MONITORING THE IMPACT OF DISSOLVED OXYGEN AND NITRITE ON ANOXIC BIOFILM IN CONTINUOUS DENITRIFICATION PROCESS

AM JANG, YEONGHEE AHN and IN S. KIM∗
Bio-Environmental Engineering Laboratory (BEEL), Department of Environmental Science and Engineering, Kwangju Institute of Science and Technology (K-JIST), 1 Oryong-dong, Gwangju, Korea (∗ author for correspondence, e-mail: iskim@kjist.ac.kr)

(Received 11 April 2002; accepted 25 September 2002)

Abstract. An anoxic biofilm involved in continuous denitrification process was monitored to investigate the effect of different concentrations of influent dissolved oxygen (DO) or nitrite on the biofilm. Microelectrode measurements evidenced nitrate removal activity of biofilm. When different concentrations of DO were applied to the reactor, generally decreased concentrations of DO were observed as bed depth increased from the bottom of the reactor. Greatest decrease of the DO was observed in the lower 20% of the bed depth. Nitrate removal efficiency was inversely proportional to influent DO concentrations (8.3–11.9 DO mg L−1 ) or nitrite loading rates (0–5.5 N-NO− kg m−3 2 day−1 ) employed in this study. Nitrite loading rates to achieve more than 90% of nitrate removal efficiency were 1.46 N-NO− kg m−3 day−1 or less at pH 7.5 and 0.34 N-NO− kg m−3 day−1 or 2 2 less at pH 6.8. Nitrate removal efficiency was 63% or more within the lower 20% of the bed depth at the nitrite loading rates that allowed more than 90% of nitrate removal efficiency of the reactor. The results of this study provide first quantitative data that nitrate removal performance of an anoxic biofilm is inhibited by DO or nitrite, reported to be a limiting factor in the suspended biological denitrification process. Keywords: anoxic biofilm, denitrification, dissolved oxygen, microelectrode, nitrite

1. Introduction It is commonly known that direct discharge of wastewaters containing nitrogenous nutrients into rivers causes negative environmental problems such as eutrophication in aquatic systems and public health concerns. Successive nitrification and denitrification is one of the most effective biological processes to remove nitrogenous chemicals from wastewaters. These two reactions are carried out by two different functional groups of microorganisms (Madigan et al., 2000). The nitrification process converts ammonium ions to nitrite or nitrate ions, whereas the denitrification process changes nitrite or nitrate ions to nitrogen gas. Since the denitrification process actually removed the nitrogenous nutrients from wastewaters, the denitrification is as important as the nitrification. Generally biological denitrification is a facultative trait based on a competitive advantage in low oxygen environments. Active denitrification occurs under anoxic
Environmental Monitoring and Assessment 87: 133–144, 2003. © 2003 Kluwer Academic Publishers. Printed in the Netherlands.

1994. 1997). The reactor was filled with frames with filamentous substrata.). Lie and Welander. × 110 cm height) with 95 cm of fibrous-loop substratum was used as a submerged anoxic biofilm reactor (Figure 1). and aerobic conditions repress enzymes involved in denitrification (Zumft. reducing the quality of effluents from wastewater treatment plants. 1995. the feeding solution was aerated using air or pure oxygen (purity. However.2:1 (substratum vol. 1993. EPA. to actual column vol.. 1994). the results from such batch tests are difficult to apply to the biofilm in a continuous process. nitrate. When it is necessary. 99%) to increase the DO concentration before introduced into the anoxic reactor. but rather chemical forms (e. Glass et al.. an anoxic biofilm involved in continuous denitrification process was monitored under different concentrations of influent DO and nitrite to provide quantitative data that the nitrate removal performance of the biofilm was inhibited by nitrite or DO. DO and nitrite are limiting factors in the denitrification process. Inhibition of denitrification by nitrite has been studied using batch tests containing suspended cells (van Versefeld et al. 2. Hagedorn-Olsen. the anoxic biofilm reactor was operated at pH 7.5 and 8 DO mg L−1 was introduced to the reactor.d. 1998). and mineral nutrients (Table I). The packing ratio of the substratum was 0. 1996. A NOXIC BIOFILM REACTOR DESIGN A Plexiglas column (10 cm i. Therefore. In these regards.134 AM JANG ET AL. Almedia et al. The reactor was seeded with excess sludge from a secondary sedimentation tank in an anoxic condition. Kornaros et al. 2. Although high DO concentrations in the biofilm reactor are necessary to enhance the activity of nitrifying bacteria. sulfate.S. Glass and Silverstein. The reactor was flushed initially with nitrogen gas .. buffer.1. conditions that do not involve molecular oxygen. nitrate. Dissolved oxygen (DO) can inhibit the denitrification reaction because oxygen functions as the electron acceptor for microorganisms over nitrate. The synthetic wastewater contained glucose. Methods 2. 1977. which are loops with 50 µm-thick nylon fibers attached to 4 mm polypropylene thread. etc) with combined oxygen atoms. R EACTOR O PERATION C ONDITIONS The bioreactor was fed with a synthetic wastewater in the upflow mode and was operated at 25 ± 4 ◦ C.2. Accumulated nitrite also inhibits the denitrification process. 1997.. nitrite. Korea). denitrification is inhibited by oxygen (U. The column surface was covered with a black sheet to eliminate possible growth of oxygenic phototrophic microorganisms. It is not known what were the DO concentration and nitrate removal efficiency at different bed-depths of an anoxic reactor when various concentrations of influent DO were introduced.g.. Hyosung BCplus (Hyosung company. Unless otherwise specified.

