Reaction Kinetics of Immobilized-Cell Denitrification. II: Experimental Study

Laboratory experiments were conducted to characterize the performance of an immobilized-cell denitrification process treating nitrate-contaminated groundwater and to provide supporting data for the validation of a quasi-steady state model based on half-order reaction kinetics. The treatment process consists of laboratory-scale, plug-flow reactors packed with biocatalyst particles. Pseudomonas denitrificans (American Type Culture Collection 13867), a heterotrophic denitrifier, was cultured and immobilized in calcium alginate particles. Ethanol was used as the source of organic carbon. Thirty concentration profiles were obtained at four levels of nitrate concentration and at three ranges of flow rate. An analysis of the nitrate and nitrite concentration profiles suggested that a half-order reaction rate model could be used to describe the reduction of both nitrate and nitrite. The half-order reaction rate constants for nitrate and nitrite reduction were dependent on the age of the biocatalyst particles and the nitrate loading history. The validity of the half-order denitrification model was satisfactorily tested with experimental data. As illustrated by preliminary calculations, the immobilized-cell denitrification process is feasible for practical applications, particularly for small drinking water systems.     

Biodegradation of benzene, toluene, ethylbenzene, and o-xylene by a coculture of Pseudomonas putida and Pseudomonas fluorescens immobilized in a fibrous-bed bioreactor

A fibrous-bed bioreactor containing the coculture of Pseudomonas putida and P. fluorescens immobilized in a fibrous matrix was developed to degrade benzene (B), toluene (T), ethylbenzene (E), and o-xylene (X) in synthetic waste streams. The kinetics of BTEX biodegradation by immobilized cells adapted in the fibrous-bed bioreactor and free cells grown in serum bottles were studied. In general, the BTEX biodegradation rate increased with increasing substrate concentration and then decreased after reaching a maximum, showing substrate-inhibition kinetics. However, for immobilized cells, the degradation rate was much higher than that of free cells. Compared to free cells, immobilized cells in the bioreactor tolerated higher concentrations (> 1000 mg l-1) of benzene and toluene, and gave at least 16-fold higher degradation rates for benzene, ethylbenzene, and o-xylene, and a 9-fold higher degradation rate for toluene. Complete and simultaneous degradation of BTEX mixture was achieved in the bioreactor under hypoxic conditions. Cells in the bioreactor were relatively insensitive to benzene toxicity; this insensitivity was attributed to adaptation of the cells in the bioreactor. Compared to the original seeding culture, the adapted cells from the fibrous-bed bioreactor had higher specific growth rate, benzene degradation rate, and cell yield when the benzene concentration was higher than 100 mg l-1. Cells in the fibrous bed had a long, slim morphology, which is different from the normal short-rod shape found for suspended cells in solution.     

Anaerobic benzene biodegradation linked to nitrate reduction

Benzene oxidation to carbon dioxide linked to nitrate reduction was observed in enrichment cultures developed from soil and groundwater microcosms. Benzene biodegradation occurred concurrently with nitrate reduction at a constant ratio of 10 mol of nitrate consumed per mol of benzene degraded. Benzene biodegradation linked to nitrate reduction was associated with cell growth; however, the yield, 8.8 g (dry weight) of cells per mol of benzene, was less than 15% of the predicted yield for benzene biodegradation linked to nitrate reduction. In experiments performed with [14C]benzene, approximately 92 to 95% of the label was recovered in 14CO2, while the remaining 5 to 8% was incorporated into the nonvolatile fraction (presumably biomass), which is consistent with the low measured yield. In benzene-degrading cultures, nitrite accumulated stoichiometrically as nitrate was reduced and then was slowly reduced to nitrogen gas. When nitrate was depleted and only nitrite remained, the rate of benzene degradation decreased to almost zero. Based on electron balances, benzene biodegradation appears to be coupled more tightly to nitrate reduction to nitrite than to further reduction of nitrite to nitrogen gas.     

