AnalyticalMethods

PAPER

Microfluidic flow

aDepartement de Chimie, Faculte des scien

City, QC, Canada. E-mail: jesse.greener@chbCHU de Quebec, Centre de recherche, Unive

QC, Canada

† Electronic supplementary informa10.1039/c8ay01513a

Cite this:Anal.Methods, 2018, 10, 4579

Received 8th July 2018Accepted 31st August 2018

DOI: 10.1039/c8ay01513a

rsc.li/methods

This journal is © The Royal Society of C

confinement to avoidchemotaxis-based upstream growth in a biofilmflow cell reactor†

Farnaz Asayesh,a Mir Pouyan Zarabadi,a Nahid Babaei Aznaveha

and Jesse Greener *ab

Introduction of a bacterial inoculant into a chemostat bioreactor can lead to unwanted contamination of

upstream elements via chemotaxis. This can result in biofilm growth in connective tubing, valves and

even the medium source reservoir itself, thus complicating the conditions of the applied liquid phase and

impeding proper chemostat functionality. Applied to biofilm forming Pseudomonas fluorescens bacteria,

we tested two different microfluidic flow confinement methods designed to impede upstream

contamination. The first isolated biofilm growth from the relatively stagnant zones within the

microchannel corners, and in the second a flow enhancement element was introduced to increase flow

velocities and shear forces. Both methods showed improvement over a control design, but flow

enhancement showed the best performance by delaying or preventing bacterial contamination of

upstream elements, ensuring stability of the applied liquid media conditions for the entire duration of the

experiments. This simple passive element has the potential for wide use as it is easy to implement and

can be optimised for different experimental requirements.

Introduction

Bacterial biolms are currently under increasing scienticscrutiny due to their roles in biofouling of industrial compo-nents and as robust virulence vectors with enhanced resistanceto antibiotics.1–4 At the same time, interest is growing in bio-lms as functional materials that can operate at ambientconditions5 for applications including wastewater treatment,6

bioltration,7 environmental bioremediation8 and bio-catalyticchemical or biological synthesis.9 One advantage of biolmsover planktonic bacteria is that they are surface-adhered, soreactors can be operated in continuous-owmode. Continuous-ow bioreactors are oen preferred over batch reactors becauseconstant ux of fresh media maintains consistent reactorconditions. In addition, the reaction conditions can be easilycontrolled by changing the ow rates and mixing concentra-tions. This provides an easy approach to study growth andkinetics of bio-catalytic conversion. Microuidic bioreactorshave high surface-area-to-volume ratios that enable efficientcontact between the wall-adhered biolm and the liquid phase.Even at high ow rates, the small liquid volumes required

ces et de genie, Universite Laval, Quebec

m.ulaval.ca

rsite Laval, 10 Rue de l'Espinay, Quebec,

tion (ESI) available. See DOI:

hemistry 2018

enable long-term studies without the need to frequently rellnutrient sources. A number of preparation steps can be con-ducted on-chip such as splitting and recombination of liquidstreams, separations, formation of stable concentration gradi-ents and manipulations of ow rates. Thus microuidic owcells offer excellent chemical and hydrodynamic reactionconditions including the possibility for precisely timed multi-step reactions. Moreover, the exibility in typical micro-fabrication techniques offers a straightforward method formodication of ow cell geometry.

Bacterial cells are introduced into the bioreactor via aninoculant solution, which is usually manually injected viasyringe by a needle passed through an elastomeric device or thefeed-line tubing attached to the device. Otherwise, inoculantsolutions can be pumped on chip through an inlet froma dedicated upstream source. Aer the bacteria have seeded theow channel, a sterile nutrient solution can be injected into thedevice to continue the growth process. Typically, bioow cellreactors are used to study biolm growth kinetics, microbialactivity and bio-catalytic conversion kinetics in response todifferent applied concentrations, which should be preciselyknown. An oen unaddressed challenge in operation of biolmchemostat reactors is that the initially sterile upstream portionsof the bioreactor can become contaminated due to bacterialchemotaxis, with the tendency for motile bacteria to movetowards nutrient sources. For example, crawling sessile bacteriaor swimming planktonic bacteria ejected from a sessile biolmare guided by their chemosensory systems to activate their

