Environ. Sci. Technol. 2005, 39, 5196-5202
Three-Dimensional Mapping of Oxygen Distribution in Wastewater Biofilms Using an Automation System and Microelectrodes CARLOS DE LA ROSA* AND TONG YU Department of Civil and Environmental Engineering, University of Alberta, 3-133 Natural Resources Engineering Facility, Edmonton, Alberta, T6G 2W2 Canada
The three-dimensional oxygen distribution in wastewater biofilms was evaluated using combined oxygen microelectrodes and an automation system. The biofilms were sampled from rotating biological contactors treating domestic wastewater. The samples studied were mature biofilms with a thickness from 630 to 1600 µm. It was demonstrated that the dissolved oxygen concentration could be depleted at the biofilm surface. The heterogeneity of the dissolved oxygen distribution was high in sections further away from the biofilm surface in the water layer. The study showed that the concentration and level of heterogeneity of dissolved oxygen inside the biofilms decreased with depth, forming stratification. The oxygen concentration in biofilms changed generally from a high degree of heterogeneity near the biofilm surface to a low degree of heterogeneity in deep sections of biofilms, indicating a cell-clusterlike structure near the surface and more compact base layer close to the substratum. The three-dimensional oxygen distribution maps revealed pockets of dissolved oxygen in deep sections of biofilms. The dissolved oxygen concentrations of these pockets in the biofilm samples ranged from 0.4 to 1.0 mg/L at 760 µm depth. The threedimensional oxygen distribution maps produced relevant knowledge of functional and structural characteristics of biofilms used for the treatment of wastewater.
Introduction The purpose of this study was to evaluate the threedimensional oxygen distribution in wastewater biofilms using combined oxygen microelectrodes and an automation system. Biofilm systems have become an important process for the treatment of industrial and municipal wastewater. The evaluation of the oxygen distribution in wastewater biofilms will produce a better understanding of important functional and structural characteristics of biofilms. The generation of this knowledge is important for the optimization of biofilm reactor design and operation. To study the oxygen distribution in biofilms, it is important to carry out measurements inside biofilm samples grown in municipal wastewater treatment plants that use biofilm systems. A reliable analytical tool available to do this type of measurement is the combined oxygen microelectrode. The application of oxygen microelectrodes has played an important role in the evaluation of biofilm systems (1, 2). * Corresponding author phone: (403)220-4335; fax: (403)282-6855; e-mail:
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FIGURE 1. Schematic diagram of the combined oxygen microelectrode. The main advantage of the combined oxygen microelectrode is that it is less subject to electromagnetic interference and that it has the potential to be used for field measurements (3). One-dimensional oxygen profiles in biofilms measured using oxygen microelectrodes have shown that oxygen is depleted at about 200-800 µm depth (1, 4, 5). However, biofilms are often highly heterogeneous in nature, and a onedimensional oxygen profile provides limited information about biofilm structure. To have a better understanding of oxygen penetration and heterogeneity in biofilms, it is necessary to map oxygen distribution in biofilms in three dimensions. The experimental data generated in threedimensional mapping can be beneficial to biofilm models that evaluate the importance of three-dimensional heterogeneity of oxygen distribution. Some new biofilm models provide sophisticated two- and three-dimensional descriptions of biofilms and incorporate not only mass transport but also hydrodynamics and population dynamics (6-9). An automation system was developed for the threedimensional mapping of oxygen distribution in biofilms using microelectrodes because the experiments require precise positioning of the microelectrode as well as acquisition of a large amount of data. 10.1021/es0484449 CCC: $30.25
2005 American Chemical Society Published on Web 06/08/2005
FIGURE 2. Components and connections of the automation system.
