Microfluidic System for Observation of Bacterial Culture and Effects On

micromachines

Communication Microfluidic System for Observation of Bacterial Culture and Effects on Biofilm Formation at Microscale

Xiao-Yan Zhang 1, Kai Sun 1,*, Aliya Abulimiti 1, Pian-Pian Xu 1 and Zhe-Yu Li 2,* 1 School of Environment, Harbin Institute of Technology, Harbin 150090, China 2 State Key Laboratory of Urban Water Resource and Environment, Harbin Institute of Technology, Harbin 150090, China * Correspondence: [email protected] (K.S.); [email protected] (Z.-Y.L.); Tel.: +86-451-8628-9126 (K.S.); +86-451-8628-6808 (Z.-Y.L.) Received: 26 August 2019; Accepted: 10 September 2019; Published: 12 September 2019

Abstract: Biofilms exist in the natural world and applied to many industries. However, due to the variety of characteristics caused by their complex components, biofilms can also lead to membrane fouling and recurrent infections which pose threats to human health. So, to make the best use of their advantages and avoid their disadvantages, knowing the best time and methods for improving or preventing biofilm formation is important. In situ observation without fluorescence labeling in microscale and according to a time scale is useful to research biofilm and confine its formation. In this study, we developed a microfluidic system for real-time observation of bacteria culture and biofilms development at microscale. We cultured E. coli ATCC 25922 on a chip at continuous flow of the velocity, which could promote bacterial formation. Biofilms formation under the condition of adding amoxicillin at different times is also discussed. In addition, the mixed strains from sludge were also cultured on chip, and possible factors in biofilm formation are discussed. Our results show that a microfluidic device could culture microorganisms in continuous flow and accelerate them to adhere to the surface, thereby promoting biofilm formation. Overall, this platform is a useful tool in research on initial biofilm formation, which can contribute to preventing biofouling and infections.

Keywords: microfluidics; biofilm; continuous flow; antibiotic; microscale; E. coli

1. Introduction Biofilm is secreted by bacteria and widely exists in nature. It aggregates free-floating microorganisms to form a sessile community, which enables bacteria to inhabit the surface. The biofilm is one of the most important factors leading to membrane fouling in membrane bioreactors (MBRs) in water treatment industry [1]. However, on the other hand, biofilms comprising microorganisms grown on carriers can remove some micropollutants and biological phosphate [2,3]. In order to optimize the positive effects and minimize the negative effects, actions should be taken at initial stage to promote or inhibit biofilm formation. Therefore, it is essential to study biofilm formation at time and spatial scales. In the process of biofilm formation, changes occur in genotype and phenotype in spatial scales. At nanoscale, planktonic bacteria communicate and coordinate with each other by enzymes and molecular signals. At the microscale, bacteria adhere to the solid surface and aggregate to form microcolonies, which secrete extracellular polymeric substances (EPS) for building biofilms structures. At the mesoscale, mature biofilms can be observed and used in practical application at the macroscale [4]. Biofilms at mesoscale and macroscale are strongly associated with practical applications, so most studies focus on the factors of biofilm development at these scales. However, intensive studies of effects on the initial formation process at microscale are also important, which are the best stage

Micromachines 2019, 10, 606; doi:10.3390/mi10090606 www.mdpi.com/journal/micromachines Micromachines 2019, 10, 606 2 of 12 to facilitate or inhibit the biofilm formation. In a traditional shake flask, most bacteria are in the planktonic state, so the biofilms are hard to culture [5]. Culturing biofilms by shake flask is not only labor-intensive, but also the characteristics of biofilms formed cannot remain the same. The reason is that the bacterial heterogeneity of spatial distribution [6] leads biofilms to vary widely in physiological conditions [7] and cellular physiologies [8]. Moreover, because biofilms are settled in different regions, mass transfer passing through the biofilm cannot be controlled. All the above factors result in different biology characteristics. Microfluidic technology can bear the organisms to culture at microscale space, and the cultural conditions can be precisely controlled. For example, Hogan et al. prevented S. aureus biofilm formation on a plastic biochip which was modified by polyurethane [9]. With the remarkable advantage of rapid analysis [10], low cost [11], low sample consumption [12], high throughput [13], controllable conditions [14], microfluidics has been a promising method for bioanalysis. In the forming process of biofilms, real-time observation is crucial to obtain the information of morphology at microscale. Undoubtedly, the microfluidic system based on a miniaturized chip is the best method to study the initial biofilm formation and the effects on the biofilms imposed by environmental conditions, including nutrient concentration [15], surface properties [16], shear stress [17], quorum sensing [18]. In the meanwhile, microfluidic device is able to confine the bacterial growth in micrometers and to mimic the practical environmental conditions to probe the complex interplay at different time scales [19,20]. Recently, researchers focus microfluidic system coupled with different detection modes to study biofilms. Funari et al. monitored microbial biofilm formation by nanoplasmonics in situ [21]. Li et al. studied biofilm accumulation and adhesive strength in microfluidics coupled with a microscope [22]. In this work, we designed and fabricated microfluidic chips for bacteria culture at continuous flow and real-time investigation on the initial formation of biofilm at microscale. To determine the hydrodynamic factor of biofilm formation, E. coli ATCC 25922 were cultured on the chip as model bacteria at different velocities. We also studied the influence of adding amoxicillin at different stages on initial biofilm formation with the microfluidic device. In addition, we analyzed the biofilm formation of mixed strains, which were cultured from the sludge of secondary sedimentation tank. The influence of amoxicillin on mixed microorganisms with biofilms was also discussed.

