Recent Advances in High Cell Density Cultivation for Production of Recombinant Protein

Document Type: Review Paper

Authors

1 Biotechnology Group, Department of Chemical Engineering, Faculty of Engineering, Tarbiat Modares University, P.O. Box 14115-143, Tehran, I.R. Iran

2 Biotechnology Group, Department of Chemical Engineering, Faculty of Engineering, Tarbiat Modares University, P.O. Box 14115-143, Tehran and epartment of Biotechnology, University College of Science, University of Tehran, P.O. Box 14155-6455, Tehran

Abstract

This paper reviews recent strategies used for increasing specific yield and productivity in high cell density cultures. High cell density cultures offer an efficient means for the economical production of recombinant proteins. However, there are still some challenges associated with high cell density cultivation (HCDC) techniques. A variety of strategies in several aspects including host design consideration, tuning recombinant protein expression, medium composition, growth methodologies, and even control and analysis of the process have been successfully employed by biotechnologists to increase yield in high cell density cultures. Although most researches have focused on Escherichia coli, other microorganisms have the potential to be grown at high density and need further investigation. In recent years, information on physiological changes of hosts during different phases of cultivation derived from functional genomics, transcriptomics and proteomics is being used to overcome the obstacles encountered in high cell density cultivation and hence increase productivity.

Keywords


Table of Contents:
1. Introduction
2. Expression system improvement
3. Culture condition improvement
   3.1. Medium composition
   3.2. Phusical conditions
4. Growth techniques imporvements
    4.1. Fed-batch processes
    4.2. Two stage cyclic fed-batch process
    4.3. Temperature-limited fed-batch (TLFB)
    4.4.  A-stat
    4.5. Dialysis fermentation
    4.6. Pressurized cultivation
    4.7. Perfusion techniques
5. Induction conditions
    5.1. Quality of inducer
    5.2. Quantity of inducer
    5.3. Induction time
    5.4. Medium condition at induction phase
6. Process analysios and contro
7. Concluding remarks and future prospects


1. INTRODUCTION

High cell density cultivation (HCDC) is a powerful technique for production of recombinant proteins, the annual market growth of which is expected to increase at a rate of 10-15% per annum (Werner, 2004). The combination of large scale culture processes with recombinant DNA technology has enabled proteins such as interferons, interleukins, colony-stimulating factors and growth hormones to be produced in quantities that might otherwise have been difficult, if not impossible, to obtain from natural sources. Productivity is a function of cell density and specific productivity (i.e. the amount of product formed per unit cell mass per unit time); so increasing the cell density as well as specific productivity increases productivity. Increasing productivity is the major objective of fermentation in research and industry and as metioned by Lee (1996) and Riesenberg and Guthke (1999), HCDCs are a prerequisite to maximize the amount of product in a given volume within a certain time. HCDC enables the researchers to reach a higher dry cell weight and as a result a higher product concentration which is not possible in conventional batch and continuous processes. So far, an exact dry cell weight per liter has not been considered as a representative of high cell density and different studies have considered different values of dry cell weight like 50 g/l (Shokri and Larsson, 2004; Rozkov, 2001) and even values in the range of 20 g/l  for a culture to be named HCDC.
    The first step for producing protein in HCDC systems is choosing a suitable expression system, well adapted to HCDC. Once the expression system is developed, fermentation is carried out to increase the protein product titer. Nutrient composition, feeding strategy and growth conditions should be optimized in order to reach HCDC. The advantages and disadvantages of HCDC are mentioned in Table 1. It should be mentioned that despite such disadvantages, the economical advantages of HCDC over conventional methods of fermentation are often so great that it is usually just a matter of how to overcome these disadvantages and set up a HCDC. However, for large-scale processes concerns like using pure oxygen, pressurized bioreactor, high mechanical load on the agitation system and sensing and probing limitations should also be considered (Shiloach and Fass, 2005).
     This review focuses on various approaches and recent advances in solving the problems associated with HCDC and increasing productivity via increasing cell density and/or specific productivity.

