Macrocystis pyrifera source of nutrients for the production of carotenoids by a marine yeast Rhodotorula mucilaginosa.

AIMS: To evaluate an aqueous extract of Macrocystis pyrifera as a nutrient source for the production of carotenoids by a marine Rhodotorula mucilaginosa isolated from seaweed samples. MATERIALS AND RESULTS: The effect of different culture conditions on the concentration of biomass and total pigments was evaluated using a Box-Behnken experimental design. The seaweed extract contained 15% w w-1 of protein and 20% w w-1 of carbohydrate; the main sugar in this fraction was trehalose (78%). The culture conditions that maximize the total pigment concentration (1·84 ± 0·03 mg l-1 ) were initial pH equal to 7, yeast extract as nitrogen source at a concentration of 4 g l-1 , seaweed extract concentration at 25% v v-1 , incubation performed at 25°C and 150 rev min-1 during 6 days. Under optimal growth conditions, three carotenoids were identified among the pigments produced by R. mucilaginosa, lycopene (38·4 ± 9·4%), ß-carotene (21·8 ± 1·5%) and astaxanthin (1·8 ± 0·3%). CONCLUSIONS: Carotenoids of commercial interest (lycopene, ß-carotene and astaxanthin) can be produced using a marine R. mucilaginosa cultivated with an aqueous extract of M. pyrifera as nutrient source. The total pigment concentration in the culture ranged between 0·82 and 1·84 mg l-1 , and was significantly affected by the concentration of the seaweed extract, and yeast extract. SIGNIFICANCE AND IMPACT OF THE STUDY: This work demonstrates that M. pyrifera can be used as a nutrient source for the production of carotenoids by the marine yeast.

Analyzing redox balance in a synthetic yeast platform to improve utilization of brown macroalgae as feedstock.

Macroalgae have high potential to be an efficient, and sustainable feedstock for the production of biofuels and other more valuable chemicals. Attempts have been made to enable the co-fermentation of alginate and mannitol by Saccharomyces cerevisiae to unlock the full potential of this marine biomass. However, the efficient use of the sugars derived from macroalgae depends on the equilibrium of cofactors derived from the alginate and mannitol catabolic pathways. There are a number of strong metabolic limitations that have to be tackled before this bioconversion can be carried out efficiently by engineered yeast cells. An analysis of the redox balance during ethanol fermentation from alginate and mannitol by Saccharomyces cerevisiae using metabolic engineering tools was carried out. To represent the strain designed for conversion of macroalgae carbohydrates to ethanol, a context-specific model was derived from the available yeast genome-scale metabolic reconstructions. Flux balance analysis and dynamic simulations were used to determine the flux distributions. The model indicates that ethanol production is determined by the activity of 4-deoxy-l-erythro-5-hexoseulose uronate (DEHU) reductase (DehR) and its preferences for NADH or NADPH which influences strongly the flow of cellular resources. Different scenarios were explored to determine the equilibrium between NAD(H) and NADP(H) that will lead to increased ethanol yields on mannitol and DEHU under anaerobic conditions. When rates of mannitol dehydrogenase and DehRNADH tend to be close to a ratio in the range 1-1.6, high growth rates and ethanol yields were predicted. The analysis shows a number of metabolic limitations that are not easily identified through experimental procedures such as quantifying the impact of the cofactor preference by DEHU reductase in the system, the low flux into the alginate catabolic pathway, and a detailed analysis of the redox balance. These results show that production of ethanol and other chemicals can be optimized if a redox balance is achieved. A possible methodology to achieve this balance is presented. This paper shows how metabolic engineering tools are essential to comprehend and overcome this limitation.

Electrochemical treatment of tannery effluent using a battery integrated DC-DC converter and solar PV power supply--an approach towards environment and energy management.

Electrochemical oxidation of tannery effluent was carried out in batch, batch recirculation and continuous reactor configurations under different conditions using a battery-integrated DC-DC converter and solar PV power supply. The effect of current density, electrolysis time and fluid flow rate on chemical oxygen demand (COD) removal and energy consumption has been evaluated. The results of batch reactor show that a COD reduction of 80.85% to 96.67% could be obtained. The results showed that after 7 h of operation at a current density of 2.5 A dm(-2) and flow rate of 100 L h(-1) in batch recirculation reactor, the removal of COD is 82.14% and the specific energy consumption was found to be 5.871 kWh (kg COD)(-1) for tannery effluent. In addition, the performance of single pass flow reactors (single and multiple reactors) system of various configurations are analyzed.

