Appl Biochem Biotechnol DOI 10.1007/s12010-014-1317-0
Denitrification of High Strength Nitrate Waste from a Nuclear Industry Using Acclimatized Biomass in a Pilot Scale Reactor Pradip B. Dhamole & Rashmi R. Nair & Stanislaus F. D’Souza & Aniruddha B. Pandit & S. S. Lele
Received: 4 August 2014 / Accepted: 15 October 2014 # Springer Science+Business Media New York 2014
Abstract This work investigates the performance of acclimatized biomass for denitrification of high strength nitrate waste (10,000 mg/L NO3) from a nuclear industry in a continuous laboratory scale (32 L) and pilot scale reactor (330 L) operated over a period of 4 and 5 months, respectively. Effect of substrate fluctuations (mainly C/NO3-N) on denitrification was studied in a laboratory scale reactor. Incomplete denitrification (95–96 %) was observed at low C/NO3-N (≤2), whereas at high C/NO3-N (≥2.25) led to ammonia formation. Ammonia production increased from 1 to 9 % with an increase in C/NO3-N from 2.25 to 6. Complete denitrification and no ammonia formation were observed at an optimum C/NO3-N of 2.0. Microbiological studies showed decrease in denitrifiers and increase in nitrite-oxidizing bacteria and ammonia-oxidizing bacteria at high C/NO3-N (≥2.25). Pilot scale studies were carried out with optimum C/NO3-N, and sustainability of the process was checked on the pilot scale for 5 months. Keywords Denitrification . Nuclear industry . DNRA . CSTR . Nitrate
Introduction High strength nitrate waste is generated by various industries such as nuclear, metal finishing, explosives, and ion exchange [1, 2]. Processes like uranium extraction and regeneration of ion exchange, resins release highly concentrated nitrate waste [1–3]. Different methods are reported for treatment of nitrate waste which includes physicochemical methods. However,
P. B. Dhamole (*) Department of Chemical Engineering, Birla Institute of Technology & Science, Pilani, Hyderabad Campus, Hyderabad 500078, India e-mail: [email protected] R. R. Nair : S. F. D’Souza Nuclear Agriculture and Biotechnology Division, Bhabha Atomic Research Centre, Mumbai 400085, India A. B. Pandit : S. S. Lele Institute of Chemical Technology, Matunga, Mumbai 400019, India
Appl Biochem Biotechnol
physicochemical methods are suitable for low strength nitrate waste, and it leads to secondary effluent which is more concentrated than the initial effluent [4]. Biological denitrification is the widely used method for treatment of nitrate waste. However, very few studies deal with biological treatment of nitrate waste [1, 2, 5–8]. Biological treatment of high strength nitrate waste is a challenging task as microbes are sensitive to high concentrations of nitrate. Recent studies show that stepwise adaptation of microbes to increasing concentration of nitrate leads to the development of consortium capable of degrading high strength nitrate waste [6, 7, 9]. Similar acclimatization strategy was developed for treatment of highly alkaline nitrate waste [10]. Complete denitrification of high strength simulated nitrate waste using acclimatized biomass was attributed to the enrichment or growth of nitrate tolerant strain that can degrade nitrate at extreme conditions [6]. Also, it was observed that enzyme levels increased due to adaptation process consequently enhancing the rate of denitrification [7]. Earlier studies investigated the performance of mixed consortium developed through acclimatization process for synthetic nitrate waste in batch systems [6, 9–11]. Acclimatized biomass could degrade 40,000 mg/L nitrate (9032 mg/L NO3N) and also highly alkaline (pH 10.5) nitrate waste under simulated conditions. Recently, Shahabi et al. [12] have compared the performance of acclimatized and non-acclimatized biomass for denitrification of drinking water and obtained improved rate of denitrification using acclimatized biomass. However, the capacity of the biomass thus developed is not investigated for industrial effluent which is highly complex. Nitrate waste generated during the uranium extraction process in a nuclear industry is highly concentrated in nitrate (10,000 to 110,000 mg/L NO3) and has various heavy metals (Al, As, B, Ca, As, Hg, Ce, CO, Cr, and Fe) and radioactivity [8]. Earlier efforts to treat this waste using unadapted biomass were unsuccessful (data not shown). Further, the sustainability of the biomass for long term needs to be explored. In view of this, the present work was undertaken with an objective of treating high strength nitrate waste from nuclear industry using acclimatized biomass in a laboratory scale and pilot scale continuous stirred tank reactor (CSTR). Effect of fluctuations in substrate (carbon/nitrogen) on denitrification was studied in a laboratory scale reactor. Microbiological changes in the sludge were monitored during the process. Denitrification studies were carried out in pilot scale reactor over a period of 5 months.
Materials and Methods Acclimatization Fresh sludge from an effluent treatment plant (ETP) of a fertilizer industry was inoculated in a 40-L sequencing batch reactor and acclimatized to increasing nitrate concentrations (567, 1134, 1701, and 2268 mg/L NO3-N) in a stepwise manner over a period of 2 months [6, 9, 10]. Reactor was operated in a cycle of 24 h with reaction time of 22 h for denitrification and 2 h for settling, decantation, and refilling. Half of the reactor volume (20 L) was replaced by fresh synthetic waste after every 24 h. Defined synthetic medium consisting of NaNO3 (13.65 g/L), CH3COONa (13.12 g/L), Na2HPO4 (7.0 g/L), K 2HPO 4 (1.5 g/L), MgSO4 (0.1 g/L), and NH4Cl (0.1 g/L) was used during the acclimatization studies to high strength nitrate [6]. Amount of carbon was determined based on inlet nitrate.
