online assessment of biofilm development, sloughing and forced detachment in tube reactor by means...

ARTICLE

Online Assessment of Biofilm Development,Sloughing and Forced Detachment in Tube Reactorby Means of Magnetic Resonance Microscopy

Michael Wagner,1 Bertram Manz,2 Frank Volke,2 Thomas R. Neu,3 Harald Horn1

1Institute of Water Quality Control, Technische Universitat Munchen, Am Coulombwall,

D-85748 Garching, Germany; telephone: þ49(0)89-289-13700; fax: þ49(0)89-289-13718;

e-mail: [email protected] Institute for Biomedical Engineering IBMT, St. Ingbert, Germany3Department of River Ecology, Helmholtz Centre of Environmental Research (UFZ),

Magdeburg, Germany

Received 23 February 2010; revision received 20 April 2010; accepted 21 April 2010

Published online 7 May 2010 in Wiley InterScience (www.interscience.wiley.com). DO
I 10.1002/bit.22784
ABSTRACT: Magnetic resonance microscopy (MRM) wassuccessfully applied for non-invasive online monitoring ofbiofilm development, sloughing, and forced detachment.Biofilm cultivation was performed in a tube reactor directlyplaced in the MRM scanner. Based on the differences inrelaxation time of free and bound protons, the distributedwater signal was allocated to the bulk and the biofilm phase.The velocity of the flowing water in the tube reactor wasmeasured in all three directions (x, y, and z) at spatialresolutions of 78mm. From the velocity data, maps of flowgradients (shear rates) were derived. The experimentsshowed that a more compact biofilm structure is sloughedoff in total with nearly no biomass left on the substratum.Continued biofilm cultivation resulted in filamentous bio-film structures, which did not show any sloughing. Experi-ments at higher Reynolds numbers were performed in orderto force biofilm detachment. Continuous measuring ofproton velocity and biomass was used to characterize thedifferent stages of biofilm development. The measurementsrevealed that biofilms are able to resist extremely high localshear stress being raised up to factor of 20 compared to themean local shear stress acting on the complete biofilmsurface. The maximum local shear stress of single biofilmstructures exposed to flow was found to be on average seventimes higher compared to the mean local shear stress of theentire biofilm surface. MRM was able to visualize andquantify the development of biofilms and interaction ofbiofilms with the surrounding fluid at the meso-scale. It issuggested that detachment and sloughing depends on bothinternal and external structural parameters.

Biotechnol. Bioeng. 2010;107: 172–181.

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KEYWORDS: biofilm; development; detachment; magneticresonance microscopy; sloughing; structure

Correspondence to: M. Wagner

Contract grant sponsor: German Research Foundation

Contract grant number: HO 1910/5-2

172 Biotechnology and Bioengineering, Vol. 107, No. 1, September 1, 2010

Introduction

Biofilms are the major form of microbial life in theenvironment and techno sphere (Bryers, 2000; Ghannoumand O’Toole, 2004; Wuertz et al., 2008). Nevertheless, thereis still a lack of knowledge about the way biofilms grow, alter,and detach. Magnetic resonance microscopy (MRM) is aunique, non-invasive, high resolution nuclear magneticresonance (NMR) imaging technique. MRM is able toacquire images of living, hydrated biological samples at themeso-scale and provides spatial information about a varietyof parameters. For example, the distribution of 1H-nuclei(protons), their displacement (diffusion, flow) or theirrelaxation times, which depend on molecular mobility and/or concentrations of paramagnetic ions (Callaghan, 1991).As biofilms may consist of over 95% water, images ofthe proton distribution alone show only very little or nocontrast at all. However, since the mobility of theintracellular water is restricted by the relatively impermeablecell walls, the self-diffusion of intracellular water moleculesis reduced significantly compared to molecules in the bulkfluid. Using diffusion-weighted NMR, Potter et al. (1996)were able to suppress the signal from the extracellularwater and measured profiles of the bacteria distribution ina water-saturated sand column. This restricted molecularmobility, along with the accumulation of paramagnetic ions(e.g., Cu2þ, Fe3þ, Mn2þ, and Gd3þ) in the biofilm matrix,leads to a reduction of the so-called longitudinal (T1) andtransverse (T2) relaxation times in the biofilm comparedto the bulk water. Hoskins et al. (1999) were the firstto employ the relaxation time contrast method to selectivelyimage and identify biofilms using magnetic resonanceimaging (MRI). By the controlled addition of specificparamagnetic relaxation agents it is possible to enhance thecontrast between biofilm and bulk fluid (Seymour et al.,2004a) or to observe the temporal evolution of labeled tracer

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molecules (Graf von der Schulenburg et al., 2008a; Lens andvan As, 2003; Nestle and Kimmich, 1996; Nott et al., 2001;van As and Lens, 2001).