Continuous sucrose hydrolysis by yeast cells immobilized to wool

A novel immobilized biocatalyst with invertase activity was prepared by adhesion of yeast cells to wool using-glutaraldehyde. Yeast cells could be immobilized onto wool by treating either the yeast cells or wool or both with glutaraldehyde. Immobilized cells were not desorbed by washing with 1 M KCl or 0.1 M buffers. pH 3.5-7.5. The biocatalyst shows a maximum enzyme activity when immobilized at pH 4.2-4.6 and 7.5-8.0. The immobilized biocatalyst was tested in a tubular fixed-bed reactor to investigate its possible application for continuous full-scale sucrose hydrolysis. The influence of temperature, sugar concentration and flow rate on the productivity of the reactor and on the specific productivity of the biocatalyst was studied. The system demonstrates a very good productivity at a temperature of 70 degrees C and a sugar concentration of 2.0 M. The increase of the volume of the biocatalyst layer exponentially increases the productivity. The productivity of the immobilized biocatalyst decreases no more than 50% during 60 days of continuous work at 70 degrees C and 2.0 M sucrose, but during the first 30 days it remains constant. The cumulative biocatalyst productivity for 60 days was 4.8 x 10(3) kg inverted sucrose/kg biocatalyst. The biocatalyst was proved to be fully capable of continuous sucrose hydrolysis in fixed-bed reactors.     

Modeling Enhanced In Situ Denitrification in Groundwater

A two-dimensional numerical solute transport model was developed for simulating an enhanced in situ denitrification experiment performed in a nitrate-contaminated aquifer on Cape Cod, Massachusetts. In this experiment, formate (HCOO?) was injected for a period of 26 days into the carbon-limited aquifer to stimulate denitrification. Calibration of the vertical-profile site model was demonstrated through error analysis and comparison with formate, nitrate, and nitrite concentration data monitored along a transect of three multilevel groundwater sampling wells for 75 days after initial injection. Formate utilization rates were approximately 142 and 38 μM/day for nitrate and nitrite reduction, respectively. Nitrate and nitrite utilization rates were approximately 29 and 8 μM/day, respectively. Nitrate utilization rates under enhanced conditions were 1 order of magnitude greater than previously reported naturally occurring rates. The nitrite production rate was approximately 29 μM/day. Persistence of nitrite was attributed to a combination of factors, including electron donor (formate) limitation late in the experiment, preferential utilization of nitrate as an electron acceptor, and greater nitrite production relative to nitrite utilization.     

Comparative analysis of the dissolution kinetics of galena in binary solutions of HCl/FeCl3 and HCl/H2O2

A comparative study of the dissolution kinetics of galena ore in binary solutions of FeCl3/HCl and H2O2/HCl has been undertaken.The dissolution kinetics of the galena was found to depend on leachant concentration,reaction temperature,stirring speed,solid-to-liquid ratio,and particle diameter.The dissolution rate of galena ore increases with the increase of leachant concentration,reaction temperature,and stirring speed,while it decreases with the increase of solid-to-liquid ratio and particle diameter.The activation energy (Ea) of 26.5 kJ/mol was obtained for galena ore dissolution in 0.3 M FeCl3/8.06 M HCl,and it suggests the surface diffusion model for the leaching reaction,while the Ea value of 40.6 kJ/mol was obtained for its dissolution in 8.06 M H202/8.06 M HC1,which suggests the surface chemical reaction model for the leaching reaction.Furthermore,the linear relationship between rate constants and the reciprocal of particle radius supports the fact that dissolution is controlled by the surface reaction in the two eases.Finally,the rate of reaction based on the reaction-controUed process has been described by a semiempirical mathematical model.The Arrhenius and reaction constants of 11.023 s-1,1.25×104 and 3.65×102 s-1,8.02×106 were calculated for the 0.3 M FeCl3/8.06 M HCl and 8.06 M H202/8.06 M HCl binary solutions,respectively.     

