Fed-batch Operation Research Articles

Biosorption of copper(II) ions onto powdered waste sludge in a completely mixed fed-batch reactor: Estimation of design parameters

Biosorption of Cu(II) ions onto pre-treated powdered waste sludge (PWS) was investigated using a fed-batch operated completely mixed reactor. Fed-batch adsorption experiments were performed by varying the feed flow rate ( 0.075–0.325 l h −1), feed copper (II) ion concentrations (50–300 mg l −1) and the amount of adsorbent (1–6 g PWS) using fed-batch operation. Breakthrough curves describing the variations of effluent copper ion concentrations with time were determined for different operating conditions. Percent copper ion removals from the aqueous phase decreased, but the biosorbed (solid phase) copper ion concentrations increased with increasing the feed flow rate and Cu(II) concentration. A modified Bohart–Adams equation was used to determine the biosorption capacity of PWS and the rate constant for Cu(II) ion biosorption. Adsorption rate constant in fed-batch operation was an order of magnitude larger than those obtained in adsorption columns because of elimination of mass transfer limitations encountered in the column operations while the biosorption capacity of PWS was comparable with powdered activated (PAC) in column operations. Therefore, a completely mixed reactor operated in fed-batch mode was proven to be more advantageous as compared to adsorption columns due to better contact between the phases yielding faster adsorption rates.

Kinetics of Agrobacterium tumefaciens ubiquinone-10 batch production

A kinetic model of ubiquinone-10 production by batch fermentation with Agrobacterium tumefaciens ATCC4452 was firstly studied. After observation of experimental data, a model based on the logistic equation of ATCC4452 growth, ubiquinone-10 accumulation combined non-growth-associated and growth-associated contributions, and consumption of sugar for biomass formation and the maintenance of biomass, was developed. The optimal set of parameters was estimated by fitting the model to experimental data. The results predicted by the model were in good agreement with the experimental observations. Based on estimated kinetic parameters and characteristics of product formation and substrate utilization, a strategy of fed-batch operation was adopted in order to obtain more ubiquinone-10 accumulation. The final production of ubiquinone-10 reached 71.5 mg l −1 in 96 h of incubation, corresponding to more 3.5 times than that of batch operation, in fed-batch fermentation using four stage feeding strategy.

Reactor Engineering in Large Scale Animal Cell Culture

This article mainly addresses the issues associated with the engineering of large-scale free suspension culture in agitated bioreactors >10,000 L because they have become the system of choice industrially. It is particularly concerned with problems that become increasingly important as the scale increases. However, very few papers have been written that are actually based on such large-scale studies and the few that do rarely address any of the issues quantitatively. Hence, it is necessary very often to extrapolate from small-scale work and this review tries to pull the two types of study together. It is shown that 'shear sensitivity' due to agitation and bursting bubbles is no longer considered a major problem. Homogeneity becomes increasingly important with respect to pH and nutrients at the largest scale and sub-surface feeding is recommended despite 'cleaning in place' concerns. There are still major questions with cell retention/recycle systems at these scales, either because of fouling, of capacity or of potential and different 'shear sensitivity' questions. Fed-batch operation gives rise to cell densities that have led to the use of oxygen and enriched air to meet oxygen demands. This strategy, in turn, gives rise to a CO(2) evolution rate that impacts on pH control, pCO(2) and osmolality. These interactions are difficult to resolve but if higher sparge and agitation intensities could be used to achieve the necessary oxygen transfer, the problem would largely disappear. Thus, the perception of 'shear sensitivity' is still impacting on the development of animal cell culture at the commercial scale. Microcarrier culture is also briefly addressed. Finally, some recommendations for bioreactor configuration and operating strategy are given.

Optimization of Fed‐Batch Saccharomyces cerevisiae Fermentation Using Dynamic Flux Balance Models

We developed a dynamic flux balance model for fed-batch Saccharomyces cerevisiae fermentation that couples a detailed steady-state description of primary carbon metabolism with dynamic mass balances on key extracellular species. Model-based dynamic optimization is performed to determine fed-batch operating policies that maximize ethanol productivity and/or ethanol yield on glucose. The initial volume and glucose concentrations, the feed flow rate and dissolved oxygen concentration profiles, and the final batch time are treated as decision variables in the dynamic optimization problem. Optimal solutions are generated to analyze the tradeoff between maximal productivity and yield objectives. We find that for both cases the prediction of a microaerobic region is significant. The optimization results are sensitive to network model parameters for the growth associated maintenance and P/O ratio. The results of our computational study motivate continued development of dynamic flux balance models and further exploration of their application to productivity optimization in biochemical reactors.