Appl Biochem Biotechnol
Denitrification in Batch and Continuous Stirred Tank Reactor Acclimatized biomass was used to treat nitrate waste (2268 mg/L NO3-N) generated during the uranium extraction process in a 40-L batch reactor. Desired nitrate level was obtained after diluting the waste. Wastewater consisted of different heavy metals (Al, As, B, Ca, As, Hg, Ce, CO, Cr, and Fe) with total concentration of 12–15 mg/L. The waste also had a radioactivity of 0.4 Bq/mL. Further studies were conducted in a 36-L CSTR (length=0.4 m, breadth=0.3 m, and height=0.3 m) (working volume=32 L) with a hydraulic retention time (HRT) of 48 h (Fig. 1). Nitrate waste from a nuclear plant diluted to 2268 mg/L NO3-N (10,000 mg/L NO3) was pumped at a rate of 14 L/d using a peristaltic pump. pH of the feed was adjusted to 7.5 before charging the feed to the feed tank. Settled biomass was recycled into a biomass container and was pumped at a rate of 2 L/d. Overhead motor (1/2 hp) was used to keep the biomass in suspension. Pilot scale studies were carried out in a 360-L CSTR (length=1.0 m, breadth=0.6 m, and height=0.6 m) (working volume=330 L) for 5 months. Schematics of the pilot scale reactor are mentioned elsewhere [8]. Nitrate, nitrite, ammonia, biomass (measured as mixed liquor suspended solids, i.e., MLSS), pH, and chemical oxygen demand (COD) were analyzed on daily basis. Laboratory scale and pilot scale reactor were operated for 4 and 5 months, respectively. Analysis Samples were analyzed for various parameters NO3, NO2, NH3, biomass, and COD. Bacteria enumeration was carried out using reported methods [7]. Nitrate and nitrite were analyzed using Dionex ion chromatograph, AS11 (2 mm) column, and guard column. Twelve millimolar of NaOH was used as an eluent. Samples were centrifuged and filtered before analysis and diluted using deionized water. Ammonia was determined following standard method of Nessler’s reagent. Sodium tartarate (10 %) (2 mL) and Nessler’s reagent (0.5 mL) were added to a sample (1 mL), and the volume was made up to 20 mL (by distilled water). Absorbance was measured at 410 nm after incubating for 30 min. Biomass was determined by dry weight method [13], and COD was estimated using potassium dichromate reflux method [14]. A sample (10 mL) was added with HgSO4 (1 g) followed by sulfuric acid reagent (5 mL) (5.5 g Ag2SO4/kg of H2SO4) added slowly and mixed to dissolve HgSO4. The mixture is cooled to
Fig. 1 Laboratory scale set up for treatment of nuclear industry effluent (T1 tank containing recycled biomass, T2 nuclear industry effluent, R reactor, M motor, T3 tank for collecting treated effluent, P1 and P2, peristaltic pumps) (Biomass from T3 is recovered and recycled to T1)
Appl Biochem Biotechnol
avoid possible loss of volatile materials. Ten milliliters of sample followed by addition of 0.04167 M K2Cr2O7 solution. The entire mixture is refluxed for 2 h, and the condenser is washed with distilled water at the end and cooled. The diluted mixture is then titrated for excess K2Cr2O7 with ferrous ammonium sulphate reagent (0.25 M) using ferroin indicator (0.1–0.15 mL). The color change from blue-green to reddish brown was taken as end point of titration.
