended bacteria. At longer hydraulic retention times, a low amount of attached biomass can be present on the carrier material as patchy biofilms. During the start-up at short hydraulic retention times the bare carrier concentration decreases, the amount of biomass per biofilm particle remains constant, and biomass increase in the reactor is due to increasing numbers of biofilm particles. The substrate surface loading rate has effect only on the amount of biomass on the biofilm particle. A higher surface load leads to a thicker biofilm.A strong nonlinear increase of the concentration of attached biomass in time was observed. This can be explained by a decreased abrasion of the biofilm particles due to the decreasing concentration of bare carriers. The detachment rate per biofilm area during the start-up is independent of the substrate loading rate, but depends strongly upon the bare carrier concentration.The Pirt-maintenance concept is applicable to BAS reactors. Surplus biomass production is diminished at high biomass concentrations. The average maximal yield of biomass on substrate during the experiments presented in this article was 0.44 +/- 0.08 C-mol/C-mol, the maintenance value 0.019 +/- 0.012 C-mol/(C-mol h). The lowest actual biomass yield measured in this study was 0.15 C-mol/C-mol. (c) 1994 John Wiley & Sons, Inc.     

A dynamic mathematical model for microbial removal of pyritic sulfur from coal

A dynamic mathematical model has been developed to describe microbial desulfurization of coal by Thiobacillus ferrooxidans. The model considers adsorption and desorption of cells on coal particles and microbial oxidation of pyritic sulfur on particle surfaces. The influence of certain parameters, such as microbial growth rate constants, adsorption-descrption constants, pulp density, coal particle size, initial cell and solid phase substrate concentration on the maximum rate of pyritic sulfur removal, have been elucidated. The maximum rate of pyritic sulfur removal was strongly dependent upon the number of attached cells per coal particle. At sufficiently high initial cell concentrations, the surfaces of coal particles are nearly saturated by the cells and the maximum leaching rate is limited either by total external surface area of coal particles or by the concentration of pyritic sulfur in the coal phase. The maximum volumetric rate of pyritic sulfur removal (mg S/h cm(3) mixture) increases with the pulp density of coal and reaches a saturation level at high pulp densities (e.g. 45%). The maximum rate also increases with decreasing particle diameter in a hyperbolic form. Increases in adsorption coefficient or decreases in the desorption coefficient also result in considerable improvements in this rate. The model can be applied to other systems consisting of suspended solid substrate particles in liquid medium with microbial oxidation occurring on the particle surfaces (e.g., bacterial ore leaching). The results obtained from this model are in good agreement with published experimental data on microbial desulfurization of coal and bacterial ore leaching.     

Kinetics of growth and elemental sulfur oxidation in batch culture of thiobacillus ferrooxidans

The kinetics of oxidation of elemental sulfur by Thiobacillus ferrooxidans in a batch reactor was followed by measuring the concentration of adsorbed cells on the sulfur surface, the concentration of free cells in liquid medium, and the amount of sulfur oxidized. As the elemental sulfur was oxidized to sulfate, the liquid-phase concentration of free cells continued to increase with time, whereas the surface concentration of adsorbed cells per unit weight of sulfur approached a limiting value, i.e., the maximum adsorption capacity. During sulfur oxidation, there was a close correlation between the concentrations of adsorbed and free cells, and these data were well correlated with the Langmuir isotherm. The observed rates of batch growth and sulfur oxidation were consistent with a kinetic model, assuming that the growth rate of batch growth and sulfur oxidation were consistent with a kinetic model. Assuming that the growth rate of adsorbed bacteria is proportional to the product of the concentration of adsorbed cells and the fraction of adsorption sites unoccupied by cells. The kinetic and stoichiometric parameters appearing in the model were evaluated using the experimental data and were compared with parameters determined previously for a few metal sulfides. (c) 1994 John Wiley & Sons, Inc.     

Growth of Thiobacillus ferrooxidans on Elemental Sulfur

Growth kinetics of Thiobacillus ferrooxidans in batch cultures, containing prills of elementary sulfur as the sole energy source, were studied by measuring the incorporation of radioactive phosphorus in free and adsorbed bacteria. The data obtained indicate an initial exponential growth of the attached bacteria until saturation of the susceptible surface was reached, followed by a linear release of free bacteria due to successive replication of a constant number of adsorbed bacteria. These adsorbed bacteria could continue replication provided the colonized prills were transferred to fresh medium each time the stationary phase was reached. The bacteria released from the prills were unable to multiply, and in the medium employed they lost viability with a half-life of 3.5 days. The spreading of the progeny on the surface was followed by staining the bacteria on the prills with crystal violet; this spreading was not uniform but seemed to proceed through distortions present in the surface. The specific growth rate of T. ferrooxidans ATCC 19859 was about 0.5 day⁻¹, both before and after saturation of the sulfur surface. The growth of adsorbed and free bacteria in medium containing both ferrous iron and elementary sulfur indicated that T. ferrooxidans can simultaneously utilize both energy sources.     