2. Materials and Methods

2.1. Materials and Reagents Luria Bertani (LB) broth are composed of 0.5% yeast extract, 1% peptone, 1% NaCl and deionized water, all of which were purchased from Beijing Ao Xing Bio-tech (Beijing, China). Ethanol and amoxicillin were obtained from Sigma-Aldrich (Shanghai, China). SU-8 2050 negative photoresist was purchased from Micro Chem Corp. (Newton, MA, USA). Polydimethylsiloxane (PDMS) Sylgard 184 was obtained from Dow Corning (Midland, MI, USA). Borosilicate glass was achieved from Matsunami (Kyoto, Japan). FilmTracer TM SYPRO® Ruby Bioﬁlm Matrix Stain was purchased from Invitrogen (Waltham, MA, USA).

2.2. Sample Preparation E. coli ATCC 25922 with ﬂagella was used as a model strain in this work. E. coli BL 21 as control was purchased from the China Center of Industrial Culture Collection (CICC). Prior to culture on the chip, bacteria from a LB agar Petri dish were transferred to LB medium and incubated overnight. E. coli was diluted in LB medium to obtain OD600 in the range of 0.112–0.115, then injected into chip to culture. The mixed strains were obtained from sludge of a secondary sedimentation tank; 1 mL suspended sludge were incubated in pretreated piggery wastewater for 24 h. Then the mixed strains were diluted in pretreated piggery wastewater to obtain OD600 in the range of 0.112–0.115 and used to culture on chip. Micromachines 2019, 10, 606 3 of 12

2.3. Bacteria Culture and Biofilm Formation in Microfluidic Device The microfluidic device was firstly sterilized by flushing with 75% alcohol overnight and washed by deionized water in sequence. After ultraviolet (UV) sterilization for 30 min, diluted E. coli in LB medium was injected into the culture chamber at the constant flow rate of 5 µL/min by syringe pumps. The fresh medium was continuously injected into the chamber after balancing for 2 min, so the free flow bacteria was washed away. Fewer bacteria were able to adhere to the glass, then they grow under the continuous flow at the rate of 0.5 µL/min and form the biofilm. Bacteria were cultured on the chip at room temperature of 25 °C, which was controlled by constant temperature and humidity air conditioner. The temperature nearby microfluidic system was monitored by the electronic thermometer in the whole experiment, and the temperature difference was 1 C. The antibiotics were added in ± ◦ different periods of bacterial growth. For bacteria from sludge on chip culture, the medium was pretreated piggery wastewater by high-temperature sterilization.

2.4. The Process of Biofilm Stain on Chip Bacteria were cultured on chip under the continuous flow at the rate of 0.5 µL/min. After 11 h, water was injected into the culture chamber and LB medium was flowed away. On account of LB medium interfering with fluorescent staining, we injected water. Then SYPRO Ruby was injected into the culture chamber at the rate of 0.5 µL/min. At the continuous flow, the samples were incubated for up 1 h, protected from light. In the end, the samples were observed by an inversion microscope.

2.5. Microﬂuidic Device Fabrication The standard soft lithographic process was used to fabricate this chip. The depth of channel and chamber was 50 µm. PDMS prepolymer and curing agent by the ratio of 10:1 were mixed and degassed, then poured onto a SU-8 mold. After baking at 70 ◦C in the oven for 2 h, the PDMS mold was peeled oﬀ and bonded with glass. In order to avoid the organic and metallic contamination, the borosilicate glass was sequentially cleaned in following solutions, H2SO4:H2O2 (4:1), H2O:H2O2:NH4OH (50:10:1), H2O:H2O2:HCl (5:1:1). After washing, the glass plate was immersed in H2O/H2O2/NH4OH solution (7:2:1) to increase the hydrophily. The PDMS layer and the glass layer were bounded by using UV O-zone treatment (Model 42-UVO-Cleaner, Jelight, Irvine, CA, USA) for 5 min and baking for 70 ◦C for 15 min.

2.6. Microfluidic System and Optical Image Analysis This system consisted of one chip to culture bacteria; two pumps (AL-1000, World Precision Instruments, Sarasota, FL, USA), two syringes (Gastight 1001, Hamilton, Reno, NV, USA) and one three-way valve (MV201, LabSmith, Livermore, CA, USA) to control the fresh medium and antibiotic. The optical images were acquired by an inversion microscope (Olympus, IX 71, Tokyo, Japan) with a charge coupled device (CCD) camera (Olympus, DP80, Tokyo, Japan). The images of bacteria magnified 400 times were taken every hour, and were analyzed by Image J software. The steps of image processing and the number of cells were stated in Figure A1. In suspension culture, the growth rate is determined by optical density (OD600). For the bacterial culture on a chip, bacteria are able to adhere to the glass and grow in a monolayer. Hence, bacterial mass is proportional to superficial area and increment ratio R = Si/S0 [23]. In this study, we use the relative growth rate ln R to quantify the speed of bacterial growth.

3. Results and Discussion

3.1. Chip Design and Operation We developed a PDMS chip to culture bacteria and form bioﬁlm under continuous ﬂow. The process of bacterial culture formation was observed by an inversion microscope. The detailed design of the chip Micromachines 2019, 10, x FOR PEER REVIEW 4 of 12