Native black Michuñe potato variety: characterization, frying conditions and sensory evaluation

The aim of this study was to evaluate the chemical and nutritional composition, to establish frying processing conditions and to determine the sensory profile and acceptability of Black Michuñe (BM) potato chips. BM had a higher protein content, half the ether extract content and nitrogen-free extract, a lower caloric intake (70 kcal/100g) and amylose content (17.5%) than Desirée (DES). To set the frying conditions, the Taguchi method was applied using a matrix design L9 (32,22). The variables studied were: temperature, time, potato variety (raw material) and pretreatment. The responses evaluated were: the color difference as well as the content of reducing sugars and total polyphenols. The best frying conditions were BM with a pretreatment at 160°C for 7 min for color, DES without pretreatment at 140°C for 7 min for reducing sugar content, and BM without pretreatment at 180°C for 4 min for polyphenol content. Then, sensory profiles of potato chips from BM, DES and a commercial package were determined by selecting the descriptors texture, firmness, color, salty taste and oiliness. Significant differences in color and oiliness were found. Finally, the acceptability test was applied to BM potato chips where color (64%) was the only attribute not as accepted as texture (95%), salty taste (87%) and product (97.3%). This study demonstrated that BM potato chips have a satisfactory acceptability by consumers.

Papa nativa variedad Michuñe negra: caracterización, condiciones de fritura y evaluación sensorial.. El presente trabajo tiene por objetivo evaluar la composición química y nutricional de papa Michuñe negro (BM), establecer condiciones de proceso de fritura, determinar el perfil sensorial y aceptabilidad de papas fritas chips. BM presentó un mayor contenido proteico, la mitad de extracto etéreo y extracto libre de nitrógeno, una menor ingesta calórica (70 kcal/100g) y un menor contenido de amilosa (17,5%) comparada con la variedad Desirée (DES). Para establecer condiciones de fritura se utilizó metodología Taguchi empleando una matriz de diseño L9 (32,22). Las variables seleccionadas fueron: temperatura, tiempo, variedad de papa (materia prima) y pretratamiento. Las respuestas evaluadas fueron: diferencia de color, contenido de azúcares reductores y polifenoles totales. Las mejores condiciones de fritura fueron: BM con pretratamiento a 160ºC por 7 min para color, DES sin pretratamiento a 140ºC por 7 min para azúcares reductores y BM sin pretratamiento a 180ºC por 4 min para polifenoles totales. Luego, los perfiles sensoriales de las papas chips de BM, DES y comercial fueron determinados seleccionando los descriptores textura, firmeza, sasbor salado y aceitoso. Se encontraron diferencias significativas en los descriptores color y aceitoso. El color (64%) fue el único atributo que obtuvo un nivel de aceptación más bajo entre los otros como textura (95%), sabor salado (87%) y producto (97.3%). Este estudio demostró que los chips de BM fueron satisfactoriamente aceptados por los consumidores.

Native black Michuñe potato variety: characterization, frying conditions and sensory evaluation.

The aim of this study was to evaluate the chemical and nutritional composition, to establish frying processing conditions and to determine the sensory profile and acceptability of Black Michuñe (BM) potato chips. BM had a higher protein content, half the ether extract content and nitrogen-free extract, a lower caloric intake (70 kcal/100g) and amylose content (17.5%) than Desirée (DES). To set the frying conditions, the Taguchi method was applied using a matrix design L9 (3(2),2(2)). The variables studied were: temperature, time, potato variety (raw material) and pretreatment. The responses evaluated were: the color difference as well as the content of reducing sugars and total polyphenols. The best frying conditions were BM with a pretreatment at 160 degrees C for 7 min for color, DES without pretreatment at 140 degrees C for 7 min for reducing sugar content, and BM without pretreatment at 180 degrees C for 4 min for polyphenol content. Then, sensory profiles of potato chips from BM, DES and a commercial package were determined by selecting the descriptors texture, firmness, color, salty taste and oiliness. Significant differences in color and oiliness were found. Finally, the acceptability test was applied to BM potato chips where color (64%) was the only attribute not as accepted as texture (95%), salty taste (87%) and product (97.3%). This study demonstrated that BM potato chips have a satisfactory acceptability by consumers.