Results and Discussion Denitrification of Nitrate Waste from Nuclear Plant in a Batch Reactor Nitrate waste generated during the process of uranium extraction was treated using acclimatized sludge. The waste was highly complex containing different heavy metals and with slight radioactivity (0.04 Bq/mL). Figure 2 shows the nitrate and nitrite profiles during the denitrification of 2268 mg/L NO3-N in a 40-L batch reactor. It can be seen that complete denitrification of 2268 mg/L NO3-N was achieved in less than 4.5 h with specific rate of nitrate and nitrite reduction as 48 mg NO3-N/g MLSS/h and 30 mg NO2-N/g MLSS/h, respectively. Further studies were carried out in a 32-L laboratory scale CSTR. Treatment of Nitrate Waste in a Laboratory Scale CSTR Continuous treatment of nitrate waste (2268 mg/L) from a nuclear industry was carried out at different C/NO3-N ratio (Fig. 3). Nitrate and nitrite in the outlet were zero at high C/NO3-N ratio of 6. However, COD levels in the outlet were significantly high (Fig. 3) (ammonia and nitrate, nitrite profile reproduced from [8]), and ammonia was also detected in the outlet (in the range of 200–250 mg/L). Under strict anaerobic environments in soil, nitrate is reduced to ammonium. This process is called as dissimilatory nitrate reduction to ammonium (DNRA) [15, 16]. The selection of DNRA pathway by microbes depends on the carbon source and 1500
NO3-N
NO2-N
NO3-N, NO2-N, mg/L
1200
900
600
300
0 0
1
2
Time, h
3
4
5
Fig. 2 Nitrate and nitrite profiles during the denitrification of nuclear industry effluent containing 2268 mg/L NO3-N in a 40-L batch reactor (alpha activity=0.04 Bq/mL) MLSS=8.5 g/L (standard error=±5 %)
12000
300
10000
250
8000
200
6000
150
4000
100
2000
50
NH3, mg/L
COD, mg/L
Appl Biochem Biotechnol
0
0 0
20
40
60
80
100
120
Time, day COD out
NH3,Out
Fig. 3 COD and ammonia profile in a CSTR during the denitrification of nitrate waste from a nuclear plant (feed concentration=2268 mg/L NO3-N, HRT=48 h, reactor volume=32 L) (standard error=±5 %)
prevailing conditions. The nature and amount of carbon source plays an important role in the choice of pathway. It is argued that excess amount of carbon would favor organisms that used electron acceptors most efficiently [17]. Hence, further studies were carried out with low C/NO3-N ratio (3), and surface aeration was provided to prevent strict anaerobic conditions. Under these conditions, ammonia formation was reduced but was not inhibited completely (Table 1). Therefore, C/NO3-N ratio was reduced further to 1.5 that resulted into incomplete denitrification and prevented ammonia formation completely (Fig. 4). Increase in C/NO3-N ratio from 1.5 to 2.5 again resulted into ammonia formation. Minimum ammonia formation was observed at C/NO3-N of 2.25. Ammonia formation was completely prevented at C/NO3-N ratio of 2.0. Also, complete denitrification was achieved at the same C/NO3-N ratio (2). Hence, for pilot scale studies, C/NO3-N ratio of 2 was used. Material balance for nitrogen at different C/NO3-N ratio showed that with increase in C/NO3-N ratio, conversion of nitrate to ammonia increased (Table 1). Maximum conversion Table 1 Material balance calculations for conversion of NO3-N into nitrogen and ammonia during denitrification of nuclear industry effluent in a 32-L CSTR (NO3-N=2268 mg/L) C/ NO3N
NO3- NO2- NH3- N in the % Conversion of NO3 into NH3-N % Conversion of NO3 into N2 outlet N N N (C/2268×100) (D/2268×100) (D = A + (A) (B) (C) B+C)
1.5
34
88
0
122
0
2
24
78
0
102
0
96
2.25
0
0
20
20
1
99
95
2.5
0
0
20
20
1
99
3
0
0
45
45
2
98
6
0
0
221
221
10
90
Appl Biochem Biotechnol
(a)
Outlet NO3-N, NO2-N (mg/L)
140 120 100 80 60 40 20
NO3-N NO2-N
0 60
63
66
69
72
75
Time, d
(b)
Outlet NO3-N, NO2-N (mg/L)
140
NO3-N NO2-N
120 100 80 60
40 20 0 76
79
82
Time, d
85
88
91
Fig. 4 Outlet nitrate and nitrite profiles during the denitrification of nitrate waste from a nuclear plant (a) C/NO3N=1.5; (b) C/NO3-N=2.0 (CSTR=32 L, inlet NO3-N=2268 mg/L, HRT=48 h) (standard error=±5 %)
(9 %) of nitrate to ammonia was observed at C/NO3-N of 6. Nitrite was detected in the outlet sample at C/NO3-N ≤2. Optimum C/NO3-N for complete denitrification was found to be 2.0. Additional step (aeration) was provided to reduce the COD reduced (data not shown). Microbial Consortium Changes During DNRA The reasons behind ammonia formation could be the relative abundance of fermentative and ammonium formers in anaerobic environments compared to true denitrifiers capable of O2 respiration or denitrification only. Most probable number (MPN) was estimated for sludge at different point of time in the process. Three main populations (denitrifiers, ammoniumoxidizing bacteria, and nitrate-oxidizing bacteria) generally found in such consortium were studied at each point of change in C/NO3-N. From Table 2, it can be seen that when the C/NO3-N in the reactor was more than 2.5 (2.5–6), ammonium-oxidizing bacteria (AOB) population increased. The excess ammonium formed during that phase was rapidly acted upon by the AOB population in the reactor, which increased to form nitrite. Nitrite-oxidizing bacteria (NOB) population acts upon the nitrite to form nitrate. The absence of nitrite and nitrate showed that the NOB and denitrifying population immediately took up the substrate to
