Utilization of an external loop bioreactor for the isolation of a flocculating strain ofKluyveromyces marxianus

A continuous open loop bioreactor was used to induce flocculation in an originally nonflocculent strain ofKluyveromyces marxianus. The sedimentation capacity of the isolated strain was of such a magnitude that the cell concentration inside the fermentor was 50 times larger than in the effluent. Also, a batch system was used with the same objective, but no flocculation was obtained.The kinetic parameters of the flocculent strain were compared with those of the mother strain. It was shown that both maximum specific growth rate and maximum specific ethanol production rate were lower in the flocculent strain. Ethanol had a larger inhibitory effect on the kinetic parameters of the isolated strain. Also, the batch fermentations with this strain presented a larger final biomass concentration and a reduced ethanol yield.     

Methods for cell separation based on sedimentation

Summary A modified Erlenmeyer flask and a sedimentation chamber have been used for separation of cells from suspension. Preliminary investigations show an efficiency in the separation of more than 80% with yeast cells. Fermentations with the Erlenmeyer flask as separator show a 4–5 times increased cell density for both hybridomas and yeast cells.     

Mechanistic simulation of batch acetone–butanol–ethanol (ABE) fermentation with in situ gas stripping using Aspen Plus™

Bioprocess and Biosystems Engineering - Process simulations of batch fermentations with in situ product separation traditionally decouple these interdependent steps by simulating a separate...     

Enhanced submerged Aspergillus ficuum phytase production by implementation of fed-batch fermentation

Phytase is an important feed and food additive, which is both used in animal and human diets. Phytase has been used to increase the absorption of several divalent ions, amino acids, and proteins in the bodies and to decrease the excessive phosphorus release in the manure to prevent negative effects on the environment. To date, microbial phytase has been mostly produced in solid-state fermentations with insignificant production volumes. There are only a few studies in the literature that phytase productions were performed in submerged bench-top reactor scale. In our previous studies, growth parameters (temperature, pH, and aeration) and important fermentation medium ingredients (glucose, Na-phytate, and CaSO₄) were optimized. This study was undertaken for further enhancement of phytase production with Aspergillus ficuum in bench-top bioreactors by conducting fed-batch fermentations. The results showed that addition of 60 g of glucose and 10 g of Na-phytate at 96 h of fermentation increased phytase activity to 3.84 and 4.82 U/ml, respectively. Therefore, the maximum phytase activity was further enhanced with addition of glucose and Na-phytate by 11 and 40 %, respectively, as compared to batch phytase fermentations. It was also reported that phytase activity increased higher in early log stage additions than late log stage additions because of higher microbial activity. In addition, the phytase activity in fed-batch fermentation did not drop significantly as compared to the batch fermentation. Overall, this study shows that fungal phytase can be successfully produced in submerged fed-batch fermentations.     

Pullulan from peat hydrolyzate fermentation kinetics

The Luedeking-Piret equation was used to fit the kinetic data of pullulan fermentations from peat hydrolyzate substrate. In batch mode, the kinetic parameters m, n, alpha, and beta varied as a function of fermentation conditions: aeration rate, agitation speed, and temperature. In constant-feed fed-batch mode, the parameters Varied according to the feed rates. In peat hydrolyzate medium, the polysaccharide synthesis was strongly growth associated in batch and continuous fermentations but entirely growth associated in fedbatch fermentations. The fed-batch mode of fermentation with an appropriate feed rate is more advantageous with respect to batch and continuous fermentations. Therefore, if the fermentation is started batchwise and then followed by fed-batch mode at a constant feed rate, the overall polysaccharide productivity (g pullulan/L h) is significantly higher than those obtained with batch or continuous fermentations using the same total medium volume.     