Fluid flow along a biofilm surface and the resultingimpact of the boundary layer on the axial flow profile wasfirst studied by Lewandowski et al. (1993). More recently,the structure/flow relationship of various biofilm systemshas been investigated using MRM (Gjersing et al., 2005;Manz, 2004; Manz et al., 2003, 2005; Metzger et al., 2006;Nott et al., 2005; Seymour et al., 2004a,b). For example, thehydrodynamic forces acting on the biofilm surface can bequantified by measuring the bulk fluid velocity and thebiofilm structure at the same time (Manz, 2004). Thismethod can be used to observe and quantify the process offorced detachment as the bulk flow rate is increased (Manzet al., 2005). Biofilm growth in a capillary bioreactor and theresulting temporal development of the flow field wasstudied by Gjersing et al. (2005). Their results proved thesignificance of the interaction between biomass and flow onadvective transport processes. However, they focused onsingle species biofilm of medical importance.

In this study, we are presenting onlineMRM results of multispecies heterotrophic biofilm development. Measurementswere performed online with respect to meso-scale character-ization of biomass accumulation, detachment, and interactionwith surrounding fluid at maximal resolvable flow velocity. Toour understanding the meso-scale is in the range of severalmillimeters, filling the gap between the micro-scale that isinvestigated using microscopic methods (micrometer range)and the macro-scale that is observable by eye (centimeterrange). This definition is in adaption to Morgenroth andMilferstedt (2009).

The influence of growth conditions (carbon source,organic loading rate, culture duration, and culture shearstress) on the cohesive strength and detachment behavior ofbiofilms was investigated by the group of Etienne Paul(Coufort et al., 2007; Derlon et al., 2008; Ochoa et al., 2007).Their results revealed that biofilms cultivated under lowshear stress of 0.01 Pa are characterized by a layeredstructure and a basal layer of high cohesive strengthwithstanding shear stress up to 13 Pa (Coufort et al., 2007;Derlon et al., 2008). Additionally, Ochoa et al. (2007)showed the dependency of the angle the shear stress acts onthe biofilm leading whether to erosion or sloughing.Further, detachment studies revealed the significance oflocal fluctuations of the fluid velocity/shear stress todetachment phenomena (Manz et al., 2003; Stoodleyet al., 1999). With respect to the accuracy of detachmentsimulations, local shear stress fluctuations need to bequantified (Picioreanu et al., 2001) as the global shear stresscalculated based on the mean fluid velocity does notnecessarily be affected by local variations (Lelievre et al.,2002). Hence, there is a need to apply measurementtechniques like MRM to investigate local flow/shearfluctuations related to the biofilm structure.

Data demonstrates that biofilm development triggers theflow field heterogeneity and that sloughing can be linked to

Wagner et al.: Asse

increased shear forces at the biofilm surface. Additionally,information about the base biofilm was generated by Forceddetachment (FD) experiments visualized by MRM.

Furthermore, different stages of biofilm development weredocumented by confocal laser scanning microscopy (CLSM)in addition to the T2 measurements. Compared to MRM,CLSM allows visualization and differentiation of intra- andextracellular biofilm structures (Lawrence et al., 1998).

Materials and Methods

Biofilm Cultivation

The test tubes were made of transparent acrylic glass(PMMA) with an inner diameter of 10mm and outerdiameter of 12mm. The inner surfaces of the tubes wereroughened by grinding (paper No. 320) to promotemicrobial adhesion. The tubes were inoculated with50mL of activated sludge supernatant from the wastewatertreatment plant in Garching (Germany). They wereincubated for 5 days outside the NMR until a base biofilmof approximately 40mm was achieved (detected withCLSM). One test tube of 20 cm length was placed centrallyin the RF coil of the NMR system. Outside the NMR systema number of additional segments (separated tubes of 5 cm oflength and connected by short pieces of silicon tubing) wereintegrated in the cultivation setup for subsequent CLSMinvestigation. These CLSM segments were mountedvertically to ensure identical growth conditions comparableto the MRM test tube (see Fig. 1 for setup). The biofilmstructure already present at the beginning of the MRMexperiment was defined as day 0.

