Chapter 1 Applications of Metabolic Engineering in the Production of
7.4 Types of Fermenters
Laboratory-scale submerged fermentations are carried out in shaker flasks, and large-scale fermentations are carried out in glass or stainless-steel tank fermenters.
The prerequisites for a good fermentation vessel are that it must be inexpensive, not allow contamination of the contents, be nontoxic to the microorganism used for the process, be easy to sterilize, be easy to operate, be robust and reliable, be leak proof, allow visual monitoring of the fermentation process, and allow sampling. The scale-up of the fermentation process is usually the final step in any research-and-development program, leading to the large-scale industrial manufacture of products by fermentation (Einsele 1978). It needs to be understood that the process of scal-ing up a fermentation system is not simply a matter of increasscal-ing culture and vessel volume, but it is frequently governed by a number of important engineering con-siderations. Therefore, usually a large-scale process does not perform as well as a small-scale laboratory process. It is often observed that the biomass yield and any growth-associated products are often decreased with the scale-up of an aerobic pro-cess (Enfors et al. 2001).
7.4.1 Shake FlaSkS
These are conical vessels made of glass and are available in different sizes. The typical volume of these flasks is 250 mL. Erlenmeyer flasks with a cotton plug are
the classic small-scale liquid fermentation system (Figure 7.1a). When the vessel is round-based and the motion is orbital, mixing is poor. Some modifications in the basic shake-flask system have been made to increase mixing and mass transfer with indentations (baffles) and corners (Figure 7.1b). Baffles have been used mainly to increase the turbulence of mixing to increase the liquid surface area and, therefore, the gas transfer. The upper limit on the volume of small-scale batch fermentations is typically set by the oxygen-uptake requirements of the culture to be fermented.
Generally, the gas transfer rate at the gas–liquid interface is lesser as the volume gets larger. Shaker beds or shaker tables are used to allow oxygen transfer by their continuous rotary motion. Although higher oxygen transfer rates can be achieved with shake flasks than with standing cultures, oxygen transfer limitations will still be unavoidable particularly when trying to achieve high cell densities. The rate of oxygen transfer in shake flasks is dependent on the shaking speed, the liquid volume, and the shake-flask design.
7.4.2 Stirred tank Fermenter
Stirred tank fermenters (STF) are some of the most commonly used fermenter types because of their flexibility (Chisti 2010). They are cylindrical vessels with a motor-driven central shaft with impellers or an agitator to stir the contents in the tank (Figure 7.2). The shaft supports three to four impellers placed approximately one impeller-diameter apart (Chisti 2010). The agitator may be top driven or bot-tom driven, depending on the scale of operation and other operational aspects.
The top-entry–stirrer (agitator) model is most commonly used because it has many advantages, such as ease of operation, reliability, and robustness, and the bottom-entry–stirrer (agitator) model is rarely used. The fermenter has an aspect (working height to weight) ratio of 2:1 and 6:1. 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 fermenter. There are four equally spaced vertical baffles that extend from near the walls into the vessel to avoid vortex forma-tion and improve mixing. The STR offers the advantages of high oxygen transfer rates required for high biomass productivity coupled with low investment and oper-ating costs, which form the basis for any successful aerobic fermentation process.
Laboratory-scale STRs are made of borosilicate glass with a stainless-steel lid and top-entry stirrer. The typical volume of these fermenters is 1 to 100 L. Stainless-steel fermenters are also used in laboratories and have special requirements. They should be made of high-grade stainless steel, have an internal surface that is polished
(a) (b)
FIgure 7.1 Shake flasks. (a) Standard (Erlenmeyer flask) and (b) with baffles.
to reduce adhesion of the contents to the walls of the fermenter, and have joints that are smooth and free from pinholes.
Traditionally, fermentation of grape juice was conducted in large wooden barrels or concrete tanks, but most modern wineries now use sophisticated stainless-steel tanks with temperature control and various other features for process management (Divies 1993).