Materials and Methods Oxygen Microelectrode. The combined oxygen microelectrodes used in this study were constructed following the procedure reported by Lu and Yu (10), which is based on the design by Revsbech (11) with some modifications. Figure 1 is a schematic diagram of the microelectrode with its components. The device consisted of a sensing electrode (the working cathode) made of platinum and plated with gold; a silver/silver chloride (Ag/AgCl) reference electrode (the anode); a guard cathode made of silver; an oxygen permeable membrane; and an electrolyte solution. The tip diameters of the combined oxygen microelectrodes were about 15-30 µm. Each oxygen microelectrode was calibrated before the experiment was run, and it was calibrated again after the experiment was ended to check for the stability of the microelectrode. The calibration curve at the end of the experiment was normal, and no damage to the microelectrode tip was found. The fabrication and calibration details are described previously (10, 12). Automation System. The automation system consisted of three parts. The first part was a data acquisition system, which was the group of components used to collect, compute, and store the data gathered from the oxygen microelectrode. The second part was a motion control system, which was the group of components used to accurately position the microelectrode at any location in a biofilm sample. The third part was a computer program, developed in National Instruments’ LabVIEW, that automatically synchronized the data acquisition system and the motion control system. The diagram in Figure 2 shows the components of the automation system. The data acquisition system consisted of a picoammeter (Unisense, Denmark; model PA2000), which received the signal from the oxygen microelectrode. The picoammeter amplified the signal, converted it to voltage, and sent it to the analog-to-digital converter (Pico Technology Ltd., Cambridgeshire, UK; model ADC-101). The analog-to-digital converter converted the voltage signal into a digital signal that was then sent to the computer. The motion control system consisted of a motorized two-dimensional stage (Phytron Inc. Waltham, MA; model MT-65) and a motorized
micromanipulator (Unisense, Denmark; model MM33M), allowing the positioning of the oxygen microelectrode in three axes. The two-dimensional stage was formed by two fixed one-dimensional bases, each with a stepper motor that drove the linear movement of each base and provided the capability of moving the bases in two horizontal axes. The motorized micromanipulator was a micromanipulator with a stepper motor that allows the movement along a vertical axis. Both the two-dimensional stage and the motorized micromanipulator were connected to two separate controllers, which were in turn connected to the computer. The computer program had the capability of displaying and storing the data gathered from the data acquisition system and controlling the movements of the motion control system. The program was designed in LabVIEW, with a user-friendly interface so that the user can easily program the software and control the entire automation system to execute complicated tasks involving data collection and positioning of the oxygen microelectrode in three dimensions. Experiment. The biofilm samples for this study were taken from the municipal wastewater treatment plant situated in the Town of Devon in Alberta, Canada. The treatment plant uses a rotating biological contactor (RBC) system with four stages. The biofilm samples were grown for 6 weeks on 2 cm × 5 cm coupons that were fixed on the RBC disks. The coupons were made of high-density polyethylene (HDPE). Before the experiment was started, all the necessary equipment was set up properly. The components of the automation system were connected as shown in Figure 2. A measuring chamber used to place the biofilm sample was clamped on the two-dimensional stage. The measuring chamber had inlet and outlet tubing used to drain and fill the measuring chamber with wastewater. A horizontal microscope (Carl Zeiss, Jena, Germany; model Stemi SV11) was placed close to the measuring chamber. With this microscope, the microelectrode tip and the biofilm surface could be clearly observed. A container filled with 2 L of filtered wastewater was placed in a stand above the level of the measuring chamber. The wastewater in the container was constantly aerated. The container was connected to the inlet VOL. 39, NO. 14, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 3. Physical structure (a) and dissolved oxygen concentration (b) at the biofilm surface. tube of the measuring chamber. The purpose of the container was to supply the measuring chamber with wastewater during the experiment. The container was placed above the level of the measuring chamber to generate a water flow from the container through the measuring chamber driven by gravity. To run the experiment, one coupon was taken off from an RBC disk and 6 L of wastewater from the wastewater treatment plant was collected at the same time. Both coupon and wastewater were then taken to the laboratory. The biofilm sample was transported in a cooler with wet sponges to keep the temperature constant and the humidity high enough to prevent desiccation of the sample during transport. The biofilm sample was prepared and analyzed immediately upon reaching the laboratory. In the laboratory, the coupon was taken out of the cooler using tweezers, and the surface of the coupon that touched the sponges was cleaned with an absorbent paper. A 1 cm × 1 cm piece of the coupon covered with the biofilm sample was cut and mounted in a measuring chamber using doublesided tape and specially designed clips. Once the sample was mounted, the measurement chamber was filled with filtered wastewater from the treatment plant. The oxygen microelectrode was fastened on the motorized micromanipulator, and its tip was set manually at about 1000 µm above the biofilm surface. This position of the microelectrode tip was called the initial position. 5198
