Fungi in cleanrooms: Reasons and factors for survival

Technical Review Article | Open Access | Published 2nd April 2025

Fungi in cleanrooms: Reasons and factors for survival

Tim Sandle, Tommaso Paoli | EJPPS | 301 (2025) Click to download

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Introduction

Fungi are not expected to be detected within cleanrooms. While occasional filamentous fungal recoveries may occur in lower-grade cleanrooms, the recovery of filamentous fungi (mould) in higher-grade cleanrooms is normally indicative of a serious breakdown of control and extreme risk due to the sporigene nature of these species. This is because such fungi, such as Aspergillus, Penicillium, Fusarium, Alternaria and other species common in the northern hemisphere, are associated with the outside environment¹ or, where they have a closer association with the built environment (such as with the melanized mould Cladosporium)² the conditions conducive to growth invariably indicate a weakness with cleanroom control parameters. In the most serious cases, fungi are detected in the drug substance or drug product³. The fungi likely to be detected are members of the classes zygomycetes, ascomycetes, and deuteromycetes.

Of the different microorganisms, filamentous fungi are the microorganisms most adapted to growth on solid substrates. This ability relates to their filamentous network and apical growth patterns that enable the effective colonization of a variety of solid particles formed from varied materials in order to obtain the necessary carbon source and mineral nutrients required. The fungi will utilize the carbon and nutrients through enzymatic substrate degradation⁴.

The growth of fungi on a substrate is dependent upon the climatic conditions on and around the substrate and the substrate itself. Fungi are capable of growing and surviving on a vast range of surfaces; amongst the determinant factors are those that influence the rate of growth and what influences metabolic activity. Where carbon and nutrient supply is sufficient, the fungal tips will elongate, and new branches will form. The rate at which this happens will vary according to the rate of metabolic activity along different hyphal segments⁵.

In this article, we look at filamentous fungi and growth within the in-built environment and consider the factors that influence their survival and dissemination. Considerations on transfer and disinfection measures will be the subject for a separate review; however, we conclude with some remediation measures.

Origins

While some filamentous fungi, in addition to yeasts, are part of the human mycobiome, the natural habitat of filamentous fungi is plant matter⁶, especially decomposing vegetation. Fungi exist on decaying plant matter during all seasons although the concentration of airborne spores varies seasonally depending on the fungal species and the time of year. The size of the spores varies according to the species, with fungi that form fruiting bodies being the largest at 20 microns; in contrast, filamentous fungi tend to be in the 4-5 micron range)⁷. Hence, most fungi found in buildings have originated from the outside environment, either introduced by people (such as on clothing) or from the items transferred in, or in cases of very weak design, via air handling systems and/or leaks into the classified areas. If inadequate quality or non-cleanroom-appropriate building materials have been used, notably gypsum or plasterboard as supporting walls, fungi can be present in such materials from the outset and spread unnoticed for a while inside the walls and ceilings until a major environmental breakdown is detected.

Requirements for fungal growth

When fungi enter a cleanroom there are different things that can happen:

The fungi may die as a consequence of the environmental conditions.
The fungi may die as a result of a lack of nutrients.
The fungi may produce spores equipped for survival and remain for a longer period, until conditions are appropriate.
The fungi may survive until killed with a disinfectant.
The fungi may attach to a substrate and grow under favourable conditions. Here, the fungus depends on water for its growth, substrate degradation, nutrient uptake, reproduction and spore dispersal. The extent of growth will also be influenced by different conditions and the length of time that passes (such as until cleaning and disinfection occur).

With filamentous fungi, growth occurs at the hyphal tips as ‘construction materials’ are transported through the fungal cells, supplied in vesicles, small bubbles on motor proteins running on long rails, and used at their outermost tip. Calcium concentration at the end of the cell defines when this happens. When a calcium pulse occurs, the vesicles merge with the cell membrane and release their contents. Under optimal conditions, despite only having a typical diameter of three micrometres, hyphae can grow by more than one micrometre per minute. As a parallel, this is equivalent to an adult human gaining ten centimetres in thickness every minute)⁸. When a fungus reaches a channel smaller than the diameter of the hyphae it undergoes plasticity, reducing in size in order to penetrate. At the same time, the rate of growth slows⁹.

Different fungi have different growth requirements, leading to the likelihood of different fungi being influenced by the type of substrate and different environmental conditions. Fungal growth can occur in as little as an hour under favourable conditions of temperature and pH.