Butanol recovery from fermentations by liquid-liquid extraction and membrane solvent extraction

Extraction can successfully be used for in-situ alcohol recovery in butanol fermentations to increase the substrate conversion. An advantage of extraction over other recovery methods may be the high capacity of the solvent and the high selectivity of the alcohol/water separation. Extraction, however, is a comprehensive operation, and the design of an extraction apparatus can be complex. The aim of this study is to assess the practical applicability of liquid-liquid extraction and membrane solvent extraction in butanol fermentations. In this view various aspects of extraction processes were investigated.Thirty-six chemicals were tested for the distribution coefficient for butanol, the selectivity of alcohol/water separation and the toxicity towards Clostridia. Convenient extractants were found in the group of esters with high molar mass.Liquid-liquid extraction was carried out in a stirred fermenter and a spray column. The formation of emulsions and the fouling of the solvent in a fermentation broth causes problems with the operation of this type of equipment. With membrane solvent extraction, in which the solvent is separated from the broth by a membrane, a dispersion-free extraction is possible, leading to an easy operation of the equipment. In this case the mass transfer in the membrane becomes important.With membrane solvent extraction the development of a process is emphasized in which the extraction characteristics of the solvent are combined with the property of silicone rubber membranes to separate butanol from water. In the case of apolar solvents with a high molar mass, the characteristics of the membrane process are determined completely by the solvent. In the case of polar solvents (e.g. ethylene glycol), the permselectivity of the membrane can profitably be used. This concept leads to a novel type of extraction process in which alcohol is extracted with a water-soluble solvent via a hydrophobic semipermeable membrane. This extraction process has been investigated for the recovery of butanol and ethanol from water. A major drawback in all processes with membrane solvent extraction was the permeation of part of the solvent to the aqueous phase.The extraction processes were coupled to batch, fed batch and continuous butanol fermentations to affirm the applicability of the recovery techniques in the actual process. In the batch and fed batch fermentations a three-fold increase in the substrate consumption could be achieved, in the continuous fermentation about 30% increase.     

Room and low temperature brewing with yeast immobilized on gluten pellets

A biocatalyst prepared by the immobilization of a cryotolerant strain of Saccharomyces cerevisiae on gluten pellets was used for batch and continuous fermentation at low temperatures. The immobilized yeast showed important operational stability in repeated batch fermentations without a decrease of activity even at 0 and 5°C. Repeated batch fermentations using the biocatalyst resulted in improvement of ethanol productivity in comparison with bottom brewing fermentation and free cells using the same yeast strain. At 0 and 10°C, the fermentation rate was four and seven times higher than that of free cells, respectively. For immobilized yeast, diacetyl and polyphenol contents were lower and the alcohol concentration higher at low temperatures (0–7°C) when compared to free cells. Fine clarity was also observed in the beers. Continuous brewing using gluten-supported biocatalyst had an operational stability of 3 months with relatively high productivity and without contamination. Polyphenol and bitterness contents were lower in the continuous process than those of batch fermentations, but at low temperature (5°C) they were higher. The diacetyl content was higher than in batch fermentations and beers had a fine aroma and taste.     

High density cell culture by membrane-based cell recycle

Enhancement of productivity of a bioprocess necessitates continuous operation of bioreactors with high biomass concentrations than are possible in conventional batch, fedbatch or continuous modes of culture. Membrane-based cell recycle has been effectively used to maintain high cell concentrations in bioreactors. This review compares membranebased cell recycle operation with other such high density cell culture systems as immobilized cell reactors and reactors with cell recycle by centrifugation or gravity sedimentation. A theoretical of production of primary and secondary metabolites in membrane-based recycle systems is presented. Operation of this type of system is discussed with examples from aerobic and anaerobic fermentations.     

Separation of human metaphase chromosomes at neutral pH by velocity sedimentation at 20 times gravity

A method for the separation of large quantities of human metaphase chromosomes at neutral pH is described. The separation was performed in a specially designed sedimentation chamber which was placed in a bucket of a speed-controlled centrifuge. Flow deflectors in the chamber allowed both the undisturbed introduction of gradient and chromosomes, as well as the undisturbed fractionation of the content of the chamber. The centrifuge was run at 20 g for 1 h taking care of slow acceleration and deceleration. The slow morphological changes of the chromosomes due to ageing at neutral pH do not affect the resolving power of the sedimentation technique within this hour. This in contrast to separation of chromosomes at neutral pH by velocity sedimentation at unit gravity during 20 h, as described before. The main advantage of chromosome sorting at neutral pH is that the fractionated chromosomes are more suitable for gene transfer and gene mapping experiments.     