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The computer program of the automation system was pre-set to measure 100 profiles in a 10 × 10 array. The distance between each profile was 100 µm. To execute one of the 100 profile measurements, the motorized micromanipulator was programmed to move down a total of 1600 µm, stopping every 80 µm to collect two readings from the analog-to-digital converter output. This means that the motorized micromanipulator had to move 20 steps down (80 µm each step) to cover the 1600 µm (in the computer program, a step is a movement of a pre-set distance). When the 20 downward steps were completed, the micromanipulator was programmed to move up 1600 µm to its original position in one single step. Then, the two-dimensional stage was moved 100 µm to the next position, where a new profile measurement was executed. Each profile measurement took 8 min to complete, and each step took 0.4 min to complete. Since the system was automated, all the measurements were taken at the same time after the start of each profile measurement. When the program was run, the automation system started the profile measurements in series of rows until the pre-set array of profile measurements (10 × 10) was completed. During the experiment, the biofilm sample was alternately exposed to air and wastewater by draining and filling the measuring chamber with the wastewater. Before a new profile measurement was automatically started, the measuring chamber was drained, exposing the biofilm to air, and filled
FIGURE 4. Dissolved oxygen concentrations at different locations above the biofilm surface. The locations are (a) 520 µm above the biofilm surface, (b) 280 µm above the biofilm surface, and (c) 40 µm above the biofilm surface. again with new wastewater. The process of draining and filling the chamber with wastewater took 1 min to complete. Once the chamber was filled with wastewater, the new profile measurement was started. The chamber was kept filled during the profile measurement. This means that each profile measurement was taken by the automation system while the biofilm sample was being exposed to wastewater. The reason for the alternate exposure of the biofilm sample to wastewater and air was to simulate the real conditions present in a RBC system. The disks of a RBC system rotate continuously under normal operation, exposing the biofilm to wastewater and air alternately. The physical surface of the biofilm was determined before each profile measurement was taken. This procedure was done by using the microscope, the motorized micromanipulator, and the microelectrode. Using the microscope (to identify the microelectrode tip and the biofilm surface) and the manual gauge on the motorized micromanipulator, the microelectrode was moved down to measure the distance between the initial position of the microelectrode and the biofilm surface, and then the
microelectrode was moved back to its original position to let the automation system begin the new profile measurement automatically. The detailed experimental procedure has been reported before (13, 14). The biofilm thickness was also measured at the end of the experiment in a similar manner. The data were processed using MS Excel, and the maps were plotted using LabVIEW.
Results and Discussion This section describes and analyzes the physical structure of the biofilm surface, the dissolved oxygen concentration at the biofilm surface, and the dissolved oxygen concentration both above the biofilm in the water layer and inside the biofilm, using the physical biofilm surface as a reference. Biofilm Surface Description. This study demonstrated that the biofilm surface is physically heterogeneous and that the dissolved oxygen concentration can be depleted at the biofilm surface. Figure 3 shows the physical structure of the biofilm surface and the dissolved oxygen concentration at VOL. 39, NO. 14, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 5. Dissolved oxygen concentrations at different depths below the biofilm surface. The depths are (a) 40 µm, (b) 280 µm, and (c) 760 µm. the biofilm surface. In Figure 3a, it can be observed that the surface of the biofilm was heterogeneous. The biofilm thickness was from 1300 µm in the right part of the graph to 1590 µm in some areas in the left part of the graph. The heterogeneity of the biofilm surface is an important characteristic of biofilms because the thickness and roughness of the biofilms can affect significantly the distribution of dissolved oxygen inside the biofilms. It has been reported that an increase in surface roughness appeared to decrease the thickness of the boundary layer; therefore, the rougher the biofilm, the less external mass transfer resistance (15). On the basis of the thickness of the biofilm sample, it can be stated that this sample was a mature biofilm. Many wastewater biofilm systems operate with a biofilm thickness from 500 to 2000 µm (16). The dissolved oxygen concentration at the biofilm surface, shown in Figure 3b, was in the range of 0-3.8 mg/L. As seen in this graph, the dissolved oxygen concentration can reach depletion on the biofilm surface. The dissolved oxygen depletion at the biofilm surface could be due to the metabolic 5200