All solutions used were prepared with local tapwater (St. Ingbert, Germany). Growth medium:140mg L�1 CaCl2, 730mg L�1 MgSO4�7H2O, 300mg L�1

NaNO3 including the substrate glucose (120mg L�1,surface loading rate¼ 3.1 gm�2 day�1). Trace elements:300mg L�1 H3BO3, 130mg L�1 CoSO4�7H2O, 8mg L�1

CuCl, 20mg L�1MnSO4�1H2O, 26mg L�1 Na2MoO4�2H2O,10mg L�1 NiCl2�6H2O, 56mg L�1 ZnSO4� 7H2O. Phosphatebuffer K2HPO4/NaH2PO4 mixed to pH 6.5 including FeSO4

(5mg L�1 FeSO4�7H2O). The solutions were mixed togetherin a glass vessel and re-circulated by using a peristalticpump. The cultivation medium was aerated continuously.The mixing vessel was cleaned every day during T2

measurements to swap out extend biomass on the wall,input tubes and aeration device. Extended submersedmicrobial growth was suppressed by adjusting a mediumexchange rate of 5 day�1. The total duration of the biofilmcultivation was 21 days.

Forced Biofilm Detachment

Detachment experiments were carried out after day 17 byapplying higher Reynolds number compared to theReynolds number during growth (360). The higherReynolds number for forced detachment was applied for

ssment of Biofilm Development, Sloughing and Forced Detachment 173

Biotechnology and Bioengineering

Figure 1. Experimental setup of biofilm cultivation and online MRM measure-

ment. Cultivation was performed in recirculation mode.

Figure 2. Color allocation: (a) flow field in x-direction, white lines indicate the MRM scanning region (21 sections¼ 256 imaginary slices), (b) flow field in y-direction, (c) flow

field in z-direction and (d) biomass distribution (T2 map) of one cross-section, respectively. Data sets of (a) and (b) have been resliced using ImageJ to image the flow of the

complete MRM scanning region (10 mm� 20mm). Applied color fields are valid for Figure 3 with exception of Figure 3c, day 10 before sloughing where the intensity values are

between 0 and 185mm s�1.

Figure 3. Monitoring of biofilm cultivation using MRM. a and b: Present data

from the center of the tube. a: Shows the velocity map in x-direction, (b) shows the

velocity map in y-direction. c and d: Present data of slice 35 in z-direction. c: Shows the

velocity map, (d) shows the T2 map. Further detailed explanations are given in Figure 2.


Table I. Hydrodynamic conditions present during biofilm cultivation and forced detachment.

Re Shear stress, twall [mPa] Redetachment/Recultivation Fluid velocity, w [mms�1] Volumetric flow rate [mL s�1]

Cultivation 360 28.8 36 2.9

Day 17, FD Re¼ 1,100 1,100 83.3 3 107 8.7

Day 19, FD Re¼ 1,400 1,400 115 4 142 11.6

Day 21, FD Re¼ 3,600 3,600 662 10 360 29.0

Reynolds numbers were calculated using the mean fluid velocity w.

2min. Afterwards the flow was reduced back to thecultivation conditions allowing comparable MRMmeasurements. For further details see Table I. Notice:Reynolds numbers were calculated using the mean fluidvelocity w.

MRM Measurements

The MRM experiments were performed using a BrukerAvance NMR spectrometer (Bruker, Rheinstetten, Germany)operating at a 1H resonance frequency of 400MHz withstandardMicro2.5microimaging equipment and amaximumgradient strength of 0.95 Tm�1. Tube segments withcultivated biofilms were connected to the flow loop andplaced inside the NMR magnet as shown in Figure 1. Thefluid flow velocity was adjusted to a volumetric flow rate of250 L day�1 corresponding to a Reynolds number of Re¼ 360in the tube.

The T2 maps were recorded once a day with flowturned off using a multi-echo imaging pulse sequencewith 16 echoes, an echo time Te¼ 10ms per echo, and arepetition time of 250ms (Edzes et al., 1998). Each mapconsisted of 128� 128� 256 voxels over a transverse field ofview of 10� 10mm2 and an axial section of 20mm length,resulting in an image resolution of 78mm transverse andaxially. The overall experiment time for a 3D T2 map was2.25 h.

It should be noted that this sequence does not measure thereal T2 value, but rather a T

02, which is additionally weighted

by the local self-diffusion coefficient (Manz, 2004). Due toobtain good contrast between biofilm and bulk liquid, onlyT 02 was acquired as the difference to real T2 is not important

in this case.Velocity maps were recorded with flow encoding in all

three directions in order to measure the full flow velocityvector. Details for orientation of data sets in x, y, and zdirections as well as further explanations are given inFigure 2. The transverse image resolution was the same as forthe T2 maps. For each map, 21 slices were recorded with athickness of 1mm and a slice separation of 1mm (seeFig. 2a). Subsequent data interpolation along the axialdirection yielded a 3D image of the velocity vector with anisotropic spatial resolution of 78mm. The overall measure-ment time for a set of three velocity maps was 1.75 h. Sets ofvelocity maps were acquired continuously except for thetime during T2 mapping.