Control of High-Solids Saccharification using a Model-Based Methodology for Fed-Batch Operation

This work utilizes both insights obtained from experimental work and kinetic modeling to develop an optimization strategy for high-solids cellulose saccharification, which presents a problem for the operation of stirred tank reactors (STRs) at insoluble solids levels greater than ~15%. In this work, a previously developed model for enyzmatic hydrolysis of cellulose was modified to consider the effects of feeding a stream of pretreated corn stover (PCS) solids and cellulase for a fed-batch process. By solving the set of model differential equations while controlling for a feed stream, it was possible to develop an open-loop control feeding profile that controls the insoluble solids at a constant or manageable level throughout the course of the reaction. This prevents any difficulties that would otherwise be encountered in mixing high-solids slurries. Experimental application of two fed-batch feeding policies in bench-scale STRs resulted in similar cellulose conversion profiles to batch shake flasks, with final cellulose conversions reaching ~80% for fed-batch STRs fed at the equivalent of 25% initial insoluble solids.

An anaerobic bioreactor allows the efficient degradation of HCH isomers in soil slurry

The insecticide gamma-hexachlorocyclohexane (γ-HCH or lindane), which has been extensively used for agricultural and medical purposes, presents high persistence and toxicity to the environment and low solubility. This study intends to assess the efficiency of an anaerobic reactor to degrade HCH isomers contained in soil slurry cultures. This study was developed in two phases: experiments in flasks to optimize the process parameters, and assessment of the slurry process in the anaerobic slurry reactor operated for an approximate period of a year. The influence of different environmental conditions was evaluated: the HCH concentration (25–100 mg HCH kg −1), the type of substrate (volatile fatty acids or starch), the sludge concentration (2–8 g VSS l −1) and the replacement of spiked soil to simulate a fed-batch operation (10–50%). The best results were obtained when the reactor was operated with a sludge concentration of 8 g VSS l −1, starch concentration of 2 g COD l −1 and soil replacements of 10–20%. Under these conditions, α- and γ-HCH were completely degraded after 10 d while nearly 90% β- and δ-HCH were removed only after 50 d. According to the obtained results related to the total degradation of the HCH isomers and the degradation rates, especially high for α- and γ-HCH, the anaerobic slurry reactor appears to be a good alternative for the degradation of the HCH isomers present in polluted soil.

Production of Alkaline Protease with Teredinobacter turnirae in Controlled Fed-batch Fermentation

By using our previously optimized media and a fed-batch operation controlled by LabVIEW Software, the key parameter for a high production of alkaline protease using the marine bacterium, Teredinobacter turnirae, was to maintain a low concentration of C and N-sources ( < 2 g sucrose l(-1) and < 0.2 g NH4C l l(-1)) using an appropriate fed-batch culture system. A maximum protease activity of 8250 U ml(-1 )was thus achieved.

Biogas production from crop residues on a farm-scale level: is it economically feasible under conditions in Sweden?

Anaerobic digestion would enable the energy potential of agricultural crop residues such as sugar beet tops and straw to be harnessed. Sweden is so spread out that full utilisation of this potential by centralised slurry-based technology is difficult. It appears that simple but effective high-solids reactor systems have a better chance of being economically viable on a farm-scale level (50-500 kW). In the present study, the financial prospects of high-solids digestion, using either single-stage fed-batch or two-stage batch reactor systems, are compared on a farm-scale level (50 kW) with those of conventional slurry digestion, on the basis of experimental results and observations on a laboratory- and pilot-scale. The gas produced can be used for heat, combined heat and power or as vehicle fuel. The results indicate high-solids single-stage fed-batch operations to stand the best chances of being competitive, particularly in connection with organic farming. The methane yield, degree of gas utilisation, and operational costs were found to have the strongest impact on the financial success of the process.