Extension of the selection of protein chromatography and the rate model to affinity chromatography.

The rational selection of optimal protein purification sequences, as well as mathematical models that simulate and allow optimization of chromatographic protein purification processes have been developed for purification procedures such as ion-exchange, hydrophobic interaction and gel filtration chromatography. This paper investigates the extension of such analysis to affinity chromatography both in the selection of chromatographic processes and in the use of the rate model for mathematical modelling and simulation. Two affinity systems were used: Blue Sepharose and Protein A. The extension of the theory developed previously for ion-exchange and HIC chromatography to affinity separations is analyzed in this paper. For the selection of operations two algorithms are used. In the first, the value of η, which corresponds to the efficiency (resolution) of the actual chromatography and, Σ, which determines the amount of a particular contaminant eliminated after each separation step, which determines the purity, have to be determined. It was found that the value of both these parameters is not generic for affinity separations but will depend on the type of affinity system used and will have to be determined on a case by case basis. With Blue Sepharose a salt gradient was used and with Protein A, a pH gradient. Parameters were determined with individual proteins and simulations of the protein mixtures were done. This approach allows investigation of chromatographic protein purification in a holistic manner that includes ion-exchange, HIC, gel filtration and affinity separations for the first time.

Mathematical modeling of elution curves for a protein mixture in ion exchange chromatography applied to high protein concentration.

Protein elution curves in ion exchange chromatography (IEC) were simulated with a rate model. Three pure proteins and their mixture were used (alpha-lactalbumin, BSA, and conalbumin) under different operational conditions. The anionic matrix Q-Sepharose FF was used packed in a 1 mL column. A high protein concentration (37.5 mg/mL of total protein injected into the column) was used in order to extend the utility of the model. Mass transfer parameters were calculated using empiric correlations, where the axial dispersion was negligible (Pe > 300) and the mass transfer was controlled by the intraparticle diffusion (Bi > 10). The model assumes a modulator-eluite relationship were the equilibrium constant of the Langmuir isotherm was a function of salt concentration. Adsorption kinetic parameters were estimated from experimental data. The parameters for pure proteins were determined, and elution curves for changes in flow rate, ionic strength gradient, concentration, and sample size were predicted by the model. Then the kinetic parameters of the mixture were determined under the same operational conditions and some of the parameters had to be modified to take into account effects such as protein-protein interactions, competition, and displacement. Experimental elution curves obtained for changes in operational conditions such as flow rate and ionic strength gradient were simulated by the rate model for the protein mixture with a relative error in retention time of visible peaks <5%. IEC operational conditions and the peak fraction collection can be selected using a cost function of the production process which considers yield, purity, concentration, and process time that are obtained from simulations. Operational conditions that gave the minimum cost were selected. Simulations allows to diminish experimental time and cost.

Production of the exopolysaccharides by Streptococcus thermophilus: effect of growth conditions on fermentation kinetics and intrinsic viscosity.

Production of exopolysaccharides (EPS) by a commercial Streptococcus thermophilus strain was evaluated at different growth conditions [temperature (32-45 degrees C), carbon source and initial nitrogen (N) content]. Lactose from deproteinized whey and sucrose allowed to obtain EPS yields higher than 1200 mg/mM of the consumed carbon source. Intrinsic viscosity of the EPS was significantly reduced by ionic strength indicating a polyelectrolyte behavior. Growth conditions used for the production of the EPS had a significant effect (p<0.05) on the intrinsic viscosity. This was attributed to the effect of growth conditions on the molecular properties of the EPS [stiffness and molecular weight (MW)]. High MW EPS were produced when the bacteria grew at a high specific growth rate; however MW of the EPS and specific growth rate were not linearly associated. In the lactose fermentations carried out at different temperatures specific EPS synthesis rate was positive and linearly associated with the specific lactose consumption rate (R2=0.967) and specific galactose production rate (R2=0.967). Critical coil overlap parameter, [eta]C*, for the EPS produced in the lactose fermentations carried out at 43 and 45 degrees C was determined to be approximately 7.6, and their critical overlap concentrations (C*) were 0.45 and 0.87 g/dL, respectively.