shown in Figure 1B. This design was aimed to culture bacteria and form biofilms by reducing fluidic impact and supporting steady culture conditions. The flow from the inlet was separated by annular Micromachineschannels, which2019, 10 was, 606 used to keep mean velocity inside of the octagon chamber. The inner octagon4 of 12 area is designed as the culture chamber, and surrounded by the battlements arrays of micro pillars, the top view of each pillar is 54 μm × 40 μm, and the gap between the two neighboring pillars is 30 structure is shown in Figure1A, and the schematic diagram of biofilm formation is shown in Figure1B. μm. Moreover, the pillars could also prevent the gas bubble from entering into the culture chamber. This design was aimed to culture bacteria and form biofilms by reducing fluidic impact and supporting The width of all the channel is 200 μm, the depth of both the channels and the chamber is 50 μm. steady culture conditions. The flow from the inlet was separated by annular channels, which was used The sample of diluted bacteria should be loaded into the chip through the outlet of the culture to keep mean velocity inside of the octagon chamber. The inner octagon area is designed as the culture chamber. After balancing for 2 min, the medium was injected continuously from the inlet of the chamber, and surrounded by the battlements arrays of micro pillars, the top view of each pillar is culture chamber. Due to the asymmetric structure, this injection mode enables bacteria to distribute 54 µm 40 µm, and the gap between the two neighboring pillars is 30 µm. Moreover, the pillars could randomly× in the culture chamber. In order to flow the planktonic bacteria in chip, the medium was also prevent the gas bubble from entering into the culture chamber. The width of all the channel is injected at the 10 μL/min for 5 min, then the velocity was reduced to 0.5 μL/min. 200 µm, the depth of both the channels and the chamber is 50 µm.

Figure 1. Schematic illustrations of chip structures for bacteria culture and biofilm formation. (A) The diameterFigure 1. of Schematic inscribed illustrations circle of regular of chip octagon structures area for is 2 bacteria mm. The culture size of and a single biofilm micropillar formation. is ( 54A)µ Them 40 µm (top view) and the gap between pillars is 30 µm. The depth of both the channel and the culture ×diameter of inscribed circle of regular octagon area is 2 mm. The size of a single micropillar is 54 μm chamber×40 μm is(top 50 view)µm. ( Band) Schematic the gap illustrationsbetween pillars and is optical 30 μm. images The depth of stages of both of biofilm the channel development: and the (1)culture Loose chamber adhesion is to 50 the μ surface.m. (B) Schematic (2) Irreversible illustrations attachment. and (3)optical Bacteria images secrete of stages the extracellular of biofilm polymericdevelopment: substances (1) Loose (EPS) adhesion to form to the the biofilm surface. structure, (2) Irreversible and the attachment. biofilm matures. (3) Bacteria (4) Biofilms secreteare the completelyextracellular mature. polymeric substances (EPS) to form the biofilm structure, and the biofilm matures. (4) Biofilms are completely mature. The sample of diluted bacteria should be loaded into the chip through the outlet of the culture chamber.3.2. Bacterial After Adhesion balancing and for Biofilm 2 min, Formation the medium on the was Chip injected continuously from the inlet of the culture chamber. Due to the asymmetric structure, this injection mode enables bacteria to distribute randomly in theIn culture this chamber.study, we In challenged order to flow to theculture planktonic E. coli bacteria as model in chip, microorganis the mediumm to was observe injected biofilm at the 10formationµL/minfor on 5the min, chip. then Bacteria the velocity biofilms was are reduced difficul tot to 0.5 cultureµL/min. and obtain in traditional shake flasks, although nearly all kind of bacteria secrete viscous substrates to form biofilms. At present, few 3.2.studies Bacterial have Adhesion investigated and Biofilm the microbe Formation except on the for Chip Pseudomonas Aeruginosa (P. Aeruginosa), which is a model microorganism. Because the pili of P. Aeruginosa contribute greatly to its ability to adhere to In this study, we challenged to culture E. coli as model microorganism to observe biofilm formation the surface and form biofilm easily. Due to this characteristic, minimal biofilm eradication on the chip. Bacteria biofilms are difficult to culture and obtain in traditional shake flasks, although concentration (MBEC) of P. Aeruginosa could be measured in most studies [24]. However, it is hard to nearly all kind of bacteria secrete viscous substrates to form biofilms. At present, few studies obtain the biofilms of E. coli in laboratory by traditional methods. Taking advantage of the large have investigated the microbe except for Pseudomonas Aeruginosa (P. Aeruginosa), which is a model microorganism. Because the pili of P. Aeruginosa contribute greatly to its ability to adhere to the surface and form biofilm easily. Due to this characteristic, minimal biofilm eradication concentration (MBEC) of P. Aeruginosa could be measured in most studies [24]. However, it is hard to obtain the biofilms of Micromachines 2019, 10, 606 5 of 12

