Fermentation continuous cultivation

A high level of poly(3HB) accumulation is also obtained if the cells are grown under carbon substrate limitation, and the cultivation in the second fermenter is also carried out under carbon limitation. In this case, a substrate flow rate (F2) below that corresponding to the maximum specific poly(3HB) formation rate should be chosen [114]. This cultivation strategy is especially convenient when using toxic substrates like acetic acid. Low substrate concentrations are more conveniently maintained in continuous cultivation than in fed-batch cultivation. The only additional equipment needed is a system to ensure constant working volumes and flow rates. 

The biofermenter BF-F500 system consisted of a 1.5 1 culture vessel, 2 1 medium reservoir and effluent bottle (2 1 glass vessels) for fresh and expended media which were connected to the perfusion (culture) vessel by a peristaltic pump. As shown in Fig. 14, the fermenter systems have a conical shape sedimentation column in the center of the fermenter, and an impeller on the bottom of the sedimentation column. The Namalwa cells, KJM-1, were cultivated by continuous cultivation in the biofermenter. In Fig. 15, the culture has been inoculated at 1 to 2 x 10 cells/ml with an initial flow rate of approximately 10 ml/h, sufficient to support the population growth. At densities of 7 x 10 -1.5 x 10" cells/ml, we have used a nutrient flow rate of 1340 ml/h using ITPSG and ITPSG-F68 serum-free media. The flow rate of fresh media was increased step-wise from 240 to 960 ml/d in proportion to the increase in cell density. This resulted in an increase of 4 to 10 fold in cell density compared to the conventional batch culture systems. This system was then scaled up to a 45 1 SUS316L unit mounted on an auto-sterilization sequence system with a medium reservoir and an effluent vessel of 901 each. 

Beshay et al. reported conditions for the short-term continuous cultivation of D. discoideum cells on porous supports (SIRAN beads) in HL-5C medium ]108]. D. discoideum cells actively colonized the porous carrier (Fig. 5.5), after which the colonized beads can be freely suspended in medium. Gell densities of free amoebae remained at about 10 per mL for most of the cultivation time, whereas the cell density on the SIRAN beads reached up to 10 per mL and remained constant for at least 16 days of fermentation [108, 109]. By using broken pumice or CeramTec (a ceramic catalyst support), the immobilized cells reach... 

A prerequisite for industrial strains to be used for continuous cultivations is the ability to remain stable for extended periods of time when grown at large scale (see also the Introduction and Part IV, Chapters 1 and 12). This has previously been shown to be the case for S. cerevisiae strains based on a POT-selection plasmid and an insuHn precursor transcribed by the constitutive TPIl-promoter [6]. During growth of S. cerevisiae on carbohydrates as the major carbon source, ethanol formation should be avoided since formation of ethanol leads to lower biomass yields and consequently decreasing product titers. This issue of fermentative metabolism is often solved by monitoring the oxygen... 

Erickson et al (1969) developed a mathematical model for the description of fermentations with two liquid phases in both batch and continuous cultivation. The considerations embrace growth at the surface of the drops and in the aqueous phase. Three spedal cases have been examined in the first case it is assumed that growth occurs only at the surface of the dispersed phase in the second and third cases, growth takes place both at the interface and in the continuous phase (water). The second case assumes that the substrate equilibrium is continuously maintained between the two phases, while in the third case the consumption of substrate is limited by the transport path to the aqueous phase. Fig. 13 shows a comparison between the model and experimental data. It is assumed that growth takes place only at the... 

SMF has three main approaches batch, fed-batch, and continuous cultivation. In all cultivations, the accretion of mycelia was observed on baffles, impellers, fermenter lid, and wall of fermenter (i.e., above the surface of medium). This problem has been solved by varying the inoculum size, changing the agitation speed, varying aeration, making the fermenter with hydrophobic materials, and changing the pH and medium composition. However, the results showed no more than minute improvements of the problem [21]. 

