Biofilm Removal Protocol for Water Circuits

Biofilm is a film of microorganisms protected by an organic matrix that adheres to the walls of pipes, tanks, heat exchangers and pasteurisers. It is the main cause of recontamination in food-industry CIP circuits and in industrial water circuits. This protocol chains together detection, alkaline cleaning, enzymatic treatment, acid phase, disinfection and verification with the doses, temperatures and times from the products' technical data sheets.

Materials required

• Insta Check Pro: biofilm detector for surfaces
• Alcalino Pro 6: very low-foam liquid alkaline detergent for CIP (0.5-5 %, 45-70 ºC, 20-45 min)
• Alcalino Pro 3 as a phosphate-free alternative with high sequestering capacity (1-3 %, 60-70 ºC, 20-45 min; do not use on aluminium)
• Ten Cip + Enzim Pro: enzymatic treatment by recirculation (0.2-0.5 % + 0.05 %, 40-55 ºC, 20-60 min)
• Nitracip Pro: strongly acidic non-foaming descaler for CIP (0.5-1 %, 40-60 ºC, 15-20 min)
• Acid Pro as a controlled-foam acid alternative (1-3 % in CIP, 40-70 ºC)
• Oxa Per: peracetic acid-based disinfectant for CIP (1 %, 15 min, 20-50 ºC)
• Clor NF Pro as an alternative non-foaming chlorinated alkaline disinfectant detergent (2-8 %, 20-60 ºC, 5-15 min; do not use on aluminium)
• Bacter 200: biocide for legionella treatment in cooling towers and evaporative condensers
• Clor Active 15%: sodium hypochlorite for the disinfection of public drinking water (maximum dose 3 mg/l of active chlorine)
• Recirculation pump or CIP unit with temperature control
• Thermometer and pH meter
• Chlorine and peroxide test strips
• Graduated containers and dispensers
• HACCP cleaning and disinfection record

Step-by-step procedure

1. Biofilm detection: on the accessible surfaces of the circuit (inside of tanks, seals, elbows, nozzles and dismantled end points) spray Insta Check Pro and wait approximately one minute. If no effervescence is observed the result is negative; effervescence indicates the presence of biofilm. Mark the positive points as control points for the final check.
2. System preparation: isolate the circuit with the shut-off valves, empty it, dismantle and clean filters and strainers, and inspect seals and dead zones. Check the materials: Alcalino Pro 3 and Clor NF Pro must not be used on aluminium.
3. Prior rinse with water: circulate water to flush out loose soil; the data sheets of Alcalino Pro 6, Alcalino Pro 3 and Nitracip Pro specify this prior rinse.
4. Alkaline cleaning by recirculation: prepare Alcalino Pro 6 at 0.5 to 5 % and recirculate at a temperature of 45 to 70 ºC for 20-45 minutes; the 1 % solution has a pH of 12.8 ± 0.5. If a product with high sequestering capacity that keeps circuits free of scale is preferred, use Alcalino Pro 3 at 1 to 3 % at 60-70 ºC for 20-45 minutes. Rinse with water.
5. Intermediate rinse: circulate water until the outlet pH matches that of the inlet water; no residual alkalinity may remain before the enzymatic or acid phase.
6. Enzymatic treatment (established biofilm): prepare Ten Cip diluted to 0.2 to 0.5 % and add Enzim Pro at a dose of 0.05 %. Circulate for 20-60 minutes at 40-55 ºC and rinse with water. Enzim Pro is a concentrated enzyme blend for the prevention and removal of biofilms in the food industry: it degrades the matrix that protects the microorganisms.
7. Acid phase: with the circuit rinsed, recirculate Nitracip Pro at a concentration of 0.5-1 % for around 15-20 minutes at a temperature of 40 to 60 ºC, and rinse with plenty of water. It removes limescale, mineral deposits and residues left by alkaline detergents. Controlled-foam alternative: Acid Pro at 1 to 3 % in CIP, at 40-70 ºC; it does not attack chrome-plated or enamelled materials or rubber and plastic seals.
8. Rinse: circulate water until the outlet pH is neutral. Mandatory before any chlorinated product: in contact with acids, Clor NF Pro and hypochlorites release toxic gases.
9. Disinfection by recirculation: apply Oxa Per diluted to 1 %, allow to act for around 15 minutes at a use temperature of 20-50 ºC and rinse thoroughly with water. Oxa Per contains 5 % peracetic acid and 23 % hydrogen peroxide; it complies with standard UNE-EN 13697 under clean conditions against Escherichia coli, Staphylococcus aureus, Enterococcus hirae, Pseudomonas aeruginosa and Saccharomyces cerevisiae, and holds registration number 17-20/40-05521-HA. As a single-phase chlorinated alkaline alternative, Clor NF Pro is used diluted at 2 to 8 % at 20-60 ºC, leaving it to act for 5-15 minutes and rinsing with plenty of water; it complies with UNE-EN 13697 under dirty conditions (including Pseudomonas aeruginosa) and UNE-EN 14476, with registration number 18-20/40-09617-HA.
10. Final rinse and verification: circulate water until the strips detect neither chlorine nor peroxide and the water runs colourless and odourless. Repeat Insta Check Pro at the control points: no effervescence means the treatment was effective. If any point remains positive, repeat from the enzymatic phase.
11. Industrial water circuits and Legionella: in cooling towers and evaporative condensers, dose Bacter 200 at 0.2 to 0.4 % every 7 days at the start of the treatment (slime present) and, once it has taken effect, reduce to 0.1 to 0.2 % weekly as maintenance. Application by specialised professional personnel, within the programme of Royal Decree 487/2022. Bacter 200 passes the UNE-EN 13623 test against Legionella pneumophila for 60 minutes at 30 ºC (registration 21-100-04598).
12. Record and maintenance plan: record date, phases, products, doses, temperatures, times, biofilm check result and person responsible. The frequency is set by the HACCP plan; a positive biofilm result or an out-of-limit analysis requires the cycle to be repeated.

