Chemistry: molecular biology and microbiology – Carrier-bound or immobilized enzyme or microbial cell;... – Enzyme or microbial cell is immobilized on or in an...
Reexamination Certificate
1999-10-21
2004-09-07
Swartz, Rodney P (Department: 1645)
Chemistry: molecular biology and microbiology
Carrier-bound or immobilized enzyme or microbial cell;...
Enzyme or microbial cell is immobilized on or in an...
C435S174000, C435S177000, C435S182000, C435S243000, C435S261000, C435S283100, C435S286100, C435S286500, C435S289100, C435S297100, C435S308100
Reexamination Certificate
active
06787340
ABSTRACT:
TECHNICAL FIELD
The present invention is in the field of biological separations and processes.
BACKGROUND
In a variety of liquids containing cellular material, such as those used in fermentation processes, it is often desirable to be able to efficiently separate cellular products, such as cell bodies, lysed cells, and their breakdown products. It is often desirable to be able to remove or separate cellular products even in instances involving viscous liquids.
Accordingly, it is a general object of the present invention to be able to have available, a process for efficiently and completely removing or separating cellular products from liquid media, even in instances involving viscous liquids.
The process of the present invention can be applied inter alia to fermentation processes. Current industrial fermentations in conventional stirred tank fermentors for production of xanthan gum and other polysaccharides are energy-intensive and costly, mainly because the high broth viscosity causes agitation and aeration to be difficult and limits the final product concentration and productivity.
The production of xanthan gum is described here as an example of some of the problems encountered in biological processes that are addressed by the present invention, although the invention is not limited to that application.
Xanthan gum is a microbial polysaccharide widely used as a suspending, stabilizing, or thickening agent in the food industry. It is also used as a lubricant, emulsifier, or mobility-control agent in the oil-drilling industry. Presently, commercial xanthan gum is produced from glucose or dextrose by batch fermentation with the bacterium
Xanthomonas campestris
; the produced xanthan gum is then recovered and partially purified using alcohol precipitation. The final product usually also contains some cells and cell debris; however, it is desirable to produce xanthan gum product that is free of any particulates or cells, particularly for applications in oil recovery. The production of cell-free xanthan broth also allows for efficient concentration of xanthan fermentation broth by ultrafiltration without significant membrane fouling caused by cells and their debris (e.g., DNA and RNA) that would otherwise be present in the xanthan broth.
The present industrial process for xanthan gum production is energy-intensive and costly, mainly because the highly viscous xanthan broth causes agitation and aeration to be difficult in conventional stirred tank fermentors. Consequently, conventional xanthan gum fermentation has low xanthan concentration (usually below 3% wt/v) and low productivity (usually below 0.5 g/L×h). There have been many attempts to increase xanthan productivity and to lower energy costs by using new agitation designs, and new types of bioreactors. Fermentation with water-in-oil emulsion and cell immobilization using porous Celite beads, which reduces broth viscosity and improves aeration and oxygen transfer, have also been studied. Although a high xanthan concentration of ~5% was achieved in these processes, separating and recovering xanthan gum from the oil emulsion or Celite particles, though feasible, was difficult.
There have been only a few studies of xanthan fermentation using immobilized cells. Robinson and Wang (1988) used porous Celite beads to immobilize cells in xanthan fermentation. It is not clear, however, if the xanthan broth so produced was free of cells. Furthermore, a large portion of the xanthan product was trapped in the beads and could not be easily separated from the cells. Lebrun et al. (1994) studied polysaccharide production by cells immobilized in composite agar layer/microporous membrane structures, but concluded that the immobilized-cell system was not appropriate for xanthan gum production. It is clear that cell entrapment is not an appropriate cell immobilization method for xanthan gum fermentation because of the high viscosity of xanthan solution. The viscosity of the xanthan solution is high even at a low concentration. In batch xanthan fermentation, the broth viscosity has been found to reach more than 3000 cp at 2% (wt/v) xanthan concentration. The high viscosity of xanthan broth presents a major challenge in separating cells and cell debris from broth at industrial scale using conventional separation techniques, such as microfiltration, flocculation, and centrifugation. Thus, one of the objects of the present invention is to find an economical way to produce cell-free xanthan broth by either cell immobilization during fermentation or cell removal after fermentation.
One of the objects of the present invention is to provide a fermentation method which allows for the efficient and substantially complete removal of cells and cell debris from fermentation broths, even in instances involving viscous fermentation broths.
It is also an object of the present invention to allow for the removal of cellular products from liquids used as media for cellular reactions, such as fermentation broths, even those that are unusually viscous.
It is also an object of the present invention to produce an apparatus for carrying out the separation/removal process and cellular reactions of the present invention.
In view of the present disclosure, other advantages and the solutions to related problems may become apparent to one of ordinary skill in the art.
SUMMARY OF THE INVENTION
The present invention includes a process for separating cells form a liquid media, a method of fermentation using such a separation, and an apparatus for conducting such a separation or for facilitating a cellular reaction.
In broadest terms, the separation process of the present invention is a process for separating cells from a liquid containing cells. The process comprises the steps of: (a) bringing the liquid containing the cells into contact with one or more microbial polysaccharide and a fibrous material so as to adsorb the cells onto the fibrous material; and (b) separating the liquid from the fibrous material so as to remove the cells from the liquid. This may be done either by having the microbial polysaccharide(s) be in the liquid or by having the fibrous material be pre-treated with the microbial polysaccharide(s). Either way, a polysaccharide-mediated adsorption of the cells onto the fibrous material is brought about.
The process of the present invention may be used to remove cells from any liquid, but such liquids typically will be aqueous solutions, such as growth media, biological fluids, diagnostic samples, aqueous test samples, etc.
As referred to with respect to the present invention, the term “cells” shall be understood to include, without limitation, cells—alive, dead or attenuated—and cell portions such as lysed cell walls, cell bodies, organelles, chromosome material and mixtures thereof.
The microbial polysaccharide(s) may be of any type. Naturally, there are a wide variety of microbial polysaccharides, such as the more well characterized and named polysaccharides selected from the group consisting of xanthan gum, dextran, pullulan, the polysaccharide types they represent and mixtures thereof.
The liquid may be brought into contact with the microbial polysaccharide and a fibrous material through any appropriate means, such as through the use of tanks and vats, liquid flows, etc.
The fibrous material used in accordance with the present invention may be any natural or synthetic fiber, and may for instance be selected from the group consisting of looped cotton terry cloth, cotton fabric sheet cloth, 50% cotton-50% polyester fabric sheet cloth, and polyester fabric sheet cloth. It has been found that cotton, particularly looped cotton terry cloth and cotton fabric sheet cloth, works best, with looped cotton terry cloth being most preferred.
The fibrous material may be in any non-woven, woven or geometrical arrangement (e.g., sheets, rolls, strands, threads, etc.), generally referred to as the “fibrous matrix,” and naturally may be produced and arranged so as to afford efficient contact with the liquid.
The liquid may be brought into contact with the microbial p
Standley Law Group LLP
Swartz Rodney P
The Ohio State University
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