Production of Galanthamine by Leucojum aestivum Shoots Grown in Different Bioreactor Systems

The production of galanthamine by shoots of Leucojum aestivum grown in different bioreactor systems (shaking and nonshaking batch culture, temporary immersion system, bubble bioreactor, continuous and discontinuous gassing bioreactor) under different culture conditions was studied. The influence of the nutrient medium, weight of inoculum, and size of bioreactor on both growth and galanthamine production was studied. The maximal yield of galanthamine (19.416?mg) was achieved by cultivating the L. aestivum shoots (10?g of fresh inoculum) in a temporary immersion system in a 1-L bioreactor vessel which was used as an airlift culture vessel, gassing 12 times per day (5?min).     

Continuous Production of Oxytetracycline in Fluidized Bed Bioreactor by Immobilized Cells of Streptomyces varsoviensis MTCC-1537: Effect of Dilution and Glucose Loading Rates

Oxytetracycline (OT) production using glutaraldehyde cross-linked calcium alginate immobilized cells of Streptomyces varsoviensis in continuous fluidized bed reactor (FBR) was investigated. Initially, batch experiments were carried in stirred tank reactor (STR) and FBR using calcium alginate immobilized cells. Higher OT production of 0.45 gm/L was achieved by FBR when compared with 0.33 g/L of OT in STR. All subsequent studies were carried out in continuous mode of operation in FBR. During 21 days of operation, effect of glucose concentration and different dilution rates were studied. A maximum of 0.75 g/L OT was achieved in the medium having 10 g/L of glucose concentration. The highest OT concentration of 0.92 g/L and the highest yield of OT with respect to biomass at 0.1713 g/g were obtained at the dilution rate of 0.25 day⁻¹.     

Estimation of Fundamental Kinetic Parameters of Polyhydroxybutyrate Fermentation Process of Azohydromonas australica Using Statistical Approach of Media Optimization

Polyhydroxybutyrate or PHB is a biodegradable and biocompatible thermoplastic with many interesting applications in medicine, food packaging, and tissue engineering materials. The present study deals with the enhanced production of PHB by Azohydromonas australica using sucrose and the estimation of fundamental kinetic parameters of PHB fermentation process. The preliminary culture growth inhibition studies were followed by statistical optimization of medium recipe using response surface methodology to increase the PHB production. Later on batch cultivation in a 7-L bioreactor was attempted using optimum concentration of medium components (process variables) obtained from statistical design to identify the batch growth and product kinetics parameters of PHB fermentation. A. australica exhibited a maximum biomass and PHB concentration of 8.71 and 6.24?g/L, respectively in bioreactor with an overall PHB production rate of 0.75?g/h. Bioreactor cultivation studies demonstrated that the specific biomass and PHB yield on sucrose was 0.37 and 0.29?g/g, respectively. The kinetic parameters obtained in the present investigation would be used in the development of a batch kinetic mathematical model for PHB production which will serve as launching pad for further process optimization studies, e.g., design of several bioreactor cultivation strategies to further enhance the biopolymer production.     

Effect of yeast extract on growth kinetics of Monascus purpureus

Growth kinetics and red pigment production of Monascus purpureus CCT 3802 was studied. A reproducible inoculum with extremely dispersed hyphae for bioreactor runs was obtained through a two-step cultivation in a shaker. First, the spores were cultivated in a complex medium rendering a suspension of vegetative cells. In the second step these cells were grown in a semisynthetic medium. Two types of media were employed in the bioreactor runs: a semisynthetic (glucose, salts, and yeast extract), and a synthetic, without yeast extract. The inclusion of yeast extract, caused an increase in cell yield on glucose (Y_s/s) as high as 40%. Also, yeast extract probably yielded a higher proportion of red pigment associated with the cell, relative to the synthetic medium. On the other hand, cells grown on the synthetic medium were slightly higher producers of red soluble pigments.     

Influence of agitation rate on growth and ribonuclease production by free and immobilizedAspergillus clavatus cells

Aspergillus clavatus spores have been immobilized in poly(vinyl alcohol) criogel (PVAC) for ribonucleae (RNase) production. The enzyme productivity and growth of free (FC) and immobilized cells (IC) were studied in a bioreactor with agitation under different cultivation conditions. The influence of some medium compunds, aeration, and agitation speeds were investigated. Production of RNase was stimulated by the presence of reducing glucose, peptone, and soybean concentrations in the medium. From 42,000 to 45,000 U/L were produced by IC using optimized batch fermentation conditions. The RNase prouction by IC was 2.3 and 2.5 times greater than the same by FC in a bioreactor and control flasks, respectively.     