Ovarian pregnancy after in vitro fertilization.

We report a rare ovarian pregnancy after in vitro fertilization and transcervical embryo transfer. Ultrasound guidance of embryo transfer does not eliminate the risk of this potentially lethal complication.

Mathematical modeling of elution curves for a protein mixture in ion exchange chromatography and for the optimal selection of operational conditions.

Elution curves in ionic exchange chromatography (IEC) for a three-protein mixture (alpha-lactoalbumin, ovalbumin, and beta-lactoglobulin), carried out under different flow rates and ionic strength conditions, were simulated using two different mathematical models. These models were the Plate Model and the more fundamentally based Rate Model. Relatively low protein concentrations were used to avoid protein-protein interactions. Simulated elution curves were compared with experimental data not used for parameter identification. Deviation between experimental data and the simulated curves using the Plate Model was less than 0.0189 (absorbance units); a slightly higher deviation [0.0252 (absorbance units)] was obtained when the Rate Model was used. A cost function was built that included the effect of the different production stages, namely fermentation, purification, and concentration. These considered the effect on the performance of IEC; yield, purity, concentration and the time needed to accomplish the separation. Operational conditions in the IEC such as flow rate, ionic strength gradient and the operational time can be selected using this model in order to find the minimum cost for the protein production process depending on the characteristics of the final product desired such as purity and yield. This cost function was successfully used for the selection of the operational conditions as well as the fraction of the product to be collected (peak cutting) in IEC. It can be used for protein products with different characteristics and qualities, such as purity and yield, by choosing the appropriate parameters.

Kinetics of Bifidobacterium longum ATCC 15707 fermentations: effect of the dilution rate and carbon source.

The effect of the dilution rate on biomass and product synthesis in fermentations of glucose, fructose and a commercial mixture of fructooligosaccharides (FOS) by Bifidobacterium longum ATCC 15707 was studied. Kinetic parameters (maximum specific growth rate, Monod constant, maintenance, and yield coefficients) in the mathematical model of the fermentation were estimated from experimental data. In the FOS mixture fermentations, approximately 12% of the total reducing sugars (mainly fructose) in the feed were not metabolized by the bacterium. In fermentations of fructose and the FOS mixture, biomass concentration increased as the dilution rate increased and, once maximum values were reached [3.90 (D=0.20 h(-1)) and 2.54 g l(-1) (D=0.15 h(-1)), respectively], decreased rapidly as the culture was washed out. Formic acid was detected at low dilution rates in glucose and fructose fermentations. The main products in fermentations of the three carbon sources were lactic and acetic acids. Average values of the molar ratio between acetic and lactic acids of 1.18, 1.21 and 0.83 mol mol(-1) were obtained in glucose, fructose and FOS mixture fermentations, respectively. In batch fermentations carried out without pH control this molar ratio was lower than 1.5 only when fructose was used as the carbon source.

Study of recombinant micro-organism populations characterized by their plasmid content per cell using a segregated model.

Numerous observations from recombinant systems have shown that properties such as the specific cell growth rate and the plasmid-free cell formation rate are related, not only to the average plasmid content per cell, but also to the plasmid distribution within a population. The plasmid distribution in recombinant cultures can have an effect on the culture productivity that cannot be modelled using average values of the overall culture. The prediction of the behaviour of a plasmid content distribution and its causes and effects can only be studied using segregated models. A segregated model that describes populations of recombinant cells characterized by their plasmid content distribution has been developed. This model includes critical causes of recombinant culture instability such as the plasmid partition mechanism at cell division, plasmid replication kinetics and the effect of the plasmid content on the specific growth rate. The segregated model allows investigation of the effect of each of these causes and that of the plasmid content distribution on the observable behaviour of a recombinant culture. The effect of two partitioning mechanisms (Gaussian distribution and binomial distribution) on culture stability was investigated. The Gaussian distribution is slightly more stable. A small plasmid replication rate constant results in a very unstable culture even after short periods of time. This instability is dramatically improved for a larger value of this constant, hence improving protein synthesis. For a very narrow initial plasmid distribution, a given plasmid replication rate and partitioning mechanism can become broad even after a relatively short period of time. In contrast, a very "broad" initial distribution gave rise to a "Gamma-like" distribution profile. If we compare the results obtained in the simulations of the segregated model with those of the non-segregated one (average model), the latter model predicts much more stable behaviour, thus these average models cannot predict culture instability with the same precision. When compared with the experimental results, the segregated model was able to predict the practical behaviour with accuracy even in a system with a high plasmid content per cell and a high rate of plasmid-free cell formation which could not be achieved with a non-segregated model.