2. Expression system improvement: Although, most HCDCS are associated with Escherichia coli as listed by Choi et al. (2006), other microorganisms have the ability to be grown to high cell densities (Table 2). For example, bacteria such as Bacillus subtilis (Vuolanto et al., 2001), Lactobacillus plantarum (Barreto et al., 1991), Pseudomonas putida (Lee et al., 2000), Methylobacterium extorquens (Belanger et al., 2004), Ralstonia eutropha (Srinivasan et al., 2003), yeasts such as Saccharomyces cerevisiae (Shang et al., 2006), Kluyveromyces marxianus (Hensing et al., 1994), Pichia pastoris (Daly and  Hearn, 2005), Hansenula polymorpha (Moon et al., 2004), Trigonopsis variabilis (Kim et al.,1997), insect cells like Spodoptera frugiperda (Elias et al., 2000), animal cells like Chinese hamster ovary cells (Lim et al., 2006), diatom Nitzschia laevis (Wen et al., 2002), Protozoon Colpidium campylum (Scheidgen-Kleyboldt et al., 2003), Tetrahymena thermophila (Kiy and Tiedtke, 1992) and even herbs such as Panax notoginseng (Zhong et al., 1999) and Galdieria sulphuraria (Schmidt et al., 2005) and other eukaryotic cells  have been reported which can grow to a high cell density.
      Microorganisms frequently experience different kinds of limiting conditions during HCDC. Cells in high density cultures are exposed to adverse conditions such as lack of nutrients, elevated osmotic pressure and other problems which have been mentioned previously, so selecting and designing a suitable host with a higher specific growth rate, increased biomass yield, reduced secretion of overflow metabolites and increased resistance to osmotic stress and nutrient deprivation is the primary step in designing a HCDC for producing recombinant proteins.
    The traditional approach for obtaining a suitable host is isolation and selection of mutants. Weikert et al. (1998) reported a three fold increase in expressing Bacillus stearothermophilus amylase using the E. coli mutant CWML2:pCSS4-p which had been isolated from a mixed culture.
     Powerful tools of genetics and cellular engineering have enabled researchers to design a better host for HCDC by rational instead of trial-and-error methods. Jena and Deb (2005) and Sorensen et al. (2005) listed genetic parameters to be considered for designing a better expression system. Moreover, redirecting the metabolic pathways has become more common recently. Especially that proteome and transcriptome profiling of microorganisms make it possible to generate invaluable information that can be used for the development of metabolic and cellular engineering strategies. Chips and microarrays are becoming standard tools for the high-throughput analysis at the level of gene expression. Chip systems also enable the rapid characterization of the desired recombinant product even in solutions from process intermediates (Forrer et al., 2004, Vasilyeva et al., 2004).
     Analyzing the transcriptome profiles by DNA microarrays shows that the growth phase can significantly affect the transcriptome profiles of E. coli during well-controlled synchronized high-cell-density fed-batch cultures (Haddadian and Harcum, 2005). Hermann (2004) analyzed transcriptome profiles of recombinant E. coli producing the human insulin-like growth factor I fusion protein during HCDC fed-batch culture using DNA microarrays. The expression levels of 529 genes were significantly altered after induction. About 200 genes were significantly downregulated during the production of protein after induction. Physiological and metabolic changes of E. coli observed by proteome analysis via gel electrophoresis (2-DE) are summarized as follows: The levels of TCA cycle enzymes (isocitrate dehydrogenase, malate dehydrogenase, succinate dehydrogenase and succinyl-CoA synthetase) increased during the exponential phase of HCDC, while the levels of glycolytic enzymes, (enolase, fructose-bisphosphate aldolase, phosphoglycerate mutase 1, triose-phosphate isomerase) decreased during the stationary phase. (Hermann 2004). The synthesis of isocitrate dehydrogenase increased considerably (up to four-fold) in the exponential growth phase. On the other hand, levels of most amino acid biosynthetic enzymes decreased during this phase of growth.
      Raman et al. (2005) used proteome analysis to evaluate the differences in protein expression of recombinant E. coli in glucose limited fed-batch fermentation. The authors reported that gene up-regulation in glucose limited fed-batch cultures equips cells for the scavenging of glucose (which is present at low concentrations), transporting and metabolizing of a wide range of substrates, tackling energy deficiency and coping with stressful conditions. Yoon et al. (2003) used combined transcriptome and proteome analysis during high cell density fed batch culture of E. coli in order to understand physiological and metabolic changes during HCDC. The authors reported that the expression of genes involved in translation, ATP synthesis and amino acid synthesis was downregulated after feeding but expression of most genes of the TCA cycle and genes which are involved in overcoming undesirable intracellular conditions was upregulated. Another interesting phenomenon observed by proteome profiling was the change in the permeability of the outer membrane as cell density increased. The expression of chaperone genes increased with cell density, which is an inevitable consequence of the stress imposed on the cell at high cell densities, which may also turn out to be beneficial for the production of correctly formed heterologous proteins (Makrides, 1996).
The use of these pioneering analyses is not limited to E. coli, although high cell density cultures of other microorganisms have rarely been studied. Examples concerning the use of high throughput analyses for other microorganisms like: Lactococcus lactis (Vido et al., 2004), B. subtilis (Helmann et al., 2003), Corynebacterium glutamicum (Ruckert et al., 2003), Aspergillus terreus (Askenazi et al., 2003), S. cerevisiae (Salusjarvi et al., 2003) and P.  putida (Heim et al., 2003) can be found in literature. 
These findings should be invaluable in designing metabolic pathways and fermentation strategies for the production of recombinant proteins and metabolites by HCDC of E. coli. Unfortunately, there is little information on the transcriptome and proteome of other microorganisms.
Another problem associated with HCDC is filamentation which is a response to the high density of cells. Filamentation of cells lowers the final achievable cell concentration and the productivity of the target protein. The expression of foreign proteins enhances the biosynthesis of the repressor of the cell division proteins FtsZ and FtsA and has been found to hamper the productivity. Over-expression of FtsZ or FtsA allows unconditional cell division and consequently, high density growth and high productivity (Jeong and Lee, 2003; Wang and Lee, 1998; Lee, 1994).
3. Culture condition improvement: In order to develop an optimized condition in terms of medium composition and physical conditions for reaching higher productivity via higher cell density and/or specific productivity, there are some points which should be considered:

