GB2346379A

GB2346379A - Growing organisms in a multi-compartment vessel

Info

Publication number: GB2346379A
Application number: GB9902757A
Authority: GB
Inventors: Neil Porter; Frances Mary Giaquinto
Original assignee: BioDiversity Ltd
Current assignee: BioDiversity Ltd
Priority date: 1999-02-08
Filing date: 1999-02-08
Publication date: 2000-08-09
Anticipated expiration: 2019-02-08
Also published as: JP2002536010A; EP1165747A1; GB2346379B; AU2311200A; WO2000047709A1; GB9902757D0

Abstract

Described is a method for growing an organism, preferably a fungi, wherein the organism is grown on a material in a first vessel and is entrained to grow in a predetermined direction into a medium in a second vessel. Single cylindrical receptacles 30 are joined by collars 56 and may have mesh inserts 40 which allow filamentous organisms to grow therebetween but prevent the egress of growth medium contained in adjacent receptacles. The technique is devised to provide a substantially homogenous sample of the organism across the medium and overcomes sectoring. The culture of the basidiomycetes, Schizospora paradoxa and Phlebia delfectens, and Asperigillus terreus is specifically described. The method can be used to provide fungal-derived pharmaceutical products.

Description

APPARATUS FOR PRESERVING MICROORGANISMS The present invention relates to preparing and maintaining cultures of microorganisms, and is particularly applicable to fungi and bacteria.

Microorganisms are essential to many important biotechnical processes including the production of foods, fine chemicals such as vitamins and organic acids, pharmaceuticals, enzymes, agrochemicals and biological control agents. In the pharmaceutical field, microorganisms have yielded drugs which are used for treating infections, disorders of the central nervous system, cardiovascular disease and for suppression of the immune system to prevent rejection following organ transplantation. They hold enormous potential for producing new pharmaceutical compounds.

As a consequence of the economic and academic importance of microorganisms, microbial genetic resource collections have been established to provide cultures of microorganisms for research. The World Data Center for Microorganisms has been assigned the role of characterising, cataloguing and most importantly preserving microorganisms in a pure, viable and genetically stable condition. Over 500 collections of microorganisms have been registered with the World Data Center.

In industry, where collections can exceed 50, 000 microorganisms, and each has the potential to yield a valuable new product or process, it is imperative that microorganism cultures are stored under conditions that maintain genetic stability. Genetic deterioration of a microorganism during storage can result in a reduction or total loss of its biotechnological properties (known or as yet undiscovered). This can result in a significant financial loss to a company.

There are two main approaches to microbial preservation.

Firstly, a culture can be maintained on a growth substrate by means of repeated sub-culture onto a new substrate as the growth substrate deteriorates.

Secondly, it is possible to create an environment where metabolism of a culture is severely reduced or halted (Smith, D & Onions, AHS, (1994)"The Preservation and Maintenance of Living Fungi", 2nd ed. Wallingford, CAB International).

A method of sub-culturing will now be described with reference to Figures 1 and 2 of the drawings.

Figure 1 shows a Petri dish 10 which is filled with a layer of agar 12 through which is dispersed a nutrient medium containing sources of carbon, nitrogen, phosphorus, essential vitamins and other elements required for growth. Agar is a natural carbohydrate substance extracted from seaweed. A sample of an organism 14 is inoculated onto the agar at the centre of the Petri dish 10. The Petri dish 10 is then left in a clean environment for a period of 10 days to 2 weeks, or longer if necessary, and maintained at a temperature suitable to promote growth, e. g. 15-25 C. Following that period, the dish will have the appearance as illustrated in Figure 2. As illustrated in Figure 2, a colony of the organism 14 has developed by the growth of filamentous strands along the surface of the agar 12 in all directions from the original sample.

Then, as illustrated in Figure 2, a sub-culture sample 16 can be taken from the growing edge, so as to sample the youngest and most viable part of the filaments. That sub-culture sample can then be inoculated at the centre of a further Petri dish of agar for further culturing.

The sample 16 can be taken by means of a sterile scalpel.

The original Petri dish can then be discarded.

