WO2000045860A1

WO2000045860A1 - Sterile substrate and process for producing it

Info

Publication number: WO2000045860A1
Application number: PCT/GB2000/000340
Authority: WO
Inventors: Peter Hawton
Original assignee: Mycocell Technologies Limited
Priority date: 1999-02-05
Filing date: 2000-02-04
Publication date: 2000-08-10
Also published as: AU2446500A; EP1148890A1; GB9902639D0

Abstract

A process for producing a sterile substrate suitable for use in the cultivation of microorganisms, especially mushrooms, comprises exposing to heat or radiation, or submitting to chemical sterilisation, a substrate which has a moisture content of 30 % by weight or less and comprises a particulate base material which is capable of absorbing or entrapping water such as sawdust, cereal grain, a porous ceramics material or a water-absorbing polymer. A sterile substrate which has a moisture content of 30 % by weight or less and comprises a particulate base material as defined above is also provided.

Description

STERILE SUBSTRATE AND PROCESS FOR PRODUCING IT

The present invention relates to sterile substrates suitable for use in microorganism cultivation, especially mushroom cultivation, and a process for their production.

The production of mushrooms worldwide is a vast business worth hundreds of millions of US dollars per annum. Mushroom cultivation usually involves, as a first step, generating a mushroom spawn which consists of a carrier material inoculated with mushroom mycelium. The purpose of the spawn is to boost the mycelium to a state of vigour where it can be launched into bulk substrates. The carrier material is typically cereal grain or enriched sawdust. It serves both as a vehicle for evenly distributing the mycelium and as a nutritional source which can support the expansion of the mycelial mass. The mushroom spawn is then transferred to a suitable moistened substrate for further growth.

The intended substrate material must be sterilised, before mushroom spawn is transferred to it, in order to avoid contamination by competitor microorganisms such as other fungi or bacteria. Sterilisation is defined in the textbook "General Microbiology" by Stanier, Adelberg and Ingraham (4^th Edn), page 25, as a treatment thatβ-ees the treated object of all living organisms.

The sterilisation of substrates for mushroom cultivation is usually carried out using steam. In a conventional method using sawdust as the substrate, the sawdust is moistened until its water content is from 65 to 70% by weight. It is then filled into bags made of a polymeric material, such as polypropylene, which can withstand the temperature of sterilisation. The bags of moistened sawdust are typically subjected to steam sterilization in autoclaves which can be filled with steam at a pressure greater than atmospheric. Steam sterilisation is therefore usually achieved at temperatures considerably above the boiling point of water. The particular sterilisation conditions used will depend partly on the nature of the substrate. For instance, a fresh hardwood sawdust substrate would typically be sterilized for two to three hours at 17 psi whereas the same sawdust supplemented with bran (as a source of nitrogen) would need a longer period. This is because, as the percentage of nitrogen supplement relative to the base substrate increases, the likelihood of contamination increases. The need for thorough sterilization is therefore even more critical. There are many drawbacks associated with this conventional steam sterilization technique. For instance, prolonged steam sterilization at high temperatures can give rise to complex chemical transformations in the substrate material. Sawdust, for example, takes on a dark brown colour and a distinctly different odour signature when it has been subjected to steam sterilization under pressure for more than 5 hours. The sawdust tends as a result to resist decomposition by the mushroom mycelium, which is detrimental to mushroom growth. In addition, the sawdust is likely to be depleted in important minerals and trace elements as a result of steam sterilization. It is difficult to replace such substances following sterilization, though, because by then the substrate is wet and cannot readily absorb further additives.

Another disadvantage of steam sterilisation is that when the hot bags of sawdust are withdrawn from the autoclave they cool down and the air inside them contracts. This encourages external air to be drawn into the bags, which may lead to contamination of the contents. The present invention aims to overcome these and other drawbacks of the conventional process of steam sterilization. It accordingly provides a process for producing a sterile substrate suitable for use in microorganism cultivation, which process comprises exposing to radiation or heat, or submitting to chemical sterilisation, a substrate which has a moisture content throughout of 30% by weight or less and comprises a particulate base material capable of absorbing or entrapping water. In one embodiment of the invention the process comprises exposing to radiation a substrate which has a uniform moisture content throughout of 12 % by weight or less and comprises a particulate base material capable of absorbing or entrapping water. The sterile substrate produced by the process of the invention as defined above has a long shelf life. It has the added advantage that, since it is relatively dry, it is capable of absorbing additives and can thus be enriched, as will be described in more detail later on.