E. coli in laboratory by traditional methods. Taking advantage of the large special surface area of chip, bacteria are able to adhere to the chip wall and grow to form the biofilm easily. E. coli is with peritrichous flagella, which propel bacteria forward by rotation motion under the controlMicromachines of the 2019 flagella, 10, x FOR bundle PEER REVIEW [25]. Under the action of the thrust force generated 5 ofby 12 flagella, E. coli is difficult to be confined on chip in liquid. For on chip culture, the medium was injected after bacteriaspecial loading, surface so free-floating area of chip, bacteria bacteria are can able be to washedadhere to away the chip and wall the and bacteria grow to with form the the slime-encasedbiofilm substanceseasily. can adhere to the glass wall. E. coli is with peritrichous flagella, which propel bacteria forward by rotation motion under the As shown in Figure2, only a few microorganisms were observed adhering to the glass wall and control of the flagella bundle [25]. Under the action of the thrust force generated by flagella, E. coli is were confirmeddifficult to tobe distributeconfined on inchip a monolayerin liquid. For aton 0chip h. culture, With the the continuousmedium was injected flow passing after bacteria through the adherentloading, bacteria, so free-floating sufficient nutrientbacteria can can be be washed supplied, away and and at the the bacteria same timewith metabolitesthe slime-encased were taken away. Insubstances addition, can bacteria adhere to of the loose glass adhesion wall. can be flowed away by the continuous flow. After 4 h incubation, bacteriaAs shown irreversibly in Figure 2, only adhered a few microorganisms to the surface, were which observed is presented adhering in to Figure the glass2. Forwall8 and h culture, bacteriawere colonies confirmed were to formed distribute and in kept a monolayer in a monolayer. at 0 h. With During the continuous this period, flow semitransparent passing through substancesthe adherent bacteria, sufficient nutrient can be supplied, and at the same time metabolites were taken began to occur in periphery of colonies. At 10 h, with bacterial growth, a single colony became larger, away. In addition, bacteria of loose adhesion can be flowed away by the continuous flow. After 4 h and bacteriaincubation, grew bacteria into multilayers. irreversibly adhered In themeantime, to the surface, the which semitransparent is presented in substances Figure 2. For enclosing 8 h the coloniesculture, could bacteria be observed colonies obviously. were formed According and kept in to a monolayer. the micrographs, During this the period, process semitransparent of bacteria growth and biofilmsubstances formation beganis to consistent occur in periphery with the of distinctcolonies. stages At 10 h, of with biofilm bacterial life cyclegrowth, [26 a]. single Firstly, colony planktonic bacteriabecame loose adherelarger, and to thebacteria surface, grew and into thismultilayers. process In is the reversible. meantime, Secondly, the semitransparent bacteria beginsubstances to undergo division,enclosing and adhesion the colonies is irreversible. could be observed Thirdly, obviousl bacteriay. secreteAccording the to EPS, the whichmicrographs, is a significant the process component of bacteria growth and biofilm formation is consistent with the distinct stages of biofilm life cycle [26]. of biofilm.Firstly, Fourthly, planktonic the bacteria biofilms loose approach adhere to maturely. the surface, After and 11this h process culture, is thereversible. crystal Secondly, materials with three-dimensionalbacteria begin structure to undergo obviously division, and appeared, adhesion which is irreversible. is shown Thirdly, in Figure bacteria3A. secrete According the EPS, to bright filed observation,which is a significant the bacteria component colonies of were biofilm. wrapped Fourthly, by the crystal biofilms materials. approach In maturely. order to verifyAfter 11 the h crystal materialsculture, are biofilms, the crystal we materials stained with the three-dimensio biofilms andnal observed structure in obviously fluorescence. appeared, SYPRO which Ruby is shown stain labels most classesin Figure ofproteins, 3A. According which to arebright the filed component observation, of biofilms.the bacteria The colonies fluorescence were wrapped image by is crystal in Figure 3B, materials. In order to verify the crystal materials are biofilms, we stained the biofilms and observed which shows the location of crystal materials forming in a bright field (Figure3A) are consistent with in fluorescence. SYPRO Ruby stain labels most classes of proteins, which are the component of fluorescencebiofilms. image. The fluorescence However, image for long is in term Figure biofilm 3B, which formation, shows the fluorescence location of crystal observation materials couldn’t be persistent.forming Wein a studiedbright field biofilms (Figure 3A) in theare consistent bright field with instead fluorescence of fluorescence, image. However, which for long contributes term to end-pointbiofilm observation. formation, fluorescence observation couldn’t be persistent. We studied biofilms in the bright field instead of fluorescence, which contributes to end-point observation.

Figure 2.FigureThe 2. photomicrographThe photomicrograph of of bacteriabacteria culture culture on onchip chip in continuous in continuous flow at 0 ﬂow h, 4 h, at 8 h, 0 h,10 h. 4 At h, 8 h, 10 h. At 00 h, h, bacteriabacteria distribute in inmonolayer. monolayer. At 4 Ath, 4bacteria h, bacteria adhere adhere to the surface to the surfaceand grow. and At grow.8 h, At 8 h, semitransparentsemitransparent substances substances surroundingsurrounding the the bacteria bacteria begin begin to form. to form. At 10 At h, 10bacteria h, bacteria grow into grow into multilayers,multilayers, and bioﬁlms and biofilms can can be observedbe observed obviously. obviously. The scale scale bar bar is 5 is μ 5m.µ m.

