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The heterotrophic-combined-with-autotrophic denitrification process: performance and interaction mechanisms Guihua Xu, Cuijie Feng, Fang Fang, Shaohua Chen, Yuanjian Xu and Xingzu Wang
ABSTRACT In this work, the interaction mechanisms between an autotrophic denitrification (AD) and heterotrophic denitrification (HD) process in a heterotrophic-autotrophic denitrification (HAD) system were investigated, and the performance of the HAD system under different S/Ac molar ratios was also evaluated. The results demonstrated that the heterotrophic-combined-with-autotrophic denitrification process is a promising technology which can remove chemical oxygen demand (COD), sulfide and nitrate simultaneously. The reduction rate of NO 3 to NO2 by the HD process was much faster than that of reducing NO 2 to N2, while the reduction rate of NO3 to NO2 by the AD process was slower than that of NO 2 to N2. Therefore, the AD process could use the surplus NO2 produced
by the HD process. This could alleviate the NO 2 –N accumulation and increase the denitrification rate. In addition, the inhibition effects of acetate on AD bacteria and sulfide on HD were observed, and the inhibition was compensated by the promotion effects on NO 2 . Therefore, the processes of AD and HD seem to react in parallel, without disturbing each other, in our HAD system. Key words
Guihua Xu Yuanjian Xu Xingzu Wang Key Laboratory of Reservoir Aquatic Environment, Chongqing Institute of Green and Intelligent Technology, Chinese Academy of Sciences, Chongqing 400714, China Guihua Xu Cuijie Feng Fang Fang Shaohua Chen (corresponding author) Key Laboratory of Urban Environment and Health, Institute of Urban Environment, Chinese Academy of Sciences, Xiamen 361021, China E-mail: [email protected]
| autotrophic denitrification, heterotrophic denitrification, interaction mechanism
INTRODUCTION The disposal of waters contaminated by nitrate or nitrite plays an important role in wastewater treatment because improperly treated wastewaters may cause serious ecological damage to the receiving water bodies (e.g., eutrophication) (Moraes & Foresti ). Previous studies have reported that nitrate caused severe health issues, such as methemoglobinemia (blue baby syndrome), and the increased risk of the formation of carcinogens (Kim et al. ). 5CH3 COOH þ 8NO 3 ! 10CO2 þ 4N2 þ 9H2 O þ 12OH
(1) Owing to the high-denitrification efficiency of the heterotrophic denitrification (HD) process, which was widely used to treat waters contaminated by nitrate, the HD process needs organic carbon sources to reduce nitrate to N2 (Equation (1)); therefore, the HD removal efficiency depends on the biochemical oxygen demand (BOD5)/N doi: 10.2166/wst.2015.097
ratio in the wastewater (Soares & Abeliovich ). Furthermore, the lack of carbon sources often results in the failure of nitrate removal. In the case of organic deficient (low BOD5/N) wastewater, the addition of external carbon sources is needed to increase the nitrate removal. However, this will increase the complexity of the operations and treatment costs. Therefore, some researchers have turned to the autotrophic denitrification (AD) process (Flere & Zhang ; Koenig & Liu ; Sierra-Alvarez et al. ; Moraes et al. ; Sun & Nemati ), which does need not any organic carbon sources and produces less biosludge (Campos et al. ; Nanda et al. ; Fajardo et al. ). However, the AD process also has shortcomings, such as a slow denitrification rate, SO2 in the effluent 4 exceeding the discharge standard, alkalinity consumption and so on (Equations (2)–(4)). Thus, the AD process cannot completely replace the traditional HD technology. þ 5S2 þ 2NO 3 þ 12H ! 5S þ N2 þ 6H2 O
(2)
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2 þ 5S þ 6NO 3 þ 2H2 O ! 5SO4 þ 3N2 þ 4H
(3)
2 þ 5S2 þ 8NO 3 þ 8H ! 5SO4 þ 4N2 þ 4H2 O
(4)
In practice, some industrial wastewater contains carbon, nitrogen and sulfur compounds, such as that from the oil industry, gourmet powder factories, the pharmaceutical industry and so on (Wang et al. ). Moreover, some features of the AD and HD processes may be complementary to each other, such as reducing the demand for organic carbon and basicity. These make it possible to combine the AD and HD processes to overcome their respective defects, and combining the AD and HD processes may be used for the above-mentioned wastewater to remove carbon, nitrogen and sulfur simultaneously. In the last 10 years, researchers have begun to study the heterotrophic-combined-with-autotrophic denitrification (HAD) process (Beristain-Cardoso et al. ; Chen et al. ; Sahinkaya & Kilic ). In 2001, Oh et al. () proved that the AD and HD processes can coexist and complete the denitrification process together. After that, the influence factors (e.g., S/N, chemical oxygen demand (COD)/N ratio) and operation parameters (e.g., residence times) were studied (Reyes-Avila et al. ; Rocca et al. ; Liu et al. ). However, the performance of the HAD process is not completely understood, and the interaction mechanisms between the HD and AD processes in the HAD system are unclear. These unclear problems limit the application of HAD technology in wastewater treatment. Based on these problems, this work aims to investigate the interaction mechanisms between the HD and AD processes in the HAD system for the simultaneous removal of carbon, nitrogen and sulfide. Moreover, the performance of the HAD system under different S/Ac molar ratios was evaluated.