Computer simulated fed-batch cultivation for over production of PHB: A comparison of simultaneous and alternate feeding of carbon and nitrogen

Batch kinetics for cultivation of Ralstonia eutropha, NRRL B14690 featured 19.7 g/l biomass and 10.89 g/l PHB accumulation in 60 h under statistically optimized culture conditions. For in-depth understanding of culture dynamics and for suitable reactor design a mathematical model was developed. The model was highly instrumental in guiding the appropriate experiments to optimize the process with minimum efforts. Offline computer simulated fed-batch cultivations allowed selection of two different nutrient (nitrogen (as urea) and fructose) feeding strategies. Fed-batch fermentation, conducted by feeding nitrogen (as urea) (8 g/l) at the rate of 70 ml/h and fructose (300 g/l) at the rate of 80 ml/h simultaneously yielded 17.57 g/l PHB in 50 h with an overall productivity of 0.35 g/l h. In another strategy, alternate feeding of the nitrogen (as urea) (15 g/l) and fructose (350 g/l) at a flow rate of 80 ml/h resulted in PHB accumulation of 18.46 g/l in 40 h resulting in overall productivity of 0.46 g/l h. Model-based fed-batch operation was very helpful for over producing PHB by R. eutropha. The increase in PHB productivity in fed-batch cultivation was almost 2.6 times as compared to batch.

H 2S removal with an immobilized cell hybrid reactor

When hydrogen sulphide gas was introduced into a single reactor, in which the cells were mixed with iron solution in the absence of the immobilization particles, the cells were damaged by the high concentration of H 2S and the H 2S removal rate was rapidly decreased. Therefore, a hybrid reactor was developed, by combining a chemical reduction reactor and a biological oxidation reactor, to remove the toxic effect of H 2S on the cells and to improve the H 2S removal rate. The microbial cells were immobilized on the surface of curdlan particles, in order to enhance the Fe(II) oxidation rate through repeated fed-batch operation. As a result, the iron oxidation rate was four times faster than that obtained with the free cells. Iron solution, oxidized in an oxidation reactor by Thiobacillus ferrooxidans, was fed into the iron reduction reactor, and the reduced iron solution was recycled into the iron oxidation reactor. The X-ray diffractometer (XRD) data indicated that iron was precipitated along with elemental sulphur at the high concentration of H 2S, resulting in the iron oxidation rate being decreased with increasing reaction time.

Production of xylitol by recombinant Saccharomyces cerevisiae containing xylose reductase gene in repeated fed-batch and cell-recycle fermentations

Xylitol is a well-known sugar substitute with low-calorie and anti-cariogenic characteristics. An effort of biological production of xylitol from xylose was made in repeated fed-batch and cell-recycle fermentations of recombinant Saccharomyces cerevisiae BJ3505/δXR harboring the xylose reductase gene from Pichia stipitis. Batch fermentation with 20 g/l xylose and 18 g/l glucose resulted in 9.52 g/l dry cell mass, 20.1 g/l xylitol concentration and approximately 100% conversion yield. Repeated fed-batch operation to remove 10% of culture broth and to supplement an equal volume of 200 g/l xylose was designed to improve xylitol production. In spite of a sudden drop of cell concentration, an increase in dry cell mass led to high accumulation of xylitol at 48.7 g/l. To overcome loss of xylitol-producing biocatalysts in repeated fed-batch fermentation, cell-recycle equipment of hollow fiber membrane was implemented into a xylitol production system. Cell-recycle operation maintained concentration of the recombinant cells high inside a bioreactor. Final dry cell mass of 22.0 g/l, 116 g/l xylitol concentration, 2.34 g/l h overall xylitol productivity were obtained in cell-recycle fermentation supplemented with xylose and yeast extract solution, which were equivalent to 2.3-, 5.8- and 3.8-fold increases compared with the corresponding values of batch-type xylitol production parameters.