The common growth requirements and influencing factors can be summarized as¹⁰⁻¹⁶:

pH.
- Moulds differ in their pH requirements, but the most common pH range is 3 to 7 (with growth possible for some species within a pH range of 3 to 9). Most moulds require slightly acidic conditions to flourish. The optimum pH range for most fungi is between 5 and 7. It is of interest that painted surfaces, which should be avoided in cleanrooms, have a typical pH of 5.
Moisture.
- As with any lifeform, fungi require water for survival and any water present needs to be able to cross the cell membrane, a process aided by fungal aquaporins.
- Moisture needs to be considered in relation to water activity, which considers the minimal requirements of free water for growth (see below). The minimal moisture requirements for growth change with temperature and increase significantly with a corresponding decrease in temperature.
Air.
- While airflow is a key factor in encouraging mould spore distribution, many fungi can thrive where there is little or no airflow.
- When items are first introduced into the facility, such as items from a warehouse, ensuring they are dry and subjected to sufficient airflow is particularly important. In terms of fungi growing and surviving in distinct parts of the facility, poor ventilation encourages fungal growth. Hence, there is an association between inadequate ventilation and fungal growth.
- While fungi require oxygen, many can grow within a low oxygen concentration (this can be as low as 0.14% to 0.25%).
Nutrients.
- Like any other living organism, to survive, mould needs to be fed. Chemical elements such as phosphorus, sulphur, potassium, magnesium, and small quantities of iron, zinc, manganese, and copper are needed by most fungi for vigorous growth; elements such as calcium, molybdenum, and gallium are additionally required by other species.
- In terms of cleanroom control, specifying materials that contain none of these nutrients or lower concentrations is important. In terms of on-going controls, cleanliness is important since many of these nutrients are found in textile lint, traces of grease, varnish, dust and dirt.
Temperature.
- The majority of fungi likely to be found within the cleanroom are mesophile. The most common temperature range where moulds will grow is between 15 and 30oC. At its optimal growth temperature, a fungus needs to be exposed to this temperature for a period of time for the necessary enzymatic activity to begin and to permit growth.
- However, some species can grow as low as 0oC and some up to 50oC. In the external environment, the increase in temperature as the result of climate change causes many fungi to turn on their adaptative responses in terms of increasing genetic changes, which leads to higher heat resistance. Above 50oC, most filamentous fungi (either vegetative or spores) begin to be killed, and none are known to survive beyond 80oC.
- Fungi are more adaptable to cold than to heat stress. Some cryophilic species, although they stop growing, maintain survivability down to (-)10oC.
- Since most cleanrooms operate between 15 and 25, and most fungi have optimum growth at ‘room temperature’, this means temperature is not an additional factor for control since this cannot be adjusted to discourage fungi.
Humidity.
- Fungi prefer humid conditions although the required levels of humidity are significantly lower than those required by bacteria. Humidity is an indirect measure (again, refer to water activity below). That said, as a ‘rule of thumb,’ many fungi can grow where there is a relative humidity of 70% or greater.
- When considering humidity, the length of time that the fungus is exposed to conditions of suitable humidity is important. This is for more than 3 days. As well as the number of days, humidity levels need to exceed 70% for 12-15 hours of each day. This time requirement decreases as the humidity increases, i.e., exposure times to 80% humidity can be shorter than the 12 hours minimum required under 70% humidity, based on generating similar levels of fungal biomass.
- As a risk factor, humidity is at its greatest when climatic conditions lead to condensation formation or to other forms of moisture being present indoors.
- Consistent humidity (and temperature) is an important part of cleanroom control. Thermal bridges should be avoided. These are characterized by a surface temperature which is lower on one room side compared with another. The relative humidity increases at such places. The corners of the room are the areas where thermal bridges are most likely to be found.
Light.
- Many moulds flourish in dark spaces but some prefer an alternate light pattern – sometimes light, and some dark. Typically, a low quantity of light, although not necessary for growth, is essential for sporulation of several species.

The above factors need to be considered in combination (as hygrothermal factors) to understand the extent of the likelihood of fungal prevalence and the probability of triggering growth.

Water activity

The influence of water activity (aw) of a given substrate affects the expression, secretion, and properties of the extracellular fungal enzymes and the secretion of other metabolites. The ability of the fungus to secrete enzymes is part of the mechanism by which the fungus extracts the nutrients it needs to break down the substrate.

Water activity concerns the amount of available water. It is defined as the partial vapor pressure of water in a solution divided by the standard state partial vapour pressure of water. Pure water has a water activity of 1.0

An assessment of water activity can be used as a useful predictor of the possibility of microbial growth¹⁷. Fungi tend to require lower levels of water activity to enable growth than bacteria, within the 0.7 to 0.85 range, although some xerophilic moulds can function at lower levels. The longer a material's water activity is over 0.75, the greater the risk of fungal growth.