In order to reduce the overall cost, there has been many fermentation techniques developed to produce PHAs with high productivity and high yield such as batch, fed-batch and continuous cultivations [5, 13]. Two stage fermentation is the most common and highly productive method to generate high density culture as well as increased amount of PHAs. 

Continuous cultivation is the third mode of operation for fermentation of hydrolyzates. In spite of a number of potential advantages in terms of productivity, this method has not been much developed in the fermentation of the acid hydrolyzates. The major drawback of the continuous fermentation is that inhibitors present in the medium will limit the specific growth rate of the cells. This will result in the wash-out of the bioreactor, unless a veiy low dilution rate is applied, giving a very low productivity. Furthermore, at a very low dilution rate the conversion rate of the inhibitors can be expected to decrease due to the decreased specific growth rate of the biomass. Thus, wash-out may occur even at very low dilution rate. Chung and Lee (67) faced this problem when they tried to continuously ferment acid hydrolyzates. They reported 90% decrease in the viable cell number after only three residence times. A straightforward continuous cultivation is therefore not an option, but some kind of cell retention is necessary... 

Seholten E, Renz T, Thomas J. (2009). Continuous cultivation approach for fermentative succinie acid production from crude glycerol by Basfia succiniciproducens DDl. Biotechnol Lett, 31,1947-1951. 

Figure 3 Fermentation modes for recombinant bacteria, yeast, and animal cells. On the left-hand side, the feed streams and harvesting streams are schematically shown for the batch, fed-batch, and continuous cultivation of microorganisms. On the right-hand side, the product concentration and the cell density are shown. For batch and fed-batch a discontinuous product concentration profile is obtained. With constitutive expression of a product, the product concentration is dependent on the cell density. Product is present in the culture supernatant during the whole production cycle and thus more susceptible to degradation. When the product formation is induction controlled, the production concentration raises sharply after addition of the inductor. The residence time of the product in the bioreactor is reduced. For continuous culture a constant product concentration profile is maintained over the entire production cycle. The residence time of the product in the bioreactor depends on the harvesting time and is shortest tor all fermentation modes when harvesting is continuously performed. Changing the mode of production can highly influence the composition of feed for preparative chromatography.

Ergot alkaloids can be manufactured also by alternative fermentation processes, e.g. by those using nontraditional substrates or immobilized cells of Claviceps spp. or their subunits. Semicontinuous or continuous cultivations represent other alternatives of the saprophytic cultivation. 

Fig. 6 shows a fed batch fermentation of sweet sorghum juice (SSJ) by Bacillus aryabhattai in 3 L fermentor under cultivating condition with agitation rate at 200 rpm, air rate of 1.5 1/min, at 30° C and feeding time at 18 and 24 hr during log phase of the culture. It was found that the cell could continuously produce both biomass and PHAs. Maximum cells were obtained at about 14.20 g/1 at 54 hr when PHAs content reached 4.84 g/1 after 66 hr (Tanamool et al., 2011). In addition, in Table 2, fed batch fermentation by A, latus was used for the production of PHAs (Yamane et al, 1996 Wang Lee, 1997). It could yield high productivity with the use of cheap carbon sources. 

Insect cell systems represent multiple advantages compared with mammalian cell cultures (1) they are easier to handle (Table 2.1) (2) cultivation media are usually cheaper (3) they need only minimum safety precautions, as baculovirus is harmless for humans (4) they provide most higher eukaryotic posttranslational modifications and heterologous eukaryotic proteins are usually obtained in their native conformation (5) the baculovirus system is easily scalable to the bioreactor scale. However, because of the viral nature of the system, continuous fermentation for transient expression is not possible - the cells finally die. 

A continuous centrifugal bioreactor, in which cells are fluidized in balance with centrifugal forces, has been designed to allow high density cell cultivation and superior aeration without elutriation of the suspended cells (van Wie et al., 1991). Reactor performance was hampered by elutriation of biomass by evolved gas in an anaerobic fermentation, indicating that it may not be suitable in its present state for three-phase fermentations. Immobilization of the cells on denser particles may overcome this problem. 

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