Kinetics of membrane-bound nitrate reductase A from Escherichia coli with analogues of physiological electron donors--different reaction sites for menadiol and duroquinol

We have compared the steady-state kinetics of wild-type nitrate reductase A and two mutant forms with altered beta subunits. To mimic conditions in vivo as closely as possible, we used analogues of the physiological quinols as electron donors and membranes with overexpressed nitrate reductase A in preference to a purified alpha beta gamma complex. With the wild-type enzyme both menadiol and duroquinol supply their electrons for the reduction of nitrate at rates that depend on the square of the quinol concentration, menadiol having the higher catalytic constant. The results as a whole are consistent with a substituted-enzyme mechanism for the reduction of nitrate by the quinols. Kinetic experiments suggest that duroquinol and menadiol deliver their electrons at different sites on nitrate reductase, with cross-inhibition. Menadiol inhibits the duroquinol reaction strongly, suggesting that menaquinol may be the preferred substrate in vivo. To examine whether electron transfer from menadiol and duroquinol for nitrate reduction requires the presence of all of the Fe-S centres, we have studied the steady-state kinetics of mutants with beta subunits that lack an Fe-S centre. The loss of the highest-potential Fe-S centre results in an enzyme without menadiol activity, but retaining duroquinol activity; the kinetic parameters are within a factor of two of those of the wild-type enzyme, indicating that this centre is not required for the duroquinol activity. The loss of a low-potential Fe-S centre affects the activity with both quinols: the enzyme is still active but the catalytic constants for both quinols are decreased by about 75%, indicating that this centre is important but not essential for the activity. The existence of a specific site of reaction on nitrate reductase for each quinol, together with the differences in the effects on the two quinols produced by the loss of the Fe-S centre of +80 mV, suggests that the pathways for transfer of electrons from duroquinol and menadiol are not identical.     

Denitrification in a sporulating thermophilic bacterium

Denitrification in a thermophile isolated on nitrite containing-medium (5 g/l) was studied by means of Warburg respirometry and gas chromatography. This strain seems to denitrify nitrite more rapidly than nitrate. Extracts of cells grown anaerobically on nitrate have dissimilatory nitrate reductase (type A); extracts of cells grown aerobically without nitrate have raised levels of the two types of nitrate reductase A and B. The optimal temperature for enzyme A activity is 60 degrees C. Nitrite reductase activity was measured using yeast extract as electron donor. For nitric oxide reductase activity, yeast extract is as efficient an electron donor as sodium lactate. Nitrous oxide reductase activity was found only in the 4 000 g supernatant showing the particulate nature of the enzyme. A mixture of FAD, FMN and NADH served as electron donor. Using acetylene as an inhibitor of nitrous oxide reduction in both whole cells and extracts, we showed that this gas is an intermediate compound in the reduction of NO to N2.     

Reduction Kinetics of Metal Oxides by Hydrogen

In this paper, a new kinetic model has been developed for reduction of metal oxides with hydrogen under both isothermal and non‐isothermal conditions. This model describes the kinetics of single reductive reaction and double reductive reactions by considering the diffusion and chemical reaction controlling mechanisms. In particular, the model is in the analytic form of expressing the reduction extent as an explicit function of time, temperature, radius of the particle, and hydrogen partial pressure, which is convenient for using and theoretical analysis. The reduction kinetics of nickel oxide, natural ilmenite, and Fe₂MoO₄ agree well with the theoretical results by the present model.     

首钢烧结矿还原动力学

采用热重法在1173~1373 K、全CO气氛条件下,对首钢烧结矿进行还原动力学实验,确定了还原反应的表观活化能,进而推断在还原反应的前期烧结矿还原速率均由界面反应控制,还原反应后期的控制环节为固相扩散.分别由未反应核模型和固相反应动力学模型,分段给出不同温度下控制环节突变的时间点;通过动力学公式计算,得出不同温度下的反应速率常数和固相扩散系数.利用光学显微镜观察了烧结矿在各还原阶段的微观形貌,验证了烧结矿还原动力学的机理,同时也证明了扩散控制阶段使用体积缩小的未反应核模型与实际情况是吻合的.      

Kinetics of reduction of MnO in powder mixtures with carbon

Experimental data on the kinetics of the reaction between MnO and graphite in a mixture of fine powders, obtained earlier by one of the authors, were reassessed. The principal motive was to test on this system a new kinetic model, the “intrinsic transport” model, published previously by th present authors. In this model, the reaction-rate-limiting step is assumed to be transport of a gaseous intermediate by pore diffusion between reaction sites on the surfaces of the reactant particles. Various other model formalisms potentially applicable to this reaction also were tested. It is shown that the intrinsic transport of CO₂ gave good to excellent fits to the experimental data over a wide range of reaction conditions. Other models gave poor agreement. It is shown, furthermore, that for the case where the CO₂ pore-diffusion path lengths were made small (fine MnO and coarse graphite particle size), thus speeding up rapid intrinsic transport, the graphite-surface reaction became rate controlling.     