Appl Biochem Biotechnol Table 2 Plate count and MPN enumeration from the sludge during the various stages of C/NO3-N changes in a 32-L CSTR of total bacteria, denitrifying bacteria (DB, i.e., bacteria reducing N anion without any accumulation of NO2 or NH3), ammonium-oxidizing bacteria (AOB, i.e., bacteria oxidizing NH3 to NO2), and nitrite-oxidizing bacteria (NOB, i.e., bacteria oxidizing NO2 to NO3) C/NO3-Na
DB
AOB
NOB
6 3
4.8×102 3.7×103
3.0×105 5.4×104
2.7×104 1.0×104
1.5
2.4×103
5.0×102
4.5×102
2.5
1.8×103
3.0×103
1.7×101
2.0
3.5×107
1.8×102
2.0×101
2.25
2.7×107
1.0×101
1.9×101
a
Confidence limit (95 %) for sludge during different stages of adaptation
form ammonium which was oxidized again to nitrite by AOB. Similarly, when the C/NO3-N was decreased to 1.5 to reduce ammonia formation, incomplete denitrification was observed. Table 2 shows that though there was no increase in the denitrifying bacteria population, the AOB population decreased drastically. With increase in C/NO3-N from 1.5 to 2.0 and 2.25, complete denitrification was observed which is due to increase in the denitrifying population. The AOB population decreased with decrease in C/NO3-N from 6.0 to 2.25. Thus, it can be concluded that at a C/NO3-N of 2.0–2.25, the microbial consortium degrades nitrate wastes completely via denitrification pathway without accumulation of ammonium or nitrite. It takes the DNRA pathway, when the C/NO3-N is increased above 2.5. Hence, for complete denitrification of the nitrate waste, the C/NO3-N of 2.0–2.25 can be used. C/NO3-N can also be optimized within this range to have maximum denitrification activity. Sustainability of the Denitrification Process on a Pilot Scale Studies in a 32-L CSTR resulted in optimum C/NO3-N ratio of 2.0. Hence, the pilot scale reactor (330 L) was operated for influent nitrate concentration of 2268 and C/NO3-N ratio of 2.0 to study if ammonia formation occurs on a larger scale and also to study the sustainability of the process. Complete denitrification was observed for initial 20 days, and sudden increase in outlet nitrate (0 to 1647 mg/L) and nitrite (0 to 792 mg/L) levels were seen from day 21 onwards (Fig. 5). This could be attributed to the increase in heavy metal concentration in the influent (from 12 to 21 mg/L). Hence, influent concentration was decreased to 1129 mg/L NO3-N, and the reactor was operated for the next 20 days with the same nitrate load till a steady outlet nitrate and nitrite profiles were obtained. It was then increased to 1693 mg/L NO3-N and operated for another 30 days at the same influent concentration. Increase in nitrate load resulted into outlet nitrate and nitrite levels for the first 10 days and subsequently reduced to zero and remained steady for the next 20 days. After steady performance at 1693 mg/L, nitrate concentration was increased to initial level of 2268 mg/L which resulted into small nitrite buildup that diminished after 10 days of continuous operation, and a steady performance (nitrate and nitrite below permissible limits) was observed for the remaining days. MLSS were in the range of 8–10 g/L throughout the studies. Ammonia formation did not take place during the nitrate waste treatment. Thus, the optimum C/NO3-N ratio on both laboratory scale and pilot scale was estimated as 2.0, and the sustainability of the process was verified on a pilot scale level.
Appl Biochem Biotechnol
2500
2000 NO3-N, NO2-N, mg/L
NO3-Nin NO3-Nout NO2-Nout
1500
1000
500
0
0
20
40
60
80
100
120
140
160
Time, d
Fig. 5 Nitrate and nitrite profiles during the denitrification of 2268 mg/L NO3-N in a 330-L CSTR (HRT=30 h, MLSS=1.5–4.0 g/L) (standard error=±5 %)
Conclusions Acclimatized biomass denitrified high strength nitrate waste (2268 mg/L NO3-N) (containing various heavy metals and radioactivity 0.04 Bq/mL) in a laboratory scale (40-L batch and 32-L CSTR) and pilot scale reactor (330 L) over a long term. High C/NO3-N ratio resulted in ammonia formation due to microbiological changes in the sludge consortium. Ammonia formation was prevented by optimizing the C/NO3-N ratio (2.0). Pilot scale studies showed no ammonia formation, and complete denitrification of the nitrate was obtained. Acknowledgments The authors would like to thank the Department of Atomic Energy (DAE), Government of India, for funding this work and Rashtriya Chemicals and Fertilizers (RCF), Mumbai, India, for providing the sludge for the experiments.
References 1. 2. 3. 4. 5. 6. 7. 8.
Glass, C., & Silverstien, J. (1999). Water Research, 33, 223–229. Glass, C., & Silverstein, J. (1998). Water Research, 32, 831–839. Dhamole, P.B. (2006). PhD Thesis, University of Mumbai. Kapoor, A., & Viraraghavan, T. (1997). Journal of Environmental Engineering, 123, 371–380. Foglar, L., & Briski, F. (2003). Process Biochemistry, 39, 95–103. Dhamole, P. B., Nair, R. R., D’Souza, S. F., & Lele, S. S. (2007). Bioresource Technology, 98, 247–252. Nair, R. R., Dhamole, P. B., Lele, S. S., & D’Souza, S. F. (2007). Chemosphere, 97, 1612–1617. Biradar, P. M., Dhamole, P. B., Nair, R. R., Roy, S. B., Satpati, S. B., D’Souza, S. F., Lele, S. S., & Pandit, A. B. (2008). Environmental Progress, 27, 365–372. 9. Dhamole, P. B., Nair, R. R., D’Souza, S. F., & Lele, S. S. (2009). Bioresource Technology, 100, 1082–1086. 10. Dhamole, P. B., Nair, R. R., D’Souza, S. F., & Lele, S. S. (2008). Applied Biochemistry and Biotechnology, 151, 433–440.
