Comparison of a quadroma and its parent hybridomas in fed batch culture

A quadroma (#22 × 63), formed by the fusion of two hybridomas, and its parent hybridomas (#22 and FMC 63) were each grown in fed batch cultures in order to examine the change in antibody productivity over time of the quadroma compared to its parent hybridomas. The growth rate, glucose uptake rate and lactate production rate of the quadroma were found to be intermediate between those of its parent cells of origin. The specific antibody productivity and internal antibody content of the quadroma followed the same decreasing trends over time as those seen in both parent hybridomas. Losses in specific antibody production rate and antibody content, however, occurred at a faster rate for the quadroma than for either of its parent hybridomas. Although the growth of a non-producing subpopulation is presumed to account for the drop in antibody production, there was no direct correlation between the percentage of high antibody containing cells, as determined by flow cytometry, and the specific antibody production rate.     

Investigation of metabolic variability observed in extended fed batch cell culture

A 13‐day fed‐batch IgG1 production process was developed by applying our proprietary chemically defined platform process. The process was highly reproducible with respect to cell growth and titer, but the cultures exhibited metabolic variability after 12 days of cultivation. This metabolic variability consisted of a subset of cultures exhibiting increased cell‐specific glucose uptake rates and high lactate production rates (LPR) despite identical operating conditions. We investigated the causes of the metabolic variability by manipulating the rate at which feed medium was delivered. Overfeeding directly led to increased LPR. High LPR was found to be associated with increased mitochondrial membrane potential in a subset of cells, as measured through fluorescent staining, and feeding TCA cycle intermediates was found to prevent the high LPR phenotype. This supports the hypothesis that mitochondrial pathways are involved in inducing metabolic variability. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1519–1527, 2013     

Effect of glucose flow on the acetone butanol fermentation in fed batch culture

Summary Clostridium acetobutylicum was grown in fed-batch cultures at different feeding rates of glucose. The sugar converted to butanol and acetone increased with increasing the glucose flow, on the contrary the conversion to butyric acid was highest at slow glucose feeding rate. The acetic acid concentration was constant at the different flows of glucose. The solventogenesis was not inhibited at high flow of sugar.     

Biodegradation of phenol and m-cresol in a batch and fed batch operated internal loop airlift bioreactor by indigenous mixed microbial culture predominantly Pseudomonas sp

An internal loop airlift reactor (ILALR) is developed and studied for biodegradation of phenol/m-cresol as single and dual substrate systems under batch and fed batch operation using an indigenous mixed microbial strain, predominantly Pseudomonas sp. The results showed that the culture could degrade phenol/m-cresol completely at a maximum concentration of 600mgl(-1) and 400mgl(-1), respectively. Batch ILALR study has revealed that phenol has been preferentially degraded by the microbial culture rather than m-cresol probably owing to the toxic effect of the later. Sum kinetic model evaluated the interaction between the phenol/m-cresol in dual substrate system, which resulted in a high coefficient of determination (R(2)) value >0.98). The fed batch results showed that the strain was able to degrade phenol/m-cresol with maximum individual concentrations 600mgl(-1) each in 26h and 37h, respectively. Moreover for fed batch operation, degradation rates increased with increase in feed concentration without any lag in the degradation profile.     

Xylitol production in repeated fed batch cultivation

The ability of Candida parapsilosis to produce xylitol was tested using successive substrate supplies, and the importance of the amount of viable cells in enhancing the conversion rate was demonstrated. The suitability of this yeast for the production of xylitol was investigated in repeated fed-batch cultivation, using pure xylose or mixtures of xylose and glucose. The use of this process increased productivity by about 40% compared with simple batch cultivation without loss of yield of product on substrate. The presence of glucose in the culture medium seemed to stimulate the specific growth rate, but had no influence over other fermentative parameters.     

