Chemistry: molecular biology and microbiology – Micro-organism – per se ; compositions thereof; proces of... – Protozoa – media therefor
Reexamination Certificate
2000-03-17
2004-04-06
Marx, Irene (Department: 1651)
Chemistry: molecular biology and microbiology
Micro-organism, per se ; compositions thereof; proces of...
Protozoa, media therefor
C435S244000, C435S947000, C424S780000
Reexamination Certificate
active
06716617
ABSTRACT:
The invention relates to a fermentation process with continuous mass cultivation of ciliates (protozoa) for producing biogenous valuable substances, where the biomass containing the desired biogenous valuable substances is obtained by continuous (permanent) cell extraction.
To date, only initial attempts have been made in the biotechnological utilization of ciliates—a class of protozoa—, although a large number of metabolites of these organisms are of economical interest, for example lysosomal enzymes. As yet, only few biotechnological processes for obtaining biogenous valuable substances from ciliates have been described, —mainly for the ciliate Tetrahymena (Kiy & Tiedtke, 1991, Appl. Microbiol. Biotechnol., 35, 14; Kiy et al., 1996, Enzyme Microb. Technol., 18, 268; Kiy & Tiedtke, 1992, Appl. Microbiol. Biotechnol., 38, 141), —and these are exclusively processes for obtaining excreted cell products, i.e. those cell products which are given off by the ciliate cells into the culture medium. In this kind of process, the ciliates are cultivated in fermenters and the culture medium containing the excreted biogenous valuable substances is removed periodically, in more or less regular intervals, and exchanged for fresh medium. During the exchange of the medium, the ciliates are retained in the fermenter using certain methods—for example the use of membranes, cell immobilization and the like—, so that virtually no cell material is lost and the cell culture exists in principle permanently. However, to obtain biogenous valuable substances which are attached to the cell it is necessary to harvest the total of the cells, the so-called biomass. To this end, in general—i.e. in the generally known fermentation processes with bacteria or fungi as producers of valuable substances—a so-called batch fermentation is carried out, where the fermenter is seeded and the cells are cultivated until the maximum biomass or product concentration has been reached. The biomass is then harvested. Such processes have already been described for various ciliates such as Paramecium, Colpoda and Tetrahymena (Proper & Garver, 1966, Biotechnol. Bioeng., 8, 287, Schönefeld et al., 1986, J. Protozool., 33, 222; Kiy & Tiedtke, 1992, Appl. Microbiol. Biotechnol. 37, 576).
However, batch fermentation processes have the fundamental disadvantage that, at intervals, cleaning, re-seeding of the fermenter and an intensive monitoring and looking after of the cell culture is required—in particular during the critical growth phase.
From the fermentation technique with bacteria or yeasts as producers of valuable substances, in addition to the bath fermentation process, the “continuous fermentation process” is also known. In this process, the cells are cultivated in the fermenter until they have reached a certain cell density and then permanently harvested by continuous cell extraction from the fermenter, while at the same time fresh culture medium is added to the same extent. The amount of cells extracted per time unit (the cell extract) is such that the cells which remain in the fermenter can easily recompensate the reduction of the cell density due to the harvest, by continuous cell divisions. In the range if a certain cell extraction rate or dilution rate “D”, the cell density in the fermenter thus remains constant, although culture and accordingly the desired product are harvested continuously.
In principle, this continuous fermentation process is economically far superior to batch fermentation; however, its realization requires that the cultivated or bred organisms grow and multiply relatively quickly and uniformly, and that they are insensitive to the stirring and shear forces which are encountered in a continuous fermentation process.
Of ciliates, however, it is generally known that frequently they grow and multiply only very slowly, that they pass through different growth phases and that they react very sensitively to stirring and shear forces (Curds & Cockburn, 1971, Journal of General Microbiology 66, 95-109; Middler & Finn, 1966, Biotechnology and Bioengineering 8, 71-84). Attempted continuous mass cultivations of ciliates have indeed been described, but exclusively with the use of bacteria-containing culture media and with resulting maximum cell densities of a few ten thousand cells per ml, despite a cultivation of 10 days or more (Curds & Cockburn, supra.). For use on an industrial scale, such cell densities are totally insufficient. In addition, the cultivation described by Curds & Cockburn is therefore also completely unsuitable for industrial use because it prescribes the use of a medium containing prey organisms, i.e. bacteria. In a bacteria-containing culture medium, the bacteria do, of course, also multiply permanently, the extent depending on how many ciliates are present. The coexistence equilibrium of ciliate population and bacteria population is very labile, and even a slight intervention can cause substantial changes in both populations.
Furthermore, the experiments of Curds & Cockburn were carried out more than 25 years, ago, and they have apparently confirmed the opinion of those skilled in the art that ciliates are unsuitable for a continuous fermentation process on an industrial scale.
It is an object of the present invention to provide a process for continuous fermentation of ciliates with cell extraction, which avoids the abovementioned disadvantages and is very suitable, in particular, for industrial use.
This object is achieved by a process of the type mentioned at the outset, where the ciliate cells are cultivated in a complex axenic medium.
For the purpose of this invention, a complex medium is a nutrient medium in aqueous solution of natural products or extracts obtained therefrom for cultivating microorganisms.
For the purpose of this invention, axenic medium is a nutrient medium which is free of feed and prey organisms (so-called food organisms).
The process according to the invention is based on the surprising finding that a continuous fermentation process with cell extraction using complex axenic media can also be carried out successfully and in an economically highly rewarding manner using pure ciliate cultures. Continuous cell densities in an order of magnitude of 1 million cells per ml can be realized without any problems, in the case of Tetrahymena as early as from the third day after the beginning of the cultivation. Thus, the prejudice of those skilled in the art that ciliates are unsuitable for continuous mass cultivation with cell densities of several hundred thousand to millions of cells per ml using known fermenters and in the presence of the shear forces which are usually encountered, in axenic medium—i.e. without living feed or prey organisms, is overcome since:
they grow too slowly and not uniformly enough,
they show only little resistance to stirring and shear forces and are very easily and rapidly damaged and/or destroyed by such forces, and
in the cultivation attempts that have been carried out so far, in spite of a prey-organism-containing media being used, and thus a substantially natural diet, only relatively very low maximum cell densities have been reached.
Using the process according to the invention, it is possible for the first time to employ ciliates for the industrial production of biogenous valuable substances attached to the cell, and thus to obtain, on an economically important scale, in particular those valuable substances which are only known from ciliates, such as, for example, taurolipids and tetrahymanol, or those which are produced extensively specifically by ciliates, such as, for example, gamma-linolenic acid, docosahexaenoic acid, eicosapentaenoic acid, octatetraenoic acid and arachidonic acid.
Since the ciliates are kept as a pure culture—i.e. free from other living organisms—, important disturbing factors are avoided a priori, and even the technical expense is limited to a minimum: fermenters for regrowing the prey organisms, for example, are completely redundant.
The products which can be obtained from the extracted biomass include peptides and pr
Marx Irene
Nutrinova
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