New calculation method for intrinsic rate constant of boudouard reaction

For obtaining intrinsic rate constant of boudouard reaction during coal based reduction of iron, the kinetic model of CO2 conversion by gasification during CO2 passing through coal layers in fixed bed was established according to the features of national standards testing method for carboxyreactivity of coal. Using the model and the testing results of carboxyreactivity of coal, the formulae of intrinsic rate constant of for gas coal and semi coke were expressed respectively. Then, the formula of boudouard reaction and CO2 conversion kinetics based on CO concentration was obtained for coal based reduction of iron.     

A nitrite biosensor based on a maltose binding protein nitrite reductase fusion immobilized on an electropolymerized film of a pyrrole-derived bipyridinium

The preparation and electrochemical characterization of glassy carbon electrodes (GCEs) modified with electropolymerized films of the cation N-(3-pyrrol-1-yl-propyl)-4,4'-bipyridine (PPB) are described. The behavior of a new biosensor, which exhibits a high catalytic activity for nitrite reduction and which consists of a maltose binding protein nitrite reductase fusion (MBP-Nir) immobilized on an electropolymerized film of PPB as an electrocatalyst, is also described. The insoluble perchlorate salt of the poly(benzyl viologen) dication was used to immobilize MBP-Nir onto an electrode previously modified with an electropolymerized film of PPB. The electropolymerized film of PPB on the GCE is redox active and exhibits special electron-transfer properties toward the MBP-Nir layer but not toward Nir (Nir without MBP fusion attached), suggesting an intimate interaction between the PPB film and the MBP-Nir layer. The kinetics of the catalytic reaction between the biosensor and nitrite anion were characterized using cyclic voltammetry and rotated disk electrode techniques, and a value of (4.6 +/- 0.5) x 10(3) M-1 S-1 was obtained for the rate constant.     

Resolution limits for optical transillumination of abnormalities deeply embedded in tissues

For the quantification of nitrite and nitrate, the stable metabolites of L-arginine-derived nitric oxide (NO) in human urine and plasma, we developed a gas chromatographic-mass spectrometric (GC-MS) method in which [15N]nitrite and [15N]nitrate were used as internal standards. Endogenous nitrite and [15N]nitrite added to acetone-treated plasma and urine samples were converted into their pentafluorobenzyl (PFB) derivatives using PFB bromide as the alkylating agent. For the analysis of endogenous nitrate and [15N]nitrate they were reduced to nitrite and [15N]nitrite, respectively, by cadmium in acidified plasma and urine samples prior to PFB alkylation. Reaction products were extracted with toluene and 1-microliter aliquots were analyzed by selected-ion monitoring at m/z 46 for endogenous nitrite (nitrate) and m/z 47 for [15N]nitrite ([15N]nitrate). The intra- and inter-assay relative standard deviations for the determination of nitrite and nitrate in urine and plasma were below 3.8%. The detection limit of the method was 22 fmol of nitrite. Healthy subjects (n = 12) excreted into urine 0.49 +/- 0.25 of nitrite and 109.5 +/- 61.7 of nitrate (mean +/- S.D., mumol/mmol creatinine) with a mean 24-h output of 5.7 mumol for nitrite and 1226 mumol for nitrate. The concentrations of nitrite and nitrate in the plasma of these volunteers were determined to be (mean +/- S.D., mumol/l) 3.6 +/- 0.8 and 68 +/- 17, respectively.     

