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Autotrophic denitrification using hydrogen generated from metallic iron corrosion
Bioresource Technology 100 (2009) 4077–4082
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Autotrophic denitrification using hydrogen generated from metallic iron corrosion Neha Sunger, Purnendu Bose * Environmental Engineering and Management Programme, Department of Civil Engineering, Indian Institute of Technology Kanpur, Kanpur 208 016, India
a r t i c l e
i n f o
Article history: Received 10 December 2008 Received in revised form 2 March 2009 Accepted 3 March 2009 Available online 26 April 2009 Keywords: Hydrogenotrophic denitrification Nitrate Metallic iron Hydrogen
a b s t r a c t Hydrogenotrophic denitrification was demonstrated using hydrogen generated from anoxic corrosion of metallic iron. For this purpose, a mixture of hydrogenated water and nitrate solution was used as reactor feed. A semi-batch reactor with nitrate loading of 2000 mg m3 d1 and hydraulic retention time (HRT) of 50 days produced effluent with nitrate concentration of 0.27 mg N L1 (99% nitrate removal). A continuous flow reactor with nitrate loading of 28.9 mg m3 d1 and HRT of 15.6 days produced effluent with nitrate concentration of 0.025 mg N L1 (95% nitrate removal). In both cases, the concentration of nitrate degradation by-products, viz., ammonia and nitrite, were below detection limits. The rate of denitrification in the reactors was controlled by hydrogen availability, and hence to operate such reactors at higher nitrate loading rates and/or lower HRT than reported in the present study, hydrogen concentration in the hydrogenated water must be significantly increased. Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction The most common human health impacts of high nitrate levels in drinking water are methemoglobinemia, gastric cancer and nonHodgkin’s lymphoma (Fan et al., 1987; Gangolli et al., 1994; Ward et al., 1994; Fan and Steinberg, 1996). The drinking water standard for nitrate recommended by the European Economic Community (EEC) and World Health Organization (WHO) is 11.3 mg L1 N, the standard set by the United States Environmental Protection Agency (USEPA) is 10 mg L1 N, while the Indian standard is 10 mg L1 N (CPHEO, 1999). In engineered systems, nitrate is commonly removed from water by the heterotrophic denitrification process. However, this process requires the constant supply of organic carbon and results in excessive biomass production. In contrast, hydrogenotrophic denitrification offers two major advantages; first, almost no denitrification by-products are present in the treated water, and second, hydrogen costs 3–15 times less than the common organic supplements for removal of the same amount of nitrate (Kurt et al., 1987). Pilot-scale and commercial-scale studies have used fixed-bed and fluidized-bed biofilm processes for hydrogenotrophic denitrification and showed that the nitrate concentration can be lowered below 0.1 mg L1 N (Dries et al., 1988; Kurt et al., 1987). In another study, Alcaligenes eutrophus, a hydrogenotrophic denitrifier was immobilized in polyacrylamide and alginate copolymer to evaluate denitrification in fluidized-bed and as well as batch reactors (Chang et al., 1999). Smith et al. (2005) reported the use of flow-through, packed-bed bioreactors for cost effective treatment * Corresponding author. Tel.: +91 512 2597403; fax: +91 512 2597395. E-mail address: [email protected] (P. Bose). 0960-8524/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2009.03.008
of nitrate-contaminated drinking water supplies. However, the main hindrance in the widespread use of hydrogenotrophic denitrification reactors as above for nitrate removal from water is the technical difficulty in the efficient supply of hydrogen gas. Some researchers (e.g., Schnobrich et al., 2007) have reported the use of gas-permeable membranes for supply to hydrogen gas to denitrification reactors. In this regard, Rittmann (2006) advocated the use of membrane biofilm reactors. Ergas and Reuss (2001) investigated the performance of a hollow-fiber membrane bioreactor (HFMB) for hydrogenotrophic denitrification of nitrate-contaminated drinking water. Hydrogen utilization efficiencies averaged 40% in this process. Mansell and Schroeder (2002) studied hydrogenotrophic denitrification in a micro-porous membrane reactor. Lee and Rittmann (2003) also reported the application of a hollow-fiber membrane biofilm reactor for denitrification of drinking water. Other researchers who have reported the use of denitrification reactors based on similar principles include Rezania et al. (2007), Celmer et al. (2006), Cowman et al. (2005), Shin et al. (2005) and Mo et al. (2005). Another trend reported in the literature is the bio-electrochemical process for nitrate removal, which involves in situ production of hydrogen gas required for the denitrification process. Komori and Sakakibara (2008) reported the use of a fluidized-bed biofilm reactor equipped with a Solid-Polymer-Electrolyte Membrane Electrode (SPEME) cell for hydrogenotrophic denitrification of groundwater. Tan et al. (2006) reported the use of a coated electrode of immobilized denitrificants for denitrification. Wan et al. (2009) reported the use of a combined bio-electrochemical and sulfur autotrophic denitrification system to treat a groundwater with nitrate contamination. Ghafari et al. (2008), in a review of bio-electrochemical nitrate removal processes concluded that
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while such processes have many advantages and are promising, they are not well established and documented. In the present study, the feasibility of using hydrogen generated from anaerobic corrosion of metallic iron for autotrophic denitrification has been examined. Till et al. (1998) indicated that in situ production of hydrogen in this manner for denitrification seems to be a promising option. Subsequently, based on the results of various studies (Biswas and Bose, 2005; Lavania and Bose, 2006), it was concluded that autotrophic denitrification of nitrate supported by hydrogen gas produced from iron corrosion is possible using both pure culture (Paracoccus Denitrificans) and a mixed culture of autotrophic denitrifying microorganisms. However design of denitrification reactors based on the above principle is fraught with technical challenges. Most importantly, to prevent abiotic reduction of nitrate to ammonia, nitrate-contaminated water cannot be directly contacted with metallic iron. Thus, the overall objective of this study was the development of reactor configurations where nitrate removal occurred through hydrogenotrophic denitrification, assisted by hydrogen generated from anoxic corrosion of metallic iron. It was also desirable that the product water produced through the above processes have negligible concentrations of undesirable by-products like nitrite and ammonia. 2. Methods 2.1. Stock culture preparation A conical flask of 2-L capacity was used for stock culture preparation. A mixed culture of autotrophic denitrifying microorganisms was developed and maintained. The composition of the mineral medium was as described by Till et al. (1998). The detailed procedure for preparation and maintenance of stock culture reactor is described fully in Biswas and Bose (2005), Lavania and Bose (2006) and Smith et al. (1994). 2.2. Hydrogen generation Electrolytic iron powder (Loba Cheme Pvt. Ltd., Mumbai, India) was taken in a 1 L glass bottle which was filled with anoxic and sterilized tap water such that no headspace existed. The tap water had negligible concentrations of nitrate and no nitrite and ammonia. This bottle was connected to a reservoir containing anoxic and sterilized tap water. This whole setup shall henceforth be called the ‘hydrogen generating system’. 2.3. Semi-batch reactor A sealed conical flask of 1000 mL volume was used as the semibatch reactor. The reactor was filled with 1000 mL of mineral medium containing 40 mg L1 N nitrate, prepared using sterilized tap water. The composition of the mineral medium was as specified by Till et al. (1998). Contents of the reactor were purged with nitrogen gas for 15 min. Hydrogen gas was then passed through the reactor for 5 min. The reactor was then seeded with 75 mL of seed from stock culture. Hydrogen gas was applied to the reactor for 5 min every 3 days. Twenty five milliliter of reactor contents were withdrawn once a week followed by addition of 25 mL of mineral medium containing 40 mg L1 N nitrate. Reactor contents were maintained in fully mixed condition. After 8 weeks, daily feeding was started. In phase I, feeding involved withdrawal of 25 mL of the reactor contents once a day and addition of 25 mL nitrate solution (40 mg L1 N) prepared in sterilized tap water. During phase II, feeding involved withdrawal of 20 mL of the reactor contents once a day and adding 20 mL nitrate solution (100 mg L1 N). Reactor operations was exactly similar in
phase III, however, the concentration of nitrate in the feed solution was 150 mg L1 N. In all cases, hydrogen was bubbled through the reactor for 2 min after daily feeding. During phase IV experiments, the ‘hydrogen generation system’ was attached to the denitrification reactor. At the time of daily feeding, 20 mL of the denitrification reactor contents was withdrawn and 10 mL of nitrate solution (containing 300 mg N L1) was added to the denitrification reactor through the funnel. Also, 10 mL of hydrogenated water from the hydrogen generating apparatus was added to the reactor. External hydrogen gas was also intermittently purged into reactor. The external hydrogen supply was gradually stopped, and in phase V experiments, the reactor operated only with the hydrogen supplied with the hydrogenated water. Reactor pH was periodically adjusted by addition of phosphate buffer during all phases of experiments to counter pH increase due to denitrification. The pH, ammonia, nitrite and nitrate concentration was measured in the reactor effluent withdrawn everyday. 2.4. Continuous reactors The description of glass columns used as continuous flow reactors are given elsewhere (see Biswas and Bose, 2005). The media in the column consisted of sand graded to 1–2 mm diameter. Graded sand was thoroughly washed with 0.1 N HCl acid and water before drying. Then the sand was loaded into the columns and the columns were sterilized by autoclaving. The height of media in each column was 10 cm, which corresponded to a volume of 125 cm3. Porosity of the media was approximately 50%. Each column was sealed at the top by a rubber stopper, with two glass tubes pierced though it. 2.4.1. Establishment of anoxic condition The experimental setup consisted of three sterilized up flow columns as described above, connected in parallel to a reservoir containing sterilized nitrogen-sparged distilled water. All three columns were fully flooded with water from the reservoir and maintained in sealed condition for 7 days. Then the columns were disconnected from the distilled water reservoir and connected to a another reservoir containing nitrogen-sparged mineral medium (composition as specified in Till et al., 1998) prepared using sterilized IIT Kanpur tap water, and containing 40 mg L1 N nitrate. Columns were flushed with the mineral medium such that the nitrate concentrations in the effluent from the columns were approximately same as the nitrate concentration in the reservoir. The apparatus was maintained in sealed condition for seven days. No degradation of nitrate was observed in the columns during this period. 2.4.2. Seeding with denitrifying microorganisms The columns were disconnected from the reservoir containing mineral medium and connected to the stock culture reactor. A peristaltic pump (Miclins Model PP 10, Chennai, India) was employed to continuously re-circulate the contents of the stock culture reactor through the columns. The flow rate through each column was 1 mL h1. The biomass in the stock culture reactor was maintained in viable condition during this period through periodic addition of nitrate and application of hydrogen gas. Based on results of previous experiments carried out under similar conditions (see Biswas and Bose, 2005), colonization of the sand media in the columns by microorganisms was determined to have occurred after 15 days of recirculation. The columns were again connected to the reservoir containing sterilized nitrogen-sparged distilled water. The columns were flushed with approximately four bed volumes of distilled water such that the nitrate concentration in the column effluent after flushing was negligible.