MATERIALS AND METHODS Experimental setup and operations To study the interaction mechanisms between the HD and AD processes in the HAD system, a Plexiglas-made reactor was established. Acetate and sulfide were used as electron donors for the HD and AD processes in the HAD system. The effects of sulfide on the HD process and acetate on the AD process need to be further investigated; therefore, the AD and HD reactors were established to investigate
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the effects of sulfide and acetate. Five liters of seeding sludge for the AD and HD reactors was collected from the Shiweitou Municipal Wastewater Treatment Plant (Xiamen, China), and the concentration of suspended solids (SS) was 3,776 ± 352 mg/L. The AD and HD reactors were operated stably for 1 year, and the HAD reactor was inoculated with 0.5 L of sludge collected on day 140 from the AD reactor and 0.5 L of sludge from the HD reactor. After the three reactors reached steady state, the SS concentration of the AD, HD and HAD reactors varied slightly and remained at 1,480 ± 157 mg L1, 2,736 ± 271 mg L1 and 2,239 ± 182 mg L1, respectively. The sludge retention times (SRT) of the AD, HD and HAD reactors were kept at 30 d by discharging excess sludge. The detailed experimental setup and operation conditions are listed in the Supplementary Materials (available online at http://www. iwaponline.com/wst/071/097.pdf).
Experimental conditions The performance of the HAD system under five S/Ac molar ratios (5:0.7, 5:1.8, 5:3.5, 5:7.1 and 5:17.6) was first evaluated, and the detailed experimental conditions are listed in Table S1 in the Supplementary Materials (available online at http://www.iwaponline.com/wst/071/097.pdf). Furthermore, to investigate the interaction mechanisms between the HD and AD processes in the HAD system, nitrite accumulation and the effects of electron donors (acetate and sulfide) were studied (Table S1). Nitrite accumulations under different S/N and Ac/N ratios were conducted in the HAD reactor, as summarized in Table S1. For the S/N molar ratio tests, the HAD sludge used sulfide as the sole electron donor (without acetate) and four S/N molar ratios were applied to the HAD influents: 5:2, 5:5, 5:8 and 5:12 (Table S1). For the different Ac/N molar ratio experiments, similar to the S/N molar ratio tests, the experiments were conducted without sulfide, and four Ac/N molar ratios were applied to the influents of the HAD reactor: 5:3.4, 5:5.9, 5:10.1 and 5:20.2 (Table S1). In addition, nitrite accumulation validation tests 1, 2 and 3 were conducted to compare the nitrite concentration in the HAD reactor under different electron donor conditions. Three different electron donors were applied to the HAD reactor for tests 1, 2 and 3: sodium sulfide (2.0 g/L), sodium acetate (0.5 g/L) and sodium sulfide (2.0 g/L) with sodium acetate (0.5 g/L), respectively (Table S1). Inhibition tests A and B were used to study the effects of sulfide on the HD process and acetate on the AD process,
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respectively (Table S1). For the effects of sulfide on the HD process (test A), 2 g L1 sodium sulfide was imposed on the HD reactor while keeping the other conditions unchanged. For the effects of acetate on the AD process (test B), 0.5 g L1 sodium acetate was conducted to the AD reactor while keeping the other conditions unchanged (Table S1). Chemical analysis Samples were filtrated by 0.45-μm filters before taking measurements. The pH, SRT, SS concentration and temperature were determined by Standard Methods (APHA ). 2 The Ac, NO 2 , NO3 and SO4 were measured by ion chromatography (ICS3000, DIONEX, USA) according to the manufacturer protocol. The ion chromatography equipment used an Ion Pac column (As15A 4 × 250 mm, AG15), and the injection volume of the sample was 10 μL. The S2 concentration was measured according to the method of a previous study (Cord-Ruwisch ).
Figure 1
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The performance of the HAD process under different S/Ac molar ratios In the HAD reactor, after 22 h of reaction, at the end-point of the denitrification, the eventual nitrate removal efficiency was 89.0%, 87.7%, 85.4%, 83.1% and 82.6% at an S/Ac ratio of 5:0.7, 5:1.8, 5:3.5, 5:7.1 and 5:17.6, respectively (Figure 1(a)). The consumption of acetate was completed in 2.25 h, 2 h, 1.5 h, 1.25 h and 0.5 h at an S/Ac of 5:17.6, 5:7.1, 5:3.5, 5:1.8 and 5:0.7, respectively (Figure 1(b)), and the consumption of sulfide was finished after 0.75 h, 1.5 h, 2.0 h, 2.5 h and 3.0 h, respectively (Figure 1(c)). Meanwhile, a continuous decrease of nitrate was also obtained, which indicated that the AD process and HD process occurred simultaneously in the HAD reactor. The results showed that the HAD process is a feasible process for simultaneous removal of COD, sulfide and nitrate.
