Conference Programme & Proceedings




The 5th International Conference on Advanced Model Measurement Technology for the Maritime Industry (AMT’17)

11th – 13th October 2017, Glasgow, UK. AMT’17 is organised by the Hydro-Testing Forum (HTF) members and will be hosted by Strathclyde University.

Conference Programme & Proceedings

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Session 3B A MARINE BIOFILM FLOW-CELL FOR SCREENING ANTIFOULING MARINE COATINGS USING OPTICAL COHERENCE TOMOGRAPHY Stefania Fabbri, Akzonobel - International Paint, UK Simon Dennington, University of Southampton, UK Paul Stoodley, University of Southampton, UK Jennifer Longyear, Akzonobel - International Paint, UK A novel fouling marine flow-cell was designed and fitted with a top clear 5 mm thick plastic lid to allow real time imaging of the biofilm using optical coherence tomography (OCT). The OCT was used to analyse biofilm removal and mechanical properties during shear-stress experiments. The OCT measures intensity depth profiles from translucent samples such as biofilms. Consecutive scans provide a crosssectional view of the biofilm structure which are then combined to give volumetric representations. The scanning speed of the OCT reached up to 30,000 scans/s and covers a field of view of 9x9 mm2. The bottom plate of the flow-cell was machined to allow the insertion of fouled microscope slides (25 x 55 x 1 mm). Marine biofilms were grown on spray coated (inert coating) slides in seawater for up to 2 years to test mechanical properties (triplicates). Marine biofilms were grown dynamically on 6 different antifouling coatings (A, B, C, D, E, F) for 8 weeks to test biofilm removal (duplicates). Marine biofilms were also grown statically and dynamically on an antifouling coating G to assess biofilm removal. Biofilm mechanical behaviour and removal were assessed by increasing (load cycle) or decreasing (unload cycle) the flow velocity (and therefore shear stress) in a stepwise manner over the entire pump range. Each step interval lasted 30 s except at the highest flow which was held for 5 min before starting the unloading cycle. The OCT was set to measure 10 xz-cross sections along the flow for each velocity step. 3D C-scans were also acquired before the loading cycle and at the end of the unloading cycle. The OCT images were analysed using ImageJ and Matlab. The angle of deformation of individual biofilm clusters were measured for each shear stress to obtain a stress/strain curve. Stress/strain curves showed classic viscoelastic biofilm behaviour. From the initial linear region of the load cycle the shear modulus (G) was estimated to be G = 46.2 ±5.43 Pa (n = 3). The biofilm also showed a residual strain εR = 0.28 ± 0.01 (n = 2). The % cross-sectional area removed (%A) as a function of the shear stress was measured from the OCT images for each antifouled slide. The %A value increases exponentially for all the antifouling coatings until a shear stress of ~25 Pa, when it reached a plateau. Considering a shear stress of 15 Pa, %A of coating C (A% = 75%) was significantly higher than the value of the other coatings showing best performance. The %A of the biofilm grown on coating G statically (A% = 68%) was lower than the value of the biofilm grown dynamically (A% = 82%). These results show that the marine biofilm flow-cell combined with OCT can be used to assess mechanical properties of marine biofilms and detect differences (in terms of removal) in biofilms grown on different coatings. Future testing will focus on assessing how mechanical properties of biofilms interact with their physical properties (roughness, thickness, extent) to produce drag. FULL PAPER TO BE INCLUDED IN UPDATED PROCEEDINGS 250

