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Microbiologique testing

Célia Martinez
In Microbiologique testing, Page IW process EN

Master your microbial issues in the paper industry

As a significant water consumer, the paper industry has adapted by reusing its industrial water. Yet, it still faces significant microbiological challenges leading to various direct and indirect consequences. These include the quality of the final product, microbial safety, environmental concerns, staff well-being…
How can the paper industry address these problems, and what solutions are available?

Microbiological Challenges in the Paper Industry

The paper industry’s water demand is substantial, estimated at 500 liters per kilogram of paper produced [1]. To address environmental concerns related to resource scarcity and better manage their effluents, the industry has adapted by recycling 95% to 98% of water used internally [2]. However, the closed-loop systems have intensified microbiological issues.

Indeed, microorganisms such as bacteria, yeast, and fungi find an ideal environment for rapid growth within the paper production process. Various factors, including abundant nutrients, suspended solids (MES), temperatures ranging from 30 to 60°C, and a neutral pH level (6.5 to 7.5), provide favorable conditions for microbial development.

When microorganisms grow uncontrollably, they form biofilms (slime) that adhere to machinery surfaces. This biofilm accumulation can lead to several detrimental effects:

  • Weakened paper sheets causing breaks: the biofilm deposited on the paper web during the drying process creates holes, reducing paper strength and resulting in costly downtime. 
  • Unwanted discoloration or staining of the paper: different microorganisms present can cause grey, yellow, or orange stains, diminishing the product’s value and even rendering it unsellable.
  • Unpleasant odors: microorganisms produce foul-smelling gases, leading to workplace discomfort, health issues, and neighborhood complaints.
  • Microbiological corrosion: development of corrosive bacteria, like sulfite-reducing bacteria, damages networks, leading to significant maintenance costs.

 

Existing Curative Solutions 

To prevent or address these issues, paper industry operators resort to heavy treatments that have significant environmental implications. The two main curative actions are:

  • Circuit cleaning using biodispersant products, followed by total network drainage.
  • Disinfection with biocidal products, which can be harmful to the environment.

However, these methods come with consequences:

Curative actions Consequences
Cleaning Filamentous bacteria discharge in wastewater treatment plants
An excessive growth of filamentous bacteria leads to bulking phenomena, affecting sludge settling. This degrades the quality of the discharged effluent.
Biodispersants discharge in wastewater treatment plants
In large quantities, these products are difficult to eliminate and/or neutralize. Their release into the natural environment can cause environmental problems.
Disinfection Biocide discharge in wastewater treatment plants
Biocidal substances are challenging to eliminate completely, resulting in their release into the natural environment, causing environmental issues.
Residues in mists enveloping the machine

A health risk (Legionellosis) for employees working in the manufacturing facility.
Significant economic cost in biocide products for the company

 

Mastering Product Treatment Usage is Achievable!

While product treatment remains essential for ensuring high-quality production and safety, controlling the quantity of used products is necessary to limit toxic agent discharge into the environment.

One solution to reduce product usage is to prevent biofilm development proactively. Regularly monitoring the water network allows operators to anticipate microbiological issues, biofilm accumulation, and subsequent product contamination. In this regard, having a total flora indicator is essential. Quantitative ATP-metry provides on-site results in just 2 minutes, indicating the overall microbiological load of water. This enables operators to take action before defects appear in the final product.

ATP tests offer several advantages, including:

  • Identifying process elements that are sources of contamination or microbial growth, facilitating targeted antimicrobial treatments.
  • Evaluating the effectiveness of existing biocidal treatments.
  • Improving treatment efficiency through better selection of active molecules, injection points, and frequencies.
  • Anticipating potential microbiological issues.

ATP tests thus provide numerous benefits:

  • Reduced production defects.
  • Decreased installation downtime.
  • Limited use of environmentally harmful products, promoting eco-responsible management of biocidal and biodispersant products – use only what is necessary!
  • Cost savings on treatment products.

In conclusion, by employing advanced techniques such as ATP-metry, the paper industry can overcome its microbiological challenges more effectively, ensuring high-quality production while maintaining a sustainable approach to environmental and economic factors.

Célia Martinez
In ATP test, Microbiologique testing

HOW TO PROPERLY COMPARE ATP TESTING AND CULTURE?