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Confocal Laser Scanning Microscopy

CLSM was carried out using a LSM510 META controlled byAIM software version 3.2 (Carl Zeiss MicroImaging GmbH,Jena, Germany). The diameter of each pinhole was adjustedin the software depending on the wavelength range of thefluorescence emission to ensure an optical slice thickness of0.78mm for each detection channel acquired (North, 2006).The biofilm on the tube segments was analyzed using a waterimmersion lens (40� magnification, NA¼ 0.8). For thispurpose a small piece of approximately 2.5� 0.5 cm2 wascut out of a tube segment. This part was then fixed withsilicon glue in a small Petri dish. Standard staining protocolsdescribed by Neu and Lawrence (1999) and Staudt et al.(2003) were applied to analyze the distribution of lectin-specific EPS glycoconjugates and nucleic acids within thebiofilm. Glycoconjugates were stained using the lectinisolated from Aleuria aurantia (LINARIS BiologischeProdukte GmbH, Wertheim-Bettingen, Germany). Thelectin was labeled with the fluorochrome AlexaFluor1

488 (Molecular Probes, Eugene, Oregon). Nucleic acidstaining was done by using SYTO 601 (Molecular Probes).

Digital Image Analysis and Visualization

CLSM image stacks were analyzed with the softwareJImageAnalyzer version 1.1 (Institute of Water QualityControl, Technische Universitat Munchen, Garching,Germany). The software is based on the core features ofImageJ (http://rsb.info.nih.gov/ij/index.html). MRM datasets were handled and analyzed using ImageJ (version1.37v).

Visualization and three-dimensional reconstruction ofdata sets was performed using Amira version 4.2 and Avizoversion 5.1 (VSG Visualization Science Group, Burlington,MA).

Results and Discussion

Evolution of Biofilm Structure and Velocity Maps DuringGrowth and Forced Detachment

A set of MRM images showing biofilm development ispresented in Figure 3. Subimage (c) and (d) represent slice35 of the whole data set. Red represents high and blue/blacklow values of the parameter presented. Subimages (a) and



(b) show the complete resliced flow profile in x- and y-direction in the center of the tube, respectively.

To remind: The beginning of the MRM experiment wasdefined as day 0 including the existing biofilm structure.Continued incubation showed biofilm growth visually byincreased opacity of the CLSM segments. Ongoing cultiva-tion was characterized by filamentous structures/streamersand partly detached biomass became visible. During everystage of the experiment disturbances in the velocity mapsindicated biomass development. After 6 days of cultivationthe biofilm covered nearly the complete inner wall (seeFig. 3d, days 0–5).

Figure 3d of day 2 reveals that biofilm growth wastriggered by an attached aggregate located in þx direction.As a result, the laminar flow profiles were altered as mappedby Figure 3c. Flow of the bulk phase was pushed in directionof �x leading to raised flow velocities in the center of thetube with approximately 85mm s�1 (white pixels). Hence,the biofilm grown in direction of �x was thinner comparedto the biofilm developing on the opposite wall. Additionalbiofilm growth was continually monitored (see, e.g.,Fig. 3d, day 5) with a maximal biomass concentration inþx direction. Rapid biofilm growth was observed for thefollowing 4 days resulting in a heterogeneous structureaccessible to shear stress and shear forces applied by themoving fluid. At day 9 of the experiment (Fig. 3c and d) thebiofilm partly detached from the wall in direction of�y anddrifted into the moving fluid creating vortexes. Hence, theproton velocity in x-direction varied between �6mm s�1 inthe center of the tube and 6mm s�1 at the wall, whereas thevelocity in x-direction at day 0 was ranged between�1.5 and0mm s�1. The accumulation of biomass is in goodagreement with the results reported by Gjersing et al.(2005). They investigated by means of MRM a medicalrelevant bacterial biofilm of Staphylococcus epidermidis(strain 35984) grown in a square capillary bioreactor.Furthermore, their results revealed a significant influence ofaccumulated biomass on the velocity maps in xy-direction,which is in contrast to the results presented in this study. Incase of our study, secondary flows reached maximal 5% ofthe axial flow velocity. A reason therefore might be the ratioof biofilm thickness and tube diameter that in our case wasseveral times higher since the diameter of the squarecapillary bioreactor used by Gjersing et al. (2005) was 1mm.Hornemann et al. (2009) quantified secondary flows inx- and y-direction up to 20% of the bulk fluid velocityin square capillary bioreactors of 0.9mm diameter.Furthermore, their results revealed that the quantity ofsecondary flows is inversely proportional to the diameter ofthe investigated system. Despite of the different results,MRM is suitable for subsequent detection of protonvelocities and biomass (Graf von der Schulenburg et al.,2008b; Vrouwenvelder et al., 2009).

