The principle of parabolic feed for a fed-batch culture of baker’s yeast

A parabolic nutrient supplementation has been derived by the specific growth rate, sugar conversion rate, and Pasteur effect, followed by high qualitative and quantitative biomass production with the lowest sugar consumption. The method produced about 95% cells in the G1 phase that were more resistant to drying and aging. These features are particularly important in the process of making dry yeast. It appears that the parabolic feed method may be used to species culture that show the Pasteur effect or produce byproducts from sugar. This may be because the supplementation is in conformity with reproducible kinetic growth during the fed-batch culture.     

Optimized Fed-Batch Fermentation of Scheffersomyces stipitis for Efficient Production of Ethanol from Hexoses and Pentoses

Scheffersomyces stipitis was cultivated in an optimized, controlled fed-batch fermentation for production of ethanol from glucose–xylose mixture. Effect of feed medium composition was investigated on sugar utilization and ethanol production. Studying influence of specific cell growth rate on ethanol fermentation performance showed the carbon flow towards ethanol synthesis decreased with increasing cell growth rate. The optimum specific growth rate to achieve efficient ethanol production performance from a glucose-xylose mixture existed at 0.1 h^?1. With these optimized feed medium and cell growth rate, a kinetic model has been utilized to avoid overflow metabolism as well as to ensure a balanced feeding of nutrient substrate in fed-batch system. Fed-batch culture with feeding profile designed based on the model resulted in high titer, yield, and productivity of ethanol compared with batch cultures. The maximal ethanol concentration was 40.7 g/L. The yield and productivity of ethanol production in the optimized fed-batch culture was 1.3 and 2 times higher than those in batch culture. Thus, higher efficiency ethanol production was achieved in this study through fed-batch process optimization. This strategy may contribute to an improvement of ethanol fermentation from lignocellulosic biomass by S. stipitis on the industrial scale.     

Assessment of various carbon sources and nutrient feeding strategies for Panax ginseng cell culture

Ginseng (root of Panax ginseng C. A. Meyer) cells were cultivated on medium supplemented with various carbohydrates including sucrose, glucose, and fructose, at initial concentrations ranging from 10 to 110 g/L. Sucrose was shown to be the superior carbon source to the monosaccharides for ginseng cell growth and the optimal concentration was between 30 and 50 g/L. An increase in the initial concentration within this range increased the maximum cell density and growth index significantly, whereas much higher concentrations inhibited cell growth. Feeding of sucrose and some other medium components during the growth (fed-batch mode) was more effective in enhancing the cell growth and biomass productivity, increasing the growth index by more than 60–70% and biomass productivity by more than 50%.     

Alcoholic Fermentation with Flocculant Saccharomyces cerevisiae in Fed-Batch Process

Studies have been conducted on selecting yeast strains for use in fermentation for ethanol production to improve the performance of industrial plants and decrease production costs. In this paper, we study alcoholic fermentation in a fed-batch process using a Saccharomyces cerevisiae yeast strain with flocculant characteristics. Central composite design (CCD) was used to determine the optimal combination of the variables involved, with the sucrose concentration of 170 g/L, a cellular concentration in the inoculum of 40 % (v/v), and a filling time of 6 h, which resulted in a 92.20 % yield relative to the theoretical maximum yield, a productivity of 6.01 g/L h and a residual sucrose concentration of 44.33 g/L. With some changes in the process such as recirculation of medium during the fermentation process and increase in cellular concentration in the inoculum after use of the CCD was possible to reduce the residual sucrose concentration to 2.8 g/L in 9 h of fermentation and increase yield and productivity for 92.75 % and 9.26 g/L h, respectively. A model was developed to describe the inhibition of alcoholic fermentation kinetics by the substrate and the product. The maximum specific growth rate was 0.103 h^?1, with K _I and K _s values of 109.86 and 30.24 g/L, respectively. The experimental results from the fed-batch reactor show a good fit with the proposed model, resulting in a maximum growth rate of 0.080 h^?1.     