the concentration was maintained at least a week to acclimate microorganisms in the reactor. sampling port.4 L day−1 ) during all experiments. Concentrations of total organic carbon (TOC) and nitrate were maintained at 70 TOC mg L−1 and 16 N-NO− mg L−1 . 70 and 95 cm. 6. 3. 4. Liquid samples were taken three times a week from the feed tank and four ports at the heights of 20.5 N-NO− kg m−3 day−1 by 2 changing the influent concentration (40–780 N-NO− mg L−1 ) of nitrite while keep2 ing the HRT constant in order to study nitrite effect on nitrate removal. and 3 hydraulic retention time (HRT) was adjusted to 3. 8. plexiglas column (anoxic reactor). from the bottom of the reactor. 2. effluent. 7. The reactor reached a steady state from day 28 of operation. respectively. respectively. as determined by more than 90% efficiency of nitrate removal. feeding tank (4 ◦ C). Samples were filtered . The reactor was switched to a continuous mode after 3 days of operation. 45. and operated in a batch mode for two days to allow microorganisms to adhere on the substratum in the reactor. supporting substratum with biofilm. 1. 9. Schematic diagram of anoxic biofilm reactor.3 to 5. Whenever a new concentration of nitrite was employed.4 hr (Q = 42. The nitrite load was increased stepwise from 0. influent (pump). air/pure oxygen supply. DO meter.MONITORING THE IMPACT OF DISSOLVED OXYGEN AND NITRITE 135 Figure 1. 5. magnetic stirrer.

Biofilm was sampled after 4 weeks of operation and used for microelectrode measurements.S. Dionex Co. 3. Dionex Co. The microelectrodes were prepared and used according to Zhang and Bishop (1994). MA. Organic compounds were analyzed by a TOC analyzer (Model DC-180. All working 3 microelectrodes were used along with an Ag/AgCl milli-electrode as the reference electrode throughout the experiments.2 mL min−1 .136 AM JANG ET AL.3.. Rosemount Analytical Inc. More than 10 microelectrode profiles were produced at different positions along the biofilm axis during the investigation. YSI Inc.) with an automatic sampler..A. B IOFILM FORMATION ON THE SUBSTRATUM AND MICROELECTRODE MEASUREMENTS Light microscopy showed that microbial biofilm was developed in the shape of flocks (Kim et al. 2000). CG12A guard column (2 × 250 mm). 2. CA. A NALYTICAL METHODS Concentrations of nitrate and nitrite were analyzed by an ion chromatography unit (Model DX-120. Eluent was (3.S.5 mM Na2 CO3 ) (2. respectively.A.5 mM NaHCO3)−1 at a flow rate of 1. Results and Discussions 3. ORP and pH were analyzed with a model 250A probe and model 97–78 redox probe (Thermo Orion.) equipped with an ASRS-II suppressor and AS14 column packed with an analytical column (Model Ionpack CS12A.S. U. M ICROELECTRODE PREPARATIONS AND MEASUREMENTS Biofilm samples were taken on day 28 of operation by cutting off a section of fibrous-loop substratum with biofilm and then immediately transferring it to a cell containing medium (31 N-NO− mg L−1 at pH 8. Sizes of microbial flocks increased gradually and reached a plateau of 300-µm thickness by day 24.. .S.A. and AS40 automated sampler. Microelectrodes having a high spatial resolution were used to measure the profiles of pH and NO− as a function of depth in the biofilm. U. CDM-3 detector of electrical conductivity.).).0) for microelectrode measure3 ments. CA. 2.A.) with a 5905 BOD probe. 0. Ion selective microelectrodes for NO− and pH were employed to measure 3 microgradients of nitrate and pH in the denitrifying biofilm from the anoxic reactor. The DO probe was placed at different bed-depths in the reactor to determine DO concentration at the positions... OH.4. Small flocks of biomass attached to the fiber were observed on day 7.S.1. U.45 µm) for chemical analyses. DO was measured by a DO meter (Model YSI 59.A. U. immediately using cellulose ester membranes (pore size. U.

