Cell-sedimentation column

We have developed several new perfusion systems which do not use filtration methods for cell propagation. When the flow rate of the continuous supplying medium is minimized, for example, when it is 1 to 3 times its working volume per day, the system has the ability to separate the suspended cells from the supernatant fluid. This is accomplished by means of an internal cell-sedimentation column in which the cells settle by gravity. The shape and length of the column are sufficient to ensure complete separation of cells fi om the medium. Cells remain in culture whereas the effluent medium is continuously withdrawn at a rate less than that of the cell sedimentation velocity. We experimented with several shapes for the sedimentation column and found that the cone and two jacketed types work best. 

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. 

Since the influence of different additives on cell sedimentation based on literature data is contradictory it is necessary to determine the cell sedimentation rates experimentally. For cells with low sedimentation rates batch runs were carried out and the displacements of the interfaces between layers of different cell concentrations along the column were determined optically. 

In bubble columns, sterile air enters through the bottom of the bioreactor and pushes the culture medium and cells by preventing cell sedimentation and causing the diffusion of nutrients and oxygen in the culture medium. The problem is when the bioreactor tank is very big, in which case the air enters too quickly and forcefully in the tank of the bioreactor, and the air bubbles break cells by crashing into them. One modification of the bubble column is the airlift bioreactor (Fig. 89.12). In this case, inside the tank, there is another mbe, the draft tube. 

In Chapter 21 on box models no distinction was made between a compound being present as a dissolved species or sorbed to solid surfaces (e.g., suspended particles, sediment-water interface). In Boxes 18.5 and 19.1, and also in Illustrative Example 19.6, we learned that several of the transport and transformation processes may selectively act on either the dissolved or the sorbed form of a constituent. For instance, a molecule sitting on the surface of a sedimentaiy particle at the lake bottom does not feel the effect of turbulent flow in the lake water, while the dissolved chemical species is passively moved around by the currents. In contrast, a molecule sorbed to a suspended particle (e.g., an algal cell) can sink through the water column because of gravity, unlike its dissolved counterpart. 

Basic Sedimentation Equilibrium Equation. Sedimentation equilibrium experiments are performed at constant temperature. The condition for sedimentation equilibrium is that the total molar potential, m, for all components i be constant everywhere in the solution column of the ultracentrifuge cell. Mathematically this can be expressed as... 

Fig. 4. Compartmental model describing the cycling of nitrogen in a planktonic community in the mixed layer of a water column. Flow pathways are represented by arrows and numbers which correspond to mathematical expressions described in Table 2. The nitrogen pool represents all abiotic nitrogen (nitrate, ammonia and urea), and other compartments represent bacteria, zooflagellates, larger protozoa, and micro-mesozooplankton, giving off waste products (F+U). Arrows (13) and (14) depict sedimentation of zooplankton faeces and phytoplankton cells, respectively (After Moloney et al., 1985).

Few studies exist where mass fluxes of Phaeocystis cell carbon have been quantified along with POC export. We compiled the existing data sets to evaluate the contribution of Phaeocystis spp. cell carbon to vertical carbon flux. Phaeocystis pouchetii blooms are regularly observed in North Norwegian fjords (Heimdal 1974 Eilertsen et al. 1981 Riebesell et al. 1995 Reigstad et al. 2000 Wassmann et al. 2005). Due to the close vicinity of Tromsp, many years of observations from the water column as well as from sediment trap measurements (without fixatives) are available from these localities. 

Despite occasionally high concentrations and dominance of Phaeocystis spp. in the water column, calculations based on sediment-trap measurements suggest low daily loss rates, implying high retention of Phaeocystis cell carbon in the upper 50-100 m. Unless deep mixing accelerates vertical export, the contribution to the vertical carbon flux at 100 m is on average 3%. The conclusion is that Phaeocystis cell carbon does not contribute significantly to vertical carbon export. 

Ammonification is another process that can result in N release. The simplest definition of the process is the release ofNH4 from organic matter (e.g., Herbert, 1999). It can occur by a number of different processes including remineralization by bacteria in the water column and sediments. Photochemical ammonification occurs abiotically when NH4+ is released from organic matter as a result of exposure to UV radiation (reviewed in Bronk, 2002 and Chapter 10 by Gryz-bowski and Tranvik, this volume). Ammonium efflux from cells has also been observed following urea uptake in a number of culture experiments (e.g., Price and Harrison, 1988 Rees and Bekheet, 1982 Uchida, 1976). The release may be due to passive diffusion through the cell membrane and is likely unavoidable because NH3 is lipid soluble. 

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