The T2 relaxation maps revealed the structural char-acteristics of the biofilm beginning to slough. Figure 3c ofday 9 visualizes a biofilm of a certain stability withstandingthe shear during cultivation (Re¼ 360) indicated by black


pixels (biofilm-bound water) within the velocity map in z-direction. This structure was stable during T2 relaxationmeasurement although flow was stopped (Dt¼ 2 h).Furthermore, the velocity maps in z-direction of day 10before sloughing (see Fig. 3c) revealed the structure of thebiofilm before it detached completely. This event occurredbefore the T2 map of day 10 was recorded. The sequence inFigure 3 (days 9 and 10) shows that the process of sloughingis not occurring within a short time; it rather may take hoursor even days. Three-dimensional projections in Figure 4 (seedays 9 and 10) of the MRM data of these days present indetail the process of sloughing.

After the complete detachment of the biofilm withinthe MRM scanning region no base biofilm could bedetected (see Fig. 3, day 10 after sloughing). Figure 3c and dof day 10 after sloughing neither revealed disturbanceswithin the velocity maps nor signals of biomass in theT2 relaxation maps. Thus, the corresponding velocitymap (see Fig. 3c, day 10 after sloughing) matched thetheoretical parabolic profile for laminar flow reportedby Sederman et al. (2004). This observation indicated thatsloughing is not only restricted to the MRM scanningregion. After the sloughing event biofilm growth was sloweddown compared to the period monitored between days 0and 10 (see Fig. 3, days 11, 14, and 18). The biofilmvisualized consisted of various single aggregates that did notcover the entire wall of the tube during 8 days of cultivation.Furthermore, the onset of transverse flow could not beaddressed completely to biofilm structures measured in theMRM scanning region. It was supposed that biofilmstructures, which remained in regions below the MRMscanning region influenced the flow fields of all directions(e.g., Fig. 3, day 21).

Forced detachment experiments (FD) were performed inorder to estimate biofilm strength. The stepwise increasedflow velocities applied for 2min resulted finally in Reynoldsnumbers up to 3,600 (see Table I). Detailed three-dimensional views presenting the interaction of flow (streamribbons) and biofilm structures (green isosurfaces) arepresented in Figure 4 (see days 14–19). Additionally, the redisosurfaces indicate the regions of highest shear (see Fig. 4)leading to stepwise loss of biomass. Alterations within thefluid flow originated from biomass below the NMRscanning are also visualized. First effects on the biofilmwere measured after applying a Reynolds number of 1,100(see Fig. 3, day 17, FD Re¼ 1,100). The velocity maps of flowin x-, y-, and z-direction revealed a completely different flowfield if compared to the situation before FD. Especially, theshape of the proton velocity in z-direction was dominated byindividual biomass spots (compare Fig. 3c, days 14 and 17,FD Re¼ 1,100). These spots originate from biofilm partssticking together and being attached to the wall. Connectedbiofilm parts were visualized in red, violet, and blue pixels inthe center of the tube indicating a certain cohesive strengthof the biofilm that might be comparable to those of thebiofilm cultivated within the first 9 days (see Fig. 3d, days17–19). The decrease of the maximal proton velocity in

Figure 4. 3D reconstruction of MRM data sets projected as isosurfaces representing the combination of measured flow velocity in z-direction and biomass (T2 maps). Color

allocation: blue¼PMMA tube, green¼ biofilm, red¼ high shear forces. Take notice of biofilm development (days 0–9), situation after sloughing event (day 11), subsequent biofilm

regrowth and forced detachment (days 14–21). Flow of the moving fluid is indicated using so-called ‘‘stream ribbons.’’ Direction of flow is towards the origin of these stream ribbons

(ring). The subimages illustrate the biomass distribution (T2 maps) acquired each day of the experiment without fluid flow through the tube. Shear forces resulting from the

interaction of the moving fluid (stream ribbons) and the biofilm surface (green isosurfaces) are visualized as red isosurfaces. Furthermore, the stepwise process of sloughing is

represented since remained biomass below the NMR scanning region altered the velocity map in z-direction at day 10 (before and after sloughing) until these biomass was finally

detached at day 11 (unaltered stream ribbons).

z-direction between days 14 and 18 indicated biomass belowthe MRM scanning region, which was partly detachedbut not visualized. After applying a Reynolds number of3,600 no biomass was detectable by T2 relaxation measure-ment (see Fig. 3d, day 21), although the velocity maps showdisturbances, which originate from biomass below theMRMscanning region. Recently, the effect of biomass on thevelocity profiles within membrane systems was studied byVrouwenvelder et al. (2009) using nuclear magnetic imaging(MRI). MRI results revealed that biofilm growth waspromoted in regions of reduced shear. Based on their resultsthe necessity of improved understanding of biofilmdevelopment is clearly pointed out since the performanceof technical systems can depend on that. Furthermore,MRI/MRM data can be used to model those processes interms of optimization and prevention (Picioreanu et al.,2009). Additionally, our results revealed the long-distanceeffect of attached and partly detached biomass on thevelocity profiles of the surrounding fluid. Thus, localvelocities can be originated from local and global biofilmstructures. This could be of relevance in future biofoulingmodels.