Zymomonas mobilis CP4 fed-batch fermentations of glucose-fructose mixtures to ethanol and sorbitol.

Zymomonas mobilis CP4 fed-batch fermentations of glucose-fructose mixtures were carried out at different operational conditions (aeration, feed rate and substrate concentration) to test their effects on the system productivity. In these fermentations, the main products were ethanol and sorbitol. Kinetic parameters were calculated using the experimental data. However, parameters in the sorbitol synthesis rate were estimated from data recorded in different experiments in order to avoid the effect of the simultaneous cell growth and ethanol synthesis. In this case, the crude cell extract was used as source of the enzyme responsible for the sorbitol synthesis. The highest degree of conversion of fructose into sorbitol obtained with the extract was equal to 71% in a sugar mixture with an initial concentration of 200 g/l. Results obtained in the fed-batch fermentations showed that aeration of the culture has a positive effect on the final biomass concentration. However, final ethanol concentration is lower under aerated conditions. The best sugar yields to biomass and ethanol were 0.032 and 0.411 g/g, respectively. On the other hand, the highest sorbitol yield in the fed-batch fermentations was 0.148 g/g.

Effect of feeding strategy on Zymomonas mobilis CP4 fed-batch fermentations and mathematical modeling of the system.

In this work, the effect of the feeding strategy in Zymomonas mobilis CP4 fed-batch fermentations on the final biomass and ethanol concentrations was studied. Highest glucose yields to biomass (0.018 g/g) and to ethanol (0.188 g/g) were obtained in fed-batch fermentations carried out using different feeding rates with a glucose concentration in the feed equal to 100 g/l. Lower values (0.0102 g biomass/g glucose and 0.085 g ethanol/ g glucose) were obtained when glucose accumulated to levels higher than 60 g/l. On the other hand, the highest biomass (5 g/l) and ethanol (39 g/l) concentrations were obtained using a glucose concentration in the feed equal to 220 g/l and exponentially varied feeding rates. Experimental data were used to validate the mathematical model of the system. The prediction errors of the model are 0.39, 14.36 and 3.24 g/l for the biomass, glucose and ethanol concentrations, respectively. Due to the complex relationship for describing the specific growth rate, a fed-batch culture in which glucose concentration is constant would not optimize the process.

Effect of the growth conditions on the synthesis of a recombinant beta-1,4-endoglucanase in continuous and fed-batch culture.

Continuous culture and fed-batch fermentations were used to test the behavior of the system Bacillus subtilis DN1885(pCH7) that synthesizes a recombinant beta-1,4-endoglucanase. Continuous culture experiments were focused on the study of the instability aspects of the system as well as determination of the biomass growth rate range at which the recombinant enzyme synthesis was improved. Fed-batch fermentations were carried out to study the possibility of enhancing recombinant enzyme synthesis through the control of medium addition. It was found that, in continuous culture fermentations, the culture is less unstable at low dilution rates (dilution rate < 0.1 h(-)(1)). Also, low dilution rates give a higher specific recombinant enzyme concentration (10 times more than that obtained at high dilution rates). In fed-batch fermentation, the final recombinant enzyme concentration can be manipulated through the medium addition strategy. To increase the recombinant enzyme concentration, the carbon source has to be fed slowly, otherwise enzyme synthesis is impaired due to catabolite repression. Therefore, an increase in the biomass concentration does not necessarily imply an increase in the recombinant enzyme concentration. Higher recombinant enzyme concentrations were found in fed-batch fermentations compared to those obtained in continuous culture.