Modeling and simulation of cephalosporin C production in a fed-batch tower-type bioreactor

Immobilized cell utilization in tower-type bioreactor is one of the main alternatives being studied to improve the industrial bioprocess. Other alternatives for the production of β-lactam antibiotics, such as a cephalosporin C fed-batch process in an aerated stirred-tank bioreactor with free cells of Cephalosporium acremonium, or a tower-type bioreactor with immobilized cells of this fungus, have proven to be more efficient than the batch process. In the fed-batch process, it is possible to minimize the catabolite repression exerted by the rapidly utilization of carbon sources (such as glucose) in the synthesis of antibiotics by utilizing a suitable flow rate of supplementary medium. In this study, several runs for cephalosporin C production, each lasting 200 h, were conducted in a fed-batch tower-type bioreactor using different hydrolyzed sucrose concentrations. For this study's model, modifications were introduced to take intoaccount the influence of supplementary medium flow rate. The balance equations considered the effect of oxygen limitation inside the bioparticles. In the Monod-type rate equations, cell concentrations, substrate concentrations, and dissolved oxygen were included as reactants affecting the bioreaction rate. The set of differential equations was solved by the numerical method, and the values of the parameters were estimated by the classic nonlinear regression method following Marquardt's procedure with a 95% confidence interval. The simulation results showed that the proposed model fit well with the experimental data, and based on the experimental data and the mathematical model, an optimal mass flow rate to maximize the bioprocess productivity could be proposed.

     

Continuous Enzymatic Hydrolysis of Lignocellulosic Biomass with Simultaneous Detoxification and Enzyme Recovery

Recovering hydrolysis enzymes and/or alternative enzyme addition strategies are two potential mechanisms for reducing the cost during the biochemical conversion of lignocellulosic materials into renewable biofuels and biochemicals. Here, we show that enzymatic hydrolysis of acid-pretreated pine wood with continuous and/or fed-batch enzyme addition improved sugar conversion efficiencies by over sixfold. In addition, specific activity of the hydrolysis enzymes (cellulases, hemicellulases, etc.) increased as a result of continuously washing the residual solids with removal of glucose (avoiding the end product inhibition) and other enzymatic inhibitory compounds (e.g., furfural, hydroxymethyl furfural, organic acids, and phenolics). As part of the continuous hydrolysis, anion exchange resin was tested for its dual application of simultaneous enzyme recovery and removal of potential enzymatic and fermentation inhibitors. Amberlite IRA-96 showed favorable adsorption profiles of inhibitors, especially furfural, hydroxymethyl furfural, and acetic acid with low affinity toward sugars. Affinity of hydrolysis enzymes to adsorb onto the resin allowed for up to 92 % of the enzymatic activity to be recovered using a relatively low-molar NaCl wash solution. Integration of an ion exchange column with enzyme recovery into the proposed fed-batch hydrolysis process can improve the overall biorefinery efficiency and can greatly reduce the production costs of lignocellulosic biorenewable products.

At-line determination of formaldehyde in bioprocesses by sequential injection analysis

A sequential injection analysis (SIA) method for the at-line determination of formaldehyde in a cultivation process of Pichia pastoris is presented. A genetically modified yeast strain was used for cultivation processes wherein methanol feed induced the production of the recombinant protein 1-3del I-TAC. Recurring measurements of culture medium, its blank and including standard addition were performed with Nash reagent using an automated syringe device and photometric detection. The apparatus was coupled via a laboratory-made flow-through adapter to a continuous filtered and cell-medium flow from the bioreactor. At-line monitoring of formaldehyde was performed at two cultivations, each of 250 h during fed-batch phases with glycerol and methanol as carbon sources. High reliability, robustness and reproducibility of the method, the software and the instrumentation as well as the high selectivity of the reaction were demonstrated.     