In a study by Ali et al. (2002), a laboratory-scale stirred fermenter of 15-L capacity with a working volume of 9 L was used for citric acid production using Aspergillus niger GCBT7. Batch fermentation was employed in a New Brunswick bioreactor for obtaining probiotic biomass from Lactobacillus plantarum BS1 and BS3 strains. The conditions of fermentation were 37°C, pH 5.5, stir 100 rpm once at every 12 h of fermentation in order to homogenize the medium for 15 min (Vamanu 2009). The production of α-amylase by Bacillus amyloliquefaciens was performed in 5-L stirred-tank bioreactor (Biostat B-5, B. Braun Biotech-Sartorius, Melsungen, Germany). It is a baffled cylindrical acrylic vessel with a working volume of 3 L and a working volume to space ratio of 1:1.66, having an internal diameter of 160 mm and a height of 250 mm with dual impellers mounted on the shaft. The baffles with
Inlet ports for media, antifoam, etc. Exit air port
Baffles
Probes
Impeller
Air sparger
Drain line Inlet air port
FIgure 7.2 Typical stirred-tank fermenter.
a width of 12 mm were placed perpendicular to the vessel. The system was equipped with a six-bladed Rushton turbine impeller for agitation with a diameter of 64 mm, a blade height of 13 mm, and a width of 19 mm. The spacing between the impel-lers was maintained at 110 mm, and the lower impeller was located at a distance of 80 mm from the bottom of the vessel. The sparger was located at a distance of 5 mm from the bottom of the vessel through which air was sparged to the tank. The ring sparger was 52 mm in diameter and had 16 symmetrically drilled holes 1 mm in diameter. The flow rate of sparged air was fixed at 1.5 vvm. The fermentation was carried out at 37°C and monitored by a temperature probe that was controlled by circulating the chilled water. Foaming in the fermentation broth was monitored by a ceramic-coated antifoam probe, and coconut oil was used as an antifoaming agent. The dissolved oxygen (DO) was maintained at a 100% saturation level, which was continuously monitored by a sterilized polarographic electrode (Mettler-Toledo InPro 6000 Series, Greifensee, Switzerland). The experiments indicated a require-ment of high rates of aeration to enhance the enzyme yield (Gangadharan et al. 2011).
The production of rennet was carried out in submerged fermentation by Rhizomucor miehei NRRL-3420 using two types of media for 40 h at 380 rpm, 1 vvm aeration, and 30 ± 1°C (De Lima et al. 2008).
7.4.3 tower Fermenter
Tower fermenters are modified stirred-tank reactors, which are simple in design and easy to construct. They consist of a long, cylindrical vessel with an inlet at the bot-tom, an exhaust at the top, and a jacket to control the temperature. They do not require agitation; hence, there are no shafts, impellers, or blades. Tower fermenters are used for continuous fermentation of beer, yeast, and Single Cell Protein (SCP). In brewing, cylindroconical fermenters have become the vessels of choice since the 1970s (Maule 1986). Traditionally, open rectangular tanks of 2–3 m depth were used (Hough et al.
1982). An enclosed vessel reduces the risk of contamination over open vessels and also helps to capture the carbon dioxide produced. The cylindroconical fermenter has a conical lower section that facilitates the sedimentation and recovery of bottom yeast, which settles out late in the fermentation process. The shape also encourages a vigor-ous mixing of yeast cells in the fermentation wort, resulting in a faster fermentation.
No aeration is required as the gas produced by the yeast cells contributes to mixing.
7.4.4 airliFt Fermenter
An airlift fermenter is a cylindrical fermentation vessel in which the cells are mixed by air introduced at the base of the vessel and that rises through the column of cul-ture medium. The working aspect ratio of these fermenters is six (height/weight of 6:1) or more. These fermenters do not have mechanical agitation systems (motor, shaft, impeller blades), but the contents are agitated by injecting air from the bottom.