3.1. Medium composition
It is desirable to make the feed solution as simple as possible by including the essential non-carbon, non-nitrogen components in the medium. But it should be borne in mind that some nutrients can inhibit cell growth when present above a certain concentration (Lee, 1996). High amounts of substrates are needed to achieve high cell density but these substrates should be fed in a controlled manner because they may have adverse effects on cell growth and production. Excess carbon source leads to metabolic by-products which are inhibitory and can be prevented by feeding a limited supply of carbon source. The main metabolic by-products are acetate for E. coli, propionate for B. subtilis, lactate for L. lactis and ethanol for S. cerevisiae (Riesenberg and Guthke, 1999).
     Another point is the precipitation of media ingredients, especially when they are present at high concentrations, which is usually the case when the cells are to be grown to high densities (Shiloach and Fass, 2005). Precipitation can affect downstream recovery, purification operations and monitoring devices. For example precipitation of mineral salts which may occur during medium preparation hampers the determination of the actual concentration of minerals in the medium; it can also complicate the measurement of cell densities (Cereghino et al., 2002). Seeking a solution to the above mentioned problems,  Brady et al. (2001) cut the concentration of all salts in the medium to one quarter of the original recipe. Another concern is the osmotic pressure and conductivity caused by high ion concentrations in the growth media that may affect membrane potential and activate different stress mechanisms that induce reduction in growth rate or termination of the growth cycle (Winzer et al., 2002). Generally, defined media are used to obtain high cell density because the nutrient concentrations are known and can be controlled during culture (van Hoek et al., 2000). Complex media such as peptone and yeast extract can vary in composition and quality making fermentation less reproducible. However, semi-defined or complex media are sometimes necessary to boost product formation. The use of a defined medium with a single or a few amino-acids to achieve higher cell or/and recombinant protein yields would be attractive for industrial conditions. It has been reported that adding a dose of leucine at the beginning of an E. coli culture with continuous feeding of glucose, threonine, tryptophan, and histidine improved productivity of b-isopropylmalate dehydrogenase (Rozkov et al., 2001).  Li et al. (1998) reported that the addition of precursor amino acids (glutamate, cysteine and glycine) and ATP improved intracellular glutathione accumulation in HCDC of    E. coli Addition of certain amino acids has also been shown to be fruitful in yeasts such as S. cerevisiae. Gorgens et al. (2005) supplemented the medium with a balanced mixture of alanine, arginine, aspargine, glutamic acid, glutamine and glycine to enhance heterologous protein production in a defined medium, such an approach has also been shown to be useful in another study (Jin and Shimizu, 1997). But, it is worth mentioning that sometimes the addition of amino acids which are present in the biomass and recombinant protein in similar amounts may even decrease the yield. For example, increasing concentration of phenylalanine resulted in a lower chloramphnicoleacetyl transferase (CAT) concentration, presumably due to feedback inhibition of biosynthesis of this amino acid and sharing common biosynthetic pathways (Ramirez and Bentley, 1993). Lee et al. (2000) applied phosphorus limitation during fed-batch culture by reducing the initial KH2PO4 concentration in order to increase the polyhydroxy alkanoate concentration. Cell density of P. putida also increased with this modification to 141 g/l. Lau et al. (2004) increased the maximum cell density by two-fold, and the final titer of product (6-deoxyerythronolide B) by 11-fold by doubling the concentration of phosphate and continuous feeding of propionate and maintaining a low propionate concentration (5-10 mM) in the medium.
     For fed-batch process, which is the most common strategy for HCDC, it is desirable to simplify the feed solution as much as possible by including sufficient non-carbon and non-nitrogen nutrients in the starting medium (Lee, 1996). However, different studies report that the addition of some materials to the feeding solution can significantly improve the productivity. Oh et al. (2002) controlled the density of B. subtilis by controlling the ratio of glucose and peptone concentrations in the feeding medium. Jeong et al. (2004) investigated chemically defined-, yeast extract-containing, and casamino acid-containing-feeding solutions for the production of human leptin by fed-batch culture of recombinant E. coli. Among these solutions, casamino acids led to the highest productivity.
      In short, new medium optimizations are necessary for the production of new recombinant proteins which seem to differ with respect to the type of microorganism and the product. It appears that enhancing amino acids and other compositions are still a good choice which have been used by many researchers. The basic approaches used to develop optimal media were trial-and-error processes. However, the use of statistical techniques for experimental design has provided a more elegant means of designing.