It has been recognised that the above method can lead to sampling errors. Microorganisms, and particularly fungi, are inherently genetically variable. For example, as illustrated in Figure 2, whereas the microorganism normally has a green appearance, the zone 18 of the culture identified by chain lines could have genetically segregated so that it has a red appearance. This phenomenon is known as sectoring.

By taking a sample 16 from the Petri dish as illustrated, only genetically segregated red material would be taken from the Petri dish. Therefore, the process of subculturing as described above would, under those circumstances, result in the sub-cultured sample having a different overall genetic make-up from the culture from which the sub-culture was selected. It could be that only the green part of the culture exhibited the biological and/or physiological features which might have an advantage suitable for pharmaceutical or agrochemical application. Therefore, by only taking the one sample, the benefit might have been eliminated. This problem was identified in Smith & Onions (1994), referred to above.

Accordingly, a further sample 16'could be taken as illustrated in Figure 2. This second sample 16'would maintain the full genetic composition of the microorganism, which would reduce the problems resulting from genetic segregation. However, some sampling error may remain, since a technologist may not be able to identify all genetic modifications by observation, and so important genetic material may be discarded as a result of the sub-culturing method described above.

Moreover, the proportion of materials of different types to be sampled would be a matter for a sampler to identify, which could introduce further errors.

Additionally, it should be emphasised that all microorganism populations arq genetically heterogeneous.

Consequently, ongoing sub-culture and growth on a synthetic agar medium can aCt as a selective pressure ensuring that a proportion of the population best suited to those particular condition$ of growth become dominant.

Desirable properties of microorganisms can be lost as a result. Long term storage of microorganisms by repeated sub-culture is therefore not desirable.

Avoiding repeated sub-culture by covering cultures grown on agar slopes with mineral oil is a traditional method still widely used. The mineral oil (liquid paraffin) prevents dehydration and slows down metabolism by reducing oxygen availability (Smith & Onions 1994 previously identified ; Smith, D & Kolkowski, J (1996) "Preservation and Maintenance of Cultures used in Biotechnology and Industry", San Diego, CA Academic Press). Although fungi have been successfully stored for 40 years using this method, it has a number of serious disadvantages which include retarded growth of the microorganism on retrieval and an increased risk of contamination.

One of the most widely used methods of creating an environment for storing microorganisms in such a way that their metabolic rate is reduced or halted involves the use of cryopreservation at ultra-low temperatures (Smith, D (1993)"Tolerance to freezing and thawing", Tolerance of Fungi, Editor-Jennings, DH pp 145-171 published by Marcel Dekker Inc, New York Smith, D (1998)"The use of cryopreservation in the ex-situ conservation of fungi" Cryoletters 19,79-90). Little metabolic activity occurs below-70 C but recrystallisation of ice, which can cause cell damage, can occur above-130 C. Consequently, microorganisms are stored at temperatures below -130 C ; in refrigerators (-135 C to-180 C) or in liquid nitrogen vapour at-196 C. Cellular damage due to ice crystal formation can occur if the freezing and thawing rates are not carefully controlled and the use of cryoprotectant chemicals is important to minimise this damage (Smith (1998) previously referred to; Smith D & Thomas VE (1998)"Cryogenic light microscopy and the development of cooling protocols for the cryopreservation of filamentous fungi", World Journal of Microbiology and Biotechnology, 14,49-57).

Genetic selection can also be a problem with this method of storage; only a small amount of culture biomass is taken for cryopreservation and only a proportion of that small amount may be viable when the material is thawed.

Cryopreservation is the most expensive in terms of capital equipment required, its running costs and preparation of cultures for storage.

For organisms that sporulate in culture, various methods of drying and freeze drying can be employed. Removal of water reduces cell metabolism and many fungal spores can remain dormant but viable in this way for a number of years. Storage in silica gel is a cheap and effective method for fungi that produce thick walled spores and it maintains good genetic stability. Storage in sterilised soil is used successfully for some soil fungi but loss of genetic integrity is common and there is a high risk of contamination. Freeze drying involves removing water from frozen cell suspensions by sublimation under reduced pressure (Mellor J. D."Fundamentals of Freeze Dring", Academic Press 1978). It is a widely used method, but it is unsuitable for non-sporulating fungi, there is often a low percentage viability, genetic damage frequently occurs and it requires expensive equipment.