it is evident that denitrifying microorganisms were active only in the upper region of the biofilm. However. Surface of biofilm was indicated as 0 µm. As the microelectrode moved inside biofilm. The nitrate concentration (31 N-NO− mg L−1 ) in the bulk liquid 3 started to decrease 150 µm above the biofilm surface. Nitrate removal activity was only found within the upper 250 µm 3 of the biofilm. . Further study with in situ hybridization combined with confocal laser scanning microscopy will give us more information on spatial distribution of denitrifiers within the biofilm. The pH values in the biofilm slightly increased from 8.3 as the microelectrode moved inside the biofilm from the bulk solution. Considering the findings that decrease of NO− concentration only occurred in the range from 150 µm above the 3 biofilm to 250 µm into the upper biofilm. reaching 22 N-NO− mg L−1 3 at the interface between the biofilm and the bulk solution. Microprofiles of nitrate concentration and pH in the biofilm.MONITORING THE IMPACT OF DISSOLVED OXYGEN AND NITRITE 137 Figure 2. the nitrate concentration decreased to 5 N-NO− mg L−1 3 until it reached the upper 200-µm of depth and then further decreased slightly by the upper 250-µm of depth. The thickness of the biofilm was counted from the point (interface of biofilm and bulkliquid) where a small and sudden drift was observed when the microelectrode moved from the bulk solution to the interface of the biofilm. the nitrate removal efficiency observed in the upper 100–200 µm of the biofilm was higher than that shown in the upper 0–100 µm of the biofilm. The profiles of pH and NO− concentration were produced as a function of distance from the sur3 face of the biofilm (Figure 2). On the other hand. the NO− concentration significantly dropped from 31 to 4 N3 NO− mg L−1 . but never reached zero.0 to 8.

Regardless of the influent DO concentrations employed in this study. Approximately 60% of the nitrate removal was achieved in this part of the reactor when 8. DO concentrations remained below 2 mg L−1 at the lower 70% (70 cm) of bed depth.6 mg L−1 . respectively. Figure 3 (a) suggests that facultatively aerobic microorganisms were present throughout the bed depth. denitrifiers in the upper 100–200 µm of the biofilm were more active. almost all DO was consumed in the lower 20 cm of the bed depth. When influent DO concentrations were 8. However.9 mg of DO was supplied to the reactor. I NHIBITORY EFFECT OF ELEVATED DO CONCENTRATION ON NITRATE REMOVAL Influents with different concentrations of DO were supplied to the anoxic reactor to investigate the effect of DO on nitrate removal. the highest rate of DO removal was observed at the lower 20% of the bed depth (Figure 3a).3–11. as DO concentrations of the influent increased. The increased DO at the region near the watersurface might be caused by the reactor top that was open. Taken together.3 and 11.9 mg L−1 . judging from the results shown in Figure 2 the sampling method described in the Methods section.3 or 11. It is technically difficult to apply in situ microelectrode measurements to biofilm in a reactor or to sample anoxic biofilm in an anoxic mode from a continuous reactor such as the one used in this study. DO concentrations at the depth of the reactor were more than 5 mg L−1 when influent DO concentrations were increased to 17. Figure 3(b) shows the profiles of nitrate removal under various concentrations of influent DO. maximum nitrate removal rates were 81 and 30 N-NO− g m−2 3 day−1 . The lower level of the nitrate removal efficiency observed in the upper 0–100 µm of the biofilm might be due to penetration of oxygen to the layer of the biofilm during the sampling event while denitrifiers in the upper 100–200 µm of the biofilm could be protected from the oxygen by microbial respiration occurred in the outer layer of the biofilm.9 mg L−1 . 3.6 mg L−1 of influent DO was applied. When 8. On the other hand. and their oxygen respiration was most active at the lower bed-depth of the anoxic reactor. The results of microelectrode measurements directly supported the finding that biofilm formed in the reactor actually contributed to nitrate removal performance of the reactor. Nitrate removal decreased. However.4 and 19.138 AM JANG ET AL. most reduction of nitrate concentration occurred in the lower 45% of the bed depth when 19. increasing the height of an . When influent DO concentrations were 8. Considering the results that DO concentration decreased as bed depth increased from the bottom of the reactor and that elevated DO concentrations inhibited nitrate removal reactions as shown in Figure 3. The profiles of DO in the biofilm reactor (Figure 3) show that DO concentration decreased as bed depth increased from the bottom of the reactor. except at the depth near the watersurface.3 mg L−1 of influent DO was employed. suggesting occurrence of active oxygen-consuming microorganisms in this part of the reactor (Figure 3a).2.