A MARINE BIOFILM FLOW CELL FOR IN SITU DETERMINATION OF DRAG, STRUCTURE AND VISCOELASTIC PROPERTIES Stefania Fabbri, AkzoNobel, UK, University of Liverpool, UK Simon Dennington, University of Southampton, UK Jennifer Longyear, AkzoNobel, UK, University of Southampton, UK Paul Stoodley, University of Southampton, UK, The Ohio State University, USA It is not straightforward to link biofilm parameters to frictional drag, likely because of the heterogeneous nature of slime. Here we present the design and calibration of a pragmatic, small scale flow-cell in which biofilms can either be cultured under flow or grown statically and then assessed under flow for drag and other properties. The flow cell test section comprised a rectangular channel (870x10x55mm) constructed by sandwiching together rigid opaque PVC panels and side panels of clear acrylic, which allow natural light to enter. A maze-like entry section evened out the inlet flow. Seawater flow rate through the channel was monitored by an in-line digital flowmeter, and pressure drop (ΔP) along the test section was measured using a differential pressure sensor. The friction coefficient (Cf) of the flow cell was found by measuring the ΔP at various flow velocities (u) over the entire pump range (maximum Re ~22,000). Flow cell calibration was carried out using a clean inert marine coating, and various roughness grades (P40, P80 and P120) of waterproof sandpaper sheets, fixed to the wide faces of the channel, to find Cf for each rigid roughness. ΔP was proportional to u2, indicating flow was turbulent in this region (R = 99%). When fouled panels are used as the channel floor and a clear acrylic panel as the ceiling, the flow cell allows for simultaneous measurement of Cf of the lower surface and biofilm physico-mechanical properties (e.g. thickness, roughness, viscoelasticity) by optical coherence tomography (OCT) imaging, which generates depth profiles of translucent samples. Changes to biofilm physico-mechanical properties during flow loading/unloading cycles, determined by image analysis, can be compared to simultaneously collected ΔP measurements scaled to a one-sided sandpaper Cf calibration. Future experiments will assess physico-mechanical and drag properties of marine fouling biofilms in flow using ΔP and OCT.

1. Introduction A well-recognised challenge in the shipping industry is frictional drag penalty associated with microbial slime fouling on ship hulls [1]. Various estimates of the magnitude of the drag penalty have been presented in the literature (for review see [1]) and a commonly cited figure for the ship powering penalty of biofilms is up to 20% dependent on the thickness of the biofilm and vessel speed [2]. Biofilm drag estimates can be blanket-applied to calculate global biofilm fuel penalties for the entire fleet, etc., but calculations such as these necessarily neglect any variability in the characteristics of biofilms and how biofilms might change with coating choice or vessel operational profile. However, it is well documented that fouling biofilms contain diverse species assemblages and have diverse physical mechanical properties including adhesion, thickness, and surface texture [3, 4]. As fouling control coatings are developed to further reduce slime fouling, metrics and methods that can differentiate coatings by the drag penalties of whatever slime does accrue will be valuable.

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It is difficult to measure biofilm fouling and drag. Ship slime is heterogeneous, patchy, and a dynamic viscoelastic material [5]. If ship performance measurements can be obtained, e.g. before and after a hull clean, the challenge is measuring the character of the biofilm across the full hull. The default metric is surface area coverage with an estimate of biofilm thickness. Ship measurements are also only point measurements or case studies, and while informative, it is difficult to extrapolate from a single study. Typically, instead, fouled test pieces (scale on the order of 1 m) are used to measure drag, and scaling similarity laws are applied to extend results to ship-scale fouling consequences. Large hydrodynamic testing facilities with the high sensitivity and high flow rates required for biofilm drag measurements at ship speeds, such as towing tanks [6, 7], cavitation tunnels [8, 9], large flow cells [10] and rotating discs [11, 12], have yielded high quality measurements of fouling biofilm drag for large panels that were fouled in static or dynamic conditions. However, these facilities are often expensive to use and can only process a small number of panels each day, necessitating low experimental replication, and the “biological contamination” of any test facility after testing live fouling may be an issue. It is also typically difficult to observe the changes to fouling during testing. By moving to smaller test pieces (scale on the order of 0.01 - 0.1m), more replicates can be considered and a better understanding of the biological, physical, mechanical, or chemical properties of the microbial fouling can be obtained. Examples include testing in microfluidic chambers [13] or by rotating disc torque measurements [14]. However, with such small samples it is not straightforward to extrapolate drag measurements to a larger scale, or it is not possible to observe the fouling appropriately during testing. This study presents the design of a small, pragmatic pressure drop testing flow cell for measuring biofilm drag that can also allow for simultaneous measurement of biofilm structure using optical coherence tomography, a tissue imaging system that has been relatively recently adopted in biofilm science [15, 16]. The flow cell has an additional advantage in that it can be used for in-situ biofilm culturing in flow, so as to create conditions more similar to ship conditions in service than static immersion. The flow cell was calibrated with rigid sandpaper surfaces to confirm the sensitivity of the pressure sensors and that flow through the channel was turbulent. Finally, we demonstrate the flow cell can be used to culture biofilms and image microbial fouling during hydrodynamic drag measurements.