A model of xylitol production by the yeast Candida mogii

Production of xylitol from xylose in batch fermentations of Candida mogii ATCC 18364 is discussed in the presence of glucose as the cosubstrate. Various initial ratios of glucose and xylose concentrations are assessed for their impact on yield and rate of production of xylitol. Supplementation with glucose at the beginning of the fermentation increased the specific growth rate, biomass yield and volumetric productivity of xylitol compared with fermentation that used xylose as the sole carbon source. A mathematical model is developed for eventual use in predicting the product formation rate and yield. The model parameters were estimated from experimental observations, using a genetic algorithm. Batch fermentations, which were carried out with xylose alone and a mixture of xylose and glucose, were used to validate the model. The model fitted well with the experimental data of cell growth, substrate consumption and xylitol production.     

Monitoring of Saccharomyces cerevisiae in commercial bakers' yeast fermentation

In the highly competitive market of commercial bakers' yeast, fermentations are operated for maximum efficiency and minimum production cost. In order to maintain competitiveness, the fermentations must be highly consistent with minimum variation in yeast performance, maximum yield on raw materials, and minimum production of undesirable side products. The use of advanced instrumentation is of critical importance to achieving these goals by the production engineer. An in situ optical density probe was used to determine the yeast cell density in full-scale commercial bakers' yeast fermentations. The optical density probe results were compared with oxygen uptake rate analyses, packed cell volume, and off-line measured cell dry weights. The most accurate measurement of cell density was found to be the optical density probe. This instrument allowed the on-line determination of cell density with highly consistent results from fermentation batch to batch and with out the need for intermittent recalibration. (c) 1995 John Wiley & Sons, Inc.     

Effects of ptb knockout on butyric acid fermentation by Clostridium tyrobutyricum

Clostridium tyrobutyricum ATCC 25755 is an anaerobic, rod-shaped, gram-positive bacterium that produces butyrate, acetate, hydrogen, and carbon dioxide from various saccharides, including glucose and xylose. Phosphotransbutyrylase (PTB) is a key enzyme in the butyric acid synthesis pathway. In this work, effects of ptb knockout by homologous recombination on metabolic flux and product distribution were investigated. When compared with the wild type, the activities of PTB and butyrate kinase in ptb knockout mutant decreased 76 and 42%, respectively; meanwhile, phosphotransacetylase and acetate kinase increased 7 and 29%, respectively. However, ptb knockout did not significantly reduce butyric acid production from glucose or xylose in batch fermentations. Instead, it increased acetic acid and hydrogen production 33.3-53.8% and ≈ 11%, respectively. Thus, the ptb knockout did increase the carbon flux toward acetate synthesis, resulting in a significant decrease (28-35% reduction) in the butyrate/acetate ratio in ptb mutant fermentations. In addition, the mutant displayed a higher specific growth rate (0.20 h(-1) vs. 0.15 h(-1) on glucose and 0.14 h(-1) vs. 0.10 h(-1) on xylose) and tolerance to butyric acid. Consequently, batch fermentation with the mutant gave higher fermentation rate and productivities (26-48% increase for butyrate, 81-100% increase for acetate, and 38-46% increase for hydrogen). This mutant thus can be used more efficiently than the parental strain in fermentations to produce butyrate, acetate, and hydrogen from glucose and xylose.     

