development of an automation system to evaluate the three-dimensional oxygen distribution in...

Sensors and Actuators B 113 (2006) 47–54

Development of an automation system to evaluate the three-dimensionaloxygen distribution in wastewater biofilms using microsensors

Carlos de la Rosa∗, Tong YuDepartment of Civil and Environmental Engineering, University of Alberta, 3-133 Natural Resources Engineering Facility,

Edmonton, Alberta, Canada T6G 2W2

Received 29 April 2004; received in revised form 25 January 2005; accepted 7 February 2005Available online 3 March 2005

Abstract

In this paper the authors describe the development of an automation system applicable to environmental biofilm studies. The automa-tion system controls a combined oxygen microsensor to measure the three-dimensional dissolved oxygen distribution in a wastewaterbiofilm sample. The biofilm is sampled from a rotating biological contactor in a municipal wastewater treatment plant. The automationsystem consists of a data acquisition system, a motion control system, and a computer program. The combined oxygen microsensor con-s solution. Thea positioningo ro-fi dissolvedo to be toolst astewatert©

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ists of a sensing electrode, a reference electrode, a guard cathode, an oxygen permeable membrane, and an electrolyteutomation system allows the acquisition and storage of data from 4000 measurements from the microsensor and the precisef the microsensor in order to measure 100 dissolved oxygen profiles in a 1000�m× 1000�m biofilm area. The three-dimensional ple shows that the dissolved oxygen concentration in the biofilm sample is highly heterogeneous and it revealed “pockets” ofxygen in deep sections of the biofilm sample. The automation system and the combined oxygen microsensor were proven

hat improve the quantity and quality of experimental results needed to understand important functions in biofilms used in wreatment.

2005 Elsevier B.V. All rights reserved.

eywords: Automation; Microsensor; Microelectrode; Oxygen; Three-dimensional; Biofilm; Wastewater; Environmental

. Introduction

Biofilms are widely used in the treatment of industrial andunicipal wastewater. A biofilm can be defined as a group of

iving cells, dead cells and cell debris in a matrix of extracel-ular polymeric substances attached to a surface[2]. Under-tanding the complex operation of biofilms is essential for theptimization of biofilm reactor design and operation. Differ-nt microsensors have been used as powerful tools to evalu-te different parameters, such as oxygen, pH, redox potential,mmonium and sulfide in biofilms[1,3,6,12,14,16,17].

One-dimensional dissolved oxygen profiles measured us-ng oxygen microsensors have always shown that oxygen is

∗ Corresponding author. Present address: Electrical and Computer Engi-eering, University of Calgary, 2500 University Drive NW, Calgary AB,anada T2N 1N4. Tel.: +1 403 2204335; fax: +1 403 2826855.

E-mail address: [email protected] (C. de la Rosa).

depleted at about 200–800�m depth in the biofilms[1,6,16].In order to evaluate the biofilm heterogeneity in terms of ogen, it is necessary to map the three-dimensional dissoxygen distribution in biofilms. This can be achieved by msuring many dissolved oxygen profiles in a specific biosection. Due to the heterogeneous nature of biofilmsthree-dimensional dissolved oxygen distribution could sfunctional and structural characteristics that might not beserved with the typical one-dimensional dissolved oxyprofiles. The data generated can be incorporated in bimodels that evaluate the importance of the three-dimensheterogeneity of oxygen in biofilms[5,10,11,13].

The amount of data needed to map the three-dimensdissolved oxygen distribution in biofilms using oxygencrosensors requires the development of an automation sthat allow precise microsensor positioning and the recorof large quantities of data.

925-4005/$ – see front matter © 2005 Elsevier B.V. All rights reserved.oi:10.1016/j.snb.2005.02.025

48 C. de la Rosa, T. Yu / Sensors and Actuators B 113 (2006) 47–54

Fig. 1. Components and connections of the automation system.

2. Experimental

2.1. Automation system

The automation system consisted of three parts: a dataacquisition system, a motion control system, and a computerprogram. InFig. 1, a schematic diagram showing all the com-ponents and connections is presented. The data acquisitionsystem is the group of components used to collect, computeand store the data gathered from the oxygen microsensor.The components of this system are the combined oxygen mi-crosensor, a picoammeter, and an analog-to-digital converter.The motion control system is the group of components usedto accurately position the microsensor at any location in abiofilm sample. The main components of this system are amotorized two-dimensional stage and a motorized microma-nipulator. The software is a computer program developed inNational Instruments’ LabVIEW® and used to automaticallysynchronize the data acquisition system and the motion con-trol system through a computer.