Aerobic Sludge Digestion under Low Dissolved Oxygen Concentrations

Low dissolved oxygen (DO) concentrations occur commonly in aerobic digesters treating thickened sludge, with benefits of smaller digester size, much reduced aeration cost, and higher digestion temperature (especially important for plants in colder areas). The effects of low DO concentrations on digestion kinetics were studied using the sludge from municipal wastewater treatment plants in Akron, Ohio, and Los Lunas, New Mexico. The experiments were conducted in both batch digestion and a mixed mode of continuous, fed-batch, and batch operations. The low DO condition was clearly advantageous in eliminating the need for pH control because of the simultaneous occurrence of nitrification and denitrification. However, when compared with fully aerobic (high DO) systems under constant pH control (rare in full-scale plants), low DO concentrations and a higher solids loading had a negative effect on the specific volatile solids (VS) digestion kinetics. Nonetheless, the overall (volumetric) digestion performance depends not only on the specific digestion kinetics, but also the solids concentration, pH, and digester temperature. All of the latter factors favor the low DO digestion of thickened sludge. The significant effect of temperature on low DO digestion was confirmed in the mixed-mode study with the Akron sludge. When compared with the well-known empirical correlation between VS reduction and the product (temperature x solids retention time), the experimental data followed the same trend, but were lower than the correlation predictions. The latter was attributed to the lower digestible VS in the Akron sludge, the slower digestion at low DO concentrations, or both. Through model simulation, the first-order decay constant (kd) was estimated as 0.004 h(-1) in the mixed-mode operations, much lower than those (0.011 to 0.029 h(-1)) obtained in batch digestion. The findings suggested that the interactions among sludges with different treatment ages may have a substantially negative effect on digestion kinetics. The use of multistage digesters, especially with small front-end reactors, may be advantageous in both "process" kinetics and "biological reaction" kinetics for sludge digestion.

Substrate replenishment extends protein synthesis with an in vitro translation system designed to mimic the cytoplasm

Cytoplasmic mimicry has recently led to the development of a novel method for cell-free protein synthesis called the "Cytomim" system. In vitro translation with this new system produced more than a 5-fold yield increase of chloramphenicol acetyl transferase (CAT) relative to a conventional method using pyruvate as an energy substrate. Factors responsible for activating enhanced protein yields, and causes leading to protein synthesis termination have been assessed in this new system. Enhanced yields were caused by the combination of three changes: growing the extract source cells on 2x YTPG media versus 2x YT, replacing polyethylene glycol with spermidine and putrescine, and reducing the magnesium concentration from conventional levels. Cessation of protein synthesis was primarily caused by depletion of cysteine, serine, CTP, and UTP. Substrate replenishment of consumed amino acids, CTP, and UTP extended the duration of protein synthesis to 24 h in fed-batch operation and produced 1.2 mg/mL of CAT. By also adding more T7 RNA polymerase and plasmid DNA, yields were further improved to 1.4 mg/mL of CAT. These results underscore the critical role that nucleotides play in the combined transcription-translation reaction and highlight the importance of understanding metabolic processes influencing substrate depletion.

Microbial population dynamics during fed-batch operation of commercially available garbage composters.

Microbial populations in terms of quantity, quality, and activity were monitored during 2 months of start-up operation of commercially available composters for fed-batch treatment of household biowaste. All the reactors, operated at a waste-loading rate of 0.7 kg day(-1) (wet wt), showed a mass reduction efficiency of 88-93%. The core temperature in the reactors fluctuated between 31 degrees C and 58 degrees C due to self-heating. The pH declined during the early stage of operation and steadied at pH 7.4-9.3 during the fully acclimated stage. The moisture content was 48-63% early in the process and 30-40% at the steady state. Both direct total counts and plate counts of bacteria increased via two phases (designated phases I, II) and reached an order of magnitude of 10(11) cells g(-1) (dry wt) at the steady state. Microbial community changes during the start-up period were studied by culture-independent quinone profiling and denatured gradient gel electrophoresis (DGGE) of PCR-amplified 16S rDNA. In all the reactors, ubiquinones predominated during phase I, whereas partially saturated menaquinones became predominant during phase II. This suggested that there was a drastic population shift from ubiquinone-containing Proteobacteria to Actinobacteria during the start-up period. The DGGE analysis of the bacterial community in one of the reactors also demonstrated a drastic population shift during phase I and the predominance of members of the phyla Proteobacteria and Bacteroidetes during the overall period. But this molecular analysis failed to detect actinobacterial clones from the reactor at any stage.