N. Sunger, P. Bose / Bioresource Technology 100 (2009) 4077–4082
2.4.3. Column operation The experimental apparatus employed for column operation is shown in Fig. 1. The hydrogen generation system was attached to the denitrification columns through a 125 mL capacity mixer bottle. The mixer bottle was filled with hydrogenated water from the ‘hydrogen generation system’. The mixer bottle was also equipped with a tube through which nitrate solution was added once a day. Thus hydrogenated water from the hydrogen generation system was mixed with nitrate solution in the mixer bottle. This mixture was fed to the denitrification reactors using a peristaltic pump (Miclins Model PP 10, Chennai, India). Simultaneously, the mixer bottle was refilled at the same rate with water from the hydrogen generation reactor. The pH, ammonia, nitrite and nitrate concentration in the effluent from the denitrification reactors was analyzed on a daily basis. Column experiments were carried out in six phases. At the start of phase I, the nitrate concentration in mixer bottle was adjusted to 1.000 mg L1 N nitrate. The contents of the mixer bottle were pumped through the denitrification columns such that the flow rate through each column was 0.667 mL h1. This resulted in a gradual decrease in nitrate concentration in the mixer bottle and hence the nitrate concentration in the feed to the reactors from 1.000 mg L1 N at the start to 0.616 mg L1 N at the end of a 24-h cycle. After 24 h, 2.4 mL of nitrate solution containing 20 mg L1 N nitrate was added to the mixer bottle such that the nitrate concentration in the mixer bottle was restored to 1.000 mg L1 N nitrate. Phase I experiments were carried out for five 24-h cycles. In phase II experiments, the flow rate through each denitrification column was reduced to 0.333 mL h1. As in phase I experiments, the nitrate concentration in the mixer bottle was 1.000 mg L1 N nitrate at the start of a cycle. This value declined gradually to 0.808 mg L1 N at the end of a 24-h cycle. After 24 h, 1.200 mL of nitrate solution containing 20 mg L1 N nitrate was added to the mixer bottle such that the nitrate concentration in the mixer bottle was restored to 1.000 mg L1 N. Phase II experiments were carried out for 20 24h cycles. Before start of phase III experiments, the nitrate concentration in the mixer bottle was adjusted to 0.500 mg L1 N. Flow rate through each denitrification column was 0.333 mL h1. This nitrate concentration declined gradually from 0.500 to 0.404 mg L1 N at the end of a 24-h cycle. Nitrate concentration in the mixer bottle at the start of each 24-h cycle was adjusted to 0.5 mg L1 N through addition of 0.6 mL of nitrate solution containing 20 mg L1 N nitrate directly to the mixer bottle. Phase III experiments were carried out for 30 24-h cycles. Phase IV experiments were similar to phase III experiments, except that the entire effluent from the denitrification columns were recycled back in the hydrogen generation system using peristaltic pumps (Miclins Model PP 10, Chennai, India), as shown in Fig. 1. Phase V experi-
PP PP Effluent
PP Influent Nitrate Hydrogenated Water
Hydrogen Generation System
1 Mixer
PP: Peristaltic Pumps
2
3
PP
Two-Way Valves
Recycling of Effluent to Hydrogen Generation System
Fig. 1. Schematic of the continuous reactor system. The recycling of effluent to the hydrogen generation system was only done in phase IV experiments.
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ments were similar to phase III experiments, except the flow rate through column 3 was increased from 0.333 mL h1 to 0.667 mL h1, while keeping the flow rates through columns 1 and 2 same as in phase III. In phase V experiments, nitrate concentration in mixer bottle declined from 0.500 to 0.372 mg L1 N nitrate over a 24-h period. Phase V experiments were carried out for 10 24-h cycles. In Phase VI experiments, the flow in column 3 was again reduced to 0.333 mL h1, while flow rates through columns 1 and 2 remained same as before. Thus phase VI experiments were same in all respects to phase IV experiments. Phase VI experiments were carried out for 10 24-h cycles. Before phase VII experiment, column 3 was detached from experimental set up, autoclaved, and re-attached. Otherwise, phase VII experiments were similar in all respects to phase VI experiments. 2.5. Analytical methods Nitrate and nitrite was measured using an Ion Chromatograph (Metrohm 761) equipped with a Phenomenex STAR ION A 300 IC anion column and a conductivity detector with ion suppression. The method used for this purpose was specified in the relevant company literature. Detection limit for both nitrate and nitrite was 0.01 mg L1. All samples for nitrate and nitrite determination were filtered through 0.2 lm filter paper and sterilized before storage under refrigeration until analysis. The samples stored for nitrate determination were also used for ammonia determination. Ammonia was measured colorimetrically by nesslerization (Method No. 417 B, APHA et al., 1985). A spectophotometer (Spectronic, 20 D+, India) with Borosil glass absorbance cells having 1 cm path length were used for this purpose. The detection limit for ammonia determination was 0.02 mg L1. The pH was also measured in all the samples. pH was measured using a combination pH electrode (Toshniwal CL-51, India) connected to a digital pH meter (Toshniwal CL-54, India). The optical density of the stock culture contents was determined as a surrogate measure for biomass concentration (Till et al., 1998). For this purpose absorbance @600 nm of the stock culture was measured using 4 cm path length spectroscopic cell. 3. Results and discussion 3.1. Denitrification in semi-batch reactors The results of the five phases of the semi-batch experiments are presented in Table 1. In phase I, the average steady-state effluent nitrate concentration was 0.20 mg L1 N. Nitrate loading and HRT were 1000 mg N m3 d1 and 40 days, respectively, while steadystate nitrate removal in the reactor was 99.50% (see Table 1). Assuming assimilatory nitrate uptake by denitrifying microorganisms to be negligible in comparison to denitrification, and the fact that ammonia and nitrite concentration in the reactor effluent was below detection limits, the entire nitrate removal may be attributed to denitrification. In phase II, the average steady-state effluent nitrate concentration was 0.27 mg L1 N, while no nitrite or ammonia was detected in the effluent. In this phase, nitrate loading and HRT were 2000 mg N m3 d1 and 50 days, respectively, while steady-state nitrate removal in the reactor was 99.73% (see Table 1). In phase III, the average steady-state effluent nitrate concentration was 3.9 mg L1 N. In this phase, nitrate loading and HRT were 3000 mg N m3 d1 and 50 days, respectively, while the steady-state nitrate removal was 97.54% (see Table 1). Nitrite and ammonia concentrations were below detection limits in the effluent from phase II and III experiments. Effluent pH in all three phases was in the 7.8–8.2 range. In phase IV experiments, a transition was made from gaseous hydrogen to hydrogenated water as the source of hydrogen for denitrification. The average steady-state effluent nitrate concentra-
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Table 1 Summary of the performance of semi-batch reactor.