For the data acquisition system, as illustrated inFig. 1,the oxygen microsensor was connected to a picoammeter(Unisense, Denmark, Model No. PA2000), the picoammeterwas connected to an external analog-to-digital converter card(Pico Technology Ltd., Cambridgeshire, UK; Model ADC-101), and the analog-to-digital converter card was connected

to a computer (Dell Model Inspiron 5000) using serial com-munication (LPT1 port).

In order to record data from the oxygen microsensor intothe computer, the analog signal produced by the oxygen mi-crosensor was converted to a digital signal. This signal pro-cess followed a series of steps. The oxygen microsensor con-verted a chemical signal, proportional to the dissolved oxygenconcentration in the biofilm sample, into an electrical signal(current). That electrical signal was then transmitted to thepicoammeter. Due to the small microsensor tip, the resultingelectrical signal is very small (in the order of picoamperes)and needs to be amplified. The picoammeter amplifies the cur-rent signal and converts it to voltage. Then, the output voltagesignal from the picoammeter was sent to the analog-to-digitalconverter to be converted to a digital signal. The analog-to-digital converter constantly receives an input voltage signalfrom the picoammeter and generates a digital output codeproportional to the input voltage signal. Finally, the digitalsignal from the analog-to-digital converter was sent to thecomputer where it can be recorded by the computer programdeveloped in LabWIEW®. The analog-to-digital converterwas a 12-bit converter.

The motion control system consisted of three actuators thatallowed the three-dimensional positioning of the microsen-sor. Two actuators were part of a two-dimensional stage forhorizontal control (Phytron Inc., Waltham, MA, USA; Model

C. de la Rosa, T. Yu / Sensors and Actuators B 113 (2006) 47–54 49

MT-65) and one actuator was part of a motorized microma-nipulator for vertical control (Unisense, Denmark; Model No.MM33M). The components and connections of the motioncontrol system are shown inFig. 1. The two-dimensionalstage consisted of two fixed one-dimensional stages withstepper motors that allow the linear movement of two bases;the motors have the capability of moving the bases in twohorizontal axes (X andY) in a range of up to 50 mm with anaccuracy of±8�m, a bi-directional repeatability of±0.2�mand a resolution of 0.3�m. The motorized micromanipulatoris a micromanipulator with a stepper motor. The motor canachieve vertical movements in a 100 mm range with a max-imum resolution of up to 0.1�m. The stepper motors of thetwo-dimensional stage and the motorized micromanipulatorhave micro-stepping technology that can produce movementsin the order of micrometers.

The motors of the two-dimensional stage and the motor-ized micromanipulator needed to be controlled by the com-puter program. Therefore, there was the need for controllersthat can establish communication between the computer andthe actuators. The computer program sends movement com-mands in the form of digital signal to the controllers. Thecontrollers interpret this signal and send an analog (elec-trical) signal to the motors, which finally move. The two-dimensional stage was connected to a two-axes microstep-controller (Phytron Inc., Waltham, MA, USA; Model SMCb erialp ma-n tru-m llerw f thec

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[8], which is based on the design by Rebsvech[15] withsome modifications.Fig. 2 shows a schematic diagram ofthe combined oxygen microsensor. The device consisted ofa sensing electrode (the working cathode) made of platinumand plated with gold; a silver/silver chloride (Ag/AgCl) ref-erence electrode (the anode); a guard cathode made of silver;an oxygen permeable membrane; and an electrolyte solution.The fabrication details are described previously[7,8]. The tipdiameter of the combined oxygen microsensor was 15�m.

2.3. Experiment

The biofilm sample for this study was taken from a mu-nicipal wastewater treatment plant situated in the Town ofDevon in Alberta, Canada. The treatment plant uses a Rotat-ing Biological Contactor (RBC) system. The treatment plantuses the RBC system as a secondary treatment to removecarbonaceous BOD. The RBC system is made of a series ofclosely spaced disks partially immersed in a tank throughwhich wastewater flows. Its biological principle is based onthe development of thick and complex biofilms on the disksurfaces[9].

The biofilm sample was grown on a High DensityPolyethylene (HDPE) coupon that was fixed on one ofthe RBC disks in the wastewater treatment plant. The2 cm× 5 cm coupon was fixed in the RBC disk for 6 weeksu ss.

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asic 7160-9-010), which was connected to one of the sorts (RS-232) of the computer. The motorized microipulator was also connected to a controller (Oriel Insents, Stratford, CT, USA; Model 18011). This controas also connected to one of the serial ports (RS-232) oomputer.