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Profiles of nitrite removal efficiency and acetate, sulfide and sulfate concentrations in the HAD reactor under various S/Ac acetate; (c) sulfide; (d) sulfate; all with an initial 194 mg L
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molar ratios. (a) Nitrite removal efficiency; (b)
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The denitrification process of HAD could be divided into two stages: S2 and Ac as electron donors (stage I, Equations (1) and (2)) and S0 as an electron donor (stage II, Equation (3)). In stage I, with 5:0.7, 5:1.8, 5:3.5, 5:7.1 and 5:17.6 as the S/Ac ratio, the sulfide concentration in the HAD reactor decreased sharply from the initial value of 333.2, 266.6, 200.1, 133.3 and 66.7 mg L1 to 227.6, 175.5, 127.1, 80.8 and 0.0 mg L1 in 0.75 h, respectively (Figure 1(c)), while the sulfate concentration increased slightly (Figure 1(d)). During this stage, the color of the liquid in the HAD reactor changed from yellow to a creamy white, an indication of bio-oxidation of S2 to S0. After the S2 concentration decreased to zero, a continuous increase of sulfate was observed (Figure 1(d)). This indicated the bio-oxidation of S0 ! SO2 4 (stage II), which was consistent with the result of the previous study (Beristain Cardoso et al. ; An et al. ). Interaction mechanisms between the HD and AD processes in the HAD system NO 2 –N accumulation in the AD process The results of NO 2 –N accumulation for the AD process without acetate in the HAD reactor are shown in Figure 2(a). The NO 2 –N concentration was close to zero at the beginning (after 5 h); the NO 2 –N concentration then increased rapidly after 7 h for S/N ¼ 5:12 and after 10 h for S/N ¼ 5:5, and 5:8. NO 2 –N was not detected throughout the test of S/N ¼ 5:2. The results showed that the reduction rate of NO 2 to N2 was 2 faster than that of NO ! S0 stage, which 3 to NO2 in the S resulted in no NO 2 –N accumulation at the beginning of the AD process (5 h). The results were consistent with the findings of B.S. Moraes in which the half-order kinetic coefficient was 0.425–0.658 mg N (L h)1 for NO 2 to N2 and 0.190– 0.609 mg N (L h)1 for denitrification via nitrate (Moraes & Foresti ). However, when S2 was exhausted and S0 was used as the electron donor (stage II), the reduction rate of NO 2 to N2 became slower than that of. NO3 to NO2 . There fore, the accumulation of NO2 occurred in this stage. NO 2 –N accumulation in the HD process Nitrite accumulation experiments for the HD process without sulfide in the HAD reactor under different Ac/N molar ratios were conducted. The concentrations of NO 2– N increased dramatically when the acetate concentration in the influent increased, and the maximum value of NO 2 –N accumulated was 101.5 mg L1, 85.2 mg L1, 55.1 mg L1 and 25.3 mg L1 at Ac/N of 5:3.4, 5:5.9, 5:10.1 and 5:20.2,
Figure 2
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Nitrite accumulation in the HAD reactor under different substrates. (a) Sulfide as an electron donor under various S/N molar ratios; (b) acetate as an electron
donor under various Ac /N molar ratios; (c) comparisons of nitrite accumulation under acetate, sulfide and the two substrates together, all with an initial 194 mg L 1 NO3 –N.
respectively (Figure 2(b)). The results indicated that the HD process reduction rate of NO 3 to NO2 was much faster than the reduction rate of NO 2 to N2. Therefore, a large
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amount of nitrite accumulation occurred (Figure S1 in the Supplementary Materials, available online at http://www. iwaponline.com/wst/071/097.pdf). In the HAD system, the reduction of NO 2 to N2 was the limited step for the HD pro cess, while the reduction of NO 3 to NO2 was slower than NO2 to N2 for the AD process. The AD process could use the surplus nitrite production from the HD process (Figure S1). Therefore, the combination of the AD and HD processes might increase the denitrification rate and decrease the amount of nitrite accumulation. Comparison of nitrite accumulation between the AD, HD and HAD processes Three different electron donors were applied to the HAD reactor: 2.0 g/L sodium sulfide (test 1), 0.5 g/L sodium acetate (test 2) and the two electron donors together (test 3) (Table S1 in the Supplementary Materials, available online at http://www.iwaponline.com/wst/071/097.pdf). The results of NO 2 –N accumulation are presented in Figure 2(c). After 2 h of reaction, with test 1, 2 and 3, the concentration of NO 2 –N was 0.2 mg L1, 32.6 mg L1 and 7.6 mg L1, respectively. Moreover, after 12 h, the concentration of NO 2 –N changed to 17.1 mg L1, 31.6 mg L1 and 19.5 mg L1, respectively. The NO 2 –N accumulation of the HAD process was higher than that of the AD process and much lower than that of the single HD process. Compared with the HD process, the combination of the AD and HD processes produced less nitrite and could alleviate the NO 2 –N accumulation phenomenon. As discussed above, in the HAD system, the HD process produced nitrite faster than the reduction rate of nitrite, which provided surplus nitrite for the AD process and reduced the total denitrification time. Thus, it might increase the denitrification rate of the HAD process. The verification tests were needed to perform comparisons of the NO 3 –N removal from the experimental data and theoretical calculation results.
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and (6), respectively, where CNO3–N0 is the initial concentration of NO3–N, CNO3–N is the concentration of NO 3 –N at time t, and Ksulfide and Kacetate is the zero-order rate constant of the AD and HD processes, respectively. CNO3 N ¼ CNO3 N0 Ksulphide × t
(5)
CNO3 N ¼ CNO3 N0 Kacetate × t
(6)
If the processes of AD and HD are performed in parallel in the HAD system without disturbing each other, the theoretical concentration of NO 3 –N in the HAD reactor can be calculated by Equation (7). CNO3 N ¼ CNO3 N0 (Kacetate þ Ksulphide ) × t
(7)
The results of the theoretical calculation and experimental data for nitrates are shown in Figure 3(a). The results
Comparison of nitrate removal between the experimental data and theoretical calculation results in the HAD process In the first 2 h of the HAD process (stage I), the substrates (i.e., sulfide and acetate) were sufficient for nitrate removal. The denitrification rate was not related to the concentrations of nitrate, acetate and sulfide and was determined by the activities and quantities of denitrifiers. Therefore, both the AD and HD processes followed the zero-order kinetic model, and the NO 3 –N concentration variations of the AD and HD processes could be calculated by Equations (5)
Figure 3
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Experiment data of nitrate concentration compared with the calculation results in the HAD reactor. (a) Experimental data and calculated data; (b) AD, HD, HAD and calculated data.