Phase-I Phase-II Phase-III Phase-IV Phase-V
Nitrate loading (mg N m3 d1)
HRT (days)
Steady-state effluent nitrate concentration (mg N L1)
Steady-state nitrate removal percentage
pH
Duration of operation (days)
1000 2000 3000 3000 3000
40 50 50 50 50
0.20 0.27 3.69 3.89 4.42
99.50 99.73 97.54 97.41 97.05
7.8–8.2 7.8–8.2 7.8–8.2 7.2–8.0 7.2–8.0
40 50 50 50 50
tion was 3.885 mg L1 N during phase IV experiments, which corresponded to 97.41% nitrate removal (see Table 1). During phase V experiments, the average steady-state effluent nitrate concentration was 4.424 mg L1 N, which corresponds to 97.05% nitrate removal. As in previous phases, nitrite and ammonia concentrations were below detection limits in the effluent from phase IV and V experiments. Effluent pH in phases IV and V were in the 7.2–8.0 range. Comparison of the phase V results with the phase IV and phase III results indicate that extent of denitrification and effluent nitrate concentrations are comparable in the three phases. It was thus concluded that during phase V experiments, the hydrogen supplied through hydrogenated water was capable of sustaining denitrification at the same rate as in Phases III and IV experiments. 3.2. Denitrification in continuous flow reactors Results of the experiments with continuous flow reactors are summarized in Table 2. From the results of phase I experiments (nitrate loading: 103 mg N m3 d1; HRT: 7.8 days) it was determined that the effluent nitrate concentration measured at the end of each 24-h cycle showed an increasing trend, with the effluent nitrate concentrations being 0.342, 0.376 and 0.368 mg L1 N in columns 1, 2 and 3, respectively, after the fifth 24-h cycle. These results indicated that the nitrate loading in the columns was too high and HRT too low. Hence, nitrate loading to the columns were reduced and HRT increased (nitrate loading: 57.8 mg N m3 d1; HRT: 15.6 days) in phase II experiments. In phase II experiments, steady-state effluent nitrate concentration was observed after 15 days of column operations in this mode. The average steadystate effluent nitrate concentration (21st–25th days) was 0.501,
0.465 and 0.384 mg L1 N in columns 1, 2 and 3, respectively. This corresponded to nitrate removal of 44.5%, 48.5% and 57.5%, respectively. To further improve the column performance, the nitrate loading to the columns was further reduced (nitrate loading: 28.9 mg N m3 d1; HRT: 15.6 days) in phase III experiments. During phase III experiments, steady-state effluent nitrate concentration was observed after 10 days of column operations in this mode. The average steady-state effluent nitrate concentration (36th–55th days) was 0.025, 0.031 and 0.031 mg L1 N in columns 1, 2 and 3, respectively. This corresponded to nitrate removal of 94.5%, 93.1% and 93.1%, respectively. Ammonia and nitrite concentrations were below detection limits in the effluent from the denitrification reactors at all times during phases I, II or III experiments. Effluent pH was also below 8 in all reactors at all times. Phase IV experiments were similar to phase III, except that the effluent from the denitrification reactors were recycled back to the hydrogen generation system (see Fig. 1). Steady-state effluent nitrate concentration was observed during the entire duration of this phase of column operation. The average steady-state effluent nitrate concentration (56th–75th days) was 0.024, 0.030 and 0.022 mg L1 N in columns 1, 2 and 3, respectively. This corresponded to nitrate removal of 94.7%, 93.4% and 95.1%, respectively. Further, ammonia and nitrite concentrations were below detection limits in the effluent from the denitrification reactors at all times during phase IV experiments. Effluent pH was also below 8 in all reactors at all times. The results of this phase of experiments indicated that, provided nitrate removal is nearly complete, effluent from the denitrification reactors could be recycled to the hydrogen generating system for production of hydrogenated water required for denitrification column operations without any impact on ammonia and nitrite concentrations in the effluent.
Table 2 Summary of the performance of continuous flow reactors.
Phase I
Phase II
Phase III
Phase IV
Phase V
Phase VI
Phase VII
Reactor Reactor Reactor Reactor Reactor Reactor Reactor Reactor Reactor Reactor Reactor Reactor Reactor Reactor Reactor Reactor Reactor Reactor Reactor Reactor Reactor
1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3
Average influent nitrate loading (mg N m3 d1)
HRT (days)
Duration of operation
Effluent nitrate concentration (mg N L1)
Percent nitrate removal
Trend of effluent nitrate concentration
103 103 103 57.8 57.8 57.8 28.9 28.9 28.9 28.9 28.9 28.9 27.9 27.9 55.8 28.9 28.9 28.9 28.9 28.9 28.9
7.8 7.8 7.8 15.6 15.6 15.6 15.6 15.6 15.6 15.6 15.6 15.6 15.6 15.6 7.8 15.6 15.6 15.6 15.6 15.6 15.6
1st–5th days
0.342 0.376 0.368 0.501 0.465 0.384 0.025 0.031 0.031 0.024 0.030 0.022 0.019 0.023 0.322 0.024 0.026 0.036 0.024 0.026 0.416
57.7 53.5 54.4 44.5 48.5 57.5 94.5 93.1 93.1 94.7 93.4 95.1 95.6 94.7 26.1 94.7 94.2 92.0 94.7 94.2 8.00
" " " M M M M M M M M M M M " M M ; M M "
": increasing trend; ;: decreasing trend; M: steady-state.
6th–25th days
26th–55th days
56th–75th days
76th–85th days
86th–95th days
96th–135th days
(average (average (average (average (average (average (average (average (average (average (average
of of of of of of of of of of of
21st–25th days) 21st-25th days) 21st–25th days) 36th–55th days) 36th–55th days) 36th–55th days) 56th–75th days) 56th–75th days) 56th–75th days) 76th–85th days) 76th–85th days)
(average of 86th–95th days) (average of 86th–95th days) (average of 96th–135th days) (average of 96th–135th days)
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In phase V experiments, nitrate loading and HRT in columns 1 and 2 were almost unchanged from phase III (nitrate loading: 27.9 mg N m3 d1; HRT: 15.6 days), while nitrate loading and HRT values for column 3 were 55.8 mg N m3 d1 and 7.8 days, respectively. While performance of columns 1 and 2 remained essentially unchanged with respect to effluent nitrate concentration, the effluent nitrate concentration from column 3 showed an increasing trend, and increased to 0.322 mg L1 N nitrate (see Table 2) after 10 days of reactor operation. These results clearly show the detrimental effect of increase in nitrate loading rate and reduction in HRT on the column performance. In phase VI experiments, nitrate loading and HRT in columns 1, 2 and 3 were restored to earlier values of 28.9 mg N m3 d1 and 15.6 days, respectively. While the performance of columns 1 and 2 remained essentially unchanged, the nitrate concentration in the effluent from column 3 showed a decreasing trend, and after 10 days of operation was reduced to 0.036 mg L1 N (see Table 2). Column 3 was sterilized by autoclaving before phase VII experiments. Afterwards, all three columns were operated under conditions similar to that in phase VI (see Table 2). While the performance of columns 1 and 2 remained essentially unchanged, effluent nitrate concentration from column 3 showed an increasing trend and the concentration after 40 days of column operation was 0.416 mg L1 N (see Table 2), which corresponded to only 8% nitrate removal in the column. This proved that biological denitrification was responsible for reduction in nitrate concentration in all columns during this study. Ammonia and nitrite concentration in the effluent from all columns in phases V–VII of experiments was below the detection limits and effluent pH was always below 8. 3.3. Discussion In a study designed to investigate the performance of a hollowfiber membrane bioreactor (HFMB) for hydrogenotrophic denitrification of contaminated drinking water, Ergas and Reuss (2001) reported nitrate utilization rates up to 770 g N m3 d1. Influent nitrate concentrations of up to 200 mg N L1 were almost completely denitrified in a reactor with 4.1 h HRT. In another study (Chang et al., 1999) A. eutrophus, a hydrogenotrophic denitrifier, was immobilized in polyacrylamide and alginate copolymer to evaluate denitrification in continuous mode and batch mode in a fluidized-bed reactor. The total nitrogen removal rate in a continuous test reached a maximum rate of 0.6–0.7 kg N m3 day1. In another study (Sierra-Alvarez et al., 2005) on autotrophic denitrification using sulfide as electron donor, 89% nitrate removal was reported with nitrate loading rate of 1.24 kg m3 d1 and HRT of 13.4 h. In comparison, the nitrate loading rates applied in continuous reactors during the present study are several orders of magnitude lower (28.9 mg N m3 d1) and HRT higher (15.6 days). The results presented in Table 2 also show that any increase in nitrate loading rate or any decrease in HRT resulted in a decline in effluent quality, possibly because of hydrogen limitation. Limitations in the experimental setup used in the present study precluded application of large hydrogen concentrations to the reactor and consequently the applied nitrate loading rates had to be kept low to maintain the nitrate limiting conditions required for high percentage nitrate removal. It is, however, postulated that the nitrate loading rates in such reactors could be significantly increased by designing the hydrogen generation system as a pressure vessel and the denitrification columns as pressure filters, wherein higher hydrogen partial pressures and hence higher hydrogen solubility in water could be maintained. Contrary to expectations, pH measurements during continuous column operations showed that pH never increased beyond 8 at