2. Materials and Methods 2.1. Design of the enclosed marine biofilm culturing channel system The body of the flow cell was made of anodized aluminium and consisted of a base plate, with studs attached as hollow frame, and an adjustable inlet/outlet (Figure 1). The test section was 870 mm in length (L), 10 mm in height (H) and 50 in width (W) (5:1 aspect ratio). The section was constructed by sandwiching together four panels into the studs of the base. A rubber gasket was inserted between each panel for a better seal. The four panels are held together by fixed bolts with nuts and washers on the top. The bottom and the top panels (870 mm in length and 100 mm in width) are made of rigid PVC, whereas the side panels (870 mm in length,10 mm in height and 25 mm in width) are of clear acrylic to allow natural light to enter from either side. Two pressure sensor ports were machined into the top panel at 3 mm and 84 mm from the inlet, giving a pressure drop test length of 81 mm. 252

The inlet manifold was composed of a 1.5 inch male camlock fitting which flowed down into a 50 mm x 10 mm form which was the same cross-sectional area. Inside, a walled-lamina type maze was built to break up the water flow (Fig. 1, inset). The flow cell outlet consisted of an open fitting to deflect the flow 90° downwards into the recirculating water reservoir. The mating faces between the inlet/outlet and the main channel had a soft rubber seal that compressed as the adjustable inlet/outlet fittings are tightened up against the mating faces.

Fig. 1 – Exploded schematic of flow cell highlighting sandwich design and maze design of inlet manifold (inset). Source: Safire Associates. The fully installed enclosed marine biofilm culturing channel system consisted of a recirculating tank, a rectangular flow cell and a submersible pump (Fig. 2). An interchangeable submersible pump (low range: Aqua Medic DC Runner 3.1, max flow rate= 5000L/h; high range: Aqua Medic DC Runner 9.1, max flow rate= 9000 L/h), was immersed in the tank and used to drive fresh seawater through the flow cell via a recirculation pipe system made of flexible PVC hoses. The water exited the flow cell freely into the tank as a waterfall. The flow cell was firmly fixed on top of the tank’s lid with spacers to raise it 30mm above the surface.

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Fig. 2 – Fully installed, the enclosed marine biofilm culturing channel system consisted of a recirculating tank, a rectangular flow cell and a submersible pump immersed in the tank. Fresh sea water entered the flow cell inlet through a recirculation pipe system connected to the pump and exited the flow cell (outlet) into the tank as a waterfall. 2.2. Flow measurements An analogue rotating vane flowmeter with digital readout (PRS Corrente+, AQUAfair) placed at the flow cell inlet monitored the flow rate (Q) and the water temperature (T) through the system. The flow and temperature accuracies of the flowmeter were ±2% and ±1% of the reading over the design range. The flow rate was varied via the pump controller and an additional by-pass ball valve on the recirculation loop. The average flow velocity (u) for each velocity setting was measured from the cross-sectional area of the channel. The Reynolds number (Re) was calculated using:

Re

Dh u v

(1)

where Dh = the hydraulic diameter = 0.017 m (based on the cross-sectional area and wetted perimeter), and ν = kinematic viscosity of seawater (1.31 × 10−6 m2/s at 10°C).