Growth models of the continuous bacterial leaching of iron pyrite by Thiobacillus ferrooxidans

The leaching of iron pyrite by Thiobacillus ferrooxidans was studied in a continuous stirred tank reactor at a variety of dilution rates (0.012-0.22 h(-1)), pyrite surface areas (18-194 m(2)/L), and inlet soluble substrate (Fe(2+)) concentrations (0-3000 ppm). The bacterial leaching rate was found to increase with increasing pyrite surface area, dilution rate, and inlet Fe(2+) concentration. The concentration of bacteria in solution was related to the concentration of bacteria attached to the pyrite surface by a Langmuir-type adsorption-desorption relation. Fitting the experimental data to this relation yielded a value for the area occupied per bacterium of 86 mum(2). This result is consistent with the concept of preferential bacterial attachment of certain sites on the solid. A bacterial growth model was developed that included both bacterial growth in solution and growth of bacteria attached to the pyrite surface. The specific growth rate of the attached bacteria was calculated from this model and was found to increase with increasing solid dilution rate and to decrease with increasing pyrite surface area and soluble substance concentration. An explanation of these results based on an active-inactive site mechanisms was also developed.     

Influence of substrate wettability on the attachment of marine bacteria to various surfaces

The effect of the initial substrate surface condition, as indicated by the critical surface tension for wetting, on the rate of attachment of marine bacteria to a variety of solid surfaces has been measured. The techniques used to determine the number of bacteria attached per unit surface area were a lipopolysaccharide test utilizing Limulus lysate and direct examination of the surface by scanning electron microscopy. The results obtained by the two techniques are compared and their significance to the control of microbiological slime film formation (microfouling) is discussed.     

Growth Kinetics of Thiobacillus thiooxidans on the Surface of Elemental Sulfur

The growth kinetics of Thiobacillus thiooxidans on elemental sulfur in batch cultures at 30(deg)C and pH 1.5 was studied by measuring the time courses of the concentration of adsorbed cells on sulfur, the concentration of free cells suspended in liquid medium, and the amount of sulfur oxidized. As the elemental sulfur was oxidized to sulfate ions, the surface concentration of adsorbed cells per unit mass of sulfur approached a maximum value (maximum adsorption capacity of sulfur particles) whereas the concentration of free cells continued to increase with time. There was a close relationship between the concentrations of free and adsorbed cells during the microbial sulfur oxidation, and the two cell concentrations were well correlated by the Langmuir isotherm with adsorption equilibrium constant K(infA) and maximum adsorption capacity X(infAm) of 2.10 x 10(sup-9) ml per cell and 4.57 x 10(sup10) cells per g, respectively. The total concentration of free and adsorbed cells increased in parallel with the amount of sulfate formed. The total growth on elemental sulfur gave a characteristic growth curve in which a linear-growth phase followed the period of an initial exponential phase. The batch rate data collected under a wide variety of inoculum levels (about 10(sup5) to 10(sup8) cells per ml) were consistent with a kinetic model assuming that the growth rate of adsorbed bacteria is proportional to the product of the concentration, X(infA), of adsorbed cells and the fraction, (theta)(infV), of adsorption sites unoccupied by cells. The kinetic and stoichiometric parameters appearing in the model were estimated from the experimental data, and the specific growth rate, (mu)(infA), and growth yield, Y(infA), were 2.58 day(sup-1) and 2.05 x 10(sup11) cells per g, respectively. The proposed model and the parameter values allowed us to predict quantitatively the surface attachment of T. thiooxidans cells on elemental sulfur and the bacterial growth in both initial exponential and subsequent linear phases. The transition from exponential to linear growth was a result of two competing factors: an increase in the adsorbed-cell concentration, X(infA), permitted a decrease in the unoccupied-site fraction, (theta)(infV).     