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any time. It appears that the natural buffering capacity of the influent feed was sufficient to prevent any pH increase in these columns. However, in cases where denitrification occurs at a higher rate, substantial pH increase inside the column is expected, with consequent adverse effects on denitrification rate and the product water quality (Jha and Bose, 2005). In such cases, the inherent pH buffering capacity of the porous media must be increased through addition of buffering agents. In this context, Jha and Bose (2005) have demonstrated that addition of pyrite in porous media columns was effective in controlling pH increase with no detrimental effect on the denitrification process. 4. Summary and conclusions Biological denitrification using hydrogen derived from anoxic corrosion of metallic iron as energy source was demonstrated in laboratory scale reactors. Following are the main conclusions from this study, Denitrification up to 99% and 95% was demonstrated in semibatch and continuous reactors, respectively. Under steady-state conditions, a semi-batch reactor with nitrate loading and HRT of 2000 mg m3 d1 and 50 days, respectively, produced effluent with nitrate concentration of 0.27 mg N L1. Continuous reactors with nitrate loading and HRT of 28.9 mg m3 d1 and 15.6 days produced effluent with nitrate concentration of 0.025 mg N L1. In all cases, the effluent contained negligible concentrations of nitrite and ammonia. It was further demonstrated that the effluent from the continuous flow reactor could be recycled to produce hydrogenated water required for the denitrification process, with no adverse impact on the effluent quality. However, significant unresolved technical challenges related to reactor scale up must be resolved before such reactors can be commercially applied for denitrification of real water/wastewater. Most importantly, to operate such reactors at higher nitrate loading rates and lower HRT than demonstrated in this study, the concentration of hydrogen in the hydrogenated water required for reactor operation must be significantly increased. Acknowledgements The authors gratefully acknowledge the financial help provided by the Department of Science and Technology of the Government of India through Project No. III.5(119)2001-SERC Engg. for carrying out this study. References APHA, WEF, AWWA, 1985. Standard Methods for Examination of Water and Wastewater, 16th ed. APHA, Washington, DC, USA. Biswas, S., Bose, P., 2005. Zero-valent iron-assisted autotrophic denitrification. Journal Environmental Engineering ASCE 131, 1212–1220. Celmer, D., Oleszkiewicz, J., Cicek, N., Husain, H., 2006. Hydrogen limitation – a method for controlling the performance of membrane biofilm reactor for autotrophic denitrification of wastewater. Water Science and Technology 54, 165–172. Chang, C.C., Tseng, S.K., Huang, H.K., 1999. Hydrogenotrophic denitrification with immobilized Alcaligenes eutrophus for drinking water treatment. Bioresource Technology 69, 53–58. Cowman, J., Torres, C.I., Rittmann, B.E., 2005. Total nitrogen removal in an aerobic/ anoxic membrane biofilm reactor system. Water Science and Technology 52, 115–120. CPHEO, 1999. Manual on Water Supply and Treatment. Third ed., Ministry of Urban Development, Govt. of India, New Delhi, India. Dries, D., Liessens, J., Verstraere, W., Stevens, P., De Vost, P., De Ley, J., 1988. Nitrate removal from drinking water by means of hydrogenotrophic denitrifiers in a polyurethane carrier reactor. Water Supply 6, 181–192.
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Ergas, S.J., Reuss, A.F., 2001. Hydrogenotrophic denitrification of drinking water using a hollow fibre membrane bioreactor. Journal of Water Supply: Research and Technology – AQUA 50, 161–171. Fan, A.M., Steinberg, V.E., 1996. Health implications of nitrate and nitrite in drinking water: an update on methemoglobinemia occurrence and reproductive and developmental toxicity. Regulatory Toxicology and Pharmacology 23, 35–43. Fan, A.M., Willhite, C.C., Book, S.A., 1987. Evaluation of the nitrate drinking water standard with reference to infant methemoglobinemia and potential reproductive toxicity. Regulatory Toxicology and Pharmacology 7, 135–148. Gangolli, S.D., Van der Brandt, P.A., Feron, V.J., 1994. Nitrate, nitrite and N-nitroso compounds. European Journal of Pharmacology-Environmental Toxicology and Pharmacology 292, 1–38. Ghafari, S., Hasan, M., Aroua, M.K., 2008. Bio-electrochemical removal of nitrate from water and wastewater – a review. Bioresource Technology 99, 3965–3974. Jha, D., Bose, P., 2005. Use of pyrite for pH control during hydrogenotrophic denitrification using metallic iron as the ultimate electron donor. Chemosphere 61, 1020–1031. Komori, M., Sakakibara, Y., 2008. High-rate hydrogenotrophic denitrification in a fluidized-bed biofilm reactor using solid-polymer-electrolyte membrane electrode (SPEME). Water Science and Technology 58, 1441–1446. Kurt, M., Dunn, I.J., Bourne, J.R., 1987. Biological denitrification of drinking water using autotrophic organisms with H2 in a fluidized-bed biofilm reactor. Biotechnology and Bioengineering 29, 493–501. Lavania, A., Bose, P., 2006. Effect of porous media composition on end-product distribution during metallic iron assisted autotrophic denitrification. Journal of Environmental Engineering, ASCE 132, 994–1000. Lee, K.C., Rittmann, B.E., 2003. Effects of pH and precipitation on autohydrogenotrophic denitrification using the hollow-fiber membranebiofilm reactor. Water Research 37, 1551–1556. Mansell, B.O., Schroeder, E.D., 2002. Biological denitrification in a continuous flow membrane reactor. Water Research 33, 1845–1850. Mo, H., Oleszkiewicz, J.A., Cicek, N., Rezania, B., 2005. Incorporating membrane gas diffusion into a membrane bioreactor for hydrogenotrophic denitrification of groundwater. Water Science and Technology 51, 357–364.
Rezania, B., Oleszkiewicz, J.A., Cicek, N., 2007. Hydrogen-dependent denitrification of water in an anaerobic submerged membrane bioreactor coupled with a novel hydrogen delivery system. Water Research 41, 1074–1080. Rittmann, B.E., 2006. The membrane biofilm reactor: the natural partnership of membranes and biofilm. Water Science and Technology 53, 219–225. Schnobrich, M.R., Chaplin, B.P., Semmens, M.J., Novak, P.J., 2007. Stimulating hydrogenotrophic denitrification in simulated groundwater containing high dissolved oxygen and nitrate concentrations. Water Research 41, 1869– 1876. Shin, J.H., Sang, B.I., Chung, Y.C., Choung, Y.K., 2005. The removal of nitrogen using an autotrophic hybrid hollow-fiber membrane biofilm reactor. Desalination 183, 447–454. Sierra-Alvarez, R., Guerrero, F., Rowlette, P., Freeman, S., Field, J.A., 2005. Comparison of chemo-, hetero- and mixotrophic denitrification in laboratoryscale UASBs. Water Science and Technology 52, 337–342. Smith, R.L., Buckwalter, S.P., Repert, D.A., Miller, D.N., 2005. Small-scale, hydrogenoxidizing-denitrifying bioreactor for treatment of nitrate-contaminated drinking water. Water Research 39, 2014–2023. Smith, R.L., Ceazan, M.L., Brooks, M.H., 1994. Autotrophic, hydrogen-oxidizing, denitrifying bacteria in groundwater, potential agents for bioremediation of nitrate contamination. Applied and Environmental Microbiology 60, 1949– 1955. Tan, Y.M., Liu, Y.Q., Su, F., Wang, L., Luo, Q.F., 2006. Experimental study on denitrification using coated electrode of immobilized denitrifying bacteria. Biomedical and Environmental Sciences 19, 47–52. Till, B.A., Weathers, L.J., Alvarez, P.J., 1998. Fe(0)-supported autotrophic denitrification. Environmental Science and Technology 32, 634–639. Wan, D.J., Liu, H.J., Qu, J.H., Lei, P.J., Mao, S.H., Hou, Y.N., 2009. Using the combined bioelectrochemical and sulfur autotrophic denitrification system for groundwater denitrification. Bioresource Technology 100, 142–148. Ward, M.H., Zahm, S.H., Blair, A., 1994. Dietary factors and non-Hodgkin’s lymphoma. Cancer Causes and Control 5, 422–432.
