The fermentor is the heart of any biochemical process in which microbial, mammalian, or plant cell systems are employed for the economic production of fermentation products. A properly designed fermentor should be used to provide an aseptic, controlled environment to facilitate optimal growth and product formation of a particular cell system. In view of the broad scope of the fermentor for cultivation of microbial, mammalian, or plant cells, it is more commonly referred to as a bioreactor; this term will be used because of its global application.
The efficiency of performance of a bioreactor is dependent on concentration of biomass, maintenance of aseptic conditions, efficient mass and heat transfer, and operation at optimum process conditions. Bioreactors can be classified into three groups based on the type of biochemical process employed (8):
1. Bioreactor with no agitation and aeration (anaerobic processes, e.g., production of wine and beer)
2. Bioreactor with agitation and aeration (aerobic submerged fermentation processes, e.g., production of citric acid and penicillin)
3. Bioreactor with aeration, but no agitation (aerobic solid state fermentation processes, e.g., production of food enzymes)
However, in industrial practice, bioreactors are distinguished by their configuration and design. The common modes of bioreactor configurations are discussed below.
The most commonly used bioreactor for industrial applications is the conventional stirred tank reactor (STR). The STR offers the advantages of high oxygen transfer rates required for high biomass productivity coupled with low investment and operating costs, which form the basis for any successful aerobic fermentation process. A schematic of a stirred tank bioreactor is shown in Figure 3.1. STRs typically have height to diameter ratios of 1:3 to 1:6. The agitator may be top driven or bottom driven depending on the scale of operation and other operational aspects. The choice of impeller depends on the physical and biological characteristics of the fermentation broth. Usually, a ring-type sparger with perforations is used to supply air to the fermentor. Baffles are provided to avoid vortex formation and improve mixing.
Most fermentation processes use complex medium ingredients like corn steep liquor, molasses, and soybean flour as inexpensive nutritional sources (for carbon and nitrogen), supplemented with vital growth factors (amino acids, proteins, and vitamins) (9). The high turbulence imparted by the impellers in an STR can result in foaming due to the presence of proteinaceous substrates. Although chemical antifoaming agents (silicone or polypropylene glycol) can be added to control the foam, these can have detrimental effects on microbial growth and product recovery. In order to overcome this, mechanical methods of foam suppression such as rakes on the stirrer shaft mounted above the critical
level have also been adopted. The emphasis on asepsis of the bioreactor, right from the end of the sterilization cycle to the end of the fermentation, has led to the maintenance of a minimum positive pressure in the fermentor to ensure sterility. A most important aspect of sterility is the point of contact between agitator shaft and vessel, which can be effectively sealed with a lubricated double mechanical seal. The sampling devices and injection ports must be contained in steam sterilizable closures.
For fermentations that have low shear and energy requirements, an air lift reactor can be useful. The amount of air required for the fermentation process is usually sufficient to act as the sole source of liquid mixing. In this process, air pumped from the bottom of the reactor creates buoyant, bubbles, which exert a drag on the surrounding fluid. A riser and a "down comer" inside the bioreactor impose a circulating fluid pattern of movement, which provides for oxygenation and mixing of the fermentation broth. The bottlenecks associated with large scale air lift bioreactors are inadequate sterilization, higher capital investment, and aeration requirements. Since mixing in an air lift is solely caused by aeration, the power required for fluid circulation and dispersion can be higher than that needed by an agitator in a stirred tank bioreactor. Air lift bioreactors have also been used for highly viscous fermentations, but the volumetric mass transfer coefficients are predictably low (10). An air lift bioreactor equipped with a draft tube results in better liquid circulation and larger gas to liquid interfacial areas, and gives higher mixing efficiency and oxygen transfer capability (11). A significant example of an air lift bioreactor that has been successfully scaled up to very large commercial levels is the ICI pressure cycle fermentor (12). In this process, methanol is used as substrate to produce single cell protein (SCP) using Methylophilus methylotrophus at a production capacity of 60,000 tons per year.