Growth Kinetics of Attached Iron-Oxidizing Bacteria

A model of growth and substrate utilization for ferrous-iron-oxidizing bacteria attached to the disks of a rotating biological contactor was developed and tested. The model describes attached bacterial growth as a saturation function in which the rate of substrate utilization is determined by a maximum substrate oxidation rate constant (P), a half-saturation constant (K_s), and the concentration of substrate within the rotating biological contactor (S₁). The maximum oxidation rate constant was proportional to flow rate, and the substrate concentration in the reactor varied with influent substrate concentration (S₀). The model allowed the prediction of metabolic constants and included terms for both constant and growth-rate-dependent maintenance energies. Estimates for metabolic constants of the attached population of acidophilic, chemolithotrophic, iron-oxidizing bacteria limited by ferrous iron were: maximum specific growth rate (μ_max), 1.14 h⁻¹; half-saturation constant (K_s) for ferrous iron, 54.9 mg/liter; constant maintenance energy coefficient (m₁), 0.154 h⁻¹; growth-rate-dependent maintenance energy coefficient (m′), 0.07 h⁻¹; maximum yield (Y_g), 0.063 mg of organic nitrogen per mg of Fe(II) oxidized.     

Oxidation of Ferrous Iron and Elemental Sulfur by Thiobacillus ferrooxidans

The oxidation of ferrous iron and elemental sulfur by Thiobacillus ferrooxidans that was absorbed and unabsorbed onto the surface of sulfur prills was studied. Unadsorbed sulfur-grown cells oxidized ferrous iron at a rate that was 3 to 7 times slower than that of ferrous iron-grown cells, but sulfur-grown cells were able to reach the oxidation rate of the ferrous iron-adapted cells after only 1.5 generations in a medium containing ferrous iron. Bacteria that were adsorbed to sulfur prills oxidized ferrous iron at a rate similar to that of unadsorbed sulfur-grown bacteria. They also showed the enhancement of ferrous iron oxidation activity in the presence of ferrous iron, even though sulfur continued to be available to the bacteria in this case. An increase in the level of rusticyanin together with the enhancement of the ferrous iron oxidation rate were observed in both sulfur-adsorbed and unadsorbed cells. On the other hand, sulfur oxidation by the adsorbed bacteria was not affected by the presence of ferrous iron in the medium. When bacteria that were adsorbed to sulfur prills were grown at a higher pH (ca. 2.5) in the presence of ferrous iron, they rapidly lost both ferrous iron and sulfur oxidation capacities and became inactive, apparently because of the deposition of a jarosite-like precipitate onto the surface to which they were attached.     

Continuous bacterial coal desulfurization employing Thiobacillus ferrooxidans

The leaching of pyrite sulfur from coal employing Thiobacillus Ferrooxidans was studied in a continuous stirred tank reactor at a variety of dilution rates (0.02-0.11 h(-1)) and coal surface areas (0.25-1.0 m(2)/mL). The bacterial leaching rate was found to increase with increasing coal surface area concentration and increasing dilution rate. The bacterial concentration on the coal surface was related to the bacterial concentration in solution by a irreversible second-order (of the second kind) kinetic equation. The concentration of bacteria on the coal in all experiments was the concentration at saturation. Step changes in the coal concentration were observed to result in dramatic declines in bacterial concentration in solution. A bacterial mass balance model was employed to calculate the specific growth rate on the solid which was observed to increase with increasing dilution rate.     

Ecological significance of acetate assimilation by Chlorobium phaeobacteroides

Abstract The effect of different concentrations of sulfide and sulfur on the assimilation of acetate by Chlorobium phaeobacteroides was investigated in batch and continuous cultures.
In batch cultures the assimilation of acetate strictly depends on the initial concentration of sulfide. In continuous cultures the uptake of acetate depends not only on the reservoir concentration of sulfide but also on the dilution rate. The more severe the limitation of sulfide the higher the incorporation of acetate.
The very efficient uptake of acetate was also observed in batch cultures, but only immediately prior to sulfide depletion. After sulfide depletion, with sulfur still available, the uptake of acetate per mmol reducing power increased even further. This phenomenon, which has been overlooked since growth decreases drastically after sulfide depletion due to incapacity for assimilatory sulfate reduction is of ecological importance in the formation of blooms of brown Chlorobium species.     

Degradation of Adsorbed Protein by Attached Bacteria in Relationship to Surface Hydrophobicity

The relationships among surface energy, adsorbed organic matter, and attached bacterial growth were examined by measuring the degradation of adsorbed ribulose-1,5-bisphosphate carboxylase (a common algal protein) by attached bacteria (Pseudomonas strain S9). We found that surface energy (work of adhesion of water) determined the amount and availability of adsorbed protein and, consequently, the growth of attached bacteria. Percent degradation of adsorbed ribulose-1,5-bisphosphate carboxylase decreased with increasing hydrophobicity of the surface (decreasing work of adhesion). As a result, growth rates of attached bacteria were initially higher on hydrophilic glass than on hydrophobic polyethylene. However, during long (6-h) incubations, growth rates increased with surface hydrophobicity because of increasing amounts of adsorbed protein. Together with previous studies, these results suggest that the number of attached bacteria over time will be a complex function of surface energy. Whereas both protein adsorption and bacterial attachment decrease with increasing surface energy, availability of adsorbed protein and consequently initial bacterial growth rates increase with surface energy.     