ATP testing and bacterial culture on solid agar plates are two completely different techniques. While culture method only measures culturable bacteria, i.e., the ones able to grow on a given media, ATP testing measures the quantity of ATP in a sample. As this molecule is produced and found in all living bacteria, ATP testing measures all the bacteria, culturable or not. This major difference makes them difficult to compare. 

However, when validating a new technique, it is natural to compare it with the conventional method. To avoid bias in result interpretation, here are some general tips.

General tips, not limited to these two techniques

 

  • Be aware of what each technique measures: ATP test measures the ATP and thus indirectly the total bacteria, while culture only measures culturable bacteria.
    .
  • Each technology has its own limits. ATP test uses a defined convention to estimate the number of bacteria in the sample (1 pgATP ≈ 1 000 bacteria). As for the culture method, it does not count the VBNC (Viable But Nonculturable) bacteria. Yet, according to literature, only 0.01 to 1% of bacteria grow on HPC media. Bacterial culture is limited by the choice of culture media, of incubation time and incubation temperature.
    .
  • It is necessary to work on a large concentration range, i.e., on several LOG.
    .
  • Perform each measurement at least in triplicate to obtain a significative value for each method.
    .
  • All the samples must be treated in the same way, whatever the analysis method. One of the most frequent mistakes is to collect the sample in a bottle with sodium thiosulfate for culture analysis and without sodium thiosulfate for ATP analysis. In the first case, the biocide action will be stopped, while in the second case, the biocide will keep its action, eliminating the biomass. The comparison would then be distorted. Therefore, it is essential to perform the analyses on the same sampling bottle. Likewise, if a dilution is necessary, it must be diluted in sterile water or physiological serum for both methods.
    .
  • Last but not least: it is primordial to look at the results with a critical eye. You should be able to identify outlying results.

Specific tips on ATP tests vs Culture comparison

On top of the previous tips, here are some advice specific to these two technologies:

  • Liquid culture media bias ATP results. Indeed, there is a large amount of free ATP and inhibitors in culture media. To avoid misleading results, dilute the samples in water or rinse the filter membrane.
    .
  • Bacteria cultures are not representative of the actual sample. As those bacteria are prepared for growth on culture media, a large part of them will grow and form colonies, whereas we know that only 0.01 to 1% of environmental bacteria are able to grow on culture media. Thus, it is important to compare the methods on real samples with complex ecosystems.
    .
  • Even if ATP test has a very high sensitivity, it cannot demonstrate sterility.

ARTICLES AVAILABLE ONLINE

Several quantitative ATP tests vs culture comparisons have been published over the last few years:

Célia Martinez
In ATP test, Microbiologique testing

HOW TO ASSESS THE EFFICACY OF UV DISINFECTION USING ATP TESTING?

UV disinfection

How does it work?

Nowadays, UV disinfection is commonly used for drinking water treatment. UV radiations alter nucleic acids (DNA and RNA) of most cells such as bacteria, viruses or protozoans. They damage the genetic material of microorganisms preventing them from replicating or ensuring part of their metabolic functions. UV radiations inactivate microorganisms.

Depending on the type of microorganism and its physiological state, the inactivation will have:

  • a bactericidal effect leading to the death of the cell. Indeed, if the UV dose is high enough, radiations will alter the membrane integrity and lead to the immediate destruction of the cell.
  • a bacteriostatic effect which will momentarily stop the growth and development of the cell. However, microorganisms have the ability to repair UV-induced damage and restore infectivity.

UV doses

The UV doses required to permanently inactivate a cell vary from one microorganism to another. The UV dose is the fundamental parameter to properly size a UV system. It corresponds to the product of UV light intensity (irradiance) and exposure time, which directly depends on the water flow.

The graph below shows the effectiveness of different UV systems depending on the water flow. The data are collected 2h after treatment using the ATP tests DENDRIDIAG SW. The graph highlights the effect of the water flow on UV disinfection efficiency. 

According to many studies on the subject, the minimal UV dose is 40 mJ/cm² to inactivate all microorganisms.  Usually, UV-C are used at a wavelength of 254nm.