The goal of the computer program was to synchronizeunctionalities of the data acquisition system and the moontrol system. The program needed to have the capabiontrol the components of the data acquisition system anotion control system in a way that three-dimensional ments and data acquisition tasks previously program

ould be accomplished. A computer program with this cility was developed in National Instruments’ LabVIEW®.he computer program development for this study consf two main parts. The first part was the construction

ront panel or user interface. This part contains all therols and indicators needed to automatically synchronizutomation system. The front panel construction was a

o provide the user with a logical and easy way to preseonduct different microsensor movement and data acqion tasks with the automation system. The second parhe creation of the block diagram, which is the source cf the program. This part contains the algorithm used tohronize the components of the automation system.

.2. Combined oxygen microsensor

The combined oxygen microsensor used in this studyabricated following the procedure reported by Lu and

ntil there was a biofilm growth of about 1.5 mm in thickneBefore the experiment was started all the necessary e

ent was set up properly. The components of the aution system were connected as shown inFig. 1. A measuringhamber used to place the biofilm sample was fixed owo-dimensional stage. The measuring chamber has annd outlet tubing used to drain and fill the measuring cher with wastewater. A horizontal microscope (Carl Zeena, Germany; Model: Stemi SV11) was placed close teasuring chamber. With this microscope, the microse

ip and the biofilm surface can be seen. In order to runxperiment, the coupon with the biofilm sample was takerom the RBC disk. A volume of 4 l of wastewater fromastewater treatment plant were collected and taken t

aboratory together with the sample. The biofilm sampleransported in a cooler with wet sponges to keep the humigh and the temperature constant. This measuremen

he biofilm sample from desiccation during transportathe transportation time from the wastewater treatment

o the laboratory was about 45 min. Once in the laborahe biofilm sample was prepared immediately for analhe sample preparation and the equipment set up tookne more hour. Thus, it took about 1 h 45 min to trans

he biofilm sample from the RBC to the laboratory andnalysis.

During the months from September to October 2002, whe experiments were conducted, the plant received aicipal wastewater influent with an average of 230 mg/OD, and produced an effluent with an average of 4.8 mgOD. The average oxygen concentration of the influent


Fig. 2. Schematic diagram of the combined oxygen microsensor.

7.3 mg/L. The average removal percentage for BOD duringthese months was 97.7%.

In the laboratory, the coupon with the biofilm was cut in apiece of 1 cm× 1 cm and the piece was placed on the measur-ing chamber using specially designed clips. The measuringchamber was filled with wastewater. The oxygen microsensorwas fastened on the motorized micromanipulator and its tipwas set manually at about 1000�m above the biofilm surfaceby using manual gauge of the motorized micromanipulator.

The computer program of the automation system waspreset to measure 100 profiles in a 10× 10 array. Thedistance required between each profile was 100�m. Inorder to execute each profile measurement, the motorizedmicromanipulator moved downward a total of 1600�m,stopping every 80�m to collect two readings from theanalog-to-digital converter output. This means that themotorized micromanipulator had to move twenty “steps”downward (80�m each step) to cover the 1600�m distance


required (in this computer program a “step” is a movementof a preset distance). When the twenty downward stepswere completed, then the micromanipulator moved upward1600�m to its original position in one single step. Then, thetwo-dimensional stage moved 100�m to the next position,where the new profile measurement was executed. Eachprofile measurement took 8 min to complete, and each steptook 0.4 min to complete. Since the system was automated,all the measurements were taken at the same time after thestart of each profile measurement. When the program wasrun, the automation system started the profile measurementsin series of rows until the preset array of profile measure-ments (10× 10) was completed. During the experiment, thebiofilm sample was alternately exposed to air and wastewaterby draining and filling the measuring chamber with thewastewater brought from the treatment plant. Before a newprofile measurement was started, the measuring chamberwas drained, exposing the biofilm to air, and filled againwith fresh wastewater. The process of draining and fillingthe chamber with wastewater took 1 min to complete. Oncethe chamber was filled with wastewater, the new profilemeasurement was started. The chamber was kept filledduring the profile measurement. This means that each profilemeasurement was taken by the automation system while thebiofilm sample was being exposed to wastewater. The reasonfor the alternate exposure of the biofilm sample to wastewatera RBCs uslyu osest icals ofilem usingt them ensort them vedd n oft nsorw ewp dureh