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indicate that the experimental data are in good agreement with the calculated values (Figure 3(a)). Furthermore, the calculated data and experimental data of the nitrate removal efficiency were compared, whereas the calculated value of the nitrate removal efficiency was calculated as a simple sum of the HD process value and the AD process value; these results also show that the calculated data are close to the experimental data (Figure 3(b)). These results confirm the hypothesis: the processes of AD and HD were performed in parallel in the HAD system without disturbing each other. The results contradicted the conclusion in the above paragraph that stated that the HAD process had promotional effects on NO 2 and increased the denitrification rate. Therefore, there should exist inhibition factors in the HAD system to offset the promotion. The effects of sulfide on the HD process and acetate on the AD process in the HAD system To find the inhibiting factors in the HAD system, the operational and experimental conditions of the AD, HD and HAD reactors were examined. The operational and experimental conditions of the AD, HD and HAD reactors were identical, except for the electron donors. The HAD process used acetate and sulfide as electron donors, while the AD and HD processes used sulfide and acetate, respectively. Therefore, the effects of sulfide on the HD process and acetate on the AD process might be the inhibiting factors in the HAD system. However, the HAD sludge used acetate and sulfide simultaneously, and the tests of the sulfide and acetate effects on the HAD system could not be conducted in the HAD reactor. The HD reactor and AD reactor were substitutes for investigating the effects of sulfide and acetate on the HAD process. The results are shown in Figure 4; after 10 h of denitrification time, the NO 3 –N concentration in the AD reactor with added acetate was 148.0 mg L1 and the AD reactor without acetate was 108.3 mg L1 (Figure 4(a)). The results demonstrated that acetate had a restraining impact on the activity of the autotrophic denitrifiers. The effect of sulfide on the HD process had similar results, as shown in Figure 4(b). The denitrification rate with sulfide was slower than that without sulfide. The results revealed that the sulfide had an inhibiting impact on the activity of the heterotrophic denitrifiers (Figure S1 in the Supplementary Materials, available online at http://www. iwaponline.com/wst/071/097.pdf). The inhibiting impact reasons are unclear in this work and need further study. `In this study, the inhibition effects caused by acetate on the AD process and sulfide on the HD process in the HAD
Figure 4
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Inhibition factors in the HAD process. (a) The effects of acetate on the AD sludge; (b) the effects of sulfide on the HD sludge.
system offset the promotion effects on NO 2 (Figure S1). Thus, the AD and HD processes looked like they were reacting in parallel without disturbing each other in our HAD system.
CONCLUSIONS This study demonstrated that the HAD process could simultaneous remove acetate, sulfide and nitrate. Sulfide biooxidation in the HAD system occurred in two distinct stages: S2 ! S0 and S0 ! SO2 4 . The AD process used the surplus NO 2 produced by the HD process, which alleviated the NO 2 –N accumulation and increased the denitrification rate of the HAD process. The inhibition effects caused by acetate on AD and sulfide on HD in the HAD process offset the promotional effects on NO 2 . Therefore, the AD and HD processes appeared to react in parallel without disturbing each other in the HAD system.
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ACKNOWLEDGEMENTS This research was supported by the National Natural Science Foundation of China (51108439, 50778156 and 51008025) and the Natural Science Foundation of Chongqing (cstc2014jcyjA20010).
REFERENCES An, S., Tang, K. & Nemati, M. Simultaneous biodesulphurization and denitrification using an oil reservoir microbial culture: effects of sulphide loading rate and sulphide to nitrate loading ratio. Water Research 44 (5), 1531–1541. APHA Standard Methods for the Examination of Water and Wastewater. 20th edn, American Public Health Association/ American Water Works Association/Water Environment Federation, Washington, DC, USA. Beristain Cardoso, R., Sierra-Alvarez, R., Rowlette, P., Razo Flores, E., Gomez, J. & Field, J. A. Sulfide oxidation under chemolithoautotrophic denitrifying conditions. Biotechnology and Bioengineering 95 (6), 1148–1157. Beristain-Cardoso, R., Texier, A. -C., Sierra-Alvarez, R., Field, J. A., Razo-Flores, E. & Gomez, J. Simultaneous sulfide and acetate oxidation under denitrifying conditions using an inverse fluidized bed reactor. Journal of Chemical Technology and Biotechnology 83 (9), 1197–1203. Campos, J. L., Carvalho, S., Portela, R., Mosquera-Corral, A. & Méndez, R. Kinetics of denitrification using sulphur compounds: effects of S/N ratio, endogenous and exogenous compounds. Bioresource Technology 99 (5), 1293–1299. Chen, C., Wang, A., Ren, N., Deng, X. & Lee, D. -J. Optimal process pattern for simultaneous sulfur, nitrogen and carbon removal. Water Science and Technology 59 (4), 833–837. Cord-Ruwisch, R. A quick method for the determination of dissolved and precipitated sulfides in cultures of sulfatereducing bacteria. Journal of Microbiological Methods 4 (1), 33–36. Fajardo, C., Mora, M., Fernández, I., Mosquera-Corral, A., Campos, J. L. & Méndez, R. Cross effect of temperature, pH and free ammonia on autotrophic denitrification process with sulphide as electron donor. Chemosphere 97, 10–15. Flere, J. M. & Zhang, T. C. Sulfur-based autotrophic denitrification pond systems for in-situ remediation of nitratecontaminated surface water. Water Science and Technology 38 (1), 15–22. Kim, I. S., Oh, S. E., Bum, M. S., Lee, J. L. & Lee, S. T. Monitoring the denitrification of wastewater containing high concentrations of nitrate with methanol in a sulfur-packed
