Extension of logarithmic growth of Thiobacillus ferrooxidans by potential controlled electrochemical reduction of Fe(III).

In this study, we demonstrated that the period of logarithmic growth for Thiobacillus ferrooxidans could be extended when optimal conditions for cell growth were maintained using potential controlled electrochemical cultivation with sufficient aeration. The optimal pH and Fe(II) concentration for the electrolytic cultivation were determined to be 2.0 and 150 mM, respectively. When the potential was set to 0.0V vs Ag/AgCl, the Pt electrode reduced Fe(III) to Fe(II) with an efficiency of 95%. A porous glass microbubble generator was used to maintain adequate levels of dissolved oxygen, which was the electron acceptor for T. ferrooxidans when the cell density in the medium was high. Under these conditions, cells at an initial density of 10(7) cells/mL grew logarithmically for 4days until the cell density was 4 x 10(9) cells/mL. This corresponded to a period of logarithmic growth that was 3 times longer than was observed in batch cultures without electrolysis. In addition, the final cell density reached 10(10) cells/mL after 6 days of electrochemical cultivation, which was a 50-fold increase over conventional batch culture. Under conditions of increasing cell density, potentiostatic electrolysis made it possible to remove Fe(III), which causes product inhibition, at an increasing rate and to correspondingly increase the production rate of Fe(II), which is the electron donor for T. ferrooxidans. Thus, our cultivation system provides a sufficient supply of electron donor and acceptor for T. ferrooxidans, thereby elongating the period of logarithmic growth and producing very high cell densities.     

Modeling enzyme production with Aspergillus oryzae in pilot scale vessels with different agitation, aeration, and agitator types

The purpose of this article is to demonstrate how a model can be constructed such that the progress of a submerged fed-batch fermentation of a filamentous fungus can be predicted with acceptable accuracy. The studied process was enzyme production with Aspergillus oryzae in 550 L pilot plant stirred tank reactors. Different conditions of agitation and aeration were employed as well as two different impeller geometries. The limiting factor for the productivity was oxygen supply to the fermentation broth, and the carbon substrate feed flow rate was controlled by the dissolved oxygen tension. In order to predict the available oxygen transfer in the system, the stoichiometry of the reaction equation including maintenance substrate consumption was first determined. Mainly based on the biomass concentration a viscosity prediction model was constructed, because rising viscosity of the fermentation broth due to hyphal growth of the fungus leads to significant lower mass transfer towards the end of the fermentation process. Each compartment of the model was shown to predict the experimental results well. The overall model can be used to predict key process parameters at varying fermentation conditions.     

Improvement in enzyme productivities from mold cultivations using the liquid-phase oxygen supply strategy

Cultivations of Aspergillus niger cells in which oxygen was provided through the liquid-phase oxygen supply strategy (which involves the need-based decomposition of H2O2 pulses to yield the necessary oxygen) were studied. Concentrations of H2O2 in the range of 1 to 5 mM were found to be suitable for use in cultivations. The growth rate constant of 0.17 h(-1) obtained in the H2O2-based cultivation, was comparable to that obtained in the cultivation with aeration; however, the maximum cell concentration in the H2O2-based cultivation was 124% of that obtained in cultivation with aeration. Maximum concentrations of catalase, protease and glucose oxidase obtained in the H2O2-based cultivation, were 240%, 172%, and 124% respectively, of those obtained in the cultivation with aeration. Further, the specific enzyme levels (units per gram cell) of catalase and protease obtained in the H2O2-based cultivation were 172% and 156% of those obtained in the cultivation with aeration, whereas, the specific glucose oxidase levels were comparable. In addition, the oxygen profiles inside the pellets ofA. niger during both the modes of oxygen supply were described using mathematical models. Studies to elucidate the mechanism of oxygen availability showed that the decomposition of the H2O2 supplied in the extracellular space occurred intracellularly, as well as extracellularly, to yield oxygen. Also, the proton motive force (PMF) was found to be involved in the process of oxygen availability from H2O2 to A. niger cells.     