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Analytical Methods Paper

agella to swim or their pili to crawl or even “walk” upright tofollow nutrient concentration gradients.10,11 Though nota problem for a growing class of studies on electroactive bio-lms which must can only grow on a localized electrodesurface,12 this process leads to contamination of connectivetubing and valves or even the medium source itself for themajority of biolm studies. Once established, the upstreambiolms can produce a constant ux of planktonic bacteria,increase resistance to ow, and change the chemical concen-trations and composition of the passing medium, which thenows downstream to the measurement area.13 The result isuncertainty in the reactor conditions. These problems aremagnied in microuidic channels because high surface-area-to-volume ratios amplify the contact between fouled surfacesand the liquid medium passing by them. Anti-fouling surfacescan temporarily impede contamination, but eventually, they canbe biologically reconditioned to support bacterial adhesion andgrowth.14 Active approaches to limiting upstream contamina-tion include local application of heat, light irradiation, elec-trochemical potential, or antimicrobial treatment.15 Apart fromcontributing to device complexity, these approaches can beenergy intensive, induce unintended chemical reactions in thenutrient medium, and cause thermal perturbations in thedownstream measurement areas, among other side effects. Inthis work, we proposed and tested two straight-forward uidicapproaches to avoid upstream contamination via the additionof ow connement elements and compared them to a controlchannel. The rst element isolated bacterial inoculant fromaccessing the microchannel corners and the second increasedthe local uid velocity v (m s�1), (eqn (1)), and wall shear force s(Pa) (eqn (2a) and (2b)).

Fig. 1 Microfluidic fabrication. (a) Three designs used in this study. Thsolutions (i). The second has an added inlet (I3) for double flow confinelement. All designs have width w ¼ 2000 mm, whereas the width of theoutlets (not to scale). (b) Mould with design described in (ii) with three ddesign was used in this study. (c) A PDMS/glass device from the mould d

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v ¼ Q/A (1)

where Q (m3 s�1) is the volumetric ow rate, and the channelcross-section area depends on the channel width (w) and height(h), A ¼ w$h (m2).

s ¼ 6mQ/h2w (2a)

s ¼ 6mv/h (2b)

where h is dened as the smaller of the two edges in a rectan-gular ow cell.

In this study, a Pseudomonas uorescens biolm was inocu-lated and grown in microuidic ow cells with and withouta ow connement element. The devices with ow connementwere able to retard or even eliminate upstream contaminationdepending on the ow conditions applied.

Materials and MethodsFlow cell design

Three different microuidic ow cell designs were used in thiswork, one control design and two different ow connementdesigns. The rst design, shown in Fig. 1a(i), was for controlmeasurements, consisting of a simple straight channel (width w¼ 2000 mm) with separated inoculation and nutrient inlets (I1and I2, respectively) and one outlet (O). As this was a controldesign, it included no special ow connement element. Thesecond design, shown Fig. 1a(ii), was the same as the rst butfeatured a third inlet (I3) for ow connement via “laminar owtemplating”. The third design, shown in Fig. 1a(iii), was basedon a published design16a that applied a “ow enhancement”

e first contains two separate inlets (I1, I2) for inoculation and nutrientement to stop corner growth. The third includes a flow confinementneck in (ii) is 250 mm. White circles indicate the positions of inlets andifferent neck lengths encased in PDMS in a Petri dish. The highlightedesign highlighted (red square) in (b). Channel height was 50 mm.

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Paper Analytical Methods

element in place of I3 in the second design. In this design, thereduction in channel width from w¼ 2000 mm to w¼ 250 mm inthe ow enhancement element caused the ow velocities toincrease by a factor of 8. Microuidic devices were fabricatedwith polydimethylsiloxane (PDMS) using casting moulds. Themoulds were made using a benchtop photolithography methodthat could be implemented outside of a cleanroom. First,a photomask design was constructed using computer-aideddesign soware (DraSightTM, Dassault Systemes, France).The mould was fabricated by adhering a laminate photoresist(SY300 lm, Fortex, UK) on a 75 mm � 50 mm � 1 mm glassslide (12550C, Fisher Scientic, Canada) using a thermalbenchtop dry lm laminator (FL-0304-01, Fortex, UK). Theadhered photoresist with a thickness of 50 mm was exposed toUV light with a wavelength of 360–365 nm for 24 secondsthrough a mask with a vacuum exposure unit (AY-315, Fortex,UK). The excess photoresist was removed using a developer andrinse solutions (SY300 Developer/Rinse, Fortex, UK). The heightof all features in the mould, which denes the height of allmicrochannels, was measured as 50 mm.