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reactor. Applied Microbiology and Biotechnology 59 (1), 91–96. Koenig, A. & Liu, L. H. Kinetic model of autotrophic denitrification in sulphur packed-bed reactors. Water Research 35 (8), 1969–1978. Liu, H., Jiang, W., Wan, D. & Qu, J. Study of a combined heterotrophic and sulfur autotrophic denitrification technology for removal of nitrate in water. Journal of Hazardous Materials 169 (1–3), 23–28. Moraes, B. S. & Foresti, E. Determination of the intrinsic kinetic parameters of sulfide-oxidizing autotrophic denitrification in differential reactors containing immobilized biomass. Bioresource Technology 104, 250–256. Moraes, B. S., Souza, T. S. O. & Foresti, E. Characterization and kinetics of sulfide-oxidizing autotrophic denitrification in batch reactors containing suspended and immobilized cells. Water Science and Technology 64 (3), 731–738. Nanda, J., Whiteley, C. G., Chang, J.-S. & Lee, D.-J. Production of elemental sulfur from sulfide and nitrate-laden wastewaters by methanogenic culture via sulfide denitrifying removal process. Biochemical Engineering Journal 78, 128–131. Oh, S. E., Yoo, Y. B., Young, J. C. & Kim, I. S. Effect of organics on sulfur-utilizing autotrophic denitrification under mixotrophic conditions. Journal of Biotechnology 92 (1), 1–8. Reyes-Avila, J., Razo-Flores, E. & Gomez, J. Simultaneous biological removal of nitrogen, carbon and sulfur by denitrification. Water Research 38 (14–15), 3313–3321. Rocca, C. D., Belgiorno, V. & Meriç, S. Heterotrophic/ autotrophic denitrification (HAD) of drinking water: prospective use for permeable reactive barrier. Desalination 210 (1–3), 194–204. Sahinkaya, E. & Kilic, A. Heterotrophic and elementalsulfur-based autotrophic denitrification processes for simultaneous nitrate and Cr(VI) reduction. Water Research 50, 278–286. Sierra-Alvarez, R., Beristain-Cardoso, R., Salazar, M., Gómez, J., Razo-Flores, E. & Field, J. A. Chemolithotrophic denitrification with elemental sulfur for groundwater treatment. Water Research 41 (6), 1253–1262. Soares, M. I. M. & Abeliovich, A. Wheat straw as substrate for water denitrification. Water Research 32 (12), 3790–3794. Sun, Y. & Nemati, M. Evaluation of sulfur-based autotrophic denitrification and denitritation for biological removal of nitrate and nitrite from contaminated waters. Bioresource Technology 114, 207–216. Wang, J., Lu, H., Chen, G. -H., Lau, G. N., Tsang, W. L. & van Loosdrecht, M. C. M. A novel sulfate reduction, autotrophic denitrification, nitrification integrated (SANI) process for saline wastewater treatment. Water Research 43 (9), 2363–2372.
First received 5 November 2014; accepted in revised form 12 February 2015. Available online 26 February 2015
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© IWA Publishing 2015 Water Science & Technology
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The heterotrophic-combined-with-autotrophic denitrification process: performance and interaction mechanisms Guihua Xu, Cuijie Feng, Fang Fang, Shaohua Chen, Yuanjian Xu and Xingzu Wang
ABSTRACT In this work, the interaction mechanisms between an autotrophic denitrification (AD) and heterotrophic denitrification (HD) process in a heterotrophic-autotrophic denitrification (HAD) system were investigated, and the performance of the HAD system under different S/Ac molar ratios was also evaluated. The results demonstrated that the heterotrophic-combined-with-autotrophic denitrification process is a promising technology which can remove chemical oxygen demand (COD), sulfide and nitrate simultaneously. The reduction rate of NO 3 to NO2 by the HD process was much faster than that of reducing NO 2 to N2, while the reduction rate of NO3 to NO2 by the AD process was slower than that of NO 2 to N2. Therefore, the AD process could use the surplus NO2 produced
by the HD process. This could alleviate the NO 2 –N accumulation and increase the denitrification rate. In addition, the inhibition effects of acetate on AD bacteria and sulfide on HD were observed, and the inhibition was compensated by the promotion effects on NO 2 . Therefore, the processes of AD and HD seem to react in parallel, without disturbing each other, in our HAD system. Key words
Guihua Xu Yuanjian Xu Xingzu Wang Key Laboratory of Reservoir Aquatic Environment, Chongqing Institute of Green and Intelligent Technology, Chinese Academy of Sciences, Chongqing 400714, China Guihua Xu Cuijie Feng Fang Fang Shaohua Chen (corresponding author) Key Laboratory of Urban Environment and Health, Institute of Urban Environment, Chinese Academy of Sciences, Xiamen 361021, China E-mail: [email protected]
| autotrophic denitrification, heterotrophic denitrification, interaction mechanism
INTRODUCTION The disposal of waters contaminated by nitrate or nitrite plays an important role in wastewater treatment because improperly treated wastewaters may cause serious ecological damage to the receiving water bodies (e.g., eutrophication) (Moraes & Foresti ). Previous studies have reported that nitrate caused severe health issues, such as methemoglobinemia (blue baby syndrome), and the increased risk of the formation of carcinogens (Kim et al. ). 5CH3 COOH þ 8NO 3 ! 10CO2 þ 4N2 þ 9H2 O þ 12OH
(1) Owing to the high-denitrification efficiency of the heterotrophic denitrification (HD) process, which was widely used to treat waters contaminated by nitrate, the HD process needs organic carbon sources to reduce nitrate to N2 (Equation (1)); therefore, the HD removal efficiency depends on the biochemical oxygen demand (BOD5)/N doi: 10.2166/wst.2015.097
ratio in the wastewater (Soares & Abeliovich ). Furthermore, the lack of carbon sources often results in the failure of nitrate removal. In the case of organic deficient (low BOD5/N) wastewater, the addition of external carbon sources is needed to increase the nitrate removal. However, this will increase the complexity of the operations and treatment costs. Therefore, some researchers have turned to the autotrophic denitrification (AD) process (Flere & Zhang ; Koenig & Liu ; Sierra-Alvarez et al. ; Moraes et al. ; Sun & Nemati ), which does need not any organic carbon sources and produces less biosludge (Campos et al. ; Nanda et al. ; Fajardo et al. ). However, the AD process also has shortcomings, such as a slow denitrification rate, SO2 in the effluent 4 exceeding the discharge standard, alkalinity consumption and so on (Equations (2)–(4)). Thus, the AD process cannot completely replace the traditional HD technology. þ 5S2 þ 2NO 3 þ 12H ! 5S þ N2 þ 6H2 O