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Figure 3. The image of biofilms in bright field (A) and in fluorescence (B). After 11 h culture, biofilms wereFigure stained 3. The byimage SYPRO of biofilms Ruby in in culture bright chamber.field (A) and The in scale fluorescence bar is 20 µ (m.B). After 11 h culture, biofilms were stained by SYPRO Ruby in culture chamber. The scale bar is 20 μm. 3.3. The Effect of Velocity on Bacteria Growth and Biofilms Formation 3.3. The Effect of Velocity on Bacteria Growth and Biofilms Formation High shear rates or an extended period of time exposed to strong shear force, which was generated by velocity,High shear may leadrates to or bacterial an extended death [period27]. However, of time underexposed no shearto strong force, shear bacteria force, will which grow was in a planktonicgenerated by condition velocity, instead may lead of sessile to bacterial communities. death [27]. Therefore, However, it isunder necessary no shear for theforce, on-chip bacteria culture will growto choose in a an planktonic appropriate condition velocity instead of constant of sessile flow, whichcommunities. can promote Therefore, bacteria it tois formnecessary biofilms for andthe on-chipavoid bacteria culture death. to choose The shearan appr rateopriate of bacteria velocity in the of microfluidicconstant flow chip, which was calculatedcan promote by bacteria to form biofilms and avoid bacteria death. The shear rate of bacteria in the microfluidic chip was Q calculated by γ = wh2 γ= where γ is shear rate, Q is flow rate, w is the width of channel, h is the depth of channel [28]. whereBacteria γ is shear were rate, cultured Q is flow at dirate,fferent w is velocitiesthe width of channel, 0.05, 0.25, h is 0.5, the 1.0 depthµL/min, of channel and the [28]. results are 1 1 1 1 shownBacteria in Figure were4A. cultured The shear at rate different is 1.67 velocities s − , 8.33 sof− ,0.05, 83.3 s0.25,− , 166.6 0.5, s1.0− ,μ respectively.L/min, and Forthe 7results h culture, are shownthe bacterial in Figure growth 4A. rateThe atshear 0.05 rateµL/ minis 1.67 is muchs−1, 8.33 larger s−1, 83.3 than s− other1, 166.6 higher s−1, respectively. velocities. It For illustrates 7 h culture, that the velocitybacterial ofgrowth 0.05 µ Lrate/min at cannot0.05 μL/min flow away is much free-floating larger than bacteria other higher and the velocities. bacteria of It looseillustrates adhesion that theto the velocity surface, of so 0.05 the bacteriaμL/min arecannot cultured flow ataway the condition free-floating of almost bacteria no shearand the force. bacteria At the of velocity loose adhesionof 0.05 µL to/min, the bacteriasurface, growso the inbact theeria planktonic are cultured state at instead the condition of in biofilms. of almost It isno proved shear force. in Figure At the4B velocitythat after of 10 0.05 h incubation, μL/min, bacteria bacteria grow do not in formthe planktonic communities state at instead 0.05 µL of/min. in biofilms. At 0.25 and It is 0.5 provedµL/min, in Figurethe bacterial 4B that growth after rates10 h incubation, are similar, andbact lowereria do than not the form velocity communities of 1 µL/min. at 0.05 Because μL/min. shear At stress0.25 and can promote0.5 μL/min, bacteria the bacterial adhering growth to and dispersingrates are similar, on the interfaceand lower at than the initial the velocity stage of of forming 1 μL/min. biofilms Because [29]. Withshear largerstress velocitycan promote of continuous bacteria flow,adhering bacteria to and suff dieredspersing higher on shear the stress.interface It leadsat the to initial faster stage biofilm of formingformation biofilms and higher [29]. bacteriaWith larger growth velocity rate. of As cont showninuous in Figure flow, 4bacteriaB, bacteria suffered could higher form communities shear stress. Itat leads 0.25, 0.5,to faster 1 µL/ min.biofilm However, formation it is and obvious higher that bact bacteriaeria growth have grownrate. As into shown multilayers in Figure at 4B, 1 µ Lbacteria/min at 10could h, which form impliescommunities biofilms at 0.25, are more 0.5, 1 mature μL/min. than However, other cases. it is Theobvious multilayer that bacteria bacteria have were grown caused into by multilayershigh velocity, at which 1 μL/min leads at to the10 h, failure which of counting.implies biofilms Therefore, are themore velocity mature of 1thanµL/min other is notcases. suitable The multilayerfor the investigation bacteria were of the caused influence by high factors velocity, on biofilm which formation. leads to the Finally, failure we of selected counting. 0.5 µTherefore,L/min for the followingvelocity of experiments. 1 μL/min is not suitable for the investigation of the influence factors on biofilm formation. Finally, we selected 0.5 μL/min for the following experiments.

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Figure 4. Growing situations at continuous flow of different velocities. (A) bacterial growth rate during incubationFigure 4. Growing at velocity situations of 0.05 µL at/min, continuous 0.25 µL/min, flow 0.5 ofµ differentL/min, 1 µvelocities.L/min. (B )( TheA) bacterial photomicrographs growth rate of bacteriaduring cultureincubation on chip at forvelocity 10 h at of velocity 0.05 ofμL/min, 0.05 µL /0.25min, 0.25μL/min,µL/min, 0.5 0.5μL/min,µL/min, 1 1 µμLL/min./min. The(B) scaleThe barphotomicrographs is 5 µm. Error bars of bacteria are standard culture deviations. on chip for 10 h at velocity of 0.05 μL/min, 0.25 μL/min, 0.5 μL/min, 1 μL/min. The scale bar is 5 μm. Error bars are standard deviations. 3.4. The Effects of Antibiotic on Biofilm Formation during the On-Chip Growth 3.4. TheBased Effects on massof Antibiotic transport, on Bio thefilm process Formation of biofilms during the formation On-Chip occursGrowth in three characteristic time scales:Based (1) hydrodynamic on mass transport, process. the Duringprocess this of biofilms period, momentum formation occurs balance in is three achieved characteristic in seconds time by momentumscales: (1) hydrodynamic transfer including process. viscous During convection this peri andod, momentum dissipation. balance (2) Mass is transport achieved solute in seconds process. by Duringmomentum this transfer period, theincluding substrate viscous mass convection balance is and achieved dissipation. in minutes (2) Mass by transport substrate solute diffusion. process. (3) GrowthDuring this process. period, During the substrate this period, mass the balance biomass is balanceachieved is achievedin minutes in by hours substrate or days diffusion. in biomass (3) growthGrowth scaleprocess. [30]. During In this this report, peri onod, thethe basisbiomass of di balancefferent characteristicsis achieved in inhours the processor days ofin biofilmsbiomass formation,growth scale we [30]. studied In this the ereport,ffects ofon adding the basis amoxicillin of different at di characteristicsfferent stages of in the the bacteria process growth. of biofilms formation,Bacteria we were studied injected the effects into the of cultureadding chamberamoxicillin and at cultured different for stages 0, 2, of 4, the 6, 8 bacteria h respectively, growth. then 4 mg/BacteriaL amoxicillin were injected was started into tothe be culture added chamber continuously. and cultured According for to0, 2, the 4, bacterial 6, 8 h respectively, growth curves, then the4 mg/L minimal amoxicillin inhibition was concentration started to be added (MIC) continuo of E. coli usly.ATCC According 25922 to amoxicillinto the bacterial is 4 mggrowth/L, which curves, is shownthe minimal in Figure inhibition A2. Therefore, concentration we studied (MIC) the of eE.ff ectscoli ATCC of 4 mg 25922/L amoxicillin to amoxicillin on the is process 4 mg/L, of which biofilm is formation.shown in Figure Amoxicillin A2. Therefore, is a kind ofwe beta-lactam studied the antibiotic, effects of preventing4 mg/L amoxicillin cell wall on from the synthesis process of to biofilm inhibit bacteriaformation. division, Amoxicillin leading is bacteria a kind toof barely beta-lactam grow. Theantibiotic, curves ofpreventing the bacteria cell growth wall from rate aresynthesis shown into Figureinhibit5 bacteria. Adding division, the antibiotic leading at bacteria the beginning to barely (black grow. solid The line), curves bacteria of the growth bacteria rate growth is the lowest.rate are Comparedshown in Figure to the 5. bacterial Adding growth the antibiotic curve withoutat the beginning amoxicillin, (black it is solid obvious line), that bacteria bacterial growth growth rate isis inhibitedthe lowest. by Compared amoxicillin, to whichthe bacterial was added growth at curve the beginning. without amoxicillin, However, theit is inhibition obvious that did bacterial not last duringgrowth the is inhibited whole process. by amoxicillin, The bacterial which growth was curveadded shows at the the beginning. growth rate However, approaches the inhibition to zero in did the firstnot last 3 h. during After 3 the h, bacteria whole process. began to The grow bacterial slowly. growth This is mainly curve shows because the bacteria growth loosely rate approaches adhere to the to glass.zero in Though the first a 3 small h. After amount 3 h, bacteria of bacteria began can to adhere grow theslowly. glass, This viscous is mainly substrates because surrounding bacteria loosely them areadhere rare, to and the the glass. antibiotic Though is easily a small permeated amount to of aff baectcteria bacteria. can Withadhere the the viscous glass, substrates viscous increasingsubstrates andsurrounding biofilm forming, them are antibiotic rare, and is the absorbed antibiotic to the is easily biofilms permeated so that bacteria to affect in bacteria. them can With grow. the After viscous 4 h, undersubstrates the protectionincreasing of and biofilms, biofilm bacteria forming, begin antibiotic to grow. is absorbed to the biofilms so that bacteria in them can grow. After 4 h, under the protection of biofilms, bacteria begin to grow.