The particulate base material should have a moisture content of 30% by weight or less, typically 25 % by weight or less, preferably 20 % by weight or less, more preferably 12 % by weight or less. It is frequently preferable for the moisture content to be uniform throughout the body of the substrate, especially when radiation is used to sterilise the substrate. If the specified moisture level instead represents an average value there will be areas within the material where the moisture content is higher than the specified level (as well as areas where the moisture content is much lower). Water is an insulator to radiation, so a radiation dose which can satisfactorily penetrate sawdust having a moisture content of, say, 30 % by weight would not fully sterilise these wetter areas. A higher dose would therefore be needed, which may be undesirable in terms of the cost and thus overall economics of the process. It has been found, for instance, that a dose of about 35 killer Grey (kG) of radiation is typically suitable for sterilising a substrate material with a water content of about 30

% by weight, whereas a dose of about 10 killer Grey (kGy) of radiation is suitable for sterilising a substrate material with a water content of about 10% by weight.

The process of the invention is applicable to the cultivation of any type of microorganism. Mushroom cultivation is particularly preferred but the process is equally applicable to other microorganisms such as yoghurt cultures, yeast, moulds and bacteria. The substrate is selected according to the type of microorganism which it is desired to cultivate, subject to the requirement that it should comprise a particulate base material capable of absorbing or entrapping water. In the case of mushroom cultivation the substrate may be a carrier material which is to be inoculated with mushroom mycelium to form a mushroom spawn, or it may be a bulk substrate into which a mushroom culture, for instance in the form of spawn, is subsequently injected for further growth.

Typical examples of the substrate material for mushroom cultivation include wood fibre, usually in the form of sawdust or wood shavings; other fibrous cellulose- based natural materials such as coconut fibre; and cereal products. The term "cereal products" as used herein refers to the grain or straw of cereal and also to the seeds of other food crops such as peas, beans, maize and clover. Suitable cereals include rye, oats, millet, barley, wheat, rice, hemp and flax. Preferred examples of cereal products include the straw of hemp, flax and wheat.

Alternatively the particulate base material may be, for example, a material known as a superabsorber which is a water-absorbing polymer such as a cross-linked polyacrylate. Such a material may, for instance, be potassium-based or sodium- based.

The base material may instead be a water-absorbing or water-entrapping mineral or ceramics substance, such as vermiculite or perlite. Vermiculite is a member of the phyllosilicate group of minerals and has the ability to expand to many times its original volume on heating. Perlite is a naturally occurring siliceous rock which, when heated to a suitable temperature in its softening range, expands to from 4 to 20 times its original volume.

Alternatively the base material may be any other natural or synthetic material which is porous or which is capable of absorbing or entrapping water, and which is in the form of particles.

When wood fibre is used as the substrate, the wood is preferably a fast decomposing hardwood. Suitable examples include alder, poplar, ash, elm, beech, birch and cottonwood. Sawdust of these woods which is suitable for use in the process of the invention may be obtained, for instance, from furniture manufacturers or sawmills. The wood may alternatively be a composted softwood.

When the substrate material is other than one which contains intrinsic nutrients, such as a cereal product, it may if desired include an added source of nitrogen or protein. This may be, for instance, cereal bran, soybean bran, yeast, soy oil or peptone. The cereal bran is preferably the bran of rice, rye, wheat, corn or oats.

It has long been known that growth of the mushroom mycelium can be promoted by the addition of a calcium salt to the substrate, for instance as taught in US patent 2044861. It is therefore possible to add a source of Ca²⁺ ions to the substrate employed in the process of the present invention. Various calcium salts may be used for this purpose, for instance calcium chloride, calcium nitrate, calcium hydroxide, calcium carbonate or calcium sulphate. The sterile substrate produced in accordance with the process of the present invention is moistened with water when the microorganism culture is finally added to it, since water is required to support growth. The process of the present invention as defined above may therefore further comprise adding water and a microorganism culture to the sterilized substrate. In the field of mushroom cultivation the water and microorganism culture may conveniently be added in the form of liquid mushroom culture inoculum. This typically comprises mushroom mycelium in an enriched liquid suspension. In a preferred embodiment, therefore, the process of the invention as defined above further comprises adding liquid mushroom culture inoculum to the sterilised substrate.

