Continuous chemostat

Estimation Procedure for Fed-Batch Continuous (Chemostat) Experiments... 

Much effort has also been directed toward the development of fully continuous chemostat-type fermentations in which a tank produces a steady stream of enzymes. In this type of operation, product-containing fermentation broth is drawn off while new media is fed to the tank to maintain a constant volume. Reasonable successes with this method have been reported in the biopharmaceutical industry. However, similar examples of successful application of this technique have not yet appeared in the industrial biotechnology literature. 

Continuous (Chemostat) Perfusion Cell Culture System [8]... 

To attain consistent and stable production of FVIII from CHO cells adapted to culture in a protein-free medium, continuous (chemostat) perfusion is used in the bioreactor culture system. An important advantage of the system is that the culture conditions can be continuously controlled, monitored, and optimized. 

When the cells in the largest bioreactor have reached an optimum density, continuous (chemostat) perfusion culture is begun. Fresh medium is added, and rFVlll-containing conditioned medium is removed at the same rate. This continuous perfusion culture is maintained for several weeks. Monitoring of culture conditions, cell growth, and microbial sterility continues and provides consistency and stability of cell density and expression of rAHF-PFM over time. The conditioned medium is filtered and rFVIII is then purified. 

Process validation studies are performed during bioreactor build-up and continuous (chemostat) cell culture. CHO cell culture conditions (e.g., viability, density, sterility) are monitored carefully during each production cycle. The parameters developed for chemostat cell culture provide consistency and stabihty of both cell density and expression of rAHF-PFM. 

Continuous (chemostat) perfusion cell 430 Contract manufacturing... 

There ate different approaches and pathways for the synthesis of PHAs. Zimm et al. [14] dis-tingitished four biosynthethic approaches to produce PHA in vitro via PHA-pofymerase catalysed polymerization, and in vivo with batch, fed-batch, and continuous (chemostat) crrltures. 

The multi-stream multi-stage system is a valuable means for obtaining steady-state growth when, in a simple chemostat, the steady-state is unstable eg when the growth-limiting substrate is also a growth inhibitor. This system can also be used to achieve stable conditions with maximum growth rate, an achievement that is impossible in a simple chemostat (substrate-limited continuous culture). 

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. 

Fig. 5.3. Schematic diagram of continuous culture with control units in a constant volume chemostat.

The continuous cultures of chemostat and biostat systems have the following criteria ... 

The material balance for cells in a continuous culture chemostat is defined as ... 

Continuous Stirred Tanks Without Biomass Recycle. The chemostat without biomass recycle is a classic CSTR. The reactor is started in the batch mode. 

In this case we assume that we know the dilution rate (D-F/V) precisely as a function of time. In a chemostat D is often constant since the feed flowrate and the volume are kept constant. In a fed-batch culture the volume is continuously increasing. The dilution rate generally varies with respect to time although it could also be kept constant if the operator provides an exponentially varying feeding rate. 

As a third example let us consider the growth kinetics in a chemostat used by Kalogerakis (1984) to evaluate sequential design procedures for model discrimination in dynamic systems. We consider the following four kinetic models for biomass growth and substrate utilization in the continuous baker s yeast fermentation. 

Figure 17.6 Dialyzed Chemostat Monoclonal antibody concentration (raw and smoothed measurements) during initial batch start-up and subsequent dialyzed continuous operation with a dialysis flow rate of 5 L/d. [reprinted from the Journal of Biotechnology Bioengineering with permission from J. Wiley],...

At time t=212 h the continuous feeding was initiated at 5 L/d corresponding to a dilution rate of 0.45 d . Soon after continuous feeding started, a sharp increase in the viability was observed as a result of physically removing dead cells that had accumulated in the bioreactor. The viable cell density also increased as a result of the initiation of direct feeding. At time t 550 h a steady state appeared to have been reached as judged by the stability of the viable cell density and viability for a period of at least 4 days. Linardos et al. (1992) used the steady state measurements to analyze the dialyzed chemostat. Our objective here is to use the techniques developed in Chapter 7 to determine the specific monoclonal antibody production rate in the period 212 to 570 h where an oscillatory behavior of the MAb titer is observed and examine whether it differs from the value computed during the start-up phase. 