Microuidic devices weremade by casting PDMS and a cross-linker solution (Sylgard184, Dow Corning, Canada) at a 10 : 1ratio in the mould and curing overnight at 70 �C (Fig. 1b). Thecross-linked PDMS device was cut and peeled from the mould,and inlets/outlets were punched. The PDMS device was sealedwith a 75 mm � 50 mm � 200 mm glass coverslip (12550C,Fisher Scientic, Canada) via exposure to air plasma (PDC-001,Harrick Plasma, USA).17 To avoid transfer of molecularcontaminants to the glass slide (which is known to affect bio-lm growth dynamics), the glass slide was not treated in theplasma cleaner.18 An image of the nalised device of design 3 isshown in Fig. 1c with its channel portion lled with dye forvisualisation. We note that PDMS is known to be highly gaspermeable, thereby eliminating concerns that O2 depletion orCO2 accumulation could impact biolm respiration.19

Fluidic control

Liquids were delivered to the microuidic system via per-uoroalkoxy connective tubing (outer diameter 1.6 mm) (U-1148, IDEX, WA, USA) connected to all inlets. The upstreamside of the tubing had threaded connectors (P-200x, P-658,IDEX, WA, USA) that interfaced with 60 mL Luer-Lok syringes(BD Scientic, NJ, USA) driven by individual pumps (PHD 2000,Harvard Apparatus, Holliston, MA, USA). All solutions weremixed using ultrapure water with a resistivity of 18.1 MU cm.Teon tubing was rst lled with pure ethanol, subsequentlysonicated for 10 minutes, and exposed to a ow of 70 : 30ethanol : water for 2 hours at a ow rate of Q ¼ 2 mL h�1. Thetubing was connected to the device, and sterilisation of themicrochannels was continued by ushing the device through allinlets with a 70 : 30 ethanol : water solution for 30 min at1 mL h�1. Residual ethanol solution was washed out of thesystem with sterile water for an additional 2 hours at Q ¼1 mL h�1 applied to each inlet. A tight-tting metal elbowcapillaries with a 90� bend was used to connect the Teontubing to the inlets and outlets, thus avoiding the need for

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epoxy and keeping the optical path of the microscope free. All ofthe liquids applied to the device were vacuum degassed prior tothe experiment to minimise air bubble formation in themicrochannel due to the recent observation that temporarilyadhered bubbles during the biolm lag phase can affect growthkinetics, even if they are released before the biolm is visual-ised.16 Syringe pumps can sometimes slip when back pressurein the tubing or device builds up, as a result of high ow ratesapplied against large hydrodynamic resistances. Therefore, withthe use of a digital ow meter (Flow-unit-M, Fluigent, Lowell,MA) and soware (AIO, Fluigent, Lowell, MA) we veried that inthe range of ow rates used in this work, ow rate was notattenuated by any of the microuidic devices used.

Biological protocol

In this study, Pseudomonas uorescens CT07 (motile, Gram-negative, rod-shaped) was used. In the literature, this bacteriais reported as Pseudomonas sp. strain CT07.20 All strains weremaintained as glycerol stocks at �80 �C. A pre-culture ofplanktonic Pseudomonas sp. was used as inoculum and wasobtained by shaking cultures of planktonic bacteria in 3 mL of5 mM AB growth media at 300 rpm for 18 h at 30 �C. The growthmedia used in cultivation in the microuidic ow cell wasmodied AB consisting of 1.51 mM (NH4)2SO4, 3.37 mMNa2HPO4, 2.20 mM KH2PO4, 179 mM NaCl, 0.1 mM MgCl2-$6H2O, 0.01 mM CaCl2$2H2O and 0.001 mM FeCl3 with 10 mMNa-citrate$6H2O as the sole carbon source.