An Enhanced Process for the Production of a Highly Purified Extracellular Lipase in the Non-conventional Yeast <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis>

Yarrowia lipolytica LgX64.81 is a non-genetically modified mutant that was previously identified as a promising microorganism for extracellular lipase production. In this work, the development of a fed-batch process for the production of this enzyme in this strain was described. A lipolytic activity of 2,145 U/mL was obtained after 32 h of batch culture in a defined medium supplemented with 10 g/L of tryptone, an enhancer of lipase expression. To maximize the volumetric productivity, two different fed-batch strategies had been investigated. In comparison to batch process, the intermittent fed-batch strategy had not improved the volumetric lipase productivity. In contrast, the stepwise feeding strategy combined with uncoupled cell growth and lipase production phases resulted in a 2-fold increase in the volumetric lipase productivity, namely, the lipase activity reached 10,000 U/mL after 80 h of culture. Furthermore, this lipase was purified to homogeneity by anion exchange chromatography on MonoQ resin followed by gel filtration on Sephacryl S-100. This process resulted in an overall yield of 72% and a 3.5-fold increase of the specific lipase activity. The developed process offers a great potential for an economic production of Lip2 at large scale in Y. lipolytica LgX64.81.     

Fed-Batch production of glucose 6-Phosphate dehydrogenase using recombinant saccharomyces cerevisiae

The strain Saccharomyces cerevisiae W303-181, having the plasmid YEpPGK-G6P (built by coupling the vector YEPLAC 181 with the promoter phosphoglycerate kinase 1), was cultured by fed-batch process in order to evaluate its capability in the formation of glucose 6-phosphate dehydrogenase (EC.1.1.1.49). Two liters of culture medium (10.0 g/L glucose, 3.7 g/L yeast nitrogen broth (YNB), 0.02 g/L L-tryptophan, 0.02 g/L L-histidine, 0.02 g/L uracil, and 0.02 g/L adenine) were inoculated with 1.5 g dry cell/L and left fermenting in the batch mode at pH 5.7, aeration of 2.2 vvm, 30 degrees C, and agitation of 400 rpm. After glucose concentration in the medium was lower than 1.0 g/L, the cell culture was fed with a solution of glucose (10.0 g/L) or micronutrients (L-tryptophan, L-histidine, uracil, and adenine each one at a concentration of 0.02 g/L) following the constant, linear, or exponential mode. The volume of the culture medium in the fed-batch process was varied from 2 L up to 3 L during 5 h. The highest glucose 6-phosphate dehydrogenase activity (350 U/L; 1 U=1 micromol of NADP/min) occurred when the glucose solution was fed into the fermenter through the decreasing linear mode.     

Microbial conversion of 4-oxoisophorone by thermomonospora curvata using an air-bubbling hollow fiber reactor

Microbial conversion of 4-oxoisophorone (OIP) by thermophilic bacteriumThermomonospora curvata was attempted in a continuous process. The correlation between cell growth and microbial conversion was first examined in a batch culture. The results indicated that this microbial conversion was strongly dependent upon cell growth. In a continuous microbial conversion of OIP using a continuous stirred tank reactor, the cell density in the reactor seemed to be the limiting factor in the OIP conversion. Therefore, we developed an air-bubbling hollow fiber reactor to achieve a high density culture. By using this bioreactor, more than 3.3 times higher productivity was achieved. In addition, during the process, only a slight cell contamination to the product was observed. Therefore, this bioreactor is suitable for the continuous microbial conversion, considering further downstream processes and high productivity.     