Production of elemental sulfur by green and purple sulfur bacteria

The utilization of sulfide by phototrophic sulfur bacteria temporarily results in the accumulation of elemental sulfur. In the green sulfur bacteria (Chlorobiaceae), the sulfur is deposited outside the cells, whereas in the purple sulfur bacteria (Chromatiaceae) sulfur is found intracellularly. Consequently, in the latter case, sulfur is unattainable for other individuals. Attempts were made to analyze the impact of the formation of extracellular elemental sulfur compared to the deposition of intracellular sulfur.According to the theory of the continuous cultivation of microorganisms, the steady-state concentration of the limiting substrate is unaffected by the reservoir concentration (S _R).It was observed in sulfide-limited continuous cultures ofChlorobium limicola f.thiosulfatophilum that higherS _R values not only resulted in higher steady-state population densities, but also in increased steady-state concentrations of elemental sulfur. Similar phenomena were observed in sulfide-limited cultures ofChromatium vinosum.It was concluded that the elemental sulfur produced byChlorobium, althouth being deposited extracellularly, is not easily available for other individuals, and apparently remains (in part) attached to the cells. The ecological significance of the data is discussed.Non-standard abbreviations RP reducing power - BChl bacteriochlorophyll - N_cell cell material - specific growth rate - {ie52-1} maximal specific growth rate - D dilution rate - K _s saturation constant - s concentration of limiting substrate - S _R same ass but in reservoir bottle - Y yield factor - iS^o intracellular elemental sulfur - eS^o extracellular elemental sulfur - PHB poly-beta-hydroxybutyric acid     

Isolation and Characterization of Strains CVO and FWKO B, Two Novel Nitrate-Reducing, Sulfide-Oxidizing Bacteria Isolated from Oil Field Brine

Bacterial strains CVO and FWKO B were isolated from produced brine at the Coleville oil field in Saskatchewan, Canada. Both strains are obligate chemolithotrophs, with hydrogen, formate, and sulfide serving as the only known energy sources for FWKO B, whereas sulfide and elemental sulfur are the only known electron donors for CVO. Neither strain uses thiosulfate as an energy source. Both strains are microaerophiles (1% O₂). In addition, CVO grows by denitrification of nitrate or nitrite whereas FWKO B reduces nitrate only to nitrite. Elemental sulfur is the sole product of sulfide oxidation by FWKO B, while CVO produces either elemental sulfur or sulfate, depending on the initial concentration of sulfide. Both strains are capable of growth under strictly autotrophic conditions, but CVO uses acetate as well as CO₂ as its sole carbon source. Neither strain reduces sulfate; however, FWKO B reduces sulfur and displays chemolithoautotrophic growth in the presence of elemental sulfur, hydrogen, and CO₂. Both strains grow at temperatures between 5 and 40°C. CVO is capable of growth at NaCl concentrations as high as 7%. The present 16s rRNA analysis suggests that both strains are members of the epsilon subdivision of the division Proteobacteria, with CVO most closely related to Thiomicrospira denitrifcans and FWKO B most closely related to members of the genus Arcobacter. The isolation of these two novel chemolithotrophic sulfur bacteria from oil field brine suggests the presence of a subterranean sulfur cycle driven entirely by hydrogen, carbon dioxide, and nitrate.     

Thienamycin production by immobilized cells of Streptomyces cattleya in a bubble column

A novel 2.0-L columnar reactor has been developed for the production of thienamycin by cells of Streptomyces cattleya attached to celite particles. Successful immobilization of cells was achieved by operating the column continuously at a high dilution rate during the growth phase. Scanning electron micrographs of the celite particles indicate the involvement of subcellular fibrils in the attachment of cells to the solid surfaces. Reactor operation was divided into two distinct phases-a growth phase and a production phase. The kinetics of attached growth and thienamycin production were found to be strongly influenced by nutrient concentrations. The influences of nutrient concentration on CO(2) production and thienamycin production during both the growth phase and the production phase are discussed.     