Influence of trace elements supplementation on the production of recombinant frutalin by Pichia pastoris KM71H in fed-batch process

Frutalin, a galactose-specific lectin used to detect specific tumour markers, is a protein with low expression level in breadfruit. In the present study, fed-batch fermentation in a stirred tank bioreactor was used as a strategy to enhance protein production by a recombinant Pichia pastoris KM71H. By using this process, the production of recombinant frutalin was 4-fold higher than the value obtained in shaker flasks batch assays. Supplementation of the fermentation medium with trace elements (Pichia trace minerals, PTM) was also evaluated in order to stimulate production of the recombinant protein. The addition of PTM to the minimum medium afforded a recombinant protein production of 13.4 mg L^?1, which was 2.5-fold higher than that achieved from the culture medium without PTM supplementation. These results are significant as the development of strategies to improve the production of recombinant frutalin may broaden its application in cancer diagnosis.     

Simultaneous production and purification ofBacillus subtilis α-amylase

Production of α-amylase withB. subtilis CCM 2722 in an aqueous two-phase polyethylene glycol/dextran system integrated with product purification by affinity chromatography on crosslinked starch during cultivation was studied. The medium was drawn from the bioreactor to the external settler during fermentation. After phase separation in the settler the dextran-rich bottom phase with cells was returned to the bioreactor. The PEG-rich top phase was pumped to the column with crosslinked starch and returned to the bioreactor after α-amylase adsorption. The same volumetric productivities, 0.53 U/mL/h, were reached in both batch and described process, but total productivity of the latter method was much higher owing to shortening upstream and downstream processing time. The enzyme of 98% homogenity in 95% yield was obtained after its elution from the column.     

Production of Fumaric Acid by Bioconversion of Corncob Hydrolytes Using an Improved <Emphasis Type="Italic">Rhizopus oryzae</Emphasis> Strain

The use of microorganism fermentation for production of fumaric acid (FA), which is widely used in food, medicine, and other fields, can provide technical support for the FA industry. In this study, we aimed to increase the titer of FA production by using an improved Rhizopus oryzae WHT5, which was domesticated to obtain a furfural-resistant strain in corncob hydrolytes. The metabolic pathways and metabolic network of this strain were investigated, and the related enzymes and metabolic flux were analyzed. Metabolic pathway analysis showed that the R. oryzae WHT5 strain produced FA mainly through two pathways. One occurred in the cytoplasm and the other was a mitochondrial pathway. The key parameters of the fermentation process were analyzed. The FA titer was 49.05 g/L from corncob hydrolytes using R. oryzae WHT5 in a 7-L bioreactor. The use of a furfural-resistant strain developed through domestication effectively increased the titer of FA. This capacity of the microorganisms to produce high amounts of FA by bioconverting corncob hydrolyte can be further applied for industrial production of FA.     

Efficient kefiran production by Lactobacillus kefiranofaciens ATCC 43761 in submerged cultivation: Influence of osmotic stress and nonionic surfactants,and potential bioactivities

Kefiran is a water soluble polysaccharide produced by Lactobacillus kefiranofaciens ATCC 43761. It has wide potential applications in food, pharmaceutical and nutraceutical industries. To the best of our knowledge, there have been no previous reports on the effect of osmotic stress and ionic surfactants on kefiran production by L. kefiranofaciens ATCC 43761. Accordingly, the current work aimed at optimizing kefiran production as affected by osmotic stress and nonionic surfactants in submerged cultivation system. Afterwards, the work was extended to investigate cytotoxic as well as antioxidant potentials of kefiran. Firstly, different osmolarities, different ionic surfactants (Triton X-100, Tween 20, Tween 80) as well as their concentrations and addition time were evaluated. The kinetics of cell growth and kefiran production were evaluated before and after the addition of surfactants. Results clearly demonstrated that osmotic stress and surfactant addition had a stimulatory effect on kefiran production. Using the optimal medium osmolality, 550 mOsmol.kg⁻¹, kefiran production was enhanced from 1.29 to about 1.38 g.L⁻¹. Furthermore, Triton X-100 was found to be the best surfactant stimulating kefiran production when added at a concentration of 1.0 g.L⁻¹ at the onset of cultivation process (0 h). This increased kefiran production from 1.38 g.L⁻¹ to 1.62 g.L⁻¹. To summarize, the maximal kefiran production can be enhanced using 550 mOsmol.kg⁻¹ and by adding 1.0 g.L⁻¹ of Triton X-100 at 0 h. The new optimized medium showed an increase of about 25.6% in kefiran production (1.29 up to 1.62 g.L⁻¹). After this step, the process was further optimized in 16-L stirred tank bioreactor. Maximal kefiran production reached 2.32 g.L⁻¹ and 1.87 g.L⁻¹ in bioreactor under control and un-controlled pH conditions, respectively, corresponding to 72.9 and 45.0% increase from the initial production titer, respectively. The produced kefiran exhibited promising anticancer activity against breast cancer (MCF-7) cells, with an IC₅₀ value of 193.89 μg.mL⁻¹. Also, kefiran showed 96.58% radical scavenging activity at 100 μg/mL, with an ED₅₀ recorded of 12.29 ± 0.98 μg.mL⁻¹.     

