Increases in alginate production and transcription levels of alginate lyase (alyA1) by control of the oxygen transfer rate in Azotobacter vinelandii cultures under diazotrophic conditions

BACKGROUND: Alginates are polysaccharides used in a wide range of industrial applications, with their functional properties depending on their molecular weight. In this study, alginate production and the expression of genes involved in polymerization and depolymerization in batch cultures of Azotobacter vinelandii were evaluated under controlled and noncontrolled oxygen transfer rate (OTR) conditions. RESULTS: Using an oxygen transfer rate (OTR) control system, a constant OTR (20.3 ± 1.3 mmol L 1 h 1 ) was maintained during cell growth and stationary phases. In cultures subjected to a controlled OTR, alginate concentrations were higher (5.5 ± 0.2 g L 1 ) than in cultures under noncontrolled OTR. The molecular weight of alginate decreased from 475 to 325 kDa at the beginning of the growth phase and remained constant until the end of the cultivation period. The expression level of alyA1, which encodes an alginate lyase, was more affected by OTR control than those of other genes involved in alginate biosynthesis. The decrease in alginate molecular weight can be explained by a higher relative expression level of alyA1 under the controlled OTR condition. CONCLUSIONS: This report describes the first time that alginate production and alginate lyase (alyA1) expression levels have been evaluated in A. vinelandii cultures subjected to a controlled OTR. The results show that automatic control of OTR may be a suitable strategy for improving alginate production while maintaining a constant molecular weight.

Mathematical Modeling of Macroscale Phenomena: Oxygen Transfer for Solid-state Fermentation in Static Tray Bioreactor.

Aims: To develop a mathematical model for prediction of variation in oxygen concentration inside the bed of tray type bioreactor for solid state fermentation and comparison of oxygen profile of unsteady state and pseudo steady state approximation. Place and Duration of Study: All the simulations were performed at the Applied and Industrial Microbiology laboratory, Indian Institute of Technology, Madras, from October 2013 to September 2014. Methodology: Models for various reaction kinetics (zero order, first order and Monod’s kinetics) were derived from the general model. Ordinary differential equations (Pseudo steady state approximation) and partial differential equations (unsteady state assumption) were solved by numerical techniques – Finite difference method (FDM) and Runge-kutta method. Simulation runs were carried out for various parameters such as bed height gas phase oxygen concentration, saturation constant, and porosity of the bed. Results: Oxygen profiles of unsteady state and pseudo steady state assumption were compared and results show lower oxygen concentration in case of unsteady state assumption. Concentration of oxygen was low for the organism following first order when compared to zero order and Monod kinetics. Results of simulation runs revealed that the oxygen concentration decreases as the bed height increases irrespective of the kinetics of the reaction. And it increases with increasing gas phase oxygen concentration, saturation constant and porosity. Conclusion: Mathematical model with unsteady state assumption was reported and it can be employed in calculating the design and operational parameters for solid state fermentors to yield optimal productivity.

Effect of kLa and Fed-batch Strategies for Enhanced Production of Xylitol by Debaryomyces nepalensis NCYC 3413.

Aims: To evaluate the effect of volumetric oxygen transfer coefficient on the production of xylitol by Debaryomyces nepalensis and to enhance the yield and productivity of xylitol by fed-batch fermentation using xylose as substrate. Place and Duration of Study: All experiments were performed at the Applied and Industrial Microbiology Laboratory, Indian Institute of Technology Madras, from March 2013 to May 2014. Methodology: Batch cultivations were carried out in a 7.5 L fermentor under various oxygen transfer coefficients in the range 12 to 39.6 h-1 in order to understand the effect of oxygen on xylitol production. Fed-batch studies were performed in 2.5 L bioreactor with a working volume of 1 L. The cultures were initially grown as batch cultures. Feed containing xylose and nitrogen source was added to the medium intermittently. Samples were periodically collected at regular intervals of time and the concentrations of xylose, xylitol and glycerol were determined by HPLC. Results: Maximal xylitol yield (0.64 g/g) and productivity (0.43 g/L·h) were obtained at kLa 13.68 h-1. The effect of pH was also studied at this kLa. A pH value of 6.0 was found to be favorable for xylitol accumulation. Fed-batch fermentation involving feeding of xylose and nitrogen source was used for xylitol production by D. nepalensis. Within the fed-batch phase, the yield of xylitol was 0.83 g/g and the productivity was increased to 0.83 g/L.h with a final product concentration of 90 g/L. Conclusion: Higher kLa favors biomass production whereas product formation was favored at lower kLa. Fed-batch process resulted in enhancement of final product concentration by 73%.