(2)
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2 þ 5S þ 6NO 3 þ 2H2 O ! 5SO4 þ 3N2 þ 4H
(3)
2 þ 5S2 þ 8NO 3 þ 8H ! 5SO4 þ 4N2 þ 4H2 O
(4)
In practice, some industrial wastewater contains carbon, nitrogen and sulfur compounds, such as that from the oil industry, gourmet powder factories, the pharmaceutical industry and so on (Wang et al. ). Moreover, some features of the AD and HD processes may be complementary to each other, such as reducing the demand for organic carbon and basicity. These make it possible to combine the AD and HD processes to overcome their respective defects, and combining the AD and HD processes may be used for the above-mentioned wastewater to remove carbon, nitrogen and sulfur simultaneously. In the last 10 years, researchers have begun to study the heterotrophic-combined-with-autotrophic denitrification (HAD) process (Beristain-Cardoso et al. ; Chen et al. ; Sahinkaya & Kilic ). In 2001, Oh et al. () proved that the AD and HD processes can coexist and complete the denitrification process together. After that, the influence factors (e.g., S/N, chemical oxygen demand (COD)/N ratio) and operation parameters (e.g., residence times) were studied (Reyes-Avila et al. ; Rocca et al. ; Liu et al. ). However, the performance of the HAD process is not completely understood, and the interaction mechanisms between the HD and AD processes in the HAD system are unclear. These unclear problems limit the application of HAD technology in wastewater treatment. Based on these problems, this work aims to investigate the interaction mechanisms between the HD and AD processes in the HAD system for the simultaneous removal of carbon, nitrogen and sulfide. Moreover, the performance of the HAD system under different S/Ac molar ratios was evaluated.
MATERIALS AND METHODS Experimental setup and operations To study the interaction mechanisms between the HD and AD processes in the HAD system, a Plexiglas-made reactor was established. Acetate and sulfide were used as electron donors for the HD and AD processes in the HAD system. The effects of sulfide on the HD process and acetate on the AD process need to be further investigated; therefore, the AD and HD reactors were established to investigate
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the effects of sulfide and acetate. Five liters of seeding sludge for the AD and HD reactors was collected from the Shiweitou Municipal Wastewater Treatment Plant (Xiamen, China), and the concentration of suspended solids (SS) was 3,776 ± 352 mg/L. The AD and HD reactors were operated stably for 1 year, and the HAD reactor was inoculated with 0.5 L of sludge collected on day 140 from the AD reactor and 0.5 L of sludge from the HD reactor. After the three reactors reached steady state, the SS concentration of the AD, HD and HAD reactors varied slightly and remained at 1,480 ± 157 mg L1, 2,736 ± 271 mg L1 and 2,239 ± 182 mg L1, respectively. The sludge retention times (SRT) of the AD, HD and HAD reactors were kept at 30 d by discharging excess sludge. The detailed experimental setup and operation conditions are listed in the Supplementary Materials (available online at http://www. iwaponline.com/wst/071/097.pdf).
Experimental conditions The performance of the HAD system under five S/Ac molar ratios (5:0.7, 5:1.8, 5:3.5, 5:7.1 and 5:17.6) was first evaluated, and the detailed experimental conditions are listed in Table S1 in the Supplementary Materials (available online at http://www.iwaponline.com/wst/071/097.pdf). Furthermore, to investigate the interaction mechanisms between the HD and AD processes in the HAD system, nitrite accumulation and the effects of electron donors (acetate and sulfide) were studied (Table S1). Nitrite accumulations under different S/N and Ac/N ratios were conducted in the HAD reactor, as summarized in Table S1. For the S/N molar ratio tests, the HAD sludge used sulfide as the sole electron donor (without acetate) and four S/N molar ratios were applied to the HAD influents: 5:2, 5:5, 5:8 and 5:12 (Table S1). For the different Ac/N molar ratio experiments, similar to the S/N molar ratio tests, the experiments were conducted without sulfide, and four Ac/N molar ratios were applied to the influents of the HAD reactor: 5:3.4, 5:5.9, 5:10.1 and 5:20.2 (Table S1). In addition, nitrite accumulation validation tests 1, 2 and 3 were conducted to compare the nitrite concentration in the HAD reactor under different electron donor conditions. Three different electron donors were applied to the HAD reactor for tests 1, 2 and 3: sodium sulfide (2.0 g/L), sodium acetate (0.5 g/L) and sodium sulfide (2.0 g/L) with sodium acetate (0.5 g/L), respectively (Table S1). Inhibition tests A and B were used to study the effects of sulfide on the HD process and acetate on the AD process,
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respectively (Table S1). For the effects of sulfide on the HD process (test A), 2 g L1 sodium sulfide was imposed on the HD reactor while keeping the other conditions unchanged. For the effects of acetate on the AD process (test B), 0.5 g L1 sodium acetate was conducted to the AD reactor while keeping the other conditions unchanged (Table S1). Chemical analysis Samples were filtrated by 0.45-μm filters before taking measurements. The pH, SRT, SS concentration and temperature were determined by Standard Methods (APHA ). 2 The Ac, NO 2 , NO3 and SO4 were measured by ion chromatography (ICS3000, DIONEX, USA) according to the manufacturer protocol. The ion chromatography equipment used an Ion Pac column (As15A 4 × 250 mm, AG15), and the injection volume of the sample was 10 μL. The S2 concentration was measured according to the method of a previous study (Cord-Ruwisch ).