A three phase internal loop gas lift bioreactor was used for experimental studies on beer fermentation (13). This reactor has the advantages of high solid loading and better mass transfer properties than fluidized beds. Low density carriers of yeast cells, such as alginate and carrageenan gel particles, are typically used in three phase gas lift reactors for beer fermentations. The positive attributes of gas lift bioreactors are simple construction, low risk of contamination, easy adjustment and control of the operational parameters, and simple capacity enlargement (14,15). Research studies on air lift bioreactors have been carried out for the production of itaconic acid (16) and ^-polylysine (17).
In the last few decades, there has been a significant increase reported in the application of fluidized bed reactor systems. These have been mainly used for cells that have been immobilized onto particulate matter. This has the advantage that a high density of particles can be used, and that the flow velocity required for the fluidization can be achieved independently of the reactor throughput.
The main advantages of a fluidized bioreactor system as observed in ethanol production from S.cerevisiae (18) are superior mass and heat transfer characteristics, very good mixing between the three phases, relatively low energy requirements, and low shear rates (which makes a fluidized bed reactor suitable also for shear sensitive cells such as mammalian and plant cells).
Fluidized bed reactors have been used with cells adsorbed inside the carrier, made either of glass or of ceramics (19-21). The upward feed flow rate in a fluidized bed biore-actor is high enough to provide fluidization of carriers, resulting in improved mixing properties and medium distribution; but this can also induce carrier abrasion and damage.
In addition, fluidization of glass and ceramic carriers may require high medium flow rates that could result in higher pumping costs and eventually cell leakage. Gas liquid solid fluidized bed bioreactors have been employed for production of ligninolytic enzymes (22), treatment of wastewater from refineries (23), and raw wastewater (24).
The majority of mammalian cells need a solid surface such as a microcarrier or a packed bed upon which to grow. The initial idea of culturing anchorage dependent mammalian cells in microcarriers was developed by van Wezel (25). The growth of cells on microcarrier beads depends directly on the surface available for growth up to the point where the microcarrier particles reach sufficient concentration to inhibit the cells and thus reduce cell yield. The microcarriers should have a density between 1.02 and 1.10 kg/m3 to enable easy suspension in stirred reactors. The toxicity of the support can cause long lag phases, death of the cells in the early stages of development, and limited cell yields. Microcarrier biore-actor systems have been used for cultivation of human fibroblast cells to produce cell mass (26) and in the production of interferon (27). A great advantage of microcarriers is the high surface area for cell growth provided under low shear conditions, while still allowing conventional fermentor equipment to be used. However, bead to bead and bead to impeller collisions, and hydrodynamic shear forces, may cause reduced viability.
Membrane bioreactors comprising hollow fiber systems have been developed and tested for the growth of mammalian and plant cells, and for the immobilization of bacteria, yeast and enzymes. Hollow fiber reactors have been used in the enzymatic hydrolysis of cellulose (28), penicillin (29), starch (30), hemoglobin (31), protein synthesis (32), and the culture of plant cells (33) and mammalian cells (34).
Hollow fibers can be made from cellulose acetate with a uniform wall matrix, or from acrylic copolymers or polysulphone fibers with asymmetric wall configurations. These hollow fibers have a highly porous surface wall about 70^m thick upon which the cells grow, and a cylindrical lumen of about 200^m in diameter. The surface of the lumen is covered with a thin ultrafiltration layer, which separates the immobilized cells and the lumen. Free diffusion of ions and molecules take place through the ultrafiltration layer, which may have nominal molecular weight cut offs (NMWCO) in the range of 10 kDa to 100 kDa. A hollow fiber bioreactor consists of a cylindrical bundle of a large number of individual fibers, which are held together in a shell and tube-type heat exchanger arrangement. Commercial hollow fiber bioreactor units are available with a luminal surface capacity between 0.01m2 and 1.0m2.