3.2. Physical conditions
Temperature: For high cell density cultures, temperature control is much more important due to significant heat release in spite of limited heat transfer because of high viscosity. Temperature should support cell growth as well as product formation. Since in most fermentation processes, growth phase is separated from production phase, temperature should be optimized for each phase while maintaining nutrient characteristics. It has been reported that temperature affects plasmid stability and consequently the yield of protein production in culture (Donovan et al., 1996). It has been demonstrated that the rate of mRNA degradation is a first order reaction and decreases with temperature. Thus it is possible that lowering culture temperature could be a simple and a potentially important method for increasing protein production (Shin et al., 1997).

Oxygen: In high cell density cultivation, a high capacity of oxygen supply is required. Oxygen often becomes limiting in HCDCs owing to its low solubility. The saturated dissolved oxygen (DO) concentration in water at 25ºC and 1 atm is ~7 mg/l, but oxygen supply can be increased by increasing the aeration rate or agitation speed (Lee, 1996). Oxygen-enriched air or pure oxygen has also been used to prevent oxygen limitation. Cells can also be cultured under pressurized conditions to increase oxygen transfer (Belo and Mota, 1998; Lee, 1996). By increasing oxygen transfer capacity of the bioreactor, it is possible to achieve higher cell productivity and final biomass concentration; because oxygen limitation results in formation of several metabolites of the mixed acid metabolism such as succinate, acetate, lactate, ethanol, and hydrogen which are undesirable and decrease the productivity of the bioreactor. (Castan et al., 2002; Enfors et al., 2001). However, when oxygen enriched air or pure oxygen is used to achieve high feed rate, the impact of high oxygen concentrations on the productivity and quality of recombinant proteins production needs to be investigated. Also it should be considered that oxygen itself is potentially toxic to some microorganisms.