Optical imaging and analysis

Optical micrographs of the biolms were acquired using aninverted light microscope (Bruker, IX73, MA, USA) with anautomatic stage (MS2, Applied Scientic Instruments, USA).The automatic stage was used to reposition the eld of view andcreate stitched images spanning larger segments of the micro-channel. Images were recorded at 2� magnication (numericalaperture 0.06) with a condenser stage (numerical aperture 0.9).The objective working distance was 475 mm, which was longerthan necessary to observe the entire vertical range of themicrochannel. Monochrome images were collected using anuncooled, 5 megapixel, 8 bit greyscale CCD camera (LumeneraInnity 3-1, Ottawa, Canada). Colour images were acquiredusing an uncooled 6 megapixel, 8 bit colour CCD camera(Lumenera Innity 3–6URC, Ottawa, Canada). Averaged OD andimage stitching were conducted using open access imageanalysis soware (ImageJ V1.47). The average OD was calculatedas follows:

OD ¼ �log(Intt/Int0) (3)

where Intt and Int0 are the average pixel intensities at an arbi-trary time, t, and at time t ¼ 0, respectively. During all phases ofthe experiments, the total ow rate of solutions applied to theinlets resulted in liquid residence times of <60 s, which weresignicantly shorter than the maximum specic planktonicdoubling time (>1 h), thus ensuring that all changes to OD werethe result of attached biolms and not planktonic bacteria.

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Analytical Methods Paper

Numerical simulation

Three-dimensional ow simulations were conducted via so-ware (COMSOL Multiphysics 4.2a) for computational uiddynamics within different components of the microuidicchannel. A ne mesh was used with physics for laminar ow ofan incompressible liquid.

Results

This section is divided into 3 sections: (i) control measurementswith no ow connement, (ii) downstream connement bylaminar ow templating, and (iii) downstream connement byow enhancement. A discussion section follows.

(i) Control measurement with no ow connement

The goal of this work was to avoid biolm growth in theupstream uidic components of microuidic bioreactors. In therst (control) channel design, a separate inlet was used tointroduce inoculum solution into the main measurementchannel (I1 in Fig. 1a(i)) which was separated from the nutrientinlet (I2). In the rst set of experiments, a stable ow of sterilenutrient solution was established from the upstream inlet, I2,(ow rate Q2 ¼ 0.3 mL h�1) and then inoculum was introducedto downstream inlet, I1, (ow rate Q1 ¼ 0.3 mL h�1). Theupstream ow was rst stabilised to ensure that the inoculantcould not back-ow in the wrong direction. Otherwise, nospecial ow connement was implemented in this design. Aer1 hour of inoculation, the inoculant in I1 was switched off, andnutrient solution continued to ow through I2, marking thebeginning of the growth phase. Based on the continuous ow ofnutrient solution from I2, the total ow rate upstream of I1 was0.3 mL h�1 (equivalent to v ¼ 830 mm s�1 and s ¼ 0.1 Pa) duringboth inoculation and growth phases, whereas the total ow ratedownstream of I1 was 0.6 mL h�1 (equivalent to v¼ 1660 mmh�1

and s ¼ 0.2 Pa) during inoculation. Initially no biolm wasobserved either up- or downstream of I1 (Fig. 2a). Although theinoculum only initially contacted the downstream positions of

Fig. 2 Transmission microscopy of the growth of a Pseudomonas sp.biofilm in a microchannel with the design shown in Fig. 1a(i). (a)Channel after one hour of inoculant flow from I1 (Q1 ¼ 0.3 mL h�1) andnutrients flowed from I2 (Q2 ¼ 0.3 mL h�1). (b) Channel after 10 hoursfollowing a 1 hour inoculation then 9 hours flow of nutrients from I1 (Q1

¼ 0.3 mL h�1) and while a constant flow of sterile water flowed from I2(Q2 ¼ 0.3 mL h�1). (c) Channel 8 hours after (b) following a switch fromsterile water to nutrient solution from I2 and blocking I1. I1 is indicatedfor all images with a red box. Red arrows point to the direction of theupstream inlet, I2, and the downstream outlet, O. Blue arrow shows thedirection of flow. The scale bar represents 2 mm.