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Figure 5. The effects of antibiotic on biofilm formation during the on-chip culture. 4 mg/L amoxicillin Figure 5. The effects of antibiotic on biofilm formation during the on-chip culture. 4 mg/L amoxicillin were added after 0, 1, 3, 5, 7 h incubation with continuous flow at the velocity of 0.5 µL/min. Error bars were added after 0, 1, 3, 5, 7 h incubation with continuous flow at the velocity of 0.5 μL/min. Error are standard deviations. bars are standard deviations. It is very interesting that the result of adding amoxicillin after 1 h incubation is completely It is very interesting that the result of adding amoxicillin after 1 h incubation is completely different from the above description, the bacteria growth rate is larger than all the other cases including different from the above description, the bacteria growth rate is larger than all the other cases with adding amoxicillin and without adding amoxicillin. It illustrates that biofilms are formed in the including with adding amoxicillin and without adding amoxicillin. It illustrates that biofilms are initial stage. After 1 h culture, amoxicillin was added, at this time the biofilms outside of the bacteria formed in the initial stage. After 1 h culture, amoxicillin was added, at this time the biofilms outside are able to resistant to it. According to the previous report, antibiotics can stimulate the bacteria to of the bacteria are able to resistant to it. According to the previous report, antibiotics can stimulate release EPS, which belong to the major construction material of biofilms, leading to membrane fouling the bacteria to release EPS, which belong to the major construction material of biofilms, leading to of bioreactors [31,32]. EPS consists of polysaccharides, proteins and other macromolecules, such as membrane fouling of bioreactors [31,32]. EPS consists of polysaccharides, proteins and other DNA, lipids, which is like a biological glue immobilizing bacteria to attach to and aggregate to the macromolecules, such as DNA, lipids, which is like a biological glue immobilizing bacteria to attach surfaces. Relying on EPS, biofilms possess strong viscoelasticity and adhesive strength [33]. Compared to and aggregate to the surfaces. Relying on EPS, biofilms possess strong viscoelasticity and to 4, 6, 8 h amoxicillin added, bacterial growth rate increases rapidly after adding antibiotics. This is adhesive strength [33]. Compared to 4, 6, 8 h amoxicillin added, bacterial growth rate increases principally because EPS protect bacteria from flowing away. From the growth curves of amoxicillin rapidly after adding antibiotics. This is principally because EPS protect bacteria from flowing away. adding at different periods, it illustrates that the bacterial growth rate increases rapidly with adding From the growth curves of amoxicillin adding at different periods, it illustrates that the bacterial amoxicillin earlier. Therefore, adding antibiotics earlier will shorten the time of loose adhesion. growth rate increases rapidly with adding amoxicillin earlier. Therefore, adding antibiotics earlier will3.5. Eshortenffect of Antibioticthe time of on loose Mixed adhesion. Microorganism by On-Chip Culture

