Chemistry: molecular biology and microbiology – Apparatus – Bioreactor
Reexamination Certificate
2000-12-15
2003-07-08
Lankford, Jr., Leon B. (Department: 1651)
Chemistry: molecular biology and microbiology
Apparatus
Bioreactor
C435S293200, C435S410000, C047S05810R
Reexamination Certificate
active
06589780
ABSTRACT:
BACKGROUND
1. Field of the Invention
The present invention relates a novel bio-reactor and more particularly to an apparatus or system for cultivation and up-scaling of genetically transformed plant organ (root) or parts in a sterile artificial environment, e.g. aqueous growth medium and to novel methods of employing the apparatus. The apparatus or vessel of the invention affords high aeration capacity through novel air dispersing and sparging systems. The high dispersion of air in the liquid medium is attainable at low agitation speed with the advantage of creating low shear thereby being favorable for the growth of highly entangled hairy root cultures.
2. Prior Art of the Invention
Considerable efforts are now being paid towards up-scaling of transgenic hairy root cultures of diverse plant species. Several publications are now available describing the use of different kinds of bio-reactors with or without design modification such as Singh, G. and Curtis, W. R. Reactor design for plant root cultures In: Ngo that T. and Sbargool Peter D. (eds.) Biotechnological application of plant cell cultures 1994 pp.-185-206. CRC Press, Inc.; Toivonen L., Utilization of hairy root cultures for production of secondary metabolites. Biotechnol. Prog. 9: 12-20 (1993), and Curtis, W. R., Cultivation of roots in bio-reactors. Curr. Opinion in Biotechnol. 4; 205-210 (1993).
The number of reports of hairy root culture in bio-reactors has increased significantly within the last few years. From a detailed literature survey it has been found that hairy roots have been cultivated in a wide range of reactor configurations varying from novel construction to simple modifications of the existing ones. Mainly two types of reactors have been used for hairy root up-scaling, i.e. bubble column reactors have been successfully used to culture hairy roots of
Tagetes patula, H. mulicus
and
Lithospermum erythrorhizon
(Buitelaar et al;1991, Mckelvey et al;1993;1992, Shimomura et al; 1991). However, when compared with other types of reactors, the performance of the bubble column reactors was rather poor (Taya et al; 1989, Weathers et al; 1989). The poor performance may be due to low oxygen supply because of channeling or tissue damage caused by sparging.
Although the other type of reactor, i.e. mechanically agitated one performed well in case of
Tagetes patula
(Buitelaar et al; 1991) the final tissue densities were limited in these systems due to mechanical damage of tissue caused mostly by the use of turbine impeller (Taya et al; 1989 and Wilson et al;1990).
Isolated impeller reactors in which the impeller is prevented from coming in contact with the root mass by using stainless steel mesh, polyurethane foam, etc., performed quite well in a number of cases (Muranaka et al; 1993, Inomata et al; 1993, Taya et al; 1989, Jung et al; 1987 & Hilton et al; 1990).
However, as with other reactor constructions, it can be anticipated that there will be numerous problems associated with high-density culture with regard to providing sufficient mixing in such systems (Singh & Curtis, 1994).
There are certain other reactor configurations which substantially differ from the conventional sparged or mechanically agitated type. Such reactors are mainly of two types- tickle bed and ebb- and -flow type, both of which are tried to provide both wetting and gas-contacting steps (Flores et al, 1992, Wilson et al; 1987, Taya et al; 1989, Cuello et al 1991, Dilorio et al; 1992a,b). Similar problems of gas we well as liquid phase channeling, as normally been encountered in case of the bubble column and stirred tank reactors, interfere with the final biomass yield. On the basis of the overall analysis, it becomes clear that the performance of all these reactors will be dependent on the ability to provide sufficient wetting of the densely packed, interconnected hairy root matrix for both nutrients as well as oxygen availability.
On the background of this information, it was felt essential to invent some modifications which would enable us to overcome the shortcomings keeping the other advantages of isolated impeller reactors constant.
OBJECTS OF THE INVENTION
The main object of this invention is to provide a novel bio-reactor to maximize genetically transformed “hairy root” biomass yield through up-scaling.
Another object of the present invention is to provide novel and improved systems/apparatus for the growth and cultivation of “hairy root” cultures affording efficient oxygen and nutrient distribution capacity throughout the reactor vessel, superior to conventional fermentors or bio-reactors.
Yet another object of the present invention is to provide improved oxygen and nutrient supply at low shear to prevent damage of shear-sensitive tissues of “hairy root” cultures.
Still another object of the present invention is to provide means for suppression of air-pocket formation so as to avoid the problem of gas channeling.
One more object of the present invention is to facilitate the supply of both oxygen and nutrients to the regions of highest tissue density so as to avoid starvation and death of “hairy root” tissues leading to poor productivity.
These and other objects, features and advantages of the invention will be seen from the following description and accompanying figures.
SUMMARY OF THE INVENTION
To meet the above objects, the present invention provides a novel bio-reactor comprising special type of impellers and sparges located at particular locations and an uniform support matrix present in the bio-reactor.
DETAILED DESCRIPTION OF THE INVENTION
Considerable interest is now being shown in
Agrabacterium rhizogenes
induced hairy root cultures for the production of high value plant secondary products. These cultures show a high degree of genetic stability, grow rapidly in hormone free culture medium with profusion of lateral branching and most importantly produce the same spectrum of secondary metabolites in quantities equivalent to or even in higher amounts than that of the parent plant (Hamill et al., 1986). So far several attempts have been reported for up-scaling of hairy roots in fermentors (Singh and Curtis 1994; Banerjee et al., 1995). Almost all such studies demonstrated the ability to grow hairy root cultures in bio-reactors and at the same time, discussed briefly the problems encountered which comprehensively indicate the scope for further improvement. In order to meet this need, the present invention is a process for up-scaling of hairy roots in a modified bio-reactor in order to take care of the earlier encountered problems.
In addition to the tissue culture multiplication steps and growth media constituents which are known in the art, an essential step of the preferred embodiment of the invention includes selection of a special type of impeller from the group consisting of turbine or rouston blade-2, 4, 6; marine blade, helical blade and pitched blade turbine impeller. In all embodiments of the invention, however, it is believed that the effectiveness of the present invention is attributable, at least in part, to the position and number of the special type of impeller as well as that of the spargers. The invention also describes a bio-reactor using low cost, autoclavable support matrix in order to isolate the impeller from growing roots as well as for uniform distribution and growth of the hairy root. The hairy roots preferably be obtained from
Hyoscyamus muticus, H. albus, R. seripentina
and
V. wallichii, Atropa belladonna
and
A. accuminata.
Agrobactrium rhizogenes
induced hairy root cultures of a wide variety of plant systems have already proved to be an excellent alternative production system for plant-derived chemical. However both advantages and disadvantages have been found to be associated with their higher level of culture differentiation. The primary advantage of hairy root cultures is that they display the tremendous biosynthetic capacity of normal roots with stable-long-term product profile, which is attributed to their higher level of cellular differentiation as compared to plant cell/suspension cultur
Banerjee Suchitra
Kahol Atul Prakash
Kukreja Arun Kumar
Kumar Sushil
Verma Praveen Chandra
Council of Scientific and Industrial Research
Greenblum & Bernstein P.L.C.
Lankford , Jr. Leon B.
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