A New Biotechnology Method of Bioelements’ Accumulation Monitoring in In Vitro Culture of Agaricus bisporus

Agaricus bisporus (J.E. Lange) Imbach is one the most popular species of edible mushrooms in the world because of its taste and nutritional properties. In the research, repeatability of accumulation of bioelements and biomass yield in experimentally chosen in vitro culture medium, was confirmed. The in vitro cultures were conducted on the modified Oddoux medium enriched with bioelements (Mg, Zn, Cu, Fe). The aim of the study was to create an effective method of sampling, which enabled non-invasive monitoring of metals concentrations changes in the medium, during increase of biomass in in vitro cultures. The first, indirect method of sampling was applied. The non-invasive probe (a dipper) for in vitro culture was used; hence, the highest biomass increase and metals accumulation were gained. The method also guaranteed culture sterility. The second method, a direct one, interfered the in vitro culture conditions and growth of mycelium, and as a consequence the lower biomass increase and metals’ accumulation were observed. Few cases of contaminations of mycelium in in vitro cultures were observed. The proposed method of non-invasive sampling of the medium can be used to monitor changes in the concentrations of metals in the medium and their accumulation in the mycelium in natural environment. Changes in concentrations of the selected metals over time, determined by the method of atomic absorption spectrometry, made it possible to correlate the obtained results with the specific stages of A. bisporus mycelium development and to attempt to explain the mechanism of sampling metals from the liquid substrate.     

On-line simultaneous monitoring of glucose and acetate with FIA during high cell density fermentation of recombinant E. coli

A two-channel flow injection analysis (FIA) system was developed for the simultaneous on-line monitoring of acetate and glucose during high cell density fed-batch fermentations of recombinant Escherichia coli. Acetate measurement was performed with a modified and optimised version of an existing method, based on acetate diffusion through a gas-diffusion chamber into a stream containing an acid-base indicator. The subsequent decrease in the absorbance was detected with an incorporated photometer. After method optimisation, it was possible to achieve linearity until 10 g/kg with no dilution step and with a detection level of 0.05 g/kg. Although some interferences were found, the performance of the method proved to be sufficiently reliable for on-line control purposes Commercially packed glucose oxidase (GOD) was used for the amperometric measurement of glucose. The method was linear up to 5 g/kg and it was possible to detect concentrations lower than 0.06 g/kg. For these measurements, no significant interferences were detected when the results were compared with other reference methods. The application of a simultaneous parallel configuration of the methods to a high cell density fed-batch E. coli fermentation was tested and reliable results were obtained within a 3 min delay. This information was made available to a supervisory computer running a developed LabVIEW™ programme via an Ethernet network, allowing the immediate implementation of control actions, improving the process performance.     

Biotransformation from geraniol to nerol by immobilized grapevine cells (V. vinifera)

The abilities of two grapevine cell suspensions (Vitis vinifera L. cv. Gamay Fréaux andVitis vinifera L. cv. Monastrell) to biotransform geraniol into nerol in a biphasic system based on the culture medium and Miglyol 812 were compared. The Gamay grape cell suspension was able to transform higher concentrations of geraniol into nerol than the Monastrell one. Gamay grape cells were immobilized in both calcium alginate beads and polyurethane foams. The cytotoxic effect of increasing concentrations of geraniol, as well as the ability of the immobilized cells to biotransform geraniol into nerol, was checked. Immobilization proved to be advantageous in protecting cells against the toxicity of the substrate. Furthermore, immobilization also seemed to have an effect on the secondary metabolism, the cells immobilized in polyurethane foams being more efficient at performing the isomerization process (40% conversion of geraniol into nerol) than both the freely suspended and calcium alginate immobilized cells (20% conversion).     

An Effective and Simplified Fed-Batch Strategy for Improved 2,3-Butanediol Production by <Emphasis Type="BoldItalic">Klebsiella oxytoca</Emphasis>

