Method for culturing ciliates

Details Method for culturing ciliates Method for culturing ciliates

1996-07-08

2003-07-15

Weber, Jon P. (Department: 1651)

Chemistry: molecular biology and microbiology

Micro-organism, per se ; compositions thereof; proces of...

Protozoa, media therefor

C435S243000

Reexamination Certificate

active

06593128

ABSTRACT:

FIELD OF THE INVENTION
The invention relates to a method for culturing ciliates comprising stirring ciliates in culture medium.
BACKGROUND OF THE INVENTION
Ciliates, protozoans belonging to the phylum Ciliophora, are a promising source of useful biogenic materials, such as, for example, enzymes (I. Munro, 1985
, Process Biochem
. 20: 139), unsaturated fatty acids (Y. Gosselin et al., 1989
, Biotechnol. Lett
., 11: 423) and “single cell protein” (A. Ayerbe, 1980
, Enzyme Microb. Technol
., 2: 54).
A reason for the fact that ciliates, despite their potential, have not been used industrially as productive organisms until now, lies in the often poor culturability of these organisms. The central problems in the fermentation of ciliates include the lack of suitable, cost-effective culture media and the often high sensitivity to shear forces (Y. Gosselin et al., 1989
, Biotechnol. Lett
., 11: 423; M. Midler & R. K. Finn, 1966
, Biotechnol. Bioeng
., 8: 71). While some ciliates such as, for example, Tetrahymena and Paramecium can be cultured successfully in fermenters (T. Kiy & A. Tiedtke, 1992
, Appl. Microbiol. Biotechnol
., 38: 141; U. Schönefeld et al.,
J. Protozool
., 33: 222), for the majority of ciliates there is to date no method for mass culturing. However, even the above-mentioned genera cannot be cultured undamaged in conventional fermenters. This can be recognized, inter alia, in that the degree of damage to Tetrahymena cells has been used as a measure of the shear forces in stirred fermenters (M. Midler & R. K. Finn, 1966
, Biotechnol. Bioeng
. 8: 71). Many ciliates were until now propagated only on a very small scale, at very low cell densities. Thus the customary method for the culture of colpodid soil ciliates is still the “flooded petri dish” method, in which a few ml of culture are incubated in Petri dishes (W. Foissner, 1993
, Protozoenfauna: [Protozoan fauna
] Vol. 4/1: Colpodia (Ciliophora), Gustav Fischer Verl.).
A method for culturing ciliates in mass cultures would be desirable. Such a system is to be regarded as a prerequisite for the assessment or use of these organisms as a source of useful biogenic materials.
The shear forces occurring in conventional fermenters are not tolerated by many ciliates. A system which produces relatively low shear forces, but nevertheless guarantees adequate mixing of the culture, is the so-called spinner flask (FIG.
1
). Typical of this system is the stirrer with magnetic core, driven by a magnetic stirrer located under the culture vessel. The stirrer brings about gentle and low shear-stress mixing.
Although this culturing method had already been developed many years ago (T. Lidl & J. Bauer, 1987, Zell—und Gewebekultur [
Cell and tissue culture
], Gustav-Fischer Verl.), for culturing animal cell cultures (e.g. hybridoma cells), the transfer of this culturing method to free-living ciliates did not occur. Only distantly related eukaryotes such as Euglena from the Euglenophyceae, Chlamydomonas from the Chlorophyceae group (Vogels et al., 1978
, J. Phycol
., 14, 403) and
Entamoeba histolytica
from the Amoebida group (Said-Fernandez, et al., 1992, Arch. Med. Res., 23, 57) have already been cultured in spinner flasks.
SUMMARY OF THE INVENTION
It is an object of the invention to provide a method for culturing ciliates comprising the steps of placing ciliates and medium therefor in a culture flask; providing said culture flask with a stirrer having a magnetic core, wherein said stirrer is suspended in the top part of the culture flask such that the stirrer does not touch the flask bottom, and wherein the motion of said stirrer is driven by means of a magnetic field; and stirring said ciliates in said culture medium. It is a further object of the invention to provide a culturing method comprising stirring ciliates in culture medium, wherein the stirrer is a membrane aeration stirrer.
It is another object of the invention to provide a culturing method comprising stirring ciliates in culture medium, wherein the culturing comprises batch fermentation, fed-batch fermentation, or cyclic medium exchange.
It is a further object of the invention to provide a culturing method comprising stirring ciliates in culture medium, wherein the stirring speed is from about 1 to about 70 rpm. It is also an object of the invention to provide such a method wherein the stirring speed is from 1 to 70 rpm.
It is yet a further object of the invention to provide a culturing method comprising stirring ciliates in culture medium, wherein the stirring speed is from about 10 to about 40 rpm. It is also an object of the invention to provide such a method wherein the stirring speed is from 10 to 40 rpm.
It is yet a further object of the invention to provide a culturing method comprising stirring ciliates in culture medium, wherein the stirring mode is a reciprocatory mixing technique.
It is yet a further object of the invention to provide a method for culturing ciliates, wherein such a method comprises any combination of the foregoing objects.
Finally, it is an object of the invention to provide a method for culturing ciliates, comprising any combination of the foregoing objects, wherein the ciliates belong to the group Colpodia, Holotrichia, Peritrichia, Spirotrichia or Suctoria.