The time taken for microorganisms to recover from storage where their cellular metabolic activity has been reduced can take three weeks or longer. It is not possible to do anything with the microorganism before the end of this period. In addition to the inconvenience caused, this time delay can add significantly to the costs of biotechnological processes.

There is a need for a simple, cost effective system for the storage and sub-culture of microorganisms which maintains cultures in a viable, metabolically active and genetically stable state.

Therefore, a first aspect of the invention has as its object the improvement of sampling techniques to maintain reliability of sub-culturing as a method of maintaining a sample of an organism.

However, the use of agar is somewhat undesirable, in that it is a synthetic growth environment which is in some respects different from the nutrient environment which microorganisms would naturally encounter. Whereas agar media are designed to simulate, as closely as possible, the combination of nutrients most amenable to the growth of microorganisms, they remain approximations. The main advantage of agar is that it provides a solid substrate that is not broken down by the microorganism.

Therefore, it is a further object of an aspect of the invention to provide a technique of culturing which makes use of naturally occurring pubstrates.

The invention provides, in a first aspect, a method of sub-culturing which involves maintenance of microbiological material without selection of specific samples thereof.

The invention provides, in a second aspect, sub-culturing apparatus for presenting a sub-culture across substantially an entire population of a culture.

The invention provides, in a third aspect, a method of generating a metabolite from an organism maintained according to the first aspect of the invention. The invention also provides, in a further aspect, a method of manufacturing a chemical composition from the metabolite generated in accordance with the third aspect of the invention.

A specific embodiment of the invention will now be described, by way of example only, with reference to the accompanying drawings, in which : Figure 1 is a perspective view of a Petri dish in accordance with an example of an existing technique; Figure 2 is a plan view of a culture on the Petri dish illustrated in Figure 1; Figure 3 is a perspective view of a receptacle in accordance with a specific embodiment of the present invention; Figure 4 is a longitudinal section of the receptacle illustrated in Figure 3 in an initial condition; Figure 5 is a perspective view of an insert of the receptacle illustrated in Figure 3; Figure 6 is a perspective view of an alternative insert to that illustrated in Figure 5; Figure 7 shows a longitudinal section of an end portion of receptacle in accordance with an alternative and specific embodiment of the invention; Figure 8 is a perspective view of a receptacle of a further alternative and specific embodiment of the present invention; Figure 9 is a longitudinal sectional view of the receptacle illustrated in Figure 3 in a first stage of use in accordance with a specific exemplary method; Figure 10 is a longitudinal sectional view of the receptacle illustrated in Figure 3 in a second stage of use in accordance with a specific exemplary method; Figure 11 is a longitudinal sectional view of the receptacle illustrated in Figure 3 in a third stage of use in accordance with a specific exemplary method; Figure 12 is a longitudinal sectional view of the receptacle illustrated in Figure 3 in a fourth stage of use in accordance with a specific exemplary method; Figure 13 is a longitudinal sectional view of the receptacle illustrated in Figure 3 in a fifth stage of use in accordance with a specific exemplary method; Figure 14 is a longitudinal sectional view of the receptacle illustrated in Figure 3 in a sixth stage of use in accordance with a specific exemplary method; and Figure 15 is a longitudinal sectional view of an arrangement of receptacles as illustrated in Figure 3 for use in accordance with an alternative specific exemplary method.

Referring to Figure 3, a receptacle 30 has a generally hollow cylindrical body 32 whch is open at both ends.

As shown in Figure 4, each end of the body 32 has an external screw thread 34, and is closed by a cap 36 having a cooperating internal screw thread 38. The caps 36 may be fitted on the body 32 sufficiently tightly that a seal is formed to prevent ingress of microscopic contaminants into the receptacle 30.

The material of the body 32 and the caps 36 is of material which is readily sterilisable for use in biotechnological applications. Moreover, the material is preferably transparent, which allows for observation of the interior of the receptacle 30. A suitable material could be glass, or plastics such as polystyrene, polyethylene, polyamide, polyacrylate. Especially important examples of a suitable material are polycarbonate or polypropylene, which can withstand sterilisation by means of hot water vapour at temperatures up to 121 C.

