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Are biofilms secretly lurking in your cooling water systems?

Water has excellent heat transfer efficiency, which is the driving force behind medium to large commercial and manufacturing facilities’ preference for water-cooled systems over their air-cooled counterparts. Ensuring optimal ambient air temperatures for guest comfort and precise process temperature control, water-cooled systems play a crucial role in various industries. 

Microbiological fouling is an ongoing concern in open recirculating cooling water systems. Biofouling can lead to corrosion damage, losses in heat transfer efficiency, and Legionella development. By detecting, eradicating, and monitoring biofilms using modern DNA tools, effective biocontrol methods will achieve superior results at lower cost.

Credit: H.Wang (1)

Key Underlying Principles

Legionella is a parasitic organism that requires the presence of certain amoeba and protozoa host organisms in a cooling water system to survive and thrive. Legionella infects and multiplies in host organisms until such point that they lyse, exploding out of the host often in thousands of new Legionella organisms, which then search for new hosts and repeat the process.

Legionella hosts require the presence of biofilms (slime layers) which can form in cooling water systems and adhere to metal surfaces. Biofilms have a diverse ecology, providing food for protozoa and amoeba. Biofilms facilitate Microbial Influenced Corrosion (MIC) and are more thermally insulating than calcium carbonate scale. When biofilms form on heat exchange surfaces they can cause significant increases in energy costs. Biofilms provide a layer of protection from biocides and disinfectants, enabling more resistant and resilient microbial communities.

Credit: B. Hayes/NIST

Example DNA Insights in 3 Cooling Water Samples

Microbe Detectives analyzed 3 cooling water samples using next gen 16S and 18S DNA sequencing and qPCR for an experienced industrial water treatment company and specialist in treating cooling water systems. Each sample was collected from a different cooling water system operating during a warm summer day and treated to prevent scaling, biofouling, and MIC.

Microbial control was described as poor for sample #1, excellent for sample #2, and good for sample #3. Summary bio-characteristics of these samples are provided in the below table.

% Relative Abundance of Microbe Group Domains

Archaea: 0.0%

No archaea were detected in any of the samples.

Bacteria: 90% – 99%

Bacteria were the primary domain observed in all three samples, with a % relative abundance of 99%, 92%, and 90% for samples #1, #2, and #3 respectively, measured as 16S gene copies/mL. The ecology of bulk recirculating cooling water with good to excellent microbial control should mainly, if not entirely, consist of bacteria. Higher life forms are indicative of biofilms.

Eukarya: 1% – 10%

Eukarya are higher life forms that rely on bacteria, archaea, and nutrients to survive and thrive in cooling water systems. Their presence in cooling water systems is generally indicative of the presence of biofilms. There are four kingdoms of Eukarya including Protists, Fungi, Plants, and Animals. Eukarya were detected in all three samples, with an estimated % rel. abundance ranging from 1% to 10%.

% Relative Abundance of Eukarya

The % rel. abundance of Eukarya groups observed is summarized in the below chart. Protista is the kingdom of known Legionella hosts Acanthamoeba, Naegleria, and Vermamoeba (2). The observed presence of Protista in all three samples was the indicator that biofilm may be present.

Identities: 150 to 216 Estimated Unique Genera

209 unique genera were observed in sample #1, 216 in sample #2, and 150 in sample #3, measured as Operational Taxonomic Units (OTUs).

Quantities: 0.2 to 39 Million Estimated

The largest estimated quantity of microbes observed was in sample #1 with ~39 million 16S and 18S gene copies/mL. Approximately 220 thousand were observed in sample #2, and 514 thousand in sample #3. These values correlate with the descriptions provided by the customer about the degree of microbial control in each sample.

Biodiversity: 2.4 to 2.9 (Range = 0 – 5)

SDI biodiversity observed was similar across the samples in the mid-to-high 2’s, on a scale of 0 – 5, with 5 representing the highest possible biodiversity value. This was the second clue that there may be more to the story. A greater difference in biodiversity between excellent microbial control of a cooling water system and poor control was expected. Specifically, excellent microbial control was expected to correlate to a lower SDI biodiversity value compared to a poorly controlled system. There are always site specific considerations to account for.