While a substrate may have constant water activity, environmental exposure can affect water levels. Fungi are adaptable to harsher conditioners. For example, if one part of a substrate is too dry, the fungus will seek to transport water from moist areas to arid areas by hydraulic redistribution.

Substrates

Fungi are capable of growth on a range of the types of substrates found in the ‘as built’ environment. No building substrate will contain the range or quantity of nutrients that are present in decomposing plant material and hence the growth of fungi indoors is far slower compared with outdoors. Yet over time, and under the optimal conditions described above, fungal growth on substrates occurs.

Some substrates are more suited for fungal growth than others. The general determinants are carbon and nitrogen; nutrient availability; organic materials; and surface topography:

a). Carbon and nitrogen

How well a fungus grows on a building material depends on the levels of carbon and nitrogen present. If carbonaceous and nitrogenous nutrients are present in suitably high concentrations, most fungi will be adept at using enzymes to decompose substrates and transform the nutrients into utilizable compounds that the fungus can absorb.

Many fungi are well-adapted to growth under low nutrient environments. To enable growth, or simple survival, fungi are capable of adjusting the carbon to nitrogen ratios needed as a fungal biomass develops. The advantages enable the conserving of energy and nutrient elements allowing for the extent of mycelial growth to be controlled¹⁸.

b). Other nutrients

As discussed above, there are a range of nutrients that fungi require for growth. Some materials will contain more available nutrients than others and hence are more likely to support growth. It also follows that materials with a greater abundance of the required nutrients will be degraded faster.

When the nutrients have been processed, a point will be reached where an insufficient level of nutrients are available and therefore growth slows down. If the fungus can disseminate spores, the objective is to find a new substrate to colonize.

c). Organic additives

Some building materials that contain organic additives are more prone to fungal growth and survival. For example, minerals are added to plaster, which is another reason to avoid plaster behind walls to be coated in polyvinyl chloride within the cleanroom.

d). Surface roughness

Surface roughness is a factor that can encourage attachment, colonization and growth but this appears less significant than with bacteria since fungi are able to penetrate surfaces. It is more likely that if a surface is damp, this presents a bigger risk factor than surface topography.

At risk substrates

Some substrates can become contaminated, and some are most likely to be already contaminated before installation. With the latter, control of cleanroom design to the level of specifying all building materials is important. With building materials these considerations need to extend to under surfaces since development of fungal growth will eventually lead to ingress into the cleanroom, especially when outer surface damage occurs. For example, wet gypsum wallboard is commonly contaminated as a consequence of the paper/carton layer surrounding the gypsum core¹⁹. Other materials likely to be contaminated include materials that should never be found in the cleanroom: wood, wallpaper, and plywood, but also in materials that are inescapable like concrete.

Fungal growth can occur in the materials used to manufacture PVC and fungi can grow in many of the adhesives used to fix materials to surfaces. While most PVC begins free of fungi, PVC can readily grow and contain fungi if it is not subject to regular cleaning and disinfection. PVC building materials contain plasticizers to enhance their flexibility, but most plasticizers are esters of fatty acids and hence contribute to supporting mould growth. The degradation of the plasticizers causes material discoloration, odour, and diminished performance over time. Risks to PVC can be reduced where phthalic and phosphoric acid derivatives are used to impart flexible properties²⁰.

Getting a grip

How well a fungus grows on or within a substrate depends on the fungus entering the first few millimetres of the substratum. If the fungus enters and survives, its ability to grow and undertake material biodeterioration, and its survival over the longer term increases. Certain coatings on materials create conditions more conducive to fungal survival, such as many types of paints. This is one reason painted surfaces should be avoided within the cleanroom. Although some antifungal paints are available, these can be difficult to evaluate and the length of time that the active ingredient remains potent for is uncertain. In addition, some disinfectants will adversely interact with the chemicals in the paint, leading to disinfectant inactivation.

Deposits on a substrate

The ability of a fungus to grow on a substrate is also influenced by the cleanliness of the environment. Where there are certain forms of dust (especially skin detritus), human perspiration, and fatty deposits, this provides additional nutrients for fungal growth. With perspiration, sweat is an excellent culture medium: sweat contains water, salt, sugar and nitrogenous compounds²¹.

In relation to debris, the greater risks can arise when the fungi grow in hidden areas and are released under various conditions when these areas are not subject to regular cleaning and disinfection.