Competitive Adsorption removal of Congo red and Rhodamine B over alkaline membrane from in situ polymerization of Gemini cationic molecule

An alkaline membrane with full interpenetrating network (Full-IPN) with positive charge groups of uniform distribution was prepared as adsorbent for removal of Rhodamine B (RB) and Congo red (CR) in single and binary dye systems. Compared with single dye system, in binary dye systems a synergistic effect is due to the interaction between RB (cationic dyes) and CR (anionic dyes), which will impede the adsorption of CR or RB. Moreover, under the same experimental conditions, the magnitude of CR removal is better than that of RB in binary dye systems and that in the single system. The aforementioned phenomenon has resulted from one CR molecule bound to one RB molecule; the RB–CR binding occurred spontaneously, and the main binding forces between CR and RB were hydrogen bond and van der Waals interactions. Pseudo-second-order rate equation and Freundlich adsorption isotherm are with the better fit in single and binary dye systems for fitting the kinetic data. The results of ΔG, ΔH and ΔS revealed that the adsorption process for single and binary systems is endothermic and spontaneous. The electrostatic interaction between the dye and the quaternized ammonium groups present in membrane was identified as a major mechanism of the adsorption process.     

Photodynamic Inactivation of <Emphasis Type="BoldItalic">E. coli</Emphasis> PTCC 1276 Using Light Emitting Diodes: Application of Rose Bengal and Methylene Blue as Two Simple Models

The lack of a comparative study about potential of high-power light emitting diodes (LEDs) for photodynamic inactivation (PDI) of pathogenic microorganisms has remained as a challenging issue for researchers. Therefore, the aim of this study is to fill this gap through introduction of an efficient model for in vitro PDI in an aqueous medium. For this purpose, two individual 30 mW/cm² irradiation systems were designed using suitable sets of green and red LEDs. At another work, Methylene blue (MB) and Rose bengal (RB) as two simple models in the range of 5–150 μM were used in order to compare PDI of E. coli PTCC 1276 using red and green LED systems. Our results showed that a first-order mathematical model has the strength to describe the temporal variation of survival curves. Based on our results, when concentration of photosensitizer increased, the rate of inactivation for RB increased while MB depicted a maximum rate value at 25 μM. In a comparative study, optimum inactivation of E. coli PTCC 1276 obtained during 2- and 10-min irradiation of the LED systems using RB and MB at 150 and 25 μM, respectively. With regard to lower value of inactivation time and higher rate of inactivation for RB, use of simultaneous green high-power LEDs and RB is proposed as an efficient approach for PDI of pathogenic bacteria in future industrial applications.     

Ex Vitro Expansion of Human Placenta-Derived Mesenchymal Stem Cells in Stirred Bioreactor

The high demand of human placenta-derived mesenchymal stem cells (hPDMSCs) for therapeutic applications requires reproducible production of large numbers of well-characterized cells under well-controlled conditions. However, no method for fast hPDMSCs proliferation has yet been reported. In the present study, the feasibility of using a stirred bioreactor system to expand hPDMSCs was examined. hPDMSCs were cultured either in stirred bioreactors or in tissue culture flasks (T-flasks) for 5 days. Total cell density and several parameters of physical microenvironments were monitored in the two culture systems every 24 h. The maintenance of the antigenic phenotype of hPDMSCs before and after culturing in the stirred bioreactor system was cytometrically assessed. Data suggested that the physical microenvironment in the stirred bioreactors was much more favorable than that of the T-flasks. At the end of 144 h culturing, the total cell number was increased 1.73 times from the T-flasks to the stirred bioreactors. In addition, hPDMSCs could maintain their antigenic phenotype when cultured in stirred bioreactors. These results provide the initial assessment for large-scale hPDMSCs production using suspension culture bioreactors.     