The cell suspension circulates around the column as a consequence of the gradient of air bubbles in different parts of the reactor. Thus, the fluid of volume is divided into two interconnected zones by means of a baffle or draft tube. Only one of the two zones is sparged with air or gas. The sparged zone is known as the riser, and
the zone that receives no gas is the downcomer (Chisti and Moo-Young 2001). These come in two models: the internal-loop and external-loop designs. In the internal-loop configuration (Figure 7.3a), the aerated riser and the unaerated downcomer are con-tained in the same shell, and in the external-loop design (Figure 7.3b), the riser and the downcomer are separate tubes linked near the top and the bottom (Chisti 1999).
The external loop design has not been used frequently in industry.
Sterile atmospheric air is used if the microorganisms are aerobic and inert gas is used if the microorganisms are anaerobic. This is a gentle method of mixing the con-tents and is most suitable for fermentation of molds because the mechanical agitation produces high shearing stress that may damage the cells. An airlift fermenter differs from bubble column bioreactors by the presence of a draft tube, which provides bet-ter mass and heat transfer efficiencies and low shear conditions. Other advantages include increased oxygen solubility and easier maintenance of sterility. The major disadvantages of airlift fermenters are high capital cost, high energy requirements, excessive foaming, and cell damage resulting from bubbles bursting.
Airlift fermenters have been used for citric acid production with 900 m3 volume using A. niger. The fermenters must be resistant to acidity and are made of stainless steel because ordinary steel can be dissolved at pH 1–2, inhibiting the fermentation.
In fermenters with a capacity of less than 1 m3, because of increased surface-to-volume ratio, corrosion becomes significant, and even steel chambers must be pro-tected with a layer of plastic material. An aeration rate of 0.2–1 vvm is often used during the production phase to avoid dissolved oxygen levels lower than 20%–25%
of the saturation (Honecker et al. 1989). The production of phytase and glucoamylase by Sporotrichum thermophile (Singh and Satyanarayana 2008) and Thermomucor
(a) (b)
FIgure 7.3 Airlift fermenter (a) with internal draft tube and (b) with external draft tube.
indicae-seudaticae (Kumar and Satyanarayana 2007) have been recently reported in the airlift fermenter, respectively.
Quorn is the leading brand of mock meat mycoprotein food product in the United Kingdom and Ireland. The mycoprotein used to produce Quorn is extracted from a fungus, Fusarium graminearum, which is grown in a very large airlift fermenter in a continuous-culture mode. F. graminearum A315 biomass is produced in an airlift or pressure-cycle fermenter at Billingham. A continuous-flow culture system is cho-sen for the process because the growth conditions in such cultures, unlike those in batch cultures, can be maintained as constant throughout the production phase and because much higher productivities can be achieved in continuous culture than in batch culture (Trinci 1994).
The technology of creating SCP from methanol has been well studied, and the most advanced process belongs to Imperial Chemical Industries (ICI, UK). The fer-mentation was carried out in a big airlift fermenter with the bacterium Methylophilus methylotrophus. This organism was selected from among other methanol utilized after screening tests for pathogenicity and toxicity. As a nitrogen source, ammonia was used and the product was named Pruteen. The pruteen contained 72% crude protein and was marketed as feed that contains a source of energy, vitamins, and minerals as well as a highly balanced protein source. The methionine and lysine content of Pruteen compared very favorably with whitefish meal. The ICI commis-sioned a 60,000 ton/year plant utilizing the single largest fermenter in the world (2 × 10,000,000 L) (Nasseri et al. 2011).
7.4.5 BuBBle Column reaCtor
A bubble column reactor is basically a cylindrical vessel with a gas distributor at the bottom. It was first applied by Helmut Gerstenberg. The gas is sparged in the form of bubbles into either a liquid phase or a liquid–solid suspension (Figure 7.4). The
FIgure 7.4 Bubble column reactor.