Metabolic and process engineering for biodesulfurization in Gram-negative bacteria.

Microbial desulfurization or biodesulfurization (BDS) is an attractive low-cost and environmentally friendly complementary technology to the hydrotreating chemical process based on the potential of certain bacteria to specifically remove sulfur from S-heterocyclic compounds of crude fuels that are recalcitrant to the chemical treatments. The 4S or Dsz sulfur specific pathway for dibenzothiophene (DBT) and alkyl-substituted DBTs, widely used as model S-heterocyclic compounds, has been extensively studied at the physiological, biochemical and genetic levels mainly in Gram-positive bacteria. Nevertheless, several Gram-negative bacteria have been also used in BDS because they are endowed with some properties, e.g., broad metabolic versatility and easy genetic and genomic manipulation, that make them suitable chassis for systems metabolic engineering strategies. A high number of recombinant bacteria, many of which are Pseudomonas strains, have been constructed to overcome the major bottlenecks of the desulfurization process, i.e., expression of the dsz operon, activity of the Dsz enzymes, retro-inhibition of the Dsz pathway, availability of reducing power, uptake-secretion of substrate and intermediates, tolerance to organic solvents and metals, and other host-specific limitations. However, to attain a BDS process with industrial applicability, it is necessary to apply all the knowledge and advances achieved at the genetic and metabolic levels to the process engineering level, i.e., kinetic modelling, scale-up of biphasic systems, enhancing mass transfer rates, biocatalyst separation, etc. The production of high-added value products derived from the organosulfur material present in oil can be regarded also as an economically viable process that has barely begun to be explored.

1,3-Propanediol production from glycerol with a novel biocatalyst Shimwellia blattae ATCC 33430: Operational conditions and kinetics in batch cultivations.

Shimwellia blattae ATCC 33430 as biocatalyst in the conversion of 1,3-propanediol from glycerol is herein evaluated. Several operational conditions in batch cultivations, employing pure and raw glycerol as sole carbon source, were studied. Temperature was studied at shaken bottle scale, while pH control strategy, together with the influence of raw glycerol and its impurities during fermentation were studied employing a 2L STBR. Thereafter, fluid dynamic conditions were considered by changing the stirring speed and the gas supply (air or nitrogen) in the same scale-up experiments. The best results were obtained at a temperature of 37°C, an agitation rate of 200rpm, with free pH evolution from 6.9 and subsequent control at 6.5 and no gas supply during the fermentation, employing an initial concentration of 30g/L of raw glycerol. Under these conditions, the biocatalyst is competitive, leading to results in line with other previous works in the literature in batch conditions, reaching a final concentration of 1,3-propanediol of 13.84g/L, with a yield of 0.45g/g and a productivity of 1.19g/(Lh) from raw glycerol.

The effect of hydrodynamic stress on the growth of Xanthomonas campestris cultures in a stirred and sparged tank bioreactor.

The specific growth and the xanthan production rates by the bacterium Xanthomonas campestris under different shear levels in shake flasks and in a stirred and sparged tank bioreactor have been studied. The shake flask has been used as a reference for studying the shear effects. An effectiveness factor expressed by the ratio of the observed growth rate and the growth rate without oxygen limitation or cell damage was calculated in both modes of cultures. It was observed that the effectiveness factor was strongly dependent on the operational conditions. A strong oxygen transfer limitation at low stirring rates, indicated by a 54 % decrease in the effectiveness factor was observed. In contrast, at higher stirrer speed, cell damage was caused by hydrodynamic stress in the turbulent bulk of the broth, yielding again a decrease in the effectiveness factor values for stirrer speeds higher than 500 rpm. Cell morphological changes were also observed depending on the agitation conditions, differences in morphology being evident at high shear stress.

Oxidation and mineralisation of substituted phenols by Fenton's reagent and catalytic wet oxidation.