Hyperproduction of l-glutamate oxidase in submerged fermentation of Streptomyces sp. N1 with culture pH control and calcium addition

Production of l-glutamate oxidase (GluOx) by Streptomyces sp. N1 was investigated by controlling culture pH at 6.2, 6.7, 7.0, and 7.3 in a 5-l stirred fermentor. The corresponding GluOx activities obtained were 2.8, 4.2, 6.0, and 5.3 U/mL, respectively. Microbial growth was inhibited by increasing the medium pH from 6.2 to 7.0. The inhibitory effect was also observed in plate colony growth under incubation with a different initial pH value. The effect of calcium on GluOx production was also studied in the pH-controlled bioreactor. When the culture pH was controlled at 6.2 or 7.0, GluOx production could not be improved or was only improved slightly by initial addition of calcium to the medium. However, when the culture pH was kept at 6.7, initial Ca²⁺ addition (60 mM) conspicuously enhanced GluOx production up to 9.3 U/mL, which was about twofold of that without Ca²⁺ addition. The enzyme production level was the highest ever reported in the literature. During fermentation the inhibition of cell growth by Ca²⁺ addition was observed. For the morphological changes, the cells mostly existed as pellets in the medium without Ca²⁺ addition, whereas few pellets were found and almost all the cells were dispersed mycelia in the broth with Ca²⁺ addition.     

Strain screening and sodium lactate effect on spiramycin production in <Emphasis Type="Italic">Streptomyces spiramyceticus</Emphasis>

Strain improvement and addition of sodium lactate to fermentation medium to enhance the productivity of spiramycin were performed. Of the sodium lactate tolerant mutants that were screened, one mutant, Streptomyces spiramyceticus 16-10-12, produced 23 % more spiramycin than the original strain, Streptomyces spiramyceticus 5-1. The effect of sodium lactate on spiramycin production with S. spiramyceticus 16-10-12 was studied. The titer was improved by 16.9 % with the addition of 15 g L^?1 sodium lactate in the fermentation medium at the beginning. The results from using the new process in a 15 L bioreactor showed that there were more precursors in fermentation broth with a sodium lactate tolerant mutant, and that these precursors were used more than with the original strain. After adding sodium lactate, the titer was increased by 23.4 %, because the flux to TCA circulation was increased, more precursors had been produced and the activities of Acyl-CoA synthetases, Acylphosphotransferases and Acylkinases in synthesis phase were also increased.     

Direct Fermentation of Gelatinized Cassava Starch to Acetone,Butanol, and Ethanol Using Clostridium acetobutylicum Mutant Obtained by Atmospheric and Room Temperature Plasma

The mutant strain designated as ART18, obtained from the wild-type strain Clostridium acetobutylicum PW12 treated by atmospheric and room temperature plasma, showed higher solvent tolerance and butanol production than that of the wild-type strain. The production of butanol was 11.3?±?0.5 g/L, 31 % higher than that of the wild-type strain when it was used for acetone, butanol, and ethanol fermentation in P2 medium. Furthermore, the effects of cassava flour concentration, pH regulators, and vitamins on the ABE production were also investigated. The highest butanol production of 15.8?±?0.8 g/L and butanol yield (0.31 g/g) were achieved after the above factors were optimized. When acetone, butanol, and ethanol fermentation by ART18 was carried out in a 15-L bioreactor, the butanol production, the productivity of butanol, and the total solvent were 16.3?±?0.9, 0.19, and 0.28 g/L^/h, respectively. These results indicate that ART18 is a promising industrial producer in ABE fermentation.     

Continuous ethanol extraction by pervaporation from a membrane bioreactor

In order to obtain a high productivity of ethanol, a membrane bioreactor consisting of a fermentor and a pervaporation system was applied to the continuous alcoholic fermentation process. A microporous hydrophobic polytetrafluoroethylene membrane was used for pervaporation. Glucose medium and baker's yeast were used for the fermentation. Three types of continuous fermentation experiment were carried out: conventional free-cell fermentation as the standard process; a fermentation in which product ethanol was extracted continuously by pervaporation from the membrane bioreactor; and a fermentation in which ethanol was extracted by pervaporation and part of the culture broth was simultaneously removed from the fermentation system.