REFERENCES:
patent: 4649118 (1987-03-01), Anderson
patent: 5008197 (1991-04-01), Wergeland et al.
patent: 56095394 (1981-01-01), None
patent: 57074082 (1982-10-01), None
patent: 08133980 (1996-05-01), None
Hofmann et al., External factors limiting the multiplication potential of Tetrahymena, J. Cell Sci., 50:407-418, 1981.*
Griffiths, Scaling up of animal cell cultures, in Animal Cell Culture, A Practical Approach, R.I. Freshney, Ed., IRL Press, Oxford, England, Chapter 3, pp. 33-69, 1986.*
Gosselin et al., Improvement of fed batch mass culture for gamma linolenic biosynthesis by Tetrahymena Rostrata (Protozoa), Biotech. Letters, 11:423-426, 1989.*
W. Foissner,Protozoenfauna, vol. 4/1: Colpodia (Ciliophora), Gustav Fishcer Verlag, 1993.
Schönefeld et al., J. Protozool., vol. 33, No. 1, p. 22, 1986.
Vogel et al., “Qualitative Assay of Dissolved Amino Acids and Sugars Excreted byChlamydomonas reinhardtii(Chlorophyceae) andEuglena Gracilis(Euglenophyceae)”, J. Phycol. vol. 14., pp. 403-406, 1978.
Fernandez et al., “Axenic Massive Cultivation ofEntamoeba histolyticaTrophozoites in Spinner Flasks and in a Microfermentor”, Arch. of Med. Res., vol. 23, No. 2, pp. 57-58, 1992.
Midler, Jr., et al., “A Model System for Evaluating Shear in the Design of Stirred Fermentors”, Biotechnology and Bioengineering, vol. VIII, pp. 71-84, 1966.
Gosselin et al., “Improvement of Fed Batch Mass Culture For &ggr; Linolenic Biosynthesis by Tetrahymena Rostrata (Protozoa)”, Biotech. Ltrs., vol. 11, No. 6, pp. 423-426, 1989.
Kiy et al., “Continuous high-cell-density fermentation of the ciliated protozoon Tetraymena in a perfused bioreactor”, Appl. Microbiol. Biotechnol., vol. 38, pp. 141-146, 1992.
Munro, “Protozoa as Sources of Commercially Producted Enzymes—A Review”, Process Biochemistry, pp. 139-144, 1985.
Ayerbe, “ProtozoaTetrahymena pyriformisas single cell protein: fermentation and nutritional aspects”, Enzyme Microb. Technol., vol. 2, pp. 54-58, 1980.

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