Evaluación de la transferencia de oxígeno en un biorreactor convencional con aireador externo / Evaluation of oxygen transfer in a conventional bioreactor with external aerator

En el cultivo de microorganismos aerobios se ha observado que los biorreactores convencionales conducen a inconvenientes asociados a la limitación de oxígeno. Para dar solución a esta problemática se diseñó, construyó y evaluó un prototipo de biorreactor con aireador externo. Este sistema permitió incrementar la velocidad de transferencia del oxígeno desde la fase gaseosa a la fase líquida, superando la limitación de oxígeno. Sin embargo, esta nueva configuración carece de modelos empíricos o teóricos para predecir la transferencia de oxígeno a través del cálculo del coeficiente global de transferencia de oxígeno (KLa). En ese sentido, en este trabajo se propuso el uso de análisis dimensional (Teorema Pi de Buckingham), como herramienta para evaluar el efecto sobre el KLa de las diferentes variables involucradas en el proceso, como las de operación, las propiedades de los fluidos y dimensiones geométricas. Luego de desarrollar el análisis propuesto, se encontró una correlación de 0.89 entre los valores generados por el modelo y los datos experimentales disponibles. Se encontró que las variables con mayor incidencia sobre el coeficiente KLa son el flujo de medio de cultivo que circula por el aireador externo denominado flujo de recirculación, la presión en el aireador externo y la velocidad de agitación.

In aerobic microorganism culture, it has been observed that conventional bioreactors lead to disadvantages associated with oxygen limitation. To solve this problem it was designed, built and tested a bioreactor prototype with external aerator. This system allowed increasing the speed of oxygen transfer from gas phase to liquid phase, overcoming the limitation of oxygen. However, this new configuration doesn't have theoretical or empirical models to predict the oxygen transfer by calculating the global volumetric coefficient of transfer of oxygen (KLa).In that way, this paper proposed the use of dimensional analysis (Buckingham's Pi Theorem), as a tool to evaluate the effect of various operating variables, fluid properties and geometrical dimensions involved in the process over KLa. After developing the proposed analysis, we found a correlation of 0.89 between the values generated by the model and the available experimental data. It was found that the main variables which affect the coefficient KLa are: the medium flow flowing through the external aerator called recirculation flow, the pressure in the external aerator, and the stirring speed.

Evaluación de la transferencia de oxígeno en cultivos con lactococcus lactis empleando un sistema de fermentación con aireación externa / Evaluating oxygen transfer in a Lactococcus lactis cultures using an external aeration fermentation system (EAFS)

En fermentaciones aerobias el oxígeno, como aceptor terminal de electrones en el proceso de respiración, comúnmente se constituye en limitante debido entre otros factores al diseño del biorreactor (factores geométricos), a las condiciones de operación de los fermentadores (condiciones ambientales requeridas en el cultivo, potencia transferida al cultivo por el sistema de agitación, propiedades del medio líquido), demanda de oxígeno por parte del microorganismo, sistema de aireación (concentración de oxígeno en el gas, solubilidad del oxígeno). La limitación de oxígeno se refleja en la fermentación con Lactococcus lactis cepa IBUN 34.1, en que presenta una baja disponibilidad de oxígeno desde muy temprano en la fase exponencial del cultivo. Para superar estas limitaciones se diseñó y desarrolló un sistema de suministro de oxígeno de alta tasa de transferencia, consistente en un sistema de fermentación con aireación externa (SFAE), el cual es comparado en este trabajo con el sistema tradicional de fermentador agitado dotado con dos turbinas tipo Rushton y aireación por difusor interno. En este trabajo se evalúa la operación del SFAE, se seleccionan y estudian algunas variables operacionales y su efecto sobre la transferencia de oxígeno gas-líquido. Los resultados indican que las variables que tienen efecto significativo sobre el coeficiente volumétrico global de transferencia de masa kLa son la agitación y el flujo de medio de cultivo que circula por el aireador externo denominado flujo de recirculación. Los valores de kLa obtenidos indican que con el fermentador convencional con aireación interna el mayor valor de kLa alcanzado fue de 40,68 (h-1), en tanto que con el SFAE se alcanzaron valores de 63,18 (h-1).