Substrate concentration in 2,3-butanediol (2,3-BD) fermentation could not be controlled well in traditional feeding strategies, such as constant, impulse, and exponential feeding strategies. In the present study, fermentative 2,3-BD production by Klebsiella oxytoca was investigated under different batch and fed-batch strategies. The glucose-feedback fed-batch strategy was proved to be not effective for economical 2,3-BD production for the inability of timely feeding, leading that the bacteria reused 2,3-BD as carbon source for cell growth. Based on the phenomena that the byproducing acids caused the pH declining and the requirement of maintaining the pH at a proper level for both cell growth and 2,3-BD accumulation, an improved strategy of pH-stat fed-batch culture with glucose and sodium hydrate fed at the same time was established. Thus, the residual glucose concentration could be controlled through the adjustment of pH automatically. At last, efficient 2,3-BD production was fulfilled under this fed-batch strategy, and the highest 2,3-BD concentration, productivity, and yield were 127.9 g/l, 1.78 g/(l•h), and 0.48 g/g (2,3-BD/glucose), respectively, compared to 98.5 g/l, 1.37 g/(l•h), and 0.43 g/g obtained in glucose-feedback fed-batch strategy. This feeding strategy was simple and easy to operate and could be feasible for industrial 2,3-BD production in the future.     

Comparison of Alcoholic Fermentation Performance of the Free and Immobilized Yeast on Water Hyacinth Stem Pieces in Medium with Different Glucose Contents

Ethanol fermentation with Saccharomyces cerevisiae cells was performed in medium with different glucose concentrations. As the glucose content augmented from 200 to 250 g/L, the growth of the immobilized cells did not change while that of the free cells was reduced. At higher glucose concentration (300, 350, and 400 g/L), the cell proliferation significantly decreased and the residual sugar level sharply augmented for both the immobilized and free yeast. The specific growth rate of the immobilized cells was 27–65 % higher than that of the free cells, and the final ethanol concentration in the immobilized yeast cultures was 9.7–18.5 % higher than that in the free yeast cultures. However, the immobilized yeast demonstrated similar or slightly lower ethanol yield in comparison with the free yeast. High fermentation rate of the immobilized yeast was associated with low unsaturation degree of fatty acids in cellular membrane. Adsorption of S. cerevisiae cells on water hyacinth stem pieces in the nutritional medium decreased the unsaturation degree of membrane lipid and the immobilized yeast always exhibited lower unsaturation degree of membrane lipid than the free yeast in ethanol fermentation.     

The use of an automatic on-line system for monitoring penicillin cultivation in a bubble-colum loop reactor

Penicillium chrysogenum is cultivated by a fed-batch mode to produce penicillin V. During fermentation, the concentrations of the medium components must be held at predetermined levels, which will change during fermentation, e.g., in the growth phase the concentration of the carbon sources and the nitrogen sources (urea and ammonium) must be high enough to maximize biomass production, whereas in the production stage these sources should be limited. To achieve optimal substrate concentrations, continuous measurement of various components in the fermentation broth is necessary. This is done by using a sterilizable ultrafiltation sampling probe and an air-segmented automatic flow analysis system to determine reducing sugars, dissolved organic carbon, ammonium, urea, sulfate, phosphate and penicillin V concentrations; spectrophotometric and gas-sensing electrodes are used in order to guarantee dependable results throughout the 290-h fermentation process, the analysis system is automatically cleaned and calibrated, and blanks are determined. The results are stored and evaluated by computer.     

Kinetic Modeling of Fermentative Production of 1, 3-Propanediol by Klebsiella pneumoniae HR526 with Consideration of Multiple Product Inhibitions

During the fermentative production of 1, 3-propanediol (1,3-PD), the multiple product inhibitions cannot be negligible to accurately describe the kinetics of fermentation process. A kinetic model for fermentative production of 1,3-PD by Klebsiella pneumoniae HR526 with glycerol as carbon source under aerobic condition was proposed. The inhibitions of multiple products including 1,3-PD, 2, 3-butanediol (2,3-BD), acetate, and succinate were considered in the model. It was found that 1,3-PD, 2,3-BD, and acetate showed strong inhibitions to cell growth depending on their concentrations. The kinetic model was relatively accurate to predict the experimental data of batch, fed-batch, and continuous fermentations. The model thus can serve as a tool for further controlling and optimizing the fermentation process.     

