Variables affecting microbial contamination of water systems
Microorganisms are present in potable water supplies to pharmaceutical water systems and microorganisms will be present in water regeneration processes. Organisms can also be detected in poorly maintained or operated pharmaceutical grade systems. Where contamination occurs, gram-negative non-fermenting bacteria predominate (such as organisms of the genera Pseudomonas, Flavobacterium and Acinetobacter and other taxonomically related bacteria) (1). Whether microorganisms are present within purified water systems will depend on various factors, including design weaknesses that create conditions favourable for bacterial attachment to surfaces (such as low water flow or the presence of dead legs). The main risk to a water system is with biofilm formation (a microbial community enclosed in exocellular polymers that adheres to a surface).
There are other important variables that regulate microbial survival, regrowth (the increase in the number of bacterial cells in a system when proper nutrients and environment are provided) and the potential for biofilm formation: the type and concentrations of available substrates, the environment, bacterial community structure and bacteria concentration.
Limitations of plate counting
Such assessments of microbial survival are limited by what can be achieved using growth media (heterotrophic plate counts using media like R2A) due to many aquatic bacteria not being culturable (2).
It is understood there are significantly more microbial cells in water than what can be cultured on growth media, as can be ascertained from technologies like flow cytometry or next-generation sequencing (3). Flow cytometry can be used for online monitoring, although the bulk of pharmaceutical water system monitoring remains culture dependent. This means any assessment of microbial numbers will be an underestimation. There are other variables that can influence assessments, including seasonality and spatiotemporal variations (4).
Influence of organic matter
The availability of organic matter can act as both a variable for growth and potential biofilm formation, which can serve as a predictor for growth potential. Understanding the dynamic interactions between organic matter and microbial communities can be an important contamination control measure.
Assimilable organic carbon
A positive correlation exists between the assimilable organic carbon concentration and the density of heterotrophic bacteria - this is the most important measure of organic materials. Therefore, whether microorganisms have sufficient nutrients for survival and growth in a water system corelates with the levels of AOC.
AOC is made up of low molecular weight organic molecules, generated either via lysis of bacterial cells or biological and chemical hydrolysis of organic matter. AOC does not simply equate to organic carbon - AOC is the fraction of carbon most easily consumed by bacteria (5). The levels of AOC are a factor of the relationship total organic carbon (TOC), dissolved organic carbon, UV254 (a measurement of the organic matter in water using ultraviolet light at 254 nm) and total phosphorous (microbial growth increases at up to a phosphate concentration of 10 µg of PO4-P (phosphate measured as phosphorus) per litre-1 (6).
What level of organic carbon presents a water system risk?
The relationship between AOC concentrations and the propensity for microbial growth are uncertain as the biomass: carbon yield ratio is based on several variables. The required concentrations of AOC that can be utilised by bacteria vary by species and over time, although the region of between ≤10 – 100 μg/L is considered less conducive for growth and between 100 and 500 μg/L upwards is cited in literature as being potentially growth supporting (7, 8). This is evidenced by the geometric mean of microbial counts and AOC concentrations. AOC is assessed using acetate carbon equivalents per litre.
Some microorganisms can adapt to require lower AOC levels. This has been observed when disinfectants are used to treat water systems (such as with chlorination). In some circumstances, disinfectants can potentially select microbes that are resistant to oxidants (9).
Determining AOC concentrations
Determining AOC can be a useful measure when assessing contamination risk, especially when results are obtained faster than is possible using a culture-dependent method. Many pharmaceutical quality control laboratories will assess TOC levels. This provides a measure of the total amount of carbon in organic compounds in water systems. In general, sustaining low TOC levels can aid with the control of biofouling (less than 2 mg/litre is theoretically sufficient, although pharmacopeia specify an additional safety feature with a limit of <0.5 mg/L). However, the levels are not directly in tandem with the potential for microbial survival and the potential for microbial growth cannot be simply drawn from measuring TOC. This is because organic carbon divides into biodegradable dissolved organic carbon (BDOC) and AOC. The approximate ratio of as low as 99:1 and up to 91:9 (hence, AOC typically compromises a small fraction and simply relying on TOC may cause an underestimation of the contamination potential).
It is possible to indirectly assess AOC. This is by assessing the linear relationship between the maximum growth of bacteria in a water sample and the AOC concentration. This requires a reference preparation to be prepared, where a given yield factor is produced by examining data from a test organism in a pure solution of acetate-carbon or oxalate-carbon. Pick et al used the nutritionally versatile species Pseudomonas fluorescens strain P-17 as a reference organism. This factor enables a conversion from nutrient concentration to cell counts to be made (10). With both TOC and AOC determinations, samples need to be tested within a relatively short time window (within 24 to 48 hours) for reliable data to be obtained.
Controlling AOC levels
AOC levels can be controlled through biological activated carbon filters, as can coagulation–sedimentation processes and rapid filters (11). However, poorly functioning filters can also increase the potential for survival of AOC (12). It is important that the frequent backwashing of granular activated carbon occurs (13). A risk exists of AOC being formed during ozonation processes as complex natural organic matter is broken down into low molecular weight by-products (14). Ironically, while ozonation kills bacteria, it also degrades organic matter in water. This increases the bioavailability of organic matter for surviving or newly contaminating bacteria. Therefore, post-ozonation assessments might be useful.
Summary
This article has looked at one area of pharmaceutical water system control based on the assessment of assimilable organic carbon. While this variable is not easy to measure, it should be considered when assessing systems as part of contamination remediation investigations and when TOC levels spike.
References
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