Various concentrations of influent DO were employed for the experiments. Concentrations of (a) DO and (b) nitrate at different bed-depths of the anoxic reactor. Relative bed-depth 100% indicates 100 cm from the bottom of the reactor.MONITORING THE IMPACT OF DISSOLVED OXYGEN AND NITRITE 139 Figure 3. .

Organic carbon removal showed a similar trend to the nitrate removal along the bed depth. microorganisms in the lower 20% of bed depth removed 65% of the influent organic carbon when 8.3 DO mg L−1 was employed (data not shown).91 N-NO− kg m−3 day−1 2 (Figure 4 and 5). As nitrite concentration increased.5 (Figure 4). However the lower pH had more negative effect on nitrate removal when the nitrate loading rate was increased from 0.5 (data not shown). showing gradual 2 reductions in nitrate concentrations along the height of the reactor. 3.90 N-NO− kg m−3 2 day−1 .5 and 6. suggesting the activity of .8 was only one third of that observed at pH 7.8 (Figure 4).34 to 0. Similar values of nitrate removal were observed when nitrite loading rates were in the range of 0.91 or 0. Therefore. Greatly decreased activity of nitrate removal was observed when nitrite loading rate was 2. This pattern of nitrate removal was different from the trend that nitrate removal occurred mostly in the lower part of the reactor when lower concentrations of DO or nitrite were supplied.34 N-NO− kg m−3 day−1 .0 or over 8.0.8. and the concentration of suspended solids decreased (data not shown).8 (Figure 4).0. where anoxic reactors designed for nitrate removal are generally operated (Halling-Sorensen and Jorgensen.0–8. When the nitrite loading rate was 0. whereas the denitrification rate showed only relatively slight changes between pH 6. This suggests that the lower pH could enhance inhibition of nitrate removal by nitrite. The organic carbon removal trend was very similar to that for nitrate removal. When the nitrate loading rate was 0. anoxic reactor or installing a degasification facility might be a solution to relieve the inhibition of nitrate removal by DO. Approximately 65% of 2 the nitrate was removed in the lower 20% of the bed depth under these conditions. suggesting heterotrophic denitrifiers were mostly active in the lower part of the bed-depth.140 AM JANG ET AL. when different loading rates of nitrite were employed at pH 7.46 N-NO− kg m−3 day−1 at pH 7.DEPTHS The denitrification rate decreased sharply when pH was below 6.2 N-NO− kg m−3 day−1 or more (Figure 4a and 5). As the nitrite concentration increased.35–1. 1993). Different concentrations of nitrite were supplied to examine the effect of nitrite loading rate on nitrate removal occurring at different bed-depths. as shown in Figures 3 (b) and 4. E FFECT OF NITRITE LOADING RATE ON NITRATE REMOVAL AT DIFFERENT BED .5 and 6. The effect was investigated at pH 7. While maintaining a specific nitrate removal rate of 81 N-NO− 3 g m−2 day−1 . the color of the biofilm formed on the substratum changed from black to light brown. the removal efficiencies of organics and nitrate decreased. maintenance of proper pH in the anoxic reactor is critical to achieve successful removal of nitrate. 2 similar values and trends of nitrate removal were observed at both pH 7. The effect of nitrite loading rate on nitrate removal at different bed-depths was tested at pH 6.5.3. the nitrate removal efficiency achieved in the lower 20% of the bed depth at pH 6.