Appl Biochem Biotechnol 11. Nair, R. R., Dhamole, P. B., Lele, S. S., & D’Souza, S. F. (2008). Applied Biochemistry and Biotechnology, 151, 193–200. 12. Shahabi, Z. A., & Naeimpoor, F. (2014). Applied Biochemistry and Biotechnology, 73, 741–752. 13. APHA, AWWA, WEF. (1992). Washington, DC: APHA. 14. APHA, AWWA, WEF. (1997). Washington, DC: APHA. 15. Yin, S. X., Chen, D., Chen, L. M., & Edis, R. (2002). Soil Biology and Biochemistry, 34, 1131–1137. 16. Ma, H., & Aelion, C. M. (2005). Soil Biology and Biochemistry, 37, 1869–1878. 17. Tiedje, J. M. (1988). In A. J. B. Zehnder (Ed.), Biology of anaerobic microorganisms. New York: Wiley.
Denitrification of High Strength Nitrate Waste from a Nuclear Industry Using Acclimatized Biomass in a Pilot Scale Reactor Pradip B. Dhamole & Rashmi R. Nair & Stanislaus F. D’Souza & Aniruddha B. Pandit & S. S. Lele
Received: 4 August 2014 / Accepted: 15 October 2014 # Springer Science+Business Media New York 2014
Abstract This work investigates the performance of acclimatized biomass for denitrification of high strength nitrate waste (10,000 mg/L NO3) from a nuclear industry in a continuous laboratory scale (32 L) and pilot scale reactor (330 L) operated over a period of 4 and 5 months, respectively. Effect of substrate fluctuations (mainly C/NO3-N) on denitrification was studied in a laboratory scale reactor. Incomplete denitrification (95–96 %) was observed at low C/NO3-N (≤2), whereas at high C/NO3-N (≥2.25) led to ammonia formation. Ammonia production increased from 1 to 9 % with an increase in C/NO3-N from 2.25 to 6. Complete denitrification and no ammonia formation were observed at an optimum C/NO3-N of 2.0. Microbiological studies showed decrease in denitrifiers and increase in nitrite-oxidizing bacteria and ammonia-oxidizing bacteria at high C/NO3-N (≥2.25). Pilot scale studies were carried out with optimum C/NO3-N, and sustainability of the process was checked on the pilot scale for 5 months. Keywords Denitrification . Nuclear industry . DNRA . CSTR . Nitrate
Introduction High strength nitrate waste is generated by various industries such as nuclear, metal finishing, explosives, and ion exchange [1, 2]. Processes like uranium extraction and regeneration of ion exchange, resins release highly concentrated nitrate waste [1–3]. Different methods are reported for treatment of nitrate waste which includes physicochemical methods. However,
P. B. Dhamole (*) Department of Chemical Engineering, Birla Institute of Technology & Science, Pilani, Hyderabad Campus, Hyderabad 500078, India e-mail: [email protected] R. R. Nair : S. F. D’Souza Nuclear Agriculture and Biotechnology Division, Bhabha Atomic Research Centre, Mumbai 400085, India A. B. Pandit : S. S. Lele Institute of Chemical Technology, Matunga, Mumbai 400019, India
Appl Biochem Biotechnol
physicochemical methods are suitable for low strength nitrate waste, and it leads to secondary effluent which is more concentrated than the initial effluent [4]. Biological denitrification is the widely used method for treatment of nitrate waste. However, very few studies deal with biological treatment of nitrate waste [1, 2, 5–8]. Biological treatment of high strength nitrate waste is a challenging task as microbes are sensitive to high concentrations of nitrate. Recent studies show that stepwise adaptation of microbes to increasing concentration of nitrate leads to the development of consortium capable of degrading high strength nitrate waste [6, 7, 9]. Similar acclimatization strategy was developed for treatment of highly alkaline nitrate waste [10]. Complete denitrification of high strength simulated nitrate waste using acclimatized biomass was attributed to the enrichment or growth of nitrate tolerant strain that can degrade nitrate at extreme conditions [6]. Also, it was observed that enzyme levels increased due to adaptation process consequently enhancing the rate of denitrification [7]. Earlier studies investigated the performance of mixed consortium developed through acclimatization process for synthetic nitrate waste in batch systems [6, 9–11]. Acclimatized biomass could degrade 40,000 mg/L nitrate (9032 mg/L NO3N) and also highly alkaline (pH 10.5) nitrate waste under simulated conditions. Recently, Shahabi et al. [12] have compared the performance of acclimatized and non-acclimatized biomass for denitrification of drinking water and obtained improved rate of denitrification using acclimatized biomass. However, the capacity of the biomass thus developed is not investigated for industrial effluent which is highly complex. Nitrate waste generated during the uranium extraction process in a nuclear industry is highly concentrated in nitrate (10,000 to 110,000 mg/L NO3) and has various heavy metals (Al, As, B, Ca, As, Hg, Ce, CO, Cr, and Fe) and radioactivity [8]. Earlier efforts to treat this waste using unadapted biomass were unsuccessful (data not shown). Further, the sustainability of the biomass for long term needs to be explored. In view of this, the present work was undertaken with an objective of treating high strength nitrate waste from nuclear industry using acclimatized biomass in a laboratory scale and pilot scale continuous stirred tank reactor (CSTR). Effect of fluctuations in substrate (carbon/nitrogen) on denitrification was studied in a laboratory scale reactor. Microbiological changes in the sludge were monitored during the process. Denitrification studies were carried out in pilot scale reactor over a period of 5 months.