Insights into adenoviral vector production kinetics in acoustic filter-based perfusion cultures.

One of the major limitations in the production of adenoviral vectors is the reduction in cell-specific productivity observed for increasing cell density at infection in batch cultures. This observation strongly suggests some nutrient depletion and/or metabolite inhibition in the media. These limitations have been partially overcome through other feeding strategies, such as fed-batch and sequential batch operations. To improve these results, we evaluated perfusion as a strategy to increase the volumetric productivity of HEK-293 cell cultures, by allowing productive infection at higher cell densities. An acoustic cell separator was employed in consideration of the increased shear sensitivity of the cells during the infection phase. The effects of perfusion rate and cell density at infection on the production of a recombinant adenovirus expressing the GFP were investigated. The perfusion mode allowed successful infection at cell densities in the range of 2.4-3 x 10(6) cell/mL, while maintaining a similar cell specific productivity (17,900 +/- 2400 VP/cell) to that of a batch infected at a low cell density (5 x 10(5) cell/mL). The highest virus concentrations (4.1 +/- 0.6 x 10(10) VP/mL) were attained for a feed rate of 2 vol/d and constituted a fivefold increase compared to a batch with medium replacement. Rapid assessment of the infection status was achieved through the use of on-line monitoring of respiration, fluorescence, and biovolume. Analysis of the kinetics of nutrient consumption and metabolite production revealed that a reduction in specific productivity is correlated with reduced metabolic activity.

Fed-batch optimization of alpha-amylase and protease-producing Bacillus subtilis using Markov chain methods.

A stoichiometry-based model for the fed-batch culture of the recombinant bacterium Bacillus subtilis ATCC 6051a, producing extracellular alpha-amylase as a desirable product and proteases as undesirable products, was developed and verified. The model was then used for optimizing the feeding schedule in fed-batch culture. To handle higher-order model equations (14 state variables), an optimization methodology for the dual-enzyme system is proposed by integrating Pontryagin's optimum principle with fermentation measurements. Markov chain Monte Carlo (MCMC) procedures were appropriate for model parameter and decision variable estimation by using a priori parameter distributions reflecting the experimental results. Using a simplified Metropolis-Hastings algorithm, the specific productivity of alpha-amylase was maximized and the optimum path was confirmed by experimentation. The optimization process predicted a further 14% improvement of alpha-amylase productivity that could not be realized because of the onset of sporulation. Among the decision variables, the switching time from batch to fed-batch operation (t(s)) was the most sensitive decision variable.

Adsorbent supplemented biological treatment of pre-treated landfill leachate by fed-batch operation

Biological treatment of landfill leachate usually results in low COD removals because of high chemical oxygen demand (COD), high ammonium-N content and presence of toxic compounds. Coagulation–flocculation with lime addition and air stripping of ammonia were used as pre-treatment in this study in order to improve biological treatability of the leachate. Pre-treated leachate was subjected to adsorbent supplemented biological treatment in an aeration tank operated in fed-batch mode. COD and NH 4-N removal performances of powdered activated carbon (PAC) and powdered zeolite (PZ) were compared during biological treatment. Adsorbent concentrations varied between 0 and 5 g l −1. Percent COD and ammonium-N removals increased with increasing adsorbent concentrations. Percent COD removals with PAC addition were significantly higher than those obtained with the zeolite. However, zeolite performed better than the PAC in ammonium-N removal from the leachate. Nearly 87% and 77% COD removals were achieved with PAC and zeolite concentrations of 2 g l −1, respectively. Ammonium-N removals were 30% and 40% with PAC and zeolite concentrations of 5 g l −1, respectively at the end of 30 h of fed-batch operation.