Figure 17.13 Dialyzed Chemostat Estimated values of specific MAb production rate versus time during the period of continuous operation. A 5% standard error in the raw data was assumed for data smoothing.

Carbon limiting is also used to encourage enzyme induction, place the population under selective pressure for degradation of recalcitrant substrates, and favor the simultaneous rather than sequential metabolism of a mixed carbon source.33 Carbon-limiting conditions can be achieved either through continuous culture (chemostat) or through a fed batch reaction. 

The unlimited growth-related type of poly(3HB) synthesis is most suited to one-stage continuous cultivation. This can be performed in a chemostat with carbon substrate limitation. The poly(3HB) content of the cells can be further... 

Continuous culture systems have been widely used to culture microorganisms for industrial and research purposes (Kubitschek 1970 Tempest 1970 Veldkamp 1976 Rhee 1980). In recent years, these culture techniques have found their way into the bioassay methods of ecotoxicology and allelopathy (Rhee 1980). The early development of a continuous culture system can be traced back to the work of Novik and Szilard (1950 a,b) who developed the first chemostat. In a continuous culture system, nutrients are supplied to the cell culture at a constant rate and to maintain a constant volume, an equal volume of cell culture is removed. This allows the cell population to reach a steady state, where the growth rate and the total number of cells/ml of culture remains constant. Two kind of continuous culture systems can be distinguished turbidostat and chemostat. ... 

Rhodobacter capsulatus B10 Chemostat wih ammonium limitation, lactate, continuous argon flow (100 ml/min), light saturation 88 0.115 97 Tsygankov et al., 1998... 

Reaction times of fermentation range from a few hours to several days. Batch processes are common, but continuous stirred tanks also are used either singly or in stages. A continuous stirred tank reactor (CSTR) also is called a chemostat. Figure 8.4 is a schematic of a fermentor with representative dimensions from the literature. 

The term chemostat refers to a tank fermentation which is operated continuously. This bioreactor mode of operation normally involves sterile feed (Xo=0), constant volume and steady state conditions, meaning that dV/dt=0, d(VSd/ dt=0, d VX1)/dt=0. 

A continuous fermenter with sterile feed is referred to as a chemostat. For constant volume operation, the inlet volumetric flow rate is equal to that at the output. With this model chemostat start-up, resultant steady state behaviour and cell washout phenomena are easily investigated by simulation. 

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]. 

Many reviews and several books [61,62] have appeared on the theoretical and experimental aspects of the continuous, stirred tank reactor - the so-called chemostat. Properties of the chemostat are not discussed here. The concentrations of the reagents and products can not be calculated by the algebraic equations obtained for steady-state conditions, when ji = D (the left-hand sides of Eqs. 27-29 are equal to zero), because of the double-substrate-limitation model (Eq. 26) used. These values were obtained from the time course of the concentrations obtained by simulation of the fermentation. It was assumed that the dispersed organic phase remains in the reactor and the dispersed phase holdup does not change during the process. The inlet liquid phase does not contain either organic phase or biomass. 

Similar in principle to chemostat configuration except the cells are retained within the fermenter and roller bottles using inline or external cell separation devices. The hollow fiber filter allows continuous separation of cells from tissue culture fluid containing products. In this configuration, the recombinant product is often designed to be excreted into medium allowing it to be collected in perfusate. 

Fermentation and alternative production techniques, such as roller bottles, can be carried out in four different ways. They are (1) batch process, (2) fed-batch process, (3) chemostat process, and (4) perfusion process. Batch and fed-batch processes require termination of cell growth while chemostat and perfusion processes allow continuous cell cultivation. 

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