Carbon dioxide: Carbon dioxide can also affect cell growth and recombinant protein production especially in high cell densities (Lee, 1996). High feed rate of the limiting substrate results in high carbon dioxide production rates and thus a high carbon dioxide concentration in the bioreactor. The dissolved carbon dioxide concentration depends on the partial pressure of the carbon dioxide according to Henry’s law. Growth inhibition and toxic effects of carbon dioxide have been reported (Castan et al., 2002). High partial pressure of CO2 (>0.3 atm) decreases growth rate and stimulates acetate formation (Lee, 1996). Therefore, the pressurized culture regime which has been used to increase oxygen transfer may also enhance the detrimental effect of CO2 (Matsui et al., 2006).

Mixing: Reduced mixing efficiency of the bioreactor is another physical limitation of HCDC due to high viscosity. This problem intensifies with increasing bioreactor size (Lee, 1996). In large scale bioreactors there are fluctuations in the concentration of the limiting substrate due to difficulties in mixing. In these processes, zones of high and low substrate concentrations are formed. In high concentration zones cells may produce toxic by-products and are prone to oxygen limitation but in low concentration zones cells may be starved of substrate. Another problem associated with this situation is that cells also have to face an imposed stress because of continuously passing through zones of high and low substrate concentrations. Increasing the rate of agitation is the main solution of these problems, this method can enhance protein formation and the volumetric oxygen transfer coefficient (Zhang et al., 2005; Kapat et al., 1998) but it may have detrimental effects on cells which are sensitive to shear stress like animal cells (Pan et al., 2000). Considering these disadvantages feeding in several points in the reactor and reducing the concentration of the feed have been proposed as possible solutions (Enfors et al. 2001).

Foaming: Foam formation may cause serious operational difficulties in aerated stirred bioreactors, especially in high cell density cultivation for recombinant protein production. Because with increasing cell density, cell lysis and consequently, protein concentration in the medium increases thus enhancing foam formation. Various procedures have been used in industry to reduce foam formation rate, with each of them having its own advantages and disadvantages. Stirring as foam disruption (SAFD) technique is a novel method to reduce foam in fermentation processes. The principle of this method is to reduce the foam layer with liquid flow generated by a stirrer placed just under the gas-liquid interface (Hoeks et al., 2003).

4. Growth technique improvement: Method of cultivation is important to the success of high cell density and recombinant protein production, because it affects environmental and nutritional conditions that are effective in microorganism’s growth and recombinant protein production. For this reason different methods, focusing on nutrient feeding strategies, have been developed to grow cells to high cell densities and to overproduce protein. The most important function of every strategy is to prevent overfeeding in which inhibitory concentrations of the feed components accumulate in the fermentor, or underfeeding in which the organism is starved for essential nutrients. The method of choice depends on many different factors, including the metabolism of the organism, the potential for production of inhibitory substrates, induction conditions and the capacity to measure parameters. Batch (Castrillo et al., 1996), continuous (Domingues et al., 2005 and 2000), semi-continuous (Elias et al., 2000), continuous with recycling (Tashiro et al., 2005) and  a variety of fed-batch processes (see below for examples) have been reported for growing cells to high densities. Fed-batch is the most commonly used method to produce recombinant proteins by HCDCs.