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Two interchangeable pumps were used to generate low and high flow velocity ranges (low range uavg = 0.7 to 1.13 m/s. Re = 8.9x103 to 1.4x104, R2 = 0.995; high range uavg = 1.3 to 2.02 m/s, Re = 1.7x104 to 2.6x104, R2 = 0.998). The pressure drop (ΔP) between the measurement ports was measured using a differential pressure sensor (PL-692, Omni Instruments) for liquids and gases. The sensor was connected to the pressure ports through 6 mm diameter tubes full of water. The pressure sensor recorded the pressure over time through a data acquisition (DAQ) unit (DI-149, DataQ) connected to a laptop computer. 2.3. Determination of flow cell hydrodynamics Waterproof sandpaper sheets of different roughness grades were fixed to the face of the bottom and top panels using a water-based polyurethane adhesive to create sandpaper-coated panels for calibration purposes. The grades of red aluminium oxide sandpaper used were P120, P80, P40 (Toolstation). Panels sprayed with an inert marine primer (dry coating film thickness= 100 μm, International Paint, UK) served as the blank surfaces for the calibration. The arithmetical mean surface roughness height (Sa) of the PVC and each sandpaper sheet was assessed by blue light interferometry (MikroCAD, LMI Technologies) (Table 1). The hydrodynamic characteristics for the flow cell with clean coated plates (blank) and sandpaper-coated panels was determined through a series of pressure drop measurements carried out over the entire flow range and the relationship between the Fanning friction factor (Cf) and Re was found. Care was taken to bleed all the air from the pressure sensor tubing to zero the instrument prior to taking the measurements. Cf is a dimensionless number given by the following equation: 𝐶 =8

(2)

where = density of seawater = 999.73 kg/ m3 at 10°C, P = pressure drop along length L (Pa), and L= length between pressure ports = 0.8 m. Rearranging equation (2) we find that ΔP is related to u2 according to: Δ𝑃 = 𝐶

𝑢 = 𝑠𝑙𝑜𝑝𝑒 𝑢

(3)

The Cf values were found for the flow cell with clean coated plates and sandpaper-coated panels by linear regression of the ∆P vs. u2 curves. 3. Results Calibration of the blank and sandpaper-coated panels consisted of a series of ΔP measurements carried out over the entire flow range. The Fanning friction factor, Cf, for the blank plotted against the Re numbers are presented in Fig. 3. As can be observed in the plot, the Cf values as a function of Reynolds number closely followed the trend predicted by the Blasius formula for turbulent flow in circular smooth pipes but were approximately 1.2 higher than those predicted from Blasius formula for turbulent flow in smooth pipes (dotted lines). This could be caused by an inadequate flow cell entry length or the corner

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effects since pressure gradients are greater across entry length regions than section of the channel where the flow is fully developed. 0.014 y = 0.292x-0.25

Friction Factor, Cf

0.012

y = 0.1549x-0.305 R² = 0.96778

0.010 0.008 0.006 0.004 0.002 0.000 0

5,000

10,000

15,000

20,000

25,000

30,000

35,000

Re

Fig. 3 - Fanning friction factor, Cf, vs Reynolds number, Re, for the flow cell prior to biofilm exposure. The dotted red line is the least squares power law fit of the present results. The black dashed line shows the Blasius formula for turbulent flow in smooth pipes. The Fanning friction factor values, Cf, for the sandpapers are presented in Fig. 4. There was a significant increase of the Cf for all the sandpapers compared to the blank. At the highest Reynolds number tested (Re ~ 22,000), Cf of the sandpapers P120, P40 and P80 were 1.9, 2.2 and 4 times the C f of blank respectively. 0.045 BLANK P120

0.04

P80 P40

Friction Factor, Cf

0.035 0.03 0.025 0.02 0.015 0.01 0.005 0 0

5,000

10,000

15,000

20,000

25,000

30,000

Re

Fig. 4 - The instantaneous Fanning friction factor, Cf, vs Reynolds number, Re, for the blank surface and three sandpaper roughness grades. 256

The pressure drop along the flow cell for blank and the sandpapers was plotted against the square of the flow velocity (Fig. 5).

Fig. 5 - Pressure drop vs u2 for the flow cell with sandpaper-covered panels. The slope was found from the linearization of the curves and used to measure the Cf according to Eq. 2. Linear regression gave the slope which was used to measure Cf according to Eq. 2 (Table 1). The data for the P120 and P80 grade sandpaper surfaces were close to each other, and the measured S a values confirmed that the two grades had a similar surface roughness of 39.3 μm and 46.1 μm respectively. Table 1 – Roughness, friction coefficients for each tested rigid surface (see Figures 4, 5) BLANK P120 P80 P40 Sa (μm) 4.13 39.3 46.1 96.7 Slope (Fig. 5) Cf (Fig. 4)

634.8 0.11

1412.5 0.24

1661 0.28

2950.9 0.49

The value of Cf calculated from the pressure drop data can be fit to the surface area mean roughness height (Sa) of the sandpapers by the linear relation (Fig. 6, R2=99%) 𝐶 = 0.0042 × 𝑆 + 0.082

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(4)

0.6 0.5

Friction Factor, Cf

0.4 y = 0.0042x+ 0.082 R² = 0.99547 0.3 0.2 0.1 0 0

10

20

30

40

50

60

70

80

90

100

Sa[µm]

Fig. 6 - Friction factor, Cf, vs measured surface roughness, Sa.