The fermented ethanol was continuously extracted, and simultaneously concentrated by pervaporation, from the membrane bioreactor, and the extracted ethanol concentration was 6 to 8 times higher than in the broth. A high concentration of microorganisms was realized by immobilizing cells in the membrane bioreactor. When the ethanol concentration in the broth was kept low by pervaporation, the specific rate of ethanol production increased. However, the fraction of viable cells decreased because of the accumulation of inorganic salts fed as a nutrient, of nonvolatile by-products and of aged cells, which were not extracted by pervaporation from the fermentation solution. In order to achieve a high ethanol productivity, part of the fermentation broth must be removed from the membrane bioreactor.     

Production of the Antimicrobial Protein Weisselin A by Weissella paramesenteroides DX in Batch Fermentations: the Type of Carbohydrate Used as the C-Source in the Substrate Affects the Association of Production with Growth

The effect of the type of carbohydrate (glucose, fructose, sucrose or galactose) used as the carbon source in the substrate on weissellin A production by Weissella paramesenteroides DX was evaluated on a solid-state cultivation procedure and conventional batch fermentations. Solid-state cultivation was done on M17-based medium over 3- and 6-h incubation periods. Experimental data showed that glucose supports the highest production levels on a per cell basis. Stirred tank bioreactor fermentations carried out at 50?% dissolved oxygen tension revealed the superiority of glucose over the other carbohydrates. Glucose supports growth-associated production and increased production rates and productivities (1,120 AU/ml). Growth-associated production was maintained with fructose but with lower fermentation rates and productivities. Sucrose cannot support this type of production. Fermentations with sucrose were characterized of lower sugar uptake rates, and lower specific growth and production rates, with bacteriocin titres not exceeding 630 AU/ml, while product formation kinetics were of the intermediate type.     

Optimization of Multiparameters for Increased Yields of Cytochrome B5 in Bioreactors

The production of recombinant proteins is gaining increasing importance as the market requests high quality proteins for several applications. However, several process parameters affect both the growth of cells and product yields. This study uses high throughput systems and statistical methods to assess the influence of fermentation conditions in lab-scale bioreactors. Using this methodology, it was possible to find the best conditions to produce cytochrome b5 with recombinant cells of Escherichia coli. Using partial least squares, the height-to-diameter ratio of the bioreactor, aeration rate, and PID controller parameters were found to contribute significantly to the final biomass and cytochrome concentrations. Hence, we could use this information to fine-tune the process parameters, which increased cytochrome production and yield several-fold. Using aeration of 1 vvm, a bioreactor with a height-to-ratio of 2.4 and tuned PID parameters, a production of 72.72 mg/L of cytochrome b5 in the culture media, and a maximum of product to biomass yield of 24.97 mg/g could be achieved.     

Simple Strategy of Repeated Batch Cultivation for Enhanced Production of 1,3-Propanediol Using Clostridium diolis

Repeated batch cultivation (empty-and-fill protocol) using obligate anaerobe Clostridium diolis was attempted in the present study to improve the production of 1,3-propanediol (1,3-PD). In repeated batch operation, 20?% (v/v) culture broth was removed from the bioreactor and supplemented with an equal volume of fresh nutrient medium when the residual glycerol concentration in the bioreactor decreased below 15?g/L. Four cycles of culture broth withdrawal and subsequent replacement resulted in achieving a 1,3-PD concentration of 67.8?g/L with a productivity of 1.04?g/L/h at the end of 65?h. This represented a 1,3-PD concentration and productivity enhancement by 2.6-fold and 1.5-fold, respectively, as compared to batch 1,3-PD fermentation. This is the first report on the use of repeated batch mode of bioreactor operation for enhanced 1,3-PD production.     

Production of citric acid using immobilized conidia of Aspergillus niger

Conidia of Aspergillus niger were immobilized in calcium alginate gel for the production of citric acid. First, the type of the preactivation medium, together with the preactivation period, was investigated. It was found that A. niger requires a 2-d preactivation period at a 0.05 g/L NH₄NO₃ concentration. Second, preactivated cells were used to determine the effects of nitrogen concentration and the flow rate of oxygen and air on the production of citric acid. Maximum citric acid production was attained with medium containing 0.01 g/L of NH₄NO₃. The rate of citric acid production in the nitrogenous medium was 33% higher when oxygen was used instead of air during the production phase. This corresponds to an increase of 85% when compared to production when neither oxygen nor air was fed into the system. In the nonnitrogenous medium citric acid concentration remained similar regardless of the use of air or oxygen. However, in the nonnitrogenous production medium, citric acid production was not influenced considerably when oxygen was used instead of air. The advantage of using immobilized cells is that production is achieved easily in the continuous system. Therefore, citric acid production was also tested using a packed-bed bioreactor, and an increase in productivity by a factor of 22 was achieved compared to the batch system.