Elimination of the formation of biofilm in industrial pipes using enzyme cleaning technique

Graphical abstract


Method details
In the water treatment process, traditional CIP techniques can usually remove or sterilize microbes on the surface of pipes. Taking the advantages of low cost and low energy consumption, these strategies were universally used in food, fermentation, and water treatment industry [1,3]. However, when the biofilm forms in pipelines, the traditional methods would not be available to eliminate it completely [2]. By contrast, the strategy of using muramidase to remove the biofilm in pipes is more effective and in-depth. The comparison of effectiveness between the traditional CIP and enzyme cleaning technique is shown in Fig. 1 and Table 1.

Preparation of material
In this new strategy, N-acetylmuramide glycanohydrolase is introduced as the critical enzyme which will react with the polymeric matrix of the biofilm, reduce its adherence and make the biofilm detach from the surface. In this study, the optimal pH and temperature for reaction is 10 AE 1 and 45 AE 2 8C, respectively. The temperature of 45 AE 2 8C is used throughout the whole application procedure. The material was processed in the following manner.  Bart test kit (HACH Corporation) ATP 1G kit (3M Corporation) 75% Alcohol

Preparation of enzyme solution for cleaning
In Fig. 2, the detergent 1 is the initial enzyme solution, and the detergent 2 represents soda or acid which were used to adjust the pH and clean the pipes. The cleaning process can be proceeded with according to the following steps: 1. Drain the retention water in all pipes and equipment.
2. Remove the in-line micro filter (d < 50 mm) unless they are necessary to protect equipment or be cleaned. 3. Pump detergent 2 (200 g/L soda) into water tank to keep the pH at 10 AE 1. 4. Pump detergent 1 (enzyme) into water tank to make the enzyme concentration reach 0.25%.
In this step, we need to test whether the concentration of enzyme solution meets the cleaning requirement using Aller test kit (Fig. 3a). Firstly, sample 500 mL enzyme solution, and then get 5 mL sample into flask using a disposable syringe. Please note that both flask and syringe need to be washed using samples previously. Add two drops of Cl-1 reagent into flask, and the sample will change into purple. Then, drop Cl-2 reagent into flask slowly and shake it simultaneously until the sample turned from purple to yellow. After that, drop Cl-3 reagent into flask slowly and shake it simultaneously until the sample changed from yellow to purple, and record the consumed volume of Cl-3 reagent as V 1 (Fig. 3b). The initial water was also tested according to the above process. The concentration of enzyme solution could be calculated following the formula below: Note: (a) V 1 represents the volume of Cl-3 reagent consumed by enzyme solution. (b) V 0 represents the volume of Cl-3 reagent consumed by the initial water. (c) E c represents the concentration of  5. Lastly, circulate the prepared enzyme solution in all pipes and equipment which need to be cleaned.

Test of ATP in process water
ATP is the essential energy molecule which universally exists in all organisms. Therefore, one can quickly judge whether there are any living organisms or the biofilm was completely eliminated by testing the amount of ATP (<10 cATP) in water (Fig. 4a). Here, we introduce ATP luciferin test technique which is based on the positive relationship between fluorescence intensity and ATP concentration while the fluorescence reaction is catalyzed by luciferase (Fig. 4b).
The AIP test can be carried out by the following steps (shown in Fig. 5):  1. Get a water quality sampling rod out of the bag (storage at 2-8 8C) and keep it in balance for 10 min at room temperature before use. 2. Pull rod core and immerse it into the sample for a few seconds, then take it out and ensure there is a drop of liquid at the bottom of the rod core. 3. Carefully insert the rod core back into test bars (make sure the core do not recline on wall and the sample rods can enter into the reaction liquid at the bottom). Then press the red part of the sample rods to make it fully inserted into the reaction liquid. 4. Shake the test bar for 5 s to mix the liquid more completely. 5. Turn on the Clean-Trace TM ATP detector. When self-check process is completed, open the slot and insert test bar.

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[ ( F i g . _ 4 ) T D $ F I G ] 6. Lastly, close the slot and start to test for about 10 s, record the results (unit: RLU). For enzyme cleaning, if the result is below 100 RLU, the cleaning effect is good, otherwise it should continue.

Bart test for biofilm-forming bacteria
The SLYM-BARTs can be used as a P/A test capable of indicating to some extent the possible organisms present in the water sample. Slime-forming bacteria are able to produce copious amounts of slime without necessarily having to use any iron. Iron bacteria also produce slime but usually it is thinner and involves the accumulation of various forms of iron (http://65.87.233.94/BARTs/ SLYM.html?).
After enzyme cleaning, one should test the water samples again, to assess the efficacy of the procedure. If the BARTs reveal there are still biofilm-forming bacteria, enzyme cleaning should be carried out again. The slime-forming bacteria should be tested to evaluate the micro-ecology of the pipelines after enzyme cleaning (see Table 1). The potentially slime-forming bacteria can be tested following the steps below (Fig. 6): Slime-forming bacteria generally produce the thickest slime formations under aerobic (oxidative) conditions, which develop around the floating ball. Growth may be recognized as a cloudy or gel-like growth, which can be localized or occur throughout the sample. These growths are usually white, gray, yellow, or beige in color and can darken over time (http://65.87.233.94/BARTs/SLYM.html?). Meanwhile, they can usually have fluorescence when incubated in BART (Fig. 7). For these samples, safely dispose using a dedicated microwave oven or by autoclaving them.