4. Discussion We have presented the design of a practical small scale turbulent flow cell. Surfaces to be tested form the base and lid of the cell, although the lid can be replaced by an optically transparent cover to allow real-time observation of the base surface. Measuring pressure drop across the cell at different flow rates allowed friction factors of control surfaces and cultivated biofilms to be determined. 4.1 Advantages of the design There are several notable advantages in using the described pragmatic flow cell: 1. The flow cell is modular, and so can be adapted to take different test pieces including biofilm panels grown elsewhere (e.g. through static sea immersion, attached to a vessel, etc.). 2.

The flow cell is relatively inexpensive to build.

3. Light can be admitted or excluded by choice of side spacers so there is potential for in situ photosynthetic marine fouling biofilm culture (as expected on a vessel) or for examining biofilms from other industrial settings grown in darkness. 4. Rheometer recesses can be milled in to the top surface of the cell and discs inserted before in situ biofilm culture. In this way the flow cell can allow for comparison of biofilm impact through linear (bottom plate) and rotational (discs from top plate) drag metrics [14] (see Fig 7.) 258

Fig. 7 – Panel with recessed rheometer discs for comparative study of drag measured by the coefficient of momentum (Cm)[14] and Cf. 5. Calibration was performed with two major channel faces covered in sandpaper. However, it was possible to determine an alternative calibration with only one face covered in sandpaper and one face a smooth, clear acrylic lid. In this configuration, the fouling removal can be observed during flow using conventional means (visual observation, photography) or using an OCT system (Fig. 8) which images the biofilm in cross-section. An example of a biofilm cross-section collected from the flow cell during a pressure drop experiment is shown in Fig. 9 below. Using image analysis, various physical properties of the biofilm, such as thickness, and surface area coverage can be determined. With video or time-lapse imaging, biofilm viscoelastic behaviour [15, 17], or drag reducing forms such as streamers or ripples [18] can be studied.

Fig. 8 – The top of the flow cell can be replaced with a clear acrylic lid for real time imaging of the biofilm in flow by optical coherence tomography (OCT) or digital photography.

Fig. 9 – Cross section of fouling biofilm on a coated panel fully immersed in the flow cell as imaged by optical coherence tomography (OCT). Biofilm thickness, roughness, volume, surface coverage, etc. can be determined non-destructively from these images. Scale bar is 100 m. 259

4.2 Limitations of the design There are likewise limitations in use of the flow cell as a testing apparatus: 1. In this design, the pressure sensors were placed close to the inlet and outlet of the cell to generate the maximum pressure drop. However, to ensure that fully turbulent flow has been developed at the highpressure sensor, lead distances of 30H or 60H (where H is the height of the cell) have both been referenced as appropriate for the development of fully turbulent flow in flow cells [19, 20]. Either lead distance would have decreased the magnitude of the observable pressure drop and/or necessitated extending the length of the flow cell. Placing the pressure drop sensors at the flow cell extremes, combined with the complex flow-equalising inlet, was considered an acceptable compromise for pragmatic testing. 2. It is uncertain how to treat one-sided calibrations with academic rigour. However, for such heterogonous surfaces as biofilms in flux during exposure to flow, one-sided data combined with real time observations of how the biofilm is changing (unobtainable in double-sided flow cells) are valuable. 4.3 Future Work In future work we will use the flow cell to relate physical properties of biofilm (thickness, roughness, surface cover and viscoelastic parameters) to drag. It has been hypothesized that the viscoelastic nature of biofilm is responsible for the higher than the predicted drag from rigid roughness but this has not been directly verified. We can also use the system to determine if different classes of marine coatings influence the viscoelastic properties and whether this might be an important consideration in the development of antifouling coatings. Finally, we will use the system to determine how the biofilm is removed as a function of shear stress and whether there is a critical shear stress at which biofilm detaches or whether there is steady erosion which increases as a function of shear stress. By relating drag to the physical interactions between the fluid flowing over the surface and the biofilm we will better understand the role that biofilm formation plays in increasing the drag penalty which in turn will lead to insights into the design of antifouling surfaces.