The advantages of using a hollow fiber reactor for microbial systems include high density of cell growth, using a perfusion system for simultaneous separation of product and biomass, and biocatalyst regeneration. However, a major disadvantage is the difficulty in monitoring and controlling the growth and metabolism of the culture. Other process constraints associated with microbial hollow fiber reactors are low oxygen transfer rates at high cell density and blockage, and rupture of the membranes due to excessive growth. The accumulation of toxic products in the hollow fiber might also inhibit the metabolic activity of the cell system. Further, the effect of microbial containment on physiology, long term viability, and productivity remains unclear. The technique has been used in the production of lactic acid (35), conversion of l-histidine and biosynthesis of ^-galactosidase.
Membrane bioreactors have been used extensively for microbial, plant, and animal cell cultivations (36). A high performance membrane bioreactor has been studied for use in ethanol (37) and organic acid fermentation (38). Continuous fermentation in a membrane bioreactor performed at a very high dilution rate enhanced productivity (39). A double vessel membrane bioreactor was reportedly employed for the production of wine from grape juice (40), wherein the low residual sugar level maintained favored higher wine production compared to a single vessel in continuous fermentation. The production of tissue plasminogen activator (tPA) in microfiltration hollow fiber (MFHF) bioreactors for mammalian cell culture has been reported (41).
Microalgae have been used successfully, with high productivity compared to higher plants. The high productivity in these systems is due to the high biomass produced in the bioreac-tor. Microalgae have been used for preparation of vitamins, pigments, antioxidants, and fatty acids, and as feed for aquaculture. The cultivation techniques employed are open systems and closed or semiclosed outdoor photobioreactors. The common photobioreac-tors used are tubular-type and plate-type reactors (42). The cyanobacterium Spirulina platensis has been studied in batch and continuous photobioreactors under varying conditions of incident light energy and nutrient limitations (43).
220.127.116.11.1 Space Bioreactor The transfer of knowledge from conventional biore-actor technology to microscale space bioreactors for cultivation of cells required in space is an emerging field of space life science research and applications. The first space biore-actors were developed and flown at the end of the last century (44). With the development of an international space station, special attention has been focused on the development of life support systems that allow recycling of expendable materials (i.e., water, air), the treatment of waste byproducts (45), and cultivation of microorganisms, mammalian cells, and tissues for food production. The bioreactors normally used on Earth may not be suitable in space for several reasons:
1. Materials presently utilized for the fabrication of a bioreactor are not accept-eable in space for safety and environmental reasons.
2. The space bioreactor equipment has to comply with size, weight, and power requirements.
3. The microgravity conditions in space create a significant hindrance for the operation of a bioreactor due to alteration in the physical factors governing cell sedimentation, nutrient mixing, and byproduct dispersion.
4. Nutrients and oxygen and waste products should be efficiently transported by means of medium exchange, perfusion, or slow mixing, as convection currents are also reduced to near zero.
For these reasons, new types of bioreactors specifically adapted to space investigations had to be developed. Space based bioreactors provide an opportunity to understand how fermentation processes could occur in the absence of gravity in a cell and its surrounding environment. This unique research environment opens new horizons for exploring unconventional bioprocessing techniques (46). Several types of cultivation systems have been designed or are currently under development as given in Table 3.2.
18.104.22.168.2 Tissue Bioreactor Tissue bioreactors are expected to play a dominant role in the production of tissues for healthcare applications. In the twenty-first century,
Characteristics of the different space cultivation systems reported (47).