However, several parameters have an impact on UV disinfection efficiency:

  • water clarity,
  • turbidity,
  • total suspended solids,
  • colour,
  • dirtiness of the lamps (scaling, high iron or manganese content in water…),
  • water film thickness,
  • aging of lamps…

Unlike biocidal treatment such as chlorination, UV radiations have no residual disinfection. If the nucleic acids show little damage, microorganisms have the ability to repair their genetic material and can grow again. This phenomenon is known as reactivation. Therefore, water disinfected by UV light should not be stored, at the risk of further proliferation. UV disinfection is particularly efficient and relevant when used:

  • at the point of use,
  • in addition with other treatements,
  • on clear water poorly contaminated.

UV treatment efficiency assessed by ATP testing

Quantitative ATP tests measure the amount of ATP in microorganisms. It measures the total flora after UV disinfection, there are 3 main scenarios:

  • Immediate decrease: the UV system has an immediate bactericidal effect that destroys the cells. The ATP is freed in the water. The membrane filtration step of the ATP test eliminates the free ATP.
  • Decrease after a two-hour delay: the UV system efficiently damaged the cells, but did not alter the membrane integrity. Therefore, this time, the filtration step does not eliminate these damaged cells. After 2 hours, the cells will be destroyed and the bactericide effect will be visible by ATP testing.
  • No decrease observed 2h after treatment: the UV system has little to no bactericidal effect. It is then important to assess whether the UV system has a bacteriostatic effect. Indeed, the risk of reactivation and regrowth is high. If the bacteriostatic effect is shown, it is then possible to use the treated water quickly, without storage.
How to assess the bacteriostatic effect of the UV disinfection?

Following UV disinfection, sample one litre of water. Using ATP test, measure the sample 2 hours after treatment, then every 24h for 3 to 4 days. This study will show you the biomass evolution in time, such as depicted in the following graph.

Keep in mind that, by culture method, this bacteriostatic effect can be mistaken with a bactericidal effect. Indeed, the inactivation leads to an increase of the latent period and thus to a decrease or absence of colony.

If the UV disinfection is not satisfactory, various options should be considered:

  • Increase the lamp intensity,
  • Decrease the water flow,
  • Check the lamp state and the quartz scaling
  • Check the water clarity…

Total biomass evolution after UV disinfection

Célia Martinez
In Microbiologique testing, News

Who are the coliphages, new parameter of the Drinking Water Directive?

Coliphages are viruses able to infect coliform bacteria such as Escherichia coli or less frequently Shigella spp or Klebsiella spp.
E. coli is the most abundant bacteria in the lower intestine of humans and animals. Therefore, coliphages, which are non-pathogenic viruses, are the most common in the intestine.
Besides, it has been shown that coliphages do not replicate in the environment because of unfavourable conditions. Thus, coliphages found in the environment mainly come from faecal contaminations and can be used as an indicator of water microbiological quality.

Jebri, S., Muniesa, M. and Jofre, J. (2017)

Main characteristics of coliphages

In water, we mainly focus on two kinds of coliphages: somatic coliphages and F-specific RNA bacteriophages.

Somatic coliphages F-specific RNA bacteriophages
Infection method Through the cell wall Through fertility (F) pili
Size Variable (≈ 50-120 nm) 21-30 nm
Genome Single or double-stranded DNA Single-stranded RNA
Most known family Myoviridae, Podoviridae or Microviridae Leviviridae
Most used surrogate ϕX174 MS2

The term « total coliphages », that we can find in some regulations, means both somatic coliphages and F-specific RNA bacteriophages. 

What is the best indicator of faecal contamination / treatment efficiency?

Are somatic coliphages a better indicator of faecal contamination than F-specific RNA bacteriophages?

It is a matter of debate. Scientific studies appear to show that somatic coliphages are generally more abundant in water than F-specific RNA bacteriophages. However, the opposite seems to be true in ground water or in recycled water treated with UV light. Besides, from a purely technical point of view, enumeration of somatic coliphages is much easier.

What is certain is that in comparison with bacterial indicators, coliphages are less sensitive to disinfection processes and survive longer in the environment. Furthermore, viruses migrate farther and faster in soils than bacteria. Thus, water can be contaminated with human enteric viruses, even in the absence of traditional bacterial indicators (coliforms, E. coli). The report from French Agency for Food, Environmental and Occupational Health & Safety (ANSES, n° 2018-SA-0027), published in 2018, points out that bacteriophages are excellent indicators of treatment efficacy against viruses.