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Autotrophic denitrification using hydrogen generated from metallic iron corrosion Neha Sunger, Purnendu Bose * Environmental Engineering and Management Programme, Department of Civil Engineering, Indian Institute of Technology Kanpur, Kanpur 208 016, India
a r t i c l e
i n f o
Article history: Received 10 December 2008 Received in revised form 2 March 2009 Accepted 3 March 2009 Available online 26 April 2009 Keywords: Hydrogenotrophic denitrification Nitrate Metallic iron Hydrogen
a b s t r a c t Hydrogenotrophic denitrification was demonstrated using hydrogen generated from anoxic corrosion of metallic iron. For this purpose, a mixture of hydrogenated water and nitrate solution was used as reactor feed. A semi-batch reactor with nitrate loading of 2000 mg m3 d1 and hydraulic retention time (HRT) of 50 days produced effluent with nitrate concentration of 0.27 mg N L1 (99% nitrate removal). A continuous flow reactor with nitrate loading of 28.9 mg m3 d1 and HRT of 15.6 days produced effluent with nitrate concentration of 0.025 mg N L1 (95% nitrate removal). In both cases, the concentration of nitrate degradation by-products, viz., ammonia and nitrite, were below detection limits. The rate of denitrification in the reactors was controlled by hydrogen availability, and hence to operate such reactors at higher nitrate loading rates and/or lower HRT than reported in the present study, hydrogen concentration in the hydrogenated water must be significantly increased. Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction The most common human health impacts of high nitrate levels in drinking water are methemoglobinemia, gastric cancer and nonHodgkin’s lymphoma (Fan et al., 1987; Gangolli et al., 1994; Ward et al., 1994; Fan and Steinberg, 1996). The drinking water standard for nitrate recommended by the European Economic Community (EEC) and World Health Organization (WHO) is 11.3 mg L1 N, the standard set by the United States Environmental Protection Agency (USEPA) is 10 mg L1 N, while the Indian standard is 10 mg L1 N (CPHEO, 1999). In engineered systems, nitrate is commonly removed from water by the heterotrophic denitrification process. However, this process requires the constant supply of organic carbon and results in excessive biomass production. In contrast, hydrogenotrophic denitrification offers two major advantages; first, almost no denitrification by-products are present in the treated water, and second, hydrogen costs 3–15 times less than the common organic supplements for removal of the same amount of nitrate (Kurt et al., 1987). Pilot-scale and commercial-scale studies have used fixed-bed and fluidized-bed biofilm processes for hydrogenotrophic denitrification and showed that the nitrate concentration can be lowered below 0.1 mg L1 N (Dries et al., 1988; Kurt et al., 1987). In another study, Alcaligenes eutrophus, a hydrogenotrophic denitrifier was immobilized in polyacrylamide and alginate copolymer to evaluate denitrification in fluidized-bed and as well as batch reactors (Chang et al., 1999). Smith et al. (2005) reported the use of flow-through, packed-bed bioreactors for cost effective treatment * Corresponding author. Tel.: +91 512 2597403; fax: +91 512 2597395. E-mail address: [email protected] (P. Bose). 0960-8524/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2009.03.008
of nitrate-contaminated drinking water supplies. However, the main hindrance in the widespread use of hydrogenotrophic denitrification reactors as above for nitrate removal from water is the technical difficulty in the efficient supply of hydrogen gas. Some researchers (e.g., Schnobrich et al., 2007) have reported the use of gas-permeable membranes for supply to hydrogen gas to denitrification reactors. In this regard, Rittmann (2006) advocated the use of membrane biofilm reactors. Ergas and Reuss (2001) investigated the performance of a hollow-fiber membrane bioreactor (HFMB) for hydrogenotrophic denitrification of nitrate-contaminated drinking water. Hydrogen utilization efficiencies averaged 40% in this process. Mansell and Schroeder (2002) studied hydrogenotrophic denitrification in a micro-porous membrane reactor. Lee and Rittmann (2003) also reported the application of a hollow-fiber membrane biofilm reactor for denitrification of drinking water. Other researchers who have reported the use of denitrification reactors based on similar principles include Rezania et al. (2007), Celmer et al. (2006), Cowman et al. (2005), Shin et al. (2005) and Mo et al. (2005). Another trend reported in the literature is the bio-electrochemical process for nitrate removal, which involves in situ production of hydrogen gas required for the denitrification process. Komori and Sakakibara (2008) reported the use of a fluidized-bed biofilm reactor equipped with a Solid-Polymer-Electrolyte Membrane Electrode (SPEME) cell for hydrogenotrophic denitrification of groundwater. Tan et al. (2006) reported the use of a coated electrode of immobilized denitrificants for denitrification. Wan et al. (2009) reported the use of a combined bio-electrochemical and sulfur autotrophic denitrification system to treat a groundwater with nitrate contamination. Ghafari et al. (2008), in a review of bio-electrochemical nitrate removal processes concluded that
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while such processes have many advantages and are promising, they are not well established and documented. In the present study, the feasibility of using hydrogen generated from anaerobic corrosion of metallic iron for autotrophic denitrification has been examined. Till et al. (1998) indicated that in situ production of hydrogen in this manner for denitrification seems to be a promising option. Subsequently, based on the results of various studies (Biswas and Bose, 2005; Lavania and Bose, 2006), it was concluded that autotrophic denitrification of nitrate supported by hydrogen gas produced from iron corrosion is possible using both pure culture (Paracoccus Denitrificans) and a mixed culture of autotrophic denitrifying microorganisms. However design of denitrification reactors based on the above principle is fraught with technical challenges. Most importantly, to prevent abiotic reduction of nitrate to ammonia, nitrate-contaminated water cannot be directly contacted with metallic iron. Thus, the overall objective of this study was the development of reactor configurations where nitrate removal occurred through hydrogenotrophic denitrification, assisted by hydrogen generated from anoxic corrosion of metallic iron. It was also desirable that the product water produced through the above processes have negligible concentrations of undesirable by-products like nitrite and ammonia. 2. Methods 2.1. Stock culture preparation A conical flask of 2-L capacity was used for stock culture preparation. A mixed culture of autotrophic denitrifying microorganisms was developed and maintained. The composition of the mineral medium was as described by Till et al. (1998). The detailed procedure for preparation and maintenance of stock culture reactor is described fully in Biswas and Bose (2005), Lavania and Bose (2006) and Smith et al. (1994). 2.2. Hydrogen generation Electrolytic iron powder (Loba Cheme Pvt. Ltd., Mumbai, India) was taken in a 1 L glass bottle which was filled with anoxic and sterilized tap water such that no headspace existed. The tap water had negligible concentrations of nitrate and no nitrite and ammonia. This bottle was connected to a reservoir containing anoxic and sterilized tap water. This whole setup shall henceforth be called the ‘hydrogen generating system’. 2.3. Semi-batch reactor A sealed conical flask of 1000 mL volume was used as the semibatch reactor. The reactor was filled with 1000 mL of mineral medium containing 40 mg L1 N nitrate, prepared using sterilized tap water. The composition of the mineral medium was as specified by Till et al. (1998). Contents of the reactor were purged with nitrogen gas for 15 min. Hydrogen gas was then passed through the reactor for 5 min. The reactor was then seeded with 75 mL of seed from stock culture. Hydrogen gas was applied to the reactor for 5 min every 3 days. Twenty five milliliter of reactor contents were withdrawn once a week followed by addition of 25 mL of mineral medium containing 40 mg L1 N nitrate. Reactor contents were maintained in fully mixed condition. After 8 weeks, daily feeding was started. In phase I, feeding involved withdrawal of 25 mL of the reactor contents once a day and addition of 25 mL nitrate solution (40 mg L1 N) prepared in sterilized tap water. During phase II, feeding involved withdrawal of 20 mL of the reactor contents once a day and adding 20 mL nitrate solution (100 mg L1 N). Reactor operations was exactly similar in
phase III, however, the concentration of nitrate in the feed solution was 150 mg L1 N. In all cases, hydrogen was bubbled through the reactor for 2 min after daily feeding. During phase IV experiments, the ‘hydrogen generation system’ was attached to the denitrification reactor. At the time of daily feeding, 20 mL of the denitrification reactor contents was withdrawn and 10 mL of nitrate solution (containing 300 mg N L1) was added to the denitrification reactor through the funnel. Also, 10 mL of hydrogenated water from the hydrogen generating apparatus was added to the reactor. External hydrogen gas was also intermittently purged into reactor. The external hydrogen supply was gradually stopped, and in phase V experiments, the reactor operated only with the hydrogen supplied with the hydrogenated water. Reactor pH was periodically adjusted by addition of phosphate buffer during all phases of experiments to counter pH increase due to denitrification. The pH, ammonia, nitrite and nitrate concentration was measured in the reactor effluent withdrawn everyday. 2.4. Continuous reactors The description of glass columns used as continuous flow reactors are given elsewhere (see Biswas and Bose, 2005). The media in the column consisted of sand graded to 1–2 mm diameter. Graded sand was thoroughly washed with 0.1 N HCl acid and water before drying. Then the sand was loaded into the columns and the columns were sterilized by autoclaving. The height of media in each column was 10 cm, which corresponded to a volume of 125 cm3. Porosity of the media was approximately 50%. Each column was sealed at the top by a rubber stopper, with two glass tubes pierced though it. 2.4.1. Establishment of anoxic condition The experimental setup consisted of three sterilized up flow columns as described above, connected in parallel to a reservoir containing sterilized nitrogen-sparged distilled water. All three columns were fully flooded with water from the reservoir and maintained in sealed condition for 7 days. Then the columns were disconnected from the distilled water reservoir and connected to a another reservoir containing nitrogen-sparged mineral medium (composition as specified in Till et al., 1998) prepared using sterilized IIT Kanpur tap water, and containing 40 mg L1 N nitrate. Columns were flushed with the mineral medium such that the nitrate concentrations in the effluent from the columns were approximately same as the nitrate concentration in the reservoir. The apparatus was maintained in sealed condition for seven days. No degradation of nitrate was observed in the columns during this period. 2.4.2. Seeding with denitrifying microorganisms The columns were disconnected from the reservoir containing mineral medium and connected to the stock culture reactor. A peristaltic pump (Miclins Model PP 10, Chennai, India) was employed to continuously re-circulate the contents of the stock culture reactor through the columns. The flow rate through each column was 1 mL h1. The biomass in the stock culture reactor was maintained in viable condition during this period through periodic addition of nitrate and application of hydrogen gas. Based on results of previous experiments carried out under similar conditions (see Biswas and Bose, 2005), colonization of the sand media in the columns by microorganisms was determined to have occurred after 15 days of recirculation. The columns were again connected to the reservoir containing sterilized nitrogen-sparged distilled water. The columns were flushed with approximately four bed volumes of distilled water such that the nitrate concentration in the column effluent after flushing was negligible.