4.1. Fed-batch processes
The fed-batch process is a suitable strategy for production in high cell density culture due to (1) extension of working time (particularly important in the production of growth-associated products), (2) controlled conditions for the provision of substrates during fermentation and (3) control over the production of by-products, or catabolite repression effects, due to limited provision of only those substrates which are solely required for product formation.
     In fed-batch cultivation, feeding strategy is the most important factor in success of the process. Different feeding strategies including constant-rate feeding, stepwise increase of the feeding rate, and exponential feeding have been used to obtain high cell densities in fed-batch cultures (Shiloach and Fass, 2005; Lee, 1996). In constant-rate feeding, concentrated nutrients are fed into the bioreactor at a predetermined rate. Because of the increase in culture volume and cell concentration in the bioreactor, the specific growth rate continuously decreases, and the increase in cell concentration slows down over time (Jensen and Carlsen, 1990). Variable feeding rates can be controlled with feedback or without feedback. The stepwise (or gradual) increase of the feeding rate can enhance cell growth by supplying more nutrients at higher cell concentrations (Jensen and Carlsen, 1990; Konstantinov et al., 1990). Cells can grow exponentially during the entire culture period if the feed rate of the growth-limiting substrate is increased in proportion to growth (Shiloach and Fass, 2005; Yee and Blanch, 1993; Strandberg and Enfors, 1991). The exponential-feeding method has been developed to allow cells to grow at constant or variable specific growth rates; it also provides the advantage that acetate production, a serious problem associated with the process, can be minimized by controlling the specific growth rate below the critical value of acetate formation (Table 3). Exponential feeding is a simple but efficient method that has been successfully used for high cell density cultivation of several non-recombinant and recombinant microorganisms; the specific growth rate is usually maintained between attainable maximum and minimum values. Maintaining the specific growth rate at an appropriate range can provide a desirable metabolic condition and results in maximum productivity (Babaeipour et al., 2007). Therefore, exponential feeding can be used as a convenient method to avoid by-product formation and to obtain maximum attainable cell density (Shiloach and Fass, 2005; Khalilzadeh et al., 2004 and 2003; Tabandeh et al., 2004; Thuesen et al., 2003; Lee, 1996; Yee and Blanch, 1993) but, the details of such feeding are still a matter of debate and new researches aim at optimizing the feeding method (Babaeipour et al., 2008; Bahrami et al., 2008; Ting et al., 2008).
      In addition to conventional fed-batch processes, there are some modified fed-batch cultivation techniques, mentioned below, which use special strategies to control the process.
4.2. Two stage, cyclic fed-batch process
Two stages, cyclic fed-batch process is a modified fed-batch process that entails transfer of a portion of the whole fermentation broth from the growth stage to the production stage while leaving a smaller fraction of the broth for continued cell growth in the growth stage. The volume of broth in the growth stage can then be replenished to its pre-transfer volume at a predetermined optimal rate while induction of gene expression and production are taking place in the production stage. The optimal process conditions in the production stage, such as pH, temperature, cell growth rate and medium composition can be controlled and maintained independently from the optimal conditions in the growth stage (Chang et al. 1998; Curless et al. 1991). Chang et al. (1998) obtained a two fold increase in volumetric productivity of rice α-amylase productivity by the yeast Yarrowia lipolytica SMY2 in comparision with a conventional fed-batch process. Choi et al. (2001) used a two-stage fed-batch process for the production of human granulocyte-colony stimulating factor. They optimized the pre-induction growth rate and the feeding strategy during the production stage. Genetic stability of the recombinant strain and the design of optimal media for growth and production stages are also critically important to a successful implementation of the two-stage, cyclic fed-batch process for production of heterologous protein and when working in large scale. Thus the risk of contamination and economical concerns will also become an issue.

4.3. Temperature-limited fed-batch (TLFB) process
The temperature-limited fed-batch process is a technique where the oxygen consumption rate is controlled by a gradually declining temperature profile rather than a growth-limiting glucose-feeding profile. Two mechanisms that may contribute to the much higher accumulation of product in the TLFB process are:       1) reduced proteolysis due to lower temperature,         2) reduced proteolysis due to lower cell death and protease release to the medium (Jahic et al., 2003).
     In E. coli cultures, this method has been proved to prevent an extensive release of endotoxins, i.e. lipopolysaccharides, which occur in glucose-limited fed-batch processes at specific growth rates below 0.1 h-1 (Svensson et al. 2005; Han and Zhong, 2003). This technique stabilizes the cell membrane towards osmotic shock which results in reduced contamination of the considered periplasmic protein extract with cytoplasmic proteins and DNA (Svensson et al., 2005).
      Mare et al. (2005) used a cultivation strategy combining the advantages of temperature-limited fed-batch and probing feeding control. The temperature was decreased to lower the O2 demand and the growth rate. A mid-ranging controller structure was used to manipulate the temperature and the stirrer speed to control the dissolved O2 tension. The probing feeding strategy is changed when the maximum stirrer speed is reached to obtain a slight excess of glucose. The resulting strategy thus limits the growth rate without the risk of acetate accumulation. A 20% increase in cell mass was achieved and the usual decrease in specific enzyme activity normally observed during the late production phase diminished with the new technique.