Repeated fed-batch biological treatment of pre-treated landfill leachate by powdered activated carbon addition

High chemical oxygen demand (COD), high ammonium-N content and presence of toxic compounds such as heavy metals result in low removal efficiencies in direct biological treatment of landfill leachate. Raw landfill leachate was subjected to pre-treatment by coagulation-flocculation and air stripping of ammonia before biological treatment. Pre-treated leachate was subjected to biological treatment in an aeration tank operated in repeated fed-batch mode in the absence and presence of powdered activated carbon (PAC) as adsorbent. Three different cycles of 1×30 h, 3×10 h, and 5×6 h were used in the absence and presence of 2 g l −1 PAC. The results clearly indicated that PAC addition has improved COD removal significantly as compared to PAC-free biological treatment. Repeated fed-batch treatment also resulted in lower effluent COD and higher COD removal efficiencies as compared to single-cycle operation. The feed COD of 7000 mg l −1 was reduced to final COD values of 365 mg l −1 and 875 mg l −1 in repeated fed-batch experiments with 5×6 h cycles in the presence (2 g l −1 PAC) and absence of PAC, respectively. Repeated fed-batch operation with 3×30 h cycle length resulted in percent COD removal and final COD content of 94% and 285 mg l −1, respectively, with 2 g l −1 PAC addition.

High-Cell-Density Cultivation of &lt;I&gt;Pseudomonas putida&lt;/I&gt; IPT 046 and Medium-Chain-Length Polyhydroxyalkanoate Production From Sugarcane Carbohydrates

We studied high-density cultures of Pseudomonas putida IPT 046 for the production of medium-chain-length polyhydroxyalkanoates (PHAMCL) using an equimolar mixture of glucose and fructose as carbon sources. Kinetics studies of P. putida growth resulted in a maximum specific growth rate of 0.65 h(-1). Limitation and inhibition owing to NH4+ ions were observed, respectively, at 400 and 3500 mg of NH4+/L. The minimum concentration of dissolved oxygen in the broth must be 15% of saturation. Fed-batch strategies for high-cell-density cultivation were proposed. Pulse feed followed by constant feed produced a cell concentration of 32 g/L in 18 h of fermentation and low PHAMCL content. Constant feed produced a cell concentration of 35 g/L, obtained in 27 h of fermentation, with up to 15% PHAMCL. Exponential feed produced a cell concentration of 30 g/L in 20 h of fermentation and low PHAMCL content. Using the last strategy, 21% PHAMCL was produced during a period of 34 h of fed-batch operation, with a final cell concentration of 40 g/L and NH4+ limitation. Using phosphate limitation, 50 g/L cell concentration, 63% PHAMCL and a productivity of 0.8 g/(L x h) were obtained in 42 h of fed-batch operation. The PHAMCL yield factors from consumed carbohydrate for N-limited and P-limited experiments were, respectively, 0.15 and 0.19 g/g.

Mass transfer and kinetics of the three-phase hydrogenation of a dinitrile over a Raney-type nickel catalyst

The hydrogenation kinetics of a dinitrile over a Raney-type nickel catalyst was evaluated from experiments performed in a fed-batch operating autoclave at 320– 355 K and 2– 7 MPa hydrogen pressure. This complex catalytic reaction consists of two main parts: almost 100% selective hydrogenation of the dinitrile to the corresponding aminonitrile and consecutive hydrogenation to either the desired primary diamine or to pyrrolidine via ring formation. An extensive study has been made on the effects of mass transfer in the applied slurry-type reactor for this reaction. The gas–liquid mass transfer is enhanced by the presence of catalyst particles, and at typical hydrogenation conditions, k L a values up to 1.0 s −1 can be reached. A Sherwood correlation for the three-phase reactor showed that important parameters in the gas–liquid mass transfer are stirrer speed and the density and viscosity of the solvent. The kinetic experiments were performed in absence of mass and heat transfer limitations. The kinetic data were modeled using two rate models based on Langmuir–Hinshelwood kinetics, assuming the reaction of dissociatively adsorbed hydrogen and nitrile compound as rate-limiting step. The first model involved competitive adsorption between hydrogen and organic compound and the second model was based on non-competitive adsorption. Both models successfully described both reaction parts. The reaction of dinitrile to aminonitrile is nearly 100% selective due to the relatively strong adsorption of the dinitriles as compared to the aminonitriles. By increasing the hydrogen partial pressure, higher yields of primary amine can be obtained. The models predict that operating in the mass-transfer regime at relatively high temperatures reduces the formation of the primary diamine.