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the ow channel, biolm was always observed to grow in boththe upstream and downstream positions with no differences inlag phase time (typically 5–25 hours depending on the experi-ment). The simultaneous growth in both positions likelyoccurred because bacteria could move toward the nutrientsolution injection point at I2, during and aer inoculation,either by swimming against the ow or by crawling along thewalls.10,11,21 The result was unchanged for growth solution owrates from I2 up to Q2¼ 4mL h�1. In another set of experiments,where a sterile water stream was applied to I2 during the 1 hourinoculation period and then switched to a nutrient solutionaer the inoculum ow was stopped, simultaneous biolmgrowth up- and downstream of I1 was still observed, again withthe same lag phase in both positions. This could have been theresult of two different processes occurring aer the introduc-tion of nutrient through I2. Either bacteria which initiallydeposited downstream could have immediately undergonechemotaxial response and moved upstream or some dormantbacteria which had randomly moved to upstream positionsdespite the initially zero local nutrient concentrations couldhave become reactivated and started growing. In anotherexperiment, we demonstrated that the upstream portion of thechannel could be maintained biolm-free indenitely whilepure water owed through I2 (Fig. 2b) but that a biolm quicklydeveloped aer switching to a nutrient source (Fig. 2c). Weconclude that simply isolating the inoculation inlet from the(initially sterile) nutrient inlet, I2, was not sufficient to preventupstream biolms from rapidly forming aer nutrients wereswitched to I2, even in the case that a pure water connementsolution through I2 initially eliminated biolm growth fromupstream locations.

(ii) Downstream connement by laminar ow templating

In the second approach, we investigated the possibility thatpreventing bacterial growth within the slow ow elds of themicrochannel corners could limit their ability to moveupstream and contaminate those positions. This goal wasaccomplished using a reported method for “laminar ow tem-plating” to pattern the biolm growth in the microchannels.22,23

In this experiment, we took advantage of the laminar owproperties of uids in the microchannels to limit mixingbetween otherwise miscible streams. A nutrient-depleted solu-tion (the connement solution) was introduced at the furthestupstream position (I2), whereas the inoculum stream (thetemplate solution) was introduced at the furthest point down-stream (I1). The template solution was conned to ow in thecentre of the channel, whereas the connement solution occu-pied the channel corners. As reported previously, this methodrequires matched ionic strengths of the template and conne-ment solutions to minimise diffusive mixing. In this work, wemodied the ow templating design by adding a third inlet, I3(Fig. 3a). In this conguration, we used separate inlets for theinoculant (I1) and the nutrient solution (I3) while achieving owconnement away from the corners using a connement solu-tion (I2). The inset in Fig. 3a shows a connement solution fromI2 conning two streams from I1 or I3 at the same time using

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Fig. 3 (a) Schematic of microchannel with a 3-inlet microflow system.The first inlet (red) introduces the confinement solution to I2. Twodifferent templated solutions are introduced in I1 and I3 for inoculum(yellow) and nutrient solution (blue), respectively. (Inset) Image ofmicrochannel with coloured fluid to show laminar flow inside of thechannel with multiple flow streams. (b) Transmission image (greyscale)of a microchannel with growth of biofilm on the downstream side of I1at 16 hours after inoculation with nutrient solution injected into I3 withflow rate 0.3 mL h�1 and a confinement flow rate of injected into I2with flow rate 0.3 mL h�1 (i). Fluorescent image (green) of the samemicrochannel with backward growth of biofilm 13 hours after theimage in (i) was acquired (ii). All scale bars are 2000 mm.

Fig. 4 (a) Schematic of the shear confinement device showing inlets 1and 2 with flow ratesQ1 andQ2, respectively, and outlet with total flowrateQO ¼Q1 +Q2. The location-specific shear confinement stress andvelocity are vx and sx, where x¼ h (head andmeasurement area 1), x¼ n(neck) and x ¼ m (measurement area, upstream of I1). Arrows show thedirection of flow. Results fromnumerical simulationwithQT¼ 0.3mL h�1

showing cross-sections in themeasurement area (b) and in the neck area(c) for flow velocity (i) and shear stress (ii) with colour scale defined in thecolour bars. Insets in (b) give zoom view of certain segments of thechannel at the same scale as the view of the neck in (c).