Photoprecipitation and the banded iron-formations — Some quantitative aspects

The oxidative deposition of iron in the Banded iron-Formations can be quantitatively accounted for by direct abiotic photo-oxidation, by extrapolating from laboratory conditions and making reasonable assumptions about the early Earth and its oceans. Within this model, iron supply was the limiting factor, the Precambrian ocean surfaces were iron-depleted, and hydrogen would have been released into the atmosphere at a rate controlled by Fe(II) mixing. Other mechanisms operating in parallel are not excluded, and the Fe(II) budget suggests that recirculation by reaction with reduced carbon was important by ca. 2.5 b.y.b.p.     

Iron limits primary productivity during spring bloom development in the central North Atlantic

We present in situ biophysical measurements and bioassay experiments that demonstrate iron limitation of primary productivity during the spring bloom in the central North Atlantic. Mass balance calculations indicate that nitrate drawdown is iron (Fe)-limited and that aeolian Fe supply to this region cannot support maximal phytoplankton growth during the bloom. Using a simple simulation model, we show that relief of Fe limitation during the spring bloom can increase nitrate drawdown and, hence, new primary production, by 70%. We conclude that the episodic nature of iron supplied by dust deposition is an important factor controlling the dynamics of the spring bloom. From this, we hypothesize that variability in the timing and magnitude of the spring bloom in response to aeolian Fe supply will affect carbon drawdown and food web dynamics in the central North Atlantic.     

Rhamnolipid production by Pseudomonas aeruginosa under denitrification: effects of limiting nutrients and carbon substrates

Being biosurfactants, rhamnolipids create severe foaming when produced in aerobic Pseudomonas aeruginosa fermentation. The necessary reduction of aeration causes oxygen limitation and restricts cell and product concentrations. In this study, we evaluate the new strategy of rhamnolipid production under denitrification conditions. Because hydrocarbons used in earlier aerobic fermentations were not metabolizable in the absence of oxygen, other potential C substrates were examined, including palmitic acid, stearic acid, oleic acid, linoleic acid, glycerol, vegetable oil, and glucose. All were found able to support cell growth under anaerobic denitrification. The growth on the two solid substrates (palmitic acid and stearic acid) was slower but could be enhanced substantially by initial addition of rhamnolipids (0.06 g/L). The effects of different limiting nutrients (N, P, S, Mg, Ca, and Fe) were also investigated. The commonly used N limitation could not be adopted in the denitrifying fermentation because the nitrate added for anaerobic respiration would also be assimilated for growth. P limitation was most effective, giving four- to fivefold higher specific productivity than the conventional N limitation. S limitation was comparable to N limitation; Mg limitation was much poorer. Ca and Fe were ineffective in limiting cell growth. The new strategy was further evaluated in a P-limited fermentation with palmitic acid as the substrate. The fermentation was first carried out under denitrification and later switched to aerobic condition. The specific productivity under denitrification was found to be about one-third that of the aerobic condition. The denitrification process was, however, free of foaming or respiratory limitation. Much higher cell concentrations may be employed to attain higher volumetric productivity and product concentrations, for more economical product recovery and/or purification.     

Modelling of agrobacteran production in a batch culture ofAgrobacterium radiobacter

A deterministic model of production of the exopolysaccharide agrobacteran, including the effect of the product on oxygen transfer into the medium, was used for evaluation of a batch cultivation ofAgrobacterium radiobacter. Application of mass-energy balance made it possible to reduce the number of identified parameters. The resulting yield coefficients and rate constants were largely independent of the method of aeration of the culture, with the exception of the maximum specific rate of agrobacteran production which was lower if the culture in an exponential growth phase was subjected to oxygen limitation.     

Determination of non-protein-bound iron in rat tissue by ion chromatography with electrochemical detection

A new, rapid, sensitive, and specific method combining ion chromatography with electrochemical detection was developed for measuring non-protein-bound Fe(II) and Fe(III) in biological samples. The procedure was based on the separation of the iron-diethylenetriaminepentaacetic acid complex formed directly on the chromatographic column with anion-exchange resin followed by electrochemical detection. The method enabled more than 0.5 microM Fe(II) and Fe(III) to be determined for injection volumes of 10 microliters. This method was applicable for the determination of Fe(II) and Fe(III) in ultrafiltrates of the rat liver cytosolic fraction. It was found that release of iron from iron-bound proteins was pH dependent and that non-protein-bound iron in the tissues was determined in a ferrous state at low pH values.     