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Structural Parameters and Shear

T2 relaxation maps were used to determine the biofilmthickness and the surface enlargement factor a (see Eq. 3) asdescribed by Manz et al. (2003). Values of a greater than1 indicate a rough, heterogeneous biofilm surface whereas ahomogeneous biofilm typically has a surface enlargementfactor a below 1. The development of the biofilm thicknessand the surface enlargement is presented in Figure 5. Initialbiofilm growth was accelerated within the first 8 days andreached a maximal biofilm thickness of 300mm. Two daysbefore the sloughing event occurred, biofilm growth sloweddown. It has been proposed, that biofilm sloughingand detachment is typically coupled with a certain biofilmthickness (Picioreanu et al., 2001). In our case, it took 2 daysuntil the biofilm was completely sloughed. T2 relaxationmaps revealed a spatially heterogeneous biofilm develop-ment and thus, as expected, the surface enlargement factor aincreased. The maximal surface enlargement was calculatedwhen the biofilm started to detach from the wall of thetube at day 9. At this stage of cultivation the surface areaof the partly detached biofilm was 2.5 times larger than



Figure 5. Surface enlargement and biofilm thickness calculated fromMRM data

sets. Dotted line indicates linear part of biomass accumulation. The surface enlarge-

ment factor a indicates the surface roughness of the biofilm.

the theoretical value (a¼Abiofilm/Atheoretical¼ 2.5). Aftersloughing the biofilm thickness was reduced to approxi-mately 30mm and the surface enlargement factor a

decreased to the initial value of 1.1. Both parameters keptnearly constant for additional 7 days and increased at day 17of the cultivation due to forced detachment. Established(multi-species) biofilm models calculate sloughing as ashort-term event and suppose that continual biofilm growthis enhanced based on the improved supply with nutrientsand substrates at the opened biofilm-bulk interface (Wannerand Gujer, 1986). Indeed,MRMmeasurements revealed thatsloughing can be a process of several hours duration and thatgrowth does not need to be enhanced after sloughing. It issupposed that these results could be beneficial due to theperformance of sophisticated (three-dimensional) biofilmmodels.

As already shown in Figure 4 (days 17, 19, and 21) theexperiments with forced detachment had different effects onthe observed biofilm. The first experiment with Re¼ 1,100led to an increase in biofilm thickness, which can beexplained by partly detached biofilm areas reaching into thetube segment. The second experiment with Re¼ 1,400 partlyremoves the biofilm but still spots of biomass can bedetected in the middle of the tube (see Fig. 4). During thesetwo experiments the biofilm thickness and the surfaceenlargement factor a kept nearly constant. The finaldetachment experiment at a Reynolds number of 3,600(day 21) reduced a to the initial value of 1.1 again and abiofilm thickness of about 50mm. A fluctuating surfaceroughness was already described by Lewandowski et al.(2007). The authors used the areal porosity as parameter,which is the ratio of the area covered by voids (empty space)to the total area of a CLSM image. Both methods CLSM and


MRM provide different scales of observation. Nevertheless,the outcome with respect to heterogeneity is comparable. Anadvantage of MRM is the possibility to combine the biofilmstructure with flow patterns shaping it.

Therefore, local shear tlocal stress and shear force Fshearwere calculated from the velocity maps:

tlocal ¼ Grad z � h ¼ dv

dz� h ½mPa� (1)

where h is the dynamic viscosity of water. For comparisonthe wall shear stress for the empty tube was estimatedaccording to Characklis et al. (1989) as:

twall ¼ f �rH2O � w2

2

f ¼ 16Re

Re < 2100

f ¼ 0:0791Re0:25

Re > 2100

(½mPa� (2)

in laminar and turbulent flow conditions, respectively,where w is the mean fluid flow velocity through the tube. Toderive the mean local shear force Fshear acting on the biofilmsurface, the surface enlargement factor a was used tocalculate the real biofilm surface area ABT:

ABT ¼ a � dT � p � lT with a ¼ usdT � p

½m2� (3)

where us is the real biofilm perimeter describing theboundary between moving fluid and biofilm, dT is the tubeinner diameter (10mm) and lT is the length of the MRMscanning region (20mm). Since the local shear stress is aforce acting on a area, the mean local shear force Fshear wascalculated as:

Fshear ¼ tlocal � ABT ½mN� (4)

The calculated values of tlocal and Fshear are given inTable II. Values calculated by the use of a represent meanvalues valid for the biofilm within the complete tube.Accordingly the maximal shear force was calculated for thevoxel of highest shear stress (ABT¼ 78mm� 78mm).

