EP1165747A1

EP1165747A1 - Apparatus for preserving microorganisms

Info

Publication number: EP1165747A1
Application number: EP00901817A
Authority: EP
Inventors: Neil BioDiversity Limited PORTER; Frances Mary BioDiversity Limited GIAQUINTO
Original assignee: BioDiversity Ltd
Current assignee: BioDiversity Ltd
Priority date: 1999-02-08
Filing date: 2000-02-08
Publication date: 2002-01-02
Also published as: GB2346379A; GB9902757D0; WO2000047709A1; JP2002536010A; GB2346379B; AU2311200A

Abstract

Subculturing apparatus (30) comprises a receptacle for supporting a growth medium (5) for culturing of a microorganism. The microorganism is entrained to grow in a predetermined direction, towards an end of the growth medium (50), where a further apparatus (30) can be placed adjacent thereto. The microorganism can then grow into the growth medium of the further apparatus. The method of storing a microorganism for use in microbiological processes is also described, as is a method of fermentation of a stored microorganism for the production of biochemicals such as pharmaceuticals or agrochemicals.

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", 2^nd 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 sub- culturing 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 are 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 conditions 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 Drying", 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.

Moreover, 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 substrates.

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 which 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 42' 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 push-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:

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. Peptone is a hydrolysed protein which can

originate from animal or plant products.

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 induced 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 has 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 a moderate rate in the sub-

culturing 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. Connection and disconnection of receptacles 30 is to be

carried out in a sterile manner, for instance in the

presence of a sterile airflow, or in the near vicinity of

a naked flame, for example a Bunsen burner flame.

The above described apparatus and procedures for sub-

culturing 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 low speed so that the heavier

solid substrate materials are sedimented and

removed leaving a suspension of biomass which is

used as an inoculum.

iϋ) 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%-

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

Thorn 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 sub¬

cultures 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:

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.

The foregoing description demonstrates that the

illustrated embodiment is capable of being used to

maintain an organism, of a filamentous nature, by

repeated subculturing without suffering from the effects

of genetic segregation. Moreover, contamination can be

limited by maintaining sterility of the body and the

caps, and by exercising caution when connecting and

disconnecting bodies from each other.

A wide range of growth media can be used with the

apparatus of the illustrated embodiment. However, it

will be appreciated that further growth media, including

synthetic growth media, could also be used in conjunction

with the apparatus. As synthetic growth media improve,

their use may be desirable in terms of cost, reliability

and sterility. By using vessels of different lengths, sub-samples of the

entire population can be effected easily. By using a

particularly short vessel at a particular point in a

chain of vessels, a small section of growth medium can be

removed from the chain for further analysis of the

organism residing therein. By taking a sample at or near

^• to the growing end of the organism, the most viable

biomass can be removed and used in inoculation of further

apparatus .

Claims

CLAIMS :

1. A process for growing an organism, comprising: providing a first vessel (30) containing a first body of growth supporting material (50) and causing the organism to grow in said material in said first vessel towards a first location; providing a second vessel (30) containing a second body of growth supporting material (50); and permitting said organism to grow from the body of material (50) in the first vessel (30) into the body of material (50) in the second vessel (30) through said first location.

2. A process according to claim 1, comprising connecting said second vessel (30) to said first vessel (30) and permitting said organism to grow into said second body (50) of material while said vessels are connected together.

3. A process according to claim 2 wherein said connecting step is performed in a sterile manner.

4. A process according to claim 2 or claim 3, comprising disconnecting said first vessel (30) from said second vessel (30) after said organism has begun to grow in said second body of material.

5. A process according to claim 4 wherein said disconnecting step is performed in a sterile manner.

6. A process according to any one of claims 1 to 5 wherein said step of causing said organism to grow comprises causing said organism to grow in said first vessel (30) and into said second vessel (30) in a predetermined growing direction.

7. A process according to claim 6 wherein said first and second vessels (30) are, in the predetermined direction, of different lengths.

8. A process according to claim 7 wherein said second vessel (30) is shorter than said first vessel (30), said process further comprising said step of removing said second vessel, after said step of permitting said organism to grow therein, for sub-sampling thereof.

9. A process according to any one of claims 1 to 8, comprising causing said organism to grow in said second body of material (50) towards a second location therein, providing a third vessel (30) containing a third body of growth supporting material (50) and permitting said organism to grow from said second body of material (50) into said third body of material (50) through said second location.