3.5 EffectIn aforementioned of Antibiotic on experiments,Mixed Microorganism single strain by On-Chip bacteria Culture have been cultured on chip and the effects of biofilm formation studied. However, biofilms existing in natural environment are formed by sessile communitiesIn aforementioned of microorganisms. experiments, Therefore, single strain we use bacter bacteriaia have from been sludge cultured of secondary on chip and sedimentation the effects oftank biofilm for on-chip formation culture studied. and biofilm However, observation. biofilms existing in natural environment are formed by sessileThe communities medium for of mixed microorganisms. microorganism Therefore, culture was we pretreated use bacteria piggery from wastewater, sludge of whichsecondary was sedimentationsterilized before tank loading for on-chip into theculture chip. and The biofilm results observation. of 8 h on-chip culture are shown in Figure6. By comparingThe medium curves for mixed between microorganism pure bacteria culture (orange wa lines pretreated in Figure piggery5) and wastewater, mixed microorganism which was sterilized(black line before in Figure loading6), it isinto obvious the chip. that The the growthresults of rate 8 h of on-chip mixed strainsculture increases are shown larger in Figure than single 6. By comparingstrain. For on-chipcurves between culture underpure bacteria continuous (orange flow, line growth in Figure relies 5) on and biofilms, mixed whichmicroorganism can immobilize (black lineand in protect Figure the 6), microorganism.it is obvious that Mixedthe growth microorganism rate of mixed are strains more likelyincreases to form larger biofilms, than single leading strain. to Formicroorganism on-chip culture attach under to the continuous surface rapidly. flow, The growth main reasonrelies on is thatbiofilms, some strainswhich ofcan the immobilize microorganism and protectin the sludge the microorganism. is regulated by Mixed genes tomicroorganism form biofilms ar [34e ].more In addition, likely to cell-cell form biofilms, communication leading can to microorganismpromote bacteria attach to form to biofilms the surface [35]. The rapidly. cell-to-cell The communication main reason mechanismis that some is known strains as quorumof the microorganismsensing (QS), which in the allows sludge bacteria is regulated to communicate by genes byto form direct biofilms cell-to-cell [34]. contact In addition, or extracellular cell-cell communicationmessenger molecules can [promote18]. For example,bacteria quorumto form sensing biofilms in P. [35]. aeruginosa The cancell-to-cell upregulate communication the secretion mechanism is known as quorum sensing (QS), which allows bacteria to communicate by direct cell-to-cell contact or extracellular messenger molecules [18]. For example, quorum sensing in P.

MicromachinesMicromachines 20192019,, 1010,, x 606 FOR PEER REVIEW 9 9 of of 12 12 aeruginosa can upregulate the secretion of EPS. Hence, the growth rate of mixed strains increases of EPS. Hence, the growth rate of mixed strains increases more rapidly than a single strain under the more rapidly than a single strain under the conditions of gene expression and quorum sensing. conditions of gene expression and quorum sensing.

Figure 6. Effect of antibiotic on mixed microorganism for on-chip culture. Mixed strains microorganism Figurefrom the 6. sludgeEffect ofof secondary antibiotic sedimentation on mixed microorga tank werenism cultured for inon-chip pretreated culture. piggery Mixed water, strains and 4 microorganismmg/L amoxicillin from were the added sludge after of 0, 1,secondary 3 h incubation. sedimentation Error bars tank are standardwere cultured deviations. in pretreated piggery water, and 4 mg/L amoxicillin were added after 0, 1, 3 h incubation. Error bars are standard deviations.Similar to the above experiment, mixed microorganisms were loaded, then culture medium with 4 mg/L amoxicillin was added into the culture chamber at 0, 2, 4 h. The total culture time was 8 h, becauseSimilar biofilms to the have above formed experiment, multilayers. mixed As microor we mentioned,ganisms were counting loaded, bacteria then becomesculture medium difficult within multilayers. 4 mg/L amoxicillin In contrast was to added single into strain the ofculture bacteria, chamber it is obvious at 0, 2, 4 that h. The mixed total microorganisms culture time was are 8 h,not because influenced biofilms by thehave antibiotic formed (blackmultilayers. line in As Figure we mentioned,6). This illustrates counting that bacteria some becomes strains in difficult mixed inmicroorganisms multilayers. In arecontrast resistant to single to amoxicillin. strain of bacteria, At the same it is time, obvious the growththat mixed rate ofmicroorganisms mixed microorganisms are not influencedon a chip increasesby the antibiotic rapidly under (black the line influence in Figure of the6). antibiotic.This illustrates As shown that some in Figure strains6, the in growth mixed microorganismscurves are close are after resistant amoxicillin to addedamoxicillin. at 0, 2,At 4 h.the Thissame is becausetime, the the growth colonies rate aggregate of mixed and microorganismsmultilayers bacteria on a leadschip increases to large errorsrapidly in under counting. the influence Therefore, of biofilmsthe antibiotic. of mixed As shown microorganisms in Figure 6,approach the growth maturely curves earlier are close than theafter single amoxicillin stain. added at 0, 2, 4 h. This is because the colonies aggregateIn a naturaland multilayers environment, bacteria microorganisms leads to large are errors existed in counting. in community Therefore, instead biofilms of single of mixed strain. microorganismsConsequently, the approach biofilms maturely formation earlier are influencedthan the single by various stain. factors, such as QS and antibiotics. EspeciallyIn a natural in membrane environment, bioreactors, microorganisms there are complex are existed reasons in forcommuni membranety instead fouling of causedsingle bystrain. EPS Consequently,has and this takes the abiofilms few days formation or even longerare influenced to study by the various influence factors, factors. such This as platformQS and antibiotics. provides a Especiallyrapid method in membrane to observe bioreactors, the biofilms there formation are complex influenced reasons by singlefor membrane or multiple fouling factors. caused by EPS has and this takes a few days or even longer to study the influence factors. This platform provides a rapid4. Conclusions method to observe the biofilms formation influenced by single or multiple factors. We have developed a novel microfluidic system to observe biofilm formation under continuous 4. Conclusions flow. The platform provided a simple and fast way at microscale to study the effects on the bacterial biofilmWe formation,have developed including a novel hydrodynamic microfluidic factor,system antibiotic to observe and biofilm quorum formation sensing. under The continuous chip is very flow.useful The for platform bacterial provided culture, anda simple enables and bacteria fast way to at adhere microscale to the to surfacestudy the and effects form on biofilm. the bacterial In the biofilmcontinuous formation, flow, free-floating including hydrodynamic bacteria were washed factor, away,antibiotic and weakand quorum shear force sensing. could The promote chip is biofilm very usefulformation. for bacterial We also culture, found that and amoxicillin enables bacteria adding to into adhere the cultureto the surface chamber and at diformfferent biofilm. periods In hadthe continuousdifferent eff flow,ects on free-floating the E. coli adhesion bacteria andwere biofilm washed formation. away, and Di ffweakerent shear phenomena force could were observedpromote biofilmwhen using formation. mixed microorganismsWe also found fromthat theamoxicillin sludge of adding the secondary into the sedimentation culture chamber tank. Complicatedat different periodsfactors includinghad different quorum effects sensing on the and E. antibioticscoli adhesion have and to biofilm be considered formation. in this Different case. All phenomena the above wereexperiments observed indicated when using that themixed microfluidic microorganisms platform from is a prospect the sludge tool of to the study secondary the biofilms sedimentation influenced tank.by single Complicated or multiple factors factors including for rapid quorum observation sensing at microscale.and antibiotics The futurehave to potential be considered application in this of case.this systemAll the canabove be valuableexperiments for controlling indicated that biofilm, the microfluidic such as in clinics platform or the is water a prospect treatment tool industry.to study the biofilms influenced by single or multiple factors for rapid observation at microscale. The future