Hydrogen reduction of cobalt ferrite

The kinetics of reduction of cobalt ferrite by hydrogen as a function of reduction temperature and pressure have been measured by thermogravimetric analysis. A minimum in the rate as a function of temperature has been observed and its cause attributed to the formation of a cobalt-wiistite subscale at higher reduction temperatures. A mathematical model, based on one derived by Spitzer, Manning, and Philbrook,¹ has been used to interpret the results in terms of the rate constants for the individual steps in the reaction. Optical microscopy has been used to characterize the morphology of the reduction product and, additionally, partially reduced single crystals of cobalt ferrite have been examined by transmission electron microscopy to characterize the microstructure of the reaction interface. A fine network of pores in the reduced scale was shown to allow the reducing and product gases to reach the immediate vicinity of the chemical reaction. The structure of the porosity and consequently the effective gaseous diffusion coefficient in the scale were both shown to be functions of the reduction temperature and pressure. The interface reaction was shown to follow Langmuir-Hinshelwood kinetics. A model was developed to explain such kinetics by incorporating a solid-state diffusion step. Such a step was considered necessary to explain the development of the observed microstructures. An ‘incubation time’ for the development of a continuous cobalt-wüstite subscale at higher reduction temperatures was attributed to the different growth kinetics for the spinel-metal and spinel-wüstite interfaces.     

Kinetic studies of a soluble alpha beta complex of nitrate reductase A from Escherichia coli. Use of various alpha beta mutants with altered beta subunits

A soluble alpha beta complex of nitrate reductase can be obtained from a strain of Escherichia coli that lacks the narI gene and expresses only the alpha and beta subunits. The beta subunit contains four Fe-S centres and the alpha subunit contains the molybdenum cofactor, which is the site at which nitrate is reduced. Despite the lack of the gamma subunit of the complete enzyme, this complex can still catalyse the reduction of nitrate with artificial electron donors such as benzyl viologen, so that it is suitable for studying the transfer of electrons between these two types of redox centre. To examine whether the electrons from reduced benzyl viologen are initially delivered to the Fe-S centres, or directly to the molybdenum cofactor, or both, we have studied the steady-state kinetics and the binding of benzyl viologen to the alpha beta complex and mutants alpha beta* with altered beta subunits. Reduction of the enzyme by reduced benzyl viologen in the absence of nitrate showed that all four Fe-S centres and the molybdenum cofactor could be reduced. Two classes of site with different equilibrium constants could be distinguished. The kinetic results suggest that benzyl viologen supplies its electrons directly to the molybdenum cofactor, at a rate showing a hyperbolic dependence on the square of the concentration of the electron donor. A reaction mechanism is proposed for the reduction of nitrate catalysed by the alpha beta complex of nitrate reductase with artificial electron donors.     

Investigation of Leaching Kinetics of Copper from Malachite Ore in Ammonium Nitrate Solutions

The production of metallic copper from low-grade copper ores is generally carried out by hydrometallurgical methods. Leaching is the first prerequisite of any hydrometallurgical process. Solutions containing ammonia may allow for selective leaching of the copper from the ore. In this study, the leaching kinetics of malachite, which is an oxidized copper ore, in ammonium nitrate solutions was examined. The effects of some experimental parameters on the leaching process were investigated, and a kinetic model to represent the effects of these parameters on the leaching rate was developed. It was determined that the leaching rate increased with increasing solution concentration, temperature, and agitation speed, as well as decreasing particle size. It was found that the leaching reaction followed the mixed kinetic controlled model, which includes two different leaching processes including the surface chemical reaction (303?K to 323?K [30?°C to 50?°C]) and diffusion through a porous product layer (323?K to 343?K [50?°C to 70?°C]). The activation energies of these sequential steps were determined to be 95.10 and 29.50?kJ/mol, respectively.     

Leaching kinetics of natural cobalt triarsenide in chlorine solutions

The influence of stirring speed, particle size, chlorine and hydrochloric acid concentrations, and temperature on the kinetics of chlorine leaching of skutterudite particles in a stirred vessel has been investigated. The reaction rate is limited by transport in the aqueous phase with significant resistance of the chemical reaction at temperatures below 20 °C. This is supported by (a) the rate constant increases with decrease in particle size, (b) the first-order dependence of the leaching rate with the total chlorine concentration and the insignificant effect of the hydrochloric acid concentration, (c) an apparent activation energy of 18 kJ/mole for transport control and an approximate value of 85 kJ/mole for the chemical reaction, and (d) the agreement of the experimental transport constants with the expected values for mass transfer coefficients. The reaction kinetics were analyzed by a shrinking core model in which [(Sh) _p − 2] (D/d) was considered virtually independent of particle size, which is consistent with the experimental data.