10. A process of storing a microorganism comprising the steps of: providing a growth medium (50); growing a population of a microorganism on or in the growth medium (50); and sampling said population for subculture; characterised in that said step of sampling includes sampling across substantially the whole population of the microorganism.

11. A process of storing a microorganism including the steps of: providing a growth medium (50); and causing a microorganism to grow on or in the growth medium; characterised in that said step of causing said organism to grow includes training said microorganism substantially in a predetermined direction.

12. A process in accordance with claim 11 wherein said step of causing said organism to grow includes causing said organism to grow towards a predetermined location, and said method further comprising sampling at said predetermined location to obtain a sample of said organism across substantially the entire population thereof.

13. A process in accordance with claim 12, wherein said sampling step includes the step of placing a sampling medium (50) adjacent said predetermined location for continuing growth of said microorganism thereon or therein.

14. A process of manufacturing a metabolite comprising the steps of: storing a microorganism in accordance with the method of any of claims 1 to 13; extracting a sample of said microorganism; subjecting said sample to conditions suitable for metabolism; and extracting metabolite from said sample.

15. A method of preparing a pharmaceutical preparation including the step of isolating a metabolite produced in accordance with claim 14.

16. A pharmaceutical preparation including a metabolite the product of a method in accordance with claim 14.

17. A storage device for use in the process of any of claims 1 to 15 including a housing (30), growth medium (50) within the housing (32), 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.

18. A storage device in accordance with claim 17, wherein the housing (32) is tubular.

19. A storage device in accordance with claim 18, wherein the housing (32) is cylindrical.

20. A storage device in accordance with any one of claims 17 to 19 wherein the housing (32) is of a sterilizable material.

21. A storage device in accordance with any one of claims 17 to 20 wherein said housing (32) has formations (34) at said first and second locations of the growth medium each formation being suitable to engage with a cooperating formation of another of said storage device (30), for propagation of microorganism therebetween.

22. A storage device in accordance with any one of claims 17 to 21 wherein said housing (32) comprises means (40) for retaining said growth medium.

23. A storage device in accordance with claim 22 wherein said retaining means (40) has at least one aperture defined therein for passage of microorganism therethrough .

24. A storage device in accordance with claim 23 wherein said retaining means (40) comprises at least one retaining member (42') across said vessel.

25. A storage device in accordance with claim 24 wherein said retaining means (40) comprises a reticular member (42) across said vessel.

26. A storage device in accordance with any one of claims 17 to 25 wherein said growth medium (50) comprises a natural foodstuff.

27. A storage device in accordance with claim 26 wherein said foodstuff is a vegetal foodstuff.

28. A storage device in accordance with claim 27 wherein said growth medium (50) comprises a quantity of a cereal.

29. A storage device in accordance with claim 27 or claim 28 wherein said growth medium (50) comprises a quantity of seed.

30. A storage device in accordance with any one of claims 27, 28 or 29 wherein said growth medium (50) comprises a quantity of a pulse.

31. A storage device in accordance with any one of claims 27 to 30 wherein said growth medium (50) comprises an agricultural crop byproduct.

32. A storage device in accordance with claim 31 wherein said agricultural crop byproduct comprises at least one of ground corn cobs, peanut shells, tea leaves and straw.

33. A storage device in accordance with any one of claims 26 to 32 wherein said growth medium (50) comprises at least one of calcium sulphate, soy oil, yeast extract and peptone .

34. A storage device in accordance with any one of claims 17 to 33 wherein said growth medium (50) is sterile.

35. A storage container for use in the storage device (30) of any one of claims 17 to 35, defining a cavity within which growth medium can be contained, said container (32) comprising first and second access means between which growth medium can extend in use, for growth of an organism between said first and second access means in use.

36. A container in accordance with claim 35 including first and second closure means (30), removably closing said access means in use.

37. A storage device for use in the process of any one of claims 1 to 15 comprising: growth medium (50) for viably supporting a microorganism; characterised in that said storage device includes a facility for presenting said population substantially in its entirety for subculture.

38. A storage device in accordance with claim 37 and further comprising a receptacle (32) supporting the growth medium.

39. A storage device in accordance with claim 38, wherein the growth medium defines a substantially elongate growing path.

40. A storage device in accordance with claim 38 or claim 39, wherein the receptacle (32) includes attachment means (34) for attachment of said device to further culturing apparatus .

41. A storage device in accordance with claim 40, wherein the attachment means (34) is operable to engage the growth medium with growth medium of a further storage device in accordance with any one of claims 37 to 40.