Materials and Methods Acclimatization Fresh sludge from an effluent treatment plant (ETP) of a fertilizer industry was inoculated in a 40-L sequencing batch reactor and acclimatized to increasing nitrate concentrations (567, 1134, 1701, and 2268 mg/L NO3-N) in a stepwise manner over a period of 2 months [6, 9, 10]. Reactor was operated in a cycle of 24 h with reaction time of 22 h for denitrification and 2 h for settling, decantation, and refilling. Half of the reactor volume (20 L) was replaced by fresh synthetic waste after every 24 h. Defined synthetic medium consisting of NaNO3 (13.65 g/L), CH3COONa (13.12 g/L), Na2HPO4 (7.0 g/L), K 2HPO 4 (1.5 g/L), MgSO4 (0.1 g/L), and NH4Cl (0.1 g/L) was used during the acclimatization studies to high strength nitrate [6]. Amount of carbon was determined based on inlet nitrate.
Appl Biochem Biotechnol
Denitrification in Batch and Continuous Stirred Tank Reactor Acclimatized biomass was used to treat nitrate waste (2268 mg/L NO3-N) generated during the uranium extraction process in a 40-L batch reactor. Desired nitrate level was obtained after diluting the waste. Wastewater consisted of different heavy metals (Al, As, B, Ca, As, Hg, Ce, CO, Cr, and Fe) with total concentration of 12–15 mg/L. The waste also had a radioactivity of 0.4 Bq/mL. Further studies were conducted in a 36-L CSTR (length=0.4 m, breadth=0.3 m, and height=0.3 m) (working volume=32 L) with a hydraulic retention time (HRT) of 48 h (Fig. 1). Nitrate waste from a nuclear plant diluted to 2268 mg/L NO3-N (10,000 mg/L NO3) was pumped at a rate of 14 L/d using a peristaltic pump. pH of the feed was adjusted to 7.5 before charging the feed to the feed tank. Settled biomass was recycled into a biomass container and was pumped at a rate of 2 L/d. Overhead motor (1/2 hp) was used to keep the biomass in suspension. Pilot scale studies were carried out in a 360-L CSTR (length=1.0 m, breadth=0.6 m, and height=0.6 m) (working volume=330 L) for 5 months. Schematics of the pilot scale reactor are mentioned elsewhere [8]. Nitrate, nitrite, ammonia, biomass (measured as mixed liquor suspended solids, i.e., MLSS), pH, and chemical oxygen demand (COD) were analyzed on daily basis. Laboratory scale and pilot scale reactor were operated for 4 and 5 months, respectively. Analysis Samples were analyzed for various parameters NO3, NO2, NH3, biomass, and COD. Bacteria enumeration was carried out using reported methods [7]. Nitrate and nitrite were analyzed using Dionex ion chromatograph, AS11 (2 mm) column, and guard column. Twelve millimolar of NaOH was used as an eluent. Samples were centrifuged and filtered before analysis and diluted using deionized water. Ammonia was determined following standard method of Nessler’s reagent. Sodium tartarate (10 %) (2 mL) and Nessler’s reagent (0.5 mL) were added to a sample (1 mL), and the volume was made up to 20 mL (by distilled water). Absorbance was measured at 410 nm after incubating for 30 min. Biomass was determined by dry weight method [13], and COD was estimated using potassium dichromate reflux method [14]. A sample (10 mL) was added with HgSO4 (1 g) followed by sulfuric acid reagent (5 mL) (5.5 g Ag2SO4/kg of H2SO4) added slowly and mixed to dissolve HgSO4. The mixture is cooled to
Fig. 1 Laboratory scale set up for treatment of nuclear industry effluent (T1 tank containing recycled biomass, T2 nuclear industry effluent, R reactor, M motor, T3 tank for collecting treated effluent, P1 and P2, peristaltic pumps) (Biomass from T3 is recovered and recycled to T1)
Appl Biochem Biotechnol
avoid possible loss of volatile materials. Ten milliliters of sample followed by addition of 0.04167 M K2Cr2O7 solution. The entire mixture is refluxed for 2 h, and the condenser is washed with distilled water at the end and cooled. The diluted mixture is then titrated for excess K2Cr2O7 with ferrous ammonium sulphate reagent (0.25 M) using ferroin indicator (0.1–0.15 mL). The color change from blue-green to reddish brown was taken as end point of titration.