A flow cytometry analysis of batch and continuous culture transient diauxic growth in Hansenula polymorpha

A flow cytometry analysis and in vitro enzyme activity study is carried out on the methylotrophic yeast, Hansenula polymorpha, during both (a) batch growth and (b) continuous cultures subjected to single perturbations in either system dilution rate or influent carbon substrate composition. Flow cytometry of yeasts growing diauxically on a glucose: methanol mixture during exponential growth, exhibit DNA and RNA distributions indicative of the S-synthesis-phase of the cell cycle. Cells at the stationary growth stage exhibit DNA and RNA distributions that indicate one portion of the population in the G ₀/G₁ resting phase and another in the M-mitosis-phase.Yeast cells grown at a steady-state of D=0.2 h¹, then shifted to D=0.35 h^–1, at a constant influent substrate mixture, are also examined with both flow cytometry and in vitro enzyme assays. Distributions of DNA, RNA, and total protein at either steady state and during the shift between dilution rates did not resemble any observed in batch culture. Flow cytometry indicates significant changes in cell composition within 20 min of the imposed dilution rate shift. In vitro enzyme assays show a response time in decreasing methanol oxidase activity of 2.5–3 h upon a dilution rate shift-up, while hexokinase activity increases to its steady-state level in less than 3 h. Similar cell compositional changes are reported for shifts in influent substrate methanol: glucose ratio at a constant dilution rate of D=0.35 h ^–1. Results suggest that an unsteady-state regime, oscillating between conditions that promote maximum enzyme activity of either glucose- or methanol-metabolizing enzymes, may allow simultaneous enhanced time-averaged production of both sets of enzymes.     

Quantitative description of microbial growth in a batch culture depending on the physiologic state of inocula]

The dynamics of biomass production and the respiration rate of five microorganisms grown as batch cultures were studied in detail. Cell suspensions with a known physiological state, i.e. chemostat cultures grown at a particular D value, as well as quasi steady-state populations cultivated with slow feeding and long energy-source starvation were used as inocula. The microorganisms were arbitrary subdivided into two groups. The biomass of Pseudomonas fluorescens, Bacillus subtilis and Debaryomyces formicarius accumulated smoothly with a monotonic rise of the specific growth rate to the upper level of microns. A distinct inverse correlation was established between the duration of the lag phase and the specific growth rate of the inoculum. Arthrobacter globiformis and Lipomyces tetrasporus were characterised by biphase growth referred to as false diauxia: glucose was accumulated without its oxidation during the first phase, and actual growth (just as in the first group of microorganism) occurred during the second phase. Most of the results are satisfactorily described by a simplified modification of the synthetic chemostat model.     

Robust operation of fed batch fermenters

Optimisation of fed batch fermenters can substantially increase the profitability of these processes. Optimal control of a fed batch fermenter is usually based on a nominal process model. Parameter uncertainties are not taken into account. Simulation studies show that results obtained with fixed nominal model parameters can be quite sensitive to the uncertainty in parameter values. This paper presents a method for obtaining robust optimal control profiles in the presence of uncertainty in the model parameters. The proposed approach is illustrated with a case study. It is also shown that feedback controllers can reduce the effect of the uncertainties.     

Mathematical modeling of biological sulfide removal in a fed batch bioreactor

In this study, biological sulfide removal is investigated in a fed batch bioreactor. In this process, sulfide is converted into elemental sulfur particles as an intermediate in the oxidation of hydrogen sulfide to sulfate. The main product is sulfur at low dissolved oxygen or at high sulfide concentrations and also more sulfates are produced at high dissolved oxygen. According to the carried out reactions, a mathematical model is developed. The model parameters are estimated and the model is validated by comparing with some experimental data. The results show that, the proposed model is in a good agreement with experimental data. According to the experimental result and mathematical model, sulfate and sulfur selectivity are sensitive to the concentration of dissolved oxygen. For sulfide concentration 0.2 (mM) in the bioreactor and dissolved oxygen of 0.5 ppm, only 10% of sulfide load is converted to sulfate, while it is 60% at the same sulfide concentration and dissolved oxygen of 4.5 ppm. At high sulfide load to the bioreactor, the concentration of uneliminated sulfide increases; it leads to more sulfur particle selectivity and consequently, less sulfate selectivity.     