Paper Analytical Methods

coloured dyes. Using this approach, we inoculated the channelin a linear pattern through I1 (Q1 ¼ 0.3 mL h�1) while applyinga connement stream through I2 (Q2 ¼ 0.3 mL h�1). Followinginoculation, the inoculum ow was stopped, and a nutrientsolution was introduced into the channel through I3 (Q3 ¼0.3 mL h�1). Thus, during inoculation and growth phases thetotal ow rate upstream of the templated biolm was 0.3 mLh�1 (equivalent to v ¼ 830 mm s�1, s ¼ 0.1 Pa) and 0.6 mL h�1

(equivalent to v ¼ 1660 mm h�1, s ¼ 0.2 Pa), respectively.Fig. 3b(i) shows a biolm growing along a linear pattern only inthe downstream portion of the channel 15 hours aer switchingthe nutrient solution to the upstream position (I2). Thus,exclusion of biolm growth from the microchannel cornersimproved the ability to halt upstream biolm growth. However,the protection against upstream growth was only short-term.Fig. 3b(ii) shows that within 29 hours, a new biolm followedthe templated nutrient stream backwards to I3 and presumablyinto the upstream tubing and beyond. We note that increasingow rates up to 4 mL h�1 did not seem to have an impact indelaying further this connement effect.

(iii) Downstream connement by ow enhancement

Finally, in the third experiment, we attempted to isolate biolmgrowth from the upstream components using a ow enhance-ment technique, in which a constriction caused localisedincreases to the ow velocity and shear force. For this purpose,a ow connement element (neck) was introduced into thechannel design (Fig. 1a(iii) and 4a) between the nutrient inlet I2and the biolmmeasurement area. Based on eqn (1) and (2), theoverall reduction in neck width caused local enhancements to

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the ow velocity and shear force (vn and sn) compared withthose values in the head (vh and sh) and with those of themeasurement area before I1 (vm and sm). We note that duringbiolm growth using this third channel design, the only owsource was the nutrient solution from I2, and therefore, thevalues of v and s before and aer I1 were the same. In this case,the reduction in the neck width to 250 mm from 2000 mm else-where resulted in increases to both v and s by a factor of 8. Theresults of a numerical simulation of v along the neck and themeasurement channel cross-section are presented in Fig. 4b fora ow rate of Q2 ¼ 1 mL h�1. Overall, v was higher in the neck,and importantly, the stagnant regions in the corners werestrongly reduced compared with those of the measurementchannel.

To test the performance of the ow enhancement element,we inoculated and grew biolms under different ow condi-tions while monitoring the biolm growth in the target regionsof the channel. The solution assignments for channel inlets I1and I2 are given in Table 1. Please see the ESI† for the volumetricow rates (Table S1†) and the calculated local average velocities

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Table 1 Inlet assignments during different growth phases

Growth phase I1 solution I2 solution

Phasei (inoculation) Inoculant WaterPhased (downstream growth) Nutrient WaterPhaseu (upstream growth) Blocked Nutrient

Fig. 6 Optical density versus time during phaseu for different flowrates Q2. Blue colour indicates the downstream of the neck, andorange colour denotes the upstream of the neck, matching the colourcoding in Fig. 4a. Time 0 is the start of second part of the experimentwith nutrient flow from l2. Nutrient flow rates were (a)Q2¼ 0.3 mL h�1,(b) Q2 ¼ 1 mL h�1, (c) Q2 ¼ 3 mL h�1 and (d) Q2 ¼ 4 mL h�1.

Analytical Methods Paper

(Table S2†) and shear forces (Table S3†) at each stage for allexperiments conducted. In brief, a typical experiment included3 different phases: an inoculation phase (phasei), a downstreamgrowth phase (phased) and an upstream growth phase (phaseu).During phased, biolms were observed to grow downstream ofI1 (not shown), whereas all portions upstream of I1 remainedcompletely free of biolm growth due to a ow of sterile purewater from I2 (Fig. 5a). Aer the downstream biolms reachedmaturity, phaseu was started by switching the nutrient solutionto I2 and blocking I1. In this phase, bacteria were free to movevia chemotaxis towards the new nutrient source in I2 and growbiolms in the upstream portions of the channel.

As expected, aer the nutrient solution passed through I2,biolm growth was observed between the downstream portionof the neck and I1, independent of Q2. This situation is essen-tially the same as for the control experiment in which there wasno ow connement element. For low values of Q2 (#1 mL h�1),biolms could grow at the upstream side of the neck with nodetectible difference in lag phase compared with the growth atthe downstream side (Fig. 5b). As Q2 increased, biolm growthat the upstream side of the neck could be slowed relative to thegrowth on the downstream side or even stopped (Fig. 5c).