Competition with bacteria

Bacteria and fungi will often share a common substrate, whether this is within a facility or in the external environment²². This spatial proximity will lead to either synergistic or antagonistic interactions. In synergistic systems, there will be a trade-off between fungal growth and tolerance towards bacteria.

Typically, fungal strains that grow best in the absence of bacteria will be most severely affected by bacterial presence, whereas fungal species less suppressed during co-existence with bacteria will see lower maximal growth rates in bacterial absence²³. Where the relationship is antagonistic and is connected to competition for substrate, if fungi are given an opportunity to establish a community on the substrate, first they will tend to out-compete the bacteria, as measured by a greater biomass²⁴.

Spore liberation

Fungi will release spores at various times based on the species and prevailing conditions; spore release is an essential part of the fungal lifecycle²⁵ and hence an ever-present risk is with the colonization of new surfaces. Fungi produce many thousands of spores, an evolutionary mechanism since most spores will not survive, dying where they land due to a lack of water and food. Whether spores are dispersed in a way that reaches other surfaces or are removed from the cleanroom depends on the airflow and air change rates. Whether spores survive depends on environmental factors. Spore survival is greater in lower, warmer latitudes and spores will eventually die from prolonged exposure to light and air; equally, spores have a greater chance of survival if they can rapidly settle onto a suitable substrate and the resultant fungal cells are able to grow, which links to the time, temperature, humidity, pH and other factors described earlier. Spore production does not mean the end of the spore-producing fungus; the mycelium does not die off after spore formation but continues to grow and forms spores again.

Detection

In severe cases of fungal contamination, the growth of fungi on a substrate will reach a level where the presence of mould is visible. In most cases, the fungal numbers will not have reached levels that are directly detectable by the human eye. Detection, and hence an assessment of the control measures put in place, requires a sound environmental monitoring program that has considered potential areas where fungi could be transferred into the cleanroom and where fungi might reside, including behind equipment, areas with reduced airflow, and areas that might remain dark or potentially wet. Consideration also needs to be given to the sampling methods, frequency, and the appropriate agar²⁶ and sample incubation regime (e.g. one or dual temperature)²⁷. Assessing fungal levels in the air inside a building can be challenging since spore liberation from a surface is sporadic and spore distribution in the air is random²⁸, therefore an appropriate frequency of monitoring must be in place, as well as a sound risk-based approach for the selection of sampling locations.

Data from environmental monitoring, where fungi are periodically recovered, should feedback into the contamination control program and enable remediation measures to be instigated. This may require adaptations to cleanroom design, improved controls for the transfer of items into cleanrooms, and a review of disinfectant efficacy studies, targeted to the endemic species identified in the areas²⁹.

Remediation

Prevention is the essential objective of any contamination control strategy and in the context of fungi, selection of appropriate building materials and careful control of climatic conditions is especially important, especially minimising areas that might become or remain damp and where there is reduced airflow. Avoiding secondary materials from becoming wet is an important aspect of ongoing maintenance, for instance, avoiding rain penetrating through a damaged roof sealing or moisture from leaky pipelines.

Attention also needs to be paid to the mechanical control of the cleanroom, as with the air quantity that is exchanged per hour. Where such areas cannot be eliminated, a greater focus on inspection and cleaning and disinfection is required. Control of incoming materials is also paramount, as is the development of a clear and risk-based material transfer protocol, both into the facility and for moving items through the cleanroom cascade.

Other measures need to be considered. According to Cundell common concerns faced by pharmaceutical manufacturers include³⁰:

Disinfectant efficacy studies may not adequately address disinfectant activity against fungal spores.
A lack of appreciation as to water damage promotion of fungal growth within pharmaceutical facilities.

Cundell also notes that most pharmaceutical microbiology laboratories lack the capability to reliably identify fungi to genus, and especially species, and often insufficient attention is given to fungal isolation and trending from the environmental monitoring program.

Summary

In this article we have considered the factors for fungal survival and growth within the cleanroom. This has required an understanding of the likelihood of growth on different substrates within different environments, under different conditions. While there are some commonalities, it is important to note that mould growth does not always follow the predicted behaviour described by mould growth models³¹. This is often explained by uncertainty in the real conditions of exposure. Hence, the most important measure remains prevention of fungi from entering the facility in the first place and developing sound material selection and transfer control.

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Author Information

Authors:

Tim Sandle¹, Tommaso Paoli²

01.PhD. QA Compliance and Sterility Assurance, Kedrion Elstree

02. PhD. Senior Vice President of Global Quality, Kedrion Biopharma

Corresponding Author:

Tim Sandle

Email: timsandle@btinternet.com