Catalytic abatement of solutions of 1,000 mg/L in phenol, ortho and para nitrophenol and ortho and para cresols was acomplished by using two catalytic systems. Fenton's reagent was used at 50 degrees C by adding 10 mg/L of ferrous cation and different dosages of H2O2. The mixture was reacting isothermically in a batch way during 3 hours. Catalytic wet oxidation (CWO) was carried out by using a commercial Activated Carbon, Industrial React FE01606A, CWO runs were carried out in a fixed bed reactor (FBR) with concurrent upflow. Temperature and oxygen pressure of the reactor were set to 160 degrees C and 16 bar, respectively. While phenols are quicky oxidised by the Fenton reagent higher mineralisation was obtained in the CWO process.

Abatement of phenolic mixtures by catalytic wet oxidation enhanced by Fenton's pretreatment: effect of H2O2 dosage and temperature.

Catalytic wet oxidation (CWO) of a phenolic mixture containing phenol, o-cresol and p-cresol (500mg/L on each pollutant) has been carried out using a commercial activated carbon (AC) as catalyst, placed in a continuous three-phase reactor. Total pressure was 16 bar and temperature was 127 degrees C. Pollutant conversion, mineralization, intermediate distribution, and toxicity were measured at the reactor outlet. Under these conditions no detoxification of the inlet effluent was found even at the highest catalyst weight (W) to liquid flow rate (Q(L)) ratio used. On the other hand, some Fenton Runs (FR) have been carried out in a batch way using the same phenolic aqueous mixture previously cited. The concentration of Fe(2+) was set to 10mg/L. The influence of the H(2)O(2) amount (between 10 and 100% of the stoichiometric dose) and temperature (30, 50, and 70 degrees C) on phenols conversion, mineralization, and detoxification have been analyzed. Phenols conversion was near unity at low hydrogen peroxide dosage but mineralization and detoxification achieved an asymptotic value at each temperature conditions. The integration of Fenton reagent as pretreatment of the CWO process remarkably improves the efficiency of the CWO reactor and allows to obtain detoxified effluents at mild temperature conditions and relatively low W/Q(L) values. For a given phenolic mixture a temperature range of 30-50 degrees C in the Fenton pretreatment with a H(2)O(2) dosage between 20 and 40% of the stoichiometric amount required can be proposed.

Evolution of toxicity upon wet catalytic oxidation of phenol.

This work reports on the evolution of the toxicity of phenol-containing simulated wastewater upon catalytic wet oxidation with a commercial copper-based catalyst (Engelhard Cu-0203T). The results of the study show that this catalyst enhances detoxification, in addition to its effect on the oxidation rate. The EC50 values of the intermediates identified throughout the oxidation route of phenol have been determined and used to predict the evolution of toxicity upon oxidation. The predicted values have been compared with the ones measured directly from the aqueous solution during the oxidation process. To learn about the evolution of toxicity through out the routes of phenol oxidation, experiments have been performed with simulated wastewaters containing separately phenol, catechol, and hydroquinone as original pollutants. The significant increase of toxicity observed during the early stages of phenol oxidation is not directly related to the development of the brown color that derives mainly from catechol oxidation. This increase of toxicity is caused by the formation of hydroquinone and p-benzoquinone as intermediates, the former showing the highest toxicity. Furthermore, synergistic effects, giving rise to a significant increase of toxicity, have been observed. These effects derive from the interactions among copper leached from the catalyst and catechol, hydroquinone, and p-benzoquinone and demand that close attention be paid to this potential problem in catalytic wet oxidation.

Hydrolysis of lactose by free and immobilized beta-galactosidase from Thermus sp. strain T2.