Each cap 36 extends over its respective end of the body 32 to a depth of not less than 25 mm to ensure that the ends of the body remain sterile when one or both of the caps 36 are removed.

The thickness of the wall of the cylindrical body 32 is 1 mm. Alternative embodiments may have thicker walls, for instance 4 mm, but it is preferable that the wall is sufficiently transparent that the contents of the receptacle 30 can be observed therethrough. Moreover, the body may be of any other suitable shape other than that of a cylinder.

The internal diameter of the body 32 is 22mm, although this can be varied in alternative embodiments, for example up to 100 mm. Moreover, in the present example, the body 32 is 90 mm in length, but other lengths of body 32 are also envisaged, for instance 50,150 or 250 mm.

An insert 40, as illustrated in Figure 5, comprises a fine mesh 42, supported on a circular collar 44. The insert 40 is placed at one end of the interior of the body 32 (the right hand end as illustrated in Figure 4).

The mesh 42 retains the contents of the receptacle 30 in the event of removal of the cap 36 at that end. The collar 44 is of a size suitable for it to form a tight fit within the body 32, to reduce the risk of the insert 40 accidentally falling out of place. The mesh 42 is sufficiently fine as to prevent egress of any growth medium contained in the receptacle therethrough, but not so fine that a filamentous microorganism is impeded from growing therethrough.

Figure 6 shows an alternative insert 40'having two crosspieces 42'supported on a collar 44'. The collar 44'is identical with the collar 44 illustrated in Figure 5. The crosspieces 42'extend diametrically and mutually perpendicularly across the collar 44'. The crosspieces 42'are operative, in use, to retain any contents of the receptacle 30 to the extent that the cap 36 at that end can be removed and replaced without significant shifting of the medium between the crosspieces 42'. It will be understood that the crosspieces 42'act to impede bulk movement rather than actively preventing it.

The use of an insert 40'with crosspieces 42t as illustrated in Figure 6 is particularly appropriate where the receptacle 30 is used to contain a highly particulate growth medium.

An alternative example of a receptacle 30'including a push-type fitting between a body 32'and a cap 36'is illustrated in Figure 7 of the drawings. In that example, the second embodiment of the insert 40', as illustrated in Figure 6, has been fitted at the end of the body 32'. The end of the body 32'is tapered on its exterior surface, and a corresponding interior tapered surface is formed on the cap 36'. The cap 36'can then be urged onto the end of the body 32'and, by means of friction and selection of suitable taper angles, the cap 36'can be retained on the body 32', forming a tight seal. Other than this pus-fit lid fitting arrangement, alternative arrangements are also envisaged, for example a bayonet fitting, and a push and twist fitting.

An alternative embodiment of the receptacle 30' illustrated in part in Figure 7 is further illustrated in Figure 8. A rectangular membrane 46'is incorporated into the wall of the body 32'. The membrane 46'is of a hydrophobic material, such as polytetrafluoroethylene or polysiloxane, which allows the transfer of gases therethrough, for example oxygen, which is required in many circumstances for the growth of microorganisms. Alternatively, or in addition, the membrane may be located within one or other of the caps 36'. As noted with respect to other components of the receptacle 30', the material selected for the membrane 46'should be suitable for withstanding sterilisation by means of hot water vapour at temperatures up to 121 C.

The membrane extends along the length of the body 32', up to a distance of 25 mm from each end of the body 32'.

With reference to Figures 9 to 14, a specific method of storing a microorganism will now be described.

As illustrated in Figure 9, the receptacle 30 previously described is filled with a suitable growing medium 50.

A growing medium should contain assimilable sources of carbon, nitrogen and mineral salts.

Assimilable sources of carbon, nitrogen and minerals may be provided by simple or complex nutrient sources.

Preferably, complex nutrient sources are used since they reflect more accurately the natural substrates on which the microorganisms grow. The great variety of nutrients present in complex sources may prevent the unwanted selection of genetic variants existing in a microorganism population which can occur by the placement of the population in an unnatural environment.