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2.4.3. Column operation The experimental apparatus employed for column operation is shown in Fig. 1. The hydrogen generation system was attached to the denitrification columns through a 125 mL capacity mixer bottle. The mixer bottle was filled with hydrogenated water from the ‘hydrogen generation system’. The mixer bottle was also equipped with a tube through which nitrate solution was added once a day. Thus hydrogenated water from the hydrogen generation system was mixed with nitrate solution in the mixer bottle. This mixture was fed to the denitrification reactors using a peristaltic pump (Miclins Model PP 10, Chennai, India). Simultaneously, the mixer bottle was refilled at the same rate with water from the hydrogen generation reactor. The pH, ammonia, nitrite and nitrate concentration in the effluent from the denitrification reactors was analyzed on a daily basis. Column experiments were carried out in six phases. At the start of phase I, the nitrate concentration in mixer bottle was adjusted to 1.000 mg L1 N nitrate. The contents of the mixer bottle were pumped through the denitrification columns such that the flow rate through each column was 0.667 mL h1. This resulted in a gradual decrease in nitrate concentration in the mixer bottle and hence the nitrate concentration in the feed to the reactors from 1.000 mg L1 N at the start to 0.616 mg L1 N at the end of a 24-h cycle. After 24 h, 2.4 mL of nitrate solution containing 20 mg L1 N nitrate was added to the mixer bottle such that the nitrate concentration in the mixer bottle was restored to 1.000 mg L1 N nitrate. Phase I experiments were carried out for five 24-h cycles. In phase II experiments, the flow rate through each denitrification column was reduced to 0.333 mL h1. As in phase I experiments, the nitrate concentration in the mixer bottle was 1.000 mg L1 N nitrate at the start of a cycle. This value declined gradually to 0.808 mg L1 N at the end of a 24-h cycle. After 24 h, 1.200 mL of nitrate solution containing 20 mg L1 N nitrate was added to the mixer bottle such that the nitrate concentration in the mixer bottle was restored to 1.000 mg L1 N. Phase II experiments were carried out for 20 24h cycles. Before start of phase III experiments, the nitrate concentration in the mixer bottle was adjusted to 0.500 mg L1 N. Flow rate through each denitrification column was 0.333 mL h1. This nitrate concentration declined gradually from 0.500 to 0.404 mg L1 N at the end of a 24-h cycle. Nitrate concentration in the mixer bottle at the start of each 24-h cycle was adjusted to 0.5 mg L1 N through addition of 0.6 mL of nitrate solution containing 20 mg L1 N nitrate directly to the mixer bottle. Phase III experiments were carried out for 30 24-h cycles. Phase IV experiments were similar to phase III experiments, except that the entire effluent from the denitrification columns were recycled back in the hydrogen generation system using peristaltic pumps (Miclins Model PP 10, Chennai, India), as shown in Fig. 1. Phase V experi-
PP PP Effluent
PP Influent Nitrate Hydrogenated Water
Hydrogen Generation System
1 Mixer
PP: Peristaltic Pumps
2
3
PP
Two-Way Valves
Recycling of Effluent to Hydrogen Generation System
Fig. 1. Schematic of the continuous reactor system. The recycling of effluent to the hydrogen generation system was only done in phase IV experiments.
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ments were similar to phase III experiments, except the flow rate through column 3 was increased from 0.333 mL h1 to 0.667 mL h1, while keeping the flow rates through columns 1 and 2 same as in phase III. In phase V experiments, nitrate concentration in mixer bottle declined from 0.500 to 0.372 mg L1 N nitrate over a 24-h period. Phase V experiments were carried out for 10 24-h cycles. In Phase VI experiments, the flow in column 3 was again reduced to 0.333 mL h1, while flow rates through columns 1 and 2 remained same as before. Thus phase VI experiments were same in all respects to phase IV experiments. Phase VI experiments were carried out for 10 24-h cycles. Before phase VII experiment, column 3 was detached from experimental set up, autoclaved, and re-attached. Otherwise, phase VII experiments were similar in all respects to phase VI experiments. 2.5. Analytical methods Nitrate and nitrite was measured using an Ion Chromatograph (Metrohm 761) equipped with a Phenomenex STAR ION A 300 IC anion column and a conductivity detector with ion suppression. The method used for this purpose was specified in the relevant company literature. Detection limit for both nitrate and nitrite was 0.01 mg L1. All samples for nitrate and nitrite determination were filtered through 0.2 lm filter paper and sterilized before storage under refrigeration until analysis. The samples stored for nitrate determination were also used for ammonia determination. Ammonia was measured colorimetrically by nesslerization (Method No. 417 B, APHA et al., 1985). A spectophotometer (Spectronic, 20 D+, India) with Borosil glass absorbance cells having 1 cm path length were used for this purpose. The detection limit for ammonia determination was 0.02 mg L1. The pH was also measured in all the samples. pH was measured using a combination pH electrode (Toshniwal CL-51, India) connected to a digital pH meter (Toshniwal CL-54, India). The optical density of the stock culture contents was determined as a surrogate measure for biomass concentration (Till et al., 1998). For this purpose absorbance @600 nm of the stock culture was measured using 4 cm path length spectroscopic cell. 3. Results and discussion 3.1. Denitrification in semi-batch reactors The results of the five phases of the semi-batch experiments are presented in Table 1. In phase I, the average steady-state effluent nitrate concentration was 0.20 mg L1 N. Nitrate loading and HRT were 1000 mg N m3 d1 and 40 days, respectively, while steadystate nitrate removal in the reactor was 99.50% (see Table 1). Assuming assimilatory nitrate uptake by denitrifying microorganisms to be negligible in comparison to denitrification, and the fact that ammonia and nitrite concentration in the reactor effluent was below detection limits, the entire nitrate removal may be attributed to denitrification. In phase II, the average steady-state effluent nitrate concentration was 0.27 mg L1 N, while no nitrite or ammonia was detected in the effluent. In this phase, nitrate loading and HRT were 2000 mg N m3 d1 and 50 days, respectively, while steady-state nitrate removal in the reactor was 99.73% (see Table 1). In phase III, the average steady-state effluent nitrate concentration was 3.9 mg L1 N. In this phase, nitrate loading and HRT were 3000 mg N m3 d1 and 50 days, respectively, while the steady-state nitrate removal was 97.54% (see Table 1). Nitrite and ammonia concentrations were below detection limits in the effluent from phase II and III experiments. Effluent pH in all three phases was in the 7.8–8.2 range. In phase IV experiments, a transition was made from gaseous hydrogen to hydrogenated water as the source of hydrogen for denitrification. The average steady-state effluent nitrate concentra-
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Table 1 Summary of the performance of semi-batch reactor.