The distribution of iron oxidation states in a constructed wetland as an indicator of its redox properties

Wastewater-treatment processes taking place inside constructed wetlands are closely connected with chemical properties of these systems. The aeration of a wetland via the roots of the vegetation (and a subsequent formation of redox-potential gradients) strongly influences the wastewater treatment efficiency, and thus it represents one of the most important characteristics of the wetland. The concentration ratios of individual iron oxidation states (Fe(II) and Fe(III)) were determined as the indicator of the redox properties of the constructed wetland reed bed during this study. Interstitial water from the wetland was sampled eleven times throughout the year 2005. The spectrophotometric method using 1,10-phenanthroline was properly optimized (limits of detection and quantification, sensitivity, linear dynamic range, repeatability, and accuracy values were assessed) and applied for iron determination. Most of iron, ca. 98%, is reduced to the Fe(II) form in raw wastewater and water from the inflow zone of the constructed wetland, however, at the outflow and in the vegetation bed both iron oxidation states are usually detected. The presence of Fe(III) in the reed bed (ca. 10-30% for some samples) demonstrates the aeration of the wetland by the vegetation (Phragmites australis) resulting in a re-oxidation of Fe(II).     

Effect of oxygen supply on the suspension culture of genetically modified tobacco cells.

The effect of oxygen supply on the cultivation of the genetically modified tobacco cells and the formation of a foreign protein, beta-glucuronidase (GUS), was investigated in 250-mL Erlenmeyer flasks, a 5-L stirred tank fermenter, and a 7-L air-lift fermenter. The oxygen supply was varied by using different volumes of medium in the case of the 250-mL Erlenmeyer flask culture or by the different aeration rate in the case of the two types of fermenters tested. Higher oxygen supply stimulated cell growth and increased oxygen consumption rate, the level of phenolics, and GUS productions.     

Development of a constructed subsurface-flow wetland simulation model

This paper presents a mechanistic, compartmental simulation model of subsurface-flow constructed wetlands. The model consists of six submodels, including the nitrogen and carbon cycles, both autotrophic and heterotrophic bacteria growth and metabolism, and water and oxygen balances. Data from an existing constructed wetland in Maryland were used to calibrate the model. Model results reproduced seasonal trends well. Interactions between the carbon, nitrogen, and oxygen cycles were evident in model output. In general, effluent biochemical oxygen demand, organic nitrogen, ammonium and nitrate concentrations were predicted well. Because little is known about rootzone aeration by wetland plants, oxygen predictions were fair. The model is generally insensitive to changes in individual parameters. This is due to the complexity of the ecosystem and the model, as well as the numerous feedback mechanisms. The model is most sensitive to changes in parameters that affect microbial growth and substrate use directly. This dynamic, compartmental, simulation model is an effective tool for evaluating the performance of subsurface-flow constructed wetlands. The model provided insights into treatment problems at an existing constructed wetland. With further evaluation and refinement, the model will be a useful design tool for subsurface-flow constructed wetlands.     

Quantitative Aspects of Growth of the Methane Oxidizing Bacterium Methylococcus capsulatus on Methane in Shake Flask and Continuous Chemostat Culture

S ummary : Experiments were directed towards the production of high biomass concentrations in cultures of Methylococcus capsulatus. In shake flasks the effects of ammonium ion, phosphate ion and various trace metals on growth were studied. In the chemostat the effects on growth of methane limitation, oxygen limitation, aeration, dilution rate, pH value and temperature were studied and carbon balances were made in steady state conditions. Growth in the fermenter was stimulated by the use of Amberlite CG–120 ion exchange resin in the medium. The probability that Amberlite removed a growth, inhibitor is discussed.     