Figure 1
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The performance of the HAD process under different S/Ac molar ratios In the HAD reactor, after 22 h of reaction, at the end-point of the denitrification, the eventual nitrate removal efficiency was 89.0%, 87.7%, 85.4%, 83.1% and 82.6% at an S/Ac ratio of 5:0.7, 5:1.8, 5:3.5, 5:7.1 and 5:17.6, respectively (Figure 1(a)). The consumption of acetate was completed in 2.25 h, 2 h, 1.5 h, 1.25 h and 0.5 h at an S/Ac of 5:17.6, 5:7.1, 5:3.5, 5:1.8 and 5:0.7, respectively (Figure 1(b)), and the consumption of sulfide was finished after 0.75 h, 1.5 h, 2.0 h, 2.5 h and 3.0 h, respectively (Figure 1(c)). Meanwhile, a continuous decrease of nitrate was also obtained, which indicated that the AD process and HD process occurred simultaneously in the HAD reactor. The results showed that the HAD process is a feasible process for simultaneous removal of COD, sulfide and nitrate.
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Profiles of nitrite removal efficiency and acetate, sulfide and sulfate concentrations in the HAD reactor under various S/Ac acetate; (c) sulfide; (d) sulfate; all with an initial 194 mg L
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molar ratios. (a) Nitrite removal efficiency; (b)
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The denitrification process of HAD could be divided into two stages: S2 and Ac as electron donors (stage I, Equations (1) and (2)) and S0 as an electron donor (stage II, Equation (3)). In stage I, with 5:0.7, 5:1.8, 5:3.5, 5:7.1 and 5:17.6 as the S/Ac ratio, the sulfide concentration in the HAD reactor decreased sharply from the initial value of 333.2, 266.6, 200.1, 133.3 and 66.7 mg L1 to 227.6, 175.5, 127.1, 80.8 and 0.0 mg L1 in 0.75 h, respectively (Figure 1(c)), while the sulfate concentration increased slightly (Figure 1(d)). During this stage, the color of the liquid in the HAD reactor changed from yellow to a creamy white, an indication of bio-oxidation of S2 to S0. After the S2 concentration decreased to zero, a continuous increase of sulfate was observed (Figure 1(d)). This indicated the bio-oxidation of S0 ! SO2 4 (stage II), which was consistent with the result of the previous study (Beristain Cardoso et al. ; An et al. ). Interaction mechanisms between the HD and AD processes in the HAD system NO 2 –N accumulation in the AD process The results of NO 2 –N accumulation for the AD process without acetate in the HAD reactor are shown in Figure 2(a). The NO 2 –N concentration was close to zero at the beginning (after 5 h); the NO 2 –N concentration then increased rapidly after 7 h for S/N ¼ 5:12 and after 10 h for S/N ¼ 5:5, and 5:8. NO 2 –N was not detected throughout the test of S/N ¼ 5:2. The results showed that the reduction rate of NO 2 to N2 was 2 faster than that of NO ! S0 stage, which 3 to NO2 in the S resulted in no NO 2 –N accumulation at the beginning of the AD process (5 h). The results were consistent with the findings of B.S. Moraes in which the half-order kinetic coefficient was 0.425–0.658 mg N (L h)1 for NO 2 to N2 and 0.190– 0.609 mg N (L h)1 for denitrification via nitrate (Moraes & Foresti ). However, when S2 was exhausted and S0 was used as the electron donor (stage II), the reduction rate of NO 2 to N2 became slower than that of. NO3 to NO2 . There fore, the accumulation of NO2 occurred in this stage. NO 2 –N accumulation in the HD process Nitrite accumulation experiments for the HD process without sulfide in the HAD reactor under different Ac/N molar ratios were conducted. The concentrations of NO 2– N increased dramatically when the acetate concentration in the influent increased, and the maximum value of NO 2 –N accumulated was 101.5 mg L1, 85.2 mg L1, 55.1 mg L1 and 25.3 mg L1 at Ac/N of 5:3.4, 5:5.9, 5:10.1 and 5:20.2,
Figure 2
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Nitrite accumulation in the HAD reactor under different substrates. (a) Sulfide as an electron donor under various S/N molar ratios; (b) acetate as an electron
donor under various Ac /N molar ratios; (c) comparisons of nitrite accumulation under acetate, sulfide and the two substrates together, all with an initial 194 mg L 1 NO3 –N.
respectively (Figure 2(b)). The results indicated that the HD process reduction rate of NO 3 to NO2 was much faster than the reduction rate of NO 2 to N2. Therefore, a large
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amount of nitrite accumulation occurred (Figure S1 in the Supplementary Materials, available online at http://www. iwaponline.com/wst/071/097.pdf). In the HAD system, the reduction of NO 2 to N2 was the limited step for the HD pro cess, while the reduction of NO 3 to NO2 was slower than NO2 to N2 for the AD process. The AD process could use the surplus nitrite production from the HD process (Figure S1). Therefore, the combination of the AD and HD processes might increase the denitrification rate and decrease the amount of nitrite accumulation. Comparison of nitrite accumulation between the AD, HD and HAD processes Three different electron donors were applied to the HAD reactor: 2.0 g/L sodium sulfide (test 1), 0.5 g/L sodium acetate (test 2) and the two electron donors together (test 3) (Table S1 in the Supplementary Materials, available online at http://www.iwaponline.com/wst/071/097.pdf). The results of NO 2 –N accumulation are presented in Figure 2(c). After 2 h of reaction, with test 1, 2 and 3, the concentration of NO 2 –N was 0.2 mg L1, 32.6 mg L1 and 7.6 mg L1, respectively. Moreover, after 12 h, the concentration of NO 2 –N changed to 17.1 mg L1, 31.6 mg L1 and 19.5 mg L1, respectively. The NO 2 –N accumulation of the HAD process was higher than that of the AD process and much lower than that of the single HD process. Compared with the HD process, the combination of the AD and HD processes produced less nitrite and could alleviate the NO 2 –N accumulation phenomenon. As discussed above, in the HAD system, the HD process produced nitrite faster than the reduction rate of nitrite, which provided surplus nitrite for the AD process and reduced the total denitrification time. Thus, it might increase the denitrification rate of the HAD process. The verification tests were needed to perform comparisons of the NO 3 –N removal from the experimental data and theoretical calculation results.