Complex sources of carbon, nitrogen and minerals may be provided by clean (not containing chemical residues such as fungicides or other pesticides) grains, cereals and seeds. Examples of such sources are tabulated below:

Cereals and Seeds Pulses Agricultural Other grains waste Quinoa Rye grass Aduki bcans Ground corn Wood chips cobs Maize Sunflower Whole Icntils Peanut shells Wheat bran Millet Linseed Soya Tea leaves Oat bran Rye Straw Other brans (especially including from wheat, corn, soybean, rye, rice, oat rye, rice and sorghum) Wheat Sawdust (especially hardwood) Oats Soil Rice Peat moss Ideally, a medium is formed of a mixture of the above materials, to provide optimum conditions for microorganism storage. Moreover, supplements may be added to the mixture, for example calcium sulphate (which separates individual grains), soy oil, yeast extract or peptone.

For example, for basidiomycetes such as Schizospora paradoxa, collected and isolated from British woodland, a medium consisting of quinoa is suggested. The use of the receptacle 30 to store a sample of that microorganism will now be described with reference to Figures 9 to 14 of the drawings.

Quinoa is soaked in boiling water, in the proportion of 1 kg quinoa to 1 litre of water. The mixture is left until it has absorbed all of the water. Then, the receptacle 30 is filled with the soaked mixture to a density of 0.8 g/cm3. Generally, a density within the range 0.6-1.0 g/cm3 would be acceptable.

Excessive compression of the mixture could inhibit filamentous growth of the microorganism, which could lead to differentiation of the microorganism. Insufficient compression could lead to voids appearing in the growth medium as water and medium is consumed by the microorganism, which would result in unsatisfactory growth of the microorganism. Once the receptacle 30 has been filled with the quinoa medium to the appropriate density and sealed by fitting the cap 36, the whole unit is sterilised by exposure to hot water vapour at 121 C for 40 minutes.

A new microorganism population is inoculated into the left hand end of the growth medium 50 as illustrated in Figure 9, i. e. the end not made inaccessible by the insert 40. Inoculation is effected by aseptically placing a sample 52 taken from an originating population grown in agar, or on grain or other nutrient source, and directly placing that sample 52 into the growth medium 50.

Inoculation could also be effected by injection of a liquid carrying the microorganism into the growth medium 50.

Once inoculation has taken place, the receptacle 30 is placed in conditions that allow optimum growth of the microorganism. These conditions include temperatures ranging from 10 C to 27 C, but ideally 18-25 C for filamentous fungi, in a clean environment such as a specially designed growth cabinet room or incubator so as to minimise risk of contamination from other microorganisms or invertebrate pests such as mites. The incubation conditions can include humidity and light regulation. A moderately humid environment will reduce the risk of medium in each receptacle from drying out.

Sporulation of some microorganisms is included by diurnal light cycles so light regulation may be advantageous if sporulation of a stored microorganism is required.

Figure 10 illustrates the-expected appearance of a receptacle 30 after this step has been performed.

Once colonisation of the receptacle 30 by the microorganism has been initiated the receptacle is transferred to storage conditions that reduce growth to a minimum. These conditions can be created in purposefully designed storage cabinets, rooms or incubators to provide clean conditions so as to minimise risk of contamination from other microorganisms or invertebrate pests such as mites. The temperature required to reduce growth should be above freezing and may vary from 4 C to 12 C but ideally 6-10 C. The storage conditions may include humidity and light regulation.

Figure 11 shows a receptacle 30 after storage thereof for a period of about six months.

When, as shown in Figure 11, the filamentous growth of the microorganism is nearing the end of the receptacle 30 in which is placed the insert 40, the cap 36 at that end is removed, and a left hand end of a further receptacle 30 is placed in abutment with that end. As shown in Figure 12, in order to retain the receptacles 30 in abutment, a collar 54 having internal thread 56 cooperable with the external threads 34 of the bodies 32 is engaged with those external threads 34. At this stage it should be noted that there may be benefit in making the threads at each end of the bodies 32 of opposite sense, i. e. one being left handed and the other being right handed. In that way, a simple twisting action of the collar 54 is sufficient in one direction to draw the receptacles 30 into close abutment, or in the opposite direction to urge the receptacles 30 apart.