Growth of mixed cultures of Actinomyces viscosus and Streptococcus mutans under dual limitation of glucose and oxygen

Abstract Streptococcus mutans and Actinomyces viscosus are among the dominant species in human dental plaque. In their natural environment, carbohydrate- and oxygen-limited conditions are likely to occur frequently. Therfore, mixed cultures of the 2 species were studied under dual limitation of glucose and oxygen. Over a wide range of oxygen-supply rates, coexistence of A. viscosus and S. mutans was observed, within this range A. viscosus increased almost linearly with oxygen supply. A mathematical model based on Monod-type type kinetics and accounting for uncompetitive inhibition of growth by oxygen was developed to simulate these mixed cultures. The model predicted coexistence over a fairly large range of aeration rates. This finding, in combination with the results of the chemostat experiments, led to the conclusion that coexistence of the two species     

Degradation of ferrous(II) cyanide complex ions by Pseudomonas fluorescens

Degradation of ferrous(II) cyanide complex (ferrocyanide) ions by free cells of P. fluorescens in the presence of glucose and dissolved oxygen was investigated as a function of initial pH, initial ferrocyanide and glucose concentrations and aeration rate in a batch fermenter. The microorganism used the ferrocyanide ions as the sole source of nitrogen. The ferrocyanide biodegradation rate was 30.7 mg g⁻¹ h⁻¹ under the conditions of initial pH: 5, stirring rate: 150 rpm, aeration rate: 0.15 vvm, initial ferrous(II) cyanide complex ion and glucose concentrations: 100 mg l⁻¹ and 0.465 g l⁻¹, respectively. The culture utilized glucose as the main substrate following the non-competitive toxic component inhibition model in the presence of 100 mg l⁻¹ initial ferrous(II) cyanide complex ion concentration. The inhibition of ferrous(II) cyanide complex ions as a secondary substrate began at very low concentrations. A mathematical model, based on non-competitive substrate inhibition was used to describe the inhibitory effect of ferrous(II) cyanide complex ions on the growth of microorganism and the best fitted model parameters were determined by non-linear regression techniques.     

Effect of oxygen transfer on lipase production by Acinetobacter radioresistens

The influence of oxygen on alkaline lipase production by Acinetobacter radioresistens was studied under two operating modes: controlled dissolved oxygen (DO) concentration and controlled aeration rate. Compared with cell growth, the lipase production depended more extensively on oxygen. The intrinsic factor determining cell growth and lipase production was oxygen transfer rate (OTR) rather than DO concentration. Improvements in OTR, either by aeration or agitation, resulted in an increase in lipase yield and/or a reduction in fermentation time. The formation of A. radioresistens lipase could be described by a mixed-growth-associated model, and the enzyme was mainly a growth-associated product. The overall productivity for the lipase, which depended more strongly on agitation than aeration, could be related with kLa. DO concentration could not be employed in this correlation, though it has been useful as a criterion for ensuring no oxygen limitation in an aerobic fermentation.     

镜泊湖藻类生长和湖泊富营养化预测初探

作者在分析了影响镜泊湖藻类生长的主要因素的基础上,建立了镜泊湖藻类生长模型,利用实验数据和有关资料对镜泊湖藻类生长和湖泊富营养化趋势迸行了预测。作者认为：在镜泊湖藻类的生长季节（５—１０月）,Ｎ浓度始终不是主要限制因子;在藻类生长旺盛的７、８两月,Ｐ浓度成为主要限制因子;在５、６、９、１０四个月中,Ｐ浓度和温度均可能成为主要限制因子,但随着Ｐ浓度的提高,温度成为主要限制因子,客观上河湖泊富营养化的发展起着遏制作用。通过检验,本文模型的输出能反映镜泊湖藻类生物量随时间变化的一般规律。     

Dynamics of oxygen evolution and biomass production during cultivation of Agardhiella subulata microplantlets in a bubble-column photobioreactor under medium perfusion