Predicting microbial growth dynamics in response to nutrient availability

Developing mathematical models to accurately predict microbial growth dynamics remains a key challenge in ecology, evolution, biotechnology, and public health. To reproduce and grow, microbes need to take up essential nutrients from the environment, and mathematical models classically assume that the nutrient uptake rate is a saturating function of the nutrient concentration. In nature, microbes experience different levels of nutrient availability at all environmental scales, yet parameters shaping the nutrient uptake function are commonly estimated for a single initial nutrient concentration. This hampers the models from accurately capturing microbial dynamics when the environmental conditions change. To address this problem, we conduct growth experiments for a range of micro-organisms, including human fungal pathogens, baker’s yeast, and common coliform bacteria, and uncover the following patterns. We observed that the maximal nutrient uptake rate and biomass yield were both decreasing functions of initial nutrient concentration. While a functional form for the relationship between biomass yield and initial nutrient concentration has been previously derived from first metabolic principles, here we also derive the form of the relationship between maximal nutrient uptake rate and initial nutrient concentration. Incorporating these two functions into a model of microbial growth allows for variable growth parameters and enables us to substantially improve predictions for microbial dynamics in a range of initial nutrient concentrations, compared to keeping growth parameters fixed.     

Inorganic sulfur oxidizing system in green sulfur bacteria

Green sulfur bacteria use various reduced sulfur compounds such as sulfide, elemental sulfur, and thiosulfate as electron donors for photoautotrophic growth. This article briefly summarizes what is known about the inorganic sulfur oxidizing systems of these bacteria with emphasis on the biochemical aspects. Enzymes that oxidize sulfide in green sulfur bacteria are membrane-bound sulfide-quinone oxidoreductase, periplasmic (sometimes membrane-bound) flavocytochrome c sulfide dehydrogenase, and monomeric flavocytochrome c (SoxF). Some green sulfur bacteria oxidize thiosulfate by the multienzyme system called either the TOMES (thiosulfate oxidizing multi-enzyme system) or Sox (sulfur oxidizing system) composed of the three periplasmic proteins: SoxB, SoxYZ, and SoxAXK with a soluble small molecule cytochrome c as the electron acceptor. The oxidation of sulfide and thiosulfate by these enzymes in vitro is assumed to yield two electrons and result in the transfer of a sulfur atom to persulfides, which are subsequently transformed to elemental sulfur. The elemental sulfur is temporarily stored in the form of globules attached to the extracellular surface of the outer membranes. The oxidation pathway of elemental sulfur to sulfate is currently unclear, although the participation of several proteins including those of the dissimilatory sulfite reductase system etc. is suggested from comparative genomic analyses.     

Effects of Inorganic Particles on Metabolism by a Periphytic Marine Bacterium

Measurements were made of adsorption of a periphytic marine bacterium, glucose, and glutamic acid to inorganic particles in seawater and defined bacterial growth medium. Measurements of the metabolism of bacteria were made in the presence and absence of particles by microcalorimetry and radiorespirometry. It was found that hydroxyapatite adsorbs glutamic acid, but not glucose, from the experimental medium. It was also found that hydroxyapatite adsorbs essentially all of the bacteria from the medium when the bacterial concentration is approximately 6 × 10⁵ bacteria per ml. If the bacterial concentration is approximately 6 × 10⁷, then only a small fraction of cells become attached. It was therefore possible to select bacterial concentrations and organic nutrients so that bacterial attachment, organic nutrient adsorption, or both would occur in different experiments. In this experimental system the metabolism by attached and nonattached bacteria of adsorbing and nonadsorbing organic nutrients was measured. The results show that bacterial activity in this model system was not enhanced by the particles, regardless of whether the bacteria, the organic nutrient, or both were associated with the surface. In fact, the respiratory activity of the attached bacteria was diminished in comparison with that of free bacteria.     

Kinetic studies on elemental sulfur oxidation by Acidithiobacillus ferrooxidans: sulfur limitation and activity of free and adsorbed bacteria

The kinetics of sulfur oxidation by Acidithiobacillus ferrooxidans in shaking flasks and a 10-L reactor was studied. The observed linearity of growth and sulfur oxidation was explained by sulfur limitation. Total cell yield was not significantly different for exponential growth as compared to growth during the sulfur-limiting phase. Kinetic studies of sulfur oxidation by growing and nongrowing bacteria indicated that both free and adsorbed bacteria oxidize sulfur. Changes in the number of free bacteria rather than cells adsorbed on sulfur were better predictors of the kinetics of sulfur oxidation, indicating that the free bacteria were performing sulfur oxidation. The active growth phase always followed adsorption of bacteria on sulfur; however, the special metabolic role of adsorbed bacteria was unclear. Their activity in sulfur solubilization was considered.