The hydrolysis of lactose by a beta-galactosidase from the thermophilic microorganism Thermus sp. strain T2, both in solution and immobilized on a commercial silica-alumina, has been studied. The enzyme has been previously produced by Escherichia coli JM101 harboring the plasmid pBGT1, which contains the codifying gene under the promoters lpp(P) and lac(PQ). The enzyme was immobilized on the support activated with tris-hydroxymethylphosphine (THP). Activity and stability of the free and the immobilized enzyme towards pH and temperature were tested. To study the activity at different pH and temperature values, lactose was used as substrate. To check the stability, the enzyme was incubated either in buffer BP or in a solution of lactose in buffer BM at different pH and temperatures, being the remaining activity tested by withdrawing samples and determining their activity toward ONPG at 70 degrees C in buffer BP. Afterward, runs were performed to obtain kinetic models adequate for the description of the hydrolysis of lactose by the free and the immobilized enzyme. These data were fitted to the kinetic models proposed (all based on the Michaelis-Menten mechanism) by non-linear regression, being the models and their parameters compared to determine the effect of the immobilization on the kinetic behavior of the enzyme. Both the free and the immobilized enzyme are competitively inhibited by galactose, while glucose inhibited only the action of the free enzyme, in an uncompetitive way. The immobilization step seems to eliminate the inhibition by glucose. Moreover, the immobilization reduced to a half the inhibitory action of galactose. In general, the immobilization reduced the activity of the enzyme, but increased its thermal stability. Finally, a comparison between the kinetic behavior of this thermophilic enzyme and enzymes of mesophile microorganisms previously studied by us (E. coli and K. fragilis) and by other authors (Aspergillus niger) is performed.

Catalytic wet oxidation of phenol: kinetics of phenol uptake.

Catalytic phenol oxidation in aqueous phase under intermediate temperature and pressure has been carried out in order to determine the kinetic model of phenol uptake rate. The catalyst employed here was a commercial one based on copper supplied by Engelhard (Cu-0203T). Operational variables have been studied in the following ranges: temperature from 127 to 180 degrees C, oxygen pressure from 3.2 to 16 bar, initial phenol concentration from 680 to 1200 ppm, and catalyst concentration from 0 to 1550 g/L of liquid phase. Because of the wide interval here employed for the catalyst concentration, two experimental setups have been used: a basket stirred tank reactor (BSTR) with the liquid phase in batch and an integral fixed-bed reactor (FBR) with co-current upflow of gas and liquid phases. An important influence of the reaction in the bulk liquid was obtained in both types of reactor. This fact has been taken into account in the kinetic model according to different approaches. The first approach was a breakup of the reaction rate in two kinetic expressions, considering the homogeneous and heterogeneous contribution separately; the second approach was empirical where the reaction rate is a potential function of the catalyst concentration. It was found that the extent of reaction in the bulk liquid is also influenced by the catalyst concentration and that the first approach is not able to adequately predict the experimental results. Finally a kinetic model, based on the second approach, was discriminated, with a power law for the catalyst concentration with an order about 0.4. This model fits quite well the experimental data obtained in both experimental setups, BSTR and FBR, throughout the wide range of variables studied.

Estimation of oxygen mass transfer coefficient in stirred tank reactors using artificial neural networks.

The estimation of volumetric mass transfer coefficient, k(L)a, in stirred tank reactors using artificial neural networks has been studied. Several operational conditions (N and V(s)), properties of fluid (µ(a)) and geometrical parameters (D and T) have been taken into account. Learning sets of input-output patterns were obtained by k(L)a experimental data in stirred tank reactors of different volumes. The inclusion of prior knowledge as an approach which improves the neural network prediction has been considered. The hybrid model combining a neural network together with an empirical equation provides a better representation of the estimated parameter values. The outputs predicted by the hybrid neural network are compared with experimental data and some correlations previously proposed in the literature for tanks of different sizes.

Diffusion and chemical reaction rates with nonuniform enzyme distribution: an experimental approach.