Production of recombinant human growth hormone in Escherichia coli: expression of different precursors and physiological effects of glucose, acetate, and salts

The constitutive cytoplasmic expression in E. coli of human growth hormone (hGH) with different N-terminal extensions (3 or 4 amino acids) has been studied. These hGH precursors were used for in vitro cleavage to obtain the mature, authentic hormone. Small changes in the amino acid extensions of the hGH precursors led to three-fold differences in specific expression rates. The specific expression rate of the hGH precursors was inversely proportional to the ratios of the specific growth rates of plasmid containing and plasmid free cells (micro(+)/micro(-)) and also to the genetic stability. To ensure a satisfactory genetic stability in production fermentors, an hGH precursor with a moderate expression efficiency was chosen.The medium composition and growth conditions were studied, resulting in the choice of a glucose fed batch fermentation process using a complex medium. In this process a yield of 2000 mg/L of met-ala-glu-hGH (MAE-hGH) was obtained. The fermentation process comprised a glucose-limited growth phase followed by a second phase with increased glucose feed and exhaustion of phosphate from the medium. The second phase is characterized by an MAE-hGH production, whereas further biomass formation is blocked. High concentrations of glucose led to reduced specific expression of MAE-hGH--the specific and total yield in batch glucose fermentations is only about 30% of the yield in optimized fed batch fermentations. The physiological background for this was investigated. Chemostat experiments showed that the glucose concentration and the metabolic condition of the cells--i.e. with or without formation of acetate--was not critical per se in order to obtain a high specific yield of MAE-hGH. Therefore it is unlikely that formation of MAE-hGH is catabolite repressed by glucose. Furthermore it was shown that the specific production rate of MAE-hGH was independent of the specific growth rate and it was further demonstrated that the decrease in expression efficiency in glucose batch fermentation was a result of an inhibitory effect of acetic acid. In batch fermentations this inhibitory effect was enhanced by a salt effect caused by increased consumption of acid and base used to control pH. The identity of the acid and the base used are not important in this context. From studies of the expression of other proteins in E. coli. with constitutive as well as inducible promoters we conclude that glucose fed batch processes are often superior to batch processes in the production of heterologous proteins E. coli.     

Protease production by Bacillus subtilis in oxygen-controlled,glucose fed-batch fermentations

Summary An open-loop, on-off control system using the dissolved oxygen level to control a glucose feed was used in a study of growth and production of protease by Bacillus subtilis CNIB 8054. With this system, both glucose and oxygen were controlled at low concentrations. In batch fermentations, protease activity in the fermentation broth was maximum when growth had stopped. During oxygen-controlled, glucose fed-batch fermentations, growth and the production of protease activity continued during glucose feeding. Oxygen-controlled, glucose fed-batch fermentations produced more protease activity than batch fermentations, depending upon the set point for dissolved oxygen. These results indicate that control of glucose and oxygen concentrations can result in improvements in protease production.     

Optimization of cyclodextrin glycosyltransferase production from Klebsiella pneumoniae AS-22 in batch, fed-batch, and continuous cultures

Production of a novel cyclodextrin glycosyltransferase (CGTase) from Klebsiella pneumoniae AS-22 strain, which converts starch predominantly to alpha-CD at high conversion yields, in batch, fed-batch, and continuous cultures, is presented. In batch fermentations, optimization of different operating parameters such as temperature, pH, agitation speed, and carbon-source concentration resulted in more than 6-fold increase in CGTase activity. The enzyme production was further improved by two fed-batch approaches. First, using glucose-based feed to increase cell density, followed by starch-based feed to induce enzyme production, resulted in high cell density of 76 g dry cell weight/L, although the CGTase production was low. Using the second approach of a single dextrin-based feed, 20-fold higher CGTase was produced compared to that in batch fermentations with media containing tapioca starch. In continuous operation, more than 8-fold increase in volumetric CGTase productivity was obtained using dextrin-based media compared to that in batch culture using starch-based media.     