The water is typically enriched in order to provide an environment for optimum microorganism growth. An important advantage of the process of the present invention is the fact that the substrate is relatively dry following sterilization. This means that its capacity to absorb water at the time of inoculation with the microorganism culture, for instance liquid inoculum, is much greater than that of conventional wet-sterilized substrates. The added water is able to penetrate inside the individual particles of the substrate material rather than residing on the surface only. This in turn facilitates the introduction of nutrient substances which will support microorganism growth, since such substances can simply be dissolved or dispersed in the water which is added to the sterile substrate at the time of inoculation. The added nutrient substances are drawn with the water into the interior of each substrate particle, thereby enabling subsequent growth of the microorganism, for instance mushroom mycelium, to take place inside each substrate particle as well as on the particle surface. A far greater food source is therefore made available for the microorganism than is possible with substrates sterilised by conventional processes.

It is possible to select the additives for the water in such a way as to mimic the particular profile of nutrients which is present in the microorganism's natural growing environment, for instance compost in the case of field mushrooms or wood in the case of wood-decayer mushrooms such as shi-itake (oyster) mushrooms. The sterile substrate produced in accordance with the present invention, being relatively dry, can therefore be customised to meet the requirements of the particular microorganism being cultivated in a way which is not possible when the substrate particles are already saturated with water. Indeed, a substrate which would not normally support microorganism growth can be transformed into an attractive environment for a particular species simply by an appropriate formulation of the

"recipe" of nutrients which is added to the substrate via the water. As indicated above, those nutrients will end up evenly distributed throughout the interior of the individual particles of the substrate, thereby encouraging uniform growth of the microorganism at not only the surface but also the interior of the particles. The source of nitrogen and source of Ca²⁺ salts, which as described above may be added to the substrate material prior to sterilization, may alternatively be introduced into the substrate as part of the recipe of nutrients which is added with the water after sterilization by the process of the invention. One advantage of this is that it opens up the possibility of using sources of nitrogen which readily disperse or dissolve in water but are not used in conventional processes because they cannot conveniently be added in a dry form to the substrate material prior to sterilisation.

Another related advantage of generating a dry sterile substrate by the process of the invention is that the acidity of the microorganism growing environment can be controlled. Different types of microorganism, for instance different mushroom species, require different pH values to flourish, but if the substrate is wet to start with it is very difficult to achieved a particular target pH throughout the bulk of the material. However, if the pH of the water added to the sterile substrate produced by the process of the invention is modified, the pH at the interior of the substrate particles will be correspondingly modified when the water is absorbed. In one aspect of the invention a mushroom culture is added to the sterile substrate produced by the process of the invention in the form of a liquid inoculum. In that case the mycelium can be drawn into the interior of the relatively dry substrate particles in the same way as the added water and "recipe" of nutrients, as described above. This promotes subsequent mycelial growth inside the particles of the substrate as well as on their surface.

When the base material used in the process of the invention is a cereal product the substrate can advantageously further comprise, in addition to the cereal product, a second particulate base material which is porous and capable of preferentially absorbing water. Cereal grain is relatively slow at absorbing water, so when water is added to a bag of sterilized dry substrate which comprises cereal grain it is likely to accumulate initially at the bottom of the bag and only be absorbed gradually. A disadvantage of this is that the lying water tends to attract contamination. This problem can be alleviated, however, by adding a second porous base material, which has a greater capacity to absorb water than the cereal product, to the substrate at the beginning of the process of the invention. This second material absorbs most of the water which is added to the sterile substrate at the time of inoculating the substrate with mushroom culture, and retains the water until it can be absorbed by the cereal product. One example of a suitable material for use in this context is vermiculite, which is a super- heated mineral in finely divided form and is a conventional component of potting compost. The process of the present invention is particularly applicable to the cultivation of all kinds of mushroom including white mushrooms, gourmet mushrooms and medicinal mushrooms. Examples of the more popular mushrooms include the following: White button mushroom (Agaricus), Shi-itake (L edodes), oyster (Pleurotus), oyster (P. pulmonarius), oyster (P. ostreatus), oyster (P. eryngii), oyster (P. djamor), oyster