Micromachines 2019, 10, x FOR PEER REVIEW 10 of 12 Micromachines 2019, 10, x FOR PEER REVIEW 10 of 12 potential application of this system can be valuable for controlling biofilm, such as in clinics or the potential application of this system can be valuable for controlling biofilm, such as in clinics or the water treatment industry. water treatment industry. Micromachines 2019, 10, 606 10 of 12 Author Contributions: Conceptualization, K.S. and X.-Y.Z.; methodology, X.-Y.Z.; software, A.A.; validation, K.S.,Author X.-Y.Z. Contributions: and Z.-Y.L.; Conceptualization, formal analysis, K.S.X.-Y.Z.; and X.-Y.Z.;investigation, method X.-Y.Z.;ology, X.-Y.Z.;resources, software, P.-P.X.; A.A.; data validation, curation, X.-Y.Z.;K.S., X.-Y.Z. writing—original and Z.-Y.L.; formaldraft preparation,analysis, X.-Y.Z.; X.-Y.Z investigation,.; writing—review X.-Y.Z.; andresources, editing, P.-P.X.; X.-Y.Z. data and curation, K.S.; AuthorX.-Y.Z.; Contributions:writing—originalConceptualization, draft preparation, K.S. andX.-Y.Z X.-Y.Z.;.; writing—review methodology, X.-Y.Z.;and editing, software, X.-Y.Z. A.A.; validation,and K.S.; K.S.,visualization, X.-Y.Z. and X.-Y.Z..; Z.-Y.L.; supervision, formal analysis, Z.-Y.L.; X.-Y.Z.; project investigation, administration, X.-Y.Z.; K.S. resources, and Z.-Y.L.; P.-P.X.; funding data curation,acquisition, X.-Y.Z.; K.S. writing—originalandvisualization, Z.-Y.L. X.-Y.Z..; draft preparation,supervision, X.-Y.Z.;Z.-Y.L.; writing—review project administration, and editing, K.S. X.-Y.Z.and Z.-Y.L.; and K.S.; funding visualization, acquisition, X.-Y.Z..; K.S. supervision,and Z.-Y.L. Z.-Y.L.; project administration, K.S. and Z.-Y.L.; funding acquisition, K.S. and Z.-Y.L. Funding: This research was funded by the National Natural Science Foundation of China (grant number Funding:61674046);Funding: ThisState researchKey Laboratory was funded of Urban by theWater NationalNational Resource NaturalNatu andral Environment ScienceScience FoundationFoundation (Harbin ofInstitute ChinaChina of (grant(grant Technology), numbernumber 61674046); State Key Laboratory of Urban Water Resource and Environment (Harbin Institute of Technology), grant61674046); number State 2017TS04. Key Laboratory of Urban Water Resource and Environment (Harbin Institute of Technology), grantgrant numbernumber 2017TS04.2017TS04. Acknowledgments: TheThe authors authors acknowledge Jian Jianzhengzheng Li (School of Environment,Environment, Harbin Institute of Technology)Acknowledgments: to provide provide The the theauthors sludge sludge acknowledge of of secondary secondary Jiansedime sedimentationzhengntation Li (School tank tank and of and pretreatedEnvironm pretreated ent,piggery piggeryHarbin wastewater wastewaterInstitute forof forexperiments.Technology) experiments. to provide the sludge of secondary sedimentation tank and pretreated piggery wastewater for experiments. ConﬂictsConflicts ofof Interest:Interest: The authors declare nono conﬂictconflict ofof interest.interest. Conflicts of Interest: The authors declare no conflict of interest. Appendix A Appendix A Appendix A

Figure A1. Image processing: (A) original image ;(B) the RGB image was transformed into grey FigureFigure A1.A1.Image Image processing: processing: (A ()A original) original image; image (B );( theB) RGBthe RGB image image was transformed was transformed into grey into format grey format image; (C) image after subtracting background; (D) binary image. The scale bar is 40 μm. image;format (image;C) image (C) after image subtracting after subtracting background; background; (D) binary (D) image. binary Theimage. scale The bar scale is 40 barµm. is 40 μm.

Figure A2. The growth curve of bacteria in 96 well-plate. E. coli were inhibited by 1, 2, 3, 4, 5 Figure A2. The growth curve of bacteria in 96 well-plate. E. coli were inhibited by 1, 2, 3, 4, 5 mg/L amoxicillin. Figuremg/L A2. amoxicillin. The growth curve of bacteria in 96 well-plate. E. coli were inhibited by 1, 2, 3, 4, 5 mg/L amoxicillin.

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