Batch-culture bioreactors

Fed-batch culture A cell cultivation technique in which one or more nutrients are supplied to the bioreactor in a given sequence during the growth or bioconversion process while the products remain in the vessel until the end of the run. 

The production-scale fermentation unit, with a projected annual capacity of over50,000 tonnes was fully commissioned in 1980. The bioreactor (Figure 4.8) is 60 m high, with a 7 m base diameter and working volume 1,500 m3. There are two downcomers and cooling bundles at the base. Initial sterilisation is with saturated steam at 140°C followed by displacement with heat sterilised water. Air and ammonia are filter sterilised as a mixture, methanol filter sterilised and other nutrients heat sterilised. Methanol is added through many nozzles, placed two per square metre. For start-up, 20 litres of inoculum is used and the system is operated as a batch culture for about 30 h. After this time the system is operated as a chemostat continuous culture, with methanol limitation, at 37°C and pH 6.7. Run lengths are normally 100 days, with contamination the usual cause of failure. 

Suspension systems can be operated in different modes batch, fed-batch, chemostat, and perfusion (Fig. 1). These operation modes differ basically in the way nutrient supply and metabolite removal are accomplished, which in turn determines cell concentration, product titer and volumetric productivity that can be achieved [8]. The intrinsic limitation of batch processes, where cells are exposed to a constantly changing environment, limits full expression of growth and metabolic potentials. This aspect is partially overcome in fed-batch cultures, where a special feeding strategy prolonges the culture and allows an increase in cell concentration to be achieved. In perfusion and chemostat processes nutrients are continuously fed to the bioreactor, while the same amount of spent medium is withdrawn. However, in perfusion cultures the cells are retained within the bioreactor, as opposed to continuous-flow culture (chemostat), which washes cells out with the withdrawn medium [9]. 

Figure 13.1 Schematic representation of a fully instrumented bioreactor system for an industrial-scale fed-batch culture (solid line - fluid flow and air flow dotted line - ana-log/digital signals).

Techne sell a bioreactor for growing cells in suspension in volumes up to 3 1 (Fig. 3.10) and New Brunswick has a model which will take 5 1. The Techne bioreactor maintains the cells or microcarriers in suspension with a floating stirrer but such a mechanism becomes increasingly less efficient up to 20 1. pH, p02 and temperature are monitored and controlled and the reactor can be used for batch cultures or in continuous mode. 

Biosensors. Sensors are required to adequately monitor bioreactor performance. Ideally, one would like to have online sensors to minimize the number of samples to be taken from the bioreactor and to automate the bioreactor process. Most bioreactors have autoclavable pH and dissolved oxygen (D.O.) electrodes as online sensors, and use offline detectors to measure other critical parameters such as glucose and glutamine concentration, cell density, and carbon dioxide partial pressure (pC02). An online fiber-optic-based pC02 sensor is commercially available and appears to be robust.37 Probes are also commercially available that determine viable cell density by measuring the capacitance of a cell suspension. Data from perfusion and batch cultures indicate that these probes are reasonably accurate at cell concentrations greater than 0.5 X 106 cells/mL.38,39... 

Cyanobacteria can be fairly cultured under laboratory conditions, ranging from small batch cultures to high-volume bioreactors (Marxen et al, 2005 Richmond et al, 1993), but they can also be grown outdoors, in tubular bioreactors or large ponds. For example, the nontoxic species Arthrospira platensis (also known as Spirulina) has been cultured in... 

For SCP production continuous rather than batch cultures are preferred because continuous culture systems give higher outputs for a bioreactor of given size ... 

Gluco-oligosaccharides (GlcOS) can be made by the action of an enzyme, dextran dextrinase produced by Gluconobacter oxydans on maltodextrins. They have been made in whole-cell bioreactors [104] and evaluated in fecal batch cultures [105] and in three-stage gut models [106]. Mountzouris et al. [104] used methylation analysis to determine the linkages of... 

Batch cultures are the simplest to perform, either in shake flasks or small bioreactors. However, as the concentration of all the nutrients and metabolites changes simultaneously with time, it is relatively difficult to assess the precise influence of a single medium component on cell kinetics... 

The batch culture was carried out in a bioreactor (B. Braun) with a working volume of 11. Forty-eight hours after the last Roux bottle inoculation, the cells were inoculated at about 2 xlO viable cells H in the bioreactor. 

Cells are inoculated, grown up to a desired cell or product concentration and harvested. The duration of batch culture depends on inoculation density, cell line and characteristics such as growth rate and antibody-production kinetics of the cell line. After every run the bioreactor must be cleaned and autoclaved for the next run. 

A new bioreactor, consisting of a liquid-gas two-phase system, was devised for utilization with berberine-secreting T. minus cells immobilized in calcium alginate beads. The cells were alternately soaked in medium, and then exposed to air, and the maximum yield of berberine production was 875 mg/L. The berberine productivity of immobilized cells was as high as that of freely suspended cells under such conditions of batch culture. In addition, the rate of production of berberine by the immobilized cells remained constant at a high value (50 mg/L/day) for 60 days of semicontinuous culture, achieved by the renewal of medium at 10 day intervals [154]. 

Bhushan and Joshi (2006) used apple pomace extract as a carbon source in an aerobic-fed batch culture for the production of baker s yeast. The fermentable sugar concentration in the bioreactor was regulated at 1-2%, and a biomass yield of 0.48 g/g of sugar was obtained. Interestingly, the dough-raising capacity of the baker s yeast grown on the apple pomace extract was apparently the same as that of commercial yeast. The use of apple pomace extract as substrate is a useful alternative to molasses, traditionally used as a carbon source for baker s yeast production. 

Feal, Advanced Transport Phenomena Fluid Mechanics and Convective Transport Fim and Shin, Fed-Batch Cultures Fundamentals, Modeling, Optimization, and Control of Semi-Batch Bioreactors... 

As the consequence of this, the metabolic waste products are initially distributed evenly in the bulk medium but accumulated with time in batch culture systems. Furthermore, as a part of normal culture operations, the vessels are taken out from the incubator for microscopic observations, which can cause movement of the bulk medium and formation of temperature gradients, consequently contributing to forced and natural convection, respectively. In industrial-scale bioreactors, the applied perfusion creates forced convection, which is the primary mode of mass transfer. The common feature of all the conventional systems, laboratory- or industrial scale, is that due to the long distances and the large medium volume in comparison with the cell volume, the significance of convection is pronounced. Consequently, mass transfer in such systems takes place in all directions (x-, y- and z-direction) with a comparable magnitude, as is schematically shown in Fig. 2a. 

Fig. 3.12 (A) Cell and (B) antibody profiles for batch (open symbols) and fed-batch cultures for a PER.C5 cell-line expressing antibody A. The data represent an average of eight 2-L bioreactor runs.

---