We quantied the biolm growth rate via ODmeasurementsat the positions highlighted in Fig. 5a for nearly 2 days. Wecommenced phaseu (t ¼ 0) by introducing the nutrient solutioninto I2 at different Q2 values aer the downstream biolm hadreached maturity. As observed in Fig. 6a and b, during phaseu,

Fig. 5 Stitched images centered at the flow confinement element showinchannel during the inoculation phase (phasei) with polygons representinpositions of the flow confinement element. The same channel region asno confinement (b), or Q2 ¼ 4 mL h�1 showing confinement (c). Scale bmination of the field of view after manual image stitching. Each image wfrom left to right.

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the biolm in the upstream and downstream side of the neckbegan to grow at nearly the same time and with nearly the samerate for Q2 ¼ 0.3 or 1 mL h�1. At higher ow rates, we observedan increase in the lag in growth and a reduction in the overallbiolm OD at the upstream side of the neck (Fig. 6c). Increasingthe ow rate to Q2 ¼ 4 mL h�1 during phaseu resulted in nodetectible increases in upstream OD during the entire experi-ment (Fig. 6d). Setting the ow rate to Q2 > 4 mL h�1 had noadditional effects during the time period evaluated. Therefore,we conclude that the enhanced hydrodynamic properties in theneck (vn and sn) were effective in limiting biolm developmentto the downstream components only during a typical experi-ment (2 days).

Discussion

Though highly dependent on its biochemical environment, theswimming velocity of P. uorescens can reach approximate

g portions immediately upstream and downstream. (a) An initially cleang the analysis regions at the upstream (orange) and downstream (blue)in (a) after the phaseu (approximately 45 h) withQ2 ¼ 1 mL h�1 showingar is 2 mm. Slight mismatches in images are due to non-uniform illu-as acquired by transmission microscopy with 2� magnification. Flow is

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Paper Analytical Methods

mean swimming speeds of 30 mm s�1,21 which is similar to otherPseudomonas species.10a,11b Compared to the calculated averagevelocities of owing solution in the microchannel, even theslowest moving liquids had average velocities of 830 mm s�1.Therefore, swimming was unlikely the mechanism for theobserved upstream contamination. Rather, crawling along themicrochannel walls and through the corners wasmore likely thecause, despite the slow speeds associated with this type ofmotility (0.04 to 0.08 mm s�1).11b Therefore, we take s as the mostimportant parameter in controlling upstream contamination inthis study. Critical values of shear stress are discussed in rela-tion to the results for the ow connement approaches.

Compared to the control channel design, both the laminarow templating method (Section ii) and the ow enhancementmethod (Section iii) showed improved downstream biolmconnement capabilities. Both methods were passive, avoidingdevice complexity associated with integrated components,energy consumption or the use of chemical compounds andrelated side-effects on the quality of the nutrient solution. In therst, connement of the inoculum and nutrient solution pre-vented biolm formation in the microchannel corners, whichresulted in a noticeable delay in upstream biolm growth.Therefore, it appears that the corners are major bacterial traf-cking areas that efficiently connect up- and downstreamportions of the microchannel. This is unsurprising since therelatively low ow velocities in the corners will not inducestrong wall shear stresses to dislodge or slow crawling sessilebacteria and may even permit some upstream swimming. Inaddition to reduced shear effects from the liquid ow, somestudies have shown that the mere presence of a solid wall canenable faster motility due to the physics of the planktonicpropulsion systems.10b As the junction between the horizontaland vertical sidewalls in a microchannel corner leads to anincrease in the local surface area, these effects can be ampli-ed.10a,24 However, despite the noted improvements in down-stream connement by templating biolm growth in the centreof the channel, linear biolm patterns were eventually observedupstream within the conned nutrient stream, regardless of theapplied ow rate (up to 4 mL h�1). Therefore, we can assert thatbacteria were able to move upstream against at wall segments,albeit less efficiently than in the corners.