What are the regulations?

For some years now, many regulations around the world have introduced the analysis of coliphages to monitor the water quality (e.g. Australia, Europe, some states of the USA…). These new microbiological criteria concern the water intended for human consumption as well as treated wastewater. The existing regulations recommends analysing either somatic coliphages or F-specific RNA bacteriophages, or both.

In Europe, the enumeration of somatic coliphages is introduced in the new EU Drinking Water directive 2020/2184. They must be checked for in raw water. If the result is above 50 PFU per 100 ml, then, the water after treatment must be analysed.

Furthermore, this new directive introduces Water Safety Plans (WSPs). These management plans require the implementation of a new global strategy of risk prevention. Therefore, managers must have new indicators, such as somatic coliphages. This revision was published on December 23th, 2020.

Enumeration of coliphages is also required by the EU regulation on minimum requirements for water reuse published in June 2020. In this case, total coliphages should be analysed in the intake and output water of the treatment plant. A 6-log reduction is required for water intended for agricultural irrigation.

How to detect coliphages?

To meet these new regulatory requirements, laboratories must implement suitable analytical methods. Based on the ANSES report, in 2018 in France, only one laboratory was accredited for coliphage analysis, and only for F-specific RNA bacteriophages.

EN ISO 10705-1 and 10705-2 set out the method to enumerate respectively F-specific RNA bacteriophages and somatic coliphages using double agar layer.

However, these Standards only offer to inoculate 5 ml of water on 20 different petri dishes to analyse 100 ml samples. This method is extremely time and effort consuming, as well as costly. Therefore, it cannot be used as a routine method.

However, part 3 of this standard recommends a few solutions. Based on several studies, concentration on membrane filter is the easiest and cheapest to implement. It is particularly well adapted to observe the 4 to 6 LOG reductions required for the analysis of low turbidity water such as drinking water or reuse water.

This is why GL BIOCONTROL offers the concentration kit VIRAPREP® already used by several analysis laboratories. 

Célia Martinez
In ATP test, Drinking water, Microbiologique testing

ATP testing: A predictive indicator of bacteriological non-compliance in water

HPC (Heterophoric Plate Count) culture mediums such as YEA, PCA or R2A, frequently used for environmental bacteria counting, detect less than 1% of the total flora (WHO, 2003). Indeed, a large proportion of bacteria cannot multiply on those mediums. For example, there are:

  • Anaerobic organisms (tolerant or obligate): oxygen presence slows down or inhibits their growth.
  • Bacteria requiring a specific temperature to grow such as psychrophile (low temperature) or thermophile (high temperature).
  • Germs requiring a specific environment such as acidophiles (high acidic conditions) or halophiles (high salinity).
  • Germs requiring specific elements such as rare amino acids, complex sugars, vitamins, cations…
  • Non-culturable bacteria whose culture is impossible using traditional methods.
Total bacterial flora

Diagram of total bacterial flora

  • VBNC bacteria (viable but non-culturable) which have momentarily lost their growing capacities as a response to stress. The use of biocides, physical treatments (ex: UV) or the environmental parameter modifications (temperature, pH…) can cause that change of state.

Furthermore, the bacteria needs to form a colony to be detected by the technicians’ eye. Which means growing from one to several billions within the allocated time period. That implies a short lag phase and a fast growth. Those parameters partly depend on the incubating temperature and the type of medium used.

 

In the end, we only detect aerobic mesophilic flora able to grow between 20°C (68°F) and 45°C (113°F) in the given time and for which the nutrients are adequate.
Why you should not speak of “total flora” with culture method?

Each culture medium will only detect a part of the bacteria depending on chosen conditions.
Thus, to be thorough, we should speak of “culturable flora” indicating the medium, temperature and incubation time chosen.

Diagram ATP vs cultural method

Example of bacterial growth monitored by culture and ATP-metry

As for ATP testing, it detects the whole living bacterial flora, even non culturable bacteria. For all these reasons, it is often observed an increase of total flora by ATP testing long before the appearance of the first colonies on the culture medium.

ATP-metry is an early indicator of microbiological contamination.
ATP tests for industrial waterATP tests for industrial water
ATP tests for drinking waterATP tests for drinking water
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