Microbial biomass product from apple pomace in batch and fed batch cultures

Summary Apple pomace, a solid waste from ap-ple processing industries, was used as the raw ma-terial for production of a protein enriched pro-duct. The experiments were conducted on a small scale in Erlenmeyer flasks or 4-1 fermentors in batch and fed-batch processes usingSaccharo-mycopsis lipolytica and Trichoderma reesei in sin-gle and mixed cultures. The results obtained indi-cate the technical feasibility of the process for ob-taining products containing 13% to 15% protein, on dry basis, which is adequate for cattle feed-ing.     

Multi-objective optimization of glycopeptide antibiotic production in batch and fed batch processes

Fermentation optimization involves potentially conflicting multiple objectives such as product concentration and production media cost. Simultaneous optimization of these objectives would result in a multiobjective optimization problem, which is characterized by a set of multiple solutions, knows as pareto optimal solutions. These solutions gives flexibility in evaluating the trade-offs and selecting the most suitable operating policy. Here, ε-constraint approach was used to generate the pareto solutions for two objectives: product concentration and product per unit cost of media, for batch and fed batch operations using process model for Amycolatopsis balhimycina, a glycopeptide antibiotic producer. This resulted in a set of several pareto optimal solutions with the two objectives ranging from (0.75 g l⁻¹, 3.97 g $^-1) to (0.44 g l⁻¹, 5.19 g $^-1) for batch and from (1.5 g l⁻¹, 5.46 g $^-1) to (1.1 g l⁻¹, 6.34 g $^-1) for fed batch operations. One pareto solution each for batch and for fed batch mode was experimentally validated.     

Community dynamics of a mixed-bacterial culture growing on petroleum hydrocarbons in batch culture

The effects of various hydrocarbon substrates, and a chemical surfactant capable of enhancing crude-oil biodegradation, on the community structure of a mixed-bacterial inoculum were examined in batch culture. Of 1000 TSA-culturable isolates, 68.6% were identified at the genus level or better by phospholipid fatty acid analysis over 7-day time course experiments. Cultures were exposed to 20 g/L Bow River crude oil with and without 0.625 g/L Igepal CO-630 (a nonylphenol ethoxylate surfactant), 5 g/L saturates, 5 g/L aromatics, or 125 g/L refinery sludge. A group of six genera dominated the cultures: Acinetobacter, Alcaligenes, Ochrobactrum, Pseudomonas/Flavimonas, Stenotrophomonas, and Yersinia. Species from four of the genera were shown to be capable of hydrocarbon degradation, and counts of hydrocarbon degrading and total heterotrophic bacteria over time were nearly identical. Pseudomonas/Flavimonas and Stenotrophomonas normally dominated during the early portions of cultures, although the lag phase of Stenotrophomonas appears to have been increased by surfactant addition. Acinetobacter calcoaceticus was the most frequently isolated microorganism during exposure to the saturate fraction of crude oil. Regardless of substrate, the culture medium supported a greater variety of organisms during the latter portions of cultures. Understanding the community structure and dynamics of mixed bacterial cultures involved in treatment of heterogeneous waste substrates may assist in process development and optimization studies.     

Serum free fed batch production of IgM

Summary Fed batch cultivation of a murine hybridoma secreting IgM in a serum free medium was successfully attempted. Cell growth and IgM productivity remained high for over a month, and compared well to a similar fed batch culture in medium supplemented with 10% fetal calf serum. The average specific secretion rates of antibody in both media were the same. Sufficient inoculum cell density was crucial to the establishment of a viable culture in serum free medium for the cell line, NS6.3, used.     