Cell and tissue cultures derived from macrophytic marine red algae are potential platforms for unique secondary metabolites. This work presents the first successful bioreactor cultivation study of an in vitro tissue culture derived from a macrophytic marine red alga. Specifically, the photosynthetic growth characteristics of a novel microplantlet suspension culture established from the macrophytic marine red alga Agardhiella subulata were studied. A bubble-column bioreactor with external illumination (43 microE m(-2) s(-1), 10:14 LD photoperiod), liquid medium perfusion, and 3800 ppm CO(2) in the aeration gas provided sufficient light and nutrient delivery for sustained growth of the microplantlet suspension at 24 degrees C and pH 8. Microplantlets, which consisted of shoot tissues of 3-5 mm length branching out from a common center, were not friable in a bubble-aerated suspension of about 1100 plantlets per liter. Since the microplantlet tissues were not friable, only batch and fed-batch cultivation modes were considered. Batch cultivation was phosphate-limited in ASP12 artificial seawater medium. However, cultivation at a medium perfusion rate of 20% per day avoided phosphate limitation and extended the growth phase to provide plantlet mass densities exceeding 14 g FW L(-1) (3.7 g DW L(-1)) after 50 days of cultivation if the suspension was not sampled. The specific oxygen evolution rate vs cultivation time profile possessed a significant pulse within the 14 days following inoculation and then leveled off at longer times. In recognition of this nonexponential growth pattern, a new photobioreactor growth model was developed that used the oxygen evolution rate vs time profile to predict the biomass growth curve in perfusion culture. Model predictions agreed reasonably with the measured growth curves.     

Characteristics of the freshwater cyanobacterium Microcystis aeruginosa grown in iron-limited continuous culture

A continuous culturing system (chemostat) made of metal-free materials was successfully developed and used to maintain Fe-limited cultures of Microcystis aeruginosa PCC7806 at nanomolar iron (Fe) concentrations (20 to 50 nM total Fe). EDTA was used to maintain Fe in solution, with bioavailable Fe controlled by absorption of light by the ferric EDTA complex and resultant reduction of Fe(III) to Fe(II). A kinetic model describing Fe transformations and biological uptake was applied to determine the biologically available form of Fe (i.e., unchelated ferrous iron) that is produced by photoreductive dissociation of the ferric EDTA complex. Prediction by chemostat theory modified to account for the light-mediated formation of bioavailable Fe rather than total Fe was in good agreement with growth characteristics of M. aeruginosa under Fe limitation. The cellular Fe quota increased with increasing dilution rates in a manner consistent with the Droop theory. Short-term Fe uptake assays using cells maintained at steady state indicated that M. aeruginosa cells vary their maximum Fe uptake rate (ρ(max)) depending on the degree of Fe stress. The rate of Fe uptake was lower for cells grown under conditions of lower Fe availability (i.e., lower dilution rate), suggesting that cells in the continuous cultures adjusted to Fe limitation by decreasing ρ(max) while maintaining a constant affinity for Fe.     

Iron autoxidation and free radical generation: effects of buffers, ligands, and chelators.

The pH of the solution along with chelation and consequently coordination of iron regulate its reactivity. In this study we confirmed that, in general, the rate of Fe(II) autoxidation increases as the pH of the solution is increased, but chelators that provide oxygen ligands for the iron can override the affect of pH. Additionally, the stoichiometry of the Fe(II) autoxidation reaction varied from 2:1 to 4:1, dependent upon the rate of Fe(II) autoxidation, which is dependent upon the chelator. No partially reduced oxygen species were detected during the autoxidation of Fe(II) by ESR using DMPO as the spin trap. However, upon the addition of ethanol to the assay, the DMPO:hydroxyethyl radical adduct was detected. Additionally, the hydroxylation of terephthalic acid by various iron-chelator complexes during the autoxidation of Fe(II) was assessed by fluorometric techniques. The oxidant formed during the autoxidation of EDTA:Fe(II) was shown to have different reactivity than the hydroxyl radical, suggesting that some type of hypervalent iron complex was formed. Ferrous iron was shown to be able to directly reduce some quinones without the reduction of oxygen. In conclusion, this study demonstrates the complexity of iron chemistry, especially the chelation of iron and its subsequent reactivity.