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position above the biofilm surface and inside the biofilm.Due to special requirements for biofilm studies, it was im-portant to use the right combination of actuators to obtainthe desired microsensor control. Two special factors wereconsidered for this type of application. The first factor wasthe size of the microsensor tip and the biofilm sample. Themicrosensor tip was 15�m in diameter and the area ofthe biofilm sample studied was 1000�m× 1000�m. Theactuators used under these small dimensions require elec-tronic micro-stepping technology. The advantage of micro-stepping is multiplication of the numbers of steps per rev-olution in the motor, thereby increasing the resolution of astepper motor system. The two-dimensional stage and themotorized micromanipulator used in this study were actua-tors with micro-stepping technology that were implementedto develop an automation system with the capability to per-form linear movements in the order of micrometers. Thesecond factor considered was space. Biofilm studies requirecertain experimental conditions. For instance, the biofilmsample had to be immersed in a measuring chamber filledwith wastewater in order to keep the biofilm characteristicsas similar as possible to the ones in the original wastew-ater treatment plant. The small dimensions of the experi-ment set up require a horizontal microscope to be placedclose to the measuring chamber. All this equipment had tobe close to the biofilm sample. Therefore, the actuators re-q meetc de-s ace,t sys-t cane h theb mi-c axis.U ree-d houtc nt re-q ina-t ree-d wasa

quisi-t rop-e thed exe-c wello ations ctiona en-s

om-b ree-d aterb ands thec ng of

nd air was to simulate the real conditions present in anystem. The disks of an RBC system rotate continuonder normal operation. The rotation alternately exp

he biofilm to air and wastewater constantly. The physurface of the biofilm was determined before each preasurement was taken. This procedure was done by

he microscope, the motorized micromanipulator andicrosensor. Using the microscope (to see the micros

ip and the biofilm surface) and the manual gauge onotorized micromanipulator, the microsensor was moown to measure the distance between the initial positio

he microsensor and the biofilm surface, then the microseas moved back to its original position to start the nrofile measurement. The detailed experimental proceas been reported before[4].

. Results and discussion

The automation system developed in this study maossible to map the three-dimensional dissolved oxygen

ribution in wastewater biofilms using combined oxygenrosensors. The application of automation and microseechnologies improved the quality and quantity of datauired to have a better understanding of important func

n environmental biofilms.The application of actuators made the automated co

f the microsensor movement and positioning possibleng the two-dimensional stage and the motorized microipulator, it was possible to place the microsensor at

uired for the automation system development had toertain physical characteristics. In order to achieve theired microsensor positioning without compromising sphe three-dimensional movement of the motion controlem was divided in two parts. The two-dimensional stagexecute movements of the fixed measuring chamber witiofilm sample in two horizontal axes, and a motorizedromanipulator can move the microsensor in a verticalsing this layout of the motion control system, the thimensional movement of the system was achieved witompromising the space necessary for other equipmeuired in biofilm studies. Therefore, the type and comb

ion of actuators used in this experiment to study the thimensional oxygen distribution in wastewater biofilmsdequate.

The computer program used to automate the data acion system and the motion control system functioned prly during the experiments, controlling the tasks thatata acquisition and the motion control systems had toute. The controls and indicators of the program wererganized, so that the user can easily program the automystem to execute complicated tasks involving data collend positioning of the oxygen microsensor in three dimions.

The combination of the automation system and the cined oxygen microsensor permitted to map the thimensional dissolved oxygen distribution in wastewiofilms. The automation system allowed the acquisitiontorage of data from a total of 4000 measurements fromombined oxygen microsensor and the precise positioni


Fig. 3. Calibration curve of the oxygen microsensor used for the measure-ment of the biofilm sample.

the microsensor in order to measure the 100 dissolved oxygenprofiles in a 1000�m× 1000�m biofilm area.

The calibration curve of the oxygen microsensor used tointerpolate the dissolved oxygen readings obtained from thebiofilm sample is shown inFig. 3. The three-point calibration

curve (0, 10.5 and 21% oxygen) indicates that the fabricationprocedure of the combined oxygen microsensor was effec-tive. The linear regression of this curve producedR2 = 1. Theoxygen microsensor was calibrated before the experimentwas run and it was calibrated again after the experiment wasended to check for any damage or fouling at the microsen-sor tip. The calibration curve at the end of the experimentwas normal and no damage to the tip was found. In previousstudies involving the fabrication of separate and combinedoxygen microsensors, the authors have stated that excellentcalibration curves are relatively easy to obtain for most oxy-gen microsensors and two-point calibration curves are suffi-cient for normal sample measurements[7,17].