Close abutment of the receptacles 30 is preferred, particularly because the growth medium 50 of the further receptacle 30 should contact the insert 40 of the original receptacle 30. In that way, the filamentous growth of the microorganism may continue uninterrupted.

Once filamentous growth ha$ established itself in the further receptacle 30 as shown in Figure 13, the original receptacle is discarded and a sterile cap 36 is placed at the left hand end of the further receptacle. The further receptacle 30 is then stored in the same way, which will result in a filamentous microorganism as illustrated in Figure 14.

The present invention is particularly applicable to organisms which do not produce a resting state such as spores. For these organisms, such as the basidiomycotina, ascomycotina and other sterile mycelia, the mycelium is the source of inoculum. Therefore, by collecting the youngest part of the colony, as a whole, in a further receptacle, the most vigorous part of the colony, and therefore the most viable part, is maintained.

A further example will now be described of use of the apparatus illustrated in Figures 3 to 14 for the maintenance and sub-culture of a microorganism. Phlebia deflectens is a basidiomycete which is commonly found growing on rotten deciduous wood in British woodland.

In preparation, the sub-culturing apparatus 30 is filled with a growth medium 50 consisting of swollen quinoa grain to a density of 0.8 gm/cm3. The quinoa grain is swollen by soaking for twelve hours in boiling deionised water in the proportion 1 kg of grain to 1 litre of water. Once the apparatus is filled, it is sterilised by the application of hot water vapour at 121 C for 40 minutes, followed by cooling under sterile conditions.

The spores of Phlebia deflectens are collected from fruit bodies in the autumn and germinated on agar containing suitable nutrients. Once a viable colony has been established on the agar, a sample can be taken from the growing edge of the colony using a sterile scalpel. The sample 52 is placed at one end of the sub-culturing receptacle 30 prepared as described above using standard microbiological techniques under sterile conditions. The apparatus 30 is then labelled and stored at 22 C for four weeks. After this time, it has been observed that mycelium grows uniformly through the medium 50 to a total length of 15 mm and the apparatus 30 can then be transferred to a controlled dark environment at 10 C.

After a further four weeks, it has been observed that the mycelium grows a further 5 to 8 mm. This indicates that a drop of temperature from 22 C to 10 C reduces the growth of the mycelium by at least 50%. After 24 weeks in the controlled dark environment, the mycelium is likely to have reached the end of the growth medium 50 and a further receptacle 30 can then be added for further sub-culture.

Phlebia deflectens is a basidiomycete that does not produce any resting stage, such as spores, in laboratory culture. Moreover, its growth and viability are observed to become curtailed when grown and sub-cultured on agar for several months. The method described above using the sub-culturing receptacle 30 maintains the vigour and viability of the Phlebia deflectens microorganism, and rapid growth has been observed when a sub-culture of the mycelium is then transferred to a Petri dish containing agar medium for viability testing.

Phlebia deflectens grows at moderate rate in the subculturing receptacle 30. On the other hand, other basidiomycetes and ascomycetes grow at a faster rate than the above example, for example 250 mm in four weeks at 22 C. For such robust and fast growing microorganisms, a cooler storage temperature of 6 C is necessary to reduce growth sufficiently to prevent the microorganism from reaching the end of the growth medium 50, and thus requiring further sub-culture, at intervals of less than six months. Otherwise, the storage method involving the use of the receptacle 30 would become quite labour intensive.

Moreover, since the apparatus may be of relatively simple construction, it provides an ideal solution to the problem of preservation of a local biology, especially in developing countries. In recent times, ecological campaigns here have resulted in countries becoming more aware of their diverse local biology, and efforts have been made for the preservation thereof. The preservation of local microbiology is an integral part of that process.

The illustrated embodiment can be stored easily on racks within an incubator. No sampling takes place and so there is less danger of a reduction in heterogeneity of a microorganism stored in accordance with the invention.