The study of the effects of nonuniform distributions of immobilized beta-galactosidase on the overall reaction rate of the hydrolysis of lactose are presented. Diffusion inside the particles has been characterized by measuring the diffusion rates of two beta-galactosidase substrates: lactose and ONPG in a commercial silica-alumina support. Effective diffusivities have been determined by the chromatographic method under inert conditions. The results obtained for tortuosity can be explained assuming that the transport only takes place in the macropores. The distribution of the immobilized enzyme has been measured by means of confocal microscopy technique. The enzyme has been tagged with FITC and immobilized in particles of different diameters, the internal local concentrations of the enzyme have been determined with the aid of an image computer program. As expected, a more nonuniform internal profile of the enzyme was found when the particle diameter was bigger. Experiments under reaction conditions were carried out in batch reactors using lactose and ONPG as substrates and particles of the immobilized beta-galactosidase of different diameter (1 x 10(-4) to 5 x 10(-3) m) as catalyst, employing a temperature of 40 degrees C for lactose and 25 and 40 degrees C for ONPG, respectively. The mass balance inside the particle for the substrates has been solved for the internal profiles of the immobilized enzyme inside particles of different size and the enzymatic reactions considered. The calculated and the experimental effectiveness factor values were similar when particles under 2.75 x 10(-3) m in diameter were employed. For the same Thiele modulus, a particle with nonuniform distribution of enzyme showed a higher effectiveness as a catalyst than particles with a more uniform distribution.

Oxygen transfer and uptake rates during xanthan gum production.

Oxygen uptake rate and oxygen mass transfer rate have been studied during xanthan gum production process in stirred tank bioreactor. Empirical equations for the oxygen mass transfer coefficient have been obtained taking into account several variables such as air flow rate, stirrer speed and apparent viscosity. Oxygen uptake rate evolution in the course fermentation has been measured, obtaining an equation as a function of biomass concentration, including overall growth and non growth-associated oxygen uptake. A metabolic kinetic model has been employed for xanthan gum production description including oxygen mass transfer and uptake rates. The results point out that this model is able to describe adequately not only oxygen dissolved evolution, but also of the production of xanthan and substrate consumption. Also, the influence of several parameters (k(L)a, air flow rate and dissolved oxygen) in the evolution of the key compounds of the system have been studied. The results of the simulation shown that an increasing of dissolved oxygen concentration favor the xanthan gum production.

Kinetic modeling of lactose hydrolysis with an immobilized beta-galactosidase from Kluyveromyces fragilis.

The kinetic model of the hydrolysis of lactose with a beta-galactosidase from Kluyveromyces fragilis immobilized on a commercial silica-alumina (KA-3, from Südchemie) has been determined. A wide experimental range of the main variables has been employed: temperature, concentrations of substrate, and products and concentration of enzyme. The runs were performed in a complex buffer with the salt composition of milk. The effect of pH and temperature on the stability and the activity of the enzyme have been studied. The optimum pH for the enzyme activity was, approximately, seven. The immobilized enzyme was more stable than the free one at acidic pH, but more instable at basic pH. The maximum temperature used for the hydrolysis runs performed to select the kinetic model was 40 degrees C, so inactivation of the enzyme during the kinetic runs has been avoided. Agitation, concentration of enzyme in the solid and particle size were selected to ensure that the overall rate was that of the chemical reaction. Eleven kinetic models were proposed to fit experimental data, from first order to more complex ones, such as those taking into account inhibition by one of the compounds involved in the hydrolysis reaction. Applying statistical and physical criteria, a Michaelis-Menten model with a competitive inhibition by galactose has been selected. The model is able to fit the experimental data correctly in the wide experimental range studied. Finally, the model obtained is compared to the one selected in a previous work for the hydrolysis of lactose with the free enzyme.

Xanthan gum production under several operational conditions: molecular structure and rheological properties*

Xanthan gum production under several operational conditions has been studied. Temperature, initial nitrogen concentration and oxygen mass transfer rate have been changed and average molecular weight, pyruvilation and acetylation degree of xanthan produced have been measured in order to know the influence of these variables on the synthesised xanthan molecular structure. Also, xanthan gum solution viscosity has been measured, and rheological properties of the solutions have been related to molecular structure and operational conditions. The Casson model has been employed to describe the rheological behaviour. The parameter values of the Casson model, tau(0) and K(c), have been obtained for each polysaccharide synthesised under different operational conditions. Both pyruvilation and acetylation degrees and average molecular weight of xanthan increase with fermentation time at any operating conditions. Xanthan molecules with the highest average molecular weight have been obtained at 25 degrees C. Nevertheless, at this temperature acetate and pyruvate radical concentration are lowest. Nitrogen concentration in broth does not show any clear influence over xanthan average molecular weight, although with high nitrogen source concentration xanthan with low pyruvilation degree is produced.