In aerobic fermentations, oxygen as terminal electron acceptor in respiration process, is commonly a limiting due to factors like its low solubility in aqueous solutions, bioreactors’ geometric factors and operating characteristics, liquid media properties, oxygen concentration in gas supply, microorganisms’ characteristics, environmental culture conditions, power supply by agitation system, etc. Oxygen limitation is present in cultures using the IBUN 34.1 Lactococcus lactis strain where oxygen availability is low some minutes after the exponential phase starts. A high transfer rate oxygen supply system was thus designed to overcome such limitations; it consisted of an external aeration fermentation system (EAFS) which was compared in this work with a conventional agitated tank fermenter equipped with two Rushton turbines and internal diffuser aeration flute mechanism.This paper evaluates the EAFS; some operational variables were selected and studied as well as their effect on oxygen transfer. Our results showed that agitation and culture medium flow through the aerator (called external recirculation flow) were the variables having the main effect on overall volumetric mass transfer coefficient (kLa). The highest kLa value in the conventional bioreactor having internal aeration was 40.68 kLa (h-1), while the EAFS reached 90 (h-1).

Transferência de oxigênio em reatores de leito fluidizado com circulação em tubos concêntricos em meios bifásico e trifásico com variação da relação entre diâmetros / Oxygen transfer in two and three-phase internal-loop airlift reactor with riser diameter variation

A eficiência do reator de leito fluidizado com circulação em tubos concêntricos depende das condições hidrodinâmicas que influem na transferência de oxigênio ao biofilme. Este trabalho investigou a influência da relação entre diâmetros dos tubos e da concentração de meio suporte (areia), sobre o coeficiente global de transferência de oxigênio (K La). Os ensaios - em reatores de 2,6 m de altura, com diâmetro externo de 250 mm e internos de 100, 125, 150 e 200 mm - empregaram vazões de ar até 2.500 L.h-1 e concentrações de até 150 g.L-1 de areia. O K La aumentou ligeiramente com 30 g.L-1 e diminuiu para concentrações maiores, confirmando relatos da literatura em condições semelhantes. Um modelo para K La em meio bifásico foi ajustado para as diversas relações ensaiadas entre a área externa e a interna, postulando-se uma redução na razão entre a transferência na fase líquida e o diâmetro da bolha com o aumento da vazão de ar.

The efficiency of the concentric tubes internal-loop airlift reactor depends on the hydrodynamic conditions that affect oxygen transfer to the biofilm. This work studied the effects of the relation between diameters of the tubes and of the carrier (sand) concentration on the global oxygen transfer coefficient (K La). The tests - in 2,6 m high reactors with 250 mm external diameter and 100, 125, 150 and 200 mm internal diameters - were performed with air flow taxes up to 2,500 L.h-1 and sand concentrations up to 150 g.L-1. The K La increased slightly for 30 g.L-1 and decreased for higher concentrations, in accordance with related data for similar conditions. A model for K La in biphasic medium was fitted embracing all the external/internal area relationships tested, based on the reduction of the liquid phase transfer coefficient and the bubble diameter ratio with increasing air flow rates.

Enhancement of Lycopene Production by Blakeslea trispora Using Oxygen-vectors and Surface Active Agents / 微生物学通报

Addition of oxygen-vectors(n-dodecane,n-hexane,hydrogen peroxide) to fermentation medium was recognized as a method of enhancing oxygen transfer and promoting lycopene yield by Blakeslea trispora fermentation.Higher lycopene production was observed in shake flask containing 1%(v/v) n-hexane,n-dodecane and 150 L/100mL hydrogen peroxide as compared to shake flasks without oxygen-vectors.The result of assays indicated that when oxygen-vectors (n-dodecane,n-hexane,1%,v/v) and hydrogen peroxide(50 L/100mL) were added to the 0-day and 1-day old culture of Blakeslea trispora the production of lycopene were 25.32%,72.84% and 40.35% higher than those without oxygen-vectors addition respectively.The production of lycopene increased 114.83% when n-dodecane and surface active agents were used at the same time.