Although the embodiment illustrated in Figures 3 to 14 demonstrates how a single receptacle 30 can be connected to another receptacle 30 on a temporary basis to allow for the propagation of a microorganism through growth medium, the invention also contemplates the arrangement illustrated in Figure 15. In that arrangement, a plurality of receptacles 30 of various lengths are connected together by means of collars 56, each receptacle 30 has an insert 40 as previously described, and the end receptacles are closed by means of caps 36.

A microorganism as shown in Figure 15 can be allowed to grow from one end of the arrangement towards the other, and samples may be taken from the colony of the microorganism by disconnecting the arrangement and removing one or more of the receptacles 30 as required.

The above described apparatus and procedures for subculturing and maintaining microorganisms in a genetically stable state can be used to improve the process of culturing microorganisms in a liquid fermentation system either as a surface culture or as a submerged culture or any other type of fermentation for use in a biotechnological process.

Fermentation procedures normally employ one or more growth or seed stages to increase microbial biomass to a level which can be used to inoculate the final production medium designed to yield optimal levels of a desired metabolite. Inoculation levels of biomass to subsequent fermentations is critical to the optimal growth of a microorganism and overall productivity (grams product/unit biomass/unit time) of the process.

A growth stage is typically initiated by the introduction of a small amount of microorganism, which has been cultured on an agar medium, to a 250 ml Erhlenmeyer flask containing 30-50 ml of liquid growth medium. The organism is then cultivated by agitation at a desired temperature (20-40 C) for a period dependent on the growth rate of the organism (range 2-10 days). This culture volume can be increased by a factor of ten at each stage by transferring to ten times the volume of fresh medium and so on.

The seed stage is used to inoculate production medium in which the organism is cultivated to produce the desired product which may be extracted and purified and which may have pharmaceutical, agrochemical or other properties.

A problem encountered with the above described fermentation process is that several seed stages are required to generate sufficient biomass to inoculate the production medium.

The sub-culturing receptacle 30 described above is advantageous in that it can be used to generate sufficient microbial biomass for direct inoculation of production medium. The inoculum may be prepared in a number of ways prior to inoculating the production medium, for example: i) The microbial biomass mixed with substrate (from the sub-culturing apparatus) can be used as a direct inoculum. ii) The microbial biomass mixed with substrate can be gently agitated with an aliquot of production medium or other suitable liquid medium. This suspension is then allowed to settle or is centrifuged at very ldw speed so that the heavier solid substrate materials are sedimented and removed leaving a suspension of biomass which is used as an inoculum. iii) The microbial biomass mixed with substrate can be suspended in an aliquot of production medium or other suitable liquid medium and gently blended under aseptic conditions using a Waring (or similar) blender. This procedure produces a substantially homogeneous suspension of biomass and substrate with a much higher inoculum potential i. e. the gentle blending step releases all the biomass from the substrate and breaks it up to produce more growing points.

In each case the optimal inoculation level will be in the range 1% to 10% weight of biomass combined with substrate (from sub-culturing apparatus) to volume or weight of reproduction medium. An ideal inoculation level is 3%- 5%.

The following example describes the use of microbial biomass generated in the sub-culturing apparatus as a direct inoculum for the production of the pharmaceutical compound mevinolin by the organism Aspergillus terreus Thom ATCC 20542.

A terreus can be maintained and sub-cultured using the above described receptacle 30 using the grain quinoa as a growth medium. As described previously, the receptacle 30 is prepared by mixing quinoa with boiling deionised water and leaving the mixture for 12 hours. The swollen grain is then packed into the sub-culturing receptacle 30 to a density of 0.8 g per cm3. The organism can be maintained in this growth medium 50 at a temperature of 10 C until required for initiating a fermentation process. By raising the temperature to 25 C, the growth rate of the organism can be increased and multiple subcultures made onto fresh grain (in separate sub-culturing receptacles) as a means of quickly generating microbial biomass for inoculation purposes.