5. References 1. 2. 3. 4. 5. 6.

Townsin, R.L., The ship hull fouling penalty. Biofouling, 2003. 19(S1): p. 9-15. Schultz, M.P. and G.W. Swain, The influence of biofilms on skin friction drag. Biofouling, 2000. 15: p. 129-139. Flemming, H.-C., et al., Biofilms: an emergent form of bacterial life. Nature Reviews Microbiology, 2016. 14(9): p. 563-575. Fabbri, S. and P. Stoodley, Mechanical properties of biofilms, in The Perfect Slime: Microbial Extracellular Polymeric Substances (EPS), H.-C. Flemming, T.R. Neu, and J. Wingender, Editors. 2016, IWA Publishing. Salta, M., et al., Marine biofilms on artificial surfaces: structure and dynamics. Environmental Microbiology, 2013. 15: p. 2879-2893. Schultz, M.P., Frictional resistance of antifouling coating systems. Transactions of the ASME-IJournal of Fluids Engineering, 2004. 126(6): p. 1039-1047. 260

7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.

Schultz, M.P., Effects of coating roughness and biofouling on ship resistance and powering. Biofouling, 2007. 23(5): p. 331-341. Atlar, M. and M. Callow, The development of foul-release coatings for seagoing vessels. Journal of Marine Design and Operations B, 2003. 4(11). Korkut, E. and M. Atlar, An experimental investigation of the effect of foul release coating application on performance, noise and cavitation characteristics of marine propellers. Ocean Engineering, 2012. 41: p. 1-12. Schultz, M.P., et al., Impact of diatomaceous biofilms on the frictional drag of fouling-release coatings. Biofouling, 2015. 31(9-10): p. 759-773. Schultz, M.P. and A. Myers, Comparison of three roughness function determination methods. Experiments in fluids, 2003. 35(4): p. 372-379. Holm, E., et al., Evaluation of hydrodynamic drag on experimental fouling-release surfaces, using rotating disks. Biofouling, 2004. 20(4-5): p. 219-226. Salta, M., et al., Life under flow: a novel microfluidic device for the assessment of anti-biofilm technologies. Biomicrofluidics, 2013. 7(6): p. 064118. Dennington, S., et al., Miniaturized rotating disc rheometer test for rapid screening of drag reducing marine coatings. Surface Topography: Metrology and Properties, 2015. 3(3): p. 034004. Blauert, F., H. Horn, and M. Wagner, Time‐resolved biofilm deformation measurements using optical coherence tomography. Biotechnology and bioengineering, 2015. 112(9): p. 1893-1905. Wagner, M. and H. Horn, Optical coherence tomography in biofilm research: A comprehensive review. Biotechnology and Bioengineering, 2017. Stoodley, P., et al., Structural deformation of bacterial biofilms caused by short-term fluctuations in fluid shear: an in situ investigation of biofilm rheology. Biotechnology and Bioengineering, 1999. 65(1): p. 83-92. Fabbri, S., et al., Fluid‐driven Interfacial instabilities and turbulence in bacterial biofilms. Environmental Microbiology, 2017. Schultz, M.P., et al., A turbulent channel flow apparatus for the determination of the adhesion strength of microfouling organisms. Biofouling, 2000. 15(4): p. 243-251. Hong, J., J. Katz, and M.P. Schultz, Near-wall turbulence statistics and flow structures over three-dimensional roughness in a turbulent channel flow. Journal of Fluid Mechanics, 2011. 667: p. 1-37.

6. Acknowledgements The authors would like to thank Adrian Walker of Safire Associates for contributions to the practical design of the flow cell and an excellent build. This work was funded by a joint InnovateUK and BBSRC grant to International Paint Ltd and University of Southampton, respectively, under the Management and Use of Biofilms Programme.

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