In-situ recovery of butanol during fermentation

End product inhibition can be reduced by the in situ removal of inhibitory fermentation products as they form. Extractive fermentation, in which an immiscible organic solvent is added to the fermentor in order to extract inhibitory products, was applied to the acetone-butanol fermentation. Six solvents or solvent mixtures were tested in batch extractive fermentations: kerosene, 30 wt% tetradecanol in kerosene, 50 wt% dodecanol in kerosene, oleyl alcohol, 50 wt% oleyl alcohol in a decane fraction and 50 wt% oleyl alcohol in benzyl benzoate. The best results were obtained with oleyl alcohol or a mixture of oleyl alcohol and benzyl benzoate. In normal batch fermentation of Clostridium acetobutylicum, glucose consumption is limited to about 80 kg/m³ due to the accumulation of butanol in the broth. In extractive fermentation using oleyl alcohol or a mixture of oleyl alcohol and benzyl benzoate, over 100 kg/m³ of glucose can be fermented. Removal of butanol from the broth as it formed also increased the rate of butanol production. Maximum volumetric butanol productivity was increased by as much as 60% in extractive fermentation compared to batch fermentation. Butanol productivities obtained in extractive fermentation compare favorably with other in situ product removal fermentations.     

Optimization of conditions and cell feeding procedures for alcohol fermentation

Alcohol fermentation was studied with an emphasis on the separation of cell growth and alcohol production stages. Experiments were conducted to establish the optimal conditions for alcohol production in batch fermentations and to simulate continuous fermentations with cell feeding at various stages. It was found that the glucose concentration should be kept under 10% (w/v), and the temperature should be between 40 and 42.5 degrees C for maximum specific alcohol productivity. If the cell concentration is increased, a decrease in specific alcohol productivity is observed. Higher cell concentrations are needed for higher final alcohol concentrations. Among the cell feeding procedures into alcohol production stages, a cocurrent design was found to be better than recycle and countercurrent designs.     

Bacterial cellulose production by fed-batch fermentation in molasses medium

Batch and fed-batch fermentations for bacterial cellulose (BC) production using molasses as a carbon source by Acetobacter xylinum BPR2001 were carried out in a jar fermentor. For improvement of BC production, molasses was subjected to H2SO4-heat treatment. The maximum BC concentration by this treated molasses increased 76%, and the specific growth rate increased 2-fold compared with that by untreated molasses. In batch fermentation, when the initial sugar concentrations of H2SO4-heat-treated molasses were varied from 20 to 70 g/L, the highest value of maximum BC concentration of 5.3 g/L was observed at 20 g/L. BC production in intermittent fed-batch (IFB) fermentation was conducted referring to the data in batch fermentation, and the highest BC production of 7.82 g/L was obtained when 0.2 L of molasses medium was added five times. When continuous fed-batch (CFB) fermentations were conducted, maximum BC concentration was obtained with a feeding rate of 6.3 g-sugar/h, which was derived from the optimal IFB experiment.     

High cell density fed-batch fermentations for lipase production: feeding strategies and oxygen transfer

This review is focused on the production of microbial lipases by high cell density fermentation. Lipases are among the most widely used of the enzyme catalysts. Although lipases are produced by animals and plants, industrial lipases are sourced almost exclusively from microorganisms. Many of the commercial lipases are produced using recombinant species. Microbial lipases are mostly produced by batch and fed-batch fermentation. Lipases are generally secreted by the cell into the extracellular environment. Thus, a crude preparation of lipases can be obtained by removing the microbial cells from the fermentation broth. This crude cell-free broth may be further concentrated and used as is, or lipases may be purified from it to various levels. For many large volume applications, lipases must be produced at extremely low cost. High cell density fermentation is a promising method for low-cost production: it allows a high concentration of the biomass and the enzyme to be attained rapidly and this eases the downstream recovery of the enzyme. High density fermentation enhances enzyme productivity compared with the traditional submerged culture batch fermentation. In production of enzymes, a high cell density is generally achieved through fed-batch operation, not through perfusion culture which is cumbersome. The feeding strategies used in fed-batch fermentations for producing lipases and the implications of these strategies are discussed. Most lipase-producing microbial fermentations require oxygen. Oxygen transfer in such fermentations is discussed.     

Increased lovastatin formation by Aspergillus terreus using repeated fed-batch process

Lovastatin biosynthesis with Aspergillus terreus in batch fermentation reached 160 U/l in 161 h at pH 6.8 and a dissolved O tension maintained at 70%. At the end of repeated fed batch fermentations, the yield of lovastatin was increased by 37% though this took over twice as long as in the batch fermentation.