Dynamic Cell culture System Swiss Bioreactor (SBR 1)
Biokosmos 9 (1989), Shuttle (1992) 2 Shuttle flights (1994,1996)
Cultivation chamber with medium exchange (osmotic pump), no regulation Zero headspace bioreactor for yeast cells, flexible continuous medium exchange (piezo-electric pump), sampling port, flow rate and pressure sensor. pH regulation, online data transfer.
tissue engineering and regenerative medicine are expected to be powerful tools for repairing damaged or diseased tissues and organs, because human donor tissue cannot meet the demand (48). Tissue engineering is the development of biological substitutes to restore, maintain, or improve tissue function (49). A clinically useful bioreactor system will need to be compact and capable of maintaining a large number of cells at relatively high densities over a prolonged period. The cultivation of hepatoma cells used for the production of bioartificial livers has been attempted in tissue bioreactors utilizing fluidized bed and hollow fiber setup (50). With the focus on automation and standardization of tissue manufacture in controlled systems, bioreactors have the potential to reduce production costs, facilitating global use of engineered tissues. The role of bioreactors in processes that are key for the ex vivo engineering of three dimensional tissues based on cells and scaffolds include cell seeding of porous scaffolds, nutrition of cells in resulting constructs, and mechanical stimulation of the developing tissues (51). A new challenge for bioprocess engineering in cell culture involves the development of highly sophisticated cultivation techniques for hematopoietic stem cells in novel bioreactors (52,53). The interdisciplinary research associated with tissue engineering will provide a basis for identifying process conditions required for generation of a specific tissue.
22.214.171.124.3 Nuclear Magnetic Resonance Bioreactor Nuclear Magnetic Resonance (NMR) spectroscopy has been used as a tool for noninvasive, real time studies of metabolic processes of cell suspensions in bioreactors (54). The NMR bioreactor is used for online NMR analysis of metabolic reactions in fermentation processes. One of the critical parameters in the evaluation of bioreactors for product formation is the oxygenation state of the cells, which can be determined using NMR (55). An NMR reactor (manufactured by M/s Bioengineering AG, Switzerland) is an autoclavable, miniaturized stirred vessel with ports to measure pH, pO2, temperature, speed, and aeration, and for the addition of medium. The fully equipped NMR reactor can be inserted into an NMR unit.
126.96.36.199.4 Mass Spectrometer Coupled Bioreactor A minibioreactor with membrane inlet mass spectrometer probe is reported for biological processes with online analysis of volatile compounds such as H2, CH4, O2, N2, CO2, ethanol, and methanol (56). The reactor comprises a small, stirred reaction vessel with a thermocouple, a pH probe, agitation, temperature control, and an option for aeration. The reactor is coupled to the mass spectrometer with the help of silicon rubber or fluorohydrocarbon membranes separating the bioreactor from the high vacuum in the mass spectrometer. Volatile compounds are selectively introduced into the mass spectrometer, where they are quantified according to their mass to charge ratio. Typical measurement ranges are from percent to parts per trillion levels in a few seconds to minutes. This bioreactor offers continuous, sensitive detection of small changes in the concentrations of dissolved gases, permitting fast kinetic measurements and in depth metabolic studies.
188.8.131.52.5 Integrated Bioreactor An integrated bioreactor is aimed at improving the productivity of the fermentation process by integration of fermentation and product recovery, for the continuous removal of a potentially inhibitory product. Two phase partitioning bioreactors have a great scope for increasing the productivity of a bioprocess (57). The concept of the two phase partitioning bioreactor can be applied to controlled delivery of a toxic substrate like phenol or benzene dissolved in an organic phase to a cell containing aqueous phase (58). A laboratory scale bioreactor coupling conventional electrodialy-sis and bipolar membrane electrodialysis was developed for in situ product removal and pH control in lactic acid fermentation (59). This electrokinetic process enabled removal of concentrated lactic acid directly from the bioreactor system, enabling good pH control and reduced end product inhibition of glucose catabolism. A membrane distillation integrated bioreactor for ethanol production using S.cerevisiae resulted in a higher productivity of 5.3 gm ethanol/dm3/h, compared to 2.6 gm ethanol/dm3/h without membrane distillation (60). The morphological and biochemical characteristics of transformed Nicotiana glauca roots demand integrated bioreactors for root growth and product extraction. A novel root tube bioreactor separator was used for the increased alkaloid productivity (61).
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