(P. cystidiosus),. nameko (Pholiota), bunashimeji (Hypsizygus), enoki (Flammulina velutipes), black poplar mushroom (Agrocybe aegerita), Ganoderma lucidum, Psilocybe, Grifola frondosa, lion's mane (Hericium) and wood ears (Auricular id). If the substrate material to be used in the process of the invention is sawdust obtained from a furniture manufacturer, the sawdust will usually derive from a solid piece of wood which has been seasoned and dried under controlled conditions. The moisture content of the sawdust in percentage by weight terms will therefore be known. However, if the substrate material is not already sufficiently dry to start with, or if its moisture content is not known, it must be dried until the moisture content is 30 % by weight throughout or less. In that case the substrate material is preferably dried in an oven until there is no further weight loss, indicating that all the water has been expelled and the material is completely dry.

The moisture content of a substrate material can be determined by taking a sample and weighing it, drying it in an oven for at least one hour and then re- weighing it. The reduction in weight corresponds to the amount of water lost. Thus, if the sample weighed lOOg before drying and only 40g after drying, 60g of water was lost and the water content of the starting material was 60% by weight. In this example further drying of the starting material would be required before it could be used in the process of the invention. The desired target weight loss for a sample weighing lOOg is 30g or less, indicating that the moisture content of the material has reached 30 % by weight or less.

When the dried substrate material is to be exposed to radiation to effect sterilisation it is usually placed within a lining material which resists the ingress of microorganisms, for instance inside a plastic bag. Such a bag may be made of any polymeric material which is stable towards radiation, for instance polyethylene. The bags are usually chosen so that their volume is several times bigger than the volume of the contents, so that the bag can be folded and sealed in such a way as to avoid post-sterilisation contamination. If desired, one or more bags may then be placed for additional protection inside a second bag, which is also folded and sealed. The or each bag is then typically placed in a cardboard box. The cardboard box is closed and may then be exposed to radiation. Any form of radiation capable of effecting sterilisation may be used. Typically the radiation is electron beam or gamma radiation, but in practice gamma rays are preferred because they achieve a higher degree of penetration of the substrate material. A preferred source of gamma rays is Co⁶⁰. The dose of radiation is typically from 5 kGy to 100 kGy, for example from 8 kGy to 100 kGy, more preferably from 10 kGy to 100 kGy. In one preferred aspect of the invention the upper limit is 60 kGy.

The process thus described yield a sterile dry substrate in a sealed bag, which is very convenient for further handling. The substrate may, for instance, be wetted and inoculated directly with mushroom culture. The process of the invention may thus include the further step of adding water and a mushroom culture to the sterilised substrate. As indicated above, the added water may be enriched with a mushroom growth promoting gas such as oxygen, or with one or more minerals, nutrients, enzymes or bacteria which support the growth of mushrooms. In cases where the substrate has a moisture content of 12% by weight or less the shelf-life is almost indefinite so the bags of dry sterile substrate may be stored for future use or transported elsewhere to be inoculated with a mushroom culture.

When the dried substrate is to be exposed to heat, the same procedure is followed as would be used for conventional steam sterilisation. The dried substrate is therefore placed in an autoclave or an oven. The temperature in the autoclave is typically of the order of 120°C. The time required for the heating will depend on the nature of the substrate, for instance its density and its bio-count. Typically the time period is from 5 to 20 hours, more usually from 10 to 20 hours, it being understood that the specific time period must be individually selected depending on not only the nature of the substrate but also the equipment being used for heating.

When the dried substrate is to be submitted to chemical sterilisation, it may be treated with a chemical such as sodium hypochlorite (bleach) or formaldehyde and subsequently dried. Alternatively the substrate may be treated with a gas or with a mixture of gases. Suitable examples of gases include formalin, ozone and methyl bromide.

As used herein the term "sterile" means substantially free, more typically totally free, of all living organisms. It may also mean substantially free of competitor microorganisms. Preferably it means, in accordance with the definition quoted in the introduction above, that the probability that the substrate material contains even one surviving microorganism is infinitesimally small.

