Balancing the gradient: Water activity, risk and microbial growth

BY DR TIM SANDLE | 05 January 2024

 

All microorganisms require some amount of water in order to reproduce (1). Microorganisms take up water by moving it through their cell membranes. This is why, even with all the necessary growth factors, water is proportionately the largest ingredient within culture media (2).  Water is required for metabolic activity and as a biophysical factor. The amount of water and the amount of ‘available’ water to a microorganism are not the same thing. For example, it is possible for a substance to have a high moisture content but have very little water activity. This happens when various components of a substance bind water, making it unavailable to microorganisms.

 

The water movement process depends on a water gradient (a high water activity outside of the cell to a lower water activity within the cell). If the amount of available water decreases, conditions of osmotic stress would be created. In this situation, the cell would no longer be able to take in water and would therefore be incapable of growth. Different microorganisms have different water activity requirements.

 

Therefore, microbiologists describe the water requirements of microorganisms in terms of the water activity (aw). It is this ‘availability’ of water that is the critical factor affecting the growth of all cells - not simply how much water there is.

Water activity

 

The availability of water for a cell depends upon its presence in the atmosphere (relative humidity) or its presence in a solution or a substance (water activity). The water activity (aw) of pure H2O is 1.0 (100% water) in the aqueous state. Water activity is defined as the ratio of water vapor pressure of a substrate to the vapour pressure of pure water at the same temperature, typically measured at 25oC ±1oC. (3). This can be expressed as:

 

aw = p/po

 

Where:

p = vapor pressure of the solution and po = vapor pressure of the solvent (in this case, water).

Putting this into context, the aw of pure water is 1.00 and the aw of a completely dehydrated substance is 0.00. The aw of materials on this scale from 0.00 - 1.00 is related to the equilibrium relative humidity above the material on a scale of 0 - 100%. Thus, the percentage Equilibrium Relative Humidity (ERH) = aw x 100.

 

The aw of a material describes the degree to which water is "bound" in the material, its availability to participate in chemical/biochemical reactions and its availability to facilitate growth of microorganisms.

 

The measurement of aw can be performed using an electric hygrometer. This consists of a potentiometer, a sample/sensor holder and a sensor (4). Guidance on measuring water activity can be found in the United States Pharmacopeia: Chapter <1112> Application of water activity determination to non-sterile pharmaceutical products. There is also a standard that can be applied from the food industry - ISO 18787:2017 Foodstuffs: Determination of water activity.

 

 

 

Different microbial requirements

 

Different microorganisms generally have optimum and minimum levels of aw for growth. Some examples are (5):

Vibrio cholerae                 -    0.98 aw
Staphylococcus aureus    -    0.98 aw
Escherichia coli                -    0.95 aw
Salmonella spp.               -    0.94 aw
Bacillus cereus                 -    0.93 aw
Candida albicans              -    0.85 aw
Aspergillus spp.                -    0.82 aw

Organisms capable of growing below these levels include halophiles (0.75 aw), xerophilies (0.65 aw) and osmophilic organisms (0.60 aw).

 

 

 

Significance for understanding product risks

 

We can take the concept of water activity and apply it to different products, be that something that is complete (like food for human consumption) or a raw material (that we might use to make food or to manufacture pharmaceuticals). If the substance is sufficiently low in water activity, it will not support microbial growth - if it is sufficiently high, it will. Between these extremes, there are different states of water activity and here some types of microorganisms will be able to reproduce, while others will not. Therefore, understanding the likely contaminants we might come across becomes important in anticipating the likelihood of growth should a particular contaminant be present (or where the potential for contamination exists).

 

Generally, the growth of most bacteria and fungi occurs at aw values above 0.90. If the aw is below 0.8, the only organisms likely to grow are xerophilic moulds and osmophilic yeasts.

 

Whilst the concept of water activity is common in the food industry, there is scope for applying it to pharmaceutical raw materials and to some non-sterile drug products when it comes to assessing the risk of the microbial stability of a product, for developing a specification or for understanding related risks across a stability testing programme (6).

 

 

 

Significance for microbial culture media

 

This means of water availability is significant for the development and formulation of culture media, an important consideration is not so much ensuring there is sufficient water in the material but more with the amount of available water. Hence microbial growth, and, in some cases, the production of microbial metabolites, is particularly sensitive to alterations in aw. 

 

Water activity alters with the presence of solutes, such as salts or sugars. The higher the solute concentration of a substance, the lower the water activity and vice-versa. Most microorganisms live over a range of aw from 1.0 to 0.75 (7). Those organisms that can survive towards the lower end of the water activity scale are called xerophilies. The aw of soil, for example, is typically between 0.9 and 1.0. The biggest influence on water activity is salt (NaCl) – bacteria able to grow at moderate salt concentrations are termed ‘halotolerant’. 

 

 

 

Summary

 

Different species of microorganisms have different minimum levels of aw that permit growth. The water activity of a product can be used to predict microbial growth and to determine the microbial stability of a material or finished product (7). Understanding what is required is also important when selecting culture media in order to maximise our chances of recovering contaminants.

References

 

  1. Postgate, J.R. and Hunter, J. R. (1962) The survival of starved bacteria, Journal of General  Microbiology, 29: 233-263
  2. Schlegel, H. G. (1993) General Microbiology, Cambridge University, Cambridge, U.K. pp. 204-207; 459
  3. Jay, J.M. (2000) Modern food microbiology. 6th Ed. Gaithersburg (MD): Aspen. p 679
  4. Gauvry E, Mathot AG, Couvert O, et al. (2021) Effects of temperature, pH and water activity on the growth and the sporulation abilities of Bacillus subtilis BSB1. Int J Food Microbiol 337:108915
  5. Public Health England. Determination of water activity in food, National Infection Service, 2017
  6. Cundell, A. M. (2009) Effects of water activity on microorganisms. In Water Activity Applications in the Pharmaceutical Industry A. M. Cundell and A. J. Fontana, Jr (Eds,) PDA/DHI, pp175-204
  7. Sandle T. (2016) The Importance of Water Activity for Risk Assessing Pharmaceutical Products, J Pharm Microbiol., 2 (1): 1-2
  8. Cundlell, T. The role of water activity in the microbial stability of non-sterile pharmaceutical drug products, European Pharmaceutical Review, Issue 1, 2015 

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