Results and Discussion Denitrification of Nitrate Waste from Nuclear Plant in a Batch Reactor Nitrate waste generated during the process of uranium extraction was treated using acclimatized sludge. The waste was highly complex containing different heavy metals and with slight radioactivity (0.04 Bq/mL). Figure 2 shows the nitrate and nitrite profiles during the denitrification of 2268 mg/L NO3-N in a 40-L batch reactor. It can be seen that complete denitrification of 2268 mg/L NO3-N was achieved in less than 4.5 h with specific rate of nitrate and nitrite reduction as 48 mg NO3-N/g MLSS/h and 30 mg NO2-N/g MLSS/h, respectively. Further studies were carried out in a 32-L laboratory scale CSTR. Treatment of Nitrate Waste in a Laboratory Scale CSTR Continuous treatment of nitrate waste (2268 mg/L) from a nuclear industry was carried out at different C/NO3-N ratio (Fig. 3). Nitrate and nitrite in the outlet were zero at high C/NO3-N ratio of 6. However, COD levels in the outlet were significantly high (Fig. 3) (ammonia and nitrate, nitrite profile reproduced from [8]), and ammonia was also detected in the outlet (in the range of 200–250 mg/L). Under strict anaerobic environments in soil, nitrate is reduced to ammonium. This process is called as dissimilatory nitrate reduction to ammonium (DNRA) [15, 16]. The selection of DNRA pathway by microbes depends on the carbon source and 1500
NO3-N
NO2-N
NO3-N, NO2-N, mg/L
1200
900
600
300
0 0
1
2
Time, h
3
4
5
Fig. 2 Nitrate and nitrite profiles during the denitrification of nuclear industry effluent containing 2268 mg/L NO3-N in a 40-L batch reactor (alpha activity=0.04 Bq/mL) MLSS=8.5 g/L (standard error=±5 %)
12000
300
10000
250
8000
200
6000
150
4000
100
2000
50
NH3, mg/L
COD, mg/L
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0
0 0
20
40
60
80
100
120
Time, day COD out
NH3,Out
Fig. 3 COD and ammonia profile in a CSTR during the denitrification of nitrate waste from a nuclear plant (feed concentration=2268 mg/L NO3-N, HRT=48 h, reactor volume=32 L) (standard error=±5 %)
prevailing conditions. The nature and amount of carbon source plays an important role in the choice of pathway. It is argued that excess amount of carbon would favor organisms that used electron acceptors most efficiently [17]. Hence, further studies were carried out with low C/NO3-N ratio (3), and surface aeration was provided to prevent strict anaerobic conditions. Under these conditions, ammonia formation was reduced but was not inhibited completely (Table 1). Therefore, C/NO3-N ratio was reduced further to 1.5 that resulted into incomplete denitrification and prevented ammonia formation completely (Fig. 4). Increase in C/NO3-N ratio from 1.5 to 2.5 again resulted into ammonia formation. Minimum ammonia formation was observed at C/NO3-N of 2.25. Ammonia formation was completely prevented at C/NO3-N ratio of 2.0. Also, complete denitrification was achieved at the same C/NO3-N ratio (2). Hence, for pilot scale studies, C/NO3-N ratio of 2 was used. Material balance for nitrogen at different C/NO3-N ratio showed that with increase in C/NO3-N ratio, conversion of nitrate to ammonia increased (Table 1). Maximum conversion Table 1 Material balance calculations for conversion of NO3-N into nitrogen and ammonia during denitrification of nuclear industry effluent in a 32-L CSTR (NO3-N=2268 mg/L) C/ NO3N
NO3- NO2- NH3- N in the % Conversion of NO3 into NH3-N % Conversion of NO3 into N2 outlet N N N (C/2268×100) (D/2268×100) (D = A + (A) (B) (C) B+C)
1.5
34
88
0
122
0
2
24
78
0
102
0
96
2.25
0
0
20
20
1
99
95
2.5
0
0
20
20
1
99
3
0
0
45
45
2
98
6
0
0
221
221
10
90
Appl Biochem Biotechnol
(a)
Outlet NO3-N, NO2-N (mg/L)
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NO3-N NO2-N
0 60
63
66
69
72
75
Time, d
(b)
Outlet NO3-N, NO2-N (mg/L)
140
NO3-N NO2-N
120 100 80 60
40 20 0 76
79
82
Time, d
85
88
91
Fig. 4 Outlet nitrate and nitrite profiles during the denitrification of nitrate waste from a nuclear plant (a) C/NO3N=1.5; (b) C/NO3-N=2.0 (CSTR=32 L, inlet NO3-N=2268 mg/L, HRT=48 h) (standard error=±5 %)
(9 %) of nitrate to ammonia was observed at C/NO3-N of 6. Nitrite was detected in the outlet sample at C/NO3-N ≤2. Optimum C/NO3-N for complete denitrification was found to be 2.0. Additional step (aeration) was provided to reduce the COD reduced (data not shown). Microbial Consortium Changes During DNRA The reasons behind ammonia formation could be the relative abundance of fermentative and ammonium formers in anaerobic environments compared to true denitrifiers capable of O2 respiration or denitrification only. Most probable number (MPN) was estimated for sludge at different point of time in the process. Three main populations (denitrifiers, ammoniumoxidizing bacteria, and nitrate-oxidizing bacteria) generally found in such consortium were studied at each point of change in C/NO3-N. From Table 2, it can be seen that when the C/NO3-N in the reactor was more than 2.5 (2.5–6), ammonium-oxidizing bacteria (AOB) population increased. The excess ammonium formed during that phase was rapidly acted upon by the AOB population in the reactor, which increased to form nitrite. Nitrite-oxidizing bacteria (NOB) population acts upon the nitrite to form nitrate. The absence of nitrite and nitrate showed that the NOB and denitrifying population immediately took up the substrate to