Xanthan gum: production, recovery, and properties.

Xanthan gum is a microbial polysaccharide of great commercial significance. This review focuses on various aspects of xanthan production, including the producing organism Xanthomonas campestris, the kinetics of growth and production, the downstream recovery of the polysaccharide, and the solution properties of xanthan.

Sophorolipid production by Candida bombicola: medium composition and culture methods.

Candida bombicola is able to produce sophorolipid molecules with surfactant properties when grown in a medium composed of two different carbon sources (usually sugar and oil) and a nitrogen source (frequently yeast extract). In this work, the composition of the medium and the culture method employed have been studied. The influences of glucose concentration, properties of oil and yeast extract concentration have been taken into account. Accordingly, a production medium composition is proposed (100 g/l glucose, 100 g/l sunflower oil and 1 g/l yeast extract). The most frequent culture methods reported in literature, i.e. batch, medium pulse mode and resting-cell methods, have been tested. The resting-cell method was found to produce the highest final concentration of sophorolipid, obtaining a good yield of carbon sources in a relatively short time. Under the best operational conditions and using the resting-cell method, 120 g/l sophorolipid was obtained in 8 d, with a carbon source yield of 0.60. Product distribution has also been investigated and the sophorolipid molecular structure of opened or cycled molecules has been determined under different operational conditions. Low yeast extract concentration and long fermentation time enhance the production of cycled structures by all the production methods studied.

Intracellular compounds quantification by means of flow cytometry in bacteria: application to xanthan production by Xanthomonas campestris.

The use of flow cytometry (FCM) to quantitatively analyze intracellular compounds is studied. FCM is a very useful technique for individual cell studies in microbial systems, and gives access to information which cannot be obtained in any other way. Nevertheless, it provides data in arbitrary units, that is, relative data. This analytical technique could be employed for kinetic modeling of microbial systems and even for internal phenomena analysis, but for this purpose, absolute data-that is concentration of intracellular compounds-must be used. In this work, relative flow cytometry data are transformed into absolute data by means of calibrations employing the same fluorochromes with another technique: spectrofluorymetry. Calibrations of DNA, RNA, and protein intracellular concentrations are presented for the bacteria, Xanthomonas campestris. Other analytical methods, based on biochemical determinations, were also employed to quantify intracellular compounds, but the results obtained are very poor compared with those achieved by means of spectrofluorymetry (SFM). Calibration equations and data obtained by both techniques are given. Evolutions of protein and nucleic acids during Xanthomonas campestris growth and xanthan gum production are shown.

[Kinetic model of micro-organism growth: the case of Xanthomonas campestris]. / Modelo cinético del crecimiento de microorganismos: el caso de Xanthomonas campestris.

Microbial growth is studied and kinetic models to describe the process rate useful in the scale-up are proposed. The growth of Xanthomonas campestris NRRL B-1459, a bacterium producing xanthan, a major industrial gum, is studied. Experimental data are arranged by means of different methods, and linear and non-linear regression techniques are applied in several ways (i.e. fixing or not fixing the values of certain parameters) and they are compared. To obtain parameter values with statistical meaning, two parameters must be calculated (namely, the maximum specific growth rate and the maximum biomass concentration available) by means of a non-linear regression technique employing the logistic equation. The maximum specific growth rate is related to temperature by means of different equations, but that of Ratkowsky et al. is the most suitable for X. campestris growth. Studied variables present no tendency to error and the reproduction of experimental data is very good.