Inoculation of the sterile substrate is typically carried out in a laminar flow cabinet, which is a conventional structure that allows an operator to work in a sterile environment. The box is opened and each of the bags inside it is opened. A measured amount of mushroom culture, for instance spawn, is then injected into the sterile substrate and shaken. Water is added to achieve a moisture content within the range of 45-50% by weight if the substrate is grain or 65 to 70% by weight if the substrate is other than grain. As indicated above, minerals and nutrients may, if desired, be dissolved or dispersed in the water before it is added to the inoculated substrate. Certain bacteria which promote mushroom growth may also be added to the water.

In an alternative embodiment, the mushroom inoculum in liquid form may be added to the water prior to addition of the latter to the dry substrate. In either case, the bag is sealed once the moisture content has reached the desired level.

The process of the invention as defined above may also be conducted in-line as a continuous process. This process is conveniently carried out in a machine which is totally enclosed in concrete in order to provide protection against the radiation used. The machine contains a conveyer belt or a screw elevator with an adjacent radiation source, for example a rod of Co⁶⁰. The substrate material is dried and then passed along the conveyer belt or screw elevator where it is exposed to radiation from the source. The substrate may then be retrieved and bagged. Alternatively, a mushroom culture and water with optional added nutrients may be added directly to the sterilised substrate, prior to bagging, as part of the continuous process. This latter embodiment has the advantage that intervention by human operators is minimised, thereby reducing the risk of further contamination. In the continuous process embodiment the radiation dose is related to the speed of the conveyer belt or the screw elevator.

The present invention further provides a sterile substrate suitable for use in microorganism cultivation, especially mushroom cultivation, which has a moisture content of 30 %.by weight or less and comprises a particulate base material which is capable of absorbing or entrapping water. In a preferred embodiment the substrate has a uniform moisture content throughout of 12 % by weight or less. The substrate may further comprise a source of nitrogen, for instance a source of nitrogen as described above.

The invention also provides a sterile substrate suitable for use in microorganism cultivation, especially mushroom cultivation, which has a moisture content throughout of 30 % by weight or less and comprises a cereal product and a second particulate base material which is capable of preferentially absorbing or entrapping water. In a preferred embodiment the substrate has a uniform moisture content throughout of 12 % by weight or less. Preferably the second base material is a porous ceramics material, a water-absorbing mineral or a water absorbing polymer. Preferred examples of the second base material include vermiculite and perlite. It is advantageous to seal a sterile substrate of the invention as defined above within a lining material which resists the ingress of microorganisms. The sterile substrate of the invention comprises a sterile, dry particulate base material. The individual particles of the base material, being dry as well as sterile, can readily absorb moisture. The substrate is thus particularly well suited to being treated with liquid mushroom culture inoculum. As noted above, liquid inoculum may comprise mushroom mycelium in an enriched liquid suspension. When the sterile, dry particulate base material of the substrate is exposed to inoculum the capillary action of the fluid component causes the enriched free liquid of the inoculum to be drawn inside the dry particles. This leaves the mycelium residing on the surface of the particles where they are able to feed off the enriched liquid trapped inside the particles. In these circumstances the mycelium can flourish and will typically grow within a few days to cover the surface of the particle almost completely. The net effect of exposing the sterile substrate of the invention to liquid mushroom culture inoculum is thus to convert the liquid inoculum into a solid-state inoculum. This, in turn, may be used as mushroom spawn since it can subsequently be launched into bulk substrates. The invention accordingly further provides a process for producing a solid- state mushroom, culture inoculum, which process comprises exposing a sterile substrate as of the invention as defined above to a liquid mushroom culture inoculum comprising mushroom mycelium in suspension, such that free liquid is drawn inside the particles of the substrate and the mycelium is disposed on the surface of the particles, and recovering the resulting mycelium-coated particles.

The present invention will be further illustrated in the following examples.

Example 1: Preparation of sterilised substrate

Sawdust and bran (a source of nitrogen) were mixed together, and the resulting mixture was checked to see that its moisture content was 10% by weight. If not, the mixture was dried in an oven until its weight loss on further drying was 10% or less. The dry mixture was divided into 5 kg samples. Each sample was placed in a plastic bag having a volume approximately five times that of the sample.

Two of the bags containing a 5 kg sample were then put inside a second bag and a dosimeter (which is an instrument that changes colour on exposure to radiation, the degree of colour change being related to the radiation dose) was placed in the centre. The bag was sealed and placed inside a cardboard carton. The carton was then exposed to a Co⁶⁰ source of gamma-radiation. The carton was retrieved and the dosimeter checked to determine the radiation level at the centre of the box.