Development of an In Situ Detachment Protocol of Vero Cells Grown on Cytodex1 Microcarriers Under Animal Component-Free Conditions in Stirred Bioreactor

Subcultivation of Vero cells grown in a proprietary animal component-free medium named IPT-AFM, on microcarriers, was studied. TrypLE Select, a non-animal-derived protease, was used as an alternative to trypsin for cell passaging. We first studied the effect of increasing concentrations of TrypLE Select toward cell growth and then studied the inactivation of the protease using either soybean trypsin inhibitor (STI) or the soy hydrolysate Hypep 1510, in six-well plates. Data showed that cell growth was impaired by residual level of TrypLE Select; STI was identified as an efficient agent to neutralize this effect. To restore cell growth and inactivate TrypLE Select, STI should be added to the medium at least at 0.2 g L^?1. Cells were also grown in spinner flask on 2 g L^?1 Cytodex1 in IPT-AFM. In these conditions, the cell detachment yield was equal to 78?±?8 %. Furthermore, cells exhibited a typical growth profile when using the dislodged cells to seed a new culture. A cell detachment yield of 70?±?19 % was also achieved when the cells were grown in a 2-L stirred bioreactor in IPT-AFM, on 3 g L^?1 Cytodex1. This protocol can be of great interest to scale-up the process of Vero cells cultivation in IPT-AFM on Cytodex1 from one stirred bioreactor culture to another.     

Use of glucose feeding to produce concentrated yeast cells

A defined medium and fed-batch feeding process for the production of a yeast biocatalyst, developed at the 23-L scale, was scaled up to the 600-L pilot scale. Presterilized 100-L-vol plastic bags were implemented for the pilot-scale nutrient feeding. Medium of increased concentration Oqs implemented at the pilot scale, and equivalent dry cell weights were reached with a medium 80% more concentrated than that used at the laboratory scale. The higher medium concentration was believed to be necessary at the pilot scale owing to the additional heat stresses on key components (e.g., complexing of magnesium sulfate with phosphate), increased dilution during sterilization, lower evaporation rate owing to the lower vessel volume per minute air flow rate, and increased dilution owing to nutrient feeding or shot additions. Peak cell density was found to be somewhat insensitive to variations in residual glucose levels. These results suggest that defined medium developed at the laboratory scale may need to be further optimized at the pilot scale for equivalent performance.     

Potential of Biocellulose Nanofibers Production from Agricultural Renewable Resources: Preliminary Study

In the present preliminary study, we report results for the biocellulose nanofibres production by Gluconacetobacter xylinus. Production was examined by utilizing different feedstocks of single sugars and sugar mixtures with compositions similar to the acid hydrolyzates of different agriculture residues. Profiles for cell proliferation, sugar consumption, and the subsequent pH changes were thoroughly analyzed. Highest biocellulose production of 5.65 g/L was achieved in fructose medium with total sugar consumption of 95.57%. Moreover, the highest production using sugar mixtures was 5.2 g/L, which was achieved in feedstock with composition identical to the acid hydrolyzate of wheat straws. This represented the highest biocellulose yield of 17.72 g/g sugars compared with 14.77 g/g fructose. The lowest production of 1.1 and 1.75 g/L were obtained in xylose and glucose media, respectively, while sucrose and arabinose media achieved relatively higher production of 4.7 and 4.1 g/L, respectively. Deviation in pH of the fermentation broths from the optimum value of 4–5 generally had marked effect on biocellulose production with single sugars in feedstock. However, the final pH values recorded in the different sugar mixtures were ～3.3–3.4, which had lower effect on production hindrance. Analyzing profiles for sugars' concentrations and cell growth showed that large amount of the metabolized sugars were mainly utilized for bacterial cell growth and maintenance, rather than biocellulose production. This was clearly observed with single sugars of low production, while sugar consumption was rather utilized for biocellulose production with sugar mixtures. Results reported in this study demonstrate that agriculture residues might be used as potential feedstocks for the biocellulose nanofibres production. Not only this represents a renewable source of feedstock, but also might lead to major improvements in production if proper supplements and control were utilized in the fermentation process.