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activity inside the biofilms. Oxygen first diffuses through the boundary layer and then into the biofilm. If the metabolic activity of microorganisms inside the biofilms is high, the microorganisms will consume the oxygen available before it can diffuse to the biofilm surface. In this study, dissolved oxygen was not present at some spots on the biofilm surface, indicating high metabolic activity inside those portions of the biofilm, consuming the dissolved oxygen available in the water layer immediately above those portions. Dissolved Oxygen Distribution Above the Biofilm Surface. The three-dimensional profiles of the dissolved oxygen concentration above the biofilm surface in the water layer showed that the heterogeneity of the dissolved oxygen distribution was higher in sections further away from the biofilm surface than in sections closer to the biofilm surface. The general trend was a change from high degree of heterogeneity in sections further away from the biofilm surface to a low degree of heterogeneity in sections closer to the biofilm surface. Figure 4 shows the oxygen distribution above the biofilm sample in the water layer. The three graphs
FIGURE 6. Pockets of dissolved oxygen in two different biofilm samples at 760 µm below the biofilm surface. represent the dissolved oxygen concentration at three different planes parallel to the actual biofilm surface. The planes represent the dissolved oxygen concentration at 520, 280, and 40 µm above the biofilm surface. Figure 4a shows that the oxygen distribution was highly heterogeneous at 520 µm above the biofilm surface, where most of the measured dissolved oxygen concentrations were from 3.6 to 6.0 mg/L. Figure 4c shows that this variability was reduced 40 µm above the biofilm surface, where most of the measured dissolved oxygen concentrations ranged only from 0.2 to 1.7 mg/L. The heterogeneity of the dissolved oxygen concentration above the biofilm surface could be due to the surface structure of the biofilm and the hydraulic conditions. As mentioned before, the biofilm surface can affect significantly the distribution of dissolved oxygen inside and above the biofilms. In previous studies by several authors, the thickness of the concentration boundary layer, where the oxygen diffuses from the bulk liquid to the biofilm, has been found in a range of 200-800 µm, depending on surface roughness and fluid velocity above the biofilm (15, 17). Low flow velocities increase the concentration boundary layer. In flow regimes where there is virtually no flow above the biofilm, such as the one in this study, the boundary layer is thick indeed and is better described as a diffusion gradient in the bulk fluid. The
dissolved oxygen variability in planes situated above the biofilm surface is difficult to determine using one-dimensional profiles. The three-dimensional profiles showed a better picture of the oxygen concentration variability in the boundary layer. This approach allows us to study the effects of the biofilm surface and hydraulic conditions on biofilm boundary layers. Dissolved Oxygen Distribution Inside Biofilms. This study showed that the concentration and level of heterogeneity of dissolved oxygen inside the biofilms decreased with depth, forming stratification. The heterogeneity of the dissolved oxygen was higher in the top portions of the biofilm sample, close to the biofilm surface. The three-dimensional maps show that dissolved oxygen concentration in biofilms changed generally from a high degree of heterogeneity near the biofilm surface to a low degree of heterogeneity toward deeper sections of the biofilms. Figure 5 shows the oxygen distribution inside the biofilm sample. The three graphs represent the dissolved oxygen concentration at three different planes parallel to the actual biofilm surface. The planes represent the dissolved oxygen concentration at 40, 280, and 760 µm below the biofilm surface. The general trend of the three-dimensional profile was the reduction of dissolved oxygen with depth, forming stratification. A high heterogeneity was found 40 µm below the biofilm surface. VOL. 39, NO. 14, 2005 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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Figure 5a shows a dissolved oxygen concentration from 0 to 2.7 mg/L. In the next depth, 280 µm below the biofilm surface, the dissolved oxygen concentration ranged only from 0 to 0.8 mg/L in 94% of the area, and only a few locations had some peaks with a dissolved oxygen concentration of 1.4 mg/L. This can be seen in Figure 5b. This indicates that the high dissolved oxygen heterogeneity found 40 µm below the biofilm surface decreased considerably at the depth of 280 µm below the biofilm surface. Finally, Figure 5c shows that at 760 µm depth, the dissolved oxygen had been depleted. As seen in this figure, 80% of the dissolved oxygen concentration measured at 760 µm below the biofilm surface was in the range of 0-0.2 mg/L. The high variability suggests a cell-cluster-like structure near biofilm surface (18), and the reduced variability implies a more compact base layer close to the substratum (16). Although the general trend of the dissolved oxygen distribution in the biofilm samples was a decrease of dissolved oxygen concentration with depth, the three-dimensional mapping of the dissolved oxygen distribution in wastewater biofilms showed pockets of high dissolved oxygen concentrations in deep sections of biofilms. Figure 6 shows the presence of the dissolved oxygen pockets in two different biofilm samples at 760 µm below the biofilm surface. The dissolved oxygen pockets in these samples were 1.0 and 0.4 mg/L. Although it was once observed in biofilms in a gasphase trickle-bed biofilter (19), this phenomenon has not been reported in studies on wastewater biofilms with onedimensional dissolved oxygen profile measurements. It demonstrates that three-dimensional mapping can provide a more complete picture of the distribution of a chemical species in biofilms. This is an interesting phenomenon because the presence of oxygen in deeper sections of biofilms can have important impacts on the metabolic activities inside biofilms.