The data obtained from this procedure were as follows:

Dose Range Specification: Min 10.0 kGy Max 60.0 kGy Dimensions of Carton: 430x420x260 mm Weight of Carton: 1 1.2 kg Density: 0.24 g per cc Dwell Time: 223 Sees Current Cobalt Loading: 1.742651 Mega Curies Standard Plant Dwell Time: 223 Sees

Dosimetry Results

Minimum Dose Reading: 20.2 kGy Maximum Dose Reading: 29.0 kGy Routine Dosimeter Reading: 25.3 kGy Dose Required at Routine Dosimeter 12.5 kGy To achieve Min 10.0 kGy Dose Required at Routine Dosimeter 52.3 kGy To achieve Max 60.0 kGy

Example 2: Inoculation of sterile substrate

The carton containing the bags of sterile dry substrate, produced as described in Example 1, was transported to a laminar flow cabinet where it was opened to reveal the second bag. This in turn was opened to reveal the two inner bags, which were removed and injected with 1.5% by weight of a mushroom culture inoculum. The bags were shaken and then water was added to each one until the moisture content was within the range of 65% to 70% by weight. Each bag was then sealed and left to enable the mycelium to grow.

Claims

1. A process for producing a sterile substrate suitable for use in microorganism cultivation, which process comprises exposing to radiation or heat, or submitting to chemical sterilisation, a substrate which has a moisture content of 30 % by weight or less and which comprises a particulate base material which is capable of absorbing or entrapping water.

2. A process according to claim 1 wherein the radiation is gamma radiation or electron beam radiation.

3. A process according to claim 1 wherein the chemical sterilisation comprises treatment with a gas or mixture of gases.

4. A process according to claim 1 or 2 wherein the radiation is gamma radiation applied at a dose of from 8 kGy to 100 kGy.

5. A process according to any one of the preceding claims wherein the base material is wood sawdust or wood shavings, coconut fibre or the straw of hemp, flax or wheat.

6. A process according to claim 5 wherein the wood is a fast decomposing hardwood or a composted softwood.

7. A process according to any one of claims 1 to 4 wherein the base material is a porous ceramics material, a water-absorbing mineral or a water-absorbing polymer.

8. A process according to any one of the preceding claims wherein the substrate further comprises a source of nitrogen.

9. A process according to claim 8 wherein the source of nitrogen is cereal bran, soybean bran, yeast, soy oil or peptone.

10. A process according to any one of claims 1 to 4 wherein the base material is a cereal product.

11. A process according to claim 10 wherein the substrate further comprises, in addition to the cereal product, a second particulate base material which is capable of preferentially absorbing or entrapping water.

12. A process according to claim 1 1 wherein the said second base material is a porous ceramics material, a water-absorbing mineral or a water-absorbing polymer.

13. A process according to any one of the preceding claims wherein the particulate base material of the substrate comprises a material suitable for use as inoculum, spawn or bulk substrate.

14. A process according to any one of the preceding claims which further comprises adding water and a microorganism culture to the sterilised substrate.

15. A process according to claim 14 wherein the water is enriched with a microorganism growth promoting gas or with one or more minerals, nutrients, enzymes or bacteria which support the growth of microorganisms.

16. A process according to any one of the preceding claims which is carried out as a continuous process.

17. A sterile substrate suitable for use in microorganism cultivation, which substrate has a moisture content of 30 % by weight or less and comprises a particulate base material which is capable of absorbing or entrapping water.

18. A sterile substrate suitable for use in microorganism cultivation, which substrate has a moisture content of 30 % by weight or less and comprises a cereal product and a second particulate base material which is capable of preferentially absorbing or entrapping water.

19. A substrate according to claim 18 wherein the said second base material is a porous ceramics material, a water-absorbing mineral or a water-absorbing polymer.

20. A process for producing a solid-state mushroom culture inoculum, which process comprises exposing a sterile substrate as defined in any one of claims 17 to

19 to a liquid mushroom culture inoculum comprising mushroom mycelium in suspension, such that free liquid is drawn inside the particles of the substrate and the mycelium is disposed on the surface of the particles, and recovering the resulting mycelium-coated particles.

21. A substrate according to any one of claims 17 to 19 which is sealed within a lining material which resists the ingress of microorganisms.