Table II clearly points out that biofilm growth itselfintroduced high shear stress and hence high shear forcesin the system of a constant Reynolds number of 360.Consequently, detachment or sloughing occurred when theshear stress reached the critical level (i.e., day 10). The meanlocal shear stress (average of 256 slices) did not changelargely during the whole cultivation. One exception wasobserved for the measurements of day 10 before sloughingwhere the mean local shear stress was raised about 10mPawithin 24 h to reach nearly 40mPa. Manz et al. (2003)reported similar values of local shear stress that correspondsto detachment of multi species biofilm. However, Ochoaet al. (2007) found that the basic biofilm layer of biofilmsgrown at low shear stress (0.01 Pa) can resist a shear stress upto 13 Pa before complete detachment. Consequently, theresults of this study revealed that even low local fluctuationsof shear stress could significantly affect the biofilm structure.

Table II. Shear conditions calculated from velocity maps in z-direction.

Shear stress, tlocal Shear force, Fshear

Meana [mPa] Maxb [mPa] SD [mPa] Meana [mN] Maxb [mN]

Day 0 19.2 37.1 5.93 0.013 0.226

Day 2 21.0 80.2 8.17 0.016 0.488

Day 5 24.6 188 18.6 0.024 1.15

Day 9 28.9 217 26.2 0.046 1.32

Day 10 before sloughing 39.9 709 49.5 — 4.32

Day 11 20.7 115 10.1 0.014 0.698

Day 14 21.3 81.1 14.4 0.015 0.493

Day 17 after FD Re¼ 1,100 16.4 133 13.2 0.015 0.809

Day 18 11.4 73.4 9.46 0.010 0.446

Day 19 after FD Re¼ 1,400 17.5 162 17.6 0.016 0.983

Day 21 after FD Re¼ 3,600 19.6 86.8 14.1 0.014 0.528

Calculations are based on the tube cross-section (diameter¼ 128 pixels). The mean values are averaged values of the complete data set of 256 slices.aThe mean shear stress or force acting on the complete biofilm.bMaximal shear stress or force of a single voxel with maximal Grad z.

The maximal local shear stress was several times highercompared to the mean local shear stress during the completemonitoring of growth and detachment. The development ofshear is in good agreement with the T2 measurements andcalculated surface enlargement factor a that indicated spatialheterogeneous growth for the first growth period and morehomogeneous growth for the second period, respectively. Asthe biofilm thickness developed mainly linearly until day 5and then between days 5 and 8 with a lower rate (see Fig. 5),the maximal shear stress increased significantly betweendays 9 and 10 (see Table II). It can be concluded that evensmall changes of the mean biofilm thickness can trigger hugechanges in surface structure causing detachment. Thus,sloughing might not only be a function of the biofilmthickness as reported by Wanner and Gujer (1986) andPicioreanu et al. (2001). Speitel and DiGiano (1987)introduced a dependency on both biofilm thickness andgrowth rate of the microorganisms involved. Biofilm growthsurely will influence sloughing but the type of growth shouldbe considered as more relevant. As can be seen from theresults there is a strong relation between surface structureand sloughing.

Figure 5 shows that the sloughing event slowed down thegrowth of the biofilm, which resulted in a stable phasebetween days 11 and 14 without significant changes (seeTable II). The first attempt with forced detachment onday 17 led to an increase of the maximum shear stress from81 to 133mPa. This happened again on day 19. From theorya forced detachment should produce a more homogenoussurface structure, as exposed structures should be detached(Bol et al., 2009). Bol et al. (2009) derived the biofilmstructure from signals of lectin-specific EPS glycoconjugatesand nucleic acids, respectively. Thus, their biofilm detach-ment model is based onmicroscopic examinations by CLSMincluding all limitations of this technique (e.g., limitedspecificity of applied stains for biofilm constituents, laserpenetration depth and field of view). Hence, the meso-scale

Wagner et al.: Asse

process of sloughing and detachment beginning with partlydetached biofilm structures was not considered in thedescribed model. Furthermore, structural properties at themicro-scale estimated by CLSM seem to be of minorrelevance for processes at the macro-scale.