4.4. A-stat
The A-stat technique is a combination of continuous and fed-batch techniques (Paalme et al., 1995; Paalme and Vilu, 1992). It is basically a continuous culture with a smooth change of the desired growth rate. At first, like in a chemostat, a steady-state culture is obtained. After that, the computer controlled smooth change of dilution rate, while keeping its time derivative constant, is started and continued up to almost complete washout. This technique showed to be a powerful technique for the quantitative study of cell physiology, being at the same time considerably less time consuming and more informative than the conventional chemostat. Also, cultures seem to react better to a smooth rather than an abrupt change in the dilution rate (Paalme et al., 1997; Paalme et al., 1995). However, the system is more suitable for academic purposes and no reports about using this system in industry have been reported to date.

4.5. Dialysis fermentation
Dialysis fermentation is a way to overcome the inhibitory effect of acetate and other nutrients and to obtain high cell density growth. Dialysis is defined as the separation of solute molecules by their unequal diffusion through a semi-permeable membrane based on a concentration gradient. Two configurations of vessel arrangement as mentioned by Shiloach and Fass (2005) were proposed for dialysis reactors: 1) two-vessel reactor consisting of a culture reactor that had  a medium reservoir connected by a dialysis device; 2) a single-vessel dialysis reactor consisting of two chambers separated by a dialysis membrane. The single vessel arrangement is less preferable because it is difficult to sterilize and sensitive to mechanical stress and oxygen limitation (Fuchs et al., 2002; Markl et al., 1993). The highest cell density recorded by membrane dialysis reactors is 190 g/l for E. coli (Nakano et al., 1997). Because of successful high cell density cultivations of E. coli in a laboratory dialysis reactor, a scale-up of the process was investigated by Fuchs et al. (2002). Seeking to provide sufficient membrane area for dialysis in a technical scale fermentor, they used an external membrane module, which was also applied for oxygen supply to the culture in the external loop. Cell densities exceeding 190 g/l, previously obtained in laboratory dialysis fermentation, were also produced with external dialysis modules. Protein concentration in a 300-L reactor was increased to 3.8-fold of industrial fed-batch-fermentations. However, despite the promising results obtained in this study, no further reports about the academic or industrial usage of this technique for HCDC have been reported to date.

4.6. Pressurized cultivation
Matsui et al. (2006) showed that an air-pressurized culture is able to meet the high demand for oxygen in the HCDC of E. coli Carbon dioxide generated by the cells under increased pressure was inhibitory and as a result, cellular growth stopped in the air-pressurized culture at a constant mass flow rate. Increasing the flow rate along with the pressure in the reactor enabled the E. coli cells to grow to 130 (non-recombinant) and 104 (recombinant) g/l due to the release of the CO2. In addition, the specific activity of the product, tryptophan synthase, was increased.

4.7. Perfusion techniques
The basic characteristics of perfusion systems are constant medium flow, cell retention and in some cases, selective removal of dead cells.  Cell retention is usually achieved by membranes or screens, or by a centrifuge capable of selective cell removal. Perfusion systems are most often used for animal cell culture. Advantages and disadvantages of using this technique are shown in Table 4.
      Kiy et al. (1996) by continuous exchange (at an optimized perfusion rate) of the medium, after an initial batch phase, obtained cell densities and enzyme activity,  20 and 50 times, respectively higher than standard batch fermentations of Tetrahymena thermophila. Scheidgen-Kleyboldt et al. (2003) applied the same strategy for producing hydrolytic enzymes by continuous high cell density cultivation of Colpidium campylum. Yang et al. (2000) increased the volumetric antibody productivity by using a “controlled-fed perfusion” approach, nearly twofold over the p

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