Phase-I Phase-II Phase-III Phase-IV Phase-V
Nitrate loading (mg N m3 d1)
HRT (days)
Steady-state effluent nitrate concentration (mg N L1)
Steady-state nitrate removal percentage
pH
Duration of operation (days)
1000 2000 3000 3000 3000
40 50 50 50 50
0.20 0.27 3.69 3.89 4.42
99.50 99.73 97.54 97.41 97.05
7.8–8.2 7.8–8.2 7.8–8.2 7.2–8.0 7.2–8.0
40 50 50 50 50
tion was 3.885 mg L1 N during phase IV experiments, which corresponded to 97.41% nitrate removal (see Table 1). During phase V experiments, the average steady-state effluent nitrate concentration was 4.424 mg L1 N, which corresponds to 97.05% nitrate removal. As in previous phases, nitrite and ammonia concentrations were below detection limits in the effluent from phase IV and V experiments. Effluent pH in phases IV and V were in the 7.2–8.0 range. Comparison of the phase V results with the phase IV and phase III results indicate that extent of denitrification and effluent nitrate concentrations are comparable in the three phases. It was thus concluded that during phase V experiments, the hydrogen supplied through hydrogenated water was capable of sustaining denitrification at the same rate as in Phases III and IV experiments. 3.2. Denitrification in continuous flow reactors Results of the experiments with continuous flow reactors are summarized in Table 2. From the results of phase I experiments (nitrate loading: 103 mg N m3 d1; HRT: 7.8 days) it was determined that the effluent nitrate concentration measured at the end of each 24-h cycle showed an increasing trend, with the effluent nitrate concentrations being 0.342, 0.376 and 0.368 mg L1 N in columns 1, 2 and 3, respectively, after the fifth 24-h cycle. These results indicated that the nitrate loading in the columns was too high and HRT too low. Hence, nitrate loading to the columns were reduced and HRT increased (nitrate loading: 57.8 mg N m3 d1; HRT: 15.6 days) in phase II experiments. In phase II experiments, steady-state effluent nitrate concentration was observed after 15 days of column operations in this mode. The average steadystate effluent nitrate concentration (21st–25th days) was 0.501,
0.465 and 0.384 mg L1 N in columns 1, 2 and 3, respectively. This corresponded to nitrate removal of 44.5%, 48.5% and 57.5%, respectively. To further improve the column performance, the nitrate loading to the columns was further reduced (nitrate loading: 28.9 mg N m3 d1; HRT: 15.6 days) in phase III experiments. During phase III experiments, steady-state effluent nitrate concentration was observed after 10 days of column operations in this mode. The average steady-state effluent nitrate concentration (36th–55th days) was 0.025, 0.031 and 0.031 mg L1 N in columns 1, 2 and 3, respectively. This corresponded to nitrate removal of 94.5%, 93.1% and 93.1%, respectively. Ammonia and nitrite concentrations were below detection limits in the effluent from the denitrification reactors at all times during phases I, II or III experiments. Effluent pH was also below 8 in all reactors at all times. Phase IV experiments were similar to phase III, except that the effluent from the denitrification reactors were recycled back to the hydrogen generation system (see Fig. 1). Steady-state effluent nitrate concentration was observed during the entire duration of this phase of column operation. The average steady-state effluent nitrate concentration (56th–75th days) was 0.024, 0.030 and 0.022 mg L1 N in columns 1, 2 and 3, respectively. This corresponded to nitrate removal of 94.7%, 93.4% and 95.1%, respectively. Further, ammonia and nitrite concentrations were below detection limits in the effluent from the denitrification reactors at all times during phase IV experiments. Effluent pH was also below 8 in all reactors at all times. The results of this phase of experiments indicated that, provided nitrate removal is nearly complete, effluent from the denitrification reactors could be recycled to the hydrogen generating system for production of hydrogenated water required for denitrification column operations without any impact on ammonia and nitrite concentrations in the effluent.
Table 2 Summary of the performance of continuous flow reactors.
Phase I
Phase II
Phase III
Phase IV
Phase V
Phase VI
Phase VII
Reactor Reactor Reactor Reactor Reactor Reactor Reactor Reactor Reactor Reactor Reactor Reactor Reactor Reactor Reactor Reactor Reactor Reactor Reactor Reactor Reactor
1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3
Average influent nitrate loading (mg N m3 d1)
HRT (days)
Duration of operation
Effluent nitrate concentration (mg N L1)
Percent nitrate removal
Trend of effluent nitrate concentration
103 103 103 57.8 57.8 57.8 28.9 28.9 28.9 28.9 28.9 28.9 27.9 27.9 55.8 28.9 28.9 28.9 28.9 28.9 28.9
7.8 7.8 7.8 15.6 15.6 15.6 15.6 15.6 15.6 15.6 15.6 15.6 15.6 15.6 7.8 15.6 15.6 15.6 15.6 15.6 15.6
1st–5th days
0.342 0.376 0.368 0.501 0.465 0.384 0.025 0.031 0.031 0.024 0.030 0.022 0.019 0.023 0.322 0.024 0.026 0.036 0.024 0.026 0.416
57.7 53.5 54.4 44.5 48.5 57.5 94.5 93.1 93.1 94.7 93.4 95.1 95.6 94.7 26.1 94.7 94.2 92.0 94.7 94.2 8.00
" " " M M M M M M M M M M M " M M ; M M "
": increasing trend; ;: decreasing trend; M: steady-state.
6th–25th days
26th–55th days
56th–75th days
76th–85th days
86th–95th days
96th–135th days
(average (average (average (average (average (average (average (average (average (average (average
of of of of of of of of of of of
21st–25th days) 21st-25th days) 21st–25th days) 36th–55th days) 36th–55th days) 36th–55th days) 56th–75th days) 56th–75th days) 56th–75th days) 76th–85th days) 76th–85th days)
(average of 86th–95th days) (average of 86th–95th days) (average of 96th–135th days) (average of 96th–135th days)
N. Sunger, P. Bose / Bioresource Technology 100 (2009) 4077–4082
In phase V experiments, nitrate loading and HRT in columns 1 and 2 were almost unchanged from phase III (nitrate loading: 27.9 mg N m3 d1; HRT: 15.6 days), while nitrate loading and HRT values for column 3 were 55.8 mg N m3 d1 and 7.8 days, respectively. While performance of columns 1 and 2 remained essentially unchanged with respect to effluent nitrate concentration, the effluent nitrate concentration from column 3 showed an increasing trend, and increased to 0.322 mg L1 N nitrate (see Table 2) after 10 days of reactor operation. These results clearly show the detrimental effect of increase in nitrate loading rate and reduction in HRT on the column performance. In phase VI experiments, nitrate loading and HRT in columns 1, 2 and 3 were restored to earlier values of 28.9 mg N m3 d1 and 15.6 days, respectively. While the performance of columns 1 and 2 remained essentially unchanged, the nitrate concentration in the effluent from column 3 showed a decreasing trend, and after 10 days of operation was reduced to 0.036 mg L1 N (see Table 2). Column 3 was sterilized by autoclaving before phase VII experiments. Afterwards, all three columns were operated under conditions similar to that in phase VI (see Table 2). While the performance of columns 1 and 2 remained essentially unchanged, effluent nitrate concentration from column 3 showed an increasing trend and the concentration after 40 days of column operation was 0.416 mg L1 N (see Table 2), which corresponded to only 8% nitrate removal in the column. This proved that biological denitrification was responsible for reduction in nitrate concentration in all columns during this study. Ammonia and nitrite concentration in the effluent from all columns in phases V–VII of experiments was below the detection limits and effluent pH was always below 8. 3.3. Discussion In a study designed to investigate the performance of a hollowfiber membrane bioreactor (HFMB) for hydrogenotrophic denitrification of contaminated drinking water, Ergas and Reuss (2001) reported nitrate utilization rates up to 770 g N m3 d1. Influent nitrate concentrations of up to 200 mg N L1 were almost completely denitrified in a reactor with 4.1 h HRT. In another study (Chang et al., 1999) A. eutrophus, a hydrogenotrophic denitrifier, was immobilized in polyacrylamide and alginate copolymer to evaluate denitrification in continuous mode and batch mode in a fluidized-bed reactor. The total nitrogen removal rate in a continuous test reached a maximum rate of 0.6–0.7 kg N m3 day1. In another study (Sierra-Alvarez et al., 2005) on autotrophic denitrification using sulfide as electron donor, 89% nitrate removal was reported with nitrate loading rate of 1.24 kg m3 d1 and HRT of 13.4 h. In comparison, the nitrate loading rates applied in continuous reactors during the present study are several orders of magnitude lower (28.9 mg N m3 d1) and HRT higher (15.6 days). The results presented in Table 2 also show that any increase in nitrate loading rate or any decrease in HRT resulted in a decline in effluent quality, possibly because of hydrogen limitation. Limitations in the experimental setup used in the present study precluded application of large hydrogen concentrations to the reactor and consequently the applied nitrate loading rates had to be kept low to maintain the nitrate limiting conditions required for high percentage nitrate removal. It is, however, postulated that the nitrate loading rates in such reactors could be significantly increased by designing the hydrogen generation system as a pressure vessel and the denitrification columns as pressure filters, wherein higher hydrogen partial pressures and hence higher hydrogen solubility in water could be maintained. Contrary to expectations, pH measurements during continuous column operations showed that pH never increased beyond 8 at