Verification and control

Three pieces of evidence: the biofilm check with Insta Check Pro at the control points before and after (no effervescence after approximately one minute means a negative result); the final rinse water (pH equal to the inlet, no chlorine or peroxide on the test strip, colourless and odourless); and the microbiological analyses of the HACCP plan or, in towers and condensers, the Legionella controls of Royal Decree 487/2022. If an ATP luminometer is used, the thresholds are those of the equipment manufacturer. Everything is entered in the cleaning and disinfection record with date, values and the signature of the person responsible.

Safety and PPE

• Protective gloves, clothing and eye protection throughout handling: Alcalino Pro 6, Alcalino Pro 3, Nitracip Pro, Acid Pro, Oxa Per and Clor NF Pro cause severe skin burns and serious eye damage
• Adequate ventilation when handling Nitracip Pro (toxic if inhaled) and Oxa Per (may cause respiratory irritation)
• Never bring Clor NF Pro, Clor Active 15 % or any hypochlorite into contact with acid products: in contact with acids they release toxic gases
• Enzim Pro may cause allergy or asthma symptoms if inhaled: avoid breathing the vapour and, if ventilation is insufficient, use respiratory protection
• Emergency shower and eye-wash station within reach; in case of eye contact, rinse with water for several minutes and seek medical attention

Recommended Instaquim products

For the alkaline cleaning of circuits, Alcalino Pro 6 (very low foam, pH 12.8 at 1 %) and Alcalino Pro 3 (phosphate-free, high sequestering capacity) cover CIP recirculation. For the biofilm matrix, the Ten Cip + Enzim Pro pairing is the enzymatic treatment for CIP systems. For the acid phase, Nitracip Pro (nitric acid-based) and Acid Pro. For disinfection by recirculation, Oxa Per (peracetic acid) and Clor NF Pro (chlorinated alkaline) hold registration numbers and UNE-EN 13697 tests; Hipoclorito 15% is also used in the circulation cleaning of circuits and pasteurisers, at doses of 0.8 to 1.6 % for surface sanitisation. For Legionella control in cooling towers and evaporative condensers, Bacter 200; and for the disinfection of drinking water, Clor Active 15 %, which complies with UNE-EN 901:2013.

Applicable regulations: Regulation (EC) No 852/2004 (food hygiene and HACCP), Regulation (EU) No 528/2012 (biocides: use according to registration and user category), Royal Decree 487/2022 (prevention and control of legionellosis), Regulation (EC) No 1272/2008 CLP (hazards and PPE on the data sheets) and Regulation (EC) No 648/2004 (detergents).

Frequently asked questions

How do I know whether there is biofilm in the circuit before treating it?

On accessible surfaces (inside of tanks, seals, elbows or dismantled nozzles) Insta Check Pro can be used: spray it, wait approximately one minute and, if effervescence appears, biofilm is present. In inaccessible sections, the reference is the microbiological analyses of the HACCP plan.

Why is disinfecting without cleaning first not enough?

The biofilm matrix protects the microorganisms and the disinfectant does not penetrate it well. That is why alkaline cleaning (organic matter), enzymatic treatment (biofilm matrix), acid phase (mineral deposits) and only then disinfection are chained together.

Which alkaline detergent should I use in a CIP circuit?

Alcalino Pro 6, very low-foam, is recirculated at 0.5 to 5 % at 45-70 ºC for 20-45 minutes, with a prior rinse with water. Alcalino Pro 3 is recirculated at 1 to 3 % at 60-70 ºC for 20-45 minutes; it must not be used on aluminium.

What is the enzymatic phase for?

Enzim Pro is a concentrated enzyme blend for the prevention and removal of biofilms in the food industry. In CIP circuits it is combined with Ten Cip (0.2-0.5 %) at a dose of 0.05 %, recirculating for 20-60 minutes at 40-55 ºC and then rinsing with water.

Can I mix the chlorinated disinfectant with the acid descaler to save a step?

No. Clor NF Pro and hypochlorites release toxic gases in contact with acids. Each phase is separated by a rinse with water.

Which product should I use for Legionella in a cooling tower?

Bacter 200, to be applied by specialised professional personnel. At the start of the treatment, with slime present, it is dosed at 0.2 to 0.4 % every 7 days; for maintenance, at 0.1 to 0.2 % weekly. It passes the UNE-EN 13623 test against Legionella pneumophila for 60 minutes at 30 ºC.

What if the circuit carries drinking water?

The products in this protocol are rinsed out completely before the circuit is returned to service. For the disinfection of the water itself, Clor Active 15 % is a hypochlorite suitable for public drinking water that complies with standard UNE-EN 901:2013, with a maximum dose of 3 mg/l of active chlorine (1 L per 50 m³ of water).