The tip diameter of the microsensor used for the exper-iment was 15�m. The microsensor completed 100 profilemeasurements, and a total of 4000 dissolved oxygen read-ings were taken from the biofilm sample. The microsensorworked properly during the whole experiment. This showedthat the combined oxygen microsensor is an affective tool forstudies where large numbers of dissolved oxygen measure-

F
ig. 4. The dissolved oxygen concentrations at (a) 680�m above the biofilm surf ace, (b) at the biofilm surface, and (c) 680�m below the biofilm surface.


ments are needed, such as determining the three-dimensionaldissolved oxygen distribution in wastewater biofilms.

The use of the automation system and the combined oxy-gen microsensors provided information about the dissolvedoxygen distribution above the biofilm surface, at the biofilmsurface and inside the biofilm.Fig. 4 shows the dissolvedoxygen concentration at 680�m above the biofilm surface,at the biofilm surface and at 680�m below the biofilm sur-face. The three-dimensional profile shows that the dissolvedoxygen concentration in the biofilm sample is highly hetero-geneous. The three-dimensional profile also shows that thedissolved oxygen concentration decreases with depth insidethe biofilm. The three-dimensional measurements revealed“pockets” of dissolved oxygen in deep sections of the biofilmsample, where the dissolved oxygen concentration was ashigh as 1 mg/L. At 680�m above the biofilm surface 94% ofthe measured dissolved oxygen concentration was in a rangefrom 4.0 to 6.0 mg/L. At the biofilm surface 60% of the mea-sured dissolved oxygen concentration was in a range from 0.4to 0.8 mg/L. And at 680�m below the biofilm surface 94% ofthe measured dissolved oxygen concentration was 0.1 mg/L.

4. Conclusions

This study showed the development of an automation sys-t au-t sorsw uan-t por-t ent.B bed

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Acknowledgments

Our special thanks go to Mr. Andy Bebbington and Mr.Jim Hepler at the Water and Wastewater Treatment Plant inthe Town of Devon in Alberta, Canada, for their cooperationin growing the biofilm samples. We gratefully acknowledgeMs. Debra Long, Mr. Richard Lu and Mr. Omar Lopez of theUniversity of Alberta for their assistance in the completionof the experimental work. This project was partially fundedby the Natural Sciences and Engineering Research Council(NSERC) of Canada and a scholarship from the NationalCouncil of Science and Technology (CONACyT) of Mexico.

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[ ents999)

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[ Lim-

[ diedol. 37

em applicable to environmental biofilm studies. Theomation system and the combined oxygen microsenere proven to be tools that improve the quality and q

ity of experimental results needed to understand imant properties in biofilms used in wastewater treatmased on the results, the following conclusions canrawn:

The automation system developed in this study maossible to map the three-dimensional dissolved oxygen

ribution in wastewater biofilms using combined oxygenrosensors. The data acquisition system allowed the acion and storage of large numbers of data from the combxygen microsensor. The motion control system allowedrecise movement and positioning of the microsensor iiofilm sample. The computer program effectively contro

he data acquisition system and the motion control systexecute tasks involving data collection and microsensoitioning in three dimensions.

The fabrication procedure of the combined oxygenrosensors was successful. This type of oxygen microsan be used in biofilm studies where large numbers ofinuous dissolved oxygen readings are needed.

The three-dimensional dissolved oxygen distributionle obtained with the application of the automation sem and the combined oxygen microsensor showed thaissolved oxygen distribution in the biofilm sample wighly heterogeneous. The three-dimensional measureevealed “pockets” of dissolved oxygen in deep sectionhe biofilm sample, where the dissolved oxygen conceion was as high as 1 mg/L.

Three-dimensional numerical study on the correlation of spstructure, hydrodynamic conditions, and mass transfer and cosion in biofilms, Chem. Eng. Sci. 55 (24) (2000) 6209–6222.

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[17] T. Yu, Stratification of microbial processes and redox potentialchanges in biofilms., PhD Dissertation., University of Cincinnati,Cincinnati, OH, USA, 2000, p. 166.

Biographies

Carlos de la Rosa received his BSc in Biology from the National Au-tonomous University of Mexico (UNAM), and his MS in Environmental

Science from the University of Alberta in Canada. His research interestsare the research and development of biosensors.

Tong Yu is an associate professor of Environmental Engineering at theUniversity of Alberta in Canada. He received his BEng and MEngfrom Tsinghua University in China and his PhD from Universityof Cincinnati in USA. His research interests are environmental pro-cesses involving biofilms and environmental applications of microsensortechnology.

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