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any time. It appears that the natural buffering capacity of the influent feed was sufficient to prevent any pH increase in these columns. However, in cases where denitrification occurs at a higher rate, substantial pH increase inside the column is expected, with consequent adverse effects on denitrification rate and the product water quality (Jha and Bose, 2005). In such cases, the inherent pH buffering capacity of the porous media must be increased through addition of buffering agents. In this context, Jha and Bose (2005) have demonstrated that addition of pyrite in porous media columns was effective in controlling pH increase with no detrimental effect on the denitrification process. 4. Summary and conclusions Biological denitrification using hydrogen derived from anoxic corrosion of metallic iron as energy source was demonstrated in laboratory scale reactors. Following are the main conclusions from this study, Denitrification up to 99% and 95% was demonstrated in semibatch and continuous reactors, respectively. Under steady-state conditions, a semi-batch reactor with nitrate loading and HRT of 2000 mg m3 d1 and 50 days, respectively, produced effluent with nitrate concentration of 0.27 mg N L1. Continuous reactors with nitrate loading and HRT of 28.9 mg m3 d1 and 15.6 days produced effluent with nitrate concentration of 0.025 mg N L1. In all cases, the effluent contained negligible concentrations of nitrite and ammonia. It was further demonstrated that the effluent from the continuous flow reactor could be recycled to produce hydrogenated water required for the denitrification process, with no adverse impact on the effluent quality. However, significant unresolved technical challenges related to reactor scale up must be resolved before such reactors can be commercially applied for denitrification of real water/wastewater. Most importantly, to operate such reactors at higher nitrate loading rates and lower HRT than demonstrated in this study, the concentration of hydrogen in the hydrogenated water required for reactor operation must be significantly increased. Acknowledgements The authors gratefully acknowledge the financial help provided by the Department of Science and Technology of the Government of India through Project No. III.5(119)2001-SERC Engg. for carrying out this study. References APHA, WEF, AWWA, 1985. Standard Methods for Examination of Water and Wastewater, 16th ed. APHA, Washington, DC, USA. Biswas, S., Bose, P., 2005. Zero-valent iron-assisted autotrophic denitrification. Journal Environmental Engineering ASCE 131, 1212–1220. Celmer, D., Oleszkiewicz, J., Cicek, N., Husain, H., 2006. Hydrogen limitation – a method for controlling the performance of membrane biofilm reactor for autotrophic denitrification of wastewater. Water Science and Technology 54, 165–172. Chang, C.C., Tseng, S.K., Huang, H.K., 1999. Hydrogenotrophic denitrification with immobilized Alcaligenes eutrophus for drinking water treatment. Bioresource Technology 69, 53–58. Cowman, J., Torres, C.I., Rittmann, B.E., 2005. Total nitrogen removal in an aerobic/ anoxic membrane biofilm reactor system. Water Science and Technology 52, 115–120. CPHEO, 1999. Manual on Water Supply and Treatment. Third ed., Ministry of Urban Development, Govt. of India, New Delhi, India. Dries, D., Liessens, J., Verstraere, W., Stevens, P., De Vost, P., De Ley, J., 1988. Nitrate removal from drinking water by means of hydrogenotrophic denitrifiers in a polyurethane carrier reactor. Water Supply 6, 181–192.
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Ergas, S.J., Reuss, A.F., 2001. Hydrogenotrophic denitrification of drinking water using a hollow fibre membrane bioreactor. Journal of Water Supply: Research and Technology – AQUA 50, 161–171. Fan, A.M., Steinberg, V.E., 1996. Health implications of nitrate and nitrite in drinking water: an update on methemoglobinemia occurrence and reproductive and developmental toxicity. Regulatory Toxicology and Pharmacology 23, 35–43. Fan, A.M., Willhite, C.C., Book, S.A., 1987. Evaluation of the nitrate drinking water standard with reference to infant methemoglobinemia and potential reproductive toxicity. Regulatory Toxicology and Pharmacology 7, 135–148. Gangolli, S.D., Van der Brandt, P.A., Feron, V.J., 1994. Nitrate, nitrite and N-nitroso compounds. European Journal of Pharmacology-Environmental Toxicology and Pharmacology 292, 1–38. Ghafari, S., Hasan, M., Aroua, M.K., 2008. Bio-electrochemical removal of nitrate from water and wastewater – a review. Bioresource Technology 99, 3965–3974. Jha, D., Bose, P., 2005. Use of pyrite for pH control during hydrogenotrophic denitrification using metallic iron as the ultimate electron donor. Chemosphere 61, 1020–1031. Komori, M., Sakakibara, Y., 2008. High-rate hydrogenotrophic denitrification in a fluidized-bed biofilm reactor using solid-polymer-electrolyte membrane electrode (SPEME). Water Science and Technology 58, 1441–1446. Kurt, M., Dunn, I.J., Bourne, J.R., 1987. Biological denitrification of drinking water using autotrophic organisms with H2 in a fluidized-bed biofilm reactor. Biotechnology and Bioengineering 29, 493–501. Lavania, A., Bose, P., 2006. Effect of porous media composition on end-product distribution during metallic iron assisted autotrophic denitrification. Journal of Environmental Engineering, ASCE 132, 994–1000. Lee, K.C., Rittmann, B.E., 2003. Effects of pH and precipitation on autohydrogenotrophic denitrification using the hollow-fiber membranebiofilm reactor. Water Research 37, 1551–1556. Mansell, B.O., Schroeder, E.D., 2002. Biological denitrification in a continuous flow membrane reactor. Water Research 33, 1845–1850. Mo, H., Oleszkiewicz, J.A., Cicek, N., Rezania, B., 2005. Incorporating membrane gas diffusion into a membrane bioreactor for hydrogenotrophic denitrification of groundwater. Water Science and Technology 51, 357–364.
Rezania, B., Oleszkiewicz, J.A., Cicek, N., 2007. Hydrogen-dependent denitrification of water in an anaerobic submerged membrane bioreactor coupled with a novel hydrogen delivery system. Water Research 41, 1074–1080. Rittmann, B.E., 2006. The membrane biofilm reactor: the natural partnership of membranes and biofilm. Water Science and Technology 53, 219–225. Schnobrich, M.R., Chaplin, B.P., Semmens, M.J., Novak, P.J., 2007. Stimulating hydrogenotrophic denitrification in simulated groundwater containing high dissolved oxygen and nitrate concentrations. Water Research 41, 1869– 1876. Shin, J.H., Sang, B.I., Chung, Y.C., Choung, Y.K., 2005. The removal of nitrogen using an autotrophic hybrid hollow-fiber membrane biofilm reactor. Desalination 183, 447–454. Sierra-Alvarez, R., Guerrero, F., Rowlette, P., Freeman, S., Field, J.A., 2005. Comparison of chemo-, hetero- and mixotrophic denitrification in laboratoryscale UASBs. Water Science and Technology 52, 337–342. Smith, R.L., Buckwalter, S.P., Repert, D.A., Miller, D.N., 2005. Small-scale, hydrogenoxidizing-denitrifying bioreactor for treatment of nitrate-contaminated drinking water. Water Research 39, 2014–2023. Smith, R.L., Ceazan, M.L., Brooks, M.H., 1994. Autotrophic, hydrogen-oxidizing, denitrifying bacteria in groundwater, potential agents for bioremediation of nitrate contamination. Applied and Environmental Microbiology 60, 1949– 1955. Tan, Y.M., Liu, Y.Q., Su, F., Wang, L., Luo, Q.F., 2006. Experimental study on denitrification using coated electrode of immobilized denitrifying bacteria. Biomedical and Environmental Sciences 19, 47–52. Till, B.A., Weathers, L.J., Alvarez, P.J., 1998. Fe(0)-supported autotrophic denitrification. Environmental Science and Technology 32, 634–639. Wan, D.J., Liu, H.J., Qu, J.H., Lei, P.J., Mao, S.H., Hou, Y.N., 2009. Using the combined bioelectrochemical and sulfur autotrophic denitrification system for groundwater denitrification. Bioresource Technology 100, 142–148. Ward, M.H., Zahm, S.H., Blair, A., 1994. Dietary factors and non-Hodgkin’s lymphoma. Cancer Causes and Control 5, 422–432.