2 .MONITORING THE IMPACT OF DISSOLVED OXYGEN AND NITRITE 141 Figure 4.5 (a) and 6. Effect of nitrite loading rates on nitrate removal at different bed-depths. The effect was tested at pH 7. Different rates of nitrite loading were achieved by changing influent nitrite concentration while keeping HRT constant (The unit of legend is kg N-NO− m−3 day−1 ).8 (b).

it is beyond the scope of our study to determine whether the enhanced inhibition was . 1997. This result agreed with results previously produced from batch experiments (Glass. heterotrophic denitrifiers might be inhibited and no more organic carbon might be required as an electron donor.142 AM JANG ET AL.5 and 6. pH 7. Nitrite loading rates to achieve more than 90% nitrate removal efficiency were 1.5 AND 6. E FFECT OF NITRITE LOADING RATE ON NITRATE REMOVAL EFFICIENCY AT pH 7. The efficiency (%) was calculated based on nitrate concentrations of the influent and effluent.8 until the nitrite loading rate was increased from 0 to 0. Nitrate removal efficiency was 2 not significantly affected at pH 6.34 NNO− kg m−3 day−1 at pH 7.8. Figure 5. followed by a significant decrease of the activity.34 N-NO− kg m−3 day−1 .4. Effect of nitrite loading rate on nitrate removal efficiency at pH 7.8 Figure 5 shows nitrate removal efficiency depending on nitrite loading rate under two different pH conditions.5 and 6.5 and 6.8. Although increased concentration of nitrite could 2 enhance inhibition of nitrate removal at a lower pH. It was evident that the lower pH enhanced inhibition of nitrate removal by nitrite. respectively.46 and 0. as shown in Figures 4–5. As nitrite loading rate increased. 1998).8. eventually reaching a plateau of 20% nitrate removal efficiency. 3. Glass and Silverstein. nitrate removal activity initially decreased slightly to 90% efficiency.

Glass and Silverstein. except the depth near the watersurface.6 mg DO L−1 .34 N-NO− 2 kg m−3 day−1 at pH 7. Conclusions 1. As the nitrite loading rate increased.34–0. 1998). Microelectrode measurements showed nitrate removal activity of biofilm taken from the continuously operated anoxic reactor used for denitrification. decreased concentrations of DO was observed as bed depth increased from the bottom of the reactor.6 mg DO L−1 .46 and 0. Nitrate removal occurred mostly within the lower 20% of the bed depth at the nitrite loading rates that allowed more than 90% of nitrate removal efficiency. regardless of influent DO concentrations (8.5 and 6. respectively. the lower 45% of the bed depth showed the highest removal efficiency when the influent DO was 19. Acknowledgements This work was supported in part by the Korea Science and Engineering Foundation (KOSEF) through the Advanced Environmental Monitoring Research Center at . Nitrate concen3 tration (31 N-NO− mg L−1 ) in the bulk started to decrease 150 µm above the 3 biofilm surface. The lower 20% of the bed depth showed the highest removal of nitrate when influent DO concentrations were 8.56 N-NO− kg m−3 day−1 ) were 2 employed at pH 7. Nitrate removal activity of the 400-µm thick biofilm was only observed within the upper 250 µm of the biofilm. nitrate concentration significantly dropped from 31 to 4 N-NO− mg L−1 . Free nitrous acid was thought to cause inhibition of nitrate removal by other researchers (Glass.3–19. and was followed by a significantly decreased efficiency.9 mg DO L−1 ) employed in this study. The lower 20% of the bed depth showed the highest rate of oxygen consumption activity. 3. nitrate removal was inversely proportional to nitrite loading rate. 1997. eventually reaching a plateau of 20% nitrate removal efficiency.3–11. When different concentrations of DO were applied to the anoxic reactor. As the microelectrode moved inside the biofilm from the bulk solution.5 and 6. 2. 4.8. 5. initially nitrate removal activity decreased slightly to 90% nitrate removal efficiency.9 DO mg L−1 ) employed in this study. This suggests that nitrite could enhance inhibition of nitrate removal efficiency at the lower pH.8. However. The maximum nitrite loading rates to achieve more than 90% nitrate removal efficiency were 1. When various nitrite loading rates (0.MONITORING THE IMPACT OF DISSOLVED OXYGEN AND NITRITE 143 directly caused by nitrite ion itself or nitrous acid (HNO2 ).3–11. 4. Nitrate removal efficiency was inversely proportional to influent DO concentrations (8.

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