Acknowledgments Special thanks go to Andy Bebbington and Jim Hepler at the Water and Wastewater Treatment Plant in the Town of Devon in Alberta, Canada for their cooperation in growing the biofilm samples. We gratefully acknowledge Debra Long, Richard Lu, and Omar Lopez of the University of Alberta for their assistance in the completion of the experimental work. This project was partially funded by the Natural Sciences and Engineering Research Council (NSERC) of Canada and a scholarship from the National Council of Science and Technology (CONACyT) of Mexico.
Literature Cited (1) Bishop, P. L.; Yu, T. A microelectrode study of redox potential change in biofilms. Water Sci. Technol. 1999, 39 (7), 179-185.
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(2) de Beer, D.; Schramm, A. Microenvironments and mass transfer phenomena in biofilms studied with microsensors. Water Sci. Technol. 1999, 39 (7), 173-178. (3) Lu, R.; Yu, T. A field study on oxygen penetration in wastewater biofilms; 75th Annual Water Environment Federation Conference: Chicago, IL, 2002. (4) Santegoeds, C. M.; Muyzer, G.; de Beer, D. Biofilm dynamics studied with microsensors and molecular techniques. Water Sci. Technol. 1998, 37 (4-5), 125-129. (5) Fu, Y. C.; Zhang, T. C.; Bishop, P. L. Determination of effective oxygen diffusivity in biofilms grown in a completely mixed biodrum reactor. Water Sci. Technol. 1994, 455-462. (6) Noguera, D. R.; Okabe, S.; Picioreanu, C. Biofilm modeling: Presents status and future directions. Water Sci. Technol. 1999, 39 (7), 273-278. (7) Eberl, H. J.; Picioreanu, C.; Heijnen, J. J.; van Loosdrecht, M. C. M. Three-dimensional numerical study on the correlation of spatial structure, hydrodynamic conditions, and mass transfer and conversion in biofilms. Chem. Eng. Sci. 2000, 55 (24), 62096222. (8) Morgenroth, E.; van Loosdrecht, M. C. M.; Wanner, O. Biofilm models for the practitioner. Water Sci. Technol. 2000, 41 (4), 509-512. (9) Morgenroth, E.; Eberl, H.; van Loosdrecht, M. C. M. Evaluating 3-D and 1-D mathematical models for mass transport in heterogeneous biofilms. Water Sci. Technol. 2000, 41 (4), 347356. (10) Lu, R.; Yu, T. Fabrication and evaluation of an oxygen microelectrode applicable to environmental engineering and science. J. Environ. Eng. Sci. 2002, 1 (1), 225-235. (11) Revsbech, N. P. An oxygen microsensor with a guard cathode. Limnol. Oceanogr. 1989, 34 (2), 474-478. (12) Lu, R. M.S. Thesis, University of Alberta, Edmonton, Alberta, Canada, 2001. (13) de la Rosa, C. M.S. Thesis, University of Alberta, Edmonton, Alberta, Canada, 2003. (14) de la Rosa, C.; Yu, T. Development of an automation system to evaluate the three-dimensional oxygen distribution in wastewater biofilms using microsensors. Sens. Actuators, B 2005, in press. (15) Zhang, T. C.; Bishop, P. L. Experimental determination of the dissolved oxygen boundary layer and mass transfer resistance near the fluid-biofilm interface. Water Sci. Technol. 1994, 30 (11), 47-58. (16) Bishop, P. L. Biofilm structure and kinetics. Water Sci. Technol. 1997, 36 (1), 287-294. (17) Wasche, S.; Horn, H.; Hempel Dietmar, C. Influence of growth conditions on biofilm development and mass transfer at the bulk/biofilm interface. Water Res. 2002, 36 (19), 4775-4784. (18) de Beer, D.; Stoodley, P.; Roe, F.; Lewandowski, Z. Effects of biofilm structures on oxygen distribution and mass transport. Biotechnol. Bioeng. 1994, 43 (11), 1131-1138. (19) Zhu, X.; Suidan, M. T.; Alonso, C.; Yu, T.; Kim, B. J.; Kim, B. R. Biofilm structure and mass transfer in a gas-phase trickle-bed biofilter. Water Sci. Technol. 2001, 43 (1), 285-293.
Received for review October 3, 2004. Revised manuscript received May 6, 2005. Accepted May 10, 2005. ES0484449