The presence of Legionella hosts and other Eukarya are an indication of biofilm(s) lurking in the cooling water system.

Legionella was detected by a standard culture test and by 16S DNA sequencing in all three samples. Est. quantities of Legionella were about 7,300 in sample #1, 300 in #2, and 6,600 in #3. Legionella hosts, specifically Vermamoeba, were observed in all 3 samples. Est. quantities were about 500 in sample #1; 70 in #2, and 90 in #3 (18S gene copies/mL). 

Other Biofilm Indicators

As shown in the above table, slime forming and corrosion associated microbes were observed in all three samples. Slime formers help produce biofilms. MIC microbes often thrive underneath biofilms and cause underdeposit corrosion. The best way to interpret this data is by monitoring trends and observing results. In general, the goal is to minimize or eliminate slime forming and MIC microbes from the cooling water system.

Key Performance Indicators (KPIs) of Biofim Removal

Shannon Diversity Index (SDI)

  1. One way to verify biofilm removal from open recirculating cooling water systems is to first measure the SDI of the bulk recirculating water prior to shutdown for scheduled maintanance.
  2. During this scheduled maintenance, thoroughly review the system for potential biofilm locations in deadlegs, strainers, filters, and any low or no-flow areas.
  3. Locate and completely remove the biofilm(s) from the system.
  4. Return the cooling system to operation and apply a strong biocontrol treatment to provide further assurance that the system has been properly restored.
  5. Once returned to normal treatment operation, re-measure the SDI of bulk recirculating water. You should find SDI values to be lower. Those values can be used as an indicator for excellent microbial control of the cooling water system when biofilms are known to have been eradicated.

Direct Measurement of Legionella Hosts

Generally speaking, if biofilms do not develop, Legionella hosts are not likely to develop because they need the diverse ecology of microbes and nutrients provided by biofilms to survive and thrive. If Legionella hosts do not develop in the system, Legionella are not likely to develop in the system.

Therefore, by detecting and tracking Legionella hosts, you are one step closer to a more predictive indicator of biofilm and Legionella development.

The Takeaway

This data demonstrates that Legionella hosts can secretly develop in a cooling water system, even when microbial control is believed to be excellent. As demonstrated, if Legionella hosts are present, Legionella bacteria are likely to be present. Making things worse, underdeposit corrosion beneath biofilms, and loss of heat transfer efficiency are also threats lurking in the system.

By detecting, eradicating, and monitoring Legionella hosts and biofilms using modern DNA tools, effective biocontrol methods will achieve superior results at lower cost.


  1. ACS Appl. Bio Mater. 2023, 6, 8, 3213–3220, Publication Date: July 10, 2023, Copyright © 2023, American Chemical Society
  2. Boamah DK, Zhou G, Ensminger AW, O’Connor TJ. From Many Hosts, One Accidental Pathogen: The Diverse Protozoan Hosts of Legionella. Front Cell Infect Microbiol. 2017 Nov 30;7:477. doi: 10.3389/fcimb.2017.00477. PMID: 29250488; PMCID: PMC5714891.
  3. Chadee, Amanda and Skovhus, Torben Lund. “Linking Microbiologically Influenced Corrosion to Microbiological Activity UsingMolecular Microbiological Methods.” Materials Performance, May 2020.
  4. “Mapping the microbiome of… ” The Forefront, University of Chicago, November 2017.
  5. Thompson, , Sanders, J., McDonald, D. et al. “A communal catalogue reveals Earth’s multiscale microbial diversity.” Nature, November 2017.
  6. Keele, “Using eDNA to test for pathogens in reused water.” U.S. Department of the Interior, Bureau of Reclamation, September 2016.
  7. Ghylin, “DNA based microbial analysis detects and locates potential contamination in distribution systems.” Journal AWWA, March 2014.


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