Production of toluene cis-glycol using recombinant Escherichia coli strains in glucose-limited fed batch culture

Production of toluene cis-glycol (TCG) was investigated using recombinant Escherichia coli strains that express toluene dioxygenase under the tac promoter. E. coli TG2 was selected as the host for the recombinant plasmid, pTAC365, because the TCG yield was 64% higher than with strain JM105 as the host. By using fed-batch culture, TCG production could be improved by 4.4-fold compared with batch cultures, with a toluene vapor feed. A further improvement of 1.2-fold was obtained by using a two-liquid phase culture system, but the improvement was 1.6-fold when the feed rate of toluene vapor was reduced by 75%. When the period of growth in fed-batch culture was extended and the reduced vapor feed was used, the maximum TCG concentration increased to 4 g/l, an overall improvement of 10-fold compared with the batch culture system used initially. However, product consumption was observed during the late stages of fed-batch growth and in stationary phase, resulting in the formation of o-cresol, 3-methylcatechol and benzyl alcohol. We conclude that the key to optimizing TCG production by recombinants is to prolong growth of the cells to ensure sustained growth-linked product formation, and to optimize the supply of the toxic substrate, toluene, but further work is needed to eliminate by-product formation.     

Autostabilization of the pH in a batch culture of Pseudomonas

Ranges within which the pH of the environment can be self-regulated have been determined for microorganisms belonging to the Pseudomonas genus. The ability of these microorganisms to self-regulate the pH was detected both at an increase and a decrease in the pH of the environment as compared to its optimal value. The rate of self-regulation appears to be proportional to the concentration of viable cells in a suspension. The results may be used for creating an economical system for maintaining the pH of the environment in industrial fermentation.     

The effect of controlled feeding of glycerol on beta-galactosidase production by Escherichia coli in batch culture

A mutant of E. coli constitutive for β-galactosidase has been grown in batch culture with the carbon source, glycerol, fed at various fixed rates to the culture. High feeding rates where growth was only slightly restricted gave final enzyme levels similar to those obtained in cultures where all the glycerol was added initially. Low feeding rates resulted in breakdown of the β-galactosidase formed and gave reduced final levels of the enzyme.     

Modelling of agrobacteran production in a batch culture ofAgrobacterium radiobacter

A deterministic model of production of the exopolysaccharide agrobacteran, including the effect of the product on oxygen transfer into the medium, was used for evaluation of a batch cultivation ofAgrobacterium radiobacter. Application of mass-energy balance made it possible to reduce the number of identified parameters. The resulting yield coefficients and rate constants were largely independent of the method of aeration of the culture, with the exception of the maximum specific rate of agrobacteran production which was lower if the culture in an exponential growth phase was subjected to oxygen limitation.     

Induction of a metabolic switch in insect cells by substrate-limited fed batch cultures

Insect cell metabolism was studied in substrate-limited fed batch cultures of Spodoptera frugiperda (Sf-9) cells. Results from a glucose-limited culture, a glutamine-limited culture, a culture limited in both glucose and glutamine and a batch culture were compared. A stringent relation between glucose excess and alanine formation was found. In contrast, glucose limitation induced ammonium formation, while, at the same time, alanine formation was completely suppressed. Simultaneous glucose and glutamine limitation suppressed both alanine and ammonium formation. Although the metabolism was influenced by substrate limitation, the specific growth rate was similar in all cultures. Alanine formation must involve incorporation of free ammonium, if ammonium formation is mediated by glutaminase and glutamate dehydrogenase, as our data suggest. On the basis of the results, two possible pathways for the formation of alanine in the intermediary metabolism in insect cells are suggested. The cellular yield on glucose was increased 6.6 times during glucose limitation, independently of the cellular yield on glutamine, which was increased 50–100 times during glutamine limitation. The results indicate that alanine overflow metabolism is energetically wasteful and that glutamine is a dispensable amino acid for cultured Sf-9 cells. Preliminary data confirm that glutamine can be synthesised by the cells themselves in amounts sufficient to support growth.     

Production of citric acid in batch culture

Work has been carried out on the production of citric acid in batch culture using Aspergillus foetidus. It is shown that the dry weight increases throughout the fermentation. The supply of nitrogen is exhausted early and the subsequent increase in dry weight is due to accumulation of carbon by the cells. It appears that the exhaustion of nitrogen is a necessary prerequisite for the production of citric acid, which is produced by carbon-storing cells. The pH history in the early part of the fermentation did not affect the final yields.