However, in our case the meso-scale surface structure wasmore or less disturbed by the increased shear forces duringthe forced detachment. Maybe the time period of 2min forthe forced detachment was too small to detach all elements,which were exposed to higher shear forces. The samehappened on day 19 where the forced detachment againincreased the maximum shear stress. Finally, after applying avery high Re of 3,600 on day 21 most parts of the biofilmwere finally detached and the maximum shear stress wasreduced to a value, which was similar to the one found onday 2 of the growth experiment.

Stoodley et al. (2002) proposed a ratio of 2.3 for wall shearstress/cohesive strength leading to detachment. In ourbiofilm the ratio between maximum and mean shear stresscalculated from the MRM experiments was on average 7� 4during the entire experiment. From gauging results it hasbeen calculated that the cohesive strength of biofilms ismuch higher than the wall shear stress applied duringgrowth. Ratios of 200–1,100 have been measured (Mohleet al., 2007). Such findings and the results presented in thisstudy show clearly that there is no unified value, which canbe used for a detachment model. The biofilm surfacestructure is shaped by hydrodynamic forces and microbialgrowth. As the hydrodynamic forces can be calculated forknown structures it is still not easy to predict the shape ofmicrobial structures to be developed under defined growthconditions. Nevertheless, MRM is a unique method, whichcan be used to measure the interaction of shear forces andmeso-scale biofilm structures. However, it does not revealthe microscopic organization of the biofilm on a cellularlevel. This is the reason why CLSMwas also employed in thisstudy.



Confocal Laser Microscopic Characterization

CLSM was applied to reveal the microscopic structure of thecultivated biofilm. Those results linked the structuralbehavior of the biofilm during growth, sloughing, anddetachment to the constitution of the biofilm. Results arepresented as three-dimensional isosurface reconstructionsin Figure 6. The initial biofilm from pre-cultivation (day 0)was characterized by both single bacteria attached to thetube and small aggregates consisting of EPS glycoconjugatesand bacteria. Further cultivation formed a highly connectedframework of partly filamentous structures coveringproduced EPS glycoconjugates (see Fig. 6, day 7). Thistime period delivered the most compact biofilm structureof the entire experiment, which on day 9 was sloughedoff as complete biofilm from the tube surface (see Fig. 4,day 9).

After the sloughing event the surface of the tube wasalmost completely free of microorganisms. In order todemonstrate the complete biomass loss the reflection of thesubstratum was recorded additionally (see Fig. 6, day 11). Itcan be concluded that the adhesion to the substratum wasnot that strong in this case. Typically, a base biofilm in therange of several 10mm is found after forced detachmentevents (Manz et al., 2005). It seems that somehow the waterflow found its way between biofilm and wall and step by stepripped off the biofilm (see Fig. 4, day 9).

Compared to that, a CLSM image is shown for the biofilmafter the final forced detachment experiment (see Fig. 6, day21). It can be seen that the hydrodynamic shear forcesmainly act on the surface, which seemed to be governed by

Figure 6. 3D imaging of CLSM data presented as isosurface projection.

Color allocation: red¼ nucleic acids, green¼ lectin-specific EPS glycoconjugates,

white¼ reflection. Box dimension in xy¼ 230mm� 230mm. For improved imaging the

data set was down-sampled (2� 2� 2).


filamentous structures. Compared to day 11 the substratumis still covered with EPS glycoconjugates and filamentousbacteria, which have resisted the forced detachment. Wesuggest a permanent re-inoculation with microorganismsoriginated in the local tap water similar to the study of Garnyet al. (2009). After the sloughing event (day 11) secondarycolonizers were able to settle on the substratum leading to acompact biofilm of enhanced cohesive and adhesive strengthwith improved resistance against applied shear.

Conclusion

The experiment proved the ability of MRM to studythe biofilm development, sloughing, and forced detach-ment non-invasively, online and in situ. Furthermore,the recorded and calculated parameters were three-dimensionally visualized and thereby revealed the interac-tion of flow, shear stress, and biofilm structure. Biomass ormore precisely the biofilm structure at meso-scale wasdirectly linked to disturbances in the velocity profiles. Thelocal exposure to shear seemed to be the main reason forbiofilm sloughing and forced detachment. By means ofMRM the forced detachment applied in form of a shortpulse and subsequent biofilm response revealed that localbiofilm structures exposed to high flow velocity and shearare responsible for detachment events. It was possible toquantify and visualize three-dimensionally that detachmentis a process triggered by local hydrodynamic conditions inrelation to biofilm structure. It is suggested that there is astructural link between exposed and base biofilm structures.Obviously, internal micro structures may directly affect themacroscopic biofilm response. MRM is the method ofchoice in order to investigate these interactions online athigh resolution at the meso-scale.

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