For fermentation, a liquid medium A is prepared in accordance with the following composition:

grams/litre Sheftone N-Z soy peptone (Sheffield Products) 10 Malt extract (Oxoid L39) 21 Glycerol (Sigma-AIdrich) 1 : Deionised water add and make up to 1 litre Adjust pH to 6.3 using 2N NaOH/2N HCI 60 ml of the above medium is transferred to a 250 ml Erhlenmeyer flask, stoppered with a polystyrene foam bung and autoclaved at 121 C for 20 minutes. 20g of A terreus culture growing on quinoa substrate is then removed from the sub-culturing receptacle 30, mixed with 20 ml of sterile medium A and aseptically blended (up to five 2 second bursts) using a Waring blender. 6 ml of the homogenised inoculum is then aseptically transferred to the Erhlenmeyer flask which is incubated at 25 C under static conditions for a further 15 days.

Mevinolin is assayed in both the broth and methanolic extracts of the separated fungal biomass using established procedures employing high performance liquid chromatography (HPLC). Using this method, average levels of mevinolin in the biomass extracts have been recorded as 414 mg/1, while the average level measured in the cell-free fermentation broth was 224 mg/1.

The recorded levels of mevinolin produced using this procedure are comparable with standard inoculation procedures using separate liquid fermentations (usually using a different medium composition) to generate inoculum for the production medium. The mevinolin can then be isolated and encapsulated for human consumption in accordance with established procedures.

Claims

CLAIMS: 1. A process for growing an organism, comprising: providing a first vessel containing a first body of growth supporting material and causing the organism to grow in said material in said first vessel towards a first location; providing a second vessel containing a second body of growth supporting material ; and permitting said organise to grow from the body of material in the first vessel into the body of material in the second vessel through said first location.
2. A process according to claim 1, comprising connecting said second vessel to said first vessel and permitting said organism to grow into said second body of material while said vessels are connected together.
3. A process according to claim 2, comprising disconnecting said first vessel from said second vessel after said organism has begun to grow in said second body of material.
4. A process according to claim 1,2 or 3, comprising causing said organism to grow in said second body of material towards a second location therein, providing a third vessel containing a third body of growth supporting material and permitting said organism to grow from said second body of material into said third body of material through said second location.
5. A process according to claim 4, wherein said first vessel is used as said third vessel.
6. A storage device for a microorganism comprising: growth means for viably supporting a microorganism; characterised in that said storage device includes a facility for presenting said population substantially in its entirety for subculture.
7. A storage device in accordance with claim 6 and further comprising a receptacle supporting the growth medium.
8. A storage device in accordance with claim 7, wherein the growth medium defines a substantially elongate growing path.
9. A storage device in accordance with claim 7 or claim 8, wherein the receptacle includes attachment means for attachment of said device to further culturing apparatus.
10. A storage device in accordance with claim 9, wherein the attachment means is operable to engage the growth medium with growth medium of a further storage device in accordance with claim 6.
11. A method of storing a microorganism comprising the steps of: providing a growth medium ; growing a population of a microorganism on or in the growth medium; and sampling said population for subculture; characterised in that said step of sampling includes sampling across substantially the whole population of the microorganism.
12. A method of storing a microorganism including the steps of: providing a growth medium ; and growing a microorganism on or in the growth medium; characterised in that said step of growing includes training said microorgani$m substantially in a predetermined direction.
13. A method in accordance with claim 12 including the step of sampling across said predetermined direction for subculture of said microorgantsm.
14. A method in accordance with claim 11 or claim 13, wherein said growing step includes the step of growing said population towards a predetermined location in the growth medium.
15. A method in accordance with claim 14, wherein said sampling step includes the step of placing a sampling medium adjacent said predetermined location for continuing growth of said microorganism thereon.
16. A method of manufacturing a metabolite comprising the steps of : storing a microorganism in accordance with the method of any of claims 1 to 5 or 11 to 15; extracting a sample of said microorganism; and subjecting said sample to conditions suitable for metabolism; and extracting metabolite from said sample.
17. A pharmaceutical preparation including a metabolite the product of a method in accordance with claim 16.
18. A method of preparing a pharmaceutical preparation including the step of isolating a metabolite produced in accordance with claim 16.
19. A storage device for a microorganism including a housing, growth medium within the housing, and first and second locations on the growth medium, such that a microorganism can be grown from the first location towards the second location where subculturing of substantially the entire population can be effected.
20. A storage device in accordance with claim 19, wherein the housing is tubular.
21. A storage device in accordance with claim 20, wherein the housing is cylindrical.