Appl Biochem Biotechnol Table 2 Plate count and MPN enumeration from the sludge during the various stages of C/NO3-N changes in a 32-L CSTR of total bacteria, denitrifying bacteria (DB, i.e., bacteria reducing N anion without any accumulation of NO2 or NH3), ammonium-oxidizing bacteria (AOB, i.e., bacteria oxidizing NH3 to NO2), and nitrite-oxidizing bacteria (NOB, i.e., bacteria oxidizing NO2 to NO3) C/NO3-Na
DB
AOB
NOB
6 3
4.8×102 3.7×103
3.0×105 5.4×104
2.7×104 1.0×104
1.5
2.4×103
5.0×102
4.5×102
2.5
1.8×103
3.0×103
1.7×101
2.0
3.5×107
1.8×102
2.0×101
2.25
2.7×107
1.0×101
1.9×101
a
Confidence limit (95 %) for sludge during different stages of adaptation
form ammonium which was oxidized again to nitrite by AOB. Similarly, when the C/NO3-N was decreased to 1.5 to reduce ammonia formation, incomplete denitrification was observed. Table 2 shows that though there was no increase in the denitrifying bacteria population, the AOB population decreased drastically. With increase in C/NO3-N from 1.5 to 2.0 and 2.25, complete denitrification was observed which is due to increase in the denitrifying population. The AOB population decreased with decrease in C/NO3-N from 6.0 to 2.25. Thus, it can be concluded that at a C/NO3-N of 2.0–2.25, the microbial consortium degrades nitrate wastes completely via denitrification pathway without accumulation of ammonium or nitrite. It takes the DNRA pathway, when the C/NO3-N is increased above 2.5. Hence, for complete denitrification of the nitrate waste, the C/NO3-N of 2.0–2.25 can be used. C/NO3-N can also be optimized within this range to have maximum denitrification activity. Sustainability of the Denitrification Process on a Pilot Scale Studies in a 32-L CSTR resulted in optimum C/NO3-N ratio of 2.0. Hence, the pilot scale reactor (330 L) was operated for influent nitrate concentration of 2268 and C/NO3-N ratio of 2.0 to study if ammonia formation occurs on a larger scale and also to study the sustainability of the process. Complete denitrification was observed for initial 20 days, and sudden increase in outlet nitrate (0 to 1647 mg/L) and nitrite (0 to 792 mg/L) levels were seen from day 21 onwards (Fig. 5). This could be attributed to the increase in heavy metal concentration in the influent (from 12 to 21 mg/L). Hence, influent concentration was decreased to 1129 mg/L NO3-N, and the reactor was operated for the next 20 days with the same nitrate load till a steady outlet nitrate and nitrite profiles were obtained. It was then increased to 1693 mg/L NO3-N and operated for another 30 days at the same influent concentration. Increase in nitrate load resulted into outlet nitrate and nitrite levels for the first 10 days and subsequently reduced to zero and remained steady for the next 20 days. After steady performance at 1693 mg/L, nitrate concentration was increased to initial level of 2268 mg/L which resulted into small nitrite buildup that diminished after 10 days of continuous operation, and a steady performance (nitrate and nitrite below permissible limits) was observed for the remaining days. MLSS were in the range of 8–10 g/L throughout the studies. Ammonia formation did not take place during the nitrate waste treatment. Thus, the optimum C/NO3-N ratio on both laboratory scale and pilot scale was estimated as 2.0, and the sustainability of the process was verified on a pilot scale level.
Appl Biochem Biotechnol
2500
2000 NO3-N, NO2-N, mg/L
NO3-Nin NO3-Nout NO2-Nout
1500
1000
500
0
0
20
40
60
80
100
120
140
160
Time, d
Fig. 5 Nitrate and nitrite profiles during the denitrification of 2268 mg/L NO3-N in a 330-L CSTR (HRT=30 h, MLSS=1.5–4.0 g/L) (standard error=±5 %)
Conclusions Acclimatized biomass denitrified high strength nitrate waste (2268 mg/L NO3-N) (containing various heavy metals and radioactivity 0.04 Bq/mL) in a laboratory scale (40-L batch and 32-L CSTR) and pilot scale reactor (330 L) over a long term. High C/NO3-N ratio resulted in ammonia formation due to microbiological changes in the sludge consortium. Ammonia formation was prevented by optimizing the C/NO3-N ratio (2.0). Pilot scale studies showed no ammonia formation, and complete denitrification of the nitrate was obtained. Acknowledgments The authors would like to thank the Department of Atomic Energy (DAE), Government of India, for funding this work and Rashtriya Chemicals and Fertilizers (RCF), Mumbai, India, for providing the sludge for the experiments.
References 1. 2. 3. 4. 5. 6. 7. 8.
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Appl Biochem Biotechnol 11. Nair, R. R., Dhamole, P. B., Lele, S. S., & D’Souza, S. F. (2008). Applied Biochemistry and Biotechnology, 151, 193–200. 12. Shahabi, Z. A., & Naeimpoor, F. (2014). Applied Biochemistry and Biotechnology, 73, 741–752. 13. APHA, AWWA, WEF. (1992). Washington, DC: APHA. 14. APHA, AWWA, WEF. (1997). Washington, DC: APHA. 15. Yin, S. X., Chen, D., Chen, L. M., & Edis, R. (2002). Soil Biology and Biochemistry, 34, 1131–1137. 16. Ma, H., & Aelion, C. M. (2005). Soil Biology and Biochemistry, 37, 1869–1878. 17. Tiedje, J. M. (1988). In A. J. B. Zehnder (Ed.), Biology of anaerobic microorganisms. New York: Wiley.