The second approach used a ow enhancement element,which consisted of a narrowed channel segment that increasedthe v and s. At high ow rates (4 mL h�1), this method out-performed the ow templating method by completely stoppingupstream biolm growth for at least two days. This was nearlyenough time to allow downstream biolms to reach maturity,opening the door to experiments under true chemostaticconditions that can be conducted at all biolm life cycle stages.The calculated s in the neck at this ow rate gives an approxi-mate threshold of 10.65 Pa for impeding upstream contami-nation. We note that increasing the ow rate in 2 mm-widechannels with no neck (e.g., Fig. 1a(i)) to 32 mL h�1 would resultin the same ow velocity and shear stress in a 250 mm neck withow rate of 4 mL h�1. However, this would be impractical duethe need for frequent syringe replacement. Moreover, the entiremeasurement area would be subjected to such elevated

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hydrodynamic conditions. Thus, without a neck design, thegoal of downstream ow connement could only be achieved atthe expense of serious constraints on the range of applicableow conditions applied to experiments. Future development ofthis approach should focus on optimising constriction dimen-sions to improve performance. For example, smaller constric-tion widths, longer lengths and even modied cross-sectionalshapes (e.g., round) could be used to increase s while keepingtotal ow rates low. Practical advantages can be longer experi-ment durations without frequently needing to change thesyringes as well as enabling a larger dynamic range of hydro-dynamic properties to be applied to biolms growing in thedownstream measurement areas. Rapid changes to pressureduring the transition between the neck and the measurementsection, could result in local formation of bubbles. In this worka combination of vacuum degassing of all liquids (see theMaterials and methods section) and a gradual transition inchannel widths prevented any bubble formation. As futurestudies may optimise performance with even smaller neckwidths, additional approaches could be taken to suppressbubble formation, including elongation of the transitionlengths between channel segments with different widths andthe use of in-line or on-chip debubblers.16d

We note that a fundamental limitation to the approach maystem from lateral biomass expansion during biolm growth. Asseen in Fig. S1 in the ESI,† a micro colony has a net tendency togrow downstream due to its viscous response to the imposedow in the microchannel.25 However, there is typically alsoa non-zero velocity component in the upstream direction,measuring 1–10 mm h�1 (0.3 to 3 nm s�1) for typical conditionsused in this work. While much slower than crawling motility ofisolated bacteria, the biolm offers enhanced protection to thebacteria within it. Thus, it is likely to better resist shear stressthat remove isolated sessile bacteria, potentially underminingconnement by the ow enhancement element.

At this point we pause to summarize the nature of thebacterial ux that can cause upstream contamination. Theprimary source of bacterial ux occurs through the channelcorners (Jcorner), as observed from the rapid growth of biolmsusing control device with design shown in Fig. 1a(i). Next mostimportant is due to migration along at microchannel surfaces(Jwall) as demonstrated by the delayed upstream biolm forma-tion when the corners were not accessible for bacterial coloni-zation, using the device shown in Fig. 1a(ii). As discussed above,colony expansion in the upstream direction (Jcolony) was observedto push the biolm upstream at a slower speed than reported forcrawling bacteria. Lastly, despite the relatively high maximumspeed of planktonic P. uorescens (30 mm s�1) bacteria, swim-ming movement through the liquid (Jliquid) should be the leasteffective upstream transport mode since downstream liquid owvelocities in microchannels will usually be much faster. Thisrelation is summarized qualitatively in eqn (4).

Jcorner > Jwall > Jcolony > Jliquid (4)

Lastly, while the goal of this work was to address the prac-tical problem of upstream contamination, the same device can

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Analytical Methods Paper

also serve as a tool to study chemotaxis under ow elds. Todate, microuidic devices have been exploited to investigatebacterial response to well-dened concentration gradients thatcan be formed and controlled by manipulations of microowstreams.26 However, apart from some interesting numericalstudies,27 studies of chemotaxis under variable liquid ow eldsare scarce. Therefore, this work can also form the basis offundamental studies into the limits of bacterial chemotaxis toovercome applied ow rates.

Conclusions

Two microuidic ow connement methods were evaluatedand compared to a control system. Both methods demonstratedan enhanced capacity to localise Pseudomonas uorescens bio-lm growth to a downstream measurement area. The mostpromising results were achieved from the inclusion of a owenhancement element consisting of a local reduction in thechannel width. With this method operating at elevated owrates, upstream contamination was completely eliminated forthe duration of the two-day experiments. Further developmentto modify the geometry of the ow enhancement constrictioncan enable downstream biolm connement under lowerow rates. This approach can also be an effective method tostudy bacterial chemotaxis against different hydrodynamicconditions.

Conflicts of interest

There are no conicts to declare.

Acknowledgements

The authors thank Muhammad Safdar for assistance with 3Dgraphics and Molly Gregas for her copy edits.

Notes and references

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