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and (6), respectively, where CNO3–N0 is the initial concentration of NO3–N, CNO3–N is the concentration of NO 3 –N at time t, and Ksulfide and Kacetate is the zero-order rate constant of the AD and HD processes, respectively. CNO3 N ¼ CNO3 N0 Ksulphide × t
(5)
CNO3 N ¼ CNO3 N0 Kacetate × t
(6)
If the processes of AD and HD are performed in parallel in the HAD system without disturbing each other, the theoretical concentration of NO 3 –N in the HAD reactor can be calculated by Equation (7). CNO3 N ¼ CNO3 N0 (Kacetate þ Ksulphide ) × t
(7)
The results of the theoretical calculation and experimental data for nitrates are shown in Figure 3(a). The results
Comparison of nitrate removal between the experimental data and theoretical calculation results in the HAD process In the first 2 h of the HAD process (stage I), the substrates (i.e., sulfide and acetate) were sufficient for nitrate removal. The denitrification rate was not related to the concentrations of nitrate, acetate and sulfide and was determined by the activities and quantities of denitrifiers. Therefore, both the AD and HD processes followed the zero-order kinetic model, and the NO 3 –N concentration variations of the AD and HD processes could be calculated by Equations (5)
Figure 3
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Experiment data of nitrate concentration compared with the calculation results in the HAD reactor. (a) Experimental data and calculated data; (b) AD, HD, HAD and calculated data.
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indicate that the experimental data are in good agreement with the calculated values (Figure 3(a)). Furthermore, the calculated data and experimental data of the nitrate removal efficiency were compared, whereas the calculated value of the nitrate removal efficiency was calculated as a simple sum of the HD process value and the AD process value; these results also show that the calculated data are close to the experimental data (Figure 3(b)). These results confirm the hypothesis: the processes of AD and HD were performed in parallel in the HAD system without disturbing each other. The results contradicted the conclusion in the above paragraph that stated that the HAD process had promotional effects on NO 2 and increased the denitrification rate. Therefore, there should exist inhibition factors in the HAD system to offset the promotion. The effects of sulfide on the HD process and acetate on the AD process in the HAD system To find the inhibiting factors in the HAD system, the operational and experimental conditions of the AD, HD and HAD reactors were examined. The operational and experimental conditions of the AD, HD and HAD reactors were identical, except for the electron donors. The HAD process used acetate and sulfide as electron donors, while the AD and HD processes used sulfide and acetate, respectively. Therefore, the effects of sulfide on the HD process and acetate on the AD process might be the inhibiting factors in the HAD system. However, the HAD sludge used acetate and sulfide simultaneously, and the tests of the sulfide and acetate effects on the HAD system could not be conducted in the HAD reactor. The HD reactor and AD reactor were substitutes for investigating the effects of sulfide and acetate on the HAD process. The results are shown in Figure 4; after 10 h of denitrification time, the NO 3 –N concentration in the AD reactor with added acetate was 148.0 mg L1 and the AD reactor without acetate was 108.3 mg L1 (Figure 4(a)). The results demonstrated that acetate had a restraining impact on the activity of the autotrophic denitrifiers. The effect of sulfide on the HD process had similar results, as shown in Figure 4(b). The denitrification rate with sulfide was slower than that without sulfide. The results revealed that the sulfide had an inhibiting impact on the activity of the heterotrophic denitrifiers (Figure S1 in the Supplementary Materials, available online at http://www. iwaponline.com/wst/071/097.pdf). The inhibiting impact reasons are unclear in this work and need further study. `In this study, the inhibition effects caused by acetate on the AD process and sulfide on the HD process in the HAD
Figure 4
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Inhibition factors in the HAD process. (a) The effects of acetate on the AD sludge; (b) the effects of sulfide on the HD sludge.
system offset the promotion effects on NO 2 (Figure S1). Thus, the AD and HD processes looked like they were reacting in parallel without disturbing each other in our HAD system.
CONCLUSIONS This study demonstrated that the HAD process could simultaneous remove acetate, sulfide and nitrate. Sulfide biooxidation in the HAD system occurred in two distinct stages: S2 ! S0 and S0 ! SO2 4 . The AD process used the surplus NO 2 produced by the HD process, which alleviated the NO 2 –N accumulation and increased the denitrification rate of the HAD process. The inhibition effects caused by acetate on AD and sulfide on HD in the HAD process offset the promotional effects on NO 2 . Therefore, the AD and HD processes appeared to react in parallel without disturbing each other in the HAD system.
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ACKNOWLEDGEMENTS This research was supported by the National Natural Science Foundation of China (51108439, 50778156 and 51008025) and the Natural Science Foundation of Chongqing (cstc2014jcyjA20010).
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First received 5 November 2014; accepted in revised form 12 February 2015. Available online 26 February 2015
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